Discovery of Immunogenic Neopeptides from Chimeric RNAs to Develop Peptide and mRNA Vaccines for Lung Cancer Treatment

Non–small cell lung cancer (NSCLC) accounts for nearly 80% of lung cancer cases and exhibits a median survival of less than one year following diagnosis. A relatively unexplored avenue of immunotherapy for specific cancers may lie in chimeric RNAs that separate cancerous cells from their non-cancerous neighbors. RNA from fusion genes resulting from deletions, duplications, inversions, translocations, or aberrant splicing events, can be used as vaccine targets due to the neoantigenic peptide junctions formed in the translated proteins. RNA sequencing data from Lung Adenocarcinoma and Squamous Cell Carcinoma patients was analyzed for chimeric RNA content. The KIF5B [exon 1-15] -RET [exon 12-19] fusion was found and chosen as the primary candidate fusion for this study based on previous studies that established it to be an actionable cancer driver. The affinity of the neopeptide sequences formed by the translated junction of KIF5B-RET to Major Histocompatibility Complex (MHC) Class I molecules was assessed using the in-silico prediction pipelines MHCNuggets and MixMHCPred 2. HLA-C07:02 was found to be the highest affinity binder to 9-mer peptides within the KIF5B-RET junction region. I then investigated binding affinity from a structural approach using HLA-Arena to dock peptides and calculate binding energies, revealing NNDVKEDPK as the strongest binder. Furthermore, peptides predicted as strong binders tended to have regions with positive electrostatic potentials oriented toward the binding pocket of HLA-C07:02. The immunogenicity of these neoantigenic peptides to HLA-C*07:02 matched Peripheral Blood Mononuclear Cells (PBMCs) was assessed by the IFN-γ Enzyme Linked Immunosorbent Spot (ELISpot) assay. Effects across donors were identified, as PBMC-isolated CD8+ T cells from donor 1 and 2 showed the highest responses to KEDPKWEFP and a pool of all junction peptides, respectively. The Single-cell 5’ gene expression and T Cell Receptor (TCR) mapping assay then identified a subset of TCR sequences responsive to peptide stimulation, along with critical positions and glycine residues in the CDR3-α and CDR3-β chains that may be responsible for this heightened immune response. This study establishes a robust pipeline to discover chimeric RNAs from RNA-sequencing performed on tumor samples, extract and validate immunogenic neoantigens from fusion junctions, and design vaccines for the treatment of cancer.