MedImmune, Granta Park, Cambridge, UK.
Protein Eng Des Sel. 2011 Sep;24(9):671-8. doi: 10.1093/protein/gzr015. Epub 2011 May 18.
Ribosome display was applied to the Fc region of human immunoglobulin G (IgG1) to select for improved binding to human FcγRIIIa, the receptor expressed on human natural killer cells that mediates antibody-dependent cellular cytotoxicity (ADCC). A library of human Fcγ1 variants was generated using error-prone polymerase chain reaction, and subjected to multiple rounds of ribosome display selection against progressively decreasing concentrations of soluble human FcγRIIIa, to enrich for improved binders. Radioimmunoassay and alphascreen analyses of the aglycosylated IgG-Fc output revealed variants with improved binding to FcγRIIIa relative to wild-type IgG-Fc. Subsequent expression in human (HEK-EBNA) cells generated glycosylated IgGs with modified activity in ADCC assays. One particular variant, 125_B01 triggered enhanced ADCC (EC(50) up to four-fold reduced with increased maximal lysis) relative to wild-type antibody, having more equal levels of ADCC for each allotype (V158/F158) of FcγRIIIa. Deconvolution of individual replacements within the variant showed that improved function arose from the Phe243Leu replacement within the CH2 domain, rather than the CH3 domain replacements Thr393Ala or His433Pro. Surprisingly, the oligosaccharide profiles of 125_B01 indicated more oligosaccharide chains lacking fucose, or with bisecting N-acetylglucosamine relative to wild-type IgG1, which correlates with improved function and the replacement Phe243Leu that is a carbohydrate contact residue within the C(H)2 domain.
核糖体展示技术被应用于人免疫球蛋白 G（IgG1）的 Fc 区域，以筛选对人 FcγRIIIa 具有更好结合力的变体，FcγRIIIa 是表达于自然杀伤细胞表面的受体，介导抗体依赖的细胞毒性（ADCC）。使用易错聚合酶链反应生成了人 Fcγ1 变体文库，并对其进行了多轮核糖体展示筛选，以降低可溶性人 FcγRIIIa 的浓度，从而富集具有更好结合力的变体。未经糖基化的 IgG-Fc 的放射免疫分析和 alphascreen 分析显示，与野生型 IgG-Fc 相比，变体与 FcγRIIIa 的结合得到了改善。随后在人（HEK-EBNA）细胞中表达，产生了具有修饰的 ADCC 活性的糖基化 IgG。特别是变体 125_B01 与野生型抗体相比，触发了增强的 ADCC（EC50 降低了四倍，最大裂解增加），对于 FcγRIIIa 的每种同种型（V158/F158），ADCC 水平更加均等。对变体中单个替换的剖析表明，功能的改善源于 CH2 结构域中的 Phe243Leu 替换，而不是 CH3 结构域中的 Thr393Ala 或 His433Pro 替换。令人惊讶的是，125_B01 的寡糖图谱表明，更多的寡糖链缺乏岩藻糖，或者具有双分支 N-乙酰葡萄糖胺，与野生型 IgG1 相比，这与功能的改善以及与 Phe243Leu 替换相关，该替换是 CH（H）2 结构域中的一个碳水化合物接触残基。
