Targeting a Homogeneously Glycosylated Antibody Fc To Bind Cancer Cells Using a Synthetic Receptor Ligand

Xiao, Junhua; Chen, Rui; Pawlicki, Mark A.; Tolbert, Thomas J.

doi:10.1021/ja9045179

Cited by 26 publications

(53 citation statements)

References 23 publications

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“…The high affinity polymorph (V158) of FcγRIIIA was cloned and expressed in yeast P. pastoris and purified using Ni 2+ -NTA affinity and phenyl sepharose chromatographies, as previously described (Okbazghi et al, 2016; Xiao et al, 2009). Different IgG3 Fc glycoforms were assessed for FcγRIIIA binding with BioLayer Interferometry (BLI) using BLItz.…”

Section: Methodsmentioning

confidence: 99%

Structural characterization of the Man5 glycoform of human IgG3 Fc

Shah

Lovell

Mehzabeen

et al. 2017

Molecular Immunology

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Immunoglobulin G (IgG) consists of four subclasses in humans: IgG1, IgG2, IgG3 and IgG4, which are highly conserved but have unique differences that result in subclass-specific effector functions. Though IgG1 is the most extensively studied IgG subclass, study of other subclasses is important to understand overall immune function and for development of new therapeutics. When compared to IgG1, IgG3 exhibits a similar binding profile to Fcγ receptors and stronger activation of complement. All IgG subclasses are glycosylated at N297, which is required for Fcγ receptor and C1q complement binding as well as maintaining optimal Fc conformation. We have determined the crystal structure of homogenously glycosylated human IgG3 Fc with a GlcNAc2Man5 (Man5) high mannose glycoform at 1.8 Å resolution and compared its structural features with published structures from the other IgG subclasses. Although the overall structure of IgG3 Fc is similar to that of other subclasses, some structural perturbations based on sequence differences were revealed. For instance, the presence of R435 in IgG3 (and H435 in the other IgG subclasses) has been implicated to result in IgG3-specific properties related to binding to protein A, protein G and the neonatal Fc receptor (FcRn). The IgG3 Fc structure helps to explain some of these differences. Additionally, protein-glycan contacts observed in the crystal structure appear to correlate with IgG3 affinity for Fcγ receptors as shown by binding studies with IgG3 Fc glycoforms. Finally, this IgG3 Fc structure provides a template for further studies aimed at engineering the Fc for specific gain of function.

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Section: Methodsmentioning

confidence: 99%

Structural characterization of the Man5 glycoform of human IgG3 Fc

Shah

Lovell

Mehzabeen

et al. 2017

Molecular Immunology

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“…58 was utilized) using glycerol (growth phase) and methanol (induction phase) as carbon sources in a NBS BioFlo 415 fermenter (Eppendorf). The starter culture (2 mL) was allowed to grow at 25°C for about 72 hrs in YPD media (1% yeast extract, 2% peptone, 5% glucose and zeocin 100 µg/ml).…”

Section: Methodsmentioning

confidence: 99%

Production, Characterization, and Biological Evaluation of Well-Defined IgG1 Fc Glycoforms as a Model System for Biosimilarity Analysis

Okbazghi

White

et al. 2016

Journal of Pharmaceutical Sciences

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Four different well-defined IgG1 Fc glycoforms are proposed as a model system to examine important biological and physicochemical features for protein drug biosimilar analyses. The IgG1 Fc glycoforms were produced by yeast expression combined with in vitro enzymatic synthesis as a series of sequentially truncated, high mannose IgG1 Fc glycoforms with an anticipated range of biological activity and structural stability. Initial characterization with mass spectrometry, SDS-PAGE, SEC, and cIEF confirmed the glycoproteins are overall highly similar with the only major difference being glycosylation state. Binding to the activating Fc receptor FcγRIIIa was used to evaluate the potential biological activity of the IgG1 Fc glycoproteins. Two complementary methods utilizing biolayer interferometry (BLI), one with protein G immobilized IgG1 Fc and the other with streptavidin immobilized FcγRIIIa, were developed to assess FcγRIIIa affinity in kinetic binding studies. The HM-Fc and Man5-Fc were highly similar to one another with high affinity for FcγRIIIa, while GlcNAc-Fc had weak affinity, and the non-glycosylated N297Q-Fc had no measurable affinity for FcγRIIIa. These four IgG1 Fc glycoforms were also evaluated in terms of physical and chemical stability profiles, and then used as model system to mathematically assess overall biosimilarity, as described in a series of companion papers.

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“…Furthermore, N-terminal fusion of a synthetic receptor ligand to IgG1-Fc and targeting of this mAb fragment to cancer cells was shown by Xiao et al (2009b). A completely different approach in functionalization of Fc fragments was introduced recently by Wozniak- Knopp et al (2010).…”

Section: Introductionmentioning

confidence: 99%