CN113214396B - 抗tim3的单链抗体及其在制备***的药物中的用途 - Google Patents

抗tim3的单链抗体及其在制备***的药物中的用途 Download PDF

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CN113214396B
CN113214396B CN202010760969.4A CN202010760969A CN113214396B CN 113214396 B CN113214396 B CN 113214396B CN 202010760969 A CN202010760969 A CN 202010760969A CN 113214396 B CN113214396 B CN 113214396B
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CN113214396A (zh
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张伟
江涛
翟优
李冠璋
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Beijing Neurosurgical Institute
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Beijing Neurosurgical Institute
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Abstract

本公开涉及一种抗TIM3的单链抗体，该抗TIM3的单链抗体的氨基酸序列包括SEQ ID NO.1所示的序列。表达抗TIM3的单链抗体的T淋巴细胞能够有效杀伤肿瘤细胞。

Description

抗TIM3的单链抗体及其在制备***的药物中的用途
技术领域
本公开涉及医药生物技术领域，具体地，涉及一种靶向TIM3的嵌合抗原受体、其编码核酸、表达载体、细胞、药物组合物和它们的用途。
背景技术
嵌合抗原受体(Chimeric antigen receptor，CAR)是人工合成的T细胞受体，由抗原结合结构域、跨膜结构域和胞内信号传导结构域组成。抗原结合结构域位于T细胞膜外，包括单链抗体或配体，用于特异性地结合靶抗原。胞内信号传导结构域位于T细胞膜内，用于向T细胞内传导信号以刺激T细胞产生免疫反应。
CAR能够靶向识别肿瘤细胞表面的靶抗原，因此，表达CAR的T细胞能够用于靶向杀伤肿瘤细胞。然而，现有的表达嵌合抗原受体CAR的T细胞对肿瘤细胞的杀伤能力仍然较弱。
发明内容
本公开的目的是克服现有的表达嵌合抗原受体CAR的T细胞对肿瘤细胞的杀伤能力仍然较弱的问题，提供一种抗TIM3的单链抗体。
为了实现上述目的，第一方面，本公开提供一种抗TIM3的单链抗体，该抗TIM3的单链抗体的氨基酸序列包括SEQ ID NO.1所示的序列。
第二方面，本公开提供一种核酸，该核酸编码第一方面所述的抗TIM3的单链抗体；优选地，所述核酸具有SEQ ID NO.2所示的核苷酸序列。
第三方面，本公开提供一种融合蛋白，所述融合蛋白含有依次连接的抗原结合结构域、跨膜结构域和胞内信号传导结构域；所述胞内信号传导结构域的氨基酸序列包括第一方面所述的抗TIM3的单链抗体的氨基酸序列；
优选地，所述抗原结合结构域包括抗CD44的单链抗体和/或抗CD133的单链抗体，所述胞内信号传导结构域包括抗TIM3的单链抗体；
更优选地，所述融合蛋白具有SEQ ID NO.3所示的氨基酸序列。
第四方面，本公开提供一种融合核酸，所述融合核酸编码第三方面所述的融合蛋白；
优选地，所述融合核酸具有SEQ ID NO.4所示的核苷酸序列。
第五方面，本公开提供一种表达载体，所述表达载体***有表达框，所述表达框包括编码抗原结合分子的第一核酸片段和编码胞内信号传导分子的第二核酸片段，所述胞内信号传导分子中含有第一方面所述的抗TIM3的单链抗体，所述第一核酸片段与所述第二核酸片段之间***有IRES元件或2A肽编码序列；
优选地，所述表达框为第四方面所述的融合核酸。
第六方面，本公开提供一种表达嵌合抗原受体的细胞，该表达嵌合抗原受体的细胞由宿主细胞转染第五方面所述的表达载体后得到，所述嵌合抗原受体中含有第一方面所述的抗TIM3的单链抗体。
可选地，所述宿主细胞为T细胞。
第七方面，本公开提供第一方面所述的抗TIM3的单链抗体、第二方面所述的核酸、第三方面所述的融合蛋白、第四方面所述的融合核酸、第五方面所述的表达载体、第六方面所述的表达嵌合抗原受体的细胞在制备***的药物中的用途。
可选地，所述肿瘤为脑胶质瘤。
第八方面，本公开提供一种药物组合物，该药物组合物的有效成分包括第六方面所述的表达抗TIM3嵌合抗原受体的细胞。
通过上述技术方案，本公开提供的抗TIM3的单链抗体，能够有效提升T淋巴细胞对肿瘤细胞的杀伤能力，因此，表达抗TIM3的单链抗体的T淋巴细胞能够有效杀伤肿瘤细胞。
本公开的其他特征和优点将在随后的具体实施方式部分予以详细说明。
附图说明
附图是用来提供对本公开的进一步理解，并且构成说明书的一部分，与下面的具体实施方式一起用于解释本公开，但并不构成对本公开的限制。在附图中：
图1是本公开实施例提供的CAR-T 1细胞的流式细胞仪检测结果图；
图2是本公开实施例提供的CAR-T 2细胞的流式细胞仪检测结果图；
图3是本公开实施例提供的CAR-T 3细胞的流式细胞仪检测结果图。
具体实施方式
以下结合附图对本公开的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本公开，并不用于限制本公开。
本公开的第一方面提供一种抗TIM3的单链抗体，该抗TIM3的单链抗体的氨基酸序列包括SEQ ID NO.1所示的序列。
其中，SEQ ID NO.1所示的序列如下所示：
QVQLQQSGPGLVKPSHTLSLTCTVSGGSISSGGYYWSWTRQHPGMGLEWIGYISYSGSIYYTPSLKSRLTISVDTSKNQFSLKLSSVTAADTAVYYCASLDSWGSNRDYWGQGTLVTVSSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIK。
T细胞免疫球蛋白粘蛋白-3(TIM3)是一个重要的肿瘤免疫检测点，广泛表达于肿瘤浸润T淋巴细胞上，能够抑制肿瘤浸润T淋巴细胞的肿瘤免疫活性。针对TIM3设计的抗TIM3的单链抗体，能够有效阻断T淋巴细胞中的TIM3途径，提升T淋巴细胞的肿瘤免疫活性，增强T淋巴细胞对肿瘤细胞的杀伤力。
本公开的发明人发现，表达上述抗TIM3的单链抗体的T淋巴细胞能够有效杀伤肿瘤细胞。
本公开的第二方面提供一种核酸，该核酸编码第一方面所述的抗TIM3的单链抗体；优选地，所述核酸具有SEQ ID NO.2所示的核苷酸序列。
其中，SEQ ID NO.2所示的核苷酸序列如下所示：
caggtgcagctacagcagtcgggcccaggactggtgaagccttcacacaccctgtccctcacctgcactgtctctggtggctccatcagcagtggtggttactactggagttggacccgtcagcacccagggatgggcctggagtggattggatacatctcttacagtgggagtatctattacactccgtccctcaagagtcgacttaccatatcagtggacacgtctaagaaccagttctccctgaagctgagctctgtgactgccgcggacacggccgtatattactgtgcgagtttggattcctggggatctaaccgtgactactggggccagggaaccctggtcaccgtctcgagtggaggtggtggatccgaaattgtgttgacgcagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggccgctcactttcggcggagggaccaaggtggagattaag。
本公开的第三方面提供一种融合蛋白，所述融合蛋白含有依次连接的抗原结合结构域、跨膜结构域和胞内信号传导结构域；所述胞内信号传导结构域的氨基酸序列包括第一方面所述的抗TIM3的单链抗体的氨基酸序列。优选地，所述抗原结合结构域包括抗CD44的单链抗体和/或抗CD133的单链抗体，所述胞内信号传导结构域包括抗TIM3的单链抗体。更优选地，所述融合蛋白具有SEQ ID NO.3所示的氨基酸序列。
其中，SEQ ID NO.3所示的融合蛋白由抗CD44的单链抗体、抗CD133的单链抗体、CD28、CD3、T2A和抗TIM3的单链抗体组成。
其中，SEQ ID NO.3所示的氨基酸序列如下所示：
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGL EWVAVIWYDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRSDYRGYYGMDVWGQGTTVTVSSGSTSGGGSGGGSGGGGSSEIVLTQSPATLSLSPGERATLSCRASQSVINYLAWYQQKPGQAPRLLIYDASNRASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRRNWPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDFEMHWVRQAPGQGLEWMGDIDPGTGDTAYNLKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCALGAFVYWGQGTLVTVSSGSTSGGGSGGGSGGGGSSDVVMTQSPLSLPVTPGEPASISCRSSQSLANSYGNTYLSWYLQKPGQSPQLLIYGISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGTHQPYTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPMTNKCLLQIALLLCFSTTALSQVQLQQSGPGLVKPSHTLSLTCTVSGGSISSGGYYWSWTRQHPGMGLEWIGYISYSGSIYYTPSLKSRLTISVDTSKNQFSLKLSSVTAADTAVYYCASLDSWGSNRDYWGQGTLVTVSSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIK。
本公开的第四方面提供一种融合核酸，所述融合核酸编码第三方面所述的融合蛋白。优选地，所述融合核酸具有SEQ ID NO.4所示的核苷酸序列。
其中，SEQ ID NO.4所示的核苷酸序列用于编码SEQ ID NO.3所示的氨基酸序列。
其中，SEQ ID NO.4所示的核苷酸序列如下所示：
caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcgtctggattcaccttcagtagctatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggtatgatggaagtaataaattctatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgaggagaagtgactacaggggctactacggtatggacgtctggggccaagggaccacggtcaccgtctcctcaggcagtactagcggtggtggctccgggggcggttccggtgggggcggcagcagcgaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttatcaactacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggcctctggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtcgcaactggccgctcactttcggcggagggaccaaggtggagatcaaaggaggtggtggatccggaggtggtggctccggaggtggtggatcccaggttcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggttacacctttaccgactttgaaatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggagatattgatcctggaactggtgatactgcctacaatctgaagttcaagggcagagtcaccatgaccacagacacatccacgagcacagcctacatggagctgaggagcctgaggtctgacgacacggccgtgtattactgtgcgttgggggcctttgtttactggggccagggaaccctggtcaccgtctcctcaggcagtactagcggtggtggctccgggggcggttccggtgggggcggcagcagcgatgttgtgatgactcagtctccactctccctgcccgtcacccctggagagccggcctccatctcctgcaggtctagtcagagtcttgcaaacagttatgggaacacctatttgtcttggtacctgcagaagccagggcagtctccacagctcctgatctatgggatttccaacagattttctggggtccctgacaggttcagtggcagtggatcaggcacagattttacactgaaaatcagcagagtggaggctgaggacgttggggtttattactgcttacaaggtacacatcagccgtacacgtttggccaggggaccaagctggagatcaaaaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctcccgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgggagggaaggggttctctcctgacctgcggcgatgtggaggagaaccccggacccatgaccaacaagtgtctcctccaaattgctctcctgttgtgcttctccactacagctctttcccaggtgcagctacagcagtcgggcccaggactggtgaagccttcacacaccctgtccctcacctgcactgtctctggtggctccatcagcagtggtggttactactggagttggacccgtcagcacccagggatgggcctggagtggattggatacatctcttacagtgggagtatctattacactccgtccctcaagagtcgacttaccatatcagtggacacgtctaagaaccagttctccctgaagctgagctctgtgactgccgcggacacggccgtatattactgtgcgagtttggattcctggggatctaaccgtgactactggggccagggaaccctggtcaccgtctcgagtggaggtggtggatccgaaattgtgttgacgcagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggccgctcactttcggcggagggaccaaggtggagattaag。
本公开的第五方面提供一种表达载体，所述表达载体***有表达框，所述表达框包括编码抗原结合分子的第一核酸片段和编码胞内信号传导分子的第二核酸片段，所述胞内信号传导分子中含有第一方面所述的抗TIM3的单链抗体，所述第一核酸片段与所述第二核酸片段之间***有IRES元件或2A肽编码序列；优选地，所述表达框为第四方面所述的融合核酸。
本公开的第六方面提供一种表达嵌合抗原受体的细胞，该表达嵌合抗原受体的细胞由宿主细胞转染第五方面所述的表达载体后得到，所述嵌合抗原受体中含有第一方面所述的抗TIM3的单链抗体。可选地，所述宿主细胞为T细胞。
本公开的第七方面提供第一方面所述的抗TIM3的单链抗体、第二方面所述的核酸、第三方面所述的融合蛋白、第四方面所述的融合核酸、第五方面所述的表达载体、第六方面所述的表达嵌合抗原受体的细胞在制备***的药物中的用途。可选地，所述肿瘤为脑胶质瘤。
本公开的第八方面提供一种药物组合物，该药物组合物的有效成分包括第六方面所述的表达抗TIM3嵌合抗原受体的细胞。
下面通过实施例来进一步说明本公开，但是本公开并不因此而受到任何限制。
实施例1
本实施例用于说明表达载体的构建。
(1)采用全序列合成方法，分别合成如SEQ ID NO.4和SEQ ID NO.5所示的核苷酸序列。SEQ ID NO.4所示的核苷酸序列用于编码SEQ ID NO.3所示的融合蛋白；SEQ ID NO.5所示的核苷酸序列用于编码SEQ ID NO.6所示的融合蛋白。其中，SEQ ID NO.3和SEQ IDNO.6所示的融合蛋白的组成如下：
G1：SEQ ID NO.3所示的融合蛋白由抗CD44的单链抗体、抗CD133的单链抗体、CD28、CD3、T2A和抗TIM3的单链抗体组成；
G2：SEQ ID NO.6所示的融合蛋白由抗CD44的单链抗体、抗CD133的单链抗体、CD28和CD3组成。
其中，SEQ ID NO.5所示的核苷酸序列如下所示：
caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcgtctggattcaccttcagtagctatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggtatgatggaagtaataaattctatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgaggagaagtgactacaggggctactacggtatggacgtctggggccaagggaccacggtcaccgtctcctcaggcagtactagcggtggtggctccgggggcggttccggtgggggcggcagcagcgaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttatcaactacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggcctctggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtcgcaactggccgctcactttcggcggagggaccaaggtggagatcaaaggaggtggtggatccggaggtggtggctccggaggtggtggatcccaggttcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggttacacctttaccgactttgaaatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggagatattgatcctggaactggtgatactgcctacaatctgaagttcaagggcagagtcaccatgaccacagacacatccacgagcacagcctacatggagctgaggagcctgaggtctgacgacacggccgtgtattactgtgcgttgggggcctttgtttactggggccagggaaccctggtcaccgtctcctcaggcagtactagcggtggtggctccgggggcggttccggtgggggcggcagcagcgatgttgtgatgactcagtctccactctccctgcccgtcacccctggagagccggcctccatctcctgcaggtctagtcagagtcttgcaaacagttatgggaacacctatttgtcttggtacctgcagaagccagggcagtctccacagctcctgatctatgggatttccaacagattttctggggtccctgacaggttcagtggcagtggatcaggcacagattttacactgaaaatcagcagagtggaggctgaggacgttggggtttattactgcttacaaggtacacatcagccgtacacgtttggccaggggaccaagctggagatcaaaaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctcccgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg。
SEQ ID NO.6所示的融合蛋白的氨基酸序列如下所示：
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRSDYRGYYGMDVWGQGTTVTVSSGSTSGGGSGGGSGGGGSSEIVLTQSPATLSLSPGERATLSCRASQSVINYLAWYQQKPGQAPRLLIYDASNRASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRRNWPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDFEMHWVRQAPGQGLEWMGDIDPGTGDTAYNLKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCALGAFVYWGQGTLVTVSSGSTSGGGSGGGSGGGGSSDVVMTQSPLSLPVTPGEPASISCRSSQSLANSYGNTYLSWYLQKPGQSPQLLIYGISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGTHQPYTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。
(2)采用pLVX-IRES-ΔNGFR(购自Clontech公司，货号为631982)作为载体，采用常规方法分别***步骤(1)中合成的2种核苷酸序列，得到本实施例的2种慢病毒表达载体。
实施例2
本实施例用于说明表达嵌合抗原受体的T细胞的制备和检测。
(1)将实施例1构建的2种慢病毒表达载体和pLVX-IRES-ΔNGFR空载体分别进行慢病毒包装，然后按照下述方法分别进行T细胞的体外培养、转染和扩增。
(2)按照下述方法分离血液中的T细胞：将1mL无菌PBS与1mL血液混匀，然后缓慢加入到淋巴细胞分离液Ficoll的上层，并于4℃、400g条件下离心30min，加减速分别设置为0。离心结束后，去掉上层血浆，吸取中间白膜层细胞，加入PBS重悬洗涤，并于100g条件下离心10min，加减速正常。离心结束后，去掉上层洗涤液，加入1mL的1640+10％FBS+1％双抗+1×Glutamine培养基重悬细胞，然后利用抗人CD3/CD28磁珠(购自Thermo Fisher公司)刺激扩增，其中，重悬后细胞浓度为1×106细胞/mL，磁珠加入量为100μL，再加入100IU/mL rhIL-2(Peprotech)，刺激培养2天，得到T细胞。
(3)按照下述方法进行慢病毒转染：取3份上述分离得到的T细胞，分别加入步骤(1)包装好的慢病毒，然后加入终浓度为6μg/mL的polybrene，混匀，并于32℃、800g的条件下离心100min。离心结束后放入培养箱中继续培养24h。培养结束后将培养物于1500rpm的条件下离心15min，并将离心得到的细胞以1×106/mL的密度接种于培养板内，以rhIL2-100IU/mL刺激培养，以后每2-3天换液一次，直至2-4周，得到转染后的T淋巴细胞CAR-T1、CAR-T 2和CAR-T 3。其中，CAR-T 1转染有SEQ ID NO.4所示的核苷酸序列，能够表达SEQ IDNO.3所示的融合蛋白；CAR-T 2转染有SEQ ID NO.5所示的核苷酸序列，能够表达SEQ IDNO.6所示的融合蛋白；CAR-T 3转染有空载体。
培养结束后，用PBS重悬细胞，并用流式细胞仪检测上述3种CAR-T细胞的比例及表面CAR蛋白的表达。检测方法为：分别离心收集转染后的待检测T细胞，PBS洗涤1次后弃上清，按抗体说明书加入相应检测量的单抗避光30min后，利用PBS洗涤、重悬，过膜后采用流式细胞仪进行夹心法检测，检测时使用的抗体为His-tag标记的CD44和PE标记的抗His-tag的抗体的混合物。结果如图1～3所示。
图1是本公开实施例提供的CAR-T 1细胞的流式细胞仪检测结果图；
图2是本公开实施例提供的CAR-T 2细胞的流式细胞仪检测结果图；
图3是本公开实施例提供的CAR-T 3细胞的流式细胞仪检测结果图。
从图1和图2可以看出，CAR-T 1细胞和CAR-T 2细胞中均检测出区别于正常T淋巴细胞的其它细胞；由图3可以看出，CAR-T 3细胞并未检测出区别于正常T淋巴细胞的其它细胞。由此说明本实施例中转染有融合基因的CAR-T细胞均已成功表达目标融合蛋白。
实施例3
本实施例用于验证实施例2构建的CAR-T细胞对肿瘤细胞的杀伤能力。
分别取实施例2中转染并培养得到的3种CAR-T细胞，分别与CD44和CD133阳性的胶质瘤干细胞GSC20按照不同的效靶比(T细胞数量：胶质瘤干细胞数量)混合。将混合后的细胞置于96孔板中进行培养，其中，每孔中含有胶质瘤干细胞4×104个，每孔反应体系为200μL。培养条件包括：37℃，5％CO2，饱和湿度孵箱孵育4小时。
乳酸脱氢酶活性测定：离心结束后，每孔吸取上清液100μL置于96孔酶标板中，同时，每孔加入100μL LDH底物，室温避光反应30min。反应结束后，每孔加50μL终止液终止酶促反应。在酶标仪490nm测定光密度值(OD)。计算各组平均光密度值(OD)，按下式计算每种T细胞对胶质瘤干细胞的裂解率。结果如表1所示。
裂解率％＝(实验组OD-胶质瘤干细胞自发释放OD-效应细胞自然释放OD)/(胶质瘤干细胞最大释放OD-胶质瘤干细胞自发释放OD)
表1
Figure BDA0002613066500000121
由表1可以看出，本公开提供的抗TIM3的单链抗体能够有效提升T淋巴细胞的肿瘤免疫活性，增强T淋巴细胞对肿瘤细胞的杀伤力。
对比例
采用靶向经典肿瘤靶点EGFR vIII的传统第二代CAR-T细胞(EGFR vIII-CD28-CD3)替换实施例3中的T细胞，然后按照实施例3的方法检测不同效靶比下该CAR-T对胶质瘤干细胞GSC20的杀伤率。检测结果见表2。
表2
Figure BDA0002613066500000122
由表2可以看出，靶向经典肿瘤干细胞靶点EGFR vIII的传统第二代CAR-T细胞对胶质瘤干细胞的杀伤力不如本公开提供的表达抗TIM3的单链抗体的T细胞。
以上结合附图详细描述了本公开的优选实施方式，但是，本公开并不限于上述实施方式中的具体细节，在本公开的技术构思范围内，可以对本公开的技术方案进行多种简单变型，这些简单变型均属于本公开的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本公开对各种可能的组合方式不再另行说明。
此外，本公开的各种不同的实施方式之间也可以进行任意组合，只要其不违背本公开的思想，其同样应当视为本公开所公开的内容。
序列表
<110> 北京市神经外科研究所
<120> 抗TIM3的单链抗体及其在制备***的药物中的用途
<130> 17157BJNI-LGZ
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 232
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser His
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly
20 25 30
Gly Tyr Tyr Trp Ser Trp Thr Arg Gln His Pro Gly Met Gly Leu Glu
35 40 45
Trp Ile Gly Tyr Ile Ser Tyr Ser Gly Ser Ile Tyr Tyr Thr Pro Ser
50 55 60
Leu Lys Ser Arg Leu Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe
65 70 75 80
Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Ser Leu Asp Ser Trp Gly Ser Asn Arg Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu Ile Val
115 120 125
Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala
130 135 140
Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp
145 150 155 160
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala
165 170 175
Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser
180 185 190
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe
195 200 205
Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu Thr Phe Gly
210 215 220
Gly Gly Thr Lys Val Glu Ile Lys
225 230
<210> 2
<211> 696
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
caggtgcagc tacagcagtc gggcccagga ctggtgaagc cttcacacac cctgtccctc 60
acctgcactg tctctggtgg ctccatcagc agtggtggtt actactggag ttggacccgt 120
cagcacccag ggatgggcct ggagtggatt ggatacatct cttacagtgg gagtatctat 180
tacactccgt ccctcaagag tcgacttacc atatcagtgg acacgtctaa gaaccagttc 240
tccctgaagc tgagctctgt gactgccgcg gacacggccg tatattactg tgcgagtttg 300
gattcctggg gatctaaccg tgactactgg ggccagggaa ccctggtcac cgtctcgagt 360
ggaggtggtg gatccgaaat tgtgttgacg cagtctccag ccaccctgtc tttgtctcca 420
ggggaaagag ccaccctctc ctgcagggcc agtcagagtg ttagcagcta cttagcctgg 480
taccaacaga aacctggcca ggctcccagg ctcctcatct atgatgcatc caacagggcc 540
actggcatcc cagccaggtt cagtggcagt gggtctggga cagacttcac tctcaccatc 600
agcagcctag agcctgaaga ttttgcagtt tattactgtc agcagcgtag caactggccg 660
ctcactttcg gcggagggac caaggtggag attaag 696
<210> 3
<211> 998
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Phe Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Ser Asp Tyr Arg Gly Tyr Tyr Gly Met Asp Val Trp Gly
100 105 110
Gln Gly Thr Thr Val Thr Val Ser Ser Gly Ser Thr Ser Gly Gly Gly
115 120 125
Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Glu Ile Val Leu Thr
130 135 140
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu
145 150 155 160
Ser Cys Arg Ala Ser Gln Ser Val Ile Asn Tyr Leu Ala Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn
180 185 190
Arg Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val
210 215 220
Tyr Tyr Cys Gln Gln Arg Arg Asn Trp Pro Leu Thr Phe Gly Gly Gly
225 230 235 240
Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
245 250 255
Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
260 265 270
Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
275 280 285
Thr Phe Thr Asp Phe Glu Met His Trp Val Arg Gln Ala Pro Gly Gln
290 295 300
Gly Leu Glu Trp Met Gly Asp Ile Asp Pro Gly Thr Gly Asp Thr Ala
305 310 315 320
Tyr Asn Leu Lys Phe Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser
325 330 335
Thr Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr
340 345 350
Ala Val Tyr Tyr Cys Ala Leu Gly Ala Phe Val Tyr Trp Gly Gln Gly
355 360 365
Thr Leu Val Thr Val Ser Ser Gly Ser Thr Ser Gly Gly Gly Ser Gly
370 375 380
Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Val Val Met Thr Gln Ser
385 390 395 400
Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys
405 410 415
Arg Ser Ser Gln Ser Leu Ala Asn Ser Tyr Gly Asn Thr Tyr Leu Ser
420 425 430
Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gly
435 440 445
Ile Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly
450 455 460
Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp
465 470 475 480
Val Gly Val Tyr Tyr Cys Leu Gln Gly Thr His Gln Pro Tyr Thr Phe
485 490 495
Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg
500 505 510
Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg
515 520 525
Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly
530 535 540
Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr
545 550 555 560
Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Ser
565 570 575
Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg
580 585 590
Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg
595 600 605
Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp
610 615 620
Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn
625 630 635 640
Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg
645 650 655
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly
660 665 670
Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
675 680 685
Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu
690 695 700
Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His
705 710 715 720
Met Gln Ala Leu Pro Pro Arg Glu Gly Arg Gly Ser Leu Leu Thr Cys
725 730 735
Gly Asp Val Glu Glu Asn Pro Gly Pro Met Thr Asn Lys Cys Leu Leu
740 745 750
Gln Ile Ala Leu Leu Leu Cys Phe Ser Thr Thr Ala Leu Ser Gln Val
755 760 765
Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser His Thr Leu
770 775 780
Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly Gly Tyr
785 790 795 800
Tyr Trp Ser Trp Thr Arg Gln His Pro Gly Met Gly Leu Glu Trp Ile
805 810 815
Gly Tyr Ile Ser Tyr Ser Gly Ser Ile Tyr Tyr Thr Pro Ser Leu Lys
820 825 830
Ser Arg Leu Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu
835 840 845
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
850 855 860
Ser Leu Asp Ser Trp Gly Ser Asn Arg Asp Tyr Trp Gly Gln Gly Thr
865 870 875 880
Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr
885 890 895
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu
900 905 910
Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln
915 920 925
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn
930 935 940
Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr
945 950 955 960
Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val
965 970 975
Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu Thr Phe Gly Gly Gly
980 985 990
Thr Lys Val Glu Ile Lys
995
<210> 4
<211> 2994
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60
tcctgtgcag cgtctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120
ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaagtaa taaattctat 180
gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gaggagaagt 300
gactacaggg gctactacgg tatggacgtc tggggccaag ggaccacggt caccgtctcc 360
tcaggcagta ctagcggtgg tggctccggg ggcggttccg gtgggggcgg cagcagcgaa 420
attgtgttga cacagtctcc agccaccctg tctttgtctc caggggaaag agccaccctc 480
tcctgcaggg ccagtcagag tgttatcaac tacttagcct ggtaccaaca gaaacctggc 540
caggctccca ggctcctcat ctatgatgca tccaacaggg cctctggcat cccagccagg 600
ttcagtggca gtgggtctgg gacagacttc actctcacca tcagcagcct agagcctgaa 660
gattttgcag tttattactg tcagcagcgt cgcaactggc cgctcacttt cggcggaggg 720
accaaggtgg agatcaaagg aggtggtgga tccggaggtg gtggctccgg aggtggtgga 780
tcccaggttc agctggtgca gtctggagct gaggtgaaga agcctggggc ctcagtgaag 840
gtctcctgca aggcttctgg ttacaccttt accgactttg aaatgcactg ggtgcgacag 900
gcccctggac aagggcttga gtggatggga gatattgatc ctggaactgg tgatactgcc 960
tacaatctga agttcaaggg cagagtcacc atgaccacag acacatccac gagcacagcc 1020
tacatggagc tgaggagcct gaggtctgac gacacggccg tgtattactg tgcgttgggg 1080
gcctttgttt actggggcca gggaaccctg gtcaccgtct cctcaggcag tactagcggt 1140
ggtggctccg ggggcggttc cggtgggggc ggcagcagcg atgttgtgat gactcagtct 1200
ccactctccc tgcccgtcac ccctggagag ccggcctcca tctcctgcag gtctagtcag 1260
agtcttgcaa acagttatgg gaacacctat ttgtcttggt acctgcagaa gccagggcag 1320
tctccacagc tcctgatcta tgggatttcc aacagatttt ctggggtccc tgacaggttc 1380
agtggcagtg gatcaggcac agattttaca ctgaaaatca gcagagtgga ggctgaggac 1440
gttggggttt attactgctt acaaggtaca catcagccgt acacgtttgg ccaggggacc 1500
aagctggaga tcaaaaccac taccccagca ccgaggccac ccaccccggc tcctaccatc 1560
gcctcccagc ctctgtccct gcgtccggag gcatgtagac ccgcagctgg tggggccgtg 1620
catacccggg gtcttgactt cgcctgcgat atctacattt gggcccctct ggctggtact 1680
tgcggggtcc tgctgctttc actcgtgatc actctttact gtaggagtaa gaggagcagg 1740
ctcctgcaca gtgactacat gaacatgact ccccgccgcc ccgggcccac ccgcaagcat 1800
taccagccct atgccccacc acgcgacttc gcagcctatc gctcccgcgt gaaattcagc 1860
cgcagcgcag atgctccagc ctacaagcag gggcagaacc agctctacaa cgaactcaat 1920
cttggtcgga gagaggagta cgacgtgctg gacaagcgga gaggacggga cccagaaatg 1980
ggcgggaagc cgcgcagaaa gaatccccaa gagggcctgt acaacgagct ccaaaaggat 2040
aagatggcag aagcctatag cgagattggt atgaaagggg aacgcagaag aggcaaaggc 2100
cacgacggac tgtaccaggg actcagcacc gccaccaagg acacctatga cgctcttcac 2160
atgcaggccc tgccgcctcg ggagggaagg ggttctctcc tgacctgcgg cgatgtggag 2220
gagaaccccg gacccatgac caacaagtgt ctcctccaaa ttgctctcct gttgtgcttc 2280
tccactacag ctctttccca ggtgcagcta cagcagtcgg gcccaggact ggtgaagcct 2340
tcacacaccc tgtccctcac ctgcactgtc tctggtggct ccatcagcag tggtggttac 2400
tactggagtt ggacccgtca gcacccaggg atgggcctgg agtggattgg atacatctct 2460
tacagtggga gtatctatta cactccgtcc ctcaagagtc gacttaccat atcagtggac 2520
acgtctaaga accagttctc cctgaagctg agctctgtga ctgccgcgga cacggccgta 2580
tattactgtg cgagtttgga ttcctgggga tctaaccgtg actactgggg ccagggaacc 2640
ctggtcaccg tctcgagtgg aggtggtgga tccgaaattg tgttgacgca gtctccagcc 2700
accctgtctt tgtctccagg ggaaagagcc accctctcct gcagggccag tcagagtgtt 2760
agcagctact tagcctggta ccaacagaaa cctggccagg ctcccaggct cctcatctat 2820
gatgcatcca acagggccac tggcatccca gccaggttca gtggcagtgg gtctgggaca 2880
gacttcactc tcaccatcag cagcctagag cctgaagatt ttgcagttta ttactgtcag 2940
cagcgtagca actggccgct cactttcggc ggagggacca aggtggagat taag 2994
<210> 5
<211> 2181
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60
tcctgtgcag cgtctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120
ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaagtaa taaattctat 180
gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gaggagaagt 300
gactacaggg gctactacgg tatggacgtc tggggccaag ggaccacggt caccgtctcc 360
tcaggcagta ctagcggtgg tggctccggg ggcggttccg gtgggggcgg cagcagcgaa 420
attgtgttga cacagtctcc agccaccctg tctttgtctc caggggaaag agccaccctc 480
tcctgcaggg ccagtcagag tgttatcaac tacttagcct ggtaccaaca gaaacctggc 540
caggctccca ggctcctcat ctatgatgca tccaacaggg cctctggcat cccagccagg 600
ttcagtggca gtgggtctgg gacagacttc actctcacca tcagcagcct agagcctgaa 660
gattttgcag tttattactg tcagcagcgt cgcaactggc cgctcacttt cggcggaggg 720
accaaggtgg agatcaaagg aggtggtgga tccggaggtg gtggctccgg aggtggtgga 780
tcccaggttc agctggtgca gtctggagct gaggtgaaga agcctggggc ctcagtgaag 840
gtctcctgca aggcttctgg ttacaccttt accgactttg aaatgcactg ggtgcgacag 900
gcccctggac aagggcttga gtggatggga gatattgatc ctggaactgg tgatactgcc 960
tacaatctga agttcaaggg cagagtcacc atgaccacag acacatccac gagcacagcc 1020
tacatggagc tgaggagcct gaggtctgac gacacggccg tgtattactg tgcgttgggg 1080
gcctttgttt actggggcca gggaaccctg gtcaccgtct cctcaggcag tactagcggt 1140
ggtggctccg ggggcggttc cggtgggggc ggcagcagcg atgttgtgat gactcagtct 1200
ccactctccc tgcccgtcac ccctggagag ccggcctcca tctcctgcag gtctagtcag 1260
agtcttgcaa acagttatgg gaacacctat ttgtcttggt acctgcagaa gccagggcag 1320
tctccacagc tcctgatcta tgggatttcc aacagatttt ctggggtccc tgacaggttc 1380
agtggcagtg gatcaggcac agattttaca ctgaaaatca gcagagtgga ggctgaggac 1440
gttggggttt attactgctt acaaggtaca catcagccgt acacgtttgg ccaggggacc 1500
aagctggaga tcaaaaccac taccccagca ccgaggccac ccaccccggc tcctaccatc 1560
gcctcccagc ctctgtccct gcgtccggag gcatgtagac ccgcagctgg tggggccgtg 1620
catacccggg gtcttgactt cgcctgcgat atctacattt gggcccctct ggctggtact 1680
tgcggggtcc tgctgctttc actcgtgatc actctttact gtaggagtaa gaggagcagg 1740
ctcctgcaca gtgactacat gaacatgact ccccgccgcc ccgggcccac ccgcaagcat 1800
taccagccct atgccccacc acgcgacttc gcagcctatc gctcccgcgt gaaattcagc 1860
cgcagcgcag atgctccagc ctacaagcag gggcagaacc agctctacaa cgaactcaat 1920
cttggtcgga gagaggagta cgacgtgctg gacaagcgga gaggacggga cccagaaatg 1980
ggcgggaagc cgcgcagaaa gaatccccaa gagggcctgt acaacgagct ccaaaaggat 2040
aagatggcag aagcctatag cgagattggt atgaaagggg aacgcagaag aggcaaaggc 2100
cacgacggac tgtaccaggg actcagcacc gccaccaagg acacctatga cgctcttcac 2160
atgcaggccc tgccgcctcg g 2181
<210> 6
<211> 727
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Phe Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Ser Asp Tyr Arg Gly Tyr Tyr Gly Met Asp Val Trp Gly
100 105 110
Gln Gly Thr Thr Val Thr Val Ser Ser Gly Ser Thr Ser Gly Gly Gly
115 120 125
Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Glu Ile Val Leu Thr
130 135 140
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu
145 150 155 160
Ser Cys Arg Ala Ser Gln Ser Val Ile Asn Tyr Leu Ala Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn
180 185 190
Arg Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val
210 215 220
Tyr Tyr Cys Gln Gln Arg Arg Asn Trp Pro Leu Thr Phe Gly Gly Gly
225 230 235 240
Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
245 250 255
Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
260 265 270
Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
275 280 285
Thr Phe Thr Asp Phe Glu Met His Trp Val Arg Gln Ala Pro Gly Gln
290 295 300
Gly Leu Glu Trp Met Gly Asp Ile Asp Pro Gly Thr Gly Asp Thr Ala
305 310 315 320
Tyr Asn Leu Lys Phe Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser
325 330 335
Thr Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr
340 345 350
Ala Val Tyr Tyr Cys Ala Leu Gly Ala Phe Val Tyr Trp Gly Gln Gly
355 360 365
Thr Leu Val Thr Val Ser Ser Gly Ser Thr Ser Gly Gly Gly Ser Gly
370 375 380
Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Val Val Met Thr Gln Ser
385 390 395 400
Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys
405 410 415
Arg Ser Ser Gln Ser Leu Ala Asn Ser Tyr Gly Asn Thr Tyr Leu Ser
420 425 430
Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gly
435 440 445
Ile Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly
450 455 460
Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp
465 470 475 480
Val Gly Val Tyr Tyr Cys Leu Gln Gly Thr His Gln Pro Tyr Thr Phe
485 490 495
Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg
500 505 510
Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg
515 520 525
Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly
530 535 540
Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr
545 550 555 560
Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Ser
565 570 575
Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg
580 585 590
Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg
595 600 605
Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp
610 615 620
Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn
625 630 635 640
Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg
645 650 655
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly
660 665 670
Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
675 680 685
Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu
690 695 700
Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His
705 710 715 720
Met Gln Ala Leu Pro Pro Arg
725

Claims (15)

1.一种抗TIM3的单链抗体，其特征在于，该抗TIM3的单链抗体的氨基酸序列为SEQ IDNO.1所示的序列。
2.一种核酸，其特征在于，该核酸编码权利要求1所述的抗TIM3的单链抗体。
3.根据权利要求2所述的核酸，其中，所述核酸具有SEQ ID NO.2所示的核苷酸序列。
4.一种融合蛋白，其特征在于，所述融合蛋白含有依次连接的抗原结合结构域、跨膜结构域和胞内信号传导结构域；所述胞内信号传导结构域的氨基酸序列包括权利要求1所述的抗TIM3的单链抗体的氨基酸序列。
5.根据权利要求4所述的融合蛋白，其中，所述抗原结合结构域包括抗CD44的单链抗体和/或抗CD133的单链抗体，所述胞内信号传导结构域包括抗TIM3的单链抗体。
6.根据权利要求4所述的融合蛋白，其中，所述融合蛋白具有SEQ ID NO.3所示的氨基酸序列。
7.一种融合核酸，其特征在于，所述融合核酸编码权利要求4所述的融合蛋白。
8.根据权利要求7所述的融合核酸，其中，所述融合核酸具有SEQ ID NO.4所示的核苷酸序列。
9.一种表达载体，其特征在于，所述表达载体***有表达框，所述表达框包括编码抗原结合分子的第一核酸片段和编码胞内信号传导分子的第二核酸片段，所述胞内信号传导分子中含有权利要求1所述的抗TIM3的单链抗体，所述第一核酸片段与所述第二核酸片段之间***有IRES元件或2A肽编码序列。
10.根据权利要求9所述的表达载体，其中，所述表达框为权利要求7所述的融合核酸。
11.一种表达嵌合抗原受体的细胞，其特征在于，该表达嵌合抗原受体的细胞由宿主细胞转染权利要求9所述的表达载体后得到，所述嵌合抗原受体中含有权利要求1所述的抗TIM3的单链抗体。
12.根据权利要求11所述的细胞，其特征在于，所述宿主细胞为T细胞。
13.权利要求1所述的抗TIM3的单链抗体、权利要求2所述的核酸、权利要求4所述的融合蛋白、权利要求7所述的融合核酸、权利要求9所述的表达载体、权利要求11或12所述的表达嵌合抗原受体的细胞在制备治疗胶质瘤的药物中的用途。
14.根据权利要求13所述的用途，其中，所述胶质瘤为脑胶质瘤。
15.一种药物组合物，其特征在于，该药物组合物的有效成分包括权利要求11或12所述的表达抗TIM3嵌合抗原受体的细胞。
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