A very powerful acidic alpha-glucosidase that increases carbohydrates

Abstract

To provide a more optimized glycan composition consisting of a higher amount of rhGAA containing N-glycans carrying mannose-6-phosphate (M6P) or bis-M6P than conventional rhGAAs.SOLUTION: A composition comprises rhGAA, wherein: 40% to 60% of N-glycan on the rhGAA are complex-type N-glycans; and each rhGAA molecule contains at least one bis-phosphorylated mannose-6-phosphate (bis-M6 P) glycan. A pharmaceutical composition comprising the composition and at least one pharmaceutically acceptable carrier or additive are also provided.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 057,842, filed September 30, 2014; U.S. Provisional Patent Application No. 62 / 057,847, filed September 30, 2014; U.S. Provisional Patent Application No. 62 / 112,463, filed February 5, 2015; and U.S. Provisional Patent Application No. 62 / 135,345, filed March 19, 2015, each of which is invoked by whole or in whole.

[0002] This invention relates to a recombinant human alpha-glucosidase (rhGAA) composition with a higher total content of mannose-6-phosphate-supported glycans, which efficiently target CIMPR on muscle cells and subsequently deliver rhGAA to lysosomes, where rhGAA can degrade abnormally high levels of accumulated glycogen. The rhGAA of this invention exhibits superior targeting to muscle cells and subsequent delivery to lysosomes compared to conventional rhGAA products, and also exhibits other pharmacokinetic properties that make the rhGAA of this invention particularly effective for enzyme replacement therapy in subjects with Pompe disease. [Background technology]

[0003] Existing enzyme replacement therapies for Pompe disease use conventional rhGAA products with low total content of M6P-supported glycans and bis-M6P-supported glycans. These conventional products are known as Lumizyme®, Myozyme®, and alglucosidase alpha. "Lumizyme" and "Myozyme" are conventional rhGAAs manufactured or marketed as biologics by Genzyme and approved by the U.S. Food and Drug Administration, and are described with reference to the Physician's Desk Reference (2014) (which is incorporated herein by reference) or as products named "Lumizyme®" or "Myozyme®" approved for use in the United States by the FDA as of October 1, 2014. Alglucosidase alpha has the chemical name [199-arginine,223-histidine]prepro-α-glucosidase (human); molecular formula, C 4758 H 7262 N 1274 O 1369 S 35 It is identified as CAS number 420794-05-0. These products are administered to subjects with Pompe disease, also known as glycogen storage disorder type II (GSD-II) or acid maltase deficiency. Enzyme replacement therapy attempts to treat Pompe disease by administering rhGAA to replace the deficient GAA in lysosomes, thereby restoring the cellular ability to degrade lysosomal glycogen.

[0004] Pompe disease is a hereditary lysosomal storage disorder caused by a deficiency in acid alpha-glucosidase (GAA) activity. Individuals with Pompe disease lack acid alpha-glucosidase (GAA), the enzyme that breaks down glycogen, and substances that the body uses as an energy source, or their levels are reduced. This enzyme deficiency leads to excessive glycogen storage in lysosomes, which are intracellular organelles containing enzymes that normally break down glycogen and other cellular debris or waste products. Glycogen storage in certain tissues (particularly muscle) of individuals with Pompe disease impairs the ability of normally functioning cells. In Pompe disease, glycogen is not properly metabolized and gradually accumulates in lysosomes, particularly in skeletal muscle cells, and in cardiac muscle cells in infant-onset forms of the disease. Glycogen storage damages muscle and nerve cells, as well as cells in other affected tissues.

[0005] Traditionally, Pompe disease is clinically recognized as either early infantile or late-onset, depending on the age of onset. Age of onset tends to correspond to the severity of the gene mutation causing Pompe disease. The most severe gene mutations cause complete loss of GAA activity, which manifests as early-onset disease in infancy. Gene mutations that reduce but do not completely eliminate GAA activity are associated with forms of Pompe disease where onset and progression are delayed. Infantile Pompe disease appears shortly after birth and is characterized by muscle weakness, respiratory failure, and heart failure. If left untreated, it usually results in death within two years. Juvenile and adult-onset Pompe disease appear later in life and usually progress more slowly than the infantile form. These forms of the disease generally do not affect the heart, but can be fatal due to weakness of skeletal muscles and skeletal muscles involved in respiration.

[0006] Current non-symptomatic treatments for Pompe disease include enzyme replacement therapy (ERT) using recombinant human GAA (rhGAA), such as Lumizyme® or Myozyme®. This rhGAA is administered to replace or supplement GAA that is deficient or lacking in subjects with Pompe disease. However, since most rhGAA in conventional rhGAA products does not target muscle tissue, the rhGAA is unproductively removed after administration.

[0007] This occurs because the total content of M6P-supported glycans and bis-M6P-supported glycans is not high in conventional rhGAA, which targets the CIMPR on target muscle cells (the rhGAA molecule is then transported into the cell's lysosomes). This cellular uptake of rhGAA for enzyme replacement therapy is facilitated by a specific carbohydrate (mannose-6-phosphate (M6P)) that binds to the cation-independent mannose-6-phosphate receptor (CIMPR) present on the cell surface for subsequent delivery of the exogenous enzyme to the lysosome.

[0008] There are seven potential N-linked glycosylation sites on rhGAA. Because the glycosylation sites are heterogeneous among the types of N-linked oligosaccharides (N-glycans) present, rhGAA is a complex mixture of protein and N-glycans with varying binding affinities to M6P receptors and other carbohydrate receptors. rhGAA containing a high-mannose N-glycan with one M6P group (mono-M6P) binds to CIMPR with low affinity (approximately 6,000 nM), while rhGAA containing two M6P groups (bis-M6P) on the same N-glycan binds with high affinity (approximately 2 nM). Representative structures of non-phosphorylated glycans, mono-M6P glycans, and bis-M6P glycans are shown in Figure 1A. The mannose-6-P group is shown in Figure 1B. Once inside the lysosome, rhGAA can enzymatically degrade stored glycogen. However, conventional rhGAA has low total levels of M6P-supported glycans and bis-M6P-supported glycans, making it difficult for target muscle cells to achieve inferior delivery of rhGAA to lysosomes. The majority of rhGAA molecules in these conventional products lack phosphorylated N-glycans and therefore lack affinity for CIMPR. Non-phosphorylated high-mannose glycans can also be removed by mannose receptors, leading to unproductive clearance of ERT (Figure 2).

[0009] Other types of N-glycans, which are complex carbohydrates containing galactose and sialic acid, are also present on rhGAA. Since these complex N-glycans are not phosphorylated, they do not have affinity for CIMPR. However, complex N-glycans with exposed galactose residues have a moderate to high affinity for asialoglycoprotein receptors on hepatocytes in the liver, leading to rapid nonproductive clearance of rhGAA (Figure 2).

[0010] Glycosylation of GAA or rhGAA can be enzymatically modified in vitro by phosphotransferases and uncovering enzymes, as described in U.S. Patent No. 6,534,300 by Canfield et al., to generate M6P groups. Enzymatic glycosylation cannot be adequately controlled, resulting in rhGAA with undesirable immunological and pharmacological properties. Enzymatically modified rhGAA may consist solely of high-mannose N-glycans that can be potentially and enzymatically phosphorylated in vitro by phosphotransferases / uncovering enzymes, and may contain an average of 5-6 M6P groups per GAA molecule. The glycosylation patterns resulting from enzymatic treatment of GAA in vitro are problematic because additional terminal mannose residues (particularly unphosphorylated terminal mannose residues) negatively impact the pharmacokinetics of modified rhGAA. When such enzymatically modified products are administered in vivo, the mannose group increases the unproductive clearance of GAA, increases the uptake of enzymatically modified GAA by immune cells, and reduces the therapeutic effect of rhGAA due to less GAA reaching target tissues such as myocardial or skeletal muscle cells. For example, terminal unphosphorylated mannose residues are known ligands for mannose receptors in the liver and spleen, resulting in rapid clearance of enzymatically modified rhGAA and reduced targeting of rhGAA to target tissues. Furthermore, the glycosylation pattern of enzymatically modified GAA with high mannose N-glycans having terminal unphosphorylated mannose residues is similar to the glycosylation patterns of glycoproteins occurring in yeast, mold, and functionally, increasing the risk of triggering immune or allergic responses (e.g., life-threatening severe allergic (anaphylactic) reactions or hypersensitivity reactions) to this enzymatically modified rhGAA.

[0011] As explained above, conventional rhGAA products such as Lumizyme® have low levels of monophosphorylated glycans, and even lower levels of bisphosphorylated glycans. For the treatment of Pompe disease to be effective, rhGAA needs to be delivered to lysosomes in muscle cells. The low total amount of mono-M6P and bis-M6P targeting groups in conventional rhGAA limits cellular uptake and lysosomal delivery by CIMPR, thus making conventional enzyme replacement therapy inefficient. For example, conventional rhGAA products at doses of 20 mg / kg or higher may alleviate some aspects of Pompe disease, but they cannot sufficiently reduce accumulated glycogen in many target tissues (especially skeletal muscle) to reverse disease progression.

[0012] Due to the inefficiency of lysosome delivery in conventional enzyme replacement therapy, such therapies often involve other problems, such as the development of immune responses to GAA. The majority of GAA in conventional rhGAA does not contain mono-M6P or bis-M6P-supported glycans, which target muscle cells. The target immune system is exposed to this excess non-phosphorylated GAA, potentially leading to a harmful immune response that recognizes GAA. Induction of an immune response to non-phosphorylated GAA that does not penetrate target tissue and is not delivered to lysosomes increases the risk of treatment failure due to immunological inactivation of administered rhGAA, and increases the risk of the patient experiencing adverse autoimmune or allergic reactions to rhGAA treatment. The rhGAA according to the present invention contains significantly less non-phosphorylated rhGAA that is not targeted, thus reducing the patient's immune system's exposure to this non-phosphorylated rhGAA.

[0013] Logistically, higher doses place an additional burden on the subjects and the healthcare professionals treating them (e.g., longer infusion times required for intravenous administration of rhGAA). This is because conventional rhGAA contains a higher proportion of non-phosphorylated rhGAA that does not target CIMPR on muscle cells. RhGAA that does not bind to CIMPR on muscle cells and enter lysosomes does not enzymatically degrade glycogen in lysosomes. When conventional rhGAA and the rhGAA according to the present invention are administered at the same dose, more rhGAA in the composition according to the present invention binds to CIMPR on muscle cells and is then delivered to lysosomes. The rhGAA of the present invention provides physicians with the option of administering a smaller amount of rhGAA but delivering the same or more rhGAA to lysosomes. [Overview of the project] [Problems that the invention aims to solve]

[0014] In conventional rhGAA production processes such as Myozyme®, Lumizyme®, or alglucosidase alpha, the processing of cellular carbohydrates is inherently complex and extremely difficult to manipulate, resulting in no significant increase in M6P or bis-M6P content. Considering these shortcomings of conventional rhGAA products, the inventors sought and identified a method to efficiently target rhGAA to muscle cells, deliver rhGAA to lysosomes, minimize unproductive clearance of administered rhGAA, and consequently more productively target rhGAA to muscle tissue. [Means for solving the problem]

[0015] In response to the problems associated with targeting and administering conventional forms of rhGAA, and the difficulties associated with producing such well-targeted forms of rhGAA, the inventors have researched and developed means for producing rhGAA that more efficiently targets CIMPR in muscle tissue and is delivered to lysosomes, by having a higher content of M6P glycan and bis-M6P glycan compared to conventional rhGAA compositions. Furthermore, the rhGAA of the present invention has a well-treated complex N-glycan that minimizes unproductive clearance of rhGAA by non-target tissues.

[0016] Considering the problems associated with current enzyme replacement therapy using conventional rhGAA products such as Lumizyme®, and after diligent research and investigation, the inventors have developed a method for producing rhGAA in CHO cells with significantly higher total content of mono-M6P glycans and bis-M6P glycans that target CIMPR on muscle cells and subsequently deliver rhGAA to lysosomes.

[0017] The rhGAA produced by this method also possesses favorable pharmacokinetic properties due to its overall glycosylation pattern, which increases target tissue uptake and reduces unproductive clearance after administration to subjects with Pompe disease. We demonstrate that the rhGAA of the present invention is more potent and efficient in targeting skeletal muscle tissue compared to conventional rhGAAs such as Lumizyme®, as exemplified by the rhGAA represented as ATB-200. The rhGAA according to the present invention has excellent ability to productively target muscle tissue in patients with Pompe disease and reduce unproductive clearance of rhGAA, as shown in Figure 2.

[0018] The excellent rhGAA according to the present invention can be further completed by a chaperone, or can be combined with a chaperone, or can be conjugated to other groups targeting CIMPR in muscle tissue such as the portion binding to CIMPR in IGF2. The following examples show that the rhGAA of the present invention (exemplified by ATB-200 rhGAA) exceeds the existing standard treatment of enzyme replacement therapy by providing significantly better glycogen clearance in skeletal muscle compared to an existing regimen using the conventional rhGAA product Lumizyme®.

[0019] The file of this application includes at least one drawing made in color.

Brief Description of Drawings

[0020] [Figure 1A] Figure 1A shows non-phosphorylated high-mannose glycan, mono-M6P glycan and bis-M6P glycan. Figure 1B shows the chemical structure of the M6P group. [Figure 1B] Figure 1A shows non-phosphorylated high-mannose glycan, mono-M6P glycan and bis-M6P glycan. Figure 1B shows the chemical structure of the M6P group. [Figure 2A] Figure 2A shows productive targeting of rhGAA to target tissues (such as muscle tissue of a subject with Pompe disease) by glycan carrying M6P. Figure 2B explains non-productive drug clearance for non-target tissues (such as liver and spleen) or non-productive drug clearance by binding of non-M6P glycan to non-target tissues. [Figure 2B] Figure 2A shows productive targeting of rhGAA to target tissues (such as muscle tissue of a subject with Pompe disease) by glycan carrying M6P. Figure 2B explains non-productive drug clearance for non-target tissues (such as liver and spleen) or non-productive drug clearance by binding of non-M6P glycan to non-target tissues. [Figure 3A]Figure 3A illustrates the CIMPR receptor (also known as the IGF2 receptor) and the domains of this receptor. Figure 3B is a table showing the binding affinities (n molar concentration) of glycans carrying bis-M6P and glycans carrying mono-M6P for CIMPR, the binding affinity of high-mannose type glycans for the mannose receptor, and the binding affinity of desialylated complex glycans for the asialoglycoprotein receptor. RhGAA having glycans carrying M6P and glycans carrying bis-M6P can productively bind to CIMPR on the target cells of muscle. RhGAA having high-mannose glycans and desialylated glycans may unproductively bind to non-target cells carrying the corresponding receptors. [Figure 3B] Figure 3A illustrates the CIMPR receptor (also known as the IGF2 receptor) and the domains of this receptor. Figure 3B is a table showing the binding affinities (n molar concentration) of glycans carrying bis-M6P and glycans carrying mono-M6P for CIMPR, the binding affinity of high-mannose type glycans for the mannose receptor, and the binding affinity of desialylated complex glycans for the asialoglycoprotein receptor. RhGAA having glycans carrying M6P and glycans carrying bis-M6P can productively bind to CIMPR on the target cells of muscle. RhGAA having high-mannose glycans and desialylated glycans may unproductively bind to non-target cells carrying the corresponding receptors. [Figure 4A]Figures 4A and 4B show the results of CIMPR affinity chromatography for Lumizyme® and Myozyme®, respectively. The dashed line represents the M6P elution gradient. Elution by M6P replaces GAA molecules bound to CIMPR via M6P-containing glycans. As shown in Figure 4A, 78% of the GAA activity in Lumizyme® eluted before the addition of M6P. Figure 4B shows that 73% of the GAA Myozyme® activity eluted before the addition of M6P. In Lumizyme® and Myozyme, only 22% or 27% of the rhGAA eluted by M6P, respectively. These figures indicate that the majority of rhGAA in these two conventional rhGAA products lacks the M6P-containing glycans necessary to target CIMPR in target muscle tissue. [Figure 4B] Figures 4A and 4B show the results of CIMPR affinity chromatography for Lumizyme® and Myozyme®, respectively. The dashed line represents the M6P elution gradient. Elution by M6P replaces GAA molecules bound to CIMPR via M6P-containing glycans. As shown in Figure 4A, 78% of the GAA activity in Lumizyme® eluted before the addition of M6P. Figure 4B shows that 73% of the GAA Myozyme® activity eluted before the addition of M6P. In Lumizyme® and Myozyme, only 22% or 27% of the rhGAA eluted by M6P, respectively. These figures indicate that the majority of rhGAA in these two conventional rhGAA products lacks the M6P-containing glycans necessary to target CIMPR in target muscle tissue. [Figure 5] This is a DNA construct for transforming CHO cells using DNA encoding rhGAA. CHO cells were transformed with the DNA construct encoding rhGAA (SEQ ID NO: 4). [Figure 6A]Figures 6A and 6B show the results of CIMPR affinity chromatography of Myozyme and ATB-200 rhGAA. As is clear from Figure 6B, approximately 70% of the rhGAA in ATB-200 rhGAA contained M6P. [Figure 6B] Figures 6A and 6B show the results of CIMPR affinity chromatography of Myozyme and ATB-200 rhGAA. As is clear from Figure 6B, approximately 70% of the rhGAA in ATB-200 rhGAA contained M6P. [Figure 7A] The purification of ATB-200 rhGAA is described in Embodiments 1 and 2. [Figure 7B] The purification of ATB-200 rhGAA is described in Embodiments 1 and 2. [Figure 8] This shows the polywax elution profiles of Lumizyme® and ATB-200.rhGAA. [Figure 9] This is an overview of the N-glycan structure of Lumizyme® compared to three different preparations of ATB-200 rhGAA, identified as BP-rhGAA, ATB200-1, and ATB200-2. [Figure 10A] Figure 10A compares the CIMPR binding affinity of ATB-200 rhGAA (left line) and Lumizyme® (right line). Figure 10B illustrates the bis-M6P content of Lumizyme® and ATB-200 rhGAA. [Figure 10B] Figure 10A compares the CIMPR binding affinity of ATB-200 rhGAA (left line) and Lumizyme® (right line). Figure 10B illustrates the bis-M6P content of Lumizyme® and ATB-200 rhGAA. [Figure 11A]Figure 11A compares ATB-200 rhGAA activity (left line) and Lumizyme® rhGAA activity (right line) in normal fibroblasts at various GAA concentrations. Figure 11B compares ATB-200 rhGAA activity (left line) and Lumizyme® rhGAA activity (right line) in fibroblasts from subjects with Pompe disease at various GAA concentrations. Figure 11C compares fibroblasts from normal subjects (Kuptake) and fibroblasts from subjects with Pompe disease (Kuptake). [Figure 11B] Figure 11A compares ATB-200 rhGAA activity (left line) and Lumizyme® rhGAA activity (right line) in normal fibroblasts at various GAA concentrations. Figure 11B compares ATB-200 rhGAA activity (left line) and Lumizyme® rhGAA activity (right line) in fibroblasts from subjects with Pompe disease at various GAA concentrations. Figure 11C compares fibroblasts from normal subjects (Kuptake) and fibroblasts from subjects with Pompe disease (Kuptake). [Figure 11C] Figure 11A compares ATB-200 rhGAA activity (left line) and Lumizyme® rhGAA activity (right line) in normal fibroblasts at various GAA concentrations. Figure 11B compares ATB-200 rhGAA activity (left line) and Lumizyme® rhGAA activity (right line) in fibroblasts from subjects with Pompe disease at various GAA concentrations. Figure 11C compares fibroblasts from normal subjects (Kuptake) and fibroblasts from subjects with Pompe disease (Kuptake). [Figure 12A]Figure 12A shows the amount of glycogen relative to protein in myocardium after contact with the vehicle (negative control), 20 mg / ml of alglucosidase alpha, or 5, 10, or 20 mg / kg of ATB-200 rhGAA. Figure 12B shows the amount of glycogen relative to protein in quadriceps after contact with the vehicle (negative control), 20 mg / ml of Lumizyme®, or 5, 10, or 20 mg / kg of ATB-200 rhGAA. Figure 12C shows the amount of glycogen relative to protein in triceps after contact with the vehicle (negative control), 20 mg / ml of Lumizyme®, or 5, 10, or 20 mg / kg of ATB-200 rhGAA. ATB-200 rhGAA resulted in a significant reduction of glycogen in the quadriceps and triceps compared to the negative control and Lumizyme®. [Figure 12B] Figure 12A shows the amount of glycogen relative to protein in myocardium after contact with the vehicle (negative control), 20 mg / ml of alglucosidase alpha, or 5, 10, or 20 mg / kg of ATB-200 rhGAA. Figure 12B shows the amount of glycogen relative to protein in quadriceps after contact with the vehicle (negative control), 20 mg / ml of Lumizyme®, or 5, 10, or 20 mg / kg of ATB-200 rhGAA. Figure 12C shows the amount of glycogen relative to protein in triceps after contact with the vehicle (negative control), 20 mg / ml of Lumizyme®, or 5, 10, or 20 mg / kg of ATB-200 rhGAA. ATB-200 rhGAA resulted in a significant reduction of glycogen in the quadriceps and triceps compared to the negative control and Lumizyme®. [Figure 12C]Figure 12A shows the amount of glycogen relative to protein in myocardium after contact with the vehicle (negative control), 20 mg / ml of alglucosidase alpha, or 5, 10, or 20 mg / kg of ATB-200 rhGAA. Figure 12B shows the amount of glycogen relative to protein in quadriceps after contact with the vehicle (negative control), 20 mg / ml of Lumizyme®, or 5, 10, or 20 mg / kg of ATB-200 rhGAA. Figure 12C shows the amount of glycogen relative to protein in triceps after contact with the vehicle (negative control), 20 mg / ml of Lumizyme®, or 5, 10, or 20 mg / kg of ATB-200 rhGAA. ATB-200 rhGAA resulted in a significant reduction of glycogen in the quadriceps and triceps compared to the negative control and Lumizyme®. [Figure 13A] The stability of ATB-200 rhGAA is improved in the presence of the chaperone AT2221. The first left line in Figure 13A shows the percentage of unfolded ATB-200 rhGAA protein at various temperatures at pH 7.4 (blood pH). The last right line shows the percentage of unfolded ATB-200 rhGAA protein at various temperatures at pH 5.2 (lysosomal pH). The three middle lines show the effect of 10 μg, 30 μg, or 100 μg of the AT2221 chaperone on protein folding. These data show that AT2221 prevents ATB-200 rhGAA unfolding at blood pH compared to the control sample. The improvement of Tm at neutral pH by AT2221 is summarized in Figure 13B. [Figure 13B]The stability of ATB-200 rhGAA is improved in the presence of the chaperone AT2221. The first left line in Figure 13A shows the percentage of unfolded ATB-200 rhGAA protein at various temperatures at pH 7.4 (blood pH). The last right line shows the percentage of unfolded ATB-200 rhGAA protein at various temperatures at pH 5.2 (lysosomal pH). The three middle lines show the effect of 10 μg, 30 μg, or 100 μg of the AT2221 chaperone on protein folding. These data show that AT2221 prevents ATB-200 rhGAA unfolding at blood pH compared to the control sample. The improvement of Tm at neutral pH by AT2221 is summarized in Figure 13B. [Figure 14] This table shows that the combination of ATB-200 rhGAA and the chaperone AT2221 results in significantly better glycogen clearance in GAA knockout mice compared to treatment with Lumizyme® and AT2221, or with a control of either Lumizyme® or ATB200 rhGAA without the AT2221 chaperone. [Figure 15] This refers to residual glycogen in the quadriceps muscle after treatment with Lumizyme, ATB-200 rhGAA, or ATB-200 rhGAA and various concentrations of AT2221 chaperone. [Figure 16A] This shows the improvement in skeletal muscle pathology in mice treated with ATB-200 + miglustat (AT2221) compared to mice treated with ERT alone. Figure 16A shows PAS glycogen staining (Figure 16B) and EM (Figure 16B) of muscle tissue from GAA KO mice treated with conventional rhGAA or ATB-200 rhGAA and miglustat (AT-2221). Figure 16C shows the evaluation of lysosomal proliferation using the LAMP-1 marker. Figure 16D shows the identification of type I and type II muscle fibers. [Figure 16B]This shows the improvement in skeletal muscle pathology in mice treated with ATB-200 + miglustat (AT2221) compared to mice treated with ERT alone. Figure 16A shows PAS glycogen staining (Figure 16B) and EM (Figure 16B) of muscle tissue from GAA KO mice treated with conventional rhGAA or ATB-200 rhGAA and miglustat (AT-2221). Figure 16C shows the evaluation of lysosomal proliferation using the LAMP-1 marker. Figure 16D shows the identification of type I and type II muscle fibers. [Figure 16C] This shows the improvement in skeletal muscle pathology in mice treated with ATB-200 + miglustat (AT2221) compared to mice treated with ERT alone. Figure 16A shows PAS glycogen staining (Figure 16B) and EM (Figure 16B) of muscle tissue from GAA KO mice treated with conventional rhGAA or ATB-200 rhGAA and miglustat (AT-2221). Figure 16C shows the evaluation of lysosomal proliferation using the LAMP-1 marker. Figure 16D shows the identification of type I and type II muscle fibers. [Figure 16D] This shows the improvement in skeletal muscle pathology in mice treated with ATB-200 + miglustat (AT2221) compared to mice treated with ERT alone. Figure 16A shows PAS glycogen staining (Figure 16B) and EM (Figure 16B) of muscle tissue from GAA KO mice treated with conventional rhGAA or ATB-200 rhGAA and miglustat (AT-2221). Figure 16C shows the evaluation of lysosomal proliferation using the LAMP-1 marker. Figure 16D shows the identification of type I and type II muscle fibers. [Figure 17A] This shows the improvement in skeletal muscle pathology in mice treated with ATB-200 + miglustat (AT2221) compared to mice treated with ERT alone. Figure 17A shows PAS glycogen staining of muscle tissue from GAA KO mice treated with conventional rhGAA or ATB-200 rhGAA and miglustat (AT-2221). Figure 17B shows the evaluation of lysosomal proliferation using the LAMP-1 marker. [Figure 17B]This shows the improvement in skeletal muscle pathology in mice treated with ATB-200 + miglustat (AT2221) compared to mice treated with ERT alone. Figure 17A shows PAS glycogen staining of muscle tissue from GAA KO mice treated with conventional rhGAA or ATB-200 rhGAA and miglustat (AT-2221). Figure 17B shows the evaluation of lysosomal proliferation using the LAMP-1 marker. [Modes for carrying out the invention]

[0021] Definitions: Terms used herein generally have their common meanings in the art in relation to the present invention and in the specific context in which each term is used. Certain terms are discussed below or elsewhere in this specification in order to provide further guidance to those skilled in the art in the description of the compositions and methods of the present invention and the methods for producing and using the compositions of the present invention.

[0022] The term "GAA" refers to human acid α-glucosidase (GAA), an enzyme that catalyzes the hydrolysis of α-1,4-glycosidic and α-1,6-glycosidic bonds in lysosomal glycogen, as well as insertion, relational variant, or substitutional variants of the GAA amino acid sequence and longer GAA sequences that exhibit enzymatic activity. The term "rhGAA" is used to distinguish endogenous GAA from synthetic or recombinant GAA, such as GAA produced by transformation of CHO cells with GAA-encoding DNA. An exemplary DNA sequence encoding GAA is NP_000143.2 (SEQ ID NO: 4), referenced by reference. GAA and rhGAA can be present in compositions containing mixtures of GAA molecules with various glycosylation patterns (e.g., mixtures of rhGAA molecules supporting mono-M6P or bis-M6P groups on glycans and GAA molecules not supporting M6P or bis-M6P). GAA and rhGAA can also be completed with other compounds such as chaperones, or conjugated to other parts of the GAA conjugate or rhGAA conjugate. For example, the target of this conjugate can be set to CIMPR, and then this conjugate can be conjugated to the IGF2 portion that delivers to the lysosome.

[0023] Other animals and non-human animals with glycogen storage disorders can also be treated, but the “subject” or “patient” is preferably human. The subject may be a fetus, neonatal, child, adolescent, or adult with Pompe disease or other glycogen storage disorder or glycogen storage disorder. An example of an individual to be treated is an individual (fetus, neonatal, child, adolescent, youth, or adult) with GSD-II (e.g., infantile GSD-II, juvenile GSD-II, or adult-onset GSD-II). This individual may have residual GAA activity or may not have measurable activity. For example, an individual with GSD-II may have GAA activity of less than about 1% of normal GAA activity (infantile GSD-II), GAA activity of about 1-10% of normal GAA activity (juvenile GSD-II), or GAA activity of about 10-40% of normal GAA activity (adult GSD-II).

[0024] The terms “to treat” and “treatment,” as used herein, mean the remission of one or more symptoms associated with the disease, the prevention or delay of the onset of one or more symptoms of the disease, and / or the mitigation of the severity or frequency of one or more symptoms of the disease. For example, treatment can mean improvement of cardiac condition (e.g., increased end-diastolic and / or end-systolic volume, or reduction, remission, or prevention of progressive cardiomyopathy typically seen in GSD-II) or improvement of pulmonary function (e.g., increased crying vital capacity above baseline and / or normalization of oxygen saturation during crying); improvement of neurodevelopment and / or motor skills (e.g., increased AIMS score); reduction of glycogen levels in the tissues of the diseased individual; or any combination of these effects. In one preferred embodiment, treatment includes improvement of cardiac condition, particularly improvement of cardiac condition in the reduction or prevention of GSD-II-associated cardiomyopathy.

[0025] When used herein, the terms “improve,” “increase,” or “reduce” refer to values ​​relative to baseline measurements, such as measurements taken in the same individual before the commencement of the treatment described herein, or measurements taken in a control individual (or multiple control individuals) in the absence of the treatment described herein. A control individual is an individual with the same form of GSD-II (infant-onset, juvenile-onset, or adult-onset) as the individual being treated, and is approximately the same age as the individual being treated (to ensure that the disease stage in the individual being treated is equivalent to that in the control individual).

[0026] The term “purified,” as used herein, means a substance isolated under conditions that reduce or eliminate the presence of unrelated substances (i.e., impurities), including the naturally occurring substance from which the isolated substance is obtained. For example, a purified protein preferably contains substantially no other proteins or nucleic acids associated with it in cells, and a purified nucleic acid molecule preferably contains substantially no proteins or other unrelated nucleic acid molecules found together with it in cells. As used herein, the term “substantially free” is used operationally in relation to analytical testing of the substance. Preferably, a substantially free purified substance is at least 95% pure, more preferably at least 97% pure, and more preferably even more than 99% pure. Purity can be assessed by chromatography, gel electrophoresis, immunoassay, compositional analysis, biological assay, enzyme assay, and other methods known in the art. In specific embodiments, purified means that the level of impurities is below a level acceptable to regulatory authorities for safe administration to humans or non-human animals. Recombinant proteins can be isolated or purified from CHO cells using methods known in the art, such as chromatographic size separation, affinity chromatography, or anion exchange chromatography.

[0027] The terms “genetically modified” or “recombinant” mean cells (e.g., CHO cells) that express a particular gene product (e.g., rhGAA or ATB-200 rhGAA) after the introduction of a nucleic acid containing a coding sequence that encodes this particular gene product, along with regulatory elements that control the expression of this coding sequence. The introduction of nucleic acid can be achieved by any method known in the art, such as gene targeting and homologous recombination. As used herein, the term also includes cells that have been engineered, for example, by gene activation techniques, to express or overexpress endogenous genes or gene products that are not normally expressed.

[0028] Pompe disease refers to autosomal recessive LSD characterized by insufficient acid alpha-glucosidase (GAA) activity that impairs lysosomal glycogen metabolism. Enzyme deficiency leads to lysosomal glycogen accumulation, resulting in progressive skeletal muscle weakness, decreased cardiac function, respiratory failure, and / or CNS dysfunction in later stages of the disease. Genetic mutations in the GAA gene can result in either decreased expression or the creation of mutant enzymes with altered stability and / or biological activity, ultimately leading to disease. (See generally Hirschhorn R, 1995, Glycogen Storage Disease Type II: Acid α-Glucosidase (Acid Maltase) Deficiency, The Metabolic and Molecular Bases of Inherited Disease, Scriver et al., eds., McGraw-Hill, New York, 7th ed., pages 2443-2464). The three recognized clinical forms of Pompe disease (infant, juvenile, and adult) correlate with levels of residual α-glucosidase activity (Reuser AJ et al., 1995, Glycogenosis Type II (Acid Maltase Deficiency), Muscle & Nerve Supplement 3, S61-S69). Infant Pompe disease (Type I or A) is the most common and severe, characterized by growth retardation within the first two years of life, generalized hypotonia, cardiac hypertrophy, and cardiopulmonary failure. Juvenile Pompe disease (Type II or B) is of intermediate severity and is characterized by dominant muscular symptoms without cardiac enlargement. Individuals with juvenile Pompe disease usually die before reaching 20 years of age due to respiratory failure. Adult-onset Pompe disease (type III or C) often presents as a slowly progressive myopathy in the teens or late 60s (Felicia KJ et al., 1995, Clinical Variability in Adult-Onset Acid Maltase Deficiency: Report of Affected Sibs and Review of the Literature, Medicine 74, 131-135).In Pompe disease, it has been found that α-glucosidase is extensively modified post-translationally by glycosylation, phosphorylation, and proteolytic processing. Optimal glycogen catalysis requires conversion of the 110 kilodalton (kDa) precursor to the 76- and 70-kDa mature forms by proteolysis in the lysosome. As used herein, the term "Pompe disease" means all types of Pompe disease. The formulations and dosing regimens disclosed herein can be used, for example, in the treatment of type I, II, or III Pompe disease.

[0029] Non-limiting embodiments of the present invention An rhGAA composition derived from CHO cells that contains more rhGAA carrying mono-mannose-6-phosphate (M6P) or bis-M6P N-glycans compared to conventional rhGAA exemplified by Lumizyme® (alglucosidase alfa; CAS 420794-05-0). An exemplary rhGAA composition according to the present invention is ATB-200 (also sometimes designated as ATB-200, ATB-200, or CBP-rhGAA) as described in the Examples. The rhGAA (ATB-200) of the present invention binds to CIMPR with high affinity (K D ~2 - 4 nM) and is efficiently internalized by Pompe fibroblasts and skeletal muscle myoblasts (K uptake ~7 - 14 nM). It has been found that ATB-200 is characterized in vivo and has an apparent plasma half-life that is shorter (t 1 / 2 ~60 minutes) compared to current rhGAA ERT (t 1 / 2 ~45 minutes).

[0030] The amino acid sequence of rhGAA may be at least 70%, 75%, 80%, 85%, 95%, or 99% identical to the amino acid sequence described in SEQ ID NOs: 1, 3, or 4, or may contain one, two, three, four, five, six, seven, eight, nine, ten, or more deletions, substitutions, or additions. In some embodiments of the GAA or rhGAA of the present invention, e.g., ATB-200 rhGAA, the GAA or rhGAA may contain a wild-type GAA amino acid sequence, such as the wild-type GAA amino acid sequence of SEQ ID NOs: 1 or 3. In other non-limiting embodiments, rhGAA may contain a subset of amino acid residues present in wild-type GAA, which may include amino acid residues that form active sites for substrate binding and / or substrate reduction in wild-type GAA. In one embodiment, rhGAA is glucosidase alpha (human enzyme acid α-glucosidase (GAA)) encoded by the most dominant of the nine observed haplotypes of this gene. The rhGAA of the present invention, such as ATB-200 rhGAA, may include amino acid sequences that are 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of human alpha-glucosidase, such as the amino acid sequence (SEQ ID NO: 1) shown by accession number AHE24104.1 (GI: 568760974) and referenced by U.S. Patent No. 8,592,362, or the amino acid sequence of NP_000143.2 (SEQ ID NO: 4). The nucleotide and amino acid sequences of the GAA are also shown by SEQ ID NOs: 2 and 3, respectively. Variants of this amino acid sequence may include those having one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more amino acid deletions, insertions, or substitutions to the GAA amino acid sequence below. Polynucleotide sequences encoding GAA and such variant human GAA can also be considered, and these polynucleotide sequences can be used to recombinantly express rhGAA according to the present invention.

[0031] The identity between two sequences can be calculated using various alignment algorithms and / or alignment programs (e.g., FASTA or BLAST, available as part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.) and usable in default settings). For example, consider polypeptides and polynucleotides encoding such polypeptides that have at least 70%, 85%, 90%, 95%, 98%, or 99% identity with the specific polypeptides described herein and preferably exhibit substantially the same function. Unless otherwise specified, similarity scores will be based on the use of BLOSUM62. When using BLASTP, the similarity rate is based on the BLASTP positive score, and the sequence identity rate is based on the BLASTP identity score. BLASTP "identity" indicates the number and percentage of total residues in high-scoring sequence pairs that are identical, while BLASTP "positive" indicates the number and percentage of residues that have a positive alignment score and are similar to each other. This disclosure intends to include and encompasses amino acid sequences having these degrees of identity or similarity, or any moderate degree of identity or similarity, to the amino acid sequences disclosed herein. The polynucleotide sequences of similar polynucleotides can be predicted using the genetic code, and these polynucleotide sequences can be obtained by conventional means, in particular by back-translation of amino acid sequences using the genetic code.

[0032] Preferably, 70, 65, 60, 55, 45, 40, 35, 30, 25, 20, 15, 10, or less than 5% of the total rhGAA in the composition according to the present invention lacks an N-glycan supporting M6P or bis-M6P, or lacks the ability to bind to the cation-independent manose-6-phosphate acceptor (CIMPR). Alternatively, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99%, <100%, or more of the rhGAA in the composition contains at least one N-glycan supporting M6P and / or N-glycan supporting bis-M6P, or has the ability to bind to CIMPR.

[0033] The rhGAA molecules in the rhGAA composition of the present invention may have one, two, three, or four M6P groups on the glycan of the rhGAAb molecule. For example, at least one N-glycan on the rhGAA molecule may support M6P (mono-phosphorylation), a single N-glycan may support two M6P groups (bis-phosphorylation), or two different N-glycans on the same rhGAA molecule may support a common M6P group. The rhGAA molecules in this rhGAA composition may also have N-glycans that do not support M6P groups. In another embodiment, on average, the N-glycan contains more than 3 mol / mol of M6P and more than 4 mol / mol of sialic acid. On average, at least about 3, 4, 5, 6, 7, 8, 9, or 10% of the total glycans on rhGAA can be in the form of mono-M6P glycans, for example, about 6.25% of the total glycans can support a single M6P group, on average at least about 0.5, 1, 1.5, 2.0, 2.5, or 3.0% of the total glycans on rhGAA can be in the form of bis-M6P glycans, and on average less than 25% of the total rhGAA of the present invention does not contain phosphorylated glycans bound to CIMPR.

[0034] The rhGAA composition according to the present invention may have an average content of M6P-supported N-glycans in the range of 0.5 to 7.0 mol / mol rhGAA, or may have any intermediate value within a subrange such as 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, or 7.0 mol / mol rhGAA. As shown in the examples, the rhGAA of the present invention can be fractionated to produce rhGAA compositions with different average numbers of M6P-supported glycans or bis-M6P-supported glycans on the rhGAA, thereby enabling further customization of rhGAA targeting to lysosomes in target tissues by selecting specific fractions or by selectively combining various fractions.

[0035] Up to 60% of the N-glycans on rhGAA can be completely sialylated; for example, up to 10%, 20%, 30%, 40%, 50%, or 60% of these N-glycans can be completely sialylated. In some embodiments, 4–20% of the total N-glycans in the rhGAA composition are completely sialylated.

[0036] In other embodiments, 5%, 10%, 20%, or 30% or less of the N-glycans on the rhGAA may support sialic acid and terminal gal. This range includes all intermediate and partial ranges; for example, 7-30% of the total N-glycans on the rhGAA in the composition may support sialic acid and terminal gal.

[0037] In further embodiments, 5, 10, 15, 16, 17, 18, 19, or 20% or less of the N-glycans on the rhGAA have only terminal Gal and do not contain sialic acid. This range includes all intermediate and partial ranges; for example, 8-19% of the total N-glycans on the rhGAA in the composition may have only terminal Gal and do not contain sialic acid.

[0038] In other embodiments of the present invention, 40, 45, 50-60% of the total N-glycans on the rhGAA in the composition are complex N-glycans, or 1, 2, 3, 4, 5, 6, or 7% or less of the total N-glycans on the rhGAA in the composition are hybrid N-glycans, 5, 10, or 15% or less of the high-mannose N-glycans on the rhGAA in the composition are unphosphorylated, at least 5% or 10% of the high-mannose N-glycans on the rhGAA in the composition are mono-M6P phosphorylated, and / or at least 1 or 2% of the high-mannose N-glycans on the rhGAA in the composition are bis-M6P phosphorylated. These values ​​include all intermediate and partial ranges. The rhGAA composition according to the present invention may satisfy one or more of the content ranges described above.

[0039] In some embodiments, the rhGAA compositions of the present invention can, on average, support 2.0 to 8.0 sialic acid residues per mole of rhGAA. This range includes all intermediate and partial ranges such as 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0 residues / mol rhGAA. Sialic acid residues can prevent unproductive clearance by the asialoglycoprotein receptor.

[0040] Preferably, the rhGAA composition of the present invention is produced by CHO cells such as the CHO cell line GA-ATB-200, or by subcultures or derivatives of such CHO cell cultures. DNA constructs expressing an amino acid sequence that is at least 90%, 95%, or 99% identical to the amino acid sequence of an allele variant of GAA or other variant GAA, for example, SEQ ID NO: 1, can be constructed and expressed in CHO cells. Those skilled in the art can select a different vector suitable for transforming CHO cells for the production of such DNA constructs.

[0041] The inventors have discovered that rhGAA possessing superior ability to target CIMPR and cellular lysosomes, as well as a glycosylation pattern that reduces unproductive clearance of rhGAA in vivo, can be produced using Chinese hamster ovary (CHO) cells. These cells can be induced to express rhGAA with significantly higher levels of total M6P and total bis-M6P compared to conventional rhGAA products. Recombinant human GAA produced by these cells has been shown to have significantly more myocyte-targeting M6P groups and myocyte-targeting bis-M6P groups compared to conventional GAAs such as Lumizyme®, and to efficiently bind to CIMPR and be efficiently taken up by skeletal and cardiac muscle. This recombinant human GAA has also been shown to offer a favorable pharmacokinetic profile and possess a glycosylation pattern that reduces unproductive clearance in vivo.

[0042] The rhGAA according to the present invention can be formulated as a pharmaceutical composition or used in the manufacture of drugs for the treatment of Pompe disease or other conditions associated with GAA deficiency. The composition can be formulated with a physiologically acceptable carrier or additive. The carrier and composition can be sterile and otherwise adapted to the mode of administration.

[0043] Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline solutions (e.g., NaCl), physiological saline, buffered physiological saline, alcohol, glycerol, ethanol, gum arabic, vegetable oil, benzyl alcohol, polyethylene glycol, gelatin, carbohydrates (e.g., lactose, amylose, or starch), sugars (e.g., mannitol, sucrose, etc.), glucose, magnesium stearate, talc, silicic acid, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, etc., and combinations thereof. Pharmaceutical preparations can be mixed with adjuvants as needed, for example, surfactants such as polysorbates like polysorbate 80, lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, colorants, flavorings and / or aromatic substances, etc., which do not react harmfully with the active compound. In preferred embodiments, water-soluble carriers suitable for intravenous administration are used.

[0044] The composition or drug may optionally contain small amounts of wetting agents, emulsifiers, or pH buffers. The composition may be in the form of a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained-release formulation, or powder. The composition may also be formulated as a suppository with conventional binders and carriers (e.g., triglycerides). Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, or magnesium carbonate. In preferred embodiments, rhGAA is administered by IV infusion.

[0045] The composition or drug can be formulated as a pharmaceutical composition suitable for administration to humans according to standard procedures. For example, in a preferred embodiment, the composition for intravenous administration is a solution in sterile isotonic aqueous buffer. If necessary, the composition may also contain a solubilizer and a local anesthetic to alleviate pain at the injection site. Typically, the components are supplied separately or mixed together in a unit dosage form, for example, as a lyophilized powder or water-free concentrate in a sealed container such as an ampoule or pouch indicating the amount of the active drug. When the composition is administered by infusion, it can be dispensed from an infusion bottle containing sterile pharmaceutical-grade water, saline, or glucose / water. When the composition is administered by injection, an ampoule of sterile water or saline for injection can be provided so that the components can be mixed before administration.

[0046] rhGAA can be formulated in neutral or salt form. Examples of pharmaceutically acceptable salts include salts formed by free amino groups derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed by free carboxyl groups derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0047] rhGGA (or a composition or drug containing GAA) is administered via an appropriate route. In one embodiment, GAA is administered intravenously. In other embodiments, GAA is administered directly to target tissue such as the heart or skeletal muscle (e.g., intramuscularly) or to the nervous system (e.g., directly into the brain, ventricles, or spinal cavity). Multiple routes may be used simultaneously as needed.

[0048] rhGAA (or a composition or drug containing GAA) is administered in a therapeutically effective amount (for example, a dose sufficient to treat the disease by preventing or delaying the onset of the disease, and / or by reducing the severity or frequency of disease symptoms, for example, by improving disease-related symptoms, as described above, when administered at regular intervals). The amount that would be therapeutically effective in treating the disease may depend on the nature and extent of the disease's effects and can be determined by standard clinical techniques. In addition, appropriate dosage ranges can be easily determined by optionally using in vitro or in vivo assays. The exact dose to be used may also depend on the route of administration and the severity of the disease and should be determined according to the judgment of those skilled in the art and the circumstances of each patient. Effective doses can be estimated from dose-response curves derived from in vitro or animal model test systems. In preferred embodiments, the therapeutically effective amount is 20 mg or less of enzyme per kg of body weight, preferably in the range of about 1 to 10 mg of enzyme per kg of body weight, and more preferably about 10 mg or about 5 mg of enzyme per kg of body weight. The effective dose for a particular individual can be adjusted over time (increased or decreased) according to the individual's needs. For example, this amount can be increased in cases of physical illness or stress, when anti-GAA antibodies are present or increase, or when symptoms of a disease worsen.

[0049] A therapeutically effective amount of GAA (or a composition or drug containing GAA) is administered regularly and continuously, depending on the nature or degree of the disease's effect. “Regular intervals” as used herein means administering a therapeutically effective amount regularly (distinguishable from a single dose). This interval can be determined by standard clinical techniques. In preferred embodiments, GAA is administered once a month, once every two months, once a week, twice a week, or daily. The administration interval for a single individual does not need to be fixed and may vary over time depending on the individual's needs. For example, the interval between doses can be shortened if anti-GAA antibodies become present or increase during physical illness or stress, or if the symptoms of the disease worsen. In some embodiments, a therapeutically effective amount of 5, 10, 20, 50, 100, or 200 mg enzyme / kg body weight is administered twice a week, once a week, or every other week, with or without a chaperone.

[0050] The GAA or rhGAA of the present invention can be prepared for later use, for example, in unit dose vials or syringes or in bottles or pouches for intravenous administration. Kits containing the GAA or rhGAA and other active ingredients such as optional additives or chaperones or other agents can be enclosed in packaging materials, and instructions for reconstitution, dilution, or administration for treating subjects requiring treatment, such as patients with Pompe disease, may be included.

[0051] GAA (or a composition or drug containing GAA) can be administered alone or in combination with other drugs such as chaperones. RhGAAs with different degrees of glycosylation by mono-M6P or bis-M6P can be administered, or combinations of rhGAAs with different degrees of M6P glycosylation or bis-M6P glycosylation can be administered.

[0052] In some embodiments, the rhGAA composition of the present invention can be complexed with or mixed with a chaperone such as AT2200 or AT-2221. A chaperone (sometimes referred to as a "pharmacological chaperone") is a compound that, when complexed with or co-administered with rhGAA, alters the pharmacokinetics and other pharmacological properties of rhGAA. Representative chaperones exemplified herein include AT2221 (miglustat, N-butyl-deoxynojirimycin) and AT2220 (duvoglustat HCl, 1-deoxynojirimycin). Such complexing or mixing can occur in vitro or in vivo, for example, by administering separate doses of rhGAA and the chaperone. For example, the targeting of the active rhGAA of the present invention, its fraction, or a derivative to CIMPR and subsequent targeting to cellular lysosomes can be improved by combining the active rhGAA with duvoglustat-HCl (AT2220, deoxynojirimycin, AT2220) or miglustat (AT2221, N-butyl-deoxynojirimycin). The following examples demonstrate a significant reduction in glycogen substrates in key skeletal muscle of GAA knockout mice administered with the well-targeted rhGAA of the present invention in combination with a chaperone.

[0053] Another aspect of the present invention relates to CHO cells or derivatives or other equivalents that produce rhGAA according to the present invention. An example of such a CHO cell line is GA-ATB-200 or its subcultures that produce the rhGAA composition described herein. Such a CHO cell line may contain multiple copies (e.g., 5, 10, 15, or 20 or more copies) of the gene comprising the polynucleotide encoding GAA.

[0054] By transforming CHO cells (Chinese hamster ovary cells) with a DNA construct encoding GAA, high-M6P rhGAA and high-bis-M6P rhGAA (e.g., ATB-200 rhGAA) can be produced according to the present invention. Although CHO cells have already been used for the production of rhGAA, it was not recognized that transformed CHO cells could be cultured and selected to produce rhGAA with high content of CIMPR-targeting M6P glycans and bis-M6P glycans.

[0055] Surprisingly, the inventors have discovered that it is possible to transform a CHO cell line, select transformants that produce rhGAA containing a high content of M6P or bis-M6P-supporting glycans targeting CIMPR, and stably express this high-M6P rhGAA. Therefore, a relevant aspect of the present invention relates to a method for producing this CHO cell line. This method comprises transforming CHO cells with DNA encoding GAA or a GAA variant; selecting CHO cells that stably incorporate the DNA encoding GAA into the chromosomes of the CHO cells and stably express GAA; selecting CHO cells that express GAA with a high content of M6P or bis-M6P-supporting glycans; and optionally selecting CHO cells that have N-glycans with a high sialic acid content and / or N-glycans with a low content of non-phosphorylated high-mannose.

[0056] By culturing this CHO cell line and recovering the rhGAA composition according to the present invention from the culture of CHO cells, rhGAA and the rhGAA composition according to the present invention can be produced using this CHO cell line.

[0057] The present invention provides a way to treat subjects with conditions, disorders, or diseases associated with insufficient lysosomal GAA by advantageously using the rhGAA composition or a fraction or derivative thereof and administering the rhGAA composition. Subjects requiring treatment include those with glycogen storage disorder type II (Pompe disease), as well as those with other conditions, disorders, or diseases that would benefit from the administration of rhGAA.

[0058] The following examples demonstrate that the rhGAA (ATB-200) of the present invention, when administered at significantly lower doses compared to conventional rhGAA products, is taken up by skeletal muscle cells, binds to CIMPR, and efficiently removes glycogen from skeletal muscle cells. In GAA knockout mice using a bi-weekly intravenous administration regimen of ATB-200, a reduction of up to 75% in glycogen in skeletal muscle myoblasts was achieved. This reduction surpassed that achieved by the same amount of Lumizyme®, indicating that the rhGAA of the present invention, with its increased content of M6P-supported N-glycans and bis-M6P-supported glycans, resulted in superior reduction of glycogen substrates. Due to improved targeting, the pharmacokinetics and effects of the rhGAA composition of the present invention can be obtained at lower doses compared to conventional rhGAA products such as Lumizyme® or Myozyme®.

[0059] The rhGAA of the present invention can be used to break down, reduce, or remove glycogen from cardiac muscle, smooth muscle, or striated muscle. Examples of skeletal or striated muscles targeted for treatment include: abductor digiti minimi (foot), abductor digiti minimi (hand), abductor hallucis, abductor pollicis brevis, abductor pollicis longus, adductor brevis, adductor hallucis, adductor longus, adductor magnus, adductor pollicis, anconeus, articularis genu, aryepiglotticus, aryjordanicus, auricularis, biceps brachii, biceps femoris, brachialis, brachioradialis, buccinator, bulbocavernosus, hypopharyngeal constrictor, oropharynx constrictor, suprapharynx constrictor, coracobrachialis, corrugator supercilii, cremaster, cricothyroid, arrhizomatous, and deep transverse perineal muscle. Perinei), deltoid, depressor anguli oris, depressor labii inferioris, diaphragm, digastric muscle, digastric muscle (anterior view), erector spinae - spinalis, erector spinae - iliocostalis, erector spinae - longissimus, extensor carpi radialis brevis, extensor carpi radialis longus, extensor carpi ulnaris, extensor digiti minimi (hand), extensor digitorum (hand), extensor digitorum brevis (foot), extensor digitorum longus (foot), extensor hallucis longus, extensor indicis, extensor hallucis brevis, extensor hallucis longus, external oblique Abdominis), flexor carpi radialis, flexor carpi ulnaris, flexor digiti minimi brevis (foot), flexor digiti minimi brevis (hand), flexor digitorum brevis, flexor digitorum longus (foot), flexor digitorum profundus, flexor digitorum superficialis, flexor hallucis brevis, flexor hallucis longus, flexor hallucis brevis, flexor hallucis longus, frontalis, gastrocnemius, inferior gemellus, superior gemellus, genioglossus, geniohyoid, gluteus maximus, gluteus medius, gluteus minimus, gracilis, hyoglossus, iliacus, inferior oblique, inferior rectus inferior, infraspinatus, external intercostal muscles, internalst intercostal muscles, internal intercostal muscles, internal obliqueAbdominis), dorsal interosseous muscles of the hand, dorsal interosseous muscles of the foot, palmar interosseous muscles of the hand, plantar interosseous muscles of the foot, interspinous muscles, intertransverse muscles, intrinsic muscles of the tongue, ischiocavernosus muscle, lateral cricoarytenoid muscle, lateral pterygoid muscle, lateral rectus muscle, latissimus dorsi, levator anguli oris muscle, levator ani-coccyx, levator ani-iliococcygeus muscle, levator ani-pubococcygeus muscle, levator ani-puborectalis muscle, levator ani-pubovaginal muscle, levator labii superioris muscle, levator labii superioris alaeque nasi muscle, levator palpebral superioris muscle, levator scapulae muscle, levator veli palatini muscle, levator costalis muscle, longus capitis muscle, longus colli muscle, medialis muscles of the foot (4), medialis muscles of the hand, masseter muscle Medial pterygoid, medial rectus, mentalis, uvula, mylohyoid, nasal muscle, oblique arytenoid, inferior oblique capitis, superior oblique capitis, obturator externus, obturator internus (A), obturator internus (B), omohyoid, opponens digiti minimi (hand), opponens pollicis, orbicularis oculi, orbicularis oris, lingual palatine, palatopharynx, palmaris brevis, palmaris longus, pectineus, pectoralis major, pectoralis minor, peroneus brevis, peroneus longus, peroneus tertius, piriformis (A), piriformis (B), plantaris, platysma, popliteus, posterior cricoarytenoid, procerus, pronator quadratus, pronator teres, psoas major, psoas minor, pyramidalis, femur Quadratus, quadratus lumborum, quadratus plantaris, rectus abdominis, rectus capitis anterior lateralis, rectus capitis lateralis, rectus capitis posterior major, rectus capitis posterior minor, rectus femoris, rhomboid major, rhomboid minor, risorius, tubopharyngeal, sartorius, anterior scalene, middle scalene, minor scalene, posterior scalene, semimembranosus, semitendinosus, serratus anterior, inferior serratus posterior, superior serratus posterior, soleus, anal sphincter, urethral sphincter, splenius capitis, splenius cervicis, stapedius, sternocleidomastoid, sternohyoid, sternothyroid, styloglossus, stylohyoid, stylohyoid (anterior view), stylopharyngeus, subclavius, subcostal This includes at least one muscle selected from the group consisting of the subscapularis, superficial transverse perineum, superior oblique, superior rectus, supinator, supraspinatus, temporalis, temporoparietal, tensor fasciae latae, tensor tympani, tensor veli palatini, teres major, teres minor, thyroarytenoid and vocal cord muscles, thyroepiglottis, thyrohyoid, tibialis anterior, tibialis posterior, transverse arytenoid, transversospinalis-multifidus, transversospinalis-rotator, transversospinalis-semispinalis, transversus abdominis, transversus thoracis, trapezius, triceps, vastus intermedius, vastus lateralis, vastus medialis, zygomaticus major, and zygomaticus minor.

[0060] The GAA composition of the present invention may be administered to subjects with type 1 (slow contraction) muscle fibers or type 2 (fast contraction) muscle fibers or subjects accumulating glycogen in such muscle fibers, or the GAA composition of the present invention may be used to treat subjects with type 1 (slow contraction) muscle fibers or type 2 (fast contraction) muscle fibers or subjects accumulating glycogen in such muscle fibers. Type 1 (slow contraction) muscle, or red muscle, is densely packed with capillaries and rich in mitochondria and myoglobin, giving muscle tissue its characteristic red color. Type 1 muscle can carry more oxygen and can sustain aerobic activity using fat or carbohydrates as fuel. Slow contraction fibers contract for a long period of time but with little force. Type 2 (fast contraction) muscle has three main subtypes (IIa, IIx and IIb) that differ in both contraction speed and the force produced. Fast contraction fibers contract rapidly and powerfully but fatigue very rapidly, maintaining anaerobic explosive activity for only a short period before muscle contraction becomes painful. Fast-contracting fibers contribute most to muscle strength and have a greater potential for mass increase. Type IIb is an anaerobic, glycolytic "white" muscle with a slightly denser concentration of mitochondria and myboglobin. In small animals (e.g., rodents), type IIB is the dominant fast-twitch muscle type, explaining the light color of the meat in these animals.

[0061] The rhGAA composition, fraction thereof, or derivative of the present invention can be administered systemically, for example, by intravenous (IV) infusion, or directly into a desired site (e.g., myocardium, or skeletal muscles such as the quadriceps or triceps, or the diaphragm). The rhGAA composition of the present invention can be administered to muscle cells, and in particular to muscle tissue, muscle, or muscle groups. For example, in such a procedure, the rhGAA composition can be directly administered intramuscularly into the target quadriceps or triceps or diaphragm.

[0062] As described above, the rhGAA composition, fraction thereof, or derivative of the present invention can be complexed with a chaperone such as AT-2220 (duvoglustat HCl, 1-deoxynojirimycin) or AT-2221 (miglustat, N-butyl-deoxynojirimycin) or salts thereof, or mixed with such a chaperone, to improve the pharmacokinetics of rhGAA administration. rhGAA and the chaperone can be administered together or separately. When administered simultaneously, the chaperone can be pre-incorporated into the GAA in the composition. Alternatively, GAA and the chaperone can be administered simultaneously or separately at different times.

[0063] Typical dosages of AT2221 range from 0.25 to 400 mg / kg, preferably from 0.5 to 200 mg / kg, and most preferably from 2 to 50 mg / kg. Specific dosages of AT2221 include 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 mg / kg. These dosages can be combined with rhGAA (e.g., ATB-200 rhGAA) in a molar ratio of AT2221 to rhGAA in the range of 15:1 to 150:1. Specific ratios include 15:1, 20:1, 25:1, 50:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 100:1, 125:1, and 150:1. rhGAA and AT2221 can be co-administered simultaneously, sequentially, or separately in these amounts or molar ratios. The above range includes all intermediate subranges and values, such as all integer values ​​between the endpoints of this range.

[0064] Typical dosages of AT2220 range from 0.1 to 120 mg / kg, preferably from 0.25 to 60 mg / kg, and most preferably from 0.6 to 15 mg / kg. Specific dosages of AT2220 include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, and 30 mg / kg. These dosages can be combined with rhGAA (e.g., ATB-200 rhGAA) in a molar ratio of AT2220 to rhGAA in the range of 15:1 to 150:1. Specific ratios include 15:1, 20:1, 25:1, 50:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 100:1, 125:1, and 150:1. rhGAA and AT2220 can be co-administered simultaneously, sequentially, or separately in these amounts or molar ratios. The above range includes all intermediate subranges and values, such as all integer values ​​between the endpoints of this range.

[0065] The rhGAA composition, fraction thereof, or derivatives of the present invention can also be used to metabolize, degrade, remove, or otherwise reduce glycogen in tissues, muscles, muscle fibers, muscle cells, lysosomes, organelles, intracellular compartments, or cytoplasm. This can be optionally achieved by administering the rhGAA composition to the target in combination with a chaperone or an agent that reduces the immune response to rhGAA.

[0066] In another embodiment of this method of use, rhGAA of the present invention can be used to modulate lysosome proliferation, autophagy, or exocryosis in cells by administering rhGAA, its fraction, or derivatives to cells, tissues, or subjects requiring regulation of lysosome proliferation, autophagy, or exocryosis in cells, optionally in combination with a chaperone, or optionally as a conjugate with another targeting moiety. Autophagy is a catabolic mechanism that allows cells to degrade glycogen or other unnecessary or dysfunctional cellular components through the action of their lysosomes. This method may also include systemic or topical administration of the GAA composition to subjects requiring treatment.

[0067] The rhGAA according to the present invention (enriched with mono-M6P and bis-M6P compared to Lumizyme® and Myozyme, and possessing advantageous pharmacokinetic properties conferred by the glycosylation pattern of this rhGAA) can also be used to treat other conditions requiring the breakdown of complex carbohydrates, for example, other disorders (e.g., glycogen storage disorder type III) in which glycogen or other carbohydrates broken down by rhGAA accumulate in lysosomes or in other parts of cells accessible to rhGAA (e.g., cytoplasm). The rhGAA according to the present invention can also be used for non-therapeutic purposes, for example, in the manufacture of foods, beverages, chemical products and pharmaceuticals that require the breakdown of complex carbohydrates such as starch and glycogen into these monomers. [Examples]

[0068] The following non-limiting embodiments illustrate aspects of the present invention.

[0069] Section I: ATB-200 rhGAA and its properties Limitations of existing Myozyme® rhGAA products and Lumizyme® rhGAA products To evaluate the rhGAA activity in Myozyme® and Lumizyme®, the only currently approved treatments for Pompe disease, these rhGAA preparations were injected onto a CIMPR column (which binds to rhGAA with an M6P group) and subsequently eluted with a free M6 gradient. The fractions were collected in 96-well plates and assayed for GAA activity with a 4MU-α-glucose substrate. The relative amounts of bound and unbound rhGAA were determined based on GAA activity and reported as a percentage of the total enzyme.

[0070] Figure 5 illustrates the problems associated with conventional ERTs (Myozyme® and Lumizyme®), where 73% of rhGAA in Myozyme® (Figure 5B) and 78% of rhGAA in Lumizyme® (Figure 5A) did not bind to CIMPR (see the leftmost peak in each figure). A small percentage, 27% of rhGAA in Myozyme® and 22% of rhGAA in Lumizyme®, contained M6P, which can productively target rhGAA to CIMPR on muscle cells (see Figure 2, which illustrates productive drug targeting and unproductive drug clearance).

[0071] The effective dose of Myozyme® and Lumizyme® corresponds to the amount of rhGAA containing M6P that targets CIMPR on muscle cells. However, the majority of rhGAA in these two conventional products does not target CIMPR receptors on target muscle cells. Administration of conventional rhGAA, in which the majority does not target muscle cells, increases the risk of allergic reactions or immune induction to untargeted rhGAA.

[0072] Preparation of CHO cells that produce ATB-200 rhGAA with a high content of mono-M6P-supported N-glycan or bis-M6P-supported N-glycan. After transfecting CHO cells with rh-GAA-expressing DNA, transformants that produce rhGAA were selected. The DNA constructs for transforming CHO cells with rh-GAA-encoding DNA are shown in Figure 5. After transfecting CHO cells with rh-GAA-expressing DNA, transformants that produce rhGAA were selected.

[0073] After transfection, DG44 CHO(DHFR-) cells containing stably incorporated GAA genes were selected in hypoxanthine / thymidine-deficient (-HT) medium. Amplification of GAA expression in these cells was induced by methotrexate treatment (MTX, 500 nM). A cell pool expressing high levels of GAA was identified by GAA enzyme activity assay, and individual clones producing rhGAA were established using this cell pool. Individual clones were generated on semi-solid medium plates, harvested using the ClonePix system, and transferred to 24-deep-well plates. Individual clones were assayed for GAA enzyme activity to identify clones expressing high levels of GAA. A 4-MU-α-glucosidase substrate was used as the conditional medium for measuring GAA activity. Clones producing higher levels of GAA as measured by GAA enzyme assay were further evaluated for viability, proliferation, GAA production capacity, N-glycan structure, and stable protein expression. Using this procedure, we isolated CHO cell lines expressing rhGAA with increased mono-M6P N-glycan or bis-M6P N-glycan levels (e.g., CHO cell line GA-ATB-200).

[0074] Purification of rhGAA ATB-200 Using the CHO cell line GA-ATB-200, multiple batches of rhGAA according to the present invention were produced in shaking flasks and perfusion bioreactors, and CIMPR binding was measured. For purified ATB-200 rhGAA from various production batches, CIMPR receptor binding (approximately 70%) was observed similar to that shown in Figures 6B and 7, indicating that ATB-200 rhGAA can be consistently produced. As shown in Figures 6A and 6B, rhGAA from Myozyme® and Lumizyme® showed significantly less CIMPR binding compared to ATB-200 rhGAA.

[0075] Analysis comparison of ATB-200 and Lumizyme ATB-200 rhGAA was fractionated according to terminal phosphate using weak anion exchange ("WAX") liquid chromatography. Elution profiles were created by increasing the amount of salt to elute ERT. These profiles were monitored under UV (A280nm). ATB-200 rhGAA was obtained from CHO cells and purified. Lumizyme® was obtained from a commercial source. Lumizyme® showed a high peak on the left side of its elution profile. ATB-200 rhGAA showed four prominent peaks eluting to the right of Lumizyme® (Figure 8). This confirms that ATB-200 rhGAA was phosphorylated to a greater extent than Lumizyme®, because this evaluation is based on terminal charge rather than CIMPR affinity.

[0076] Characterization of the oligosaccharide ATB-200 rhGAA Purified ATB-200 rhGAA and Lumizyme® glycans were evaluated by MALDI-TOF, and the individual glycan structures found on each ERT were determined (Figure 9). The ATB-200 sample was found to contain slightly lower amounts of non-phosphorylated high-mannose N-glycans compared to Lumizyme®. The higher M6P glycan content in ATB-200 compared to Lumizyme suggests that ATB-200 rhGAA targets muscle cells more efficiently. The high proportion of mono-phosphorylated and bis-phosphorylated structures determined by MALDI is consistent with the CIMPR profile showing significantly higher binding of ATB-200 to the CIMPR receptor. N-glycan analysis by MALDI-TOF mass spectrometry confirmed that, on average, each ATB200 molecule contains at least one native bis-M6P N-glycan structure. Higher bis-M6P N-glycan content in ATB-200 rhGAA indicates high affinity binding to CIMPR in M6P receptor plate-binding assays (K D It was directly correlated with approximately 2-4 nM (Figure 10A).

[0077] Characterization of CIMPR affinity of ATB-200 In addition to having a higher proportion of rhGAA capable of binding to CIMPR, understanding the quality of this interaction is crucial. Receptor binding of Lumizyme® and ATB200 rhGAA was measured using a CIMPR plate binding assay. In short, GAA was captured using a CIMPR-coated plate. Various concentrations of rhGAA were applied to the immobilized receptor, and unbound rhGAA was washed away. The amount of remaining rhGAA was measured by GAA activity. As shown in Figure 10A, ATB-200 rhGAA bound to CIMPR significantly more than Lumizyme.

[0078] Figure 10B shows the relative content of bis-M6P glycan in Lumizyme, a conventional rhGAA, and in ATB-200 according to the present invention. In the case of Lumizyme®, on average only 10% of molecules contain bis-phosphorylated glycan. In contrast, in ATB-200, on average every rhGAA molecule contains at least one bis-phosphorylated glycan.

[0079] ATB-200 rhGAA was internalized more efficiently by fibroblasts compared to Lumizyme. The relative cellular uptake of rhGAA from ATB-200 and Lumizyme® was compared using normal fibroblast cell lines and Pompe fibroblast cell lines. The comparison included ATB-200 rhGAA according to the present invention at concentrations of 5–100 nM and conventional rhGAA Lumizyme® at concentrations of 10–500 nM. After 16 hours of incubation, the external rhGAA was inactivated with TRIS bases, and the cells were collected after washing them three times with PBS. Internalized GAA was measured by 4MU-α-glucoside hydrolysis and graphed against total cellular protein; the results are shown in Figure 11.

[0080] It was also found that ATB-200 rhGAA is efficiently internalized in each cell (Figures 11A and 11B), indicating that ATB-200 rhGAA is internalized in both normal fibroblasts and Pompe fibroblasts, and that it is internalized to a greater extent than conventional Lumizyme® rhGAA. ATB-200 rhGAA saturates the cell receptor at approximately 20 nM, while Lumizyme® requires approximately 250 nM. The uptake efficiency constant (K) can be estimated from these results. uptake As shown in Figure 11C, the rhGAA is 2-3 nm for ATB-200 and 56 nM for Lumizyme®. These results suggest that ATB-200 rhGAA is a good targeted treatment for Pompe disease.

[0081] Section II: Preclinical studies ATB-200 rhGAA, which possesses superior glycosylation properties, showed significantly better glycogen clearance in the skeletal muscle of GAA KO mice compared to standard treatment ERT. As described above, enzyme replacement therapy (ERT) using recombinant human rhGAA (rhGAA) is the only approved treatment available for Pompe disease. This ERT requires a specific carbohydrate, mannose 6-phosphate (M6P), for cellular uptake and subsequent delivery to lysosomes via the cellular cation-independent M6P receptor (CIMPR). However, current rhGAA ERTs contain only small amounts of M6P, thus limiting drug targeting and efficacy in disease-related tissues. To improve drug targeting, we have developed a cell line and production method for rhGAA (referred to as ATB-200 rhGAA) that has superior glycosylation and a high M6P content compared to conventional rhGAA (particularly compared to the high-affinity bis-M6P N-glycan structure). ATB-200 rhGAA binds to CI-MPR with high affinity (KD approximately 2-4 nM) and is efficiently internalized by Pompe fibroblasts and skeletal muscle myoblasts (K uptake approximately 7-14nM).

[0082] ATB-200 rhGAA significantly better removes glycogen from skeletal muscle compared to Lumizyme®. The effects of Lumizyme® and ATB-200 rhGAA administration on glycogen clearance in GAA KO mice were evaluated. Animals received two IV bolus doses (every other week), and tissue was extracted two weeks after the last dose and analyzed for GAA activity and glycogen content (Figure 12). ATB-200 rhGAA and Lumizyme® rhGAA were equally effective in removing glycogen from the heart (Figure 12A). As shown in Figures 12B and 12C, ATB-200 rhGAA at 5 mg / kg was equivalent to Lumizyme® rhGAA at 20 mg / kg in terms of reducing glycogen in skeletal muscle, and ATB-200 administered at 10 and 20 mg / kg was significantly better than Lumizyme® in terms of removing glycogen from skeletal muscle.

[0083] Theoretical basis for co-administration of ATB-200 rhGAA and AT2221 (CHART technology) Chaperones bind to and stabilize rhGAA ERT, increasing the uptake of reactive oxygen species into tissues, improving tolerability, and potentially reducing immunogenicity. As shown above, CHART® was used to significantly improve the protein stability of ERT under unfavorable conditions. See CHART: Chaperone-Improved Replacement Therapy, referenced by reference http: / / _www.amicusrx.com / chaperone.aspx (last accessed September 22, 2015). As shown in Figures 13A and 13B, AT2221 (miglustat, N-butyl-deoxynojirimycin) significantly improved the stability of ATB-200. RhGAA protein folding was monitored at 37°C by thermal denaturation in neutral (pH 7.4 - plasma environment) buffer or acidic (pH 5.2 - lysosomal environment) buffer. AT2220 stabilized the rhGAA protein for more than 24 hours in neutral pH buffer.

[0084] Co-administration of Myozyme® with miglustat compared to co-administration of ATB-200 rhGAA with AT2221 (miglustat). 12-week-old GAA KO mice were treated with Lumizyme® or ATB200 (20 mg / kg IV injection every other week for four times), and miglustat was co-administered at 10 mg / kg PO 30 minutes prior to rhGAA administration where indicated. Tissue was collected 14 days after the last enzyme dose for glycogen measurement. Figure 14 shows the relative reduction in glycogen in the skeletal muscle of the quadriceps and triceps.

[0085] Tissue glycogen reduction by ATB-200 rhGAA co-administration with the pharmacological chaperone AT2221 (miglustat). The combination of a pharmacological chaperone and ATB-200 rhGAA was found to enhance glycogen clearance in vivo. GAA KO mice were administered rhGAA twice as an IV bolus at 20 mg / kg every other week. The pharmacological chaperone AT2221 was orally administered at doses of 0, 1, 2, and 10 mg / kg 30 minutes prior to rhGAA administration. Tissue was excised two weeks after the last dose of the ERT and analyzed for GAA activity, glycogen content, cell-specific glycogen, and lysosomal proliferation.

[0086] As shown in Figure 15, animals administered ATB200 + chaperone AT2221 showed enhanced glycogen clearance from the quadriceps muscle. ATB-200 rhGAA (20 mg / kg) reduced glycogen compared to the same dose of Lumizyme®, and when ATB-200 rhGAA was combined with AT2220 at 10 mg / kg, near-normal levels of muscle glycogen were achieved.

[0087] As shown in Figures 16A and 16B, unlike conventional rhGAA which showed limited glycogen reduction (indicated by a large number of punctate PAS signals), ATB-200rhGAA alone showed a significant reduction in PAS signals. Co-administration with 10 mg / kg miglustat resulted in a substantial further reduction of substrate. TEM revealed that the majority of glycogen in lysosomes was membrane-bound, electron-dense material, corresponding to punctate PAS signals. Co-administration of ATB-200 rhGAA with miglustat reduced the number, size, and density of substrate-containing lysosomes, suggesting targeted delivery of ATB-200 rhGAA to muscle cells and subsequent delivery to lysosomes.

[0088] From the study described above (two IV bolus injections every other week), tissue was treated with the LAMP1 marker for lysosomal proliferation, and this upregulation is another feature of Pompe disease. LAMP: Lysosome-associated membrane protein. From the study described above (two IV bolus EOW injections), soleus muscle tissue was treated with LAMP1 staining in the adjacent compartment and with type I fiber-specific antibody (NOQ7.5.4D) in the adjacent compartment (Figures 16C and 16D). ATB-200 rhGAA resulted in a significant reduction in LAMP1 compared to conventional rhGAA, reducing it to levels seen in wild-type animals (Figure 16C).

[0089] In addition, unlike rhGAA, whose effects are largely limited to type I fibers (slow contraction, marked with asterisks), ATB-200 rhGAA significantly reduced LAMP1 signaling in some type II (fast contraction) fibers (red arrowheads) as well (Figure 16D). Importantly, co-administration with miglustat further improved the ATB-200-mediated reduction of LAMP1 proliferation in most type II fibers (Figures 16C and 16D). As a result, no significant differences specific to fiber type were observed at the level of LAMP1 signaling. Similar conclusions were drawn from the quadriceps and diaphragm (data not shown).

[0090] Another similarly designed study investigated the effects of ATB-200 ± AT2221 over a longer period using four bi-weekly IV bolus injections. In the heart, repeated administration of either rhGAA or ATB-200 readily removed major glycogen stores in cardiomyocytes to levels seen in wild-type (WT) animals (Figure 17A). However, substrates in cardiac smooth muscle cells appeared to be preferably removed by ATB-200 rhGAA, suggesting a potentially broader biodistribution of ATB-200 compared to rhGAA (asterisks indicate vascular lumens in the heart). Importantly, co-administration with miglustat further improved the ATB-200-mediated reduction of LAMP1 proliferation.

[0091] These results indicate that ATB-200 rhGAA (with higher levels of M6P and bis-M6P on this N-glycan) efficiently targets CIMPR in skeletal muscle. ATB-200 rhGAA also possesses a well-processed complex N-glycan that minimizes unproductive clearance in vivo, has pharmacokinetic properties suitable for in vivo use, and exhibits good targeting to key muscle tissues in vivo. These findings suggest that ATB-200 rhGAA is superior to conventional standard treatment Lumizyme in reducing glycogen in muscle tissue, and that the combination of ATB-200 rhGAA with the chaperone AT2221 further improves glycogen removal from target tissues and improves muscle pathology. <Sequence Listing> SEQUENCE LISTING <110> AMICUS THERAPEUTICS GOTSCHALL, Russell DO, Hung <120> HIGHLY POTENT MODIFIED ACID ALPHA-GLUCOSIDASE WITH ENHANCED CARBOHYDRATES <130> 442420WO <150> US 62 / 135,345 <151> 2015-03-19 <150> US 62 / 112,643 <151> 2015-02-05 <150> US 62 / 057,847 <151> 2014-09-30 <150> US 62 / 057,842 <151> 2014-09-30 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 952 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <222> (1)..(952) <223> Sequence 4 from patent US 8592362GenBank: AHE24104.1 <400> 1 Met Gly Val Arg His Pro Pro Cys Ser His Arg Leu Leu Ala Val Cys 1 5 10 15 Ala Leu Val Ser Leu Ala Thr Ala Ala Leu Leu Gly His Ile Leu Leu 20 25 30 His Asp Phe Leu Leu Val Pro Arg Glu Leu Ser Gly Ser Ser Pro Val 35 40 45 Leu Glu Glu Thr His Pro Ala His Gln Gln Gly Ala Ser Arg Pro Gly 50 55 60 Pro Arg Asp Ala Gln Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr 65 70 75 80 Gln Cys Asp Val Pro Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys 85 90 95 Ala Ile Thr Gln Glu Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro 100 105 110 Ala Lys Gln Gly Leu Gln Gly Ala Gln Met Gly Gln Pro Trp Cys Phe 115 120 125 Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser 130 135 140 Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe 145 150 155 160 Pro Lys Asp Ile Leu Thr Leu Arg Leu Asp Val Met Met Glu Thr Glu 165 170 175 Asn Arg Leu His Phe Thr Ile Lys Asp Pro Ala Asn Arg Arg Tyr Glu 180 185 190 Val Pro Leu Glu Thr Pro Arg Val His Ser Arg Ala Pro Ser Pro Leu 195 200 205 Tyr Ser Val Glu Phe Ser Glu Glu Pro Phe Gly Val Ile Val His Arg 210 215 220 Gln Leu Asp Gly Arg Val Leu Leu Asn Thr Thr Val Ala Pro Leu Phe 225 230 235 240 Phe Ala Asp Gln Phe Leu Gln Leu Ser Thr Ser Leu Pro Ser Gln Tyr 245 250 255 Ile Thr Gly Leu Ala Glu His Leu Ser Pro Leu Met Leu Ser Thr Ser 260 265 270 Trp Thr Arg Ile Thr Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly 275 280 285 Ala Asn Leu Tyr Gly Ser His Pro Phe Tyr Leu Ala Leu Glu Asp Gly 290 295 300 Gly Ser Ala His Gly Val Phe Leu Leu Asn Ser Asn Ala Met Asp Val 305 310 315 320 Val Leu Gln Pro Ser Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile 325 330 335 Leu Asp Val Tyr Ile Phe Leu Gly Pro Glu Pro Lys Ser Val Val Gln 340 345 350 Gln Tyr Leu Asp Val Val Gly Tyr Pro Phe Met Pro Pro Tyr Trp Gly 355 360 365 Leu Gly Phe His Leu Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr 370 375 380 Arg Gln Val Val Glu Asn Met Thr Arg Ala His Phe Pro Leu Asp Val 385 390 395 400 Gln Trp Asn Asp Leu Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe 405 410 415 Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala Met Val Gln Glu Leu His 420 425 430 Gln Gly Gly Arg Arg Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser 435 440 445 Ser Gly Pro Ala Gly Ser Tyr Arg Pro Tyr Asp Glu Gly Leu Arg Arg 450 455 460 Gly Val Phe Ile Thr Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys Val 465 470 475 480 Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu 485 490 495 Ala Trp Trp Glu Asp Met Val Ala Glu Phe His Asp Gln Val Pro Phe 500 505 510 Asp Gly Met Trp Ile Asp Met Asn Glu Pro Ser Asn Phe Ile Arg Gly 515 520 525 Ser Glu Asp Gly Cys Pro Asn Asn Glu Leu Glu Asn Pro Pro Tyr Val 530 535 540 Pro Gly Val Val Gly Gly Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser 545 550 555 560 Ser His Gln Phe Leu Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly 565 570 575 Leu Thr Glu Ala Ile Ala Ser His Arg Ala Leu Val Lys Ala Arg Gly 580 585 590 Thr Arg Pro Phe Val Ile Ser Arg Ser Thr Phe Ala Gly His Gly Arg 595 600 605 Tyr Ala Gly His Trp Thr Gly Asp Val Trp Ser Ser Trp Glu Gln Leu 610 615 620 Ala Ser Ser Val Pro Glu Ile Leu Gln Phe Asn Leu Leu Gly Val Pro 625 630 635 640 Leu Val Gly Ala Asp Val Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu 645 650 655 Leu Cys Val Arg Trp Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg 660 665 670 Asn His Asn Ser Leu Leu Ser Leu Pro Gln Glu Pro Tyr Ser Phe Ser 675 680 685 Glu Pro Ala Gln Gln Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala 690 695 700 Leu Leu Pro His Leu Tyr Thr Leu Phe His Gln Ala His Val Ala Gly 705 710 715 720 Glu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser 725 730 735 Thr Trp Thr Val Asp His Gln Leu Leu Trp Gly Glu Ala Leu Leu Ile 740 745 750 Thr Pro Val Leu Gln Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro 755 760 765 Leu Gly Thr Trp Tyr Asp Leu Gln Thr Val Pro Ile Glu Ala Leu Gly 770 775 780 Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser 785 790 795 800 Glu Gly Gln Trp Val Thr Leu Pro Ala Pro Leu Asp Thr Ile Asn Val 805 810 815 His Leu Arg Ala Gly Tyr Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr 820 825 830 Thr Thr Glu Ser Arg Gln Gln Pro Met Ala Leu Ala Val Ala Leu Thr 835 840 845 Lys Gly Gly Glu Ala Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser 850 855 860 Leu Glu Val Leu Glu Arg Gly Ala Tyr Thr Gln Val Ile Phe Leu Ala 865 870 875 880 Arg Asn Asn Thr Ile Val Asn Glu Leu Val Arg Val Thr Ser Glu Gly 885 890 895 Ala Gly Leu Gln Leu Gln Lys Val Thr Val Leu Gly Val Ala Thr Ala 900 905 910 Pro Gln Gln Val Leu Ser Asn Gly Val Pro Val Ser Asn Phe Thr Tyr 915 920 925 Ser Pro Asp Thr Lys Val Leu Asp Ile Cys Val Ser Leu Leu Met Gly 930 935 940 Glu Gln Phe Leu Val Ser Trp Cys 945 950 <210> 2 <211> 3624 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (220)..(3078) <223> H.sapiens GAA mRNA for lysosomal alpha-glucosidase (acid maltase); GenBank: Y00839.1 <400> 2 cagttgggaa agctgaggtt gtcgccgggg ccgcgggtgg aggtcgggga tgaggcagca 60 ggtaggacag tgacctcggt gacgcgaagg accccggcca cctctaggtt ctcctcgtcc 120 gcccgttgtt cagcgaggga ggctctgggc ctgccgcagc tgacggggaa actgaggcac 180 ggagcgggcc tgtaggagct gtccaggcca tctccaacc atg gga gtg agg cac 234 Met Gly Val Arg His 1 5 ccg ccc tgc tcc cac cgg ctc ctg gcc gtc tgc gcc ctc gtg tcc ttg 282 Pro Pro Cys Ser His Arg Leu Leu Ala Val Cys Ala Leu Val Ser Leu 10 15 20 gca acc gct gca ctc ctg ggg cac atc cta ctc cat gat ttc ctg ctg 330 Ala Thr Ala Ala Leu Leu Gly His Ile Leu Leu His Asp Phe Leu Leu 25 30 35 gtt ccc cga gag ctg agt ggc tcc tcc cca gtc ctg gag gag act cac 378 Val Pro Arg Glu Leu Ser Gly Ser Ser Pro Val Leu Glu Glu Thr His 40 45 50 cca gct cac cag cag gga gcc agc aga cca ggg ccc cgg gat gcc cag 426 Pro Ala His Gln Gln Gly Ala Ser Arg Pro Gly Pro Arg Asp Ala Gln 55 60 65 gca cac ccc ggc cgt ccc aga gca gtg ccc aca cag tgc gac gtc ccc 474 Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr Gln Cys Asp Val Pro 70 75 80 85 ccc aac agc cgc ttc gat tgc gcc cct gac aag gcc atc acc cag gaa 522 Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys Ala Ile Thr Gln Glu 90 95 100 cag tgc gag gcc cgc ggc tgc tgc tac atc cct gca aag cag ggg ctg 570 Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro Ala Lys Gln Gly Leu 105 110 115 cag gga gcc cag atg ggg cag ccc tgg tgc ttc ttc cca ccc agc tac 618 Gln Gly Ala Gln Met Gly Gln Pro Trp Cys Phe Phe Pro Pro Ser Tyr 120 125 130 ccc agc tac aag ctg gag aac ctg agc tcc tct gaa atg ggc tac acg 666 Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser Glu Met Gly Tyr Thr 135 140 145 gcc acc ctg acc cgt acc acc ccc acc ttc ttc ccc aag gac atc ctg 714 Ala Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe Pro Lys Asp Ile Leu 150 155 160 165 acc ctg cgg ctg gac gtg atg atg gag act gag aac cgc ctc cac ttc 762 Thr Leu Arg Leu Asp Val Met Met Glu Thr Glu Asn Arg Leu His Phe 170 175 180 acg atc aaa gat cca gct aac agg cgc tac gag gtg ccc ttg gag acc 810 Thr Ile Lys Asp Pro Ala Asn Arg Arg Tyr Glu Val Pro Leu Glu Thr 185 190 195 ccg cgt gtc cac agc cgg gca ccg tcc cca ctc tac agc gtg gag ttc 858 Pro Arg Val His Ser Arg Ala Pro Ser Pro Leu Tyr Ser Val Glu Phe 200 205 210 tcc gag gag ccc ttc ggg gtg atc gtg cac cgg cag ctg gac ggc cgc 906 Ser Glu Glu Pro Phe Gly Val Ile Val His Arg Gln Leu Asp Gly Arg 215 220 225 gtg ctg ctg aac acg acg gtg gcg ccc ctg ttc ttt gcg gac cag ttc 954 Val Leu Leu Asn Thr Thr Val Ala Pro Leu Phe Phe Ala Asp Gln Phe 230 235 240 245 ctt cag ctg tcc acc tcg ctg ccc tcg cag tat atc aca ggg ctc gcc 1002 Leu Gln Leu Ser Thr Ser Leu Pro Ser Gln Tyr Ile Thr Gly Leu Ala 250 255 260 gag cac ctc agt ccc ctg atg ctc agc acc agc tgg acc agg atc acc 1050 Glu His Leu Ser Pro Leu Met Leu Ser Thr Ser Trp Thr Arg Ile Thr 265 270 275 ctg tgg aac cgg gac ctt gcg ccc acg ccc ggt gcg aac ctc tac ggg 1098 Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly Ala Asn Leu Tyr Gly 280 285 290 tct cac cct ttc tac ctg gcg ctg gag gac ggc ggg tcg gca cac ggg 1146 Ser His Pro Phe Tyr Leu Ala Leu Glu Asp Gly Gly Ser Ala His Gly 295 300 305 gtg ttc ctg cta aac agc aat gcc atg gat gtg gtc ctg cag ccg agc 1194 Val Phe Leu Leu Asn Ser Asn Ala Met Asp Val Val Leu Gln Pro Ser 310 315 320 325 cct gcc ctt agc tgg agg tcg aca ggt ggg atc ctg gat gtc tac atc 1242 Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile Leu Asp Val Tyr Ile 330 335 340 ttc ctg ggc cca gag ccc aag agc gtg gtg cag cag tac ctg gac gtt 1290 Phe Leu Gly Pro Glu Pro Lys Ser Val Val Gln Gln Tyr Leu Asp Val 345 350 355 gtg gga tac ccg ttc atg ccg cca tac tgg ggc ctg ggc ttc cac ctg 1338 Val Gly Tyr Pro Phe Met Pro Pro Tyr Trp Gly Leu Gly Phe His Leu 360 365 370 tgc cgc tgg ggc tac tcc tcc acc gct atc acc cgc cag gtg gtg gag 1386 Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr Arg Gln Val Val Glu 375 380 385 aac atg acc agg gcc cac ttc ccc ctg gac gtc caa tgg aac gac ctg 1434 Asn Met Thr Arg Ala His Phe Pro Leu Asp Val Gln Trp Asn Asp Leu 390 395 400 405 gac tac atg gac tcc cgg agg gac ttc acg ttc aac aag gat ggc ttc 1482 Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe Asn Lys Asp Gly Phe 410 415 420 cgg gac ttc ccg gcc atg gtg cag gag ctg cac cag ggc ggc cgg cgc 1530 Arg Asp Phe Pro Ala Met Val Gln Glu Leu His Gln Gly Gly Arg Arg 425 430 435 tac atg atg atc gtg gat cct gcc atc agc agc tcg ggc cct gcc ggg 1578 Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser Ser Gly Pro Ala Gly 440 445 450 agc tac agg ccc tac gac gag ggt ctg cgg agg ggg gtt ttc atc acc 1626 Ser Tyr Arg Pro Tyr Asp Glu Gly Leu Arg Arg Gly Val Phe Ile Thr 455 460 465 aac gag acc ggc cag ccg ctg att ggg aag gta tgg ccc ggg tcc act 1674 Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys Val Trp Pro Gly Ser Thr 470 475 480 485 gcc ttc ccc gac ttc acc aac ccc aca gcc ctg gcc tgg tgg gag gac 1722 Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu Ala Trp Trp Glu Asp 490 495 500 atg gtg gct gag ttc cat gac cag gtg ccc ttc gac ggc atg tgg att 1770 Met Val Ala Glu Phe His Asp Gln Val Pro Phe Asp Gly Met Trp Ile 505 510 515 gac atg aac gag cct tcc aac ttc atc aga ggc tct gag gac ggc tgc 1818 Asp Met Asn Glu Pro Ser Asn Phe Ile Arg Gly Ser Glu Asp Gly Cys 520 525 530 ccc aac aat gag ctg gag aac cca ccc tac gtg cct ggg gtg gtt ggg 1866 Pro Asn Asn Glu Leu Glu Asn Pro Pro Tyr Val Pro Gly Val Val Gly 535 540 545 ggg acc ctc cag gcg gcc acc atc tgt gcc tcc agc cac cag ttt ctc 1914 Gly Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser Ser His Gln Phe Leu 550 555 560 565 tcc aca cac tac aac ctg cac aac ctc tac ggc ctg acc gaa gcc atc 1962 Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly Leu Thr Glu Ala Ile 570 575 580 gcc tcc cac agg gcg ctg gtg aag gct cgg ggg aca cgc cca ttt gtg 2010 Ala Ser His Arg Ala Leu Val Lys Ala Arg Gly Thr Arg Pro Phe Val 585 590 595 atc tcc cgc tcg acc ttt gct ggc cac ggc cga tac gcc ggc cac tgg 2058 Ile Ser Arg Ser Thr Phe Ala Gly His Gly Arg Tyr Ala Gly His Trp 600 605 610 acg ggg gac gtg tgg agc tcc tgg gag cag ctc gcc tcc tcc gtg cca 2106 Thr Gly Asp Val Trp Ser Ser Trp Glu Gln Leu Ala Ser Ser Val Pro 615 620 625 gaa atc ctg cag ttt aac ctg ctg ggg gtg cct ctg gtc ggg gcc gac 2154 Glu Ile Leu Gln Phe Asn Leu Leu Gly Val Pro Leu Val Gly Ala Asp 630 635 640 645 gtc tgc ggc ttc ctg ggc aac acc tca gag gag ctg tgt gtg cgc tgg 2202 Val Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu Leu Cys Val Arg Trp 650 655 660 acc cag ctg ggg gcc ttc tac ccc ttc atg cgg aac cac aac agc ctg 2250 Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg Asn His Asn Ser Leu 665 670 675 ctc agt ctg ccc cag gag ccg tac agc ttc agc gag ccg gcc cag cag 2298 Leu Ser Leu Pro Gln Glu Pro Tyr Ser Phe Ser Glu Pro Ala Gln Gln 680 685 690 gcc atg agg aag gcc ctc acc ctg cgc tac gca ctc ctc ccc cac ctc 2346 Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala Leu Leu Pro His Leu 695 700 705 tac aca ctg ttc cac cag gcc cac gtc gcg ggg gag acc gtg gcc cgg 2394 Tyr Thr Leu Phe His Gln Ala His Val Ala Gly Glu Thr Val Ala Arg 710 715 720 725 ccc ctc ttc ctg gag ttc ccc aag gac tct agc acc tgg act gtg gac 2442 Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser Thr Trp Thr Val Asp 730 735 740 cac cag ctc ctg tgg ggg gag gcc ctg ctc atc acc cca gtg ctc cag 2490 His Gln Leu Leu Trp Gly Glu Ala Leu Leu Ile Thr Pro Val Leu Gln 745 750 755 gcc ggg aag gcc gaa gtg act ggc tac ttc ccc ttg ggc aca tgg tac 2538 Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro Leu Gly Thr Trp Tyr 760 765 770 gac ctg cag acg gtg cca ata gag gcc ctt ggc agc ctc cca ccc cca 2586 Asp Leu Gln Thr Val Pro Ile Glu Ala Leu Gly Ser Leu Pro Pro Pro 775 780 785 cct gca gct ccc cgt gag cca gcc atc cac agc gag ggg cag tgg gtg 2634 Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser Glu Gly Gln Trp Val 790 795 800 805 acg ctg ccg gcc ccc ctg gac acc atc aac gtc cac ctc cgg gct ggg 2682 Thr Leu Pro Ala Pro Leu Asp Thr Ile Asn Val His Leu Arg Ala Gly 810 815 820 tac atc atc ccc ctg cag ggc cct ggc ctc aca acc aca gag tcc cgc 2730 Tyr Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr Thr Thr Glu Ser Arg 825 830 835 cag cag ccc atg gcc ctg gct gtg gcc ctg acc aag ggt gga gag gcc 2778 Gln Gln Pro Met Ala Leu Ala Val Ala Leu Thr Lys Gly Gly Glu Ala 840 845 850 cga ggg gag ctg ttc tgg gac gat gga gag agc ctg gaa gtg ctg gag 2826 Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser Leu Glu Val Leu Glu 855 860 865 cga ggg gcc tac aca cag gtc atc ttc ctg gcc agg aat aac acg atc 2874 Arg Gly Ala Tyr Thr Gln Val Ile Phe Leu Ala Arg Asn Asn Thr Ile 870 875 880 885 gtg aat gag ctg gta cgt gtg acc agt gag gga gct ggc ctg cag ctg 2922 Val Asn Glu Leu Val Arg Val Thr Ser Glu Gly Ala Gly Leu Gln Leu 890 895 900 cag aag gtg act gtc ctg ggc gtg gcc acg gcg ccc cag cag gtc ctc 2970 Gln Lys Val Thr Val Leu Gly Val Ala Thr Ala Pro Gln Gln Val Leu 905 910 915 tcc aac ggt gtc cct gtc tcc aac ttc acc tac agc ccc gac acc aag 3018 Ser Asn Gly Val Pro Val Ser Asn Phe Thr Tyr Ser Pro Asp Thr Lys 920 925 930 gtc ctg gac atc tgt gtc tcg ctg ttg atg gga gag cag ttt ctc gtc 3066 Val Leu Asp Ile Cys Val Ser Leu Leu Met Gly Glu Gln Phe Leu Val 935 940 945 agc tgg tgt tag ccgggcggag tgtgttagtc tctccagagg gaggctggtt 3118 Ser Trp Cys 950 ccccagggaa gcagagcctg tgtgcgggca gcagctgtgt gcgggcctgg gggttgcatg 3178 tgtcacctgg agctgggcac taaccattcc aagccgccgc atcgcttgtt tccacctcct 3238 gggccggggc tctggccccc aacgtgcta ggagagcttt ctccctagat cgcactgtgg 3298 gccggggcct ggagggctgc tctgtgttaa taagattgta aggtttgccc tcctcacctg 3358 ttgccggcat gcgggtagta ttagccaccc ccctccatct gttcccagca ccggagaagg 3418 gggtgctcag gtggaggtgt ggggtatgca cctgagctcc tgcttcgcgc ctgctgctct 3478 gccccaacgc gaccgcttcc cggctgccca gagggctgga tgcctgccgg tcccgagca 3538 agcctgggaa ctcaggaaaa ttcacaggac ttgggagatt ctaaatctta agtgcaatta 3598 ttttaataaa aggggcattt ggaatc 3624 <210> 3 <211> 952 <212> PRT <213> Homo sapiens <400> 3 Met Gly Val Arg His Pro Pro Cys Ser His Arg Leu Leu Ala Val Cys 1 5 10 15 Ala Leu Val Ser Leu Ala Thr Ala Ala Leu Leu Gly His Ile Leu Leu 20 25 30 His Asp Phe Leu Leu Val Pro Arg Glu Leu Ser Gly Ser Ser Pro Val 35 40 45 Leu Glu Glu Thr His Pro Ala His Gln Gln Gly Ala Ser Arg Pro Gly 50 55 60 Pro Arg Asp Ala Gln Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr 65 70 75 80 Gln Cys Asp Val Pro Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys 85 90 95 Ala Ile Thr Gln Glu Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro 100 105 110 Ala Lys Gln Gly Leu Gln Gly Ala Gln Met Gly Gln Pro Trp Cys Phe 115 120 125 Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser 130 135 140 Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe 145 150 155 160 Pro Lys Asp Ile Leu Thr Leu Arg Leu Asp Val Met Met Glu Thr Glu 165 170 175 Asn Arg Leu His Phe Thr Ile Lys Asp Pro Ala Asn Arg Arg Tyr Glu 180 185 190 Val Pro Leu Glu Thr Pro Arg Val His Ser Arg Ala Pro Ser Pro Leu 195 200 205 Tyr Ser Val Glu Phe Ser Glu Glu Pro Phe Gly Val Ile Val His Arg 210 215 220 Gln Leu Asp Gly Arg Val Leu Leu Asn Thr Thr Val Ala Pro Leu Phe 225 230 235 240 Phe Ala Asp Gln Phe Leu Gln Leu Ser Thr Ser Leu Pro Ser Gln Tyr 245 250 255 Ile Thr Gly Leu Ala Glu His Leu Ser Pro Leu Met Leu Ser Thr Ser 260 265 270 Trp Thr Arg Ile Thr Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly 275 280 285 Ala Asn Leu Tyr Gly Ser His Pro Phe Tyr Leu Ala Leu Glu Asp Gly 290 295 300 Gly Ser Ala His Gly Val Phe Leu Leu Asn Ser Asn Ala Met Asp Val 305 310 315 320 Val Leu Gln Pro Ser Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile 325 330 335 Leu Asp Val Tyr Ile Phe Leu Gly Pro Glu Pro Lys Ser Val Val Gln 340 345 350 Gln Tyr Leu Asp Val Val Gly Tyr Pro Phe Met Pro Pro Tyr Trp Gly 355 360 365 Leu Gly Phe His Leu Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr 370 375 380 Arg Gln Val Val Glu Asn Met Thr Arg Ala His Phe Pro Leu Asp Val 385 390 395 400 Gln Trp Asn Asp Leu Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe 405 410 415 Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala Met Val Gln Glu Leu His 420 425 430 Gln Gly Gly Arg Arg Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser 435 440 445 Ser Gly Pro Ala Gly Ser Tyr Arg Pro Tyr Asp Glu Gly Leu Arg Arg 450 455 460 Gly Val Phe Ile Thr Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys Val 465 470 475 480 Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu 485 490 495 Ala Trp Trp Glu Asp Met Val Ala Glu Phe His Asp Gln Val Pro Phe 500 505 510 Asp Gly Met Trp Ile Asp Met Asn Glu Pro Ser Asn Phe Ile Arg Gly 515 520 525 Ser Glu Asp Gly Cys Pro Asn Asn Glu Leu Glu Asn Pro Pro Tyr Val 530 535 540 Pro Gly Val Val Gly Gly Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser 545 550 555 560 Ser His Gln Phe Leu Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly 565 570 575 Leu Thr Glu Ala Ile Ala Ser His Arg Ala Leu Val Lys Ala Arg Gly 580 585 590 Thr Arg Pro Phe Val Ile Ser Arg Ser Thr Phe Ala Gly His Gly Arg 595 600 605 Tyr Ala Gly His Trp Thr Gly Asp Val Trp Ser Ser Trp Glu Gln Leu 610 615 620 Ala Ser Ser Val Pro Glu Ile Leu Gln Phe Asn Leu Leu Gly Val Pro 625 630 635 640 Leu Val Gly Ala Asp Val Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu 645 650 655 Leu Cys Val Arg Trp Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg 660 665 670 Asn His Asn Ser Leu Leu Ser Leu Pro Gln Glu Pro Tyr Ser Phe Ser 675 680 685 Glu Pro Ala Gln Gln Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala 690 695 700 Leu Leu Pro His Leu Tyr Thr Leu Phe His Gln Ala His Val Ala Gly 705 710 715 720 Glu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser 725 730 735 Thr Trp Thr Val Asp His Gln Leu Leu Trp Gly Glu Ala Leu Leu Ile 740 745 750 Thr Pro Val Leu Gln Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro 755 760 765 Leu Gly Thr Trp Tyr Asp Leu Gln Thr Val Pro Ile Glu Ala Leu Gly 770 775 780 Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser 785 790 795 800 Glu Gly Gln Trp Val Thr Leu Pro Ala Pro Leu Asp Thr Ile Asn Val 805 810 815 His Leu Arg Ala Gly Tyr Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr 820 825 830 Thr Thr Glu Ser Arg Gln Gln Pro Met Ala Leu Ala Val Ala Leu Thr 835 840 845 Lys Gly Gly Glu Ala Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser 850 855 860 Leu Glu Val Leu Glu Arg Gly Ala Tyr Thr Gln Val Ile Phe Leu Ala 865 870 875 880 Arg Asn Asn Thr Ile Val Asn Glu Leu Val Arg Val Thr Ser Glu Gly 885 890 895 Ala Gly Leu Gln Leu Gln Lys Val Thr Val Leu Gly Val Ala Thr Ala 900 905 910 Pro Gln Gln Val Leu Ser Asn Gly Val Pro Val Ser Asn Phe Thr Tyr 915 920 925 Ser Pro Asp Thr Lys Val Leu Asp Ile Cys Val Ser Leu Leu Met Gly 930 935 940 Glu Gln Phe Leu Val Ser Trp Cys 945 950 <210> 4 <211> 952 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <222> (1)..(952) <223> Lysosomal alpha-glucosidase preproprotein [Homo sapiens]; NCBI Reference Sequence: NP_000143.2 <400> 4 Met Gly Val Arg His Pro Pro Cys Ser His Arg Leu Leu Ala Val Cys 1 5 10 15 Ala Leu Val Ser Leu Ala Thr Ala Ala Leu Leu Gly His Ile Leu Leu 20 25 30 His Asp Phe Leu Leu Val Pro Arg Glu Leu Ser Gly Ser Ser Pro Val 35 40 45 Leu Glu Glu Thr His Pro Ala His Gln Gln Gly Ala Ser Arg Pro Gly 50 55 60 Pro Arg Asp Ala Gln Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr 65 70 75 80 Gln Cys Asp Val Pro Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys 85 90 95 Ala Ile Thr Gln Glu Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro 100 105 110 Ala Lys Gln Gly Leu Gln Gly Ala Gln Met Gly Gln Pro Trp Cys Phe 115 120 125 Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser 130 135 140 Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe 145 150 155 160 Pro Lys Asp Ile Leu Thr Leu Arg Leu Asp Val Met Met Glu Thr Glu 165 170 175 Asn Arg Leu His Phe Thr Ile Lys Asp Pro Ala Asn Arg Arg Tyr Glu 180 185 190 Val Pro Leu Glu Thr Pro His Val His Ser Arg Ala Pro Ser Pro Leu 195 200 205 Tyr Ser Val Glu Phe Ser Glu Glu Pro Phe Gly Val Ile Val Arg Arg 210 215 220 Gln Leu Asp Gly Arg Val Leu Leu Asn Thr Thr Val Ala Pro Leu Phe 225 230 235 240 Phe Ala Asp Gln Phe Leu Gln Leu Ser Thr Ser Leu Pro Ser Gln Tyr 245 250 255 Ile Thr Gly Leu Ala Glu His Leu Ser Pro Leu Met Leu Ser Thr Ser 260 265 270 Trp Thr Arg Ile Thr Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly 275 280 285 Ala Asn Leu Tyr Gly Ser His Pro Phe Tyr Leu Ala Leu Glu Asp Gly 290 295 300 Gly Ser Ala His Gly Val Phe Leu Leu Asn Ser Asn Ala Met Asp Val 305 310 315 320 Val Leu Gln Pro Ser Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile 325 330 335 Leu Asp Val Tyr Ile Phe Leu Gly Pro Glu Pro Lys Ser Val Val Gln 340 345 350 Gln Tyr Leu Asp Val Val Gly Tyr Pro Phe Met Pro Pro Tyr Trp Gly 355 360 365 Leu Gly Phe His Leu Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr 370 375 380 Arg Gln Val Val Glu Asn Met Thr Arg Ala His Phe Pro Leu Asp Val 385 390 395 400 Gln Trp Asn Asp Leu Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe 405 410 415 Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala Met Val Gln Glu Leu His 420 425 430 Gln Gly Gly Arg Arg Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser 435 440 445 Ser Gly Pro Ala Gly Ser Tyr Arg Pro Tyr Asp Glu Gly Leu Arg Arg 450 455 460 Gly Val Phe Ile Thr Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys Val 465 470 475 480 Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu 485 490 495 Ala Trp Trp Glu Asp Met Val Ala Glu Phe His Asp Gln Val Pro Phe 500 505 510 Asp Gly Met Trp Ile Asp Met Asn Glu Pro Ser Asn Phe Ile Arg Gly 515 520 525 Ser Glu Asp Gly Cys Pro Asn Asn Glu Leu Glu Asn Pro Pro Tyr Val 530 535 540 Pro Gly Val Val Gly Gly Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser 545 550 555 560 Ser His Gln Phe Leu Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly 565 570 575 Leu Thr Glu Ala Ile Ala Ser His Arg Ala Leu Val Lys Ala Arg Gly 580 585 590 Thr Arg Pro Phe Val Ile Ser Arg Ser Thr Phe Ala Gly His Gly Arg 595 600 605 Tyr Ala Gly His Trp Thr Gly Asp Val Trp Ser Ser Trp Glu Gln Leu 610 615 620 Ala Ser Ser Val Pro Glu Ile Leu Gln Phe Asn Leu Leu Gly Val Pro 625 630 635 640 Leu Val Gly Ala Asp Val Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu 645 650 655 Leu Cys Val Arg Trp Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg 660 665 670 Asn His Asn Ser Leu Leu Ser Leu Pro Gln Glu Pro Tyr Ser Phe Ser 675 680 685 Glu Pro Ala Gln Gln Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala 690 695 700 Leu Leu Pro His Leu Tyr Thr Leu Phe His Gln Ala His Val Ala Gly 705 710 715 720 Glu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser 725 730 735 Thr Trp Thr Val Asp His Gln Leu Leu Trp Gly Glu Ala Leu Leu Ile 740 745 750 Thr Pro Val Leu Gln Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro 755 760 765 Leu Gly Thr Trp Tyr Asp Leu Gln Thr Val Pro Val Glu Ala Leu Gly 770 775 780 Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser 785 790 795 800 Glu Gly Gln Trp Val Thr Leu Pro Ala Pro Leu Asp Thr Ile Asn Val 805 810 815 His Leu Arg Ala Gly Tyr Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr 820 825 830 Thr Thr Glu Ser Arg Gln Gln Pro Met Ala Leu Ala Val Ala Leu Thr 835 840 845 Lys Gly Gly Glu Ala Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser 850 855 860 Leu Glu Val Leu Glu Arg Gly Ala Tyr Thr Gln Val Ile Phe Leu Ala 865 870 875 880 Arg Asn Asn Thr Ile Val Asn Glu Leu Val Arg Val Thr Ser Glu Gly 885 890 895 Ala Gly Leu Gln Leu Gln Lys Val Thr Val Leu Gly Val Ala Thr Ala 900 905 910 Pro Gln Gln Val Leu Ser Asn Gly Val Pro Val Ser Asn Phe Thr Tyr 915 920 925 Ser Pro Asp Thr Lys Val Leu Asp Ile Cys Val Ser Leu Leu Met Gly 930 935 940 Glu Gln Phe Leu Val Ser Trp Cys 945 950

Claims

1. A composition comprising recombinant human acid alpha-glucosidase (rhGAA), A composition in which 40% to 60% of the N-glycans on the rhGAA are complex N-glycans, and at least 17% of the total glycans on the rhGAA are bis-M6P glycans.

2. The composition according to claim 1, wherein the rhGAA contains at least 3.0 mol of mannose-6-phosphate (M6P) residues per mol of rhGAA.

3. The composition according to claim 1, wherein the rhGAA contains at least 4.0 mol of M6P residues per mol of rhGAA.

4. The composition according to claim 1, wherein the rhGAA contains at least 5.0 mol of M6P residues per mol of rhGAA.

5. The composition according to claim 1, wherein the rhGAA contains at least 6.0 mol of M6P residues per mol of rhGAA.

6. The composition according to claim 1, wherein the rhGAA contains at least 7.0 mol of M6P residues per mol of rhGAA.

7. The composition according to claim 1, wherein the rhGAA contains 3.0 to 7.0 mol of M6P residues per mol of rhGAA.

8. The composition according to claim 1, wherein the rhGAA contains 3.0 to 6.0 mol of M6P residues per mol of rhGAA.

9. The composition according to claim 1, wherein rhGAA comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

4.

10. A pharmaceutical composition comprising the rhGAA composition described in claim 1, and at least one pharmaceutically acceptable carrier or additive.

11. The pharmaceutical composition according to claim 10, wherein the pharmaceutically acceptable carrier is water.

12. The pharmaceutical composition according to claim 10, wherein the composition is in the form of a freeze-dried powder.

13. The pharmaceutical composition according to claim 10, wherein the composition comprises mannitol.

14. The pharmaceutical composition according to claim 10, wherein the composition comprises polysorbate 80.

15. The pharmaceutical composition according to claim 10, wherein the composition comprises a pH buffering agent.

16. The composition according to claim 1, wherein the rhGAA contains at least 4 mol of sialic acid residues per mol of rhGAA.

17. The composition according to claim 1, wherein the rhGAA contains at least 1.3 mol of bis-M6P per 1 mol of rhGAA.

18. The pharmaceutical composition according to claim 10, wherein the rhGAA contains at least 4 mol of sialic acid residues per 1 mol of rhGAA.

19. The pharmaceutical composition according to claim 10, wherein the rhGAA contains at least 1.3 mol of bis-M6P per 1 mol of rhGAA.

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