Design and Engineering of Recombinant Oncolytic Adenoviruses Expressing an Anti-EpCAM/Anti-CD3 BiTE for Targeted Immunotherapy of A549 Lung Cancer Cells

Background: Oncolytic virotherapy is a novel therapeutic approach in oncology that uses viruses to target and eradicate cancer cells specifically. To improve therapeutic effectiveness, we investigated the application of genetically modified oncolytic adenoviruses (OVs) engineered to express bispecific T-cell engager (BiTE) antibodies. Methods: In particular, we engineered Ad5-delta24 to express an EpCAM-targeting BiTE under the major late promoter, yielding the recombinant virus Ad5D24-Anti-EpCAM-Anti-CD3-scFv (rOAd5-BiTE). Following infection of A549 lung cancer cells, rOAd5-BiTE promoted the expression and secretion of BiTE antibodies targeting EpCAM and CD3. Results: In co-culture experiments, rOAd5-BiTE elicited significant T-cell activation and proliferation, characterized by increased secretion of IFN-γ and IL-2, as well as augmented cytotoxic activity from neighboring bystander cells. Moreover, the combination of rOAd5-BiTE with peripheral blood mononuclear cells (PBMCs) markedly enhanced the antitumor activity against A549 cells. Conclusion: The results indicate that equipping OVs with BiTE molecules may address critical challenges in virotherapy by enhancing and redirecting T-cell activity within the tumor microenvironment. This strategy offers a promising avenue for developing targeted immunovirotherapy approaches for the treatment of solid tumors.