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<p>Cholangiocarcinoma (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.</p>

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Identification of internalizing ScFvs for EGFR inhibition and apoptosis induction in cholangiocarcinoma cells

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年11月19日

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  • Apinya Sayinta,
  • Jinchutha Duangdara,
  • Thitima Sumphanapai,
  • Kuntalee Rangnoi,
  • Boonyakorn Boonsri,
  • Kittiya Supradit,
  • Nopporn Jongkamonwiwat,
  • Kiattawee Choowongkomon,
  • Veerachai Thitapakorn,
  • Montarop Yamabhai,
  • Kanokpan Wongprasert

摘要

Cholangiocarcinoma (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.