Insulin Receptor AntibodyâÎ±-N-Acetylglucosaminidase Fusion Protein

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Insulin Receptor Antibody-#-N-Acetylglucosaminidase Fusion Protein Penetrates the Primate Blood-Brain Barrier and Reduces Glycosoaminoglycans in Sanfilippo Type B Fibroblasts Ruben J. Boado, Jeff Zhiqiang Lu, Eric Ka-Wai Hui, Huilan Lin, and William M. Pardridge Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00037 • Publication Date (Web): 24 Feb 2016 Downloaded from http://pubs.acs.org on March 1, 2016

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Molecular Pharmaceutics

Insulin Receptor Antibody-α α-N-Acetylglucosaminidase Fusion Protein Penetrates the Primate Blood-Brain Barrier and Reduces Glycosoaminoglycans in Sanfilippo Type B Fibroblasts Ruben J. Boado Jeff Zhiqiang Lu Eric Ka-Wai Hui Huilan Lin 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]

ACS Paragon Plus Environment

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Table of Contents Graphic:


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Abstract Mucopolysaccharidosis Type IIIB (MPSIIIB) is caused by mutations in the gene encoding the lysosomal enzyme, α-N-acetylglucosaminidase (NAGLU). MPSIIIB presents with severe disease of the central nervous system, but intravenous NAGLU enzyme replacement therapy has not been developed, because the NAGLU enzyme does not cross the blood-brain barrier (BBB). A BBB-penetrating form of the enzyme was produced by re-engineering NAGLU as an IgG-enzyme fusion protein, where the IgG domain is a monoclonal antibody (MAb) against the human insulin receptor (HIR). The HIRMAb traverses the BBB via transport on the endogenous insulin receptor, and acts as a molecular Trojan horse to ferry the fused NAGLU across the BBB from blood. The NAGLU was fused to the carboxyl terminus of each heavy chain of the HIRMAb via an extended 31-amino acid linker, and the fusion protein is designated HIRMAb-LL-NAGLU. The fusion protein retains high affinity binding to the HIR, and on a molar basis has an enzyme activity equal to that of recombinant human NAGLU. Treatment of MPSIIIB fibroblasts with the fusion protein normalizes intracellular NAGLU enzyme activity, and reduces sulfate incorporation into intracellular glycosoaminoglycan. The fusion protein is targeted to the lysosomal compartment of the cells as shown by confocal microscopy. The fusion protein was radiolabeled with the [125I]-Bolton-Hunter reagent, and injected intravenously in the adult Rhesus monkey. The fusion protein was rapidly cleared from plasma by all major peripheral organs. The high brain uptake of the fusion protein, 1% injected dose/brain, enables normalization of brain NAGLU enzyme activity with a therapeutic dose of 1 mg/kg. The HIRMAb-LL-NAGLU fusion protein is a new treatment of the brain in MPSIIIB which can be administered by non-invasive intravenous infusion.


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Keywords: blood-brain barrier, Sanfilippo syndrome, IgG fusion protein, lysosomal enzyme, insulin receptor, Rhesus monkey


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Introduction Mucopolysaccharidosis (MPS) Type III, also called Sanfilippo syndrome, or MPSIII, is an inherited disease caused by mutations in one of 4 lysosomal enzymes, i.e., heparan Nsulfatase also called N-sulfoglucosamine sulfohydrolase (SGSH) is mutated in MPS Type IIIA, N-acetyl-α-D-glucosaminidase (NAGLU) is mutated in MPS Type IIIB, heparan-αglucosaminide N-acetyltransferase (HGNAT) is mutated in MPS Type IIIC, and Nacetylglucosamine-6-sulfatase (GNS) is mutated in MPS Type IIID. About 90% of MPSIII cases are comprised of either Type IIIA or Type IIIB. In the case of MPSIIIB, the NAGLU enzyme degrades heparan sulfate (HS) by hydrolysis of terminal N-acetyl-D-glucosamine residues. The absence of NAGLU activity leads to the accumulation of glycosaminoglycans (GAGs) in tissues, including the brain.1 While peripheral tissues are affected in MPSIIIB, the clinical features of the disorder are primarily neurological. 1 The gene encoding NAGLU was isolated 20 years ago. 2,3 However, treatment of MPSIIIB with the recombinant enzyme and Enzyme Replacement Therapy (ERT) has not been developed, because the NAGLU enzyme incorporates mannose 6phosphate (M6P) poorly.4,5 Therefore, NAGLU uptake into target cells via the M6P receptor (M6PR) is minimal. The M6PR is also a high affinity receptor for insulin-like growth factor (IGF)-2. Following the re-engineering of the NAGLU enzyme as an NAGLU-IGF2 fusion protein, the enzyme is taken up by MPSIIIB fibroblasts via a process that is inhibited by IGF2.6 However, the NAGLU-IGF2 fusion protein does not cross the blood-brain barrier (BBB), and reduction in HS in brain in MPSIIIB mice requires invasive injection of the fusion protein into the cerebrospinal fluid (CSF) compartment of the mouse brain.7 Intravenous (IV) infusion of the enzyme, the preferred form of chronic treatment of MPSIIIB, is not possible, because NAGLU does not cross the BBB.8 BBB penetration by a


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lysosomal enzyme is possible following the re-engineering as an IgG-enzyme fusion protein. The IgG domain targets an endogenous receptor on the BBB, such as the insulin receptor or the transferrin receptor (TfR), and binding of the IgG-enzyme fusion protein to the BBB receptor triggers receptor-mediated transport from blood to brain, as well as receptor-mediated endocytosis of the fusion protein by target cells in brain.9,10 IgG-enzyme fusion proteins are targeted to the lysosomal compartment, as shown by confocal microscopy.9,10 Previously, IgGenzyme fusion proteins were engineered with lysosomal enzymes such as iduronidase (IDUA) or iduronate 2-sulfatase (IDS) that are mannose 6-phosphorylated. It was unknown whether an IgGNAGLU fusion protein would be targeted to the lysosome, or reduce GAGs in MPSIIIB cells, since the NAGLU enzyme barely incorporates M6P. Also unknown was whether the NAGLU enzyme activity would be preserved following the re-engineering of the enzyme as an IgGNAGLU fusion protein. IDUA or IDS enzyme activity is preserved following fusion of the enzyme to the carboxyl terminus of the heavy chain of a monoclonal antibody (MAb) to the human insulin receptor (HIR).9,10 However, the enzyme activity of another lysosomal enzyme, βglucuronidsase (GUSB), is >95% decreased following fusion to the carboxyl terminus of the HIRMAb.11 In the present study, human NAGLU is fused to the carboxyl terminus of the heavy chain of the HIRMAb (Figure 1) to produce a treatment of the brain in MPSIIIB, designated the HIRMAb-NAGLU fusion protein.


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Experimental Section Genetic engineering and production of HIRMAb-NAGLU fusion protein. A synthetic gene encoding human NAGLU was custom synthesized by GenScript (Piscataway Township, NJ) with the following sequence: (a) nucleotides (nt) 410 through 2572 of Genbank accession number NM_000263, which encoded for the 720 amino acid mature human NAGLU plus the TGA stop codon; (b) a StuI site (AGGCCT) followed by ‘CA’ was inserted at the 5’end, to maintain the open reading frame of the NAGLU cDNA with the CH3 region of the HIRMAb heavy chain, and to insert a Ser-Ser-Ser-Ser short linker between the C-terminus of the heavy chain and the amino terminus of the NAGLU domain. The NAGLU synthetic gene was released from the vendor plasmid by restriction endonuclease digestion and purified by agarose gel electrophoresis, followed by subcloning at the 3’-terminal end of the open reading frame of the HIRMAb heavy chain expression plasmid to produce a new expression plasmid DNA, designated the pCD-HC-NAGLU. The identity of this plasmid DNA was confirmed by bidirectional DNA sequence analysis using custom DNA sequencing primers, which allowed for deduction of the amino acid (AA) sequence of the HIRMAb-NAGLU fusion protein. The fusion protein of the HIRMAb heavy chain and NAGLU was comprised of 1,184 AA, which included a 19 AA signal peptide, the 441 AA HIRMAb heavy chain, a 4 AA linker (Ser-Ser-Ser-Ser), and the 720 AA mature NAGLU enzyme. The predicted pI of the heavy chain is 7.80, and the predicted molecular weight (MW) of the heavy chain, without glycosylation, is 129,130 Da, which includes 80,353 Da within the NAGLU domain. The AA sequence of the NAGLU domain is 100% identical with the sequence of mature human NAGLU (Genbank NP_000254), and includes 6 predicted N-linked glycosylation sites.


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The HIRMAb-NAGLU fusion protein with the (Ser)4 linker was expressed in COS cells following co-lipofection with pCD-HC-NAGLU and the antibody light chain expression plasmid, pCD-LC, and serum free conditioned medium was collected at 3 and 7 days following lipofection. Medium fusion protein was detected with an ELISA specific for the human IgG1 constant region. However, the expression levels of the HIRMAb-NAGLU fusion protein were undetectable and no fusion protein could be isolated for biochemical characterization. In an attempt to increase secretion of the HIRMAb-NAGLU fusion protein, a new fusion protein was engineered with a 23 AA linker between the antibody heavy chain carboxyl-terminus and the NAGLU amino-terminus. The fusion protein with this 23-amino acid linker is designated the HIRMAb-L-NAGLU fusion protein. The 23 AA linker corresponds to the 17 amino acids from the hinge region of human IgG3, and is derived from the 12 amino acids of the upper hinge region, followed by 5 amino acids of the first part of the core hinge region, and is flanked by a Ser-Ser-Ser sequence on the amino terminus and a Ser-Ser-Ser sequence on the carboxyl terminus. The 2 cysteine residues of the first part of the core hinge region are mutated to serine residues, so as to eliminate disulfide bonding. The identity of the expression plasmid, pCD-HCL-NAGLU, was confirmed by bidirectional DNA sequencing. The expression of the new fusion protein, designated HIRMAb-L-NAGLU, was investigated in COS cells by co-lipofection using both the pCD-HC-L-NAGLU plasmid and the pCD-LC plasmid. The expression levels in COS cells of the HIRMAb-L-NAGLU fusion protein were still low, albeit increased relative to the expression of the HIRMAb-NAGLU fusion protein. In an attempt to further increase secretion of the fusion protein, a new fusion protein with a 31 amino acid extended linker was engineered, and this fusion protein is designated HIRMAb-LL-NAGLU. The 31 AA linker includes 25 AA from the human IgG3 hinge region, and is derived from the 12 amino acids of the upper hinge


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region, followed by 5 amino acids of the first part of the core hinge region, followed by 8 amino acids of the lower hinge region, and is flanked by a Ser-Ser-Ser sequence on the amino terminus and a Ser-Ser-Ser sequence on the carboxyl terminus. The 2 cysteine residues of the first part of the core hinge region are mutated to serine residues, so as to eliminate disulfide bonding. The new heavy chain expression plasmid is designated pCD-HC-LL-NAGLU, and the identity of this plasmid was confirmed by bidirectional DNA sequencing, and was used for co-lipofection of COS cells with the light chain expression plasmid, pCD-LC. The expression levels in COS cell conditioned medium of the HIRMAb-LL-NAGLU fusion protein was 5-fold higher than the expression levels of the HIRMAb-L-NAGLU fusion protein, as determined with a human IgGspecific ELISA. To enable stable expression in Chinese hamster ovary (CHO) cells of the HIRMAb-LLNAGLU fusion protein, a tandem vector (TV) was engineered, which contained genes for i) the HIRMAb light chain, ii) the HIRMAb-LL-NAGLU heavy chain fusion protein, and iii) a dihydrofolate reductase (DHFR) selection gene, and this TV is designated the pTV-HIRMAbLL-NAGLU. The identity of the 3 expression cassettes was confirmed by bi-directional DNA sequence analysis, which encompassed 10,795 nt. The pTV-HIRMAb-LL-NAGLU was linearized by restriction endonuclease followed by electroporation of CHO cells, followed by selection in serum free medium with methotrexate, which inhibits endogenous DHFR. High producing lines were isolated by limited dilution cloning, which expressed medium fusion protein levels >10 mg/L at a viable cell density of about 2x106 cells/mL. The fusion protein was affinity purified by protein A chromatography and analyzed for purity, identity, and potency. Biochemical properties of HIRMAb-LL-NAGLU fusion protein. Fusion protein purity was assessed with reducing and non-reducing sodium dodecyl sulfatate polyacrylamide


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gel electrophoresis (SDS-PAGE). Fusion protein identity was determined by Western blot analysis using a primary goat antibody against either human IgG heavy and light chains (Bethyl Labs, Montgomery, TX), or primary rabbit antibody against human NAGLU (Abcam, Cambridge, MA). SDS-PAGE compared migration of the fusion protein with the non-fused HIRMAb produced in CHO cells. Western blotting compared immunoreactivity of the fusion protein with either non-fused HIRMAb or recombinant human NAGLU (R&D Systems, Minneapolis, MN). The recombinant NAGLU was produced in CHO cells, and is assumed to have very low M6P similar to all other forms of human NAGLU produced in CHO cells.4,5 Fusion protein affinity for the HIR extracellular domain (ECD) was determined by ELISA. The capture agent was a complex of a murine MAb against the HIR (Abcam) and the HIR ECD, which was purified by lectin affinity chromatography from serum free medium conditioned by CHO cells stably transfected with the gene encoding the HIR ECD. The detection reagent was a conjugate of alkaline phosphatase and a goat anti-human IgG-Fc antibody (Abcam). Fusion protein binding in the ELISA was compared to the non-fused HIRMAb and the human IgG1k isotype control antibody (Sigma Chemical Co., St. Louis, MO). NAGLU enzyme activity was determined at 37○C and pH=4.3 using the method of Marsh and Fensom,12 which uses as the assay substrate 4-methylumbelliferyl-N-acetyl-α-D-glucosaminide (4MUαGlcNAc) (Calbiochem Life Science Research, Billerica, MA) at a final concentration of 1 mM. The NAGLU enzyme converts this substrate to 4-methylumbelliferone (4-MU). The standard curve was produced with 4-MU (Sigma), and fluorescence was determined with an emission and excitation wavelength of 450 nm and 365 nm, respectively. NAGLU enzyme activity was expressed as units/mg protein, where 1 unit = 1 nmol/hr. The enzyme activity of recombinant human NAGLU (R&D Systems) was determined in parallel.


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Treatment of MPSIIIB fibroblasts with HIRMAb-LL-NAGLU fusion protein. MPSIIIB fibroblasts (GM02931) were obtained from the Coriell Institute for Medical Research (Camden, NJ), and were used for 3 types of experiments: (a) time-response study of intracellular NAGLU enzyme activity, (b) incorporation of [35S]-sulfate into intracellular GAGs, and (c) confocal microscopy. For the time-response study, the cells were grown to confluence in DMEM medium with 10% fetal bovine serum. After removal of the medium, the cells were wash with phosphate buffered saline (PBS), followed by the addition of 1 mL per well of a 35 mm dish with serum free DMEM containing 20 nM HIRMAb-LL-NAGLU fusion protein. At the end of 2-30 hour incubation, the medium was aspirated, and the monolayer was washed 5 times with cold PBS, followed by cell lysis in 0.01 M phosphate/pH=5.8/0.2% Triton X-100/0.1 mM dithiothreitol and 5 cycles of repeat freeze/thaw. The lysate was microfuged at 4C for 10 min and the supernatant was taken for both NAGLU enzyme activity and protein content using the bicinchoninic acid assay (BCA, Pierce Thermo/Fisher Scientific, Pittsburgh, PA). Intracellular enzyme activity is expressed as units per mg cell protein. The incorporation of [35S]-sulfate into intracellular GAGs was assayed with a method that eliminates the incorporation of the sulfate into extracellular GAGs. MPSIIIB fibroblasts were grown to confluency in DMEM with 10% serum. For the ‘pulse’ phase of the study, the medium was aspirated, the cells were washed with PBS, and 1 mL of Ham’s F12 low sulfate medium with 10% dialyzed fetal bovine serum and 28 uCi/mL of carrier-free [35S]-sodium sulfate (PerkinElmer) was added to each dish followed by incubation at 37C for 48 hours. For the ‘chase’ phase of the study, the medium was aspirated, the cells washed with PBS, and 1 mL/well of DMEM medium was added, which contained 2.4 nM of either the HIRMAb-LL-NAGLU fusion protein or recombinant NAGLU. Following a 48 hr incubation at 37C, the medium was


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aspirated and the cells washed with PBS. The adherent cells were removed from the plate by a 4 min incubation with trypsin-EDTA, and then centrifuged at 1000g for 6 min. The purpose of the trypsin step is to eliminate the secreted GAGs from the intracellular GAGs.13 The cell pellet was then solubilized in 1N NaOH for determination of intracellular [35S] radioactivity and cell protein using the BCA assay. Radioactivity was counted in the Ultima-gold cocktail (PerkinElmer) with a liquid scintillation counter detecting radioactivity between 2 and 1700 kev (PerkinElmer TriCarb 2800TR). The results were expressed as CPM/mg protein. The confocal microscopy was performed with the MPSIIIB fibroblasts. Sterile glass coverslips were placed in 6-well cluster dishes, followed by addition of MPSIIIB fibroblasts in DMEM with serum so that the wells were >80% confluent the next day. The medium was aspirated, the monolayer was washed with PBS and MEM without serum was added to each well containing 10 ug/mL of the HIRMAb-LL-NAGLU fusion protein. At 24 hours, the medium was aspirated, the monolayer washed with PBS and then fixed with cold acetone for 10 min at -20○C. The cells were blocked with 10% donkey serum, and were reacted with 5 ug/mL concentrations of either a mouse antibody against human lysosomal associated membrane protein (LAMP)-1 (Developmental Studies Hybridoma Bank, Iowa City, IA), or a rabbit antibody against human NAGLU (Abcam). The secondary antibody (5 ug/mL) used for LAMP1 and NAGLU staining was an Alexa Fluor-488 conjugated donkey anti-mouse IgG (Invitrogen-Thermo-Fisher), and an Alexa Fluor-555 conjugated donkey anti-rabbit IgG (Invitrogen-Thermo-Fisher), respectively. The inverted coverslips were mounted on to glass slides with Vectashield-DAPI (Vector Labs, Burlingame, CA). The cells were viewed with a confocal microscope in 3 channels: green (LAMP1), red (NAGLU), and yellow (LAMP1/NAGLU overlap). Confocal microscopy was performed with a LSM 710 Zeiss inverted fluorescence microscope with a Zeiss Plan-


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Apochromat 40X objective and a Ziess confocal laser scanning adapter utilizing argon (488 nm), DPSS (561 nm), and Coherent Chameleon (405 nm) lasers, respectively. A single optical section was obtained. Plasma pharmacokinetics and brain uptake in the Rhesus monkey. The HIRMAbLL-NAGLU fusion protein was custom radiolabeled with the [125I]-Bolton-Hunter reagent by PerkinElmer (Waltham, MA) to a specific activity of 5.6 uCi/ug, and a radiochemical purity of >95%. Although iodination of proteins may have some effect on the lipophilicity of a protein, prior work shows that iodination of an IgG molecule with no receptor specificity has no effect on BBB transport.14 Following radiolabeling, the fusion protein was shipped to MPI Research, Inc. (Mattawan, MI) for IV injection in an adult, 4.2 kg, female Rhesus monkey. The injection dose (ID) was 1900 uCi, which is equivalent to an ID of 81 ug/kg. Plasma was sampled from a femoral vein at 2, 5, 15, 30, 60, 90, and 120 min, followed by euthanasia at 120 minutes after injection. Plasma radioactivity (DPM/mL), which was precipitated by 10% cold trichloroacetic acid (TCA), was converted to ng/mL based on the specific activity of the fusion protein, and the plasma concentration was fit to a 2-exponential model by non-linear regression analysis using the PAR subroutine of the BMDP Statistical Software (Statistical Solutions, Boston, MA). The plasma pharmacokinetic (PK) parameters, plasma half-life (T1/2), mean residence time (MRT), central volume of distribution (Vc), steady state volume of distribution (Vss), steady state area under the concentration curve (AUCss), and the plasma clearance, CL, were computed from the slopes and intercepts of the 2 curves defining the plasma decay curve. Radioactivity (DPM/gram) for frontal cortex, cerebellar cortex, choroid plexus, liver, spleen, lung, heart, omental fat, and skeletal muscle, was determined and expressed as a percent of injected dose per 100 grams tissue. Organ uptake was normalized per 100 grams, because the brain weight in the adult


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Rhesus monkey is 100 grams. Transport of the fusion protein across the BBB and into brain parenchyma was confirmed with the capillary depletion method, which separates the vascular and post-vascular compartments of brain.15 The method measures the volume of distribution (VD) of the fusion protein in the total brain homogenate, the vascular pellet, and the postvascular supernatant, as described previously.16 A molecule that was completely sequestered within the capillary compartment of brain would have a VD in the post-vascular supernatant equal to the homogenate VD for a molecule that is confined to the plasma compartment of brain, such as the human IgG1k isotype control antibody. The metabolic stability of the HIRMAb-LL-NAGLU fusion protein in Rhesus monkey plasma was assessed by NAGLU Western blotting. Rhesus monkey plasma (50%) in Tris buffered saline/pH=7.0 were incubated in 100 uL volumes at 37C with 25 ug of the HIRMAbLL-NAGLU fusion protein for 0, 1, 2, and 4 hours. Aliquots were then separated by 4-12% gradient SDS-PAGE, blotted to nitrocellulose, and the filter probed with 0.5 ug/mL of the rabbit antibody against human NAGLU.


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Results The HIRMAb-LL-NAGLU fusion protein was purified to homogeneity based on reducing SDS-PAGE (Figure 2). The heavy chain of the fusion protein and the HIRMAb migrate at a MW of 140 kDa and 55 kDa, respectively, whereas both proteins share a common light chain that migrates at 27 kDa, based on either SDS-PAGE (Figure 2), or human IgG or NAGLU Western blotting (Figure 3). Based on migration in SDS-PAGE, the MW of the hetero-tetrameric HIRMAb-LL-NAGLU fusion protein is 340 kDa. The NAGLU migrates as a 85 kDa monomer on the NAGLU Western blot (Figure 3, lane 4), and the HIRMAb alone does not react with the anti-NAGLU antibody (Figure 3, lane 5). The HIR ELISA shows high affinity binding of either the HIRMAb or the HIRMAb-LL-NAGLU fusion protein to the HIR ECD, with no binding by the human IgG1k isotype control (Figure 4). Based on the MW of the HIRMAb, 150 kDa, and the HIRMAb-LL-NAGLU fusion protein, 340 kDa, the ED50 of binding of the HIRMAb or the HIRMAb-LL-NAGLU fusion protein to the HIR ECD is 0.29 ± 0.02 nM and 0.44 ± 0.03 nM, respectively (Figure 4). The NAGLU enzyme activity assay was linear with respect to incubation time, 10-60 min, and mass of fusion protein, 1 to 100 ng/tube. The NAGLU enzyme activity of the recombinant NAGLU and the HIRMAb-LL-NAGLU fusion protein was 139,400 ± 24,300 units/mg protein and 72,400 ± 3,800 units/mg protein, respectively. Based on a MW of 85 kDa for the NAGLU enzyme and 170 kDa for half of the HIRMAb-LL-NAGLU tetramer, the NAGLU specific activity is equal to 11,800 ± 2,060 and 12,300 ± 660 units/nmol of protein, respectively, for the NAGLU enzyme and the HIRMAb-LL-NAGLU fusion protein. Confocal microscopy showed the HIRMAb-LL-NAGLU fusion protein was targeted to the lysosomal compartment in MPSIIIB fibroblasts, and there is overlap of the NAGLU and LAMP1 immunoreactivity in these cells after incubation with the fusion protein (Figure 5). The


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time-response study shows the intracellular NAGLU enzyme activity exceeds 5 units/mg protein (Table 1). The intracellular NAGLU enzyme activity was undetectable following incubation with a comparable amount of recombinant NAGLU. The incorporation of [35S]-sulfate into intracellular GAGs was reduced 74 % by exposure of the MPSIIIB fibroblasts to 2.4 nM HIRMAb-LL-NAGLU fusion protein, whereas exposure of these cells to the same concentration of recombinant NAGLU had no effect on sulfate incorporation into the cells (Figure 6). The [125I]-HIRMAb-LL-NAGLU fusion protein was rapidly cleared from plasma following the IV injection of the fusion protein in an adult Rhesus monkey (Figure 7). The plasma concentration of the fusion protein was determined from the plasma radioactivity that was TCA precipitable, and the specific activity of the fusion protein, and was fit to a biexponential decay function to compute the PK parameters shown in Table 2. Consistent with the rapid clearance from plasma, the percent of plasma radioactivity that was precipitable by 10% TCA declined with time, and was 97 ± 1, 95 ± 1, 89 ± 1, 80 ± 1, 65 ± 1, 61 ± 1, and 61 ± 1, at 2, 5, 15, 30, 60, 90, and 120 min, respectively. The decay in the fractional radioactivity in plasma that was TCA precipitable reflects degradation of the fusion protein in peripheral tissues, and not in the plasma compartment. The HIRMAb-LL-NAGLU fusion protein was stable following incubation in 50% Rhesus monkey plasma at 37○C for up to 4 hours; the NAGLU Western blot of primate plasma shows no cleavage of the NAGLU enzyme from the HIRMAb (Figure 8). The organ uptake of the [125I]-HIRMAb-LL-NAGLU fusion protein is given in Table 3. The uptake was high in liver and spleen, moderate in lung, brain, heart, and fat, and low in skeletal muscle (Table 3). The uptake in brain represents transport across the BBB with distribution into the post-vascular supernatant, as shown by the capillary depletion method (Table 4). The VD of the [125I]-HIRMAb-LL-NAGLU fusion protein in the post-vascular


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supernatant, 214 ± 9 uL/gram, is >10-fold greater than the homogenate VD for the IgG1 isotype control antibody (Table 4). The TCA-precipitable radioactivity in the post-vascular supernatant at 120 min after injection, 93 ± 1%, is high relative to the plasma TCA-precipitable radioactivity at 120 min, 61 ± 1%, which indicates the low molecular weight [125I]-Bolton-Hunter radiolabeled metabolites generated in plasma with time after injection do not cross the BBB.


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Discussion The results of this study are consistent with the following conclusions. First, a novel IgG-NAGLU fusion protein has been engineered, wherein the IgG domain is the HIRMAb, and the fusion protein exhibits the same properties found in both the HIRMAb alone, and the NAGLU enzyme alone, ie, high affinity binding to the HIR (Figure 4) and high NAGLU enzyme activity (Results). Second, the HIRMAb-LL-NAGLU fusion protein is taken up by MSPIIIB fibroblasts to normalize intracellular NAGLU enzyme activity (Table 1), which is associated with targeting of the enzyme to the lysosomal compartment (Figure 5), and a reduction in intracellular GAGs (Figure 6). Third, the HIRMAb-LL-NAGLU fusion protein is rapidly cleared from plasma in the primate (Figure 7, Table 2), which is associated with high organ uptake by peripheral tissues (Table 3). Fourth, the uptake of the HIRMAb-LL-NAGLU fusion protein by the brain in the monkey is high, 1% ID/brain (Table 3), and the capillary depletion method shows the fusion protein broadly distributes into the parenchyma of brain. The treatment of MPSIIIB with recombinant NAGLU is made difficult for 2 reasons. First, the enzyme incorporates mannose 6-phosphate poorly, so that the enzyme is not targeted to cells via the M6PR.4,5 Second, the enzyme does not cross the BBB,8 which is a problem, because the major clinical problems of the disease are of brain origin.1 Both problems are solved by reengineering the enzyme as an IgG-NAGLU fusion protein, wherein the IgG domain targets the HIR, and thereby acts as a molecular Trojan horse to ferry the enzyme into cells via the insulin receptor, which is broadly expressed in tissues, including the BBB. IgG-NAGLU fusion proteins have not been previously engineered. The present study shows re-engineering the enzyme as the fusion protein shown in Figure 1 results in retention of both the HIRMAb and the NAGLU functionalities. A single IgG fusion protein both binds the HIR with high affinity (Figure 4) and


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retains high NAGLU enzyme activity (Results). The enzyme activity of recombinant NAGLU and the HIRMAb-LL-NAGLU fusion protein is 139,400 ± 24,300 units/mg protein and 72,400 ± 3,800 units/mg protein, respectively. However, on a molar basis, the NAGLU enzyme activity is comparable, as the effective MW of half of the hetero-tetrameric HIRMAb-LL-NAGLU fusion protein is 170 kDa, as opposed to the MW of 85 kDa for the NAGLU (Results). The NAGLU activity for the HIRMAb-LL-NAGLU fusion protein and the NAGLU is 12,000 units/nmol for either form of the enzyme (Results). The HIRMAb-LL-NAGLU fusion protein is taken up by MPSIIIB fibroblasts, and the maximal intracellular NAGLU enzyme activity is over 5 units/mg protein. This level of intracellular NAGLU enzyme activity is comparable either to the NAGLU in healthy fibroblasts,5 or the intracellular NAGLU enzyme activity in MPSIIIB fibroblasts exposed to the NAGLU-IGF2 fusion protein.6 The HIRMAb-LL-NAGLU fusion protein is targeted to the lysosomal compartment of MPSIIIB fibroblasts (Figure 5), despite the absence of M6P incorporation in the fusion protein. The lysosomal targeting of the fusion protein is associated with a reduction in intracellular sulfate accumulation (Figure 6). In contrast, recombinant NAGLU does not reduce sulfate accumulation in intracellular GAGs (Figure 6), and this is attributed to the poor distribution of the enzyme into the cells owing to the minimal incorporation of M6P into the enzyme.4,5 Following targeting of lysosomal enzymes to human cells via HIRMAb-mediated delivery, the intracellular T1/2 of the enzyme is 3 days.10 The HIRMAb-LL-NAGLU fusion protein is rapidly cleared from the plasma following IV injection in the adult Rhesus monkey (Figure 7). The clearance of the fusion protein is 2.8 ± 0.1 mL/min/kg, which is more than 10-fold faster than the plasma clearance of the non-fused HIRMAb in the Rhesus monkey.18 However, despite the near absence of M6P incorporation in


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NAGLU, the enzyme is rapidly removed from blood following the IV injection in MPSIIIB mice, where the T1/2 of clearance of the alpha and beta phases is 2 and 18 minutes, respectively.19 Therefore, mechanisms exist for rapid plasma clearance of the NAGLU enzyme by certain organs. In the mouse, the NAGLU is rapidly cleared by liver and spleen, with minimal clearance by lung, kidney, or heart, and there is no uptake by brain.19 The HIRMAb-LL-NAGLU fusion protein is also rapidly cleared by liver and spleen, but also penetrates into peripheral organs such as lung and heart, in the Rhesus monkey (Table 3). However, the important distinction between the HIRMAb-LL-NAGLU fusion protein and recombinant NAGLU is the uptake of the fusion protein by brain in the primate (Table 3). Capillary depletion analysis shows the brain uptake of the fusion protein does not represent sequestration by the microvascular insulin receptor, but represent transport of the fusion protein across the BBB with penetration into brain parenchyma (Table 4). Following uptake by target organs, the IgG-lysosomal enzyme is targeted to lysosomes, since chronic treatment of MPS mice with the IgG-enzyme fusion protein causes reduction in GAGs in both brain and peripheral tissues.20 The brain uptake of the HIRMAb-LL-NAGLU fusion protein is sufficiently high as to enable normalization of brain NAGLU enzyme activity following the IV infusion of the fusion protein at a dose of 1 mg/kg. Given a specific activity of 72,000 units/mg (Results), and a body weight of 50 kg, the infusion dose (ID) of 1 mg/kg of the fusion protein is equal to a dose of 3.6 million units. Given a brain weight in humans of 1,000 grams, and a brain uptake of 1% ID/brain, the brain NAGLU enzyme activity is 36,000 units/brain, or 36 units/gram brain. Assuming 100 mg protein per gram brain, the ID of 1 mg/kg of the fusion protein generates an enzyme activity in brain of 0.36 units/mg protein, which is comparable to the NAGLU enzyme


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activity in either the monkey brain, 0.7 units/mg protein,21 or in the mouse brain, 0.56 units/mg protein.19 The present study describes the use of a 31 amino acid linker, derived from the human IgG3 hinge region, between the carboxyl terminus of the heavy chain of the IgG domain and the amino terminus of the NAGLU domain (Methods). This extended linker conferred a degree of flexibility between the 2 major domains of the fusion protein, which resulted in a marked increase in secretion of the fusion protein by the transfected host cell (Methods). The IgG3 hinge sequence was chosen as a linker since this linker provides flexibility around the hinge region of the IgG3 isotype antibody, but is stable from proteases in the plasma. Similarly, the hinge region of the HIRMAb-LL-NAGLU fusion protein is resistant to proteases in primate plasma (Figure 8). However, the stability of the hinge linker does not prevent the enzyme domain from being targeted to the lysosomal compartment of target cells (Figure 5), or from reduction in intracellular GAGs in target cells (Figure 6). In conclusion, the present study describes the engineering, expression, and validation of a new recombinant HIRMAb-NAGLU fusion protein that is targeted to cells via a M6Pindependent mechanism, and is transported across the BBB in the primate in vivo. HIRMAblysosomal enzyme fusion proteins have been administered chronically for 6 months to primates at doses as high as 30 mg/kg.22,23 The brain uptake of the HIRMAb-enzyme fusion protein is sufficiently high so as to normalize brain NAGLU enzyme activity following the intravenous infusion of a 1 mg/kg dose of the fusion protein. BBB-penetrating forms of the NAGLU enzyme provide for a new treatment of the brain in MPSIIIB.


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Acknowledgement The confocal microscopy part of this work was performed at the USC/Norris Cell and Tissue Imaging Core, which is supported by NIH grants NCI 5 P30 CA014089 and NEI EY03040).


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Table 1. Intracellular NAGLU enzyme activity in MPSIIIB fibroblasts treated with the HIRMAb-LL-NAGLU fusion protein Incubation time (hours)

Intracellular NAGLU activity (units/mg protein)

none

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