CN110121505B - 嵌合抗原受体和表达其的自然杀伤细胞 - Google Patents

嵌合抗原受体和表达其的自然杀伤细胞 Download PDF

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CN110121505B
CN110121505B CN201780081622.3A CN201780081622A CN110121505B CN 110121505 B CN110121505 B CN 110121505B CN 201780081622 A CN201780081622 A CN 201780081622A CN 110121505 B CN110121505 B CN 110121505B
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黃琉炅
赵诚唯
元星湧
林虎勇
许正贤
郑美永
金炫娥
权秀贤
李恩率
金汉率
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GC Cell Corp
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Abstract

本发明涉及一种嵌合抗原受体和表达其的自然杀伤细胞，更具体地涉及一种包括细胞内信号传导结构域的嵌合抗原受体(CAR)以及表达其的免疫细胞，所述细胞内信号传导结构域包括OX40配体(CD252)的全部或一部分，因此具有优异的对免疫细胞的抗癌活性增强效果。

Description

嵌合抗原受体和表达其的自然杀伤细胞
技术领域
本发明涉及一种嵌合抗原受体和表达其的自然杀伤细胞。
背景技术
人自然杀伤细胞(natural killer cells,NK细胞)在针对恶性淋巴瘤细胞的先天免疫防御中起着重要作用，并且因此可用于过继免疫治疗(即过继细胞免疫治疗，adoptiveimmune therapy,adoptive cellular immunotherapy)。然而，由于离体细胞扩增的困难和NK细胞的活性在个体患者中的差异，难以使用NK细胞。
在使用T细胞的免疫细胞疗法的领域，即T细胞治疗领域，已开发用于自身免疫细胞治疗的嵌合抗原受体。已知嵌合抗原受体通过重新编程T细胞来帮助T细胞激活，以改善对特定癌症的治疗效果或克服对T细胞治疗的抗性。
OX40配体(CD252)是一种属于TNFR超家族的蛋白，并且公知在抗原呈递细胞(APC)、部分NK细胞和部分B细胞中表达，还已知其在这些细胞激活后数小时至数天内表达。
作为OX40配体(CD252)的受体，OX40(CD134)已知在T细胞中表达，并且还已知其在通过抗原和CD28激活T细胞后在24小时内表达。已知CD134的表达可通过CD28激活进一步增强T细胞的应答，从而增加T细胞的增殖以及细胞因子的分泌和存活。
然而，尚不清楚OX40配体在增强NK细胞的抗癌活性中的作用，并且尚未报道试图将OX40配体用于嵌合抗原受体的尝试。
为了研究CD252在NK细胞的抗肿瘤应答中的作用，本发明人构建了两种不同的含有CAR的NK受体。首先，对已知在信号传导结构域中增加NK细胞的细胞毒性的多种刺激辅助因子进行遗传工程化来产生编码高亲和力重组FCRG3A V158变体(CD16V)的慢病毒。将包括不同的细胞间信号分子的CD16V受体在NK细胞中进行转导，接着检测其表达。此外，在对CD20分子特异的利妥昔单抗的存在下，在体外使用淋巴瘤细胞系研究了转导的NK细胞的抗肿瘤应答。
作为第二种方法，试验了CD252在NKG2D介导的抗肿瘤活性中的功能作用。作为NK细胞激活的关键受体的NKG2D可识别MHC 1类链相关A(MICA)、MICB以及其它不同的UL 16结合蛋白(ULBP)，它们在遗传毒性应激、感染和/或细胞毒性后优先表达。
将多种含有不同信号分子的NKG2D嵌合抗原受体遗传工程化、转导和表达于NK92MI细胞中。此后，对转导的细胞进行体外抗肿瘤效果的评价。
本发明人发现，作为克服当前的免疫细胞治疗如NK细胞的限制的一种方法，与已知的其它信号传导结构域相比，含有OX40配体的嵌合抗原受体具有显著改进的NK细胞抗癌活性，从而完成了本发明。
发明内容
发明要解决的问题
因此，本发明的目的是提供一种嵌合抗原受体，其能够使用自然杀伤(NK)细胞来提高抗癌免疫细胞治疗效率。
用于解决问题的方案
1.一种嵌合抗原受体，其包括：包含OX40配体(CD252)的全部或一部分的细胞内信号传导结构域。
2.根据上述1所述的嵌合抗原受体，其进一步包括：与细胞内信号传导结构域(intracellular signaling domain)连接的跨膜结构域(transmembrane domain)；与跨膜结构域连接的间隔区结构域(spacer domain)；和与间隔区连接的胞外结构域(extracellular domain)。
3.根据上述2所述的嵌合抗原受体，其进一步包括与胞外结构域连接的信号序列。
4.根据上述3所述的嵌合抗原受体，其中所述信号序列包括CD16的全部或一部分。
5.根据上述2所述的嵌合抗原受体，其中所述胞外结构域包括选自由抗体的抗原结合片段、Fc受体、自然细胞毒性受体、NKG2D、2B4和DNAM-1组成的组的任一种的全部或一部分。
6.根据上述5所述的嵌合抗原受体，其中抗原结合片段为Fab片段、F(ab')片段、F(ab')2片段或Fv片段。
7.根据上述6所述的嵌合抗原受体，其中Fv片段是单链可变片段(single-chainvariable fragment,ScFv)。
8.根据上述5所述的嵌合抗原受体，其中Fc受体选自由CD16、CD32、CD64、CD23、CD89及其变体组成的组。
9.根据上述8所述的嵌合抗原受体，其中Fc受体为CD16或其变体。
10.根据上述5所述的嵌合抗原受体，其中自然细胞毒性受体选自由NKp46、NKp30、NKp44、NKp80和NKp65受体组成的组。
11.根据上述2所述的嵌合抗原受体，其中间隔区结构域包括选自由CD8α和CD28组成的组的任一种的全部或一部分。
12.根据上述2所述的嵌合抗原受体，其中跨膜结构域包括选自由CD8α和CD28组成的组的任一种的全部或一部分。
13.根据上述1所述的嵌合抗原受体，其中细胞内信号传导结构域进一步包括CD3-zeta的全部或一部分。
14.根据上述13所述的嵌合抗原受体，其中CD3-zeta的全部或一部分和OX40配体的全部或一部分从细胞膜向细胞的内部顺序排列。
15.一种嵌合抗原受体，其包括细胞内信号传导结构域，所述细胞内信号传导结构域包括：包含选自由CD28和4-1BB组成的组的任一种的全部或一部分的第一细胞内信号传导结构域；包含选自由OX40配体、OX40和4-1BB组成的组的任一种的全部或一部分的第二细胞内信号传导结构域；和包含CD3-zeta的全部或一部分的第三细胞内信号传导结构域，其中第一、第二和第三细胞内信号传导结构域从细胞膜向细胞的内部顺序排列。
16.根据上述15所述的嵌合抗原受体，其进一步包括：与细胞内信号传导结构域连接的跨膜结构域；与跨膜结构域连接的间隔区结构域；和与间隔区结构域连接的胞外结构域。
17.根据上述16所述的嵌合抗原受体，其进一步包括与胞外结构域连接的信号序列。
18.根据上述17所述的嵌合抗原受体，其中信号序列包括CD16的全部或一部分。
19.根据上述16所述的嵌合抗原受体，其中胞外结构域包括选自由抗体的抗原结合片段、Fc受体、自然细胞毒性受体、NKG2D、2B4和DNAM-1组成的组的任一种的全部或一部分。
20.根据上述16所述的嵌合抗原受体，其中间隔区结构域包括选自由CD8α和CD28组成的组的任一种的全部或一部分。
21.根据上述16所述的嵌合抗原受体，其中跨膜结构域包括选自由CD8α和CD28组成的组的任一种的全部或一部分。
22.根据上述15所述的嵌合抗原受体，其中第一细胞内信号传导结构域包括CD28的全部或一部分；第二细胞内信号传导结构域包括OX40配体的全部或一部分；并且第三细胞内信号传导结构域包括CD3-zeta的全部或一部分。
23.一种免疫细胞，其表达根据上述1至22中任一项所述的嵌合抗原受体。
24.根据上述23所述的免疫细胞，其中所述免疫细胞是自然杀伤细胞(NK细胞)。
25.一种用于治疗肿瘤的药物组合物，其包括根据上述23所述的免疫细胞作为活性成分。
26.根据上述25所述的药物组合物，当胞外结构域为Fc受体时，所述药物组合物进一步包含抗体作为活性成分。
27.一种核酸序列，其编码根据上述1至22中任一项所述的嵌合抗原受体。
28.根据上述27所述的核酸序列，其中所述核酸序列编码选自由SEQ ID NO：33、41、43、45、47、49、51、53、55、69、71、77、81、83、85、87、89、91和93组成的组的一个或多个氨基酸序列，或其具有80％以上的序列同一性的变体。
29.根据上述28所述的核酸序列，其中所述核酸序列包括选自由SEQ ID NO：32、40、42、44、46、48、50、52、54、68、70、76、80、82、84、86、88、90和92组成的组的一个或多个核酸序列，或其具有80％以上的序列同一性的变体。
30.一种治疗肿瘤的方法，其包括向受试者施用根据上述23所述的免疫细胞。
发明的效果
根据本发明的嵌合抗原受体的NK细胞激活效率优异。
取决于待靶向的癌症的类型，根据本发明的嵌合抗原受体可与各种癌症靶抗体一起使用。
根据本发明的嵌合抗原受体通过应用不同的抗原识别位点适用于不同的癌症类型。
表达根据本发明的嵌合抗原受体的NK细胞对癌细胞的细胞毒性优异。
表达根据本发明的嵌合抗原受体的NK细胞可有效用于免疫细胞治疗。
附图说明
图1A示出根据本发明的一个实施方案，分别用CD16V-Z CAR(第1代)、CD16V-28ZCAR(第2代)、CD16V-BBZ CAR(第2代)、CD16V-OX40Z CAR(第2代)或CD16V-28OX40LZ CAR(第3代)转导的NK92MI细胞的CAR表达水平。
图1B示出通过对K562细胞的细胞毒性评估获得的分别用CD16V-Z CAR(第1代)、CD16V-28Z CAR(第2代)、CD16V-BBZ CAR(第2代)、CD16V-OX40Z CAR(第2代)或CD16V-28OX40LZ CAR(第3代)转导的NK92MI细胞的内在细胞杀伤能力(即，‘细胞毒性’)。
图1C示出当与针对Ramos细胞的抗体组合时，根据本发明的一个实施方案，分别用CD16V-Z CAR(第1代)、CD16V-28Z CAR(第2代)、CD16V-BBZ CAR(第2代)、CD16V-OX40Z CAR(第2代)或CD16V-28OX40LZ CAR(第3代)转导的NK92MI细胞的NK细胞介导的细胞毒性的评估。
图1D示出根据本发明的一个实施方案，分别用CD16V-Z CAR(第1代)和CD16V-OX40LZ CAR(第2代)转导的NK92MI细胞的CAR表达水平。
图1E示出当与针对Ramos细胞的抗体组合时，根据本发明的一个实施方案，分别用CD16V-Z CAR(第1代)和CD16V-OX40LZ CAR(第2代)转导的NK92MI细胞的NK细胞介导的细胞毒性的评估。
图1F示出根据本发明的一个实施方案，分别用CD16V-Z CAR(第1代)和CD16V-ZOX40L CAR(第2代)转导的NK92MI细胞的CAR表达水平。
图1G示出当与针对Ramos细胞的抗体组合时，根据本发明的一个实施方案，分别用CD16V-Z CAR(第1代)或CD16V-ZOX40L CAR(第2代)转导的NK92MI细胞的NK细胞介导的细胞毒性的评估。
图2A示出根据本发明的一个实施方案，分别用CD16V-Z CAR(第1代)、CD16V-BBZCAR(第2代)或CD16V-BBOX40LZ CAR(第3代)转导的NK92MI细胞的CAR表达水平。
图2B示出当与针对Ramos细胞的抗体组合时，根据本发明的一个实施方案，分别表达CD16V-Z CAR(第1代)、CD16V-BBZ CAR(第2代)或CD16V-BBOX40LZ CAR(第3代)的NK92MI细胞的NK细胞介导的细胞毒性的评估。
图3A示出根据本发明的一个实施方案，分别用CD16V-28OX40LZ CAR、CD16V-28OX40Z CAR或CD16V-28BBZ CAR转导的NK92MI细胞的CAR表达水平，其中全部为第3代CAR。
图3B示出当与针对Ramos细胞的抗体组合时，根据本发明的一个实施方案，分别表达CD16V-28OX40LZ CAR、CD16V-28OX40Z CAR或CD16V-28BBZ CAR的NK92MI细胞的NK细胞介导的细胞毒性的评估，其中全部为第3代CAR。
图4A示出根据本发明的一个实施方案，分别用CD16V-28(H)BBZ CAR、CD16V-28(H)OX40Z CAR或CD16V-28(H)OX40LZ CAR转导的NK92MI细胞的CAR表达水平，其中全部为各自在铰链包含CD28的第3代CAR。
图4B示出当与针对Ramos细胞的抗体组合时，根据本发明的实施方案，分别表达CD16V-28(H)BBZ CAR、CD16V-28(H)OX40Z CAR或CD16V-28(H)OX40LZ CAR的NK92MI细胞的NK细胞介导的细胞毒性的评估，其中全部为各自在铰链包含CD28的第3代CAR。
图5A示出根据本发明的一个实施方案，分别用NKG2D-Z CAR(第1代)、NKG2D-28ZCAR(第2代)、NKG2D-BBZ CAR(第2代)或NKG2D-OX40Z CAR(第2代)转导的NK92MI细胞的CAR表达水平。
图5B示出根据本发明的一个实施方案，分别用NKG2D-Z CAR(第1代)、NKG2D-28ZCAR(第2代)、NKG2D-BBZ CAR(第2代)或NKG2D-OX40Z CAR(第2代)转导的NK92MI细胞对人乳腺癌细胞系MCF-7的NK细胞介导的细胞毒性的评估。
图6A示出根据本发明，分别用NKG2D-Z CAR(第1代)、包括CD28信号传导结构域的NKG2D-28BBZ CAR(第3代)、或NKG2D-28OX40Z CAR(第3代)转导的NK92MI细胞的CAR表达水平。
图6B示出根据本发明的一个实施方案，分别用NKG2D-Z CAR(第1代)、NKG2D-28BBZCAR(第3代)或NKG2D-28OX40Z CAR(第3代)转导的NK92MI细胞对人乳腺癌细胞系MCF-7的NK细胞介导的细胞毒性的评估。
图7A示出根据本发明的一个实施方案，分别用NKG2D-Z CAR(第1代)、NKG2D-28ZCAR(第2代)、NKG2D-28(H)OX40LZ CAR(第3代)或包括NKG2D胞外结构域和CD28铰链之间的AAA序列的NKG2D-AAA-28(H)OX40LZ CAR(第3代)转导的NK92MI细胞的CAR表达水平。
图7B示出根据本发明的一个实施方案，分别用NKG2D-Z CAR(第1代)、NKG2D-28ZCAR(第2代)、NKG2D-28(H)OX40LZ CAR(第3代)或包括NKG2D胞外结构域和CD28铰链之间的AAA序列的NKG2D-AAA-28(H)OX40LZ CAR(第3代)转导的NK92MI细胞对人乳腺癌细胞系MCF-7的NK细胞介导的细胞毒性的评估。
图8A示出根据本发明的一个实施方案，分别用NKG2D-Z CAR(第1代)或包括NKG2D胞外结构域和CD28铰链之间的AAA序列的NKG2D-AAA-28(H)OX40LZ CAR(第3代)转导的NK92MI细胞的CAR表达水平。
图8B示出根据本发明的一个实施方案，人肺癌细胞系H1299和H1944中不同NKG2D配体的表达水平。
图8C示出根据本发明的一个实施方案，分别用NKG2D-Z CAR(第1代)或包括NKG2D胞外结构域和CD28铰链之间的AAA序列的NKG2D-AAA-28(H)OX40LZ CAR(第3代)转导的NK92MI细胞对人肺癌细胞系H1299和H1944的NK介导的细胞毒性的评估。
具体实施方式
本发明公开了嵌合抗原受体和表达其的NK细胞，更具体地，公开了通过包括含有OX40配体(CD252)的全部或一部分的细胞内信号传导结构域而具有优异的免疫细胞的抗癌活性的增加效果的嵌合抗原受体(chimeric antigen receptor,CAR)、以及表达其的免疫细胞。
在下文中，将详细描述本发明。
本发明的嵌合抗原受体可包括含有OX40配体(CD252)的全部或一部分的细胞内信号传导结构域。
根据本发明的一个实施方案，嵌合抗原受体可以为进一步包括与细胞内信号传导结构域连接的跨膜结构域；与跨膜结构域连接的间隔区结构域；和与间隔区结构域连接的胞外结构域的嵌合抗原受体。另外，根据本发明的一个实施方案，嵌合抗原受体可以为进一步包括与胞外结构域的、未与间隔区结构域连接的末端相连接的信号序列的嵌合抗原受体。根据本发明的一个实施方案，上述结构域可以彼此直接连接，或者可通过接头连接。
根据本发明的一个实施方案，当表达嵌合抗原受体时，信号序列可允许胞外结构域位于免疫细胞(例如，NK细胞)的细胞膜外。例如，信号序列可以包括CD16的全部或一部分。
根据本发明的一个实施方案，胞外结构域是与抗体特异性连接或特异性识别抗原的结构域，并且可以包括，例如Fc受体、例如单链可变区(ScFv)等抗体中的抗原结合片段、自然细胞毒性受体、NKG2D、2B4或DNAM-1。因此，在本公开中，术语“胞外结构域”以与“抗原识别位点”、“抗原结合片段”和/或“抗体结合位点”相同的含义使用。
根据本发明的一个实施方案的嵌合抗原受体可包括作为胞外结构域的Fc受体，因此与根据待治疗的癌症细胞类型而不同的抗体相关联。根据一个实施方案，Fc受体可为选自由CD16、CD32、CD64、CD23和CD89组成的组的任一种。根据一个更具体的实施方案，Fc受体可以是包括CD16 V158变体(CD16V)的全部或一部分的Fc受体。
根据另一个实施方案，本发明的嵌合抗原受体还可进一步包括作为胞外结构域的抗体的抗原结合片段，其可在不与抗体共同施用的情况下直接识别抗原。根据一个实施方案，抗原结合片段可以是Fab片段、F(ab')片段、F(ab')2片段或Fv片段。根据本发明的一个实施方案，抗体可以是能够特异性结合抗原的、简言之为具有抗原特异性结合能力的各种类型的抗体中的任一种。例如，抗体可以是其中一条轻链和一条重链彼此键合的抗体，或者其中两条轻链和两条重链彼此键合的抗体。例如，当两条轻链和两条重链彼此键合时，抗体可以是其中包括彼此键合的第一轻链和第一重链的第一单元和包括彼此键合的第二轻链和第二重链的第二单元彼此组合的抗体。在此，所成的键可以是二硫键，但不限于此。根据本发明的实施方案，上述的两个单元可以彼此相同或不同。例如，包括第一轻链和第一重链的第一单元和包括第二轻链和第二重链的第二单元可以彼此相同或不同。因此，所制备的分别通过第一单元和第二单元识别两种不同抗原的抗体通常被称为“双特异性抗体”。另外，例如，抗体可以是其中三个以上的上述单元彼此组合的抗体。本发明的抗原结合片段可衍生于如上所述各种类型的抗体，但不限于此。
根据本发明的另一实施方案，此处使用的胞外结构域可以是自然杀伤受体(也称为“自然细胞毒性受体”)。根据一个具体的实施方案，NK受体可包括NKp46、NKp30、NKp44、NKp80和NKp65受体，但不限于此。
根据本发明的一个实施方案，跨膜结构域穿透细胞膜，并且只要该结构域可在不干扰胞外结构域和细胞内信号传导结构域功能的情况下穿透细胞膜，其可以是任何一种而没有限制。例如，跨膜结构域可包括选自由CD8α和CD28组成的组的任一种的全部或一部分。
根据本发明的一个实施方案，胞外结构域和跨膜结构域可以通过间隔区结构域彼此连接。例如，该间隔区结构域可以是铰链结构域。根据一个具体的实施方案，间隔区结构域可以包括选自由CD8α和CD28组成的组的任一种的全部或一部分。
根据本发明的一个实施方案，细胞内信号传导结构域为位于NK细胞的细胞膜内侧、即位于其细胞质中的一部分，并且可以包括当与胞外结构域连接的抗体与靶抗原相结合时，可以传递信号来激活NK细胞的序列。
根据本发明的一个实施方案，嵌合抗原受体可包括一个以上的细胞内信号传导结构域。当包括两个以上的细胞内信号传导结构域时，细胞内结构域可以彼此串联连接。例如，当包括三个细胞内信号传导结构域时，第一细胞内信号传导结构域的末端可与跨膜结构域的、未与间隔区结构域连接的末端相连接，第二细胞内信号传导结构域的末端可与第一细胞内信号传导结构域的、未与跨膜结构域连接的末端相连接，并且第三细胞内信号传导结构域的末端可与第二细胞内信号传导结构域的、未与第一胞内信号传导结构域连接的末端相连接。即，第一、第二和第三细胞内信号传导结构域可以从细胞膜向细胞的内部的顺序排列。此外，即使当包括两个、四个或更多个细胞内信号传导结构域时，这些结构域可以以如上所述相同的方式彼此连接。根据本发明的一个实施方案，这些相应的结构域可以彼此直接连接，或者可以通过接头连接。
根据本发明的一个实施方案，嵌合抗原受体可包括两个细胞内信号传导结构域。例如，嵌合抗原受体可包括与跨膜结构域连接的第一细胞内信号传导结构域，和与第一细胞内信号传导结构域的、未与跨膜结构域连接的末端相连接的第二细胞内信号传导结构域。根据一个更具体的实施方案，第一细胞内信号传导结构域可包括选自由OX40(CD134)、OX40配体(OX40L、CD252)、4-1BB(CD137)、CD28、DAP10、CD3-zeta(CD3ζ)和DAP12组成的组的任一种的的全部或一部分，并且第二细胞内信号传导结构域可包括选自由OX40配体、CD3-zeta和DAP12组成的组的任一种的全部或一部分。在这种情况下，第一细胞内信号传导结构域和第二细胞内信号传导结构域中的至少一个包括OX40配体的全部或一部分。例如，嵌合抗原受体可包括含有OX40配体的全部或一部分的第一细胞内信号传导结构域和含有选自CD3-zeta和DAP12的任一种的全部或一部分的第二细胞内信号传导结构域。此外，例如，嵌合抗原受体可包括含有选自由CD3-zeta和DAP12组成的组的任一种的全部或一部分的第一细胞内信号传导结构域和含有OX40配体的全部或一部分的第二细胞内信号传导结构域。
根据本发明的另一个实施方案，嵌合抗原受体可包括三个细胞内信号传导结构域。例如，嵌合抗原受体可以包括：与跨膜结构域连接的第一细胞内信号传导结构域；与第一细胞内信号传导结构域的、未与跨膜结构域连接的末端相连接的第二细胞内信号传导结构域；和与第二细胞内信号传导结构域的、未与第一细胞内信号传导结构域连接的末端相连接的第三细胞内信号传导结构域。根据一个更具体的实施方案，第一细胞内信号传导结构域可包括选自由4-1BB、OX40、OX40配体、CD28和DAP10组成的组的任一种的全部或一部分，第二细胞内信号传导结构域可包括选自由OX40配体、OX40和4-1BB组成的组的任一种的全部或一部分，并且第三细胞内信号传导结构域可包括选自由OX40配体、CD3-zeta和DAP12组成的组的任一种的全部或一部分。在这种情况下，第一细胞内信号传导结构域、第二细胞内信号传导结构域和第三细胞内信号传导结构域中的至少一个可包括OX40配体的全部或一部分。
另一方面，本发明可提供嵌合抗原受体，其包括：包含选自由CD28和4-1BB组成的组的任一种的全部或一部分的第一细胞内信号传导结构域；包含选自由OX40配体、OX40和4-1BB组成的组的任一种的的全部或一部分的第二细胞内信号传导结构域；和包含CD3-zeta的全部或一部分的第三细胞内信号传导结构域，其中第一、第二和第三细胞内信号传导结构域从细胞膜向细胞的内部顺序排列。根据本发明的一个实施方案，上述各个结构域可以彼此直接连接，或者可以通过接头连接。
根据本发明的一个实施方案，嵌合抗原受体可以进一步包括：与第一细胞内信号传导结构域连接的跨膜结构域；与跨膜结构域连接的间隔区结构域；和与间隔区结构域连接的胞外结构域。另外，嵌合抗原受体还可进一步包括与胞外结构域连接的信号序列。根据本发明的一个实施方案，上述各个结构域可以彼此直接连接，或者可以通过接头连接。
根据本发明的一个实施方案，胞外结构域是用于与抗体特异性结合或特异性识别抗原的结构域，例如，Fc受体、例如单链可变片段(ScFv)等的抗体的抗原结合片段、NK受体(自然细胞毒性受体)、NKG2D、2B4或DNAM-1等。因此，在本公开中，术语“胞外结构域”以与“抗原识别位点”、“抗原结合片段”和/或“抗体结合位点”相同的含义使用。
根据本发明实施方案所述的嵌合抗原受体可包括作为胞外结构域的Fc受体，并且因此，可取决于待治疗的癌症细胞类型与各种抗体一起使用。根据一个实施方案，Fc受体可包括选自由CD16、CD32、CD64、CD23和CD89及其变体组成的组的任一种。根据一个更具体的实施方案，Fc受体可包括CD16或其变体，并且最具体地，可包括CD16 V158变体(CD16V)的全部或一部分。
根据另一个实施方案，本发明的嵌合抗原受体可包括作为胞外结构域的抗体的抗原结合片段，其在不与抗体共同施用的情况下直接识别抗原。根据一个实施方案，抗原结合片段可以是Fab片段、F(ab')片段、F(ab')2片段或Fv片段。根据本发明的一个实施方案，抗体可以是能够特异性结合抗原的各种类型的抗体中的任一种。例如，抗体可以是其中一条轻链和一条重链彼此键合的抗体，或者其中两条轻链和两条重链彼此键合的抗体。例如，当两条轻链和两条重链彼此键合时，抗体可以是其中包括彼此键合的第一轻链和第一重链的第一单元和包括彼此键合的第二轻链和第二重链的第二单元彼此组合的抗体。所成的键可以是二硫键，但不限于此。根据本发明的一个实施方案，上述两个单元可以彼此相同或不同。例如，包括第一轻链和第一重链的第一单元和包括第二轻链和第二重链的第二单元可以彼此相同或不同。因此，在相关技术领域中，所制备的分别通过第一单元和第二单元识别两种不同抗原的抗体通常被称为“双特异性抗体”。另外，例如，抗体可以是其中三个以上的上述单元相互组合的抗体。本发明的抗原结合片段可衍生自如上所述的各种抗体，但不限于此。
根据本发明的另一个实施方案，本文使用的胞外结构域可以是NK受体(自然细胞毒性受体)。根据一个具体实施方案，NK受体可包括NKp46、NKp30、NKp44、NKp80和NKp65受体，但不限于此。
根据一个实施方案，信号序列可以包括CD16的全部或一部分。根据另一个实施方案，胞外结构域可包括CD16 V158变体(CD16V)的全部或一部分。根据另一个实施方案，间隔区结构域可包括选自由CD8α(CD8-alpha)和CD28组成的组的任一种的全部或一部分。根据另一个实施方案，跨膜结构域可包括选自由CD8α和CD28组成的组的任一种的全部或一部分。
根据一个具体实施方案，嵌合抗原受体可包括选自由SEQ ID NO：33、41、43、45、47、49、51、53、55、69、71、77、81、83、85、87、89、91和93组成的组的一个或多个的氨基酸序列，或其具有80％以上的序列同一性的变体。
根据另一方面，本发明提供免疫细胞(例如，NK细胞)来表达根据本发明的上述嵌合抗原受体。
本发明的免疫细胞可对肿瘤细胞表现出毒性。已确定根据本发明的嵌合抗原受体对哪些类型的肿瘤细胞表现出特异性毒性，取决于哪些类型的抗体与胞外结构域结合。因此，表达根据本发明的嵌合抗原受体的免疫细胞可以对其表现出特定毒性的肿瘤细胞的类型并不特别限定。根据一个实施方案，当本发明的免疫细胞(例如，NK细胞)与利妥昔单抗一起使用时，细胞可对恶性淋巴瘤细胞表现出毒性。例如，恶性淋巴瘤细胞可表达CD20。此外，例如，恶性淋巴瘤可以是B细胞淋巴瘤。
根据另一方面，本发明进一步提供了一种用于预防或治疗肿瘤或肿瘤转移的药物组合物，其包括表达上述根据本发明的嵌合抗原受体的免疫细胞(例如，NK细胞)，其数量为待治疗的受试者(“治疗靶”)中的肿瘤细胞(例如恶性淋巴瘤细胞)的数量的2至7.5倍。
根据本发明的一个实施方案，在本发明的药物组合物中包括的单次剂量的免疫细胞(例如，NK细胞)的数量的范围可为治疗靶中肿瘤细胞(例如，恶性淋巴瘤细胞)的数量的0.75至10倍。例如，单次剂量的免疫细胞(例如，NK细胞)的数量的范围可为治疗靶中肿瘤细胞(例如，恶性淋巴瘤细胞)数量的2至7.5倍。
根据另一方面，本发明进一步提供编码上述根据本发明的嵌合抗原受体的核酸序列。
根据本发明的一个实施方案，核酸序列可包括选自由SEQ ID NO：32、40、42、44、46、48、50、52、54、68、70、76、80、82、84、86、88、90和92组成的组的一个或多个核苷酸序列，或其具有80％或更多序列同一性的变体。
根据另一方面，本发明进一步提供了包括上述根据本发明的核酸序列的载体。
根据另一方面，本发明进一步提供一种治疗肿瘤的方法，其包括向受试者施用上述的免疫细胞。
根据另一方面，本发明进一步提供一种预防肿瘤转移的方法，其包括向受试者施用上述免疫细胞。
受试者可以是具有肿瘤的哺乳动物，具体地，是人类，但并不限于此。
给药可以通过如下方式进行：表达根据本发明的嵌合抗原受体的免疫细胞(例如，NK细胞)的数量的范围可以是治疗靶中肿瘤细胞(例如，恶性淋巴瘤细胞)的数量的2至7.5倍。
给药方法没有特别限制，例如，可以包括通过口服或胃肠外途径给药。
肿瘤没有特别限制，但可以包括恶性淋巴瘤、白血病、乳腺癌、肺癌等，并且更具体地是B细胞淋巴瘤。
在下文中，将通过以下实施例更详细地描述本发明。这些实施例仅用于详细描述本发明，并且本发明的范围不限于实施例中所示的内容。
实施例1：方法和试剂
细胞系
人B系细胞系Ramos、人红白血病细胞系K562、人乳腺癌细胞系MCF-7、人肺癌细胞系H1299和H1944、以及NK-92MI由美国典型培养物保藏中心(American Type CultureCollection)(ATCC，Manassas，VA，USA)提供。K562在含有10％FBS的RPMI-1640(Gibco，Grand Island，NY，USA)中维持。Ramos在含有10％FBS(胎牛血清；Gibco，Grand Island，NY，USA)的RPMI-1640(ATCC)(Manassas，VA)中维持。MCF-7在EMEM(ATCC)+10％FBS(Gibco)培养基中维持，同时H1299和H1944在RPMI-1640(ATCC)+10％FBS(Gibco)培养基中维持。另外，NK-92MI和转导的NK-92MI细胞在含1％人血浆的无血清培养基中维持。人胚肾成纤维细胞，即293T细胞系由ATCC提供。该细胞系在含有10％FBS(Gibco，Grand Island，NY，USA)的DMEM中维持。
质粒
分别人工合成FCRG3A V158突变体(CD16V)的信号序列和胞外结构域，NKG2D的胞外结构域；CD8α的信号序列，CD8α的铰链和跨膜结构域；CD28的铰链和跨膜结构域；以及4-1BB、OX40、OX40配体(OX40L)和CD3ζ的细胞内信号传导结构域。上述材料通过PCR重叠延伸剪接(splicing by overlapping extension by PCR,‘SOE-PCR’)以各种组合进行组装。通过直接测序确认PCR产物。将每个PCR产物以Nhe1和EcoR1切割，然后分别插入(即，连接至)例如MSCV-EF1α-GFP载体或EF1a-MCS载体等第3代自失活慢病毒表达载体的Nhe1和EcoRI位点。
已在下表1中总结根据本发明实施方案的嵌合抗原受体(CAR)。根据本发明实施方案的所有CAR的结构域已彼此串联(级联)并且框内(in frame)连接。
[表1]
CD16V-Z CAR(第1代)是通过连接以下产生的：CD16的信号序列结构域(核苷酸34-84，GenBank登录号No.X52645)；CD16V(FCRG3A V158)的胞外结构域(核苷酸85-651，在GenBank登录号No.X52645中第559位核苷酸G突变)；人CD8α衍生的铰链和跨膜结构域(核苷酸1292-1507，GenBank NM 001768.6)；CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBank NM 000734.3)；以及终止密码子TGA。
CD16V-BBZ CAR(第2代)是通过连接以下产生的：CD16的信号序列结构域(核苷酸34-84，GenBank登录号No.X52645)；CD16V(FCRG3A V158)的胞外结构域(核苷酸85-651，GenBank登录号No.X52645中第559位核苷酸G突变)；人CD8α衍生的铰链和跨膜结构域(核苷酸1292-1507，GenBank NM 001768.6)；CD137衍生的细胞内信号传导结构域(核苷酸901-1026，GenBank NM 001561.5)；CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBank NM 000734.3)；以及终止密码子TGA。
CD16V-OX40Z CAR(第2代)是通过连接以下产生的：CD16的信号序列结构域(核苷酸34-84，GenBank登录号No.X52645)；CD16V(FCRG3A V158)的胞外结构域(核苷酸85-651，GenBank登录号No.X52645中第559位核苷酸G突变)；人CD8α衍生的铰链和跨膜结构域(核苷酸1292-1507，GenBank NM 001768.6)；CD134衍生的细胞内信号传导结构域(核苷酸733-840，GenBank AB590584.1)；CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBankNM 000734.3)；以及终止密码子TGA。
CD16V-OX40LZ CAR(第2代)是通过连接以下提供的：CD16的信号序列结构域(核苷酸34-84，GenBank登录号No.X52645)；CD16V(FCRG3A V158)的胞外结构域(核苷酸85-651，GenBank登录号No.X52645中第559位核苷酸G突变)；人CD8α衍生的铰链和跨膜结构域(核苷酸1292-1507，GenBank NM 001768.6)；CD252衍生的细胞内信号传导结构域(核苷酸141-206，GenBank NM 003326.4)；CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBankNM 000734.3)；以及终止密码子TGA。
CD16V-ZOX40L CAR(第2代)是通过连接以下产生的：CD16的信号序列结构域(核苷酸34-84，GenBank登录号No.X52645)；CD16V(FCRG3A V158)的胞外结构域(核苷酸85-651，GenBank登录号No.X52645中第559位核苷酸G突变)；人CD8α衍生的铰链和跨膜结构域(核苷酸1292-1507，GenBank NM 001768.6)；CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBank NM 000734.3)；CD252衍生的细胞内信号传导结构域(核苷酸141-206，GenBank NM 003326.4)；以及终止密码子TGA。CD16V-28Z CAR(第2代)是通过连接以下产生的：CD16的信号序列结构域(34-84核苷酸，GenBank登录号No.X52645)；CD16V(FCRG3AV158)的胞外结构域(核苷酸85-651，GenBank登录号No.X52645中第559位核苷酸G突变)；人CD8α衍生的铰链结构域(核苷酸1292-1435，GenBank NM 001768.6)；CD28衍生的跨膜和细胞内信号传导结构域(核苷酸679-882，GenBank NM 006139.3)；CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBank NM 000734.3)；以及终止密码子TGA。
CD16V-28(H)Z CAR(第2代)是通过连接以下产生的：CD16的信号序列结构域(核苷酸34-84，GenBank登录号No.X52645)；CD16V(FCRG3A V158)的胞外结构域(核苷酸85-651，GenBank登录号No.X52645中第559位核苷酸G突变)；CD28衍生的铰链、跨膜和细胞内信号传导结构域(核苷酸562-882，GenBank NM 006139.3)；CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBank NM 000734.3)；以及终止密码子TGA。
CD16V-BBOX40Z CAR(第3代)是通过连接以下产生的：CD16的信号序列结构域(核苷酸34-84，GenBank登录号No.X52645)；CD16V(FCRG3A V158)的胞外结构域(核苷酸85-651，GenBank登录号No.X52645中第559位核苷酸G突变)；人CD8α衍生的铰链和跨膜结构域(核苷酸1292-1507，GenBank NM 001768.6)；CD137衍生的细胞内信号传导结构域(核苷酸901-1026，GenBank NM 001561.5)；CD134衍生的细胞内信号传导结构域(核苷酸733-840，GenBank AB590584.1)；CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBank NM000734.3)；以及终止密码子TGA。
CD16V-OX40BBZ CAR(第3代)是通过连接以下产生的：CD16的信号序列结构域(核苷酸34-84，GenBank登录号No.X52645)；CD16V(FCRG3A V158)的胞外结构域(核苷酸85-651，GenBank登录号No.X52645中第559位核苷酸G突变)；人CD8α衍生的铰链和跨膜结构域(核苷酸1292-1507，GenBank NM 001768.6)；CD134衍生的细胞内信号传导结构域(核苷酸733-840，GenBank AB590584.1)；CD137衍生的细胞内信号传导结构域(核苷酸901-1026，GenBank NM 001561.5)；CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBank NM000734.3)；以及终止密码子TGA。
CD16V-28BBZ CAR(第3代)是通过连接以下产生的：CD16的信号序列结构域(核苷酸34-84，GenBank登录号No.X52645)；CD16V(FCRG3A V158)的胞外结构域(核苷酸85-651，GenBank登录号No.X52645中第559位核苷酸G突变)；CD8α衍生的铰链(核苷酸1292-1435，GenBank NM 001768.6)；CD28衍生的跨膜和细胞内信号传导结构域(核苷酸679-882，GenBank NM 006139.3)；CD137衍生的细胞内信号传导结构域(核苷酸901-1026，GenBankNM 001561.5)；CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBank NM000734.3)；以及终止密码子TGA。
CD16V-28OX40Z CAR(第3代)是通过连接以下产生的：CD16的信号序列结构域(核苷酸34-84，GenBank登录号No.X52645)；CD16V(FCRG3A V158)的胞外结构域(核苷酸85-651，GenBank登录号No.X52645中第559位核苷酸G突变)；CD8α衍生的铰链结构域(核苷酸1292-1435，GenBank NM 001768.6)；CD28衍生的跨膜和细胞内信号传导结构域(核苷酸679-882，GenBank NM 006139.3)；CD134衍生的细胞内信号传导结构域(核苷酸733-840，GenBank AB590584.1)；CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBank NM000734.3)；以及终止密码子TGA。
CD16V-28OX40LZ CAR(第3代)是通过连接以下产生的：CD16的信号序列结构域(核苷酸34-84，GenBank登录号No.X52645)；CD16V(FCRG3A V158)的胞外结构域(核苷酸85-651，GenBank登录号No.X52645中第559位核苷酸G突变)；CD8α衍生的铰链结构域(核苷酸1292-1435，GenBank NM 001768.6)；CD28衍生的跨膜和细胞内信号传导结构域(核苷酸679-882，GenBank NM 006139.3)；CD252衍生的细胞内信号传导结构域(核苷酸141-206，GenBank NM 003326.4)；CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBank NM000734.3)；以及终止密码子TGA。
CD16V-28(H)BBZ CAR(第3代)是通过连接以下产生的：CD16的信号序列结构域(核苷酸34-84，GenBank登录号No.X52645)；CD16V(FCRG3A V158)的胞外结构域(核苷酸85-651，GenBank登录号No.X52645中第559位核苷酸G突变)；CD28衍生的铰链、跨膜和细胞内信号传导结构域(核苷酸562-882，GenBank NM 006139.3)；CD137衍生的细胞内信号传导结构域(核苷酸901-1026，GenBank NM 001561.5)；CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBank NM 000734.3)；以及终止密码子TGA。
CD16V-28(H)OX40Z CAR(第3代)是通过连接以下产生的：CD16的信号序列结构域(核苷酸34-84，GenBank登录号No.X52645)；CD16V(FCRG3A V158)的胞外结构域(核苷酸85-651，GenBank登录号No.X52645中第559位核苷酸G突变)；CD28衍生的铰链、跨膜和细胞内信号传导结构域(核苷酸562-882，GenBank NM 006139.3)；CD134衍生的细胞内信号传导结构域(核苷酸733-840，GenBank AB590584.1)；CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBank NM 000734.3)；以及终止密码子TGA。
CD16V-28(H)OX40LZ CAR(第3代)是通过连接以下产生的：CD16的信号序列结构域(核苷酸34-84，GenBank登录号No.X52645)；CD16V(FCRG3A V158)的胞外结构域(核苷酸85-651，GenBank登录号No.X52645中第559位核苷酸G突变)；CD28衍生的铰链、跨膜和细胞内信号传导结构域(核苷酸562-882，GenBank NM 006139.3)；CD252衍生的细胞内信号传导结构域(核苷酸141-206，GenBank NM 003326.4)；和CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBank NM 000734.3)以及终止密码子TGA。
CD16V-BBOX40LZ CAR(第3代)是通过连接以下产生的：CD16的信号序列结构域(核苷酸34-84，GenBank登录号No.X52645)；CD16V(FCRG3A V158)的胞外结构域(核苷酸85-651，GenBank登录号No.X52645中第559位核苷酸G突变)；人CD8α衍生的铰链和跨膜结构域(核苷酸1292-1507，GenBank NM 001768.6)；CD137衍生的细胞内信号传导结构域(核苷酸901-1026，GenBank NM 001561.5)；CD252衍生的细胞内信号传导结构域(核苷酸141-206，GenBank NM 003326.4)；CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBank NM000734.3)；以及终止密码子TGA。
用于根据本发明实施方案的嵌合抗原受体(CAR)及其制备方法的结构域的序列表总结于下表2。
[表2]
另外，根据本发明实施方案的嵌合抗原受体(CAR)总结于下表3。根据本发明实施方案的全部CAR中各个CAR的结构域与另一个CAR串联(级联)并且也框内连接。
[表3]
NKG2D-Z CAR(第1代)是通过连接以下产生的：CD8α的信号序列结构域(核苷酸890-952，GenBank NM 001768.6)；NKG2D的胞外结构域(核苷酸788-1192，GenBank ID:AF461811.1)；人CD8α衍生的铰链和跨膜结构域(核苷酸1292-1507，GenBank NM001768.6)；CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBank NM 000734.3)；以及终止密码子TGA。
NKG2D-BBZ CAR(第2代)是通过连接以下产生的：CD8α的信号序列结构域(核苷酸890-952，GenBank NM 001768.6)；NKG2D的胞外结构域(核苷酸788-1192，GenBank ID:AF461811.1)；人CD8α衍生的铰链和跨膜结构域(核苷酸1292-1507，GenBank NM001768.6)；CD137衍生的细胞内信号传导结构域(核苷酸901-1026，GenBank NM001561.5)；CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBank NM 000734.3)；以及终止密码子TGA。
NKG2D-OX40Z CAR(第2代)是通过连接以下产生的：CD8α的信号序列结构域(核苷酸890-952，GenBank NM 001768.6)；NKG2D的胞外结构域(核苷酸788-1192，GenBank ID:AF461811.1)；人CD8α衍生的铰链和跨膜结构域(核苷酸1292-1507，GenBank NM001768.6)；CD134衍生的细胞内信号传导结构域(核苷酸733-840，GenBank AB590584.1)；CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBank NM 000734.3)；以及终止密码子TGA。
NKG2D-ZOX40L CAR(第2代)是通过连接以下产生的：CD8α的信号序列结构域(核苷酸890-952，GenBank NM 001768.6)；NKG2D的胞外结构域(核苷酸788-1192，GenBank ID:AF461811.1)；人CD8α衍生的铰链和跨膜结构域(核苷酸1292-1507，GenBank NM001768.6)；CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBank NM 000734.3)；CD252衍生的细胞内信号传导结构域(核苷酸141-206，GenBank NM 003326.4)；以及CD3ζ终止密码子TGA。
NKG2D-28Z CAR(第2代)是通过连接以下产生的：CD8α的信号序列结构域(核苷酸890-952，GenBank NM 001768.6)；NKG2D的胞外结构域(核苷酸788-1192，GenBank ID:AF461811.1)；人CD8α衍生的铰链结构域(核苷酸1292-1435，GenBank NM 001768.6)；CD28衍生的跨膜和细胞内信号传导结构域(核苷酸679-882，GenBank NM 006139.3)；CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBank NM 000734.3)；以及终止密码子TGA。
NKG2D-28(H)Z CAR(第2代)是通过连接以下产生的：CD8α的信号序列结构域(核苷酸890-952，GenBank NM 001768.6)；NKG2D的胞外结构域(核苷酸788-1192，GenBank ID:AF461811.1)；CD28衍生的铰链、跨膜和细胞内信号传导结构域(核苷酸562-882，GenBankNM 006139.3)；CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBank NM000734.3)；以及终止密码子TGA。
NKG2D-BBOX40Z CAR(第3代)是通过连接以下产生的：CD8α的信号序列结构域(核苷酸890-952，GenBank NM 001768.6)；NKG2D的胞外结构域(核苷酸788-1192，GenBank ID:AF461811.1)；人CD8α衍生的铰链和跨膜结构域(核苷酸1292-1507，GenBank NM001768.6)；CD137衍生的细胞内信号传导结构域(核苷酸901-1026，GenBank NM001561.5)；CD134衍生的细胞内信号传导结构域(核苷酸733-840，GenBank AB590584.1)；CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBank NM 000734.3)；以及终止密码子TGA。
NKG2D-BBOX40LZ CAR(第3代)是通过连接以下产生的：CD8α的信号序列结构域(核苷酸890-952，GenBank NM 001768.6)；NKG2D的胞外结构域(核苷酸788-1192，GenBank ID:AF461811.1)；人CD8α衍生的铰链和跨膜结构域(核苷酸1292-1507，GenBank NM001768.6)；CD137衍生的细胞内信号传导结构域(核苷酸901-1026，GenBank NM001561.5)；CD252衍生的细胞内信号传导结构域(核苷酸141-206，GenBank NM 003326.4)；CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBank NM 000734.3)；以及终止密码子TGA。
NKG2D-OX40BBZ CAR(第3代)是通过连接以下产生的：CD8α的信号序列结构域(核苷酸890-952，GenBank NM 001768.6)；NKG2D的胞外结构域(核苷酸788-1192，GenBank ID:AF461811.1)；人CD8α衍生的铰链和跨膜结构域(核苷酸1292-1507，GenBank NM001768.6)；CD134衍生的细胞内信号传导结构域(核苷酸733-840，GenBank AB590584.1)；CD137衍生的细胞内信号传导结构域(核苷酸901-1026，GenBank NM 001561.5)；CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBank NM 000734.3)；以及终止密码子TGA。
NKG2D-28BBZ CAR(第3代)是通过连接以下产生的：CD8α的信号序列结构域(核苷酸890-952，GenBank NM 001768.6)；NKG2D的胞外结构域(核苷酸788-1192，GenBank ID:AF461811.1)；CD8α衍生的铰链结构域(核苷酸1292-1435，GenBank NM 001768.6)；CD28衍生的跨膜和细胞内信号传导结构域(核苷酸679-882，GenBank NM 006139.3)；CD137衍生的细胞内信号传导结构域(核苷酸901-1026，GenBank NM 001561.5)；CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBank NM 000734.3)；以及终止密码子TGA。
NKG2D-28OX40Z CAR(第3代)是通过连接以下产生的：CD8α的信号序列结构域(核苷酸890-952，GenBank NM 001768.6)；NKG2D的胞外结构域(核苷酸788-1192，GenBank ID:AF461811.1)；CD8α衍生的铰链结构域(核苷酸1292-1435，GenBank NM 001768.6)；CD28衍生的跨膜和细胞内信号传导结构域(核苷酸679-882，GenBank NM 006139.3)；CD134衍生的细胞内信号传导结构域(核苷酸733-840，GenBank AB590584.1)；CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBank NM 000734.3)；以及终止密码子TGA。
NKG2D-28OX40LZ CAR(第3代)是通过连接以下产生的：CD8α的信号序列结构域(核苷酸890-952，GenBank NM 001768.6)；NKG2D的胞外结构域(核苷酸788-1192，GenBank ID:AF461811.1)；CD8α衍生的铰链结构域(核苷酸1292-1435，GenBank NM 001768.6)；CD28衍生的跨膜和细胞内信号传导结构域(核苷酸679-882，GenBank NM 006139.3)；CD252衍生的细胞内信号传导结构域(核苷酸141-206，GenBank NM 003326.4)；CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBank NM 000734.3)；以及终止密码子TGA。
NKG2D-28(H)BBZ CAR(第3代)是通过连接以下产生的：CD8α的信号序列结构域(核苷酸890-952，GenBank NM 001768.6)；NKG2D的胞外结构域(核苷酸788-1192，GenBank ID:AF461811.1)；CD28衍生的铰链、跨膜和细胞内信号传导结构域(核苷酸562-882，GenBankNM 006139.3)；CD137衍生的细胞内信号传导结构域(核苷酸901-1026，GenBank NM001561.5)；CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBank NM 000734.3)；以及终止密码子TGA。
NKG2D-28(H)OX40Z CAR(第3代)是通过连接以下产生的：CD8α的信号序列结构域(核苷酸890-952，GenBank NM 001768.6)；NKG2D的胞外结构域(核苷酸788-1192，GenBankID:AF461811.1)；CD28衍生的铰链、跨膜和细胞内信号传导结构域(核苷酸562-882，GenBank NM 006139.3)；CD134衍生的细胞内信号传导结构域(核苷酸733-840，GenBankAB590584.1)；CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBank NM000734.3)；以及终止密码子TGA。
NKG2D-28(H)OX40LZ CAR(第3代)是通过连接以下产生的：CD8α的信号序列结构域(核苷酸890-952，GenBank NM 001768.6)；NKG2D的胞外结构域(核苷酸788-1192，GenBankID:AF461811.1)；CD28衍生的铰链、跨膜和细胞内信号传导结构域(核苷酸562-882，GenBank NM 006139.3)；CD252衍生的细胞内信号传导结构域(核苷酸141-206，GenBank NM003326.4)；CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBank NM 000734.3)；以及终止密码子TGA。
NKG2D-AAA-28(H)OX40LZ CAR(第3代)是通过连接以下产生的：CD8α的信号序列结构域(核苷酸890-952，GenBank NM 001768.6)；NKG2D的胞外结构域(核苷酸788-1192，GenBank ID:AF461811.1)；AAA(三丙氨酸)；CD28衍生的铰链、跨膜和细胞内信号传导结构域(核苷酸562-882，GenBank NM 006139.3)；CD252衍生的细胞内信号传导结构域(核苷酸141-206，GenBank NM 003326.4)；CD3ζ衍生的细胞内信号传导结构域(核苷酸299-634，GenBank NM 000734.3)；以及终止密码子TGA。
用于根据本发明实施方案的嵌合抗原受体(CAR)及其制备方法的结构域的序列表总结于下表4。
[表4]
病毒的产生和基因转移
为了制备VSVG假型慢病毒，在DMEM培养基中培养的293T细胞与例如PCDH1-MSCV-CD16-构建体-EF1-copGFP载体、EF1a-NKG2D构建体载体、PCDH1-MSCV-EF1-copGFP对照载体、或EF1a-GFP对照载体(用于使用空载体制备Mock感染病毒)等各种类型的载体；以及基于HIV的pPACKH1慢病毒包装试剂盒(System Biosciences)共转染。为此，使用了Lipofectamine 2000试剂(Invitrogen，Carlsbad，CA)。各种类型的CD16V构建体如下：CD16V-Z CAR,CD16V-BBZ CAR,CD16V-OX40Z CAR,CD16V-OX40LZ CAR,CD16V-ZOX40L,CD16V-28Z CAR,CD16V-28(H)Z CAR,CD16V-BBOX40Z CAR,CD16V-BBOX40LZ CAR,CD16V-OX40BBZ CAR,CD16V-28BBZ CAR,CD16V-28OX40Z CAR,CD16V-28OX40LZ CAR,CD16V-28(H)BBZ CAR,CD16V-28(H)OX40Z CAR,CD16V-28(H)OX40LZ CAR。此外，各种类型的NKG2D构建体如下：NKG2D-Z CAR,NKG2D-BBZ CAR,NKG2D-OX40Z CAR,NKG2D-ZOX40L CAR,NKG2D-28ZCAR,NKG2D-28(H)Z CAR,NKG2D-BBOX40Z CAR,NKG2D-BBOX40LZ CAR,NKG2D-OX40BBZ CAR,NKG2D-28BBZ CAR,NKG2D-28OX40Z CAR,NKG2D-28OX40LZ CAR,NKG2D-28(H)BBZ CAR,NKG2D-28(H)OX40Z CAR,NKG2D-28(H)OX40LZ CAR,NKG2D-AAA-28(H)OX40LZ CAR。通过在烧瓶中用以下转染80％致密的HEK293T细胞来制备各个慢病毒：与pPACKH1慢病毒包装质粒一起的各种类型的CD16V构建体表达载体、各种类型的NKG2D构建体表达载体或对照质粒。6小时后，培养基用含10％FBS的DMEM培养基替换。在转染48小时后，收集含有慢病毒的条件培养基，接着用0.45μm的过滤单元(Milliopore，Billerica，MA，USA)过滤来去除细胞碎片。通过使用Amicon Filter(Millipore)在4℃下以3000rpm离心20分钟以将含有病毒的病毒上清液浓缩约50倍。将浓缩的病毒储存于-80℃。
对于慢病毒感染，使用含1％人血浆的Cellgro(CellGenix)将指数生长期的NK92MI细胞调节至1x106个细胞/ml的浓度，然后在8μg/ml聚凝胺(polybrene)的存在下添加50至100MOI的慢病毒上清液，接着以1800g离心90分钟。离心后，将细胞置于37℃、5％CO2条件下的加湿培养箱中48小时。其后，用RPMI-1640清洗细胞两次，然后置于含10％FBS的RPMI-1640中供将来使用。对照细胞仅用载体转导。
含有CD16V或NKG2D的受体的表达的检测
用FACS缓冲液清洗两次CD16V CAR转导的NK92MI细胞、NKG2D CAR转导的NK92MI细胞、对照载体转导的NK92MI(NK92MI-Mock)或NK-92MI亲本细胞，使用7-AAD(Beckmancoulter)、抗CD3、抗CD56和抗CD16(BD Biosciences)mAb染色清洗过的细胞。使用BDLSRFortessa测定染色细胞的表达比率和平均荧光强度(MFI)。
使用NKG2D构建体的转导效率通过流式细胞术分析CD3-CD56+细胞中表达NKG2D的细胞来确定。首先，将NK92MI细胞以单线态进行门控，然后以7AAD-和CD3-CD56+进行门控。CD16构建体的转化效率通过流式细胞术分析CD3-CD56+细胞中表达GFP和CD16的细胞来确定。
钙黄绿素释放细胞毒性试验
在37℃下用3μM钙黄绿素-乙酰氧基甲酯(Calcein-AM；分子探针)标记靶细胞1小时。清洗后，将标记的靶细胞以1x104细胞每孔分配至96孔板中。收集NK92MI细胞、洗涤，然后在具有或不具有不同浓度的利妥昔单抗的条件下，添加不同E/T(效应物对靶)比的NK92MI细胞。作为独立于利妥昔单抗的对照组，使用抗人抗体(Sigma aldrich)。2小时后，以2000rpm离心板3分钟，并且收集100μL上清液，使用荧光酶标仪(fluorescencemicroplate reader)(Victor3，PerkinElmer)在485nm激发波长和535nm发射波长下进行钙调绿素释放的测定。通过下式计算具体的钙黄绿素释放量：特异性裂解百分比＝(试验释放-自发释放)x100/(最大释放-自发释放)。对于最大裂解，使用1％Triton X-100。
实施例2：表达含有OX40配体(CD252)的嵌合抗原受体(CAR)的NK92MI细胞对CD20阳性淋巴瘤细胞的细胞毒性的评估
含有与共刺激基序相关的CD16V的嵌合抗原受体的转导与表达
FCRG3A(CD16)的V158变体(多态性)是一种高亲和力的免疫球蛋白Fc受体并且认为在抗体治疗中具有良好的效果。本发明人制备了FCRG3A(CD 16)的V158变体，并将制备的变体与CD8α的铰链和跨膜结构域；T细胞刺激分子，即CD3ζ；以及例如CD28、4-1BB、OX-40和OX-40配体等不同的辅助刺激分子的细胞内结构域以其各种组合进行结合(表1)。将所制备的含有CD16V的嵌合抗原受体(例如，CD16V-Z CAR(第1代)、CD16V-28Z CAR(第2代)、CD16V-BBZ CAR(第2代)、CD16V-OX40Z CAR(第2代)或CD16V-28OX40LZ CAR(第3代)通过包含MSCV启动子的慢病毒载体在NK92MI细胞中表达。我们通过使用单克隆小鼠抗人抗体检测人CD16确认了各个CAR在NK92MI细胞表面上的表达。通过涉及流式细胞术分析的重复实验，已证实CAR以超过90％的效率在NK92MI细胞中转导(图1A)。本文使用的慢病毒的量等于50以上的感染复数(MOI)。
表达与CD252(OX40配体)相结合的CD16V受体的NK92MI细胞对CD20阳性淋巴瘤的肿瘤杀伤的效果增加
为了确定根据本发明的遗传修饰是否会诱导癌细胞死亡的增加，通过钙黄绿素-AM释放试验评价用空载体转导的NK92MI细胞和表达含有CD16受体的NK92MI细胞对CD20阳性淋巴瘤细胞(Ramos细胞)的细胞毒性。
在淋巴瘤细胞试验之前，将K562人红白血病细胞系用作标准对照来评价转导的NK92MI细胞的内在细胞杀伤能力(即细胞毒性)。通过使用具有50以上的感染复数(MOI)的慢病毒转导，根据本发明的各个CAR(CD16V-Z CAR(第1代)、CD16V-28Z CAR(第2代)、CD16V-BBZ CAR(第2代)、CD16V-OX40Z CAR(第2代)或CD16V-28OX40LZ CAR(第3代)在90％以上的NK92MI细胞中高水平表达(图1A)。
已证明，表达根据本发明的CAR的NK92MI细胞对K562的细胞毒性与用空载体转导的对照组(Mock)的细胞毒性相似。这意味着NK92MI细胞对靶的固有细胞毒性不通过遗传修饰本身引起的副作用而增加(图1B)。在图1B中，作为效应细胞(effector cell)的NK92MI细胞的数量与作为靶细胞(target cell)的K562细胞的数量之比表示为5:1、2.5:1、1:1和0.5:1。
为了评估各种辅助刺激分子如何在体外影响表达含有CD16V受体的NK92MI细胞的抗原特异性抗肿瘤活性，对转导的NK92MI细胞和癌细胞(B细胞淋巴瘤细胞系，即Ramos)进行共培养，然后根据钙黄绿素-AM释放试验评估癌细胞的裂解(图1C)。在图1C中，NK92MI细胞(效应细胞)的数量与Ramos细胞(靶细胞)的数量之比表示为5:1、2.5:1、1:1和0.5:1。已示出由于NK92MI不表达CD16，利妥昔单抗并不显著增加NK-92MI介导的细胞毒性。
如图1C所示，我们发现，在没有利妥昔单抗的情况下，相比于由对照(Mock)(即用仅含GFP的载体转导的NK-92MI细胞)诱导的细胞毒性，根据本发明的各种CAR的表达并不显著增加NK-92MI介导的细胞毒性。
CD16V-Z CAR(第1代)，其先前显示在各种CAR应用中增强细胞毒性，用作阳性对照。为了增加含有CD16V的受体的细胞毒性，本发明人将CD28、CD134(OX40)、CD137(4-1BB)或CD252(OX40配体)引入CD16V-Z-CAR中。在2小时体外细胞毒性检测中，表达CD16V-Z CAR(第1代)、CD16V-28Z CAR(第2代)、CD16V-BBZ CAR(第2代)、CD16V-OX40Z CAR(第2代)或CD16V-28OX40LZ CAR(第3代)的NK-92MI细胞在利妥昔单抗的存在下表现出高细胞毒性，并且效应细胞与靶细胞比为5:1和2.5:1。其中，特别是包括OX40配体的第3代CAR，即CD16V-28OX40LZ CAR，相比于其他CAR，表现最高的细胞毒性(图1C)。
为了探讨含有OX40配体的第2代CAR是否可增强NK92MI细胞的抗癌活性，制备CD16V-OX40LZ CAR并且对其进行细胞毒性的评价。当使用慢病毒载体用CD16-OX40LZ转导NK92MI细胞时，CD16V-OX40LZ CAR未表达(图1D)。即使在利妥昔单抗的存在下，也未表现由CD16V-OX40LZ CAR介导的对Ramos细胞的细胞毒性(图1E)。因此，本发明人注意到OX40配体是II型蛋白、而与OX40配体连接的CD3ζ是I型蛋白的事实，并且通过改变上述两个结构域的顺序来制备具有在N-末端的CD3ζ和在C末端的OX40配体CD16V-ZOX40L CAR。当使用具有MSCV启动子的慢病毒载体用CD16V-ZOX40L转导NK92MI细胞时，与CD16V-OX40LZ相比，NK92MI细胞有效表达CD16V-OX40LZ CAR(图1F)。此外，作为具有其中引入CD16V-ZOX40L的第2代CAR的抗癌活性的评估结果，在利妥昔单抗的存在下，在效应细胞与靶细胞之比为10:1、5:1和2.5:1时，显示出对Ramos细胞的高细胞毒性(图1G)。已确认，除含有OX40配体的CD16V-28OX40LZ CAR(第3代)外，CD16-ZOX40L CAR(第2代)也具有优于用作阳性对照的CD16V-Z CAR的抗癌活性。
CD16V-BBOX40LZ CAR的效力的评估
通过将OX40配体引入基于CD137(41BB)信号传导结构域的CAR中，对NK92MI细胞进行表达和细胞毒性的评估。CD16V-BBZ CAR(第2代)和CD16V-BBOX40LZ CAR(第3代)都在NK92MI细胞中高水平表达(图2A)。当利妥昔单抗不存在于体外细胞毒性试验中时，CD16V-Z、CD16V-BBZ和CD16V-BBOX40LZ都对Ramos细胞表现出低细胞毒性。另一方面，在利妥昔单抗的存在下，与OX40配体组合的CD16V-BBOX40 LZ在效应细胞与靶细胞的甚至任何比例中都表现出最高细胞毒性(图2B)。
CD16V-28OX40LZ CAR与其他CAR之间的效力的比较
接下来，本发明人比较了表达CD16V-28OX40LZ CAR的NK92MI细胞的细胞毒性和表达具有其他细胞内信号传导结构域的第3代CAR的NK92MI细胞的细胞毒性。本实验中使用的第3代CAR(CD16V-28OX40LZ CAR、CD16V-28OX40Z CAR和CD16V-28BBZ CAR)都已证明在NK92MI细胞中高水平表达(图3A)。
在比较了根据本发明的各种第3代CAR(即第3代嵌合抗原受体)的NK细胞激活效力后，在图3B中总结了其结果。如图3B所示，当不存在利妥昔单抗时，这些CAR全都对Ramos细胞表现出低细胞毒性，并且细胞毒性程度全部相似。另一方面，在利妥昔单抗的存在下，CD16V-28OX40LZ CAR、CD16V-28OX40Z CAR和CARCD16V-28BBZ都显示出对Ramos细胞的高细胞毒性。其中，特别是含有OX40配体的CD16V-28OX40LZ CAR表现出最优异的细胞毒性(图3B)。
含有在铰链具有CD28的OX40配体的第3代CAR的效力的评估
为了使表达NK细胞的CAR在最优化的条件下识别癌细胞抗原，不仅癌细胞抗原识别受体本身是重要的，而且抗原特异性受体和细胞膜之间的铰链序列和组成的识别也是重要的。取决于靶分子，可需要不同地设计铰链序列和组成。
在上述实验中使用的CAR使用CD8α的片段作为间隔区结构域(“铰链”)。与此相比，评估了当在铰链处使用CD28片段时CAR的细胞毒性的变化并且在图4中总结了其结果。本发明人已生产了包括CD16V的胞外结构域(胞外域)和CD28的铰链的慢病毒载体。在含有CD28片段作为间隔区的第3代CAR中，将CD28衍生的铰链、跨膜和细胞内信号传导结构域与CD134(OX40)、CD137(4-1BB)或OX40配体(CD252)的信号传导模块(signaling module)连接。使用慢病毒载体在NK92MI细胞上表达如此生产的第3代CAR。已证明，转导的NK92MI细胞可以高水平分别表达上述第3代CAR，即CD16V-28(H)BBZ CAR、CD16V-28(H)OX40Z CAR或CD16V-28(H)OX40LZ CAR(图4A)。
如图4B所示，当利妥昔单抗不存在时，CD16V-28(H)BBZ CAR、CD16V-28(H)OX40ZCAR和CD16V-28(H)OX40LZ CAR都不表现细胞毒性。另一方面，在利妥昔单抗的存在下，CD16V-28(H)BBZ CAR、CD16V-28(H)OX40Z CAR和CD16V-28(H)OX40LZ CAR都表现出高细胞毒性。其中，特别是包含OX40配体的CD16V-28(H)OX40LZ CAR具有最高的细胞毒性。
上述实验结果表明，当由本发明人发现的、特别包含OX40配体作为细胞内信号传导结构域的新CAR在自然杀伤细胞中表达时，可以获得优异的抗肿瘤效果。
实施例3：表达含有OX40配体(CD252)的NKG2D嵌合抗原受体(NKG2D-CAR)的NK92MI细胞对人乳腺癌细胞和肺癌细胞的细胞毒性的评估
包含与共刺激基序相关联的NKG2D的嵌合抗原受体的转导与表达
本发明人合成了人NKG2D基因，并且以各种组合将其与CD8α的铰链和跨膜结构域；以及T细胞刺激分子CD3ζ的胞内结构域和包括CD28、4-1BB、OX-40和OX-40配体的辅因子相结合来显著提高T细胞或NK细胞的活性(表3)。使用慢病毒载体在NK92MI细胞中表达这样的NKG2D CAR构建体。本发明人通过使用单克隆小鼠抗人抗体检测人NKG2D的方法来鉴定NK92MI细胞中各个NKG2D CAR的表面表达。通过涉及流式细胞术的重复实验，已证明CAR以70％以上的效率在NK92MI细胞中转导。
表达含有多种辅助刺激信号传导结构域的NKG2D受体的NK92MI细胞对MCF7乳腺癌细胞系的肿瘤杀伤的效果增加
为了研究遗传修饰导致的杀伤活性的变化，通过钙黄绿素-AM释放试验比较了表达NKG2D CAR的NK-92MI细胞对MCF7乳腺癌细胞的细胞毒性。
为了评价各种辅助刺激信号传导结构域对表达NKG2D CAR的NK-92MI细胞的抗原特异性抗肿瘤效果的体外效果，将转导的NK-92MI细胞和癌细胞共培养，接着通过钙黄绿素-AM释放测定肿瘤细胞的裂解。根据以前的研究，已知将辅助刺激信号传导结构域添加至嵌合受体可增加T淋巴细胞和NK淋巴细胞的细胞毒性。为了提高NKG2D CAR的细胞毒性，本发明人将最公知的三个辅因子即CD28、CD134(OX-40)和CD137(4-1BB)的信号传导结构域引入NKG2D CAR中。含有引入的NKG2D-Z CAR(第1代)、NKG2D-28Z CAR、NKG2D-BBZ CAR、NKG2D-OX40Z CAR(第2代)的NK92MI细胞显示出NKG2D的有效表达(图5A)。进行2小时体外细胞毒性分析，其证明表达NKG2D-Z CAR(第1代)、NKG2D-28Z CAR、NKG2D-BBZ CAR和NKG2D-OX40ZCAR(第2代)的NK-92MI细胞对MCF7乳腺癌细胞表现出细胞毒性。然而，添加CD134(OX-40)或CD137(4-1BB)信号传导结构域至NKG2D-Z第1代CAR的主链中未表现出比NKG2D-Z CAR(第1代)更高的细胞毒性。通过包括NKG2D-28Z CAR(第2代)的NK-92MI细胞的癌细胞杀伤优于阳性对照--表达NKG2D-Z CAR(第1代)的NK-92MI细胞(图5B)。
表达含有CD28胞内结构域的NKG2D受体的NK92MI细胞对MCF7乳腺癌细胞系的肿瘤杀伤的效果增加
已知第3代CAR增加抗肿瘤活性。在生理学的T细胞应答中，最佳的淋巴细胞激活需要一个或多个与例如CD28等辅助刺激分子相结合的辅因子受体。在辅因子受体中，最重要的受体为CD137(4-1BB)和OX40(CD134)，其为肿瘤坏死因子(TNFR)的成员。
为了研究表达NKG2D CAR(第3代)的NK-92MI细胞中不同辅因子的效果，本发明人生产了NKG2D-28OX40Z CAR(第3代)和NKG2D-28BBZ CAR(第3代)。使用慢病毒载体在NK92MI细胞中表达NKG2D CAR(第3代)构建体。转导的NK92MI细胞高效表达含有第3代CAR的各种NKG2D受体(图6A)。与用对照载体转导的NK92MI细胞相比，表达各种NKG2D CAR(第3代)的NK92MI细胞在体外有效杀伤MCF7细胞。然而，第3代NKG2D-28OX40Z CAR和NKG2D-28BBZCAR，其各自在NKG2D-28Z CAR(第2代)主链中包含有CD134或CD137辅助刺激信号传导结构域，未表现出比NKG2D-Z CAR(第1代)更高的细胞毒性(图6B)。
NKG2D-AAA-28(H)OX40LZ CAR(第3代)与其它受体的比较
为了评价CD252(OX40配体)辅助刺激信号传导结构域的效果，将OX40配体的信号传导结构域融合至NKG2D-28Z CAR(第2代)的主链，从而构建NKG2D-28(H)OX40LZ CAR(第3代)。在肿瘤杀伤试验中，通过NKG2D-28(H)OX40LZ CAR(第3代)诱导的对MCF7细胞的肿瘤特异性细胞毒性与NKG2D-Z CAR(第1代)相比大幅增强，然而，其实质上与NKG2D-28Z CAR(第2代)相似(图7B)。
为了最佳的CAR T细胞识别，不仅肿瘤抗原识别受体本身是重要的，而且抗原特异性受体和细胞膜之间的铰链和接头序列也是重要的。本实验中，分析了间隔区对NKG2D-28(H)OX40LZ CAR(第3代)的功能的附加影响。本发明人已在NKG2D胞外结构域和CD28铰链之间引入了AAA(三丙氨酸)接头。使用慢病毒载体在NK92MI细胞中表达包含在此引入的AAA接头的NKG2D-AAA-28(H)OX40LZ CAR(第3代)(图7A)。本实验中，将通过表达NKG2D-AAA-28(H)OX40LZ CAR(第3代)的NK-92MI细胞的MCF7细胞杀伤与通过表达NKG2D-Z CAR(第1代)、NKG2D-28Z CAR(第2代)或NKG2D-28(H)OX40LZ CAR(第3代)的NK-92MI细胞的MCF7细胞杀伤相比较。特别地，在NKG2D胞外结构域和NKG2D-28(H)OX40LZ CAR(第3代)的CD28铰链之间引入AAA接头的引入，导致比无AAA的亲本NKG2D-28(H)OX40LZ CAR(第3代)、NKG2D-Z CAR(第1代)和/或NKG2D-28Z CAR(第2代)更好的靶细胞杀伤(图7B)。
表达NKG2D-AAA-28(H)OX40LZ CAR(第3代)的NK92MI细胞对肺癌细胞系的肿瘤杀伤效果的增加
为了评价用NKG2D-AAA-28(H)OX40LZ CAR(第3代)转导的NK92MI细胞是否可识别肺癌细胞中的NKG2D配体，使用NKG2D配体阳性肿瘤细胞(H1299和H1944)作为包含NKG2D-AAA-28(H)OX40L CAR(第3代)的NK92MI细胞的靶。转导的NK92MI细胞有效表达NKG2D-Z CAR(第1代)或NKG2D-AAA-28(H)OX40L CAR(第3代)(图8A)。接着，评价H1299和H1944细胞中NKG2D配体的表达。本发明人通过流式细胞术分析鉴定了H1299和H1944细胞中检测到的NKG2D配体的表达(图8B)。如图8C所示，包含NKG2D-AAA-28(H)OX40L CAR(第3代)的NK92MI细胞能够比作为对照组的NK92MI细胞和表达NKG2D-Z CAR(第1代)的NK92MI细胞更有效地在体外杀伤表达NKG2D配体的靶细胞。此外，由于H1299和H1944细胞不被通过对照空载体转导的NKG2D非表达的NK92MI细胞所杀伤，因此明显确证了靶细胞杀伤的特异性。
<110> 株式会社绿十字细胞治疗
<120> 嵌合抗原受体和表达其的自然杀伤细胞
<130> 16P10085
<150> KR 10-2016-0181119
<151> 2016-12-28
<160> 93
<170> KoPatentIn 3.0
<210> 1
<211> 618
<212> DNA
<213> 人工序列
<220>
<223> CD16 核苷酸
<400> 1
atgtggcagc tgctcctccc aactgctctg ctacttctag tttcagctgg catgcggact 60
gaagatctcc caaaggctgt ggtgttcctg gagcctcaat ggtacagggt gctcgagaag 120
gacagtgtga ctctgaagtg ccagggagcc tactcccctg aggacaattc cacacagtgg 180
tttcacaatg agagcctcat ctcaagccag gcctcgagct acttcattga cgctgccaca 240
gtcgacgaca gtggagagta caggtgccag acaaacctct ccaccctcag tgacccggtg 300
cagctagaag tccatatcgg ctggctgttg ctccaggccc ctcggtgggt gttcaaggag 360
gaagacccta ttcacctgag gtgtcacagc tggaagaaca ctgctctgca taaggtcaca 420
tatttacaga atggcaaagg caggaagtat tttcatcata attctgactt ctacattcca 480
aaagccacac tcaaagacag cggctcctac ttctgcaggg ggctttttgg gagtaaaaat 540
gtgtcttcag agactgtgaa catcaccatc actcaaggtt tggcagtgtc aaccatctca 600
tcattctttc cacctggg 618
<210> 2
<211> 206
<212> PRT
<213> 人工序列
<220>
<223> CD16 氨基酸
<400> 2
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
195 200 205
<210> 3
<211> 618
<212> DNA
<213> 人工序列
<220>
<223> CD16V 核苷酸
<400> 3
atgtggcagc tgctcctccc aactgctctg ctacttctag tttcagctgg catgcggact 60
gaagatctcc caaaggctgt ggtgttcctg gagcctcaat ggtacagggt gctcgagaag 120
gacagtgtga ctctgaagtg ccagggagcc tactcccctg aggacaattc cacacagtgg 180
tttcacaatg agagcctcat ctcaagccag gcctcgagct acttcattga cgctgccaca 240
gtcgacgaca gtggagagta caggtgccag acaaacctct ccaccctcag tgacccggtg 300
cagctagaag tccatatcgg ctggctgttg ctccaggccc ctcggtgggt gttcaaggag 360
gaagacccta ttcacctgag gtgtcacagc tggaagaaca ctgctctgca taaggtcaca 420
tatttacaga atggcaaagg caggaagtat tttcatcata attctgactt ctacattcca 480
aaagccacac tcaaagacag cggctcctac ttctgcaggg ggcttgttgg gagtaaaaat 540
gtgtcttcag agactgtgaa catcaccatc actcaaggtt tggcagtgtc aaccatctca 600
tcattctttc cacctggg 618
<210> 4
<211> 618
<212> DNA
<213> 人工序列
<220>
<223> CD16V 核苷酸密码子最优化
<400> 4
atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60
gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120
gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180
ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240
gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300
cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360
gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420
tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480
aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540
gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600
tctttctttc cccctggg 618
<210> 5
<211> 206
<212> PRT
<213> 人工序列
<220>
<223> CD16V 氨基酸
<400> 5
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 Val
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
195 200 205
<210> 6
<211> 216
<212> DNA
<213> 人工序列
<220>
<223> CD8a 核苷酸
<400> 6
gcgaagccca ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 60
cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 120
agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 180
gtccttctcc tgtcactggt tatcaccctt tactgc 216
<210> 7
<211> 216
<212> DNA
<213> 人工序列
<220>
<223> CD8a 核苷酸密码子最优化
<400> 7
gccaaaccta ccacaactcc tgctccaaga ccacccacac ccgctcctac tattgcatct 60
cagccactga gtctgcgacc agaggcctgc cggcccgccg ccggcggggc cgtgcatacc 120
aggggcctgg acttcgcctg tgatatctac atttgggctc cactggctgg gacttgcggc 180
gtgctgctgc tgtctctggt catcactctg tattgt 216
<210> 8
<211> 72
<212> PRT
<213> 人工序列
<220>
<223> CD8a 氨基酸
<400> 8
Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro
1 5 10 15
Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro
20 25 30
Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 45
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
50 55 60
Ser Leu Val Ile Thr Leu Tyr Cys
65 70
<210> 9
<211> 321
<212> DNA
<213> 人工序列
<220>
<223> CD28 核苷酸
<400> 9
attgaagtta tgtatcctcc tccttaccta gacaatgaga agagcaatgg aaccattatc 60
catgtgaaag ggaaacacct ttgtccaagt cccctatttc ccggaccttc taagcccttt 120
tgggtgctgg tggtggttgg tggagtcctg gcttgctata gcttgctagt aacagtggcc 180
tttattattt tctgggtgag gagtaagagg agcaggctcc tgcacagtga ctacatgaac 240
atgactcccc gccgccccgg gcccacccgc aagcattacc agccctatgc cccaccacgc 300
gacttcgcag cctatcgctc c 321
<210> 10
<211> 321
<212> DNA
<213> 人工序列
<220>
<223> CD28 核苷酸密码子最优化
<400> 10
atcgaggtca tgtacccccc tccatatctg gacaacgaaa agtccaatgg aactatcatt 60
cacgtgaagg gcaaacatct gtgcccatct cccctgttcc ctgggccaag taaacctttt 120
tgggtcctgg tggtcgtggg aggggtgctg gcatgttact cactgctggt caccgtggcc 180
ttcatcatct tctgggtgcg gagcaagagg tcccgcctgc tgcacagcga ctatatgaac 240
atgaccccac ggagacccgg ccctacacgg aaacattacc agccctatgc tccaccccgg 300
gacttcgcag cttacagaag t 321
<210> 11
<211> 107
<212> PRT
<213> 人工序列
<220>
<223> CD28 氨基酸
<400> 11
Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn
1 5 10 15
Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu
20 25 30
Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly
35 40 45
Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe
50 55 60
Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn
65 70 75 80
Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr
85 90 95
Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser
100 105
<210> 12
<211> 336
<212> DNA
<213> 人工序列
<220>
<223> CD3z 核苷酸
<400> 12
agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300
tacgacgccc ttcacatgca ggccctgccc cctcgc 336
<210> 13
<211> 336
<212> DNA
<213> 人工序列
<220>
<223> CD3z 核苷酸密码子最优化
<400> 13
cgagtgaagt tcagcaggtc cgccgacgct cctgcatacc agcagggaca gaaccagctg 60
tataacgagc tgaatctggg ccggagagag gaatacgacg tgctggacaa aaggcggggc 120
cgggaccccg aaatgggagg gaagccacga cggaaaaacc cccaggaggg cctgtacaat 180
gagctgcaaa aggacaaaat ggccgaggct tattctgaaa tcgggatgaa gggagagaga 240
aggcgcggaa aaggccacga tggcctgtac caggggctga gcaccgctac aaaggacacc 300
tatgatgcac tgcacatgca ggccctgccc cctcgg 336
<210> 14
<211> 112
<212> PRT
<213> 人工序列
<220>
<223> CD3z 氨基酸
<400> 14
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> 15
<211> 66
<212> DNA
<213> 人工序列
<220>
<223> OX40L (CD252) 核苷酸
<400> 15
gaaagggtcc aacccctgga agagaatgtg ggaaatgcag ccaggccaag attcgagagg 60
aacaag 66
<210> 16
<211> 66
<212> DNA
<213> 人工序列
<220>
<223> OX40L (CD252) 核苷酸密码子最优化
<400> 16
gaaagagtgc agcccctgga agagaatgtc gggaatgccg ctcgcccaag atttgaaagg 60
aacaaa 66
<210> 17
<211> 22
<212> PRT
<213> 人工序列
<220>
<223> OX40L (CD252) 氨基酸
<400> 17
Glu Arg Val Gln Pro Leu Glu Glu Asn Val Gly Asn Ala Ala Arg Pro
1 5 10 15
Arg Phe Glu Arg Asn Lys
20
<210> 18
<211> 108
<212> DNA
<213> 人工序列
<220>
<223> OX40 (CD134) 核苷酸
<400> 18
agggaccaga ggctgccccc cgatgcccac aagccccctg ggggaggcag tttccggacc 60
cccatccaag aggagcaggc cgacgcccac tccaccctgg ccaagatc 108
<210> 19
<211> 108
<212> DNA
<213> 人工序列
<220>
<223> OX40 (CD134) 核苷酸密码子最优化
<400> 19
cgggaccaga gactgccacc tgatgcacac aagccaccag gaggaggcag cttcaggacc 60
cccatccagg aggaacaggc cgacgctcat tccacactgg ccaaaatt 108
<210> 20
<211> 36
<212> PRT
<213> 人工序列
<220>
<223> OX40 (CD134) 氨基酸
<400> 20
Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly
1 5 10 15
Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr
20 25 30
Leu Ala Lys Ile
35
<210> 21
<211> 126
<212> DNA
<213> 人工序列
<220>
<223> 4-1BB (CD137) 核苷酸
<400> 21
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactg 126
<210> 22
<211> 126
<212> DNA
<213> 人工序列
<220>
<223> 4-1BB (CD137) 核苷酸密码子最优化
<400> 22
aagcggggaa gaaagaaact gctgtacatc ttcaaacagc cctttatgag gcctgtgcag 60
accacacagg aggaagacgg ctgctcctgc cggttccccg aggaagagga aggcgggtgc 120
gagctg 126
<210> 23
<211> 42
<212> PRT
<213> 人工序列
<220>
<223> 4-1BB (CD137) 氨基酸
<400> 23
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> 24
<211> 1173
<212> DNA
<213> 人工序列
<220>
<223> CD16V-Z 核苷酸
<400> 24
atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60
gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120
gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180
ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240
gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300
cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360
gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420
tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480
aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540
gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600
tctttctttc cccctggggc caaacctacc acaactcctg ctccaagacc acccacaccc 660
gctcctacta ttgcatctca gccactgagt ctgcgaccag aggcctgccg gcccgccgcc 720
ggcggggccg tgcataccag gggcctggac ttcgcctgtg atatctacat ttgggctcca 780
ctggctggga cttgcggcgt gctgctgctg tctctggtca ttactctgta ttgtcgagtg 840
aagttcagca ggtccgccga cgctcctgca taccagcagg gacagaacca gctgtataac 900
gagctgaatc tgggccggag agaggaatac gacgtgctgg acaaaaggcg gggccgggac 960
cccgaaatgg gagggaagcc acgacggaaa aacccccagg agggcctgta caatgagctg 1020
caaaaggaca aaatggccga ggcttattct gaaatcggga tgaagggaga gagaaggcgc 1080
ggaaaaggcc acgatggcct gtaccagggg ctgagcaccg ctacaaagga cacctatgat 1140
gcactgcaca tgcaggccct gccccctcgg tga 1173
<210> 25
<211> 390
<212> PRT
<213> 人工序列
<220>
<223> CD16V-Z 氨基酸
<400> 25
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 Val
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 Ala Lys
195 200 205
Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile
210 215 220
Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala
225 230 235 240
Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr
245 250 255
Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu
260 265 270
Val Ile Thr Leu Tyr Cys Arg Val Lys Phe Ser Arg Ser Ala Asp Ala
275 280 285
Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu
290 295 300
Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp
305 310 315 320
Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu
325 330 335
Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile
340 345 350
Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
355 360 365
Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met
370 375 380
Gln Ala Leu Pro Pro Arg
385 390
<210> 26
<211> 1299
<212> DNA
<213> 人工序列
<220>
<223> CD16V-BBZ 核苷酸
<400> 26
atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60
gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120
gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180
ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240
gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300
cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360
gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420
tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480
aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540
gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600
tctttctttc cccctggggc caaacctacc acaactcctg ctccaagacc acccacaccc 660
gctcctacta ttgcatctca gccactgagt ctgcgaccag aggcctgccg gcccgccgcc 720
ggcggggccg tgcataccag gggcctggac ttcgcctgtg atatctacat ttgggctcca 780
ctggctggga cttgcggcgt gctgctgctg tctctggtca ttactctgta ttgtaagcgg 840
ggaagaaaga aactgctgta catcttcaaa cagcccttta tgaggcctgt gcagaccaca 900
caggaggaag acggctgctc ctgccggttc cccgaggaag aggaaggcgg gtgcgagctg 960
cgagtgaagt tcagcaggtc cgccgacgct cctgcatacc agcagggaca gaaccagctg 1020
tataacgagc tgaatctggg ccggagagag gaatacgacg tgctggacaa aaggcggggc 1080
cgggaccccg aaatgggagg gaagccacga cggaaaaacc cccaggaggg cctgtacaat 1140
gagctgcaaa aggacaaaat ggccgaggct tattctgaaa tcgggatgaa gggagagaga 1200
aggcgcggaa aaggccacga tggcctgtac caggggctga gcaccgctac aaaggacacc 1260
tatgatgcac tgcacatgca ggccctgccc cctcggtga 1299
<210> 27
<211> 432
<212> PRT
<213> 人工序列
<220>
<223> CD16V-BBZ 氨基酸
<400> 27
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 Val
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 Ala Lys
195 200 205
Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile
210 215 220
Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala
225 230 235 240
Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr
245 250 255
Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu
260 265 270
Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile
275 280 285
Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp
290 295 300
Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
305 310 315 320
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
325 330 335
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
340 345 350
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
355 360 365
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
370 375 380
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
385 390 395 400
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
405 410 415
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
420 425 430
<210> 28
<211> 1281
<212> DNA
<213> 人工序列
<220>
<223> CD16V-OX40Z 核苷酸
<400> 28
atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60
gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120
gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180
ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240
gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300
cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360
gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420
tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480
aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540
gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600
tctttctttc cccctggggc caaacctacc acaactcctg ctccaagacc acccacaccc 660
gctcctacta ttgcatctca gccactgagt ctgcgaccag aggcctgccg gcccgccgcc 720
ggcggggccg tgcataccag gggcctggac ttcgcctgtg atatctacat ttgggctcca 780
ctggctggga cttgcggcgt gctgctgctg tccctggtca tcactctgta ttgtcgggac 840
cagagactgc cacctgatgc acacaagcca ccaggaggag gcagcttcag gacccccatc 900
caggaggaac aggccgacgc tcattccaca ctggccaaaa ttcgagtgaa gttcagcagg 960
tccgccgacg ctcctgcata ccagcaggga cagaaccagc tgtataacga gctgaatctg 1020
ggccggagag aggaatacga cgtgctggac aaaaggcggg gccgggaccc cgaaatggga 1080
gggaagccac gacggaaaaa cccccaggag ggcctgtaca atgagctgca aaaggacaaa 1140
atggccgagg cttattctga aatcgggatg aagggagaga gaaggcgcgg aaaaggccac 1200
gatggcctgt accaggggct gagcaccgct acaaaggaca cctatgatgc actgcacatg 1260
caggccctgc cccctcggtg a 1281
<210> 29
<211> 426
<212> PRT
<213> 人工序列
<220>
<223> CD16V-OX40Z 氨基酸
<400> 29
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 Val
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 Ala Lys
195 200 205
Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile
210 215 220
Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala
225 230 235 240
Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr
245 250 255
Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu
260 265 270
Val Ile Thr Leu Tyr Cys Arg Asp Gln Arg Leu Pro Pro Asp Ala His
275 280 285
Lys Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln
290 295 300
Ala Asp Ala His Ser Thr Leu Ala Lys Ile Arg Val Lys Phe Ser Arg
305 310 315 320
Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn
325 330 335
Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
340 345 350
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro
355 360 365
Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
370 375 380
Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His
385 390 395 400
Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp
405 410 415
Ala Leu His Met Gln Ala Leu Pro Pro Arg
420 425
<210> 30
<211> 1239
<212> DNA
<213> 人工序列
<220>
<223> CD16V-OX40LZ 核苷酸
<400> 30
atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60
gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120
gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180
ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240
gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300
cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360
gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420
tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480
aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540
gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600
tctttctttc cccctggggc caaacctacc acaactcctg ctccaagacc acccacaccc 660
gctcctacta ttgcatctca gccactgagt ctgcgaccag aggcctgccg gcccgccgcc 720
ggcggggccg tgcataccag gggcctggac ttcgcctgtg atatctacat ttgggctcca 780
ctggctggga cttgcggcgt gctgctgctg tctctggtca ttactctgta ttgtgaaaga 840
gtgcagcccc tggaagagaa tgtcgggaat gccgctcgcc caagatttga aaggaacaaa 900
cgagtgaagt tcagcaggtc cgccgacgct cctgcatacc agcagggaca gaaccagctg 960
tataacgagc tgaatctggg ccggagagag gaatacgacg tgctggacaa aaggcggggc 1020
cgggaccccg aaatgggagg gaagccacga cggaaaaacc cccaggaggg cctgtacaat 1080
gagctgcaaa aggacaaaat ggccgaggct tattctgaaa tcgggatgaa gggagagaga 1140
aggcgcggaa aaggccacga tggcctgtac caggggctga gcaccgctac aaaggacacc 1200
tatgatgcac tgcacatgca ggccctgccc cctcggtga 1239
<210> 31
<211> 412
<212> PRT
<213> 人工序列
<220>
<223> CD16V-OX40LZ 氨基酸
<400> 31
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 Val
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 Ala Lys
195 200 205
Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile
210 215 220
Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala
225 230 235 240
Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr
245 250 255
Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu
260 265 270
Val Ile Thr Leu Tyr Cys Glu Arg Val Gln Pro Leu Glu Glu Asn Val
275 280 285
Gly Asn Ala Ala Arg Pro Arg Phe Glu Arg Asn Lys Arg Val Lys Phe
290 295 300
Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu
305 310 315 320
Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp
325 330 335
Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys
340 345 350
Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
355 360 365
Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
370 375 380
Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
385 390 395 400
Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
405 410
<210> 32
<211> 1305
<212> DNA
<213> 人工序列
<220>
<223> CD16V-28Z 核苷酸
<400> 32
atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60
gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120
gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180
ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240
gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300
cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360
gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420
tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480
aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540
gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600
tctttctttc cccctggggc caaacctacc acaactcctg ctccaagacc acccacaccc 660
gctcctacta ttgcatctca gccactgagt ctgcgaccag aggcctgccg gcccgccgcc 720
ggcggggccg tgcataccag gggcctggac ttcgcctgtg atttttgggt cctggtggtc 780
gtgggagggg tgctggcatg ttactcactg ctggtcaccg tggccttcat catcttctgg 840
gtgcggagca agaggtcccg cctgctgcac agcgactata tgaacatgac cccacggaga 900
cccggcccta cacggaaaca ttaccagccc tatgctccac cccgggactt cgcagcttac 960
agaagtcgag tgaagttcag caggtccgcc gacgctcctg cataccagca gggacagaac 1020
cagctgtata acgagctgaa tctgggccgg agagaggaat acgacgtgct ggacaaaagg 1080
cggggccggg accccgaaat gggagggaag ccacgacgga aaaaccccca ggagggcctg 1140
tacaatgagc tgcaaaagga caaaatggcc gaggcttatt ctgaaatcgg gatgaaggga 1200
gagagaaggc gcggaaaagg ccacgatggc ctgtaccagg ggctgagcac cgctacaaag 1260
gacacctatg atgcactgca catgcaggcc ctgccccctc ggtga 1305
<210> 33
<211> 434
<212> PRT
<213> 人工序列
<220>
<223> CD16V-28Z 氨基酸
<400> 33
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 Val
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 Ala Lys
195 200 205
Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile
210 215 220
Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala
225 230 235 240
Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp
245 250 255
Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val
260 265 270
Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu
275 280 285
Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr
290 295 300
Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr
305 310 315 320
Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
325 330 335
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
340 345 350
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
355 360 365
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
370 375 380
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
385 390 395 400
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
405 410 415
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
420 425 430
Pro Arg
<210> 34
<211> 1278
<212> DNA
<213> 人工序列
<220>
<223> CD16V-28(H)Z 核苷酸
<400> 34
atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60
gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120
gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180
ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240
gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300
cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360
gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420
tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480
aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540
gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600
tctttctttc cccctgggat cgaggtcatg tacccccctc catatctgga caacgaaaag 660
tccaatggaa ctatcattca cgtgaagggc aaacatctgt gcccatctcc cctgttccct 720
gggccaagta aacctttttg ggtcctggtg gtcgtgggag gggtgctggc atgttactca 780
ctgctggtca ccgtggcctt catcatcttc tgggtgcgga gcaagaggtc ccgcctgctg 840
cacagcgact atatgaacat gaccccacgg agacccggcc ctacacggaa acattaccag 900
ccctatgctc caccccggga cttcgcagct tacagaagtc gagtgaagtt cagcaggtcc 960
gccgacgctc ctgcatacca gcagggacag aaccagctgt ataacgagct gaatctgggc 1020
cggagagagg aatacgacgt gctggacaaa aggcggggcc gggaccccga aatgggaggg 1080
aagccacgac ggaaaaaccc ccaggagggc ctgtacaatg agctgcaaaa ggacaaaatg 1140
gccgaggctt attctgaaat cgggatgaag ggagagagaa ggcgcggaaa aggccacgat 1200
ggcctgtacc aggggctgag caccgctaca aaggacacct atgatgcact gcacatgcag 1260
gccctgcccc ctcggtga 1278
<210> 35
<211> 425
<212> PRT
<213> 人工序列
<220>
<223> CD16V-28(H)Z 氨基酸
<400> 35
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 Val
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 Ile Glu
195 200 205
Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr
210 215 220
Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro
225 230 235 240
Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu
245 250 255
Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val
260 265 270
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr
275 280 285
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
290 295 300
Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser
305 310 315 320
Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu
325 330 335
Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg
340 345 350
Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln
355 360 365
Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr
370 375 380
Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp
385 390 395 400
Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala
405 410 415
Leu His Met Gln Ala Leu Pro Pro Arg
420 425
<210> 36
<211> 1407
<212> DNA
<213> 人工序列
<220>
<223> CD16V-BBOX40Z 核苷酸
<400> 36
atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60
gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120
gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180
ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240
gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300
cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360
gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420
tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480
aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540
gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600
tctttctttc cccctggggc caaacctacc acaactcctg ctccaagacc acccacaccc 660
gctcctacta ttgcatctca gccactgagt ctgcgaccag aggcctgccg gcccgccgcc 720
ggcggggccg tgcataccag gggcctggac ttcgcctgtg atatctacat ttgggctcca 780
ctggctggga cttgcggcgt gctgctgctg tctctggtca ttactctgta ttgtaagcgg 840
ggaagaaaga aactgctgta catcttcaaa cagcccttta tgaggcctgt gcagaccaca 900
caggaggaag acggctgctc ctgccggttc cccgaggaag aggaaggcgg gtgcgagctg 960
cgggaccaga gactgccacc tgatgcacac aagccaccag gaggaggcag cttcaggacc 1020
cccatccagg aggaacaggc cgacgctcat tccacactgg ccaaaattcg agtgaagttc 1080
agcaggtccg ccgacgctcc tgcataccag cagggacaga accagctgta taacgagctg 1140
aatctgggcc ggagagagga atacgacgtg ctggacaaaa ggcggggccg ggaccccgaa 1200
atgggaggga agccacgacg gaaaaacccc caggagggcc tgtacaatga gctgcaaaag 1260
gacaaaatgg ccgaggctta ttctgaaatc gggatgaagg gagagagaag gcgcggaaaa 1320
ggccacgatg gcctgtacca ggggctgagc accgctacaa aggacaccta tgatgcactg 1380
cacatgcagg ccctgccccc tcggtga 1407
<210> 37
<211> 468
<212> PRT
<213> 人工序列
<220>
<223> CD16V-BBOX40Z 氨基酸
<400> 37
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 Val
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 Ala Lys
195 200 205
Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile
210 215 220
Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala
225 230 235 240
Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr
245 250 255
Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu
260 265 270
Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile
275 280 285
Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp
290 295 300
Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
305 310 315 320
Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly
325 330 335
Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr
340 345 350
Leu Ala Lys Ile Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala
355 360 365
Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg
370 375 380
Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu
385 390 395 400
Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn
405 410 415
Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met
420 425 430
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
435 440 445
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala
450 455 460
Leu Pro Pro Arg
465
<210> 38
<211> 1407
<212> DNA
<213> 人工序列
<220>
<223> CD16V-OX40BBZ 核苷酸
<400> 38
atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60
gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120
gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180
ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240
gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300
cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360
gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420
tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480
aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540
gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600
tctttctttc cccctggggc caaacctacc acaactcctg ctccaagacc acccacaccc 660
gctcctacta ttgcatctca gccactgagt ctgcgaccag aggcctgccg gcccgccgcc 720
ggcggggccg tgcataccag gggcctggac ttcgcctgtg atatctacat ttgggctcca 780
ctggctggga cttgcggcgt gctgctgctg tccctggtca tcactctgta ttgtcgggac 840
cagagactgc cacctgatgc acacaagcca ccaggaggag gcagcttcag gacccccatc 900
caggaggaac aggccgacgc tcattccaca ctggccaaaa ttaagcgggg aagaaagaaa 960
ctgctgtaca tcttcaaaca gccctttatg aggcctgtgc agaccacaca ggaggaagac 1020
ggctgctcct gccggttccc cgaggaagag gaaggcgggt gcgagctgcg agtgaagttc 1080
agcaggtccg ccgacgctcc tgcataccag cagggacaga accagctgta taacgagctg 1140
aatctgggcc ggagagagga atacgacgtg ctggacaaaa ggcggggccg ggaccccgaa 1200
atgggaggga agccacgacg gaaaaacccc caggagggcc tgtacaatga gctgcaaaag 1260
gacaaaatgg ccgaggctta ttctgaaatc gggatgaagg gagagagaag gcgcggaaaa 1320
ggccacgatg gcctgtacca ggggctgagc accgctacaa aggacaccta tgatgcactg 1380
cacatgcagg ccctgccccc tcggtga 1407
<210> 39
<211> 468
<212> PRT
<213> 人工序列
<220>
<223> CD16V-OX40BBZ 氨基酸
<400> 39
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 Val
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 Ala Lys
195 200 205
Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile
210 215 220
Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala
225 230 235 240
Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr
245 250 255
Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu
260 265 270
Val Ile Thr Leu Tyr Cys Arg Asp Gln Arg Leu Pro Pro Asp Ala His
275 280 285
Lys Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln
290 295 300
Ala Asp Ala His Ser Thr Leu Ala Lys Ile Lys Arg Gly Arg Lys Lys
305 310 315 320
Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr
325 330 335
Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly
340 345 350
Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala
355 360 365
Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg
370 375 380
Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu
385 390 395 400
Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn
405 410 415
Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met
420 425 430
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
435 440 445
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala
450 455 460
Leu Pro Pro Arg
465
<210> 40
<211> 1431
<212> DNA
<213> 人工序列
<220>
<223> CD16V-28BBZ 核苷酸
<400> 40
atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60
gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120
gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180
ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240
gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300
cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360
gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420
tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480
aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540
gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600
tctttctttc cccctggggc caaacctacc acaactcctg ctccaagacc acccacaccc 660
gctcctacta ttgcatctca gccactgagt ctgcgaccag aggcctgccg gcccgccgcc 720
ggcggggccg tgcataccag gggcctggac tttgcctgcg atttttgggt cctggtggtc 780
gtgggagggg tgctggcatg ttactcactg ctggtcaccg tggccttcat catcttctgg 840
gtgcggagca agaggtcccg cctgctgcac agcgactata tgaacatgac cccacggaga 900
cccggcccta cacggaaaca ttaccagccc tatgctccac cccgggactt cgcagcttac 960
agaagtaagc ggggaagaaa gaaactgctg tacatcttca aacagccctt tatgaggcct 1020
gtgcagacca cacaggagga agacggctgc tcctgccggt tccccgagga agaggaaggc 1080
gggtgcgagc tgcgagtgaa gttcagcagg tccgccgacg ctcctgcata ccagcaggga 1140
cagaaccagc tgtataacga gctgaatctg ggccggagag aggaatacga cgtgctggac 1200
aaaaggcggg gccgggaccc cgaaatggga gggaagccac gacggaaaaa cccccaggag 1260
ggcctgtaca atgagctgca aaaggacaaa atggccgagg cttattctga aatcgggatg 1320
aagggagaga gaaggcgcgg aaaaggccac gatggcctgt accaggggct gagcaccgct 1380
acaaaggaca cctatgatgc actgcacatg caggccctgc cccctcggtg a 1431
<210> 41
<211> 476
<212> PRT
<213> 人工序列
<220>
<223> CD16V-28BBZ 氨基酸
<400> 41
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 Val
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 Ala Lys
195 200 205
Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile
210 215 220
Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala
225 230 235 240
Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp
245 250 255
Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val
260 265 270
Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu
275 280 285
Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr
290 295 300
Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr
305 310 315 320
Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro
325 330 335
Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys
340 345 350
Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe
355 360 365
Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu
370 375 380
Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp
385 390 395 400
Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys
405 410 415
Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
420 425 430
Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
435 440 445
Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
450 455 460
Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
465 470 475
<210> 42
<211> 1413
<212> DNA
<213> 人工序列
<220>
<223> CD16V-28OX40Z 核苷酸
<400> 42
atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60
gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120
gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180
ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240
gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300
cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360
gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420
tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480
aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540
gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600
tctttctttc cccctggggc caaacctacc acaactcctg ctccaagacc acccacaccc 660
gctcctacta ttgcatctca gccactgagt ctgcgaccag aggcctgccg gcccgccgcc 720
ggcggggccg tgcataccag gggcctggac tttgcctgcg atttttgggt cctggtggtc 780
gtgggagggg tgctggcatg ttactcactg ctggtcaccg tggccttcat catcttctgg 840
gtgcggagca agaggtcccg cctgctgcac agcgactata tgaacatgac cccacggaga 900
cccggcccta cacggaaaca ttaccagccc tatgctccac cccgggactt cgcagcttac 960
agaagtcggg accagagact gccacctgat gcacacaagc caccaggagg aggcagcttc 1020
aggaccccca tccaggagga acaggccgac gctcattcca cactggccaa aattcgagtg 1080
aagttcagca ggtccgccga cgctcctgca taccagcagg gacagaacca gctgtataac 1140
gagctgaatc tgggccggag agaggaatac gacgtgctgg acaaaaggcg gggccgggac 1200
cccgaaatgg gagggaagcc acgacggaaa aacccccagg agggcctgta caatgagctg 1260
caaaaggaca aaatggccga ggcttattct gaaatcggga tgaagggaga gagaaggcgc 1320
ggaaaaggcc acgatggcct gtaccagggg ctgagcaccg ctacaaagga cacctatgat 1380
gcactgcaca tgcaggccct gccccctcgg tga 1413
<210> 43
<211> 470
<212> PRT
<213> 人工序列
<220>
<223> CD16V-28OX40Z 氨基酸
<400> 43
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 Val
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 Ala Lys
195 200 205
Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile
210 215 220
Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala
225 230 235 240
Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp
245 250 255
Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val
260 265 270
Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu
275 280 285
Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr
290 295 300
Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr
305 310 315 320
Arg Ser Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly
325 330 335
Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His
340 345 350
Ser Thr Leu Ala Lys Ile Arg Val Lys Phe Ser Arg Ser Ala Asp Ala
355 360 365
Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu
370 375 380
Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp
385 390 395 400
Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu
405 410 415
Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile
420 425 430
Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
435 440 445
Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met
450 455 460
Gln Ala Leu Pro Pro Arg
465 470
<210> 44
<211> 1371
<212> DNA
<213> 人工序列
<220>
<223> CD16V-28OX40LZ 核苷酸
<400> 44
atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60
gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120
gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180
ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240
gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300
cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360
gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420
tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480
aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540
gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600
tctttctttc cccctggggc caaacctacc acaactcctg ctccaagacc acccacaccc 660
gctcctacta ttgcatctca gccactgagt ctgcgaccag aggcctgccg gcccgccgcc 720
ggcggggccg tgcataccag gggcctggac ttcgcctgtg atttttgggt cctggtggtc 780
gtgggagggg tgctggcatg ttactcactg ctggtcaccg tggccttcat catcttctgg 840
gtgcggagca agaggtcccg cctgctgcac agcgactata tgaacatgac cccacggaga 900
cccggcccta cacggaaaca ttaccagccc tatgctccac cccgggactt cgcagcttac 960
agaagtgaaa gagtgcagcc cctggaagag aatgtcggga atgccgctcg cccaagattt 1020
gaaaggaaca aacgagtgaa gttcagcagg tccgccgacg ctcctgcata ccagcaggga 1080
cagaaccagc tgtataacga gctgaatctg ggccggagag aggaatacga cgtgctggac 1140
aaaaggcggg gccgggaccc cgaaatggga gggaagccac gacggaaaaa cccccaggag 1200
ggcctgtaca atgagctgca aaaggacaaa atggccgagg cttattctga aatcgggatg 1260
aagggagaga gaaggcgcgg aaaaggccac gatggcctgt accaggggct gagcaccgct 1320
acaaaggaca cctatgatgc actgcacatg caggccctgc cccctcggtg a 1371
<210> 45
<211> 456
<212> PRT
<213> 人工序列
<220>
<223> CD16V-28OX40LZ 氨基酸
<400> 45
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 Val
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 Ala Lys
195 200 205
Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile
210 215 220
Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala
225 230 235 240
Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp
245 250 255
Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val
260 265 270
Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu
275 280 285
Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr
290 295 300
Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr
305 310 315 320
Arg Ser Glu Arg Val Gln Pro Leu Glu Glu Asn Val Gly Asn Ala Ala
325 330 335
Arg Pro Arg Phe Glu Arg Asn Lys Arg Val Lys Phe Ser Arg Ser Ala
340 345 350
Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu
355 360 365
Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
370 375 380
Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu
385 390 395 400
Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser
405 410 415
Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly
420 425 430
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
435 440 445
His Met Gln Ala Leu Pro Pro Arg
450 455
<210> 46
<211> 1404
<212> DNA
<213> 人工序列
<220>
<223> CD16V-28(H)BBZ 核苷酸
<400> 46
atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60
gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120
gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180
ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240
gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300
cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360
gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420
tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480
aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540
gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600
tctttctttc cccctgggat cgaggtcatg tacccccctc catatctgga caacgaaaag 660
tccaatggaa ctatcattca cgtgaagggc aaacatctgt gcccatctcc cctgttccct 720
gggccaagta aacctttttg ggtcctggtg gtcgtgggag gggtgctggc atgttactca 780
ctgctggtca ccgtggcctt catcatcttc tgggtgcgga gcaagaggtc ccgcctgctg 840
cacagcgact atatgaacat gaccccacgg agacccggcc ctacacggaa acattaccag 900
ccctatgctc caccccggga cttcgcagct tacagaagta agcggggaag aaagaaactg 960
ctgtacatct tcaaacagcc ctttatgagg cctgtgcaga ccacacagga ggaagacggc 1020
tgctcctgcc ggttccccga ggaagaggaa ggcgggtgcg agctgcgagt gaagttcagc 1080
aggtccgccg acgctcctgc ataccagcag ggacagaacc agctgtataa cgagctgaat 1140
ctgggccgga gagaggaata cgacgtgctg gacaaaaggc ggggccggga ccccgaaatg 1200
ggagggaagc cacgacggaa aaacccccag gagggcctgt acaatgagct gcaaaaggac 1260
aaaatggccg aggcttattc tgaaatcggg atgaagggag agagaaggcg cggaaaaggc 1320
cacgatggcc tgtaccaggg gctgagcacc gctacaaagg acacctatga tgcactgcac 1380
atgcaggccc tgccccctcg gtga 1404
<210> 47
<211> 467
<212> PRT
<213> 人工序列
<220>
<223> CD16V-28(H)BBZ 氨基酸
<400> 47
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 Val
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 Ile Glu
195 200 205
Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr
210 215 220
Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro
225 230 235 240
Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu
245 250 255
Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val
260 265 270
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr
275 280 285
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
290 295 300
Pro Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu
305 310 315 320
Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln
325 330 335
Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly
340 345 350
Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr
355 360 365
Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
370 375 380
Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met
385 390 395 400
Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu
405 410 415
Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys
420 425 430
Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu
435 440 445
Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu
450 455 460
Pro Pro Arg
465
<210> 48
<211> 1386
<212> DNA
<213> 人工序列
<220>
<223> CD16V-28(H)OX40Z 核苷酸
<400> 48
atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60
gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120
gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180
ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240
gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300
cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360
gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420
tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480
aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540
gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600
tctttctttc cccctgggat cgaggtcatg tacccccctc catatctgga caacgaaaag 660
tccaatggaa ctatcattca cgtgaagggc aaacatctgt gcccatctcc cctgttccct 720
gggccaagta aacctttttg ggtcctggtg gtcgtgggag gggtgctggc atgttactca 780
ctgctggtca ccgtggcctt catcatcttc tgggtgcgga gcaagaggtc ccgcctgctg 840
cacagcgact atatgaacat gaccccacgg agacccggcc ctacacggaa acattaccag 900
ccctatgctc caccccggga cttcgcagct tacagaagtc gggaccagag actgccacct 960
gatgcacaca agccaccagg aggaggcagc ttcaggaccc ccatccagga ggaacaggcc 1020
gacgctcatt ccacactggc caaaattcga gtgaagttca gcaggtccgc cgacgctcct 1080
gcataccagc agggacagaa ccagctgtat aacgagctga atctgggccg gagagaggaa 1140
tacgacgtgc tggacaaaag gcggggccgg gaccccgaaa tgggagggaa gccacgacgg 1200
aaaaaccccc aggagggcct gtacaatgag ctgcaaaagg acaaaatggc cgaggcttat 1260
tctgaaatcg ggatgaaggg agagagaagg cgcggaaaag gccacgatgg cctgtaccag 1320
gggctgagca ccgctacaaa ggacacctat gatgcactgc acatgcaggc cctgccccct 1380
cggtga 1386
<210> 49
<211> 461
<212> PRT
<213> 人工序列
<220>
<223> CD16V-28(H)OX40Z 氨基酸
<400> 49
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 Val
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 Ile Glu
195 200 205
Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr
210 215 220
Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro
225 230 235 240
Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu
245 250 255
Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val
260 265 270
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr
275 280 285
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
290 295 300
Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Asp Gln Arg Leu Pro Pro
305 310 315 320
Asp Ala His Lys Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln
325 330 335
Glu Glu Gln Ala Asp Ala His Ser Thr Leu Ala Lys Ile Arg Val Lys
340 345 350
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
355 360 365
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
370 375 380
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
385 390 395 400
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
405 410 415
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
420 425 430
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
435 440 445
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
450 455 460
<210> 50
<211> 1344
<212> DNA
<213> 人工序列
<220>
<223> CD16V-28(H)OX40LZ 核苷酸
<400> 50
atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60
gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120
gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180
ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240
gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300
cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360
gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420
tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480
aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540
gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600
tctttctttc cccctgggat cgaggtcatg tacccccctc catatctgga caacgaaaag 660
tccaatggaa ctatcattca cgtgaagggc aaacatctgt gcccatctcc cctgttccct 720
gggccaagta aacctttttg ggtcctggtg gtcgtgggag gggtgctggc atgttactca 780
ctgctggtca ccgtggcctt catcatcttc tgggtgcgga gcaagaggtc ccgcctgctg 840
cacagcgact atatgaacat gaccccacgg agacccggcc ctacacggaa acattaccag 900
ccctatgctc caccccggga cttcgcagct tacagaagtg aaagagtgca gcccctggaa 960
gagaatgtcg ggaatgccgc tcgcccaaga tttgaaagga acaaacgagt gaagttcagc 1020
aggtccgccg acgctcctgc ataccagcag ggacagaacc agctgtataa cgagctgaat 1080
ctgggccgga gagaggaata cgacgtgctg gacaaaaggc ggggccggga ccccgaaatg 1140
ggagggaagc cacgacggaa aaacccccag gagggcctgt acaatgagct gcaaaaggac 1200
aaaatggccg aggcttattc tgaaatcggg atgaagggag agagaaggcg cggaaaaggc 1260
cacgatggcc tgtaccaggg gctgagcacc gctacaaagg acacctatga tgcactgcac 1320
atgcaggccc tgccccctcg gtga 1344
<210> 51
<211> 447
<212> PRT
<213> 人工序列
<220>
<223> CD16V-28(H)OX40LZ 氨基酸
<400> 51
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 Val
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 Ile Glu
195 200 205
Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr
210 215 220
Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro
225 230 235 240
Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu
245 250 255
Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val
260 265 270
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr
275 280 285
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
290 295 300
Pro Arg Asp Phe Ala Ala Tyr Arg Ser Glu Arg Val Gln Pro Leu Glu
305 310 315 320
Glu Asn Val Gly Asn Ala Ala Arg Pro Arg Phe Glu Arg Asn Lys Arg
325 330 335
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
340 345 350
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
355 360 365
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
370 375 380
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
385 390 395 400
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
405 410 415
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
420 425 430
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
435 440 445
<210> 52
<211> 1365
<212> DNA
<213> 人工序列
<220>
<223> CD16V-BBOX40LZ 核苷酸
<400> 52
atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60
gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120
gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180
ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240
gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300
cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360
gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420
tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480
aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540
gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600
tctttctttc cccctggggc caaacctacc acaactcctg ctccaagacc acccacaccc 660
gctcctacta ttgcatctca gccactgagt ctgcgaccag aggcctgccg gcccgccgcc 720
ggcggggccg tgcataccag gggcctggac ttcgcctgtg atatctacat ttgggctcca 780
ctggctggga cttgcggcgt gctgctgctg tctctggtca ttactctgta ttgtaagcgg 840
ggaagaaaga aactgctgta catcttcaaa cagcccttta tgaggcctgt gcagaccaca 900
caggaggaag acggctgctc ctgccggttc cccgaggaag aggaaggcgg gtgcgagctg 960
gaaagagtgc agcccctgga agagaatgtc gggaatgccg ctcgcccaag atttgaaagg 1020
aacaaacgag tgaagttcag caggtccgcc gacgctcctg cataccagca gggacagaac 1080
cagctgtata acgagctgaa tctgggccgg agagaggaat acgacgtgct ggacaaaagg 1140
cggggccggg accccgaaat gggagggaag ccacgacgga aaaaccccca ggagggcctg 1200
tacaatgagc tgcaaaagga caaaatggcc gaggcttatt ctgaaatcgg gatgaaggga 1260
gagagaaggc gcggaaaagg ccacgatggc ctgtaccagg ggctgagcac cgctacaaag 1320
gacacctatg atgcactgca catgcaggcc ctgccccctc ggtga 1365
<210> 53
<211> 454
<212> PRT
<213> 人工序列
<220>
<223> CD16V-BBOX40LZ 氨基酸
<400> 53
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 Val
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 Ala Lys
195 200 205
Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile
210 215 220
Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala
225 230 235 240
Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr
245 250 255
Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu
260 265 270
Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile
275 280 285
Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp
290 295 300
Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
305 310 315 320
Glu Arg Val Gln Pro Leu Glu Glu Asn Val Gly Asn Ala Ala Arg Pro
325 330 335
Arg Phe Glu Arg Asn Lys Arg Val Lys Phe Ser Arg Ser Ala Asp Ala
340 345 350
Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu
355 360 365
Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp
370 375 380
Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu
385 390 395 400
Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile
405 410 415
Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
420 425 430
Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met
435 440 445
Gln Ala Leu Pro Pro Arg
450
<210> 54
<211> 1239
<212> DNA
<213> 人工序列
<220>
<223> CD16V-ZOX40L 核苷酸
<400> 54
atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60
gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120
gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180
ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240
gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300
cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360
gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420
tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480
aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540
gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600
tctttctttc cccctggggc caaacctacc acaactcctg ctccaagacc acccacaccc 660
gctcctacta ttgcatctca gccactgagt ctgcgaccag aggcctgccg gcccgccgcc 720
ggcggggccg tgcataccag gggcctggac ttcgcctgtg atatctacat ttgggctcca 780
ctggctggga cttgcggcgt gctgctgctg tctctggtca ttactctgta ttgtcgagtg 840
aagttcagca ggtccgccga cgctcctgca taccagcagg gacagaacca gctgtataac 900
gagctgaatc tgggccggag agaggaatac gacgtgctgg acaaaaggcg gggccgggac 960
cccgaaatgg gagggaagcc acgacggaaa aacccccagg agggcctgta caatgagctg 1020
caaaaggaca aaatggccga ggcttattct gaaatcggga tgaagggaga gagaaggcgc 1080
ggaaaaggcc acgatggcct gtaccagggg ctgagcaccg ctacaaagga cacctatgat 1140
gcactgcaca tgcaggccct gccccctcgg gaaagagtgc agcccctgga agagaatgtc 1200
gggaatgccg ctcgcccaag atttgaaagg aacaaatga 1239
<210> 55
<211> 412
<212> PRT
<213> 人工序列
<220>
<223> CD16V-ZOX40L 氨基酸
<400> 55
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 Val
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 Ala Lys
195 200 205
Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile
210 215 220
Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala
225 230 235 240
Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr
245 250 255
Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu
260 265 270
Val Ile Thr Leu Tyr Cys Arg Val Lys Phe Ser Arg Ser Ala Asp Ala
275 280 285
Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu
290 295 300
Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp
305 310 315 320
Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu
325 330 335
Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile
340 345 350
Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
355 360 365
Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met
370 375 380
Gln Ala Leu Pro Pro Arg Glu Arg Val Gln Pro Leu Glu Glu Asn Val
385 390 395 400
Gly Asn Ala Ala Arg Pro Arg Phe Glu Arg Asn Lys
405 410
<210> 56
<211> 63
<212> DNA
<213> 人工序列
<220>
<223> CD8 α 核苷酸
<400> 56
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccg 63
<210> 57
<211> 63
<212> DNA
<213> 人工序列
<220>
<223> CD8 α 核苷酸密码子最优化
<400> 57
atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60
cct 63
<210> 58
<211> 21
<212> PRT
<213> 人工序列
<220>
<223> CD8 α 氨基酸
<400> 58
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro
20
<210> 59
<211> 405
<212> DNA
<213> 人工序列
<220>
<223> NKG2D 核苷酸
<400> 59
ttattcaacc aagaagttca aattcccttg accgaaagtt actgtggccc atgtcctaaa 60
aactggatat gttacaaaaa taactgctac caattttttg atgagagtaa aaactggtat 120
gagagccagg cttcttgtat gtctcaaaat gccagccttc tgaaagtata cagcaaagag 180
gaccaggatt tacttaaact ggtgaagtca tatcattgga tgggactagt acacattcca 240
acaaatggat cttggcagtg ggaagatggc tccattctct cacccaacct actaacaata 300
attgaaatgc agaagggaga ctgtgcactc tatgcctcga gctttaaagg ctatatagaa 360
aactgttcaa ctccaaatac atacatctgc atgcaaagga ctgtg 405
<210> 60
<211> 405
<212> DNA
<213> 人工序列
<220>
<223> NKG2D 核苷酸密码子最优化
<400> 60
ctgttcaacc aggaggtcca gatcccactg accgaaagtt actgcggacc atgtcccaag 60
aactggatct gctacaagaa caactgttac cagttctttg acgagtctaa gaactggtat 120
gaatctcagg ccagttgcat gtcacagaat gcttcactgc tgaaggtgta cagcaaagag 180
gaccaggatc tgctgaagct ggtgaaatcc tatcactgga tgggcctggt ccatatccca 240
accaacgggt cttggcagtg ggaggacgga agcattctgt cccccaatct gctgacaatc 300
attgaaatgc agaagggcga ttgtgctctg tacgcaagct ccttcaaagg gtatatcgag 360
aactgctcca cccccaatac atacatttgt atgcagagga cagtg 405
<210> 61
<211> 135
<212> PRT
<213> 人工序列
<220>
<223> NKG2D 氨基酸
<400> 61
Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys Gly
1 5 10 15
Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln Phe
20 25 30
Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met Ser
35 40 45
Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp Leu
50 55 60
Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile Pro
65 70 75 80
Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn
85 90 95
Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala
100 105 110
Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr
115 120 125
Ile Cys Met Gln Arg Thr Val
130 135
<210> 62
<211> 1023
<212> DNA
<213> 人工序列
<220>
<223> NKG2D-Z 核苷酸
<400> 62
atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60
cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120
aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180
tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240
gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300
ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360
atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420
gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtggc aaaacctacc 480
acaactcctg caccacgccc ccctactcca gcacctacca tcgcatctca gccactgagt 540
ctgcgaccag aggcctgccg gcccgccgcc ggcggggccg tccataccag agggctggac 600
tttgcctgcg atatctacat ttgggcccct ctggctggaa catgtggcgt gctgctgctg 660
tccctggtca ttactctgta ttgtcgagtg aagttcagca ggtccgccga cgctcctgca 720
taccagcagg gacagaacca gctgtataac gagctgaatc tgggccggag agaggaatac 780
gacgtgctgg acaaaaggcg gggccgggac cccgaaatgg gagggaagcc acgacggaaa 840
aacccccagg agggcctgta caatgagctg caaaaggaca aaatggccga ggcttattct 900
gaaatcggga tgaagggaga gagaaggcgc ggaaaaggcc acgatggcct gtaccagggg 960
ctgagcaccg ctacaaagga cacctatgat gcactgcaca tgcaggccct gccccctcgg 1020
tga 1023
<210> 63
<211> 340
<212> PRT
<213> 人工序列
<220>
<223> NKG2D-Z 氨基酸
<400> 63
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr
20 25 30
Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn
35 40 45
Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln
50 55 60
Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys
65 70 75 80
Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly
85 90 95
Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser
100 105 110
Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp
115 120 125
Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser
130 135 140
Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ala Lys Pro Thr
145 150 155 160
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
165 170 175
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
180 185 190
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp
195 200 205
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
210 215 220
Thr Leu Tyr Cys Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala
225 230 235 240
Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg
245 250 255
Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu
260 265 270
Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn
275 280 285
Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met
290 295 300
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
305 310 315 320
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala
325 330 335
Leu Pro Pro Arg
340
<210> 64
<211> 1149
<212> DNA
<213> 人工序列
<220>
<223> NKG2D-BBZ 核苷酸
<400> 64
atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60
cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120
aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180
tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240
gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300
ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360
atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420
gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtggc aaaacctacc 480
acaactcctg caccacgccc ccctactcca gcacctacca tcgcatctca gccactgagt 540
ctgcgaccag aggcctgccg gcccgccgcc ggcggggccg tccataccag agggctggac 600
tttgcctgcg atatctacat ttgggcccct ctggctggaa catgtggcgt gctgctgctg 660
tccctggtca ttactctgta ttgtaagcgg ggaagaaaga aactgctgta catcttcaaa 720
cagcccttta tgaggcctgt gcagaccaca caggaggaag acggctgctc ctgccggttc 780
cccgaggaag aggaaggcgg gtgcgagctg cgagtgaagt tcagcaggtc cgccgacgct 840
cctgcatacc agcagggaca gaaccagctg tataacgagc tgaatctggg ccggagagag 900
gaatacgacg tgctggacaa aaggcggggc cgggaccccg aaatgggagg gaagccacga 960
cggaaaaacc cccaggaggg cctgtacaat gagctgcaaa aggacaaaat ggccgaggct 1020
tattctgaaa tcgggatgaa gggagagaga aggcgcggaa aaggccacga tggcctgtac 1080
caggggctga gcaccgctac aaaggacacc tatgatgcac tgcacatgca ggccctgccc 1140
cctcggtga 1149
<210> 65
<211> 382
<212> PRT
<213> 人工序列
<220>
<223> NKG2D-BBZ 氨基酸
<400> 65
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr
20 25 30
Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn
35 40 45
Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln
50 55 60
Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys
65 70 75 80
Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly
85 90 95
Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser
100 105 110
Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp
115 120 125
Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser
130 135 140
Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ala Lys Pro Thr
145 150 155 160
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
165 170 175
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
180 185 190
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp
195 200 205
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
210 215 220
Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys
225 230 235 240
Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys
245 250 255
Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val
260 265 270
Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn
275 280 285
Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val
290 295 300
Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg
305 310 315 320
Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys
325 330 335
Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg
340 345 350
Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
355 360 365
Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
370 375 380
<210> 66
<211> 1131
<212> DNA
<213> 人工序列
<220>
<223> NKG2D-OX40Z 核苷酸
<400> 66
atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60
cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120
aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180
tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240
gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300
ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360
atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420
gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtggc aaaacctacc 480
acaactcctg caccacgccc ccctactcca gcacctacca tcgcatctca gccactgagt 540
ctgcgaccag aggcctgccg gcccgccgcc ggcggggccg tccataccag agggctggac 600
tttgcctgcg atatctacat ttgggcccct ctggctggaa catgtggcgt gctgctgctg 660
tccctggtca ttactctgta ttgtcgggac cagagactgc cacctgatgc acacaagcca 720
ccaggaggag gcagcttcag gacccccatc caggaggaac aggccgacgc tcattccaca 780
ctggccaaaa ttcgagtgaa gttcagcagg tccgccgacg ctcctgcata ccagcaggga 840
cagaaccagc tgtataacga gctgaatctg ggccggagag aggaatacga cgtgctggac 900
aaaaggcggg gccgggaccc cgaaatggga gggaagccac gacggaaaaa cccccaggag 960
ggcctgtaca atgagctgca aaaggacaaa atggccgagg cttattctga aatcgggatg 1020
aagggagaga gaaggcgcgg aaaaggccac gatggcctgt accaggggct gagcaccgct 1080
acaaaggaca cctatgatgc actgcacatg caggccctgc cccctcggtg a 1131
<210> 67
<211> 376
<212> PRT
<213> 人工序列
<220>
<223> NKG2D-OX40Z 氨基酸
<400> 67
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr
20 25 30
Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn
35 40 45
Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln
50 55 60
Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys
65 70 75 80
Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly
85 90 95
Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser
100 105 110
Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp
115 120 125
Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser
130 135 140
Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ala Lys Pro Thr
145 150 155 160
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
165 170 175
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
180 185 190
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp
195 200 205
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
210 215 220
Thr Leu Tyr Cys Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro
225 230 235 240
Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp
245 250 255
Ala His Ser Thr Leu Ala Lys Ile Arg Val Lys Phe Ser Arg Ser Ala
260 265 270
Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu
275 280 285
Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
290 295 300
Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu
305 310 315 320
Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser
325 330 335
Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly
340 345 350
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
355 360 365
His Met Gln Ala Leu Pro Pro Arg
370 375
<210> 68
<211> 1089
<212> DNA
<213> 人工序列
<220>
<223> NKG2D-ZOX40L 核苷酸
<400> 68
atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60
cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120
aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180
tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240
gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300
ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360
atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420
gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtggc aaaacctacc 480
acaactcctg caccacgccc ccctactcca gcacctacca tcgcatctca gccactgagt 540
ctgcgaccag aggcctgccg gcccgccgcc ggcggggccg tccataccag agggctggac 600
tttgcctgcg atatctacat ttgggcccct ctggctggaa catgtggcgt gctgctgctg 660
tccctggtca ttactctgta ttgtcgagtg aagttcagca ggtccgccga cgctcctgca 720
taccagcagg gacagaacca gctgtataac gagctgaatc tgggccggag agaggaatac 780
gacgtgctgg acaaaaggcg gggccgggac cccgaaatgg gagggaagcc acgacggaaa 840
aacccccagg agggcctgta caatgagctg caaaaggaca aaatggccga ggcttattct 900
gaaatcggga tgaagggaga gagaaggcgc ggaaaaggcc acgatggcct gtaccagggg 960
ctgagcaccg ctacaaagga cacctatgat gcactgcaca tgcaggccct gccccctcgg 1020
gaaagagtgc agcccctgga agagaatgtc gggaatgccg ctcgcccaag atttgaaagg 1080
aacaaatga 1089
<210> 69
<211> 362
<212> PRT
<213> 人工序列
<220>
<223> NKG2D-ZOX40L 氨基酸
<400> 69
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr
20 25 30
Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn
35 40 45
Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln
50 55 60
Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys
65 70 75 80
Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly
85 90 95
Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser
100 105 110
Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp
115 120 125
Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser
130 135 140
Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ala Lys Pro Thr
145 150 155 160
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
165 170 175
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
180 185 190
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp
195 200 205
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
210 215 220
Thr Leu Tyr Cys Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala
225 230 235 240
Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg
245 250 255
Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu
260 265 270
Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn
275 280 285
Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met
290 295 300
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
305 310 315 320
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala
325 330 335
Leu Pro Pro Arg Glu Arg Val Gln Pro Leu Glu Glu Asn Val Gly Asn
340 345 350
Ala Ala Arg Pro Arg Phe Glu Arg Asn Lys
355 360
<210> 70
<211> 1155
<212> DNA
<213> 人工序列
<220>
<223> NKG2D-28Z 核苷酸
<400> 70
atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60
cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120
aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180
tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240
gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300
ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360
atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420
gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtggc aaaacctacc 480
acaactcctg caccacgccc ccctactcca gcacctacca tcgcatctca gccactgagt 540
ctgcgaccag aggcctgccg gcccgccgcc ggcggggccg tccataccag agggctggac 600
tttgcctgcg atttttgggt cctggtggtc gtgggagggg tgctggcatg ttactcactg 660
ctggtcaccg tggccttcat catcttctgg gtgcggagca agaggtcccg cctgctgcac 720
agcgactata tgaacatgac cccacggaga cccggcccta cacggaaaca ttaccagccc 780
tatgctccac cccgggactt cgcagcttac agaagtcgag tgaagttcag caggtccgcc 840
gacgctcctg cataccagca gggacagaac cagctgtata acgagctgaa tctgggccgg 900
agagaggaat acgacgtgct ggacaaaagg cggggccggg accccgaaat gggagggaag 960
ccacgacgga aaaaccccca ggagggcctg tacaatgagc tgcaaaagga caaaatggcc 1020
gaggcttatt ctgaaatcgg gatgaaggga gagagaaggc gcggaaaagg ccacgatggc 1080
ctgtaccagg ggctgagcac cgctacaaag gacacctatg atgcactgca catgcaggcc 1140
ctgccccctc ggtga 1155
<210> 71
<211> 384
<212> PRT
<213> 人工序列
<220>
<223> NKG2D-28Z 氨基酸
<400> 71
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr
20 25 30
Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn
35 40 45
Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln
50 55 60
Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys
65 70 75 80
Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly
85 90 95
Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser
100 105 110
Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp
115 120 125
Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser
130 135 140
Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ala Lys Pro Thr
145 150 155 160
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
165 170 175
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
180 185 190
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val Leu
195 200 205
Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val
210 215 220
Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His
225 230 235 240
Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys
245 250 255
His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser
260 265 270
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
275 280 285
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
290 295 300
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
305 310 315 320
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
325 330 335
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
340 345 350
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
355 360 365
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
370 375 380
<210> 72
<211> 1128
<212> DNA
<213> 人工序列
<220>
<223> NKG2D-28(H)Z 核苷酸
<400> 72
atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60
cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120
aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180
tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240
gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300
ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360
atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420
gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtgat cgaggtcatg 480
tacccccctc catatctgga caacgaaaag tccaatggaa ctatcattca cgtgaagggc 540
aaacatctgt gcccatctcc cctgttccct gggccaagta aacctttttg ggtcctggtg 600
gtcgtgggag gggtgctggc atgttactca ctgctggtca ccgtggcctt catcatcttc 660
tgggtgcgga gcaagaggtc ccgcctgctg cacagcgact atatgaacat gaccccacgg 720
agacccggcc ctacacggaa acattaccag ccctatgctc caccccggga cttcgcagct 780
tacagaagtc gagtgaagtt cagcaggtcc gccgacgctc ctgcatacca gcagggacag 840
aaccagctgt ataacgagct gaatctgggc cggagagagg aatacgacgt gctggacaaa 900
aggcggggcc gggaccccga aatgggaggg aagccacgac ggaaaaaccc ccaggagggc 960
ctgtacaatg agctgcaaaa ggacaaaatg gccgaggctt attctgaaat cgggatgaag 1020
ggagagagaa ggcgcggaaa aggccacgat ggcctgtacc aggggctgag caccgctaca 1080
aaggacacct atgatgcact gcacatgcag gccctgcccc ctcggtga 1128
<210> 73
<211> 375
<212> PRT
<213> 人工序列
<220>
<223> NKG2D-28(H)Z 氨基酸
<400> 73
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr
20 25 30
Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn
35 40 45
Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln
50 55 60
Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys
65 70 75 80
Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly
85 90 95
Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser
100 105 110
Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp
115 120 125
Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser
130 135 140
Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ile Glu Val Met
145 150 155 160
Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile
165 170 175
His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro
180 185 190
Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys
195 200 205
Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser
210 215 220
Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg
225 230 235 240
Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg
245 250 255
Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp
260 265 270
Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn
275 280 285
Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg
290 295 300
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly
305 310 315 320
Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
325 330 335
Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu
340 345 350
Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His
355 360 365
Met Gln Ala Leu Pro Pro Arg
370 375
<210> 74
<211> 1257
<212> DNA
<213> 人工序列
<220>
<223> NKG2D-BBOX40Z 核苷酸
<400> 74
atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60
cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120
aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180
tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240
gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300
ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360
atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420
gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtggc aaaacctacc 480
acaactcctg caccacgccc ccctactcca gcacctacca tcgcatctca gccactgagt 540
ctgcgaccag aggcctgccg gcccgccgcc ggcggggccg tccataccag agggctggac 600
tttgcctgcg atatctacat ttgggcccct ctggctggaa catgtggcgt gctgctgctg 660
tccctggtca ttactctgta ttgtaagcgg ggaagaaaga aactgctgta catcttcaaa 720
cagcccttta tgaggcctgt gcagaccaca caggaggaag acggctgctc ctgccggttc 780
cccgaggaag aggaaggcgg gtgcgagctg cgggaccaga gactgccacc tgatgcacac 840
aagccaccag gaggaggcag cttcaggacc cccatccagg aggaacaggc cgacgctcat 900
tccacactgg ccaaaattcg agtgaagttc agcaggtccg ccgacgctcc tgcataccag 960
cagggacaga accagctgta taacgagctg aatctgggcc ggagagagga atacgacgtg 1020
ctggacaaaa ggcggggccg ggaccccgaa atgggaggga agccacgacg gaaaaacccc 1080
caggagggcc tgtacaatga gctgcaaaag gacaaaatgg ccgaggctta ttctgaaatc 1140
gggatgaagg gagagagaag gcgcggaaaa ggccacgatg gcctgtacca ggggctgagc 1200
accgctacaa aggacaccta tgatgcactg cacatgcagg ccctgccccc tcggtga 1257
<210> 75
<211> 418
<212> PRT
<213> 人工序列
<220>
<223> NKG2D-BBOX40Z 氨基酸
<400> 75
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr
20 25 30
Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn
35 40 45
Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln
50 55 60
Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys
65 70 75 80
Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly
85 90 95
Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser
100 105 110
Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp
115 120 125
Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser
130 135 140
Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ala Lys Pro Thr
145 150 155 160
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
165 170 175
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
180 185 190
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp
195 200 205
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
210 215 220
Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys
225 230 235 240
Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys
245 250 255
Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Asp
260 265 270
Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly Ser Phe
275 280 285
Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr Leu Ala
290 295 300
Lys Ile Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
305 310 315 320
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
325 330 335
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
340 345 350
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
355 360 365
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
370 375 380
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
385 390 395 400
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
405 410 415
Pro Arg
<210> 76
<211> 1215
<212> DNA
<213> 人工序列
<220>
<223> NKG2D-BBOX40LZ 核苷酸
<400> 76
atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60
cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120
aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180
tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240
gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300
ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360
atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420
gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtggc aaaacctacc 480
acaactcctg caccacgccc ccctactcca gcacctacca tcgcatctca gccactgagt 540
ctgcgaccag aggcctgccg gcccgccgcc ggcggggccg tccataccag agggctggac 600
tttgcctgcg atatctacat ttgggcccct ctggctggaa catgtggcgt gctgctgctg 660
tccctggtca ttactctgta ttgtaagcgg ggaagaaaga aactgctgta catcttcaaa 720
cagcccttta tgaggcctgt gcagaccaca caggaggaag acggctgctc ctgccggttc 780
cccgaggaag aggaaggcgg gtgcgagctg gaaagagtgc agcccctgga agagaatgtc 840
gggaatgccg ctcgcccaag atttgaaagg aacaaacgag tgaagttcag caggtccgcc 900
gacgctcctg cataccagca gggacagaac cagctgtata acgagctgaa tctgggccgg 960
agagaggaat acgacgtgct ggacaaaagg cggggccggg accccgaaat gggagggaag 1020
ccacgacgga aaaaccccca ggagggcctg tacaatgagc tgcaaaagga caaaatggcc 1080
gaggcttatt ctgaaatcgg gatgaaggga gagagaaggc gcggaaaagg ccacgatggc 1140
ctgtaccagg ggctgagcac cgctacaaag gacacctatg atgcactgca catgcaggcc 1200
ctgccccctc ggtga 1215
<210> 77
<211> 404
<212> PRT
<213> 人工序列
<220>
<223> NKG2D-BBOX40LZ 氨基酸
<400> 77
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr
20 25 30
Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn
35 40 45
Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln
50 55 60
Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys
65 70 75 80
Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly
85 90 95
Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser
100 105 110
Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp
115 120 125
Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser
130 135 140
Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ala Lys Pro Thr
145 150 155 160
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
165 170 175
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
180 185 190
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp
195 200 205
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
210 215 220
Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys
225 230 235 240
Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys
245 250 255
Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Glu Arg
260 265 270
Val Gln Pro Leu Glu Glu Asn Val Gly Asn Ala Ala Arg Pro Arg Phe
275 280 285
Glu Arg Asn Lys Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala
290 295 300
Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg
305 310 315 320
Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu
325 330 335
Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn
340 345 350
Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met
355 360 365
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
370 375 380
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala
385 390 395 400
Leu Pro Pro Arg
<210> 78
<211> 1257
<212> DNA
<213> 人工序列
<220>
<223> NKG2D-OX40BBZ 核苷酸
<400> 78
atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60
cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120
aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180
tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240
gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300
ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360
atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420
gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtggc aaaacctacc 480
acaactcctg caccacgccc ccctactcca gcacctacca tcgcatctca gccactgagt 540
ctgcgaccag aggcctgccg gcccgccgcc ggcggggccg tccataccag agggctggac 600
tttgcctgcg atatctacat ttgggcccct ctggctggaa catgtggcgt gctgctgctg 660
tccctggtca ttactctgta ttgtcgggac cagagactgc cacctgatgc acacaagcca 720
ccaggaggag gcagcttcag gacccccatc caggaggaac aggccgacgc tcattccaca 780
ctggccaaaa ttaagcgggg aagaaagaaa ctgctgtaca tcttcaaaca gccctttatg 840
aggcctgtgc agaccacaca ggaggaagac ggctgctcct gccggttccc cgaggaagag 900
gaaggcgggt gcgagctgcg agtgaagttc agcaggtccg ccgacgctcc tgcataccag 960
cagggacaga accagctgta taacgagctg aatctgggcc ggagagagga atacgacgtg 1020
ctggacaaaa ggcggggccg ggaccccgaa atgggaggga agccacgacg gaaaaacccc 1080
caggagggcc tgtacaatga gctgcaaaag gacaaaatgg ccgaggctta ttctgaaatc 1140
gggatgaagg gagagagaag gcgcggaaaa ggccacgatg gcctgtacca ggggctgagc 1200
accgctacaa aggacaccta tgatgcactg cacatgcagg ccctgccccc tcggtga 1257
<210> 79
<211> 418
<212> PRT
<213> 人工序列
<220>
<223> NKG2D-OX40BBZ 氨基酸
<400> 79
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr
20 25 30
Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn
35 40 45
Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln
50 55 60
Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys
65 70 75 80
Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly
85 90 95
Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser
100 105 110
Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp
115 120 125
Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser
130 135 140
Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ala Lys Pro Thr
145 150 155 160
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
165 170 175
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
180 185 190
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp
195 200 205
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
210 215 220
Thr Leu Tyr Cys Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro
225 230 235 240
Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp
245 250 255
Ala His Ser Thr Leu Ala Lys Ile Lys Arg Gly Arg Lys Lys Leu Leu
260 265 270
Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu
275 280 285
Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys
290 295 300
Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
305 310 315 320
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
325 330 335
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
340 345 350
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
355 360 365
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
370 375 380
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
385 390 395 400
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
405 410 415
Pro Arg
<210> 80
<211> 1281
<212> DNA
<213> 人工序列
<220>
<223> NKG2D-28BBZ 核苷酸
<400> 80
atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60
cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120
aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180
tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240
gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300
ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360
atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420
gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtggc aaaacctacc 480
acaactcctg caccacgccc ccctactcca gcacctacca tcgcatctca gccactgagt 540
ctgcgaccag aggcctgccg gcccgccgcc ggcggggccg tccataccag agggctggac 600
tttgcctgcg atttttgggt cctggtggtc gtgggagggg tgctggcatg ttactcactg 660
ctggtcaccg tggccttcat catcttctgg gtgcggagca agaggtcccg cctgctgcac 720
agcgactata tgaacatgac cccacggaga cccggcccta cacggaaaca ttaccagccc 780
tatgctccac cccgggactt cgcagcttac agaagtaagc ggggaagaaa gaaactgctg 840
tacatcttca aacagccctt tatgaggcct gtgcagacca cacaggagga agacggctgc 900
tcctgccggt tccccgagga agaggaaggc gggtgcgagc tgcgagtgaa gttcagcagg 960
tccgccgacg ctcctgcata ccagcaggga cagaaccagc tgtataacga gctgaatctg 1020
ggccggagag aggaatacga cgtgctggac aaaaggcggg gccgggaccc cgaaatggga 1080
gggaagccac gacggaaaaa cccccaggag ggcctgtaca atgagctgca aaaggacaaa 1140
atggccgagg cttattctga aatcgggatg aagggagaga gaaggcgcgg aaaaggccac 1200
gatggcctgt accaggggct gagcaccgct acaaaggaca cctatgatgc actgcacatg 1260
caggccctgc cccctcggtg a 1281
<210> 81
<211> 426
<212> PRT
<213> 人工序列
<220>
<223> NKG2D-28BBZ 氨基酸
<400> 81
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr
20 25 30
Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn
35 40 45
Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln
50 55 60
Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys
65 70 75 80
Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly
85 90 95
Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser
100 105 110
Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp
115 120 125
Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser
130 135 140
Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ala Lys Pro Thr
145 150 155 160
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
165 170 175
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
180 185 190
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val Leu
195 200 205
Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val
210 215 220
Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His
225 230 235 240
Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys
245 250 255
His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser
260 265 270
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
275 280 285
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
290 295 300
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg
305 310 315 320
Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn
325 330 335
Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
340 345 350
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro
355 360 365
Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
370 375 380
Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His
385 390 395 400
Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp
405 410 415
Ala Leu His Met Gln Ala Leu Pro Pro Arg
420 425
<210> 82
<211> 1263
<212> DNA
<213> 人工序列
<220>
<223> NKG2D-28OX40Z 核苷酸
<400> 82
atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60
cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120
aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180
tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240
gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300
ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360
atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420
gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtggc aaaacctacc 480
acaactcctg caccacgccc ccctactcca gcacctacca tcgcatctca gccactgagt 540
ctgcgaccag aggcctgccg gcccgccgcc ggcggggccg tccataccag agggctggac 600
tttgcctgcg atttttgggt cctggtggtc gtgggagggg tgctggcatg ttactcactg 660
ctggtcaccg tggccttcat catcttctgg gtgcggagca agaggtcccg cctgctgcac 720
agcgactata tgaacatgac cccacggaga cccggcccta cacggaaaca ttaccagccc 780
tatgctccac cccgggactt cgcagcttac agaagtcggg accagagact gccacctgat 840
gcacacaagc caccaggagg aggcagcttc aggaccccca tccaggagga acaggccgac 900
gctcattcca cactggccaa aattcgagtg aagttcagca ggtccgccga cgctcctgca 960
taccagcagg gacagaacca gctgtataac gagctgaatc tgggccggag agaggaatac 1020
gacgtgctgg acaaaaggcg gggccgggac cccgaaatgg gagggaagcc acgacggaaa 1080
aacccccagg agggcctgta caatgagctg caaaaggaca aaatggccga ggcttattct 1140
gaaatcggga tgaagggaga gagaaggcgc ggaaaaggcc acgatggcct gtaccagggg 1200
ctgagcaccg ctacaaagga cacctatgat gcactgcaca tgcaggccct gccccctcgg 1260
tga 1263
<210> 83
<211> 420
<212> PRT
<213> 人工序列
<220>
<223> NKG2D-28OX40Z 氨基酸
<400> 83
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr
20 25 30
Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn
35 40 45
Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln
50 55 60
Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys
65 70 75 80
Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly
85 90 95
Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser
100 105 110
Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp
115 120 125
Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser
130 135 140
Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ala Lys Pro Thr
145 150 155 160
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
165 170 175
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
180 185 190
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val Leu
195 200 205
Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val
210 215 220
Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His
225 230 235 240
Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys
245 250 255
His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser
260 265 270
Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly
275 280 285
Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr
290 295 300
Leu Ala Lys Ile Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala
305 310 315 320
Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg
325 330 335
Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu
340 345 350
Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn
355 360 365
Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met
370 375 380
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
385 390 395 400
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala
405 410 415
Leu Pro Pro Arg
420
<210> 84
<211> 1221
<212> DNA
<213> 人工序列
<220>
<223> NKG2D-28OX40LZ 核苷酸
<400> 84
atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60
cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120
aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180
tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240
gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300
ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360
atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420
gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtggc aaaacctacc 480
acaactcctg caccacgccc ccctactcca gcacctacca tcgcatctca gccactgagt 540
ctgcgaccag aggcctgccg gcccgccgcc ggcggggccg tccataccag agggctggac 600
tttgcctgcg atttttgggt cctggtggtc gtgggagggg tgctggcatg ttactcactg 660
ctggtcaccg tggccttcat catcttctgg gtgcggagca agaggtcccg cctgctgcac 720
agcgactata tgaacatgac cccacggaga cccggcccta cacggaaaca ttaccagccc 780
tatgctccac cccgggactt cgcagcttac agaagtgaaa gagtgcagcc cctggaagag 840
aatgtcggga atgccgctcg cccaagattt gaaaggaaca aacgagtgaa gttcagcagg 900
tccgccgacg ctcctgcata ccagcaggga cagaaccagc tgtataacga gctgaatctg 960
ggccggagag aggaatacga cgtgctggac aaaaggcggg gccgggaccc cgaaatggga 1020
gggaagccac gacggaaaaa cccccaggag ggcctgtaca atgagctgca aaaggacaaa 1080
atggccgagg cttattctga aatcgggatg aagggagaga gaaggcgcgg aaaaggccac 1140
gatggcctgt accaggggct gagcaccgct acaaaggaca cctatgatgc actgcacatg 1200
caggccctgc cccctcggtg a 1221
<210> 85
<211> 406
<212> PRT
<213> 人工序列
<220>
<223> NKG2D-28OX40LZ 氨基酸
<400> 85
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr
20 25 30
Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn
35 40 45
Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln
50 55 60
Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys
65 70 75 80
Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly
85 90 95
Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser
100 105 110
Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp
115 120 125
Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser
130 135 140
Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ala Lys Pro Thr
145 150 155 160
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
165 170 175
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
180 185 190
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val Leu
195 200 205
Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val
210 215 220
Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His
225 230 235 240
Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys
245 250 255
His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser
260 265 270
Glu Arg Val Gln Pro Leu Glu Glu Asn Val Gly Asn Ala Ala Arg Pro
275 280 285
Arg Phe Glu Arg Asn Lys Arg Val Lys Phe Ser Arg Ser Ala Asp Ala
290 295 300
Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu
305 310 315 320
Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp
325 330 335
Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu
340 345 350
Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile
355 360 365
Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
370 375 380
Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met
385 390 395 400
Gln Ala Leu Pro Pro Arg
405
<210> 86
<211> 1254
<212> DNA
<213> 人工序列
<220>
<223> NKG2D-28(H)BBZ 核苷酸
<400> 86
atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60
cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120
aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180
tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240
gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300
ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360
atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420
gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtgat cgaggtcatg 480
tacccccctc catatctgga caacgaaaag tccaatggaa ctatcattca cgtgaagggc 540
aaacatctgt gcccatctcc cctgttccct gggccaagta aacctttttg ggtcctggtg 600
gtcgtgggag gggtgctggc atgttactca ctgctggtca ccgtggcctt catcatcttc 660
tgggtgcgga gcaagaggtc ccgcctgctg cacagcgact atatgaacat gaccccacgg 720
agacccggcc ctacacggaa acattaccag ccctatgctc caccccggga cttcgcagct 780
tacagaagta agcggggaag aaagaaactg ctgtacatct tcaaacagcc ctttatgagg 840
cctgtgcaga ccacacagga ggaagacggc tgctcctgcc ggttccccga ggaagaggaa 900
ggcgggtgcg agctgcgagt gaagttcagc aggtccgccg acgctcctgc ataccagcag 960
ggacagaacc agctgtataa cgagctgaat ctgggccgga gagaggaata cgacgtgctg 1020
gacaaaaggc ggggccggga ccccgaaatg ggagggaagc cacgacggaa aaacccccag 1080
gagggcctgt acaatgagct gcaaaaggac aaaatggccg aggcttattc tgaaatcggg 1140
atgaagggag agagaaggcg cggaaaaggc cacgatggcc tgtaccaggg gctgagcacc 1200
gctacaaagg acacctatga tgcactgcac atgcaggccc tgccccctcg gtga 1254
<210> 87
<211> 417
<212> PRT
<213> 人工序列
<220>
<223> NKG2D-28(H)BBZ 氨基酸
<400> 87
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr
20 25 30
Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn
35 40 45
Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln
50 55 60
Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys
65 70 75 80
Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly
85 90 95
Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser
100 105 110
Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp
115 120 125
Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser
130 135 140
Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ile Glu Val Met
145 150 155 160
Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile
165 170 175
His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro
180 185 190
Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys
195 200 205
Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser
210 215 220
Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg
225 230 235 240
Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg
245 250 255
Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr
260 265 270
Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu
275 280 285
Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu
290 295 300
Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln
305 310 315 320
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
325 330 335
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
340 345 350
Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
355 360 365
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
370 375 380
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
385 390 395 400
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
405 410 415
Arg
<210> 88
<211> 1236
<212> DNA
<213> 人工序列
<220>
<223> NKG2D-28(H)OX40Z 核苷酸
<400> 88
atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60
cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120
aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180
tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240
gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300
ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360
atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420
gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtgat cgaggtcatg 480
tacccccctc catatctgga caacgaaaag tccaatggaa ctatcattca cgtgaagggc 540
aaacatctgt gcccatctcc cctgttccct gggccaagta aacctttttg ggtcctggtg 600
gtcgtgggag gggtgctggc atgttactca ctgctggtca ccgtggcctt catcatcttc 660
tgggtgcgga gcaagaggtc ccgcctgctg cacagcgact atatgaacat gaccccacgg 720
agacccggcc ctacacggaa acattaccag ccctatgctc caccccggga cttcgcagct 780
tacagaagtc gggaccagag actgccacct gatgcacaca agccaccagg aggaggcagc 840
ttcaggaccc ccatccagga ggaacaggcc gacgctcatt ccacactggc caaaattcga 900
gtgaagttca gcaggtccgc cgacgctcct gcataccagc agggacagaa ccagctgtat 960
aacgagctga atctgggccg gagagaggaa tacgacgtgc tggacaaaag gcggggccgg 1020
gaccccgaaa tgggagggaa gccacgacgg aaaaaccccc aggagggcct gtacaatgag 1080
ctgcaaaagg acaaaatggc cgaggcttat tctgaaatcg ggatgaaggg agagagaagg 1140
cgcggaaaag gccacgatgg cctgtaccag gggctgagca ccgctacaaa ggacacctat 1200
gatgcactgc acatgcaggc cctgccccct cggtga 1236
<210> 89
<211> 411
<212> PRT
<213> 人工序列
<220>
<223> NKG2D-28(H)OX40Z 氨基酸
<400> 89
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr
20 25 30
Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn
35 40 45
Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln
50 55 60
Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys
65 70 75 80
Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly
85 90 95
Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser
100 105 110
Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp
115 120 125
Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser
130 135 140
Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ile Glu Val Met
145 150 155 160
Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile
165 170 175
His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro
180 185 190
Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys
195 200 205
Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser
210 215 220
Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg
225 230 235 240
Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg
245 250 255
Asp Phe Ala Ala Tyr Arg Ser Arg Asp Gln Arg Leu Pro Pro Asp Ala
260 265 270
His Lys Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu
275 280 285
Gln Ala Asp Ala His Ser Thr Leu Ala Lys Ile Arg Val Lys Phe Ser
290 295 300
Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr
305 310 315 320
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys
325 330 335
Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn
340 345 350
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
355 360 365
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
370 375 380
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
385 390 395 400
Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
405 410
<210> 90
<211> 1194
<212> DNA
<213> 人工序列
<220>
<223> NKG2D-28(H)OX40LZ 核苷酸
<400> 90
atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60
cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120
aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180
tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240
gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300
ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360
atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420
gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtgat cgaggtcatg 480
tacccccctc catatctgga caacgaaaag tccaatggaa ctatcattca cgtgaagggc 540
aaacatctgt gcccatctcc cctgttccct gggccaagta aacctttttg ggtcctggtg 600
gtcgtgggag gggtgctggc atgttactca ctgctggtca ccgtggcctt catcatcttc 660
tgggtgcgga gcaagaggtc ccgcctgctg cacagcgact atatgaacat gaccccacgg 720
agacccggcc ctacacggaa acattaccag ccctatgctc caccccggga cttcgcagct 780
tacagaagtg aaagagtgca gcccctggaa gagaatgtcg ggaatgccgc tcgcccaaga 840
tttgaaagga acaaacgagt gaagttcagc aggtccgccg acgctcctgc ataccagcag 900
ggacagaacc agctgtataa cgagctgaat ctgggccgga gagaggaata cgacgtgctg 960
gacaaaaggc ggggccggga ccccgaaatg ggagggaagc cacgacggaa aaacccccag 1020
gagggcctgt acaatgagct gcaaaaggac aaaatggccg aggcttattc tgaaatcggg 1080
atgaagggag agagaaggcg cggaaaaggc cacgatggcc tgtaccaggg gctgagcacc 1140
gctacaaagg acacctatga tgcactgcac atgcaggccc tgccccctcg gtga 1194
<210> 91
<211> 397
<212> PRT
<213> 人工序列
<220>
<223> NKG2D-28(H)OX40LZ 氨基酸
<400> 91
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr
20 25 30
Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn
35 40 45
Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln
50 55 60
Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys
65 70 75 80
Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly
85 90 95
Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser
100 105 110
Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp
115 120 125
Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser
130 135 140
Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ile Glu Val Met
145 150 155 160
Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile
165 170 175
His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro
180 185 190
Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys
195 200 205
Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser
210 215 220
Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg
225 230 235 240
Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg
245 250 255
Asp Phe Ala Ala Tyr Arg Ser Glu Arg Val Gln Pro Leu Glu Glu Asn
260 265 270
Val Gly Asn Ala Ala Arg Pro Arg Phe Glu Arg Asn Lys Arg Val Lys
275 280 285
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
290 295 300
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
305 310 315 320
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
325 330 335
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
340 345 350
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
355 360 365
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
370 375 380
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
385 390 395
<210> 92
<211> 1203
<212> DNA
<213> 人工序列
<220>
<223> NKG2D-AAA-28(H)OX40LZ 核苷酸
<400> 92
atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60
cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120
aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180
tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240
gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300
ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360
atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420
gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtggc cgccgctatc 480
gaggtcatgt acccccctcc atatctggac aacgaaaagt ccaatggaac tatcattcac 540
gtgaagggca aacatctgtg cccatctccc ctgttccctg ggccaagtaa acctttttgg 600
gtcctggtgg tcgtgggagg ggtgctggca tgttactcac tgctggtcac cgtggccttc 660
atcatcttct gggtgcggag caagaggtcc cgcctgctgc acagcgacta tatgaacatg 720
accccacgga gacccggccc tacacggaaa cattaccagc cctatgctcc accccgggac 780
ttcgcagctt acagaagtga aagagtgcag cccctggaag agaatgtcgg gaatgccgct 840
cgcccaagat ttgaaaggaa caaacgagtg aagttcagca ggtccgccga cgctcctgca 900
taccagcagg gacagaacca gctgtataac gagctgaatc tgggccggag agaggaatac 960
gacgtgctgg acaaaaggcg gggccgggac cccgaaatgg gagggaagcc acgacggaaa 1020
aacccccagg agggcctgta caatgagctg caaaaggaca aaatggccga ggcttattct 1080
gaaatcggga tgaagggaga gagaaggcgc ggaaaaggcc acgatggcct gtaccagggg 1140
ctgagcaccg ctacaaagga cacctatgat gcactgcaca tgcaggccct gccccctcgg 1200
tga 1203
<210> 93
<211> 400
<212> PRT
<213> 人工序列
<220>
<223> NKG2D-AAA-28(H)OX40LZ 氨基酸
<400> 93
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr
20 25 30
Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn
35 40 45
Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln
50 55 60
Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys
65 70 75 80
Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly
85 90 95
Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser
100 105 110
Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp
115 120 125
Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser
130 135 140
Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ala Ala Ala Ile
145 150 155 160
Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly
165 170 175
Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe
180 185 190
Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val
195 200 205
Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp
210 215 220
Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met
225 230 235 240
Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala
245 250 255
Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Glu Arg Val Gln Pro Leu
260 265 270
Glu Glu Asn Val Gly Asn Ala Ala Arg Pro Arg Phe Glu Arg Asn Lys
275 280 285
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
290 295 300
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
305 310 315 320
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
325 330 335
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
340 345 350
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
355 360 365
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
370 375 380
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
385 390 395 400

Claims (9)

1.一种嵌合抗原受体，其包括：
包括OX40配体(CD252)和CD3-zeta的细胞内信号传导结构域；与所述细胞内信号传导结构域连接的跨膜结构域；与所述跨膜结构域连接的间隔区结构域；与所述间隔区结构域连接的胞外结构域，和与所述胞外结构域连接的信号序列，
其中所述细胞内信号传导结构域为从细胞膜向所述细胞的内部顺序排列的第一细胞内信号传导结构域和第二细胞内信号传导结构域，或从细胞膜向所述细胞的内部顺序排列的第一细胞内信号传导结构域、第二细胞内信号传导结构域和第三细胞内信号传导结构域，和其中所述OX40配体不是第一细胞内信号传导结构域。
2.根据权利要求1所述的嵌合抗原受体，其中所述信号序列为CD16。
3.根据权利要求1所述的嵌合抗原受体，其中所述胞外结构域为选自由抗体的抗原结合片段、Fc受体、自然细胞毒性受体、NKG2D、2B4和DNAM-1组成的组的一种。
4.根据权利要求3所述的嵌合抗原受体，其中所述抗原结合片段为Fab片段、F(ab')片段、F(ab')2片段、Fv片段或单链可变片段(ScFv)。
5.根据权利要求4所述的嵌合抗原受体，其中所述抗原结合片段是单链可变片段(ScFv)。
6.根据权利要求1所述的嵌合抗原受体，其中所述间隔区结构域为选自由CD8α和CD28组成的组的任一种。
7.根据权利要求1所述的嵌合抗原受体，其中所述跨膜结构域为选自由CD8α和CD28组成的组的一种。
8.一种嵌合抗原受体，其包括细胞内信号传导结构域、与所述细胞内信号传导结构域连接的跨膜结构域；与所述跨膜结构域连接的间隔区结构域；与所述间隔区结构域连接的胞外结构域，和与所述胞外结构域连接的信号序列，
其中所述细胞内信号传导结构域由以下组成：
CD28作为第一细胞内信号传导结构域；
OX40配体作为第二细胞内信号传导结构域；和
CD3-zeta作为第三细胞内信号传导结构域，
其中所述第一、第二和第三细胞内信号传导结构域从细胞膜向所述细胞的内部顺序排列。
9.一种核酸分子，其编码根据权利要求1至8中任一项所述的嵌合抗原受体。
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