IgG4亚型Fc突变体的构建及其对血液半衰期的影响

Construction of IgG4 Fc variants and their serum half-lives

摘要:

本研究旨在构建一系列IgG4 (immunoglobulin G4)亚型的Fc (fragment crystallizable)重组蛋白突变体，筛选出血液半衰期显著提高的Fc突变体，分析基因突变位点与半衰期的关系，为IgG4亚型的抗体及Fc融合蛋白药物的开发提供理论依据。选取9个基因突变位点进行突变，并将不同的突变位点进行组合，通过分子克隆及点突变技术构建不同的突变子表达质粒pET24b-Fc。将以上质粒转化至大肠杆菌BL21(DE3)，通过蛋白表达技术成功表达了具有不同突变的Fc重组蛋白，其中Fc2和Fc3突变体重组蛋白产量略低，其他突变体蛋白的表达未受影响；通过体外细胞计数试剂盒(cell counting kit-8, CCK-8)、钙黄绿素乙酰氧基甲酯/碘化丙啶(calcein acetoxymethyl ester/propidium iodide, calcein AM/PI)试剂盒及体内酶联免疫吸附测定(enzyme-linked immunosorbent assay, ELISA)检测不同Fc突变体的毒性，结果表明突变后的重组蛋白Fc具有良好的生物相容性和安全性。最后，利用荧光标记Fc突变体，通过体内实验探究不同突变对血液半衰期的影响，成功筛选出血液半衰期显著提高的Fc5突变体。本研究为IgG4亚型的抗体及Fc融合蛋白药物的优化设计提供了理论依据与实践基础。

Abstract:

In this study, we constructed a series of recombinant Fc variants of immunoglobulin G4 (IgG4), screened the fragment crystallizable (Fc) variants with significantly prolonged serum half-lives, and analyzed the relationship between mutation site and half-life, aiming to provide a theoretical basis for the development of IgG4 antibodies and Fc fusion protein-based drugs. Nine gene sites were selected for mutation, and different mutation sites were combined. The variant expression plasmids pET24b-Fc were constructed by molecular cloning and point mutation. The plasmids were transformed into Escherichia coli BL21(DE3) for the expression of different recombinant proteins of Fc. Fc2 and Fc3 variants had slightly lower recombinant protein yields, and the expression of other variants was not affected. The toxicity of different Fc variants was determined by cell counting kit-8 (CCK-8) and calcein acetoxymethyl ester/ propidium iodide (calcein AM/PI) in vitro and enzyme-linked immuno sorbent assay (ELISA) in vivo. The results showed that the recombinant Fc variants had good biocompatibility and safety. Finally, the Fc variants were labeled with fluorescent markers, and the effects of different mutations on their serum half-lives were investigated by in vivo experiments. The Fc5 variant with prolonged serum half-life was successfully screened out, which provided a theoretical and practical basis for the optimal design of IgG4 subtype antibody and Fc fusion protein drugs.