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Abstract

The field of immunotherapy has advanced greatly in the past decades and brought forward numerous treatment modalities, including antibody therapy and oncolytic virus (OV) therapy. Different antibody formats have been engineered for more potent anti-tumor effects, such as T-cell engagers (TCEs) that simultaneously bind to tumor-associated antigens (TAAs) and the T-cell receptor, redirecting T-cells to kill cancer cells. Numerous viruses have been explored for OV therapy, including oncolytic Adenoviruses (oAds), the most commonly used OVs in clinical development and an attractive platform for delivery of transgenes to tumors. Tumor heterogeneity, immune evasion and systemic toxicity pose challenges for effective TCE therapy, which could be overcome by local delivery of TCEs targeting multiple antigens via oAds. Here, I generated different TCE formats, bispecific T-cell engagers (BiTEs), bispecific single-chain Diabodies (scDbs) and trispecific scDb single-chain variable fragments (scDb-scFvs) for multitargeting of four TAAs, focusing on the epidermal growth factor receptor (EGFR) and the cellular mesenchymal epithelial transition factor (c-MET). I conducted a first of its kind comparison of scDbs and BiTEs directed at the chosen targets, analyzing binding properties and induction of T-cell mediated cytotoxicity. My results showed equal efficacies of the scDb-cMET and the BiTE-cMET, allowing for the generation of a scDb-cMET-based trispecific scDb-scFv, which was more favorable for genetic delivery by oAds due to its smaller size and fewer repetitive sequences than two distinct TCEs. I could show equal binding and induction of cytotoxicity on single target-positive cells compared to corresponding bispecifics and superior efficacy on double target-positive cells for the scDb-cMET-scFv-EGFR. I therefore produced a panel of oAds expressing one or two TCEs to characterize dual-targeting strategies of EGFR and c-MET. I was able to identify an expression strategy that retained oncolytic activity of oAds while driving expression of functional TCEs. My T-cell activation data revealed secretion of IL-2 and IFNγ and upregulation of CD69 on T-cells in cocultures of oAd-infected tumor cells with PBMCs only when cross-linking TCEs were produced. In addition, tumor cell killing was enhanced by TCEs when PBMCs were present. Ad5/3-CMV-scDb-cMET-scFv-EGFR, encoding the trispecific TCE, exhibited the highest potency regarding T-cell activation and killing throughout all tested cell lines compared to other dual-targeting strategies with bispecific TCEs via co-expression or co-infection approaches. This oAd showed efficacy in both single and double target-positive cell lines, indicating potential in treatment of heterogeneous tumors and reducing the risk of immune evasion of tumor cells. In summary, I generated and showed the efficacy of the novel scDb-cMET-scFv-EGFR and successfully incorporated it into a viro-antibody-therapy approach. Future experiments have to be conducted to elucidate the therapeutic potential of the Ad5/3-CMV-scDb-cMET-scFv-EGFR for different tumor entities in vivo.