Characterization of the complex formed between a potent neutralizing ovine-derived polyclonal anti-TNFα Fab fragment and human TNFα

W Mark Abbott,Gareth Davies,Peter Ceuppens,Peter Newham,Joris J Benschop,Sonia Eckersley,Melanie Snow,Jerry Slootstra,Jessica E Lee,Richard A Norman,Jonathan Renshaw,Yoshitomo Hamuro

doi:10.1042/bsr20130044

W Mark Abbott, Gareth Davies + Show 10 more

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https://doi.org/10.1042/bsr20130044

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Abstract

TNFα (tumour necrosis factor α) is an early mediator in the systemic inflammatory response to infection and is therefore a therapeutic target in sepsis. AZD9773 is an ovine-derived, polyclonal anti-TNFα Fab fragment derived from a pool of serum and currently being developed as a treatment for severe sepsis and septic shock. In the present study, we show that although AZD9773 has a modest affinity for TNFα in a binding assay, the Ki in a cell-based assay is approximately four orders of magnitude lower. We show using SEC (size exclusion chromatography) that the maximum size of the complex between AZD9773 and TNFα is consistent with approximately 12 Fabs binding to one TNFα trimer. A number of approaches were taken to map the epitopes recognized by AZD9773. These revealed that a number of different regions on TNFα are involved in binding to the polyclonal Fab. The data suggest that there are probably three epitopes per monomer that are responsible for most of the inhibition by AZD9773 and that all three can be occupied at the same time in the complex. We conclude that AZD9773 is clearly demonstrated to bind to multiple epitopes on TNFα and suggest that the polyclonal nature may account, at least in part, for the very high potency observed in cell-based assays.

Highlights

tumour necrosis factor α (TNFα) is a potent pro-inflammatory cytokine that is the first to be released in response to infection and can trigger the complete spectrum of responses characteristic of endotoxic shock [1,2,3]
The key role that TNFα plays in the inflammatory response has made it a very attractive target in inflammatory diseases, and biologic agents are widely used to suppress its action in rheumatoid arthritis, psoriasis and Crohn’s disease [3,4,5]
A TNFα column, this was not done as it has two disadvantages: firstly, it would have required very large quantities of TNFα, and secondly, the elution conditions would likely elute some TNFα as it is a non-covalent trimer

Summary

Introduction

TNFα (tumour necrosis factor α) is a potent pro-inflammatory cytokine that is the first to be released in response to infection and can trigger the complete spectrum of responses characteristic of endotoxic shock [1,2,3]. The affinity of AZD9773 was determined by immobilizing the Fab directly onto a sensor chip, injecting different concentrations of hTNFα, and allowing the system to reach steady-state binding after 360 s.

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