Delivery of Β-galactosidase to Mouse Brain via the Blood-brain Barrier Transferrin Receptor

Enzyme replacement therapy of lysosomal storage disorders is complicated by the lack of enzyme transport across the bloodbrain barrier (BBB). The present studies evaluate the delivery of a model enzyme across the BBB following enzyme conjugation to a BBB receptor-specific monoclonal antibody (mAb). Bacterial -galactosidase (116 kDa) was conjugated to the rat 8D3 mAb to the rat transferrin receptor (TfR) via a streptavidin-biotin linkage. The unconjugated -galactosidase or the -galactosidase-8D3 conjugate was injected intravenously in adult mice, and enzyme activity was measured at 1 and 4 h in brain and peripheral organs (liver, spleen, kidney, and heart). Unconjugated -galactosidase was rapidly removed from the blood compartment owing to avid uptake by liver and spleen. There was minimal uptake of the unconjugated -galactosidase by brain. Following conjugation of the enzyme to the 8D3 TfRmAb, there was a 10-fold increase in brain uptake of the enzyme based on measurement of enzyme activity. Histochemistry of brain showed localization of the enzyme in the intraendothelial compartment of brain following intravenous injection of the enzyme-mAb conjugate. The capillary depletion technique showed that more than 90% of the enzyme-8D3 conjugate that entered into the endothelial compartment of brain passed through the BBB to enter brain parenchyma. In conclusion, high molecular weight enzymes, such as bacterial -galactosidase, can be conjugated to BBB targeting antibodies for effective delivery across the BBB in vivo. Fusion proteins comprised of BBB targeting antibodies and recombinant enzymes could be therapeutic in the treatment of the brain in human lysosomal storage disorders. Lysosomal storage disorders are treated with recombinant enzyme replacement therapy. The majority of lysosomal storage disorders affect the brain (Cheng and Smith, 2003). A major limitation in the enzyme replacement therapy of lysosomal storage disorders is the lack of transport of the therapeutic enzyme across the brain capillary wall, which forms the blood-brain barrier (BBB). The involvement of the central nervous system is generally severe in lysosomal storage disorders (Cheng and Smith, 2003), and it is important to develop BBB drug delivery strategies for therapeutic enzymes. Recombinant proteins as large as 40,000 Da have been delivered across the BBB in vivo with molecular Trojan horses that access endogenous BBB receptor-mediated transport systems (Pardridge, 2001). A peptidomimetic monoclonal antibody (mAb) to the BBB transferrin receptor (TfR) mediated the delivery of several peptides and recombinant proteins across the BBB with in vivo central nervous system pharmacological effects following intravenous administration (Pardridge, 2002). The recombinant protein is attached to the TfRmAb via avidin-biotin technology. In this approach, the nontransportable protein drug is monobiotinylated in parallel with the production of a TfRmAb-streptavidin (SA) conjugate. Owing to the very high affinity of SA binding of biotin, there is instantaneous formation of the protein-TfRmAb conjugate following mixing of the monobiotinylated drug and the TfRmAb-SA (Pardridge, 2001). Lysosomal enzymes have a molecular weight of 50 to 100 kDa, and it is not clear if BBB molecular Trojan horses such as a receptor-specific mAb can deliver a protein of this size across the BBB in vivo. Molecular Trojan horses have delivered liposomes carrying genes across the BBB (Pardridge, 2002). However, to date, the largest protein delivered across the BBB with a molecular Trojan horse is pegylated brain derived neurotrophic factor, which has a molecular weight of about 40,000 Da (Pardridge et al., 1998). The present studies use bacterial -galactosidase, which has a molecular weight Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.104.082974. ABBREVIATIONS: BBB, blood-brain barrier; mAb, monoclonal antibody; TfR, transferrin receptor; SA, streptavidin; NHS, N-hydroxysuccinimide; LC, long-chain; S-SMPB, sulfosuccinimidyl-4-(p-malimidophenyl)butyrate; BCA, bicinchoninic acid; PBS, phosphate-buffered saline; HABA, 2-(4 -hydroxyazobenzene)benzoic acid; -gal, -galactosidase; ID, injected dose; GTP, -glutamyl transpeptidase; 8D3-SA, conjugate of 8D3 TfRmAb and SA; HIR, human insulin receptor. 0022-3565/05/3133-1075–1081$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 313, No. 3 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 82974/1199541 JPET 313:1075–1081, 2005 Printed in U.S.A. 1075 at A PE T Jornals on O cber 5, 2017 jpet.asjournals.org D ow nladed from of 116,000 Da, as a model enzyme for delivery to mouse brain. The rat 8D3 mAb to the mouse TfR, which enters brain via the BBB TfR (Lee et al., 2000), is used in the present studies to deliver enzyme to the brain. Materials and Methods Materials. Recombinant Escherichia coli -galactosidase ( -Dgalactoside galactohydrolase, EC 3.2.1.23) with a specific activity of 700 to 1200 units/mg protein was purchased from Roche Diagnostics (Indianapolis, IN). Sulfo-N-hydroxysuccinimide (NHS)-long-chain (LC)-LC-biotin, EZ biotin quantitation kit, Traut’s reagent, sulfosuccinimidyl-4-(p-malimidophenyl)butyrate (S-SMPB) and the bicinchoninic acid (BCA) protein assay reagents were from Pierce (Rockford, IL). [H]Biotin (60 Ci/mmol) was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). Protein G-Sepharose and Sephacryl S-300HR were obtained from Amersham Biosciences (Piscataway, NJ). Sephadex G-25, recombinant streptavidin, and dextran (60,000–90,000 Da) were obtained from Sigma-Aldrich (St. Louis, MO). The -galactosidase histochemistry kit was from Invitrogen (Carlsbad, CA). The Beta-Glo -galactosidase luminescence assay system, lysis buffer, and the -galactosidase enzyme assay system were purchased from Promega (Madison, WI). Synthesis of 8D3-SA Conjugate. The rat hybridoma line secreting the 8D3 mAb to the mouse TfR was cultured on a feeder layer of mouse thymocytes and peritoneal cells in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (Lee et al., 2000). The hybridoma cells were propagated as ascites in nude mice, and the 8D3 mAb was purified by protein G affinity chromatography. A 1:1 conjugate of the 8D3 mAb and SA was prepared by stable thiol-ether linkage using 8D3 thiolated with Traut’s reagent at a 40:1 molar ratio of Traut’s reagent. The SA was activated with S-SMPB at a 24:1 molar ratio, and the 8D3-SA conjugate was purified with a 2.5 95-cm column of Sephacryl S-300HR in PBS 0.05% Tween 20 (0.01 M Na2HPO4, 0.15 M NaCl, pH 7.4, 0.05% Tween 20). The elution of the 8D3-SA conjugate and unconjugated SA were monitored by adding a trace amount of [H]biotin to the mixture prior to addition to the column. The fractions containing the 8D3-SA conjugate (Fig. 1A, reaction I) were pooled and stored at 20°C. Monobiotinylation of -Galactosidase and Biotin Quantitation. Bacterial -galactosidase was homogeneous on SDS-polyacrylamide gel electrophoresis and migrated with a molecular weight of 116,00 Da (Fig. 1B). The -galactosidase was dissolved in 0.05 M NaHCO3/8.5, and the protein concentration was determined with the BCA assay. The sulfo-NHS-LC-LC-biotin (45 nmol/ l) was prepared in 0.05 M NaHCO3/8.5, and 19 l of sulfo-NHS-LC-LC-biotin solution (855 nmol) was added to 5 mg (43 nmol) of -galactosidase, which was a 20:1 molar ratio of biotin/ -galactosidase. The mixture was capped and rocked end over end for 60 min at room temperature. The sample was applied to a 0.7 15-cm Sephadex G-25 column, eluted with 10 ml of 0.01 M PBS/7.4 at 0.5 ml/min, and 0.5-ml fractions were collected. The three fractions comprising the first A280 peak were pooled, the protein concentration was determined, and the biotin-LC-LC-galactosidase (Fig. 1A, reaction II) was stored at 20C. The enzymatic activity of -galactosidase or biotinylated -gal (biotin-LC-LC-galactosidase) was measured with either the spectrophotometric method or the Beta-Glo luminescence assay system. The molar ratio of sulfo-NHS-LC-LC-biotin to -galactosidase was determined to yield 1 to 1.5 biotin moieties per enzyme molecule. The degree of biotinylation was determined with Pierce EZ biotin quantitation kit per the manufacturer’s instructions, which monitors the binding of 2-(4 -hydroxyazobenzene)benzoic acid (HABA) to avidin by absorbance at 500 nm with an extinction coefficient of 34 mM . The displacement of HABA from avidin is proportional to the biotin content in the biotin-LC-LC-galactosidase. The -galactosidase-8D3 conjugate, also designated -gal-8D3 (Fig. 1, reaction III), was formed by mixing a 1:1 molar ratio of biotin-LC-LC-galactosidase and the 8D3-SA conjugate at 15 min at room temperature. There was no loss in -galactosidase enzyme activity following monobiotinylation and attachment to the 8D3-SA conjugate (Fig. 1C). Brain Delivery of -Galactosidase and -Galactosidase-8D3 in Adult Mice. Adult female BALB/c mice weighing 20 to 25 g (Charles River Laboratories, Wilmington, MA) were anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine intraperitoneal. The mice were injected via the jugular vein with either unconjugated -galactosidase or the -gal-8D3 conjugate. In the high dose treatment, mice were administered either 150 g/mouse of unconjugated -galactosidase or 150 g/mouse of biotinylated -galactosidase conjugated to 300 g/mouse of 8D3-SA. In the low dose treatment, mice were administered either 15 g/mouse of unconjugated -galactosidase or 15 g/mouse of biotinylated -galactosidase conjugated to 30 g/mouse of 8D3-SA. The mice were sacrificed at either 1 or 4 h after intravenous (i.v.) injection. The brain, liver, spleen, heart, and kidney were removed, weighed, and frozen on dry ice. The blood from each mouse was collected, heparinized, and stored at 20°C. Organs and blood were also removed from uninjected mice to determine the activity of endogenous -galactosidase at neutral pH. -Galactosidase Enzyme Activity Measurements. The spectrophotometric assay for -galactosidase enzyme activity ( -galactosidase enzyme assay system; Promega) was not used owing to interference in the absorbance readings by endogenous tissue pigments. Enzyme activity was measured with Promega Beta-Glo luminescence assay system. The tissue was extracted with Promega lysis buffer at a ratio of 2 ml of buffer to 0.5 g of tissue, followed by homogenization with a Brinkmann Polytron PT3000. The homogenate was centrifuged for 10 min at 12,000g, and the supernatant was used to measure -galactosidase activity with the Promega Beta-Glo assay solution at pH 7.6. The mixture was incubated in the dark at room temperature for 1 h. The relative light units were measured Fig. 1. Conjugate synthesis. A, reaction I, thiolation of the 8D3 mAb with Traut’s reagent is performed in parallel with the activation of recombinant SA with S-SMPB. The thiolated 8D3 mAb and activated SA are conjugated to form a stable thiol-ether linkage between the 8D3 mAb and SA. Reaction II, bacterial -galactosidase is monobiotinylated with sulfoNHS-LC-LC-biotin. The double LC linker provides a 14-atom spacer between the biotin moiety and the -amino group of surface lysine residues on the enzyme. Reaction III, the -galactosidase-8D3 conjugate is formed upon mixing the monobiotinylated -galactosidase ( -gal-LC-LCbiotin) and the 8D3-SA conjugate. B, SDS-polyacrylamide gel electrophoresis of molecular weight standards (left lane) and E. coli -galactosidase (right lane). The size of the molecular weight standards is shown in the figure. The -galactosidase migrates at a molecular weight of 116 kDa. C, the -galactosidase enzyme activity is unchanged following conjugation to the 8D3 monoclonal antibody. Data are mean S.E. (n 3) and are normalized for milligrams of -galactosidase protein. 1076 Zhang and Pardridge at A PE T Jornals on O cber 5, 2017 jpet.asjournals.org D ow nladed from with a luminometer (PerkinElmer Life and Analytical Sciences, Boston, MA), and the relative light units were converted to milliunits (mU) of enzyme activity based on a -galactosidase standard curve. The protein content in the organ extract was measured with the BCA reagent. Organ enzyme activity was measured as milliunits per milligram of protein, milliunits per gram of organ weight, or percent of injected dose (ID) per gram of organ weight. The ID was computed from the known specific activity (milliunits of microgram) of the unconjugated -galactosidase or the -gal-8D3 conjugate. The endogenous -galactosidase enzyme activity in uninjected mice was also measured in each organ. Brain -Galactosidase Histochemistry. Mice were anesthetized and injected with maximal doses of either unconjugated -galactosidase (300 g/mouse) or the -gal-8D3 conjugate (300 g/ mouse of biotin-LC-LC-galactosidase mixed with 600 g/mouse of 8D3-SA conjugate) via the jugular vein. At 60 min after i.v. injection, the brain plasma volume was cleared with a 4-min infusion of 4 ml of ice-cold PBS into the ascending aorta at a rate of 1 ml/min, followed by a 20-min perfusion of 20 ml of fixative (2% paraformaldehyde in 0.01 M PBS/7.4 with 0.5% glutaraldehyde and 2 mM MgCl2) at a rate of 1 ml/min. The brain was removed and divided into four coronal slabs, and the slabs were immersion-fixed in the same fixative at 4°C for 4 h. The tissue was washed briefly in 0.1 M phosphate-buffered water/7.4 and then placed in 30% sucrose/0.1 M PBS/7.4 for 24 h at 4°C. The brain slab was frozen in Tissue-Tek O.C.T. compound and stored at 70°C until sectioning. Frozen section of 40 m was prepared on an HM505 microtome (Mikron, San Diego, CA), and -galactosidase histochemistry was performed with the Invitrogen -galactosidase staining kit. The frozen section was fixed with 2% formaldehyde and 0.2% glutaraldehyde in 0.01 M PBS/7.4 for 5 min. After washing in PBS, the section was incubated in X-gal staining solution (4 mM potassium ferricyanide, 4 mM potassium ferrocyanide, 2 mM MgCl2, 0.02% IGEPAL CA-630, 0.01% sodium deoxycholate, and 1 mg/ml X-gal, pH 7.4) at 37°C overnight, where X-gal 5-bromo-4-chloro-3-indoyl-D-galactoside. The pH of the incubation was maintained at 7.4 throughout the incubation. After staining with X-gal, the section was briefly washed in distilled water, mounted without counter-staining, and photographed. A dot blot assay was developed to determine the minimal -galactosidase enzyme activity that could be detected with a colorimetric histochemical assay. Enzyme (100 l) was spotted with a Bio-Rad dot blot apparatus in a 3-mm circle to nitrocellulose filter paper in the following amounts: 68, 6.8, 0.68, 0.068, and 0.0068 mU with or without fixation of the blotted filter paper in 0.2% glutaraldehyde in 0.1 M Na2HPO4/7.4/2 mM MgCl2 for 2 min. Enzyme activity in the filter paper was measured with the colorimetric technique ( -galactosidase enzyme assay system; Promega). The amount of enzyme that was barely detected by eye was 2 mU with fixation and 1 mU without fixation. A 40m section of mouse brain weighs approximately 1 mg. Therefore, it would be necessary to achieve a -galactosidase enzyme activity 2000 mU/g brain to visualize the enzyme in brain parenchyma with a colorimetric technique such as histochemistry. Brain Capillary Depletion Method. Mice were anesthetized and injected with the -gal-8D3 conjugate (150 g/mouse of biotinLC-LC-galactosidase mixed with 300 g/mouse of 8D3-SA conjugate) via the jugular vein. At 60 min after i.v. injection, the brain plasma volume was cleared with a 4-min infusion of 4 ml of ice-cold PBS into the ascending aorta at a rate of 1 ml/min. The brain was removed, weighed, and homogenized in an ice-cold physiological buffer (10 mM HEPES, 141 mM NaCl, 4 mM KCl, 2.8 mM CaCl2, 1 mM MgSO4, 1 mM NaH2O4, and 10 mM D-glucose, pH 7.4) with a glass tissue grinder, followed by the addition of ice-cold dextran to a final concentration of 40%. After removal of an aliquot of the homogenate, the remainder was centrifuged at 3200g for 10 min at 4°C, and the supernatant was carefully separated from the capillary pellet with the capillary depletion technique described previously (Triguero et al., 1990). The homogenate, postvascular supernatant, and the capillary pellet were solubilized in Promega lysis buffer. The -galactosidase enzymatic activity was measured with the Promega Beta-Glo assay system as described above and reported as milliunits per gram of brain for each of the three fractions. The enzyme activity of a brain microvascular-enriched enzyme, -glutamyl transpeptidase (GTP), was measured with a spectrophotometric assay using L-glutamyl-p-nitroanilide as a substrate (Diagnostic Chemicals Ltd., Charlottetown, PE, Canada). GTP is highly enriched at the brain microvasculature and is also expressed in brain parenchymal cells. The volume of the microvascular endothelium in brain is 0.1% of the total intracellular volume in brain. Therefore, GTP enzyme activity in the postvascular supernatant approximates the GTP enzyme activity in the starting brain homogenate; however, the ratio of GTP enzyme activity in the vascular pellet, relative to the brain homogenate, is a quantitative measure of the extent to which the postvascular supernatant has been depleted of the microvascular component of brain (Triguero et al., 1990).