(M-502) Increased Efficacy of a Melanoma mRNA-LNP Vaccine Containing Co-stimulatory Molecules

With the recent FDA approval of the mRNA-LNP vaccines for COVID-19, there has been an increased interest in using the mRNA-LNP platform for therapeutic cancer vaccines to treat pancreatic cancer. Discovery of neoantigens, newly formed antigens generated by tumor cells, have improved the cancer vaccine field because they are recognized as non-self and are excellent targets for immunotherapies. However, when delivered on their own, many of these neoantigens lack sufficient immunogenicity to create effective anti-tumor responses. For proper anti-tumor T-cell activation, two stimuli are required: presentation of the neoantigen through the major histocompatibility complex and co-stimulation through secondary signaling. Several co-stimulatory molecules have been studied in the context of cancer; however, little information regarding their efficacy in mRNA-LNP vaccines is known. We screened three co-stimulatory molecules – OX40L, CD70, and GITRL – to determine which molecule, or combination of molecules, increased the immunogenicity of a low immunogenic pancreatic cancer neoantigen. By co-delivering the neoantigen and a co-stimulatory molecule in a mRNA-LNP vaccine, each co-stimulatory molecule’s effect on the anti-neoantigen T-cell response was quantified.

We have determined a pancreatic cancer neoantigen (MUT77) which has low immunogenicity when delivered via a mRNA-LNP vaccine. This neoantigen was used throughout this study. Plasmids for each of the co-stimulatory molecules were designed: OX40L, CD70 and GITRL. Plasmid linearization and HiScribe T7 High Yield RNA Synthesis Kit was used to synthesize IVT mRNA for all co-stimulatory molecules. To confirm the synthesized IVT mRNA could be translated to the corresponding protein with proper localization to the plasma membrane, HEK293 cells were transfected using Lipofectamine MessengerMAX reagent. Immunofluorescence confirmed the presence of each co-stimulatory molecule protein in the plasma membrane of the transfected cells. After confirming the proper expression of co-stimulatory molecules, an IFNγ IL-4 dual ELISpot (enzyme-linked immunosorbent spot) assay was used to determine the adjuvant properties of each co-stimulatory molecule. C57BL/6 mice were co-vaccinated with co-stimulatory mRNA LNPs and neoantigen mRNA LNPs on days 0, 3, 7, and 14. Six days post-the fourth vaccination, T-cells were isolated from the spleens of mice with an EasySep T Cell Isolation kit. The vaccination and T-cell isolation process was completed for each co-stimulatory molecule (6 mice per group). T-cells were plated on a dual spot plate for detection of IL-4 and IFNγ production. The resulting spots were analyzed using the Cytation 7 imager and a code developed for spot quantification using ImageJ Software.

The production of IVT mRNA and proper protein translation for both co-stimulatory molecules were confirmed using western blot and immunofluorescence assays. Neoantigen and co-stimulatory molecule mRNA was then encapsulated in LNPs for vaccination in mice. Mice were vaccinated with the neoantigen mRNA-LNP alone as a control, and it was shown to have low immunogenicity. When neoantigen mRNA was co-delivered with co-stimulatory molecule mRNA it was found that both co-stimulatory molecules could act as an adjuvant and increase anti-neoantigen T-cell responses. We were particularly interested in co-stimulatory molecules that increased IFNγ as this corresponds to increase in CD8+ T-cell effector function and Th1 responses, as these are essential for the anti-tumor immune response. The co-stimulatory molecules increased both CD4+ and CD8+ T-cell anti-neoantigen responses. This dual spot ELIspot assay was performed to quantify the production of IL-4 and IFNγ. IL-4 production is upregulated in Th2 responses and IFNγ production is upregulated in Th1 responses. In the context of cancer, it is desired to increase Th1, cellular immunity, responses as T-cells are primarily responsible for tumor death. Both co-stimulatory molecules had the ability to increase Th1 responses while retaining or downregulating Th2 T-cells.

Incorporating co-stimulatory molecules in a mRNA-LNP cancer vaccine helped increase the adaptive immune response toward a weak pancreatic cancer neoantigen. Future plans for this work are to combine the optimal co-stimulatory molecules, those that increased CD8+ and Th1 responses, together to determine if any synergistic effects to the immune response are observed. Additionally, the top mRNA-LNP vaccine formulation (neoantigen + co-stimulatory molecule/s) will be compared to the neoantigen mRNA-LNP vaccine alone in the Pan02 pancreatic cancer mouse model. Additionally, flow cytometry analysis will be completed on the tumor microenvironment: quantification of regulatory T cells, memory T cells, effector T cells, and various markers of T cell exhaustion.