CN113698489B - 融合蛋白及其靶向psca的car及其应用 - Google Patents

融合蛋白及其靶向psca的car及其应用 Download PDF

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CN113698489B
CN113698489B CN202110556140.7A CN202110556140A CN113698489B CN 113698489 B CN113698489 B CN 113698489B CN 202110556140 A CN202110556140 A CN 202110556140A CN 113698489 B CN113698489 B CN 113698489B
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CN113698489A (zh
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洪娟
沈俊杰
杨智
黄霞
赵永春
赵文旭
陈军
徐艳敏
齐亚男
梅恩典
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Chongqing Jingzhun Biological Industrial Technology Institute Co ltd
Chongqing Precision Biotech Co ltd
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Chongqing Precision Biotech Co ltd
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Abstract

本发明属于肿瘤细胞免疫治疗技术领域，具体涉及一种融合蛋白及其靶向PSCA的CAR及其应用。所述融合蛋白为SIRPγ融合蛋白，包含胞外段、跨膜结构和胞内信号区。该融合蛋白和靶向PSCA的CAR结构连接共同或单独转染免疫细胞,该免疫细胞在缺氧环境下活性增强，而且以逆转肿瘤微环境，破除肿瘤组织中抑制性信号对CAR‑T功能的影响，实现了CAR‑T治疗有效性，同时能够保证一定的安全性。

Description

融合蛋白及其靶向PSCA的CAR及其应用
技术领域
本发明属于肿瘤细胞免疫治疗技术领域，具体涉及一种融合蛋白及其靶向PSCA的CAR及其应用。
背景技术
CAR-T全称是嵌合抗原受体T细胞免疫疗法，嵌合抗原受体(chimeric antigenreceptor，CAR)是模拟TCR功能的人工受体，由抗原识别域、铰链区和跨膜区及胞内信号域依次连接组成，胞内信号域通常为CD3ζ链或FcRγ，或与一种或多种共刺激分子相连，如4-1BB，CD28，ICOS(CD278)。CAR分子包含的ScFv对肿瘤抗原具有特异性识别的特点，通过铰链区和跨膜区进行T细胞激活细胞传递。目前临床上CAR-T治疗主要运用于血液瘤系统的肿瘤治疗。
CD47作为继PD-1/PD-L1,CTLA-4之后新的免疫检查点，通过向巨噬细胞传递“不要吃我”信号，抑制固有免疫的进行。目前有文献报道采用靶向CD47 ScFv制备CAR-T进行肿瘤治疗。CD47可以促成肿瘤逃逸的免疫微环境，不过由于肿瘤的异质性，单独靶向CD47疗效有限，并且外源的ScFv由于其强的亲和力可能会导致CAR-T细胞过度活化存续性差和非特异性引起安全隐患。因此，采用更安全有效的方案去识别CD47破除肿瘤微环境的逃逸信号十分重要。信号调节蛋白α(SIRPα)是CD47的配体之一，可与CD47结合，抑制巨噬细胞的吞噬作用，目前绝大多数关于CD47的研究均集中于SIRPα。SIRPγ也可与CD47结合，其存在于T细胞表面，胞外区由一个V结构域和两个C1结构域组成，无胞内信号，仅通过CD47传递单向信号，目前很少有研究者针对SIRPγ进行改造，而我们认为其是更适合进行针对CD47进行肿瘤微环境破除的配体。因此，我们选择SIRPγ蛋白的胞外段进行改造设计，并进一步设计肿瘤免疫抑制抵抗型的CAR，并且CD47作为继PD-1/PD-L1,CTLA-4之后新的免疫检查点，通过向巨噬细胞传递“不要吃我”信号，抑制固有免疫的进行。目前有文献报道采用靶向CD47ScFv制备CAR-T进行肿瘤治疗。CD47可以促成肿瘤逃逸的免疫微环境，不过由于肿瘤的异质性，单独靶向CD47疗效有限，并且外源的ScFv由于其强的亲和力可能会导致CAR-T细胞过度活化存续性差和非特异性引起安全隐患。因此，采用更安全有效的方案去识别CD47破除肿瘤微环境的逃逸信号十分重要。
前列腺干细胞抗原(PSCA)是最早发现于前列腺癌的GPI锚定的细胞膜蛋白,PSCA在前列腺癌,胰腺癌,膀胱癌等肿瘤中表达增加,相反在食管癌和胃癌中表达降低可能在胃上皮中发挥肿瘤抑制功能,PSCA发挥致瘤和抑瘤两种截然不同的作用与细胞所处的环境不同有关。除此之外,PSCA作为肿瘤的免疫治疗的靶点也显示出其良好的临床应用潜力。因此PSCA不仅成为肿瘤诊断和预后判断的生物学标记也是肿瘤免疫治疗重要的候选靶蛋白。
发明内容
有鉴于此，本发明在于提供一种逆转肿瘤微环境的融合蛋白，其与靶向PSCA的CAR结构a构成一种含有该融合蛋白的免疫细胞，该免疫细胞可以靶向杀伤CD47阳性的的肿瘤细胞；破除肿瘤组织中抑制性信号对免疫细胞功能的影响，实现了CAR-T治疗有效性，同时能够保证一定的安全性，其中含有融合蛋白的免疫细胞与不含有融合蛋白的免疫细胞相比较，含有融合蛋白的免疫细胞的体内存续时间会更长并且安全性更高。。
进一步，所述融合蛋白为SIRPγ融合蛋白，所述SIRPγ融合蛋白结构包含胞外段、跨膜结构和胞内信号区，所述SIRPγ胞外段氨基酸序列如SEQ ID NO:5或其功能性变体所示；优选地，所述跨膜结构来源于人CD28跨膜区或人CD8来源跨膜区；优选地，所述跨膜结构氨基酸序列如SEQ ID NO:3所示或其功能性变体；或如SEQ ID NO:4所示或其功能性变体。
进一步，所述SIRPγ融合蛋白的结构为SIRPγ-CD28TM-CD28或SIRPγ-CD8TM-4-1BB；优选地，所述SIRPγ融合蛋白SIRPγ-CD28TM-CD28的氨基酸序列如SEQ ID NO:6所示或其功能性变体；或所述SIRPγ融合蛋白SIRPγ-CD8TM-4-1BB的氨基酸序列如SEQ ID NO:7所示或其功能性变体。
进一步，编码所述SIRPγ融合蛋白SIRPγ-CD28TM-CD28的核苷酸序列如SEQ IDNO:8所示；或编码所述SIRPγ融合蛋白SIRPγ-CD8TM-4-1BB的核苷酸序列如SEQ ID NO:9所示。
还进一步提供一种包含前述融合蛋白的表达载体。
进一步，所述表达载体为慢病毒表达载体、逆转录病毒表达载体、腺病毒表达载体、腺相关病毒表达载体、DNA载体，RNA载体中的任一种。
还进一步提供一种包含前述表达载体的免疫细胞，所述免疫细胞是T细胞、T细胞前体或者NK细胞。
进一步，所述免疫细胞包含具有识别肿瘤抗原的嵌合抗原受体结构，所述嵌合抗原受体包含识别肿瘤抗原的胞外段、hinge区、跨膜区和胞内信号区，所述肿瘤抗原为PSCA。
进一步还提供一种制备所述免疫细胞的方法，将不包含所述SIRPγ融合蛋白的CAR结构和所述SIRPγ融合蛋白共同表达于一个载体转染免疫细胞；或将不包含所述SIRPγ融合蛋白的CAR结构和所述SIRPγ融合蛋白分别表达于两个载体转染免疫细胞。
本发明目的在于还提供一种包含前述融合蛋白的肿瘤免疫抑制抵抗型CAR，所述肿瘤免疫抑制抵抗型CAR是融合蛋白与CAR结构a结合而成。发明人认为SIRPγ蛋白更适合进行针对CD47进行肿瘤微环境破除的配体。因此，选择SIRPγ蛋白的胞外段进行改造设计，并进一步设计肿瘤免疫抑制抵抗型的CAR；因此本发明创造性的将SIRPγ蛋白联合CAR-T细胞技术应用到肿瘤的免疫治疗当中，即进一步对传统的CAR结构进行改造，提高CAR-T在实体瘤上的治疗药效及CAR-T的安全性。
进一步，所述CAR结构还包括CAR结构a。所述CAR结构a可以是常规的第一代、第二代、第三代CAR结构，也可以是改进的双CAR、可调控CAR结构(如FRB/FKBP12调控)等新型CAR结构。
进一步，CAR结构a中的铰链区序列可以来源于：IgG、CD8、CD7、CD4；CAR结构中跨膜区可以来源于：CD8、CD28、CD3ε、CD4、CD16、CD137、CD80以及CD86；CAR结构中胞内信号区可来源于：CD3、CD137、CD28、CD27、OX40、ICOS、GITR、CD2、CD40、PD-1、PD1L、B7-H3、淋巴细胞功能相关抗原-1(LFA-1)、ICAM-1、CD7、NKG2C、CD83、CD86以及CD127。
优选地，所述CAR结构a包括PSCA单链抗体、铰链区、CD28跨膜区、CD28、CD137和CD3ξ三刺激信号；所述铰链区为G4H或7H。优选地，所述CAR结构a氨基酸序列如SEQ ID NO：1所示或其功能性变体；或如SEQ ID NO：2所示或其功能性变体。
具体地，所述融合蛋白通过多顺反子结构与CAR结构a连接，所述多顺反子结构为自剪切多肽或内部核糖体进入位点IRES，所述自剪切多肽为T2A、P2A、E2A或F2A。所述CAR结构a和融合蛋白组成的结构为ScFv-hinge-TM-CD3ζ-自剪切肽-SIRPγ融合蛋白或ScFv-hinge-TM-4-1BB-CD3ζ-自剪切肽-SIRPγ融合蛋白，即为ScFv-hinge-TM-CD3ζ-自剪切肽-SIRPγ-CD28TM-CD28或ScFv-hinge-TM-4-1BB-CD3ζ-自剪切肽-SIRPγ-CD28TM-CD28。
在某些实施例中，所述CAR结构a带有截短的EGFRt调控标签；在某些实施例中，所述CAR结构a为通用型CAR结构；在某些实施例中，CAR结构a带有自杀基因如iCasp9。
在某些实施例中，所述CAR结构a包含天然杀伤细胞受体(NKR)的一种或多种组分，因而形成NKR-CAR。NKR组分可以是来自以下任何天然杀伤细胞受体的跨膜结构域、铰链结构域或胞质结构域：杀伤细胞免疫球蛋白样受体(KIR)，例如KIR2DL1、KIR2DL2/L3、KIR2DL4、KIR2DL5A、KIR2DL5B、KIR2DS1、KIR2DS2、KIR2DS3、KIR2DS4、DIR2DS5、KIR3DL1/S1、KIR3DL2、KIR3DL3、KIR2DP1和KIR3DP1；天然细胞毒性受体(NCR)，例如，NKp30、NKp44、NKp46；免疫细胞受体的信号传导淋巴细胞活化分子(SLAM)家族，例如，CD48、CD229、2B4、CD84、NTB-A、CRA、BLAME和CD2F-10；Fc受体(FcR)，例如，CD16、和CD64；和Ly49受体，例如，LY49A、LY49C。所述的NKR-CAR分子可以与衔接分子或胞内信号结构域(例如，DAP12)相互作用。
本发明目的在于提供一种核酸序列，所述核酸序列编码前任一项所述的CAR结构，所述核酸序列包含如SEQ ID NO：10或SEQ ID NO：11所示的序列。
在某些具体实施例中，所述SEQ ID NO：10所示的序列编码所述的免疫细胞中的CAR结构为：PSCA ScFv-G4H铰链区-CD8跨膜区-CD28-CD137-CD3ξ-P2A-SIRPγ-CD28TM-CD28；所述SEQ ID NO：11所示的序列编码的所述免疫细胞中的CAR结构为CAR结构为：PSCAScFv-7H铰链区-CD8跨膜区-CD28-CD137-CD3ξ-P2A-SIRPγ-CD28TM-CD28。
本发明目的在于还提供一种包含上述的核酸序列的重组质粒。所述重组质粒还包括表达载体，所述所述表达载体为慢病毒表达载体、逆转录病毒表达载体、腺病毒表达载体、腺相关病毒表达载体、DNA载体，RNA载体、质粒中的任一种。
在某些实施例中，所述慢病毒载体选自基本上由以下组成的群组：人免疫缺陷病毒1(HIV-1)、人免疫缺陷病毒2(HIV-2)、维斯纳-梅迪病毒(visna-maedi virus，VMV)病毒、山羊关节炎-脑炎病毒(CAEV)、马传染性贫血病毒(EIAV)、猫免疫缺陷病毒(FIV)、牛免疫缺陷病毒(BIV)和猿猴免疫缺陷病毒(SIV)。
在某些实施例中，载体包含左(5')逆转录病毒LTR、Psi(Ψ)包装信号、中心多嘌呤段/DNA瓣(cPPT/FLAP)、逆转录病毒导出元件、可操作地连接到编码本文所涵盖的CAR的多核苷酸的启动子和右(3')逆转录病毒LTR。
在某些实施例中，CAR包含乙型肝炎病毒转录后调节元件(HPRE)或土拔鼠转录后调节元件(WPRE)以及优化的土拔鼠转录后调节元件(oPRE)。
在某些实施例中，所述5'LTR的启动子经异源启动子置换。
在某些实施例中，所述异源启动子是巨细胞病毒(CMV)启动子、劳斯肉瘤病毒(Rous Sarcoma Virus，RSV)启动子或猿猴病毒40(SV40)启动子。
在某些实施例中，所述5'LTR或3'LTR是慢病毒LTR。
在某些实施例中，所述3'LTR是自我失活(SIN)LTR。
在某些实施例中，所述CAR结构的核酸序列包含优化的Kozark序列。
在某些实施例中，可操作地连接到编码本文所涵盖的CAR的多核苷酸的所述启动子以及以下组成的群组：巨细胞病毒立即早期基因启动子(CMV)、延伸因子1α启动子(EF1-α)、磷酸甘油酸激酶-1启动子(PGK)、泛素-C启动子(UBQ-C)、巨细胞病毒增强子/鸡β-肌动蛋白启动子(CAG)、多瘤病毒增强子/单纯疱疹胸苷激酶启动子(MC1)、β肌动蛋白启动子(β-ACT)、猿猴病毒40启动子(SV40)和骨髓增生肉瘤病毒增强子，阴性对照区缺失的、dl587rev引物结合位点取代的(MND)启动子。
在某些实施例中，包含CAR的载体可以包含分泌型抗PD-1ScFv；在某些实施例中，包含CAR的载体包含PD-1共轭转导肽(如PD-1-CD28-CD137-CD3信号结构)；在某些实施例中，包含CAR的载体多个CAR组合，如2个靶向不同抗原或同一抗原的不同识别位点的CAR组合。
进一步，还提供一种包含上述重组质粒的免疫细胞，所述免疫细胞是T细胞、T细胞前体或者NK细胞。
在某些实施例中，所述免疫细胞可以表达其它活性剂，例如，增强CAR表达细胞活性的活性剂。活性剂可以是阻断抑制性分子的活性剂。抑制性分子如PD1可以在一些实施方案中降低CAR表达细胞发动免疫效应子反应的能力。抑制性分子包括PD1、PD-L1、CTLA4、TIM3、LAG3、VISTA、BTLA、TIGIT,LAIR1、CD160、2B4、CEACAM(CEACAM-1、CEACAM-3、CEACAM-5)、LAG3、VISTA、BTLA、TIG、LAIR1、CD160、2B4、CD80、CD86、B7-H3(CD276)、B7-H4(VTCN1)、HVEM(TNFRSF14或CD270)、KIR、A2aR、MHC I类、MHC II类、GAL9、腺苷、TGFR(TGFRβ)和TGFRβ。所述抑制性分子的胞外结构域可以融合到跨膜结构域和胞内信号传导结构域，比如PD1CAR。
本发明还提供一种前任一所述的免疫细胞或肿瘤免疫抑制抵抗型CAR及其核酸序列、重组质粒在制备肿瘤药物中的应用。
进一步，所述肿瘤为恶性肿瘤，包括急性淋巴样白血病、慢性淋巴细胞白血病、慢性髓性白血病、非霍奇金淋巴瘤、霍奇金淋巴瘤、前列腺癌、结直肠癌、乳腺癌、卵巢癌、宫颈癌、胰腺癌、肺癌、肾癌、肝癌、脑癌和皮肤癌。
本发明目的在于还提供一种药物组合物，所述药物组合物包括：前任一项所述的免疫细胞或CAR结构和可增强CAR表达活性的活性剂和/或治疗剂。
进一步，所述活性剂和/或治疗剂包括环孢素(cyclosporin)、硫唑嘌呤(azathioprine)、甲氨蝶呤(methotrexate)、霉酚酸酯(mycophenolate)和FK506、抗体或其它免疫清除剂(immunoablativeagents)例如CAMPATH、抗CD3抗体或其它抗体治疗、环磷酰胺(cytoxan)、氟达拉滨(fludarabine)、环孢素(cyclosporin)、FK506、雷帕霉素(rapamycin)、霉酚酸(mycophenolicacid)、类固醇(steroids)、FR901228、细胞因子。
本发明中，所述“功能性变体”通常是指包括与其具有基本上相同的功能(例如，可以具备所述嵌合抗原受体的性质)，且与其具有至少85％(例如，至少85％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％，或至少100％)序列同一性的氨基酸序列。在某些实施方式中，所述氨基酸序列的变体为与其具有基本上相同的功能。
本发明有益效果在于
本发明提供的包含逆转肿瘤微环境的融合蛋白的CAR-T细胞或免疫细胞可以逆转肿瘤微环境，靶向PSCA靶点杀伤CD47阳性的的肿瘤细胞；破除肿瘤组织中抑制性信号对CAR-T功能的影响，实现了CAR-T治疗有效性，同时能够保证一定的安全性，其中含有融合蛋白的CAR与不含有融合蛋白的CAR相比较，含有融合蛋白的CAR的结构的体内存续时间会更长并且安全性更高。
附图说明
图1为PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28分泌IFN-γ因子情况。
图2为PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28分泌IFN-γ因子情况。
图3为PSCA-28BBZ-G4H-28TM+SIRPγ-28分泌IFN-γ因子情况。
图4为PSCA-28BBZ-7H-28TM+SIRPγ-28分泌IFN-γ因子情况。
图5为PSCA-28BBZ-G4H-28TM、PSCA-28BBZ-G4H-28TM+SIRPγ-28分泌IFN-γ因子情况。
图6为PSCA-28BBZ-7H-28TM、PSCA-28BBZ-7H-28TM+SIRPγ-28分泌IFN-γ因子情况。
图7为PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28分别在阳性/阴性细胞中细胞杀伤情况。
图8为PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28分别在阳性/阴性细胞中细胞杀伤情况。
图9为PSCA-28BBZ-G4H-28TM+SIRPγ-28分别在阳性/阴性细胞中细胞杀伤情况。
图10为PSCA-28BBZ-7H-28TM+SIRPγ-28分别在阳性/阴性细胞中细胞杀伤情况。
图11为PSCA-28BBZ-G4H-28TM、PSCA-28BBZ-G4H-28TM+SIRPγ-28中细胞杀伤情况。
图12为PSCA-28BBZ-7H-28TM、PSCA-28BBZ-7H-28TM+SIRPγ-28中细胞杀伤情况。
图中，RT4-Luc-GFP为阳性细胞，PC-3-Luc-GFP为阴性细胞；SIRPγ-28TM-28(SIRPγ-28)是指胞内仅有来源CD28胞内域信号的融合肽；PSCA-28BBZ-G4H-28TM+SIRPγ-28或PSCA-28BBZ-7H-28TM+SIRPγ-28指的是将PSCA-28BBZ-G4H-28TM或PSCA-28BBZ-7H-28TM和SIRPγ-28与融合蛋白SIRPγ-28分别表达于两个载体转染免疫细胞获得的产品。PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28或PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28指的是将PSCA-28BBZ-G4H-28TM或PSCA-28BBZ-7H-28TM和融合蛋白SIRPγ-28共同表达于一个载体转染免疫细胞获得的产品。
具体实施方式
所举实施例是为了更好地对本发明进行说明，但并不是本发明的内容仅局限于所举实施例。所以熟悉本领域的技术人员根据上述发明内容对实施方案进行非本质的改进和调整，仍属于本发明的保护范围。
本发明实施例中感染T细胞的方法为：采用磷酸钙法包装慢病毒，具体为：用含10％FBS(w/v)的DMEM培养基培养293T细胞至较佳状态，包装质粒(RRE:REV:2G)和表达质粒按一定比列加入到1.5的离心管中，加入CaCl2和2×HBS，混匀后室温静置后加入到处理好的293T细胞培养液中，3-5h后再次换液至10mL含10％FBS的DMEM培养基，48h或72h后收集细胞上清，纯化病毒。
本发明实施例中，IFN-γ检测采用BD IFN-γ试剂盒检测，实验步骤依据产品说明书进行；IL-2检测采用inritrogen IL-2试剂盒检测，实验步骤依据产品说明书进行；TNF-α检测采用Biolegend试剂盒检测，实验步骤依据产品说明书进行。
本发明实施例中，检测不同CAR-T对靶细胞杀伤能力的方法为：利用ACEAxCELLigence RTCA MP仪器进行，实验步骤依据仪器说明书进行。ACEA xCELLigence RTCAMP原理为对附着于孔底的肿瘤细胞以电阻指数为数据每15分钟记录一次，通过电阻指数判断贴壁的靶细胞的增殖或者死亡情况。利用电阻指数分析结果公式为：CAR-T细胞杀伤率＝基线电阻指数-实时电阻指数。
本发明实施例中，PSCA-28BBZ-G4H-28TM+SIRPγ-28或PSCA-28BBZ-7H-28TM+SIRPγ-28指的是将PSCA-28BBZ-G4H-28TM或PSCA-28BBZ-7H-28TM和SIRPγ-28与融合蛋白SIRPγ-28分别表达于两个载体转染免疫细胞获得的产品。PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28或PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28指的是将PSCA-28BBZ-G4H-28TM或PSCA-28BBZ-7H-28TM和SIRPγ-28与融合蛋白SIRPγ-28共同表达于一个载体转染免疫细胞获得的产品。
实施例1质粒构建及感染T细胞
(1)PBKL1-5H1P-CEA-OPRE构建
慢病毒表达载体、PSCA ScFv-G4H铰链区-CD28跨膜区-CD28-CD137-CD3ξ-P2A-SIRPγ-28(PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28)的CAR结构利用双酶切分别切割并回收片段，基因片段进行连接、转化并挑单克隆。
慢病毒表达载体、PSCA ScFv-7H铰链区-CD28跨膜区-CD28-CD137-CD3ξ-P2A-SIRPγ-28(PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28)的CAR结构利用双酶切分别切割并回收片段，基因片段进行连接、转化并挑单克隆。
(2)感染T细胞
将获得的质粒感染T细胞，得CAR-T细胞。
实施例2IFN-γ因子分泌情况
以Control T为对照组，实验组分别设置PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28、PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28、PSCA-28BBZ-G4H-28TM+SIRPγ-28、PSCA-28BBZ-7H-28TM+SIRPγ-28、PSCA-28BBZ-G4H-28TM、PSCA-28BBZ-7H-28TM，以RT4-Luc-GFP(PSCA阳性)为靶细胞，通过体外因子分泌验证体外有效性。结果如表1-表6及图1-图6所示。采用一个共表达的载体转染免疫细胞获得的产品PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28或PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28与采用两个分别表达的载体共转染免疫细胞获得的产品PSCA-28BBZ-G4H-28TM+SIRPγ-28或PSCA-28BBZ-7H-28TM+SIRPγ-28的IFN-γ因子分泌显著高于对照组和PSCA-28BBZ-G4H-28TM组或PSCA-28BBZ-7H-28TM组，对阴性细胞无杀伤
表1 PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28分泌IFN-γ因子情况
结构 IFN-γ(pg/ml)
PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28 4060.33
Control T 234.23
表2 PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28分泌IFN-γ因子情况
结构 IFN-γ(pg/ml)
PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28 4024.67
Control T 234.23
表3 PSCA-28BBZ-G4H-28TM+SIRPγ-28分泌IFN-γ因子情况
结构 IFN-γ(pg/ml)
PSCA-28BBZ-G4H-28TM+SIRPγ-28 6862.33
Control T 234.23
表4 PSCA-28BBZ-7H-28TM+SIRPγ-28分泌IFN-γ因子情况
结构 IFN-γ(pg/ml)
PSCA-28BBZ-7H-28TM+SIRPγ-28 8550.67
Control T 234.23
表5 PSCA-28BBZ-G4H-28TM、PSCA-28BBZ-G4H-28TM+SIRPγ-28分泌IFN-γ因子情况
结构 IFN-γ(pg/ml)
PSCA-28BBZ-G4H-28TM 6998.67
PSCA-28BBZ-G4H-28TM+SIRPγ-28 6862.33
Control T 234.23
表6 PSCA-28BBZ-7H-28TM、PSCA-28BBZ-7H-28TM+SIRPγ-28分泌IFN-γ因子情况
结构 IFN-γ(pg/ml)
PSCA-28BBZ-7H-28TM 6660.33
PSCA-28BBZ-7H-28TM+SIRPγ-28 8550.67
Control T 234.23
实施例3细胞杀伤情况
实验组分别设置PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28、PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28、PSCA-28BBZ-G4H-28TM+SIRPγ-28、PSCA-28BBZ-7H-28TM+SIRPγ-28、PSCA-28BBZ-G4H-28TM、PSCA-28BBZ-7H-28TM，分别以RT4-Luc-GFP(PSCA阳性)、PC-3-Luc-GFP(PSCA阴性)为靶细胞，结果如表7-表12及图7-图12所示。采用一个共表达的载体转染免疫细胞获得的产品PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28或PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28与采用两个分别表达的载体共转染免疫细胞获得的产品PSCA-28BBZ-G4H-28TM+SIRPγ-28或PSCA-28BBZ-7H-28TM+SIRPγ-28的体外杀伤显著高于对照组和PSCA-28BBZ-G4H-28TM组或PSCA-28BBZ-7H-28TM组，对阴性细胞无杀伤。
表7 PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28分别在阳性/阴性细胞中细胞杀伤情况
表8 PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28分别在阳性/阴性细胞中细胞杀伤情况
表9 PSCA-28BBZ-G4H-28TM+SIRPγ-28分别在阳性/阴性细胞中细胞杀伤情况
表10 PSCA-28BBZ-7H-28TM+SIRPγ-28分别在阳性/阴性细胞中细胞杀伤情况
表11 PSCA-28BBZ-G4H-28TM、PSCA-28BBZ-G4H-28TM+SIRPγ-28中细胞杀伤情况
表12 PSCA-28BBZ-7H-28TM、PSCA-28BBZ-7H-28TM+SIRPγ-28中细胞杀伤情况
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的宗旨和范围，其均应涵盖在本发明的权利要求范围当中。
序列表
<110> 重庆精准生物技术有限公司、重庆精准生物产业技术研究院有限公司
<120> 融合蛋白及其靶向PSCA的CAR及其应用
<130> 2021-3-4
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 696
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Asp Ile Gln Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala
1 5 10 15
Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val
20 25 30
Arg Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg
35 40 45
Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe
50 55 60
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
65 70 75 80
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Ser
85 90 95
Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Ser Thr
100 105 110
Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Ser
115 120 125
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
130 135 140
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr
145 150 155 160
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
165 170 175
Ala Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Phe Val Pro Lys Phe
180 185 190
Gln Gly Arg Ala Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
195 200 205
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
210 215 220
Lys Thr Gly Gly Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
225 230 235 240
Leu Glu Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
245 250 255
Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
260 265 270
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
275 280 285
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
290 295 300
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
305 310 315 320
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
325 330 335
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
340 345 350
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
355 360 365
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
370 375 380
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
385 390 395 400
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
405 410 415
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
420 425 430
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
435 440 445
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
450 455 460
Leu Ser Leu Ser Leu Gly Lys Glu Phe Phe Trp Val Leu Val Val Val
465 470 475 480
Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile
485 490 495
Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr
500 505 510
Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln
515 520 525
Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Val Lys Arg
530 535 540
Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro
545 550 555 560
Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu
565 570 575
Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala
580 585 590
Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu
595 600 605
Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
610 615 620
Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu
625 630 635 640
Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser
645 650 655
Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly
660 665 670
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
675 680 685
His Met Gln Ala Leu Pro Pro Arg
690 695
<210> 2
<211> 503
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Asp Ile Gln Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala
1 5 10 15
Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val
20 25 30
Arg Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg
35 40 45
Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe
50 55 60
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
65 70 75 80
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Ser
85 90 95
Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Ser Thr
100 105 110
Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Ser
115 120 125
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
130 135 140
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr
145 150 155 160
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
165 170 175
Ala Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Phe Val Pro Lys Phe
180 185 190
Gln Gly Arg Ala Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
195 200 205
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
210 215 220
Lys Thr Gly Gly Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
225 230 235 240
Leu Glu Ala Pro Pro Arg Ala Ser Ala Leu Pro Ala Pro Pro Thr Gly
245 250 255
Ser Ala Leu Pro Asp Pro Gln Thr Ala Ser Ala Leu Pro Asp Pro Pro
260 265 270
Ala Ala Ser Ala Leu Pro Glu Phe Phe Trp Val Leu Val Val Val Gly
275 280 285
Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile
290 295 300
Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met
305 310 315 320
Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro
325 330 335
Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Val Lys Arg Gly
340 345 350
Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val
355 360 365
Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu
370 375 380
Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp
385 390 395 400
Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn
405 410 415
Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg
420 425 430
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly
435 440 445
Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
450 455 460
Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu
465 470 475 480
Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His
485 490 495
Met Gln Ala Leu Pro Pro Arg
500
<210> 3
<211> 27
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
1 5 10 15
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val
20 25
<210> 4
<211> 24
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
1 5 10 15
Ser Leu Val Ile Thr Leu Tyr Cys
20
<210> 5
<211> 362
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Met Pro Val Pro Ala Ser Trp Pro His Pro Pro Gly Pro Phe Leu Leu
1 5 10 15
Leu Thr Leu Leu Leu Gly Leu Thr Glu Val Ala Gly Glu Glu Glu Leu
20 25 30
Gln Met Ile Gln Pro Glu Lys Leu Leu Leu Val Thr Val Gly Lys Thr
35 40 45
Ala Thr Leu His Cys Thr Val Thr Ser Leu Leu Pro Val Gly Pro Val
50 55 60
Leu Trp Phe Arg Gly Val Gly Pro Gly Arg Glu Leu Ile Tyr Asn Gln
65 70 75 80
Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Leu Thr Lys
85 90 95
Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Ser Ile Thr Pro Ala
100 105 110
Asp Val Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser Pro Glu
115 120 125
Asn Val Glu Phe Lys Ser Gly Pro Gly Thr Glu Met Ala Leu Gly Ala
130 135 140
Lys Pro Ser Ala Pro Val Val Leu Gly Pro Ala Ala Arg Thr Thr Pro
145 150 155 160
Glu His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro Arg
165 170 175
Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp Phe
180 185 190
Gln Thr Asn Val Asp Pro Thr Gly Gln Ser Val Ala Tyr Ser Ile Arg
195 200 205
Ser Thr Ala Arg Val Val Leu Asp Pro Trp Asp Val Arg Ser Gln Val
210 215 220
Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg Gly
225 230 235 240
Thr Ala Asn Leu Ser Glu Ala Ile Arg Val Pro Pro Thr Leu Glu Val
245 250 255
Thr Gln Gln Pro Met Arg Val Gly Asn Gln Val Asn Val Thr Cys Gln
260 265 270
Val Arg Lys Phe Tyr Pro Gln Ser Leu Gln Leu Thr Trp Ser Glu Asn
275 280 285
Gly Asn Val Cys Gln Arg Glu Thr Ala Ser Thr Leu Thr Glu Asn Lys
290 295 300
Asp Gly Thr Tyr Asn Trp Thr Ser Trp Phe Leu Val Asn Ile Ser Asp
305 310 315 320
Gln Arg Asp Asp Val Val Leu Thr Cys Gln Val Lys His Asp Gly Gln
325 330 335
Leu Ala Val Ser Lys Arg Leu Ala Leu Glu Val Thr Val His Gln Lys
340 345 350
Asp Gln Ser Ser Asp Ala Thr Pro Leu Glu
355 360
<210> 6
<211> 430
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Met Pro Val Pro Ala Ser Trp Pro His Pro Pro Gly Pro Phe Leu Leu
1 5 10 15
Leu Thr Leu Leu Leu Gly Leu Thr Glu Val Ala Gly Glu Glu Glu Leu
20 25 30
Gln Met Ile Gln Pro Glu Lys Leu Leu Leu Val Thr Val Gly Lys Thr
35 40 45
Ala Thr Leu His Cys Thr Val Thr Ser Leu Leu Pro Val Gly Pro Val
50 55 60
Leu Trp Phe Arg Gly Val Gly Pro Gly Arg Glu Leu Ile Tyr Asn Gln
65 70 75 80
Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Leu Thr Lys
85 90 95
Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Ser Ile Thr Pro Ala
100 105 110
Asp Val Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser Pro Glu
115 120 125
Asn Val Glu Phe Lys Ser Gly Pro Gly Thr Glu Met Ala Leu Gly Ala
130 135 140
Lys Pro Ser Ala Pro Val Val Leu Gly Pro Ala Ala Arg Thr Thr Pro
145 150 155 160
Glu His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro Arg
165 170 175
Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp Phe
180 185 190
Gln Thr Asn Val Asp Pro Thr Gly Gln Ser Val Ala Tyr Ser Ile Arg
195 200 205
Ser Thr Ala Arg Val Val Leu Asp Pro Trp Asp Val Arg Ser Gln Val
210 215 220
Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg Gly
225 230 235 240
Thr Ala Asn Leu Ser Glu Ala Ile Arg Val Pro Pro Thr Leu Glu Val
245 250 255
Thr Gln Gln Pro Met Arg Val Gly Asn Gln Val Asn Val Thr Cys Gln
260 265 270
Val Arg Lys Phe Tyr Pro Gln Ser Leu Gln Leu Thr Trp Ser Glu Asn
275 280 285
Gly Asn Val Cys Gln Arg Glu Thr Ala Ser Thr Leu Thr Glu Asn Lys
290 295 300
Asp Gly Thr Tyr Asn Trp Thr Ser Trp Phe Leu Val Asn Ile Ser Asp
305 310 315 320
Gln Arg Asp Asp Val Val Leu Thr Cys Gln Val Lys His Asp Gly Gln
325 330 335
Leu Ala Val Ser Lys Arg Leu Ala Leu Glu Val Thr Val His Gln Lys
340 345 350
Asp Gln Ser Ser Asp Ala Thr Pro Leu Glu Phe Trp Val Leu Val Val
355 360 365
Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe
370 375 380
Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp
385 390 395 400
Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr
405 410 415
Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser
420 425 430
<210> 7
<211> 428
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Pro Val Pro Ala Ser Trp Pro His Pro Pro Gly Pro Phe Leu Leu
1 5 10 15
Leu Thr Leu Leu Leu Gly Leu Thr Glu Val Ala Gly Glu Glu Glu Leu
20 25 30
Gln Met Ile Gln Pro Glu Lys Leu Leu Leu Val Thr Val Gly Lys Thr
35 40 45
Ala Thr Leu His Cys Thr Val Thr Ser Leu Leu Pro Val Gly Pro Val
50 55 60
Leu Trp Phe Arg Gly Val Gly Pro Gly Arg Glu Leu Ile Tyr Asn Gln
65 70 75 80
Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Leu Thr Lys
85 90 95
Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Ser Ile Thr Pro Ala
100 105 110
Asp Val Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser Pro Glu
115 120 125
Asn Val Glu Phe Lys Ser Gly Pro Gly Thr Glu Met Ala Leu Gly Ala
130 135 140
Lys Pro Ser Ala Pro Val Val Leu Gly Pro Ala Ala Arg Thr Thr Pro
145 150 155 160
Glu His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro Arg
165 170 175
Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp Phe
180 185 190
Gln Thr Asn Val Asp Pro Thr Gly Gln Ser Val Ala Tyr Ser Ile Arg
195 200 205
Ser Thr Ala Arg Val Val Leu Asp Pro Trp Asp Val Arg Ser Gln Val
210 215 220
Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg Gly
225 230 235 240
Thr Ala Asn Leu Ser Glu Ala Ile Arg Val Pro Pro Thr Leu Glu Val
245 250 255
Thr Gln Gln Pro Met Arg Val Gly Asn Gln Val Asn Val Thr Cys Gln
260 265 270
Val Arg Lys Phe Tyr Pro Gln Ser Leu Gln Leu Thr Trp Ser Glu Asn
275 280 285
Gly Asn Val Cys Gln Arg Glu Thr Ala Ser Thr Leu Thr Glu Asn Lys
290 295 300
Asp Gly Thr Tyr Asn Trp Thr Ser Trp Phe Leu Val Asn Ile Ser Asp
305 310 315 320
Gln Arg Asp Asp Val Val Leu Thr Cys Gln Val Lys His Asp Gly Gln
325 330 335
Leu Ala Val Ser Lys Arg Leu Ala Leu Glu Val Thr Val His Gln Lys
340 345 350
Asp Gln Ser Ser Asp Ala Thr Pro Leu Glu Ile Tyr Ile Trp Ala Pro
355 360 365
Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu
370 375 380
Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro
385 390 395 400
Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys
405 410 415
Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
420 425
<210> 8
<211> 1290
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atgcctgtcc cagcctcctg gccccatcct cctggtcctt tcctgcttct gactctactg 60
ctgggactta cagaagtggc aggtgaggag gagctacaga tgattcagcc tgagaagctc 120
ctgttggtca cagttggaaa gacagccact ctgcactgca ctgtgacctc cctgcttccc 180
gtgggacccg tcctgtggtt cagaggagtt ggaccaggcc gggaattaat ctacaatcaa 240
aaagaaggcc acttccccag ggtaacaaca gtttcagacc tcacaaagag aaacaacatg 300
gacttttcca tccgcatcag tagcatcacc ccagcagatg tcggcacata ctactgtgtg 360
aagtttcgaa aagggagccc tgagaacgtg gagtttaagt ctggaccagg cactgagatg 420
gctttgggtg ccaaaccctc tgcccccgtg gtattgggcc ctgcggcgag gaccacacct 480
gagcatacag tgagtttcac ctgtgagtcc catggcttct ctcccagaga catcaccctg 540
aaatggttca aaaatgggaa tgagctctca gacttccaga ccaacgtgga ccccacagga 600
cagagtgtgg cctacagcat ccgcagcaca gccagggtgg tactggaccc ctgggacgtt 660
cgctctcagg tcatctgcga ggtggcccat gtcaccttgc agggggaccc tcttcgtggg 720
actgccaact tgtctgaggc catccgagtt ccacccacct tggaggttac tcaacagccc 780
atgagggtgg ggaaccaggt aaacgtcacc tgccaggtga ggaagttcta cccccagagc 840
ctacagctga cctggtcgga gaatggaaac gtgtgccaga gagaaacagc ctcgaccctt 900
acagagaaca aggatggtac ctacaactgg acaagctggt tcctggtgaa catatctgac 960
caaagggatg atgtggtcct cacctgccag gtgaagcatg atgggcagct ggcggtcagc 1020
aaacgccttg ccctagaggt cacagtccac cagaaggacc agagctcaga tgctacccct 1080
ctcgagttct gggtgctggt cgtggtgggt ggcgtgctgg cctgctacag cctgctggtg 1140
acagtggcct tcatcatctt ttgggtgagg agcaagcgga gcagaggcgg ccacagcgac 1200
tacatgaaca tgactccccg ccgccccggg cccacccgca agcattacca gccctatgcc 1260
ccaccacgcg acttcgcagc ctatcgctcc 1290
<210> 9
<211> 1284
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
atgcctgtcc cagcctcctg gccccatcct cctggtcctt tcctgcttct gactctactg 60
ctgggactta cagaagtggc aggtgaggag gagctacaga tgattcagcc tgagaagctc 120
ctgttggtca cagttggaaa gacagccact ctgcactgca ctgtgacctc cctgcttccc 180
gtgggacccg tcctgtggtt cagaggagtt ggaccaggcc gggaattaat ctacaatcaa 240
aaagaaggcc acttccccag ggtaacaaca gtttcagacc tcacaaagag aaacaacatg 300
gacttttcca tccgcatcag tagcatcacc ccagcagatg tcggcacata ctactgtgtg 360
aagtttcgaa aagggagccc tgagaacgtg gagtttaagt ctggaccagg cactgagatg 420
gctttgggtg ccaaaccctc tgcccccgtg gtattgggcc ctgcggcgag gaccacacct 480
gagcatacag tgagtttcac ctgtgagtcc catggcttct ctcccagaga catcaccctg 540
aaatggttca aaaatgggaa tgagctctca gacttccaga ccaacgtgga ccccacagga 600
cagagtgtgg cctacagcat ccgcagcaca gccagggtgg tactggaccc ctgggacgtt 660
cgctctcagg tcatctgcga ggtggcccat gtcaccttgc agggggaccc tcttcgtggg 720
actgccaact tgtctgaggc catccgagtt ccacccacct tggaggttac tcaacagccc 780
atgagggtgg ggaaccaggt aaacgtcacc tgccaggtga ggaagttcta cccccagagc 840
ctacagctga cctggtcgga gaatggaaac gtgtgccaga gagaaacagc ctcgaccctt 900
acagagaaca aggatggtac ctacaactgg acaagctggt tcctggtgaa catatctgac 960
caaagggatg atgtggtcct cacctgccag gtgaagcatg atgggcagct ggcggtcagc 1020
aaacgccttg ccctagaggt cacagtccac cagaaggacc agagctcaga tgctacccct 1080
ctcgagatct acatctgggc gcccttggcc gggacttgtg gggtccttct cctgtcactg 1140
gttatcaccc tttactgcaa acggggcaga aagaaactcc tgtatatatt caaacaacca 1200
tttatgagac cagtacaaac tactcaagag gaagatggct gtagctgccg atttccagaa 1260
gaagaagaag gaggatgtga actg 1284
<210> 10
<211> 1509
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
gacatccagg atatccagct gacacagtcc cctagctccc tgagcgcctc cgtgggcgac 60
agggtgacca tcacatgcag cgcctctagc tccgtgagat tcatccactg gtaccagcag 120
aagcccggca aggcccctaa gaggctgatc tatgatacct ctaagctggc cagcggagtg 180
ccctcccgct tctctggcag cggctccggc accgacttta ccctgacaat ctctagcctg 240
cagccagagg attttgccac atactattgc cagcagtggt cctctagccc cttcaccttt 300
ggccagggca caaaggtgga gatcaaggga tctaccagcg gatccggcaa gcctggatct 360
ggagagggca gcacaaaggg ctccgaggtg cagctggtgg agtccggagg aggactggtg 420
cagccaggag gatctctgag gctgagctgt gcagcctccg gcttcaacat caaggactac 480
tatatccact gggtgagaca ggcacctggc aagggactgg agtgggtggc atggatcgac 540
cctgagaatg gcgataccga gttcgtgcca aagtttcagg gcagggccac aatctctgcc 600
gacaccagca agaacacagc ctacctgcag atgaatagcc tgcgcgccga ggataccgcc 660
gtgtactatt gtaagacagg aggattttgg ggacagggca ccctggtgac agtgtcctct 720
ctcgaggcac cacctcgggc cagcgccctg cctgcaccac ccaccggctc cgccctgcca 780
gaccctcaga cagcatctgc cctgccagat cctccagcag caagcgccct gcccgaattc 840
ttctgggtgc tggtcgtggt gggtggcgtg ctggcctgct acagcctgct ggtgacagtg 900
gccttcatca tcttttgggt gaggagcaag cggagcagag gcggccacag cgactacatg 960
aacatgactc cccgccgccc cgggcccacc cgcaagcatt accagcccta tgccccacca 1020
cgcgacttcg cagcctatcg ctccgttaaa cggggcagaa agaaactcct gtatatattc 1080
aaacaaccat ttatgagacc agtacaaact actcaagagg aagatggctg tagctgccga 1140
tttccagaag aagaagaagg aggatgtgaa ctgagagtga agttcagcag gagcgcagac 1200
gcccccgcgt accagcaggg ccagaaccag ctctataacg agctcaatct aggacgaaga 1260
gaggagtacg atgttttgga caagagacgt ggccgggacc ctgagatggg gggaaagccg 1320
agaaggaaga accctcagga aggcctgtac aatgaactgc agaaagataa gatggcggag 1380
gcctacagtg agattgggat gaaaggcgag cgccggaggg gcaaggggca cgatggcctt 1440
taccagggtc tcagtacagc caccaaggac acctacgacg cccttcacat gcaggccctg 1500
ccccctcgc 1509
<210> 11
<211> 2088
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gacatccagg atatccagct gacacagtcc cctagctccc tgagcgcctc cgtgggcgac 60
agggtgacca tcacatgcag cgcctctagc tccgtgagat tcatccactg gtaccagcag 120
aagcccggca aggcccctaa gaggctgatc tatgatacct ctaagctggc cagcggagtg 180
ccctcccgct tctctggcag cggctccggc accgacttta ccctgacaat ctctagcctg 240
cagccagagg attttgccac atactattgc cagcagtggt cctctagccc cttcaccttt 300
ggccagggca caaaggtgga gatcaaggga tctaccagcg gatccggcaa gcctggatct 360
ggagagggca gcacaaaggg ctccgaggtg cagctggtgg agtccggagg aggactggtg 420
cagccaggag gatctctgag gctgagctgt gcagcctccg gcttcaacat caaggactac 480
tatatccact gggtgagaca ggcacctggc aagggactgg agtgggtggc atggatcgac 540
cctgagaatg gcgataccga gttcgtgcca aagtttcagg gcagggccac aatctctgcc 600
gacaccagca agaacacagc ctacctgcag atgaatagcc tgcgcgccga ggataccgcc 660
gtgtactatt gtaagacagg aggattttgg ggacagggca ccctggtgac agtgtcctct 720
ctcgaggaga gcaagtacgg ccctccctgc cccccttgcc ctgcccccga gttcctgggc 780
ggacccagcg tgttcctgtt cccccccaag cccaaggaca ccctgatgat cagccggacc 840
cccgaggtga cctgtgtggt ggtggacgtg tcccaggagg accccgaggt ccagttcaac 900
tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agccccggga ggagcagttc 960
aatagcacct accgggtggt gtccgtgctg accgtgctgc accaggactg gctgaacggc 1020
aaggaataca agtgtaaggt gtccaacaag ggcctgccca gcagcatcga gaaaaccatc 1080
agcaaggcca agggccagcc tcgggagccc caggtgtaca ccctgccccc tagccaagag 1140
gagatgacca agaatcaggt gtccctgacc tgcctggtga agggcttcta ccccagcgac 1200
atcgccgtgg agtgggagag caacggccag cccgagaaca actacaagac caccccccct 1260
gtgctggaca gcgacggcag cttcttcctg tacagcaggc tgaccgtgga caagagccgg 1320
tggcaggagg gcaacgtctt tagctgctcc gtgatgcacg aggccctgca caaccactac 1380
acccagaaga gcctgtccct gagcctgggc aaggaattct tctgggtgct ggtcgtggtg 1440
ggtggcgtgc tggcctgcta cagcctgctg gtgacagtgg ccttcatcat cttttgggtg 1500
aggagcaagc ggagcagagg cggccacagc gactacatga acatgactcc ccgccgcccc 1560
gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 1620
tccgttaaac ggggcagaaa gaaactcctg tatatattca aacaaccatt tatgagacca 1680
gtacaaacta ctcaagagga agatggctgt agctgccgat ttccagaaga agaagaagga 1740
ggatgtgaac tgagagtgaa gttcagcagg agcgcagacg cccccgcgta ccagcagggc 1800
cagaaccagc tctataacga gctcaatcta ggacgaagag aggagtacga tgttttggac 1860
aagagacgtg gccgggaccc tgagatgggg ggaaagccga gaaggaagaa ccctcaggaa 1920
ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg 1980
aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc 2040
accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgc 2088

Claims (7)

1.一种肿瘤免疫抑制抵抗型 CAR，其特征在于，包含融合蛋白和CAR结构a，所述CAR结构a包含识别PSCA的胞外段、hinge区、跨膜区和胞内信号区；所述融合蛋白为SIRPγ融合蛋白，所述SIRPγ融合蛋白结构包含胞外段、跨膜结构和胞内信号区，所述SIRPγ的胞外段氨基酸序列如SEQ ID NO:5所示；所述SIRPγ融合蛋白的结构为SIRPγ- CD28TM-CD28或SIRPγ- CD8TM-4-1BB； SIRPγ融合蛋白SIRPγ- CD28TM-CD28的氨基酸序列如SEQ ID NO:6所示；或SIRPγ融合蛋白SIRPγ- CD8TM-4-1BB的氨基酸序列如SEQ ID NO:7所示。
2.根据权利要求1所述的肿瘤免疫抑制抵抗型 CAR，其特征在于，编码所述SIRPγ融合蛋白SIRPγ- CD28TM-CD28的核苷酸序列如SEQ ID NO:8所示；或编码所述SIRPγ融合蛋白SIRPγ- CD8TM-4-1BB的核苷酸序列如SEQ ID NO:9所示。
3.根据权利要求1所述的肿瘤免疫抑制抵抗型CAR，其特征在于，所述CAR结构a包括PSCA单链抗体、铰链区、CD28跨膜区、CD28、CD137和CD3ξ三刺激信号；所述铰链区为G4H或7H。
4.根据权利要求3所述的肿瘤免疫抑制抵抗型CAR，其特征在于，所述CAR结构a氨基酸序列如SEQ ID NO：1所示；或如SEQ ID NO：2所示。
5.一种制备免疫细胞的方法，其特征在于，免疫细胞包含分离的核酸分子，分离的核酸分子编码权利要求2或3所述的肿瘤免疫抑制抵抗型 CAR，所述核酸序列包含如SEQ ID NO：10或SEQ ID NO：11所示的序列；将CAR结构a和所述SIRPγ融合蛋白共同表达于一个载体转染免疫细胞；或将CAR结构a和所述SIRPγ融合蛋白分别表达于两个载体转染免疫细胞。
6.权利要求1、2、3或4所述的肿瘤免疫抑制抵抗型CAR在制备治疗肿瘤的药物中的应用，所述肿瘤为前列腺癌或膀胱癌。
7.一种药物组合物，其特征在于，所述药物组合物包括：权利要求1、2、3或4所述的肿瘤免疫抑制抵抗型CAR和可增强CAR表达活性的活性剂和/或治疗剂。
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