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Theranostics 2021; 11(4):1901-1917. doi:10.7150/thno.51299 This issue Cite

Research Paper

A general Fc engineering platform for the next generation of antibody therapeutics

Da Chen¹, Yingjie Zhao^2,3, Mingyu Li¹, Hang Shang¹, Na Li¹, Fan Li¹, Wei Wang⁴, Yuan Wang¹, Ruina Jin¹, Shiyu Liu¹, Xun Li⁵, Shan Gao¹, Yujie Tian¹, Ruonan Li¹, Huanhuan Li¹, Yongyan Zhang¹, Mingjuan Du⁴, Youjia Cao¹, Yan Zhang^2,3, Xin Li¹, Yi Huang⁶, Liaoyuan A. Hu^5✉, Fubin Li^2,3✉, Hongkai Zhang^1,4✉

1. State Key Laboratory of Medicinal Chemical Biology and College of Life Sciences, Nankai University, 94 Weijin Road, Tianjin, 300071, China.
2. Shanghai Institute of Immunology, Faculty of Basic Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
3. Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
4. Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, 201210, China.
5. Amgen Research, Amgen Biopharmaceutical R&D (Shanghai) Co., Ltd, Shanghai, 201210, China.
6. Department of Analytical Science, Zhenge Biotech, Shanghai, 201318, China.

✉ Corresponding authors: E-mail: hongkaiedu.cn; fubin.liedu.cn; liaoyuancom.

Abstract

Rationale: Fc engineering has become the focus of antibody drug development. The current mutagenesis and in silico 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.

Methods: 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.

Results: 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 in vitro and in vivo models.

Conclusions: 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.

Keywords: antibody therapeutics, Fc engineering, Glycoengineering, mammalian cell display, Fc gamma receptors

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