CN109476749A - 改良的过继性t细胞疗法 - Google Patents

改良的过继性t细胞疗法 Download PDF

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CN109476749A
CN109476749A CN201780045952.7A CN201780045952A CN109476749A CN 109476749 A CN109476749 A CN 109476749A CN 201780045952 A CN201780045952 A CN 201780045952A CN 109476749 A CN109476749 A CN 109476749A
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C·克莱因
C·祖斯特曼
G·尼德费尔纳
M·盖革
S·恩德雷斯
S·科博尔德
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F Hoffmann La Roche AG
Ludwig Maximilians Universitaet Muenchen LMU
Hoffmann La Roche Inc
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Abstract

本发明一般性涉及通过使用与融合蛋白的胞外域特异性结合/相互作用的三价双特异性抗体分子募集的,经该融合蛋白转染/转导的T细胞,诸如CD8+T细胞,CD4+T细胞,CD3+T细胞,γδT细胞或天然杀伤(NK)T细胞。更加精确地,本发明涉及包含本发明的核酸分子,载体和/或融合蛋白的试剂盒和本发明的三价双特异性抗体分子。本发明的别的方面是包含编码该融合蛋白以及该三价双特异性抗体分子的核酸分子的表达载体。而且,描述了用于生成本发明的三价双特异性抗体分子的方法和包含所述三价双特异性抗体分子的药物/药用组合物。本发明还提供所述三价双特异性抗体分子在用于治疗特定疾病的的方法以及包含所述三价双特异性抗体分子的药用组合物/药物中的用途,其中所述三价双特异性抗体分子要与包含本发明的融合蛋白的经转导T细胞组合施用。本发明还提供用于治疗特定疾病的方法。

Description

改良的过继性T细胞疗法
发明领域
本发明一般性涉及通过使用与融合蛋白的胞外域特异性结合/相互作用的三价双特异性抗体分子募集的,经该融合蛋白转染/转导的T细胞,诸如CD8+ T细胞,CD4+ T细胞,CD3+ T细胞,γδ T细胞或天然杀伤(NK)T细胞。更加精确地,本发明涉及包含本发明的核酸分子,载体和/或融合蛋白的试剂盒和本发明的三价双特异性抗体分子。本发明的别的方面是包含编码该融合蛋白以及该三价双特异性抗体分子的核酸分子的表达载体。而且,描述了用于生成本发明的三价双特异性抗体分子的方法和包含所述三价双特异性抗体分子的药物/药用组合物。本发明还提供所述三价双特异性抗体分子在用于治疗特定疾病的方法以及包含所述三价双特异性抗体分子的药用组合物/药物中的用途,其中所述三价双特异性抗体分子要与包含本发明的融合蛋白的经转导T细胞组合施用。本发明还提供用于治疗特定疾病的方法。
发明背景
过继性T细胞疗法(ACT)是一种使用癌症特异性T细胞的强有力的治疗办法(Rosenberg and Restifo,Science 348(6230)(2015),62-68)。ACT可使用天然发生肿瘤特异性细胞或T细胞,其使用T细胞或嵌合抗原受体通过遗传工程使其变得特异性的(Rosenberg and Restifo,Science 348(6230)(2015),62-68)。ACT能在甚至罹患晚期和其它方面治疗不应的疾病,诸如急性淋巴细胞性白血病,非霍奇金氏淋巴瘤或黑素瘤的患者中成功治疗和诱导消退(Dudley et al.,J Clin Oncol 26(32)(2008),5233-5239；Gruppet al.,N Engl J Med 368(16)(2013),1509-1518；Kochenderfer et al.,J Clin Oncol。(2015)33(6):540-549,doi:10.1200/JCO.2014.56.2025.Epub 2014Aug 25)。然而,长期益处局限于小子集的患者而大多数会复发及屈服于他们的顽固疾病。
认为T细胞接近肿瘤细胞或组织对于ACT的成功是至关紧要的。如此,需要开发和执行能够实现T细胞进入的策略(Gattinoni et al.,Nat Rev Immunol 6(5)(2006),383-393)。当前实现增强的T细胞浸润的最有效方法是全身辐照,它透化肿瘤组织,重塑血管系统及消减遏制性细胞(Dudley et al.,J Clin Oncol 23(10)(2005),2346-2357)。虽然这种策略在临床试验中显示功效,但是它的非特异性性质诱导严重的副作用,限制它应用并寻求更加特异性的策略(Dudley et al.,J Clin Oncol 23(10)(2005),2346-2357)。
另外,抑制剂诸如抗CTLA-4或抗PD1-抗体用于黑素瘤疗法的批准深刻地改变了治疗前景和具有转移性疾病的患者的结局(Hodi et al.,N Engl J Med 363(8)(2010),711-723；Robert et al.,N Engl J Med 372(4)(2015),320-330)。这两种模态的组合承载更加深刻的响应的希望,如通过无进展存活率和潜在延长的总体存活例示的(Larkin et al.,NEngl J Med 373(1)(2015),23-34)。然而,实质量的(如果不是大多数)患者会不受益或复发,寻求另外的治疗选项。
通过使用肿瘤浸润性淋巴细胞(TIL)进一步证明了T细胞对于黑素瘤疗法的价值,它若与全身辐照组合则诱导无比的响应率,高达77％治疗的患者(Dudley et al.,J ClinOncol 26(32)(2008),5233-5239；Rosenberg et al.,Clin Cancer Res 17(13)(2011),4550-4557)。虽然认为治愈了一些患者,但是大多数会复发且屈服于他们的疾病,指示大多数黑素瘤对T细胞攻击的天性敏感性但寻求另外的策略来维持抗肿瘤应答和预防复发。
最近显示经标志物抗原转导的抗原特异性T细胞和四价双特异性抗体分子的组合增强肿瘤识别并介导延长的肿瘤控制但未能根除肿瘤,潜在地由于局部免疫遏制和T细胞耗竭(WO 2013/113615；Kobold et al.,J Natl Cancer Inst107(1)(2015),364；Koboldet al.,J Natl Cancer Inst 107(8)(2015)1-10；Kobold et al.,Journal forImmunoTherapy of Cancer 2(Suppl.2):P42(2014))。然而,如Kobold et al.,Journalfor ImmunoTherapy of Cancer 2(Suppl.2):P42(2014)中描述的双特异性抗体分子既没有完全在结构上表征又没有保藏。如WO 2013/113615；Kobold et al.,J Natl CancerInst 107(1)(2015),364；Kobold et al.,J Natl Cancer Inst 107(8)(2015)1-10；Kobold et al.,Journal for ImmunoTherapy of Cancer 2(Suppl.2):P42(2014)中描述的策略也有不便,在于T细胞疗法依赖于MHC约束且不容许另外的T细胞刺激。而且,US2010/0256340公开三价双特异性抗体分子的构建。然而,US 2010/0256340无一处描述三价双特异性抗体分子作为用于将经融合蛋白转导的T细胞特异性募集至癌细胞的工具的用途。而且,双特异性抗体分子与经融合蛋白转导的T细胞的组合已有描述(Urbanska etal.,Journal of Translational Medicine 12(347)(2014),doi:10.1186/12967-014-0347-2)。然而,Urbanska等人出版物中描述的实验规程的唯一目的是证明通过交联两种靶向与CD8,CD28和CD3z融合的叶酸受体和肿瘤相关抗原(CD20和HER2)的单克隆抗体分子获得的抗体分子会再指引T细胞针对癌细胞。然而,由于下面的原因,Urbanska等人的数据不能排除别的概念概括:1)没有提供任何靶向与T细胞刺激域融合的不同胞外域构成的融合蛋白的抗体分子会能够活化T细胞的数据；2)没有提供在肿瘤细胞缺失下四价对T细胞活化的影响的分析；3)没有提供数据三价双特异性抗体分子会能够类似地或甚至以更强的能力实施T细胞活化。而且,Urbanska等人出版物中显示的数据可以以这样一种方式解读,即CD8域对于T细胞活化会是至关紧要的。
因而,需要改良靶向肿瘤疗法,特别是过继性T细胞疗法以满足癌症患者的需要。如此,仍然需要提供具有改善ACT的安全性和功效和克服上述缺点的潜力的改良手段。
通过提供如权利要求书中限定的实施方案,本发明解决了这种需要。
发明概述
本发明涉及一种试剂盒,其包含(A)融合蛋白,其包含在自要治疗的受试者获得的T细胞中或上并不天然发生的信号传导受体的胞外域,锚定跨膜域,和刺激性信号传导域,和(B)三价双特异性抗体分子,其结合该融合蛋白的胞外域(即在T细胞中或上并不发生的信号传导受体的胞外域)和在肿瘤细胞的表面上天然发生的肿瘤特异性抗原。在一个更加优选的实施方案中,本发明涉及一种试剂盒,其包含(A)融合蛋白,其包含在自要治疗的受试者获得的T细胞中或上并不天然发生的信号传导受体的胞外域,锚定跨膜域,至少一个共刺激性信号传导域和刺激性信号传导域,和(B)三价双特异性抗体分子,其结合该融合蛋白的胞外域(即在T细胞中或上并不发生的信号传导受体的胞外域)和在肿瘤细胞的表面上天然发生的肿瘤特异性抗原。
本发明涉及用如本文中描述的融合蛋白转导T细胞,诸如CD8+ T细胞,CD4+ T细胞,CD3+ T细胞,γδ T细胞或天然杀伤(NK)T细胞,优选CD8+ T细胞,和通过三价双特异性抗体分子将它们靶向募集至肿瘤。与WO 2013/113615的实施例中描述的具有两个针对引入该T细胞的标志物抗原的结合域和两个针对在肿瘤细胞的表面上天然发生的肿瘤特异性抗原的结合域的四价双特异性抗体形成对比,本发明基于使用具有仅仅一个针对该融合蛋白的胞外域的结合域和两个针对在肿瘤细胞的表面上天然发生的肿瘤特异性抗原的结合域,或者,两个针对该融合蛋白的胞外域的结合域和仅仅一个针对该在肿瘤细胞的表面上天然发生的肿瘤特异性抗原的结合域的三价双特异性抗体分子。如所附实施例中显示的,作为发明概念的证据,构建了一种三价双特异性抗体“BsAB EGFRvIII-EpCAM”(SEQ ID NO:233,其包含/组成为质粒/载体“EGFR vIII MR1.1VH Ck muEpCAM VH CH1EE Fc节PG LALA,pETR14953”(SEQ ID NO:22,如由SEQ ID NO:21中显示的DNA序列编码的),“EGFR vIIIMR1.1VL CH1,pETR14951”(SEQ ID NO:24(蛋白质)和23(DNA),“VL EpCAM G.8.8Ck RK,pETR14882”(SEQ ID NO:26(蛋白质)和25(DNA)和“VH muEpCAM CH1EE Fc穴PG LALA HRYF,pETR14940”(SEQ ID NO:28(蛋白质)和27(DNA)；还见图9和表1和2),其中第二结合域,即一个结合域,与(人)EGFRvIII(代表该在所述T细胞中或上并不天然发生的信号传导受体的胞外域)相互作用/结合且第一和第三结合域,即两个结合域,与鼠EpCAM(代表在肿瘤细胞的表面上天然发生的肿瘤特异性抗原)相互作用/结合。而且,构建了一种三价双特异性抗体“BsAB EGFRvIII-MSLN”(SEQ ID NO:235,其包含/组成为质粒/载体“EGFR vIII MR1.1VHCk MSLN VH CH1EE Fc节PG LALA,pETR15655”(SEQ ID NO:2,如由SEQ ID NO:1中显示的DNA序列编码的),“EGFR vIII MR1.1VL CH1,pETR15656”(SEQ ID NO:4(蛋白质)和3(DNA),“VL MSLN Ck RK,pETR15443”(SEQ ID NO:6(蛋白质)和5(DNA)和“VH MSLN CH1EE Fc穴PGLALA HRYF,pETR15667”(SEQ ID NO:8(蛋白质)和7(DNA)；还见图10和表3和4),其中第二结合域,即一个结合域,与人EGFRvIII(代表该在所述T细胞中或上并不天然发生的信号传导受体的胞外域)相互作用/结合且第一和第三结合域,即两个结合域,与人间皮素(该人间皮素的UniProt条目号是Q13421(版本号132及序列号2；SEQ ID NO:149(DNA)和150(蛋白质))相互作用/结合。此外,构建了一种三价双特异性抗体“BsAB EGFRvIII-MCSP”(SEQ ID NO:234,其包含/组成为质粒/载体“MR1.1EGFRvIII VH-Ck-(G4S)2MCSP M4-3VH CH1EE Fc节PGLALA,pETR16621”(SEQ ID NO:208(蛋白质)和207(DNA),“EGFR vIII MR1.1VL CH1,pETR15656”(SEQ ID NO:210(蛋白质)和209(DNA),“MCSP ML2VL Ck RK,pETR16619”(SEQID NO:212(蛋白质)和211(DNA)和“MCSP M4-3VH CH1EE Fc穴PG LALA HYRF,pETR16618”(SEQ ID NO:214(蛋白质)和213(DNA)；还见图11和表5和6)，其中第二结合域，即一个结合域，与人EGFRvIII(代表该在所述T细胞中或上并不天然发生的信号传导受体的胞外域)相互作用/结合且第一和第三结合域,即两个结合域，与人MCSP(黑素瘤硫酸软骨素蛋白聚糖；该人MCSP的UniProt条目号是Q6UVK1(版本号118；序列版本2；SEQ ID NO:237(蛋白质)和236(DNA))相互作用/结合。与使用具有两个与人EGFRvIII(代表该在所述T细胞中或上并不天然发生的信号传导受体的胞外域)相互作用/结合的结合域和两个与鼠EpCAM(代表该在所述T细胞中或上并不天然发生的信号传导受体的胞外域)相互作用/结合的结合域的四价双特异性抗体“BsAb EpCAM-EGFRvIII,MR1.1”(SEQ ID NO:229(轻链(无前导序列)和SEQID NO:230(重链(无前导序列))的实验相比,通过三价双特异性抗体“BsAB EGFRvIII-EpCAM”(SEQ ID NO:233),其包含/组成为质粒/载体“EGFR vIII MR1.1VH Ck muEpCAM VHCH1EE Fc节PG LALA,pETR14953”(SEQ ID NO:22,如由SEQ ID NO:21中显示的DNA序列编码的),“EGFR vIII MR1.1VL CH1,pETR14951”(SEQ ID NO:24(蛋白质)和23(DNA),“VL EpCAMG.8.8Ck RK,pETR14882”(SEQ ID NO:26(蛋白质)和25(DNA)和“VH muEpCAM CH1EE Fc穴PGLALA HRYF,pETR14940”(SEQ ID NO:28(蛋白质)和27(DNA)；还见图9和表1和2)和表达EGFRvIII-CD28-CD3z融合蛋白(SEQ ID NO:41(DNA)和42(蛋白质))的经转导肿瘤特异性T细胞(优选CD8+ T细胞)的组合治疗肿瘤令人惊讶地消除非特异性细胞毒性；见图6和7。而且,还在人肿瘤系统中显示了三价双特异性抗体“BsAB EGFRvIII-MSLN”(SEQ ID NO:235；还见表3和4)的功能性；见例如图17。因而,令人惊讶且出乎意料地发现经本发明的融合蛋白转导的T细胞,诸如CD8+ T细胞,CD4+ T细胞,CD3+ T细胞,γδ T细胞或天然杀伤(NK)T细胞,优选CD8+ T细胞能通过使用三价双特异性抗体分子来特异性刺激并通过所述三价双特异性抗体分子募集至肿瘤细胞。如此,在本发明中令人惊讶且出乎意料地显示了将三价双特异性抗体分子与经包含/组成为在T细胞中或上并不天然发生的信号传导受体的胞外域,锚定跨膜域,T细胞刺激性信号传导域(和任选的至少一个共刺激性信号传导域)的融合蛋白转导的T细胞配对会导致肿瘤细胞的特异性活化和MHC不依赖性裂解。这种办法还承载胜过常规的基于T细胞的办法的显著的安全性优点,因为T细胞在该三价双特异性抗体分子缺失下会是惰性的且它们的利用度可以通过选择的抗体分子格式来控制(即分子越小半衰期越短,反之亦然)。
因而,本发明涉及一种试剂盒,其包含(A)编码用于转导自要治疗的受试者获得的T细胞,诸如CD8+ T细胞,CD4+ T细胞,CD3+ T细胞,γδ T细胞或天然杀伤(NK)T细胞,优选CD8+ T细胞的融合蛋白的核酸分子,该融合蛋白具有下面的域:(1)在所述T细胞中或上并不天然发生的信号传导受体的胞外域；(2)锚定跨膜域；(3)任选的至少一个共刺激性信号传导域；和(4)刺激性信号传导域,和(B)三价双特异性抗体分子,其包含:(i)结合(A)中表征的融合蛋白的胞外域(1)的第一结合域；(ii)结合在肿瘤细胞的表面上天然发生的肿瘤特异性抗原的第二结合域；和(iii)结合(A)中表征的融合蛋白的胞外域(1),即在所述T细胞中或上并不天然发生的信号传导受体的胞外域或结合所述在肿瘤细胞的表面上天然发生的肿瘤特异性抗原的第三结合域。
发明详述
在本发明的语境中,“融合蛋白”涉及由来自不同来源的多肽部分构成的蛋白。因而,它可以理解为“嵌合蛋白”。通常,融合蛋白是经由连接最初编码分开的蛋白的两种或更多种基因(或优选cDNA)而创建的蛋白。这种融合基因(或融合cDNA)的翻译导致单一多肽,优选具有自每一种原始蛋白衍生的功能特性。重组融合蛋白通过重组DNA技术而人工创建,供生物学研究或治疗学中使用。本发明的融合蛋白的生成的别的详情在本文下文中描述。
在本发明的语境中,术语“多肽”,“肽”和“蛋白质”可互换使用,指氨基酸残基的聚合物。该术语还应用于其中一个或多个氨基酸残基是人工化学模拟物或相应的天然发生氨基酸的氨基酸聚合物,以及天然发生氨基酸聚合物。因而,在本发明的语境中,术语“多肽”涉及包含通过肽(酰胺)键连接的氨基酸单体的链或由其组成的分子。肽键是当一个氨基酸的羧基基团与另一个的氨基基团反应时形成的共价化学键。在本文中“多肽”不限于具有限定长度的分子。如此,在本文中术语“多肽”涉及涵盖氨基酸链的肽,寡肽,蛋白质,或多肽,其中氨基酸残基通过共价肽键而连接。然而,在本文中术语“多肽”还涵盖此类蛋白质/多肽的肽模拟物,其中氨基酸和/或肽键已经用功能性类似物替换的。术语多肽还指且不排除多肽的修饰,例如糖基化,乙酰化,磷酸化等等。此类修饰在本领域有较好描述。
术语“氨基酸”指天然发生的和合成的氨基酸,以及以与天然发生氨基酸相似的方式发挥功能的氨基酸类似物和氨基酸模拟物。天然发生氨基酸是那些由遗传密码编码的,以及那些稍后修饰的氨基酸,例如羟脯氨酸,γ-羧基谷氨酸,和O-磷酸丝氨酸。氨基酸类似物指具有与天然发生氨基酸相同的基础化学结构(即结合氢,羧基基团,氨基基团,和R基团的α-碳)的化合物,例如高丝氨酸,正亮氨酸,甲硫氨酸亚砜,甲硫氨酸甲基锍。此类类似物具有经过修饰的R基团(例如正亮氨酸)或经过修饰的肽主链,但是保留与天然发生氨基酸相同的基础化学结构。氨基酸模拟物指具有与氨基酸的一般化学结构不同的结构,但以与天然发生氨基酸相似的方式发挥功能的化学化合物。氨基酸在本文中可以通过由IUPAC-IUB生物化学命名委员会推荐的它们的公知的三字母符号或一字母符号来提及。
在本发明的语境中,融合蛋白可包含在T细胞中或上并不天然发生的信号传导受体的胞外域的片段/多肽部分。如此,包含在本文中提供的融合蛋白中的“在T细胞中或上并不天然发生的信号传导受体的胞外域”是如本文中限定的在T细胞中或上并不天然发生的信号传导受体的全长胞外域的片段/多肽部分。在本发明的语境中且如本文中上文解释的,本发明的三价双特异性抗体分子与融合蛋白的胞外域,即在T细胞中或上并不天然发生的信号传导受体的胞外域结合/相互作用。
在本发明的一个例示性的实施方案中,作为概念的证据,提供了一种融合蛋白,其包含如本文中作为SEQ ID NO:151(DNA)和152(蛋白质)显示的人EGFRvIII(人EGFRvIII的NCBI参照序列是NM_201283.1(版本:NM_201283.1；GI:41327733))的片段/多肽部分或包含如本文中作为SEQ ID NO:153(DNA)和154(蛋白质)显示的人Cripto(人Cripto的Uniprot条目号是P13385(该序列的版本号151和版本1))的片段/多肽部分。因而,在本发明的语境中,本文中描述的融合蛋白可包含/组成为如SEQ ID NO:152中显示的(如由SEQ ID NO:151中显示的DNA序列编码的)人EGFRvIII的氨基酸序列。或者,在本发明的语境中,融合蛋白可包含/组成为如SEQ ID NO:232中显示的(如由SEQ ID NO:231中显示的DNA序列编码的)人del-hEGFRvIII的氨基酸序列。此外,在本发明的语境中,本文中描述的融合蛋白可包含/组成为如SEQ ID NO:154中显示的(如由SEQ ID NO:153中显示的DNA序列编码的)人Cripto的氨基酸序列。如此,更加优选地,在T细胞中或上并不天然发生的信号传导受体的胞外域可包含或组成为如SEQ ID NO:52中显示的氨基酸序列(人EGFRvIII)(如由SEQ ID NO:51中显示的DNA序列编码的(人EGFRvIII))。在本发明的一个备选优选实施方案中，在T细胞中或上并不天然发生的信号传导受体的胞外域可包含或组成为如SEQ ID NO:232中显示的(如由SEQ ID NO:231中显示的DNA序列编码的)氨基酸序列。或者,在本发明的语境中,在T细胞中或上并不天然发生的信号传导受体的胞外域可包含或组成为如SEQ ID NO:76或78中显示的(如由SEQ ID NO:75或77中显示的DNA序列编码的)氨基酸序列。在本发明的语境中，在T细胞中或上并不天然发生的信号传导受体的胞外域可包含或组成为如SEQ ID NO:62中显示的(人Cripto)(如由SEQ ID NO:61中显示的DNA序列编码的)氨基酸序列。在本发明的语境中,还可使用更小/更短的EGFRvIII或Cripto的片段。因而,在本发明的语境中,可使用如SEQ ID NO:52,232,76或78中描绘的EGFRvIII的或如SEQ ID NO:62中描绘的Cripto更小/更短的片段。特别地,可以在本发明的融合蛋白中使用所述胞外域(即EGFRvIII或Cripto)的任何部分,前提是这个部分受到如本文中限定的双特异性抗体结合。此类片段会能够经由本发明的融合蛋白触发T细胞活化,例如CD8+ T细胞活化。优选地,本发明的融合蛋白的胞外域衍生自人EGFRvIII或人Cripto。此类胞外部分的一个例子是EGFRvIII的胞外域,例如具有如SEQ ID NO:52(如由SEQ ID NO:51中显示的DNA编码的),SEQ ID NO:232(如由SEQID NO:231中显示的DNA序列编码的)或SEQ ID NO:76(如由SEQ ID NO:75中显示的DNA编码的)中显示的氨基酸序列。此外,在本发明的语境中,Cripto的胞外域可包含/组成为如SEQID NO:62中显示的(如由SEQ ID NO:61中显示的DNA序列编码的)氨基酸序列。
在本发明的语境中,本发明的融合蛋白的锚定跨膜域可以特征在于不具有哺乳动物蛋白酶的切割位点。在本发明的语境中,蛋白酶指能够水解本发明的融合蛋白的锚定跨膜域的氨基酸序列的蛋白水解酶。术语蛋白酶包括内肽酶和外肽酶二者。在本发明的语境中,可使用格外由CD命名法规定的跨膜蛋白的任何胞外部分来生成本发明的融合蛋白,一旦结合如本文中限定的三价双特异性抗体,它活化T细胞,诸如CD8+ T细胞,CD4+ T细胞,CD3+ T细胞,γδ T细胞或天然杀伤(NK)T细胞,优选CD8+ T细胞。此类锚定跨膜域的一个例子是CD28的跨膜域,例如具有如本文中在SEQ ID NO:54中显示的(如由SEQ ID NO:53中显示的DNA序列编码的)氨基酸序列。然而,因为人序列在本发明的语境中是最优选的,所以融合蛋白的锚定跨膜域可包含或组成为如SEQ ID NO:66中显示的(如由SEQ ID NO:65中显示的DNA序列编码的)氨基酸序列。在本发明的语境中,本发明的融合蛋白的跨膜域可包含/组成为如SEQ ID NO:80中显示的(如由SEQ ID NO:79中显示的DNA序列编码的)氨基酸序列。在本文中提供的融合蛋白包含如SEQ ID NO:80中显示的EGFRvIII的跨膜域的情况中,融合蛋白可包含如SEQ ID NO:82中显示(如由SEQ ID NO:81中显示的DNA序列编码的)的锚定域。在本发明的一个例示性实施方案中,作为概念的证据,提供了一种融合蛋白,其包含或组成为如本文中作为SEQ ID NO:52(如由SEQ ID NO:51中显示的DNA序列编码的),SEQ IDNO:232(如由SEQ ID NO:231中显示的DNA序列编码的),SEQ ID NO:76(如由SEQ ID NO:75中显示的DNA序列编码的)或SEQ ID NO:78(如由SEQ ID NO:77中显示的DNA序列编码的)显示的EGFRvIII的片段/多肽部分且包含如本文中作为SEQ ID NO:156显示的(如由SEQ IDNO:155中显示的DNA序列编码的)CD28(人CD28的Uniprot条目号是P10747(该序列的版本号173和版本1))的片段/多肽部分。在本发明的语境中,可使用CD28的任何部分/片段作为锚定跨膜域。或者,可使用如格外由CD命名法提供的具有跨膜域的任何蛋白作为本发明的融合蛋白的锚定域。依照本发明,此类不具有哺乳动物蛋白酶的切割位点的锚定跨膜域可用于触发T细胞(例如CD8+ T细胞)活化。在本发明的一个别的实施方案中,融合蛋白的不具有哺乳动物蛋白酶的切割位点的锚定跨膜域可包含或组成为如SEQ ID NO:54中显示的(如由SEQ ID NO:53(小鼠)中显示的DNA序列编码的)氨基酸序列。然而,更加优选地,本发明的融合蛋白包含自人起源衍生的多肽。如此,更加优选地,包含在本发明的融合蛋白中的多肽可包含或组成为如SEQ ID NO:66中显示的(如由SEQ ID NO:65(人)中显示的DNA序列编码的)氨基酸序列。
如上文描述的,本文中提供的融合蛋白可包含CD28的锚定跨膜域,其位于如SEQID NO:156中显示的(如由SEQ ID NO:155中显示的cDNA编码的)人全长CD28蛋白质的氨基酸153至179,154至179,155至179,156至179,157至179,158至179,159至179,160至179,161至179,162至179,163至179,164至179,165至179,166至179,167至179,168至179,169至179,170至179,171至179,172至179,173至179,174至179,175至179,176至179,177至179或178至179。因而,在本发明的语境中,锚定跨膜域可包含或组成为如SEQ ID NO:66中显示的氨基酸序列。
如上文描述的,本文中提供的融合蛋白任选包含至少一个共刺激性域,它会对T细胞提供另外的活性(见下文)。本文中提供的融合蛋白可包含共刺激性信号传导域,它是鼠/小鼠或人CD28(人CD28的UniProt条目是P10747(版本号173及序列号1)；鼠/小鼠CD28的UniProt条目是P31041(版本号134及序列号2)),CD137(人CD137的UniProt条目是Q07011(版本号145及序列号1)；鼠/小鼠CD137的UniProt条目是P20334(版本号139及序列号1)),OX40(人OX40的UniProt条目是P23510(版本号138及序列号1)；鼠/小鼠OX40的UniProt条目是P43488(版本号119及序列号1)),ICOS(人ICOS的UniProt条目是Q9Y6W8(版本号126及序列号1))；鼠/小鼠ICOS的UniProt条目是Q9WV40(主要可引用登录号)或Q9JL17(次要可引用登录号)及版本号102和序列版本2)),CD27(人CD27的UniProt条目是P26842(版本号160及序列号2)；鼠/小鼠CD27的Uniprot条目是P41272(版本号137及序列版本1)),4-1-BB(鼠/小鼠4-1-BB的UniProt条目是P20334(版本号140及序列版本1)；人4-1-BB的UniProt条目是Q07011(版本号146及序列版本))或DAP10(人DAP10的UniProt条目是Q9UBJ5(版本号25及序列号1)；鼠/小鼠DAP10的UniProt条目是Q9QUJ0(主要可引用登录号)或Q9R1E7(次要可引用登录号)及版本号101和序列号1))的片段/多肽部分。在本发明的一个别的实施方案中,本发明的融合蛋白可包含一个或多个,即1,2,3,4,5,6或7个本文中限定的共刺激性信号传导域。因而,在本发明的语境中,本发明的融合蛋白可包含鼠/小鼠或优选人CD28的片段/多肽部分作为第一共刺激性信号传导域且第二共刺激性信号传导域选自由鼠/小鼠或优选人CD137,OX40,ICOS,CD27,4-1-BB和DAP10组成的组。正如所附实施例中例示的,包含在本发明的融合蛋白中的共刺激性信号传导域可包含或组成为如SEQ ID NO:56中显示的(如由SEQ ID NO:55(小鼠)中显示的DNA序列编码的)氨基酸序列和/或如SEQ ID NO:60中显示的(如由SEQ ID NO:59(小鼠)中显示的DNA序列编码的)氨基酸序列。然而,更加优选地,本发明的融合蛋白包含自人起源衍生的多肽。如此,更加优选地,本发明的融合蛋白包含自人起源衍生的多肽。如此,更加优选地,包含在本发明的融合蛋白中的共刺激性信号传导域可包含或组成为如SEQ ID NO:68中显示的(如由SEQ ID NO:67(人)中显示的DNA序列编码的)氨基酸序列和/或如SEQ ID NO:72中显示的(如由SEQ ID NO:71中显示的DNA序列编码的)氨基酸序列。
如此,可任选包含在本文中提供的融合蛋白中的共刺激性信号传导域是全长CD28,CD137,OX40,ICOS,CD27,4-1-BB或DAP10的片段/多肽部分。鼠/小鼠全长CD28,CD137,OX40,ICOS,CD27,4-1-BB或DAP10的氨基酸序列在本文中作为SEQ ID NO:158(CD28),162(CD137),166(OX40),170(ICOS),174(CD27),203(4-1-BB)或178(DAP10)显示(鼠/小鼠,如由SEQ ID NO:157(CD28),161(CD137),165(OX40),169(ICOS),173(CD27),227(4-1-BB)或177(DAP10)中显示的DNA序列编码的)。然而,因为人序列在本发明的语境中是最优选的,所以可任选包含在本文中提供的融合蛋白中的共刺激性信号传导域是人全长CD29,CD137,OX40,ICOS,CD27,4-1-BB或DAP10的片段/多肽部分。人全长CD28,CD137,OX40,ICOS,CD27,4-1-BB或DAP10的氨基酸序列在本文中作为SEQ ID NO:156(CD28),160(CD137),164(OX40),168(ICOS),172(CD27),204(4-1-BB)或176(DAP10)显示(人,如由SEQ ID NO:155(CD28),159(CD137),163(OX40),167(ICOS),171(CD27),228(4-1-BB)或175(DAP10)中显示的DNA序列编码的)。
本文中提供的融合蛋白可包含CD28的片段作为共刺激性域,前提是包含至少一个CD28的信号传导域。特别地,CD28的任何部分/片段适合于本发明的融合蛋白,只要至少包含CD28的信号传导基序之一。例如,包含在本发明的融合蛋白中的CD28多肽可包含或组成为SEQ ID NO:56中显示的(如由SEQ ID NO:55中显示的DNA序列编码的)氨基酸序列。在本发明中,作为共刺激性域发挥功能的CD28的胞内域可包含自具有序列YMNM(SEQ ID NO:122)和/或PYAP(SEQ ID NO:121)的CD28多肽的胞内域衍生的序列。然而,更加优选地,本发明的融合蛋白包含自人起源衍生的多肽。例如,可包含在本发明的融合蛋白中的人CD28的片段/多肽部分可包含或组成为SEQ ID NO:68中显示的(如由SEQ ID NO:67中显示的DNA序列编码的)氨基酸序列。因而,在本发明的语境中,融合蛋白包含如SEQ ID NO:68中显示的序列或与SEQID NO:68相比具有多至1,2,3,4,5,6,7,8,9或10处替代,删除或插入且特征在于具有共刺激性信号传导活性的序列。共刺激性信号传导活性可测定；例如通过增强的细胞因子释放(如通过ELISA(IL-2,IFNγ,TNFα)测量的),增强的增殖活性(如通过增强的细胞数测量的),或增强的裂解活性(如通过LDH释放测定法测量的)。
如上文提到的,在本发明的一个实施方案中,融合蛋白的共刺激性域可衍生自人CD28基因(Uni Prot条目号:P10747(该序列的登录号及条目版本:173和版本1))且提供CD28活性,定义为本文中描述的经转导细胞,像经转导T细胞的细胞因子生成,增殖和裂解活性。CD28活性可通过细胞因子的释放(通过细胞因子诸如干扰素-γ(IFN-γ)或白介素2(IL-2)的ELISA或流式细胞术),T细胞的增殖(例如通过ki67测量,通过流式细胞术的细胞量化测量的)(如下文在所附实施例中描述的),或裂解活性(如通过靶细胞的实时阻抗测量评估的)(通过使用例如ICELLligence仪器)来测量(如描述于例如Thakur et al.,BiosensBioelectron.35(1)(2012),503-506；Krutzik et al.,Methods Mol Biol.699(2011),179-202；Ekkens et al.,Infect Immun.75(5)(2007),2291-2296；Geet al.,Proc NatlAcad Sci U S A.99(5)(2002),2983-2988；Düwell et al.,CellDeath Differ.21(12)(2014),1825-1837,Erratum in:Cell Death Differ.21(12)(2014),161)。共刺激性信号传导域PYAP(SEQ ID NO:156(如由SEQ ID NO:155中显示的DNA序列编码的)的氨基酸(AA)208至211)和YMNM(SEQ ID NO:156的AA 191至194)有益于CD28多肽的功能和上文列举的功能效果。YMNM域的氨基酸序列在SEQ ID NO:122中显示；PYAP域的氨基酸序列在SEQ ID NO:121中显示。因而,在本发明的融合蛋白中,CD28多肽优选包含自具有序列YMNM(SEQ ID NO:122)和/或PYAP(SEQ ID NO:121)的CD28多肽的胞内域衍生的序列。在本发明的语境中,CD28多肽的胞内域具有序列YMNM(SEQ ID NO:122)和/或PYAP(SEQ ID NO:121),特征在于CD28活性,定义为本文中描述的经转导细胞,像例如经转导T细胞的细胞因子生成,增殖和裂解活性。因而,在本发明的语境中,本发明的融合蛋白的共刺激性信号传导域具有SEQ IDNO:68(人)(如由SEQ ID NO:67中显示的DNA序列编码的)或SEQ ID NO:56(小鼠/鼠)(如由SEQ ID NO:55中显示的DNA序列编码的)的氨基酸序列。然而,在本发明的融合蛋白中,这些域之一或二者可相应突变成FMNM(SEQ ID NO:123)和/或AYAA(SEQ ID NO:124)。这些突变任一降低融合蛋白释放细胞因子的能力而不影响它增殖的能力且能有利地用于延长经转导细胞的存活力和如此治疗潜力。或者,换言之,此类非功能性突变优选增强经本文中提供的融合蛋白转导的细胞在体内的持久性。然而,这些信号传导基序可存在于本文中提供的融合蛋白的胞内域内的任何位点。
因而,如上文提到的,本发明的融合蛋白可包含CD28的片段作为共刺激性域,前提是包含至少一个CD28的信号传导域。特别地,CD28的任何部分/片段适合作为共刺激性域,只要包含至少一个CD28的信号传导基序,即YMNM(SEQ ID NO:122)和/或PYAP(SEQ ID NO:121)。例如,用作共刺激性域的CD28多肽可包含或组成为SEQ ID NO:66中显示的(如由SEQID NO:65中显示的DNA序列编码的)氨基酸序列。在本发明中,作为共刺激性域发挥功能的CD28的胞内域可包含自具有序列YMNM(SEQ ID NO:122)和/或PYAP(SEQ ID NO:121)的CD28多肽的胞内域衍生的序列。在本发明的语境中,CD28多肽的共刺激性信号传导域可以是任何长度的,前提是本发明的融合蛋白的共刺激性域包含序列YMNM(SEQ ID NO:122)和/或PYAP(SEQ ID NO:121)。因而,在本发明的语境中,融合蛋白的CD28的共刺激性信号传导域可包含自具有序列YMNM(SEQ ID NO:122)和/或PYAP(SEQ ID NO:121)的CD28多肽衍生的序列。例如,包含在本发明的融合蛋白中的CD28多肽可包含或组成为如SEQ ID NO:56中显示的(如由SEQ ID NO:55中显示的DNA序列编码的)氨基酸序列。正如提到的,融合蛋白优选包含人起源的多肽。例如,包含在本发明的融合蛋白中的CD28多肽可包含或组成为如SEQ IDNO:66中显示的(如由SEQ ID NO:65中显示的DNA序列编码的)氨基酸序列。在本发明的语境中,自CD28多肽衍生的共刺激性信号传导域可以是任何长度的,前提是本发明的融合蛋白的共刺激性信号传导域包含序列YMNM(SEQ ID NO:122)和/或PYAP(SEQ ID NO:121)。因而,在本发明的语境中,融合蛋白的CD28的共刺激性域可包含自具有序列YMNM(SEQ ID NO:122)和/或PYAP(SEQ ID NO:121)的CD28多肽衍生的序列。例如,包含在本发明的融合蛋白中的CD28多肽可包含或组成为如SEQ ID NO:56(鼠/小鼠)或66(人)中显示的氨基酸序列。在本发明的语境中,融合蛋白的CD28多肽具有SEQ ID NO:66(人)的氨基酸序列。在本发明的语境中,融合蛋白包含具有序列YMNM(SEQ ID NO:122)和/或PYAP(SEQ ID NO:121)的CD28多肽的胞内域。因而,在本发明的语境中,CD28多肽具有SEQ ID NO:66(人)的氨基酸序列。
如上文描述的,本文中提供的融合蛋白包含刺激性信号传导域,其提供T细胞活化,通过与T细胞活化相同的手段来测量。本文中提供的融合蛋白可包含如下的刺激性信号传导域,它是鼠/小鼠或人CD3z(人CD3z的UniProt条目是P20963(版本号177及序列号2；鼠/小鼠CD3z的UniProt条目是P24161(主要可引用登录号)或Q9D3G3(次要可引用登录号)及版本号143和序列号1)),FCGR3A(人FCGR3A的UniProt条目是P08637(版本号178及序列号2)),或NKG2D(人NKG2D的UniProt条目是P26718(版本号151及序列号1)；鼠/小鼠NKG2D的UniProt条目是O54709(版本号132及序列号2))的片段/多肽部分。
如此,包含在本文中提供的融合蛋白中的刺激性信号传导域可以是全长CD3z,FCGR3A或NKG2D的片段/多肽部分。鼠/小鼠全长CD3z,或NKG2D的氨基酸序列在本文中作为SEQ ID NO:180(CD3z)或182(NKG2D)(鼠/小鼠,如由SEQ ID NO:179(CD3z)或181(NKG2D)中显示的DNA序列编码的)显示。人全长CD3z,FCGR3A或NKG2D的氨基酸序列在本文中作为SEQID NO:184(CD3z),186(FCGR3A)或188(NKG2D)(人,如由SEQ ID NO:183(CD3z),185(FCGR3A)或187(NKG2D)中显示的DNA序列编码的)显示。本发明的融合蛋白可包含CD3z,FCGR3A或NKG2D的片段作为刺激性域,前提是包含至少一个信号传导域。特别地,CD3z,FCGR3A,或NKG2D的任何部分/片段适合作为刺激性域,只要包含至少一个信号传导基序。然而,更加优选地,本发明的融合蛋白包含自人起源衍生的多肽。如此,更加优选地,本文中提供的融合蛋白包含如本文中作为SEQ ID NO:184(CD3z),186(FCGR3A)或188(NKG2D)(人,如由SEQ ID NO:183(CD3z),185(FCGR3A)或187(NKG2D)中显示的DNA序列编码的)显示的氨基酸序列。例如,可包含在本发明的融合蛋白中的人CD3z的片段/多肽部分可包含或组成为SEQ ID NO:70中显示的(如由SEQ ID NO:69中显示的DNA序列编码的)氨基酸序列。因而,在本发明的语境中,融合蛋白包含如SEQ ID NO:70中显示的序列或与SEQ ID NO:70相比具有多至1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,23,24,25,26,27,28,29或30处替代,删除或插入且特征在于具有刺激性信号传导活性的序列。刺激性信号传导活性可测定；例如通过增强的细胞因子释放,(如通过ELISA(IL-2,IFNγ,TNFα)测量的),增强的增殖活性(如通过增强的细胞数测量的),或增强的裂解活性(如通过LDH释放测定法测量的)。
此外,本文中提供的融合蛋白可包含接头(或“间隔物”)。接头通常是具有多至20个氨基酸的长度的肽。因而,在本发明的语境中,接头可具有1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19或20个氨基酸的长度。例如,本文中提供的融合蛋白可包含在T细胞中或上并不天然发生的信号传导受体的胞外域,不具有哺乳动物蛋白酶的切割位点的锚定跨膜域,共刺激性信号传导域和/或刺激性域之间的接头。此类接头具有它们能使得融合蛋白的不同多肽(即在T细胞中或上并不天然发生的信号传导受体的胞外域,不具有哺乳动物蛋白酶的切割位点的锚定跨膜域,共刺激性信号传导域和/或刺激性域)更有可能独立折叠且如预期的那样运转的优点。如此,在本发明的语境中,在T细胞中或上并不天然发生的信号传导受体的胞外域,不具有哺乳动物蛋白酶的切割位点的锚定跨膜域,共刺激性信号传导域和刺激性域可以包含在单链多功能多肽中。例如,单链融合构建物可以由包含一个或多个在T细胞中或上并不天然发生的信号传导受体的胞外域,一个或多个不具有哺乳动物蛋白酶的切割位点的锚定跨膜域,一个或多个共刺激性信号传导域和/或一个或多个刺激性域的一条或多条多肽组成。
而且,本文中提供的融合蛋白可含有铰链域,它作为受到抗体识别的部分和跨膜域之间的间隔物起作用。它可以是任何长度的且可以属于融合蛋白上受到三价双特异性抗体分子识别的抗原的相同或不同胞外部分。在本发明的语境中,可使用胞外蛋白的任何胞外部分作为跨膜域和抗体结合位点之间的铰链域。候选者包括CD命名法的任何成员。因而,在本发明的语境中,可使用如格外由CD命名法提供的具有胞外域的胞外部分的任何蛋白作为本发明的融合蛋白中的铰链域。此类铰链域的一个例子可以是CD8的胞外部分,例如具有如本文中在SEQ ID NO:64中显示的(如由SEQ ID NO:63(小鼠)中显示的DNA序列编码的)氨基酸序列的。然而,因为人序列在本发明的语境中是最优选的,所以融合蛋白的铰链域可包含或组成为如SEQ ID NO:74中显示的(如由SEQ ID NO:73中显示的DNA序列编码的)氨基酸序列。在本文中提供的融合蛋白包含或组成为如SEQ ID NO:64(小鼠)或74(人)中描绘的铰链域的情况中,本发明的融合蛋白不具有锚定跨膜域。因而,在本文中提供的融合蛋白包含或组成为铰链域如SEQ ID NO:64或74中描绘的情况中,融合蛋白下面的域:(1)在所述T细胞中或上并不天然发生的信号传导受体的胞外域,(2)如SEQ ID NO:64或74中描绘的铰链域,(3)任选至少一个共刺激性信号传导域；和(4)刺激性信号传导域。例示性地,如SEQ IDNO:46中描绘的(鼠/小鼠Cripto-CD28-CD3z,如由SEQ ID NO:45中显示的DNA序列编码的)融合蛋白包含如SEQ ID NO:64中描绘的铰链域。此外,如SEQ ID NO:120中描绘的融合蛋白(人Cripto-CD28-CD3z,如由SEQ ID NO:119中显示的DNA序列编码的)包含如SEQ ID NO:74中描绘的铰链域。在本发明的另一个实施方案中,本发明的融合蛋白特征在于不具有铰链域。例示性地,如SEQ ID NO:42(鼠/小鼠EGFRvIII-CD28-CD3z,如由SEQ ID NO:41中显示的DNA序列编码的),SEQ ID NO:44(鼠/小鼠EGFRvIII-CD28-4-1-BB-CD3z,如由SEQ ID NO:43中显示的DNA序列编码的),SEQ ID NO:48(人EGFRvIII-CD28-CD3z,如由SEQ ID NO:47中显示的DNA序列编码的),SEQ ID NO:50(人EGFRvIII-CD28-4-1-BB-CD3z,如由SEQ ID NO:49中显示的DNA序列编码的))中描绘的融合蛋白特征在于不具有铰链域。
本文中提供的融合蛋白可包含或组成为如SEQ ID NO:42(鼠/小鼠EGFRvIII-CD28-CD3z(如由SEQ ID NO:41中显示的DNA序列编码的)),SEQ ID NO:44(鼠/小鼠EGFRvIII-CD28-4-1-BB-CD3z(如由SEQ ID NO:43中显示的DNA序列编码的))或SEQ ID NO:46(鼠/小鼠Cripto-CD28-CD3z(如由SEQ ID NO:45中显示的DNA序列编码的))中显示的氨基酸序列。最优选地,本文中提供的融合蛋白包含或组成为如SEQ ID NO:48(人EGFRvIII-CD28-CD3z(如由SEQ ID NO:47中显示的DNA序列编码的)),SEQ ID NO:50(人EGFRvIII-CD28-4-1-BB-CD3z(如由SEQ ID NO:49中显示的DNA序列编码的))或SEQ ID NO:120(人Cripto-CD28-CD3z(如由SEQ ID NO:119中显示的DNA序列编码的))中显示的氨基酸序列。因而,本发明在一个优选的实施方案中涉及一种融合蛋白,其可具有SEQ ID NO:48,SEQ IDNO:50或SEQ ID NO:120的氨基酸序列。
如果本文中提供的融合蛋白包含Cripto的片段的话,融合蛋白可包含前导序列。此类前导序列会将蛋白质带到T细胞膜的表面。例如,在本文中提供的融合蛋白中,前导序列可具有如SEQ ID NO:206中显示的(如由SEQ ID NO:205中显示的DNA序列编码的)氨基酸序列。
因而,在本发明的语境中,试剂盒可包含或组成为与如SEQ ID NO:235中显示的三价双特异性抗体分子组合的如SEQ ID NO:42中显示的氨基酸序列(鼠/小鼠EGFRvIII-CD28-CD3z(如由SEQ ID NO:41中显示的DNA序列编码的))。或者,试剂盒可包含或组成为与如SEQ ID NO:233中显示的三价双特异性抗体分子组合的如SEQ ID NO:42中显示的氨基酸序列(鼠/小鼠EGFRvIII-CD28-CD3z(如由SEQ ID NO:41中显示的DNA序列编码的))。此外,在本发明的语境中,试剂盒可包含或组成为与如SEQ ID NO:234中显示的三价双特异性抗体分子组合的如SEQ ID NO:42中显示的氨基酸序列。此外,在本发明的语境中,试剂盒可包含或组成为与如SEQ ID NO:235中显示的三价双特异性抗体分子组合的如SEQ ID NO:44中显示的氨基酸序列(鼠/小鼠EGFRvIII-CD28-4-1-BB-CD3z(如由SEQ ID NO:43中显示的DNA序列编码的))。或者,试剂盒可包含或组成为与如SEQ ID NO:233中显示的三价双特异性抗体分子组合的如SEQ ID NO:44中显示的氨基酸序列(鼠/小鼠EGFRvIII-CD28-4-1-BB-CD3z(如由SEQ ID NO:43中显示的DNA序列编码的))。此外,试剂盒可包含或组成为与如SEQ IDNO:234中显示的三价双特异性抗体分子组合的如SEQ ID NO:44中显示的氨基酸序列。然而,因为人序列是在本发明的语境中最优选的,所以本发明的试剂盒可包含或组成为与如SEQ ID NO:235中显示的三价双特异性抗体分子组合的如SEQ ID NO:48中显示的氨基酸序列(人EGFRvIII-CD28-CD3z(如由SEQ ID NO:47中显示的DNA序列编码的))。或者,在本发明的语境中,本发明的试剂盒可包含或组成为与如SEQ ID NO:235中显示的三价双特异性抗体分子组合的如SEQ ID NO:50中显示的氨基酸序列(人EGFRvIII-CD28-4-1-BB-CD3z(如由SEQ ID NO:49中显示的DNA序列编码的))。而且,本发明的试剂盒可包含或组成为与如SEQID NO:233中显示的三价双特异性抗体分子组合的如SEQ ID NO:48中显示的氨基酸序列(人EGFRvIII-CD28-CD3z(如由SEQ ID NO:47中显示的DNA序列编码的))。或者,本发明的试剂盒可包含或组成为与如SEQ ID NO:233中显示的三价双特异性抗体分子组合的如SEQ IDNO:50中显示的氨基酸序列(人EGFRvIII-CD28-4-1-BB-CD3z(如由SEQ ID NO:49中显示的DNA序列编码的))。此外,本发明的试剂盒可包含或组成为与如SEQ ID NO:234中显示的三价双特异性抗体分子组合的如SEQ ID NO:50中显示的氨基酸序列(人EGFRvIII-CD28-4-1-BB-CD3z)。此外,本发明的试剂盒可包含或组成为与如SEQ ID NO:234中显示的三价双特异性抗体分子组合的如SEQ ID NO:48中显示的氨基酸序列(人EGFRvIII-CD28-CD3z(如由SEQID NO:47中显示的DNA序列编码的))。
而且,本发明的试剂盒的各部分可以在管形瓶或瓶中个别包装或在容器或多容器单元中组合包装。而且,本发明的试剂盒包含(闭合的)袋式细胞温育系统,其中患者细胞(优选如本文中描述的T细胞)可以在GMP(优秀制造规范,如由欧洲委员会在http://ec.europa.eu/health/documents/eudralex/index_en.htm下公布的优秀制造规范指南中描述的)条件下转导和温育。而且,本发明的试剂盒包含(闭合的)袋式细胞温育系统,其中分离的/获得的患者T细胞可以在GMP下转导和温育。而且,在本发明的语境中,试剂盒可还包含编码如本文中描述的融合蛋白的载体和/或编码如本文中上文描述的T细胞受体的核酸分子。本发明的试剂盒可有利地使用,格外用于进行本发明的方法且可以在本文中提到的多种应用中采用,例如作为研究工具或医学工具。试剂盒的制造优选遵循本领域技术人员知道的标准规程。
在此语境中,如本文中使用的术语“三价双特异性抗体分子”涉及双特异性抗体分子能够经由一个或两个结合域结合本文中描述的融合蛋白的胞外域,即在所述T细胞中或上并不天然发生的信号传导受体的胞外域,且能够经由针对在肿瘤细胞的表面上天然发生(即内源表达)的肿瘤特异性抗原的其余结合域诱导靶细胞的消除/裂解。如本文中描述的融合蛋白的胞外域的结合活化该T细胞且经由三价双特异性结合构建物使得它们与肿瘤细胞物理接触。未转导或内源T细胞(例如CD8+ T细胞)保持不受三价双特异性结合构建物影响。因而,创造性的三价双特异性抗体分子具有在体内和/或在体外裂解靶细胞(肿瘤细胞)的能力。相应的靶细胞包含表达受到创造性三价双特异性抗体分子的至少一个,优选两个结合域识别的表面分子,即在肿瘤细胞的表面上天然发生的肿瘤特异性抗原的细胞。此类表面分子在本发明的语境中表征。因而,在本发明的语境中,三价双特异性抗体分子只具有三个结合域。这意味着在本发明的语境中术语“包含”并不意味着涵盖具有超过三个结合域的双特异性抗体分子。
靶细胞的裂解可通过本领域知道的方法来检测。因而,此类方法格外包含生理学体外测定法。此类生理学测定法可监测细胞死亡,例如通过细胞膜完整性的丧失(例如基于FACS的碘化丙啶测定法,锥虫蓝流入测定法,光度测定酶释放测定法(LDH),放射测定51Cr释放测定法,荧光测定铕释放和钙黄绿素AM释放测定法)。别的测定法包含监测细胞存活力,例如通过光度测定MTT,XTT,WST-1和alamarBlue测定法,放射测定3H-Thd掺入测定法,测量细胞分裂活性的产克隆测定法,和测量线粒体跨膜梯度的荧光测定罗丹明123测定法。另外,凋亡可例如通过基于FACS的磷脂酰丝氨酸暴露测定法,基于ELISA的TUNEL测试,胱天蛋白酶活性测定法(光度测定,荧光测定或基于ELISA的)或分析变化的细胞形态(收缩,膜起泡)来监测。
如在本发明的语境中使用的术语“结合”限定至少两个“抗原相互作用位点”与彼此的结合(相互作用)。依照本发明,术语“抗原相互作用位点”限定多肽中显示与一种或一组特定抗原的特异性相互作用能力的基序。所述结合/相互作用还理解为限定“特异性识别”。依照此发明的术语“特异性识别”意味着抗体构建物能够与如本文中限定的人靶分子每一种的至少两个氨基酸特异性相互作用和/或结合。抗体能识别,相互作用和/或结合相同靶分子上的不同表位。此术语涉及抗体分子的特异性,即它在如本文中限定的人靶分子的特定区域之间区分的能力。抗原相互作用位点与它的特定抗原的特异性相互作用可导致信号的启动,例如由于诱导抗原的构象的变化,抗原的寡聚化,等。如此,作为它们的一级,二级或三级结构的结果以及所述结构的二级修饰的结果,抗原相互作用位点和抗原的氨基酸序列中的特定基序彼此结合。
如依照本发明使用的术语“特异性相互作用”意味着本发明的三价双特异性结合构建物(三价双特异性抗体分子)并不或本质上并不与相似结构的(多)肽交叉反应。因而,本发明的三价双特异性抗体分子与肿瘤标志物,细胞表面标志物,在T细胞中和/或上并不天然发生的抗原特异性结合/相互作用且能够与特定的,选择的其它化合物,抗原,细胞表面标志物,肿瘤标志物,等(在肿瘤细胞的表面上确实天然发生的)相互作用。此类三价双特异性抗体分子的具体例子在本文中下文给出。
可测试一组处于调查中的构建物的交叉反应性,例如通过评估所述组的双特异性抗体构建物在常规条件(见例如Harlow and Lane,Antibodies:A Laboratory Manual,Cold Spring Harbor Laboratory Press,(1988)和Using Antibodies:A LaboratoryManual,Cold Spring Harbor Laboratory Press,(1999))下对感兴趣(多)肽以及一些或多或少(结构上和/或功能上)密切相关的(多)肽的结合。只有那些结合感兴趣(多)肽/蛋白但并不或本质上并不结合由与感兴趣(多)肽相同的组织(例如由肿瘤组织的细胞)表达的任何其它(多)肽的构建物(即抗体,(双特异性)scFvs等等)认为是对感兴趣(多)肽/蛋白特异性的且选择用于依照本文中提供的方法的别的研究。这些方法可格外包含结合研究,与结构上和/或功能上密切相关的分子的阻断和竞争研究。这些结合研究还包含FACS分析,表面等离振子共振(SPR,例如用),分析性超速离心,等温滴定热量测定术,荧光各向异性,荧光光谱术或放射性标记的配体结合测定法。而且,可实施生理学测定法,像细胞毒性测定法和上文提到的测定法。因而,抗原相互作用位点与特定抗原的特异性相互作用的例子可包含配体对它的受体的特异性。所述定义特别包含在结合它的特异性受体后诱导信号的配体的相互作用。相应的配体的例子包含与它的特异性细胞因子受体相互作用/结合的细胞因子。所述定义还特别包含的是抗原相互作用位点对抗原诸如选择蛋白家族,整联蛋白和生长因子(像EGF)家族的抗原的结合。所述定义还特别包含的所述相互作用的另一个例子是抗原决定簇(表位)与抗体上的抗原结合位点的相互作用。
术语“结合”不仅涉及线性表位而且还可涉及由人靶分子或其部分的两个区域组成的构象表位,结构表位或不连续表位。在此发明的语境中,构象表位通过在一级序列中分开的,当多肽折叠成天然蛋白质时在分子的表面上来到一起的两个或更多个离散氨基酸序列来定义(Sela,Science 166(1969),1365and Laver,Cell 61(1990),553-536)。此外,术语“结合”在本发明的语境中与术语“相互作用”可互换使用。
因而,特异性可通过本领域知道的方法和如本文中描述的方法来实验测定。此类方法包含但不限于Western印迹,ELISA测试,RIA测试,ECL测试,IRMA测试和肽扫描。
术语(Ig衍生的)“第一结合域”,(Ig衍生的)“第二结合域”或(Ig衍生的)“第三结合域”涉及“免疫球蛋白衍生域”,具体涉及抗体分子或其片段,单链抗体,合成的抗体,抗体片段,诸如Fab,F(ab2)’,Fv或scFv片段等,或任何这些的化学修饰的衍生物。这些抗体分子可衍生自不同物种,或者可以是嵌合起源的。在本发明的语境中(正如所附实施例中例示的),本发明的双特异性抗体分子中包含的所述(Ig衍生的)第一和第三域可以是第三(Ig衍生的)“结合域”所融合的(单克隆)抗体。
本发明的“抗体”具有三个结合域且是双特异性的。抗体可以是全长来自单一物种,或者是嵌合化的或人源化的。对于具有超过两个抗原结合域的抗体,一些结合域可以是同一的,只要蛋白具有针对两种不同抗原的结合域。
如在当前申请内使用的术语“三价”表示抗体分子中存在规定数目的结合域。为此,术语“三价”表示双特异性抗体分子中存在三个结合域。三价双特异性抗体分子描述于例如Bacac et al.,Clin.Cancer Res,1-12(DOI:10.1158/1078-0432.CCR-15-1696),WO2013/026833,WO 2014/131712和WO 2016/020309。如图9,10和11中例示的,本发明的三价双特异性抗体分子可包含特异性结合第一抗原的全长抗体且包含/组成为特异性结合第二抗原的Fab片段。术语“全长抗体”表示由两条“全长抗体重链”和两条“全长抗体轻链”组成的抗体。“全长抗体重链”是以N端至C端方向由抗体重链可变域(VH),抗体恒定重链域1(CH1),抗体铰链区(HR),抗体重链恒定域2(CH2),和抗体重链恒定域3(CH3)(缩写为VH-CH1-HR-CH2-CH3)；和在亚类IgE的抗体的情况中任选的抗体重链恒定域4(CH4)组成的多肽。优选地,“全长抗体重链”是以N端至C端方向由VH,CH1,HR,CH2和CH3组成的多肽。“全长抗体轻链”是以N端至C端方向由抗体轻链可变域(VL)和抗体轻链恒定域(CL)(缩写为VL-CL)组成的多肽。抗体轻链恒定域(CL)可以是κ(卡帕)或λ(拉姆达)。两条全长抗体链经由CL域和CH1域之间的和全长抗体重链的铰链区之间的多肽间二硫键连接到一起。典型的全长抗体的例子是天然抗体,像IgG(例如IgG1和IgG2),IgM,IgA,IgD,和IgE)。依照本发明的全长抗体可来自单一物种,例如人,或者它们可以是嵌合化或人源化抗体。依照本发明的全长抗体包含两个抗原结合位点,每个由一对VH和VL形成,均特异性结合相同抗原。所述全长抗体的重或轻链的C端表示所述重或轻链的C端处的最后一个氨基酸。如本文中使用的“Fab片段”由一条轻链和一条重链的CH1和可变区构成。Fab分子的重链不能与另一重链分子形成二硫键。因而,本发明的三价双特异性抗体包括具有经由一个或多个肽接头连接别的抗原结合域,例如单链Fv,VH域和或VL域或Fab的全长抗体的恒定域结构的抗体。在本发明的一个优选实施方案中,所述全长抗体分子的CH3域可以通过“节-入-穴”技术来改变,该技术以数个实施例详细描述于例如WO 96/027011,Ridgway et al.,Protein Eng.9(1996),617-621。在“节-入-穴”技术中,(全长抗体分子的两条重链的)两个CH3域的相互作用表面可以是“节”而另一是“穴”。引入二硫桥进一步稳定异二聚体(Merchant et al.,NatureBiotech 16(1998),667-681,Atwell et al，J.Mol.Biol.270(1997)，26-35)且提高产率。
如此，在本发明的一个方面，如本文中描述的三价双特异性抗体分子可以进一步特征在于全长抗体分子的一条重链的CH3域和全长抗体分子的另一条重链的CH3域每一个在包含抗体CH3域之间的原始界面的界面处相遇；其中改变特征在于(i)一条重链的CH3域有替代，使得在一条重链的CH3域中遇到三价双特异性抗体内的另一条重链的CH3域的原始界面的原始界面内，一个氨基酸残基用具有更大侧链体积的氨基酸残基替换，由此在一条重链的CH3域的界面内生成可放置在另一条重链的CH3域的界面内的空腔中的隆起，且(ii)另一条重链的CH3域有替代，使得在第二CH3域中遇到三价双特异性抗体内的第一CH3域的原始界面的原始界面内,一个氨基酸残基用具有更小侧链体积的氨基酸残基替换,由此在第二CH3域的界面内生成其内可放置第一CH3域的界面内的隆起的空腔。
优选地,所述具有更大侧链体积的氨基酸残基选自由甘氨酸(G),精氨酸(R),苯丙氨酸(F),酪氨酸(Y),色氨酸(W)组成的组。优选地,所述具有更小侧链体积的氨基酸残基选自由丙氨酸(A),脯氨酸(P)，丝氨酸(S)，苏氨酸(T)，缬氨酸(V)组成的组。在本发明的一个方面，两个CH3域均通过引入半胱氨酸(C)作为每一个CH3域的相应位置中的氨基酸，使得两个CH3域之间的二硫桥能形成来进一步改变。
在本发明的一个优选实施方案中，如本文中描述的三价双特异性抗体分子包含“节链”的CH3域中的P329G突变和“穴链”的CH3域中的P329G突变。还可以使用CH3域之间的另外一个链间二硫桥(Merchant et al.,Nature Biotech 16(1998),667-681,Atwell etal,J.Mol.Biol.270(1997),26-35),例如通过将Y349C突变引入“节链”的CH3域并将“E356C”或“S354C”突变引入“穴链”的CH3域。
在本发明的一个备选实施方案中,如本文中描述的三价双特异性抗体分子经由CH3域中的静电互补联合而联合。静电互补联合技术描述于例如Klein et al.,LandesBioscience 4(6)(2012),653-663,Kitazawa et al.,Nat Med.18(10)(2012),1570-1574和Gunasekeran K.et al.,J Biol Chem 285(25)(2010),19637-19646。
要依照本发明采用的三价双特异性抗体分子,抗体片段,抗体衍生物(均是Ig衍生的)可以使用本领域知道的常规技术来进一步修饰,例如通过使用氨基酸删除,插入,替代,添加,和/或重组和/或本领域知道的任何其它修饰,或是单独的或是组合的。用于在免疫球蛋白链的氨基酸序列根本的DNA序列中引入此类修饰的方法是本领域技术人员公知的；见例如Sambrook(1989),见上。术语“Ig衍生域”特别涉及包含至少一个CDR的(多)肽构建物。所述Ig衍生域的片段或衍生物限定作为上文抗体分子的部分和/或通过化学/生物化学或分子生物学方法修饰的(多)肽。相应的方法是本领域知道的且格外描述于实验室手册(见Sambrook et al.,Molecular Cloning:A Laboratory Manual；Cold Spring HarborLaboratory Press,2nd edition(1989)and 3rd edition(2001)；Gerhardt et al.,Methods for General and Molecular Bacteriology ASM Press(1994)；Lefkovits,Immunology Methods Manual:The Comprehensive Sourcebook of Techniques；AcademicPress(1997)；Golemis，Protein-Protein Interactions:A Molecular Cloning ManualCold Spring Harbor Laboratory Press(2002))。
如本文中采用的术语“CDR”涉及本领域公知的“互补决定区”。CDR是免疫球蛋白中决定所述分子的特异性且与特定配体进行接触的部分。CDR是分子的最可变部分且促成这些分子的多样性。每个V域中有三个CDR区,CDR1,CDR2和CDR3。CDR-H描绘可变重链的CDR区而CDR-L涉及可变轻链的CDR区。VH意味着可变重链而VL意味着可变轻链。Ig衍生区的CDR区可以如Kabat“Sequences of Proteins of Immunological Interest”,5thedit.NIHPublication no.91-3242U.S.Department of Health and Human Services(1991)；Chothia J.Mol.Biol.196(1987),901-917或Chothia Nature 342(1989),877-883所述来确定。
因而,在本发明的语境中,术语“抗体”涉及完整免疫球蛋白分子以及此类免疫球蛋白分子的部分。而且,如上文讨论的,该术语涉及经过修饰和/或改变的抗体分子。该术语还涉及重组或合成生成的/合成的抗体。
要注意的是,在本文中限定的第一(Ig衍生)域,第二(Ig衍生)域和第三(Ig衍生)域以外,本发明的三价双特异性抗体分子可包含一个或多个另外的域,例如用于分离和/或制备重组生成的构建物。
要注意的是,依照此发明,不仅可修饰与如本文中描述的融合蛋白的胞外域,即在所述T细胞中或上并不天然发生的信号传导受体的胞外域特异性相互作用/结合的上文描述的域。还涵盖的是修饰(Ig衍生)第一域,(Ig衍生)第二域,(Ig衍生)第三域和/或连接接头区,例如人源化抗体,CDR嫁接抗体或完全人抗体。
“人源化办法”是本领域公知的且特别为抗体分子,例如Ig衍生分子描述。术语“人源化”指含有自非人抗体衍生的序列的一些部分的非人(例如鼠)抗体或其片段(诸如Fv,Fab,Fab’,F(ab’),scFvs,或抗体的其它抗原结合部分序列)的人源化形式。人源化抗体包括如下的人免疫球蛋白,其中来自人免疫球蛋白的互补决定区(CDR)的残基用来自具有期望的结合特异性,亲和力和能力的非人物种,诸如小鼠,大鼠或家兔的CDR的残基替换。一般而言,人源化抗体会包含至少一个,一般两个实质性整个如下的可变域,其中所有或实质性所有CDR区对应于非人免疫球蛋白的那些且所有或实质性所有FR区是人免疫球蛋白共有序列的那些。人源化抗体最佳地还会包含免疫球蛋白恒定区(Fc)的至少一部分,典型地是人免疫球蛋白的；格外见Jones et al.,Nature 321(1986),522-525,Presta,Curr.Op.Struct.Biol.2(1992),593-596。用于人源化非人抗体的方法是本领域公知的。一般地,人源化抗体具有引入其中的来自非人来源的一个或多个氨基酸,仍然保留抗体的原始结合活性。用于人源化抗体/抗体分子的方法进一步详述于Jones et al.,Nature 321(1986),522-525；Reichmann et al.,Nature 332(1988),323-327；和Verhoeyen et al.,Science 239(1988),1534-1536。人源化抗体,例如针对EpCAM的抗体的具体例子是本领域知道,见例如LoBuglio,Proceedings of the American Society of Clinical OncologyAbstract(1997),1562和Khor,Proceedings of the American Society of ClinicalOncology Abstract(1997),847。
因而,在此发明的语境中,特别提供了三价双特异性抗体分子,它们是人源化的且能在药用组合物中成功采用。在本发明的语境中,本文中描述的(人源化)三价双特异性抗体分子能在如本文中限定的试剂盒中采用。
在本发明的语境中,三价双特异性抗体分子(Ig衍生)结合域包含具有针对在所述T细胞中或上并不天然发生的信号传导受体的胞外域的特异性的抗原相互作用位点。
如本文中使用的,术语“在所述T细胞中或上并不天然发生的信号传导受体的胞外域”涉及并入T细胞的,在T细胞中和/或表面上并不天然呈现的且在正常(非转导)T细胞中或上并不(内源)表达的分子。如此,将在T细胞中和/或上并不天然发生的抗原/标志物人工引入T细胞。在本发明的语境中,自如本文中限定的要治疗的受试者分离/获得所述T细胞,优选CD8+ T细胞。因而,人工引入且随后在所述T细胞中和/或表面上呈现的这些分子包含对(Ig衍生的)结合域,优选抗体,抗体片段或衍生物可及的(在体外或在体内)在T细胞中和/或上并不天然发生的域或表位。在本发明的语境中,这些人工引入的分子在如本文中下文描述的(逆转录病毒)转导之后呈现在所述T细胞中和/或表面上。
在本发明的语境中,术语“在所述T细胞中或上并不天然发生的信号传导受体的胞外域”指在T细胞中和/或上并不以超过500,550,600,650,700,750,800,850,900,950或1000个抗原分子每个T细胞天然发生/内源表达的信号传导受体的胞外域。如此,信号传导受体的胞外域在T细胞中和/或上并不在正常(非转导)T细胞的群体的超过1.0,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9或2.0‰(promille)中天然发生/内源表达。在T细胞,优选CD8+ T细胞中和/或上天然发生的信号传导受体的胞外域的存在和量可通过本领域知道的方法来监测,诸如FACS分析,ELISA,共焦显微术,分析性HPLC等等。
这些分子的例子包含非免疫原性蛋白,优选人起源的。或者,所述分子可以本身是功能上惰性的蛋白分子或者会通过本领域知道的基因重组技术变成功能上惰性的(例子会是突变版本的人EGFR,如例如如SEQ ID NO:152,232,52,76或78(如由SEQ ID NO:151，231，51，75或77编码的)中描绘的EGFRvIII。EGFRvIII是在成胶质细胞瘤中,和在乳腺,卵巢和肺的癌症中找到的人表皮生长因子受体的一种突变体。突变型受体在它的胞外域中具有删除(Lorimer et al.,Proc.Natl.Acad.Sci USA 93(1996),14815-14820)。非突变型人EGFR版本在SEQ ID NO:198(如由SEQ ID NO:197中显示的DNA序列编码的)中描绘。
满足上文提到的这些标准的标志物的例子在本文中下文给出且包含但不限于Cripto(cryptic家族蛋白),CD(分化簇)家族的成员(非T细胞),EGFR,EGFRvIII,NGFR或TSH-R。
在本发明的语境中,本文中描述的三价双特异性抗体分子结合本文中描述的融合蛋白的胞外域,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域。在本发明的语境中,在T细胞中和/或上并不天然发生的信号传导受体的胞外域选自由Cripto(cryptic家族蛋白),CD(分化簇)家族的成员(非T细胞),EGFR,EGFRvIII,NGFR和TSH-R组成的组。因而,本文中描述的三价双特异性抗体分子与(唯独)在T细胞中和/或上并不天然发生(如它通过术语“非T细胞”提出的)的CD家族的成员,Cripto,EGFR,EGFRvIII,NGFR或TSH-R相互作用/结合。在本发明的语境中,本文中描述的三价双特异性抗体分子与在T细胞中和/或表面上并不内源表达(如它通过术语“非T细胞”提出的)的CD家族的成员,Cripto,EGFR,EGFRvIII,NGFR或TSH-R相互作用/结合。
Cripto(cryptic家族蛋白),CD(分化簇)家族的成员(非T细胞),EGFR,EGFRvIII,NGFR或TSH-R的(人)成员的序列在UniProtKB/Swiss-Prot数据库中可得且可以自http://www.uniprot.org/uniprot/？query＝reviewed％3Ayes取得。这些(蛋白质)序列还涉及有注解的经修饰序列。本发明还提供其中使用本文中提供的简要(concise)序列的同源序列,还有遗传等位变体等等的技术和方法。优选地，使用本文中的简要序列的此类“变体”等等。优选地，此类“变体”是遗传变体。技术人员可以容易地推导这些数据库条目(它们还可包含基因组DNA以及mRNA/cDNA的条目)中的这些(蛋白质)序列的相关编码区。例示性地，NGFR的鼠/小鼠序列可以自UniProt数据库条目Q9Z0W1(条目版本132，序列版本1)获得。NGFR的人序列可以自UniProt数据库条目P08138(条目版本182,序列版本1)获得。
如本文中与“在T细胞中和/或上并不天然发生/内源表达的信号传导受体的胞外域”联合使用的术语“CD(分化簇)家族(非T细胞)”指选自由CD9,CD10,CD11,CD12,CD13,CD14,CD15,CD16,CD17,CD18,CD19,CD20,CD21,CD22,CD23,CD24,CD25,CD26,CD27,CD28,CD29,CD30,CD31,CD32,CD33,CD34,CD35,CD36,CD37,CD38,CD39,CD40,CD41,CD43,CD46,CD48,CD49,CD50,CD51,CD54,CD55,CD56,CD57,CD59,CD61,CD63,CD64,CD66,CD67,CD68,CD70,CD72,CD74,CD75,CD76,CD77,CD79,CD81,CD82,CD83,CD84,CD87,CD88,CD89,CD90,CD91,CD92,CD93,CD94,CD95,CD97,CD98,CD99,CD100,CD101,CD102,CD103,CD104,CD105,CD106,CD107,CD108,CD109,CD110,CD111,CD112,CD113,CD114,CD115,CD116,CD117,CD118,CD119,CD121,CD123,CD124,CD125,CD126,CD130,CD131,CD133,CD134,CD135,CD136,CD137,CD138,CD140,CD141,CD142,CD143,CD144,CD146,CD147,CD148,CD151,CD153,CD155,CD156,CD157,CD158,CD159,CD160,CD161,CD162,CD163,CD164,CD166,CD167,CD168,CD169,CD170,CD171,CD172,CD177,CD178,CD179,CD180,CD181,CD182,CD183,CD184,CD185,CD186,CD191,CD192,CD193,CD200,CD201,CD204,CD206,CD207,CD208,CD209,CD217,CD218,CD220,CD221,CD222,CD223,CD224,CD225,CD226,CD227,CD228,CD230,CD231,CD232,CD233,CD234,CD236,CD238,CD239,CD241,CD242,CD243,CD244,CD246,CD248,CD249,CD252,CD253,CD254,CD256,CD257,CD258,CD261,CD262,CD263,CD264,CD265,CD266,CD267,CD268,CD269,CD270,CD271,CD276,CD277,CD280,CD281,CD282,CD283,CD284,CD286,CD288,CD289,CD290,CD292,CD294,CD295,CD296,CD297,CD298,CD299,CD300,CD301,CD302,CD303,CD304,CD305,CD306,CD309,CD312,CD314,CD315,CD316,CD317,CD318,CD319,CD320,CD321,CD322,CD324,CD325,CD326,CD327,CD328,CD329,CD331,CD332,CD333,CD334,CD335,CD336,CD337,CD338,CD339,CD340,CD344,CD349,CD350,CD351,CD352,CD353,CD354,CD355,CD357,CD358,CD360,CD361,CD362和CD363组成的组的任一CD序列。
(人)CD9(CD9抗原)的序列可以自Swiss-Prot数据库条目P21926(条目版本123,序列版本4)获得；(人)CD10(中性溶酶)的序列可以自Swiss-Prot数据库条目P08473(条目版本151,序列版本2)获得；(人)CD11(整联蛋白α-D)的序列可以自Swiss-Prot数据库条目Q13349(条目版本110,序列版本2)获得；(人)CD13(氨肽酶N)的序列可以自Swiss-Prot数据库条目P15144(条目版本145,序列版本4)获得；(人)CD14(单核细胞分化抗原CD14)的序列可以自Swiss-Prot数据库条目P08571(条目版本131,序列版本2)获得；(人)CD16(Fcγ受体IIIb)的序列可以自Swiss-Prot数据库条目Q9ULV2(条目版本51,序列版本1)获得；(人)CD18(整联蛋白β-2)的序列可以自Swiss-Prot数据库条目P05107(条目版本162,序列版本2)获得；(人)CD19(B淋巴细胞抗原CD19)的序列可以自Swiss-Prot数据库条目P15391(条目版本128,序列版本6)获得；(人)CD20(B淋巴细胞抗原CD20)的序列可以自Swiss-Prot数据库条目P11836(条目版本118,序列版本1)获得；(人)CD21(补体受体2型)的序列可以自Swiss-Prot数据库条目P20023(条目版本128,序列版本2)获得；(人)CD22(B细胞受体CD22)的序列可以自Swiss-Prot数据库条目P20273(条目版本136,序列版本2)获得；(人)CD23(低亲和力免疫球蛋白εFc受体)的序列可以自Swiss-Prot数据库条目P06734(条目版本133,序列版本1)获得；(人)CD24(信号转导物CD24)的序列可以自Swiss-Prot数据库条目P25063(条目版本106,序列版本2)获得；(人)CD26(二肽基肽酶4)的序列可以自Swiss-Prot数据库条目P27487(条目版本140,序列版本2)获得；(人)CD27(CD27抗原)的序列可以自Swiss-Prot数据库条目P26842(条目版本119,序列版本2)获得；(人)CD29(整联蛋白β-1)的序列可以自Swiss-Prot数据库条目P05556(条目版本154,序列版本2)获得；(人)CD30(肿瘤坏死因子受体超家族成员8)的序列可以自Swiss-Prot数据库条目P28908(条目版本129；序列版本1)获得；(人)CD31(血小板内皮细胞粘附分子)的序列可以自Swiss-Prot数据库条目P16284(条目版本146,序列版本1)获得；(人)CD32(低亲和力免疫球蛋白γFc区受体II-b)的序列可以自Swiss-Prot数据库条目P31994(条目版本138,序列版本2)获得；(人)CD33(髓样细胞表面抗原CD33)的序列可以自Swiss-Prot数据库条目P20138(条目版本130,序列版本2)获得；(人)CD34(造血祖细胞抗原CD34)的序列可以自Swiss-Prot数据库条目P28906(条目版本108,序列版本2)获得；(人)CD35(补体受体1型)的序列可以自Swiss-Prot数据库条目P17927(条目版本131,序列版本3)获得；(人)CD36(血小板糖蛋白4)的序列可以自Swiss-Prot数据库条目P16671(条目版本133,序列版本2)获得；(人)CD38(ADP-核糖基环化酶1)的序列可以自Swiss-Prot数据库条目P28907(条目版本126,序列版本2)获得；(人)CD39(胞外核苷三磷酸二磷酸水解酶1)的序列可以自Swiss-Prot数据库条目P49961(条目版本114,序列版本1)获得；(人)CD40(肿瘤坏死因子受体超家族成员5)的序列可以自Swiss-Prot数据库条目P25942(条目版本147,序列版本1)获得；(人)CD41(整联蛋白α-IIb)的序列可以自Swiss-Prot数据库条目P08514(条目版本158,序列版本3)获得；(人)CD43(白涎蛋白)的序列可以自Swiss-Prot数据库条目P16150(条目版本110,序列版本1)获得；(人)CD46(膜辅因子蛋白)的序列可以自Swiss-Prot数据库条目P15529(条目版本145,序列版本3)获得；(人)CD48(CD48抗原)的序列可以自Swiss-Prot数据库条目P09326(条目版本137,序列版本2)获得；(人)CD49(整联蛋白α-4)的序列可以自Swiss-Prot数据库条目P13612(条目版本128,序列版本3)获得；(人)CD50(细胞间粘附分子3)的序列可以自Swiss-Prot数据库条目P32942(条目版本128,序列版本2)获得；(人)CD51(整联蛋白α-V)的序列可以自Swiss-Prot数据库条目P06756(条目版本149,序列版本2)获得；(人)CD54(细胞间粘附分子1)的序列可以自Swiss-Prot数据库条目P05362(条目版本160,序列版本2)获得；(人)CD55(补体衰退加速因子)的序列可以自Swiss-Prot数据库条目P08174(条目版本143,序列版本4)获得；(人)CD56(神经细胞粘附分子1)的序列可以自Swiss-Prot数据库条目P13591(条目版本132,序列版本3)获得；(人)CD57(杀伤细胞凝集素样受体亚家族G成员1)的序列可以自Swiss-Prot数据库条目Q96E93(条目版本72,序列版本1)获得；(人)CD59(CD59糖蛋白)的序列可以自Swiss-Prot数据库条目P13987(条目版本139,序列信息1)获得；(人)CD61(整联蛋白β-3)的序列可以自Swiss-Prot数据库条目P05106(条目版本175,序列版本2)获得；(人)CD63(CD63抗原)的序列可以自Swiss-Prot数据库条目P08962(条目版本122,序列版本2)获得；(人)CD64(高亲和力免疫球蛋白γFc受体I)的序列可以自Swiss-Prot数据库条目P12314(条目版本128,序列版本2)获得；(人)CD66(癌胚抗原相关细胞粘附分子1)的序列可以自Swiss-prot数据库条目P13688(条目版本133,序列版本2)获得；(人)CD67(癌胚抗原相关细胞粘附分子8)的序列可以自Swiss-type prot数据库条目P31997(条目版本115,序列版本2)获得；(人)CD68(巨噬涎蛋白)的序列可以自Swiss-Prot数据库条目P34810(条目版本106,序列版本2)获得；(人)CD70(CD70抗原)的序列可以自Swiss-Prot数据库条目P32970(条目版本101,序列版本2)获得；(人)CD72(B细胞分化抗原CD72)的序列可以自Swiss-Prot数据库条目P21854(版本条目113,序列版本1)获得；(人)CD74(HLA II类组织相容性抗原γ链)的序列可以自Swiss-Prot数据库条目P04233(条目版本141,序列版本3)获得；(人)CD75(β-半乳糖苷α-2,6-唾液基转移酶1)的序列可以自Swiss-Prot数据库条目P15907(条目版本130,序列版本1)获得；(人)CD77(乳糖基酰基神经酰胺4-α-半乳糖基转移酶)的序列可以自Swiss-Prot数据库条目Q9NPC4(条目版本100,序列版本1)获得；(人)CD79(B细胞抗原受体复合物相关蛋白α链)的序列可以自Swiss-Prot数据库条目P11912(条目版本120,序列版本2)获得；(人)CD81(CD81抗原)的序列可以自Swiss-Prot数据库条目P60033(条目版本82,序列版本1)获得；(人)CD82(CD82抗原)的序列可以自Swiss-Prot数据库条目P27701(条目版本98,序列版本1)获得；(人)CD83(CD83抗原)的序列可以自Swiss-Prot数据库条目Q01151(条目版本113,序列版本1)获得；(人)CD84(SLAM家族成员5)的序列可以自Swiss-Prot数据库条目Q9UIB8(条目版本87,序列版本1)获得；(人)CD87(尿激酶纤溶酶原活化剂表面受体)的序列可以自Swiss-Prot数据库条目Q03405(条目版本129,序列版本1)获得；(人)CD88(C5a过敏毒素趋化受体)的序列可以自Swiss-Prot数据库条目P21730(条目版本116,序列版本2)获得；(人)CD89(免疫球蛋白αFc受体)的序列可以自Swiss-Prot数据库条目P24071(条目版本121,序列版本1)获得；(人)CD90(Thy-1膜糖蛋白)的序列可以自Swiss-Prot数据库条目P04216(条目版本128,序列版本2)获得；(人)CD91(原低密度脂蛋白受体相关蛋白1)的序列可以自Swiss-Prot数据库条目Q07954(条目版本133,序列版本2)获得；(人)CD92(胆碱转运蛋白样蛋白1)的序列可以自Swiss-Prot数据库条目Q8WWI5(条目版本79,序列版本1)获得；(人)CD93(补体成分C1q受体)的序列可以自Swiss-Prot数据库条目Q9NPY3(条目版本115,序列版本3)获得；(人)CD94(天然杀伤细胞抗原CD94)的序列可以自Swiss-Prot数据库条目Q13241(条目版本107,序列版本2)获得；(人)CD95(肿瘤坏死因子配体超家族成员6)的序列可以自Swiss-Prot数据库条目P48023(条目版本134,序列版本1)获得；(人)CD97(CD97抗原)的序列可以自Swiss-Prot数据库条目P48960(条目版本125,序列版本4)获得；(人)CD98(4F2细胞表面抗原重链)的序列可以自Swiss-Prot数据库条目P08195(条目版本140,序列版本3)获得；(人)CD99(CD99抗原)的序列可以自Swiss-Prot数据库条目P14209(条目版本117,序列版本1)获得；(人)CD100(脑信号蛋白-4D)的序列可以自Swiss-Prot数据库条目Q92854(条目版本125,序列版本1)获得；(人)CD101(免疫球蛋白超家族成员2)的序列可以自Swiss-Prot数据库条目Q93033(条目版本89,序列版本2)获得；(人)CD102(细胞间粘附分子2)的序列可以自Swiss-Prot数据库条目P13598(条目版本131,序列版本2)获得；(人)CD103(整联蛋白α-E)的序列可以自Swiss-Prot数据库条目P38570(条目版本118,序列版本3)获得；(人)CD104(整联蛋白β-4)的序列可以自Swiss-Prot数据库条目P16144(条目版本160,序列版本5)获得；(人)CD105(内皮糖蛋白)的序列可以自Swiss-Prot数据库条目P17813(条目版本133,序列版本2)获得；(人)CD106(血管细胞粘附蛋白1)的序列可以自Swiss-Prot数据库条目P19320(条目版本158,序列版本1)获得；(人)CD107(溶酶体相关膜糖蛋白1)的序列可以自Swiss-Prot数据库条目P11279(条目版本117,序列版本3)获得；(人)CD108(脑信号蛋白-7A)的序列可以自Swiss-Prot数据库条目O75326(条目版本107,序列版本1)获得；(人)CD109(CD109抗原)的序列可以自Swiss-Prot数据库条目Q6YHK3(条目版本64,序列版本2)获得；(人)CD110(血小板生成素受体)的序列可以自Swiss-Prot数据库条目P40238(条目版本122,序列版本1)获得；(人)CD111(脊髓灰质炎病毒受体相关蛋白1)的序列可以自Swiss-Prot数据库条目Q15223(条目版本114,序列版本3)获得；(人)CD112(脊髓灰质炎病毒受体相关蛋白2)的序列可以自Swiss-Prot数据库条目Q92692(条目版本123,序列版本1)获得；(人)CD113(脊髓灰质炎病毒受体相关蛋白3)的序列可以自Swiss-Prot数据库条目Q9NQS3(条目版本78,序列版本1)获得；(人)CD114(粒细胞集落刺激因子受体)的序列可以自Swiss-Prot数据库条目Q99062(条目版本129,序列版本1)获得；(人)CD115(巨噬细胞集落刺激因子1受体)的序列可以自Swiss-Prot数据库条目P07333(条目版本145,序列版本2)获得；(人)CD116(粒细胞-巨噬细胞集落刺激因子受体亚基α)的序列可以自Swiss-Prot数据库条目P15509(条目版本128,序列版本1)获得；(人)CD117(肥大/干细胞生长因子受体kit)的序列可以自Swiss-Prot数据库条目P10721(条目版本150,序列版本1)获得；(人)CD118(白血病抑制因子受体)的序列可以自Swiss-Prot数据库条目P42702(条目版本115,序列版本1)获得；(人)CD119(干扰素γ受体1)的序列可以自Swiss-Prot数据库条目P15260(条目版本140,序列版本1)获得；(人)CD121(白介素-1受体1型)的序列可以自Swiss-Prot数据库条目P14778(条目版本151,序列版本1)获得；(人)CD123(白介素-3受体亚基α)的序列可以自Swiss-Prot数据库条目P26951(条目版本110,序列版本1)获得；(人)CD124(白介素-4受体亚基α)的序列可以自Swiss-Prot数据库条目P24394(条目版本144,序列版本1)获得；(人)CD125(白介素-5受体亚基α)的序列可以自Swiss-Prot数据库条目Q01344(条目版本120,序列版本2)获得；(人)CD126(白介素-6受体亚基α)的序列可以自Swiss-Prot数据库条目P08887(条目版本143,序列版本1)获得；(人)CD130(白介素-6受体亚基β)的序列可以自Swiss-Prot数据库条目P40189(条目版本142,序列版本2)获得；(人)CD131(细胞因子受体共同亚基β)的序列可以自Swiss-Prot数据库条目P32927(条目版本128,序列版本2)获得；(人)CD133(Prominin-1)的序列可以自Swiss-Prot数据库条目O43490(条目版本110,序列版本1)获得；(人)CD134(肿瘤坏死因子受体超家族成员4)的序列可以自Swiss-Prot数据库条目P43489(条目版本106,序列版本1)获得；(人)CD135(受体型酪氨酸蛋白质激酶FLT-3)的序列可以自Swiss-Prot数据库条目P36888(条目版本119,序列版本2)获得；(人)CD136(巨噬细胞刺激蛋白受体)的序列可以自Swiss-Prot数据库条目Q04912(条目版本129,序列版本2)获得；(人)CD137(肿瘤坏死因子受体超家族成员9)的序列可以自Swiss-Prot数据库条目Q07011(条目版本109,序列版本1)获得；(人)CD138(黏结蛋白聚糖-1)的序列可以自Swiss-Prot数据库条目P18827(条目版本114,序列版本3)获得；(人)CD140(血小板衍生生长因子受体β)的序列可以自Swiss-Prot数据库条目P09619(条目版本154,序列版本1)获得；(人)CD141(血栓调节蛋白)的序列可以自Swiss-Prot数据库条目P07204(条目版本162,序列版本2)获得；(人)CD142(组织因子)的序列可以自Swiss-Prot数据库条目P13726(条目版本137,序列版本1)获得；(人)CD143(血管紧张肽转变酶)的序列可以自Swiss-Prot数据库条目P12821(条目版本157,序列版本1)获得；(人)CD144(钙粘蛋白-5)的序列可以自Swiss-Prot数据库条目P33151(条目版本108,序列版本5)获得；(人)CD146(细胞表面糖蛋白MUC18)的序列可以自Swiss-Prot数据库条目P43121(条目版本109,序列版本2)获得；(人)CD147(Basigin)的序列可以自Swiss-Prot数据库条目P35613(条目版本134,序列版本2)获得；(人)CD148(受体型酪氨酸蛋白质磷酸酶η)的序列可以自Swiss-Prot数据库条目Q12913(条目版本124,序列版本3)获得；(人)CD151(CD151抗原)的序列可以自Swiss-Prot数据库条目P48509(条目版本108,序列版本3)获得；(人)CD153(肿瘤坏死因子配体超家族成员8)的序列可以自Swiss-Prot数据库条目P32971(条目版本90,序列版本1)获得；(人)CD155(脊髓灰质炎病毒受体)的序列可以自Swiss-Prot数据库条目P15151(条目版本132,序列版本2)获得；(人)CD156(含有解联蛋白和金属硫蛋白酶域的蛋白8)的序列可以自Swiss-Prot数据库条目P78325(条目版本115,序列版本1)获得；(人)CD157(ADP-核糖基环化酶2)的序列可以自Swiss-Prot数据库条目Q10588(条目版本116,序列版本2)获得；(人)CD158(杀伤细胞免疫球蛋白样受体3DL3)的序列可以自Swiss-Prot数据库条目Q8N743(条目版本91,序列版本2)获得；(人)CD159(NKG2-A/NKG2-B II型膜内在蛋白)的序列可以自Swiss-Prot数据库条目P26715(条目版本116,序列版本2)获得；(人)CD160(CD160抗原)的序列可以自Swiss-Prot数据库条目O95971(条目版本98,序列版本1)获得；(人)CD161(杀伤细胞凝集素样受体亚家族B成员1)的序列可以自Swiss-Prot数据库条目Q12918(条目版本81,序列版本1)获得；(人)CD162(P-选择蛋白糖蛋白配体1)的序列可以自Swiss-Prot数据库条目Q14242(条目版本103,序列版本1)获得；(人)CD163(清除受体富半胱氨酸1型蛋白M130)的序列可以自Swiss-Prot数据库条目Q86VB7(条目版本77,序列版本2)获得；(人)CD164(唾液粘蛋白核心蛋白24)的序列可以自Swiss-Prot数据库条目Q04900(条目版本89),序列版本2)获得；(人)CD166(CD166抗原)的序列可以自Swiss-Prot数据库条目Q13740(条目版本111,序列版本2)获得；(人)CD167(含有盘菌素域的受体2)的序列可以自Swiss-Prot数据库条目Q16832(条目版本120,序列版本2)获得；(人)CD168(乙酰透明质酸介导的运动力受体)的序列可以自Swiss-Prot数据库条目O75330(条目版本99,序列版本2)获得；(人)CD169(唾液酸粘附素)的序列可以自Swiss-Prot数据库条目Q9BZZ2(条目版本103,序列版本2)获得；(人)CD170(唾液酸结合性Ig样凝集素5)的序列可以自Swiss-Prot数据库条目O15389(条目版本106,序列版本1)获得；(人)CD171(神经细胞粘附分子L1)的序列可以自Swiss-Prot数据库条目P32004(条目版本139,序列版本2)获得；(人)CD172(信号调节蛋白β-1)的序列可以自Swiss-Prot数据库条目O00241(条目版本112,序列版本5)获得；(人)CD177(CD177抗原)的序列可以自Swiss-Prot数据库条目Q8N6Q3(条目版本65,序列版本2)获得；(人)CD178(肿瘤坏死因子配体超家族成员6)的序列可以自Swiss-Prot数据库条目P48023(条目版本134,序列版本1)获得；(人)CD179(免疫球蛋白ι链)的序列可以自Swiss-Prot数据库条目P12018(条目版本115,序列版本2)获得；(人)CD180(CD180抗原)的序列可以自Swiss-Prot数据库条目Q99467(条目版本101,序列版本2)获得；(人)CD181(C-X-C趋化因子受体1型)的序列可以自Swiss-Prot数据库条目P25024(条目版本125,序列版本2)获得；(人)CD182(C-X-C趋化因子受体2型)的序列可以自Swiss-Prot数据库条目P25025(条目版本123,序列版本2)获得；(人)CD183(C-X-C趋化因子受体3型)的序列可以自Swiss-Prot数据库条目P49682(条目版本118,序列版本2)获得；(人)CD184(C-X-C趋化因子受体4型)的序列可以自Swiss-Prot数据库条目P61073(条目版本95,序列版本1)获得；(人)CD185(C-X-C趋化因子受体5型)的序列可以自Swiss-Prot数据库条目P32302(条目版本109,序列版本1)获得；(人)CD186(C-X-C趋化因子受体6型)的序列可以自Swiss-Prot数据库条目O00574(条目版本104,序列版本1)获得；(人)CD191(C-C趋化因子受体1型)的序列可以自Swiss-Prot数据库条目P32246(条目版本106,序列版本1)获得；(人)CD192(C-C趋化因子受体2型)的序列可以自Swiss-Prot数据库条目P41597(条目版本128,序列版本1)获得；(人)CD193(C-C趋化因子受体3型)的序列可以自Swiss-Prot数据库条目P51677(条目版本112,序列版本1)获得；(人)CD200(OX-2膜糖蛋白)的序列可以自Swiss-Prot数据库条目P41217(条目版本110,序列版本4)获得；(人)CD201(内皮蛋白C受体)的序列可以自Swiss-Prot数据库条目Q9UNN8(条目版本110,序列版本1)获得；(人)CD204(巨噬细胞清除受体I和II型)的序列可以自Swiss-Prot数据库条目P21757(条目版本122,序列版本1)获得；(人)CD206(巨噬细胞甘露糖受体1)的序列可以自Swiss-Prot数据库条目P22897(条目版本138,序列版本1)获得；(人)CD207(C型凝集素域家族4成员K)的序列可以自Swiss-Prot数据库条目Q9UJ71(条目版本85,序列版本2)获得；(人)CD208(溶酶体相关膜糖蛋白3)的序列可以自Swiss-Prot数据库条目Q9UQV4(条目版本69,序列版本3)获得；(人)CD209(CD209抗原)的序列可以自Swiss-Prot数据库条目Q9NNX6(条目版本103,序列版本1)获得；(人)CD217(白介素-17受体A)的序列可以自Swiss-Prot数据库条目Q96F46(条目版本94,序列版本2)获得；(人)CD218(白介素-18受体1)的序列可以自Swiss-Prot数据库条目Q13478(条目版本104,序列版本1)获得；(人)CD220(胰岛素受体)的序列可以自Swiss-Prot数据库条目P06213(条目版本175,序列版本4)获得；(人)CD221(胰岛素样生长因子1受体)的序列可以自Swiss-Prot数据库条目P08069(条目版本145,序列版本1)获得；(人)CD222(阳离子不依赖性甘露糖-6-磷酸受体)的序列可以自Swiss-Prot数据库条目P11717(条目版本137,序列版本3)获得；(人)CD223(淋巴细胞活化基因3蛋白)的序列可以自Swiss-Prot数据库条目P18627(条目版本108,序列版本5)获得；(人)CD224(γ-谷酰基转肽酶1)的序列可以自Swiss-Prot数据库条目P19440(条目版本137,序列版本2)获得；(人)CD225(干扰素诱导的跨膜蛋白1)的序列可以自Swiss-Prot数据库条目P13164(条目版本101,序列版本3)获得；(人)CD226(CD226抗原)的序列可以自Swiss-Prot数据库条目Q15762(条目版本89,序列版本2)获得；(人)CD227(粘蛋白-1)的序列可以自Swiss-Prot数据库条目P15941(条目版本136,序列版本3)获得；(人)CD228(黑素运铁蛋白)的序列可以自Swiss-Prot数据库条目P08582(条目版本124,序列版本2)获得；(人)CD230(主要朊蛋白)的序列可以自Swiss-Prot数据库条目P04156(条目版本161,序列版本1)获得；(人)CD231(四跨膜蛋白-7)的序列可以自Swiss-Prot数据库条目P41732(条目版本115,序列版本2)获得；(人)CD232(丛蛋白-C1)的序列可以自Swiss-Prot数据库条目O60486(条目版本80,序列版本1)获得；(人)CD233(条带3阴离子转运蛋白)的序列可以自Swiss-Prot数据库条目P02730(条目版本167,序列版本3)获得；(人)CD234(Duffy抗原/趋化因子受体)的序列可以自Swiss-Prot数据库条目Q16570(条目版本114,序列版本3)获得；(人)CD236(血型糖蛋白-C)的序列可以自Swiss-Prot数据库条目P04921(条目版本116,序列版本1)获得；(人)CD238(Kell血型糖蛋白)的序列可以自Swiss-Prot数据库条目P23276(条目版本124,序列版本2)获得；(人)CD239(基细胞粘附分子)的序列可以自Swiss-Prot数据库条目P50895(条目版本117,序列版本2)获得；(人)CD241(铵转运蛋白Rh A型)的序列可以自Swiss-Prot数据库条目Q02094(条目版本98,序列版本2)获得；(人)CD242(细胞间粘附分子4)的序列可以自Swiss-Prot数据库条目Q14773(条目版本106,序列版本1)获得；(人)CD243(多药抗性蛋白1)的序列可以自Swiss-Prot数据库条目P08183(条目版本146,序列版本3获得；(人)CD244(天然杀伤细胞受体2B4)的序列可以自Swiss-Prot数据库条目Q9BZW8(条目版本94,序列版本2)获得；(人)CD246(ALK酪氨酸激酶受体)的序列可以自Swiss-Prot数据库条目Q9UM73(条目版本120,序列版本3)获得；(人)CD248(内皮唾液酸蛋白)的序列可以自Swiss-Prot数据库条目Q9HCU0(条目版本87,序列版本1)获得；(人)CD249(谷酰基氨肽酶)的序列可以自Swiss-Prot数据库条目Q07075(条目版本121,序列版本3)获得；(人)CD252(肿瘤坏死因子配体超家族成员4)的序列可以自Swiss-Prot数据库条目P23510(条目版本101,序列版本1)获得；(人)CD253(肿瘤坏死因子配体超家族成员10)的序列可以自Swiss-Prot数据库条目P50591(条目版本118,序列版本1)获得；(人)CD254(肿瘤坏死因子配体超家族成员11)的序列可以自Swiss-Prot数据库条目O14788(条目版本110,序列版本1)获得；(人)CD256(肿瘤坏死因子配体超家族成员13)的序列可以自Swiss-Prot数据库条目O75888(条目版本111,序列版本1)获得；(人)CD257(肿瘤坏死因子配体超家族成员13B)的序列可以自Swiss-Prot数据库条目Q9Y275(条目版本127,序列版本1)获得；(人)CD258(肿瘤坏死因子配体超家族成员14)的序列可以自Swiss-Prot数据库条目O43557(条目版本117,序列版本2)获得；(人)CD261(肿瘤坏死因子受体超家族成员10A)的序列可以自Swiss-Prot数据库条目O00220(条目版本112,序列版本3)获得；(人)CD262(肿瘤坏死因子受体超家族成员10B)的序列可以自Swiss-Prot数据库条目O14763(条目版本133,序列版本2)获得；(人)CD263(肿瘤坏死因子受体超家族成员10C)的序列可以自Swiss-Prot数据库条目O14798(条目版本99,序列版本3)获得；(人)CD264(肿瘤坏死因子受体超家族成员10D)的序列可以自Swiss-Prot数据库条目Q9UBN6(条目版本109,序列版本1)获得；(人)CD265(肿瘤坏死因子受体超家族成员11A)的序列可以自Swiss-Prot数据库条目Q9Y6Q6(条目版本100,序列版本1)获得；(人)CD266(肿瘤坏死因子受体超家族成员12A)的序列可以自Swiss-Prot数据库条目Q9NP84(条目版本89,序列版本1)获得；(人)CD267(肿瘤坏死因子受体超家族成员13B)的序列可以自Swiss-Prot数据库条目O14836(条目版本102,序列版本1)获得；(人)CD268(肿瘤坏死因子受体超家族成员13C)的序列可以自Swiss-Prot数据库条目Q96RJ3(条目版本91,序列版本1)获得；(人)CD269(肿瘤坏死因子受体超家族成员17)的序列可以自Swiss-Prot数据库条目Q02223(条目版本125,序列版本2)获得；(人)CD270(肿瘤坏死因子受体超家族成员14)的序列可以自Swiss-Prot数据库条目Q92956(条目版本134,序列版本3)获得；(人)CD271(肿瘤坏死因子受体超家族成员16)的序列可以自Swiss-Prot数据库条目P08138(条目版本135,序列版本1)获得；(人)CD276(CD276抗原)的序列可以自Swiss-Prot数据库条目Q5ZPR3(条目版本71,序列版本1)获得；(人)CD277(嗜乳脂蛋白亚家族3成员A1)的序列可以自Swiss-Prot数据库条目O00481(条目版本102,序列版本3)获得；(人)CD280(C型甘露糖受体2)的序列可以自Swiss-Prot数据库条目Q9UBG0(条目版本79,序列版本2)获得；(人)CD281(Toll样受体1)的序列可以自Swiss-Prot数据库条目Q15399(条目版本125,序列版本3)获得；(人)CD282(Toll样受体2)的序列可以自Swiss-Prot数据库条目O60603(条目版本129,序列版本1)获得；(人)CD283(Toll样受体3)的序列可以自Swiss-Prot数据库条目O15455(条目版本120,序列版本1)获得；(人)CD284(Toll样受体4)的序列可以自Swiss-Prot数据库条目O00206(条目版本125,序列版本2)获得；(人)CD286(Toll样受体6)的序列可以自Swiss-Prot数据库条目Q9Y2C9(条目版本108,序列版本2)获得；(人)CD288(Toll样受体8)的序列可以自Swiss-Prot数据库条目Q9NR97(条目版本103,序列版本1)获得；(人)CD289(Toll样受体9)的序列可以自Swiss-Prot数据库条目Q9NR96(条目版本107,序列版本2)获得；(人)CD290(Toll样受体10)的序列可以自Swiss-Prot数据库条目Q9BXR5(条目版本105,序列版本2)获得；(人)CD292(骨形态发生蛋白受体1A型)的序列可以自Swiss-Prot数据库条目P36894(条目版本146,序列版本2)获得；(人)CD294(推定的G蛋白偶联受体44)的序列可以自Swiss-Prot数据库条目Q9Y5Y4(条目版本91,序列版本3)获得；(人)CD295(瘦蛋白受体)的序列可以自Swiss-Prot数据库条目P48357(条目版本132,序列版本2)获得；(人)CD296(GPI连接的NAD(P)(+)--精氨酸ADP-核糖基转移酶1)的序列可以自Swiss-Prot数据库条目P52961(条目版本96,序列版本2)获得；(人)CD297(胞外-ADP-核糖基转移酶4)的序列可以自Swiss-Prot数据库条目Q93070(条目版本106,序列版本2)获得；(人)CD298(钠/钾转运ATP酶亚基β-3)的序列可以自Swiss-Prot数据库条目P54709(条目版本102,序列版本1)获得；(人)CD299(C型凝集素域家族4成员M)的序列可以自Swiss-Prot数据库条目Q9H2X3(条目版本Q9H2X3(条目版本108,序列版本1))获得；(人)CD300(CMRF35样分子9)的序列可以自Swiss-Prot数据库条目Q6UXG3(条目版本67,序列版本2)获得；(人)CD301(C型凝集素域家族10成员A)的序列可以自Swiss-Prot数据库条目Q8IUN9(条目版本80,序列版本1)获得；(人)CD302(CD302抗原)的序列可以自Swiss-Prot数据库条目Q8IX05(条目版本64,序列版本1)获得；(人)CD303(C型凝集素域家族4成员C)的序列可以自Swiss-Prot数据库条目Q8WTT0(条目版本82,序列版本1)获得；(人)CD304(神经毡蛋白-1)的序列可以自Swiss-Prot数据库条目O14786(条目版本129,序列版本3)获得；(人)CD305(白细胞相关免疫球蛋白样受体1)的序列可以自Swiss-Prot数据库条目Q6GTX8(条目版本70,序列版本1)获得；(人)CD306(白细胞相关免疫球蛋白样受体2)的序列可以自Swiss-Prot数据库条目Q6ISS4(条目版本63,序列版本1)获得；(人)CD309(血管内皮生长因子受体2)的序列可以自Swiss-Prot数据库条目P35968(条目版本138,序列版本2)获得；(人)CD312(含有EGF样模块的粘蛋白样激素受体样2)的序列可以自Swiss-Prot数据库条目Q9UHX3(条目版本113,序列版本2)获得；(人)CD314(NKG2-D II型膜内在蛋白)的序列可以自Swiss-Prot数据库条目P26718(条目版本117,序列版本1)获得；(人)CD315(前列腺素F2受体负调节物)的序列可以自Swiss-Prot数据库条目Q9P2B2(条目版本98,序列版本2)获得；(人)CD316(免疫球蛋白超家族成员8)的序列可以自Swiss-Prot数据库条目Q969P0(条目版本81,序列版本1)获得；(人)CD317(骨髓基质抗原2)的序列可以自Swiss-Prot数据库条目Q10589(条目版本95,序列版本1)获得；(人)CD318(含有CUB域的蛋白1)的序列可以自Swiss-Prot数据库条目Q9H5V8(条目版本78,序列版本3)获得；(人)CD319(SLAM家族成员7)的序列可以自Swiss-Prot数据库条目Q9NQ25(条目版本92,序列版本1)获得；(人)CD320(CD320抗原)的序列可以自Swiss-Prot数据库条目Q9NPF0(条目版本86,序列版本1)获得；(人)CD321(连接粘附分子A)的序列可以自Swiss-Prot数据库条目Q9Y624(条目版本124,序列版本1)获得；(人)CD322(连接粘附分子B)的序列可以自Swiss-Prot数据库条目P57087(条目版本107,序列版本1)获得；(人)CD324(钙粘蛋白-1)的序列可以自Swiss-Prot数据库条目P12830(条目版本157,序列版本3)获得；(人)CD325(钙粘蛋白-2)的序列可以自Swiss-Prot数据库条目P19022(条目版本118,序列版本4)获得；(人)CD326(上皮细胞粘附分子)的序列可以自Swiss-Prot数据库条目P16422(条目版本118,序列版本2)获得；(人)CD327(唾液酸结合性Ig样凝集素6)的序列可以自Swiss-Prot数据库条目O43699(条目版本107,序列版本2)获得；(人)CD328(唾液酸结合性Ig样凝集素7)的序列可以自Swiss-Prot数据库条目Q9Y286(条目版本111,序列版本1)获得；(人)CD329(唾液酸结合性Ig样凝集素8)的序列可以自Swiss-Prot数据库条目Q9NYZ4(条目版本100,序列版本2)获得；(人)CD331(成纤维细胞生长因子受体1)的序列可以自Swiss-Prot数据库条目P11362(条目版本169,序列版本3)获得；(人)CD332(成纤维细胞生长因子受体2)的序列可以自Swiss-Prot数据库条目P21802(条目版本165,序列版本1)获得；(人)CD333(成纤维细胞生长因子受体3)的序列可以自Swiss-Prot数据库条目P22607(条目版本161,序列版本1)获得；(人)CD334(成纤维细胞生长因子受体4)的序列可以自Swiss-Prot数据库条目P22455(条目版本136,序列版本2)获得；(人)CD335(天然细胞毒性触发受体1)的序列可以自Swiss-Prot数据库条目O76036(条目版本98,序列版本1)获得；(人)CD336(天然细胞毒性触发受体2)的序列可以自Swiss-Prot数据库条目O95944(条目版本86,序列版本2)获得；(人)CD337(天然细胞毒性触发受体3)的序列可以自Swiss-Prot数据库条目O14931(条目版本103,序列版本1)获得；(人)CD338(ATP结合盒亚家族G成员2)的序列可以自Swiss-Prot数据库条目Q9UNQ0(条目版本120,序列版本3)获得；(人)CD339(锯齿蛋白(Jagged)-1)的序列可以自Swiss-Prot数据库条目P78504(条目版本(条目版本129；序列版本3)获得；(人)CD340(受体酪氨酸蛋白质激酶erbB-2)的序列可以自Swiss-Prot数据库条目P04626(条目版本162,序列版本1)获得；(人)CD344(卷曲蛋白(Frizzled)-4)的序列可以自Swiss-Prot数据库条目Q9ULV1(条目版本107,序列版本2)获得；(人)CD349(卷曲蛋白-9)的序列可以自Swiss-Prot数据库条目O00144(条目版本103,序列版本1)获得；(人)CD350(卷曲蛋白-10)的序列可以自Swiss-Prot数据库条目Q9ULW2(条目版本100,序列版本1)获得；(人)CD351(高亲和力免疫球蛋白α和免疫球蛋白μFc受体)的序列可以自Swiss-Prot数据库条目Q8WWV6(条目版本65,序列版本1)获得；(人)CD352(SLAM家族成员6)的序列可以自Swiss-Prot数据库条目Q96DU3(条目版本93,序列版本3)获得；(人)CD353(SLAM家族成员8)的序列可以自Swiss-Prot数据库条目Q9P0V8(条目版本80,序列版本1)获得；(人)CD354(在髓样细胞上表达的触发受体1)的序列可以自Swiss-Prot数据库条目Q9NP99(条目版本93,序列版本1)获得；(人)CD355(细胞毒性和调节T细胞分子)的序列可以自Swiss-Prot数据库条目O95727(条目版本81,序列版本2)获得；(人)CD357(肿瘤坏死因子受体超家族成员18)的序列可以自Swiss-Prot数据库条目Q9Y5U5(条目版本103,序列版本1)获得；(人)CD358(肿瘤坏死因子受体超家族成员21)的序列可以自Swiss-Prot数据库条目O75509(条目版本110,序列版本1)获得；(人)CD360(白介素-21受体)的序列可以自Swiss-Prot数据库条目Q9HBE5(条目版本104,序列版本1)获得；(人)CD361(蛋白EVI2B)的序列可以自Swiss-Prot数据库条目P34910(条目版本87,序列版本2)获得；(人)CD362(黏结蛋白聚糖-2)的序列可以自Swiss-Prot数据库条目P34741(条目版本105,序列版本2)获得；(人)CD363(鞘氨醇1-磷酸受体1)的序列可以自Swiss-Prot数据库条目P21453(条目版本116,序列版本2)获得；(人)Criptic家族蛋白(Criptic家族蛋白1-B)的序列可以自Swiss-Prot数据库条目P0CG36(条目版本12,序列版本1)获得；(人)促甲状腺素受体(TSH-R)的序列可以自Swiss-Prot数据库条目P16473(条目版本152，序列版本2)获得；或(人)表皮生长因子受体(EGFR)的序列可以自Swiss-Prot数据库条目P00533(条目版本178，序列版本2；(SEQ ID NO:198(蛋白质)和197(DNA))获得。
如上文提到的，上文描述的三价双特异性抗体分子的(Ig衍生的)域可包含对细胞表面分子，即在肿瘤细胞的表面上天然发生的肿瘤特异性抗原具有特异性的抗原相互作用位点。
如本文中使用的，术语“在肿瘤细胞的表面上天然发生的细胞表面分子”/“在肿瘤细胞的表面上天然发生的肿瘤特异性抗原”还表示在肿瘤细胞的表面上呈现的分子。术语“天然发生”涉及在肿瘤细胞的表面上内源表达的分子。术语“细胞表面分子”涉及在细胞的表面上(天然/内源)表达/呈现且包含对如本文中描述的(Ig衍生的)三价双特异性抗体的域(在体外或在体内)可及的域或表位的分子。所述细胞表面分子的例子是膜和跨膜蛋白,适应所述蛋白或细胞表面的分子等。因而,在本发明的语境中,所述细胞表面分子是肿瘤特异性标志物。在本发明的语境中,所述肿瘤特异性标志物涉及通常在肿瘤细胞的表面上内源表达的标志物。
在此发明的语境中,术语“肿瘤特异性标志物”涉及在肿瘤细胞的表面上天然/内源呈现和/或定位或在肿瘤细胞的表面上遍在表达但只对三价双特异性抗体分子,抗体片段或抗体衍生物的结合可及的分子。如本文中提到的“肿瘤特异性标志物”描述在肿瘤细胞上优先或唯独表达的蛋白。优先意味着在肿瘤上比在正常体细胞上相对更高表达而唯独意味着通过专家知道的蛋白质检测的标准手段在体细胞上没有找到的蛋白在肿瘤细胞上的表达。满足这些标准的蛋白可以例如通过本领域公知的相减或差异表达筛选来鉴定。要得到本发明的疗法方法采用所需要的肿瘤细胞特异性表达的程度可通过其中将表达感兴趣抗原的细胞和对这种抗原特异性的T细胞在一起温育并测定诱导的杀伤的特异性的细胞测定法来评估。
“优先表达”指由于由影响此类蛋白的周转的基因扩增，转录上调或mRNA稳定化或突变介导的蛋白质过表达，与正常细胞相比在肿瘤细胞上高表达的蛋白。优先还定义在肿瘤细胞上而且还在正常细胞上表达，但其中正常细胞通常对T细胞或抗体诸如人体的免疫特权区不可及的蛋白。另外,在治疗的范围内在肿瘤细胞上表达但在正常细胞上不表达的蛋白落在此定义下,诸如唯独在胚胎发育期间表达的蛋白。
“唯独表达”指在治疗过程期间仅仅在肿瘤细胞上找到的蛋白。优选地,此类蛋白展示在细胞表面上且携带在正常细胞上没有找到的在它们的胞外部分中的点突变或删除。类似地,源自脱落酶的肿瘤特异性活性的新表位属于这个范畴。唯独表达还包括唯独在肿瘤上但非在正常细胞上找到的异常糖结构。
在肿瘤细胞的表面上天然发生的肿瘤标志物的例子在本文中下文给出且包含但不限于EpCAM(上皮细胞粘附分子),MSLN(间皮素),MCSP(黑素瘤硫酸软骨素蛋白聚糖),HER-1(人表皮生长因子1),HER-2(人表皮生长因子2),HER-3(人表皮生长因子3),CD20,CD22,CD33,CD52,FMS样酪氨酸激酶3(FLT-3),叶酸受体1(FOLR1),人滋养层细胞表面抗原2(Trop-2),癌抗原12-5(CA-12-5),人白细胞抗原-抗原D相关的(HLA-DR),MUC-1(粘蛋白-1),A33-抗原,PSMA(前列腺特异性膜抗原),PSCA(前列腺干细胞抗原),运铁蛋白受体,生腱蛋白或碳酸酐酶IX(CA-IX)。
因而,在本发明的语境中,本文中描述的三价双特异性抗体分子结合选自由EpCAM(上皮细胞粘附分子),MSLN(间皮素),MCSP(黑素瘤硫酸软骨素蛋白聚糖),HER-1(人表皮生长因子1),HER-2(人表皮生长因子2),HER-3(人表皮生长因子3),CD20,CD22,CD33,CD52,FMS样酪氨酸激酶3(FLT-3),叶酸受体1(FOLR1),人滋养层细胞表面抗原2(Trop-2),癌抗原12-5(CA-12-5),人白细胞抗原-抗原D相关的(HLA-DR),MUC-1(粘蛋白-1),A33-抗原,PSMA(前列腺特异性膜抗原),PSCA(前列腺干细胞抗原),运铁蛋白受体,生腱蛋白或CA-IX(碳酸酐酶IX)组成的组的在肿瘤细胞的表面上天然发生的抗原/标志物。
EpCAM(上皮细胞粘附分子),MSLN(间皮素),MCSP(黑素瘤硫酸软骨素蛋白聚糖),HER-1(人表皮生长因子1),HER-2(人表皮生长因子2),HER-3(人表皮生长因子3),CD20,CD22,CD33,CD52,FMS样酪氨酸激酶3(FLT-3),叶酸受体1(FOLR1),人滋养层细胞表面抗原2(Trop-2),癌抗原12-5(CA-12-5),人白细胞抗原-抗原D相关的(HLA-DR),MUC-1(粘蛋白-1),A33-抗原,PSMA(前列腺特异性膜抗原),PSCA(前列腺干细胞抗原),运铁蛋白受体,生腱蛋白或CA-IX(碳酸酐酶IX)的(人)成员的序列在UniProtKB/Swiss-Prot数据库中可得且可以自http://www.uniprot.org/uniprot/？query＝reviewed％3Ayes取得。这些(蛋白质)序列还涉及有注解的经修饰序列。本发明还提供其中使用本文中提供的简要序列的同源序列,还有遗传等位变体等等的技术和方法。优选地,使用本文中的简要序列的此类“变体”等等。优选地,此类“变体”是遗传变体。技术人员可以容易地推导这些数据库条目(它们可还包含基因组DNA以及mRNA/cDNA的条目)中的这些(蛋白质)序列的相关编码区。
(人)EpCAM(上皮细胞粘附分子)的序列可以自Swiss-Prot数据库条目P16422(条目版本117,序列版本2)获得；(人)MSLN(间皮素)的序列可以自UniProt条目号Q13421(版本号132获得；序列版本2)；SEQ ID NO:(149(DNA)和150(蛋白质)获得；(人)FMS样酪氨酸激酶3(FLT-3)的序列可以自Swiss-Prot数据库条目P36888(主要可引用登录号)或Q13414(次要登录号)及版本号165和序列版本2获得；(人)MCSP(黑素瘤硫酸软骨素蛋白聚糖)的序列可以自UniProt条目号Q6UVK1(版本号118；序列版本2)获得；(人)叶酸受体1(FOLR1)的序列可以自UniProt条目号P15328(主要可引用登录号)或Q53EW2(次要登录号)及版本号153和序列版本3获得；(人)滋养层细胞表面抗原2(Trop-2)的序列可以自UniProt条目号P09758(主要可引用登录号)或Q15658(次要登录号)及版本号172和序列版本3获得；(人)PSCA(前列腺干细胞抗原)的序列可以自UniProt条目号O43653(主要可引用登录号)或Q6UW92(次要登录号)及版本号134和序列版本1获得；(人)HER-1(表皮生长因子受体1)的序列可以自Swiss-Prot数据库条目P00533(条目版本177,序列版本2)获得；(人)HER-2(表皮生长因子2)的序列可以自Swiss-Prot数据库条目P04626(条目版本161,序列版本1)获得；(人)HER-3(表皮生长因子3)的序列可以自Swiss-Prot数据库条目P21860(条目版本140,序列版本1)获得；(人)CD20(B淋巴细胞抗原CD20)的序列可以自Swiss-Prot数据库条目P11836(条目版本117,序列版本1)获得；(人)CD22(B淋巴细胞抗原CD22)的序列可以自Swiss-Prot数据库条目P20273(条目版本135,序列版本2)获得；(人)CD33(B淋巴细胞抗原CD33)的序列可以自Swiss-Prot数据库条目P20138(条目版本129,序列版本2)获得；(人)CA-12-5(粘蛋白16)的序列可以自Swiss-Prot数据库条目Q8WXI7(条目版本66,序列版本2)获得；(人)HLA-DR的序列可以自Swiss-Prot数据库条目Q29900(条目版本59,序列版本1)获得；(人)MUC-1(粘蛋白-1)的序列可以自Swiss-Prot数据库条目P15941(条目版本135,序列版本3)获得；(人)A33(细胞表面A33抗原)的序列可以自Swiss-Prot数据库条目Q99795(条目版本104,序列版本1)获得；(人)PSMA(前列腺特异性膜抗原)的序列可以自Swiss-Prot数据库条目Q04609(条目版本133,序列版本1)获得；(人)运铁蛋白受体的序列可以自Swiss-Prot数据库条目Q9UP52(条目版本99,序列版本1)和P02786(条目版本152,序列版本2)获得；(人)生腱蛋白的序列可以自Swiss-Prot数据库条目P24821(条目版本141,序列版本3)获得；或(人)CA-IX(碳酸酐酶IX)的序列可以自Swiss-Prot数据库条目Q16790(条目版本115,序列版本2)获得。
本文中提供的分子或构建物(即本文中描述的三价双特异性抗体分子)在医学背景中特别有用。例如,恶性疾病可以用本文中描述的三价双特异性抗体分子来治疗。在本发明的语境中,恶性疾病可以是上皮,内皮或间皮起源的癌症/癌或血液的癌症。在本发明的语境中,癌症/癌选自由胃肠癌,胰腺癌,胆管细胞癌,肺癌,乳腺癌,卵巢(ovarian)癌,皮肤癌,口腔癌,胃癌,宫颈癌,B和T细胞淋巴瘤,髓样白血病,卵巢(ovarial)癌,白血病,淋巴细胞性白血病,鼻咽癌,结肠癌,前列腺癌,肾细胞癌,头和颈癌,皮肤癌(黑素瘤),生殖泌尿道癌,例如睾丸癌,卵巢癌,内皮癌,宫颈癌和肾癌,胆管癌,食管癌,唾液腺癌和甲状腺癌或其它肿瘤性疾病像血液学肿瘤,胶质瘤,肉瘤或骨肉瘤组成的组。
本文中提供的分子或构建物(即本文中描述的双特异性抗体分子)在医学背景中特别有用。例如,肿瘤性疾病和/或淋巴瘤可以用针对这些医学适应证的双特异性构建物来治疗。三价双特异性抗体/分子的适应证通过肿瘤抗原的表达来给出。在实体中表达的肿瘤抗原实际上可以与任何上文提到的T细胞标志物(代表在肿瘤细胞的表面上天然发生/内源表达的抗原)组合。例如,胃肠癌,胰腺癌,胆管细胞癌,肺癌,乳腺癌,卵巢癌,皮肤癌和/或口腔癌可以用针对(人)EpCAM(作为在肿瘤细胞的表面上天然发生的肿瘤特异性抗原)且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域之一的结合域的三价双特异性分子或构建物(即本文中描述的三价双特异性抗体分子)来治疗。因而,在本发明的语境中,三价双特异性抗体构建物针对EpCAM,优选人EpCAM,且进一步包含一个或两个与之相互作用/结合/针对Cripto的结合域,可用于治疗胃肠癌,例如胃肠起源的腺癌。如此,在本发明的语境中,三价双特异性抗体构建物/分子经由一个结合域针对EpCAM,优选人EpCAM,且包含两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域。在本发明的一个备选实施方案中,三价双特异性抗体分子还可以以这样一种方式设计,即它经由两个结合域针对EpCAM,优选人EpCAM,且一个结合域针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域。
胃肠癌,胰腺癌,胆管细胞癌,肺癌,乳腺癌,卵巢癌,皮肤癌和/或口腔癌可以用经由一个或两个结合域针对HER1,优选人HER1(作为在肿瘤细胞的表面上天然发生的肿瘤特异性抗原)且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的三价双特异性分子或构建物(即本文中描述的三价双特异性抗体分子)来治疗。这意味着胃肠癌,胰腺癌,胆管细胞癌,肺癌,乳腺癌,卵巢癌,皮肤癌和/或口腔癌可以用经由两个结合域针对HER1,优选人HER1(作为在肿瘤细胞的表面上天然发生的肿瘤特异性抗原)且包含一个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的三价双特异性分子或构建物(即本文中描述的三价双特异性抗体分子)来治疗。或者,胃肠癌,胰腺癌,胆管细胞癌,肺癌,乳腺癌,卵巢癌,皮肤癌和/或口腔癌可以用经由一个结合域针对HER1,优选人HER1(作为在肿瘤细胞的表面上天然发生的肿瘤特异性抗原)且包含两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的三价双特异性分子或构建物(即本文中描述的三价双特异性抗体分子)来治疗。而且,胃肠癌,胰腺癌,胆管细胞癌,肺癌,乳腺癌,卵巢癌,皮肤癌,成胶质细胞瘤和/或口腔癌可以用经由一个或两个结合域针对MCSP,优选人MCSP(作为在肿瘤细胞的表面上天然发生的肿瘤特异性抗原)且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的三价双特异性分子或构建物(即本文中描述的三价双特异性抗体分子)来治疗。胃肠癌,胰腺癌,胆管细胞癌,肺癌,乳腺癌,卵巢癌,皮肤癌,成胶质细胞瘤和/或口腔癌可以用经由一个或两个结合域针对FOLR1,优选人FOLR1(作为在肿瘤细胞的表面上天然发生的肿瘤特异性抗原)且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的三价双特异性分子或构建物(即本文中描述的三价双特异性抗体分子)来治疗。胃肠癌,胰腺癌,胆管细胞癌,肺癌,乳腺癌,卵巢癌,皮肤癌,成胶质细胞瘤和/或口腔癌可以用经由一个或两个结合域针对Trop-2,优选人Trop-2(作为在肿瘤细胞的表面上天然发生的肿瘤特异性抗原)且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的三价双特异性分子或构建物(即本文中描述的三价双特异性抗体分子)来治疗。胃肠癌,胰腺癌,胆管细胞癌,肺癌,乳腺癌,卵巢癌,皮肤癌,成胶质细胞瘤和/或口腔癌可以用经由一个或两个结合域针对PSCA,优选人PSCA(作为在肿瘤细胞的表面上天然发生的肿瘤特异性抗原)且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的三价双特异性分子或构建物(即本文中描述的三价双特异性抗体分子)来治疗。胃肠癌,胰腺癌,胆管细胞癌,肺癌,乳腺癌,卵巢癌,皮肤癌,成胶质细胞瘤和/或口腔癌可以用经由一个或两个结合域针对EGFRvIII,优选人EGFRvIII(作为在肿瘤细胞的表面上天然发生的肿瘤特异性抗原)且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的三价双特异性分子或构建物(即本文中描述的三价双特异性抗体分子)来治疗。胃肠癌,胰腺癌,胆管细胞癌,肺癌,乳腺癌,卵巢癌,皮肤癌,成胶质细胞瘤和/或口腔癌可以用经由一个或两个结合域针对MSLN,优选人MSLN(作为在肿瘤细胞的表面上天然发生的肿瘤特异性抗原)且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的三价双特异性分子或构建物(即本文中描述的三价双特异性抗体分子)来治疗。胃癌,乳腺癌和/或宫颈癌可以用经由一个或两个结合域针对HER2,优选人HER2(作为在肿瘤细胞的表面上天然发生的肿瘤特异性抗原)且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的三价双特异性分子或构建物(即本文中描述的三价双特异性抗体分子)来治疗。胃癌和/或肺癌可以用经由一个或两个结合域针对HER3,优选人HER3(作为在肿瘤细胞的表面上天然发生的肿瘤特异性抗原)且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的三价双特异性分子或构建物(即本文中描述的三价双特异性抗体分子)来治疗。B细胞淋巴瘤和/或T细胞淋巴瘤可以用经由一个或两个结合域针对CD20,优选人CD20(作为在肿瘤细胞的表面上天然发生的肿瘤特异性抗原)且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的三价双特异性分子或构建物(即本文中描述的三价双特异性抗体分子)来治疗。B细胞淋巴瘤和/或T细胞淋巴瘤可以用经由一个或两个结合域针对CD22,优选人CD22(作为在肿瘤细胞的表面上天然发生的肿瘤特异性抗原)且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的三价双特异性分子或构建物(即本文中描述的三价双特异性抗体分子)来治疗。髓样白血病可以用经由一个或两个结合域针对CD33,优选人CD33(作为在肿瘤细胞的表面上天然发生的肿瘤特异性抗原)且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的三价双特异性构建物来治疗。卵巢癌,肺癌,乳腺癌和/或胃肠癌可以用经由一个或两个结合域针对CA12-5,优选人CA12-5(作为在肿瘤细胞的表面上天然发生的肿瘤特异性抗原)且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的三价双特异性分子或构建物(即本文中描述的三价双特异性抗体分子)来治疗。胃肠癌,白血病和/或鼻咽癌可以用经由一个或两个结合域针对HLA-DR,优选人HLA-DR(作为在肿瘤细胞的表面上天然发生的肿瘤特异性抗原)且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的三价双特异性分子或构建物(即本文中描述的三价双特异性抗体分子)来治疗。结肠癌,乳腺癌,卵巢癌,肺癌和/或胰腺癌可以用经由一个或两个结合域针对MUC-1,优选人MUC-1(作为在肿瘤细胞的表面上天然发生的肿瘤特异性抗原)且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的三价双特异性分子或构建物(即本文中描述的三价双特异性抗体分子)来治疗。结肠癌可以用经由一个或两个结合域针对A33,优选人A33(作为在肿瘤细胞的表面上天然发生的肿瘤特异性抗原)且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的三价双特异性分子或构建物(即本文中描述的三价双特异性抗体分子)来治疗。前列腺癌可以用经由一个或两个结合域针对PSMA,优选人PSMA(作为在肿瘤细胞的表面上天然发生的肿瘤特异性抗原)且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的三价双特异性分子或构建物(即本文中描述的三价双特异性抗体分子)来治疗。胃肠癌,胰腺癌,胆管细胞癌,肺癌,乳腺癌,卵巢癌,皮肤癌和/或口腔癌可以用经由一个或两个结合域针对运铁蛋白受体,优选人运铁蛋白受体(作为在肿瘤细胞的表面上天然发生的肿瘤特异性抗原)且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的三价双特异性分子或构建物(即本文中描述的三价双特异性抗体分子)来治疗。胰腺癌,肺癌和/或乳腺癌可以用经由一个或两个结合域针对运铁蛋白受体,优选人运铁蛋白受体(作为在肿瘤细胞的表面上天然发生的肿瘤特异性抗原)且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的三价双特异性分子或构建物(即本文中描述的三价双特异性抗体分子)来治疗。肾癌可以用经由一个或两个结合域针对CA-IX,优选人CA-IX(作为在肿瘤细胞的表面上天然发生的肿瘤特异性抗原)且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的三价双特异性分子或构建物(即本文中描述的三价双特异性抗体分子)来治疗。
还如所附实施例中例示的,作为本发明的概念的证据,本发明的特异性三价双特异性抗体分子包含上文限定的与之相互作用/针对/结合人EGFRvIII的第一和第二(Ig衍生的)域和与之相互作用/针对/结合鼠EpCAM的第二(Ig衍生的)域(见图9)。而且,图10例示本发明的一种特异性三价双特异性抗体分子,其包含上文限定的与之相互作用/针对/结合人EGFRvIII的第一和第二(Ig衍生的)域和与之相互作用/针对/结合人MSLN的第二(Ig衍生的)域。此外,图11例示本发明的一种特异性三价双特异性抗体分子,其包含上文限定的与之相互作用/针对/结合人EGFRvIII的第一和第二(Ig衍生的)域和与之相互作用/针对/结合人MCSP的第二(Ig衍生的)域。可通过使用针对EGFRvIII和EpCAM的三价双特异性抗体,通过使用针对EGFRvIII和MSLN的三价双特异性抗体,或通过使用针对EGFRvIII和MCSP的三价双特异性抗体分子来治疗的疾病包括胃肠癌,胰腺癌,胆管细胞癌,肺癌,乳腺癌,卵巢癌,皮肤癌,成胶质细胞瘤和/或口腔癌。
上皮细胞粘附分子(EpCAM,还称作17-1A抗原,KSA,EGP40,GA733-2,ks1-4或esa)是一种314个氨基酸的40kDa膜整合糖蛋白,具有某些上皮中和许多人癌上的特异性表达(综述见Balzar,J.Mol.Med.(1999),77,699-712)。EpCAM是经由鼠单克隆抗体17-1A/edrecolomab对它的识别而发现和随后克隆的(Goettlinger,Int J Cancer 38(1986),47-53和Simon,Proc.Natl.Acad.Sci.USA 87(1990),2755-2759)。EpCAM用来以定向且高度有序的方式粘附上皮细胞(Litvinov,J Cell Biol.139(1997),1337-1348)。上皮细胞一旦恶性转化,快速生长的肿瘤细胞放弃上皮的高细胞秩序。因而,EpCAM的表面分布变成更少受到约束且该分子变成更好地在肿瘤细胞上暴露且在肿瘤细胞的表面上对抗体,抗体片段或抗体衍生物结合可及。由于它们的上皮细胞起源,来自大多数癌的肿瘤细胞仍然在它们的表面上表达EpCAM。
在体内,EpCAM的表达与升高的上皮增殖有关且与细胞分化负相关(综述见Balzar,J.Mol.Med.77(1999),699-712)。本质上对所有重大癌看到EpCAM的表达(综述见Balzar,J.Mol.Med.77(1999),699-712或格外记录于De Bree,Nucl Med Commun.15(1994),613-27；Zhang,Clin Cancer Res.4(1998),295-302)。因为它的广泛表达,EpCAM称作“泛癌”抗原。在许多情况中,观察到肿瘤细胞表达EpCAM至比它们的亲本上皮或所述癌症的更低攻击性形式高得多的程度。例如,升高的EpCAM表达代表前列腺癌的发生中的一个早期事件(Poczatek,J.Urol.162(1999),1462-1644)。另外,在宫颈的大多数鳞和腺癌二者中,强EpCAM表达与升高的增殖和终末分化的标志物的消失有关(Litvinov,Am.J.Pathol.148(1996),865-75)。在乳腺癌中,EpCAM在肿瘤细胞上的过表达是存活的一项预测器(Gastl,Lancet 356(2000),1981-1982)。EpCAM是检测罹患头,颈和肺的鳞状细胞癌的患者中播散性肿瘤细胞的一项标志物(Chaubal,Anticancer Res.19(1999),2237-2242和Piyathilake,Hum.Pathol.31(2000),482-487)。如在表皮,口腔,会厌,咽,喉和食管中找到的,正常鳞状上皮并不显著表达EpCAM(Quak,Hybridoma 9(1990),377-387)。已经显示EpCAM在大多数原发性，转移性，和播散性NSCLC(非小细胞肺癌细胞(Passlick，Int JCancer 87(2000)，548-552))上,在胃和胃-食管连接腺癌上(Martin，J.Clin.Pathol.52(1999)，701-4)和在自结肠直肠，胰腺癌和乳腺癌衍生的细胞系中(Szala,Proc.Natl.Acad.Sci.USA 87(1990),3542-6和Packeisen，Hybridoma 18(1999)，37-40)表达。
如所附实施例中例示性显示的,作为本发明的概念的证据,构建了三价双特异性抗体分子“BsAB EGFRvIII-EpCAM”(SEQ ID NO:233,其包含/组成为质粒/载体“EGFR vIIIMR1.1VH Ck muEpCAM VH CH1EE Fc节PGLALA,pETR14953”,“EGFR vIII MR1.1VH CkmuEpCAM VH CH1EE Fc节PG LALA,pETR14953”,“EGFR vIII MR1.1VL CH1,pETR14951”,“VLEpCAM G.8.8Ck RK,pETR14882”和“VH muEpCAM CH1EE Fc穴PGLALA HRYF,pETR14940”),其包含一个与之相互作用/针对/结合人EGFRvIII的域和两个与之相互作用/针对/结合鼠EpCAM的域。三价双特异性抗体分子“BsAB EGFRvIII-EpCAM”的序列(氨基酸和cDNA)在下文表1和2中显示。
表1:
表2:
而且,如图10中例示的,作为本发明的概念的别的证据,构建了三价双特异性抗体“BsAB EGFRvIII-MSLN”(SEQ ID NO:235,其包含/组成为质粒/载体“EGFRvIII MR1.1VH CkMSLN CH CH1EE Fc节PG LALA,pETR15655”,“EGFR vIII MR1.1VL CH1,pETR15656”,“VLMSLN Ck RK,pETR15443”和“VH MSLN CH1EE Fc穴PG LALA HRYF,pETR15667”),其包含与之相互作用/针对/结合人MSLN的两个域和与之相互作用/针对/结合人EGFRvIII的一个域。三价双特异性抗体分子“BsAB EGFRvIII-MSLN”的序列(氨基酸和DNA)在表3和4中显示。
表3:
表4:
而且,如图11中例示的,作为本发明的概念的别的证据,构建了三价双特异性抗体“BsAB EGFRvIII-MCSP”(SEQ ID NO:234,其包含/组成为质粒“MR1.1EGFRvIII VH-Ck-(G4S)2MCSP M4-3 VH CH1 EE Fc节PG LALA,pETR16621”,“EGFR vIII MR1.1 VL CH1,pETR15656”,“MCSP ML2 VL Ck RK,pETR16619”和“MCSP M4-3 VH CH1EE Fc穴PG LALAHYRF,pETR16618”),其包含两个与之相互作用/针对/结合人MCSP的域和一个与之相互作用/针对/结合人EGFRvIII的域。三价双特异性抗体分子“BsABEGFRvIII-MCSP”的序列(氨基酸和DNA)在表5和6中显示。
表5:
表6:
本发明还提供一种或多种编码本发明的三价双特异性抗体分子的核酸分子。本发明还涵盖的是一种或多种编码本发明的融合蛋白的核酸分子。
术语“核酸分子”涉及由多核苷酸包含的碱基的序列,包含基于嘌呤和嘧啶的碱基,由此所述碱基代表核酸分子的一级结构。在本文中,术语“核酸分子”包括DNA,cDNA,基因组DNA,RNA,合成形式的DNA和包含两个或更多个这些分子的混合聚合物。另外,术语“核酸分子”包括有义和反义链二者。此外,本文中描述的“核酸分子”可含有非天然的或衍生化的核苷酸碱基,如本领域技术人员会容易领会的。例示性的编码本发明的融合蛋白的核酸分子在SEQ ID NO:41,43,45,47,49或119中显示。而且,例示性的本发明的三价双特异性抗体分子的重和/或轻链的核酸分子编码区在SEQ ID NO:1,3,5,7(见表1和2),SEQ ID NO:21,23,25,27(见表3和4)和SEQ ID NO:207,209,211和213(见表5和6)中显示。
本发明的核酸分子可以在调节序列的控制下。例如,可采用启动子,转录增强子和/或容许诱导本发明的融合蛋白表达的序列。在本发明的语境中,核酸分子在组成性或诱导型启动子控制下表达。合适的启动子是例如CMV启动子(Qin et al.,PLoS One 5(5)(2010),e10611),UBC启动子(Qin et al.,PLoS One 5(5)(2010),e10611),PGK(Qin etal.,PLoS One 5(5)(2010),e10611),EF1A启动子(Qin et al.,PLoS One 5(5)(2010),e10611),CAGG启动子(Qin et al.,PLoS One 5(5)(2010),e10611),SV40启动子(Qin etal.,PLoS One 5(5)(2010),e10611),COPIA启动子(Qin et al.,PLoS One 5(5)(2010),e10611),ACT5C启动子(Qin et al.,PLoS One 5(5)(2010),e10611),TRE启动子(Qin etal.,PLoS One 5(5)(2010),e10611),Oct3/4启动子(Chang et al.,Molecular Therapy 9(2004),S367-S367(doi:10.1016/j.ymthe.2004.06.904)),或Nanog启动子(Wu et al.,Cell Res.15(5)(2005),317-24)。
术语“调节序列”指实现与它们连接的编码序列表达所必需的DNA序列。此类控制序列的性质取决于宿主生物体而不同。在原核生物中,控制序列一般包括启动子,核糖体结合位点,和终止子。在真核生物中,一般控制序列包括启动子,终止子,和一些情况中的增强子,反式激活蛋白或转录因子。术语“控制序列”意图最低限度包括其存在是表达所必需的所有构件,而且还可包括另外的有利的构件。
所述核酸分子可以是重组生成的单独地或组合地包含任何上述核酸分子的嵌合核酸分子。在本发明的语境中,核酸分子是载体的部分。
本发明因此还涉及包含本发明中描述的核酸分子的载体。在本文中术语“载体”涉及能在它所导入的宿主细胞中(即在经转导细胞中)自主复制的环形或线性核酸分子。许多合适的载体是分子生物学技术人员知道,其选择会取决于期望的功能且包括质粒,粘粒,病毒,噬菌体和遗传工程中常规使用的其它载体。可使用本领域技术人员公知的方法来构建各种质粒和载体；见例如Sambrook等人(见上文)和Ausubel,Current Protocols inMolecular Biology,Green Publishing Associates and Wiley Interscience,N.Y.(1989),(1994)中描述的技术。或者,本发明的多核苷酸和载体可重建入脂质体,用于投递至靶细胞。如下文进一步详细讨论的,使用克隆载体来分离各DNA序列。在需要表达特定多肽的情况中,可将有关序列转移入表达载体。典型的克隆载体包括pBluescript SK,pGEM,pUC9,pBR322,pGA18和pGBT9。典型的表达载体包括pTRE,pCAL-n-EK,pESP-1,pOP13CAT。
本发明还涉及包含如下核酸分子的载体,该核酸分子是与所述编码本文中限定的三价双特异性抗体构建物(分子)的核酸分子可操作连接的调节序列。在本发明的语境中,载体可以是多顺反子的。如所附实施例中显示的,可以在至少三种不同核酸分子上表达三价双特异性抗体分子,其中每一种核酸分子与调节序列可操作连接。
此类调节序列(控制元件)是技术人员知道的且可包括启动子,剪接盒,翻译起始密码子,翻译和插入位点,用于将插入物引入载体。在本发明的语境中,所述核酸分子与所述容许真核或原核细胞中的表达的表达控制序列可操作连接。
涵盖的是所述载体是包含编码本文中限定的三价双特异性抗体构建物(分子)的核酸分子的表达载体。
术语“调节序列”指实现与它们连接的编码序列表达所必需的DNA序列。此类控制序列的性质取决于宿主生物体而不同。在原核生物中,控制序列一般包括启动子,核糖体结合位点,和终止子。在真核生物中,一般控制序列包括启动子,终止子,和一些情况中的增强子,反式激活蛋白或转录因子。术语“控制序列”意图最低限度包括其存在是表达所必需的所有构件,而且还可包括另外的有利的构件。
术语“可操作连接”指如下的并列,其中如此描述的构件处于允许它们以它们的预定方式发挥功能的关系的。与编码序列“可操作连接”的控制序列以这样一种方式连接,即在与控制序列相容的条件下实现编码序列的表达。在控制序列是启动子的情况中,对技术人员显而易见的是优选使用双链核酸。
在本发明的语境中,所述载体是表达载体(s)。“表达载体”是可用于转化所选择的宿主及提供编码序列在所选择的宿主中的表达的构建物。表达载体可以是例如克隆载体,二元载体或整合型载体。表达优选包含核酸分子转录成可翻译mRNA。确保原核生物和/或真核细胞中的表达的调节元件是本领域技术人员公知的。在真核细胞的情况中,它们包含正常情况下确保转录起始的启动子和任选的确保转录终止及稳定转录物的poly-A信号。可能的允许原核宿主细胞中的表达的调节元件包含例如大肠杆菌中的PL,lac,trp或tac启动子,而允许真核宿主细胞中的表达的调节元件的例子是酵母中的AOX1或GAL1启动子或CMV,SV40,RSV启动子(劳斯肉瘤病毒),CMV增强子,SV40增强子或哺乳动物和其它动物细胞中的珠蛋白内含子。
在负责转录起始的元件以外,此类调节元件还可包含多核苷酸下游的转录终止信号,诸如SV40-poly-A位点或tk-poly-A位点。而且,取决于所使用的表达系统,能够将多肽指引至细胞隔室或将它分泌入培养基的前导序列可添加至所述核酸序列的编码序列且是本领域公知的；还见例如所附实施例。
前导序列以适宜相与翻译,起始和终止序列装配,而且优选地,前导序列能够指引所翻译的蛋白质或其部分分泌入周质空间或胞外培养基。任选地,异源序列可编码融合蛋白，其包括给予期望特征的N端鉴定肽，例如所表达的重组产物的稳定化或简化纯化；见上文。在此语境中，合适的表达载体是本领域知道的，诸如Okayama-Berg cDNA表达载体pcDV1(Pharmacia)，pCDM8，pRc/CMV，pcDNA1，pcDNA3(In-vitrogene)，pEF-DHFR，pEF-ADA或pEF-neo(Raum et al.,Cancer Immunol Immunother 50(2001),141-150)或pSPORT1(GIBCOBRL)。
在本发明的语境中,表达控制序列会是能够转化或转染真核宿主细胞的载体中的真核启动子系统,但是还可使用原核宿主的控制序列。一旦载体已经导入适宜的宿主，在适合于核苷酸序列高水平表达的条件下维持宿主，而且在期望时，可继以本发明的多肽的收集和纯化；见例如所附实施例。
可用来表达细胞周期相互作用蛋白的一种备选表达系统是昆虫系统。在一种此类系统中，使用苜蓿银纹夜蛾(Autographa californica)核型多角体病毒(AcNPV)作为载体在草地贪夜蛾(Spodoptera frugiperda)细胞中或在粉夜蛾(Trichoplusia)幼虫中表达外来基因。所述核酸分子的编码序列可克隆入病毒的非必需区，诸如多角体蛋白基因且置于多角体蛋白启动子控制下。所述编码序列的成功插入会使得多角体蛋白基因失活及生成缺乏外壳蛋白外壳的重组病毒。然后使用重组病毒感染其中表达本发明的蛋白的草地贪夜蛾细胞或粉夜蛾幼虫(Smith,J.Virol.46(1983),584；Engelhard，Proc.Nat.Acad.Sci.USA91(1994)，3224-3227)。
另外的调节元件可包括转录以及翻译增强子。有利的是，上文描述的本发明的载体包含可选择和/或可打分标志物。
对于选择经转化细胞和例如植物组织和植株有用的可选择标志物基因是本领域技术人员公知的且包含例如抗代谢物抗性，作为针对赋予对甲氨蝶呤的抗性的dhfr(Reiss，Plant Physiol.(Life Sci.Adv.)13(1994)，143-149)，赋予对氨基糖苷新霉素，卡那霉素和巴龙霉素的抗性的npt(Herrera-Estrella，EMBO J.2(1983),987-995)和赋予对潮霉素的抗性的hygro(Marsh,Gene 32(1984),481-485)的选择的基础。另外的可选择基因已有描述,即容许细胞利用吲哚代替色氨酸的trpB；容许细胞利用组胺醇(histinol)代替组氨酸的hisD(Hartman,Proc.Natl.Acad.Sci.USA 85(1988),8047)；容许细胞利用甘露糖的甘露糖-6-磷酸异构酶(WO 94/20627)和赋予对鸟氨酸脱羧酶抑制剂2-(二氟甲基)-DL-鸟氨酸DFMO的抗性的ODC(鸟氨酸脱羧酶)(McConlogue,1987,In:Current Communicationsin Molecular Biology,Cold Spring Harbor Laboratory ed.)或来自土曲霉(Aspergillus terreus)的赋予对杀稻瘟素S的抗性的脱氨酶(Tamura,Biosci.Biotechnol.Biochem.59(1995),2336-2338)。
有用的可打分标志物也是本领域技术人员知道的且是可商购的。有利的是,所述标志物是编码萤光素酶(Giacomin,Pl.Sci.116(1996),59-72；Scikantha,J.Bact.178(1996),121),绿色荧光蛋白(Gerdes,FEBS Lett.389(1996),44-47)或β-葡糖醛酸糖苷酶(Jefferson,EMBO J.6(1987),3901-3907)的基因。这个实施方案对于简单且快速筛选含有所述载体的细胞,组织和生物体是特别有用的。
如上文描述的,所述核酸分子可单独或作为载体的部分使用,在细胞中表达所编码的三价双特异性构建物,用于例如纯化,还有基因疗法目的,优选与经转导T细胞组合。将含有编码上文描述的任一种三价双特异性抗体分子的DNA序列的核酸分子或载体导入细胞,继而生成感兴趣多肽。基于通过离体或体内技术将治疗性基因导入细胞的基因疗法是基因转移的最重要应用之一。用于体外或体内基因疗法的方法或基因投递系统中的合适的载体,方法或基因投递系统在文献中有描述且是本领域技术人员知道的；见例如Giordano,Nature Medicine 2(1996),534-539；Schaper,Circ.Res.79(1996),911-919；Anderson,Science 256(1992),808-813；Verma,Nature 389(1994),239；Isner,Lancet 348(1996),370-374；Muhlhauser,Circ.Res.77(1995),1077-1086；Onodera,Blood 91(1998),30-36；Verma,Gene Ther.5(1998),692-699；Nabel,Ann.N.Y.Acad.Sci.811(1997),289-292；Verzeletti,Hum.Gene Ther.9(1998),2243-51；Wang,Nature Medicine 2(1996),714-716；WO 94/29469；WO 97/00957；US 5,580,859；US 5,589,466；或Schaper,CurrentOpinion in Biotechnology 7(1996),635-640。可设计用于直接或经由脂质体或病毒载体(例如腺病毒,逆转录病毒)导入细胞的所述核酸分子和载体。在本发明的语境中,所述细胞是种系细胞,胚胎细胞,或卵细胞或自其衍生的,最优选所述细胞是干细胞。胚胎干细胞的一个例子可以格外是如Nagy,Proc.Natl.Acad.Sci.USA 90(1993),8424-8428中描述的干细胞。
依照上文,本发明涉及衍生包含编码本文中限定的双特异性抗体构建物的多肽序列的核酸分子的遗传工程中常规使用的载体,特别是质粒,粘粒和噬菌体的方法。在本发明的语境中,所述载体是表达载体和/或基因转移或打靶载体。可使用自病毒,诸如逆转录病毒,牛痘病毒,腺伴随病毒,疱疹病毒,或牛乳头状瘤病毒衍生的表达载体将所述多核苷酸或载体投递入靶定细胞群。
可使用本领域技术人员公知的方法来构建重组载体；见例如Sambrook等人(见上文),Ausubel(1989,见上文)或其它标准教科书中描述的技术。或者,可以将所述核酸分子和载体重建入脂质体以投递至靶细胞。可通过公知方法将含有本发明的核酸分子的载体转移入宿主细胞,随细胞宿主的类型而变化。例如,氯化钙转染常常用于原核细胞,而磷酸钙处理或电穿孔可用于其它细胞宿主；见Sambrook,见上文。所述载体格外可以是pEF-DHFR,pEF-ADA或pEF-neo。载体pEF-DHFR,pEF-ADA和pEF-neo在本领域中已有描述,例如Mack etal.Proc.Natl.Acad.Sci.USA 92(1995),7021-7025和Raum et al.Cancer ImmunolImmunother 50(2001),141-150。
本发明还提供经如本文中描述的载体转化或转染的宿主。所述宿主可通过将至少一种上文描述的载体或至少一种上文描述的核酸分子导入宿主来生成。宿主中所述至少一种载体或至少一种核酸分子的存在可介导编码上文描述的三价双特异性抗体分子或构建物(即本文中描述的三价双特异性抗体分子)的基因的表达。本发明的载体可以是多顺反子的。
在本发明的三价双特异性抗体分子的表达的情况中,轻链的重复可容许相对于其中轻链编码区与重链编码区在宿主细胞中以1:1比率存在的情况改良的完整三价双特异性抗体分子的装配和/或表达。如此,本发明提供其中轻链构件与重链构件的编码区比率是1:1或大于1:1的构建物和方法。例如,在一个实施方案中,轻链构件与重链构件的比率是2:1或大于2:1,例如3:1,3:2,4:1或大于4:1。在本发明的三价双特异性抗体分子包含改变的CH3域的情况中,可以以1:2:1:1比率(“载体重链穴(VH-CH1-CH2-CH3)”:“轻链(LC)”:“载体重链节(VH-CK-VH-CH1-CH2-CH3)”:“交叉轻链(VL-CH1)”)用相应的表达载体转染细胞。
导入宿主的所描述的核酸分子或载体可整合入宿主的基因组,或者它可在染色体外维持。
宿主可以是任何原核或真核细胞。
术语“原核生物”意图包括能用DNA或DNA或RNA分子转化,转导或转染来表达本发明的蛋白的所有细菌。原核宿主可包括革兰氏阴性以及革兰氏阳性细菌,诸如例如大肠埃希氏菌/大肠杆菌(E.coli),鼠伤寒沙门氏菌(S.typhimurium),粘质沙雷氏菌(Serratiamarcescens)和枯草芽孢杆菌(Bacillus subtilis)。术语“真核”意图包括酵母,高等植物,昆虫和优选哺乳动物细胞。取决于重组生成规程中采用的宿主,由本发明的多核苷酸编码的蛋白可以是糖基化的或非糖基化的。尤其优选的是含有本发明的多肽和与之遗传融合的N端FLAG标签和/或C端His标签的编码序列的质粒或病毒的用途。优选地,所述FLAG标签的长度是约4至8个氨基酸,最优选8个氨基酸。可使用本领域普通技术人员普遍知道的任何技术使用上文描述的多核苷酸转化或转染宿主。而且,用于在例如哺乳动物细胞和细菌中制备融合的,可操作连接的基因和表达它们的方法是本领域公知的(Sambrook,见上文)。
在本发明的语境中,宿主(细胞)是细菌,昆虫,真菌,植物或动物细胞。
特别涵盖的是所述宿主可以是哺乳动物细胞,更加优选人细胞或人细胞系。
特别优选的宿主细胞包含HEK293,CHO细胞,COS细胞,骨髓瘤细胞系像SP2/0或NS/0。正如所附实施例中例示的,特别优选的是HEK293细胞和CHO细胞作为宿主。
本发明如此涉及一种用于生成上文描述的三价双特异性抗体分子或构建物(即本文中描述的三价双特异性抗体分子)的方法,其包含在容许三价双特异性抗体分子或构建物(即本文中描述的三价双特异性抗体分子)表达的条件下培养本发明的细胞和/或宿主细胞并自细胞和/或培养培养基回收分子或构建物(即本文中描述的三价双特异性抗体分子)。
经转化宿主可以在发酵罐生长并依照本领域知道的技术培养以实现最佳细胞生长。然后可以自生长培养基分离本发明的多肽。例如微生物表达的本发明的多肽的分离和纯化可以通过任何常规手段,诸如例如制备性层析分离和免疫学分离,诸如那些牵涉使用针对例如本发明的多肽的标签的单克隆或多克隆抗体的或如所附实施例中描述的。
而且,本发明提供一种组合物(药物),其包含一种或多种如本文中限定的三价双特异性(单克隆)抗体分子或一种或多种如通过上文公开的方法生成的(人)三价双特异性抗体分子,一种或多种编码本发明的三价双特异性抗体分子的核酸分子,一种或多种载体或包含如本文中描述的融合蛋白的经转导T细胞。在本发明的语境中,所述组合物是药用组合物,其进一步包含任选的合适的载剂,稳定剂和/或赋形剂的配制剂。
而且,本发明提供一种或多种如本文中上文限定的三价双特异性抗体分子,供作为药物使用,其中所述三价双特异性抗体分子要在施用包含如本文中描述的融合蛋白的经转导T细胞之前,同时或之后施用且其中所述T细胞是自要治疗的受试者获得的。
在本发明的语境中,提供了一种药用组合物/药物,其包含如本文中限定的上文三价双特异性抗体分子,要与包含如本文中描述的融合蛋白的经转导T细胞组合施用,其中所述双特异性抗体分子要在施用包含在T细胞中或上并不天然发生的抗原的经转导T细胞之前,同时或之后施用且其中所述T细胞是自要治疗的受试者获得的。
在本发明的语境中,用编码如本文中上文限定的融合蛋白的核酸分子和/或用包含此类核酸分子的载体转导T细胞。在用本文中限定的融合蛋白转染T细胞的语境中,术语“载体”涉及能在已经接受它导入的宿主细胞中(即在经转导细胞中)自主复制的环形或线性核酸分子。如本文中使用的“载体”特别指质粒,粘粒,病毒,噬菌体和遗传工程中常常使用的其它载体。在一个优选实施方案,本发明的载体适合于转化细胞,优选T细胞,诸如CD8+T细胞,CD4+ T细胞,CD3+ T细胞,γδ T细胞或天然杀伤(NK)T细胞,最优选CD8+ T细胞。因而,在本发明的一个方面,如本文中提供的载体是表达载体。表达载体在文献中已有广泛描述。特别地,本文中提供的载体优选包含重组多核苷酸(即编码本发明的融合蛋白的核酸分子)以及与要表达的核苷酸序列可操作连接的表达控制序列。如本文中提供的载体优选进一步包含启动子。本文中描述的用于转导T细胞的载体还可包含选择标志物基因和复制起点,确保宿主(即经转导细胞)中的复制。此外,本文中提供的用于转导T细胞的载体还可包含转录终止信号。启动子和终止信号之间优选有至少一种限制性位点或多接头,能够插入期望要表达的核酸分子(例如编码本发明的融合蛋白的核酸分子)。技术人员知道如何能实施此类插入。适合包含本发明的核酸分子以形成本发明的载体来转染T细胞的载体的例子是本领域知道的。例如,在本发明的语境中,合适的载体包括并入本发明的核酸分子(即编码本发明的融合蛋白的核酸分子)的粘粒,质粒(例如裸的或包含在脂质体中的)和病毒(例如慢病毒,逆转录病毒,腺病毒,和腺伴随病毒)。优选地,本发明的载体是病毒载体。更加优选地,本发明的载体是慢病毒载体,而且甚至更加优选地,本发明的载体是逆转录病毒载体(例如pMP71载体)。因而,在本发明的语境中,载体是慢病毒载体或逆转录病毒载体。本发明的载体容许编码本发明的融合蛋白的核酸分子的组成性或条件性表达。在此语境中,适合于本发明的融合蛋白表达的逆转录病毒载体是本领域知道的,诸如SAMEN CMV/SRa(Clayet al.,J.Immunol.163(1999),507-513),LZRS-id3-IHRES(Heemskerk et al.,J.Exp.Med.186(1997),1597-1602),FeLV(Neil et al.,Nature 308(1984),814-820),SAX(Kantoff et al.,Proc.Natl.Acad.Sci.USA 83(1986),6563-6567),pDOL(Desiderio,J.Exp.Med.167(1988),372-388),N2(Kasid et al.,Proc.Natl.Acad.Sci.USA 87(1990),473-477),LNL6(Tiberghien et al.,Blood 84(1994),1333-1341),pZipNEO(Chen etal.,J.Immunol.153(1994),3630-3638),LASN(Mullen et al.,Hum.Gene Ther.7(1996),1123-1129),pG1XsNa(Taylor et al.,J.Exp.Med.184(1996),2031-2036),LCNX(Sun etal.,Hum.Gene Ther.8(1997),1041-1048),SFG(Gallardo et al.,Blood 90(1997),LXSN(Sun et al.,Hum.Gene Ther.8(1997),1041-1048),SFG(Gallardo et al.,Blood 90(1997),952-957),HMB-Hb-Hu(Vieillard et al.,Proc.Natl.Acad.Sci.USA 94(1997),11595-11600),pMV7(Cochlovius et al.,Cancer Immunol.Immunother.46(1998),61-66),pSTITCH(Weitjens et al.,Gene Ther 5(1998),1195-1203),pLZR(Yang et al.,Hum.Gene Ther.10(1999),123-132),pBAG(Wu et al.,Hum.Gene Ther.10(1999),977-982),rKat.43.267bn(Gilham et al.,J.Immunother.25(2002),139-151),pLGSN(Engelset al.,Hum.Gene Ther.14(2003),1155-1168),pMP71(Engels et al.,Hum.Gene Ther.14(2003),1155-1168),pGCSAM(Morgan et al.,J.Immunol.171(2003),3287-3295),pMSGV(Zhao et al.,J.Immunol.174(2005),4415-4423),或pMX(de Witte et al.,J.Immunol.181(2008),5128-5136)。而且,在本发明的语境中,适合于表达本发明的融合蛋白的慢病毒载体是例如PL-SIN慢病毒载体(Hotta et al.,Nat Methods.6(5)(2009),370-376),p156RRL-sinPPT-CMV-GFP-PRE/NheI(Campeau et al.,PLoSOne 4(8)(2009),e6529),pCMVR8.74(Addgene目录No.:22036),FUGW(Lois et al.,Science 295(5556)(2002),868-872),pLVX-EF1(Addgene目录No.:64368),pLVE(Brunger et al.,Proc NatlAcad Sci U S A111(9)(2014),E798-806),pCDH1-MCS1-EF1(Hu et al.,Mol CancerRes.7(11)(2009),1756-1770),pSLIK(Wang et al.,Nat Cell Biol.16(4)(2014),345-356),pLJM1(Solomon et al.,Nat Genet.45(12)(2013),1428-30),pLX302(Kang et al.,Sci Signal.6(287)(2013),rs13),pHR-IG(Xie et al.,J Cereb Blood Flow Metab.33(12)(2013),1875-85),pRRLSIN(Addgene目录No.:62053),pLS(Miyoshi et al.,JVirol.72(10)(1998),8150-8157),pLL3.7(Lazebnik et al.,J Biol Chem.283(7)(2008),11078-82),FRIG(Raissi et al.,Mol Cell Neurosci.57(2013),23-32),pWPT(Ritz-Laser et al.,Diabetologia.46(6)(2003),810-821),pBOB(Marr et al.,J MolNeurosci.22(1-2)(2004),5-11),或pLEX(Addgene目录No.:27976)。
本发明还涉及表达由编码本发明的融合蛋白的核酸分子编码的融合蛋白的经转导T细胞,诸如CD8+ T细胞,CD4+ T细胞,CD3+ T细胞,γδ T细胞或天然杀伤(NK)T细胞,优选CD8+ T细胞。因而,在本发明的语境中,经转导细胞可包含编码本发明的融合蛋白的核酸分子或表达本发明的融合蛋白的本发明的载体。
在本发明的语境中,术语“经转导细胞”涉及经遗传修饰的细胞(即其中已经有意导入核酸分子的细胞)。本文中提供的经转导细胞可包含本发明的载体。优选地,本文中提供的经转导细胞包含编码本发明的融合蛋白的核酸分子和/或本发明的载体。本发明的经转导细胞可以是瞬时或稳定表达外来DNA(即已经导入细胞的核酸分子)的细胞。特别地,编码本发明的融合蛋白的核酸分子可以通过使用逆转录病毒或慢病毒转导稳定整合入细胞的基因组。通过使用mRNA转染,编码本发明的融合蛋白的核酸分子可以瞬时表达。优选地,本文中提供的经转导细胞通过经由病毒载体(例如逆转录病毒载体或慢病毒载体)在细胞中导入核酸分子已经遗传修饰。因而,融合蛋白的表达可以是组成性的且融合蛋白的胞外域可以是在细胞表面上可检测的。融合蛋白的这个胞外域可包含如本文中限定的在T细胞中或上并不天然发生的信号传导受体的完整胞外域,还有其部分。所需要的最小尺寸是在融合蛋白的一侧上受到三价双特异性抗体分子结合的表位。
在可诱导或可遏制启动子控制下将融合蛋白引入T细胞,诸如CD8+ T细胞,CD4+ T细胞,CD3+ T细胞,γδ T细胞或天然杀伤(NK)T细胞,优选CD8+T细胞的情况中,表达还可以是条件性的或诱导型的。此类可诱导或可遏制启动子的例子可以是含有醇脱氢酶I(alcA)基因启动子和反式激活蛋白的蛋白AlcR的转录系统。使用基于不同农业醇的配制剂来控制与alcA启动子连接的感兴趣基因的表达。而且,四环素响应性启动子系统能在四环素存在下发挥激活或遏制基因表达系统的功能。该系统的一些元件包括四环素阻抑蛋白(TetR),四环素操纵基因序列(tetO)和四环素反式激活蛋白融合蛋白(tTA),它是TetR和单纯疱疹病毒蛋白16(VP16)激活序列的融合物。而且,可使用类固醇响应性启动子,金属调节的或发病相关(PR)蛋白相关启动子。
表达可以是组成性的或组成型的,取决于所使用的系统。本发明的融合蛋白可以在本文中提供的经转导细胞的表面上表达。融合蛋白的胞外部分(即在T细胞中或上并不天然发生的信号传导受体的胞外域)能在细胞表面是检测到,而胞内部分(即融合蛋白的锚定跨膜域,共刺激性信号传导域和刺激性信号传导域)与膜结合但在细胞表面上检测不到。融合蛋白的胞外域的检测可通过使用特异性结合这种胞外域的抗体来进行。胞外域可使用这些抗体通过流式细胞术或显微术来检测。本发明的经转导细胞可以是任何免疫细胞。这些包括但不限于B细胞,T细胞,天然杀伤(NK)细胞,天然杀伤(NK)T细胞,γδ T细胞,先天髓样细胞,巨噬细胞,单核细胞,树突细胞,或嗜中性细胞。优选地,所述免疫细胞会是淋巴细胞,优选NK或T细胞。所述T细胞包括CD4 T细胞和CD8 T细胞,最优选CD4+ T细胞和CD8+ T细胞。白细胞的表面上本发明的融合蛋白的触发会使得细胞变成针对它的靶细胞是细胞毒性的,不管细胞起源的谱系。细胞毒性会发生,不管为融合蛋白选择的刺激性信号传导域或共刺激性信号传导域且不依赖于另外的细胞因子的外源供应。因而,本发明的经转导细胞可以是例如CD4+ T细胞,CD8+ T细胞,γδ T细胞,天然杀伤(NK)T细胞,天然杀伤(NK)细胞,肿瘤浸润性淋巴细胞(TIL)细胞,髓样细胞,或间充质干细胞。优选地,本文中提供的经转导细胞是T细胞(例如自体T细胞),更加优选地,经转导细胞是CD8+ T细胞。因而,在本发明的语境中,经转导细胞是CD8+ T细胞。而且,在本发明的语境中,经转导细胞是自体T细胞。因而,在本发明的语境中,经转导细胞优选是自体CD8+ T细胞。在使用自受试者分离的自体细胞(例如T细胞)以外,本发明还涵盖使用同种异基因细胞。因而,在本发明的语境中,经转导细胞还可以是同种异基因细胞,诸如同种异基因CD8+ T细胞。同种异基因细胞的使用基于细胞(优选T细胞)能识别由外来抗原呈递细胞(APC)呈递的特定抗原表位的事实,前提是该APC表达该特定响应细胞群(即T细胞群)受到约束的I类或II类MHC分子连同受到该T细胞识别的抗原表位。如此,术语同种异基因指来自与会通过例如本文中描述的表达融合蛋白的经转导细胞来治疗的个体/受试者是人白细胞抗原(HLA)相容的无关供体个体/受试者的细胞。自体细胞指如本文中上文描述的自要用本文中描述的经转导细胞治疗的受试者分离/获得的细胞。
如上文描述的,用表达本文中提供的融合蛋白的核酸分子转导本发明的经转导细胞。在携带天然抗肿瘤特异性的细胞诸如肿瘤浸润性淋巴细胞的细胞(TIL,Dudley etal.,J Clin Oncol.31(17)(2013),2152-2159(doi:10.1200/JCO.2012.46.6441))或通过流式细胞术通过它们的肿瘤特异性自患者的外周血分选的抗原特异性细胞(Hunsucker etal.,Cancer Immunol Res.3(3)(2015),228-235(doi:10.1158/2326-6066.CIR-14-0001))的情况中,本文中描述的细胞只会用本发明的融合蛋白转导。然而,可以用别的核酸分子,例如编码T细胞受体或嵌合抗原受体的核酸分子共转导本发明的经转导细胞。而且,在本发明的语境中,可以用别的核酸分子,例如编码Fas配体(FasL)的核酸分子共转导本发明的经转导细胞。已知FasL与Fas相互作用(Nagata et al.,Science 267(5203)(1995),1449-1456；Walker et al.,J Immunol.158(10)(1997),4521-4524)。Fas和它的配体FasL分别是I型和II型跨膜蛋白和肿瘤坏死因子/神经生长因子受体和肿瘤坏死因子家族蛋白的成员(人Fas在UniProt条目号P25445(条目版本218,序列版本1；SEQ ID NO:241(蛋白质)和240(DNA))下可得；人FasL具有UniProt条目号P48023(条目版本190,序列版本1；SEQ ID NO:245(蛋白质)和244(DNA))；鼠FAS具有UniProt条目号P41047(条目版本169,序列版本1；SEQID NO:239(蛋白质)和238(DNA))；鼠FasL具有UniProt条目号P41047(条目版本169,序列版本1；SEQ ID NO:243(蛋白质)和242(DNA)))。在本发明的语境中,已经令人惊讶且出乎意料发现FasL对于作用的模式是重要的。特别地,显示了经本文中描述的融合蛋白转染/转导的T细胞,诸如CD8+ T细胞,CD4+ T细胞,CD3+ T细胞,γδ T细胞或天然杀伤(NK)T细胞的杀伤能力可通过阻断FasL-Fas相互作用而削弱(见图13)。因而,令人惊讶地发现了(过)表达FasL的经转导细胞可用于治疗特征在于所具有的肿瘤细胞具有Fas(过)表达性细胞的疾病。
依照此发明,术语“T细胞受体”是本领域普遍知道的。特别地,本文中的术语“T细胞受体”指任何T细胞受体,前提是满足下面三项标准:(i)肿瘤特异性,(ii)识别(大多数)肿瘤细胞,这意味着抗原或靶物应当在(大多数)肿瘤细胞中表达,和(iii)TCR匹配要治疗的受试者的HLA型。在此语境中,满足上文提到的三项标准的合适的T细胞受体是本领域知道的,诸如识别WT1(维尔姆斯肿瘤特异性抗原1；序列信息见例如Sugiyama,JapaneseJournal of Clinical Oncology 40(2010),377-87),MAGE(序列见例如WO-A1 2007/032255和PCT/US2011/57272),SSX(美国临时申请No.61/388,983),NY-ESO-1(序列信息见例如PCT/GB2005/001924)和/或HER2neu(序列信息见WO-A1 2011/0280894)的受体。
术语“嵌合抗原受体”或“嵌合受体”是本领域知道的且指由单链抗体域的胞外部分与CD3z和CD28的信号域通过间隔物序列融合而构成的受体。再次,这种嵌合抗原受体应当提供肿瘤特异性且容许识别大多数肿瘤细胞。合适的嵌合受体包括:抗EGFRvIII-CAR(序列见WO-A1 2012/138475),抗CD22-CAR(见WO-A1 2013/059593),抗BCMA-CAR(见WO-A12013/154760),抗CD19-CAR(见WO-A1 2012/079000或US-A1 2014/0271635),抗CD123-CAR(见US-A1 2014/0271582),抗CD30-CAR(见WO-A1 2015/028444)或抗间皮素-CAR(见WO-A12013/142034)。
本发明还涉及一种用于生成表达本发明的融合蛋白的经转导细胞的方法,其包含用本发明的载体转导细胞,在容许在所述经转导细胞中或上表达该融合蛋白的条件下培养经转导细胞并回收所述经转导细胞的步骤。
在本发明的语境中,本发明的经转导细胞优选通过下面的过程来生成:自受试者(优选人患者)分离/获得细胞(例如T细胞,优选CD8+ T细胞)。用于自患者或供体分离/获得细胞(例如T细胞,优选CD8+ T细胞)的方法是本领域公知的,而且在本发明的语境中,来自患者或供体的细胞(例如T细胞,优选CD8+ T细胞)可以通过采集血液取出骨髓来分离。在作为患者的样品分离/获得细胞之后,将细胞(例如T细胞)与样品的其它组分分开。知道数种用于自样品分出细胞(例如T细胞)的方法,包括但不限于例如用于自来自患者或供体的外周血样品获得细胞的白细胞分离法,通过使用FACSort装置来分离/获得细胞,自包含活细胞的新鲜活检标本手工或通过使用显微操作器在死细胞中挑取活细胞(见例如Dudley,Immunother.26(2003),332-342；Robbins,Clin.Oncol.29(201 1),917-924或Leisegang,J.Mol.Med.86(2008),573-58)。本文中的术语“新鲜患者活检”指通过手术或任何其它已知手段自受试者取出的组织(优选肿瘤组织)以及肿瘤细胞系或来自肿瘤组织/肿瘤细胞的(分离的)细胞。随后培养并扩充分离的/获得的细胞T细胞,优选CD8+ T细胞,例如通过使用抗CD3抗体,通过使用抗CD3和抗CD28单克隆抗体和/或通过使用抗CD3抗体,抗CD28抗体和白介素-2(IL-2)(见例如Dudley,Immunother.26(2003),332-342或Dudley,Clin.Oncol.26(2008),5233-5239)。
在一个后续步骤中,通过本领域知道的方法人工/遗传修饰/转导细胞(例如T细胞)(见例如Lemoine,J Gene Med 6(2004),374-386)。用于转导细胞(例如T细胞)的方法是本领域知道的且包括但不限于转导核酸或重组核酸的情况中的例如电穿孔方法,磷酸钙方法,阳离子脂质方法或脂质体方法。要转导的核酸可以通过使用可商购的转染试剂常规和高效转导,例如Lipofectamine(由Invitrogen制造,目录号:11668027)。在使用载体的情况中,载体可以以与上文提到的核酸相同的方式转导,只要载体是质粒载体(即并非病毒载体的载体)。在本发明的语境中,用于转导细胞(例如T细胞)的方法包括逆转录病毒或慢病毒T细胞转导以及mRNA转染。“mRNA转染”指本领域技术人员公知的用于在要转导的细胞中瞬时表达感兴趣蛋白(像本情况中的本发明的融合蛋白)的一种方法。简言之,可以通过使用电穿孔系统(诸如例如Gene Pulser,Bio-Rad)用编码本发明的融合蛋白的mRNA电穿孔细胞,之后通过如上文描述的标准细胞(例如T细胞)培养方案(见Zhao et al.,Mol Ther.13(1)(2006),151-159)培养。本发明的经转导细胞是T细胞,最优选CD8+ T细胞,而且通过慢病毒,最优选逆转录病毒T细胞转导来生成。
在此语境中,适合于转导T细胞的逆转录病毒载体是本领域知道的,诸如SAMENCMV/SRa(Clay et al.,J.Immunol.163(1999),507-513),LZRS-id3-IHRES(Heemskerk etal.,J.Exp.Med.186(1997),1597-1602),FeLV(Neil et al.,Nature 308(1984),814-820),SAX(Kantoff et al.,Proc.Natl.Acad.Sci.USA83(1986),6563-6567),pDOL(Desiderio,J.Exp.Med.167(1988),372-388),N2(Kasid et al.,Proc.Natl.Acad.Sci.USA87(1990),473-477),LNL6(Tiberghien et al.,Blood 84(1994),1333-1341),pZipNEO(Chen et al.,J.Immunol.153(1994),3630-3638),LASN(Mullen et al.,Hum.Gene Ther.7(1996),1123-1129),pG1XsNa(Taylor et al.,J.Exp.Med.184(1996),2031-2036),LCNX(Sun et al.,Hum.Gene Ther.8(1997),1041-1048),SFG(Gallardo et al.,Blood 90(1997),和LXSN(Sun et al.,Hum.Gene Ther.8(1997),1041-1048),SFG(Gallardo et al.,Blood 90(1997),952-957),HMB-Hb-Hu(Vieillard et al.,Proc.Natl.Acad.Sci.USA 94(1997),11595-11600),pMV7(Cochlovius et al.,Cancer Immunol.Immunother.46(1998),61-66),pSTITCH(Weitjenset al.,Gene Ther 5(1998),1195-1203),pLZR(Yang et al.,Hum.Gene Ther.10(1999),123-132),pBAG(Wu et al.,Hum.Gene Ther.10(1999),977-982),rKat.43.267bn(Gilhamet al.,J.Immunother.25(2002),139-151),pLGSN(Engels et al.,Hum.Gene Ther.14(2003),1155-1168),pMP71(Engels et al.,Hum.Gene Ther.14(2003),1155-1168),pGCSAM(Morgan et al.,J.Immunol.171(2003),3287-3295),pMSGV(Zhao et al.,J.Immunol.174(2005),4415-4423),或pMX(de Witte et al.,J.Immunol.181(2008),5128-5136)。在本发明的语境中,适合于转导细胞(例如T细胞)的慢病毒载体是例如PL-SIN慢病毒载体(Hotta et al.,Nat Methods.6(5)(2009),370-376),p156RRL-sinPPT-CMV-GFP-PRE/NheI(Campeau et al.,PLoS One 4(8)(2009),e6529),pCMVR8.74(Addgene目录号:22036),FUGW(Lois et al.,Science 295(5556)(2002),868-872),pLVX-EF1(Addgene目录号:64368),pLVE(Brunger et al.,Proc Natl Acad Sci U S A111(9)(2014),E798-806),pCDH1-MCS1-EF1(Hu et al.,Mol Cancer Res.7(11)(2009),1756-1770),pSLIK(Wang et al.,Nat Cell Biol.16(4)(2014),345-356),pLJM1(Solomon et al.,NatGenet.45(12)(2013),1428-30),pLX302(Kang et al.,Sci Signal.6(287)(2013),rs13),pHR-IG(Xie et al.,J Cereb Blood Flow Metab.33(12)(2013),1875-85),pRRLSIN(Addgene目录号:62053),pLS(Miyoshi et al.,J Virol.72(10)(1998),8150-8157),pLL3.7(Lazebnik et al.,J Biol Chem.283(7)(2008),11078-82),FRIG(Raissi et al.,Mol Cell Neurosci.57(2013),23-32),pWPT(Ritz-Laser et al.,Diabetologia.46(6)(2003),810-821),pBOB(Marr et al.,J Mol Neurosci.22(1-2)(2004),5-11),或pLEX(Addgene目录号:27976)。
本发明的经转导T细胞/T细胞优选在它们的天然环境以外,在受控条件下生长。特别地,术语“培养”意味着自多细胞真核生物(优选人患者)衍生的细胞(例如本发明的经转导细胞)在体外生长。培养细胞是保持与它们的原始组织来源分开的细胞活着的一种实验室技术。在本文中,在容许本发明的融合蛋白在所述经转导细胞中或上表达的条件下培养本发明的经转导细胞。容许转基因(即本发明的融合蛋白)表达的条件是本领域普遍知道的且包括例如激动性抗CD3和抗CD28抗体和添加细胞因子诸如白介素2(IL-2),白介素7(IL-7),白介素12(IL-12)和/或白介素15(IL-15)。在培养的经转导细胞中表达本发明的融合蛋白之后,自培养物(即培养培养基)回收(即再提取)经转导细胞。
本发明还涵盖的是通过该本发明的方法可获得的表达由本发明的核酸分子编码的融合蛋白的经转导细胞。
而且,本发明提供一种药用组合物/药物,其包含本发明的三价双特异性抗体分子或如通过上文公开的方法获得/生成的三价双特异性抗体分子。在本发明的语境中,所述组合物是药用组合物,其进一步包含任选的合适的载剂,稳定剂和/或赋形剂的配制剂。
依照此发明,术语“药物”与术语“药用组合物”可互换使用且涉及用于施用于患者,优选人患者的组合物。在本发明的语境中,药物/药用组合物要施用于自其分离/获得T细胞,最优选CD8+ T细胞的患者。在本发明的语境中,患者指人患者。而且,在本发明的语境中,患者罹患疾病,其中所述疾病是恶性疾病,尤其是上皮,内皮或间皮起源的癌症/癌或血液的癌症。在本发明的语境中,癌症/癌选自由胃肠癌,胰腺癌,胆管细胞癌,肺癌,乳腺癌,卵巢癌,皮肤癌,口腔癌,胃癌,宫颈癌,B和T细胞淋巴瘤,髓样白血病,卵巢癌,白血病,淋巴细胞性白血病,鼻咽癌,结肠癌,前列腺癌,肾细胞癌,头和颈癌,皮肤癌(黑素瘤),生殖泌尿道癌,例如睾丸癌,内皮癌,宫颈癌和肾癌,胆管癌,食管癌,唾液腺癌和甲状腺癌或其它肿瘤性疾病像血液学肿瘤,胶质瘤,肉瘤或骨肉瘤组成的组。
在一个优选实施方案中,药用组合物/药物包含如本文中限定的三价双特异性抗体分子,供胃肠外,经皮,腔内,动脉内,鞘内施用或直接注射入组织或肿瘤。在本发明的语境中,组合物/药物包含如本文中限定的三价双特异性抗体分子,它要在施用包含如本文中限定的融合蛋白的经转导T细胞之前,同时或之后施用。在本发明的语境中,包含如本文中限定的三价双特异性抗体分子的药用组合物/药物要与包含如本文中限定的融合蛋白的经转导T细胞组合施用,其中所述T细胞是自要治疗的受试者获得的。
术语“与……组合”的使用并不约束治疗方案的各成分要施用于受试者的次序。因而,本文中描述的药用组合物/药物涵盖在施用包含本发明的融合蛋白的经转导T细胞之前,同时或之后施用如本文中限定的三价双特异性抗体分子。如本文中使用的“与……组合”也并不约束施用如本文中之前限定的三价双特异性抗体分子和包含如本文中限定的融合蛋白的经转导T细胞之间的时机。如此,当两种成分并非同时/并行施用时,施用可以相隔1分钟,5分钟,15分钟,30分钟,45分钟,1小时,2小时,4小时,6小时,12小时,24小时,48小时或72小时或本领域技术人员容易确定的和/或本文中描述的任何合适的时间差异。
在本发明的语境中,术语“与......组合”还涵盖其中如本文中限定的三价双特异性抗体分子和包含融合蛋白的经转导T细胞在施用于受试者之前在一起预温育的情况。如此,两种成分可以在施用之前预温育例如1分钟,5分钟,10分钟,15分钟,30分钟,45分钟或1小时或本领域技术人员容易确定的任何合适的时间。在另一个优选实施方案中,本发明涉及一种治疗方案,其中要同时/并行施用如本文中限定的三价双特异性抗体分子和包含如本文中限定的融合蛋白的经转导T细胞。在本发明的语境中,如本文中限定的三价双特异性抗体分子可以在已经施用包含融合蛋白的经转导T细胞之后施用。
而且,如本文中使用的“与......组合”并不将所公开的治疗方案约束于以紧临的顺序(即施用两种成分之一继以(在某时间间隔之后)施用另一种,中间没有施用和/或实施任何其它治疗方案)施用如本文中限定的三价双特异性抗体分子和包含融合蛋白的经转导T细胞,优选CD8+ T细胞。因此,本治疗方案还涵盖分开施用如本文中限定的三价双特异性抗体分子和包含融合蛋白的经转导T细胞,优选CD8+ T细胞,其中该施用相隔一个或多个对于治疗或预防疾病或其症状必需和/或合适的治疗方案。此类居间治疗方案的例子包括但不限于施用疼痛药疗；施用化疗剂,疾病或其症状的手术操作。因而,本文中公开的治疗方案涵盖施用如本文中限定的三价双特异性抗体分子和包含如本文中限定的融合蛋白的经转导T细胞,优选CD8+ T细胞,连同适合于治疗或预防疾病或其症状的无一,一个,或超过一个如本文中描述的或如本领域知道的治疗方案。
特别涵盖的是所述药用组合物/药物要经由输注或注射来施用于患者。在本发明的语境中,包含如限定的融合蛋白的经转导T细胞要经由输注或注射来施用于患者。合适的组合物/药物的施用可通过不同方式,静脉内,腹膜内,皮下,肌肉内,表面或皮内施用来实现。
本发明的药用组合物/药物可进一步包含药学可接受载剂。合适的药用载剂的例子是本领域公知的且包括磷酸盐缓冲盐水溶液,水,乳状液,诸如油/水乳状液,各种类型的润湿剂,无菌溶液,等。包含此类载剂的组合物可通过公知的常规方法来配制。这些药用组合物可以以合适的剂量施用于受试者。剂量方案会由主治医师和临床因素确定。正如医学领域公知的,用于任一患者的剂量取决于许多因素,包括患者的体型,体表面积,年龄,要施用的具体化合物,性别,施用的时间和路径,一般健康,和并行施用的其它药物。一般地,作为定期施用药用组合物的方案应当在1μg至5g单位每天的范围中。然而,用于连续输注的一个更加优选剂量可以在0.01μg至2mg,优选0.01μg至1mg,更加优选0.01μg至100μg,甚至更加优选0.01μg至50μg和最优选0.01μg至10μg单位每千克体重每小时的范围中。特别优选的剂量在本文中有描述。可通过周期性评估来监测进展。剂量会变化但用于静脉内施用DNA的一个优选剂量是大约106至1012个拷贝的DNA分子。本发明的组合物可局部或系统施用。施用一般会是胃肠外,例如静脉内；DNA还可以定向靶位点施用,例如通过生物弹道投递至内部或外部靶位点或通过导管投递至动脉中的位点。用于胃肠外施用的制剂包括无菌水性或非水性溶液,悬浮液,和乳状液。非水性溶剂的例子是丙二醇,聚乙二醇,植物油诸如橄榄油,和可注射有机酯诸如油酸乙酯。水性载剂包括水,醇/水溶液,乳状液或悬浮液,包括盐水和缓冲介质。胃肠外媒介包括氯化钠溶液,林格氏右旋糖,右旋糖和氯化钠,乳酸林格氏,或不挥发性油。静脉内媒介包括流体和营养物补充剂,电解质补充剂(诸如那些基于林格氏右旋糖的),等等。还可以存在防腐剂和其它添加剂,诸如例如抗微生物剂,抗氧化剂,螯合剂,和惰性气体等等。另外,本发明的药用组合物可包含蛋白质性质载剂,像例如血清清蛋白或免疫球蛋白,优选人起源的。涵盖的是,在蛋白质性质双特异性抗体构建物或编码它的核酸分子或载体(如此发明中描述的)以外,本发明的药用组合物可包含别的生物学活性剂,取决于药用组合物的预定用途。此类药剂可以是作用于胃肠系统的药物,作为细胞抑制剂起作用的药物,预防高尿酸血的药物,抑制免疫反应的药物(例如皮质类固醇),作用于循环系统的药物和/或诸如本领域知道的T细胞共刺激性分子或细胞因子等药剂。
施用本发明的组合物/药物的可能的适应证是恶性疾病,尤其是上皮癌症/癌,诸如乳腺癌,结肠癌,前列腺癌,头和颈癌,皮肤癌(黑素瘤),生殖泌尿道癌,例如卵巢癌,睾丸癌,内皮癌,宫颈癌和肾癌,肺癌,胃癌,胆管癌,食管癌,唾液腺癌和甲状腺癌或其它肿瘤性疾病像血液学肿瘤,胶质瘤,肉瘤或骨肉瘤。
本发明进一步涵盖与其它化合物,例如能够为免疫效应细胞,细胞增殖或细胞刺激提供激活信号的分子的共施用方案。所述分子可以是例如别的用于T细胞的主要激活信号(例如别的共刺激性分子:B7家族的分子,Ox40L,4.1BBL,CD40L,抗CTLA-4,抗PD-1),或别的细胞因子白介素(例如IL-2)。
如上文描述的本发明的组合物还可以是诊断性组合物,其进一步包含任选的用于检测的手段和方法。
本文中提供的三价双特异性结合分子或构建物(即本文中描述的三价双特异性抗体分子)还适合于在其中能以液相或结合至固相载体利用它们的免疫测定法中使用。能利用本发明的多肽的免疫测定法的例子是直接或间接格式的竞争性或非竞争性免疫测定法。此类免疫测定法的例子是酶联免疫吸附测定法(ELISA),酶免疫测定法(EIA),放射免疫测定法(RIA),夹心式(免疫计量测定法)和Western印迹测定法。
本发明的三价双特异性结合分子或构建物(即本文中描述的三价双特异性抗体分子)可结合至许多不同载体并用于分离特异性结合至所述多肽的细胞。公知载剂的例子包括玻璃,聚苯乙烯,聚氯乙烯,聚丙烯,聚乙烯,聚碳酸酯,右旋糖苷,尼龙,直链淀粉,天然和改性纤维素,聚丙烯酰胺,琼脂糖,和磁铁矿。出于本发明的目的,载体的性质可以是可溶性的或不溶性的,例如如珠。
有本领域普通技术人员知道的许多不同标记物和标记方法。能在本发明中使用的标记物的类型的例子包括酶,放射性同位素,胶态金属,荧光化合物,化学发光化合物,和生物发光化合物。
在本发明的一个最优选实施方案,涵盖供作为药物使用的本发明的三价双特异性抗体构建物/分子。在本发明的语境中,描述了供作为药物使用的三价双特异性抗体分子,其中所述三价双特异性抗体分子要在施用包含如本文中限定的融合蛋白的经转导T细胞,优选CD8+ T细胞之前,同时或之后施用且其中所述T细胞,优选CD8+ T细胞是自要治疗的受试者获得的。所述药物可以在一种治疗恶性疾病的方法中采用,尤其是上皮,内皮或间皮起源的或血液的癌症/癌。在本发明的语境中,癌症/癌选自由胃肠癌,胰腺癌,胆管细胞癌,肺癌,乳腺癌,卵巢癌,皮肤癌,口腔癌,胃癌,宫颈癌,B和T细胞淋巴瘤,髓样白血病,卵巢癌,白血病,淋巴细胞性白血病,鼻咽癌,结肠癌,前列腺癌,肾细胞癌,头和颈癌,皮肤癌(黑素瘤),生殖泌尿道癌,例如睾丸癌,卵巢癌,内皮癌,宫颈癌和肾癌,胆管癌,食管癌,唾液腺癌和甲状腺癌或其它肿瘤性疾病像血液学肿瘤,胶质瘤,肉瘤或骨肉瘤组成的组。
而且,在本发明的语境中,涵盖供一种治疗恶性疾病的方法中使用的如本文中描述的三价双特异性抗体分子,其包含(i)结合融合蛋白的胞外域,即在T细胞中或上并不天然发生的信号传导受体的胞外域的第一结合域,(ii)结合在肿瘤细胞的表面上天然发生的肿瘤特异性抗原的第二结合域；和(iii)结合融合蛋白的胞外域,即在T细胞中或上并不天然发生的信号传导受体的胞外域,或结合在肿瘤细胞的表面上天然发生的所述肿瘤特异性抗原的第三结合域,其中所述双特异性抗体分子要在施用包含如本文中限定的融合蛋白的经转导T细胞之前,同时或之后施用,其中所述T细胞,优选CD8+ T细胞是自要治疗的受试者获得的。
而且,在本发明的语境中,一种治疗恶性疾病的方法,该方法包含将本发明的三价双特异性抗体分子施用于有需要的受试者,其包含(i)结合融合蛋白的胞外域,即在T细胞中或上并不天然发生的信号传导受体的胞外域的第一结合域,(ii)结合在肿瘤细胞的表面上天然发生的肿瘤特异性抗原的第二结合域；和(iii)结合融合蛋白的胞外域,即在T细胞中或上并不天然发生的信号传导受体的胞外域,或结合在肿瘤细胞的表面上天然发生的所述肿瘤特异性抗原的第三结合域,其中所述三价双特异性抗体分子要在施用来自所述受试者的包含如本文中限定的融合蛋白的经转导T细胞,优选CD8+ T细胞之前,同时或之后施用。在本发明的语境中,癌症/癌选自由胃肠癌,胰腺癌,胆管细胞癌,肺癌,乳腺癌,卵巢癌,皮肤癌,口腔癌,胃癌,宫颈癌,B和T细胞淋巴瘤,髓样白血病,卵巢癌,白血病,淋巴细胞性白血病,鼻咽癌,结肠癌,前列腺癌,肾细胞癌,头和颈癌,皮肤癌(黑素瘤),生殖泌尿道癌,例如睾丸癌,卵巢癌,内皮癌,宫颈癌和肾癌,胆管癌,食管癌,唾液腺癌和甲状腺癌或其它肿瘤性疾病像血液学肿瘤,胶质瘤,肉瘤或骨肉瘤组成的组。
而且,依照本发明,包含一个或两个与之相互作用/结合/针对EpCAM,优选人EpCAM(作为在肿瘤细胞的表面上天然发生的肿瘤特异性抗原)的结合域且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的分子或构建物(即本文中描述的三价双特异性抗体分子)可在一种用于治疗胃肠癌,胰腺癌,胆管细胞癌,肺癌,乳腺癌,卵巢癌,皮肤癌和/或口腔癌的方法中使用。如此,在本发明的语境中,包含两个与之相互作用/结合/针对EpCAM,优选人EpCAM的结合域且包含一个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的三价双特异性抗体分子可在一种用于治疗胃肠癌,胰腺癌,胆管细胞癌,肺癌,乳腺癌,卵巢癌,皮肤癌和/或口腔癌的方法中使用。在本发明的一个备选实施方案中,包含一个与之相互作用/结合/针对EpCAM,优选人EpCAM的结合域且包含两个结合域与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的三价双特异性抗体分子可在一种用于治疗胃肠癌,胰腺癌,胆管细胞癌,肺癌,乳腺癌,卵巢癌,皮肤癌和/或口腔癌的方法中使用。在本发明的语境中,包含一个或两个针对EpCAM,优选人EpCAM的结合域且包含一个或两个与之相互作用/结合/针对Cripto的结合域的三价双特异性抗体分子可在治疗胃肠癌,例如胃肠起源的腺癌中使用。包含一个或两个针对HER1,优选人HER1的结合域和一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的本文中描述的三价双特异性抗体分子可在一种用于治疗胃肠癌,胰腺癌,胆管细胞癌,肺癌,乳腺癌,卵巢癌,皮肤癌和/或口腔癌的方法中使用。包含一个或两个与之相互作用/结合/针对HER2,优选人HER2的结合域且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的分子或构建物(即本文中描述的三价双特异性抗体分子)可在用于治疗胃癌,乳腺癌和/或宫颈癌一种的方法中使用。包含一个或两个与之相互作用/结合/针对HER3,优选人HER3的结合域且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的分子或构建物(即本文中描述的三价双特异性抗体分子)可在一种用于治疗胃癌,乳腺癌和/或宫颈癌的方法中使用。包含一个或两个与之相互作用/结合/针对CD20,优选人CD20的结合域且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的分子或构建物(即本文中描述的三价双特异性抗体分子)可在一种用于治疗胃癌,乳腺癌和/或宫颈癌的方法中使用。包含一个或两个与之相互作用/结合/针对CD22,优选人CD22的结合域且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的分子或构建物(即本文中描述的三价双特异性抗体分子)可在一种用于治疗胃癌,乳腺癌和/或宫颈癌的方法中使用。包含一个或两个与之相互作用/结合/针对CD33,优选人CD33的结合域且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的分子或构建物(即本文中描述的三价双特异性抗体分子)可在一种用于治疗胃癌,乳腺癌和/或宫颈癌的方法中使用。包含一个或两个与之相互作用/结合/针对CA12-5,优选人CA12-5的结合域且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的分子或构建物(即本文中描述的三价双特异性抗体分子)可在一种用于治疗胃癌,乳腺癌和/或宫颈癌的方法中使用。包含一个或两个与之相互作用/结合/针对HLA-DR,优选人HLA-DR的结合域且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的分子或构建物(即本文中描述的三价双特异性抗体分子)可在一种用于治疗胃癌,乳腺癌和/或宫颈癌的方法中使用。包含一个或两个与之相互作用/结合/针对MUC-1,优选人MUC-1的结合域且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的分子或构建物(即本文中描述的三价双特异性抗体分子)可在一种用于治疗胃癌,乳腺癌和/或宫颈癌的方法中使用。包含一个或两个与之相互作用/结合/针对A33,优选人A33的结合域且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的分子或构建物(即本文中描述的三价双特异性抗体分子)可在一种用于治疗胃癌,乳腺癌和/或宫颈癌的方法中使用。包含一个或两个与之相互作用/结合/针对PSMA,优选人PSMA的结合域且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的分子或构建物(即本文中描述的三价双特异性抗体分子)可在一种用于治疗胃癌,乳腺癌和/或宫颈癌的方法中使用。包含一个或两个与之相互作用/结合/针对运铁蛋白受体,优选人运铁蛋白受体的结合域且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的分子或构建物(即本文中描述的三价双特异性抗体分子)可在一种用于治疗胃癌,乳腺癌和/或宫颈癌的方法中使用。包含一个或两个与之相互作用/结合/针对CA-IX,优选人CA-IX的结合域且包含一个或两个与之相互作用/结合/针对本文中限定的融合蛋白的胞外域之一,即在T细胞中和/或上并不天然发生的信号传导受体的胞外域的结合域的分子或构建物(即本文中描述的三价双特异性抗体分子)可在一种用于治疗胃癌,乳腺癌和/或宫颈癌的方法中使用。
本发明还涉及一种用于治疗疾病,恶性疾病诸如上皮,内皮或间皮起源的癌症和/或血液的癌症的方法。此类疾病会格外是:食管,胃,结肠,小肠,肝,胰腺,乳腺,肺,脑,肾,睾丸的癌症,皮肤癌,白血病和/或淋巴瘤,其包含将经转导T细胞施用于受试者。在本发明的语境中,所述受试者是人。
在本发明的语境中,描述了一种用于治疗疾病的方法,其包含下述步骤:
(a)自受试者分离T细胞,诸如CD8+ T细胞,CD4+ T细胞,CD3+ T细胞,γδ T细胞或天然杀伤(NK)T细胞,优选CD8+ T细胞；
(b)用如本文中上文描述的融合蛋白转导所述分离的T细胞,诸如CD8+ T细胞,CD4+ T细胞,CD3+ T细胞,γδ T细胞或天然杀伤(NK)T细胞,优选CD8+ T细胞；并
(c)将经转导T细胞,诸如CD8+ T细胞,CD4+ T细胞,CD3+ T细胞,γδ T细胞或天然杀伤(NK)T细胞,优选CD8+ T细胞施用于所述受试者。
在本发明的语境中,通过静脉内输注将所述经转导T细胞,优选CD8+ T细胞施用于所述受试者。
此外,本发明提供一种用于治疗疾病的方法,其包含下述步骤:
(a)自受试者分离T细胞,诸如CD8+ T细胞,CD4+ T细胞,CD3+ T细胞,γδ T细胞或天然杀伤(NK)T细胞,优选CD8+ T细胞；
(b)用如本文中上文描述的融合蛋白转导所述分离的T细胞,诸如CD8+ T细胞,CD4+ T细胞,CD3+ T细胞,γδ T细胞或天然杀伤(NK)T细胞,优选CD8+ T细胞；
(c)用T细胞受体共转导所述分离的T细胞,诸如CD8+ T细胞,CD4+ T细胞,CD3+ T细胞,γδ T细胞或天然杀伤(NK)T细胞,优选CD8+ T细胞；
(d)扩充T细胞,诸如CD8+ T细胞,CD4+ T细胞,CD3+ T细胞,γδ T细胞或天然杀伤(NK)T细胞,优选CD8+ T细胞,通过抗CD3和抗CD28抗体；并
(e)将经转导T细胞,诸如CD8+ T细胞,CD4+ T细胞,CD3+ T细胞,γδ T细胞或天然杀伤(NK)T细胞,优选CD8+ T细胞施用于所述受试者。
本发明涉及分离的T细胞,它们通过方法进行分析以确保在分离的T细胞上天然发生的(肿瘤)抗原与本文中描述的三价双特异性抗体经由一个或两个结合域结合的肿瘤抗原相同。在本发明的语境中,通过导入包含在T细胞中和/或上并不天然发生/天然表达的信号传导受体的胞外域的融合蛋白来人工修饰分离的/获得的包含如本文中限定的融合蛋白的T细胞。在本发明的语境中,分离的/获得的T细胞的人工修饰涉及本文中描述的转导方法。因而,在本发明的语境中,要治疗的受试者涉及特征在于罹患特征在于具有如本文中上文描述的在肿瘤细胞的表面上天然发生的肿瘤特异性抗原的疾病的受试者。在本发明的语境中,自要治疗的受试者获得/分离的经转导T细胞的施用会通过静脉内输注来实施。
在又一个实施方案中,本发明涉及一种用于治疗疾病的方法,其包含下述步骤:
(a)自来自患者的切除肿瘤分离肿瘤浸润淋巴细胞(TIL)；
(b)培养并用如本文中上文描述的融合蛋白转导TIL；
(c)基于功能性肿瘤识别测定法选择TIL培养物；
(d)通过抗CD3和/或抗CD28抗体扩充TIL；并
(e)将经转导TIL施用于所述受试者。
术语“功能性肿瘤识别”测定法意味着TIL与自体(例如患者的)肿瘤细胞或相同HLA型的细胞系的共培养。读出是对肿瘤细胞的细胞毒性活性(LDH,钙黄绿素释放)。别的读出可以是细胞因子分泌,T细胞的关于胞内细胞因子的存在的流式细胞术,ELISPOT测定法。
上文提到的步骤(d)(指通过抗CD3和/或抗CD28抗体扩充T细胞诸如TIL的步骤)也可以在(刺激性)细胞因子诸如白介素-2和/或白介素-15(IL-15)存在下实施。在本发明的语境中,上文提到的步骤(d)(指通过抗CD3和/或抗CD28抗体扩充T细胞诸如TIL的步骤)也可以在白介素-12(IL-12),白介素-7(IL-7)和/或白介素-21(IL-21)存在下实施。
用于治疗的方法还可另外包含施用本发明的三价双特异性抗体。所述三价双特异性抗体可以在施用经转导T细胞之前,同时或之后施用。在本发明的语境中,经转导T细胞的施用会通过静脉内输注来实施。在本发明的语境中,经转导T细胞是自该要治疗的受试者分离/获得的。
本发明的说明书和实施例公开并涵盖这些和其它实施方案。关注要依照本发明采用的抗体,方法,用途和化合物任一的别的文献可以使用例如电子设备自公众图书馆和数据库取得。例如,可利用在因特网上可得的公众数据库“Medline”,例如在http://www.ncbi.nlm.nih.gov/PubMed/medline.html下。别的数据库和地址,诸如http://www.ncbi.nlm.nih.gov/,http://www.infobiogen.fr/,http://www.fmi.ch/biology/research_tools.html,http://www.tigr.org/是本领域技术人员知道的且也能获得的,例如使用http://www.lycos.com。
附图简述
图1:表达肿瘤抗原EpCAM(EpCAM+)或不表达肿瘤抗原EpCAM(EpCAM-)的经转导T细胞和鼠胰腺癌肿瘤细胞(Panc02-OVA)的共培养物
为了证明经转导T细胞经由活化性融合蛋白EGFRvIII-CD28-CD3z(SEQ ID NO:42(如由SEQ ID NO:41中显示的DNA编码的))的活化,用EGFRvIII-CD28-CD3z融合蛋白转导T细胞(以下称作“E3 T细胞”)。在有或没有四价双特异性抗体(bsAb)“BsAb EpCAM-EGFRvIII,MR1.1”(SEQ ID NO:229(轻链(无前导序列)和SEQ ID NO:230(重链(无前导序列))的情况下以10:1比率将经转导T细胞与表达肿瘤抗原EpCAM(EpCAM+)的胰腺癌(Panc02-OVA)细胞或不表达肿瘤抗原EpCAM(EpCAM-)的胰腺癌(Panc02-OVA)细胞一起温育12小时。另外,将有E3 T细胞有相应浓度的四价双特异性抗体“BsAb EpCAM-EGFRvIII,MR1.1”的条件用含20％FCS(Gibco Products,Grand Island,美国)的PBS于37℃预处理30分钟以封闭非特异性结合。封闭的概念是本领域技术人员公知的,一种通过添加过量的多克隆蛋白质(这里是FCS)来防止给定蛋白质(这里是抗体)的非特异性结合的手段。作为阴性对照,使用用非信号传导标志物抗原转导的T细胞。使用ELISA作为INF-γ分泌来测量T细胞活化。结果显示抗原阳性(EpCAM+)胜过抗原阴性肿瘤细胞的增强的肿瘤细胞识别。四价双特异性抗体(bsAb)“BsAb EpCAM-EGFRvIII,MR1.1”将E3 T细胞募集至胰腺癌(Panc02-OVA)细胞并诱导朝向表达肿瘤细胞的EpCAM的特异性再定向T细胞活化。在经EGFR转导的T细胞中检测不到活化。这些结果指示可使用人EGFRvIII-CD28-CD3z融合蛋白来经由四价双特异性抗体“BsAb EpCAM-EGFRvIII,MR1.1”触发T细胞活化。然而,在四价双特异性抗体“BsAb EpCAM-EGFRvIII,MR1.1”存在下的T细胞活化在四价双特异性抗体分子“BsAbEpCAM-EGFRvIII,MR1.1”的肿瘤靶缺失下也发生,指示非特异性T细胞活化。图1中的术语“封闭的”指为了评估非特异性T细胞活化的程度,其中在用FCS封闭的板上将经转导T细胞与四价双特异性抗体一起共温育的条件。在这种条件中,活化,如果有的话,应当源自非定向T细胞交联。术语“PancO2-OVA”指为了评估非特异性(非肿瘤定向的)活化和裂解,其中将经转导T细胞与四价双特异性抗体和EpCAM-肿瘤细胞一起共温育的条件。术语“Panc02-OVA-EpCAM”指为了评估特异性(肿瘤上)活化和裂解,其中将经转导T细胞与四价双特异性抗体和EpCAM+肿瘤细胞一起共温育的条件。
图2:经转导T细胞和肿瘤细胞的共培养物中的双特异性抗体滴定
基于上文关于图1描述的实验设置,实施其中四价双特异性抗体(bsAb)“BsAbEpCAM-EGFRvIII,MR1.1”(SEQ ID NO:229(轻链(无前导序列)和SEQ ID NO:230(重链(无前导序列))的浓度改变的实验。通过自1ng/mL至1μg/mL滴定双特异性抗体分子(bsAb),能看到通过ELISA测量的分泌的IFN-γ的量的增加。发现EpCAM-特异性T细胞活化和非特异性T细胞活化均是剂量依赖性的。
图3:由板结合的三价较之四价双特异性抗体提供的T细胞活化的比较
将经EGFRvIII-CD28-CD3z融合蛋白(SEQ ID NO:42(蛋白质)和41(DNA))转导的T细胞(以下称作“E3 T细胞”)或野生型(WT)T细胞用浓度递增的或是包被在板上或是添加至T细胞培养物的(i)四价双特异性抗体“BsAbEpCAM-EGFRvIII,MR1.1”(SEQ ID NO:229(轻链(无前导序列)和SEQ IDNO:230(重链(无前导序列)),(ii)三价双特异性抗体“BsABEGFRvIII-EpCAM”(SEQ ID NO:233,其包含/组成为质粒/载体“EGFR vIII MR1.1 VH CkmuEpCAM VH CH1EE Fc节PG LALA,pETR14953”(SEQ ID NO:22(蛋白质)和21(DNA),“EGFRvIII MR1.1 VL CH1,pETR14951”(SEQ ID NO:24(蛋白质)和23(DNA),“VL EpCAM G.8.8 CkRK,pETR14882”(SEQ ID NO:26(蛋白质)和25(DNA)和“VH muEpCAM CH1EE Fc穴PG LALAHRYF,pETR14940”(SEQ ID NO:28(蛋白质)和27(DNA)；还见图9和表1和2)或(iii)作为阳性对照的西妥昔单抗(Merck,德国)刺激48小时。在第一种条件中,用抗体包被测定板,在第二种条件中,将抗体可溶解的添加至T细胞。通过ELISA来测定IFN-γ释放。结果显示在包被的条件中所有抗体相当的经E3转导T细胞活化。图3中的术语“封闭的”指为了评估非特异性T细胞活化的程度,其中在用FCS封闭的板上将经转导T细胞与四价双特异性抗体一起共温育的条件。在这种条件中,活化,如果有的话,应当源自非定向T细胞交联。
图4:与可溶性三价较之四价双特异性抗体一起温育的经转导T细胞的活化的比较
为了调查在可溶性条件中经EGFRvIII-CD28-CD3z融合蛋白(SEQ ID NO:42(蛋白质)和41(DNA))转导的T细胞(以下称作“E3 T细胞”)的非特异性活化,在IFN-γELISA中不稀释地使用如图3所述的样品。四价双特异性抗体分子“BsAb EpCAM-EGFRvIII,MR1.1”(SEQID NO:229(轻链(无前导序列)和SEQ ID NO:230(重链(无前导序列))显示T细胞的非特异性活化,可能是由于两个针对T细胞的结合位点。这可导致两个T细胞之间的交联和由此细胞的IFN-γ分泌。与之相反,凭借三价双特异性抗体(bsAb)分子“BsAB EGFRvIII-EpCAM”(SEQ ID NO:233,其包含/组成为质粒/载体“EGFR vIIIMR1.1 VH Ck muEpCAM VH CH1 EEFc节PG LALA,pETR14953”(SEQ ID NO:22(蛋白质)和21(DNA),“EGFR vIII MR1.1 VL CH1,pETR14951”(SEQ ID NO:24(蛋白质)和23(DNA),“VL EpCAM G.8.8 Ck RK,pETR14882”(SEQID NO:26(蛋白质)和25(DNA)和“VH muEpCAM CH1 EE Fc穴PG LALA HRYF,pETR14940”(SEQID NO:28(蛋白质)和27(DNA)；还见图9和表1和2),这种非特异性活化因丧失一个EGFR结合位点而消除。
图5:作为抗体浓度的函数的表面抗原结合的剂量依赖性的分析
为了了解抗体效价对四价双特异性抗体分子“BsAb EpCAM-EGFRvIII,MR1.1”(SEQID NO:229(轻链(无前导序列)和SEQ ID NO:230(重链(无前导序列))和三价双特异性抗体(bsAb)分子“BsAB EGFRvIII-EpCAM”(SEQ ID NO:233,其包含/组成为质粒/载体“EGFRvIII MR1.1VH Ck muEpCAM VH CH1EE Fc节PG LALA,pETR14953”(SEQ ID NO:22(蛋白质)和21(DNA),“EGFR vIII MR1.1VL CH1,pETR14951”(SEQ ID NO:24(蛋白质)和23(DNA),“VLEpCAM G.8.8 Ck RK,pETR14882”(SEQ ID NO:26(蛋白质)和25(DNA)和“VH muEpCAM CH1EEFc穴PG LALA HRYF,pETR14940”(SEQ ID NO:28(蛋白质)和27(DNA)；还见图9和表1和2)对经EGFRvIII-CD28-CD3z融合蛋白(SEQ ID NO:42(蛋白质)和41(DNA))转导的T细胞的表面饱和的影响,将细胞与浓度递增的四价或三价/bsAb(10ng/mL；100ng/mL；500ng/mL；1μg/mL；5μg/mL；10μg/mL；20μg/mL；25μg/mL)一起温育并通过流式细胞术测定二抗(FITCAffiniPure F(ab')2片段山羊抗人IgG,F(ab')2片段特异性:109-096-097)染色的表面饱和。受益于另外的针对EGFR的结合位点,四价双特异性抗体(bsAb)在低浓度显示更高的饱和(曲线与三价双特异性抗体(bsAb)相比左移)。
图6:作为抗体浓度的函数的由三价较之四价双特异性抗体(bsAb)介导的经转导T细胞针对EpCAM+肿瘤细胞的再定向裂解能力的比较
为了比较这两种抗体的细胞毒性潜力,将bsAb预加载的经人EGFRvIII-CD28-CD3z融合蛋白(SEQ ID NO:42(蛋白质)和41(DNA))转导的T细胞(以下称作“E3 T细胞”)(分别用三价或四价双特异性抗体(bsAb))与表达肿瘤抗原EpCAM的胰腺癌细胞(Panc02-OVA)一起共培养9小时。通过肿瘤细胞的LDH释放来测量杀伤功效。抗体(浓度为250ng/ml,125ng/ml或62.5ng/ml)具有或多或少相同的针对EpCAM表达性(EpCAM+)胰腺癌细胞的裂解能力。然而,在较低浓度(即浓度为31.25ng/ml或15.63ng/ml),三价bsAb特征在于针对EpCAM+癌细胞的细胞毒性作用与四价双特异性抗体分子相比升高。
图7:作为抗体浓度的函数的由三价较之四价双特异性抗体介导的经转导T细胞针对EpCAM-肿瘤细胞的非特异性裂解能力的比较
将不表达EpCAM(EpCAM-)的胰腺癌细胞(Panc02-OVA)与bsAb预加载的T细胞(用三价或四价双特异性抗体)一起共培养9小时。通过LDH释放来测定裂解能力。在高抗体浓度(即抗体浓度为250ng/ml或62.5ng/ml),四价双特异性抗体分子“BsAb EpCAM-EGFRvIII,MR1.1”显示非特异性背景裂解,它在较低抗体浓度时降低。与之相反,三价双特异性抗体(bsAb)分子“BsABEGFRvIII-EpCAM”(SEQ ID NO:233,其包含/组成为质粒/载体“EGFR vIIIMR1.1 VH Ck muEpCAM VH CH1 EE Fc节PG LALA,pETR14953”(SEQ ID NO:22(蛋白质)和21(DNA),“EGFR vIII MR1.1VL CH1,pETR14951”(SEQ ID NO:24(蛋白质)和23(DNA),“VLEpCAM G.8.8 Ck RK,pETR14882”(SEQ ID NO:26(蛋白质)和25(DNA)和“VH muEpCAM CH1EE Fc穴PG LALA HRYF,pETR14940”(SEQ ID NO:28(蛋白质)和27(DNA)；还见图9和表1和2)在每种测试的浓度不显示非特异性背景裂解。对三价bsAb显示的非特异性靶细胞裂解在四价bsAb时消除。如此,三价双特异性抗体(bsAb)分子“BsAB EGFRvIII-EpCAM”(SEQ ID NO:233)和经人EGFRvIII-CD28-CD3z融合蛋白(SEQ ID NO:42(蛋白质)和41(DNA))转导的T细胞(称作“E3 T细胞”)的组合特异性裂解EpCAM表达性(EpCAM+)胰腺癌细胞。
图8:融合蛋白的示意性概览
Cripto融合蛋白(SEQ ID NO:46(鼠)和120(人))由Ig前导序列(SEQ ID NO:206),Cripto的胞外域(SEQ ID NO:62),CD8的铰链域(SEQ ID NO:64(鼠)和74(人)),和CD28(SEQID NO:56(鼠)和68(人))和CD3z(SEQ ID NO:58(鼠)和70(人))的共刺激性信号传导域构成。EGFRvIII融合蛋白(SEQ ID NO:42(鼠)和48(人))由EGFRvIII胞外域(SEQ ID NO:76),CD28的锚定跨膜域(SEQ ID NO:54(鼠)和66(人)),CD28的共刺激性信号传导域(SEQ IDNO:56(鼠)和68(人))和CD3z的信号传导域(SEQ ID NO:58(鼠)和70(人))构成。或者,EGFRvIII融合蛋白(SEQ ID NO:44(鼠)和50(人))由EGFRvIII胞外域(SEQ ID NO:76),CD28的锚定跨膜域(SEQ ID NO:54(鼠)和66(人)),CD28(SEQ ID NO:56(鼠)或66(人))和4-1-BB(SEQ ID NO:60(鼠)或72(人))的共刺激性信号传导域和CD3z的刺激性信号传导域(SEQ IDNO:58(鼠)或70(人))构成。
图9:三价双特异性抗体(bsAb)分子“BsAB EGFRvIII-EpCAM”的示意性结构
三价双特异性抗体(bsAb)分子“BsAB EGFRvIII-EpCAM”(SEQ ID NO:233,其包含/组成为质粒/载体“EGFR vIII MR1.1 VH Ck muEpCAM VH CH1 EE Fc节PG LALA,pETR14953”,“EGFR vIII MR1.1 VL CH1,pETR14951”,“VL EpCAM G.8.8 Ck RK,pETR14882”和“VH muEpCAM CH1 EE Fc穴PG LALA HRYF,pETR14940”；还见表1和2)的示意性结构。将muEpCAM/EGFRvIII的可变域与预插入相应受体哺乳动物表达载体的恒定链同框亚克隆。蛋白质表达由CMV启动子驱动且合成的polyA信号序列存在于编码序列(CDS)的3’端。另外,每种载体含有EBV OriP序列。如下生成分子,即用哺乳动物表达载体共转染悬浮生长的CHO细胞。瞬时转染在Evitria AG(瑞士)进行。以1:2:1:1比率(“载体重链穴(VH-CH1-CH2-CH3)”:“轻链(LC)”:“载体重链节(VH-CK-VH-CH1-CH2-CH3)”:“交叉轻链(VL-CH1)”)用相应的表达载体转染细胞。将过滤后的上清液保持于4℃直至纯化。通过使用蛋白A亲和层析的亲和层析,接着是一个至两个大小排阻层析(SEC)步骤自细胞培养物上清液纯化分泌的蛋白质。通过测量280nm处的光密度(OD),除以基于氨基酸序列计算的摩尔消光系数来测定纯化后的蛋白质样品的蛋白质浓度。在还原剂存在和缺失下通过CE-SDS分析来分析最终的纯化步骤之后分子的纯度和分子量(图9A和9B,右边)。依照制造商的用法说明书使用Caliper LabChip GXII系统(Caliper Lifescience)。于25℃在25mM K2HPO4,125mMNaCl,200mM L-精氨酸单氢氯化物,0.02％(w/v)NaN3,pH 6.7运行缓冲液中使用TSKgelG3000 SW XL分析性大小排阻柱(Tosoh)分析分子的聚集物含量。所有分子的最终质量较好,具有≥96％的单体含量。分子1和2指在体外批次中生成的(分子1)或在体内批次中生成的(分子2)三价双特异性抗体(bsAb)分子“BsAB EGFRvIII-EpCAM”(SEQ ID NO:233,其包含/组成为质粒/载体“EGFR vIII MR1.1 VH Ck muEpCAM VH CH1 EE Fc节PG LALA,pETR14953”(SEQ ID NO:22(蛋白质)和21(DNA),“EGFR vIII MR1.1 VL CH1,pETR14951”(SEQ ID NO:24(蛋白质)和23(DNA),“VL EpCAM G.8.8 Ck RK,pETR14882”(SEQ ID NO:26(蛋白质)和25(DNA)和“VH muEpCAM CH1 EE Fc穴PG LALA HRYF,pETR14940”(SEQ ID NO:28(蛋白质)和27(DNA)；还见表1和2)。muEpCAM/EGFRvIII分子的生成和纯化的汇总:
分子 滴度[mg/l] 产率[mg/l] 分析性SEC(HMW/单体/LMW)[％]
1(体外批次) 18 6.98 0％/100％/0％
2(体内批次) 18 11.6 3.68％/96.32％/0％
HMW＝高分子量
LMW＝低分子量
图10:三价双特异性抗体(bsAb)分子“BsAB EGFRvIII-MSLN”的示意性结构
三价双特异性抗体(bsAb)分子“BsAB EGFRvIII-MSLN”(SEQ ID NO:235,其包含/组成为质粒/载体“EGFR vIII MR1.1 VH Ck MSLN VH CH1 EE Fc节PG LALA,pETR15655”,“EGFR vIII MR1.1 VL CH1,pETR15656”,“VL MSLN Ck RK,pETR15443”和“VH MSLN CH1 EEFc穴PG LALA HRYF,pETR15667”；还见表3和4)的示意性结构。将MSLN/EGFRvIII的可变域与预插入相应受体哺乳动物表达载体的恒定链同框亚克隆。蛋白质表达由MPSV启动子驱动且合成的polyA信号序列存在于CDS的3’端。另外,每种载体含有EBV OriP序列。如下生成分子,即使用聚乙烯亚胺(PEI)用哺乳动物表达载体共转染悬浮生长的HEK293-EBNA细胞。以1:2:1:1比率(“载体重链穴(VH-CH1-CH2-CH3)”:“轻链(LC)”:“载体重链节(VH-CK-VH-CH1-CH2-CH3)”:“交叉轻链(VL-CH1)”)用相应的表达载体转染细胞。将溶液无菌过滤(0.22μm滤器)并以0.01％w/v的终浓度添加叠氮化钠。将溶液保持于4℃直至纯化。通过使用蛋白A亲和层析的亲和层析,接着是一个至两个大小排阻层析(SEC)步骤自细胞培养物上清液纯化分泌的蛋白质。在还原剂存在和缺失下通过CE-SDS分析来分析最终的纯化步骤之后分子的纯度和分子量(图10A,右边)。依照制造商的用法说明书使用Caliper LabChip GXII系统(Caliper Lifescience)。于25℃在25mM K2HPO4,125mM NaCl,200mM L-精氨酸单氢氯化物,0.02％(w/v)NaN3,pH 6.7运行缓冲液中使用TSKgel G3000 SW XL分析性大小排阻柱(Tosoh)分析分子的聚集物含量(图10A)。所有分子的最终质量较好,具有≥96％的单体含量。分子1指三价双特异性抗体(bsAb)分子“BsAB EGFRvIII-MSLN”(SEQ ID NO:235,其包含/组成为质粒/载体“EGFR vIII MR1.1 VH Ck MSLN VH CH1 EE Fc节PG LALA,pETR15655”(SEQ ID NO:2(蛋白质)和1(DNA),“EGFR vIII MR1.1 VL CH1,pETR15656”(SEQID NO:4(蛋白质)和3(DNA),“VL MSLN Ck RK,pETR15443”(SEQ ID NO:6(蛋白质)和5(DNA)和“VH MSLN CH1EE Fc穴PG LALA HRYF,pETR15667”(SEQ ID NO:8(蛋白质)和7(DNA)；还见图10和表3和4)。MSLN/EGFRvIII分子的生成和纯化的汇总:
分子 滴度[mg/l] 产率[mg/l] 分析性SEC(HMW/单体/LMW)[％]
1 65 1.16 3.31/96.3/0.4
HMW＝高分子量
LMW＝低分子量
(A)三价双特异性抗体(bsAb)分子“BsAB EGFRvIII-MSLN”(SEQ ID NO:235,其包含/组成为质粒/载体“EGFR vIII MR1.1 VH Ck MSLN VH CH1 EE Fc节PG LALA,pETR15655”,“EGFR vIII MR1.1 VL CH1,pETR15656”,“VL MSLN Ck RK,pETR15443”和“VHMSLN CH1 EE Fc穴PG LALA HRYF,pETR15667”；还见表3和4)的CE-SDS分析:为电子凝胶电泳显示了蛋白质标准品,处于非还原条件的蛋白质和处于还原条件的蛋白质。右边的图显示处于非还原和还原条件的蛋白质的荧光。(B)三价双特异性抗体(bsAb)分子“BsABEGFRvIII-MSLN”(SEQ ID NO:235；还见表3和4)的用于测定纯度的分析性大小排阻层析分析。
图11:三价双特异性抗体(bsAb)分子“BsAB EGFRvIII-MCSP的示意性结构
三价双特异性抗体(bsAb)分子“BsAB EGFRvIII-MCSP(SEQ ID NO:234,其包含/组成为质粒/载体“MR1.1 EGFRvIII VH-Ck-(G4S)2 MCSP M4-3 VH CH1 EE Fc节PG LALA,pETR16621(SEQ ID NO:208,如由SEQ ID NO:207中显示的DNA序列编码的),“EGFR vIIIMR1.1 VL CH1,pETR15656”(SEQ ID NO:210(蛋白质)和209(DNA),“MCSP ML2 VL Ck RK,pETR16619”(SEQ ID NO:212(蛋白质)和211(DNA)和“MCSP M4-3 VH CH1 EE Fc穴PG LALAHYRF,pETR16618”(SEQ ID NO:214(蛋白质)和213(DNA))；还见表5和6)的示意性结构。(A)三价双特异性抗体(bsAb)分子“BsAB EGFRvIII-MCSP(SEQ ID NO:234；还见表5和6)的CE-SDS分析:为电子凝胶电泳显示了蛋白质标准品,处于非还原条件的蛋白质和处于还原条件的蛋白质。
图12:表达肿瘤抗原EpCAM(EpCAM+)的鼠癌症肿瘤细胞(B16EpCAM和4T1肿瘤模型)与四价双特异性抗体(bsAb)“BsAb EpCAM-EGFRvIII,MR1.1”(SEQ ID NO:229(轻链(无前导序列)和SEQ ID NO:230(重链(无前导序列))和三价双特异性抗体“BsAB EGFRvIII-EpCAM”(SEQ ID NO:233；还见表1和2)的共培养物
为了测定双特异性抗体(bsAb)的剂量-响应曲线,头对头地向下滴定四价双特异性抗体(bsAb)“BsAb EpCAM-EGFRvIII,MR1.1”(SEQ ID NO:229(轻链(无前导序列)和SEQID NO:230(重链(无前导序列))和三价双特异性抗体“BsAB EGFRvIII-EpCAM”(SEQ ID NO:233；还见表1和2)二者并通过乳酸脱氢酶(LDH)释放来测量细胞毒性。因此,在B16EpCAM肿瘤模型中(图12A)以及在4T1肿瘤模型中(图12B),相应地对较低剂量的抗体能观察到递减的细胞毒性。而且,两种抗体格式均显示足够且相当的肿瘤细胞杀伤。
图13:用于确定E3转导的鼠T细胞针对Panc02-EpCAM肿瘤细胞的杀伤的机制的实时细胞毒性测定法
经EGFRvIII-CD28-CD3z融合蛋白(SEQ ID NO:42(蛋白质)和41(DNA))转导的T细胞(称作“E3”)的杀伤能力可通过用FasL封闭性抗体(CD178(Fas配体)单克隆抗体,克隆MFL3(目录号16-5911-85(ThermoFisher ScientificTM)阻断肿瘤细胞和T细胞之间的FasL-Fas相互作用而削弱。这一发现通过使用来自ACEA Bioscience的iCELLigence仪器在图13中显示。该装置测量阻抗的量级随时间的变化,这取决于贴壁细胞的数目。与活化性融合蛋白EGFRvIII-CD28-CD3z(SEQ ID NO:42(蛋白质)和41(DNA))形成对比,经EpCAM特异性嵌合抗原受体(CAREpcam；SEQ ID NO:249(蛋白质)和248(DNA))转导的T细胞仍然能够在FasL封闭性抗体存在下诱导肿瘤细胞裂解。条件PancOVAEpCAM指只有肿瘤细胞的条件。条件T细胞指只有T细胞的条件。条件CAREpCAM指CAREpCAM(SEQ ID NO:249(蛋白质)和248(DNA))转导的T细胞与PancOVA-EpCAM的共培养物。
图14:使用浓度变化的三价双特异性抗体分子“BsAb EGFRvIII-MSLN”(SEQ IDNO:235；还见表3和4)的抗体结合测定法
将经人版本的EGFRvIII-CD28-CD3z融合蛋白(SEQ ID NO:48(蛋白质),如由SEQID NO:47(DNA)编码的)转导的T细胞(以下称作“E3 T细胞”)用三价双特异性抗体“EGFRvIII-MSLN”(SEQ ID NO:235；还见表3和4)以bsAB浓度1.0g/ml刺激48小时。“EGFRvIII-MSLN”bsAb特异性刺激E3转导的T细胞而缺乏完整E3构建物的T细胞(E3del(SEQID NO:247(蛋白质)和246(DNA))和UT)在“EGFRvIII-MSLN”bsAb存在下没有得到刺激。这种T细胞刺激以E3-bsAb经由它的Fc样部分结合至板为条件,所以可溶性E3-bsAb不刺激E3转导的T细胞。E3del是缺乏胞内域的截短版本的E3,而UT指未转导的T细胞。
图15:重组间皮素(MSLN)刺激:在重组间皮素存在下经转导T细胞和三价双特异性抗体分子“BsAb EGFRvIII-MSLN”(SEQ ID NO:235；还见表3和4)的共培养物
经人版本的EGFRvIII-CD28-CD3z融合蛋白(SEQ ID NO:48(蛋白质),如由SEQ IDNO:47(DNA)编码的)转导的T细胞(称作“E3 T细胞”或“E3”)用三价双特异性抗体(bsAb)“EGFRvIII-MSLN”(SEQ ID NO:235；还见表3和4)以bsAb浓度1.0μg/ml刺激48小时。“EGFRvIII-MSLN”bsAb特异性刺激E3 T细胞而缺乏完整E3构建物的T细胞(E3del(SEQ IDNO:247(蛋白质)和246(DNA))和UT)在“EGFRvIII-MSLN”bsAb(可溶性)和重组间皮素(包被至孔–浓度为5μg/ml)存在下没有得到刺激。对照是T细胞(E3 T细胞较之未转导的T细胞较之E3del构建物转导的T细胞)。E3del是缺乏胞内域的截短版本的E3,而UT指未转导的T细胞。
图16:HEK293-FLIPin-MSLN E3转导的人T细胞的共培养物
将经人版本的EGFRvIII-CD38-CD3z融合蛋白(SEQ ID NO:48(蛋白质),如由SEQID NO:47(DNA)编码的)转导的T细胞(以下称作“E3 T细胞”或“E3”)用三价双特异性抗体“EGFRvIII-MSLN”(SEQ ID NO:235；还见表3和4)以“EGFRvIII-MSLN”bsAB浓度1.0μg/ml刺激48小时。“EGFRvIII-MSLN”bsAb特异性刺激E3 T细胞而缺乏完整E3构建物的T细胞(E3del(SEQ ID NO:247(蛋白质)和246(DNA))和UT)在“EGFRvIII-MSLN”bsAb(可溶性)和HEK293-FLPin-MSLN细胞(HEK293)存在下没有得到刺激。当用单克隆(C12)以及用多克隆设置建立测定法时观察到这一点。UT指未转导的T细胞。BsAB指只有双特异性抗体(bsAb)的条件。共培养以效应对靶比率10:1运行48小时。在共培养之前6小时接种肿瘤细胞；在共培养之前30分钟用bsAb预加载T细胞(bsAb浓度1μg/ml)。E3del是缺乏胞内域的截短版本的E3,而UT指未转导的T细胞。
图17:Suit-OE-MSLN刺激测定法:使用MSLN过表达性胰腺细胞系来测试三价双特异性抗体“EGFRvIII-MSLN”(SEQ ID NO:235；还见表3和4)对于在所述肿瘤细胞存在或缺失下T细胞的条件性刺激的有效性
将经人版本的EGFRvIII-CD38-CD3z融合蛋白(SEQ ID NO:48(蛋白质),如由SEQID NO:47(DNA)编码的)转导的间皮素(MSLN)过表达性胰腺细胞系的T细胞(在图中称作“Suits007OE”)(以下称作“E3 T细胞”或“E3”)用三价双特异性抗体(bsAb)“EGFRvIII-MSLN”(SEQ ID NO:235；还见表3和4)以“EGFRvIII-MSLN”bsAB浓度1.0μg/ml刺激48小时。术语“Suits007OE”指胰腺细胞系。“EGFRvIII-MSLN”bsAb特异性刺激E3 T细胞而缺乏完整E3构建物的T细胞(E3del(SEQ ID NO:247(蛋白质)和246(DNA))和UT)在“EGFRvIII-MSLN”bsAb(可溶性)和Suit007OE细胞存在下没有得到刺激。对肿瘤细胞以效应对靶比率19:1实施测定法。在共培养之前6小时接种肿瘤细胞；在共培养之前30分钟用“EGFRvIII-MSLN”bsAb预加载T细胞(bsAb浓度1μg/ml)。结果证明该策略识别和活化癌细胞的能力。E3del是缺乏胞内域的截短版本的E3,而UT指未转导的T细胞。
实施例
下面的实施例例示本发明。
实施例1:四价双特异性抗体“BsAb EpCAM-EGFRvIII,MR1.1”(SEQ ID NO:229(轻链(无前导序列)和SEQ ID NO:230(重链(无前导序列))的制备
通过WO 2013/113615的实施例1,2和4中描述的克隆策略制备四价双特异性抗体分子“BsAb EpCAM-EGFRvIII,MR1.1”(SEQ ID NO:229(轻链(无前导序列)和SEQ ID NO:230(重链(无前导序列))。例示性地,作为概念的证据,在下面的实施例中,依照WO 2013/113615(据此通过援引将其收录)的实施例4构建具有一条臂上的两个针对del-hEGFRvIII(SEQ ID NO:232(蛋白质)和231(核酸(DNA))的抗原结合位点/结合域和另一条臂上的两个针对(鼠)EpCAM(SEQ ID NO:83(核酸(DNA))和84(蛋白质))的抗原结合位点/结合域的四价双特异性抗体分子“BsAb EpCAM-EGFRvIII,MR1.1”(SEQ ID NO:229和230)。
实施例2:三价双特异性抗体的制备
2.1三价双特异性抗体(bsAb)分子“BsAB EGFRvIII-EpCAM”(SEQ ID NO:233,其包含/组成为质粒/载体“EGFR vIII MR1.1 VH Ck muEpCAM VH CH1 EE Fc节PG LALA,pETR14953”,“EGFR vIII MR1.1 VL CH1,pETR14951”,“VL EpCAM G.8.8 Ck RK,pETR14882”和“VH muEpCAM CH1 EE Fc穴PG LALA HRYF,pETR14940”；还见表1和2)的制备
在这个实施例中制备三价双特异性抗体分子“BsAB EGFRvIII-EpCAM”(SEQ IDNO:233,其包含/组成为质粒/载体“EGFR vIII MR1.1 VH Ck muEpCAM VH CH1 EE Fc节PGLALA,pETR14953”,“EGFR vIII MR1.1 VL CH1,pETR14951”,“VL EpCAM G.8.8 Ck RK,pETR14882”和“VH muEpCAM CH1 EE Fc穴PG LALA HRYF,pETR14940”；还见表1和2)；其示意图在图9中显示；muEpCAM/EGFRvIII 2+1 IgG，经典格式(SEQ ID NO:233；还见表1和2)。与预插入相应受体哺乳动物表达载体的恒定链同框亚克隆muEpCAM/EGFRvIII的可变域。蛋白质表达由CMV启动子驱动且合成的polyA信号序列存在于CDS的3’端。另外，每种载体含有EBV OriP序列。如下生成分子,即用哺乳动物表达载体共转染悬浮生长的CHO细胞。在Evitria AG(瑞士)进行瞬时转染。以1:2:1:1比率(“载体重链穴(VH-CH1-CH2-CH3)”:“轻链(LC)”:“载体重链节(VH-CK-VH-CH1-CH2-CH3)”:“交叉轻链(VL-CH1)”)用相应的表达载体转染细胞。将过滤后的上清液保持于4℃直至纯化。通过使用蛋白A亲和层析的亲和层析,接着是一个至两个大小排阻层析步骤自细胞培养物上清液纯化分泌的蛋白质。对于亲和层析,将上清液加载到用25ml 20mM磷酸钠，20mM柠檬酸钠，0.5M氯化钠，0.01％Tween-20 pH7.5平衡的HiTrap蛋白A FF柱(CV＝5mL，GE Healthcare)上。通过用至少10个柱体积的20mM磷酸钠，20mM柠檬酸钠，0.5M氯化钠,0.01％Tween-20 pH 7.5清洗来去除未结合的蛋白并在20个柱体积(0％-100％梯度)的20mM柠檬酸钠,0.5M氯化钠,0.01％Tween-20 pH 2.5中洗脱靶蛋白质。通过添加1/10 2MTris pH 10.5来中和蛋白质溶液。用Ultra-15Ultracel 30K(Merck Millipore Ltd.)将靶蛋白浓缩至最大4ml的体积,之后加载到用20mM组氨酸,140mM氯化钠,pH 6.0,0.01％Tween20平衡的HiLoad Superdex 200柱(GEHealthcare)上。对于大小排阻层析之后的分析,在还原剂缺失下通过SDS-PAGE和考马斯(InstantBlueTM,Expedeon)染色来分析单一级分中分子的纯度和分子量。依照制造商的用法说明书使用预制凝胶系统(4-12％Bis-Tris,Invitrogen或3-8％Tris-乙酸盐,Invitrogen)。通过测量280nm处的光密度(OD),除以基于氨基酸序列计算的摩尔消光系数来测定纯化后的蛋白质样品的蛋白质浓度。在还原剂存在和缺失下通过CE-SDS分析来分析最终的纯化步骤之后分子的纯度和分子量。依照制造商的用法说明书使用CaliperLabChip GXII系统(Caliper Lifescience)(图9A,9B)。于25℃在25mM K2HPO4,125mM NaCl,200mM L-精氨酸单氢氯化物,0.02％(w/v)NaN3,pH 6.7运行缓冲液中使用TSKgel G3000SW XL分析性大小排阻柱(Tosoh)分析分子的聚集物含量(图9B)。所有分子的最终质量较好,具有≥96％的单体含量。下面的表7汇总muEpCAM/EGFRvIII分子的生成和纯化。表7中的分子1和2指在体外批次中生成的(分子1)或在体内批次中生成的(分子2)三价双特异性抗体分子“BsAB EGFRvIII-EpCAM”(SEQ ID NO:233),其包含/组成为质粒/载体“EGFR vIIIMR1.1 VH Ck muEpCAM VH CH1 EE Fc节PG LALA,pETR14953”,“EGFR vIII MR1.1 VL CH1,pETR14951”,“VL EpCAM G.8.8 Ck RK,pETR14882”和“VH muEpCAM CH1 EE Fc穴PG LALAHRYF,pETR14940”；还见表1和2)。
表7:
分子 滴度[mg/l] 产率[mg/l] 分析性SEC(HMW/单体/LMW)[％]
1(体外批次) 18 6.98 0％/100％/0％
2(体内批次) 18 11.6 3.68％/96.32％/0％
2.2三价双特异性抗体(bsAb)分子“BsAB EGFRvIII-MSLN”(SEQ ID NO:235,其包含/组成为质粒/载体“EGFRvIII MR1.1 VH Ck MSLN CH CH1 EE Fc节PG LALA,pETR15655”,“EGFR vIII MR1.1 VL CH1,pETR15656”,“VL MSLN Ck RK,pETR15443”和“VHMSLN CH1EE Fc穴PG LALA HRYF,pETR15667”；还见表3和4)的制备
在这个实施例中制备三价双特异性抗体分子“BsAB EGFRvIII-MSLN”(SEQ ID NO:235,其包含/组成为质粒/载体“EGFRvIII MR1.1 VH Ck MSLN CH CH1 EE Fc节PG LALA，pETR15655”,“EGFR vIII MR1.1 VL CH1,pETR15656”,“VL MSLN Ck RK,pETR15443”和“VHMSLN CH1EE Fc穴PG LALA HRYF,pETR15667”；还见表3和4)；其示意图在图10中显示(MSLN/EGFRvIII 2+1IgG,经典格式(SEQ ID NO:235；还见表3和4)。与预插入相应受体哺乳动物表达载体的恒定链同框亚克隆MSLN/EGFRvIII的可变域。蛋白质表达由MPSV启动子驱动且合成的polyA信号序列存在于CDS的3’端。另外,每种载体含有EBV OriP序列。如下生成分子,即使用聚乙烯亚胺(PEI)用哺乳动物表达载体共转染悬浮生长的HEK293-EBNA细胞。以1:2:1:1比率(“载体重链穴(VH-CH1-CH2-CH3)”:“轻链(LC)”:“载体重链节(VH-CK-VH-CH1-CH2-CH3)”:“交叉轻链(VL-CH1)”)用相应的表达载体转染细胞。将过滤后的上清液保持于4℃直至纯化。对于转染,在含有6mM L-谷氨酰胺和250mg/l G418的无血清ExCell培养培养基中培养HEK293 EBNA细胞。对于600ml管型转瓶(最大工作体积400mL)中的生成,在没有G418的情况下在转染之前24小时接种800x 106个HEK293 EBNA细胞。对于转染,将800x 106个细胞以210x g离心5分钟并用40ml预温热的含有6mM L-谷氨酰胺的CD-CHO培养基替换上清液。将表达载体与40ml含有6mM L-谷氨酰胺的CD-CHO培养基混合至400μg DNA的总量。在添加1080μl PEI溶液(2.7μg/ml)之后,将混合物漩涡振荡15秒,随后于室温温育10分钟。之后,将细胞与DNA/PEI溶液混合,转移至600ml管型转瓶并在具有5％CO2气氛的温箱中于37℃温育3小时。在温育之后,添加320ml ExCell+6mM L-谷氨酰胺+5g/L Pepsoy+1.0mM VPA+3g/l葡萄糖培养基并将细胞培养24小时,之后补料7％补料7。6-7天后,收集培养上清液用于纯化,即以210x g离心20-30分钟(Sigma 8K离心机)。将溶液无菌过滤(0.22μm滤器)并以0.01％w/v的终浓度添加叠氮化钠。将溶液保持于4℃直至纯化。通过使用蛋白A亲和层析的亲和层析,接着是一个至两个大小排阻层析步骤自细胞培养物上清液纯化分泌的蛋白质。对于亲和层析,将上清液加载到用25ml 20mM磷酸钠,20mM柠檬酸钠,0.5M氯化钠,0.01％Tween-20pH 7.5平衡的HiTrap蛋白A FF柱(CV＝5mL,GE Healthcare)上。通过用至少10个柱体积的20mM磷酸钠,20mM柠檬酸钠,0.5M氯化钠,0.01％Tween-20pH 7.5清洗来去除未结合的蛋白质并在20个柱体积(0％-100％梯度)的20mM柠檬酸钠,0.5M氯化钠,0.01％Tween-20pH 2.5中洗脱靶蛋白。通过添加1/10的2M Tris pH 10.5来中和蛋白质溶液。用Ultra-15 Ultracel 30K(Merck Millipore Ltd.)将靶蛋白浓缩至最大4ml的体积,之后加载到用20mM组氨酸,140mM氯化钠,pH 6.0,0.01％Tween20平衡的HiLoadSuperdex 200柱(GE Healthcare)上。
在还原剂存在和缺失下通过CE-SDS分析来分析最终的纯化步骤之后分子的纯度和分子量。依照制造商的用法说明书使用Caliper LabChip GXII系统(CaliperLifescience)(图10B)。于25℃在25mM K2HPO4,125mM NaCl,200mM L-精氨酸单氢氯化物,0.02％(w/v)NaN3,pH 6.7运行缓冲液中使用TSKgel G3000 SW XL分析性大小排阻柱(Tosoh)分析分子的聚集物含量(HMW)(图10A)。表8中的分子1指如上文描述的三价双特异性抗体分子“BsAB EGFRvIII-MSLN”(SEQ ID NO:235,其包含/组成为质粒/载体“EGFRvIIIMR1.1 VH Ck MSLN CH CH1 EE Fc节PG LALA,pETR15655”,“EGFR vIII MR1.1 VL CH1,pETR15656”,“VL MSLN Ck RK,pETR15443”和“VH MSLN CH1 EE Fc穴PG LALA HRYF,pETR15667”；还见表3和4)。所有分子的最终质量较好,具有≥96％的单体含量。下面的表8汇总MSLN/EGFRvIII分子的生成和纯化。
表8:
分子 滴度[mg/l] 产率[mg/l] 分析性SEC(HMW/单体/LMW)[％]
1 65 1.16 3.31/96.3/0.4
2.3三价双特异性抗体(bsAb)分子“BsAB EGFRvIII-MCSP(SEQ ID NO:234，其包含/组成为质粒/载体“MR1.1 EGFRvIII VH-Ck-(G4S)2 MCSP M4-3 VH CH1 EE Fc节PGLALA,pETR16621，“EGFR vIII MR1.1 VL CH1，pETR15656”,“MCSP ML2 VL Ck RK,pETR16619”和“MCSP M4-3 VH CH1 EE Fc穴PG LALA HYRF，pETR16618”；还见表5和6)的制备
在这个实施例中制备三价双特异性抗体分子“BsAB EGFRvIII-MCSP(SEQ ID NO:234,其包含/组成为质粒/载体“MR1.1 EGFRvIII VH-Ck-(G4S)2 MCSP M4-3 VH CH1 EE Fc节PG LALA,pETR16621,“EGFR vIII MR1.1 VL CH1,pETR15656”,“MCSP ML2 VL Ck RK,pETR16619”和“MCSP M4-3 VH CH1 EE Fc穴PG LALA HYRF,pETR16618”；还见表5和6)；其示意图在图11中显示(MCSP/EGFRvIII 2+1 IgG,经典格式(SEQ ID NO:234；还见表5和6)。与预插入相应受体哺乳动物表达载体的恒定链同框亚克隆MCSP/EGFRvIII的可变域。蛋白质表达由MPSV启动子驱动且合成的polyA信号序列存在于CDS的3’端。另外,每种载体含有EBVOriP序列。如下生成分子,即使用聚乙烯亚胺(PEI)用哺乳动物表达载体共转染悬浮生长的HEK293-EBNA细胞。以1:2:1:1比率(“载体重链穴(VH-CH1-CH2-CH3)”:“轻链(LC)”:“载体重链节(VH-CK-VH-CH1-CH2-CH3)”:“交叉轻链(VL-CH1)”)用相应的表达载体转染细胞。将过滤后的上清液保持于4℃直至纯化。对于转染,在含有6mM L-谷氨酰胺和250mg/l G418的无血清ExCell培养培养基中培养HEK293 EBNA细胞。对于600ml管型转瓶(最大工作体积400mL)中的生成,在没有G418的情况下在转染之前24小时接种800x 106个HEK293 EBNA细胞。对于转染,将800x 106个细胞以210x g离心5分钟并用40ml预温热的含有6mM L-谷氨酰胺的CD-CHO培养基替换上清液。将表达载体与40ml含有6mM L-谷氨酰胺的CD-CHO培养基混合至400μg DNA的总量。在添加1080μl PEI溶液(2.7μg/ml)之后,将混合物漩涡振荡15秒,随后于室温温育10分钟。之后,将细胞与DNA/PEI溶液混合,转移至600ml管型转瓶并在具有5％CO2气氛的温箱中于37℃温育3小时。在温育之后,添加320ml ExCell+6mM L-谷氨酰胺+5g/L Pepsoy+1.25mM VPA+3g/l葡萄糖培养基并将细胞培养24小时,之后补料12％补料7。6-7天后,收集培养上清液用于纯化,即以210xg离心20-30分钟(Sigma 8K离心机)。将溶液无菌过滤(0.22μm滤器)并以0.01％w/v的终浓度添加叠氮化钠。将溶液保持于4℃直至纯化。通过亲和层析,接着是一个至两个大小排阻层析步骤自细胞培养物上清液纯化分泌的蛋白质。对于亲和层析,将上清液加载到用25ml 20mM柠檬酸钠,20mM磷酸钠,pH 7.5平衡的蛋白A MabSelectSure GE Healthcare(CV＝5mL,GE Healthcare)上。通过用至少10个柱体积的20mM柠檬酸钠,20mM磷酸钠,pH 7.5清洗来去除未结合的蛋白质并在20个柱体积(0％-100％梯度)的20mM柠檬酸钠,100mM氯化钠,100mM甘氨酸,pH 3.0中洗脱靶蛋白。通过添加0.5M Na2HPO4pH 8.0(1:10)来中和蛋白质溶液。用Ultra-15 Ultracel 30K(Merck Millipore Ltd.)将靶蛋白浓缩至最大4ml的体积,之后加载到用20mM组氨酸,140mM NaCl,0.01％Tween pH 6.0平衡的HiLoad 16/600 S200,120ml柱(GE Healthcare)上。在还原剂存在和缺失下通过CE-SDS分析来分析最终的纯化步骤之后分子的纯度和分子量。依照制造商的用法说明书使用Caliper LabChip GXII系统(Caliper Lifescience)。于25℃在25mM K2HPO4,125mM NaCl,200mM L-精氨酸单氢氯化物,0.02％(w/v)NaN3,pH 6.7运行缓冲液中使用TSKgel G3000 SW XL分析性大小排阻柱(Tosoh)分析分子的聚集物含量。所有分子的最终质量较好,具有≥98％的单体含量。下面的表9汇总三价双特异性抗体(bsAb)“BsAB EGFRvIII-MCSP(SEQ ID NO:234)分子的生成和纯化。
表9:
分子 滴度[mg/l] 产率[mg/l] 分析性SEC(HMW/单体/LMW)[％]
1 34 1.58 1.8/98.2/0
实施例3:融合蛋白的克隆和表达
3.1融合蛋白EGFRvIII-CD28-CD3z(SEQ ID NO:42(蛋白质)和41(DNA))，EGFRvIII-CD28-4-1-BB-CD3z(SEQ ID NO:44(蛋白质)和43(DNA))和Cripto-CD28-4-CD3z(SEQ ID NO:46(蛋白质)和45(DNA))的克隆
通过交叠延伸PCR和重组表达克隆入逆转录病毒pMP71载体来生成EGFRvIII和Cripto融合蛋白(Schambach et al.,Mol Ther 2(5)(2000),435-45；EP-B1 0 955 374)。通过PCR扩增来生成EGFRvIII-CD28-CD3z融合蛋白(SEQ ID NO:42(如由SEQ ID NO:41中显示的DNA编码的))的构建。以四个步骤进行扩增:第一个,通过使用下面的引物用针对CD28胞内域的部分交叠来扩增人EGFRvIII-胞外和跨膜域:5’-AGCTTGCTCG CGGCCGCGCCACCATGCGAC CCTCCG-3’(SEQ ID NO:103；EGFRvIIINotIfwd)和5’-TCTGTTCCTT CTACTATTCATGAAGAGGCC GATCCC-3’(SEQ ID NO:104；EGFRtm CD28iz rev)。在同一时间,通过使用下面的引物用针对人EGFRvIII-跨膜域和鼠CD3z域的部分交叠来扩增鼠CD28的胞内域:5’-GGGATCGGCC TCTTCATGAA TAGTAGAAGG AACAGA-3’(SEQ ID NO:105；EGFRtm CD28iz fwd)和5’-CTGCTGAATT TTGCTCTGGG GCGGTACGCT GCAA-3’(SEQ ID NO:106；CD28in/CD3zetarev)。在第三个反应步骤中,通过使用该下面的引物用针对鼠CD28胞内域的部分交叠来扩增鼠CD3z:5’-TTGCAGCGTA CCGCCCCAGA GCAAAATTCA GCAG-3’(SEQ ID NO:107；CD3zeta/CD28fwd)和5’-TAATGAATTC TTAGCGAGGGGCCAGGGTC-3’(SEQ ID NO:108；CD3zetaEcoRIrev)。在第四个和最终的步骤中,使用所有产物作为扩增模板,使用EGFRvIII引物(5′-AGCTTGCTCG CGGCCGCGCC ACCATGCGAC CCTCCG；SEQ ID NO:103；EGFRvIIINotIfwd)和CD3z引物(5’-TAATGAATTC TTAGCGAGGG GCCAGGGTC-3’；CD3zetaEcoRIrev(SEQ ID NO:108))。
对于Cripto融合蛋白,如下以五个步骤进行克隆:第一个,用下面的引物扩增人:5’-ATTAGCGGCC GCGCCACCAT GGAAACAGAT ACAC-3’(SEQ ID NO:109；Leader_NotI_fwd)和5’-AAATTCCTGA TGGCCCAGGC TTCTAGCAGG CTGGGC-3’(SEQ ID NO:110；LeaderCriptoIsorev)。用下面的引物进行IgK前导序列和人Cripto的交叠:5’-GCCCAGCCTGCTAGAAGCCT GGGCCATCAG GAATTT-3’(SEQ ID NO:111；LeaderCriptoIsofwd)和5’-CAGCACTGGC TTGGTAGTAT CACAGCCGGG TAGAAA-3’(SEQ ID NO:112；Cripto CD8aex rev)。随后,用下面的引物进行人Cripto和鼠CD8的交叠:5’-TTTCTACCCG GCTGTGATACTACCAAGCCA GTGCTG-3`(SEQ ID NO:113；CriptoCD8aex fwd)和5’-TCTGTTCCTTCTACTATTGA TGAGAGTGAT GATCAA-3’(SEQ ID NO:114；CD8tm-CD28iz rev)。随后,用下面的引物进行鼠CD8和鼠CD28的交叠:5’-TTGATCATCA CTCTCATCAA TAGTAGAAGG AACAGA-3’(SEQID NO:115；CD8tm-CD28izfwd)和5’-CTGCTGAATT TTGCTCTGGG GCGGTACGCT GCAA-3’(SEQID NO:116；CD28in/CD3zeta rev)。用下面的引物进行鼠CD28和鼠CD3z的交叠:5’-TTGCAGCGTA CCGCCCCAGA GCAAAATTCA GCAG-3’(SEQ ID NO:117；CD3zeta/CD28fwd)和5’-TAATGAATTC TTAGCGAGGG GCCAGGGTC-3’(SEQ ID NO:118；CD3zetaEcoRIre)。在第五个和最终的反应中,与下面的引物一起,使用所有产物作为模板:5’-ATTAGCGGCC GCGCCACCATGGAAACAGAT ACAC-3’(SEQ ID NO:109；Leader_NotI_fwd)和5’-TAATGAATTC TTAGCGAGGGGCCAGGGTC-3’(SEQ ID NO:118；CD3zetaEcoR1rev)。
在扩增之后,使用EcoRI和NotI限制酶切割和DNA连接将插入物连接入pMP71载体。
3.2融合蛋白EGFRvIII-CD28-CD3z(SEQ ID NO:48(蛋白质)和47(DNA))和EGFRvIII-CD28-4-1-BB-CD3z(SEQ ID NO:50(蛋白质)和49(DNA))的克隆
3.2.1 EGFRvIII-CD28-CD3z(SEQ ID NO:48(蛋白质)和47(DNA))
在第一个反应中,通过使用下面的引物创建针对CD28的3’-交叠:EGFRvIII fwd(5’-AGCTTGCTCG CGGCCGCGCC ACCATGCGAC CC-3’(SEQ ID NO:125))和引物EGFRvIII(-人CD28)rev(5’-CCACCAGCAC CCAAAAGGAC GGGATCTTAG GCCCA-3’(SEQ ID NO:126))。在第二个反应中,通过使用下面的引物进行针对CD28的5’-交叠和针对CD3Z的3’-交叠:(EGFRvIII-)人CD28fwd(5’-TGGGCCTAAG ATCCCGTCCT TTTGGGTGCT GGTGG-3’(SEQ ID NO:127))和人CD3z rev(5’-TAATGAATTC TTAGCGAGGG GGCAGG-3’(SEQ ID NO:128))。使用上述产物的第三个反应包含引物EGFRvIII fwd(5’-AGCTTGCTCG CGGCCGCGCC ACCATGCGAC CC-3’(SEQ ID NO:129))和人CD3z rev(5’-TAATGAATTC TTAGCGAGGG GGCAGG-3’(SEQ ID NO:130))。
在扩增之后,使用EcoRI和NotI限制酶切割和DNA连接将插入物连接入pMP71载体。
3.2.2 EGFRvIII-CD28-4-1-BB-CD3z(SEQ ID NO:50(蛋白质)和49(DNA))
在第一个反应中,通过使用下面的引物进行针对CD28的3’-交叠:5’-AGCTTGCTCGCGGCCGCGCC ACCATGCGAC CC-3’(SEQ ID NO:131；EGFRvIII)和5’-CCACCAGCAC CCAAAAGGACGGGATCTTAG GCCCA-3’(SEQ ID NO:132；人CD28rev)。在第二个反应中,通过使用下面的引物进行针对EGFRv3的5’-交叠和针对4-1-BB的3’-交叠:(EGFRvIII-)人CD28fwd(5’-TGGGCCTAAG ATCCCGTCCT TTTGGGTGCT GGTGG-3’(SEQ ID NO:133)和引物人CD28(-人4-1-BB)rev(5’-CTTTCTGCCC CGTTTGGAGC GATAGGCTGC GA-3’(SEQ ID NO:134))。在第三个反应中,用下面的引物进行针对CD28的5’-交叠和针对CD3z的3’-交叠:(人CD28-)人4-1-BB fwd(5’-TCGCAGCCTA TCGCTCCAAA CGGGGCAGAA AG-3’(SEQ ID NO:135))和人4-1-BB(-人CD3z)rev(5’-TGCTGAACTT CACTCTCAGT TCACATCCTC CT-3’(SEQ ID NO:136))。在第四个反应中,使用下面的引物进行针对41BB的5’-交叠和针对CD3z的3’-交叠:(人4-1-BB-)人CD3z fwd(5’-GGAGGATGTG AACTGAGAGT GAAGTTCAGC AGGA-3’(SEQ ID NO:137))和引物人CD3z rev(5’-TAATGAATTC TTAGCGAGGG GGCAGG-3’(SEQ ID NO:138))。在第四个和最终的反应中,与5’-前导引物EGFRvIII fwd(5’-AGCTTGCTCG CGGCCGCGCC ACCATGCGAC CC-3’(SEQ ID NO:139))和人CD3z rev(5’-TAATGAATTC TTAGCGAGGG GGCAGG-3’(SEQ ID NO:140))一起,使用所有产物作为模板。
在扩增之后,使用EcoRI和NotI限制酶切割和DNA连接将插入物连接入pMP71载体。
3.2.3 CAR1人(Cripto-CD8aex/tm-CD28iz-CD3z)(SEQ ID NO:120(蛋白质)和119 (DNA))
在第一个反应中,通过使用下面的引物进行针对CD8的3’-交叠:Cripto fwd(5’-ATTAGCGGCC GCGCCACCAT GGAAACAGAT ACAC-3’(SEQ ID NO:141))和Cripto(-人CD8a)rev(5’-ACACCCGGAA CTGGCTATCA CAGCCGGGTA GA-3’(SEQ ID NO:142))。在第二个反应中,通过使用下面的引物进行针对Cripto的5’-交叠和针对CD28的3’-交叠:(Cripto-)人CD8afwd(5’-TCTACCCGGC TGTGATAGCC AGTTCCGGGT G-3’(SEQ ID NO:143))和人CD8a(-人CD28)rev(5’-CTCCTCTTAC TCCTGGTGAT AACCAGTGAC AGG-3’(SEQ ID NO:144))。在第三个反应中,通过使用下面的引物扩增针对CD8的5’-交叠和针对CD3z的3’-交叠:(人CD8a-)人CD28fwd(5’-CCTGTCACTG GTTATCACCA GGAGTAAGAG GAGCAGG-3’(SEQ ID NO:145))和人CD3zrev(5’-TAATGAATTC TTAGCGAGGG GGCAGG-3’(SEQ ID NO:146))。在第四个和最终的反应中,通过使用下面的引物与5’-前导引物一起,使用所有产物作为模板:Cripto fwd(5’-ATTAGCGGCC GCGCCACCAT GGAAACAGAT ACA-3’(SEQ ID NO:147))和人CD3z rev(5’-TAATGAATTC TTAGCGAGGG GGCAGG-3’(SEQ ID NO:148))。
在扩增之后,使用EcoRI和NotI限制酶切割和DNA连接将插入物连接入pMP71载体。
3.2.4
通过交叠延伸聚合酶链式反应(PCR)生成构建物E3del(SEQ ID NO:247(蛋白质)和246(DNA))并克隆入逆转录病毒pMP71载体。依照EGFRvIII序列,用SnapGene软件套组设计特异性交叠引物。用来自New England BioLabs(NEB)的在线Tm Calculator V 1.8.1计算退火部分的解链温度。唯独用来自NEB的Q5聚合酶执行所有PCR反应。构建物E3del由人EGFRvIII胞外域,人EGFRvIII跨膜域和10个胞内氨基酸组成以改善E3del在细胞膜中的锚定。
实施例4:T细胞转导和细胞毒性杀伤测定法
4.1细胞培养
4.1.1鼠癌细胞系
鼠胰腺癌细胞系Panc02和它的卵清蛋白转染对应物Panc02-OVA先前已有描述(Jacobs et al.,Int J Cancer 128(4)(2011),897-907)。经由将致癌物甲基胆蒽A注射入野生型C57Bl/6小鼠的胰腺以诱导癌发生而生成Panc02细胞系。通过用含有全长鼠EpCAM(SEQ ID NO:83(核酸(DNA))和84(蛋白质))的pMXs-puro(Kitamura et al.,Exp.Hematol.31(2003),1007-1014)转导并用终浓度10μg/ml的嘌呤霉素选择而生成Panc02-OVA-EpCAM。包装细胞系Plat-E先前已有描述(Morita et al.,Gene Ther 7(2000),1063-6)。所有细胞在含10％胎牛血清(FBS,Life Technologies,美国),1％青霉素和链霉素(PS)和1％L-谷氨酰胺(均来自PAA,德国)的DMEM中培养。将10μg/ml嘌呤霉素和1μg/ml杀稻瘟素(Sigma,德国)添加至Plat-E培养基。原代鼠T细胞(关于培养见下文2.5节)在含10％FBS,1％PS和1％L-谷氨酰胺的RPMI 1640中培养。将1％丙酮酸钠,1mM HEPES和50μMβ-巯基乙醇添加至T细胞培养基。
4.1.2人癌细胞系
人胰腺癌细胞系SUIT-2先前已有描述(Iwamora et al.,Jpn J Cancer Res.78(1)(1987),54-62)。SUIT-2细胞系衍生自人胰腺癌的转移性肝肿瘤。通过用含有全长人MSLN(SEQ ID NO:83(核酸(DNA))和84(蛋白质))的pMP71-amp(Kitamura et al.,Exp.Hematol.31(2003),1007-1014)转导并用终浓度10μg/ml的氨苄青霉素选择而生成SUIT-2-OE-MSLN。Flp-HEK 293人胚肾上皮细胞先前已有描述(Thankamony et al.,TheJournal of Biological Chemistry 281(45)(2006),34601-34609)。通过用含有全长人MSLN(SEQ ID NO:83(核酸(DNA))和84(蛋白质))的pMP71-amp(Kitamura et al.,Exp.Hematol.31(2003),1007-1014)转导并用终浓度10μg/ml的氨苄青霉素选择而生成细胞系HEK293-FLPin-MSLN。包装细胞系Plat-A先前已有描述(Wu et al.,J BiomedBiotechnol.2009(2009),901079)。所有细胞在含10％胎牛血清(FBS,Life Technologies,美国),1％青霉素和链霉素(PS)和1％L-谷氨酰胺(均来自PAA,德国)的DMEM中培养。将10μg/ml嘌呤霉素和1μg/ml杀稻瘟素(Sigma,德国)添加至Plat-A培养基。原代人T细胞(关于培养见下文2.5节)在含2.5％人血清,1％PS,1％L-谷氨酰胺,1％丙酮酸钠,和1％非必需氨基酸的VLE RPMI 1640中培养。
4.1.3
人胰腺癌细胞系SUIT-2先前已有描述(Iwamora et al.,Jpn J Cancer Res.78(1)(1987),54-62)。该细胞系经由不同储存库诸如澳大利亚细胞库(代码:JCRB1094)可得。SUIT-2细胞系衍生自人胰腺癌的转移性肝肿瘤。通过用含有全长人MSLN/CAK1/MPF(HGNCID:HGNC:7371(Chang et al.PNAS.93(1)(1996),136-40)的pMXs-amp(Kitamura et al.,Exp.Hematol.31(2003),1007-1014)转导而生成SUIT-2-OE-MSLN。人MSLN衍生自HeLa细胞的cDNA(Macville et al.,Cancer Res.59(1)(1999),141-50)。MSLN基因编码一种71kDa前体蛋白质,进一步加工成称作间皮素的一种40kDa糖基-磷脂酰肌醇锚定细胞表面蛋白和自细胞释放的称作巨核细胞强化因子的一种NH2端31kDa片段(Ho et al.,Clin CancerRes.13(5)(2007),1571-75)。
4.2 T细胞转导
4.2.1鼠T细胞转导
使用逆转录病毒载体pMP71(Schambach et al.,Mol Ther 2(5)(2000),435-45；EP-B1 0 955 374)来转染亲嗜性包装细胞系Plat-E。依照Leisegang et al.,J Mol Med86(2008),573-83；Mueller et al.,J Virol.86(2012),10866-10869；Kobold et al.,JNatl Cancer Inst(2014),印刷中描述的方法实施转导。简言之,在6孔板中接种包装细胞系Plat E(如Morita et al.,Gene Ther 7(2000),1063-6描述的)并生长过夜至70-80％汇合。在第1天,将16μg DNA与100mM CaCl2(Merck,德国)和126.7μM氯喹(Sigma,美国)混合在一起。将Plat-E细胞在低血清培养基(3％)中饥饿30分钟,然后与沉淀的DNA一起温育6小时。然后去除培养基并用培养培养基交换。在第2天,自C57Bl/6小鼠(HarlanLaboratories,荷兰)收获原代脾细胞。将脾细胞的单细胞悬浮液用T细胞培养基中的抗CD3(克隆145-2c11 BD Pharmingen,美国),抗CD28(克隆37.51,BD Pharmingen,美国)和重组鼠IL-2(Peprotech,德国)刺激过夜。在第3天,将24孔板用12.5μg/ml重组retronectin(Takara Biotech,日本)于室温包被2小时,用2％牛血清清蛋白(Roth,德国)于37℃封闭30分钟并用PBS清洗。收获Plat E的上清液并通过滤器(40μm,Milipore,美国)。然后将新鲜T细胞培养基添加至Plat E细胞。在每个孔中分配1ml过滤后的上清液并于4℃旋转感染2小时。然后自24孔板去除上清液。每个孔在补充有10U IL-2和400000个抗CD3和抗CD28珠(Invitrogen,德国)的1ml T细胞培养基中接种106个T细胞并以800g于32℃旋转感染30分钟。在第4天,再次收获Plat E上清液并过滤。将1ml添加至24孔板的每个孔并以800g于32℃旋转感染90分钟。随后将细胞于37℃温育另外6小时。用含IL-2的T细胞培养基替换1ml上清液。在第5天,收获细胞,计数并在补充有10ng/ml IL-15(Peprotech,德国)的T细胞培养基中以106个细胞/ml密度再接种。将T细胞保持于这个密度直至第10天,此时实施细胞分析或功能测定法。
4.2.2人T细胞转导
使用逆转录病毒载体pMP71(Schambach et al.,Mol Ther 2(5)(2000),435-45；EP-B1 0 955 374)来转染亲嗜性包装细胞系Plat-A。依照Leisegang et al.,J Mol Med86(2008),573-83；Mueller et al.,J Virol.86(2012),10866-10869；Kobold et al.,JNatl Cancer Inst(2014)描述的方法实施转导。简言之,在6孔板中接种包装细胞系Plat A(如Morita et al.,Gene Ther 7(2000),1063-6描述的)并生长过夜至70-80％汇合。在第1天,将18μg DNA与100mM CaCl2(Merck,德国)混合在一起。将Plat-A细胞在低血清培养基(3％)中饥饿30分钟,然后与沉淀的DNA一起温育6小时。然后去除培养基并用培养培养基交换。另外,在第2天为T细胞准备6孔板,即用抗人CD3和CD28抗体(分别是克隆HIT3a和CD28.2)(eBiosciences,德国)包被它们。在第2天,自健康供体采集全血。然后使用密度梯度离心来分离PBMC。在与人CD3微珠一起温育之后并遵循MACS CD3正选择试剂盒LS-柱方案(Miltenyi Biotec,德国)进行CD3+细胞的分离。然后如4.1节中所述培养CD3+ T细胞,添加T细胞培养基中的IL-2,IL-15和β-巯基乙醇(均来自Peprotech,德国)和8.25μl/106个细胞的人CD3/CD28dynabead,过夜。在第3天,用12.5μg/ml重组retronectin(Takara Biotech,日本)包被24孔板并于4℃温育过夜。在第4天,将板用2％牛血清清蛋白(Roth,德国)于37℃封闭30分钟并用含2.5％HEPES的PBS清洗。收获Plat A的上清液并通过滤器(40μm,Milipore,美国)。然后将新鲜DMEM培养培养基添加至Plat A细胞。在每个孔中添加1ml过滤后的病毒上清液,随后于32℃离心1小时30分钟。然后自24孔板去除上清液。在有关病毒上清液所在处在1ml补充有IL-2,IL-15和β-巯基乙醇的T细胞培养基中接种106个T细胞。在第5天,重复第4天的方案,此时给予T细胞第二次和最终的转导打击。在第6天,收获细胞,计数并在补充有IL-2,IL-15和β-巯基乙醇(Peprotech,德国)的T细胞培养基中以106个细胞/ml密度再接种。然后使用FACS分析对T细胞检查它们的转导效率。如果转导是成功的话,隔天再培养T细胞并维持于106个细胞/ml的浓度。
4.3杀伤测定法
在96孔平底板(Corning)中接种20.000个稳定表达卵清蛋白(SEQ ID NO:200(蛋白质)和199(DNA))且用EpCAM(SEQ ID NO:202(蛋白质)和201(DNA))转导的PancOVAEpCAM鼠胰腺癌细胞。将100.000个经EGFRvIII-CD28-CD3z融合蛋白(SEQ ID NO:42(蛋白质)和41(DNA))转导的T细胞用bsAb(或是三价双特异性抗体分子“EGFRvIII MR1.1VH Ck MSLN CHCH1EE Fc节PG LALA,pETR15655”(SEQ ID NO:2(如由SEQ ID NO:1中显示的DNA序列编码的)或是四价双特异性抗体分子“BsAb EpCAM-EGFRvIII,MR1.1”(SEQ ID NO:229(轻链氨基酸序列)和230(重链氨基酸序列))预加载30分钟并与肿瘤细胞(Panc02-OVA-EpCAM或Panc02-OVA)一起共培养8-12小时(E:T＝5:1)。依照制造商的用法说明书(CytoTox非放射性细胞毒性测定法,Promega)量化上清液中的乳酸脱氢酶(LDH)水平。简言之,通过乳酸氧化成丙酮酸,LDH催化NAD+还原成NADH和H+。接着,心肌黄酶与NADH和H+反应以催化四唑盐(INT)还原成甲月替,它在490nm处吸收。依照下面的公式计算比裂解(％):
(LDH感兴趣–LDH背景–LDH仅效应器)/(LDH完全裂解–LDH最小裂解–LDH背景)x100％。
4.4干扰素-γ(IFN-γ)释放测定法
将96孔平底板(Corning)用浓度递增(0μg/mL；0.1μg/mL；1μg/mL；10μg/mL)的四价双特异性抗体分子“BsAb EpCAM-EGFRvIII,MR1.1”,三价双特异性抗体分子“EGFRvIIIMR1.1 VH Ck MSLN CH CH1 EE Fc节PG LALA,pETR15655”(SEQ ID NO:2(蛋白质)和1(DNA))或西妥昔单抗(Merck)于4℃包被12小时。将孔用20％胎牛血清(FBS,LifeTechnologies,美国)于37℃封闭30分钟并分别添加0.25x 106个具有EGFRvIII-CD28-CD3z融合蛋白(SEQ ID NO:42(蛋白质)和41(DNA)的T细胞(以下称作E3细胞)或野生型(WT)T细胞。48小时后,收集上清液并通过酶联免疫吸附测定法(ELISA；BD)来量化IFN-γ释放。通过Mithras LB 940多模微量板读数仪(Software MicroWin 2000)测量吸光度。
4.5 iCELLigence的杀伤测定法
在E-板L8(OLS)上接种50.000个PancOVAEpCAM肿瘤细胞并在20小时的时间范围里每20分钟测量肿瘤细胞增殖。分别将500.000个经EGFRvIII-CD28-CD3z融合蛋白(SEQ IDNO:42(蛋白质)和41(DNA))或EpCam scVf-CD3z嵌合抗原受体(CAREpCAM；SEQ ID NO:249(蛋白质)和248(DNA))转导的C57Bl6野生型T细胞添加至肿瘤细胞。如先前所述用1μg/mLbsAb预加载经EGFRvIII-CD28-CD3z融合蛋白(SEQ ID NO:42(蛋白质)和41(DNA))转导的T细胞。对于FasL封闭条件,立即对孔添加10μg/mL CD178(Fas配体)单克隆抗体(克隆:MFL3；目录号16-5911-85(ThermoFisher ScientificTM),Functional Grade(eBioscience)。在24小时里每6分钟测量T细胞杀伤。
4.6抗体结合测定法
将0.25x 106个/150μL经EGFRvIII-CD28-CD3z融合蛋白(SEQ ID NO:42(蛋白质)和41(DNA))转导的T细胞与浓度递增(10ng/mL；100ng/mL；500ng/mL；1μg/mL；5μg/mL；10μg/mL；20μg/mL；25μg/mL)的bsAb(三或四特异性,在50μL PBS中)一起于37℃温育30分钟。添加1μL二抗FITC缀合的AffiniPure F(ab’)2片段山羊抗人IgG(Jackson Laboratories；FITCAffiniPure F(ab')2片段山羊抗人IgG,F(ab')2片段特异性:109-096-097)或Cy2缀合的AffiniPure山羊抗小鼠IgG(Jackson Laboratories；Cy2AffiniPure山羊抗小鼠IgG:115-225-006)并于4℃温育30分钟。通过流式细胞术来量化FITC均值荧光强度(FITC MFI)。使用BD FACS Canto II(BD,德国)来分析染色。作为最大值的百分比计算表面饱和(FITC MFI感兴趣/FITC MFI最高浓度)x 100％。用FlowJo7.6.1实施数据分析。
4.7统计分析
对于统计学,使用GraphPad Prism软件版本5.0b。所有报告的变量是连续的。使用不配对双侧Student氏t检验来分析实验条件之间的差异。对于小鼠个体的实验条件的比较,使用Mann-Whitney检验。p值<0.05认为是显著的。
4.8 T细胞刺激测定法
在96孔平底板(Corning)中在T细胞培养基中接种Suit-OE-MSLN肿瘤细胞达6小时。5个半小时后,将T细胞与三价双特异性抗体(bsAb)分子“BsAbEGFRvIII-MSLN”(SEQ IDNO:235；还见表3和4)一起共温育30分钟。在这之后,将T细胞/双特异性抗体缀合物添加至该肿瘤细胞,并于37℃,5％CO2温育48小时。在这个时段之后,收集上清液并通过酶联免疫吸附测定法(ELISA；BD)来量化IFN-γ释放。通过Mithras LB 940多模微量板读数仪(Software MicroWin 2000)来测量吸光度。
4.9重组间皮素-T细胞刺激测定法
在96孔平底板(Corning)中包被人重组间皮素蛋白(5μg/ml)(Sino BiologicalInc.,德国)并于4℃温育过夜。用含10％FBS的PBS封闭板。将T细胞与三价双特异性抗体(bsAb)分子“BsAb EGFRvIII-MSLN”(SEQ ID NO:235；还见表3和4)一起共温育30分钟。在这之后,将T细胞/双特异性抗体缀合物添加至重组间皮素包被的孔,并与37℃,5％CO2温育48小时。在这个时段之后,收集上清液并通过酶联免疫吸附测定法(ELISA；BD)来量化IFN-γ释放。通过Mithras LB 940多模微量板读数仪(Software MicroWin 2000)来测量吸光度。
实施例5:特定实施方案的例子
本公开文本的某些非限制性实施方案的例子在下文列出。特别地,本发明涉及下面的项:
1.一种试剂盒,其包含
(A)编码用于转导自要治疗的受试者获得的T细胞的融合蛋白的核酸分子,该融合蛋白具有下面的域:
(1)在所述T细胞中或上并不天然发生的信号传导受体的胞外域；
(2)锚定跨膜域；和
(3)刺激性信号传导域,和
(B)三价双特异性抗体分子,其包含:
(i)结合(A)中表征的融合蛋白的胞外域(1)的第一结合域；
(ii)结合在肿瘤细胞的表面上天然发生的肿瘤特异性抗原的第二结合域；和
(iii)结合(A)中表征的融合蛋白的胞外域(1)或结合所述在肿瘤细胞的表面上天然发生的肿瘤特异性抗原的第三结合域。
2.项1的试剂盒,其中所述融合蛋白进一步包含至少一个共刺激性信号传导域。
3.项1或项2的试剂盒,其中所述锚定跨膜域不具有哺乳动物蛋白酶的切割位点。
4.项1至3任一项的试剂盒,其中所述融合蛋白进一步包含铰链域。
5.一种三价双特异性抗体分子,其包含:
(i)结合项1(A)中表征的融合蛋白的胞外域(1)的第一结合域；
(ii)结合在肿瘤细胞的表面上天然发生的肿瘤特异性抗原的第二结合域；和
(iii)结合项1(A)中表征的融合蛋白的胞外域(1)或结合所述在肿瘤细胞的表面上天然发生的肿瘤特异性抗原的第三结合域,
其供作为药物使用,其中所述双特异性抗体分子要在施用包含项1(A)中
表征的融合蛋白的经转导T细胞之前,同时或之后施用且其中所述T细
胞是自该要治疗的受试者获得的。
6.一种包含三价双特异性抗体分子的药用组合物,该三价双特异性抗体分子包含:
(i)结合项1(A)中表征的融合蛋白的胞外域(1)的第一结合域；
(ii)结合在肿瘤细胞的表面上天然发生的肿瘤特异性抗原的第二结合域；和
(iii)结合项1(A)中表征的融合蛋白的胞外域(1)或结合所述在肿瘤细胞的表面上天然发生的肿瘤特异性抗原的第三结合域,
其要与包含项1(A)中表征的融合蛋白的经转导T细胞组合施用且其中所述T细胞是自该要治疗的受试者获得的。
7.一种三价双特异性抗体分子,其包含:
(i)结合项1(A)中表征的融合蛋白的胞外域(1)的第一结合域；
(ii)结合在肿瘤细胞的表面上天然发生的肿瘤特异性抗原的第二结合域；和
(iii)结合项1(A)中表征的融合蛋白的胞外域(1)或结合所述在肿瘤细胞的表面上天然发生的肿瘤特异性抗原的第三结合域,
其供治疗恶性疾病的方法中使用,其中所述三价双特异性抗体分子要在施用包含项1(A)中表征的融合蛋白的经转导T细胞之前,同时或之后施用且其中所述T细胞是自该要治疗的受试者获得的。
8.一种治疗恶性疾病的方法,该方法包含将三价双特异性抗体分子施用于有需要的受试者,该三价双特异性抗体分子包含:
(i)结合项1(A)中表征的融合蛋白的胞外域(1)的第一结合域；
(ii)结合在肿瘤细胞的表面上天然发生的肿瘤特异性抗原的第二结合域；和
(iii)结合项1(A)中表征的融合蛋白的胞外域(1)或结合在肿瘤细胞的表面上天然发生的肿瘤特异性抗原的第三结合域,
其中所述三价双特异性抗体分子在施用包含项1(A)中表征的融合蛋白的来自所述受试者的经转导T细胞之前,同时或之后施用。
9.项7的三价双特异性抗体分子,或项8的用于治疗恶性疾病的方法,其中所述恶性疾病选自上皮,内皮或间皮起源的癌症和血液的癌症。
10.项1至4任一项的试剂盒,项6的药用组合物,项5或7的三价双特异性抗体分子,或项8或9的方法,其中所述在肿瘤细胞的表面上天然发生的抗原选自由EpCAM,MSLN,MCSP,HER-1,HER-2,HER-3,CD20,CD22,CD33,CD52,FLT-3,FOLR1,Trop-2,CA-12-5,HLA-DR,MUC-1(粘蛋白),A33-抗原,PSMA PSCA,(前列腺特异性膜抗原),运铁蛋白受体,生腱蛋白和CA-IX(碳酸酐酶IX)组成的组。
11.项1至4或10任一项的试剂盒,项6或项10的药用组合物,项5,7或10任一项的三价双特异性抗体分子,项8,9或10任一项的方法,其中所述在所述T细胞中或上并不天然发生的信号传导受体的胞外域选自由Cripto(cryptic家族蛋白),CD(分化簇)家族的成员(非T细胞),EGFR,EGFRvIII和TSH-R组成的组。
12.项1至4,10或11任一项的试剂盒,项6,10或11任一项的药用组合物,项5,7,10或11任一项的三价双特异性抗体分子,或项8至11任一项的方法,其中所述经转导T细胞进一步包含在所述T细胞上天然发生的T细胞受体和/或遗传导入所述T细胞的T细胞受体。
13.一种表达载体,其包含编码如项1(B)和5至8任一项中限定的三价双特异性抗体的核酸序列。
14.项13的载体,其是多顺反子的。
15.项13或项14的该载体,其中所述载体进一步包含与项13的所述核酸序列可操作连接的调节序列。
16.一种宿主细胞,其经如项13至15任一项中限定的载体转化。
17.一种用于生成如项1(B)和5至8任一项中限定的三价双特异性抗体分子的方法,所述方法包含
(a)在容许如项1(B)和5至8任一项中限定的三价双特异性抗体分子表达的条件下培养如项16中限定的宿主细胞；和
(b)自培养物回收生成的三价双特异性抗体分子。
18.如项1(B)和5至8任一项中限定的三价双特异性抗体分子,其包含
(i)结合项1(A)中表征的融合蛋白的胞外域(1)的第一结合域；
(ii)结合在肿瘤细胞的表面上天然发生的肿瘤特异性抗原的第二结合域；和
(iii)结合项1(A)中表征的融合蛋白的胞外域(1)或结合所述在肿瘤细胞的表面上天然发生的肿瘤特异性抗原的第三结合域,
其中所述抗体分子是通过项17的方法可获得的。
19.一种用于在受试者中治疗疾病的方法,其包含下述步骤:
(a)自受试者分离T细胞；
(b)用项1(A)中表征的融合蛋白转导所述分离的T细胞；和
(c)将所述经转导T细胞施用于所述受试者。
20.项19的方法,其中所述经转导T细胞通过静脉内输注施用于所述受试者。
21.项19或项20的方法,其中该经转导T细胞经T细胞受体共转导。
22.项19至21任一项的方法,其中所述经转导T细胞通过抗CD3和抗CD28抗体扩充。
23.项19至22任一项的方法,其中该经转导T细胞的扩充在细胞因子,优选白介素-2(IL-2)和/或白介素-15(IL-15)存在下实施。
24.项19至23任一项的方法,其进一步包含
(d)施用如项1(B)和5至8或18任一项中限定的三价双特异性抗体。
25.项19至24任一项的用于治疗疾病的方法,其中所述三价双特异性抗体要在施用该经转导T细胞之前,同时或之后施用。
26.项19至25任一项的方法,其中所述疾病是恶性疾病。
27.项19至26任一项的方法,其中所述恶性疾病选自上皮,内皮或间皮起源的癌症和血液的癌症。
序列表
<110> 豪夫迈·罗氏有限公司（F. Hoffmann-La Roche AG）
豪夫迈·罗氏有限公司（Hoffmann-La Roche Inc.）
慕尼黑路德韦格马克西米利安斯大学（LMU München）
<120> 改良的过继性T细胞疗法
<130> X3673 PCT S3
<150> EP 16 17 7203.3
<151> 2016-06-30
<160> 249
<170> BiSSAP 1.3
<210> 1
<211> 2061
<212> DNA
<213> 人工序列
<220>
<223> EGFR vIII MR1.1 VH Ck MSLN VH CH1 EE Fc 节 PG LALA, pETR15655
<220>
<221> CDS
<222> 1..2061
<223> /翻译表=1
<400> 1
caa gtg aag ctg cag cag agt ggg ggc gga ctc gtg aaa cct ggc gcc 48
Gln Val Lys Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Ala
1 5 10 15
tct ctg aag ctg agc tgc gtg acc agc ggc ttc acc ttc aga aag ttc 96
Ser Leu Lys Leu Ser Cys Val Thr Ser Gly Phe Thr Phe Arg Lys Phe
20 25 30
ggc atg agc tgg gtg cgc cag acc agc gac aag cgg ctg gaa tgg gtg 144
Gly Met Ser Trp Val Arg Gln Thr Ser Asp Lys Arg Leu Glu Trp Val
35 40 45
gcc agc atc agc acc ggc ggc tac aac acc tac tac agc gac aac gtg 192
Ala Ser Ile Ser Thr Gly Gly Tyr Asn Thr Tyr Tyr Ser Asp Asn Val
50 55 60
aag ggc cgg ttc acc atc agc aga gag aac gcc aag aac acc ctg tac 240
Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
ctg cag atg agc agc ctg aag tcc gag gac acc gcc ctg tac tac tgc 288
Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
acc aga ggc tac agc ccc tac agc tac gcc atg gac tat tgg ggc cag 336
Thr Arg Gly Tyr Ser Pro Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
ggc acc acc gtg acc gtg tca tct gct agc gtg gcc gct ccc tcc gtg 384
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val
115 120 125
ttc atc ttc cca cct tcc gac gag cag ctg aag tcc ggc acc gct tct 432
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser
130 135 140
gtc gtg tgc ctg ctg aac aac ttc tac ccc cgc gag gcc aag gtg cag 480
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln
145 150 155 160
tgg aag gtg gac aac gcc ctg cag tcc ggc aac agc cag gaa tcc gtg 528
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val
165 170 175
acc gag cag gac tcc aag gac agc acc tac tcc ctg tcc tcc acc ctg 576
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu
180 185 190
acc ctg tcc aag gcc gac tac gag aag cac aag gtg tac gcc tgc gaa 624
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu
195 200 205
gtg acc cac cag ggc ctg tct agc ccc gtg acc aag tct ttc aac cgg 672
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg
210 215 220
ggc gag tgc ggt ggc gga ggt tcc gga ggc gga gga tcc cag gtg cag 720
Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln
225 230 235 240
ctg gtg cag tct ggc gcc gaa gtg aag aaa cca ggc gcc agc gtg aag 768
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys
245 250 255
gtg tcc tgc aag gcc agc ggc tac agc ttc acc ggc tac acc atg aac 816
Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn
260 265 270
tgg gtg cgc cag gct cct gga cag ggc ctg gaa tgg atg ggc ctg atc 864
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Leu Ile
275 280 285
acc ccc tac aac ggc gcc agc agc tac aac cag aag ttc cgg ggc aag 912
Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe Arg Gly Lys
290 295 300
gcc acc atg acc gtg gac acc agc acc tcc acc gtg tat atg gaa ctg 960
Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu
305 310 315 320
agc agc ctg cgg agc gag gac acc gcc gtg tac tat tgt gcc aga ggc 1008
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly
325 330 335
ggc tac gac ggc aga ggc ttc gat tat tgg ggc cag ggc acc ctc gtg 1056
Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
340 345 350
acc gtg tcc agc gct agc acc aag ggc ccc tcc gtg ttc ccc ctg gcc 1104
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
355 360 365
ccc agc agc aag agc acc agc ggc ggc aca gcc gct ctg ggc tgc ctg 1152
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
370 375 380
gtc gag gac tac ttc ccc gag ccc gtg acc gtg tcc tgg aac agc gga 1200
Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
385 390 395 400
gcc ctg acc tcc ggc gtg cac acc ttc ccc gcc gtg ctg cag agt tct 1248
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
405 410 415
ggc ctg tat agc ctg agc agc gtg gtc acc gtg cct tct agc agc ctg 1296
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
420 425 430
ggc acc cag acc tac atc tgc aac gtg aac cac aag ccc agc aac acc 1344
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
435 440 445
aag gtg gac gag aag gtg gag ccc aag agc tgc gac aaa act cac aca 1392
Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
450 455 460
tgc cca ccg tgc cca gca cct gaa gct gca ggg gga ccg tca gtc ttc 1440
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
465 470 475 480
ctc ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc cgg acc cct 1488
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
485 490 495
gag gtc aca tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc 1536
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
500 505 510
aag ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag aca 1584
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
515 520 525
aag ccg cgg gag gag cag tac aac agc acg tac cgt gtg gtc agc gtc 1632
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
530 535 540
ctc acc gtc ctg cac cag gac tgg ctg aat ggc aag gag tac aag tgc 1680
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
545 550 555 560
aag gtc tcc aac aaa gcc ctc ggc gcc ccc atc gag aaa acc atc tcc 1728
Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser
565 570 575
aaa gcc aaa ggg cag ccc cga gaa cca cag gtg tac acc ctg ccc cca 1776
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
580 585 590
tgc cgg gat gag ctg acc aag aac cag gtc agc ctg tgg tgc ctg gtc 1824
Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val
595 600 605
aaa ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg 1872
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
610 615 620
cag ccg gag aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac 1920
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
625 630 635 640
ggc tcc ttc ttc ctc tac agc aag ctc acc gtg gac aag agc agg tgg 1968
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
645 650 655
cag cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac 2016
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
660 665 670
aac cac tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa tga 2061
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
675 680 685
<210> 2
<211> 686
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..2061, 来自SEQ ID NO 1
<400> 2
Gln Val Lys Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Leu Lys Leu Ser Cys Val Thr Ser Gly Phe Thr Phe Arg Lys Phe
20 25 30
Gly Met Ser Trp Val Arg Gln Thr Ser Asp Lys Arg Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Thr Gly Gly Tyr Asn Thr Tyr Tyr Ser Asp Asn Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Thr Arg Gly Tyr Ser Pro Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val
115 120 125
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser
130 135 140
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln
145 150 155 160
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val
165 170 175
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu
180 185 190
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu
195 200 205
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg
210 215 220
Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln
225 230 235 240
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys
245 250 255
Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn
260 265 270
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Leu Ile
275 280 285
Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe Arg Gly Lys
290 295 300
Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu
305 310 315 320
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly
325 330 335
Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
340 345 350
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
355 360 365
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
370 375 380
Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
385 390 395 400
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
405 410 415
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
420 425 430
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
435 440 445
Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
450 455 460
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
465 470 475 480
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
485 490 495
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
500 505 510
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
515 520 525
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
530 535 540
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
545 550 555 560
Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser
565 570 575
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
580 585 590
Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val
595 600 605
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
610 615 620
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
625 630 635 640
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
645 650 655
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
660 665 670
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
675 680 685
<210> 3
<211> 639
<212> DNA
<213> 人工序列
<220>
<223> EGFR vIII MR1.1 VL CH1, pETR15656
<220>
<221> CDS
<222> 1..639
<223> /翻译表=1
<400> 3
gat atc gag ctg aca cag agc ccc gcc agc ctg tct gtg gcc acc ggc 48
Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ser Val Ala Thr Gly
1 5 10 15
gag aaa gtg acc atc cgg tgc atg acc agc acc gac atc gac gac gac 96
Glu Lys Val Thr Ile Arg Cys Met Thr Ser Thr Asp Ile Asp Asp Asp
20 25 30
atg aac tgg tat cag cag aag ccc ggc gag ccc ccc aag ttc ctg atc 144
Met Asn Trp Tyr Gln Gln Lys Pro Gly Glu Pro Pro Lys Phe Leu Ile
35 40 45
agc gag ggc aac aca ctg cgg cct ggc gtg cca agc aga ttc agc agc 192
Ser Glu Gly Asn Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Ser
50 55 60
tct ggc acc ggc acc gac ttc gtg ttt acc atc gag aat acc ctg agc 240
Ser Gly Thr Gly Thr Asp Phe Val Phe Thr Ile Glu Asn Thr Leu Ser
65 70 75 80
gag gac gtg ggc gac tac tac tgc ctg cag agc tgg aac gtg ccc ctg 288
Glu Asp Val Gly Asp Tyr Tyr Cys Leu Gln Ser Trp Asn Val Pro Leu
85 90 95
acc ttt ggc gac ggc acc aag ctg gaa atc aag agc agc gct agc acc 336
Thr Phe Gly Asp Gly Thr Lys Leu Glu Ile Lys Ser Ser Ala Ser Thr
100 105 110
aaa ggc cct tcc gtg ttt cct ctg gct cct agc tcc aag tcc acc tct 384
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
115 120 125
gga ggc acc gct gct ctc gga tgc ctc gtg aag gat tat ttt cct gag 432
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
130 135 140
cct gtg aca gtg tcc tgg aat agc gga gca ctg acc tct gga gtg cat 480
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
145 150 155 160
act ttc ccc gct gtg ctg cag tcc tct gga ctg tac agc ctg agc agc 528
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
165 170 175
gtg gtg aca gtg ccc agc agc agc ctg ggc acc cag acc tac atc tgc 576
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
180 185 190
aac gtg aac cac aag ccc agc aac acc aag gtg gac aag aag gtg gaa 624
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
195 200 205
ccc aag tct tgt tga 639
Pro Lys Ser Cys
210
<210> 4
<211> 212
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..639, 来自SEQ ID NO 3
<400> 4
Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ser Val Ala Thr Gly
1 5 10 15
Glu Lys Val Thr Ile Arg Cys Met Thr Ser Thr Asp Ile Asp Asp Asp
20 25 30
Met Asn Trp Tyr Gln Gln Lys Pro Gly Glu Pro Pro Lys Phe Leu Ile
35 40 45
Ser Glu Gly Asn Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Ser
50 55 60
Ser Gly Thr Gly Thr Asp Phe Val Phe Thr Ile Glu Asn Thr Leu Ser
65 70 75 80
Glu Asp Val Gly Asp Tyr Tyr Cys Leu Gln Ser Trp Asn Val Pro Leu
85 90 95
Thr Phe Gly Asp Gly Thr Lys Leu Glu Ile Lys Ser Ser Ala Ser Thr
100 105 110
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
115 120 125
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
130 135 140
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
145 150 155 160
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
165 170 175
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
180 185 190
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
195 200 205
Pro Lys Ser Cys
210
<210> 5
<211> 642
<212> DNA
<213> 人工序列
<220>
<223> VL MSLN Ck RK, pETR15443
<220>
<221> CDS
<222> 1..642
<223> /翻译表=1
<400> 5
gac atc cag atg acc cag agc ccc agc agc ctg tct gcc agc gtg ggc 48
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
gac aga gtg acc atc acc tgt agc gcc agc agc agc gtg tcc tac atg 96
Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
20 25 30
cac tgg tat cag cag aag tcc ggc aag gcc ccc aag ctg ctg atc tac 144
His Trp Tyr Gln Gln Lys Ser Gly Lys Ala Pro Lys Leu Leu Ile Tyr
35 40 45
gac acc agc aag ctg gcc tcc ggc gtg ccc agc aga ttt tct ggc agc 192
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
ggc tcc ggc acc gac ttc acc ctg aca atc agc tcc ctc cag ccc gag 240
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu
65 70 75 80
gac ttc gcc acc tac tac tgc cag cag tgg tcc aag cac ccc ctg acc 288
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Lys His Pro Leu Thr
85 90 95
ttt ggc cag ggc acc aag ctg gaa atc aag cgt acg gtg gct gca cca 336
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro
100 105 110
tct gtc ttc atc ttc ccg cca tct gat cgg aag ttg aaa tct gga act 384
Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys Ser Gly Thr
115 120 125
gcc tct gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc aaa 432
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140
gta cag tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag gag 480
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160
agt gtc aca gag cag gac agc aag gac agc acc tac agc ctc agc agc 528
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175
acc ctg acg ctg agc aaa gca gac tac gag aaa cac aaa gtc tac gcc 576
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190
tgc gaa gtc acc cat cag ggc ctg agc tcg ccc gtc aca aag agc ttc 624
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
195 200 205
aac agg gga gag tgt tag 642
Asn Arg Gly Glu Cys
210
<210> 6
<211> 213
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..642, 来自SEQ ID NO 5
<400> 6
Asp 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 Ser Ala Ser Ser Ser Val Ser Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Ser Gly Lys Ala Pro Lys Leu Leu Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu
65 70 75 80
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Lys His Pro Leu Thr
85 90 95
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro
100 105 110
Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys Ser Gly Thr
115 120 125
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
195 200 205
Asn Arg Gly Glu Cys
210
<210> 7
<211> 1350
<212> DNA
<213> 人工序列
<220>
<223> VH MSLN CH1 EE Fc 穴 PG LALA HRYF, pETR15657
<220>
<221> CDS
<222> 1..1350
<223> /翻译表=1
<400> 7
cag gtg cag ctg gtg cag tct ggc gcc gaa gtg aag aaa cca ggc gcc 48
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
agc gtg aag gtg tcc tgc aag gcc agc ggc tac agc ttc acc ggc tac 96
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
acc atg aac tgg gtg cgc cag gct cct gga cag ggc ctg gaa tgg atg 144
Thr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
ggc ctg atc acc ccc tac aac ggc gcc agc agc tac aac cag aag ttc 192
Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe
50 55 60
cgg ggc aag gcc acc atg acc gtg gac acc agc acc tcc acc gtg tat 240
Arg Gly Lys Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80
atg gaa ctg agc agc ctg cgg agc gag gac acc gcc gtg tac tat tgt 288
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
gcc aga ggc ggc tac gac ggc aga ggc ttc gat tat tgg ggc cag ggc 336
Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Gln Gly
100 105 110
acc ctc gtg acc gtg tcc tct gct agc acc aag ggc ccc tcc gtg ttc 384
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
ccc ctg gcc ccc agc agc aag agc acc agc ggc ggc aca gcc gct ctg 432
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
ggc tgc ctg gtc gag gac tac ttc ccc gag ccc gtg acc gtg tcc tgg 480
Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
aac agc gga gcc ctg acc tcc ggc gtg cac acc ttc ccc gcc gtg ctg 528
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
cag agt tct ggc ctg tat agc ctg agc agc gtg gtc acc gtg cct tct 576
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
agc agc ctg ggc acc cag acc tac atc tgc aac gtg aac cac aag ccc 624
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
agc aac acc aag gtg gac gag aag gtg gag ccc aag agc tgc gac aaa 672
Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
act cac aca tgc cca ccg tgc cca gca cct gaa gct gca ggg gga ccg 720
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro
225 230 235 240
tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc 768
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc cac gaa gac 816
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat 864
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
gcc aag aca aag ccg cgg gag gag cag tac aac agc acg tac cgt gtg 912
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat ggc aag gag 960
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
tac aag tgc aag gtc tcc aac aaa gcc ctc ggc gcc ccc atc gag aaa 1008
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys
325 330 335
acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca cag gtg tgc acc 1056
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr
340 345 350
ctg ccc cca tcc cgg gat gag ctg acc aag aac cag gtc agc ctc tcg 1104
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser
355 360 365
tgc gca gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag 1152
Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
agc aat ggg cag ccg gag aac aac tac aag acc acg cct ccc gtg ctg 1200
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
gac tcc gac ggc tcc ttc ttc ctc gtg agc aag ctc acc gtg gac aag 1248
Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys
405 410 415
agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag 1296
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
gct ctg cac aac cac tac acg cag aag agc ctc tcc ctg tct ccg ggt 1344
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
aaa tga 1350
Lys
<210> 8
<211> 449
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..1350, 来自SEQ ID NO 7
<400> 8
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 Ser Phe Thr Gly Tyr
20 25 30
Thr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe
50 55 60
Arg Gly Lys Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Gln 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 Glu 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 Glu 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 Cys Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser
355 360 365
Cys Ala 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 Val 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> 9
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> EGFRvIII MR1.1 CDR H1 Kabat
<400> 9
Lys Phe Gly Met Ser
1 5
<210> 10
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> EGFRvIII MR1.1 CDR H2 Kabat
<400> 10
Ser Ile Ser Thr Gly Gly Tyr Asn Thr Tyr Tyr Ser Asp Asn Val Lys
1 5 10 15
Gly
<210> 11
<211> 11
<212> PRT
<213> 人工序列
<220>
<223> EGFRvIII MR1.1 CDR H3 Kabat
<400> 11
Gly Tyr Ser Pro Tyr Ser Tyr Ala Met Asp Tyr
1 5 10
<210> 12
<211> 11
<212> PRT
<213> 人工序列
<220>
<223> EGFRvIII MR1.1 CDR1 L1 Kabat
<400> 12
Met Thr Ser Thr Asp Ile Asp Asp Asp Met Asn
1 5 10
<210> 13
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> EGFRvIII MR1.1 CDR L2 Kabat
<400> 13
Glu Gly Asn Thr Leu Arg Pro
1 5
<210> 14
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> EGFRvIII MR1.1 CDR L3 Kabat
<400> 14
Leu Gln Ser Trp Asn Val Pro Leu Thr
1 5
<210> 15
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> aMSLN CDR H1 Kabat
<400> 15
Gly Tyr Thr Met Asn
1 5
<210> 16
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> aMSLN CDR H2 Kabat
<400> 16
Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe Arg
1 5 10 15
Gly
<210> 17
<211> 10
<212> PRT
<213> 人工序列
<220>
<223> aMSLN CDR H3 Kabat
<400> 17
Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr
1 5 10
<210> 18
<211> 10
<212> PRT
<213> 人工序列
<220>
<223> aMSLN CDR1 L1 Kabat
<400> 18
Ser Ala Ser Ser Ser Val Ser Tyr Met His
1 5 10
<210> 19
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> aMSLN CDR L2 Kabat
<400> 19
Asp Thr Ser Lys Leu Ala Ser
1 5
<210> 20
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> aMSLN CDR L3 Kabat
<400> 20
Gln Gln Trp Ser Lys His Pro Leu Thr
1 5
<210> 21
<211> 2067
<212> DNA
<213> 人工序列
<220>
<223> EGFR vIII MR1.1 VH Ck muEpCAM VH CH1 EE Fc 节 PG LALA,
pETR14953
<220>
<221> CDS
<222> 1..2067
<223> /翻译表=1
<400> 21
caa gtg aag ctg cag cag agt ggg ggc gga ctc gtg aaa cct ggc gcc 48
Gln Val Lys Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Ala
1 5 10 15
tct ctg aag ctg agc tgc gtg acc agc ggc ttc acc ttc aga aag ttc 96
Ser Leu Lys Leu Ser Cys Val Thr Ser Gly Phe Thr Phe Arg Lys Phe
20 25 30
ggc atg agc tgg gtg cgc cag acc agc gac aag cgg ctg gaa tgg gtg 144
Gly Met Ser Trp Val Arg Gln Thr Ser Asp Lys Arg Leu Glu Trp Val
35 40 45
gcc agc atc agc acc ggc ggc tac aac acc tac tac agc gac aac gtg 192
Ala Ser Ile Ser Thr Gly Gly Tyr Asn Thr Tyr Tyr Ser Asp Asn Val
50 55 60
aag ggc cgg ttc acc atc agc aga gag aac gcc aag aac acc ctg tac 240
Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
ctg cag atg agc agc ctg aag tcc gag gac acc gcc ctg tac tac tgc 288
Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
acc aga ggc tac agc ccc tac agc tac gcc atg gac tat tgg ggc cag 336
Thr Arg Gly Tyr Ser Pro Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
ggc acc acc gtg acc gtg tca tct gct agc gtg gcc gct ccc tcc gtg 384
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val
115 120 125
ttc atc ttc cca cct tcc gac gag cag ctg aag tcc ggc acc gct tct 432
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser
130 135 140
gtc gtg tgc ctg ctg aac aac ttc tac ccc cgc gag gcc aag gtg cag 480
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln
145 150 155 160
tgg aag gtg gac aac gcc ctg cag tcc ggc aac agc cag gaa tcc gtg 528
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val
165 170 175
acc gag cag gac tcc aag gac agc acc tac tcc ctg tcc tcc acc ctg 576
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu
180 185 190
acc ctg tcc aag gcc gac tac gag aag cac aag gtg tac gcc tgc gaa 624
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu
195 200 205
gtg acc cac cag ggc ctg tct agc ccc gtg acc aag tct ttc aac cgg 672
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg
210 215 220
ggc gag tgc ggt ggc gga ggt tcc gga ggc gga gga tcc gaa gtg cag 720
Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln
225 230 235 240
ctg gcc gag agc ggc gga ggc ctg gtg cag cct ggc aga tcc atg aag 768
Leu Ala Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg Ser Met Lys
245 250 255
ctg agc tgc gcc gcc agc ggc ttc acc ttc agc aac ttc ccc atg gcc 816
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe Pro Met Ala
260 265 270
tgg gtc cga cag gcc ccc acc aag tgc ctg gaa tgg gtg gcc acc atc 864
Trp Val Arg Gln Ala Pro Thr Lys Cys Leu Glu Trp Val Ala Thr Ile
275 280 285
agc acc agc ggc ggc agc acc tac tac cgg gac agc gtg aag ggc cgg 912
Ser Thr Ser Gly Gly Ser Thr Tyr Tyr Arg Asp Ser Val Lys Gly Arg
290 295 300
ttc acc atc agc cgg gac aac gcc aag agc acc ctg tac ctg cag atg 960
Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu Tyr Leu Gln Met
305 310 315 320
aac agc ctg cgg agc gag gac acc gcc acc tac tac tgc acc cgg acc 1008
Asn Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys Thr Arg Thr
325 330 335
ctg tat atc ctg cgg gtg ttc tac ttc gac tac tgg ggc cag ggc gtg 1056
Leu Tyr Ile Leu Arg Val Phe Tyr Phe Asp Tyr Trp Gly Gln Gly Val
340 345 350
atg gtc acc gtg tct agc gct agc acc aag ggc ccc tcc gtg ttt cct 1104
Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
355 360 365
ctg gcc cct tcc agc aag tcc acc tct ggc gga act gcc gct ctg ggc 1152
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
370 375 380
tgc ctg gtg gaa gat tac ttc ccc gag ccc gtg acc gtg tcc tgg aat 1200
Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
385 390 395 400
tct ggc gct ctg acc tcc ggc gtg cac acc ttt cca gct gtg ctg cag 1248
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
405 410 415
tcc tcc ggc ctg tac tcc ctg tcc tcc gtc gtg aca gtg ccc tcc agc 1296
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
420 425 430
tct ctg ggc acc cag acc tac atc tgc aac gtg aac cac aag ccc tcc 1344
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
435 440 445
aac acc aag gtg gac gag aag gtg gaa ccc aag tcc tgc gac aag acc 1392
Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
450 455 460
cac acc tgt ccc ccc tgc cct gct cct gaa gct gct ggt ggc cct agc 1440
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
465 470 475 480
gtg ttc ctg ttc ccc cca aag ccc aag gac acc ctg atg atc tcc cgg 1488
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
485 490 495
acc ccc gaa gtg acc tgc gtg gtg gtg gat gtg tcc cac gag gac cct 1536
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
500 505 510
gaa gtg aag ttc aat tgg tac gtg gac ggc gtg gaa gtg cac aac gcc 1584
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
515 520 525
aag acc aag cct aga gag gaa cag tac aac tcc acc tac cgg gtg gtg 1632
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
530 535 540
tcc gtg ctg aca gtg ctg cac cag gac tgg ctg aac ggc aaa gag tac 1680
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
545 550 555 560
aag tgc aag gtg tcc aac aag gcc ctg ggc gct ccc atc gaa aag acc 1728
Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr
565 570 575
atc tcc aag gcc aag ggc cag ccc cgg gaa ccc cag gtg tac acc ctg 1776
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
580 585 590
ccc cca tgc cgg gat gag ctg acc aag aac cag gtc agc ctg tgg tgc 1824
Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys
595 600 605
ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag agc 1872
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
610 615 620
aat ggg cag ccg gag aac aac tac aag acc acg cct ccc gtg ctg gac 1920
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
625 630 635 640
tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc gtg gac aag agc 1968
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
645 650 655
agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag gct 2016
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
660 665 670
ctg cac aac cac tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa 2064
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
675 680 685
tga 2067
<210> 22
<211> 688
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..2067, 来自SEQ ID NO 21
<400> 22
Gln Val Lys Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Leu Lys Leu Ser Cys Val Thr Ser Gly Phe Thr Phe Arg Lys Phe
20 25 30
Gly Met Ser Trp Val Arg Gln Thr Ser Asp Lys Arg Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Thr Gly Gly Tyr Asn Thr Tyr Tyr Ser Asp Asn Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Thr Arg Gly Tyr Ser Pro Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val
115 120 125
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser
130 135 140
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln
145 150 155 160
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val
165 170 175
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu
180 185 190
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu
195 200 205
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg
210 215 220
Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln
225 230 235 240
Leu Ala Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg Ser Met Lys
245 250 255
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe Pro Met Ala
260 265 270
Trp Val Arg Gln Ala Pro Thr Lys Cys Leu Glu Trp Val Ala Thr Ile
275 280 285
Ser Thr Ser Gly Gly Ser Thr Tyr Tyr Arg Asp Ser Val Lys Gly Arg
290 295 300
Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu Tyr Leu Gln Met
305 310 315 320
Asn Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys Thr Arg Thr
325 330 335
Leu Tyr Ile Leu Arg Val Phe Tyr Phe Asp Tyr Trp Gly Gln Gly Val
340 345 350
Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
355 360 365
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
370 375 380
Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
385 390 395 400
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
405 410 415
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
420 425 430
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
435 440 445
Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
450 455 460
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
465 470 475 480
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
485 490 495
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
500 505 510
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
515 520 525
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
530 535 540
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
545 550 555 560
Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr
565 570 575
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
580 585 590
Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys
595 600 605
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
610 615 620
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
625 630 635 640
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
645 650 655
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
660 665 670
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
675 680 685
<210> 23
<211> 639
<212> DNA
<213> 人工序列
<220>
<223> EGFR vIII MR1.1 VL CH1, pETR14951
<220>
<221> CDS
<222> 1..639
<223> /翻译表=1
<400> 23
gat atc gag ctg aca cag agc ccc gcc agc ctg tct gtg gcc acc ggc 48
Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ser Val Ala Thr Gly
1 5 10 15
gag aaa gtg acc atc cgg tgc atg acc agc acc gac atc gac gac gac 96
Glu Lys Val Thr Ile Arg Cys Met Thr Ser Thr Asp Ile Asp Asp Asp
20 25 30
atg aac tgg tat cag cag aag ccc ggc gag ccc ccc aag ttc ctg atc 144
Met Asn Trp Tyr Gln Gln Lys Pro Gly Glu Pro Pro Lys Phe Leu Ile
35 40 45
agc gag ggc aac aca ctg cgg cct ggc gtg cca agc aga ttc agc agc 192
Ser Glu Gly Asn Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Ser
50 55 60
tct ggc acc ggc acc gac ttc gtg ttt acc atc gag aat acc ctg agc 240
Ser Gly Thr Gly Thr Asp Phe Val Phe Thr Ile Glu Asn Thr Leu Ser
65 70 75 80
gag gac gtg ggc gac tac tac tgc ctg cag agc tgg aac gtg ccc ctg 288
Glu Asp Val Gly Asp Tyr Tyr Cys Leu Gln Ser Trp Asn Val Pro Leu
85 90 95
acc ttt ggc gac ggc acc aag ctg gaa atc aag agc agc gct agc acc 336
Thr Phe Gly Asp Gly Thr Lys Leu Glu Ile Lys Ser Ser Ala Ser Thr
100 105 110
aaa ggc cct tcc gtg ttt cct ctg gct cct agc tcc aag tcc acc tct 384
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
115 120 125
gga ggc acc gct gct ctc gga tgc ctc gtg aag gat tat ttt cct gag 432
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
130 135 140
cct gtg aca gtg tcc tgg aat agc gga gca ctg acc tct gga gtg cat 480
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
145 150 155 160
act ttc ccc gct gtg ctg cag tcc tct gga ctg tac agc ctg agc agc 528
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
165 170 175
gtg gtg aca gtg ccc agc agc agc ctg ggc acc cag acc tac atc tgc 576
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
180 185 190
aac gtg aac cac aag ccc agc aac acc aag gtg gac aag aag gtg gaa 624
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
195 200 205
ccc aag tct tgt tga 639
Pro Lys Ser Cys
210
<210> 24
<211> 212
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..639, 来自SEQ ID NO 23
<400> 24
Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ser Val Ala Thr Gly
1 5 10 15
Glu Lys Val Thr Ile Arg Cys Met Thr Ser Thr Asp Ile Asp Asp Asp
20 25 30
Met Asn Trp Tyr Gln Gln Lys Pro Gly Glu Pro Pro Lys Phe Leu Ile
35 40 45
Ser Glu Gly Asn Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Ser
50 55 60
Ser Gly Thr Gly Thr Asp Phe Val Phe Thr Ile Glu Asn Thr Leu Ser
65 70 75 80
Glu Asp Val Gly Asp Tyr Tyr Cys Leu Gln Ser Trp Asn Val Pro Leu
85 90 95
Thr Phe Gly Asp Gly Thr Lys Leu Glu Ile Lys Ser Ser Ala Ser Thr
100 105 110
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
115 120 125
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
130 135 140
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
145 150 155 160
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
165 170 175
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
180 185 190
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
195 200 205
Pro Lys Ser Cys
210
<210> 25
<211> 645
<212> DNA
<213> 人工序列
<220>
<223> VL EpCAM G8.8 Ck RK, pETR14882
<220>
<221> CDS
<222> 1..645
<223> /翻译表=1
<400> 25
gac atc cag atg aca cag agc ccc gcc agc ctg agc gcc tct ctg ggc 48
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
gag aca gtg tcc atc gag tgc ctg gcc agc gag ggc atc agc aac gac 96
Glu Thr Val Ser Ile Glu Cys Leu Ala Ser Glu Gly Ile Ser Asn Asp
20 25 30
ctg gcc tgg tat cag cag aag tcc ggc aag agc ccc cag ctg ctg atc 144
Leu Ala Trp Tyr Gln Gln Lys Ser Gly Lys Ser Pro Gln Leu Leu Ile
35 40 45
tac gcc acc agc aga ctg cag gac ggc gtg ccc agc aga ttc agc ggc 192
Tyr Ala Thr Ser Arg Leu Gln Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
agc ggc tcc ggc acc cgg tac agc ctg aag atc agc ggc atg cag ccc 240
Ser Gly Ser Gly Thr Arg Tyr Ser Leu Lys Ile Ser Gly Met Gln Pro
65 70 75 80
gag gac gag gcc gac tac ttc tgc cag cag agc tac aag tac ccc tgg 288
Glu Asp Glu Ala Asp Tyr Phe Cys Gln Gln Ser Tyr Lys Tyr Pro Trp
85 90 95
acc ttc ggc tgc ggc acc aag ctg gaa ctg aag cgt acg gtg gct gca 336
Thr Phe Gly Cys Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala
100 105 110
cca tct gtc ttc atc ttc ccg cca tct gat cgg aag ttg aaa tct gga 384
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys Ser Gly
115 120 125
act gcc tct gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc 432
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
aaa gta cag tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag 480
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
gag agt gtc aca gag cag gac agc aag gac agc acc tac agc ctc agc 528
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
agc acc ctg acg ctg agc aaa gca gac tac gag aaa cac aaa gtc tac 576
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
gcc tgc gaa gtc acc cat cag ggc ctg agc tcg ccc gtc aca aag agc 624
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
ttc aac agg gga gag tgt tag 645
Phe Asn Arg Gly Glu Cys
210
<210> 26
<211> 214
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..645, 来自SEQ ID NO 25
<400> 26
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Glu Thr Val Ser Ile Glu Cys Leu Ala Ser Glu Gly Ile Ser Asn Asp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Ser Gly Lys Ser Pro Gln Leu Leu Ile
35 40 45
Tyr Ala Thr Ser Arg Leu Gln Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Arg Tyr Ser Leu Lys Ile Ser Gly Met Gln Pro
65 70 75 80
Glu Asp Glu Ala Asp Tyr Phe Cys Gln Gln Ser Tyr Lys Tyr Pro Trp
85 90 95
Thr Phe Gly Cys Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 27
<211> 1356
<212> DNA
<213> 人工序列
<220>
<223> VH muEpCAM CH1 EE Fc 穴 PG LALA HRYF, pETR14940
<220>
<221> CDS
<222> 1..1356
<223> /翻译表=1
<400> 27
gaa gtg cag ctg gcc gag agc ggc gga ggc ctg gtg cag cct ggc aga 48
Glu Val Gln Leu Ala Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
tcc atg aag ctg agc tgc gcc gcc agc ggc ttc acc ttc agc aac ttc 96
Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe
20 25 30
ccc atg gcc tgg gtc cga cag gcc ccc acc aag tgc ctg gaa tgg gtg 144
Pro Met Ala Trp Val Arg Gln Ala Pro Thr Lys Cys Leu Glu Trp Val
35 40 45
gcc acc atc agc acc agc ggc ggc agc acc tac tac cgg gac agc gtg 192
Ala Thr Ile Ser Thr Ser Gly Gly Ser Thr Tyr Tyr Arg Asp Ser Val
50 55 60
aag ggc cgg ttc acc atc agc cgg gac aac gcc aag agc acc ctg tac 240
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu Tyr
65 70 75 80
ctg cag atg aac agc ctg cgg agc gag gac acc gcc acc tac tac tgc 288
Leu Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
acc cgg acc ctg tat atc ctg cgg gtg ttc tac ttc gac tac tgg ggc 336
Thr Arg Thr Leu Tyr Ile Leu Arg Val Phe Tyr Phe Asp Tyr Trp Gly
100 105 110
cag ggc gtg atg gtc acc gtg tct agc gct agc acc aag ggc ccc tcc 384
Gln Gly Val Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125
gtg ttc ccc ctg gcc ccc agc agc aag agc acc agc ggc ggc aca gcc 432
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140
gct ctg ggc tgc ctg gtc gag gac tac ttc ccc gag ccc gtg acc gtg 480
Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
tcc tgg aac agc gga gcc ctg acc tcc ggc gtg cac acc ttc ccc gcc 528
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
gtg ctg cag agt tct ggc ctg tat agc ctg agc agc gtg gtc acc gtg 576
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
cct tct agc agc ctg ggc acc cag acc tac atc tgc aac gtg aac cac 624
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
195 200 205
aag ccc agc aac acc aag gtg gac gag aag gtg gag ccc aag agc tgc 672
Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys
210 215 220
gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa gct gca ggg 720
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly
225 230 235 240
gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg 768
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255
atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc cac 816
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270
gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag gtg 864
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285
cat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc acg tac 912
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300
cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat ggc 960
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320
aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc ggc gcc ccc atc 1008
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile
325 330 335
gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca cag gtg 1056
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350
tgc acc ctg ccc cca tcc cgg gat gag ctg acc aag aac cag gtc agc 1104
Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
355 360 365
ctc tcg tgc gca gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag 1152
Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380
tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg cct ccc 1200
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
385 390 395 400
gtg ctg gac tcc gac ggc tcc ttc ttc ctc gtg agc aag ctc acc gtg 1248
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val
405 410 415
gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg 1296
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430
cat gag gct ctg cac aac cgc ttc acg cag aag agc ctc tcc ctg tct 1344
His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445
ccg ggt aaa tga 1356
Pro Gly Lys
450
<210> 28
<211> 451
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..1356, 来自SEQ ID NO 27
<400> 28
Glu Val Gln Leu Ala Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe
20 25 30
Pro Met Ala Trp Val Arg Gln Ala Pro Thr Lys Cys Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Thr Ser Gly Gly Ser Thr Tyr Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Thr Arg Thr Leu Tyr Ile Leu Arg Val Phe Tyr Phe Asp Tyr Trp Gly
100 105 110
Gln Gly Val Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140
Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys
210 215 220
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly
225 230 235 240
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile
325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350
Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
355 360 365
Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val
405 410 415
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430
His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445
Pro Gly Lys
450
<210> 29
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> EGFRvIII MR1.1 CDR H1 Kabat
<400> 29
Lys Phe Gly Met Ser
1 5
<210> 30
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> EGFRvIII MR1.1 CDR H2 Kabat
<400> 30
Ser Ile Ser Thr Gly Gly Tyr Asn Thr Tyr Tyr Ser Asp Asn Val Lys
1 5 10 15
Gly
<210> 31
<211> 11
<212> PRT
<213> 人工序列
<220>
<223> EGFRvIII MR1.1 CDR H3 Kabat
<400> 31
Gly Tyr Ser Pro Tyr Ser Tyr Ala Met Asp Tyr
1 5 10
<210> 32
<211> 11
<212> PRT
<213> 人工序列
<220>
<223> EGFRvIII MR1.1 CDR1 L1 Kabat
<400> 32
Met Thr Ser Thr Asp Ile Asp Asp Asp Met Asn
1 5 10
<210> 33
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> EGFRvIII MR1.1 CDR L2 Kabat
<400> 33
Glu Gly Asn Thr Leu Arg Pro
1 5
<210> 34
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> EGFRvIII MR1.1 CDR L3 Kabat
<400> 34
Leu Gln Ser Trp Asn Val Pro Leu Thr
1 5
<210> 35
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> muEpCAM CDR H1 Kabat
<400> 35
Asn Phe Pro Met Ala
1 5
<210> 36
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> muEpCAM CDR H2 Kabat
<400> 36
Thr Ile Ser Thr Ser Gly Gly Ser Thr Tyr Tyr Arg Asp Ser Val Lys
1 5 10 15
Gly
<210> 37
<211> 12
<212> PRT
<213> 人工序列
<220>
<223> muEpCAM CDR H3 Kabat
<400> 37
Thr Leu Tyr Ile Leu Arg Val Phe Tyr Phe Asp Tyr
1 5 10
<210> 38
<211> 11
<212> PRT
<213> 人工序列
<220>
<223> muEpCAM CDR1 L1 Kabat
<400> 38
Leu Ala Ser Glu Gly Ile Ser Asn Asp Leu Ala
1 5 10
<210> 39
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> muEpCAM CDR L2 Kabat
<400> 39
Ala Thr Ser Arg Leu Gln Asp Gly
1 5
<210> 40
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> muEpCAM CDR L3 Kabat
<400> 40
Gln Gln Ser Tyr Lys Tyr Pro Trp Thr
1 5
<210> 41
<211> 1713
<212> DNA
<213> 人工序列
<220>
<223> E3 鼠 (EGFRex-CD28tm/iz-CD3z)
<400> 41
agcttgctcg cggccgcgcc accatgcgac cctccgggac ggccggggca gcgctcctgg 60
cgctgctggc tgcgctctgc ccggcgagtc gggctctgga ggaaaagaaa ggtaattatg 120
tggtgacaga tcacggctcg tgcgtccgag cctgtggggc cgacagctat gagatggagg 180
aagacggcgt ccgcaagtgt aagaagtgcg aagggccttg ccgcaaagtg tgtaacggaa 240
taggtattgg tgaatttaaa gactcactct ccataaatgc tacgaatatt aaacacttca 300
aaaactgcac ctccatcagt ggcgatctcc acatcctgcc ggtggcattt aggggtgact 360
ccttcacaca tactcctcct ctggatccac aggaactgga tattctgaaa accgtaaagg 420
aaatcacagg gtttttgctg attcaggctt ggcctgaaaa caggacggac ctccatgcct 480
ttgagaacct agaaatcata cgcggcagga ccaagcaaca tggtcagttt tctcttgcag 540
tcgtcagcct gaacataaca tccttgggat tacgctccct caaggagata agtgatggag 600
atgtgataat ttcaggaaac aaaaatttgt gctatgcaaa tacaataaac tggaaaaaac 660
tgtttgggac ctccggtcag aaaaccaaaa ttataagcaa cagaggtgaa aacagctgca 720
aggccacagg ccaggtctgc catgccttgt gctcccccga gggctgctgg ggcccggagc 780
ccagggactg cgtctcttgc cggaatgtca gccgaggcag ggaatgcgtg gacaagtgca 840
accttctgga gggtgagcca agggagtttg tggagaactc tgagtgcata cagtgccacc 900
cagagtgcct gcctcaggcc atgaacatca cctgcacagg acggggacca gacaactgta 960
tccagtgtgc ccactacatt gacggccccc actgcgtcaa gacctgcccg gcaggagtca 1020
tgggagaaaa caacaccctg gtctggaagt acgcagacgc cggccatgtg tgccacctgt 1080
gccatccaaa ctgcacctac ggatgcactg ggccaggtct tgaaggctgt ccaacgaatg 1140
ggcctaagat cccgtccttt tgggcactgg tcgtggttgc tggagtcctg ttttgttatg 1200
gcttgctagt gacagtggct ctttgtgtta tctggacaaa tagtagaagg aacagactcc 1260
ttcaaagtga ctacatgaac atgactcccc ggaggcctgg gctcactcga aagccttacc 1320
agccctacgc ccctgccaga gactttgcag cgtaccgccc cagagcaaaa ttcagcagga 1380
gtgcagagac tgctgccaac ctgcaggacc ccaaccagct ctacaatgag ctcaatctag 1440
ggcgaagaga ggaatatgac gtcttggaga agaagcgggc tcgggatcca gagatgggag 1500
gcaaacagca gaggaggagg aacccccagg aaggcgtata caatgcactg cagaaagaca 1560
agatggcaga agcctacagt gagatcggca caaaaggcga gaggcggaga ggcaaggggc 1620
acgatggcct ttaccagggt ctcagcactg ccaccaagga cacctatgat gccctgcata 1680
tgcagaccct ggcccctcgc taagaattca tta 1713
<210> 42
<211> 566
<212> PRT
<213> 人工序列
<220>
<223> E3 鼠 (EGFRex-CD28tm/iz-CD3z)
<400> 42
Leu Ala Arg Gly Arg Ala Thr Met Arg Pro Ser Gly Thr Ala Gly Ala
1 5 10 15
Ala Leu Leu Ala Leu Leu Ala Ala Leu Cys Pro Ala Ser Arg Ala Leu
20 25 30
Glu Glu Lys Lys Gly Asn Tyr Val Val Thr Asp His Gly Ser Cys Val
35 40 45
Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu Asp Gly Val Arg
50 55 60
Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val Cys Asn Gly Ile
65 70 75 80
Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile
85 90 95
Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu
100 105 110
Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp
115 120 125
Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe
130 135 140
Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe
145 150 155 160
Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe
165 170 175
Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser
180 185 190
Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn
195 200 205
Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser
210 215 220
Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys
225 230 235 240
Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp
245 250 255
Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly
260 265 270
Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu
275 280 285
Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro
290 295 300
Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile
305 310 315 320
Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro
325 330 335
Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp
340 345 350
Ala Gly His Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys
355 360 365
Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro
370 375 380
Ser Phe Trp Ala Leu Val Val Val Ala Gly Val Leu Phe Cys Tyr Gly
385 390 395 400
Leu Leu Val Thr Val Ala Leu Cys Val Ile Trp Thr Asn Ser Arg Arg
405 410 415
Asn Arg Leu Leu Gln Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro
420 425 430
Gly Leu Thr Arg Lys Pro Tyr Gln Pro Tyr Ala Pro Ala Arg Asp Phe
435 440 445
Ala Ala Tyr Arg Pro Arg Ala Lys Phe Ser Arg Ser Ala Glu Thr Ala
450 455 460
Ala Asn Leu Gln Asp Pro Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly
465 470 475 480
Arg Arg Glu Glu Tyr Asp Val Leu Glu Lys Lys Arg Ala Arg Asp Pro
485 490 495
Glu Met Gly Gly Lys Gln Gln Arg Arg Arg Asn Pro Gln Glu Gly Val
500 505 510
Tyr Asn Ala Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile
515 520 525
Gly Thr Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
530 535 540
Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met
545 550 555 560
Gln Thr Leu Ala Pro Arg
565
<210> 43
<211> 1857
<212> DNA
<213> 人工序列
<220>
<223> E4 鼠 (EGFRex-CD28tm/iz-4-1BB-CD3z)
<400> 43
agcttgctcg cggccgcgcc accatgcgac cctccgggac ggccggggca gcgctcctgg 60
cgctgctggc tgcgctctgc ccggcgagtc gggctctgga ggaaaagaaa ggtaattatg 120
tggtgacaga tcacggctcg tgcgtccgag cctgtggggc cgacagctat gagatggagg 180
aagacggcgt ccgcaagtgt aagaagtgcg aagggccttg ccgcaaagtg tgtaacggaa 240
taggtattgg tgaatttaaa gactcactct ccataaatgc tacgaatatt aaacacttca 300
aaaactgcac ctccatcagt ggcgatctcc acatcctgcc ggtggcattt aggggtgact 360
ccttcacaca tactcctcct ctggatccac aggaactgga tattctgaaa accgtaaagg 420
aaatcacagg gtttttgctg attcaggctt ggcctgaaaa caggacggac ctccatgcct 480
ttgagaacct agaaatcata cgcggcagga ccaagcaaca tggtcagttt tctcttgcag 540
tcgtcagcct gaacataaca tccttgggat tacgctccct caaggagata agtgatggag 600
atgtgataat ttcaggaaac aaaaatttgt gctatgcaaa tacaataaac tggaaaaaac 660
tgtttgggac ctccggtcag aaaaccaaaa ttataagcaa cagaggtgaa aacagctgca 720
aggccacagg ccaggtctgc catgccttgt gctcccccga gggctgctgg ggcccggagc 780
ccagggactg cgtctcttgc cggaatgtca gccgaggcag ggaatgcgtg gacaagtgca 840
accttctgga gggtgagcca agggagtttg tggagaactc tgagtgcata cagtgccacc 900
cagagtgcct gcctcaggcc atgaacatca cctgcacagg acggggacca gacaactgta 960
tccagtgtgc ccactacatt gacggccccc actgcgtcaa gacctgcccg gcaggagtca 1020
tgggagaaaa caacaccctg gtctggaagt acgcagacgc cggccatgtg tgccacctgt 1080
gccatccaaa ctgcacctac ggatgcactg ggccaggtct tgaaggctgt ccaacgaatg 1140
ggcctaagat cccgtccttt tgggcactgg tcgtggttgc tggagtcctg ttttgttatg 1200
gcttgctagt gacagtggct ctttgtgtta tctggacaaa tagtagaagg aacagactcc 1260
ttcaaagtga ctacatgaac atgactcccc ggaggcctgg gctcactcga aagccttacc 1320
agccctacgc ccctgccaga gactttgcag cgtaccgccc ctctgtgctc aaatggatca 1380
ggaaaaaatt cccccacata ttcaagcaac catttaagaa gaccactgga gcagctcaag 1440
aggaagatgc ttgtagctgc cgatgtccac aggaagaaga aggaggagga ggaggctatg 1500
agctgagagc aaaattcagc aggagtgcag agactgctgc caacctgcag gaccccaacc 1560
agctctacaa tgagctcaat ctagggcgaa gagaggaata tgacgtcttg gagaagaagc 1620
gggctcggga tccagagatg ggaggcaaac agcagaggag gaggaacccc caggaaggcg 1680
tatacaatgc actgcagaaa gacaagatgg cagaagccta cagtgagatc ggcacaaaag 1740
gcgagaggcg gagaggcaag gggcacgatg gcctttacca gggtctcagc actgccacca 1800
aggacaccta tgatgccctg catatgcaga ccctggcccc tcgctaagaa ttcatta 1857
<210> 44
<211> 614
<212> PRT
<213> 人工序列
<220>
<223> E4 鼠 (EGFRex-CD28tm/iz-4-1BB-CD3z)
<400> 44
Leu Ala Arg Gly Arg Ala Thr Met Arg Pro Ser Gly Thr Ala Gly Ala
1 5 10 15
Ala Leu Leu Ala Leu Leu Ala Ala Leu Cys Pro Ala Ser Arg Ala Leu
20 25 30
Glu Glu Lys Lys Gly Asn Tyr Val Val Thr Asp His Gly Ser Cys Val
35 40 45
Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu Asp Gly Val Arg
50 55 60
Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val Cys Asn Gly Ile
65 70 75 80
Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile
85 90 95
Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu
100 105 110
Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp
115 120 125
Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe
130 135 140
Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe
145 150 155 160
Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe
165 170 175
Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser
180 185 190
Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn
195 200 205
Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser
210 215 220
Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys
225 230 235 240
Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp
245 250 255
Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly
260 265 270
Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu
275 280 285
Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro
290 295 300
Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile
305 310 315 320
Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro
325 330 335
Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp
340 345 350
Ala Gly His Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys
355 360 365
Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro
370 375 380
Ser Phe Trp Ala Leu Val Val Val Ala Gly Val Leu Phe Cys Tyr Gly
385 390 395 400
Leu Leu Val Thr Val Ala Leu Cys Val Ile Trp Thr Asn Ser Arg Arg
405 410 415
Asn Arg Leu Leu Gln Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro
420 425 430
Gly Leu Thr Arg Lys Pro Tyr Gln Pro Tyr Ala Pro Ala Arg Asp Phe
435 440 445
Ala Ala Tyr Arg Pro Ser Val Leu Lys Trp Ile Arg Lys Lys Phe Pro
450 455 460
His Ile Phe Lys Gln Pro Phe Lys Lys Thr Thr Gly Ala Ala Gln Glu
465 470 475 480
Glu Asp Ala Cys Ser Cys Arg Cys Pro Gln Glu Glu Glu Gly Gly Gly
485 490 495
Gly Gly Tyr Glu Leu Arg Ala Lys Phe Ser Arg Ser Ala Glu Thr Ala
500 505 510
Ala Asn Leu Gln Asp Pro Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly
515 520 525
Arg Arg Glu Glu Tyr Asp Val Leu Glu Lys Lys Arg Ala Arg Asp Pro
530 535 540
Glu Met Gly Gly Lys Gln Gln Arg Arg Arg Asn Pro Gln Glu Gly Val
545 550 555 560
Tyr Asn Ala Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile
565 570 575
Gly Thr Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
580 585 590
Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met
595 600 605
Gln Thr Leu Ala Pro Arg
610
<210> 45
<211> 1132
<212> DNA
<213> 人工序列
<220>
<223> CAR1 鼠 (Cripto-CD8aex/tm-CD28iz-CD3z)
<400> 45
attagcggcc gcgccaccat ggaaacagat acactgctgc tgtgggtgct gctgctgtgg 60
gtgccaggat ctacagggga tggcgcccag cctgctagaa gcctgggcca tcaggaattt 120
gctcgtccat ctcggggata cctggccttc agagatgaca gcatttggcc ccaggaggag 180
cctgcaattc ggcctcggtc ttcccagcgt gtgccgccca tggggataca gcacagtaag 240
gagctaaaca gaacctgctg cctgaatggg ggaacctgca tgctggggtc cttttgtgcc 300
tgccctccct ccttctacgg acggaactgt gagcacgatg tgcgcaaaga gaactgtggg 360
tctgtgcccc atgacacctg gctgcccaag aagtgttccc tgtgtaaatg ctggcacggt 420
cagctccgct gctttcctca ggcatttcta cccggctgtg atactaccaa gccagtgctg 480
cgaactccct cacctgtgca ccctaccggg acatctcagc cccagagacc agaagattgt 540
cggccccgtg gctcagtgaa ggggaccgga ttggacttcg cctgtgatat ttacatctgg 600
gcacccttgg ccggaatctg cgtggccctt ctgctgtcct tgatcatcac tctcatcaat 660
agtagaagga acagactcct tcaaagtgac tacatgaaca tgactccccg gaggcctggg 720
ctcactcgaa agccttacca gccctacgcc cctgccagag actttgcagc gtaccgcccc 780
agagcaaaat tcagcaggag tgcagagact gctgccaacc tgcaggaccc caaccagctc 840
tacaatgagc tcaatctagg gcgaagagag gaatatgacg tcttggagaa gaagcgggct 900
cgggatccag agatgggagg caaacagcag aggaggagga acccccagga aggcgtatac 960
aatgcactgc agaaagacaa gatggcagaa gcctacagtg agatcggcac aaaaggcgag 1020
aggcggagag gcaaggggca cgatggcctt taccagggtc tcagcactgc caccaaggac 1080
acctatgatg ccctgcatat gcagaccctg gcccctcgct aagaattcat ta 1132
<210> 46
<211> 373
<212> PRT
<213> 人工序列
<220>
<223> CAR1 鼠 (Cripto-CD8aex/tm-CD28iz-CD3z)
<400> 46
Ile Ser Gly Arg Ala Thr Met Glu Thr Asp Thr Leu Leu Leu Trp Val
1 5 10 15
Leu Leu Leu Trp Val Pro Gly Ser Thr Gly Asp Gly Ala Gln Pro Ala
20 25 30
Arg Ser Leu Gly His Gln Glu Phe Ala Arg Pro Ser Arg Gly Tyr Leu
35 40 45
Ala Phe Arg Asp Asp Ser Ile Trp Pro Gln Glu Glu Pro Ala Ile Arg
50 55 60
Pro Arg Ser Ser Gln Arg Val Pro Pro Met Gly Ile Gln His Ser Lys
65 70 75 80
Glu Leu Asn Arg Thr Cys Cys Leu Asn Gly Gly Thr Cys Met Leu Gly
85 90 95
Ser Phe Cys Ala Cys Pro Pro Ser Phe Tyr Gly Arg Asn Cys Glu His
100 105 110
Asp Val Arg Lys Glu Asn Cys Gly Ser Val Pro His Asp Thr Trp Leu
115 120 125
Pro Lys Lys Cys Ser Leu Cys Lys Cys Trp His Gly Gln Leu Arg Cys
130 135 140
Phe Pro Gln Ala Phe Leu Pro Gly Cys Asp Thr Thr Lys Pro Val Leu
145 150 155 160
Arg Thr Pro Ser Pro Val His Pro Thr Gly Thr Ser Gln Pro Gln Arg
165 170 175
Pro Glu Asp Cys Arg Pro Arg Gly Ser Val Lys Gly Thr Gly Leu Asp
180 185 190
Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Ile Cys Val
195 200 205
Ala Leu Leu Leu Ser Leu Ile Ile Thr Leu Ile Asn Ser Arg Arg Asn
210 215 220
Arg Leu Leu Gln Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly
225 230 235 240
Leu Thr Arg Lys Pro Tyr Gln Pro Tyr Ala Pro Ala Arg Asp Phe Ala
245 250 255
Ala Tyr Arg Pro Arg Ala Lys Phe Ser Arg Ser Ala Glu Thr Ala Ala
260 265 270
Asn Leu Gln Asp Pro Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg
275 280 285
Arg Glu Glu Tyr Asp Val Leu Glu Lys Lys Arg Ala Arg Asp Pro Glu
290 295 300
Met Gly Gly Lys Gln Gln Arg Arg Arg Asn Pro Gln Glu Gly Val Tyr
305 310 315 320
Asn Ala Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly
325 330 335
Thr Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln
340 345 350
Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln
355 360 365
Thr Leu Ala Pro Arg
370
<210> 47
<211> 1700
<212> DNA
<213> 人工序列
<220>
<223> E3 人 (EGFRex-CD28tm/iz-CD3z)
<400> 47
agcttgctcg cggccgcgcc accatgcgac cctccgggac ggccggggca gcgctcctgg 60
cgctgctggc tgcgctctgc ccggcgagtc gggctctgga ggaaaagaaa ggtaattatg 120
tggtgacaga tcacggctcg tgcgtccgag cctgtggggc cgacagctat gagatggagg 180
aagacggcgt ccgcaagtgt aagaagtgcg aagggccttg ccgcaaagtg tgtaacggaa 240
taggtattgg tgaatttaaa gactcactct ccataaatgc tacgaatatt aaacacttca 300
aaaactgcac ctccatcagt ggcgatctcc acatcctgcc ggtggcattt aggggtgact 360
ccttcacaca tactcctcct ctggatccac aggaactgga tattctgaaa accgtaaagg 420
aaatcacagg gtttttgctg attcaggctt ggcctgaaaa caggacggac ctccatgcct 480
ttgagaacct agaaatcata cgcggcagga ccaagcaaca tggtcagttt tctcttgcag 540
tcgtcagcct gaacataaca tccttgggat tacgctccct caaggagata agtgatggag 600
atgtgataat ttcaggaaac aaaaatttgt gctatgcaaa tacaataaac tggaaaaaac 660
tgtttgggac ctccggtcag aaaaccaaaa ttataagcaa cagaggtgaa aacagctgca 720
aggccacagg ccaggtctgc catgccttgt gctcccccga gggctgctgg ggcccggagc 780
ccagggactg cgtctcttgc cggaatgtca gccgaggcag ggaatgcgtg gacaagtgca 840
accttctgga gggtgagcca agggagtttg tggagaactc tgagtgcata cagtgccacc 900
cagagtgcct gcctcaggcc atgaacatca cctgcacagg acggggacca gacaactgta 960
tccagtgtgc ccactacatt gacggccccc actgcgtcaa gacctgcccg gcaggagtca 1020
tgggagaaaa caacaccctg gtctggaagt acgcagacgc cggccatgtg tgccacctgt 1080
gccatccaaa ctgcacctac ggatgcactg ggccaggtct tgaaggctgt ccaacgaatg 1140
ggcctaagat cccgtccttt tgggtgctgg tggtggttgg tggagtcctg gcttgctata 1200
gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg agcaggctcc 1260
tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc aagcattacc 1320
agccctatgc cccaccacgc gacttcgcag cctatcgctc cagagtgaag ttcagcagga 1380
gcgcagacgc ccccgcgtac cagcagggcc agaaccagct ctataacgag ctcaatctag 1440
gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct gagatggggg 1500
gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag aaagataaga 1560
tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc aaggggcacg 1620
atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc cttcacatgc 1680
aggccctgcc ccctcgctaa 1700
<210> 48
<211> 565
<212> PRT
<213> 人工序列
<220>
<223> E3 人 (EGFRex-CD28tm/iz-CD3z)
<400> 48
Leu Ala Arg Gly Arg Ala Thr Met Arg Pro Ser Gly Thr Ala Gly Ala
1 5 10 15
Ala Leu Leu Ala Leu Leu Ala Ala Leu Cys Pro Ala Ser Arg Ala Leu
20 25 30
Glu Glu Lys Lys Gly Asn Tyr Val Val Thr Asp His Gly Ser Cys Val
35 40 45
Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu Asp Gly Val Arg
50 55 60
Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val Cys Asn Gly Ile
65 70 75 80
Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile
85 90 95
Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu
100 105 110
Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp
115 120 125
Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe
130 135 140
Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe
145 150 155 160
Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe
165 170 175
Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser
180 185 190
Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn
195 200 205
Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser
210 215 220
Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys
225 230 235 240
Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp
245 250 255
Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly
260 265 270
Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu
275 280 285
Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro
290 295 300
Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile
305 310 315 320
Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro
325 330 335
Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp
340 345 350
Ala Gly His Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys
355 360 365
Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro
370 375 380
Ser Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser
385 390 395 400
Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg
405 410 415
Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro
420 425 430
Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe
435 440 445
Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro
450 455 460
Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly
465 470 475 480
Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro
485 490 495
Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr
500 505 510
Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly
515 520 525
Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln
530 535 540
Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln
545 550 555 560
Ala Leu Pro Pro Arg
565
<210> 49
<211> 1826
<212> DNA
<213> 人工序列
<220>
<223> E4 人 (EGFRex-CD28tm/iz-4-1BB-CD3z)
<400> 49
agcttgctcg cggccgcgcc accatgcgac cctccgggac ggccggggca gcgctcctgg 60
cgctgctggc tgcgctctgc ccggcgagtc gggctctgga ggaaaagaaa ggtaattatg 120
tggtgacaga tcacggctcg tgcgtccgag cctgtggggc cgacagctat gagatggagg 180
aagacggcgt ccgcaagtgt aagaagtgcg aagggccttg ccgcaaagtg tgtaacggaa 240
taggtattgg tgaatttaaa gactcactct ccataaatgc tacgaatatt aaacacttca 300
aaaactgcac ctccatcagt ggcgatctcc acatcctgcc ggtggcattt aggggtgact 360
ccttcacaca tactcctcct ctggatccac aggaactgga tattctgaaa accgtaaagg 420
aaatcacagg gtttttgctg attcaggctt ggcctgaaaa caggacggac ctccatgcct 480
ttgagaacct agaaatcata cgcggcagga ccaagcaaca tggtcagttt tctcttgcag 540
tcgtcagcct gaacataaca tccttgggat tacgctccct caaggagata agtgatggag 600
atgtgataat ttcaggaaac aaaaatttgt gctatgcaaa tacaataaac tggaaaaaac 660
tgtttgggac ctccggtcag aaaaccaaaa ttataagcaa cagaggtgaa aacagctgca 720
aggccacagg ccaggtctgc catgccttgt gctcccccga gggctgctgg ggcccggagc 780
ccagggactg cgtctcttgc cggaatgtca gccgaggcag ggaatgcgtg gacaagtgca 840
accttctgga gggtgagcca agggagtttg tggagaactc tgagtgcata cagtgccacc 900
cagagtgcct gcctcaggcc atgaacatca cctgcacagg acggggacca gacaactgta 960
tccagtgtgc ccactacatt gacggccccc actgcgtcaa gacctgcccg gcaggagtca 1020
tgggagaaaa caacaccctg gtctggaagt acgcagacgc cggccatgtg tgccacctgt 1080
gccatccaaa ctgcacctac ggatgcactg ggccaggtct tgaaggctgt ccaacgaatg 1140
ggcctaagat cccgtccttt tgggtgctgg tggtggttgg tggagtcctg gcttgctata 1200
gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg agcaggctcc 1260
tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc aagcattacc 1320
agccctatgc cccaccacgc gacttcgcag cctatcgctc caaacggggc agaaagaaac 1380
tcctgtatat attcaaacaa ccatttatga gaccagtaca aactactcaa gaggaagatg 1440
gctgtagctg ccgatttcca gaagaagaag aaggaggatg tgaactgaga gtgaagttca 1500
gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat aacgagctca 1560
atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg gaccctgaga 1620
tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag 1680
ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg aggggcaagg 1740
ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac gacgcccttc 1800
acatgcaggc cctgccccct cgctaa 1826
<210> 50
<211> 607
<212> PRT
<213> 人工序列
<220>
<223> E4 人 (EGFRex-CD28tm/iz-4-1BB-CD3z)
<400> 50
Leu Ala Arg Gly Arg Ala Thr Met Arg Pro Ser Gly Thr Ala Gly Ala
1 5 10 15
Ala Leu Leu Ala Leu Leu Ala Ala Leu Cys Pro Ala Ser Arg Ala Leu
20 25 30
Glu Glu Lys Lys Gly Asn Tyr Val Val Thr Asp His Gly Ser Cys Val
35 40 45
Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu Asp Gly Val Arg
50 55 60
Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val Cys Asn Gly Ile
65 70 75 80
Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile
85 90 95
Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu
100 105 110
Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp
115 120 125
Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe
130 135 140
Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe
145 150 155 160
Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe
165 170 175
Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser
180 185 190
Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn
195 200 205
Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser
210 215 220
Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys
225 230 235 240
Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp
245 250 255
Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly
260 265 270
Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu
275 280 285
Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro
290 295 300
Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile
305 310 315 320
Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro
325 330 335
Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp
340 345 350
Ala Gly His Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys
355 360 365
Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro
370 375 380
Ser Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser
385 390 395 400
Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg
405 410 415
Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro
420 425 430
Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe
435 440 445
Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe
450 455 460
Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly
465 470 475 480
Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg
485 490 495
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
500 505 510
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
515 520 525
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
530 535 540
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
545 550 555 560
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
565 570 575
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
580 585 590
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
595 600 605
<210> 51
<211> 1134
<212> DNA
<213> 人工序列
<220>
<223> EGFRvIII ex
<220>
<221> CDS
<222> 1..1134
<223> /翻译表=1
<400> 51
atg cga ccc tcc ggg acg gcc ggg gca gcg ctc ctg gcg ctg ctg gct 48
Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala
1 5 10 15
gcg ctc tgc ccg gcg agt cgg gct ctg gag gaa aag aaa ggt aat tat 96
Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Gly Asn Tyr
20 25 30
gtg gtg aca gat cac ggc tcg tgc gtc cga gcc tgt ggg gcc gac agc 144
Val Val Thr Asp His Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser
35 40 45
tat gag atg gag gaa gac ggc gtc cgc aag tgt aag aag tgc gaa ggg 192
Tyr Glu Met Glu Glu Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly
50 55 60
cct tgc cgc aaa gtg tgt aac gga ata ggt att ggt gaa ttt aaa gac 240
Pro Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp
65 70 75 80
tca ctc tcc ata aat gct acg aat att aaa cac ttc aaa aac tgc acc 288
Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr
85 90 95
tcc atc agt ggc gat ctc cac atc ctg ccg gtg gca ttt agg ggt gac 336
Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp
100 105 110
tcc ttc aca cat act cct cct ctg gat cca cag gaa ctg gat att ctg 384
Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu
115 120 125
aaa acc gta aag gaa atc aca ggg ttt ttg ctg att cag gct tgg cct 432
Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro
130 135 140
gaa aac agg acg gac ctc cat gcc ttt gag aac cta gaa atc ata cgc 480
Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg
145 150 155 160
ggc agg acc aag caa cat ggt cag ttt tct ctt gca gtc gtc agc ctg 528
Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu
165 170 175
aac ata aca tcc ttg gga tta cgc tcc ctc aag gag ata agt gat gga 576
Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly
180 185 190
gat gtg ata att tca gga aac aaa aat ttg tgc tat gca aat aca ata 624
Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile
195 200 205
aac tgg aaa aaa ctg ttt ggg acc tcc ggt cag aaa acc aaa att ata 672
Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile
210 215 220
agc aac aga ggt gaa aac agc tgc aag gcc aca ggc cag gtc tgc cat 720
Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His
225 230 235 240
gcc ttg tgc tcc ccc gag ggc tgc tgg ggc ccg gag ccc agg gac tgc 768
Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys
245 250 255
gtc tct tgc cgg aat gtc agc cga ggc agg gaa tgc gtg gac aag tgc 816
Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys
260 265 270
aac ctt ctg gag ggt gag cca agg gag ttt gtg gag aac tct gag tgc 864
Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys
275 280 285
ata cag tgc cac cca gag tgc ctg cct cag gcc atg aac atc acc tgc 912
Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys
290 295 300
aca gga cgg gga cca gac aac tgt atc cag tgt gcc cac tac att gac 960
Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp
305 310 315 320
ggc ccc cac tgc gtc aag acc tgc ccg gca gga gtc atg gga gaa aac 1008
Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn
325 330 335
aac acc ctg gtc tgg aag tac gca gac gcc ggc cat gtg tgc cac ctg 1056
Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu
340 345 350
tgc cat cca aac tgc acc tac gga tgc act ggg cca ggt ctt gaa ggc 1104
Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly
355 360 365
tgt cca acg aat ggg cct aag atc ccg tcc 1134
Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser
370 375
<210> 52
<211> 378
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..1134, 来自SEQ ID NO 51
<400> 52
Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala
1 5 10 15
Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Gly Asn Tyr
20 25 30
Val Val Thr Asp His Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser
35 40 45
Tyr Glu Met Glu Glu Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly
50 55 60
Pro Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp
65 70 75 80
Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr
85 90 95
Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp
100 105 110
Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu
115 120 125
Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro
130 135 140
Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg
145 150 155 160
Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu
165 170 175
Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly
180 185 190
Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile
195 200 205
Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile
210 215 220
Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His
225 230 235 240
Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys
245 250 255
Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys
260 265 270
Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys
275 280 285
Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys
290 295 300
Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp
305 310 315 320
Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn
325 330 335
Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu
340 345 350
Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly
355 360 365
Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser
370 375
<210> 53
<211> 81
<212> DNA
<213> 人工序列
<220>
<223> CD28TM (小鼠)
<220>
<221> CDS
<222> 1..81
<223> /翻译表=1
<400> 53
ttt tgg gca ctg gtc gtg gtt gct gga gtc ctg ttt tgt tat ggc ttg 48
Phe Trp Ala Leu Val Val Val Ala Gly Val Leu Phe Cys Tyr Gly Leu
1 5 10 15
cta gtg aca gtg gct ctt tgt gtt atc tgg aca 81
Leu Val Thr Val Ala Leu Cys Val Ile Trp Thr
20 25
<210> 54
<211> 27
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..81, 来自SEQ ID NO 53
<400> 54
Phe Trp Ala Leu Val Val Val Ala Gly Val Leu Phe Cys Tyr Gly Leu
1 5 10 15
Leu Val Thr Val Ala Leu Cys Val Ile Trp Thr
20 25
<210> 55
<211> 123
<212> DNA
<213> 人工序列
<220>
<223> CD28IC (小鼠)
<220>
<221> CDS
<222> 1..123
<223> /翻译表=1
<400> 55
aat agt aga agg aac aga ctc ctt caa agt gac tac atg aac atg act 48
Asn Ser Arg Arg Asn Arg Leu Leu Gln Ser Asp Tyr Met Asn Met Thr
1 5 10 15
ccc cgg agg cct ggg ctc act cga aag cct tac cag ccc tac gcc cct 96
Pro Arg Arg Pro Gly Leu Thr Arg Lys Pro Tyr Gln Pro Tyr Ala Pro
20 25 30
gcc aga gac ttt gca gcg tac cgc ccc 123
Ala Arg Asp Phe Ala Ala Tyr Arg Pro
35 40
<210> 56
<211> 41
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..123, 来自SEQ ID NO 55
<400> 56
Asn Ser Arg Arg Asn Arg Leu Leu Gln Ser Asp Tyr Met Asn Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Leu Thr Arg Lys Pro Tyr Gln Pro Tyr Ala Pro
20 25 30
Ala Arg Asp Phe Ala Ala Tyr Arg Pro
35 40
<210> 57
<211> 342
<212> DNA
<213> 人工序列
<220>
<223> CD3Z (小鼠)
<220>
<221> CDS
<222> 1..342
<223> /翻译表=1
<400> 57
aga gca aaa ttc agc agg agt gca gag act gct gcc aac ctg cag gac 48
Arg Ala Lys Phe Ser Arg Ser Ala Glu Thr Ala Ala Asn Leu Gln Asp
1 5 10 15
ccc aac cag ctc tac aat gag ctc aat cta ggg cga aga gag gaa tat 96
Pro Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
gac gtc ttg gag aag aag cgg gct cgg gat cca gag atg gga ggc aaa 144
Asp Val Leu Glu Lys Lys Arg Ala Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
cag cag agg agg agg aac ccc cag gaa ggc gta tac aat gca ctg cag 192
Gln Gln Arg Arg Arg Asn Pro Gln Glu Gly Val Tyr Asn Ala Leu Gln
50 55 60
aaa gac aag atg gca gaa gcc tac agt gag atc ggc aca aaa ggc gag 240
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Thr Lys Gly Glu
65 70 75 80
agg cgg aga ggc aag ggg cac gat ggc ctt tac cag ggt ctc agc act 288
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
85 90 95
gcc acc aag gac acc tat gat gcc ctg cat atg cag acc ctg gcc cct 336
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Thr Leu Ala Pro
100 105 110
cgc taa 342
Arg
<210> 58
<211> 113
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..342, 来自SEQ ID NO 57
<400> 58
Arg Ala Lys Phe Ser Arg Ser Ala Glu Thr Ala Ala Asn Leu Gln Asp
1 5 10 15
Pro Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Glu Lys Lys Arg Ala Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Gln Gln Arg Arg Arg Asn Pro Gln Glu Gly Val Tyr Asn Ala Leu Gln
50 55 60
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Thr Lys Gly Glu
65 70 75 80
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
85 90 95
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Thr Leu Ala Pro
100 105 110
Arg
<210> 59
<211> 144
<212> DNA
<213> 人工序列
<220>
<223> 41BB (小鼠)
<220>
<221> CDS
<222> 1..144
<223> /翻译表=1
<400> 59
tct gtg ctc aaa tgg atc agg aaa aaa ttc ccc cac ata ttc aag caa 48
Ser Val Leu Lys Trp Ile Arg Lys Lys Phe Pro His Ile Phe Lys Gln
1 5 10 15
cca ttt aag aag acc act gga gca gct caa gag gaa gat gct tgt agc 96
Pro Phe Lys Lys Thr Thr Gly Ala Ala Gln Glu Glu Asp Ala Cys Ser
20 25 30
tgc cga tgt cca cag gaa gaa gaa gga gga gga gga ggc tat gag ctg 144
Cys Arg Cys Pro Gln Glu Glu Glu Gly Gly Gly Gly Gly Tyr Glu Leu
35 40 45
<210> 60
<211> 48
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..144, 来自SEQ ID NO 59
<400> 60
Ser Val Leu Lys Trp Ile Arg Lys Lys Phe Pro His Ile Phe Lys Gln
1 5 10 15
Pro Phe Lys Lys Thr Thr Gly Ala Ala Gln Glu Glu Asp Ala Cys Ser
20 25 30
Cys Arg Cys Pro Gln Glu Glu Glu Gly Gly Gly Gly Gly Tyr Glu Leu
35 40 45
<210> 61
<211> 360
<212> DNA
<213> 人工序列
<220>
<223> CRIPTO (人)
<220>
<221> CDS
<222> 1..360
<223> /翻译表=1
<400> 61
ctg ggc cat cag gaa ttt gct cgt cca tct cgg gga tac ctg gcc ttc 48
Leu Gly His Gln Glu Phe Ala Arg Pro Ser Arg Gly Tyr Leu Ala Phe
1 5 10 15
aga gat gac agc att tgg ccc cag gag gag cct gca att cgg cct cgg 96
Arg Asp Asp Ser Ile Trp Pro Gln Glu Glu Pro Ala Ile Arg Pro Arg
20 25 30
tct tcc cag cgt gtg ccg ccc atg ggg ata cag cac agt aag gag cta 144
Ser Ser Gln Arg Val Pro Pro Met Gly Ile Gln His Ser Lys Glu Leu
35 40 45
aac aga acc tgc tgc ctg aat ggg gga acc tgc atg ctg ggg tcc ttt 192
Asn Arg Thr Cys Cys Leu Asn Gly Gly Thr Cys Met Leu Gly Ser Phe
50 55 60
tgt gcc tgc cct ccc tcc ttc tac gga cgg aac tgt gag cac gat gtg 240
Cys Ala Cys Pro Pro Ser Phe Tyr Gly Arg Asn Cys Glu His Asp Val
65 70 75 80
cgc aaa gag aac tgt ggg tct gtg ccc cat gac acc tgg ctg ccc aag 288
Arg Lys Glu Asn Cys Gly Ser Val Pro His Asp Thr Trp Leu Pro Lys
85 90 95
aag tgt tcc ctg tgt aaa tgc tgg cac ggt cag ctc cgc tgc ttt cct 336
Lys Cys Ser Leu Cys Lys Cys Trp His Gly Gln Leu Arg Cys Phe Pro
100 105 110
cag gca ttt cta ccc ggc tgt gat 360
Gln Ala Phe Leu Pro Gly Cys Asp
115 120
<210> 62
<211> 120
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..360, 来自SEQ ID NO 61
<400> 62
Leu Gly His Gln Glu Phe Ala Arg Pro Ser Arg Gly Tyr Leu Ala Phe
1 5 10 15
Arg Asp Asp Ser Ile Trp Pro Gln Glu Glu Pro Ala Ile Arg Pro Arg
20 25 30
Ser Ser Gln Arg Val Pro Pro Met Gly Ile Gln His Ser Lys Glu Leu
35 40 45
Asn Arg Thr Cys Cys Leu Asn Gly Gly Thr Cys Met Leu Gly Ser Phe
50 55 60
Cys Ala Cys Pro Pro Ser Phe Tyr Gly Arg Asn Cys Glu His Asp Val
65 70 75 80
Arg Lys Glu Asn Cys Gly Ser Val Pro His Asp Thr Trp Leu Pro Lys
85 90 95
Lys Cys Ser Leu Cys Lys Cys Trp His Gly Gln Leu Arg Cys Phe Pro
100 105 110
Gln Ala Phe Leu Pro Gly Cys Asp
115 120
<210> 63
<211> 195
<212> DNA
<213> 人工序列
<220>
<223> CD8 (鼠)
<220>
<221> CDS
<222> 1..195
<223> /翻译表=1
<400> 63
act acc aag cca gtg ctg cga act ccc tca cct gtg cac cct acc ggg 48
Thr Thr Lys Pro Val Leu Arg Thr Pro Ser Pro Val His Pro Thr Gly
1 5 10 15
aca tct cag ccc cag aga cca gaa gat tgt cgg ccc cgt ggc tca gtg 96
Thr Ser Gln Pro Gln Arg Pro Glu Asp Cys Arg Pro Arg Gly Ser Val
20 25 30
aag ggg acc gga ttg gac ttc gcc tgt gat att tac atc tgg gca ccc 144
Lys Gly Thr Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro
35 40 45
ttg gcc gga atc tgc gtg gcc ctt ctg ctg tcc ttg atc atc act ctc 192
Leu Ala Gly Ile Cys Val Ala Leu Leu Leu Ser Leu Ile Ile Thr Leu
50 55 60
atc 195
Ile
65
<210> 64
<211> 65
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..195, 来自SEQ ID NO 63
<400> 64
Thr Thr Lys Pro Val Leu Arg Thr Pro Ser Pro Val His Pro Thr Gly
1 5 10 15
Thr Ser Gln Pro Gln Arg Pro Glu Asp Cys Arg Pro Arg Gly Ser Val
20 25 30
Lys Gly Thr Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro
35 40 45
Leu Ala Gly Ile Cys Val Ala Leu Leu Leu Ser Leu Ile Ile Thr Leu
50 55 60
Ile
65
<210> 65
<211> 81
<212> DNA
<213> 人工序列
<220>
<223> CD28TM (人)
<220>
<221> CDS
<222> 1..81
<223> /翻译表=1
<400> 65
ttt tgg gtg ctg gtg gtg gtt ggt gga gtc ctg gct tgc tat agc ttg 48
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
1 5 10 15
cta gta aca gtg gcc ttt att att ttc tgg gtg 81
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val
20 25
<210> 66
<211> 27
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..81, 来自SEQ ID NO 65
<400> 66
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
1 5 10 15
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val
20 25
<210> 67
<211> 123
<212> DNA
<213> 人工序列
<220>
<223> CD28IC (人)
<220>
<221> CDS
<222> 1..123
<223> /翻译表=1
<400> 67
agg agt aag agg agc agg ctc ctg cac agt gac tac atg aac atg act 48
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr
1 5 10 15
ccc cgc cgc ccc ggg ccc acc cgc aag cat tac cag ccc tat gcc cca 96
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
20 25 30
cca cgc gac ttc gca gcc tat cgc tcc 123
Pro Arg Asp Phe Ala Ala Tyr Arg Ser
35 40
<210> 68
<211> 41
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..123, 来自SEQ ID NO 67
<400> 68
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
20 25 30
Pro Arg Asp Phe Ala Ala Tyr Arg Ser
35 40
<210> 69
<211> 336
<212> DNA
<213> 人工序列
<220>
<223> CD3Z (人)
<220>
<221> CDS
<222> 1..336
<223> /翻译表=1
<400> 69
aga gtg aag ttc agc agg agc gca gac gcc ccc gcg tac cag cag ggc 48
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 15
cag aac cag ctc tat aac gag ctc aat cta gga cga aga gag gag tac 96
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
gat gtt ttg gac aag aga cgt ggc cgg gac cct gag atg ggg gga aag 144
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
ccg aga agg aag aac cct cag gaa ggc ctg tac aat gaa ctg cag aaa 192
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
gat aag atg gcg gag gcc tac agt gag att ggg atg aaa ggc gag cgc 240
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
cgg agg ggc aag ggg cac gat ggc ctt tac cag ggt ctc agt aca gcc 288
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
acc aag gac acc tac gac gcc ctt cac atg cag gcc ctg ccc cct cgc 336
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 70
<211> 112
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..336, 来自SEQ ID NO 69
<400> 70
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 71
<211> 126
<212> DNA
<213> 人工序列
<220>
<223> 41BB (人)
<220>
<221> CDS
<222> 1..126
<223> /翻译表=1
<400> 71
aaa cgg ggc aga aag aaa ctc ctg tat ata ttc aaa caa cca ttt atg 48
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
1 5 10 15
aga cca gta caa act act caa gag gaa gat ggc tgt agc tgc cga ttt 96
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
20 25 30
cca gaa gaa gaa gaa gga gga tgt gaa ctg 126
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
35 40
<210> 72
<211> 42
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..126, 来自SEQ ID NO 71
<400> 72
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
1 5 10 15
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
20 25 30
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
35 40
<210> 73
<211> 546
<212> DNA
<213> 人工序列
<220>
<223> CD8 (人)
<220>
<221> CDS
<222> 1..546
<223> /翻译表=1
<400> 73
agc cag ttc cgg gtg tcg ccg ctg gat cgg acc tgg aac ctg ggc gag 48
Ser Gln Phe Arg Val Ser Pro Leu Asp Arg Thr Trp Asn Leu Gly Glu
1 5 10 15
aca gtg gag ctg aag tgc cag gtg ctg ctg tcc aac ccg acg tcg ggc 96
Thr Val Glu Leu Lys Cys Gln Val Leu Leu Ser Asn Pro Thr Ser Gly
20 25 30
tgc tcg tgg ctc ttc cag ccg cgc ggc gcc gcc gcc agt ccc acc ttc 144
Cys Ser Trp Leu Phe Gln Pro Arg Gly Ala Ala Ala Ser Pro Thr Phe
35 40 45
ctc cta tac ctc tcc caa aac aag ccc aag gcg gcc gag ggg ctg gac 192
Leu Leu Tyr Leu Ser Gln Asn Lys Pro Lys Ala Ala Glu Gly Leu Asp
50 55 60
acc cag cgg ttc tcg ggc aag agg ttg ggg gac acc ttc gtc ctc acc 240
Thr Gln Arg Phe Ser Gly Lys Arg Leu Gly Asp Thr Phe Val Leu Thr
65 70 75 80
ctg agc gac ttc cgc cga gag aac gag ggc tac tat ttc tgc tcg gcc 288
Leu Ser Asp Phe Arg Arg Glu Asn Glu Gly Tyr Tyr Phe Cys Ser Ala
85 90 95
ctg agc aac tcc atc atg tac ttc agc cac ttc gtg ccg gtc ttc ctg 336
Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe Val Pro Val Phe Leu
100 105 110
cca gcg aag ccc acc acg acg cca gcg ccg cga cca cca aca ccg gcg 384
Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala
115 120 125
ccc acc atc gcg tcg cag ccc ctg tcc ctg cgc cca gag gcg tgc cgg 432
Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg
130 135 140
cca gcg gcg ggg ggc gca gtg cac acg agg ggg ctg gac ttc gcc tgt 480
Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys
145 150 155 160
gat atc tac atc tgg gcg ccc ctg gcc ggg act tgt ggg gtc ctt ctc 528
Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu
165 170 175
ctg tca ctg gtt atc acc 546
Leu Ser Leu Val Ile Thr
180
<210> 74
<211> 182
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..546, 来自SEQ ID NO 73
<400> 74
Ser Gln Phe Arg Val Ser Pro Leu Asp Arg Thr Trp Asn Leu Gly Glu
1 5 10 15
Thr Val Glu Leu Lys Cys Gln Val Leu Leu Ser Asn Pro Thr Ser Gly
20 25 30
Cys Ser Trp Leu Phe Gln Pro Arg Gly Ala Ala Ala Ser Pro Thr Phe
35 40 45
Leu Leu Tyr Leu Ser Gln Asn Lys Pro Lys Ala Ala Glu Gly Leu Asp
50 55 60
Thr Gln Arg Phe Ser Gly Lys Arg Leu Gly Asp Thr Phe Val Leu Thr
65 70 75 80
Leu Ser Asp Phe Arg Arg Glu Asn Glu Gly Tyr Tyr Phe Cys Ser Ala
85 90 95
Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe Val Pro Val Phe Leu
100 105 110
Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala
115 120 125
Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg
130 135 140
Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys
145 150 155 160
Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu
165 170 175
Leu Ser Leu Val Ile Thr
180
<210> 75
<211> 1260
<212> DNA
<213> 人工序列
<220>
<223> EGFRvIII (人)
<400> 75
agcttgctcg cggccgcgcc accatgcgac cctccgggac ggccggggca gcgctcctgg 60
cgctgctggc tgcgctctgc ccggcgagtc gggctctgga ggaaaagaaa ggtaattatg 120
tggtgacaga tcacggctcg tgcgtccgag cctgtggggc cgacagctat gagatggagg 180
aagacggcgt ccgcaagtgt aagaagtgcg aagggccttg ccgcaaagtg tgtaacggaa 240
taggtattgg tgaatttaaa gactcactct ccataaatgc tacgaatatt aaacacttca 300
aaaactgcac ctccatcagt ggcgatctcc acatcctgcc ggtggcattt aggggtgact 360
ccttcacaca tactcctcct ctggatccac aggaactgga tattctgaaa accgtaaagg 420
aaatcacagg gtttttgctg attcaggctt ggcctgaaaa caggacggac ctccatgcct 480
ttgagaacct agaaatcata cgcggcagga ccaagcaaca tggtcagttt tctcttgcag 540
tcgtcagcct gaacataaca tccttgggat tacgctccct caaggagata agtgatggag 600
atgtgataat ttcaggaaac aaaaatttgt gctatgcaaa tacaataaac tggaaaaaac 660
tgtttgggac ctccggtcag aaaaccaaaa ttataagcaa cagaggtgaa aacagctgca 720
aggccacagg ccaggtctgc catgccttgt gctcccccga gggctgctgg ggcccggagc 780
ccagggactg cgtctcttgc cggaatgtca gccgaggcag ggaatgcgtg gacaagtgca 840
accttctgga gggtgagcca agggagtttg tggagaactc tgagtgcata cagtgccacc 900
cagagtgcct gcctcaggcc atgaacatca cctgcacagg acggggacca gacaactgta 960
tccagtgtgc ccactacatt gacggccccc actgcgtcaa gacctgcccg gcaggagtca 1020
tgggagaaaa caacaccctg gtctggaagt acgcagacgc cggccatgtg tgccacctgt 1080
gccatccaaa ctgcacctac ggatgcactg ggccaggtct tgaaggctgt ccaacgaatg 1140
ggcctaagat cccgtccatc gccactggga tggtgggggc cctcctcttg ctgctggtgg 1200
tggccctggg gatcggcctc ttcatgcgaa ggcgccacat cgttcggaag cgctgagaat 1260
<210> 76
<211> 417
<212> PRT
<213> 人工序列
<220>
<223> EGFRvIII (人)
<400> 76
Leu Ala Arg Gly Arg Ala Thr Met Arg Pro Ser Gly Thr Ala Gly Ala
1 5 10 15
Ala Leu Leu Ala Leu Leu Ala Ala Leu Cys Pro Ala Ser Arg Ala Leu
20 25 30
Glu Glu Lys Lys Gly Asn Tyr Val Val Thr Asp His Gly Ser Cys Val
35 40 45
Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu Asp Gly Val Arg
50 55 60
Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val Cys Asn Gly Ile
65 70 75 80
Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile
85 90 95
Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu
100 105 110
Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp
115 120 125
Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe
130 135 140
Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe
145 150 155 160
Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe
165 170 175
Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser
180 185 190
Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn
195 200 205
Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser
210 215 220
Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys
225 230 235 240
Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp
245 250 255
Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly
260 265 270
Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu
275 280 285
Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro
290 295 300
Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile
305 310 315 320
Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro
325 330 335
Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp
340 345 350
Ala Gly His Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys
355 360 365
Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro
370 375 380
Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val
385 390 395 400
Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg His Ile Val Arg Lys
405 410 415
Arg
<210> 77
<211> 1134
<212> DNA
<213> 人工序列
<220>
<223> EGFRvIII 细胞外
<220>
<221> CDS
<222> 1..1134
<223> /翻译表=1
<400> 77
atg cga ccc tcc ggg acg gcc ggg gca gcg ctc ctg gcg ctg ctg gct 48
Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala
1 5 10 15
gcg ctc tgc ccg gcg agt cgg gct ctg gag gaa aag aaa ggt aat tat 96
Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Gly Asn Tyr
20 25 30
gtg gtg aca gat cac ggc tcg tgc gtc cga gcc tgt ggg gcc gac agc 144
Val Val Thr Asp His Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser
35 40 45
tat gag atg gag gaa gac ggc gtc cgc aag tgt aag aag tgc gaa ggg 192
Tyr Glu Met Glu Glu Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly
50 55 60
cct tgc cgc aaa gtg tgt aac gga ata ggt att ggt gaa ttt aaa gac 240
Pro Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp
65 70 75 80
tca ctc tcc ata aat gct acg aat att aaa cac ttc aaa aac tgc acc 288
Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr
85 90 95
tcc atc agt ggc gat ctc cac atc ctg ccg gtg gca ttt agg ggt gac 336
Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp
100 105 110
tcc ttc aca cat act cct cct ctg gat cca cag gaa ctg gat att ctg 384
Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu
115 120 125
aaa acc gta aag gaa atc aca ggg ttt ttg ctg att cag gct tgg cct 432
Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro
130 135 140
gaa aac agg acg gac ctc cat gcc ttt gag aac cta gaa atc ata cgc 480
Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg
145 150 155 160
ggc agg acc aag caa cat ggt cag ttt tct ctt gca gtc gtc agc ctg 528
Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu
165 170 175
aac ata aca tcc ttg gga tta cgc tcc ctc aag gag ata agt gat gga 576
Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly
180 185 190
gat gtg ata att tca gga aac aaa aat ttg tgc tat gca aat aca ata 624
Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile
195 200 205
aac tgg aaa aaa ctg ttt ggg acc tcc ggt cag aaa acc aaa att ata 672
Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile
210 215 220
agc aac aga ggt gaa aac agc tgc aag gcc aca ggc cag gtc tgc cat 720
Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His
225 230 235 240
gcc ttg tgc tcc ccc gag ggc tgc tgg ggc ccg gag ccc agg gac tgc 768
Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys
245 250 255
gtc tct tgc cgg aat gtc agc cga ggc agg gaa tgc gtg gac aag tgc 816
Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys
260 265 270
aac ctt ctg gag ggt gag cca agg gag ttt gtg gag aac tct gag tgc 864
Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys
275 280 285
ata cag tgc cac cca gag tgc ctg cct cag gcc atg aac atc acc tgc 912
Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys
290 295 300
aca gga cgg gga cca gac aac tgt atc cag tgt gcc cac tac att gac 960
Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp
305 310 315 320
ggc ccc cac tgc gtc aag acc tgc ccg gca gga gtc atg gga gaa aac 1008
Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn
325 330 335
aac acc ctg gtc tgg aag tac gca gac gcc ggc cat gtg tgc cac ctg 1056
Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu
340 345 350
tgc cat cca aac tgc acc tac gga tgc act ggg cca ggt ctt gaa ggc 1104
Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly
355 360 365
tgt cca acg aat ggg cct aag atc ccg tcc 1134
Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser
370 375
<210> 78
<211> 378
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..1134, 来自SEQ ID NO 77
<400> 78
Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala
1 5 10 15
Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Gly Asn Tyr
20 25 30
Val Val Thr Asp His Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser
35 40 45
Tyr Glu Met Glu Glu Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly
50 55 60
Pro Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp
65 70 75 80
Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr
85 90 95
Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp
100 105 110
Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu
115 120 125
Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro
130 135 140
Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg
145 150 155 160
Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu
165 170 175
Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly
180 185 190
Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile
195 200 205
Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile
210 215 220
Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His
225 230 235 240
Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys
245 250 255
Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys
260 265 270
Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys
275 280 285
Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys
290 295 300
Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp
305 310 315 320
Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn
325 330 335
Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu
340 345 350
Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly
355 360 365
Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser
370 375
<210> 79
<211> 69
<212> DNA
<213> 人工序列
<220>
<223> EGFRvIII TM
<220>
<221> CDS
<222> 1..69
<223> /翻译表=1
<400> 79
atc gcc act ggg atg gtg ggg gcc ctc ctc ttg ctg ctg gtg gtg gcc 48
Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala
1 5 10 15
ctg ggg atc ggc ctc ttc atg 69
Leu Gly Ile Gly Leu Phe Met
20
<210> 80
<211> 23
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..69, 来自SEQ ID NO 79
<400> 80
Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala
1 5 10 15
Leu Gly Ile Gly Leu Phe Met
20
<210> 81
<211> 27
<212> DNA
<213> 人工序列
<220>
<223> EGFRvIII IZ 锚
<220>
<221> CDS
<222> 1..27
<223> /翻译表=1
<400> 81
cga agg cgc cac atc gtt cgg aag cgc 27
Arg Arg Arg His Ile Val Arg Lys Arg
1 5
<210> 82
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..27, 来自SEQ ID NO 81
<400> 82
Arg Arg Arg His Ile Val Arg Lys Arg
1 5
<210> 83
<211> 945
<212> DNA
<213> 人工序列
<220>
<223> Epcam (小鼠)
<220>
<221> CDS
<222> 1..945
<223> /翻译表=1
<400> 83
atg gcg ggc ccg cag gcg ctg gcg ttt ggc ctg ctg ctg gcg gtg gtg 48
Met Ala Gly Pro Gln Ala Leu Ala Phe Gly Leu Leu Leu Ala Val Val
1 5 10 15
acc gcg acc ctg gcg gcg gcg cag cgc gat tgc gtg tgc gat aac tat 96
Thr Ala Thr Leu Ala Ala Ala Gln Arg Asp Cys Val Cys Asp Asn Tyr
20 25 30
aaa ctg gcg acc agc tgc agc ctg aac gaa tat ggc gaa tgc cag tgc 144
Lys Leu Ala Thr Ser Cys Ser Leu Asn Glu Tyr Gly Glu Cys Gln Cys
35 40 45
acc agc tat ggc acc cag aac acc gtg att tgc agc aaa ctg gcg agc 192
Thr Ser Tyr Gly Thr Gln Asn Thr Val Ile Cys Ser Lys Leu Ala Ser
50 55 60
aaa tgc ctg gcg atg aaa gcg gaa atg acc cat agc aaa agc ggc cgc 240
Lys Cys Leu Ala Met Lys Ala Glu Met Thr His Ser Lys Ser Gly Arg
65 70 75 80
cgc att aaa ccg gaa ggc gcg att cag aac aac gat ggc ctg tat gat 288
Arg Ile Lys Pro Glu Gly Ala Ile Gln Asn Asn Asp Gly Leu Tyr Asp
85 90 95
ccg gat tgc gat gaa cag ggc ctg ttt aaa gcg aaa cag tgc aac ggc 336
Pro Asp Cys Asp Glu Gln Gly Leu Phe Lys Ala Lys Gln Cys Asn Gly
100 105 110
acc gcg acc tgc tgg tgc gtg aac acc gcg ggc gtg cgc cgc acc gat 384
Thr Ala Thr Cys Trp Cys Val Asn Thr Ala Gly Val Arg Arg Thr Asp
115 120 125
aaa gat acc gaa att acc tgc agc gaa cgc gtg cgc acc tat tgg att 432
Lys Asp Thr Glu Ile Thr Cys Ser Glu Arg Val Arg Thr Tyr Trp Ile
130 135 140
att att gaa ctg aaa cat aaa gaa cgc gaa agc ccg tat gat cat cag 480
Ile Ile Glu Leu Lys His Lys Glu Arg Glu Ser Pro Tyr Asp His Gln
145 150 155 160
agc ctg cag acc gcg ctg cag gaa gcg ttt acc agc cgc tat aaa ctg 528
Ser Leu Gln Thr Ala Leu Gln Glu Ala Phe Thr Ser Arg Tyr Lys Leu
165 170 175
aac cag aaa ttt att aaa aac att atg tat gaa aac aac gtg att acc 576
Asn Gln Lys Phe Ile Lys Asn Ile Met Tyr Glu Asn Asn Val Ile Thr
180 185 190
att gat ctg atg cag aac agc agc cag aaa acc cag gat gat gtg gat 624
Ile Asp Leu Met Gln Asn Ser Ser Gln Lys Thr Gln Asp Asp Val Asp
195 200 205
att gcg gat gtg gcg tat tat ttt gaa aaa gat gtg aaa ggc gaa agc 672
Ile Ala Asp Val Ala Tyr Tyr Phe Glu Lys Asp Val Lys Gly Glu Ser
210 215 220
ctg ttt cat agc agc aaa agc atg gat ctg cgc gtg aac ggc gaa ccg 720
Leu Phe His Ser Ser Lys Ser Met Asp Leu Arg Val Asn Gly Glu Pro
225 230 235 240
ctg gat ctg gat ccg ggc cag acc ctg att tat tat gtg gat gaa aaa 768
Leu Asp Leu Asp Pro Gly Gln Thr Leu Ile Tyr Tyr Val Asp Glu Lys
245 250 255
gcg ccg gaa ttt agc atg cag ggc ctg acc gcg ggc att att gcg gtg 816
Ala Pro Glu Phe Ser Met Gln Gly Leu Thr Ala Gly Ile Ile Ala Val
260 265 270
att gtg gtg gtg agc ctg gcg gtg att gcg ggc att gtg gtg ctg gtg 864
Ile Val Val Val Ser Leu Ala Val Ile Ala Gly Ile Val Val Leu Val
275 280 285
att agc acc cgc aaa aaa agc gcg aaa tat gaa aaa gcg gaa att aaa 912
Ile Ser Thr Arg Lys Lys Ser Ala Lys Tyr Glu Lys Ala Glu Ile Lys
290 295 300
gaa atg ggc gaa att cat cgc gaa ctg aac gcg 945
Glu Met Gly Glu Ile His Arg Glu Leu Asn Ala
305 310 315
<210> 84
<211> 315
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..945, 来自SEQ ID NO 83
<400> 84
Met Ala Gly Pro Gln Ala Leu Ala Phe Gly Leu Leu Leu Ala Val Val
1 5 10 15
Thr Ala Thr Leu Ala Ala Ala Gln Arg Asp Cys Val Cys Asp Asn Tyr
20 25 30
Lys Leu Ala Thr Ser Cys Ser Leu Asn Glu Tyr Gly Glu Cys Gln Cys
35 40 45
Thr Ser Tyr Gly Thr Gln Asn Thr Val Ile Cys Ser Lys Leu Ala Ser
50 55 60
Lys Cys Leu Ala Met Lys Ala Glu Met Thr His Ser Lys Ser Gly Arg
65 70 75 80
Arg Ile Lys Pro Glu Gly Ala Ile Gln Asn Asn Asp Gly Leu Tyr Asp
85 90 95
Pro Asp Cys Asp Glu Gln Gly Leu Phe Lys Ala Lys Gln Cys Asn Gly
100 105 110
Thr Ala Thr Cys Trp Cys Val Asn Thr Ala Gly Val Arg Arg Thr Asp
115 120 125
Lys Asp Thr Glu Ile Thr Cys Ser Glu Arg Val Arg Thr Tyr Trp Ile
130 135 140
Ile Ile Glu Leu Lys His Lys Glu Arg Glu Ser Pro Tyr Asp His Gln
145 150 155 160
Ser Leu Gln Thr Ala Leu Gln Glu Ala Phe Thr Ser Arg Tyr Lys Leu
165 170 175
Asn Gln Lys Phe Ile Lys Asn Ile Met Tyr Glu Asn Asn Val Ile Thr
180 185 190
Ile Asp Leu Met Gln Asn Ser Ser Gln Lys Thr Gln Asp Asp Val Asp
195 200 205
Ile Ala Asp Val Ala Tyr Tyr Phe Glu Lys Asp Val Lys Gly Glu Ser
210 215 220
Leu Phe His Ser Ser Lys Ser Met Asp Leu Arg Val Asn Gly Glu Pro
225 230 235 240
Leu Asp Leu Asp Pro Gly Gln Thr Leu Ile Tyr Tyr Val Asp Glu Lys
245 250 255
Ala Pro Glu Phe Ser Met Gln Gly Leu Thr Ala Gly Ile Ile Ala Val
260 265 270
Ile Val Val Val Ser Leu Ala Val Ile Ala Gly Ile Val Val Leu Val
275 280 285
Ile Ser Thr Arg Lys Lys Ser Ala Lys Tyr Glu Lys Ala Glu Ile Lys
290 295 300
Glu Met Gly Glu Ile His Arg Glu Leu Asn Ala
305 310 315
<210> 85
<211> 339
<212> DNA
<213> 人工序列
<220>
<223> CD3Z UniProtKB - P20963
<220>
<221> CDS
<222> 1..339
<223> /翻译表=1
<400> 85
cgc gtg aaa ttt agc cgc agc gcg gat gcg ccg gcg tat cag cag ggc 48
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 15
cag aac cag ctg tat aac gaa ctg aac ctg ggc cgc cgc gaa gaa tat 96
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
gat gtg ctg gat aaa cgc cgc ggc cgc gat ccg gaa atg ggc ggc aaa 144
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
ccg cag cgc cgc aaa aac ccg cag gaa ggc ctg tat aac gaa ctg cag 192
Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
50 55 60
aaa gat aaa atg gcg gaa gcg tat agc gaa att ggc atg aaa ggc gaa 240
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
65 70 75 80
cgc cgc cgc ggc aaa ggc cat gat ggc ctg tat cag ggc ctg agc acc 288
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
85 90 95
gcg acc aaa gat acc tat gat gcg ctg cat atg cag gcg ctg ccg ccg 336
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
100 105 110
cgc 339
Arg
<210> 86
<211> 113
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..339, 来自SEQ ID NO 85
<400> 86
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
50 55 60
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
65 70 75 80
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
85 90 95
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
100 105 110
Arg
<210> 87
<211> 75
<212> DNA
<213> 人工序列
<220>
<223> Fcgr3a: UniProtKB - P08637
<220>
<221> CDS
<222> 1..75
<223> /翻译表=1
<400> 87
aaa acc aac att cgc agc agc acc cgc gat tgg aaa gat cat aaa ttt 48
Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp Lys Asp His Lys Phe
1 5 10 15
aaa tgg cgc aaa gat ccg cag gat aaa 75
Lys Trp Arg Lys Asp Pro Gln Asp Lys
20 25
<210> 88
<211> 25
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..75, 来自SEQ ID NO 87
<400> 88
Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp Lys Asp His Lys Phe
1 5 10 15
Lys Trp Arg Lys Asp Pro Gln Asp Lys
20 25
<210> 89
<211> 153
<212> DNA
<213> 人工序列
<220>
<223> NKG2D UniProtKB - P26718
<220>
<221> CDS
<222> 1..153
<223> /翻译表=1
<400> 89
atg ggc tgg att cgc ggc cgc cgc agc cgc cat agc tgg gaa atg agc 48
Met Gly Trp Ile Arg Gly Arg Arg Ser Arg His Ser Trp Glu Met Ser
1 5 10 15
gaa ttt cat aac tat aac ctg gat ctg aaa aaa agc gat ttt agc acc 96
Glu Phe His Asn Tyr Asn Leu Asp Leu Lys Lys Ser Asp Phe Ser Thr
20 25 30
cgc tgg cag aaa cag cgc tgc ccg gtg gtg aaa agc aaa tgc cgc gaa 144
Arg Trp Gln Lys Gln Arg Cys Pro Val Val Lys Ser Lys Cys Arg Glu
35 40 45
aac gcg agc 153
Asn Ala Ser
50
<210> 90
<211> 51
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..153, 来自SEQ ID NO 89
<400> 90
Met Gly Trp Ile Arg Gly Arg Arg Ser Arg His Ser Trp Glu Met Ser
1 5 10 15
Glu Phe His Asn Tyr Asn Leu Asp Leu Lys Lys Ser Asp Phe Ser Thr
20 25 30
Arg Trp Gln Lys Gln Arg Cys Pro Val Val Lys Ser Lys Cys Arg Glu
35 40 45
Asn Ala Ser
50
<210> 91
<211> 123
<212> DNA
<213> 人工序列
<220>
<223> UniProtKB - P10747 CD28
<220>
<221> CDS
<222> 1..123
<223> /翻译表=1
<400> 91
cgc agc aaa cgc agc cgc ctg ctg cat agc gat tat atg aac atg acc 48
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr
1 5 10 15
ccg cgc cgc ccg ggc ccg acc cgc aaa cat tat cag ccg tat gcg ccg 96
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
20 25 30
ccg cgc gat ttt gcg gcg tat cgc agc 123
Pro Arg Asp Phe Ala Ala Tyr Arg Ser
35 40
<210> 92
<211> 41
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..123, 来自SEQ ID NO 91
<400> 92
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
20 25 30
Pro Arg Asp Phe Ala Ala Tyr Arg Ser
35 40
<210> 93
<211> 126
<212> DNA
<213> 人工序列
<220>
<223> UniProtKB - Q07011 CD137
<220>
<221> CDS
<222> 1..126
<223> /翻译表=1
<400> 93
aaa cgc ggc cgc aaa aaa ctg ctg tat att ttt aaa cag ccg ttt atg 48
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
1 5 10 15
cgc ccg gtg cag acc acc cag gaa gaa gat ggc tgc agc tgc cgc ttt 96
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
20 25 30
ccg gaa gaa gaa gaa ggc ggc tgc gaa ctg 126
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
35 40
<210> 94
<211> 42
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..126, 来自SEQ ID NO 93
<400> 94
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
1 5 10 15
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
20 25 30
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
35 40
<210> 95
<211> 129
<212> DNA
<213> 人工序列
<220>
<223> UniProtKB - P23510 OX40
<220>
<221> CDS
<222> 1..129
<223> /翻译表=1
<400> 95
cag gtg agc cat cgc tat ccg cgc att cag agc att aaa gtg cag ttt 48
Gln Val Ser His Arg Tyr Pro Arg Ile Gln Ser Ile Lys Val Gln Phe
1 5 10 15
acc gaa tat aaa atg gaa cgc gtg cag ccg ctg gaa gaa aac gtg ggc 96
Thr Glu Tyr Lys Met Glu Arg Val Gln Pro Leu Glu Glu Asn Val Gly
20 25 30
aac gcg gcg cgc ccg cgc ttt gaa cgc aac aaa 129
Asn Ala Ala Arg Pro Arg Phe Glu Arg Asn Lys
35 40
<210> 96
<211> 43
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..129, 来自SEQ ID NO 95
<400> 96
Gln Val Ser His Arg Tyr Pro Arg Ile Gln Ser Ile Lys Val Gln Phe
1 5 10 15
Thr Glu Tyr Lys Met Glu Arg Val Gln Pro Leu Glu Glu Asn Val Gly
20 25 30
Asn Ala Ala Arg Pro Arg Phe Glu Arg Asn Lys
35 40
<210> 97
<211> 114
<212> DNA
<213> 人工序列
<220>
<223> UniProtKB - Q9Y6W8 ICOS
<220>
<221> CDS
<222> 1..114
<223> /翻译表=1
<400> 97
tgc tgg ctg acc aaa aaa aaa tat agc agc agc gtg cat gat ccg aac 48
Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn
1 5 10 15
ggc gaa tat atg ttt atg cgc gcg gtg aac acc gcg aaa aaa agc cgc 96
Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg
20 25 30
ctg acc gat gtg acc ctg 114
Leu Thr Asp Val Thr Leu
35
<210> 98
<211> 38
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..114, 来自SEQ ID NO 97
<400> 98
Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn
1 5 10 15
Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg
20 25 30
Leu Thr Asp Val Thr Leu
35
<210> 99
<211> 144
<212> DNA
<213> 人工序列
<220>
<223> UniProtKB - P26842 CD27
<220>
<221> CDS
<222> 1..144
<223> /翻译表=1
<400> 99
cag cgc cgc aaa tat cgc agc aac aaa ggc gaa agc ccg gtg gaa ccg 48
Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser Pro Val Glu Pro
1 5 10 15
gcg gaa ccg tgc cat tat agc tgc ccg cgc gaa gaa gaa ggc agc acc 96
Ala Glu Pro Cys His Tyr Ser Cys Pro Arg Glu Glu Glu Gly Ser Thr
20 25 30
att ccg att cag gaa gat tat cgc aaa ccg gaa ccg gcg tgc agc ccg 144
Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro Ala Cys Ser Pro
35 40 45
<210> 100
<211> 48
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..144, 来自SEQ ID NO 99
<400> 100
Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser Pro Val Glu Pro
1 5 10 15
Ala Glu Pro Cys His Tyr Ser Cys Pro Arg Glu Glu Glu Gly Ser Thr
20 25 30
Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro Ala Cys Ser Pro
35 40 45
<210> 101
<211> 72
<212> DNA
<213> 人工序列
<220>
<223> UniProtKB - Q9UBK5 DAP10
<220>
<221> CDS
<222> 1..72
<223> /翻译表=1
<400> 101
ctg tgc gcg cgc ccg cgc cgc agc ccg gcg cag gaa gat ggc aaa gtg 48
Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln Glu Asp Gly Lys Val
1 5 10 15
tat att aac atg ccg ggc cgc ggc 72
Tyr Ile Asn Met Pro Gly Arg Gly
20
<210> 102
<211> 24
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..72, 来自SEQ ID NO 101
<400> 102
Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln Glu Asp Gly Lys Val
1 5 10 15
Tyr Ile Asn Met Pro Gly Arg Gly
20
<210> 103
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> /注释="EGFRvIIINot1fwd"
/注释="引物"
<400> 103
agcttgctcg cggccgcgcc accatgcgac cctccg 36
<210> 104
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> /注释="引物"
/注释="EGFRtm CD28iz rev"
<400> 104
tctgttcctt ctactattca tgaagaggcc gatccc 36
<210> 105
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> /注释="引物"
/注释="EGFRtm CD28iz fwd"
<400> 105
gggatcggcc tcttcatgaa tagtagaagg aacaga 36
<210> 106
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> /注释="引物"
/注释="CD28in/CD3zeta rev"
<400> 106
ctgctgaatt ttgctctggg gcggtacgct gcaa 34
<210> 107
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> /注释="引物"
/注释="CD3zeta/CD28fwd"
<400> 107
ttgcagcgta ccgccccaga gcaaaattca gcag 34
<210> 108
<211> 29
<212> DNA
<213> 人工序列
<220>
<223> /注释="CD3zetaEcoR1rev"
/注释="引物"
<400> 108
taatgaattc ttagcgaggg gccagggtc 29
<210> 109
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> /注释="引物"
/注释="前导_Not1_fwd"
<400> 109
attagcggcc gcgccaccat ggaaacagat acac 34
<210> 110
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> 前导CriptoIsorev
<400> 110
aaattcctga tggcccaggc ttctagcagg ctgggc 36
<210> 111
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> /注释="引物"
/注释="前导CriptoIsofwd"
<400> 111
gcccagcctg ctagaagcct gggccatcag gaattt 36
<210> 112
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> /注释="Cripto CD8aex rev"
/注释="引物"
<400> 112
cagcactggc ttggtagtat cacagccggg tagaaa 36
<210> 113
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> /注释="CriptoCD8aex fwd"
/注释="引物"
<400> 113
tttctacccg gctgtgatac taccaagcca gtgctg 36
<210> 114
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> /注释="引物"
/注释="CD8tm-CD28iz rev"
<400> 114
tctgttcctt ctactattga tgagagtgat gatcaa 36
<210> 115
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> /注释="CD8tm-CD28izfwd"
/注释="引物"
<400> 115
ttgatcatca ctctcatcaa tagtagaagg aacaga 36
<210> 116
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> /注释="引物"
/注释="CD28in/CD3zeta rev"
<400> 116
ctgctgaatt ttgctctggg gcggtacgct gcaa 34
<210> 117
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> /注释="引物"
/注释="CD3zeta/CD28fwd"
<400> 117
ttgcagcgta ccgccccaga gcaaaattca gcag 34
<210> 118
<211> 29
<212> DNA
<213> 人工序列
<220>
<223> /注释="引物"
/注释="CD3zetaEcoR1rev"
<400> 118
taatgaattc ttagcgaggg gccagggtc 29
<210> 119
<211> 1470
<212> DNA
<213> 人工序列
<220>
<223> CAR1 鼠 (Cripto-CD8aex/tm-CD28iz-CD3z
<400> 119
attagcggcc gcgccaccat ggaaacagat acactgctgc tgtgggtgct gctgctgtgg 60
gtgccaggat ctacagggga tggcgcccag cctgctagaa gcctgggcca tcaggaattt 120
gctcgtccat ctcggggata cctggccttc agagatgaca gcatttggcc ccaggaggag 180
cctgcaattc ggcctcggtc ttcccagcgt gtgccgccca tggggataca gcacagtaag 240
gagctaaaca gaacctgctg cctgaatggg ggaacctgca tgctggggtc cttttgtgcc 300
tgccctccct ccttctacgg acggaactgt gagcacgatg tgcgcaaaga gaactgtggg 360
tctgtgcccc atgacacctg gctgcccaag aagtgttccc tgtgtaaatg ctggcacggt 420
cagctccgct gctttcctca ggcatttcta cccggctgtg atagccagtt ccgggtgtcg 480
ccgctggatc ggacctggaa cctgggcgag acagtggagc tgaagtgcca ggtgctgctg 540
tccaacccga cgtcgggctg ctcgtggctc ttccagccgc gcggcgccgc cgccagtccc 600
accttcctcc tatacctctc ccaaaacaag cccaaggcgg ccgaggggct ggacacccag 660
cggttctcgg gcaagaggtt gggggacacc ttcgtcctca ccctgagcga cttccgccga 720
gagaacgagg gctactattt ctgctcggcc ctgagcaact ccatcatgta cttcagccac 780
ttcgtgccgg tcttcctgcc agcgaagccc accacgacgc cagcgccgcg accaccaaca 840
ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc cagaggcgtg ccggccagcg 900
gcggggggcg cagtgcacac gagggggctg gacttcgcct gtgatatcta catctgggcg 960
cccctggccg ggacttgtgg ggtccttctc ctgtcactgg ttatcaccag gagtaagagg 1020
agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1080
aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cagagtgaag 1140
ttcagcagga gcgcagacgc ccccgcgtac cagcagggcc agaaccagct ctataacgag 1200
ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 1260
gagatggggg gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag 1320
aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 1380
aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 1440
cttcacatgc aggccctgcc ccctcgctaa 1470
<210> 120
<211> 489
<212> PRT
<213> 人工序列
<220>
<223> CAR1 鼠 (Cripto-CD8aex/tm-CD28iz-CD3z
<400> 120
Ile Ser Gly Arg Ala Thr Met Glu Thr Asp Thr Leu Leu Leu Trp Val
1 5 10 15
Leu Leu Leu Trp Val Pro Gly Ser Thr Gly Asp Gly Ala Gln Pro Ala
20 25 30
Arg Ser Leu Gly His Gln Glu Phe Ala Arg Pro Ser Arg Gly Tyr Leu
35 40 45
Ala Phe Arg Asp Asp Ser Ile Trp Pro Gln Glu Glu Pro Ala Ile Arg
50 55 60
Pro Arg Ser Ser Gln Arg Val Pro Pro Met Gly Ile Gln His Ser Lys
65 70 75 80
Glu Leu Asn Arg Thr Cys Cys Leu Asn Gly Gly Thr Cys Met Leu Gly
85 90 95
Ser Phe Cys Ala Cys Pro Pro Ser Phe Tyr Gly Arg Asn Cys Glu His
100 105 110
Asp Val Arg Lys Glu Asn Cys Gly Ser Val Pro His Asp Thr Trp Leu
115 120 125
Pro Lys Lys Cys Ser Leu Cys Lys Cys Trp His Gly Gln Leu Arg Cys
130 135 140
Phe Pro Gln Ala Phe Leu Pro Gly Cys Asp Ser Gln Phe Arg Val Ser
145 150 155 160
Pro Leu Asp Arg Thr Trp Asn Leu Gly Glu Thr Val Glu Leu Lys Cys
165 170 175
Gln Val Leu Leu Ser Asn Pro Thr Ser Gly Cys Ser Trp Leu Phe Gln
180 185 190
Pro Arg Gly Ala Ala Ala Ser Pro Thr Phe Leu Leu Tyr Leu Ser Gln
195 200 205
Asn Lys Pro Lys Ala Ala Glu Gly Leu Asp Thr Gln Arg Phe Ser Gly
210 215 220
Lys Arg Leu Gly Asp Thr Phe Val Leu Thr Leu Ser Asp Phe Arg Arg
225 230 235 240
Glu Asn Glu Gly Tyr Tyr Phe Cys Ser Ala Leu Ser Asn Ser Ile Met
245 250 255
Tyr Phe Ser His Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr
260 265 270
Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln
275 280 285
Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala
290 295 300
Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala
305 310 315 320
Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr
325 330 335
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr
340 345 350
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
355 360 365
Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser
370 375 380
Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu
385 390 395 400
Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg
405 410 415
Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln
420 425 430
Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr
435 440 445
Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp
450 455 460
Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala
465 470 475 480
Leu His Met Gln Ala Leu Pro Pro Arg
485
<210> 121
<211> 4
<212> PRT
<213> 人工序列
<220>
<223> CD28 域 (未突变)
<400> 121
Pro Tyr Ala Pro
1
<210> 122
<211> 4
<212> PRT
<213> 人工序列
<220>
<223> CD28 域 (未突变)
<400> 122
Tyr Met Asn Met
1
<210> 123
<211> 4
<212> PRT
<213> 人工序列
<220>
<223> CD28 域 (已突变)
<400> 123
Phe Met Asn Met
1
<210> 124
<211> 4
<212> PRT
<213> 人工序列
<220>
<223> CD28 域 (已突变)
<400> 124
Ala Tyr Ala Ala
1
<210> 125
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> /注释="EGFRvIII fwd"
/注释="引物"
<400> 125
agcttgctcg cggccgcgcc accatgcgac cc 32
<210> 126
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> /注释="引物"
/注释="EGFRvIII (-人 CD28) rev"
<400> 126
ccaccagcac ccaaaaggac gggatcttag gccca 35
<210> 127
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> /注释="(EGFRvIII-) 人 CD28 fwd"
/注释="引物"
<400> 127
tgggcctaag atcccgtcct tttgggtgct ggtgg 35
<210> 128
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> /注释="引物"
/注释="人 CD3z rev"
<400> 128
taatgaattc ttagcgaggg ggcagg 26
<210> 129
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> /注释="引物"
/注释="EGFRvIII fwd"
<400> 129
agcttgctcg cggccgcgcc accatgcgac cc 32
<210> 130
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> /注释="人 CD3z rev"
/注释="引物"
<400> 130
taatgaattc ttagcgaggg ggcagg 26
<210> 131
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> /注释="EGFRvIII fwd"
/注释="引物"
<400> 131
agcttgctcg cggccgcgcc accatgcgac cc 32
<210> 132
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> /注释="引物"
/注释="EGFRvIII (-人 CD28) rev"
<400> 132
ccaccagcac ccaaaaggac gggatcttag gccca 35
<210> 133
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> /注释="(EGFRvIII-) 人 CD28 fwd"
/注释="引物"
<400> 133
tgggcctaag atcccgtcct tttgggtgct ggtgg 35
<210> 134
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> /注释="人 CD28 (-人 41BB) rev"
/注释="引物"
<400> 134
ctttctgccc cgtttggagc gataggctgc ga 32
<210> 135
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> /注释="(人 CD28-) 人 41BB fwd"
/注释="引物"
<400> 135
tcgcagccta tcgctccaaa cggggcagaa ag 32
<210> 136
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> /注释="人 41BB (-人 CD3z) rev"
/注释="引物"
<400> 136
tgctgaactt cactctcagt tcacatcctc ct 32
<210> 137
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> /注释="(人 41BB-) 人 CD3z fwd"
/注释="引物"
<400> 137
ggaggatgtg aactgagagt gaagttcagc agga 34
<210> 138
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> /注释="引物"
/注释="人 CD3z rev"
<400> 138
taatgaattc ttagcgaggg ggcagg 26
<210> 139
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> /注释="引物"
/注释="EGFRvIII fwd "
<400> 139
agcttgctcg cggccgcgcc accatgcgac cc 32
<210> 140
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> /注释="引物"
/注释="人 CD3z rev"
<400> 140
taatgaattc ttagcgaggg ggcagg 26
<210> 141
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> /注释="引物"
/注释="Cripto fwd"
<400> 141
attagcggcc gcgccaccat ggaaacagat acac 34
<210> 142
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> /注释="Cripto (-人 CD8a) rev"
/注释="引物"
<400> 142
acacccggaa ctggctatca cagccgggta ga 32
<210> 143
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> /注释="(Cripto-) 人 CD8a fwd"
/注释="引物"
<400> 143
tctacccggc tgtgatagcc agttccgggt g 31
<210> 144
<211> 33
<212> DNA
<213> 人工序列
<220>
<223> /注释="人 CD8a (-人 CD28) rev"
/注释="引物"
<400> 144
ctcctcttac tcctggtgat aaccagtgac agg 33
<210> 145
<211> 37
<212> DNA
<213> 人工序列
<220>
<223> /注释="引物"
/注释="(人 CD8a-) 人 CD28 fwd"
<400> 145
cctgtcactg gttatcacca ggagtaagag gagcagg 37
<210> 146
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> /注释="人 CD3z rev"
/注释="引物"
<400> 146
taatgaattc ttagcgaggg ggcagg 26
<210> 147
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> /注释="Cripto fwd "
/注释="引物"
<400> 147
attagcggcc gcgccaccat ggaaacagat acac 34
<210> 148
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> /注释="引物"
/注释="人 CD3z rev"
<400> 148
taatgaattc ttagcgaggg ggcagg 26
<210> 149
<211> 1890
<212> DNA
<213> 人工序列
<220>
<223> 人 间皮素
<220>
<221> CDS
<222> 1..1890
<223> /翻译表=1
<400> 149
atg gcg ctg ccg acc gcg cgc ccg ctg ctg ggc agc tgc ggc acc ccg 48
Met Ala Leu Pro Thr Ala Arg Pro Leu Leu Gly Ser Cys Gly Thr Pro
1 5 10 15
gcg ctg ggc agc ctg ctg ttt ctg ctg ttt agc ctg ggc tgg gtg cag 96
Ala Leu Gly Ser Leu Leu Phe Leu Leu Phe Ser Leu Gly Trp Val Gln
20 25 30
ccg agc cgc acc ctg gcg ggc gaa acc ggc cag gaa gcg gcg ccg ctg 144
Pro Ser Arg Thr Leu Ala Gly Glu Thr Gly Gln Glu Ala Ala Pro Leu
35 40 45
gat ggc gtg ctg gcg aac ccg ccg aac att agc agc ctg agc ccg cgc 192
Asp Gly Val Leu Ala Asn Pro Pro Asn Ile Ser Ser Leu Ser Pro Arg
50 55 60
cag ctg ctg ggc ttt ccg tgc gcg gaa gtg agc ggc ctg agc acc gaa 240
Gln Leu Leu Gly Phe Pro Cys Ala Glu Val Ser Gly Leu Ser Thr Glu
65 70 75 80
cgc gtg cgc gaa ctg gcg gtg gcg ctg gcg cag aaa aac gtg aaa ctg 288
Arg Val Arg Glu Leu Ala Val Ala Leu Ala Gln Lys Asn Val Lys Leu
85 90 95
agc acc gaa cag ctg cgc tgc ctg gcg cat cgc ctg agc gaa ccg ccg 336
Ser Thr Glu Gln Leu Arg Cys Leu Ala His Arg Leu Ser Glu Pro Pro
100 105 110
gaa gat ctg gat gcg ctg ccg ctg gat ctg ctg ctg ttt ctg aac ccg 384
Glu Asp Leu Asp Ala Leu Pro Leu Asp Leu Leu Leu Phe Leu Asn Pro
115 120 125
gat gcg ttt agc ggc ccg cag gcg tgc acc cgc ttt ttt agc cgc att 432
Asp Ala Phe Ser Gly Pro Gln Ala Cys Thr Arg Phe Phe Ser Arg Ile
130 135 140
acc aaa gcg aac gtg gat ctg ctg ccg cgc ggc gcg ccg gaa cgc cag 480
Thr Lys Ala Asn Val Asp Leu Leu Pro Arg Gly Ala Pro Glu Arg Gln
145 150 155 160
cgc ctg ctg ccg gcg gcg ctg gcg tgc tgg ggc gtg cgc ggc agc ctg 528
Arg Leu Leu Pro Ala Ala Leu Ala Cys Trp Gly Val Arg Gly Ser Leu
165 170 175
ctg agc gaa gcg gat gtg cgc gcg ctg ggc ggc ctg gcg tgc gat ctg 576
Leu Ser Glu Ala Asp Val Arg Ala Leu Gly Gly Leu Ala Cys Asp Leu
180 185 190
ccg ggc cgc ttt gtg gcg gaa agc gcg gaa gtg ctg ctg ccg cgc ctg 624
Pro Gly Arg Phe Val Ala Glu Ser Ala Glu Val Leu Leu Pro Arg Leu
195 200 205
gtg agc tgc ccg ggc ccg ctg gat cag gat cag cag gaa gcg gcg cgc 672
Val Ser Cys Pro Gly Pro Leu Asp Gln Asp Gln Gln Glu Ala Ala Arg
210 215 220
gcg gcg ctg cag ggc ggc ggc ccg ccg tat ggc ccg ccg agc acc tgg 720
Ala Ala Leu Gln Gly Gly Gly Pro Pro Tyr Gly Pro Pro Ser Thr Trp
225 230 235 240
agc gtg agc acc atg gat gcg ctg cgc ggc ctg ctg ccg gtg ctg ggc 768
Ser Val Ser Thr Met Asp Ala Leu Arg Gly Leu Leu Pro Val Leu Gly
245 250 255
cag ccg att att cgc agc att ccg cag ggc att gtg gcg gcg tgg cgc 816
Gln Pro Ile Ile Arg Ser Ile Pro Gln Gly Ile Val Ala Ala Trp Arg
260 265 270
cag cgc agc agc cgc gat ccg agc tgg cgc cag ccg gaa cgc acc att 864
Gln Arg Ser Ser Arg Asp Pro Ser Trp Arg Gln Pro Glu Arg Thr Ile
275 280 285
ctg cgc ccg cgc ttt cgc cgc gaa gtg gaa aaa acc gcg tgc ccg agc 912
Leu Arg Pro Arg Phe Arg Arg Glu Val Glu Lys Thr Ala Cys Pro Ser
290 295 300
ggc aaa aaa gcg cgc gaa att gat gaa agc ctg att ttt tat aaa aaa 960
Gly Lys Lys Ala Arg Glu Ile Asp Glu Ser Leu Ile Phe Tyr Lys Lys
305 310 315 320
tgg gaa ctg gaa gcg tgc gtg gat gcg gcg ctg ctg gcg acc cag atg 1008
Trp Glu Leu Glu Ala Cys Val Asp Ala Ala Leu Leu Ala Thr Gln Met
325 330 335
gat cgc gtg aac gcg att ccg ttt acc tat gaa cag ctg gat gtg ctg 1056
Asp Arg Val Asn Ala Ile Pro Phe Thr Tyr Glu Gln Leu Asp Val Leu
340 345 350
aaa cat aaa ctg gat gaa ctg tat ccg cag ggc tat ccg gaa agc gtg 1104
Lys His Lys Leu Asp Glu Leu Tyr Pro Gln Gly Tyr Pro Glu Ser Val
355 360 365
att cag cat ctg ggc tat ctg ttt ctg aaa atg agc ccg gaa gat att 1152
Ile Gln His Leu Gly Tyr Leu Phe Leu Lys Met Ser Pro Glu Asp Ile
370 375 380
cgc aaa tgg aac gtg acc agc ctg gaa acc ctg aaa gcg ctg ctg gaa 1200
Arg Lys Trp Asn Val Thr Ser Leu Glu Thr Leu Lys Ala Leu Leu Glu
385 390 395 400
gtg aac aaa ggc cat gaa atg agc ccg cag gcg ccg cgc cgc ccg ctg 1248
Val Asn Lys Gly His Glu Met Ser Pro Gln Ala Pro Arg Arg Pro Leu
405 410 415
ccg cag gtg gcg acc ctg att gat cgc ttt gtg aaa ggc cgc ggc cag 1296
Pro Gln Val Ala Thr Leu Ile Asp Arg Phe Val Lys Gly Arg Gly Gln
420 425 430
ctg gat aaa gat acc ctg gat acc ctg acc gcg ttt tat ccg ggc tat 1344
Leu Asp Lys Asp Thr Leu Asp Thr Leu Thr Ala Phe Tyr Pro Gly Tyr
435 440 445
ctg tgc agc ctg agc ccg gaa gaa ctg agc agc gtg ccg ccg agc agc 1392
Leu Cys Ser Leu Ser Pro Glu Glu Leu Ser Ser Val Pro Pro Ser Ser
450 455 460
att tgg gcg gtg cgc ccg cag gat ctg gat acc tgc gat ccg cgc cag 1440
Ile Trp Ala Val Arg Pro Gln Asp Leu Asp Thr Cys Asp Pro Arg Gln
465 470 475 480
ctg gat gtg ctg tat ccg aaa gcg cgc ctg gcg ttt cag aac atg aac 1488
Leu Asp Val Leu Tyr Pro Lys Ala Arg Leu Ala Phe Gln Asn Met Asn
485 490 495
ggc agc gaa tat ttt gtg aaa att cag agc ttt ctg ggc ggc gcg ccg 1536
Gly Ser Glu Tyr Phe Val Lys Ile Gln Ser Phe Leu Gly Gly Ala Pro
500 505 510
acc gaa gat ctg aaa gcg ctg agc cag cag aac gtg agc atg gat ctg 1584
Thr Glu Asp Leu Lys Ala Leu Ser Gln Gln Asn Val Ser Met Asp Leu
515 520 525
gcg acc ttt atg aaa ctg cgc acc gat gcg gtg ctg ccg ctg acc gtg 1632
Ala Thr Phe Met Lys Leu Arg Thr Asp Ala Val Leu Pro Leu Thr Val
530 535 540
gcg gaa gtg cag aaa ctg ctg ggc ccg cat gtg gaa ggc ctg aaa gcg 1680
Ala Glu Val Gln Lys Leu Leu Gly Pro His Val Glu Gly Leu Lys Ala
545 550 555 560
gaa gaa cgc cat cgc ccg gtg cgc gat tgg att ctg cgc cag cgc cag 1728
Glu Glu Arg His Arg Pro Val Arg Asp Trp Ile Leu Arg Gln Arg Gln
565 570 575
gat gat ctg gat acc ctg ggc ctg ggc ctg cag ggc ggc att ccg aac 1776
Asp Asp Leu Asp Thr Leu Gly Leu Gly Leu Gln Gly Gly Ile Pro Asn
580 585 590
ggc tat ctg gtg ctg gat ctg agc atg cag gaa gcg ctg agc ggc acc 1824
Gly Tyr Leu Val Leu Asp Leu Ser Met Gln Glu Ala Leu Ser Gly Thr
595 600 605
ccg tgc ctg ctg ggc ccg ggc ccg gtg ctg acc gtg ctg gcg ctg ctg 1872
Pro Cys Leu Leu Gly Pro Gly Pro Val Leu Thr Val Leu Ala Leu Leu
610 615 620
ctg gcg agc acc ctg gcg 1890
Leu Ala Ser Thr Leu Ala
625 630
<210> 150
<211> 630
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..1890, 来自SEQ ID NO 149
<400> 150
Met Ala Leu Pro Thr Ala Arg Pro Leu Leu Gly Ser Cys Gly Thr Pro
1 5 10 15
Ala Leu Gly Ser Leu Leu Phe Leu Leu Phe Ser Leu Gly Trp Val Gln
20 25 30
Pro Ser Arg Thr Leu Ala Gly Glu Thr Gly Gln Glu Ala Ala Pro Leu
35 40 45
Asp Gly Val Leu Ala Asn Pro Pro Asn Ile Ser Ser Leu Ser Pro Arg
50 55 60
Gln Leu Leu Gly Phe Pro Cys Ala Glu Val Ser Gly Leu Ser Thr Glu
65 70 75 80
Arg Val Arg Glu Leu Ala Val Ala Leu Ala Gln Lys Asn Val Lys Leu
85 90 95
Ser Thr Glu Gln Leu Arg Cys Leu Ala His Arg Leu Ser Glu Pro Pro
100 105 110
Glu Asp Leu Asp Ala Leu Pro Leu Asp Leu Leu Leu Phe Leu Asn Pro
115 120 125
Asp Ala Phe Ser Gly Pro Gln Ala Cys Thr Arg Phe Phe Ser Arg Ile
130 135 140
Thr Lys Ala Asn Val Asp Leu Leu Pro Arg Gly Ala Pro Glu Arg Gln
145 150 155 160
Arg Leu Leu Pro Ala Ala Leu Ala Cys Trp Gly Val Arg Gly Ser Leu
165 170 175
Leu Ser Glu Ala Asp Val Arg Ala Leu Gly Gly Leu Ala Cys Asp Leu
180 185 190
Pro Gly Arg Phe Val Ala Glu Ser Ala Glu Val Leu Leu Pro Arg Leu
195 200 205
Val Ser Cys Pro Gly Pro Leu Asp Gln Asp Gln Gln Glu Ala Ala Arg
210 215 220
Ala Ala Leu Gln Gly Gly Gly Pro Pro Tyr Gly Pro Pro Ser Thr Trp
225 230 235 240
Ser Val Ser Thr Met Asp Ala Leu Arg Gly Leu Leu Pro Val Leu Gly
245 250 255
Gln Pro Ile Ile Arg Ser Ile Pro Gln Gly Ile Val Ala Ala Trp Arg
260 265 270
Gln Arg Ser Ser Arg Asp Pro Ser Trp Arg Gln Pro Glu Arg Thr Ile
275 280 285
Leu Arg Pro Arg Phe Arg Arg Glu Val Glu Lys Thr Ala Cys Pro Ser
290 295 300
Gly Lys Lys Ala Arg Glu Ile Asp Glu Ser Leu Ile Phe Tyr Lys Lys
305 310 315 320
Trp Glu Leu Glu Ala Cys Val Asp Ala Ala Leu Leu Ala Thr Gln Met
325 330 335
Asp Arg Val Asn Ala Ile Pro Phe Thr Tyr Glu Gln Leu Asp Val Leu
340 345 350
Lys His Lys Leu Asp Glu Leu Tyr Pro Gln Gly Tyr Pro Glu Ser Val
355 360 365
Ile Gln His Leu Gly Tyr Leu Phe Leu Lys Met Ser Pro Glu Asp Ile
370 375 380
Arg Lys Trp Asn Val Thr Ser Leu Glu Thr Leu Lys Ala Leu Leu Glu
385 390 395 400
Val Asn Lys Gly His Glu Met Ser Pro Gln Ala Pro Arg Arg Pro Leu
405 410 415
Pro Gln Val Ala Thr Leu Ile Asp Arg Phe Val Lys Gly Arg Gly Gln
420 425 430
Leu Asp Lys Asp Thr Leu Asp Thr Leu Thr Ala Phe Tyr Pro Gly Tyr
435 440 445
Leu Cys Ser Leu Ser Pro Glu Glu Leu Ser Ser Val Pro Pro Ser Ser
450 455 460
Ile Trp Ala Val Arg Pro Gln Asp Leu Asp Thr Cys Asp Pro Arg Gln
465 470 475 480
Leu Asp Val Leu Tyr Pro Lys Ala Arg Leu Ala Phe Gln Asn Met Asn
485 490 495
Gly Ser Glu Tyr Phe Val Lys Ile Gln Ser Phe Leu Gly Gly Ala Pro
500 505 510
Thr Glu Asp Leu Lys Ala Leu Ser Gln Gln Asn Val Ser Met Asp Leu
515 520 525
Ala Thr Phe Met Lys Leu Arg Thr Asp Ala Val Leu Pro Leu Thr Val
530 535 540
Ala Glu Val Gln Lys Leu Leu Gly Pro His Val Glu Gly Leu Lys Ala
545 550 555 560
Glu Glu Arg His Arg Pro Val Arg Asp Trp Ile Leu Arg Gln Arg Gln
565 570 575
Asp Asp Leu Asp Thr Leu Gly Leu Gly Leu Gln Gly Gly Ile Pro Asn
580 585 590
Gly Tyr Leu Val Leu Asp Leu Ser Met Gln Glu Ala Leu Ser Gly Thr
595 600 605
Pro Cys Leu Leu Gly Pro Gly Pro Val Leu Thr Val Leu Ala Leu Leu
610 615 620
Leu Ala Ser Thr Leu Ala
625 630
<210> 151
<211> 1595
<212> DNA
<213> 人工序列
<220>
<223> 人 EGFRvIII
<400> 151
ccccggcgca gcgcggccgc agcagcctcc gccccccgca cggtgtgagc gcccgacgcg 60
gccgaggcgg ccggagtccc gagctagccc cggcggccgc cgccgcccag accggacgac 120
aggccacctc gtcggcgtcc gcccgagtcc ccgcctcgcc gccaacgcca caaccaccgc 180
gcacggcccc ctgactccgt ccagtattga tcgggagagc cggagcgagc tcttcgggga 240
gcagcgatgc gaccctccgg gacggccggg gcagcgctcc tggcgctgct ggctgcgctc 300
tgcccggcga gtcgggctct ggaggaaaag aaagtttgcc aaggcacgag taacaagctc 360
acgcagttgg gcacttttga agatcatttt ctcagcctcc agaggatgtt caataactgt 420
gaggtggtcc ttgggaattt ggaaattacc tatgtgcaga ggaattatga tctttccttc 480
ttaaagacca tccaggaggt ggctggttat gtcctcattg ccctcaacac agtggagcga 540
attcctttgg aaaacctgca gatcatcaga ggaaatatgt actacgaaaa ttcctatgcc 600
ttagcagtct tatctaacta tgatgcaaat aaaaccggac tgaaggagct gcccatgaga 660
aatttacagg aaatcctgca tggcgccgtg cggttcagca acaaccctgc cctgtgcaac 720
gtggagagca tccagtggcg ggacatagtc agcagtgact ttctcagcaa catgtcgatg 780
gacttccaga accacctggg cagctgccaa aagtgtgatc caagctgtcc caatgggagc 840
tgctggggtg caggagagga gaactgccag aaactgacca aaatcatctg tgcccagcag 900
tgctccgggc gctgccgtgg caagtccccc agtgactgct gccacaacca gtgtgctgca 960
ggctgcacag gcccccggga gagcgactgc ctggtctgcc gcaaattccg agacgaagcc 1020
acgtgcaagg acacctgccc cccactcatg ctctacaacc ccaccacgta ccagatggat 1080
gtgaaccccg agggcaaata cagctttggt gccacctgcg tgaagaagtg tccccgtaat 1140
tatgtggtga cagatcacgg ctcgtgcgtc cgagcctgtg gggccgacag ctatgagatg 1200
gaggaagacg gcgtccgcaa gtgtaagaag tgcgaagggc cttgccgcaa agtgtgtaac 1260
ggaataggta ttggtgaatt taaagactca ctctccataa atgctacgaa tattaaacac 1320
ttcaaaaact gcacctccat cagtggcgat ctccacatcc tgccggtggc atttaggggt 1380
gactccttca cacatactcc tcctctggat ccacaggaac tggatattct gaaaaccgta 1440
aaggaaatca caggtttgag ctgaattatc acatgaatat aaatgggaaa tcagtgtttt 1500
agagagagaa cttttcgaca tatttcctgt tcccttggaa taaaaacatt tcttctgaaa 1560
ttttaccgtt aaaaaaaaaa aaaaaaaaaa aaaaa 1595
<210> 152
<211> 405
<212> PRT
<213> 人工序列
<220>
<223> 人 EGFRvIII
<400> 152
Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala
1 5 10 15
Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln
20 25 30
Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe
35 40 45
Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn
50 55 60
Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys
65 70 75 80
Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val
85 90 95
Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr
100 105 110
Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn
115 120 125
Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu
130 135 140
His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu
145 150 155 160
Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met
165 170 175
Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro
180 185 190
Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln
195 200 205
Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg
210 215 220
Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys
225 230 235 240
Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp
245 250 255
Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro
260 265 270
Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly
275 280 285
Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His
290 295 300
Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu
305 310 315 320
Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val
325 330 335
Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn
340 345 350
Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp
355 360 365
Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr
370 375 380
Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu
385 390 395 400
Ile Thr Gly Leu Ser
405
<210> 153
<211> 564
<212> DNA
<213> 人工序列
<220>
<223> 人 Cripto
<220>
<221> CDS
<222> 1..564
<223> /翻译表=1
<400> 153
atg gat tgc cgc aaa atg gcg cgc ttt agc tat agc gtg att tgg att 48
Met Asp Cys Arg Lys Met Ala Arg Phe Ser Tyr Ser Val Ile Trp Ile
1 5 10 15
atg gcg att agc aaa gtg ttt gaa ctg ggc ctg gtg gcg ggc ctg ggc 96
Met Ala Ile Ser Lys Val Phe Glu Leu Gly Leu Val Ala Gly Leu Gly
20 25 30
cat cag gaa ttt gcg cgc ccg agc cgc ggc tat ctg gcg ttt cgc gat 144
His Gln Glu Phe Ala Arg Pro Ser Arg Gly Tyr Leu Ala Phe Arg Asp
35 40 45
gat agc att tgg ccg cag gaa gaa ccg gcg att cgc ccg cgc agc agc 192
Asp Ser Ile Trp Pro Gln Glu Glu Pro Ala Ile Arg Pro Arg Ser Ser
50 55 60
cag cgc gtg ccg ccg atg ggc att cag cat agc aaa gaa ctg aac cgc 240
Gln Arg Val Pro Pro Met Gly Ile Gln His Ser Lys Glu Leu Asn Arg
65 70 75 80
acc tgc tgc ctg aac ggc ggc acc tgc atg ctg ggc agc ttt tgc gcg 288
Thr Cys Cys Leu Asn Gly Gly Thr Cys Met Leu Gly Ser Phe Cys Ala
85 90 95
tgc ccg ccg agc ttt tat ggc cgc aac tgc gaa cat gat gtg cgc aaa 336
Cys Pro Pro Ser Phe Tyr Gly Arg Asn Cys Glu His Asp Val Arg Lys
100 105 110
gaa aac tgc ggc agc gtg ccg cat gat acc tgg ctg ccg aaa aaa tgc 384
Glu Asn Cys Gly Ser Val Pro His Asp Thr Trp Leu Pro Lys Lys Cys
115 120 125
agc ctg tgc aaa tgc tgg cat ggc cag ctg cgc tgc ttt ccg cag gcg 432
Ser Leu Cys Lys Cys Trp His Gly Gln Leu Arg Cys Phe Pro Gln Ala
130 135 140
ttt ctg ccg ggc tgc gat ggc ctg gtg atg gat gaa cat ctg gtg gcg 480
Phe Leu Pro Gly Cys Asp Gly Leu Val Met Asp Glu His Leu Val Ala
145 150 155 160
agc cgc acc ccg gaa ctg ccg ccg agc gcg cgc acc acc acc ttt atg 528
Ser Arg Thr Pro Glu Leu Pro Pro Ser Ala Arg Thr Thr Thr Phe Met
165 170 175
ctg gtg ggc att tgc ctg agc att cag agc tat tat 564
Leu Val Gly Ile Cys Leu Ser Ile Gln Ser Tyr Tyr
180 185
<210> 154
<211> 188
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..564, 来自SEQ ID NO 153
<400> 154
Met Asp Cys Arg Lys Met Ala Arg Phe Ser Tyr Ser Val Ile Trp Ile
1 5 10 15
Met Ala Ile Ser Lys Val Phe Glu Leu Gly Leu Val Ala Gly Leu Gly
20 25 30
His Gln Glu Phe Ala Arg Pro Ser Arg Gly Tyr Leu Ala Phe Arg Asp
35 40 45
Asp Ser Ile Trp Pro Gln Glu Glu Pro Ala Ile Arg Pro Arg Ser Ser
50 55 60
Gln Arg Val Pro Pro Met Gly Ile Gln His Ser Lys Glu Leu Asn Arg
65 70 75 80
Thr Cys Cys Leu Asn Gly Gly Thr Cys Met Leu Gly Ser Phe Cys Ala
85 90 95
Cys Pro Pro Ser Phe Tyr Gly Arg Asn Cys Glu His Asp Val Arg Lys
100 105 110
Glu Asn Cys Gly Ser Val Pro His Asp Thr Trp Leu Pro Lys Lys Cys
115 120 125
Ser Leu Cys Lys Cys Trp His Gly Gln Leu Arg Cys Phe Pro Gln Ala
130 135 140
Phe Leu Pro Gly Cys Asp Gly Leu Val Met Asp Glu His Leu Val Ala
145 150 155 160
Ser Arg Thr Pro Glu Leu Pro Pro Ser Ala Arg Thr Thr Thr Phe Met
165 170 175
Leu Val Gly Ile Cys Leu Ser Ile Gln Ser Tyr Tyr
180 185
<210> 155
<211> 660
<212> DNA
<213> 人工序列
<220>
<223> 人 CD28
<220>
<221> CDS
<222> 1..660
<223> /翻译表=1
<400> 155
atg ctg cgc ctg ctg ctg gcg ctg aac ctg ttt ccg agc att cag gtg 48
Met Leu Arg Leu Leu Leu Ala Leu Asn Leu Phe Pro Ser Ile Gln Val
1 5 10 15
acc ggc aac aaa att ctg gtg aaa cag agc ccg atg ctg gtg gcg tat 96
Thr Gly Asn Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr
20 25 30
gat aac gcg gtg aac ctg agc tgc aaa tat agc tat aac ctg ttt agc 144
Asp Asn Ala Val Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser
35 40 45
cgc gaa ttt cgc gcg agc ctg cat aaa ggc ctg gat agc gcg gtg gaa 192
Arg Glu Phe Arg Ala Ser Leu His Lys Gly Leu Asp Ser Ala Val Glu
50 55 60
gtg tgc gtg gtg tat ggc aac tat agc cag cag ctg cag gtg tat agc 240
Val Cys Val Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser
65 70 75 80
aaa acc ggc ttt aac tgc gat ggc aaa ctg ggc aac gaa agc gtg acc 288
Lys Thr Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr
85 90 95
ttt tat ctg cag aac ctg tat gtg aac cag acc gat att tat ttt tgc 336
Phe Tyr Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys
100 105 110
aaa att gaa gtg atg tat ccg ccg ccg tat ctg gat aac gaa aaa agc 384
Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser
115 120 125
aac ggc acc att att cat gtg aaa ggc aaa cat ctg tgc ccg agc ccg 432
Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro
130 135 140
ctg ttt ccg ggc ccg agc aaa ccg ttt tgg gtg ctg gtg gtg gtg ggc 480
Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly
145 150 155 160
ggc gtg ctg gcg tgc tat agc ctg ctg gtg acc gtg gcg ttt att att 528
Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile
165 170 175
ttt tgg gtg cgc agc aaa cgc agc cgc ctg ctg cat agc gat tat atg 576
Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met
180 185 190
aac atg acc ccg cgc cgc ccg ggc ccg acc cgc aaa cat tat cag ccg 624
Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro
195 200 205
tat gcg ccg ccg cgc gat ttt gcg gcg tat cgc agc 660
Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser
210 215 220
<210> 156
<211> 220
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..660, 来自SEQ ID NO 155
<400> 156
Met Leu Arg Leu Leu Leu Ala Leu Asn Leu Phe Pro Ser Ile Gln Val
1 5 10 15
Thr Gly Asn Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr
20 25 30
Asp Asn Ala Val Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser
35 40 45
Arg Glu Phe Arg Ala Ser Leu His Lys Gly Leu Asp Ser Ala Val Glu
50 55 60
Val Cys Val Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser
65 70 75 80
Lys Thr Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr
85 90 95
Phe Tyr Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys
100 105 110
Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser
115 120 125
Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro
130 135 140
Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly
145 150 155 160
Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile
165 170 175
Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met
180 185 190
Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro
195 200 205
Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser
210 215 220
<210> 157
<211> 654
<212> DNA
<213> 人工序列
<220>
<223> 鼠 CD28
<220>
<221> CDS
<222> 1..654
<223> /翻译表=1
<400> 157
atg acc ctg cgc ctg ctg ttt ctg gcg ctg aac ttt ttt agc gtg cag 48
Met Thr Leu Arg Leu Leu Phe Leu Ala Leu Asn Phe Phe Ser Val Gln
1 5 10 15
gtg acc gaa aac aaa att ctg gtg aaa cag agc ccg ctg ctg gtg gtg 96
Val Thr Glu Asn Lys Ile Leu Val Lys Gln Ser Pro Leu Leu Val Val
20 25 30
gat agc aac gaa gtg agc ctg agc tgc cgc tat agc tat aac ctg ctg 144
Asp Ser Asn Glu Val Ser Leu Ser Cys Arg Tyr Ser Tyr Asn Leu Leu
35 40 45
gcg aaa gaa ttt cgc gcg agc ctg tat aaa ggc gtg aac agc gat gtg 192
Ala Lys Glu Phe Arg Ala Ser Leu Tyr Lys Gly Val Asn Ser Asp Val
50 55 60
gaa gtg tgc gtg ggc aac ggc aac ttt acc tat cag ccg cag ttt cgc 240
Glu Val Cys Val Gly Asn Gly Asn Phe Thr Tyr Gln Pro Gln Phe Arg
65 70 75 80
agc aac gcg gaa ttt aac tgc gat ggc gat ttt gat aac gaa acc gtg 288
Ser Asn Ala Glu Phe Asn Cys Asp Gly Asp Phe Asp Asn Glu Thr Val
85 90 95
acc ttt cgc ctg tgg aac ctg cat gtg aac cat acc gat att tat ttt 336
Thr Phe Arg Leu Trp Asn Leu His Val Asn His Thr Asp Ile Tyr Phe
100 105 110
tgc aaa att gaa ttt atg tat ccg ccg ccg tat ctg gat aac gaa cgc 384
Cys Lys Ile Glu Phe Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Arg
115 120 125
agc aac ggc acc att att cat att aaa gaa aaa cat ctg tgc cat acc 432
Ser Asn Gly Thr Ile Ile His Ile Lys Glu Lys His Leu Cys His Thr
130 135 140
cag agc agc ccg aaa ctg ttt tgg gcg ctg gtg gtg gtg gcg ggc gtg 480
Gln Ser Ser Pro Lys Leu Phe Trp Ala Leu Val Val Val Ala Gly Val
145 150 155 160
ctg ttt tgc tat ggc ctg ctg gtg acc gtg gcg ctg tgc gtg att tgg 528
Leu Phe Cys Tyr Gly Leu Leu Val Thr Val Ala Leu Cys Val Ile Trp
165 170 175
acc aac agc cgc cgc aac cgc ctg ctg cag agc gat tat atg aac atg 576
Thr Asn Ser Arg Arg Asn Arg Leu Leu Gln Ser Asp Tyr Met Asn Met
180 185 190
acc ccg cgc cgc ccg ggc ctg acc cgc aaa ccg tat cag ccg tat gcg 624
Thr Pro Arg Arg Pro Gly Leu Thr Arg Lys Pro Tyr Gln Pro Tyr Ala
195 200 205
ccg gcg cgc gat ttt gcg gcg tat cgc ccg 654
Pro Ala Arg Asp Phe Ala Ala Tyr Arg Pro
210 215
<210> 158
<211> 218
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..654, 来自SEQ ID NO 157
<400> 158
Met Thr Leu Arg Leu Leu Phe Leu Ala Leu Asn Phe Phe Ser Val Gln
1 5 10 15
Val Thr Glu Asn Lys Ile Leu Val Lys Gln Ser Pro Leu Leu Val Val
20 25 30
Asp Ser Asn Glu Val Ser Leu Ser Cys Arg Tyr Ser Tyr Asn Leu Leu
35 40 45
Ala Lys Glu Phe Arg Ala Ser Leu Tyr Lys Gly Val Asn Ser Asp Val
50 55 60
Glu Val Cys Val Gly Asn Gly Asn Phe Thr Tyr Gln Pro Gln Phe Arg
65 70 75 80
Ser Asn Ala Glu Phe Asn Cys Asp Gly Asp Phe Asp Asn Glu Thr Val
85 90 95
Thr Phe Arg Leu Trp Asn Leu His Val Asn His Thr Asp Ile Tyr Phe
100 105 110
Cys Lys Ile Glu Phe Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Arg
115 120 125
Ser Asn Gly Thr Ile Ile His Ile Lys Glu Lys His Leu Cys His Thr
130 135 140
Gln Ser Ser Pro Lys Leu Phe Trp Ala Leu Val Val Val Ala Gly Val
145 150 155 160
Leu Phe Cys Tyr Gly Leu Leu Val Thr Val Ala Leu Cys Val Ile Trp
165 170 175
Thr Asn Ser Arg Arg Asn Arg Leu Leu Gln Ser Asp Tyr Met Asn Met
180 185 190
Thr Pro Arg Arg Pro Gly Leu Thr Arg Lys Pro Tyr Gln Pro Tyr Ala
195 200 205
Pro Ala Arg Asp Phe Ala Ala Tyr Arg Pro
210 215
<210> 159
<211> 765
<212> DNA
<213> 人工序列
<220>
<223> 人 CD137
<220>
<221> CDS
<222> 1..765
<223> /翻译表=1
<400> 159
atg ggc aac agc tgc tat aac att gtg gcg acc ctg ctg ctg gtg ctg 48
Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu
1 5 10 15
aac ttt gaa cgc acc cgc agc ctg cag gat ccg tgc agc aac tgc ccg 96
Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro
20 25 30
gcg ggc acc ttt tgc gat aac aac cgc aac cag att tgc agc ccg tgc 144
Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys
35 40 45
ccg ccg aac agc ttt agc agc gcg ggc ggc cag cgc acc tgc gat att 192
Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile
50 55 60
tgc cgc cag tgc aaa ggc gtg ttt cgc acc cgc aaa gaa tgc agc agc 240
Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser
65 70 75 80
acc agc aac gcg gaa tgc gat tgc acc ccg ggc ttt cat tgc ctg ggc 288
Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly
85 90 95
gcg ggc tgc agc atg tgc gaa cag gat tgc aaa cag ggc cag gaa ctg 336
Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu
100 105 110
acc aaa aaa ggc tgc aaa gat tgc tgc ttt ggc acc ttt aac gat cag 384
Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln
115 120 125
aaa cgc ggc att tgc cgc ccg tgg acc aac tgc agc ctg gat ggc aaa 432
Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys
130 135 140
agc gtg ctg gtg aac ggc acc aaa gaa cgc gat gtg gtg tgc ggc ccg 480
Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro
145 150 155 160
agc ccg gcg gat ctg agc ccg ggc gcg agc agc gtg acc ccg ccg gcg 528
Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala
165 170 175
ccg gcg cgc gaa ccg ggc cat agc ccg cag att att agc ttt ttt ctg 576
Pro Ala Arg Glu Pro Gly His Ser Pro Gln Ile Ile Ser Phe Phe Leu
180 185 190
gcg ctg acc agc acc gcg ctg ctg ttt ctg ctg ttt ttt ctg acc ctg 624
Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu
195 200 205
cgc ttt agc gtg gtg aaa cgc ggc cgc aaa aaa ctg ctg tat att ttt 672
Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe
210 215 220
aaa cag ccg ttt atg cgc ccg gtg cag acc acc cag gaa gaa gat ggc 720
Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly
225 230 235 240
tgc agc tgc cgc ttt ccg gaa gaa gaa gaa ggc ggc tgc gaa ctg 765
Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
245 250 255
<210> 160
<211> 255
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..765, 来自SEQ ID NO 159
<400> 160
Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu
1 5 10 15
Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro
20 25 30
Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys
35 40 45
Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile
50 55 60
Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser
65 70 75 80
Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly
85 90 95
Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu
100 105 110
Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln
115 120 125
Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys
130 135 140
Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro
145 150 155 160
Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala
165 170 175
Pro Ala Arg Glu Pro Gly His Ser Pro Gln Ile Ile Ser Phe Phe Leu
180 185 190
Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu
195 200 205
Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe
210 215 220
Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly
225 230 235 240
Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
245 250 255
<210> 161
<211> 768
<212> DNA
<213> 人工序列
<220>
<223> 鼠 CD137
<220>
<221> CDS
<222> 1..768
<223> /翻译表=1
<400> 161
atg ggc aac aac tgc tat aac gtg gtg gtg att gtg ctg ctg ctg gtg 48
Met Gly Asn Asn Cys Tyr Asn Val Val Val Ile Val Leu Leu Leu Val
1 5 10 15
ggc tgc gaa aaa gtg ggc gcg gtg cag aac agc tgc gat aac tgc cag 96
Gly Cys Glu Lys Val Gly Ala Val Gln Asn Ser Cys Asp Asn Cys Gln
20 25 30
ccg ggc acc ttt tgc cgc aaa tat aac ccg gtg tgc aaa agc tgc ccg 144
Pro Gly Thr Phe Cys Arg Lys Tyr Asn Pro Val Cys Lys Ser Cys Pro
35 40 45
ccg agc acc ttt agc agc att ggc ggc cag ccg aac tgc aac att tgc 192
Pro Ser Thr Phe Ser Ser Ile Gly Gly Gln Pro Asn Cys Asn Ile Cys
50 55 60
cgc gtg tgc gcg ggc tat ttt cgc ttt aaa aaa ttt tgc agc agc acc 240
Arg Val Cys Ala Gly Tyr Phe Arg Phe Lys Lys Phe Cys Ser Ser Thr
65 70 75 80
cat aac gcg gaa tgc gaa tgc att gaa ggc ttt cat tgc ctg ggc ccg 288
His Asn Ala Glu Cys Glu Cys Ile Glu Gly Phe His Cys Leu Gly Pro
85 90 95
cag tgc acc cgc tgc gaa aaa gat tgc cgc ccg ggc cag gaa ctg acc 336
Gln Cys Thr Arg Cys Glu Lys Asp Cys Arg Pro Gly Gln Glu Leu Thr
100 105 110
aaa cag ggc tgc aaa acc tgc agc ctg ggc acc ttt aac gat cag aac 384
Lys Gln Gly Cys Lys Thr Cys Ser Leu Gly Thr Phe Asn Asp Gln Asn
115 120 125
ggc acc ggc gtg tgc cgc ccg tgg acc aac tgc agc ctg gat ggc cgc 432
Gly Thr Gly Val Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Arg
130 135 140
agc gtg ctg aaa acc ggc acc acc gaa aaa gat gtg gtg tgc ggc ccg 480
Ser Val Leu Lys Thr Gly Thr Thr Glu Lys Asp Val Val Cys Gly Pro
145 150 155 160
ccg gtg gtg agc ttt agc ccg agc acc acc att agc gtg acc ccg gaa 528
Pro Val Val Ser Phe Ser Pro Ser Thr Thr Ile Ser Val Thr Pro Glu
165 170 175
ggc ggc ccg ggc ggc cat agc ctg cag gtg ctg acc ctg ttt ctg gcg 576
Gly Gly Pro Gly Gly His Ser Leu Gln Val Leu Thr Leu Phe Leu Ala
180 185 190
ctg acc agc gcg ctg ctg ctg gcg ctg att ttt att acc ctg ctg ttt 624
Leu Thr Ser Ala Leu Leu Leu Ala Leu Ile Phe Ile Thr Leu Leu Phe
195 200 205
agc gtg ctg aaa tgg att cgc aaa aaa ttt ccg cat att ttt aaa cag 672
Ser Val Leu Lys Trp Ile Arg Lys Lys Phe Pro His Ile Phe Lys Gln
210 215 220
ccg ttt aaa aaa acc acc ggc gcg gcg cag gaa gaa gat gcg tgc agc 720
Pro Phe Lys Lys Thr Thr Gly Ala Ala Gln Glu Glu Asp Ala Cys Ser
225 230 235 240
tgc cgc tgc ccg cag gaa gaa gaa ggc ggc ggc ggc ggc tat gaa ctg 768
Cys Arg Cys Pro Gln Glu Glu Glu Gly Gly Gly Gly Gly Tyr Glu Leu
245 250 255
<210> 162
<211> 256
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..768, 来自SEQ ID NO 161
<400> 162
Met Gly Asn Asn Cys Tyr Asn Val Val Val Ile Val Leu Leu Leu Val
1 5 10 15
Gly Cys Glu Lys Val Gly Ala Val Gln Asn Ser Cys Asp Asn Cys Gln
20 25 30
Pro Gly Thr Phe Cys Arg Lys Tyr Asn Pro Val Cys Lys Ser Cys Pro
35 40 45
Pro Ser Thr Phe Ser Ser Ile Gly Gly Gln Pro Asn Cys Asn Ile Cys
50 55 60
Arg Val Cys Ala Gly Tyr Phe Arg Phe Lys Lys Phe Cys Ser Ser Thr
65 70 75 80
His Asn Ala Glu Cys Glu Cys Ile Glu Gly Phe His Cys Leu Gly Pro
85 90 95
Gln Cys Thr Arg Cys Glu Lys Asp Cys Arg Pro Gly Gln Glu Leu Thr
100 105 110
Lys Gln Gly Cys Lys Thr Cys Ser Leu Gly Thr Phe Asn Asp Gln Asn
115 120 125
Gly Thr Gly Val Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Arg
130 135 140
Ser Val Leu Lys Thr Gly Thr Thr Glu Lys Asp Val Val Cys Gly Pro
145 150 155 160
Pro Val Val Ser Phe Ser Pro Ser Thr Thr Ile Ser Val Thr Pro Glu
165 170 175
Gly Gly Pro Gly Gly His Ser Leu Gln Val Leu Thr Leu Phe Leu Ala
180 185 190
Leu Thr Ser Ala Leu Leu Leu Ala Leu Ile Phe Ile Thr Leu Leu Phe
195 200 205
Ser Val Leu Lys Trp Ile Arg Lys Lys Phe Pro His Ile Phe Lys Gln
210 215 220
Pro Phe Lys Lys Thr Thr Gly Ala Ala Gln Glu Glu Asp Ala Cys Ser
225 230 235 240
Cys Arg Cys Pro Gln Glu Glu Glu Gly Gly Gly Gly Gly Tyr Glu Leu
245 250 255
<210> 163
<211> 831
<212> DNA
<213> 人工序列
<220>
<223> 人 OX40
<220>
<221> CDS
<222> 1..831
<223> /翻译表=1
<400> 163
atg tgc gtg ggc gcg cgc cgc ctg ggc cgc ggc ccg tgc gcg gcg ctg 48
Met Cys Val Gly Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu
1 5 10 15
ctg ctg ctg ggc ctg ggc ctg agc acc gtg acc ggc ctg cat tgc gtg 96
Leu Leu Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Leu His Cys Val
20 25 30
ggc gat acc tat ccg agc aac gat cgc tgc tgc cat gaa tgc cgc ccg 144
Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His Glu Cys Arg Pro
35 40 45
ggc aac ggc atg gtg agc cgc tgc agc cgc agc cag aac acc gtg tgc 192
Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln Asn Thr Val Cys
50 55 60
cgc ccg tgc ggc ccg ggc ttt tat aac gat gtg gtg agc agc aaa ccg 240
Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val Ser Ser Lys Pro
65 70 75 80
tgc aaa ccg tgc acc tgg tgc aac ctg cgc agc ggc agc gaa cgc aaa 288
Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly Ser Glu Arg Lys
85 90 95
cag ctg tgc acc gcg acc cag gat acc gtg tgc cgc tgc cgc gcg ggc 336
Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg Cys Arg Ala Gly
100 105 110
acc cag ccg ctg gat agc tat aaa ccg ggc gtg gat tgc gcg ccg tgc 384
Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp Cys Ala Pro Cys
115 120 125
ccg ccg ggc cat ttt agc ccg ggc gat aac cag gcg tgc aaa ccg tgg 432
Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala Cys Lys Pro Trp
130 135 140
acc aac tgc acc ctg gcg ggc aaa cat acc ctg cag ccg gcg agc aac 480
Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln Pro Ala Ser Asn
145 150 155 160
agc agc gat gcg att tgc gaa gat cgc gat ccg ccg gcg acc cag ccg 528
Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro Ala Thr Gln Pro
165 170 175
cag gaa acc cag ggc ccg ccg gcg cgc ccg att acc gtg cag ccg acc 576
Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr Val Gln Pro Thr
180 185 190
gaa gcg tgg ccg cgc acc agc cag ggc ccg agc acc cgc ccg gtg gaa 624
Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr Arg Pro Val Glu
195 200 205
gtg ccg ggc ggc cgc gcg gtg gcg gcg att ctg ggc ctg ggc ctg gtg 672
Val Pro Gly Gly Arg Ala Val Ala Ala Ile Leu Gly Leu Gly Leu Val
210 215 220
ctg ggc ctg ctg ggc ccg ctg gcg att ctg ctg gcg ctg tat ctg ctg 720
Leu Gly Leu Leu Gly Pro Leu Ala Ile Leu Leu Ala Leu Tyr Leu Leu
225 230 235 240
cgc cgc gat cag cgc ctg ccg ccg gat gcg cat aaa ccg ccg ggc ggc 768
Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly
245 250 255
ggc agc ttt cgc acc ccg att cag gaa gaa cag gcg gat gcg cat agc 816
Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser
260 265 270
acc ctg gcg aaa att 831
Thr Leu Ala Lys Ile
275
<210> 164
<211> 277
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..831, 来自SEQ ID NO 163
<400> 164
Met Cys Val Gly Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu
1 5 10 15
Leu Leu Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Leu His Cys Val
20 25 30
Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His Glu Cys Arg Pro
35 40 45
Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln Asn Thr Val Cys
50 55 60
Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val Ser Ser Lys Pro
65 70 75 80
Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly Ser Glu Arg Lys
85 90 95
Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg Cys Arg Ala Gly
100 105 110
Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp Cys Ala Pro Cys
115 120 125
Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala Cys Lys Pro Trp
130 135 140
Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln Pro Ala Ser Asn
145 150 155 160
Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro Ala Thr Gln Pro
165 170 175
Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr Val Gln Pro Thr
180 185 190
Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr Arg Pro Val Glu
195 200 205
Val Pro Gly Gly Arg Ala Val Ala Ala Ile Leu Gly Leu Gly Leu Val
210 215 220
Leu Gly Leu Leu Gly Pro Leu Ala Ile Leu Leu Ala Leu Tyr Leu Leu
225 230 235 240
Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly
245 250 255
Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser
260 265 270
Thr Leu Ala Lys Ile
275
<210> 165
<211> 816
<212> DNA
<213> 人工序列
<220>
<223> 鼠 OX40
<220>
<221> CDS
<222> 1..816
<223> /翻译表=1
<400> 165
atg tat gtg tgg gtg cag cag ccg acc gcg ctg ctg ctg ctg gcg ctg 48
Met Tyr Val Trp Val Gln Gln Pro Thr Ala Leu Leu Leu Leu Ala Leu
1 5 10 15
acc ctg ggc gtg acc gcg cgc cgc ctg aac tgc gtg aaa cat acc tat 96
Thr Leu Gly Val Thr Ala Arg Arg Leu Asn Cys Val Lys His Thr Tyr
20 25 30
ccg agc ggc cat aaa tgc tgc cgc gaa tgc cag ccg ggc cat ggc atg 144
Pro Ser Gly His Lys Cys Cys Arg Glu Cys Gln Pro Gly His Gly Met
35 40 45
gtg agc cgc tgc gat cat acc cgc gat acc ctg tgc cat ccg tgc gaa 192
Val Ser Arg Cys Asp His Thr Arg Asp Thr Leu Cys His Pro Cys Glu
50 55 60
acc ggc ttt tat aac gaa gcg gtg aac tat gat acc tgc aaa cag tgc 240
Thr Gly Phe Tyr Asn Glu Ala Val Asn Tyr Asp Thr Cys Lys Gln Cys
65 70 75 80
acc cag tgc aac cat cgc agc ggc agc gaa ctg aaa cag aac tgc acc 288
Thr Gln Cys Asn His Arg Ser Gly Ser Glu Leu Lys Gln Asn Cys Thr
85 90 95
ccg acc cag gat acc gtg tgc cgc tgc cgc ccg ggc acc cag ccg cgc 336
Pro Thr Gln Asp Thr Val Cys Arg Cys Arg Pro Gly Thr Gln Pro Arg
100 105 110
cag gat agc ggc tat aaa ctg ggc gtg gat tgc gtg ccg tgc ccg ccg 384
Gln Asp Ser Gly Tyr Lys Leu Gly Val Asp Cys Val Pro Cys Pro Pro
115 120 125
ggc cat ttt agc ccg ggc aac aac cag gcg tgc aaa ccg tgg acc aac 432
Gly His Phe Ser Pro Gly Asn Asn Gln Ala Cys Lys Pro Trp Thr Asn
130 135 140
tgc acc ctg agc ggc aaa cag acc cgc cat ccg gcg agc gat agc ctg 480
Cys Thr Leu Ser Gly Lys Gln Thr Arg His Pro Ala Ser Asp Ser Leu
145 150 155 160
gat gcg gtg tgc gaa gat cgc agc ctg ctg gcg acc ctg ctg tgg gaa 528
Asp Ala Val Cys Glu Asp Arg Ser Leu Leu Ala Thr Leu Leu Trp Glu
165 170 175
acc cag cgc ccg acc ttt cgc ccg acc acc gtg cag agc acc acc gtg 576
Thr Gln Arg Pro Thr Phe Arg Pro Thr Thr Val Gln Ser Thr Thr Val
180 185 190
tgg ccg cgc acc agc gaa ctg ccg agc ccg ccg acc ctg gtg acc ccg 624
Trp Pro Arg Thr Ser Glu Leu Pro Ser Pro Pro Thr Leu Val Thr Pro
195 200 205
gaa ggc ccg gcg ttt gcg gtg ctg ctg ggc ctg ggc ctg ggc ctg ctg 672
Glu Gly Pro Ala Phe Ala Val Leu Leu Gly Leu Gly Leu Gly Leu Leu
210 215 220
gcg ccg ctg acc gtg ctg ctg gcg ctg tat ctg ctg cgc aaa gcg tgg 720
Ala Pro Leu Thr Val Leu Leu Ala Leu Tyr Leu Leu Arg Lys Ala Trp
225 230 235 240
cgc ctg ccg aac acc ccg aaa ccg tgc tgg ggc aac agc ttt cgc acc 768
Arg Leu Pro Asn Thr Pro Lys Pro Cys Trp Gly Asn Ser Phe Arg Thr
245 250 255
ccg att cag gaa gaa cat acc gat gcg cat ttt acc ctg gcg aaa att 816
Pro Ile Gln Glu Glu His Thr Asp Ala His Phe Thr Leu Ala Lys Ile
260 265 270
<210> 166
<211> 272
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..816, 来自SEQ ID NO 165
<400> 166
Met Tyr Val Trp Val Gln Gln Pro Thr Ala Leu Leu Leu Leu Ala Leu
1 5 10 15
Thr Leu Gly Val Thr Ala Arg Arg Leu Asn Cys Val Lys His Thr Tyr
20 25 30
Pro Ser Gly His Lys Cys Cys Arg Glu Cys Gln Pro Gly His Gly Met
35 40 45
Val Ser Arg Cys Asp His Thr Arg Asp Thr Leu Cys His Pro Cys Glu
50 55 60
Thr Gly Phe Tyr Asn Glu Ala Val Asn Tyr Asp Thr Cys Lys Gln Cys
65 70 75 80
Thr Gln Cys Asn His Arg Ser Gly Ser Glu Leu Lys Gln Asn Cys Thr
85 90 95
Pro Thr Gln Asp Thr Val Cys Arg Cys Arg Pro Gly Thr Gln Pro Arg
100 105 110
Gln Asp Ser Gly Tyr Lys Leu Gly Val Asp Cys Val Pro Cys Pro Pro
115 120 125
Gly His Phe Ser Pro Gly Asn Asn Gln Ala Cys Lys Pro Trp Thr Asn
130 135 140
Cys Thr Leu Ser Gly Lys Gln Thr Arg His Pro Ala Ser Asp Ser Leu
145 150 155 160
Asp Ala Val Cys Glu Asp Arg Ser Leu Leu Ala Thr Leu Leu Trp Glu
165 170 175
Thr Gln Arg Pro Thr Phe Arg Pro Thr Thr Val Gln Ser Thr Thr Val
180 185 190
Trp Pro Arg Thr Ser Glu Leu Pro Ser Pro Pro Thr Leu Val Thr Pro
195 200 205
Glu Gly Pro Ala Phe Ala Val Leu Leu Gly Leu Gly Leu Gly Leu Leu
210 215 220
Ala Pro Leu Thr Val Leu Leu Ala Leu Tyr Leu Leu Arg Lys Ala Trp
225 230 235 240
Arg Leu Pro Asn Thr Pro Lys Pro Cys Trp Gly Asn Ser Phe Arg Thr
245 250 255
Pro Ile Gln Glu Glu His Thr Asp Ala His Phe Thr Leu Ala Lys Ile
260 265 270
<210> 167
<211> 597
<212> DNA
<213> 人工序列
<220>
<223> 人 ICOS
<220>
<221> CDS
<222> 1..597
<223> /翻译表=1
<400> 167
atg aaa agc ggc ctg tgg tat ttt ttt ctg ttt tgc ctg cgc att aaa 48
Met Lys Ser Gly Leu Trp Tyr Phe Phe Leu Phe Cys Leu Arg Ile Lys
1 5 10 15
gtg ctg acc ggc gaa att aac ggc agc gcg aac tat gaa atg ttt att 96
Val Leu Thr Gly Glu Ile Asn Gly Ser Ala Asn Tyr Glu Met Phe Ile
20 25 30
ttt cat aac ggc ggc gtg cag att ctg tgc aaa tat ccg gat att gtg 144
Phe His Asn Gly Gly Val Gln Ile Leu Cys Lys Tyr Pro Asp Ile Val
35 40 45
cag cag ttt aaa atg cag ctg ctg aaa ggc ggc cag att ctg tgc gat 192
Gln Gln Phe Lys Met Gln Leu Leu Lys Gly Gly Gln Ile Leu Cys Asp
50 55 60
ctg acc aaa acc aaa ggc agc ggc aac acc gtg agc att aaa agc ctg 240
Leu Thr Lys Thr Lys Gly Ser Gly Asn Thr Val Ser Ile Lys Ser Leu
65 70 75 80
aaa ttt tgc cat agc cag ctg agc aac aac agc gtg agc ttt ttt ctg 288
Lys Phe Cys His Ser Gln Leu Ser Asn Asn Ser Val Ser Phe Phe Leu
85 90 95
tat aac ctg gat cat agc cat gcg aac tat tat ttt tgc aac ctg agc 336
Tyr Asn Leu Asp His Ser His Ala Asn Tyr Tyr Phe Cys Asn Leu Ser
100 105 110
att ttt gat ccg ccg ccg ttt aaa gtg acc ctg acc ggc ggc tat ctg 384
Ile Phe Asp Pro Pro Pro Phe Lys Val Thr Leu Thr Gly Gly Tyr Leu
115 120 125
cat att tat gaa agc cag ctg tgc tgc cag ctg aaa ttt tgg ctg ccg 432
His Ile Tyr Glu Ser Gln Leu Cys Cys Gln Leu Lys Phe Trp Leu Pro
130 135 140
att ggc tgc gcg gcg ttt gtg gtg gtg tgc att ctg ggc tgc att ctg 480
Ile Gly Cys Ala Ala Phe Val Val Val Cys Ile Leu Gly Cys Ile Leu
145 150 155 160
att tgc tgg ctg acc aaa aaa aaa tat agc agc agc gtg cat gat ccg 528
Ile Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro
165 170 175
aac ggc gaa tat atg ttt atg cgc gcg gtg aac acc gcg aaa aaa agc 576
Asn Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser
180 185 190
cgc ctg acc gat gtg acc ctg 597
Arg Leu Thr Asp Val Thr Leu
195
<210> 168
<211> 199
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..597, 来自SEQ ID NO 167
<400> 168
Met Lys Ser Gly Leu Trp Tyr Phe Phe Leu Phe Cys Leu Arg Ile Lys
1 5 10 15
Val Leu Thr Gly Glu Ile Asn Gly Ser Ala Asn Tyr Glu Met Phe Ile
20 25 30
Phe His Asn Gly Gly Val Gln Ile Leu Cys Lys Tyr Pro Asp Ile Val
35 40 45
Gln Gln Phe Lys Met Gln Leu Leu Lys Gly Gly Gln Ile Leu Cys Asp
50 55 60
Leu Thr Lys Thr Lys Gly Ser Gly Asn Thr Val Ser Ile Lys Ser Leu
65 70 75 80
Lys Phe Cys His Ser Gln Leu Ser Asn Asn Ser Val Ser Phe Phe Leu
85 90 95
Tyr Asn Leu Asp His Ser His Ala Asn Tyr Tyr Phe Cys Asn Leu Ser
100 105 110
Ile Phe Asp Pro Pro Pro Phe Lys Val Thr Leu Thr Gly Gly Tyr Leu
115 120 125
His Ile Tyr Glu Ser Gln Leu Cys Cys Gln Leu Lys Phe Trp Leu Pro
130 135 140
Ile Gly Cys Ala Ala Phe Val Val Val Cys Ile Leu Gly Cys Ile Leu
145 150 155 160
Ile Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro
165 170 175
Asn Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser
180 185 190
Arg Leu Thr Asp Val Thr Leu
195
<210> 169
<211> 600
<212> DNA
<213> 人工序列
<220>
<223> 鼠 ICOS
<220>
<221> CDS
<222> 1..600
<223> /翻译表=1
<400> 169
atg aaa ccg tat ttt tgc cgc gtg ttt gtg ttt tgc ttt ctg att cgc 48
Met Lys Pro Tyr Phe Cys Arg Val Phe Val Phe Cys Phe Leu Ile Arg
1 5 10 15
ctg ctg acc ggc gaa att aac ggc agc gcg gat cat cgc atg ttt agc 96
Leu Leu Thr Gly Glu Ile Asn Gly Ser Ala Asp His Arg Met Phe Ser
20 25 30
ttt cat aac ggc ggc gtg cag att agc tgc aaa tat ccg gaa acc gtg 144
Phe His Asn Gly Gly Val Gln Ile Ser Cys Lys Tyr Pro Glu Thr Val
35 40 45
cag cag ctg aaa atg cgc ctg ttt cgc gaa cgc gaa gtg ctg tgc gaa 192
Gln Gln Leu Lys Met Arg Leu Phe Arg Glu Arg Glu Val Leu Cys Glu
50 55 60
ctg acc aaa acc aaa ggc agc ggc aac gcg gtg agc att aaa aac ccg 240
Leu Thr Lys Thr Lys Gly Ser Gly Asn Ala Val Ser Ile Lys Asn Pro
65 70 75 80
atg ctg tgc ctg tat cat ctg agc aac aac agc gtg agc ttt ttt ctg 288
Met Leu Cys Leu Tyr His Leu Ser Asn Asn Ser Val Ser Phe Phe Leu
85 90 95
aac aac ccg gat agc agc cag ggc agc tat tat ttt tgc agc ctg agc 336
Asn Asn Pro Asp Ser Ser Gln Gly Ser Tyr Tyr Phe Cys Ser Leu Ser
100 105 110
att ttt gat ccg ccg ccg ttt cag gaa cgc aac ctg agc ggc ggc tat 384
Ile Phe Asp Pro Pro Pro Phe Gln Glu Arg Asn Leu Ser Gly Gly Tyr
115 120 125
ctg cat att tat gaa agc cag ctg tgc tgc cag ctg aaa ctg tgg ctg 432
Leu His Ile Tyr Glu Ser Gln Leu Cys Cys Gln Leu Lys Leu Trp Leu
130 135 140
ccg gtg ggc tgc gcg gcg ttt gtg gtg gtg ctg ctg ttt ggc tgc att 480
Pro Val Gly Cys Ala Ala Phe Val Val Val Leu Leu Phe Gly Cys Ile
145 150 155 160
ctg att att tgg ttt agc aaa aaa aaa tat ggc agc agc gtg cat gat 528
Leu Ile Ile Trp Phe Ser Lys Lys Lys Tyr Gly Ser Ser Val His Asp
165 170 175
ccg aac agc gaa tat atg ttt atg gcg gcg gtg aac acc aac aaa aaa 576
Pro Asn Ser Glu Tyr Met Phe Met Ala Ala Val Asn Thr Asn Lys Lys
180 185 190
agc cgc ctg gcg ggc gtg acc agc 600
Ser Arg Leu Ala Gly Val Thr Ser
195 200
<210> 170
<211> 200
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..600, 来自SEQ ID NO 169
<400> 170
Met Lys Pro Tyr Phe Cys Arg Val Phe Val Phe Cys Phe Leu Ile Arg
1 5 10 15
Leu Leu Thr Gly Glu Ile Asn Gly Ser Ala Asp His Arg Met Phe Ser
20 25 30
Phe His Asn Gly Gly Val Gln Ile Ser Cys Lys Tyr Pro Glu Thr Val
35 40 45
Gln Gln Leu Lys Met Arg Leu Phe Arg Glu Arg Glu Val Leu Cys Glu
50 55 60
Leu Thr Lys Thr Lys Gly Ser Gly Asn Ala Val Ser Ile Lys Asn Pro
65 70 75 80
Met Leu Cys Leu Tyr His Leu Ser Asn Asn Ser Val Ser Phe Phe Leu
85 90 95
Asn Asn Pro Asp Ser Ser Gln Gly Ser Tyr Tyr Phe Cys Ser Leu Ser
100 105 110
Ile Phe Asp Pro Pro Pro Phe Gln Glu Arg Asn Leu Ser Gly Gly Tyr
115 120 125
Leu His Ile Tyr Glu Ser Gln Leu Cys Cys Gln Leu Lys Leu Trp Leu
130 135 140
Pro Val Gly Cys Ala Ala Phe Val Val Val Leu Leu Phe Gly Cys Ile
145 150 155 160
Leu Ile Ile Trp Phe Ser Lys Lys Lys Tyr Gly Ser Ser Val His Asp
165 170 175
Pro Asn Ser Glu Tyr Met Phe Met Ala Ala Val Asn Thr Asn Lys Lys
180 185 190
Ser Arg Leu Ala Gly Val Thr Ser
195 200
<210> 171
<211> 780
<212> DNA
<213> 人工序列
<220>
<223> 人 CD27
<220>
<221> CDS
<222> 1..780
<223> /翻译表=1
<400> 171
atg gcg cgc ccg cat ccg tgg tgg ctg tgc gtg ctg ggc acc ctg gtg 48
Met Ala Arg Pro His Pro Trp Trp Leu Cys Val Leu Gly Thr Leu Val
1 5 10 15
ggc ctg agc gcg acc ccg gcg ccg aaa agc tgc ccg gaa cgc cat tat 96
Gly Leu Ser Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His Tyr
20 25 30
tgg gcg cag ggc aaa ctg tgc tgc cag atg tgc gaa ccg ggc acc ttt 144
Trp Ala Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr Phe
35 40 45
ctg gtg aaa gat tgc gat cag cat cgc aaa gcg gcg cag tgc gat ccg 192
Leu Val Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp Pro
50 55 60
tgc att ccg ggc gtg agc ttt agc ccg gat cat cat acc cgc ccg cat 240
Cys Ile Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro His
65 70 75 80
tgc gaa agc tgc cgc cat tgc aac agc ggc ctg ctg gtg cgc aac tgc 288
Cys Glu Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn Cys
85 90 95
acc att acc gcg aac gcg gaa tgc gcg tgc cgc aac ggc tgg cag tgc 336
Thr Ile Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln Cys
100 105 110
cgc gat aaa gaa tgc acc gaa tgc gat ccg ctg ccg aac ccg agc ctg 384
Arg Asp Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu
115 120 125
acc gcg cgc agc agc cag gcg ctg agc ccg cat ccg cag ccg acc cat 432
Thr Ala Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His
130 135 140
ctg ccg tat gtg agc gaa atg ctg gaa gcg cgc acc gcg ggc cat atg 480
Leu Pro Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His Met
145 150 155 160
cag acc ctg gcg gat ttt cgc cag ctg ccg gcg cgc acc ctg agc acc 528
Gln Thr Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser Thr
165 170 175
cat tgg ccg ccg cag cgc agc ctg tgc agc agc gat ttt att cgc att 576
His Trp Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg Ile
180 185 190
ctg gtg att ttt agc ggc atg ttt ctg gtg ttt acc ctg gcg ggc gcg 624
Leu Val Ile Phe Ser Gly Met Phe Leu Val Phe Thr Leu Ala Gly Ala
195 200 205
ctg ttt ctg cat cag cgc cgc aaa tat cgc agc aac aaa ggc gaa agc 672
Leu Phe Leu His Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser
210 215 220
ccg gtg gaa ccg gcg gaa ccg tgc cat tat agc tgc ccg cgc gaa gaa 720
Pro Val Glu Pro Ala Glu Pro Cys His Tyr Ser Cys Pro Arg Glu Glu
225 230 235 240
gaa ggc agc acc att ccg att cag gaa gat tat cgc aaa ccg gaa ccg 768
Glu Gly Ser Thr Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro
245 250 255
gcg tgc agc ccg 780
Ala Cys Ser Pro
260
<210> 172
<211> 260
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..780, 来自SEQ ID NO 171
<400> 172
Met Ala Arg Pro His Pro Trp Trp Leu Cys Val Leu Gly Thr Leu Val
1 5 10 15
Gly Leu Ser Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His Tyr
20 25 30
Trp Ala Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr Phe
35 40 45
Leu Val Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp Pro
50 55 60
Cys Ile Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro His
65 70 75 80
Cys Glu Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn Cys
85 90 95
Thr Ile Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln Cys
100 105 110
Arg Asp Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu
115 120 125
Thr Ala Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His
130 135 140
Leu Pro Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His Met
145 150 155 160
Gln Thr Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser Thr
165 170 175
His Trp Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg Ile
180 185 190
Leu Val Ile Phe Ser Gly Met Phe Leu Val Phe Thr Leu Ala Gly Ala
195 200 205
Leu Phe Leu His Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser
210 215 220
Pro Val Glu Pro Ala Glu Pro Cys His Tyr Ser Cys Pro Arg Glu Glu
225 230 235 240
Glu Gly Ser Thr Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro
245 250 255
Ala Cys Ser Pro
260
<210> 173
<211> 750
<212> DNA
<213> 人工序列
<220>
<223> 鼠 CD27
<220>
<221> CDS
<222> 1..750
<223> /翻译表=1
<400> 173
atg gcg tgg ccg ccg ccg tat tgg ctg tgc atg ctg ggc acc ctg gtg 48
Met Ala Trp Pro Pro Pro Tyr Trp Leu Cys Met Leu Gly Thr Leu Val
1 5 10 15
ggc ctg agc gcg acc ctg gcg ccg aac agc tgc ccg gat aaa cat tat 96
Gly Leu Ser Ala Thr Leu Ala Pro Asn Ser Cys Pro Asp Lys His Tyr
20 25 30
tgg acc ggc ggc ggc ctg tgc tgc cgc atg tgc gaa ccg ggc acc ttt 144
Trp Thr Gly Gly Gly Leu Cys Cys Arg Met Cys Glu Pro Gly Thr Phe
35 40 45
ttt gtg aaa gat tgc gaa cag gat cgc acc gcg gcg cag tgc gat ccg 192
Phe Val Lys Asp Cys Glu Gln Asp Arg Thr Ala Ala Gln Cys Asp Pro
50 55 60
tgc att ccg ggc acc agc ttt agc ccg gat tat cat acc cgc ccg cat 240
Cys Ile Pro Gly Thr Ser Phe Ser Pro Asp Tyr His Thr Arg Pro His
65 70 75 80
tgc gaa agc tgc cgc cat tgc aac agc ggc ttt ctg att cgc aac tgc 288
Cys Glu Ser Cys Arg His Cys Asn Ser Gly Phe Leu Ile Arg Asn Cys
85 90 95
acc gtg acc gcg aac gcg gaa tgc agc tgc agc aaa aac tgg cag tgc 336
Thr Val Thr Ala Asn Ala Glu Cys Ser Cys Ser Lys Asn Trp Gln Cys
100 105 110
cgc gat cag gaa tgc acc gaa tgc gat ccg ccg ctg aac ccg gcg ctg 384
Arg Asp Gln Glu Cys Thr Glu Cys Asp Pro Pro Leu Asn Pro Ala Leu
115 120 125
acc cgc cag ccg agc gaa acc ccg agc ccg cag ccg ccg ccg acc cat 432
Thr Arg Gln Pro Ser Glu Thr Pro Ser Pro Gln Pro Pro Pro Thr His
130 135 140
ctg ccg cat ggc acc gaa aaa ccg agc tgg ccg ctg cat cgc cag ctg 480
Leu Pro His Gly Thr Glu Lys Pro Ser Trp Pro Leu His Arg Gln Leu
145 150 155 160
ccg aac agc acc gtg tat agc cag cgc agc agc cat cgc ccg ctg tgc 528
Pro Asn Ser Thr Val Tyr Ser Gln Arg Ser Ser His Arg Pro Leu Cys
165 170 175
agc agc gat tgc att cgc att ttt gtg acc ttt agc agc atg ttt ctg 576
Ser Ser Asp Cys Ile Arg Ile Phe Val Thr Phe Ser Ser Met Phe Leu
180 185 190
att ttt gtg ctg ggc gcg att ctg ttt ttt cat cag cgc cgc aac cat 624
Ile Phe Val Leu Gly Ala Ile Leu Phe Phe His Gln Arg Arg Asn His
195 200 205
ggc ccg aac gaa gat cgc cag gcg gtg ccg gaa gaa ccg tgc ccg tat 672
Gly Pro Asn Glu Asp Arg Gln Ala Val Pro Glu Glu Pro Cys Pro Tyr
210 215 220
agc tgc ccg cgc gaa gaa gaa ggc agc gcg att ccg att cag gaa gat 720
Ser Cys Pro Arg Glu Glu Glu Gly Ser Ala Ile Pro Ile Gln Glu Asp
225 230 235 240
tat cgc aaa ccg gaa ccg gcg ttt tat ccg 750
Tyr Arg Lys Pro Glu Pro Ala Phe Tyr Pro
245 250
<210> 174
<211> 250
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..750, 来自SEQ ID NO 173
<400> 174
Met Ala Trp Pro Pro Pro Tyr Trp Leu Cys Met Leu Gly Thr Leu Val
1 5 10 15
Gly Leu Ser Ala Thr Leu Ala Pro Asn Ser Cys Pro Asp Lys His Tyr
20 25 30
Trp Thr Gly Gly Gly Leu Cys Cys Arg Met Cys Glu Pro Gly Thr Phe
35 40 45
Phe Val Lys Asp Cys Glu Gln Asp Arg Thr Ala Ala Gln Cys Asp Pro
50 55 60
Cys Ile Pro Gly Thr Ser Phe Ser Pro Asp Tyr His Thr Arg Pro His
65 70 75 80
Cys Glu Ser Cys Arg His Cys Asn Ser Gly Phe Leu Ile Arg Asn Cys
85 90 95
Thr Val Thr Ala Asn Ala Glu Cys Ser Cys Ser Lys Asn Trp Gln Cys
100 105 110
Arg Asp Gln Glu Cys Thr Glu Cys Asp Pro Pro Leu Asn Pro Ala Leu
115 120 125
Thr Arg Gln Pro Ser Glu Thr Pro Ser Pro Gln Pro Pro Pro Thr His
130 135 140
Leu Pro His Gly Thr Glu Lys Pro Ser Trp Pro Leu His Arg Gln Leu
145 150 155 160
Pro Asn Ser Thr Val Tyr Ser Gln Arg Ser Ser His Arg Pro Leu Cys
165 170 175
Ser Ser Asp Cys Ile Arg Ile Phe Val Thr Phe Ser Ser Met Phe Leu
180 185 190
Ile Phe Val Leu Gly Ala Ile Leu Phe Phe His Gln Arg Arg Asn His
195 200 205
Gly Pro Asn Glu Asp Arg Gln Ala Val Pro Glu Glu Pro Cys Pro Tyr
210 215 220
Ser Cys Pro Arg Glu Glu Glu Gly Ser Ala Ile Pro Ile Gln Glu Asp
225 230 235 240
Tyr Arg Lys Pro Glu Pro Ala Phe Tyr Pro
245 250
<210> 175
<211> 279
<212> DNA
<213> 人工序列
<220>
<223> 人 DAP10
<220>
<221> CDS
<222> 1..279
<223> /翻译表=1
<400> 175
atg att cat ctg ggc cat att ctg ttt ctg ctg ctg ctg ccg gtg gcg 48
Met Ile His Leu Gly His Ile Leu Phe Leu Leu Leu Leu Pro Val Ala
1 5 10 15
gcg gcg cag acc acc ccg ggc gaa cgc agc agc ctg ccg gcg ttt tat 96
Ala Ala Gln Thr Thr Pro Gly Glu Arg Ser Ser Leu Pro Ala Phe Tyr
20 25 30
ccg ggc acc agc ggc agc tgc agc ggc tgc ggc agc ctg agc ctg ccg 144
Pro Gly Thr Ser Gly Ser Cys Ser Gly Cys Gly Ser Leu Ser Leu Pro
35 40 45
ctg ctg gcg ggc ctg gtg gcg gcg gat gcg gtg gcg agc ctg ctg att 192
Leu Leu Ala Gly Leu Val Ala Ala Asp Ala Val Ala Ser Leu Leu Ile
50 55 60
gtg ggc gcg gtg ttt ctg tgc gcg cgc ccg cgc cgc agc ccg gcg cag 240
Val Gly Ala Val Phe Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln
65 70 75 80
gaa gat ggc aaa gtg tat att aac atg ccg ggc cgc ggc 279
Glu Asp Gly Lys Val Tyr Ile Asn Met Pro Gly Arg Gly
85 90
<210> 176
<211> 93
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..279, 来自SEQ ID NO 175
<400> 176
Met Ile His Leu Gly His Ile Leu Phe Leu Leu Leu Leu Pro Val Ala
1 5 10 15
Ala Ala Gln Thr Thr Pro Gly Glu Arg Ser Ser Leu Pro Ala Phe Tyr
20 25 30
Pro Gly Thr Ser Gly Ser Cys Ser Gly Cys Gly Ser Leu Ser Leu Pro
35 40 45
Leu Leu Ala Gly Leu Val Ala Ala Asp Ala Val Ala Ser Leu Leu Ile
50 55 60
Val Gly Ala Val Phe Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln
65 70 75 80
Glu Asp Gly Lys Val Tyr Ile Asn Met Pro Gly Arg Gly
85 90
<210> 177
<211> 237
<212> DNA
<213> 人工序列
<220>
<223> 鼠 DAP10
<220>
<221> CDS
<222> 1..237
<223> /翻译表=1
<400> 177
atg gat ccg ccg ggc tat ctg ctg ttt ctg ctg ctg ctg ccg gtg gcg 48
Met Asp Pro Pro Gly Tyr Leu Leu Phe Leu Leu Leu Leu Pro Val Ala
1 5 10 15
gcg agc cag acc agc gcg ggc agc tgc agc ggc tgc ggc acc ctg agc 96
Ala Ser Gln Thr Ser Ala Gly Ser Cys Ser Gly Cys Gly Thr Leu Ser
20 25 30
ctg ccg ctg ctg gcg ggc ctg gtg gcg gcg gat gcg gtg atg agc ctg 144
Leu Pro Leu Leu Ala Gly Leu Val Ala Ala Asp Ala Val Met Ser Leu
35 40 45
ctg att gtg ggc gtg gtg ttt gtg tgc atg cgc ccg cat ggc cgc ccg 192
Leu Ile Val Gly Val Val Phe Val Cys Met Arg Pro His Gly Arg Pro
50 55 60
gcg cag gaa gat ggc cgc gtg tat att aac atg ccg ggc cgc ggc 237
Ala Gln Glu Asp Gly Arg Val Tyr Ile Asn Met Pro Gly Arg Gly
65 70 75
<210> 178
<211> 79
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..237, 来自SEQ ID NO 177
<400> 178
Met Asp Pro Pro Gly Tyr Leu Leu Phe Leu Leu Leu Leu Pro Val Ala
1 5 10 15
Ala Ser Gln Thr Ser Ala Gly Ser Cys Ser Gly Cys Gly Thr Leu Ser
20 25 30
Leu Pro Leu Leu Ala Gly Leu Val Ala Ala Asp Ala Val Met Ser Leu
35 40 45
Leu Ile Val Gly Val Val Phe Val Cys Met Arg Pro His Gly Arg Pro
50 55 60
Ala Gln Glu Asp Gly Arg Val Tyr Ile Asn Met Pro Gly Arg Gly
65 70 75
<210> 179
<211> 492
<212> DNA
<213> 人工序列
<220>
<223> CD3z 小鼠
<220>
<221> CDS
<222> 1..492
<223> /翻译表=1
<400> 179
atg aaa tgg aaa gtg agc gtg ctg gcg tgc att ctg cat gtg cgc ttt 48
Met Lys Trp Lys Val Ser Val Leu Ala Cys Ile Leu His Val Arg Phe
1 5 10 15
ccg ggc gcg gaa gcg cag agc ttt ggc ctg ctg gat ccg aaa ctg tgc 96
Pro Gly Ala Glu Ala Gln Ser Phe Gly Leu Leu Asp Pro Lys Leu Cys
20 25 30
tat ctg ctg gat ggc att ctg ttt att tat ggc gtg att att acc gcg 144
Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Ile Thr Ala
35 40 45
ctg tat ctg cgc gcg aaa ttt agc cgc agc gcg gaa acc gcg gcg aac 192
Leu Tyr Leu Arg Ala Lys Phe Ser Arg Ser Ala Glu Thr Ala Ala Asn
50 55 60
ctg cag gat ccg aac cag ctg tat aac gaa ctg aac ctg ggc cgc cgc 240
Leu Gln Asp Pro Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
65 70 75 80
gaa gaa tat gat gtg ctg gaa aaa aaa cgc gcg cgc gat ccg gaa atg 288
Glu Glu Tyr Asp Val Leu Glu Lys Lys Arg Ala Arg Asp Pro Glu Met
85 90 95
ggc ggc aaa cag cag cgc cgc cgc aac ccg cag gaa ggc gtg tat aac 336
Gly Gly Lys Gln Gln Arg Arg Arg Asn Pro Gln Glu Gly Val Tyr Asn
100 105 110
gcg ctg cag aaa gat aaa atg gcg gaa gcg tat agc gaa att ggc acc 384
Ala Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Thr
115 120 125
aaa ggc gaa cgc cgc cgc ggc aaa ggc cat gat ggc ctg tat cag ggc 432
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
130 135 140
ctg agc acc gcg acc aaa gat acc tat gat gcg ctg cat atg cag acc 480
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Thr
145 150 155 160
ctg gcg ccg cgc 492
Leu Ala Pro Arg
<210> 180
<211> 164
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..492, 来自SEQ ID NO 179
<400> 180
Met Lys Trp Lys Val Ser Val Leu Ala Cys Ile Leu His Val Arg Phe
1 5 10 15
Pro Gly Ala Glu Ala Gln Ser Phe Gly Leu Leu Asp Pro Lys Leu Cys
20 25 30
Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Ile Thr Ala
35 40 45
Leu Tyr Leu Arg Ala Lys Phe Ser Arg Ser Ala Glu Thr Ala Ala Asn
50 55 60
Leu Gln Asp Pro Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
65 70 75 80
Glu Glu Tyr Asp Val Leu Glu Lys Lys Arg Ala Arg Asp Pro Glu Met
85 90 95
Gly Gly Lys Gln Gln Arg Arg Arg Asn Pro Gln Glu Gly Val Tyr Asn
100 105 110
Ala Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Thr
115 120 125
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
130 135 140
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Thr
145 150 155 160
Leu Ala Pro Arg
<210> 181
<211> 696
<212> DNA
<213> 人工序列
<220>
<223> NKG2d 小鼠
<220>
<221> CDS
<222> 1..696
<223> /翻译表=1
<400> 181
atg gcg ctg att cgc gat cgc aaa agc cat cat agc gaa atg agc aaa 48
Met Ala Leu Ile Arg Asp Arg Lys Ser His His Ser Glu Met Ser Lys
1 5 10 15
tgc cat aac tat gat ctg aaa ccg gcg aaa tgg gat acc agc cag gaa 96
Cys His Asn Tyr Asp Leu Lys Pro Ala Lys Trp Asp Thr Ser Gln Glu
20 25 30
cag cag aaa cag cgc ctg gcg ctg acc acc agc cag ccg ggc gaa aac 144
Gln Gln Lys Gln Arg Leu Ala Leu Thr Thr Ser Gln Pro Gly Glu Asn
35 40 45
ggc att att cgc ggc cgc tat ccg att gaa aaa ctg aaa att agc ccg 192
Gly Ile Ile Arg Gly Arg Tyr Pro Ile Glu Lys Leu Lys Ile Ser Pro
50 55 60
atg ttt gtg gtg cgc gtg ctg gcg att gcg ctg gcg att cgc ttt acc 240
Met Phe Val Val Arg Val Leu Ala Ile Ala Leu Ala Ile Arg Phe Thr
65 70 75 80
ctg aac acc ctg atg tgg ctg gcg att ttt aaa gaa acc ttt cag ccg 288
Leu Asn Thr Leu Met Trp Leu Ala Ile Phe Lys Glu Thr Phe Gln Pro
85 90 95
gtg ctg tgc aac aaa gaa gtg ccg gtg agc agc cgc gaa ggc tat tgc 336
Val Leu Cys Asn Lys Glu Val Pro Val Ser Ser Arg Glu Gly Tyr Cys
100 105 110
ggc ccg tgc ccg aac aac tgg att tgc cat cgc aac aac tgc tat cag 384
Gly Pro Cys Pro Asn Asn Trp Ile Cys His Arg Asn Asn Cys Tyr Gln
115 120 125
ttt ttt aac gaa gaa aaa acc tgg aac cag agc cag gcg agc tgc ctg 432
Phe Phe Asn Glu Glu Lys Thr Trp Asn Gln Ser Gln Ala Ser Cys Leu
130 135 140
agc cag aac agc agc ctg ctg aaa att tat agc aaa gaa gaa cag gat 480
Ser Gln Asn Ser Ser Leu Leu Lys Ile Tyr Ser Lys Glu Glu Gln Asp
145 150 155 160
ttt ctg aaa ctg gtg aaa agc tat cat tgg atg ggc ctg gtg cag att 528
Phe Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val Gln Ile
165 170 175
ccg gcg aac ggc agc tgg cag tgg gaa gat ggc agc agc ctg agc tat 576
Pro Ala Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ser Leu Ser Tyr
180 185 190
aac cag ctg acc ctg gtg gaa att ccg aaa ggc agc tgc gcg gtg tat 624
Asn Gln Leu Thr Leu Val Glu Ile Pro Lys Gly Ser Cys Ala Val Tyr
195 200 205
ggc agc agc ttt aaa gcg tat acc gaa gat tgc gcg aac ctg aac acc 672
Gly Ser Ser Phe Lys Ala Tyr Thr Glu Asp Cys Ala Asn Leu Asn Thr
210 215 220
tat att tgc atg aaa cgc gcg gtg 696
Tyr Ile Cys Met Lys Arg Ala Val
225 230
<210> 182
<211> 232
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..696, 来自SEQ ID NO 181
<400> 182
Met Ala Leu Ile Arg Asp Arg Lys Ser His His Ser Glu Met Ser Lys
1 5 10 15
Cys His Asn Tyr Asp Leu Lys Pro Ala Lys Trp Asp Thr Ser Gln Glu
20 25 30
Gln Gln Lys Gln Arg Leu Ala Leu Thr Thr Ser Gln Pro Gly Glu Asn
35 40 45
Gly Ile Ile Arg Gly Arg Tyr Pro Ile Glu Lys Leu Lys Ile Ser Pro
50 55 60
Met Phe Val Val Arg Val Leu Ala Ile Ala Leu Ala Ile Arg Phe Thr
65 70 75 80
Leu Asn Thr Leu Met Trp Leu Ala Ile Phe Lys Glu Thr Phe Gln Pro
85 90 95
Val Leu Cys Asn Lys Glu Val Pro Val Ser Ser Arg Glu Gly Tyr Cys
100 105 110
Gly Pro Cys Pro Asn Asn Trp Ile Cys His Arg Asn Asn Cys Tyr Gln
115 120 125
Phe Phe Asn Glu Glu Lys Thr Trp Asn Gln Ser Gln Ala Ser Cys Leu
130 135 140
Ser Gln Asn Ser Ser Leu Leu Lys Ile Tyr Ser Lys Glu Glu Gln Asp
145 150 155 160
Phe Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val Gln Ile
165 170 175
Pro Ala Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ser Leu Ser Tyr
180 185 190
Asn Gln Leu Thr Leu Val Glu Ile Pro Lys Gly Ser Cys Ala Val Tyr
195 200 205
Gly Ser Ser Phe Lys Ala Tyr Thr Glu Asp Cys Ala Asn Leu Asn Thr
210 215 220
Tyr Ile Cys Met Lys Arg Ala Val
225 230
<210> 183
<211> 492
<212> DNA
<213> 人工序列
<220>
<223> 人 CD3z
<220>
<221> CDS
<222> 1..492
<223> /翻译表=1
<400> 183
atg aaa tgg aaa gtg agc gtg ctg gcg tgc att ctg cat gtg cgc ttt 48
Met Lys Trp Lys Val Ser Val Leu Ala Cys Ile Leu His Val Arg Phe
1 5 10 15
ccg ggc gcg gaa gcg cag agc ttt ggc ctg ctg gat ccg aaa ctg tgc 96
Pro Gly Ala Glu Ala Gln Ser Phe Gly Leu Leu Asp Pro Lys Leu Cys
20 25 30
tat ctg ctg gat ggc att ctg ttt att tat ggc gtg att att acc gcg 144
Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Ile Thr Ala
35 40 45
ctg tat ctg cgc gcg aaa ttt agc cgc agc gcg gaa acc gcg gcg aac 192
Leu Tyr Leu Arg Ala Lys Phe Ser Arg Ser Ala Glu Thr Ala Ala Asn
50 55 60
ctg cag gat ccg aac cag ctg tat aac gaa ctg aac ctg ggc cgc cgc 240
Leu Gln Asp Pro Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
65 70 75 80
gaa gaa tat gat gtg ctg gaa aaa aaa cgc gcg cgc gat ccg gaa atg 288
Glu Glu Tyr Asp Val Leu Glu Lys Lys Arg Ala Arg Asp Pro Glu Met
85 90 95
ggc ggc aaa cag cag cgc cgc cgc aac ccg cag gaa ggc gtg tat aac 336
Gly Gly Lys Gln Gln Arg Arg Arg Asn Pro Gln Glu Gly Val Tyr Asn
100 105 110
gcg ctg cag aaa gat aaa atg gcg gaa gcg tat agc gaa att ggc acc 384
Ala Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Thr
115 120 125
aaa ggc gaa cgc cgc cgc ggc aaa ggc cat gat ggc ctg tat cag ggc 432
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
130 135 140
ctg agc acc gcg acc aaa gat acc tat gat gcg ctg cat atg cag acc 480
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Thr
145 150 155 160
ctg gcg ccg cgc 492
Leu Ala Pro Arg
<210> 184
<211> 164
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..492, 来自SEQ ID NO 183
<400> 184
Met Lys Trp Lys Val Ser Val Leu Ala Cys Ile Leu His Val Arg Phe
1 5 10 15
Pro Gly Ala Glu Ala Gln Ser Phe Gly Leu Leu Asp Pro Lys Leu Cys
20 25 30
Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Ile Thr Ala
35 40 45
Leu Tyr Leu Arg Ala Lys Phe Ser Arg Ser Ala Glu Thr Ala Ala Asn
50 55 60
Leu Gln Asp Pro Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
65 70 75 80
Glu Glu Tyr Asp Val Leu Glu Lys Lys Arg Ala Arg Asp Pro Glu Met
85 90 95
Gly Gly Lys Gln Gln Arg Arg Arg Asn Pro Gln Glu Gly Val Tyr Asn
100 105 110
Ala Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Thr
115 120 125
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
130 135 140
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Thr
145 150 155 160
Leu Ala Pro Arg
<210> 185
<211> 762
<212> DNA
<213> 人工序列
<220>
<223> 人 FCGR3a
<220>
<221> CDS
<222> 1..762
<223> /翻译表=1
<400> 185
atg tgg cag ctg ctg ctg ccg acc gcg ctg ctg ctg ctg gtg agc gcg 48
Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala
1 5 10 15
ggc atg cgc acc gaa gat ctg ccg aaa gcg gtg gtg ttt ctg gaa ccg 96
Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro
20 25 30
cag tgg tat cgc gtg ctg gaa aaa gat agc gtg acc ctg aaa tgc cag 144
Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln
35 40 45
ggc gcg tat agc ccg gaa gat aac agc acc cag tgg ttt cat aac gaa 192
Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu
50 55 60
agc ctg att agc agc cag gcg agc agc tat ttt att gat gcg gcg acc 240
Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr
65 70 75 80
gtg gat gat agc ggc gaa tat cgc tgc cag acc aac ctg agc acc ctg 288
Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu
85 90 95
agc gat ccg gtg cag ctg gaa gtg cat att ggc tgg ctg ctg ctg cag 336
Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln
100 105 110
gcg ccg cgc tgg gtg ttt aaa gaa gaa gat ccg att cat ctg cgc tgc 384
Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys
115 120 125
cat agc tgg aaa aac acc gcg ctg cat aaa gtg acc tat ctg cag aac 432
His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn
130 135 140
ggc aaa ggc cgc aaa tat ttt cat cat aac agc gat ttt tat att ccg 480
Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro
145 150 155 160
aaa gcg acc ctg aaa gat agc ggc agc tat ttt tgc cgc ggc ctg ttt 528
Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe
165 170 175
ggc agc aaa aac gtg agc agc gaa acc gtg aac att acc att acc cag 576
Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln
180 185 190
ggc ctg gcg gtg agc acc att agc agc ttt ttt ccg ccg ggc tat cag 624
Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln
195 200 205
gtg agc ttt tgc ctg gtg atg gtg ctg ctg ttt gcg gtg gat acc ggc 672
Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly
210 215 220
ctg tat ttt agc gtg aaa acc aac att cgc agc agc acc cgc gat tgg 720
Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp
225 230 235 240
aaa gat cat aaa ttt aaa tgg cgc aaa gat ccg cag gat aaa 762
Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys
245 250
<210> 186
<211> 254
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..762, 来自SEQ ID NO 185
<400> 186
Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala
1 5 10 15
Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro
20 25 30
Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln
35 40 45
Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu
50 55 60
Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr
65 70 75 80
Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu
85 90 95
Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln
100 105 110
Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys
115 120 125
His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn
130 135 140
Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro
145 150 155 160
Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe
165 170 175
Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln
180 185 190
Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln
195 200 205
Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly
210 215 220
Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp
225 230 235 240
Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys
245 250
<210> 187
<211> 648
<212> DNA
<213> 人工序列
<220>
<223> 人 NKg2d
<220>
<221> CDS
<222> 1..648
<223> /翻译表=1
<400> 187
atg ggc tgg att cgc ggc cgc cgc agc cgc cat agc tgg gaa atg agc 48
Met Gly Trp Ile Arg Gly Arg Arg Ser Arg His Ser Trp Glu Met Ser
1 5 10 15
gaa ttt cat aac tat aac ctg gat ctg aaa aaa agc gat ttt agc acc 96
Glu Phe His Asn Tyr Asn Leu Asp Leu Lys Lys Ser Asp Phe Ser Thr
20 25 30
cgc tgg cag aaa cag cgc tgc ccg gtg gtg aaa agc aaa tgc cgc gaa 144
Arg Trp Gln Lys Gln Arg Cys Pro Val Val Lys Ser Lys Cys Arg Glu
35 40 45
aac gcg agc ccg ttt ttt ttt tgc tgc ttt att gcg gtg gcg atg ggc 192
Asn Ala Ser Pro Phe Phe Phe Cys Cys Phe Ile Ala Val Ala Met Gly
50 55 60
att cgc ttt att att atg gtg gcg att tgg agc gcg gtg ttt ctg aac 240
Ile Arg Phe Ile Ile Met Val Ala Ile Trp Ser Ala Val Phe Leu Asn
65 70 75 80
agc ctg ttt aac cag gaa gtg cag att ccg ctg acc gaa agc tat tgc 288
Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys
85 90 95
ggc ccg tgc ccg aaa aac tgg att tgc tat aaa aac aac tgc tat cag 336
Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln
100 105 110
ttt ttt gat gaa agc aaa aac tgg tat gaa agc cag gcg agc tgc atg 384
Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met
115 120 125
agc cag aac gcg agc ctg ctg aaa gtg tat agc aaa gaa gat cag gat 432
Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp
130 135 140
ctg ctg aaa ctg gtg aaa agc tat cat tgg atg ggc ctg gtg cat att 480
Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile
145 150 155 160
ccg acc aac ggc agc tgg cag tgg gaa gat ggc agc att ctg agc ccg 528
Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro
165 170 175
aac ctg ctg acc att att gaa atg cag aaa ggc gat tgc gcg ctg tat 576
Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr
180 185 190
gcg agc agc ttt aaa ggc tat att gaa aac tgc agc acc ccg aac acc 624
Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr
195 200 205
tat att tgc atg cag cgc acc gtg 648
Tyr Ile Cys Met Gln Arg Thr Val
210 215
<210> 188
<211> 216
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..648, 来自SEQ ID NO 187
<400> 188
Met Gly Trp Ile Arg Gly Arg Arg Ser Arg His Ser Trp Glu Met Ser
1 5 10 15
Glu Phe His Asn Tyr Asn Leu Asp Leu Lys Lys Ser Asp Phe Ser Thr
20 25 30
Arg Trp Gln Lys Gln Arg Cys Pro Val Val Lys Ser Lys Cys Arg Glu
35 40 45
Asn Ala Ser Pro Phe Phe Phe Cys Cys Phe Ile Ala Val Ala Met Gly
50 55 60
Ile Arg Phe Ile Ile Met Val Ala Ile Trp Ser Ala Val Phe Leu Asn
65 70 75 80
Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys
85 90 95
Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln
100 105 110
Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met
115 120 125
Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp
130 135 140
Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile
145 150 155 160
Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro
165 170 175
Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr
180 185 190
Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr
195 200 205
Tyr Ile Cys Met Gln Arg Thr Val
210 215
<210> 189
<211> 2979
<212> DNA
<213> 人工序列
<220>
<223> 人 Flt3
<220>
<221> CDS
<222> 1..2979
<223> /翻译表=1
<400> 189
atg ccg gcg ctg gcg cgc gat ggc ggc cag ctg ccg ctg ctg gtg gtg 48
Met Pro Ala Leu Ala Arg Asp Gly Gly Gln Leu Pro Leu Leu Val Val
1 5 10 15
ttt agc gcg atg att ttt ggc acc att acc aac cag gat ctg ccg gtg 96
Phe Ser Ala Met Ile Phe Gly Thr Ile Thr Asn Gln Asp Leu Pro Val
20 25 30
att aaa tgc gtg ctg att aac cat aaa aac aac gat agc agc gtg ggc 144
Ile Lys Cys Val Leu Ile Asn His Lys Asn Asn Asp Ser Ser Val Gly
35 40 45
aaa agc agc agc tat ccg atg gtg agc gaa agc ccg gaa gat ctg ggc 192
Lys Ser Ser Ser Tyr Pro Met Val Ser Glu Ser Pro Glu Asp Leu Gly
50 55 60
tgc gcg ctg cgc ccg cag agc agc ggc acc gtg tat gaa gcg gcg gcg 240
Cys Ala Leu Arg Pro Gln Ser Ser Gly Thr Val Tyr Glu Ala Ala Ala
65 70 75 80
gtg gaa gtg gat gtg agc gcg agc att acc ctg cag gtg ctg gtg gat 288
Val Glu Val Asp Val Ser Ala Ser Ile Thr Leu Gln Val Leu Val Asp
85 90 95
gcg ccg ggc aac att agc tgc ctg tgg gtg ttt aaa cat agc agc ctg 336
Ala Pro Gly Asn Ile Ser Cys Leu Trp Val Phe Lys His Ser Ser Leu
100 105 110
aac tgc cag ccg cat ttt gat ctg cag aac cgc ggc gtg gtg agc atg 384
Asn Cys Gln Pro His Phe Asp Leu Gln Asn Arg Gly Val Val Ser Met
115 120 125
gtg att ctg aaa atg acc gaa acc cag gcg ggc gaa tat ctg ctg ttt 432
Val Ile Leu Lys Met Thr Glu Thr Gln Ala Gly Glu Tyr Leu Leu Phe
130 135 140
att cag agc gaa gcg acc aac tat acc att ctg ttt acc gtg agc att 480
Ile Gln Ser Glu Ala Thr Asn Tyr Thr Ile Leu Phe Thr Val Ser Ile
145 150 155 160
cgc aac acc ctg ctg tat acc ctg cgc cgc ccg tat ttt cgc aaa atg 528
Arg Asn Thr Leu Leu Tyr Thr Leu Arg Arg Pro Tyr Phe Arg Lys Met
165 170 175
gaa aac cag gat gcg ctg gtg tgc att agc gaa agc gtg ccg gaa ccg 576
Glu Asn Gln Asp Ala Leu Val Cys Ile Ser Glu Ser Val Pro Glu Pro
180 185 190
att gtg gaa tgg gtg ctg tgc gat agc cag ggc gaa agc tgc aaa gaa 624
Ile Val Glu Trp Val Leu Cys Asp Ser Gln Gly Glu Ser Cys Lys Glu
195 200 205
gaa agc ccg gcg gtg gtg aaa aaa gaa gaa aaa gtg ctg cat gaa ctg 672
Glu Ser Pro Ala Val Val Lys Lys Glu Glu Lys Val Leu His Glu Leu
210 215 220
ttt ggc acc gat att cgc tgc tgc gcg cgc aac gaa ctg ggc cgc gaa 720
Phe Gly Thr Asp Ile Arg Cys Cys Ala Arg Asn Glu Leu Gly Arg Glu
225 230 235 240
tgc acc cgc ctg ttt acc att gat ctg aac cag acc ccg cag acc acc 768
Cys Thr Arg Leu Phe Thr Ile Asp Leu Asn Gln Thr Pro Gln Thr Thr
245 250 255
ctg ccg cag ctg ttt ctg aaa gtg ggc gaa ccg ctg tgg att cgc tgc 816
Leu Pro Gln Leu Phe Leu Lys Val Gly Glu Pro Leu Trp Ile Arg Cys
260 265 270
aaa gcg gtg cat gtg aac cat ggc ttt ggc ctg acc tgg gaa ctg gaa 864
Lys Ala Val His Val Asn His Gly Phe Gly Leu Thr Trp Glu Leu Glu
275 280 285
aac aaa gcg ctg gaa gaa ggc aac tat ttt gaa atg agc acc tat agc 912
Asn Lys Ala Leu Glu Glu Gly Asn Tyr Phe Glu Met Ser Thr Tyr Ser
290 295 300
acc aac cgc acc atg att cgc att ctg ttt gcg ttt gtg agc agc gtg 960
Thr Asn Arg Thr Met Ile Arg Ile Leu Phe Ala Phe Val Ser Ser Val
305 310 315 320
gcg cgc aac gat acc ggc tat tat acc tgc agc agc agc aaa cat ccg 1008
Ala Arg Asn Asp Thr Gly Tyr Tyr Thr Cys Ser Ser Ser Lys His Pro
325 330 335
agc cag agc gcg ctg gtg acc att gtg gaa aaa ggc ttt att aac gcg 1056
Ser Gln Ser Ala Leu Val Thr Ile Val Glu Lys Gly Phe Ile Asn Ala
340 345 350
acc aac agc agc gaa gat tat gaa att gat cag tat gaa gaa ttt tgc 1104
Thr Asn Ser Ser Glu Asp Tyr Glu Ile Asp Gln Tyr Glu Glu Phe Cys
355 360 365
ttt agc gtg cgc ttt aaa gcg tat ccg cag att cgc tgc acc tgg acc 1152
Phe Ser Val Arg Phe Lys Ala Tyr Pro Gln Ile Arg Cys Thr Trp Thr
370 375 380
ttt agc cgc aaa agc ttt ccg tgc gaa cag aaa ggc ctg gat aac ggc 1200
Phe Ser Arg Lys Ser Phe Pro Cys Glu Gln Lys Gly Leu Asp Asn Gly
385 390 395 400
tat agc att agc aaa ttt tgc aac cat aaa cat cag ccg ggc gaa tat 1248
Tyr Ser Ile Ser Lys Phe Cys Asn His Lys His Gln Pro Gly Glu Tyr
405 410 415
att ttt cat gcg gaa aac gat gat gcg cag ttt acc aaa atg ttt acc 1296
Ile Phe His Ala Glu Asn Asp Asp Ala Gln Phe Thr Lys Met Phe Thr
420 425 430
ctg aac att cgc cgc aaa ccg cag gtg ctg gcg gaa gcg agc gcg agc 1344
Leu Asn Ile Arg Arg Lys Pro Gln Val Leu Ala Glu Ala Ser Ala Ser
435 440 445
cag gcg agc tgc ttt agc gat ggc tat ccg ctg ccg agc tgg acc tgg 1392
Gln Ala Ser Cys Phe Ser Asp Gly Tyr Pro Leu Pro Ser Trp Thr Trp
450 455 460
aaa aaa tgc agc gat aaa agc ccg aac tgc acc gaa gaa att acc gaa 1440
Lys Lys Cys Ser Asp Lys Ser Pro Asn Cys Thr Glu Glu Ile Thr Glu
465 470 475 480
ggc gtg tgg aac cgc aaa gcg aac cgc aaa gtg ttt ggc cag tgg gtg 1488
Gly Val Trp Asn Arg Lys Ala Asn Arg Lys Val Phe Gly Gln Trp Val
485 490 495
agc agc agc acc ctg aac atg agc gaa gcg att aaa ggc ttt ctg gtg 1536
Ser Ser Ser Thr Leu Asn Met Ser Glu Ala Ile Lys Gly Phe Leu Val
500 505 510
aaa tgc tgc gcg tat aac agc ctg ggc acc agc tgc gaa acc att ctg 1584
Lys Cys Cys Ala Tyr Asn Ser Leu Gly Thr Ser Cys Glu Thr Ile Leu
515 520 525
ctg aac agc ccg ggc ccg ttt ccg ttt att cag gat aac att agc ttt 1632
Leu Asn Ser Pro Gly Pro Phe Pro Phe Ile Gln Asp Asn Ile Ser Phe
530 535 540
tat gcg acc att ggc gtg tgc ctg ctg ttt att gtg gtg ctg acc ctg 1680
Tyr Ala Thr Ile Gly Val Cys Leu Leu Phe Ile Val Val Leu Thr Leu
545 550 555 560
ctg att tgc cat aaa tat aaa aaa cag ttt cgc tat gaa agc cag ctg 1728
Leu Ile Cys His Lys Tyr Lys Lys Gln Phe Arg Tyr Glu Ser Gln Leu
565 570 575
cag atg gtg cag gtg acc ggc agc agc gat aac gaa tat ttt tat gtg 1776
Gln Met Val Gln Val Thr Gly Ser Ser Asp Asn Glu Tyr Phe Tyr Val
580 585 590
gat ttt cgc gaa tat gaa tat gat ctg aaa tgg gaa ttt ccg cgc gaa 1824
Asp Phe Arg Glu Tyr Glu Tyr Asp Leu Lys Trp Glu Phe Pro Arg Glu
595 600 605
aac ctg gaa ttt ggc aaa gtg ctg ggc agc ggc gcg ttt ggc aaa gtg 1872
Asn Leu Glu Phe Gly Lys Val Leu Gly Ser Gly Ala Phe Gly Lys Val
610 615 620
atg aac gcg acc gcg tat ggc att agc aaa acc ggc gtg agc att cag 1920
Met Asn Ala Thr Ala Tyr Gly Ile Ser Lys Thr Gly Val Ser Ile Gln
625 630 635 640
gtg gcg gtg aaa atg ctg aaa gaa aaa gcg gat agc agc gaa cgc gaa 1968
Val Ala Val Lys Met Leu Lys Glu Lys Ala Asp Ser Ser Glu Arg Glu
645 650 655
gcg ctg atg agc gaa ctg aaa atg atg acc cag ctg ggc agc cat gaa 2016
Ala Leu Met Ser Glu Leu Lys Met Met Thr Gln Leu Gly Ser His Glu
660 665 670
aac att gtg aac ctg ctg ggc gcg tgc acc ctg agc ggc ccg att tat 2064
Asn Ile Val Asn Leu Leu Gly Ala Cys Thr Leu Ser Gly Pro Ile Tyr
675 680 685
ctg att ttt gaa tat tgc tgc tat ggc gat ctg ctg aac tat ctg cgc 2112
Leu Ile Phe Glu Tyr Cys Cys Tyr Gly Asp Leu Leu Asn Tyr Leu Arg
690 695 700
agc aaa cgc gaa aaa ttt cat cgc acc tgg acc gaa att ttt aaa gaa 2160
Ser Lys Arg Glu Lys Phe His Arg Thr Trp Thr Glu Ile Phe Lys Glu
705 710 715 720
cat aac ttt agc ttt tat ccg acc ttt cag agc cat ccg aac agc agc 2208
His Asn Phe Ser Phe Tyr Pro Thr Phe Gln Ser His Pro Asn Ser Ser
725 730 735
atg ccg ggc agc cgc gaa gtg cag att cat ccg gat agc gat cag att 2256
Met Pro Gly Ser Arg Glu Val Gln Ile His Pro Asp Ser Asp Gln Ile
740 745 750
agc ggc ctg cat ggc aac agc ttt cat agc gaa gat gaa att gaa tat 2304
Ser Gly Leu His Gly Asn Ser Phe His Ser Glu Asp Glu Ile Glu Tyr
755 760 765
gaa aac cag aaa cgc ctg gaa gaa gaa gaa gat ctg aac gtg ctg acc 2352
Glu Asn Gln Lys Arg Leu Glu Glu Glu Glu Asp Leu Asn Val Leu Thr
770 775 780
ttt gaa gat ctg ctg tgc ttt gcg tat cag gtg gcg aaa ggc atg gaa 2400
Phe Glu Asp Leu Leu Cys Phe Ala Tyr Gln Val Ala Lys Gly Met Glu
785 790 795 800
ttt ctg gaa ttt aaa agc tgc gtg cat cgc gat ctg gcg gcg cgc aac 2448
Phe Leu Glu Phe Lys Ser Cys Val His Arg Asp Leu Ala Ala Arg Asn
805 810 815
gtg ctg gtg acc cat ggc aaa gtg gtg aaa att tgc gat ttt ggc ctg 2496
Val Leu Val Thr His Gly Lys Val Val Lys Ile Cys Asp Phe Gly Leu
820 825 830
gcg cgc gat att atg agc gat agc aac tat gtg gtg cgc ggc aac gcg 2544
Ala Arg Asp Ile Met Ser Asp Ser Asn Tyr Val Val Arg Gly Asn Ala
835 840 845
cgc ctg ccg gtg aaa tgg atg gcg ccg gaa agc ctg ttt gaa ggc att 2592
Arg Leu Pro Val Lys Trp Met Ala Pro Glu Ser Leu Phe Glu Gly Ile
850 855 860
tat acc att aaa agc gat gtg tgg agc tat ggc att ctg ctg tgg gaa 2640
Tyr Thr Ile Lys Ser Asp Val Trp Ser Tyr Gly Ile Leu Leu Trp Glu
865 870 875 880
att ttt agc ctg ggc gtg aac ccg tat ccg ggc att ccg gtg gat gcg 2688
Ile Phe Ser Leu Gly Val Asn Pro Tyr Pro Gly Ile Pro Val Asp Ala
885 890 895
aac ttt tat aaa ctg att cag aac ggc ttt aaa atg gat cag ccg ttt 2736
Asn Phe Tyr Lys Leu Ile Gln Asn Gly Phe Lys Met Asp Gln Pro Phe
900 905 910
tat gcg acc gaa gaa att tat att att atg cag agc tgc tgg gcg ttt 2784
Tyr Ala Thr Glu Glu Ile Tyr Ile Ile Met Gln Ser Cys Trp Ala Phe
915 920 925
gat agc cgc aaa cgc ccg agc ttt ccg aac ctg acc agc ttt ctg ggc 2832
Asp Ser Arg Lys Arg Pro Ser Phe Pro Asn Leu Thr Ser Phe Leu Gly
930 935 940
tgc cag ctg gcg gat gcg gaa gaa gcg atg tat cag aac gtg gat ggc 2880
Cys Gln Leu Ala Asp Ala Glu Glu Ala Met Tyr Gln Asn Val Asp Gly
945 950 955 960
cgc gtg agc gaa tgc ccg cat acc tat cag aac cgc cgc ccg ttt agc 2928
Arg Val Ser Glu Cys Pro His Thr Tyr Gln Asn Arg Arg Pro Phe Ser
965 970 975
cgc gaa atg gat ctg ggc ctg ctg agc ccg cag gcg cag gtg gaa gat 2976
Arg Glu Met Asp Leu Gly Leu Leu Ser Pro Gln Ala Gln Val Glu Asp
980 985 990
agc 2979
Ser
<210> 190
<211> 993
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..2979, 来自SEQ ID NO 189
<400> 190
Met Pro Ala Leu Ala Arg Asp Gly Gly Gln Leu Pro Leu Leu Val Val
1 5 10 15
Phe Ser Ala Met Ile Phe Gly Thr Ile Thr Asn Gln Asp Leu Pro Val
20 25 30
Ile Lys Cys Val Leu Ile Asn His Lys Asn Asn Asp Ser Ser Val Gly
35 40 45
Lys Ser Ser Ser Tyr Pro Met Val Ser Glu Ser Pro Glu Asp Leu Gly
50 55 60
Cys Ala Leu Arg Pro Gln Ser Ser Gly Thr Val Tyr Glu Ala Ala Ala
65 70 75 80
Val Glu Val Asp Val Ser Ala Ser Ile Thr Leu Gln Val Leu Val Asp
85 90 95
Ala Pro Gly Asn Ile Ser Cys Leu Trp Val Phe Lys His Ser Ser Leu
100 105 110
Asn Cys Gln Pro His Phe Asp Leu Gln Asn Arg Gly Val Val Ser Met
115 120 125
Val Ile Leu Lys Met Thr Glu Thr Gln Ala Gly Glu Tyr Leu Leu Phe
130 135 140
Ile Gln Ser Glu Ala Thr Asn Tyr Thr Ile Leu Phe Thr Val Ser Ile
145 150 155 160
Arg Asn Thr Leu Leu Tyr Thr Leu Arg Arg Pro Tyr Phe Arg Lys Met
165 170 175
Glu Asn Gln Asp Ala Leu Val Cys Ile Ser Glu Ser Val Pro Glu Pro
180 185 190
Ile Val Glu Trp Val Leu Cys Asp Ser Gln Gly Glu Ser Cys Lys Glu
195 200 205
Glu Ser Pro Ala Val Val Lys Lys Glu Glu Lys Val Leu His Glu Leu
210 215 220
Phe Gly Thr Asp Ile Arg Cys Cys Ala Arg Asn Glu Leu Gly Arg Glu
225 230 235 240
Cys Thr Arg Leu Phe Thr Ile Asp Leu Asn Gln Thr Pro Gln Thr Thr
245 250 255
Leu Pro Gln Leu Phe Leu Lys Val Gly Glu Pro Leu Trp Ile Arg Cys
260 265 270
Lys Ala Val His Val Asn His Gly Phe Gly Leu Thr Trp Glu Leu Glu
275 280 285
Asn Lys Ala Leu Glu Glu Gly Asn Tyr Phe Glu Met Ser Thr Tyr Ser
290 295 300
Thr Asn Arg Thr Met Ile Arg Ile Leu Phe Ala Phe Val Ser Ser Val
305 310 315 320
Ala Arg Asn Asp Thr Gly Tyr Tyr Thr Cys Ser Ser Ser Lys His Pro
325 330 335
Ser Gln Ser Ala Leu Val Thr Ile Val Glu Lys Gly Phe Ile Asn Ala
340 345 350
Thr Asn Ser Ser Glu Asp Tyr Glu Ile Asp Gln Tyr Glu Glu Phe Cys
355 360 365
Phe Ser Val Arg Phe Lys Ala Tyr Pro Gln Ile Arg Cys Thr Trp Thr
370 375 380
Phe Ser Arg Lys Ser Phe Pro Cys Glu Gln Lys Gly Leu Asp Asn Gly
385 390 395 400
Tyr Ser Ile Ser Lys Phe Cys Asn His Lys His Gln Pro Gly Glu Tyr
405 410 415
Ile Phe His Ala Glu Asn Asp Asp Ala Gln Phe Thr Lys Met Phe Thr
420 425 430
Leu Asn Ile Arg Arg Lys Pro Gln Val Leu Ala Glu Ala Ser Ala Ser
435 440 445
Gln Ala Ser Cys Phe Ser Asp Gly Tyr Pro Leu Pro Ser Trp Thr Trp
450 455 460
Lys Lys Cys Ser Asp Lys Ser Pro Asn Cys Thr Glu Glu Ile Thr Glu
465 470 475 480
Gly Val Trp Asn Arg Lys Ala Asn Arg Lys Val Phe Gly Gln Trp Val
485 490 495
Ser Ser Ser Thr Leu Asn Met Ser Glu Ala Ile Lys Gly Phe Leu Val
500 505 510
Lys Cys Cys Ala Tyr Asn Ser Leu Gly Thr Ser Cys Glu Thr Ile Leu
515 520 525
Leu Asn Ser Pro Gly Pro Phe Pro Phe Ile Gln Asp Asn Ile Ser Phe
530 535 540
Tyr Ala Thr Ile Gly Val Cys Leu Leu Phe Ile Val Val Leu Thr Leu
545 550 555 560
Leu Ile Cys His Lys Tyr Lys Lys Gln Phe Arg Tyr Glu Ser Gln Leu
565 570 575
Gln Met Val Gln Val Thr Gly Ser Ser Asp Asn Glu Tyr Phe Tyr Val
580 585 590
Asp Phe Arg Glu Tyr Glu Tyr Asp Leu Lys Trp Glu Phe Pro Arg Glu
595 600 605
Asn Leu Glu Phe Gly Lys Val Leu Gly Ser Gly Ala Phe Gly Lys Val
610 615 620
Met Asn Ala Thr Ala Tyr Gly Ile Ser Lys Thr Gly Val Ser Ile Gln
625 630 635 640
Val Ala Val Lys Met Leu Lys Glu Lys Ala Asp Ser Ser Glu Arg Glu
645 650 655
Ala Leu Met Ser Glu Leu Lys Met Met Thr Gln Leu Gly Ser His Glu
660 665 670
Asn Ile Val Asn Leu Leu Gly Ala Cys Thr Leu Ser Gly Pro Ile Tyr
675 680 685
Leu Ile Phe Glu Tyr Cys Cys Tyr Gly Asp Leu Leu Asn Tyr Leu Arg
690 695 700
Ser Lys Arg Glu Lys Phe His Arg Thr Trp Thr Glu Ile Phe Lys Glu
705 710 715 720
His Asn Phe Ser Phe Tyr Pro Thr Phe Gln Ser His Pro Asn Ser Ser
725 730 735
Met Pro Gly Ser Arg Glu Val Gln Ile His Pro Asp Ser Asp Gln Ile
740 745 750
Ser Gly Leu His Gly Asn Ser Phe His Ser Glu Asp Glu Ile Glu Tyr
755 760 765
Glu Asn Gln Lys Arg Leu Glu Glu Glu Glu Asp Leu Asn Val Leu Thr
770 775 780
Phe Glu Asp Leu Leu Cys Phe Ala Tyr Gln Val Ala Lys Gly Met Glu
785 790 795 800
Phe Leu Glu Phe Lys Ser Cys Val His Arg Asp Leu Ala Ala Arg Asn
805 810 815
Val Leu Val Thr His Gly Lys Val Val Lys Ile Cys Asp Phe Gly Leu
820 825 830
Ala Arg Asp Ile Met Ser Asp Ser Asn Tyr Val Val Arg Gly Asn Ala
835 840 845
Arg Leu Pro Val Lys Trp Met Ala Pro Glu Ser Leu Phe Glu Gly Ile
850 855 860
Tyr Thr Ile Lys Ser Asp Val Trp Ser Tyr Gly Ile Leu Leu Trp Glu
865 870 875 880
Ile Phe Ser Leu Gly Val Asn Pro Tyr Pro Gly Ile Pro Val Asp Ala
885 890 895
Asn Phe Tyr Lys Leu Ile Gln Asn Gly Phe Lys Met Asp Gln Pro Phe
900 905 910
Tyr Ala Thr Glu Glu Ile Tyr Ile Ile Met Gln Ser Cys Trp Ala Phe
915 920 925
Asp Ser Arg Lys Arg Pro Ser Phe Pro Asn Leu Thr Ser Phe Leu Gly
930 935 940
Cys Gln Leu Ala Asp Ala Glu Glu Ala Met Tyr Gln Asn Val Asp Gly
945 950 955 960
Arg Val Ser Glu Cys Pro His Thr Tyr Gln Asn Arg Arg Pro Phe Ser
965 970 975
Arg Glu Met Asp Leu Gly Leu Leu Ser Pro Gln Ala Gln Val Glu Asp
980 985 990
Ser
<210> 191
<211> 771
<212> DNA
<213> 人工序列
<220>
<223> 人 FOLR1
<220>
<221> CDS
<222> 1..771
<223> /翻译表=1
<400> 191
atg gcg cag cgc atg acc acc cag ctg ctg ctg ctg ctg gtg tgg gtg 48
Met Ala Gln Arg Met Thr Thr Gln Leu Leu Leu Leu Leu Val Trp Val
1 5 10 15
gcg gtg gtg ggc gaa gcg cag acc cgc att gcg tgg gcg cgc acc gaa 96
Ala Val Val Gly Glu Ala Gln Thr Arg Ile Ala Trp Ala Arg Thr Glu
20 25 30
ctg ctg aac gtg tgc atg aac gcg aaa cat cat aaa gaa aaa ccg ggc 144
Leu Leu Asn Val Cys Met Asn Ala Lys His His Lys Glu Lys Pro Gly
35 40 45
ccg gaa gat aaa ctg cat gaa cag tgc cgc ccg tgg cgc aaa aac gcg 192
Pro Glu Asp Lys Leu His Glu Gln Cys Arg Pro Trp Arg Lys Asn Ala
50 55 60
tgc tgc agc acc aac acc agc cag gaa gcg cat aaa gat gtg agc tat 240
Cys Cys Ser Thr Asn Thr Ser Gln Glu Ala His Lys Asp Val Ser Tyr
65 70 75 80
ctg tat cgc ttt aac tgg aac cat tgc ggc gaa atg gcg ccg gcg tgc 288
Leu Tyr Arg Phe Asn Trp Asn His Cys Gly Glu Met Ala Pro Ala Cys
85 90 95
aaa cgc cat ttt att cag gat acc tgc ctg tat gaa tgc agc ccg aac 336
Lys Arg His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn
100 105 110
ctg ggc ccg tgg att cag cag gtg gat cag agc tgg cgc aaa gaa cgc 384
Leu Gly Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg
115 120 125
gtg ctg aac gtg ccg ctg tgc aaa gaa gat tgc gaa cag tgg tgg gaa 432
Val Leu Asn Val Pro Leu Cys Lys Glu Asp Cys Glu Gln Trp Trp Glu
130 135 140
gat tgc cgc acc agc tat acc tgc aaa agc aac tgg cat aaa ggc tgg 480
Asp Cys Arg Thr Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly Trp
145 150 155 160
aac tgg acc agc ggc ttt aac aaa tgc gcg gtg ggc gcg gcg tgc cag 528
Asn Trp Thr Ser Gly Phe Asn Lys Cys Ala Val Gly Ala Ala Cys Gln
165 170 175
ccg ttt cat ttt tat ttt ccg acc ccg acc gtg ctg tgc aac gaa att 576
Pro Phe His Phe Tyr Phe Pro Thr Pro Thr Val Leu Cys Asn Glu Ile
180 185 190
tgg acc cat agc tat aaa gtg agc aac tat agc cgc ggc agc ggc cgc 624
Trp Thr His Ser Tyr Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg
195 200 205
tgc att cag atg tgg ttt gat ccg gcg cag ggc aac ccg aac gaa gaa 672
Cys Ile Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu
210 215 220
gtg gcg cgc ttt tat gcg gcg gcg atg agc ggc gcg ggc ccg tgg gcg 720
Val Ala Arg Phe Tyr Ala Ala Ala Met Ser Gly Ala Gly Pro Trp Ala
225 230 235 240
gcg tgg ccg ttt ctg ctg agc ctg gcg ctg atg ctg ctg tgg ctg ctg 768
Ala Trp Pro Phe Leu Leu Ser Leu Ala Leu Met Leu Leu Trp Leu Leu
245 250 255
agc 771
Ser
<210> 192
<211> 257
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..771, 来自SEQ ID NO 191
<400> 192
Met Ala Gln Arg Met Thr Thr Gln Leu Leu Leu Leu Leu Val Trp Val
1 5 10 15
Ala Val Val Gly Glu Ala Gln Thr Arg Ile Ala Trp Ala Arg Thr Glu
20 25 30
Leu Leu Asn Val Cys Met Asn Ala Lys His His Lys Glu Lys Pro Gly
35 40 45
Pro Glu Asp Lys Leu His Glu Gln Cys Arg Pro Trp Arg Lys Asn Ala
50 55 60
Cys Cys Ser Thr Asn Thr Ser Gln Glu Ala His Lys Asp Val Ser Tyr
65 70 75 80
Leu Tyr Arg Phe Asn Trp Asn His Cys Gly Glu Met Ala Pro Ala Cys
85 90 95
Lys Arg His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn
100 105 110
Leu Gly Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg
115 120 125
Val Leu Asn Val Pro Leu Cys Lys Glu Asp Cys Glu Gln Trp Trp Glu
130 135 140
Asp Cys Arg Thr Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly Trp
145 150 155 160
Asn Trp Thr Ser Gly Phe Asn Lys Cys Ala Val Gly Ala Ala Cys Gln
165 170 175
Pro Phe His Phe Tyr Phe Pro Thr Pro Thr Val Leu Cys Asn Glu Ile
180 185 190
Trp Thr His Ser Tyr Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg
195 200 205
Cys Ile Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu
210 215 220
Val Ala Arg Phe Tyr Ala Ala Ala Met Ser Gly Ala Gly Pro Trp Ala
225 230 235 240
Ala Trp Pro Phe Leu Leu Ser Leu Ala Leu Met Leu Leu Trp Leu Leu
245 250 255
Ser
<210> 193
<211> 969
<212> DNA
<213> 人工序列
<220>
<223> 人 Trop-2
<220>
<221> CDS
<222> 1..969
<223> /翻译表=1
<400> 193
atg gcg cgc ggc ccg ggc ctg gcg ccg ccg ccg ctg cgc ctg ccg ctg 48
Met Ala Arg Gly Pro Gly Leu Ala Pro Pro Pro Leu Arg Leu Pro Leu
1 5 10 15
ctg ctg ctg gtg ctg gcg gcg gtg acc ggc cat acc gcg gcg cag gat 96
Leu Leu Leu Val Leu Ala Ala Val Thr Gly His Thr Ala Ala Gln Asp
20 25 30
aac tgc acc tgc ccg acc aac aaa atg acc gtg tgc agc ccg gat ggc 144
Asn Cys Thr Cys Pro Thr Asn Lys Met Thr Val Cys Ser Pro Asp Gly
35 40 45
ccg ggc ggc cgc tgc cag tgc cgc gcg ctg ggc agc ggc atg gcg gtg 192
Pro Gly Gly Arg Cys Gln Cys Arg Ala Leu Gly Ser Gly Met Ala Val
50 55 60
gat tgc agc acc ctg acc agc aaa tgc ctg ctg ctg aaa gcg cgc atg 240
Asp Cys Ser Thr Leu Thr Ser Lys Cys Leu Leu Leu Lys Ala Arg Met
65 70 75 80
agc gcg ccg aaa aac gcg cgc acc ctg gtg cgc ccg agc gaa cat gcg 288
Ser Ala Pro Lys Asn Ala Arg Thr Leu Val Arg Pro Ser Glu His Ala
85 90 95
ctg gtg gat aac gat ggc ctg tat gat ccg gat tgc gat ccg gaa ggc 336
Leu Val Asp Asn Asp Gly Leu Tyr Asp Pro Asp Cys Asp Pro Glu Gly
100 105 110
cgc ttt aaa gcg cgc cag tgc aac cag acc agc gtg tgc tgg tgc gtg 384
Arg Phe Lys Ala Arg Gln Cys Asn Gln Thr Ser Val Cys Trp Cys Val
115 120 125
aac agc gtg ggc gtg cgc cgc acc gat aaa ggc gat ctg agc ctg cgc 432
Asn Ser Val Gly Val Arg Arg Thr Asp Lys Gly Asp Leu Ser Leu Arg
130 135 140
tgc gat gaa ctg gtg cgc acc cat cat att ctg att gat ctg cgc cat 480
Cys Asp Glu Leu Val Arg Thr His His Ile Leu Ile Asp Leu Arg His
145 150 155 160
cgc ccg acc gcg ggc gcg ttt aac cat agc gat ctg gat gcg gaa ctg 528
Arg Pro Thr Ala Gly Ala Phe Asn His Ser Asp Leu Asp Ala Glu Leu
165 170 175
cgc cgc ctg ttt cgc gaa cgc tat cgc ctg cat ccg aaa ttt gtg gcg 576
Arg Arg Leu Phe Arg Glu Arg Tyr Arg Leu His Pro Lys Phe Val Ala
180 185 190
gcg gtg cat tat gaa cag ccg acc att cag att gaa ctg cgc cag aac 624
Ala Val His Tyr Glu Gln Pro Thr Ile Gln Ile Glu Leu Arg Gln Asn
195 200 205
acc agc cag aaa gcg gcg ggc gat gtg gat att ggc gat gcg gcg tat 672
Thr Ser Gln Lys Ala Ala Gly Asp Val Asp Ile Gly Asp Ala Ala Tyr
210 215 220
tat ttt gaa cgc gat att aaa ggc gaa agc ctg ttt cag ggc cgc ggc 720
Tyr Phe Glu Arg Asp Ile Lys Gly Glu Ser Leu Phe Gln Gly Arg Gly
225 230 235 240
ggc ctg gat ctg cgc gtg cgc ggc gaa ccg ctg cag gtg gaa cgc acc 768
Gly Leu Asp Leu Arg Val Arg Gly Glu Pro Leu Gln Val Glu Arg Thr
245 250 255
ctg att tat tat ctg gat gaa att ccg ccg aaa ttt agc atg aaa cgc 816
Leu Ile Tyr Tyr Leu Asp Glu Ile Pro Pro Lys Phe Ser Met Lys Arg
260 265 270
ctg acc gcg ggc ctg att gcg gtg att gtg gtg gtg gtg gtg gcg ctg 864
Leu Thr Ala Gly Leu Ile Ala Val Ile Val Val Val Val Val Ala Leu
275 280 285
gtg gcg ggc atg gcg gtg ctg gtg att acc aac cgc cgc aaa agc ggc 912
Val Ala Gly Met Ala Val Leu Val Ile Thr Asn Arg Arg Lys Ser Gly
290 295 300
aaa tat aaa aaa gtg gaa att aaa gaa ctg ggc gaa ctg cgc aaa gaa 960
Lys Tyr Lys Lys Val Glu Ile Lys Glu Leu Gly Glu Leu Arg Lys Glu
305 310 315 320
ccg agc ctg 969
Pro Ser Leu
<210> 194
<211> 323
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..969, 来自SEQ ID NO 193
<400> 194
Met Ala Arg Gly Pro Gly Leu Ala Pro Pro Pro Leu Arg Leu Pro Leu
1 5 10 15
Leu Leu Leu Val Leu Ala Ala Val Thr Gly His Thr Ala Ala Gln Asp
20 25 30
Asn Cys Thr Cys Pro Thr Asn Lys Met Thr Val Cys Ser Pro Asp Gly
35 40 45
Pro Gly Gly Arg Cys Gln Cys Arg Ala Leu Gly Ser Gly Met Ala Val
50 55 60
Asp Cys Ser Thr Leu Thr Ser Lys Cys Leu Leu Leu Lys Ala Arg Met
65 70 75 80
Ser Ala Pro Lys Asn Ala Arg Thr Leu Val Arg Pro Ser Glu His Ala
85 90 95
Leu Val Asp Asn Asp Gly Leu Tyr Asp Pro Asp Cys Asp Pro Glu Gly
100 105 110
Arg Phe Lys Ala Arg Gln Cys Asn Gln Thr Ser Val Cys Trp Cys Val
115 120 125
Asn Ser Val Gly Val Arg Arg Thr Asp Lys Gly Asp Leu Ser Leu Arg
130 135 140
Cys Asp Glu Leu Val Arg Thr His His Ile Leu Ile Asp Leu Arg His
145 150 155 160
Arg Pro Thr Ala Gly Ala Phe Asn His Ser Asp Leu Asp Ala Glu Leu
165 170 175
Arg Arg Leu Phe Arg Glu Arg Tyr Arg Leu His Pro Lys Phe Val Ala
180 185 190
Ala Val His Tyr Glu Gln Pro Thr Ile Gln Ile Glu Leu Arg Gln Asn
195 200 205
Thr Ser Gln Lys Ala Ala Gly Asp Val Asp Ile Gly Asp Ala Ala Tyr
210 215 220
Tyr Phe Glu Arg Asp Ile Lys Gly Glu Ser Leu Phe Gln Gly Arg Gly
225 230 235 240
Gly Leu Asp Leu Arg Val Arg Gly Glu Pro Leu Gln Val Glu Arg Thr
245 250 255
Leu Ile Tyr Tyr Leu Asp Glu Ile Pro Pro Lys Phe Ser Met Lys Arg
260 265 270
Leu Thr Ala Gly Leu Ile Ala Val Ile Val Val Val Val Val Ala Leu
275 280 285
Val Ala Gly Met Ala Val Leu Val Ile Thr Asn Arg Arg Lys Ser Gly
290 295 300
Lys Tyr Lys Lys Val Glu Ile Lys Glu Leu Gly Glu Leu Arg Lys Glu
305 310 315 320
Pro Ser Leu
<210> 195
<211> 369
<212> DNA
<213> 人工序列
<220>
<223> 人 PSCA
<220>
<221> CDS
<222> 1..369
<223> /翻译表=1
<400> 195
atg aaa gcg gtg ctg ctg gcg ctg ctg atg gcg ggc ctg gcg ctg cag 48
Met Lys Ala Val Leu Leu Ala Leu Leu Met Ala Gly Leu Ala Leu Gln
1 5 10 15
ccg ggc acc gcg ctg ctg tgc tat agc tgc aaa gcg cag gtg agc aac 96
Pro Gly Thr Ala Leu Leu Cys Tyr Ser Cys Lys Ala Gln Val Ser Asn
20 25 30
gaa gat tgc ctg cag gtg gaa aac tgc acc cag ctg ggc gaa cag tgc 144
Glu Asp Cys Leu Gln Val Glu Asn Cys Thr Gln Leu Gly Glu Gln Cys
35 40 45
tgg acc gcg cgc att cgc gcg gtg ggc ctg ctg acc gtg att agc aaa 192
Trp Thr Ala Arg Ile Arg Ala Val Gly Leu Leu Thr Val Ile Ser Lys
50 55 60
ggc tgc agc ctg aac tgc gtg gat gat agc cag gat tat tat gtg ggc 240
Gly Cys Ser Leu Asn Cys Val Asp Asp Ser Gln Asp Tyr Tyr Val Gly
65 70 75 80
aaa aaa aac att acc tgc tgc gat acc gat ctg tgc aac gcg agc ggc 288
Lys Lys Asn Ile Thr Cys Cys Asp Thr Asp Leu Cys Asn Ala Ser Gly
85 90 95
gcg cat gcg ctg cag ccg gcg gcg gcg att ctg gcg ctg ctg ccg gcg 336
Ala His Ala Leu Gln Pro Ala Ala Ala Ile Leu Ala Leu Leu Pro Ala
100 105 110
ctg ggc ctg ctg ctg tgg ggc ccg ggc cag ctg 369
Leu Gly Leu Leu Leu Trp Gly Pro Gly Gln Leu
115 120
<210> 196
<211> 123
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..369, 来自SEQ ID NO 195
<400> 196
Met Lys Ala Val Leu Leu Ala Leu Leu Met Ala Gly Leu Ala Leu Gln
1 5 10 15
Pro Gly Thr Ala Leu Leu Cys Tyr Ser Cys Lys Ala Gln Val Ser Asn
20 25 30
Glu Asp Cys Leu Gln Val Glu Asn Cys Thr Gln Leu Gly Glu Gln Cys
35 40 45
Trp Thr Ala Arg Ile Arg Ala Val Gly Leu Leu Thr Val Ile Ser Lys
50 55 60
Gly Cys Ser Leu Asn Cys Val Asp Asp Ser Gln Asp Tyr Tyr Val Gly
65 70 75 80
Lys Lys Asn Ile Thr Cys Cys Asp Thr Asp Leu Cys Asn Ala Ser Gly
85 90 95
Ala His Ala Leu Gln Pro Ala Ala Ala Ile Leu Ala Leu Leu Pro Ala
100 105 110
Leu Gly Leu Leu Leu Trp Gly Pro Gly Gln Leu
115 120
<210> 197
<211> 1854
<212> DNA
<213> 人工序列
<220>
<223> EGFR
<220>
<221> CDS
<222> 1..1854
<223> /翻译表=1
<400> 197
atg cgc ccg agc ggc acc gcg ggc gcg gcg ctg ctg gcg ctg ctg gcg 48
Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala
1 5 10 15
gcg ctg tgc ccg gcg agc cgc gcg ctg gaa gaa aaa aaa gtg tgc cag 96
Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln
20 25 30
ggc acc agc aac aaa ctg acc cag ctg ggc acc ttt gaa gat cat ttt 144
Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe
35 40 45
ctg agc ctg cag cgc atg ttt aac aac tgc gaa gtg gtg ctg ggc aac 192
Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn
50 55 60
ctg gaa att acc tat gtg cag cgc aac tat gat ctg agc ttt ctg aaa 240
Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys
65 70 75 80
acc att cag gaa gtg gcg ggc tat gtg ctg att gcg ctg aac acc gtg 288
Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val
85 90 95
gaa cgc att ccg ctg gaa aac ctg cag att att cgc ggc aac atg tat 336
Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr
100 105 110
tat gaa aac agc tat gcg ctg gcg gtg ctg agc aac tat gat gcg aac 384
Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn
115 120 125
aaa acc ggc ctg aaa gaa ctg ccg atg cgc aac ctg cag gaa att ctg 432
Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu
130 135 140
cat ggc gcg gtg cgc ttt agc aac aac ccg gcg ctg tgc aac gtg gaa 480
His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu
145 150 155 160
agc att cag tgg cgc gat att gtg agc agc gat ttt ctg agc aac atg 528
Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met
165 170 175
agc atg gat ttt cag aac cat ctg ggc agc tgc cag aaa tgc gat ccg 576
Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro
180 185 190
agc tgc ccg aac ggc agc tgc tgg ggc gcg ggc gaa gaa aac tgc cag 624
Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln
195 200 205
aaa ctg acc aaa att att tgc gcg cag cag tgc agc ggc cgc tgc cgc 672
Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg
210 215 220
ggc aaa agc ccg agc gat tgc tgc cat aac cag tgc gcg gcg ggc tgc 720
Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys
225 230 235 240
acc ggc ccg cgc gaa agc gat tgc ctg gtg tgc cgc aaa ttt cgc gat 768
Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp
245 250 255
gaa gcg acc tgc aaa gat acc tgc ccg ccg ctg atg ctg tat aac ccg 816
Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro
260 265 270
acc acc tat cag atg gat gtg aac ccg gaa ggc aaa tat agc ttt ggc 864
Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly
275 280 285
gcg acc tgc gtg aaa aaa tgc ccg cgc aac tat gtg gtg acc gat cat 912
Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His
290 295 300
ggc agc tgc gtg cgc gcg tgc ggc gcg gat agc tat gaa atg gaa gaa 960
Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu
305 310 315 320
gat ggc gtg cgc aaa tgc aaa aaa tgc gaa ggc ccg tgc cgc aaa gtg 1008
Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val
325 330 335
tgc aac ggc att ggc att ggc gaa ttt aaa gat agc ctg agc att aac 1056
Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn
340 345 350
gcg acc aac att aaa cat ttt aaa aac tgc acc agc att agc ggc gat 1104
Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp
355 360 365
ctg cat att ctg ccg gtg gcg ttt cgc ggc gat agc ttt acc cat acc 1152
Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr
370 375 380
ccg ccg ctg gat ccg cag gaa ctg gat att ctg aaa acc gtg aaa gaa 1200
Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu
385 390 395 400
att acc ggc ttt ctg ctg att cag gcg tgg ccg gaa aac cgc acc gat 1248
Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp
405 410 415
ctg cat gcg ttt gaa aac ctg gaa att att cgc ggc cgc acc aaa cag 1296
Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln
420 425 430
cat ggc cag ttt agc ctg gcg gtg gtg agc ctg aac att acc agc ctg 1344
His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu
435 440 445
ggc ctg cgc agc ctg aaa gaa att agc gat ggc gat gtg att att agc 1392
Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser
450 455 460
ggc aac aaa aac ctg tgc tat gcg aac acc att aac tgg aaa aaa ctg 1440
Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu
465 470 475 480
ttt ggc acc agc ggc cag aaa acc aaa att att agc aac cgc ggc gaa 1488
Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu
485 490 495
aac agc tgc aaa gcg acc ggc cag gtg tgc cat gcg ctg tgc agc ccg 1536
Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro
500 505 510
gaa ggc tgc tgg ggc ccg gaa ccg cgc gat tgc gtg agc tgc cgc aac 1584
Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn
515 520 525
gtg agc cgc ggc cgc gaa tgc gtg gat aaa tgc aac ctg ctg gaa ggc 1632
Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly
530 535 540
gaa ccg cgc gaa ttt gtg gaa aac agc gaa tgc att cag tgc cat ccg 1680
Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro
545 550 555 560
gaa tgc ctg ccg cag gcg atg aac att acc tgc acc ggc cgc ggc ccg 1728
Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro
565 570 575
gat aac tgc att cag tgc gcg cat tat att gat ggc ccg cat tgc gtg 1776
Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val
580 585 590
aaa acc tgc ccg gcg ggc gtg atg ggc gaa aac aac acc ctg gtg tgg 1824
Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp
595 600 605
aaa tat gcg gat gcg ggc cat gtg tgc cat 1854
Lys Tyr Ala Asp Ala Gly His Val Cys His
610 615
<210> 198
<211> 618
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..1854, 来自SEQ ID NO 197
<400> 198
Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala
1 5 10 15
Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln
20 25 30
Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe
35 40 45
Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn
50 55 60
Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys
65 70 75 80
Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val
85 90 95
Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr
100 105 110
Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn
115 120 125
Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu
130 135 140
His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu
145 150 155 160
Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met
165 170 175
Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro
180 185 190
Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln
195 200 205
Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg
210 215 220
Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys
225 230 235 240
Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp
245 250 255
Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro
260 265 270
Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly
275 280 285
Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His
290 295 300
Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu
305 310 315 320
Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val
325 330 335
Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn
340 345 350
Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp
355 360 365
Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr
370 375 380
Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu
385 390 395 400
Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp
405 410 415
Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln
420 425 430
His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu
435 440 445
Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser
450 455 460
Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu
465 470 475 480
Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu
485 490 495
Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro
500 505 510
Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn
515 520 525
Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly
530 535 540
Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro
545 550 555 560
Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro
565 570 575
Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val
580 585 590
Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp
595 600 605
Lys Tyr Ala Asp Ala Gly His Val Cys His
610 615
<210> 199
<211> 1158
<212> DNA
<213> 人工序列
<220>
<223> 卵清蛋白
<220>
<221> CDS
<222> 1..1158
<223> /翻译表=1
<400> 199
atg ggc agc att ggc gcg gcg agc atg gaa ttt tgc ttt gat gtg ttt 48
Met Gly Ser Ile Gly Ala Ala Ser Met Glu Phe Cys Phe Asp Val Phe
1 5 10 15
aaa gaa ctg aaa gtg cat cat gcg aac gaa aac att ttt tat tgc ccg 96
Lys Glu Leu Lys Val His His Ala Asn Glu Asn Ile Phe Tyr Cys Pro
20 25 30
att gcg att atg agc gcg ctg gcg atg gtg tat ctg ggc gcg aaa gat 144
Ile Ala Ile Met Ser Ala Leu Ala Met Val Tyr Leu Gly Ala Lys Asp
35 40 45
agc acc cgc acc cag att aac aaa gtg gtg cgc ttt gat aaa ctg ccg 192
Ser Thr Arg Thr Gln Ile Asn Lys Val Val Arg Phe Asp Lys Leu Pro
50 55 60
ggc ttt ggc gat agc att gaa gcg cag tgc ggc acc agc gtg aac gtg 240
Gly Phe Gly Asp Ser Ile Glu Ala Gln Cys Gly Thr Ser Val Asn Val
65 70 75 80
cat agc agc ctg cgc gat att ctg aac cag att acc aaa ccg aac gat 288
His Ser Ser Leu Arg Asp Ile Leu Asn Gln Ile Thr Lys Pro Asn Asp
85 90 95
gtg tat agc ttt agc ctg gcg agc cgc ctg tat gcg gaa gaa cgc tat 336
Val Tyr Ser Phe Ser Leu Ala Ser Arg Leu Tyr Ala Glu Glu Arg Tyr
100 105 110
ccg att ctg ccg gaa tat ctg cag tgc gtg aaa gaa ctg tat cgc ggc 384
Pro Ile Leu Pro Glu Tyr Leu Gln Cys Val Lys Glu Leu Tyr Arg Gly
115 120 125
ggc ctg gaa ccg att aac ttt cag acc gcg gcg gat cag gcg cgc gaa 432
Gly Leu Glu Pro Ile Asn Phe Gln Thr Ala Ala Asp Gln Ala Arg Glu
130 135 140
ctg att aac agc tgg gtg gaa agc cag acc aac ggc att att cgc aac 480
Leu Ile Asn Ser Trp Val Glu Ser Gln Thr Asn Gly Ile Ile Arg Asn
145 150 155 160
gtg ctg cag ccg agc agc gtg gat agc cag acc gcg atg gtg ctg gtg 528
Val Leu Gln Pro Ser Ser Val Asp Ser Gln Thr Ala Met Val Leu Val
165 170 175
aac gcg att gtg ttt aaa ggc ctg tgg gaa aaa gcg ttt aaa gat gaa 576
Asn Ala Ile Val Phe Lys Gly Leu Trp Glu Lys Ala Phe Lys Asp Glu
180 185 190
gat acc cag gcg atg ccg ttt cgc gtg acc gaa cag gaa agc aaa ccg 624
Asp Thr Gln Ala Met Pro Phe Arg Val Thr Glu Gln Glu Ser Lys Pro
195 200 205
gtg cag atg atg tat cag att ggc ctg ttt cgc gtg gcg agc atg gcg 672
Val Gln Met Met Tyr Gln Ile Gly Leu Phe Arg Val Ala Ser Met Ala
210 215 220
agc gaa aaa atg aaa att ctg gaa ctg ccg ttt gcg agc ggc acc atg 720
Ser Glu Lys Met Lys Ile Leu Glu Leu Pro Phe Ala Ser Gly Thr Met
225 230 235 240
agc atg ctg gtg ctg ctg ccg gat gaa gtg agc ggc ctg gaa cag ctg 768
Ser Met Leu Val Leu Leu Pro Asp Glu Val Ser Gly Leu Glu Gln Leu
245 250 255
gaa agc att att aac ttt gaa aaa ctg acc gaa tgg acc agc agc aac 816
Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Glu Trp Thr Ser Ser Asn
260 265 270
gtg atg gaa gaa cgc aaa att aaa gtg tat ctg ccg cgc atg aaa atg 864
Val Met Glu Glu Arg Lys Ile Lys Val Tyr Leu Pro Arg Met Lys Met
275 280 285
gaa gaa aaa tat aac ctg acc agc gtg ctg atg gcg atg ggc att acc 912
Glu Glu Lys Tyr Asn Leu Thr Ser Val Leu Met Ala Met Gly Ile Thr
290 295 300
gat gtg ttt agc agc agc gcg aac ctg agc ggc att agc agc gcg gaa 960
Asp Val Phe Ser Ser Ser Ala Asn Leu Ser Gly Ile Ser Ser Ala Glu
305 310 315 320
agc ctg aaa att agc cag gcg gtg cat gcg gcg cat gcg gaa att aac 1008
Ser Leu Lys Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile Asn
325 330 335
gaa gcg ggc cgc gaa gtg gtg ggc agc gcg gaa gcg ggc gtg gat gcg 1056
Glu Ala Gly Arg Glu Val Val Gly Ser Ala Glu Ala Gly Val Asp Ala
340 345 350
gcg agc gtg agc gaa gaa ttt cgc gcg gat cat ccg ttt ctg ttt tgc 1104
Ala Ser Val Ser Glu Glu Phe Arg Ala Asp His Pro Phe Leu Phe Cys
355 360 365
att aaa cat att gcg acc aac gcg gtg ctg ttt ttt ggc cgc tgc gtg 1152
Ile Lys His Ile Ala Thr Asn Ala Val Leu Phe Phe Gly Arg Cys Val
370 375 380
agc ccg 1158
Ser Pro
385
<210> 200
<211> 386
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..1158, 来自SEQ ID NO 199
<400> 200
Met Gly Ser Ile Gly Ala Ala Ser Met Glu Phe Cys Phe Asp Val Phe
1 5 10 15
Lys Glu Leu Lys Val His His Ala Asn Glu Asn Ile Phe Tyr Cys Pro
20 25 30
Ile Ala Ile Met Ser Ala Leu Ala Met Val Tyr Leu Gly Ala Lys Asp
35 40 45
Ser Thr Arg Thr Gln Ile Asn Lys Val Val Arg Phe Asp Lys Leu Pro
50 55 60
Gly Phe Gly Asp Ser Ile Glu Ala Gln Cys Gly Thr Ser Val Asn Val
65 70 75 80
His Ser Ser Leu Arg Asp Ile Leu Asn Gln Ile Thr Lys Pro Asn Asp
85 90 95
Val Tyr Ser Phe Ser Leu Ala Ser Arg Leu Tyr Ala Glu Glu Arg Tyr
100 105 110
Pro Ile Leu Pro Glu Tyr Leu Gln Cys Val Lys Glu Leu Tyr Arg Gly
115 120 125
Gly Leu Glu Pro Ile Asn Phe Gln Thr Ala Ala Asp Gln Ala Arg Glu
130 135 140
Leu Ile Asn Ser Trp Val Glu Ser Gln Thr Asn Gly Ile Ile Arg Asn
145 150 155 160
Val Leu Gln Pro Ser Ser Val Asp Ser Gln Thr Ala Met Val Leu Val
165 170 175
Asn Ala Ile Val Phe Lys Gly Leu Trp Glu Lys Ala Phe Lys Asp Glu
180 185 190
Asp Thr Gln Ala Met Pro Phe Arg Val Thr Glu Gln Glu Ser Lys Pro
195 200 205
Val Gln Met Met Tyr Gln Ile Gly Leu Phe Arg Val Ala Ser Met Ala
210 215 220
Ser Glu Lys Met Lys Ile Leu Glu Leu Pro Phe Ala Ser Gly Thr Met
225 230 235 240
Ser Met Leu Val Leu Leu Pro Asp Glu Val Ser Gly Leu Glu Gln Leu
245 250 255
Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Glu Trp Thr Ser Ser Asn
260 265 270
Val Met Glu Glu Arg Lys Ile Lys Val Tyr Leu Pro Arg Met Lys Met
275 280 285
Glu Glu Lys Tyr Asn Leu Thr Ser Val Leu Met Ala Met Gly Ile Thr
290 295 300
Asp Val Phe Ser Ser Ser Ala Asn Leu Ser Gly Ile Ser Ser Ala Glu
305 310 315 320
Ser Leu Lys Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile Asn
325 330 335
Glu Ala Gly Arg Glu Val Val Gly Ser Ala Glu Ala Gly Val Asp Ala
340 345 350
Ala Ser Val Ser Glu Glu Phe Arg Ala Asp His Pro Phe Leu Phe Cys
355 360 365
Ile Lys His Ile Ala Thr Asn Ala Val Leu Phe Phe Gly Arg Cys Val
370 375 380
Ser Pro
385
<210> 201
<211> 945
<212> DNA
<213> 人工序列
<220>
<223> EpCAM
<220>
<221> CDS
<222> 1..945
<223> /翻译表=1
<400> 201
atg gcg ggc ccg cag gcg ctg gcg ttt ggc ctg ctg ctg gcg gtg gtg 48
Met Ala Gly Pro Gln Ala Leu Ala Phe Gly Leu Leu Leu Ala Val Val
1 5 10 15
acc gcg acc ctg gcg gcg gcg cag cgc gat tgc gtg tgc gat aac tat 96
Thr Ala Thr Leu Ala Ala Ala Gln Arg Asp Cys Val Cys Asp Asn Tyr
20 25 30
aaa ctg gcg acc agc tgc agc ctg aac gaa tat ggc gaa tgc cag tgc 144
Lys Leu Ala Thr Ser Cys Ser Leu Asn Glu Tyr Gly Glu Cys Gln Cys
35 40 45
acc agc tat ggc acc cag aac acc gtg att tgc agc aaa ctg gcg agc 192
Thr Ser Tyr Gly Thr Gln Asn Thr Val Ile Cys Ser Lys Leu Ala Ser
50 55 60
aaa tgc ctg gcg atg aaa gcg gaa atg acc cat agc aaa agc ggc cgc 240
Lys Cys Leu Ala Met Lys Ala Glu Met Thr His Ser Lys Ser Gly Arg
65 70 75 80
cgc att aaa ccg gaa ggc gcg att cag aac aac gat ggc ctg tat gat 288
Arg Ile Lys Pro Glu Gly Ala Ile Gln Asn Asn Asp Gly Leu Tyr Asp
85 90 95
ccg gat tgc gat gaa cag ggc ctg ttt aaa gcg aaa cag tgc aac ggc 336
Pro Asp Cys Asp Glu Gln Gly Leu Phe Lys Ala Lys Gln Cys Asn Gly
100 105 110
acc gcg acc tgc tgg tgc gtg aac acc gcg ggc gtg cgc cgc acc gat 384
Thr Ala Thr Cys Trp Cys Val Asn Thr Ala Gly Val Arg Arg Thr Asp
115 120 125
aaa gat acc gaa att acc tgc agc gaa cgc gtg cgc acc tat tgg att 432
Lys Asp Thr Glu Ile Thr Cys Ser Glu Arg Val Arg Thr Tyr Trp Ile
130 135 140
att att gaa ctg aaa cat aaa gaa cgc gaa agc ccg tat gat cat cag 480
Ile Ile Glu Leu Lys His Lys Glu Arg Glu Ser Pro Tyr Asp His Gln
145 150 155 160
agc ctg cag acc gcg ctg cag gaa gcg ttt acc agc cgc tat aaa ctg 528
Ser Leu Gln Thr Ala Leu Gln Glu Ala Phe Thr Ser Arg Tyr Lys Leu
165 170 175
aac cag aaa ttt att aaa aac att atg tat gaa aac aac gtg att acc 576
Asn Gln Lys Phe Ile Lys Asn Ile Met Tyr Glu Asn Asn Val Ile Thr
180 185 190
att gat ctg atg cag aac agc agc cag aaa acc cag gat gat gtg gat 624
Ile Asp Leu Met Gln Asn Ser Ser Gln Lys Thr Gln Asp Asp Val Asp
195 200 205
att gcg gat gtg gcg tat tat ttt gaa aaa gat gtg aaa ggc gaa agc 672
Ile Ala Asp Val Ala Tyr Tyr Phe Glu Lys Asp Val Lys Gly Glu Ser
210 215 220
ctg ttt cat agc agc aaa agc atg gat ctg cgc gtg aac ggc gaa ccg 720
Leu Phe His Ser Ser Lys Ser Met Asp Leu Arg Val Asn Gly Glu Pro
225 230 235 240
ctg gat ctg gat ccg ggc cag acc ctg att tat tat gtg gat gaa aaa 768
Leu Asp Leu Asp Pro Gly Gln Thr Leu Ile Tyr Tyr Val Asp Glu Lys
245 250 255
gcg ccg gaa ttt agc atg cag ggc ctg acc gcg ggc att att gcg gtg 816
Ala Pro Glu Phe Ser Met Gln Gly Leu Thr Ala Gly Ile Ile Ala Val
260 265 270
att gtg gtg gtg agc ctg gcg gtg att gcg ggc att gtg gtg ctg gtg 864
Ile Val Val Val Ser Leu Ala Val Ile Ala Gly Ile Val Val Leu Val
275 280 285
att agc acc cgc aaa aaa agc gcg aaa tat gaa aaa gcg gaa att aaa 912
Ile Ser Thr Arg Lys Lys Ser Ala Lys Tyr Glu Lys Ala Glu Ile Lys
290 295 300
gaa atg ggc gaa att cat cgc gaa ctg aac gcg 945
Glu Met Gly Glu Ile His Arg Glu Leu Asn Ala
305 310 315
<210> 202
<211> 315
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..945, 来自SEQ ID NO 201
<400> 202
Met Ala Gly Pro Gln Ala Leu Ala Phe Gly Leu Leu Leu Ala Val Val
1 5 10 15
Thr Ala Thr Leu Ala Ala Ala Gln Arg Asp Cys Val Cys Asp Asn Tyr
20 25 30
Lys Leu Ala Thr Ser Cys Ser Leu Asn Glu Tyr Gly Glu Cys Gln Cys
35 40 45
Thr Ser Tyr Gly Thr Gln Asn Thr Val Ile Cys Ser Lys Leu Ala Ser
50 55 60
Lys Cys Leu Ala Met Lys Ala Glu Met Thr His Ser Lys Ser Gly Arg
65 70 75 80
Arg Ile Lys Pro Glu Gly Ala Ile Gln Asn Asn Asp Gly Leu Tyr Asp
85 90 95
Pro Asp Cys Asp Glu Gln Gly Leu Phe Lys Ala Lys Gln Cys Asn Gly
100 105 110
Thr Ala Thr Cys Trp Cys Val Asn Thr Ala Gly Val Arg Arg Thr Asp
115 120 125
Lys Asp Thr Glu Ile Thr Cys Ser Glu Arg Val Arg Thr Tyr Trp Ile
130 135 140
Ile Ile Glu Leu Lys His Lys Glu Arg Glu Ser Pro Tyr Asp His Gln
145 150 155 160
Ser Leu Gln Thr Ala Leu Gln Glu Ala Phe Thr Ser Arg Tyr Lys Leu
165 170 175
Asn Gln Lys Phe Ile Lys Asn Ile Met Tyr Glu Asn Asn Val Ile Thr
180 185 190
Ile Asp Leu Met Gln Asn Ser Ser Gln Lys Thr Gln Asp Asp Val Asp
195 200 205
Ile Ala Asp Val Ala Tyr Tyr Phe Glu Lys Asp Val Lys Gly Glu Ser
210 215 220
Leu Phe His Ser Ser Lys Ser Met Asp Leu Arg Val Asn Gly Glu Pro
225 230 235 240
Leu Asp Leu Asp Pro Gly Gln Thr Leu Ile Tyr Tyr Val Asp Glu Lys
245 250 255
Ala Pro Glu Phe Ser Met Gln Gly Leu Thr Ala Gly Ile Ile Ala Val
260 265 270
Ile Val Val Val Ser Leu Ala Val Ile Ala Gly Ile Val Val Leu Val
275 280 285
Ile Ser Thr Arg Lys Lys Ser Ala Lys Tyr Glu Lys Ala Glu Ile Lys
290 295 300
Glu Met Gly Glu Ile His Arg Glu Leu Asn Ala
305 310 315
<210> 203
<211> 255
<212> PRT
<213> 人（Homo sapiens）
<220>
<223> 肿瘤坏死因子受体超家族成员9 (4-1-BB)
<400> 203
Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu
1 5 10 15
Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro
20 25 30
Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys
35 40 45
Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile
50 55 60
Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser
65 70 75 80
Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly
85 90 95
Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu
100 105 110
Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln
115 120 125
Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys
130 135 140
Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro
145 150 155 160
Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala
165 170 175
Pro Ala Arg Glu Pro Gly His Ser Pro Gln Ile Ile Ser Phe Phe Leu
180 185 190
Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu
195 200 205
Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe
210 215 220
Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly
225 230 235 240
Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
245 250 255
<210> 204
<211> 256
<212> PRT
<213> 小鼠（Mus musculus）
<220>
<223> 肿瘤坏死因子受体超家族成员9 (4-1-BB)
<400> 204
Met Gly Asn Asn Cys Tyr Asn Val Val Val Ile Val Leu Leu Leu Val
1 5 10 15
Gly Cys Glu Lys Val Gly Ala Val Gln Asn Ser Cys Asp Asn Cys Gln
20 25 30
Pro Gly Thr Phe Cys Arg Lys Tyr Asn Pro Val Cys Lys Ser Cys Pro
35 40 45
Pro Ser Thr Phe Ser Ser Ile Gly Gly Gln Pro Asn Cys Asn Ile Cys
50 55 60
Arg Val Cys Ala Gly Tyr Phe Arg Phe Lys Lys Phe Cys Ser Ser Thr
65 70 75 80
His Asn Ala Glu Cys Glu Cys Ile Glu Gly Phe His Cys Leu Gly Pro
85 90 95
Gln Cys Thr Arg Cys Glu Lys Asp Cys Arg Pro Gly Gln Glu Leu Thr
100 105 110
Lys Gln Gly Cys Lys Thr Cys Ser Leu Gly Thr Phe Asn Asp Gln Asn
115 120 125
Gly Thr Gly Val Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Arg
130 135 140
Ser Val Leu Lys Thr Gly Thr Thr Glu Lys Asp Val Val Cys Gly Pro
145 150 155 160
Pro Val Val Ser Phe Ser Pro Ser Thr Thr Ile Ser Val Thr Pro Glu
165 170 175
Gly Gly Pro Gly Gly His Ser Leu Gln Val Leu Thr Leu Phe Leu Ala
180 185 190
Leu Thr Ser Ala Leu Leu Leu Ala Leu Ile Phe Ile Thr Leu Leu Phe
195 200 205
Ser Val Leu Lys Trp Ile Arg Lys Lys Phe Pro His Ile Phe Lys Gln
210 215 220
Pro Phe Lys Lys Thr Thr Gly Ala Ala Gln Glu Glu Asp Ala Cys Ser
225 230 235 240
Cys Arg Cys Pro Gln Glu Glu Glu Gly Gly Gly Gly Gly Tyr Glu Leu
245 250 255
<210> 205
<211> 84
<212> DNA
<213> 人工序列
<220>
<223> IgK 前导序列
<220>
<221> CDS
<222> 1..84
<223> /翻译表=1
<400> 205
atg gaa aca gat aca ctg ctg ctg tgg gtg ctg ctg ctg tgg gtg cca 48
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
gga tct aca ggg gat ggc gcc cag cct gct aga agc 84
Gly Ser Thr Gly Asp Gly Ala Gln Pro Ala Arg Ser
20 25
<210> 206
<211> 28
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..84, 来自SEQ ID NO 205
<400> 206
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Asp Gly Ala Gln Pro Ala Arg Ser
20 25
<210> 207
<211> 2055
<212> DNA
<213> 人工序列
<220>
<223> MR1.1 EGFRvIII VH-Ck-(G4S)2 MCSP M4-3 VH CH1 EE Fc 节 PG LALA,
pETR16621
<220>
<221> CDS
<222> 1..2055
<223> /翻译表=1
<400> 207
caa gtg aag ctg cag cag agt ggg ggc gga ctc gtg aaa cct ggc gcc 48
Gln Val Lys Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Ala
1 5 10 15
tct ctg aag ctg agc tgc gtg acc agc ggc ttc acc ttc aga aag ttc 96
Ser Leu Lys Leu Ser Cys Val Thr Ser Gly Phe Thr Phe Arg Lys Phe
20 25 30
ggc atg agc tgg gtg cgc cag acc agc gac aag cgg ctg gaa tgg gtg 144
Gly Met Ser Trp Val Arg Gln Thr Ser Asp Lys Arg Leu Glu Trp Val
35 40 45
gcc agc atc agc acc ggc ggc tac aac acc tac tac agc gac aac gtg 192
Ala Ser Ile Ser Thr Gly Gly Tyr Asn Thr Tyr Tyr Ser Asp Asn Val
50 55 60
aag ggc cgg ttc acc atc agc aga gag aac gcc aag aac acc ctg tac 240
Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
ctg cag atg agc agc ctg aag tcc gag gac acc gcc ctg tac tac tgc 288
Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
acc aga ggc tac agc ccc tac agc tac gcc atg gac tat tgg ggc cag 336
Thr Arg Gly Tyr Ser Pro Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
ggc acc acc gtg acc gtg tca tct gct agc gtg gcc gct ccc tcc gtg 384
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val
115 120 125
ttc atc ttc cca cct tcc gac gag cag ctg aag tcc ggc acc gct tct 432
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser
130 135 140
gtc gtg tgc ctg ctg aac aac ttc tac ccc cgc gag gcc aag gtg cag 480
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln
145 150 155 160
tgg aag gtg gac aac gcc ctg cag tcc ggc aac agc cag gaa tcc gtg 528
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val
165 170 175
acc gag cag gac tcc aag gac agc acc tac tcc ctg tcc tcc acc ctg 576
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu
180 185 190
acc ctg tcc aag gcc gac tac gag aag cac aag gtg tac gcc tgc gaa 624
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu
195 200 205
gtg acc cac cag ggc ctg tct agc ccc gtg acc aag tct ttc aac cgg 672
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg
210 215 220
ggc gag tgc ggt ggc gga ggt tcc gga ggc gga gga tcc gga gga ggg 720
Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
gga tct cag gtg caa ttg cag gaa agc ggc cct ggc ctg gtc aag ccc 768
Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro
245 250 255
agc cag acc ctg agc ctg acc tgc acc gtg tcc ggc ggc agc atc acc 816
Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Thr
260 265 270
agc ggc tat tat tgg aac tgg att cgg cag cac ccc ggc aag ggc ctg 864
Ser Gly Tyr Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu
275 280 285
gaa tgg atc ggc tac atc act ttc gac ggc tct aac aac tac aac ccc 912
Glu Trp Ile Gly Tyr Ile Thr Phe Asp Gly Ser Asn Asn Tyr Asn Pro
290 295 300
agc ctg aag tcc aga gtg acc atc agc cgg gac acc agc aag aac cag 960
Ser Leu Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln
305 310 315 320
ttc agc ctg aag ctg tcc agc gtg aca gcc gcc gac acc gcc gtg tac 1008
Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
325 330 335
tac tgc gcc gac ttc gac tac tgg ggc cag ggc acc ctg gtc acc gtg 1056
Tyr Cys Ala Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
340 345 350
tcc agc gct agc acc aag ggc ccc tcc gtg ttc ccc ctg gcc ccc agc 1104
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
355 360 365
agc aag agc acc agc ggc ggc aca gcc gct ctg ggc tgc ctg gtc gag 1152
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Glu
370 375 380
gac tac ttc ccc gag ccc gtg acc gtg tcc tgg aac agc gga gcc ctg 1200
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
385 390 395 400
acc tcc ggc gtg cac acc ttc ccc gcc gtg ctg cag agt tct ggc ctg 1248
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
405 410 415
tat agc ctg agc agc gtg gtc acc gtg cct tct agc agc ctg ggc acc 1296
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
420 425 430
cag acc tac atc tgc aac gtg aac cac aag ccc agc aac acc aag gtg 1344
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
435 440 445
gac gag aag gtg gag ccc aag agc tgc gac aaa act cac aca tgc cca 1392
Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
450 455 460
ccg tgc cca gca cct gaa gct gca ggg gga ccg tca gtc ttc ctc ttc 1440
Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe
465 470 475 480
ccc cca aaa ccc aag gac acc ctc atg atc tcc cgg acc cct gag gtc 1488
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
485 490 495
aca tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc aag ttc 1536
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
500 505 510
aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag aca aag ccg 1584
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
515 520 525
cgg gag gag cag tac aac agc acg tac cgt gtg gtc agc gtc ctc acc 1632
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
530 535 540
gtc ctg cac cag gac tgg ctg aat ggc aag gag tac aag tgc aag gtc 1680
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
545 550 555 560
tcc aac aaa gcc ctc ggc gcc ccc atc gag aaa acc atc tcc aaa gcc 1728
Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
565 570 575
aaa ggg cag ccc cga gaa cca cag gtg tac acc ctg ccc cca tgc cgg 1776
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg
580 585 590
gat gag ctg acc aag aac cag gtc agc ctg tgg tgc ctg gtc aaa ggc 1824
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly
595 600 605
ttc tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg cag ccg 1872
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
610 615 620
gag aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc 1920
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
625 630 635 640
ttc ttc ctc tac agc aag ctc acc gtg gac aag agc agg tgg cag cag 1968
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
645 650 655
ggg aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac aac cac 2016
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
660 665 670
tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa tga 2055
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
675 680
<210> 208
<211> 684
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..2055, 来自SEQ ID NO 207
<400> 208
Gln Val Lys Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Leu Lys Leu Ser Cys Val Thr Ser Gly Phe Thr Phe Arg Lys Phe
20 25 30
Gly Met Ser Trp Val Arg Gln Thr Ser Asp Lys Arg Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Thr Gly Gly Tyr Asn Thr Tyr Tyr Ser Asp Asn Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Thr Arg Gly Tyr Ser Pro Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val
115 120 125
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser
130 135 140
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln
145 150 155 160
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val
165 170 175
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu
180 185 190
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu
195 200 205
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg
210 215 220
Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro
245 250 255
Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Thr
260 265 270
Ser Gly Tyr Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu
275 280 285
Glu Trp Ile Gly Tyr Ile Thr Phe Asp Gly Ser Asn Asn Tyr Asn Pro
290 295 300
Ser Leu Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln
305 310 315 320
Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
325 330 335
Tyr Cys Ala Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
340 345 350
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
355 360 365
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Glu
370 375 380
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
385 390 395 400
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
405 410 415
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
420 425 430
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
435 440 445
Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
450 455 460
Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe
465 470 475 480
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
485 490 495
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
500 505 510
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
515 520 525
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
530 535 540
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
545 550 555 560
Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
565 570 575
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg
580 585 590
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly
595 600 605
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
610 615 620
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
625 630 635 640
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
645 650 655
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
660 665 670
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
675 680
<210> 209
<211> 639
<212> DNA
<213> 人工序列
<220>
<223> EGFR vIII MR1.1 VL CH1, pETR15656
<220>
<221> CDS
<222> 1..639
<223> /翻译表=1
<400> 209
gat atc gag ctg aca cag agc ccc gcc agc ctg tct gtg gcc acc ggc 48
Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ser Val Ala Thr Gly
1 5 10 15
gag aaa gtg acc atc cgg tgc atg acc agc acc gac atc gac gac gac 96
Glu Lys Val Thr Ile Arg Cys Met Thr Ser Thr Asp Ile Asp Asp Asp
20 25 30
atg aac tgg tat cag cag aag ccc ggc gag ccc ccc aag ttc ctg atc 144
Met Asn Trp Tyr Gln Gln Lys Pro Gly Glu Pro Pro Lys Phe Leu Ile
35 40 45
agc gag ggc aac aca ctg cgg cct ggc gtg cca agc aga ttc agc agc 192
Ser Glu Gly Asn Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Ser
50 55 60
tct ggc acc ggc acc gac ttc gtg ttt acc atc gag aat acc ctg agc 240
Ser Gly Thr Gly Thr Asp Phe Val Phe Thr Ile Glu Asn Thr Leu Ser
65 70 75 80
gag gac gtg ggc gac tac tac tgc ctg cag agc tgg aac gtg ccc ctg 288
Glu Asp Val Gly Asp Tyr Tyr Cys Leu Gln Ser Trp Asn Val Pro Leu
85 90 95
acc ttt ggc gac ggc acc aag ctg gaa atc aag agc agc gct agc acc 336
Thr Phe Gly Asp Gly Thr Lys Leu Glu Ile Lys Ser Ser Ala Ser Thr
100 105 110
aaa ggc cct tcc gtg ttt cct ctg gct cct agc tcc aag tcc acc tct 384
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
115 120 125
gga ggc acc gct gct ctc gga tgc ctc gtg aag gat tat ttt cct gag 432
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
130 135 140
cct gtg aca gtg tcc tgg aat agc gga gca ctg acc tct gga gtg cat 480
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
145 150 155 160
act ttc ccc gct gtg ctg cag tcc tct gga ctg tac agc ctg agc agc 528
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
165 170 175
gtg gtg aca gtg ccc agc agc agc ctg ggc acc cag acc tac atc tgc 576
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
180 185 190
aac gtg aac cac aag ccc agc aac acc aag gtg gac aag aag gtg gaa 624
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
195 200 205
ccc aag tct tgt tga 639
Pro Lys Ser Cys
210
<210> 210
<211> 212
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..639, 来自SEQ ID NO 209
<400> 210
Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ser Val Ala Thr Gly
1 5 10 15
Glu Lys Val Thr Ile Arg Cys Met Thr Ser Thr Asp Ile Asp Asp Asp
20 25 30
Met Asn Trp Tyr Gln Gln Lys Pro Gly Glu Pro Pro Lys Phe Leu Ile
35 40 45
Ser Glu Gly Asn Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Ser
50 55 60
Ser Gly Thr Gly Thr Asp Phe Val Phe Thr Ile Glu Asn Thr Leu Ser
65 70 75 80
Glu Asp Val Gly Asp Tyr Tyr Cys Leu Gln Ser Trp Asn Val Pro Leu
85 90 95
Thr Phe Gly Asp Gly Thr Lys Leu Glu Ile Lys Ser Ser Ala Ser Thr
100 105 110
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
115 120 125
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
130 135 140
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
145 150 155 160
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
165 170 175
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
180 185 190
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
195 200 205
Pro Lys Ser Cys
210
<210> 211
<211> 645
<212> DNA
<213> 人工序列
<220>
<223> MCSP ML2 VL Ck RK, pETR16619
<220>
<221> CDS
<222> 1..645
<223> /翻译表=1
<400> 211
gac atc cag atg acc cag agc ccc agc agc ctg agc gcc agc gtg ggc 48
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
gac aga gtg acc atc acc tgc cgg gcc agc cag ggc atc cgg aac tac 96
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr
20 25 30
ctg aac tgg tat cag cag aag ccc ggc aag gcc ccc aag ctg ctg atc 144
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
tac tac acc agc agc ctg cac agc ggc gtg cct agc cgg ttt agc ggc 192
Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
agc ggc tcc ggc acc gac tac acc ctg acc att agc tcc ctg cag ccc 240
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
gag gac ttc gcc acc tac tac tgc cag cag tac tct gct ctg ccg tgg 288
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ala Leu Pro Trp
85 90 95
acc ttc ggc cag gga aca aag gtg gag atc aag cgt acg gtg gct gca 336
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
cca tct gtc ttc atc ttc ccg cca tct gat cgg aag ttg aaa tct gga 384
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys Ser Gly
115 120 125
act gcc tct gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc 432
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
aaa gta cag tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag 480
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
gag agt gtc aca gag cag gac agc aag gac agc acc tac agc ctc agc 528
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
agc acc ctg acg ctg agc aaa gca gac tac gag aaa cac aaa gtc tac 576
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
gcc tgc gaa gtc acc cat cag ggc ctg agc tcg ccc gtc aca aag agc 624
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
ttc aac agg gga gag tgt tag 645
Phe Asn Arg Gly Glu Cys
210
<210> 212
<211> 214
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..645, 来自SEQ ID NO 211
<400> 212
Asp 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 Gly Ile Arg Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr 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 Ala Leu Pro Trp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 213
<211> 1329
<212> DNA
<213> 人工序列
<220>
<223> MCSP M4-3 VH CH1 EE Fc 穴 PG LALA HYRF, pETR16618
<220>
<221> CDS
<222> 1..1329
<223> /翻译表=1
<400> 213
cag gtg caa ttg cag gaa agc ggc cct ggc ctg gtc aag ccc agc cag 48
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
acc ctg agc ctg acc tgc acc gtg tcc ggc ggc agc atc acc agc ggc 96
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Thr Ser Gly
20 25 30
tat tat tgg aac tgg att cgg cag cac ccc ggc aag ggc ctg gaa tgg 144
Tyr Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp
35 40 45
atc ggc tac atc act ttc gac ggc tct aac aac tac aac ccc agc ctg 192
Ile Gly Tyr Ile Thr Phe Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu
50 55 60
aag tcc aga gtg acc atc agc cgg gac acc agc aag aac cag ttc agc 240
Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
ctg aag ctg tcc agc gtg aca gcc gcc gac acc gcc gtg tac tac tgc 288
Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
gcc gac ttc gac tac tgg ggc cag ggc acc ctg gtc acc gtg tcc agc 336
Ala Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
100 105 110
gct agc acc aag ggc ccc tcc gtg ttc ccc ctg gcc ccc agc agc aag 384
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
115 120 125
agc acc agc ggc ggc aca gcc gct ctg ggc tgc ctg gtc gag gac tac 432
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Glu Asp Tyr
130 135 140
ttc ccc gag ccc gtg acc gtg tcc tgg aac agc gga gcc ctg acc tcc 480
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
145 150 155 160
ggc gtg cac acc ttc ccc gcc gtg ctg cag agt tct ggc ctg tat agc 528
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
165 170 175
ctg agc agc gtg gtc acc gtg cct tct agc agc ctg ggc acc cag acc 576
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
180 185 190
tac atc tgc aac gtg aac cac aag ccc agc aac acc aag gtg gac gag 624
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Glu
195 200 205
aag gtg gag ccc aag agc tgc gac aaa act cac aca tgc cca ccg tgc 672
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
210 215 220
cca gca cct gaa gct gca ggg gga ccg tca gtc ttc ctc ttc ccc cca 720
Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
225 230 235 240
aaa ccc aag gac acc ctc atg atc tcc cgg acc cct gag gtc aca tgc 768
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
245 250 255
gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc aag ttc aac tgg 816
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
260 265 270
tac gtg gac ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg gag 864
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
275 280 285
gag cag tac aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg 912
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
290 295 300
cac cag gac tgg ctg aat ggc aag gag tac aag tgc aag gtc tcc aac 960
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
305 310 315 320
aaa gcc ctc ggc gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggg 1008
Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
325 330 335
cag ccc cga gaa cca cag gtg tgc acc ctg ccc cca tcc cgg gat gag 1056
Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu
340 345 350
ctg acc aag aac cag gtc agc ctc tcg tgc gca gtc aaa ggc ttc tat 1104
Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr
355 360 365
ccc agc gac atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac 1152
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
370 375 380
aac tac aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc 1200
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
385 390 395 400
ctc gtg agc aag ctc acc gtg gac aag agc agg tgg cag cag ggg aac 1248
Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
405 410 415
gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac aac cgc ttc acg 1296
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr
420 425 430
cag aag agc ctc tcc ctg tct ccg ggt aaa tga 1329
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440
<210> 214
<211> 442
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..1329, 来自SEQ ID NO 213
<400> 214
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Thr Ser Gly
20 25 30
Tyr Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp
35 40 45
Ile Gly Tyr Ile Thr Phe Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
100 105 110
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
115 120 125
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Glu Asp Tyr
130 135 140
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
145 150 155 160
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
165 170 175
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
180 185 190
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Glu
195 200 205
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
210 215 220
Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
225 230 235 240
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
245 250 255
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
260 265 270
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
275 280 285
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
290 295 300
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
305 310 315 320
Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
325 330 335
Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu
340 345 350
Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr
355 360 365
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
370 375 380
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
385 390 395 400
Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
405 410 415
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr
420 425 430
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440
<210> 215
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> EGFRvIII MR1.1 CDR H1 Kabat
<400> 215
Lys Phe Gly Met Ser
1 5
<210> 216
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> EGFRvIII MR1.1 CDR H2 Kabat
<400> 216
Ser Ile Ser Thr Gly Gly Tyr Asn Thr Tyr Tyr Ser Asp Asn Val Lys
1 5 10 15
Gly
<210> 217
<211> 11
<212> PRT
<213> 人工序列
<220>
<223> EGFRvIII MR1.1 CDR H3 Kabat
<400> 217
Gly Tyr Ser Pro Tyr Ser Tyr Ala Met Asp Tyr
1 5 10
<210> 218
<211> 11
<212> PRT
<213> 人工序列
<220>
<223> EGFRvIII MR1.1 CDR1 L1 Kabat
<400> 218
Met Thr Ser Thr Asp Ile Asp Asp Asp Met Asn
1 5 10
<210> 219
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> EGFRvIII MR1.1 CDR L2 Kabat
<400> 219
Glu Gly Asn Thr Leu Arg Pro
1 5
<210> 220
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> EGFRvIII MR1.1 CDR L3 Kabat
<400> 220
Leu Gln Ser Trp Asn Val Pro Leu Thr
1 5
<210> 221
<211> 6
<212> PRT
<213> 人工序列
<220>
<223> MCSP CDR H1 Kabat
<400> 221
Ser Gly Tyr Tyr Trp Asn
1 5
<210> 222
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> MCSP CDR H2 Kabat
<400> 222
Tyr Ile Thr Phe Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu Lys Ser
1 5 10 15
<210> 223
<211> 4
<212> PRT
<213> 人工序列
<220>
<223> MCSP CDR H3 Kabat
<400> 223
Phe Asp Tyr Tyr
1
<210> 224
<211> 11
<212> PRT
<213> 人工序列
<220>
<223> MCSP CDR1 L1 Kabat
<400> 224
Arg Ala Ser Gln Gly Ile Arg Asn Tyr Leu Asn
1 5 10
<210> 225
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> MCSP CDR L2 Kabat
<400> 225
Tyr Thr Ser Ser Leu His Ser
1 5
<210> 226
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> MCSP CDR L3 Kabat
<400> 226
Gln Gln Tyr Ser Ala Leu Pro Trp Thr
1 5
<210> 227
<211> 768
<212> DNA
<213> 人（Homo sapiens）
<220>
<223> 肿瘤坏死因子受体超家族成员9 (4-1-BB)
<400> 227
atgggcaaca actgctataa cgtggtggtg attgtgctgc tgctggtggg ctgcgaaaaa 60
gtgggcgcgg tgcagaacag ctgcgataac tgccagccgg gcaccttttg ccgcaaatat 120
aacccggtgt gcaaaagctg cccgccgagc acctttagca gcattggcgg ccagccgaac 180
tgcaacattt gccgcgtgtg cgcgggctat tttcgcttta aaaaattttg cagcagcacc 240
cataacgcgg aatgcgaatg cattgaaggc tttcattgcc tgggcccgca gtgcacccgc 300
tgcgaaaaag attgccgccc gggccaggaa ctgaccaaac agggctgcaa aacctgcagc 360
ctgggcacct ttaacgatca gaacggcacc ggcgtgtgcc gcccgtggac caactgcagc 420
ctggatggcc gcagcgtgct gaaaaccggc accaccgaaa aagatgtggt gtgcggcccg 480
ccggtggtga gctttagccc gagcaccacc attagcgtga ccccggaagg cggcccgggc 540
ggccatagcc tgcaggtgct gaccctgttt ctggcgctga ccagcgcgct gctgctggcg 600
ctgattttta ttaccctgct gtttagcgtg ctgaaatgga ttcgcaaaaa atttccgcat 660
atttttaaac agccgtttaa aaaaaccacc ggcgcggcgc aggaagaaga tgcgtgcagc 720
tgccgctgcc cgcaggaaga agaaggcggc ggcggcggct atgaactg 768
<210> 228
<211> 765
<212> DNA
<213> 小鼠（Mus musculus）
<220>
<223> 肿瘤坏死因子受体超家族成员9 (4-1-BB)
<400> 228
atgggcaaca gctgctataa cattgtggcg accctgctgc tggtgctgaa ctttgaacgc 60
acccgcagcc tgcaggatcc gtgcagcaac tgcccggcgg gcaccttttg cgataacaac 120
cgcaaccaga tttgcagccc gtgcccgccg aacagcttta gcagcgcggg cggccagcgc 180
acctgcgata tttgccgcca gtgcaaaggc gtgtttcgca cccgcaaaga atgcagcagc 240
accagcaacg cggaatgcga ttgcaccccg ggctttcatt gcctgggcgc gggctgcagc 300
atgtgcgaac aggattgcaa acagggccag gaactgacca aaaaaggctg caaagattgc 360
tgctttggca cctttaacga tcagaaacgc ggcatttgcc gcccgtggac caactgcagc 420
ctggatggca aaagcgtgct ggtgaacggc accaaagaac gcgatgtggt gtgcggcccg 480
agcccggcgg atctgagccc gggcgcgagc agcgtgaccc cgccggcgcc ggcgcgcgaa 540
ccgggccata gcccgcagat tattagcttt tttctggcgc tgaccagcac cgcgctgctg 600
tttctgctgt tttttctgac cctgcgcttt agcgtggtga aacgcggccg caaaaaactg 660
ctgtatattt ttaaacagcc gtttatgcgc ccggtgcaga ccacccagga agaagatggc 720
tgcagctgcc gctttccgga agaagaagaa ggcggctgcg aactg 765
<210> 229
<211> 214
<212> PRT
<213> 人工序列
<220>
<223> 轻链, 无前导序列
<400> 229
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Glu Thr Val Ser Ile Glu Cys Leu Ala Ser Glu Gly Ile Ser Asn Asp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Ser Gly Lys Ser Pro Gln Leu Leu Ile
35 40 45
Tyr Ala Thr Ser Arg Leu Gln Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Arg Tyr Ser Leu Lys Ile Ser Gly Met Gln Pro
65 70 75 80
Glu Asp Glu Ala Asp Tyr Phe Cys Gln Gln Ser Tyr Lys Tyr Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala
100 105 110
Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly
115 120 125
Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile
130 135 140
Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu
145 150 155 160
Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser
165 170 175
Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr
180 185 190
Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser
195 200 205
Phe Asn Arg Asn Glu Cys
210
<210> 230
<211> 703
<212> PRT
<213> 人工序列
<220>
<223> 重链, 无前导序列
<400> 230
Glu Val Gln Leu Ala Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe
20 25 30
Pro Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Thr Ser Gly Gly Ser Thr Tyr Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Thr Arg Thr Leu Tyr Ile Leu Arg Val Phe Tyr Phe Asp Tyr Trp Gly
100 105 110
Gln Gly Val Met Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser
115 120 125
Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val
130 135 140
Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu
145 150 155 160
Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr
180 185 190
Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro
195 200 205
Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr
210 215 220
Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly
225 230 235 240
Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met
245 250 255
Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu
260 265 270
Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val
275 280 285
His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu
290 295 300
Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly
305 310 315 320
Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile
325 330 335
Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val
340 345 350
Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr
355 360 365
Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu
370 375 380
Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro
385 390 395 400
Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val
405 410 415
Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val
420 425 430
His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr
435 440 445
Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Lys
450 455 460
Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Ala Ser Leu Lys
465 470 475 480
Leu Ser Cys Val Thr Ser Gly Phe Thr Phe Arg Lys Phe Gly Met Ser
485 490 495
Trp Val Arg Gln Thr Ser Asp Lys Arg Leu Glu Trp Val Ala Ser Ile
500 505 510
Ser Thr Gly Gly Tyr Asn Thr Tyr Tyr Ser Asp Asn Val Lys Gly Arg
515 520 525
Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met
530 535 540
Ser Ser Leu Lys Ser Glu Asp Thr Ala Leu Tyr Tyr Cys Thr Arg Gly
545 550 555 560
Tyr Ser Pro Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr
565 570 575
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
580 585 590
Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ser
595 600 605
Val Ala Thr Gly Glu Lys Val Thr Ile Arg Cys Met Thr Ser Thr Asp
610 615 620
Ile Asp Asp Asp Met Asn Trp Tyr Gln Gln Lys Pro Gly Glu Pro Pro
625 630 635 640
Lys Phe Leu Ile Ser Glu Gly Asn Thr Leu Arg Pro Gly Val Pro Ser
645 650 655
Arg Phe Ser Ser Ser Gly Thr Gly Thr Asp Phe Val Phe Thr Ile Glu
660 665 670
Asn Thr Leu Ser Glu Asp Val Gly Asp Tyr Tyr Cys Leu Gln Ser Trp
675 680 685
Asn Val Pro Leu Thr Phe Gly Asp Gly Thr Lys Leu Glu Ile Lys
690 695 700
<210> 231
<211> 1253
<212> DNA
<213> 人工序列
<220>
<223> DNA序列, del-hEGFRvIII
<400> 231
gcggccgcgc caccatgcga ccctccggga cggccggggc agcgctcctg gcgctgctgg 60
ctgcgctctg cccggcgagt cgggctctgg aggaaaagaa aggtaattat gtggtgacag 120
atcacggctc gtgcgtccga gcctgtgggg ccgacagcta tgagatggag gaagacggcg 180
tccgcaagtg taagaagtgc gaagggcctt gccgcaaagt gtgtaacgga ataggtattg 240
gtgaatttaa agactcactc tccataaatg ctacgaatat taaacacttc aaaaactgca 300
cctccatcag tggcgatctc cacatcctgc cggtggcatt taggggtgac tccttcacac 360
atactcctcc tctggatcca caggaactgg atattctgaa aaccgtaaag gaaatcacag 420
ggtttttgct gattcaggct tggcctgaaa acaggacgga cctccatgcc tttgagaacc 480
tagaaatcat acgcggcagg accaagcaac atggtcagtt ttctcttgca gtcgtcagcc 540
tgaacataac atccttggga ttacgctccc tcaaggagat aagtgatgga gatgtgataa 600
tttcaggaaa caaaaatttg tgctatgcaa atacaataaa ctggaaaaaa ctgtttggga 660
cctccggtca gaaaaccaaa attataagca acagaggtga aaacagctgc aaggccacag 720
gccaggtctg ccatgccttg tgctcccccg agggctgctg gggcccggag cccagggact 780
gcgtctcttg ccggaatgtc agccgaggca gggaatgcgt ggacaagtgc aaccttctgg 840
agggtgagcc aagggagttt gtggagaact ctgagtgcat acagtgccac ccagagtgcc 900
tgcctcaggc catgaacatc acctgcacag gacggggacc agacaactgt atccagtgtg 960
cccactacat tgacggcccc cactgcgtca agacctgccc ggcaggagtc atgggagaaa 1020
acaacaccct ggtctggaag tacgcagacg ccggccatgt gtgccacctg tgccatccaa 1080
actgcaccta cggatgcact gggccaggtc ttgaaggctg tccaacgaat gggcctaaga 1140
tcccgtccat cgccactggg atggtggggg ccctcctctt gctgctggtg gtggccctgg 1200
ggatcggcct cttcatgcga aggcgccaca tcgttcggaa gcgctgagaa ttc 1253
<210> 232
<211> 420
<212> PRT
<213> 人工序列
<220>
<223> 蛋白质序列, del-hEGFRvIII
<400> 232
Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala
1 5 10 15
Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Gly Asn Tyr
20 25 30
Val Val Thr Asp His Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser
35 40 45
Tyr Glu Met Glu Thr Glu Glu Asp Gly Val Arg Lys Cys Lys Lys Cys
50 55 60
Glu Gly Pro Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe
65 70 75 80
Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn
85 90 95
Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg
100 105 110
Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp
115 120 125
Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala
130 135 140
Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile
145 150 155 160
Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val
165 170 175
Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser
180 185 190
Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn
195 200 205
Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys
210 215 220
Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val
225 230 235 240
Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg
245 250 255
Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp
260 265 270
Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser
275 280 285
Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Glu Thr
290 295 300
Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala
305 310 315 320
His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val
325 330 335
Met Glu Thr Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala
340 345 350
Gly His Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr
355 360 365
Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser
370 375 380
Ile Ala Thr Gly Met Glu Thr Val Gly Ala Leu Leu Leu Leu Leu Val
385 390 395 400
Val Ala Leu Gly Ile Gly Leu Phe Met Glu Thr Arg Arg Arg His Ile
405 410 415
Val Arg Lys Arg
420
<210> 233
<211> 1779
<212> PRT
<213> 人工序列
<220>
<223> BsAB EGFRvIII-EpCAM
<400> 233
Gln Val Lys Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Leu Lys Leu Ser Cys Val Thr Ser Gly Phe Thr Phe Arg Lys Phe
20 25 30
Gly Met Ser Trp Val Arg Gln Thr Ser Asp Lys Arg Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Thr Gly Gly Tyr Asn Thr Tyr Tyr Ser Asp Asn Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Thr Arg Gly Tyr Ser Pro Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val
115 120 125
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser
130 135 140
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln
145 150 155 160
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val
165 170 175
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu
180 185 190
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu
195 200 205
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg
210 215 220
Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln
225 230 235 240
Leu Ala Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg Ser Met Lys
245 250 255
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe Pro Met Ala
260 265 270
Trp Val Arg Gln Ala Pro Thr Lys Cys Leu Glu Trp Val Ala Thr Ile
275 280 285
Ser Thr Ser Gly Gly Ser Thr Tyr Tyr Arg Asp Ser Val Lys Gly Arg
290 295 300
Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu Tyr Leu Gln Met
305 310 315 320
Asn Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys Thr Arg Thr
325 330 335
Leu Tyr Ile Leu Arg Val Phe Tyr Phe Asp Tyr Trp Gly Gln Gly Val
340 345 350
Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
355 360 365
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
370 375 380
Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
385 390 395 400
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
405 410 415
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
420 425 430
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
435 440 445
Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
450 455 460
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
465 470 475 480
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
485 490 495
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
500 505 510
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
515 520 525
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
530 535 540
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
545 550 555 560
Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr
565 570 575
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
580 585 590
Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys
595 600 605
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
610 615 620
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
625 630 635 640
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
645 650 655
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
660 665 670
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
675 680 685
Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ser Val Ala Thr Gly
690 695 700
Glu Lys Val Thr Ile Arg Cys Met Thr Ser Thr Asp Ile Asp Asp Asp
705 710 715 720
Met Asn Trp Tyr Gln Gln Lys Pro Gly Glu Pro Pro Lys Phe Leu Ile
725 730 735
Ser Glu Gly Asn Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Ser
740 745 750
Ser Gly Thr Gly Thr Asp Phe Val Phe Thr Ile Glu Asn Thr Leu Ser
755 760 765
Glu Asp Val Gly Asp Tyr Tyr Cys Leu Gln Ser Trp Asn Val Pro Leu
770 775 780
Thr Phe Gly Asp Gly Thr Lys Leu Glu Ile Lys Ser Ser Ala Ser Thr
785 790 795 800
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
805 810 815
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
820 825 830
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
835 840 845
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
850 855 860
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
865 870 875 880
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
885 890 895
Pro Lys Ser Cys Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser
900 905 910
Ala Ser Leu Gly Glu Thr Val Ser Ile Glu Cys Leu Ala Ser Glu Gly
915 920 925
Ile Ser Asn Asp Leu Ala Trp Tyr Gln Gln Lys Ser Gly Lys Ser Pro
930 935 940
Gln Leu Leu Ile Tyr Ala Thr Ser Arg Leu Gln Asp Gly Val Pro Ser
945 950 955 960
Arg Phe Ser Gly Ser Gly Ser Gly Thr Arg Tyr Ser Leu Lys Ile Ser
965 970 975
Gly Met Gln Pro Glu Asp Glu Ala Asp Tyr Phe Cys Gln Gln Ser Tyr
980 985 990
Lys Tyr Pro Trp Thr Phe Gly Cys Gly Thr Lys Leu Glu Leu Lys Arg
995 1000 1005
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys
1010 1015 1020
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
1025 1030 1035 1040
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
1045 1050 1055
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
1060 1065 1070
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
1075 1080 1085
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
1090 1095 1100
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Asp Ile Gln Met Thr Gln
1105 1110 1115 1120
Ser Pro Ala Ser Leu Ser Ala Ser Leu Gly Glu Thr Val Ser Ile Glu
1125 1130 1135
Cys Leu Ala Ser Glu Gly Ile Ser Asn Asp Leu Ala Trp Tyr Gln Gln
1140 1145 1150
Lys Ser Gly Lys Ser Pro Gln Leu Leu Ile Tyr Ala Thr Ser Arg Leu
1155 1160 1165
Gln Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Arg
1170 1175 1180
Tyr Ser Leu Lys Ile Ser Gly Met Gln Pro Glu Asp Glu Ala Asp Tyr
1185 1190 1195 1200
Phe Cys Gln Gln Ser Tyr Lys Tyr Pro Trp Thr Phe Gly Cys Gly Thr
1205 1210 1215
Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe
1220 1225 1230
Pro Pro Ser Asp Arg Lys Leu Lys Ser Gly Thr Ala Ser Val Val Cys
1235 1240 1245
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val
1250 1255 1260
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln
1265 1270 1275 1280
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser
1285 1290 1295
Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His
1300 1305 1310
Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
1315 1320 1325
Glu Val Gln Leu Ala Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1330 1335 1340
Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe
1345 1350 1355 1360
Pro Met Ala Trp Val Arg Gln Ala Pro Thr Lys Cys Leu Glu Trp Val
1365 1370 1375
Ala Thr Ile Ser Thr Ser Gly Gly Ser Thr Tyr Tyr Arg Asp Ser Val
1380 1385 1390
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu Tyr
1395 1400 1405
Leu Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
1410 1415 1420
Thr Arg Thr Leu Tyr Ile Leu Arg Val Phe Tyr Phe Asp Tyr Trp Gly
1425 1430 1435 1440
Gln Gly Val Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
1445 1450 1455
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
1460 1465 1470
Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val
1475 1480 1485
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
1490 1495 1500
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
1505 1510 1515 1520
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
1525 1530 1535
Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys
1540 1545 1550
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly
1555 1560 1565
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
1570 1575 1580
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
1585 1590 1595 1600
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
1605 1610 1615
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
1620 1625 1630
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
1635 1640 1645
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile
1650 1655 1660
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
1665 1670 1675 1680
Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
1685 1690 1695
Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
1700 1705 1710
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
1715 1720 1725
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val
1730 1735 1740
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
1745 1750 1755 1760
His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser
1765 1770 1775
Pro Gly Lys
<210> 234
<211> 1766
<212> PRT
<213> 人工序列
<220>
<223> BsAB EGFRvIII-MCSP
<400> 234
Gln Val Lys Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Leu Lys Leu Ser Cys Val Thr Ser Gly Phe Thr Phe Arg Lys Phe
20 25 30
Gly Met Ser Trp Val Arg Gln Thr Ser Asp Lys Arg Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Thr Gly Gly Tyr Asn Thr Tyr Tyr Ser Asp Asn Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Thr Arg Gly Tyr Ser Pro Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val
115 120 125
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser
130 135 140
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln
145 150 155 160
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val
165 170 175
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu
180 185 190
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu
195 200 205
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg
210 215 220
Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro
245 250 255
Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Thr
260 265 270
Ser Gly Tyr Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu
275 280 285
Glu Trp Ile Gly Tyr Ile Thr Phe Asp Gly Ser Asn Asn Tyr Asn Pro
290 295 300
Ser Leu Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln
305 310 315 320
Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
325 330 335
Tyr Cys Ala Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
340 345 350
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
355 360 365
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Glu
370 375 380
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
385 390 395 400
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
405 410 415
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
420 425 430
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
435 440 445
Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
450 455 460
Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe
465 470 475 480
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
485 490 495
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
500 505 510
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
515 520 525
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
530 535 540
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
545 550 555 560
Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
565 570 575
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg
580 585 590
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly
595 600 605
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
610 615 620
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
625 630 635 640
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
645 650 655
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
660 665 670
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Asp Ile Gln Met
675 680 685
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr
690 695 700
Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr Leu Asn Trp Tyr
705 710 715 720
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser
725 730 735
Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
740 745 750
Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala
755 760 765
Thr Tyr Tyr Cys Gln Gln Tyr Ser Ala Leu Pro Trp Thr Phe Gly Gln
770 775 780
Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe
785 790 795 800
Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys Ser Gly Thr Ala Ser Val
805 810 815
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp
820 825 830
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr
835 840 845
Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr
850 855 860
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val
865 870 875 880
Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly
885 890 895
Glu Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
900 905 910
Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg
915 920 925
Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
930 935 940
Leu Ile Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe
945 950 955 960
Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu
965 970 975
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ala Leu
980 985 990
Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val
995 1000 1005
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys
1010 1015 1020
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
1025 1030 1035 1040
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
1045 1050 1055
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
1060 1065 1070
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
1075 1080 1085
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
1090 1095 1100
Lys Ser Phe Asn Arg Gly Glu Cys Asp Ile Glu Leu Thr Gln Ser Pro
1105 1110 1115 1120
Ala Ser Leu Ser Val Ala Thr Gly Glu Lys Val Thr Ile Arg Cys Met
1125 1130 1135
Thr Ser Thr Asp Ile Asp Asp Asp Met Asn Trp Tyr Gln Gln Lys Pro
1140 1145 1150
Gly Glu Pro Pro Lys Phe Leu Ile Ser Glu Gly Asn Thr Leu Arg Pro
1155 1160 1165
Gly Val Pro Ser Arg Phe Ser Ser Ser Gly Thr Gly Thr Asp Phe Val
1170 1175 1180
Phe Thr Ile Glu Asn Thr Leu Ser Glu Asp Val Gly Asp Tyr Tyr Cys
1185 1190 1195 1200
Leu Gln Ser Trp Asn Val Pro Leu Thr Phe Gly Asp Gly Thr Lys Leu
1205 1210 1215
Glu Ile Lys Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
1220 1225 1230
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
1235 1240 1245
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
1250 1255 1260
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
1265 1270 1275 1280
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
1285 1290 1295
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
1300 1305 1310
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gln Val Gln Leu
1315 1320 1325
Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu
1330 1335 1340
Thr Cys Thr Val Ser Gly Gly Ser Ile Thr Ser Gly Tyr Tyr Trp Asn
1345 1350 1355 1360
Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile
1365 1370 1375
Thr Phe Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val
1380 1385 1390
Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser
1395 1400 1405
Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Asp Phe Asp
1410 1415 1420
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
1425 1430 1435 1440
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
1445 1450 1455
Gly Thr Ala Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro
1460 1465 1470
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
1475 1480 1485
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
1490 1495 1500
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
1505 1510 1515 1520
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro
1525 1530 1535
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
1540 1545 1550
Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
1555 1560 1565
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
1570 1575 1580
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
1585 1590 1595 1600
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
1605 1610 1615
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
1620 1625 1630
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly
1635 1640 1645
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
1650 1655 1660
Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
1665 1670 1675 1680
Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile
1685 1690 1695
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
1700 1705 1710
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys
1715 1720 1725
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
1730 1735 1740
Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu
1745 1750 1755 1760
Ser Leu Ser Pro Gly Lys
1765
<210> 235
<211> 1773
<212> PRT
<213> 人工序列
<220>
<223> BsAB EGFRvIII-MSLN
<400> 235
Gln Val Lys Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Leu Lys Leu Ser Cys Val Thr Ser Gly Phe Thr Phe Arg Lys Phe
20 25 30
Gly Met Ser Trp Val Arg Gln Thr Ser Asp Lys Arg Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Thr Gly Gly Tyr Asn Thr Tyr Tyr Ser Asp Asn Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Thr Arg Gly Tyr Ser Pro Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val
115 120 125
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser
130 135 140
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln
145 150 155 160
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val
165 170 175
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu
180 185 190
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu
195 200 205
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg
210 215 220
Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln
225 230 235 240
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys
245 250 255
Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn
260 265 270
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Leu Ile
275 280 285
Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe Arg Gly Lys
290 295 300
Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu
305 310 315 320
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly
325 330 335
Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
340 345 350
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
355 360 365
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
370 375 380
Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
385 390 395 400
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
405 410 415
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
420 425 430
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
435 440 445
Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
450 455 460
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
465 470 475 480
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
485 490 495
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
500 505 510
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
515 520 525
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
530 535 540
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
545 550 555 560
Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser
565 570 575
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
580 585 590
Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val
595 600 605
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
610 615 620
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
625 630 635 640
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
645 650 655
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
660 665 670
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Asp Ile
675 680 685
Glu Leu Thr Gln Ser Pro Ala Ser Leu Ser Val Ala Thr Gly Glu Lys
690 695 700
Val Thr Ile Arg Cys Met Thr Ser Thr Asp Ile Asp Asp Asp Met Asn
705 710 715 720
Trp Tyr Gln Gln Lys Pro Gly Glu Pro Pro Lys Phe Leu Ile Ser Glu
725 730 735
Gly Asn Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Ser Ser Gly
740 745 750
Thr Gly Thr Asp Phe Val Phe Thr Ile Glu Asn Thr Leu Ser Glu Asp
755 760 765
Val Gly Asp Tyr Tyr Cys Leu Gln Ser Trp Asn Val Pro Leu Thr Phe
770 775 780
Gly Asp Gly Thr Lys Leu Glu Ile Lys Ser Ser Ala Ser Thr Lys Gly
785 790 795 800
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
805 810 815
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
820 825 830
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
835 840 845
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
850 855 860
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
865 870 875 880
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
885 890 895
Ser Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
900 905 910
Val Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser
915 920 925
Tyr Met His Trp Tyr Gln Gln Lys Ser Gly Lys Ala Pro Lys Leu Leu
930 935 940
Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser
945 950 955 960
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
965 970 975
Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Lys His Pro
980 985 990
Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala
995 1000 1005
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys Ser
1010 1015 1020
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
1025 1030 1035 1040
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
1045 1050 1055
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
1060 1065 1070
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
1075 1080 1085
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
1090 1095 1100
Ser Phe Asn Arg Gly Glu Cys Asp Ile Gln Met Thr Gln Ser Pro Ser
1105 1110 1115 1120
Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Ala
1125 1130 1135
Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Gly Lys
1140 1145 1150
Ala Pro Lys Leu Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val
1155 1160 1165
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
1170 1175 1180
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
1185 1190 1195 1200
Trp Ser Lys His Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
1205 1210 1215
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
1220 1225 1230
Arg Lys Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
1235 1240 1245
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
1250 1255 1260
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
1265 1270 1275 1280
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
1285 1290 1295
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
1300 1305 1310
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gln Val Gln Leu
1315 1320 1325
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val
1330 1335 1340
Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp
1345 1350 1355 1360
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Leu Ile Thr
1365 1370 1375
Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe Arg Gly Lys Ala
1380 1385 1390
Thr Met Thr Val Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu Ser
1395 1400 1405
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Gly
1410 1415 1420
Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
1425 1430 1435 1440
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
1445 1450 1455
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
1460 1465 1470
Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
1475 1480 1485
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
1490 1495 1500
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
1505 1510 1515 1520
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
1525 1530 1535
Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
1540 1545 1550
Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu
1555 1560 1565
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
1570 1575 1580
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
1585 1590 1595 1600
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
1605 1610 1615
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
1620 1625 1630
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
1635 1640 1645
Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys
1650 1655 1660
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser
1665 1670 1675 1680
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys
1685 1690 1695
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
1700 1705 1710
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
1715 1720 1725
Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
1730 1735 1740
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
1745 1750 1755 1760
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
1765 1770
<210> 236
<211> 6966
<212> DNA
<213> 人（Homo sapiens）
<220>
<223> MSCP
<220>
<221> CDS
<222> 1..6966
<223> /翻译表=1
<400> 236
atg cag agc ggc ccg cgc ccg ccg ctg ccg gcg ccg ggc ctg gcg ctg 48
Met Gln Ser Gly Pro Arg Pro Pro Leu Pro Ala Pro Gly Leu Ala Leu
1 5 10 15
gcg ctg acc ctg acc atg ctg gcg cgc ctg gcg agc gcg gcg agc ttt 96
Ala Leu Thr Leu Thr Met Leu Ala Arg Leu Ala Ser Ala Ala Ser Phe
20 25 30
ttt ggc gaa aac cat ctg gaa gtg ccg gtg gcg acc gcg ctg acc gat 144
Phe Gly Glu Asn His Leu Glu Val Pro Val Ala Thr Ala Leu Thr Asp
35 40 45
att gat ctg cag ctg cag ttt agc acc agc cag ccg gaa gcg ctg ctg 192
Ile Asp Leu Gln Leu Gln Phe Ser Thr Ser Gln Pro Glu Ala Leu Leu
50 55 60
ctg ctg gcg gcg ggc ccg gcg gat cat ctg ctg ctg cag ctg tat agc 240
Leu Leu Ala Ala Gly Pro Ala Asp His Leu Leu Leu Gln Leu Tyr Ser
65 70 75 80
ggc cgc ctg cag gtg cgc ctg gtg ctg ggc cag gaa gaa ctg cgc ctg 288
Gly Arg Leu Gln Val Arg Leu Val Leu Gly Gln Glu Glu Leu Arg Leu
85 90 95
cag acc ccg gcg gaa acc ctg ctg agc gat agc att ccg cat acc gtg 336
Gln Thr Pro Ala Glu Thr Leu Leu Ser Asp Ser Ile Pro His Thr Val
100 105 110
gtg ctg acc gtg gtg gaa ggc tgg gcg acc ctg agc gtg gat ggc ttt 384
Val Leu Thr Val Val Glu Gly Trp Ala Thr Leu Ser Val Asp Gly Phe
115 120 125
ctg aac gcg agc agc gcg gtg ccg ggc gcg ccg ctg gaa gtg ccg tat 432
Leu Asn Ala Ser Ser Ala Val Pro Gly Ala Pro Leu Glu Val Pro Tyr
130 135 140
ggc ctg ttt gtg ggc ggc acc ggc acc ctg ggc ctg ccg tat ctg cgc 480
Gly Leu Phe Val Gly Gly Thr Gly Thr Leu Gly Leu Pro Tyr Leu Arg
145 150 155 160
ggc acc agc cgc ccg ctg cgc ggc tgc ctg cat gcg gcg acc ctg aac 528
Gly Thr Ser Arg Pro Leu Arg Gly Cys Leu His Ala Ala Thr Leu Asn
165 170 175
ggc cgc agc ctg ctg cgc ccg ctg acc ccg gat gtg cat gaa ggc tgc 576
Gly Arg Ser Leu Leu Arg Pro Leu Thr Pro Asp Val His Glu Gly Cys
180 185 190
gcg gaa gaa ttt agc gcg agc gat gat gtg gcg ctg ggc ttt agc ggc 624
Ala Glu Glu Phe Ser Ala Ser Asp Asp Val Ala Leu Gly Phe Ser Gly
195 200 205
ccg cat agc ctg gcg gcg ttt ccg gcg tgg ggc acc cag gat gaa ggc 672
Pro His Ser Leu Ala Ala Phe Pro Ala Trp Gly Thr Gln Asp Glu Gly
210 215 220
acc ctg gaa ttt acc ctg acc acc cag agc cgc cag gcg ccg ctg gcg 720
Thr Leu Glu Phe Thr Leu Thr Thr Gln Ser Arg Gln Ala Pro Leu Ala
225 230 235 240
ttt cag gcg ggc ggc cgc cgc ggc gat ttt att tat gtg gat att ttt 768
Phe Gln Ala Gly Gly Arg Arg Gly Asp Phe Ile Tyr Val Asp Ile Phe
245 250 255
gaa ggc cat ctg cgc gcg gtg gtg gaa aaa ggc cag ggc acc gtg ctg 816
Glu Gly His Leu Arg Ala Val Val Glu Lys Gly Gln Gly Thr Val Leu
260 265 270
ctg cat aac agc gtg ccg gtg gcg gat ggc cag ccg cat gaa gtg agc 864
Leu His Asn Ser Val Pro Val Ala Asp Gly Gln Pro His Glu Val Ser
275 280 285
gtg cat att aac gcg cat cgc ctg gaa att agc gtg gat cag tat ccg 912
Val His Ile Asn Ala His Arg Leu Glu Ile Ser Val Asp Gln Tyr Pro
290 295 300
acc cat acc agc aac cgc ggc gtg ctg agc tat ctg gaa ccg cgc ggc 960
Thr His Thr Ser Asn Arg Gly Val Leu Ser Tyr Leu Glu Pro Arg Gly
305 310 315 320
agc ctg ctg ctg ggc ggc ctg gat gcg gaa gcg agc cgc cat ctg cag 1008
Ser Leu Leu Leu Gly Gly Leu Asp Ala Glu Ala Ser Arg His Leu Gln
325 330 335
gaa cat cgc ctg ggc ctg acc ccg gaa gcg acc aac gcg agc ctg ctg 1056
Glu His Arg Leu Gly Leu Thr Pro Glu Ala Thr Asn Ala Ser Leu Leu
340 345 350
ggc tgc atg gaa gat ctg agc gtg aac ggc cag cgc cgc ggc ctg cgc 1104
Gly Cys Met Glu Asp Leu Ser Val Asn Gly Gln Arg Arg Gly Leu Arg
355 360 365
gaa gcg ctg ctg acc cgc aac atg gcg gcg ggc tgc cgc ctg gaa gaa 1152
Glu Ala Leu Leu Thr Arg Asn Met Ala Ala Gly Cys Arg Leu Glu Glu
370 375 380
gaa gaa tat gaa gat gat gcg tat ggc cat tat gaa gcg ttt agc acc 1200
Glu Glu Tyr Glu Asp Asp Ala Tyr Gly His Tyr Glu Ala Phe Ser Thr
385 390 395 400
ctg gcg ccg gaa gcg tgg ccg gcg atg gaa ctg ccg gaa ccg tgc gtg 1248
Leu Ala Pro Glu Ala Trp Pro Ala Met Glu Leu Pro Glu Pro Cys Val
405 410 415
ccg gaa ccg ggc ctg ccg ccg gtg ttt gcg aac ttt acc cag ctg ctg 1296
Pro Glu Pro Gly Leu Pro Pro Val Phe Ala Asn Phe Thr Gln Leu Leu
420 425 430
acc att agc ccg ctg gtg gtg gcg gaa ggc ggc acc gcg tgg ctg gaa 1344
Thr Ile Ser Pro Leu Val Val Ala Glu Gly Gly Thr Ala Trp Leu Glu
435 440 445
tgg cgc cat gtg cag ccg acc ctg gat ctg atg gaa gcg gaa ctg cgc 1392
Trp Arg His Val Gln Pro Thr Leu Asp Leu Met Glu Ala Glu Leu Arg
450 455 460
aaa agc cag gtg ctg ttt agc gtg acc cgc ggc gcg cgc cat ggc gaa 1440
Lys Ser Gln Val Leu Phe Ser Val Thr Arg Gly Ala Arg His Gly Glu
465 470 475 480
ctg gaa ctg gat att ccg ggc gcg cag gcg cgc aaa atg ttt acc ctg 1488
Leu Glu Leu Asp Ile Pro Gly Ala Gln Ala Arg Lys Met Phe Thr Leu
485 490 495
ctg gat gtg gtg aac cgc aaa gcg cgc ttt att cat gat ggc agc gaa 1536
Leu Asp Val Val Asn Arg Lys Ala Arg Phe Ile His Asp Gly Ser Glu
500 505 510
gat acc agc gat cag ctg gtg ctg gaa gtg agc gtg acc gcg cgc gtg 1584
Asp Thr Ser Asp Gln Leu Val Leu Glu Val Ser Val Thr Ala Arg Val
515 520 525
ccg atg ccg agc tgc ctg cgc cgc ggc cag acc tat ctg ctg ccg att 1632
Pro Met Pro Ser Cys Leu Arg Arg Gly Gln Thr Tyr Leu Leu Pro Ile
530 535 540
cag gtg aac ccg gtg aac gat ccg ccg cat att att ttt ccg cat ggc 1680
Gln Val Asn Pro Val Asn Asp Pro Pro His Ile Ile Phe Pro His Gly
545 550 555 560
agc ctg atg gtg att ctg gaa cat acc cag aaa ccg ctg ggc ccg gaa 1728
Ser Leu Met Val Ile Leu Glu His Thr Gln Lys Pro Leu Gly Pro Glu
565 570 575
gtg ttt cag gcg tat gat ccg gat agc gcg tgc gaa ggc ctg acc ttt 1776
Val Phe Gln Ala Tyr Asp Pro Asp Ser Ala Cys Glu Gly Leu Thr Phe
580 585 590
cag gtg ctg ggc acc agc agc ggc ctg ccg gtg gaa cgc cgc gat cag 1824
Gln Val Leu Gly Thr Ser Ser Gly Leu Pro Val Glu Arg Arg Asp Gln
595 600 605
ccg ggc gaa ccg gcg acc gaa ttt agc tgc cgc gaa ctg gaa gcg ggc 1872
Pro Gly Glu Pro Ala Thr Glu Phe Ser Cys Arg Glu Leu Glu Ala Gly
610 615 620
agc ctg gtg tat gtg cat cgc ggc ggc ccg gcg cag gat ctg acc ttt 1920
Ser Leu Val Tyr Val His Arg Gly Gly Pro Ala Gln Asp Leu Thr Phe
625 630 635 640
cgc gtg agc gat ggc ctg cag gcg agc ccg ccg gcg acc ctg aaa gtg 1968
Arg Val Ser Asp Gly Leu Gln Ala Ser Pro Pro Ala Thr Leu Lys Val
645 650 655
gtg gcg att cgc ccg gcg att cag att cat cgc agc acc ggc ctg cgc 2016
Val Ala Ile Arg Pro Ala Ile Gln Ile His Arg Ser Thr Gly Leu Arg
660 665 670
ctg gcg cag ggc agc gcg atg ccg att ctg ccg gcg aac ctg agc gtg 2064
Leu Ala Gln Gly Ser Ala Met Pro Ile Leu Pro Ala Asn Leu Ser Val
675 680 685
gaa acc aac gcg gtg ggc cag gat gtg agc gtg ctg ttt cgc gtg acc 2112
Glu Thr Asn Ala Val Gly Gln Asp Val Ser Val Leu Phe Arg Val Thr
690 695 700
ggc gcg ctg cag ttt ggc gaa ctg cag aaa cag ggc gcg ggc ggc gtg 2160
Gly Ala Leu Gln Phe Gly Glu Leu Gln Lys Gln Gly Ala Gly Gly Val
705 710 715 720
gaa ggc gcg gaa tgg tgg gcg acc cag gcg ttt cat cag cgc gat gtg 2208
Glu Gly Ala Glu Trp Trp Ala Thr Gln Ala Phe His Gln Arg Asp Val
725 730 735
gaa cag ggc cgc gtg cgc tat ctg agc acc gat ccg cag cat cat gcg 2256
Glu Gln Gly Arg Val Arg Tyr Leu Ser Thr Asp Pro Gln His His Ala
740 745 750
tat gat acc gtg gaa aac ctg gcg ctg gaa gtg cag gtg ggc cag gaa 2304
Tyr Asp Thr Val Glu Asn Leu Ala Leu Glu Val Gln Val Gly Gln Glu
755 760 765
att ctg agc aac ctg agc ttt ccg gtg acc att cag cgc gcg acc gtg 2352
Ile Leu Ser Asn Leu Ser Phe Pro Val Thr Ile Gln Arg Ala Thr Val
770 775 780
tgg atg ctg cgc ctg gaa ccg ctg cat acc cag aac acc cag cag gaa 2400
Trp Met Leu Arg Leu Glu Pro Leu His Thr Gln Asn Thr Gln Gln Glu
785 790 795 800
acc ctg acc acc gcg cat ctg gaa gcg acc ctg gaa gaa gcg ggc ccg 2448
Thr Leu Thr Thr Ala His Leu Glu Ala Thr Leu Glu Glu Ala Gly Pro
805 810 815
agc ccg ccg acc ttt cat tat gaa gtg gtg cag gcg ccg cgc aaa ggc 2496
Ser Pro Pro Thr Phe His Tyr Glu Val Val Gln Ala Pro Arg Lys Gly
820 825 830
aac ctg cag ctg cag ggc acc cgc ctg agc gat ggc cag ggc ttt acc 2544
Asn Leu Gln Leu Gln Gly Thr Arg Leu Ser Asp Gly Gln Gly Phe Thr
835 840 845
cag gat gat att cag gcg ggc cgc gtg acc tat ggc gcg acc gcg cgc 2592
Gln Asp Asp Ile Gln Ala Gly Arg Val Thr Tyr Gly Ala Thr Ala Arg
850 855 860
gcg agc gaa gcg gtg gaa gat acc ttt cgc ttt cgc gtg acc gcg ccg 2640
Ala Ser Glu Ala Val Glu Asp Thr Phe Arg Phe Arg Val Thr Ala Pro
865 870 875 880
ccg tat ttt agc ccg ctg tat acc ttt ccg att cat att ggc ggc gat 2688
Pro Tyr Phe Ser Pro Leu Tyr Thr Phe Pro Ile His Ile Gly Gly Asp
885 890 895
ccg gat gcg ccg gtg ctg acc aac gtg ctg ctg gtg gtg ccg gaa ggc 2736
Pro Asp Ala Pro Val Leu Thr Asn Val Leu Leu Val Val Pro Glu Gly
900 905 910
ggc gaa ggc gtg ctg agc gcg gat cat ctg ttt gtg aaa agc ctg aac 2784
Gly Glu Gly Val Leu Ser Ala Asp His Leu Phe Val Lys Ser Leu Asn
915 920 925
agc gcg agc tat ctg tat gaa gtg atg gaa cgc ccg cgc cat ggc cgc 2832
Ser Ala Ser Tyr Leu Tyr Glu Val Met Glu Arg Pro Arg His Gly Arg
930 935 940
ctg gcg tgg cgc ggc acc cag gat aaa acc acc atg gtg acc agc ttt 2880
Leu Ala Trp Arg Gly Thr Gln Asp Lys Thr Thr Met Val Thr Ser Phe
945 950 955 960
acc aac gaa gat ctg ctg cgc ggc cgc ctg gtg tat cag cat gat gat 2928
Thr Asn Glu Asp Leu Leu Arg Gly Arg Leu Val Tyr Gln His Asp Asp
965 970 975
agc gaa acc acc gaa gat gat att ccg ttt gtg gcg acc cgc cag ggc 2976
Ser Glu Thr Thr Glu Asp Asp Ile Pro Phe Val Ala Thr Arg Gln Gly
980 985 990
gaa agc agc ggc gat atg gcg tgg gaa gaa gtg cgc ggc gtg ttt cgc 3024
Glu Ser Ser Gly Asp Met Ala Trp Glu Glu Val Arg Gly Val Phe Arg
995 1000 1005
gtg gcg att cag ccg gtg aac gat cat gcg ccg gtg cag acc att agc 3072
Val Ala Ile Gln Pro Val Asn Asp His Ala Pro Val Gln Thr Ile Ser
1010 1015 1020
cgc att ttt cat gtg gcg cgc ggc ggc cgc cgc ctg ctg acc acc gat 3120
Arg Ile Phe His Val Ala Arg Gly Gly Arg Arg Leu Leu Thr Thr Asp
1025 1030 1035 1040
gat gtg gcg ttt agc gat gcg gat agc ggc ttt gcg gat gcg cag ctg 3168
Asp Val Ala Phe Ser Asp Ala Asp Ser Gly Phe Ala Asp Ala Gln Leu
1045 1050 1055
gtg ctg acc cgc aaa gat ctg ctg ttt ggc agc att gtg gcg gtg gat 3216
Val Leu Thr Arg Lys Asp Leu Leu Phe Gly Ser Ile Val Ala Val Asp
1060 1065 1070
gaa ccg acc cgc ccg att tat cgc ttt acc cag gaa gat ctg cgc aaa 3264
Glu Pro Thr Arg Pro Ile Tyr Arg Phe Thr Gln Glu Asp Leu Arg Lys
1075 1080 1085
cgc cgc gtg ctg ttt gtg cat agc ggc gcg gat cgc ggc tgg att cag 3312
Arg Arg Val Leu Phe Val His Ser Gly Ala Asp Arg Gly Trp Ile Gln
1090 1095 1100
ctg cag gtg agc gat ggc cag cat cag gcg acc gcg ctg ctg gaa gtg 3360
Leu Gln Val Ser Asp Gly Gln His Gln Ala Thr Ala Leu Leu Glu Val
1105 1110 1115 1120
cag gcg agc gaa ccg tat ctg cgc gtg gcg aac ggc agc agc ctg gtg 3408
Gln Ala Ser Glu Pro Tyr Leu Arg Val Ala Asn Gly Ser Ser Leu Val
1125 1130 1135
gtg ccg cag ggc ggc cag ggc acc att gat acc gcg gtg ctg cat ctg 3456
Val Pro Gln Gly Gly Gln Gly Thr Ile Asp Thr Ala Val Leu His Leu
1140 1145 1150
gat acc aac ctg gat att cgc agc ggc gat gaa gtg cat tat cat gtg 3504
Asp Thr Asn Leu Asp Ile Arg Ser Gly Asp Glu Val His Tyr His Val
1155 1160 1165
acc gcg ggc ccg cgc tgg ggc cag ctg gtg cgc gcg ggc cag ccg gcg 3552
Thr Ala Gly Pro Arg Trp Gly Gln Leu Val Arg Ala Gly Gln Pro Ala
1170 1175 1180
acc gcg ttt agc cag cag gat ctg ctg gat ggc gcg gtg ctg tat agc 3600
Thr Ala Phe Ser Gln Gln Asp Leu Leu Asp Gly Ala Val Leu Tyr Ser
1185 1190 1195 1200
cat aac ggc agc ctg agc ccg cgc gat acc atg gcg ttt agc gtg gaa 3648
His Asn Gly Ser Leu Ser Pro Arg Asp Thr Met Ala Phe Ser Val Glu
1205 1210 1215
gcg ggc ccg gtg cat acc gat gcg acc ctg cag gtg acc att gcg ctg 3696
Ala Gly Pro Val His Thr Asp Ala Thr Leu Gln Val Thr Ile Ala Leu
1220 1225 1230
gaa ggc ccg ctg gcg ccg ctg aaa ctg gtg cgc cat aaa aaa att tat 3744
Glu Gly Pro Leu Ala Pro Leu Lys Leu Val Arg His Lys Lys Ile Tyr
1235 1240 1245
gtg ttt cag ggc gaa gcg gcg gaa att cgc cgc gat cag ctg gaa gcg 3792
Val Phe Gln Gly Glu Ala Ala Glu Ile Arg Arg Asp Gln Leu Glu Ala
1250 1255 1260
gcg cag gaa gcg gtg ccg ccg gcg gat att gtg ttt agc gtg aaa agc 3840
Ala Gln Glu Ala Val Pro Pro Ala Asp Ile Val Phe Ser Val Lys Ser
1265 1270 1275 1280
ccg ccg agc gcg ggc tat ctg gtg atg gtg agc cgc ggc gcg ctg gcg 3888
Pro Pro Ser Ala Gly Tyr Leu Val Met Val Ser Arg Gly Ala Leu Ala
1285 1290 1295
gat gaa ccg ccg agc ctg gat ccg gtg cag agc ttt agc cag gaa gcg 3936
Asp Glu Pro Pro Ser Leu Asp Pro Val Gln Ser Phe Ser Gln Glu Ala
1300 1305 1310
gtg gat acc ggc cgc gtg ctg tat ctg cat agc cgc ccg gaa gcg tgg 3984
Val Asp Thr Gly Arg Val Leu Tyr Leu His Ser Arg Pro Glu Ala Trp
1315 1320 1325
agc gat gcg ttt agc ctg gat gtg gcg agc ggc ctg ggc gcg ccg ctg 4032
Ser Asp Ala Phe Ser Leu Asp Val Ala Ser Gly Leu Gly Ala Pro Leu
1330 1335 1340
gaa ggc gtg ctg gtg gaa ctg gaa gtg ctg ccg gcg gcg att ccg ctg 4080
Glu Gly Val Leu Val Glu Leu Glu Val Leu Pro Ala Ala Ile Pro Leu
1345 1350 1355 1360
gaa gcg cag aac ttt agc gtg ccg gaa ggc ggc agc ctg acc ctg gcg 4128
Glu Ala Gln Asn Phe Ser Val Pro Glu Gly Gly Ser Leu Thr Leu Ala
1365 1370 1375
ccg ccg ctg ctg cgc gtg agc ggc ccg tat ttt ccg acc ctg ctg ggc 4176
Pro Pro Leu Leu Arg Val Ser Gly Pro Tyr Phe Pro Thr Leu Leu Gly
1380 1385 1390
ctg agc ctg cag gtg ctg gaa ccg ccg cag cat ggc gcg ctg cag aaa 4224
Leu Ser Leu Gln Val Leu Glu Pro Pro Gln His Gly Ala Leu Gln Lys
1395 1400 1405
gaa gat ggc ccg cag gcg cgc acc ctg agc gcg ttt agc tgg cgc atg 4272
Glu Asp Gly Pro Gln Ala Arg Thr Leu Ser Ala Phe Ser Trp Arg Met
1410 1415 1420
gtg gaa gaa cag ctg att cgc tat gtg cat gat ggc agc gaa acc ctg 4320
Val Glu Glu Gln Leu Ile Arg Tyr Val His Asp Gly Ser Glu Thr Leu
1425 1430 1435 1440
acc gat agc ttt gtg ctg atg gcg aac gcg agc gaa atg gat cgc cag 4368
Thr Asp Ser Phe Val Leu Met Ala Asn Ala Ser Glu Met Asp Arg Gln
1445 1450 1455
agc cat ccg gtg gcg ttt acc gtg acc gtg ctg ccg gtg aac gat cag 4416
Ser His Pro Val Ala Phe Thr Val Thr Val Leu Pro Val Asn Asp Gln
1460 1465 1470
ccg ccg att ctg acc acc aac acc ggc ctg cag atg tgg gaa ggc gcg 4464
Pro Pro Ile Leu Thr Thr Asn Thr Gly Leu Gln Met Trp Glu Gly Ala
1475 1480 1485
acc gcg ccg att ccg gcg gaa gcg ctg cgc agc acc gat ggc gat agc 4512
Thr Ala Pro Ile Pro Ala Glu Ala Leu Arg Ser Thr Asp Gly Asp Ser
1490 1495 1500
ggc agc gaa gat ctg gtg tat acc att gaa cag ccg agc aac ggc cgc 4560
Gly Ser Glu Asp Leu Val Tyr Thr Ile Glu Gln Pro Ser Asn Gly Arg
1505 1510 1515 1520
gtg gtg ctg cgc ggc gcg ccg ggc acc gaa gtg cgc agc ttt acc cag 4608
Val Val Leu Arg Gly Ala Pro Gly Thr Glu Val Arg Ser Phe Thr Gln
1525 1530 1535
gcg cag ctg gat ggc ggc ctg gtg ctg ttt agc cat cgc ggc acc ctg 4656
Ala Gln Leu Asp Gly Gly Leu Val Leu Phe Ser His Arg Gly Thr Leu
1540 1545 1550
gat ggc ggc ttt cgc ttt cgc ctg agc gat ggc gaa cat acc agc ccg 4704
Asp Gly Gly Phe Arg Phe Arg Leu Ser Asp Gly Glu His Thr Ser Pro
1555 1560 1565
ggc cat ttt ttt cgc gtg acc gcg cag aaa cag gtg ctg ctg agc ctg 4752
Gly His Phe Phe Arg Val Thr Ala Gln Lys Gln Val Leu Leu Ser Leu
1570 1575 1580
aaa ggc agc cag acc ctg acc gtg tgc ccg ggc agc gtg cag ccg ctg 4800
Lys Gly Ser Gln Thr Leu Thr Val Cys Pro Gly Ser Val Gln Pro Leu
1585 1590 1595 1600
agc agc cag acc ctg cgc gcg agc agc agc gcg ggc acc gat ccg cag 4848
Ser Ser Gln Thr Leu Arg Ala Ser Ser Ser Ala Gly Thr Asp Pro Gln
1605 1610 1615
ctg ctg ctg tat cgc gtg gtg cgc ggc ccg cag ctg ggc cgc ctg ttt 4896
Leu Leu Leu Tyr Arg Val Val Arg Gly Pro Gln Leu Gly Arg Leu Phe
1620 1625 1630
cat gcg cag cag gat agc acc ggc gaa gcg ctg gtg aac ttt acc cag 4944
His Ala Gln Gln Asp Ser Thr Gly Glu Ala Leu Val Asn Phe Thr Gln
1635 1640 1645
gcg gaa gtg tat gcg ggc aac att ctg tat gaa cat gaa atg ccg ccg 4992
Ala Glu Val Tyr Ala Gly Asn Ile Leu Tyr Glu His Glu Met Pro Pro
1650 1655 1660
gaa ccg ttt tgg gaa gcg cat gat acc ctg gaa ctg cag ctg agc agc 5040
Glu Pro Phe Trp Glu Ala His Asp Thr Leu Glu Leu Gln Leu Ser Ser
1665 1670 1675 1680
ccg ccg gcg cgc gat gtg gcg gcg acc ctg gcg gtg gcg gtg agc ttt 5088
Pro Pro Ala Arg Asp Val Ala Ala Thr Leu Ala Val Ala Val Ser Phe
1685 1690 1695
gaa gcg gcg tgc ccg cag cgc ccg agc cat ctg tgg aaa aac aaa ggc 5136
Glu Ala Ala Cys Pro Gln Arg Pro Ser His Leu Trp Lys Asn Lys Gly
1700 1705 1710
ctg tgg gtg ccg gaa ggc cag cgc gcg cgc att acc gtg gcg gcg ctg 5184
Leu Trp Val Pro Glu Gly Gln Arg Ala Arg Ile Thr Val Ala Ala Leu
1715 1720 1725
gat gcg agc aac ctg ctg gcg agc gtg ccg agc ccg cag cgc agc gaa 5232
Asp Ala Ser Asn Leu Leu Ala Ser Val Pro Ser Pro Gln Arg Ser Glu
1730 1735 1740
cat gat gtg ctg ttt cag gtg acc cag ttt ccg agc cgc ggc cag ctg 5280
His Asp Val Leu Phe Gln Val Thr Gln Phe Pro Ser Arg Gly Gln Leu
1745 1750 1755 1760
ctg gtg agc gaa gaa ccg ctg cat gcg ggc cag ccg cat ttt ctg cag 5328
Leu Val Ser Glu Glu Pro Leu His Ala Gly Gln Pro His Phe Leu Gln
1765 1770 1775
agc cag ctg gcg gcg ggc cag ctg gtg tat gcg cat ggc ggc ggc ggc 5376
Ser Gln Leu Ala Ala Gly Gln Leu Val Tyr Ala His Gly Gly Gly Gly
1780 1785 1790
acc cag cag gat ggc ttt cat ttt cgc gcg cat ctg cag ggc ccg gcg 5424
Thr Gln Gln Asp Gly Phe His Phe Arg Ala His Leu Gln Gly Pro Ala
1795 1800 1805
ggc gcg agc gtg gcg ggc ccg cag acc agc gaa gcg ttt gcg att acc 5472
Gly Ala Ser Val Ala Gly Pro Gln Thr Ser Glu Ala Phe Ala Ile Thr
1810 1815 1820
gtg cgc gat gtg aac gaa cgc ccg ccg cag ccg cag gcg agc gtg ccg 5520
Val Arg Asp Val Asn Glu Arg Pro Pro Gln Pro Gln Ala Ser Val Pro
1825 1830 1835 1840
ctg cgc ctg acc cgc ggc agc cgc gcg ccg att agc cgc gcg cag ctg 5568
Leu Arg Leu Thr Arg Gly Ser Arg Ala Pro Ile Ser Arg Ala Gln Leu
1845 1850 1855
agc gtg gtg gat ccg gat agc gcg ccg ggc gaa att gaa tat gaa gtg 5616
Ser Val Val Asp Pro Asp Ser Ala Pro Gly Glu Ile Glu Tyr Glu Val
1860 1865 1870
cag cgc gcg ccg cat aac ggc ttt ctg agc ctg gtg ggc ggc ggc ctg 5664
Gln Arg Ala Pro His Asn Gly Phe Leu Ser Leu Val Gly Gly Gly Leu
1875 1880 1885
ggc ccg gtg acc cgc ttt acc cag gcg gat gtg gat agc ggc cgc ctg 5712
Gly Pro Val Thr Arg Phe Thr Gln Ala Asp Val Asp Ser Gly Arg Leu
1890 1895 1900
gcg ttt gtg gcg aac ggc agc agc gtg gcg ggc att ttt cag ctg agc 5760
Ala Phe Val Ala Asn Gly Ser Ser Val Ala Gly Ile Phe Gln Leu Ser
1905 1910 1915 1920
atg agc gat ggc gcg agc ccg ccg ctg ccg atg agc ctg gcg gtg gat 5808
Met Ser Asp Gly Ala Ser Pro Pro Leu Pro Met Ser Leu Ala Val Asp
1925 1930 1935
att ctg ccg agc gcg att gaa gtg cag ctg cgc gcg ccg ctg gaa gtg 5856
Ile Leu Pro Ser Ala Ile Glu Val Gln Leu Arg Ala Pro Leu Glu Val
1940 1945 1950
ccg cag gcg ctg ggc cgc agc agc ctg agc cag cag cag ctg cgc gtg 5904
Pro Gln Ala Leu Gly Arg Ser Ser Leu Ser Gln Gln Gln Leu Arg Val
1955 1960 1965
gtg agc gat cgc gaa gaa ccg gaa gcg gcg tat cgc ctg att cag ggc 5952
Val Ser Asp Arg Glu Glu Pro Glu Ala Ala Tyr Arg Leu Ile Gln Gly
1970 1975 1980
ccg cag tat ggc cat ctg ctg gtg ggc ggc cgc ccg acc agc gcg ttt 6000
Pro Gln Tyr Gly His Leu Leu Val Gly Gly Arg Pro Thr Ser Ala Phe
1985 1990 1995 2000
agc cag ttt cag att gat cag ggc gaa gtg gtg ttt gcg ttt acc aac 6048
Ser Gln Phe Gln Ile Asp Gln Gly Glu Val Val Phe Ala Phe Thr Asn
2005 2010 2015
ttt agc agc agc cat gat cat ttt cgc gtg ctg gcg ctg gcg cgc ggc 6096
Phe Ser Ser Ser His Asp His Phe Arg Val Leu Ala Leu Ala Arg Gly
2020 2025 2030
gtg aac gcg agc gcg gtg gtg aac gtg acc gtg cgc gcg ctg ctg cat 6144
Val Asn Ala Ser Ala Val Val Asn Val Thr Val Arg Ala Leu Leu His
2035 2040 2045
gtg tgg gcg ggc ggc ccg tgg ccg cag ggc gcg acc ctg cgc ctg gat 6192
Val Trp Ala Gly Gly Pro Trp Pro Gln Gly Ala Thr Leu Arg Leu Asp
2050 2055 2060
ccg acc gtg ctg gat gcg ggc gaa ctg gcg aac cgc acc ggc agc gtg 6240
Pro Thr Val Leu Asp Ala Gly Glu Leu Ala Asn Arg Thr Gly Ser Val
2065 2070 2075 2080
ccg cgc ttt cgc ctg ctg gaa ggc ccg cgc cat ggc cgc gtg gtg cgc 6288
Pro Arg Phe Arg Leu Leu Glu Gly Pro Arg His Gly Arg Val Val Arg
2085 2090 2095
gtg ccg cgc gcg cgc acc gaa ccg ggc ggc agc cag ctg gtg gaa cag 6336
Val Pro Arg Ala Arg Thr Glu Pro Gly Gly Ser Gln Leu Val Glu Gln
2100 2105 2110
ttt acc cag cag gat ctg gaa gat ggc cgc ctg ggc ctg gaa gtg ggc 6384
Phe Thr Gln Gln Asp Leu Glu Asp Gly Arg Leu Gly Leu Glu Val Gly
2115 2120 2125
cgc ccg gaa ggc cgc gcg ccg ggc ccg gcg ggc gat agc ctg acc ctg 6432
Arg Pro Glu Gly Arg Ala Pro Gly Pro Ala Gly Asp Ser Leu Thr Leu
2130 2135 2140
gaa ctg tgg gcg cag ggc gtg ccg ccg gcg gtg gcg agc ctg gat ttt 6480
Glu Leu Trp Ala Gln Gly Val Pro Pro Ala Val Ala Ser Leu Asp Phe
2145 2150 2155 2160
gcg acc gaa ccg tat aac gcg gcg cgc ccg tat agc gtg gcg ctg ctg 6528
Ala Thr Glu Pro Tyr Asn Ala Ala Arg Pro Tyr Ser Val Ala Leu Leu
2165 2170 2175
agc gtg ccg gaa gcg gcg cgc acc gaa gcg ggc aaa ccg gaa agc agc 6576
Ser Val Pro Glu Ala Ala Arg Thr Glu Ala Gly Lys Pro Glu Ser Ser
2180 2185 2190
acc ccg acc ggc gaa ccg ggc ccg atg gcg agc agc ccg gaa ccg gcg 6624
Thr Pro Thr Gly Glu Pro Gly Pro Met Ala Ser Ser Pro Glu Pro Ala
2195 2200 2205
gtg gcg aaa ggc ggc ttt ctg agc ttt ctg gaa gcg aac atg ttt agc 6672
Val Ala Lys Gly Gly Phe Leu Ser Phe Leu Glu Ala Asn Met Phe Ser
2210 2215 2220
gtg att att ccg atg tgc ctg gtg ctg ctg ctg ctg gcg ctg att ctg 6720
Val Ile Ile Pro Met Cys Leu Val Leu Leu Leu Leu Ala Leu Ile Leu
2225 2230 2235 2240
ccg ctg ctg ttt tat ctg cgc aaa cgc aac aaa acc ggc aaa cat gat 6768
Pro Leu Leu Phe Tyr Leu Arg Lys Arg Asn Lys Thr Gly Lys His Asp
2245 2250 2255
gtg cag gtg ctg acc gcg aaa ccg cgc aac ggc ctg gcg ggc gat acc 6816
Val Gln Val Leu Thr Ala Lys Pro Arg Asn Gly Leu Ala Gly Asp Thr
2260 2265 2270
gaa acc ttt cgc aaa gtg gaa ccg ggc cag gcg att ccg ctg acc gcg 6864
Glu Thr Phe Arg Lys Val Glu Pro Gly Gln Ala Ile Pro Leu Thr Ala
2275 2280 2285
gtg ccg ggc cag ggc ccg ccg ccg ggc ggc cag ccg gat ccg gaa ctg 6912
Val Pro Gly Gln Gly Pro Pro Pro Gly Gly Gln Pro Asp Pro Glu Leu
2290 2295 2300
ctg cag ttt tgc cgc acc ccg aac ccg gcg ctg aaa aac ggc cag tat 6960
Leu Gln Phe Cys Arg Thr Pro Asn Pro Ala Leu Lys Asn Gly Gln Tyr
2305 2310 2315 2320
tgg gtg 6966
Trp Val
<210> 237
<211> 2322
<212> PRT
<213> 人（Homo sapiens）
<220>
<223> [CDS]:1..6966, 来自SEQ ID NO 236
<400> 237
Met Gln Ser Gly Pro Arg Pro Pro Leu Pro Ala Pro Gly Leu Ala Leu
1 5 10 15
Ala Leu Thr Leu Thr Met Leu Ala Arg Leu Ala Ser Ala Ala Ser Phe
20 25 30
Phe Gly Glu Asn His Leu Glu Val Pro Val Ala Thr Ala Leu Thr Asp
35 40 45
Ile Asp Leu Gln Leu Gln Phe Ser Thr Ser Gln Pro Glu Ala Leu Leu
50 55 60
Leu Leu Ala Ala Gly Pro Ala Asp His Leu Leu Leu Gln Leu Tyr Ser
65 70 75 80
Gly Arg Leu Gln Val Arg Leu Val Leu Gly Gln Glu Glu Leu Arg Leu
85 90 95
Gln Thr Pro Ala Glu Thr Leu Leu Ser Asp Ser Ile Pro His Thr Val
100 105 110
Val Leu Thr Val Val Glu Gly Trp Ala Thr Leu Ser Val Asp Gly Phe
115 120 125
Leu Asn Ala Ser Ser Ala Val Pro Gly Ala Pro Leu Glu Val Pro Tyr
130 135 140
Gly Leu Phe Val Gly Gly Thr Gly Thr Leu Gly Leu Pro Tyr Leu Arg
145 150 155 160
Gly Thr Ser Arg Pro Leu Arg Gly Cys Leu His Ala Ala Thr Leu Asn
165 170 175
Gly Arg Ser Leu Leu Arg Pro Leu Thr Pro Asp Val His Glu Gly Cys
180 185 190
Ala Glu Glu Phe Ser Ala Ser Asp Asp Val Ala Leu Gly Phe Ser Gly
195 200 205
Pro His Ser Leu Ala Ala Phe Pro Ala Trp Gly Thr Gln Asp Glu Gly
210 215 220
Thr Leu Glu Phe Thr Leu Thr Thr Gln Ser Arg Gln Ala Pro Leu Ala
225 230 235 240
Phe Gln Ala Gly Gly Arg Arg Gly Asp Phe Ile Tyr Val Asp Ile Phe
245 250 255
Glu Gly His Leu Arg Ala Val Val Glu Lys Gly Gln Gly Thr Val Leu
260 265 270
Leu His Asn Ser Val Pro Val Ala Asp Gly Gln Pro His Glu Val Ser
275 280 285
Val His Ile Asn Ala His Arg Leu Glu Ile Ser Val Asp Gln Tyr Pro
290 295 300
Thr His Thr Ser Asn Arg Gly Val Leu Ser Tyr Leu Glu Pro Arg Gly
305 310 315 320
Ser Leu Leu Leu Gly Gly Leu Asp Ala Glu Ala Ser Arg His Leu Gln
325 330 335
Glu His Arg Leu Gly Leu Thr Pro Glu Ala Thr Asn Ala Ser Leu Leu
340 345 350
Gly Cys Met Glu Asp Leu Ser Val Asn Gly Gln Arg Arg Gly Leu Arg
355 360 365
Glu Ala Leu Leu Thr Arg Asn Met Ala Ala Gly Cys Arg Leu Glu Glu
370 375 380
Glu Glu Tyr Glu Asp Asp Ala Tyr Gly His Tyr Glu Ala Phe Ser Thr
385 390 395 400
Leu Ala Pro Glu Ala Trp Pro Ala Met Glu Leu Pro Glu Pro Cys Val
405 410 415
Pro Glu Pro Gly Leu Pro Pro Val Phe Ala Asn Phe Thr Gln Leu Leu
420 425 430
Thr Ile Ser Pro Leu Val Val Ala Glu Gly Gly Thr Ala Trp Leu Glu
435 440 445
Trp Arg His Val Gln Pro Thr Leu Asp Leu Met Glu Ala Glu Leu Arg
450 455 460
Lys Ser Gln Val Leu Phe Ser Val Thr Arg Gly Ala Arg His Gly Glu
465 470 475 480
Leu Glu Leu Asp Ile Pro Gly Ala Gln Ala Arg Lys Met Phe Thr Leu
485 490 495
Leu Asp Val Val Asn Arg Lys Ala Arg Phe Ile His Asp Gly Ser Glu
500 505 510
Asp Thr Ser Asp Gln Leu Val Leu Glu Val Ser Val Thr Ala Arg Val
515 520 525
Pro Met Pro Ser Cys Leu Arg Arg Gly Gln Thr Tyr Leu Leu Pro Ile
530 535 540
Gln Val Asn Pro Val Asn Asp Pro Pro His Ile Ile Phe Pro His Gly
545 550 555 560
Ser Leu Met Val Ile Leu Glu His Thr Gln Lys Pro Leu Gly Pro Glu
565 570 575
Val Phe Gln Ala Tyr Asp Pro Asp Ser Ala Cys Glu Gly Leu Thr Phe
580 585 590
Gln Val Leu Gly Thr Ser Ser Gly Leu Pro Val Glu Arg Arg Asp Gln
595 600 605
Pro Gly Glu Pro Ala Thr Glu Phe Ser Cys Arg Glu Leu Glu Ala Gly
610 615 620
Ser Leu Val Tyr Val His Arg Gly Gly Pro Ala Gln Asp Leu Thr Phe
625 630 635 640
Arg Val Ser Asp Gly Leu Gln Ala Ser Pro Pro Ala Thr Leu Lys Val
645 650 655
Val Ala Ile Arg Pro Ala Ile Gln Ile His Arg Ser Thr Gly Leu Arg
660 665 670
Leu Ala Gln Gly Ser Ala Met Pro Ile Leu Pro Ala Asn Leu Ser Val
675 680 685
Glu Thr Asn Ala Val Gly Gln Asp Val Ser Val Leu Phe Arg Val Thr
690 695 700
Gly Ala Leu Gln Phe Gly Glu Leu Gln Lys Gln Gly Ala Gly Gly Val
705 710 715 720
Glu Gly Ala Glu Trp Trp Ala Thr Gln Ala Phe His Gln Arg Asp Val
725 730 735
Glu Gln Gly Arg Val Arg Tyr Leu Ser Thr Asp Pro Gln His His Ala
740 745 750
Tyr Asp Thr Val Glu Asn Leu Ala Leu Glu Val Gln Val Gly Gln Glu
755 760 765
Ile Leu Ser Asn Leu Ser Phe Pro Val Thr Ile Gln Arg Ala Thr Val
770 775 780
Trp Met Leu Arg Leu Glu Pro Leu His Thr Gln Asn Thr Gln Gln Glu
785 790 795 800
Thr Leu Thr Thr Ala His Leu Glu Ala Thr Leu Glu Glu Ala Gly Pro
805 810 815
Ser Pro Pro Thr Phe His Tyr Glu Val Val Gln Ala Pro Arg Lys Gly
820 825 830
Asn Leu Gln Leu Gln Gly Thr Arg Leu Ser Asp Gly Gln Gly Phe Thr
835 840 845
Gln Asp Asp Ile Gln Ala Gly Arg Val Thr Tyr Gly Ala Thr Ala Arg
850 855 860
Ala Ser Glu Ala Val Glu Asp Thr Phe Arg Phe Arg Val Thr Ala Pro
865 870 875 880
Pro Tyr Phe Ser Pro Leu Tyr Thr Phe Pro Ile His Ile Gly Gly Asp
885 890 895
Pro Asp Ala Pro Val Leu Thr Asn Val Leu Leu Val Val Pro Glu Gly
900 905 910
Gly Glu Gly Val Leu Ser Ala Asp His Leu Phe Val Lys Ser Leu Asn
915 920 925
Ser Ala Ser Tyr Leu Tyr Glu Val Met Glu Arg Pro Arg His Gly Arg
930 935 940
Leu Ala Trp Arg Gly Thr Gln Asp Lys Thr Thr Met Val Thr Ser Phe
945 950 955 960
Thr Asn Glu Asp Leu Leu Arg Gly Arg Leu Val Tyr Gln His Asp Asp
965 970 975
Ser Glu Thr Thr Glu Asp Asp Ile Pro Phe Val Ala Thr Arg Gln Gly
980 985 990
Glu Ser Ser Gly Asp Met Ala Trp Glu Glu Val Arg Gly Val Phe Arg
995 1000 1005
Val Ala Ile Gln Pro Val Asn Asp His Ala Pro Val Gln Thr Ile Ser
1010 1015 1020
Arg Ile Phe His Val Ala Arg Gly Gly Arg Arg Leu Leu Thr Thr Asp
1025 1030 1035 1040
Asp Val Ala Phe Ser Asp Ala Asp Ser Gly Phe Ala Asp Ala Gln Leu
1045 1050 1055
Val Leu Thr Arg Lys Asp Leu Leu Phe Gly Ser Ile Val Ala Val Asp
1060 1065 1070
Glu Pro Thr Arg Pro Ile Tyr Arg Phe Thr Gln Glu Asp Leu Arg Lys
1075 1080 1085
Arg Arg Val Leu Phe Val His Ser Gly Ala Asp Arg Gly Trp Ile Gln
1090 1095 1100
Leu Gln Val Ser Asp Gly Gln His Gln Ala Thr Ala Leu Leu Glu Val
1105 1110 1115 1120
Gln Ala Ser Glu Pro Tyr Leu Arg Val Ala Asn Gly Ser Ser Leu Val
1125 1130 1135
Val Pro Gln Gly Gly Gln Gly Thr Ile Asp Thr Ala Val Leu His Leu
1140 1145 1150
Asp Thr Asn Leu Asp Ile Arg Ser Gly Asp Glu Val His Tyr His Val
1155 1160 1165
Thr Ala Gly Pro Arg Trp Gly Gln Leu Val Arg Ala Gly Gln Pro Ala
1170 1175 1180
Thr Ala Phe Ser Gln Gln Asp Leu Leu Asp Gly Ala Val Leu Tyr Ser
1185 1190 1195 1200
His Asn Gly Ser Leu Ser Pro Arg Asp Thr Met Ala Phe Ser Val Glu
1205 1210 1215
Ala Gly Pro Val His Thr Asp Ala Thr Leu Gln Val Thr Ile Ala Leu
1220 1225 1230
Glu Gly Pro Leu Ala Pro Leu Lys Leu Val Arg His Lys Lys Ile Tyr
1235 1240 1245
Val Phe Gln Gly Glu Ala Ala Glu Ile Arg Arg Asp Gln Leu Glu Ala
1250 1255 1260
Ala Gln Glu Ala Val Pro Pro Ala Asp Ile Val Phe Ser Val Lys Ser
1265 1270 1275 1280
Pro Pro Ser Ala Gly Tyr Leu Val Met Val Ser Arg Gly Ala Leu Ala
1285 1290 1295
Asp Glu Pro Pro Ser Leu Asp Pro Val Gln Ser Phe Ser Gln Glu Ala
1300 1305 1310
Val Asp Thr Gly Arg Val Leu Tyr Leu His Ser Arg Pro Glu Ala Trp
1315 1320 1325
Ser Asp Ala Phe Ser Leu Asp Val Ala Ser Gly Leu Gly Ala Pro Leu
1330 1335 1340
Glu Gly Val Leu Val Glu Leu Glu Val Leu Pro Ala Ala Ile Pro Leu
1345 1350 1355 1360
Glu Ala Gln Asn Phe Ser Val Pro Glu Gly Gly Ser Leu Thr Leu Ala
1365 1370 1375
Pro Pro Leu Leu Arg Val Ser Gly Pro Tyr Phe Pro Thr Leu Leu Gly
1380 1385 1390
Leu Ser Leu Gln Val Leu Glu Pro Pro Gln His Gly Ala Leu Gln Lys
1395 1400 1405
Glu Asp Gly Pro Gln Ala Arg Thr Leu Ser Ala Phe Ser Trp Arg Met
1410 1415 1420
Val Glu Glu Gln Leu Ile Arg Tyr Val His Asp Gly Ser Glu Thr Leu
1425 1430 1435 1440
Thr Asp Ser Phe Val Leu Met Ala Asn Ala Ser Glu Met Asp Arg Gln
1445 1450 1455
Ser His Pro Val Ala Phe Thr Val Thr Val Leu Pro Val Asn Asp Gln
1460 1465 1470
Pro Pro Ile Leu Thr Thr Asn Thr Gly Leu Gln Met Trp Glu Gly Ala
1475 1480 1485
Thr Ala Pro Ile Pro Ala Glu Ala Leu Arg Ser Thr Asp Gly Asp Ser
1490 1495 1500
Gly Ser Glu Asp Leu Val Tyr Thr Ile Glu Gln Pro Ser Asn Gly Arg
1505 1510 1515 1520
Val Val Leu Arg Gly Ala Pro Gly Thr Glu Val Arg Ser Phe Thr Gln
1525 1530 1535
Ala Gln Leu Asp Gly Gly Leu Val Leu Phe Ser His Arg Gly Thr Leu
1540 1545 1550
Asp Gly Gly Phe Arg Phe Arg Leu Ser Asp Gly Glu His Thr Ser Pro
1555 1560 1565
Gly His Phe Phe Arg Val Thr Ala Gln Lys Gln Val Leu Leu Ser Leu
1570 1575 1580
Lys Gly Ser Gln Thr Leu Thr Val Cys Pro Gly Ser Val Gln Pro Leu
1585 1590 1595 1600
Ser Ser Gln Thr Leu Arg Ala Ser Ser Ser Ala Gly Thr Asp Pro Gln
1605 1610 1615
Leu Leu Leu Tyr Arg Val Val Arg Gly Pro Gln Leu Gly Arg Leu Phe
1620 1625 1630
His Ala Gln Gln Asp Ser Thr Gly Glu Ala Leu Val Asn Phe Thr Gln
1635 1640 1645
Ala Glu Val Tyr Ala Gly Asn Ile Leu Tyr Glu His Glu Met Pro Pro
1650 1655 1660
Glu Pro Phe Trp Glu Ala His Asp Thr Leu Glu Leu Gln Leu Ser Ser
1665 1670 1675 1680
Pro Pro Ala Arg Asp Val Ala Ala Thr Leu Ala Val Ala Val Ser Phe
1685 1690 1695
Glu Ala Ala Cys Pro Gln Arg Pro Ser His Leu Trp Lys Asn Lys Gly
1700 1705 1710
Leu Trp Val Pro Glu Gly Gln Arg Ala Arg Ile Thr Val Ala Ala Leu
1715 1720 1725
Asp Ala Ser Asn Leu Leu Ala Ser Val Pro Ser Pro Gln Arg Ser Glu
1730 1735 1740
His Asp Val Leu Phe Gln Val Thr Gln Phe Pro Ser Arg Gly Gln Leu
1745 1750 1755 1760
Leu Val Ser Glu Glu Pro Leu His Ala Gly Gln Pro His Phe Leu Gln
1765 1770 1775
Ser Gln Leu Ala Ala Gly Gln Leu Val Tyr Ala His Gly Gly Gly Gly
1780 1785 1790
Thr Gln Gln Asp Gly Phe His Phe Arg Ala His Leu Gln Gly Pro Ala
1795 1800 1805
Gly Ala Ser Val Ala Gly Pro Gln Thr Ser Glu Ala Phe Ala Ile Thr
1810 1815 1820
Val Arg Asp Val Asn Glu Arg Pro Pro Gln Pro Gln Ala Ser Val Pro
1825 1830 1835 1840
Leu Arg Leu Thr Arg Gly Ser Arg Ala Pro Ile Ser Arg Ala Gln Leu
1845 1850 1855
Ser Val Val Asp Pro Asp Ser Ala Pro Gly Glu Ile Glu Tyr Glu Val
1860 1865 1870
Gln Arg Ala Pro His Asn Gly Phe Leu Ser Leu Val Gly Gly Gly Leu
1875 1880 1885
Gly Pro Val Thr Arg Phe Thr Gln Ala Asp Val Asp Ser Gly Arg Leu
1890 1895 1900
Ala Phe Val Ala Asn Gly Ser Ser Val Ala Gly Ile Phe Gln Leu Ser
1905 1910 1915 1920
Met Ser Asp Gly Ala Ser Pro Pro Leu Pro Met Ser Leu Ala Val Asp
1925 1930 1935
Ile Leu Pro Ser Ala Ile Glu Val Gln Leu Arg Ala Pro Leu Glu Val
1940 1945 1950
Pro Gln Ala Leu Gly Arg Ser Ser Leu Ser Gln Gln Gln Leu Arg Val
1955 1960 1965
Val Ser Asp Arg Glu Glu Pro Glu Ala Ala Tyr Arg Leu Ile Gln Gly
1970 1975 1980
Pro Gln Tyr Gly His Leu Leu Val Gly Gly Arg Pro Thr Ser Ala Phe
1985 1990 1995 2000
Ser Gln Phe Gln Ile Asp Gln Gly Glu Val Val Phe Ala Phe Thr Asn
2005 2010 2015
Phe Ser Ser Ser His Asp His Phe Arg Val Leu Ala Leu Ala Arg Gly
2020 2025 2030
Val Asn Ala Ser Ala Val Val Asn Val Thr Val Arg Ala Leu Leu His
2035 2040 2045
Val Trp Ala Gly Gly Pro Trp Pro Gln Gly Ala Thr Leu Arg Leu Asp
2050 2055 2060
Pro Thr Val Leu Asp Ala Gly Glu Leu Ala Asn Arg Thr Gly Ser Val
2065 2070 2075 2080
Pro Arg Phe Arg Leu Leu Glu Gly Pro Arg His Gly Arg Val Val Arg
2085 2090 2095
Val Pro Arg Ala Arg Thr Glu Pro Gly Gly Ser Gln Leu Val Glu Gln
2100 2105 2110
Phe Thr Gln Gln Asp Leu Glu Asp Gly Arg Leu Gly Leu Glu Val Gly
2115 2120 2125
Arg Pro Glu Gly Arg Ala Pro Gly Pro Ala Gly Asp Ser Leu Thr Leu
2130 2135 2140
Glu Leu Trp Ala Gln Gly Val Pro Pro Ala Val Ala Ser Leu Asp Phe
2145 2150 2155 2160
Ala Thr Glu Pro Tyr Asn Ala Ala Arg Pro Tyr Ser Val Ala Leu Leu
2165 2170 2175
Ser Val Pro Glu Ala Ala Arg Thr Glu Ala Gly Lys Pro Glu Ser Ser
2180 2185 2190
Thr Pro Thr Gly Glu Pro Gly Pro Met Ala Ser Ser Pro Glu Pro Ala
2195 2200 2205
Val Ala Lys Gly Gly Phe Leu Ser Phe Leu Glu Ala Asn Met Phe Ser
2210 2215 2220
Val Ile Ile Pro Met Cys Leu Val Leu Leu Leu Leu Ala Leu Ile Leu
2225 2230 2235 2240
Pro Leu Leu Phe Tyr Leu Arg Lys Arg Asn Lys Thr Gly Lys His Asp
2245 2250 2255
Val Gln Val Leu Thr Ala Lys Pro Arg Asn Gly Leu Ala Gly Asp Thr
2260 2265 2270
Glu Thr Phe Arg Lys Val Glu Pro Gly Gln Ala Ile Pro Leu Thr Ala
2275 2280 2285
Val Pro Gly Gln Gly Pro Pro Pro Gly Gly Gln Pro Asp Pro Glu Leu
2290 2295 2300
Leu Gln Phe Cys Arg Thr Pro Asn Pro Ala Leu Lys Asn Gly Gln Tyr
2305 2310 2315 2320
Trp Val
<210> 238
<211> 981
<212> DNA
<213> 小鼠（Mus musculus）
<220>
<223> FAS
<220>
<221> CDS
<222> 1..981
<223> /翻译表=1
<400> 238
atg ctg tgg att tgg gcg gtg ctg ccg ctg gtg ctg gcg ggc agc cag 48
Met Leu Trp Ile Trp Ala Val Leu Pro Leu Val Leu Ala Gly Ser Gln
1 5 10 15
ctg cgc gtg cat acc cag ggc acc aac agc att agc gaa agc ctg aaa 96
Leu Arg Val His Thr Gln Gly Thr Asn Ser Ile Ser Glu Ser Leu Lys
20 25 30
ctg cgc cgc cgc gtg cgc gaa acc gat aaa aac tgc agc gaa ggc ctg 144
Leu Arg Arg Arg Val Arg Glu Thr Asp Lys Asn Cys Ser Glu Gly Leu
35 40 45
tat cag ggc ggc ccg ttt tgc tgc cag ccg tgc cag ccg ggc aaa aaa 192
Tyr Gln Gly Gly Pro Phe Cys Cys Gln Pro Cys Gln Pro Gly Lys Lys
50 55 60
aaa gtg gaa gat tgc aaa atg aac ggc ggc acc ccg acc tgc gcg ccg 240
Lys Val Glu Asp Cys Lys Met Asn Gly Gly Thr Pro Thr Cys Ala Pro
65 70 75 80
tgc acc gaa ggc aaa gaa tat atg gat aaa aac cat tat gcg gat aaa 288
Cys Thr Glu Gly Lys Glu Tyr Met Asp Lys Asn His Tyr Ala Asp Lys
85 90 95
tgc cgc cgc tgc acc ctg tgc gat gaa gaa cat ggc ctg gaa gtg gaa 336
Cys Arg Arg Cys Thr Leu Cys Asp Glu Glu His Gly Leu Glu Val Glu
100 105 110
acc aac tgc acc ctg acc cag aac acc aaa tgc aaa tgc aaa ccg gat 384
Thr Asn Cys Thr Leu Thr Gln Asn Thr Lys Cys Lys Cys Lys Pro Asp
115 120 125
ttt tat tgc gat agc ccg ggc tgc gaa cat tgc gtg cgc tgc gcg agc 432
Phe Tyr Cys Asp Ser Pro Gly Cys Glu His Cys Val Arg Cys Ala Ser
130 135 140
tgc gaa cat ggc acc ctg gaa ccg tgc acc gcg acc agc aac acc aac 480
Cys Glu His Gly Thr Leu Glu Pro Cys Thr Ala Thr Ser Asn Thr Asn
145 150 155 160
tgc cgc aaa cag agc ccg cgc aac cgc ctg tgg ctg ctg acc att ctg 528
Cys Arg Lys Gln Ser Pro Arg Asn Arg Leu Trp Leu Leu Thr Ile Leu
165 170 175
gtg ctg ctg att ccg ctg gtg ttt att tat cgc aaa tat cgc aaa cgc 576
Val Leu Leu Ile Pro Leu Val Phe Ile Tyr Arg Lys Tyr Arg Lys Arg
180 185 190
aaa tgc tgg aaa cgc cgc cag gat gat ccg gaa agc cgc acc agc agc 624
Lys Cys Trp Lys Arg Arg Gln Asp Asp Pro Glu Ser Arg Thr Ser Ser
195 200 205
cgc gaa acc att ccg atg aac gcg agc aac ctg agc ctg agc aaa tat 672
Arg Glu Thr Ile Pro Met Asn Ala Ser Asn Leu Ser Leu Ser Lys Tyr
210 215 220
att ccg cgc att gcg gaa gat atg acc att cag gaa gcg aaa aaa ttt 720
Ile Pro Arg Ile Ala Glu Asp Met Thr Ile Gln Glu Ala Lys Lys Phe
225 230 235 240
gcg cgc gaa aac aac att aaa gaa ggc aaa att gat gaa att atg cat 768
Ala Arg Glu Asn Asn Ile Lys Glu Gly Lys Ile Asp Glu Ile Met His
245 250 255
gat agc att cag gat acc gcg gaa cag aaa gtg cag ctg ctg ctg tgc 816
Asp Ser Ile Gln Asp Thr Ala Glu Gln Lys Val Gln Leu Leu Leu Cys
260 265 270
tgg tat cag agc cat ggc aaa agc gat gcg tat cag gat ctg att aaa 864
Trp Tyr Gln Ser His Gly Lys Ser Asp Ala Tyr Gln Asp Leu Ile Lys
275 280 285
ggc ctg aaa aaa gcg gaa tgc cgc cgc acc ctg gat aaa ttt cag gat 912
Gly Leu Lys Lys Ala Glu Cys Arg Arg Thr Leu Asp Lys Phe Gln Asp
290 295 300
atg gtg cag aaa gat ctg ggc aaa agc acc ccg gat acc ggc aac gaa 960
Met Val Gln Lys Asp Leu Gly Lys Ser Thr Pro Asp Thr Gly Asn Glu
305 310 315 320
aac gaa ggc cag tgc ctg gaa 981
Asn Glu Gly Gln Cys Leu Glu
325
<210> 239
<211> 327
<212> PRT
<213> 小鼠（Mus musculus）
<220>
<223> [CDS]:1..981, 来自SEQ ID NO 238
<400> 239
Met Leu Trp Ile Trp Ala Val Leu Pro Leu Val Leu Ala Gly Ser Gln
1 5 10 15
Leu Arg Val His Thr Gln Gly Thr Asn Ser Ile Ser Glu Ser Leu Lys
20 25 30
Leu Arg Arg Arg Val Arg Glu Thr Asp Lys Asn Cys Ser Glu Gly Leu
35 40 45
Tyr Gln Gly Gly Pro Phe Cys Cys Gln Pro Cys Gln Pro Gly Lys Lys
50 55 60
Lys Val Glu Asp Cys Lys Met Asn Gly Gly Thr Pro Thr Cys Ala Pro
65 70 75 80
Cys Thr Glu Gly Lys Glu Tyr Met Asp Lys Asn His Tyr Ala Asp Lys
85 90 95
Cys Arg Arg Cys Thr Leu Cys Asp Glu Glu His Gly Leu Glu Val Glu
100 105 110
Thr Asn Cys Thr Leu Thr Gln Asn Thr Lys Cys Lys Cys Lys Pro Asp
115 120 125
Phe Tyr Cys Asp Ser Pro Gly Cys Glu His Cys Val Arg Cys Ala Ser
130 135 140
Cys Glu His Gly Thr Leu Glu Pro Cys Thr Ala Thr Ser Asn Thr Asn
145 150 155 160
Cys Arg Lys Gln Ser Pro Arg Asn Arg Leu Trp Leu Leu Thr Ile Leu
165 170 175
Val Leu Leu Ile Pro Leu Val Phe Ile Tyr Arg Lys Tyr Arg Lys Arg
180 185 190
Lys Cys Trp Lys Arg Arg Gln Asp Asp Pro Glu Ser Arg Thr Ser Ser
195 200 205
Arg Glu Thr Ile Pro Met Asn Ala Ser Asn Leu Ser Leu Ser Lys Tyr
210 215 220
Ile Pro Arg Ile Ala Glu Asp Met Thr Ile Gln Glu Ala Lys Lys Phe
225 230 235 240
Ala Arg Glu Asn Asn Ile Lys Glu Gly Lys Ile Asp Glu Ile Met His
245 250 255
Asp Ser Ile Gln Asp Thr Ala Glu Gln Lys Val Gln Leu Leu Leu Cys
260 265 270
Trp Tyr Gln Ser His Gly Lys Ser Asp Ala Tyr Gln Asp Leu Ile Lys
275 280 285
Gly Leu Lys Lys Ala Glu Cys Arg Arg Thr Leu Asp Lys Phe Gln Asp
290 295 300
Met Val Gln Lys Asp Leu Gly Lys Ser Thr Pro Asp Thr Gly Asn Glu
305 310 315 320
Asn Glu Gly Gln Cys Leu Glu
325
<210> 240
<211> 1005
<212> DNA
<213> 人（Homo sapiens）
<220>
<223> FAS
<220>
<221> CDS
<222> 1..1005
<223> /翻译表=1
<400> 240
atg ctg ggc att tgg acc ctg ctg ccg ctg gtg ctg acc agc gtg gcg 48
Met Leu Gly Ile Trp Thr Leu Leu Pro Leu Val Leu Thr Ser Val Ala
1 5 10 15
cgc ctg agc agc aaa agc gtg aac gcg cag gtg acc gat att aac agc 96
Arg Leu Ser Ser Lys Ser Val Asn Ala Gln Val Thr Asp Ile Asn Ser
20 25 30
aaa ggc ctg gaa ctg cgc aaa acc gtg acc acc gtg gaa acc cag aac 144
Lys Gly Leu Glu Leu Arg Lys Thr Val Thr Thr Val Glu Thr Gln Asn
35 40 45
ctg gaa ggc ctg cat cat gat ggc cag ttt tgc cat aaa ccg tgc ccg 192
Leu Glu Gly Leu His His Asp Gly Gln Phe Cys His Lys Pro Cys Pro
50 55 60
ccg ggc gaa cgc aaa gcg cgc gat tgc acc gtg aac ggc gat gaa ccg 240
Pro Gly Glu Arg Lys Ala Arg Asp Cys Thr Val Asn Gly Asp Glu Pro
65 70 75 80
gat tgc gtg ccg tgc cag gaa ggc aaa gaa tat acc gat aaa gcg cat 288
Asp Cys Val Pro Cys Gln Glu Gly Lys Glu Tyr Thr Asp Lys Ala His
85 90 95
ttt agc agc aaa tgc cgc cgc tgc cgc ctg tgc gat gaa ggc cat ggc 336
Phe Ser Ser Lys Cys Arg Arg Cys Arg Leu Cys Asp Glu Gly His Gly
100 105 110
ctg gaa gtg gaa att aac tgc acc cgc acc cag aac acc aaa tgc cgc 384
Leu Glu Val Glu Ile Asn Cys Thr Arg Thr Gln Asn Thr Lys Cys Arg
115 120 125
tgc aaa ccg aac ttt ttt tgc aac agc acc gtg tgc gaa cat tgc gat 432
Cys Lys Pro Asn Phe Phe Cys Asn Ser Thr Val Cys Glu His Cys Asp
130 135 140
ccg tgc acc aaa tgc gaa cat ggc att att aaa gaa tgc acc ctg acc 480
Pro Cys Thr Lys Cys Glu His Gly Ile Ile Lys Glu Cys Thr Leu Thr
145 150 155 160
agc aac acc aaa tgc aaa gaa gaa ggc agc cgc agc aac ctg ggc tgg 528
Ser Asn Thr Lys Cys Lys Glu Glu Gly Ser Arg Ser Asn Leu Gly Trp
165 170 175
ctg tgc ctg ctg ctg ctg ccg att ccg ctg att gtg tgg gtg aaa cgc 576
Leu Cys Leu Leu Leu Leu Pro Ile Pro Leu Ile Val Trp Val Lys Arg
180 185 190
aaa gaa gtg cag aaa acc tgc cgc aaa cat cgc aaa gaa aac cag ggc 624
Lys Glu Val Gln Lys Thr Cys Arg Lys His Arg Lys Glu Asn Gln Gly
195 200 205
agc cat gaa agc ccg acc ctg aac ccg gaa acc gtg gcg att aac ctg 672
Ser His Glu Ser Pro Thr Leu Asn Pro Glu Thr Val Ala Ile Asn Leu
210 215 220
agc gat gtg gat ctg agc aaa tat att acc acc att gcg ggc gtg atg 720
Ser Asp Val Asp Leu Ser Lys Tyr Ile Thr Thr Ile Ala Gly Val Met
225 230 235 240
acc ctg agc cag gtg aaa ggc ttt gtg cgc aaa aac ggc gtg aac gaa 768
Thr Leu Ser Gln Val Lys Gly Phe Val Arg Lys Asn Gly Val Asn Glu
245 250 255
gcg aaa att gat gaa att aaa aac gat aac gtg cag gat acc gcg gaa 816
Ala Lys Ile Asp Glu Ile Lys Asn Asp Asn Val Gln Asp Thr Ala Glu
260 265 270
cag aaa gtg cag ctg ctg cgc aac tgg cat cag ctg cat ggc aaa aaa 864
Gln Lys Val Gln Leu Leu Arg Asn Trp His Gln Leu His Gly Lys Lys
275 280 285
gaa gcg tat gat acc ctg att aaa gat ctg aaa aaa gcg aac ctg tgc 912
Glu Ala Tyr Asp Thr Leu Ile Lys Asp Leu Lys Lys Ala Asn Leu Cys
290 295 300
acc ctg gcg gaa aaa att cag acc att att ctg aaa gat att acc agc 960
Thr Leu Ala Glu Lys Ile Gln Thr Ile Ile Leu Lys Asp Ile Thr Ser
305 310 315 320
gat agc gaa aac agc aac ttt cgc aac gaa att cag agc ctg gtg 1005
Asp Ser Glu Asn Ser Asn Phe Arg Asn Glu Ile Gln Ser Leu Val
325 330 335
<210> 241
<211> 335
<212> PRT
<213> 人（Homo sapiens）
<220>
<223> [CDS]:1..1005, 来自SEQ ID NO 240
<400> 241
Met Leu Gly Ile Trp Thr Leu Leu Pro Leu Val Leu Thr Ser Val Ala
1 5 10 15
Arg Leu Ser Ser Lys Ser Val Asn Ala Gln Val Thr Asp Ile Asn Ser
20 25 30
Lys Gly Leu Glu Leu Arg Lys Thr Val Thr Thr Val Glu Thr Gln Asn
35 40 45
Leu Glu Gly Leu His His Asp Gly Gln Phe Cys His Lys Pro Cys Pro
50 55 60
Pro Gly Glu Arg Lys Ala Arg Asp Cys Thr Val Asn Gly Asp Glu Pro
65 70 75 80
Asp Cys Val Pro Cys Gln Glu Gly Lys Glu Tyr Thr Asp Lys Ala His
85 90 95
Phe Ser Ser Lys Cys Arg Arg Cys Arg Leu Cys Asp Glu Gly His Gly
100 105 110
Leu Glu Val Glu Ile Asn Cys Thr Arg Thr Gln Asn Thr Lys Cys Arg
115 120 125
Cys Lys Pro Asn Phe Phe Cys Asn Ser Thr Val Cys Glu His Cys Asp
130 135 140
Pro Cys Thr Lys Cys Glu His Gly Ile Ile Lys Glu Cys Thr Leu Thr
145 150 155 160
Ser Asn Thr Lys Cys Lys Glu Glu Gly Ser Arg Ser Asn Leu Gly Trp
165 170 175
Leu Cys Leu Leu Leu Leu Pro Ile Pro Leu Ile Val Trp Val Lys Arg
180 185 190
Lys Glu Val Gln Lys Thr Cys Arg Lys His Arg Lys Glu Asn Gln Gly
195 200 205
Ser His Glu Ser Pro Thr Leu Asn Pro Glu Thr Val Ala Ile Asn Leu
210 215 220
Ser Asp Val Asp Leu Ser Lys Tyr Ile Thr Thr Ile Ala Gly Val Met
225 230 235 240
Thr Leu Ser Gln Val Lys Gly Phe Val Arg Lys Asn Gly Val Asn Glu
245 250 255
Ala Lys Ile Asp Glu Ile Lys Asn Asp Asn Val Gln Asp Thr Ala Glu
260 265 270
Gln Lys Val Gln Leu Leu Arg Asn Trp His Gln Leu His Gly Lys Lys
275 280 285
Glu Ala Tyr Asp Thr Leu Ile Lys Asp Leu Lys Lys Ala Asn Leu Cys
290 295 300
Thr Leu Ala Glu Lys Ile Gln Thr Ile Ile Leu Lys Asp Ile Thr Ser
305 310 315 320
Asp Ser Glu Asn Ser Asn Phe Arg Asn Glu Ile Gln Ser Leu Val
325 330 335
<210> 242
<211> 837
<212> DNA
<213> 小鼠 <小鼠, 属>
<220>
<223> FASL
<220>
<221> CDS
<222> 1..837
<223> /翻译表=1
<400> 242
atg cag cag ccg atg aac tat ccg tgc ccg cag att ttt tgg gtg gat 48
Met Gln Gln Pro Met Asn Tyr Pro Cys Pro Gln Ile Phe Trp Val Asp
1 5 10 15
agc agc gcg acc agc agc tgg gcg ccg ccg ggc agc gtg ttt ccg tgc 96
Ser Ser Ala Thr Ser Ser Trp Ala Pro Pro Gly Ser Val Phe Pro Cys
20 25 30
ccg agc tgc ggc ccg cgc ggc ccg gat cag cgc cgc ccg ccg ccg ccg 144
Pro Ser Cys Gly Pro Arg Gly Pro Asp Gln Arg Arg Pro Pro Pro Pro
35 40 45
ccg ccg ccg gtg agc ccg ctg ccg ccg ccg agc cag ccg ctg ccg ctg 192
Pro Pro Pro Val Ser Pro Leu Pro Pro Pro Ser Gln Pro Leu Pro Leu
50 55 60
ccg ccg ctg acc ccg ctg aaa aaa aaa gat cat aac acc aac ctg tgg 240
Pro Pro Leu Thr Pro Leu Lys Lys Lys Asp His Asn Thr Asn Leu Trp
65 70 75 80
ctg ccg gtg gtg ttt ttt atg gtg ctg gtg gcg ctg gtg ggc atg ggc 288
Leu Pro Val Val Phe Phe Met Val Leu Val Ala Leu Val Gly Met Gly
85 90 95
ctg ggc atg tat cag ctg ttt cat ctg cag aaa gaa ctg gcg gaa ctg 336
Leu Gly Met Tyr Gln Leu Phe His Leu Gln Lys Glu Leu Ala Glu Leu
100 105 110
cgc gaa ttt acc aac cag agc ctg aaa gtg agc agc ttt gaa aaa cag 384
Arg Glu Phe Thr Asn Gln Ser Leu Lys Val Ser Ser Phe Glu Lys Gln
115 120 125
att gcg aac ccg agc acc ccg agc gaa aaa aaa gaa ccg cgc agc gtg 432
Ile Ala Asn Pro Ser Thr Pro Ser Glu Lys Lys Glu Pro Arg Ser Val
130 135 140
gcg cat ctg acc ggc aac ccg cat agc cgc agc att ccg ctg gaa tgg 480
Ala His Leu Thr Gly Asn Pro His Ser Arg Ser Ile Pro Leu Glu Trp
145 150 155 160
gaa gat acc tat ggc acc gcg ctg att agc ggc gtg aaa tat aaa aaa 528
Glu Asp Thr Tyr Gly Thr Ala Leu Ile Ser Gly Val Lys Tyr Lys Lys
165 170 175
ggc ggc ctg gtg att aac gaa acc ggc ctg tat ttt gtg tat agc aaa 576
Gly Gly Leu Val Ile Asn Glu Thr Gly Leu Tyr Phe Val Tyr Ser Lys
180 185 190
gtg tat ttt cgc ggc cag agc tgc aac aac cag ccg ctg aac cat aaa 624
Val Tyr Phe Arg Gly Gln Ser Cys Asn Asn Gln Pro Leu Asn His Lys
195 200 205
gtg tat atg cgc aac agc aaa tat ccg gaa gat ctg gtg ctg atg gaa 672
Val Tyr Met Arg Asn Ser Lys Tyr Pro Glu Asp Leu Val Leu Met Glu
210 215 220
gaa aaa cgc ctg aac tat tgc acc acc ggc cag att tgg gcg cat agc 720
Glu Lys Arg Leu Asn Tyr Cys Thr Thr Gly Gln Ile Trp Ala His Ser
225 230 235 240
agc tat ctg ggc gcg gtg ttt aac ctg acc agc gcg gat cat ctg tat 768
Ser Tyr Leu Gly Ala Val Phe Asn Leu Thr Ser Ala Asp His Leu Tyr
245 250 255
gtg aac att agc cag ctg agc ctg att aac ttt gaa gaa agc aaa acc 816
Val Asn Ile Ser Gln Leu Ser Leu Ile Asn Phe Glu Glu Ser Lys Thr
260 265 270
ttt ttt ggc ctg tat aaa ctg 837
Phe Phe Gly Leu Tyr Lys Leu
275
<210> 243
<211> 279
<212> PRT
<213> 小鼠 <小鼠, 属>
<220>
<223> [CDS]:1..837, 来自SEQ ID NO 242
<400> 243
Met Gln Gln Pro Met Asn Tyr Pro Cys Pro Gln Ile Phe Trp Val Asp
1 5 10 15
Ser Ser Ala Thr Ser Ser Trp Ala Pro Pro Gly Ser Val Phe Pro Cys
20 25 30
Pro Ser Cys Gly Pro Arg Gly Pro Asp Gln Arg Arg Pro Pro Pro Pro
35 40 45
Pro Pro Pro Val Ser Pro Leu Pro Pro Pro Ser Gln Pro Leu Pro Leu
50 55 60
Pro Pro Leu Thr Pro Leu Lys Lys Lys Asp His Asn Thr Asn Leu Trp
65 70 75 80
Leu Pro Val Val Phe Phe Met Val Leu Val Ala Leu Val Gly Met Gly
85 90 95
Leu Gly Met Tyr Gln Leu Phe His Leu Gln Lys Glu Leu Ala Glu Leu
100 105 110
Arg Glu Phe Thr Asn Gln Ser Leu Lys Val Ser Ser Phe Glu Lys Gln
115 120 125
Ile Ala Asn Pro Ser Thr Pro Ser Glu Lys Lys Glu Pro Arg Ser Val
130 135 140
Ala His Leu Thr Gly Asn Pro His Ser Arg Ser Ile Pro Leu Glu Trp
145 150 155 160
Glu Asp Thr Tyr Gly Thr Ala Leu Ile Ser Gly Val Lys Tyr Lys Lys
165 170 175
Gly Gly Leu Val Ile Asn Glu Thr Gly Leu Tyr Phe Val Tyr Ser Lys
180 185 190
Val Tyr Phe Arg Gly Gln Ser Cys Asn Asn Gln Pro Leu Asn His Lys
195 200 205
Val Tyr Met Arg Asn Ser Lys Tyr Pro Glu Asp Leu Val Leu Met Glu
210 215 220
Glu Lys Arg Leu Asn Tyr Cys Thr Thr Gly Gln Ile Trp Ala His Ser
225 230 235 240
Ser Tyr Leu Gly Ala Val Phe Asn Leu Thr Ser Ala Asp His Leu Tyr
245 250 255
Val Asn Ile Ser Gln Leu Ser Leu Ile Asn Phe Glu Glu Ser Lys Thr
260 265 270
Phe Phe Gly Leu Tyr Lys Leu
275
<210> 244
<211> 843
<212> DNA
<213> 人（Homo sapiens）
<220>
<223> FASL
<220>
<221> CDS
<222> 1..843
<223> /翻译表=1
<400> 244
atg cag cag ccg ttt aac tat ccg tat ccg cag att tat tgg gtg gat 48
Met Gln Gln Pro Phe Asn Tyr Pro Tyr Pro Gln Ile Tyr Trp Val Asp
1 5 10 15
agc agc gcg agc agc ccg tgg gcg ccg ccg ggc acc gtg ctg ccg tgc 96
Ser Ser Ala Ser Ser Pro Trp Ala Pro Pro Gly Thr Val Leu Pro Cys
20 25 30
ccg acc agc gtg ccg cgc cgc ccg ggc cag cgc cgc ccg ccg ccg ccg 144
Pro Thr Ser Val Pro Arg Arg Pro Gly Gln Arg Arg Pro Pro Pro Pro
35 40 45
ccg ccg ccg ccg ccg ctg ccg ccg ccg ccg ccg ccg ccg ccg ctg ccg 192
Pro Pro Pro Pro Pro Leu Pro Pro Pro Pro Pro Pro Pro Pro Leu Pro
50 55 60
ccg ctg ccg ctg ccg ccg ctg aaa aaa cgc ggc aac cat agc acc ggc 240
Pro Leu Pro Leu Pro Pro Leu Lys Lys Arg Gly Asn His Ser Thr Gly
65 70 75 80
ctg tgc ctg ctg gtg atg ttt ttt atg gtg ctg gtg gcg ctg gtg ggc 288
Leu Cys Leu Leu Val Met Phe Phe Met Val Leu Val Ala Leu Val Gly
85 90 95
ctg ggc ctg ggc atg ttt cag ctg ttt cat ctg cag aaa gaa ctg gcg 336
Leu Gly Leu Gly Met Phe Gln Leu Phe His Leu Gln Lys Glu Leu Ala
100 105 110
gaa ctg cgc gaa agc acc agc cag atg cat acc gcg agc agc ctg gaa 384
Glu Leu Arg Glu Ser Thr Ser Gln Met His Thr Ala Ser Ser Leu Glu
115 120 125
aaa cag att ggc cat ccg agc ccg ccg ccg gaa aaa aaa gaa ctg cgc 432
Lys Gln Ile Gly His Pro Ser Pro Pro Pro Glu Lys Lys Glu Leu Arg
130 135 140
aaa gtg gcg cat ctg acc ggc aaa agc aac agc cgc agc atg ccg ctg 480
Lys Val Ala His Leu Thr Gly Lys Ser Asn Ser Arg Ser Met Pro Leu
145 150 155 160
gaa tgg gaa gat acc tat ggc att gtg ctg ctg agc ggc gtg aaa tat 528
Glu Trp Glu Asp Thr Tyr Gly Ile Val Leu Leu Ser Gly Val Lys Tyr
165 170 175
aaa aaa ggc ggc ctg gtg att aac gaa acc ggc ctg tat ttt gtg tat 576
Lys Lys Gly Gly Leu Val Ile Asn Glu Thr Gly Leu Tyr Phe Val Tyr
180 185 190
agc aaa gtg tat ttt cgc ggc cag agc tgc aac aac ctg ccg ctg agc 624
Ser Lys Val Tyr Phe Arg Gly Gln Ser Cys Asn Asn Leu Pro Leu Ser
195 200 205
cat aaa gtg tat atg cgc aac agc aaa tat ccg cag gat ctg gtg atg 672
His Lys Val Tyr Met Arg Asn Ser Lys Tyr Pro Gln Asp Leu Val Met
210 215 220
atg gaa ggc aaa atg atg agc tat tgc acc acc ggc cag atg tgg gcg 720
Met Glu Gly Lys Met Met Ser Tyr Cys Thr Thr Gly Gln Met Trp Ala
225 230 235 240
cgc agc agc tat ctg ggc gcg gtg ttt aac ctg acc agc gcg gat cat 768
Arg Ser Ser Tyr Leu Gly Ala Val Phe Asn Leu Thr Ser Ala Asp His
245 250 255
ctg tat gtg aac gtg agc gaa ctg agc ctg gtg aac ttt gaa gaa agc 816
Leu Tyr Val Asn Val Ser Glu Leu Ser Leu Val Asn Phe Glu Glu Ser
260 265 270
cag acc ttt ttt ggc ctg tat aaa ctg 843
Gln Thr Phe Phe Gly Leu Tyr Lys Leu
275 280
<210> 245
<211> 281
<212> PRT
<213> 人（Homo sapiens）
<220>
<223> [CDS]:1..843, 来自SEQ ID NO 244
<400> 245
Met Gln Gln Pro Phe Asn Tyr Pro Tyr Pro Gln Ile Tyr Trp Val Asp
1 5 10 15
Ser Ser Ala Ser Ser Pro Trp Ala Pro Pro Gly Thr Val Leu Pro Cys
20 25 30
Pro Thr Ser Val Pro Arg Arg Pro Gly Gln Arg Arg Pro Pro Pro Pro
35 40 45
Pro Pro Pro Pro Pro Leu Pro Pro Pro Pro Pro Pro Pro Pro Leu Pro
50 55 60
Pro Leu Pro Leu Pro Pro Leu Lys Lys Arg Gly Asn His Ser Thr Gly
65 70 75 80
Leu Cys Leu Leu Val Met Phe Phe Met Val Leu Val Ala Leu Val Gly
85 90 95
Leu Gly Leu Gly Met Phe Gln Leu Phe His Leu Gln Lys Glu Leu Ala
100 105 110
Glu Leu Arg Glu Ser Thr Ser Gln Met His Thr Ala Ser Ser Leu Glu
115 120 125
Lys Gln Ile Gly His Pro Ser Pro Pro Pro Glu Lys Lys Glu Leu Arg
130 135 140
Lys Val Ala His Leu Thr Gly Lys Ser Asn Ser Arg Ser Met Pro Leu
145 150 155 160
Glu Trp Glu Asp Thr Tyr Gly Ile Val Leu Leu Ser Gly Val Lys Tyr
165 170 175
Lys Lys Gly Gly Leu Val Ile Asn Glu Thr Gly Leu Tyr Phe Val Tyr
180 185 190
Ser Lys Val Tyr Phe Arg Gly Gln Ser Cys Asn Asn Leu Pro Leu Ser
195 200 205
His Lys Val Tyr Met Arg Asn Ser Lys Tyr Pro Gln Asp Leu Val Met
210 215 220
Met Glu Gly Lys Met Met Ser Tyr Cys Thr Thr Gly Gln Met Trp Ala
225 230 235 240
Arg Ser Ser Tyr Leu Gly Ala Val Phe Asn Leu Thr Ser Ala Asp His
245 250 255
Leu Tyr Val Asn Val Ser Glu Leu Ser Leu Val Asn Phe Glu Glu Ser
260 265 270
Gln Thr Phe Phe Gly Leu Tyr Lys Leu
275 280
<210> 246
<211> 1233
<212> DNA
<213> 人工序列
<220>
<223> E3del
<220>
<221> CDS
<222> 1..1233
<223> /翻译表=1
<400> 246
atg cga ccc tcc ggg acg gcc ggg gca gcg ctc ctg gcg ctg ctg gct 48
Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala
1 5 10 15
gcg ctc tgc ccg gcg agt cgg gct ctg gag gaa aag aaa ggt aat tat 96
Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Gly Asn Tyr
20 25 30
gtg gtg aca gat cac ggc tcg tgc gtc cga gcc tgt ggg gcc gac agc 144
Val Val Thr Asp His Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser
35 40 45
tat gag atg gag gaa gac ggc gtc cgc aag tgt aag aag tgc gaa ggg 192
Tyr Glu Met Glu Glu Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly
50 55 60
cct tgc cgc aaa gtg tgt aac gga ata ggt att ggt gaa ttt aaa gac 240
Pro Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp
65 70 75 80
tca ctc tcc ata aat gct acg aat att aaa cac ttc aaa aac tgc acc 288
Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr
85 90 95
tcc atc agt ggc gat ctc cac atc ctg ccg gtg gca ttt agg ggt gac 336
Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp
100 105 110
tcc ttc aca cat act cct cct ctg gat cca cag gaa ctg gat att ctg 384
Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu
115 120 125
aaa acc gta aag gaa atc aca ggg ttt ttg ctg att cag gct tgg cct 432
Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro
130 135 140
gaa aac agg acg gac ctc cat gcc ttt gag aac cta gaa atc ata cgc 480
Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg
145 150 155 160
ggc agg acc aag caa cat ggt cag ttt tct ctt gca gtc gtc agc ctg 528
Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu
165 170 175
aac ata aca tcc ttg gga tta cgc tcc ctc aag gag ata agt gat gga 576
Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly
180 185 190
gat gtg ata att tca gga aac aaa aat ttg tgc tat gca aat aca ata 624
Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile
195 200 205
aac tgg aaa aaa ctg ttt ggg acc tcc ggt cag aaa acc aaa att ata 672
Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile
210 215 220
agc aac aga ggt gaa aac agc tgc aag gcc aca ggc cag gtc tgc cat 720
Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His
225 230 235 240
gcc ttg tgc tcc ccc gag ggc tgc tgg ggc ccg gag ccc agg gac tgc 768
Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys
245 250 255
gtc tct tgc cgg aat gtc agc cga ggc agg gaa tgc gtg gac aag tgc 816
Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys
260 265 270
aac ctt ctg gag ggt gag cca agg gag ttt gtg gag aac tct gag tgc 864
Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys
275 280 285
ata cag tgc cac cca gag tgc ctg cct cag gcc atg aac atc acc tgc 912
Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys
290 295 300
aca gga cgg gga cca gac aac tgt atc cag tgt gcc cac tac att gac 960
Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp
305 310 315 320
ggc ccc cac tgc gtc aag acc tgc ccg gca gga gtc atg gga gaa aac 1008
Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn
325 330 335
aac acc ctg gtc tgg aag tac gca gac gcc ggc cat gtg tgc cac ctg 1056
Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu
340 345 350
tgc cat cca aac tgc acc tac gga tgc act ggg cca ggt ctt gaa ggc 1104
Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly
355 360 365
tgt cca acg aat ggg cct aag atc ccg tcc atc gcc act ggg atg gtg 1152
Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val
370 375 380
ggg gcc ctc ctc ttg ctg ctg gtg gtg gcc ctg ggg atc ggc ctc ttc 1200
Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe
385 390 395 400
atg cga agg cgc cac atc gtt cgg aag cgc tga 1233
Met Arg Arg Arg His Ile Val Arg Lys Arg
405 410
<210> 247
<211> 410
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..1233, 来自SEQ ID NO 246
<400> 247
Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala
1 5 10 15
Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Gly Asn Tyr
20 25 30
Val Val Thr Asp His Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser
35 40 45
Tyr Glu Met Glu Glu Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly
50 55 60
Pro Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp
65 70 75 80
Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr
85 90 95
Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp
100 105 110
Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu
115 120 125
Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro
130 135 140
Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg
145 150 155 160
Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu
165 170 175
Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly
180 185 190
Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile
195 200 205
Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile
210 215 220
Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His
225 230 235 240
Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys
245 250 255
Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys
260 265 270
Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys
275 280 285
Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys
290 295 300
Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp
305 310 315 320
Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn
325 330 335
Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu
340 345 350
Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly
355 360 365
Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val
370 375 380
Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe
385 390 395 400
Met Arg Arg Arg His Ile Val Arg Lys Arg
405 410
<210> 248
<211> 1410
<212> DNA
<213> 人工序列
<220>
<223> CAREpCam
<220>
<221> CDS
<222> 1..1410
<223> /翻译表=1
<400> 248
atg gac atc aga ctg agc ctg gcc ttc ctg gtg ctg ttc atc aag ggc 48
Met Asp Ile Arg Leu Ser Leu Ala Phe Leu Val Leu Phe Ile Lys Gly
1 5 10 15
gtg cag tgc gag gtg cag ctg gcc gaa tct ggc ggc gga ctg gtg cag 96
Val Gln Cys Glu Val Gln Leu Ala Glu Ser Gly Gly Gly Leu Val Gln
20 25 30
ccc ggc aga tcc atg aag ctg agc tgc gct gcc agc ggc ttc acc ttc 144
Pro Gly Arg Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
35 40 45
agc aac ttc ccc atg gcc tgg gtg cgc cag gcc ccc acc aag tgt ctg 192
Ser Asn Phe Pro Met Ala Trp Val Arg Gln Ala Pro Thr Lys Cys Leu
50 55 60
gaa tgg gtg gcc acc atc agc acc agc ggc ggc agc acc tac tac cgg 240
Glu Trp Val Ala Thr Ile Ser Thr Ser Gly Gly Ser Thr Tyr Tyr Arg
65 70 75 80
gac agc gtg aag ggc cgg ttc acc atc agc cgg gac aac gcc aag agc 288
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser
85 90 95
acc ctg tac ctg cag atg aac agc ctg cgg agc gag gac acc gcc acc 336
Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Thr
100 105 110
tac tac tgc acc cgg acc ctg tac atc ctg cgg gtg ttc tac ttc gac 384
Tyr Tyr Cys Thr Arg Thr Leu Tyr Ile Leu Arg Val Phe Tyr Phe Asp
115 120 125
tac tgg ggc cag ggc gtg atg gtg aca gtg tct agc ggc gga ggc ggc 432
Tyr Trp Gly Gln Gly Val Met Val Thr Val Ser Ser Gly Gly Gly Gly
130 135 140
agc gga ggt gga gga agt ggc ggc gga gga tcc gac atc cag atg acc 480
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
145 150 155 160
cag tct ccc gcc agc ctg agc gcc tct ctg ggc gag aca gtg tcc atc 528
Gln Ser Pro Ala Ser Leu Ser Ala Ser Leu Gly Glu Thr Val Ser Ile
165 170 175
gag tgc ctg gcc agc gag ggc atc agc aac gac ctg gcc tgg tat cag 576
Glu Cys Leu Ala Ser Glu Gly Ile Ser Asn Asp Leu Ala Trp Tyr Gln
180 185 190
cag aag tcc ggc aag agc ccc cag ctg ctg atc tac gcc acc agc aga 624
Gln Lys Ser Gly Lys Ser Pro Gln Leu Leu Ile Tyr Ala Thr Ser Arg
195 200 205
ctg cag gac ggc gtg ccc agc aga ttc agc ggc agc ggc tcc ggc acc 672
Leu Gln Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
210 215 220
cgg tac agc ctg aag atc agc ggc atg cag ccc gag gac gag gcc gac 720
Arg Tyr Ser Leu Lys Ile Ser Gly Met Gln Pro Glu Asp Glu Ala Asp
225 230 235 240
tac ttc tgc cag cag agc tac aag tac ccc tgg acc ttc ggc tgc ggc 768
Tyr Phe Cys Gln Gln Ser Tyr Lys Tyr Pro Trp Thr Phe Gly Cys Gly
245 250 255
aca aag ctg gaa ctg aag ggc gga ggg ggc tct ggg gga ggc gga tct 816
Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
260 265 270
ctc gag gaa cag aag ctg atc agc gaa gag gac ctg act act acc aag 864
Leu Glu Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Thr Thr Thr Lys
275 280 285
cca gtg ctg cga act ccc tca cct gtg cac cct acc ggg aca tct cag 912
Pro Val Leu Arg Thr Pro Ser Pro Val His Pro Thr Gly Thr Ser Gln
290 295 300
ccc cag aga cca gaa gat tgt cgg ccc cgt ggc tca gtg aag ggg acc 960
Pro Gln Arg Pro Glu Asp Cys Arg Pro Arg Gly Ser Val Lys Gly Thr
305 310 315 320
gga ttg gac ttc gcc tgt gat att tac atc tgg gca ccc ttg gcc gga 1008
Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly
325 330 335
atc tgc gtg gcc ctt ctg ctg tcc ttg atc atc act ctc atc tgc tac 1056
Ile Cys Val Ala Leu Leu Leu Ser Leu Ile Ile Thr Leu Ile Cys Tyr
340 345 350
cac agg agc cga aga gca aaa ttc agc agg agt gca gag act gct gcc 1104
His Arg Ser Arg Arg Ala Lys Phe Ser Arg Ser Ala Glu Thr Ala Ala
355 360 365
aac ctg cag gac ccc aac cag ctc tac aat gag ctc aat cta ggg cga 1152
Asn Leu Gln Asp Pro Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg
370 375 380
aga gag gaa tat gac gtc ttg gag aag aag cgg gct cgg gat cca gag 1200
Arg Glu Glu Tyr Asp Val Leu Glu Lys Lys Arg Ala Arg Asp Pro Glu
385 390 395 400
atg gga ggc aaa cag cag agg agg agg aac ccc cag gaa ggc gta tac 1248
Met Gly Gly Lys Gln Gln Arg Arg Arg Asn Pro Gln Glu Gly Val Tyr
405 410 415
aat gca ctg cag aaa gac aag atg gca gaa gcc tac agt gag atc ggc 1296
Asn Ala Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly
420 425 430
aca aaa ggc gag agg cgg aga ggc aag ggg cac gat ggc ctt tac cag 1344
Thr Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln
435 440 445
ggt ctc agc act gcc acc aag gac acc tat gat gcc ctg cat atg cag 1392
Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln
450 455 460
acc ctg gcc cct cgc taa 1410
Thr Leu Ala Pro Arg
465
<210> 249
<211> 469
<212> PRT
<213> 人工序列
<220>
<223> [CDS]:1..1410, 来自SEQ ID NO 248
<400> 249
Met Asp Ile Arg Leu Ser Leu Ala Phe Leu Val Leu Phe Ile Lys Gly
1 5 10 15
Val Gln Cys Glu Val Gln Leu Ala Glu Ser Gly Gly Gly Leu Val Gln
20 25 30
Pro Gly Arg Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
35 40 45
Ser Asn Phe Pro Met Ala Trp Val Arg Gln Ala Pro Thr Lys Cys Leu
50 55 60
Glu Trp Val Ala Thr Ile Ser Thr Ser Gly Gly Ser Thr Tyr Tyr Arg
65 70 75 80
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser
85 90 95
Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Thr
100 105 110
Tyr Tyr Cys Thr Arg Thr Leu Tyr Ile Leu Arg Val Phe Tyr Phe Asp
115 120 125
Tyr Trp Gly Gln Gly Val Met Val Thr Val Ser Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
145 150 155 160
Gln Ser Pro Ala Ser Leu Ser Ala Ser Leu Gly Glu Thr Val Ser Ile
165 170 175
Glu Cys Leu Ala Ser Glu Gly Ile Ser Asn Asp Leu Ala Trp Tyr Gln
180 185 190
Gln Lys Ser Gly Lys Ser Pro Gln Leu Leu Ile Tyr Ala Thr Ser Arg
195 200 205
Leu Gln Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
210 215 220
Arg Tyr Ser Leu Lys Ile Ser Gly Met Gln Pro Glu Asp Glu Ala Asp
225 230 235 240
Tyr Phe Cys Gln Gln Ser Tyr Lys Tyr Pro Trp Thr Phe Gly Cys Gly
245 250 255
Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
260 265 270
Leu Glu Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Thr Thr Thr Lys
275 280 285
Pro Val Leu Arg Thr Pro Ser Pro Val His Pro Thr Gly Thr Ser Gln
290 295 300
Pro Gln Arg Pro Glu Asp Cys Arg Pro Arg Gly Ser Val Lys Gly Thr
305 310 315 320
Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly
325 330 335
Ile Cys Val Ala Leu Leu Leu Ser Leu Ile Ile Thr Leu Ile Cys Tyr
340 345 350
His Arg Ser Arg Arg Ala Lys Phe Ser Arg Ser Ala Glu Thr Ala Ala
355 360 365
Asn Leu Gln Asp Pro Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg
370 375 380
Arg Glu Glu Tyr Asp Val Leu Glu Lys Lys Arg Ala Arg Asp Pro Glu
385 390 395 400
Met Gly Gly Lys Gln Gln Arg Arg Arg Asn Pro Gln Glu Gly Val Tyr
405 410 415
Asn Ala Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly
420 425 430
Thr Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln
435 440 445
Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln
450 455 460
Thr Leu Ala Pro Arg
465

Claims (15)

1.一种试剂盒,其包含
(A)编码用于转导自要治疗的受试者获得的T细胞的融合蛋白的核酸分子,该融合蛋白具有下面的域:
(1)在所述T细胞中或上并不天然发生的信号传导受体的胞外域；
(2)锚定跨膜域；和
(3)刺激性信号传导域,和
(B)三价双特异性抗体分子,其包含:
(i)结合(A)中表征的融合蛋白的胞外域(1)的第一结合域；
(ii)结合在肿瘤细胞的表面上天然发生的肿瘤特异性抗原的第二结合域；和
(iii)结合(A)中表征的融合蛋白的胞外域(1)或结合所述在肿瘤细胞的表面上天然发生的肿瘤特异性抗原的第三结合域。
2.权利要求1的试剂盒,其中所述融合蛋白进一步包含至少一个共刺激性信号传导域。
3.一种三价双特异性抗体分子,其包含:
(i)结合权利要求1(A)中表征的融合蛋白的胞外域(1)的第一结合域；
(ii)结合在肿瘤细胞的表面上天然发生的肿瘤特异性抗原的第二结合域；和
(iii)结合权利要求1(A)中表征的融合蛋白的胞外域(1)或结合所述在肿瘤细胞的表面上天然发生的肿瘤特异性抗原的第三结合域,
供作为药物使用,其中所述双特异性抗体分子要在施用包含权利要求1(A)中表征的融合蛋白的经转导T细胞之前,同时或之后施用且其中所述T细胞是自该要治疗的受试者获得的。
4.一种包含三价双特异性抗体分子的药用组合物,其包含:
(i)结合权利要求1(A)中表征的融合蛋白的胞外域(1)的第一结合域；
(ii)结合在肿瘤细胞的表面上天然发生的肿瘤特异性抗原的第二结合域；和
(iii)结合权利要求1(A)中表征的融合蛋白的胞外域(1)或结合所述在肿瘤细胞的表面上天然发生的肿瘤特异性抗原的第三结合域,
其要与包含权利要求1(A)中表征的融合蛋白的经转导T细胞组合施用且其中所述T细胞是自该要治疗的受试者获得的。
5.一种三价双特异性抗体分子,其包含:
(i)结合权利要求1(A)中表征的融合蛋白的胞外域(1)的第一结合域；
(ii)结合在肿瘤细胞的表面上天然发生的肿瘤特异性抗原的第二结合域；和
(iii)结合权利要求1(A)中表征的融合蛋白的胞外域(1)或结合所述在肿瘤细胞的表面上天然发生的肿瘤特异性抗原的第三结合域,
其供治疗恶性疾病的方法中使用,其中所述三价双特异性抗体分子要在施用包含权利要求1(A)中表征的融合蛋白的经转导T细胞之前,同时或之后施用且其中所述T细胞是自该要治疗的受试者获得的。
6.权利要求5的三价双特异性抗体分子,其中所述恶性疾病选自上皮,内皮或间皮起源的癌症和血液的癌症。
7.权利要求1或权利要求2的试剂盒,权利要求4的药用组合物,权利要求3或5的三价双特异性抗体分子,其中所述在肿瘤细胞的表面上天然发生的抗原选自由EpCAM,MSLN,MCSP,HER-1,HER-2,HER-3,CD20,CD22,CD33,CD52,FLT-3,FOLR1,Trop-2,CA-12-5,HLA-DR,MUC-1(粘蛋白),A33-抗原,PSMA,PSCA,(前列腺特异性膜抗原),运铁蛋白受体,生腱蛋白和CA-IX(碳酸酐酶IX)组成的组。
8.权利要求1,权利要求2或权利要求7的试剂盒,权利要求4或权利要求7的药用组合物,权利要求3,5或7的三价双特异性抗体分子,其中所述在所述T细胞中或上并不天然发生的信号传导受体的胞外域选自由Cripto(cryptic家族蛋白),CD(分化簇)家族的成员(非T细胞),EGFR,EGFRvIII和TSH-R组成的组成的组。
9.权利要求1,2,7或8任一项的试剂盒,权利要求4,7或8任一项的药用组合物,权利要求3,5,7或8任一项的三价双特异性抗体分子,其中所述经转导T细胞进一步包含在所述T细胞上天然发生的T细胞受体和/或遗传导入所述T细胞的T细胞受体。
10.一种表达载体,其包含编码权利要求1(B)和3至5任一项中限定的三价双特异性抗体的核酸序列。
11.权利要求10的载体,其是多顺反子的。
12.权利要求10或权利要求11的载体,其中所述载体进一步包含与权利要求10的所述核酸序列可操作连接的调节序列。
13.一种宿主细胞,其经权利要求10至12任一项中限定的载体转化。
14.一种用于生成权利要求1(B)和3至5任一项中限定的三价双特异性抗体分子的方法,所述方法包含
(a)在容许权利要求1(B)和3至5任一项中限定的三价双特异性抗体分子表达的条件下培养权利要求13中限定的宿主细胞；和
(b)自培养物回收生成的三价双特异性抗体分子。
15.权利要求1(B)和3至5任一项中限定的三价双特异性抗体分子,其包含
(i)结合权利要求1(A)中表征的融合蛋白的胞外域(1)的第一结合域；
(ii)结合在肿瘤细胞的表面上天然发生的肿瘤特异性抗原的第二结合域；和
(iii)结合权利要求1(A)中表征的融合蛋白的胞外域(1)或结合在肿瘤细胞的表面上天然发生的所述肿瘤特异性抗原的第三结合域,
其中所述抗体分子是通过权利要求14的方法可获得的。
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