Bioinformatics, Molecular Docking analysis and Cell Culture Testing of Mutated p53 and Overexpressed CEA in Lung Cancer for mRNA Vaccine Development

Background: Lung cancer, particularly non-small cell lung cancer (NSCLC), remains the leading cause of cancer-related deaths globally. Mutations in TP53 and overexpression of carcinoembryonic antigen (CEA) are common in NSCLC, providing attractive targets for immunotherapy. Recent advances in messenger RNA (mRNA) vaccine technology offered new opportunities to elicit targeted immune responses against tumor-specific antigens. Aim of the study: To design novel therapeutic mRNA vaccine containing mRNA of mutated p53 and overexpressed CEA versus NSCLC using bioinformatics and molecular docking. Methods: A comprehensive immunoinformatics pipeline was employed to identify cytotoxic (CTL) and helper T lymphocyte (HTL) epitopes from the mutant p53 (R248Q) and overexpressed CEA proteins. Antigenicity, allergenicity, toxicity, and population coverage of predicted epitopes were assessed. High-affinity epitopes were validated via molecular docking against HLA-A02:01 and HLA-DRB101:01 alleles. A multi-epitope mRNA vaccine construct was designed, codon-optimized, and structurally modeled. Lipid nanoparticle (LNP) delivery parameters and immune simulations were conducted in silico. Results: Fourteen immunogenic, non-allergenic, and non-toxic T-cell epitopes were selected based on MHC binding affinity and global population coverage (˃ 85%). Molecular docking analysis confirmed stable epitope-HLA interactions with binding energies ranging from -9.2 kcal/mol. The mRNA vaccine construct demonstrated high codon adaptation index (0.92), ideal GC content (55.3%), and stable secondary structure. In silico immune simulations predicted potent induction of IFN-ɣ and IL-2, effective clonal expansion of T cells, and memory formation. LNP modeling confirmed efficient encapsulation and delivery characteristics. During the cell culturing testing of the the test vaccine, the protection power of the test immunizing agent was detected to be 71± 2.04%. Tumor size was reduced by approximately 62± 0.83%. Metastasis was hindered by 80%. Conclusion: The present study presented a rationally designed LNP-mRNA vaccine targeting two key lung cancer antigens (mutantp53 and overexpressed CEA). The findings provided a promising framework for the development of therapeutic mRNA immunotherapies in NSCLC and warranted further experimental validation in preclinical and clinical trials.