Laboratory of Oncology, G. Gaslini Institute, Genova, Italy.
Sirius-biotech, c/o Advanced Biotechnology Center, Genova, Italy.
PLoS One. 2016 Feb 11;11(2):e0148103. doi: 10.1371/journal.pone.0148103. eCollection 2016.
Fibronectin (FN) is a large multidomain molecule that is involved in many cellular processes. Different FN isoforms arise from alternative splicing of the pre-mRNA including, most notably, the FN isoform that contains the "extra-domain-B" (ED-B). The FN isoform containing ED-B (known as B-FN) is undetectable in healthy adult tissues but is present in large amounts in neoplastic and foetal tissues as well as on the blood vessels during angiogenesis. Thus, antibodies specific for B-FN can be useful for detecting and targeting neoplastic tissues in vivo. We previously characterised C6, a new monoclonal antibody specific for human B-FN and we suggested that it reacts with the B-C loop of the type III repeat 8 which is masked in FN isoforms lacking ED-B and that the insertion of ED-B in FN molecules unmasked it. Here we have now consolidated and refined the characterization of this B-FN specific antibody demonstrating that the epitope recognized by C6 also includes loop E-F of ED-B.
We built the three dimensional model of the variable regions of the mAb C6 and of the FN fragment EDB-III8 and performed protein:protein docking simulation using the web server ClusPro2.0. To confirm the data obtained by protein:protein docking we generated mutant fragments of the recombinant FN fragment EDB-III8 and tested their reactivity with C6.
The monoclonal antibody C6 reacts with an epitope formed by the B-C loop of domain III8 and the E-F loop of ED-B. Both loops are required for an immunological reaction, thus this monoclonal is strictly specific for B-FN but the part of the epitope on III8 confers the human specificity.
纤连蛋白（FN）是一种参与多种细胞过程的大型多功能分子。前 mRNA 的选择性剪接产生不同的 FN 同工型，其中最显著的是包含“额外结构域-B”（ED-B）的 FN 同工型。含有 ED-B 的 FN 同工型（称为 B-FN）在健康成人组织中无法检测到，但在肿瘤和胎儿组织以及血管生成过程中的血管中大量存在。因此，针对 B-FN 的特异性抗体可用于体内检测和靶向肿瘤组织。我们之前描述了 C6，一种针对人 B-FN 的新型单克隆抗体，我们推测它与 III 型重复 8 的 B-C 环反应，该环在缺乏 ED-B 的 FN 同工型中被掩盖，而 ED-B 在 FN 分子中的插入使它暴露。在这里，我们现在整合并细化了对这种 B-FN 特异性抗体的表征，证明 C6 识别的表位还包括 ED-B 的 E-F 环。
我们构建了 mAb C6 的可变区和 FN 片段 EDB-III8 的三维模型，并使用 ClusPro2.0 网络服务器进行了蛋白质：蛋白质对接模拟。为了确认蛋白质：蛋白质对接获得的数据，我们生成了重组 FN 片段 EDB-III8 的突变片段，并测试了它们与 C6 的反应性。
单克隆抗体 C6 与 III8 结构域的 B-C 环和 ED-B 的 E-F 环形成的表位反应。两个环都是免疫反应所必需的，因此该单克隆抗体严格针对 B-FN，但表位的 III8 部分赋予了其人类特异性。
