Identification of internalizing ScFvs for EGFR inhibition and apoptosis induction in cholangiocarcinoma cells.
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| Název: | Identification of internalizing ScFvs for EGFR inhibition and apoptosis induction in cholangiocarcinoma cells. |
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| Autoři: | Sayinta A; Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, Thailand.; Division of Basic and Medical Sciences, Faculty of Allied Health Sciences, Pathumthani University, Pathum Thani, Thailand., Duangdara J; Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, Thailand., Sumphanapai T; Division of Clinical Hematology and Microscopy, Department of Medical Technology, School of Allied Health Sciences, University of Phayao, Phayao, Thailand.; Molecular Biotechnology Laboratory, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, Thailand., Rangnoi K; Molecular Biotechnology Laboratory, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, Thailand., Boonsri B; Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, Thailand.; Division of Health and Applied Sciences, Faculty of Science, Prince of Songkla University, Songkhla, Thailand., Supradit K; Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, Thailand.; Department of Radiological Technology, Faculty of Science, Ramkhamhaeng University, Bangkok, Thailand., Jongkamonwiwat N; Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, Thailand., Choowongkomon K; Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok, Thailand., Thitapakorn V; Chulabhorn International College of Medicine, Thammasat University, Pathum Thani, Thailand.; Research Unit in Opisthorchiasis, Cholangiocarcinoma, and Neglected Parasitic Diseases, Thammasat University, Pathum Thani, Thailand., Yamabhai M; Molecular Biotechnology Laboratory, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, Thailand., Wongprasert K; Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, Thailand. kanokpan.won@mahidol.ac.th. |
| Zdroj: | Scientific reports [Sci Rep] 2025 Nov 19; Vol. 15 (1), pp. 40648. Date of Electronic Publication: 2025 Nov 19. |
| Způsob vydávání: | Journal Article |
| Jazyk: | English |
| Informace o časopise: | Publisher: Nature Publishing Group Country of Publication: England NLM ID: 101563288 Publication Model: Electronic Cited Medium: Internet ISSN: 2045-2322 (Electronic) Linking ISSN: 20452322 NLM ISO Abbreviation: Sci Rep Subsets: MEDLINE |
| Imprint Name(s): | Original Publication: London : Nature Publishing Group, copyright 2011- |
| Výrazy ze slovníku MeSH: | ErbB Receptors*/antagonists & inhibitors , ErbB Receptors*/metabolism , ErbB Receptors*/chemistry , Cholangiocarcinoma*/pathology , Cholangiocarcinoma*/metabolism , Cholangiocarcinoma*/drug therapy , Apoptosis*/drug effects , Single-Chain Antibodies*/pharmacology , Single-Chain Antibodies*/metabolism , Single-Chain Antibodies*/chemistry , Single-Chain Antibodies*/immunology , Bile Duct Neoplasms*/pathology , Bile Duct Neoplasms*/metabolism, Humans ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Molecular Docking Simulation ; Phosphorylation/drug effects ; Protein Binding ; Peptide Library ; Protein Kinase Inhibitors/pharmacology |
| Abstrakt: | Competing Interests: Declarations. Competing interests: The authors declare no competing interests. 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. (© 2025. The Author(s).) |
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| Grant Information: | Grant No. N41D640036 National Research Council of Thailand (NRCT) under the Young Researcher Development Program 2021; Research Grant No. NDFR26/2564 Mahidol University |
| Contributed Indexing: | Keywords: Antibodies internalization; Apoptosis; Cholangiocarcinoma; EGFR tyrosine kinase; Single-chain variable fragment (scFv) |
| Substance Nomenclature: | EC 2.7.10.1 (ErbB Receptors) 0 (Single-Chain Antibodies) EC 2.7.10.1 (EGFR protein, human) 0 (Peptide Library) 0 (Protein Kinase Inhibitors) |
| Entry Date(s): | Date Created: 20251119 Date Completed: 20251119 Latest Revision: 20251122 |
| Update Code: | 20251122 |
| PubMed Central ID: | PMC12630736 |
| DOI: | 10.1038/s41598-025-24324-w |
| PMID: | 41257876 |
| Databáze: | MEDLINE |
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Nájsť tento článok vo Web of Science | Abstrakt: | Competing Interests: Declarations. Competing interests: The authors declare no competing interests.<br />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.<br /> (© 2025. The Author(s).) |
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| ISSN: | 2045-2322 |
| DOI: | 10.1038/s41598-025-24324-w |