Reduction in Brain Heparan Sulfate with Systemic Administration of an

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Reduction in Brain Heparan Sulfate with Systemic Administration of an IgG Trojan Horse-Sulfamindase Fusion Protein in the Mucopolysaccharidosis Type IIIA Mouse Ruben J. Boado, Jeff Zhiqiang Lu, Eric Ka-Wai Hui, and William M. Pardridge Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00958 • Publication Date (Web): 18 Dec 2017 Downloaded from http://pubs.acs.org on December 19, 2017

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Molecular Pharmaceutics

Reduction in Brain Heparan Sulfate with Systemic Administration of an IgG Trojan Horse-Sulfamindase Fusion Protein in the Mucopolysaccharidosis Type IIIA Mouse

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

ArmaGen, Inc. Calabasas, California 91302, United States

*

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] ORCID: 0000-0002-2664-1338

ACS Paragon Plus Environment

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Table of Contents Graphic:


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Abstract Mucopolysaccharidosis Type IIIA (MPSIIIA), also known as Sanfilippo A syndrome, is an inherited neurodegenerative disease caused by mutations in the lysosomal enzyme, Nsulfoglucosamine sulfohydrolase (SGSH), also known as sulfamidase. Mutations in the SGSH enzyme, the only mammalian heparan N-sulfatase, causes accumulation of lysosomal inclusion bodies in brain cells comprised of heparan sulfate (HS) glycosaminoglycans (GAGs). Treatment of MPSIIIA with intravenous recombinant SGSH is not possible because this large molecule does not cross the blood-brain barrier (BBB). BBB penetration by SGSH was enabled in the present study by re-engineering this enzyme as an IgG-SGSH fusion protein, where the IgG domain is a chimeric monoclonal antibody (MAb) against the mouse transferrin receptor (TfR), designated the cTfRMAb. The IgG domain of the fusion protein acts as a molecular Trojan horse to deliver the enzyme into brain via transport on the endogenous BBB TfR. The cTfRMAbSGSH fusion protein bound to the mouse TfR with high affinity, ED50 = 0.74 ± 0.07 nM, and retained high SGSH enzyme activity, 10,043 ± 1,003 units/mg protein, which is comparable to recombinant human SGSH. Male and female MPSIIIA mice, null for the SGSH enzyme, were treated for 6 weeks with thrice-weekly intra-peritoneal injections of vehicle, 5 mg/kg of the cTfRMAb alone, or 5 mg/kg of the cTfRMAb-SGSH fusion protein, starting at the age of 2 weeks, and were euthanized 1 week after the last injection. Brain and liver HS, and dermatan sulfate, as determined by liquid chromatography-mass spectrometry, were elevated 30-fold and 36-fold, respectively, in the MPSIIIA mouse. Treatment of the mice with the cTfRMAb-SGSH fusion protein caused a 70% and 85% reduction in brain and liver HS, respectively. The reduction in brain HS was associated with a 28% increase in latency on the rotarod test of motor activity in male mice. The mice exhibited no injection related reactions and only a low titer end


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of study anti-drug antibody response was observed. In conclusion, substantial reductions in brain pathologic GAGs in a murine model of MPSIIIA are produced by chronic systemic administration of an IgG-SGSH fusion protein engineered to penetrate the BBB via receptormediated transport.

Keywords: blood-brain barrier, Sanfilippo A syndrome, IgG fusion protein, lysosomal enzyme, sulfamidase, transferrin receptor, Rhesus monkey, mouse


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Introduction Mucopolysaccharidosis (MPS) Type IIIA (MPSIIIA), also known as Sanfilippo A syndrome, is a lysosomal storage disease caused by mutations in the lysosomal enzyme, Nsulfoglucosamine sulfohydrolase (SGSH), also known as sulfamidase or heparan N-sulfatase, the only mammalian heparan N-sulfatase.1 The primary clinical manifestation of MPSIIIA is neurodegeneration starting in infancy to childhood, leading to mental retardation, and early death at a mean age of 18 years.2 Mutations in the sulfamidase enzyme lead to the accumulation of heparan sulfate (HS), a glycosaminoglycan (GAG).3 GAG buildup causes the deposition of lysosomal inclusion bodies in brain cells leading to CNS dysfunction.2 Enzyme replacement therapy (ERT) with recombinant sulfamidase is not possible via the intravenous (IV) route, owing to lack of transport of sulfamidase across the blood-brain barrier (BBB). Intrathecal administration of sulfamidase reduces brain HS when administered to MPSIIIA mice via the intra-cisternal route.4 However, chronic cisternal injections in humans are not feasible, and it is not clear if there is adequate distribution of the enzyme to the large brain of humans following enzyme injection into the cerebrospinal fluid (CSF).5 Chronic injections of recombinant sulfamidase into the lumbar CSF have been attempted in patients with MPSIIIA.6 Sulfamidase can be delivered across the BBB for intravenous ERT if the enzyme is reengineered to penetrate the BBB. This is possible by engineering the biologic drug as an IgG fusion protein, where the IgG domain is a monoclonal antibody (MAb) against an endogenous BBB receptor-transporter such as the human insulin receptor (HIR).7 The HIRMAb acts as a molecular Trojan horse to ferry across the BBB the fused biologic via transport on the endogenous BBB insulin receptor. An HIRMAb-SGSH fusion protein has been engineered, and the brain uptake in the Rhesus monkey, 1% of injected dose (ID)/brain, is sufficient to normalize


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brain sulfamidase enzyme activity following the IV infusion of therapeutic doses of 1-3 mg/kg.8 The efficacy of the HIRMAb-SGSH fusion protein cannot be tested in the MPSIIIA mouse model, because the HIRMAb domain of the fusion protein does not cross react with the mouse insulin receptor.9 Therefore, a surrogate Trojan horse is used in mouse treatment studies, which is a genetically engineered chimeric MAb against the mouse transferrin receptor (TfR), designated the cTfRMAb.10 The present studies describe the genetic engineering and expression of the cTfRMAb-SGSH fusion protein, and the chronic treatment of MPSIIIA neonatal mice by systemic (intra-peritoneal) administration of the cTfRMAb-SGSH fusion protein. Therapeutic effects in brain are assessed with measurements of brain HS by liquid chromatography-mass spectrometry (LC-MS). Since chronic administration of recombinant human SGSH to MPSIIIA mice leads to severe immune reactions,11 the present studies also examine the formation of antidrug antibodies (ADA) against the cTfRMAb-SGSH fusion protein.


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Experimental Section Genetic engineering and production of cTfRMAb-SGSH fusion protein. A 1.5 kb cDNA encoding the human SGSH was produced by the polymerase chain reaction (PCR), as described previously,8 and subcloned into the HpaI site of a tandem vector (TV) encoding the heavy chain (HC) and the light chain (LC) of the cTfRMAb. The engineering of the HC and LC genes of the cTfRMAb have been described previously.10 The SGSH cDNA was inserted at the 3’-end of the HC expression cassette with a Ser-Ser linker between the carboxyl terminus of the HC and the amino terminus of the SGSH, minus the enzyme signal peptide, to produce a new expression plasmid DNA, designated the TV-cTfRMAb-SGSH. The identity of this plasmid DNA was confirmed by bi-directional DNA sequence analysis using custom DNA sequencing primers, which allowed for deduction of the amino acid (AA) sequence of the cTfRMAb-SGSH fusion protein. The fusion protein of the cTfRMAb heavy chain and human SGSH was comprised of 926 amino acids (AA), which included the following domains: a 19 AA signal peptide, the 118 AA variable region of the cTfRMAb heavy chain (VH), the 324 AA mouse IgG1 constant region, a 2 AA linker (Ser-Ser), and the 482 AA mature human SGSH enzyme, minus the enzyme signal peptide. The predicted isoelectric point (pI) of the heavy chain is 6.25, and the predicted molecular weight (MW) of the heavy chain, without glycosylation, is 103,612 Da. The AA sequence of the SGSH domain is 100% identical with AA 21-502 of mature human SGSH (Genbank NP_000190), and includes 5 predicted N-linked glycosylation sites. The light chain (LC) is comprised of 234 AA, and is formed by a 20 AA signal peptide, a 108 AA variable region of the LC (VL), and a 106 mouse kappa constant region. The hetero-tetramer of the cTfRMAb-SGSH fusion protein has a MW of 254,278 Da, without glycosylation, and a pI of 6.20. In addition to the HC and LC expression cassettes, the TV-cTfRMAb-SGSH encodes the


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dihydrofolate reductase (DHFR) gene for selection and amplification of host cell lines with methotrexate (MTX). The HC, LC, and DHFR expression cassettes were comprised of 10,009 nucleotides. Chinese hamster ovary (CHO) cells were stably transfected with TV-cTfRMAbSGSH, and cultured in serum free medium (SFM) and high producing lines were selected with MTX, and screened with a mouse IgG ELISA. The CHO line was cultured in a 25L bioreactor in SFM, and the fusion protein was purified from the conditioned medium with protein A affinity chromatography. The purified protein was diafiltered against 0.01 M Tris/0.15M NaCl/pH=7.5/0.01% polysorbate-80 (TBST), sterilized by 0.22 micron filtration, vialed, and stored at 4C or -70C at a concentration of 0.5 to 1 mg/mL. Biochemical properties of the cTfRMAb-SGSH fusion protein. Fusion protein purity was assessed with reducing sodium dodecyl sulfatate polyacrylamide gel electrophoresis (SDSPAGE). Fusion protein identity was determined by Western blot (WB) analysis using a primary goat antibody against mouse IgG heavy and light chains (Bethyl Labs, Montgomery, TX), or primary rabbit antibody against human SGSH (Thermo-Fisher, Carlsbad, CA). SDS-PAGE and WB compared migration of the fusion protein with the non-fused cTfRMAb also produced in CHO cells. Fusion protein affinity for the mouse TfR extracellular domain (ECD) was determined by ELISA. The capture agent of the mouse TfR ELISA was murine TfR1 ECD (Sino Biological, Beijing, China). The detection reagent was a conjugate of alkaline phosphatase and a goat anti-mouse kappa antibody (Bethyl Labs, Montgomery, TX). Fusion protein binding in the ELISA was compared to a mouse IgG1k isotype control antibody (Sigma). The SGSH enzyme activity of the cTfRMAb-SGSH fusion protein was determined by a fluorometric 2-step enzyme assay using 4-methylumbelliferyl-alpha-N-sulpho-D-glucosaminide (MU-αGlcNS) as substrate.12 In the first step, the MU-αGlcNS is hydrolyzed by SGSH to 4-methylumbelliferyl-α-


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D-glucosaminide (MU-αGlcNH2), which is then hydrolyzed to the fluorescent product, 4methylumbelliferone (4-MU), by the α-glucosaminidase side-activity in commercial yeast αglucosidase. The fusion protein was incubated in 30 uL of McIlvaine’s buffer and 3.3 mM substrate for 17 hours at 37C, followed by the addition of 30 uL of 2X McIlvaine’s buffer and 0.1 units of α-glucosidase (Sigma) and a 24 hr incubation at 37C. The reaction was terminated by the addition of 200 uL stop buffer (0.5 M Na2CO3, pH=10.7, 0.025% Triton X-100). A standard curve was prepared with 4-MU (Sigma), and enzyme activity was expressed as units/mg protein of the cTfRMAb-SGSH fusion protein, where 1 unit=1 nmol of 4-MU product formed during the 17 hour first step incubation.8 MPSIIIA mouse study. A colony of MPSIIIA mice [B6.Cg-Sgshmps3a/PstJ, stock# 003780) was maintained at The Jackson Laboratory (JAX, Sacramento, CA). Pups born to pregnant mice were genotyped by 1 week of age, and homozygous null mice were enrolled in the treatment study. The cTfRMAb-SGSH, the cTfRMAb, and the TBST test articles were shipped on dry ice to JAX, for storage at -70C until use. The project was reviewed and approved by the Institutional Animal Care and Use Committee of The Jackson Laboratory. A total of 8 wild type mice (2 weeks old) and 24 MPSIIIA mice (2 weeks old) were tested, with 8 mice (4 males, 4 females) in each of 4 treatment groups: (1) MPSIIIA mice treated with intra-peritoneal (IP) saline vehicle three times per week for 6 weeks; (2) MPSIIIA mice treated with IP cTfRMAb alone (5 mg/kg/dose) three times per week for 6 weeks; (3) MPSIIIA mice treated with IP cTfRMAb-SGSH (5 mg/kg/dose) three times per week for 6 weeks; (4) wild type mice with no treatment. The IP injection volume was 120-150 uL/mouse. The mice were euthanized one week after the last dose when the mice were 9 weeks old. In the last week of the study, after cessation of drug treatment, motor activity was assessed in the MPSIIIA mice with a Ugo Basile (Varese,


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Italy) model 47600 accelerating rotarod. The rotarod was accelerated from 4 to 40 RPM over 5 minutes and latency, in seconds, on the beam was averaged over 3 consecutive trials. At euthanasia, the brain and liver from each mouse was removed, weighed, and homogenized in 5 volumes of cold 0.05 M sodium acetate/0.2 M NaCl/pH=6.0. Injection related reactions (IRR) following drug treatment were scored as follows: (+1), mild reduction in mobility clearing in 10 minutes; (+2), moderate reduction in mobility clearing in 30 minutes; (+3) severe reaction with suppressed activity and hunched posture. Unused cTfRMAb-SGSH fusion protein was returned by JAX at the end of the study, and showed no change in SGSH enzyme activity relative to the cTfRMAb-SGSH fusion protein reference standard. LC-MS of heparan sulfate and dermatan sulfate. The heparan sulfate (HS) and dermatan sulfate (DS) concentrations in the brain and liver homogenates were assayed at IAS, Inc. (Berkeley, CA) by liquid chromatography/tandem mass spectrometry (LC-MS) in negative electrospray ionization mode with a graphite reverse phase column and an Applied Biosystems/MDS Sciex API 4000 tandem quadrupole mass spectrometer. Tissue HS or DS was converted to disaccharides with recombinant heparinase-I, -II, -III, and chondroitinase B (Ibex Pharmaceuticals, Montreal, Canada), respectively. A total of 4 HS disaccharide standards, and 1 DS disaccharide standard, were used, as described previously,13 and all disaccharide standards (HD002, HD004, HD005, HD006, CD002, and CD004), as well as the internal HS standard (HD009), were purchased from Iduron (Macclesfield, UK). The disaccharide structure code (DSC) of these HS standards correspond to D2S0, D0S6, D0S0, D0A0, respectively, and were chosen as the sum of these disaccharides comprises 89% of the total HS, whereas the DS standards (D0A4, D0A10) comprise 100% of total DS.13 The D2S0 and D0S6 standards were not resolved on LC and were treated as a single standard. The calibration range of the disaccharide


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standards was 4 to 3000 ng/mL. The LC-MS measurements reported the HS or DS in ng/mL of homogenate for each standard, and these were converted to pmol/mL based on the MW of the disaccharide standard, and then combined to express the tissue concentration of HS or DS, which was normalized per mg wet weight tissue. The fractional reduction in tissue GAG was computed from {1-[A-C]/[B-C]}, where A=tissue HS or DS after treatment of the MPSIIIA mouse with the fusion protein, B=tissue GAG after treatment of the MPSIIIA mouse with saline, and C=tissue GAG in the wild type mouse. Anti-drug antibody (ADA) ELISA. ADA responses against the cTfRMAb-SGSH fusion protein were quantified with a sandwich ELISA. The cTfRMAb-SGSH fusion protein was the capture agent and biotinylated cTfRMAb-SGSH fusion protein was the detector agent in the sandwich ELISA. The biotinylation of the cTfRMAb-SGSH fusion protein was confirmed by Western blot as described previously.14 The cTfRMAb-SGSH fusion protein was plated (100 uL of 2.5µg/mL) in 96-well plates overnight at 4ºC. The wells were blocked with PBSB (0.01 M Na2HPO4/0.15 M NaCl/1% BSA/pH7.4) for 1h at room temperature (RT) and washed with PBSB. Plasma samples (diluted 1:50 in PBS; 100µL/well) were added to the wells and incubated for 1h at 37C followed by washing with PBSB. Wells were incubated with 50 ng/well of biotinylated cTfRMAb-SGSH fusion protein for 1h at 37C followed by washing with PBSB. Wells were then incubated with 100 µL/well of 5 µg/mL of streptavidin-peroxidase conjugate (Vector Laboratories, Burlingame, CA) and incubated for 30 min at RT. After washing with PBSB, wells were incubated with o-phenylenediamine-H2O2 developing solution (Sigma) 15 min in the dark at RT and the reaction was stopped by adding 100 µL of 1M HCl per well. Absorbance was measured at 492 nm (A492) and 650 nm (A650). The A650 was subtracted from the A492. Sample (A492 − A650) values were corrected with value of the PBSB blank.


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Results The cTfRMAb-SGSH fusion protein was purified to homogeneity on reducing SDSPAGE (Figure 1). Based on migration of MW standards, the estimated MW of the cTfRMAbSGSH fusion protein heavy chain, the cTfRMAb heavy chain, and the light chain is 119 kDa, 54 kDa, and 24 kDa, respectively, which predicts a MW of 286 kDa for the glycosylated heterotetrameric cTfRMAb-SGSH fusion protein. The identity of the fusion protein was confirmed by mouse IgG and human SGSH Western blotting (Figure 2). The anti-mouse IgG (H+L) antibody reacts with both HC and LC of both the cTfRMAb-SGSH fusion protein and the cTfRMAb alone (Figure 2, left panel), and the anti-human SGSH antibody reacts with only the HC of the cTfRMAb-SGSH fusion protein, and does not react with the cTfRMAb alone (Figure 2, right panel). The cTfRMAb-SGSH fusion protein binds to the mouse TfR, whereas there is no binding of the mouse IgG1k control antibody (Figure 3). The ED50 of fusion protein binding to the mouse TfR is 215 ± 20 ng/mL, which corresponds to an ED50 of 0.74 ± 0.07 nM based on a MW of 286 kDa. This ED50 is not significantly different from the ED50 of mouse TfR binding of the cTfRMAb alone, 0.78 ± 0.05 nM.15 The SGSH enzyme specific activity of the cTfRMAbSGSH fusion protein averaged 10,043 ± 1,003 units/mg protein (mean ± SE) over 4 separate assays, and no loss of enzyme activity was observed after a freeze/thaw cycle. MPSIIIA mice, 2 weeks of age, were treated for 6 weeks with thrice-weekly IP injections of TBST vehicle, the 5 mg/kg of the cTfRMAb alone, or 5 mg/kg of the cTfRMAb-SGSH fusion protein. The body weight increased 3-fold from 6-7 grams, at 2 weeks, to 19-20 grams, at 8 weeks, equally in all treatment groups. No mice in any treatment group exhibited IRRs at any time during the study, and there were no mortalities in any group.


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The liver and brain HS concentrations at the end of the treatment study are shown in Table 1. In the saline treated MPSIIIA mice, the brain and liver HS were increased 30-fold and 36-fold above the values in wild type mice, respectively. Treatment with the cTfRMAb alone had no effect on the HS concentration in brain or liver (Table 1). However, treatment with the cTfRMAb-SGSH fusion protein caused a 70% and 85% reduction in HS concentration in brain and liver, respectively (Table 1). There were no differences in brain or liver HS between male and female mice. Dermatan sulfate (DS) was not elevated in brain in the MPSIIIA mice (Table 2). However, DS was increased 2-fold in liver in the MPSIIIA mouse, and liver DS was selectively decreased 94% by treatment with the cTfRMAb-SGSH fusion protein (Table 2). The reduction in brain HS was associated with a 28% increase in latency in the rotarod test in male mice (Figure 4), with no significant change in rotarod latency in female mice. A low titer ADA response against the cTfRMAb-SGSH fusion protein was observed with 1:50 dilutions of terminal serum (Figure 5). The average titer, expressed as optical density (OD) per uL undiluted serum is 0.1.


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Discussion The results of this study are consistent with the following conclusions. First, the cTfRMAb-SGSH fusion protein is a bifunctional IgG-enzyme fusion protein, which binds the mouse TfR with the same avidity as the cTfRMAb (Figure 3), and which exhibits high SGSH enzyme activity comparable to recombinant SGSH (Results). Second, systemic administration of MPSIIIA mice with the cTfRMAb-SGSH fusion protein results in a 70% reduction in brain HS, which is paralleled by a 85% reduction in HS in liver (Table 1). Third, MPSIIIA mice are immune tolerant to chronic treatment with the cTfRMAb-SGSH fusion protein for at least 6 weeks by the IP route, and show no injection related reactions and only a low titer ADA response (Figure 5). The treatment of MPSIIIA mice by systemic injection is limited by the lack of transport of SGSH across the BBB. Even in the newborn, the BBB is fully developed.16 The volume of distribution of SGSH in mouse brain is no different from the volume of distribution of albumin, a cerebral blood volume marker, in the 2 day old mouse,17 which indicates that SGSH does not cross the BBB. Attempts have been made to increase the BBB penetration of SGSH by fusion of a apolipoprotein B (apoB) binding domain to SGSH, in an effort to target the BBB low density lipoprotein receptor (LDLR).18 However, the activity of the BBB LDLR may be low, since there is minimal transport of LDL-bound cholesterol into the brain from blood.19 The BBB TfR is an active transport system for transferrin, as well as for certain MAb’s against exofacial epitopes of the TfR.20 An MAb specific for the mouse TfR is the rat 8D3 antibody,21 which selectively penetrates the BBB in the mouse.22 The VH and VL of the 8D3 mouse TfRMAb form the cTfRMAb used in these studies,10 and fusion of human SGSH to the cTfRMAb enables access of the enzyme to brain from blood via transport on the BBB TfR. The cTfRMAb-SGSH fusion


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protein produced in these studies has high SGSH enzyme activity even though the host cell was not co-transfected with the sulfatase modifying factor type 1 (SUMF1). SUMF1 converts a near N-terminal cysteine to an N-formyl glycine residue to enable sulfatase enzymatic activity.23 The SGSH enzyme activity of the cTfRMAb-SGSH fusion protein, which has a MW of 286 kDa, is 10,043 units/mg protein (Results). When expressed on a molar basis, the SGSH enzyme activity of the cTfRMAb-SGSH fusion protein is comparable to the activity, 15,000 units/mg protein, of recombinant SGSH.17 The cTfRMAb-SGSH fusion protein was administered via the IP route in this study, as the mice at the start of the study had a body weight of only 6 grams (Results). The peritoneal membrane is semi-permeable and does not restrict the transfer of IgG molecules from the peritoneal cavity into the circulation.24 In the mouse, the brain uptake of cTfRMAb fusion proteins following IP administration is 1.2% ID/gram brain at an injection dose (ID) of 3 mg/kg.25 In the present work, postnatal mice with an average body weight of 12 grams were administered 5 mg/kg IP of the cTfRMAb-SGSH fusion protein, which is equal to 60 ug of fusion protein per mouse. This produces a brain concentration of the fusion protein of 0.6 ug/gram. Based on the SGSH specific activity of the cTfRMAb-SGSH fusion protein of 10,000 units/mg protein, the brain SGSH enzyme activity in the null mice is 6.0 units/gram brain, or 0.060 units/mg brain protein, given 100 mg protein per gram brain.26 This level of SGSH enzyme activity is 50% of the endogenous SGSH activity in the normal mouse brain, 0.12 units/mg protein.27 Therefore, the IP administration of 5 mg/kg of the cTfRMAb-SGSH fusion protein is expected to produce a therapeutic level of SGSH enzyme activity in the brains of the MPSIIIA mice.


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Chronic treatment of 2 week old MPSIIIA mice with the IP administration of the cTfRMAb-SGSH fusion protein at 5 mg/kg for 6 weeks produces a 70% reduction of HS in brain of MPSIIIA mice, with a 85% reduction in liver HS (Table 1). The reduction in brain HS observed in this study may under-estimate the therapeutic effect, because the mice were euthanized 1 week after the last dose of fusion protein (Methods). Following a single intracisternal injection of SGSH in 6 week old MPSIIIA mice, the nadir of brain HS levels is reached at 3 days after the injection, but the brain HS is twice as high as the nadir by 7 days after the single IC injection of SGSH.4 The reduction in brain HS observed in the present study was associated with a 28% increase in latency on the rotarod test in male mice (Figure 4). No significant difference in rotarod performance was observed in female mice in this study, which parallels other reports showing that female mice have superior performance on the rotarod test of motor activity as compared to male mice.28 Dermatan sulfate is generally not elevated in MPSIIIA,3 and DS is not elevated in the brain of the MPSIIIA mouse (Table 2). However, there is a 2-fold increase in DS in liver in the MPSIIIA mouse, and the DS elevation in liver is reduced 94% by treatment with the cTfRMAbSGSH fusion protein (Table 2). DS is a known secondary metabolite in MPSIII, and elevations in DS are caused by the HS-mediated inhibition of cellular iduronate 2-sulfatase.29 Lysosomal enzymes are highly immunogenic in the respective null mouse. Severe immune reactions leading to anaphylaxis and mortality are observed in ASA null mice following the 3rd or 4th IV injection of human arylsulfatase A (ASA),30 a sulfatase related to SGSH. Similarly, immune reactions comprised of altered respiration, lethargy, and reduced activity were observed in MPSIIIA mice following the 5th or 6th administration of recombinant human SGSH at an IV dose of 1 mg/kg.11 Pretreatment with antihistamines was not sufficient to enable


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treatment of the MPSIIIA mice beyond 6 weeks.11 In the present study, no immune reactions were observed in any MPSIIIA mice administered the cTfRMAb alone or the cTfRMAb-SGSH fusion protein following IP administration. Only a low titer, OD/uL=0.1, ADA response against the cTfRMAb-SGSH fusion protein was observed at the end of the 6 week treatment study of the MPSIIIA mice (Figure 5). No ADA response was detected in the wild type mice as this group of mice was not administered fusion protein (Methods). The findings of a low titer ADA response to the IgG-SGSH fusion protein in the MPSIIIA mice suggest that re-engineering the SGSH lysosomal enzyme as an IgG-enzyme fusion protein induces immune tolerance even in mice null for the lysosomal enzyme. The constant region of the IgG contains certain amino acid sequences termed Tregitopes, which induce T cell immune tolerance.31 In conclusion, the present study shows systemic administration of MPSIIIA mice with the cTfRMAb-SGSH fusion protein causes a 70% reduction in brain heparan sulfate, the pathologic GAG of the CNS in MPSIIIA. Treatment of humans with MPSIIIA with an IgG-SGSH fusion protein, such as the HIRMAb-SGSH fusion protein,8 may treat the brain with non-invasive administration of the enzyme via intravenous infusion. In addition to mediating transport across the BBB, the IgG domain of the fusion protein may enhance immune tolerance to the lysosomal enzyme.


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2008 Oct 15;112(8):3303-11.


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Table 1 Brain and liver heparan sulfate (HS) in 9 week old MPSIIIA mice Mouse

MPSIIIA

Wild type

treatment

Brain HS

Liver HS

(nmol/gram)

(nmol/gram)

saline IP

1,120 ± 15

6,080 ± 534

cTfRMAb IP

1,070 ± 30

4,880 ± 448

cTfRMAb-SGSH IP

360 ± 100 *

1,060 ± 444 *

Wild type

37 ± 4

167 ± 11

Mean ± SD (N=8 mice/group). MPSIIIA mice were 2 weeks of age at the start of a 6 week treatment study, and were 9 weeks old at euthanasia for measurement of brain and liver HS. * P

Reduction in Brain Heparan Sulfate with Systemic Administration of an

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