Effect of trastuzumab interchain disulfide bond cleavage on Fcγ receptor binding and antibody-dependent tumour cell phagocytosis

Suzuki, Mami; Yamanoi, Ayaka; Machino, Yusuke; Ootsubo, Michiko; Izawa, Ken-ichi; Kohroki, Junya; Masuho, Yasuhiko

doi:10.1093/jb/mvv074

Cited by 7 publications

(3 citation statements)

References 31 publications

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“…The functional roles of allosteric disulfide bonds in blood and cancer cells have been extensively reviewed by Hogg et al [3, 21]. A recent report showed that reduction and alkylation of some disulfide bonds in rituximab and trastuzumab, IgG1-based drugs, increased the binding affinity of the modified drug to some Fc gamma receptor isotypes [22, 23], but also led to decreased binding to other Fc gamma receptors [23]. In some cases, mutation of Cys residues involved in disulfide bonds may have no effect on the protein’s biological activity, as is the case of an anti-CD44 IgG2 antibody where Cys to Ser mutations led to structural changes but had no impact on the binding of the protein to receptors and to complement C1 [24].…”

Section: Introductionmentioning

confidence: 99%

Recent mass spectrometry-based techniques and considerations for disulfide bond characterization in proteins

2017

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Disulfide bonds are important structural moieties of proteins: they ensure proper folding, provide stability, and ensure proper function. With the increasing use of proteins for biotherapeutics, particularly monoclonal antibodies, which are highly disulfide bonded, it is now important to confirm the correct disulfide bond connectivity and to verify the presence, or absence, of disulfide bond variants in the protein therapeutics. These studies help to ensure safety and efficacy. Hence, disulfide bonds are among the critical quality attributes of proteins that have to be monitored closely during the development of biotherapeutics. However, disulfide bond analysis is challenging because of the complexity of the biomolecules. Mass spectrometry (MS) has been the go-to analytical tool for the characterization of such complex biomolecules, and several methods have been reported to meet the challenging task of mapping disulfide bonds in proteins. In this review, we describe the relevant, recent MS-based techniques and provide important considerations needed for efficient disulfide bond analysis in proteins. The review focuses on methods for proper sample preparation, fragmentation techniques for disulfide bond analysis, recent disulfide bond mapping methods based on the fragmentation techniques, and automated algorithms designed for rapid analysis of disulfide bonds from liquid chromatography-MS/MS data. Researchers involved in method development for protein characterization can use the information herein to facilitate development of new MS-based methods for protein disulfide bond analysis. In addition, individuals characterizing biotherapeutics, especially by disulfide bond mapping in antibodies, can use this review to choose the best strategies for disulfide bond assignment of their biologic products. Graphical Abstract This review, describing characterization methods for disulfide bonds in proteins, focuses on three critical components: sample preparation, mass spectrometry data, and software tools.

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Section: Introductionmentioning

confidence: 99%

Recent mass spectrometry-based techniques and considerations for disulfide bond characterization in proteins

2017

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“…Previous studies have shown that the affinity of trastuzumab for its target antigen were unchanged following chemical (DTT) reduction and subsequent alkylation, whereas for rituximab, they were slightly increased (44, 45). Here, using more physiologically relevant enzymatic Trx reduction, we see a similar trend of increased antigen binding for rituximab and no change in antigen binding for trastuzumab, again indicating that the same disulfide bonds are reduced by DTT and Trx.…”

Section: Discussionmentioning

confidence: 99%

The impact of thioredoxin reduction of allosteric disulfide bonds on the therapeutic potential of monoclonal antibodies

Gurjar

Wheeler

Wadhwa

et al. 2019

Journal of Biological Chemistry

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Therapeutic mAbs are used to manage a wide range of cancers and autoimmune disorders. However, mAb-based treatments are not always successful, highlighting the need for a better understanding of the factors influencing mAb efficacy. Increased levels of oxidative stress associated with several diseases are counteracted by the activities of various oxidoreductase enzymes, such as thioredoxin (Trx), which also reduces allosteric disulfide bonds in proteins, including mAbs. Here, using an array of in vitro assays, we explored the functional effects of Trx-mediated reduction on the mechanisms of action of six therapeutic mAbs. We found that Trx reduces the interchain disulfide bonds of the mAbs, after which they remain intact but have altered function. In general, this reduction increased antigen-binding capacity, resulting in, for example, enhanced tumor necrosis factor (TNF) neutralization by two anti-TNF mAbs. Conversely, Trx reduction decreased the antiproliferative activity of an anti-tyrosine kinase-type cell-surface receptor HER2 mAb. In all of the mAbs, Fc receptor binding was abrogated by Trx activity, with significant loss in both complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) activity of the mAbs tested. We also confirmed that without alkylation, Trx-reduced interchain disulfide bonds reoxidize, and ADCC activity is restored. In summary, Trx-mediated reduction has a substantial impact on the functional effects of an mAb, including variable effects on antigen binding and Fc function, with the potential to significantly impact mAb efficacy in vivo.

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“…One end of FccR could combine with immune cells, while the other end with specific antibodies, thus triggering a series of functions of cellular effector molecules, like phagocytosis, antibody-dependent cell-mediated cytotoxicity, the production of super-oxygen ions, antigen presentation, the releasing of cytokines and the generation of regulatory antibodies [12][13][14][15]. According to their affinity to IgG subtypes, FccR could be categorized into FccRI, FccRII and FccRIII, with the middle one showing low affinity to IgG [16]. Based on its function, FccRII could be further classified into three subtypes, A, B and C [17].…”

Section: Introductionmentioning

confidence: 99%

CD32a polymorphism rs1801274 affects the risk of Kawasaki disease

Wang

Geng

2020

Artificial Cells, Nanomedicine, and Biotechnology

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Aim: To analyze the impact of CD32a polymorphism rs1801274 on the occurrence of Kawasaki disease (KD) through the meta-analysis. Methods: The correlation between CD32a polymorphism rs1801274 and the susceptibility to KD was appraised using summarized odds ratios (ORs) with their 95% confidence intervals (95% CIs). Besides, stratification analyses were further implemented on the basis of ethnicity and control source, respectively. Between-study heterogeneity was checked adopting chi-square-based Q test, with p < .05 as significant level. And results from Q test determined which model would be employed for OR calculation, fixed-or random-effects. Sensitivity analysis was accomplished to test the stability of final results. Potential publication bias among included studies was investigated using Begg's funnel plot and Egger's test. If publication bias was significant, its influence on overall estimates would be measured adopting the trim-and-fill method. Results: CD32a polymorphism rs1801274 significantly increased KD risk in total analysis under the comparisons of AA vs. GG, AA þ AG vs. GG, AA vs. GG þ AG, A vs. G and AG vs. GG (OR ¼ 2.69, 95% CI ¼ 1.39-5.20; OR ¼ 2.00, 95% CI ¼ 1.23-3.26; OR ¼ 1.90, 95% CI ¼ 1.23-2.94; OR ¼ 1.77, 95% CI ¼ 1.34-2.34; OR ¼ 1.53, 95% CI ¼ 1.07-2.19). After stratification analysis by ethnicity, similar tendency was also observed in Caucasian and Asian subgroups under corresponding genetic models. And parallel results were replicated in population-based and other-source subgroups after stratified analysis by control source, under some contrasts. Conclusion: CD32a polymorphism rs1801274 has strong relation to KD onset, and the presence of its A allele could elevate the disease incidence.

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Effect of trastuzumab interchain disulfide bond cleavage on Fcγ receptor binding and antibody-dependent tumour cell phagocytosis

Cited by 7 publications

References 31 publications

Recent mass spectrometry-based techniques and considerations for disulfide bond characterization in proteins

Recent mass spectrometry-based techniques and considerations for disulfide bond characterization in proteins

The impact of thioredoxin reduction of allosteric disulfide bonds on the therapeutic potential of monoclonal antibodies

CD32a polymorphism rs1801274 affects the risk of Kawasaki disease

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