Multivalent Small Circular mRNA Vaccines for Melanoma Combination Immunotherapy

Abstract Melanoma is the most serious type of skin cancer. Although immune checkpoint blockade (ICB) therapy has benefited many melanoma patients, most patients do not respond to current ICB. Vaccines can promote ICB therapeutic efficacy by generating or amplifying tumor-reactive T cells. Conventional vaccines are associated

with limitations such as low stability and bioavailability, preexisting anti-viral-vector immunity, weak antigenicity, or concerns over genomic integration or virulent reversion. mRNA vaccines hold a great potential for cancer immunotherapy. Yet, current linear mRNA vaccines are still associated with 1) limited biostability, despite

structural and nucleoside modifications, and the resulting limited shelf-life and moderate antigen translation efficiency, 2) complicated, time-consuming, and error-prone enzymatic mRNA production, 3) limited loading capacity in nanocarriers, and 4) short immune memory. To address these limitations, we propose developing

novel, highly stable, modification-free multivalent small circular mRNA (circRNA) to elicit potent and durable antitumor immunity for ICB combination immunotherapy of melanoma. Small circRNA is comprised of minimal RNA elements to translate peptide antigens. We showed that 1) small circRNA has high loading capacity in

nanocarriers and efficiently accumulates in lymph nodes and antigen-presenting cells; 2) terminus-free small circRNA, either free or loaded in nanoparticles, are highly stable relative to current state-of-the-art modified mRNA vaccine; 3) circRNA vaccines are self-adjuvanted due to intrinsic activation of intracellular pattern

recognition receptors; 4) circRNA prolong antigen translation accompanied by innate immunostimulation, which promotes T cell responses; 5) circRNA may produce concatemeric long peptide antigens that, relative to minimal antigens, undergo proteolytic processing for optimal antitumor T cell responses; and 6) low-dose small circRNA

vaccines outperform several current state-of-the-art modified mRNA vaccines to generate potent and durable T cell immunity. Further, a multivalent circRNA vaccine elicited significantly potentiated ICB therapeutic efficacy of melanoma in mice. The objective of this project is to develop and test multivalent melanoma circRNA to elicit

multivalent antimelanoma T cell responses and reduce melanoma tumor immunosuppression, elucidate their molecular and cellular mechanisms to elicit innate and adaptive antitumor immunity, and assess their melanoma therapeutic efficacies and toxicity in mice. Our highly collaborative team has complementary expertise in circRNA

vaccine, melanoma vaccinology, and clinical melanoma immunotherapy for accomplishing our scientific goals. If successful, a significant deliverable from this study is to establish a scientific framework for using this novel mRNA vaccines in melanoma combination immunotherapy.