Genetic engineering of a bifunctional IgG fusion protein with iduronate-2-sulfatase.
Iduronate-2-sulfatase (IDS) is a lysosomal sulfatase that prevents the accumulation within the brain of glycosoaminoglycans. However, IDS does not cross the blood-brain barrier (BBB). To enable BBB transport, human IDS, minus its signal peptide, was fused to the carboxyl terminus of the heavy chain of a chimeric monoclonal antibody (mAb) to the human insulin receptor (HIR). The HIRMAb crosses the BBB on the endogenous insulin receptor and acts as a molecular Trojan horse to ferry the IDS into brain. The HIRMAb-IDS fusion protein was expressed in COS cells and purified with protein A affinity chromatography. The size of the fusion heavy chain, as measured with Western blotting and antibodies to either human IDS or human IgG, was increased about 80 kDa, relative to the size of the heavy chain of the parent HIRMAb. The HIRMAb-IDS fusion protein retained high-affinity binding for the HIR. The IDS enzyme specific activity of the fusion protein was 51 +/- 7 nmol/h per microgram of protein, which is comparable to the enzyme activity of recombinant IDS. The fusion protein was taken up by human fibroblasts, and the accumulation of glycosoaminoglycans in fibroblasts null for the sulfatase was decreased 84% by treatment with the fusion protein. The HIRMAb-IDS fusion protein is a bifunctional IgG-sulfatase fusion protein, which has been specifically engineered for targeted drug delivery across the human BBB.
Lu
Jeff Zhiqiang
JZ
ArmaGen Technologies, Inc., Santa Monica, CA 90401, USA.
Hui
Eric Ka-Wai
EK
Boado
Ruben J
RJ
Pardridge
William M
WM
D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D001812:Blood-Brain Barrier / Q000378:metabolism*; D015153:Blotting, Western; D019556:COS Cells; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity; D006224:Cricetinae; D016503:Drug Delivery Systems; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000737:chemistry / Q000378:metabolism; D005818:Genetic Engineering; D006025:Glycosaminoglycans / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007066:Iduronate Sulfatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000493:pharmacokinetics*; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D011972:Receptor, Insulin / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000493:pharmacokinetics*; D013379:Substrate Specificity
D006025:Glycosaminoglycans; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D011972:Receptor, Insulin; D007066:Iduronate Sulfatase