Cell-based glycoengineering for production of homogeneous and specific glycoform-enriched antibodies with improved effector functions

• 成果类型: Article
• 署名作者: Huang, Han-Wen; Zeng, Yi-Fang; Shivatare, Vidya S.; Tseng, Tzu-Hao; Wong, Chi-Huey
• 署名单位: Scripps Research Institute
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-13378
• DOI: 10.1073/pnas.2423853122
• 发表日期: 2025-02-25
• 关键词: antiinflammatory activity; immunoglobulin-g; glycosylation; binding; expression; mutants; site; igm

摘要

Glycosylation of humanized antibody at Fc- Asn297 significantly affects the Fc- mediated killing of target cells through effector functions, especially antibody- dependent cellular cytotoxicity (ADCC), antibody- dependent cell- mediated phagocytosis (ADCP), and antibody- dependent vaccinal effect (ADVE). Previous studies showed that therapeutic immunoglobulin G (IgG) antibodies with alpha 2,6- sialyl complex type (SCT) glycan attached to Fc- Asn297 exhibited optimal binding to the Fc receptors on effector cells associated with ADCC, ADCP, and ADVE. However, the production of antibodies with homogeneous Fc-SCT glycan requires multiple in vitro enzymatic and purification steps. In this study, we report two cell- based methods to produce Fc- GlcNAc antibody and Fc-SCT- enriched antibodies with improved effector functions. First, we expressed endoglycosidase S2 in Expi293F GnT1- cells to trim all N- glycans to Fc- GlcNAc antibody for in vitro transglycosylation to generate homogeneous antibodies with well- defined Fc glycan. Second, we engineered the glycosylation pathway of HEK293T cells through knock- out of undesired glycosyltransferases and knock- in of desired glycosyltransferases to produce Fc-SCT- enriched antibodies and evaluated their binding to Fc receptors, and we found that the Fc-SCT- enriched antibody is like or better than the homogeneous Fc-SCT antibody in binding to the Fc receptors associated with ADCC, ADCP, and ADVE.