Very High Plasma Concentrations of a Monoclonal Antibody against

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Very high plasma concentrations of a monoclonal antibody against the human insulin receptor are produced by subcutaneous injection in the Rhesus monkey Ruben J. Boado, Eric Ka-Wai Hui, Jeff Zhiqiang Lu, and William M. Pardridge Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00456 • Publication Date (Web): 11 Aug 2016 Downloaded from http://pubs.acs.org on August 17, 2016

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Molecular Pharmaceutics

Very high plasma concentrations of a monoclonal antibody against the human insulin receptor are produced by subcutaneous injection in the Rhesus monkey

Ruben J. Boado Eric Ka-Wai Hui Jeff Zhiqiang Lu William M. Pardridge

ArmaGen, Inc. Calabasas, California 91302, United States

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Address correspondence to: Dr. William M. Pardridge ArmaGen, Inc. 26679 Agoura Road, Suite 100 Calabasas, CA 91302 Ph: 818-252-8200 Fax: 818-252-8214 Email: [email protected]

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Table of Contents Graphic:


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Abstract Brain penetration of recombinant protein drugs is possible following the re-engineering of the drug as an IgG fusion protein. The IgG domain is a monoclonal antibody (MAb) against an endogenous blood-brain barrier (BBB) receptor transporter, such as the insulin receptor. One such MAb targets the human insulin receptor (HIR), and is active in Rhesus monkeys. Prior work has measured the plasma pharmacokinetics of HIRMAb-derived fusion proteins following intravenous (IV) infusion. However, an alternative method of administration for chronic treatment of brain disease is the subcutaneous (SQ) route. The extent to which an antibody against the insulin receptor undergoes systemic distribution and clearance is unknown. Therefore, in the present study, the rate of plasma clearance of the HIRMAb is measured in Rhesus monkeys following IV or SQ administration of 3, 10, and 30 mg/kg doses of the antibody. The HIRMAb is readily absorbed into the systemic circulation following SQ injection with a 42% plasma bioavailability. The rate of plasma clearance of the antibody, 0.04-0.06 mL/min/kg, is the same following either IV or SQ administration. Owing to the slow rate of plasma clearance of the antibody, high concentrations of the HIRMAb are sustained in plasma for days after the SQ injection. The plasma concentration of the HIRMAb exceeds 0.8 mg/mL, which is 9% of the entire plasma IgG pool in the primate, after the SQ injection of the high dose, 30 mg/kg, of the antibody. In summary, the pharmacokinetics of plasma clearance of the HIRMAb are such that HIRMAb-derived fusion proteins can be developed as protein therapeutics for the brain with chronic SQ administration on a weekly or twice-weekly regimen. Keywords: monoclonal antibody, insulin receptor, blood-brain barrier, IgG fusion protein, pharmacokinetics


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Introduction Recombinant protein drugs are large molecule pharmaceuticals that do not cross the blood-brain barrier (BBB). Therefore, development of such biologics as drugs for the brain requires re-engineering of the drug with a BBB delivery system.1 One approach is the use of molecular Trojan horse technology, where the biologic drug is re-engineered as an IgG fusion protein.2 The IgG domain binds an endogenous receptor-mediated transport system on the BBB, such as the insulin receptor or transferrin receptor. The receptor-specific antibody undergoes receptor-mediated transport across the BBB and carries into brain the fused biologic drug. Prior work has described the genetic engineering of IgG-lysosomal enzyme fusion proteins, where the IgG domain is a monoclonal antibody (MAb) against the human insulin receptor (HIR), and the enzyme domain is either iduronidase (IDUA)3 or iduronate 2-sulfatase (IDS).4 The HIRMAbIDUA and HIRMAb-IDS fusion proteins are designed for the treatment of the brain in lysosomal enzyme storage disorders by weekly intravenous (IV) infusions, similar to standard enzyme replacement therapy. However, an alternative mode of chronic treatment with biologic agents is weekly or twice-weekly subcutaneous (SQ) injections of the drug.5 The extent to which the HIRMAb undergoes systemic distribution and plasma clearance following SQ administration is unknown. The present study was designed to measure the plasma pharmacokinetics (PK) of the HIRMAb following repeat SQ dosing, and to determine the plasma bioavailability of the antibody. The HIRMAb cross reacts with the insulin receptor of Old World primates,6 such as the Rhesus monkey, but does not cross react with the rodent insulin receptor.7 Therefore, these PK studies were performed in Rhesus monkeys. The PK analysis was performed at days 1 and 21 of chronic dosing, where the HIRMAb was administered by SQ injection of doses of 3, 10, and


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30 mg/kg given every 3-4 days for 3 weeks. The PK of plasma clearance of the HIRMAb was also measured following IV infusion of the antibody in monkeys at doses of 3, 10, and 30 mg/kg.


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Experimental Section Production of HIRMAb. Chinese hamster ovary (CHO) cells were stably transfected with the genes encoding the heavy chain and the light chain of the HIRMAb, a chimeric antibody derived from the variable region of a murine antibody against the human insulin receptor, and the constant regions of human IgG1κ.8 The transfected CHO cells were adapted to serum free medium, and propagated in a 50L perfusion mode bioreactor with a 25L bed volume. The bioreactor was maintained for 34 days, the viable cell density reached 16-20 million cells/mL, the medium IgG concentration was >200 mg/L, and the IgG production in pg/cell/day averaged 12. Following purification with protein A affinity chromatography, SP Sepharose cation exchange chromatography, and nano-filtration, a total of 58 grams was produced, vialed at 13 mg/mL, and stored at 4°C in 10 mM sodium acetate/140 mM NaCl/0.01% Tween-80/pH=5.5. The final drug product passed acceptance criteria for 15 test methods of concentration, quality, identity, potency, and purity. The affinity of the HIRMAb for the HIR extracellular domain (ECD) was measured by ELISA, and the ED50 was 0.48 ± 0.03 nM. The CHO cell host protein was 0.3 parts per million (PPM), recombinant protein A was 13 PPM, and the CHO cell host DNA was

Very High Plasma Concentrations of a Monoclonal Antibody against

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