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

ABSTRACT: TNF? (tumour necrosis factor ?) 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-TNF? 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 TNF? 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 TNF? is consistent with approximately 12 Fabs binding to one TNF? trimer. A number of approaches were taken to map the epitopes recognized by AZD9773. These revealed that a number of different regions on TNF? 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 TNF? and suggest that the polyclonal nature may account, at least in part, for the very high potency observed in cell-based assays.

SUBMITTER: Abbott WM 

PROVIDER: S-EPMC3755337 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature