Soluble multi-epitope protein vaccination leverages antigen availability to drive CD8⁺ T cell immunity and tumor control

The shift toward precision medicine in cancer immunotherapy has increased demand for rapid, scalable production platforms for personalized vaccine antigens targeting neoantigens and tumor-associated antigens. Escherichia coli-based recombinant protein production represents a globally established system offering speed and cost-effectiveness, yet the immunogenic potential of soluble antigens produced via this platform remains incompletely characterized. Here, we systematically evaluated the capacity of E. coli-derived soluble antigen formulations to elicit anti-tumor T cell responses. Using ClearColi BL21 (DE3), a lipopolysaccharide (LPS)-truncated strain free of endotoxic contamination, we produced soluble formulations containing murine OVA-derived epitopes. In vitro antigen presentation assays revealed minimal T cell activation despite high numbers of CD8+ and CD4+ T cells, indicating compromised antigen presentation capacity. However, in vivo adoptive transfer experiments demonstrated strongly inducible T cell expansion in draining and non-draining lymph nodes, with high frequencies of antigen-specific CD8+ T cells producing IFN-γ and TNF-α. Prime-boost vaccination strategies in experimental melanoma models achieved protective efficacy and durable survival with persistent antigen-specific T cell responses throughout the observation period. Under stringent single-prime conditions, transient T cell frequencies were observed without long-term tumor control.