Development of a Multi-Neoepitope Vaccine Targeting Non-Small Cell Lung Cancer through Reverse Vaccinology and Bioinformatics Approaches

Elahe Asadollahi¹Alireza Zomorodipour^2*Zahra-Soheila Soheili²Babak Jahangiri²Majid Sadeghizadeh^1*

• ¹Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
• ²Department of Molecular Medicine, Institute of Medical Biotechnology, Institute of Genetic Engineering and Biotechnology, Tehran, Alborz, Iran

Lung cancer, predominantly non-small cell lung cancer (NSCLC), is the leading cause of cancer-related mortality worldwide. Among immunotherapeutic strategies, the personalized multi-neoepitope vaccine (MNEV) is a promising approach for managing advanced-stage NSCLC. We used reverse vaccinology, immunoinformatics, and bioinformatics to design an MNEV targeting lung cancer in mouse (LL/2) cells. Whole exome sequencing (WES) and RNA sequencing data human and mouse NSCLC were analyzed to select neoantigens, evaluated for their ability to stimulate B cells, helper T lymphocytes (HTLs), and cytotoxic T lymphocytes (CTLs). Molecular docking studies estimated the binding affinity of mouse neoepitopes with MHC class I, MHC class II, and B-cell receptors. Suitable linkers were selected to construct the MNEV, with the 50S L7/L12 ribosomal protein sequence added as an adjuvant to enhance immune responses. The Igκ chain signal peptide was incorporated to improve secretion efficiency. The stability of the final construction of vaccine/TLR3,4, and 9 complex was confirmed through binding analysis and adjustment of the best-predicted 3D model. To evaluate the immunological efficacy of the MNEV, female C57BL/6 mice were immunized subcutaneously. The assessment involved measuring total IgG levels in serum using enzymelinked immunosorbent assay (ELISA) and quantifying IFN-γ and granzyme B levels in the supernatant of cultured splenocytes. The proportions of CD19+ B cells and CD4+ and CD8+ T cells were assessed using flow cytometric analysis. In silico evaluations indicated that the MNEV is non-toxic, non-allergenic, and stable, exhibiting high-affinity interactions with B lymphocytes, CTLs, and HTLs. Immunization with the designed MNEV led to a significant increase in serum IgG levels. Flow cytometry analysis revealed elevated percentages of CD19+ B cells and CD4+ and CD8+ T cells. Furthermore, splenocytes from immunized mice showed a marked increase in IFN-γ and granzyme B secretion compared to control groups. This study demonstrates that the multi-neoepitope vaccine (MNEV) can induce a strong immune response, highlighting its potential as a promising approach for cancer prevention and immunotherapy, particularly for lung cancer. Furthermore, it provides a foundation for developing neoepitopebased vaccines against various malignancies, guiding future research in cancer vaccine development through advanced computational methods in immunology and oncology.