Abstract
Erythropoietin (EPO) is a neurotrophic factor that could be developed as a new drug for brain disorders. However, EPO does not cross the blood-brain barrier (BBB). In the present study, human EPO was re-engineered by fusion to the carboxyl terminus of the heavy chain of a chimeric monoclonal antibody (MAb) to the human insulin receptor (HIR). The HIRMAb acts as a molecular Trojan horse to ferry the EPO into the brain via receptor-mediated transport on the endogenous BBB insulin receptor. The HIRMAb-EPO fusion protein was immunoreactive with antibodies to both human IgG and EPO. The HIRMAb-EPO fusion protein bound with high affinity to the extracellular domain of both the HIR (ED50 = 0.21 ± 0.05 nM) and the EPO receptor (ED50 = 0.30 ± 0.01 nM) and activated thymidine incorporation into human TF-1 cells with an ED50 of 0.1 nM. Differentially radiolabeled EPO and the HIRMAb-EPO fusion protein were injected intravenously into adult rhesus monkeys. Whereas EPO did not cross the primate BBB, the HIRMAb-EPO fusion protein was rapidly transported into brain, at levels that produce pharmacologic elevations in brain EPO at small systemic doses. The HIRMAb fusion protein selectively targeted the brain relative to peripheral organs. In conclusion, a novel IgG-EPO fusion protein has been engineered, expressed, and shown to be bifunctional with retention of high-affinity binding to both the insulin and EPO receptors. The IgG-EPO fusion protein represents a new class of EPO neurotherapeutics that has been specifically re-engineered to penetrate the human BBB.
Footnotes
This work was supported by ArmaGen Technologies.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.109.165092.
↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- EPO
- erythropoietin
- EPOR
- EPO receptor
- BBB
- blood-brain barrier
- MAb
- monoclonal antibody
- PD
- Parkinson's disease
- HIR
- human insulin receptor
- ID
- injected dose
- HC
- heavy chain
- LC
- light chain
- TV
- tandem vector
- SFM
- serum-free medium
- CHO
- Chinese hamster ovary
- TCA
- trichloroacetic acid
- VD
- volume of distribution
- PK
- pharmacokinetics
- PS
- permeability-surface area
- AUC
- area under the plasma concentration curve
- AUCss
- steady-state AUC
- AA
- amino acid
- ECD
- extracellular domain
- ELISA
- enzyme-linked immunosorbent assay
- DHFR
- dihydrofolate reductase
- PAGE
- polyacrylamide gel electrophoresis
- SEC
- size exclusion chromatography
- CSF
- cerebrospinal fluid
- PBS
- phosphate-buffered saline
- HPLC
- high-performance liquid chromatography.
- Received December 21, 2009.
- Accepted March 15, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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