Combined glyco- and protein-Fc engineering simultaneously enhance cytotoxicity and half-life of a therapeutic antibody

Monnet, Céline; Jorieux, Sylvie; Souyris, Nathalie; Zaki, Ouafa; Jacquet, Alexandra; Fournier, Nathalie; Crozet, Fabien; Romeuf, Christophe de; Bouayadi, Khalil; Urbain, Rémi; Behrens, Christian K.; Mondon, Philippe; Fontayne, Alexandre

doi:10.4161/mabs.27854

Cited by 41 publications

(52 citation statements)

References 67 publications

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“…IgG1/FcRn binding. 25,27 Using the residue numbering scheme of mAb-A or mAb-E, these mutations are P233S/T, F244L, T253Q, V267A/E, N318D, A333V, N364D, A381V/T, M431L). 25,27 Several of these previously reported mutations (e.g., F244L, T253Q, V267A/E, N318D, and A333V) are in the peptide segments (HC 244-255, HC 269-280, HC 320-332) that showed increased local flexibility due to the YTE-mutations in our work.…”

Section: Discussionmentioning

confidence: 99%

“…25,27 Using the residue numbering scheme of mAb-A or mAb-E, these mutations are P233S/T, F244L, T253Q, V267A/E, N318D, A333V, N364D, A381V/T, M431L). 25,27 Several of these previously reported mutations (e.g., F244L, T253Q, V267A/E, N318D, and A333V) are in the peptide segments (HC 244-255, HC 269-280, HC 320-332) that showed increased local flexibility due to the YTE-mutations in our work. Hence, it can be inferred that residues in the segments with increased flexibility in the C H 2 domain and the C Furthermore, recent studies have shown that mAbs with identical F c but differing F ab sequences, 52 as well as mAbs and F c fusion proteins with the same F c sequences, 53 can bind FcRn differentially with varying pK properties.…”

Section: Discussionmentioning

confidence: 99%

“…14,[22][23][24][25][26][27][28] For example, the increased FcRn binding at pH 6.0 by a YTE triple-mutant mAb is mediated by the creation of one additional salt bridge between Glu 256 (E) of Fc-YTE and Gln 2 (Q) of the b2-microglobulin chain of FcRn compared to the original IgG1 Fc structure. 29 The YTE mutations (M252Y/ S254T/T256E) in the C H 2 domain of an IgG1 mAb resulted in a nearly four-fold higher in vivo half-life in cynomolgus monkeys, the largest increase in IgG mAb half-life reported to-date for non-human primates.…”

Section: Introductionmentioning

confidence: 99%

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Correlations between changes in conformational dynamics and physical stability in a mutant IgG1 mAb engineered for extended serum half-life

Majumdar

Esfandiary²,

Bishop³

et al. 2015

mAbs

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(2015) Correlations between changes in conformational dynamics and physical stability in a mutant IgG1 mAb engineered for extended serum half-life, mAbs, 7:1, 84-95, DOI: 10.4161/19420862.2014.985494 To link to this article: https://doi.org/10. 4161/19420862.2014 This study compares the local conformational dynamics and physical stability of an IgG1 mAb (mAb-A) with its corresponding YTE (M255Y/S257T/T259E) mutant (mAb-E), which was engineered for extended half-life in vivo. Structural dynamics was measured using hydrogen/deuterium (H/D) exchange mass spectrometry while protein stability was measured with differential scanning calorimetry (DSC) and size exclusion chromatography (SEC). The YTE mutation induced differences in H/D exchange kinetics at both pH 6.0 and 7.4. Segments covering the YTE mutation sites and the FcRn binding epitopes showed either subtle or no observable differences in local flexibility. Surprisingly, several adjacent segments in the C H 2 and distant segments in the V H , C H 1, and V L domains had significantly increased flexibility in the YTE mutant. Most notable among the observed differences is increased flexibility of the 244-254 segment of the C H 2 domain, where increased flexibility has been shown previously to correlate with decreased conformational stability and increased aggregation propensity in other IgG1 mAbs (e.g., presence of destabilizing additives as well as upon de-glycosylation or methionine oxidation). DSC analysis showed decreases in both thermal onset (T onset ) and unfolding (T m 1) temperatures of 7 C and 6.7 C, respectively, for the C H 2 domain of the YTE mutant. In addition, mAb-E aggregated faster than mAb-A under accelerated stability conditions as measured by SEC analysis. Hence, the relatively lower physical stability of the YTE mutant correlates with increased local flexibility of the 244-254 segment, providing a site-directed mutant example that this segment of the C H 2 domain is an aggregation hot spot in IgG1 mAbs.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Correlations between changes in conformational dynamics and physical stability in a mutant IgG1 mAb engineered for extended serum half-life

Majumdar

Esfandiary²,

Bishop³

et al. 2015

mAbs

View full text Add to dashboard Cite

show abstract

“…[43][44][45][46][47] More recently, mutations in these residues have exemplified the complex binding relationship of FcRn and IgG that modulates in vivo half-life, while highlighting the importance of both association at acidic pH (6.0) and dissociation at physiological pH (7.4). 11,18,23,37,38,[48][49][50][51][52][53][54][55] Independent of the direct mAb-FcRn contact within the IgG constant region (Fc), additional studies have determined the antigen binding fragment (Fab) can have a significant effect on the mAb-FcRn interaction and in vivo half-life. 25,56,57 This phenomenon explains why circulating IgGs with identical Fc regions can vary 2-3 fold in serum half-life even when the same antigen is targeted.…”

Section: Introductionmentioning

confidence: 99%

A novel in vitro assay to predict neonatal Fc receptor-mediated human IgG half-life

Souders

Nelson²,

Wang

et al. 2015

mAbs

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Immunoglobulin G (IgG) has an unusually long serum half-life in comparison to proteins of a similar size. It is wellknown that this phenomenon is due to IgG's ability to bind the neonatal Fc receptor (FcRn) in a pH-dependent manner. FcRn binding properties can vary among IgGs, resulting in altered in vivo half-lives, and therefore it would be beneficial to accurately predict the FcRn binding properties of therapeutic IgG monoclonal antibodies (mAbs). Here we describe the development of an in vitro model capable of predicting the in vivo half-life of human IgG. Using a high-throughput biolayer interferometry (BLI) platform, the human FcRn association rate at acidic pH and subsequent dissociation rate at physiological pH was determined for 5 human IgG1 mAbs. Comparing the combined FcRn association and dissociation rates to the Phase 1 clinical study half-lives of the mAbs resulted in a strong correlation. The correlation was also verified in vivo using mice transgenic for human FcRn. The model was used to characterize various factors that may influence FcRn-mAb binding, including mAb variable region sequence differences and constant region glycosylation patterns. Results indicated that the complementarity-determining regions of the heavy chain significantly influence the mAb's FcRn binding properties, while the absence of glycosylation does not alter mAb-FcRn binding. Development of this high-throughput FcRn binding model could potentially predict the half-life of therapeutic IgGs and aid in selection of lead candidates while also serving as a screening tool for the development of mAbs with desired pharmacokinetic properties.

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“…For instance, antibody glyco-engineering, to improve their ADCC and cytotoxicity, and protein-engineering, to increase the Fc domain affinity for the neonatal Fc receptor (FcRn) and thus the antibody half-life, are promising approaches to optimize the direct therapeutic effects of mAbs. 14 However, a new concept has recently emerged. In parallel to their direct short-term effects, mAbs are now also considered immunomodulatory molecules that can recruit Fc-receptorexpressing innate immune cells to induce a long-term endogenous adaptive immune response (vaccine-like effect) that is responsible for the better and sustained control of tumor development observed in some patients.…”

Section: Ta-targeting Mabs: More Than Just Direct Effectsmentioning

confidence: 99%

Tumor antigen-targeting monoclonal antibody-based immunotherapy: Orchestrating combined strategies for the development of long-term antitumor immunity

et al. 2014

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Combined glyco- and protein-Fc engineering simultaneously enhance cytotoxicity and half-life of a therapeutic antibody

Cited by 41 publications

References 67 publications

Correlations between changes in conformational dynamics and physical stability in a mutant IgG1 mAb engineered for extended serum half-life

Correlations between changes in conformational dynamics and physical stability in a mutant IgG1 mAb engineered for extended serum half-life

A novel in vitro assay to predict neonatal Fc receptor-mediated human IgG half-life

Tumor antigen-targeting monoclonal antibody-based immunotherapy: Orchestrating combined strategies for the development of long-term antitumor immunity

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