αβ TCR基因转染Vγ9Vδ2T细胞协同融合细胞疫苗过继性免疫治疗骨肉瘤的研究

Our group previous study showed that osteosarcoma cells were killed by Vγ9Vδ2T cells expanded in vitro, but the in vivo experiments suggested that Vγ9Vδ2T cells have limited survival time and specific recognition ability to osteosarcoma cell, which represent obstacles for the adoptive immunotherapy of osteosarcoma. Therefore, it is urgent to modified Vγ9Vδ2T cells to increase their activity and specificity to osteosarcoma cells. The human epidermal growth factor receptor 2 (HER2) is expressed in up to 60% of primary osteosarcoma samples and correlates with poor outcome. Furthermore, immunotherapy against HER2 is effective in osteosarcoma, which suggests that HER2 plays an important role in the immune regulation of osteosarcoma. Recently, tumor antigen presenting cell (APC)-tumor fusion vaccine showed satisfactory anti-tumor effects, and typical APC features of Vγ9Vδ2T cells were newly discovered, but there are no reports on the development of Vγ9Vδ2T-tumor fusion vaccine. This project aims to identify the HER2 αβ TCR specific epitope in osteosarcoma, and stably transfects it to Vγ9Vδ2T cells; Then, the Vγ9Vδ2T-osteosarcoma fusion vaccine is produced by a process termed electrofusion; In the end, we will synergistically combine HER2 αβ TCR gene transferred Vγ9Vδ2T cells with Vγ9Vδ2T-osteosarcoma fusion vaccine, two methods of adoptive immunotherapy, to treat osteosarcoma in vitro and in vivo, which may provide new strategy in clinical immunotherapy of cancer.

本课题组研究发现体外扩增的Vγ9Vδ2T细胞对骨肉瘤细胞有较好的体外杀伤作用，但Vγ9Vδ2T细胞对在体骨肉瘤细胞识别特异性不足且存活时间有限。因此，寻找一种同时特异性识别骨肉瘤细胞且激活机体自身主动免疫的Vγ9Vδ2T细胞是亟需解决的关键问题。HER2在60%以上的骨肉瘤标本有阳性表达，且常常与患者不良预后相关，尤其是针对HER2的免疫治疗有效，提示HER2在骨肉瘤免疫调节中起重要作用。近来，APC与肿瘤细胞融合制作的肿瘤疫苗具有确切的杀伤效果，尽管新近发现Vγ9Vδ2T细胞具有典型的APC特性，但国内外尚未报道Vγ9Vδ2T与肿瘤细胞融合制作的肿瘤疫苗。本课题旨在确定特异性识别骨肉瘤表达的HER2的αβTCR表位，构建αβ TCR基因转染Vγ9Vδ2T细胞并制作与骨肉瘤细胞的融合细胞疫苗，联用HER2 αβ 基因转染Vγ9Vδ2T细胞，探讨过继性免疫治疗两大方法协同治疗骨肉瘤的机制。

本课题组此前的研究表明Vγ9Vδ2T细胞对骨肉瘤细胞有较好的杀伤作用，但在体内试验中效果有限。因此构建能特异性识别抑制骨肉瘤的Vγ9Vδ2T细胞是当务之急。我们在针对骨肉瘤样本的检测中发现，HER2抗原在有较高的阳性表达，且研究表明针对HER2的相关免疫治疗有不错的成效，因而构建针对HER2的基因工程T细胞将会有较好的应用前景。此外，由抗原呈递细胞与肿瘤细胞构成的肿瘤疫苗具有确切的杀伤效果，虽然Vγ9Vδ2T细胞也具有抗原呈递细胞的特性， 但是尚未有关于其参与肿瘤疫苗制作的报道。因此本课题通过构建能特异性识别HER2抗原的HER2 αβ TCR基因转染Vγ9Vδ2T细胞，探究其在体内外的肿瘤抑制效果。同时应用Vγ9Vδ2T细胞肿瘤疫苗，探究其能否有效地预防骨肉瘤的发生与发展。最后探讨过继性免疫治疗两大方法协同治疗骨肉瘤的疗效与可能机制。通过四年的研究，我们发现HER2αβTCR-γδT细胞对骨肉瘤细胞系的杀伤效果较普通Vγ9Vδ2T细胞有显著提升，验证了此基因工程化的T细胞产品具有一定的临床应用前景。与此同时，由Vγ9Vδ2T细胞和肿瘤细胞融合所得的肿瘤疫苗也能极大地提升CTL细胞的肿瘤杀伤能力。这些都为今后相关的临床试验提供了宝贵的前期数据。

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