A circRNA neoantigen vaccine elicits potent antitumor immunity and synergizes with checkpoint blockade in melanoma

Cancer immunotherapy harnesses and amplifies the host’s anti-tumor immune responses to eradicate malignant cells. Tumor neoantigens serve as ideal targets due to high specificity and immunogenicity. Circular RNA (circRNA), with a stable covalently closed structure, can be engineered as a novel vector for neoantigen expression. Here, we developed a circRNA-based vaccine encoding multiple melanoma neoantigens (circRNA MNA ). circRNA MNA significantly suppressed tumor growth in both prophylactic and therapeutic murine models and induced robust anti-tumor immunity. Mechanistically, using single-cell RNA sequencing, we elucidated the antitumor mechanisms and alterations in the tumor and immune microenvironment following vaccination. circRNA MNA immunization nearly eliminated the Ccl6 + Mel subcluster—highly active in chemokine signaling, leukocyte recruitment, and antigen presentation pathways. While residual Cdk1 + Mel and Cdhr4 + Mel subclusters exhibited downregulation of immune-response-related pathways. Furthermore, circRNA MNA vaccination reshaped myeloid cell composition, markedly increasing infiltration of monocytes/macrophages (Monocyte/MФ) with antitumor phenotypes, including classical inflammatory monocytes (Mono-Vcan), Macro-C1qc, and activated M1-like macrophages (Macro-Ifng). Chemokine signaling was enriched in Ccl6 + Mel and Monocyte/MФ clusters. Moreover, circRNA MNA enhanced T cell-mediated antitumor responses, elevating T cell receptor (TCR) clonotype diversity and expansion of TCR clonotypes especially in CD4 + effector (CD4 Eff) and CD8 + cytotoxic (CD8 Cyto) T cells. Notably, Tigit was identified as the most highly expressed checkpoint receptor in CD8 + exhausted T cells (CD8 Tex), and combining circRNA MNA with anti-TIGIT/PD1 blockade produced synergistic antitumor effects. Collectively, this study systematically elucidates the therapeutic potential and immune mechanisms of the neoantigen circRNA vaccine in melanoma, providing a mechanistic framework for developing personalized RNA vaccines and rational combination immunotherapies. • circRNA MNA suppresses tumor growth in prophylactic and therapeutic murine models. • circRNA MNA reprograms monocyte/MФ functions to promote melanoma cell elimination. • circRNA MNA vaccination boosts T and NK cell activity to enhance antitumor immunity. • circRNA MNA vaccine synergizes with ICB to cooperatively inhibit tumor growth in vivo.