CD4 T cell activating neoantigens enhance personalized cancer vaccine efficacy

Personalized cancer vaccines aim to activate and expand cytotoxic anti-tumor CD8+ T cells to recognize and kill tumor cells. However, the role of CD4+ T cell activation in the clinical benefit of these vaccines is not well defined. We previously established a personalized neoantigen vaccine (PancVAX) for the pancreatic cancer cell line Panc02, which activates tumor-specific CD8+ T cells but required combinatorial checkpoint modulators to achieve therapeutic efficacy. To determine the effects of neoantigen-specific CD4+ T cell activation, we generated a new vaccine (PancVAX2) targeting both MHCI- and MHCII-specific neoantigens. Tumor-bearing mice vaccinated with PancVAX2 had significantly improved control of tumor growth and long-term survival benefit without concurrent administration of checkpoint inhibitors. PancVAX2 significantly enhanced priming and recruitment of neoantigen-specific CD8+ T into the tumor with lower PD1 expression after reactivation compared to the CD8+ vaccine alone. Vaccine-induced neoantigen- specific Th1 CD4+ T cells in the tumor were associated with decreased T regulatory cells (Tregs). Consistent with this, PancVAX2 was associated with more pro-immune myeloid-derived suppressor cells and M1-like macrophages in the tumor demonstrating a less immunosuppressive tumor microenvironment. This study demonstrates the biological importance of prioritizing and including CD4 T cell-specific neoantigens for personalized cancer vaccine modalities. Subcutaneous Panc02 tumors were harvested from C57BL/6 mice treated with combinations of PancVAX neoantigen peptide vaccine, Anti-PD-1 antibody, and Anti-CTLA-4 antibody. Single cell suspensions were obtained from dissociated whole tumors and enrichment for immune cells by Percoll density separation and subjected to single-cell RNA-sequencing. Cell suspensions from multiple mice receiving the same treatment were pooled into one sample for library preparation. Samples that underwent immune cell enrichment were also subjected to paired single cell T cell receptor sequencing.