Targeting NRAS-Derived Public Neoantigens in T Cell-Based Cancer Immunotherapy 2261145

Abstract Introduction T cells mediate anti-cancer immune responses through recognition of neoantigens - cancer-specific, mutation-derived peptides presented by MHC proteins. “Public” neoantigens are a subset of neoantigens derived from recurrently mutated driver genes and presented by common HLA alleles, making them ideal immunotherapy targets. NRAS is the second most frequently mutated RAS isoform, and its common hotspot mutations (Q61R, Q61K, and Q61L) give rise to a family of HLA-A*01:01 (A1)-restricted public neoantigens. Methods This study aims to elucidate how the NRAS Q61 neoantigens are structurally and biophysically distinct from their wild-type (WT) counterpart and to resolve the mechanisms that enable a panel of patient-derived TCRs to achieve neoantigen specificity. Results Differential scanning fluorimetry shows that each neoantigen and the WT peptide bind A1 with comparable affinity. X-ray crystal structures of the neoantigen/A1 and WT/A1 complexes reveal that the p7Q side chain of the WT peptide is “tethered” to the α2 helix of A1 via a network of hydrogen bonds. This network is disrupted with all neoantigens, increasing the solvent accessibility of the mutated p7 side chains and facilitating potential TCR interactions. In surface plasmon resonance (SPR) binding experiments, one TCR (TCR11L) binds all three neoantigens (Q61R, Q61K, Q61L) and the WT peptide with high affinity. Crystal structures reveal that TCR11L primarily contacts the penultimate p9E of the peptides, bypassing the p7 mutation site. However, T cell-based functional assays show that TCR11L responds only to the Q61K and Q61L neoantigens. Intriguingly, SPR kinetic analyses reveal significantly slower dissociation rates for TCR11L complexed with Q61K and Q61L compared to Q61R and WT, suggesting that the functional specificity of TCR11L is kinetically driven. Conclusion Collectively, these biophysical, structural, and functional studies reveal how TCR promiscuity can be exploited to enhance scalability in neoantigen-targeting therapies. Funding Source NIH R01CA286507 Topic Categories Vaccines and Immunotherapy (VAC)