Characterization of different soluble TNF receptor (TNFR80) derivatives: positive influence of the intracellular domain on receptor/ligand interaction and TNF neutralization capacity.
Different soluble human TNFR80 derivatives, a solubilized form of the complete TNFR80, the TNFR80 extracellular domain, a secretory TNFR80 mutant (TR80TM-) with a deleted transmembrane region, and a TNFR80 immunoadhesin were produced in insect cells and characterized side by side with a recombinant human TNFR60 extracellular domain with respect to TNF binding affinity and neutralization of TNF bioactivity. The construct TR80TM- and the solubilized complete TNFR80 revealed a similar TNF binding and neutralization capacity, which was superior to the monovalent TNFR80 extracellular domain and comparable to the bivalent TNFR80 immunoadhesin, already known as a potent TNF antagonist. Determination of ligand off rate constants of the various receptor constructs by surface plasmon resonance revealed a correlation of low off rates with a high TNF neutralization capacity. We propose that the high TNF binding and neutralization capacity of the solubilized complete TNFR80 and TR80TM- in comparison with the monovalent extracellular TNR80 domain is due to a noncovalent self-aggregation of the receptors via their intracellular domain. This finding suggests that efficient soluble TNF antagonists can be derived from TNFR themselves without the need of construction of TNFR Ig Fc fusion proteins.
Moosmayer
D
D
Institute of Cell Biology and Immunology, University of Stuttgart, Germany.
Wajant
H
H
Gerlach
E
E
Schmidt
M
M
Brocks
B
B
Pfizenmaier
K
K
D003602:Cytotoxicity, Immunologic; D006801:Humans; D008024:Ligands; D009154:Mutation; D017434:Protein Structure, Tertiary; D018124:Receptors, Tumor Necrosis Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D012995:Solubility; D013329:Structure-Activity Relationship; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors*
D008024:Ligands; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha