FUNCTIONAL COMPARISONS OF DIFFERENT TUMOR-NECROSIS-FACTOR RECEPTOR IGG FUSION PROTEINS

Nine different IgG fusion proteins and one non-fusion protein, all con taining sequences from the extracellular domain of either of two human TNF receptors, were compared for their ability to bind and inhibit hu man TNF-alpha or TNF-beta. The fusion proteins differed with respect t o TNF receptor type (p55 or p75 TNF receptor), receptor valency (one, two or four receptor domains per molecule), the presence or absence of a CH1 domain in the IgG constant region, and the proportion of the ex tracellular domain included in the construct. In vitro TNF binding ass ays and cytotoxicity assays indicated that, of the constructs that bou nd TNF, the greatest difference in affinity and neutralizing capabilit y was between monovalent and bivalent receptor constructs. Differences were also noted between tetravalent and bivalent versions of p55 fusi on proteins, as well as between a p75 fusion protein comprising the co mplete extracellular domain and one lacking the C-terminal 53 amino ac ids of the extracellular domain, p55 constructs that included only the second cysteine-rich domain (CRD) or only the second and third CRDs s howed no TNF binding activity. The presence or absence of an IgG CH1 d omain made no difference in the ability of fusion proteins to neutrali ze TNF-alpha or TNF-beta. Animal experiments comparing the tetravalent and bivalent p55 fusions and the effects of the CH1 domain did not sh ow significant differences in their ability to protect mice from endot oxin-induced lethality, although the p55 fusion proteins appeared to b e more protective than the p75 fusion proteins. Thus, this study has i dentified structural modifications to TNF receptor/IgG fusion proteins which have differing effects on their neutralizing ability towards TN F-alpha or TNF-beta.