State Key Laboratory of Medicinal Chemical Biology and College of Life Sciences, Nankai University, 94 Weijin Road, Tianjin, 300071, China.
Shanghai Institute of Immunology, Faculty of Basic Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
Theranostics. 2021 Jan 1;11(4):1901-1917. doi: 10.7150/thno.51299. eCollection 2021.
Fc engineering has become the focus of antibody drug development. The current mutagenesis and protein design methods are confined by the limited throughput and high cost, while the high-throughput phage display and yeast display technologies are not suitable for screening glycosylated Fc variants. Here we developed a mammalian cell display-based Fc engineering platform. By using mammalian cell display and next generation sequencing, we screened millions of Fc variants for optimized affinity and specificity for FcγRIIIa or FcγRIIb. The identified Fc variants with improved binding to FcγRIIIa were substituted into trastuzumab and rituximab and the effector function of antibodies were examined in the PBMC-based assay. On the other hand, the identified Fc variants with selectively enhanced FcγRIIb binding were applied to CD40 agonist antibody and the activities of the antibodies were measured on different cell assays. The immunostimulatory activity of CD40 antibodies was also evaluated by OVA-specific CD8 T cell response model in FcγR/CD40-humanized mice. Using this approach, we screened millions of Fc variant and successfully identified several novel Fc variants with enhanced FcγRIIIa or FcγRIIb binding. These identified Fc variants displayed a dramatic increase in antibody-dependent cellular cytotoxicity in PBMC-based assay. Novel variants with selectively enhanced FcγRIIb binding were also identified. CD40 agonist antibodies substituted with these Fc variants displayed activity more potent than the parental antibody in the and models This approach increased the throughput of Fc variant screening from thousands to millions magnitude, enabled screening variants containing multiple mutations and could be integrated with glycoengineering technology, represents an ideal platform for Fc engineering. The initial efforts demonstrated the capability of the platform and the novel Fc variants could be substituted into nearly any antibody for the next generation of antibody therapeutics.
Fc 工程已成为抗体药物开发的焦点。目前的突变和蛋白质设计方法受到通量有限和成本高的限制，而高通量噬菌体展示和酵母展示技术不适用于筛选糖基化 Fc 变体。在这里，我们开发了一种基于哺乳动物细胞展示的 Fc 工程平台。通过使用哺乳动物细胞展示和下一代测序，我们筛选了数百万个 Fc 变体，以优化对 FcγRIIIa 或 FcγRIIb 的亲和力和特异性。鉴定出的与 FcγRIIIa 结合增强的 Fc 变体被替换到曲妥珠单抗和利妥昔单抗中，并在基于 PBMC 的测定中检查了抗体的效应功能。另一方面，鉴定出的与 FcγRIIb 结合选择性增强的 Fc 变体被应用于 CD40 激动剂抗体，并在不同的细胞测定中测量了抗体的活性。FcγR/CD40 人源化小鼠中的 OVA 特异性 CD8 T 细胞反应模型也评估了 CD40 抗体的免疫刺激活性。使用这种方法，我们筛选了数百万个 Fc 变体，并成功鉴定出几种与增强的 FcγRIIIa 或 FcγRIIb 结合的新型 Fc 变体。在基于 PBMC 的测定中，这些鉴定出的 Fc 变体显示出抗体依赖性细胞毒性的显著增加。还鉴定出与 FcγRIIb 结合选择性增强的新型变体。用这些 Fc 变体替换的 CD40 激动剂抗体在 和 模型中显示出比亲本抗体更强的活性。这种方法将 Fc 变体筛选的通量从数千增加到数百万数量级，能够筛选包含多个突变的变体，并可与糖基工程技术集成，代表了 Fc 工程的理想平台。初步努力证明了该平台的能力，新型 Fc 变体可替代为下一代抗体治疗药物的几乎任何抗体。
