Highly parallel characterization of IgG Fc binding interactions

Boesch, Austin W.; Brown, Eric P.; Cheng, Hao; Ofori, Maame Ofua; Normandin, Erica; Nigrović, Peter A.; Alter, Galit; Ackerman, Margaret E.

doi:10.4161/mabs.28808

Cited by 76 publications

(88 citation statements)

References 54 publications

(60 reference statements)

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“…Furthermore, fucosylation levels of the antibody have an impact on the binding to FcγRIIIa and FcγRIIIb, whereas no differences in binding were measured on any of the other Fc receptors. These results are in agreement with the literature 13, 16, 17, 18, 19. None of the cited references studied the effect of DG on fraction bound at neutral pH, and we have demonstrated that there is a significant impact.…”

Section: Discussionsupporting

confidence: 93%

“…Hardly any IgG binding to the low‐affinity Fcγ receptors and FcγRI was measured after DG, which is in agreement with results from others 12, 13, 15, 40. The binding to FcRn receptor is not or only moderately influenced by the glycan occupancy, as similar affinity at pH 6 was measured using deglycosylated IgG1 compared to the glycosylated reference IgG.…”

Section: Discussionsupporting

confidence: 92%

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Rapid screening of IgG quality attributes – effects on Fc receptor binding

Geuijen¹,

Oppers-Tiemissen

Egging

et al. 2017

FEBS Open Bio

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The interactions of therapeutic antibodies with fragment crystallizable γ (Fcγ) receptors and neonatal Fc receptors (FcRn) are measured in vitro as indicators of antibody functional performance. Antibodies are anchored to immune cells through the Fc tail, and these interactions are important for the efficacy and safety of therapeutic antibodies. High‐throughput binding studies on each of the human Fcγ receptor classes (FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb) as well as FcRn have been developed and performed with human IgG after stress‐induced modifications to identify potential impact in vivo. Interestingly, we found that asparagine deamidation (D‐N) reduced the binding of IgG to the low‐affinity Fcγ receptors (FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb), while FcγRI and FcRn binding was not impacted. Deglycosylation completely inhibited binding to all Fcγ receptors, but showed no impact on binding to FcRn. On the other hand, afucosylation only impacted binding to FcγRIIIa and FcγRIIIb. Methionine oxidation at levels below 7%, multiple freeze/thaw cycles and short‐term thermal/shake stress did not influence binding to any of the Fc receptors. The presence of high molecular weight species, or aggregates, disturbed measurements in these binding assays; up to 5% of aggregates in IgG samples changed the binding and kinetics to each of the Fc receptors. Overall, the screening assays described in this manuscript prove that rapid and multiplexed binding assays may be a valuable tool for lead optimization, process development, in‐process controls, and biosimilarity assessment of IgGs during development and manufacturing of therapeutic IgGs.

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Section: Discussionsupporting

confidence: 93%

Section: Discussionsupporting

confidence: 92%

Rapid screening of IgG quality attributes – effects on Fc receptor binding

Geuijen¹,

Oppers-Tiemissen

Egging

et al. 2017

FEBS Open Bio

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“…Number 12 December 2017 mutation (27). Modification of the Fc region of VRC01 to alter or negate FcγR engagement did not impair the capacity of VRC01 to suppress IFN production, demonstrating that FcγR engagement is not required for Ab-mediated suppression of IFN production by pDCs ( Figure 3B).…”

Section: Introductionmentioning

confidence: 93%

“…4E10 mAb was also purchased from Polymun Scientific. Gp120 mAb VRC01 and VRC01 FcR-binding variants were produced as previously described (27).…”

Section: Methodsmentioning

confidence: 99%

HIV-antibody complexes enhance production of type I interferon by plasmacytoid dendritic cells

Veenhuis¹,

Freeman²,

Korleski³

et al. 2017

Journal of Clinical Investigation

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“…For example, recombinant soluble FcγR ectodomains with c-terminal hexahistidine tags have been immobilised on plates via antihexahistidine mAb [166] or nickel [18] to produce an array of receptors for binding IgG ligands. A powerful high through-put assay used receptor coupled beads with antibody binding detected using PE-conjugated anti-human IgG [167]. While these assays give valuable information about immune complex:FcγR binding, the stoichiometries of the interactions are not well defined.…”

Section: Fcγr Binding Assaysmentioning

confidence: 99%

Antibody functional assays as measures of Fc receptor-mediated immunity to HIV - new technologies and their impact on the HIV vaccine field.

Wines¹,

Billings²,

Mcleand³

et al. 2017

CHR

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Background:There is now intense interest in the role of HIV-specific antibodies and the engagement of FcγR functions in the control and prevention of HIV infection. The analyses of the RV144 vaccine trial, natural progression cohorts, and macaque models all point to a role for Fc-dependent effector functions, such as cytotoxicity (ADCC) or phagocytosis (ADCP), in the control of HIV. However, reliable assays that can be reproducibly used across different laboratories to measure Fcdependent functions, such as antibody dependent cellular cytotoxicity (ADCC) are limited. Method: This brief review highlights the importance of Fc properties for immunity to HIV, particularly via FcγR diversity and function. We discuss assays used to study FcR mediated functions of HIV-specific Ab, including our recently developed novel cell-free ELISA using homo-dimeric FcγR ectodomains to detect functionally relevant viral antigen-specific antibodies. Results: The binding of these dimeric FcγR ectodomains, to closely spaced pairs of IgG Fc, mimics the engagement and cross-linking of Fc receptors by IgG opsonized virions or infected cells as the essential prerequisite to the induction of Ab-dependent effector functions. The dimeric FcγR ELISA reliably correlates with ADCC in patient responses to influenza. The assay is amenable to high throughput and could be standardized across laboratories. Conclusion: We propose the assay has broader implications for the evaluation of the quality of antibody responses in viral infections and for the rapid evaluation of responses in vaccine development campaigns for HIV and other viral infections.

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Highly parallel characterization of IgG Fc binding interactions

Cited by 76 publications

References 54 publications

Rapid screening of IgG quality attributes – effects on Fc receptor binding

Rapid screening of IgG quality attributes – effects on Fc receptor binding

HIV-antibody complexes enhance production of type I interferon by plasmacytoid dendritic cells

Antibody functional assays as measures of Fc receptor-mediated immunity to HIV - new technologies and their impact on the HIV vaccine field.

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