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

Shalom A Gurjar,Jun X Wheeler,Meenu Wadhwa,Robin Thorpe,Ian Kimber,Jeremy P Derrick,Rebecca J Dearman,Clive Metcalfe

doi:10.1074/jbc.ra119.010637

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.

Highlights

The antiproliferative effect of trastuzumab is largely determined by HER2 antigen binding; we examined this in vitro using the HER2-expressing BT474 cell line
We have reported previously that the J9 (CD16-expressing) cell line– based luciferase reporter gene assay is a specific and sensitive surrogate method for the measurement of antibody-dependent cellular cytotoxicity (ADCC) activity [36]
Fc-mediated mAb function, such as ADCC or complement-dependent cytotoxicity (CDC), results in the killing of the target cell, and we have shown here that for all of the mAbs where assays were available, ADCC activity is diminished after Trx reduction

Summary

Introduction

In direct contrast to these observations, increased flexibility in the hinge region upon Trx reduction could account for the remarkable increase in TNF neutralization potency for infliximab and adalimumab. With full-length bivalent mAbs, large immune complexes can form between infliximab or adalimumab and trimeric TNF [53, 54]. The more rigid the mAb structure, the greater role that steric hindrance plays in restricting the size of these complexes. It is likely, that more flexible Trx-reduced mAbs could form larger complexes, effectively increasing the number of TNF molecules neutralized per molecule of mAb, effectively increasing the TNF neutralization potenc

Methods

Results

Conclusion