(502d) Co-Stimulatory Molecules Increase the Immunogenicity of Tumor-Specific Neoantigens

The advancement of next-generation sequencing technologies has led to the identification of patient-specific tumor antigens. These tumor-specific antigens, termed neoantigens, arise from genomic mutations and can be used to create personalized cancer immunotherapies. In particular, neoantigen based vaccines are clinically being studied as a means to increase and diversify the anti-tumor T-cell responses in patients. Sequencing has allowed for large libraries of neoantigen sequences to be identified; however, only a small population of neoantigens are able to mount strong immune responses in vivo. This greatly limits the efficacy of personalized neoantigen based vaccines. There is a clinical need to increase the anti-tumor T-cell responses, or immunogenicity, that lowly immunogenic neoantigens produce. The goal of this study was to determine if co-stimulatory molecules could increase the immunogenicity of a low immunogenic neoantigen. Co-stimulatory molecules provide a secondary signal to T-cells and are required for full T-cell activation. We identified neoantigens in the murine Pan02 tumor model and ranked their immunogenicity using an enzyme-linked immunospot (ELISPOT) assay. A low immunogenic neoantigen, called Neoantigen 77, was unable to produce a significant immune response and was chosen as the model neoantigen for completion of this work.

We set up a screen of six co-stimulatory molecules predicted in literature to increase the Th1 cellular immune response when expressed on antigen presenting cells. Each individual co-stimulatory molecule and every pairing combination of molecules were co-delivered with Neoantigen 77 and the neoantigen-specific T-cell response determined. Specifically, mice were vaccinated intramuscularly with lipid nanoparticles (LNPs) co-delivering mRNA encoding Neoantigen 77 and encoding co-stimulatory molecules. Sixteen days after the first vaccine dose, splenocytes were isolated from mice and analyzed in an IFNy, IL-4 ELISPOT. ELISPOT data revealed that three of the individual co-stimulatory molecules significantly increased the number of activated anti-neoantigen 77 T-cells. Additionally, after screening every pairing combination of co-stimulatory molecules, it was found that several significantly increased the immunogenicity of Neoantigen 77. One pairing in particular had a 3.5 fold greater number of IFNy secreting Neoantigen 77 specific T-cells compared to mice injected with Neoantigen 77 alone. The optimal co-stimulatory pairing was dose optimized for further studies. Splenocytes were isolated from mice treated with Neoantigen 77 plus the optimized pairing, restimulated with dendritic cells presenting Neoantigen 77, and then stained for flow cytometry. Flow analysis revealed mice receiving the optimized co-stimulatory molecules had an increase in both IFNy secreting CD4+ and CD8+ T-cells, as well as higher ratios of CD8+ to regulatory T-cells (compared to control mice receiving Neoantigen 77 alone). Toxicity studies revealed that cytotoxicity remained low even after repeated doses of the optimal co-stimulatory pairing. Mouse body weight was measured, and all biological replicates remained within 10% of starting weight, even after four injections of co-stimulatory molecules given over a two-week period. Liver enzymes, AST and ALT, did not increase after vaccination, indicating low liver toxicity. Additionally, H&E staining of liver, lungs, kidney, and brain of mice treated with the optimized dose of co-stimulatory molecules did not show any significant organ toxicity.

In conclusion, this is the first study to our knowledge that has tested co-stimulatory molecules in the context of a mRNA-LNP cancer vaccine. It was shown that co-stimulatory molecules can increase the immunogenicity of a low immunogenic neoantigen while maintaining low toxicity. Future work for this research will test a neoantigen mRNA-LNP vaccine with the optimized co-stimulatory pair in the Pan02 pancreatic mouse model.