Frost, Sarah Chaney; (2024) Interchain disulfide bond reduction alters the structure and function of therapeutic monoclonal antibodies. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Anti-TNF IgG biotherapeutics are used to treat immune disorders such as rheumatoid arthritis. They neutralise cytokine TNF-α to dampen the inflammatory response. These drugs are hugely successful, for instance Humira manufactured by AbbVie was the second bestselling drug of 2021 globally. However in approximately one-third of patients, the efficacy of anti-TNF treatment decreases with time. There are several proposed hypotheses, such as increased production of anti-drug antibodies (ADAs), however there is no definitive explanation. It is now proposed that elevated levels of the enzyme thioredoxin (Trx), which increases with inflammation, reduces IgG interchain disulfide bonds, therefore altering the drug structure and efficacy. Trx is known to reduce these disulfides during biotherapeutic manufacture, which can lead to product loss. Additionally, in vitro reduction of anti-TNF biotherapeutics by Trx increases potency to antigen TNF, but simultaneously abrogates Fc-mediated effector functions. These mysterious findings were postulated to result from the reduction causing increased flexibility around the IgG hinge, which could explain the observed decrease in efficacy in vivo. This project uses infliximab (IFX) as a model anti-TNF biotherapeutic to explore the altered drug structure following reduction. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) shows the only structural change is in the IgG CH2 domain, a common destabilisation observed from many chemical modifications that explains knockout of Fc receptor binding. Transmission electron microscopy (TEM) proves increased flexibility of reduced IFX, with differing and more compact structures than native IFX. Importantly, a covalent interaction between reduced IFX and complement protein C1q is also identified in vitro. This is a novel finding of huge significance as it may have relevance for biotherapeutic mAbs in vivo. Overall this project contributes important structural information of reduced IgG1 mAbs and progresses the hypothesis for lack of efficacy of IgG biotherapeutics; Trx reduces interchain disulfides, causing immune complex formation with complement.

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