title: An anti-TNFR1 scFv-HSA fusion protein as selective antagonist of TNF action.
abstract: IZI-06.1 is a humanized anti-TNFR1 single-chain fragment variable (scFv) that selectively inhibits binding of tumor necrosis factor (TNF) and lymphotoxin alpha to tumor necrosis factor receptor 1 (TNFR1) but not TNFR2. Recently, IZI-06.1 was converted into a fully human IgG1 antibody (ATROSAB) for the treatment of inflammatory diseases. Here, we compare the bivalent ATROSAB with a monovalent scFv-human serum albumin (HSA) fusion protein lacking any antibody-associated effector functions and possessing approximately only half the molecular mass of an IgG, which should facilitate accumulation in inflamed tissues. Furthermore, the half-life of the scFv should be strongly extended while maintaining monovalent binding, avoiding a possible signal transduction by receptor cross-linking in the absence of TNF. The scFv-HSA fusion protein was produced by stably transfected Chinese hamster ovary cells and purified by affinity chromatography. The fusion protein bound specifically to TNFR1 in enzyme-linked immunosorbent assay and TNFR1-transfected mouse embryonic fibroblasts. Affinity determined by quartz crystal microbalance was reduced compared with ATROSAB, which resulted also in a reduced inhibitory activity. Compared with the scFv fragment, the half-life of the fusion protein was significantly increased, although not reaching the long half-life of ATROSAB. In summary, the scFv-HSA may provide an alternative to the full-length IgG1 with the ability to selectively inhibit TNFR1 and exploiting the pharmacokinetic properties of albumin.
authors.affiliation: Celonic, Karl-Heinz-Beckurts-Straße 13, 52428 Jülich, Germany.
authors.forename: Verena
authors.initials: V
authors.lastname: Berger
authors.forename: Fabian
authors.initials: F
authors.lastname: Richter
authors.forename: Kirstin
authors.initials: K
authors.lastname: Zettlitz
authors.forename: Felix
authors.initials: F
authors.lastname: Unverdorben
authors.forename: Peter
authors.initials: P
authors.lastname: Scheurich
authors.forename: Andreas
authors.initials: A
authors.lastname: Herrmann
authors.forename: Klaus
authors.initials: K
authors.lastname: Pfizenmaier
authors.forename: Roland E
authors.initials: RE
authors.lastname: Kontermann
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D006801:Humans; D051379:Mice; D015202:Protein Engineering; D055550:Protein Stability; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000493:pharmacokinetics; D012709:Serum Albumin / Q000235:genetics*; D015398:Signal Transduction; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000493:pharmacokinetics; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D047888:Receptors, Tumor Necrosis Factor, Type I; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D057127:Single-Chain Antibodies; D014409:Tumor Necrosis Factor-alpha
keywords: antagonist; human serum albumin; single-chain Fv; tumor necrosis factor; tumor necrosis factor receptor 1