We previously described the construction of a highly engineered, tumour-selective virotherapy, Ad5_NULL-A20, devoid of all native tropisms, but able to infect via αvβ6 integrin expressed in multiple aggressively transformed epithelial cancers. To enhance the activation of tumor infiltrating T-cells and NK cells at tumor sites we engineered the Ad5_NULL-A20 platform to express bi-specific molecules targeting either CD3 or CD16, and the tumour-antigen Epidermal growth factor receptor (EGFR) to encourage immune cell activation and redirect an effective immune response to tumour sites.

Analysis of bispecific transgene function was assessed in co-culture assays with cancer cell lines and healthy donor T-cells demonstrating significant increases in CD4+ and CD8+ T-cell activation, intracellular IFN-γ production and immune cell proliferation in response to cancer lines transduced with Ad5_NULL-A20 secreting CD3-EGFR bispecific constructs. In addition, NK cell CD107a degranulation was increased in cells secreting CD16-EGFR bi-specifics. All constructs induced immune mediated cancer killing between 1-5 days of co-culture.  Furthermore, patient derived pancreatic tumour organoids transduced with oncolytic Ad5_NULL-A20 expressing CD3-EGFR or CD16-EGFR demonstrated immune mediated cell killing of cancerous cells within 48 hours of co-culture. The bispecific constructs tested demonstrated both their specificity to the target antigen and proficiency to induce activation pathways and immune mediated cell killing in vitro.  In summary, these data demonstrate the arming of a highly tumour selective agent, Ad5_NULL-A20 to express bispecific immune cell engagers can immunologically “heat up” the tumour microenvironment. This approach has significant translational potential.