Poster CC10 - Multi-amine core ionizable lipids for mRNA lipid nanoparticle cancer vaccines

Introduction: As demonstrated by the success of the SARS-CoV-2 mRNA vaccines, lipid nanoparticles (LNPs) are a promising drug delivery carrier for antigen mRNA in vaccine applications. The need for safer and more efficient LNP technology will continue to grow as we prepare for future diseases including cancer. To achieve this, designing new structures of ionizable lipids that enable selective mRNA delivery to immune cells, efficient endosomal escape, and activation of immunity against antigens will be crucial for the development of next-generation vaccine delivery systems. In this study, we designed a new class of ionizable lipid with crosslinkers. These crosslinkers were used to generate novel structures of lipid amine cores through coupling reactions. By adding lipid tails following the coupling of the amines, the one-pot two step synthesis was able to generate multi-amine core ionizable lipid libraries in a fast and facile manner. The multi-amine core LNP was able to improve mRNA delivery to dendritic cells and induced potent vaccine efficacy.

Materials and

Methods: Multi-amine core ionizable lipids were prepared using crosslinkers and formulated with phospholipid, cholesterol, and lipid-PEG via microfluidic mixing (Fig. 1A). Transfection efficiency of LNPs was evaluated on dendritic cells by delivering reporter gene mRNA. Antigen presentation was evaluated on dendritic cells by delivering ovalbumin (OVA) mRNA followed by flow cytometry. For evaluation of cancer vaccine efficacy, C57BL/6 mice were immunized with OVA mRNA encapsulated LNPs by intramuscular injection on day 0 and day 5. Serum and spleen were collected on day 10 to assess humoral and cellular immune response against OVA model antigen. For activation of the humoral immune response, anti-OVA antibody titer was evaluated. For activation of the cellular immune response, OVA specific CD8 T cell population in the spleen was evaluated. On day 12, B16-OVA cells were inoculated, and the tumor growth was monitored.

Results, Conclusions, and Discussions: The multi-amine core ionizable lipids “C-A” showed improved in vitro and in vivo mRNA delivery compared to single amine ionizable lipids “A”. Specifically, the C-A-based LNP efficiently delivered mRNA to dendritic cells (Fig. 1B), thereby enabling efficient antigen presentation for vaccine application (Fig. 1C, 1D). In a B16-OVA tumor mouse model, the intramuscularly injected C-A-LNPs showed significantly higher mRNA translation compared to A-LNP (Fig. 1E). This led to the production of OVA-specific antibodies in serum (Fig. 1F) and OVA-specific T cells in the spleen (Fig. 1G), demonstrating the establishment of humoral and cellular immunity against OVA antigen. Finally, the establishment of immunity by C-A-LNP led to significant tumor growth inhibition compared to untreated and A-LNP treated mice (Fig. 1H). Overall, the multi-amine core ionizable lipids demonstrated great potential as a next-generation cancer mRNA vaccine delivery system, showing efficient mRNA delivery and immune activation. Currently, we are expanding this research towards various vaccine applications including infectious disease to prove its versatility as a platform.

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