Abstract 847: Small circular RNA for cancer immunotherapy

Abstract Introduction: Messenger RNA (mRNA) has emerged as a novel vaccine platform and has proven highly effective during the pandemic. This platform had also found application in the development of cancer vaccines. Despite the encouraging outcomes observed in early-phase clinical trials, certain concerns are still associated with current mRNA-based cancer vaccines such as limited stability, suboptimal tumor therapy potency and durability, and limited safety. Here, we report novel highly stable antigen-encoding small circular mRNA (circRNA) vaccines that elicit potent and durable T cell responses for robust tumor immunotherapy. Method: Small circRNAs comprise minimal elements: codon-optimized peptide antigen-encoding RNA, a short IRES, and a Kozak consensus sequence that recruits ribosomes for peptide translation. Using MHC-I-restricted ovalbumin257-264 (OVA257-264 or SIINFEKL) as a model antigen, we prepared circRNA as follows: 1) circularizing 5’-phosphate-RNA using T4 RNA ligases and complementary 30-mer DNA splints, 2) removing linear/lariat RNA and DNA using DNase I and exonuclease T or RNase R, 3) purification to remove immunostimulatory small DNA/RNA fragments, enzymes, and nucleotides, and 4) verification by Sanger sequencing and gel electrophoresis. Results: Compared to modified mRNA and current long circRNA, small circRNA shows superior stability, minimal protein kinase R activation, and low cytotoxicity. This allows small circRNA vaccines to sustain efficient antigen translation over an extended period while activating pattern recognition receptors for innate immunostimulation. Relative to several modified mRNAs, nanocarrier-delivered small circRNA vaccines show superior safety and elicit up to 10-fold antigen-specific T cells, accounting for 25%-75% total peripheral CD8+ T cells over 6 months in mice. circRNA vaccines are applicable for various major histocompatibility complex I/II-restricted tumor and viral (neo)antigens to elicit CD8+ and CD4+ T cell responses, in young adult and immunosenescentaged mice. In mice, mono-/multi-valent circRNA vaccines plus immune checkpoint blockade reduced tumor immunosuppression and eradicated multiple tumors, including B16F10, MC38, TC1, and immunotherapy-resistant BrafV600E melanoma. Overall, small circRNA vaccines represent a promising advancement in the field of tumor immunotherapy, offering optimal stability, safety, and the potential to generate potent and enduring immune responses against a variety of tumors. Citation Format: Xiang Liu. Small circular RNA for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 847.