A novel strategy to improve current immunotherapies is the enhancement of cytotoxic activity by bispecific antibodies (bsAb), which selectively target immune cells to tumor-associated antigens such as human epidermal growth factor receptor 2 (HER2/neu). An enhanced expression of HER2/neu on the cell surface of high-grade differentiated tumors or recurrent tumors has contributed to the selection of HER2 as a clinically relevant tumor-target. A polyclonal activation of all T cells as e.g. by the bsAb [HER2xCD3] could have fatal consequences, for instance due to a cytokine storm. The hypothesis of our study is that it could be beneficial to selectively activate γδ T cells by the tribody [(HER2)2xVγ9] instead of activating all T cells. We observed that Vγ9Vδ2 and to our surprise Vγ9Vδ1 γδ T cells infiltrate in high numbers in primary ovarian tumors and pancreatic ductal adenocarcinomas (PDAC) in comparison to the autologous blood. The preliminary observation that a certain percentage of Vγ9 γδ T cells are not associated with Vδ2 within the tumor arise the speculation of tumor-antigen specific Vγ9Vδ1 γδ T cells. Regarding this preliminary observation, we will characterize the tumor-specific TCRγδ -repertoire in fresh tumor tissue in comparison to autologous blood of ovarian cancer and PDAC patients in more detail (immunophenotyping with self-made anti-TCRVγ monoclonal Ab, TCR repertoire by single cell RNA analysis). To provide more insights in the molecular mechanisms of signaling pathways involved in cytotoxicity, proliferation and cytokine release, Vγ9Vδ1 compared to Vγ9δ2 or other Vδ1 γδ T cell subsets in the blood versus tumor-infiltrating T cells will be co-cultured with different (autologous) primary tumors. The induction of the signaling pathways by bsAb [HER2xCD3], tribody [(HER2)2xVγ9], [(HER2)2×Vγ9] and UCHT1 diabodies in resting versus short-term expanded Vγ9Vδ1and Vγ9Vδ2 γδ T cell subsets as well as costimulatory requirements of these subsets in interaction with primary ovarian tumors and PDAC cells will provide information about the best strategy to activate γδ T cells by bsAb in vivo or clarify whether an adoptive γδ T cell-transfer together with bsAb is required. The ensuing question whether the tribody [(HER2)2xVγ9] can overcome an immunosuppressive microenvironment will be investigated in a preclinical humanized mouse model to pave the way for therapeutic intervention. The individual research projects within the FOR2799 (”Receiving and Translating Signals via the γδ T Cell Receptor”) will benefit from the collaborations among each other and provide a better insight in the role of γδ T cells in anti-tumor and anti-viral immunity.