隐形嵌合抗原受体及其减少对正常细胞的细胞毒性的用途
文献发布时间:2023-06-19 19:28:50
本申请要求于2020年8月19日提交的标题为“靶标依赖性开关嵌合抗原受体”的美国临时专利申请号63/067,870的权益;将该专利的内容通过援引以其全文并入本文。
技术领域
本申请涉及生物制药领域,并且特别地涉及隐形嵌合抗原受体(CAR)及其相关多核苷酸、载体、组合物、制备方法和用途。
背景技术
CAR T细胞疗法在血液恶性肿瘤治疗中的初步成功预示了免疫治疗性治疗策略的新领域的快速发展。CAR是设计受体,其典型地具有衍生自CD3ζITAM基序的模块化激活结构域和例如来自4-1BB或CD28的共刺激结构域。由这些受体进行的靶标识别通常通过CAR的细胞外末端的scFv结构域提供,尽管也设计了可替代方法。
安全性是阻碍CAR-T疗法发展的主要障碍。CAR分子通常组成型地表达在T细胞表面上。因此,当将CAR-T细胞注射到患者体内时,它们将迁移到表达靶抗原的不同组织,通常对正常组织以及肿瘤细胞具有非预期的毒性和损害。此外,CAR分子的组成型表达促进紧张性信号传导和T细胞耗竭。
迄今为止,已经开发了几种类型的开关来控制CAR-T活性。这些开关中的一些开关是基于将“自杀基因”掺入到CAR构建体中。例如,已将HSV-TK转基因用于过继细胞疗法以允许在用更昔洛韦治疗时经修饰细胞的诱导型细胞凋亡。另一方法依赖于使用诱导型半胱天冬酶9(iCasp9)。在这些策略中,通过靶向消除CAR-T细胞本身来终止治疗性应答。
其他开关系统依赖于受体的修饰,例如,衔接子介导的CAR(也称为“通用CAR”)。这些CAR T细胞共享共同的受体,该受体可以经修饰以通过使该受体与结合CAR和肿瘤靶标两者的衔接子分子连接来识别多种肿瘤靶标。类似地,已经提出了经由药物诱导的二聚体将细胞内信号传导结构域募集到CAR的跨膜组分,作为控制给药和激活的手段。虽然目前临床上批准的CAR被设计为具有组成型活性,但衔接子依赖性CAR T细胞只能在施用衔接子时识别和杀伤,从而允许对CAR-T细胞进行可滴定的和可逆控制。
对于这些类型的开关,CAR的表达和功能性是通过应用小分子药物或其他治疗剂进行外部调节的。这些类型的开关的主要挑战包括:可以应用的现有FDA批准的开关分子的可用性、开关分子的安全性特性、生物利用度、生物分布、以及由于开关分子和CAR-T细胞的复杂动力学和分布而导致的体内调节复杂性。
通过靶向单个肿瘤相关抗原(TAA)对靶细胞群产生的选择性压力最终导致靶抗原的下调和CAR T细胞免疫应答的逃逸
高亲和力IL2受体是一种跨膜受体,其由三种不同的非共价缔合的组分构成:α、β和γ链。已经表明,在α和γ亚基不存在的情况下,β链被组成型地内吞并降解
LDLR的C-末端残基的分析表明存在内体分选基序NDXY。研究表明,该基序通过与ARH的相互作用和与AP-2复合物的结合介导间接内体分选机制
本申请旨在通过提供用于减少对正常细胞的细胞毒性的隐形CAR来解决CAR-T的安全性问题。
发明内容
在本申请的新颖的CAR系统中,CAR分子的表达/稳定化取决于靶抗原的可用性。当没有可用的抗原时,T细胞表面上没有可检测的CAR,并且没有CAR功能。当工程化的T细胞识别其靶抗原时,CAR被稳定化并且可以启动信号传导。这是一种靶标依赖性开关。通过调节应答抗原的CAR表达,本申请可以减少CAR T细胞毒性和耗竭。由于CAR在没有抗原的情况下是“不可见”的,因此本申请的发明人选择将其命名为“隐形CAR”。隐形CAR可以应用于单特异性和双特异性CAR的情况中、以及自体和同种异体的情况中。它可以在T细胞、NK细胞、γδT细胞、和iPSC衍生的T细胞的背景下应用。
内吞隐形CAR系统也可以应用于解决CAR T疗法面临的特定挑战,即“中靶、脱肿瘤”效应。由于TAA在肿瘤组织上通常会上调,但在正常组织上也以较低水平表达,因此,CART疗法可能具有靶向非癌细胞的不想要的效应。通过使用由抗原表达稳定的CAR,表达高水平靶抗原的细胞(例如,肿瘤细胞)为我们的CAR系统提供了更强的激活。因此,本申请可以促进CAR T细胞的肿瘤特异性杀伤,同时最大程度减少对正常组织的影响(图2)。在组合模型中,两种CAR都可以是内吞类型的,最大程度减少中靶、脱肿瘤效应,并解决了抗原逃逸。
通过在双重特异性的情况中应用隐形CAR,本申请预期该系统可用于解决抗原逃逸问题。通过在细胞表面处表达仅瞬时稳定的CAR变体,本申请提供了减少附带靶向和损伤正常组织的机制。肿瘤细胞上次级靶抗原的上调提供了更强的信号并促进CAR表面稳定性和下游激活。通过用一种CAR靶向初级TAA并用第二隐形CAR靶向次级TAA,本申请可以引起强烈的初始肿瘤应答并且仅在次级TAA上调时才引起靶向的次级应答。
本申请的发明人设计并制备了几种具有修饰的跨膜和近膜序列的CAR变体,这些序列促进受体的细胞内运输。这些序列衍生自IL2受体β链(IL2Rβ或IL2Rb)、低密度脂蛋白受体(LDLR)和人癫痫发作6样蛋白2(SEZ6L2)。这些受体的跨膜和细胞内序列中的氨基酸基序经由内体途径促进细胞内运输。
本申请提供了新颖的隐形CAR,用于减少对正常细胞的细胞毒性和改善CAR-T安全性、以及减少由组成型CAR表面表达引起的炎症性细胞因子和紧张性信号传导。本申请还提供了包含隐形CAR和第二CAR的双重CAR,以及包含编码隐形CAR或双重CAR的多核苷酸的相关核酸、载体、宿主细胞和药物组合物。本申请进一步提供了一种在有需要的受试者中治疗疾病的方法,该方法包括向该受试者施用有效量的药物组合物;一种减少CAR-T细胞对正常细胞的细胞毒性的方法;以及一种产生对正常细胞的细胞毒性减少的CAR-T细胞的方法。
在一个方面,本申请提供了一种嵌合抗原受体(CAR),该嵌合抗原受体包含:
(1)细胞外配体结合结构域,其包含与预定抗原特异性结合的单链可变片段(scFv);其中优选地,该预定抗原是肿瘤相关抗原(TAA);更优选地,该TAA选自以下中的一种或多种:CEA、密封蛋白18.2、CGC3、受体酪氨酸激酶样孤儿受体1(ROR1)、CD38、CD19、CD20、CD22、BCMA、CAIX、CD446、CD13、EGFR、EGFRvIII、EpCam、GD2、EphA2、HER1、HER2、ICAM-1、IL13Ra2、间皮素、MUC1、MUC16、NKG2D、PSCA、NY-ESO-1、MART-1、WT1、MAGE-A10、MAGE-A3、MAGE-A4、EBV、PD1、PD-L1、CD25、IL-2、和CD3;
(2)跨膜(tm)连接近膜(jm)结构域;
其中该跨膜连接近膜结构域包含IL2受体β链(IL2Rβ)跨膜结构域和IL2Rβ近膜结构域,并且该跨膜连接近膜结构域与IL2Rβ降解序列(DT)相邻;其中优选地,该IL2Rβ降解序列在该跨膜连接近膜结构域的C-末端处;其中优选地,该IL2Rβ降解序列包含与由SEQ IDNO:2表示的氨基酸序列具有至少约95%、96%、97%、98%、99%或100%同一性的氨基酸序列;
其中该跨膜连接近膜结构域包含低密度脂蛋白受体(LDLR)跨膜结构域和LDLR近膜结构域;或
其中该跨膜连接近膜结构域包含癫痫发作6样蛋白2(SEZ6L2)跨膜结构域和SEZ6L2近膜结构域;
(3)细胞内结构域;其中优选地,该细胞内结构域包含信号传导结构域;更优选地,该信号传导结构域包含一个或多个选自由以下组成的组的信号传导结构域:4-1BB信号传导结构域、CD28信号传导结构域和CD3ζ信号传导结构域。
在另外的方面,CAR从N-末端到C-末端包含:TAA scFv-CD8铰链-IL2Rβtm jm DT-4-1BB-CD3ζ、TAA scFv-CD8铰链-IL2Rβtm jm DT-CD28-CD3ζ、TAA scFv-CD8铰链-LDLR tmjm-4-1BB-CD3ζ、或TAA scFv-CD8铰链-SEZ6L2 tm jm-4-1BB-CD3ζ。
在另外的方面,TAA scFv选自CEA scFv、密封蛋白18.2scFv和HER2 scFv中的一种或多种;更优选地,CEA scFv为MN14op CEA scFv,或密封蛋白18.2scFv是841密封蛋白18.2scFv;最优选地,MN14op CEA scFv、841密封蛋白18.2scFv、或HER2scFv包含与分别由SEQ ID NO.5、30或31表示的氨基酸序列具有至少约95%、96%、97%、98%、99%或100%同一性的氨基酸序列。
在另外的方面,CAR的N-末端进一步含有前导序列和/或HA序列。
在另外的方面,CAR从N-末端到C-末端包含:
HA-MN14op CEA scFv-CD8铰链-IL2Rβtm jm DT-4-1BB-CD3ζ、
HA-MN14op CEA scFv-CD8铰链-IL2Rβtm jm DT-4-1BB-CD3ζ-P2A-GFP、
HER2 scFv-CD8铰链-IL2Rβtm jm DT-4-1BB-CD3ζ-P2A-GFP、
HER2 scFv-CD8铰链-IL2Rβtm jm DT-CD28-CD3ζ-P2A-GFP、
MN14op CEA scFv-CD8铰链-IL2Rβtm jm DT-4-1BB-CD3ζ-P2A-GFP、
MN14op CEA scFv-CD8铰链-LDLR tm jm-4-1BB-CD3ζ-P2A-GFP、
MN14op CEA scFv-CD8铰链-SEZ6L2 tm jm-4-1BB-CD3ζ-P2A-GFP、
841密封蛋白18.2scFv-CD8铰链-SEZ6L2 tm jm-4-1BB-CD3ζ-P2A-GFP、或
HER2 scFv-CD8铰链-SEZ6L2 tm jm-4-1BB-CD3ζ-P2A-GFP,任选地该CAR不包括P2A-GFP和/或HA;
在另外的方面,CAR包含与分别由SEQ ID NO.9、14、15、17、18、19、21、22或24表示的氨基酸序列具有至少约95%、96%、97%、98%、99%或100%同一性的氨基酸序列,任选地该CAR的氨基酸序列不包括P2A-GFP、前导序列、和/或HA的氨基酸序列。
在另外的方面,CAR包含铰链结构域;优选地,该铰链结构域包含衍生自细胞外区域的CD8或CD28铰链、或IgG铰链。
在另一方面,本申请还提供了一种嵌合抗原受体(CAR),该嵌合抗原受体包含:
(1)细胞外配体结合结构域,其包含与预定抗原特异性结合的单链可变片段(scFv);其中优选地,该预定抗原是肿瘤相关抗原(TAA);更优选地,该TAA选自以下中的一种或多种:CEA、密封蛋白18.2、CGC3、受体酪氨酸激酶样孤儿受体1(ROR1)、CD38、CD19、CD20、CD22、BCMA、CAIX、CD446、CD13、EGFR、EGFRvIII、EpCam、GD2、EphA2、HER1、HER2、ICAM-1、IL13Ra2、间皮素、MUC1、MUC16、NKG2D、PSCA、NY-ESO-1、MART-1、WT1、MAGE-A10、MAGE-A3、MAGE-A4、EBV、NKG2D、PD1、PD-L1、CD25、IL-2、和CD3;
(2)跨膜结构域,以及
(3)包含IL2Rβ降解序列(DT)和至少一个信号传导结构域的细胞质区段;其中优选地,该IL2Rβ降解序列包含与由SEQ ID NO:2表示的氨基酸序列具有至少约95%、96%、97%、98%、99%或100%同一性的氨基酸序列;其中优选地,该IL2Rβ降解序列在该细胞质区段的C-末端处。
在另外的方面,CAR从N-末端到C-末端包含:TAA scFv-CD8铰链-CD8tm-4-1BB-CD3ζ-IL2RβDT。
在另外的方面,CAR的N-末端进一步含有前导序列和/或HA序列。
在另外的方面,该TAA scFv是CEA scFv;更优选地,该TAA scFv是MN14op CEAscFv;最优选地,MN14op CEA scFv包含与由SEQ ID NO.5表示的氨基酸序列具有至少约95%、96%、97%、98%、99%或100%同一性的氨基酸序列;最优选地,该CAR包含与分别由SEQ ID NO.8或16表示的氨基酸序列具有至少约95%、96%、97%、98%、99%或100%同一性的氨基酸序列,任选地该CAR的氨基酸序列不包括P2A-GFP、前导序列、和/或HA的氨基酸序列。
在另外的方面,IL2Rβ、LDLR、或SEZ6L2的跨膜(tm)包含与分别由SEQ ID NO.1、38或40表示的氨基酸序列具有至少约95%、96%、97%、98%、99%或100%同一性的氨基酸序列;
该IL2Rβ、LDLR、或SEZ6L2的近膜(jm)包含与分别由SEQ ID NO.4、39或41表示的氨基酸序列具有至少约95%、96%、97%、98%、99%或100%同一性的氨基酸序列;
该IL2Rβ、LDLR、或SEZ6L2的跨膜(tm)连接近膜(jm)包含与分别由SEQ IDNO.27、28或29表示的氨基酸序列具有至少约95%、96%、97%、98%、99%或100%同一性的氨基酸序列;
该MN14op CEA scFv、841密封蛋白18.2scFv、HER2 scFv、PD-L1 scFv、HA、CD8铰链、CD3ζ、4-1BB、CD28、CD8 tm、GFP、或前导序列包含与分别由SEQ IDNO.5、30、31、32、33、34、35、36、37、42、45或46表示的氨基酸序列具有至少约95%、96%、97%、98%、99%或100%同一性的氨基酸序列;并且/或者
该P2A包含与由SEQ ID NO.43或44表示的氨基酸序列具有至少约95%、96%、97%、98%、99%或100%同一性的氨基酸序列。
在另外的方面,本申请提供了一种嵌合抗原受体(CAR),该嵌合抗原受体包含:
(1)细胞外配体结合结构域,其包含与预定抗原特异性结合的单链可变片段(scFv);(2)跨膜(tm)连接近膜(jm)结构域;
其中该跨膜连接近膜结构域包含IL2受体β链(IL2Rβ)跨膜结构域和IL2Rβ近膜结构域,并且该跨膜连接近膜结构域与IL2Rβ降解序列(DT)相邻;其中优选地,该IL2Rβ降解序列在该跨膜连接近膜结构域的C-末端处;其中优选地,该IL2Rβ降解序列包含与由SEQ IDNO:2表示的氨基酸序列具有至少约95%、96%、97%、98%、99%或100%同一性的氨基酸序列;
其中该跨膜连接近膜结构域包含低密度脂蛋白受体(LDLR)跨膜结构域和LDLR近膜结构域;或
其中该跨膜连接近膜结构域包含人癫痫发作6样蛋白2(SEZ6L2)跨膜结构域和SEZ6L2近膜结构域;
该IL2Rβ、LDLR、或SEZ6L2的跨膜(tm)包含与分别由SEQ ID NO.1、38或40表示的氨基酸序列具有至少约95%、96%、97%、98%、99%或100%同一性的氨基酸序列;
该IL2Rβ、LDLR、或SEZ6L2的近膜(jm)包含与分别由SEQ ID NO.4、39或41表示的氨基酸序列具有至少约95%、96%、97%、98%、99%或100%同一性的氨基酸序列;
(3)细胞内结构域。
在另外的方面,本申请还提供了一种嵌合抗原受体(CAR),该嵌合抗原受体包含:
(1)细胞外配体结合结构域,其包含与预定抗原特异性结合的单链可变片段(scFv);
(2)跨膜结构域,以及
(3)包含IL2Rβ降解序列(DT)和至少一个信号传导结构域的细胞质区段;该IL2Rβ降解序列包含与由SEQ ID NO:2表示的氨基酸序列具有至少约95%、96%、97%、98%、99%或100%同一性的氨基酸序列;该IL2Rβ降解序列在该细胞质区段的C-末端处。
在特别的实施例中,从N-末端到C-末端包含IL2Rβ跨膜(tm)、近膜(jm)和降解序列(DT)的CAR包含:
(1)MLB003 CAR:HA-MN14op CEA scFv-CD8铰链-IL2Rβtm jm DT-4-1BB-CD3ζ(由SEQ ID NO.9表示的氨基酸序列);
(2)MLB013 CAR:HA-MN14op CEA scFv-CD8铰链-IL2Rβtm jm DT-4-1BB-CD3ζ-P2A-GFP(由SEQ ID NO.15表示的氨基酸序列);
(3)MLB020 CAR:MN14op CEA scFv-CD8铰链-IL2Rβtm jm DT-4-1BB-CD3ζ-P2A-GFP(由SEQ ID NO.14表示的氨基酸序列);
(4)MLB038 CAR:HER2 scFv-CD8铰链-IL2Rβtm jm DT-4-1BB-CD3ζ-P2A-GFP(由SEQ ID NO.21表示的氨基酸序列);或
(5)MLB039 CAR:HER2 scFv-CD8铰链-IL2Rβtm jm DT-CD28-CD3ζ-P2A-GFP(由SEQID NO.22表示的氨基酸序列);
任选地该CAR不包括P2A-GFP、前导序列、和/或HA。
在特别的实施例中,从N-末端到C-末端包含LDLR跨膜(tm)和近膜(jm)的CAR包含:
(1)MLB048 CAR:MN14op CEA scFv-CD8铰链-LDLR tm jm-4-1BB-CD3ζ-P2A-GFP(由SEQ ID NO.18表示的氨基酸序列);
任选地该CAR不包括P2A-GFP、前导序列、和/或HA。
在特别的实施例中,从N-末端到C-末端包含SEZ6L2跨膜(tm)和近膜(jm)的CAR包含:
(1)MLB047 CAR:MN14op CEA scFv-CD8铰链-SEZ6L2 tm jm-4-1BB-CD3ζ-P2A-GFP(由SEQ ID NO.17表示的氨基酸序列);
(2)MLB054 CAR:841密封蛋白18.2scFv-CD8铰链-SEZ6L2 tm jm-4-1BB-CD3ζ-P2A-GFP(由SEQ ID NO.19表示的氨基酸序列);或
(3)MLB080 CAR:HER2 scFv-CD8铰链-SEZ6L2 tm jm-4-1BB-CD3ζ-P2A-GFP(由SEQID NO.24表示的氨基酸序列);
任选地该CAR不包括P2A-GFP、前导序列、和/或HA。
在特别的实施例中,包含细胞质区段(其从N-末端到C-末端包含IL2Rβ降解序列(DT)和至少一个信号传导结构域)的CAR包含:
(1)MLB025 CAR:MN14op CEA scFv-CD8铰链-CD8 tm-4-1BB-CD3ζ-IL2RβDT-P2A-GFP(由SEQ ID NO.16表示的氨基酸序列);或
(2)MLB002 CAR:HA-MN14op scFv-CD8铰链-CD8 tm-4-1BB-CD3ζ-IL2RβDT(由SEQID NO.8表示的氨基酸序列);
任选地该CAR不包括P2A-GFP、前导序列、和/或HA。
在另一方面,本申请还提供了一种双重CAR,该双重CAR包含:根据上述实施例中任一个所述的第一CAR,以及
第二CAR,该第二CAR包含:
(1)细胞外配体结合结构域,其包含与预定抗原特异性结合的scFv;
(2)跨膜结构域;其中优选地,该跨膜结构域是CD8跨膜结构域;
(3)细胞内结构域;其中优选地,该细胞内结构域包含信号传导结构域;更优选地,该信号传导结构域包含一个或多个选自由以下组成的组的信号传导结构域:4-1BB信号传导结构域、CD28信号传导结构域和CD3ζ信号传导结构域;
其中该第一CAR靶向抗原,并且该第二CAR靶向另一抗原。
在另外的方面,第一CAR和第二CAR由P2A连接。
在另外的方面,该P2A包含与由SEQ ID NO.43或44表示的氨基酸序列具有至少约95%、96%、97%、98%、99%或100%同一性的氨基酸序列。
在另外的方面,双重CAR从N-末端到C-末端包含:
TAA scFv-CD8铰链-IL2Rβtm jm DT-4-1BB-CD3ζ-P2A-另一TAA scFv-CD8铰链-CD8 tm-CD28-CD3ζ、TAA scFv-CD8铰链-CD8 tm-4-1BB-CD3ζ-P2A-另一TAA scFv-CD8铰链-IL2Rβtm jm DT-CD28-CD3ζ、TAA scFv-CD8铰链-LDLR tm jm-4-1BB-CD3ζ-P2A-另一TAAscFv-CD8铰链-CD8 tm-CD28-CD3ζ、TAA scFv-CD8铰链-CD8 tm-4-1BB-CD3ζ-P2A-另一TAAscFv-CD8铰链-LDLR tm jm-CD28-CD3ζ、TAA scFv-CD8铰链-SEZ6L2 tm jm-4-1BB-CD3ζ-P2A-另一TAA scFv-CD8铰链-CD8tm-CD28、或TAA scFv-CD8铰链-CD8 tm-4-1BB-CD3ζ-P2A-另一TAA scFv-CD8铰链-SEZ6L2 tm jm-CD3ζ。
在另外的方面,双重CAR从N-末端到C-末端分别包含:841密封蛋白18.2scFv-CD8铰链-SEZ6L2 tm jm-4-1BB-CD3ζ-P2A-PD-L1 scFv-CD8铰链-CD8 tm-CD28-(G
在特别的实施例中,双重CAR从N-末端到C-末端包含:
(1)MLB055 CAR:841密封蛋白18.2scFv-CD8铰链-SEZ6L2 tm jm-4-1BB-CD3ζ-P2A-PD-L1 scFv-CD8铰链-CD8 tm-CD28-(G4S)2-GFP(由SEQ ID NO.20表示的氨基酸序列);
(2)MLB040 CAR:841密封蛋白18.2scFv-CD8铰链-CD8tm-4-1BB-CD3ζ-P2A-HER2scFv-IL2Rβtm jm DT-CD28-CD3ζ(由SEQ ID NO.25表示的氨基酸序列);或
(3)MLB108 CAR:841密封蛋白18.2scFv-CD8铰链-CD8tm-4-1BB-CD3ζ-P2A-HER2scFv-SEZ6L2 tm jm-CD3ζ(由SEQ ID NO.26表示的氨基酸序列);
任选地该双重CAR不包括(G
在另一方面,本申请还提供了包含编码上述CAR或双重CAR的多核苷酸的核酸。
在另一方面,本申请还提供了包含上述编码上述CAR或双重CAR的多核苷酸的载体。
在另一方面,本申请还提供了包含至少一种上述核酸或至少一种上述载体的组合物。
在另一方面,本申请还提供了一种宿主细胞,该宿主细胞包含本申请的一种或多种核酸或载体或组合物。
在另外的方面,宿主细胞是自体细胞或同种异体细胞。
在另外的方面,宿主细胞是哺乳动物细胞,优选地灵长类动物细胞,更优选地人细胞。
在另外的方面,宿主细胞选自T细胞、NK细胞、iNKT细胞、脐带血NK细胞、γδT细胞(γδT-细胞)、TCR敲除T细胞、病毒特异性T细胞、单核细胞、巨噬细胞、或iPSC衍生的T细胞。
在另一方面,本申请还提供了一种药物组合物,该药物组合物包含本申请的CAR或双重CAR、一种或多种核酸、一种或多种载体、或宿主细胞。
在另外的方面,药物组合物进一步包含一种或多种药学上可接受的赋形剂。
在另一方面,本申请涉及一种在有需要的受试者中治疗疾病的方法,该方法包括向该受试者施用(例如治疗有效量的)本申请的药物组合物、CAR、双重CAR。
在另外的方面,疾病是包含血液恶性肿瘤或一种或多种实体瘤的癌症。
在另外的方面,癌症是卵巢癌、胰腺癌、结肠癌、结直肠癌、淋巴瘤、食管癌、肺癌、肝癌、头颈癌、或胆囊癌。
在另一方面,本申请涉及使用本申请的CAR或双重CAR、核酸、或载体、或组合物减少CAR-T细胞对正常细胞的细胞毒性的方法。
在一个方面,本申请涉及一种产生对正常细胞的细胞毒性减少的CAR-T细胞的方法,该方法包括:
(1)将本申请的核酸或载体引入宿主细胞,以及
(2)在引入之后分离和/或扩增这些CAR-T细胞。
在另外的方面,宿主细胞是哺乳动物细胞,优选地灵长类动物细胞,更优选地人细胞。
在另外的方面,宿主细胞选自T细胞、NK细胞、iNKT细胞、脐带血NK细胞、γδT细胞(γδT-细胞)、TCR敲除T细胞、病毒特异性T细胞、单核细胞、巨噬细胞、或iPSC衍生的T细胞。
本申请的技术方案至少具有以下优点:
1.上述嵌合抗原受体(CAR)是靶标依赖性开关。在靶抗原不存在的情况下,无法检测到CAR的表面表达。由于该系统在抗原不存在的情况下可检测性有限,因此,本申请将其称为“隐形CAR”。
2.隐形CAR可以通过使用IL2Rβ、LDLR、SEZ6L2的跨膜和近膜基序和/或IL2Rβ的降解序列(PSKFFSQL)来减少对正常细胞的细胞毒性并提高CAR-T的安全性,这导致在抗原不存在的情况下细胞表面处的CAR表达大幅降低,同时保留应答表达抗原的靶细胞的下游激活能力。
3.本申请已表明,表达这些表面不稳定的隐形CAR的原代T细胞能够引起抗原依赖性靶细胞杀伤。通过以这种方式限制CAR的活性,本申请可以减少治疗性毒性和T细胞耗竭。
附图说明
本申请的新颖特征在所附权利要求书中具体阐述。在以下具体实施方式中以实施例和实例的方式解释了本申请的一些特征和优点。
图1.用于解决抗原逃逸的内吞CAR系统的草图表示。通过经由初级CAR靶向初级肿瘤抗原对肿瘤细胞构成的选择性压力导致受体下调和“抗原逃逸”。然后,可以经由次级(内吞)CAR识别的次级肿瘤抗原靶向存活的肿瘤细胞。
图2.CAR的表面稳定性降低有助于减少治疗性毒性的草图表示。肿瘤抗原通常是在癌组织上上调但仍在正常组织上以较低程度表达的蛋白标志物。CAR只在对应抗原存在的情况下在T细胞表面稳定化,这促进对表达较大量靶蛋白的细胞的杀伤。
图3.内吞CAR构建体的示意图。CAR构建体有两种变体,一种使用CAR序列的C-末端处的PSKFFSQL降解序列(DT),而另一种使用IL2Rβ跨膜结构域和27个氨基酸的近膜结构域以及IL2Rβ降解序列(DT)替代CD8跨膜结构域。
图4.Jurkat E6.1 NFAT报告细胞显示了电穿孔后的CAR表达。Jurkat细胞用PiggyBac转座酶mRNA和每种CAR构建体变体电镀,该变体包括MN14op CAR(LBC001)、HA-MN14op CAR(MLB001)、HA-MN14op CAR-DT(MLB002)、和HA-MN14op CAR-IL2Rb-tm(MLB003)。使用抗人Fab’(AF488)观察受体的表达。通过掺入DAPI排除死细胞。
图5.HA标签和抗人Fab(AF488)染色显示线性相关。将表达内吞CAR变体(MLB002和MLB003)的Jurkat细胞针对HA(PE)和人Fab(AF488)共染色,显示了两种染色之间的线性关系。具有可替代的染色策略使得能够进行脉冲追踪实验以测量受体内化速率。
图6.原代T细胞中的内吞CAR表达。将供体PBMC衍生的T(原代T细胞)细胞用PiggyBac转座酶mRNA和每种内吞CAR构建体变体(MLB002和MLB003)进行电穿孔。通过抗人Fab’(AF488)染色评价受体的表面表达。
图7.内吞CAR(MLB002和MLB003)驱动下游应答肿瘤抗原的信号传导。将JurkatNFAT荧光素酶报告细胞与表达CEA靶抗原的LOVO细胞1:1共培养。与LOVO细胞一起孵育的Jurkat细胞显示出荧光素酶活性,而在靶细胞不存在的情况下(LOVO阴性)生长的Jurkat细胞在6小时时显示出最少的报告基因表达。
图8.长期刺激显示CAR变体之间的活性相当。将表达内吞CAR构建体的JurkatNFAT荧光素酶报告细胞与表达CEA抗原的LOVO细胞1:1共培养。24小时刺激时间段后,表达受体的IL2Rβ变体(MLB002和MLB003)的Jurkat显示出与原始MN14op-CAR相当的激活。
图9.内吞CAR构建体引导Kato-III细胞的靶细胞杀伤。将供体衍生的原代T细胞与表达CEA抗原的Kato-III荧光素酶报告细胞1:1(5e5:5e5)孵育。24小时后,通过发光测量细胞毒性,表明CAR的两种IL2Rβ变体都能够引导比未转染的对照细胞的抗原特异性细胞毒性高的抗原特异性细胞毒性。
图10.CAR构建体(MLB047和MLB048)的示意图展示了对跨膜和近膜序列的改变。MLB047利用MN14op CEA ScFv抗原识别结构域。MLB047设计有癫痫发作6样蛋白2(SEZ6L2)跨膜和来自近膜序列的33个氨基酸。MLB048利用LDLR跨膜和近膜序列。这些CAR不包括P2A-GFP。
图11.CAR构建体(MLB054和MLB055)的示意图展示了对跨膜和近膜序列的改变。MLB054利用841密封蛋白18.2ScFv(CN02)抗原识别结构域。MLB054设计有癫痫发作6样蛋白2(SEZ6L2)跨膜和来自近膜序列的33个氨基酸。MLB055是双组分CAR(双重CAR),其基于MLB054密封蛋白18.2CAR序列以及以与原始CAR相同的CD8铰链和跨膜结构域偶联至CD28信号传导结构域的抗PDL1。这些CAR不包括P2A-GFP。
图12.新颖的隐形CAR构建体(MLB025、MLB020、MLB048和MLB047)在模型T细胞系中的表达特性。MLB010是一种对照CAR构建体。用PiggyBAC CAR构建体和PiggyBAC转座酶mRNA转导Jurkat细胞。通过用抗人F(ab’)
图13.隐形CAR构建体驱动NFAT依赖性T细胞激活。将经转导以表达隐形CAR构建体的Jurkat荧光素酶报告细胞与CEA阳性LoVo细胞共培养24小时,并通过NFAT驱动的荧光素酶报告子的表达评价T细胞的激活。将25,000个CAR阳性Jurkat报告细胞与用于该测定的靶细胞1:1培养。通过在CAR载体上共表达GFP来确定CAR阳性Jurkat细胞的数目。
图14A-14F.隐形CAR形式显示出比基于CD8的CAR形式弱的激活特性。将表达MLB010、MLB020、MLB025、MLB048和MLB047的Jurkat T细胞与LoVo细胞共培养3小时、6小时或24小时,并使用PE缀合的抗CD69抗体通过由流式细胞术检测CD69的表达来测量Jurkat细胞的激活。基于GFP表达检测CAR阳性细胞,同时通过CD3表达鉴定T细胞,以描绘转导和未转导的群体。此处显示了CD69表达的散点图和对应的直方图。图14A显示了MLB010(M10)和MLB020(M20)的CAR阳性群的结果。图14B显示了MLB025(M25)和MLB048(M48)的CAR阳性群的结果。图14C显示了MLB047(M47)和未转导(UTD)的CAR阳性群的结果。图14D显示了MLB010(M10)和MLB020(M20)的总体CD69上调程度的结果。图14E显示了MLB025(M25)和MLB048(M48)的总体CD69上调程度的结果。图14F显示了MLB047(M47)和未转导(UTD)的总体CD69上调程度的结果。
图15.Jurkat隐形CAR激活的定量显示与抗原表达细胞共培养后激活状态降低。将图15中的直方图定量以获得CAR T细胞群中激活细胞的分数和CD69表达的程度。如在活化细胞分数中见到的,我们观察到应答抗原的激活动力学相当,然而,相对于原始MLB010(M10)CAR形式,隐形CAR MLB020(M20)、MLB025(M25)、MLB048(M48)和MLB047(M47)显示出低得多的CD69表达。
图16.新颖的隐形CAR构建体(MLB020、MLB047、MLB048)在原代细胞中的表达特性。用PiggyBAC CAR构建体和PiggyBAC转座酶mRNA转导原代人PBMC衍生的T细胞。通过用抗人F(ab’)
图17A-17C.隐形CAR(MLB013、MLB048、MLB047)驱动依赖于靶细胞抗原表达的可变细胞介导的细胞毒性。使用三种代表性CEA阳性靶细胞系:LoVo(CEA
图18A-18C.隐形CAR(MLB013、MLB048、MLB047)T细胞显示炎症性细胞因子的表达特性大幅降低。收获来自针对以下三种代表性CEA阳性靶细胞系的细胞毒性测定的上清液:LoVo(CEA
图19.隐形CAR(MLB013、MLB048、MLB047)T细胞显示IL-2的表达特性大幅降低。收获来自针对以下两种代表性CEA阳性靶细胞系的细胞毒性测定的上清液:A549(CEA
图20.在Jurkat T细胞模型系统中密封蛋白18.2特异性隐形CAR(MLB026、MLB054、MLB055)T细胞的表达。用针对LBC010、MLB026、MLB054、和MLB055的PiggyBAC CAR构建体和PiggyBAC转座酶mRNA转导Jurkat细胞。通过用抗人F(ab’)
图21A-21D.密封蛋白18.2隐形CAR变体显示抗原特异性激活。将表达MLB026、MLB054和MLB055的Jurkat T细胞与HEK293T、HEK293T-cldn18.2、或NUGC4-cldn18.2细胞共培养过夜,并使用PE缀合的抗CD19抗体通过流式细胞术测量激活。通过CD3表达鉴定T细胞。我们观察到CD8 CAR形式的紧张性信号传导增加,如通过应答阴性细胞系的CD69上调所证明的。MLB054和MLB055两者均显示出基线激活降低和CD69的抗原特异性上调。图21A-21D显示内吞(MLB054、MLB055)和非内吞CAR(MLB026)两者都具有CD69上调。图21A-21B显示CAR阳性群,并且图21C-21D显示总体CD69上调程度。
图22.CD69表达的定量显示隐形CAR变体的激活降低。图22中CD69表达的定量显示CAR变体MLB054和MLB055的CD69表达降低并且应答表达密封蛋白18.2的靶细胞的应答细胞减少,尽管MLB054与MLB055两者均显示出相对于MLB026的基线激活降低。图22的左侧表示CD69表达,并且图22的右侧表示CD69+%。
图23.用构建体MLB026和MLB054经由电穿孔转导供体衍生的T细胞,并在电穿孔后第7天评价CAR的表达。将细胞用AF647缀合的抗人F(ab’)
图24.将表达MLB026或MLB054的供体衍生的T细胞与表达HEKcldn18.2荧光素酶的细胞共培养24小时,之后通过添加等体积的NeoLite底物来定量发光。将细胞毒性百分比评价为相对于未处理的对照细胞的降低。
图25.将表达MLB026或MLB054的供体衍生的T细胞与表达NUGC4cldn18.2荧光素酶的细胞共培养24小时,之后通过添加等体积的NeoLite底物来定量发光。将细胞毒性百分比评价为相对于未处理的对照细胞的降低。
图26.从与表达HEKcldn18.2荧光素酶的细胞共培养的MLB026和MLB054的24小时细胞毒性测定中收获上清液,并通过ELISA评价IFN-γ的表达。
图27.从与表达NUGC4cldn18.2荧光素酶的细胞共培养的MLB026和MLB054的24小时细胞毒性测定中收获上清液,并通过ELISA评价IFN-γ的表达。
图28A-28B.用构建体MLB038、MLB039、MLB079、和MLB080经由电穿孔转导供体衍生的T细胞,并在电穿孔后第7天评价CAR的表达。将细胞用AF647缀合的抗人F(ab’)2抗体染色。图28A是UTD、MLB038、和MLB039的结果。图28B是MLB079和MLB080的结果。
图29.将MDA-MB-231和SK-BR-3细胞用荧光缀合的抗人HER2或非特异性对照抗体染色。将LoVo细胞用荧光缀合的抗人HER2染色。将所有染色的细胞与未染色的对照进行比较。
图30.将表达MLB038、MLB039、MLB079、或MLB080的供体衍生的T细胞与表达MDA-MB-231荧光素酶的细胞共培养24小时,之后通过添加等体积的NeoLite底物来定量发光。将细胞毒性百分比评价为相对于未处理的对照细胞的降低。
图31.将表达MLB038、MLB039、MLB079、或MLB080的供体衍生的T细胞与表达LoVo荧光素酶的细胞共培养24小时,之后通过添加等体积的NeoLite底物来定量发光。将细胞毒性百分比评价为相对于未处理的对照细胞的降低。
图32.将表达MLB038、MLB039、MLB079、或MLB080的供体衍生的T细胞与表达SK-BR-3荧光素酶的细胞共培养24小时,之后通过添加等体积的NeoLite底物来定量发光。将细胞毒性百分比评价为相对于未处理的对照细胞的降低。
图33.从与表达MDA-MB-231荧光素酶的细胞共培养的MLB038、MLB039、MLB079、或MLB080的24小时细胞毒性测定中收获上清液,并通过ELISA评价IFN-γ的表达。
图34.从与表达LoVo荧光素酶的细胞共培养的MLB038、MLB039、MLB079、或MLB080的24小时细胞毒性测定中收获上清液,并通过ELISA评价IFN-γ的表达。
图35.从与表达SK-BR-3荧光素酶的细胞共培养的MLB038、MLB039、MLB079、或MLB080的24小时细胞毒性测定中收获上清液,并通过ELISA评价IFN-γ的表达。
图36.将表达HEKcldn18.2荧光素酶的细胞针对抗人HER2或密封蛋白18.2染色。由841克隆和808克隆两者检测密封蛋白18.2。当使用808克隆时,观察到密封蛋白18.2的更强染色,尽管841仍然可以检测到密封蛋白18.2。当针对密封蛋白18.2染色时,首先用未缀合的人源化841和808抗体对细胞进行染色,并使用荧光缀合的小鼠抗人IgG二级抗体检测结合。
图37.将密封蛋白18.2/HER2双重CAR构建体与MLB026密封蛋白18.2单CAR对照比较的示意图。将MLB108用与MLB026相同的显性密封蛋白18.2CAR和第二非显性CAR进行工程化,该第二非显性CAR基于赫赛汀抗HER2抗体结构域、SEZ6L2跨膜和近膜结构域、以及CD3ζITAM信号传导基序。将MLB040双重CAR用与MLB026相同的显性密封蛋白18.2CAR和第二非显性CAR进行工程化,该第二非显性CAR基于赫赛汀抗HER2抗体结构域、IL2Rβ的跨膜和近膜结构域、CD28共受体信号传导结构域、以及CD3ζ细胞内ITAM信号传导基序。
图38.用构建体MLB026和MLB108经由电穿孔转导供体衍生的T细胞,并在电穿孔后第7天评价CAR的表达。将细胞用AF594缀合的抗人F(ab’)
图39.将表达MLB026和MLB108的供体衍生的T细胞与表达HEKcldn18.2荧光素酶的细胞共培养24小时,之后通过添加等体积的NeoLite底物来定量发光。将细胞毒性百分比评价为相对于未处理的对照细胞的降低。
图40.从与表达HEKcldn18.2荧光素酶的细胞共培养的MLB026和MLB108的24小时细胞毒性测定中收获上清液,并通过ELISA评价IFN-γ的表达。
图41A-41B.用构建体MLB026、MLB038、MLB039、和MLB040经由电穿孔转导供体衍生的T细胞,并在电穿孔后第7天评价CAR的表达。将细胞用AF647缀合的抗人F(ab’)
图42.将表达MLB026、MLB038、MLB039、或MLB040的供体衍生的T细胞与表达HEKcldn18.2荧光素酶的细胞共培养24小时,之后通过添加等体积的NeoLite底物来定量发光。将细胞毒性百分比评价为相对于未处理的对照细胞的降低。
图43.从与表达HEKcldn18.2荧光素酶的细胞共培养的MLB026、MLB038、MLB039、和MLB040的24小时细胞毒性测定中收获上清液,并通过ELISA评价IFN-γ的表达。
图44.将表达MLB026、MLB038、MLB039、或MLB040的供体衍生的T细胞与表达SK-BR-3荧光素酶的细胞共培养24小时,之后通过添加等体积的NeoLite底物来定量发光。将细胞毒性百分比评价为相对于未处理的对照细胞的降低。
具体实施方式
除非另外定义,否则本文所用的技术与科学术语具有如本披露所属领域通常所用的相同含义。出于解释本说明书的目的,以下定义将适用并且在适当时,以单数形式使用的术语也包括复数并且反之亦然。如本文和所附权利要求书中所用,除非上下文另有明确规定,否则单数形式“一个/种(a/an)”和“该(the)”也指复数形式,例如,提及“宿主细胞”包括多个此类宿主细胞。
如本文所用,术语“嵌合抗原受体(CAR)”意指包含能够与预定抗原结合的细胞外结构域、含有信号传导结构域的细胞内结构域、和跨膜结构域的融合蛋白。短语“与预定抗原结合”意指能够与预定抗原特异性结合的任何蛋白质分子或其部分。“信号传导结构域”意指任何寡肽或多肽结构域,已知其发挥作用以传递信号,从而导致激活或抑制细胞中的生物学过程,例如激活免疫细胞(如T细胞或NK细胞)。实例包括4-1BB、CD28和/或CD3ζ信号传导结构域。
如本文所用,术语“IL2Rβ”或“IL2Rb”意指IL2受体β链。高亲和力IL2受体是一种跨膜受体,其由三种不同的非共价缔合的组分构成:α、β和γ链。已经表明,在α和γ亚基不存在的情况下,β链被组成型地内吞并降解。
如本文所用,术语“隐形CAR”或“内吞CAR”最广义上是指嵌合抗原受体(CAR),该CAR包含:(1)细胞外配体结合结构域,其包含与预定抗原特异性结合的单链可变片段(scFv);(2)跨膜(tm)连接近膜(jm)结构域;其中该跨膜连接近膜结构域包含IL2受体β链(IL2Rβ)跨膜结构域和IL2Rβ近膜结构域,并且该跨膜连接近膜结构域与IL2Rβ降解序列(DT)相邻;其中优选地,该IL2Rβ降解序列在该跨膜连接近膜结构域的C-末端处;其中优选地,该IL2Rβ降解序列包含与由SEQ ID NO:2表示的氨基酸序列具有至少约95%、96%、97%、98%、99%或100%同一性的氨基酸序列;其中该跨膜连接近膜结构域包含低密度脂蛋白受体(LDLR)跨膜结构域和LDLR近膜结构域;或其中该跨膜连接近膜结构域包含癫痫发作6样蛋白2(SEZ6L2)跨膜结构域和SEZ6L2近膜结构域;(3)细胞内结构域;其中优选地,该细胞内结构域包含信号传导结构域;更优选地,该信号传导结构域包含一个或多个选自由以下组成的组的信号传导结构域:4-1BB信号传导结构域、CD28信号传导结构域和CD3ζ信号传导结构域。或者嵌合抗原受体(CAR)包含:(1)细胞外配体结合结构域,其包含与预定抗原特异性结合的单链可变片段(scFv);(2)跨膜结构域,以及(3)包含IL2Rβ降解序列(DT)和至少一个信号传导结构域的细胞质区段;其中优选地,该IL2Rβ降解序列包含与由SEQ ID NO:2表示的氨基酸序列具有至少约95%、96%、97%、98%、99%或100%同一性的氨基酸序列;其中优选地,该IL2Rβ降解序列在该细胞质区段的C-末端处。或者一种双重CAR,该双重CAR包含根据上述实施例中任一个所述的第一CAR,以及第二CAR。
如本文所用,术语“抗原结合片段”或“抗原结合分子”最广义上是指特异性地结合抗原决定簇的分子。抗原结合分子的实例为抗体、抗体片段和支架抗原结合蛋白。本文中术语“抗体”以最广义使用,并且涵盖多种抗体结构,这些抗体结构包括但不限于单克隆抗体、多克隆抗体、单特异性和多特异性抗体(例如,双特异性抗体)、以及抗体片段,只要它们表现出所需的抗原结合活性即可。
如本文所用,术语“单链可变片段(scFv)”是抗体的重链可变区(VH)和轻链可变区(VL)的融合蛋白,用十至约25个氨基酸的短接头肽连接。该接头通常富含甘氨酸(针对灵活性)、以及丝氨酸或苏氨酸(针对溶解度),并且可以将VH的N-末端和VL的C-末端连接起来,或反之亦然。尽管去除了恒定区并引入了接头,但这种蛋白质保留了原始抗体的特异性。另外,包含单链多肽的抗体片段具有VH结构域的特征,即能够与VL结构域一起组装到功能性抗原结合位点;或这些包含单链多肽的抗体片段具有VL结构域的特征,即能够与VH结构域一起组装到功能性抗原结合位点,从而提供全长抗体的抗原结合特性。
如本文所用,术语药剂例如药物组合物的“治疗有效量”是指在必要的剂量和时间段内有效达到所希望的治疗结果或预防结果的量。治疗有效量的药剂例如消除、减少、延迟、最小化或预防疾病的不利影响。
如本文所用,术语“个体”或“受试者”是哺乳动物。哺乳动物包括但不限于驯养动物(例如牛、羊、猫、狗和马)、灵长类动物(例如人和非人灵长类动物,如猴子)、兔和啮齿动物(例如小鼠和大鼠)。特别地,个体或受试者是人。术语“药物组合物”是指这样的制剂,其呈允许包含其中的活性成分的生物学活性有效的形式,并且不含对接受配制品施用的受试者具有不可接受的毒性的另外的组分。“药学上可接受的赋形剂”是指药物组合物中除活性成分以外的对受试者无毒的成分。药学上可接受的赋形剂包括但不限于缓冲剂、稳定剂或防腐剂。
如本文所用,“治疗(treatment)”(及其语法变体,如“治疗(treat或treating)”)是指试图改变被治疗个体的自然过程的临床干预,并且可以用于预防或在临床病理过程期间进行。治疗的期望效果包括但不限于预防疾病的发生或复发、减轻症状、减少疾病的任何直接或间接病理后果、预防转移、降低疾病进展速率、改善或缓和疾病状态、以及缓解或改善预后。在一些实施例中,本申请的分子用于延迟疾病的发展或减缓疾病的进展。
如本文所用,术语“肿瘤相关抗原(TAA)”或“肿瘤抗原”意指具有抗原性的生物分子,其表达被认为与细胞的恶性改变相关。本披露中的肿瘤抗原包括肿瘤特异性抗原(仅在肿瘤细胞中存在而未在其他正常细胞中发现的抗原)和肿瘤相关抗原(也存在于其他器官和组织、或异质和同种异体正常细胞中的抗原,或在发育和/或分化期间表达的抗原)。
如在本申请中所用,术语“氨基酸”表示天然存在的羧基α-氨基酸的组,这些羧基α-氨基酸包含丙氨酸(三字母代码:Ala,一字母代码:A)、精氨酸(Arg,R)、天冬酰胺(Asn,N)、天冬氨酸(Asp,D)、半胱氨酸(Cys,C)、谷氨酰胺(Gln,Q)、谷氨酸(Glu,E)、甘氨酸(Gly,G)、组氨酸(His,H)、异亮氨酸(Ile,I)、亮氨酸(Leu,L)、赖氨酸(Lys,K)、甲硫氨酸(Met,M)、苯丙氨酸(Phe,F)、脯氨酸(Pro,P)、丝氨酸(Ser,S)、苏氨酸(Thr,T)、色氨酸(Trp,W)、酪氨酸(Tyr,Y)、和缬氨酸(Val,V)。相对于参考多肽(蛋白质)序列的“氨基酸序列同一性百分比(%)”定义为在比对序列并引入空位(如有必要)以达到最大序列同一性百分比、并且不考虑任何保守取代作为序列同一性的一部分后,候选序列中与参考多肽序列的氨基酸残基相同的氨基酸残基的百分比。出于确定氨基酸序列同一性百分比的目的,可以用本领域技术中的多种方式来实现比对,例如使用公众可得的计算机软件,如BLAST、BLAST-2、ALIGN、SAWI或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适当参数,包括在被比较序列的全长范围实现最大比对所需的任何算法。然而,出于本文的目的,使用序列比较计算机程序ALIGN-2生成氨基酸序列同一性%值。ALIGN-2序列比较计算机程序由基因泰克公司(Genentech,Inc.)编写,并且源代码已经与美国版权局(U.S.Copyright Office)(华盛顿特区(Washington D.C.),20559)中的用户文档一起提交,其在美国版权登记号TXU510087下注册。ALIGN-2程序从基因泰克公司(加利福尼亚州,南旧金山)公开获得,或者可从源代码编译。应编译ALIGN-2程序以在UNIX操作系统(包括数字UNIX V4.0D)上使用。所有序列比较参数均由ALIGN-2程序设置并且不变。
术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可互换使用,并且是指已引入外源核酸的细胞,包括此类细胞的子代。宿主细胞包括“转化体”和“转化细胞”,其包括原代转化细胞和从其衍生的子代,而与传代次数无关。
如本文所用,术语“癌症”或“肿瘤”是指增殖性疾病,例如卵巢癌、胰腺癌、结肠癌、结直肠癌、淋巴瘤、淋巴细胞白血病、肺癌、非小细胞肺(NSCL)癌、细支气管肺泡细胞肺癌、骨癌、胰腺癌、皮肤癌、头颈癌、皮肤或眼内黑色素瘤、子宫癌、直肠癌、肛门区域癌、胃部癌症(stomach cancer)、胃癌(gastric cancer)、乳腺癌、输卵管癌、子宫内膜癌、子宫颈癌、阴道癌、外阴癌、霍奇金病、食道癌、小肠癌、内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、阴茎癌、前列腺癌、膀胱癌、肾癌或输尿管癌、肾细胞癌、肾盂癌、间皮瘤、肝细胞癌、胆管癌、中枢神经系统(CNS)肿瘤、脊髓轴肿瘤、脑干神经胶质瘤、多形性胶质母细胞瘤、星形细胞瘤、神经鞘瘤、室管膜瘤、髓母细胞瘤、脑膜瘤、鳞状细胞癌、垂体腺瘤和尤文氏肉瘤,包括上述癌症中的任一种的难治性型式,或上述癌症中的一种或多种的组合。
实例
实例1.内吞CAR构建体(MLB003和MLB002)的设计和生成
CAR构建体的制备是本领域的常用技术方法。例如,首先,通过基因合成技术制备CAR基因片段,然后构建CAR PiggyBac转座子表达载体,将CAR构建体电穿孔到靶细胞中,并通过流式细胞术或总蛋白分析评估构建体表达。
在本实例1中,设计了两种基于IL2Rβ链跨膜和近膜基序的新颖CAR构建体(图3)。
(1)含有包含IL2Rβ降解序列(DT)的细胞质区段的CAR构建体:
HA-MN14op-CAR-DT(MLB002:HA-MN14ops cFv-CD8铰链-CD8tm-4-1BB-CD3ζ-DT,SEQ ID NO:8);
(2)含有IL2Rβ跨膜结构域、IL2Rβ近膜结构域并进一步含有IL2Rβ降解序列(DT)的CAR构建体:
HA-MN14op-CAR-IL2Rb-tm(MLB003:HA-MN14op CEA scFv-CD8铰链-IL2Rβtm jmDT-4-1BB-CD3ζ,SEQ ID NO.9)
(3)使用CD8跨膜结构域的原始CAR构建体:
MN14op-CAR(LBC001:MN14op CEA scFv-CD8铰链-CD8tm-4-1BB-CD3ζ)。
(4)使用CD8跨膜结构域和HA序列的对照CAR构建体:
HA-MN14op CAR(MLB001:HA-MN14op CEA scFv-CD8铰链-CD8tm-4-1BB-CD3ζ)。
pMAX-GFP是用于确定电穿孔效率的对照质粒,特别是当荧光标志物在测试构建体中不可用时。
通过T细胞激活测定(包括但不限于NFAT报告子测定和细胞毒性测定)评价功能性功效。
表1.实例1-5中使用的序列
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*前导序列加下划线,HA标签为斜体,IL2Rβ序列为粗体,CD8铰链加下划线且为斜体。
实例2.使用转座子递送CAR构建体以修饰原代人T细胞和Jurkat E6.1细胞
实例1中内吞CAR构建体的转座子介导递送:使用EF1α启动子驱动在转座子载体中的我们的CAR构建体的表达。
用CAR构建体转导原代T细胞和Jurkat E6.1 T细胞(Jurkat E6.1细胞):使用电穿孔用携带我们在实例1构建体中的CAR的转座子载体转导PBMC衍生的CD3+纯化的T细胞(原代T细胞)或Jurkat E6.1 T细胞。
实例3.表达内吞CAR(MLB002和MLB003)的CAR T细胞的表征
通过流式细胞术分析转导的CAR-T细胞,以测量构建体与宿主细胞的整合。使用与AlexaFluor 488(AF488)缀合的抗人Fab’对细胞进行染色。也用细胞外N-末端HA标签对内吞CAR进行工程化,以帮助检测受体并进行脉冲追踪实验。将构建体电穿孔到Jurkat E6.1细胞后,可检测到CAR的表面表达(图4)。在图4中,纵坐标的SSC是细胞复杂性的相对测量,并且pMAX-GFP是用于确定电穿孔效率的对照质粒,特别是当荧光标志物在测试构建体中不可用时。检测到抗人Fab’AF488和HA染色之间的线性相关性(图5)。使用抗人Fab’AF488染色也证实了我们的内吞CAR构建体在供体PBMC衍生的T细胞中的表达。原代T细胞和JurkatE6.1细胞之间的受体表达趋势相当(图6)。图4-6展示的结论是HA-MN14op-CAR-IL2Rb-tm(MLB003)与未转导的(UTD)类似,其中抗人Fab’AF488的染色值低于对照CAR(HA-MN14op-CAR,MLB001),这反映了细胞表面上的表达最少,从而表明通过使用IL2Rβ链跨膜、近膜基序和DT序列(PSKFFSQL)获得了良好的内吞作用。与无DT序列的构建体(HA-MN14op-CAR,MLB001)相比,HA-MN14op-CAR-DT(MLB002)的表达较低,并且还表现出内吞,这在无DT序列的构建体中没有观察到。
实例4.针对内吞CAR(MLB002和MLB003)的NFAT荧光素酶报告子测定将表达CAR构建体的Jurkat E6.1 NFAT-荧光素酶报告细胞与表达抗原的靶细胞(LoVo)1:1混合,并在共培养6小时(图7)或24小时(图8)后测量荧光素酶活性。LoVo阴性意指没有LoVo细胞与Jurkat E6.1细胞混合。NFAT是一种与T细胞激活相关的T细胞转录因子。NFAT活性增加与信号强度增加相关。因此,如果我们见到荧光素酶信号增加,我们就知道NFAT活性增加并且CAR信号强度增加。图7和8显示,在表达靶抗原的细胞中,LoVo靶抗原是CEA,并且内吞CAR(MLB002或MLB003)被激活,其具有NFAT驱动的荧光素酶活性。内吞CAR的发光值高于未转导的(UTD)。尽管共培养6小时后MLB002和MLB003的发光值低于对照组的那些,但共培养24小时后MLB002与MLB003的值高于对照组的那些。尽管流式细胞仪无法检测到内吞CAR构建体的表面表达(图4和5),但用内吞CAR构建体转导的细胞显示出NFAT驱动的基因表达和荧光素酶活性的变化,证实了CAR的功能性。
实例5.针对内吞CAR(MLB002和MLB003)细胞毒性测定
将经转导以表达我们的CAR构建体的供体衍生的原代CD3+细胞(原代细胞)与表达抗原的荧光素酶+靶细胞(Kato-III,其表达靶抗原CEA)1:1混合,并共培养24小时。通过荧光素酶活性的增加来测量细胞毒性(图9)。尽管通过流式细胞术无法检测到原代细胞中的内吞CAR(MLB002和MLB003)表达(图6),但相对于未转导的对照细胞(UTD),用内吞CAR转导的细胞显示出增强的细胞介导杀伤。
实例6.用内吞CAR构建体(MLB020、MLB013、MLB025、MLB048、MLB047、MLB054和MLB055)对Jurkat和原代CD3+PBMC进行非慢病毒转染
在本实例6中,将CAR构建体设计如下:
(1)含有IL2Rβ跨膜结构域、IL2Rβ近膜结构域并进一步含有IL2Rβ降解序列(DT)的CAR构建体:
(a)MN14op CEA scFv-CD8铰链-IL2Rβtm jm DT-4-1BB-CD3ζ-P2A-GFP(MLB020);
(b)HA-MN14op CEA scFv-CD8铰链-IL2Rβtm jm DT-4-1BB-CD3ζ-P2A-GFP(MLB013)。
(2)含有包含IL2Rβ降解序列(DT)的细胞质区段的CAR构建体:
(a)MN14op CEA scFv-CD8铰链-CD8 tm-4-1BB-CD3ζ-IL2RβDT-P2A-GFP(MLB025)。
(3)含有LDLR跨膜结构域和LDLR近膜结构域的CAR构建体:
(a)MN14op CEA scFv-CD8铰链-LDLR tm jm-4-1BB-CD3ζ-P2A-GFP(MLB048)。
(4)含有SEZ6L2跨膜结构域和SEZ6L2近膜结构域的CAR构建体:
(a)MN14op CEA scFv-CD8铰链-SEZ6L2 tm jm-4-1BB-CD3ζ-P2A-GFP(MLB047);
(b)841密封蛋白18.2scFv-CD8铰链-SEZ6L2 tm jm-4-1BB-CD3ζ-P2A-GFP(MLB054)。
(5)双重CAR构建体:
(a)841密封蛋白18.2scFv-CD8铰链-SEZ6L2 tm jm-4-1BB-CD3ζ-P2A-PD-L1scFv-CD8铰链-CD8 tm-CD28-(G
(6)使用CD8跨膜结构域的对照CAR构建体:
(a)MN14op CEA scFv-CD8铰链-CD8 tm-4-1BB-CD3ζ-P2A-GFP(MLB010);
(b)841密封蛋白18.2scFv-CD8铰链-CD8 tm-4-1BB-CD3ζ-P2A-GFP(MLB026)。
pMAX-GFP是用于确定电穿孔效率的对照质粒,特别是当荧光标志物在测试构建体中不可用时。
表2.实例6-10中使用的序列
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通过电穿孔Piggy Bac转座子质粒(具有处于EF1α启动子控制下的CAR构建体并具有转座酶mRNA)诱导表达NFAT驱动的荧光素酶报告子的T细胞系Jurkat E6.1和供体衍生的T细胞,以表达我们的CAR构建体。图10和11显示了CAR构建体的两幅示意图,展示了对跨膜和近膜序列的改变。CAR MLB047和MLB054分别利用MN14op CEA ScFv和841密封蛋白18.2ScFv(CN02)抗原识别结构域。这些CAR设计有癫痫发作6样蛋白2(SEZ6L2)跨膜和来自近膜序列的33个氨基酸。MLB048利用LDLR跨膜和近膜序列。MLB055是双组分CAR(双重CAR),其基于MLB054密封蛋白18.2CAR序列以及以与原始CAR相同的CD8铰链和跨膜结构域偶联至CD28信号传导结构域的抗PDL1。
通过用AF647缀合的α人Fab’抗体染色,通过流式细胞术评价CAR的表面表达;通过处于P2A序列下的GFP的共表达来确定CAR构建体的总体表达(图12)。鉴于无法检测到使用IL2Rβ、LDLR和癫痫发作6样蛋白2跨膜和近膜结构域设计的CAR构建体的表面表达,使用GFP表达使所有后续测定的CAR阳性细胞分数归一化。图12显示当用AF674染色时,使用IL2Rβ、LDLR和SEZ6L2跨膜和近膜结构域的CAR构建体(MLB025、MLB020、MLB048和MLB047)表现出低于对照CAR构建体MLB010的染色值,证明了当没有抗原靶标时,内吞CAR不在细胞表面上表达。对照CAR构建体MLB010的AF674和GFP染色值较高。
实例7.通过荧光素酶报告子活性和CD69上调评价CAR构建体的活性
将表达CEACAM-5靶抗原的LoVo细胞在1μg/mL丝裂霉素C存在下在白色、不透明的平底96孔板中培养以抑制细胞增殖。在时程测定启动前6-8小时,每孔接种25,000个LoVo靶细胞以允许粘附。用CAR+Jurkat T细胞以1:1的E:T比率进行测定。孵育8小时后,洗涤孔,并补充培养基。在发光读数前24、6、3和1小时,洗涤孔,并将表达CAR构建体的Jurkat T细胞添加到每个孔中。在24小时的时间段结束时,将Bio-Glo
对发光信号的观察表明,所有CAR变体的激活动力学与原始CAR构建体类似并且相当,NFAT驱动的基因表达在激活6小时左右强烈发生。尽管激活动力学相当,但CAR的原始(MLB010)和c-末端(MLB025)加标签版本的细胞激活程度最强。基于LDLR和SEZ6L2的变体(MLB047和MLB048)两者均显示出低得多的总体激活水平,并且基于IL2Rβ的构建体(MLB020)显示出最低的表达水平(图13)。
为了进一步确定各种跨膜结构域对Jurkat T细胞总体激活状态的影响,我们进行了第二共培养测定以确定刺激后CD69上调的程度。将50,000个LoVo靶细胞与Jurkat CAR阳性T细胞以1:1的比率共培养3、6或24小时,并通过流式细胞术评价CD69的表达。通过对GFP表达的细胞进行设门,我们能够通过评估荧光强度来确定CAR阳性群中表达CD69的细胞的分数和总体CD69上调程度(图14A-14F)。图14A-14F显示了内吞(MLB020、MLB025、MLB048和MLB047)和非内吞CAR(MLB010)两者都具有CD69上调。图14A-14C显示了CAR阳性群,并且图14D-14F显示了总体CD69上调程度。使用该信息,我们能够生成展示CAR阳性细胞的激活状态的曲线以更好地在构建体之间进行比较(图15)。在图14A-14F和图15中,M10代表MLB010,M20代表MLB020,M25代表MLB025,M48代表MLB048,并且M47代表MLB047。我们的数据表明,尽管所有CAR构建体之间的CD69激活动力学相似,但表达IL2Rβ、LDLR或SEZ6L2结构域的那些(MLB020、MLB025、MLB048和MLB047)在任何给定时间点都显示出较低数目的应答细胞以及较低的总体CD69表达(图15)。这些结果表明,尽管在抗原不存在的情况下没有检测到CAR的表面表达,但新颖的受体变体能够驱动T细胞激活。这些发现还表明,相对于MLB010,MLB020、MLB025、MLB047和MLB048的激活应答减弱,这是由受体在细胞内的独特分布引起的。
实例8.针对CAR MLB013、MLB020、MLB047和MLB048的细胞毒性测定
为了评估IL2Rβ(MLB013)、SEZ6L2(MLB047)和LDLR(MLB048)结构域对下游T细胞活性的影响,建立了针对CEACAM-5阳性靶细胞系LoVo、A549和HT-29的细胞毒性测定。选择这些靶细胞系是因为它们分别具有高、中和低水平的CEA表达。鉴于CAR靶抗原在肿瘤组织和非癌组织之间通常具有可变的表达,因此该测定允许我们评估可在临床情况下引起严重的副作用的中靶、脱肿瘤CAR活性的可能性。
使用PiggyBac表达系统转导供体衍生的CD3+PBMC以表达CAR构建体。电穿孔后,通过在CAR表达载体上共表达GFP经由流式细胞术评价CAR的表达,并使用该CAR的表达将细胞数目归一化以直接比较CAR-T细胞的活性(图16)。图16显示了用AF674对使用IL2Rβ、LDLR和SEZ6L2跨膜和近膜结构域的MLB020、MLB047、和MLB048 CAR构建体进行染色的结果,展现了低于对照CAR构建体MLB010的染色值,这证明了当没有抗原靶标时,内吞CAR不在细胞表面上表达。对照CAR构建体MLB010的AF674染色值较高。将表达抗原的荧光素酶阳性靶细胞以10,000个细胞/孔的密度接种,并以三种E:T比率(3:1、1:1、0.3:1,CAR阳性细胞与靶细胞)进行测定。将效应细胞和靶细胞共培养24小时,并将细胞毒性确定为荧光素酶活性降低、同时靶细胞死亡。
如所预期的,细胞毒性活性与靶细胞系上的抗原表达量直接相关,针对LoVo细胞检测到的细胞毒性的量最高,并且针对HT-29细胞检测到的细胞毒性的量最低(图17A-17C)。同样如所预期的,细胞毒性随效应细胞数目/孔而缩放,较高的E:T比率导致更大的细胞毒性。有趣的是,在3:1的E:T比率处,MLB013、MLB047和MLB048针对高表达靶抗原的细胞的表现与亲本CAR(MLB010和MLB011,MLB011在MLB010的MN14op CEA scFv N-末端处具有添加的HA序列)类似,但随着E:T比率降低,MLB013、MLB047和MLB048变体的细胞毒性潜力降低更快。注意,只有具有SEZ6L2的MLB048保留了针对低抗原HT-29细胞的细胞毒性活性。这些结果给出了以下信心:向CAR构建体添加内吞靶向基序可以扩大CAR构建体的治疗窗。
实例9.CAR MLB013、MLB047和MLB048的细胞因子表达分析
已经证明新颖的跨膜CAR构建体能够保留针对表达抗原的靶细胞的细胞毒性活性,我们希望测量另外的CAR T细胞活性读数。两种常见的激活标志物是IL-2和IFN-γ。为了测定细胞因子分泌,在过夜细胞毒性测定后收获培养基,并经由ELISA确定可溶性细胞因子的表达。
在细胞因子分泌中观察到的趋势反映了我们在细胞毒性中观察到的那些,针对高CEA靶细胞检测到的细胞因子分泌水平最高,并且针对HT-29细胞检测到的细胞因子表达大幅降低(图18A-18C和图19)。有趣的是,对MLB013、MLB047和MLB048几乎无法检测到细胞因子分泌,其中MLB047相对于MLB013和MLB048显示出略高的细胞因子表达。
这些发现是重要的,因为已知CAR T细胞疗法会促进潜在致命的细胞因子释放综合征(CRS)。出于这个原因,这些结果具有临床意义,并且表明CAR T细胞的细胞因子特性可以通过使用可替代的跨膜和近膜结构域来调节。通过使用已知靶向内体途径的受体的跨膜结构域和相关的运输基序,结果表明可以调节CAR T细胞的活性,从而扩大治疗窗。
实例10.Jurkat E6.1细胞中密封蛋白18.2特异性的和增强的CAR T细胞激活
将表达NFAT驱动的荧光素酶报告子的Jurkat E6.1 T细胞用表达密封蛋白18.2特异性CAR构建体(MLB026、MLB054、MLB055)的Piggy Bac载体和Piggy Bac转座酶mRNA电穿孔。如之前一样,通过用AF647缀合的α人Fab’抗体进行表面染色并经由P2A序列在CAR载体上共表达GFP来评价CAR的表达(图20)。如之前一样,在CAR序列中包括SEZ6L2跨膜结构域(MLB054)导致CAR的表面表达降低,而由GFP确定的总体CAR表达仍然高。由于构建体中存在scFv PD-L1-CD28-GFP共受体,还观察到MLB055中GFP表达与α人Fab’染色之间的强相关性。
为了针对CAR T细胞活性进行筛选,将CLDN18.2(密封蛋白18.2)特异性CAR T细胞与抗原阴性HEK293T细胞或经转导以表达CLDN18.2(密封蛋白18.2)的HEK293T和NUGC4细胞共培养。如之前一样,用表达CAR的Jurkat以1:1的比率培养50,000个靶细胞。如αCEA CAR变体那样(图21A-21D),观察到表达CAR变体的Jurkat的抗原特异性激活,其中在表达SEZ6L2跨膜结构域的变体中检测到降低的激活(图22)。图21A-21D显示内吞(MLB054、MLB055)和非内吞CAR(MLB026)两者都具有CD69上调。图21A-21B显示CAR阳性群,并且图21C-21D显示总体CD69上调程度。
实例11.SEZ6L2修饰的CAR用于减弱靶向密封蛋白18.2的T细胞中的细胞因子特性
经由菲科帕克(Ficoll-Paque)分离将PBMC分离,并通过CD3阴性选择进行纯化以富集T细胞群。在IL-2、IL-7和IL-15存在下,将分离的T细胞用StemCell T细胞激活剂(CD2/CD3/CD28)刺激三天,然后用过度活跃的PiggyBac转座酶mRNA和PiggyBak载体MLB026和MLB054电穿孔,以分别诱导基于CD8α跨膜结构域或SEZ6L2跨膜和近膜结构域的CAR表达。这些CAR构建体利用对密封蛋白18.2特异的841scFv。
通过用荧光缀合的抗人F(ab’)
为了测试抗密封蛋白18.2、SEZ6L2修饰的隐形CAR的功能性能力,针对经转导以过表达密封蛋白18.2亚型以及荧光素酶的HEK293T或NUGC4细胞进行了基于荧光素酶的细胞毒性测定。通过流式细胞术评价CAR阳性细胞的数目,并将计数相对于CAR阳性细胞的数目归一化(图23)。将HEK-cldn18.2 Luc或NUGC4-cldn18.2 Luc细胞以10,000个细胞/孔的密度接种在不透明96孔板中的100μL ImmunoCult培养基中。然后将CAR T细胞或未转导的对照细胞以3:1、1:1和0.3:1的效应子:靶标(E:T)比率添加到另外的100μL培养基中,使总反应体积为200μL。然后将细胞在37℃、5% CO
经由夹心ELISA分析共培养上清液中由CAR T细胞产生的IFN-γ的表达。将上清液以1比3稀释在ELISA稀释液中,并且HRP检测底物的比色活性表明SEZ6L2修饰的隐形CAR(MLB054)针对HEK-cldn18.2(图26)和NUGC4-cldn18.2(图27)细胞系均具有降低的细胞因子分泌特性。这些数据表明这些细胞的抗原识别和细胞毒性潜力得到了保留,同时产生较低的炎症性应答。我们预计,在解决现有CAR技术中见到的细胞因子释放综合征(CRS)的问题方面,这可以是期望的。
实例12.SEZ6L2和IL2Rβ修饰的CAR(MLB038、MLB039和MLB080)用于减弱靶向HER2的T细胞中的细胞因子特性
在本实例12中,将CAR构建体设计如下:
(1)包含IL2Rβ跨膜结构域、IL2Rβ近膜结构域并进一步包含IL2Rβ降解序列(DT)的CAR构建体:
(a)HER2 scFv-CD8铰链-IL2Rβtm jm DT-4-1BB-CD3ζ-P2A-GFP(MLB038);
(b)HER2 scFv-CD8铰链-IL2Rβtm jm DT-CD28-CD3ζ-P2A-GFP(MLB039);
(2)包含SEZ6L2跨膜结构域和SEZ6L2近膜结构域的CAR构建体:
(a)HER2 scFv-CD8铰链-SEZ6L2 tm jm-4-1BB-CD3ζ-P2A-GFP(MLB080)。
(3)双重CAR构建体:
(a)841密封蛋白18.2scFv-CD8铰链-CD8 tm-4-1BB-CD3ζ-P2A-HER2 scFv-IL2Rβtm jm DT-CD28-CD3ζ(MLB040)
(b)841密封蛋白18.2scFv-CD8铰链-CD8 tm-4-1BB-CD3ζ-P2A-HER2 scFv-SEZ6L2tm jm-CD3ζ(MLB108)。
(4)使用CD8跨膜结构域的对照CAR构建体:
(a)HER2 scFv-CD8铰链-CD8 tm-4-1BB-CD3ζ-P2A-GFP(MLB079)。
表3.实例12-13中使用的序列
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内化基序为粗体并加下划线。
表4.本申请的CAR和抗体中使用的序列
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经由菲科帕克分离将PBMC分离,并通过CD3阴性选择进行纯化以富集T细胞群。在IL-2、IL-7和IL-15存在下,将分离的T细胞用StemCell T细胞激活剂(CD2/CD3/CD28)刺激三天,然后用过度活跃的PiggyBac转座酶mRNA和PiggyBac载体MLB038、MLB039、MLB079和MLB080电穿孔。将所有四种CAR变体用衍生自赫赛汀抗体的4D5V8 scFv工程化,该4D5V8scFv识别HER2(4D5V8 Her2 scFv的氨基酸序列为SEQ ID NO.31)。基于IL2受体β跨膜和近膜序列设计MLB038和MLB039 CAR。MLB038和MLB039可由它们的共受体信号传导结构域来区分,MLB038使用CD28共受体结构域设计,并且MLB039用4-1BB信号传导结构域设计。这两种形式也具有CD3ζ细胞内ITAM信号传导基序。MLB079被设计为对照并基于CD8α跨膜结构域,而MLB080用SEZ6L2跨膜和近膜序列工程化。
通过用荧光缀合的抗人F(ab’)
为了测试抗HER2隐形CAR变体的功能性能力,针对细胞系SK-BR-3、LoVo和MDA-MB-231(临床上描述为三阴性乳腺癌细胞系)进行了基于荧光素酶的细胞毒性测定(图29)。图29显示了荧光素酶靶细胞系上不同程度的HER2表达。MDA-MB-231在临床上为HER2阴性,并显示出低HER2染色。LoVo细胞也显示出低水平的HER2抗体染色。SK-BR-3细胞显示出高水平的HER2染色。靶细胞的HER2表达特性的差异允许我们评价我们的HER2 CAR变体的功效。然后将CAR T细胞或未转导的对照细胞以3:1、1:1和0.3:1的效应子:靶标(E:T)比率添加到另外的100μL培养基中,使总反应体积为200μL。然后将细胞在37℃、5% CO2下共培养24小时。在该时间段结束时,经由离心收获100μL培养基用于细胞因子分析,并将NeoLite底物添加到每个孔中以确定活细胞率。将特异性细胞裂解百分比确定为治疗组相对于未治疗对照组的发光信号强度降低。所有四种CAR变体(MLB038、MLB039、MLB079、MLB080)分别针对所有三种靶细胞系:MDA-MB-231、LoVo、SKBR3都可检测到抗原特异性细胞裂解(图30、图31、图32)。每种CAR变体的细胞毒性活性大致相当并具有剂量依赖性,即随着E:T比率降低,靶细胞杀伤也降低。变体MLB038和MLB039的比较表明共受体结构域不影响体外杀伤效率。
经由夹心ELISA分析共培养上清液中由CAR T细胞产生的IFN-γ的表达。将上清液以1比3稀释在ELISA稀释液中,并且HRP检测底物的比色活性表明,针对所有测试的细胞系:MDA-MB-231、LoVo、SKBR3,隐形CAR变体MLB038、MLB039和MLB080相对于CD8α跨膜对照CARMLB079具有减少的细胞因子分泌(图33、图34、图35)。在HER2高表达细胞系中,IFN-γ在经转导以表达MLB080的细胞中的表达高于MLB038和MLB039两者,但仍低于原始CAR形式MLB079(图35)。这表明当靶向具有高肿瘤相关抗原表达的细胞时,SEZ6L2修饰的CAR仍然能够保留细胞因子信号传导,尽管程度较低。所有细胞系中细胞因子的表达随E:T比率的降低呈剂量依赖性降低。这些结果表明CAR共受体信号传导结构域没有极大的影响细胞因子表达,并且隐形CAR(MLB038、MLB039、MLB080)相对于对照CAR(MLB079)具有降低的炎症性细胞因子特性。
实例13.靶向密封蛋白18.2/HER2的双CAR显示出针对表达一种或两种肿瘤相关抗原的肿瘤细胞系的细胞毒性潜力以及增强的功能性
使用可商购HER2抗体或内部开发的密封蛋白18.2抗体克隆hCLDN18.2-808抗体(VH的氨基酸序列为SEQ ID NO:47;VL的氨基酸顺序为SEQ ID NO:48)和hCLDN18.2-841抗体(VH的氨基酸序列为SEQ ID NO:49;VL的氨基酸序列为SEQ ID NO:50)分析HEKcldn18.2细胞的HER2和密封蛋白18.2表达(图36)。图36的左分图显示HER2染色。阴性峰和阳性峰之间的距离很小,因此HER2的表达低。该数据表明了强烈的密封蛋白18.2染色,HER2的表达低。用所谓“显性CAR”和“非显性CAR”设计双重CAR,该“显性CAR”基于CD8α跨膜结构域并且对密封蛋白18.2具有特异性,该“非显性CAR”用SEZ6L2跨膜和近膜结构域修饰、缺乏共受体结构域并且对HER2具有特异性(MLB108)。可替代的非显性CAR使用IL2Rβ跨膜和近膜序列设计,但包括CD28共受体结构域(MLB040)(图37)。将双重CAR的功能性与密封蛋白18.2CAR(MLB026)进行比较。
经由菲科帕克分离将PBMC分离,并通过CD3阴性选择进行纯化以富集T细胞群。在IL-2、IL-7和IL-15存在下,将分离的T细胞用StemCell T细胞激活剂(CD2/CD3/CD28)刺激三天,然后用过度活跃的PiggyBac转座酶mRNA和PiggyBac载体MLB026和MLB108电穿孔。通过用荧光缀合的抗人F(ab’)
功能性测定表明双重CAR MLB108能够以剂量依赖性方式引起HEKcldn18.2细胞的抗原特异性杀伤(图39)。细胞毒性测定上清液中IFN-γ释放的比较表明表达MLB108双重CAR的细胞的强IFN-γ释放,这与显性CAR对密封蛋白18.2的靶向一致(图40)。
在另外的双重CAR测定中,将MLB040双重CAR形式与密封蛋白18.2特异性显性CAR(MLB026)和HER2隐形CAR(MLB038和MLB039)进行比较。经由菲科帕克分离将PBMC分离,并通过CD3阴性选择进行纯化以富集T细胞群。在IL-2、IL-7和IL-15存在下,将分离的T细胞用StemCell T细胞激活剂(CD2/CD3/CD28)刺激三天,然后用过度活跃的PiggyBac转座酶mRNA和PiggyBac载体MLB026、MLB038、MLB039和MLB040电穿孔。通过用荧光缀合的抗人F(ab’)
功能性测定表明了针对HEKcldn18.2细胞的细胞毒性略有增强,这些HEKcldn18.2细胞表达高水平的密封蛋白18.2和低水平的HER2(图42)。在该测定中,我们比较了单密封蛋白18.2(MLB026)和具有密封蛋白18.2/HER2双重CAR(MLB040)的HER2 CAR构建体的功能性活性。MLB040由显性密封蛋白18.2CAR(MLB026)和具有CD28共受体结构域的IL2Rβ隐形CAR(MLB039)构成,因此我们针对HEK293Tcldn18.2细胞比较了MLB040与MLB026、MLB038和MLB039的活性以确定分离和串联的CAR构建体的活性。我们观察到单个CAR(MLB026、MLB038和MLB039)针对HEK293T细胞的强细胞毒性,其随着E:T比率的降低而降低,显示出抗原特异性裂解。此外,我们观察到CLDN18.2/HER2双重CAR(MLB040)在所有测试的E:T比率下的增强的靶细胞杀伤。这些结果证明隐形CAR(MLB040)在双重CAR系统中仍然具有功能性。细胞毒性测定上清液的ELISA分析显示从MLB026和MLB040两者释放的IFN-γ水平相当,与显性CAR对密封蛋白18.2的靶向一致。从HER2单隐形CAR(MLB038和MLB039)中的任一个中未检测到细胞因子释放,尽管可检测到细胞毒性(图43)。当针对HER2高表达细胞系SK-BR-3进行测试时,由表达MLB038、MLB039和MLB040的T细胞观察到强烈的抗原特异性细胞毒性,而在MLB026单CAR中观察到与背景激活一致的弱细胞毒性。这些结果表明,双重靶向CAR能够识别密封蛋白18.2和HER2两者(图44)。
其他实施例
应当理解,虽然已经结合具体实施方式对本披露进行了描述,但前面的描述旨在说明而非限制本披露的范围,本披露的范围由所附的权利要求书的范围限定。其他方面、优点以及修改在以下权利要求书的范围内。
上文描述的各种实施例可以进行组合以提供另外的实施例。本说明书中提及的和/或申请数据表中列出的所有美国专利、美国专利申请出版物、美国专利申请、国外专利、国外专利申请和非专利出版物,均通过援引以其全文并入本文。这些实施例的各方面可以经修改而采用各个专利、申请和出版物的概念来提供又另外的实施例。
鉴于以上详细描述,可以对实施例做出这些和其他改变。总的来说,在以下权利要求中,所使用的术语不应被解释为将权利要求限制于说明书和权利要求书中披露的特别实施例,而是应被解释为包括所有可能的实施例以及这些权利要求有权具有的等同物的全部范围。因此,权利要求书不受本披露的限制。
引文列表:
1.Ruella,M.,&Maus,M.V.(2016).Catch me if you can:Leukemia Escapeafter CD19-Directed T Cell Immunotherapies.In Computational and StructuralBiotechnology Journal(Vol.14,pp.357–362).Elsevier B.V.https://doi.org/10.1016/j.csbj.2016.09.003.
2.Fousek,K.,Watanabe,J.,Joseph,S.K.,George,A.,An,X.,Byrd,T.T.,Morris,J.S.,Luong,A.,Martínez-Paniagua,M.A.,Sanber,K.,Navai,S.A.,Gad,A.Z.,Salsman,V.S.,Mathew,P.R.,Kim,H.N.,Wagner,D.L.,Brunetti,L.,Jang,A.,Baker,M.L.,…Ahmed,N.(2020).CAR T-cells that target acute B-lineage leukemia irrespective ofCD19 expression.Leukemia,1,4.https://doi.org/10.1038/s41375-020-0792-2.
3.Hémar,A.,Lieb,M.,Subtil,A.,Disanto,J.P.,&Dautry-Varsat,A.(1994).Endocytosis of theβchain of interleukin-2receptor requires neitherinterleukin-2 nor theγchain.European Journal of Immunology,24(9),1951–1955.https://doi.org/10.1002/eji.1830240902.
4.Tao,W.,Moore,R.,Meng,Y.,Smith,E.R.&Xu,X.X.Endocytic adaptors Arhand Dab2 control homeostasis of circulatory cholesterol.J.Lipid Res.57,809-817.
5.Fasano,T.,Sun,X.M.,Patel,D.D.&Soutar,A.K.Degradation of LDLRprotein mediated by‘gain of function’PCSK9 mutants in normal and ARHcells.Atherosclerosis 203,166-171.
6.Bonifacino,J.S.&Traub,L.M.Signals for sorting of transmembraneproteins to endosomes and lysosomes.Annual Review of Biochemistry vol.72 395-447(2003).
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Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Trp Thr Ser Thr Arg His
210 215 220
Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
225 230 235 240
Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr
245 250 255
Cys Gln Gln Tyr Ser Leu Tyr Arg Ser Phe Gly Gly Gly Thr Lys Val
260 265 270
Glu Ile Lys Gly Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala
275 280 285
Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg
290 295 300
Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys
305 310 315 320
Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu
325 330 335
Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu
340 345 350
Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln
355 360 365
Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly
370 375 380
Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr
385 390 395 400
Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
405 410 415
Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met
420 425 430
Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu
435 440 445
Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys
450 455 460
Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu
465 470 475 480
Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu
485 490 495
Pro Pro Arg Pro Ser Lys Phe Phe Ser Gln Leu
500 505
<210>9
<211>527
<212>PRT
<213>人工序列
<220>
<223>HA-MN14op CEA scFv-CD8铰链-IL2Rβ tm jm DT-4-1BB-CD3ζ(MLB003)
<400>9
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 1015
His Ala Ala Arg Pro Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Glu Val
202530
Gln Leu Val Glu Ser Gly Gly Gly Val Val Lys Pro Gly Gly Ser Leu
354045
Arg Leu Ser Cys Ser Ala Ser Gly Phe Asp Phe Thr Thr Tyr Trp Met
505560
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu
65707580
Ile His Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu Lys Asp
859095
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln
100 105 110
Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Tyr Cys Ala Ser
115 120 125
Leu Tyr Phe Gly Phe Pro Trp Phe Ala Tyr Trp Gly Gln Gly Thr Thr
130 135 140
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
145 150 155 160
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser
165 170 175
Pro Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Thr Cys
180 185 190
Lys Ala Ser Gln Asp Val Gly Thr Ser Val Ala Trp Tyr Gln Gln Lys
195 200 205
Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Trp Thr Ser Thr Arg His
210 215 220
Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
225 230 235 240
Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr
245 250 255
Cys Gln Gln Tyr Ser Leu Tyr Arg Ser Phe Gly Gly Gly Thr Lys Val
260 265 270
Glu Ile Lys Gly Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala
275 280 285
Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg
290 295 300
Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys
305 310 315 320
Asp Ile Pro Trp Leu Gly His Leu Leu Val Gly Leu Ser Gly Ala Phe
325 330 335
Gly Phe Ile Ile Leu Val Tyr Leu Leu Ile Asn Cys Arg Asn Thr Gly
340 345 350
Pro Trp Leu Lys Lys Val Leu Lys Cys Asn Thr Pro Asp Pro Ser Lys
355 360 365
Phe Phe Ser Gln Leu Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe
370 375 380
Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly
385 390 395 400
Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg
405 410 415
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln
420 425 430
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
435 440 445
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
450 455 460
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
465 470 475 480
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
485 490 495
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
500 505 510
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
515 520 525
<210>10
<211>231
<212>PRT
<213>人工序列
<220>
<223>CAR-DT△scFv
<400>10
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 1015
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
202530
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile
354045
Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val
505560
Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe
65707580
Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly
859095
Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg
100 105 110
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln
115 120 125
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
130 135 140
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
145 150 155 160
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
165 170 175
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
180 185 190
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
195 200 205
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Pro
210 215 220
Ser Lys Phe Phe Ser Gln Leu
225 230
<210>11
<211>251
<212>PRT
<213>人工序列
<220>
<223>CAR- IL2Rβ TM △scFv
<400>11
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 1015
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
202530
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Pro Trp
354045
Leu Gly His Leu Leu Val Gly Leu Ser Gly Ala Phe Gly Phe Ile Ile
505560
Leu Val Tyr Leu Leu Ile Asn Cys Arg Asn Thr Gly Pro Trp Leu Lys
65707580
Lys Val Leu Lys Cys Asn Thr Pro Asp Pro Ser Lys Phe Phe Ser Gln
859095
Leu Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
100 105 110
Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
115 120 125
Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser
130 135 140
Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr
145 150 155 160
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys
165 170 175
Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn
180 185 190
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
195 200 205
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
210 215 220
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
225 230 235 240
Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
245 250
<210>12
<211>751
<212>PRT
<213>人工序列
<220>
<223>MN14op CEA scFv -CD8铰链-CD8 tm-4-1BB-CD3ζ-P2A-GFP(MLB010)
<400>12
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 1015
His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val
202530
Val Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe
354045
Asp Phe Thr Thr Tyr Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys
505560
Gly Leu Glu Trp Ile Gly Glu Ile His Pro Asp Ser Ser Thr Ile Asn
65707580
Tyr Ala Pro Ser Leu Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala
859095
Lys Asn Thr Leu Tyr Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr
100 105 110
Gly Val Tyr Tyr Cys Ala Ser Leu Tyr Phe Gly Phe Pro Trp Phe Ala
115 120 125
Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
145 150 155 160
Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
165 170 175
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ser
180 185 190
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
195 200 205
Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly
210 215 220
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
225 230 235 240
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Leu Tyr Arg Ser
245 250 255
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Thr Thr Thr Pro Ala
260 265 270
Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser
275 280 285
Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr
290 295 300
Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala
305 310 315 320
Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys
325 330 335
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
340 345 350
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
355 360 365
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg
370 375 380
Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn
385 390 395 400
Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
405 410 415
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro
420 425 430
Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
435 440 445
Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His
450 455 460
Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp
465 470 475 480
Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Ala Thr Asn
485 490 495
Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro
500 505 510
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
515 520 525
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
530 535 540
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile
545 550 555 560
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
565 570 575
Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys
580 585 590
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
595 600 605
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
610 615 620
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
625 630 635 640
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
645 650 655
Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn
660 665 670
Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser
675 680 685
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly
690 695 700
Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu
705 710 715 720
Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
725 730 735
Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
740 745 750
<210>13
<211>754
<212>PRT
<213>人工序列
<220>
<223>841密封蛋白18.2 scFv-CD8铰链-CD8 tm-4-1BB-CD3ζ-P2A-GFP
(MLB026)
<400>13
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 1015
His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
202530
Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly
354045
Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln
505560
Gly Leu Glu Trp Met Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn
65707580
Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Val Thr Ala Asp Lys Ser
859095
Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
100 105 110
Ala Val Tyr Tyr Cys Ala Arg Glu Arg Asp Asn Trp Asp Pro Tyr Tyr
115 120 125
Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val
130 135 140
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
145 150 155 160
Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val
165 170 175
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn Ser
180 185 190
Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
195 200 205
Ile Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser
210 215 220
Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln
225 230 235 240
Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Phe Pro
245 250 255
Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Thr Thr
260 265 270
Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln
275 280 285
Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala
290 295 300
Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala
305 310 315 320
Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr
325 330 335
Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
340 345 350
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
355 360 365
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
370 375 380
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln
385 390 395 400
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
405 410 415
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
420 425 430
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
435 440 445
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
450 455 460
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
465 470 475 480
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly
485 490 495
Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn
500 505 510
Pro Gly Pro Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val
515 520 525
Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser
530 535 540
Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu
545 550 555 560
Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu
565 570 575
Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp
580 585 590
His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr
595 600 605
Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr
610 615 620
Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu
625 630 635 640
Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys
645 650 655
Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys
660 665 670
Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu
675 680 685
Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile
690 695 700
Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln
705 710 715 720
Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu
725 730 735
Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu
740 745 750
Tyr Lys
<210>14
<211>779
<212>PRT
<213>人工序列
<220>
<223>MN14op CEA scFv-CD8铰链-IL2Rβ tm jm DT-4-1BB-CD3ζ-P2A-GFP
(MLB020)
<400>14
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 1015
His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val
202530
Val Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe
354045
Asp Phe Thr Thr Tyr Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys
505560
Gly Leu Glu Trp Ile Gly Glu Ile His Pro Asp Ser Ser Thr Ile Asn
65707580
Tyr Ala Pro Ser Leu Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala
859095
Lys Asn Thr Leu Tyr Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr
100 105 110
Gly Val Tyr Tyr Cys Ala Ser Leu Tyr Phe Gly Phe Pro Trp Phe Ala
115 120 125
Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
145 150 155 160
Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
165 170 175
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ser
180 185 190
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
195 200 205
Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly
210 215 220
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
225 230 235 240
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Leu Tyr Arg Ser
245 250 255
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Thr Thr Thr Pro Ala
260 265 270
Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser
275 280 285
Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr
290 295 300
Arg Gly Leu Asp Phe Ala Cys Asp Ile Pro Trp Leu Gly His Leu Leu
305 310 315 320
Val Gly Leu Ser Gly Ala Phe Gly Phe Ile Ile Leu Val Tyr Leu Leu
325 330 335
Ile Asn Cys Arg Asn Thr Gly Pro Trp Leu Lys Lys Val Leu Lys Cys
340 345 350
Asn Thr Pro Asp Pro Ser Lys Phe Phe Ser Gln Leu Lys Arg Gly Arg
355 360 365
Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln
370 375 380
Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu
385 390 395 400
Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala
405 410 415
Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu
420 425 430
Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp
435 440 445
Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu
450 455 460
Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile
465 470 475 480
Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
485 490 495
Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met
500 505 510
Gln Ala Leu Pro Pro Arg Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu
515 520 525
Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Val Ser Lys
530 535 540
Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp
545 550 555 560
Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly
565 570 575
Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly
580 585 590
Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly
595 600 605
Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe
610 615 620
Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe
625 630 635 640
Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu
645 650 655
Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys
660 665 670
Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser
675 680 685
His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val
690 695 700
Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala
705 710 715 720
Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu
725 730 735
Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro
740 745 750
Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala
755 760 765
Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
770 775
<210>15
<211>786
<212>PRT
<213>人工序列
<220>
<223>HA-MN14op CEA scFv-CD8铰链-IL2Rβ tm jm DT-4-1BB-CD3ζ-P2A-GFP
(MLB013)
<400>15
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 1015
His Ala Ala Arg Pro Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Met Glu
202530
Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Lys Pro Gly Gly Ser
354045
Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Asp Phe Thr Thr Tyr Trp
505560
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly
65707580
Glu Ile His Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu Lys
859095
Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu
100 105 110
Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Tyr Cys Ala
115 120 125
Ser Leu Tyr Phe Gly Phe Pro Trp Phe Ala Tyr Trp Gly Gln Gly Thr
130 135 140
Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
145 150 155 160
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln
165 170 175
Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Thr
180 185 190
Cys Lys Ala Ser Gln Asp Val Gly Thr Ser Val Ala Trp Tyr Gln Gln
195 200 205
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Trp Thr Ser Thr Arg
210 215 220
His Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
225 230 235 240
Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr
245 250 255
Tyr Cys Gln Gln Tyr Ser Leu Tyr Arg Ser Phe Gly Gly Gly Thr Lys
260 265 270
Val Glu Ile Lys Gly Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro
275 280 285
Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys
290 295 300
Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala
305 310 315 320
Cys Asp Ile Pro Trp Leu Gly His Leu Leu Val Gly Leu Ser Gly Ala
325 330 335
Phe Gly Phe Ile Ile Leu Val Tyr Leu Leu Ile Asn Cys Arg Asn Thr
340 345 350
Gly Pro Trp Leu Lys Lys Val Leu Lys Cys Asn Thr Pro Asp Pro Ser
355 360 365
Lys Phe Phe Ser Gln Leu Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile
370 375 380
Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp
385 390 395 400
Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
405 410 415
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly
420 425 430
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
435 440 445
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
450 455 460
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
465 470 475 480
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
485 490 495
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
500 505 510
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
515 520 525
Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn
530 535 540
Pro Gly Pro Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val
545 550 555 560
Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser
565 570 575
Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu
580 585 590
Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu
595 600 605
Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp
610 615 620
His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr
625 630 635 640
Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr
645 650 655
Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu
660 665 670
Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys
675 680 685
Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys
690 695 700
Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu
705 710 715 720
Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile
725 730 735
Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln
740 745 750
Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu
755 760 765
Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu
770 775 780
Tyr Lys
785
<210>16
<211>759
<212>PRT
<213>人工序列
<220>
<223>MN14op CEA scFv-CD8铰链- CD8 tm-4-1BB-CD3ζ-IL2Rβ DT-P2A-GFP
(MLB025)
<400>16
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 1015
His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val
202530
Val Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe
354045
Asp Phe Thr Thr Tyr Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys
505560
Gly Leu Glu Trp Ile Gly Glu Ile His Pro Asp Ser Ser Thr Ile Asn
65707580
Tyr Ala Pro Ser Leu Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala
859095
Lys Asn Thr Leu Tyr Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr
100 105 110
Gly Val Tyr Tyr Cys Ala Ser Leu Tyr Phe Gly Phe Pro Trp Phe Ala
115 120 125
Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
145 150 155 160
Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
165 170 175
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ser
180 185 190
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
195 200 205
Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly
210 215 220
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
225 230 235 240
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Leu Tyr Arg Ser
245 250 255
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Thr Thr Thr Pro Ala
260 265 270
Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser
275 280 285
Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr
290 295 300
Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala
305 310 315 320
Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys
325 330 335
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
340 345 350
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
355 360 365
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg
370 375 380
Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn
385 390 395 400
Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
405 410 415
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro
420 425 430
Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
435 440 445
Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His
450 455 460
Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp
465 470 475 480
Ala Leu His Met Gln Ala Leu Pro Pro Arg Pro Ser Lys Phe Phe Ser
485 490 495
Gln Leu Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly
500 505 510
Asp Val Glu Glu Asn Pro Gly Pro Met Val Ser Lys Gly Glu Glu Leu
515 520 525
Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn
530 535 540
Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr
545 550 555 560
Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val
565 570 575
Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe
580 585 590
Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala
595 600 605
Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp
610 615 620
Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu
625 630 635 640
Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn
645 650 655
Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr
660 665 670
Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile
675 680 685
Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln
690 695 700
Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His
705 710 715 720
Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg
725 730 735
Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu
740 745 750
Gly Met Asp Glu Leu Tyr Lys
755
<210>17
<211>781
<212>PRT
<213>人工序列
<220>
<223>MN14op CEA scFv-CD8铰链-SEZ6L2 tm jm-4-1BB-CD3ζ-P2A-GFP
(MLB047)
<400>17
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 1015
His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val
202530
Val Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe
354045
Asp Phe Thr Thr Tyr Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys
505560
Gly Leu Glu Trp Ile Gly Glu Ile His Pro Asp Ser Ser Thr Ile Asn
65707580
Tyr Ala Pro Ser Leu Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala
859095
Lys Asn Thr Leu Tyr Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr
100 105 110
Gly Val Tyr Tyr Cys Ala Ser Leu Tyr Phe Gly Phe Pro Trp Phe Ala
115 120 125
Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
145 150 155 160
Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
165 170 175
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ser
180 185 190
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
195 200 205
Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly
210 215 220
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
225 230 235 240
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Leu Tyr Arg Ser
245 250 255
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Thr Thr Thr Pro Ala
260 265 270
Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser
275 280 285
Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr
290 295 300
Arg Gly Leu Asp Phe Ala Cys Asp Leu Ala Leu Ala Ile Leu Leu Pro
305 310 315 320
Leu Gly Leu Val Ile Val Leu Gly Ser Gly Val Tyr Ile Tyr Tyr Thr
325 330 335
Lys Leu Gln Gly Lys Ser Leu Phe Gly Phe Ser Gly Ser His Ser Tyr
340 345 350
Ser Pro Ile Thr Val Glu Ser Asp Phe Ser Asn Pro Leu Tyr Lys Arg
355 360 365
Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro
370 375 380
Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu
385 390 395 400
Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala
405 410 415
Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu
420 425 430
Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
435 440 445
Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu
450 455 460
Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser
465 470 475 480
Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly
485 490 495
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
500 505 510
His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Ala Thr Asn Phe Ser
515 520 525
Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Val
530 535 540
Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu
545 550 555 560
Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly
565 570 575
Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr
580 585 590
Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr
595 600 605
Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His
610 615 620
Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr
625 630 635 640
Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys
645 650 655
Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp
660 665 670
Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr
675 680 685
Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile
690 695 700
Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln
705 710 715 720
Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val
725 730 735
Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys
740 745 750
Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr
755 760 765
Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
770 775 780
<210>18
<211>767
<212>PRT
<213>人工序列
<220>
<223>MN14op CEA scFv-CD8铰链-LDLR tm jm-4-1BB-CD3ζ-P2A-GFP(MLB048)
<400>18
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 1015
His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val
202530
Val Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe
354045
Asp Phe Thr Thr Tyr Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys
505560
Gly Leu Glu Trp Ile Gly Glu Ile His Pro Asp Ser Ser Thr Ile Asn
65707580
Tyr Ala Pro Ser Leu Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala
859095
Lys Asn Thr Leu Tyr Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr
100 105 110
Gly Val Tyr Tyr Cys Ala Ser Leu Tyr Phe Gly Phe Pro Trp Phe Ala
115 120 125
Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
145 150 155 160
Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
165 170 175
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ser
180 185 190
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
195 200 205
Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly
210 215 220
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
225 230 235 240
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Leu Tyr Arg Ser
245 250 255
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Thr Thr Thr Pro Ala
260 265 270
Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser
275 280 285
Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr
290 295 300
Arg Gly Leu Asp Phe Ala Cys Asp Ala Leu Ser Ile Val Leu Pro Ile
305 310 315 320
Val Leu Leu Val Phe Leu Cys Leu Gly Val Phe Leu Leu Trp Lys Asn
325 330 335
Trp Arg Leu Lys Asn Ile Asn Ser Ile Asn Phe Asp Asn Pro Val Tyr
340 345 350
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
355 360 365
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
370 375 380
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg
385 390 395 400
Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn
405 410 415
Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
420 425 430
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro
435 440 445
Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
450 455 460
Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His
465 470 475 480
Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp
485 490 495
Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Ala Thr Asn
500 505 510
Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro
515 520 525
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
530 535 540
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
545 550 555 560
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile
565 570 575
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
580 585 590
Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys
595 600 605
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
610 615 620
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
625 630 635 640
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
645 650 655
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
660 665 670
Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn
675 680 685
Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser
690 695 700
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly
705 710 715 720
Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu
725 730 735
Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
740 745 750
Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
755 760 765
<210>19
<211>784
<212>PRT
<213>人工序列
<220>
<223>841密封蛋白18.2 scFv-CD8铰链-SEZ6L2 tm jm-4-1BB-CD3ζ-P2A-GFP
(MLB054)
<400>19
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 1015
His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
202530
Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly
354045
Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln
505560
Gly Leu Glu Trp Met Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn
65707580
Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Val Thr Ala Asp Lys Ser
859095
Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
100 105 110
Ala Val Tyr Tyr Cys Ala Arg Glu Arg Asp Asn Trp Asp Pro Tyr Tyr
115 120 125
Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val
130 135 140
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
145 150 155 160
Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val
165 170 175
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn Ser
180 185 190
Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
195 200 205
Ile Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser
210 215 220
Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln
225 230 235 240
Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Phe Pro
245 250 255
Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Thr Thr
260 265 270
Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln
275 280 285
Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala
290 295 300
Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Leu Ala Leu Ala Ile
305 310 315 320
Leu Leu Pro Leu Gly Leu Val Ile Val Leu Gly Ser Gly Val Tyr Ile
325 330 335
Tyr Tyr Thr Lys Leu Gln Gly Lys Ser Leu Phe Gly Phe Ser Gly Ser
340 345 350
His Ser Tyr Ser Pro Ile Thr Val Glu Ser Asp Phe Ser Asn Pro Leu
355 360 365
Tyr Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
370 375 380
Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
385 390 395 400
Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser
405 410 415
Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr
420 425 430
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys
435 440 445
Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn
450 455 460
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
465 470 475 480
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
485 490 495
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
500 505 510
Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Ala Thr
515 520 525
Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly
530 535 540
Pro Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile
545 550 555 560
Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser
565 570 575
Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe
580 585 590
Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr
595 600 605
Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met
610 615 620
Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln
625 630 635 640
Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala
645 650 655
Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys
660 665 670
Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu
675 680 685
Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys
690 695 700
Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly
705 710 715 720
Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp
725 730 735
Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala
740 745 750
Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu
755 760 765
Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
770 775 780
<210>20
<211>1169
<212>PRT
<213>人工序列
<220>
<223>841密封蛋白18.2 scFv-CD8铰链-SEZ6L2 tm jm-4-1BB-CD3ζ-P2A-
PD-L1 scFv-CD8铰链-CD8 tm-CD28-(G4S)2GFP(MLB055)
<400>20
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 1015
His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
202530
Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly
354045
Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln
505560
Gly Leu Glu Trp Met Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn
65707580
Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Val Thr Ala Asp Lys Ser
859095
Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
100 105 110
Ala Val Tyr Tyr Cys Ala Arg Glu Arg Asp Asn Trp Asp Pro Tyr Tyr
115 120 125
Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val
130 135 140
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
145 150 155 160
Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val
165 170 175
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn Ser
180 185 190
Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
195 200 205
Ile Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser
210 215 220
Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln
225 230 235 240
Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Phe Pro
245 250 255
Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Thr Thr
260 265 270
Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln
275 280 285
Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala
290 295 300
Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Leu Ala Leu Ala Ile
305 310 315 320
Leu Leu Pro Leu Gly Leu Val Ile Val Leu Gly Ser Gly Val Tyr Ile
325 330 335
Tyr Tyr Thr Lys Leu Gln Gly Lys Ser Leu Phe Gly Phe Ser Gly Ser
340 345 350
His Ser Tyr Ser Pro Ile Thr Val Glu Ser Asp Phe Ser Asn Pro Leu
355 360 365
Tyr Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
370 375 380
Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
385 390 395 400
Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser
405 410 415
Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr
420 425 430
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys
435 440 445
Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn
450 455 460
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
465 470 475 480
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
485 490 495
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
500 505 510
Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Ala Thr
515 520 525
Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly
530 535 540
Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu
545 550 555 560
Leu His Ala Ala Arg Pro Glu Val Gln Leu Val Gln Ser Gly Gly Gly
565 570 575
Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
580 585 590
Phe Thr Phe Ser Asn Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly
595 600 605
Lys Gly Leu Glu Trp Val Ala Ile Ile Thr Tyr Ala Gly Ser Asn Glu
610 615 620
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
625 630 635 640
Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp
645 650 655
Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Ile Trp Val Asp Tyr Trp
660 665 670
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
675 680 685
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser
690 695 700
Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys
705 710 715 720
Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu
725 730 735
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr
740 745 750
Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser
755 760 765
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu
770 775 780
Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Ser Thr Pro Leu Thr
785 790 795 800
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Thr Thr Thr Pro Ala Pro
805 810 815
Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu
820 825 830
Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg
835 840 845
Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly
850 855 860
Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg
865 870 875 880
Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro
885 890 895
Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro
900 905 910
Arg Asp Phe Ala Ala Tyr Arg Ser Gly Gly Gly Gly Ser Gly Gly Gly
915 920 925
Gly Ser Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro
930 935 940
Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val
945 950 955 960
Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys
965 970 975
Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val
980 985 990
Thr Thr Leu Thr Tyr Gly Val GlnCys Phe Ser Arg TyrPro Asp His
995 1000 1005
Met LysGln His Asp Phe PheLys Ser Ala Met ProGlu Gly Tyr
1010 1015 1020
Val GlnGlu Arg Thr Ile PhePhe Lys Asp Asp GlyAsn Tyr Lys
1025 1030 1035
Thr ArgAla Glu Val Lys PheGlu Gly Asp Thr LeuVal Asn Arg
1040 1045 1050
Ile GluLeu Lys Gly Ile AspPhe Lys Glu Asp GlyAsn Ile Leu
1055 1060 1065
Gly HisLys Leu Glu Tyr AsnTyr Asn Ser His AsnVal Tyr Ile
1070 1075 1080
Met AlaAsp Lys Gln Lys AsnGly Ile Lys Val AsnPhe Lys Ile
1085 1090 1095
Arg HisAsn Ile Glu Asp GlySer Val Gln Leu AlaAsp His Tyr
1100 1105 1110
Gln GlnAsn Thr Pro Ile GlyAsp Gly Pro Val LeuLeu Pro Asp
1115 1120 1125
Asn HisTyr Leu Ser Thr GlnSer Ala Leu Ser LysAsp Pro Asn
1130 1135 1140
Glu LysArg Asp His Met ValLeu Leu Glu Phe ValThr Ala Ala
1145 1150 1155
Gly IleThr Leu Gly Met AspGlu Leu Tyr Lys
1160 1165
<210>21
<211>777
<212>PRT
<213>人工序列
<220>
<223>HER2 scFv-CD8铰链-IL2Rβ tm jm DT-4-1BB-CD3ζ-P2A-GFP(MLB038)
<400>21
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 1015
His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
202530
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
354045
Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
505560
Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro
65707580
Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile
859095
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His
100 105 110
Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
130 135 140
Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
145 150 155 160
Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn
165 170 175
Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly
180 185 190
Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr
195 200 205
Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys
210 215 220
Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
225 230 235 240
Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp
245 250 255
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro
260 265 270
Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu
275 280 285
Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His
290 295 300
Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Pro Trp Leu Gly His Leu
305 310 315 320
Leu Val Gly Leu Ser Gly Ala Phe Gly Phe Ile Ile Leu Val Tyr Leu
325 330 335
Leu Ile Asn Cys Arg Asn Thr Gly Pro Trp Leu Lys Lys Val Leu Lys
340 345 350
Cys Asn Thr Pro Asp Pro Ser Lys Phe Phe Ser Gln Leu Lys Arg Gly
355 360 365
Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val
370 375 380
Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu
385 390 395 400
Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp
405 410 415
Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn
420 425 430
Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg
435 440 445
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly
450 455 460
Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
465 470 475 480
Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu
485 490 495
Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His
500 505 510
Met Gln Ala Leu Pro Pro Arg Ala Thr Asn Phe Ser Leu Leu Lys Gln
515 520 525
Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Val Ser Lys Gly Glu
530 535 540
Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp
545 550 555 560
Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala
565 570 575
Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu
580 585 590
Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln
595 600 605
Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys
610 615 620
Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys
625 630 635 640
Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp
645 650 655
Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp
660 665 670
Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn
675 680 685
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe
690 695 700
Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His
705 710 715 720
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp
725 730 735
Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu
740 745 750
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
755 760 765
Thr Leu Gly Met Asp Glu Leu Tyr Lys
770 775
<210>22
<211>776
<212>PRT
<213>人工序列
<220>
<223>HER2 scFv-CD8铰链-IL2Rβ tm jm DT- CD28-CD3ζ-P2A-GFP(MLB039)
<400>22
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 1015
His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
202530
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
354045
Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
505560
Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro
65707580
Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile
859095
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His
100 105 110
Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
130 135 140
Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
145 150 155 160
Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn
165 170 175
Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly
180 185 190
Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr
195 200 205
Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys
210 215 220
Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
225 230 235 240
Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp
245 250 255
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro
260 265 270
Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu
275 280 285
Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His
290 295 300
Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Pro Trp Leu Gly His Leu
305 310 315 320
Leu Val Gly Leu Ser Gly Ala Phe Gly Phe Ile Ile Leu Val Tyr Leu
325 330 335
Leu Ile Asn Cys Arg Asn Thr Gly Pro Trp Leu Lys Lys Val Leu Lys
340 345 350
Cys Asn Thr Pro Asp Pro Ser Lys Phe Phe Ser Gln Leu Arg Ser Lys
355 360 365
Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg
370 375 380
Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp
385 390 395 400
Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala
405 410 415
Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu
420 425 430
Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp
435 440 445
Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu
450 455 460
Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile
465 470 475 480
Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
485 490 495
Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met
500 505 510
Gln Ala Leu Pro Pro Arg Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala
515 520 525
Gly Asp Val Glu Glu Asn Pro Gly Pro Met Val Ser Lys Gly Glu Glu
530 535 540
Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val
545 550 555 560
Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr
565 570 575
Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro
580 585 590
Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys
595 600 605
Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser
610 615 620
Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp
625 630 635 640
Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr
645 650 655
Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly
660 665 670
Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val
675 680 685
Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys
690 695 700
Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr
705 710 715 720
Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn
725 730 735
His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys
740 745 750
Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr
755 760 765
Leu Gly Met Asp Glu Leu Tyr Lys
770 775
<210>23
<211>752
<212>PRT
<213>人工序列
<220>
<223>HER2 scFv-CD8铰链-CD8 tm-4-1BB-CD3ζ-P2A-GFP(MLB079)
<400>23
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 1015
His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
202530
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
354045
Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
505560
Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro
65707580
Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile
859095
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His
100 105 110
Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
130 135 140
Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
145 150 155 160
Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn
165 170 175
Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly
180 185 190
Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr
195 200 205
Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys
210 215 220
Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
225 230 235 240
Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp
245 250 255
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro
260 265 270
Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu
275 280 285
Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His
290 295 300
Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu
305 310 315 320
Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr
325 330 335
Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
340 345 350
Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
355 360 365
Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser
370 375 380
Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr
385 390 395 400
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys
405 410 415
Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn
420 425 430
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
435 440 445
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
450 455 460
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
465 470 475 480
Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Ala Thr
485 490 495
Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly
500 505 510
Pro Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile
515 520 525
Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser
530 535 540
Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe
545 550 555 560
Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr
565 570 575
Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met
580 585 590
Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln
595 600 605
Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala
610 615 620
Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys
625 630 635 640
Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu
645 650 655
Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys
660 665 670
Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly
675 680 685
Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp
690 695 700
Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala
705 710 715 720
Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu
725 730 735
Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
740 745 750
<210>24
<211>782
<212>PRT
<213>人工序列
<220>
<223>HER2 scFv-CD8铰链-SEZ6L2 tm jm-4-1BB-CD3ζ-P2A-GFP(MLB080)
<400>24
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 1015
His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
202530
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
354045
Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
505560
Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro
65707580
Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile
859095
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His
100 105 110
Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
130 135 140
Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
145 150 155 160
Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn
165 170 175
Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly
180 185 190
Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr
195 200 205
Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys
210 215 220
Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
225 230 235 240
Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp
245 250 255
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro
260 265 270
Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu
275 280 285
Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His
290 295 300
Thr Arg Gly Leu Asp Phe Ala Cys Asp Leu Ala Leu Ala Ile Leu Leu
305 310 315 320
Pro Leu Gly Leu Val Ile Val Leu Gly Ser Gly Val Tyr Ile Tyr Tyr
325 330 335
Thr Lys Leu Gln Gly Lys Ser Leu Phe Gly Phe Ser Gly Ser His Ser
340 345 350
Tyr Ser Pro Ile Thr Val Glu Ser Asp Phe Ser Asn Pro Leu Tyr Lys
355 360 365
Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg
370 375 380
Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro
385 390 395 400
Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser
405 410 415
Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu
420 425 430
Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg
435 440 445
Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln
450 455 460
Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr
465 470 475 480
Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp
485 490 495
Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala
500 505 510
Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Ala Thr Asn Phe
515 520 525
Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met
530 535 540
Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val
545 550 555 560
Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu
565 570 575
Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys
580 585 590
Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu
595 600 605
Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln
610 615 620
His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg
625 630 635 640
Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val
645 650 655
Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile
660 665 670
Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn
675 680 685
Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly
690 695 700
Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val
705 710 715 720
Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro
725 730 735
Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser
740 745 750
Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val
755 760 765
Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
770 775 780
<210>25
<211>1033
<212>PRT
<213>人工序列
<220>
<223>841密封蛋白18.2 scFv-CD8铰链-CD8 tm-4-1BB-CD3ζ-P2A-HER2
scFv-IL2Rβ tm jm DT-CD28-CD3ζ(MLB040)
<400>25
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 1015
His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
202530
Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly
354045
Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln
505560
Gly Leu Glu Trp Met Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn
65707580
Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Val Thr Ala Asp Lys Ser
859095
Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
100 105 110
Ala Val Tyr Tyr Cys Ala Arg Glu Arg Asp Asn Trp Asp Pro Tyr Tyr
115 120 125
Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val
130 135 140
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
145 150 155 160
Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val
165 170 175
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn Ser
180 185 190
Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
195 200 205
Ile Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser
210 215 220
Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln
225 230 235 240
Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Phe Pro
245 250 255
Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Thr Thr
260 265 270
Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln
275 280 285
Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala
290 295 300
Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala
305 310 315 320
Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr
325 330 335
Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
340 345 350
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
355 360 365
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
370 375 380
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln
385 390 395 400
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
405 410 415
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
420 425 430
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
435 440 445
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
450 455 460
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
465 470 475 480
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly
485 490 495
Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn
500 505 510
Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala
515 520 525
Leu Leu Leu His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro
530 535 540
Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg
545 550 555 560
Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro
565 570 575
Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser
580 585 590
Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr
595 600 605
Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
610 615 620
Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val
625 630 635 640
Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
645 650 655
Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly
660 665 670
Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser
675 680 685
Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro
690 695 700
Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr
705 710 715 720
Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp
725 730 735
Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
740 745 750
Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr
755 760 765
Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr
770 775 780
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
785 790 795 800
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
805 810 815
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Pro Trp Leu
820 825 830
Gly His Leu Leu Val Gly Leu Ser Gly Ala Phe Gly Phe Ile Ile Leu
835 840 845
Val Tyr Leu Leu Ile Asn Cys Arg Asn Thr Gly Pro Trp Leu Lys Lys
850 855 860
Val Leu Lys Cys Asn Thr Pro Asp Pro Ser Lys Phe Phe Ser Gln Leu
865 870 875 880
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr
885 890 895
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
900 905 910
Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser
915 920 925
Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu
930 935 940
Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg
945 950 955 960
Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln
965 970 975
Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr
980 985 990
Ser Glu Ile Gly Met Lys Gly GluArg Arg Arg Gly LysGly His Asp
995 1000 1005
Gly LeuTyr Gln Gly Leu SerThr Ala Thr Lys AspThr Tyr Asp
1010 1015 1020
Ala LeuHis Met Gln Ala LeuPro Pro Arg
1025 1030
<210>26
<211>994
<212>PRT
<213>人工序列
<220>
<223>841 密封蛋白 18.2 scFv-CD8铰链-CD8 tm-4-1BB-CD3ζ-P2A-HER2
scFv-SEZ6L2 tm jm-CD3ζ(MLB108)
<400>26
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 1015
His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
202530
Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly
354045
Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln
505560
Gly Leu Glu Trp Met Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn
65707580
Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Val Thr Ala Asp Lys Ser
859095
Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
100 105 110
Ala Val Tyr Tyr Cys Ala Arg Glu Arg Asp Asn Trp Asp Pro Tyr Tyr
115 120 125
Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val
130 135 140
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
145 150 155 160
Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val
165 170 175
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn Ser
180 185 190
Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
195 200 205
Ile Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser
210 215 220
Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln
225 230 235 240
Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Phe Pro
245 250 255
Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Thr Thr
260 265 270
Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln
275 280 285
Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala
290 295 300
Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala
305 310 315 320
Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr
325 330 335
Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
340 345 350
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
355 360 365
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
370 375 380
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln
385 390 395 400
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
405 410 415
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
420 425 430
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
435 440 445
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
450 455 460
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
465 470 475 480
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly
485 490 495
Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn
500 505 510
Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala
515 520 525
Leu Leu Leu His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro
530 535 540
Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg
545 550 555 560
Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro
565 570 575
Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser
580 585 590
Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr
595 600 605
Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
610 615 620
Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val
625 630 635 640
Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
645 650 655
Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly
660 665 670
Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser
675 680 685
Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro
690 695 700
Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr
705 710 715 720
Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp
725 730 735
Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
740 745 750
Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr
755 760 765
Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr
770 775 780
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
785 790 795 800
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
805 810 815
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Leu Ala Leu Ala
820 825 830
Ile Leu Leu Pro Leu Gly Leu Val Ile Val Leu Gly Ser Gly Val Tyr
835 840 845
Ile Tyr Tyr Thr Lys Leu Gln Gly Lys Ser Leu Phe Gly Phe Ser Gly
850 855 860
Ser His Ser Tyr Ser Pro Ile Thr Val Glu Ser Asp Phe Ser Asn Pro
865 870 875 880
Leu Tyr Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys
885 890 895
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
900 905 910
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
915 920 925
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
930 935 940
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
945 950 955 960
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
965 970 975
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
980 985 990
Pro Arg
<210>27
<211>44
<212>PRT
<213>人工序列
<220>
<223>IL2Rβ的跨膜连接近膜
(IL2Rβ tm jm)
<400>27
Ile Pro Trp Leu Gly His Leu Leu Val Gly Leu Ser Gly Ala Phe Gly
1 5 1015
Phe Ile Ile Leu Val Tyr Leu Leu Ile Asn Cys Arg Asn Thr Gly Pro
202530
Trp Leu Lys Lys Val Leu Lys Cys Asn Thr Pro Asp
3540
<210>28
<211>40
<212>PRT
<213>人工序列
<220>
<223>LDLR的跨膜连接近膜
(LDLR tm jm)
<400>28
Ala Leu Ser Ile Val Leu Pro Ile Val Leu Leu Val Phe Leu Cys Leu
1 5 1015
Gly Val Phe Leu Leu Trp Lys Asn Trp Arg Leu Lys Asn Ile Asn Ser
202530
Ile Asn Phe Asp Asn Pro Val Tyr
3540
<210>29
<211>54
<212>PRT
<213>人工序列
<220>
<223>SEZ6L的跨膜连接近膜(SEZ6L2 tm jm)
<400>29
Leu Ala Leu Ala Ile Leu Leu Pro Leu Gly Leu Val Ile Val Leu Gly
1 5 1015
Ser Gly Val Tyr Ile Tyr Tyr Thr Lys Leu Gln Gly Lys Ser Leu Phe
202530
Gly Phe Ser Gly Ser His Ser Tyr Ser Pro Ile Thr Val Glu Ser Asp
354045
Phe Ser Asn Pro Leu Tyr
50
<210>30
<211>249
<212>PRT
<213>人工序列
<220>
<223>841密封蛋白18.2scFv
<400>30
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 1015
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr
202530
Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
354045
Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe
505560
Gln Gly Arg Val Thr Val Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65707580
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
859095
Ala Arg Glu Arg Asp Asn Trp Asp Pro Tyr Tyr Tyr Tyr Tyr Gly Met
100 105 110
Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met
130 135 140
Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly Asp Arg Val Thr
145 150 155 160
Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn Ser Trp Leu Ala Trp Tyr
165 170 175
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys Ala Ser
180 185 190
Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
195 200 205
Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala
210 215 220
Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Phe Pro Leu Thr Phe Gly Gly
225 230 235 240
Gly Thr Lys Val Glu Ile Lys Arg Thr
245
<210>31
<211>247
<212>PRT
<213>人工序列
<220>
<223>HER2 scFv
<400>31
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 1015
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
202530
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
354045
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
505560
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65707580
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
859095
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
115 120 125
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser
130 135 140
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr
145 150 155 160
Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala
165 170 175
Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys
180 185 190
Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu
195 200 205
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser
210 215 220
Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly
225 230 235 240
Thr Leu Val Thr Val Ser Ser
245
<210>32
<211>244
<212>PRT
<213>人工序列
<220>
<223>PD-L1 scFv
<400>32
Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 1015
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
202530
Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
354045
Ala Ile Ile Thr Tyr Ala Gly Ser Asn Glu Tyr Tyr Ala Asp Ser Val
505560
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65707580
Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys
859095
Ala Arg Asp Arg Ile Trp Val Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
115 120 125
Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Asp Ser Leu Ala Val
130 135 140
Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val
145 150 155 160
Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys
165 170 175
Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu
180 185 190
Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
195 200 205
Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr
210 215 220
Cys Gln Gln Tyr Tyr Ser Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys
225 230 235 240
Val Glu Ile Lys
<210>33
<211>9
<212>PRT
<213>人工序列
<220>
<223>HA
<400>33
Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
1 5
<210>34
<211>45
<212>PRT
<213>人工序列
<220>
<223>CD8铰链
<400>34
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 1015
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
202530
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
354045
<210>35
<211>112
<212>PRT
<213>人工序列
<220>
<223>CD3ζ
<400>35
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly
1 5 1015
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
202530
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
354045
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
505560
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65707580
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
859095
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210>36
<211>42
<212>PRT
<213>人工序列
<220>
<223>4-1BB
<400>36
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
1 5 1015
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
202530
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
3540
<210>37
<211>41
<212>PRT
<213>人工序列
<220>
<223>CD28
<400>37
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr
1 5 1015
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
202530
Pro Arg Asp Phe Ala Ala Tyr Arg Ser
3540
<210>38
<211>22
<212>PRT
<213>人工序列
<220>
<223>LDLR的跨膜(LDLR tm)
<400>38
Ala Leu Ser Ile Val Leu Pro Ile Val Leu Leu Val Phe Leu Cys Leu
1 5 1015
Gly Val Phe Leu Leu Trp
20
<210>39
<211>18
<212>PRT
<213>人工序列
<220>
<223>LDLR的近膜(LDLR jm)
<400>39
Lys Asn Trp Arg Leu Lys Asn Ile Asn Ser Ile Asn Phe Asp Asn Pro
1 5 1015
Val Tyr
<210>40
<211>21
<212>PRT
<213>人工序列
<220>
<223>SEZ6L2的跨膜(SEZ6L2 tm)
<400>40
Leu Ala Leu Ala Ile Leu Leu Pro Leu Gly Leu Val Ile Val Leu Gly
1 5 1015
Ser Gly Val Tyr Ile
20
<210>41
<211>33
<212>PRT
<213>人工序列
<220>
<223>SEZ6L2的近膜(SEZ6L2 jm)
<400>41
Tyr Tyr Thr Lys Leu Gln Gly Lys Ser Leu Phe Gly Phe Ser Gly Ser
1 5 1015
His Ser Tyr Ser Pro Ile Thr Val Glu Ser Asp Phe Ser Asn Pro Leu
202530
Tyr
<210>42
<211>24
<212>PRT
<213>人工序列
<220>
<223>CD8跨膜(CD8 tm)
<400>42
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
1 5 1015
Ser Leu Val Ile Thr Leu Tyr Cys
20
<210>43
<211>22
<212>PRT
<213>人工序列
<220>
<223>P2A-1
<400>43
Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val
1 5 1015
Glu Glu Asn Pro Gly Pro
20
<210>44
<211>19
<212>PRT
<213>人工序列
<220>
<223>P2A-2
<400>44
Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn
1 5 1015
Pro Gly Pro
<210>45
<211>239
<212>PRT
<213>人工序列
<220>
<223>GFP
<400>45
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
1 5 1015
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
202530
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile
354045
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
505560
Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys
65707580
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
859095
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
100 105 110
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
115 120 125
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
130 135 140
Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn
145 150 155 160
Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser
165 170 175
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly
180 185 190
Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu
195 200 205
Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
210 215 220
Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
225 230 235
<210>46
<211>21
<212>PRT
<213>人工序列
<220>
<223>前导序列
<400>46
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 1015
His Ala Ala Arg Pro
20
<210>47
<211>124
<212>PRT
<213>人工序列
<220>
<223>hCLDN18.2-808抗体的VH
<400>47
Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 1015
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
202530
Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
354045
Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
505560
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65707580
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
859095
Ala Arg Glu Leu Arg Phe Phe Asp Trp Leu Leu Gly Ser Ala Phe Asp
100 105 110
Ile Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210>48
<211>107
<212>PRT
<213>人工序列
<220>
<223>hCLDN18.2-808抗体的VL
<400>48
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 1015
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp
202530
Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
354045
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
505560
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65707580
Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Asp Tyr Asn Tyr Pro Arg
859095
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210>49
<211>125
<212>PRT
<213>人工序列
<220>
<223>hCLDN18.2-841抗体的VH
<400>49
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 1015
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr
202530
Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
354045
Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe
505560
Gln Gly Arg Val Thr Val Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65707580
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
859095
Ala Arg Glu Arg Asp Asn Trp Asp Pro Tyr Tyr Tyr Tyr Tyr Gly Met
100 105 110
Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 125
<210>50
<211>107
<212>PRT
<213>人工序列
<220>
<223>hCLDN18.2-841抗体的VL
<400>50
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
1 5 1015
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn Ser Trp
202530
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
354045
Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
505560
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65707580
Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Phe Pro Leu
859095
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105