Post-translational Modifications Differentially Affect IgG1 Conformation and Receptor Binding

Houde, Damian J.; Peng, Yucai; Berkowitz, Steven A.; Engen, John R.

doi:10.1074/mcp.m900540-mcp200

Cited by 370 publications

(376 citation statements)

References 52 publications

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“…No difference in binding was observed after oxidative stress (Table 3), which is generally found in the literature as well 14, 20. Only Bertolotti‐Ciarlet et al .…”

Section: Resultssupporting

confidence: 73%

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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“…No difference in binding was observed after oxidative stress (Table 3), which is generally found in the literature as well 14, 20. Only Bertolotti‐Ciarlet et al .…”

Section: Resultssupporting

confidence: 73%

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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“…Although it is known that amino acid oxidation can occur during sample preparation, it is tempting to speculate that the oxidative modification of certain circulating IgGs may have resulted from in vivo posttranslational modifications rather than processing artifacts, because plasma cells experience high levels of oxidative stress (34), and the t 1/2 of circulating Igs in serum is >1 wk, resulting in extensive exposure of antibodies to oxidizing conditions. L-Methionine oxidation has also been observed repeatedly in recombinantly expressed therapeutic antibodies from CHO cells (35,36). Additional studies will be needed to determine the extent of L-Methionine oxidation during sample processing and the frequency of in vivo modification.…”

Section: Discussionmentioning

confidence: 99%

Molecular deconvolution of the monoclonal antibodies that comprise the polyclonal serum response

Wine

Boutz

Lavinder

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

129

156

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We have developed and validated a methodology for determining the antibody composition of the polyclonal serum response after immunization. Pepsin-digested serum IgGs were subjected to standard antigen-affinity chromatography, and resulting elution, wash, and flow-through fractions were analyzed by bottom-up, liquid chromatography-high-resolution tandem mass spectrometry. Identification of individual monoclonal antibodies required the generation of a database of IgG variable gene (V-gene) sequences constructed by NextGen sequencing of mature B cells. Antibody V-gene sequences are characterized by short complementarity determining regions (CDRs) of high diversity adjacent to framework regions shared across thousands of IgGs, greatly complicating the identification of antigen-specific IgGs from proteomically observed peptides. By mapping peptides marking unique V H CDRH3 sequences, we identified a set of V-genes heavily enriched in the affinity chromatography elution, constituting the serum polyclonal response. After booster immunization in a rabbit, we find that the antigenspecific serum immune response is oligoclonal, comprising antibodies encoding 34 different CDRH3s that group into 30 distinct antibody V H clonotypes. Of these 34 CDRH3s, 12 account for ∼60% of the antigen-specific CDRH3 peptide mass spectral counts. For comparison, antibodies with 18 different CDRH3s (12 clonotypes) were represented in the antigen-specific IgG fraction from an unimmunized rabbit that fortuitously displayed a moderate titer for BSA. Proteomically identified antibodies were synthesized and shown to display subnanomolar affinities. The ability to deconvolute the polyclonal serum response is likely to be of key importance for analyzing antibody responses after vaccination and for more completely understanding adaptive immune responses in health and disease.antibody proteomics | antibody repertoire | serum immunoprofiling | B-cell response | humoral response T he first Nobel Prize in Medicine was awarded to Emil von Behring, who in collaboration with Kitasato Shibasaburo and Paul Ehrlich discovered serum antitoxins (1, 2). Remarkably, after more than 100 y of intense research in immunology, little is known about the clonality, relative concentrations, and binding properties of the monoclonal antibodies that constitute the antigen-specific Ig pool in serum.At steady state, circulating antibodies are produced by terminally differentiated B lymphocytes (plasma cells) within the bone marrow, and thus cannot be accessed in living individuals (3). Although recent single B-cell cloning methods (4, 5) have led to the identification of peripheral antigen-specific B memory and/or antibody-secreting cells (plasmablasts), it is generally unknown whether the Igs encoded by peripheral blood B cells correspond to the antibodies present in circulation and especially whether they are present at physiologically relevant levels (i.e., at serum concentrations above K D corresponding to >1 μg/mL for an average affinity of individual antibodies of 5 nM)...

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“…, 67-69 By carrying out enzymatic hyper-galactosylation across four batches of monoclonal antibodies produced from standard manufacturing processes in CHO cells, Thomann et al. demonstrated that hyper-galactosylation of antibody samples consistently leads to improvement in FcγRIIIa binding and ADCC 68 .…”

Section: Fc Galactosylation and Sialylation Also Modulate Igg1 Interamentioning

confidence: 99%

The “less-is-more” in therapeutic antibodies: Afucosylated anti-cancer antibodies with enhanced antibody-dependent cellular cytotoxicity

Pereira

Chan

Lin

et al. 2018

mAbs

267

211

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Therapeutic monoclonal antibodies are the fastest growing class of biological therapeutics for the treatment of various cancers and inflammatory disorders. In cancer immunotherapy, some IgG1 antibodies rely on the Fc-mediated immune effector function, antibody-dependent cellular cytotoxicity (ADCC), as the major mode of action to deplete tumor cells. It is well-known that this effector function is modulated by the N-linked glycosylation in the Fc region of the antibody. In particular, absence of core fucose on the Fc N-glycan has been shown to increase IgG1 Fc binding affinity to the FcγRIIIa present on immune effector cells such as natural killer cells and lead to enhanced ADCC activity. As such, various strategies have focused on producing afucosylated antibodies to improve therapeutic efficacy. This review discusses the relevance of antibody core fucosylation to ADCC, different strategies to produce afucosylated antibodies, and an update of afucosylated antibody drugs currently undergoing clinical trials as well as those that have been approved.

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Post-translational Modifications Differentially Affect IgG1 Conformation and Receptor Binding

Cited by 370 publications

References 52 publications

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

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

Molecular deconvolution of the monoclonal antibodies that comprise the polyclonal serum response

The “less-is-more” in therapeutic antibodies: Afucosylated anti-cancer antibodies with enhanced antibody-dependent cellular cytotoxicity

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