(M-514) Engineering a mRNA-LNP Vaccine for Efficacy in a Pancreatic Mouse Model

In 2023, cancer remains one of the leading causes of death globally. In particular, pancreatic cancer is considered to be one of the most fatal and difficult forms of cancer to treat with a 5-year relative survival rate of 12%. Multiple cancer immunotherapies have been developed to better enable the human immune system to detect and combat the disease. One of the most commonly used immunotherapies are checkpoint inhibitors such as anti-PD1 monoclonal antibodies which help prolong the adaptive immune system’s ability to kill tumor cells. However, in cases of pancreatic ductal adenocarcinoma (PDAC), one of the most prevalent and aggressive forms of pancreatic cancer, checkpoint inhibitor therapy has an extremely low response rate of 1-2%, demonstrating the need for additional forms of treatment. Previous literature has identified neoantigens (antigens arising in cancer cells) present in the pan02 mouse model of PDAC. The goal of this study was to test pan02 neoantigens in mRNA form. Neoantigen mRNA was encapsulated in lipid nanoparticles (LNP) and their immunogenicity assessed. Neoantigens found to elicit an immune response were then linked into one mRNA sequence and delivered as a vaccine for the treatment of pan02 tumors. Additionally, this treatment could be used in conjunction with checkpoint inhibitor therapy to potentially increase the efficacy of anti-PD1 therapy in this model.

Eight pan02 pancreatic cancer neoantigens were tested in vivo to determine their immunogenicity. Plasmids encoding the eight neoantigens were designed and in-vitro transcribed mRNA was produced using the HiScribe T7 High Yield RNA Kit. The mRNA and lipids were resuspended in 100mM citrate buffer and ethanol, respectively. LNPs were formed and mRNA encapsulated by adding the mRNA to the lipids and pipetting vigorously. The eight neoantigens were encapsulated in LNPs and all eight LNP batches were characterized for diameter (dynamic light scattering) and mRNA encapsulation efficiency.

To test the immunogenicity of the neoantigens an IFNγ ELISpot was conducted. C57BL/6 mice were intramuscularly injected with one of the LNP formulations (4 mice per neoantigen) on Day 0, 3, 7, and 14. Spleens from] vaccinated mice were removed followed by T-cell isolation. Isolated ] T-cells were co-incubated with dendritic cells that had been transfected with neoantigen mRNA. The ELISpot was performed following the manufacturer's protocol. The ELISpot plate was imaged and an ImageJ macro was ran to quantify the number of spots in each sample. Neoantigens that produced significantly greater spots than negative control were considered immunogenic and used in future studies.

Immunogenic neoantigens were linked into one mRNA sequence for easy co-delivery. Additionally, pan02 cells were subcutaneously injected (500,000 cells/mice) in the right flank of C57BL/6 mice. Mice were vaccinated with the mRNA-LNP vaccine (or PBS control) through intramuscular injections on days 3, 6, 9, and 12. Tumor volume and mouse weight was measured every three days.

All eight neoantigen LNP batches were injected in C57BL/6 mice and their immunogenicity determined using an IFNγ ELISpot. The ELISpot was imaged and the spots in each sample were quantified using an ImageJ code, where each spot corresponds to an IFNγ secreting T-cell. Of the eight tested pan02 neoantigens, two had significantly higher spot counts compared to the negative control group, indicating significant immunogenicity. An mRNA sequence was designed to link the two neoantigens together so that they could be easily co-delivered in one LNP formulation. Mice inoculated with tumors were vaccinated with the mRNA-LNP vaccine or a PBS control and tumor volume and growth rate were monitored over time. Results indicated that treatment with the vaccine showed a significant reduction in tumor growth rate and increased survival compared to PBS control mice. These results indicate that the mRNA-LNP vaccine holds therapeutic potential in the pan02 model of pancreatic ductal adenocarcinoma.

Based on the experimental results, two of the tested neoantigens were significantly immunogenic and generated an adaptive immune response when injected into C57BL/6 mice. The optimized mRNA-LNP vaccine significantly decreased tumor growth rate and volume, indicating its therapeutic potential in the pan02 model. In future studies, this vaccine should be tested in conjunction with current checkpoint inhibitor therapies to determine any synergistic effects.