Abstract
The fundamental issue in immunotherapy is the lack of tumor-specific antigens in most types of tumors, leading to immune tolerance. For approximately 85% of patients with microsatellite stable (MSS) colorectal cancer (CRC), the absence of tumor neoantigens results in poor immunotherapy efficacy. Our previous study demonstrated that the misincorporation of non-proteinogenic proline (Pro) analog azetidine-2-carboxylic acid (AZE) could generate mutated proteins that significantly enhance tumor cell antigenicity and anti-tumor immune responses. Methods: To activate more specific anti-tumor immune responses with fewer side effects, we utilized the non-proteinogenic serine (Ser) analog β-N-methylamino-L-alanine (BMAA), which can be misincorporated into proteins as a Ser substitute by seryl tRNA synthetase at an appropriate rate. BMAA misincorporated neoantigens were detected using mass spectrometry (MS), and cancer cell-enriched peptides with high antigenicity were selected in a murine CRC model for the preparation of BMAA-based self-assembling nanoparticles (SAN). Single-cell sequencing was performed to analyze immune responses induced by SAN vaccination combined with a toll-like receptor 7 agonist (TLRa) adjuvant and BMAA treatment. Results: SAN-TLRa vaccination with BMAA treatment induced an anti-tumor immune microenvironment. This combination stimulated the generation of specific CD8+ T cells and IgG targeting BMAA misincorporated neoepitopes, ultimately promoting immune activation, tumor suppression, and prolonged survival in the CRC murine model. Additionally, BMAA combined with SAN vaccine significantly enhanced the efficacy of the immune checkpoint inhibitor anti-PD-1 antibody. Conclusion: Our findings provide a promising strategy for artificially introducing neoantigens using BMAA, which can break immune tolerance without disrupting systemic immune balance. This approach offers novel avenues for CRC immunotherapy.