Engineering Aglycosylated IgG Variants with Wild-Type or Improved Binding Affinity to Human Fc Gamma RIIA and Fc Gamma RIIIAs. Issue 16 (4th August 2017)

• Record Type: Journal Article
• Authors: Chen, Tiffany F.; Sazinsky, Stephen L.; Houde, Damian; DiLillo, David J.; Bird, Julie; Li, Kevin K.; Cheng, George T.; Qiu, Huawei; Engen, John R.; Ravetch, Jeffrey V.; Wittrup, K. Dane
• Is Part Of: Journal of molecular biology. Volume 429:Issue 16(2017)
• Page Start: 2528
• Page End: 2541
• Publication Date: 2017-08-04
• Subjects: hFcγR human Fc gamma receptor -- WT wild-type -- SGTA S298G/T299A -- DTT N297D/S298T -- DAT N297D/S298A -- HAT N297H/S298A -- IYG K326I/A327Y/L328G -- DLS dynamic light scattering -- GM-CSF granulocyte macrophage colony-stimulating factor -- PBS phosphate-buffered saline -- H/DX MS hydrogen–deuterium exchange mass spectrometry -- DSC differential scanning calorimetry; directed evolution -- yeast display -- antibody engineering -- Fc-gamma receptor; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2017.07.001
• Languages: English
• ISSNs: 0022-2836

Abstract

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. Graphical abstract: Highlights: Previous Fc-engineered aglycosylated antibodies restored binding to FcγRI or FcγRII. We have aglycosylated variants that restore binding to all low-affinity FcγRs. In vitro phagocytosis and in vivo tumor control confirm activity of Fc variants. Mathematical modeling and H/DX were used to characterize the Fc variants. We have an aglycosylated variant that may effectively substitute for WT Fc.