title: Characterization of the complex formed between a potent neutralizing ovine-derived polyclonal anti-TNFα Fab fragment and human TNFα.
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.
authors.affiliation: AstraZeneca, Alderley Park, Macclesfield, Cheshire SK10 4TG, U.K. mark.abbott@astrazeneca.com
authors.forename: W Mark
authors.initials: WM
authors.lastname: Abbott
authors.forename: Melanie
authors.initials: M
authors.lastname: Snow
authors.forename: Sonia
authors.initials: S
authors.lastname: Eckersley
authors.forename: Jonathan
authors.initials: J
authors.lastname: Renshaw
authors.forename: Gareth
authors.initials: G
authors.lastname: Davies
authors.forename: Richard A
authors.initials: RA
authors.lastname: Norman
authors.forename: Peter
authors.initials: P
authors.lastname: Ceuppens
authors.forename: Jerry
authors.initials: J
authors.lastname: Slootstra
authors.forename: Joris J
authors.initials: JJ
authors.lastname: Benschop
authors.forename: Yoshitomo
authors.initials: Y
authors.lastname: Hamuro
authors.forename: Jessica E
authors.initials: JE
authors.lastname: Lee
authors.forename: Peter
authors.initials: P
authors.lastname: Newham
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D034561:Sheep, Domestic; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000276:immunology
chemical_list: C587873:AZD9773; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; C439524:TNF protein, human; D014409:Tumor Necrosis Factor-alpha