基于肿瘤结合特异性CDR3δ序列的CARγδ-T细胞治疗实体肿瘤的研究

Chimeric antigen receptor (CAR) -T cells has made a major breakthrough in tumor immunotherapy in the treatment of B cell lymphocytoma . However, so far, CAR-T cells in the treatment of solid tumors is still no substantive progress, mainly due to the lack of specific therapeutic target molecules like CD19 in highly heterogeneous solid tumors. Without MHC restriction, gamma delta T cell receptor (TCRγδ) recognizes a broad spectrum of solid tumor antigens directly in a manner similar to the antibody, and can effectively compensate for the lack of CAR-T cells in the treatment of solid tumors. In this study, we tried to combine the advantages of TCRγδ in tumor antigen recognition and classic CAR-T cells. First CARγδ -T cells with tumor specific CDR3δ sequence OT3 and GTM would be constructed that the extracellular molecular structure of CARs were replaced by single chain TCRγδ molecules based on above CDR3δ sequences; And combined with a variety of strategies that beneficial for cell activation, invasion and to eliminate the microenvironment of immune suppression, the therapeutic effects of CARγδ-T cells of solid tumor in vivo were observed. In addition, by the high-throughput immune repertoire sequencing technology, we tried to obtain new tumor-specific CDR3δ sequences that dominant in tumor patients and specific binding to tumor cells. The study is a new attempt for combination of γδ T cells and CAR-T cells in tumor immunotherapy. The results would provide reference and suggestions for CAR-T cells adoptive immunotherapy to solid tumors.

嵌合抗原受体（CAR）-T细胞在B细胞肿瘤的免疫治疗中取得了很好的效果，但由于高度异质性的，缺乏像CD19分子一样特异性的治疗靶点，CAR-T细胞在实体瘤的治疗中一直无实质性进展。γδT细胞受体，能直接识别完整抗原，且对实体瘤识别谱广，能有效的弥补CAR-T细胞治疗实体瘤上的不足。鉴于此，本研究拟基于前期已验证的，具有肿瘤结合特异性CDR3δ序列（OT3和GTM），用包含该序列的单链γδTCR结构，作为CAR分子的胞外区，构建CARγδ-T细胞；并在体内外实验中联合多种辅助策略，观察CARγδ-T细胞对包括非小细胞肺癌、肝癌、胃癌、卵巢癌在内的实体瘤的治疗效果。此外，我们还将利用高通量免疫组库测序技术，继续筛选验证更多的具有肿瘤结合特异性和亲和力的CDR3δ序列。该研究是结合γδT细胞和CAR-T细胞的肿瘤免疫治疗新的尝试，将为解决实体肿瘤的CAR-T细胞过继免疫治疗提供参考和建议。

CAR-T细胞在B细胞肿瘤的免疫治疗中取得了很好的效果，但由于高度异质性的，缺乏像CD19分子一样特异性的治疗靶点，CAR-T细胞在实体瘤的治疗中一直无实质性进展。γδT细胞受体，能直接识别完整抗原，且对实体瘤识别谱广，能有效的弥补CAR-T细胞治疗实体瘤上的不足。. 鉴于此，本研究首先基于前期已验证的，具有肿瘤结合特异性CDR3δ序列（OT3和GTM），用包含该序列的单链γδTCR结构作为CAR分子的胞外区，构建了一种新的、具有更广谱的肿瘤识别特异性的CARγδ-T细胞，并在体内外验证了其的抗肿瘤活性；在此基础上，充分利用IL-7和CCL19在T细胞活性维持和免疫细胞在实体瘤组织浸润方面的优势，进一步优化CARγδ-T细胞的功能，构建了两种自分泌IL-7和CCL19的超级CARγδ-T细胞，并在体内外验证了其抗肿瘤的疗效；最后，利用高通量免疫组库测序技术，筛选与验证了更多具有肿瘤结合特异性的CDR3δ序列，为构建更多具有抗肿瘤效果的CARγδ-T细胞奠定了基础。. 综上所述，本研究综合考虑γδT细胞识别肿瘤抗原的特性和细胞因子、趋化因子的功能，构建了一类具有抗肿瘤疗效的新型CARγδ-T细胞，在肿瘤的细胞过继免疫治疗方面有很大的应用前景，将为解决实体肿瘤的CAR-T细胞过继免疫治疗提供参考和建议。