Kassab, M. M. S.

Background: Breast cancer remains a significant therapeutic challenge due to the heterogeneity of tumor antigens and the presence of immunologically cold tumor microenvironments (TME) that resist conventional immunotherapy. mRNA vaccines offer a versatile platform for multiepitope targeting, but their clinical utility is often limited by inherent instability and poor cellular internalization. Objective: To design, characterize, and evaluate an R8-stabilized multi-epitope mRNA vaccine targeting HER2, MUC1, and Survivin for the treatment of aggressive breast cancer. Methods: A multiepitope mRNA construct (R8-CTL1,7-HTL1,2) was designed and synthesized via in vitro transcription (IVT). The mRNA was complexed with an octaarginine (R8) domain at an N/P ratio of 4:1 to form stable nanoparticles. Characterization included Minimum Free Energy (MFE) modeling and Dynamic Light Scattering (DLS). In vitro uptake and antigen expression were quantified in breast cancer cell lines. In vivo efficacy was assessed in female BALB/c mice (n=6) challenged with 4T1 cells, focusing on tumor growth inhibition, CD8+ T cell cytotoxicity, and intratumoral T cell infiltration (counts/mm2) over a 28-day period. Results: The mRNA construct exhibited high structural stability (MFE = -450 kcal/mol) and formed uniform nanoparticles (mean diameter ~92 nm). R8-complexation significantly enhanced cellular uptake to 88%, resulting in robust relative expression of HER2 and MUC1. In vivo results demonstrated potent systemic immunity with a marked increase in CD8+ T cell cytotoxicity (p<0.05). Most notably, vaccinated mice showed a 65% increase in intratumoral T cell recruitment (from 1.4 to 2.3 counts/mm), correlating with significant tumor growth suppression compared to the control group by Day 28. Conclusion: The R8 stabilized mRNA platform effectively overcomes the delivery barriers and warms up the immunosuppressive tumor microenvironment. By inducing high-density T cell infiltration and systemic cytotoxicity, this multiepitope approach provides a promising therapeutic strategy for converting cold breast tumors into immunologically active, treatable targets.