Dataset Information

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

ABSTRACT: The binding of human IgG1 to human Fc gamma receptors (hFc?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?R binding. Previously, we demonstrated that aglycosylated human IgG1 Fc variants can engage the human Fc?RII class of the low-affinity hFc?Rs, demonstrating that N-linked glycosylation of the Fc is not a strict requirement for hFc?R engagement. In the present study, we demonstrate that aglycosylated IgG variants can be engineered to productively engage with Fc?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?RI and hFc?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.

SUBMITTER: Chen TF

PROVIDER: S-EPMC5584586 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Publications

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

Chen Tiffany F TF Sazinsky Stephen L SL Houde Damian D DiLillo David J DJ Bird Julie J Li Kevin K KK Cheng George T GT Qiu Huawei H Engen John R JR Ravetch Jeffrey V JV Wittrup K Dane KD

Journal of molecular biology 20170708 16

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 demon ...[more]

PMID: 28694069

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Project description:The neonatal Fc receptor (FcRn) is a key membrane protein that plays an integral role in serum immunoglobulin (IgG) recycling, which extends the half-life of antibody. In addition, FcRn is known to traffic antigen-bound immunoglobulins (Ag-IgGs), and to interact with immune complexes to facilitate the antigen cross-presentation of peptides derived from the immune complexes in antigen-presenting cells (APCs). Studies on the IgG-FcRn molecular interactions have primarily focused on the Fc region, and only recently have shown the potential impact of the antigen-binding fragment physiochemical properties on FcRn binding. However, the effect of the antigen physiochemical properties on IgG structure as it relates to Ag-IgG-FcRn binding is not well understood. Here we used an IgG-peptide antigen complex as a model system to investigate the structural effects of the antigen's physiochemical properties on the IgG structure, and the subsequent effects of Ag-IgG-FcRn interactions. We used hydroxyl radical footprinting-mass spectrometry to investigate the structural impact on an IgG upon antigen binding, and observed that the physicochemical properties of the antigen differentially induce conformational changes in the IgG FcRn binding region. The extent of these structural changes directly correlates to the magnitude of the affinity differences between the Ag-IgG complexes and FcRn. Moreover, the antigen's physicochemical properties differentially induce structural differences within the Ag-IgG-FcRn ternary complex. We also provide electron microscopy data that shows corroborating Fab-FcRn interactions, and confirms the hypothesis of potential 2:1 FcRn:IgG binding stoichiometry. These data demonstrate antigen-induced Fc structural rearrangements affect both the affinity toward FcRn and the trimeric antigen-IgG-FcRn complex, providing novel molecular insights in the first steps toward understanding interactions of FcRn-containing large(r)-sized immune complex.

Dataset Information

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

Publications

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

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