Engineering Aglycosylated IgG Variants with Wild-Type or Improved Binding Affinity to Human Fc Gamma RIIA and Fc Gamma RIIIAs

Tiffany F. Chen, Stephen L. Sazinsky, Damian Houde, David J. DiLillo, Julie Bird, Kevin K. Li, George T. Cheng, Huawei Qiu, John R. Engen, Jeffrey V. Ravetch, K. Dane Wittrup

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

Abstract

The binding of human IgG1 to human Fc gamma receptors (hFcγRs) is highly sensitive to the presence of a single N-linked glycosylation site at asparagine 297 of the Fc, with deglycosylation resulting in a complete loss of hFcγR binding. Previously, we demonstrated that aglycosylated human IgG1 Fc variants can engage the human FcγRII class of the low-affinity hFcγRs, demonstrating that N-linked glycosylation of the Fc is not a strict requirement for hFcγR engagement. In the present study, we demonstrate that aglycosylated IgG variants can be engineered to productively engage with FcγRIIIA, as well as the human Fc gamma RII subset. We also assess the biophysical properties and serum half-life of the aglycosylated IgG variants to measure stability. Aglycosylated constructs N297D/S298T (DTT)–K326I/A327Y/L328G (IYG) and N297D/S298A–IYG optimally drove tumor cell phagocytosis. A mathematical model of phagocytosis suggests that hFcγRI and hFcγRIIIA dimers were the main drivers of phagocytosis. In vivo tumor control of B16F10 lung metastases further confirmed the variant DTT–IYG to be the best at restoring wild-type-like properties in prevention of lung metastases. While deuterium incorporation was similar across most of the protein, several peptides within the CH2 domain of DTT–IYG showed differential deuterium uptake in the peptide region of the FG loop as compared to the aglycosylated N297Q. Thus, in this study, we have found an aglycosylated variant that may effectively substitute for wild-type Fc. These aglycosylated variants have the potential to allow therapeutic antibodies to be produced in virtually any expression system and still maintain effector function.

Original languageEnglish
Pages (from-to)2528-2541
Number of pages14
JournalJournal of Molecular Biology
Volume429
Issue number16
DOIs
StatePublished - Aug 4 2017
Externally publishedYes

Keywords

  • antibody engineering
  • directed evolution
  • Fc-gamma receptor
  • yeast display

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