{"database":"biostudies-literature","file_versions":[],"scores":null,"additional":{"submitter":["Kazemzadeh H"],"funding":["Research Center for Pharmaceutical Nanotechnology","Iran National Science Foundation"],"pagination":["21964-21973"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC11112697"],"repository":["biostudies-literature"],"omics_type":["Unknown"],"volume":["9(20)"],"pubmed_abstract":["Angiogenesis, as a tumor hallmark, plays an important role in the growth and development of the tumor vasculature system. There is a huge amount of evidence suggesting that the vascular endothelial growth factor receptor (VEGFR-2)/VEGF-A axis is one of the main contributors to tumor angiogenesis and metastasis. Thus, inhibition of the VEGFR-2 signaling pathway by anti-VEGFR-2 mAb can retard tumor growth. In this study, we employ phage display technology and solution-phase biopanning (SPB) to isolate specific single-chain variable fragments (scFvs) against VEGFR-2 and report on the receptor binding characteristics of the candidate scFvs A semisynthetic phage antibody library to isolate anti-VEGFR-2 scFvs through an SPB performed with decreasing concentrations of the VEGFR-2-His tag and VEGFR-2-biotin. After successful expression and purification, the specificity of the selected scFv clones was further analyzed by enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunoblotting. The competition assay was undertaken to identify the VEGFR-2 receptor-blocking properties of the scFvs. Furthermore, the molecular binding characteristics of candidate scFvs were extensively studied by peptide-protein docking. Polyclonal ELISA analysis subsequent to four rounds of biopanning showed a significant enrichment of VEGFR-2-specific phage clones by increasing positive signals from the first round toward the fourth round of selection. The individual VEGFR-2-reactive scFv phage clones were identified by monoclonal phage ELISA. The sequence analysis and complementarity-determining region alignment identified the four unique anti-VEGFR-2-scFv clones. The soluble and purified scFvs displayed binding activity against soluble and cell-associated forms of VEGFR-2 protein in the ELISA and flow cytometry assays. Based on the inference from the molecular docking results, scFvs D3, E1, H1, and E9 recognized domains 2 and 3 on the VEGFR-2 protein and displayed competition with VEGF-A for binding to VEGFR-2. The competition assay confirmed that scFvs H1 and D3 can block the VEGFR-2/VEGF-A interaction. In conclusion, we identified novel VEGFR-2-blocking scFvs that perhaps exhibit the potential for angiogenesis inhibition in VEGFR-2-overexpressed tumor cells."],"journal":["ACS omega"],"pubmed_title":["Isolation and Characterization of the Vascular Endothelial Growth Factor Receptor Targeting ScFv Antibody Fragments Derived from Phage Display Technology."],"pmcid":["PMC11112697"],"funding_grant_id":["96001856","65956"],"pubmed_authors":["Kheirabadi M","Haider S","Wang H","Bagheri M","Sepehri M","Kazemzadeh H","Ebrahimi E","Tohidkia MR"],"additional_accession":[]},"is_claimable":false,"name":"Isolation and Characterization of the Vascular Endothelial Growth Factor Receptor Targeting ScFv Antibody Fragments Derived from Phage Display Technology.","description":"Angiogenesis, as a tumor hallmark, plays an important role in the growth and development of the tumor vasculature system. There is a huge amount of evidence suggesting that the vascular endothelial growth factor receptor (VEGFR-2)/VEGF-A axis is one of the main contributors to tumor angiogenesis and metastasis. Thus, inhibition of the VEGFR-2 signaling pathway by anti-VEGFR-2 mAb can retard tumor growth. In this study, we employ phage display technology and solution-phase biopanning (SPB) to isolate specific single-chain variable fragments (scFvs) against VEGFR-2 and report on the receptor binding characteristics of the candidate scFvs A semisynthetic phage antibody library to isolate anti-VEGFR-2 scFvs through an SPB performed with decreasing concentrations of the VEGFR-2-His tag and VEGFR-2-biotin. After successful expression and purification, the specificity of the selected scFv clones was further analyzed by enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunoblotting. The competition assay was undertaken to identify the VEGFR-2 receptor-blocking properties of the scFvs. Furthermore, the molecular binding characteristics of candidate scFvs were extensively studied by peptide-protein docking. Polyclonal ELISA analysis subsequent to four rounds of biopanning showed a significant enrichment of VEGFR-2-specific phage clones by increasing positive signals from the first round toward the fourth round of selection. The individual VEGFR-2-reactive scFv phage clones were identified by monoclonal phage ELISA. The sequence analysis and complementarity-determining region alignment identified the four unique anti-VEGFR-2-scFv clones. The soluble and purified scFvs displayed binding activity against soluble and cell-associated forms of VEGFR-2 protein in the ELISA and flow cytometry assays. Based on the inference from the molecular docking results, scFvs D3, E1, H1, and E9 recognized domains 2 and 3 on the VEGFR-2 protein and displayed competition with VEGF-A for binding to VEGFR-2. The competition assay confirmed that scFvs H1 and D3 can block the VEGFR-2/VEGF-A interaction. In conclusion, we identified novel VEGFR-2-blocking scFvs that perhaps exhibit the potential for angiogenesis inhibition in VEGFR-2-overexpressed tumor cells.","dates":{"release":"2024-01-01T00:00:00Z","publication":"2024 May","modification":"2026-04-08T19:51:15.795Z","creation":"2026-04-08T14:33:45.361Z"},"accession":"S-EPMC11112697","cross_references":{"pubmed":["38799304"],"doi":["10.1021/acsomega.3c10158"]}}