Targeting TP53 pathologic variants for cancer immunotherapy using a self-replicating mRNA vaccine strategy.

3085 Background: TP53 pathogenic variants (PVs) are highly prevalent in human malignancies as a whole and especially in patients with pancreatic ductal adenocarcinoma (PDAC). Approximately 80% of TP53 PVs are missense mutations that may be targeted for cancer therapy. Methods: We have developed a self-replicating mRNA (srRNA) vaccine (TMG-mut TP53 srRNA SA-LNP) encoding concatenated p53 mutant peptides encompassing 14 hotspot TP53 missense mutations in PDAC, encapsulated in sialic acid (SA)-modified lipid nanoparticles (LNPs). These 14 hotspot TP53 PVs were selected by examining four public available datasets and from our previous studies. Results: TMG-mut TP53 srRNA SA-LNPs incorporated Venezuelan equine encephalitis virus replicase which enabled higher and prolonged in vivo protein expression compared to conventional mRNA construct. Encapsulation with SA-modified LNPs led to significantly better efficiency in targeting dendritic cells (DCs) in vitro and in vivo compared to conventional LNPs. We used in vitro (cell lines) and in vivo assays (PDX and human engineered mouse genetic models) to test the efficacy of our RNA vaccine. We validated the detection of TP53 neoantigen by mass spectrometry generated by our srRNA vaccine. We then tested its anti-tumor immunity by itself alone and in combination with anti-PD1 inhibition using multiple cell lines and PDX that carry different TP53 PV (R175H and R273H). TMG-mut TP53 srRNA SA-LNP stimulated potent and specific anti-tumor CD8 + T cell responses in in vitro and in vivo assays using autologous DCs, T cells and cancer cells from multiple PDAC patients. Using both Panc02 syngeneic PDAC cell line and patient-derived PDAC xenografts (PDX), we show that TMG-mut TP53 srRNA SA-LNP stimulated potent and specific anti-tumor immunity against PDAC tumors that harbor different corresponding p53 PVs. Panc02- TP53 R175H tumor-bearing transgenic mice expressing human HLA-A*02:01 treated with TMG-mut TP53 srRNA SA-LNP showed significantly suppressed tumor growth and prolonged survival with increased tumor infiltration of activated CD8 + T cells as well as increased production of cytokines including type II interferon, TNFalpha, and other biomarkers. We also show our vaccine stimulates increased tumor cell apoptosis. In addition, TMG-mut TP53 srRNA SA-LNP dramatically enhanced the anti-tumor immunity of anti-PD1 by synergistically suppressing tumor growth and prolonging the survival of tumor-bearing mice in both low and high-tumor burden animal model. Conclusions: Our results demonstrate for the first time that missense p53 PVs are valid targets for developing novel cancer immunotherapy using srRNA vaccine approach enhanced by SA-LNPs encapsulation. Our data potentially opens a new approach for developing mRNA vaccine-based immunotherapy for a large population of cancer patients as TP53 PVs are highly prevalent in nearly every type of malignant tumors.