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

Ahn, S.; Oh, T.-S.; Suh, S.; Jeon, J.-y.; Lah, S.; Ryu, K.; Kim, H.; Lee, E. G.; Lee, H.; Lee, J.; Kim, D.-K.; Ku, B. M.; Jung, W.; Ahn, M.-J.; Jung, J. U.; Kim, Y.-S.; Oh, B.-H.; Jeong, B.-S.

doi:10.1101/2025.08.31.673313

bioRxiv · 10.1101/2025.08.31.673313

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

Ahn, S.·Oh, T.-S.·Suh, S.·Jeon, J.-y.·Lah, S.·Ryu, K.·Kim, H.·Lee, E. G.·Lee, H.·Lee, J.·Kim, D.-K.·Ku, B. M.·Jung, W.·Ahn, M.-J.·Jung, J. U.·Kim, Y.-S.·Oh, B.-H.·Jeong, B.-S.

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Abstract

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.

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Ahn, S., Oh, T.-S., Suh, S., Jeon, J.-y., Lah, S., Ryu, K., Kim, H., Lee, E. G., Lee, H., Lee, J., Kim, D.-K., Ku, B. M., Jung, W., Ahn, M.-J., Jung, J. U., Kim, Y.-S., Oh, B.-H., Jeong, B.-S.. 2025-09-04. An integrated in silico-in vitro workflow for discovering high-affinity, selective antibodies to the KRAS(G12D)-MHC I complex. https://doi.org/10.1101/2025.08.31.673313

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