Riet Tobias, Holzinger Astrid, Dörrie Jan, Schaft Niels, Schuler Gerold, Abken Hinrich
Department I of Internal Medicine, Tumor Genetics, and Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany.
Methods Mol Biol. 2013;969:187-201. doi: 10.1007/978-1-62703-260-5_12.
Redirecting T cells with a chimeric antigen receptor (CAR) of predefined specificity showed remarkable efficacy in the adoptive therapy trials of malignant diseases. The CAR consists of a single chain fragment of variable region (scFv) antibody targeting domain covalently linked to the CD3ζ signalling domain of the T cell receptor complex to mediate T cell activation upon antigen engagement. By using an antibody-derived targeting domain a CAR can potentially redirect T cells towards any target expressed on the cell surface as long as a binding domain is available. Antibody-mediated targeting moreover circumvents MHC restriction of the targeted antigen, thereby broadening the potential of applicability of adoptive T cell therapy. While T cells were so far genetically modified by viral transduction, transient modification with a CAR by RNA transfection gained increasing interest during the last years. This chapter focuses on methods to modify human T cells from peripheral blood with a CAR by electroporation of in vitro transcribed RNA and to test modified T cells for function for use in adoptive immunotherapy.
利用具有预定义特异性的嵌合抗原受体（CAR）重定向T细胞在恶性疾病的过继性治疗试验中显示出显著疗效。CAR由靶向结构域的可变区单链片段（scFv）抗体与T细胞受体复合物的CD3ζ信号结构域共价连接组成，以在抗原结合时介导T细胞活化。通过使用抗体衍生的靶向结构域，只要有结合结构域，CAR就有可能将T细胞重定向至细胞表面表达的任何靶标。此外，抗体介导的靶向作用规避了靶向抗原的MHC限制，从而拓宽了过继性T细胞疗法的潜在应用范围。虽然到目前为止T细胞是通过病毒转导进行基因改造的，但在过去几年中，通过RNA转染用CAR进行瞬时改造越来越受到关注。本章重点介绍通过体外转录RNA电穿孔用CAR改造外周血人T细胞的方法，以及测试改造后的T细胞在过继性免疫治疗中的功能。
