Sayinta A.Duangdara J.Sumphanapai T.Rangnoi K.Boonsri B.Supradit K.Jongkamonwiwat N.Choowongkomon K.Thitapakorn V.Yamabhai M.Wongprasert K.Mahidol University2025-11-272025-11-272025-11-19Scientific Reports Vol.15 No.1 (2025) , 40648https://repository.li.mahidol.ac.th/handle/123456789/113256Cholangiocarcinoma (CCA) is an aggressive malignancy with limited treatment options. EGFR overexpression is associated with tumor recurrence and poor prognosis, yet current EGFR-targeted therapies show limited efficacy. To identify alternative therapeutic candidates, we performed subtractive bio-panning of a naïve human single-chain variable fragment (scFv) phage display library against CCA cell lysates. Three novel scFv antibodies-E1, G8, and H2-were selected based on preferential binding to CCA cells with minimal cross-reactivity to unrelated cancers and normal fibroblasts. Surface plasmon resonance and kinase inhibition assays demonstrated that G8 and H2 bound the EGFR tyrosine kinase (EGFR-TK) domain with nanomolar affinities and suppressed kinase activity, whereas E1 showed weak binding and no kinase inhibition. All three scFvs exhibited efficient internalization into EGFR-overexpressing HuCCA-1 cells. Functional analyses revealed distinct effects on cell growth: G8 and H2 reduced EGFR phosphorylation, decreased cell viability, and induced apoptosis accompanied by S/G₂-M phase accumulation, whereas E1 primarily inhibited proliferation through G₁-phase arrest without significantly affecting EGFR phosphorylation. Molecular docking predicted that G8 interacts near the ATP-binding pocket and H2 at the dimerization interface of EGFR-TK. These interactions may contribute to EGFR inhibition in HuCCA-1 cells. These findings suggest that G8 and H2 function as intracellular inhibitors of EGFR-TK activity and are promising candidates for antibody-based therapy in EGFR-driven CCA.MultidisciplinaryArticleSCOPUS10.1038/s41598-025-24324-w2-s2.0-1050222311642045232241257876