CN117050195B - 一种靶向baffr嵌合抗原受体、car-t细胞和应用 - Google Patents
一种靶向baffr嵌合抗原受体、car-t细胞和应用 Download PDFInfo
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Abstract
本发明公开了一种靶向BAFFR嵌合抗原受体、CAR‑T细胞和应用,靶向BAFFR嵌合抗原受体包括从氨基端到羧基端依次连接的信号肽区域、靶向BAFFR的抗原结合域、铰链区、跨膜结构域、共刺激结构域和信号转导结构域,共刺激结构域包括相连的CD28共刺激结构域和4‑1BB共刺激结构域,信号转导结构域为CD3ζ的ITAM1。本发明通过BAFFR构建了CAR‑T细胞,利用抗原抗体结合的机制,能有效地消耗自身免疫性疾病或一些肿瘤患者体内的自身免疫性B细胞或恶行增殖B细胞,为人类自身免疫相关疾病以及一些血液肿瘤的治疗提供了新的选择。
Description
技术领域
本发明涉及分子生物学和医学技术领域,尤其涉及一种靶向BAFFR嵌合抗原受体、CAR-T细胞和应用。
背景技术
嵌合抗原受体(Chimeric Antigen Receptors,CARs)T细胞治疗(CAR-T)主要是利用基因工程构建嵌合抗原受体表达载体转染T细胞,使T细胞表面能够表达一个能识别靶细胞的嵌合抗体,通过对靶细胞表面抗原进行特异性识别,达到对靶细胞特异性杀伤的效果。
与其它的免疫细胞治疗相比,CAR-T细胞技术有更多的优势。首先,CAR-T细胞拉近了与靶细胞的距离,直接精确地攻击靶细胞。其次,由于CAR-T细胞杀伤靶细胞不依赖于MHC,不需要抗原提呈机制来识别,这样就克服了靶细胞通过MHC的下调和抗原提呈的降低而介导的免疫逃脱,更有效地杀伤靶细胞。再次,CAR基因的构建是基于靶细胞表达的靶抗原,CAR-T既可以利用蛋白质抗原,又可利用糖脂类非蛋白质抗原,扩大了靶抗原靶点范围。最后,治疗效果更持久。CAR-T结构中可以加入促进T细胞增殖与活化的基因序列,能保证T细胞进入体内后还可以增殖,CAR-T细胞具有免疫记忆功能,可以长期在体内存活。
近年来,该疗法多用于肿瘤治疗,其有很多其他疗法无法比拟的优势,被认为是最有希望攻克癌症的疗法之一,但是在其它疾病如自身免疫性疾病方面,CAR-T免疫治疗却受到很大的限制,其中一个最主要的原因是新靶点的发现。研究表明BAFF参与多种自身免疫性疾病的进展,在很多自身免疫性疾病中表达水平明显升高,例如在系统性红斑狼疮患者中BAFF持续高表达,患者血清中BAFF的浓度与抗dsDNA抗体(anti-dsDNA antibodies)的滴度呈正相关。此外,在干燥综合征、类风湿性关节炎以及多发性硬化疾患者的血清中,BAFF表达水平也显著升高。BAFF可与B细胞表面TNFR家族的三种膜受体结合,分别是BAFFR、TACI和BCMA,但是BAFF和BAFFR的结合在外周B细胞成熟过程中发挥着最主要的作用。为此针对BAFFR构建CAR-T细胞将是自身免疫性疾病和\或血液型肿瘤治疗的一个重要突破。
发明内容
鉴于现有技术中的上述缺陷或不足,期望提供一种靶向BAFFR嵌合抗原受体、CAR-T细胞和应用。通过噬菌体展示技术结合高通量表达的平台,筛选出了靶向BAFFR的纳米抗体,具有亲和力高、稳定性高、分子量小、免疫原性低、渗透力强等优势。根据所得的BAFFR抗体序列构建了CAR-T细胞,能够特定针对自身免疫性B细胞,避免了全B细胞删除造成的免疫缺陷和病人的感染。另一方面,全B细胞删除可能降低患者对疫苗,例如COVID-19疫苗的反应性,而CAR-T细胞只针对自身反应性B细胞,可以有效避免全B细胞删除造成的疫苗低反应现象,为人类自身免疫相关疾病的治疗提供了新的选择。
本发明提供的一种靶向BAFFR嵌合抗原受体,包括从氨基端到羧基端依次连接的信号肽区域、靶向BAFFR的抗原结合域、铰链区、跨膜结构域、共刺激结构域和信号转导结构域,所述共刺激结构域包括相连的CD28共刺激结构域和4-1BB共刺激结构域,所述信号转导结构域为CD3ζ的ITAM1;
所述靶向BAFFR的抗原结合域为靶向BAFFR的纳米抗体,所述靶向BAFFR的纳米抗体的氨基酸序列如SEQ ID No:2所示,或如SEQ ID No:3所示,或如SEQ ID No:4所示。
进一步的,所述信号肽区域的氨基酸序列如SEQ ID No:1所示。
进一步的,所述铰链区为氨基酸序列如SEQ ID No:5所示的CD8铰链结构域;
和/或,所述跨膜结构域为氨基酸序列如SEQ ID No:6所示的CD28跨膜结构域;
和/或,所述CD28共刺激结构域的氨基酸序列如SEQ ID No:7所示;
和/或,所述4-1BB共刺激结构域的氨基酸序列如SEQ ID No:8所示;
和/或,所述CD3ζ的ITAM1的氨基酸序列如SEQ ID No:9所示。
另,本发明还提供一种分离的核酸,所述分离的核酸包括用于表达上述的靶向BAFFR嵌合抗原受体的核苷酸序列。
进一步的,所述分离的核酸包括核苷酸序列如SEQ ID No:10所示的核酸片段。
另,本发明还提供一种重组载体,所述重组载体包括上述的分离的核酸。
另,本发明还提供一种CAR-T细胞,所述CAR-T细胞含有上述的分离的核酸,或所述CAR-T细胞为被上述的重组载体所转化的细胞。
另,本发明还提供一种靶向BAFFR嵌合抗原受体在制备药物中的应用,包括如上述的靶向BAFFR嵌合抗原受体、如上述的分离的核酸、如上述的重组载体或如上述的CAR-T细胞在制备用于治疗免疫相关疾病的药物中的应用;所述免疫相关疾病为自身免疫病或肿瘤;
所述自身免疫病选自多发性硬化、视神经脊髓炎、重症肌无力、风湿性关节炎、系统性红斑狼疮、干燥综合征;
所述肿瘤为B细胞淋巴瘤。
另,本发明还提供一种药物组合物,包括用于表达上述的靶向BAFFR嵌合抗原受体的表达载体或上述的CAR-T细胞。
相对于现有技术而言,本发明的有益效果是:
(1)目前应用CD19或CD20抗体的B细胞删除是自身免疫病的主要治疗方式,但是治疗效果并不理想。一方面,CD19或CD20抗体会针对全部幼稚和成熟B细胞,容易造成免疫缺陷甚至病人的感染。另一方面,CD19或CD20抗体可能降低患者对疫苗,例如COVID-19疫苗的反应性。本发明通过BAFFR构建了CAR-T细胞,可以靶向自身反应性B细胞特别是浆细胞,而不是删除所有B细胞,保留了患者的基本自身免疫能力以及疫苗反应能力。
(2)自身免疫病患者中存在自身抗体,如系统性红斑狼疮患者中的dsDNA抗体,或者多发病硬化患者中的MOG抗体,视神经脊髓炎患者中的AQP4抗体等自身抗体,而CD20单抗并不能很好地降低患者体内这些致病性自身抗体的浓度(30-70%)。本发明通过BAFFR构建了CAR-T细胞,主要靶向产生自身抗体的浆细胞,可以有效降低患者体内自身抗体的浓度。
(3)本申请通过噬菌体展示技术结合高通量表达的平台,筛选出了靶向BAFFR的纳米抗体,具有亲和力高、稳定性高、分子量小、免疫原性低、渗透力强等优势。
应当理解,发明内容部分中所描述的内容并非旨在限定本发明的实施例的关键或重要特征,亦非用于限制本发明的范围。本发明的其它特征将通过以下的描述变得容易理解。
附图说明
通过阅读参照以下附图所作的对非限制性实施例所作的详细描述,本发明的其它特征、目的和优点将会变得更明显。
图1为ELISA测定抗BAFFR抗体与BAFFR的结合活性示意图。
图2为CAR-T细胞的结构示意图;其中:BAFFR-CAR表示靶向BAFFR嵌合抗原受体多肽,从N端到C端包含CD8a信号肽区域、抗BAFFR scFv、CD8铰链区、CD28跨膜区、CD28共刺激结构域、4-1BB共刺激结构域和CD3ζ信号转导结构域。
图3为流式细胞仪检测CAR质粒转染T细胞的比例示意图。
图4为实施例中的BAFFR-CAR-T和对照BAFFR-CAR-T在不同效靶比时对Nalm-6-Luc细胞的杀伤效果对比图。
图5为实施例中BAFFR-CAR-T和对照BAFFR-CAR-T在不同效靶比时IL-2和IFN-γ的分泌水平对比图。
具体实施方式
下面结合附图和实施例对本发明作进一步的详细说明。可以理解的是,此处所描述的具体实施例仅仅用于解释相关发明,而非对该发明的限定。另外还需要说明的是,为了便于描述,附图中仅示出了与发明相关的部分。
需要说明的是,在不冲突的情况下,本发明中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本发明。
本发明的实施例提供了一种靶向BAFFR嵌合抗原受体,包括从氨基端到羧基端依次连接的信号肽区域、靶向BAFFR的抗原结合域、铰链区、跨膜结构域、共刺激结构域和信号转导结构域,共刺激结构域包括相连的CD28共刺激结构域和4-1BB共刺激结构域,信号转导结构域为CD3ζ的ITAM1。
在一些实施例中,铰链区为至少选自以下蛋白的一种铰链区:CD28、CD3ζ、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD134、CD137、ICOS和CD154;
在一些实施例中,跨膜结构域为至少选自以下蛋白的一种跨膜区:T细胞受体的α、β或ζ链、CD28、CD3ζ、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、KIRDS2、OX40、CD2、CD27、ICOS、GITR、CD40、BAFFR、HVEM、SLAMF7、NKp80、CD160、CD19、IL2Rβ、IL2Rγ、IL7Rα、ITGA1、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、ITGB7、TNFR2、DNAM1、SLAMF4、CD84、CD96、CEACAM1、CRTAM、Ly9、PSGL1、CD100、SLAMF6、SLAM、BLAME、SELPLG、LTBR、PAG/Cbp、NKp44、NKp30、NKp46、NKG2D和NKG2C。
在一优选实施例中,信号肽区域的氨基酸序列如SEQ ID No:1所示。
SEQ ID No:1(信号肽区域的氨基酸序列)为:
MALPVTALLLPLALLLHAARP。
在一优选实施例中,靶向BAFFR的抗原结合域为靶向BAFFR的纳米抗体,靶向BAFFR的纳米抗体的氨基酸序列如SEQ ID No:2所示,或如SEQ ID No:3所示,或如SEQ ID No:4所示。
其中,SEQ ID No:2(NB467-7的氨基酸序列)为:
QVQLVESGGGSVHPGGSLRLSCAGSGFTLAGYAIGWFRQAPGKEREGVSCINSSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADVYYSGSYLYRSCNPTESKYWGQGTQVTVSS;
SEQ ID No:3(NB467-71的氨基酸序列)为:
EVQVVESGGGLVQSGGSLRLSCVASGFNLDHYAIGWFRQIPGKEREGVSCISSGGDSTFYIDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYHCAADVYYSGNYLYRSCNPHESKYWGQGTQVTVSS;
SEQ ID No:4(NB467-88的氨基酸序列)为:
EVQVVESGGGLVQPGGSLRLSCAASGFRLNYYAIGWFRQAPGKEREGVSCISSGGDRIYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLYYSGSYYYRSCNPAESGYWGQGTQVTVSS。
在一优选实施例中,铰链区为氨基酸序列如SEQ ID No:5所示的CD8铰链结构域;
和/或,跨膜结构域为氨基酸序列如SEQ ID No:6所示的CD28跨膜结构域;
和/或,CD28共刺激结构域的氨基酸序列如SEQ ID No:7所示;
和/或,4-1BB共刺激结构域的氨基酸序列如SEQ ID No:8所示;
和/或,CD3ζ的ITAM1的氨基酸序列如SEQ ID No:9所示。
其中,SEQ ID No:5(CD8 铰链结构域)为:
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD;
SEQ ID No:6(CD28跨膜结构域)为:
FWVLVVVGGVLACYSLLVTVAFIIFWV;
SEQ ID No:7(CD28共刺激结构域的氨基酸序列)为:
RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS;
SEQ ID No:8(4-1BB共刺激结构域的氨基酸序列)为:
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL;
SEQ ID No:9(CD3ζ的ITAM1的氨基酸序列)为:
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。
另,本发明的实施例还提供了一种分离的核酸,该分离的核酸包括用于表达上述的靶向BAFFR嵌合抗原受体的核苷酸序列。
在一优选实施例中,分离的核酸包括核苷酸序列如SEQ ID No:10所示的核酸片段。
其中,SEQ ID No:10为:
ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCC
GAGGTGCAGGTTGTGGAATCTGGCGGAGGACTGGTTCAGTCTGGCGGCTCTCTGAGACTGAGCTGTGTGGCCAGCGGCTTCAACCTGGATCACTATGCCATCGGCTGGTTCAGACAGATCCCCGGCAAAGAGAGAGAGGGCGTCAGCTGTATCAGCAGCGGCGGAGATAGCACCTTCTACATCGACAGCGTGAAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAAGCCTGAGGACACCGCCGTGTATCATTGTGCCGCCGACGTGTACTACAGCGGCAACTACCTGTACAGAAGCTGCAACCCTCACGAGAGCAAGTACTGGGGCCAGGGCACACAAGTGACCGTGTCATCT
ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAAGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT
TTCTGGGTGCTGGTCGTTGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTGGTGACAGTGGCCTTCATCATCTTTTGGGTG
AGGAGCAAGCGGAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCCCGGAGGCCTGGCCCCACCCGGAAGCACTACCAGCCCTACGCCCCTCCCAGGGATTTCGCCGCCTACCGGAGC
AAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTG
CGCGTGAAGTTCAGCAGATCAGCCGATGCTCCTGCCTACCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAGGGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTACCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA。
另,本发明的实施例还还提供了一种重组载体,该重组载体包括上述的分离的核酸。
另,本发明的实施例还提供了一种CAR-T细胞,该CAR-T细胞含有上述的分离的核酸,或该CAR-T细胞为被上述的重组载体所转化的细胞。
另,本发明的实施例还提供了一种靶向BAFFR嵌合抗原受体在制备药物中的应用,包括如上述的靶向BAFFR嵌合抗原受体、如上述的分离的核酸、如上述的重组载体或如上述的CAR-T细胞在制备用于治疗免疫相关疾病的药物中的应用。
免疫相关疾病为自身免疫病或肿瘤。
其中,自身免疫病选自神经系统免疫性疾病如多发性硬化、视神经脊髓炎、重症肌无力等,或其它自身免疫性疾病如风湿性关节炎、系统性红斑狼疮、干燥综合征等疾病的患者等。
肿瘤为B细胞淋巴瘤,如以霍奇金淋巴瘤和结节性淋巴细胞为主型的霍奇金淋巴瘤,以及非霍奇金淋巴瘤,如弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、黏膜相关淋巴组织淋巴瘤(MALT)、小淋巴细胞淋巴瘤/慢性淋巴细胞白血病、套细胞淋巴瘤(MCL)。
另,本发明的实施例还提供了一种药物组合物,包括用于表达上述的靶向BAFFR嵌合抗原受体的表达载体或上述的CAR-T细胞。
实施例1:通过噬菌体展示获得抗BAFFR的纳米抗体
在本实施例中,通过用人BAFFR作为抗原对羊驼进行免疫获得了多种抗体编码片段,随后通过噬菌体文库和哺乳动物表达系统筛选出了候选抗体克隆。具体而言,抗BAFFR纳米抗体的筛选过程的具体步骤如下所示。
a.抗原的制备
依据人BAFFR的氨基酸序列和核苷酸序列,分析并设计可有效诱导羊驼产生针对人BAFFR的特异性抗体的抗原,并在其C端连接Human IgG1 Fc,获得经修饰的抗原,记作“人BAFFR hFC抗原”,其氨基酸序列如SEQ ID No:11所示,核苷酸序列如SEQ ID No:12所示。
其中,SEQ ID No:11(人BAFFR hFC抗原的氨基酸序列)为:
MGWSCIILFLVATATGVHSSLRGRDAPAPTPCVPAECFDLLVRHCVACGLLRTPRPKPAGASSPAPRTALQPQESVGAGAGEAALPLPGLEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;
SEQ ID No:12(人BAFFR hFC抗原的核苷酸序列)为:
ATGGGCTGGTCCTGCATCATCCTGTTTCTGGTCGCTACCGCCACAGGCGTCCACTCCTCCCTGAGGGGCAGAGACGCCCCCGCTCCAACACCTTGCGTGCCTGCCGAGTGCTTCGATCTGCTGGTGAGGCACTGCGTGGCCTGCGGCCTGCTGAGGACCCCTAGGCCTAAGCCCGCCGGCGCTAGCAGCCCAGCTCCTAGAACAGCCCTGCAGCCCCAGGAGAGCGTGGGCGCTGGAGCTGGAGAGGCCGCTCTGCCCCTGCCTGGCCTGGAACCAAAATCTTGTGACAAAACCCACACATGCCCACCTTGTCCCGCCCCTGAACTGCTGGGCGGACCTTCTGTCTTTCTGTTCCCCCCCAAACCCAAGGATACACTGATGATCTCTAGAACCCCCGAGGTCACATGTGTCGTCGTGGATGTGTCCCATGAGGACCCTGAAGTGAAATTCAACTGGTACGTGGACGGAGTGGAAGTCCATAACGCCAAAACCAAACCACGAGAGGAACAGTACAATAGCACATATCGAGTCGTGTCTGTGCTGACTGTGCTGCATCAGGATTGGCTGAACGGCAAGGAATACAAATGTAAAGTGTCCAATAAGGCACTGCCCGCTCCTATTGAAAAAACAATTTCAAAGGCAAAAGGCCAGCCTCGTGAACCTCAGGTGTACACACTGCCACCTTCTCGGGAGGAAATGACCAAAAACCAGGTGTCACTGACCTGTCTGGTCAAGGGCTTTTACCCTTCCGATATTGCTGTCGAGTGGGAGAGTAACGGCCAGCCCGAAAACAACTACAAAACCACCCCTCCTGTGCTGGATTCCGATGGCTCATTCTTCCTGTACTCTAAACTGACCGTGGATAAGAGTCGCTGGCAGCAGGGCAATGTGTTTTCTTGCTCCGTGATGCATGAGGCACTGCACAACCACTACACCCAGAAATCCCTGTCACTGTCTCCCGGAAAATAA。
b.免疫羊驼
将步骤a获得的人BAFFR hFC抗原与等体积的弗氏佐剂完全混合均匀,并用于对羊驼进行皮下注射。
具体地,在第1天,使用500μg的人BAFFR hFC抗原与等体积弗氏完全佐剂的乳化混合物对羊驼进行初免,并分别在第21天、42天、63天、84天用250μg的人 BAFFR hFC抗原与等体积弗氏完全佐剂的乳化混合物进行4次加强免疫。在前4次免疫的7天后,分别采10ml羊驼外周血以通过ELISA检测血中的抗BAFFR血清效价。
ELISA检测的具体步骤如下:将人 BAFFR his抗原用0.05M碳酸盐缓冲液(pH=9.6)稀释至2μg/mL,并按100μL/孔的量添加至培养板中。将培养板在4℃包被过夜。弃包被液,并用PBST洗涤3次。每孔加入5%脱脂牛奶300µL,于37℃封闭1h。用PBST缓冲液洗涤3次。加入100μL/孔的血清稀释液(从1:2000开始倍比稀释),并于37℃孵育45min。用PBST洗涤5次,向每孔加入100μL山羊抗羊驼IgG(Goat anti-Alpaca IgG)(H+L)HRP(成都阿帕克生物科技有限公司,Cat#:S001H,成都阿帕克生物科技有限公司,用PBS按1:1W稀释),37℃孵育45min。用PBST洗板5次。加入TMB显色液显色(100μL/孔),并在37℃孵育5min。加入终止液终止反应(50μL/孔),并于450nm下测光密度。
在第5次免疫的1周后,采羊驼外周血50mL并分离单个核细胞。
c.文库构建
提取步骤b获得的PBMC的RNA,并在反转录之后通过巢式PCR获得目的基因片段。将目的基因片段克隆至真核表达载体中,并将获得的表达载体转化进感受态细胞以构建BAFFR-VHH噬菌体展示文库。具体步骤如下所示。
以步骤b筛选得到的PBMC细胞为模板,利用RNAiso Plus提取细胞总RNA,并利用PrimeScript™ II 1st Strand cDNA Synthesis Kit试剂盒将RNA反转录为cDNA。通过巢式PCR获得目的片段。目的片段的扩增体系如下表1所示,扩增程序如下表2所示。
表1巢式PCR一轮反应体系
表2巢式PCR二轮反应体系
将扩增获得的编码羊驼VHH的核苷酸片段克隆至真核表达载体pComb3XSS(成都阿帕克生物科技有限公司)中。将生成的重组载体通过电击转化至TG1的感受态细胞中,得到VHH噬菌体展示文库。为了进一步鉴定BAFFR-VHH噬菌体展示文库是否构建成功,将文库培养在2-YT-A平板上,并在形成的菌落中挑选48个克隆进行测序。
测序结果显示,克隆的成功插入率为100%。根据文库转化子数、库插入率和多样性测序分析结果,计算出的文库库容为1.92×109,说明得到的BAFFR-VHH噬菌体展示文库的文库库容和多样性良好。
d.纳米抗体的获得
使用步骤c得到的噬菌体展示文库进行淘选并选出阳性克隆。将选出的VHH抗体的编码序列与Human IgG1 Fc编码序列融合,并构建到pTT5 (成都阿帕克生物科技有限公司)表达载体中以转染真核细胞,从而进行表达和纯化。最终,获得了抗人BAFFR纳米抗体。具体方法如下。
取出SA-磁珠(苏州纳薇科技有限公司,MPHTSA-300),PBS清洗2次备用;将Bio-Human BAFFR-his(Acro,BAR-H82E3)抗原用PBS稀释至终浓度为5μg/ml,加入2×1011噬菌体文库,37℃孵育1h;将孵育后的混合物加入磁珠管中,4℃振摇作用45min;在磁力架作用下吸出上清,用PBST洗涤3次,PBS洗涤2次;加入800µL Gly-HCl 洗脱液,37℃孵育8min,洗脱特异性结合的噬菌体;将该洗脱液转移至1.5mL无菌离心管中,迅速用160μL Tris-HCl中和缓冲液中和;取10µL进行梯度稀释,测定滴度,计算淘选回收率,其余洗脱物混合后进行扩增和纯化,用于下一轮亲和淘选。
经过两轮筛选,在第二轮筛选后进行单克隆噬菌体上清ELISA鉴定。将Bio-HumanBAFFR-his抗原按照2μg/ml*100μl规格固定在96孔酶标板中,于4℃孵育过夜。弃包被液,并用PBST 洗涤3次。每孔加入5%脱脂牛奶300µL,37℃封闭1h。用PBST缓冲液洗涤3次,以100μL/孔加入二倍稀释的噬菌体上清液,并在37℃孵育45min。用PBST洗涤5次,以100μL/孔向各孔中分别加入小鼠抗M13抗体HRP (mouse anti-M13 antibody HRP,成都阿帕克生物科技有限公司),并在37℃孵育45min。用PBST洗板5次。以100μL/孔加入TMB显色液显色,37℃孵育5min。
以50μL/孔加入1M HCl终止液终止反应,并于450nm波长下检测光密度。通过检测结果筛选阳性克隆(OD450>1)。将与阳性克隆融合Human IgG1 Fc构建到pTT5质粒中,对应的质粒转染至哺乳动物细胞HEK293T中,于37℃、5%二氧化碳的摇床中表达7天。培养后收集细胞上清液,采用Protein A亲和填料(苏州纳薇科技有限公司,17010-050100)分离纯化得到了3株目的抗体,分别命名为NB467-7、NB467-71、NB467-88。
纳米抗体NB467-7、NB467-71、NB467-88的VHH氨基酸序列和核苷酸序列如下所示:
SEQ ID No:2(NB467-7的氨基酸序列)为:
QVQLVESGGGSVHPGGSLRLSCAGSGFTLAGYAIGWFRQAPGKEREGVSCINSSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADVYYSGSYLYRSCNPTESKYWGQGTQVTVSS;
SEQ ID No:3(NB467-71的氨基酸序列)为:
EVQVVESGGGLVQSGGSLRLSCVASGFNLDHYAIGWFRQIPGKEREGVSCISSGGDSTFYIDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYHCAADVYYSGNYLYRSCNPHESKYWGQGTQVTVSS;
SEQ ID No:4(NB467-88的氨基酸序列)为:
EVQVVESGGGLVQPGGSLRLSCAASGFRLNYYAIGWFRQAPGKEREGVSCISSGGDRIYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLYYSGSYYYRSCNPAESGYWGQGTQVTVSS;
SEQ ID No:18(NB467-7的核苷酸序列)为:
CAGGTGCAGCTGGTTGAATCTGGCGGAGGATCTGTGCACCCTGGCGGATCTCTGAGACTGTCTTGTGCCGGCAGCGGCTTTACACTGGCCGGATATGCCATCGGCTGGTTCAGACAGGCCCCTGGCAAAGAGAGAGAGGGCGTCAGCTGCATCAATAGCTCTGGCGGCAGCACCTACTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAAGCCTGAGGACACCGCCGTGTACTATTGTGCCGCCGACGTGTACTACAGCGGCAGCTACCTGTACAGAAGCTGCAACCCCACCGAGAGCAAGTATTGGGGCCAGGGCACACAAGTGACCGTGTCTAGT;
SEQ ID No:19(NB467-71的核苷酸序列)为:
GAGGTGCAGGTTGTGGAATCTGGCGGAGGACTGGTTCAGTCTGGCGGCTCTCTGAGACTGAGCTGTGTGGCCAGCGGCTTCAACCTGGATCACTATGCCATCGGCTGGTTCAGACAGATCCCCGGCAAAGAGAGAGAGGGCGTCAGCTGTATCAGCAGCGGCGGAGATAGCACCTTCTACATCGACAGCGTGAAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAAGCCTGAGGACACCGCCGTGTATCATTGTGCCGCCGACGTGTACTACAGCGGCAACTACCTGTACAGAAGCTGCAACCCTCACGAGAGCAAGTACTGGGGCCAGGGCACACAAGTGACCGTGTCATCT;
SEQ ID No:20(NB467-88的核苷酸序列)为:
GAGGTGCAGGTTGTGGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCCGGCTGAATTACTATGCCATCGGCTGGTTCAGACAGGCCCCTGGCAAAGAGAGAGAGGGCGTCAGCTGTATCAGCTCTGGCGGCGACAGAATCTACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAAGCCTGAGGACACCGCCGTGTATTACTGTGCCGCCGACCTGTACTACAGCGGCAGCTACTACTACAGAAGCTGCAACCCTGCCGAGAGCGGCTATTGGGGACAGGGAACACAAGTGACCGTGTCCTCT。
实施例2:抗BAFFR纳米抗体与过表达BAFFR的293T细胞的结合
将实施例1中获得的3种抗BAFFR纳米抗体NB467-7、NB467-71和NB467-88通过流式细胞仪进行结合亲和力动力学的表征性测定。
具体地,从细胞培养瓶中收集HEK-293T-BAFFR细胞,并用FACS缓冲液洗两次并离心。将细胞重悬于合适体积的流式细胞分析染色液,使每管细胞终浓度达到2×105细胞/mL。
将实施例1中生成的抗体NB467-7、NB467-71和NB467-88,以及对照抗体分别稀释至200nM。将50μl抗体溶液加入带有HEK-293T-BAFFR细胞的试管中。混合均匀,在4℃孵育60分钟。孵育完成,利用FACS缓冲液洗涤细胞一次。
根据说明书稀释抗人FC-647二抗(Jackson,109-605-003),并将100μl二抗稀释液加入到样品管中。混合均匀之后,在4℃孵育60分钟。孵育完成之后,用FACS缓冲液洗涤细胞3次,并将离心获得的细胞沉淀重悬于200μl PBS中,用流式细胞仪(Sony,SA3800)检测。检测结果见表3和图1。
表3.纳米抗体与过表达BAFF的293T细胞的结合(EC50)
图1中显示了各抗体的平均荧光强度(MFI)值,证实与对照抗体相比,本发明的3株纳米抗体与过表达人BAFFR的293T细胞的结合能力更强。表3显示,本发明的纳米抗体EC50值。
实施例3:BAFFR-CAR-T细胞的构建
一、CAR慢病毒表达载体构建
将获得的BAFFR抗体序列信息密码子优化后,与相关的铰链区、跨膜区及胞内信号区基因片段合成构建嵌合抗原受体慢病毒表达载体。并选定构建载体为PCDH慢病毒载体(pCDH-EF1a),如图2所示。经sanger测序确证插入序列无误后,进行慢病毒包装。
二、CAR慢病毒包装
1) 准备15cm细胞培养皿,接种1×107个293T细胞,加入完全培养基(DMEM高糖、10%FBS),置于37℃、5%CO2培养箱,过夜培养。
2) 从冰箱中取出LVTransm及慢病毒表达质粒(CAR质粒)、慢病毒包装质粒mix,室温解冻后,用移液枪上下吹打完全混匀。取出PBS缓冲液,温热至室温。取2mL PBS至6孔板的一个孔,分别加入20μg慢病毒表达质粒、30μL慢病毒包装质粒Mix,移液枪上下吹打充分混匀后,加入150μL LVTransm,立即用移液器上下吹打混匀,室温下静置10分钟。
3) 将上述DNA/LVTransm复合物逐滴加入到15cm培养皿中,轻轻晃动培养皿,充分混匀。将培养皿置于37℃、5%CO2培养箱,培养6~8小时后,将含有转染试剂的培养基去掉,更换为新鲜的完全培养基。
4) 连续培养48小时后,收集培养皿中含有病毒的培养基上清,用0.45μm的滤膜过滤,转至离心管中,配平后,40000×g、4℃离心1.5小时。离心结束后,在生物安全柜中,小心将离心管中的液体吸去,加入1mL PBS缓冲液将沉淀重悬,将病毒置于-80℃保存。
三、外周血T细胞分离
1) 将抗凝血样转移至50mL无菌离心管内,旋紧盖子,800×g离心20分钟。
2) 离心结束后,从离心机中取出离心管,避免剧烈晃动或颠倒离心管,去掉上层浅黄色血清层,然后向下层的外周血细胞层加入等体积的PBS,轻轻上下颠倒混匀。
3) 取出淋巴细胞分离液,并上下颠倒数次,充分混匀。向50mL离心管中加入20mL淋巴细胞分离液,然后使用移液器小心将第2步骤中稀释后的血样按照等体积沿管壁缓慢加至淋巴细胞分离试剂的上层,避免分离试剂与血样的混合,800×g离心20分钟。
4) 离心结束后,轻轻的取出离心管,将处于中间的白色单核细胞层吸至一个新的无菌离心管中,加入等体积的生理盐水轻轻混匀,800×g,离心5分钟,离心结束后,去除上清,重复清洗一次PBMC,调整细胞密度至5×107个细胞/mL,转移至2ml细胞冻存管,按照1ml/管进行冻存。
四、CAR-T细胞制备
1) 使用PBS洗涤CD3/CD28 Dynabeads 2遍。
2) 取适量的Dynabeads加入到PBMC中,轻轻混匀,室温孵育20min。
3) 将2mL细胞冻存管插入磁极,室温静置1min,保持冻存管插在磁极的孔内,轻轻倒置,将管内的液体倒出。
4) 将细胞冻存管从磁极中取出,加入适量X-Vivo 15培养基(含200IU/mL IL2,10ng/mL IL7,5ng/mL IL15),用移液器重悬细胞和beads混合物,并调整细胞密度至0.5~1×106个细胞/mL,转移至6孔板中。
5) 将细胞置于37℃、5%CO2培养箱中连续48h后,将细胞密度调整至1×106个/mL。
6) 从-80℃超低温冰箱中,取出慢病毒快速解冻。
7) 向准备好的T细胞(1×106细胞/ml),共2ml,加入polybrene至终浓度为6μg/mL,加入300μL慢病毒,用移液器轻轻吹打充分混匀,使用封口膜将培养器皿密封,800×g室温离心1小时。
8) 离心结束后,继续培养24小时,对T细胞进行换液。
9) 继续培养24小时后,通过孵育EGFR抗体流式检测CAR-T细胞阳性率。
10) 剩余CAR-T细胞继续培养,每天轻轻吹打体系中的beads/细胞团至完全分开,当细胞密度大于1×106个/mL时,添加X-Vivo 15培养基(含200IU/mL IL-2,10ng/mL IL-7,5ng/mL IL-15)调整细胞密度至0.5-0.7×106个/mL。
实施例4:CAR-T细胞的检测
一、CAR阳性率的检测
1) 500×g离心5分钟,收集细胞沉淀(准备2管细胞,5×105个细胞/管)。
2) 用含0.5% BSA的PBS缓冲液洗涤细胞沉淀3次,每次500×g离心5分钟。
3) 使用100μL稀释后的EGFR抗体(1ug/管)重悬细胞沉淀,室温孵育60分钟。500×g离心5分钟,去掉上清(一管细胞留作对照,此步仅加入含0.5% BSA的PBS)。
4) 用含0.5% BSA的PBS缓冲液洗涤细胞沉淀3次,每次500×g离心5分钟。
5) 使用100μL稀释后的PE anti human IgG(1:500稀释)重悬细胞沉淀(所有细胞都孵育二抗),避光孵育45分钟。500×g离心5分钟,收集细胞沉淀。
6) 用含0.5% BSA的PBS缓冲液洗涤细胞沉淀3次,每次500×g离心5分钟。
7) 最后用400μL PBS重悬细胞沉淀,进行流式分析。具体结果见图3。
图3结果显示,使用EGFR抗体进行检测,CAR阳性率均大于30%,可以进行CAR-T的体外杀伤检测,其中NB467-7 CAR-T阳性率为53.22%,NB467-77 CAR-T阳性率为69.65%,NB467-88 CAR-T阳性率为51.93%。
二、CAR-T细胞对靶细胞的裂解
1)将靶细胞Nalm-6-Luc重悬于完全培养基中(RPMI1640+10%FBS),调整细胞密度至2×105个/mL,取一块新的96孔板,按照100μL/孔的量接种靶细胞。96孔板四周未用的孔,每孔加入100μL无菌水,以防中间的实验孔水分蒸发。将孔板置于5%CO2、37℃培养箱中,过夜培养。
2)离心收集制备的CAR-T细胞,使用10%FBS的1640培养基重悬;从培养箱中取出96孔板,将孔中的培养基完全吸出,用无菌PBS轻轻将细胞洗涤一遍,然后按照不同E/T比例(1:1,2.5:1,5:1,10:1)加入CAR-T细胞,并将最终体积补至200μL/孔;Maxi lysis为接种同样数量的靶细胞,但是不加CAR-T细胞。将孔板置于5% CO2、37℃培养箱中,培养18小时。
3)培养结束后,将孔板从培养箱中取出,离心收集上清保存在-80℃冰箱用于Elisa检测IL2和IFN-γ的表达。向细胞中加入Bright-GloTM通过检测Luciferase活性反映重组CAR-T细胞对靶细胞的裂解能力。
4)靶细胞裂解百分数计算公式:
;
5)具体结果见图4。
图4结果显示,取靶细胞Nalm-6-Luc按照效靶比E/T=1:1,2.5:1,5:1,10:1和CAR-T细胞共培养,和空白T细胞相比,NB467-7 CAR-T,NB467-71 CAR-T和NB467-88 CAR-T细胞对Nalm-6-Luc有强的杀伤活性。
三、CAR-T细胞因子分泌水平检测
取出共培养的上清,分别取100ul原液用于IL2和IFN-γ的检测。按照IL2和IFN-γ的ELISA检测试剂盒操作说明,检测IL2和IFN-γ的分泌情况。具体结果见图5。
图5结果显示,与空白T相比,NB467-7 CAR-T,NB467-71 CAR-T和NB467-88 CAR-T细胞受Nalm-6-Luc细胞刺激后,IL-2和IFN-γ分泌显著增强。
本发明提供了一种针对BAFFR的CAR-T细胞,主要靶向自身反应性B细胞,为自身免疫性疾病和某些血液肿瘤的治疗提供了新的选择。
在本说明书的描述中,术语“一个实施例”、“一些实施例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或特点包含于本申请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一定指的是相同的实施例或实例。而且,描述的具体特征、结构、材料或特点可以在任何的一个或多个实施例或示例中以合适的方式结合。
以上仅为本申请的优选实施例而已,并不用于限制本申请,对于本领域的技术人员来说,本申请可以有各种更改和变化。凡在本申请的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本申请的保护范围之内。
Claims (9)
1.一种靶向BAFFR嵌合抗原受体,其特征在于,包括从氨基端到羧基端依次连接的信号肽区域、靶向BAFFR的抗原结合域、铰链区、跨膜结构域、共刺激结构域和信号转导结构域,所述共刺激结构域包括相连的CD28共刺激结构域和4-1BB共刺激结构域,所述信号转导结构域为CD3ζ的ITAM1;
所述靶向BAFFR的抗原结合域为靶向BAFFR的纳米抗体,所述靶向BAFFR的纳米抗体的氨基酸序列如SEQ ID No:2所示,或如SEQ ID No:3所示,或如SEQ ID No:4所示。
2.根据权利要求1所述的靶向BAFFR嵌合抗原受体,其特征在于,所述信号肽区域的氨基酸序列如SEQ ID No:1所示。
3.根据权利要求1所述的靶向BAFFR嵌合抗原受体,其特征在于,所述铰链区为氨基酸序列如SEQ ID No:5所示的CD8铰链结构域;
和/或,所述跨膜结构域为氨基酸序列如SEQ ID No:6所示的CD28跨膜结构域;
和/或,所述CD28共刺激结构域的氨基酸序列如SEQ ID No:7所示;
和/或,所述4-1BB共刺激结构域的氨基酸序列如SEQ ID No:8所示;
和/或,所述CD3ζ的ITAM1的氨基酸序列如SEQ ID No:9所示。
4.一种分离的核酸,其特征在于,所述分离的核酸包括用于表达如权利要求1-3任一项所述的靶向BAFFR嵌合抗原受体的核苷酸序列。
5.根据权利要求4所述的分离的核酸,其特征在于,所述分离的核酸包括核苷酸序列如SEQ ID No:10所示的核酸片段。
6.一种重组载体,其特征在于,所述重组载体包括如权利要求4或5任一项所述的分离的核酸。
7.一种CAR-T细胞,其特征在于,所述CAR-T细胞含有如权利要求4或5所述的分离的核酸,或所述CAR-T细胞为被如权利要求6所述的重组载体所转化的细胞。
8.一种靶向BAFFR嵌合抗原受体在制备药物中的应用,其特征在于,为如权利要求1-3中任一项所述的靶向BAFFR嵌合抗原受体、如权利要求4或5所述的分离的核酸、如权利要求6所述的重组载体或如权利要求7所述的CAR-T细胞在制备用于治疗免疫相关疾病的药物中的应用;所述免疫相关疾病为自身免疫病或肿瘤;
所述自身免疫病选自多发性硬化、视神经脊髓炎、重症肌无力、风湿性关节炎、系统性红斑狼疮、干燥综合征;
所述肿瘤为B细胞淋巴瘤。
9.一种药物组合物,其特征在于,包括用于表达如权利要求1-3任一项所述的靶向BAFFR嵌合抗原受体的表达载体或如权利要求7所述的CAR-T细胞。
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