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

Abstract

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.

The NRF2, Thioredoxin, and Glutathione System in Tumorigenesis and Anticancer Therapies

“…TRX alters the function of therapeutic monoclonal antibodies by reducing the antibodies' interchain disulfide bonds. [219] Joint inhibition of TRX, GSH, and NRF2 promotes intracellular ROS and suppresses the growth of head and neck cancer cells. [220] TRX phosphorylation at T100 attributes to its anti-apoptotic effects in tumor cells.…”

HLAII peptide presentation of infliximab increases when complexed with TNF

“…Overall, measurements of size of the TNF antagonists alone were consistent with previous reports based on DLS and other biophysical assessments ( 50 – 55 ). Of note, the distinct difference in size observed for the bivalent antibodies ADAL and INFL (10.5 ± 1.0 nm and 17.5 ± 2.4 nm, respectively), despite their equivalent molecular weight (MW, 56 ), may result from differential physicochemical properties as previously suggested ( 50 – 55 , 57 – 60 ). Reversible self-association of INFL has been associated with an apparent size increase ( 61 , 62 ).…”

A Review of Methodologies for the Detection, Quantitation, and Localization of Free Cysteine in Recombinant Proteins: A Focus on Therapeutic Monoclonal Antibodies

“…More recently, we developed a differential alkylation strategy to investigate mAbs that does not require stable isotope pairs ( Gurjar et al, 2019 ). Instead, IAA is used to initially alkylate native free-Cys, and then NEM is subsequently used to alkylate free-Cys liberated upon denaturation and reduction.…”

“…Disulfide bonds are represented by bars, with green being disulfide bonds buried within Ig domains and red being exposed interchain disulfide bonds. Reproduced with permission from Gurjar et al, 2019 .…”

“…The majority of detected free-Cys arises from incomplete processing within the host cell during manufacture where high conditions of cellular stress are encountered or through extracellular reduction by intercellular host proteins such as thioredoxin in the harvest and purification of mAbs. Free-Cys arising from disulfide bond reduction in mAbs is undesirable due to the negative effects this has on affinity ( Harris, 2005 ), functions ( Gurjar et al, 2019 ), aggregation ( Trivedi et al, 2009 ; Buchanan et al, 2013 ; Chung et al, 2017 ), and stability ( Lacy et al, 2008 ); manufacturers go to great lengths to minimize the amount of free-Cys in therapeutic mAb preparations ( Trexler-Schmidt et al, 2010 ). Although as yet there are no guidelines from regulators on acceptable levels, manufacturers justify the levels on a safety and efficacy basis for each product.…”