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.