Discovery of Immunogenic Neo-peptides from Actionable RNA Fusion CD74-NRG1 for Developing Cancer Vaccines
This study investigates the CD74-NRG1 fusion protein as a tumor-specific neoantigen source and presents a comprehensive pipeline for identifying and validating immunogenic neoantigens from chimeric RNAs, with promising implications for personalized cancer vaccines. The CD74-NRG1 fusion, resulting from a translocation between chromosomes 5 and 8, creates a unique fusion junction that disrupts cell signaling and drives tumorigenesis. This novel antigenic sequence, absent in healthy cells, represents an attractive target for tumor-specific immunotherapy with minimal side effects. To evaluate the neoantigenic potential of CD74-NRG1, 36 peptides (9, 10, and 11 amino acids) derived from the fusion junction and wild-type sequences were tested for HLA binding using in-silico tools (NetMHCpan4.1, MixMHCpred3.0, BigMHC) and an in-vitro flow-cytometry based biochemical HLA assay. NetMHCpan4.1 showed a 88% overlap with the 10 neopeptides with strong binding to HLA-B*07:02 established through the flow-cytometry based assay and was therefore found to have the best predictive accuracy of these computational methods. Further immunogenicity testing of the 36 peptides using an ELISpot assay revealed that 7 of 10 validated binders to HLA-B*07:02 effectively activated CD8 T cells, indicating their potential to elicit robust immune responses. The dCODE Dextramer assay was used to identify specific immune cell states induced by these peptides, including naïve, early activated, early exhausted, and effector memory CD8 T cells. Clonal analysis found 21 distinct CD8 T cell clones expanded by 6 of the top binding neopeptides which were validated to be immunogenic through the ELISpot assay. Conserved amino acid residues in the CDR3 sequences of these TCRs supported their structural stability and specificity. Additionally, CrossDome bioinformatics analysis evaluated off-target effects, identifying APPKATSTS as having the highest off-target potential with 13 self-antigen matches. Overall, these findings underscore the CD74-NRG1 fusion as a promising neoantigen for TCR-based immunotherapy. Six fusion-derived peptides, APPKATSTS, PPKATSTST, QKPTDAPPKA, KPTDAPPKATS, DAPPKATSTST, and APPKATSTSTT emerged as potential vaccine candidates, though further validation is recommended to assess off-target risks.
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