Ashoor Dana N, Ben Khalaf Noureddine, Bourguiba-Hachemi Sonia, Marzouq Maryam H, Fathallah M Dahmani
Health Biotechnology Program, Department of Life Sciences, College of Graduate Studies, Arabian Gulf University, Manama, Kingdom of Bahrain.
Protein Eng Des Sel. 2018 Jun 1;31(6):205-212. doi: 10.1093/protein/gzy019.
The interaction between antibodies and Immune cells surface FcγRIIIa (CD16a) receptor triggers a variety of immune responses including antibody-dependent cell-mediated cytotoxicity, antibody neutralization, phagocytosis, inflammation and tissue injury. Recent studies showed that IgG1 upper hinge region and FcγRs polymorphism play a major role in the interaction with Fcγ receptors and in the stability of the immune complex hence, in mounting strong inflammatory response. To further investigate this issue, we developed a tool box of IgG1 Fc isoforms to depict the affinity between mutated IgG1 Fc regions and extracellular domain variants (V158F) of CD16a. Our strategy consisted of designing different random upper-hinge mutated variants of IgG1 Fc domain, reproducing the naturally occurring two variants of CD16a and producing all of them as recombinant fusion proteins in Pichia Pastoris. The interactions were assayed using the Surface Plasmon Resonance (Biacore) method along with an in silico analysis to identify the major interaction and key residues that underline the affinity between the Fc region and CD16a variants. Our data showed that the affinity of the Fc region to the CD16a is strongly correlated to polar interactions. This molecular engineering approach yielded an IgG1Fc mutant with enhanced binding affinity to CD16a F158 variant.
抗体与免疫细胞表面的FcγRIIIa（CD16a）受体之间的相互作用会引发多种免疫反应，包括抗体依赖性细胞介导的细胞毒性、抗体中和、吞噬作用、炎症和组织损伤。最近的研究表明，IgG1上铰链区和FcγRs多态性在与Fcγ受体的相互作用以及免疫复合物的稳定性中起主要作用，因此在引发强烈的炎症反应中也起主要作用。为了进一步研究这个问题，我们开发了一个IgG1 Fc异构体工具箱，以描述突变的IgG1 Fc区域与CD16a的细胞外结构域变体（V158F）之间的亲和力。我们的策略包括设计IgG1 Fc结构域的不同随机上铰链突变变体，复制CD16a的天然存在的两种变体，并在毕赤酵母中作为重组融合蛋白生产所有这些变体。使用表面等离子体共振（Biacore）方法以及计算机分析来测定相互作用，以识别强调Fc区域与CD16a变体之间亲和力的主要相互作用和关键残基。我们的数据表明，Fc区域与CD16a的亲和力与极性相互作用密切相关。这种分子工程方法产生了一种对CD16a F158变体具有增强结合亲和力的IgG1Fc突变体。
