A tumor necrosis factor (TNF) receptor-IgG heavy chain chimeric protein as a bivalent antagonist of TNF activity.
Using a multistep polymerase chain reaction method, we have produced a construct in which a cDNA sequence encoding the extracellular domain of the human 55-kD tumor necrosis factor (TNF) receptor is attached to a sequence encoding the Fc portion and hinge region of a mouse IgG1 heavy chain through an oligomer encoding a thrombin-sensitive peptide linker. This construct was placed downstream from a cytomegalovirus promoter sequence, and expressed in Chinese hamster ovary cells. A secreted protein, capable of binding TNF and inactivating it, was produced by the transfected cells. Molecular characterization revealed that this soluble version of the TNF receptor was dimeric. Moreover, the protein could be quantitatively cleaved by treatment with thrombin. However, the monovalent extracellular domain prepared in this way has a greatly reduced TNF inhibitory activity compared with that of the bivalent inhibitor. Perhaps because of its high affinity for TNF, the chimeric protein is far more effective as a TNF inhibitor than are neutralizing monoclonal antibodies. This molecule may prove very useful as a reagent for the antagonism and assay of TNF and lymphotoxin from diverse species in health and disease, and as a means of deciphering the exact mechanism through which TNF interacts with the 55-kD receptor.
Peppel
K
K
Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75235.
Crawford
D
D
Beutler
B
B
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D016466:CHO Cells; D002846:Chromatography, Affinity; D006224:Cricetinae; D006801:Humans; D007074:Immunoglobulin G / Q000502:physiology*; D007143:Immunoglobulin Heavy Chains / Q000502:physiology*; D008969:Molecular Sequence Data; D011956:Receptors, Cell Surface / Q000502:physiology*; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D013917:Thrombin / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D011956:Receptors, Cell Surface; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D013917:Thrombin