TW201125580A - Anti- α -enolase I antibodies for diagnosis and treatment of α -enolase I-associated diseases - Google Patents

Anti- α -enolase I antibodies for diagnosis and treatment of α -enolase I-associated diseases Download PDF

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TW201125580A
TW201125580A TW99102465A TW99102465A TW201125580A TW 201125580 A TW201125580 A TW 201125580A TW 99102465 A TW99102465 A TW 99102465A TW 99102465 A TW99102465 A TW 99102465A TW 201125580 A TW201125580 A TW 201125580A
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antibody
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cancer
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TW99102465A
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TWI468176B (en
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Sy-Jye Leu
Yi-Yuan Yang
Neng-Yao Shih
I-Jen Huang
Ko-Jiunn Liu
Yu-Chin Lee
Yung-Luen Shih
Bor-Yu Tsai
Yu-Jia Chang
Yuan-Soon Ho
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Univ Taipei Medical
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Abstract

The invention relates to antibodies against α -enolase I, their pharmaceutical compositions and diagnosis and treatment uses. Particularly, the invention provides polyclonal anti- α -enolase I antibodies and monoclonal single-chain variable fragment (scFv) anti- α -enolase antibodies, pharmaceutical compositions containing the same and their uses in uses in diagnosis and treatment of cancers, autoimmune disorders, ischemia and bacterial infection.

Description

201125580 六、發明說明: 【發明所屬之技術領域】 本發明係關於抗α-異烯醇酶1(或稻发 、為α-異烯醇酶)之抗 體、其醫藥組合物以及其診斷及治療用途。特定言之,本 發明提供多株抗《_異婦醇酶!抗體及單株單鍵可^區片段 (scFv)抗α·異稀醇酶抗體、含其之醫藥組合物以及直用於 診斷及治療癌症、自體免疫病症、局部缺血及細菌感染之 用途。 【先前技術】 癌症之特徵在於源自正常組織之異常或贅生性細胞數目 增加’該等細胞增殖形成腫瘤塊’此等 入相鄰組織,以及產生惡性細胞,該等惡性細胞最終㈣ 血液或:巴系統擴散至局部淋巴結且經由稱作轉移之過程 擴散至遠處部位。對該等腫瘤相關細胞表面抗原多肤之鑑 別已產生特異絲向癌細胞之能力以經由基於抗體之療法 進行破壞。在發現癌症療法或_之有效細胞縣之嘗試 中,研究人員已設法鐘別出相較於—或多種正常非癌細胞 而特異性過度表現於特定類型之癌細胞表面上的多肽。在 發現癌症療法之有效細胞標靶之其他嘗試中,研究人員已 設法鑑別出由特定類型之癌細胞產生且分泌之多肽,其表 現量高於由-或多種正常非癌細胞產生且分泌之多狀了儘 官在哺乳動物癌症療法中已取得上述確認之進展,但非常 需要分別能_測哺乳動物體内腫瘤之存在及用於治療癌 症之其他診斷劑及治療劑。 ' 143761.doc 201125580 異烯醇酶原先被描述的特性為涉及糖解代謝、催化2-磷 酸甘油酸酯轉化為磷酸烯醇丙酮酸酯的酶。在哺乳動物 中,異烯醇酶存在三種同功異型物,稱作α-ΕΝΟΙ、β-ΕΝ03及γ-ΕΝ02。α-異烯醇酶為異烯醇酶的主要形式,其 存在於胚胎發育之早期階段,普遍表現於各類型組織中, 而γ-ΕΝ02及β-ΕΝ03僅發現於神經元及肌肉細胞中 (Antikainen等人,2007, FEMS Immunol Med Microbiol 51, 526-534 ; Chang 等人,2006,Clin Cancer Res 12, 5746-5754)。關於a-異烯醇酶之核酸及胺基酸序列的資訊可自 NCBI 網站 http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi? ORG=Hs&CID = 517145獲得。據報導異烯醇酶為一種展 現酶催化、結構及受體功能之多功能蛋白(Chang等人, 2006,Clin Cancer Res 12,5746-5754 ; Lee 等人,2003, Arthritis Rheum 48, 2025-2035)。除 了其糖解功能之外, 已發現α-異烯醇酶在多種生物學及病理生理學過程中扮演 重要作用。特定言之,α-異烯醇酶被認為在腫瘤形成中扮 演重要作用。該蛋白質發現於細胞表面上,充當一種可在 腫瘤侵入中發揮作用的纖維蛋白溶酶原受體(Redlitz等 人,1995, Eur J Biochem 227,407-415)。已報導在多種高 致瘤性或轉移性細胞株中α-異烯醇酶受到上游調控(Chang 等人,2006,Clin Cancer Res 12, 5746-5754 ; Peebles 等 人,2003, Carcinogenesis 24, 65 1-657 ; Satoshi Ito, 2007, Cancer Science 98,499-505 ; Wu 等人,2002, Clin Exp Metastasis 19,319-326 ; Zhang等人,2000,J Surg Res 93, 143761.doc 201125580 108-119)。α-異烯醇酶過度表現與導致多類型癌症的致瘤 性相關,表明其在癌症形成中之病理生理學作用 (Altenberg及 Greulich,2004,Genomics 84,1014-1020)。此 外,在非小細胞肺癌中鑑別出(X-異烯醇酶之自體抗原,且 其過度表現與不良存活結果高度相關(Chang等人,2006, Clin Cancer Res 12,5746-5754)。除 了在癌症中之作用之 外,α-異烯醇酶已涉及眾多疾病,包括自體免疫病症、局 部缺血及細菌感染。(Antikainen等人,2007, FEMS Immunol Med Microbiol 51, 526-534 ; Bogdanos 等人 > 2004,J Autoimmune Dis 1,4 ; Gitlits 等人,2001,J Investig Med 49,138-145 ; Jiang等人,1997,Cancer Res 57,5328-5335 ; Kinloch等人,2005,Arthritis Res Ther 7, R1421-1429 ; Saulot等人,2002, Arthritis Rheum 46, 1196-1201) 〇 因此,a-異烯醇酶為用於治療或預防癌症發展之治療劑 或用於偵測癌症之潛在標靶。仍需要偵測、治療、預防及 逆轉癌症之發展。 【發明内容】 本發明之一目的在於提供一種特異性結合α-異烯醇酶I 之禽類抗α-異烯醇酶I多株抗體。 本發明之另一目的在於提供一種純化單株抗體或其抗原 結合片段,其包含結合α-異烯醇酶I之重鏈免疫球蛋白可 變域及輕鏈免疫球蛋白可變域,其中該輕鏈免疫球蛋白可 Γ 變域包含以下胺基酸序列:(i)CDRl中之SGGSGSYG(SEQl 143761.doc 201125580 ID NO: 1)、SGGSSSYGYG(SEQ ID NO: 2)、SGSSGSYG (SEQ ID NO: 3)、SGGSSSYGYS(SEQ ID NO: 4)或 SGSSGYGYG(SEQ ID NO: 5) ; (ii)CDR2 中之 ANTNRPS (SEQ ID NO: 6)、NDNQRPS(SEQ ID NO: 7)、RDDKRPS (SEQ ID NO: 8)、SNNQRPS(SEQ ID NO: 9)或 SNDKRPS (SEQ ID NO: 10);及(iii)CDR3 中之 GGYDSSAGI(SEQ ID NO: 11)、GSGDSSTGM(SEQ ID NO: 12)、GSGESSTNNGI (SEQ ID NO: 13)、GSMDSSNSGV(SEQ ID NO: 14)或 GGYDSSASYVGI(SEQ ID NO: 15);且其中該重鏈免疫球 蛋白可變域包含以下胺基酸序列:(i)CDRl中之SFNMF (SEQ ID NO: 16)、SHDMG(SEQ ID NO: 17)、DYCVQ (SEQ ID NO: 18)、SFYMF(SEQ ID NO: 19)或 SYAMH(SEQ ID NO: 20) ; (ii)CDR2 中之 GINNAGSTTNHGAAVKG(SEQ ID NO: 21) ' GIENAAGIGTFYGAAVKG(SEQ ID NO: 22) ' AISNTGRYTGYGSAVKG(SEQ ID NO: 23) > GISGDGRYTGYGAAVDG (SEQ ID NO: 24)或 GISRDGGSSTRYYGAAVKG(SEQ ID NO: 25);及(iii)CDR3 中之SPGGIDGIDG(SEQ ID NO: 26)、 GADTGGWPAANIDA (SEQ ID NO: 27) ' DGCAGCCGSYYIDG (SEQ ID NO: 28) ' ESGSGCCNGDNIDA(SEQ ID NO: 29)或 DSDNGGYYCDDIDA(SEQ ID NO: 30)。 本發明之另一目的在於提供一種醫藥組合物,其包含本 發明之多株抗體及醫藥學上可接受之載劑。 本發明之另一目的在於提供一種治療或預防α-異烯醇酶 I相關病症之方法,其包含投與治療有效量之本發明之多 143761.doc 201125580 株抗體或單株抗體。 本發明之另一目的在於提供一種用於偵測樣品中異烤 醇酶I之存在的活體外診斷方法,其包含:⑴使樣品與本 發明之抗α-異烯醇酶I抗體接觸;及(ii)偵測該α_異烯醇酶工 抗aa與该樣品之間的複合物的形成。 本發明之另一目的在於提供一種用於偵測樣品中α_異烯 醇酶I之存在的套組,其包含本發明之抗心異烯醇酶I抗體 或其片段,及視情況存在之資訊材料。 【實施方式】 本發明藉由噬菌體呈現系統產生且表徵免疫雞的多株抗 =異烯醇_抗體及單株單鏈可變區片段(心如異稀醇 酶I抗體。此等抗體可有助於開發用於癌症、自體免疫病 症、局部缺血及細@感染之分子診斷劑及治療劑。 定義 應注意到,術語 個抗異烯醇酶抗體 體。因而,術語「 可互換使用。 一」係指一或多個;舉例而言,「一 」應理解為表示一或多個抗異烯醇酶抗 」'「一或多」及「至少一」在本文中 如本文中所用,術註·「八,, _ 衍。口刀離」或「純化」意謂多肽已自 其自然環境之組份中鋏别月八 〒鉸別及分離及/或回收。其自然環境 之污染組份為通常合不振夕„丄 曰干擾多肽之診斷或治療用途的物質, 且可包括酶、激素及其他蛋 貝或非蛋白質溶質。 如本文中所用,術語「枋 a」係指能夠結合所選標靶的 完整抗體或抗體片段。句把p ( c 括 Fv、ScFv、Fab•及 F(ab,)2、單 143761.doc 201125580 株及多株抗體、經工程改造抗體(包括嵌合抗體、CDR移 植及人類化抗體、完全人類抗體,及人工選擇抗體),及 使用嗟體呈現或替代技術產生之合成或半合成抗體。諸 如Fv及scFv之小片段因其小尺寸及隨之產生之優良組織分 布而具有用於診斷及治療應用之有利特性。 術語「抗體」進一步意欲涵蓋抗體、其消化片段、特定 部分及變異體,包括抗體模擬物或包含模擬抗體或其特定 片段或部分之結構及/或功能的抗體部分,包括單鏈抗體 及其片段。功能性片段包括結合α_異烯醇酶之抗原結合片 段。舉例而言,本發明涵蓋能夠結合心異烯醇酶或其部分 之抗體片段,包括(但不限於)Fab(例如,藉由木瓜酶消化 所產生)、Fab’(例如,藉由胃蛋白酶消化及部分還原所產 生)及F(ab,)2(例如,藉由胃蛋白酶消化所產生)、(例 如,藉由纖維蛋白溶酶消化所產生)、pFcI(例如,藉由胃 蛋白酶或纖維蛋白溶酶消化所產生)、Fd(例如,藉由胃蛋201125580 VI. Description of the Invention: [Technical Field] The present invention relates to an anti-α-isoenolase 1 (or rice cultivar, α-isoenolase) antibody, a pharmaceutical composition thereof, and a diagnosis and treatment thereof use. In particular, the present invention provides multiple strains of anti-"isokinase! Antibody and single-stranded single-stranded fragment (scFv) anti-α-isololase antibody, pharmaceutical composition containing the same, and use for direct diagnosis and treatment of cancer, autoimmune disease, ischemia and bacterial infection . [Prior Art] Cancer is characterized by an abnormality in the normal tissue or an increase in the number of neoplastic cells, which form a tumor mass, into the adjacent tissues, and the production of malignant cells, which eventually (4) blood or: The bar system spreads to the local lymph nodes and spreads to distant sites via a process called metastasis. The identification of these tumor-associated cell surface antigens has produced the ability of specific filaments to cancer cells to be destroyed via antibody-based therapies. In an attempt to discover cancer therapies or effective cell counts, researchers have managed to discern peptides that are specifically overexpressed on the surface of specific types of cancer cells compared to - or multiple normal non-cancer cells. In other attempts to discover effective cellular targets for cancer therapy, researchers have sought to identify polypeptides produced and secreted by certain types of cancer cells that are expressed in a higher amount than that produced by or - a variety of normal non-cancer cells. The above-mentioned confirmed progress has been made in mammalian cancer therapy, but it is highly desirable to separately detect the presence of tumors in mammals and other diagnostic agents and therapeutic agents for treating cancer. ' 143761.doc 201125580 Isoenolase was originally described as an enzyme involved in glycolytic metabolism and catalyzing the conversion of 2-phosphoglycerate to phosphoenolpyruvate. In mammals, isoenolase has three isoforms, called α-ΕΝΟΙ, β-ΕΝ03, and γ-ΕΝ02. Α-isoenolase is the main form of isoenolase, which is present in the early stages of embryonic development and is commonly found in various types of tissues, while γ-ΕΝ02 and β-ΕΝ03 are found only in neurons and muscle cells ( Antikainen et al, 2007, FEMS Immunol Med Microbiol 51, 526-534; Chang et al, 2006, Clin Cancer Res 12, 5746-5754). Information on the nucleic acid and amino acid sequences of a-isoenolase is available on the NCBI website at http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi? ORG=Hs&CID = 517145. Isoenolase is reported to be a multifunctional protein exhibiting enzyme catalysis, structure and receptor function (Chang et al., 2006, Clin Cancer Res 12, 5746-5754; Lee et al., 2003, Arthritis Rheum 48, 2025-2035 ). In addition to its glycolytic function, alpha-isoenolase has been found to play an important role in a variety of biological and pathophysiological processes. In particular, α-isoenolase is thought to play an important role in tumor formation. This protein is found on the cell surface and acts as a plasminogen receptor that can play a role in tumor invasion (Redlitz et al., 1995, Eur J Biochem 227, 407-415). Alpha-isoenolase has been previously regulated in a variety of highly tumorigenic or metastatic cell lines (Chang et al, 2006, Clin Cancer Res 12, 5746-5754; Peebles et al, 2003, Carcinogenesis 24, 65 1 -657; Satoshi Ito, 2007, Cancer Science 98, 499-505; Wu et al, 2002, Clin Exp Metastasis 19, 319-326; Zhang et al, 2000, J Surg Res 93, 143761.doc 201125580 108-119) . Overexpression of α-isoenolase is associated with tumorigenicity leading to multiple types of cancer, indicating its pathophysiological role in cancer formation (Altenberg and Greulich, 2004, Genomics 84, 1014-1020). Furthermore, autoantigens of X-isoenolase have been identified in non-small cell lung cancer, and their overexpression is highly correlated with poor survival outcomes (Chang et al, 2006, Clin Cancer Res 12, 5746-5754). In addition to its role in cancer, alpha-isoenolase has been implicated in a number of diseases, including autoimmune disorders, ischemia and bacterial infections. (Antikainen et al., 2007, FEMS Immunol Med Microbiol 51, 526-534; Bogdanos Et al. 2004, J Autoimmune Dis 1, 4; Gitlits et al, 2001, J Investig Med 49, 138-145; Jiang et al, 1997, Cancer Res 57, 5328-5335; Kinloch et al, 2005, Arthritis Res Ther 7, R1421-1429; Saulot et al., 2002, Arthritis Rheum 46, 1196-1201) 〇 Therefore, a-isoenolase is a therapeutic agent for the treatment or prevention of cancer development or a potential marker for detecting cancer There is still a need to detect, treat, prevent, and reverse the development of cancer. SUMMARY OF THE INVENTION One object of the present invention is to provide a multi-strain of avian anti-α-isoenolase I that specifically binds α-isoenolase I. Another object of the present invention is Provided is a purified monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain immunoglobulin variable domain and a light chain immunoglobulin variable domain which binds to α-isoenolase I, wherein the light chain immunoglobulin is Γ The variable domain comprises the following amino acid sequence: (i) SGGSGSYG (SEQ ID 143761.doc 201125580 ID NO: 1), SGGSSSYGYG (SEQ ID NO: 2), SGSSSGSYG (SEQ ID NO: 3), SGGSSSYGYS (SEQ ID) in CDR1 NO: 4) or SGSSGYGYG (SEQ ID NO: 5); (ii) ANTNRPS (SEQ ID NO: 6), NDNQRPS (SEQ ID NO: 7), RDDKRPS (SEQ ID NO: 8), SNNQRPS (SEQ) in CDR2 ID NO: 9) or SNDDRPS (SEQ ID NO: 10); and (iii) GGYDSSAGI (SEQ ID NO: 11), GSGDSSTGM (SEQ ID NO: 12), GSGESSTNNGI (SEQ ID NO: 13), GSMDSSNSGV in CDR3 (SEQ ID NO: 14) or GGYDSSASYVGI (SEQ ID NO: 15); and wherein the heavy chain immunoglobulin variable domain comprises the following amino acid sequence: (i) SFNMF (SEQ ID NO: 16) in CDR1, SHDMG (SEQ ID NO: 17), DYCVQ (SEQ ID NO: 18), SFYMF (SEQ ID NO: 19) or SYAMH (SEQ ID NO: 20); (ii) GINNAGSTTNHGAAVKG (SEQ ID NO: 21) in CDR2 ' GIENAAGIGTFYGAAVKG (SEQ ID NO: 22) ' AISN TGRYTGYGSAVKG (SEQ ID NO: 23) > GISGDGRYTGYGAAVDG (SEQ ID NO: 24) or GISRDGGSSTRYYGAAVKG (SEQ ID NO: 25); and (iii) SPGGIDGIDG (SEQ ID NO: 26), GADTGGWPAANIDA (SEQ ID NO: 27) 'DGCAGCCGSYYIDG (SEQ ID NO: 28) 'ESGSGCCNGDNIDA (SEQ ID NO: 29) or DSDNGGYYCDDIDA (SEQ ID NO: 30). Another object of the present invention is to provide a pharmaceutical composition comprising the polyclonal antibody of the present invention and a pharmaceutically acceptable carrier. Another object of the present invention is to provide a method for treating or preventing an α-isoenolase I-related disorder comprising administering a therapeutically effective amount of the 143761.doc 201125580 strain antibody or monoclonal antibody of the present invention. Another object of the present invention is to provide an in vitro diagnostic method for detecting the presence of isobeolase I in a sample, which comprises: (1) contacting a sample with the anti-α-isoenolase I antibody of the present invention; (ii) detecting the formation of a complex between the α-isoenolase anti-aa and the sample. Another object of the present invention is to provide a kit for detecting the presence of α-isoenolase I in a sample comprising the anti-cardinolase I antibody of the present invention or a fragment thereof, and optionally Information materials. [Embodiment] The present invention generates and characterizes multiple strains of anti-isoenol-antibody and single-stranded variable-chain fragment of a single chicken by a phage display system (hearts such as isoamylase I antibody. These antibodies may have Helps to develop molecular diagnostics and therapeutics for cancer, autoimmune disorders, ischemia and fine infections. Definitions It should be noted that the term anti-isoenolase antibody bodies. Thus, the term "is used interchangeably. And "at least one" as used herein, as used herein, The "Notes", "Eight, _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ A substance that is generally ambiguous to interfere with the diagnostic or therapeutic use of the polypeptide, and may include enzymes, hormones, and other eggshell or non-protein solutes. As used herein, the term "枋a" refers to the ability to combine Targeted intact antibody or antibody fragment. sentence put p ( c including Fv, ScFv, Fab• and F(ab,)2, single 143761.doc 201125580 strain and multiple antibodies, engineered antibodies (including chimeric antibodies, CDR grafts and humanized antibodies, fully human antibodies, and labor) Selection of antibodies), and synthetic or semi-synthetic antibodies produced using steroid rendering or alternative techniques. Small fragments such as Fv and scFv have advantageous properties for diagnostic and therapeutic applications due to their small size and consequent excellent tissue distribution. The term "antibody" is further intended to encompass antibodies, digested fragments, specific portions and variants thereof, including antibody mimetics or antibody portions comprising mimicking the structure and/or function of a particular fragment or portion thereof, including single chain antibodies and Fragments. Functional fragments include antigen-binding fragments that bind to alpha-isoenolase. For example, the invention encompasses antibody fragments capable of binding to cardinolase or a portion thereof, including but not limited to Fabs (eg, Produced by papain digestion), Fab' (for example, by pepsin digestion and partial reduction) and F(ab,) 2 (for example, by pepsin elimination) Generated), (e.g., by plasmin digestion generated), pFcI (e.g., by pepsin or plasmin digestion generated), Fd (e.g., by pepsin

製劑不同, 不同抗體的習知(多株)抗體 具有針對單一抗原位點的高特異性。此 對不同決定基(抗原決定基)之不同抗體 上之單一決定基。 各單株抗體係針對抗原上之單 143761.doc 201125580 因此,修飾語「單株」指示自一群由實質上同源族群之 抗體獲得之抗體的特徵,且不應理解為需要藉由任何特定 方法產生抗體。舉例而言,根據本發明使用之單株抗體可 由此項技術中已知之融合瘤方法製備,或可由重組〇1^八方 法(諸如美國專利第4,816,567號中所述者)製備。舉例而 言,「單株抗體」亦可自例如使用McCafferty等人, Nature,348:552-554 (199〇)中所述之技術產生的噬菌體文 庫分離。 術語「特異性結合」一般意謂抗體經由其抗原結合域结 合抗原決定基’且結合要求抗原結合域與抗原決定基之; 有-些互補性。根據此定義’當抗體經由其抗原結合域結 合抗原決定基比抗體結合隨機無關抗原決定基更容易時',。 稱抗體「特異性結合」該抗原決定基。術語「特显性、在 本文中用於限定某種抗體結合某種抗原決定基之相對親和 如本文中所用之術語「治療」及「療法」係指治療性 法及預防性療法。 灯往 術§吾「癌症」及「癌性 或述特徵通常為細胞 ^ ° ?㉟生理學病狀。癌症之實例包括(但不 於)癌瘤、淋巴瘤、母細胞瘤、肉瘤及白血病。該等痒 之更特定實例包括鱗狀細 ^ 痒、田 小細胞肺癌、非小細胞 ^ 甘細胞瘤、胃腸癌、瞥杳 D. 工 癌、神經膠母細胞瘤 甲、,工母、、,田胞瘤、子宮頸癌、卵 田Different formulations, conventional (multi-strain) antibodies of different antibodies have high specificity for a single antigenic site. This is a single determinant on different antibodies to different determinants (antigenic determinants). Each monoclonal antibody is directed against the antigen on a single 143761.doc 201125580. Thus, the modifier "single plant" indicates the characteristics of an antibody obtained from a population of antibodies from a substantially homologous population and should not be construed as requiring any particular method. Produce antibodies. For example, monoclonal antibodies for use in accordance with the present invention can be prepared by the fusion tumor method known in the art, or can be prepared by recombinant methods such as those described in U.S. Patent No. 4,816,567. For example, "monoclonal antibodies" can also be isolated, for example, from phage libraries generated using the techniques described in McCafferty et al, Nature, 348:552-554 (199). The term "specifically binds" generally means that an antibody binds to an epitope via its antigen-binding domain and binds to an antigen-binding domain and an epitope; there is some complementarity. According to this definition, when an antibody binds to an epitope via its antigen-binding domain, it is easier to bind a random unrelated epitope than the antibody. The antibody is said to "specifically bind" to the epitope. The term "specificity, as used herein, to define the relative affinity of an antibody for binding to an epitope" as used herein, as used herein, refers to both therapeutic and prophylactic therapies. Lights § My "cancer" and "cancerous or characterized are usually cells ^ ° 35 physiological conditions. Examples of cancer include (but not) cancer, lymphoma, blastoma, sarcoma and leukemia. More specific examples of such itch include squamous itch, small cell lung cancer, non-small cell glandoma, gastrointestinal cancer, sputum D. cancer, glioma cell, A, M, Field tumor, cervical cancer, egg field

癌、肝細胞瘤、乳痒 "4、月癌、膀J 礼癌、、,》知癌、結腸直腸癌、子宮内/ 143761.doc 201125580 癌、唾液腺癌、腎癌、肝癌、前列腺癌、外陰癌、甲狀腺 癌、肝癌及各種類型之頭頸癌。 術語「個體」或「動物」或「患者」或「哺乳動物」意 謂需要診斷或治療之任何個體,尤其哺乳動物個體。哺乳 動物個體包括人類、家畜、農畜,及動物園、競技或寵物 動物,諸如犬、I苗、天竺鼠、兔、大鼠、小鼠、馬、牛、 奶牛等。 抗體 本發明係關於特異性結合α_異烯醇酶〗多肽之抗體 '其 抗原結合抗體片段以及抗體變異體及月段。此等抗體可為 例如多株或單株抗體。更佳為單株抗體。仍更佳為嵌合或 人類化抗體,且仍更佳為人類抗體。 多株抗體 在一態樣中,本發明提供一種特異性結合心異烯醇酶丁 之禽類抗α_異烯醇酶多株抗體。在本發明之一實施例中, 。亥禽為雞且多株抗體為多株抗α_異烯醇酶〗“Υ抗體。多株 抗體較佳為抗α_異烯醇酶以隹匕丫抗體。 此項技術中已知多株抗體之產生。首先,利用人類心異 烯醇酶I多肽之多次注射使禽物種(例如雞、鴨、火雞及其 頜似物)免疫以在該禽中引起免疫原性反應。在經歷適合 時間以在禽體内形成高力價之抗α_異烯醇酶1抗體後,自 經α_異烯醇酶I免疫之禽採集血清或卵。 免疫禽之血清及卵黃含有對許多天然暴露抗原具特異性 、夕種抗體。5亥方法之下一步驟為自血清或卵黃中分離免 14376l.doc 201125580 疫球蛋白之IgY溶離伤(已知該溶離份含有α-異婦醇酶特異 性抗體)。此可藉由利用例如Promega Corporation之 EGGstract® IgY純化系統(Promega Corporation,Madison, Wis. U.S.A.)及 HiTrap™ IgY純化 HP 管柱(GE Healthcare, U.S.A.)之商業產品用卵黃來達成。亦存在自卵黃中分離免 疫球蛋白之其他多種方法,諸如其他依序沈澱方法,其為 熟習此項技術者所熟知。(參見例如Sc〇pes, R κ 「Protein Purification: Principles andPractice」,Springer_Cancer, hepatocellular carcinoma, itching "4, monthly cancer, bladder cancer, cancer, colorectal cancer, intrauterine / 143761.doc 201125580 cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, Vulvar cancer, thyroid cancer, liver cancer and various types of head and neck cancer. The term "individual" or "animal" or "patient" or "mammal" means any individual, particularly a mammalian subject, in need of diagnosis or treatment. Mammalian animals include humans, livestock, farm animals, and zoos, athletics, or pet animals such as dogs, I seedlings, guinea pigs, rabbits, rats, mice, horses, cows, cows, and the like. Antibody The present invention relates to an antibody which specifically binds to an α-isoolase polypeptide, an antigen-binding antibody fragment thereof, and an antibody variant and a segment. Such antibodies may be, for example, multiple strains or monoclonal antibodies. More preferably, it is a monoclonal antibody. Still more preferred are chimeric or humanized antibodies, and still better as human antibodies. Multi-Strain Antibodies In one aspect, the present invention provides a multi-strain antibody against avian anti-α-isoenolase that specifically binds to cardinolase. In an embodiment of the invention, . The hai poultry is chicken and the multi-strain antibody is a plurality of anti-α-isoenolase Υ Υ antibody. The multi-antibody antibody is preferably an anti-α-isoenolase 隹匕丫 antibody. A plurality of antibodies are known in the art. First, multiple injections of the human heart isoenolase I polypeptide are used to immunize avian species (eg, chickens, ducks, turkeys, and their jaws) to cause an immunogenic response in the bird. Time to collect serum or eggs from avian mice immunized with α_isoenolase I after forming a high-valence anti-α-isoenolase 1 antibody in poultry. The immunological avian serum and yolk contain many natural exposures. The antigen is specific and the antibody is used. The next step of the 5H method is to isolate the IgY-dissolved wound of 14376l.doc 201125580 phage globulin from serum or egg yolk (the lysate is known to contain α-isochelinase specificity). Antibody) This can be achieved by using the egg yolk of a commercial product such as Promega Corporation's EGGstract® IgY Purification System (Promega Corporation, Madison, Wis. USA) and HiTrapTM IgY Purified HP Column (GE Healthcare, USA). Isolation and immunity from egg yolk Other methods of globulin, such as other sequential precipitation methods, are well known to those skilled in the art (see, for example, Sc〇pes, R κ "Protein Purification: Principles and Practice", Springer_

Verlag New York, 1994,其關於蛋白質純化方法之教示係 以引用的方式併入本文中)。完全令人滿意地實施本發明 」出蛋白質溶離份,此係Verlag New York, 1994, its teachings on protein purification methods are incorporated herein by reference). Fully satisfactorily implementing the present invention, "the protein is dissolved, this system

的習知蛋白質分離方法為「鹽析」出4 藉由使蛋白質自鹽溶液中沈澱來進行, 自禽金清分離出IgY多株抗體。此外, 方法。The conventional protein separation method is "salting out" 4 by precipitating the protein from the salt solution, and isolating the IgY antibody from the avian gold. In addition, the method.

單株抗體 I4376I.doc 201125580 在另一態樣中,本發明提供一種結合〇t -異稀醇酶I之純 化單株抗體或其抗原結合片段,其包含重鏈免疫球蛋白可 變域及輕鏈免疫球蛋白可變域,其中該輕鏈免疫球蛋白可 變域包含以下胺基酸序列:(i)CDRl中之SGGSGSYG(SEQ ID NO: 1)、SGGSSSYGYG(SEQ ID NO: 2)、SGSSGSYG (SEQ ID NO: 3)、SGGSSSYGYS(SEQ ID NO: 4)或 SGSSGYGYG(SEQ ID NO: 5) ; (ii)CDR2 中之 ANTNRPS (SEQ ID NO: 6)、NDNQRPS(SEQ ID NO: 7)、RDDKRPS (SEQ ID NO: 8)、SNNQRPS(SEQ ID NO: 9)或 SNDKRPS (SEQ ID NO: 10);及(iii)CDR3 中之 GGYDSSAGI(SEQ ID NO: 11)、GSGDSSTGM(SEQ ID NO: 12)、GSGESSTNNGI (SEQ ID NO: 13)、GSMDSSNSGV(SEQ ID NO: 14)或 GGYDSSASYVGI(SEQ ID NO: 15);且其中該重鏈免疫球 蛋白可變域包含以下胺基酸序列:(i)CDRl中之SFNMF (SEQ ID NO: 16) 、 SHDMG(SEQ ID NO: 17)、 DYCVQ(SEQ ID NO: 18)、SFYMF(SEQ ID NO: 19)或 SYAMH(SEQ ID NO: 20) ; (ii)CDR2 中之 GINNAGSTTNHGAAVKG (SEQ ID NO: 21) > GIENAAGIGTFYGAAVKG(SEQ ID NO: 22) ' AISNTGRYTGYGSAVKG(SEQ ID NO: 23)、 GISGDGRYTGYGAAVDG(SEQ ID NO: 24)或Monoclonal antibody I4376I.doc 201125580 In another aspect, the present invention provides a purified monoclonal antibody or antigen-binding fragment thereof comprising 〇t-isololase I, comprising a heavy chain immunoglobulin variable domain and light A chain immunoglobulin variable domain, wherein the light chain immunoglobulin variable domain comprises the following amino acid sequence: (i) SGGSGSYG (SEQ ID NO: 1), SGGSSSYGYG (SEQ ID NO: 2), SGSSGSYG in CDR1 (SEQ ID NO: 3), SGGSSSYGYS (SEQ ID NO: 4) or SGSSGYGYG (SEQ ID NO: 5); (ii) ANTNRPS (SEQ ID NO: 6), NDNQRPS (SEQ ID NO: 7) in CDR2, RDDKRPS (SEQ ID NO: 8), SNNQRPS (SEQ ID NO: 9) or SNDDRPS (SEQ ID NO: 10); and (iii) GGYDSSAGI (SEQ ID NO: 11), GSGDSSTGM (SEQ ID NO: 12) in CDR3 , GSGESSTNNGI (SEQ ID NO: 13), GSMDSSNSGV (SEQ ID NO: 14) or GGYDSSASYVGI (SEQ ID NO: 15); and wherein the heavy chain immunoglobulin variable domain comprises the following amino acid sequence: (i) SFNMF (SEQ ID NO: 16), SHDMG (SEQ ID NO: 17), DYCVQ (SEQ ID NO: 18), SFYMF (SEQ ID NO: 19) or SYAMH (SEQ ID NO: 20) in CDR1; GINNAGSTTNHGAAVKG in CDR2 (SEQ ID NO: 2) 1) > GIENAAGIGTFYGAAVKG (SEQ ID NO: 22) ' AISNTGRYTGYGSAVKG (SEQ ID NO: 23), GISGDGRYTGYGAAVDG (SEQ ID NO: 24) or

GISRDGGSSTRYYGAAVKG(SEQ ID NO: 25);及(iii)CDR3 中之 SPGGIDGIDG(SEQ ID NO: 26) ' GADTGGWPAANIDA (SEQ ID NO: 27)、DGCAGCCGSYYIDG(SEQ ID NO: 28)、 ESGSGCCNGDNIDA(SEQ ID NO: 29)或 DSDNGGYYCDDIDA 143761.doc 201125580 (SEQ ID NO: 30)。GISRDGGSSTRYYGAAVKG (SEQ ID NO: 25); and (iii) SPGGIDGIDG (SEQ ID NO: 26) in CDR3 ' GADTGGWPAANIDA (SEQ ID NO: 27), DGCAGCCGSYYIDG (SEQ ID NO: 28), ESGSGCCNGDNIDA (SEQ ID NO: 29 ) or DSDNGGYYCDDIDA 143761.doc 201125580 (SEQ ID NO: 30).

在一實施例中,本發明之純化單株抗體或其抗原結合片 段包含選自由雞免疫球蛋白之生殖系基因序列編碼之VH * 及VL FR1、FR2、FR3及FR4構架區的構架區(關於VL及 * VH,請分別參閱 www.ncbi.nlm.nih.gov/nuccore/16902088 及 http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=protein& dopt=GenPept&RID=MS2P2YSH012&log%24=prottop&blast_rank=l &list_uids=104726)或胺基酸序列與由雞免疫球蛋白之生殖系 基因序列編碼之VH及VL FR1、FR2及FR3構架區至少85% 相同的構架區(framework region)。純化抗體或其抗原結合 片段較佳包含胺基酸序列與由雞免疫球蛋白之生殖系基因 序列編碼之VH及VL FR1、FR2及FR3構架區至少87%、 88%、89%、90%、91%、92%、93%、94%、95%、97%、 98%或99%相同的構架區。在某些實施例中,本發明之單 株抗體之VH及VL結構域可經生殖系化,亦即可使用習知 分子生物學技術改變此等結構域之構架區(FR)以在一或多 個位置(例如,至少 70%、80%、85%、87%、88%、89%、 90%、91%、92%、93%、94。/〇或 95%、97%、98%或 99%的 ‘ 構架位置)匹配人類生殖系基因或人類生殖系基因產物之 共同胺基酸序列。在其他實施例中,構架序列不同於生殖 系。 在一實施例中,本發明之純化單株抗體或其抗原結合片 段包含具有選自由以下胺基酸序列組成之群之胺基酸序列 Γ 的VL互補決定區:i)CDRl中之SEQ ID NO:l ' CDR2中之^ •13· 143761.doc 201125580 SEQ ID NO: 6及 CDR3 中之 SEQ ID NO: 11 ; ii)CDRl 中之 SEQ ID NO: 2、CDR2 中之 SEQ ID NO: 7及 CDR3 中之 SEQ ID NO: 12 ; iii)CDRl 中之 SEQ ID NO: 3、CDR2 中之 SEQ ID NO: 8及 CDR3 中之 SEQ ID NO: 1 3 ; iv)CDRl 中之 SEQ ID NO: 4、CDR2 中之 SEQ ID NO: 9及 CDR3 中之 SEQ ID NO: 14及 v)CDRl 中之 SEQ ID NO: 5、CDR2 中之 SEQ ID NO: 10及CDR3中之SEQ ID NO: 15,及具有選自由以下胺 基酸序列組成之群之胺基酸序列的VH互補決定區: i)CDRl 中之 SEQ ID NO: 16、CDR2 中之 SEQ ID NO: 21及 CDR3 中之 SEQ ID NO: 26 ; ii)CDRl 中之 SEQ ID NO: 17、 CDR2 中之 SEQ ID NO: 22及 CDR3 中之 SEQ ID NO: 27; iii)CDRl 中之 SEQ ID NO: 18、CDR2 中之 SEQ ID NO: 23及 CDR3 中之 SEQ ID NO: 28 ; iv)CDRl 中之 SEQ ID NO: 19、 CDR2 中之 SEQ ID NO: 24及 CDR3 中之 SEQ ID NO: 29及 v)CDRl 中之 SEQ ID NO: 20、CDR2 中之 SEQ ID NO: 25及 CDR3中之SEQ ID NO: 30。較佳地,VL互補決定區具有ii) 或v)中所提及之胺基酸序列且VH互補決定區具有ii)或v)中 所提及之胺基酸序列。 在另一實施例中,本發明之純化單株抗體或其抗原結合 片段包含具有選自由SEQ ID NO: 3 1-35組成之群之胺基酸 序列的輕鏈免疫球蛋白可變域及具有選自由SEQ ID NO: 36-40組成之群之胺基酸序列的重鏈免疫球蛋白可變域。 在一實施例中,抗體或其片段之CDR序列與本文中所述 抗體之CDR序列僅有非實質性差異。非實質性差異包括較 143761.doc -14- 201125580 少胺基酸改變,諸如CDR(例如,Chothia或Kabat CDR)序 列中通常任何5-7個胺基酸中之1或2個胺基酸取代。通常 胺基酸係由具有類似電荷、疏水性或立體化學特性之相關 胺基酸取代。該等取代屬於一般技術者之技能範圍内。與 CDR中不同,可在對抗體之結合性質無不利影響的情況下 對結構構架區(FR)作出更多實質性改變。對FR之改變包括 (但不限於)使非人源構架人類化或工程改造某些對抗原接 觸或對結合位點穩定化重要的構架殘基,例如,改變恆定 區之種類或次類,改變可能改變效應子(effector)功能(諸 如Fc受體結合)之特定胺基酸殘基(Lund等人,(1991) J. Immunol. 147:2657-62 ; Morgan等人,(1995) Immunology 86:319-24),或改變衍生自恆定區之物種。 本發明之單株抗體或其抗原結合片段可藉由噬菌體呈現 (phage display)技術獲得。嗟菌體呈現涉及使肽以與鞘蛋 白(例如噬菌體粒子之pill、pIIV)末端融合的形式定位。參 見 Scott,J· K.及 G. P. Smith (1990) Science 249(4967):386-390 ;及Lowman,Η. Β·等人,(1991) Biochem. 30 (45): 10832-10838。一般而言,具有特異性結合功能之多肽係 如下分離:與標靶一起培育,洗掉非結合噬菌體,溶離已 結合嗤菌體,且隨後藉由感染新鮮細菌培養物而使嗤菌體 群體再擴增。其他呈現形式及方法包括mRNA呈現、核糖 體或多核糖體呈現、真核病毒呈現,及細菌、酵母及哺乳 動物細胞表面呈現。參見Mattheakis, L. C.等人,(1994) Γ PNAS USA 91 (19): 9022-9026 ; Wilson,D. S.等人,‘ 143761.doc -15· 201125580 (2001) PNAS USA 98(7):3750-3755 ; Shusta,E. V.等人, (1999) Curr. Opin. Biotech. 10(2):117-122 ;以及Boder,E. T.及 K. D. Wittrup (1997) Nature Biotech. 15(6):553-557。 已開發且報導用於在微生物表面上呈現的多種替代呈現技 術且作為分離蛋白質結合肽之一般策略加以推行,但尚未 報導成功。參見 Maurer, J.等人,(1997) J. Bacteriol. 179(3):794-804 ; Samuelson, P.等人,(1995) J. Bacteriol. 177(6):1470-1476 ; Robert,A.等人,(1996) FEBS Letters 390(3): 327-333 ; Stathopoulos,C. #Λ,(1996)Αρρ1· Microbiol. & Biotech· 45(1-2): 112-119 ; Georgiou,G.等 人,(1996) Protein Engineering 9(2):239-247 ; Haddad, D. 等人’(1995)?丑:\18 11111111111〇1.&]^(11。31^^(;1*〇1^〇1.12(3- 4):175-186 ; Pallesen,L.等人,(1995) Microbiol. 141(Pt 11):2839-2848 ; Xu,Z·及 S. Y. Lee (1999) Appl. Environ. Microbiol. 65(11):5142-5147 ; Wernerus,H.及 S_ Stahl (2002) FEMS Microbiol, Lett. 212(1): 47-54 ;及In one embodiment, the purified monoclonal antibody or antigen-binding fragment thereof of the present invention comprises a framework region selected from the VH* and VL FR1, FR2, FR3 and FR4 framework regions encoded by the germline gene sequence of chicken immunoglobulin (about VL and * VH, please refer to www.ncbi.nlm.nih.gov/nuccore/16902088 and http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=protein& dopt= GenPept&RID=MS2P2YSH012&log%24=prottop&blast_rank=l &list_uids=104726) or the amino acid sequence and at least the VH and VL FR1, FR2 and FR3 framework regions encoded by the germline gene sequence of chicken immunoglobulin 85% of the same framework region. Preferably, the purified antibody or antigen-binding fragment thereof comprises an amino acid sequence and at least 87%, 88%, 89%, 90% of the VH and VL FR1, FR2 and FR3 framework regions encoded by the germline gene sequence of chicken immunoglobulin, 91%, 92%, 93%, 94%, 95%, 97%, 98% or 99% identical framework regions. In certain embodiments, the VH and VL domains of the monoclonal antibodies of the invention can be germlined, or the framework regions (FR) of such domains can be altered using conventional molecular biology techniques. Multiple locations (eg, at least 70%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94./〇 or 95%, 97%, 98%) Or 99% of the 'framework position' matches the common amino acid sequence of the human germline gene or human germline gene product. In other embodiments, the framework sequence is different from the reproductive system. In one embodiment, the purified monoclonal antibody or antigen-binding fragment thereof of the present invention comprises a VL complementarity determining region having an amino acid sequence 选自 selected from the group consisting of: i) SEQ ID NO in CDR1 : l ' CDR2 ^ 13 143761.doc 201125580 SEQ ID NO: 6 and SEQ ID NO: 11 in CDR3; ii) SEQ ID NO: 2 in CDR1, SEQ ID NO: 7 and CDR3 in CDR2 SEQ ID NO: 12; iii) SEQ ID NO: 3 in CDR1, SEQ ID NO: 8 in CDR2 and SEQ ID NO: 1 3 in CDR3; iv) SEQ ID NO: 4, CDR2 in CDR1 SEQ ID NO: 14 in SEQ ID NO: 9 and CDR3, SEQ ID NO: 5 in CDR1, SEQ ID NO: 10 in CDR2 and SEQ ID NO: 15 in CDR3, and having The VH complementarity determining region of the amino acid sequence of the following amino acid sequence: i) SEQ ID NO: 16 in CDR1, SEQ ID NO: 21 in CDR2 and SEQ ID NO: 26 in CDR3; ii) SEQ ID NO: 17 in CDR1, SEQ ID NO: 22 in CDR2 and SEQ ID NO: 27 in CDR3; iii) SEQ ID NO: 18 in CDR1, SEQ ID NO: 23 and CDR3 in CDR2 SEQ ID NO: 28; iv) CDR1 SEQ ID NO: 19, SEQ ID NO: 24 in CDR2 and SEQ ID NO: 29 and v in CDR3 SEQ ID NO: 20 in CDR1, SEQ ID NO: 25 in CDR2 and SEQ ID NO in CDR3 : 30. Preferably, the VL complementarity determining region has the amino acid sequence mentioned in ii) or v) and the VH complementarity determining region has the amino acid sequence mentioned in ii) or v). In another embodiment, the purified monoclonal antibody or antigen-binding fragment thereof of the present invention comprises a light chain immunoglobulin variable domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 1-35 and having The heavy chain immunoglobulin variable domain of the amino acid sequence of the group consisting of SEQ ID NO: 36-40 is selected. In one embodiment, the CDR sequences of the antibodies or fragments thereof are only insubstantially different from the CDR sequences of the antibodies described herein. Non-substantial differences include less amino acid changes than 143761.doc -14-201125580, such as one or two amino acid substitutions in any of the 5-7 amino acids in the CDR (eg, Chothia or Kabat CDR) sequence. . Typically, the amino acid is substituted with an associated amino acid having similar charge, hydrophobicity or stereochemical properties. Such substitutions are within the skill of those of ordinary skill. Unlike in CDRs, more substantial changes can be made to the structural framework regions (FR) without adversely affecting the binding properties of the antibodies. Alterations to FR include, but are not limited to, humanizing or engineering non-human frameworks for certain framework residues that are important for antigen contact or stabilization of binding sites, for example, changing the type or subclass of constant regions, changing Specific amino acid residues that may alter the effector function (such as Fc receptor binding) (Lund et al, (1991) J. Immunol. 147: 2657-62; Morgan et al, (1995) Immunology 86: 319-24), or change the species derived from the constant region. The monoclonal antibodies or antigen-binding fragments thereof of the present invention can be obtained by phage display technology. The bacterium cell presentation involves localization of the peptide in a form fused to the end of the sheath protein (e.g., pill, pIIV) of phage particles. See Scott, J. K. and G. P. Smith (1990) Science 249 (4967): 386-390; and Lowman, Η. Β· et al., (1991) Biochem. 30 (45): 10832-10838. In general, a polypeptide having a specific binding function is isolated by incubation with a target, washing off the non-binding phage, dissolving the bound sputum, and then cultivating the sputum population by infecting the fresh bacterial culture. Amplification. Other presentation formats and methods include mRNA presentation, ribosome or polysome display, eukaryotic virus presentation, and bacterial, yeast, and mammalian cell surface presentation. See Mattheakis, LC et al., (1994) Γ PNAS USA 91 (19): 9022-9026; Wilson, DS et al., '143761.doc -15· 201125580 (2001) PNAS USA 98(7): 3750-3755; Shusta, EV et al, (1999) Curr. Opin. Biotech. 10(2): 117-122; and Boder, ET and KD Wittrup (1997) Nature Biotech. 15(6): 553-557. A variety of alternative presentation techniques for presentation on the surface of microorganisms have been developed and reported as general strategies for isolating protein binding peptides, but have not been reported successfully. See Maurer, J. et al., (1997) J. Bacteriol. 179(3): 794-804; Samuelson, P. et al., (1995) J. Bacteriol. 177(6): 1470-1476; Robert, A Et al., (1996) FEBS Letters 390(3): 327-333; Stathopoulos, C. #Λ, (1996)Αρρ1·Microbiol. & Biotech· 45(1-2): 112-119 ; Georgiou, G Et al., (1996) Protein Engineering 9(2): 239-247; Haddad, D. et al. (1995)? Ugly: \18 11111111111〇1.&]^(11.31^^(;1 *〇1^〇1.12(3- 4): 175-186; Pallesen, L. et al., (1995) Microbiol. 141(Pt 11): 2839-2848; Xu, Z· and SY Lee (1999) Appl. Environ. Microbiol. 65(11): 5142-5147; Wernerus, H. and S_ Stahl (2002) FEMS Microbiol, Lett. 212(1): 47-54;

Westerlund-Wikstrom, B. (2000) Int. J. Med. Microbiol. 290(3):223-230 ° 本文中所述之多株或單株抗體亦可標記可偵測或功能性 標記。可偵測標記包括諸如ηι〗或99Tc之放射性標記,其 可使用此項技術中習知之化學方法連接至本文中所述之抗 體。標記亦包括酶標記’諸如辣根過氧化酶或鹼性磷酸 酶。標記進一步包括諸如生物素之化學部分,其可經由結 合特疋同源可偵測部分(例如經標記之抗生物素蛋白)加以 143761.doc -16· 201125580 偵測。 本文中所揭示抗體之結合特性可由任何適合方法量測, 包括以下方法:Biacore分析、酶結合免疫吸附分析 (ELIS A)、X射線結晶學、序列分析及掃描突變誘發,及此 項技術中熟知之其他方法。 本發明之醫藥組合物 抗ex-異烯醇酶I抗體可併入醫藥組合物中,例如與醫藥 學上可接受之載劑組合H且合物亦可含有例如多種稀釋 劑、填充劑、鹽、緩衝劑、穩定劑、增溶劑,及此項技術 中熟知之其他物質。術語「醫藥學上可接受」意謂不干擾 活性成份之生物活性效力的無毒物質。載劑之特性可視投 藥途徑而定。 如本文中所用,術語「治療有效量」意謂醫藥組合物或 方法之各種活性組份之足以顯示患者有意義受益(例如改 善該等病狀之症狀、治療該等病狀或提高該等病狀之治疥 速旬的總量。當應用於單獨投與之個別活性成份時,該 術語係指單獨彼成份。當應用於組合時’該術語係指產生 治療效果之活性成份的組合量,不論組合、依序或同時投 與。 在實施本文中所述之例示性治療方法或用途時,可將治 療有效量之結合α_異烯醇酶〗 ㈣墓…‘ 功能性^異烯醇酶1信 =複合物形成(且例如中和或抑制一或 每相關活性)之抗體投與個體,例如哺乳動物(例如人〇 可根據所述方法將抗體單獨或與其他療法組合❹。舍, 143761.doc 201125580 -或多種藥劑共投與時’抗體可與第二藥劑同時投與,或 分別(例如依序)投與。若分別(例如依序)投與,則主治醫 師將決定投與抗體與其他藥劑之適當順序。 醫藥組合物(例如,含有可結合α_異烯醇酶之抗體的醫 藥組合物)之投與可以多種習知方式進行,諸如經口攝 入、吸入,或經皮、皮下或靜脈内注射。皮下投與患者為 較佳。 當治療有效量之結合α_異烯醇如且干擾功能性α•異烯醇 酶I信號傳導複合物形成之抗體係經口投與時,結合劑將 王錠劑$囊 '散劑、溶液或驰劑形式。當呈鍵劑形式投 與時’醫藥組合物可另外含有諸如明膠之固體载劑或: 劑。錠劑、膠囊及散劑含有約5%至95%結合劑,且較佳約 25%至9G%結合劑。當呈液體形式投與時,可添加液體载 劑’諸如水、石油、動物或植物來源之油(諸如花生油、 礦物油、大丑油或芝麻油)或合成油。醫藥組合物之液體 形式可進一步含有生理鹽水溶液、右旋糖或其他醣類溶 液,或諸如乙二醇、丙二醇或聚乙二醇之二醇類。 當治療有效量之結合異烯醇酶I之抗體係藉由靜脈 内、經皮或皮下注射投與時,結合劑將呈無熱原質、非經 腸可接丈之水洛液形式。製備該等非經腸可接受之蛋白質 溶液(具有預定pH值、等張性、穩定性及其類似性質)在此 項技術範圍内已知。用#起^ 用於靜脈内、經皮或皮下注射之較佳 醫藥組合物除了含有結合劑之外,亦應含有等張性媒劑, 諸如氯化鈉庄射劑、林袼氏注射劑(r㈣响如。^、右 143761.doc -18- 201125580 旋糖;主射劑、右旋糖及氯化鈉注射劑、乳酸化林格氏注射 背J或此項技術中已知之其他媒劑。醫藥組合物亦可含有穩 定劑、防腐劑、緩衝劑、抗氧化劑或熟習此項技術者已知 的其他添加劑。 醫藥組合物中抗體之量可視所治療病狀之性質及嚴重程 度以及患者已經歷之先前治療的性質而定。最終,主治醫 師將決定用於治療各個別患者之抗體量。最初,主治醫師 將投與低劑量之抗體且觀察患者之反應。可投與較大劑量 之抗體直至獲得對患者之最佳治療效果,且此時一般不再 進一步增加劑量。舉例而言,可投與劑量之範圍為每公斤 體重 0.1-50 mg、0.5-50 mg ' 1-100 mg、〇 5_25 mg、〇 卜15 mg或 1-8 mg。 抗α-異烯醇酶1抗體之治療及預防用途 本發明之抗體可用於治療α-異烯醇酶I相關病症,例 如,選自以下一或多者之病症:癌症、自體免疫病症、局 部缺血及細菌感染。 α·異婦醇酶I及其受體可能涉及至少—些類型之癌症(例 如’源自造灰細胞之癌症或源自肺癌之癌症)的發展。癌 症較佳為肺癌、乳癌、肛門癌、膀胱癌、骨癌、腸癌、腦 腫瘤、腎癌、白血病、肝癌、胰癌、前列腺癌、直腸癌。 肺癌更佳為非小細胞肺癌。癌症係指—或多種細胞喪失對 正常生長控制之反應性,且通常隨調節減低(相對於相應 正常細胞而言)而增殖。 抗α-異烯醇酶I抗體之診斷用途 [ 143761.doc 19 201125580 在另一悲樣中’本發明提供一種活體外偵測α-異烯醇酶 I之存在的#斷方法(例如生物樣品(諸如組織)活體檢查)。 該方法包括.(1)使樣品與本發明之抗α_異烯醇抗體接 觸,及(η)偵測α·異烯醇酶“充體與樣品之間之複合物的形 成。该方法亦可包括使參考樣品(例如對照樣品)與配位體 接觸,及相對&參考樣品測定配位體與樣品之間之複合物 形成的程度。相對於對照樣品或個體,樣品或個體中之複 合物形成之變化(例如統計學顯著變化)可指示樣品中存在 (X-異烯醇酶I。 可藉由量測或顯現異烯醇_結合之配位體或未結 合配位體來偵測《_異烯醇酶!抗體與α_異料酶【之間的複 合物形成。可使用習知偵測檢^,例如酶結合免疫吸附分 析(ELISA)、放射性免疫分析(RIA)或組織免疫組織化學。 除標記α-異烯醇_;E抗體外,亦可利用標記可偵測物質之 標準物及未標記之α_異烯醇❸抗體藉由競爭免疫分析法 分析樣品中α-異稀醇酶I之存在。 套組 抗異稀醇酶!抗體或其片段可例如作為套組組份提供 :套組中。舉例而言’套組包括(伽異烯醇酶!抗體或 〃片段,例如包括抗α-異烯醇抗體或其片段之^且入 物,及視情況存在之(b)資訊材料。資訊材料可 所述方法及/或抗α_異烯醇,抗體或其片段用於: 述方法之㈣有關的描述性、指導性、銷售或其他材料。 套組之貧訊材料形式不受限制。在—實施例中,資訊材 I4376I.doc •20· 201125580 料可包括關於化合物生產、化合物分子量、濃度、有效曰 期、批次之資訊,或生產地點資訊等。在一實施例中,資 訊材料係關於使用配位體治療、預防或診斷本文中所述之 病症。 套組可包括一或多個用於合古 夕调用於3有抗《-異烯醇酶I抗體或1 二之組合物的容器。在-些實施例令,套組含有用於組 貧輯科之各別容11、間隔物或隔室。舉例而古, 組合物可包含於瓶、小瓿或注射器中,且資訊材^ 於塑膠封套或封包中。在其他實施例中,套組之各別要辛 包t於早—未分隔容器中。舉例而言,組合物包含於附有 標戴形式之貧訊材料的瓶、小觀或注射器中。在―些垂扩 例中,套組包括複數個(例如一包)個別容器,各容器:有 一或多個抗α異婦醇酶1抗體或其片段之單位劑型(例如本 文中所述之劑型)。舉例而言,套組包括複數個注射哭、 安瓶、落包、霧化器或吸入裝置,其各含有一個單位劑量 之抗心異料酶1抗體或其片段,或多個單位劑量。 t組視情況包括適於投與組合物之裝置,例如注射器、 吸官、錄子、置匙、滴管(例如滴眼管)、藥簽(例如棉質藥 簽或木製藥簽),或任何此等傳遞裝置。在一較佳實施例 中’裝置為分配定劑量之配位體的可植入裝置。 下述實例有助於理解本發明,但不意欲且不應理解為以 任何方式限制本發明之範疇。 實例 實例1表徵_化之重組α·異烯醇酶及多株抗α_異稀醇酶[s 143761.doc -21 · 201125580Westerlund-Wikstrom, B. (2000) Int. J. Med. Microbiol. 290(3): 223-230 ° The multi-plant or monoclonal antibodies described herein may also be labeled with detectable or functional markers. Detectable labels include radioactive labels such as ηι or 99Tc, which can be linked to the antibodies described herein using chemical methods known in the art. The label also includes an enzyme label 'such as horseradish peroxidase or alkaline phosphatase. The label further includes a chemical moiety, such as biotin, which can be detected by binding to a homologous detectable moiety (e.g., labeled avidin) 143761.doc -16·201125580. The binding properties of the antibodies disclosed herein can be measured by any suitable method, including the following methods: Biacore analysis, enzyme-bound immunosorbent assay (ELIS A), X-ray crystallography, sequence analysis, and scanning mutation induction, and are well known in the art. Other methods. The pharmaceutical composition of the present invention may be incorporated into a pharmaceutical composition, such as in combination with a pharmaceutically acceptable carrier, and may also contain, for example, various diluents, fillers, salts. Buffers, stabilizers, solubilizers, and other materials well known in the art. The term "pharmaceutically acceptable" means a non-toxic substance that does not interfere with the biologically active efficacy of the active ingredient. The nature of the carrier can be determined by the route of administration. The term "therapeutically effective amount" as used herein means that the various active ingredients of a pharmaceutical composition or method are sufficient to demonstrate meaningful benefit to the patient (eg, to ameliorate the symptoms of the condition, to treat the condition, or to ameliorate the condition The total amount of 疥 旬 。. When applied to individual active ingredients administered separately, the term refers to a single component. When applied to a combination, the term refers to the combined amount of active ingredients that produce a therapeutic effect, regardless of In combination, sequential or simultaneous administration. In the practice of the exemplary therapeutic methods or uses described herein, a therapeutically effective amount of a combination of alpha-isoenolase can be administered (4) tomb... 'functional ^isoenolase 1 The antibody that the letter = complex forms (and, for example, neutralizes or inhibits one or each of the relevant activities), is administered to an individual, such as a mammal (e.g., human sputum can be used alone or in combination with other therapies according to the methods described. 舍, 143,761. Doc 201125580 - or when multiple agents are administered together, antibodies can be administered simultaneously with the second agent, or separately (eg, sequentially). If administered separately (eg, sequentially), the attending physician will decide to vote Appropriate sequence of body and other agents. Administration of pharmaceutical compositions (e.g., pharmaceutical compositions containing antibodies that bind to alpha-isoenolase) can be carried out in a variety of conventional manners, such as oral ingestion, inhalation, or via Intradermal, subcutaneous or intravenous injection. Subcutaneous administration of the patient is preferred. When a therapeutically effective amount of the combination of α-isoenol, such as and interferes with the formation of functional α•isoenolase I signaling complex, is orally administered. In the meantime, the binding agent will be in the form of a sachet, a powder, a solution or a medicinal agent. When administered as a keying agent, the pharmaceutical composition may additionally contain a solid carrier such as gelatin or a: a tablet, a capsule, and The powder contains from about 5% to 95% binder, and preferably from about 25% to 9G% binder. When administered in liquid form, a liquid carrier such as an oil of water, petroleum, animal or vegetable origin may be added (such as Peanut oil, mineral oil, large oil or sesame oil) or synthetic oil. The liquid form of the pharmaceutical composition may further comprise a physiological saline solution, dextrose or other saccharide solution, or such as ethylene glycol, propylene glycol or polyethylene glycol. Glycols When the therapeutically effective amount of the combination of the isoenolase I is administered by intravenous, transdermal or subcutaneous injection, the binding agent will be in the form of a pyrogen-free, non-enteral extractable water solution. Such parenterally acceptable protein solutions (having predetermined pH, isotonicity, stability, and the like) are known in the art. They are used for intravenous, percutaneous or subcutaneous injection. Preferably, the pharmaceutical composition should contain an isotonic vehicle in addition to the binding agent, such as sodium chloride smear, Linyi injection (r (four) ringing. ^, right 143761.doc -18- 201125580 Main injection, dextrose and sodium chloride injection, lactated Ringer's injection back J or other vehicles known in the art. Pharmaceutical compositions may also contain stabilizers, preservatives, buffers, antioxidants Or other additives known to the skilled artisan. The amount of antibody in a pharmaceutical composition will depend on the nature and severity of the condition being treated and the nature of the prior treatment that the patient has experienced. Ultimately, the attending physician will determine the amount of antibody used to treat each individual patient. Initially, the attending physician will administer a low dose of antibody and observe the patient's response. A larger dose of antibody can be administered until the optimal therapeutic effect is obtained for the patient, and at this time no further dose increase is generally made. For example, the dose can be administered in the range of 0.1-50 mg per kg body weight, 0.5-50 mg ' 1-100 mg, 〇 5_25 mg, 〇 15 mg or 1-8 mg. Therapeutic and prophylactic use of an anti-α-isoenolase 1 antibody The antibody of the present invention can be used for the treatment of an α-isoenolase I-related disorder, for example, a condition selected from one or more of the following: cancer, autoimmune disorder, Ischemia and bacterial infection. Alpha-isokinase I and its receptors may be involved in the development of at least some types of cancers, such as 'cancers derived from ash-producing cells or cancers derived from lung cancer. The cancer is preferably lung cancer, breast cancer, anal cancer, bladder cancer, bone cancer, intestinal cancer, brain tumor, kidney cancer, leukemia, liver cancer, pancreatic cancer, prostate cancer, and rectal cancer. Lung cancer is more preferably non-small cell lung cancer. Cancer refers to the loss of reactivity to normal growth control, or a variety of cells, and usually proliferate as regulation decreases (relative to the corresponding normal cells). Diagnostic use of anti-α-isoenolase I antibody [143761.doc 19 201125580] In another sad example, the present invention provides an in vitro method for detecting the presence of α-isoenolase I (eg, biological samples) (such as organization) biopsy). The method comprises: (1) contacting the sample with the anti-α-isoenol antibody of the present invention, and (η) detecting the formation of a complex between the enrichment and the sample by the α-isoenolase. This may include contacting a reference sample (eg, a control sample) with a ligand, and determining the extent of complex formation between the ligand and the sample relative to the & reference sample. Composites in the sample or individual relative to the control sample or individual Changes in the formation of the substance (eg, statistically significant changes) may indicate the presence of (X-isoenolase I in the sample. It can be detected by measuring or visualizing the isoenol-bound ligand or unbound ligand). Complex formation between "isoenolase!antibody and alpha-heterologous enzyme. A conventional detection assay such as enzyme-binding immunosorbent assay (ELISA), radioimmunoassay (RIA) or tissue immunization can be used. Histochemistry. In addition to labeling α-isoenol _; E antibody, it is also possible to analyze the α-isotype in the sample by competitive immunoassay using the standard of the label detectable substance and the unlabeled α-isoenol oxime antibody. The presence of dilute alcoholase I. Kit anti-isoxanolase! antibody or The segment can be provided, for example, as a kit component: in a kit. For example, a 'set includes (a galenolase! antibody or a guanidine fragment, for example, including an anti-alpha-isoenol antibody or a fragment thereof) And (b) informational material as appropriate. The informational material may be described by the method and/or anti-alpha_isoenol, antibody or fragment thereof for: descriptive, instructional, sales or other The form of the poor information material of the kit is not limited. In the embodiment, the information material I4376I.doc •20· 201125580 may include information on the production of the compound, the molecular weight of the compound, the concentration, the effective period, the batch, or Production location information, etc. In one embodiment, the informational material relates to the use of a ligand to treat, prevent, or diagnose a condition as described herein. The kit may include one or more for the combination of the ancient and the old for 3 - a container of an isoenolase I antibody or a combination of two. In some embodiments, the kit contains individual compartments 11, spacers or compartments for use in the group of poor families. Can be included in bottles, small bottles or syringes, and information materials ^ In a plastic envelope or package, in other embodiments, the kits are each packaged in an early-un-separated container. For example, the composition is contained in a bottle with a poorly-formed material in the form of a logo, small In a syringe or a syringe, in some of the dimples, the kit includes a plurality (eg, a pack) of individual containers, each container: one or more unit dosage forms of an anti-alpha iso-livalase 1 antibody or fragment thereof (eg, The dosage form described herein. For example, the kit includes a plurality of injection crying, ampoules, drop bags, nebulizers or inhalation devices each containing a unit dose of an anti-heart dismutase 1 antibody or fragment thereof, Or multiple unit doses. The t group includes, as appropriate, devices suitable for administration of the composition, such as syringes, sputum, recordings, spoons, droppers (eg, eye drops), swabs (eg, cotton swabs or Wood Pharmaceuticals sign), or any such transfer device. In a preferred embodiment, the device is an implantable device that dispenses a dose of ligand. The following examples are provided to aid the understanding of the invention, but are not intended to be construed as limiting the scope of the invention in any way. Example 1 characterization of recombinant α-isoenolase and multiple anti-α-isoglycolase [s 143761.doc -21 · 201125580

IgY抗體 藉由反轉錄-PCR、使用基因特異性引子5·-GGTGGAATTCTATCTATTCTCAAGATCCATGCC-3\^q ID NO·· 41)(前置引子)及5,-ACTCCATGGTTAC7TCGCC4y4GGGG77TCT-3' (SEQ ID NO: 42)(反置引子)自PE089細胞選殖出編碼α-異 烯醇酶蛋白之基因。所得PCR片段選殖於pGEX-KG載體中 EcoRI及Ncol位點上且轉型於大腸桿菌BL-21(DE3)菌株中 以便其表現。該基因亦使用5’- CCGCGTGAATTCGGGGATCCATGTCTATTCTCAAGATCC-3,(SEQ ID NO: 43)(前置引子)及5’- CATGGAGTCGACCTCGXGCTTGGCCAAGGGGTTTCTG-3'(SEQ ID NO: 44)(反置引子)次選殖於pET21a載體中且以 His融合α-異烯醇酶形式表現。在37°C下使個別純系在5 ml 含有安比西林(ampicillin)(l 00 pg/ml)之LB培養基中生長隔 夜。用相同LB培養基將細菌培養物稀釋10倍且使其進一步 生長,直至OD6G()達到0.6與1_0之間。為誘導GST融合α-異 烯醇酶或His融合α-異烯醇酶蛋白質表現,添加異丙基-β-D-硫代哌喃半乳糖苷(IPTG)直至其在培養物中之最終濃度 為0.5 mM。將細胞小球再懸浮於2 ml含有1% Triton X-100 的lxPBS中且利用三輪冷凍(-70°C)及解凍(37。〇溶胞。離 心後,所得細胞溶胞物根據製造商之說明(General Electronics,Piscataway, NJ,USA)與楚胱甘肽瓊脂糖 4B(Glutathione Sepharose 4B)或 Ni2+-帶電荷樹脂管柱 γ 起 培育以純化α-異烯醇酶蛋白。 143761.doc -22- 201125580 在電泳及庫馬斯藍(Coomassie blue)染色後,經純化之 His融合α-異烯醇酶及GST融合α-異烯醇酶顯現為48kD及 75kD之單色帶(圖1A中分別為泳道2及3)。如圖1B泳道3中 所示,使用抗GST抗體證實GST融合α-異烯醇酶之身分。 類似地,經純化His融合α-異烯醇酶免疫之雞中產生的多 株IgY抗體能夠明確識別西方墨點上固定之His融合α-異烯 醇酶與GST融合α-異烯醇酶(圖1C中之泳道2及3)。 實例2雞的免疫 藉由肌肉内注射、以含於等體積之弗氏完全佐劑 (Freund's complete adjuvant)中之 100 pg純化 α-異稀醇酶使 雖性白萊亨雞(white leghorn)(家雞(Ga//"·? 免 疫。以7天之時間間隔用不完全佐劑進行另外三次免疫。 在各次免疫後,採集血清及卵黃中之IgY抗體且藉由酶聯 免疫吸附檢定(ELISA)進行滴定以測定體液抗α-異烯醇酶 免疫反應之存在。如先前所述(Akita及Nakai, 1993a, b)使 用10%硫酸葡聚糖將卵黃與卵白分離以進行IgY純化。將 來自各卵之經純化總IgY抗體溶解於5 ml含有0.05%疊氮化 鈉的TBS中且儲存於-20°C下。 在各次免疫之前及之後採集雞之血清及印。將印黃中之 總IgY抗體純化且使用結合辣根過氧化酶之抗雞IgY抗體偵 測重鏈與輕鏈片段的存在(資料未展示)。使用純化IgY測 試其對固定於硝化纖維素膜(圖1C)或ELISA板孔上之α-異 烯醇酶的結合活性。如圖2中所示,在第4次免疫後自卵黃 製備之IgY抗體在以1:16,000稀釋滴定時特異性結合α-異稀[ 143761.doc -23- 201125580 醇酶而非牛血清白蛋白,表明在雞宿主中引起強體液抗體 反應。與之相反,來自免疫前雞卵之IgY抗體對兩種抗原 均展示極小的結合信號。 實例3 scFv抗體文庫之建構及生物淘選 基於先前報導建立抗體文庫:Andris-Widhopf,J·,IgY antibody by reverse transcription-PCR, using gene-specific primer 5·-GGTGGAATTCTATCTATTCTCAAGATCCATGCC-3\^q ID NO·· 41) (pre-priming) and 5,-ACTCCATGGTTAC7TCGCC4y4GGGG77TCT-3' (SEQ ID NO: 42) Inverse primer) A gene encoding an α-isoenolase protein was selected from PE089 cells. The resulting PCR fragment was cloned into the EcoRI and Ncol sites in the pGEX-KG vector and transformed into E. coli BL-21 (DE3) strain for its expression. This gene was also subcloned into the pET21a vector using 5'-CCGCGTGAATTCGGGGATCCATGTCTATTCTCAAGATCC-3, (SEQ ID NO: 43) (pre-priming) and 5'-CATGGAGTCGACCTCGXGCTTGGCCAAGGGGTTTCTG-3' (SEQ ID NO: 44) (reverse primer). And expressed in the form of His fusion α-isoenolase. Individual pure lines were grown overnight in 5 ml of LB medium containing ampicillin (100 pg/ml) at 37 °C. The bacterial culture was diluted 10-fold with the same LB medium and allowed to grow further until OD6G() reached between 0.6 and 1_0. To induce GST fusion α-isoenolase or His fusion α-isoenolase protein expression, add isopropyl-β-D-thiogalactopyranoside (IPTG) until its final concentration in culture It is 0.5 mM. The cell pellet was resuspended in 2 ml of 1x Triton X-100 in lxPBS and thawed in three rounds (-70 ° C) and thawed (37. lysed. After centrifugation, the resulting cell lysate was according to the manufacturer. Description (General Electronics, Piscataway, NJ, USA) was incubated with glutathione Sepharose 4B or Ni2+-charged resin column γ to purify the α-isoenolase protein. 143761.doc -22 - 201125580 After electrophoresis and Coomassie blue staining, the purified His fusion α-isoenolase and GST fusion α-isoenolase appeared as a 48kD and 75kD monochromatic band (Fig. 1A Lanes 2 and 3). As shown in lane 3 of Figure 1B, the GST-fused α-isoenolase was confirmed using an anti-GST antibody. Similarly, it was produced in a chicken immunized with purified His fusion α-isoenolase. Multiple IgY antibodies can clearly recognize the His-fused α-isoenolase immobilized on Western blots and GST-fused α-isoenolase (lanes 2 and 3 in Figure 1C). Example 2 Chicken Immunity by Muscle Intra-injection, purified at 100 pg in an equal volume of Freund's complete adjuvant Alpha-isosalination enzymes make white leghorns (house chickens (Ga//&) immunization. Three additional immunizations with incomplete adjuvants at 7-day intervals. Thereafter, IgY antibodies in serum and yolk were collected and titrated by enzyme-linked immunosorbent assay (ELISA) to determine the presence of a humoral anti-α-isoenolase immunoreactivity as previously described (Akita and Nakai, 1993a, b) The egg yolk was separated from the egg white using 10% dextran sulfate for IgY purification. The purified total IgY antibody from each egg was dissolved in 5 ml of TBS containing 0.05% sodium azide and stored at -20 °C. The serum and print of chicken were collected before and after each immunization. The total IgY antibody in the yellow was purified and the anti-chicken IgY antibody combined with horseradish peroxidase was used to detect the presence of heavy and light chain fragments (data not shown). The binding activity of α-isoenolase immobilized on the pores of nitrocellulose membrane (Fig. 1C) or ELISA plate was tested using purified IgY. As shown in Fig. 2, prepared from egg yolk after the fourth immunization. IgY antibody specifically binds α-isosaturated at a titration of 1:16,000 [143761.d Oc -23- 201125580 Alcoholase, not bovine serum albumin, indicates a strong humoral antibody response in chicken hosts. In contrast, IgY antibodies from pre-immune chicken eggs exhibit minimal binding signals to both antigens. Example 3 Construction and Biopanning of the scFv Antibody Library Establishment of an antibody library based on previous reports: Andris-Widhopf, J.,

Rader,C·,Steinberger,P.,Fuller, R.,Barbas, C.F.,第 3 版 > 2000, Methods for the generation of chicken monoclonal antibody fragments by phage display. J Immunol Methods 242,15 9-181。簡言之,在最終免疫後採集雞脾且立即置 於Trizol試劑中均質化7天。使用Superscript RT套組 (Invitrogen,USA)將 10 pg總 RNA反轉錄為第一股 cDNA。 使用雞特異性因子擴增後,用短或長連接子使重鏈及輕鏈 可變(VH及VL)區之PCR產物進行第二輪PCR以形成全長 scFv片段,用進一步消化且選殖入pComb3X載體中。 重組DNA藉由電穿孔轉型於大腸桿菌XL-1 blue菌株中。藉 由添加VCS-M13辅助嗟菌體開始產生重組嗤菌體,以4〇/。 聚乙二醇8000及3% NaCl(w/v)沈澱,且最終再次懸浮於含 有1%牛血清白蛋白(BSA)之磷酸鹽緩衝鹽水(pbS)中且儲 存於4°C下。接著’將1011個溶菌斑形成單位(plaque_ forming units,pfu)之來自scFv抗體文庫之重組嗤菌體添 加至預塗α-異烯醇酶蛋白(0.5微克/孔)的孔中,且在37^下 培育2小時。移除未結合噬菌體後,已結合噬菌體用〇丨Μ HC1/甘胺酸(pH 2.2)/0.1% BSA溶離,用2 M Tris驗緩衝液 中和且用於感染XL-1 blue菌株。經擴增嗟菌體如上所述、尤 143761.doc •24· 201125580 澱且回收以進行第二輪選擇。重複淘洗程序三次或四次。 利用最終淘遂過程隨機選擇一組純系且使其生長。在0.5 mM IPTG謗導6小時後,收集細菌細胞且藉由三輪冷凍及 解;東及/或音波處理溶胞。使用西方墨點法及ELIS A分析上 清液之scFv抗體表現及其對心異烯醇酶之結合反應性。在 流動式細胞量測及免疫螢光分析中亦製備ScFv抗體’該等 抗體表現於TOP 10F'大腸桿菌(Invitr〇gen’非抑制菌株)中 且如製造商(Amersham Biosciences,UK)所述使用Ni2+-帶 電荷瓊脂糖純化° 在最後免疫之後8週將雞處死且自腫大之脾提取總RNA 以建構抗體文庫。使用2個連續的PCR步驟擴增全長scFv基 因片段。在第一個PCR中’使用含有短連接子 (GGSSRSS)(SEQ ID NO: 45)及長連接子(GGSSRSSSSGGGGSGGGG) (SEQ ID NO: 46)之引子將VH基因產物擴增為400 bp大 小,分別如EnVH.S(圖3A,泳道2)及EnVH.L(圖3A,泳道 3)所顯示。相應地,VL基因擴增為350 bp之色帶且負載於 圖3A中之泳道4(EnVL)中。隨後,將經擴增VH與VL連接 以形成約750 bp之全長scFv基因片段,如圖3B中之 EnscFv.S(泳道2)及EnscFv.L(泳道3)所顯示。建構多個喔菌 體呈現抗體文庫且用於篩檢特異性抗α-異烯醇酶scFv抗 體。 實例4表徵抗α-異烯醇酶scFv純系 為偵測scFv抗體表現,對細胞溶胞物進行十二烷基硫酸 Γ 鈉聚丙烯醯胺凝膠電泳(SDS-PAGE)。將所有蛋白質轉移^ 143761.doc -25- 201125580 至石肖化纖維素膜(Amersham Biosciences, UK)上,隨後用 5%脫脂牛奶之TBST溶液阻斷1小時。添加1:3000稀釋之多 株山羊抗雞 IgY輕鍵抗體(Bethyl Laboratories, Montgomery, TX,USA)且再培育1小時。用TBST洗滌膜三次,每次5分 鐘。藉由添加1:3000稀釋之辣根過氧化酶(HRP)結合驢抗 山羊Ig抗體(Sigma,St. Louis, MO, USA)來偵測結合抗體。 三次洗滌後,用二胺基聯苯胺(DAB)受質使膜顯影,直至 達到所需強度。為研究其結合反應性,將來自第4次免疫 之雞的純化IgY或經表現scFv抗體與固定於硝化纖維素膜 或ELIS A板孔上之純化α-異烯醇酶一起培育,且隨後藉由 添加如上所述之山羊抗雞IgY輕鏈抗體及HRP結合驢抗山 羊Ig抗體來偵測。各樣品均於雙重複孔中進行ELISA測 試。 如上所述進行四輪淘選。在各次淘選後,隨機選擇15種 純系且對pCom3_X選殖載體中之750 bp片段插入物及其scFv 抗體蛋白表現進行分析。資料(未示)指示各輪淘選之純系 67%(10/15)、87%(13/15)、100%(15/15)及 100%(15/15)具 有全長插入物。此外,最後一輪淘選之10種具有750 bp插 入物之純系經誘導以表現其scFv抗體。如圖4中所示,使 用山羊抗雞IgY輕鏈抗體及辣根過氧化酶(HRP)結合驢抗山 羊Ig抗體,對純系EnL2、EnL4、EnL5、EnL6及EnL7明轉 偵測至|J scFv表現,而對純系EnLl 、EnL3 、EnL8、EnL9及 EnL 1 0極少或未偵測到表現。此等結果指示具有高度保守 序列之免疫球蛋白基因的表現量可顯著不同,即使在相同 143761.doc -26- 201125580 實驗條件下。 實例5基因定序及ELISA分析 藉由自動定序機(ABI PRISM 377 ; Perkin-Elmer, National Health Research Institute)使用 ompseq(5'-AAGACAGCTATCGCGATTGCAGTG-3')(SEQ ID NO: 47)及 HRML-F(5'-GGTGGTTCCTCTAGATCTTCC-3')(SEQ ID NO: 48)引子對所選純系之重鏈及輕鏈可變區進行核苷酸序列 測定。使用比對程式BLAST及載體NTI (http://www.ncbi.nlm.nih.gov/BLAST)分析結果。 測定1 0種純系之重鏈及輕鏈基因之可變區的核苷酸序列 且與雞免疫球蛋白之生殖系基因序列進行比對。結果揭示 3 0%之定序純系(EnL5、EnL6及EnL7)共有相同之重鏈及輕 鏈基因,導致其scFv抗體表現概況相似,如圖4之泳道5、 6及7中所見。與生殖系基因序列相比之總體突變率在重鏈 及輕鏈可變區中分別為18.6°/。至27.4%及13·5%至23.1%(圖 5)。使用£1^13八分析所表現之£11乙1至£111^7 3〇?丫抗體針對〇1-異烯醇酶的結合活性。發現與2種先前已表徵且已知特異 性識別SARS-CoV棘蛋白(spike protein)之其他scFv抗體相 比,彼等scFv抗體片段展現針對α-異烯醇酶的顯著結合活 性。特定言之,與來自經人類α-異烯醇酶分子免疫之雞的 純化多株IgY相比,EnL2、EnL4、EnL5及EnL6 scFv抗體 展示更強的正反應性(圖6(A)及(B))。 實例6流動式細胞量測術(Flowcytometry)分析 PE089細胞株最初獲自患有IV期肺腺癌之36歲患者的積^ 143761.doc -27- 201125580 液腫瘤細胞,其係由來自National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan之Neng-Yao Shih博士友情提供。該等細胞已活體外 於補充有5%胎牛血清、2 mmol/L麩醯胺酸及抗生素之 RPMI 1640中培養至少40個繼代。採集總共2χ106個細胞且 如先前所述用2%三聚甲醛固定。用經純化scFv EnL2及 EnL5抗體偵測表現於PE089細胞中之α-異烯醇酶,以小鼠 抗HA(1:200)及結合Cy-2之山羊抗小鼠抗體(1:200)(Jackson ImmunoResearch Laboratories, West Grove,PA,USA)顯 現,且使用FAC Scan流動式細胞量測儀(Becton Dickinson, Franklin Lakes,NJ,USA)進行分析。在不使用一次scFv EnL2及EnL5抗體的情況下如上所述進行陰性對照分析, 同時使用兔多株抗人類異烯醇酶抗體(1:200)替代scFv EnL2及EnL5抗體來進行陽性對照分析。 為測試該等經選殖scFv抗體之結合反應性,將人類α-異 烯醇酶基因轉染於ΡΕ089腫瘤細胞中以便其於膜表面上表 現,隨後藉由流動式細胞量測術進行分析。SDS-PAGE上 為單色帶之經純化EnL2及EnL5 scFv抗體(資料未示)能夠 偵測表現於PE089細胞中之重組α-異烯醇酶蛋白,其結合 信號與對α-異烯醇酶具特異性之市售兔多株抗體之結合信 號類似,如圖7中所示。 實例7免疫螢光及雷射掃描顯微法 將ΡΕ089細胞(2 xlO5個細胞/毫升)接種於蓋玻片上且藉由 用等體積之冰冷8%三聚甲醛(新鮮製備)於冰上培育15分鐘 143761.doc -28- 201125580 進行固定。固定後,以70%、95%及99%曱醇依序處理使 細胞脫水且用95%及70%曱醇再水合。隨後在室溫下用阻 斷緩衝液(含有1% BSA之lxPBS)覆蓋載片1小時。以lxPBS 洗滌後,將scFv抗體與細胞在室溫下再培育1小時。最 後,依次藉由小鼠抗HA抗體及結合Cy-2之山羊抗小鼠抗 體來偵測scFv抗體與α-異烯醇酶蛋白之結合。亦用所推薦 之ΡΙ溶液(Invitrogen, USA)對核進行對比染色。利用共焦 光譜顯微鏡成像系統(Confocal Spectral Microscope Imaging System)(TCS SP5, Leica)對載片進行觀察。 亦應用免疫細胞化學染色來評估經純化EnL2及EnL5 scFv抗體針對表現於PE089細胞中之α-異烯醇酶分子的結 合能力。已證實α-異烯醇酶分子主要表現且移位於ΡΕ089 細胞之核膜上(與N-Y Shih博士之個人通信)。因此,重組 EnL2及EnL5 scFv抗體在細胞核膜周圍展現顯著結合信 號,如圖8中所示。與之相比,兩種陰性對照物(包括結合 Cy-2或4L8純系(表現對SARS-CoV棘蛋白具特異性之scFv 抗體)之山羊抗小鼠抗體)未展示任何反應性。光學顯微鏡 下之細胞形態及分布包括於最左圖中以進行比較。總而言 之,結果進一步表明噬菌體呈現技術可為選殖及產生抗特 異性抗原之scFv抗體的更佳替代方法。 【圖式簡單說明】 圖1為重組α-異烯醇酶及多株抗α-異烯醇酶IgY抗體之表 徵。各圖中之樣品為蛋白質標記(泳道M)、純化GST(泳道 1)、純化α-異烯醇酶(泳道2)及純化GST-α-異烯醇酶融合蛋[ 143761.doc -29- 201125580 白(冰道3)將藉由庫馬斯藍染色而顯現之純化蛋白(圖a) 點潰於硝化纖維素紙上,且用抗咖抗體(圖B)或第4次免 疫雞之血清(圖C)探測。重組α_異烯醇酶蛋白之分子量為 約 48 kD ; 圖2為藉由ELIS A分析的第4次免疫後之雞中的體液 反應。將純化α-異烯醇酶蛋白及牛血清抗原(bsa)塗於板 孔上。檢驗一系列經稀釋IgY抗體對a-異烯醇酶或BSA之 特異性結合活性。實心及空心條柱分別表示第4次免疫之 雞或免疫前之雞之IgY與心異烯醇酶的結合。另外,由深 灰色及淺灰色條柱表示之第4次免疫之雞或免疫前之雞之 IgY與BSA的結合係作為陰性對照並行展示; 圖3為雞免疫球蛋白基因中可變區之pcR擴增。輕鏈 (EnVL)及具有短連接子之重鏈(EnVHS)或具有長連接子之 重鏈(EnVH.L)的可變區經成功擴增(圖A)。第二輪pcR產 生具有紐連接子之全長scFv*因片段(EnseFvS)或具有長 連接子之全長scFv基因片段(EnscFv L)(圖b); 圖4為藉由西方墨點法分析之3(;卜抗體表現。將來自% 種純系之相同量之總細胞溶胞物負載於SDS_pAGE上且轉 移至硝化纖維素紙上。依次以丨:3〇〇〇稀釋之山羊抗雞輕鏈 抗體、HRP結合驢抗山羊IgG偵測scfv抗體的存在。scF^ 段之預測分子量約為35 kDa。墨點為1〇種所選純系中scFv 表現之代表性結果; 圖5為藉由ELISA分析的scFv抗體對純化α-異烯醇酶之結 合活性。檢驗第4輪淘選隨機所選之含有scFv抗體的細胞 143761.doc •30- 201125580 溶胞物與塗於板孔上之純化…異烯醇酶的結合。依次使用 1··3000稀釋之山羊抗雞輕鏈抗體、HRp結合驢抗山羊偵 測偵測結合活性且在450 rnn下量測。使用兩種抗SARS_ CoV scFv抗體(SC〇S-S8及SC〇S-L22)作為陰性對照物。在 不添加一次重組scFv抗體之情況下如所述進行另一對照實 驗。使用經純化α-異烯醇酶免疫之雞的多株IgY抗體作為 陽性對照物。ELISA資料以雙重複實驗之平均值表示; 圖6為scFv抗體之VL(圖6(A))及VH(圖6(B))序列之序列分 析。測定10種純系之VH及VLi核苷酸序列且轉譯成胺基酸 序列以與雞生殖系基因之胺基酸序列比對。FR :構架區; CDR :互補決定區。序列缺口經引入以最大化比對且由空 白區表示。圓點表示共同序列。構架區(FR)及互補決定區 (CDR)邊界指示於生殖系序列上方; 圖7為藉由’瓜動式細胞罝測術分析之scfv抗體對純化 異烯醇酶的結合活性。使用純化EnL2&EnL5 scFv抗體、 小鼠抗HA(1:200)及Cy-2結合山羊抗小鼠抗體(1:2〇〇)偵測 PE089細胞表面結合之α•異烯醇酶。灰色細線表示陰性對 照,其為經單獨DMS0處理且經螢光標記之抗表面標記 Abs染色的細胞;灰色實線表示經螢光標記之“同型對照 物染色的細胞;且黑色實線表示經抗表面α_異烯醇酶之 scFv抗體EnL2及EnL5螢光標記Abs染色的細胞。展示三個 獨立實驗中之一代表性實驗之結果;及 圖8為PE089細胞中α-異烯醇酶蛋白之免疫螢光染色。如 Materials and Methods」中所述將細胞固定於玻璃板[b 143761.doc -31. 201125580 上。使用純化EnL2及EnL5 scFv抗體、接著使用小鼠抗HA 及Cy-2結合山羊抗小鼠抗體偵測a-異烯醇酶表現。細胞核 (紅色)藉由PI顯現。EnL2與EnL5 scFv抗體均使PE089細胞 核膜清晰染色(綠色)。抗SARS-CoV scFv抗體4L8未展示與 核膜之任何反應性。 143761.doc •32· 201125580 序列表 <110〉»僵P大學 <120〉診斷及治療α-異烯醇酶(α-enolase)相關疾病之抗α-異烯醇酶抗體 <130〉 T54267/US1358 <160> 50 <170> Patentln version 3. 5 <210〉 1 <211〉 8 <212〉 PRT <213〉人工序列 <220〉 <223〉合成序列 <400> 1Rader, C., Steinberger, P., Fuller, R., Barbas, C.F., 3rd edition > 2000, Methods for the generation of chicken monoclonal antibody fragments by phage display. J Immunol Methods 242, 15 9-181. Briefly, chicken spleens were collected after final immunization and immediately homogenized for 7 days in Trizol reagent. 10 pg of total RNA was reverse transcribed into the first strand using the Superscript RT kit (Invitrogen, USA). After amplification with chicken-specific factors, the PCR products of the heavy and light chain variable (VH and VL) regions are subjected to a second round of PCR using short or long linkers to form a full-length scFv fragment, which is further digested and colonized. In the pComb3X vector. Recombinant DNA was transformed into E. coli XL-1 blue strain by electroporation. Recombinant bacillus was produced by the addition of VCS-M13 to assist the bacillus to 4 〇/. Polyethylene glycol 8000 and 3% NaCl (w/v) were precipitated and finally resuspended in phosphate buffered saline (pbS) containing 1% bovine serum albumin (BSA) and stored at 4 °C. Then, 1011 plaque_forming units (pfu) of recombinant sputum cells from the scFv antibody library were added to the wells pre-coated with α-isoenolase protein (0.5 μg/well), and at 37 ^ Cultivate for 2 hours. After removal of unbound phage, the bound phage was eluted with 〇丨ΜHC1/glycine (pH 2.2)/0.1% BSA, neutralized with 2 M Tris buffer and used to infect XL-1 blue strain. The amplified bacillus was collected as described above, especially 143761.doc •24·201125580, and recovered for the second round of selection. Repeat the panning process three or four times. A set of pure lines were randomly selected and grown using the final panning process. After 6 hours of incubation at 0.5 mM IPTG, bacterial cells were harvested and resolved by three rounds of freezing; lysate was treated with East and/or sonication. The scFv antibody expression of the supernatant and its binding reactivity to cardinolase were analyzed using Western blotting and ELIS A. ScFv antibodies were also prepared in flow cytometry and immunofluorescence assays. These antibodies were expressed in TOP 10F' E. coli (Invitr〇gen' non-inhibitory strain) and used as described by the manufacturer (Amersham Biosciences, UK). Ni2+-charged agarose purification ° Chickens were sacrificed 8 weeks after the last immunization and total RNA was extracted from the swollen spleens to construct an antibody library. The full length scFv gene fragment was amplified using 2 consecutive PCR steps. In the first PCR, the VH gene product was amplified to a size of 400 bp using a primer containing a short linker (GGSSRSS) (SEQ ID NO: 45) and a long linker (GGSSRSSSSGGGGSGGGG) (SEQ ID NO: 46), respectively As shown by EnVH.S (Fig. 3A, lane 2) and EnVH.L (Fig. 3A, lane 3). Accordingly, the VL gene was amplified into a 350 bp band and loaded in lane 4 (EnVL) in Figure 3A. Subsequently, the amplified VH was ligated with VL to form a full-length scFv gene fragment of about 750 bp as shown by EnscFv.S (lane 2) and EnscFv.L (lane 3) in Figure 3B. A plurality of sputum strains were constructed to present antibody libraries and used to screen for specific anti-α-isoenolase scFv antibodies. Example 4 Characterization of anti-α-isoenolase scFv pure line To detect the expression of scFv antibody, the cell lysate was subjected to sodium dodecyl sulfate sodium polyacrylamide gel electrophoresis (SDS-PAGE). All proteins were transferred to 143761.doc -25-201125580 onto a stone xiaohua cellulose membrane (Amersham Biosciences, UK) and subsequently blocked with 5% skim milk TBST solution for 1 hour. A goat anti-chicken IgY light-chain antibody (Bethyl Laboratories, Montgomery, TX, USA) diluted 1:3000 was added and incubated for an additional hour. The membrane was washed three times with TBST for 5 minutes each time. Bound antibodies were detected by the addition of 1:3000 dilution of horseradish peroxidase (HRP) in combination with donkey anti-goat Ig antibody (Sigma, St. Louis, MO, USA). After three washes, the film was developed with diaminobenzidine (DAB) until the desired strength was achieved. To study their binding reactivity, purified IgY from a fourth immunized chicken or a scFv-expressing antibody was incubated with purified alpha-isoenolase immobilized on a nitrocellulose membrane or ELIS A plate well, and subsequently borrowed It was detected by adding a goat anti-chicken IgY light chain antibody as described above and HRP-conjugated donkey anti-goat Ig antibody. Each sample was subjected to an ELISA test in double replicate wells. Four rounds of panning were performed as described above. After each panning, 15 pure lines were randomly selected and the 750 bp fragment insert and its scFv antibody protein expression in the pCom3_X selection vector were analyzed. Information (not shown) indicates that the pure lines of each round of panning are 67% (10/15), 87% (13/15), 100% (15/15) and 100% (15/15) with full length inserts. In addition, 10 pure lines with 750 bp inserts from the last round of panning were induced to express their scFv antibodies. As shown in Figure 4, goat anti-chicken IgY light chain antibody and horseradish peroxidase (HRP) were combined with donkey anti-goat Ig antibody to detect pure line EnL2, EnL4, EnL5, EnL6 and EnL7 to |J scFv Performance, with little or no performance detected for pure EnLl, EnL3, EnL8, EnL9 and EnL 1 0. These results indicate that the amount of immunoglobulin genes with highly conserved sequences can vary significantly, even under the same experimental conditions of 143761.doc -26-201125580. Example 5 Gene sequencing and ELISA analysis using ompseq (5'-AAGACAGCTATCGCGATTGCAGTG-3') (SEQ ID NO: 47) and HRML-F by an automated sequencer (ABI PRISM 377; Perkin-Elmer, National Health Research Institute) (5'-GGTGGTTCCTCTAGATCTTCC-3') (SEQ ID NO: 48) The primers were subjected to nucleotide sequence determination of the heavy and light chain variable regions of the selected pure line. The results were analyzed using the alignment program BLAST and the vector NTI (http://www.ncbi.nlm.nih.gov/BLAST). The nucleotide sequences of the variable regions of the 10 pure heavy and light chain genes were determined and aligned with the germline gene sequences of chicken immunoglobulin. The results revealed that 30% of the sequenced lines (EnL5, EnL6 and EnL7) share the same heavy and light chain genes, resulting in similar profiles of their scFv antibodies, as seen in lanes 5, 6 and 7 of Figure 4. The overall mutation rate compared to the germline gene sequence was 18.6°/ in the heavy and light chain variable regions, respectively. To 27.4% and 13.5% to 23.1% (Figure 5). The binding activity of the £11 B1 to £111^7 3〇?丫 antibody against 〇1-isoenolase was analyzed using £1^13. These scFv antibody fragments were found to exhibit significant binding activity against alpha-isoenolase compared to two other scFv antibodies previously characterized and known to specifically recognize the SARS-CoV spike protein. In particular, EnL2, EnL4, EnL5 and EnL6 scFv antibodies exhibited stronger positive reactivity than purified multiple IgY from chickens immunized with human alpha-isoenolase (Figure 6(A) and B)). Example 6 Flowcytometry Analysis PE089 cell line was originally obtained from a 36 year old patient with stage IV lung adenocarcinoma. 143761.doc -27- 201125580 fluid tumor cells from the National Institute of Dr. Neng-Yao Shih from Cancer Research, National Health Research Institutes, Tainan, Taiwan. These cells have been cultured in vitro for at least 40 passages in RPMI 1640 supplemented with 5% fetal calf serum, 2 mmol/L branic acid and antibiotics. A total of 2χ106 cells were collected and fixed with 2% paraformaldehyde as previously described. The α-isoenolase expressed in PE089 cells was detected with purified scFv EnL2 and EnL5 antibodies, and mouse anti-HA (1:200) and Cy-2 goat anti-mouse antibody (1:200) ( Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was visualized and analyzed using a FAC Scan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Negative control analysis was performed as described above without using the scFv EnL2 and EnL5 antibodies once, and a positive control analysis was performed using rabbit anti-human isoenolase antibody (1:200) instead of scFv EnL2 and EnL5 antibodies. To test the binding reactivity of these selected scFv antibodies, the human alpha-isoenolase gene was transfected into ΡΕ089 tumor cells for expression on the membrane surface, followed by flow cytometry. Purified EnL2 and EnL5 scFv antibodies (data not shown) on SDS-PAGE are capable of detecting recombinant α-isoenolase protein expressed in PE089 cells, binding signals and α-isoenolase The binding signal of the specific rabbit polyclonal antibody of specificity was similar, as shown in FIG. Example 7 Immunofluorescence and Laser Scanning Microscopy ΡΕ089 cells (2 x 10 5 cells/ml) were seeded on coverslips and incubated on ice by using an equal volume of ice-cold 8% paraformaldehyde (freshly prepared). Minutes 143761.doc -28- 201125580 Fixed. After fixation, the cells were dehydrated by sequential treatment with 70%, 95% and 99% sterol and rehydrated with 95% and 70% sterols. The slides were then covered with blocking buffer (lx PBS containing 1% BSA) for 1 hour at room temperature. After washing with lxPBS, the scFv antibody was incubated with the cells for an additional hour at room temperature. Finally, the binding of the scFv antibody to the α-isoenolase protein was detected by mouse anti-HA antibody and Cy-2 goat anti-mouse antibody in combination. The nuclei were also comparatively stained using the recommended sputum solution (Invitrogen, USA). The slides were observed using a Confocal Spectral Microscope Imaging System (TCS SP5, Leica). Immunocytochemical staining was also used to assess the binding ability of the purified EnL2 and EnL5 scFv antibodies to the alpha-isoenolase molecules expressed in PE089 cells. The alpha-isoenolase molecule has been shown to be predominantly expressed and located on the nuclear membrane of ΡΕ089 cells (communication with Dr. N-Y Shih). Thus, recombinant EnL2 and EnL5 scFv antibodies exhibited significant binding signals around the nuclear membrane, as shown in Figure 8. In contrast, two negative controls, including goat anti-mouse antibodies that bind to the Cy-2 or 4L8 pure line (scFv antibodies that are specific for SARS-CoV spiny protein), did not exhibit any reactivity. Cell morphology and distribution under light microscopy are included in the leftmost panel for comparison. Taken together, the results further indicate that phage display technology can be a better alternative to the selection and production of anti-specific antigen scFv antibodies. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a graph showing the expression of recombinant α-isoenolase and a plurality of anti-α-isoenolase IgY antibodies. The samples in each figure are protein label (lane M), purified GST (lane 1), purified α-isoenolase (lane 2), and purified GST-α-isoenolase fusion egg [143761.doc -29- 201125580 White (Ice 3) The purified protein (Fig. a) developed by Coomassie blue staining was spotted on nitrocellulose paper, and anti-cafe antibody (Fig. B) or the fourth immunized chicken serum (Fig. B) Figure C) Detection. The molecular weight of the recombinant α-isoenolase protein was about 48 kD; Figure 2 shows the humoral response in chickens after the fourth immunization by ELIS A analysis. The purified α-isoenolase protein and bovine serum antigen (bsa) were applied to the wells. The specific binding activity of a series of diluted IgY antibodies to a-isoenolase or BSA was examined. The solid and open bars indicate the binding of IgY to cardioenolase in the 4th immunized chicken or pre-immune chicken, respectively. In addition, the binding of IgY to BSA in the 4th immunized chicken or pre-immune chicken indicated by dark gray and light gray bars was displayed as a negative control in parallel; Figure 3 is the pcR of the variable region in the chicken immunoglobulin gene. Amplification. The light chain (EnVL) and the heavy chain with a short linker (EnVHS) or the heavy chain with a long linker (EnVH.L) were successfully amplified (panel A). The second round of pcR produces a full-length scFv* factor fragment (EnseFvS) with a ligated linker or a full-length scFv gene fragment with a long linker (EnscFv L) (Fig. b); Figure 4 shows the analysis by Western blotting method 3 ( Bubo antibody performance. The same amount of total cell lysate from % pure lines was loaded onto SDS_pAGE and transferred to nitrocellulose paper. The goat anti-chicken light chain antibody and HRP binding were diluted with 丨:3〇〇〇 in turn. The anti-goat IgG detects the presence of the scfv antibody. The predicted molecular weight of the scF^ segment is approximately 35 kDa. The ink dot is a representative result of the scFv expression in the selected pure line; Figure 5 is the scFv antibody pair analyzed by ELISA. Purification of the binding activity of α-isoenolase. Purification of the 4th round of panning randomly selected cells containing scFv antibody 143761.doc •30- 201125580 Lysate and purification on plate wells...isoenolase Binding. The goat anti-chicken light chain antibody diluted with 1··3000 was used in sequence, and HRp-conjugated anti-goat was used to detect the binding activity and measured at 450 rnn. Two anti-SARS_CoV scFv antibodies were used (SC〇S-S8). And SC〇S-L22) as a negative control. Do not add a reconstitution s In the case of the cFv antibody, another control experiment was performed as described. A plurality of IgY antibodies of chickens immunized with purified α-isoenolase were used as positive controls. The ELISA data were expressed as the average of the double replicate experiments; Sequence analysis of VL (Fig. 6 (A)) and VH (Fig. 6 (B)) sequences of scFv antibodies. Ten pure VH and VLi nucleotide sequences were determined and translated into amino acid sequences to interact with chicken germline genes. Amino acid sequence alignment. FR: framework region; CDR: complementarity determining region. Sequence gaps are introduced to maximize alignment and are represented by blank regions. Dots indicate common sequences. Framework regions (FR) and complementarity determining regions ( The CDR) borders are indicated above the germline sequence; Figure 7 is the binding activity of the scfv antibody to the purified isoenolase as analyzed by 'Guap cell cytometry. Purified EnL2 & EnL5 scFv antibody, mouse anti-HA ( 1:200) and Cy-2 combined with goat anti-mouse antibody (1:2〇〇) to detect α•isoenolase bound to PE089 cells. Gray thin line indicates negative control, which was treated with DMS0 alone and passed through firefly. Light-labeled cells stained with surface-labeled Abs; gray solid lines indicate the cursor The cells stained with the isotype control were recorded; and the solid black line indicates cells stained with anti-surface α_isoenolase scFv antibody EnL2 and EnL5 fluorescently labeled Abs. One of three independent experiments was shown. The results; and Figure 8 is the immunofluorescence staining of the α-isoenolase protein in PE089 cells. The cells were fixed to glass plates as described in Materials and Methods [b 143761.doc -31. 201125580. The a-isoenolase expression was detected using purified EnL2 and EnL5 scFv antibodies followed by mouse anti-HA and Cy-2 binding goat anti-mouse antibodies. The nucleus (red) appears by PI. Both EnL2 and EnL5 scFv antibodies clearly stained the PE089 cell nuclear membrane (green). The anti-SARS-CoV scFv antibody 4L8 did not exhibit any reactivity with the nuclear envelope. 143761.doc •32· 201125580 Sequence Listing <110>»Zombie University<120>Anti-α-isoenolase antibody for diagnosis and treatment of α-isoolase-related diseases <130〉 T54267/US1358 <160> 50 <170> Patentln version 3. 5 <210> 1 <211> 8 <212> PRT < 213 > artificial sequence < 220 < 223 > 223 > synthetic sequence <400> 1

Ser Gly Gly Ser Gly Ser Tyr Gly <210〉 2 <211〉 10 <212> PRT <213〉人工序列 <220> <223〉 EnL2之VL之CDR1 <400〉 2Ser Gly Gly Ser Gly Ser Tyr Gly <210〉 2 <211> 10 <212> PRT <213>Artificial Sequence <220><223> EnL2 VL CDR1 <400> 2

Ser Gly Gly Ser Ser Ser Tyr Gly Tyr Gly 15 10 <210〉 3 <211〉 8 <212> PRT <213〉人工序列 <220〉 <223〉 EnL3之VL之CDK1 <400> 3Ser Gly Gly Ser Ser Serrr Gly Tyr Gly 15 10 <210> 3 <211> 8 <212> PRT < 213 > 213 > Artificial Sequence <220 < 223 > 223 > VL of EnL3 CDK1 <400> 3

Ser Gly Ser Ser Gly Ser Tyr Gly 1 5 <210> 4 <211> 10 <212> PRT <213〉人工序列 <220〉 <223〉 EnL4之VL之CDR1 <400〉 4Ser Gly Ser Ser Gly Ser Tyr Gly 1 5 <210> 4 <211> 10 <212> PRT <213>Artificial Sequence <220> <223> EnL4 VL CDR1 <400> 4

Ser Gly Gly Ser Ser Ser Tyr Gly Tyr Ser 1 5 10 <210〉 5 <211〉 9 <212> PRT <213〉人工序列 <220〉 143761.doc 201125580 <223〉 EnL5之VL之CDRl <400〉 5Ser Gly Gly Ser Ser Ser Tyr Gly Tyr Ser 1 5 10 <210> 5 <211> 9 <212> PRT <213>Artificial Sequence <220> 143761.doc 201125580 <223> EnL5 VL CDRl <400〉 5

Ser Gly Ser Ser Gly Tyr Gly Tyr Gly 1 5 <210〉 6 <211〉 7 <212〉 PRT <213〉人工序列 <220> <223〉 EnLl之VL之CDR2 <400> 6Ser Gly Ser Ser Gly Tyr Gly Tyr Gly 1 5 <210〉 6 <211> 7 <212> PRT <213>Artificial Sequence <220><223> EnLl VL CDR2 <400> 6

Ala Asn Thr Asn Arg Pro Ser 1 5 <210〉 7 <211〉 7 <212> PRT <213> 人工序列 <220> <223〉 EnL3 之 VL 之 CDR2 <400〉 7LAC2 <220〉 7

Asn Asp Asn Gin Arg Pro Ser <210> 8 <211> 7 <212> PRT <213〉人工序列 <220〉 <223〉 EnL3之VL之CDR2 <400> 8Asn Asp Asn Gin Arg Pro Ser <210> 8 <211> 7 <212> PRT <213>Artificial Sequence <220> <223> EnL3 VL CDR2 <400>

Arg Asp Asp Lys Arg Pro Ser 1 5 <210> 9 <211> 7 <212> PRT <213〉人工序列 <220> <223〉 EnL4之VL之CDR2 <400> 9Arg Asp Asp Lys Arg Pro Ser 1 5 <210> 9 <211> 7 <212> PRT <213>Artificial Sequence <220><223> EnL4 VL CDR2 <400>

Ser Asn Asn Gin Arg Pro Ser 1 5 <210> 10 <211〉 7 <212〉 PRT <213〉人工序列 <220〉 <223〉 EnL5之VL之CDR2 <400> 10Ser Asn Asn Gin Arg Pro Ser 1 5 <210> 10 <211> 7 <212> PRT <213>Artificial Sequence <220> <223> EnL5 VL CDR2 <400>

Ser Asn Asp Lys Arg Pro Ser 143761.doc 201125580 1 5 <210〉 11 <211〉 9 <212〉 PRT <213〉人工序列 <220〉 <223〉 EnLl之VL之CDR3 <400> 11Ser Asn Asp Lys Arg Pro Ser 143761.doc 201125580 1 5 <210> 11 <211> 9 <212> PRT <213>Artificial Sequence <220> <223> EnLl VL CDR3 <400&gt ; 11

Gly Gly Tyr Asp Ser Ser Ala Gly He 1 5 <210〉 12 <211〉 9 <212〉 PRT <213〉人工序列 <220〉 <223〉 EnL2之VL之CDR3 <400> 12Gly Gly Tyr Asp Ser Ser Ala Gly He 1 5 <210> 12 <211> 9 <212> PRT <213>Artificial Sequence <220> <223> EnL2 VL CDR3 <400> 12

Gly Ser Gly Asp Ser Ser Thr Gly Met 1 5 <210> 13 <211〉 11 <212〉 PRT <213> 人工序列 <220> <223> EnL3之VL之CDK3 <400> 13Gly Ser Gly Asp Ser Ser Thr Gly Met 1 5 <210> 13 <211> 11 <212> PRT <213> Artificial sequence <220><223> EnL3 VL CDK3 <400> 13

Gly Ser Gly Glu Ser Ser Thr Asn Asn Gly lie 1 5 10 <210> 14 <211> 10 <212〉 PRT <213> 人工序列 <220〉 <223> EnL4 之 VL 之 CDR3 <400> 14 1 5 <210> 15 <211> 12 <212〉 PRT <213> 人工序列 <220〉 <223〉 EnL5 之 VL 之 CDR3 <400> 15Gly Ser Gly Glu Ser Ser Thr Asn Asn Gly lie 1 5 10 <210> 14 <211> 10 <212> PRT <213> Artificial Sequence <220><223> EnL4 VL CDR3 <400> 14 1 5 <210> 15 <211> 12 <212> PRT <213> Artificial sequence <220><223> VL3 of VL of EnL5 <400>

Gly Ser Met Asp Ser Ser Asn Ser Gly ValGly Ser Met Asp Ser Ser Asn Ser Gly Val

Gly Gly Tyr Asp Ser Ser Ala Ser Tyr Val Gly He 1 5 10 <210> 16 <211〉 5 143761.doc 201125580 <212> PRT <213〉 人工序列 <220〉 <223〉 EnLl 之 VH 之 CDR1 <400> 16 Ser Phe Asn Met Phe 1 5 <210> 17 <211〉 5 <212> PRT <213〉 人工序列 <220〉 <223> EnL2 之 VH 之 CDR1 <400〉 17 Ser His Asp Met Gly 1 5 <210〉 18 <211〉 5 <212〉 PRT <213> 人工序列 <220〉 <223〉 EnL3 之 VH 之 CDR1 <400> 18 Asp Tyr Cys Val Gin 1 5 <210〉 19 <211〉 5 <212〉 PRT <213〉 人工序列 <220〉 <223> EnL4 之 VH 之 CDR1 <400〉 19 Ser Phe Tyr Met Phe 1 5 <210〉 20 <211〉 5 <212〉 PRT <213> 人工序列 <220〉 <223> EnL5 之 VH 之 CDR1 <400> 20 Ser Tyr Ala Met His 1 5 <210〉 21 <211〉 17 <212> PRT <213〉 人工序列 <220> <223> EnLl 之 VH 之 CDR2 143761.doc 201125580 <400〉 21Gly Gly Tyr Asp Ser Ser Ala Ser Tyr Val Gly He 1 5 10 <210> 16 <211> 5 143761.doc 201125580 <212> PRT <213> Artificial Sequence <220> <223> EnLl CDR1 <400> 16 Ser Phe Asn Met Phe 1 5 <210> 17 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> LV1 <223>;400> 17 Ser His Asp Met Gly 1 5 <210> 18 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> LR1 <400> of VH of EnL3 Asp Tyr Cys Val Gin 1 5 <210> 19 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> EnL4 VH CDR1 <400> 19 Ser Phe Tyr Met Phe 1 5 <210> 20 <211> 5 <212> PRT <213> Artificial sequence <220><223> EnL5 VH CDR1 <400> 20 Ser Tyr Ala Met His 1 5 &lt ;210> 21 <211> 17 <212> PRT <213> Artificial sequence <220><223> EnLl VH CDR2 143761.doc 201125 580 <400〉 21

Gly lie Asn Asn Ala Gly Ser Thr Thr Asn His Gly Ala Ala Val Lys 15 10 15Gly lie Asn Asn Ala Gly Ser Thr Thr Asn His Gly Ala Ala Val Lys 15 10 15

Gly <210> 22 <211> 18 <212〉 PRT <213〉人工序列 <220 <223〉 EnL2之VH之CDR2 <400〉 22Gly <210> 22 <211> 18 <212> PRT < 213 > 213 > artificial sequence <220 < 223 > 223 & CDR 2 of VH of EnL2 <400 > 22

Gly lie Glu Asn Ala Ala Gly lie Gly Thr Phe Tyr Gly Ala Ala Val 15 10 15Gly lie Glu Asn Ala Ala Gly lie Gly Thr Phe Tyr Gly Ala Ala Val 15 10 15

Lys Gly <210> 23 <211> 17 <212〉 PRT <213〉人工序列 <220> <223〉 EnL3之VH之CDR2 <400> 23Lys Gly <210> 23 <211> 17 <212> PRT <213>Artificial sequence <220><223> LV2 <400> of VH of EnL3 23

Ala lie Ser Asn Thr Gly Arg Tyr Thr Gly Tyr Gly Ser Ala Val Lys 15 10 15Ala lie Ser Asn Thr Gly Arg Tyr Thr Gly Tyr Gly Ser Ala Val Lys 15 10 15

Gly <210> 24 <211> 17 <212> PRT <213> 人工序列 <220> <223〉 EnL4之VH之CDR2 <400> 24Gly <210> 24 <211> 17 <212> PRT <213> Artificial sequence <220><223> EnL4 VH CDR2 <400>

Gly lie Ser Gly Asp Gly Arg Tyr Thr Gly Tyr Gly Ala Ala Val Asp 15 10 15Gly lie Ser Gly Asp Gly Arg Tyr Thr Gly Tyr Gly Ala Ala Val Asp 15 10 15

Gly <210〉 25 <211〉 19 <212> PRT <213〉人工序列 <220〉 <223〉 EnL5之VH之CDR2 <400> 25 143761.doc 201125580Gly <210> 25 <211> 19 <212> PRT < 213 > 213 > artificial sequence <220 < 223 > LR 2 of VH of EnL5 <400> 25 143761.doc 201125580

Gly lie Ser Arg Asp Gly Gly Ser Ser Thr Arg Tyr Tyr Gly Ala Ala 15 10 15Gly lie Ser Arg Asp Gly Gly Ser Ser Thr Arg Tyr Tyr Gly Ala Ala 15 10 15

Val Lys Gly <210> 26 <211> 10 <212〉 PRT <213〉人工序列 <220> <223〉 EnLl之VH之CDR3 <400〉 26Val Lys Gly <210> 26 <211> 10 <212> PRT <213>Artificial sequence <220><223> EnLl VH CDR3 <400> 26

Ser Pro Gly Gly lie Asp Gly lie Asp Gly 1 5 10 <210〉 27 <211〉 14 <212> PRT <213〉人工序列 <220〉 <223〉 EnL2之VH之CDR3 <4〇0> 27Ser Pro Gly Gly lie Asp Gly lie Asp Gly 1 5 10 <210> 27 <211> 14 <212> PRT <213>Artificial Sequence <220> <223> EnL2 VH CDR3 <4 〇0> 27

Gly Ala Asp Thr Gly Gly Trp Pro Ala Ala Asn lie Asp Ala 1 5 10 <210〉 28 <211〉 14 <212〉 PRT <213〉人工序列 <220> <223〉 EnL3之VH之CDR3 <400> 28Gly Ala Asp Thr Gly Gly Trp Pro Ala Ala Asn lie Asp Ala 1 5 10 <210> 28 <211> 14 <212> PRT <213>Artificial Sequence <220><223> EnL3 VH CDR3 <400> 28

Asp Gly Cys Ala Gly Cys Cys Gly Ser Tyr Tyr lie Asp Gly 15 10 <210> 29 <211> 14 <212〉 PRT <213〉人工序列 <220〉 <223〉 EnL4之VH之CDR3 <400> 29Asp Gly Cys Ala Gly Cys Cys Gly Ser Tyr Tyr lie Asp Gly 15 10 <210> 29 <211> 14 <212> PRT <213>Artificial Sequence <220> <223> EnL4 VH CDR3 <400> 29

Glu Ser Gly Ser Gly Cys Cys Asn Gly Asp Asn lie Asp Ala 1 5 10 <210> 30 <211> 14 <212> PRT <213〉人工序列 <220〉 <223〉 EnL5之VH之CDR3 <400〉 30Glu Ser Gly Ser Gly Cys Cys Asn Gly Asp Asn lie Asp Ala 1 5 10 <210> 30 <211> 14 <212> PRT <213>Artificial Sequence <220> <223> EnL5 VH CDR3 <400〉 30

Asp Ser Asp Asn Gly Gly Tyr Tyr Cys Asp Asp lie Asp Ala -6- 143761.doc 201125580 5 10 <210> 31 <211> 102 <212〉 PRT <213〉人工序列 <220〉 <223〉EuLl之胺基酸 <400> 31Asp Ser Asp Asn Gly Gly Tyr Tyr Cys Asp Asp lie Asp Ala -6- 143761.doc 201125580 5 10 <210> 31 <211> 102 <212> PRT <213>Artificial Sequence <220> < 223>EuLl Amino Acid <400> 31

Ala Leu Thr Gin Pro Ser Ser Val Ser Ala Asn Pro Gly Gly Thr Val 15 10 15Ala Leu Thr Gin Pro Ser Ser Val Ser Ala Asn Pro Gly Gly Thr Val 15 10 15

Glu lie Thr Cys Ser Gly Gly Ser Gly Ser Tyr Gly Trp Phe Gin Gin 20 25 30Glu lie Thr Cys Ser Gly Gly Ser Gly Ser Tyr Gly Trp Phe Gin Gin 20 25 30

Lys Ala Pro Gly Ser Ala Pro Val Thr Pro He Tyr Ala Asn Thr Asn 35 40 45Lys Ala Pro Gly Ser Ala Pro Val Thr Pro He Tyr Ala Asn Thr Asn 35 40 45

Arg Pro Ser Asp lie Pro Ser Arg Phe Ser Gly Ser Lys Ser Gly Ser 50 55 60Arg Pro Ser Asp lie Pro Ser Arg Phe Ser Gly Ser Lys Ser Gly Ser 50 55 60

Thr Ala Thr Leu Thr lie Thr Gly Val Gin Ala Asp Asp Glu Ala Val 65 70 75 80Thr Ala Thr Leu Thr lie Thr Gly Val Gin Ala Asp Asp Glu Ala Val 65 70 75 80

Tyr Phe Cys Gly Gly Tyr Asp Ser Ser Ala Gly lie Phe Gly Ala Gly 85 90 95Tyr Phe Cys Gly Gly Tyr Asp Ser Ser Ala Gly lie Phe Gly Ala Gly 85 90 95

Thr Thr Leu Thr Val Leu 100 <210〉 32 <211〉 104 <212〉 PRT <213〉人工序列 <220〉 <223〉EnL2之VL之胺基酸 <400> 32Thr Thr Leu Thr Val Leu 100 <210> 32 <211> 104 <212> PRT < 213 > 213 > 221 > 223 < 223 > 223 > LL > LL <400>

Ala Leu Thr Gin Pro Ser Ser Val Ser Ala Asn Pro Gly Gly Thr Val 15 10 15Ala Leu Thr Gin Pro Ser Ser Val Ser Ala Asn Pro Gly Gly Thr Val 15 10 15

Glu lie Thr Cys Ser Gly Gly Ser Ser Ser Tyr Gly Tyr Gly Trp Tyr 20 25 30Glu lie Thr Cys Ser Gly Gly Ser Ser Ser Tyr Gly Tyr Gly Trp Tyr 20 25 30

Gin Gin Lys Ser Pro Gly Ser Pro Pro Val Thr Val lie Tyr Asn Asp 35 40 45Gin Gin Lys Ser Pro Gly Ser Pro Pro Val Thr Val lie Tyr Asn Asp 35 40 45

Asn Gin Arg Pro Ser Asp lie Pro Ser Arg Phe Ser Gly Ala Leu Ser 50 55 60Asn Gin Arg Pro Ser Asp lie Pro Ser Arg Phe Ser Gly Ala Leu Ser 50 55 60

Gly Ser Thr Ala Thr Leu Thr lie Thr Gly Val Gin Ala Asp Asp Glu 65 70 75 80Gly Ser Thr Ala Thr Leu Thr lie Thr Gly Val Gin Ala Asp Asp Glu 65 70 75 80

Ala Val Tyr Phe Cys Gly Ser Gly Asp Ser Ser Thr Gly Met Phe Gly 85 90 95 143761.doc 201125580Ala Val Tyr Phe Cys Gly Ser Gly Asp Ser Ser Thr Gly Met Phe Gly 85 90 95 143761.doc 201125580

Ala Gly Thr Thr Leu Thr Val Leu 100 <210〉 33 <211〉 104 <212> PRT <213〉人工序列 <220〉 <223〉EnL3之VL之胺基酸 <400〉 33Ala Gly Thr Thr Leu Thr Val Leu 100 <210> 33 <211> 104 <212> PRT < 213 > 213 > Artificial Sequence <220 < 223 > 223 > EnL3 VL Amino Acid <400> 33

Ala Leu Thr Gin Pro Ser Ser Val Ser Ala Asn Pro Gly Gly Thr Val 15 10 15Ala Leu Thr Gin Pro Ser Ser Val Ser Ala Asn Pro Gly Gly Thr Val 15 10 15

Glu He Thr Cys Ser Gly Ser Ser Gly Ser Tyr Gly Trp Tyr Gin Gin 20 25 30Glu He Thr Cys Ser Gly Ser Ser Gly Ser Tyr Gly Trp Tyr Gin Gin 20 25 30

Lys Ser Pro Gly Ser Ala Pro Val Thr Val lie Tyr Arg Asp Asp Lys 35 40 45Lys Ser Pro Gly Ser Ala Pro Val Thr Val lie Tyr Arg Asp Asp Lys 35 40 45

Arg Pro Ser Asp lie Pro Ser Arg Phe Ser Gly Ser Leu Ser Gly Ser 50 55 60Arg Pro Ser Asp lie Pro Ser Arg Phe Ser Gly Ser Leu Ser Gly Ser 50 55 60

Thr Ala Thr Leu Thr lie Thr Gly Val Gin Ala Glu Asp Glu Ala Val 65 70 75 80Thr Ala Thr Leu Thr lie Thr Gly Val Gin Ala Glu Asp Glu Ala Val 65 70 75 80

Tyr Phe Tyr Gly Ser Gly Glu Ser Ser Thr Asn Asn Gly lie Phe Gly 85 90 95Tyr Phe Tyr Gly Ser Gly Glu Ser Ser Thr Asn Asn Gly lie Phe Gly 85 90 95

Ala Gly Thr Thr Leu Thr Val Leu 100 <210> 34 <211〉 105 <212〉 PRT <213〉人工序列 <220> <223〉EnL4之VL之胺基酸 <400〉 34Ala Gly Thr Thr Leu Thr Val Leu 100 <210> 34 <211> 105 <212> PRT <213>Artificial Sequence <220><223>EnL4 VL Amino Acid <400> 34

Ala Leu Thr Gin Pro Ser Ser Val Ser Ala Asn Pro Gly Gly Thr Val 15 10 15Ala Leu Thr Gin Pro Ser Ser Val Ser Ala Asn Pro Gly Gly Thr Val 15 10 15

Glu He Thr Cys Ser Gly Gly Ser Ser Ser Tyr Gly Tyr Ser Arg His 20 25 30Glu He Thr Cys Ser Gly Gly Ser Ser Ser Tyr Gly Tyr Ser Arg His 20 25 30

Gin Gin Lys Ser Pro Gly Ser Ala Pro Val Thr Ala He Tyr Ser Asn 35 40 45Gin Gin Lys Ser Pro Gly Ser Ala Pro Val Thr Ala He Tyr Ser Asn 35 40 45

Asn Gin Arg Pro Ser Asp lie Pro Ser Arg Phe Ser Gly Ser Thr Ser 50 55 60Asn Gin Arg Pro Ser Asp lie Pro Ser Arg Phe Ser Gly Ser Thr Ser 50 55 60

Gly Ser Thr Ala Thr Leu Thr lie Thr Gly Val Gin Ala Asp Asp Glu 65 70 75 80Gly Ser Thr Ala Thr Leu Thr lie Thr Gly Val Gin Ala Asp Asp Glu 65 70 75 80

Ala Val Tyr Phe Cys Gly Ser Met Asp Ser Ser Asn Ser Gly Val Phe 85 90 95 143761.doc 201125580Ala Val Tyr Phe Cys Gly Ser Met Asp Ser Ser Asn Ser Gly Val Phe 85 90 95 143761.doc 201125580

Gly Ala Gly Thr Thr Leu Thr Val Leu 100 105 <210> 35 <211〉 106 <212> PRT <213〉人工序列 <220> <223〉EnL5之VL之胺基酸 <400> 35Gly Ala Gly Thr Thr Leu Thr Val Leu 100 105 <210> 35 <211> 106 <212> PRT <213>Artificial Sequence <220><223>EnL5 VL Amino Acid <400>; 35

Ala Leu Thr Gin Pro Ser Ser Val Ser Ala Asn Pro Gly Gly Thr Val 15 10 15Ala Leu Thr Gin Pro Ser Ser Val Ser Ala Asn Pro Gly Gly Thr Val 15 10 15

Glu lie Thr Cys Ser Gly Ser Ser Gly Tyr Gly Tyr Gly Trp Tyr Gin 20 25 30Glu lie Thr Cys Ser Gly Ser Ser Gly Tyr Gly Tyr Gly Trp Tyr Gin 20 25 30

Gin Lys Ser Pro Gly Ser Ala Pro Val Thr Val lie Tyr Ser Asn Asp 35 40 45Gin Lys Ser Pro Gly Ser Ala Pro Val Thr Val lie Tyr Ser Asn Asp 35 40 45

Lys Arg Pro Ser Asp lie Pro Ser Arg Phe Ser Gly Ser Ala Ser Gly 50 55 60Lys Arg Pro Ser Asp lie Pro Ser Arg Phe Ser Gly Ser Ala Ser Gly 50 55 60

Ser Thr Ala Thr Leu Thr lie Thr Gly Val Arg Ala Glu Asp Glu Ala 65 70 75 80Ser Thr Ala Thr Leu Thr lie Thr Gly Val Arg Ala Glu Asp Glu Ala 65 70 75 80

Val Tyr Phe Cys Gly Gly Tyr Asp Ser Ser Ala Ser Tyr Val Gly lie 85 90 95Val Tyr Phe Cys Gly Gly Tyr Asp Ser Ser Ala Ser Tyr Val Gly lie 85 90 95

Phe Gly Ala Gly Thr Thr Leu Thr Val Leu 100 105 <210〉 36 <211〉 144 <212〉 PRT <213〉人工序列 <220〉 <223〉EnLl之VH之胺基酸 <400〉 36Phe Gly Ala Gly Thr Thr Leu Thr Val Leu 100 105 <210> 36 <211> 144 <212> PRT < 213 > 213 > 221 > 223 > 223 > 223 > 400> 36

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gin Thr Pro Gly Gly 15 10 15Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gin Thr Pro Gly Gly 15 10 15

Thr Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Thr Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30

Asn Met Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Asn Met Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ala Gly lie Asn Asn Ala Gly Ser Thr Thr Asn His Gly Ala Ala Val 50 55 60Ala Gly lie Asn Asn Ala Gly Ser Thr Thr Asn His Gly Ala Ala Val 50 55 60

Lys Gly Arg Ala Thr lie Ser Arg Asp Asn Gly Gin Ser Thr Leu Arg 65 70 75 80Lys Gly Arg Ala Thr lie Ser Arg Asp Asn Gly Gin Ser Thr Leu Arg 65 70 75 80

Leu Gin Leu Asn Asp Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys 85 90 95 -9- 143761.doc 201125580Leu Gin Leu Asn Asp Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys 85 90 95 -9- 143761.doc 201125580

Ala Arg Ser Pro Gly Gly He Asp Gly He Asp Gly Trp Gly His Gly 100 105 110Ala Arg Ser Pro Gly Gly He Asp Gly He Asp Gly Trp Gly His Gly 100 105 110

Thr Glu Val lie Val Ser Ser Thr Ser Gly Gin Ala Gly Gin His His 115 120 125Thr Glu Val lie Val Ser Ser Thr Ser Gly Gin Ala Gly Gin His His 115 120 125

His His His His Gly Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser 130 135 140 <210> 37 <211> 149 <212〉 PRT <213〉人工序列 <220> <223〉EnL2之VH之胺基酸 <4 ⑻ > 37His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His Amino acid of VH <4 (8) > 37

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gin Thr Pro Gly Gly 15 10 15Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gin Thr Pro Gly Gly 15 10 15

Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Asp Phe Ser Ser His 20 25 30Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Asp Phe Ser Ser His 20 25 30

Asp Met Gly Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Phe Val 35 40 45Asp Met Gly Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Phe Val 35 40 45

Ala Gly lie Glu Asn Ala Ala Gly lie Gly Thr Phe Tyr Gly Ala Ala 50 55 60Ala Gly lie Glu Asn Ala Ala Gly lie Gly Thr Phe Tyr Gly Ala Ala 50 55 60

Val Lys Gly Arg Ala Thr lie Ser Arg Asp Asn Gly Gin Ser Thr Val 65 70 75 80Val Lys Gly Arg Ala Thr lie Ser Arg Asp Asn Gly Gin Ser Thr Val 65 70 75 80

Arg Leu Gin Leu Asn Asn Leu Arg Pro Glu Asp Thr Gly Thr Tyr Phe 85 90 95Arg Leu Gin Leu Asn Asn Leu Arg Pro Glu Asp Thr Gly Thr Tyr Phe 85 90 95

Cys Ala Arg Gly Ala Asp Thr Gly Gly Trp Pro Ala Ala Asn lie Asp 100 105 110Cys Ala Arg Gly Ala Asp Thr Gly Gly Trp Pro Ala Ala Asn lie Asp 100 105 110

Ala Trp Gly His Gly Thr Glu Val lie Val Ser Ser Thr Ser Gly Gin 115 120 125Ala Trp Gly His Gly Thr Glu Val lie Val Ser Ser Thr Ser Gly Gin 115 120 125

Ala Gly Gin His His His His His His Gly Ala Tyr Pro Tyr Asp Val 130 135 140Ala Gly Gin His His His His His His His His Gly Ala Tyr Pro Tyr Asp Val 130 135 140

Pro Asp Tyr Ala Ser 145 <210〉 38 <211> 148 <212> PRT <213〉人工序列 <220〉 <223〉EnL3之VH之胺基酸 <400> 38Pro Asp Tyr Ala Ser 145 <210> 38 <211> 148 <212> PRT <213> artificial sequence <220><223><223>

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gin Thr Pro Gly Gly 10- 143761.doc 201125580 10 15Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gin Thr Pro Gly Gly 10- 143761.doc 201125580 10 15

Gly Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Ala Leu Thr Asp Tyr 20 25 30Gly Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Ala Leu Thr Asp Tyr 20 25 30

Cys Val Gin Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Phe Val 35 40 45Cys Val Gin Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Phe Val 35 40 45

Ala Ala lie Ser Asn Thr Gly Arg Tyr Thr Gly Tyr Gly Ser Ala Val 50 55 60Ala Ala lie Ser Asn Thr Gly Arg Tyr Thr Gly Tyr Gly Ser Ala Val 50 55 60

Lys Gly Arg Ala Thr lie Ser Arg Asp Asn Gly Gin Ser Thr Val Arg 65 70 75 80Lys Gly Arg Ala Thr lie Ser Arg Asp Asn Gly Gin Ser Thr Val Arg 65 70 75 80

Leu Gin Val Asn Asn Leu Arg Ala Glu Asp Met Gly Thr Tyr Phe Cys 85 90 95Leu Gin Val Asn Asn Leu Arg Ala Glu Asp Met Gly Thr Tyr Phe Cys 85 90 95

Thr Arg Asp Gly Cys Ala Gly Cys Cys Gly Ser Tyr Tyr lie Asp Gly 100 105 110Thr Arg Asp Gly Cys Ala Gly Cys Cys Gly Ser Tyr Tyr lie Asp Gly 100 105 110

Trp Gly His Gly Thr Glu Val lie Val Ser Ser Thr Ser Gly Gin Ala 115 120 125Trp Gly His Gly Thr Glu Val lie Val Ser Ser Thr Ser Gly Gin Ala 115 120 125

Gly Gin His His His His His His Gly Ala Tyr Pro Tyr Asp Val Pro 130 135 140Gly Gin His His His His His His His His His Gly Ala Tyr Pro Tyr Asp Val Pro 130 135 140

Asp Tyr Ala Ser 145 <210〉 39 <211〉 148 <212> PRT <213〉人工序列<220> <223〉EnL4之VH之胺基酸 <400> 39 Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gin Thr Pro Gly Gly 15 10 15Asp Tyr Ala Ser 145 <210> 39 <211> 148 <212> PRT < 213 > 213 > artificial sequence <220><223> 223 > AH of VH of EnL4<400> 39 Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gin Thr Pro Gly Gly 15 10 15

Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30

Tyr Met Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Tyr Met Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ala Gly lie Ser Gly Asp Gly Arg Tyr Thr Gly Tyr Gly Ala Ala Val 50 55 60Ala Gly lie Ser Gly Asp Gly Arg Tyr Thr Gly Tyr Gly Ala Ala Val 50 55 60

Asp Gly Arg Ala Thr lie Ser Arg Asp Asn Gly Gin Ser Thr Val Arg 65 70 75 80Asp Gly Arg Ala Thr lie Ser Arg Asp Asn Gly Gin Ser Thr Val Arg 65 70 75 80

Leu Gin Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly lie Tyr Tyr Cys 85 90 95Leu Gin Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly lie Tyr Tyr Cys 85 90 95

Ala Arg Glu Ser Gly Ser Gly Cys Cys Asn Gly Asp Asn lie Asp Ala 100 105 110 c 143761.doc -11 - 201125580Ala Arg Glu Ser Gly Ser Gly Cys Cys Asn Gly Asp Asn lie Asp Ala 100 105 110 c 143761.doc -11 - 201125580

Trp Gly His Gly Thr Glu Val He Val Ser Ser Thr Ser Gly Gin Ala 115 120 125Trp Gly His Gly Thr Glu Val He Val Ser Ser Thr Ser Gly Gin Ala 115 120 125

Gly Gin His His His His His His Gly Ala Tyr Pro Tyr Asp Val Pro 130 135 140Gly Gin His His His His His His His His His Gly Ala Tyr Pro Tyr Asp Val Pro 130 135 140

Asp Tyr Ala Ser 145 <210> 40 <211〉 150 <212> PRT <213〉人工序列 <220〉 <223〉EnL5之VH之胺基酸 <400> 40Asp Tyr Ala Ser 145 <210> 40 <211> 150 <212> PRT < 213 > 213 > 221 > 221 < 223 > 223 > 223 > EnL5 VH of amino acid <400> 40

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gin Thr Pro Gly Gly 15 10 15Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gin Thr Pro Gly Gly 15 10 15

Ala Leu Ser Leu Val Cys Lys Gly Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Leu Ser Leu Val Cys Lys Gly Ser Gly Phe Thr Phe Ser Ser 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

Ala Gly lie Ser Arg Asp Gly Gly Ser Ser Thr Arg Tyr Tyr Gly Ala 50 55 60Ala Gly lie Ser Arg Asp Gly Gly Ser Ser Thr Arg Tyr Tyr Gly Ala 50 55 60

Ala Val Lys Gly Arg Ala Thr lie Ser Arg Asp Asn Gly Gin Ser Ala 65 70 75 80Ala Val Lys Gly Arg Ala Thr lie Ser Arg Asp Asn Gly Gin Ser Ala 65 70 75 80

Met Arg Leu Gin Leu Asn Asn Leu Arg Ala Glu Asp Thr Ala Thr Tyr 85 90 95Met Arg Leu Gin Leu Asn Asn Leu Arg Ala Glu Asp Thr Ala Thr Tyr 85 90 95

Phe Cys Ala Lys Asp Ser Asp Asn Gly Gly Tyr Tyr Cys Asp Asp lie — _ 105 ' 110Phe Cys Ala Lys Asp Ser Asp Asn Gly Gly Tyr Tyr Cys Asp Asp lie — _ 105 ' 110

Asp Ala Trp Gly His Gly Thr Glu Val lie Val Ser Ser Thr Ser Gly 115 120 125Asp Ala Trp Gly His Gly Thr Glu Val lie Val Ser Ser Thr Ser Gly 115 120 125

Gin Ala Gly Gin His His His His His His Gly Ala Tyr Pro Tyr Asp 130 135 140Gin Ala Gly Gin His His His His His His His Gly Ala Tyr Pro Tyr Asp 130 135 140

Val Pro Asp Tyr Ala Ser 145 150 &lt;210&gt; 41 &lt;211&gt; 33 &lt;212〉 DNA &lt;213〉人工序列 &lt;220〉 &lt;223〉引子序列 &lt;400〉 41 ggtggaattc tatctattct caagatccat gcc 12- 143761.doc 201125580 &lt;210&gt; &lt;211〉 &lt;212&gt; &lt;213&gt; 42 30 DNA 人工序列 &lt;220&gt; &lt;223〉 反置引子序列 &lt;40'0&gt; 42 actccatggt tacttggcca aggggtttct 30 &lt;210〉 &lt;211〉 &lt;212〉 &lt;213〉 43 39 DNA 人工序列 &lt;220&gt; &lt;223〉 前置引子序列 &lt;400〉 43 ccgcgtgaat tcggggatcc atgtctattc tcaagatcc 39 &lt;210〉 &lt;211&gt; &lt;212〉 &lt;213&gt; 44 37 DNA 人工序列 &lt;220&gt; &lt;223&gt; 反置引子序列 &lt;400〉 44 catggagtcg acctcgagct tggccaaggg gtttctg 37 &lt;210〉 &lt;211〉 &lt;212〉 &lt;213〉 45 7 PRT 人工序列 &lt;220&gt; &lt;223〉 合成序列 &lt;400&gt; 45 Gly Gly Ser Ser Arg Ser Ser 1 5 &lt;210〉 &lt;211&gt; &lt;212〉 &lt;213〉 46 18 DNA 人工序列 &lt;220〉 &lt;223〉 引子序列 &lt;400&gt; 46 ggssrssssg gggsgggg 18 &lt;210〉 &lt;211〉 &lt;212&gt; &lt;213〉 47 24 DNA 人工序列 &lt;220〉 &lt;223&gt; 引子序列 &lt;400&gt; 47 aagacagcta tcgcgattgc agtg 24 13- 143761.doc 201125580 &lt;210&gt; 48 &lt;211〉 21 &lt;212〉 DNA &lt;213〉人工序列 &lt;220〉 &lt;223&gt;引子序列 &lt;4 ⑻ &gt; 48 ggtggttcct ctagatcttc c &lt;210〉 49 &lt;211〉 104 &lt;212〉 PRT &lt;213〉人工序列 &lt;220&gt; &lt;223〉生殖系VL &lt;400〉 49Val Pro Asp Tyr Ala Ser 145 150 &lt;210&gt; 41 &lt;211&gt; 33 &lt;212> DNA &lt;213>Artificial Sequence&lt;220&gt;&lt;223&gt;Introduction Sequence&lt;400> 41 ggtggaattc tatctattct caagatccat gcc 12- 143761.doc 201125580 &lt;210&gt;&lt;211>&lt;212&gt;&lt;213&gt; 42 30 DNA artificial sequence &lt;220&gt;&lt;223&gt; inverted primer sequence &lt;40'0&gt; 42 actccatggt tacttggcca aggggtttct 30 &lt; 210> &lt;211> &lt;212> &lt;213> 43 39 DNA artificial sequence &lt;220&gt;&lt;223> pre-priming sequence &lt;400> 43 ccgcgtgaat tcggggatcc atgtctattc tcaagatcc 39 &lt;210> &lt;211&gt;&lt;;212>&lt;213&gt; 44 37 DNA artificial sequence &lt;220&gt;&lt;223&gt; Inverted primer sequence &lt;400> 44 catggagtcg acctcgagct tggccaaggg gtttctg 37 &lt;210> &lt;211> &lt;212> &lt;213> 45 7 PRT artificial sequence &lt;220&gt;&lt;223> synthetic sequence &lt;400&gt; 45 Gly Gly Ser Ser Arg Ser Ser 1 5 &lt;210> &lt;211&gt;&lt;212&gt;&lt;213&gt;&lt;220〉&lt;223> primer sequence &lt;400&gt; 46 ggssrssssg gggsgggg 18 &lt;210> &lt;211> &lt;212&gt;&lt;213> 47 24 DNA artificial sequence &lt;220> &lt;223&gt; primer sequence &lt;400&gt; 47 aagacagcta tcgcgattgc agtg 24 13- 143761.doc 201125580 &lt;210&gt; 48 &lt;211> 21 &lt;212> DNA &lt;213>Artificial sequence &lt;220&gt;&lt;223&gt;Introduction sequence &lt;4 (8) &gt; 48 ggtggttcct ctagatcttc c &lt;210> 49 &lt;211&gt; 104 &lt;212&gt; PRT &lt; 213 &gt; 213 &gt; artificial sequence&lt;220&gt;&lt;223&gt; </ br> genital VL &lt; 400 > 49

Ala Leu Thr Gin Pro Ser Ser Val Ser Ala Asn Pro Gly Gly Thr Val 15 10 15Ala Leu Thr Gin Pro Ser Ser Val Ser Ala Asn Pro Gly Gly Thr Val 15 10 15

Lys He Thr Cys Ser Gly Asp Ser Ser Tyr Tyr Gly Trp Tyr Gin Gin 20 25 30Lys He Thr Cys Ser Gly Asp Ser Ser Tyr Tyr Gly Trp Tyr Gin Gin 20 25 30

Lys Ala Pro Gly Ser Ala Pro Val Thr Val lie Tyr Asp Asn Thr Asn 35 40 45Lys Ala Pro Gly Ser Ala Pro Val Thr Val lie Tyr Asp Asn Thr Asn 35 40 45

Arg Pro Ser Asn lie Pro Ser Arg Phe Ser Gly Ser Lys Ser Gly Ser 50 55 60Arg Pro Ser Asn lie Pro Ser Arg Phe Ser Gly Ser Lys Ser Gly Ser 50 55 60

Thr Ala Thr Leu Thr lie Thr Gly Val Arg Ala Asp Asp Asn Ala Val 65 70 75 80Thr Ala Thr Leu Thr lie Thr Gly Val Arg Ala Asp Asp Asn Ala Val 65 70 75 80

Tyr Tyr Cys Ala Ser Thr Asp Ser Ser Ser Thr Ala Gly lie Phe Gly 85 90 95Tyr Tyr Cys Ala Ser Thr Asp Ser Ser Ser Thr Ala Gly lie Phe Gly 85 90 95

Ala Gly Thr Thr Leu Thr Val Leu 100 &lt;210〉 50 &lt;211&gt; 142 &lt;212&gt; PRT &lt;213〉人工序列 &lt;220&gt; &lt;223〉生殖系VH之胺基酸序列 &lt;400&gt; 50Ala Gly Thr Thr Leu Thr Val Leu 100 &lt;210> 50 &lt;211&gt; 142 &lt;212&gt; PRT &lt; 213 &gt; 213 &gt; artificial sequence&lt;220&gt;&lt;223&gt;&gt;223&gt; Amino acid sequence of germline VH &lt;400&gt; 50

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gin Thr Pro Gly Gly 15 10 15Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gin Thr Pro Gly Gly 15 10 15

Ala Leu Arg Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Leu Arg Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Asp Met Leu Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Phe Val 35 40 45Asp Met Leu Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Phe Val 35 40 45

Ala Gly lie Asp Asn Thr Gly Ser Tyr Thr His Tyr Gly Ala Ala Val 50 55 60 14- 143761.doc 201125580Ala Gly lie Asp Asn Thr Gly Ser Tyr Thr His Tyr Gly Ala Ala Val 50 55 60 14- 143761.doc 201125580

Lys Gly Arg Ala Thr lie Ser Arg Asp Asn Gly Gin Ser Thr Gly Arg 65 70 75 80Lys Gly Arg Ala Thr lie Ser Arg Asp Asn Gly Gin Ser Thr Gly Arg 65 70 75 80

Leu Gin Leu Asn Asn Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95Leu Gin Leu Asn Asn Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95

Ala Lys Arg Thr Ala Gly Ser lie Asp Ala Trp Gly His Gly Thr Glu 100 105 110Ala Lys Arg Thr Ala Gly Ser lie Asp Ala Trp Gly His Gly Thr Glu 100 105 110

Val lie Val Ser Ser Thr Ser Gly Gin Ala Gly Gin His His His His 115 120 125Val lie Val Ser Ser Thr Ser Gly Gin Ala Gly Gin His His His His 115 120 125

His His Gly Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser 130 135 140 -15- 143761.docHis His Gly Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser 130 135 140 -15- 143761.doc

Claims (1)

201125580 七、申請專利範圍: 1. 一種禽類抗α-異烯醇酶I(a-enolase I)多株抗體,其特異 性結合α-異烯醇酶I。 2. 如請求項1之多株抗體,其中該禽為雞、鴨或火雞。 3. 如請求項1之多株抗體,其中該禽為雞。 4. 如請求項1之多株抗體,其為多株抗α-異烯醇酶I IgY抗 體。 5. 如請求項1之多株抗體,其為抗α-異烯醇酶I雞igY抗體。 6. —種純化單株抗體或其抗原結合片段,其包含結合α-異 烯醇酶I之重鏈免疫球蛋白可變域及輕鏈免疫球蛋白可變 域,其中該輕鏈免疫球蛋白可變域包含以下胺基酸序 列:(i)CDRl 中之 SGGSGSYG(SEQ ID NO: 1)、 SGGSSSYGYG(SEQ ID NO: 2)、SGSSGSYG(SEQ ID NO: 3)、SGGSSSYGYS(SEQ ID NO: 4)或 SGSSGYGYG(SEQ ID NO: 5) ; (ii)CDR2 中之 ANTNRPS(SEQ ID NO: 6)、 NDNQRPS(SEQ ID NO: 7)、RDDKRPS(SEQ ID NO: 8)、 SNNQRPS(SEQ ID NO: 9)或 SNDKRPS(SEQ ID NO: 10);及(iii)CDR3 中之 GGYDSSAGI(SEQ ID NO: 11)、 GSGDSSTGM(SEQ ID NO: 12)、GSGESSTNNGI(SEQ ID NO: 13)、GSMDSSNSGV(SEQ ID NO: 14)或GGYDSSASYVGI (SEQ ID NO: 15);且其中該重鏈免疫球蛋白可變域包含 以下胺基酸序列:(i)CDRl中之SFNMF(SEQ ID NO: 16) ' SHDMG(SEQ ID NO: 17)、DYCVQ(SEQ ID NO: 18) ' SFYMF(SEQ ID NO: 19)或 SYAMH(SEQ ID NO:[ 143761.doc 201125580 20) ; (ii)CDR2 令之 GINNAGSTTNHGAAVKG(SEQ ID NO: 21)、GIENAAGIGTFYGAAVKG(SEQ ID NO: 22)、 AISNTGRYTGYGSAVKG(SEQ ID NO: 23) 、 GISGDGRYTGYGAAVDG(SEQ ID NO: 24)或 GISRDGGSSTRYYGAAVKG(SEQ ID NO: 25);及 (iii)CDR3 中之 SPGGIDGIDG(SEQ ID NO: 26)、 GADTGGWPAANIDA(SEQ ID NO: 27)、DGCAGCCGSYYIDG (SEQ ID NO: 28)、ESGSGCCNGDNIDA(SEQ ID NO: 29) 或 DSDNGGYYCDDIDA(SEQ ID NO: 30)。 7. 如請求項6之純化單株抗體或其抗原結合片段,其包含 選自由雞免疫球蛋白之生殖系基因序列(SEQ ID NO: 31) 編碼之VH及VL FR1、FR2、FR3及FR4構架區的構架區 或胺基酸序列與由雞免疫球蛋白之該生殖系基因序列 (SEQ ID NO:31)編碼之該等 VH 及 VL FR1、FR2 及 FR3 構 架區至少85%相同的構架區。 8. 如請求項7之純化單株抗體或其抗原結合片段,其包含 選自胺基酸序列與由雞免疫球蛋白之該生殖系基因序列 (SEQ ID NO:31)編碼之該等 VH 及 VL FR1、FR2 及 FR3 構 架區至少 87%、88%、89%、90%、91%、92%、93%、 94%、95%、97%、98%或99%相同的構架區。 9. 如請求項6之純化單株抗體或其抗原結合片段,其包含 具有選自由以下胺基酸序列組成之群之胺基酸序列的VL 互補決定區:i)CDRl中之SEQ ID NO: 1、CDR2中之SEQ ID NO: 6及 CDR3 中之 SEQ ID NO: 11 ; ii)CDRl 中之 SEQ 143761-doc 201125580 ID NO: 2、CDR2 中之 SEQ ID NO: 7及 CDR3 中之 SEQ ID NO: 12 ; iii)CDRl 中之 SEQ ID NO: 3、CDR2 中之 SEQ ID NO: 8及 CDR3 中之 SEQ ID NO: 13 ; iv)CDRl 中之 SEQ ID * NO: 4、CDR2 中之 SEQ ID NO: 9及 CDR3 中之 SEQ ID - NO: 14及 v)CDRl 中之 SEQ ID NO: 5、CDR2 中之 SEQ ID NO: 10及 CDR3 中之 SEQ ID NO: 15。 1 0.如請求項6之純化單株抗體或其抗原結合片段,其包含 具有選自由以下胺基酸序列組成之群之胺基酸序列的VL 互補決定區:ii)CDRl中之SEQ ID NO: 2、CDR2中之 SEQ ID NO: 7及 CDR3 中之 SEQ ID NO: 12 ;及 v)CDRl 中 之 SEQ ID NO: 5、CDR2 中之 SEQ ID NO: 10及 CDR3 中之 SEQ ID NO: 15。 11. 如請求項6之純化單株抗體或其抗原結合片段,其包含 具有選自由SEQ ID NO: 3 1-35組成之群之胺基酸序列的 VL互補決定區及具有選自由SEQ ID NO: 36-40組成之群 之胺基酸序列的&quot;V Η互補決定區。 12. 如請求項11之純化單株抗體或其抗原結合片段,其包含 ' 具有SEQ ID NO: 32或SEQ ID NO: 35之胺基酸序列的VL ^ 互補決定區及具有SEQ ID NO: 37或SEQ ID NO: 40之胺 基酸序列的VH互補決定區。 13. —種醫藥組合物,其包含如請求項1之多株抗體或如請 求項6之單株抗體及醫藥學上可接受之載劑。 14. 一種治療或預防α-異烯醇酶I相關病症之方法,其包含投 Γ 與治療有效量之如請求項1之多株抗體或如請求項6之單^ 143761.doc 201125580 株抗體。 項14之方法’其中該病症係選自由以下組成之 症、自體免疫病症、局部缺血及 16.如請求項丨 圏饮呆 链.昧 之方法,其中该癌症係選自由以下組成之 蚁癌礼癌、肛門癌、膀胱癌、骨癌、腸癌、腦腫 Ή癌、白血病、肝癌、胰癌、前列腺癌、直腸癌。 17·如請求項16之方法,其中該癌症為肺癌。 18.如請求項17之方法,其中該肺癌為非小細胞肺癌。 I%種^測樣品中《•異料酶!之存在的活體外診斷方 烯醇酿,含:⑴使樣品與如請求項1或請求項6之抗α_異 烯知酶I抗體接觸;及(ii)偵測該α_ 樣品之間的複合物的形成。 一1抗體與該 20 :Γ用二偵測樣品中α·異烯醇酶1之存在的套組,1包 求項1或請求項6之抗心異烯醇酶!抗體或其片 段’及視情況存在之資訊材料。 21. 如請求項20之套組,且中兮ρ 求項6之單株抗體。抗異歸醇酶1抗體為如請 22. 如請求項20之套組,且中續括 求項η之單株抗體。 異㈣酶1抗體為如請 143761.doc201125580 VII. Scope of application: 1. An avian anti-α-isoolase I antibody, which specifically binds to α-isoenolase I. 2. The antibody of claim 1, wherein the bird is a chicken, duck or turkey. 3. The antibody of claim 1, wherein the bird is a chicken. 4. The multi-strain antibody of claim 1, which is a plurality of anti-α-isoenolase I IgY antibodies. 5. The multi-strain antibody of claim 1, which is an anti-α-isoenolase I chicken igY antibody. 6. A purified monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain immunoglobulin variable domain and a light chain immunoglobulin variable domain that binds to α-isoenolase I, wherein the light chain immunoglobulin The variable domain comprises the following amino acid sequence: (i) SGGSGSYG (SEQ ID NO: 1), SGGSSSYGYG (SEQ ID NO: 2), SGSSSGSYG (SEQ ID NO: 3), SGGSSSYGYS (SEQ ID NO: 4) in CDR1 Or SGSSGYGYG (SEQ ID NO: 5); (ii) ANTNRPS (SEQ ID NO: 6), NDNQRPS (SEQ ID NO: 7), RDDKRPS (SEQ ID NO: 8), SNNQRPS (SEQ ID NO: 9) or SNDDRPS (SEQ ID NO: 10); and (iii) GGYDSSAGI (SEQ ID NO: 11), GSGDSSTGM (SEQ ID NO: 12), GSGESSTNNGI (SEQ ID NO: 13), GSMDSSNSGV (SEQ ID) in CDR3 NO: 14) or GGYDSSASYVGI (SEQ ID NO: 15); and wherein the heavy chain immunoglobulin variable domain comprises the following amino acid sequence: (i) SFNMF in CDR1 (SEQ ID NO: 16) 'SHDMG (SEQ ID NO: 17), DYCVQ (SEQ ID NO: 18) 'SFYMF (SEQ ID NO: 19) or SYAMH (SEQ ID NO: [143761.doc 201125580 20); (ii) CDR2 GINNAGSTTNHGAAVKG (SEQ ID NO: 21), GIENAAGIGTFYGAAVKG (SEQ ID NO: 22), AISNT GRYTGYGSAVKG (SEQ ID NO: 23), GISGDGRYTGYGAAVDG (SEQ ID NO: 24) or GISRDGGSSTRYYGAAVKG (SEQ ID NO: 25); and (iii) SPGGIDGIDG (SEQ ID NO: 26), GADTGGWPAANIDA (SEQ ID NO: 27) in CDR3 ), DGCAGCCGSYYIDG (SEQ ID NO: 28), ESGSGCCNGDNIDA (SEQ ID NO: 29) or DSDNGGYYCDDIDA (SEQ ID NO: 30). 7. The purified monoclonal antibody or antigen-binding fragment thereof according to claim 6, which comprises a VH and VL FR1, FR2, FR3 and FR4 framework selected from the germline gene sequence (SEQ ID NO: 31) of chicken immunoglobulin. The framework region or amino acid sequence of the region is at least 85% identical to the framework regions of the VH and VL FR1, FR2 and FR3 framework regions encoded by the germline gene sequence of chicken immunoglobulin (SEQ ID NO: 31). 8. The purified monoclonal antibody or antigen-binding fragment thereof according to claim 7, which comprises an amino acid sequence selected from the VHs encoded by the germline gene sequence of chicken immunoglobulin (SEQ ID NO: 31) and At least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98% or 99% of the framework regions of the VL FR1, FR2 and FR3 framework regions. 9. The purified monoclonal antibody or antigen-binding fragment thereof according to claim 6, which comprises a VL complementarity determining region having an amino acid sequence selected from the group consisting of the following amino acid sequences: i) SEQ ID NO in CDR1: 1. SEQ ID NO: 6 in CDR2 and SEQ ID NO: 11 in CDR3; ii) SEQ 143761-doc in CDR1 201125580 ID NO: 2. SEQ ID NO: 7 in CDR2 and SEQ ID NO in CDR3 : 12 ; iii) SEQ ID NO: 3 in CDR1, SEQ ID NO: 8 in CDR2 and SEQ ID NO: 13 in CDR3; iv) SEQ ID * NO: 4 in CDR1, SEQ ID NO in CDR2 SEQ ID NO: 5 in SEQ ID NO: 14 and CDR3, SEQ ID NO: 5 in CDR2, SEQ ID NO: 10 in CDR2 and SEQ ID NO: 15 in CDR3. A purified monoclonal antibody or antigen-binding fragment thereof according to claim 6, which comprises a VL complementarity determining region having an amino acid sequence selected from the group consisting of the following amino acid sequences: ii) SEQ ID NO in CDR1 2, SEQ ID NO: 7 in CDR2 and SEQ ID NO: 12 in CDR3; and v) SEQ ID NO: 5 in CDR1, SEQ ID NO: 10 in CDR2 and SEQ ID NO: 15 in CDR3 . 11. The purified monoclonal antibody or antigen-binding fragment thereof according to claim 6, which comprises a VL complementarity determining region having an amino acid sequence selected from the group consisting of SEQ ID NOS: 3 1-35 and having a SEQ ID NO selected from the group consisting of SEQ ID NO : The &quot;V Η complementarity determining region of the amino acid sequence of the group consisting of 36-40. 12. The purified monoclonal antibody or antigen-binding fragment thereof according to claim 11, which comprises a VL^complementarity determining region having the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 35 and having SEQ ID NO: 37 Or the VH complementarity determining region of the amino acid sequence of SEQ ID NO: 40. 13. A pharmaceutical composition comprising a polyclonal antibody of claim 1 or a monoclonal antibody of claim 6 and a pharmaceutically acceptable carrier. A method for treating or preventing an α-isoenolase I-related disorder, comprising administering a therapeutically effective amount of the polyclonal antibody of claim 1 or the antibody of claim 1 143761.doc 201125580 strain of claim 6. The method of Item 14 wherein the condition is selected from the group consisting of: an autoimmune disorder, ischemia, and a method of sipping a chain, wherein the cancer is selected from the group consisting of Cancer cancer, anal cancer, bladder cancer, bone cancer, intestinal cancer, brain cancer, leukemia, liver cancer, pancreatic cancer, prostate cancer, rectal cancer. 17. The method of claim 16, wherein the cancer is lung cancer. 18. The method of claim 17, wherein the lung cancer is non-small cell lung cancer. I% species test sample "• Heterogeneous enzymes! The presence of an in vitro diagnostic enol may comprise: (1) contacting the sample with an anti-α-isoenzyme I antibody as claimed in claim 1 or claim 6; and (ii) detecting a complex between the α_ samples The formation of objects. A 1 antibody and the 20: Γ2 detection kit for the presence of α-isoenolase 1 in the sample, 1 package of the anti-cardinal enolase of claim 1 or claim 6! Antibody or its fragments&apos; and informational materials as appropriate. 21. The kit of claim 20, and the antibody of the individual strain of 6ρ. The anti-isosinase 1 antibody is a set of antibodies as described in claim 22. Iso (tetra) enzyme 1 antibody is 143761.doc
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TWI579304B (en) * 2014-12-31 2017-04-21 財團法人生物技術開發中心 Humanized alpha-enolase specific antibodies and methods of uses in cancer therapy
CN112451661A (en) * 2013-12-20 2021-03-09 财团法人生物技术开发中心 Alpha-enolase specific antibodies and methods of use thereof in the treatment of immune disorders
EP3924379A4 (en) * 2019-02-15 2022-12-21 Integral Molecular, Inc. Antibodies comprising a common light chain and uses thereof

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KR100910962B1 (en) * 2008-09-22 2009-08-05 한국생명공학연구원 Eno1-specific human antibody

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Publication number Priority date Publication date Assignee Title
CN112451661A (en) * 2013-12-20 2021-03-09 财团法人生物技术开发中心 Alpha-enolase specific antibodies and methods of use thereof in the treatment of immune disorders
TWI579304B (en) * 2014-12-31 2017-04-21 財團法人生物技術開發中心 Humanized alpha-enolase specific antibodies and methods of uses in cancer therapy
EP3924379A4 (en) * 2019-02-15 2022-12-21 Integral Molecular, Inc. Antibodies comprising a common light chain and uses thereof

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