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Delivery of a peptide radiopharmaceutical to brain with an IgG-avidin fusion protein.

The genetic engineering, host cell expression, purity, identity, and in vivo brain drug targeting properties are described for a new IgG-fusion protein, designated the cTfRMAb-AV fusion protein. Avidin (AV) is fused to the carboxyl terminus of the heavy chain of the genetically engineered chimeric monoclonal antibody (mAb) against the mouse transferrin receptor (TfR). The TfRMAb binds the endogenous TfR on the blood-brain barrier (BBB), which triggers transport into brain from blood. The cTfRMAb-AV fusion protein is produced in stably transfected Chinese hamster ovary cells, which are grown in serum free medium under conditions of biotin starvation. Following affinity purification, the purity and identity of the cTfRMAb-AV fusion protein were verified by electrophoresis and Western blotting. The affinity of the cTfRMAb for the murine TfR is high, K(I) = 4.6 ± 0.5 nM, despite fusion of avidin to the antibody heavy chain. The model peptide radiopharmaceutical used in this study is the Aβ(1-40) amyloid peptide of Alzheimer's disease (AD), which in a brain-penetrating form could be used to image the amyloid plaque in brain in AD. The BBB transport and brain uptake of the [(125)I]-Aβ(1-40) peptide was measured in mice injected intravenously (IV) with the peptide either free or conjugated to the cTfRMAb-AV fusion protein. The brain uptake of the free Aβ(1-40) peptide was very low, 0.1% of injected dose (ID)/gram brain following i.v. injection, and is comparable to the brain uptake of a brain blood volume marker. However, the brain uptake of the Aβ(1-40) peptide was high, 2.1 ± 0.2% ID/gram brain, following attachment of the biotinylated peptide to the cTfRMAb-AV fusion protein. Capillary depletion analysis showed the peptide penetrated the brain parenchyma from blood. The cTfRMAb-AV fusion protein is a new drug delivery system that can target to mouse brain monobiotinylated peptide or antisense radiopharmaceuticals.

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