Construction, purification, and characterization of new interferon gamma (IFN gamma) inhibitor proteins. Three IFN gamma receptor-immunoglobulin hybrid molecules.
Three efficient mouse interferon gamma (MoIFN gamma) inhibitors were constructed, which consist of the MoIFN gamma receptor (MoIFN gamma R) extracellular portion and constant domains of immunoglobulin (Ig) molecules. These are: 1) the constant domain of the mouse kappa chain, 2) the hinge region and the constant domains 2 and 3 of the mouse gamma 2a chain, and 3) the hinge region and the constant domains 2 and 3 of the human gamma 3 chain. The hybrid molecules were expressed in the mouse myeloma cell line J558L and recovered from the supernatants of cell cultures in one purification step. The proteins MoIFN gamma R-M gamma 2a and MoIFN gamma R-H gamma 3 form homodimers, whereas MoIFN gamma R-M kappa is a monomer. All three constructs inhibit the binding of radiolabeled MoIFN gamma to its receptor on L1210 cells. They are biologically active in vitro, neutralizing the action of MoIFN gamma in an antiviral activity assay. The fusions of Ig regions to the soluble MoIFN gamma R do not decrease the affinity of the binding site for the ligand. MoIFN gamma R-M kappa has about the same affinity as the soluble MoIFN gamma R and the cell surface receptor of L1210 cells in situ, which are also monomers, whereas the dimers MoIFN gamma R-M gamma 2a and MoIFN gamma R-H gamma 3 display a 5-10-fold higher affinity for MoIFN gamma than the monomeric molecules. This is best documented in the efficacy of the inhibitors to antagonize the antiviral activity of MoIFN gamma, as the dimeric constructs are about 10 times more active than MoIFN gamma R-M kappa and the soluble MoIFN gamma R. The hybrid constructs can be used as high efficiency MoIFN gamma inhibitors in mouse models of several pathological states in humans, where IFN gamma is thought to play a disease-promoting role.
Kürschner
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Department of Biology, Pharmaceutical Research New Technologies, F. Hoffmann-LaRoche Ltd., Basel, Switzerland.
Garotta
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Dembić
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D000818:Animals; D000998:Antiviral Agents; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D007136:Immunoglobulins / Q000235:genetics*; D007371:Interferon-gamma / Q000037:antagonists & inhibitors* / Q000378:metabolism; D007939:Leukemia L1210; D051379:Mice; D008969:Molecular Sequence Data; D009101:Multiple Myeloma; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011971:Receptors, Immunologic / Q000235:genetics*; D017471:Receptors, Interferon; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
D000998:Antiviral Agents; D007136:Immunoglobulins; D011971:Receptors, Immunologic; D017471:Receptors, Interferon; D011993:Recombinant Fusion Proteins; D007371:Interferon-gamma; D004247:DNA