Biotechnology Section, Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran.
School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran.
Breast Cancer Res Treat. 2018 Jun;169(3):427-436. doi: 10.1007/s10549-017-4641-6. Epub 2018 Feb 6.
FZD7 has a critical role as a surface receptor of Wnt/β-catenin signaling in cancer cells. Suppressing Wnt signaling through blocking FZD7 is shown to decrease cell viability, metastasis and invasion. Bioinformatic methods have been a powerful tool in epitope designing studies. Small size, high affinity and human origin of scFv antibodies have provided unique advantages for these recombinant antibodies.
Two epitopes from extracellular domain of FZD7 were designed using bioinformatic methods. Specific anti-FZD7 scFvs were selected against these epitopes through panning process. The specificity of the scFvs was assessed by phage ELISA and the ability to bind to FZD7 expressing cell line (MDA-MB-231) was determined by flowcytometry. Antiproliferative and apoptotic effects of the scFvs were evaluated by MTT and Annexin V/PI assays. The effects of selected scFvs on expression level of Surivin, c-Myc and Dvl genes were also evaluated by real-time PCR.
Results demonstrated selection of two specific scFvs (scFv-I and scFv-II) with frequencies of 35 and 20%. Both antibodies bound to the corresponding peptides and cell surface receptors as shown by phage ELISA and flowcytometry, respectively. The scFvs inhibited cell growth of MDA-MB-231 cells significantly as compared to untreated cells. Growth inhibition of 58.6 and 53.1% were detected for scFv-I and scFv-II, respectively. No significant growth inhibition was detected for SKBR-3 negative control cells. The scFvs induced apoptotic effects in the MDA-MB-231 treated cells after 48 h, which were 81.6 and 74.9% for scFv-I and scFv-II, respectively. Downregulation of Surivin, c-Myc and Dvl genes were also shown after 48h treatment of cells with either of scFvs (59.3-93.8%). ScFv-I showed significant higher antiproliferative and apoptotic effects than scFv-II.
Bioinformatic methods could effectively select potential epitopes of FZD7 protein and suggest that epitope designing by bioinformatic methods could contribute to the selection of key antigens for cancer immunotherapy. The selected scFvs, especially scFv-I, with high antiproliferative and apoptotic effects could be considered as effective agents for immunotherapy of cancers expressing FZD7 receptor including triple negative breast cancer.
FZD7 作为 Wnt/β-连环蛋白信号在癌细胞中的表面受体，具有关键作用。通过阻断 FZD7 抑制 Wnt 信号，可降低细胞活力、转移和侵袭。生物信息学方法已成为表位设计研究的有力工具。scFv 抗体的体积小、亲和力高和源自人类，为这些重组抗体提供了独特的优势。
使用生物信息学方法设计了 FZD7 胞外结构域的两个表位。通过淘选过程针对这些表位选择了特异性抗 FZD7 scFv。通过噬菌体 ELISA 评估 scFv 的特异性，并通过流式细胞术确定其与表达 FZD7 的细胞系（MDA-MB-231）的结合能力。通过 MTT 和 Annexin V/PI 测定评估 scFv 的抗增殖和促凋亡作用。还通过实时 PCR 评估了选定的 scFv 对 Survivin、c-Myc 和 Dvl 基因表达水平的影响。
结果表明，筛选出了两种特异性 scFv（scFv-I 和 scFv-II），其频率分别为 35%和 20%。噬菌体 ELISA 和流式细胞术分别显示，两种抗体均与相应的肽和细胞表面受体结合。与未经处理的细胞相比，scFv 显著抑制了 MDA-MB-231 细胞的生长。scFv-I 和 scFv-II 的细胞生长抑制率分别为 58.6%和 53.1%。SKBR-3 阴性对照细胞未检测到明显的生长抑制。scFv 在 48 小时后诱导 MDA-MB-231 细胞发生凋亡，scFv-I 和 scFv-II 的凋亡率分别为 81.6%和 74.9%。用 scFv 处理细胞 48 小时后，Surivin、c-Myc 和 Dvl 基因的表达也下调（59.3%-93.8%）。scFv-I 显示出比 scFv-II 更高的抗增殖和促凋亡作用。
生物信息学方法可以有效地选择 FZD7 蛋白的潜在表位，并表明生物信息学方法的表位设计有助于选择癌症免疫治疗的关键抗原。所选的 scFv，特别是 scFv-I，具有高抗增殖和促凋亡作用，可被视为表达 FZD7 受体的癌症（包括三阴性乳腺癌）免疫治疗的有效药物。
