 I am William Pardridge and my colleagues at Armogen Technologies, Drs. Ruben Boato, Eric Wee, and Jeff Lu, and I recently published this paper in biotechnology and bioengineering. The paper describes a novel IgG enzyme fusion protein called AGT-182, which is developed as a new treatment of the brain in a lysosomal storage disorder called mucopolysacredosis type 2 or Hunter syndrome. The lysosomal enzyme mutated in Hunter syndrome is iduronate 2 sulfatase or IDS. About 70% of Hunter syndrome patients have severe brain involvement, where common IDS does not treat the brain because this enzyme does not cross the blood-brain barrier. This paper reports on the re-engineering of the IDS enzyme as a blood-brain barrier penetrating IgG fusion protein. The lack of transport of the IDS enzyme across the blood-brain barrier, or BVB, is depicted in this scheme of blood-to-brain transport. In contrast, circulating insulin does penetrate the blood-brain barrier via receptor-mediated transport on the endogenous insulin receptor, or IR, which is expressed on the endothelial cell of the brain capillary that forms the blood-brain barrier in vivo. Similar to insulin, certain peptidobametic monoclonal antibodies directed against the human insulin receptor, or HIR, also undergo receptor-mediated transport across the blood-brain barrier. The insulin receptor antibody is designated as HIRMAB in this slide. The genetic engineering of chimeric or humanized forms of the insulin receptor antibody is the genetic engineering of BVB penetrating IgG fusion protein, such as AGT182. The IDS enzyme is fused to the carboxyl terminus of each heavy chain of the insulin receptor antibody. High IDS enzyme activity and high affinity antibody binding to the insulin receptor are both retained following genetic engineering of the fusion protein. High affinity trojan horses are desired for blood-brain barrier delivery. A low affinity trojan horse has a low-level brain uptake. This reduced brain uptake requires the use of high systemic injection doses of the fusion protein, which results in a reduced therapeutic index of the pharmaceutical. Owing to the high affinity of the fusion protein for the insulin receptor, the fusion protein rapidly traverses the blood-brain barrier following intravenous administration and monkeys. The insulin receptor is also expressed on brain cells, which enables the receptor-meeted endocytosis of the fusion protein by target cells. The IDS of the domain of the fusion protein triages the fusion protein to the lysosomal compartment, as shown by confocal microscopy of cells from hunter syndrome donors. The differential brain uptake of the fusion protein versus the recombinant IDS enzyme was demonstrated in rhesus monkeys. The fusion protein and recombinant IDS were each radioidinated with the bulk hunter reaction and separately administered rhesus monkeys. The brain scans obtained two hours after intravenous injection show the profound effect on brain uptake of the IDS enzyme following fusion to the insulin receptor antibody. The brain scans on the left show a high-level brain uptake of the fusion protein in all parts of the brain. The calcium-autoradiography of the primate brain shows the fusion protein passes through the blood-brain barrier and distributes to all cells in the brain. The brain uptake of the fusion protein is 1% of injected dose per brain, a level that is comparable to the brain uptake of lipid-cybal small molecules in the primate. In contrast, the brain scans on the right show there is no brain uptake of the IDS enzyme alone. The residual radioactivity in the brain simply reflects sequestration of the IDS enzyme inside the plasma compartment of the brain with no transfer across the blood-brain barrier. Prior to clinical trials treatment of patients with hunter syndrome with the AGT-182 fusion protein. It is necessary to form chronic dosing studies and primates over a six-month period under good laboratory practice or GLP testing conditions. A total of 30 juvenile male monkeys were divided into four dosing groups of 0, 3, 10, or 30 milligram per kilogram fusion protein per week for 26 consecutive weeks. The fusion protein was administered by 60-minute constant intravenous infusion in 5% dextrose and normal saline. The animals were euthanized at 26 or 30 weeks. The pharmacokinetics or PK of plasma clearance of the fusion protein was monitored with both a sandwich elisa that measures immunoreactive fusion protein in plasma and by a fluorometric enzyme assay that measures plasma IDS enzyme activity using a physiologic substrate. The formation of anti-drug antibodies or ADAs in plasma during the course of the study was monitored with a second sandwich elisa. The plasma profiles of the immunoreactive fusion protein or the IDS enzyme activity are shown for the three doses of fusion protein. The maximal plasma concentration or CMAX of the immunoreactive fusion protein was 26, 120, and 420 microgram per male respectively for the three doses. The CMAX of plasma IDS enzyme activity was 9,000, 38,000, and 167,000 units per male respectively for the three doses. These CMAX values are greater than three law orders of magnitude higher than therapeutic concentrations of the fusion protein. The safety pharmacology studies are summarized in this slide. No signs of toxicity were observed with respect to any parameter and the study established a no observable adverse effect level or NOAAL 30 milligrams per kilogram which was the highest dose tested in the study. No animals exhibited in fusion-related reactions or signs of immune response during the course of the study. No pathologic lesions were noted in brain or any other organ in the monkeys following six months of chronic treatment. A favorable safety profile of the IgG fusion protein observed in this study parallels similar findings from prior work. The GLP study reported in the 2009 pharmaceutical search showed no toxicity in Rhesus monkeys following acute administration of the fusion protein of Gleel-derived neurotrophic factor or GDNF and the insulin receptor antibody. The GLP study reported in the 2013 bioconscript chemistry showed no toxicity in Rhesus monkeys following six months of chronic, weekly intravenous infusions of a fusion protein of the same insulin receptor antibody and another lysosomal enzyme, iduronidase. Hypoglycemia was observed only after infusion of the high-dose 30 milligram per kilogram of the fusion protein in normal saline. The hypoglycemia was eliminated by drug infusion and saline with dextrose. An intravenous glucose tolerance test performed at the end of six months of chronic treatment showed no change in glycemic control of the monkeys including the high-dose effusion protein. Safety studies have also been performed in mice effusion proteins wherein the Trojan horse domain is a transparent receptor antibody. The study reported in the 2011 drug metabolism and disposition showed no evidence of histopathology changes in brain, pancreas or other peripheral organs following 12 weeks of chronic treatment with the GDNF transparent receptor antibody effusion protein. The study reported in the 2013 molecular pharmaceutics showed no adverse effects from subcutaneous injections of 5 milligrams per kilogram of a bispecific fusion antibody given daily for 12 consecutive weeks. No mice in these studies showed any signs of complement fixation or immune reaction following fusion protein administration. In summary, this study demonstrates the favorable safety profile of the IgG-IDS fusion protein, a form of re-engineered enzyme that rapidly crosses the blood-brain barrier. The fusion protein is a potential new treatment of the brain in a serious lysosomal storage disorder. The contact information of the authors of this study is provided. Thank you.