Dataset Information

Identification of Fc Gamma Receptor Glycoforms That Produce Differential Binding Kinetics for Rituximab.

ABSTRACT: Fc gamma receptors (Fc?R) bind the Fc region of antibodies and therefore play a prominent role in antibody-dependent cell-based immune responses such as ADCC, CDC and ADCP. The immune effector cell activity is directly linked to a productive molecular engagement of Fc?Rs where both the protein and glycan moiety of antibody and receptor can affect the interaction and in the present study we focus on the role of the Fc?R glycans in this interaction. We provide a complete description of the glycan composition of Chinese hamster ovary (CHO) expressed human Fc? receptors RI (CD64), RIIa_{Arg131/His131} (CD32a), RIIb (CD32b) and RIIIa_{Phe158/Val158} (CD16a) and analyze the role of the glycans in the binding mechanism with IgG. The interactions of the monoclonal antibody rituximab with each Fc?R were characterized and we discuss the CHO-Fc?RIIIa_{Phe158/Val158} and CHO-Fc?RI interactions and compare them to the equivalent interactions with human (HEK293) and murine (NS0) produced receptors. Our results reveal clear differences in the binding profiles of rituximab, which we attribute in each case to the differences in host cell-dependent Fc?R glycosylation. The glycan profiles of CHO expressed Fc?RI and Fc?RIIIa_{Phe158/Val158} were compared with the glycan profiles of the receptors expressed in NS0 and HEK293 cells and we show that the glycan type and abundance differs significantly between the receptors and that these glycan differences lead to the observed differences in the respective Fc?R binding patterns with rituximab. Oligomannose structures are prevalent on Fc?RI from each source and likely contribute to the high affinity rituximab interaction through a stabilization effect. On Fc?RI and Fc?RIIIa large and sialylated glycans have a negative impact on rituximab binding, likely through destabilization of the interaction. In conclusion, the data show that the IgG1-Fc?R binding kinetics differ depending on the glycosylation of the Fc?R and further support a stabilizing role of Fc?R glycans in the antibody binding interaction.

SUBMITTER: Hayes JM

PROVIDER: S-EPMC5629263 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Identification of Fc Gamma Receptor Glycoforms That Produce Differential Binding Kinetics for Rituximab.

Hayes Jerrard M JM Frostell Asa A Karlsson Robert R Müller Steffen S Martín Silvia Míllan SM Pauers Martin M Reuss Franziska F Cosgrave Eoin F EF Anneren Cecilia C Davey Gavin P GP Rudd Pauline M PM

Molecular & cellular proteomics : MCP 20170602 10

Fc gamma receptors (FcγR) bind the Fc region of antibodies and therefore play a prominent role in antibody-dependent cell-based immune responses such as ADCC, CDC and ADCP. The immune effector cell activity is directly linked to a productive molecular engagement of FcγRs where both the protein and glycan moiety of antibody and receptor can affect the interaction and in the present study we focus on the role of the FcγR glycans in this interaction. We provide a complete description of the glycan ...[more]

PMID: 28576848

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Project description:Structurally well-defined IgG-Fc glycoforms are highly demanded for understanding the effects of glycosylation on an antibody's effector functions. We report in this paper chemoenzymatic synthesis and Fcγ receptor binding of an array of homogeneous IgG-Fc glycoforms. The chemoenzymatic approach consists of the chemical synthesis of defined N-glycan oxazolines as donor substrates, the expression of the Fc domain in a CHO cell line in the presence of an α-mannosidase inhibitor kifunensine, and an endoglycosidase-catalyzed glycosylation of the deglycosylated Fc domain (GlcNAc-Fc homodimer) with the synthetic glycan oxazolines. The enzyme from Arthrobacter protophormiae (Endo-A) was found to be remarkably efficient to take various modified N-glycan core oxazolines, including the bisecting sugar-containing derivatives, for Fc glycosylation remodeling, resulting in the formation of the corresponding homogeneous Fc glycoforms. Nevertheless, neither Endo-A nor the Mucor hiemalis endoglycosidase mutants (EndoM-N175A and EndoM-N175Q) were able to transfer full-length complex-type N-glycan to the Fc domain, implicating the limitations of these two enzymes in Fc glycosylation remodeling. Surface plasmon resonance (SPR) binding studies with the synthetic IgG-Fc glycoforms unambiguously proved that the presence of a bisecting GlcNAc moiety could significantly enhance the binding of Fc to FcγRIIIa, the activating Fcγ receptor, independent of Fc core-fucosylation. Interestingly, the Fc glycoforms carrying an unusual bisecting sugar moiety such as a mannose or a LacNAc moiety also demonstrated enhanced affinity to FcγRIIIa. On the orther hand, the presence of a bisecting GlcNAc or core-fucosylation had little effect on the affinity of Fc to the inhibitory Fcγ receptor, FcγRIIb. Our experimental data also showed that the α-linked mannose residues in the pentasaccharide Man3GlcNAc2 core was essential to maintain a high affinity of Fc to both FcγRIIIa and FcγRIIb. The synthetic homogeneous Fc glycoforms thus provide a useful tool for elucidating how a fine Fc N-glycan structure precisely affects the function of the Fc domain.

Project description:Receptors recognizing the Fc part of immunoglobulin G (Fc?Rs) are key determinants in antibody-mediated immune responses. Members of the Herpesviridae interfere with this immune regulatory network by expressing viral Fc?Rs (vFc?Rs). Human cytomegalovirus (HCMV) encodes four distinct vFc?Rs that differ with respect to their IgG subtype specificity and their impact on antibody-mediated immune function in vitro The impact of vFc?Rs on HCMV pathogenesis and immunomodulation in vivo is not known. The closest evolutionary animal model of HCMV is rhesus CMV (RhCMV) infection of rhesus macaques. To enable the characterization of vFc?R function in this model, we studied IgG binding by RhCMV. We show that lysates of RhCMV-infected cells contain an IgG-binding protein of 30?kDa encoded by the gene Rh05 that is a predicted type I glycoprotein belonging to the RL11 gene family. Upon deletion of Rh05, IgG-Fc binding by RhCMV strain 68-1 is lost, whereas ectopic expression of Rh05 results in IgG binding to transfected cells consistent with Rh05 being a vFc?R. Using a set of reporter cell lines stably expressing human and rhesus Fc?Rs, we further demonstrate that Rh05 antagonizes host Fc?R activation. Compared to Rh05-intact RhCMV, RhCMV?Rh05 showed an increased activation of host Fc?R upon exposure of infected cells to IgG from RhCMV-seropositive animals, suggesting that Rh05 protects infected cells from opsonization and IgG-dependent activation of host Fc?Rs. However, antagonizing host Fc?R activation by Rh05 was not required for the establishment and maintenance of infection of RhCMV, even in a seropositive host, as shown by the induction of T cell responses to heterologous antigens expressed by RhCMV lacking the gene region encoding Rh05. In contrast to viral evasion of natural killer cells or T cell recognition, the evasion of antibody-mediated effects does not seem to be absolutely required for infection or reinfection. The identification of the first vFc?R that efficiently antagonizes host Fc?R activation in the RhCMV genome will thus permit more detailed studies of this immunomodulatory mechanism in promoting viral dissemination in the presence of natural or vaccine-induced humoral immunity.IMPORTANCE Rhesus cytomegalovirus (RhCMV) offers a unique model for studying human cytomegalovirus (HCMV) pathogenesis and vaccine development. RhCMV infection of nonhuman primates greatly broadened the understanding of mechanisms by which CMVs evade or reprogram T cell and natural killer cell responses in vivo However, the role of humoral immunity and viral modulation of anti-CMV antibodies has not been studied in this model. There is evidence from in vitro studies that HCMVs can evade humoral immunity. By gene mapping and with the help of a novel cell-based reporter assay system we characterized the first RhCMV encoded IgG-Fc? binding glycoprotein as a potent antagonist of rhesus Fc?R activation. We further demonstrate that, unlike evasion of T cell immunity, this viral Fc? receptor is not required to overcome anti-CMV immunity to establish secondary infections. These findings enable more detailed studies of the in vivo consequences of CMV evasion from IgG responses in nonhuman primate models.

Project description:BackgroundTumor necrosis factor (TNF) is an important cytokine in the pathogenesis of inflammatory bowel disease (IBD). Anti-TNF antibodies have been successfully implemented in IBD therapy, however their efficacies differ among IBD patients. Here we investigate the influence of CD64 Fc receptor on the inhibitory activity of anti-TNFs in cells of intestinal wall.MethodsIntestinal cell lines, monocytes/macrophages and peripheral blood mononuclear cells (PBMCs) were used as models. The efficacies of adalimumab, infliximab and certolizumab-pegol were assessed by RT-PCR for target genes. Protein levels and localizations were examined by Western blotting and immunofluorescence. Antibody fragments were obtained by proteolytic digestion, immunoprecipitation and protein chip analysis. Knock-down of specific gene expression was performed using siRNAs.ResultsInfliximab had limited efficacy towards soluble TNF in cell types expressing Fc gamma receptor CD64. Both adalimumab and infliximab had lower efficacies in PBMCs of IBD patients, which express elevated levels of CD64. Infliximab-TNF complexes were more potent in activating CD64 in THP-1 cells than adalimumab, which was accompanied by distinct phospho-tyrosine signals. Blocking Fc parts and isolation of Fab fragments of infliximab improved its efficacy. IFN-?-induced expression of CD64 correlated with a loss of efficacy of infliximab, whereas reduction of CD64 expression by either siRNA or PMA treatment improved inhibitory activity of this drug. Colonic mRNA expression levels of CD64 and other Fc gamma receptors were significantly increased in the inflamed tissues of infliximab non-responders.ConclusionsCD64 modulates the efficacy of infliximab both in vitro and ex vivo, whereas the presence of this receptor has no impact on the inhibitory activity of certolizumab-pegol, which lacks Fc fragment. These data could be helpful in both predicting and evaluating the outcome of anti-TNF therapy in IBD patients with elevated systemic and local levels of Fc receptors.

Dataset Information

Identification of Fc Gamma Receptor Glycoforms That Produce Differential Binding Kinetics for Rituximab.

Publications

Identification of Fc Gamma Receptor Glycoforms That Produce Differential Binding Kinetics for Rituximab.

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