TWI661096B - 功能性抗體片段的高通量篩選方法及其免疫偶聯物以及用於篩選之適體-藥物偶聯物 - Google Patents

功能性抗體片段的高通量篩選方法及其免疫偶聯物以及用於篩選之適體-藥物偶聯物 Download PDF

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TWI661096B
TWI661096B TW106119418A TW106119418A TWI661096B TW I661096 B TWI661096 B TW I661096B TW 106119418 A TW106119418 A TW 106119418A TW 106119418 A TW106119418 A TW 106119418A TW I661096 B TWI661096 B TW I661096B
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An-Suei Yang
楊安綏
Hong-sen CHEN
陳鴻森
Chung-Ming Yu
余忠銘
Shin-Chen HOU
侯信成
Wei-Ying Kuo
郭蔚瑩
Yi-Kai CHIU
邱奕凱
Yueh-liang TSOU
鄒岳良
Hung-Ju Hsu
許鴻儒
Hwei-Jiung WANG
王惠鈞
Shih-Hsien Chuang
莊士賢
Chao-Pin Lee
李照斌
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中央研究院
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Abstract

在此揭示高通量篩選用於免疫偶聯物之功能性片段的方法，此種免疫偶聯物可標的一蛋白抗原。所述方法結合了噬菌體表現的合成抗體庫以及高通量的細胞毒性篩選，篩選過程使用非共價結合的免疫毒素或細胞毒性藥物，以鑑別出具有高度功能性的合成抗體片段，其可用於遞送毒素藥物負載。

Description

功能性抗體片段的高通量篩選方法及其免疫偶聯物以及用於篩選之適體-藥物偶聯物
本發明是有關於一種篩選功能性抗體片段的方法。
免疫偶聯物(immunoconjugate)已逐漸成為抗體相關療法的主流。特別是帶有抗體片段作為標的模組以及蛋白毒素作為藥物負載的免疫偶聯物(即，免疫毒素)已取代使用抗體-藥物偶聯物(抗體-drug conjugate，ADC)的化療毒素，成為抗癌藥物的重要領域。1999年美國食品暨藥物管理局核准了第一個用於治療的蛋白毒素(即，白喉毒素)，地尼白介素(denileukin diftitox)含有白喉毒素，可用以治療表皮T細胞淋巴瘤(cutaneous T-cell lymphoma)。目前有十種左右的免疫毒素正在臨床開發階段。免疫毒素的副作用包括血管滲漏症候群以及蛋白毒素導致的免疫原性等，雖然上述副作用帶來許多挑戰，在下一代的免疫毒素開發中，對於降低蛋白毒素負載的免疫原性以及毒性已有實質進展。
目前人類臨床試驗中所用的免疫毒素大約有五成運用了源自假單胞菌屬外毒素(Pseudomonas exotoxin，PE) A的細胞毒性藥物負載。PE是由綠膿桿菌(P. aeruginosa )產生的A-B類細胞毒性蛋白，已知PE會遵循常見的逆向傳輸路徑(retrograde trafficking route)進入細胞質。PE的B次單元可和人類細胞表面受體LRP1或LRP1B (低密度脂蛋白受體-相關蛋白1/1B (low density lipoprotein receptor-related 1/1B))結合，引發受體介導之細胞吞噬作用。在胞內體中，結合於細胞膜上的內切蛋白酶—弗林蛋白酶(endoprotease furin)會將A-B次單元之間的胜肽鍵結切斷。經過剪切的毒素被傳送到高基氏體之後會和KDEL受體結合，其之後會將毒素傳送到內質網(endoplasmic reticulum，ER)，在該處由蛋白雙硫鍵異構酶(protein disulfide isomerase，PDI)切開連接A-B次單元的雙硫鍵，且只會將PE的A次單元傳送至細胞質，此一過程可能是透過Sec61易位組(translocon)來進行。在細胞質中，A次單元催化真核生物延長因子(eukaryotic elongation factor 2，eEF2)的不活化作用，上述作用是透過將ADP-核糖基由NAD+轉移至eEF2中高度保留的經轉譯後修飾之白喉醯胺-組胺酸。eEF2的不活化會使得蛋白合成作用終止，進而導致細胞凋亡。據估計，細胞質中有一個PE A次單元分子即足以誘導該細胞的凋亡。同理，由PE的A次單元與抗體片段組成的免疫毒素會藉由抗體-抗原互動而專一標的至特定的細胞表面抗原。然而，只有當免疫毒素因為抗體-抗原互動而造成其內化並經過適當處理後，PE的A次單元才能夠有效地運用和原生毒素一般的逆向傳輸路徑，並因而發揮其強烈的細胞毒性而殺死細胞。
免疫毒素的細胞毒性效果是透過含抗體之標的模組誘導受體介導之細胞吞噬作用，並將毒素藥物負載遞送至適當的次細胞位置以發揮最佳毒性。已知受體介導之細胞吞噬作用，特別是透過格形蛋白包覆小窩(clathrin-coated pits)的作用，是用以控制受體依賴性信息傳導的相關機制，此一作用會根據細胞整體的活化狀態來調整細胞表面受體分布。當抗體-受體互動誘導經由不同細胞吞噬機制的受體介導之細胞吞噬作用時，就會進行免疫毒素內化作用。雖然已經發現抗體介導之受體交聯與受體上的抗體結合位置對於免疫偶聯物誘導之細胞吞噬作用的效率扮演關鍵角色，對於免疫偶聯物之內化的機制以及毒素藥物負載的傳遞等仍然所知有限。
由上文可知，想要發現能夠透過和特定細胞表面標的之互動來遞送細胞毒性藥物負載適當抗體必須依賴篩選大量候選抗體，此一過程既耗時又費力。因此，本領域亟需提出一種能夠快速篩選大量功能性片段的方法，以找出能夠作為免疫偶聯物之標的模組的功能性片段。
發明內容旨在提供本揭示內容的簡化摘要，以使閱讀者對本揭示內容具備基本的理解。此發明內容並非本揭示內容的完整概述，且其用意並非在指出本發明實施例的重要/關鍵元件或界定本發明的範圍。
本發明的一態樣是關於一種高通量篩選方法，其可用以篩選用於免疫偶聯物之功能性片段，所述免疫偶聯物可標的一蛋白抗原。
根據本發明一實施例，所述方法包含以下步驟： (a) 提供一噬菌體表現的合成單鏈變異片段(single-chain variable fragment，scFv)抗體庫，該抗體庫包含複數個噬菌體表現的scFv，其中該些噬菌體表現的scFv之每一者的VH結構域對蛋白A有一結合親和力，且該些噬菌體表現的scFv之每一者的VL結構域對蛋白L有一結合親和力； (b) 從步驟(a)之該噬菌體表現的合成scFv抗體庫中篩選表現對該蛋白抗原專一結合的複數個噬菌體； (c) 從步驟(b)選出的該些噬菌體分別製備複數個分泌之scFv； (d) 藉由一適體-藥物偶聯物(adaptor-drug conjugate)分別與步驟(c)製備的該些分泌之scFv混合，以得到複數個scFv-適體-藥物複合物(scFv-adaptor-drug complex)，其中該適體-藥物偶聯物包含一藥物及一適體，該適體包含至少一AL蛋白模組，其包含位於N端的一蛋白A片段以及位於C端的一蛋白L片段； (e)將步驟(d)的條件下所得之該些scFv-適體-藥物複合物，分別與複數個呈現該蛋白抗原的細胞一起培養； (f) 決定該些scFv-適體-藥物複合物對步驟(e)中培養的呈現該蛋白抗原的該些細胞之的毒殺效果；以及 (g) 基於步驟(f)的結果，選擇用於該免疫偶聯物的該功能性片段，其中選擇該些scFv-適體-藥物複合物中相較於其他scFv-適體-藥物複合物展現較佳效果的一或多個scFv-適體-藥物複合物的scFv作為用於該免疫偶聯物的該功能性片段。
根據某些實施方式，所述蛋白抗原為人類表皮生長因子受體2 (human epidermal growth factor receptor 2，HER2)、麥芽糖結合蛋白、牛血清白蛋白、人類血清白蛋白、溶菌酶、介白素-1β (interleukin-1 beta，IL-1β)、流行性感冒病毒的血球凝集素、血管內皮生長因子 (vascular endothelial growth factor，VEGF)、表皮生長因子受體1 (epidermal growth factor receptor 1，EGFR1)、表皮生長因子受體3 (EGFR3)、升糖素受體、程序性死亡配體1 (programmed death-ligand 1，PD-L1)、唾液酸结合免疫球蛋白樣凝集素3 (sialic acid binding Ig-like lectin 3，SIGLEC 3)或利妥昔單抗(rituximab)。於一些實施方式中，所述蛋白抗原是HER2。
在一些視需要而採用的實施方式中，所述藥物包含一藥物部分；藥物部分可以是免疫毒素、免疫微脂體、或細胞毒性藥物。在部分實施方式中，所述藥物部分是免疫毒素，譬如外毒素。舉例來說，外毒素可以是假單胞菌屬外毒素(PE) A或由其所衍生。於一些實施方式中，免疫毒素包含截短的PE A次單元毒素。在進一步的實施方式中，所述藥物更包含內質網(ER)駐留部分，其位於PE A次單元毒素的C端。舉例來說，ER駐留部分包含KDEL序列。
在部分視需要而採用的實施方式中，於步驟(f)中測量步驟(e)中培養之呈現所述蛋白抗原的細胞的細胞存活率
於另一種態樣中，本揭示內容是關於一種高通量篩選方法，其可用於篩選免疫偶聯物之功能性片段，所述免疫偶聯物可標的人類表皮生長因子受體2 (HER2)。
根據多種實施方式，所述方法包含以下步驟： (a) 提供一噬菌體表現的合成單鏈變異片段(scFv)抗體庫，該抗體庫包含複數個噬菌體表現的scFv，其中該些噬菌體表現的scFv之每一者的VH結構域對蛋白A有一結合親和力，且該些噬菌體表現的scFv之每一者的VL結構域對蛋白L有一結合親和力； (b) 從步驟(a)之該噬菌體表現的合成scFv抗體庫中篩選表現對HER2專一結合的複數個噬菌體； (c) 從步驟(b)選出的該些噬菌體分別製備複數個分泌之scFv； (d) 藉由一適體-藥物偶聯物分別與步驟(c)製備的該些分泌之scFv混合，以得到複數個scFv-適體-藥物複合物，其中該適體-藥物偶聯物包含：一蛋白藥物，其包含外毒素；以及一適體，該適體包含至少一AL蛋白模組，其包含位於N端的一蛋白A片段以及位於C端的一蛋白L片段； (e)將步驟(d)的條件下所得之該些scFv-適體-藥物複合物，分別與複數個呈現HER2的細胞一起培養； (f) 決定該些scFv-適體-藥物複合物對步驟(e)中培養的呈現該蛋白抗原的該些細胞之的毒殺效果；以及 (g) 基於步驟(f)的結果，選擇用於該免疫偶聯物的該功能性片段，其中選擇該些scFv-適體-藥物複合物中相較於其他scFv-適體-藥物複合物展現較佳效果的一或多個scFv-適體-藥物複合物的scFv作為用於該免疫偶聯物的該功能性片段。
在一些視需要而採用的實施方式中，所述外毒素可以是假單胞菌屬外毒素(PE) A或由其所衍生；舉例來說，蛋白藥物包含截短的PE A次單元毒素。在進一步的實施方式中，蛋白藥物更包含內質網(ER)駐留部分，其位於PE A次單元毒素的C端。舉例來說，ER駐留部分包含KDEL序列。
在又另一種態樣中，本發明是關於一種標的一蛋白抗原的免疫偶聯物，或包含此種免疫偶聯物的藥學組合物。
根據某些實施方式，所述免疫偶聯物包含一藥物與一標的模組，其中標的模組包含利用上述本發明任一種態樣/實施方式所挑選出的功能性片段。此外，所述藥物包含一藥物部分，藥物部分可以是免疫毒素、免疫微脂體或細胞毒性藥物。
根據本揭示內容實施方式的藥學組合物包含有效量的免疫偶聯物以及藥學上可接受的免疫偶聯物受體。
在又另一種態樣中，本揭示內容是關於用以治療有需要個體的方法，所述方法包含以下步驟：對該患者投予有效量之根據本發明任一態樣/實施方式之免疫偶聯物或藥學組合物。
在又另一種態樣中，本揭示內容是關於一種適體-藥物偶聯物，其可運用於根據本揭示內容任一態樣/實施方式之高通量篩選方法。
根據某些實施方式，所述適體-藥物偶聯物包含：(1)一適體，其包含至少一AL蛋白模組，其中每一AL蛋白模組包含位於N端的蛋白A片段以及位於C端的蛋白L片段，以及用以連接上述蛋白A片段與蛋白L片段的一第一多肽連接物；(2)一藥物；以及(3)一第二多肽連接物，用以將藥物連接至適體C端。
在一些視需要而採用的實施方式中，所述藥物包含一藥物部分；藥物部分可以是免疫毒素、免疫微脂體、或細胞毒性藥物。在部分實施方式中，所述藥物部分是免疫毒素，譬如外毒素。舉例來說，外毒素可以是假單胞菌屬外毒素(PE) A或由其所衍生。於一些實施方式中，免疫毒素包含截短的PE A次單元毒素。在進一步的實施方式中，所述藥物更包含內質網(ER)駐留部分，其位於PE A次單元毒素的C端。舉例來說，ER駐留部分包含KDEL序列
在參閱下文實施方式後，本發明所屬技術領域中具有通常知識者當可輕易瞭解本發明之基本精神及其他發明目的，以及本發明所採用之技術手段與實施態樣。
為了使本揭示內容的敘述更加詳盡與完備，下文針對了本發明的實施態樣與具體實施例提出了說明性的描述；但這並非實施或運用本發明具體實施例的唯一形式。實施方式中涵蓋了多個具體實施例的特徵以及用以建構與操作這些具體實施例的方法步驟與其順序。然而，亦可利用其他具體實施例來達成相同或均等的功能與步驟順序。
為了便於說明，此處整理了說明書、實施例與附隨申請專利範圍中所用的某些詞彙。除非本說明書另有定義，此處所用的科學與技術詞彙之含義與本發明所屬技術領域中具有通常知識者所理解與慣用的意義相同。
在不和上下文衝突的情形下，本說明書所用的單數名詞涵蓋該名詞的複數型；而所用的複數名詞時亦涵蓋該名詞的單數型。此外，在本說明書與申請專利範圍中，「至少一」與「一或更多」等表述方式的意義相同，兩者都代表包含了一、二、三或更多。更有甚者，在本說明書與申請專利範圍中，「A、B及C其中至少一者」、「A、B或C其中至少一者」以及「A、B和/或C其中至少一者」係指涵蓋了僅有A、僅有B、僅有C、A與B兩者、B與C兩者、與C兩者、以及A、B與C三者。
雖然用以界定本發明較廣範圍的數值範圍與參數皆是約略的數值，此處已盡可能精確地呈現具體實施例中的相關數值。然而，任何數值本質上不可避免地含有因個別測試方法所致的標準偏差。在此處，「約」通常係指實際數值在一特定數值或範圍的正負10%、5%、1%或0.5%之內。或者是，「約」一詞代表實際數值落在平均值的可接受標準誤差之內，視本發明所屬技術領域中具有通常知識者的考量而定。除了實驗例之外，或除非另有明確的說明，當可理解此處所用的所有範圍、數量、數值與百分比（例如用以描述材料用量、時間長短、溫度、操作條件、數量比例及其他相似者）均經過「約」的修飾。因此，除非另有相反的說明，本說明書與附隨申請專利範圍所揭示的數值參數皆為約略的數值，且可視需求而更動。至少應將這些數值參數理解為所指出的有效位數與套用一般進位法所得到的數值。在此處，將數值範圍表示成由一端點至另一段點或介於二端點之間；除非另有說明，此處所述的數值範圍皆包含端點。
在本說明書中，「抗原」(antigen或Ag)一詞是指可引發免疫反應的分子。所述的免疫反應可能涉及抗體的產生或特定免疫勝任細胞之活化，或二者。本發明所述技術領域中具有通常知識者當可理解，任何大分子(包括幾乎所有蛋白與胜肽)皆可作為抗原。此外，抗原亦可源自重組或基因體DNA。本發明所述技術領域中具有通常知識者當可了解，任何編碼能夠引發免疫反應之蛋白的DNA(包含一核酸序列或部分核酸序列)，也都因此編碼了此處所述的「抗原」。此外，所屬技術領域具有通常知識者亦可了解，抗原不必然是由基因中單一全長核酸序列編碼而成，而是有可能由源自多個基因、經不同重組方式排列之部分核酸序列編碼而成，藉以引發所欲的免疫反應。此外，所屬技術領域具有通常知識者當可了解，抗原不必然是由一「基因」編碼而成；目前已知抗原可以是合成或源自生物檢體的物質。所述生物檢體包含，但不限於組織檢體、腫瘤檢體、細胞或生物體液。
「抗體」(antibody)一詞在本說明書泛指單株抗體(monoclonal antibody；包含全長單株抗體)、多株抗體(polyclonal antibody)、多專一或多價性抗體(multi-specific or multivalent antibody；例如雙專一性抗體)以及能夠產生特定生物活性的抗體片段。「抗體片段」(antibody fragment)一詞在本說明書中包含全長抗體的一部分，該部分通常為抗體中與抗原結合的位置或變異區域。例示性的抗體片段包含Fab、Fab′、F(ab′)2、Fv片段、雙鏈抗體(diabody)、線性抗體(linear antibody)、單鏈抗體分子(single-chain antibody molecule)以及由抗體片段形成的多專一性抗體。
「抗體庫」(antibody library)一詞在本說明書是指一群經表現的抗體及/或抗體片段，用以篩選及/或組合成完整抗體。抗體及/或抗體片段可以表現於核醣體(ribosome)、噬菌體或細胞表面，特別是酵母菌細胞表面。
在本說明書中，「單鏈變異片段」(single-chain variable fragment或scFv)是指包含一免疫球蛋白之重鏈變異區域(variable regions of the heavy chain，VH)及輕鏈變異區域(variable regions of the light chain，VL)的融合蛋白，其中所述的VH與VL透過共價鍵結以形成一VH::VL異二聚體(heterodimer)。所述VH與VL可直接連結或透過編碼一胜肽的連接物而連結，上述連接物可連接VH的N端及VL的C端，或是連接VH的C端及VL的N端。連接物通常包含多個能增加可撓性的甘胺酸(glycine)以及多個能增加溶解性的絲胺酸(serine)或蘇胺酸(threonine)。雖然移除了抗體的恒定區域且插入連接物，scFv蛋白仍保有原免疫球蛋白的專一性。可由包含編碼VH及VL序列的核酸來表現單鏈Fv多肽抗體。
「互補性決定區域」(complementarity determining region，CDR)一詞在此處是指抗體分子的高度變異區域，其可與結合抗原之三維表面形成互補表面。由N端到C端，每一抗體的重鏈及輕鏈皆包含三個CDR，分別是CDR 1、CDR 2及CDR3。因此，一個HLA-DR抗原結合位置共包含6個CDR，即，三個重鏈變異區域的CDR與三個輕鏈變異區域的CDR。各CDR中的胺基酸殘基會與結合抗原緊密接觸，其中與抗原接觸最為緊密的通常是重鏈CDR3。
在本說明書中，「EC50 」一詞是指一抗體或其抗原結合區域的濃度，該濃度在活體外或活體試驗中所引發的反應是最大反應的50%，即最大反應量與基線的中間值。
在本說明書中，「結合速率常數」(association rate constant，kon )是用以表示抗體與抗體標的抗原間之結合強度的數值，通常是以抗原-抗體的反應動力學來測定之。「解離速率常數」(dissociation rate constant，koff )則是用以表示抗體與抗體標的抗原間之解離強度的數值，一般是以抗原-抗體的反應動力學來測定之。將「解離速率常數」(koff )除以「結合速率常數」(kon )即可得到「解離常數」(dissociation constant，Kd )。以上常數可作為表示抗體對其抗原之親和力以及中和所述抗原之活性的指標。
「噬質體」(phagemid)一詞在本說明書是指一結合細菌噬菌體及質體特性的載體(vector)。細菌噬菌體是指任何一種可感染細菌的病毒。
「核酸序列」(nucleic acid sequence)、「多核苷酸」(polynucleotide)或「核酸」(nucleic acid)等詞彙在本說明書中可交替使用，可指雙股去氧核糖核酸(deoxyribonuclic acid，DNA)、單股DNA或所述DNA之轉錄產物（例如核糖核酸分子(ribonucleic acid，RNA))。需知本揭示內容無關乎自然界或自然狀態下的基因多核苷酸序列。能用以分離或純化(或部份純化)本發明之核酸、多核苷酸或核苷酸序列的方法包含，但不限於：離子交換色層分析(ion-exchange chromatography)、分子篩選色層分析(molecular size exclusion chromatography)或是可用於基因工程技術諸如擴增(amplification)、扣除雜交法(subtractive hybridization)、選殖(cloning)、次選殖(sub-cloning)、化學合成(chemical synthesis)或其組合。
本發明亦包含所有簡併核苷酸序列(degenerate nucleotide sequence)，其係用以編碼於生物體中具有特定活性或功能的胜肽/多肽/蛋白(例如本發明的CDR)。「簡併核苷酸序列」(degenerate nucleotide sequence)是指包含一或多個簡併密碼子(degenerate codon；與可編碼一多肽之參考多核苷酸分子相比)的核苷酸序列。簡併密碼子可包含不同的核苷酸三聯體(triplet)，卻可編碼出相同的胺基酸殘基(例如GAU及GAC三聯體皆可編碼天門冬胺酸(aspartic acid，Asp))。
「編碼序列」(coding sequence)及「編碼區域」(coding region)在本說明書中可交替使用，是指包含RNA及DNA的核苷酸序列及及核酸序列，其可用以編碼合成RNA、蛋白、部分RNA或部分蛋白時需要的基因訊息。一生物體基因組之自然產物以外的核苷酸序列稱為「外來核苷酸序列」(foreign nucleotide sequence)、「異源性核苷酸序列」(heterologous nucleotide sequences)或「外源性核苷酸序列」(exogenous nucleotide sequences)。「異源性蛋白」(heterologous proteins)是指由外來、異源性或外源性核苷酸編碼之蛋白，故因此在所述細胞中通常不會天然表現這些蛋白。經分離後重新引入相同種類(例如同一物種)生物體的核苷酸序列不屬於該特定生物體基因組天然產生的產物，而會視為異性或外源性核苷酸序列。
此處針對合成胜肽序列所述的「胺基酸序列相似度百分比」(percent % amino acid sequence identity)係指候選合成胜肽之胺基酸殘基與一參考多胜肽之胺基酸殘基完全相同的百分比。於進行上述比對時，可將該候選合成胜肽與該參考多胜肽並排，並於必要時引入間隙，以使二序列形成最高的序列相似度，且在計算相似度時，並未將保守性置換之胺基酸殘基納入考量。相關領域已有多種方法可供進行上述並排，譬如可公開取得的軟體如BLAST、BLAST-2、ALIGN或Megalign (DNASTAR)等。本發明所屬技術領域中具有通常知識者在進行並排時，可選擇適當的參數與計算方式，以得到最佳的排列方式。在本說明書中，二胺基酸序列間的序列比較是採用美國國家生物科技資訊中心(Nation Center for Biotechnology Information，簡稱NCBI)所提供的蛋白質-蛋白質BLAST分析軟體Blastp來進行。一候選胺基酸序列A相較於一參考胺基酸序列B的胺基酸序列相似度(在本說明書中亦稱之為序列A與序列B具有特定百分比(%)的胺基酸序列相似度)的計算方式如下:% 其中X是利用Blastp軟體對序列A、B進行排列後所得到的相同胺基酸殘基數目（identical matches），而Y是A、B二序列中較短者的胺基酸殘基總數。
在此處「藥學上可接受賦型劑」(pharmaceutically acceptable excipient)係指一種藥學上可接受的材料、組合物或媒劑(vehicle)，諸如液體或固體填充物(liquid or solid filler)、稀釋劑(diluent)、賦型劑(excipient)、溶劑(solvent)或包覆材料(encapsulating material)，其可用以攜帶或運送標的成分由身體的一器官或部分到達身體的另一器官或部份。「可接受」的載體係指其可和組合物中的其他成分相容。藥學組合物或藥物製劑(pharmaceutical formulation)包含本案所述的活性成分以及一或多種藥學組合物。載體可為固態、半固態、液態、乳霜或膠囊等形式。這些藥學配方也屬於本發明之一態樣。一般來說，活性成分的含量通常佔藥學配方重量比的0.1-95%之間，而在腸道外給藥時較佳為0.2-20% (重量百分比)。臨床上使用本發明之方法時，可將此處提出的藥學組合物製備成適合所欲給藥途徑的劑型。
本揭示內容是關於一種高通量篩選方法，其可用以鑑別功能性抗體(或其片段)，所述功能性抗體可作為標的模組，以將包含該功能性抗體之免疫偶聯物遞送至一個體體內的目標部位。在本申請案中，利用可標的HER2過度表現之細胞的免疫毒素作為模型系統，以評估使用合成抗體庫來開發免疫偶聯物的可行性。使用滿足以下條件之抗體作為免疫偶聯物中的標的模組，較有可能得到理想且具有療效的免疫偶聯物：對標的受體有適當的親和力與專一性；能夠誘導受體介導之細胞吞噬作用；能夠遞送毒素藥物負載至次細胞位置以發揮理想的細胞毒性；源自人類以降低其免疫原性；易於製造且有高度表現效率與及蛋白穩定性。為了達到以上目的，本案建構了噬菌體表現的合成抗體庫(GH2)，其具有單一種的人類變異結構域抗體種系架構：IGKV1-NL1*01/IGHV3-23*04，在此一架構上，透過電腦運算與實驗分析得出抗體-蛋白互動原則，並進一步設計了多個抗體庫。透過基於噬菌體表現的選擇與篩選，從GH2抗體庫中選出了數百個對HER2-ECD有高結合親和力與專一性的抗體。GH2抗體在HER2-ECD上的抗原決定位散佈於HER2-ECD的分子表面，且有許多新的抗原決定位。更有甚者，可以高效率地表現出非常大量的GH2抗體，不論是scFv還是IgG形式，且蛋白穩定性高。因此，進行基礎研究，以探究這些HER-ECD-專一GH2抗體作為毒素藥物負載之標的模組的可行性。
本研究的目的之一是要從大量對標的專一的抗體中，鑑別出可用於免疫毒素中的理想功能性抗體，以便瞭解影響抗體作為遞送細胞毒性藥物之標的模組的效率之原理。本案開發出適體-毒素融合蛋白AL1-PE38KDEL與AL2-PE38KDEL，其可用於高通量篩選可作為標的模組來遞送PE38KDEL之GH2抗體，所述PE38KDEL包含截短的PE A次單元毒素。利用蛋白A與蛋白L選擇來建構GH2抗體庫，以確保GH2 scFv的VH與VL結構域之IGKV1-NL1*01/IGHV3-23*04架構可同時分別連接至蛋白A與蛋白L。AL1片段包含連續的蛋白A與蛋白L，兩者間以一多肽連接物隔開，以使得蛋白A與蛋白L可同時結合至一GH2 scFv；AL2片段事由兩個連續AL1模組彼此以另一多肽連接物隔開，其長度經設計可促使兩個GH2 scFv同時連接至AL2片段。雖然在一個免疫偶聯物複合物中，將scFv-AL1-PE38KDEL設計成抗體:毒素為1:1，scFv-AL2-PE38KDEL則被設計為類似二價抗原結合，在一個免疫偶聯物複合物中，IgG有兩個scFv。測試了可和HER2-ECD以高度專一性與親和力結合的92個GH2 scFv，以分析其是否可作為標的模組，並將其與AL1-PE38KDEL與AL2-PE38KDEL適體-毒素融合蛋白結合。
由下文所示的實驗結果可以發現，當將不同的scFv和PE38KDEL偶聯之後，出人意料地會展現出不同的細胞毒性。在某些scFv中，二價結合會增進較佳的毒素遞送能力，但在其他scFv中，二價與一價結合能力對細胞毒性沒有明顯的差別。抗原決定位在標的受體上的位置某種程度上決定了受體介導之細胞吞噬作用的效率及/或毒素遞送效率；抗體對位(paratope)上的胺基酸種類也可能是重要的決定因素，這會影響次細胞毒素遞送位置。標的受體在細胞表面上的密度則決定了免疫偶聯物的細胞毒性效力。這些結果指出，標的細胞及作為標的模組之抗體和免疫毒素的細胞毒性有決定性的關連；從大量候選抗體中篩選標的模組是開發免疫偶聯物中之最佳化抗體的一個重要策略。雖然從天然抗體來源中很難得到大量的候選抗體，但利用合成抗體庫就可以得到功能性抗體，而不需受限於天然的抗體反應。將合成抗體庫和本發明提出的高通量篩選平台結合，使得研發人員掌握了較佳的開發能力，而能夠得到以功能性抗體為基礎的理想標的模組，並用以製備免疫偶聯物。
有鑑於此，本揭示內容的第一態樣是關於一種高通量篩選方法，其可用以篩選用於免疫偶聯物之功能性片段，以使得該免疫偶聯物能夠標的一蛋白抗原。下文討論此方法之步驟。
首先，在步驟(a)，提供噬菌體表現的合成單鏈變異片段(scFv)抗體庫。噬菌體表現的合成scFv抗體庫包含複數個噬菌體表現的scFv。具體來說，透過蛋白A與蛋白L的選擇邋建構上述噬菌體表現的合成scFv抗體庫，而使得這些噬菌體表現的scFv中的每一者都有能夠結合至蛋白A的VH結構域以及能夠結合至蛋白L的VL結構域。可利用申請中的PCT專利申請案PCT/US2016/19128中所述的方法來建構這種噬菌體表現的合成scFv抗體庫。
接著，在步驟(b)，從步驟(a)的噬菌體表現的合成scFv抗體庫中選擇複數個噬菌體，這些噬菌體表現出對所標的之蛋白抗原專一的scFv。舉例來說，先將所述蛋白抗原固定於一基質(譬如瓊酯糖樹脂或聚丙烯醯胺)上，並和此處提出之噬菌體表現的scFv抗體庫混合。之後，對於表現的scFv可和所述蛋白抗原結合之噬菌體，以洗提緩衝液處理(譬如，pH 2.2的甘胺酸溶液)，以便打斷經固定之蛋白抗原與噬菌體表現抗體之間的結合。如此一來，可以收集到複數個表現出對所述蛋白抗原專一之scFv的噬菌體。
其後，在步驟(c)，從步驟(b)中選擇的該些噬菌體分別製備複數個分泌之可溶性scFv。可利用本發明所述技術領域中具有通常知識者熟知的方法來完成此一製備步驟。
根據某些實施方式，上述蛋白抗原可以是人類表皮生長因子受體2 (HER2)、麥芽糖結合蛋白、牛血清白蛋白、人類血清白蛋白、溶菌酶、介白素-1β (IL-1β)、流行性感冒病毒的血球凝集素、血管內皮生長因子 (VEGF)、表皮生長因子受體1 (EGFR1)、表皮生長因子受體3 (EGFR3)、升糖素受體、程序性死亡配體1 (PD-L1)、唾液酸结合免疫球蛋白樣凝集素3 (SIGLEC 3)或利妥昔單抗。於一些實施方式中，所述蛋白抗原是HER2。
根據某些視需要而採用的實施方式，可進一步利用相同的蛋白抗原來篩選在步驟(c)所製得之分泌之可溶性scFv，以找出對所述蛋白抗原具有較高結合親和力與專一性的可溶性scFv (步驟(c1))。
接著，在步驟(d)，將來自步驟(c)或步驟(c1)的分泌之可溶性scFv分別和一適體-藥物偶聯物相接觸。
根據本揭示內容的多種實施方式，所述適體-藥物偶聯物包含一藥物與一適體。所述適體包含一AL蛋白模組，其包含位於N端的蛋白A片段以及位於C端的蛋白L片段。如此一來，適體-藥物偶聯物的適體就能夠和分泌之可溶性scFv非共價接合，因而形成複數個scFv-適體-藥物複合物。
在部分視需要而採用的實施方式中，AL蛋白模組更包含第一多肽連接物，用以連接蛋白A片段與蛋白L片段。同樣在視需要而採用的實施方式中，適體-藥物偶聯物更包含第二多肽連接物，以將藥物連接至適體C端。
在部分視需要而採用的實施方式中，適體包含兩個AL蛋白模組，且更包含第三多肽連接物，用以連接上述兩個AL蛋白模組。
當可理解，上述適體經過特別涉及而能運用於此處提出的高通量篩選方法，且因此，所述適體亦為本發明尋求保護的標的之一。
在一些視需要而採用的實施方式中，藥物包含小分子細胞毒性藥物或免疫毒素(譬如，外毒素)。舉例來說，外毒素可以是假單胞菌屬外毒素(PE) A或由其所衍生。於一些實施方式中，免疫毒素包含截短的PE A次單元毒素。在進一步的實施方式中，所述藥物更包含內質網(ER)駐留部分，其位於PE A次單元毒素的C端。舉例來說，ER駐留部分包含KDEL序列。
當在步驟(d)中形成scFv-適體-藥物複合物之後，進行本方法的步驟(e)，於此步驟中分別在帶有步驟(d)中形成之該些scFv-適體-藥物複合物存在的條件下，培養複數個呈現所述蛋白抗原的細胞。用以培養能夠呈現各種蛋白抗原之細胞的流程為所屬技術領域中具有通常知識者所熟知。
之後，在步驟(f)，決定該些scFv-適體-藥物複合物在步驟(e)所用之呈現蛋白抗原之該些細胞內個別的效果。
在部分視需要而採用的實施方式中，可藉由測量步驟(e)中培養的呈現蛋白抗原之該些細胞的個別細胞存活率，來進行步驟(f)；然而，本揭示內容不限於此。反之，可依藥物欲於細胞上所實現的療效，來進行一或多種適當的定量或定性分析。
接著，在步驟(g)，基於步驟(f)所決定的結果來挑選用於免疫偶聯物的功能性片段。具體來說，從相較於其他scFv-適體-藥物複合物能夠展現較優功效的scFv-適體-藥物複合物中，挑選出一或多個scFv-適體-藥物複合物，並選擇其所含的scFv作為免疫偶聯物的功能性片段。
此處提出的高通量篩選方法的優點至少在於其免去了耗時、費力的免疫偶聯物次選殖與純化步驟。正因如此，能夠大幅改善開發並最佳化標的抗體之通量。
在利用此處提出的高通量篩選方法鑑別出適當標的模組之後，就可以製備包含所述標的模組的免疫偶聯物。能夠將藥物(包括外毒素)和功能性片段偶聯的方法為本領域所熟知。當可理解，這些免疫偶聯物以及包含所述免疫偶聯物的藥學組合物同樣也屬於本揭示內容請求保護的範圍。
根據某些實施方式，所述的免疫偶聯物包含一藥物以及一標的模組，其中標的模組包含根據本揭示內容任一態樣/實施方式所選出的功能性片段。
根據本揭示內容的多種實施方式，所述藥學組合物包含有效量的免疫偶聯物以及對所述免疫偶聯物藥學上可接受的賦型劑。
在另一態樣中，本揭示內容是關於用以治療有需要個體的方法，所述方法包含以下步驟：對該個體投予根據本揭示內容任一態樣/實施方式所述的免疫偶聯物或藥學組合物。
下文提出多個實驗例來說明本發明的某些態樣，以利本發明所屬技術領域中具有通常知識者實作本發明，且不應將這些實驗例視為對本發明範圍的限制。據信習知技藝者在閱讀了此處提出的說明後，可在不需過度解讀的情形下，完整利用並實踐本發明。此處所引用的所有公開文獻，其全文皆視為本說明書的一部分。
材料及方法
細胞株
N87、SKBR3、BT474與MCF7細胞取美國菌種保藏中心(American Type Culture Collection，ATCC)。將N87胃癌細胞(HER2-陽性)培養於RPMI 1640培養基(Invitrogen, Carlsbad, CA)中，培養基添加10%胎牛血清(FBS)，培養環境為37°C、含5%二氧化碳的潮濕大氣。經修飾的小分子細胞毒性藥物--順丁烯二醯亞胺基己醯基-纈胺酸-瓜胺酸-一甲基澳瑞他汀E (Maleimidocaproyl-valine- citrulline-monomethyl auristatin E，mc-vc-MMAE)是購自MedChem Express (Princeton, NJ, USA)。SKBR3細胞培養於RPMI 1640 (Gibco)，添加10%胎牛血清(Gibco)及盤尼西林/鏈黴素(100X；Gibco)。BT474細胞培養於Hybri-Care培養基(ATCC)，添加10%胎牛血清及盤尼西林/鏈黴素/麩醯胺酸(100X；Gibco)。MCF7細胞培養於RPMI 1640，添加10%胎牛血清及盤尼西林/鏈黴素(100X)。懸浮HEK293 Freestyle (293-F，購自Life Technologies, USA)細胞培養於不含血清的Freestyle 293表現培養基(Gibco)中，培養於含8%二氧化碳的培育箱(Thermo Scientific)中，在37°C下以110 rpm的轉速震盪。
AL1-PE38KDEL 與 AL2-PE38KDEL 的基因建構及蛋白表現 / 純化
基於已公開的蛋白A/L片段(PDB code:4HKZ)與截短形式的綠膿桿菌外毒素A (PDB code:1IKQ)來合成AL1-PE38KDEL基因(序列編號：99)及AL2-PE38KDEL基因(序列編號：100)。
於建構質體時，將所合成的基因次轉殖到pET32a表現載體(Novagen)中，透過SfiI及NotI限制酶切位而得到在AL1-/AL2-PE38KDEL的N端帶有硫氧化還原蛋白(thioredoxin)的融合蛋白，而所得到的融合蛋白在硫氧化還原蛋白的C端帶有6XHis標記，且在6XHis標記和AL1-/AL2-PE38KDEL之間有TEV蛋白酶切位。
欲進行蛋白表現時，將所建構的質體轉型至大腸桿菌Rosetta-gami B (DE3)品系(Novagen)中，並將所選的菌落培養於2X YT培養基(胰化蛋白16 g/L、酵母抽出物10 g/L、氯化鈉5 g/L)中，培養基添加胺苄青黴素(200 µg/L)、四環黴素(12.5 µg/L)、氯黴素(37.5 µg/L)及康黴素(15 µg/L)，於37°C下培養直到細胞濃度到達OD600 為1.0為至，之後再於16°C下培養1小時。利用最終濃度為0.5 mM IPTG在16°C下培養一夜，以誘導外源蛋白合成。之後進行離心，並將收集到的細胞重新懸浮於緩衝液A (500 mM氯化鈉及5 mM咪唑於20 mM Tris-HCl中，pH 8.0)中，再以Microfluidizer (Microfluidics, MA)裂解細胞，之後再以40,000 × g離心1小時。將上清液載入經緩衝液A平衡之HisTrap HP管柱(GE Healthcare)。在以30 mL緩衝液A沖洗後，以線性梯度0–50%的緩衝液B (除了使用1000 mM咪唑之外都和緩衝液A相同)洗提管柱。收集利用0.14-0.24 M咪唑洗提之AL1-/AL2-PE38KDEL餾份，並以His-標記之TEV蛋白酶(A280 比50∶1)在30°C下處理3小時，接著再相對於緩衝液A進行透析。經剪切的硫氧化還原蛋白與TEV蛋白酶都帶有6XHis標記，可利用HisTrap HP管柱將之移除。收集流出的AL1-PE38KDEL蛋白(序列編號：101)及AL2-PE38KDEL蛋白(序列編號：102)，並利用Superdex200分子篩管柱(GE Healthcare)於SEC緩衝液(150 mM氯化鈉，溶於50 mM Tris-HCl中，pH 8.0)中進一步純化之。接著利用SDS-PAGE來分析所分離的蛋白，以確認其純度與分子量。
AL2-RFP/GFP 的基因建構及蛋白表現 / 純化
根據標準的大腸桿菌表現及純化方法來進行AL2-RFP的表現與純化。簡言之，將N端帶有6XHis標記的AL2-GGGSG-RFP/GFP (紅色螢光蛋白(序列編號：103)或綠色螢光蛋白(序列編號：104))之編碼區域進行適合大腸桿菌表現之密碼子最佳化，並將其選殖至pET-32b表現載體中。大腸桿菌BL21 (DE3)品系(Merck)的培養物以pET32b表現載體中的AL2-RFP/GFP構築體轉型後，培養於2X YT培養基(胰化蛋白16 g/L、酵母抽出物10 g/L、氯化鈉5 g/L)中，培養基添加胺苄青黴素(150 µg/L)，培養溫度37˚C直到OD600 達1.0~2.0為止，之後於16˚C下繼續培養1小時再加入最終濃度為0.5 mM IPTG。於表現一夜(至少16小時)後，將細胞離心並將沈澱物重新懸浮於溶解緩衝液(Tris-HCl 20 mM、氯化鈉150 mM、咪唑10 mM，pH 8.0)中，之後利用Microfluidizer (Microfluidics, MA)裂解懸浮的細胞。利用Ni2+ -IMAC管柱(GE Healthcare Life Sciences)並使用梯度為10 mM至500 mM的咪唑(溶於TS溶液(Tris-HCl 20 mM、氯化鈉150 mM，pH 8.0)中)，以純化重組AL2-RFP/GFP融合蛋白。利用Superdex200分子篩管柱(GE Healthcare Life Sciences)在PBS緩衝液中進一步純化AL2-RFP/GFP融合蛋白(序列編號：105、序列編號：106)，並儲存於-80˚C。利用SDS-PAGE與考瑪斯藍染色來分析純化的蛋白。
scFv 表現 / 純化
製備分泌之scFv的方法如下。以含有scFv基因且可收穫噬菌體的噬質體pCANTAB5E感染大腸桿菌ER2738品系，時間30分鐘。在2X YT(添加100 µg/mL胺苄青黴素)擴增被感染的ER2738，處理時間5-6小時，之後加入10 mM IPTG。在培養一夜之後，利用0.22 µm濾網來過濾含有分泌之可溶性scFv的培養上清液。
於製備帶有E標記之純化曲妥珠單抗scFv時，合成衍生自曲妥珠單抗Fab (PDB code:4HKZ)的基因序列，並以連接多肽(G)4 S(G)4 S(G)4 S (序列編號：91)連接兩個可片區域，且在C端引入E標記寡肽(GAPVPYPDPLEPRAA；序列編號：92)，以完成建構。之後將所得到的基因透過SfiI及NotI限制酶切位選殖到pET32a表現載體(Novagen)中。此一scFv蛋白表現及純化的詳細流程如Yu, C.M.等人在“Rationalization and design of the complementarity determining region sequences in an antibody-antigen recognition interface” (PLoS One 7, e33340 (2012))一文中所述。
培養基中 scFv 的濃度決定
藉由比較分泌之scFv與已知濃度之純化標準scFv兩者的ELISA信號，來決定分泌之scFv在培養基中的濃度。簡言之，將蛋白L抗原(每孔0.2 µg)於PBS緩衝液(pH7.4)中塗覆於NUNC 96孔Maxisorb免疫分析盤上，於4°C下反應一夜，再以5%脫脂牛奶(於PBST [0.05% (v/v) Tween 20]中)封阻1小時。利用培養ER2738之2X YT將分泌之scFv與標準scFv連續稀釋。在封阻之後，將50 µL的稀釋樣本連同50 µL的5%脫脂牛奶(於PBST [0.05% (v/v) Tween 20]中)加入每一孔中，並在溫和搖晃下培育1小時。以300 µL的PBST將分析盤沖洗6次，且之後加入100 µL的1:20000倍稀釋之辣根過氧化酶/抗E標記IgG抗體偶聯物(ICL；於帶有5%牛奶的PBST中)並培育1小時。以PBST緩衝液將分析盤沖洗六次，再以PBS沖洗兩次，以3,3’,5,5’-四甲基聯苯胺過氧化酶基質(3,3’,5,5’- tetramethyl-benzidine peroxidase substrate，購自Kirkegaard & Perry Laboratories)顯色3分鐘，之後以1.0 M的HCl終止反應，再利用分光光度儀於450 nm下進行讀取。根據由已知scFv濃度所得之標準曲線來決定分泌之scFv的濃度。
scFv 對 AL1-PE38KDEL 與 AL2-PE38KDEL 的結合動力學之表面電漿共振測量
利用BIAcore T200 (GE Healthcare)儀器來決定scFv和AL1-/AL2-PE38KDEL之間的結合親和力及動力學參數。利用胺基偶聯套組將純化的AL1-/AL2-PE38KDEL蛋白(於10 mM醋酸緩衝液(pH 4.5)中)固定於CM5感應晶片上，使其反應單位(response unit，RU)達到250-300。在HBS-EP+電泳緩衝液(10 mM HEPES pH 7.4、150 mM氯化鈉、3 mM EDTA、0.05% v/v介面活性劑P20；GE Healthcare)中，以每分鐘30 μL的流率來測定AL1-/AL2-PE38KDEL和scFv間之互動的結合(kon )與解離(koff )常數。於注入新的scFv之前，以10 mM甘胺酸(pH 2.0)使感應晶片表面還原，且將所得到的信號減去由並未塗覆配體之參考通道所得到的信號。利用Biacore評估軟體(GE Healthcare)對結合模型進行1:1的全域擬合，以決定結合動力。實驗重複三次，並以平均值與標準偏差來表示結果。
AL1-PE38KDEL 與 AL2-PE38KDEL 之 ELISA 分析的 EC50 測量值
進行ELISA分析，以決定可溶性scFv與AL1-/AL2-PE38KDEL結合的EC50 。簡言之，將HER2/ECD抗原(每孔0.3 µg)於PBS緩衝液(pH7.4)中塗覆於NUNC 96孔Maxisorb免疫分析盤上，於4°C下反應一夜，再以5%脫脂牛奶(於PBST [0.05% (v/v) Tween 20]中)封阻1小時。同一時間，將經過濾之上清液中的分泌之scFv與AL1-/AL2-PE38KDEL混合(scFv:AL片段之莫耳比1:1)。於室溫下培育1小時後，以2YT培養基將scFv或scFv-AL1-/AL2-PE38KDEL混合物進行2倍連續稀釋以製得11種濃度。將50 µL的稀釋scFv樣本和50 µL的封阻緩衝液混合，之後加入已封阻的微定量分析盤，並於溫和搖晃下再培養1小時。以300 µL的PBST將分析盤沖洗6次，再加入100 µL之以PBST (含5%牛奶)的1:4000稀釋之辣根過氧化酶/抗E標記抗體偶聯物(Abcam)。以PBST緩衝液沖洗分析盤六次，再以PBS沖洗兩次，3,3’,5,5’-四甲基聯苯胺過氧化酶基質顯色2分鐘，之後以1.0 M的HCl終止反應，再利用分光光度儀於450 nm下進行讀取。根據Stewart and Watson方法來計EC50 (ng/mL)。
AL1- 及 AL2- 藥物偶聯物的製備
為了將順丁烯二醯亞胺藥物負載偶聯至AL1或AL2適體蛋白，在AL適體蛋白的C端加入半光胺酸殘基(標示為AL1C或AL2C)。在基因合成之後，利用pET26表現載體建構AL1C或AL2C，並進一步利用HisTrap HP與Superdex G200管柱進行表現與純化。於偶聯AL1C-026時，將2.84 mL的20 mM TCEP (110.4 nmole)加入0.5 mg (31.6 nmole)的AL1C蛋白(於PBS緩衝液中)，以還原AL1C蛋白與AL1C-β-巰基乙醇(AL1C-beta-mercaptoethanol)複合物之分子間雙硫鍵，之後在20°C下培育2小時。進行還原反應之後，將8.52 μL的10 mM順丁烯二醯亞胺-vc-MMAE (於DMSO中)加入含有AL1C蛋白溶液的TCEP中並混合均勻，再於20°C下培育16小時。進一步利用3 kDa Centricon (Millipore Corp. Billerica, MA, USA)來濃縮蛋白溶液，再使用Superdex75 10/300 GL管柱(GE Healthcare)進行AL1C-vc-MMAE偶聯物的膠體過濾純化。利用BCA kit (Thermo SCIENTIFIC)來決定AL1C-vc-MMAE偶聯物的濃度。進行PAGE分析並決定EC50 ，以確認AL1C-vc-MMAE偶聯物的完整性。於合成AL1C-021時，將AL1C蛋白和Osu-一甲基澳瑞他汀F (MMAF)透過離胺酸偶聯。於製備AL2C-002與AL2C-004偶聯物時，分別將AL2C蛋白和順丁烯二醯亞胺-vc-MMAE或順丁烯二醯亞胺-MMAF偶聯。
抗體 - 藥物偶聯物之製備
根據上文所述的方法並簡單修改，以製備抗體-vcMMAE偶聯物。簡言之，加入2 mM的EDTA以及加入TCEP使其含量為相對於抗體過量3倍，使樣本抗體(2 mg/mL)在20°C下還原2小時。加入5.25當量的順丁烯二醯亞胺-vc-MMAE (使用於DMSO中的10-Mm儲備液)以偶聯還原之抗體。使偶聯反應在20°C下進行1小時，之後加入N-乙醯基半光胺酸使反應淬滅。5 mL的ZebaTM Spin Desalting管柱(7K MWCO，購自Thermo SCIENTIFIC)將淬滅後的偶聯混合物去鹽兩次，之後進行離心超過濾(Amicon Ultra 10K device；Millipore Corp. Billerica, MA, USA)使其濃縮。在利用BCA protein assay kit (Thermo SCIENTIFIC)定量其濃度之後，進行最終偶聯物的EC50 結合分析、SDS-PAGE，並利用疏水性互動色層分析(hydrophobic interaction chromatography，簡稱HIC；使用butyl-NPR管柱，購自Tosoh Bioscience, Tokyo, Japan)計算藥物-抗體比(drug-antibody-ratio，DAR)，並儲存於4°C。
細胞毒性分析
於決定免疫毒素的細胞毒性效果時，將細胞以每孔104 個細胞的濃度接種96孔盤上。將IgGs或scFv以scFv:AL莫耳比1:1的比例在室溫下預先培育1小時，以便形成非共價鍵結的免疫毒素。利用培養ER2738之2X YT將scFv-AL1-PE38KDEL/AL2-PE38KDEL混合物連續稀釋，之後將其加入不含血清的細胞培養物中。利用培養ER2738之2X YT加上AL1-PE38KDEL或AL2-PE38KDEL作為負對照組。在37°C下培養4小時之後，將抗體毒素置換為新鮮的含血清正常培養基。於37°C下培養4天後，利用WST-1 (Roche)來測量OD450 ，以定量存活之細胞數目。細胞存活率百分比的計算方式如下： 細胞存活率% = OD450nm (經抗體處理細胞)/OD450nm (負對照組細胞) × 100%。
對於AL1-PE38KDEL、AL1C-Osu-MMAF (AL1C-021)與AL1C-vc-MMAE (AL1C-026)，將細胞以每孔1×104 個細胞的量接種培養盤上，並讓細胞貼附。16-18小時之後，移除培養基，並置換為不含血清之培養基，其中含有不同濃度之AL1C偶聯物及H32、曲妥珠單抗(Herceptin)、GH2-20、GH2-41、GH2-75及GH2-61抗HER2 IgG。AL1C偶聯物與IgG的莫耳比為2:1。於37 °C下培育4-16小時之後，將抗體毒素置換為新鮮的含血清正常培養基。於37°C下培養4天後，利用WST-1 (Roche)依製造商的指示來處理細胞。利用Victor (Perkin Elmer)分析每一孔的OD450 ，並基於以培養基處理的細胞進行標準化，以分析相對細胞存活率。對於AL2C偶聯物，AL2C與IgG的莫耳比為1:1。其他步驟與上文參照AL1C偶聯物所述相同。對於IgG-vc-MMAE偶聯物，不同數量的將偶聯物直接加入培養基中培養72小時。
利用流式細胞儀測量細胞表面上 scFv 連同 AL2-RFP 之平均螢光強度測量值
進行FACS分析，以決定scFv-HER2-ECD對表現HER2之細胞的結合能力。利用胰蛋白處理使細胞懸浮，並通過40-微米的孔洞。將約2×105 個細胞和100μL的scFv在4°C下培育30分鐘，之後以0.5% FBS 1X PBS (沖洗緩衝液)沖洗一次，再和1.2 μg AL2-RFP (於50Μl的沖洗緩衝液中)於4°C下混合20分鐘，且之後以沖洗緩衝液沖洗兩次。在離心與重新懸浮之後，利用FACS (BD FACS CantoⅡ)分析細胞的RFP信號。
IgG 內化的共焦顯微分析
將生長於玻璃蓋玻片上的SKBR-3細胞和1 μg/mL的曲妥珠單抗、M32或H32 IgG於37°C下培育1小時。在沖洗之後，利用3.7%甲醛固定細胞並以0.1% Triton-X 100使細胞透化(permeabilized)。以兔抗Rab5、Rab7或Rab9抗體(1:200稀釋，於PBS/0.1% Triton-100/3% BSA中)在室溫下處理經固定的細胞1小時，且之後將其和Cy3-偶聯抗兔二級抗體(1:200稀釋，於PBS/0.1% Triton-100/3% BSA中)在室溫下處理1小時。利用Cy2-偶聯抗人二級抗體確認曲妥珠單抗及H32 IgG的存在，並以Cy2-偶聯抗鼠抗體確認M32的存在。於蓋玻片上以Gel Mount液體裝載培養基(Sigma, St.Louis, MO, USA)做封片。利用配有63倍物鏡(1.4油鏡)的Zeiss LSM 510 META共焦顯微鏡取得影像。
利用西方墨點偵測 IgG 誘導之囊泡蛋白表現
將SKBR-3細胞和1μg/mL曲妥珠單抗、M32或H32 IgG在37°C下處理1小時，之後將細胞刮下溶解於緩衝液(1% NP-40、50 mM Tris pH 7.4、150 mM氯化鈉、2 mM氯化鎂、1 mM EGTA及蛋白酶與磷酸酶抑制劑)中。利用SDS–PAGE來分離溶解物中的細胞蛋白，且之後將其轉移到PVDF膜上進行西方墨點分析，先利用抗Rab7與Rab9抗體以及抗GAPDH抗體處理後，再以適當的HRP-偶聯二級抗體浸泡呈色。利用強化的化學冷光系統使默漬顯影。
利用生物素反應分析抗體的內化
在4°C下以EZ-link Sulfo-NHS-SS-Biotin (Pierce,IL)的PBS溶液處理SKBR-3細胞30分鐘，以使其細胞膜生物素化標定。以冷PBS沖洗經標記的細胞兩次，之後和10 μg/mL的曲妥珠單抗或H32 IgG在37°C下培育以使其內化，1小時之後，利用還原緩衝液(100 mM2-巰基乙醇磺酸鈉、50 mM Tris、pH 8.6、100 mM氯化鈉)在4°C將留存在細胞表面的生物素基團移除。之後以冷PBS沖洗細胞三次，並刮下細胞於溶解緩衝液中。以PBS將細胞溶解物稀釋為10 μg/mL全蛋白，並利用ELISA分析盤上(Nunc immobilizer；Nunc, Roskilde, Denmark)的鏈霉卵白素由細胞溶解物中捕捉HER-2受體與內化抗體之生物素化的膜複合物，處理條件為室溫下1小時。之後以PBS沖洗分析盤三次，再以辣根過氧化酶(horseradish peroxidase，HRP)-偶聯抗人二級抗體培育1小時，並以PBS沖洗三次；之後和OPD呈色受質(Sigma, St. Louis, MO)一起培育，以使及HRP信號顯色。利用ELISA讀取儀讀取492 nm下的顏色信號。
負染色 EM 及影像處理
將約8 μg/Ml的抗體-抗原複合物塗覆於輝光放電之(glow-discharged)覆碳銅網上。以0.75% (重量/體積)的二甲酸鈾醯(uranyl formate)清洗並染色銅網30秒。在120 Kv下操作FEI Tecnai F20電子顯微鏡以顯影樣本。利用Gatan UltraScan 1000 CCD紀錄顯微照片(1.21 Å/像素，平均散焦1.3 μm)。對影像進行對比值傳遞函數(contrast transfer function)進行校正，並利用CTFfind3決定散焦值。在Appion 中以DoG選取器(picker)框選突起與粒子，並利用ISAC與Relion套裝軟體以每像素3.63 Å將其分類為2D類別。利用EMAN2自始公用線方法產生3D初始模型。以約60,000顆粒子利用Relion將初始模型重新定義為23 Å解析度並使用金標準FSC=0.5截止值。以UCSF Chimera軟體將Fab及HER2 ECD (PDB 3wsq)結晶結構擬合至3D模型，以對映結合區域。
IgG 表現與純化
進行IgG表現時，利用PCR從結合噬菌體的scFv質體擴增輕鏈變異結構域(VL)與重鏈(VH) cDNAs，之後將其選殖到哺乳動物表現載體pIgG (中央研究院基因體研究中心張子文博士的餽贈)。利用校對DNA聚合酶(KOD Hot Start DNA polymerase, Novagen)對VL結構域cDNA進行PCR擴增，所用的引子組為GH2-VL-F-KpnI (序列編號：93；caggtgcacgatgtgatggtacc gatattcaaatgacccagagcccgagcagcctgagc)以及GH2-VL-R (序列編號：94；TGCAGCCACCGTACGTTTGATTTCCACCTTG GTGCC)；而VH結構域所用的引子組為GH2-VH-F (序列編號：95；Cgtgtcgcatctgaagtgcagctggtggaatcgg ga)以及GH2-VH-R-NheI (序列編號：96；(GACCGATGGG CCCTTGGTGCTAGC CGAGCTCACGGTAACAAGGGTGCC)。引子中斜體標示部分表示限制酶切位。PCR反應之體積為50 µL，使用100 ng的DNA模版以及1 μL的10 μM引子，進行30個循環(95°C下30秒，56°C下30秒，72°C下30秒)之後在72°C下進行10分鐘的最後合成步驟。利用1.0%洋菜凝膠電泳取出PCR產物。利用上文所述的PCR方法，從pIgG載體得到VL 與VH 結構域之間的連接物DNA片段，所用的引子組為GH2-IgG-linker-F (序列編號：97；AAGGTGGAAATCAAAC GTACGGTGGCTGCACCATCTGTC)及GH2-IgG-linker-R (序列編號：98；CTGCACTTCAGATGCGACACGCGTAGCA ACAGC)。連接物片段包括輕鏈固定結構域、牛生長荷爾蒙(bovine growth hormone；BGH)聚A信號、及人細胞巨大病毒(cytomegalovirus；CMV)促進子，之後接著IgG重鏈的信號肽。使用GH2-VL-F-KpnI與GH2-VH-R-NheI引子組進行PCR，以將上述三種DNA片段(VL結構域、連接物及VH結構域)組合到一起，進行30個循環(95°C下30秒，58°C下30秒，72°C下90秒)。利用1.0%洋菜凝膠電泳取出PCR產物，並利用Gibson組裝法將其選殖到pIgG載體中。簡言之，將2 μL (20 ng)的線性化pIgG載體(先前以KpnI與NheI剪切)與2 μL (20 ng)的插入DNA和4 Μl的Gibson Assembly Master Mix (New England BioLabs Inc. Ipswich, MA, USA)混合，並於50°C下培育1小時。其後，以大腸桿菌JM109勝任細胞加入一半體積的接合混合物進行轉型。利用限制酶剪切與核苷酸定序，來確認定序質體的DNA插入。所建構的載體含有IgG的輕鏈與重鏈，兩者獨立地受到CMV促進子的調控。
利用以下方法進行HEK293 F細胞轉染與IgG表現。在進行500mL的培養液轉染時，將2-L艾氏燒瓶中的293-F細胞懸浮液的濃度調整為1.0 x 106 細胞/mL。以25 mL的無血清培養基稀釋質體DNA (500 μg)，並以0.2 μm注射器濾網滅菌，之後和含有1 mg陽離子聚合物聚乙烯亞胺(polyethylenimine，PEI；購自Polysciences)的25 mL培養基劇烈混合。於室溫下培育20分鐘之後，將混合物一滴一滴加入細胞並輕微搖晃，其後將細胞培養於37°C的中大型培育箱中。在轉染後第24小時加入胰化蛋白N1 (ST Bio, Inc., Taipei, Taiwan)，最終濃度為0.5%。培養5天後，以8,000 x g離心30分鐘之後以0.8-μm的濾膜(Pall Corporation, Michigan)過濾，接著收集上清液。將上清液載入HiTrap蛋白A親和管柱(GE Healthcare, Uppsala, Sweden)中，並以0.2 N甘胺酸-HCl (pH 2.50)洗提至1/10體積的1 M Tris-HCl緩衝液(pH 9.1)中。利用Superdex 200膠體過濾管柱(10/300 GL, GE Healthcare, Uppsala, Sweden)進一步純化IgG蛋白，以移除高分子量團聚物(aggregates)。
腫瘤異種移植研究
依據中央研究院機構動物照護與使用委員會的大綱進行小鼠實驗。
利用以下方法建立帶有N87腫瘤的小鼠模型。在6週大雄性NOD.CB17-Prkdcscid/NcrCrlBltw NOD/SCID小鼠(BioLASCO Taiwan Co. Ltd.)的右腹脥接種1×106 個細胞。當腫瘤體積達到約100–200 mm3 時，將小鼠指派為以下治療組別(n=6)，並以GH2-61 IgG-AL1-PE38KDEL免疫毒素處理各組小鼠，給藥劑量分別為0.375、0.75、1.5與3 mg/kg。為了監控腫瘤生長，每週兩次以數位量尺測量腫瘤最大與最小的直徑。根據下列公式計算腫瘤體積(mm3 )：腫瘤體積= (長度×寬度2 ) × 0.523。在每次治療處理前，測量小鼠體重以監控治療的全身性毒性。每週兩次對小鼠腹膜內注射PBS或GH2-61 IgG-AL1-PE38KDEL免疫毒素，每次注射總體積為100 μL。為了評估GH2-61 IgG-AL1C-026複合物與GH2-61 IgG-vcMMAE偶聯物在N87異種移植腫瘤模型中的抗腫瘤功效，每週一次給予GH2-61 IgG-AL1C-026複合物(2.5或5 mg/kg)或GH2-61 IgG-vcMMAE偶聯物(2.5或5 mg/kg)，共計三週。
實驗例 1
用於非共價結合之免疫毒素與噬菌體表現的合成抗體庫的高通量篩選平台
高通量篩選可同時測試大量單株抗體的生物活性功能。傳統免疫毒素的功能測試需要進行耗時費力的免疫毒素次選殖與純化，限制了開發與最佳化標的用抗體的產出效率。在96孔深孔盤中，以1 ml的培養基來培養大腸桿菌，其所分泌的可溶性scFv中，可以穩定地表現出濃度約為10 nM至100 nM的單株GH2合成抗體。為了要利用培養基中的可溶性scFv在不需純化scFv的情況下實現高通量的篩選平台，本發明建構了一種適體-毒素融合蛋白：AL1-PE38KDEL與AL2-PE38KDEL，此種融合蛋白採用和具有IGKV1-NL1*01/IGHV3-23*04架構(PDB編碼：4HKZ)的scFv複合之蛋白A與蛋白L。
具體來說，AL1-PE38KDEL適體-毒素融合蛋白是包含AL1-PE38KDEL免疫偶聯物與曲妥珠單抗scFv的複合結構。將曲妥珠單抗的scFv變異結構域和蛋白A與蛋白L所形成的複合結構衍生自PDB編碼：4HKZ。VL結構域的C端殘基與VH結構域的N端殘基之間的距離約為37.3 Å；而蛋白AC端殘基與蛋白LN端殘基之間的距離約為39.8 Å。PE38KDEL是一種截短的假單胞菌屬外毒素(PDB編碼：1IKQ)，其包含外毒素的結構域II與結構域III。
關於AL2-PE38KDEL適體-毒素融合蛋白，其帶有一組額外的AL片段，此一AL片段透過長度為5個殘基的連接物(連接物2)而連接至上述AL1-PE38KDEL的N端。
這些適體-毒素融合蛋白和帶有IGKV1-NL1*01/IGHV3-23*04架構的重組scFv或IgG結合，可在培養基之粗混和物中自我組裝成為非共價連接的免疫毒素複合物，而使得此種自我組裝的免疫毒素複合物可以針對培養在微定量測試盤中的細胞，進行高通量的細胞毒性分析，而不需進行會影響實驗進度的次選殖與純化步驟。
實驗例 2
與一價標的模組非共價結合之免疫毒素
AL1-PE38KDEL會和IGKV1-NL1*01/ IGHV3-23*04架構的scFv以高親和力結合。適體-毒素融合蛋白AL1-PE38KDEL的AL片段中，蛋白A及蛋白L依序由N端至C端排列對於避免連接物3和VL-VH連接物之間的衝突扮演關鍵角色，所述的VL-VH連接物長度為15個殘基，用以連接GH2抗體庫之scFv構建體中依序由N端至C端排列的VL及VH結構域。連接物3是15個殘基的多肽連接物，此一長度足以讓一個scFv分子同時結合到AL片段中的蛋白A與蛋白L上。連接物4的長度為5個殘基，用以將AL片段連接至毒素PE38KDEL。適體-毒素融合蛋白AL1-PE38KDEL具有一個高親和力的scFv結合部位，使得蛋白A能夠結合至VH結構域且蛋白L可以同時結合到VL結構域。在另一種替代性的結合方式中，蛋白A/L中只有一個會和scFv分子互動，這些組態的親和力低，且解離Kd 介於10-6 ~10-7 M之間。因此，預期這些一價複合物組態不適用於AL1-PE38KDEL複合物中。利用表面電將共振測量scFv對AL1-PE38KDEL的結合親和力與及結合動力亦證實以上推論，此測量方法使scFv溶液流經固定的AL1-PE38KDEL，所得結果如下：Kd = 5.64 ± 0.03 × 10-9 M、kon = 4.54 ± 0.06 × 105 M-1 S-1 且koff =2.56 ± 0.04 × 10-3 S-1 (圖1)。
實驗例 3
與二價標的模組非共價結合之免疫毒素
預期適體-毒素融合蛋白AL2-PE38KDEL可利用其連續的兩個AL片段和兩個IGKV1-NL1*01/IGHV3-23*04架構之scFv結合。以連接物2來連結兩個AL片段，連接物2長度為5個殘基，用以防止形成由第一個AL片段的蛋白L和第二個AL片段的蛋白A而組成的scFv-結合部位。因此，預期AL2-PE38KDEL適體-毒素融合蛋白含有兩個獨立的scFv結合部位，且其親和力為奈米莫耳等級。雖然複合物也可能透過和第一AL片段的蛋白A以及第二AL片段的蛋白L而與一個scFv結合進而形成複合物，但可知此種結構對scFv的親和力遠低於上文所述的複合物結構。此種認知係基於和中間相隔了很長的間隔片段(分為第一AL片段的蛋白L以及第二AL片段的蛋白A)之N-與C-端形成複合物時，所需的熵遠高於形成上文所述之scFv-AL複合物所需的熵。透過表面電漿共振來測量scFv對AL2-PE38KDEL的結合親和力及結合動力(使scFv流經固定的AL2-PE38KDEL)也確認了此一認知，所測得的結果如下：Kd = 5.52 ± 0.03 × 10-9 M、kon = 3.60 ± 0.06 × 105 M-1 S-1 及koff =1.99 ± 0.04 × 10-3 S-1 (圖2)。這些測量值與上文所述之scFv與AL1-PE38KDEL的結合分析結果相近(圖1)，顯示AL2-PE38KDEL有兩個獨立的scFv結合部位，每一結合部位的結合熱動力學與動力學測量值都和AL1-PE38KDEL中的單個AL片段相似；可預期上文所述的複合物結構應為scFv-AL2-PE38KDEL複合物中的主要複合物結構。
實驗例 4
AL-scFv 複合物的形成不會干擾 scFv-HER2-ECD 互動
已知蛋白A及蛋白L和IGKV1-NL1*01/ IGHV3-23*04架構的scFv之結合對於所述scFv和其相應抗原的結合力影響不大。圖3A至3E顯示在AL1-PE38KDEL與AL2-PE38KDEL存在或不存在的情況下，scFv-抗原結合親和力的定量比較。利用對於HER2-ECD親和力各異的16種HER2-ECD-專一scFv來測量其在AL1-PE38KDEL存在(圖3A)或不存在(圖3B)以及在AL2-PE38KDEL存在(圖3C)的情況下，和scFv-HER2-ECD結合之EC50 (實驗數據如表1所示)。
將scFv-AL1-PE38KDEL複合物對HER2-ECD結合之EC50 相對於同一組單獨scFv的EC50 作圖，所得斜率為1.0且R2 =0.88 (皮爾森氏相關係數0.94) (圖3D)，意味著從定量分析的角度來看，這16種scFv中每一者和AL1-PE38KDEL結合後都不會影響其和scFv-HER2-ECD的互動。將scFv-AL2-PE38KDEL複合物對HER2-ECD結合之EC50 相對於同一組單獨scFv的EC50 作圖，所得斜率為2.9且R2 =0.74 (皮爾森氏相關係數0.86) (圖3E)，代表scFv-AL2-PE38KDEL系統中的有效scFv之濃度減少了3倍，這可能是因為scFv-AL2-PE38KDEL複合物在產生等價ELISA信號強度的能力低於相應的單獨scFv或scFv-AL1-PE38KDEL複合物。scFv-AL1-PE38KDEL (圖3D)或scFv-AL2-PE38KDEL (圖3E)複合物和單獨scFv兩者之親和力有較高線性相關時，代表所述複合物的形成不會影響scFv結合至抗原分子的相對親和力。 表1
實驗例 5
非共價結合之 scFv-AL1-PE38KDEL 與 scFv-AL2-PE38KDEL 是強效免疫毒素，其 IC50 為次奈米莫耳等級
scFv-AL2-PE38KDEL複合物與scFv-AL1- PE38KDEL複合物都是強效的免疫毒素。帶有IGKV1-NL1*01/IGHV3-23*04架構之VL及VH變異結構域的IgG和AL1-PE38KDEL複合之後，其作為免疫毒素的效力與scFv-AL2-PE38KDEL (圖4A至4C)相仿。scFv(GH2-42)-AL1-PE38KDEL、scFv(GH2-61)-AL1-PE38KDEL以及scFv(GH2-75)-AL1-PE38KDEL作為免疫毒素的效力相近，其IC50 約為0.1 nM，亦即scFv-AL1-PE38KDEL作為免疫毒素而毒殺50%受測細胞之濃度約為0.1 nM (圖4A至4C)。就所示的三種代表性抗體而言，IgG-AL1-PE38KDEL免疫毒素和相應的scFv-AL2-PE38KDEL複合物的效力相等(圖4A至4C)，極有可能是因為這兩類免疫毒素都具有二價抗體標的模組。帶有二價標的模組的免疫毒素相較於帶有一價標的模組之相應免疫毒素具有更強的細胞毒性(圖4A至4C)；二價效應的程度會隨著抗體-抗原互動的細節而異(圖4A至4C)。上述結果顯示雖然scFv-AL2-PE38KDEL複合物會使得ELISA分析中測得之有效scFv濃度降低(圖3E)，但使用細胞的細胞毒性分析卻測顯示具有二價抗原結合部位的免疫毒素有較佳的功效。ELISA測量值無法用以表示免疫毒素的功效；在篩選免疫毒素效力時，使用細胞直接測量細胞毒性較為關鍵。整體來說，此處提出的技術平台能夠用於高通量篩選作為標的模組之scFv/IgG，此種標的模組可用於帶有一價或二價標的模組之非共價結合免疫毒素。
實驗例 6
非共價結合之免疫毒素的高通量篩選
從GH2合成抗體庫中選出92種HER2-ECD-專一scFv，並使用本案提出的高通量篩選平台來測試其作為非共價結合之免疫毒素之標的模組的效果。本組scFv的CDR序列以及其細胞毒性如表2所示，而部分隨機選擇之scFv的產率、EC50 及Biacore分析結果如表3所示。
具體來說，抗原決定位群組1是利用92種GH2 scFv對6種老鼠IgGs (M32、M41、M61、M62、M63與M64)的競爭分析結果所做的分群，M32-M62抗原決定位位於HER2-ECD的結構域I上，M41-M61位於結構域IV上，且M63-M64位於結構域III上；而抗原決定位群組2是利用92種GH2 scFv對5種已知抗體-抗原複合物結構的競爭分析結果所做的分群，所述5種抗體-抗原複合物結構為：曲妥珠單抗(抗原決定位：HER2-ECD結構域IV)、帕妥珠單抗(抗原決定位：HER2-ECD結構域II)、A21 (抗原決定位：HER2-ECD結構域I)、Fab37 (抗原決定位：HER2-ECD結構域III)及M32 (抗原決定位：HER2-ECD結構域I)。利用Kabat編碼來表示相應CDR的序列範圍，並依相應scFv-AL1-PE38KDEL的細胞毒性來排序。具體來說，CDR-L1區域為L30至L32殘基、CDR-L2區域為L49至L53殘基、CDR-L3區域為L91至L96殘基、CDR-H1區域為H30至H33殘基、CDR-H2區域為H50至H58殘基，且CDR-H3區域為H96至H100B殘基。 表2 Table 3
將培養基中可溶性scFv的濃度調整至0.5 nM，且培養基中有相應的AL1-PE38KDEL或AL2-PE38KDEL，其濃度為scFv:AL片段的莫耳比為1:1。在微定量分析盤中加入免疫毒素溶液以測試細胞毒性；細胞存活率讀值顯示，對於兩種類型的免疫毒素，當scFv濃度為0.5 nM時，免疫毒素可產生大範圍的細胞毒性(圖5)。對於少數效力最強的免疫毒素，使用連續稀釋後的scFv-AL1-PE38KDEL免疫毒素(圖6A)及scFv-AL2-PE38KDEL免疫毒素(圖6B)來處理細胞，以測定其細胞毒性並計算IC50 。在本組92個scFv中，於兩種類型的免疫毒素結構下，最有效的標的模組是GH2-41與GH2-20，且其二價形式優於為一價形式，前者的細胞毒性約為後者的10倍，且前者的IC50 小於10皮莫耳。相較之下，以曲妥珠單抗scFv作為標的模組時，相較於所測試的所有scFv中最佳的scFv，曲妥珠單抗scFv的IC50 差了100倍(圖6A及6B)。
受測scFv的抗原決定位與結合親和力/動力學並非免疫毒素效力的決定性因素。scFv-AL1-PE38KDEL免疫毒素的細胞毒性與相應scFv-AL2-PE38KDEL免疫毒素的細胞毒性呈現高度線性相關，其斜率為0.89且R2 = 0.57 (皮爾森氏相關係數0.75) (圖5)。資料圖中的斜率小代表二價免疫毒素通常比一價免疫毒素強效；二價效應與scFv-抗原互動的細節相關(圖4A至4C)，這解釋了圖中資料點較為分散的原因。資料圖中中度的線性相關代表scFv的內部特徵可決定一價與二價免疫毒素的效力。然而，沒有明顯可用於決定免疫毒素效力的決定性因子：來自相同抗原決定位的scFv (圖5中以相同顏色的資料點表示，同時參見表2與表3)有較廣的細胞毒性，這意味著影響免疫毒素的細胞毒性的參數不會受到抗原表面上抗原決定位的位置所限制。將scFv的EC50 、Kd 、kon 與koff 相對於相應scFv-AL1-PE38KDEL免疫毒素的細胞毒性作圖(圖7A至7D)以及相對於相應scFv-AL2-PE38KDEL免疫毒素的細胞毒性作圖(圖8A至8D)，結果顯示，相關係數接近0，這代表scFv的上述結合親和力及結合動力參數不是影響免疫毒素效力的主要決定因素。
scFv之CDR區域上的胺基酸類型可能是影響免疫毒素細胞毒性的重要決定性因素。GH2合成scFv只有CDRH3區域帶有分散的可離子化組胺酸殘基(參見圖5中帶有黑色外框的資料點)，其可能影響免疫毒素的細胞毒性效力，因為當免疫毒素從pH 7的環境進行內化而暴露於較為酸性的的環境中（如，晚期胞內體的pH值接近5）時，組胺酸側鏈可能會改變電荷狀態(pKa = 6.5)。若CDRH3帶有His殘基，且這些His殘基涉及了抗體-抗原互動，這些His殘基可能會決定在酸性環境中，免疫毒素從標的受體解離的次細胞位置。若將免疫毒素轉移到細胞質中，可以決定此種解離位置。本實驗例將92種scFv依scFv-AL1-PE38KDEL細胞毒性(表2與表3)分成4等分，並發現在細胞毒性遞減的92種scFv-AL1-PE38KDEL免疫毒素中，各組內CDRH3帶有組胺酸的scFv比例分別是6/23、8/23、10/23與13/23 (圖5及表2與表3)。此種單一性的趨勢指出涉及抗體-抗原互動的組胺酸側鏈可作為毒素藥物負載遞送路徑的決定性因素，而上述路徑又決定了免疫毒素的細胞毒性。
為了測定CDR組胺酸側鏈用以決定毒素細胞毒性的能力，分析四小組scFv-AL1-PE38KDEL之免疫毒素-HER2結合親和力的pH依存性，這些scFv-AL1-PE38KDEL的細胞毒性差異甚大，並決定其CDR區域中的His殘基分布。將選自屬於M32-M62抗原決定位群組之免疫毒素的四小組scFv-AL1-PE38KDEL隨機分組；參見圖5 (帶有紅色外框的灰色菱形資料點表示CDR中有組胺酸，而沒有紅色外框的則表示CDR中沒有組胺酸)；第一小組scFv-AL1-PE38KDEL (GH2-42、72、62、31、101)的細胞毒性為前25%，且CDR區域中沒有His殘基(圖9A)；第二小組 (GH2-61、92、75、24、103)的細胞毒性同樣是前25%，但CDR區域中帶有His殘基(圖9B)；第三小組 (GH2-11、38、4、26、及H32)的細胞毒性為後25%，且CDR區域中沒有His殘基(圖9C)；第四小組(GH2-102、40、107、8、12、55)的細胞毒性同樣是後25%，但CDR區域中帶有His殘基(圖9D)。只有來自第四小組的成員其在pH5下對HER2-ECD的親和力相較於pH7下有明顯降低(圖9A至圖9D及下表4)。第一與第三小組的CDR區域中都沒有His殘基，因此可以預見其對於個別抗原之親和力不具pH依存性。第二小組之親和力不具pH依存性(圖9B)以及第四小組之親和力具備pH依存性(圖9D)顯示在胞內體的酸性環境下，因組胺酸側鏈離子化進而干擾抗體-抗原互動是造成免疫毒素細胞毒性低落的原因之一，此一結果與前一段的結論相符。scFv(曲妥珠單抗)-AL1-PE38KDEL對HER-ECD的親和力高，且不具pH依存性(圖9C)，故因而，此種免疫毒素的低細胞毒性極有可能是由在HER2-ECD結構域IV的曲妥珠單抗之抗原決定位所造成，其位置距離在HER2-ECD的結構域I上的M32-M62抗原決定位群組較遠。 表4
從表3中隨機選取部分scFv。表4上半部的scFv選自表3四小組中的第一小組，亦即表3中依相應scFv-AL1-PE38KDEL之細胞毒性排序前25%的scFv；表4下半部的scFv選自表3四小組中的第四小組，亦即表3中依相應scFv-AL1-PE38KDEL之細胞毒性排序後25%的scFv。比例是以EC50 (pH5)除以EC50 (pH7)所得。
實驗例 7
細胞表面標的受體表現量決定免疫毒素效力
標的受體HER2之分布密度(圖10B、10D、10F及10H)影響免疫毒素細胞毒性(圖10A、10C、10E及10G)。在圖10A-10H所測試的各種細胞株中，N87細胞株細胞表面的HER2受體密度最高(圖10B)，且scFv-AL2-PE38KDEL免疫毒素對其效果最強，其對於此一細胞株的IC50 最低(圖10A)。SKBR3及BT474細胞株的HER2受體密度相較於N87來得低(圖10D及10F)；相應免疫毒素對這些細胞株的細胞毒性亦較低(圖10C及10E)。在負對照組細胞株MCF7中，在細胞表面僅能觀察到極低密度的HER2 (圖10H)，細胞對免疫毒素不敏感(圖10G)。應注意到，scFv對細胞表面受體之親和力排序和免疫毒素細胞毒性排序並無相關性(圖10A至10H)。更有甚者，scFv 作為標的模組的效力排序亦與受測細胞株相關(圖10A、10C及10E)，代表細胞見的差異也可能會影響免疫毒素細胞毒性。
實驗例 8
抗體結合至細胞表面上的 HER2-ECD 導致抗體之胞吞遞送至次級胞內體與高基氏體
根據抗體(H32、M32及曲妥珠單抗)內化位置與Rab5、Rab7及Rab9 GTP酶的關係，進一步探究抗體-HER2複合物的胞吞機制(圖11)。抗體H32為抗體M32的人源化版本，抗體M32是對照鼠抗HER2抗體，其在HER2-ECD上的抗原決定位對大多數抗體具有代表性(圖5)；表2與表3所列的92種scFv中有超過四分之三屬於此一抗原決定位群組(標記為M32-M62抗原決定位群組)。生物素化分析(參見材料與方法)結果顯示，H32 IgG與HER2結合後的內化作用的效率是曲妥珠單抗的1.4倍，這部分解釋了H32作為免疫毒素之標的模組的效力比起曲妥珠單抗來得好的原因(圖5)。圖11顯示，從相對於Rab5、Rab7及Rab9的次細胞位置來看，這兩種HER2-結合IgG的遞送路徑相同。Rab5與早期胞吞路徑相關，而Rab7與Rab9則作用於晚期胞吞路徑。可在靠近細胞質膜之格形蛋白包覆之囊泡附近以及早期胞內體附近偵測到Rab5，而Rab7與Rab9則是位於晚期胞內體附近。抗體M32、H32與曲妥珠單抗會和Rab7共同內化(圖11，圖框A)以及會和Rab9共同內化(圖11，圖框B)，但不會和Rab5共同內化(圖11，圖框C)，這代表抗體在結合至細胞表面上的細胞表面之後，會隨著晚期胞吞路徑而內化。與抗體相關的受體細胞吞噬作用亦會強化Rab9的表現，特別是抗體H32 (圖11，圖框D)。已知Rab9可調控從晚期胞內體至高基氏體之甘露糖-6-磷酸受體的回收。由抗體H32結合至HER2所造成的Rab9表現強化可以解釋scFv(H32)-AL2-PE38KDEL的高細胞毒性，此一免疫毒素的效果遠高於scFv(曲妥珠單抗)-AL2-PE38KDEL (圖5)。Rab9表現的提升亦佐證可驅動與胞吞抗體相關之受體的遞送使其依循逆向傳輸路徑而遞送至高基氏體。
實驗例 9
Fab H32 結合至 HER2-ECD 之結構域 I
利用負染色EM及單粒子重構來分析Fab H32-HER2-ECD複合物(圖12A)。2D類別平均顯示樣本的均質性相對較高，且主要是1:1的抗體-抗原複合物(圖12B)。在某些2D類別平均中可利用其方位明顯判別Fab H32的結構域。在某些2D類別平均中亦可從側面觀察而辨別HER2-ECD的結構域，進而能夠標記出結構域I、II、III與較小的尾部結構域IV。在某些2D類別中，HER2-ECD的密度，特別是結構域IV的密度，沒有Fab H32的密度來得清楚，這代表HER2-ECD結構域相對位置間的結構彈性與變異。之後將所選2D類別的粒子用於3D重構，並重新定義為23 Å的解析度(金標準樣本FSC=0.5截止值) (圖12C)。利用HER2 ECD的模型Fab及結晶結構(PDB 3wsq)來擬合3D映射圖(資料未顯示)。實驗數據，Fab H32可透過結構域I中A21抗原決定位旁邊的區域和HER2-ECD互動(資料未顯示)，此一結果與2D投射影像的分析結果一致，HDX-MS與競爭資料(參見Chen, H.S. et al. Predominant structural configuration of natural antibody repertoires enables potent antibody responses against protein antigens.Sci Rep 5 , 12411 (2015)一文圖6)。H32的抗原決定位和HER2上任何已觀察到之抗原決定位不同(資料未顯示)。抗體H32與曲妥珠單抗間抗原決定位的差異說明了利用這兩種抗體製備之免疫毒素之效力的差異。
實驗例 10
IgG-AL1-PE38KDEL 免疫毒素、 IgG-AL1C-/AL2C- 藥物複合物及 IgG-vcMMAE 偶聯物之 細胞毒性測量值比較
表5摘要整理了6種抗HER2 IgG-vc-MMAE偶聯物。產率計算方式如下：產率 = (輸出抗體-藥物偶聯物 / 輸入單獨IgG) X 100%。利用丁基-NPR疏水性管柱決定每一偶聯物的藥物-抗體比(drug-to-antibody ratio；DAR)。EC50 比的計算方式如下：EC50 比 = EC50 (偶聯) / EC50 (未偶聯)。 表5
可利用適體-毒素融合蛋白AL1-PE38KDEL來實現免疫毒素的細胞毒性。免疫毒素建構速率受限，導致通量不高。與AL1-PE38KDEL結合的抗HER2 IgG或scFv能夠驗證先導抗體的細胞毒性，且因此能夠縮短藥物開發所需時間。在三種高度HER表現的細胞株中，GH2-75 IgG對胃癌N87細胞的細胞毒性最高，而H32對乳癌SKBR3細胞的細胞毒性最高；參見圖13A至13C)。因此，不同抗HER2 IgG在不同癌細胞中會展現不同程度的細胞毒性，而可用於特定癌症治療。
抗體-藥物偶聯物是由單株抗體、細胞毒性化學藥物及合成連接物所組成。連接物與細胞毒性藥物的選擇決定了細胞毒性效力。可藉由將連接物及藥物與AL1C/AL2C複合，且之後測試IgG-AL1C/AL2C-連接物-藥物複合物在標的癌細胞中的細胞毒性，以選擇適當的連接物與化學藥物。本實驗使用與抗HER2 IgG結合的AL1C-Osu-MMAF (AL1C-021)及AL1C-vc-MMAE (AL1C-026)處理N87細胞(圖14A及14B)與SKBR3細胞(圖14C及14D)，並分析其結果。結果顯示，GH2-75 IgG在各種IgG-AL1C-Osu-MMAF中的細胞毒性最強，但在SKBR3細胞中，以H32 IgG在各種IgG-AL1C-vc-MMAE複合物中的毒性最強(圖14C及14D)。因此，當以IgG-AL2C-vc-MMAE (AL2C-002)複合物(圖15A及15B)及IgG-AL2C-MMAF (AL2C-004)複合物處理細胞時(圖15C及15D)，上述IgG細胞毒性效力的排序在N87與BT-474細胞中不同。上述IgG細胞毒性的排序和當將抗HER2 IgG與vc-MMAE偶聯後，在胃癌N87細胞株以及乳癌BT-474細胞株(圖16A及16B)中的排序一致。
因此，AL1C/AL2C-藥物適體蛋白可作為一種高通量平台，以便在進行耗時費力的IgG-藥物偶聯之前，篩選出適用於不同適應症的連接物-化學藥物。
實驗例 11
兩類 GH2-61 IgG-AL1-PE38KDEL 免疫毒素和 GH2-61 IgG-vc-MMAE 偶聯物之活體內抗腫瘤活性 的比較
首先在NOD/SCID小鼠中使用N87腫瘤模型來測試GH2-61 IgG-AL1-PE38KDEL免疫毒素的抗腫瘤活性。實驗結果顯示，對荷瘤小鼠給予GH2-61 IgG-AL1-PE38KDEL免疫毒素治療達到了顯著的腫瘤縮小結果，且呈劑量相關性(圖17A)。於治療過程中，在治療組和PBS處理組間並未發現顯著的體重差異(圖17B)。接著在相同的N87活體內模型中評估兩種ADC系統的抗腫瘤活性。實驗結果顯示，與MMAE偶聯後，其活體內抗腫瘤能力高於GH2-61 IgG-AL1C-026複合物(圖18A)。每一治療組別中，皆未觀察到體重減輕，這代表著受試對象對所述療法的耐受性良好(圖18B)。
就開發理想的功能性免疫毒素而言，以上所述的實驗結果有三個重要的面向。
首先，含有以二價抗體為基礎之標的模組的免疫毒素通優於含相應的一價標的模組者，前者細胞毒性效力提高了最高10倍(圖4A至4C及圖6A及6B)，雖然目前開發中的大多數免疫毒素都是使用一價設計。二價標的模組可能會導致細胞表面受體交聯，此一現象又會加速受體介導之細胞吞噬作用及降低免疫毒素的脫靶機率。這兩種情形都有助於將毒素遞送至細胞質。然而，相關資料顯示對免疫毒素細胞毒性的二價強化效果有抗體依存性，亦即，二價結合在某些scFv中會促進較佳的毒素遞送，但其他scFv的二價與一價結合則可能沒有明顯的差異(圖4A至4C及圖6A及6B)。有鑑於此，由於透過抗體標的模組的價數、抗原決定位及結合親和力來預測免疫毒素的效力可能並不準確，利用本案所述的高通量的實驗測試能夠提供大量的資訊，以供最佳化功能性免疫毒素。
其次，免疫毒素的效力和細胞表面抗原密度成正相關(圖10A至10H)。但由於細胞表面標的密度降低兩倍時，效力降低的相關程度可能從數倍到數百倍不等(譬如，抗體GH2-41，參見圖10A至10H)，這可能是因為細胞間的其他差異所致。由此可以推知，可建構免疫毒素使其含有大量細胞表面標的之目標細胞產生理想的細胞毒性，同時對細胞表面具有較低標的密度之脫靶細胞所產生的毒性仍為可接受的。
第三，以抗體為基礎之標的模組的抗原決定位是免疫毒素之效力的主要決定因素(圖11及12A至12C)。然而，相同抗原決定位群組中的抗體於遞送毒素藥物負載之效力可能差異極大(圖5、6A及6B)。其他因素，特別是抗體對位中所用的胺基酸類型，可能會決定毒素藥物負載的遞送路徑，並因此影響免疫毒素的效力。研究抗體-抗原互動的pH依存性顯示，抗體-抗原介面之經遺傳工程改變的組胺酸側鏈在胞內體中酸性環境下的離子化可增加回到細胞質中的抗體，因而改善抗體的藥動學特性與停留時間。相較之下，圖5、圖9A至9D與表4的結果指出，涉及在胞內體中增加之酸性環境下的抗體-抗原互動之組胺酸側鏈的離子化會擾亂免疫毒素遞送，因而減低毒素藥物負載之細胞毒性。
這些實驗數據顯示免疫毒素中作為標的模組之抗體的複雜功能，進一步點出在將標的抗體之毒素遞送能力最佳化的過程中，應考量諸多面向，包括：特定標的受體、標的細胞及細胞毒性藥物負載。現有技術長期使用天然抗體作為免疫毒素中標的模組的主要來源，但天然抗體的多樣性受到由生殖系基因使用主導之主要選殖選擇的限制，也受到因為自抗原的免疫容忍性所造成的選殖移除之限制，而所述自抗原經常是免疫毒素的標的。正因如此，使用天然抗體作為免疫毒素的標的模組可能不盡理想，因為可供選擇的天然抗體庫中適當的候選抗體數量有限。相較之下，設計與建構得當的合成抗體庫能夠提供大量的抗體，具有不同的抗原決定位-抗體對位組合，更適合用於開發針對自抗原的免疫毒素或其他類型的免疫偶聯物，且能夠展現理想的細胞毒性。本揭示內容提出的數據證實可利用此種合成抗體庫進行高通量的抗體篩選與最佳化，以作為免疫偶聯物之標的模組。
雖然上文實施方式中揭露了本發明的具體實施例，然其並非用以限定本發明，本發明所屬技術領域中具有通常知識者，在不悖離本發明之原理與精神的情形下，當可對其進行各種更動與修飾，因此本發明之保護範圍當以隨附申請專利範圍所界定者為準。
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為讓本發明的上述與其他目的、特徵、優點與實施例能更明顯易懂，所附圖式之說明如下： 圖1的折線圖摘要整理了使1、2、4、8、16、32及64 nM之曲妥珠單抗(曲妥珠單抗) scFv流經固定之AL1-PE38KDEL後進行表面電漿共振(surface plasmon resonance，SPR)分析的結果，其中SPR測量結果以黑色粗線表示，而將實驗數據進行經全域擬合(global fit)的結果以細線表示，Kd = 5.64 ± 0.03 × 10−9 M；kon = 4.54 ± 0.06 × 105 M−1 S−1 ；koff = 2.56 ± 0.04 × 10−3 S−1 。 圖2的折線圖摘要整理了使1、2、4、8、16、32及64 nM之曲妥珠單抗scFv流經固定之AL2-PE38KDEL後進行SPR分析的結果，其中SPR測量結果以黑色粗線表示，而將實驗數據進行經全域擬合的結果以細線表示，Kd = 5.52 ± 0.03 × 10−9 M；kon = 3.60 ± 0.06 × 105 M−1 S−1 ；koff = 1.99 ± 0.04 × 10−3 S−1 。 圖3A至圖3E顯示多種scFv對抗原HER2-ECD的結合親和力。使用16種帶有IGKV1-NL1*01/IGHV3-23*04種系架構(germline framework)的scFv來測量其在沒有(圖3A)以及存有AL1-PE38KDEL (圖3B)或存有AL2-PE38KDEL (圖3C)的條件下，對HER2-ECD結合之EC50 ，其中scFv:AL片段的莫耳比為1:1。每一scFv的EC50 數值如表1所示。scFv-AL1-PE38KDEL複合物(圖3D)或scFv-AL2-PE38KDEL複合物(圖3E)對HER2-ECD-結合之EC50 係以單獨的scFv為對照。 圖4A至4C顯示和適體-毒素融合蛋白AL1-PE38KDEL或AL2-PE38KDEL複合之scFv或IgG的細胞毒性。N87細胞經對照物(AL1-PE38KDEL、AL2-PE38KDEL、scFv及IgG)處理或以未共價結合之免疫毒素(IgG-AL1- PE38KDEL、scFv-AL1-PE38KDEL與scFv-AL2- PE38KDEL)處理，以決定細胞毒性。圖中y-軸為N87細胞的細胞存活率百分比，x-軸為VL-VH變異結構域或AL片段在對照物或免疫毒素複合物中的濃度；將抗體(IgG或scFv)和適體-毒素融合蛋白(AL1-PE38KDEL或AL2- PE38KDEL)以VL-VH:AL片段莫耳比為1:1的比例混合。由三次獨立重複試驗的結果計算出誤差槓。圖4A、4B與4C分別表示抗體GH2-42、G2-61與G2-75的scFv或IgG的細胞毒性測試結果。 圖5為92種HER2-ECD-專一scFv-AL1/AL2-PE38KDEL免疫毒素的細胞毒性篩選。x-軸與y-軸分別是經過scFv- AL1-PE38KDEL或scFv-AL2-PE38KDEL處理的N87細胞之細胞存活率百分比。將這些GH2 scFv的四類抗原決定位分別以灰色(M32-M62)、綠色(M41-M61)、黃色(M63-M64)及紫色(未分群)表示。在圖例符號外圍加上紅框，以表示CDR H3中帶有一或多個組胺酸的GH2 scFv。表2與表3中列出了每一scFv的詳細資料。 圖6A及圖6B分別顯示所選GH2 scFv對scFv-AL1-PE38KDEL複合物與scFv-AL2-PE38KDEL複合物的IC50 測量值，其中y-軸為經處理之N87細胞的細胞存活率百分比，而x-軸為scFv之濃度。由三次獨立重複試驗的結果計算出誤差槓。 圖7A至圖7D分別是EC50 、KD 、kon 與koff 和scFv-AL1-PE38KDEL免疫毒素的細胞毒性之關係圖。將scFv的EC50 、KD 、kon 與koff 對於對應之scFv-AL1-PE38KDEL免疫毒素的細胞毒性作圖。這些scFv (資料集S30)的詳細資訊如表2與表3所示。 圖8A至圖8D分別是EC50 、KD 、kon 與koff 和scFv-AL2-PE38KDEL免疫毒素的細胞毒性之關係圖。將scFv的EC50 、KD 、kon 與koff 對於對應之scFv-AL2-PE38KDEL免疫毒素的細胞毒性作圖。這些scFv (資料集S30)的詳細資訊如表2與表3所示。 圖9A至圖9D分別是所選scFv在pH 7及pH 5的條件下對於scFv-AL1-PE38KDEL的EC50 。圖9A：隨機選自表2前25%之scFv，其CDR-H3序列不含組胺酸；圖9B：隨機選自表2前25%之scFv，其CDR-H3序列含組胺酸；圖9C：隨機選自表2後25%之scFv，其CDR-H3序列不含組胺酸；圖9D：隨機選自表2後25%之scFv，其CDR-H3序列含組胺酸。 圖10A至圖10H顯示免疫毒素對具有不同HER2表現量之細胞的細胞毒性及平均螢光強度(Mean fluorescence intensity，MFI)。將所選GH2 scFv和AL2-PE38KDEL以scFv:AL片段之莫耳比為1:1的比例複合後，測量其對以下細胞之IC50 ：N87細胞(圖10A)、SKBR3細胞(圖10C)、BT474細胞(圖10E)及MCF7細胞(圖10G)。由三次獨立重複試驗的結果計算出誤差槓。將同一組scFv和AL2-RFP以scFv:AL片段之莫耳比為1:1的比例複合後，以流式細胞儀測量其對以下細胞之MFI：N87細胞(圖10B)、SKBR3細胞(圖10D)、BT474細胞(圖10F)及MCF7細胞(圖10H)，其中y-軸所示之MFI係以經AL2-RFP處理但未加入scFv之細胞作為背景值調整後之強度。x-軸為VL-VH變異結構域或AL片段的濃度。 圖11為M32、H32及曲妥珠單抗IgG結合至細胞表面之HER2-ECD的內化路徑比較。將SKBR-3細胞和曲妥珠單抗、M32或H32 IgG共同培育1小時，並利用免疫螢光顯微分析來觀察內化之IgG與Rab7 (圖框A)、Rab9 (圖框B)與Rab5 (圖框C)的相對位置。圖框D顯示在經過曲妥珠單抗、M32或H32 IgG處理之SKBR-3細胞中Rab7及Rab9的表現量。利用西方墨點法分析表現量，並偵測GAPDH作為內部對照。由三次獨立重複試驗的結果來計算表現量。 圖12A至12C為Fab(H32)-HER2-ECD複合物的負染色EM重構。圖12A為HER2 ECD-Fab(H32)複合物之負染色EM的代表性顯微照片。圖12B為利用ISAC軟體分析所得之代表性2D類別平均。圖12C為3D模型的黃金標準FSC曲線(Gold standard FSC curve)，0.5截止值以虛線表示。EM結構決定方法如下文方法所述。 圖13A至13C顯示非共價結合之IgG與AL1-PE38KDEL (即，IgG-AL1-PE38KDEL免疫毒素)對以下細胞為強效的免疫毒素：人類胃細胞株N87 (圖13A)、表現HER2之小鼠乳癌細胞株EMT6-HER2 (圖13B)及人類乳癌細胞株SKBR3 (圖13C)。 圖14A至14D為和適體-毒素融合蛋白AL1C-Osu-MMAF或AL1C-vc-MMAE結合之IgG (即，IgGs-AL1C- Osu-MMAF (AL1C-021)複合物與IgGs-AL1C-vc- MMAE (AL1C-026)複合物)對N87細胞(圖14A及14B)與SKBR3細胞(圖14C及14D)的細胞毒性。 圖15A至15D為和適體-毒素融合蛋白AL2C-vc-MMAE或AL2C-MMAF結合之IgG (即，IgGs-AL2C-vc-MMAE (AL2C-002)複合物與IgGs-AL2C-MMAF (AL2C-004)複合物)對N87細胞(圖15A及15B)與乳癌BT474細胞(圖15C及15D)的細胞毒性。 圖16A及16B為六種抗-HER2 IgG-vc-MMAE偶聯物對胃癌N87細胞(圖16A)及乳癌BT474細胞(圖16B)的細胞毒性；以KADCYLA (即，Herceptin-SMCC-DM1)作為正對照組。 圖17A及17B顯示GH2-61 IgG-AL1-PE38KDEL免疫毒素對腫瘤生長的劑量依賴性效果。 圖18A及18B顯示GH2-61 IgG-vc-MMAE偶聯物在N87異種移植動物模型中的活體內抗腫瘤活性。
 序列表<110> 中央研究院<120> 功能性抗體片段的高通量篩選方法及其免疫偶聯物複合物以及用於篩選之適配體-藥物偶聯物<130> P2926-TW<140> TW106119418<141> 2017-06-12<150> US62348860<151> 2016-06-11<160> 107<170> BiSSAP 1.3.6<210> 1<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-3-胺基酸序列<400> 1Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Asn Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Arg Tyr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Gly Gly Gly Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Ser Gly Gly Trp Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Trp Pro 165 170 175 Ser Gly Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Tyr Phe 210 215 220 Gly Phe Gly Asp Leu Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 2<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-4-胺基酸序列<400> 2Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Phe Gly Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Phe Asp Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Asn Ser Gly Ser Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Phe Ile Gly Pro 165 170 175 Phe Gly Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Gly Trp 210 215 220 Asn Asp Tyr Asp Phe Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 3<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-5-胺基酸序列<400> 3Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Gly Ala Pro Gly Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Phe Asn Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Asn Asn Trp Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Gly Pro 165 170 175 Phe Gly Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Gly Val 210 215 220 Val Tyr Asp Gly Ile Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 4<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-7-胺基酸序列<400> 4Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Gly Trp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Gly Thr Ala Gly Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asp Phe Pro Val 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Phe Pro Pro Leu Val Ala Gly Gly Gly Gly Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Asp Asp Tyr Phe Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Gly Pro 165 170 175 Ser Trp Gly Tyr Thr Phe Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Gly His 210 215 220 Asn Phe Val Asn Gly Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 5<211> 251<212> PRT<213> 人工序列<220> <223> 抗體GH2-8-胺基酸序列<400> 5Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Gly Trp Trp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Gly Ala Thr Gly Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Trp Asp Ser Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Gly Ser Ser Gly Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly 165 170 175 Ile Trp Pro Tyr Trp Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Phe Gly Val Gly Tyr His Tyr Tyr Met Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 6<211> 251<212> PRT<213> 人工序列<220> <223> 抗體GH2-9-胺基酸序列<400> 6Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asp Asp Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Gly Ser Ser Trp Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Gly Tyr Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Glu Phe Thr Ile Gly Ser Trp Gly Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly 165 170 175 Ile Gly Pro Tyr Gly Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Phe Asp Ile Trp Asn Tyr Phe Gly Met Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 7<211> 251<212> PRT<213> 人工序列<220> <223> 抗體GH2-11-胺基酸序列<400> 7Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Gly Asp Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Gly Trp Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Tyr Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asn Asp Trp Gly Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly 165 170 175 Ile Gly Pro Tyr Gly Gly Tyr Thr Ser Tyr Ala Asp Ser Val Arg Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Phe Val Asn Trp Asp Phe Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 8<211> 250<212> PRT<213> 人工序列<220> <223> 抗體GH2-12-胺基酸序列<400> 8Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Asn Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Pro Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asn Tyr Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asn Asp Trp Gly Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly 165 170 175 Ile Trp Pro Tyr Trp Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Tyr His Val Tyr Phe Trp Trp Met Asp Tyr Trp Gly Gln Gly Thr Leu 225 230 235 240 Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 9<211> 251<212> PRT<213> 人工序列<220> <223> 抗體GH2-13-胺基酸序列<400> 9Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Tyr Phe Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Gly Thr Thr Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Gly Ser Tyr Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Asp Gly Gly Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly 165 170 175 Ile Trp Pro Tyr Trp Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Phe Asp Asn Asn Trp Val Gly Asn Met Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 10<211> 251<212> PRT<213> 人工序列<220> <223> 抗體GH2-14-胺基酸序列<400> 10Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Gly Gly Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Gly Pro Pro Tyr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Phe Asn Tyr Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asn Asp His Gly Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly 165 170 175 Ile Gly Pro Phe Gly Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Phe Asn Phe Asn Asp Val Gly Gly Met Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 11<211> 250<212> PRT<213> 人工序列<220> <223> 抗體GH2-16-胺基酸序列<400> 11Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Tyr Ser Ser 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Tyr Thr Ser Tyr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Gly Gly Pro Met 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Leu Trp Arg Gly Ser Gly Gly Gly Gly Glu Val Gln 115 120 125 Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg 130 135 140 Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asn Asn Trp Gly Ile His 145 150 155 160 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile 165 170 175 Trp Pro Phe Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg 180 185 190 Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met 195 200 205 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe 210 215 220 Asp Tyr Leu Asn Asn Gly Gly Met Asp Tyr Trp Gly Gln Gly Thr Leu 225 230 235 240 Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 12<211> 244<212> PRT<213> 人工序列<220> <223> 抗體GH2-17-胺基酸序列<400> 12Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Phe Ser Ser 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Tyr Thr Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Gly Ser Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Val Gln Leu Val Glu Ser Gly Gly 115 120 125 Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser 130 135 140 Gly Phe Thr Ile Asn Asn Ser Gly Ile His Trp Val Arg Gln Ala Pro 145 150 155 160 Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Trp Pro Tyr Gly Gly Tyr 165 170 175 Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp 180 185 190 Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu 195 200 205 Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Asp Tyr Phe Asn Ile Gly 210 215 220 Gly Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 225 230 235 240 Ser Ala Ala Ala <210> 13<211> 247<212> PRT<213> 人工序列<220> <223> 抗體GH2-18-胺基酸序列<400> 13Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Ser Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Thr Thr Gly Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Ser Gly Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Pro Ser Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu 115 120 125 Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 130 135 140 Ala Ala Ser Gly Phe Thr Ile Ser Asn Trp Gly Ile His Trp Val Arg 145 150 155 160 Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Trp Pro Tyr 165 170 175 Gly Gly Tyr Thr Phe Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 180 185 190 Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu 195 200 205 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Asn Tyr Leu 210 215 220 Asn Leu Gly Gly Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 225 230 235 240 Ser Ser Ala Ser Ala Ala Ala 245 <210> 14<211> 251<212> PRT<213> 人工序列<220> <223> 抗體GH2-19-胺基酸序列<400> 14Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Phe Gly Trp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Arg Trp Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asn Tyr Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asn Asp Phe Phe Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly 165 170 175 Ile Gly Pro Phe Gly Gly Phe Thr Ser Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Phe Tyr Trp Gly Asp Asp Phe Asp Met Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 15<211> 251<212> PRT<213> 人工序列<220> <223> 抗體GH2-20-胺基酸序列<400> 15Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Asn Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Thr Thr Tyr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Asn Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Ile Asn Asp Tyr Gly Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser 165 170 175 Ile Gly Pro Ser Gly Gly Phe Thr Ser Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Phe Val Ile Tyr Trp Gly Phe Phe Met Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 16<211> 251<212> PRT<213> 人工序列<220> <223> 抗體GH2-21-胺基酸序列<400> 16Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Asp Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Thr Gly Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Gly Phe Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asn Asp Tyr Ser Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser 165 170 175 Ile Trp Pro Phe Gly Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Phe Asn Tyr Trp Ile Gly Ile Ile Met Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 17<211> 249<212> PRT<213> 人工序列<220> <223> 抗體GH2-23-胺基酸序列<400> 17Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Phe Trp Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Trp Ser Ala Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Phe Ser Phe Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Glu Val Gln Leu 115 120 125 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 130 135 140 Ser Cys Ala Ala Ser Gly Phe Thr Ile Asn Gly Gly Gly Ile His Trp 145 150 155 160 Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Phe Ile Gly 165 170 175 Pro Phe Gly Gly Ser Thr Phe Tyr Ala Asp Ser Val Lys Gly Arg Phe 180 185 190 Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn 195 200 205 Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Phe 210 215 220 Gly Gly Asn Ile His Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 225 230 235 240 Thr Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 18<211> 251<212> PRT<213> 人工序列<220> <223> 抗體GH2-24-胺基酸序列<400> 18Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Gly Trp Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Ser Thr Arg Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asp Asn Gly Ser Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Phe 165 170 175 Ile Gly Pro Ser Gly Gly Tyr Thr Phe Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Phe Gly Trp His Asn Val Asp Asn Met Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 19<211> 251<212> PRT<213> 人工序列<220> <223> 抗體GH2-25-胺基酸序列<400> 19Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Gly Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Tyr Thr Arg Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Gly Tyr Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Asn Tyr Phe Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Phe 165 170 175 Ile Gly Pro Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Phe Asp Leu Tyr Asn Tyr Gly Gly Met Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 20<211> 251<212> PRT<213> 人工序列<220> <223> 抗體GH2-26-胺基酸序列<400> 20Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Ser Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Thr Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Phe Asn Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Asp Tyr Gly Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Phe 165 170 175 Ile Gly Pro Tyr Trp Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Phe Tyr Phe Phe Gly Asp Gly Leu Met Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 21<211> 251<212> PRT<213> 人工序列<220> <223> 抗體GH2-28-胺基酸序列<400> 21Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Asp Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Phe Tyr Thr Arg Trp Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Phe Ser Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asn Asn Gly Gly Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser 165 170 175 Ile Gly Pro Ser Trp Gly Ser Thr Val Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Phe Gly Phe Asp Tyr Asp Gly Val Met Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 22<211> 251<212> PRT<213> 人工序列<220> <223> 抗體GH2-29-胺基酸序列<400> 22Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Phe Gly Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Tyr Pro Gly Trp Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asn Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asn Asn Tyr Gly Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Phe 165 170 175 Ile Gly Pro Ser Trp Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Phe Gly Ile His Tyr Asp Gly Val Met Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 23<211> 251<212> PRT<213> 人工序列<220> <223> 抗體GH2-30-胺基酸序列<400> 23Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Phe Gly Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Gly Thr Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Phe Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Arg 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asn Asn Phe Gly Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser 165 170 175 Ile Gly Pro Phe Trp Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Phe Gly Asn Asp Tyr Asp Gly Val Met Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 24<211> 251<212> PRT<213> 人工序列<220> <223> 抗體GH2-31-胺基酸序列<400> 24Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Trp Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Pro Pro Trp Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asn Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asn Asn Phe Gly Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser 165 170 175 Ile Gly Pro Phe Trp Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Phe Gly Asn Asp Tyr Asp Gly Val Met Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 25<211> 251<212> PRT<213> 人工序列<220> <223> 抗體GH2-32-胺基酸序列<400> 25Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Trp Gly Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Gly Pro Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Gly Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asn Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Asn Trp Gly Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly 165 170 175 Ile Gly Pro Ser Trp Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Phe Gly Asn Val Tyr Asp Gly Val Met Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 26<211> 251<212> PRT<213> 人工序列<220> <223> 抗體GH2-33-胺基酸序列<400> 26Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Gly Tyr Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Thr Thr Tyr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Arg Gly Gly Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asn Asn Phe Gly Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly 165 170 175 Ile Gly Pro Phe Trp Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Phe Gly Asp Tyr Tyr Asp Gly Val Met Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 27<211> 251<212> PRT<213> 人工序列<220> <223> 抗體GH2-35-胺基酸序列<400> 27Asp Ile Gln Val Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Gly Ser Tyr 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Ser Pro Ser Trp Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asp Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Ser Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asn Asn Phe Gly Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Tyr 165 170 175 Ile Trp Pro Phe Gly Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Phe Asn Gly Asp Tyr Val Gly Leu Met Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 28<211> 251<212> PRT<213> 人工序列<220> <223> 抗體GH2-36-胺基酸序列<400> 28Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Ser Asn 20 25 30 Val Ala Trp Tyr Gln Gln Glu Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Phe Ser Ser Gly Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Gly Gly Gly Gly Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Tyr 165 170 175 Ile Gly Pro Tyr Gly Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Phe His Asp His Ile Gly Gly Ile Met Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 29<211> 250<212> PRT<213> 人工序列<220> <223> 抗體GH2-37-胺基酸序列<400> 29Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Phe Gly Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Ser Pro Arg Trp Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asn Tyr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Gly Gly Gly Gly Glu Val Gln 115 120 125 Leu Val Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg 130 135 140 Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Gly Asn Phe Gly Ile His 145 150 155 160 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Tyr Ile 165 170 175 Gly Pro Tyr Gly Gly Tyr Thr Phe Tyr Ala Asp Ser Val Lys Gly Arg 180 185 190 Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met 195 200 205 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe 210 215 220 Gly Asp His Phe Asp Gly Val Met Asp Tyr Trp Gly Gln Gly Thr Leu 225 230 235 240 Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 30<211> 251<212> PRT<213> 人工序列<220> <223> 抗體GH2-38-胺基酸序列<400> 30Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Phe Gly Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Pro Ser Tyr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Asp Gly Gly Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly 165 170 175 Ile Gly Pro Tyr Gly Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Phe Gly Asp Asn Phe Ile Gly Val Met Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 31<211> 251<212> PRT<213> 人工序列<220> <223> 抗體GH2-39-胺基酸序列<400> 31Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Gly Asp Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Pro Gly Tyr Leu Tyr Ser Gly Val Pro Leu Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asp Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Asp Trp Gly Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly 165 170 175 Ile Gly Pro Tyr Gly Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Phe Gly Val Asn Tyr Asp Gly Asn Met Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 32<211> 251<212> PRT<213> 人工序列<220> <223> 抗體GH2-40-胺基酸序列<400> 32Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Asn Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Gly Thr Pro Trp Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Phe Asp Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys His Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asn Ser Trp Gly Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Trp 165 170 175 Ile Gly Pro Tyr Trp Gly Phe Thr Ser Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Phe Asp Gly His Phe Asp Gly Val Met Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 33<211> 251<212> PRT<213> 人工序列<220> <223> 抗體GH2-41-胺基酸序列<400> 33Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Asn Ser 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Gly Ala Ala Trp Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Gly Asn Phe Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Asn Trp Gly Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser 165 170 175 Ile Gly Pro Ser Gly Gly Phe Thr Ser Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Phe Tyr Ile Tyr Gly Gly Val Ile Met Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 34<211> 249<212> PRT<213> 人工序列<220> <223> 抗體GH2-42-胺基酸序列<400> 34Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Ser Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Gly Pro Thr Gly Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asp Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Lys Arg Gly Gly Ser Ser Arg Ser 100 105 110 Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln Leu 115 120 125 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 130 135 140 Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Asn Trp Gly Ile His Trp 145 150 155 160 Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Gly 165 170 175 Pro Tyr Gly Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe 180 185 190 Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn 195 200 205 Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Gly 210 215 220 Phe Tyr Phe Asp Gly Ile Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 225 230 235 240 Thr Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 35<211> 251<212> PRT<213> 人工序列<220> <223> 抗體GH2-43-胺基酸序列<400> 35Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Gly Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Gly Thr Pro Gly Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asn Tyr Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Gly Trp Gly Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser 165 170 175 Ile Gly Pro Phe Trp Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Phe Gly Val Asp Val Asp Gly Val Met Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 36<211> 251<212> PRT<213> 人工序列<220> <223> 抗體GH2-45-胺基酸序列<400> 36Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Gly Ser Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Trp Pro Gly Tyr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asn Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asp Asp Tyr Gly Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Phe 165 170 175 Ile Gly Pro Phe Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Phe Asp Asn Asn Asn Val Trp Gly Met Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 37<211> 251<212> PRT<213> 人工序列<220> <223> 抗體GH2-46-胺基酸序列<400> 37Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Asn Ser 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Gly Gly Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Gly Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Leu Gly Gly Gly Gly Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Gly Ser Tyr Gly Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly 165 170 175 Ile Gly Pro Tyr Gly Gly Tyr Thr Phe Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Phe Gly Tyr His Val Asp Gly Ile Met Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 38<211> 244<212> PRT<213> 人工序列<220> <223> 抗體GH2-47-胺基酸序列<400> 38Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Phe Ser Phe 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Gly Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Phe Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly 115 120 125 Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser 130 135 140 Gly Phe Thr Ile Asn Asp Tyr Gly Ile His Trp Val Arg Gln Ala Pro 145 150 155 160 Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Gly Pro Phe Gly Gly Tyr 165 170 175 Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp 180 185 190 Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu 195 200 205 Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Gly Val Leu Phe Asp Gly 210 215 220 Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 225 230 235 240 Ser Ala Ala Ala <210> 39<211> 251<212> PRT<213> 人工序列<220> <223> 抗體GH2-48-胺基酸序列<400> 39Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Trp Trp Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Ser Thr Ser Tyr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asn Trp Pro Val 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asn Gly Gly Gly Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Phe 165 170 175 Ile Trp Pro Tyr Gly Gly Phe Thr Phe Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Phe Asn Tyr Asn His Gly Trp Phe Met Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 40<211> 251<212> PRT<213> 人工序列<220> <223> 抗體GH2-49-胺基酸序列<400> 40Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Phe Ser Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Ser Ser Thr Tyr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asp Tyr Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys His Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asn Gly Phe Ser Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Trp 165 170 175 Ile Trp Pro Ser Gly Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Phe Asn His His His His Trp Gly Met Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 41<211> 251<212> PRT<213> 人工序列<220> <223> 抗體GH2-50-胺基酸序列<400> 41Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Phe Tyr Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Phe Trp Thr Thr Tyr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Phe Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asn Asn Asp Gly Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ala 165 170 175 Ile Gly Pro Phe Trp Gly Ser Thr Asp Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Thr Asn Thr Ala Tyr Leu His 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Arg 210 215 220 Phe Gly Asn Tyr Tyr Asp Gly Val Met Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 42<211> 251<212> PRT<213> 人工序列<220> <223> 抗體GH2-51-amino acid<400> 42Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Ser Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Trp Thr Thr Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Pro Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asp Gly Tyr Gly Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser 165 170 175 Ile Gly Pro Phe Trp Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Phe Gly Asp Tyr Tyr Asp Gly Val Met Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 245 250 <210> 43<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-53-胺基酸序列<400> 43Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Gly Asn Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Phe Trp Ser Arg Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Phe Asn Tyr Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Ser Asp Tyr Trp Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Gly Pro 165 170 175 Phe Gly Gly Phe Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe His Tyr 210 215 220 Tyr Trp Gly His Phe Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 44<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-54-胺基酸序列<400> 44Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Asp Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Phe Gly Ser Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Trp Asn Tyr Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Ser Asp Gly Ser Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Phe Ile Gly Pro 165 170 175 Tyr Trp Gly Phe Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Val Asn 210 215 220 Trp Val His Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 45<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-55-胺基酸序列<400> 45Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asp Gly Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Ser Ala Gly Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Trp Ser Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Asp Gly Phe Ser Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Gly Pro 165 170 175 Tyr Gly Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Asp Ile 210 215 220 Trp His Asn Phe Gly Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 46<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-56-胺基酸序列<400> 46Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asp Asn Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ala Tyr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Gly Asn Phe Pro Val 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Gly Gly Ser Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Gly Pro 165 170 175 Tyr Gly Gly Phe Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Asn Ile 210 215 220 Trp Tyr Gly Val Asn Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 47<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-57-胺基酸序列<400> 47Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Asn Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Gly Gly Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Ser Ser Gly Ser Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Gly Pro 165 170 175 Tyr Trp Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Glu Tyr Tyr Cys Ala Arg Phe Leu His 210 215 220 Gly Asp Ile Phe Gly Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 48<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-59-胺基酸序列<400> 48Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Gly Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Trp Thr Gly Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Asp Phe Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Arg Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Gly Asp Trp Tyr Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Gly Pro 165 170 175 Tyr Trp Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Val Gly 210 215 220 Asp Val Trp His Asp Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Ala 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 49<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-60-胺基酸序列<400> 49Asp Ile Gln Met Thr Gln Gly Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Gly Ser Trp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Ser Pro Pro Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Phe Asp Ser Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Ser Asp Phe Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Trp Pro 165 170 175 Tyr Trp Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Trp Asn 210 215 220 Ile Tyr Trp Asn Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 50<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-61-胺基酸序列<400> 50Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Gly Ser Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Trp Ser Thr Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Gly Gly Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Asn Asn Trp Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Trp Pro 165 170 175 Tyr Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Tyr Asn 210 215 220 His His Gly Gly Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 51<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-62-胺基酸序列<400> 51Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Gly Gly Tyr 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Phe Gly Ala Ser Gly Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Tyr Ser Ser Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Asn Asn Trp Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Trp Pro 165 170 175 Phe Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Asp Tyr 210 215 220 Leu Asn Asn Gly Gly Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 52<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-63-胺基酸序列<400> 52Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asp Ser Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Phe Thr Arg Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Gly Asn Trp Ser Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Trp Pro 165 170 175 Tyr Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe His Tyr 210 215 220 Trp Trp His Asn Ile Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 53<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-64-胺基酸序列<400> 53Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Gly Ser Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Phe Phe Ser Ser Trp Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asn Tyr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Gly Asp Trp Ser Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Trp Pro 165 170 175 Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Asn Tyr 210 215 220 Trp Ile Asn Gly Ile Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 54<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-65-胺基酸序列<400> 54Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Gly Trp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asp Phe Pro Val 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Ser Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Asp Asp Trp Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Trp Ile Trp Pro 165 170 175 Tyr Trp Gly Tyr Thr Phe Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Leu Asp 210 215 220 Trp Asn Asn Asn Trp Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 55<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-66-胺基酸序列<400> 55Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Tyr Ser Tyr 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Tyr Ala Ser Gly Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asn Ser Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Gly Asn Trp Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Tyr Ile Trp Pro 165 170 175 Tyr Trp Gly Phe Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Leu Asp 210 215 220 Trp Asn Leu Leu Trp Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 56<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-68-胺基酸序列<400> 56Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Asn Ser 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Phe Ser Pro Arg Tyr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Asn Asn Phe Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Gly Pro 165 170 175 Tyr Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Gly Ile 210 215 220 His His Asp Gly Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 57<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-69-胺基酸序列<400> 57Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Tyr Tyr Ser 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Phe Gly Thr Thr Gly Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Phe Ser Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Asn Ser Trp Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Gly Pro 165 170 175 Phe Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Gly Asp 210 215 220 Asp His Asp Gly Leu Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 58<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-71-胺基酸序列<400> 58Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Gly Ser Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Phe Ser Thr Gly Gly Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asn Trp Pro Leu 85 90 95 Thr Phe Ser Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Arg Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Ser Asn Phe Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Gly Pro 165 170 175 Ser Gly Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Asn Asn 210 215 220 Trp Asp Ile Phe Gly Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 59<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-72-胺基酸序列<400> 59Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Gly Tyr Phe 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Gly Thr Thr Trp Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Ser Asn Gly Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Tyr Ile Trp Pro 165 170 175 Ser Gly Gly Tyr Thr Phe Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Phe Asn 210 215 220 Asn Asp Trp Ile Gly Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 60<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-73-胺基酸序列<400> 60Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Ser Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Ser Pro Thr Gly Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ala Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asn Trp Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Ser Gly Gly Ser Glu Val Arg Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Ser Asn Trp Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Trp Pro 165 170 175 Phe Gly Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe His His 210 215 220 Phe Val Trp Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 61<211> 243<212> PRT<213> 人工序列<220> <223> 抗體GH2-75-胺基酸序列<400> 61Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Ile Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Gly Tyr 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr 50 55 60 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 65 70 75 80 Tyr Tyr Cys Gln Gln Tyr Tyr Asn Trp Pro Val Thr Phe Gly Gln Gly 85 90 95 Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly Ser Ile Glu Gly Arg 100 105 110 Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly 115 120 125 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 130 135 140 Ser Thr Ile Gly Asn Ser Gly Ile His Trp Val Arg Gln Ala Pro Gly 145 150 155 160 Lys Gly Leu Glu Trp Val Ala Tyr Ile Gly Pro Tyr Gly Gly Tyr Thr 165 170 175 Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr 180 185 190 Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 195 200 205 Thr Ala Val Tyr Tyr Cys Ala Arg Phe Asp Asp Tyr His Trp Asp Gly 210 215 220 Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser 225 230 235 240 Ala Ala Ala <210> 62<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-76-胺基酸序列<400> 62Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Asp Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Phe Ser Thr Ser Trp Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ala Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asn Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Gly Asn Ser Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Phe Ile Gly Pro 165 170 175 Tyr Gly Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Asn Leu 210 215 220 Asp His Gly Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 63<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-77-胺基酸序列<400> 63Asp Ile Gln Met Ser Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Gly Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Phe Ser Pro Ala Gly Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asn Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Gly Asn Trp Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Gly Pro 165 170 175 Tyr Gly Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Gly His 210 215 220 Asn Tyr Asp Gly Ile Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 64<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-78-胺基酸序列<400> 64Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Asn Ser 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Trp Pro Thr Gly Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Ser Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Asn Asn Trp Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Gly Pro 165 170 175 Tyr Gly Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Gly Val 210 215 220 Ile Ile Asp Gly Ile Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 65<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-79-胺基酸序列<400> 65Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Ala Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Tyr Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Gly Pro Ala Gly Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Asn Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Asn Ser Tyr Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Phe Ile Gly Pro 165 170 175 Phe Trp Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe His Asp 210 215 220 Asp Ile Asn Trp Gly Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 66<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-80-胺基酸序列<400> 66Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Asn Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Phe Pro Thr Gly Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asn Tyr Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Ser Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Asn Asn Trp Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Gly Pro 165 170 175 Tyr Trp Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Gly Asn 210 215 220 His His Asp Gly Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 67<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-81-胺基酸序列<400> 67Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Asp Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Phe Thr Thr Gly Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asn Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Asn Asp Tyr Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Gly Pro 165 170 175 Phe Gly Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Gly Val 210 215 220 Leu Phe Asp Gly Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 68<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-82-胺基酸序列<400> 68Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Trp Gly Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Gly Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Phe Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Ser Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Ser Gly Tyr Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Gly Pro 165 170 175 Tyr Gly Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Gly Ile 210 215 220 Asp Tyr Asp Gly Ile Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 69<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-83-胺基酸序列<400> 69Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Trp Gly Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Gly Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Phe Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Gln Gly Arg Ser Gly Ser Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ile 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Ser Gly Tyr Gly Ile His Trp Met 145 150 155 160 Arg Arg Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Gly Pro 165 170 175 Tyr Gly Gly Tyr Thr Ser Tyr Ala Tyr Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Thr Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu His Thr Ala Val Tyr Tyr Cys Ala Arg Phe Ala Ile 210 215 220 Asp Tyr Asp Gly Ile Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 70<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-84-胺基酸序列<400> 70Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Trp Phe Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Ser Thr Ser Trp Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Phe Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Ser Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Gly Asp Tyr Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Gly Pro 165 170 175 Ser Gly Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Gly Phe 210 215 220 Asp Ile Ile Gly Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 71<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-85-胺基酸序列<400> 71Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Gly Asp Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Gly Thr Ser Tyr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Asp Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Ser Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Ser Asn Tyr Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Gly Pro 165 170 175 Tyr Gly Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Tyr Val 210 215 220 Asp Leu Gly Gly His Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 72<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-87-胺基酸序列<400> 72Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Asn Ser 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Phe Gly Thr Arg Tyr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asn Tyr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Val Glu Gly Arg Ser Gly Ser Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Ser Ser Tyr Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Gly Pro 165 170 175 Phe Gly Gly Phe Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Leu Asn 210 215 220 Ile His Leu Gly Trp Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 73<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-89-胺基酸序列<400> 73Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Ser Ser 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Phe Phe Ala Gly Trp Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Arg Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Asn Asp Trp Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser Ile Gly Pro 165 170 175 Phe Trp Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Ala Tyr Tyr Cys Ala Arg Phe Gly Asn 210 215 220 Tyr Val Asp Gly Ile Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 74<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-90-胺基酸序列<400> 74Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Tyr Tyr Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Glu Ala Pro Lys Leu Leu Ile 35 40 45 Phe Phe Ser Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Ser Ser Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Val Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Asn Asp Trp Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser Ile Gly Pro 165 170 175 Phe Trp Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Gly Phe 210 215 220 Asp Val Asp Gly Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 75<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-91-胺基酸序列<400> 75Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser His Asp Val Gly Trp Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Trp Pro Thr Tyr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Phe Ser Tyr Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Asn Asn Phe Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser Ile Gly Pro 165 170 175 Phe Trp Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Gly Asn 210 215 220 Asp Tyr Asp Gly Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 76<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-92-胺基酸序列<400> 76Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Asp Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Phe Gly Ser Pro Tyr Leu Phe Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Phe Ser Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Gln Val Gly Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Ser Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Gly Asn Trp Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser Ile Gly Pro 165 170 175 Phe Trp Gly Tyr Thr Phe Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Gly Asn 210 215 220 His Tyr Asp Gly Ile Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 77<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-93-胺基酸序列<400> 77Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Trp Trp Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Phe Gly Ala Ser Gly Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Phe Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Gly Asn Trp Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Gly Pro 165 170 175 Tyr Trp Gly Ser Thr Phe Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Gly Asn 210 215 220 Ile Val Asp Gly Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 78<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-94-胺基酸序列<400> 78Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Gly Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Gly Ser Gly Tyr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asp Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Asp Asn Trp Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser Ile Gly Pro 165 170 175 Ser Trp Gly Phe Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Gly Val 210 215 220 Asp Ile Asp Gly Ile Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 79<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-95-胺基酸序列<400> 79Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Gly Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Phe Gly Ser Thr Trp Leu Tyr Ser Gly Val Pro Leu Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asp Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Asp Ser Trp Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Gly Pro 165 170 175 Phe Trp Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Gly Ile 210 215 220 His Phe Asp Gly Ile Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 80<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-96-胺基酸序列<400> 80Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Gly Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ser Ser Trp Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Phe Asp Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Ser Gly Tyr Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Gly Pro 165 170 175 Phe Trp Gly Tyr Thr Phe Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Gly His 210 215 220 Ile Val Asp Gly Leu Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 81<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-97-胺基酸序列<400> 81Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Gly Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ser Thr Trp Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asn Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Asn Asn Tyr Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Gly Pro 165 170 175 Tyr Trp Gly Tyr Thr Phe Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Gly Asp 210 215 220 Val Ile Asp Gly Ile Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 82<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-98-胺基酸序列<400> 82Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Gly Ser Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Phe Ser Ala Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Asn Asn Tyr Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Gly Pro 165 170 175 Ser Trp Gly Phe Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Gly Phe 210 215 220 Tyr Asn Asp Gly Phe Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 83<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-100-胺基酸序列<400> 83Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Tyr Ser Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Thr Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Thr Thr Tyr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Phe Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Gly Asn Phe Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Tyr Ile Gly Pro 165 170 175 Tyr Trp Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe His Asn 210 215 220 Asp Ile Gly Gly Ile Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 84<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-101-胺基酸序列<400> 84Asp Ile Gln Met Ala Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Asn Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Thr Thr Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Asn Ser Tyr Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser Ile Gly Pro 165 170 175 Tyr Trp Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Tyr Ile 210 215 220 Trp Phe Asp Gly Leu Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 85<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-102-胺基酸序列<400> 85Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Gly Ser Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Pro Tyr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Asn Gly Gly Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser Ile Gly Pro 165 170 175 Tyr Gly Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Gly His 210 215 220 His Tyr Asp Gly His Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 86<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-103-胺基酸序列<400> 86Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Phe Ser Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Gly Thr Pro Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asn Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Asp Glu Tyr Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Phe Ile Gly Pro 165 170 175 Tyr Trp Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Gly His 210 215 220 Leu His Asp Gly Leu Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 87<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-104-胺基酸序列<400> 87Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Ser Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ser Pro Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asp Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Arg Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Asp Ser Trp Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Gly Pro 165 170 175 Phe Gly Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Gly Asp 210 215 220 Tyr Ile Asp Gly Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 88<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-105-胺基酸序列<400> 88Asp Thr Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Cys Ser Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Gly Ser Pro Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Gly Trp Pro Met 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Ser Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Asp Asn Trp Trp Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Gly Pro 165 170 175 Phe Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Asn Asn 210 215 220 Cys Gly Ile Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 89<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-106-胺基酸序列<400> 89Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asn Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Tyr Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Gly Ala Gly Tyr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Ser Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Ser Ser Ser Tyr Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Gly Pro 165 170 175 Phe Gly Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Gly Trp 210 215 220 His His Leu His Leu Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 90<211> 248<212> PRT<213> 人工序列<220> <223> 抗體GH2-107-胺基酸序列<400> 90Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Asp Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Gly Ala Gly Tyr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Tyr Ser Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Ser Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Ser Asn Phe Gly Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Tyr Ile Gly Pro 165 170 175 Tyr Gly Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Ala Ala Val Tyr Tyr Cys Ala Arg Phe His Ile 210 215 220 His Asn Leu Trp Gly Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Ala Ser Ala Ala Ala 245 <210> 91<211> 15<212> PRT<213> 人工序列<220> <223> Synthetized<400> 91Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 92<211> 15<212> PRT<213> 人工序列<220> <223> Synthesized<400> 92Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu Glu Pro Arg Ala Ala 1 5 10 15 <210> 93<211> 59<212> PRT<213> 人工序列<220> <223> Synthesized<400> 93Cys Ala Gly Gly Thr Gly Cys Ala Cys Gly Ala Thr Gly Thr Gly Ala 1 5 10 15 Thr Gly Gly Thr Ala Cys Cys Gly Ala Thr Ala Thr Thr Cys Ala Ala 20 25 30 Ala Thr Gly Ala Cys Cys Cys Ala Gly Ala Gly Cys Cys Cys Gly Ala 35 40 45 Gly Cys Ala Gly Cys Cys Thr Gly Ala Gly Cys 50 55 <210> 94<211> 36<212> PRT<213> 人工序列<220> <223> Synthesized<400> 94Thr Gly Cys Ala Gly Cys Cys Ala Cys Cys Gly Thr Ala Cys Gly Thr 1 5 10 15 Thr Thr Gly Ala Thr Thr Thr Cys Cys Ala Cys Cys Thr Thr Gly Gly 20 25 30 Thr Gly Cys Cys 35 <210> 95<211> 36<212> PRT<213> 人工序列<220> <223> Synthesized<400> 95Cys Gly Thr Gly Thr Cys Gly Cys Ala Thr Cys Thr Gly Ala Ala Gly 1 5 10 15 Thr Gly Cys Ala Gly Cys Thr Gly Gly Thr Gly Gly Ala Ala Thr Cys 20 25 30 Gly Gly Gly Ala 35 <210> 96<211> 48<212> PRT<213> 人工序列<220> <223> Synthesized<400> 96Gly Ala Cys Cys Gly Ala Thr Gly Gly Gly Cys Cys Cys Thr Thr Gly 1 5 10 15 Gly Thr Gly Cys Thr Ala Gly Cys Cys Gly Ala Gly Cys Thr Cys Ala 20 25 30 Cys Gly Gly Thr Ala Ala Cys Ala Ala Gly Gly Gly Thr Gly Cys Cys 35 40 45 <210> 97<211> 39<212> PRT<213> 人工序列<220> <223> Synthesized<400> 97Ala Ala Gly Gly Thr Gly Gly Ala Ala Ala Thr Cys Ala Ala Ala Cys 1 5 10 15 Gly Thr Ala Cys Gly Gly Thr Gly Gly Cys Thr Gly Cys Ala Cys Cys 20 25 30 Ala Thr Cys Thr Gly Thr Cys 35 <210> 98<211> 33<212> PRT<213> 人工序列<220> <223> Synthesized<400> 98Cys Thr Gly Cys Ala Cys Thr Thr Cys Ala Gly Ala Thr Gly Cys Gly 1 5 10 15 Ala Cys Ala Cys Gly Cys Gly Thr Ala Gly Cys Ala Ala Cys Ala Gly 20 25 30 Cys <210> 99<211> 1491<212> DNA<213> 人工序列<220> <223> Synthesized<400> 99ggagccatgg cccagccggc cggatcctac aataaagatc agcagagcgc cttctatgaa 60attctgaata tgccgaatct gaacgaagaa cagcgcaatg gttttattca gagcctgaaa 120gatgatccga gccagagcac caatgttctg ggtgaagcaa aaaaactgaa tgaaagccag 180gcaccgaaag cagataatgg tggtggtagc ggtggtggtt caggtggtgg cagtggagga 240ggttctggcg gtagcgaagt taccattaaa gttaatctga ttttcgccga tggcaaaatt 300cagaccgcag aatttaaagg cacctttgaa gaagcaacag ccgaagcata tcgttatgca 360gcactgctgg caaaagttaa tggtgaatat accgctgatc tggaagatgg tggtaatcac 420atgaatatca aatttgcagg cggtggtagt ggtaccggag gtagcctggc agcactgacc 480gcacatcagg catgtcatct gccgctggaa acctttaccc gtcatcgtca gcctcgtggt 540tgggaacagc tggaacagtg tggttatccg gttcagcgtc tggttgcact gtatctggca 600gcccgtctga gctggaatca ggttgatcag gttattcgta atgcactggc aagtccgggt 660agcggtggtg atctgggtga agcaattcgt gaacagcctg aacaggcacg tctggcactg 720accctggcag ccgcagaaag cgaacgtttt gttcgtcagg gtacaggtaa tgatgaagcc 780ggtgcagcaa atggtccggc agatagcggt gatgcactgc tggaacgtaa ttatccgaca 840ggtgcagaat ttctgggtga tggtggtgat gttagcttta gcacccgtgg cacccagaat 900tggaccgttg aacgtctgct gcaggcacac cgtcagctgg aagaacgtgg ttatgttttt 960gttggttatc atggcacctt tctggaagca gcacagagca ttgtttttgg tggtgttcgt 1020gcacgtagcc aggatctgga tgcaatttgg cgtggtttct atattgccgg tgatccggca 1080ctggcctatg gttatgcaca ggatcaagaa ccggatgcac gtggtcgtat tcgcaatggt 1140gccctgctgc gtgtttatgt tccgcgtagc agcctgcctg gtttttatcg taccagcctg 1200acactggctg cacctgaagc agcgggtgaa gtggaacgtc tgattggtca tccgctgccg 1260ctgcgtctgg atgccattac aggtccggaa gaagaaggcg gtcgtctgga aaccattctg 1320ggttggcctc tggcagaacg taccgttgtt attccgagcg caattccgac cgatccgcgt 1380aatgttggtg gcgatctgga cccgagcagc attccggata aagaacaggc aattagcgca 1440ctgccggatt atgcaagcca gcctggtaaa ccgcctaaag atgagctgta a 1491<210> 100<211> 1920<212> DNA<213> 人工序列<220> <223> Synthesized<400> 100ggagccatgg cccagccggc cggatcctac aataaagatc agcagagcgc cttctatgaa 60attctgaata tgccgaatct gaacgaagaa cagcgcaatg gttttattca gagcctgaaa 120gatgatccga gccagagcac caatgttctg ggtgaagcaa aaaaactgaa tgaaagccag 180gcaccgaaag cagataatgg tggtggtagc ggtggtggtt caggtggtgg cagtggagga 240ggttctggcg gtagcgaagt taccattaaa gttaatctga ttttcgccga tggcaaaatt 300cagaccgcag aatttaaagg cacctttgaa gaagcaacag ccgaagcata tcgttatgca 360gcactgctgg caaaagttaa tggtgaatat accgctgatc tggaagatgg tggtaatcac 420atgaatatca aatttgcagg cggaggtagc ggtggatata acaaagatca gcagagtgcc 480ttctatgaaa ttctgaatat gccgaatctg aacgaagaac agcgcaatgg ttttattcag 540agcctgaaag atgatccgag ccagagcacc aatgttctgg gtgaagcaaa aaaactgaat 600gaaagccagg caccgaaagc agataatggt ggtggtagcg gtggtggttc aggtggtggc 660agtggaggag gttctggcgg tagcgaagtt accattaaag ttaatctgat tttcgccgat 720ggcaaaattc agaccgcaga atttaaaggc acctttgaag aagcaacagc cgaagcatat 780cgttatgcag cactgctggc aaaagttaat ggtgaatata ccgctgatct ggaagatggt 840ggtaatcaca tgaacattaa attcgccggt ggtggcagtg gtaccggagg tagcctggca 900gcactgaccg cacatcaggc atgtcatctg ccgctggaaa cctttacccg tcatcgtcag 960cctcgtggtt gggaacagct ggaacagtgt ggttatccgg ttcagcgtct ggttgcactg 1020tatctggcag cccgtctgag ctggaatcag gttgatcagg ttattcgtaa tgcactggca 1080agtccgggta gcggtggtga tctgggtgaa gcaattcgtg aacagcctga acaggcacgt 1140ctggcactga ccctggcagc cgcagaaagc gaacgttttg ttcgtcaggg tacaggtaat 1200gatgaagccg gtgcagcaaa tggtccggca gatagcggtg atgcactgct ggaacgtaat 1260tatccgacag gtgcagaatt tctgggtgat ggtggtgatg ttagctttag cacccgtggc 1320acccagaatt ggaccgttga acgtctgctg caggcacacc gtcagctgga agaacgtggt 1380tatgtttttg ttggttatca tggcaccttt ctggaagcag cacagagcat tgtttttggt 1440ggtgttcgtg cacgtagcca ggatctggat gcaatttggc gtggtttcta tattgccggt 1500gatccggcac tggcctatgg ttatgcacag gatcaagaac cggatgcacg tggtcgtatt 1560cgcaatggtg ccctgctgcg tgtttatgtt ccgcgtagca gcctgcctgg tttttatcgt 1620accagcctga cactggctgc acctgaagca gcgggtgaag tggaacgtct gattggtcat 1680ccgctgccgc tgcgtctgga tgccattaca ggtccggaag aagaaggcgg tcgtctggaa 1740accattctgg gttggcctct ggcagaacgt accgttgtta ttccgagcgc aattccgacc 1800gatccgcgta atgttggtgg cgatctggac ccgagcagca ttccggataa agaacaggca 1860attagcgcac tgccggatta tgcaagccag cctggtaaac cgcctaaaga tgagctgtaa 1920<210> 101<211> 496<212> PRT<213> 人工序列<220> <223> synthesized<400> 101Gly Ala Met Ala Gln Pro Ala Gly Ser Tyr Asn Lys Asp Gln Gln Ser 1 5 10 15 Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg 20 25 30 Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn 35 40 45 Val Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala 50 55 60 Asp Asn Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly 65 70 75 80 Gly Ser Gly Gly Ser Glu Val Thr Ile Lys Val Asn Leu Ile Phe Ala 85 90 95 Asp Gly Lys Ile Gln Thr Ala Glu Phe Lys Gly Thr Phe Glu Glu Ala 100 105 110 Thr Ala Glu Ala Tyr Arg Tyr Ala Ala Leu Leu Ala Lys Val Asn Gly 115 120 125 Glu Tyr Thr Ala Asp Leu Glu Asp Gly Gly Asn His Met Asn Ile Lys 130 135 140 Phe Ala Gly Gly Gly Ser Gly Thr Gly Gly Ser Leu Ala Ala Leu Thr 145 150 155 160 Ala His Gln Ala Cys His Leu Pro Leu Glu Thr Phe Thr Arg His Arg 165 170 175 Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln 180 185 190 Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val 195 200 205 Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp 210 215 220 Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu 225 230 235 240 Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly 245 250 255 Asn Asp Glu Ala Gly Ala Ala Asn Gly Pro Ala Asp Ser Gly Asp Ala 260 265 270 Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly 275 280 285 Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu 290 295 300 Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe 305 310 315 320 Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Gln Ser Ile Val Phe 325 330 335 Gly Gly Val Arg Ala Arg Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly 340 345 350 Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp 355 360 365 Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg 370 375 380 Val Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu 385 390 395 400 Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly 405 410 415 His Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu 420 425 430 Gly Gly Arg Leu Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr 435 440 445 Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gly 450 455 460 Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala 465 470 475 480 Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro Lys Asp Glu Leu 485 490 495 <210> 102<211> 639<212> PRT<213> 人工序列<220> <223> synthesized<400> 102Gly Ala Met Ala Gln Pro Ala Gly Ser Tyr Asn Lys Asp Gln Gln Ser 1 5 10 15 Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg 20 25 30 Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn 35 40 45 Val Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala 50 55 60 Asp Asn Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly 65 70 75 80 Gly Ser Gly Gly Ser Glu Val Thr Ile Lys Val Asn Leu Ile Phe Ala 85 90 95 Asp Gly Lys Ile Gln Thr Ala Glu Phe Lys Gly Thr Phe Glu Glu Ala 100 105 110 Thr Ala Glu Ala Tyr Arg Tyr Ala Ala Leu Leu Ala Lys Val Asn Gly 115 120 125 Glu Tyr Thr Ala Asp Leu Glu Asp Gly Gly Asn His Met Asn Ile Lys 130 135 140 Phe Ala Gly Gly Gly Ser Gly Gly Tyr Asn Lys Asp Gln Gln Ser Ala 145 150 155 160 Phe Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn 165 170 175 Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val 180 185 190 Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp 195 200 205 Asn Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly 210 215 220 Ser Gly Gly Ser Glu Val Thr Ile Lys Val Asn Leu Ile Phe Ala Asp 225 230 235 240 Gly Lys Ile Gln Thr Ala Glu Phe Lys Gly Thr Phe Glu Glu Ala Thr 245 250 255 Ala Glu Ala Tyr Arg Tyr Ala Ala Leu Leu Ala Lys Val Asn Gly Glu 260 265 270 Tyr Thr Ala Asp Leu Glu Asp Gly Gly Asn His Met Asn Ile Lys Phe 275 280 285 Ala Gly Gly Gly Ser Gly Thr Gly Gly Ser Leu Ala Ala Leu Thr Ala 290 295 300 His Gln Ala Cys His Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln 305 310 315 320 Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg 325 330 335 Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp 340 345 350 Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu 355 360 365 Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr 370 375 380 Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn 385 390 395 400 Asp Glu Ala Gly Ala Ala Asn Gly Pro Ala Asp Ser Gly Asp Ala Leu 405 410 415 Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly 420 425 430 Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg 435 440 445 Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Val 450 455 460 Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Gln Ser Ile Val Phe Gly 465 470 475 480 Gly Val Arg Ala Arg Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe 485 490 495 Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln 500 505 510 Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val 515 520 525 Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr 530 535 540 Leu Ala Ala Pro Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His 545 550 555 560 Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly 565 570 575 Gly Arg Leu Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val 580 585 590 Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp 595 600 605 Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu 610 615 620 Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro Lys Asp Glu Leu 625 630 635 <210> 103<211> 1584<212> DNA<213> 人工序列<220> <223> Synthetic<400> 103atgcatcatc accaccatca tggatcctac aataaagatc agcagagcgc cttctatgaa 60attctgaata tgccgaatct gaacgaagaa cagcgcaatg gttttattca gagcctgaaa 120gatgatccga gccagagcac caatgttctg ggtgaagcaa aaaaactgaa tgaaagccag 180gcaccgaaag cagataatgg tggtggtagc ggtggtggtt caggtggtgg cagtggagga 240ggttctggcg gtagcgaagt taccattaaa gttaatctga ttttcgccga tggcaaaatt 300cagaccgcag aatttaaagg cacctttgaa gaagcaacag ccgaagcata tcgttatgca 360gcactgctgg caaaagttaa tggtgaatat accgctgatc tggaagatgg tggtaatcac 420atgaatatca aatttgcagg cggaggtagc ggtggatata acaaagatca gcagagtgcc 480ttctatgaaa ttctgaatat gccgaatctg aacgaagaac agcgcaatgg ttttattcag 540agcctgaaag atgatccgag ccagagcacc aatgttctgg gtgaagcaaa aaaactgaat 600gaaagccagg caccgaaagc agataatggt ggtggtagcg gtggtggttc aggtggtggc 660agtggaggag gttctggcgg tagcgaagtt accattaaag ttaatctgat tttcgccgat 720ggcaaaattc agaccgcaga atttaaaggc acctttgaag aagcaacagc cgaagcatat 780cgttatgcag cactgctggc aaaagttaat ggtgaatata ccgctgatct ggaagatggt 840ggtaatcaca tgaacattaa attcgccggt ggtggcggca gcggtaccat gagcgagctg 900atcaaggaga acatgcacat gaagctgtac atggagggca ccgtgaacaa ccaccacttc 960aagtgcacat ccgagggcga aggcaagccc tacgagggca cccagaccat gaagatcaag 1020gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc tggctaccag cttcatgtac 1080ggcagcaaag ccttcatcaa ccacacccag ggcatccccg acttctttaa gcagtccttc 1140cctgagggct tcacatggga gagaatcacc acatacgaag acgggggcgt gctgaccgct 1200acccaggaca ccagcttcca gaacggctgc atcatctaca acgtcaagat caacggggtg 1260aacttcccat ccaacggccc tgtgatgcag aagaaaacac gcggctggga ggccaacacc 1320gagatgctgt accccgctga cggcggcctg agaggccaca gccagatggc cctgaagctc 1380gtgggcgggg gctacctgca ctgctccttc aagaccacat acagatccaa gaaacccgct 1440aagaacctca agatgcccgg cttccacttc gtggaccaca gactggaaag aatcaaggag 1500gccgacaaag agacctacgt cgagcagcac gagatggctg tggccaagta ctgcgacctc 1560cctagcaaac tggggcacag ataa 1584<210> 104<211> 1608<212> DNA<213> 人工序列<220> <223> Synthesized<400> 104atgcatcatc accaccatca tggatcctac aataaagatc agcagagcgc cttctatgaa 60attctgaata tgccgaatct gaacgaagaa cagcgcaatg gttttattca gagcctgaaa 120gatgatccga gccagagcac caatgttctg ggtgaagcaa aaaaactgaa tgaaagccag 180gcaccgaaag cagataatgg tggtggtagc ggtggtggtt caggtggtgg cagtggagga 240ggttctggcg gtagcgaagt taccattaaa gttaatctga ttttcgccga tggcaaaatt 300cagaccgcag aatttaaagg cacctttgaa gaagcaacag ccgaagcata tcgttatgca 360gcactgctgg caaaagttaa tggtgaatat accgctgatc tggaagatgg tggtaatcac 420atgaatatca aatttgcagg cggaggtagc ggtggatata acaaagatca gcagagtgcc 480ttctatgaaa ttctgaatat gccgaatctg aacgaagaac agcgcaatgg ttttattcag 540agcctgaaag atgatccgag ccagagcacc aatgttctgg gtgaagcaaa aaaactgaat 600gaaagccagg caccgaaagc agataatggt ggtggtagcg gtggtggttc aggtggtggc 660agtggaggag gttctggcgg tagcgaagtt accattaaag ttaatctgat tttcgccgat 720ggcaaaattc agaccgcaga atttaaaggc acctttgaag aagcaacagc cgaagcatat 780cgttatgcag cactgctggc aaaagttaat ggtgaatata ccgctgatct ggaagatggt 840ggtaatcaca tgaacattaa attcgccggt ggtggcggca gcggtaccat ggtgagcaag 900ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac 960ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc 1020ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc 1080ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc 1140ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac 1200ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc 1260gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac 1320aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg 1380aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag 1440cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc 1500cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc 1560gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaa 1608<210> 105<211> 527<212> PRT<213> 人工序列<220> <223> symthesized<400> 105Met His His His His His His Gly Ser Tyr Asn Lys Asp Gln Gln Ser 1 5 10 15 Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg 20 25 30 Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn 35 40 45 Val Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala 50 55 60 Asp Asn Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly 65 70 75 80 Gly Ser Gly Gly Ser Glu Val Thr Ile Lys Val Asn Leu Ile Phe Ala 85 90 95 Asp Gly Lys Ile Gln Thr Ala Glu Phe Lys Gly Thr Phe Glu Glu Ala 100 105 110 Thr Ala Glu Ala Tyr Arg Tyr Ala Ala Leu Leu Ala Lys Val Asn Gly 115 120 125 Glu Tyr Thr Ala Asp Leu Glu Asp Gly Gly Asn His Met Asn Ile Lys 130 135 140 Phe Ala Gly Gly Gly Ser Gly Gly Tyr Asn Lys Asp Gln Gln Ser Ala 145 150 155 160 Phe Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn 165 170 175 Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val 180 185 190 Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp 195 200 205 Asn Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly 210 215 220 Ser Gly Gly Ser Glu Val Thr Ile Lys Val Asn Leu Ile Phe Ala Asp 225 230 235 240 Gly Lys Ile Gln Thr Ala Glu Phe Lys Gly Thr Phe Glu Glu Ala Thr 245 250 255 Ala Glu Ala Tyr Arg Tyr Ala Ala Leu Leu Ala Lys Val Asn Gly Glu 260 265 270 Tyr Thr Ala Asp Leu Glu Asp Gly Gly Asn His Met Asn Ile Lys Phe 275 280 285 Ala Gly Gly Gly Gly Ser Gly Thr Met Ser Glu Leu Ile Lys Glu Asn 290 295 300 Met His Met Lys Leu Tyr Met Glu Gly Thr Val Asn Asn His His Phe 305 310 315 320 Lys Cys Thr Ser Glu Gly Glu Gly Lys Pro Tyr Glu Gly Thr Gln Thr 325 330 335 Met Lys Ile Lys Val Val Glu Gly Gly Pro Leu Pro Phe Ala Phe Asp 340 345 350 Ile Leu Ala Thr Ser Phe Met Tyr Gly Ser Lys Ala Phe Ile Asn His 355 360 365 Thr Gln Gly Ile Pro Asp Phe Phe Lys Gln Ser Phe Pro Glu Gly Phe 370 375 380 Thr Trp Glu Arg Ile Thr Thr Tyr Glu Asp Gly Gly Val Leu Thr Ala 385 390 395 400 Thr Gln Asp Thr Ser Phe Gln Asn Gly Cys Ile Ile Tyr Asn Val Lys 405 410 415 Ile Asn Gly Val Asn Phe Pro Ser Asn Gly Pro Val Met Gln Lys Lys 420 425 430 Thr Arg Gly Trp Glu Ala Asn Thr Glu Met Leu Tyr Pro Ala Asp Gly 435 440 445 Gly Leu Arg Gly His Ser Gln Met Ala Leu Lys Leu Val Gly Gly Gly 450 455 460 Tyr Leu His Cys Ser Phe Lys Thr Thr Tyr Arg Ser Lys Lys Pro Ala 465 470 475 480 Lys Asn Leu Lys Met Pro Gly Phe His Phe Val Asp His Arg Leu Glu 485 490 495 Arg Ile Lys Glu Ala Asp Lys Glu Thr Tyr Val Glu Gln His Glu Met 500 505 510 Ala Val Ala Lys Tyr Cys Asp Leu Pro Ser Lys Leu Gly His Arg 515 520 525 <210> 106<211> 535<212> PRT<213> 人工序列<220> <223> Synthesized<400> 106Met His His His His His His Gly Ser Tyr Asn Lys Asp Gln Gln Ser 1 5 10 15 Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg 20 25 30 Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn 35 40 45 Val Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala 50 55 60 Asp Asn Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly 65 70 75 80 Gly Ser Gly Gly Ser Glu Val Thr Ile Lys Val Asn Leu Ile Phe Ala 85 90 95 Asp Gly Lys Ile Gln Thr Ala Glu Phe Lys Gly Thr Phe Glu Glu Ala 100 105 110 Thr Ala Glu Ala Tyr Arg Tyr Ala Ala Leu Leu Ala Lys Val Asn Gly 115 120 125 Glu Tyr Thr Ala Asp Leu Glu Asp Gly Gly Asn His Met Asn Ile Lys 130 135 140 Phe Ala Gly Gly Gly Ser Gly Gly Tyr Asn Lys Asp Gln Gln Ser Ala 145 150 155 160 Phe Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn 165 170 175 Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val 180 185 190 Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp 195 200 205 Asn Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly 210 215 220 Ser Gly Gly Ser Glu Val Thr Ile Lys Val Asn Leu Ile Phe Ala Asp 225 230 235 240 Gly Lys Ile Gln Thr Ala Glu Phe Lys Gly Thr Phe Glu Glu Ala Thr 245 250 255 Ala Glu Ala Tyr Arg Tyr Ala Ala Leu Leu Ala Lys Val Asn Gly Glu 260 265 270 Tyr Thr Ala Asp Leu Glu Asp Gly Gly Asn His Met Asn Ile Lys Phe 275 280 285 Ala Gly Gly Gly Gly Ser Gly Thr Met Val Ser Lys Gly Glu Glu Leu 290 295 300 Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn 305 310 315 320 Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr 325 330 335 Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val 340 345 350 Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe 355 360 365 Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala 370 375 380 Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp 385 390 395 400 Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu 405 410 415 Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn 420 425 430 Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr 435 440 445 Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile 450 455 460 Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln 465 470 475 480 Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His 485 490 495 Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg 500 505 510 Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu 515 520 525 Gly Met Asp Glu Leu Tyr Lys 530 535 <210> 107<211> 249<212> PRT<213> 人工序列<220> <223> Synthesized<400> 107Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro Leu 85 90 95 Ala Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr Phe Met Tyr Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Trp Ile Asp Pro 165 170 175 Glu Asn Gly Asn Thr Ile Tyr Asp Pro Lys Phe Gln Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg Gly Tyr Tyr 210 215 220 Gly Ser Arg Val Leu Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 225 230 235 240 Thr Val Ser Ser Ala Ser Ala Ala Ala 245

Claims (29)

  1. 一種高通量篩選用於一免疫偶聯物之功能性片段的方法，其中該免疫偶聯物標的一蛋白抗原，包含以下步驟： (a) 提供一噬菌體表現的合成單鏈變異片段(synthetic single-chain variable fragment，scFv)抗體庫，該抗體庫包含複數個噬菌體表現的scFv，其中該些噬菌體表現的scFv之每一者的VH結構域對蛋白A有一結合親和力，且該些噬菌體表現的scFv之每一者的VL結構域對蛋白L有一結合親和力； (b) 從步驟(a)之該噬菌體表現的合成scFv抗體庫中篩選表現對該蛋白抗原專一結合的複數個噬菌體； (c) 從步驟(b)選出的該些噬菌體分別製備複數個分泌之scFv； (d) 藉由一適體-藥物偶聯物分別與步驟(c)製備的該些分泌之scFv混合，以得到複數個scFv-適體-藥物複合物，其中該適體-藥物偶聯物包含一藥物及一適體，該適體包含至少一AL蛋白模組，其包含位於N端的一蛋白A片段以及位於C端的一蛋白L片段； (e)將步驟(d)的條件下所得之該些scFv-適體-藥物複合物，分別與複數個呈現該蛋白抗原的細胞一起培養； (f) 決定該些scFv-適體-藥物複合物對步驟(e)中培養的呈現該蛋白抗原的該些細胞之的毒殺效果；以及 (g) 基於步驟(f)的結果，選擇用於該免疫偶聯物的該功能性片段，其中選擇該些scFv-適體-藥物複合物中相較於其他scFv-適體-藥物複合物展現較佳效果的一或多個scFv-適體-藥物複合物的scFv作為用於該免疫偶聯物的該功能性片段。
  2. 如請求項1所述之方法，其中該蛋白抗原為人類表皮生長因子受體2 (human epidermal growth factor receptor 2，HER2)、麥芽糖結合蛋白、牛血清白蛋白、人類血清白蛋白、溶菌酶、介白素-1β (interleukin-1 beta，IL-1β)、流行性感冒病毒的血球凝集素、血管內皮生長因子 (vascular endothelial growth factor，VEGF)、表皮生長因子受體1 (epidermal growth factor receptor 1，EGFR1)、表皮生長因子受體3 (EGFR3)、升糖素受體、程序性死亡配體1 (programmed death-ligand 1，PD-L1)、唾液酸结合免疫球蛋白樣凝集素3 (sialic acid binding Ig-like lectin 3，SIGLEC 3)或利妥昔單抗(rituximab)。
  3. 如請求項2所述之方法，其中該蛋白抗原為HER2。
  4. 如請求項1所述之方法，其中該藥物包含一藥物部分，其係選自以下所組成的群組：免疫毒素、免疫微脂體及細胞毒性藥物。
  5. 如請求項4所述之方法，其中該藥物更包含一內質網(endoplasmic reticulum，ER)駐留部分，與該藥物部分連接。
  6. 如請求項5所述之方法，其中該ER駐留部分包含KDEL序列。
  7. 如請求項所述之方法4，其中該藥物部分為該免疫毒素。
  8. 如請求項7所述之方法，其中該免疫毒素為一外毒素。
  9. 如請求項8所述之方法，其中該外毒素為假單胞菌屬外毒素A (Pseudomonas exotoxin A，PE A)或衍生自PE A。
  10. 如請求項9所述之方法，其中該免疫毒素包含一截短之PE A次單元毒素。
  11. 如請求項10所述之方法，其中該藥物更包含一ER駐留部分，與該PE A次單元毒素的C端連接。
  12. 如請求項11所述之方法，其中該ER駐留部分包含KDEL序列。
  13. 如請求項1所述之方法，其中該AL蛋白模組更包含一第一多肽連接物，用以連接該蛋白A片段與該蛋白L片段。
  14. 如請求項1所述之方法，其中該適體-藥物偶聯物更包含一第二多肽連接物，用以將該藥物連接至該適體C端。
  15. 如請求項1所述之方法，其中該適體包含兩個AL蛋白模組，且更包含一第三多肽連接物，用以連接該兩個AL蛋白模組。
  16. 如請求項1所述之方法，其中步驟(f)中測量步驟(e)中培養之該些呈現該蛋白抗原的細胞的細胞存活率。
  17. 一適體-藥物偶聯物，包含： 一適體，其包含至少一AL蛋白模組，其中每一AL蛋白模組包含位於N端的一蛋白A片段以及位於C端的一蛋白L片段，以及一第一多肽連接物，用以連接該蛋白A片段與該蛋白L片段； 一藥物；以及 一第二多肽連接物，用以將該藥物連接至該適體的C端。
  18. 如請求項17所述之適體-藥物偶聯物，其中該藥物包含一藥物部分，其係選自以下所組成的群組：免疫毒素、免疫微脂體及細胞毒性藥物。
  19. 如請求項18所述之適體-藥物偶聯物，其中該藥物更包含一ER駐留部分，與該藥物部分連接。
  20. 如請求項19所述之適體-藥物偶聯物，其中該ER駐留部分包含KDEL序列。
  21. 如請求項18所述之適體-藥物偶聯物，其中該藥物部分為該免疫毒素。
  22. 如請求項21所述之適體-藥物偶聯物，其中該免疫毒素為一外毒素。
  23. 如請求項22所述之適體-藥物偶聯物，其中該外毒素為假單胞菌屬外毒素A (Pseudomonas exotoxin A，PE A)或衍生自PE A。
  24. 如請求項23所述之適體-藥物偶聯物，其中該免疫毒素包含一截短之PE A次單元毒素。
  25. 如請求項24所述之適體-藥物偶聯物，其中該藥物更包含一ER駐留部分，與該PE A次單元毒素的C端連接。
  26. 如請求項25所述之適體-藥物偶聯物，其中該ER駐留部分包含KDEL序列。
  27. 如請求項17所述之適體-藥物偶聯物，其中該適體包含兩個AL蛋白模組，且更包含一第三多肽連接物，用以連接該兩個AL蛋白模組。
  28. 一種標的一蛋白抗原的免疫偶聯物，包含：一藥物以及一標的模組，其中該標的模組包含一功能性片段，該功能性片段是利用根據請求項1至16任一項所述之方法所選擇。
  29. 如請求項28所述的免疫偶聯物，其中該藥物包含一藥物部分，其係選自以下所組成的群組：免疫毒素、免疫微脂體及細胞毒性藥物。
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