Robust CD8(+) T cell responses induced by an mRNA-LNP vaccine encoding rat HER2 extracellular domain confer prophylactic tumor protection.

BACKGROUND: HER2-positive cancers present challenges of drug resistance, toxicity, and immune tolerance. HER2-targeted peptide vaccines have failed clinically, while nucleoside-modified mRNA-lipid nanoparticle (mRNA-LNP) vaccines show promise for robust antitumor immunity. This study utilized heterologous immunity (rat HER2 extracellular domain, rHER2 ECD) to circumvent HER2 tolerance, developing a safe mRNA-LNP vaccine and exploring its synergy with anti-PD-1 therapy. METHODS: We synthesized N1-methyl-pseudouridine (m1ψ)-modified mRNAs, encoding either rHER2 ECD alone (rHER2 ECD mRNA-LNP) or rHER2 ECD fused to murine IFNγ (rHER2 ECD-IFNγ mRNA-LNP), and then encapsulated them in LNPs via microfluidic mixing. BALB/c mice were immunized on days 1, 15, 29, then challenged with HER2-overexpressing 4T1 (4T1-HER2) cells 2 weeks post-final immunization. Efficacy was assessed by tumor growth, immune responses via flow cytometry, ELISA, ELISpot and cytotoxicity assays, safety via body/organ weight and serum markers. RESULTS: HER2 ECD mRNA-LNPs exhibited >80% encapsulation efficiency and narrow particle size distribution. As monotherapy, rHER2 ECD mRNA-LNPs induced high anti-HER2 antibody titers, polyfunctional CD8(+) T cells (IFNγ(+)/TNFα(+)), and durable memory T cells, achieving 87.0% tumor inhibition. This inhibition rate was significantly higher than that of the rHER2 ECD-IFNγ mRNA-LNP variant, which achieved 21.9% tumor inhibition and induced anti-IFNγ neutralizing antibodies. Combination with anti-PD-1 monoclonal antibody (mAb) further enhanced tumor inhibition, with 2/6 tumors failing to establish. This synergy was driven by tissue-resident memory T (T(RM)) cell enrichment in the tumor microenvironment (TME), which reached 15.2% in CD4(+) T cells and 12.9% in CD8(+) T cells. No systemic toxicity was observed. CONCLUSION: The rHER2 ECD mRNA-LNP effectively circumvents HER2-specific immune tolerance. As a potent and safe platform, it induces robust coordinated humoral and cellular immunity and significantly enhances checkpoint blockade efficacy via the enrichment of T(RM) cells in the TME, which underscores its promising potential for the treatment of HER2-positive cancers.