[Show abstract][Hide abstract] ABSTRACT: The CD28-specific mAb TGN1412 rapidly caused a life-threatening "cytokine storm" in all six healthy volunteers in the Phase I clinical trial of this superagonist, signaling a failure of preclinical safety testing. We report novel in vitro procedures in which TGN1412, immobilized in various ways, is presented to human white blood cells in a manner that stimulates the striking release of cytokines and profound lymphocyte proliferation that occurred in vivo in humans. The novel procedures would have predicted the toxicity of this superagonist and are now being applied to emerging immunotherapeutics and to other therapeutics that have the potential to act upon the immune system. Data from these novel procedures, along with data from in vitro and in vivo studies in nonhuman primates, suggest that the dose of TGN1412 given to human volunteers was close to the maximum immunostimulatory dose and that TGN1412 is not a superagonist in nonhuman primates.
The Journal of Immunology 10/2007; 179(5):3325-31. DOI:10.4049/jimmunol.179.5.3325 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Interleukin 6 (IL-6) is a cytokine involved in many aspects of the acute phase and immune responses. Cloning of rat IL-6 cDNA into the pET-21d expression plasmid under control of a bacteriophage T7 RNA polymerase promoter system allowed isopropylthio-galactopyranoside (IPTG)-inducible production of recombinant rat IL-6 in Escherichia coli. The cloning, expression and purification of rat IL-6 is described. In this expression system, rat IL-6 was produced in insoluble inclusion bodies. The protein was solubilized in 6 M guanidine hydrochloride and refolded in a glutathione redox system. Refolded rat IL-6 was purified to homogeneity using anion-exchange chromatography on SP-Trisacryl. The purified recombinant rat IL-6 had a molecular mass of 21 756.38+/-0.25 Da, which is within 0.01% of the predicted value, taking into account cleavage of the N-terminal methionine residue and the formation of two disulfide bridges. Recombinant rat IL-6 was 2-3-fold more bioactive than the human standard preparation in the B9 hybridoma bioassay. Purified rat IL-6 was used to raise polyclonal antibodies in sheep and these reagents were used to develop a novel rat IL-6 enzyme-linked immunosorbent assay (ELISA). The ELISA is sensitive to 10 pg/ml and has been shown to detect IL-6 in plasma from rats injected with lipopolysaccharide (LPS).