Recombinant human acid alpha-glycosidase

Abstract

To provide methods for treating Pompe's disease to exhibit superior intake into muscle cells and then delivery to lysosome as compared with current rhGAA products.SOLUTION: A method of treating Pompe's disease in a subject in need thereof comprises administering to the subject a population of recombinant human acid α-glucosidase (rhGAA) molecules derived from Chinese hamster ovary (CHO) cells; where the rhGAA molecules comprise seven potential N-glycosylation sites: where the rhGAA molecules on average comprise 3 to 4 mannose-6-phosphate (M6P) residues; where the rhGAA molecules on average comprise at least about 0.5 mole of bis-mannose-6-phosphate (bis-M6P) per mole of rhGAA at the first potential N-glycosylation site as determined using liquid chromatography-tandem mass spectrometry (LC-MS / MS); and where the population of rhGAA is administered at a dosage capable of reversing disease progression in the subject.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] Related Patent Cross-References This application claims priority to, and each of which is incorporated by reference in its entirety, U.S. Provisional Patent Application No. 62 / 506,561, filed May 15, 2017, U.S. Provisional Patent Application No. 62 / 506,569, filed May 15, 2017, U.S. Provisional Patent Application No. 62 / 506,574, filed May 15, 2017, and U.S. Provisional Patent Application No. 62 / 564,0 No. 83, filed September 27, 2017; U.S. Provisional Patent Application No. 62 / 567,334, filed October 3, 2017; U.S. Provisional Patent Application No. 62 / 618,021, filed January 16, 2018; U.S. Provisional Patent Application No. 62 / 624,638, filed January 31, 2018; and U.S. Provisional Patent Application No. 62 / 660,758, filed April 20, 2018.

[0002] FIELD OF THEINVENTION The present invention relates to the fields of medicine, genetics and recombinant glycoprotein biochemistry, and specifically relates to a recombinant human α-glucosidase (rhGAA) composition with a high total content of mannose-6-phosphate-bearing N-glycans that efficiently targets CIMPR on cells and then delivers rhGAA to lysosomes where it can degrade abnormally high levels of accumulated glycogen. The rhGAA of the present invention exhibits superior uptake into muscle cells and then delivery to lysosomes compared to conventional rhGAA products, and other pharmacokinetic properties that make it particularly effective for enzyme replacement therapy of subjects with Pompe disease.

[0003] The present invention also provides a method for treating Pompe disease, comprising administering to an individual the combination of rhGAA and pharmacological chaperone.For example, in some embodiments, the present invention provides a method for treating Pompe disease, comprising administering to an individual the combination of rhGAA and miglustat.The rhGAA of the present invention shows surprising effectiveness in treating and reversing disease progression in subjects suffering from Pompe disease. [Background technology]

[0004] background Pompe disease is an inherited lysosomal storage disease resulting from a deficiency in acid alpha-glucosidase (GAA) activity. Individuals with Pompe disease lack or have reduced levels of acid alpha-glucosidase (GAA), an enzyme that breaks down glycogen into glucose, the main source of energy for muscles. Deficiency of this enzyme results in excess glycogen accumulation in lysosomes, intracellular organelles that normally contain enzymes that break down glycogen and other cellular debris or waste products. Glycogen accumulation in certain tissues, especially muscles, of subjects with Pompe disease impairs the cells' ability to function normally. In Pompe disease, glycogen is not metabolized properly and progressively accumulates in lysosomes, especially in skeletal muscle cells and in infantile-onset forms of the disease, cardiac muscle cells. Glycogen accumulation damages muscle and nerve cells as well as those of other affected tissues.

[0005] Traditionally, depending on the age of onset, Pompe disease is recognized clinically as either early childhood or late onset. The age of onset tends to parallel the severity of the genetic mutation that causes Pompe disease. The most severe genetic mutations result in a complete loss of GAA activity and manifest as early onset disease during infancy. Genetic mutations that reduce, but do not completely eliminate, GAA activity are associated with forms of Pompe disease that are slower onset and progressive. Infantile onset Pompe disease manifests shortly after birth and is characterized by muscle weakness, respiratory failure, and cardiac failure. If untreated, it is usually fatal within two years. Childhood and adult onset Pompe disease manifest later in life and usually progresses more slowly than the infantile onset disease. This form of the disease does not generally affect the heart, but may be fatal due to weakness of skeletal muscles and those involved in breathing.

[0006] Current non-symptomatic treatments for Pompe disease include enzyme replacement therapy (ERT) using recombinant human GAA (rhGAA), known as Lumizyme®, Myozyme®, or alglucosidase alfa. This conventional enzyme replacement therapy seeks to treat Pompe disease by administering rhGAA to replenish GAA lost in lysosomes, thereby restoring the cell's ability to break down lysosomal glycogen. "Lumizyme®" and "Myozyme®" are conventional forms of rhGAA produced and marketed as biopharmaceuticals by Genzyme and approved by the U.S. Food and Drug Administration, and are described by reference in the Physician's Desk Reference (2014) (herein incorporated by reference). Alglucosidase alfa has the chemical name [199-arginine, 223-histidine] prepro-α-glucosidase (human); molecular formula: C 4758 H 7262 N 1274 O 1369 S 35 identified as CAS number 420794-05-0. These products are administered to subjects with Pompe disease, also known as Glycogen Storage Disease Type II (GSD-II) or Acid Maltase Deficiency Disease.

[0007] The cellular uptake of rhGAA molecules is facilitated by a specific sugar, mannose-6-phosphate (M6P), which binds to the cation-independent mannose-6-phosphate receptor (CIMPR) present on target cells, such as muscle cells. Upon binding, rhGAA molecules are taken up by the target cells and subsequently transported to the lysosomes within the cells. However, most of the conventional rhGAA products do not have a high total content of mono-M6P- and bis-M6P-bearing N-glycans (i.e., N-glycans bearing one M6P residue or N-glycans bearing two M6P residues, respectively), which limits their CIMPR-mediated cellular uptake and lysosomal delivery, thereby making conventional enzyme replacement therapy less effective. For example, while conventional rhGAA products at doses of 20 mg / kg or more improve some aspects of Pompe disease, they are not sufficient to reverse disease progression, particularly (i) treat the underlying cellular dysfunction, (ii) restore muscle structure, or (iii) reduce glycogen accumulation in many target tissues, such as skeletal muscle. Furthermore, high doses may impose additional burdens on subjects and medical personnel treating them, such as extending the infusion time required to administer rhGAA intravenously. There is a need for further improvements in enzyme replacement therapy for the treatment of Pompe disease, such as rhGAA with improved tissue uptake, improved enzyme activity, improved stability, and reduced immunogenicity.

[0008] The glycosylation of GAA or rhGAA can be enzymatically modified in vitro by phosphotransferases and uncovering enzymes to generate M6P groups, as described by Canfield et al., U.S. Patent No. 6,534,300. Enzymatic glycosylation may not be well controlled and may produce rhGAA with undesirable immunological and pharmacological properties. Enzymatically modified rhGAA may contain only high mannose oligosaccharides that can potentially all be enzymatically phosphorylated in vitro with phosphotransferases or uncovering enzymes, and may contain an average of 5-6 M6P groups per GAA. The glycosylation pattern produced by in vitro enzymatic treatment of GAA is problematic because the additional terminal mannose residues, especially the non-phosphorylated terminal mannose residues, adversely affect the pharmacokinetics of the modified rhGAA. When such enzymatically modified products are administered in vivo, these mannose groups increase the non-productive clearance of GAA, increase the uptake of enzymatically modified GAA by immune cells, and reduce rhGAA therapeutic efficacy due to the reduction of GAA reaching target tissues such as skeletal muscle myocytes. For example, terminal non-phosphorylated mannose residues are well-known ligands for mannose receptors in the liver and spleen, resulting in rapid clearance of enzymatically modified rhGAA and reducing the targeting of rhGAA to target tissues. Furthermore, the glycosylation pattern of enzymatically modified GAA with high mannose N-glycans with terminal non-phosphorylated mannose residues is similar to that on glycoproteins produced in yeast and mold, increasing the risk of eliciting immune and allergic responses, such as life-threatening severe allergic reactions (anaphylaxis) or hypersensitivity reactions, to enzymatically modified rhGAA.

[0009] In view of these deficiencies of conventional rhGAA products and in vitro methods for phosphorylating rhGAA, the present inventors have searched and identified a method to produce rhGAA with an optimized N-glycan profile to enhance biodistribution and lysosomal uptake and thereby minimize non-productive clearance of administered rhGAA. The present invention provides stable or declining Pompe patients with an effective treatment to reverse disease progression at the cellular level. The present inventors also report that the rhGAA of the present invention reverses disease progression - including clearing lysosomal glycogen more efficiently than the current standard treatment - and that patients treated with the rhGAA of the present invention show surprising and significant health improvements, including improvements in muscle strength, motor function and / or lung function, and / or reversal in disease progression, as demonstrated in various efficacy results (e.g., Examples 10 and 11) from clinical studies. Summary of the Invention

[0010] The present invention relates to methods of treating a disease or disorder, such as Pompe disease, in a subject comprising administering a population of recombinant human acid alpha glucosidase (rhGAA) molecules.

[0011] The rhGAA molecules described herein may be expressed in Chinese Hamster Ovary (CHO) cells and contain seven potential N-glycosylation sites. In some embodiments, the N-glycosylation profile of a population of rhGAA molecules described herein is determined using liquid chromatography tandem mass spectrometry (LC-MS / MS). In some embodiments, the rhGAA molecules contain an average of 3-4 mannose-6-phosphate (M6P) residues. In some embodiments, the rhGAA molecules contain an average of at least about 0.5 moles of bis-phosphorylated N-glycan groups (bis-M6P) per mole of rhGAA at the first potential N-glycosylation site. In some embodiments, the rhGAA comprises an amino acid sequence at least 95% identical to SEQ ID NO:1 or SEQ ID NO:5. In some embodiments, the rhGAA comprises an amino acid sequence identical to SEQ ID NO:1 or SEQ ID NO:5. In some embodiments, at least 30% of the rhGAA molecules contain one or more N-glycan units bearing one or two M6P residues. In some embodiments, rhGAA molecules contain an average of about 0.5 mol to about 7.0 mol of N-glycan units bearing one or two M6P residues per mole of rhGAA. In some embodiments, rhGAA molecules contain an average of at least 2.5 mol of M6P residues per mole of rhGAA and at least 4 mol of sialic acid residues per mole of rhGAA. In some embodiments, rhGAA molecules comprising an average of 3 to 4 M6P residues per mole and an average of about at least 0.5 moles of bis-M6P per mole of rhGAA at a first potential N-glycosylation site further comprise an average of about 0.4 to about 0.6 moles of mono-phosphorylated N-glycan (mono-M6P) per mole of rhGAA at a second potential N-glycosylation site, about 0.4 to about 0.6 moles of bis-M6P per mole of rhGAA at a fourth potential N-glycosylation site, and about 0.3 to about 0.4 moles of mono-M6P per mole of rhGAA at a fourth potential N-glycosylation site.In some embodiments, the rhGAA molecule further comprises an average of about 4 mol to about 7.3 mol of sialic acid per mole of rhGAA, including about 0.9 to about 1.2 mol of sialic acid per mole of rhGAA at the third potential N-glycosylation site, about 0.8 to about 0.9 mol of sialic acid per mole of rhGAA at the fifth potential N-glycosylation site, and about 1.5 to about 4.2 mol of sialic acid per mole of rhGAA at the sixth potential N-glycosylation site. In some embodiments, the population of rhGAA molecules is formulated into a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprising the population of rhGAA molecules further comprises at least one buffer selected from the group consisting of citrate, phosphate, and combinations thereof, and at least one excipient selected from the group consisting of mannitol, polysorbate 80, and combinations thereof. In some embodiments, the pH of the pharmaceutical composition is about 5.0 to about 7.0, about 5.0 to about 6.0, or about 6.0. In some embodiments, the pharmaceutical composition further comprises water, an acidifying agent, an alkalizing agent, or a combination thereof. In some embodiments, the pharmaceutical composition has a pH of 6.0 and comprises a population of about 5-50 mg / mL rhGAA molecules, about 10-100 mM sodium citrate buffer, about 10-50 mg / mL mannitol, about 0.1-1 mg / mL polysorbate 80, and water, optionally including an acidifying agent and / or an alkalizing agent. In some embodiments, the pharmaceutical composition has a pH of 6.0 and comprises a population of about 15 mg / mL rhGAA molecules, about 25 mM sodium citrate buffer, about 20 mg / mL mannitol, about 0.5 mg / mL polysorbate 80, and water, optionally including an acidifying agent and / or an alkalizing agent.

[0012] In some embodiments, the population of rhGAA molecules is administered at a dose of about 1 mg / kg to about 100 mg / kg. In some embodiments, the population of rhGAA molecules is administered at a dose of about 20 mg / kg. In some embodiments, the population of rhGAA molecules is given once every two months, once a month, once every two weeks, once a week, twice a week or daily, for example, once every two weeks. In some embodiments, the population of rhGAA molecules is administered intravenously.

[0013] In some embodiments, the population of rhGAA molecules is administered simultaneously or sequentially with a pharmacological chaperone such as miglustat (also referred to as AT2221) or a pharma- ceutically acceptable salt thereof. In some embodiments, miglustat or a pharma-ceutically acceptable salt thereof is orally administered at a dose of, for example, about 200 mg to about 600 mg, and optionally about 260 mg. In some embodiments, the population of rhGAA molecules is administered intravenously at a dose of about 5 mg / kg to about 20 mg / kg, and miglustat or a pharma-ceutically acceptable salt thereof is orally administered at a dose of about 233 mg to about 500 mg. In some embodiments, the population of rhGAA molecules is administered intravenously at a dose of about 5 mg / kg to about 20 mg / kg, and miglustat or a pharma-ceutically acceptable salt thereof is orally administered at a dose of about 50 mg to about 200 mg. In one embodiment, the population of rhGAA molecules is administered intravenously at a dose of about 20 mg / kg, and miglustat or a pharma- ceutically acceptable salt thereof is administered orally at a dose of about 260 mg. In some embodiments, miglustat or a pharma- ceutically acceptable salt thereof is administered prior to administration of the population of rhGAA molecules (e.g., about one hour prior). In at least one embodiment, the subject fasts for at least 2 hours before and at least 2 hours after administration of miglustat or a pharma- ceutically acceptable salt thereof.

[0014] Embodiments of the present invention demonstrate the efficacy of rhGAA as described herein for treating and reversing disease progression in subjects with Pompe disease.

[0015] In some embodiments, the population of rhGAA molecules is administered at a dosage that can reverse the disease progression of the subject.For example, after treatment, the muscle or muscle fiber in the subject shows a reduction in the size of lysosomes and / or disappearance of autophagy buildup.In some embodiments, after treatment, less than 65% of the muscle fibers analyzed in the subject have autophagy buildup.In some embodiments, after treatment, at least 36% of the muscle fibers analyzed in the subject have normal or near-normal appearance.In some embodiments, the subject that experiences a reversal in disease progression after treatment is an ERT-switched patient, for example, an ERT-switched patient that has been previously treated with alglucosidase alpha for at least 2 years.

[0016] In some embodiments, the population of rhGAA molecules is administered at a dosage that can reduce glycogen content in the muscle of a subject faster than the same dosage of alglucosidase alfa. The rhGAA can reduce glycogen content at least about 1.25, 1.5, 1.75, 2.0 or 3.0 times faster than the same dosage of alglucosidase alfa. In some embodiments, the population of rhGAA molecules is administered at a dosage that can reduce glycogen content in the muscle of a subject more effectively than alglucosidase alfa administered at the same dosage when evaluated after 1, 2, 3, 4, 5 or 6 administrations. In some embodiments, the population of rhGAA molecules reduces glycogen content at least about 10%, 20%, 30%, 50%, 75% or 90% more effectively than alglucosidase alfa administered at the same dosage. In some embodiments, after treatment, the subject shows low levels of the glycogen storage biomarker, urinary hexose tetrasaccharide (Hex4). In at least one embodiment, the Hex4 level in the subject is reduced by at least 30% 6 months after treatment compared to baseline. For example, ambulatory or non-ambulatory subjects previously treated with enzyme replacement therapy (ERT-switched patients) can show at least a 35% reduction in Hex4 levels 6 months after treatment compared to baseline. In another case, ambulatory subjects who have not previously received enzyme replacement therapy (ERT-naive patients) can show at least a 45% reduction in Hex4 levels 6 months after treatment compared to baseline.

[0017] In some embodiments, the population of rhGAA molecules is administered in a dosage that can improve motor function in subjects.The improvement in motor function can be measured by motor function tests such as 6-minute walk test (6MWT), timed up and go test, four-stair climb test, 10-meter walk test, Gowers test, gait-stair-gower-chair (GSGC) test or combinations thereof.In some embodiments, after 6 months of treatment, subjects (when compared to baseline) show an increase in 6MWT distance of at least 20 meters, a decrease in timed up and go test of at least 1 second, a decrease in four-stair climb test of at least 0.6 seconds, a decrease in 10-meter walk test of at least 0.7 seconds, a decrease in Gowers test time of at least 1 second, and / or a decrease in GCSC score of at least 1. For example, ambulatory ERT-switched patients 6 months after treatment (compared to baseline) may show an increase in 6MWT of at least 20 meters, a decrease in timed up-and-go test time of at least 1.5 seconds, a decrease in 4-step up test time of at least 0.6 seconds, and / or a decrease in Gowers test time of at least 1 second. In other cases, ambulatory ERT-naive patients 6 months after treatment (compared to baseline) may show an increase in 6MWT distance of at least 40 meters, a decrease in timed up-and-go test time of at least 1 second, a decrease in 4-step up test time of at least 0.6 seconds, a decrease in 10-meter walk test time of at least 0.7 seconds, and / or a decrease in GSGC score of at least 1. In some embodiments, ERT-switched patients show an improvement in at least one motor function test after treatment compared to the motor function test results of patients after previous ERT using alglucosidase alfa.

[0018] In some embodiments, the population of rhGAA molecules is administered in a dosage that can improve upper body strength in a subject. In some embodiments, the population of rhGAA molecules is administered to an ambulatory subject and can further improve lower body strength and / or total body strength in the subject.

[0019] In some embodiments, the improvement in upper body strength is measured using manual strength score.The manual strength score of the subject can improve by at least 1 (for ambulatory ERT-switched patients) or at least 5.5 (for non-ambulatory ERT-switched patients) 6 months after treatment compared to baseline.In some embodiments, ERT-switched patients show improvement in upper body strength after treatment compared to the upper body strength of patients after previous ERT using alglucosidase alpha.

[0020] In some embodiments, the population of rhGAA molecules is administered at a dosage that can improve upper limb strength measured by quantitative muscle testing or manual muscle testing of shoulder adduction, shoulder abduction, elbow flexion and / or elbow extension.For example, compared to baseline, 6 months after treatment, non-ambulatory ERT-switched patients can show at least 8 pounds of improvement in shoulder adduction, at least 1 pound of improvement in shoulder abduction, at least 2 pounds of improvement in elbow flexion and / or at least 5 pounds of improvement in elbow extension.

[0021] In some embodiments, the population of rhGAA molecules is administered in a dosage that can improve lung function in subjects.The improvement in motor function can be measured by pulmonary function tests, such as upright (sitting) forced vital capacity test, maximum expiratory pressure (MEP) test, maximum inspiratory pressure (MIP) test or a combination thereof.In some embodiments, the subject shows an improvement in FVC of at least 4%, an improvement in MEP of at least 16cmH2O and / or an improvement in MIP of at least 0.3cmH2O 6 months after treatment (compared to baseline).For example, ambulatory ERT-switched patients can show an improvement in MEP of at least 16cmH2O 6 months after treatment (compared to baseline).In another case, ambulatory ERT-naive patients can show an improvement in FVC of at least 4% and / or an improvement in MIP of at least 11cmH2O 6 months after treatment (compared to baseline). In some embodiments, ERT-switched patients show improvement in at least one pulmonary function test following treatment compared to the patient's pulmonary function test results following their previous ERT with alglucosidase alfa.

[0022] In some embodiments, the population of rhGAA molecules is administered in a dosage that can reduce fatigue in a subject as measured by a fatigue severity scale (FSS) score. For example, the subject is a non-ambulatory ERT-switched patient and shows a decrease in FSS score of at least 3.5 6 months after treatment compared to baseline. In another example, the subject is an ambulatory ERT-switched patient and shows a decrease in FSS score of at least 8 6 months after treatment compared to baseline. In yet another example, the subject is an ambulatory ERT-naive patient and shows a decrease in FSS score of at least 5 6 months after treatment compared to baseline. In some embodiments, the ERT-switched patient shows a lower FSS score after treatment compared to the FSS score of the patient after previous ERT using alglucosidase alfa.

[0023] In some embodiments, the population of rhGAA molecules is administered at a dosage that can reduce the level of at least one muscle damage biomarker, such as creatine kinase (CK), alanine aminotransferase (ALT), aspartate aminotransferase (AST), or a combination thereof. In some embodiments, the CK level of the subject is reduced by at least 15% after 6 months of treatment compared to baseline, the ALT level of the subject is reduced by at least 5% after 6 months of treatment compared to baseline, and / or the AST level of the subject is reduced by at least 5% after 6 months of treatment compared to baseline. For example, the subject is an ambulatory ERT-switched patient and shows at least 15% reduction in CK level, at least 15% reduction in ALT level, and / or at least 10% reduction in AST level after 6 months of treatment compared to baseline. In another example, the subject is a non-ambulatory ERT-switched patient and shows at least 20% reduction in CK, at least 5% reduction in ALT level, and / or at least 5% reduction in AST level after 6 months of treatment compared to baseline. In yet another example, the subject is an ambulatory ERT-naive patient who exhibits at least a 35% reduction in CK levels, at least a 35% reduction in ALT levels, and / or at least a 30% reduction in AST levels 6 months after treatment compared to baseline. [Brief description of the drawings]

[0024] [Figure 1] A shows non-phosphorylated high mannose N-glycans, mono-M6P N-glycans and bis-M6P N-glycans. B shows the chemical structure of an M6P group. Each box represents an N-acetylglucosamine (GlcNAc), each circle represents a mannose and each P represents a phosphate. [Diagram 2]Figure 1 shows productive targeting of rhGAA to target tissues (e.g., muscle tissue in subjects with Pompe disease) via M6P-bearing N-glycans. Figure 2 shows non-productive drug clearance to non-target tissues (e.g., liver and spleen) or by binding of non-M6P N-glycans to non-target tissues. [Diagram 3] FIG. 1 is a schematic diagram of an exemplary process for the production, capture and purification of recombinant lysosomal proteins. [Figure 4] FIG. 1 shows a DNA construct for transforming CHO cells with DNA encoding rhGAA. [Diagram 5] FIG. 5 is a graph showing the results of CIMPR affinity chromatography of ATB200 rhGAA with (embodiment 2) or without (embodiment 1) capture on an anion exchange (AEX) column. [Figure 6A] Figure 6 shows the results of site-specific N-glycosylation analysis of ATB200 rhGAA using two different LC-MS / MS analytical techniques. Figure 6A shows the site occupancy of seven potential N-glycosylation sites for ATB200. [Figure 6B] 6A and 6B show the results of site-specific N-glycosylation analysis of ATB200 rhGAA using two different LC-MS / MS analytical techniques. FIG. 6B shows two analyses of the N-glycosylation profile of the first potential N-glycosylation site for ATB200. [Figure 6C] Figure 6 shows the results of site-specific N-glycosylation analysis of ATB200 rhGAA using two different LC-MS / MS analytical techniques. Figure 6C shows two analyses of the N-glycosylation profile of the second potential N-glycosylation site for ATB200. [Figure 6D] FIG. 6B shows the results of site-specific N-glycosylation analysis of ATB200 rhGAA using two different LC-MS / MS analytical techniques. FIG. 6D shows two analyses of the N-glycosylation profile of the third potential N-glycosylation site for ATB200. [Figure 6E] FIG. 6B shows the results of site-specific N-glycosylation analysis of ATB200 rhGAA using two different LC-MS / MS analytical techniques. FIG. 6E shows two analyses of the N-glycosylation profile of the fourth potential N-glycosylation site for ATB200. [Figure 6F] FIG. 6B shows the results of site-specific N-glycosylation analysis of ATB200 rhGAA using two different LC-MS / MS analytical techniques. FIG. 6F shows two analyses of the N-glycosylation profile of the fifth potential N-glycosylation site for ATB200. [Figure 6G] FIG. 6 shows the results of site-specific N-glycosylation analysis of ATB200 rhGAA using two different LC-MS / MS analytical techniques. FIG. 6G shows two analyses of the N-glycosylation profile of the sixth potential N-glycosylation site for ATB200. [Figure 6H] Figure 6 shows the results of site-specific N-glycosylation analysis of ATB200 rhGAA using two different LC-MS / MS analytical techniques. Figure 6H summarizes the relative percentages of monophosphorylated and bis-phosphorylated species for the first, second, third, fourth, fifth and sixth potential N-glycosylation sites. [Figure 7] 1 is a graph showing the Polywax elution profiles of Lumizyme® (alglucosidase alpha, light line, eluted on the left) and ATB200 (darker line, eluted on the right). [Figure 8] 1 is a table outlining the N-glycan structures of Lumizyme® compared to three different preparations of ATB200 rhGAA identified as BP-rhGAA, ATB200-1 and ATB200-2. [Figure 9] Graphs A and B show the results of CIMPR affinity chromatography of Lumizyme® and Myozyme®. [Figure 10]A is a graph comparing the CIMPR binding affinity of ATB200 rhGAA (left trace) with that of Lumizyme® (right trace). B is a table comparing the bis-M6P content of Lumizyme® and ATB200 rhGAA. [Figure 11] A is a graph comparing ATB200 rhGAA activity (left trace) in normal fibroblasts with Lumizyme® rhGAA activity (right trace) at various GAA concentrations. B is a table comparing ATB200 rhGAA activity (left trace) in fibroblasts from subjects with Pompe disease with Lumizyme® rhGAA activity (right trace) at various GAA concentrations. C is a table comparing Kuptake in fibroblasts from normal subjects and subjects with Pompe disease. [Figure 12] 1 is a graph showing the stability of ATB200 in acidic or neutral pH buffers assessed in a thermal stability assay using SYPRO Orange as a fluorescent dye that increases as the protein unfolds. [Figure 13] Figure 1 shows tissue glycogen content in WT mice or Gaa KO mice treated with vehicle, alglucosidase alfa or ATB200 / AT2221 as determined using amyloglucosidase digestion. Bars represent mean ± SEM of 7 mice / group. *p<0.05 compared to alglucosidase alfa with multiple comparisons using Dunnett's test under one-way ANOVA analysis. [Figure 14] Figure 1 shows LAMP1-positive vesicles in muscle fibers from Gaa KO or WT mice treated with vehicle, alglucosidase alfa, or ATB200 / AT2221. Images were taken from the vastus lateralis muscle and are representative of 7 mice per group. Magnification = 200x (1,000x in insert). [Figure 15A]Figure 1 shows LC3-positive aggregates in muscle fibers from Gaa KO or WT mice treated with vehicle, alglucosidase alpha or ATB200 / AT2221. Images were taken from the vastus lateralis muscle and are representative of 7 mice per group. Magnification = 400x. [Figure 15B] Figure 1 shows Western blot analysis of LC3II protein. A total of 30 mg of protein was loaded in each lane. [Figure 16] Figure 1 shows dysferlin expression in muscle fibers from Gaa KO or WT mice treated with vehicle, alglucosidase alpha or ATB200 / AT2221. Images were taken from the vastus lateralis muscle and are representative of 7 mice per group. Magnification = 200x. [Figure 17] Figure 1 shows simultaneous immunofluorescence staining of LAMP1 (green) (see, e.g., "B") and LC3 (red) (see, e.g., "A") in single fibers isolated from white gastrocnemius muscles of Gaa KO mice treated with vehicle, alglucosidase alpha or ATB200. "C" shows the absence of autophagy debris clearance and enlarged lysosomes. A minimum of 30 fibers were examined for each animal. [Figure 18] FIG. 13 shows stabilization of ATB200 by AT2221 compared to ATB200 alone at 17 μM and 170 μM AT2221, respectively. [Figure 19A] Figure 26A shows the ATB200-02 study design. Low dose = 130 mg High dose = 260 mg In Figure 26A, "6MWT" = 6-minute walk test; "FVC" = forced vital capacity; "QOW" = every other week; "a" = safety data from two sentinel patients from cohort 1 were reviewed at each dose level prior to dosing in cohorts 2 and 3; "b" = during stages 2 and 3, AT2221 was administered orally prior to the start of ATB200 intravenous infusion. For all doses, ATB200 was infused intravenously for a duration of 4 hours. "c" = the first two patients in cohorts 2 and 3 were designated as sentinel patients for their respective cohorts. [Figure 19B] Figure 26A shows the ATB200-02 study design. Low dose = 130 mg High dose = 260 mg In Figure 26A, "6MWT" = 6-minute walk test; "FVC" = forced vital capacity; "QOW" = every other week; "a" = safety data from two sentinel patients from cohort 1 were reviewed at each dose level prior to dosing in cohorts 2 and 3; "b" = during stages 2 and 3, AT2221 was administered orally prior to the start of ATB200 intravenous infusion. For all doses, ATB200 was infused intravenously for a duration of 4 hours. "c" = the first two patients in cohorts 2 and 3 were designated as sentinel patients for their respective cohorts. [Figure 19C] Figure outlining baseline characteristics of patients enrolled across cohorts 1, 2 and 3. "NA" = not applicable. "SD" = standard deviation. "a" = Cohort 1 patients were required to receive alglucosidase alfa for 2-6 years at baseline. LOPD = late-onset Pompe disease. [Figure 20] 1 is a table showing pharmacokinetic data for AT2221. "AUC" = area under the curve; "CL / F" = plasma clearance adjusted for AT2221 oral bioavailability; "Cmax" = maximum drug concentration; "CV" = coefficient of variation; "t1 / 2" = half-life; "tmax" = time to maximum drug concentration; "VZ / F" = apparent terminal phase volume of distribution adjusted for AT2221 oral bioavailability. "a" = geometric mean (CV%); "b" = median (min-max); "c" = arithmetic mean (CV%). [Figure 21]1 is a table showing total GAA protein by signature peptide T09 for cohorts 1 and 3. "AUC" = area under the curve; "CLT" = total body clearance; "Cmax" = maximum drug concentration; "CV" = coefficient of variation; "MD" = multiple doses; "t1 / 2" = half-life; "tmax" = time to maximum drug concentration; "Frel" = AUC ratios for 20mg / kg ATB 200 alone and 10mg / kg ATB200 alone vs. 5mg / kg ATB200 alone, and 20mg / kg ATB200 + low or high dose AT2221 vs. 20mg / kg ATB200 alone. "a" = geometric mean (CV%); "b" = median (min-max); "c" = arithmetic mean (CV%); "d" = n=11; "e" = n=5 Low dose = 130mg High dose = 260mg. [Figure 22A-B] Graph showing total GAA protein by cohort. Low dose = 130 mg High dose = 260 mg. A shows the mean total GAA protein concentration-time profile for cohort 1 (single dose). B shows the mean total GAA protein concentration-time profile for cohort 1 (multiple doses). [Fig. 22C-D] Graph showing total GAA protein by cohort. Low dose = 130 mg High dose = 260 mg. C shows the mean total GAA protein concentration-time profile for cohort 1 vs. cohort 3 (single dose). D shows the mean total GAA protein concentration-time profile for cohort 1 vs. cohort 3 (multiple dose). [Fig. 22E-F] Graph showing total GAA protein by cohort. Low dose=130mg High dose=260mg. E shows total GAA protein compared to 20mg / kg ATB200 at 12 hours post-dose; *=p<0.05; **=p<0.01; ***p<0.001. F shows total GAA protein compared to 20mg / kg ATB200 at 24 hours post-dose; *=p<0.05; **=p<0.01; "ns"=not significant. [Figure 23]1 is a table showing analysis of variance (ANOVA) for total GAA protein by signature peptide T09. Area under the curve (AUC) is shown in μg·h / mL; "CI" = confidence interval. [Figure 24A] Figure showing analysis summary and available interim data for the 6 Minute Walk Test ("6MWT") showing change from baseline ("CFBL") at 6, 9 and 12 months for Cohort 1 and Cohort 3 patients. [Figure 24B] 1 is a table showing 6MWT data for individual patients in Cohort 1 and Cohort 3. [Figure 24C] FIG. 1 shows a summary of analysis and available interim data from other motor function tests: Timed Up and Go Motor Function Test, Four Step Climb, Ten Meter (10M) Walk Test, Gower's and Walk-Step-Gower-Chair ("GSGC") Motor Function Tests, showing change from baseline ("CFBL") at 6, 9 and 12 months for patients in Cohort 1 and Cohort 3. The GSGC is an observer-assessed composite score of four motor function assessments: Walk (10m walk), Four Step Climb, Gower's (floor rise) and chair rise. Each test is scored from 1 (normal) to 7 (unable to perform; maximum score of 6 for chair rise). Total scores range from 4 to 27. "a" = n = 9, missing values ​​not obtained due to patient refusal to perform test; "b" = median change from baseline was -1.5, 7 / 9 patients had a decline; "c" = median change from baseline was -0.8, 4 / 5 patients had a decline. [Diagram 25]Figure 13 shows summary analysis of muscle strength testing (QMT) and interim data available, showing change from baseline at 6 and 9 months ("CFBL") for Cohort 2 patients. QMT = Quantified Muscle Strength Test. Values ​​are shown in pounds of force for right and left sides combined. "a" = shoulder adduction was not available for one subject; "b" = scored: (1) visible muscle movement but not at the joint; (2) movement at the joint but not against gravity; (3) movement against gravity but not against added resistance; (4) movement against added resistance but below normal; (5) normal strength. [Figure 26A] Figure 1 shows an overview of the analysis of manual muscle testing (MMT) scores and available interim data in Cohort 1 patients. MMT scores were calculated for the upper body (maximum score: 40), lower body (maximum score: 40) and total body (maximum score: 80). Increases in manual muscle strength were observed at 6, 9 and 12 months in Cohort 1 patients. "SD" = standard deviation. [Figure 26B] Figure 27 shows an overview of the analysis of manual muscle testing (MMT) scores in cohort 2 patients and available interim data. MMT scores were calculated for the upper body (maximum score: 40). Increases in manual muscle strength were observed at 6 and 9 months in cohort 2 patients. "SD" = standard deviation. MMT results were generally consistent with QMT results (shown in Figure 28). [Figure 26C] Figure 1 shows an overview of the analysis of manual muscle testing (MMT) scores and available interim data in Cohort 3 patients. MMT scores were calculated for the upper body (maximum score: 40), lower body (maximum score: 40) and total body (maximum score: 80). Increases in manual muscle strength were observed at 6, 9 and 12 months for Cohort 3 patients, respectively. "SD" = standard deviation. [Figure 27]Figure showing summary analysis and available interim data from sitting forced vital capacity (FVC), maximum inspiratory pressure (MIP) and maximum expiratory pressure (MEP) showing change from baseline ("CFBL") at 6, 9 and 12 months for Cohort 1 and Cohort 3 patients. "a" = FVC was not available for one subject. MEP and MIP were measured in cmH2O. [Figure 28] FIG. 27 shows an overview of the analysis and available interim data for the Fatigue Severity Scale ("FSS"), a self-assessment questionnaire consisting of 9 questions each scored on a scale of 1 to 7. Total scores range from 9 to 63, with higher values ​​representing higher levels of fatigue due to the disease state. The normal value in a healthy population is approximately 21. FIG. 28 shows the change from baseline ("CFBL") at 6, 9 and 12 months for patients in Cohort 1, Cohort 2 and Cohort 3. [Figure 29A] FIG. 29A shows the mean percentage change from baseline in markers of injury (alanine aminotransferase, aspartate aminotransferase, and creatine kinase) in all patient cohorts. FIG. 29A shows data from Cohort 1 patients over 58 weeks. "BL" = baseline. "SE" = standard error. "WK" = weeks. "M" = months. [Figure 29B] FIG. 29B shows the mean percentage change from baseline in markers of injury (alanine aminotransferase, aspartate aminotransferase, and creatine kinase) in all patient cohorts. FIG. 29B shows data from Cohort 2 patients over 24 weeks. "BL" = baseline. "SE" = standard error. "WK" = weeks. "M" = months. [Figure 29C]FIG. 29C shows the mean percentage change from baseline in markers of injury (alanine aminotransferase, aspartate aminotransferase, and creatine kinase) across all patient cohorts. FIG. 29C shows data from Cohort 3 patients over 36 weeks. "BL" = baseline. "SE" = standard error. "WK" = weeks. "M" = months. [Figure 29D] Mean percentage change from baseline in markers of muscle damage (CK = creatine kinase) and disease substrate (Hex4 = urinary hexose tetrasaccharide) up to 12 months for Cohort 1, Cohort 2 and Cohort 3 patients are shown. "BL" = baseline. "SE" = standard error. "WK" = weeks. "M" = months. [Diagram 30] FIG. 1 summarizes safety data from the ATB200-02 study. "AE" = adverse event; "IAR" = infusion-related reaction; "a" = reported through interim data analysis (up to 20+ months); "b" = includes upper and lower abdominal pain. [Diagram 31] FIG. 1 summarizes the efficacy and safety data available from the ATB200-02 study. [Fig. 32A-H] 32A-32H show the results of site-specific N-glycosylation analysis of ATB200 rhGAA, including the N-glycosylation profile for the seventh potential N-glycosylation site, using LC-MS / MS analysis of protease-digested ATB200. Figures 32A-32H provide average data for 10 lots of ATB200 produced at different scales. [Figure 32A] The average site occupancy of the seven potential N-glycosylation sites for ATB200 is shown. The N-glycosylation sites are provided by SEQ ID NO: 1. CV = coefficient of variation. [Figure 32B]FIG. 32B shows the site-specific N-glycosylation analysis of all seven potential N-glycosylation sites for ATB200 with the site numbers presented according to SEQ ID NO: 5. The bars show the maximum and minimum percentages of N-glycan molecular species identified as specific N-glycan groups for the ten lots of ATB200 analyzed. FIG. 32B shows the N-glycosylation profile of the first potential N-glycosylation site for ATB200. [Figure 32C] FIG. 32C shows the site-specific N-glycosylation analysis of all seven potential N-glycosylation sites for ATB200 with the site numbers presented according to SEQ ID NO: 5. The bars show the maximum and minimum percentages of N-glycan molecular species identified as specific N-glycan groups for the ten lots of ATB200 analyzed. FIG. 32C shows the N-glycosylation profile of the second potential N-glycosylation site for ATB200. [Fig. 32D] FIG. 32D shows the site-specific N-glycosylation analysis of all seven potential N-glycosylation sites for ATB200 with the site numbers presented according to SEQ ID NO: 5. The bars show the maximum and minimum percentages of N-glycan molecular species identified as specific N-glycan groups for the ten lots of ATB200 analyzed. FIG. 32D shows the N-glycosylation profile of the third potential N-glycosylation site for ATB200. [Figure 32E] FIG. 32B shows the site-specific N-glycosylation analysis of all seven potential N-glycosylation sites for ATB200 with the site numbers presented according to SEQ ID NO: 5. The bars show the maximum and minimum percentages of N-glycan molecular species identified as specific N-glycan groups for the ten lots of ATB200 analyzed. FIG. 32C shows the N-glycosylation profile of the fourth potential N-glycosylation site for ATB200. [Fig. 32F]FIG. 32F shows the site-specific N-glycosylation analysis of all seven potential N-glycosylation sites for ATB200 with the site numbers presented according to SEQ ID NO: 5. The bars show the maximum and minimum percentages of N-glycan molecular species identified as specific N-glycan groups for the ten lots of ATB200 analyzed. FIG. 32F shows the N-glycosylation profile of the fifth potential N-glycosylation site for ATB200. [Fig. 32G] FIG. 32G shows the site-specific N-glycosylation analysis of all seven potential N-glycosylation sites for ATB200 with the site numbers presented according to SEQ ID NO: 5. The bars show the maximum and minimum percentages of N-glycan molecular species identified as specific N-glycan groups for the ten lots of ATB200 analyzed. FIG. 32G shows the N-glycosylation profile of the sixth potential N-glycosylation site for ATB200. [Fig. 32H] FIG. 32A shows the site-specific N-glycosylation analysis of all seven potential N-glycosylation sites for ATB200 with the site numbers presented according to SEQ ID NO: 5. The bars show the maximum and minimum percentages of N-glycan molecular species identified as specific N-glycan groups for the ten lots of ATB200 analyzed. FIG. 32H shows the N-glycosylation profile of the seventh potential N-glycosylation site for ATB200. [Figure 33A] Figures 33A-33H further characterize and summarize the N-glycosylation profile of ATB200, which is also shown in Figures 32A-32H. Figure 33A shows 2-anthranilic acid (2-AA) glycan mapping and LC / MS-MS analysis of ATB200, outlining the N-glycan species identified in ATB200 as a percentage of total fluorescence. Data from 2-AA glycan mapping and LC-MS / MS analysis are also shown in Table 5. [Figure 33B]Figure 33 further characterizes and summarizes the N-glycosylation profile of ATB200, which is also shown in Figures 32A-32H. Figure 33B summarizes the average site occupancy and the average N-glycan profile, including total phosphorylation, monophosphorylation, bis-phosphorylation and sialylation, for all seven potential N-glycosylation sites for ATB200. ND = not detected. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Before describing certain exemplary embodiments of the invention, it is to be understood that the invention is not limited to the details of construction or process steps set forth in the following description, as the invention is capable of other embodiments and of being practiced or carried out in various ways.

[0026] I. Definition The terms used herein generally have their ordinary meanings in the art in the context of the present invention and in the specific context in which each term is used. Certain terms are discussed below or elsewhere herein to describe the compositions and methods of the present invention and how to make and use them, and to provide additional guidance to practitioners. The articles "a" and "an" refer to one or more than one (i.e., at least one) of the grammatical object of the article. The term "or" means and is used interchangeably with the term "and / or" unless the context clearly indicates otherwise. In this application, the use of the singular includes the plural unless specifically stated otherwise. Furthermore, the use of the term "including" as well as other forms such as "includes" and "included" is not limiting. Any range described herein is understood to include the endpoints and all values ​​between the endpoints. Unless the context otherwise requires to express the term or necessary context, in this specification the words "comprises" or variations such as "comprising" are used in an inclusive sense, i.e., to specify the presence of the features stated in the various embodiments of the invention, but do not exclude the presence or addition of further features.

[0027] The term "GAA" refers to the human acid alpha glucosidase (GAA) enzyme, which catalyzes the hydrolysis of alpha-1,4- and alpha-1,6-glycosidic linkages of lysosomal glycogen, as well as insertion, related or substitution variants of GAA amino acid sequences and fragments of longer GAA sequences that exert enzymatic activity. Human acid alpha glucosidase is encoded by the GAA gene (National Centre for Biotechnology Information (NCBI) gene number 2548) and is mapped to the long arm of chromosome 17 (location 17q25.2-q25.3). An exemplary DNA sequence encoding GAA is NP 000143.2, which is incorporated by reference. Over 500 mutations have now been identified in the human GAA gene, many of which are associated with Pompe disease. Mutations that result in misfolding or misprocessing of the acid alpha glucosidase enzyme include T1064C (Leu355Pro) and C2104T (Arg702Cys). Additionally, GAA mutations that affect maturation and processing of the enzyme include Leu405Pro and Met519Thr. The conserved hexapeptide WIDMNE at amino acid residues 516-521 is required for activity of the acid alpha glucosidase protein. As used herein, the abbreviation "GAA" is intended to refer to the human acid alpha glucosidase enzyme, while the italicized abbreviation "GAA" is intended to refer to the human gene encoding the human acid alpha glucosidase enzyme. The italicized abbreviation "Gaa" is intended to refer to the non-human gene encoding the non-human acid alpha glucosidase enzyme, including but not limited to the rat or mouse gene, and the abbreviation "Gaa" is intended to refer to the non-human acid alpha glucosidase enzyme.

[0028] The term "rhGAA" is intended to refer to recombinant human acid alpha glucosidase enzyme and is used to distinguish endogenous GAA from synthetic or recombinantly produced GAA (e.g., GAA produced from CHO cells transformed with DNA encoding GAA). The term "rhGAA" encompasses a population of individual rhGAA molecules. Properties of populations of rhGAA molecules are provided herein. The term "conventional rhGAA production" is intended to refer to production containing alglucosidase alpha, such as Lumizyme® or Myozyme®.

[0029] The term "genetically modified" or "recombinant" refers to a cell, such as a CHO cell, that expresses a particular gene product, such as rhGAA, after introduction of a nucleic acid that includes a coding sequence that encodes the gene product together with regulatory elements that control the expression of the coding sequence. Introduction of the nucleic acid can be accomplished by any method known in the art, including gene targeting and homologous recombination. As used herein, the term also includes cells that have been engineered to express or overexpress an endogenous gene or gene product that is not normally expressed by such cells, for example, by gene activation techniques.

[0030] As used herein, the term "purified" refers to a substance that is isolated under conditions that reduce or eliminate the presence of unrelated substances, i.e., contaminants, including the natural substances from which the substance was obtained. For example, a purified protein is preferably substantially free of other proteins or nucleic acids with which it is associated in a cell; a purified nucleic acid molecule is substantially free of proteins or other unrelated nucleic acid molecules that may be found in a cell. As used herein, the term "substantially free" is used operationally in the context of analytical testing of a substance. Preferably, a purified substance that is substantially free of contaminants is at least 95% pure; more preferably at least 97% pure, and even more preferably at least 99% pure. Purity can be assessed by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, enzymatic assay, and other methods well known in the art. In a specific embodiment, purification means that the level of contaminants is below the level that is accepted by regulatory authorities for safe administration to humans or non-human animals. Recombinant proteins such as rhGAA can be isolated or purified from CHO cells using methods well known in the art, including chromatographic size separation, affinity chromatography, or anion exchange chromatography. In some embodiments, rhGAA is purified by a method including anion exchange chromatography followed by immobilized metal affinity chromatography, optionally followed by purification using a third chromatography column.

[0031] As used herein, the term "alglucosidase alfa" is intended to refer to recombinant human acid alpha glucosidase identified as [199-Arginine, 223-Histidine] prepro-alpha-glucosidase (human); Chemical Registry Number 420794-05-0. Alglucosidase alfa has been approved for sale in the United States by Genzyme under the products Lumizyme® and Myozyme®.

[0032] As used herein, the term "ATB200" is intended to refer to recombinant human acid alpha-glucosidase, as described in International Application No. PCT / US2015 / 053252, the disclosure of which is incorporated herein by reference.

[0033] As used herein, the term "glycan" is intended to refer to a polysaccharide chain that is covalently attached to an amino acid residue on a protein or polypeptide. As used herein, the term "N-glycan" or "N-linked glycan" is intended to refer to a polysaccharide chain that is attached to an amino acid residue on a protein or polypeptide through a covalent bond to the nitrogen atom of the amino acid residue. For example, an N-glycan can be covalently attached to the side chain nitrogen atom of an asparagine residue. A glycan may contain one or several monosaccharide units, and the monosaccharide units may be covalently linked to form a linear or branched chain. In at least one embodiment, the N-glycan unit attached to rhGAA may include one or more monosaccharide units independently selected from N-acetylglucosamine, mannose, galactose, fucose, mannose-6-phosphate, or sialic acid. The N-glycan units on a protein may be determined by any suitable analytical technique, such as mass spectrometry. In some embodiments, the N-glycan units attached to rhGAA are determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS) using an instrument such as a Thermo Scientific™ Orbitrap Velos Pro™ Mass Spectrometer, a Thermo Scientific™ Orbitrap Fusion™ Lumos Tribid™ Mass Spectrometer or a Waters Xevo® G2-XS QTofMass Spectrometer.

[0034] As used herein, the term "high mannose N-glycan" is intended to refer to an N-glycan having one to six or more mannose units. In some embodiments, the high mannose N-glycan unit may contain a bis(N-acetylglucosamine) chain linked to an asparagine residue and further linked to a branched polymannose chain. As used interchangeably herein, the term "M6P" or "mannose-6-phosphate" is intended to refer to a mannose unit phosphorylated at the 6-position, i.e., having a phosphate group linked to a hydroxyl group at the 6-position. In some embodiments, one or more mannose units of one or more N-glycan units are phosphorylated at the 6-position to form a mannose-6-phosphate unit. In some embodiments, the term "M6P" or "mannose-6-phosphate" refers to both mannose phosphodiester with N-acetylglucosamine (GlcNAc) as a "cap" at the phosphate group as well as mannose units with exposed phosphate groups that lack a GlcNAc cap. In at least one embodiment, the N-glycan of the protein may have multiple M6P groups, including at least one M6P group having a GlcNAc cap and at least one other M6P group lacking a GlcNAc cap.

[0035] As used herein, the term "complex N-glycan" is intended to refer to an N-glycan that contains sugar types other than GlcNac and mannose, such as one or more galactose and / or sialic acid units. In at least one embodiment, the complex N-glycan may be a high mannose N-glycan in which one or more mannose units are further linked to one or more monosaccharide units each independently selected from N-acetylglucosamine, galactose and sialic acid. As used herein, "hybrid N-glycan" is intended to refer to an N-glycan that contains at least one high mannose branch and at least one complex branch. Representative structures for non-phosphorylated, mono-M6P and bis-M6P N-glycans are shown in FIG. 1A. The mannose-6-phosphate group is shown in FIG. 1B.

[0036] As used herein, "normalization" of lysosomes in muscle refers to the process of restoring affected muscle to wild-type muscle lysosomal morphology by reducing the size and number of its stored glycogen, thereby causing the affected muscle to substantially resemble normal lysosomal morphology, ultimately resulting in reversal of disease progression.

[0037] As used herein, "reversal of disease progression" specifically refers to (i) reducing or eliminating glycogen accumulation, (ii) reducing or eliminating lysosomal swelling and / or dysfunction, and (iii) reducing or eliminating autophagy debris build-up. Reversal of disease progression may be manifested in ambulatory ERT-experienced Pompe disease patients as two or more of the following "clinical improvements": (a) a mean increase in 6-minute walking test distance of at least 20 meters, (b) a mean improvement in maximum expiratory pressure of at least 16 cmH2O, and (c) a mean reduction in Fatigue Severity Scale score of at least 7. Reversal of disease progression may be manifested in non-ambulatory ERT-experienced Pompe disease patients as two or more of the following "clinical improvements": (a) a mean improvement in shoulder adduction of at least 8 pounds of force, (b) a mean improvement in elbow extension of at least 5 pounds of force, and (c) a mean reduction in Fatigue Severity Scale score of at least 3.5. Reversal of disease progression may be manifested in ERT-naïve Pompe disease patients as two or more of the following "clinical improvement": (a) a mean increase in 6-minute walk test distance of at least 40 meters, (b) a mean improvement in upright (sitting) forced vital capacity of at least 4%, (c) a mean improvement in peak inspiratory pressure of at least 11 cmH2O, and (d) a mean reduction in Fatigue Severity Scale score of at least 5.

[0038] The advantage of the treatment method disclosed herein compared to the administration of alglucosidase alfa is that Pompe patients treated with the former show prolonged clinical improvement.For example, improvement may be observed for 2 to 3 years or more after the administration of the first treatment, including for example 4, 5 or 6 years after the administration of the first treatment.In contrast, after 2 years of enzyme replacement therapy with standard treatment (e.g., alglucosidase alfa), Pompe patients either (i) maintain the benefits from the baseline prior to treatment but do not show any discernible improvement beyond the 2- or 3-year threshold, or (ii) experience a slow decline and lose all the benefits achieved through 2- or 3-years after treatment with standard treatment. Kuperus et al., 2017. Long-term benefit of enzyme replacement therapy in Pompe disease: A 5-year prospective study. Neurology. 89:2365-2373. In contrast, rhGAA as described herein has been shown to clear lysosomal glycogen more efficiently than standard treatments and induce improvement in patients who are not expected to improve for at least two years after enzyme replacement therapy (e.g., "ERT-switched ambulatory" cohort 1 of study ATB200-02). Clinical data to date using rhGAA or pharmaceutical compositions as described herein is expected to provide continued improvement in patient outcomes even two years after treatment. Thus, in some embodiments, patients treated with rhGAA or pharmaceutical compositions as described herein continue to show progression in one or more clinical improvements more than two years after treatment (e.g., experience additional benefit beyond the benefit achieved by or at the two-year baseline).

[0039] As used herein, "reversal of lysosomal pathology" refers to the partial or complete clearance of glycogen that has accumulated in cells due to lack of optimal GAA activity.

[0040] As used herein, forced vital capacity or "FVC" is the volume of air that can be forcefully exhaled from a subject's lungs after the subject takes the deepest possible breath.

[0041] As used herein, the "6-minute walk test" (6MWT) is a test to measure the distance an individual can walk on a hard, flat surface over a total period of six minutes. The test is performed by having the individual walk as far as possible in six minutes.

[0042] As used herein, the "10 Meter Walk Test" (10MWT) is a test to measure the time it takes an individual to walk 10 meters on a flat surface in walking shoes.

[0043] As used herein, the compound miglustat, also known as N-butyl-1-deoxynojirimycin or NB-DNJ or (2R,3R,4R,5S)-1-butyl-2-(hydroxymethyl)piperidine-3,4,5-triol, has the following chemical formula: The compound has the following structure: TIFF2025016455000002.tif38170.

[0044] One formulation of miglustat is marketed under the trade name Zavesca® as monotherapy for Gaucher disease type 1. In some embodiments, miglustat is referred to as AT2221.

[0045] As discussed below, pharma- ceutically acceptable salts of miglustat may also be used in the present invention. If a salt of miglustat is used, the dosage of the salt is adjusted so that the patient receives the equivalent dose of miglustat that they would receive if the miglustat free base was used.

[0046] As used herein, the compound duvoglustat, also known as 1-deoxynojirimycin or DNJ or (2R,3R,4R,5S)-2-(hydroxymethyl)piperidine-3,4,5-triol, has the following chemical formula: The compound has the following structure: TIFF2025016455000003.tif39170.

[0047] As used herein, the term "pharmacological chaperone" or sometimes simply the term "chaperone" refers to a pharmacological agent that specifically binds to acid alpha-glucosidase and has the following effects: Enhancement of the formation of stable molecular conformations of proteins; · enhancing proper transport of proteins from the endoplasmic reticulum to another cellular location, preferably the native cellular location, thereby preventing endoplasmic reticulum-associated degradation of the protein; · Preventing the aggregation of conformationally unstable or misfolded proteins; - restoring and / or enhancing at least partial wild-type function, stability and / or activity of a protein; and / or Improving the phenotype or function of cells carrying acid α-glucosidase It is intended to refer to a molecule having one or more of:

[0048] Thus, a pharmacological chaperone for acid alpha glucosidase is a molecule that binds to acid alpha glucosidase and results in the proper folding, transport, non-aggregation and activity of acid alpha glucosidase. As used herein, this term includes, but is not limited to, active site specific chaperones (ASSCs) that bind to the active site of the enzyme, inhibitors or antagonists and agonists. In at least one embodiment, the pharmacological chaperone can be an inhibitor or antagonist of acid alpha glucosidase. As used herein, the term "antagonist" is intended to refer to any molecule that binds to acid alpha glucosidase and partially or completely blocks, inhibits, reduces or neutralizes the activity of acid alpha glucosidase. In at least one embodiment, the pharmacological chaperone is miglustat. Another non-limiting example of a pharmacological chaperone for acid alpha glucosidase is duvoglustat.

[0049] As used herein, the term "active site" is intended to refer to the region of a protein that is related to and necessary for the specific biological activity of the protein. In at least one embodiment, the active site may be the site that binds to a substrate or other binding partner and contributes amino acid residues that are directly involved in the formation and cleavage of chemical bonds. The active site in the present invention may include the catalytic site of an enzyme, the antigen binding site of an antibody, the ligand binding domain of a receptor, the binding domain of a regulator or the receptor binding domain of a secreted protein. The active site may also include the transactivation, protein-protein interaction or DNA binding domain of transcription factors and regulators.

[0050] As used herein, the term "AUC" or "area under the curve" is intended to refer to a mathematical calculation to evaluate the total exposure of the body to a given drug over time.In a graph that plots how the blood concentration of a drug administered to a subject changes over time after administration, the drug concentration variable is on the y-axis and time is on the x-axis.The area between the drug concentration curve and the x-axis of a specified time range is the AUC.AUC is used as a guide for dosing schedule and to compare the bioavailability of the effectiveness of various drugs in the body.

[0051] As used herein, the term "Cmax" is intended to refer to the maximum plasma concentration of a drug achieved following administration to a subject.

[0052] As used herein, the term "volume of distribution" or "V" is intended to refer to the theoretical volume required to contain the total amount of administered drug at the same concentration as observed in plasma, and represents the degree to which the drug is distributed in body tissues rather than in plasma. A higher value of V indicates a higher degree of tissue distribution. "Central volume of distribution" or "Vc" is intended to refer to the volume of distribution in blood and tissues that are highly perfused by blood. "Peripheral volume of distribution" or "V2" is intended to refer to the volume of distribution in peripheral tissues.

[0053] The terms "clearance", "systemic clearance" or "CL" as used interchangeably herein are intended to refer to the volume of plasma per unit time through which an administered drug is completely cleared. "Peripheral clearance" is intended to refer to the volume of peripheral tissue per unit time through which an administered drug is cleared.

[0054] As used herein, the term "pharmaceutical acceptable" is intended to refer to molecular entities and compositions that are physiologically acceptable and typically do not produce adverse reactions when administered to humans.Preferably, as used herein, the term "pharmaceutical acceptable" means approved by federal or state government regulatory agencies or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals and more specifically in humans.As used herein, the term "carrier" is intended to refer to a diluent, adjuvant, excipient, or vehicle with which a compound is administered.Suitable pharmaceutical carriers are well known in the art and, in at least one embodiment, are described in "Remington's Pharmaceutical Sciences" 18th Edition by EW Martin or other editions.

[0055] As used herein, the term "pharmaceutically acceptable salts" is intended to mean salts that are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, within the scope of sound medical judgment, are commensurate with a reasonable benefit / risk ratio, and are generally soluble or dispersible in water or oil, and are effective for their intended use. The term includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. Lists of suitable salts can be found, for example, in S. M. Berge et al., J. Pharm. Sci., 1977, 66, pp. 1-19, which is incorporated by reference. As used herein, the term "pharmaceutically acceptable acid addition salts" is intended to mean salts formed with inorganic acids that retain the biological effectiveness and properties of the free bases and are not biologically or otherwise undesirable. As used herein, the term "pharmaceutically acceptable base addition salts" is intended to mean salts formed with inorganic acids which retain the biological effectiveness and properties of the free bases and which are not biologically or otherwise undesirable.

[0056] As used herein, the term "buffer" refers to a solution containing a weak acid and its conjugated base that serves to prevent changes in pH.

[0057] As used herein, the terms "therapeutically effective dose" and "effective amount" are intended to refer to the amount of acid alpha glucosidase and / or miglustat and / or a combination thereof sufficient to produce a therapeutic response in a subject. The therapeutic response may be any response that a user (e.g., a clinician) recognizes as an effective response to treatment, including any surrogate clinical marker or symptom described herein and known in the art. Thus, in at least one embodiment, the therapeutic response may be an improvement or suppression of one or more symptoms or markers of Pompe disease, such as those known in the art. Symptoms or markers of Pompe disease include, but are not limited to, decreased acid alpha-glucosidase tissue activity; cardiac myopathy; cardiac enlargement; progressive muscle weakness, especially in the trunk or lower limbs; severe hypotonia; macroglossia (and in some cases, tongue protrusion); difficulty swallowing, sucking and / or feeding; respiratory failure; hepatomegaly (moderate); facial muscle relaxation; areflexia; exercise intolerance; dyspnea on exertion; orthopnea; sleep apnea; morning headache; somnolence; lordosis and / or scoliosis; decreased deep tendon reflexes; low back pain; and failure to meet motor development screening. It should be noted that the concentration of miglustat that has an inhibitory effect on acid alpha-glucosidase constitutes an "effective amount" for purposes of the present invention due to the dilution (and resulting shift in binding due to changes in equilibrium and pH), bioavailability and metabolism of miglustat upon in vivo administration.

[0058] Therapeutic response also includes molecular responses such as glycogen accumulation, lysosomal proliferation and the formation of autophagy zones.Therapeutic response can be evaluated by comparing the physiological and molecular responses of muscle biopsy before and after treatment with rhGAA as described herein.For example, the amount of glycogen present in biopsy sample can be used as a marker to determine therapeutic response.Another example includes biomarkers such as LAMP-1, LC3 and dysferlin, which can be used as indicators of lysosomal storage dysfunction.For example, muscle biopsy collected before and after treatment with rhGAA as described herein can be stained with an antibody that recognizes one of the biomarkers.

[0059] As used herein, the term "enzyme replacement therapy" or "ERT" is intended to refer to the introduction of non-natural, purified enzymes to individuals who are deficient in such enzymes. The administered protein can be obtained from natural materials or by recombinant expression. The term also refers to the introduction of purified enzymes in other individuals who need or will benefit from the administration of purified enzymes. In at least one embodiment, such individuals suffer from enzyme deficiency. The enzymes introduced can be purified, recombinant enzymes produced in vitro or proteins purified from isolated tissues or body fluids such as placenta or animal milk, or from plants.

[0060] As used herein, the term "combination therapy" is intended to refer to any therapy in which two or more individual therapies are administered simultaneously or sequentially. In some embodiments, the results of the combination therapy are enhanced compared to the effect of each treatment when performed individually. Enhancement can include any improvement in the effect of various therapies that can produce advantageous results compared to the results achieved by the treatment when performed alone. Enhanced effects or results include synergistic enhancement, where the enhanced effect is greater than the additive effect of each treatment when performed alone; additive enhancement, where the enhanced effect is substantially equal to the additive effect of each treatment when performed alone; or less than additive effect, where the enhanced effect is less than the additive effect of each treatment when performed alone, but still better than the effect of each treatment when performed alone. Enhanced effects can be measured by any means known in the art by which treatment efficacy or outcome can be measured.

[0061] As used herein, the term "simultaneous" is intended to mean at the same time or within a reasonably short period of time before or after, as understood by those skilled in the art.For example, when two treatments are administered simultaneously with each other, one treatment can be administered before or after the other treatment, taking into account the time required to prepare the latter of the two treatments.Thus, "simultaneous administration" of two treatments includes, but is not limited to, one treatment followed by the other treatment for about 30 minutes or less, about 30 minutes, about 20 minutes or less, about 20 minutes, about 15 minutes, about 10 minutes, about 9 minutes, about 8 minutes, about 7 minutes, about 6 minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, about 1 minute or less.

[0062] "Pompe disease" refers to an autosomal recessive LSD characterized by defective acid alpha-glucosidase (GAA) activity that impairs lysosomal glycogen metabolism. Enzyme deficiency leads to lysosomal glycogen storage, resulting in progressive skeletal muscle weakness, reduced cardiac function, respiratory failure and / or CNS dysfunction in the later stages of the disease. Genetic mutations in the GAA gene result in reduced expression or produce mutant forms of the enzyme with altered stability and / or biological activity, ultimately resulting in disease (see generally Hirschhorn R, 1995, Glycogen Storage Disease Type II: Acid a-Glucosidase (Acid Maltase) Deficiency, The Metabolic and Molecular Bases of Inherited Disease, Scriver et al., eds., McGraw-Hill, New York, 7th ed., pp. 2443-2464). The three recognized clinical forms of Pompe disease (infantile, pediatric, and adult) correlate with the level of residual α-glucosidase activity (Reuser AJ et al., 1995, Glycogenosis Type II (Acid Maltase Deficiency), Muscle & Nerve Supplement 3, S61-S69). Infantile Pompe disease (type I or A) is the most common and most severe and is characterized by failure to thrive, generalized hypotonia, cardiomegaly, and cardiopulmonary failure within the first 2 years of life. Pediatric Pompe disease (type II or B) is intermediate in severity and is characterized by a predominance of muscular manifestations without cardiomegaly. Pediatric Pompe individuals usually die before reaching the age of 20 years from respiratory failure. Adult Pompe disease (type III or C) often presents as a slowly progressive myopathy in the teens or late sixth decade (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).It has been shown that in Pompe, α-glucosidase is extensively modified post-translationally by glycosylation, phosphorylation and proteolytic processing. The conversion of 110 kilodalton (kDa) precursor to 76 and 70 KDa mature forms by proteolysis in lysosomes is required for optimal glycogen catalysis. As used herein, the term "Pompe disease" refers to all types of Pompe disease. The formulations and dosing regimens disclosed in this application can be used to treat, for example, type I, type II or type III Pompe disease.

[0063] A "subject" or "patient" is preferably a human, although other mammals and non-human animals with disorders related to glycogen accumulation may also be treated. A subject may be a fetus, neonate, child, pediatric, or adult with Pompe disease or other glycogen storage or accumulation disorder. One example of an individual to be treated is an individual (fetus, neonate, child, pediatric, adolescent, or adult human) with GSD-II (e.g., infantile GSD-II, pediatric GSD-II, or adult-onset GSD-II). The individual may have residual GAA activity or no measurable activity. For example, an individual with GSD-II may have GAA activity that is less than about 1% of normal GAA activity (infantile GSD-II), about 1-10% of normal GAA activity (pediatric GSD-II), or about 10-40% of normal GAA activity (adult GSD-II). In some embodiments, the subject or patient is an "ERT-experienced" or "ERT-switched" patient, which refers to a Pompe disease patient who has previously undergone enzyme replacement therapy. In some embodiments, the subject or patient is an "ERT-naive" patient, which refers to a Pompe disease patient who has not previously undergone enzyme replacement therapy. In certain embodiments, the subject or patient is ambulatory (e.g., an ambulatory ERT-switched patient or an ambulatory ERT-naive patient). In certain embodiments, the subject or patient is non-ambulatory (e.g., a non-ambulatory ERT-switched patient). Ambulatory or non-ambulatory status can be determined by a 6-minute walk test (6MWT). In some embodiments, an ambulatory patient is a Pompe disease patient who can walk at least 200 meters in the 6MWT. In some embodiments, a non-ambulatory patient is a Pompe disease patient who cannot walk unaided or get out of a wheelchair.

[0064] As used herein, the terms "treat" and "treatment" refer to the improvement 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 reduction of the severity or frequency of one or more symptoms of the disease.For example, treatment can refer to the improvement of cardiac condition (e.g., increasing end-diastolic and / or end-systolic volume, or reducing, improving or preventing the progressive cardiac myopathy typically found in GSD-II) or the improvement of pulmonary function (e.g., increasing vital capacity during crying above baseline capacity, and / or normalizing oxygen desaturation during crying); the improvement in neurodevelopment and / or motor skills (e.g., increasing AIMS score); the reduction of glycogen levels in tissues of individuals affected by the disease; or any combination of these effects.In a preferred embodiment, treatment includes the improvement of cardiac condition, particularly the reduction or prevention of GSD-II-related cardiac myopathy.

[0065] As used herein, the terms "improve," "increase," and "reduce" refer to values ​​compared to a baseline measurement, such as a measurement in the same individual prior to the initiation of a treatment described herein, or a measurement in a control individual (or individuals) lacking the treatment described herein. A control individual is one who suffers from the same form of GSD-II (either infantile, pediatric, or adult onset) as the individual being treated, and who is approximately the same age as the individual being treated (to ensure that the stage of the disease in the treated and control individual(s) is comparable).

[0066] As used herein, the terms "about" and "approximately" are intended to refer to the acceptable degree of error for the measured quantity, taking into account the nature and precision of the measurement. For example, the degree of error is indicated by the number of significant values ​​provided for the measurement, as understood in the art, including but not limited to ±1 variation in the most accurate and significant value reported for the measurement. Typical exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. The quantities provided herein are approximate unless otherwise stated, and the term "about" or "approximately" means that they may be inferred unless expressly stated.

[0067] II. Recombinant human acid alpha-glucosidase (rhGAA) In some embodiments, the recombinant human acid alpha glucosidase (rhGAA) is an enzyme having the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. In some embodiments, the rhGAA is encoded by the nucleotide sequence set forth in SEQ ID NO:2. TIFF2025016455000004.tif254170TIFF2025016455000005.tif237170

[0068] In some embodiments, rhGAA has the wild-type GAA amino acid sequence set forth in SEQ ID NO: 1 as set forth in U.S. Patent No. 8,592,362, and has GenBank accession number AHE24104.1 (GI:568760974). In some embodiments, rhGAA has an mRNA sequence with GenBank accession number Y00839.1, the wild-type GAA amino acid sequence encoded by SEQ ID NO: 2. In some embodiments, rhGAA has the wild-type GAA amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, rhGAA has the GAA amino acid sequence set forth in SEQ ID NO: 4, and has National Center for Biotechnology Information (NCBI) accession number NP_000143.2. In some embodiments, rhGAA is alglucosidase alpha, a human acid alpha glucosidase enzyme encoded by the most prevalent of the nine observed haplotypes of the GAA gene.

[0069] In some embodiments, rhGAA is initially expressed with the full length 952 amino acid sequence of wild type GAA set forth in SEQ ID NO:1, and rhGAA undergoes intracellular processing that removes a portion of the amino acids, for example the first 56 amino acids. Thus, rhGAA secreted by a host cell may have a shorter amino acid sequence than rhGAA initially expressed in the cell. In one embodiment, the shorter protein has the amino acid sequence set forth in SEQ ID NO:5, which differs from SEQ ID NO:1 only in that the first 56 amino acids, including the signal peptide and precursor peptide, are removed, resulting in a protein having 896 amino acids. Other variations in the number of amino acids are also possible, such as having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more deletions, substitutions and / or insertions compared to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:5. In some embodiments, the rhGAA product includes a mixture of recombinant human acid alpha glucosidase molecules having various amino acid lengths.

[0070] In some embodiments, rhGAA comprises an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to SEQ ID NO:1 or SEQ ID NO:5. Various sequence comparison algorithms and / or programs, including FASTA or BLAST, available as part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), can be used to calculate the identity between two sequences. For example, polypeptides and polynucleotides encoding such polypeptides that have at least 90%, 95%, 98% or 99% identity to a particular polypeptide described herein and preferably exhibit substantially the same function, are considered. Unless otherwise indicated, similar scores are based on the use of BLOSUM62. When BLASTP is used, the similarity percentage is based on the BLASTP positive score, and the sequence identity percentage is based on the BLASTP identity score. BLASTP "identity" indicates the number and percentage of total residues that are identical in the high-scoring sequence pair; BLASTP "positive" indicates the number and percentage of residues that have a positive sequence comparison score and are similar to each other. Amino acid sequences having these degrees of identity or similarity, or any intermediate degree of similarity, to the amino acid sequences disclosed herein are contemplated and encompassed by the present disclosure. Polynucleotide sequences of similar polypeptides can be deduced using the genetic code and obtained by conventional means, specifically by back-translating the amino acid sequence using the genetic code.

[0071] In some embodiments, rhGAA undergoes post-translational and / or chemical modifications to one or more amino acid residues in the protein. For example, methionine and tryptophan residues may undergo oxidation. As another example, N-terminal glutamine may form pyroglutamate. As another example, asparagine residues may undergo deamidation to aspartic acid. As yet another example, aspartic acid residues may undergo isomerization to isoaspartic acid. As yet another example, unpaired cysteine ​​residues in the protein may form disulfide bonds with free glutathione and / or cysteine. Thus, in some embodiments, the enzyme is initially expressed with an amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, or encoded by SEQ ID NO:2, and the enzyme undergoes one or more of these post-translational and / or chemical modifications. Such modifications are also within the scope of the present disclosure.

[0072] III. N-Linked Glycosylation of rhGAA There are seven potential N-linked glycosylation sites on a single rhGAA molecule. These potential glycosylation sites are at the following positions of SEQ ID NO:5: N84, N177, N334, N414, N596, N826 and N869. Similarly, for the full length amino acid sequence of SEQ ID NO:1, these potential glycosylation sites are at the following positions: N140, N233, N390, N470, N652, N882 and N925. Other variants of rhGAA may have similar glycosylation sites depending on the position of the asparagine residue. In general, the sequence of Asn-X-Ser or Asn-X-Thr of the amino acid sequence in a protein indicates a potential glycosylation site, with the exception that X cannot be His or Pro.

[0073] The rhGAA molecules described herein may have an average of 1, 2, 3, or 4 mannose-6-phosphate (M6P) groups on their N-glycans. For example, only one N-glycan on a rhGAA molecule may carry M6P (monophosphorylated or mono-M6P), a single N-glycan may carry two M6P groups (bis-phosphorylated or bis-M6P), or two different N-glycans on the same rhGAA molecule may each carry a single M6P group. In some embodiments, the rhGAA molecules described herein have an average of 3-4 M6P groups on their N-glycans. Recombinant human acid alpha glucosidase molecules also have N-glycans that do not carry M6P groups. In another embodiment, on average, the rhGAA contains more than 2.5 moles of M6P per mole of rhGAA and more than 4 moles of sialic acid per mole of rhGAA. In some embodiments, on average, rhGAA contains about 3-3.5 mol M6P per mole of rhGAA. In some embodiments, on average, rhGAA contains about 4-5.4 mol sialic acid per mole of rhGAA. On average, at least about 3, 4, 5, 6, 7, 8, 9, 10% or 20% of the total N-glycans on rhGAA may be in the form of mono-M6P N-glycans, for example about 6.25% of the total N-glycans may bear a single M6P group, on average, at least about 0.5, 1, 1.5, 2.0, 2.5, 3.0% of the total N-glycans on rhGAA are in the form of bis-M6P N-glycans, and on average, less than 25% of the total rhGAA does not contain phosphorylated N-glycans that bind to CIMPR. In some embodiments, on average, about 10% to about 14% of the total N-glycans on rhGAA are monophosphorylated. In some embodiments, on average about 7% to about 25% of the total N-glycans on rhGAA are bisphosphorylated, hi some embodiments, on average rhGAA contains about 1.3 mol bis-M6P per mol rhGAA.

[0074] The rhGAA described herein may have an average content of M6P-bearing N-glycans ranging from 0.5 to 7.0 mol M6P per mole rhGAA or any intermediate value or subrange thereof, including 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 M6P per mole rhGAA. The rhGAA may be fractionated to provide rhGAA preparations containing different average numbers of mono-M6P-bearing or bis-M6P-bearing N-glycans, thereby allowing further customization of rhGAA targeting to lysosomes in target tissues by selecting specific fractions or by selectively combining different fractions.

[0075] In some embodiments, up to 60% of the N-glycans on rhGAA may be fully sialylated, for example, 10%, 20%, 30%, 40%, 50% or up to 60% of the N-glycans may be fully sialylated. In some embodiments, 50% or less of the N-glycans on rhGAA are fully sialylated. In some embodiments, 4% to 20% of the total N-glycans are fully sialylated. In other embodiments, 5%, 10%, 20% or less of the N-glycans on rhGAA bear sialic acid and terminal galactose residues (Gal). This range includes all intermediate values ​​and partial ranges, for example, 7% to 30% of the total N-glycans on rhGAA may bear sialic acid and terminal galactose. In yet other embodiments, no more than 5%, 10%, 15%, 16%, 17%, 18%, 19%, or 20% of the N-glycans on the rhGAA have only terminal galactose and no sialic acid, this range including all intermediate values ​​and subranges, for example, between 8% and 19% of the total N-glycans on the rhGAA in the composition have only terminal galactose and no sialic acid.

[0076] In some embodiments, 40%, 45%, 50% or 55% to 60% of the total N-glycans on rhGAA are complex type N-glycans; or 1%, 2%, 3%, 4%, 5%, 6% or 7% or less of the total N-glycans on rhGAA are hybrid type N-glycans; 5%, 10%, 15%, 20% or 25% or less of the high mannose type N-glycans on rhGAA are non-phosphorylated; at least 5% or 10% of the high mannose type N-glycans on rhGAA are monophosphorylated; and / or at least 1% or 2% of the high mannose type N-glycans on rhGAA are bis-phosphorylated. These values ​​include all intermediate values ​​and partial ranges. rhGAA can meet one or more of the content ranges described above.

[0077] In some embodiments, rhGAA may bear an average of 2.0 to 8.0 moles of sialic acid residues per mole of rhGAA, this range including all intermediate values ​​and subranges thereof, including 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 moles of sialic acid residues per mole of rhGAA. Without being bound by theory, it is believed that the presence of N-glycan units bearing sialic acid residues may prevent non-productive clearance of rhGAA by the asialoglycoprotein receptor.

[0078] In one or more embodiments, rhGAA has a certain N-glycosylation profile at a certain potential N-glycosylation site. In some embodiments, rhGAA has seven potential N-glycosylation sites. In some embodiments, at least 20% of rhGAA is phosphorylated at the first potential N-glycosylation site (e.g., N84 for SEQ ID NO:5 and N140 for SEQ ID NO:1). For example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of rhGAA can be phosphorylated at the first potential N-glycosylation site. This phosphorylation can be the result of mono-M6P and / or bis-M6P units. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the rhGAA possesses a mono-M6P unit at the first potential N-glycosylation site. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the rhGAA possesses a bis-M6P unit at the first potential N-glycosylation site. In some embodiments, rhGAA contains an average of about 1.4 moles of M6P (mono-M6P and bis-M6P) per mole of rhGAA at the first potential N-glycosylation site. In some embodiments, rhGAA contains an average of at least about 0.5 moles of bis-M6P per mole of rhGAA at the first potential N-glycosylation site. In some embodiments, rhGAA contains an average of about 0.25 moles of mono-M6P per mole of rhGAA at the first potential N-glycosylation site. In some embodiments, rhGAA contains an average of about 0.2 moles to about 0.3 moles of sialic acid per mole of rhGAA at the first potential N-glycosylation site. In at least one embodiment, rhGAA has a first potential N-glycosylation site occupancy as shown in FIG. 6A and an N-glycosylation profile as shown in FIG. 6B.In at least one embodiment, rhGAA has a first potential N-glycosylation site occupancy as shown in Figure 32A and an N-glycosylation profile as shown in Figure 32B or Figure 33B.

[0079] In some embodiments, at least 20% of the rhGAA is phosphorylated at the second potential N-glycosylation site (e.g., N177 for SEQ ID NO:5 and N223 for SEQ ID NO:1). For example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the rhGAA may be phosphorylated at the second N-glycosylation site. This phosphorylation may be the result of mono-M6P and / or bis-M6P units. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the rhGAA possesses a mono-M6P unit at the second N-glycosylation site. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the rhGAA possesses a bis-M6P unit at the second N-glycosylation site. In some embodiments, rhGAA comprises an average of about 0.5 moles of M6P (mono-M6P and bis-M6P) per mole of rhGAA at the second potential N-glycosylation site. In some embodiments, rhGAA comprises an average of about 0.4 to about 0.6 moles of mono-M6P per mole of rhGAA at the second potential N-glycosylation site. In at least one embodiment, rhGAA comprises a second potential N-glycosylation site occupancy as shown in FIG. 6A and an N-glycosylation profile as shown in FIG. 6C. In at least one embodiment, rhGAA comprises a second potential N-glycosylation site occupancy as shown in FIG. 32A and an N-glycosylation profile as shown in FIG. 32C or FIG. 33B.

[0080] In one or more embodiments, at least 5% of rhGAA is phosphorylated at the third potential N-glycosylation site (e.g., N334 for SEQ ID NO:5 and N390 for SEQ ID NO:1). In other embodiments, less than 5%, 10%, 15%, 20% or 25% of rhGAA is phosphorylated at the third potential N-glycosylation site. For example, the third potential N-glycosylation site has a mixture of non-phosphorylated high mannose N-glycans, bi-, tri- and tetra-antennary complex N-glycans and hybrid N-glycans as the predominant molecular species. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of rhGAA is sialylated at the third potential N-glycosylation site. In some embodiments, rhGAA comprises an average of about 0.9 to about 1.2 moles of sialic acid per mole of rhGAA at the third potential N-glycosylation site. In at least one embodiment, rhGAA has a third potential N-glycosylation site occupancy as shown in Figure 6A and an N-glycosylation profile as shown in Figure 6D. In at least one embodiment, rhGAA comprises a third potential N-glycosylation site occupancy as shown in Figure 32A and an N-glycosylation profile as shown in Figure 32D or Figure 33B.

[0081] In some embodiments, at least 20% of the rhGAA is phosphorylated at the fourth potential N-glycosylation site (e.g., N414 for SEQ ID NO:5 and N470 for SEQ ID NO:1). For example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the rhGAA may be phosphorylated at the fourth potential N-glycosylation site. This phosphorylation may be the result of mono-M6P and / or bis-M6P units. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the rhGAA possesses a mono-M6P unit at the fourth potential N-glycosylation site. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the rhGAA possesses a bis-M6P unit at the fourth potential N-glycosylation site. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20% or 25% of the rhGAA is sialylated at the fourth potential N-glycosylation site. In some embodiments, the rhGAA contains an average of about 1.4 moles of M6P (mono-M6P and bis-M6P) per mole of rhGAA at the fourth potential N-glycosylation site. In some embodiments, the rhGAA contains an average of about 0.4 to about 0.6 moles of bis-M6P per mole of rhGAA at the fourth potential N-glycosylation site. In some embodiments, the rhGAA contains an average of about 0.3 to about 0.4 moles of mono-M6P per mole of rhGAA at the fourth potential N-glycosylation site. In at least one embodiment, the rhGAA has a fourth potential N-glycosylation site occupancy as shown in FIG. 6A and an N-glycosylation profile as shown in FIG. 6E. In at least one embodiment, rhGAA comprises the fourth potential N-glycosylation site occupancy shown in Figure 32A and the N-glycosylation profile shown in Figure 32E or Figure 33B.

[0082] In some embodiments, at least 5% of rhGAA is phosphorylated at the fifth potential N-glycosylation site (e.g., N596 for SEQ ID NO:5 and N692 for SEQ ID NO:1). In other embodiments, less than 5%, 10%, 15%, 20% or 25% of rhGAA is phosphorylated at the fifth potential N-glycosylation site. For example, the fifth potential N-glycosylation site may have fucosylated biantennary complex N-glycans as the predominant molecular species. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of rhGAA is sialylated at the fifth potential N-glycosylation site. In some embodiments, rhGAA comprises an average of about 0.8 to about 0.9 moles of sialic acid per mole of rhGAA at the fifth potential N-glycosylation site. In at least one embodiment, rhGAA has a fifth potential N-glycosylation site occupancy as shown in Figure 6A and an N-glycosylation profile as shown in Figure 6F. In at least one embodiment, rhGAA has a fifth potential N-glycosylation site occupancy as shown in Figure 32A and an N-glycosylation profile as shown in Figure 32F or Figure 33B.

[0083] In some embodiments, at least 5% of rhGAA is phosphorylated at the sixth N-glycosylation site (e.g., N826 for SEQ ID NO:5 and N882 for SEQ ID NO:1). In other embodiments, less than 5%, 10%, 15%, 20% or 25% of rhGAA is phosphorylated at the sixth N-glycosylation site. For example, the sixth N-glycosylation site may have a mixture of bi-, tri- and tetra-antennary complex N-glycans as the predominant molecular species. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of rhGAA is sialylated at the sixth N-glycosylation site. In some embodiments, rhGAA comprises an average of about 1.5 to about 4.2 moles of sialic acid per mole of rhGAA at the sixth potential N-glycosylation site. In some embodiments, rhGAA comprises an average of about 0.9 moles of acetylated sialic acid per mole of rhGAA at the sixth potential N-glycosylation site. In at least one embodiment, rhGAA has a sixth potential N-glycosylation site occupancy as shown in Figure 6A and an N-glycosylation profile as shown in Figure 6G. In at least one embodiment, rhGAA has a sixth potential N-glycosylation site occupancy as shown in Figure 32A and an N-glycosylation profile as shown in Figure 32G or Figure 33B.

[0084] In some embodiments, at least 5% of rhGAA is phosphorylated at the seventh potential N-glycosylation site (e.g., N869 for SEQ ID NO:5 and N925 for SEQ ID NO:1). In other embodiments, less than 5%, 10%, 15%, 20% or 25% of rhGAA is phosphorylated at the seventh potential N-glycosylation site. In some embodiments, less than 40%, 45%, 50%, 55%, 60% or 65% of rhGAA has any N-glycan at the seventh potential N-glycosylation site. In some embodiments, at least 30%, 35% or 40% of rhGAA has an N-glycan at the seventh potential N-glycosylation site. In some embodiments, rhGAA contains an average of about 0.86 moles of sialic acid per mole of rhGAA at the seventh potential N-glycosylation site. In at least one embodiment, all N-glycans identified at the seventh potential N-glycosylation site are complex N-glycans. In at least one embodiment, rhGAA has a seventh potential N-glycosylation site occupancy as shown in Figure 6A or as shown in Figure 32A, and an N-glycosylation profile as shown in Figure 32H or as shown in Figure 33B.

[0085] In some embodiments, rhGAA contains an average of 3-4 M6P residues per rhGAA molecule and about 4 to about 7.3 moles of sialic acid per mole of rhGAA molecule. In some embodiments, the rhGAA is provided with an average of at least about 0.5 moles of bis-M6P per mole rhGAA at a first potential N-glycosylation site, about 0.4 to about 0.6 moles of mono-M6P per mole rhGAA at a second potential N-glycosylation site, about 0.9 to about 1.2 moles of sialic acid per mole rhGAA at a third potential N-glycosylation site, about 0.4 to about 0.6 moles of bis-M6P per mole rhGAA at a fourth potential N-glycosylation site, about 0.3 to about 0.4 moles of mono-M6P per mole rhGAA at a fourth potential N-glycosylation site, about 0.8 to about 0.9 moles of sialic acid per mole rhGAA at a fifth potential N-glycosylation site, and about 0.8 to about 0.9 moles of sialic acid per mole rhGAA at a sixth potential N-glycosylation site. rhGAA further comprises about 1.5 to about 4.2 moles of sialic acid per mole. In at least one embodiment, rhGAA further comprises an average of about 0.86 moles of sialic acid per mole of rhGAA at the seventh potential N-glycosylation site. In at least one embodiment, rhGAA comprises seven potential N-glycosylation sites with the occupancy and N-glycosylation profile shown in Figures 6A-6H. In at least one embodiment, rhGAA comprises seven potential N-glycosylation sites with the occupancy and N-glycosylation profile shown in Figures 32A-32H and Figures 33A-33B.

[0086] Methods for making rhGAA are disclosed in U.S. Provisional Patent Application No. 62 / 057,842, filed Sep. 30, 2014, the entire contents of which are incorporated herein by reference.

[0087] Once inside the lysosome, rhGAA can enzymatically degrade accumulated glycogen. However, conventional rhGAA products have low total levels of mono-M6P- and bis-M6P-bearing N-glycans, which makes it difficult to target muscle cells and poorly deliver rhGAA to lysosomes. Many of the rhGAA molecules in these conventional products do not have phosphorylated N-glycans, which makes them lack affinity for CIMPR. Non-phosphorylated high mannose N-glycans can also be cleared by the mannose receptor, which results in non-productive clearance of ERT (Figure 2B). In contrast, the rhGAA described herein, as shown in Figure 2A, can contain high amounts of mono-M6P- and bis-M6P-bearing N-glycans, resulting in productive uptake of rhGAA into certain tissues, such as muscle.

[0088] IV. Production and Purification of N-Linked Glycosylated rhGAA As described in International Application No. PCT / US2015 / 053252, the entirety of which is incorporated herein by reference, cells such as Chinese Hamster Ovary (CHO) cells can be used to produce rhGAA as described herein. Expressing high M6P rhGAA in CHO cells is advantageous for post-translationally modifying the glycan profile of rhGAA, at least in part because only the former can be converted by glycan degradation to a form of rhGAA with optimal glycogen hydrolysis, thereby enhancing therapeutic efficacy.

[0089] In some embodiments, rhGAA is preferably produced from one or more CHO cell lines transformed with the DNA construct encoding rhGAA described herein. Such CHO cell lines may contain multiple copies of the gene, such as 5, 10, 15, or 20 or more copies of the polynucleotide encoding GAA. DNA constructs expressing allelic variants of acid alpha glucosidase or other variant acid alpha glucosidase amino acid sequences, such as those at least 90%, 95%, 98% or 99% identical to SEQ ID NO:1 or SEQ ID NO:5, can be constructed and expressed in CHO cells. Those skilled in the art can select suitable alternative vectors for transforming CHO cells to produce such DNA constructs.

[0090] Methods for producing such CHO cell lines are described in International Application No. PCT / US2015 / 053252, the entirety of which is incorporated herein by reference. Briefly, these methods include transforming CHO cells with DNA encoding GAA or a GAA variant, selecting CHO cells that have stably integrated DNA encoding GAA into their chromosome(s) and stably express GAA, and selecting CHO cells that express GAA with a high content of N-glycans carrying mono-M6P or bis-M6P, and optionally selecting CHO cells with N-glycans with a high sialic acid content and / or with N-glycans containing a low content of non-phosphorylated high mannose. The selected CHO cell lines can be used to produce rhGAA and rhGAA compositions by culturing the CHO cell lines and recovering the compositions from the culture of the CHO cells. In some embodiments, the rhGAA produced from the selected CHO cell line contains a high content of N-glycans carrying mono-M6P or bis-M6P targeting CIMPR. In some embodiments, the rhGAA produced as described herein has low levels of complex N-glycans with terminal galactose. In some embodiments, the selected CHO cell line is referred to as GA-ATB200 or ATB200-X5-14. In some embodiments, the selected CHO cell line includes a subculture or derivative of such a CHO cell culture. In some embodiments, the rhGAA produced from the selected CHO cell line is referred to as ATB200.

[0091] rhGAA produced as described herein can be purified according to the methods described in the following International Application No. PCT / US2017 / 024981 and U.S. Provisional Patent Application No. 62 / 506,569, both of which are incorporated by reference in their entireties. An exemplary process for producing, capturing and purifying rhGAA produced from a CHO cell line is shown in FIG.

[0092] Briefly, bioreactor 601 contains a culture of cells, such as CHO cells, that express and secrete rhGAA into the surrounding liquid culture medium. Bioreactor 601 may be any suitable bioreactor for culturing cells, such as a perfusion, batch, or fed-batch bioreactor. Culture medium is removed from the bioreactor after a sufficient period for the cells to produce rhGAA. Such medium removal may be continuous for a perfusion bioreactor or batch-by-batch for a batch or fed-batch bioreactor. The medium may be filtered by a filtration system 603 to remove the cells. Filtration system 603 may be any suitable filtration system, including an alternating tangential flow filtration (ATF) system, a tangential flow filtration (TFF) system, and / or a centrifugal filtration system. In various embodiments, the filtration system utilizes filters having a pore size between about 10 nanometers and about 2 micrometers.

[0093] After filtration, the filtrate is loaded into a protein capture system 605. The protein capture system 605 may include one or more chromatography columns. If more than one chromatography column is used, then the columns may be arranged in series, whereby as the first column is loaded the next column begins to load. Alternatively, the media removal process may be stopped for the time that the columns are switched.

[0094] In various embodiments, protein capture system 605 includes one or more anion exchange (AEX) columns for direct product capture of rhGAA, particularly rhGAA with high M6P content. The rhGAA captured by protein capture system 605 is eluted from the column(s) by changing the pH and / or salt content in the column. Exemplary conditions for AEX columns are provided in Table 2. TIFF2025016455000006.tif124170

[0095] The eluted rhGAA may be subjected to further purification and / or quality assurance steps. For example, the eluted rhGAA may be subjected to a virus kill step 607. Such virus kill 607 may include one or more of low pH kill, detergent kill or other techniques known in the art. The rhGAA from the virus kill step 607 may be introduced into a second chromatography system 609 for further purification of the rhGAA product. Alternatively, the rhGAA eluted from the protein capture system 605 may be fed directly to the second chromatography system 609. In various embodiments, the second chromatography system 609 includes one or more immobilized metal affinity chromatography (IMAC) columns for further removal of impurities. Exemplary conditions for the IMAC columns are provided in Table 3 below. TIFF2025016455000007.tif207170

[0096] After the rhGAA is loaded onto the second chromatography system 609, the recombinant protein is eluted from the column(s). The eluted rhGAA may be subjected to a virus kill step 611. Similar to virus kill 607, virus kill 611 may include one or more of low pH kill, detergent kill or other techniques known in the art. In some embodiments, only one of virus kill 607 or 611 is used, or the virus kill is performed at the same stage of the purification process.

[0097] The rhGAA from the virus killing step 611 may be introduced into a third chromatography system 613 for further purification of the recombinant protein product. Alternatively, the recombinant protein eluted from the second chromatography system 609 may be fed directly to the third chromatography system 613. In various embodiments, the third chromatography system 613 includes one or more cation exchange chromatography (CEX) and / or size exclusion chromatography (SEC) columns for further removal of impurities. The rhGAA product is then eluted from the third chromatography system 613. Exemplary conditions for the CEX columns are provided below in Table 4. TIFF2025016455000008.tif111170

[0098] The rhGAA product may be subjected to further processing. For example, another filtration system 615 may be used to remove viruses. In some embodiments, such filtration may utilize a filter having a pore size between 5 and 50 μm. Other product processing may include a product conditioning step 617, where the recombinant protein product may be sterilized, filtered, concentrated, stored, and / or have additional components for addition to a final product formulation.

[0099] As used herein, the term "ATB200" refers to rhGAA with a high content of N-glycans bearing mono-M6P and bis-M6P, produced from the GA-ATB200 cell line and purified using the methods described herein.

[0100] V. Pharmaceutical Compositions In various embodiments, pharmaceutical compositions are provided that include rhGAA as described herein, alone or in combination with other therapeutic agents and / or a pharma- ceutically acceptable carrier.

[0101] In one or more embodiments, the pharmaceutical compositions described herein include pharma- ceutically acceptable salts.

[0102] In some embodiments, the pharma- ceutically acceptable salts used herein are pharma- ceutically acceptable acid addition salts. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, nitric acid, phosphoric acid, and the like, and include, but are not limited to, acetic acid, trifluoroacetic acid, adipic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, butyric acid, camphorsulfonic acid, cinnamic acid, citric acid, digluconic acid, ethanesulfonic acid, glutamic acid, glycolic acid, glycerophosphoric acid, hemisulfic acid, sulphur ... acid), hexanoic acid, formic acid, fumaric acid, 2-hydroxyethanesulfonic acid (isethionic acid), lactic acid, hydroxymaleic acid, malic acid, malonic acid, mandelic acid, mesitylenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, nicotinic acid, 2-naphthalenesulfonic acid, oxalic acid, pamoic acid, pectinic acid, phenylacetic acid, 3-phenylpropionic acid, pivalic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, p-toluenesulfonic acid, and undecanoic acid.

[0103] In some embodiments, the pharma- ceutically acceptable salt used herein is a pharma- ceutically acceptable base addition salt. Pharmaceutically acceptable base addition salts include, but are not limited to, hydroxides, carbonates or bicarbonates of ammonia or metal cations such as ammonium or sodium, potassium, lithium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, etc. Salts derived from pharma- ceutically acceptable organic non-toxic bases include, but are not limited to, primary, secondary and tertiary amines, quaternary amine compounds, substituted amines including naturally occurring substituted amines, cyclic amines, and methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, isopropylamine, tripropylamine, tributylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, hydrabamine, ketone ... Examples of such salts include basic ion exchange resins such as amines, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, tetramethylammonium compounds, tetraethylammonium compounds, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine, and N,N'-dibenzylethylenediamine, and polyamine resins.

[0104] In some embodiments, rhGAA or a pharma- ceutically acceptable salt thereof may be formulated as a pharmaceutical composition adapted for intravenous administration. In some embodiments, the pharmaceutical composition is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the injection site. The components of the pharmaceutical composition may be supplied as lyophilized powders or water-free concentrates in a hermetically sealed container, such as an ampoule or sachet indicating the amount of active agent, either separately or mixed together in a unit dosage form. If the composition is administered by infusion, it may be dispensed into an infusion bottle containing sterile pharmaceutical grade pure water, saline or dextrose / water. In some embodiments, the infusion may be administered in a hospital or clinic. In some embodiments, the infusion may be administered outside of a hospital or clinic setting, for example, at the subject's residence. If the composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the components can be mixed prior to administration.

[0105] In some embodiments, rhGAA or a pharma- ceutically acceptable salt thereof may be formulated for oral administration. Orally administrable compositions may be formulated for immediate, delayed, modified, sustained, pulsed or controlled release applications in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, gels, syrups, mouthwashes or dry powders for reconstitution with water or other suitable vehicles before use, optionally including flavors and colorants. Solid compositions such as tablets, capsules, lozenges, pastilles, pills, boluses, powders, pastes, granules, burettes, dragees or premixed preparations may also be used. Solid and liquid compositions for oral use may be prepared by methods well known in the art. Such compositions may also contain one or more pharma- ceutically acceptable carriers and excipients that may be in solid or liquid form. Tablets or capsules may be prepared by conventional means using pharma- ceutically acceptable excipients, including but not limited to binders, fillers, lubricants, disintegrants or wetting agents. Suitable pharma- ceutically acceptable excipients are well known in the art and include, but are not limited to, pregelatinized starch, polyvinylpyrrolidone, povidone, hydroxypropyl methylcellulose (HPMC), hydroxypropyl ethylcellulose (HPEC), hydroxypropyl cellulose (HPC), sucrose, gelatin, acacia, lactose, microcrystalline cellulose, calcium hydrogen phosphate, magnesium stearate, stearic acid, glyceryl behenate, talc, silica, corn, potato or tapioca starch, sodium starch glycolate, sodium lauryl sulfate, sodium citrate, calcium carbonate, calcium hydrogen phosphate, glycine croscarmellose sodium and complex silicates. Tablets may be coated by methods well known in the art.

[0106] In some embodiments, the pharmaceutical compositions described herein may be formulated according to International Application No. PCT / US2017 / 024982 and US Provisional Patent Application No. 62 / 506,574, both of which are incorporated herein by reference in their entirety. For example, in some embodiments, the pH of the pharmaceutical compositions described herein is about 5.0 to about 7.0 or about 5.0 to about 6.0. In some embodiments, the pH ranges from about 5.5 to about 6.0. In some embodiments, the pH of the pharmaceutical composition is 6.0. In some embodiments, the pH may be adjusted to a target pH by using a pH adjusting agent (e.g., an alkalizing agent and an acidifying agent), such as sodium hydroxide and / or hydrochloric acid.

[0107] The pharmaceutical compositions described herein may include a buffer system, such as a citrate-based, a phosphate-based, and combinations thereof. The citrate and / or phosphate may be sodium citrate or sodium phosphate. Other salts include potassium and ammonium salts. In one or more embodiments, the buffer includes a citrate. In further embodiments, the buffer includes sodium citrate (e.g., a mixture of anhydrous sodium citrate and citric acid monohydrate). In one or more embodiments, the citrate-containing buffer solution may include sodium citrate and citric acid. In some embodiments, both citrate and phosphate buffers are present.

[0108] In some embodiments, the pharmaceutical compositions described herein include at least one excipient. An excipient can function as a tonicity agent, bulking agent, and / or stabilizer. A tonicity agent is an ingredient that helps ensure that the formulation has a similar or the same osmotic pressure as human blood. A bulking agent is an ingredient that adds volume to the formulation (e.g., lyophilized) and gives the cake the proper structure. A stabilizer is a compound that can prevent or minimize the formation of aggregates at the hydrophobic air-water interface. Some excipients can simultaneously function as a tonicity agent and bulking agent. For example, mannitol can function as both a tonicity agent and provide the benefit of a bulking agent.

[0109] Examples of tonicity agents include sodium chloride, mannitol, sucrose and trehalose. In some embodiments, the tonicity agent comprises mannitol. In some embodiments, the total amount of tonicity agent(s) is in the range of about 10 mg / mL to about 50 mg / mL. In further embodiments, the total amount of tonicity agent(s) is in the range of about 10, 11, 12, 13, 14 or 15 mg / mL to about 16, 20, 25, 30, 35, 40, 45 or 50 mg / mL.

[0110] In some embodiments, the excipient comprises a stabilizer. In some embodiments, the stabilizer is a surfactant. In some embodiments, the stabilizer is polysorbate 80. In one or more embodiments, the total amount of stabilizer ranges from about 0.1 mg / mL to about 1.0 mg / mL. In further embodiments, the total amount of stabilizer ranges from about 0.1, 0.2, 0.3, 0.4, or 0.5 mg / mL to about 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mg / mL. In yet further embodiments, the total amount of stabilizer is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mg / mL.

[0111] In some embodiments, the pharmaceutical composition comprises (a) rhGAA (such as ATB200), (b) at least one buffer selected from the group consisting of citrate, phosphate, and combinations thereof, and (c) at least one excipient selected from the group consisting of mannitol, polysorbate 80, and combinations thereof, and has a pH of (i) about 5.0 to about 6.0 or (ii) about 5.0 to about 7.0. In some embodiments, the composition further comprises water. In some embodiments, the composition may further comprise an acidifier and / or an alkalizer.

[0112] In some embodiments, the pharmaceutical composition comprises (a) rhGAA (such as ATB200) at a concentration of about 5-50 mg / mL, about 5-30 mg / mL, or about 15 mg / mL, (b) sodium citrate buffer at a concentration of about 10-100 mM or about 25 mM, (c) mannitol at a concentration of about 10-50 mg / mL or about 20 mg / mL, (d) polysorbate 80 present at a concentration of about 0.1-1 mg / mL, about 0.2-0.5 mg / mL, or about 0.5 mg / mL, and (e) water, and has a pH of about 6.0. In at least one embodiment, the pharmaceutical composition comprises (a) 15 mg / mL rhGAA (such as ATB200), (b) 25 mM sodium citrate buffer, (c) 20 mg / mL mannitol, (d) 0.5 mg / mL polysorbate 80, and (e) water, and has a pH of about 6.0. In some embodiments, the composition may further comprise an acidifying agent and / or an alkalizing agent.

[0113] In some embodiments, a pharmaceutical composition comprising rhGAA is diluted prior to administration to a subject in need thereof.

[0114] In some embodiments, the pharmaceutical compositions described herein comprise a chaperone. In some embodiments, the chaperone is miglustat or a pharma- ceutically acceptable salt thereof. In another embodiment, the chaperone is duvoglustat or a pharma- ceutically acceptable salt thereof.

[0115] In some embodiments, the rhGAA described herein is formulated in one pharmaceutical composition, while the chaperone, such as miglustat, is formulated in another pharmaceutical composition. In some embodiments, the pharmaceutical composition comprising miglustat is based on the formulation commercially available as Zavesca® (Actelion Pharmaceuticals).

[0116] In some embodiments, the pharmaceutical compositions described herein may undergo a lyophilization (freeze-drying) process to provide a cake or powder. Accordingly, another aspect of the present invention relates to the pharmaceutical composition after lyophilization. The lyophilization mixture may include rhGAA (e.g., ATB200) described herein, a buffer selected from the group consisting of citrate, phosphate, and combinations thereof, and at least one excipient selected from the group consisting of trehalose, mannitol, polysorbate 80, and combinations thereof. In some embodiments, other components (e.g., other excipients) may be added to the lyophilization mixture. The pharmaceutical composition comprising the lyophilized formulation may be provided in a vial that can be stored, shipped, reconstituted, and / or administered to a patient.

[0117] VI. Treatment Method A. Treatment of Disease Another aspect of the present invention relates to a method for treating a disease or disorder related to glycogen storage dysregulation by administering rhGAA or a pharmaceutical composition as described herein. In some embodiments, the disease is Pompe's disease (also known as acid maltase deficiency (AMD) and glycogen storage disease type II (GSD II)). In some embodiments, the rhGAA is ATB200. In some embodiments, the pharmaceutical composition comprises ATB200.

[0118] The rhGAA or pharmaceutical composition described herein is administered by suitable route. In one embodiment, the rhGAA or pharmaceutical composition is administered intravenously. In other embodiments, the rhGAA or pharmaceutical composition is administered directly to target tissue, such as the heart or skeletal muscle (e.g., intramuscularly) or the nervous system (e.g., directly into the brain; intraventricular; intrathecal injection). In some embodiments, the rhGAA or pharmaceutical composition is administered orally. If necessary, more than one route can be used simultaneously.

[0119] In some embodiments, the therapeutic effect of rhGAA or pharmaceutical compositions described herein may be assessed based on one or more of the following criteria: (1) cardiac status (e.g., increase in end-diastolic and / or end-systolic volumes or reduction, amelioration or prevention of progressive cardiac myopathy typically seen in GSD-II); (2) pulmonary function (e.g., increase in crying vital capacity over baseline capacity and / or normalization of oxygen saturation during crying); (3) neurodevelopment and / or motor skills (e.g., increase in AIMS score); and (4) reduction in glycogen levels in tissues of disease-affected individuals.

[0120] In some embodiments, the subject's cardiac condition is improved by 10%, 20%, 30%, 40% or 50% (or any percentage in between) after administration of one or more dosages of rhGAA or pharmaceutical compositions described herein, compared to that of a subject treated with a vehicle or prior to treatment. The subject's cardiac condition can be evaluated by measuring end-diastolic and / or end-systolic volume and / or clinically evaluating cardiac myopathy. In some embodiments, the subject's pulmonary function is improved by 10%, 20%, 30%, 40% or 50% (or any percentage in between) after administration of one or more dosages of ATB200 or pharmaceutical compositions comprising ATB200, compared to that of a subject treated with a vehicle or prior to treatment. In certain embodiments, the improvement is achieved after 1 week, 2 weeks, 3 weeks, 1 month, 2 months or more (or any period in between) of administration. In certain embodiments, ATB200 or a pharmaceutical composition comprising ATB200 improves a subject's lung function after 1 week, 2 weeks, 3 weeks, 1 month, 2 months or more (or any period in between) of administration.

[0121] In some embodiments, the pulmonary function of the subject is improved by 10%, 20%, 30%, 40% or 50% (or any percentage between) after administration of one or more dosages of rhGAA or pharmaceutical compositions described herein, compared to that of the subject treated with vehicle or prior to treatment. The pulmonary function of the subject can be assessed by normalization of crying vital capacity above baseline capacity and / or oxygen saturation during crying. In some embodiments, the pulmonary function of the subject is improved by 10%, 20%, 30%, 40% or 50% (or any percentage between) after administration of one or more dosages of ATB200 or pharmaceutical compositions comprising ATB200, compared to that of the subject treated with vehicle or prior to treatment. In certain embodiments, the improvement is achieved after 1 week, 2 weeks, 3 weeks, 1 month, 2 months or more (or any period between) of administration. In certain embodiments, ATB200 or a pharmaceutical composition comprising ATB200 improves a subject's lung function after 1 week, 2 weeks, 3 weeks, 1 month, 2 months or more (or any period in between) of administration.

[0122] In some embodiments, the subject's neurodevelopment and / or motor skills are improved by 10%, 20%, 30%, 40% or 50% (or any percentage therebetween) after administration of one or more dosages of rhGAA or pharmaceutical compositions described herein compared to those of subjects treated with vehicle or prior to treatment. The subject's neurodevelopment and / or motor skills can be assessed by determining the AIMS score. The AIMS is a 12-item fixed scale administered and scored by a clinician (see Rush JA Jr., Handbook of Psychiatric Measures, American Psychiatric Association, 2000, 166-168). Items 1-10 are graded on a 5-point fixed scale. Items 1-4 assess orofacial movements. Items 5-7 address limb and truncal dyskinesias. Items 8-10 address the overall severity as judged by the examiner, as well as the patient's awareness of and difficulties with movement. Items 11-12 are yes / no questions regarding problems with teeth and / or dentures (such problems may result in misdiagnosis of dyskinesia). In some embodiments, the subject's neurodevelopment and / or motor skills are improved by 10%, 20%, 30%, 40% or 50% (or any percentage in between) after administration of one or more dosages of ATB200 or a pharmaceutical composition comprising ATB200, compared to those of subjects treated with vehicle or those preceding treatment. In certain embodiments, the improvement is achieved after 1 week, 2 weeks, 3 weeks, 1 month, 2 months or more (or any period in between) after administration. In certain embodiments, ATB200 or a pharmaceutical composition comprising ATB200 improves the subject's neurodevelopment and / or motor skills after 1 week, 2 weeks, 3 weeks, 1 month, 2 months or more (or any period in between) after administration.

[0123] In some embodiments, the glycogen level of a certain tissue of a subject is reduced by 10%, 20%, 30%, 40% or 50% (or any percentage therebetween) after administration of one or more dosages of rhGAA or pharmaceutical compositions described herein, compared to that of a subject treated with vehicle or prior to treatment. In some embodiments, the tissue is muscle, such as quadriceps, triceps and gastrocnemius. Glycogen levels of tissue can be analyzed using methods well known in the art. Determination of glycogen levels is well known based on amyloglucosidase digestion and is described in publications such as: Amalfitano et al. (1999), "Systemic correction of the muscle disorder glycogen storage disease type II after hepatic targeting of a modified adenovirus vector encoding human acid-alphaglucosidase", Proc Natl Acad Sci USA, 96:8861-8866. In some embodiments, the glycogen level in the muscle of the subject is reduced by 10%, 20%, 30%, 40% or 50% (or any percentage in between) after administration of one or more dosages of ATB200 or pharmaceutical compositions comprising ATB200, compared to that of a subject treated with vehicle or that of a subject prior to treatment. In certain embodiments, the reduction is achieved after 1 week, 2 weeks, 3 weeks, 1 month, 2 months or more (or any period in between) of administration. In certain embodiments, ATB200 or pharmaceutical compositions comprising ATB200 reduce the glycogen level in the muscle of the subject after 1 week, 2 weeks, 3 weeks, 1 month, 2 months or more (or any period in between) of administration.

[0124] B. Biomarkers Biomarkers of glycogen accumulation in muscle fibers of subjects, such as urinary hexose tetrasaccharide (Hex4), can be used to evaluate and compare the therapeutic effect of enzyme replacement therapy in subjects with Pompe disease. In some embodiments, the therapeutic effect of rhGAA or a pharmaceutical composition comprising rhGAA on glycogen accumulation is evaluated by measuring the level of Hex4 in the subject.

[0125] Biomarkers of muscle damage or injury, such as creatine kinase (CK), alanine aminotransferase (ALT) and aspartate aminotransferase (AST), can be used to evaluate and compare the therapeutic effect of enzyme replacement therapy in subjects with Pompe disease. In some embodiments, the therapeutic effect of rhGAA or a pharmaceutical composition comprising rhGAA on muscle damage is evaluated by measuring the level of CK, ALT and / or AST in a subject. In at least one embodiment, the therapeutic effect of rhGAA or a pharmaceutical composition comprising rhGAA on muscle damage is evaluated by measuring the level of CK in a subject.

[0126] Biomarkers such as LAMP-1, LC3 and dysferlin can also be used to evaluate and compare the therapeutic effect of rhGAA or pharmaceutical compositions described herein. In Pompe disease, the lack of GAA to hydrolyze lysosomal glycogen results in the abnormal accumulation of large lysosomes filled with glycogen in some tissues (Raben et al., JBC 273:19086-19092, 1998). Studies in mouse models of Pompe disease have shown that enlarged lysosomes in skeletal muscle do not fully explain the reduced organic capacity, and the presence of large inclusions containing degraded myofibrils (i.e., autophagy buildup) contributes to impaired muscle function (Raben et al., Humanmol Genet 17:3897-3908, 2008). Reports also suggest that impaired autophagy flux is associated with poor treatment outcomes in Pompe patients (Nascimbeni et al., Neuropathology and Applied Neurobiology doi:10.1111 / nan.12214, 2015; Fukuda et al., Mol Ther 14:831-839, 2006). In addition, late-onset Pompe disease is commonly seen in unclassified limb-girdle muscular dystrophies (LGMDs), a group of genetically heterogeneous neuromuscular diseases that include more than 30 genetically defined subtypes of varying severity (Preisler et al., Mol Genet Metab 110:287-289, 2013). IHC examination revealed substantially elevated presence of dysferlin in the sarcoplasm of skeletal muscle fibers in Gaa KO mice.

[0127] Various well-known methods can be used to measure gene expression levels and / or protein levels of such biomarkers. For example, a sample from a subject treated with rhGAA or pharmaceutical compositions described herein can be obtained from tissue, specifically muscle biopsy, etc. In some embodiments, the sample is a muscle biopsy of the subject. In some embodiments, the muscle is selected from quadriceps, triceps and gastrocnemius. The sample obtained from the subject can be stained with one or more antibodies or other detection agents that detect such biomarkers, or can be identified and quantified by mass spectrometry. The sample can also or alternatively be processed to detect the presence of nucleic acid, such as mRNA, that encodes the biomarker, for example, via RT-qPCR.

[0128] In some embodiments, gene expression levels and / or protein levels of one or more biomarkers are measured in muscle biopsies obtained from individuals prior to and after treatment with rhGAA or pharmaceutical compositions described herein.In some embodiments, gene expression levels and / or protein levels of one or more biomarkers are measured in muscle biopsies obtained from individuals treated with vehicle.In some embodiments, gene expression levels and / or protein levels of one or more biomarkers are reduced by 10%, 20%, 30%, 40% or 50% (or any percentage between) after administration of one or more doses of rhGAA or pharmaceutical compositions described herein compared to those of subjects treated with vehicle or those of subjects prior to treatment. In some embodiments, the gene expression level and / or protein level of one or more biomarkers is reduced by 10%, 20%, 30%, 40% or 50% (or any percentage therebetween) after administration of one or more dosages of ATB200 or a pharmaceutical composition comprising ATB200, compared to that of a subject treated with vehicle or that of a subject preceding treatment. In certain embodiments, the reduction is achieved after 1 week, 2 weeks, 3 weeks, 1 month, 2 months or more (or any period therebetween) of administration. In certain embodiments, ATB200 or a pharmaceutical composition comprising ATB200 reduces the gene expression level and / or protein level of one or more biomarkers after 1 week, 2 weeks, 3 weeks, 1 month, 2 months or more (or any period therebetween) of administration.

[0129] C. rhGAA Dosage The pharmaceutical preparation or reconstituted composition is administered in a therapeutically effective amount (e.g., a dosage that, when administered at regular intervals, is sufficient to treat the disease, such as ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and / or reducing the severity or frequency of symptoms of the disease). The amount that is therapeutically effective in treating the disease may depend on the nature and extent of the disease's impact and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can be optionally used to help identify optimal dosage ranges. In at least one embodiment, the rhGAA or pharmaceutical composition comprising rhGAA described herein is administered at a dose of about 5 mg / kg to about 30 mg / kg, typically about 1 mg / kg to about 100 mg / kg, such as about 5 mg / kg to about 20 mg / kg. In at least one embodiment, the rhGAA or pharmaceutical composition described herein is administered at a dose of about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 50 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg. In some embodiments, the rhGAA is administered at a dose of 5 mg / kg, 10 mg / kg, 20 mg / kg, 50 mg / kg, 75 mg / kg, or 100 mg / kg. In at least one embodiment, the rhGAA or pharmaceutical composition is administered at a dose of about 20 mg / kg. In some embodiments, the rhGAA or pharmaceutical composition is administered simultaneously or sequentially with a pharmacological chaperone. In some embodiments, the pharmacological chaperone is miglustat. In at least one embodiment, miglustat is administered as an oral dose of about 260mg.The effective dose for a specific individual may vary (e.g., increase or decrease) over time, depending on the individual's needs.For example, during times of physical illness or stress, or when anti-acid alpha-glucosidase antibodies become present or increase, or when disease symptoms worsen, the amount may be increased.

[0130] In some embodiments, the therapeutically effective dose of the rhGAA or pharmaceutical composition described herein is lower than that of conventional rhGAA production. For example, if the therapeutically effective dose of conventional rhGAA production is 20 mg / kg, the dose of the rhGAA or pharmaceutical composition described herein required to produce the same or better therapeutic effect as that of conventional rhGAA production is lower than 20 mg / kg. The therapeutic effect can be evaluated based on one or more of the evaluation criteria discussed above (e.g., cardiac status, glycogen level, or biomarker expression). In some embodiments, the therapeutically effective dose of the rhGAA or pharmaceutical composition described herein is at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more lower than that of conventional rhGAA production.

[0131] In some embodiments, the therapeutic effects of rhGAA or pharmaceutical compositions described herein include improvement in motor function, improvement in muscle strength (upper body, lower body or total body), improvement in pulmonary function, reduced fatigue, reduced levels of at least one biomarker of muscle damage, reduced levels of at least one biomarker of glycogen accumulation, or combinations thereof. In some embodiments, the therapeutic effects of rhGAA or pharmaceutical compositions described herein include reversal of lysosomal pathology in muscle fibers, faster and / or more effective reduction of glycogen content in muscle fibers, increased 6-minute walk test distance, decreased timed up and go test time, decreased 4-step climb test time, decreased 10-meter walk test time, decreased walk-step-Gower-chair score, increased upper limb strength, improved shoulder adduction, improved shoulder abduction, improved elbow flexion, improved elbow extension, improved upper body strength, improved lower body strength, improved total body strength, improved upright (sitting) forced vital capacity, improved maximum expiratory pressure, improvement in maximum inspiratory pressure, decreased fatigue severity scale score, decreased urinary hexose tetrasaccharide levels, decreased creatine kinase levels, decreased alanine aminotransferase levels, decreased aspartate aminotransferase levels, or combinations thereof.

[0132] In some embodiments, the rhGAA or pharmaceutical composition described herein achieves the desired therapeutic effect faster than conventional rhGAA products when administered at the same dose. The therapeutic effect can be evaluated based on one or more of the evaluation criteria discussed above (e.g., cardiac status, glycogen level, or biomarker expression). For example, if a single dose of conventional rhGAA products reduces glycogen levels in the tissue of a treated individual by 10% in one week, the same degree of reduction can be achieved in less than one week when the same dose of rhGAA or pharmaceutical composition described herein is administered. In some embodiments, the rhGAA or pharmaceutical composition described herein can achieve the desired therapeutic effect at least about 1.25, 1.5, 1.75, 2.0, 3.0 or more faster than conventional rhGAA products when administered at the same dose.

[0133] In some embodiments, the therapeutically effective amount of rhGAA (or a composition or medicament comprising rhGAA) is administered more than once. In some embodiments, rhGAA or pharmaceutical compositions described herein are administered at regular intervals and continuously depending on the nature and extent of the disease's effects. As used herein, administration at "regular intervals" indicates that the therapeutically effective amount is administered periodically (as distinguished from a one-time dose). The intervals can be determined by standard clinical techniques. In certain embodiments, rhGAA is administered once every two months, once a month, once every two weeks, once a week, twice a week, or daily. In some embodiments, rhGAA is administered intravenously twice a week, weekly, or every other week. The administration interval for a single individual need not be a fixed interval and may vary over time according to the individual's needs. For example, the interval between administrations may be decreased during times of physical illness or stress, or when anti-rhGAA antibodies become present or increase, or when disease symptoms worsen.

[0134] In some embodiments, when used at the same dose, the rhGAA or pharmaceutical composition described herein may be administered less frequently than conventional rhGAA products, and still produce the same or better therapeutic effect as conventional rhGAA products. For example, when conventional rhGAA products are administered weekly at 20 mg / kg, the rhGAA or pharmaceutical composition described herein can produce the same or better therapeutic effect as conventional rhGAA products when administered at 20 mg / kg, even when the rhGAA or pharmaceutical composition is administered less frequently, for example, once every two weeks or monthly. The therapeutic effect can be evaluated based on one or more of the evaluation criteria discussed above (e.g., cardiac status, glycogen level, or biomarker expression). In some embodiments, the interval between two doses of the rhGAA or pharmaceutical composition described herein is longer than that of conventional rhGAA products. In some embodiments, the interval between two doses of the rhGAA or pharmaceutical composition is at least about 1.25, 1.5, 1.75, 2.0, 3.0 times or more longer than that of conventional rhGAA products.

[0135] In some embodiments, under the same treatment conditions (e.g., the same dose administered at the same intervals), the rhGAA or pharmaceutical composition described herein provides a therapeutic effect to a greater extent than that provided by conventional rhGAA production. The therapeutic effect can be evaluated based on one or more of the evaluation criteria discussed above (e.g., cardiac status, glycogen level, or biomarker expression). For example, compared with conventional rhGAA production administered at 20 mg / kg weekly, rhGAA or pharmaceutical composition administered at 20 mg / kg weekly can reduce glycogen levels in tissues of treated individuals to a greater extent. In some embodiments, when administered under the same treatment conditions, the rhGAA or pharmaceutical composition described herein provides a therapeutic effect that is at least about 1.25, 1.5, 1.75, 2.0, 3.0 times or more greater than that of conventional rhGAA production.

[0136] D. Combination Therapy In one or more embodiments, the rhGAA or pharmaceutical composition comprising rhGAA described herein is administered simultaneously or sequentially with a pharmacological chaperone. In some embodiments, the rhGAA or pharmaceutical composition is administered via a route different from that of the pharmacological chaperone. For example, the pharmacological chaperone is administered orally, while the rhGAA or pharmaceutical composition is administered intravenously.

[0137] In various embodiments, the pharmacological chaperone is miglustat. In some embodiments, miglustat is administered at an oral dose of about 50 mg to about 600 mg. In at least one embodiment, miglustat is administered at an oral dose of about 200 mg to about 600 mg, or at an oral dose of about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg or about 600 mg. In at least one embodiment, miglustat is administered at an oral dose of about 233 mg to about 500 mg. In at least one embodiment, miglustat is administered at an oral dose of about 250 to about 270 mg, or at an oral dose of about 250 mg, about 255 mg, about 260 mg, about 265 mg or about 270 mg. In at least one embodiment, miglustat is administered in an oral dose of about 260 mg.

[0138] It is understood by those skilled in the art that an oral dose of miglustat in the range of about 200mg to 600mg or any smaller range thereof may be suitable for an adult patient depending on his / her body weight. For example, for patients with a body weight significantly lower than about 70kg, including but not limited to infants, children or low body weight adults, a smaller dose may be considered as appropriate by a physician. Thus, in at least one embodiment, miglustat is administered at an oral dose of about 50mg to about 200mg, or at an oral dose of about 50mg, about 75mg, about 100mg, 125mg, about 150mg, about 175mg or about 200mg. In at least one embodiment, miglustat is administered at an oral dose of about 65mg to about 195mg, or at an oral dose of about 65mg, about 130mg or about 195mg.

[0139] In some embodiments, rhGAA is administered intravenously at a dose of about 5 mg / kg to about 20 mg / kg, and miglustat is administered orally at a dose of about 50 mg to about 600 mg. In some embodiments, rhGAA is administered intravenously at a dose of about 5 mg / kg to about 20 mg / kg, and miglustat is administered orally at a dose of about 50 mg to about 200 mg. In some embodiments, rhGAA is administered intravenously at a dose of about 5 mg / kg to about 20 mg / kg, and miglustat is administered orally at a dose of about 200 mg to about 600 mg. In some embodiments, rhGAA is administered intravenously at a dose of about 5 mg / kg to about 20 mg / kg, and miglustat is administered orally at a dose of about 233 mg to about 500 mg. In one embodiment, rhGAA is administered intravenously at a dose of about 20 mg / kg, and miglustat is administered orally at a dose of about 260 mg.

[0140] In some embodiments, miglustat and rhGAA are administered simultaneously.For example, miglustat can be administered within 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 minutes before or after the administration of rhGAA.In some embodiments, miglustat is administered within 5, 4, 3, 2 or 1 minutes before or after the administration of rhGAA.

[0141] In some embodiments, miglustat and rhGAA are administered sequentially. In at least one embodiment, miglustat is administered prior to administration of rhGAA. In at least one embodiment, miglustat is administered less than 3 hours prior to administration of rhGAA. In at least one embodiment, miglustat is administered about 2 hours prior to administration of rhGAA. For example, miglustat can be administered about 1.5 hours, about 1 hour, about 50 minutes, about 30 minutes, or about 20 minutes prior to administration of rhGAA. In at least one embodiment, miglustat is administered about 1 hour prior to administration of rhGAA.

[0142] In some embodiments, miglustat is administered after administration of rhGAA.In at least one embodiment, miglustat is administered within 3 hours after administration of rhGAA.In at least one embodiment, miglustat is administered within 2 hours after administration of rhGAA.For example, miglustat is administered within about 1.5 hours, about 1 hour, about 50 minutes, about 30 minutes or about 20 minutes after administration of rhGAA.

[0143] In some embodiments, the subject fasts for at least 2 hours before and at least 2 hours after administration of miglustat.

[0144] E.Kit Another aspect of the invention relates to a kit comprising the rhGAA or pharmaceutical composition described herein. In one or more embodiments, the kit comprises a container (e.g., a vial, tube, bag, etc.) that contains the rhGAA or pharmaceutical composition (either pre- or post-lyophilization) and instructions for reconstitution, dilution, and administration. EXAMPLES

[0145] Example 1: Preparation of CHO cells producing rhGAA with high content of mono- or bis-M6P-bearing N-glycans DG44 CHO (DHFR-) cells were transfected with a DNA construct expressing rhGAA. The DNA construct is shown in Figure 4. After transfection, CHO cells containing a stably integrated GAA gene were selected using hypoxanthine / thymidine deficient (-HT) medium. GAA expression in these cells was induced by methotrexate treatment (MTX, 500 nM).

[0146] Cell pools expressing high amounts of GAA were identified by GAA enzyme activity assay and used to establish individual clones producing rhGAA. Individual clones were generated on semi-solid medium plates, picked by 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. Conditioned medium for determining GAA activity used 4-MU-α-glucosidase substrate. Clones producing high levels of GAA as measured by GAA enzyme assay were further evaluated for viability, proliferation, GAA productivity, N-glycan structure and stable protein expression. CHO cell lines expressing rhGAA with enhanced mono-M6P or bis-M6P N-glycans, including the CHO cell line GA-ATB200, were isolated using this procedure.

[0147] Example 2: Purification of rhGAA Batches of rhGAA according to the present invention were produced in shake flasks and in a perfusion bioreactor using the CHO cell line GA-ATB200, and the product is referred to as "ATB200". Weak anion exchange ("WAX") liquid chromatography was used to fractionate ATB200 rhGAA by terminal phosphate and sialic acid. Elution profiles were generated by eluting ERT with increasing amounts of salt. Profiles were monitored by UV (A280 nm). Similar CIMPR receptor binding (at least about 70%) profiles were observed for purified ATB200 rhGAA from different production batches (Figure 5), indicating that ATB200 rhGAA can be produced sustainably.

[0148] Example 3: Oligosaccharide characterization of ATB200 rhGAA ATB200 rhGAA was analyzed for site-specific N-glycan profiles using a variety of LC-MS / MS analytical techniques. Results from the first two LC-MS / MS methods are shown in Figures 6A-6H. Results from the third LC-MS / MS method, along with 2-AA glycan mapping, are shown in Figures 32A-32H, Figures 33A-33B, and Table 5.

[0149] In the first LC-MS / MS analysis, proteins were denatured, reduced, alkylated and digested prior to LC-MS / MS analysis. During protein denaturation and reduction, 200 μg protein sample, 5 μL of 1 mol / L Tris-HCl (final concentration 50 mM), 75 μL of 8 mol / L guanidine HCl (final concentration 6 M), 1 μL of 0.5 mol / L EDTA (final concentration 5 mM), 2 μL of 1 mol / L DTT (final concentration 20 mM) and Milli-Q® water were added to a 1.5 mL tube to a total volume of 100 μL. The sample was mixed and incubated at 56 °C for 30 min in a dry bath. During alkylation, the denatured and reduced protein sample was mixed with 5 μL of 1 mol / L iodoacetamide (IAM, final concentration 50 mM) and then incubated at 10-30 °C in the dark for 30 min. After alkylation, 400 μL of pre-chilled acetone was added to the sample and the mixture was frozen at -80°C for 4 hours. The sample was then centrifuged at 13000 rpm, 4°C for 5 minutes and the supernatant was removed. 400 μL of pre-chilled acetone was added to the pellet and then centrifuged at 13000 rpm, 4°C for 5 minutes and the supernatant was removed. The sample was then air-dried on ice in the dark to remove acetone residues. 40 microliters of 8M urea and 160 μL of 100 mM NH4HCO3 were added to the sample to dissolve the protein. During trypsin digestion, 50 μg of protein was then added with trypsin digestion buffer to a final volume of 100 μL and 5 μL of 0.5 mg / mL trypsin (protein to enzyme ratio 20 / 1 w / w). The solution was mixed thoroughly and incubated overnight (16 ± 2 hours) at 37°C. The reaction was quenched by adding 2.5 microliters of 20% TFA (final concentration 0.5%). Samples were then analyzed using a Thermo Scientific™ Orbitrap Velos Pro™ Mass Spectrometer.

[0150] In a second LC-MS / MS analysis, ATB200 samples were prepared by similar denaturation, reduction, alkylation and digestion procedures, except that iodoacetic acid (IAA) was used as the alkylating agent instead of IAM, and then analyzed using a Thermo Scientific™ Orbitrap Fusion™ Lumos Tribid™ Mass Spectrometer.

[0151] The results of the first and second analyses are shown in Figures 6A-6H. In Figures 6A-6H, the results of the first analysis are represented by the left bars (dark grey) and the results from the second analysis are represented by the right bars (light grey). The nomenclature of symbols for glycan presentation follows Varki, A., Cummings, RD, Esko JD et al., Essentials of Glycobiology, 2nd ed. (2009).

[0152] As can be seen from Figures 6A-6H, the two analyses provided similar results with some variation between the results. This variation may be due to a number of factors including the equipment used and the completeness of the N-glycan analysis. For example, if some molecular species of phosphorylated N-glycans were not identified and / or quantified, the total number of phosphorylated N-glycans may be underestimated and the percentage of rhGAA bearing phosphorylated N-glycans at that site may be underestimated. As another example, if some molecular species of non-phosphorylated N-glycans were not identified and / or quantified, the total number of non-phosphorylated N-glycans may be underestimated and the percentage of rhGAA bearing phosphorylated N-glycans at that site may be overestimated.

[0153] Figure 6A shows the N-glycosylation site occupancy of ATB200. As can be seen in Figure 6A, both analyses detect that near or above 90% and up to about 100% of the ATB200 enzyme has N-glycans detected at each potential N-glycosylation site, with the first, second, third, fourth, fifth and sixth N-glycosylation sites being mostly occupied. However, the seventh potential N-glycosylation site is N-glycosylated about half the time.

[0154] Figure 6B shows the N-glycosylation profile of the first potential N-glycosylation site N84. As seen in Figure 6B, the main N-glycan species is bis-M6P N-glycan. Both the first and second analyses detected that more than 75% of ATB200 has bis-M6P at the first site, corresponding to an average of about 0.8 mol bis-M6P per mol ATB200 at the first site.

[0155] Figure 6C shows the N-glycosylation profile of the second potential N-glycosylation site N177. As seen in Figure 6C, the main N-glycan molecular species are mono-M6P N-glycan and non-phosphorylated high mannose N-glycan. Both the first and second analyses detected that more than 40% of ATB200 has mono-M6P at the second site, corresponding to an average of about 0.4 to about 0.6 mol mono-M6P per mol ATB200 at the second site.

[0156] Figure 6D shows the N-glycosylation profile of the third potential N-glycosylation site N334. As seen in Figure 6D, the main N-glycan molecular species are non-phosphorylated high mannose N-glycans, bi-, tri- and tetra-antennary complex N-glycans and hybrid N-glycans. Both the first and second analyses detected that more than 20% of ATB200 has sialic acid residues at the third site, corresponding to an average of about 0.9 to about 1.2 mol sialic acid per mol ATB200 at the third site.

[0157] FIG. 6E shows the N-glycosylation profile of the fourth potential N-glycosylation site N414. As seen in FIG. 6E, the main N-glycan species are bis-M6P and mono-M6P N-glycans. Both the first and second analyses detected that more than 40% of ATB200 has bis-M6P at the fourth site, corresponding to an average of about 0.4 to about 0.6 mol bis-M6P per mole of ATB200 at the fourth site. Both the first and second analyses also detected that more than 25% of ATB200 has mono-M6P at the fourth site, corresponding to an average of about 0.3 to about 0.4 mol mono-M6P per mole of ATB200 at the fourth site.

[0158] Figure 6F shows the N-glycosylation profile of the fifth potential N-glycosylation site N596. As seen in Figure 6F, the main N-glycan molecular species is a fucosylated biantennary complex N-glycan. Both the first and second analyses detected that more than 70% of ATB200 has a sialic acid residue at the fifth site, corresponding to an average of about 0.8 to about 0.9 mol sialic acid per mol ATB200 at the fifth site.

[0159] Figure 6G shows the N-glycosylation profile of the sixth potential N-glycosylation site N826. As seen in Figure 6G, the main N-glycan molecular species are bi-, tri- and tetra-antennary complex N-glycans. Both the first and second analyses detected that more than 80% of ATB200 has sialic acid residues at the sixth site, corresponding to an average of about 1.5 to about 1.8 mol sialic acid per mol ATB200 at the sixth site.

[0160] Analysis of N-glycosylation at the seventh site, N869, showed approximately 40% N-glycosylation, with the most common N-glycans being A4S3S3GF (12%), A5S3G2F (10%), A4S2G2F (8%) and A6S3G3F (8%).

[0161] Figure 6H shows a summary of phosphorylation at each of the seven potential N-glycosylation sites. As can be seen in Figure 6H, both the first and second analyses detected high phosphorylation levels at the first, second and fourth potential N-glycosylation sites. Both analyses detected that more than 80% of ATB200 was mono- or bis-phosphorylated at the first site, more than 40% of ATB200 was mono-phosphorylated at the second site, and more than 80% of ATB200 was mono- or bis-phosphorylated at the fourth site.

[0162] Another N-glycosylation analysis of ATB200 was performed by the LC-MS / MS method described below. This analysis resulted in an average N-glycosylation profile across 10 lots of ATB200 (Figures 32A-32H, Figures 33A-33B).

[0163] N-linked glycans from ATB200 were enzymatically released using PNGase-F and labeled with 2-anthranilic acid (2-AA). The 2-AA labeled N-glycans were further processed by solid phase extraction (SPE) to remove excess salts and other contaminants. The purified 2-AA N-glycans were dissolved in acetonitrile / water (20 / 80; v / v) and 10 micrograms were loaded onto an aminopolymer analytical column (apHera™, Supelco) for high performance liquid chromatography with fluorescence detection (HPLC-FLD) and high resolution mass spectrometry (HRMS) analysis.

[0164] Liquid chromatography (LC) separation was performed under normal phase conditions in gradient elution mode with mobile phase A (2% acetic acid in acetonitrile) and mobile phase B (5% acetic acid in water; 20 mM ammonium acetate, adjusted to pH 4.3 with ammonium hydroxide). The starting mobile phase composition was 70%A / 30%B. For fluorescence detection, the parameters for the detector (RF-20Axs, Shimadzu) were excitation (Ex): 320 nm; emission (Em): 420 nm. HRMS analysis was performed using a Quadrupole Time of Flight mass spectrometer (Sciex X500B QTOF) operated in Independent Data Acquisition (IDA) mode. The acquired data files were converted to mzML files using MSConvert from ProteoWizard and then GRITS Toolbox 1.2 Morning Blend software (UGA) was utilized for glycan database searching and subsequent annotation of the identified N-glycans. N-glycans were identified using both precursor monoisotopic mass (m / z) and product ion m / z. Experimental product ions and fragmentation patterns were confirmed in silico using the GlycoWorkbench 2 application.

[0165] To determine the relative amounts of N-linked glycans from ATB200, the data obtained from the HPLC-FLD-QTOF MS / MS experiment was processed as follows: all N-glycan peaks in the FLD chromatogram were integrated and each peak was assigned a percentage of the total area of ​​all peaks in the FLD chromatogram. The fluorescence signal, expressed as the peak area, is a quantitative measurement of the amount of each N-glycan in the sample (Figure 33A). However, in most cases multiple N-glycan species were contained in the same FLD peak. Therefore, mass spectrometer data was also required to obtain relative quantification of each N-glycan species (Table 5). The ion intensity signal for each N-glycan was "extracted" from the data to create a chromatographic peak called an extracted ion chromatogram (XIC). The XIC was aligned with the FLD chromatographic peak and was specific to only one N-glycan species. The XIC peak created from the ion intensity signal was then integrated and the peak area is a relative quantitative measurement of the amount of glycan present. Both FLD peak areas and mass spectrometer XIC peak areas were used to allow relative quantification of all N-linked glycan molecular species of ATB200 reported herein.

[0166] The results of this LC-MS / MS analysis are provided below in Table 5. The symbol nomenclature for glycan presentation follows: Wopereis W et al., 2006.Abnormal glycosylation with hypersialylated O-glycans in patients with Sialuria.Biochimica et Biophysica Acta.1762:598~607;Gornik O et al., 2007.Changes of serum glycans during sepsis and acute pancreatitis.Glycobiology.17:1321~1332 https: / / doi.org / 10.1093 / glycob / cwm106;Kattla JJ et al., 2011.Biologic protein glycosylation.In: Murray Moo-Young (ed.), Comprehensive Biotechnology, 2nd edition, 3:467~486;Tharmalingam-Jaikaran T et al., N-glycan profiling of bovine follicular fluid at key dominant follicle development stages.2014.Reproduction.148:569~580;Clerc F et al., Human plasma protein N-glycosylation.2015.Glycoconj J.DOI 10.1007 / s10719-015-9626-2; and Blackler RJ et al., 2016.Single-chain antibody-fragment M6P-1 possesses a mannose 6-phosphate monosaccharide-specific binding pocket that distinguishes N-glycan phosphorylation in a branch-specific manner.Glycobiology.26~2:181~192. TIFF2025016455000009.tif185170

[0167] Based on this 2-AA and LC-MS / MS analysis, and as further summarized in Figure 33C, the ATB200 examined has an average M6P content (for both mono-M6P and bis-M6P) of 3-5 mol per mol of ATB200 and a sialic acid content of 4-7 mol per mol of ATB200.

[0168] As shown in Figures 32A-32H and summarized in Figure 33B, the first potential N-glycosylation site of ATB200 has an average M6P content of about 1.4 mol M6P / mol ATB200, and is responsible for the average mono-M6P content of about 0.25 mol mono-M6P / mol ATB200 and the average bis-M6P content of about 0.56 mol bis-M6P / mol ATB200; the second potential N-glycosylation site of ATB200 has an average M6P content of about 0.5 mol M6P / mol ATB200, with the predominant phosphorylated N-glycan molecular species being mono-M6P N-glycan; the third potential N-glycosylation site of ATB200 has an average sialic acid content of about 1 mol sialic acid / mol ATB200; the fourth potential N-glycosylation site of ATB200 has an average sialic acid content of about 1.4 mol M6P / mol ATB200 has an average M6P content of about 0.35 mol mono-M6P / mol ATB200 and an average bis-M6P content of about 0.52 mol bis-M6P / mol ATB200; the fifth potential N-glycosylation site of ATB200 has an average sialic acid content of about 0.86 mol sialic acid / mol ATB200; the sixth potential N-glycosylation site of ATB200 has an average sialic acid content of about 4.2 mol sialic acid / mol ATB200; and the seventh potential N-glycosylation site of ATB200 has an average sialic acid content of about 0.86 mol sialic acid / pol ATB200.

[0169] Similarly, the 2-AA and LC-MS / MS analytical techniques reveal that on average, about 65% of the N-glycans at the first potential N-glycosylation site of ATB200 are high mannose N-glycans, about 89% of the N-glycans at the second potential N-glycosylation site of ATB200 are high mannose N-glycans, the majority of the N-glycans at the third potential N-glycosylation site of ATB200 are sialylated (approximately 20% are fully sialylated), about 85% of the N-glycans at the third potential N-glycosylation site of ATB200 are complex N-glycans, about 84% of the N-glycans at the fourth potential N-glycosylation site of ATB200 are high mannose N-glycans, and about 89% of the N-glycans at the fifth potential N-glycosylation site of ATB200 are complex N-glycans. About 70% of the N-glycans at the 5th potential N-glycosylation site of ATB200 are sialylated (about 26% are fully sialylated), about 100% of the N-glycans at the 5th potential N-glycosylation site of ATB200 are complex N-glycans, about 85% of the N-glycans at the 6th potential N-glycosylation site of ATB200 are sialylated (nearly 27% are fully sialylated), about 98% of the N-glycans at the 6th potential N-glycosylation site of ATB200 are complex N-glycans, about 87% of the N-glycans at the 7th potential N-glycosylation site of ATB200 are sialylated (nearly 8% are fully sialylated), and about 100% of the N-glycans at the 7th potential N-glycosylation site of ATB200 are complex N-glycans.

[0170] Example 4: Analytical Comparison of ATB200 with Myozyme® / Lumizyme® Purified ATB200 and Lumizyme® N-glycans were evaluated by MALDI-TOF to determine the individual N-glycan structures found in each ERT. Lumizyme® was obtained from a commercial source. As shown in FIG. 7, ATB200 showed four prominent peaks eluted to the right of Lumizyme®. This confirms that ATB200 was phosphorylated to a higher extent than Lumizyme®, as this evaluation is based on terminal charge rather than CIMPR affinity. As summarized in FIG. 8, ATB200 samples were found to contain less non-phosphorylated high mannose type N-glycans than Lumizyme®.

[0171] To assess the ability of conventional rhGAA in Myozyme® and Lumizyme® to interact with CIMPR, two conventional rhGAA preparations were injected onto a CIMPR affinity column (which binds rhGAA with M6P groups) and the flow-through was collected. Bound material was eluted with a free M6 gradient. Fractions were collected into 96-well plates and assayed for GAA activity with 4MU-α-glucosidase substrate. The relative amounts of unbound (flow-through) and bound (M6P elution) rhGAA were determined based on GAA activity and reported as a percentage of total enzyme. Figures 9A and 9B show the binding profiles of rhGAA in Myozyme® and Lumizyme®: 73% of rhGAA in Myozyme® (Figure 9B) and 78% of rhGAA in Lumizyme® (Figure 9A) did not bind to CIMPR. Indeed, only 27% of rhGAA in Myozyme® and 22% in Lumizyme® contained M6P, which may be productive for targeting it to CIMPR on muscle cells. In contrast, as shown in Figure 5, under the same conditions, more than 70% of rhGAA in ATB200 was found to bind to CIMPR.

[0172] In addition to having a higher percentage of rhGAA that can bind to CIMPR, it is important to understand the quality of the interaction. Lumizyme® and ATB200 receptor binding was determined using a CIMPR plate binding assay. Briefly, a CIMPR-coated plate was used to capture GAA. Various concentrations of rhGAA were applied to the immobilized receptor, and unbound rhGAA was washed off. The amount of remaining rhGAA was determined by GAA activity. As shown in Figure 10A, ATB200 bound to CIMPR significantly better than Lumizyme®. Figure 10B shows the relative content of bis-M6P N-glycan in Lumizyme® (conventional rhGAA production) and ATB200 according to the present invention. For Lumizyme®, on average, only 10% of the molecules have bis-phosphorylated N-glycan. In contrast, on average, all rhGAA molecules in ATB200 have at least one bis-phosphorylated N-glycan.

[0173] Overall, the higher content of M6P N-glycans in ATB200 than in Lumizyme® indicates that a greater proportion of rhGAA molecules in ATB200 are able to target muscle cells. As shown above, the higher percentage of mono- and bis-phosphorylated structures determined by MALDI is consistent with the CIMPR profile illustrating the significantly higher binding of ATB200 to the CIMPR receptor. N-glycan analysis via MALDI-TOF mass spectrometry confirmed that, on average, each ATB200 molecule contains at least one natural bis-M6P N-glycan structure. This higher bis-M6P N-glycan content in ATB200 directly correlates with higher affinity binding to CIMPR in the M6P receptor plate binding assay (K D approximately 2-4 nM).

[0174] The relative cellular uptake of ATB200 and Lumizyme® rhGAA was compared using normal and Pompe fibroblast cell lines. The comparison involved 5-100 nM ATB200 according to the invention and 10-500 nM of the conventional rhGAA producer Lumizyme®. After 16 hours of incubation, external rhGAA was inactivated with TRIS base and cells were washed three times with PBS before harvesting. Internalized GAA was measured by 4MU-α-glucoside hydrolysis and graphed relative to total intracellular protein, and the results are shown in Figures 11A-11C.

[0175] ATB200 has also been shown to be efficiently internalized by cells. As shown in Figures 11A-11B, ATB200 is internalized by both normal and Pompe fibroblasts and to a greater extent than the conventional rhGAA producer Lumizyme®. ATB200 saturates cell receptors at approximately 20 nM, while approximately 250 nM of Lumizyme® is required to saturate cell receptors. The uptake efficiency constant (K uptake ) is 2-3 nm for ATB200 and 56 nM for Lumizyme® as shown by Figure 11C. These results suggest that ATB200 is a well-targeted treatment for Pompe disease.

[0176] Example 5: ATB200 and Pharmacological Chaperones The stability of ATB200 in acidic or neutral pH buffers was evaluated in a thermal stability assay using SYPRO Orange, where the fluorescence of the dye increases when the protein is denatured. As shown in FIG. 12, compared to the stability of ATB200 at pH 5.2, a condition mimicking the acidic environment of the lysosome, the addition of AT2221 stabilized ATB200 at pH 7.4 in a concentration-dependent manner. As summarized in Table 6, the addition of AT2221 increased the melting temperature (T m ) to 10 o It rose to nearly C. TIFF2025016455000010.tif45170

[0177] Example 6: Simultaneous administration of ATB200 and AT2221 in Gaa KO mice The therapeutic effects of ATB200 and AT2221 were evaluated in Gaa KO mice and compared against alglucosidase alfa. Male Gaa KO (3 to 4 months old) and age-matched wild-type (WT) mice were used for the study. Alglucosidase alfa was administered via a bolus tail vein intravenous (IV) injection. In a co-administration regimen, AT2221 was administered via oral gavage (PO) 30 minutes prior to the IV injection of ATB200. Treatments were given once every two weeks. Treated mice were sacrificed 14 days after the last administration and various tissues were collected for further analysis. Table 7 summarizes the study design: TIFF2025016455000011.tif55170

[0178] Tissue glycogen content in tissue samples was determined using amyloglucosidase digestion as discussed above. As shown in Figure 13, the combination of 20 mg / kg ATB200 and 10 mg / kg AT2221 significantly reduced glycogen content compared to the same dosage of alglucosidase alfa in four different tissues (quadriceps, triceps, gastrocnemius and heart).

[0179] Tissue samples were also analyzed for biomarker changes according to the methods discussed in: Khanna R et al. (2012), "The pharmacological chaperone AT2220 increases recombinant human acid α-glucosidase uptake and glycogen reduction in a mouse model of Pompe disease", Plos One 7(7):e40776; and Khanna, R et al. (2014), "The Pharmacological Chaperone AT2220 Increases the Specific Activity and Lysosomal Delivery of Mutant Acid α-Glucosidase,and Promotes Glycogen Reduction in a Transgenic Mouse Model of Pompe Disease", PLoS ONE 9(7):e102092. As shown in FIG. 14, a significant increase and expansion of LAMP1-positive vesicles, an indicator of lysosomal proliferation, was seen in muscle fibers of Gaa KO animals compared to WT. Co-administration of ATB200 / AT2221 resulted in additional fibrils with normalized LAMP1 levels, while the remaining LAMP1-positive vesicles were also reduced in size (inset).

[0180] Similarly, significant LC3-positive aggregates in muscle fibers of untreated Gaa KO mice indicate the presence of autophagy zones and autophagy build-up. LC3-positive aggregates (red) were selectively reduced in mice treated with ATB200 / AT2221 co-administration compared to mice treated with alglucosidase alfa (Figure 15A). A similar observation was seen when the expression of LC3 was assessed using Western blot. As shown in Figure 15B, the majority of animals treated with ATB200 / AT2221 showed a marked decrease in the levels of LC3 II, a lipid adduct form associated with autophagosomes, suggesting improved autophagic flux. In contrast, the effect of alglucosidase alfa on autophagy was modest.

[0181] Dysferlin, a protein involved in membrane repair and whose deficiency / mistransportation is associated with multiple muscular dystrophies, was also evaluated. As shown in Figure 16, dysferlin (brown) was severely accumulated in the sarcoplasm of Gaa KO mice. Compared with alglucosidase alpha, ATB200 / AT2221 was able to transport dysferlin back to the sarcolemma of more muscle fibers.

[0182] These data are consistent with the improvements at the cellular level demonstrated in human Pompe disease patients treated with ATB200 and miglustat (e.g., patients show reduced levels of biomarkers of glycogen accumulation and muscle damage), resulting in not only effective treatment of Pompe disease, but also reversal of disease progression. Clinical data in human Pompe disease patients are summarized in Examples 8-13 below.

[0183] Example 7: Single fiber analysis As shown in FIG. 17, the majority of vehicle-treated mice showed significantly enlarged lysosomes (green) (see, e.g., “B”) and the presence of extensive autophagic buildup (red) (see, e.g., “A”). Myozyme®-treated mice did not show any notable differences compared to vehicle-treated mice. In contrast, most fibers isolated from mice treated with ATB200 showed dramatically reduced lysosome size (see, e.g., “C”). Furthermore, the area with autophagic buildup was also reduced to various degrees (see, e.g., “C”). As a result, a significant portion (36-60%) of analyzed muscle fibers from ATB200-treated mice were considered normal or near-normal. Table 8 below summarizes the single fiber analysis shown in FIG. 17. TIFF2025016455000012.tif85170

[0184] Overall, the data indicate that ATB200, with its high M6P content, both alone and further stabilized by the pharmacological chaperone AT2221 at the neutral pH of blood, is more efficient in tissue targeting and lysosomal transport compared to alglucosidase alfa when administered to Gaa KO mice, consistent with the stabilization of ATB200 by AT2221 shown in FIG. 18. As a result, administration of ATB200 and co-administration of ATB200 / AT2221 were more effective than alglucosidase alfa in correcting some of the disease-associated pathologies, such as glycogen accumulation, lysosomal proliferation and the formation of autophagy zones. These positive therapeutic effects indicate that administration of ATB200 and co-administration of ATB200 / AT2221 improves the chances of muscle fiber recovery from injury and further reverses the injury by clearing the glycogen accumulated in the cells due to the lack of optimal GAA activity. As in Example 6, these data are consistent with the improvements at the cellular level demonstrated in human Pompe disease patients, resulting in both effective treatment of Pompe disease and reversal of disease progression after administration of ATB200 and miglustat. Clinical data in human Pompe disease patients are summarized in Examples 8-13 below.

[0185] Example 8: ATB200-02 Clinical Trial: A Non-Human Study of ATB200 / AT2221 in Patients with Pompe Disease Preclinical studies were performed in Gaa knockout mice with varying ATB200 enzyme replacement therapy (ERT) and AT2221 chaperone doses to evaluate the pharmacokinetics (PK) and efficiency of glycogen reduction. These data were used to extrapolate the equivalent AT2221 chaperone dose in humans. Study ATB200-02 (NCT02675465) was then designed as an open-label, fixed-sequence, ascending-dose, first-in-human, Phase 1 / 2 study to evaluate the safety, tolerability, PK, pharmacodynamics (PD) and efficacy of ATB200 co-administered with AT2221 in patients with Pompe disease. Figures 19A-19B show the ATB200-02 study design. Ambulatory patients who previously received enzyme replacement therapy with alglucosidase alfa are referred to as ambulatory ERT-switched (or ERT-switched ambulatory) patients or Cohort 1 patients. Non-ambulatory patients who previously received enzyme replacement therapy with alglucosidase alfa are referred to as non-ambulatory ERT-switched (or ERT-switched non-ambulatory) patients or Cohort 2 patients. Ambulant patients who have not previously received enzyme replacement therapy with alglucosidase alfa are referred to as ERT-naïve (or ERT-naïve ambulatory) patients or Cohort 3 patients.

[0186] Sixteen clinical sites across five countries participated in the ATB200-02 study. The study used the following key inclusion criteria: men and women aged 18-65 years who were diagnosed with Pompe disease based on documented deficiency of GAA enzyme activity or by GAA phenotype and who had received enzyme replacement therapy with alglucosidase alfa for 2-6 years (cohort 1) (or ≥2 years for cohort 2) prior to study initiation. Eligible subjects were those who were currently receiving alglucosidase alfa every 2 weeks and had completed the last two infusions without drug-related adverse events (AEs) resulting in dose interruption (cohorts 1 and 2). Subjects were able to walk 200 to 500 meters in a 6-minute walk test (6MWT) (Cohorts 1 and 3), had an upright forced vital capacity (FVC) of 30-80% of predicted normal (Cohorts 1 and 3), or were unable to leave a wheelchair or walk unassisted (Cohort 2). Protocols for 6MWT and FVC testing can be found, for example, in Lachman and Schoser, Journal of Rare Diseases, 2013, 8:160 and Bittner and Singh, The 6 Minute Walk Test, Cardiology Advisor, 2013. Figure 19C provides baseline characteristics for 20 subjects. Safety, tolerability, and biomarkers were evaluated for Cohorts 1, 2, and 3. The following functional assessments were evaluated for cohorts 1 and 3: 6MWT, other motor function tests (timed test and gait-step-gower-chair (GSGC)), manual muscle testing and pulmonary function (FVC, maximum inspiratory pressure (MIP) / maximum expiratory pressure (MEP)). Protocols for timed test and GSGC testing can be found, for example, in Lachman and Schoser, Journal of Rare Diseases, 2013, 8:160. For cohort 2, functional assessments included muscle testing.

[0187] Example 9: Interim PK Results from the ATB200-02 Clinical Trial A summary of the pharmacokinetic data for AT2221 is shown in Figure 20. Plasma total GAA protein concentrations at 5 mg / kg, 10 mg / kg and 20 mg / kg for ATB200 were determined by validated LC-MS / MS quantification of rhGAA-specific "signature" peptide(s) T09 (primary) and T50 (confirmatory) for 11 Cohort 1 patients who completed stages 1 and 2, and for 5 Cohort 2 patients who completed the PK study in stage 3. For stage 1, blood samples for plasma total GAA protein concentrations were collected prior to the start of the ATB200 infusion and 1, 2, 3, 3.5, 4, 4.5, 5, 6, 8, 10, 12 and 24 hours after the start of the infusion. For stages 2 and 3, blood samples for plasma total GAA protein concentrations were collected prior to the start of the infusion and at 1, 2, 3, 3.5, 4, 4.5, 5, 6, 8, 10, 12 and 24 hours after the start of the infusion.

[0188] AT2221 PK analysis was performed on 11 Cohort 1 patients who completed Stages 1 and 2 and 5 Cohort 2 patients who completed the PK study in Stage 3. Blood samples for plasma AT2221 concentrations were collected immediately prior to oral AT2221 dosing (time 0) and at 1.5, 2, 2.5, 3, 4, 5, 6, 9, 11, and 25 hours after oral AT2221 dosing. Plasma AT2221 was determined by a validated LC-MS / MS assay.

[0189] As shown in Figure 21, the level of ATB200 increased in a slightly greater than dose-proportional manner when administered alone. Co-administration of ATB200 at 20 mg / kg with a single high dose (260 mg) of AT2221 increased the total GAA protein exposure area under the curve (AUC) by approximately 17% compared to ATB200 at 20 mg / kg administered alone (Figure 21, Figure 22C). Co-administration of ATB200 at 20 mg / kg with multiple high doses (260 mg) of AT2221 increased the total GAA protein exposure area under the curve (AUC) by approximately 29% compared to ATB200 administered alone at 20 mg / kg (Figure 21, Figure 22D). Increases in distribution half-life and partial AUC-24 hours were observed on a logarithmic scale during the terminal elimination phase (Figure 21, Figure 22A, Figure 22B). As shown in FIG. 21, the distribution half-life (α-phase) was increased by 40%, consistent with the stabilizing effect of high dose AT2221 on ATB200 in plasma. The increase in distribution half-life was accompanied by a 42.2% increase in partial AUC from the time of maximum plasma concentration to 24 hours post-dose (FIG. 21, FIG. 22B). Further evidence of stabilization of ATB200 by AT2221 was observed at 12 and 24 hours post-dose in a comparison of low and high dose AT2221 versus ATB200 alone (FIG. 22E and 22F). There was no statistically significant difference in plasma total GAA protein exposure between ERT-naive (cohort 3) and ERT-switched patients (cohort 1) (FIG. 23). The PK kinetics of signature peptide T50 was not different from that of signature peptide T09 (AUC ratio: 1.00).

[0190] Example 10: Interim Efficacy Results from the ATB200-02 Clinical Trial As shown in Figures 24A and 24B, 6MWT improved ambulatory ERT-switched and ERT-naive patients at 6 months, with the observed benefits continuing through 12 months. 6MWT increased in 7 / 10, 8 / 10, and 8 / 8 ERT-switched patients at 6, 9, and 12 months, respectively. 6MWT increased in 5 / 5, 5 / 5, and 2 / 2 ERT-naive patients at 6, 9, and 12 months, respectively.

[0191] As shown in Figures 24C, 26A and 26C, improvements in motor function tests and manual muscle strength along with 6MWT are consistent with overall improvements in muscle function for both ERT-switched and ERT-naive patients over the 12-month period.

[0192] As shown in Figures 25 and 26B, consistent and substantial increases were observed in upper limb strength in all non-ambulatory ERT-switched patients at 6 and 9 months.

[0193] As shown in Figure 27, FVC was stable or increased in 5 / 9, 6 / 9, and 3 / 7 ERT-switched patients at 6, 9, and 12 months, respectively, and FVC was stable or increased in 5 / 5, 5 / 5, and 2 / 2 ERT-naive patients at 6, 9, and 12 months, respectively. Also shown in Figure 27, maximum inspiratory pressure (MIP) was stable and maximum expiratory pressure (MEP) increased in ERT-switched ambulatory patients, while MIP increased and MEP was stable in ERT-naive patients.

[0194] The Fatigue Severity Scale ("FSS") is a self-assessment questionnaire consisting of 9 questions, each scored on a scale of 1 to 7. Total scores range from 9 to 63, with higher values ​​representing higher levels of fatigue due to the disease state. The normal value in a healthy population is approximately 21 (Grace J et al., Parkinsonism Relat Disord. 2007;13:443-445). As shown in Figure 28, all cohorts were significantly affected by fatigue at baseline, and all cohorts demonstrated improvements in their FSS after receiving ATB200 / AT2221.

[0195] Example 11: Interim Results from the ATB200-02 Clinical Trial: Markers of Muscle Damage The following muscle damage markers were evaluated: creatine kinase (CK) enzyme, alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Results available after 9 months of clinical trials are reported in Figures 29A-29C (data from up to 58, 24 and 36 weeks for cohorts 1, 2 and 3, respectively; low n values ​​indicate that some data could not be analyzed or have not yet been analyzed). The mean reductions from baseline observed at each of these time points were approximately 30-35% for ambulatory ERT-switched patients (n=9), 5-20% for non-ambulatory ERT-switched patients (n=4) and 40-55% for ERT-naive patients (n=5). Results for CK enzyme obtained after 12 months of clinical trials are reported in Figure 29D (data from up to 12 months for cohorts 1, 2 and 3; low n values ​​indicate that some data could not be analyzed or have not yet been analyzed).

[0196] Urinary hexose tetrasaccharide (Hex4) was evaluated as a marker of glycogen accumulation. Results for Hex4 obtained after 12 months of clinical trial are reported in Figure 29D (data obtained up to 12 months for cohorts 1, 2 and 3; low n values ​​indicate that some data could not be analyzed or have not yet been analyzed).

[0197] Example 12: Interim safety results from the ATB200-02 clinical trial The longest duration of treatment was more than 20 months. Adverse events (AEs) were generally mild and transient, with a very low rate of infusion-related reactions (<1%) after a total of more than 400 infusions across all three cohorts. The occurrence of these was controlled by standard premedication.

[0198] The most common AEs reported as treatment-related through 72 weeks were nausea (3 / 20), tremor (3 / 20), headache (3 / 20), fatigue (3 / 20), diarrhea (2 / 20), muscle spasms (2 / 20), and swollen joints (2 / 20).

[0199] The most common AEs reported as treatment-related by 20+ months were abdominal pain (including upper and lower abdominal pain) (8 / 20), diarrhea (8 / 20), nasopharyngitis (6 / 20), nausea (5 / 20), headache (5 / 20) and upper respiratory tract infection (5 / 20) (Figure 30). One severe AE that was unrelated to study drug was reported (hospitalization for lower respiratory tract infection). No patients were discontinued from the study due to an AE.

[0200] There were 3 incidents of infusion-associated reactions (IARs) in 550+ infusions that were controlled by standard premedication. One IAR event (skin discoloration) occurred in a nonambulatory ERT-switched patient (Cohort 2). Two IAR events (localized pruritus, erythema, and burning sensation) occurred in ERT-naive patients (Cohort 3) (Figure 30).

[0201] Example 13: Summary of Interim Results and Conclusions from the ATB200-02 Clinical Trial As summarized in Figure 31, there is agreement in interim data from the ATB200-02 trial showing significant and unexpected parallel improvements in markers of muscle damage and substrate accumulation, muscle function tests (timed and duration), manual muscle strength across the various cohorts, and stabilization and / or improvement in respiratory function tests. Muscle function improved in 16 / 18 and 10 / 10 patients at 6 and 9 months, respectively. Increases in 6MWT distance were consistent and durable out to 12 months in ERT-switched ambulatory and ERT-naive patients, similar to improvements in other motor function tests in ERT-switched ambulatory and ERT-naive patients. Qualitative and quantitative measures showed increases in upper limb strength in nonambulatory ERT-switched patients at 6 and 9 months. FVC, MIP, and MEP were generally stable in ERT-switched patients and increased in ERT-naive patients. Improvements in fatigue scores were observed in all cohorts. Biomarker levels (eg, CK and Hex4 levels) were decreased in all cohorts and ATB200 / AT2221 was generally well tolerated.

[0202] Therefore, the multidimensional effects of treatment suggest that ATB200 / AT2221 combination regimen may be an important treatment option for patients with Pompe disease. These clinical results support the results from the single fiber analysis study described in Example 7, demonstrating that treatment is effective in clearing pathology from muscle fibers. Further studies in clinical trials are ongoing. SEQUENCE LISTING <110> AMICUS THERAPEUTICS, INC. <120> RECOMBINANT HUMAN ACID ALPHA-GLUCOSIDASE <130> 14322.12-304 <140> <141> <150> 62 / 660,758 <151> 2018-04-20 <150> 62 / 624,638 <151> 2018-01-31 <150> 62 / 618,021 <151> 2018-01-16 <150> 62 / 567,334 <151> 2017-10-03 <150> 62 / 564,083 <151> 2017-09-27 <150> 62 / 506,574 <151> 2017-05-15 <150> 62 / 506,569 <151> 2017-05-15 <150> 62 / 506,561 <151> 2017-05-15 <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 952 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <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> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <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 tctccaacca tgggagtgag gcacccgccc 240 tgctcccacc ggctcctggc cgtctgcgcc ctcgtgtcct tggcaaccgc tgcactcctg 300 gggcacatcc tactccatga tttcctgctg gttccccgag agctgagtgg ctcctcccca 360 gtcctggagg agactcaccc agctcaccag cagggagcca gcagaccagg gccccgggat 420 gcccaggcac accccggccg tcccagagca gtgcccacac agtgcgacgt cccccccaac 480 agccgcttcg attgcgcccc tgacaaggcc atcacccagg aacagtgcga ggcccgcggc 540 tgctgctaca tccctgcaaa gcaggggctg cagggagccc agatggggca gccctggtgc 600 ttcttcccac ccagctaccc cagctacaag ctggagaacc tgagctcctc tgaaatgggc 660 tacacggcca ccctgacccg taccaccccc accttcttcc ccaaggacat cctgaccctg 720 cggctggacg tgatgatgga gactgagaac cgcctccact tcacgatcaa agatccagct 780 aacaggcgct acgaggtgcc cttggagacc ccgcgtgtcc acagccgggc accgtcccca 840 ctctacagcg tggagttctc cgaggagccc ttcggggtga tcgtgcaccg gcagctggac 900 ggccgcgtgc tgctgaacac gacggtggcg cccctgttct ttgcggacca gttccttcag 960 ctgtccacct cgctgccctc gcagtatatc acaggcctcg ccgagcacct cagtcccctg 1020 atgctcagca ccagctggac caggatcacc ctgtggaacc gggaccttgc gcccacgccc 1080 ggtgcgaacc tctacgggtc tcaccctttc tacctggcgc tggaggacgg cgggtcggca 1140 cacggggtgt tcctgctaaa cagcaatgcc atggatgtgg tcctgcagcc gagccctgcc 1200 cttagctgga ggtcgacagg tgggatcctg gatgtctaca tcttcctggg cccagagccc 1260 aagagcgtgg tgcagcagta cctggacgtt gtgggatacc cgttcatgcc gccatactgg 1320 ggcctgggct tccacctgtg ccgctggggc tactcctcca ccgctatcac ccgccaggtg 1380 gtggagaaca tgaccagggc ccacttcccc ctggacgtcc aatggaacga cctggactac 1440 atggactccc ggagggactt cacgttcaac aaggatggct tccgggactt cccggccatg 1500 gtgcaggagc tgcaccaggg cggccggcgc tacatgatga tcgtggatcc tgccatcagc 1560 agctcgggcc ctgccgggag ctacaggccc tacgacgagg gtctgcggag gggggttttc 1620 atcaccaacg agaccggcca gccgctgatt gggaaggtat ggcccgggtc cactgccttc 1680 cccgacttca ccaaccccac agccctggcc tggtgggagg acatggtggc tgagttccat 1740 gaccaggtgc ccttcgacgg catgtggatt gacatgaacg agccttccaa cttcatcaga 1800 ggctctgagg acggctgccc caacaatgag ctggagaacc caccctacgt gcctggggtg 1860 gttgggggga ccctccaggc ggccaccatc tgtgcctcca gccaccagtt tctctccaca 1920 cactacaacc tgcacaacct ctacggcctg accgaagcca tcgcctccca cagggcgctg 1980 gtgaaggctc gggggacacg cccatttgtg atctcccgct cgacctttgc tggccacggc 2040 cgatacgccg gccactggac gggggacgtg tggagctcct gggagcagct cgcctcctcc 2100 gtgccagaaa tcctgcagtt taacctgctg ggggtgcctc tggtcggggc cgacgtctgc 2160 ggcttcctgg gcaacacctc agaggagctg tgtgtgcgct ggacccagct gggggccttc 2220 taccccttca tgcggaacca caacagcctg ctcagtctgc cccaggagcc gtacagcttc 2280 agcgagccgg cccagcaggc catgaggaag gccctcaccc tgcgctacgc actcctcccc 2340 cacctctaca cactgttcca ccaggcccac gtcgcggggg agaccgtggc ccggcccctc 2400 ttcctggagt tccccaagga ctctagcacc tggactgtgg accaccagct cctgtggggg 2460 gaggccctgc tcatcacccc agtgctccag gccgggaagg ccgaagtgac tggctacttc 2520 cccttgggca catggtacga cctgcagacg gtgccaatag aggcccttgg cagcctccca 2580 cccccacctg cagctccccg tgagccagcc atccacagcg aggggcagtg ggtgacgctg 2640 ccggcccccc tggacaccat caacgtccac ctccgggctg ggtacatcat ccccctgcag 2700 ggccctggcc tcacaaccac agagtcccgc cagcagccca tggccctggc tgtggccctg 2760 accaagggtg gagaggcccg aggggagctg ttctgggacg atggagagag cctggaagtg 2820 ctggagcgag gggcctacac acaggtcatc ttcctggcca ggaataacac gatcgtgaat 2880 gagctggtac gtgtgaccag tgagggagct ggcctgcagc tgcagaaggt gactgtcctg 2940 ggcgtggcca cggcgcccca gcaggtcctc tccaacggtg tccctgtctc caacttcacc 3000 tacagccccg acaccaaggt cctggacatc tgtgtctcgc tgttgatggg agagcagttt 3060 ctcgtcagct ggtgttagcc gggcggagtg tgttagtctc tccagaggga ggctggttcc 3120 ccagggaagc agagcctgtg tgcgggcagc agctgtgtgc gggcctgggg gttgcatgtg 3180 tcacctggag ctgggcacta accattccaa gccgccgcat cgcttgtttc cacctcctgg 3240 gccggggctc tggcccccaa cgtgtctagg agagctttct ccctagatcg cactgtgggc 3300 cggggcctgg agggctgctc tgtgttaata agattgtaag gtttgccctc ctcacctgtt 3360 gccggcatgc gggtagtatt agccaccccc ctccatctgt tcccagcacc ggagaagggg 3420 gtgctcaggt ggaggtgtgg ggtatgcacc tgagctcctg cttcgcgcct gctgctctgc 3480 cccaacgcga ccgcttcccg gctgcccaga gggctggatg cctgccggtc cccgagcaag 3540 cctgggaact caggaaaatt cacaggactt gggagattct aaatcttaag tgcaattatt 3600 ttaataaaag gggcatttgg aatc 3624 <210> 3 <211> 952 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <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> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <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 <210> 5 <211> 896 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 5 Gln Gln Gly Ala Ser Arg Pro Gly Pro Arg Asp Ala Gln Ala His Pro 1 5 10 15 Gly Arg Pro Arg Ala Val Pro Thr Gln Cys Asp Val Pro Pro Asn Ser 20 25 30 Arg Phe Asp Cys Ala Pro Asp Lys Ala Ile Thr Gln Glu Gln Cys Glu 35 40 45 Ala Arg Gly Cys Cys Tyr Ile Pro Ala Lys Gln Gly Leu Gln Gly Ala 50 55 60 Gln Met Gly Gln Pro Trp Cys Phe Phe Pro Pro Ser Tyr Pro Ser Tyr 65 70 75 80 Lys Leu Glu Asn Leu Ser Ser Ser Glu Met Gly Tyr Thr Ala Thr Leu 85 90 95 Thr Arg Thr Thr Pro Thr Phe Phe Pro Lys Asp Ile Leu Thr Leu Arg 100 105 110 Leu Asp Val Met Met Glu Thr Glu Asn Arg Leu His Phe Thr Ile Lys 115 120 125 Asp Pro Ala Asn Arg Arg Tyr Glu Val Pro Leu Glu Thr Pro Arg Val 130 135 140 His Ser Arg Ala Pro Ser Pro Leu Tyr Ser Val Glu Phe Ser Glu Glu 145 150 155 160 Pro Phe Gly Val Ile Val His Arg Gln Leu Asp Gly Arg Val Leu Leu 165 170 175 Asn Thr Thr Val Ala Pro Leu Phe Phe Ala Asp Gln Phe Leu Gln Leu 180 185 190 Ser Thr Ser Leu Pro Ser Gln Tyr Ile Thr Gly Leu Ala Glu His Leu 195 200 205 Ser Pro Leu Met Leu Ser Thr Ser Trp Thr Arg Ile Thr Leu Trp Asn 210 215 220 Arg Asp Leu Ala Pro Thr Pro Gly Ala Asn Leu Tyr Gly Ser His Pro 225 230 235 240 Phe Tyr Leu Ala Leu Glu Asp Gly Gly Ser Ala His Gly Val Phe Leu 245 250 255 Leu Asn Ser Asn Ala Met Asp Val Val Leu Gln Pro Ser Pro Ala Leu 260 265 270 Ser Trp Arg Ser Thr Gly Gly Ile Leu Asp Val Tyr Ile Phe Leu Gly 275 280 285 Pro Glu Pro Lys Ser Val Val Gln Gln Tyr Leu Asp Val Val Gly Tyr 290 295 300 Pro Phe Met Pro Pro Tyr Trp Gly Leu Gly Phe His Leu Cys Arg Trp 305 310 315 320 Gly Tyr Ser Ser Thr Ala Ile Thr Arg Gln Val Val Glu Asn Met Thr 325 330 335 Arg Ala His Phe Pro Leu Asp Val Gln Trp Asn Asp Leu Asp Tyr Met 340 345 350 Asp Ser Arg Arg Asp Phe Thr Phe Asn Lys Asp Gly Phe Arg Asp Phe 355 360 365 Pro Ala Met Val Gln Glu Leu His Gln Gly Gly Arg Arg Tyr Met Met 370 375 380 Ile Val Asp Pro Ala Ile Ser Ser Ser Gly Pro Ala Gly Ser Tyr Arg 385 390 395 400 Pro Tyr Asp Glu Gly Leu Arg Arg Gly Val Phe Ile Thr Asn Glu Thr 405 410 415 Gly Gln Pro Leu Ile Gly Lys Val Trp Pro Gly Ser Thr Ala Phe Pro 420 425 430 Asp Phe Thr Asn Pro Thr Ala Leu Ala Trp Trp Glu Asp Met Val Ala 435 440 445 Glu Phe His Asp Gln Val Pro Phe Asp Gly Met Trp Ile Asp Met Asn 450 455 460 Glu Pro Ser Asn Phe Ile Arg Gly Ser Glu Asp Gly Cys Pro Asn Asn 465 470 475 480 Glu Leu Glu Asn Pro Pro Tyr Val Pro Gly Val Val Gly Gly Thr Leu 485 490 495 Gln Ala Ala Thr Ile Cys Ala Ser Ser His Gln Phe Leu Ser Thr His 500 505 510 Tyr Asn Leu His Asn Leu Tyr Gly Leu Thr Glu Ala Ile Ala Ser His 515 520 525 Arg Ala Leu Val Lys Ala Arg Gly Thr Arg Pro Phe Val Ile Ser Arg 530 535 540 Ser Thr Phe Ala Gly His Gly Arg Tyr Ala Gly His Trp Thr Gly Asp 545 550 555 560 Val Trp Ser Ser Trp Glu Gln Leu Ala Ser Ser Val Pro Glu Ile Leu 565 570 575 Gln Phe Asn Leu Leu Gly Val Pro Leu Val Gly Ala Asp Val Cys Gly 580 585 590 Phe Leu Gly Asn Thr Ser Glu Glu Leu Cys Val Arg Trp Thr Gln Leu 595 600 605 Gly Ala Phe Tyr Pro Phe Met Arg Asn His Asn Ser Leu Leu Ser Leu 610 615 620 Pro Gln Glu Pro Tyr Ser Phe Ser Glu Pro Ala Gln Gln Ala Met Arg 625 630 635 640 Lys Ala Leu Thr Leu Arg Tyr Ala Leu Leu Pro His Leu Tyr Thr Leu 645 650 655 Phe His Gln Ala His Val Ala Gly Glu Thr Val Ala Arg Pro Leu Phe 660 665 670 Leu Glu Phe Pro Lys Asp Ser Ser Thr Trp Thr Val Asp His Gln Leu 675 680 685 Leu Trp Gly Glu Ala Leu Leu Ile Thr Pro Val Leu Gln Ala Gly Lys 690 695 700 Ala Glu Val Thr Gly Tyr Phe Pro Leu Gly Thr Trp Tyr Asp Leu Gln 705 710 715 720 Thr Val Pro Ile Glu Ala Leu Gly Ser Leu Pro Pro Pro Pro Ala Ala 725 730 735 Pro Arg Glu Pro Ala Ile His Ser Glu Gly Gln Trp Val Thr Leu Pro 740 745 750 Ala Pro Leu Asp Thr Ile Asn Val His Leu Arg Ala Gly Tyr Ile Ile 755 760 765 Pro Leu Gln Gly Pro Gly Leu Thr Thr Thr Glu Ser Arg Gln Gln Pro 770 775 780 Met Ala Leu Ala Val Ala Leu Thr Lys Gly Gly Glu Ala Arg Gly Glu 785 790 795 800 Leu Phe Trp Asp Asp Gly Glu Ser Leu Glu Val Leu Glu Arg Gly Ala 805 810 815 Tyr Thr Gln Val Ile Phe Leu Ala Arg Asn Asn Thr Ile Val Asn Glu 820 825 830 Leu Val Arg Val Thr Ser Glu Gly Ala Gly Leu Gln Leu Gln Lys Val 835 840 845 Thr Val Leu Gly Val Ala Thr Ala Pro Gln Gln Val Leu Ser Asn Gly 850 855 860 Val Pro Val Ser Asn Phe Thr Tyr Ser Pro Asp Thr Lys Val Leu Asp 865 870 875 880 Ile Cys Val Ser Leu Leu Met Gly Glu Gln Phe Leu Val Ser Trp Cys 885 890 895

Claims

1. 1. A method of treating Pompe disease in a subject in need thereof comprising administering to the subject a population of recombinant human acid alpha glucosidase (rhGAA) molecules derived from Chinese hamster ovary (CHO) cells, The rhGAA molecule contains seven potential N-glycosylation sites; the rhGAA molecule contains an average of 3-4 mannose-6-phosphate (M6P) residues; the rhGAA molecule contains, on average, at least about 0.5 moles of bis-mannose-6-phosphate (bis-M6P) at the first potential N-glycosylation site per mole of rhGAA as determined using liquid chromatography-tandem mass spectrometry (LC-MS / MS); The method, wherein the population of rhGAA is administered in a dosage capable of reversing disease progression in the subject.

2. 10. The method of claim 1, wherein reversing disease progression comprises reducing the size of lysosomes in the muscle of the subject.

3. 3. The method of claim 1 or 2, wherein reversing disease progression comprises abolishing autophagy buildup in muscle of the subject.

4. 4. The method of claim 3, wherein less than 65% of muscle fibers analyzed in the subject have autophagic build-up following treatment.

5. The method of any one of claims 1 to 4, wherein the subject is an ERT-switched patient.

6. 6. The method of claim 5, wherein the ERT-switched patient has been previously treated with alglucosidase alfa for at least two years.

7. 7. The method of any one of claims 1 to 6, wherein at least 36% of muscle fibers analyzed in the subject have normal or near-normal appearance after treatment.

8. 1. A method of treating Pompe disease in a subject in need thereof comprising administering to the subject a population of recombinant human acid alpha glucosidase (rhGAA) molecules derived from Chinese hamster ovary (CHO) cells, The rhGAA molecule contains seven potential N-glycosylation sites; the rhGAA molecule contains an average of 3-4 mannose-6-phosphate (M6P) residues; the rhGAA molecule contains, on average, at least about 0.5 moles of bis-mannose-6-phosphate (bis-M6P) at the first potential N-glycosylation site per mole of rhGAA as determined using LC-MS / MS; The method, wherein glycogen content in the subject's muscle following treatment is reduced more quickly than if alglucosidase alfa was administered in the same dosage.

9. 9. The method of claim 8, wherein glycogen content in the muscle of the subject following treatment is reduced at a rate at least about 1.25, 1.5, 1.75, 2.0 or 3.0 times faster than the rate when alglucosidase alfa is administered in the same dosage.

10. 10. The method of claim 8 or 9, wherein glycogen content in the muscle of the subject after treatment is reduced more effectively than when alglucosidase alfa is administered at the same dosage, when assessed after 1, 2, 3, 4, 5 or 6 administrations.

11. 11. The method of claim 10, wherein glycogen content in the muscle of the subject after treatment is more effectively reduced by at least about 10%, 20%, 30%, 50%, 75% or 90% than when alglucosidase alfa is administered in the same dosage.

12. 12. The method of claim 10 or 11, wherein glycogen content is assessed after six administrations.

13. 13. The method of any one of claims 8 to 12, wherein the subject exhibits reduced levels of urinary hexose tetrasaccharide following treatment.

14. 14. The method of claim 13, wherein urinary hexose tetrasaccharide levels 6 months after treatment are reduced by at least 30% compared to baseline.

15. 14. The method of claim 13, wherein the subject is an ambulatory ERT-switched patient or a non-ambulatory ERT-switched patient, and the subject's urinary hexose tetrasaccharide level 6 months after treatment is reduced by at least 35% compared to baseline.

16. 14. The method of claim 13, wherein the subject is an ambulatory ERT-naive patient and the subject's urinary hexose tetrasaccharide level 6 months after treatment is reduced by at least 45% compared to baseline.

17. 1. A method of treating Pompe disease in a subject in need thereof comprising administering to the subject a population of recombinant human acid alpha glucosidase (rhGAA) molecules derived from Chinese hamster ovary (CHO) cells, The rhGAA molecule contains seven potential N-glycosylation sites; the rhGAA molecule contains an average of 3-4 mannose-6-phosphate (M6P) residues; the rhGAA molecule contains, on average, at least about 0.5 moles of bis-mannose-6-phosphate (bis-M6P) at the first potential N-glycosylation site per mole of rhGAA as determined using LC-MS / MS; The method, wherein the population of rhGAA is administered in a dosage capable of improving motor function in a subject.

18. 18. The method of claim 17, wherein the improvement in motor function in the subject is measured by at least one motor function test selected from the group consisting of the 6-minute walk test (6MWT), the timed up and go test, the four-step climb test, the 10 meter walk test, the Gower's test, the Walk-Step-Gower-Chair (GSGC) test, and combinations thereof.

19. 19. The method of claim 18, wherein, compared to baseline, the subject's 6MWT distance at 6 months after treatment is increased by at least 20 meters, the Timed Up and Go Test time at 6 months after treatment is decreased by at least 1 second, the Four Step Climb Test time at 6 months after treatment is decreased by at least 0.6 seconds, the 10 Meter Walk Test time at 6 months after treatment is decreased by at least 0.7 seconds, the Gowers Test time at 6 months after treatment is decreased by at least 1 second, or the GSGC score at 6 months after treatment is decreased by at least 1.

20. 19. The method of claim 18, wherein the subject is an ambulatory ERT-switched patient and, compared to baseline, the subject's 6MWT distance 6 months after treatment is increased by at least 20 meters, the subject's Timed Up and Go Test time is reduced by at least 1.5 seconds in the subject's Timed Four Step Climb Test time is reduced by at least 0.6 seconds in the subject's Timed Four Step Climb Test time is reduced by at least 1 second in the subject's Timed Four Step Climb Test time is reduced by at least 1 second in the subject's Timed Four Step Climb Test time is reduced by at least 1 second in the subject's Timed Six Months After Treatment.

21. 19. The method of claim 18, wherein the subject is an ambulatory ERT-naive patient and, compared to baseline, the subject's 6MWT distance at 6 months after treatment is increased by at least 40 meters, the subject's Timed Up and Go Test time at 6 months after treatment is decreased by at least 1 second, the subject's Four Step Climb Test time at 6 months after treatment is decreased by at least 0.6 seconds, the subject's 10 Meter Walk Test time at 6 months after treatment is decreased by at least 0.7 seconds, or the subject's GSGC score at 6 months after treatment is decreased by at least 1.

22. 20. The method of claim 18, wherein the subject has previously undergone alglucosidase alfa enzyme replacement therapy, and the subject exhibits improvement in at least one motor function test following treatment with the population of rhGAA compared to the subject's motor function test results following the previous alglucosidase alfa enzyme replacement therapy.

23. 1. A method of treating Pompe disease in a subject in need thereof comprising administering to the subject a population of recombinant human acid alpha glucosidase (rhGAA) molecules derived from Chinese hamster ovary (CHO) cells, The rhGAA molecule contains seven potential N-glycosylation sites; the rhGAA molecule contains an average of 3-4 mannose-6-phosphate (M6P) residues; the rhGAA molecule contains, on average, at least about 0.5 moles of bis-mannose-6-phosphate (bis-M6P) at the first potential N-glycosylation site per mole of rhGAA as determined using LC-MS / MS; The method, wherein the population of rhGAA is administered in a dosage capable of improving upper body strength in a subject.

24. 24. The method of claim 23, wherein the improvement in upper body strength in the subject is measured by manual muscle score.

25. 25. The method of claim 24, wherein the subject is an ambulatory ERT-switched patient and shows an improvement of at least 1 in upper body manual strength score compared to baseline 6 months after treatment.

26. 25. The method of claim 24, wherein the subject is a non-ambulatory ERT-switched patient and shows an improvement of at least 5.5 in upper body manual strength scores compared to baseline 6 months after treatment.

27. 24. The method of claim 23, wherein the improvement in upper body strength in the subject is improvement in upper limb strength, as measured by quantitative or manual muscle testing of at least one upper limb muscle group selected from the group consisting of shoulder adduction, shoulder abduction, elbow flexion, and elbow extension.

28. 28. The method of claim 27, wherein the subject is a non-ambulatory ERT-switched patient and, compared to baseline, the subject's shoulder adduction is improved by at least 8 pounds of force at 6 months after treatment, the subject's shoulder abduction is improved by at least 1 pound of force at 6 months after treatment, the subject's elbow flexion is improved by at least 2 pounds of force at 6 months after treatment, or the subject's elbow extension is improved by at least 5 pounds of force at 6 months after treatment.

29. 24. The method of claim 23, wherein the subject is ambulatory and further exhibits improved lower body strength and / or total body strength following treatment.

30. 24. The method of claim 23, wherein the subject has previously undergone alglucosidase alfa enzyme replacement therapy, and the subject shows an improvement in upper body strength after treatment with the population of rhGAA compared to the subject's upper body strength after the previous alglucosidase alfa enzyme replacement therapy.

31. 1. A method of treating Pompe disease in a subject in need thereof comprising administering to the subject a population of recombinant human acid alpha glucosidase (rhGAA) molecules derived from Chinese hamster ovary (CHO) cells, The rhGAA molecule contains seven potential N-glycosylation sites; the rhGAA molecule contains an average of 3-4 mannose-6-phosphate (M6P) residues; the rhGAA molecule contains, on average, at least about 0.5 moles of bis-mannose-6-phosphate (bis-M6P) at the first potential N-glycosylation site per mole of rhGAA as determined using LC-MS / MS; The method, wherein the population of rhGAA is administered in a dosage capable of improving pulmonary function in a subject.

32. 32. The method of claim 31 , wherein the improvement in pulmonary function in the subject is measured by at least one pulmonary function test selected from the group consisting of a forced vital capacity (FVC) test, a maximum expiratory pressure (MEP) test, a maximum inspiratory pressure (MIP) test, and combinations thereof.

33. The subject's FVC improves by at least 4% at 6 months after treatment compared to baseline, and the subject's MEP improves by at least 16 cmH at 6 months after treatment ２ O improvement or MIP of the subject 6 months after treatment is at least 0.3 cmH ２ 33. The method of claim 32, wherein the O is improved.

34. Subjects are ambulatory ERT-switched patients and have an MEP of at least 16 cmH at 6 months post-treatment compared to baseline. ２ 33. The method of claim 32, wherein the O is improved.

35. The subject is an ambulatory ERT-naive patient, and the subject's FVC improves by at least 4% at 6 months post-treatment compared to baseline, or the subject's MIP improves by at least 11 cmH at 6 months post-treatment. ２ 33. The method of claim 32, wherein the O is improved.

36. 33. The method of claim 32, wherein the subject has previously undergone alglucosidase alfa enzyme replacement therapy, and the subject exhibits an improvement in at least one pulmonary function test following treatment with the population of rhGAA compared to the subject's pulmonary function test results following the previous alglucosidase alfa enzyme replacement therapy.

37. 1. A method of treating Pompe disease in a subject in need thereof comprising administering to the subject a population of recombinant human acid alpha glucosidase (rhGAA) molecules derived from Chinese hamster ovary (CHO) cells, The rhGAA molecule contains seven potential N-glycosylation sites; the rhGAA molecule contains an average of 3-4 mannose-6-phosphate (M6P) residues; the rhGAA molecule contains, on average, at least about 0.5 moles of bis-mannose-6-phosphate (bis-M6P) at the first potential N-glycosylation site per mole of rhGAA as determined using LC-MS / MS; The method, wherein the population of rhGAA is administered in a dosage capable of reducing fatigue in a subject as measured by Fatigue Severity Scale (FSS) score.

38. 38. The method of claim 37, wherein the subject's FSS score 6 months after treatment is reduced by at least 3.5 compared to baseline.

39. The method of claim 38, wherein the subject is a non-ambulatory ERT-switched patient.

40. 39. The method of claim 37 or 38, wherein the subject is an ambulatory ERT-switched patient and the subject's FSS score 6 months after treatment is reduced by at least 8 compared to baseline.

41. 39. The method of claim 37 or 38, wherein the subject is an ambulatory ERT-naive patient and the subject's FSS score 6 months after treatment is reduced by at least 5 compared to baseline.

42. 38. The method of claim 37, wherein the subject has previously undergone alglucosidase alpha enzyme replacement therapy, and the subject's FSS score is reduced following treatment with the population of rhGAA compared to the subject's FSS score following the previous alglucosidase alpha enzyme replacement therapy.

43. 1. A method of treating Pompe disease in a subject in need thereof comprising administering to the subject a population of recombinant human acid alpha glucosidase (rhGAA) molecules derived from Chinese hamster ovary (CHO) cells, The rhGAA molecule contains seven potential N-glycosylation sites; the rhGAA molecule contains an average of 3-4 mannose-6-phosphate (M6P) residues; the rhGAA molecule contains, on average, at least about 0.5 moles of bis-mannose-6-phosphate (bis-M6P) at the first potential N-glycosylation site per mole of rhGAA as determined using LC-MS / MS; The method, wherein the population of rhGAA is administered in a dosage capable of reducing the level of at least one muscle damage biomarker selected from the group consisting of creatine kinase, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and combinations thereof.

44. 44. The method of claim 43, wherein at least one muscle damage biomarker is creatine kinase.

45. 44. The method of claim 43, wherein the subject's creatine kinase level is reduced by at least 15% after 6 months of treatment, the subject's ALT level is reduced by at least 5% after 6 months of treatment, or the subject's AST level is reduced by at least 5% after 6 months of treatment, compared to baseline.

46. 44. The method of claim 43, wherein the subject is an ambulatory ERT-switched patient and, compared to baseline, the subject's creatine kinase levels are reduced by at least 15% 6 months after treatment, the subject's ALT levels are reduced by at least 15% 6 months after treatment, or the subject's AST levels are reduced by at least 10% 6 months after treatment.

47. 44. The method of claim 43, wherein the subject is a non-ambulatory ERT-switched patient and, compared to baseline, the subject's creatine kinase levels are reduced by at least 20% 6 months after treatment, the subject's ALT levels are reduced by at least 5% 6 months after treatment, or the subject's AST levels are reduced by at least 5% 6 months after treatment.

48. 44. The method of claim 43, wherein the subject is an ambulatory ERT-naive patient, and the subject's creatine kinase levels are reduced by at least 35% after 6 months of treatment, the subject's ALT levels are reduced by at least 35% after 6 months of treatment, or the subject's AST levels are reduced by at least 30% after 6 months of treatment compared to baseline.

49. 49. The method of any one of claims 1 to 48, wherein the population of rhGAA molecules is administered at a dose of about 1 mg / kg to about 100 mg / kg.

50. 50. The method of any one of claims 1 to 49, wherein the population of rhGAA molecules is administered at a dose of about 20 mg / kg.

51. 51. The method of any one of claims 1 to 50, wherein the population of rhGAA molecules is administered once every two months, once a month, once every two weeks, once a week, twice a week, or daily.

52. 52. The method of claim 51, wherein the population of rhGAA molecules is administered once every two weeks.

53. 53. The method of any one of claims 1 to 52, wherein the population of rhGAA molecules is administered intravenously.

54. 54. The method of any one of claims 1 to 53, wherein the population of rhGAA molecules is administered simultaneously or sequentially with a pharmacological chaperone.

55. 55. The method of claim 54, wherein the pharmacological chaperone is miglustat or a pharma- ceutically acceptable salt thereof.

56. 56. The method of claim 55, wherein the miglustat or a pharma- ceutically acceptable salt thereof is administered orally.

57. 57. The method of claim 55 or 56, wherein miglustat or a pharma- ceutically acceptable salt thereof is administered in a dose of about 200 mg to about 600 mg.

58. 58. The method of claim 57, wherein the miglustat or a pharma- ceutically acceptable salt thereof is administered in a dose of about 260 mg.

59. 58. The method of claim 57, wherein the population of rhGAA molecules is administered intravenously at a dose of about 5 mg / kg to about 20 mg / kg and miglustat or a pharma- ceutically acceptable salt thereof is administered orally at a dose of about 233 mg to about 500 mg.

60. 57. The method of claim 56, wherein miglustat or a pharma- ceutically acceptable salt thereof is orally administered at a dose of about 50 mg to about 200 mg.

61. 57. The method of claim 56, wherein the population of rhGAA molecules is administered intravenously at a dose of about 20 mg / kg and miglustat or a pharma- ceutically acceptable salt thereof is administered orally at a dose of about 260 mg.

62. 62. The method of any one of claims 55 to 61, wherein miglustat or a pharma- ceutically acceptable salt thereof is administered prior to administration of rhGAA.

63. 63. The method of claim 62, wherein the miglustat or a pharma- ceutically acceptable salt thereof is administered about 1 hour prior to administration of the rhGAA.

64. 64. The method of claim 62 or 63, wherein the subject fasts for at least 2 hours before and at least 2 hours after administration of miglustat or a pharma- ceutically acceptable salt thereof.

65. 65. The method of any one of claims 1 to 64, wherein the rhGAA molecule comprises an amino acid sequence at least 95% identical to SEQ ID NO:1 or SEQ ID NO:

5.

66. 66. The method of any one of claims 1 to 65, wherein the rhGAA molecule comprises an amino acid sequence identical to SEQ ID NO:1 or SEQ ID NO:

5.

67. 67. The method of any one of claims 1 to 66, wherein at least 30% of rhGAA molecules contain one or more N-glycan units bearing one mannose-6-phosphate residue (mono-M6P) or bis-M6P, as determined using LC-MS / MS.

68. 68. The method of any one of claims 1 to 67, wherein the rhGAA molecule contains an average of about 0.5 mol to about 7.0 mol of mono-M6P or bis-M6P per mol of rhGAA as determined using LC-MS / MS.

69. 69. The method of any one of claims 1 to 68, wherein the rhGAA molecule contains, on average, at least 2.5 mol of M6P per mol of rhGAA and at least 4 mol of sialic acid per mol of rhGAA, as determined using LC-MS / MS.

70. The rhGAA molecules have an average of: (a) about 0.4 to about 0.6 mol of mono-M6P at a second potential N-glycosylation site; (b) about 0.4 to about 0.6 mol of bis-M6P at the fourth potential N-glycosylation site; and (c) about 0.3 to about 0.4 mol of mono-M6P at the fourth potential N-glycosylation site; Including, 70. The method of any one of claims 1 to 69, wherein (a)-(c) are determined using LC-MS / MS.

71. the rhGAA molecule further comprises about 4 mol to about 7.3 mol of sialic acid per mol of rhGAA; The rhGAA molecules have an average of: (a) about 0.9 to about 1.2 mol of sialic acid at the third potential N-glycosylation site; (b) about 0.8 to about 0.9 mol of sialic acid at the fifth potential N-glycosylation site; and (c) about 1.5 to about 4.2 mol of sialic acid at the sixth potential N-glycosylation site; Including, 71. The method of claim 70, wherein (a)-(c) are determined using LC-MS / MS.

72. 72. The method of any one of claims 1 to 71, wherein the population of rhGAA molecules is formulated into a pharmaceutical composition.

73. 73. The method of claim 72, wherein the pharmaceutical composition further comprises at least one buffer selected from the group consisting of citrate, phosphate, and combinations thereof, and at least one excipient selected from the group consisting of mannitol, polysorbate 80, and combinations thereof, and wherein the pharmaceutical composition has a pH of about 5.0 to about 7.

0.

74. 74. The method of claim 73, wherein the pharmaceutical composition has a pH of about 5.0 to about 6.

0.

75. 75. The method of claim 73 or 74, wherein the pharmaceutical composition further comprises water, an acidifying agent, an alkalizing agent, or a combination thereof.

76. 74. The method of claim 73, wherein the population of rhGAA molecules in the pharmaceutical composition is present at a concentration of about 5-50 mg / mL, the at least one buffer is a sodium citrate buffer present at a concentration of about 10-100 mM, the at least one excipient is mannitol present at a concentration of about 10-50 mg / mL and polysorbate 80 present at a concentration of about 0.1-1 mg / mL, the pharmaceutical composition further comprises water, and optionally an acidifying agent and / or an alkalizing agent; and the pharmaceutical composition has a pH of about 6.

0.

77. 77. The method of claim 76, wherein the population of rhGAA molecules is present at a concentration of about 15 mg / mL, the sodium citrate buffer is present at a concentration of about 25 mM, the mannitol is present at a concentration of about 20 mg / mL, and the polysorbate 80 is present at a concentration of about 0.5 mg / mL in the pharmaceutical composition.

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