An integrated in silico-in vitro workflow for discovering high-affinity, selective antibodies to the KRAS(G12D)-MHC I complex

Antibodies that recognize peptide–loaded class I major histocompatibility complex (pMHC I) molecules could enable therapeutic targeting of intracellular oncogenic proteins, yet their discovery has been hampered by the small size of peptide antigens and allele-specificity. We describe an integrated in silico–in vitro workflow for generating high-affinity, selective antibodies to KRAS(G12D) ₁₀ presented by HLA-C*08:02, a clinically validated cancer neoantigen. In silico, multiple human antibody-derived variable fragments (Fvs) plausibly docked to the target pMHC were generated, followed by limited complementarity-determining region (CDR) sequence design. In vitro, CDR diversity was introduced at 3–4 positions per Fv to construct yeast surface display library for iterative selections. This workflow yielded antibodies with exclusive binding to KRAS(G12D) ₁₀ /HLA-C*08:02 without cross-reactivity. Affinity maturation achieved nanomolar dissociation constants, and incorporation into chimeric antigen receptor T cells enabled specific activation against target-positive cells. This study establishes a practical design-to-function pipeline for TCR-like antibody discovery, and demonstrates the feasibility of therapeutic targeting against KRAS(G12D)-driven malignancies.