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

JOURNAL OF MOLECULAR BIOLOGY

卷:429

Article

Chen, Tiffany F.^1,4  Sazinsky, Stephen L.¹  Houde, Damian^5,8  DiLillo, David J.⁶  Bird, Julie⁷  Li, Kevin K.^1,4  Cheng, George T.³  Qiu, Huawei⁷  Engen, John R.⁵  Ravetch, Jeffrey V.⁶  Wittrup, K. Dane^1,2,4 

[1] MIT, Dept Biol Engn, 77 Massachusetts Ave, Cambridge, MA 02139 USA

[2] MIT, Dept Chem Engn, 77 Massachusetts Ave, Cambridge, MA 02139 USA

[3] MIT, Dept Elect Engn & Comp Sci, 77 Massachusetts Ave, Cambridge, MA 02139 USA

[4] MIT, Koch Inst Integrat Canc Res, 77 Massachusetts Ave, Cambridge, MA 02139 USA

[5] Northeastern Univ, Dept Chem & Chem Biol, 360 Huntington Ave, Boston, MA 02115 USA

[6] Rockefeller Univ, Lab Mol Genet & Immunol, 1230 York Ave, New York, NY 10065 USA

[7] Sanofi Genzyme, Biol Res, 49 New York Ave, Framingham, MA 01701 USA

[8] Codiak Biosci, Proc Analyt, Cambridge, MA 02142 USA

关键词: directed evolution;    yeast display;    antibody engineering;    Fc-gamma receptor;   

DOI  :  10.1016/j.jmb.2017.07.001

来源: Elsevier

【 摘 要 】

The binding of human IgG1 to human Fc gamma receptors (hFc gamma Rs) 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 hFc gamma R binding. Previously, we demonstrated that aglycosylated human IgG1 Fc variants can engage the human Fc gamma RII class of the low-affinity hFc gamma Rs, demonstrating that N-linked glycosylation of the Fc is not a strict requirement for hFc gamma R engagement. In the present study, we demonstrate that aglycosylated IgG variants can be engineered to productively engage with Fc gamma RIIIA, 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 hFc gamma R) and hFc gamma RIIIA 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. (C) 2017 Elsevier Ltd. All rights reserved.

【 授权许可】

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