Promoting improved agnostic cross presentation of tumor antigens with an oncolytic adenovirus expressing bispecific macrophage engagers

Background: Non-specific (‘agnostic') cross-presentation of tumor antigens (TAA) is essential for generating adaptive immune responses against cancer. APCs uptake TAA by phagocytozing proteins from cancer cells and present them on HLA molecules to activate T cells.To induce cancer cell uptake by the APCs, we aimed develop a bispecific macrophage engager (BiME) that can bring cancer cells and APCs into close proximity, and we hypothesized that oncolytic viruses could be used to express BiMEs in the tumor microenvironment. Methods: We generated an oncolytic adenovirus expressing a BiME (Ad-BiME) designed to bind a tumor-associated antigen on cancer cells and an activating receptor on APCs, thereby promoting interaction and triggering APC activation. The expressed bispecific protein efficiently bound ligands on both APCs and cancer cells. Co-culture assays evaluated phagocytosis, macrophage activation, and antigen cross-presentation. To analyse cross-presentation, we engineered NY-ESO-1-expressing HLA-A2-negative cancer cells and co-cultured them with HLA-mismatched HLA-A2-positive APCs. NY-ESO-1(157-165) peptide-loaded HLA-A2-specific 1G4 TCR primary T cells detected cross-presentation. Finally, freshly dissected colorectal tumor and normal colon biopsies were used to assess cancer-specific virus replication, BiME generation, and macrophage activation. Results: The BiME significantly enhanced phagocytosis of cancer cells by macrophages, surpassing the clinically tested anti-CD47 antibody. It further activated macrophages, upregulating CD86 and CD80 expression. Notably, Ad-BiME induced the strongest macrophage activation. Tumor antigen cross-presentation by macrophages was markedly increased following BiME treatment. Ex vivo experiments showed that oncolytic viruses replicated and produced BiME exclusively in colorectal tumor biopsies, where they induced endogenous macrophage activation, unlike in normal tissues. Elevated IL-1beta, TNF, IL-6, CXCL10, and IFN-gamma were detected only in tumor samples, indicating targeted BiME expression. Interestingly, free BiME treatment markedly increased CCL17, a chemokine attracting regulatory T cells (Tregs). In contrast, Ad-BiME did not elevate CCL17, likely due to gradual in situ BiME generation. Bulk RNA analysis further revealed that Ad-BiME uniquely upregulated macrophage pathways linked to antigen cross-presentation and T-cell chemotaxis. Conclusion: We generated the first oncolytic virus expressing a BiME and demonstrated its potential as a promising mechanism for antigen-agnostic cross-presentation in developing cancer vaccines for solid tumors.