IgG-Paraoxonase-1 Fusion Protein for Targeted Drug Delivery across the Human Blood-Brain Barrier

Paraoxonase (PON)-1 is the most potent human protein with organophosphatase activity against organophosphate (OP) toxins. OP compounds readily cross the blood-brain barrier (131313) and have lethal mechanisms of action within the brain. The production of a brain penetrating form of human PON1, which crosses the 131313, is possible with the re-engineering of the enzyme as a fusion protein with a monoclonal antibody (mAb) against the human insulin receptor (HIR). The HIRMAb crosses the 131313 via the endogenous insulin receptor, and acts as a molecular Trojan horse to ferry the POW into brain. The human POW was fused to the carboxyl terminus of the heavy chain of the chimeric HIRMAb. COS cells were dual transfected with the heavy chain gene and the light chain gene, and the HIRMAb-PON1 fusion protein was affinity purified with protein A chromatography. Western blotting with antibodies to human IgG or human PON1 showed the heavy chain of the HIRMAb-PON1 fusion protein was 40 kDa larger than the heavy chain of the chimeric HIRMAb. The ED50 of binding to the HIR extracellular domain was 0.55 +/- 0.07 nM and 1.1 +/- 0.1 nM, respectively, for the chimeric HIRMAb and the HIRMAb-PON1 fusion protein. The POW enzyme activity of the fusion protein was approximately 25% of the enzyme activity in human plasma, based on a fluorometric enzymatic assay. In conclusion, human POW has been re-engineered as an IgG-organophosphatase fusion protein that penetrates the human BBB.