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Genetic Engineering of a Bifunctional IgG Fusion Protein with Iduronate-2-Sulfatase Jeff Zhiqiang Lu,† Eric Ka-Wai Hui,† Ruben J. Boado,†,‡ and William M. Pardridge*,‡ ArmaGen Technologies, Inc., Santa Monica, California 90401, and Department of Medicine, University of California Los Angeles, Los Angeles, California 90024. Received August 31, 2009; Revised Manuscript Received October 5, 2009
Iduronate-2-sulfatase (IDS) is a lysosomal sulfatase that prevents the accumulation within the brain of glycosoaminoglycans. However, IDS does not cross the blood-brain barrier (BBB). To enable BBB transport, human IDS, minus its signal peptide, was fused to the carboxyl terminus of the heavy chain of a chimeric monoclonal antibody (mAb) to the human insulin receptor (HIR). The HIRMAb crosses the BBB on the endogenous insulin receptor and acts as a molecular Trojan horse to ferry the IDS into brain. The HIRMAb-IDS fusion protein was expressed in COS cells and purified with protein A affinity chromatography. The size of the fusion heavy chain, as measured with Western blotting and antibodies to either human IDS or human IgG, was increased about 80 kDa, relative to the size of the heavy chain of the parent HIRMAb. The HIRMAb-IDS fusion protein retained high-affinity binding for the HIR. The IDS enzyme specific activity of the fusion protein was 51 ( 7 nmol/h per microgram of protein, which is comparable to the enzyme activity of recombinant IDS. The fusion protein was taken up by human fibroblasts, and the accumulation of glycosoaminoglycans in fibroblasts null for the sulfatase was decreased 84% by treatment with the fusion protein. The HIRMAb-IDS fusion protein is a bifunctional IgG-sulfatase fusion protein, which has been specifically engineered for targeted drug delivery across the human BBB.
INTRODUCTION Mucopolysaccharidosis (MPS) type II (MPS-II), or Hunter’s syndrome, is a lysosomal storage disorder caused by defects in the gene encoding the lysosomal enzyme, iduronate-2-sulfatase (IDS) (1). MPS-II is treated with recombinant human IDS in enzyme replacement therapy (2). However, many cases of MPSII affect the central nervous system (3, 4), and enzyme replacement therapy is not effective for the brain, because the IDS does not cross the blood-brain barrier (BBB) (4, 5). In MPS-II cases that do affect the brain, the use of ERT is considered optional (5). IDS, and other lysosomal enzymes, can be made to cross the human BBB following the re-engineering of the enzyme as a fusion protein with a BBB molecular Trojan horse (6). The latter is an endogenous peptide, or peptidomimetic monoclonal antibody (mAb), that crosses the BBB via an endogenous receptor-mediated transport system. The molecular Trojan horse with the highest BBB permeation rate is a mAb against the R-subunit of the human insulin receptor (HIR) (7). Genetically engineered forms of the chimeric or humanized HIRMAb have been produced (8), and these cross the BBB in the adult Rhesus monkey in vivo at rates comparable to small molecule penetration of the brain. Previously, the lysosomal enzyme, iduronidase (IDUA), the enzyme mutated in MPS-I (9), was re-engineered as a fusion protein with the HIRMAb (10). The HIRMAb-IDUA fusion protein retained high binding affinity for the HIR and high IDUA enzyme activity. The fusion protein normalized intracellular IDUA enzyme activity and glycosoaminoglycan (GAG) accumulation in MPS-I fibroblasts, and confocal mi* Corresponding author. Dr. William M. Pardridge, UCLA Warren Hall 13-164, 900 Veteran Ave., Los Angeles, CA 90024, Ph: +1 310 825 8858, Fax: +1 310 206 5163, Email address: wpardridge@ mednet.ucla.edu. † ArmaGen Technologies, Inc. ‡ UCLA.
croscopy demonstrated that the HIRMAb-IDUA fusion protein was targeted to the lysosomal compartment of MPS-I fibroblasts (10). The HIRMAb-IDUA fusion protein was rapidly transported across the Rhesus monkey BBB in vivo, at a level that would normalize the brain IDUA enzyme activity at a therapeutic dose of 0.6 mg/kg intravenously (10). The re-engineering of lysosomal enzymes as fusion proteins with BBB molecular Trojan horses represents a new approach to the treatment of the brain in lysosomal storage disorders. The HIRMAb-IDUA described previously (10) is specific for MPS-I. Patients with MPS-II have mutations in IDS, which also adversely affects the brain. Therefore, the purpose of the present investigation was to engineer a new treatment of the brain in MPS-II, wherein the amino terminus of the mature human IDS is fused to the carboxyl terminus of the heavy chain of the HIRMAb. The structure of the HIRMAb-IDS fusion protein is shown in Figure 1. The HIRMAb-IDS fusion protein is a heterotetrameric protein comprising two heavy chains and two light chains. These studies examined the bifunctional properties of this IgG-enzyme fusion protein to determine if the new fusion protein both bound the HIR with high affinity and retained high IDS enzyme activity. The extent to which intracellular IDS enzyme activity and GAG accumulation were normalized in MPS-II fibroblasts was also investigated.
MATERIALS AND METHODS Engineering of HIRMAb Heavy Chain-IDS Fusion Protein Expression Vector. The human IDS cDNA, encoding Ser26-Pro550 (1), minus the 25 amino acid signal peptide (Genbank NP_000193), was produced by reverse transcription and PCR, starting with human liver polyA+ RNA (Clontech). Human liver cDNA was prepared using the SuperScript firststrand synthesis kit (Invitrogen, San Diego, CA) and oligodeoxythymidine priming. The IDS cDNA was cloned using 2 µL liver cDNA reverse transcription reaction, 0.2 µM IDS forward and reverse ODN primers, 0.2 mM deoxynucleotidetriphos-
10.1021/bc900382q 2010 American Chemical Society Published on Web 12/14/2009
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Figure 1. The HIRMAb-IDS fusion protein is formed by fusion of the amino terminus of the mature IDS to the carboxyl terminus of the CH3 region of the heavy chain of the chimeric HIRMAb. The fusion protein is a bifunctional molecule: the fusion protein binds the HIR, at the BBB, to mediate transport into the brain, and expresses IDS enzyme activity, which is deficient in MPS type II (Hunter’s syndrome).
Figure 2. (A) Ethidium bromide stain of agarose gel of human IDS cDNA (lane 1), which was produced by PCR from human liver cDNA, and IDS-specific primers (Methods). Lanes 2 and 3: PhiX174 HaeIII digested DNA standard, and Lambda HindIII digested DNA standard. (B) The heavy chain of the HIRMAb-IDS fusion protein is expressed by the pCD-HIRMAb-IDS plasmid, which is generated by subcloning the IDS cDNA into the HpaI site of the pCD-HIRMAb-HC plasmid.
phates, and 2.5 U PfuUltra DNA polymerase (Stratagene, San Diego, CA) in a 50 µL Pfu buffer (Stratagene). The amplification was performed in a Mastercycler temperature cycler (Eppendorf, Hamburg, Germany) with an initial denaturing step of 95 °C for 2 min followed by 30 cycles of denaturing at 95 °C for 30 s, annealing at 55 °C for 30 s and amplification at 72 °C for 1 min. PCR products were resolved in 1% agarose gel electrophoresis, and the expected major single band of ∼1.6 kb corresponding to the human IDS cDNA was isolated (Figure 2A). The cloned human IDS was inserted into the pCDHIRMAb HC eukaryotic expression plasmid at the HpaI site, and this expression plasmid was designated pCD-HIRMAb-IDS, as outlined in Figure 2B; the pCD-HIRMAb-HC expression plasmid encodes the heavy chain (HC) of the chimeric HIRMAb. The entire expression cassette of the plasmid was confirmed by bidirectional DNA sequencing. The IDS forward PCR primer
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was a 23-mer coding for the 7 amino acids at the beginning of the mature IDS protein. This primer introduces “CC” nucleotides to maintain the open reading frame and to introduce a Ser-Ser linker between the carboxyl terminus of the CH3 region of the HIRMAb HC and the amino terminus of the IDS minus the 25 amino acid signal peptide of the enzyme. The IDS reverse PCR primer was a 27-mer complementary to the end of the IDS cDNA including the stop codon, “TGA”, immediately after the terminal Pro of the mature human IDS protein. The fusion of the IDS monomer to the carboxyl terminus of each HC is depicted in Figure 1. Transient Expression of HIRMAb-IDS Fusion Protein in COS Cells. COS cells were plated in 6-well cluster dishes and were dual-transfected with pHIRMAb-LC and pCDHIRMAb-IDS plasmids using Lipofectamine 2000, with a ratio of 1:2.5 µg DNA:µL Lipofectamine, and conditioned serum free medium was collected at 3 and 7 days. The pHIRMAb-LC encodes for the light chain (LC) of the chimeric HIRMAb. Expression was confirmed by measurement of human IgG secreted to the medium by ELISA, as described previously (10). For production of larger amounts of fusion protein, COS cells were transfected in 10 × T500 flasks. The 3 day and 7 day media were pooled, and the 2 L of serum free conditioned medium was concentrated with tangential flow filtration (Millipore) followed by purification with protein A affinity chromatography. SDS-PAGE and Western Blotting. The purity of protein A purified fusion protein produced by COS cells was evaluated with 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with 5% β-mercaptoethanol. Immunoreactivity was tested with a primary goat antibody to human IDS (R&D Systems, Minneapolis, MN) or a primary goat antiserum against human IgG heavy and light chains (Vector Laboratories, Burlingame, CA). Immunoreactivity of the HIRMAb-IDS fusion protein was compared to human recombinant IDS expressed in myeloma cells from R&D Systems. HIR Receptor Assay. The affinity of the fusion protein for the HIR extracellular domain (ECD) was determined with ELISA. Chinese hamster ovary cells permanently transfected with the HIR ECD were grown in serum-free media, and the HIR ECD was purified with a wheat germ agglutinin affinity column, as previously described (11). The HIR ECD was plated on Nunc-Maxisorb 96 well dishes and the binding of the HIRMAb or the HIRMAb-IDS fusion protein to the HIR ECD was detected with a biotinylated goat antihuman IgG (H+L) secondary antibody, followed by avidin and biotinylated peroxidase (Vector Laboratories, Burlingame, CA). The concentration of either HIRMAb or HIRMAb-IDS fusion protein that gave 50% maximal binding was determined with a nonlinear regression analysis. IDS Enzyme Assay. The IDS enzyme activity was determined with a fluorometric assay using 4-methylumbelliferyl R-Liduronide-2-sulfate (4-MUS) (12), which was purchased from Moscerdam Substrates (Rotterdam, The Netherlands). This substrate is hydrolyzed by IDS to 4-methylumbelliferyl R-Liduronide (MUBI), and the MUBI is hydrolyzed by iduronidase (IDUA, Aldurazyme, Genzyme, Boston, MA) to 4-methylumbelliferone (4-MU), which is detected fluorometrically with a Farrand filter fluorometer using an emission wavelength of 450 nm and an excitation wavelength of 365 nm. A standard curve was constructed with known amounts of 4-MU (Sigma-Aldrich, St. Louis, MO). The assay was performed by incubation at 37 °C at pH ) 4.5 for 4 h in McIlvaine’s buffer, followed by the addition of 12 ug of IDUA and an additional 24 incubation at 37 °C. The incubation was terminated by the addition of 0.2 mL of 0.5 M sodium carbonate (pH ) 10.3). One unit ) 1 nmol/h.
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Fusion Protein Uptake and Biological Activity in MPS Type II Fibroblasts. Fibroblasts were chosen as a model system, since type II MPS Hunter fibroblasts (GM000298), as well as healthy human fibroblasts (GM000497), are available from the Coriell Institute for Medical Research (Camden, NJ). The cells were grown in 6-well cluster dishes to confluency. The medium was aspirated, wells washed with phosphate buffered saline (PBS), and incubated with 1 mL of Dulbecco’s modified Eagle medium without serum, along with a range of concentrations of the HIRMAb-IDS fusion protein, for 2 h at 37 °C. The medium was aspirated, and the wells were washed extensively (1 mL/well, 5 washes) with PBS, and the monolayer was extracted in 0.3 mL/well of lysis buffer (5 mM sodium formate, 0.2% Triton X-100, pH ) 4.0), followed by four freeze/ thaw cycles, and microfuged 10 min 4 °C. The supernatant was removed for IDS enzyme activity and bicinchoninic acid protein (BCA) assay. The uptake of the fusion protein was expressed as nanomoles per hour of IDS enzyme activity per milligram of cell protein. Sulfate incorporation into cellular glycosoaminoglycans (GAG) is increased in the absence of cellular IDS (13). Therefore, 35S incorporation into the healthy and Hunter fibroblasts was measured with or without treatment of the cells with the HIRMAb-IDS fusion protein. Type II MPS or healthy human fibroblasts were plated in 6-well cluster dishes at 250 000 cells/well and grown for 4 days in DMEM with 10% fetal bovine serum. The medium was discarded, the wells were washed with PBS, and 1 mL/well of low-sulfate F12 medium with 10% dialyzed fetal bovine serum was added, along with 5 mM CaCl2, the HIRMAb-IDS fusion protein (0.3 ug/mL), and 10 uCi/mL of 35S-sodium sulfate (Perkin-Elmer, Boston, MA). Following a 24 h incubation at 37 °C, the medium was aspirated, the wells were washed with cold PBS (1 mL, 5 washes), and the cells were lysed with 0.4 mL/well of 1 N NaOH. The lysate was heated 60 °C for 60 min to solubilize protein, an aliquot was removed for BCA protein assay, and the sample was counted for radioactivity with a Perkin-Elmer Tri-Carb 2100 liquid scintillation counter. The data were expressed as 35S counts per minute (CPM) per microgram of protein, as described previously (10). The percent normalization of GAG accumulation was computed from [(A - B/(A - C)] × 100, where A ) the 35S radioactivity incorporated in untreated Hunter fibroblasts, B ) the 35S radioactivity incorporated in Hunter fibroblasts treated with the HIRMAb-IDS fusion protein, and C ) the 35S radioactivity incorporated in healthy human fibroblasts.
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Figure 3. Reducing SDS-PAGE of molecular weight standards (STD), the purified HIRMAb, and the purified HIRMAb-IDS fusion protein.
RESULTS
Figure 4. Western blot with either antihuman (h) IgG primary antibody (A) or antihuman IDS primary antiserum (B). The immunoreactivity of the HIRMAb-IDS fusion protein is compared to the chimeric HIRMAb and recombinant human IDS. Both the HIRMAb-IDS fusion protein and the HIRMAb have identical light chains on the anti-hIgG Western. The HIRMAb-IDS fusion heavy chain reacts with both the anti-hIgG and the antihuman IDS antibody, whereas the HIRMAb heavy chain only reacts with the anti-hIgG antibody. The size of the HIRMAbIDS fusion heavy chain, 130 kDa, is about 80 kDa larger than the size of the heavy chain of the HIRMAb, owing to the fusion of the 80 kDa IDS to the 50 kDa HIRMAb heavy chain.
DNA sequencing of the expression cassette of the pCDHIRMAb-IDS plasmid encompassed 4063 nucleotides (nt), including a 714 nt cytomegalovirus promoter, a 9 nt full Kozak site (GCCGCCACC), a 2970 nt HIRMAb HC-IDS fusion protein open reading frame, and a 370 nt bovine growth hormone polyA sequence. The plasmid encoded for a 989 amino acid protein, comprising a 19 amino acid IgG signal peptide, the 443 amino acid HIRMAb HC, a 2 amino acid linker (SerSer), and the 525 amino acid human IDS minus the enzyme signal peptide. The predicted molecular weight of the heavychain fusion protein, minus glycosylation, is 108 029 Da, with a predicted isoelectric point (pI) of 6.03. Dual transfection of COS cells with the pCD-HIRMAb-IDS and the pHIRMAb-LC produced 2.3 L of serum-free conditioned medium over a 7 day incubation. The HIRMAb-IDS fusion protein was purified by protein A affinity chromatography to homogeneity as demonstrated by SDS-PAGE (Figure 3). The size of the heavy chain of the HIRMAb-IDS fusion protein, 135 kDa, is about 80 kDa larger than the heavy chain of the HIRMAb, whereas both proteins have light chains of equal size
(Figure 3). On Western blotting of the purified HIRMAb and the HIRMAb-IDS fusion protein, the antihuman IgG antibody reacts with a 135 kDa heavy chain for the fusion protein, and a 52 kDa heavy chain for the HIRMAb, and the difference in size, 80 kDa, is due to the fusion of IDS (Figure 4A). The antihuman IgG antibody reacts equally with the light chain of either the HIRMAb-IDS fusion protein or the HIRMAb, since both proteins comprise the same light chain, but the antihuman IgG antibody does not react with recombinant human IDS (Figure 4A). The anti-IDS antibody reacts with the 135 kDa heavy chain of the fusion protein and with the recombinant IDS, but not with the heavy chain of the HIRMAb (Figure 4B). The affinity of the fusion protein for the HIR extracellular domain (ECD) was determined with a ligand binding assay using lectin affinity purified HIR ECD (Materials and Methods section). There is comparable binding of either the chimeric HIRMAb or the HIRMAb-IDS fusion protein for the HIR ECD with ED50 of 0.32 ( 0.15 nM and 0.40 ( 0.04 nM, respectively (Figure 5). The IDS enzyme activity was determined with a
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Figure 5. Binding of either the chimeric HIRMAb or the HIRMAbIDS fusion protein to the HIR extracellular domain (ECD) is saturable. The ED50 of HIRMAb-IDS binding to the HIR ECD is comparable to the ED50 of the binding of the chimeric HIRMAb.
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Figure 7. Intracellular IDS enzyme activity is increased in Hunter fibroblasts in proportion to the concentration of medium HIRMAbIDS fusion protein. Data are means ( SE (n ) 3 dishes/point). The horizontal bar is the IDS enzyme activity in healthy human fibroblasts (mean ( SD).
Figure 8. Reversal of glycosaminoglycan (GAG) accumulation in Hunter fibroblasts with a single treatment of 0.3 ug/mL of HIRMAbIDS fusion protein in the medium. There is an 84% reduction in GAG accumulation, as compared to the 35S incorporation in healthy human fibroblasts (p < 0.0005). Data are means ( SE (n ) 4 dishes/point).
human fibroblasts, were incubated 24 h in the presence of 35Ssodium sulfate, which is incorporated into intracellular GAGs. Treatment with the HIRMAb-IDS fusion protein reduces GAG accumulation in Hunter fibroblasts by 84% as compared to healthy fibroblasts (p < 0.0005) (Figure 8).
DISCUSSION Figure 6. (A) Substrate (4-MUS), intermediate (MUBI), and product (4-MU) of the two-step enzymatic fluorometric assay of IDS enzyme activity. (B) The fluorometric units (FU) are proportional to the mass of purified HIRMAb-IDS fusion protein, and the average enzyme specific activity of the fusion protein is 51 ( 7 nmol/h/µg protein.
two-step enzymatic fluorometric assay outlined in Figure 6A. The fluorometric units were proportional to the mass of purified HIRMAb-IDS fusion protein, and the enzymatic activity of the fusion protein was 51 ( 7 nmol/h/µg protein (Figure 6B). The HIRMAb-IDS fusion protein was taken up by MPS type II fibroblasts (Figure 7A). The basal IDS activity in these cells without treatment is very low, 10 ( 3 nmol/h/mgp. The intracellular IDS enzyme activity increases in proportion to the concentration of medium HIRMAb-IDS (Figure 7). The normal IDS enzymatic activity in healthy human fibroblasts is shown by the horizontal bar in Figure 7. A medium concentration of the HIRMAb-IDS fusion protein of ∼500 ng/mL causes a 100% normalization of intracellular IDS enzyme activity (Figure 7). The Hunter fibroblasts, with or without treatment with 0.3 ug/ mL HIRMAb-IDS fusion protein in the medium, and the healthy
The results of this study are consistent with the following conclusions. First, a fusion gene has been genetically engineered that encodes the HIRMAb-IDS fusion protein depicted in Figure 1, and following expression in COS cells, the heavy chain of the fusion protein is about 80 kDa larger than the heavy chain of the chimeric HIRMAb (Figure 3), and the fusion protein heavy chain reacts with antibodies to both human IgG and human IDS (Figure 4). Second, the fusion protein is a bifunctional molecule and binds both the HIR ECD with comparable affinity to the chimeric HIRMAb (Figure 5) and has high IDS enzyme activity (Figure 6). Third, the HIRMAbIDS fusion protein is taken up by Hunter fibroblasts (Figure 7) and causes an 84% decline in GAG accumulation with 24 h of treatment (Figure 8). There are multiple approaches possible for the engineering of an IgG-enzyme fusion protein, as the enzyme could be fused to either the amino terminus or the carboxyl terminus of either the IgG heavy chain or light chain. If the enzyme is fused to the amino terminus of the IgG chain, then the enzyme is translated and folded before the IgG chain in the host cell, and this might ensure retention of enzyme activity in the fusion
IgG-Enzyme Fusion Protein
protein. However, the antigen binding site of the IgG chain is near the amino terminus and fusion of the enzyme at the amino terminus could impair IgG binding to the target receptor. These considerations were confirmed in the engineering of a fusion protein of the HIRMAb and human β-glucuronidase (GUSB). Fusion of the GUSB to the amino terminus of the HIRMAb heavy chain resulted in a retention in GUSB enzyme activity, but greatly impaired binding of the fusion protein to the HIR (14). With regard to another lysosomal enzyme, IDUA, a HIRMAb-IDUA fusion protein retained high HIR binding and high IDUA enzyme activity following fusion of the IDUA to the carboxyl terminus of the CH3 region of the HIRMAb heavy chain (10). A similar result is demonstrated in the present study on the engineering of the HIRMAb-IDS fusion protein shown in Figure 1. The fusion protein binds the HIR with the same high affinity as the HIRMAb (Figure 5). The IDS enzyme activity of the HIRMAb-IDS fusion protein is also retained, as the IDS enzyme specific activity, 51 ( 7 nmol/h/µg protein (Figure 6B), is comparable to the enzyme activity of human recombinant IDS (15). The engineering of the IgG-IDS fusion protein variant shown in Figure 1 places the IDS in a dimeric configuration. IDS is a member of a family of sulfatases, which includes arylsulfatase A (ASA) (16), and the natural configuration of ASA at neutral pH is a dimeric structure (17). The retention of IDS enzyme activity in the HIRMAb-IDS fusion protein indicates this fusion protein is correctly processed in the host cell. The SDS-PAGE (Figure 3) and the IgG and IDS Western blotting (Figure 4) show that the fusion protein secreted to the medium is of the correct size. IDS is a member of a family of sulfatases (16), wherein the activity of the enzyme is activated following the conversion of Cys-59 to a formylglycine residue by a sulfatase modifying factor in the endoplasmic reticulum (18). The retention of IDS enzyme activity in the HIRMAb-IDS fusion protein indicates the IDS is activated within the host cell despite fusion to the HIRMAb heavy chain. The HIRMAb-IDS heavy chain is triaged to the lysosome within the target cell following uptake via the cell membrane HIR, as evidenced by the 84% reduction in GAG accumulation within the Hunter fibroblast (Figure 8). Prior studies with the HIRMAb-IDUA fuson protein used confocal microscopy to confirm localization of the fusion protein within the lysosomal compartment of the human fibroblast (10). The reduction in GAG accumulation shown in Figure 8 was observed following exposure of the Hunter fibroblasts to a 0.3 ug/mL concentration of the HIRMAb-IDS fusion protein (Figure 8). A concentration of 0.3 ug/mL of the HIRMAb-IDS fusion protein is a subtherapeutic dose (Figure 7). This result corroborates prior studies, which suggest that partial restoration of intracellular lysosomal enzyme activity causes therapeutic effects within the cell (19). The HIRMAb is specific for the human insulin receptor and cross-reacts with the Old World primate insulin receptor in Rhesus monkeys, but does not react with the insulin receptor in other species (7). Consequently, the HIRMAb-IDS fusion protein cannot be administered to mouse models of MPS-II, as the fusion protein would not be recognized by the murine insulin receptor. There is no known mAb against the mouse insulin receptor that can be used as a BBB Trojan horse. Furthur drug development of the HIRMAb-IDS fusion protein as a new therapy for treatment of the brain in MPS-II will require evaluations of the potential toxicity and immunogenicity of this fusion protein. With respect to immunogenicity of fusion proteins, recent studies report on the administration of high doses to Rhesus monkeys of a fusion protein of the HIRMAb and either glial derived neurotrophic factor (GDNF) (20) or IDUA (21). The administration of large doses of the HIRMAb fusion proteins, 10-20 mg/kg, caused no tissue toxicity and had no effect on glycemic control in plasma or cerebrospinal fluid (20, 21).
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A 20 mg/kg dose of fusion protein is large compared to the projected therapeutic dose of HIRMAb-enzyme fusion proteins, 0.6 mg/kg (10). There was minimal immune response to the HIRMAb fusion proteins (20, 21). The re-engineering of recombinant proteins as IgG fusion proteins may reduce the potential immunogenicity of the recombinant protein, which can be a side effect of enzyme replacement therapy (22). Recent work shows that the constant region of human IgG contains amino acid sequences, called Tregitopes, which induce immune tolerance (23). Therefore, the re-engineering of human IDS as the HIRMAb fusion protein shown in Figure 1 may reduce the immunogenicity of the lysosomal enzyme in MPS-II patients, as well as enable enzyme penetration of the BBB.
ACKNOWLEDGMENT Winnie Tai and Phuong Tram provided expert technical assistance.
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