DARPin-fused T cell engager for adenovirus-mediated cancer therapy

Bispecific T cell engagers are a promising class of therapeutic proteins for cancer therapy. Their potency and small size often come with systemic toxicity and short half-life, making intravenous administration cumbersome. These limitations can be overcome by tumor-specific in situ expression, allowing high local accumulation while reducing systemic concentrations. However, encoding T cell engagers in viral or non-viral vectors and expressing them in situ ablates all forms of quality control performed during recombinant protein production. It is therefore vital to design constructs that feature minimal domain mispairing, and increased homogeneity of the therapeutic product. Here, we report a T cell engager architecture specifically designed for vector-mediated immunotherapy. It is based on a fusion of a designed ankyrin repeat protein (DARPin) to a CD3-targeting single-chain antibody fragment, termed DATE (DARPin-fused T cell Engager). The DATE induces potent T cell-mediated killing of HER2⁺ cancer cells, both as recombinantly produced therapeutic protein and as in situ expressed payload from a HER2⁺-retargeted high-capacity adenoviral vector (HC-AdV). We report remarkable tumor remission, DATE accumulation, and T cell infiltration through in situ expression mediated by a HER2⁺-retargeted HC-AdV in vivo. Our results support further investigations and developments of DATEs as payloads for vector-mediated immunotherapy.