Oncolytic adenoviruses encoding bispecific T cell engagers or a novel trispecific T cell engager for dual-targeting of c-MET and EGFR

This study reports a strategy to overcome immune escape of tumors during viro-immunotherapy due to tumor heterogeneity. We pursued a dual-targeting approach of epidermal growth factor receptor (EGFR) and cellular mesenchymal epithelial transition factor (c-MET) facilitated by two bispecific T cell engagers (TCEs) or one trispecific TCE encoded by oncolytic adenoviruses (oAds). Different bi- and trispecific TCE formats were generated and characterized. We showed efficacy of bispecific TCEs single-chain diabody (scDb)-cMET and tandem scFv (taFv)-EFGR <i>in vitro</i>, encouraging the design of the diabody-based trispecific TCE (Db-TriTE) scDb-cMET-scFv-EGFR. The Db-TriTE exhibited effective dual-targeting, i.e., the induction of cytotoxicity for single-positive cells of both targets and superior efficacy on double-positive cells. Therefore, a panel of oAds was produced to test different TCE (co-)expression strategies. Our results showed no or minor attenuation of viruses harboring the transgenes for one of the analyzed insertion strategies, expression of TCEs, and specific induction of both T cell activation and cytotoxicity in co-cultures of infected tumor cells with peripheral blood mononuclear cells (PBMCs). Importantly, we demonstrated dual-targeting by oAds encoding the two bispecific TCEs or the Db-TriTE with the latter featuring superior genomic stability and the overall highest efficacy. Taken together, we report novel bi- and trispecific TCEs and proof of principle for their application as dual-targeted viro-antibody therapy.