IgG-Single Chain Fv Fusion Protein Therapeutic for Alzheimers Disease: Expression in CHO Cells and Pharmacokinetics and Brain Delivery in the Rhesus Monkey

摘要

Monoclonal antibodies (MAb) directed against the Abeta amyloid peptide of Alzheimer's disease (AD) are potential new therapies for AD, since these antibodies disaggregate brain amyloid plaque. However, the MAb is not transported across the blood-brain barrier (BBB). To enable BBB transport, a single chain Fv (ScFv) antibody against the Abeta peptide of AD was re-engineered as a fusion protein with the MAb against the human insulin receptor (HIR). The HIRMAb acts as a molecular Trojan horse to ferry the ScFv therapeutic antibody across the BBB. Chinese hamster ovary (CHO) cells were stably transfected with a tandem vector encoding the heavy and light chains of the HIRMAb-ScFv fusion protein. A high secreting line was isolated following methotrexate amplification and dilutional cloning. The HIRMAb-ScFv fusion protein in conditioned serum free medium was purified by protein A affinity chromatography. The fusion protein was stable as a liquid formulation, and retained high affinity binding of both the HIR and the Abeta amyloid peptide. The HIRMAb-ScFv fusion protein was radiolabeled with the ¹²⁵I-Bolton-Hunter reagent, followed by measurement of the pharmacokinetics of plasma clearance and brain uptake in the adult Rhesus monkey. The HIRMAb-ScFv fusion protein was rapidly cleared from plasma and was transported across the primate BBB in vivo. In conclusion, the HIRMAb-ScFv fusion protein is a new class of antibody-based therapeutic for AD that has been specifically engineered to cross the human BBB.