CN113913379A - T淋巴细胞及其应用 - Google Patents
T淋巴细胞及其应用 Download PDFInfo
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- CN113913379A CN113913379A CN202110763406.5A CN202110763406A CN113913379A CN 113913379 A CN113913379 A CN 113913379A CN 202110763406 A CN202110763406 A CN 202110763406A CN 113913379 A CN113913379 A CN 113913379A
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Abstract
本发明提出了一种T淋巴细胞。该T淋巴细胞共表达融合蛋白以及嵌合抗原受体,所述嵌合抗原受体识别肿瘤抗原,其中,所述嵌合抗原受体包括:胞外区;跨膜区,所述跨膜区与所述胞外区相连,并且嵌入到所述转基因淋巴细胞的细胞膜中;胞内区,所述胞内区与所述跨膜区相连,并且所述胞内区包括免疫共刺激分子胞内段;所述融合蛋白包括:免疫检查点单链抗体和T细胞激活分子。该T淋巴细胞作用对肿瘤的杀伤更为有效、长效,安全性更高。
Description
技术领域
本发明涉及生物制药领域,具体地,本发明涉及T淋巴细胞及其应用,更具体地,本发明涉及T淋巴细胞、慢病毒、转基因淋巴细胞、构建体、制备T淋巴细胞或者转基因淋巴细胞的方法、治疗癌症的治疗组合物以及降低T淋巴细胞表面免疫检查点表达的方法。
背景技术
嵌合抗原受体T细胞免疫疗法,简称CAR-T技术,是通过体外改造病人的T细胞,使病人的T细胞具备识别肿瘤细胞的能力,体外扩大培养后回输到病人体内进行治疗的一种方法。目前,以CD19为靶点的CAR-T在治疗B细胞血液肿瘤方面取得了巨大的成果,但根据临床研究结果来看,CD19 CAR-T在治疗B细胞淋巴瘤方面的疗效远远不及在治疗B细胞急性淋巴细胞白血病方面的疗效,这可能是因为B细胞淋巴瘤是实体肿瘤,其细胞表面表达有大量的PD-L1分子造成的,虽然目前尚未有文献对B-ALL病人B细胞上PD-L1分子表达水平报道,但是在针对弥漫性大B细胞淋巴瘤的临床研究中发现病人B细胞淋巴瘤表面的PD-L1分子表达水平与临床疗效直接相关,B细胞淋巴瘤表面PD-L1表达低的病人,在综合疗法、单独化疗和PD-1抗体免疫疗法中均有着更高的生存率。与此同时,运用CART技术在对其它靶点实体肿瘤治疗的过程中,肿瘤细胞也会通过过表达PD-L1来逃避CAR-T对其进行杀伤,提示实体瘤治疗过程中,封闭PD-1/PD-L1信号通路将会最大程度的增加CAR-T的临床疗效,使患者受益。
IL-21由CD4 T细胞和NKT细胞产生,刺激CD8 T细胞和NK细胞的成熟并增强其细胞毒性,同时具备促进记忆性CD8 T细胞的分化等功能。IL-21的诸多效能使其成为免疫治疗的潜在靶点,但由于IL-21R广泛表达于包括T细胞、B细胞、NK细胞和骨髓细胞,所以怎样使IL-21专一性的作用于CART细胞,以控制其毒性成为免疫细胞治疗所关注的焦点。此外,IL-21本身半衰期很短,也需要有效的改造以提高半衰期。
发明内容
本发明旨在至少在一定程度上解决相关技术中的技术问题之一。
发明人开发了一种分泌PD-1抗体与IL-21融合蛋白的CART,PD-1主要表达在T细胞表面,主要是CD8+T细胞,分泌出的PD-1抗体与IL-21融合蛋白选择性地结合在T细胞和CART细胞表面,一方面,封闭PD-1/PD-L1信号通路,另一方面,又使得IL-21专一性的作用于T细胞和CART细胞,进而行使其双重功能;与此同时,融合蛋白由于分子量变大,大大地提高了药物的半衰期。
鉴于此,在本发明的第一方面,本发明提出了一种T淋巴细胞。根据本发明的实施例,所述T淋巴细胞共表达融合蛋白以及嵌合抗原受体,其中,所述嵌合抗原受体包括:胞外区,所述胞外区包括单链抗体的重链可变区和轻链可变区以及CD8铰链区,所述单链抗体特异性识别肿瘤抗原;跨膜区,所述跨膜区与所述胞外区相连,所述跨膜区包括CD8的跨膜段,并且嵌入到所述T淋巴细胞的细胞膜中;胞内区,所述胞内区与所述跨膜区相连,并且所述胞内区包括4-1BB的胞内段以及CD3ζ链;所述融合蛋白包括:免疫检查点单链抗体和T细胞激活分子。根据本发明实施例的T淋巴细胞分泌包括免疫检查点单链抗体和T细胞激活分子的融合蛋白,将免疫检查点单链抗体和T细胞激活分子的双重优越性专一性的作用于CAR-T细胞,降低肿瘤微环境对CART抑制作用的同时,使CART细胞作用更为长效;同时发明人意外而惊喜地发现,根据本发明实施例的CART细胞的细胞表面表达极低比例的免疫检查点分子,同时有更强的杀瘤效果;根据本发明实施例的CART细胞与单独分泌免疫检查点单链抗体或T细胞激活分子的CART细胞相比,大大提高了T细胞激活分子与T淋巴细胞结合的特异性,降低了药物的毒性。
根据本发明的实施例,上述T淋巴细胞还可以进一步包括如下附加技术特征至少之一:
根据本发明的实施例,所述免疫检查点包括选自PD-1、PD-L1、CTLA-4、TIM3、LAG3、BTLA和TIGIT至少之一。
根据本发明的实施例,所述T细胞激活分子包括选自IL2、IL7、IL9、IL12、IL15、IL18与IL21的至少之一。
根据本发明的实施例,所述免疫检查点为PD-1,所述T细胞激活分子为IL21。
根据本发明的实施例,所述IL21的C端与PD-1单链抗体的N端相连;优选地,所述PD-1单链抗体的C端与所述IL21的N端相连。发明人发现,当所述PD-1单链抗体的C端与所述IL21的N端相连,T淋巴细胞表面的PD-1表达量更低,T淋巴细胞对肿瘤的杀伤效果更加显著。
根据本发明的实施例,所述融合蛋白进一步包括连接肽,所述连接肽设置于所述免疫检查点单链抗体和T细胞激活分子之间。
根据本发明的实施例,所述连接肽具有SEQ ID NO:1所示的氨基酸序列。.
GGGGSGGGGSGGGGS(SEQ ID NO:1)。
根据本发明的实施例,所述连接肽的N端与所述免疫检查点单链抗体的C端相连,所述连接肽的C端与所述T细胞激活分子的N端相连。
根据本发明的实施例,所述融合蛋白具有SEQ ID NO:2或SEQ ID NO:3所示的氨基酸序列。
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSGGGGSGGGGSGGGGSMHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS(SEQ ID NO:2)。
HKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGGGGSGGGGSGGGGSMEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS(SEQ ID NO:3)。
其中,融合蛋白scFV-IL21具有SEQ ID NO:2所示的氨基酸序列,融合蛋白IL21-scFV具有SEQ ID NO:3所示的氨基酸序列,其中,scFV表示免疫检查点单链抗体,IL21表示T细胞激活分子为IL21,融合蛋白scFV-IL21中scFV和IL21的连接顺序为免疫检查点单链抗体的C端与IL21的N端相连,融合蛋白IL21-scFV中scFV和IL21的连接顺序为IL21的C端与免疫检查点单链抗体的N端相连。
在本发明的第二方面,本发明提出了一种慢病毒。根据本发明的实施例,所述慢病毒携带下列核酸分子:(a)编码融合蛋白的核酸分子,所述融合蛋白包括:免疫检查点单链抗体和T细胞激活分子;(b)编码嵌合抗原受体的核酸分子,所述嵌合抗原受体的胞外区识别肿瘤抗原。将根据本发明实施例的慢病毒导入受体细胞T淋巴细胞中,可在受体细胞中表达和分泌包括免疫检查点单链抗体和T细胞激活分子的融合蛋白以及嵌合抗原受体,降低T淋巴细胞表面免疫检查点的表达,降低肿瘤微环境对T淋巴细胞的抑制作用,T细胞作用对肿瘤的杀伤更为有效、长效,安全性更高。
根据本发明的实施例,上述慢病毒还可以进一步包括如下附加技术特征至少之一:
根据本发明的实施例,所述免疫检查点为PD-1,所述T细胞激活分子为IL21,所述融合蛋白具有SEQ ID NO:2或3所示的氨基酸序列。
根据本发明的实施例,所述肿瘤抗原为CD19,所述嵌合抗原受体具有SEQ ID NO:4所示的氨基酸序列。
DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:4)根据本发明的实施例,所述编码融合蛋白的核酸分子具有SEQ ID NO:5或6任一所示的核苷酸序列。
GAGATCGTGCTGACCCAGTCTCCAGCCACACTGAGCCTGTCTCCTGGCGAGAGAGCCACCCTGTCTTGTAGGGCCAGCCAGTCCGTGAGCTCTTACCTGGCCTGGTATCAGCAGAAGCCAGGCCAGGCCCCAAGACTGCTGATCTACGACGCCTCCAACAGAGCCACCGGCATCCCAGCCAGATTTTCTGGCTCCGGCTCTGGCACCGACTTCACACTGACCATCAGCTCTCTGGAGCCAGAGGATTTCGCCGTGTATTACTGCCAGCAGAGCTCTAACTGGCCAAGAACATTCGGGCAGGGGACCAAGGTGGAAATCAAGAGGGGCGGCGGCGGCTCTGGCGGCGGCGGCTCCGGCGGCGGCGGCTCTCAGGTGCAGCTGGTGGAGAGCGGCGGCGGAGTGGTGCAGCCAGGCAGATCTCTGAGACTGGATTGCAAGGCCAGCGGCATCACCTTCAGCAATTCCGGCATGCACTGGGTGCGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCGTGATCTGGTATGACGGCTCTAAGCGGTACTATGCCGACTCTGTGAAGGGCAGATTCACCATCTCCAGGGACAACTCCAAGAATACCCTGTTCCTGCAGATGAACAGCCTGAGGGCCGAGGATACCGCCGTGTACTATTGCGCCACCAACGACGATTACTGGGGCCAGGGCACACTGGTGACCGTGTCCAGCGGCGGCGGCGGCTCTGGCGGCGGCGGCTCCGGCGGCGGCGGCTCTATGCACAAATCAAGCTCCCAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC(SEQ ID NO:5)。
CACAAATCAAGCTCCCAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCCGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTATGGAGATCGTGCTGACCCAGTCTCCAGCCACACTGAGCCTGTCTCCTGGCGAGAGAGCCACCCTGTCTTGTAGGGCCAGCCAGTCCGTGAGCTCTTACCTGGCCTGGTATCAGCAGAAGCCAGGCCAGGCCCCAAGACTGCTGATCTACGACGCCTCCAACAGAGCCACCGGCATCCCAGCCAGATTTTCTGGCTCCGGCTCTGGCACCGACTTCACACTGACCATCAGCTCTCTGGAGCCAGAGGATTTCGCCGTGTATTACTGCCAGCAGAGCTCTAACTGGCCAAGAACATTCGGGCAGGGGACCAAGGTGGAAATCAAGAGGGGCGGCGGCGGCTCTGGCGGCGGCGGCTCCGGCGGCGGCGGCTCTCAGGTGCAGCTGGTGGAGAGCGGCGGCGGAGTGGTGCAGCCAGGCAGATCTCTGAGACTGGATTGCAAGGCCAGCGGCATCACCTTCAGCAATTCCGGCATGCACTGGGTGCGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCGTGATCTGGTATGACGGCTCTAAGCGGTACTATGCCGACTCTGTGAAGGGCAGATTCACCATCTCCAGGGACAACTCCAAGAATACCCTGTTCCTGCAGATGAACAGCCTGAGGGCCGAGGATACCGCCGTGTACTATTGCGCCACCAACGACGATTACTGGGGCCAGGGCACACTGGTGACCGTGTCCAGC(SEQ ID NO:6)。
其中,具有SEQ ID NO:5所示核苷酸序列的核酸分子编码的融合蛋白具有SEQ IDNO:2所示的氨基酸序列,具有SEQ ID NO:6所示核苷酸序列的核酸分子编码的融合蛋白具有SEQ ID NO:3所示的氨基酸序列。
根据本发明的实施例,所述编码嵌合抗原受体的核酸分子具有SEQ ID NO:7所示的核苷酸序列。
GACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATCACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:7)。
在本发明的第三方面,本发明提出了一种慢病毒。根据本发明的实施例,所述慢病毒携带具有SEQ ID NO:8或9所示核苷酸序列的核酸分子。
GACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATCACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGCCACTAACTTCTCCCTGTTGAAACAAGCAGGGGATGTCGAAGAGAATCCCGGGCCAGAGATCGTGCTGACCCAGTCTCCAGCCACACTGAGCCTGTCTCCTGGCGAGAGAGCCACCCTGTCTTGTAGGGCCAGCCAGTCCGTGAGCTCTTACCTGGCCTGGTATCAGCAGAAGCCAGGCCAGGCCCCAAGACTGCTGATCTACGACGCCTCCAACAGAGCCACCGGCATCCCAGCCAGATTTTCTGGCTCCGGCTCTGGCACCGACTTCACACTGACCATCAGCTCTCTGGAGCCAGAGGATTTCGCCGTGTATTACTGCCAGCAGAGCTCTAACTGGCCAAGAACATTCGGGCAGGGGACCAAGGTGGAAATCAAGAGGGGCGGCGGCGGCTCTGGCGGCGGCGGCTCCGGCGGCGGCGGCTCTCAGGTGCAGCTGGTGGAGAGCGGCGGCGGAGTGGTGCAGCCAGGCAGATCTCTGAGACTGGATTGCAAGGCCAGCGGCATCACCTTCAGCAATTCCGGCATGCACTGGGTGCGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCGTGATCTGGTATGACGGCTCTAAGCGGTACTATGCCGACTCTGTGAAGGGCAGATTCACCATCTCCAGGGACAACTCCAAGAATACCCTGTTCCTGCAGATGAACAGCCTGAGGGCCGAGGATACCGCCGTGTACTATTGCGCCACCAACGACGATTACTGGGGCCAGGGCACACTGGTGACCGTGTCCAGCGGCGGCGGCGGCTCTGGCGGCGGCGGCTCCGGCGGCGGCGGCTCTATGCACAAATCAAGCTCCCAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC(SEQ ID NO:8)。
GACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATCACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGCCACTAACTTCTCCCTGTTGAAACAAGCAGGGGATGTCGAAGAGAATCCCGGGCCACACAAATCAAGCTCCCAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCCGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTATGGAGATCGTGCTGACCCAGTCTCCAGCCACACTGAGCCTGTCTCCTGGCGAGAGAGCCACCCTGTCTTGTAGGGCCAGCCAGTCCGTGAGCTCTTACCTGGCCTGGTATCAGCAGAAGCCAGGCCAGGCCCCAAGACTGCTGATCTACGACGCCTCCAACAGAGCCACCGGCATCCCAGCCAGATTTTCTGGCTCCGGCTCTGGCACCGACTTCACACTGACCATCAGCTCTCTGGAGCCAGAGGATTTCGCCGTGTATTACTGCCAGCAGAGCTCTAACTGGCCAAGAACATTCGGGCAGGGGACCAAGGTGGAAATCAAGAGGGGCGGCGGCGGCTCTGGCGGCGGCGGCTCCGGCGGCGGCGGCTCTCAGGTGCAGCTGGTGGAGAGCGGCGGCGGAGTGGTGCAGCCAGGCAGATCTCTGAGACTGGATTGCAAGGCCAGCGGCATCACCTTCAGCAATTCCGGCATGCACTGGGTGCGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCGTGATCTGGTATGACGGCTCTAAGCGGTACTATGCCGACTCTGTGAAGGGCAGATTCACCATCTCCAGGGACAACTCCAAGAATACCCTGTTCCTGCAGATGAACAGCCTGAGGGCCGAGGATACCGCCGTGTACTATTGCGCCACCAACGACGATTACTGGGGCCAGGGCACACTGGTGACCGTGTCCAGC(SEQ ID NO:9)。
其中,具有SEQ ID NO:8所示核苷酸序列的核酸分子编码CD19CAR-scFV-IL21,具有SEQ ID NO:9所示核苷酸序列的核酸分子编码CD19CAR-IL21-scFV。
在本发明的第四方面,本发明提出了一种转基因淋巴细胞。根据本发明的实施例,所述转基因淋巴细胞共表达融合蛋白以及嵌合抗原受体,所述嵌合抗原受体识别肿瘤抗原,其中,所述嵌合抗原受体包括:胞外区;跨膜区,所述跨膜区与所述胞外区相连,并且嵌入到所述转基因淋巴细胞的细胞膜中;胞内区,所述胞内区与所述跨膜区相连,并且所述胞内区包括免疫共刺激分子胞内段;所述融合蛋白包括:免疫检查点单链抗体和T细胞激活分子。根据本发明实施例的转基因淋巴细胞表达嵌合抗原受体和分泌包括免疫检查点单链抗体和T细胞激活分子的融合蛋白,将免疫检查点单链抗体和T细胞激活分子的双重优越性专一性的作用于该转基因淋巴细胞,降低肿瘤微环境对该转基因淋巴细胞的抑制作用的同时,使该转基因淋巴细胞作用更为长效;同时发明人意外而惊喜地发现,根据本发明实施例的转基因淋巴细胞的细胞表面表达极低比例的免疫检查点分子,同时有更强的杀瘤效果;根据本发明实施例的转基因淋巴细胞与单独分泌免疫检查点单链抗体或T细胞激活分子的转基因淋巴细胞相比,大大提高了T细胞激活分子与T淋巴细胞结合的特异性,降低了药物的毒性。
根据本发明的实施例,上述转基因淋巴细胞还可以进一步包括如下附加技术特征至少之一:
根据本发明的实施例,所述免疫共刺激分子胞内段独立地选自4-1BB、OX-40、CD40L、CD27、CD30、CD28、CD3以及他们的衍生物的至少一种。
根据本发明的实施例,所述免疫共刺激分子胞内段是4-1BB、CD3的胞内段。
根据本发明的实施例,所述淋巴细胞是CD3+T淋巴细胞。
根据本发明的实施例,所述淋巴细胞是CD8+T淋巴细胞。
根据本发明的实施例,所述淋巴细胞是自然杀伤细胞。
根据本发明的实施例,所述淋巴细胞是自然杀伤T细胞。
根据本发明的实施例,所述免疫检查点包括选自PD-1、PD-L1、CTLA-4、TIM3、LAG3、BTLA和TIGIT的至少之一。
根据本发明的实施例,所述T细胞激活分子包括选自IL2、IL7、IL9、IL12、IL15、IL18与IL21的至少之一。
根据本发明的实施例,所述免疫检查点为PD-1,所述T细胞激活分子为IL21。
根据本发明的实施例,所述IL21的C端与PD-1单链抗体的N端相连。
根据本发明的实施例,所述PD-1单链抗体的C端与所述IL21的N端相连。进而根据本发明实施例的转基因淋巴细胞表面的PD-1的表达更低,该转基因淋巴细胞对肿瘤细胞的杀伤效果更加显著。
根据本发明的实施例,所述融合蛋白进一步包括连接肽,所述连接肽设置于所述免疫检查点单链抗体和T细胞激活分子之间。
根据本发明的实施例,所述连接肽具有SEQ ID NO:1所示的氨基酸序列。
根据本发明的实施例,所述连接肽的N端与所述免疫检查点单链抗体的C端相连,所述连接肽的C端与所述T细胞激活分子的N端相连。
根据本发明的实施例,所述融合蛋白具有SEQ ID NO:2或SEQ ID NO:3所示的氨基酸序列。
在本发明的第五方面,本发明提出了一种构建体。根据本发明的实施例,所述构建体包括:第一核酸分子,所述第一核酸分子编码融合蛋白,所述融合蛋白包括:免疫检查点单链抗体和T细胞激活分子;第二核酸分子,所述第二核酸分子编码嵌合抗原受体,所述嵌合抗原受体识别肿瘤抗原;其中,所述融合蛋白、所述嵌合抗原受体是如前面所描述的。将根据本发明实施例的构建体导入受体细胞淋巴细胞后,可在淋巴细胞表面表达嵌合抗原受体和分泌融合蛋白,淋巴对肿瘤的杀伤效果更加显著、持久和安全。
根据本发明的实施例,上述构建体还可以进一步包括如下附加技术特征至少之一:
根据本发明的实施例,所述第一核酸分子、所述第二核酸分子被设置为在前面所述的淋巴细胞中表达所述融合蛋白、嵌合抗原受体,并且所述融合蛋白与嵌合抗原受体呈非融合形式。
根据本发明的实施例,所述构建体进一步包括:第一启动子,所述第一启动子与所述第一核酸分子可操作地连接;以及第二启动子,所述第二启动子与所述第二核酸分子可操作地连接。进而可实现第一核酸分子和第二核酸分子分别独立地表达。
根据本发明的具体实施例,所述第一启动子、所述第二启动子分别独立地选自U6、H1、CMV、EF-1、LTR或RSV启动子。
根据本发明的实施例,所述构建体进一步包括:内部核糖体进入位点序列,所述内部核糖体进入位点序列设置在所述第一核酸分子与所述第二核酸分子之间,所述内部核糖体进入位点具有SEQ ID NO:10所示的核苷酸序列。进而可实现所表达的融合蛋白与嵌合抗原受体呈非融合形式。
GCCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACAACC(SEQ ID NO:10)。
内部核糖体进入位点通常位于RNA病毒基因组的5’非翻译区(UTR),这样一个病毒蛋白的翻译就可以不依赖于5‘帽子结构,另一个蛋白通常靠5’帽子结构起始翻译,IRES前后的两个基因的表达通常是成比例的。内部核糖体进入位点序列的引入使得编码编码嵌合抗原受体的核酸分子与编码融合蛋白的核酸分子分别独立的表达。
根据本发明的实施例,所述构建体进一步包括:第三核酸分子,所述第三核酸分子设置在所述第一核酸分子与所述第二核酸分子之间,并且所述第三核酸分子编码可切割连接肽,所述可切割连接肽能够在所述淋巴细胞中被切割。进而淋巴细胞中表达的融合蛋白和嵌合抗原受体在连接肽处被切割,使得嵌合抗原受体独立地表达于淋巴细胞膜表面,融合蛋白独立地从淋巴细胞中分离出来。
根据本发明的实施例,所述可切割连接肽具有SEQ ID NO:11所示的氨基酸序列。
IDATNFSLLKQAGDVEENPGP(SEQ ID NO:11)。
根据本发明的实施例,所述构建体的载体是非致病性病毒载体;
根据本发明的实施例,所述病毒载体包括选自反转录病毒载体、慢病毒载体或腺病毒相关病毒载体的至少之一。
在本发明的第六方面,本发明提出了一种制备前面所述的T淋巴细胞或者前面所述的转基因淋巴细胞的方法。根据本发明的实施例,所述方法包括:将前面所述的构建体或者前面所述的慢病毒引入到淋巴细胞中或者T淋巴细胞。根据本发明实施例的方法所制备的T淋巴细胞或者转基因淋巴细胞对肿瘤细胞的杀伤效果更加显著、持久和安全性更高。
在本发明的第七方面,本发明提出了一种用于治疗癌症的治疗组合物。根据本发明的实施例,所述治疗组合物包括:前面所述的构建体、前面所述的慢病毒、前面所述的T淋巴细胞或者前面所述的转基因淋巴细胞。根据本发明实施例的治疗组合物对肿瘤细胞的杀伤效果更加显著、持久和安全性更高。
根据本发明的实施例,所述癌症包括选自肝癌、胰腺癌、卵巢癌、胆管癌、肺癌、胃癌、肠癌、食管癌和乳腺癌的至少之一。
在本发明的第八方面,本发明提出了前面所述的T淋巴细胞、前面所述的慢病毒、前面所述的转基因淋巴细胞、前面所述的构建体或前面所述的治疗组合物在制备治疗癌症的药物中的用途。
根据本发明的实施例,所述癌症包括选自肝癌、胰腺癌、卵巢癌、胆管癌、肺癌、胃癌、肠癌、食管癌和乳腺癌的至少之一。
在本发明的第九方面,本发明提出了一种降低T淋巴细胞表面免疫检查点表达的方法。根据本发明的实施例,所述方法包括:使所述T淋巴细胞表达嵌合抗原受体和融合蛋白;或使所述T淋巴细胞与表达嵌合抗原受体和融合蛋白的T细胞共培养,所述融合蛋白包括:免疫检查点单链抗体和T细胞激活分子,其中,所述嵌合抗原受体、融合蛋白是如前面所限定的。根据本发明实施例的方法,可显著降低T淋巴细胞表面的免疫检查点表达,封闭免疫逃逸机制。
根据本发明的实施例,所述免疫检查点为PD-1,所述T细胞激活分子为IL21。
综上,本发明的创新点如下所述:
1).利用CART分泌PD-1抗体与IL-21融合蛋白,PD-1单链抗体首先特异性的选择结合T细胞表面的PD-1分子,从而使IL-21特也异性的作用于T细胞,发挥其功能,与单纯性过表达IL21相比,降低了IL21与其他细胞表面的IL21受体结合所造成的毒性;
2).与PD-1抗体和IL-21单独表达比较而言,PD-1抗体与IL-21融合表达,增加了CART药物的半衰期;
3).实验数据显示,CART分泌PD-1抗体与IL-21融合蛋白,大大降低了T细胞表面PD-1表达的比例;
4).与PD-1抗体和IL-21单独表达比较而言,PD-1抗体与IL-21融合表达,CART对实体瘤有更强的杀伤功能。
本发明的附加方面和优点将在下面的描述中部分给出,部分将从下面的描述中变得明显,或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解,其中:
图1本发明实施例1构建的核酸的结构示意图,其中,图中“Hinge”是指铰链区,“Linker”是指连接肽;
图2本发明实施例3T细胞侵染后D6的CAR19阳性率检测结果;
图3本发明实施例5流式分选单克隆建立Raji-PD-L1过表达靶细胞株的结果图,其中图中“Multi-sample”是指多重采样;
图4本发明实施例6CART杀伤阴性对照细胞K562结果图;
图5本发明实施例6CART杀伤RAJI-PD-L1-A3结果图;
图6本发明实施例7CART杀伤RAJI-PD-L1-A3过程中的细胞因子释放的结果图;
图7本发明实施例8CART体内疗效评估实验的时间轴;
图8本发明实施例8CART体内治疗后肿瘤缓解率;
图9本发明实施例8CART体内治疗后肿瘤复发率;
图10本发明实施例8CART体内治疗后组内小鼠个体肿瘤缓解及复发情况统计图;
图11本发明实施例9构建的核酸的结构示意图,其中,图中“Hinge”是指铰链区,“Linker”是指连接肽;
图12本发明实施例9感染T细胞后D7的CAR-MSLN阳性率检测结果;
图13本发明实施例10MSLN CAR-T上清液中IL-21检测;
图14本发明实施例11MSLN CAR-T肿瘤细胞杀伤检测;以及
图15本发明实施例11MSLN CAR-T与AsPC-1细胞共培养时IL-2和IFN-γ分泌检测。
具体实施方式
下面通过参考附图描述的实施例是示例性的,旨在用于解释本发明,而不能理解为对本发明的限制。
此外,术语“第一”、“第二”仅用于描述目的,而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此,限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中,“多个”的含义是至少两个,例如两个,三个等,除非另有明确具体的限定。
术语“任选地”仅用于描述目的,而不能理解为指示或暗示相对重要性。由此,限定有“任选地”的特征可以明示或者隐含地包括或不包括该特征。
单链抗体(scFv)是一种基因工程抗体,其中VH和VL域与柔性多肽连接体相连。与整体抗体的Fab区相比,单链抗体表现出更好的组织渗透药动学,并且由于抗原结合表面未被改变而具有完全的抗原结合特异性。
免疫检查点(immune cheeckpoint)是免疫系统中起抑制作用的调节分子,其对于维持自身耐受、防止自身免疫反应、以及通过控制免疫应答的时间和强度而使组织损伤最小化等至关重要。免疫检查点表达于免疫细胞上,将抑制免疫细胞功能,使机体无法产生有效的抗肿瘤免疫应答,肿瘤形成免疫逃逸。与肿瘤相关的免疫检查点分子主要有:PD1、CTLA4、Tim3和LAG3等。
本申请所述的“免疫检查点单链抗体”是具有抗免疫检查点活性,可以与免疫检查点特异性结合的单链抗体。
T淋巴细胞激活分子是指能够刺激淋巴细胞活化或分化,增强T淋巴细胞细胞毒性的分子。如IL-21。
免疫共刺激分子是指为T淋巴细胞或B淋巴细胞完全活化提供共刺激信号的细胞表面分子及其配体,如4-1BB、OX-40、CD40L、CD27、CD30、CD28、CD3以及他们的衍生物。
根据本发明的具体实施例,以构建一个慢病毒载体为例,发明人为了构建一个慢病毒载体,在某些病毒序列的位置,将目的核酸插入到病毒基因组中,从而产生复制缺陷的病毒。为了产生病毒体,发明人进而构建包装细胞系(包含gag,pol和env基因,但不包括LTR和包装成分)。发明人将含有目的基因的重组质粒,连同慢病毒LTR和包装序列,一起引入包装细胞系中。包装序列允许重组质粒RNA转录产物被包装到病毒颗粒中,然后被分泌到培养基中。进而发明人收集包含重组慢病毒的基质,有选择性地浓缩,并用于基因转移。慢载体可以感染多种细胞类型,包括可分裂细胞和不可分裂细胞。
另外,根据本发明的实施例,本发明实施例的慢病毒是复合慢病毒,除了常见的慢病毒基因gag,pol和env,还包含有调控和结构功能的其他基因。慢病毒载体是本领域技术人员所熟知的,慢病毒包括:人类免疫缺陷病毒HIV–1,HIV–2和猿猴免疫缺陷病毒SIV。慢病毒载体通过多重衰减艾滋病毒致病基因产生,例如全部删除基因env,vif,vpr,vpu和nef,使慢病毒载体形成生物安全型载体。重组慢病毒载体能够感染非分裂细胞,同时可用于体内和体外基因转移和核酸序列表达。例如:在合适的宿主细胞中,和带有包装功能(gag,pol,env,rev和tat)的两个或更多的载体一起,能够感染非分裂细胞。重组病毒的靶向性,是通过抗体或特定配体(靶向特定细胞类型受体)与膜蛋白的结合来实现的。同时,重组病毒的靶向性通过插入一个有效序列(包括调控区域)到病毒载体中,连同另一个编码了特定靶细胞上的受体的配体的基因,使载体具有了特定的靶向。各种有用的慢病毒载体,以及各种方法和操作等产生的载体,用于改变细胞的表达。
根据本发明的实施例,本发明实施例的腺关联病毒载体(AAV)可使用一种或多种为人熟知的血清类型腺关联病毒载体的DNA构建。另外,根据本发明的实施例,本发明实施例的也包含微基因。微基因意味着用组合(选定的核苷酸序列和可操作的必要的相关连接序列)来指导转化、转录和/或基因产物在体内或体外的宿主细胞中的表达。应用“可操作的连接”序列包含连续目的基因的表达控制序列,和作用于反式或远距离控制目的基因的表达控制序列。
另外,本发明实施例的载体还包括常规控制元素,在和质粒载体一起的细胞转染或/和病毒载体一起的细胞感染中。大量的表达控制序列(包括天然的,可诱导和/或特定组织的启动子)可能被使用。根据本发明的实施例,启动子为选自U6、H1、pol I、pol II和polIII的RNA聚合酶启动子。根据本发明的实施例,启动子为组织特异型启动子。根据本发明的实施例,启动子为诱导型启动子。根据本发明的实施例,启动子为选自基于所选载体的启动子。根据本发明的实施例,当选择慢病毒载体时,启动子为U6、H1、CMV IE基因、EF-1α、泛素C或磷酸甘油激酶(PGK)启动子。其他常规表达控制序列包括可选标记或报告基因,包括编码遗传霉素,潮霉素,氨苄青霉素或嘌呤霉素耐药性等的核苷酸序列。载体的其他组件包括复制起点。
构建载体的技术为本领域技术人员所熟知的,这些技术包括常规克隆技术,
根据本发明的实施例,提供给患者的本发明实施例的组合物,较好的应用于生物兼容溶液或可接受的药学运载载体。作为准备的各种治疗组合物被悬浮或溶解在医药上或生理上可接受的载体,如生理盐水;等渗的盐溶液或其他精于此道的人的比较明显的配方中。适当的载体在很大程度上取决于给药途径。其他有水和无水的等渗无菌注射液和有水和无水的无菌悬浮液,是医药上可接受的载体。
表达和分泌融合蛋白以及表达特有的针对抗原CD19嵌合抗原受体的这些方法是联合治疗的一部分。这些病毒载体和用于过继免疫治疗的抗肿瘤T细胞,可以被单独或结合其他治疗癌症的方法一起执行。在合适的条件下,一个治疗方法的包括使用一个或多个药物疗法。
下面将结合实施例对本发明的方案进行解释。
本领域技术人员将会理解,下面的实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体技术或条件的,按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著,黄培堂等译的《分子克隆实验指南》,第三版,科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1构建表达PD-1抗体与IL-21融合蛋白的CART19(表达靶向CD19的嵌合抗原受体的T细胞)慢病毒载体及其对照载体
基因合成图1所示结构的核苷酸序列,按照慢病毒载体酶切位点,将核苷酸片段构建至慢病毒载体上,设计引物,通过测序结果验证载体构建的正确性。
实施例2包装并浓缩慢病毒
将293T按照8x106个细胞/150mm2培养皿的密度接种,次日观察细胞的状态,用PEI转染的方法将3代慢病毒包装载体共转至293T,转染完6小时后换液,按照15ml/150mm2培养皿添加含有10%胎牛血清的DMEM培养基,转染完48小时与72小时收集病毒上清,2000rpm 4℃10min离心,去除细胞碎片,之后0.45微米的滤器过滤杂质,过滤后的病毒悬液在25000rpm、4℃的条件下离心2小时浓缩慢病毒,浓缩后的病毒加入适量的培养基重悬,置于-80℃保存
实施例3生产CAR-T及对照细胞
抽血20mL,Ficall梯度离心分离PBMC,用Stemcell公司T细胞阴选试剂盒(货号:19051)分离T细胞,分离后的T细胞用添加5%人AB血清及300单位/ML IL-2X-VIVO 15培养基重悬T细胞至1×106个细胞/ML,用含1%FBS X-VIVO 15清洗beads,按照磁珠:T细胞=2:1比例加入预先清洗过的磁珠(Cat#40203D,10ML,Life technology),2-3天后用新鲜的培养基重悬T细胞至3-5×106个细胞/mL,按照MOI=10值加入慢病毒,同时加入8μg/mL的Polybrene,4-6小时之后,补加培养基稀释细胞至1×106个细胞/mL,次日更换新鲜培养基,使细胞浓度维持在0.2-0.3×106PBMC/mL,之后每隔2-3天更换一次培养基,病毒侵染完72小时后流式分析细胞阳性率,图2是T细胞侵染后第六天(D6)的CAR阳性率检测结果,CAR19组(66.97%)、CAR19&scFv组(65.07%)、CAR19&scFv&IL21组(56.31%)、CAR19&scFv-IL21组(78.1%)、CAR19&IL21-scFv组(57.78%),由结果可以看出CAR19&scFv-IL21组侵染效率最高。
实施例4CART表型鉴定;
侵染后D6的各组细胞,流式细胞术检测其T、B、NK细胞分群,同时检测T细胞中CD4、CD8、PD-1的分群,结果如表1所示(其中,T cell组表示普通T细胞组,CAR19表示表达靶向CD19的嵌合抗原受体的T细胞组,CAR19&scFv表示表达scFv的CART19组,CAR19&scFv&IL21表示单独表达scFv、IL21的CART19组,CAR19&scFv-IL21表示表达融合蛋白scFv-IL21的CART19组,CAR19&scFv-IL21表示表达融合蛋白IL21-scFv的CART19组),可以看到CAR19&scFv-IL21组T细胞仅有少量比例的细胞(2.87%)表达PD-1分子,提示CAR19&scFv-IL21可能会调控T细胞表面分子PD-1的表达。PD-1分子作为免疫检查站点家族的成员,文献报道T细胞受到CD3/CD28抗体活化后会上调PD-1分子的表达水平来抑制T细胞(T cell)的活化,另外它也是T细胞衰竭的一个重要标志物。
表1:
实施例5构建Raji-PD-L1表达靶细胞株
构建慢病毒载体过表达人类PD-L1载体,包装浓缩慢病毒,之后按照MOI=10的比例侵染Raji细胞,侵染72h后使用APC anti-human PD-L1抗体检测侵染效率,流式分选阳性细胞,单克隆建立Raji-PD-L1过表达靶细胞株。结果如图3所示:成功筛选出过表达PD-L1的Raji细胞株(克隆号A3表示为Raji-PD-L1-A3),与Raji相比,Raji-PD-L1-A3细胞株CD19抗原表达没有发生变化。
实施例6CART体外杀伤功能评估
杀伤实验:将CAR-T各组与对照T细胞用CFSE染料标记,之后按照效靶比10:1、5:1、2.5:1、1.25:1和1:0与RAJI-PD-L1-A3细胞株共培养,4小时后,收集杀伤上清留作细胞因子检测,杀伤后的细胞用碘化丙碇(PI)和Annexin V染色,选择CFSE阴性的RAJI-PD-L1-A3细胞群,流式检测RAJI-PD-L1-A3细胞的晚期凋亡和早期凋亡情况;同时以表达CD19阴性的K562细胞作为杀伤对照。结果如图4和图5所示:实验各组对表达CD19阴性的K562细胞无明显杀伤功能,但是CART各组对CD19表达阳性的RAJI-PD-L1-A3均有明显杀伤功能,且CAR19&scFv-IL21杀伤效果最好,在效靶比10:1的时候,CAR19&scFv-IL21杀伤RAJI-PD-L1-A3的效率为74.33%。
实施例7CART杀伤RAJI-PD-L1-A3过程中的细胞因子释放
因子检测:检测细胞因子表达情况按照Invitrogen IL2检测说明书(货号:88-7025)、IL21检测说明书(货号:88-8218)和IFN-γ(货号88-7316)进行,PD-1单链抗体检测方法为本公司制定。细胞因子分子释放结果如图6所示:与其他各组相比,CAR19&scFv-IL21杀伤RAJI-PD-L1-A3的过程释放更多的IL21、PD-1单链抗体(scFv)与IFN-γ,释放较多的IL2,提示CAR19&scFv-IL21可能是通过高表达上述因子发挥杀伤效能的。
实施例8CART体内疗效评估
实验方案:选择5周龄,NPG雌性小鼠,第0天(Day0)皮下接种2E5 Raji-PD-L1-A3/只,共40只;第7天(DAY7)选择肿瘤体积为100mm3的小鼠入组实验,共四组,尽量使每组小鼠肿瘤体积保持一致,剔除肿瘤体积过大或者过小的小鼠;第8天(DAY8),每组小鼠尾静脉注射对应的CART(CAR19、CAR19&scFv和CAR19&scFv-IL21),对照组注射PBS;给药后每周三测量肿瘤体积与体重,对照小鼠达到肿瘤测量终点评估肿瘤缓解率,二个月评估肿瘤复发率与生存曲线。实验时间轴如图7所示,D0每只NPG小鼠按照2E6的量皮下接种Raji-PD-L1细胞,D7测量小鼠肿瘤体积并进行分组,每组8只,D8按照5E6的量尾静脉注射CART细胞,肿瘤接种后一个月内评估CART药效,两个月评估药物控制复发能力。D26各组平均肿瘤体积、肿瘤缓解率和抑瘤率如表2和图8所示,CART19&scFv-IL21组平均肿瘤体积与CART19&scFv相比差异显著,与CART19组相比差异及其显著。D65各组平均复发肿瘤体积和复发率如图9和表3所示,CART19&scFv-IL21组和CART19&scFv组平均复发肿瘤体积与CART19组相比差异显著,CART19&scFv-IL21组和CART19&scFv组相比差异不显著。组内小鼠个体肿瘤缓解及复发情况如图10所示,CART19&scFv-IL21组内小鼠有更长的无瘤生长期。
表2:
SCAR-T各组平均肿瘤体积与PBS组相比,T检验(Mann-Whitney),*P<0.05,**P<0.01,***P<0.001
表3:
S除PBS组外各组平均复发肿瘤体积与CART19组相比,T检验(Mann-Whitney),*P<0.05,**P<0.01,***p<0.001
实施例9靶向Mesothelin(MSLN)的嵌合抗原受体的T细胞的构建与生产
基于图11基因合成以下核苷酸序列,按照慢病毒载体酶切位点,将核苷酸片段构建至慢病毒载体上,设计引物,通过测序结果验证载体构建的正确性,其中SS1为抗人Mesothelin(MSLN)抗体的scFv序列,MSLN CAR质粒命名为PCDHF85,MSLN CAR+Anti PD1-IL-21质粒命名为PCDHF86。
包装慢病毒将以上2个质粒分别包装慢病毒,包装体系如下表4所示:
表4:
PCDHF-85 | PCDHF-86 | |
PCDH D(PMD2.G) | 24μg | 36μg |
PCDH M(pMDLg/pRRE) | 24μg | 36μg |
PCDH N(pRSV-Rev) | 24μg | 36μg |
PCDHF85 | 48μg | |
PCDHF86 | 72μg | |
PEI(Polysciences 636951) | 240μg | 360μg |
OPTI-MEMI(Gibco 31985070) | 12mL | 18mL |
接种293T细胞5E6于10cm细胞培养皿中,加入10mL含10%FBS的DMEM培养基(DMEMGibco,11995040-1L;FBS Gibco,10091-148),5%CO2,37℃条件下CO2培养箱中培养24h;按表4所示进行慢病毒包装。CART细胞制备,Ficoll淋巴分离液(达科为,AS1114546)从血液(菲鹏生物员工0068号志愿者献血50mL)中分离PBMC细胞,偶联CD3/CD28抗体的磁珠(Dynabeads,CD3/CD28 CTS,货号40203D,批号A2-011710E)阳选法分离获得T细胞,将慢病毒按MOI=5:1感染T细胞以制备CART细胞,CART细胞培养7天后通过检测CART细胞的MSLN抗体表达来确定CART细胞CAR阳性率,如图12所示。
实施例10 MSLN CAR-T上清液检测
CART细胞分泌IL-21,首先检测CART细胞培养上清中IL-21的表达水平,细胞起始密度均为2E5个细胞/ml,培养72h后收集上清,IL-21ELISA检测试剂盒,(Invitrogen,批号220657),结果如图13所示,结果显示86CART细胞正常分泌IL-21和PD1抗体。
实施例11 MSLN CAR-T肿瘤细胞杀伤及细胞因子释放检测
CART细胞杀伤肿瘤细胞,向平底96孔板(Costar,货号,25719016)分别加入2E4/100ul/孔AsPC-1细胞(ATCC,CRL-1682,Mesothelin表达阳性细胞),培养基为RPMI1640(Gibco,批号2215748)+10%FBS(Gibco批号2152441P),5%CO2,37℃培养48h。分别取T细胞,85CART细胞,86CART细胞,计数之后用T细胞将85CART细胞稀释到CAR+阳性率为45%,按效应细胞(CAR+CART细胞):靶细胞=1:1,5:1,10:1的比例分别加入各孔,CART细胞的培养基为X-VIVO 15,100ul/孔,CART细胞与肿瘤细胞混合培养16h,吸上清备用,用DPBS(Hyclone,批号AE29431662)轻轻清洗2次并吸弃DPBS,进一步吸弃孔中悬浮细胞,然后加入RPMI1640+10%FBS(100ul)再加入提前准备好的Promega(CellTiter-Glo LuminescentCell Viability Assay,货号,0000453271)底物与缓冲液混合液,100ul/孔,10min后用多功能酶标仪(MOLECRLAR DEVICES,SpectraMax i3X)检测各孔的荧光值,通过各孔的荧光值与AsPC-1细胞孔(未加T/CART细胞)荧光值进行比值,从而计算出各孔的活细胞和凋亡细胞的比值。检测结果如图14所示,结果显示,对于AsPC-1细胞的杀伤效果,86CART>85CART>T。
用IL-2/IFN-γELISA检测试剂盒(Human IL-21Uncoated ELISA kit,R&D,批号223086-004;IFN gamma Human Uncoated ELISA Kit,R&D,批号223086-003)检测CART与AsPC-1细胞共培养后的上清中的IL-2和IFN-γ分泌水平,检测结果如图15所示,结果显示CART细胞与AsPC-1细胞共培养时,IL-2和IFN-γ正常分泌。
在本说明书的描述中,参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外,在不相互矛盾的情况下,本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
SEQUENCE LISTING
<110> 深圳市菲鹏生物治疗股份有限公司
<120> T淋巴细胞及其应用
<130> SI4210193
<150> CN 202010648451.1
<151> 2020-07-07
<160> 11
<170> PatentIn version 3.5
<210> 1
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> 连接肽
<400> 1
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 2
<211> 390
<212> PRT
<213> Artificial Sequence
<220>
<223> 融合蛋白
<400> 2
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly
100 105 110
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val
115 120 125
Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Asp
130 135 140
Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser Gly Met His Trp Val
145 150 155 160
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile Trp Tyr
165 170 175
Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
180 185 190
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe Leu Gln Met Asn Ser
195 200 205
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Thr Asn Asp Asp
210 215 220
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
225 230 235 240
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met His Lys Ser Ser
245 250 255
Ser Gln Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp
260 265 270
Ile Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe
275 280 285
Leu Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe
290 295 300
Ser Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn
305 310 315 320
Glu Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro
325 330 335
Ser Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser
340 345 350
Cys Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe
355 360 365
Lys Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr
370 375 380
His Gly Ser Glu Asp Ser
385 390
<210> 3
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<223> 融合蛋白
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His Lys Ser Ser Ser Gln Gly Gln Asp Arg His Met Ile Arg Met Arg
1 5 10 15
Gln Leu Ile Asp Ile Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu
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Val Pro Glu Phe Leu Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu
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Trp Ser Ala Phe Ser Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn
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Thr Gly Asn Asn Glu Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys
65 70 75 80
Arg Lys Pro Pro Ser Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu
85 90 95
Thr Cys Pro Ser Cys Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe
100 105 110
Leu Glu Arg Phe Lys Ser Leu Leu Gln Lys Met Ile His Gln His Leu
115 120 125
Ser Ser Arg Thr His Gly Ser Glu Asp Ser Gly Gly Gly Gly Ser Gly
130 135 140
Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Glu Ile Val Leu Thr Gln
145 150 155 160
Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser
165 170 175
Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln
180 185 190
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg
195 200 205
Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
210 215 220
Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr
225 230 235 240
Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg Thr Phe Gly Gln Gly Thr
245 250 255
Lys Val Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
260 265 270
Gly Gly Gly Gly Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val
275 280 285
Val Gln Pro Gly Arg Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile
290 295 300
Thr Phe Ser Asn Ser Gly Met His Trp Val Arg Gln Ala Pro Gly Lys
305 310 315 320
Gly Leu Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr
325 330 335
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
340 345 350
Lys Asn Thr Leu Phe Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
355 360 365
Ala Val Tyr Tyr Cys Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr
370 375 380
Leu Val Thr Val Ser Ser
385 390
<210> 4
<211> 465
<212> PRT
<213> Artificial Sequence
<220>
<223> 嵌合抗原受体
<400> 4
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu
115 120 125
Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys
130 135 140
Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg
145 150 155 160
Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser
165 170 175
Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile
180 185 190
Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln
195 200 205
Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly
210 215 220
Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val
225 230 235 240
Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr
245 250 255
Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala
260 265 270
Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile
275 280 285
Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser
290 295 300
Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr
305 310 315 320
Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu
325 330 335
Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu
340 345 350
Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln
355 360 365
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
370 375 380
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
385 390 395 400
Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
405 410 415
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
420 425 430
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
435 440 445
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
450 455 460
Arg
465
<210> 5
<211> 1170
<212> DNA
<213> Artificial Sequence
<220>
<223> 编码融合蛋白的核酸分子
<400> 5
gagatcgtgc tgacccagtc tccagccaca ctgagcctgt ctcctggcga gagagccacc 60
ctgtcttgta gggccagcca gtccgtgagc tcttacctgg cctggtatca gcagaagcca 120
ggccaggccc caagactgct gatctacgac gcctccaaca gagccaccgg catcccagcc 180
agattttctg gctccggctc tggcaccgac ttcacactga ccatcagctc tctggagcca 240
gaggatttcg ccgtgtatta ctgccagcag agctctaact ggccaagaac attcgggcag 300
gggaccaagg tggaaatcaa gaggggcggc ggcggctctg gcggcggcgg ctccggcggc 360
ggcggctctc aggtgcagct ggtggagagc ggcggcggag tggtgcagcc aggcagatct 420
ctgagactgg attgcaaggc cagcggcatc accttcagca attccggcat gcactgggtg 480
cggcaggccc ccggcaaggg cctggagtgg gtggccgtga tctggtatga cggctctaag 540
cggtactatg ccgactctgt gaagggcaga ttcaccatct ccagggacaa ctccaagaat 600
accctgttcc tgcagatgaa cagcctgagg gccgaggata ccgccgtgta ctattgcgcc 660
accaacgacg attactgggg ccagggcaca ctggtgaccg tgtccagcgg cggcggcggc 720
tctggcggcg gcggctccgg cggcggcggc tctatgcaca aatcaagctc ccaaggtcaa 780
gatcgccaca tgattagaat gcgtcaactt atagatattg ttgatcagct gaaaaattat 840
gtgaatgact tggtccctga atttctgcca gctccagaag atgtagagac aaactgtgag 900
tggtcagctt tttcctgttt tcagaaggcc caactaaagt cagcaaatac aggaaacaat 960
gaaaggataa tcaatgtatc aattaaaaag ctgaagagga aaccaccttc cacaaatgca 1020
gggagaagac agaaacacag actaacatgc ccttcatgtg attcttatga gaaaaaacca 1080
cccaaagaat tcctagaaag attcaaatca cttctccaaa agatgattca tcagcatctg 1140
tcctctagaa cacacggaag tgaagattcc 1170
<210> 6
<211> 1170
<212> DNA
<213> Artificial Sequence
<220>
<223> 编码融合蛋白的核酸分子
<400> 6
cacaaatcaa gctcccaagg tcaagatcgc cacatgatta gaatgcgtca acttatagat 60
attgttgatc agctgaaaaa ttatgtgaat gacttggtcc ctgaatttct gccagctcca 120
gaagatgtag agacaaactg tgagtggtca gctttttcct gttttcagaa ggcccaacta 180
aagtcagcaa atacaggaaa caatgaaagg ataatcaatg tatcaattaa aaagctgaag 240
aggaaaccac cttccacaaa tgcagggaga agacagaaac acagactaac atgcccttca 300
tgtgattctt atgagaaaaa accacccaaa gaattcctag aaagattcaa atcacttctc 360
caaaagatga ttcatcagca tctgtcctct agaacacacg gaagtgaaga ttccggtggc 420
ggtggctcgg gcggtggtgg gtcgggtggc ggcggatcta tggagatcgt gctgacccag 480
tctccagcca cactgagcct gtctcctggc gagagagcca ccctgtcttg tagggccagc 540
cagtccgtga gctcttacct ggcctggtat cagcagaagc caggccaggc cccaagactg 600
ctgatctacg acgcctccaa cagagccacc ggcatcccag ccagattttc tggctccggc 660
tctggcaccg acttcacact gaccatcagc tctctggagc cagaggattt cgccgtgtat 720
tactgccagc agagctctaa ctggccaaga acattcgggc aggggaccaa ggtggaaatc 780
aagaggggcg gcggcggctc tggcggcggc ggctccggcg gcggcggctc tcaggtgcag 840
ctggtggaga gcggcggcgg agtggtgcag ccaggcagat ctctgagact ggattgcaag 900
gccagcggca tcaccttcag caattccggc atgcactggg tgcggcaggc ccccggcaag 960
ggcctggagt gggtggccgt gatctggtat gacggctcta agcggtacta tgccgactct 1020
gtgaagggca gattcaccat ctccagggac aactccaaga ataccctgtt cctgcagatg 1080
aacagcctga gggccgagga taccgccgtg tactattgcg ccaccaacga cgattactgg 1140
ggccagggca cactggtgac cgtgtccagc 1170
<210> 7
<211> 1395
<212> DNA
<213> Artificial Sequence
<220>
<223> 编码嵌合抗原受体的核酸分子
<400> 7
gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60
atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120
gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180
aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240
gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300
gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 360
ggatctgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 420
tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc 480
cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac 540
tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt 600
ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat 660
tattactacg gtggtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtc 720
tcctcaacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 780
cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 840
gggctggact tcgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc 900
cttctcctgt cactggttat caccctttac tgcaaacggg gcagaaagaa actcctgtat 960
atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1020
tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 1080
gcagacgccc ccgcgtacaa gcagggccag aaccagctct ataacgagct caatctagga 1140
cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 1200
aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1260
gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1320
ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1380
gccctgcccc ctcgc 1395
<210> 8
<211> 2622
<212> DNA
<213> Artificial Sequence
<220>
<223> 慢病毒携带的核酸分子
<400> 8
gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60
atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120
gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180
aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240
gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300
gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 360
ggatctgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 420
tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc 480
cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac 540
tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt 600
ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat 660
tattactacg gtggtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtc 720
tcctcaacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 780
cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 840
gggctggact tcgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc 900
cttctcctgt cactggttat caccctttac tgcaaacggg gcagaaagaa actcctgtat 960
atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1020
tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 1080
gcagacgccc ccgcgtacaa gcagggccag aaccagctct ataacgagct caatctagga 1140
cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 1200
aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1260
gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1320
ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1380
gccctgcccc ctcgcgccac taacttctcc ctgttgaaac aagcagggga tgtcgaagag 1440
aatcccgggc cagagatcgt gctgacccag tctccagcca cactgagcct gtctcctggc 1500
gagagagcca ccctgtcttg tagggccagc cagtccgtga gctcttacct ggcctggtat 1560
cagcagaagc caggccaggc cccaagactg ctgatctacg acgcctccaa cagagccacc 1620
ggcatcccag ccagattttc tggctccggc tctggcaccg acttcacact gaccatcagc 1680
tctctggagc cagaggattt cgccgtgtat tactgccagc agagctctaa ctggccaaga 1740
acattcgggc aggggaccaa ggtggaaatc aagaggggcg gcggcggctc tggcggcggc 1800
ggctccggcg gcggcggctc tcaggtgcag ctggtggaga gcggcggcgg agtggtgcag 1860
ccaggcagat ctctgagact ggattgcaag gccagcggca tcaccttcag caattccggc 1920
atgcactggg tgcggcaggc ccccggcaag ggcctggagt gggtggccgt gatctggtat 1980
gacggctcta agcggtacta tgccgactct gtgaagggca gattcaccat ctccagggac 2040
aactccaaga ataccctgtt cctgcagatg aacagcctga gggccgagga taccgccgtg 2100
tactattgcg ccaccaacga cgattactgg ggccagggca cactggtgac cgtgtccagc 2160
ggcggcggcg gctctggcgg cggcggctcc ggcggcggcg gctctatgca caaatcaagc 2220
tcccaaggtc aagatcgcca catgattaga atgcgtcaac ttatagatat tgttgatcag 2280
ctgaaaaatt atgtgaatga cttggtccct gaatttctgc cagctccaga agatgtagag 2340
acaaactgtg agtggtcagc tttttcctgt tttcagaagg cccaactaaa gtcagcaaat 2400
acaggaaaca atgaaaggat aatcaatgta tcaattaaaa agctgaagag gaaaccacct 2460
tccacaaatg cagggagaag acagaaacac agactaacat gcccttcatg tgattcttat 2520
gagaaaaaac cacccaaaga attcctagaa agattcaaat cacttctcca aaagatgatt 2580
catcagcatc tgtcctctag aacacacgga agtgaagatt cc 2622
<210> 9
<211> 2622
<212> DNA
<213> Artificial Sequence
<220>
<223> 慢病毒携带的核酸分子
<400> 9
gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60
atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120
gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180
aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240
gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300
gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 360
ggatctgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 420
tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc 480
cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac 540
tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt 600
ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat 660
tattactacg gtggtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtc 720
tcctcaacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 780
cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 840
gggctggact tcgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc 900
cttctcctgt cactggttat caccctttac tgcaaacggg gcagaaagaa actcctgtat 960
atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1020
tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 1080
gcagacgccc ccgcgtacaa gcagggccag aaccagctct ataacgagct caatctagga 1140
cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 1200
aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1260
gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1320
ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1380
gccctgcccc ctcgcgccac taacttctcc ctgttgaaac aagcagggga tgtcgaagag 1440
aatcccgggc cacacaaatc aagctcccaa ggtcaagatc gccacatgat tagaatgcgt 1500
caacttatag atattgttga tcagctgaaa aattatgtga atgacttggt ccctgaattt 1560
ctgccagctc cagaagatgt agagacaaac tgtgagtggt cagctttttc ctgttttcag 1620
aaggcccaac taaagtcagc aaatacagga aacaatgaaa ggataatcaa tgtatcaatt 1680
aaaaagctga agaggaaacc accttccaca aatgcaggga gaagacagaa acacagacta 1740
acatgccctt catgtgattc ttatgagaaa aaaccaccca aagaattcct agaaagattc 1800
aaatcacttc tccaaaagat gattcatcag catctgtcct ctagaacaca cggaagtgaa 1860
gattccggtg gcggtggctc gggcggtggt gggtcgggtg gcggcggatc tatggagatc 1920
gtgctgaccc agtctccagc cacactgagc ctgtctcctg gcgagagagc caccctgtct 1980
tgtagggcca gccagtccgt gagctcttac ctggcctggt atcagcagaa gccaggccag 2040
gccccaagac tgctgatcta cgacgcctcc aacagagcca ccggcatccc agccagattt 2100
tctggctccg gctctggcac cgacttcaca ctgaccatca gctctctgga gccagaggat 2160
ttcgccgtgt attactgcca gcagagctct aactggccaa gaacattcgg gcaggggacc 2220
aaggtggaaa tcaagagggg cggcggcggc tctggcggcg gcggctccgg cggcggcggc 2280
tctcaggtgc agctggtgga gagcggcggc ggagtggtgc agccaggcag atctctgaga 2340
ctggattgca aggccagcgg catcaccttc agcaattccg gcatgcactg ggtgcggcag 2400
gcccccggca agggcctgga gtgggtggcc gtgatctggt atgacggctc taagcggtac 2460
tatgccgact ctgtgaaggg cagattcacc atctccaggg acaactccaa gaataccctg 2520
ttcctgcaga tgaacagcct gagggccgag gataccgccg tgtactattg cgccaccaac 2580
gacgattact ggggccaggg cacactggtg accgtgtcca gc 2622
<210> 10
<211> 588
<212> DNA
<213> Artificial Sequence
<220>
<223> 内部核糖体进入位点具有的核苷酸序列
<400> 10
gcccctctcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt 60
gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc 120
ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag 180
gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac 240
aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc 300
tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc 360
acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca 420
aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt 480
gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg 540
gggacgtggt tttcctttga aaaacacgat gataatatgg ccacaacc 588
<210> 11
<211> 21
<212> PRT
<213> Artificial Sequence
<220>
<223> 可切割连接肽
<400> 11
Ile Asp Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu
1 5 10 15
Glu Asn Pro Gly Pro
20
Claims (20)
1.一种T淋巴细胞,其特征在于,所述T淋巴细胞共表达融合蛋白以及嵌合抗原受体,其中,
所述嵌合抗原受体包括:
胞外区,所述胞外区包括单链抗体的重链可变区和轻链可变区以及CD8铰链区,所述单链抗体特异性识别肿瘤抗原;
跨膜区,所述跨膜区与所述胞外区相连,所述跨膜区包括CD8的跨膜段,并且嵌入到所述T淋巴细胞的细胞膜中;
胞内区,所述胞内区与所述跨膜区相连,并且所述胞内区包括4-1BB的胞内段以及CD3ζ链;
所述融合蛋白包括:免疫检查点单链抗体和T细胞激活分子。
2.根据权利要求1所述的T淋巴细胞,其特征在于,所述免疫检查点包括选自PD-1、PD-L1、CTLA-4、TIM3、LAG3、BTLA和TIGIT至少之一;
任选地,所述T细胞激活分子包括选自IL2、IL7、IL9、IL12、IL15、IL18和IL21的至少之一。
3.根据权利要求1所述的T淋巴细胞,其特征在于,所述免疫检查点为PD-1,所述T细胞激活分子为IL21;
任选地,所述IL21的C端与PD-1单链抗体的N端相连;优选地,所述PD-1单链抗体的C端与所述IL21的N端相连。
4.根据权利要求1所述的T淋巴细胞,其特征在于,所述融合蛋白进一步包括连接肽,所述连接肽设置于所述免疫检查点单链抗体和T细胞激活分子之间;
任选地,所述连接肽具有SEQ ID NO:1所示的氨基酸序列;
优选地,所述连接肽的N端与所述免疫检查点单链抗体的C端相连,所述连接肽的C端与所述T细胞激活分子的N端相连。
5.根据权利要求1所述的T淋巴细胞,其特征在于,所述融合蛋白具有SEQ ID NO:2或SEQ ID NO:3所示的氨基酸序列。
6.一种慢病毒,其特征在于,所述慢病毒携带下列核酸分子:
(a)编码融合蛋白的核酸分子,所述融合蛋白包括:免疫检查点单链抗体和T细胞激活分子;
(b)编码嵌合抗原受体的核酸分子,所述嵌合抗原受体的胞外区识别肿瘤抗原。
7.根据权利要求6所述的慢病毒,其特征在于,所述免疫检查点为PD-1,所述T细胞激活分子为IL21,所述融合蛋白具有SEQ ID NO:2或3所示的氨基酸序列;
任选地,所述肿瘤抗原为CD19,所述嵌合抗原受体具有SEQ ID NO:4所示的氨基酸序列;
任选地,所述编码融合蛋白的核酸分子具有SEQ ID NO:5或6任一所示的核苷酸序列;
任选地,所述编码嵌合抗原受体的核酸分子具有SEQ ID NO:7所示的核苷酸序列。
8.一种慢病毒,其特征在于,所述慢病毒携带具有SEQ ID NO:8或9所示核苷酸序列的核酸分子。
9.一种转基因淋巴细胞,其特征在于,所述转基因淋巴细胞共表达融合蛋白以及嵌合抗原受体,所述嵌合抗原受体识别肿瘤抗原,
其中,所述嵌合抗原受体包括:
胞外区;
跨膜区,所述跨膜区与所述胞外区相连,并且嵌入到所述转基因淋巴细胞的细胞膜中;
胞内区,所述胞内区与所述跨膜区相连,并且所述胞内区包括免疫共刺激分子胞内段;
所述融合蛋白包括:免疫检查点单链抗体和T细胞激活分子。
10.根据权利要求9所述的转基因淋巴细胞,其特征在于,所述免疫共刺激分子胞内段独立地选自4-1BB、OX-40、CD40L、CD27、CD30、CD28、CD3以及他们的衍生物的至少一种;
优选地,所述免疫共刺激分子胞内段是4-1BB、CD3的胞内段;
任选地,所述淋巴细胞是CD3+T淋巴细胞;
任选地,所述淋巴细胞是CD8+T淋巴细胞;
任选地,所述淋巴细胞是自然杀伤细胞;
任选地,所述淋巴细胞是自然杀伤T细胞;
任选地,所述免疫检查点包括选自PD-1、PD-L1、CTLA-4、TIM3、LAG3、BTLA和TIGIT的至少之一;
任选地,所述T细胞激活分子包括选自IL2、IL7、IL9、IL12、IL15、IL18与IL21的的至少之一;
优选地,所述免疫检查点为PD-1,所述T细胞激活分子为IL21;
任选地,所述IL21的C端与PD-1单链抗体的N端相连;
优选地,所述PD-1单链抗体的C端与所述IL21的N端相连;
任选地,所述融合蛋白进一步包括连接肽,所述连接肽设置于所述免疫检查点单链抗体和T细胞激活分子之间;
任选地,所述连接肽具有SEQ ID NO:1所示的氨基酸序列;
优选地,所述连接肽的N端与所述免疫检查点单链抗体的C端相连,所述连接肽的C端与所述T细胞激活分子的N端相连;
任选地,所述融合蛋白具有SEQ ID NO:2或SEQ ID NO:3所示的氨基酸序列。
11.一种构建体,其特征在于,所述构建体包括:
第一核酸分子,所述第一核酸分子编码融合蛋白,所述融合蛋白包括:
免疫检查点单链抗体和T细胞激活分子;
第二核酸分子,所述第二核酸分子编码嵌合抗原受体,所述嵌合抗原受体识别肿瘤抗原;
其中,所述融合蛋白、所述嵌合抗原受体是如权利要求1~5、9或10所限定的。
12.根据权利要求11所述的构建体,其特征在于,所述第一核酸分子、所述第二核酸分子被设置为在权利要求9或10所述的淋巴细胞中表达所述融合蛋白、嵌合抗原受体,并且所述融合蛋白与嵌合抗原受体呈非融合形式。
13.根据权利要求12所述的构建体,其特征在于,进一步包括:
第一启动子,所述第一启动子与所述第一核酸分子可操作地连接;以及
第二启动子,所述第二启动子与所述第二核酸分子可操作地连接;
任选地,所述第一启动子、所述第二启动子分别独立地选自U6、H1、CMV、EF-1、LTR或RSV启动子;
任选地,进一步包括:
内部核糖体进入位点序列,所述内部核糖体进入位点序列设置在所述第一核酸分子与所述第二核酸分子之间,所述内部核糖体进入位点具有SEQ ID NO:10所示的核苷酸序列;
任选地,进一步包括:
第三核酸分子,所述第三核酸分子设置在所述第一核酸分子与所述第二核酸分子之间,并且所述第三核酸分子编码可切割连接肽,所述可切割连接肽能够在所述淋巴细胞中被切割;
任选地,所述可切割连接肽具有SEQ ID NO:11所示的氨基酸序列。
14.根据权利要求11所述的构建体,其特征在于,所述构建体的载体是非致病性病毒载体;
任选地,所述病毒载体包括选自逆转录病毒载体、慢病毒载体或腺病毒相关病毒载体的至少之一。
15.一种制备权利要求1~5任一项所述的T淋巴细胞或者权利要求9或10所述的转基因淋巴细胞的方法,其特征在于,包括:
将权利要求11~14任一项所述的构建体或者权利要求6~8任一项所述的慢病毒引入到淋巴细胞中或者T淋巴细胞。
16.一种用于治疗癌症的治疗组合物,其特征在于,包括:
权利要求11~14任一项所述的构建体、权利要求6~8任一项所述的慢病毒、权利要求1~5任一项所述的T淋巴细胞或者权利要求9或10所述的转基因淋巴细胞;
任选地,所述癌症包括选自肝癌、胰腺癌、卵巢癌、胆管癌、肺癌、胃癌、肠癌、食管癌和乳腺癌的至少之一。
17.权利要求1~5任一项所述的T淋巴细胞、权利要求6~8任一项所述的慢病毒、权利要求9或10所述的转基因淋巴细胞、权利要求11~14任一项所述的构建体或权利要求16所述的治疗组合物在制备治疗癌症的药物中的用途。
18.根据权利要求17所述的用途,其特征在于,所述癌症包括选自肝癌、胰腺癌、卵巢癌、胆管癌、肺癌、胃癌、肠癌、食管癌和乳腺癌的至少之一。
19.一种降低T淋巴细胞表面免疫检查点表达的方法,其特征在于,包括:
使所述T淋巴细胞表达嵌合抗原受体和融合蛋白;或使所述T淋巴细胞与表达嵌合抗原受体和融合蛋白的T细胞共培养,
所述融合蛋白包括:免疫检查点单链抗体和T细胞激活分子,
其中,所述嵌合抗原受体、融合蛋白是如权利要求1~5、9或10所限定的。
20.根据权利要求19所述的方法,其特征在于,所述免疫检查点为PD-1,所述T细胞激活分子为IL21。
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