A modified human alpha 2-macroglobulin derivative that binds tumor necrosis factor-alpha and interleukin-1 beta with high affinity in vitro and reverses lipopolysaccharide toxicity in vivo in mice.
The plasma protein alpha 2-macroglobulin (alpha 2M) has been reported to bind the proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta), which play a central role in the pathogenesis of chronic inflammatory disorders, including Crohn's disease and rheumatoid arthritis. In this study, we chemically modified alpha 2M to stabilize a conformation of the protein (termed MAC, Macroglobulin Activated for Cytokine binding) with greatly increased TNF-alpha- and IL-1 beta-binding activity. The equilibrium dissociation constant (KD) for the binding of TNF-alpha to MAC was 80 +/- 20 nM, reflecting a 100-fold increase in affinity compared with native alpha 2M. To test the ability of MAC to neutralize proinflammatory cytokines in vivo, we treated mice with lipopolysaccharide (LPS) by intravenous injection. When MAC (2.5 mg) was administered by intraperitoneal injection 1 hour before the LPS, 12 of 12 mice survived and were without signs of toxicity at 5 days. None of the mice survived in the untreated control group (0/26) or in the group treated with 2.5 mg of unmodified alpha 2M (0/4). MAC also prevented the large increase in expression of inducible nitric oxide synthase in the liver, kidneys, and heart of LPS-treated mice. A novel property of MAC, compared with previously studied anticytokine agents, was its ability to reverse LPS toxicity in 12 of 24 mice when administered after the plasma level of TNF-alpha was elevated. These studies demonstrate that a naturally occurring protein, alpha 2M, can be modified so that it acquires the properties of clinically active monoclonal antibodies. Thus, MAC may have therapeutic potential in the control of chronic inflammatory disorders.
Webb
D J
DJ
Department of Pathology, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
Gonias
S L
SL
D000818:Animals; D005260:Female; D006801:Humans; D007274:Injections, Intraperitoneal; D007275:Injections, Intravenous; D007375:Interleukin-1 / Q000378:metabolism*; D007457:Iodine Radioisotopes; D007700:Kinetics; D008070:Lipopolysaccharides / Q000008:administration & dosage / Q000037:antagonists & inhibitors* / Q000633:toxicity*; D051379:Mice; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II; D011485:Protein Binding / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D000511:alpha-Macroglobulins / Q000008:administration & dosage / Q000378:metabolism* / Q000494:pharmacology*
D007375:Interleukin-1; D007457:Iodine Radioisotopes; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D000511:alpha-Macroglobulins; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse