Superior in-vivo efficacy of integrated tumor neoantigen vaccination with adoptive neoantigen-reactive T-cell therapy in solid tumor.

OBJECTIVE: Neoantigens from tumor mutations are key cancer immunotherapy targets. This study evaluated the efficacy of combined immunotherapy (neoantigen vaccines + adoptive neoantigen-reactive T cells, NRT) in solid tumors to lay a foundation for clinical trials. METHODS: Candidate neoantigens were predicted via whole-exome/transcriptome sequencing of MC38, B16-MO4, and Hepa1-6 tumors, validated by ELISPOT. NRT preparation was optimized; peptide vaccine+NRT efficacy was tested in tumor-bearing mice. Neo-DC vaccines (mRNA-electroporated DCs) were evaluated prophylactically, and mRNA-DC+NRT+anti-PD1 efficacy was assessed by cytokine, tumor growth, and TME changes. RESULTS: A total of 4, 5, and 8 neoantigen epitopes were identified in MC38, B16-MO4, and Hepa1-6 tumor cells, respectively. T lymphocytes from neoantigen vaccine-immunized hosts promoted NRT induction and expansion, yielding NRT cells with higher neoantigen-specific T cell proportions and stronger tumor-killing capacity. Their tumor neoantigen-specific cytotoxicity was verified by Real-time cell killing assay (RTCA) and flow cytometry. Combined neoantigen peptide vaccine and NRT therapy inhibited tumor growth and altered the TME to favor CD8+T cell infiltration. mRNA-DC vaccines showed prophylactic efficacy and lymph node homing. Multi-combination immunotherapy in Hepa1-6 mice induced partial tumor regression, with reduced TME expression of Matrix Metalloproteinase 9 (MMP9), Ki67, and TGF-β1, increased TNF-α, elevated M1/M2 ratio, and enhanced CD8+T cell infiltration. CONCLUSION: Neoantigen vaccines combined with NRT cells and anti-PD1 therapy exhibit robust antitumor efficacy in solid tumors, serving as a safe and effective novel immunotherapeutic approach.