Engineering an aglycosylated Fc variant for enhanced FcγRI engagement and pH-dependent human FcRn binding

Sang Taek Jung, Tae Hyun Kang, Dong il Kim

Research output: Contribution to journalArticlepeer-review

13 Citations (Scopus)

Abstract

The clinical use of therapeutic antibodies has increased sharply because of their many advantages over conventional small molecule drugs, particularly with respect to their affinity, specificity, and serum stability. Tumor or infected cells are removed by the binding of antibody Fc regions to Fc gamma receptors (FcγRs), which stimulate the activation of immune effector cells. Aglycosylated full-length IgG antibodies expressed in bacteria have different Fc conformations compared to their glycosylated counterparts produced in mammalian cells. As a result, they are unable to bind FcγRs, resulting in little to no activation of immune effector cells. In this study, we created a combinatorial library randomized at the upper CH2 loops of an aglycosylated Fc variant (Fc5: E382V/M428) and used a high-throughput flow cytometry library screening method, combined with bacterial display of homodimeric Fc domains for enhanced FcγR binding affinity. The trastuzumab Fc variant containing the identified mutations (Q295R, L328W, A330V, P331A, I332Y, E382V, M428I) not only exhibited over 120 fold higher affinity of specific binding to FcγRI than wild type aglycosylated Fc, but also retained pH-dependent FcRn binding. These results show that an aglycosylated antibody expressed in bacteria can be evolved for novel FcγR affinity and specificity.

Original languageEnglish
Pages (from-to)780-789
Number of pages10
JournalBiotechnology and Bioprocess Engineering
Volume19
Issue number5
DOIs
Publication statusPublished - 2014 Nov 20
Externally publishedYes

Bibliographical note

Funding Information:
We thank Dr. George Georgiou (University of Texas at Austin) for advice on experiments and helpful discussion. This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (2013R1A1A1004576), the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. NRF-2013M3A9B6075887), and a grant from the National R&D Program for Cancer Control, Ministry of Health and Welfare, Republic of Korea (1420160).

Publisher Copyright:
© 2014, The Korean Society for Biotechnology and Bioengineering and Springer-Verlag Berlin Heidelberg.

Keywords

  • FcγRI (CD64)
  • antibody Fc
  • directed evolution
  • effector functions
  • neonatal Fc receptor (FcRn)

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology
  • Biomedical Engineering

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