Kazemzadeh Hamid, Bagheri Mahsima, Sepehri Maryam, Ebrahimi Elham, Wang Huan, Haider Shozeb, Kheirabadi Mitra, Tohidkia Mohammad Reza
Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz 51368, Iran.
Basic Science Department, Faculty of Biology, Hakim Sabzevari University, P.O. Box 96179-76487, Sabzevar 571, Iran.
ACS Omega. 2024 May 6;9(20):21964-21973. doi: 10.1021/acsomega.3c10158. eCollection 2024 May 21.
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.
血管生成作为肿瘤的一个标志，在肿瘤血管系统的生长和发育中起着重要作用。大量证据表明，血管内皮生长因子受体（VEGFR-2）/血管内皮生长因子A（VEGF-A）轴是肿瘤血管生成和转移的主要促成因素之一。因此，抗VEGFR-2单克隆抗体抑制VEGFR-2信号通路可延缓肿瘤生长。在本研究中，我们采用噬菌体展示技术和溶液相生物淘选（SPB）来分离针对VEGFR-2的特异性单链可变片段（scFv），并报告候选scFv的受体结合特性。利用一个半合成噬菌体抗体文库，通过用浓度递减的VEGFR-2-组氨酸标签和VEGFR-2-生物素进行SPB来分离抗VEGFR-2 scFv。成功表达和纯化后，通过酶联免疫吸附测定（ELISA）、流式细胞术和免疫印迹进一步分析所选scFv克隆的特异性。进行竞争试验以鉴定scFv的VEGFR-2受体阻断特性。此外，通过肽-蛋白质对接广泛研究了候选scFv的分子结合特性。四轮生物淘选后的多克隆ELISA分析显示，通过从第一轮选择到第四轮选择增加阳性信号，VEGFR-2特异性噬菌体克隆有显著富集。通过单克隆噬菌体ELISA鉴定了各个与VEGFR-2反应的scFv噬菌体克隆。序列分析和互补决定区比对确定了四个独特的抗VEGFR-2-scFv克隆。在ELISA和流式细胞术试验中，可溶性和纯化的scFv对可溶性和细胞相关形式的VEGFR-2蛋白显示出结合活性。基于分子对接结果的推断，scFv D3、E1、H1和E9识别VEGFR-2蛋白上的结构域2和3，并与VEGF-A竞争结合VEGFR-2。竞争试验证实scFv H1和D3可阻断VEGFR-2/VEGF-A相互作用。总之，我们鉴定出了新型的VEGFR-2阻断scFv，其可能在VEGFR-2过表达的肿瘤细胞中具有抑制血管生成的潜力。
