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Identification of internalizing ScFvs for EGFR inhibition and apoptosis induction in cholangiocarcinoma cells
Apinya Sayinta1,2, Jinchutha Duangdara1, Thitima Sumphanapai3,4
1Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, Thailand.
Abstract:
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.
Insights
Novel antibodies targeting EGFR tyrosine kinase (EGFR-TK) show promise for treating cholangiocarcinoma (CCA). Two antibodies, G8 and H2, effectively inhibit EGFR-TK activity, reduce cancer cell viability, and induce apoptosis, offering new therapeutic avenues for this aggressive cancer.
Area of Science:
- Oncology
- Molecular Biology
- Immunology
Background:
- Cholangiocarcinoma (CCA) is an aggressive cancer with poor prognosis.
- Epidermal Growth Factor Receptor (EGFR) overexpression correlates with CCA recurrence and poor outcomes.
- Existing EGFR-targeted therapies have limited efficacy in CCA treatment.
Purpose of the Study:
- To identify novel therapeutic antibody candidates for cholangiocarcinoma (CCA).
- To investigate the efficacy of novel single-chain variable fragment (scFv) antibodies against EGFR in CCA.
- To explore the mechanism of action of these scFvs in inhibiting EGFR activity and impacting CCA cell growth.
Main Methods:
- Subtractive bio-panning of a human scFv phage display library against CCA cell lysates.
- Surface plasmon resonance (SPR) and kinase inhibition assays to assess antibody binding and EGFR-TK activity.
- Cellular assays including internalization, viability, apoptosis, and cell cycle analysis.
- Molecular docking to predict antibody-target interactions within EGFR-TK.
Main Results:
- Three novel scFvs (E1, G8, H2) were selected for preferential binding to CCA cells.
- G8 and H2 demonstrated high-affinity binding to EGFR tyrosine kinase (EGFR-TK) and suppressed its activity.
- G8 and H2 reduced EGFR phosphorylation, decreased cell viability, induced apoptosis, and caused S/G₂-M phase arrest in CCA cells.
- E1 inhibited proliferation via G₁ arrest without affecting EGFR phosphorylation.
- Molecular docking suggested G8 and H2 interact with critical sites in EGFR-TK, potentially inhibiting its function.
Conclusions:
- The novel scFvs G8 and H2 act as intracellular inhibitors of EGFR-TK activity.
- These antibodies show significant anti-cancer effects in EGFR-overexpressing CCA cells.
- G8 and H2 represent promising candidates for developing antibody-based therapies for EGFR-driven cholangiocarcinoma.
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