Prof. Dr. Daniela Wesch

Strategies to enhance cytotoxicity of tumor-infiltrating γδ T-cell subsets against autologous tumor cells

Localization, distribution, cytotoxicity and activation state of human tumor infiltrating γδ T cells (γδ TIL) and their impact on survival of cancer patients are of great interest for an improvement of γδ T cell-based immunotherapy. High recurrence rates in epithelial ovarian cancer (EOC) - even after successful initial surgical and poly-chemotherapeutic treatment - reflects a significant problem. Chemotherapeutic standard regimens are often not of long-lasting efficacy in the palliative situation. The presence of intra-tumoral cytotoxic T cells has been correlated with a favorable clinical outcome.

Recently, we demonstrated an increased Vγ2,3,4 γδ T-cell infiltration co expressing Vδ1- or Vδ3-chains in freshly isolated EOC tissue which strongly produce cytotoxic mediators such as granzyme A/B. Further, we discovered a novel role of TNF-related apoptosis inducing ligand (TRAIL)-receptor 4 that enhance anti-tumoral Vδ1 T-cell cytotoxicity. Since central and effector memory Vδ1 TIL are increased within the EOC tissue, it is likely that they are activated at the tumor-site. The identification of site-specific occurrence of Vγ2,3,4 and Vγ9 γδ T-cell clones and a butyrophilin-dependent activation of Vγ9Vδ1and non-Vδ1/Vδ2 TIL will be investigated within the FOR2799.

Our previous results demonstrated that the bispecific T cell engager (bsTCE)[(HER)2xVγ9] enhanced Vγ9 T-cell cytotoxicity against autologous EOC cells expressing the human epidermal growth factor receptor (HER)-2. Further, bsTCE partially overcomes intrinsic tumor resistance mechanisms which prevent γδ T-cell activation. Preliminary results revealed a 30-90% antitumor efficacy of [(HER)2xVγ9] in our novel patient derived ex vivo tumor tissue model including an immunosuppressive tumor microenvironment (TME). Since Vγ2,3,4 TIL accumulate in the EOC tissue, we generated the bsTCE [(HER)2xVγ2,3,4], which allows in combination with [(HER)2xVγ9] to target up to 78% of the γδ TIL in EOC patients. The efficacy of [(HER)2xVγ2,3,4] on γδ T cell-mediated lysis of autologous EOC cells and the impact of the TME will be examined in the NSG mouse model and the patient derived ex vivo tumor tissue model. The latter is of great use when trying to reduce the translational gap between preclinical and clinical research. The use of the bsTCE in the patient-derived ex vivo tumor tissue model also allows the targeted investigation of the effector function of Vγ2,3,4 and Vγ9 TIL. Since our bsTCEs do not induce γδ T-cell death like repetitive application of phosphorylated antigens does, this is a decisive advantage of bsTCE application. We expect that our bsTCEs reflect a novel tool for a future γδ T cell-based immunotherapy.

Influence of bispecific antibodies on the interaction of γδ T cells and tumor cells 

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.