Neuroprotection in experimental stroke in the rat with an IgG–erythropoietin fusion protein

Erythropoietin (EPO) is a potent neuroprotective agent that could be developed as a new treatment for stroke. However, the blood–brain barrier (BBB) is intact in the early hours after stroke when neuroprotection is still possible, and EPO does not cross the intact BBB. To enable BBB transport, human EPO was re-engineered as an IgG–EPO fusion protein, wherein the IgG part is a monoclonal antibody (MAb) against the human insulin receptor (HIR). The HIRMAb acts as a BBB molecular Trojan horse to ferry the fused EPO across the BBB via transport on the BBB insulin receptor. The HIRMAb part of the HIRMAb–EPO fusion protein does not recognize the rat insulin receptor. However, the EPO part of the fusion protein does recognize the rat EPO receptor. Therefore, the neuroprotective properties of the HIRMAb–EPO fusion protein were investigated with a permanent middle cerebral artery occlusion model in the rat. The HIRMAb–EPO fusion protein was injected into the ipsilateral brain under stereotaxic guidance. High doses of the HIRMAb–EPO fusion protein (61 pmol) completely eliminated both cortical and sub-cortical infarction. Lower doses of the fusion protein (4.5 pmol) eliminated the cortical infarct with no significant effect on sub-cortical infarct. The neurologic deficit was reduced by 35% and 90%, respectively, by the 4.5 and 61 pmol doses of the HIRMAb–EPO fusion protein. In conclusion, these studies demonstrate the biological activity of the HIRMAb–EPO fusion protein in the brain in vivo, and that EPO retains neuroprotective properties following fusion to the HIRMAb BBB Trojan horse.

Research Highlights
► IgG–erythropoietin fusion protein reduces stroke volume in the rat.
► Improvement in neural deficit correlates with reduced stroke volume.
► IgG part is a genetically engineered monoclonal antibody to the insulin receptor.
► The IgG–erythropoietin is engineered to cross the blood–brain barrier.