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We have developed a new format of a chimeric antigen receptor for αβ T cells, in which the single-chain variable fragment recognizing the tumor antigen is directly fused to the T cell receptor, called T cell receptor fusion construct (TRuC). Here, we express an anti-CD19 εTRuC in primary γδ T cells that were expanded using zoledronate (Zol) or concanavalin A. We show that the resulting εTRuC γδ T cells were reprogrammed to better recognize CD19-positive B cell tumors and—in case of the Zol-expanded cells—a CD19-expressing colon adenocarcinoma-derived cell line in vitro. This resulted in enhanced tumor killing, upregulation of the activation marker CD25, and secretion of cytokines. We found that the transduction efficiency of the concanavalin A–expanded cells was better than the one of the Zol-expanded ones. Our in vitro cytotoxicity data suggest that the Vδ2 T cells were better killers than the Vδ1 T cells. Finally, addition of vitamin C promoted the recovery of larger γδ T cell numbers after lentiviral transduction, as used for the expression of the εTRuC. In conclusion, the generation and use of γδ εTRuC T cells might be a new approach for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Epithelial ovarian cancer displays the highest mortality of all gynecological tumors. A relapse of the disease even after successful surgical treatment is a significant problem. Resistance against the current platinum‐based chemotherapeutic standard regime requires a detailed ex vivo immune profiling of tumor‐infiltrating cells and the development of new therapeutic strategies. In this study, we phenotypically and functionally characterize tumor cells and autologous tumor‐derived αβ and γδ T lymphocyte subsets. Tumor‐infiltrating (TIL) and tumor‐ascites lymphocytes (TAL) were ex vivo isolated out of tumor tissue and ascites, respectively, from high‐grade ovarian carcinoma patients (FIGO‐stage IIIa‐IV). We observed an increased γδ T cell percentage in ascites compared to tumor‐tissue and blood of these patients, whereas CD8+ αβ T cells were increased within TAL and TIL. The number of Vδ1 and non‐Vδ1/Vδ2‐expressing γδ T cells was increased in the ascites and in the tumor tissue compared to the blood of the same donors. Commonly in PBL, the Vγ9 chain of the γδ T cell receptor is usually associated exclusively with the Vδ2 chain. Interestingly, we detected Vδ1 and non‐Vδ1/Vδ2 T cells co‐expressing Vγ9, which is so far not described for TAL and TIL. Importantly, our data demonstrated an expression of human epidermal growth factor receptor (HER)‐2 on high‐grade ovarian tumors, which can serve as an efficient tumor antigen to target CD3 TIL or selectively Vγ9‐expressing γδ T cells by bispecific antibodies (bsAbs) to ovarian cancer cells. Our bsAbs efficiently enhance cytotoxicity of TIL and TAL against autologous HER‐2‐expressing ovarian cells. [ABSTRACT FROM AUTHOR]