Functional comparisons of different tumour necrosis factor receptor/IgG fusion proteins.
Nine different IgG fusion proteins and one non-fusion protein, all containing sequences from the extracellular domain of either of two human TNF receptors, were compared for their ability to bind and inhibit human TNF-alpha or TNF-beta. The fusion proteins differed with respect to 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 extracellular domain included in the construct. In vitro TNF binding assays and cytotoxicity assays indicated that, of the constructs that bound TNF, the greatest difference in affinity and neutralizing capability was between monovalent and bivalent receptor constructs. Differences were also noted between tetravalent and bivalent versions of p55 fusion proteins, as well as between a p75 fusion protein comprising the complete extracellular domain and one lacking the C-terminal 53 amino acids of the extracellular domain. p55 constructs that included only the second cysteine-rich domain (CRD) or only the second and third CRDs showed no TNF binding activity. The presence or absence of an IgG CH1 domain made no difference in the ability of fusion proteins to neutralize TNF-alpha or TNF-beta. Animal experiments comparing the tetravalent and bivalent p55 fusions and the effects of the CH1 domain did not show significant differences in their ability to protect mice from endotoxin-induced lethality, although the p55 fusion proteins appeared to be more protective than the p75 fusion proteins. Thus, this study has identified structural modifications to TNF receptor/IgG fusion proteins which have differing effects on their neutralizing ability towards TNF-alpha or TNF-beta.
Scallon
B J
BJ
Pharmaceutical Research, Centocor, Inc, Malvern, PA 19355, USA.
Trinh
H
H
Nedelman
M
M
Brennan
F M
FM
Feldmann
M
M
Ghrayeb
J
J
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D005803:Genes, Immunoglobulin; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007074:Immunoglobulin G / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors / Q000633:toxicity*; D008233:Lymphotoxin-alpha / Q000378:metabolism* / Q000494:pharmacology*; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D018124:Receptors, Tumor Necrosis Factor / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism* / Q000494:pharmacology*
D017931:DNA Primers; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D008070:Lipopolysaccharides; D008233:Lymphotoxin-alpha; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha