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Re-engineering erythropoietin as an IgG fusion protein that penetrates the blood-brain barrier in the mouse.

Erythropoietin (EPO) is a potent neuroprotective agent that could be developed for the treatment of multiple brain disorders. However, EPO does not cross the blood-brain barrier (BBB). A brain-penetrating form of EPO, specific for the mouse, was engineered by fusion of the 166 amino acid EPO to the carboxyl terminus of the heavy chain of a chimeric monoclonal antibody (mAb) against the mouse transferrin receptor (TfR), and this new fusion protein is designated cTfRMAb-EPO. The fusion protein was expressed in stably transfected Chinese hamster ovary cells and purified by protein G chromatography. The fusion protein was homogeneous on SDS-PAGE and Western blotting, and bound the mouse EPO receptor (EPOR) with high affinity, ED50 = 0.33 ± 0.04 nM. The cTfRMAb-EPO fusion protein was radiolabeled by tritiation and injected intravenously (iv) into adult mice for measurements of the plasma pharmacokinetics and brain uptake. The (3)H-fusion protein was rapidly cleared from the blood with a clearance rate of 5.9 ± 0.3 mL/min/kg, which is 14-fold faster than the clearance of EPO in the mouse. The cTfRMAb-EPO fusion protein penetrated the BBB in vivo, as shown by the capillary depletion method. The brain uptake of the cTfRMAb-EPO fusion protein was 2.0 ± 0.1% injected dose/g of brain following iv administration. The high level of brain uptake of the fusion protein enables pharmacologic increases in exogenous EPO in the mouse brain following the systemic injection of the cTfRMAb-EPO fusion protein. In conclusion, EPO has been re-engineered as an IgG fusion protein that binds dual receptors: the mouse TfR, to enable penetration of the BBB, and the mouse EPOR, to produce neuroprotection in brain behind the intact BBB.