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.

中文翻译：

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噬菌体展示技术衍生的针对 ScFv 抗体片段的血管内皮生长因子受体的分离和表征

血管生成作为肿瘤的标志，在肿瘤血管系统的生长和发育中发挥着重要作用。大量证据表明血管内皮生长因子受体（VEGFR-2）/VEGF-A轴是肿瘤血管生成和转移的主要贡献者之一。因此，抗VEGFR-2 mAb抑制VEGFR-2信号通路可以延缓肿瘤生长。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的受体结合特征。通过使用降低浓度的 VEGFR-2-His 标签和 VEGFR-2-生物素进行的 SPB 进行抗 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。进行竞争测定来鉴定 scFv 的 VEGFR-2 受体阻断特性。此外，候选 scFv 的分子结合特征通过肽-蛋白质对接进行了广泛研究。四轮生物淘选后的多克隆 ELISA 分析显示，通过从第一轮到第四轮选择增加阳性信号，VEGFR-2 特异性噬菌体克隆显着富集。通过单克隆噬菌体 ELISA 鉴定各个 VEGFR-2 反应性 scFv 噬菌体克隆。序列分析和互补决定区比对鉴定出四个独特的抗 VEGFR-2-scFv 克隆。在 ELISA 和流式细胞术测定中，可溶性和纯化的 scFv 显示出针对可溶性和细胞相关形式的 VEGFR-2 蛋白的结合活性。根据分子对接结果推断，scFvs 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。竞争测定证实 scFvs H1 和 D3 可以阻断 VEGFR-2/VEGF-A 相互作用。总之，我们鉴定了新型 VEGFR-2 阻断性 scFv，它们可能在 VEGFR-2 过表达的肿瘤细胞中表现出抑制血管生成的潜力。