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Human IgG Fc domain engineering enhances antitoxin neutralizing antibody activity.

ABSTRACT: The effector activity of antibodies is dependent on engagement with Fc? receptors (Fc?Rs) and activation of the associated intracellular signaling pathways. Preclinical evaluation of therapeutic humanized or chimeric mAbs to study the interactions of their Fc regions with Fc?Rs is hampered by substantial structural and functional Fc?R diversity among species. In this report, we used mice expressing only human Fc?Rs to evaluate the contribution of Fc?R-mediated pathways to the neutralizing activity of an anti-anthrax toxin chimeric mAb. We observed that the protective activity of this mAb was highly dependent upon Fc?R engagement, with minimal protection against anthrax toxin observed in Fc?R-deficient mice following mAb administration. We generated anti-anthrax toxin mAbs with specific Fc domain variants with selectively enhanced affinity for particular human Fc?Rs and assessed their activity in Fc?R-humanized mice. We determined that Fc domain variants that were capable of selectively engaging activating Fc?Rs substantially enhanced the in vitro and in vivo activity of anthrax toxin-neutralizing antibodies. These findings indicate that the application of Fc domain engineering is a feasible strategy to enhance toxin-neutralizing activity and suggest that engineered antitoxin antibodies will have improved therapeutic efficacy.

SUBMITTER: Bournazos S

PROVIDER: S-EPMC3904629 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Human IgG Fc domain engineering enhances antitoxin neutralizing antibody activity.

Bournazos Stylianos S Chow Siu-Kei SK Abboud Nareen N Casadevall Arturo A Ravetch Jeffrey V JV

The Journal of clinical investigation 20140109 2

The effector activity of antibodies is dependent on engagement with Fcγ receptors (FcγRs) and activation of the associated intracellular signaling pathways. Preclinical evaluation of therapeutic humanized or chimeric mAbs to study the interactions of their Fc regions with FcγRs is hampered by substantial structural and functional FcγR diversity among species. In this report, we used mice expressing only human FcγRs to evaluate the contribution of FcγR-mediated pathways to the neutralizing activi ...[more]

PMID: 24401277

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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 Fc? 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 ?-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 Fc?RIIIa, the activating Fc? 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 Fc?RIIIa. On the orther hand, the presence of a bisecting GlcNAc or core-fucosylation had little effect on the affinity of Fc to the inhibitory Fc? receptor, Fc?RIIb. Our experimental data also showed that the ?-linked mannose residues in the pentasaccharide Man3GlcNAc2 core was essential to maintain a high affinity of Fc to both Fc?RIIIa and Fc?RIIb. 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.

Dataset Information

Human IgG Fc domain engineering enhances antitoxin neutralizing antibody activity.

Publications

Human IgG Fc domain engineering enhances antitoxin neutralizing antibody activity.

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