Assessment and Modulation of Therapeutic Monoclonal Antibody Function

Abstract

Background: Within the last 30 years, the use of therapeutic monoclonal antibodies (mAbs) has rapidly expanded. Research has broadened their scope of applicability and increasing numbers of novel and biosimilar mAbs are being approved. Antibody dependent cellular cytotoxicity (ADCC) is a key mechanism for oncology and autoimmune disease related mAbs which leads to the accelerated apoptosis of target cells, primarily via activation of natural killer (NK) effector cells. Subtle differences, particularly in the Fc region, can impact upon the level of ADCC that a mAb can elicit. Furthermore, it has been identified that the thioredoxin (Trx) system is responsible for interchain disulphide bond reduction during mAb manufacture, and is furthermore elevated in several inflammatory diseases and cancers, both of which are indicated for mAb therapy. Aims and hypothesis: As it is essential that sensitive assays are available to detect and study modifications to ADCC activity, we developed assays using two CD16 expressing transformed Jurkat T reporter gene cell lines as surrogate effector cells. As this readout is not subject to complex cellular regulation required for NK cell activation, greater signal sensitivity was expected. Use of a cell line also eliminates the use of donors, potentially conferring less variation and greater ease of use. With this, and a traditional ADCC assay using donor derived effectors, we explored the functional impact of disulphide bond reduction mediated by Trx system on mAb function. As this was likely results in a loss in structural stability in the hinge region, we expect an abrogation of Fc mediated function and thus also explored the impact on complement dependent cytotoxicity (CDC). Results: Both cell lines were demonstrated to generate a dose dependent luciferase response, exclusively in an ADCC mediating content, with both suspension and adherent cell lines. One cell line was further characterised and determined to be sensitive to mAb isotype and the presence and pattern, specifically fucosylation, of the N-lined glycan. Also, in parallel with CD16 affinity data and HPLC analysis of the N-glycan, the cell line revealed the highly similar potency of two anti-CD20 rituximab products against the innovator Mabthera. The surrogate effector cell line ased assay, alongside an optimised NK cell based assay using calcein-AM release as a readout for cytotoxicity, was used to assess the impact of Trx mediated disulphide bond reduction. They revealed that mAb ADCC activity is abrogated upon reduction. Furthermore a significant loss of complement dependent cytoxicity was observed. These functions were, however, recovered following the time dependent reformation of disulphide bonds. Conclusions: Overall I highlight the challenges of developing donor derived ADCC assays, which can in part be replaced by a sensitive and practical CD16 expressing surrogate effector cell line. These are not only useful from a regulatory perspective, but also for the development of biosimilars and research into mAb structure-function relationships to direct the design of more potent next generation mAbs. Furthermore, as Trx mediated reduction of mAbs completely abrogated both ADCC and CDC activity, this may impact mAb efficacy against diseases in which these mechanisms are relevant. However, as this effect was confirmed to be transient, it may not have an overall impact but certainly requires in vivo examination.

Date of Award	1 Aug 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Jeremy Derrick (Supervisor) & Rebecca Dearman (Supervisor)