CN114773467B - 抗SIRPα抗体或其抗原结合片段及应用 - Google Patents

抗SIRPα抗体或其抗原结合片段及应用 Download PDF

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CN114773467B
CN114773467B CN202110911478.XA CN202110911478A CN114773467B CN 114773467 B CN114773467 B CN 114773467B CN 202110911478 A CN202110911478 A CN 202110911478A CN 114773467 B CN114773467 B CN 114773467B
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屈向东
潘琴
郑翰
都业杰
陈子硕
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Abstract

涉及抗SIRPα抗体或其抗原结合片段及应用。通过免疫小鼠获得抗人SIRPα单克隆抗体，进一步获得嵌合抗体和人源化抗体。提供的抗SIRPα抗体或其抗原结合片段，能够结合人SIRPα蛋白，阻断CD47‑SIRPα信号通路，具有增强吞噬等免疫调控作用。还提供了抗SIRPα抗体或其抗原结合片段在制备用于抑制或治疗疾病的药物中的用途。

Description

抗SIRPα抗体或其抗原结合片段及应用
技术领域
本发明涉及生物医药技术领域，具体涉及抗SIRPα抗体或其抗原结合片段及应用。
背景技术
据AACR预计，2018年美国新确诊恶性肿瘤病例将达到180万例，由于癌症死亡人数将超过60万，仅次于心脏病。据中国国家癌症中心公布的数据，2014年中国新确诊癌症共计380万例，而当年由于癌症死亡人数达到230万人。细胞增殖失控是导致恶性肿瘤形成的关键因素，肿瘤细胞为了加速自身的生长，保护自己免于受到机体免疫系统的攻击，在漫长的进化过程中获得了一系列途径逃逸宿主对肿瘤细胞的免疫监控。根据Schreiber提出的癌症免疫编辑理论，肿瘤细胞与宿主免疫系统的关系可以分为3种不同阶段：清除(elimination)、平衡(equilibrium)以及逃逸(escape)。在“清除”状态下，新生肿瘤细胞具有较强抗原性，易被免疫系统识别并清除；而逃过清除作用存活下来的肿瘤细胞则会与免疫系统进入“平衡”状态，表现为宿主荷瘤生存的状态。但肿瘤细胞在宿主免疫系统的选择压力下的基因突变积累到一定程度时就会打破平衡，进入到最终的“逃逸”状态。在这个阶段的肿瘤细胞可以产生一系列恶性表型，关闭抑癌响应机制，诱导正常的免疫反应，从而被识别为正常细胞。肿瘤细胞中的细胞凋亡信号通路也发生变化，使免疫细胞诱导的肿瘤细胞凋亡机制失效。此外，由于肿瘤细胞不受抑制的快速生长，形成的组织结构会产生抑制免疫细胞的微环境。在肿瘤微环境中，肿瘤细胞会释放具有免疫抑制功能的分子，如VEGF、TGF-β、IL-10等，抑制骨髓树突状细胞(dentritic cell)的激活和分化，从而抑制适应性免疫系统。同时能在外周血和淋巴结诱导产生表达CTLA-4的调节性T细胞(Treg)，对其他免疫细胞产生抑制作用，导致免疫系统产生对肿瘤的免疫耐受。
肿瘤微环境中的肿瘤相关巨噬细胞(TAM，tumor associated macrophage)的吞噬作用受到抑制，其原因在于几乎所有肿瘤细胞表面都高表达CD47蛋白，能够与骨髓细胞表面的信号调节蛋白α(signal regulatory proteinα,SIRPα)结合向机体发出“don’t eatme”或“self”的信号，从而抑制吞噬作用。CD47又名整合素相关蛋白(IAP，intergin-associated protein)，是一种广泛表达的跨膜糖蛋白，属于免疫球蛋白(Ig)超家族。CD47分子量为50kD，结构包含了大量的糖基化的N-末端IgV可变结构域，5个高度疏水的跨膜结构域和一个短的C-末端胞质尾区，C-末端胞质尾区的4种选择性拼接形式决定了CD47在不同组织的表达。与之相对应的SIRPα又被称为SHPS-1、BIT或CD172a蛋白，它是一种跨膜蛋白，主要在骨髓细胞(myeloid cell)上表达，包括巨噬细胞、骨髓树突状细胞、粒细胞、肥大细胞及其前体细胞。SIRPα由胞外的3个Ig样结构域和胞质内的4个酪氨酸残基组成，该4个酪氨酸残基被推测认为是磷酸化位点。当发生磷酸化后，SIRPα通过结合SHP-1/2蛋白的SH2结构域并使之激活，从而激活下游信号通路。SHP-1和SHP-2蛋白的表达具有组织特异性，因此SIRPα是一种停靠蛋白，通过响应胞外刺激招募及激活下游蛋白磷酸酶。Oldenborg首先报道了成熟血红细胞(RBC，red blood cell)通过CD47与脾脏巨噬细胞SIRPα结合保护自身免遭后者的清除。随后研究人员发现RBC上的也能与单核细胞SIRPα结合从而抑制依赖Fcγ受体的吞噬作用，这是通过对吞噬作用中的关键分子肌球蛋白-IIA(myosin-IIA)去磷酸化作用实现的。临床上多种实体瘤和血液恶性肿瘤中都发现了CD47高表达的现象，包括急性髓样白血病(AML)、急性淋巴白血病(ALL)、慢性髓样白血病(CML)、非霍奇金淋巴瘤(NHL)、乳腺癌、膀胱癌、卵巢癌、结肠癌等，其本质是肿瘤细胞通过上文所述调节机制逃避巨噬细胞的细胞清除作用。CD47还通过与其他受体结合或通过其胞内细胞质区域的信号传导对其他生物过程产生影响。CD47与血小板反应蛋白-1(TSP-1，Thrombospondin-1)或血管内皮生长因子受体2(VEGFR-2)的相互作用抑制血管生成，从而限制肿瘤生长。
已有多项研究证明CD47抗体或者SIRPα-Fc重组蛋白能够在不同的小鼠PDX模型中发挥作用，而与阿糖胞苷、多柔比星、紫杉醇、顺铂等常见化疗药物或利妥昔单抗、阿仑单抗、西妥昔单抗、曲妥珠单抗联合使用的报道中也具有良好的效果。Sockolosky等则首次报道了联合使用CD47纳米抗体和PD-L1能够在黑色素瘤B16F10细胞的Syngeneic模型中表现出良好的抗肿瘤作用。近年来也有数个作用于CD47-SIRPα通路的抗体进入临床I期，包括Hu5F9-G4(恶性实体瘤：NCT02216409，恶性血液瘤：NCT02678338、NCT03248479，结肠癌：NCT02953782，B细胞非霍奇金淋巴瘤：NCT02953509)，CC-90002(AML和骨髓增生异常综合症：NCT02641002，晚期实体瘤和血液瘤：NCT02367196)，SRF231(晚期实体瘤和血液瘤：NCT03512340)，SIRPα-Fc重组蛋白TTI-662(实体瘤：NCT02890368，恶性血液瘤和实体瘤：NCT02663518，骨髓瘤和淋巴瘤：NCT03530683)，ALX148(晚期实体瘤和淋巴瘤：NCT03013218)。
CD47本身的生物学功能决定了CD47治疗性抗体和SIRPα-Fc重组蛋白可能具有血液系统毒性或者有引发贫血的风险，这在CD47基因敲除NOD鼠和使用CD47抗体进行治疗的小鼠模型中均有报道。此外，内皮细胞CD47被报道能够通过细胞黏附作用与SIRPγ相互作用促进T细胞的跨内皮迁移(transendothelial migration)，而SIRPγ主要表达于T细胞而非骨髓细胞。
因此，使用SIRPα抗体作为阻断CD47-SIRPα信号通路的更优选择。Stanford大学Weissman研究组证明其筛选出的人源化SIRPα抗体KWAR23与利妥昔单抗联用能够在敲入人源SIRPα基因的SRG鼠(Rag2-/-Il2rγ-/-)中有效抑制Burkitt淋巴瘤生长，但KWAR23单独使用则无明显药效。
发明内容
本发明的目的是提供一种抗SIRPα抗体或其抗原结合片段及应用，抗SIRPα抗体或其抗原结合片段能够结合人SIRPα蛋白，阻断CD47-SIRPα信号通路。
为了达到上述目的，本发明提供了一种抗SIRPα抗体或其抗原结合片段，其包含：重链可变区和轻链可变区；所述重链可变区包含：氨基酸序列分别如SEQ ID NO:3、4、5所示的VHCDR1、VHCDR2和VHCDR3；所述轻链可变区包含：氨基酸序列分别如以下任意一组序列所示的VLCDR1、VLCDR2和VLCDR3；
(1)SEQ ID NO:37、38、9；
(2)SEQ ID NO:39、38、9；
(3)SEQ ID NO:7、40、9；
(4)SEQ ID NO:7、8、41；
(5)SEQ ID NO:7、8、42；
(6)SEQ ID NO:7、8、43；
(7)SEQ ID NO:37、38、41；
(8)SEQ ID NO:44、38、41；
(9)SEQ ID NO:7、8、9。
VLCDR的序列可参阅下表：
可选的，所述可变区还包含：鼠源或人源FR区。
可选的，所述FR区的序列为鼠源；所述重链可变区的序列如SEQ ID NO:2所示或与其具有至少85％序列同一性，所述轻链可变区的序列如SEQ ID NO:6所示或与其具有至少85％序列同一性。
可选的，所述人源FR区包括：重链FR区序列；所述重链FR区序列来源于人种系重链IGHV1-18及IGHJ2*01的组合序列，包括人种系重链IGHV1-18的FR1、FR2、FR3区和IGHJ2*01的FR4区。
可选的，所述人源FR区包括：轻链FR区序列；所述轻链FR区序列来源于人种系轻链IGKV4-1及IGKJ2*01的组合序列，包括人种系轻链IGKV4-1的FR1、FR2、FR3区和IGKJ2*01的FR4区。
可选的，所述重链可变区的FR区序列来源于人种系，所述重链可变区的序列如SEQID NO:17所示或与其具有至少85％序列同一性。
可选的，所述轻链可变区的FR区序列来源于人种系，所述轻链可变区的序列选自SEQ ID NO:18、19、20、21、22、23、24、25中任意一项，或与其具有至少85％序列同一性。
可选的，所述抗SIRPα抗体或其抗原结合片段还包含：选自人源IgG1、IgG2、IgG3、或IgG4或其变体的重链恒定区；以及选自人源κ、λ链或其变体的轻链恒定区。
可选的，所述重链恒定区包含：Fc片段或其变体。
可选的，所述Fc片段的变体来源于IgG1，根据EU计数，包括突变位点：L234A、L235A、K338A。
可选的，所述抗SIRPα抗体或其抗原结合片段的重链序列如SEQ ID NO:26所示，或与其具有至少85％序列同一性。
可选的，所述的抗SIRPα抗体或其抗原结合片段为单克隆抗体、双特异抗体、或多特异抗体，或者，所述的抗体或其抗原结合片段用于制备抗体药物偶联物。
可选的，所述的抗SIRPα抗体或其抗原结合片段的结构形式为为Fab、F(ab’)2、Fv、或ScFv。
本发明还提供了一种药物组合物，其含有上述的抗SIRPα抗体或其抗原结合片段，以及一种或多种药学上可接受的载体、稀释剂或赋形剂。
本发明还提供了一种核酸分子，其编码上述的抗SIRPα抗体或其抗原结合片段。
本发明还提供了一种载体，其包含上述的核酸分子。
本发明还提供了一种采用上述的载体转化得到的宿主细胞。
本发明还提供了上述的抗SIRPα抗体或其抗原结合片段在制备用于抑制或治疗疾病、病症或状况的药物中的用途。
可选的，所述的药物由所述的抗SIRPα抗体或其抗原结合片段与一种或多种其他癌症治疗剂联合制备。
可选的，所述疾病、病症或状况包括：癌症、实体瘤、慢性感染、炎性疾病、多发性硬化、自身免疫性疾病、神经系统疾病、脑损伤、神经损伤、红细胞增多症、血色素沉着病、创伤、感染性休克、纤维化、动脉粥样硬化、肥胖症、II型糖尿病、移植功能障碍或关节炎。
可选的，所述癌症选自肛门癌、阑尾癌、星形细胞瘤、基底细胞癌、胆囊癌、胃癌、肺癌、支气管癌、骨癌、肝胆管癌、胰腺癌、乳腺癌、肝癌、卵巢癌、睾丸癌、肾癌、肾盂和输尿管癌、唾液腺癌、小肠癌、尿道癌、膀胱癌、头颈癌、脊柱癌、脑癌、宫颈癌、子宫癌、子宫内膜癌、结肠癌、结直肠癌、直肠癌、食道癌、胃肠道癌、皮肤癌、前列腺癌、垂体癌、阴道癌、甲状腺癌、喉癌、胶质母细胞瘤、黑素瘤、骨髓增生异常综合征、肉瘤、畸胎瘤、慢性淋巴细胞白血病(CLL)、慢性髓性白血病(CML)、急性淋巴细胞白血病(ALL)、急性髓性白血病(AML)、霍奇金淋巴瘤、非霍奇金淋巴瘤、多发性骨髓瘤、T或B细胞淋巴瘤、胃肠道间质瘤、软组织肿瘤、肝细胞癌或腺癌。
本发明还提供了上述的抗SIRPα抗体或其抗原结合片段在制备阻断SIRPα和CD47结合的制剂中的应用，所述制剂包括检测剂。
相对于现有技术，本发明具有以下有益效果：
(1)本发明提供的抗SIRPα抗体或其抗原结合片段，能够结合人SIRPα蛋白，阻断CD47-SIRPα信号通路，有望用于肿瘤治疗或制备肿瘤抗体药物。
(2)本发明提供的抗SIRPα抗体或其抗原结合片段，能够结合人SIRPα蛋白的所有亚型，有利于临床开发。
附图说明
图1至图6为Binding-ELISA检测结果。
图7为Blocking-ELISA检测结果。
图8为FACS检测SIRPα抗体结合天然表达人SIRPa的人肾透明细胞腺癌细胞786-O细胞的检测结果。
图9至图11为抗SIRPα抗体体外功能实验ADCP结果。
图12为各组肿瘤成像信号值反应的肿瘤生长曲线及D18成像信号强度结果图。
图13为各组生存曲线。
图14至图23为ELISA检测本发明的抗体CHO71及对照抗体18D5、KWAR23结合SIRPαV1/V2/V3/V4/V5/V6/V7/V8/V9/V10亚型的结果图。
图24为已知的人类SIRP alpha结合域等位基因的氨基酸序列比对图。
具体实施方式
以下结合附图和实施例对本发明的技术方案做进一步的说明。
本实验中未注明具体条件的实验方法，通常是按照常规条件，或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
术语：
“抗体”(antibody，Ab)指包含至少一个抗原结合位点并能特异性结合抗原的免疫球蛋白分子(immunoglobulin，Ig)。
“抗原”是在机体内能诱发免疫应答且与抗体特异性结合的物质。抗体与抗原的结合依靠二者间形成的相互作用来介导，包括氢键、范德华力、离子键以及疏水键。抗原表面与抗体结合的区域为“抗原决定簇”或“表位”，一般来说，每个抗原有多个决定簇。
本发明所提及的术语“抗体”以其最广泛的含义理解，并包含单克隆抗体(包括全长单克隆抗体)、多克隆抗体、抗体片段、包含至少两个不同的抗原结合结构域的多特异性抗体(例如，双特异性抗体)。抗体还包括鼠源抗体、人源化抗体、嵌合抗体、人抗体以及其它来源的抗体。本发明的抗体可以来源于任何动物，包括但不限于人、非人灵长类动物、小鼠、大鼠、牛、马、鸡、骆驼、美洲驼羊(Llama)、羊驼(Alpaca)、大羊驼(Guanaco)、骆马(Vicunas)的免疫球蛋白分子等。抗体可以含有另外的改变，如非天然氨基酸，Fc效应子功能突变和糖基化位点突变。抗体还包括翻译后修饰的抗体、包含抗体的抗原决定簇的融合蛋白，以及包含对抗原识别位点的任何其它修饰的免疫球蛋白分子，只要这些抗体展现出所期望的生物活性。
抗体的基本结构是由两条完全相同的重链(heavy chain，H)和两条完全相同的轻链(light chain，L)通过二硫键连接的呈Y形的单体。每条链分别由2～5个约含110个氨基酸，序列相似但功能不同的结构域(又称功能区)组成。抗体分子中轻链和重链靠近N端的氨基酸序列变化较大，形成的结构域称为可变区(variable region，V区)；靠近C端的氨基酸序列相对恒定的区域称为恒定区(constant region，C区)。
重链和轻链的V区分别称为VH和VL，VH和VL各有3个区域的氨基酸组成和排列顺序高度可变，称为高变区(hypervariable region，HVR)；该区域形成与抗原表位互补的空间构象，又被称为互补决定区(complementarity determining region，CDR)。VH的3个CDR分别用VHCDR1、VHCDR2、VHCDR3表示，VL的3个CDR分别用VLCDR1、VLCDR2、VLCDR3表示。VH和VL共6个CDR共同组成抗原结合部位(antigen-binding site)。CDR区氨基酸的多样性是抗体与数量庞大的不同抗原特异性结合的分子基础。V区中CDR之外的氨基酸组成和排列顺序相对变化不大，称为骨架区或框架区(framework region，FR)。VH和VL各有4个骨架区，分别用FR1、FR2、FR3、FR4表示。每个VH和VL有三个CDR和四个FR组成，从氨基端到羧基端排列顺序为：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。
根据抗体重链恒定区的氨基酸序列，可以将人免疫球蛋白分为5类：IgM、IgG、IgA、IgD、IgE。其还可以进一步分成不同的亚类(同种型)，如人IgG可以分为IgG1、IgG2、IgG3、IgG4；IgA可分为IgA1和IgA2。IgM、IgD、IgE尚未发现有亚类。根据轻链氨基酸序列，可将轻链分类为κ链和λ链。本发明的抗体可以是任何种类(如IgM、IgG、IgA、IgD、IgE)或亚类(如IgG1、IgG2、IgG3、IgG4、IgA1、IgA2)。
重链和轻链的恒定区分别称为CH和CL。IgG、IgA、IgD的重链恒定区有CH1、CH2、CH3三个结构域，IgM、IgE的重链恒定区有CH1、CH2、CH3、CH4四个结构域。
CH1和CH2之间为铰链区(hinge region)，含有丰富的脯氨酸，因此易伸展弯曲，能改变Y形两个臂之间的距离，有利于两臂同时结合抗原表位。
“抗原结合片段”指具有抗原结合活性的Fab片段、F(ab’)2片段、Fv片段、ScFv片段等。“Fab片段”(fragment of antigen binding,Fab)，意指由VL、VH、CL和CH1结构域组成的抗体片段，与单个抗原表位结合(单价)。本领域技术人员可知，木瓜蛋白酶水解IgG形成2个相同的Fab段和1个Fc段；胃蛋白酶水解IgG形成1个F(ab’)2段和若干多肽碎片(pFc’)。若F(ab’)2重链间二硫键断裂，可形成2个Fab’片段，后者可进一步被酶解成Fv片段。Fv片段含有抗体重链可变区和轻链可变区，但没有恒定区。单链可变片段scFv(single chainantibody fragment)，或称单链抗体，由抗体重链可变区和轻链可变区通过连接片段(linker)连接而成。
术语“Fc”、“Fc段”或“Fc片段”是指可结晶片段(fragment crystallizable)，无抗原结合活性，是抗体与效应分子或细胞表面Fc受体(FcR)相互作用的部位。Fc片段包含抗体除重链恒定区CH1之外的恒定区多肽。Fc片段与表面具有相应Fc受体的细胞结合，产生不同的生物学作用。在ADCC效应中(抗体依赖的细胞介导的细胞毒性作用，antibody-dependentcell-mediated cytotoxicity)，抗体的Fab段结合病毒感染的细胞或肿瘤细胞的抗原表位，其Fc段与杀伤细胞(NK细胞、巨噬细胞等)表面的FcR结合，介导杀伤细胞直接杀伤靶细胞。ADCP为抗体依赖性细胞吞噬作用(antibody-dependent cellular phagocytosis)，ADCP的机制是抗体作用的靶细胞激活巨噬细胞表面上的FcγR机制，诱导吞噬，使靶细胞内化和被吞噬体酸化降解。在某些特定情形下消除抗体Fc功能更有益。这些情形包括抗体用作：(1)受体激动剂，诱导细胞信号；(2)受体拮抗剂，阻断受体和配体的结合，抑制信号；或，(3)作为药物载体递送药物到表达相应抗原的靶细胞。如果保持着Fc功能将导致抗体药物误伤表达相应受体的细胞，以及造成抗体偶联药物脱靶情况下误伤重要的免疫细胞。
Fc变体或突变的组合不限于以下形式(根据EU计数)。
本发明所述抗体或抗原结合片段的CDR氨基酸残基在数量和位置上符合已知的Kabat编号规则。
目前鼠源抗体是抗体药物的一大来源。由于鼠源抗体的免疫原性，一般会对其作人源化处理。以下实施例中提供了鼠源抗体、嵌合抗体、人源化抗体。“嵌合抗体”是将鼠源抗体的可变区和人抗体的恒定区融合而成的抗体，可以减轻鼠源抗体诱发的免疫应答反应。人抗体的恒定区可选自人源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区，以及选自人源κ、λ链或其变体的轻链恒定区。“人源化抗体”是指将鼠源抗体的CDR序列移植到人的抗体可变区框架得到的抗体，可以克服嵌合抗体由于携带大量小鼠蛋白成分，从而诱导强烈反应。此类框架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。为避免免疫原性下降的同时引起的活性下降，可对人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。
理论上，抗体亲和力的提高有助于改善抗体的特异性和效力，有助于减少用药剂量，降低毒副作用等。虽然实际的研究工作证明亲和力的提高与抗体效价的提高并不总是线性的关系，尤其在实体瘤的治疗上，但很多情况下，这个线性关系是明显存在的。本发明的人源化抗体也包括进一步由噬菌体展示对CDR进行亲和力成熟后的人源化抗体。体外的抗体亲和力成熟的理论基础是模仿了体内抗体亲和力的过程。通过构建随机突变库，模拟B细胞在体内的高频突变，可以筛选到高亲和的抗体。
本发明提供的药物可含有“治疗有效量”的抗体或抗原结合片段。“治疗有效量”指有效预防或缓解具体疾病的治疗剂的量，可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在不同患者体内产生需要疗效的能力。
“序列同一性”指两个多核苷酸序列之间或两个多肽之间的序列相似性，是两个多核苷酸或两个多肽之间具有相同碱基或氨基酸的程度。本发明所述的“具有至少85％序列同一性”是指至少达到85％、90％、95％、97％、或99％同一性。
Antibody-Drug Conjugates(ADCs，抗体药物偶联物)，是指与一个或多个化学药物(任选地可以是治疗剂或细胞毒性剂)连接的结合蛋白。可通过永久的或者不稳定的化学连接体(linker)将细胞毒小分子(cytotoxin)和抗体连接，获得抗体药物偶联物。ADC可选择和持续性地将细胞毒性药物递送至肿瘤。
编码SIRPα的基因是多态性基因(polymorphic gene)，已知人类群体中有10种SIRPα变体。Katsuto Takenaka等对来自人类HapMap基因组计划的37个不相关的正常高加索人、非洲人、中国人和日本人的IgV编码SIRP alpha域进行了测序，发现了10个不同的SIRP alpha IgV编码等位基因(Polymorphismin Sirpa modulates engraftment ofhuman hematopoietic stem cells，NATURE IMMUNOLOGY VOLUME 8NUMBER 12DECEMBER2007)。10种SIRPα变体分别为SIRPαV1/V2/V3/V4/V5/V6/V7/V8/V9/V10亚型。虽然SIRPalpha具有高度多态性，但是ChiaChiM.Ho等对已知的人类SIRPalpha等位基因的氨基酸序列比对显示，SIRP alpha与CD47结合界面只有两个独特的序列，分别为等位基因V1(a2d1)型和V2(a1d1)型。(“Velcro”Engineering of High Affinity CD47 Ectodomain asSignal Regulatory Protein(SIRP alpha)Antagonists That Enhance Antibody-dependent Cellular Phagocytosis，JOURNAL OF BIOLOGICAL CHEMISTRY，VOLUME 290·NUMBER 20·MAY 15,2015)。
如图24所示，对已知的人类SIRP alpha结合域等位基因的氨基酸序列比对显示，在CD47接触界面只有两个变异：a1d1和a2d1。图24的第一行文本为人类最显著的SIRPalpha等位基因V1(a2d1)的氨基酸序列，图1的第二行文本为人类最显著的SIRP等位基因V2(a1d1)的氨基酸序列。黑盒子表示与CD47相互作用的残基，而阴影表示与V1序列不同的残基。Janet Sim等对2535个个体的SIRPα序列和510个样本的Sanger测序鉴定出两个SIRPα变体v1和v2，代表三个等位基因组：纯合子v1/v1,纯合子v2/v2,杂合子v1/v2。通过对在不同的群体和不相关的亚群体中确定了SIRPαv1和v2等位基因群的分布和频率。其中欧洲(EUR)、美国(AMR)、东亚(EAS)、非洲(AFR)和南亚(SAS)5个超级群体的v1/v2杂合子分布相似，分布范围为42.0％～47.2％。东亚人口v2/v2数量明显高于v1/v1，发生频率分别为42.3％和13.3％，在非洲、欧洲人、美国人、和南亚人群v1/v1数量高于v2/v2，v1和v2的发生频率范围分别为30.3-49.1％和8.9-24.2％(参考文献详见MABS，2019,VOL.11,NO.6,1036¨C1052，https://doi.org/10.1080/19420862.2019.1624123)。Aduro Biotech也研究了东亚人群v2/v2纯合子发生频率为41.3％，v1/v1纯合子发生频率为34.6％，同样证明东亚人群中有41.3％的人是V2/V2纯合子(参考文献详见Voets et al.Journal forImmunoTherapy of Cancer(2019)7:340)。
基于SIRPα多态分析的结果，抗SIRPα抗体能够同时结合SIRPαv1型和SIRPa v2型基因，这是至关重要的，以便于临床开发。
实施例1：获得抗SIRPα小鼠抗体
(1)小鼠免疫：
抗人SIRPα单克隆抗体通过免疫小鼠产生。实验用Balb/c白小鼠，雌性，6周龄。饲养环境：SPF级。小鼠购进后，实验室环境饲养1周，12/12小时光/暗周期调节，温度20-25℃；湿度40-60％。免疫Balb/c小鼠，首次用弗式完全佐剂(CFA)免疫重组蛋白QP009(SIRPα)50μg/只小鼠两周后，此后用QP009(SIRPα)加弗式不完全佐剂(IFA)或者QP009(SIRPα)加铝盐Alum+CpG ODN 1826交替免疫，25μg/只小鼠，一周一次。
QP009(SIRPα)具有如下所示氨基酸序列(SEQ ID NO:1)：
EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDYKDDDDKHHHHHH。序列参考自UNIPROT编号P78324(31-149)(SIRPA-Tyrosine-protein phosphatase non-receptortype substrate 1 precursor-Homo sapiens(Human)-SIRPA gene&protein(uniprot.org))。
(2)细胞融合：
选择血清中抗体滴度高的小鼠进行脾细胞融合。融合前72小时，腹腔注射冲刺免疫所选小鼠。采用优化的PEG介导的融合步骤将脾淋巴细胞与骨髓瘤Sp2/0细胞进行融合得到杂交瘤细胞。融合好的杂交瘤细胞用HAT完全培养基(含20％FBS、1×HAT和1×OPI的IMDM培养基)重悬，分装于96孔细胞培养板中(1×105个/150μl/孔)，37℃、5％CO2培养。融合后的第5天加入20％FBS的IMDM培养基(含2×HAT和1×OPI)，50μl/孔，37℃，5％CO2培养。融合后第7天～8天，根据细胞生长密度，全换液，培养基为HT完全培养基(含20％FBS、1×HT和1×OPI的IMDM培养基)，250μl/孔，37℃、5％CO2培养。
(3)杂交瘤细胞筛选：
根据细胞生长密度，融合后第10-14天，进行ELISA检测筛选杂交瘤上清中抗SIRPα抗体。取杂交瘤融合孔上清，通过ELISA进行96孔板整板初筛，检测上清中抗SIRPα抗体能阻断SIRPα/CD47的结合，即为初筛阳性孔。再取初筛阳性孔上清ELISA检测与QP009(SIRPα)的结合，选取结合SIRPα且能阻断SIRPα/CD47结合均为阳性的克隆，即为抗SIRPα抗体阳性克隆孔。将阳性克隆扩培并及时转移至24/6孔板中，通过ELISA再次检测细胞培养上清结合SIRPα且能阻断SIRPα/CD47的结合均为阳性的克隆孔，即为抗SIRPα抗体阳性克隆孔。将阳性克隆进行2-3轮有限稀释至单细胞克隆，冻存阳性单细胞株，获得单细胞克隆71C10。
(4)杂交瘤单抗测序获得抗体序列：
取杂交瘤阳性单克隆细胞株71C10，提取mRNA，mRNA反转录成cDNA，以cDNA为模板PCR扩增，挑选PCR阳性克隆送测序，通过序列分析得到单克隆抗体轻重链可变区序列。
71C10的重链可变区序列为SEQ ID NO:2，具体如下：
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。加粗并下划线部分分别为VHCDR1(SEQ ID NO:3)、VHCDR2(SEQ ID NO:4)、VHCDR3(SEQ ID NO:5)。
71C10的轻链可变区序列为SEQ ID NO:6，具体如下所示：
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。加粗并下划线部分分别为VLCDR1(SEQ ID NO:7)、VLCDR2(SEQ ID NO:8)、VLCDR3(SEQ ID NO:9)。
实施例2：抗SIRPα嵌合抗体SPR检测亲和力
(1)将单克隆细胞株71C10的鼠源可变区序列与人恒定区基因融合，获得嵌合抗体分子。抗体轻链采用kappa轻链恒定区CL。同时设计不同的抗原序列，用于抗体分子的性能测试。抗原及嵌合抗体分子克隆设计见表1和表2。
表1嵌合抗体分子克隆设计
注：
蛋白编号为QP026027、QP026249的抗体作为对照抗体，其均采用已知抗SIRPα抗体KWAR23的可变区序列，区别在于恒定区不同。QP163164、QP163245均采用单克隆细胞株71C10的可变区，区别在于恒定区不同。上述序列编号所示的序列分别给出各个抗体分子的重、轻链序列。
pQD为带信号肽及恒定区基因(CH1-FC/CL)片段的载体名称，其中，pQDH用于重链可变区的连接和表达，带有信号肽及恒定区基因(CH1-FC)片段；pQDK用于轻链可变区的连接和表达，带有信号肽及恒定区基因(CL)片段。“H”表示重链，“L”表示轻链。“(IgG4)”表示重链采用人IgG4的恒定区。如果未标注有“(IgG4)”，则默认采用人IgG1的恒定区。180122VH代表来源于单克隆细胞株71C10的重链可变区，180122VL代表来源于单克隆细胞株71C10的轻链可变区。
示例性的，“pQDH-KWAR23-H”表示对照序列KWAR23融合到pQDH载体上，pQDH带有信号肽及恒定区基因(CH1-FC)片段，采用人IgG1的恒定区。“pQDH-180122VH”表示重链可变区序列180122VH融合到pQDH载体上，采用人IgG1的恒定区。上述序列编号所示的序列具体如下：
>QD026(SEQ ID NO:10)
MDMRVPAQLLGLLLLWFPGSRCQIVLTQSPAIMSASPGEKVTLTCSASSSVSSSYLYWYQQKPGSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFCHQWSSYPRTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。
>QD027(SEQ ID NO:11)
MEFGLSWLFLVAILKGVQCEVQLQQSGAELVKPGASVKLSCTASGFNIKDYYIHWVQQRTEQGLEWIGRIDPEDGETKYAPKFQDKATITADTSSNTAYLHLSSLTSEDTAVYYCARWGAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。其中，MEFGLSWLFLVAILKGVQC为信号肽。
>QD249(SEQ ID NO:12)
MEFGLSWLFLVAILKGVQCEVQLQQSGAELVKPGASVKLSCTASGFNIKDYYIHWVQQRTEQGLEWIGRIDPEDGETKYAPKFQDKATITADTSSNTAYLHLSSLTSEDTAVYYCARWGAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。其中，MEFGLSWLFLVAILKGVQC为信号肽。
>QD163(SEQ ID NO:13)
其中，双下划线部分是恒定区序列。
>QD164(SEQ ID NO:14)
其中，双下划线部分是恒定区序列。
>QD245(SEQ ID NO:15)
其中，MEFGLSWLFLVAILKGVQC为信号肽，双下划线部分为重链恒定区序列。
表2抗原克隆设计
注：QP098为食蟹猴SIRPα序列(uniprot数据库序列编号I7G9Z7)，QP100为食蟹猴SIRPα序列(uniprot数据库序列编号G7PGS8)，QP271为恒河猴SIRPα序列，是发明人通过测序猴子PBMC获得，QP273为食蟹猴SIRPα序列，是发明人通过测序猴子PBMC获得。
(2)抗原及嵌合抗体表达、纯化
293E细胞培养密度维持在(0.2-3)×106/ml之间，维护阶段培养基(GIBCOFreestyle 293expression medium)进行培养，转染前一天待转染细胞离心换液，调整细胞密度为(0.5-0.8)×106/ml。转染当天，293E细胞密度为(1-1.5)×106/ml。准备质粒和转染试剂PEI，需转染质粒量为100μg/100ml细胞，使用PEI和质粒的质量比为2:1。将质粒和PEI进行混匀，静置15min，不宜超过20min。将质粒和PEI混合物缓慢加入293E的细胞中，放入8％ CO2，120rpm，37℃的摇床中培养，转染第五天，水平离心机4700rpm离心20min收集细胞上清。
Protein A亲和层析纯化：用平衡液过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致，流速1ml/min；将离心后培养液上清过柱，上样40ml，流速0.33ml/min；用平衡液过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致，流速0.33ml/min；用洗脱液过柱，UV280上升至15mAU时开始收集洗脱峰(PAC-EP)，UV280下降至15mAU时停止收集，流速1ml/min。样品收集完成后，用pH调节液将PAC-EP调至中性。
(3)表面等离子体共振(SPR)检测亲和力
通过Biacore T200(GE)测定抗SIRPα嵌合抗体QP163164与人SIRPαV1型(蛋白编号QP094)及人SIRPαV2型(蛋白编号QP096)亲和力。表3和表4展示QP163164以及QP026027的检测结果。结果显示SIRPα嵌合抗体QP163164结合人SIRPαV1型亲和力KD为5.27E-10M，结合人SIRPαV2型亲和力KD值为6.78E-10M。结合人SIRPαV1型及人SIRPαV2型亲和力明显优于对照抗体KWAR23(QP026027)。
表3抗SIRPα嵌合抗体与SIRPαV1及SIRPαV2亲和力
通过biacore测定嵌合抗体与食蟹猴SIRPα亲和力如下表所示：
表4嵌合抗体与食蟹猴SIRPα亲和力
实施例3：抗SIRPα杂交瘤单克隆抗体的人源化
通过比对IMGT人类抗体重轻链可变区种系基因数据库和MOE软件，挑选与QP163164同源性高的重轻链可变区种系基因作为模板，将鼠源抗体的CDR分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。再选择一些重要的氨基酸残基做回复突变组合。其中氨基酸残基由Kabat编号系统确定并注释。以下实施例中，重链FR区序列来源于人种系重链IGHV1-18及IGHJ2*01的组合序列，其包含人种系重链IGHV1-18的FR1、FR2、FR3区和IGHJ2*01的FR4区。轻链FR区序列来源于人种系轻链IGKV4-1及IGKJ2*01的组合序列，其包含人种系轻链IGKV4-1的FR1、FR2、FR3区和IGKJ2*01的FR4区。
(1)抗SIRPα抗体人源化分子克隆
设计引物PCR搭建各人源化抗体VH/VK基因片段，再与表达载体pQD(带信号肽及恒定区基因(CH1-FC/CL)片段)进行同源重组，构建抗体全长表达载体VH-CH1-FC-pQD/VK-CL-pQD。
利用在线软件DNAWorks(v3.2.2)(http://helixweb.nih.gov/dnaworks/)设计多条引物合成VH/VK含重组所需基因片段：5’-30bp信号肽+VH/VK+30bp CH1/CL-3’。按照TaKaRa公司Primer STAR GXL DNA聚合酶操作说明书，用上面设计的多条引物，分两步PCR扩增得到VH/VK含重组所需基因片段。表达载体pQD构建及酶切，利用一些特殊的限制性内切酶，如BsmBI识别序列与酶切位点不同的特性设计构建表达载体pQD。BsmBI酶切载体，切胶回收备用。构建重链表达载体pQD-VH-CH1-FC及轻链表达载体pQD-VL-CL：重链可变区VH基因片段与经BsmBI酶切的载体pQD(带信号肽及重链恒定区(CH1-FC)片段)按照3:1比例混合；轻链可变区VL基因片段与经BsmBI酶切的载体pQD(带信号肽及轻链恒定区(CL)片段)按照3:1比例混合；将混合物分别转入DH5a感受态细胞中，0℃冰浴30min，42℃热击90s，加入5倍体积LB介质，37℃孵育45min，涂布LB-Amp平板，37℃培养过夜，挑取单克隆送测序得到各目的克隆。
下表所示为QP163164的人源化设计的具体信息。蛋白表达编号为QP256253。此表中抗体轻链采用kappa轻链恒定区CL，抗体重链采用人IgG4恒定区(恒定区具体序列可参阅实施例2)。人源化设计轻重链可变区序列并不限于下表所示的序列。
表5人源化设计轻重链序列及蛋白表达编号
注：
QP256253的轻链可变区由编号为QD253的质粒编码。轻链可变区序列SEQ ID NO:16具体序列为：
QP256253的重链可变区由编号为QD256的质粒编码。重链可变区序列SEQ ID NO:17具体序列为：
(2)抗SIRPα抗体人源化蛋白表达
293E细胞培养密度维持在(0.2-3)×106/ml之间，维护阶段培养基(GIBCOFreestyle 293expression medium)进行培养，转染前一天待转染细胞离心换液，调整细胞密度为(0.5-0.8)×106/ml。转染当天，293E细胞密度为(1-1.5)×106/ml。准备质粒和转染试剂PEI，需转染质粒量为100μg/100ml细胞，使用PEI和质粒的质量比为2:1。将质粒和PEI进行混匀，静置15min，不宜超过20min。将质粒和PEI混合物缓慢加入293E的细胞中，放入8％ CO2，120rpm，37℃的摇床中培养，转染第五天，水平离心机4700rpm离心20min收集细胞上清。
(3)抗SIRPα抗体人源化蛋白纯化
Protein A亲和层析纯化：用平衡液过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致，流速1ml/min；将离心后培养液上清过柱，上样40ml，流速0.33ml/min；用平衡液过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致，流速0.33ml/min；用洗脱液过柱，UV280上升至15mAU时开始收集洗脱峰(PAC-EP)，UV280下降至15mAU时停止收集，流速1ml/min。样品收集完成后，用pH调节液将PAC-EP调至中性。
(4)人源化SIRPα抗体活性鉴定(Binding-ELISA)
Binding-ELISA实验方法：分别包被QP094(SIRPαV1-flag-his)、QP096(SIRPαV2-Flag-his)、QP100(cynoSIRPα-flag-his)0.5μg/ml，50μl/孔，4度过夜。PBS洗3遍，3％BSA/PBS200μl/孔，RT孵育2h；PBST洗3遍；加入不同浓度的抗体，RT孵育1h，PBST洗3遍，PBS洗3遍；孵育二抗HRP-anti Fab 1：2500稀释，RT孵育1h，PBST洗3遍，PBS洗3遍，TMB显示，2M的H2SO4终止，450nm读数。
(5)人源化SIRPα抗体SPR鉴定亲和力
通过biacore测定人源化抗体与人SIRPαV1型、人SIRPαV2型及食蟹猴SIRPα亲和力如下表6所示，结果显示抗SIRPα人源化抗体QP256253结合人SIRPαV1型亲和力KD为3.36E-10M，结合人SIRPαV2型亲和力KD值为3.19E-10M。
表6人源化抗体与人SIRPαV1型及SIRPαV2型及食蟹猴SIRPα亲和力
实施例4：抗SIRPα抗体QP163164亲和力成熟
(1)构建人源化噬菌粒载体
人源化后的QP256253分别以scFv模式(VH-3个GGGGS-VL)构建到噬菌粒载体中，作为野生型序列(即作为原始或起始序列，亲和力成熟筛选得到的为突变序列)。利用重叠PCR(over-lap PCR)拼接VH、(GGGGS)3接头、VL，采用NcoI和NotI酶切位点连接入噬菌粒载体。
(2)构建噬菌体展示文库
利用构建好的野生型scFv为模板，采用基于密码子的(codon-based)引物，在引物合成过程中，突变区域密码子有50％野生型的密码子和50％的NNK(反向引物为MNN)，在所有CDR区引入突变构建突变文库。PCR片段经过NcoI和NotI酶切，连接到噬菌粒体载体中，最后电转化大肠杆菌TG1。每条基于密码子的引物建立一个独立的文库。
(3)文库淘筛
文库经过拯救包装出淘筛用的噬菌体颗粒后，利用生物素化的QP098(cynoSIRPα(ECD))抗原和链霉亲和素磁珠进行液相法淘筛，并且每一轮筛选相对于上一轮都降低抗原浓度。三轮淘筛之后，挑取250克隆进行噬菌体ELISA检测结合活性，阳性克隆进行测序。经过对测序克隆进行比对分析，去除冗余序列之后，将非冗余序列转换成全长IG(重链恒定区选择hIgG4的CH1-CH2-CH3；轻链恒定区选择κ轻链CL)进行哺乳动物细胞表达。亲和纯化之后得到全长IG蛋白。具体序列如下表所示。此表中抗体轻链采用kappa轻链恒定区CL，抗体重链采用人IgG4恒定区(恒定区具体序列可参阅实施例2)。
表7阳性克隆序列
注：蛋白编号的命名规则为重链质粒编号和轻链质粒编号的组合。示例性的，蛋白编号为QP256279的抗体分子，其重链质粒编号为QD256，轻链质粒编号为QD279。表中序列编号所示的序列为不同抗体的重链可变区或轻链可变区序列。轻链可变区具体序列如下：
>QD279(SEQ ID NO:18)
>QD291(SEQ ID NO:19)
>QD1581(SEQ ID NO:20)
>QD1586(SEQ ID NO:21)
>QD1589(SEQ ID NO:22)
>QD1594(SEQ ID NO:23)
>QD1770(SEQ ID NO:24)
>QD1771(SEQ ID NO:25)
以上加粗并下划线部分分别为各抗体分子的VLCDR1、VLCDR2、VLCDR3，与野生型序列QP256253具体比较如下：
表8各抗体分子的LCDR区序列与野生型序列的比较
注：“/”表示序列与QP256253相同，黑体并加粗表示与QD253不同的氨基酸。
(4)ELISA检测
Binding-ELISA实验方法：分别包被QP094(SIRPαV1-flag-his)、QP096(SIRPαV2-Flag-his)、QP098(cynoSIRPα-flag-his)、QP100(cynoSIRPα-flag-his)0.5μg/ml，50μl/孔，4度过夜。PBS洗3遍，3％BSA/PBS200μl/孔，RT孵育2h；PBST洗3遍；加入不同浓度的抗体，RT孵育1h，PBST洗3遍，PBS洗3遍；孵育二抗HRP-anti Fab 1：2500稀释，RT孵育1h，PBST洗3遍，PBS洗3遍，TMB显示，2M的H2SO4终止，450nm读数。EC50值如下表所示。下表中还展示了人源化抗体QP256253、嵌合抗体QP163245、对照抗体QP026249的检测结果。结果如图1至图6所示。
表9 ELISA检测EC50值
conc.(μg/ml) QP094 QP096 QP098 QP100 QP271 QP273
QP2561589 0.001366 0.001171 0.001598 0.002009 0.0009365 0.001429
QP256291 0.001859 0.001429 0.00157 0.001178 0.001173 0.001618
QP2561586 0.001046 0.001108 0.001199 0.001235 0.0009672 0.001027
QP2561581 0.001075 0.0008623 0.0009179 0.0007578 0.0005554 0.0009209
QP2561594 0.001115 0.001055 0.001134 0.0009833 0.0008066 0.002348
QP256279 0.0006508 0.0006239 0.000619 0.0005584 0.0006008 0.0007207
QP2561770 0.001615 0.001363 0.001302 0.001005 0.001207 0.001675
QP2561771 0.001145 0.001127 0.001082 0.001128 0.001127 0.001217
QP256253 0.001189 0.001185 0.001255 0.00172 0.0009472 0.001514
QP163245 0.001106 0.001235 0.001526 0.001166 0.0008822 0.0016
QP026249 0.005285 0.003311 0.02366 0.001925 0.00193 0.002028
Blocking-ELISA实验方法：coat QP001.2 2μg/ml，4度过夜，PBS洗3遍，5％milk250μl/孔封闭，孵育Biotin-QP002 0.05μg/ml+Abs 50μg/ml 1：1混合，25度孵育1h，HRP-Strepavidin(1:5000)。结果如图7所示。
(5)表面等离子体共振(SPR)检测亲合力
通过biacore测定抗SIRPα抗体与人SIRPαV1型、人SIRPαV2型及食蟹猴SIRPα亲和力，部分结果如表10所示。由表10可知，抗SIRPα抗体QP2561589、QP2561586、QP2561581、QP256279、QP2561770均结合人SIRPαV1型和人SIRPαV2型。同时QP2561589、QP2561586、QP256279、QP2561770、QP256253均结合不同食蟹猴及恒河猴SIRPα蛋白。
表10SPR检测亲合力结果
由上表可知，亲和力成熟抗体QP2561589、QP2561586、QP256279蛋白对人SIRPαV1型及SIRPαV2型亲和力比对照抗体KWAR23(QP026249)的高50倍以上。
实施例5：FACS检测抗SIRPα抗体结合天然表达人SIRPα的人肾透明细胞腺癌细胞786-O细胞
实验步骤：收集786-O细胞2E5/孔，PBS洗1遍，300g离心3min弃上清。封闭：用2％FBS重悬，2E5/孔，200μl/孔种96孔U底板，冰浴1h。300g离心3min弃上清。抗体孵育：孵育抗体10μg/ml1:3稀释，100μl/孔，冰浴1h。离心，弃上清。加入预冷的PBS200μl/孔，300g离心5min弃上清，重复2次。二抗：PE-anti human FC(1:200)50μl/孔，冰浴0.5h。离心，弃上清。加入预冷的PBS200μl/孔，300g离心5min弃上清，重复3次。FACS读取平均荧光值。结果如图8所示，SIRPα抗体QP163245、QP256253、QP256279、QP2561586、QP2561589均结合天然表达人SIRPα的人肾透明细胞腺癌细胞786-O细胞，且结合亲和力优于对照抗体QP026249(KWAR23)。
实施例6：抗SIRPα抗体体外功能实验ADCP
(1)将抗SIRPα抗体做成不同IgG亚型，分子克隆设计如下：
表11抗SIRPα抗体体外功能实验ADCP分子克隆设计
注：蛋白编号的命名规则为重链质粒编号和轻链质粒编号的组合。重链的序列编号所示的序列为不同亚型抗体的重链序列。轻链的序列编号所示的序列为不同亚型抗体的轻链或轻链可变区序列。
其中，QP32700279的重链(SEQ ID NO:26)的具体序列如下：
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWINWVRQAPGQGLEWIAMIDPSDSETHYNQIFKDRATLTTDTSTSTAYMELRSLRSDDTAVYYCAMDYGSLYAMDYWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISAAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。
QP32700279的轻链可变区的序列如SEQ ID NO:18所示。
(2)抗SIRPα抗体体外功能实验ADCP
准备巨噬细胞(Macrophage)：复苏PBMC，用试剂盒EasySepTM Human MonocyteIsolation Kit(Stemcell-19359)分离单核细胞(monocytes)，加入Human Recombinant M-CSF(终浓度为50ng/mL)，充分混匀；37℃培养细胞6天诱导为Macrophage；收集细胞，计数备用。CFSE标记Raji细胞。将Raji重悬为2×106cells/ml，然后按50μl/well(1×105/well)加入已有巨噬细胞的96孔板中；稀释抗体：将Rituximab(利妥昔单抗)用完全培养基稀释为80μg/ml，再3倍稀释9个梯度，将anti-SIRPα用完全培养基稀释为20μg/ml；抗体混合：Combination组将稀释好的2个抗体1:1混合，Rituximab组用等体积的培养基混合，按50μl/well加入之前铺好的细胞的96孔板中；37℃培养2h；FACS检测：通过对活CFSE+/CD14+细胞进行门控来测量吞噬作用(Phagocytosis was measured by gating live CFSE+/CD14+cells)。
将亲和力成熟分子和对照抗体做了协同Rituximab的ADCP assay，实验结果显示SIRPα抗体和Rituximab联用与Rituximab单用相比，EC50变小，ADCP协同效果显著增强。结果如图9、图10、图11所示。
实施例7：QP32700279在B-NDG-hSIRPΑ鼠模型中评价抗SIRPα抗体对Raji-Luc肿瘤生长的抑制作用
为了考察抗SIRPα抗体对肿瘤的杀伤作用，采用B-NDG-hSIRPΑ静脉接种Raji-Luc肿瘤模型评价SIRPα抗体和Rituximab对肿瘤的生长抑制作用。Raji-Luc细胞培养在含10％胎牛血清的RPMI1640培养液中。将PBS重悬的Raji-Luc细胞以5×105个/0.2mL浓度，0.2mL/只体积接种到B-NDG-hSIPRa小鼠尾静脉。在接种后第0天、第3天使用小动物成像仪测量肿瘤成像信号值，当平均成像信号强度达到1×106P/S左右，根据肿瘤成像信号值、动物体重选择合适动物入组，平均分配到4个实验组中，每个实验组8只。分组当天开始给药，具体给药方案见下表：
表12
注：
a：给药体积依实验动物体重按10μL/g计算；
b：Q3D指每3天给药1次，Q2W指每2周给药1次。
分组给药当天计为D0，截止至D18，各组肿瘤成像信号值反应的肿瘤生长曲线及D18成像信号强度数据如图12和下表所示：
表13
肿瘤生长曲线结果显示，Rituximab、QP32700279及QP32700279、Rituximab联合用药组均能显著抑制Raji-Luc肿瘤生长，肿瘤抑制率(TGI)分别为58.6％、46.4％和84.5％，而且联合用药组比单独用药组表现出更强的抗肿瘤活性。
由于模型特征，试验后期小鼠会出现行动异常或瘫痪的现象，此时给予小鼠安乐死处理，并记录生存曲线。截止至G1组小鼠全部死亡(D25)，各组生存曲线如图13所示
生存分析采用Kaplan-Meier法，组间比较采用Log rank检验，p<0.05视为差异显著。与对照组相比，QP32700279和联合给药组(QP32700279+Rituximab)均可以显著延长Raji-Luc荷瘤小鼠的生存期(p＝0.0445*,p<0.001**)，而Rituximab组不能有效地延长荷瘤小鼠的生存期(p＝0.23)。该试验结果提示，QP32700279和QP32700279+Rituximab联合给药可以有效地抑制Raji-Luc荷瘤小鼠的肿瘤生长情况，进而改善小鼠的生存情况。
实施例8：ELISA检测抗SIRPα抗体对人SIRPα所有亚型结合
根据已有文献(“Velcro”Engineering of High Affinity CD47 Ectodomain asSignal Regulatory Protein(SIRP alpha)Antagonists That Enhance Antibody-dependent Cellular Phagocytosis，JOURNAL OF BIOLOGICAL CHEMISTRY，VOLUME 290·NUMBER 20·MAY 15,2015)报道的SIRPαV1/V2/V3/V4/V5/V6/V7/V8/V9/V10序列，通过基因合成将上面SIRPαC端融合小鼠IgG2a亚型的Fc(mouse IgG2a)，构建到真核表达载体pQD，通过293E瞬时转染，Protein A纯化瞬转第五天上清，分别得到SIRPαV1/V2/V3/V4/V5/V6/V7/V8/V9/V10融合Fc(mouse IgG2a)蛋白，进一步进行ELISA检测SIRPα抗体与SIRPα所有亚型的结合。序列如下所示。
>SIRPαV1(SEQ ID NO:51)
GVAGEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS
>SIRPαV2(SEQ ID NO:52)
GVAGEEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS
>SIRPαV3(SEQ ID NO:53)
GVAGEEELQVIQPDKSVSVAAGESAILLCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPD-TEFKSGAGTELSVRAKPS
>SIRPαV4(SEQ ID NO:54)
GVAGEEGLQVIQPDKSVSVAAGESAILHCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS
>SIRPαV5(SEQ ID NO:55)
GVAGEEELQVIQPDKFVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS
>SIRPαV6(SEQ ID NO:56)
GVAGEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFPIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS
>SIRPαV7(SEQ ID NO:57)
GVAGEEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPD-TEFKSGAGTELSVRGKPS
>SIRPαV8(SEQ ID NO:58)
GVAGEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPD-TEFKSGAGTELSVRAKPS
>SIRPαV9(SEQ ID NO:59)
GVAGEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRISNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS
>SIRPαV10(SEQ ID NO:60)
RVAGEEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPD-TEFKSGAGTELSVRAKPS
>FC(mouse IgG2a)(SEQ ID NO:61)
EPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK。
待检测SIRPα抗体：
将SIRPα抗体QP256279在CHOS细胞中稳定表达，CHOS稳转表达蛋白编号为CHO71。
根据专利WO2017178653提供的序列，分子克隆构建并表达纯化OSE公司抗SIRPα抗体18D5，用于实验对照。同时如前所述QP026249即为47公司(Forty Seven)的抗SIRPα抗体KWAR23，这里用KWAR23表示。
ELISA检测SIRPα抗体结合SIRPαV1/V2/V3/V4/V5/V6/V7/V8/V9/V10实验步骤：
包被SIRPαV1/V2/V3/V4/V5/V6/V7/V8/V9/V10，1μg/ml，60μl/孔，4℃过夜，PBST洗2遍；5％non-fat milk(生工)封闭，200μl/孔，室温孵育1h，PBST洗2遍；孵育抗体10μg/ml，5倍稀释，10个梯度，60μ/孔，室温孵育1h，PBST洗5遍；孵育二抗：anti-hFab1：10000，60μl/孔，室温孵育1h，PBST洗5遍；显色：TMB提前1h平衡室温，100μl/孔，显色10min，2M H2SO450ul/孔终止，酶标仪450nm读数。
实验结果如图14至图23所示，本发明的SIRPα抗体CHO71结合SIRPαV1/V2/V3/V4/V5/V6/V7/V8/V9/V10所有亚型。OSE公司的SIRPa抗体18D5不结合SIRPa V2/V3/V7/V8/V10。
尽管本发明的内容已经通过上述优选实施例作了详细介绍，但应当认识到上述的描述不应被认为是对本发明的限制。在本领域技术人员阅读了上述内容后，对于本发明的多种修改和替代都将是显而易见的。因此，本发明的保护范围应由所附的权利要求来限定。
序列表
<110> 启愈生物技术（上海）有限公司
<120> 抗SIRPα抗体或其抗原结合片段及应用
<160> 61
<170> SIPOSequenceListing 1.0
<210> 1
<211> 133
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala
1 5 10 15
Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu
100 105 110
Ser Val Arg Ala Lys Pro Ser Asp Tyr Lys Asp Asp Asp Asp Lys His
115 120 125
His His His His His
130
<210> 2
<211> 119
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Gln Val Gln Leu Gln Gln Pro Gly Thr Glu Leu Val Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Ala Met Ile Asp Pro Ser Asp Ser Glu Thr His Tyr Asn Gln Ile Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Gly Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Met Asp Tyr Gly Ser Leu Tyr Ala Met Asp Tyr Trp Gly Arg Gly
100 105 110
Thr Ser Val Thr Val Ser Ser
115
<210> 3
<211> 5
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Asn Tyr Trp Ile Asn
1 5
<210> 4
<211> 17
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Ile Asp Pro Ser Asp Ser Glu Thr His Tyr Asn Gln Ile Phe Lys
1 5 10 15
Asp
<210> 5
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Asp Tyr Gly Ser Leu Tyr Ala Met Asp Tyr
1 5 10
<210> 6
<211> 110
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Ser Ser
20 25 30
Gly Tyr Asn Tyr Ile Phe Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Asp Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg
85 90 95
Glu Leu Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 7
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Arg Ala Ser Lys Ser Val Ser Ser Ser Gly Tyr Asn Tyr Ile Phe
1 5 10 15
<210> 8
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Leu Ala Ser Asn Leu Asp Ser
1 5
<210> 9
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Gln His Ser Arg Glu Leu Pro Thr
1 5
<210> 10
<211> 237
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15
Phe Pro Gly Ser Arg Cys Gln Ile Val Leu Thr Gln Ser Pro Ala Ile
20 25 30
Met Ser Ala Ser Pro Gly Glu Lys Val Thr Leu Thr Cys Ser Ala Ser
35 40 45
Ser Ser Val Ser Ser Ser Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly
50 55 60
Ser Ser Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly
65 70 75 80
Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu
85 90 95
Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Ser Tyr Phe Cys His
100 105 110
Gln Trp Ser Ser Tyr Pro Arg Thr Phe Gly Ala Gly Thr Lys Leu Glu
115 120 125
Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
130 135 140
Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn
145 150 155 160
Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala
165 170 175
Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
180 185 190
Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
195 200 205
Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu
210 215 220
Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230 235
<210> 11
<211> 462
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys
20 25 30
Pro Gly Ala Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile
35 40 45
Lys Asp Tyr Tyr Ile His Trp Val Gln Gln Arg Thr Glu Gln Gly Leu
50 55 60
Glu Trp Ile Gly Arg Ile Asp Pro Glu Asp Gly Glu Thr Lys Tyr Ala
65 70 75 80
Pro Lys Phe Gln Asp Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn
85 90 95
Thr Ala Tyr Leu His Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Arg Trp Gly Ala Tyr Trp Gly Gln Gly Thr Leu Val
115 120 125
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
130 135 140
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
145 150 155 160
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
165 170 175
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
180 185 190
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
195 200 205
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
210 215 220
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
225 230 235 240
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
245 250 255
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
260 265 270
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
275 280 285
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
290 295 300
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
305 310 315 320
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
325 330 335
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
340 345 350
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
355 360 365
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
370 375 380
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
385 390 395 400
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
405 410 415
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
420 425 430
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
435 440 445
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455 460
<210> 12
<211> 459
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys
20 25 30
Pro Gly Ala Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile
35 40 45
Lys Asp Tyr Tyr Ile His Trp Val Gln Gln Arg Thr Glu Gln Gly Leu
50 55 60
Glu Trp Ile Gly Arg Ile Asp Pro Glu Asp Gly Glu Thr Lys Tyr Ala
65 70 75 80
Pro Lys Phe Gln Asp Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn
85 90 95
Thr Ala Tyr Leu His Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Arg Trp Gly Ala Tyr Trp Gly Gln Gly Thr Leu Val
115 120 125
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
130 135 140
Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu
145 150 155 160
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
165 170 175
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
180 185 190
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
195 200 205
Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr
210 215 220
Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro
225 230 235 240
Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
245 250 255
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
260 265 270
Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn
275 280 285
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
290 295 300
Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
305 310 315 320
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
325 330 335
Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys
340 345 350
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu
355 360 365
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
370 375 380
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
385 390 395 400
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
405 410 415
Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly
420 425 430
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
435 440 445
Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
450 455
<210> 13
<211> 217
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Ser Ser
20 25 30
Gly Tyr Asn Tyr Ile Phe Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Asp Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg
85 90 95
Glu Leu Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr
100 105 110
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
115 120 125
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
130 135 140
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
145 150 155 160
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
165 170 175
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
180 185 190
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
195 200 205
Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 14
<211> 449
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Gln Val Gln Leu Gln Gln Pro Gly Thr Glu Leu Val Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Ala Met Ile Asp Pro Ser Asp Ser Glu Thr His Tyr Asn Gln Ile Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Gly Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Met Asp Tyr Gly Ser Leu Tyr Ala Met Asp Tyr Trp Gly Arg Gly
100 105 110
Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210> 15
<211> 465
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Gln Val Gln Leu Gln Gln Pro Gly Thr Glu Leu Val Arg
20 25 30
Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45
Thr Asn Tyr Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu
50 55 60
Glu Trp Ile Ala Met Ile Asp Pro Ser Asp Ser Glu Thr His Tyr Asn
65 70 75 80
Gln Ile Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn
85 90 95
Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Gly Asp Ser Ala Val
100 105 110
Tyr Tyr Cys Ala Met Asp Tyr Gly Ser Leu Tyr Ala Met Asp Tyr Trp
115 120 125
Gly Arg Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
130 135 140
Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr
145 150 155 160
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
165 170 175
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
180 185 190
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
195 200 205
Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp
210 215 220
His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr
225 230 235 240
Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro
245 250 255
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
260 265 270
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp
275 280 285
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
290 295 300
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val
305 310 315 320
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
325 330 335
Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys
340 345 350
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
355 360 365
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
370 375 380
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
385 390 395 400
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
405 410 415
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys
420 425 430
Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
435 440 445
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
450 455 460
Lys
465
<210> 16
<211> 110
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Lys Ser Val Ser Ser Ser
20 25 30
Gly Tyr Asn Tyr Ile Phe Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Asp Ser Gly Val Pro Asp
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln His Ser Arg
85 90 95
Glu Leu Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 17
<211> 119
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Ala Met Ile Asp Pro Ser Asp Ser Glu Thr His Tyr Asn Gln Ile Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Met Asp Tyr Gly Ser Leu Tyr Ala Met Asp Tyr Trp Gly Arg Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 18
<211> 110
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Gln Ser Val Arg Ser Ser
20 25 30
Gly Tyr Asn Trp Ile Phe Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Arg Asp Ser Gly Val Pro Asp
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln His Ser Arg
85 90 95
Glu Leu Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 19
<211> 110
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Lys Ser Val Gly Ser Ser
20 25 30
Gly Tyr Asn Trp Leu Phe Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Arg Asp Ser Gly Val Pro Asp
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln His Ser Arg
85 90 95
Glu Leu Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 20
<211> 110
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Lys Ser Val Ser Ser Ser
20 25 30
Gly Tyr Asn Tyr Ile Phe Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Asp Pro Gly Val Pro Asp
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln His Ser Arg
85 90 95
Glu Leu Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 21
<211> 110
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Lys Ser Val Ser Ser Ser
20 25 30
Gly Tyr Asn Tyr Ile Phe Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Asp Ser Gly Val Pro Asp
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Glu Ser Arg
85 90 95
Glu Leu Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 22
<211> 110
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 22
Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Lys Ser Val Ser Ser Ser
20 25 30
Gly Tyr Asn Tyr Ile Phe Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Asp Ser Gly Val Pro Asp
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Glu Ser Trp
85 90 95
Glu Leu Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 23
<211> 110
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 23
Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Lys Ser Val Ser Ser Ser
20 25 30
Gly Tyr Asn Tyr Ile Phe Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Asp Ser Gly Val Pro Asp
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln His Ser Arg
85 90 95
Asp Leu Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 24
<211> 110
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 24
Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Gln Ser Val Arg Ser Ser
20 25 30
Gly Tyr Asn Trp Ile Phe Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Arg Asp Ser Gly Val Pro Asp
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Glu Ser Arg
85 90 95
Glu Leu Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 25
<211> 110
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 25
Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Lys Ser Val Ser Ser Ser
20 25 30
Gly Tyr Asn Trp Ile Phe Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Arg Asp Ser Gly Val Pro Asp
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Glu Ser Arg
85 90 95
Glu Leu Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 26
<211> 449
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 26
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Ala Met Ile Asp Pro Ser Asp Ser Glu Thr His Tyr Asn Gln Ile Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Met Asp Tyr Gly Ser Leu Tyr Ala Met Asp Tyr Trp Gly Arg Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Ala Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210> 27
<211> 449
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 27
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Ala Met Ile Asp Pro Ser Asp Ser Glu Thr His Tyr Asn Gln Ile Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Met Asp Tyr Gly Ser Leu Tyr Ala Met Asp Tyr Trp Gly Arg Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Gln Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210> 28
<211> 446
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 28
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Ala Met Ile Asp Pro Ser Asp Ser Glu Thr His Tyr Asn Gln Ile Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Met Asp Tyr Gly Ser Leu Tyr Ala Met Asp Tyr Trp Gly Arg Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val
260 265 270
Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Phe Gln Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
435 440 445
<210> 29
<211> 445
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 29
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Ala Met Ile Asp Pro Ser Asp Ser Glu Thr His Tyr Asn Gln Ile Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Met Asp Tyr Gly Ser Leu Tyr Ala Met Asp Tyr Trp Gly Arg Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu
210 215 220
Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu
290 295 300
Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335
Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 30
<211> 449
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 30
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Ala Met Ile Asp Pro Ser Asp Ser Glu Thr His Tyr Asn Gln Ile Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Met Asp Tyr Gly Ser Leu Tyr Ala Met Asp Tyr Trp Gly Arg Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210> 31
<211> 449
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 31
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Ala Met Ile Asp Pro Ser Asp Ser Glu Thr His Tyr Asn Gln Ile Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Met Asp Tyr Gly Ser Leu Tyr Ala Met Asp Tyr Trp Gly Arg Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210> 32
<211> 443
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 32
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Ile His Trp Val Gln Gln Arg Thr Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Glu Asp Gly Glu Thr Lys Tyr Ala Pro Lys Phe
50 55 60
Gln Asp Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu His Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Trp Gly Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
115 120 125
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
130 135 140
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
145 150 155 160
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
165 170 175
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
180 185 190
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
195 200 205
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
210 215 220
Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro
225 230 235 240
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
245 250 255
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
260 265 270
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
275 280 285
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
290 295 300
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
305 310 315 320
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Ala Ala Lys
325 330 335
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
340 345 350
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
355 360 365
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
370 375 380
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
385 390 395 400
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
405 410 415
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
420 425 430
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440
<210> 33
<211> 449
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 33
Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Arg Ile Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asn Tyr
20 25 30
Tyr Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Tyr Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Lys Phe
50 55 60
Lys Ala Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Pro Thr Ile Ile Ala Thr Asp Phe Asp Val Trp Gly Ala Gly
100 105 110
Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Ala Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210> 34
<211> 449
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 34
Gln Val Gln Leu Gln Gln Pro Gly Thr Glu Leu Val Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Ala Met Ile Asp Pro Ser Asp Ser Glu Thr His Tyr Asn Gln Ile Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Gly Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Met Asp Tyr Gly Ser Leu Tyr Ala Met Asp Tyr Trp Gly Arg Gly
100 105 110
Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Gln Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210> 35
<211> 440
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 35
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Ile His Trp Val Gln Gln Arg Thr Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Glu Asp Gly Glu Thr Lys Tyr Ala Pro Lys Phe
50 55 60
Gln Asp Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu His Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Trp Gly Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser
115 120 125
Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
130 135 140
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
145 150 155 160
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
165 170 175
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys
180 185 190
Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp
195 200 205
Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala
210 215 220
Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
225 230 235 240
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
245 250 255
Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val
260 265 270
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
275 280 285
Phe Gln Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
290 295 300
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
305 310 315 320
Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
325 330 335
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr
340 345 350
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
355 360 365
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
370 375 380
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
385 390 395 400
Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe
405 410 415
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
420 425 430
Ser Leu Ser Leu Ser Leu Gly Lys
435 440
<210> 36
<211> 236
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 36
Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15
Phe Pro Gly Ser Arg Cys Asp Ile Val Met Thr Gln Ser His Lys Phe
20 25 30
Met Ser Thr Ser Val Gly Asp Arg Val Asn Ile Thr Cys Lys Ala Ser
35 40 45
Gln Gly Val Gly Thr Ala Val Gly Trp Tyr Gln Gln Lys Pro Gly Gln
50 55 60
Ser Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr Gly Val
65 70 75 80
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Ala
85 90 95
Ile Ser Asn Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln
100 105 110
Tyr Ser Thr Tyr Pro Phe Thr Phe Gly Gly Gly Thr Asn Leu Glu Ile
115 120 125
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
130 135 140
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
145 150 155 160
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
165 170 175
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
180 185 190
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
195 200 205
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
210 215 220
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230 235
<210> 37
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 37
Arg Ala Ser Gln Ser Val Arg Ser Ser Gly Tyr Asn Trp Ile Phe
1 5 10 15
<210> 38
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 38
Leu Ala Ser Asn Arg Asp Ser
1 5
<210> 39
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 39
Arg Ala Ser Lys Ser Val Gly Ser Ser Gly Tyr Asn Trp Leu Phe
1 5 10 15
<210> 40
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 40
Leu Ala Ser Asn Leu Asp Pro
1 5
<210> 41
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 41
Gln Glu Ser Arg Glu Leu Pro Thr
1 5
<210> 42
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 42
Gln Glu Ser Trp Glu Leu Pro Thr
1 5
<210> 43
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 43
Gln His Ser Arg Asp Leu Pro Thr
1 5
<210> 44
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 44
Arg Ala Ser Lys Ser Val Ser Ser Ser Gly Tyr Asn Trp Ile Phe
1 5 10 15
<210> 45
<211> 376
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 45
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val
20 25 30
Leu Val Ala Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser
35 40 45
Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly
50 55 60
Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr
65 70 75 80
Thr Val Ser Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg
85 90 95
Ile Gly Asn Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys
100 105 110
Phe Arg Lys Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly
115 120 125
Thr Glu Leu Ser Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly
130 135 140
Pro Ala Ala Arg Ala Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu
145 150 155 160
Ser His Gly Phe Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn
165 170 175
Gly Asn Glu Leu Ser Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu
180 185 190
Ser Val Ser Tyr Ser Ile His Ser Thr Ala Lys Val Val Leu Thr Arg
195 200 205
Glu Asp Val His Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu
210 215 220
Gln Gly Asp Pro Leu Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg
225 230 235 240
Val Pro Pro Thr Leu Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn
245 250 255
Gln Val Asn Val Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu
260 265 270
Gln Leu Thr Trp Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala
275 280 285
Ser Thr Val Thr Glu Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp
290 295 300
Leu Leu Val Asn Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys
305 310 315 320
Gln Val Glu His Asp Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu
325 330 335
Lys Val Ser Ala His Pro Lys Glu Gln Gly Ser Asn Thr Ala Ala Glu
340 345 350
Asn Thr Gly Ser Asn Glu Arg Gly Ser Ser Asp Tyr Lys Asp Asp Asp
355 360 365
Asp Lys His His His His His His
370 375
<210> 46
<211> 375
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 46
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val
20 25 30
Ser Val Ala Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser
35 40 45
Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala
50 55 60
Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr
65 70 75 80
Thr Val Ser Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser
85 90 95
Ile Ser Asn Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys
100 105 110
Phe Arg Lys Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr
115 120 125
Glu Leu Ser Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro
130 135 140
Ala Ala Arg Ala Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser
145 150 155 160
His Gly Phe Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly
165 170 175
Asn Glu Leu Ser Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser
180 185 190
Val Ser Tyr Ser Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu
195 200 205
Asp Val His Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln
210 215 220
Gly Asp Pro Leu Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val
225 230 235 240
Pro Pro Thr Leu Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln
245 250 255
Val Asn Val Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln
260 265 270
Leu Thr Trp Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser
275 280 285
Thr Val Thr Glu Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu
290 295 300
Leu Val Asn Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln
305 310 315 320
Val Glu His Asp Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys
325 330 335
Val Ser Ala His Pro Lys Glu Gln Gly Ser Asn Thr Ala Ala Glu Asn
340 345 350
Thr Gly Ser Asn Glu Arg Gly Ser Ser Asp Tyr Lys Asp Asp Asp Asp
355 360 365
Lys His His His His His His
370 375
<210> 47
<211> 375
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 47
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Glu Glu Glu Leu Gln Val Ile Gln Pro Glu Lys Ser Val
20 25 30
Ser Val Ala Ala Gly Asp Ser Ala Thr Leu Asn Cys Thr Val Ser Ser
35 40 45
Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly
50 55 60
Arg Glu Leu Ile Tyr Asn Leu Lys Glu Gly His Phe Pro Arg Val Thr
65 70 75 80
Ala Val Ser Asp Pro Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg
85 90 95
Ile Ser Asn Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys
100 105 110
Phe Arg Lys Gly Ser Pro Asp Val Glu Leu Lys Ser Gly Ala Gly Thr
115 120 125
Glu Leu Ser Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro
130 135 140
Ala Val Arg Ala Thr Ala Glu His Thr Val Ser Phe Thr Cys Glu Ser
145 150 155 160
His Gly Phe Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly
165 170 175
Asn Glu Leu Ser Asp Val Gln Thr Asn Val Asp Pro Ala Gly Lys Ser
180 185 190
Val Ser Tyr Ser Ile Arg Ser Thr Ala Arg Val Leu Leu Thr Arg Arg
195 200 205
Asp Val His Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln
210 215 220
Gly Asp Pro Leu Arg Gly Thr Ala Asn Leu Ser Glu Ala Ile Arg Val
225 230 235 240
Pro Pro Phe Leu Glu Val Thr Gln Gln Ser Met Arg Ala Asp Asn Gln
245 250 255
Val Asn Val Thr Cys Gln Val Thr Lys Phe Tyr Pro Gln Arg Leu Gln
260 265 270
Leu Thr Trp Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Met Ala Ser
275 280 285
Ala Leu Pro Glu Asn Lys Asp Gly Thr Tyr Asn Trp Thr Ser Trp Leu
290 295 300
Leu Val Asn Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln
305 310 315 320
Val Glu His Asp Gly Gln Pro Ala Val Asn Lys Ser Phe Ser Val Lys
325 330 335
Val Ser Ala His Pro Lys Glu Gln Gly Ser Asn Thr Ala Ala Glu Asn
340 345 350
Thr Gly Thr Asn Glu Arg Gly Ser Ser Asp Tyr Lys Asp Asp Asp Asp
355 360 365
Lys His His His His His His
370 375
<210> 48
<211> 375
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 48
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Glu Glu Glu Leu Gln Val Ile Gln Pro Glu Lys Ser Val
20 25 30
Ser Val Ala Ala Gly Glu Ser Ala Thr Leu Asn Cys Thr Ala Thr Ser
35 40 45
Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Val Gly Pro Gly
50 55 60
Arg Glu Leu Ile Tyr His Gln Lys Glu Gly His Phe Pro Arg Val Thr
65 70 75 80
Pro Val Ser Asp Pro Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg
85 90 95
Ile Ser Asn Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys
100 105 110
Phe Arg Lys Gly Ser Pro Asp Val Glu Leu Lys Ser Gly Ala Gly Thr
115 120 125
Glu Leu Ser Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro
130 135 140
Ala Val Arg Ala Thr Ala Glu His Thr Val Ser Phe Thr Cys Glu Ser
145 150 155 160
His Gly Phe Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly
165 170 175
Asn Glu Leu Ser Asp Phe Gln Thr Asn Val Asp Pro Ala Gly Lys Ser
180 185 190
Val Ser Tyr Ser Ile Arg Ser Thr Ala Arg Val Val Leu Thr Arg Arg
195 200 205
Asp Val His Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln
210 215 220
Gly Asp Pro Leu Arg Gly Thr Ala Asn Leu Ser Glu Ala Ile Arg Val
225 230 235 240
Pro Pro Phe Leu Glu Phe Thr Gln Gln Ser Met Arg Ala Asp Asn Gln
245 250 255
Val Asn Val Thr Cys Gln Val Met Lys Phe Tyr Pro Gln Arg Leu Gln
260 265 270
Leu Thr Trp Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Met Ala Ser
275 280 285
Ala Leu Pro Glu Asn Lys Asp Gly Thr Tyr Asn Trp Thr Ser Trp Leu
290 295 300
Leu Val Asn Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln
305 310 315 320
Val Glu His Asp Gly Gln Pro Ala Val Asn Lys Ser Phe Ser Val Lys
325 330 335
Val Ser Ala His Pro Lys Glu Gln Gly Ser Asn Thr Ala Ala Glu Asn
340 345 350
Thr Gly Thr Asn Glu Arg Gly Ser Ser Asp Tyr Lys Asp Asp Asp Asp
355 360 365
Lys His His His His His His
370 375
<210> 49
<211> 372
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 49
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Glu Glu Glu Leu Gln Val Ile Gln Pro Glu Lys Ser Val
20 25 30
Ser Val Ala Ala Gly Asp Ser Ala Thr Leu Asn Cys Thr Val Thr Ser
35 40 45
Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly
50 55 60
Arg Glu Leu Ile Tyr His Gln Lys Glu Gly His Phe Pro Arg Val Thr
65 70 75 80
Ser Val Ser Glu Ser Thr Lys Arg Asn Asn Met Asp Phe Ser Ile His
85 90 95
Ile Ser Asn Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys
100 105 110
Phe Arg Lys Gly Ser Pro Asp Val Glu Val Lys Ser Gly Ala Gly Thr
115 120 125
Glu Leu Ser Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro
130 135 140
Ala Val Arg Ala Thr Ala Glu His Thr Val Ser Phe Thr Cys Glu Ser
145 150 155 160
His Gly Phe Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly
165 170 175
Asn Glu Leu Ser Asp Phe Gln Thr Asn Val Asp Pro Ala Gly Lys Ser
180 185 190
Val Ser Tyr Ser Ile Arg Ser Thr Ala Arg Val Val Leu Thr Arg Arg
195 200 205
Asp Val His Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln
210 215 220
Gly Asp Pro Leu Arg Gly Thr Ala Asn Leu Ser Glu Ala Ile Arg Val
225 230 235 240
Pro Pro Phe Leu Glu Val Thr Gln Gln Ser Met Arg Ala Asp Asn Gln
245 250 255
Val Asn Val Thr Cys Gln Val Thr Lys Phe Tyr Pro Gln Arg Leu Gln
260 265 270
Leu Thr Trp Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Met Ala Ser
275 280 285
Ala Leu Pro Glu Asn Lys Asp Gly Thr Tyr Asn Trp Thr Ser Trp Leu
290 295 300
Leu Val Asn Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln
305 310 315 320
Val Glu His Asp Gly Gln Pro Ala Val Asn Lys Ser Phe Ser Val Lys
325 330 335
Val Ser Ala His Pro Lys Glu Gln Gly Ser Asn Thr Ala Ala Glu Asn
340 345 350
Thr Gly Thr Asn Glu Arg Asp Tyr Lys Asp Asp Asp Asp Lys His His
355 360 365
His His His His
370
<210> 50
<211> 372
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 50
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Glu Glu Glu Leu Gln Val Ile Gln Pro Glu Lys Ser Val
20 25 30
Ser Val Ala Ala Gly Glu Ser Ala Thr Leu Asn Cys Thr Ala Thr Ser
35 40 45
Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Val Gly Pro Gly
50 55 60
Arg Glu Leu Ile Tyr Ser Gln Lys Glu Gly His Phe Pro Arg Val Thr
65 70 75 80
Pro Val Ser Asp Pro Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg
85 90 95
Ile Ser Asn Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys
100 105 110
Phe Arg Lys Gly Ser Pro Asp Val Glu Leu Lys Ser Gly Ala Gly Thr
115 120 125
Glu Leu Ser Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro
130 135 140
Ala Val Arg Ala Thr Ala Glu His Thr Val Ser Phe Thr Cys Glu Ser
145 150 155 160
His Gly Phe Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly
165 170 175
Asn Glu Leu Ser Asp Phe Gln Thr Asn Val Asp Pro Ala Gly Lys Ser
180 185 190
Val Ser Tyr Ser Ile Arg Ser Thr Ala Arg Val Val Leu Thr Arg Arg
195 200 205
Asp Val His Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln
210 215 220
Gly Asp Pro Leu Arg Gly Thr Ala Asn Leu Ser Glu Ala Ile Arg Val
225 230 235 240
Pro Pro Phe Leu Glu Val Thr Gln Gln Ser Met Arg Ala Asp Asn Gln
245 250 255
Val Asn Val Thr Cys Gln Val Thr Lys Phe Tyr Pro Gln Arg Leu Gln
260 265 270
Leu Thr Trp Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Met Ala Ser
275 280 285
Ala Leu Pro Glu Asn Lys Asp Gly Thr Tyr Asn Trp Thr Ser Trp Leu
290 295 300
Leu Val Asn Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln
305 310 315 320
Val Glu His Asp Gly Gln Pro Ala Val Asn Lys Ser Phe Ser Val Lys
325 330 335
Val Ser Ala His Pro Lys Glu Gln Gly Ser Asn Thr Ala Ala Glu Asn
340 345 350
Thr Gly Thr Asn Glu Arg Asp Tyr Lys Asp Asp Asp Asp Lys His His
355 360 365
His His His His
370
<210> 51
<211> 123
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 51
Gly Val Ala Gly Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser
1 5 10 15
Val Leu Val Ala Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr
20 25 30
Ser Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro
35 40 45
Gly Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val
50 55 60
Thr Thr Val Ser Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile
65 70 75 80
Arg Ile Gly Asn Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val
85 90 95
Lys Phe Arg Lys Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala
100 105 110
Gly Thr Glu Leu Ser Val Arg Ala Lys Pro Ser
115 120
<210> 52
<211> 122
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 52
Gly Val Ala Gly Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser
1 5 10 15
Val Ser Val Ala Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr
20 25 30
Ser Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro
35 40 45
Ala Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val
50 55 60
Thr Thr Val Ser Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile
65 70 75 80
Ser Ile Ser Asn Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val
85 90 95
Lys Phe Arg Lys Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly
100 105 110
Thr Glu Leu Ser Val Arg Ala Lys Pro Ser
115 120
<210> 53
<211> 122
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 53
Gly Val Ala Gly Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser
1 5 10 15
Val Ser Val Ala Ala Gly Glu Ser Ala Ile Leu Leu Cys Thr Val Thr
20 25 30
Ser Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro
35 40 45
Ala Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val
50 55 60
Thr Thr Val Ser Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile
65 70 75 80
Ser Ile Ser Asn Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val
85 90 95
Lys Phe Arg Lys Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly
100 105 110
Thr Glu Leu Ser Val Arg Ala Lys Pro Ser
115 120
<210> 54
<211> 123
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 54
Gly Val Ala Gly Glu Glu Gly Leu Gln Val Ile Gln Pro Asp Lys Ser
1 5 10 15
Val Ser Val Ala Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ala Thr
20 25 30
Ser Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro
35 40 45
Gly Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val
50 55 60
Thr Thr Val Ser Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile
65 70 75 80
Arg Ile Gly Asn Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val
85 90 95
Lys Phe Arg Lys Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala
100 105 110
Gly Thr Glu Leu Ser Val Arg Ala Lys Pro Ser
115 120
<210> 55
<211> 123
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 55
Gly Val Ala Gly Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Phe
1 5 10 15
Val Leu Val Ala Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr
20 25 30
Ser Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro
35 40 45
Gly Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val
50 55 60
Thr Thr Val Ser Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile
65 70 75 80
Arg Ile Gly Asn Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val
85 90 95
Lys Phe Arg Lys Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala
100 105 110
Gly Thr Glu Leu Ser Val Arg Ala Lys Pro Ser
115 120
<210> 56
<211> 123
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 56
Gly Val Ala Gly Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser
1 5 10 15
Val Leu Val Ala Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr
20 25 30
Ser Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro
35 40 45
Gly Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val
50 55 60
Thr Thr Val Ser Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Pro Ile
65 70 75 80
Arg Ile Gly Asn Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val
85 90 95
Lys Phe Arg Lys Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala
100 105 110
Gly Thr Glu Leu Ser Val Arg Ala Lys Pro Ser
115 120
<210> 57
<211> 122
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 57
Gly Val Ala Gly Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser
1 5 10 15
Val Ser Val Ala Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr
20 25 30
Ser Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro
35 40 45
Ala Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val
50 55 60
Thr Thr Val Ser Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile
65 70 75 80
Ser Ile Ser Asn Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val
85 90 95
Lys Phe Arg Lys Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly
100 105 110
Thr Glu Leu Ser Val Arg Gly Lys Pro Ser
115 120
<210> 58
<211> 122
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 58
Gly Val Ala Gly Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser
1 5 10 15
Val Leu Val Ala Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr
20 25 30
Ser Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro
35 40 45
Ala Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val
50 55 60
Thr Thr Val Ser Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile
65 70 75 80
Ser Ile Ser Asn Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val
85 90 95
Lys Phe Arg Lys Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly
100 105 110
Thr Glu Leu Ser Val Arg Ala Lys Pro Ser
115 120
<210> 59
<211> 123
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 59
Gly Val Ala Gly Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser
1 5 10 15
Val Leu Val Ala Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr
20 25 30
Ser Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro
35 40 45
Gly Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val
50 55 60
Thr Thr Val Ser Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile
65 70 75 80
Arg Ile Ser Asn Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val
85 90 95
Lys Phe Arg Lys Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala
100 105 110
Gly Thr Glu Leu Ser Val Arg Ala Lys Pro Ser
115 120
<210> 60
<211> 122
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 60
Arg Val Ala Gly Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser
1 5 10 15
Val Ser Val Ala Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr
20 25 30
Ser Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro
35 40 45
Ala Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val
50 55 60
Thr Thr Val Ser Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile
65 70 75 80
Ser Ile Ser Asn Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val
85 90 95
Lys Phe Arg Lys Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly
100 105 110
Thr Glu Leu Ser Val Arg Ala Lys Pro Ser
115 120
<210> 61
<211> 233
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 61
Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro
1 5 10 15
Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys
20 25 30
Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val
35 40 45
Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe
50 55 60
Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu
65 70 75 80
Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His
85 90 95
Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys
100 105 110
Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser
115 120 125
Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met
130 135 140
Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro
145 150 155 160
Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn
165 170 175
Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met
180 185 190
Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser
195 200 205
Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr
210 215 220
Lys Ser Phe Ser Arg Thr Pro Gly Lys
225 230

Claims (23)

1.一种抗SIRPα抗体或其抗原结合片段，其特征在于，包含：重链可变区和轻链可变区；所述重链可变区包含：氨基酸序列分别如SEQ ID NO:3、4、5所示的VHCDR1、VHCDR2和VHCDR3；所述轻链可变区包含：氨基酸序列分别如以下任意一组序列所示的VLCDR1、VLCDR2和VLCDR3；
(1)SEQ ID NO:37、38、9；
(2)SEQ ID NO:39、38、9；
(3)SEQ ID NO:7、40、9；
(4)SEQ ID NO:7、8、41；
(5)SEQ ID NO:7、8、42；
(6)SEQ ID NO:7、8、43；
(7)SEQ ID NO:37、38、41；
(8)SEQ ID NO:44、38、41；
(9)SEQ ID NO:7、8、9。
2.根据权利要求1所述的抗SIRPα抗体或其抗原结合片段，其特征在于，所述可变区还包含：鼠源或人源FR区。
3.根据权利要求2所述的抗SIRPα抗体或其抗原结合片段，其特征在于，所述FR区的序列为鼠源；所述重链可变区的序列如SEQ ID NO:2所示或与其具有至少85％序列同一性，所述轻链可变区的序列如SEQ ID NO:6所示或与其具有至少85％序列同一性。
4.根据权利要求2所述的抗SIRPα抗体或其抗原结合片段，其特征在于，所述人源FR区包括：重链FR区序列；所述重链FR区序列来源于人种系重链IGHV1-18及IGHJ2*01的组合序列，包括人种系重链IGHV1-18的FR1、FR2、FR3区和IGHJ2*01的FR4区。
5.根据权利要求2所述的抗SIRPα抗体或其抗原结合片段，其特征在于，所述人源FR区包括：轻链FR区序列；所述轻链FR区序列来源于人种系轻链IGKV4-1及IGKJ2*01的组合序列，包括人种系轻链IGKV4-1的FR1、FR2、FR3区和IGKJ2*01的FR4区。
6.根据权利要求2所述的抗SIRPα抗体或其抗原结合片段，其特征在于，所述重链可变区的FR区序列来源于人种系，所述重链可变区的序列如SEQ ID NO:17所示或与其具有至少85％序列同一性；
并且，所述轻链可变区的FR区序列来源于人种系，所述轻链可变区的序列选自SEQIDNO:18、19、20、21、22、23、24、25中任意一项，或与其具有至少85％序列同一性。
7.根据权利要求1所述的抗SIRPα抗体或其抗原结合片段，其特征在于，所述抗SIRPα抗体或其抗原结合片段还包含：选自人源IgG1、IgG2、IgG3、或IgG4或其变体的重链恒定区；以及选自人源κ、λ链或其变体的轻链恒定区。
8.根据权利要求7所述的抗SIRPα抗体或其抗原结合片段，其特征在于，所述轻链恒定区选自：SEQ ID NO:13的第111位至第217位的氨基酸序列。
9.根据权利要求8所述的抗SIRPα抗体或其抗原结合片段，其特征在于，所述重链恒定区包含：Fc片段或其变体。
10.根据权利要求9所述的抗SIRPα抗体或其抗原结合片段，其特征在于，所述Fc片段的变体来源于IgG1，根据EU计数，包括突变位点：L234A、L235A、K338A。
11.根据权利要求9所述的抗SIRPα抗体或其抗原结合片段，其特征在于，所述抗SIRPα抗体或其抗原结合片段的重链序列如SEQ ID NO:26所示，或与其具有至少85％序列同一性。
12.根据权利要求1所述的抗SIRPα抗体或其抗原结合片段，其特征在于，其为单克隆抗体、双特异抗体、或多特异抗体，或者，所述的抗体或其抗原结合片段用于制备抗体药物偶联物。
13.根据权利要求1所述的抗SIRPα抗体或其抗原结合片段，其特征在于，其结构形式为为Fab、F(ab’)2、Fv、或ScFv。
14.一种药物组合物，其含有权利要求1至13中任意一项所述的抗SIRPα抗体或其抗原结合片段，以及一种或多种药学上可接受的载体、稀释剂或赋形剂。
15.一种核酸分子，其编码权利要求1至13中任意一项所述的抗SIRPα抗体或其抗原结合片段。
16.一种载体，其包含权利要求15所述的核酸分子。
17.一种采用权利要求16所述的载体转化得到的宿主细胞。
18.权利要求1至13中任意一项所述的抗SIRPα抗体或其抗原结合片段在制备用于抑制或治疗癌症或动脉粥样硬化的药物中的用途，所述癌症选自实体瘤、骨髓增生异常综合征、急性淋巴细胞白血病(ALL)、急性髓性白血病(AML)、淋巴瘤、多发性骨髓瘤。
19.根据权利要求18所述的用途，其特征在于，所述的药物由所述的抗SIRPα抗体或其抗原结合片段与一种或多种其他癌症治疗剂联合制备。
20.根据权利要求18所述的用途，其特征在于，所述实体瘤选自胃癌、肺癌、胰腺癌、乳腺癌、肝癌、卵巢癌、肾癌、头颈癌、宫颈癌、结直肠癌、食道癌、胶质母细胞瘤、黑素瘤、肉瘤。
21.权利要求1至13中任意一项所述的抗SIRPα抗体或其抗原结合片段在制备用于抑制或阻断SIRPα的药物中的用途，所述药物用于抑制或治疗癌症或动脉粥样硬化，其中，所述癌症选自实体瘤、骨髓增生异常综合征、急性淋巴细胞白血病(ALL)、急性髓性白血病(AML)、淋巴瘤、多发性骨髓瘤。
22.权利要求1至13中任意一项所述的抗SIRPα抗体或其抗原结合片段在制备阻断SIRPα和CD47结合的制剂中的应用。
23.权利要求1至13中任意一项所述的抗SIRPα抗体或其抗原结合片段在制备SIRPα和CD47结合的检测剂中的应用。
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018190719A2 (en) * 2017-04-13 2018-10-18 Aduro Biotech Holdings, Europe B.V. Anti-sirp alpha antibodies

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