Rapid and Effective Generation of Nanobody Based CARs using PCR and Gibson Assembly
Rapid and Effective Generation of Nanobody Based CARs using PCR and Gibson Assembly
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DOI：
10.3390/ijms21030883
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发表时间：
2020-02-01
影响因子：
5.6
通讯作者：
Vandekerckhove, Bart
中科院分区：
文献类型：
--
作者：
De Munter, Stijn;Van Parys, Alexander;Vandekerckhove, Bart
欧洲药品管理局(EMA)和美国联邦药品监督管理局(FDA)最近批准了嵌合抗原受体(CAR)T细胞疗法，CAR T临床试验的显著结果表明了这种疗法的治疗潜力。虽然针对多种不同抗原的CARS正在开发和(临床前)测试，但仍有优化的需要。使用单链可变片段(ScFv)作为靶向部分阻碍了功能汽车的快速生成，并可能潜在地限制其疗效。相反，纳米机构可能会在很大程度上绕过这些困难。我们使用了一个可用的纳米体库，该文库是在骆驼通过DNA疫苗免疫分化簇(CD)20或使用可溶性蛋白免疫CD33的胞外区后产生的。用聚合酶链式反应扩增纳米体特异性序列，Gibson Assembly将其克隆到含有两种不同第二代CAR载体的逆转录病毒载体中。在T细胞中转导后，我们观察到所有病例都有高表达的细胞膜纳米CAR。在用抗原阳性细胞系刺激表达纳米CAR的T细胞后，在体外和体内都观察到了强大的T细胞激活、细胞因子产生和肿瘤细胞溶解。将纳米车身技术与聚合酶链式反应和吉布森组装技术相结合，可以快速而有效地产生紧凑型汽车。
Recent approval of chimeric antigen receptor (CAR) T cell therapy by the European Medicines Agency (EMA)/Federal and Drug Administration (FDA) and the remarkable results of CAR T clinical trials illustrate the curative potential of this therapy. While CARs against a multitude of different antigens are being developed and tested (pre)clinically, there is still a need for optimization. The use of single -chain variable fragments (scFvs) as targeting moieties hampers the quick generation of functional CARs and could potentially limit the efficacy. Instead, nanobodies may largely circumvent these difficulties. We used an available nanobody library generated after immunization of llamas against Cluster of Differentiation (CD) 20 through DNA vaccination or against the ectodomain of CD33 using soluble protein. The nanobody specific sequences were amplified by PCR and cloned by Gibson Assembly into a retroviral vector containing two different second -generation CAR constructs. After transduction in T cells, we observed high cell membrane nanoCAR expression in all cases. Following stimulation of nanoCAR-expressing T cells with antigen-positive cell lines, robust T cell activation, cytokine production and tumor cell lysis both in vitro and in vivo was observed. The use of nanobody technology in combination with PCR and Gibson Assembly allows for the rapid and effective generation of compact CARs.