Dual-degenerate TCRs target multiple KRAS hotspot and HLA-A3 family combinations.

Oncogenic KRAS mutations drive a substantial proportion of lung cancers and are linked to poor prognosis, positioning KRAS as a compelling target for cellular immunotherapy. Thirty-five percent of lung adenocarcinomas harbor the KRAS G12C, G12V, G12D mutations. Here, we developed HLA-A*03:01- and HLA-A*11:01-restricted T cell receptors (TCR) targeting the most prevalent G12C and G12V KRAS hotspot mutations in lung adenocarcinoma. Predicted high affinity peptides were screened using our TCR discovery and validation pipeline, and functional assessment was performed to determine sensitivity, specificity, and cytotoxic potential of TCR-engineered T cells. We discovered and validated 5 novel TCRs targeting KRAS G12C and G12V 9-mers, each of which demonstrated an ability to recognize and lyse tumor cells endogenously presenting mutant KRAS on HLA-A*03:01 or HLA-A*11:01. Notably, several TCRs demonstrated distinct modes of cross-reactivity, including peptide degeneracy across KRAS G12 variants, HLA degeneracy across HLA-A*03:01 and HLA-A*11:01, or dual degeneracy across both KRAS and HLA, thereby broadening the treatable target populations. TCRs that recognize the KRAS hotspot shared sequence motifs were found in several lung cancer patients. Our study highlights the successful generation of multi-valent KRAS-specific TCRs and supports the feasibility of targeting shared KRAS neoantigens through TCR engineering in lung cancer.