Methods for treating Poincare disease

Abstract

Provided herein are methods of treating Poincare disease by administering to a subject a recombinant human acidic alpha-glucosidase molecule population, or a pharmaceutical composition or formulation thereof, and an enzyme stabilizer.

Description

Technical Field

[0001] The present disclosure relates to methods of treating Pompe disease by administering recombinant human alpha-glucosidase. Background Art

[0002] Pompe disease (also known as glycogen storage disease type II (GSD-II) or acid maltase deficiency) is an inherited lysosomal storage disease caused by a deficiency in the activity of acid alpha-glucosidase (GAA). People with Pompe disease lack or have reduced levels of acid alpha-glucosidase (GAA), an enzyme that breaks down glycogen into glucose, the main energy source for muscle. This enzyme deficiency leads to the accumulation of excess glycogen in lysosomes, which are intracellular organelles that contain enzymes that normally break down glycogen and other cellular debris or waste products. The accumulation of glycogen in certain tissues (especially muscle) of subjects with Pompe disease impairs the ability of cells to function normally. In Pompe disease, glycogen is not properly metabolized and gradually accumulates in lysosomes, particularly in skeletal muscle cells, and in the infantile-onset form of the disease, in heart muscle cells. The accumulation of glycogen damages muscle cells and nerve cells, as well as those in other affected tissues.

[0003] Current non-palliative treatments for Pompe disease include the use of the trademark and Recombinant alglucosidase alfa products sold under the trademark Avalglucosidase alfa is a commercially available enzyme replacement therapy (ERT) for Pompe disease. This conventional enzyme replacement therapy seeks to treat Pompe disease by replacing missing GAA in lysosomes with rhGAA, thereby restoring the cell's ability to break down lysosomal glycogen. and

[00145] DRUG ® is a conventional form of rhGAA manufactured or sold as a biologic by Sanofi Genzyme and approved by the U.S. Food and Drug Administration and described by reference to the Physician's Desk Reference (2014), which is hereby incorporated by reference.

[0004] However, currently marketed ERTs provide, at best, limited improvements in measures of muscle function, strength, and respiratory function for a limited time, followed by a slow decline in these parameters (Toscano and Schoser, (2013) J Neurol 260, 951-959; Wyatt et al., (2012) Health Technol Assess 16(39). Pompe disease treatments that provide long-term improvements are needed. Summary of the Invention

[0005] Provided herein is a method for improving and / or stabilizing motor function and / or lung function in a subject with Pompe disease for at least 24 months, at least 36 months, or at least 48 months, the method comprising administering to the subject a population of recombinant human acid α-glucosidase (rhGAA) molecules simultaneously or sequentially with an enzyme stabilizer; wherein each rhGAA molecule comprises seven potential N-glycosylation sites; wherein 40%-60% of the N-glycans on the rhGAA molecule are complex N-glycans; wherein the rhGAA molecule comprises at least 0.5 mol bis-mannose-6-phosphate (bis-M6P) / mol rhGAA at the first potential N-glycosylation site as determined using liquid chromatography tandem mass spectrometry (LC-MS / MS); and wherein the method improves and / or stabilizes the subject's motor function, muscle strength, and / or lung function compared to baseline.

[0006] In some embodiments, the subject is an enzyme replacement therapy (ERT)-experienced subject.

[0007] In some embodiments of the method for treating a subject who has experienced ERT, motor function is measured by a 6-minute walk test; and the improvement from baseline in 6-minute walk distance (6MWD) is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 meters 12 or 24 months after initiation of treatment.

[0008] In some embodiments of the method for treating a subject who has experienced ERT, motor function is measured by a 6-minute walk test; and the improvement from baseline in 6-minute walk distance (6MWD) is at least 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 41, 42, 43, 44, 45, 46, or 47 meters 36 months after initiation of treatment.

[0009] In some embodiments of the method for treating a subject who has experienced ERT, the subject has a baseline 6MWD of: (a) at least 300 meters; or (b) less than 300 meters.

[0010] In some embodiments of the method for treating a subject who has undergone ERT, muscle strength is measured by manual muscle testing (MMT); and the improvement from baseline in the MMT lower extremity score is at least 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5 points at 12, 24, 36, 48 months after initiation of treatment.

[0011] In some embodiments of the method for treating a subject who has undergone ERT, the subject has a baseline MMT Lower Extremity score of: (a) at least 25; or (b) less than 25.

[0012] In some embodiments of the method for treating a subject who has experienced ERT, lung function is measured by a seated forced vital capacity (FVC) test, and the subject's percent predicted FVC is stable compared to baseline 24 months or 36 months after initiating treatment.

[0013] In some embodiments of the method for treating an ERT-experienced subject, the subject has a baseline percent predicted FVC of: (a) at least 50%; or (b) less than 50%.

[0014] In some embodiments, the subject who has undergone ERT has previously been treated with alglucosidase alfa. In some embodiments, the subject who has undergone ERT has previously been treated with alglucosidase alfa for about 2 to about 6 years. In some embodiments, the subject who has undergone ERT has previously been treated with alglucosidase alfa for at least about 7 years. In some embodiments, the subject who has undergone ERT is unable to walk. In some embodiments, the subject who has undergone ERT is able to walk.

[0015] In some embodiments, the subject is an ERT-naive subject.

[0016] In some embodiments of the method for treating ERT-naive subjects, motor function is measured by a 6-minute walk test; and the improvement from baseline in 6-minute walk distance (6MWD) is at least 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 meters at 12, 24, 36, or 48 months after initiation of treatment.

[0017] In some embodiments of the method for treating ERT-naive subjects, motor function is measured by a 6-minute walk test; and the improvement from baseline in 6-minute walk distance (6MWD) is at least 34, 35, 40, 41, 42, 43, 44, or 45 meters 36 months after initiating treatment.

[0018] In some embodiments of the method for treating an ERT-naive subject, the subject has a baseline 6MWD of: (a) at least 300 meters; or (b) less than 300 meters.

[0019] In some embodiments of the method for treating ERT-naive subjects, muscle strength is measured by manual muscle testing (MMT); and the improvement from baseline in the MMT lower extremity score is at least 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5 points at 12, 24, 36, or 48 months after initiation of treatment.

[0020] In some embodiments of the method for treating an ERT-naive subject, the subject has a baseline MMT Lower Extremity score of: (a) at least 25; or (b) less than 25.

[0021] In some embodiments of the method for treating an ERT-naive subject, lung function is measured by a seated forced vital capacity (FVC) test; and the subject's improvement from baseline in percent predicted FVC is at least 2.0, 2.5, 3.0, 3.5, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.5, 6.0, 6.4, 6.5, 6.6, 6.7, 6.8, 7.0, 7.5, 8.0% at 12, 24, 36, or 48 months after initiating treatment.

[0022] In some embodiments of the method for treating ERT-naive subjects, lung function is measured by a seated forced vital capacity (FVC) test; and 36 months after initiating treatment, the subject's improvement from baseline in percent predicted FVC is at least 5.7%, 5.8%, 5.9%, 6.0%, 6.1%, or 6.2%.

[0023] In some embodiments of the method for treating an ERT-naive subject, the subject has a baseline percent predicted FVC of: a) at least 50%; or (b) less than 50%.

[0024] In some embodiments of any of the methods disclosed herein, the method further reduces the level of at least one muscle damage marker and / or at least one glycogen accumulation marker in the subject, compared to baseline. In some embodiments, the at least one muscle damage marker is creatine kinase (CK), and / or the at least one glycogen accumulation marker is urinary hexotetraose (Hex4).

[0025] In some embodiments, the population of rhGAA molecules is administered at a dose of 5 mg / kg to 20 mg / kg, optionally 20 mg / kg.

[0026] In some embodiments, the population of rhGAA molecules is administered once every two weeks. In some embodiments, the population of rhGAA molecules is administered intravenously.

[0027] In some embodiments, the enzyme stabilizer is miglustat or a pharmaceutically acceptable salt thereof, wherein miglustat or a pharmaceutically acceptable salt thereof is further optionally administered orally. In some embodiments, miglustat or a pharmaceutically acceptable salt thereof is administered at a dose of 195 mg or 260 mg.

[0028] In some embodiments, miglustat or a pharmaceutically acceptable salt thereof is administered prior to administration of the rhGAA molecule population, optionally one hour prior to administration of the rhGAA molecule population. In some embodiments, the subject fasts for at least two hours prior to administration of miglustat or a pharmaceutically acceptable salt thereof and fasts for at least two hours after administration of miglustat or a pharmaceutically acceptable salt thereof.

[0029] In some embodiments, the rhGAA molecule comprises an amino acid sequence at least 95% identical to SEQ ID NO:4 or SEQ ID NO:6.

[0030] In some embodiments, the rhGAA molecule comprises the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:6.

[0031] In some embodiments, at least 30% of the rhGAA molecules comprise one or more N-glycan units with one mannose-6-phosphate residue (mono-M6P) or bis-M6P as determined using LC-MS / MS.

[0032] In some embodiments, the rhGAA molecules comprise an average of 0.5 mol to 7.0 mol mono-M6P or bis-M6P per mol rhGAA as determined using LC-MS / MS.

[0033] In some embodiments, the rhGAA molecules comprise an average of 2.0 to 8.0 mol sialic acid / mol rhGAA as determined using LC-MS / MS.

[0034] In some embodiments, the rhGAA molecules comprise an average of at least 2.5 mol M6P / mol rhGAA and at least 4 mol sialic acid / mol rhGAA as determined using LC-MS / MS.

[0035] In some embodiments, per mol of rhGAA, the rhGAA molecules comprise: (a) an average of 0.4 to 0.6 mol of mono-M6P at the second potential N-glycosylation site; (b) an average of 0.4 to 0.6 mol of di-M6P at the fourth potential N-glycosylation site; or (c) an average of 0.3 to 0.4 mol of mono-M6P at the fourth potential N-glycosylation site; wherein (a)-(c) are determined using LC-MS / MS.

[0036] In some embodiments, the rhGAA molecules further comprise 4 to 7.3 mol of sialic acid per mol of rhGAA; and, per mol of rhGAA, the rhGAA molecules comprise: (a) an average of 0.9 to 1.2 mol of sialic acid at the third potential N-glycosylation site; (b) an average of 0.8 to 0.9 mol of sialic acid at the fifth potential N-glycosylation site; or (c) an average of 1.5 to 4.2 mol of sialic acid at the sixth potential N-glycosylation site; wherein (a)-(c) are determined using LC-MS / MS.

[0037] In some embodiments, the population of rhGAA molecules is formulated in a pharmaceutical composition further comprising at least one pharmaceutically acceptable buffer, excipient, or carrier.

[0038] In some embodiments, 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; wherein the pharmaceutical composition has a pH of 5.0 to 7.0.

[0039] In some embodiments, the pharmaceutical composition has a pH of 5.0 to 6.0.

[0040] In some embodiments, the pharmaceutical composition further comprises water, an acidifying agent, an alkalizing agent, or a combination thereof.

[0041] In some embodiments, in the pharmaceutical composition, the rhGAA molecule population is present at a concentration of 5-50 mg / mL, at least one buffer is a sodium citrate buffer present at a concentration of 10-100 mM, at least one excipient is mannitol present at a concentration of 10-50 mg / mL and polysorbate 80 present at a concentration of 0.1-1 mg / mL, and the pharmaceutical composition further comprises water and optionally an acidifier and / or an alkalinizer; wherein the pharmaceutical composition has a pH of 6.0.

[0042] In some embodiments, in the pharmaceutical composition, the rhGAA molecule population is present at a concentration of 15 mg / mL, the sodium citrate buffer is present at a concentration of 25 mM, the mannitol is present at a concentration of 20 mg / mL, and the polysorbate 80 is present at a concentration of 0.5 mg / mL.

[0043] In some embodiments, rhGAA is produced by Chinese hamster ovary cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1A Non-phosphorylated high mannose N-glycans, mono-M6P N-glycans, and di-M6P N-glycans are shown. Figure 1B The chemical structure of the M6P group is shown. Each square represents N-acetylglucosamine (GlcNAc), each circle represents mannose, and each P represents phosphate.

[0045] Figure 2A Productive targeting of rhGAA to target tissues (eg, muscle tissue in subjects with Pompe disease) via M6P-bearing N-glycans is described. Figure 2B Non-productive drug clearance to non-target tissues (eg, liver and spleen) or through binding of non-M6P N-glycans to non-target tissues has been described.

[0046] Figure 3 is a schematic diagram of an exemplary method for producing, capturing, and purifying recombinant lysosomal proteins.

[0047] Figure 4 Shown are DNA constructs used to transform CHO cells with DNA encoding rhGAA.

[0048] Figure 5 Graphs showing the results of CIMPR affinity chromatography of ATB200 rhGAA that was captured (Example 2) and not captured (Example 1) on an anion exchange (AEX) column.

[0049] Figures 6A-6H Shown are the results of site-specific N-glycosylation analysis of ATB200rhGAA using two different LC-MS / MS analytical techniques. Figure 6A Site occupancy of seven potential N-glycosylation sites of ATB200 is shown. Figure 6B Two analyses of the N-glycosylation profile of the first potential N-glycosylation site of ATB200 are shown. Figure 6C Two analyses of the N-glycosylation profile of the second potential N-glycosylation site of ATB200 are shown. Figure 6D Two analyses of the N-glycosylation profile of the third potential N-glycosylation site of ATB200 are shown. Figure 6E Two analyses of the N-glycosylation profile of the fourth potential N-glycosylation site of ATB200 are shown. Figure 6F Two analyses of the N-glycosylation profile of the fifth potential N-glycosylation site of ATB200 are shown. Figure 6G Two analyses of the N-glycosylation profile of the sixth potential N-glycosylation site of ATB200 are shown. Figure 6H The relative percentages of mono- and di-phosphorylated species are summarized for the first, second, third, fourth, fifth, and sixth potential N-glycosylation sites.

[0050] Figure 7 Yes Display Figure 2 shows the Polywax elution profiles of ATB200 (thinner line, eluting to the left) and ATB200 (thicker line, eluting to the right).

[0051] Figure 8 Figure 2 shows the comparison of three different formulations of ATB200rhGAA identified as BP-rhGAA, ATB200-1, and ATB200-2. Table of the overview of N-glycan structures.

[0052] Figure 9A and Figure 9B Are displayed separately and Figure 2 shows the CIMPR affinity chromatography results.

[0053] Figure 10 compares the CIMPR binding affinity of ATB200 rhGAA (left trace) with Plot of the CIMPR binding affinity (right trace).

[0054] Figure 11A The activity of ATB200rhGAA in normal fibroblasts (left trace) was compared with that in normal fibroblasts (left trace) at different GAA concentrations. Graph of rhGAA activity (right trace). Figure 11B ATB200rhGAA activity in fibroblasts from subjects with Pompe disease (left trace) was compared with that in fibroblasts from subjects with Pompe disease at different GAA concentrations. Table of rhGAA activity (right trace). Figure 11C is a comparison of K between fibroblasts from normal subjects and subjects with Pompe disease 摄取 table.

[0055] Figure 12 Depicted is the stability of ATB200 in acidic or neutral pH buffers, which was assessed in a thermal stability assay using SYPROOrange, as the fluorescence of the dye increases upon protein denaturation.

[0056] Figure 13 Shown are tissue glycogen content in WT or Gaa KO mice treated with vehicle, alglucosidase alfa, or ATB200 / migestastat, as measured by amyloglucosidase digestion. Bars represent the mean ± SEM of 7 mice per group. *P < 0.05 compared with alglucosidase alfa using one-way ANOVA with Dunnett's multiple comparison method.

[0057] Figure 14 Depicted are LAMP1-positive vesicles in myofibers of Gaa KO or WT mice treated with vehicle, alglucosidase alfa, or ATB200 / miglustat. Images were taken from the vastus lateralis muscle and are representative of 7 mice / group. Magnification = 200x (1,000x in inset).

[0058] Figure 15A Shown are LC3-positive aggregates in myofibers of Gaa KO or WT mice treated with vehicle, alglucosidase alfa, or ATB200 / migestastat. Images were taken from the vastus lateralis muscle and are representative of 7 mice / group. Magnification = 400x. Figure 15B Western blot analysis of LC3 II protein is shown. A total of 30 mg of protein was loaded per lane.

[0059] Figure 16 Dysferlin expression in myofibers of Gaa KO or WT mice treated with vehicle, alglucosidase alfa, or ATB200 / migestastat is shown. Images were taken from the vastus lateralis muscle and are representative of 7 mice / group. Magnification = 200x.

[0060] Figure 17 Depicts co-immunofluorescence staining of LAMP1 (green) (see, e.g., "B") and LC3 (red) (see, e.g., "A") in single fibers isolated from white gastrocnemius muscle of Gaa KO mice treated with vehicle, alglucosidase α, or ATB200. "C" depicts clearance of autophagic debris and absence of enlarged lysosomes. At least 30 fibers were examined per animal.

[0061] Figure 18 Depicted is the stabilization of ATB200 by miglustat at 17 μM and 170 μM, respectively, compared to ATB200 alone.

[0062] Figures 19A-19H Shown are the results of site-specific N-glycosylation analysis of ATB200 rhGAA, including the N-glycosylation profile at the seventh potential N-glycosylation site, using LC-MS / MS analysis of protease-digested ATB200. Figures 19A-19HAverage data of ten batches of ATB200 produced at different scales are presented.

[0063] Figure 19A Shown are the average site occupancies of seven potential N-glycosylation sites of ATB200. The N-glycosylation sites are provided according to SEQ ID NO: 1. CV = coefficient of variation.

[0064] Figures 19B-19H Site-specific N-glycosylation analysis of all seven potential N-glycosylation sites of ATB200 is shown, with site numbering provided according to SEQ ID NO: 5. Bars represent the maximum and minimum percentages of N-glycan species identified as specific N-glycan groups for the ten batches of ATB200 analyzed. Figure 19B The N-glycosylation profile of the first potential N-glycosylation site of ATB200 is shown. Figure 19C The N-glycosylation profile of the second potential N-glycosylation site of ATB200 is shown. Figure 19D The N-glycosylation profile of the third potential N-glycosylation site of ATB200 is shown. Figure 19E The N-glycosylation profile of the fourth potential N-glycosylation site of ATB200 is shown. Figure 19F The N-glycosylation profile of the fifth potential N-glycosylation site of ATB200 is shown. Figure 19G The N-glycosylation profile of the sixth potential N-glycosylation site of ATB200 is shown. Figure 19H The N-glycosylation profile of the seventh potential N-glycosylation site of ATB200 is shown.

[0065] Figure 20A-Figure 20B Further characterization and summary of Figures 19A-19H N-glycosylation profile of ATB200 is shown. Figure 20A 2-Anthranilic acid (2-AA) glycan mapping and LC / MS-MS analysis of ATB200 are shown and the percentage of total fluorescence of the N-glycan species identified in ATB200 is summarized. Data from 2-AA glycan mapping and LC-MS / MS analysis are also described in Table 6. Figure 20B The average site occupancy and average N-glycan profiles for all seven potential N-glycosylation sites of ATB200 are summarized, including total phosphorylation, monophosphorylation, diphosphorylation, and sialylation. ND = Not Detected.

[0066] Figure 21 A schematic diagram of the ATB200-03 study design is shown.

[0067] Figure 22Baseline 6-minute walk distance (6MWD) and seated forced vital capacity (FVC) characteristics of 122 subjects participating in the ATB200-03 study are shown. The AT-GAA group included subjects who received ATB200 / miglustat and the alglucosidase alfa group included subjects who received alglucosidase alfa / placebo.

[0068] Figure 23A Depicted are 6MWD and FVC data showing baseline, change from baseline at Week 52 ("CFBL"), differences, and P values ​​for the total population (n=122). AT-GAA group: subjects receiving ATB200 / miglustat; alglucosidase alfa group: subjects receiving alglucosidase alfa / placebo.

[0069] Figure 23B 6MWD and FVC data are depicted, showing changes over time from baseline for the total population (n=122).Cipaglucosidase alfa / miglustat group: subjects receiving ATB200 / miglustat; alglucosidase alfa / placebo: subjects receiving alglucosidase alfa / placebo.

[0070] Figure 24 Depicted are 6MWD and FVC data showing baseline, week 52 CFBL, differences, and P values ​​for the ERT-experienced population (n=95). AT-GAA group: subjects treated with ATB200 / miglustat; alglucosidase alfa group: subjects treated with alglucosidase alfa / placebo.

[0071] Figure 25 Changes from baseline in 6MWD and FVC at Weeks 12, 26, 38, and 52 are depicted for the ERT-experienced population (n=95).

[0072] Figure 26A Depicted are 6MWD and FVC data showing baseline, week 52 CFBL, differences, and P values ​​for the ERT-naive population (n=27). AT-GAA group: subjects receiving ATB200 / miglustat; alglucosidase alfa group: subjects receiving alglucosidase alfa / placebo.

[0073] Figure 26B 6MWD and FVC data are depicted, showing changes over time from baseline in an ERT-naive population (n=27).Cipaglucosidase alfa / miglustat group: subjects receiving ATB200 / miglustat; alglucosidase alfa / placebo: subjects receiving alglucosidase alfa / placebo.

[0074] Figure 27 Baseline characteristics of key secondary endpoints and biomarkers are described for the overall population and the ERT-experienced population: AT-GAA group: subjects receiving ATB200 / miglustat; alglucosidase alfa group: subjects receiving alglucosidase alfa / placebo.

[0075] Figure 28 Depicted are changes from baseline in lower manual muscle testing (MMT) at weeks 12, 26, 38, and 52 for the total population (left) and the ERT-experienced population (right).

[0076] Figure 29 Depicted are changes in gait, stairs, gowers, and chair (GSGC) from baseline at Weeks 12, 26, 38, and 52 for the overall population (left) and the ERT-experienced population (right). Cipaglucosidase alfa / miglustat group: subjects receiving ATB200 / miglustat; alglucosidase alfa / placebo: subjects receiving alglucosidase alfa / placebo.

[0077] Figure 30 Patient-Reported Outcomes Measurement Information System (PROMIS) data depicting changes in physical function from baseline at weeks 12, 26, 38, and 52 for the total population (left) and the population that underwent ERT (right).

[0078] Figure 31 PROMIS showing the change from baseline in fatigue at Weeks 12, 26, 38, and 52 for the total population (left) and the ERT-experienced population (right) are depicted.

[0079] Figure 32 Depicted are changes from baseline in the creatine kinase (CK) biomarker at weeks 12, 26, 38, and 52 for the total population (left) and the ERT-experienced population (right).

[0080] Figure 33 Depicted are changes from baseline in urinary hexotetraose (Hex4) biomarkers at weeks 12, 26, 38, and 52 for the total population (left) and the ERT-experienced population (right).

[0081] Figure 34 Heat maps of primary, secondary, and biomarker endpoints are shown for the overall population (left) and the ERT-experienced population (right). AT-GAA group: subjects who received ATB200 / miglustat; alglucosidase alfa group: subjects who received alglucosidase alfa / placebo.

[0082] Figure 35 This paper summarizes the safety data from the ATB200-03 study. The AT-GAA group included subjects who received ATB200 / miglustat; the alglucosidase alfa group included subjects who received alglucosidase alfa / placebo. TEAEs: treatment-emergent adverse events; IARs: infusion-related reactions.

[0083] Figure 36 The results of the ATB200-03 study are summarized.

[0084] Figure 37 The study objectives and statistical methods of the ATB200-03 study are described.

[0085] Figure 38 The primary and secondary endpoints of the ATB200-03 study are described.

[0086] Figure 39 Patient disposition in the ATB200-03 study is summarized.

[0087] Figure 40 The baseline demographic characteristics of the ATB200-03 study population are summarized.

[0088] Figure 41 Shown are subgroup analyses of changes from baseline in 6MWD and FVC according to baseline status in the total population (n=122) (Group A) and in patients who underwent ERT (n=95) (Group B) in the ATB200-03 study.

[0089] Figure 42 Shown is a list of treatment-emergent adverse events (TEAEs) in ≥10% of patients in any group in the ATB200-03 study.

[0090] Figure 43 The study design of the phase I / II ATB200-02 study is shown. Asterisks indicate that the previous ERT was with 20 mg / kg alglucosidase alfa Q2W. Q2W, every 2 weeks

[0091] Figure 44 A summary of the endpoints and cohorts reported for the ATB200-02 study is shown.

[0092] Figure 45 Baseline characteristics and patient disposition in the ATB200-02 study are shown. An asterisk indicates an ERT-naive patient who received one dose of alglucosidase alfa >6 months before study entry. M denotes meter; M:F denotes male-to-female ratio; N / A denotes not applicable; SD denotes standard deviation.

[0093] Figures 46A-46DThe subjects who underwent ERT in the ATB200-02 study ( Figure 46A 、 46C ) and ERT-naive subjects ( Figure 46B 、 46D ) over time from baseline (CFBL).

[0094] Figures 47A-47B The subjects who underwent ERT in the ATB200-02 study ( Figure 47A ) and ERT-naive subjects ( Figure 47B Mean change from baseline (CFBL) in percent predicted sitting forced vital capacity (FVC) over time.

[0095] Figures 48A-48B The subjects who underwent ERT in the ATB200-02 study ( Figure 48A ) and ERT-naive subjects ( Figure 48B ) in the manual muscle test (MMT) lower extremity score over time from baseline (CFBL).

[0096] Figure 49A-49B Shown are urinary hexotetraose (Hex4) levels in ERT-experienced and ERT-naive subjects in the ATB200-02 study ( Figure 49A ) and plasma creatine kinase (CK) levels ( Figure 49B ) Mean percentage change from baseline (CFBL).

[0097] Figure 50 A summary of treatment-emergent adverse events (TEAEs) in the ATB200-02 study is shown. Asterisks indicate diffuse large B-cell lymphoma. IARs denote infusion-related reactions; TEAEs are treatment-emergent adverse events with an onset date on or after the first dose of study drug.

[0098] Figure 51 Shown is a comparison of the long-term effects of sipagnosidase alfa / miglustat and efavirenz alfa on changes from baseline in 6MWD and percent predicted FVC (sitting) in subjects who had undergone ERT.

[0099] Figure 52 Shown is a comparison of the long-term effects of sipagnosidase alfa / miglustat and efavirenz alfa on changes from baseline in 6MWD and percent predicted FVC (sitting) in ERT-naive subjects.

[0100] Figure 53A-Figure 53B Shown are the percent predicted 6-minute walk test (6MWT) during treatment with alglucosidase alfa in ERT-experienced subjects. Figure 53B Shows the Figure 53A Data replotted only from year 2 onwards.

[0101] Figure 54 Shown are the percent predicted FVC during treatment with alglucosidase alfa in ERT-experienced subjects.

[0102] Figure 55 Endpoints and cohort summaries are shown for Cohort 2 (nonambulatory ERT-experienced patients) of the ATB200-02 study.

[0103] Figure 56 Baseline characteristics and patient disposition are shown for Cohort 2 (nonambulatory ERT-experienced patients) of the ATB200-02 study. Asterisks indicate the last nonmissing result for the baseline assessment, which was administered at or before the first dose of study drug (20 mg / kg sipa-glucosidase alfa plus 260 mg miglustat co-administered). M:F denotes the male-to-female ratio; SD denotes standard deviation.

[0104] Figure 57 Shown are the mean changes in percent predicted sitting forced vital capacity (FVC) over time from baseline (CFBL) in cohort 2 (nonambulatory ERT-experienced patients).

[0105] Figure 58 Shown is a summary of treatment-emergent adverse events (TEAEs) for Cohort 2 (nonambulatory ERT-experienced patients) in the ATB200-02 study. Asterisks indicate urticaria considered an IAR. IAR denotes infusion-related reaction; TEAEs are treatment-emergent adverse events with an onset date on or after the first dose of study drug.

[0106] Figure 59 Baseline characteristics of seven clinical studies identified by a systematic literature review (SLR) are shown

[0107] Figure 60 Longitudinal efficacy results for 6MWD (m) and FVC (% predicted) as changes from baseline compared to the trials are shown for each identified study.

[0108] Figure 61 Networks for 6MWD (m) and sitting FVC (% predicted) are shown.

[0109] Figure 62 Forest plot showing the relative effect estimates of 6MWD in the base case scenario (primary analysis), with 95% confidence intervals.

[0110] Figure 63Forest plots showing the relative effect estimates for FVC in the base case scenario (primary analysis), with 95% confidence intervals.

[0111] Figure 64 Shown are forest plots of the relative effect estimates on 6MWD according to ERT duration, with 95% confidence intervals.

[0112] Figure 65 Shown are forest plots of the relative effect estimates on FVC according to ERT duration, with 95% confidence intervals.

[0113] Figure 66 Forest plot showing the relative effect estimates of 6MWD in the base case scenario (sensitivity analysis), with 95% confidence intervals.

[0114] Figure 67 Forest plots showing relative effect estimates for FVC in the base case scenario (sensitivity analysis) with 95% confidence intervals.

[0115] Figure 68 The study design and patient disposition of the ATB200-07 study are shown.

[0116] Figure 69 The baseline demographic characteristics of the ATB200-07 study population were summarized.

[0117] Figures 70A-70B The predicted 6MWD% ( Figure 70A ) and 6MWD%( Figure 70B ) mean change from baseline.

[0118] Figure 71 Shown are the mean changes from baseline in % predicted FVC for ERT-experienced and ERT-naive patients in the ATB200-07 study.

[0119] Figure 72 Shown are the mean changes from baseline in serum CK in ERT-experienced and ERT-naive patients in the ATB200-07 study.

[0120] Figure 73 Shown are the mean changes from baseline in urinary Hex4 in ERT-experienced and ERT-naive patients in the ATB200-07 study.

[0121] Figure 74 The safety summary of the ATB200-07 study is shown.

[0122] Figure 75is a graph showing the distribution of 2-AA labeled N-glycans identified by LC-FLD analysis of alglucosidase alfa and three sipa-glucosidase alfa preparations.

[0123] Figure 76 A depicts protein loading control for Western blots of mock-treated alglucosidase alpha, mock-treated sipa-glucosidase alpha, and purple acid phosphatase (PAP)-treated sipa-glucosidase alpha. Figure 76 B shows a Western blot depicting GAA protein levels for the samples shown in 76 A. 76C shows a variation of a far-western blot to determine CIMPR binding for the samples in 76 A. 76D is a graph showing 4MU-α-glucosidase activity for the samples shown in 76 A.

[0124] Figure 77 A is a graph showing the uptake of internalized rhGAA in skeletal muscle myoblasts at various rhGAA concentrations. Figure 77 B Comparison of GAA activities of mock-treated alglucosidase alfa, mock-treated sipa-glucosidase alfa, and PAP-treated sipa-glucosidase alfa in Pompe disease patient-derived fibroblasts at 20 nM GAA concentration. Figure 77 C is the image after taking Figure 77 Western blot of GAA content of cell lysates shown in B.

[0125] Figure 78 The graph shows the effect of chronic sipa-glucosidase alfa administration (20 mg / kg, 12 biweekly boluses) compared to alglucosidase alfa on myofiber size in terms of mean smallest fiber diameter (FD) of the quadriceps femoris of Gaa KO mice.

[0126] Figure 79 A schematic diagram illustrating the design of study ATB200-02.

[0127] Figure 80 Schematic diagram illustrating the design of study ATB200-03.

[0128] Figure 81 is a line graph of the LS means (SE) of the change from baseline in MMT lower extremity scores over time (ITT-LOCF population) for the ERT-naive population in Study ATB200-03, excluding subject 4005-2511.

[0129] Figure 82 is a line graph of the LS means (SE) of the change from baseline in PROMIS-Physical Function Total Score over time (ITT-LOCF population) for the ERT-naive population in Study ATB200-03, excluding subject 4005-2511.

[0130] Figure 83 is a line graph of the LS means (SE) of the change in PROMIS-Fatigue total score (ITT-LOCF population) for the ERT-naive population in Study ATB200-03, excluding subject 4005-2511.

[0131] Figure 84 is a line graph of the LS mean (SE) change from baseline in GSGC total score over time (ITT-LOCF population) for the ERT-naive population in Study ATB200-03, excluding subject 4005-2511.

[0132] Figure 85 The bar graph summarizes all endpoints for the ITT population (excluding outlier subject 4005-2511) in Study ATB200-03.

[0133] Figure 86 The bar graph illustrates overall physical health status of SGIC at Week 52 compared to baseline in Study ATB200-03.

[0134] Figure 87A The bar graph illustrates the proportion of subjects with a change from baseline in 6MWD (meters) at Week 52 by pooled range group (ITT-LOCF, excluding subjects 4005-2511) in Study ATB200-03. Figure 87B The bar graph illustrates the proportion of subjects with a change from baseline in % predicted sitting FVC at Week 52 by pooled range group (ITT-LOCF, excluding subjects 4005-2511) in Study ATB200-03. Figure 87C The bar graph illustrates the proportion of subjects with a composite response for 6MWD and % predicted FVC at Week 52 (ITT-LOCF excluding subjects 4005-2511) in Study ATB200-03.

[0135] Figure 88 is a line graph of the mean (±SE) change in 6MWD (meters) over time from Study ATB200-03 baseline for the OLE-ES population excluding Subject 4005-2511 provided by Study ATB200-07.

[0136] Figure 89 is a line graph of the mean (±SE) change in % predicted FVC sitting over time from baseline in Study ATB200-03 for the OLE-ES population excluding Subject 4005-2511 provided by Study ATB200-07.

[0137] Figure 90Ais a line graph of the mean (±SE) change in CK (U / L) over time from Study ATB200-03 baseline for the OLE-ES population excluding Subject 4005-2511 provided by Study ATB200-07. Figure 90B is a line graph of the mean (±SE) change in Hex4 (mmol / mol creatinine) over time from Study ATB200-03 baseline for the OLE-ES population excluding Subject 4005-2511 provided by Study ATB200-07. DETAILED DESCRIPTION

[0138] Provided herein are methods for treating Pompe disease, comprising administering recombinant human α-glucosidase (rhGAA) and an enzyme stabilizer to an individual, wherein the method provides long-term benefits (e.g., improvement and / or stabilization of motor function, muscle strength, and / or lung function). The methods provided herein also have a good safety profile. The long-term benefits of the methods provided herein are improvements to enzyme replacement therapy (ERT) options currently used to treat Pompe disease.

[0139] Current ERT options for treating Pompe disease provide limited improvement, followed by a slow decline. In 2012, a systematic review of all studies conducted in subjects with late-onset Pompe disease (LOPD) was conducted (Toscano and Schoser, (2013) J Neurol 260, 951-959). The review included data from 368 subjects with LOPD from published studies, including 27 adolescent subjects (age range: 2 to 17 years) and 251 adults who received alglucosidase alfa for at least 2 years. The results showed that >30% of subjects did not show initial improvement during alglucosidase alfa treatment and continued to experience worsening muscle and respiratory function despite treatment. Among subjects who initially responded to alglucosidase alfa, several additional long-term studies have shown that improvement typically lasts only approximately 2 years. Thereafter, subjects typically plateau before gradually declining.

[0140] In 2012, the UK Health Technology Assessment Programme (Wyatt et al. (2012) Health Technol Assess 16(39)), part of the UK National Institute for Health Research, issued recommendations based on a review of longitudinal data from 81 patients with Pompe disease (both infantile and late-onset (children and adults)) who received the currently approved standard of care ERT, alglucosidase alfa. Key markers of Pompe disease progression (forced vital capacity, ventilatory dependency, mobility, 6-minute walk test, muscle strength, and body mass index) were assessed and modeled in relation to the duration of treatment with alglucosidase alfa. The results of this review showed that within the first 2 years after starting ERT with alglucosidase alfa, patients with LOPD showed improvements in FVC, 6-minute walk distance, and muscle strength, which declined with continued treatment beyond this time frame. Furthermore, a 3-year study of 38 subjects with LOPD who were treated with alglucosidase alfa showed that motor function improved in the first year of treatment, remained generally stable in the second year, and began to decline in the third year (Regnery et al. (2012) Journal of Inherited Metabolic Disease 35:837-845).

[0141] In addition, a A 10-year follow-up report from a phase 3 study (Sanofi Genzyme) showed that participants experienced improvements in motor and lung function during the first few years of treatment, followed by a slow decline with continued treatment (van der Ploeg et al. (2017) European Journal of Neurology 24.6:768-e31). In this study, the average decline in the percentage predicted baseline 6-minute walk distance from year 3 to year 6 of treatment was approximately 10%, with approximately 80% of participants experiencing decline.

[0142] The most serious tolerability issue with alglucosidase alfa is the occurrence of infusion-related reactions (IARs), which in some cases may include life-threatening anaphylaxis or other severe allergic reactions ( Summary of Product Characteristics, December 2018). Management of these events includes dose reduction, reduced infusion rate, and prolonged infusion duration, as well as dose interruption or discontinuation. Premedication (before infusion) with antihistamines and steroids is also frequently used to prevent and reduce the occurrence and severity of IAR and hypersensitivity reactions associated with alglucosidase alfa infusion. Despite these measures, patients with Pompe disease may experience IAR, and some patients are unable to tolerate routine infusions of currently approved ERT.

[0143] In 2017, the European Pompe Disease Consortium, a network of experts in the field of Pompe disease from 11 European countries, conducted a systematic review of the literature (van der Ploeg et al. (2017) European Journal of Neurology 24.6:768-e31). The consortium evaluated the evidence for the effects of ERT at the group level based on data from one clinical study and 43 observational studies (covering a total of 586 adult subjects). The current consensus of the European Pompe Disease Consortium is to discontinue ERT therapy once severe IAR or progressive clinical worsening of disease symptoms occurs, as well as the presence of high neutralizing antibody (Ab) titers (which would effectively render existing ERT therapy ineffective). The European Pompe Disease Consortium's consensus recommendation also includes consideration of restarting ERT if disease progression and clinical worsening recur after ERT cessation.

[0144] The cellular uptake of rhGAA molecules is facilitated by the specialized carbohydrate mannose-6-phosphate (M6P), which binds to the cation-independent mannose-6-phosphate receptor (CIMPR) present on target cells (such as muscle cells). After binding, rhGAA molecules are taken up by the target cells and subsequently transported to the lysosomes within the cells. However, most conventional rhGAA products lack a high total content of mono-M6P- and di-M6P-bearing N-glycans (i.e., N-glycans with one M6P residue or N-glycans with two M6P residues, respectively), which limits their cellular uptake via CIMPR and lysosomal delivery, thereby rendering conventional enzyme replacement therapy less effective. For example, while conventional rhGAA products at doses of 20 mg / kg or higher can improve some aspects of Pompe disease, they are unable to adequately (i) treat the underlying cellular dysfunction; (ii) restore muscle structure; or (iii) reduce the accumulated glycogen in many target tissues (such as skeletal muscle) to reverse disease progression. Furthermore, higher doses impose additional burdens on the subject and the medical professionals treating the subject, such as the prolonged infusion time required for intravenous administration of rhGAA.

[0145] Glycosylation of GAA or rhGAA can be enzymatically modified in vitro using phosphotransferases and uncovering enzymes as described by Canfield et al., U.S. Patent No. 6,534,300 to produce M6P groups. However, enzymatic glycosylation cannot be adequately controlled and can produce rhGAA with undesirable immunological and pharmacological properties. Enzymatically modified rhGAA may contain only high-mannose oligosaccharides, all of which can potentially be enzymatically phosphorylated in vitro using phosphotransferases or uncovering enzymes. The glycosylation pattern generated by in vitro enzymatic treatment of GAA is problematic because the additional terminal mannose residues, particularly non-phosphorylated terminal mannose residues, negatively impact 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 the therapeutic efficacy of rhGAA because less GAA reaches target tissues (such as skeletal muscle myocytes). For example, terminal non-phosphorylated mannose residues are known ligands for mannose receptors in the liver and spleen, which leads to rapid clearance of enzymatically modified rhGAA and reduced targeting of rhGAA to target tissues. In addition, the glycosylation pattern of enzymatically modified GAA with high mannose N-glycans containing terminal non-phosphorylated mannose residues is similar to the glycosylation pattern on glycoproteins produced in yeast and molds, and increases the risk of triggering an immune or allergic reaction to enzymatically modified rhGAA, such as a life-threatening severe allergic (anaphylactic) or hypersensitivity reaction.

[0146] Compared to conventional recombinant rhGAA products and in vitro phosphorylated rhGAA, the rhGAA used in the two-component therapy according to the present disclosure has an optimized N-glycan profile for enhanced biodistribution and lysosomal uptake, thereby minimizing non-productive clearance of rhGAA after administration. The present disclosure provides an effective therapy for reversing disease progression at the cellular level for patients with stable or worsening Pompe disease - including more effective clearance of lysosomal glycogen than current standard of care. Patients treated with the two-component therapy of the present disclosure comprising rhGAA and an enzyme stabilizer (e.g., miglustat) showed significant health improvements, including improvements and / or stabilization of muscle strength, motor function and / or lung function, and / or including reversal of disease progression.

[0147] In addition, three randomized clinical trials (LOTS: alglucosidase alfa versus placebo; COMET: efrucisidase alfa versus alglucosidase alfa; and PROPEL: sipagliflozin / miglustat versus alglucosidase alfa) shared 6MWD and predicted FVC% as primary or secondary endpoints. Using patient-level data from the PROPEL randomized clinical trial (RCT) plus pooled published data from other RCTs, phase I / II, and open-label extension trials, a multilevel network meta-regression was performed, adjusting for various baseline covariates, including prior ERT duration. The base case scenario had covariates similar to the PROPEL population (treatment-naive and ERT-experienced), and the change from baseline in 6MWD and FVC at 52 weeks was analyzed. In terms of 6MWD and FVC, sipa-glucosidase alfa / miglustat was more favorable than alglucosidase alfa and avrilase alfa, with relative effects of 16.3 meters (95% confidence interval: 9.6-24.3) and 29.5 meters (7.4-52.6) for 6MWD, and 3.1% (2.4-3.8) and 2.8% (1.0-4.6) for FVC, respectively. For treatment-naive subjects, the relative effect of sipa-glucosidase alfa / miglustat remained significant for both endpoints, except for FVC, which was 0.4% (-0.8-1.7) for sipa-glucosidase alfa / miglustat compared to aval. These findings suggest that sipa-glucosidase alfa / miglustat may make a clinical difference in key motor and respiratory endpoints for patients with LOPD, especially in the ERT-experienced population. definition

[0148] The terms used in this specification generally have their ordinary meaning in the art in the context of the present disclosure and in the specific context of using each term. Certain terms are discussed elsewhere below or in the specification to provide additional guidance to the practitioner when describing the compositions and methods of the present disclosure and how to prepare and use them. The article "a and an" refers to the grammatical object of one or more than one (that is, at least one) of the article. Unless the context clearly states otherwise, the term "or" refers to the term "and / or" and can be used interchangeably with the term "and / or". In this application, the use of the singular includes the plural, unless clearly stated otherwise. In addition, the use of the term "including (including)", and other forms such as "including (includes)" and "including (included)" is not restrictive. Any range described herein will be understood to include all values ​​between endpoints and endpoints. In this specification, unless the context requires otherwise, due to express language or necessary meaning, the word "comprises", or variations such as "comprising" are used in an inclusive sense, that is, to specify the presence of the stated features, but not to exclude other features that may be present or added in different embodiments of the present disclosure.

[0149] The term "GAA" refers to human acid alpha-glucosidase (GAA), an enzyme that catalyzes the hydrolysis of α-1,4-glycosidic bonds and α-1,6-glycosidic bonds of lysosomal glycogen, and refers to insertions, related or substitution variants of the GAA amino acid sequence, and fragments of longer GAA sequences that exhibit enzymatic activity. Human acid alpha-glucosidase is encoded by the GAA gene (National Center for Biotechnology Information (NCBI) gene ID 2548), which has been mapped to the long arm of chromosome 17 (position 17q25.2-q25.3). An exemplary amino acid sequence of GAA is NP 000143.2, which is incorporated by reference. The present disclosure also includes a DNA sequence encoding the amino acid sequence of NP000143.2. More than 500 mutations have been identified in the human GAA gene, many of which are associated with Pompe disease. The mutation that causes the misfolding or misprocessing of this acid alpha-glucosidase includes T1064C (Leu355Pro) and C2104T (Arg702Cys). In addition, the GAA mutation that affects the maturation and processing of enzyme includes Leu405Pro and Met519Thr. The conserved hexapeptide WIDMNE (SEQ ID NO:7) at amino acid residues 516-521 is essential for the activity of acid alpha-glucosidase protein. As used herein, the abbreviation "GAA" is intended to mean human acid alpha-glucosidase, but the italic abbreviation "GAA" is intended to mean the human gene encoding human acid alpha-glucosidase. The italic abbreviation "Gaa" is intended to mean the non-human gene encoding non-human acid alpha-glucosidase, including but not limited to rat or mouse genes, and the abbreviation "Gaa" is intended to mean non-human acid alpha-glucosidase.

[0150] The term "rhGAA" is intended to mean recombinant human acid alpha-glucosidase and is used to distinguish synthetically and / or recombinantly produced GAA (e.g., GAA produced from CHO cells or other host cells transformed with DNA encoding GAA) from endogenous GAA. Thus, rhGAA does not include endogenous GAA. The term "rhGAA" encompasses a population of individual rhGAA molecules. Characterization of populations of rhGAA molecules is provided herein. The term "conventional rhGAA product" is intended to refer to products containing alglucosidase alpha (e.g., or ) or efrucidusidase alfa (e.g. ) products.

[0151] The term "genetically modified" or "recombinant" refers to cells (such as CHO cells) that express a specific gene product (such as rhGAA) after the introduction of a nucleic acid comprising a coding sequence encoding the gene product, together with regulatory elements that control the expression of the coding sequence. The introduction of nucleic acids 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 (e.g., by gene activation technology) to express or overexpress endogenous genes or gene products that are not normally expressed by such cells.

[0152] As used herein, the term "alglucosidase alfa" is intended to refer to recombinant human acid alpha-glucosidase identified as [199-arginine, 223-histidine] precursor pro-alpha-glucosidase (homologous); Chemical Abstracts Registry Number 420794-05-0. Alglucosidase alfa is approved for sale in the United States by Sanofi Genzyme as a product and

[0153] As used herein, the term "ifugosidase alfa" is intended to refer to the recombinant human acid α-glucosidase identified as ifugosidase alfa-ngpt; Chemical Abstracts Registry Number 1802558-87-7. Ifugosidase alfa is approved for sale in the United States by Sanofi Genzyme as a product

[0154] As used herein, the term "ATB200" refers to the recombinant human acid α-glucosidase described in International Patent Application No. PCT / 2015 / 053252, U.S. Patent No. 10,208,299, and U.S. Patent No. 10,961,522 (the disclosures of which are incorporated herein by reference in their entireties). ATB200 is also referred to as "sipa-glucosidase α." In some embodiments, "ATB200" refers to rhGAA having a high content of N-glycans with mono- and di-M6P, which is produced by the GA-ATB200 cell line and purified using the methods described herein.

[0155] As used herein, the term "glycan" is intended to refer to an oligosaccharide covalently bound 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 attached to an asparagine residue on a protein or polypeptide by covalently binding to the nitrogen atom of the asparagine residue. In some embodiments, the N-glycan units attached to rhGAA can be analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) using an instrument such as Thermo Scientific. TM (ThermoScientific TM )Orbitrap Velos Pro TM Mass spectrometer, Thermo Scientific TMOrbitrap Fusion TM LumosTribid TM Mass spectrometer or Waters The results were determined by G2-XS QTof mass spectrometer. As used herein, the term "mono-M6P-bearing glycan" or "bis-M6P-bearing glycan" means a monophosphorylated (mono-M6P) or diphosphorylated (bis-M6P) N-glycan unit as part of all N-glycan types, unless a high mannose N-glycan type or a hybrid N-glycan type is specifically indicated.

[0156] As used herein, forced vital capacity or "FVC" is the amount of air that can be forcefully exhaled from a subject's lungs after the subject takes the deepest possible breath.

[0157] As used herein, the "Six-Minute Walk Test" (6MWT) is a test used to measure the distance an individual can walk in six minutes on a hard, flat surface. The test is performed by having the individual walk as far as possible in six minutes.

[0158] As used herein, the "Ten Meter Walk Test" (10MWT) is a test used to measure the time it takes an individual wearing walking shoes to walk ten meters on a flat surface.

[0159] 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, is a compound having the following chemical formula: Alternatively it can be displayed as:

[0160] A formulation of Miglustat is marketed as monotherapy for type 1 Gaucher disease under the trade name In some embodiments, miglustat is referred to as AT2221.

[0161] As discussed below, pharmaceutically acceptable salts of miglustat may also be used in the present disclosure. When a salt of miglustat is used, the dosage of the salt will be adjusted so that the patient receives a dose of miglustat that is comparable to the amount received when the free base of miglustat is used.

[0162] 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, is a compound having the following chemical formula:

[0163] As used herein, the term "enzyme stabilizer" is intended to refer to a molecule that specifically binds to acid α-glucosidase and has one or more of the following effects: Enhance the formation of stable molecular conformations of proteins; Enhance the proper transport of proteins from the endoplasmic reticulum to another cellular location, preferably the native cellular location, in order to prevent endoplasmic reticulum-associated degradation of the proteins; Prevent aggregation of conformationally unstable or misfolded proteins; Restoring and / or enhancing at least part of the wild-type function, stability, and / or activity of a protein; Improving the phenotype or function of cells having acid alpha-glucosidase; and / or • Stabilization of acid alpha-glucosidase in vitro and / or in vivo (eg in the blood of a patient).

[0164] Enzyme stabilizers are sometimes also called "pharmacological chaperones."

[0165] Therefore, an enzyme stabilizer for acid α-glucosidase is a molecule that binds to acid α-glucosidase, resulting in proper folding, transport, non-aggregation and / or activity of acid α-glucosidase. In at least one embodiment, the enzyme stabilizer is miglustat. Another non-limiting example of an enzyme stabilizer for acid α-glucosidase is deoxynojirimycin.

[0166] As used herein, the term "pharmaceutically acceptable" is intended to refer to molecular entities and compositions that are physiologically tolerable and generally do not produce adverse reactions when administered to humans. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by the regulatory agencies of the federal or state governments or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeias for animals (more particularly for humans). As used herein, the term "carrier" is intended to refer to diluents, adjuvants, excipients or vehicles used together with the compound. Suitable pharmaceutical carriers are known in the art, and in at least one embodiment, are described in "Remington's Pharmaceutical Sciences" by E.W. Martin, 18th edition, or other versions.

[0167] 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, etc., commensurate with a reasonable benefit / risk ratio, generally water- or oil-soluble or dispersible, and effective for their intended use. The term includes pharmaceutically acceptable acid addition salts as well as pharmaceutically acceptable base addition salts. Lists of suitable salts are found, for example, in SM Berge et al., J. Pharm. Sci., 1977, 66, pp. 1-19, incorporated herein by reference. As used herein, the term "pharmaceutically acceptable acid addition salts" means those 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" means those salts formed with inorganic bases that retain the biological effectiveness and properties of the free acids and are not biologically or otherwise undesirable.

[0168] As used herein, the term "buffer" refers to a solution containing a weak acid and its conjugate base, or a weak base and its conjugate acid, which helps prevent changes in pH.

[0169] As used herein, the terms "therapeutically effective dose" and "effective amount" are intended to mean an amount of acid alpha-glucosidase, and / or an amount of miglustat, and / or an amount of a dual-component therapy thereof, sufficient to result in a therapeutic response in a subject.

[0170] Therapeutic response may also include molecular responses, such as glycogen accumulation, lysosomal proliferation, and the formation of autophagy zones. Therapeutic response can be assessed by comparing the physiological and molecular responses of muscle biopsies before and after treatment with rhGAA as described herein. For example, the amount of glycogen present in the biopsy sample can be used as a marker for determining therapeutic response. Another example includes biomarkers, such as lysosomal associated protein 1 (LAMP-1), microtubule-associated protein 1 light chain 3 (LC3), and desferlin, which can be used as indicators of lysosomal storage dysfunction. Other biomarkers include biomarkers of muscle damage or injury, such as creatine kinase (CK), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST), and / or markers of glycogen accumulation, such as urine hexose tetraose (Hex4). For example, muscle biopsies collected before and after treatment with rhGAA as described herein can be stained with antibodies that recognize one of the biomarkers. Therapeutic response may also include a reduction in fatigue or improvement in other patient-reported outcomes (eg, activities of daily living, well-being, etc.).

[0171] As used herein, the term "enzyme replacement therapy" or "ERT" is intended to refer to the introduction of a non-natural, purified enzyme into an individual having an enzyme deficiency. The administered protein can be obtained from a natural source or by recombinant expression. The term also refers to the introduction of a purified enzyme into an individual who would otherwise require or benefit from the administration of the purified enzyme. In at least one embodiment, such an individual suffers from an enzyme deficiency. The introduced enzyme can be a purified recombinant enzyme produced in vitro, or a protein purified from an isolated tissue or body fluid, such as, for example, the placenta or animal milk, or from a plant.

[0172] As used herein, the term "two-component therapy" is intended to refer to any therapy in which two or more individualized therapies are administered simultaneously or sequentially. In some embodiments, the results of the two-component therapy are enhanced compared to the effects of each therapy when performed alone. Enhancement can include any improvement in the effects of different treatments that can produce a favorable outcome compared to the results achieved by performing the treatments alone. The enhanced effect or outcome can include synergistic enhancement, where the enhanced effect is greater than the additive effect of each therapy when performed alone; additive enhancement, where the enhanced effect is substantially equal to the additive effect of each therapy when performed alone; or less than additive effect, where the enhanced effect is less than the additive effect of each therapy when performed alone, but still better than the effect of each therapy when performed alone. The enhanced effect can be measured by any means known in the art that can measure therapeutic efficacy or outcome.

[0173] Pompe disease refers to an autosomal recessive LSD characterized by a deficiency in acid alpha-glucosidase (GAA) activity, which impairs lysosomal glycogen metabolism. The enzyme deficiency leads to lysosomal glycogen accumulation and, in the late stages of the disease, progressive skeletal muscle weakness, decreased cardiac function, respiratory insufficiency, and / or central nervous system damage. Genetic mutations in the GAA gene result in lower expression or the production of mutant forms of the enzyme with altered stability and / or biological activity that ultimately lead to disease, (see generally, Hirschhorn R, 1995, Glycogen Storage Disease Type II: Acid a-Glucosidase (Acid Maltase) Deficiency, in 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 (infants, children and adults) are associated with residual α-glucosidase activity levels (Reuser AJ et al., 1995, Glycogen Storage Disease Type II (Acid Maltase Deficiency), Muscle & Nerve Supplement 3, S61-S69). Infantile Pompe disease (Type I or Type A) is the most common and most severe, characterized by failure to thrive, systemic hypotonicity, cardiac hypertrophy and cardiopulmonary failure within the second year of life. The severity of childhood Pompe disease (Type II or Type B) is moderate and is characterized by the absence of a preponderance of muscle symptoms with cardiac hypertrophy. Childhood Pompe disease individuals usually die before reaching the age of 20 due to respiratory failure. Adult Pompe disease (type III or C) typically presents as a slowly progressive myopathy during adolescence or as late as the 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). In Pompe disease, α-glucosidase has been shown to be extensively modified post-translationally by glycosylation, phosphorylation, and proteolytic processing.The conversion of the 110 kilodalton (kDa) precursor to the 76 and 70 kDa mature forms by proteolysis in the lysosome is required for optimal glycogenolysis. 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.

[0174] Pompe disease is now considered a continuous phenotypic spectrum, with the most clinically severe, rapidly progressive phenotype being classic infantile-onset Pompe disease (IOPD) and the less severe, slowly progressive phenotype being late-onset Pompe disease (LOPD). Late-onset Pompe disease can present in childhood or adulthood and does not present with clinically apparent cardiac involvement (Leslie and Bailey, 2017). When occurring in the pediatric subgroup of the LOPD category, late-onset Pompe disease is often referred to as juvenile-onset Pompe disease. Compared with classic IOPD, LOPD progresses more slowly, with most patients presenting with progressive limb-girdle weakness and respiratory failure due to involvement of the proximal lower and upper limb muscles, paraspinal muscles, and diaphragm. Clinical manifestations include difficulty walking, climbing stairs, and progressively limiting motor activities of daily living, progressing to the need for support for walking and then wheelchair dependence (Reuser et al., 2001). The clinical manifestations of the disease are exacerbated by respiratory involvement, initially manifesting as sleep-disordered breathing and orthopnea (shortness of breath in the supine position). The progressive nature of Pompe disease often leads to the use of invasive mechanical ventilation. Biochemical abnormalities include elevated serum creatine kinase (CK) levels, a biomarker of muscle damage, and elevated urine hexotetraose (Hex4) levels (An et al. 2005; Young et al. 2009). Life expectancy for patients with LOPD can range from early childhood to late adulthood, depending on the age of onset, rate of disease progression, extent of respiratory muscle involvement, and the presence of comorbidities (Hagemans et al. 2004). Without treatment, life expectancy for adults with Pompe disease is significantly shortened (Gungor et al. 2011).

[0175] As used herein, "significant" refers to statistical significance. This term refers to the statistical evidence that there is a difference between two treatment groups. It can be defined as the probability of rejecting the null hypothesis when the null hypothesis is actually true. This decision is typically made using a p-value < 0.05 derived from an appropriate comparative statistical analysis. See, for example, Example 9.

[0176] A "subject" or "patient" is preferably a human, although other mammals and non-human animals with conditions involving glycogen accumulation may also be treated. The subject may be a fetus, neonate, infant, child, or adult with Pompe disease or other glycogen storage or accumulation disorders. An example of a treated individual is an individual (fetus, neonate, infant, child, adolescent, or adult) 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 a GAA activity of less than about 1% of normal GAA activity (infant GSD-II), a GAA activity of about 1%-10% of normal GAA activity (pediatric GSD-II), or a GAA activity of about 10%-40% of normal GAA activity (adult GSD-II). In some embodiments, the subject or patient is an "ERT-experienced" or "converted ERT" patient, which refers to a Pompe disease patient who has previously received enzyme replacement therapy. In some embodiments, an "ERT-experienced" or "ERT-switched" patient is a Pompe disease patient who has received or is currently receiving alglucosidase alfa for greater than or equal to 24 months. In some embodiments, an "ERT-experienced" or "ERT-switched" patient is a Pompe disease patient who has been treated with a currently approved ERT (e.g., or ) is worsening. In some embodiments, the subject is an adult patient (e.g., 18 years of age or older) diagnosed with late-onset Pompe disease (acid alpha-glucosidase (GAA) deficiency) who has previously received enzyme replacement therapy (ERT). In some embodiments, the subject is an adult (e.g., 18 years of age and older) weighing ≥40 kg suffering from late-onset Pompe disease (lysosomal acid alpha-glucosidase [GAA] deficiency) whose disease progresses while receiving enzyme replacement therapy (ERT). In some embodiments, the subject or patient is an "ERT-naive" patient, which refers to a Pompe disease patient who has not previously received enzyme replacement therapy. In certain embodiments, the subject or patient is ambulatory (e.g., an ERT conversion patient who can walk or an ERT-naive patient who can walk). In certain embodiments, the subject or patient is incapable of walking (e.g., an ERT conversion patient who cannot walk). The ambulatory or incapable of walking state can be determined by a six-minute walk test (6MWT). In some embodiments, an ambulatory patient is a Pompe disease patient who can walk at least 200 meters in a 6MWT. In some embodiments, the patient who cannot walk is a Pompe disease patient who is unable to walk independently or is in a wheelchair. In some embodiments, the subject is using effective contraception. In some embodiments, the subject and / or the subject's partner is using highly effective contraception, such as contraception that results in a low failure rate (e.g., <1% per year) when used continuously and correctly. Examples of highly effective contraceptive methods include, but are not limited to: complete abstinence; combined (estrogen and progestin) hormonal contraception associated with ovulation suppression; oral, intravaginal, transdermal progestin-only hormonal contraception associated with ovulation suppression: oral, injection, implantable intrauterine device; intrauterine hormone-releasing system; bilateral fallopian tube occlusion; and vasectomy. In some embodiments, the subject is postmenopausal. In some embodiments, the subject does not have reproductive potential. In some embodiments, the subject is permanently infertile. In some embodiments, the subject is not pregnant. In some embodiments, the subject is not breastfeeding.

[0177] In some embodiments, the patient is diagnosed with late-onset Pompe disease based on documentation of at least one of: (1) GAA enzyme deficiency; and / or (2) gene encoding human acid alpha-glucosidase (GAA) genotyping. In some embodiments, the patient is 18 years of age or older. In some embodiments, the patient has previously received enzyme replacement therapy (ERT). In some embodiments, the patient is receiving a currently approved ERT (e.g., or In some embodiments, both male and female patients, if of reproductive potential, agree to use highly effective contraception throughout treatment and for up to 90 days after the last dose. In some embodiments, patients currently taking a beta2-agonist or non-selective beta-blocker (e.g., propranolol, nadolol, carvedilol) are maintained on an appropriate and stable dose as determined by the treating physician.

[0178] As used herein, the term "treat" or "treatment" refers to the improvement of one or more symptoms associated with a disease, the delaying of the onset of one or more symptoms of a disease, and / or the reduction of the severity or frequency of one or more symptoms of a disease. For example, treatment can refer to an improvement in cardiac status (e.g., an increase in end-diastolic and / or end-systolic volume, or a reduction or improvement in the progressive cardiomyopathy typically found in GSD-II) or an improvement in lung function (e.g., an increase in crying vital capacity over baseline capacity, and / or normalization of oxygen desaturation during crying); improvement in neurodevelopment and / or motor skills (e.g., an increase in AIMS score); a reduction in glycogen levels in the tissues of individuals affected by the disease; or any combination of these effects. In a preferred embodiment, treatment includes improving cardiac status, specifically reducing GSD-II associated cardiomyopathy.

[0179] As used herein, the terms "improve," "increase," and "decrease" refer to values ​​relative to a baseline measurement or a corresponding value from a control treatment (such as a measurement in the same individual before starting a treatment described herein, a measurement in a control individual (or multiple control individuals) in the absence of a treatment described herein, or a measurement after a control treatment). A control individual is an individual with the same form of GSD-II (infantile, pediatric, or adult onset) as the individual being treated, who is approximately the same age as the individual being treated (to ensure that the disease stage in the treated and control individuals is comparable). In some embodiments, the control treatment comprises administration of a placebo of alglucosidase alfa and an enzyme stabilizer (see Example 9).

[0180] As used herein, the phrases "stabilize motor function," "stabilize lung function," and similar terms refer to reducing or arresting the decline in motor and lung function, and / or restoring motor and / or lung function. Because untreated Pompe disease patients are expected to experience a significant decline in motor and / or lung function over time, an increase in the rate of decline in motor and / or lung function and / or an increase in motor and / or lung function demonstrates a benefit from the treatments described herein. Furthermore, because motor and / or lung function in Pompe disease patients who have undergone ERT typically continues to decline over time, stabilizing motor and / or lung function using the therapies described herein may include treatment with a patient who has received prior ERT (e.g., or ) patients, and reduce and / or prevent the decline in exercise and / or lung function compared to such patients.

[0181] As used herein, the terms "about" and "approximately" are intended to refer to an acceptable degree of error for a measured quantity given the nature or precision of the measurement. For example, as understood in the art, the degree of error can be indicated by the number of significant figures provided for a measurement, and includes, but is not limited to, a variation of ±1 in the most precise significant figure reported for a measurement. A typical exemplary degree of error is within 20 percent (%) of a given value or range of values, preferably within 10%, and more preferably within 5%. Unless otherwise indicated, numerical quantities given herein are approximate, meaning that the term "about" or "approximately" can be inferred when not explicitly stated.

[0182] All references, articles, publications, patents, patent publications and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, the reference to any reference, article, publication, patent, patent publication and patent application cited herein is not, and should not be taken as, an admission or any form of suggestion that it constitutes available prior art or forms part of the common general knowledge in any country in the world.

[0183] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Recombinant human acid α-glucosidase (rhGAA)

[0184] In some embodiments, recombinant human acid alpha-glucosidase (rhGAA) is an enzyme having an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In some embodiments, rhGAA is encoded by a nucleotide sequence as shown in SEQ ID NO: 2. Table 1. Nucleotide and protein sequences

[0185] In some embodiments, rhGAA has the GAA amino acid sequence set forth in SEQ ID NO: 1 as described in U.S. Patent No. 8,592,362 and has GenBank Accession No. AHE24104.1 (GI: 568760974). In some embodiments, rhGAA has the GAA amino acid sequence encoded in SEQ ID NO: 2 (mRNA sequence having GenBank Accession No. Y00839.1). In some embodiments, rhGAA has the 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 No. NP_000143.2 or UniProtKB Accession No. P10253.

[0186] In some embodiments, rhGAA is initially expressed as wild-type GAA having a full-length 952 amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4, and rhGAA undergoes intracellular processing that removes a portion of amino acids (e.g., the first 56 amino acids). Thus, the rhGAA secreted by the host cell can have a shorter amino acid sequence than the rhGAA initially expressed in the cell. In some embodiments, the shorter protein can have the amino acid sequence shown 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 of SEQ ID NO: 1, have been removed, thereby producing a protein with 896 amino acids. In some embodiments, the shorter protein can have the amino acid sequence shown in SEQ ID NO: 6, which differs from SEQ ID NO: 4 only in that the first 56 amino acids, including the signal peptide and precursor peptide of SEQ ID NO: 4, have been removed, thereby producing a protein with 896 amino acids. Other variations in the number of amino acids are also possible, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more deletions, substitutions and / or insertions relative to the amino acid sequence depicted by SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, the rhGAA product comprises a mixture of recombinant human acid α-glucosidase molecules having different amino acid lengths.

[0187] In some embodiments, rhGAA comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to SEQ ID NO: 4 or SEQ ID NO: 6. Different alignment algorithms and / or programs can be used to calculate the identity between two sequences, including FASTA or BLAST, which are available as part of the GCG sequence analysis package (University of Wisconsin, Madison, Wisconsin), and can be used with, for example, default settings. For example, polypeptides that have at least 80%, 85%, 90%, 95%, 98% or 99% identity to a particular polypeptide described herein, and preferably exhibit substantially the same function, are considered, along with polynucleotides encoding such polypeptides. Unless otherwise indicated, similarity scores will be based on the use of BLOSUM62. When BLASTP is used, the percentage similarity is based on the BLASTP positive score, and the percentage sequence identity is based on the BLASTP identity score. BLASTP "identity" shows the number and fraction of total residues that are identical in a high-scoring sequence pair; and BLASTP "positive" indicates the number and fraction of residues that have a positive alignment score and are similar to each other. The present disclosure contemplates and encompasses amino acid sequences having these degrees of identity or similarity, or any intermediate degrees of identity or similarity, to the amino acid sequences disclosed herein. The polynucleotide sequences of similar polypeptides can be derived using the genetic code and can be obtained by conventional means, particularly by reverse translating their amino acid sequences using the genetic code.

[0188] In some embodiments, rhGAA undergoes post-translational and / or chemical modifications at one or more amino acid residues in the protein. For example, methionine and tryptophan residues can undergo oxidation reactions. As another example, the N-terminal glutamine in SEQ ID NO:6 can be further modified to form pyroglutamic acid. As another example, an asparagine residue can undergo deamidation to form aspartic acid. As another example, an aspartic acid residue can undergo isomerization to form isoaspartic acid. As another example, unpaired cysteine ​​residues in the protein can form disulfide bonds with free glutathione and / or cysteine. Therefore, in some embodiments, the enzyme is initially expressed as having an amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, or an amino acid sequence encoded by SEQ ID NO:2, and the enzyme undergoes one or more of these post-translational modifications and / or chemical modifications. Such modifications are also within the scope of the present disclosure. N-linked glycosylation of rhGAA

[0189] There are seven potential N-linked glycosylation sites on a single rhGAA molecule. These potential glycosylation sites are at the following positions in SEQ ID NO:6: N84, N177, N334, N414, N596, N826, and N869. Similarly, for the full-length amino acid sequence of SEQ ID NO:4, 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. Generally, the sequence Asn-X-Ser or Asn-X-Thr in the protein amino acid sequence indicates a potential glycosylation site, except that X cannot be His or Pro.

[0190] 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, a single N-glycan on an rhGAA molecule may carry M6P (mono-phosphorylated or mono-M6P), a single N-glycan may carry two M6P groups (di-phosphorylated or di-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 mol M6P groups per mol of rhGAA on their N-glycans. Recombinant human acid α-glucosidase molecules may also have N-glycans that do not carry M6P groups. In another embodiment, the rhGAA comprises, on average, greater than 2.5 mol M6P / mol rhGAA and greater than 4 mol sialic acid / mol rhGAA. In some embodiments, the rhGAA comprises, on average, about 3-3.5 mol M6P / mol rhGAA. In some embodiments, the rhGAA comprises, on average, about 4-5.4 mol sialic acid / mol rhGAA. On average, at least about 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 20% of the N-glycans on rhGAA can be in the form of mono-M6P N-glycans, and on average, at least about 0.5%, 1%, 1.5%, 2.0%, 2.5%, 3.0%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the 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, an average of about 10% to about 14% of the total N-glycans on rhGAA are monophosphorylated. In some embodiments, an average of about 7% to about 25% of the total N-glycans on rhGAA are diphosphorylated. In some embodiments, rhGAA comprises, on average, at least about 1.0, 1.1, 1.2, or 1.3 mol of bis-M6P / mol of rhGAA.

[0191] The rhGAA described herein can have an average of 0.5 to 7.0 mol M6P / mol 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 / mol rhGAA. The rhGAA can be fractionated to provide rhGAA preparations having different average amounts of mono-M6P-bearing or di-M6P-bearing N-glycans, thereby allowing further customization of the rhGAA for lysosomal targeting in target tissues by selecting specific fractions or by selectively combining different fractions.

[0192] In some embodiments, up to 60% of the N-glycans on rhGAA can be fully sialylated, for example, up to 10%, 20%, 30%, 40%, 50% or 60% of the N-glycans can be fully sialylated. In some embodiments, no more than 50% 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, no more than 5%, 10%, 20%, or 30% of the N-glycans on rhGAA carry sialic acid and a terminal galactose residue (Gal). This range includes all intermediate values ​​and subranges, for example, 7% to 30% of the total N-glycans on rhGAA can carry sialic acid and a terminal galactose residue. In yet other embodiments, no more than 5%, 10%, 15%, 16%, 17%, 18%, 19%, or 20% of the N-glycans on rhGAA have only terminal galactose and do not contain sialic acid. This range includes all intermediate values ​​and subranges, for example, from 8% to 19% of the total N-glycans on rhGAA in the composition can have only terminal galactose and do not contain sialic acid.

[0193] In some embodiments, 30% to 60%, 35% to 60%, 40% to 60%, 45% to 60%, 50% to 60%, or 55% to 60% of the total N-glycans on rhGAA are complex N-glycans; or no more than 1%, 2%, 3%, 4%, 5%, 6%, or 7% of the total N-glycans on rhGAA are hybrid N-glycans; no more than 5%, 10%, 15%, 20%, 25%, or 30% of the total N-glycans on rhGAA are non-phosphorylated, highly mannose-type N-glycans; at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% of the total N-glycans on rhGAA are monophosphorylated high-mannose N-glycans; and / or at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or 20% of the total N-glycans on rhGAA are diphosphorylated high-mannose N-glycans. These values ​​include all intermediate values ​​and subranges. rhGAA can meet one or more of the above-recited content ranges.

[0194] In some embodiments, rhGAA may carry an average of 2.0 to 8.0 moles of sialic acid residues per mole of rhGAA. This range includes 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 the unproductive clearance of rhGAA by the asialoprotein receptor.

[0195] In one or more embodiments, rhGAA has a certain N-glycosylation profile at certain potential N-glycosylation sites. 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 of SEQ ID NO: 6 and N140 of SEQ ID NO: 4). For example, at the first potential N-glycosylation site, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of rhGAA may be phosphorylated. This phosphorylation may be the result of mono-M6P and / or di-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 rhGAA bears 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 rhGAA bears a di-M6P unit at the first potential N-glycosylation site. In some embodiments, rhGAA comprises an average of about 1.4 mol M6P (mono-M6P and di-M6P) / mol rhGAA at the first potential N-glycosylation site. In some embodiments, rhGAA comprises at least about 0.5 mol of di-M6P / mol of rhGAA at the first potential N-glycosylation site. In some embodiments, rhGAA comprises about 0.25 mol of mono-M6P / mol of rhGAA at the first potential N-glycosylation site. In some embodiments, rhGAA comprises an average of about 0.2 mol to about 0.3 mol of sialic acid / mol of rhGAA at the first potential N-glycosylation site. In at least one embodiment, rhGAA comprises Figure 6A The first potential N-glycosylation site occupancy is shown and Figure 6B In at least one embodiment, rhGAA comprises Figure 19A The first potential N-glycosylation site occupancy is shown and Figure 19B or Figure 20B N-glycosylation profiles are shown.

[0196] In some embodiments, at least 20% of rhGAA is phosphorylated at the second potential N-glycosylation site (e.g., N177 of SEQ ID NO: 6 and N223 of SEQ ID NO: 4). For example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of rhGAA may be phosphorylated at the second N-glycosylation site. This phosphorylation may be the result of mono-M6P and / or di-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 rhGAA bears 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 rhGAA bears a di-M6P unit at the second N-glycosylation site. In some embodiments, rhGAA comprises an average of about 0.5 mol M6P (mono-M6P and di-M6P) / mol rhGAA at the second potential N-glycosylation site. In some embodiments, the rhGAA comprises about 0.4 to about 0.6 mol mono-M6P / mol rhGAA at the second potential N-glycosylation site. In at least one embodiment, the rhGAA comprises Figure 6A The occupancy of the second potential N-glycosylation site is shown and Figure 6C In at least one embodiment, rhGAA comprises Figure 19A The occupancy of the second potential N-glycosylation site is shown and Figure 19C or Figure 20B N-glycosylation profiles are shown.

[0197] In one or more embodiments, at least 5% of rhGAA is phosphorylated at the third potential N-glycosylation site (e.g., N334 of SEQ ID NO: 6, and N390 of SEQ ID NO: 4). 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 may have a mixture of non-phosphorylated high mannose N-glycans, di-, tri-, and tetra-antennary complex N-glycans, and hybrid N-glycans as the predominant 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 mol sialic acid / mol rhGAA at the third potential N-glycosylation site. In at least one embodiment, rhGAA comprises Figure 6A The third potential N-glycosylation site occupancy is shown and Figure 6D In at least one embodiment, rhGAA comprises Figure 19A The third potential N-glycosylation site occupancy is shown and Figure 19D or Figure 20B N-glycosylation profiles are shown.

[0198] In some embodiments, at least 20% of rhGAA is phosphorylated at the fourth potential N-glycosylation site (e.g., N414 of SEQ ID NO: 6 and N470 of SEQ ID NO: 4). For example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of rhGAA may be phosphorylated at the fourth potential N-glycosylation site. This phosphorylation may be the result of mono-M6P and / or di-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 rhGAA bears 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 rhGAA bears a di-M6P unit at the fourth potential N-glycosylation site. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, or 25% of rhGAA is sialylated at the fourth potential N-glycosylation site. In some embodiments, rhGAA comprises an average of about 1.4 mol M6P (mono-M6P and di-M6P) / mol rhGAA at the fourth potential N-glycosylation site. In some embodiments, rhGAA comprises about 0.4 to about 0.6 mol di-M6P / mol rhGAA at the fourth potential N-glycosylation site. In some embodiments, rhGAA comprises about 0.3 to about 0.4 mol mono-M6P / mol rhGAA at the fourth potential N-glycosylation site. In at least one embodiment, rhGAA comprises Figure 6A The occupancy of the fourth potential N-glycosylation site is shown and Figure 6E In at least one embodiment, rhGAA comprises Figure 19A The occupancy of the fourth potential N-glycosylation site is shown and Figure 19E or Figure 20B N-glycosylation profiles are shown.

[0199] In some embodiments, at least 5% of rhGAA is phosphorylated at the fifth potential N-glycosylation site (e.g., N596 of SEQ ID NO: 6 and N692 of SEQ ID NO: 4). 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 di-antennary complex N-glycans as the predominant 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 mol sialic acid / mol rhGAA at the fifth potential N-glycosylation site. In at least one embodiment, rhGAA comprises Figure 6A The fifth potential N-glycosylation site occupancy is shown and Figure 6F In at least one embodiment, rhGAA comprises Figure 19A The fifth potential N-glycosylation site occupancy is shown and Figure 19F or Figure 20B N-glycosylation profiles are shown.

[0200] In some embodiments, at least 5% of rhGAA is phosphorylated at the sixth N-glycosylation site (e.g., N826 of SEQ ID NO: 6 and N882 of SEQ ID NO: 4). 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 can have a mixture of di-, tri-, and tetra-antennary complex N-glycans as the predominant 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 mol sialic acid / mol rhGAA at the sixth potential N-glycosylation site. In some embodiments, rhGAA comprises an average of about 0.9 mol acetylated sialic acid / mol rhGAA at the sixth potential N-glycosylation site. In some embodiments, rhGAA comprises an average of at least 0.05 mol glycan species having poly-N-acetyl-D-lactosamine residues (poly-LacNAc) / mol rhGAA at the sixth potential N-glycosylation site. In some embodiments, greater than 10% of rhGAA comprises glycans with poly-LacNAc residues at the sixth potential N-glycosylation site. In at least one embodiment, rhGAA comprises Figure 6A The occupancy of the sixth potential N-glycosylation site is shown and Figure 6G In at least one embodiment, rhGAA comprises Figure 19A The occupancy of the sixth potential N-glycosylation site is shown and Figure 19G or Figure 20B N-glycosylation profiles are shown.

[0201] In some embodiments, at least 5% of rhGAA is phosphorylated at the seventh potential N-glycosylation site (e.g., N869 of SEQ ID NO: 6 and N925 of SEQ ID NO: 4). 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-glycans at the seventh potential N-glycosylation site. In some embodiments, at least 30%, 35%, or 40% of rhGAA has N-glycans at the seventh potential N-glycosylation site. In some embodiments, rhGAA comprises an average of at least 0.5 mol sialic acid / mol rhGAA at the seventh potential N-glycosylation site. In some embodiments, rhGAA comprises an average of at least 0.8 mol sialic acid / mol rhGAA at the seventh potential N-glycosylation site. In some embodiments, rhGAA comprises an average of about 0.86 mol sialic acid / mol rhGAA at the seventh potential N-glycosylation site. In some embodiments, rhGAA comprises an average of at least 0.3 mol of glycan species bearing poly-LacNAc residues / mol rhGAA at the seventh potential N-glycosylation site. In some embodiments, approximately half of the rhGAA comprises glycans bearing poly-LacNAc residues 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 comprises Figure 6A or Figure 19A The occupancy of the seventh potential N-glycosylation site is shown and Figure 19H or Figure 20B N-glycosylation profiles are shown.

[0202] In some embodiments, the rhGAA comprises an average of 3-4 mol M6P residues / mol rhGAA and about 4 to about 7.3 mol sialic acid / mol rhGAA. In some embodiments, the rhGAA further comprises an average of at least about 0.5 mol bis-M6P / mol rhGAA at the first potential N-glycosylation site, an average of about 0.4 to about 0.6 mol mono-M6P / mol rhGAA at the second potential N-glycosylation site, about 0.9 to about 1.2 mol sialic acid / mol rhGAA at the third potential N-glycosylation site, about 0.4 to about 0.6 mol bis-M6P / mol rhGAA at the fourth potential N-glycosylation site, about 0.3 to about 0.4 mol mono-M6P / mol rhGAA at the fourth potential N-glycosylation site, about 0.8 to about 0.9 mol sialic acid / mol rhGAA at the fifth potential N-glycosylation site, and about 1.5 to about 4.2 mol sialic acid / mol rhGAA at the sixth potential N-glycosylation site. In some embodiments, rhGAA further comprises an average of at least 0.5 mol sialic acid / mol rhGAA at the seventh potential N-glycosylation site. In some embodiments, rhGAA further comprises an average of at least 0.8 mol sialic acid / mol rhGAA at the seventh potential N-glycosylation site. In at least one embodiment, rhGAA further comprises an average of about 0.86 mol sialic acid / mol rhGAA at the seventh potential N-glycosylation site. In at least one embodiment, rhGAA comprises seven potential N-glycosylation sites, the occupancy and N-glycosylation profile of which are as follows: Figures 6A-6H In at least one embodiment, rhGAA contains seven potential N-glycosylation sites, and their occupancy and N-glycosylation profiles are shown in FIG. Figures 19A-19H and Figures 20A-20B shown.

[0203] Methods of making rhGAA are disclosed in International Patent No. PCT / 2015 / 053252, U.S. Patent No. 10,208,299, and U.S. Patent No. 10,961,522, the entire disclosures of which are incorporated herein by reference.

[0204] Once inside the lysosomes, rhGAA can enzymatically degrade the accumulated glycogen. However, conventional rhGAA products have low total levels of N-glycans with mono-M6P and di-M6P, and therefore target myocytes poorly, resulting in poor delivery of rhGAA to lysosomes. Most rhGAA molecules in these conventional products do not have phosphorylated N-glycans, thereby lacking affinity for CIMPR. Non-phosphorylated high-mannose N-glycans can also be cleared by mannose receptors, which lead to non-productive clearance of ERT ( Figure 2B). In contrast, Figure 2A As shown, the rhGAA described herein may contain a higher amount of mono-M6P- and di-M6P-bearing N-glycans, resulting in an efficient uptake of rhGAA into specific tissues such as muscle. Production and purification of N-linked glycosylated rhGAA

[0205] As described in International Patent Application No. PCT / 2015 / 053252, U.S. Patent No. 10,208,299, and U.S. Patent No. 10,961,522 (the entire contents of which are incorporated herein by reference), cells such as Chinese hamster ovary (CHO) cells can be used to produce rhGAA as described therein. Expressing high M6P rhGAA in CHO cells is more advantageous than post-translationally modifying the glycan profile of rhGAA, at least in part because only the former can be converted to a form of rhGAA with optimal glycogenolysis through glycan degradation, thereby enhancing therapeutic efficacy.

[0206] In certain embodiments, rhGAA is preferably produced by one or more CHO cell lines, which are transformed with a DNA construct encoding rhGAA as described herein. Such CHO cell lines can comprise multiple copies of the gene encoding the polynucleotide of GAA, such as 5, 10, 15 or 20 or more copies. DNA constructs expressing allelic variants or other variant acid α-glucosidase amino acid sequences (such as those with SEQ ID NO: 4 or SEQ ID NO: 6 having at least 90%, 95%, 98% or 99% consistency) of acid α-glucosidase can be constructed and expressed in CHO cells. Those of ordinary skill in the art can select an alternative vector suitable for transforming CHO cells for the production of such DNA constructs.

[0207] Methods for preparing such CHO cell lines are described in International Patent No. PCT / 2015 / 053252, U.S. Patent No. 10,208,299, and U.S. Patent No. 10,961,522, the entire contents of which are incorporated herein by reference. Briefly, these methods involve transforming CHO cells with DNA encoding GAA or a GAA variant, selecting CHO cells that have stably integrated the GAA-encoding DNA into their chromosomes and stably express GAA, and selecting CHO cells that express GAA with a high content of mono-M6P or di-M6P-bearing N-glycans, and optionally selecting a CHO cell that has N-glycans with a high sialic acid content and / or N-glycans with a high mannose content and a low non-phosphorylated content. The selected CHO cell line can be used to produce rhGAA and rhGAA compositions by culturing the CHO cell line and recovering the composition from the CHO cell culture. In some embodiments, rhGAA produced by a selected CHO cell line comprises a high content of CIMPR-targeted N-glycans with mono-M6P or di-M6P. In some embodiments, rhGAA produced as described herein has low levels of complex N-glycans with terminal galactose. In some embodiments, the selected CHO cell line is designated GA-ATB200 or ATB200-X5-14. In some embodiments, the selected CHO cell line comprises a subculture or derivative of such a CHO cell culture. In some embodiments, the rhGAA produced by the selected CHO cell line is designated ATB200.

[0208] The rhGAA produced as described herein can be purified by the following methods described in U.S. Patent No. 10,227,577 and U.S. Provisional Application No. 62 / 506,569 (both of which are incorporated herein by reference in their entireties). An exemplary process for producing, capturing, and purifying rhGAA produced from a CHO cell line is as follows: Figure 3 shown.

[0209] In short, bioreactor 601 comprises cell culture, for example CHO cells, which express rhGAA and secrete rhGAA into the surrounding liquid culture medium. Bioreactor 601 can be any suitable bioreactor for culturing cells, such as perfusion, batch or fed-batch bioreactor. After a sufficient time for the cells to produce rhGAA, the culture medium is taken out from the bioreactor. Such culture medium removal can be continuous for perfusion bioreactors, or can be batch for batch or fed-batch reactors. The medium is filtered through filtration system 603 to remove cells. Filtration system 603 can be any suitable filtration system, including alternating tangential flow filtration (ATF) system, tangential flow filtration (TFF) system and / or centrifugal filtration system, etc. In various embodiments, the filtration system utilizes a filter with a pore size between about 10 nanometers and about 2 microns.

[0210] After filtration, the filtrate is loaded onto the protein capture system 605. The protein capture system 605 can include one or more chromatography columns. If more than one chromatography column is used, the chromatography columns can be placed in series so that the next chromatography column can be loaded immediately after the first chromatography column is loaded. Alternatively, the media removal process can be suspended during the switching of the chromatography columns.

[0211] In various embodiments, the protein capture system 605 includes one or more anion exchange (AEX) columns for directly capturing the rhGAA product, particularly rhGAA with a high M6P content. The rhGAA captured by the protein capture system 605 is eluted from the one or more columns by changing the pH and / or salt content in the column. Table 2 provides exemplary conditions for the AEX column. Table 2. Exemplary conditions for AEX columns

[0212] The eluted rhGAA can be subjected to further purification steps and / or quality assurance steps. For example, the eluted rhGAA can be subjected to a virus killing step 607. Such virus killing 607 can include one or more of low pH killing, detergent killing, or other techniques known in the art. The rhGAA product from the virus killing step 607 can be introduced into a second chromatography system 609 to further purify the rhGAA product. Alternatively, the eluted rhGAA from the protein capture system 605 can be fed directly to the second chromatography system 609. In various embodiments, the second chromatography system 609 includes one or more fixed metal affinity chromatography (IMAC) columns for further removing impurities. Exemplary conditions for the IMAC column are provided in Table 3 below. Table 3. Exemplary conditions for IMAC columns

[0213] After the rhGAA is loaded onto the second chromatography system 609, the recombinant protein is eluted from the one or more columns. The eluted rhGAA can be subjected to a viral killing step 611. As with viral killing 607, viral killing 611 can include one or more of low pH killing, detergent killing, or other techniques known in the art. In some embodiments, only one of viral killing 607 or 611 is used, or viral killing is performed at the same stage of the purification process.

[0214] The rhGAA product from the virus killing step 611 can be introduced into a third chromatography system 613 to further purify the recombinant protein product. Alternatively, the eluted recombinant protein from the second chromatography system 609 can be fed directly into the third chromatography system 613. In various embodiments, the third chromatography system 613 includes one or more cation exchange chromatography (CEX) columns and / or size exclusion chromatography (SEC) columns for further impurity removal. The rhGAA product is then eluted from the third chromatography system 613. Exemplary conditions for the CEX column are provided in Table 4 below. Table 4. Exemplary conditions for CEX columns

[0215] The rhGAA product may also 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, in which the recombinant protein product may be sterilized, filtered, concentrated, stored, and / or have additional components added for final product formulation. Pharmaceutical composition

[0216] In various embodiments, pharmaceutical compositions are provided that include rhGAA described herein (alone or in combination with other therapeutic agents) and / or a pharmaceutically acceptable carrier.

[0217] In one or more embodiments, the pharmaceutical compositions described herein comprise a pharmaceutically acceptable salt.

[0218] In some embodiments, the pharmaceutically acceptable salts used herein are pharmaceutically 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 organic acids, including but not limited to acetic acid, trifluoroacetic acid, adipic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, butyric acid, camphoric acid, camphorsulfonic acid, cinnamic acid, citric acid, digluconic acid, ethanesulfonic acid, glutamic acid, glycolic acid, glycerophosphoric acid, hemisulfuric acid, hexanoic acid, formic acid, fumaric acid, 2-hydroxyethylsulfonic acid (hydroxyethanesulfonic 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, undecanoic acid, and the like.

[0219] In some embodiments, the pharmaceutically acceptable salts used herein are pharmaceutically acceptable base addition salts. Pharmaceutically acceptable base addition salts may include, but are not limited to, hydroxides, carbonates, or bicarbonates of ammonia or ammonium or metal cations such as sodium, potassium, lithium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Salts derived from pharmaceutically acceptable organic non-toxic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, quaternary ammonium compounds, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, isopropylamine, tripropylamine, tributylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, hydrazine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, tetramethylammonium compounds, tetraethylammonium compounds, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, dibenzylamine, N,N-dibenzylphenethylamine, 1-diphenylhydroxymethylamine, N,N'-dibenzylethylenediamine, polyamine resins, and the like.

[0220] In some embodiments, rhGAA or a pharmaceutically acceptable salt thereof can be formulated into a pharmaceutical composition suitable 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 solubilizer and a local anesthetic to relieve pain at the injection site. The components of the pharmaceutical composition can be provided separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or anhydrous concentrate in an airtight container, such as an ampoule or sachet indicating the amount of active agent. When the composition is administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical grade water, saline, or dextrose / water. In some embodiments, the infusion can occur in a hospital or clinic. In some embodiments, the infusion can occur outside of a hospital or clinic setting, for example, at the subject's residence. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed prior to administration.

[0221] In some embodiments, rhGAA or a pharmaceutically acceptable salt thereof is administered every 2 weeks as an IV infusion lasting about 4 hours. In some embodiments, the total infusion volume is determined by the patient's weight. If the patient experiences IAR, the infusion rate can be reduced and the infusion duration increased.

[0222] In some embodiments, the initial infusion rate is 1 mg / kg / hour. In some embodiments, if there are no signs of an infusion-related reaction (IAR), the infusion rate is gradually increased by 2 mg / kg / hour every 30 minutes until a maximum rate of 7 mg / kg / hour is reached; then, the infusion rate is maintained at 7 mg / kg / hour until the infusion is complete. In some embodiments, the approximate total infusion duration is 4 hours.

[0223] The infusion should be administered in a stepwise manner using an infusion pump. Based on the patient's weight, the infusion rate can be increased from an initial rate of + / - 5 minutes every 30 minutes until the maximum rate is reached, as shown in Table 5 below. Table 5: Recommended infusion volume and rate for a 20 mg / kg dose

[0224] The most serious tolerability issue with rhGAA or a pharmaceutically acceptable salt thereof is the occurrence of infusion-related reactions (IARs), which in some cases may include life-threatening anaphylaxis or other severe allergic reactions. In some embodiments, prior to administration of rhGAA or a pharmaceutically acceptable salt thereof, a pretreatment with an antihistamine, antipyretic, and / or corticosteroid is administered. If pretreatment is used with prior enzyme replacement therapy (ERT), a pretreatment with an antihistamine, antipyretic, and / or corticosteroid is administered prior to administration of rhGAA or a pharmaceutically acceptable salt thereof.

[0225] In some embodiments, rhGAA or a pharmaceutically acceptable salt thereof can be formulated for oral administration. Orally administrable compositions can be formulated as tablets, capsules, ovules, elixirs, solutions or suspensions, gels, syrups, mouthwashes, or dry powders that are reconstituted with water or other suitable vehicles before use, and may optionally contain flavorings and colorants for immediate release, delayed release, modified release, sustained release, pulsed release, or controlled release applications. Solid compositions such as tablets, capsules, lozenges, pastilles, pills, boluses, powders, pastes, granules, bullets, dragees, or premixed formulations can also be used. Solid and liquid compositions for oral use can be prepared according to methods known in the art. Such compositions can also contain one or more pharmaceutically acceptable carriers and excipients that can be in solid or liquid form. Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients (including but not limited to binders, fillers, lubricants, disintegrants, or wetting agents). Suitable pharmaceutically acceptable excipients are known in the art and include, but are not limited to, pregelatinized starch, polyvinylpyrrolidone, povidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylethylcellulose (HPEC), hydroxypropylcellulose (HPC), sucrose, gelatin, gum arabic, lactose, microcrystalline cellulose, dibasic calcium phosphate, magnesium stearate, stearic acid, glyceryl behenate, talc, silica, corn, potato or tapioca starch, sodium starch glycolate, sodium lauryl sulfate, sodium citrate, calcium carbonate, dibasic calcium phosphate, croscarmellose sodium glycine, and complex silicates. The tablets may be coated by methods well known in the art.

[0226] In some embodiments, the pharmaceutical compositions described herein can be formulated according to U.S. Patent No. 10,512,676 and U.S. Provisional Application No. 62 / 506,574 (each incorporated herein by reference in its entirety). For example, in some embodiments, the pH of the pharmaceutical compositions described herein is from about 5.0 to about 7.0 or from about 5.0 to about 6.0. In some embodiments, the pH is in the range of 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 can be adjusted to the 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.

[0227] The pharmaceutical compositions described herein may comprise a buffer system, such as a citrate system, a phosphate system, and / or a combination 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 comprises citrate. In further embodiments, the buffer comprises sodium citrate (e.g., a mixture of dehydrated sodium citrate and citric acid monohydrate). In one or more embodiments, the buffer solution comprising citrate may comprise sodium citrate and citric acid. In some embodiments, both citrate and phosphate buffer are present.

[0228] In certain embodiments, the pharmaceutical compositions described herein include at least one excipient. Excipients can act as tonicity agents, swelling agents, and / or stabilizers. Tonicity agents are components that help ensure that the formulation has an osmotic pressure similar or identical to that of human blood. Swelling agents increase the quality of these formulations (e.g., lyophilized) and provide sufficient structural components for the bulk. Stabilizers are compounds that can prevent or minimize aggregate formation at the hydrophobic air-water interface surface. An excipient can serve as both a tonicity agent and a swelling agent. For example, mannitol can function as a tonicity agent and also provide benefits as a swelling agent.

[0229] 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 one or more tonicity agents ranges from about 10 mg / mL to about 50 mg / mL. In further embodiments, the total amount of one or more tonicity agents ranges from about 10, 11, 12, 13, 14, or 15 mg / mL to about 16, 20, 25, 30, 35, 40, 45, or 50 mg / mL.

[0230] In certain embodiments, the excipient comprises a stabilizer. In certain embodiments, the stabilizer is a surfactant. In certain embodiments, the stabilizer is polysorbate 80. In one or more embodiments, the total amount of the stabilizer is in the range of about 0.1 mg / mL to about 1.0 mg / mL. In a further embodiment, the total amount of the stabilizer is in the range of 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 a further embodiment, the total amount of the 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.

[0231] In some embodiments, the pharmaceutical composition comprises one or more of the following excipients: sodium citrate dihydrate, citric acid monohydrate, mannitol, or polysorbate 80.

[0232] In some embodiments, the pharmaceutical composition comprises (a) rhGAA (e.g., ATB200 or Sipaglucosidase α), (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 acidifying agent and / or an alkalizing agent.

[0233] In some embodiments, the pharmaceutical composition comprises (a) rhGAA (e.g., ATB200 or Sipaglucosidase alpha) 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 at a concentration of about 0.1-1 mg / mL, about 0.2-0.5 mg / mL, or about 0.5 mg / mL, ) water, and having a pH of about 6.0. In at least one embodiment, the pharmaceutical composition comprises (a) 15 mg / mL rhGAA (e.g., ATB200 or Sipaglucosidase α), (b) 25 mM sodium citrate buffer, (c) 20 mg / mL mannitol, (d) 0.5 mg / mL polysorbate 80, and (e) natural 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.

[0234] In some embodiments, a pharmaceutical composition comprising rhGAA (eg, ATB200 or Sipaglucosidase alfa) is diluted prior to administration to a subject in need thereof.

[0235] In some embodiments, the pharmaceutical compositions described herein may undergo a freeze-drying (freeze-drying) process to provide a cake or powder. Thus, in some embodiments, the pharmaceutical compositions described herein are freeze-dried rhGAA compositions. The freeze-dried mixture may comprise rhGAA as described herein (e.g., ATB200 or Sipaglucosidase α), 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 ingredients (e.g., other excipients) may be added to the freeze-dried mixture. The pharmaceutical composition comprising the freeze-dried formulation can be provided in vials, which can then be stored, transported, reconstituted, and / or administered to a patient.

[0236] In some embodiments, a pharmaceutical composition comprising rhGAA as described herein (e.g., ATB200 or Sipaglucosidase α) is a lyophilized powder in a glass vial. In some embodiments, each vial may contain approximately 105 mg of lyophilized rhGAA (e.g., ATB200 or Sipaglucosidase α). The powder can be reconstituted in sterile water and then diluted with 0.9% sodium chloride before administration by IV infusion. In some embodiments, after reconstitution, the concentrate obtained contains 15 mg of rhGAA (e.g., ATB200 or Sipaglucosidase α) / mL. In some embodiments, after reconstitution with 7.2 mL of diluent, the vial contains a usable volume of 7.0 mL of concentrate containing 15 mg / mL of rhGAA (e.g., ATB200 or Sipaglucosidase α). In some embodiments, the diluent is sterile water and / or 0.9% sodium chloride. In some embodiments, each vial may include an overflow to compensate for liquid loss during preparation. In some embodiments, the present disclosure provides a vial (e.g., a glass vial) containing 105 mg of a lyophilized rhGAA (e.g., ATB200 or Sipa-glucosidase alfa) composition comprising rhGAA (e.g., ATB200 or Sipa-glucosidase alfa), sodium citrate dihydrate, citric acid monohydrate, mannitol, polysorbate 80, wherein the amount / concentration of each ingredient can be selected from those described herein.

[0237] In some embodiments, the number of vials to be reconstituted is based on the patient's weight and the fact that each vial contains 105 mg rhGAA (e.g., ATB200 or sipa-glucosidase alfa). Thus, in some embodiments, patient dose (mg) = subject weight (kg) x dose (mg / kg); and the number of vials required = patient dose (in mg) / 105 (mg / vial). In some embodiments, if the number of vials includes a fraction, it is rounded to the next whole number.

[0238] For example, in a 65 kg patient with a dose of 20 mg / kg: Patient dose (mg) = 65 kg x 20 mg / kg = 1300 mg total dose; Number of vials required = 1300 / 105 mg / vial = 12.381 rounded to 13 vials for dispensing purposes. The patient dose is 20 mg / kg, so the entire volume of the first 12 vials will be withdrawn, but 2.7 ml will be withdrawn from the 13th vial and added to the infusion bag.

[0239] In some embodiments, the reconstitution method comprises or consists essentially of the following: (1) reconstitute each vial by slowly injecting 7.2 mL of sterile water for injection into the inner wall of each vial instead of directly injecting the lyophilized cake; (2) gently rolling each vial without inverting, rotating or shaking; (3) diluting a certain amount of reconstituted rhGAA (based on the patient's weight) in 0.9% sodium chloride for injection and immediately after reconstitution, diluting to a total infusion volume for a 20 mg / kg dose (based on the patient's weight); (4) before adding the reconstituted rhGAA, remove air and add a total amount equal to the reconstitution volume of the injection bag with 0.9% sodium chloride; (5) slowly remove the reconstituted rhGAA from the injection bag; Remove the reconstituted solution from each vial, avoiding foaming in the syringe; (6) slowly add the reconstituted sipa-glucosidase alfa solution directly to the 0.9% Sodium Chloride Injection bag (not directly into any air space that may remain in the bag), avoiding foaming in the bag (alternatively, an appropriately sized empty bag can be used to complete the product dilution by adding the required volume of 0.9% Sodium Chloride Injection and reconstituted rhGAA based on the patient's weight. Pre-filled 0.9% Sodium Chloride Injection bags are recommended); (7) gently invert or massage the bag to mix; and (8) cap the bag and tubing to protect the drug from light. After reconstitution, each vial will produce a concentration of 15 mg / mL. The total extractable dose per vial is 105 mg / 7 mL.

[0240] In some embodiments, only the exact amount of reconstituted rhGAA based on the patient's weight is diluted. In some embodiments, for partial withdrawals of reconstituted vials, the amount is rounded to one decimal place. In some embodiments, the infusion bag is not shaken for mixing or transported using pneumatic tubing. In some embodiments, the reconstituted and diluted solution may contain particles that appear as thin white strands or translucent fibers after initial preparation and increase in size over time.

[0241] The present disclosure also provides a pharmaceutical composition comprising an enzyme stabilizer. In some embodiments, the enzyme stabilizer is miglustat or a pharmaceutically acceptable salt thereof. In another embodiment, the enzyme stabilizer is deoxynojirimycin or a pharmaceutically acceptable salt thereof.

[0242] In some embodiments, the rhGAA described herein is formulated in one pharmaceutical composition, and an enzyme stabilizer such as miglustat is formulated in another pharmaceutical composition. In some embodiments, the pharmaceutical composition comprising miglustat is based on a (Actelion Pharmaceuticals). In some embodiments, the pharmaceutical composition comprising miglustat comprises microcrystalline cellulose, pregelatinized starch, Sucralose powder, magnesium stearate and / or colloidal silicon dioxide. In some embodiments, the pharmaceutical composition is a hard gelatin capsule for oral administration, which contains about 65 mg of miglustat, microcrystalline cellulose, pregelatinized starch, Sucralose powder, magnesium stearate and / or colloidal silicon dioxide.

[0243] In some embodiments, the pharmaceutical composition comprising miglustat comprises 20%-40% miglustat by weight, for example, 30-35% miglustat by weight. In some embodiments, the pharmaceutical composition comprising miglustat further comprises 40%-60% microcrystalline cellulose by weight, for example, 45-55% microcrystalline cellulose by weight. In some embodiments, the pharmaceutical composition comprising miglustat further comprises 5%-25% pregelatinized starch by weight, for example, 10-20% pregelatinized starch by weight. In some embodiments, the pharmaceutical composition comprising miglustat further comprises 0.1%-5% sucralose by weight, for example, 0.2-1% sucralose by weight. In some embodiments, the pharmaceutical composition comprising miglustat further comprises 0.1%-5% magnesium stearate by weight, for example, 0.2-1% magnesium stearate by weight. In some embodiments, the pharmaceutical composition comprising miglustat further comprises 0.1%-5% by weight of colloidal silicon dioxide, for example 0.2-1% by weight of colloidal silicon dioxide. In some embodiments, the pharmaceutical composition comprising miglustat is provided in a hard gelatin capsule for oral administration. In some embodiments, the pharmaceutical composition is a hard gelatin capsule for oral administration, comprising about 20%-40% (e.g., 30-35%) of miglustat by weight, 40%-60% (e.g., 45-55%) of microcrystalline cellulose by weight, 5%-25% (e.g., 10-20%) of pregelatinized starch by weight, 0.1%-5% (e.g., 0.2%-1%) of sucralose by weight, 0.1%-5% (e.g., 0.2%-1%) of magnesium stearate by weight, and / or 0.1%-5% (e.g., 0.2%-1%) of colloidal silicon dioxide by weight. In some embodiments, the pharmaceutical composition comprising miglustat is provided in an oral liquid dosage form, such as an oral solution, dispersion, or suspension. In some embodiments, the pharmaceutical composition is an oral liquid dosage form comprising about 20%-40% (e.g., 30-35%) of miglustat by weight, 40%-60% (e.g., 45-55%) of microcrystalline cellulose by weight, 5%-25% (e.g., 10-20%) of pregelatinized starch by weight, 0.1%-5% (e.g., 0.2%-1%) of sucralose by weight, 0.1%-5% (e.g., 0.2%-1%) of magnesium stearate by weight, and / or 0.1%-5% (e.g., 0.2%-1%) of colloidal silicon dioxide by weight.

[0244] In some embodiments, the pharmaceutical composition comprising miglustat comprises about 50 to about 100 mg of miglustat, for example, about 65 mg of miglustat. In some embodiments, the pharmaceutical composition comprising miglustat comprises about 50 to about 150 mg of microcrystalline cellulose, for example, about 75 to about 125 mg of microcrystalline cellulose. In some embodiments, the pharmaceutical composition comprising miglustat comprises about 20 to about 50 mg of pregelatinized starch, for example, about 30 to about 40 mg of pregelatinized starch. In some embodiments, the pharmaceutical composition comprising miglustat comprises about 0.1 to about 5 mg of sucralose, for example, about 0.5 to about 2 mg of sucralose. In some embodiments, the pharmaceutical composition comprising miglustat comprises about 0.1 to about 5 mg of magnesium stearate, for example, about 0.5 to about 2 mg of magnesium stearate. In some embodiments, the pharmaceutical composition comprising miglustat comprises about 0.1 to about 5 mg of colloidal silicon dioxide, for example, about 0.2 mg to about 1 mg of colloidal silicon dioxide. In some embodiments, the pharmaceutical composition comprising miglustat is provided in a hard gelatin capsule for oral administration. In some embodiments, the pharmaceutical composition is a hard gelatin capsule for oral administration, comprising about 50 to about 100 mg (e.g., 65 mg) of miglustat, about 50 to about 150 mg (e.g., 75 mg to about 125 mg) of microcrystalline cellulose, about 20 to about 50 mg (e.g., 30 mg to about 40 mg) of pregelatinized starch, about 0.1 to about 5 mg (e.g., 0.5 mg to about 2 mg) of sucralose, about 0.1 to about 5 mg (e.g., 0.5 mg) to about 2 mg of magnesium stearate, and / or about 0.1 to about 5 mg (e.g., 0.5 mg to about 2 mg) of colloidal silicon dioxide. In some embodiments, the pharmaceutical composition comprising miglustat is provided in an oral liquid dosage form, such as an oral solution, dispersion, or suspension. In some embodiments, the pharmaceutical composition is an oral liquid dosage form comprising about 50 to about 100 mg (e.g., 65 mg) of miglustat, about 50 to about 150 mg (e.g., 75 mg to about 125 mg) of microcrystalline cellulose, about 20 to about 50 mg (e.g., 30 mg to about 40 mg) of pregelatinized starch, about 0.1 to about 5 mg (e.g., 0.5 mg to about 2 mg) of sucralose, about 0.1 to about 5 mg (e.g., 0.5 mg) to about 2 mg of magnesium stearate, and / or about 0.1 to about 5 mg (e.g., 0.5 mg to about 2 mg) of colloidal silicon dioxide.

[0245] In some embodiments, pharmaceutical compositions comprising miglustat are provided in hard gelatin capsules for oral administration, which contain about 65 mg miglustat, about 100 mg microcrystalline cellulose, about 32.6 mg pregelatinized starch, about 1 mg sucralose powder, about 1 mg magnesium stearate, and about 0.4 mg colloidal silicon dioxide.

[0246] In some embodiments, a pharmaceutical composition comprising miglustat is provided as an oral solution, dispersion, or suspension comprising about 65 mg miglustat, about 100 mg microcrystalline cellulose, about 32.6 mg pregelatinized starch, about 1 mg sucralose powder, about 1 mg magnesium stearate, and about 0.4 mg colloidal silicon dioxide.

[0247] In some embodiments, a pharmaceutical composition comprising miglustat is provided as an oral solution, dispersion, or suspension comprising about 130 mg miglustat, about 200 mg microcrystalline cellulose, about 65.2 mg pregelatinized starch, about 2 mg sucralose powder, about 2 mg magnesium stearate, and about 0.8 mg colloidal silicon dioxide.

[0248] In some embodiments, a pharmaceutical composition comprising miglustat is provided as an oral solution, dispersion, or suspension comprising about 195 mg miglustat, about 300 mg microcrystalline cellulose, about 97.8 mg pregelatinized starch, about 3 mg sucralose powder, about 3 mg magnesium stearate, and about 1.2 mg colloidal silicon dioxide.

[0249] In some embodiments, a pharmaceutical composition comprising miglustat is provided as an oral solution, dispersion, or suspension comprising about 260 mg miglustat, about 400 mg microcrystalline cellulose, about 130.4 mg pregelatinized starch, about 4 mg sucralose powder, about 4 mg magnesium stearate, and about 1.6 mg colloidal silicon dioxide. Treatment A. Treatment of diseases

[0250] Another aspect of the present disclosure relates to methods for treating diseases or disorders associated with glycogen storage disorders by administering rhGAA or pharmaceutical compositions as described herein. In some embodiments, the disease is Pompe disease (also known as acid maltase deficiency (AMD) and glycogen storage disease type II (GSD II)). In some embodiments, the rhGAA is ATB200 or Sipaglucosidase alpha. In some embodiments, the pharmaceutical composition comprises rhGAA (e.g., ATB200 or Sipaglucosidase alpha). Also provided herein is the use of rhGAA (e.g., ATB200 or Sipaglucosidase alpha) to treat Pompe disease.

[0251] In some embodiments, the subject treated by the methods disclosed herein is a patient who has undergone ERT. For example, the subject treated by the methods disclosed herein is an adult patient 18 years of age or older diagnosed with late-onset Pompe disease (acid alpha-glucosidase (GAA) deficiency) who has previously received enzyme replacement therapy (ERT). In some embodiments, the patient who has undergone ERT is currently receiving an approved ERT (e.g., or In some embodiments, the patient is currently receiving ERT. In some embodiments, the methods disclosed herein are initiated approximately 2 weeks after the last ERT dose. In some embodiments, the subject treated by the methods disclosed herein is an ERT-naive patient.

[0252] In some embodiments, it is expected that the patient will be seen again every 3 months to ensure the clinical benefit provided to the patient and therefore continue treatment. This frequency of visits may be necessary until before the pharmaceutical product is launched on the market. In some embodiments, the patient's response to treatment is regularly assessed based on the assessment of the main clinical and laboratory parameters of the disease. In some embodiments, rhGAA (e.g., Sipaglucosidase α) and Miglustat as described herein are administered every two weeks. In some embodiments, the dosage of rhGAA, such as Sipaglucosidase α, is about 20 mg / kg body weight, given as a 4-hour infusion. In some embodiments, if the infusion is delayed, the infusion should not be started more than 3 hours after oral administration of Miglustat. In some embodiments, for patients weighing 50 kg or more, four 65 mg capsules (260 mg in total).

[0253] In some of the above embodiments, for patients weighing between 40 kg and 50 kg, a dose of three 65 mg capsules (195 mg total) is administered. In some of the above embodiments, rhGAA (eg, Sipaglucosidase alfa) is administered by infusion every two weeks.

[0254] The rhGAA or pharmaceutical compositions described herein are administered by a suitable route. In one embodiment, the rhGAA or pharmaceutical composition is administered intravenously. In some embodiments, the rhGAA or pharmaceutical composition is administered intravenously using an infusion pump. In some embodiments, when administered intravenously, the infusion bag and infusion tubing are covered to protect from light. In other embodiments, the rhGAA or pharmaceutical composition is administered by direct administration to the target tissue (such as to the heart or skeletal muscle (e.g., intramuscularly)) or the nervous system (e.g., direct injection into the brain; intraventricularly; intrathecally). In some embodiments, the rhGAA or pharmaceutical composition is administered orally. If desired, more than one route may be used simultaneously.

[0255] In some embodiments, the therapeutic effect of rhGAA or a pharmaceutical composition described herein can be assessed based on one or more of the following criteria: (1) cardiac status (e.g., an increase in end-diastolic and / or end-systolic volumes, or a reduction, improvement, or prevention of progressive cardiomyopathy typically found in GSD-II), (2) improvement in lung function (e.g., an increase in crying vital capacity over baseline capacity, and / or normalization of oxygen desaturation during crying), (3) neurodevelopment and / or motor skills / function (e.g., an increase in AIMS scores), (4) a reduction in glycogen levels in tissues of individuals affected by the disease, (5) muscle strength; and / or (6) quality of life.

[0256] In some embodiments, the therapeutic effect of rhGAA or a pharmaceutical composition described herein can be assessed by measurements of motor function, muscle strength, lung function, patient-reported outcomes (PROs), and biomarkers. In some embodiments, the therapeutic effect of rhGAA or a pharmaceutical composition described herein can be measured by a 6-minute walk test (6MWT), predicted 6MWD%, a 10-meter walk test (10MWT), GSGC, climbing 4 stairs, Gowers', chair test, and timed up and go (TUG). Treatment with rhGAA or a pharmaceutical composition described herein can also result in improvements in pulmonary function tests (PFTs), such as those measured by FVC (e.g., sitting, supine), slow vital capacity (SVC), maximum inspiratory pressure (MIP), maximum expiratory pressure (MEP), and sniff-nasal inspiratory pressure (SNIP), as well as improvements in muscle strength across all tested body parts in both ambulatory and non-ambulatory subjects, such as those measured by MMT (e.g., lower extremity MMT, upper extremity MMT, total body MMT) and quantitative muscle testing (QMT).

[0257] In some embodiments, after administering one or more doses of rhGAA or a pharmaceutical composition as described herein, the subject's cardiac state improves by 10%, 20%, 30%, 40% or 50% (or any percentage therebetween) compared to the cardiac state of a subject treated with a vehicle or the cardiac state of a subject before treatment. The subject's cardiac state can be assessed by measuring end-diastolic and / or end-systolic volumes and / or by clinically assessing cardiomyopathy. In some embodiments, after administering one or more doses of rhGAA (e.g., ATB200 or sipa-glucosidase α) or a pharmaceutical composition comprising rhGAA (e.g., ATB200 or sipa-glucosidase α), the subject's pulmonary function improves by 10%, 20%, 30%, 40% or 50% (or any percentage therebetween) compared to the pulmonary function of a subject treated with a vehicle or the pulmonary function of a subject before treatment. In certain embodiments, the improvement is achieved after administering 1 week, 2 weeks, 3 weeks, 1 month, 2 months or more (or any time period therebetween). In certain embodiments, rhGAA (e.g., ATB200 or Sipaglucosidase alfa) or a pharmaceutical composition comprising rhGAA (e.g., ATB200 or Sipaglucosidase alfa) improves and / or stabilizes lung function in a subject 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or longer after administration (or any time period therebetween).

[0258] In some embodiments, after administration of one or more doses of rhGAA or a pharmaceutical composition as described herein, the subject's lung function improves by 10%, 20%, 30%, 40% or 50% (or any percentage therebetween) compared to the lung function of a subject treated with a vehicle or the lung function of a subject before treatment. The subject's lung function can be assessed by normalization of the crying vital capacity over the baseline vital capacity and / or oxygen saturation during crying. In some embodiments, after administration of one or more doses of rhGAA (e.g., ATB200 or sipa glucosidase alpha) or a pharmaceutical composition comprising rhGAA (e.g., ATB200 or sipa glucosidase alpha), the subject's lung function improves by 10%, 20%, 30%, 40% or 50% (or any percentage therebetween) compared to the lung function of a subject treated with a vehicle or the lung function of a subject before treatment. In certain embodiments, the improvement is achieved after administration for 1 week, 2 weeks, 3 weeks, 1 month, 2 months or longer (or any time period therebetween). In certain embodiments, rhGAA (e.g., ATB200 or Sipaglucosidase alfa) or a pharmaceutical composition comprising rhGAA (e.g., ATB200 or Sipaglucosidase alfa) improves lung function in a subject 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or longer after administration (or any time period therebetween).

[0259] In some embodiments, the neurodevelopment and / or motor skills of a subject treated with a vehicle or the neurodevelopment and / or motor skills of a subject before treatment are compared, after administering one or more doses of rhGAA (e.g., ATB200 or sipa glucosidase alpha) or a pharmaceutical composition of rhGAA as described herein (e.g., ATB200 or sipa glucosidase alpha), the neurodevelopment and / or motor skills of the subject are improved by 10%, 20%, 30%, 40% or 50% (or any percentage therebetween). The neurodevelopment and / or motor skills of the subject can be assessed by determining the AIMS score. The AIMS is a 12-item anchored scale (see Rush JA Jr., Handbook of Psychiatric Measures [Manual of Neurological Scales], American Psychiatric Association (American Psychiatric Association), 2000, 166-168) performed and scored by a clinician. Items 1-10 are scored on a 5-point anchored scale. Items 1-4 assess orofacial movement. Items 5-7 relate to limb and trunk movement disorders. Items 8-10 relate to the overall severity judged by the examiner, as well as the patient's awareness of movement and the pain associated therewith. Items 11-12 are yes / no questions about dental and / or denture problems (such questions may lead to a misdiagnosis as movement disorders). In some embodiments, after administering one or more doses of rhGAA (e.g., ATB200 or Sipaglucosidase α) or a pharmaceutical composition comprising rhGAA (e.g., ATB200 or Sipaglucosidase α), the subject's neurodevelopment and / or motor skills improve by 10%, 20%, 30%, 40% or 50% (or any percentage therebetween) compared to the neurodevelopment and / or motor skills of a subject treated with a vehicle or the neurodevelopment and / or motor skills of the subject before treatment. In certain embodiments, the improvement is achieved after administering 1 week, 2 weeks, 3 weeks, 1 month, 2 months or more (or any time period therebetween). In certain embodiments, rhGAA (e.g., ATB200 or Sipa-glucosidase alfa) or a pharmaceutical composition comprising rhGAA (e.g., ATB200 or Sipa-glucosidase alfa) improves and / or stabilizes neurodevelopment and / or motor skills in a subject 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or longer after administration (or any time period therebetween).

[0260] In some embodiments, the glycogen level of a tissue of a subject is reduced by 10%, 20%, 30%, 40%, or 50% (or any percentage therebetween) after administration of one or more doses of rhGAA or a pharmaceutical composition as described herein, compared to the glycogen level of the tissue of a subject treated with vehicle or the glycogen level of the tissue of the subject before treatment. In some embodiments, the tissue is muscle, such as the quadriceps, triceps, and gastrocnemius. The glycogen level of the tissue can be analyzed using methods known in the art. As is well known, determination of glycogen levels is based on amyloglucosidase digestion and is described in the following publications: Amalfitano et al. (1999), Proc Natl Acad Sci USA, 96:8861-8866. In some embodiments, after administration of one or more doses of rhGAA (e.g., ATB200 or sipa-glucosidase alpha) or a pharmaceutical composition comprising rhGAA (e.g., ATB200 or sipa-glucosidase alpha), the glycogen level in the muscle of the subject is reduced by 10%, 20%, 30%, 40% or 50% (or any percentage therebetween) compared to the glycogen level in the muscle of a subject treated with vehicle or the glycogen level in the muscle of the subject before treatment. In certain embodiments, the reduction is achieved after 1 week, 2 weeks, 3 weeks, 1 month, 2 months or more of administration (or any time period therebetween). In certain embodiments, rhGAA (e.g., ATB200 or sipa-glucosidase alpha) or a pharmaceutical composition comprising rhGAA (e.g., ATB200 or sipa-glucosidase alpha) reduces the glycogen level in the muscle of the subject 1 week, 2 weeks, 3 weeks, 1 month, 2 months or more of administration (or any time period therebetween).

[0261] In some embodiments, the therapeutic effects of the pharmaceutical compositions of the present application, such as those described herein, are long-lasting and maintained within 12, 24, 36, 48 or >48 months of treatment. In some embodiments, the therapeutic effects of the pharmaceutical compositions of the present application are sustained to or throughout 24, 36 or 48 months of treatment, including, for example, improved motor function (e.g., as measured by 6MWD, GSGC, 10m walk, climbing 4 stairs, Gowers, chair test, TUG), pulmonary function or respiratory function, or improved or stabilized PFTs (e.g., as measured by predicted FVC% (sitting and supine), SVC, MIP, MEP and SNIP), biomarker levels (e.g., serum CK and urine Hex4), stabilized or improved muscle strength in all tested body parts (e.g., as measured by MMT (lower limbs, upper limbs, total body), QMT) and / or patient-reported outcomes (PROs, including PROMIS-Physical Function Short Form [SF] 20a and PROMIS-Fatigue SF 8a).

[0262] In some embodiments, patients treated with the pharmaceutical compositions of the present application are more likely to develop leukemia than patients treated with conventional rhGAA products (e.g., or ) achieve greater (e.g., greater clinically meaningful) improvements in motor function (e.g., as measured by 6MWD, GSGC, 10 m walk, 4 stairs climbed, Gowers, chair test, TUG), pulmonary or respiratory function, or improved or stable PFTs (e.g., as measured by % predicted FVC (sitting and supine), SVC, MIP, MEP, and SNIP), biomarker levels (e.g., serum CK and urine Hex4), stable or improved muscle strength in all tested body parts (e.g., as measured by MMT (lower extremity, upper extremity, total body), QMT), and / or patient-reported outcomes (PROs, including PROMIS-Physical Function Short Form [SF] 20a and PROMIS-Fatigue SF 8a). In some embodiments, patients treated with the pharmaceutical compositions of the present application show greater improvements in motor function (e.g., as measured by 6MWD), pulmonary or respiratory function (e.g., as measured by FVC), and / or muscle strength (e.g., as measured by MMT) than patients treated with conventional rhGAA products (e.g., or ) patients treated with a greater (e.g., greater clinical significance) improvement. In some embodiments, this greater improvement persists for or throughout 12, 24, 36, 48, or >48 months of treatment.

[0263] In some embodiments, patients who have undergone ERT (e.g., transition from conventional rhGAA products (e.g., or ) patients who were switched to the pharmaceutical composition of the present application) showed greater (e.g., greater clinically meaningful) improvement in motor function (e.g., as measured by 6MWD, GSGC, 10 m walk, 4 stairs climbed, Gowers, chair test, TUG), pulmonary function or respiratory function, or improved or stable PFTs (e.g., as measured by predicted FVC% (sitting and supine), SVC, MIP, MEP, and SNIP), biomarker levels (e.g., serum CK and urine Hex4), stable or improved muscle strength in all tested body parts (e.g., as measured by MMT (lower extremity, upper extremity, total body), QMT), and / or patient-reported outcomes (PROs, including PROMIS-Physical Function Short Form [SF] 20a and PROMIS-Fatigue SF 8a) when compared to patients who continued to use conventional rhGAA products. In some embodiments, patients receiving conventional rhGAA products (e.g., or Patients treated with the pharmaceutical composition of the present invention (whose motor function no longer improves or worsens) achieve improvements in motor function (e.g., as measured by 6MWD, GSGC, 10m walk, 4 stairs climbed, Gowers, chair test, TUG), pulmonary function or respiratory function, or improved or stable PFTs (e.g., as measured by predicted FVC% (sitting and supine), SVC, MIP, MEP, and SNIP), biomarker levels (e.g., serum CK and urine Hex4), stable or improved muscle strength in all tested body parts (e.g., as measured by MMT (lower extremity, upper extremity, total body), QMT), and / or patient-reported outcomes (PROs, including PROMIS-Physical Function Short Form [SF] 20a and PROMIS-Fatigue SF 8a). In some embodiments, such improvements persist for up to or throughout 12, 24, 36, 48, or >48 months. B. Biomarkers

[0264] Biomarkers of glycogen accumulation in a subject, such as urinary hexotetraose (Hex4), can be used to evaluate and compare the therapeutic efficacy of enzyme replacement therapy in subjects with Pompe disease. In some embodiments, the therapeutic efficacy of rhGAA or a pharmaceutical composition comprising rhGAA on glycogen accumulation is assessed by measuring the subject's urinary Hex4 level.

[0265] Biomarkers of muscle damage or injury, such as creatine kinase (CK), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST), can be used to evaluate and compare the therapeutic effects 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 assessed by measuring the levels of CK, LDH, ALT, and / or AST in the subject. In at least one embodiment, the therapeutic effect of rhGAA or a pharmaceutical composition comprising rhGAA on muscle damage is assessed by measuring the CK level in the subject.

[0266] Biomarkers such as LAMP-1, LC3 and desfulin can also be used to evaluate and compare the therapeutic effects of rhGAA or pharmaceutical compositions described herein. In Pompe disease, GAA cannot hydrolyze lysosomal glycogen, resulting in abnormal accumulation of large lysosomes filled with glycogen in certain tissues. (Raben et al., JBC [Journal of Biological Chemistry] 273: 19086-19092, 1998.) Studies on Pompe disease mouse models have shown that enlarged lysosomes in skeletal muscle cannot fully explain the decline in mechanical properties, and the presence of large inclusion bodies containing degraded myofibrils (i.e., autophagy accumulation) can lead to impaired muscle function. (Raben et al., Human Mol Genet [Human Molecular Genetics] 17: 3897-3908, 2008.) Reports also show that impaired autophagy flux is associated with poor therapeutic outcomes in Pompe disease 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 prevalent in unclassified limb-girdle muscular dystrophy (LGMD) (Preisler et al., Mol Genet Metab 110:287-289, 2013), a group of genetically heterogeneous neuromuscular diseases with more than 30 genetically defined subtypes of varying severity. IHC examination showed a significant increase in the presence of desferlin in the sarcoplasm of skeletal muscle fibers of Gaa KO mice.

[0267] Various known methods can be used to measure the gene expression level and / or protein level of such biomarkers. For example, a sample, such as a tissue biopsy, particularly a muscle biopsy, from a subject treated with rhGAA or a pharmaceutical composition described herein can be obtained. In some embodiments, the sample is a biopsy of a subject's muscle. In some embodiments, the muscle is selected from the quadriceps femoris, triceps femoris, and gastrocnemius. The sample obtained from the subject can be stained with one or more antibodies or other detection agents for detecting such biomarkers or identified and quantified by mass spectrometry. The sample can also or alternatively be processed to detect the presence of nucleic acids encoding biomarkers, such as mRNA, by, for example, RT-qPCR methods.

[0268] In some embodiments, the gene expression levels and / or protein levels of one or more biomarkers are measured in muscle biopsies obtained from individuals before and after treatment with rhGAA or a pharmaceutical composition as described herein. In some embodiments, the gene expression levels and / or protein levels of one or more biomarkers are measured in muscle biopsies obtained from individuals treated with a vehicle. In some embodiments, the gene expression levels and / or protein levels of one or more biomarkers are reduced by 10%, 20%, 30%, 40%, or 50% (or any percentage therebetween) after administration of one or more doses of rhGAA or a pharmaceutical composition as described herein, compared to the gene expression levels and / or protein levels of a subject treated with a vehicle or the gene expression levels and / or protein levels of a subject before 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 doses of rhGAA (e.g., ATB200 or Sipaglucosidase alfa) or a pharmaceutical composition comprising rhGAA (e.g., ATB200 or Sipaglucosidase alfa) compared to the gene expression level and / or protein level in a subject treated with vehicle or the gene expression level and / or protein level in the subject before treatment. In certain embodiments, the reduction is achieved after 1 week, 2 weeks, 3 weeks, 1 month, 2 months or more of administration (or any time period therebetween). In certain embodiments, rhGAA (e.g., ATB200 or Sipaglucosidase alfa) or a pharmaceutical composition comprising rhGAA (e.g., ATB200 or Sipaglucosidase alfa) reduces gene expression levels and / or protein levels of one or more biomarkers after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or more (or any time period therebetween) from administration. C. rhGAA dosage

[0269] The pharmaceutical formulation or reconstituted composition is administered in a therapeutically effective amount (e.g., a dose sufficient to treat the disease when administered at regular intervals, such as by alleviating symptoms associated with the disease, delaying the onset of the disease, and / or reducing the severity or frequency of the symptoms of the disease). The amount therapeutically effective in treating the disease depends on the nature and extent of the disease effects and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be used to help identify the optimal dosage range. In at least one embodiment, the rhGAA or a pharmaceutical composition comprising rhGAA described herein is administered in a dose of about 1 mg / kg to about 100 mg / kg, for example, about 5 mg / kg to about 30 mg / kg, typically about 5 mg / kg to about 20 mg / kg. In at least one embodiment, rhGAA or a 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, 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, rhGAA or a pharmaceutical composition is administered at a dose of about 20 mg / kg. In some embodiments, rhGAA or a pharmaceutical composition is administered simultaneously or sequentially with an enzyme stabilizer. In some embodiments, the enzyme stabilizer is miglustat. In at least one embodiment, miglustat is administered at an oral dose of about 260 mg. In at least one embodiment, miglustat is administered orally at a dose of about 195 mg. The effective dose for a particular individual can vary over time (e.g., increase or decrease), depending on the needs of the individual. For example, the amount of rhGAA and / or miglustat can be adjusted during periods of physiological illness or physical stress, or if anti-acid α-glucosidase antibodies are present or increase, or if disease symptoms worsen.

[0270] In some embodiments, the therapeutically effective dose of rhGAA or a pharmaceutical composition described herein is lower than the therapeutically effective dose of a conventional rhGAA product. For example, if the therapeutically effective dose of a conventional rhGAA product is 20 mg / kg, the dose of rhGAA or a pharmaceutical composition described herein required to produce the same or better therapeutic effect as the conventional rhGAA product may be lower than 20 mg / kg. The therapeutic effect can be assessed based on one or more criteria discussed above (e.g., cardiac status, glycogen levels, or biomarker expression). In some embodiments, the therapeutically effective dose of rhGAA or a 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 a conventional rhGAA product.

[0271] In some embodiments, the therapeutic effects of rhGAA or the pharmaceutical compositions described herein include improving and / or stabilizing motor function, improving and / or stabilizing muscle strength (upper body, lower body, or whole body), improving and / or stabilizing lung function, reducing fatigue, reducing the level of at least one muscle damage biomarker, reducing the level of at least one glycogen accumulation biomarker, or a combination thereof. In some embodiments, the therapeutic effects of rhGAA or the pharmaceutical compositions described herein include reversal of lysosomal pathology in myofibers, faster and / or more efficient reduction of glycogen content in myofibers, increase in six-minute walk test distance, decrease in timed up and go time, decrease in four-stair climb test time, decrease in ten-meter walk test time, decrease in gait-stair-Gauls-Chair score, increase in upper limb strength, improvement and / or stabilization of shoulder adduction, improvement and / or stabilization of shoulder abduction, improvement and / or stabilization of elbow flexion, improvement and / or stabilization of elbow extension, improvement and / or stabilization of upper body strength, improvement and / or stabilization of lower body strength, improvement and / or stabilization of whole body strength, improvement in upright (seated) forced vital capacity, improvement and / or stabilization of maximal expiratory pressure, improvement and / or stabilization of maximal inspiratory pressure, decrease in fatigue severity scale score, decrease in urine hexotetraose levels, decrease in creatine kinase levels, decrease in alanine aminotransferase levels, decrease in aspartate aminotransferase levels, or any combination thereof.

[0272] In some embodiments, the rhGAA or pharmaceutical compositions described herein achieve the desired therapeutic effect faster than conventional rhGAA products when administered at the same dose. The therapeutic effect can be assessed based on one or more criteria discussed above (e.g., cardiac status, glycogen levels, or biomarker expression). For example, if a single dose of a conventional rhGAA product reduces glycogen levels in the tissues of a treated individual by 10% within one week, the same degree of reduction can be achieved in less than one week when the same dose of rhGAA or pharmaceutical compositions described herein is administered. In some embodiments, the rhGAA or pharmaceutical compositions 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.

[0273] In some embodiments, a therapeutically effective amount of rhGAA (or a composition or drug comprising rhGAA) is administered more than once. In some embodiments, rhGAA or a pharmaceutical composition described herein is continuously administered at regular intervals, depending on the nature and extent of the disease impact. As used herein, administration at "regular intervals" means that the therapeutically effective amount is administered regularly (as distinguished from a one-time dose). The interval 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 once a day. In some embodiments, rhGAA is administered intravenously twice a week, once a week, or every other week. The administration interval for a single individual does not need to be a fixed interval, but can be time-varying, depending on the needs of the individual. For example, in the event of physiological illness or physical stress, if anti-rhGAA antibodies are present or increase, or if the symptoms of the disease worsen, the interval between doses can be reduced.

[0274] In some embodiments, when used at the same dose, the rhGAA or pharmaceutical compositions described herein can be administered less frequently than conventional rhGAA products, yet still produce the same or better therapeutic effect as conventional rhGAA products. For example, if a conventional rhGAA product is administered weekly at 20 mg / kg, then when administered at 20 mg / kg, the rhGAA or pharmaceutical compositions described herein can produce the same or better therapeutic effect as the conventional rhGAA product, even if the rhGAA or pharmaceutical composition is administered less frequently, such as once every two weeks or once a month. The therapeutic effect can be assessed based on one or more criteria discussed above (e.g., cardiac status, glycogen levels, or biomarker expression). In some embodiments, the interval between two doses of the rhGAA or pharmaceutical compositions described herein is longer than the interval for 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, or more longer than the interval for conventional rhGAA products.

[0275] In some embodiments, under the same treatment conditions (e.g., the same dose administered at the same intervals), the rhGAA or pharmaceutical compositions described herein provide a therapeutic effect that is superior to the therapeutic effect provided by conventional rhGAA products. The therapeutic effect can be assessed based on one or more criteria discussed above (e.g., cardiac status, glycogen levels, or biomarker expression). For example, when compared to a conventional rhGAA product administered at 20 mg / kg per week, rhGAA or pharmaceutical compositions administered at 20 mg / kg per week can reduce glycogen levels in the tissues of the treated individual to a greater extent. In some embodiments, when administered under the same treatment conditions, the rhGAA or pharmaceutical compositions described herein provide a therapeutic effect that is at least about 1.25, 1.5, 1.75, 2.0, 3.0, or more greater than the therapeutic effect of a conventional rhGAA product. D. Two-component therapy

[0276] In one or more embodiments, rhGAA or a pharmaceutical composition comprising rhGAA as described herein is administered simultaneously or sequentially with an enzyme stabilizer. In some embodiments, rhGAA or the pharmaceutical composition is administered via a different route than the enzyme stabilizer. For example, the enzyme stabilizer can be administered orally, while the rhGAA or the pharmaceutical composition can be administered intravenously.

[0277] In various embodiments, the enzyme stabilizer is miglustat. Without wishing to be bound by any theory, it is believed that when co-administered, miglustat stabilizes rhGAA (e.g., ATB200 or sipa-glucosidase alfa) from denaturation in the systemic circulation, which enhances the delivery of the active component rhGAA (e.g., ATB200 or sipa-glucosidase alfa) to lysosomes.

[0278] 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 at an oral dose of about 260 mg.

[0279] Those skilled in the art will understand that an oral dose of miglustat in the range of about 200 mg to 600 mg, or any smaller range, may be suitable for an adult patient, depending on his / her weight. For example, for patients weighing significantly less than about 70 kg, including but not limited to infants, children, or underweight adults, a physician may deem a smaller dose to be suitable. Thus, in at least one embodiment, miglustat is administered at an oral dose of about 50 mg to about 200 mg, or at an oral dose of about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 130 mg, about 150 mg, about 175 mg, about 195 mg, about 200 mg, or about 260 mg. In at least one embodiment, miglustat is administered at an oral dose of about 65 mg to about 195 mg, an oral dose of about 65 mg to about 260 mg, an oral dose of about 195 mg to about 260 mg, or an oral dose of about 65 mg, about 130 mg, about 195 mg, or about 260 mg.

[0280] In some embodiments, the starting dose of rhGAA (e.g., ATB200 or sipa-glucosidase alfa) and miglustat can be based on body weight (see, e.g., Table 1). In some embodiments, the miglustat dose can be adjusted and weight gain or loss can be confirmed on two consecutive infusion visits before changing the miglustat dose. Table 6: Exemplary rhGAA and Miglustat Dosage Regimens a Dosage: three 65 mg capsules or 195 mg oral solution b Dosage: Administered as four 65 mg capsules or as a 260 mg oral liquid dosage form

[0281] Miglustat exhibited linear pharmacokinetics, with the plasma area under the concentration-time curve (AUC) and maximum concentration (C max ) was approximately proportional to the dose increase from 130 mg (0.5 times the recommended dose of 260 mg in patients weighing ≥50 kg) to 260 mg. At the recommended dose of 260 mg, the mean C max The mean AUC is approximately 3 mcg / mL and is approximately 25 mcg*hr / mL. The mean time to maximum concentration is 2 to 3 hours.

[0282] Miglustat plasma concentrations are increased in patients with renal impairment. Therefore, the apparent clearance of miglustat decreases with decreasing renal function. Available data estimate that the AUC of miglustat is lower in patients with mild (creatinine clearance based on the Cockcroft-Gault equation, CLcr 60-89 mL / min), moderate (CLcr 30-59 mL / min), and severe (CLcr 15-29 mL / min) renal impairment compared to patients with normal renal function. 0-24hr An increase of 21%, 26% and 31% respectively. In some embodiments, the dosage of miglustat is reduced in patients with moderate or severe renal impairment compared to patients with normal renal function. Table 7 below provides exemplary dosages for patients with moderate or severe renal impairment: Table 7: Recommended Miglustat Dosage in Patients with Moderate or Severe Renal Impairment 1 Renal function classified by creatinine clearance according to the Cockcroft-Gault equation.

[0283] In some embodiments, for patients with mild renal impairment (creatinine clearance based on the Cockcroft-Gault equation, CLcr 60-89 mL / min), the recommended miglustat dose is the same as for patients with normal renal function.

[0284] In one or more embodiments, the rhGAA dose is not adjusted for patients with renal impairment.

[0285] Available animal pharmacodynamic / toxicology data indicate that sipa-glucosidase alfa is secreted in breast milk. It is unknown whether miglustat is secreted in breast milk. A risk to newborns / breastfed infants cannot be excluded. The developmental and health benefits of breastfeeding, the clinical need for the mother receiving co-administered sipa-glucosidase alfa and miglustat therapy, and any adverse reactions experienced by the breastfed child that may be related to the co-administration of sipa-glucosidase alfa and miglustat or the underlying maternal condition should be considered. In some embodiments, breastfeeding is not permitted during administration of sipa-glucosidase alfa and miglustat.

[0286] There are no clinical data demonstrating the effect of co-administration of sipa-glucosidase alfa and miglustat on fertility. Preclinical data did not reveal any significant adverse effects of sipa-glucosidase alfa.

[0287] In seven healthy adult men, 100 mg of miglustat was administered orally twice daily for 6 weeks, with no observed effects on sperm concentration, motility, or morphology. Preclinical data in rats suggest that miglustat negatively affects sperm characteristics (motility and morphology), thereby reducing fertility.

[0288] 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 200 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. 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 130 mg to about 200 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 195 mg.

[0289] The area under the plasma concentration-time curve (AUC) and maximum plasma concentration (Cmax) of sipa-glucosidase alfa following administration of 20 mg / kg sipa-glucosidase alfa with a single oral dose of 260 mg miglustat in adult patients with LOPD are summarized in Table 8 below: Table 8. Mean (CV%) Pharmacokinetic Parameters of Sipaglucosidase Alpha in Adult LOPD Patients Who Underwent ERT

[0290] 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 minute before or after administration of rhGAA. In some embodiments, miglustat is administered within 5, 4, 3, 2, or 1 minute before or after administration of rhGAA.

[0291] In some embodiments, miglustat and rhGAA are administered sequentially. In at least one embodiment, miglustat is administered prior to the administration of rhGAA. In at least one embodiment, miglustat is administered less than three hours prior to the administration of rhGAA. In at least one embodiment, miglustat is administered approximately two hours prior to the administration of rhGAA. In at least one embodiment, miglustat is administered within a range of 50 minutes to 90 minutes prior to the administration of rhGAA. For example, miglustat may be administered approximately 1.5 hours, approximately 1 hour, approximately 50 minutes, approximately 30 minutes, or approximately 20 minutes prior to the administration of rhGAA. In at least one embodiment, miglustat is administered approximately one hour prior to the administration of rhGAA. In some embodiments, miglustat is orally administered approximately one hour prior to the administration of rhGAA.

[0292] In some embodiments, miglustat is administered after administration of rhGAA. In at least one embodiment, miglustat is administered within three hours after administration of rhGAA. In at least one embodiment, miglustat is administered within two hours after administration of rhGAA. For example, miglustat can be administered within about 1.5 hours, about 1 hour, about 50 minutes, about 30 minutes, or about 20 minutes after administration of rhGAA.

[0293] In some embodiments, the subject fasts for at least two hours prior to administration of miglustat. In some embodiments, the subject fasts for at least two hours after administration of miglustat. In some embodiments, the subject fasts for at least two hours prior to administration of miglustat and for at least two hours after administration of miglustat.

[0294] In some embodiments, the subject fasts for at least two hours before and at least two hours after administration of miglustat, and miglustat is administered about one hour before administration of rhGAA. In some embodiments, fasting, administration of miglustat, and administration of rhGAA follow the following dosing timeline: Dosing schedule

[0295] In some embodiments, a two-component therapy according to the present disclosure improves one or more disease symptoms in a subject with Pompe disease compared to (1) a baseline or (2) a control treatment comprising administration of alglucosidase alfa and a placebo for an enzyme stabilizer, wherein the placebo is administered in place of the enzyme stabilizer.

[0296] Provided herein is a method for improving and / or stabilizing motor function and / or lung function in a subject with Pompe disease for at least 24 months, at least 36 months, or at least 48 months, the method comprising administering to the subject a population of recombinant human acid α-glucosidase (rhGAA) molecules simultaneously or sequentially with an enzyme stabilizer; wherein each rhGAA molecule comprises seven potential N-glycosylation sites; wherein 40%-60% of the N-glycans on the rhGAA molecule are complex N-glycans; wherein the rhGAA molecule comprises at least 0.5 mol bis-mannose-6-phosphate (bis-M6P) / mol rhGAA at the first potential N-glycosylation site as determined using liquid chromatography tandem mass spectrometry (LC-MS / MS); and wherein the method improves and / or stabilizes the subject's motor function and / or lung function compared to baseline.

[0297] In some embodiments, the subject treated by the two-component therapy is a patient who has undergone ERT. In some embodiments, the subject who has undergone ERT has previously been treated with alglucosidase alfa. In some embodiments, the subject who has undergone ERT has previously been treated with alglucosidase alfa for about 2 to about 6 years. In some embodiments, the subject who has undergone ERT has previously been treated with alglucosidase alfa for at least about 7 years. In some embodiments, the subject who has undergone ERT is unable to walk. In some embodiments, the subject who has undergone ERT is able to walk. In some embodiments, the subject is an ERT-naive subject.

[0298] In some embodiments, the subject treated by the two-component therapy is an ERT-naive patient.

[0299] In some embodiments, the two-component therapy according to the present disclosure improves and / or stabilizes the motor function of the subject as measured by a 6-minute walk test (6MWT). In some embodiments, the subject's 6-minute walk distance (6MWD) increases by at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 meters or at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% after 12, 26, 38, or 52 weeks, or after 12, 18, 24, 30, 36, or 48 months of treatment compared to baseline. In some embodiments, the subject's 6MWD increases by at least 20 meters or at least 5% after 52 weeks of treatment. In some embodiments, the subject's 6MWD is increased by at least 20 meters or at least 5% after 12, 18, 24, 30, 36, or 48 months of treatment. In some embodiments, the subject's 6MWD is improved by at least 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, or 50 meters after 12, 26, 38, or 52 weeks or after 18, 24, 30, 36, or 48 months of treatment compared to a control treatment. In some embodiments, the subject's 6MWD is improved by at least 13 meters after 52 weeks of treatment compared to a control treatment. In some embodiments of the method for treating a subject who has experienced ERT, motor function is measured by a 6-minute walk test; and the improvement in 6-minute walk distance (6MWD) relative to baseline is at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 meters 12, 18, 24, 30, 36, or 48 months after starting treatment. In some embodiments of the method for treating a subject who has experienced ERT, motor function is measured by a 6-minute walk test; and the improvement in 6-minute walk distance (6MWD) relative to baseline is at least 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 41, 42, 43, 44, 45, 46, or 47 meters 36 or 48 months after starting treatment.

[0300] In some embodiments of the method for treating ERT-naive subjects, motor function is measured by a 6-minute walk test; and at 12, 24, 36, or 48 months after starting treatment, the improvement in 6-minute walk distance (6MWD) relative to baseline is at least 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 meters. In some embodiments of the method for treating ERT-naive subjects, motor function is measured by a 6-minute walk test; and at 36 or 48 months after starting treatment, the improvement in 6-minute walk distance (6MWD) relative to baseline is at least 34, 35, 40, 41, 42, 43, 44, or 45 meters. In some embodiments, the subject has a baseline 6MWD of less than 300 meters. In some embodiments, the subject has a baseline 6MWD of greater than or equal to 300 meters.

[0301] In some embodiments, the two-component therapy according to the present disclosure stabilizes the lung function of the subject, as measured by forced vital capacity (FVC) test. In some embodiments, after 12, 26, 38 or 52 weeks, or after treating 18, 24, 30, 36 or 48 months, the predicted FVC percentage of the subject increases compared to baseline or decreases compared to baseline by less than 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%. In some embodiments, after treating 52 weeks, the predicted FVC percentage of the subject decreases by less than 1% compared to baseline. In some embodiments, after treating 12, 18, 24, 30, 36 or 48 months, the predicted FVC percentage of the subject increases by at least 1%, at least 2%, at least 3% or at least 5% compared to baseline. In some embodiments, the predicted FVC percentage of the subject is significantly improved and / or stable after treatment compared to the control treatment. In some embodiments, after 12, 26, 38 or 52 weeks, or after 18, 24, 30, 36 or 48 months of treatment, the predicted FVC percentage of the subject is significantly improved by at least 0.5%, 1%, 2%, 3%, 4%, 5% or 6% compared to the control treatment. In some embodiments, after 52 weeks of treatment, the predicted FVC percentage of the subject is significantly improved by at least 3% compared to the control treatment. In some embodiments, after 12, 18, 24, 30, 36 or 48 months of treatment, the predicted FVC percentage of the subject is significantly improved by at least 3% or at least 5% compared to the control treatment. In some embodiments of the method for treating a subject who has experienced ERT, pulmonary function is measured by a seated forced vital capacity (FVC) test, and 24, 36 or 48 months after starting treatment, the predicted FVC percentage of the subject is stable compared to baseline. In some embodiments of the method for treating ERT-naive subjects, lung function is measured by a forced vital capacity (FVC) test in a sitting position; and at 24 months after starting treatment, the subject's predicted FVC percentage relative to baseline is at least 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.5%, 6.0%, 6.4%, 6.5%, 6.6%, 6.7 or 6.8%. In some embodiments of the method for treating ERT-naive subjects, lung function is measured by a forced vital capacity (FVC) test in a sitting position; and at 36 or 48 months after starting treatment, the subject's predicted FVC percentage relative to baseline is at least 5.7%, 5.8%, 5.9%, 6.0%, 6.1% or 6.2%. In some embodiments, the subject has a baseline FVC of less than 55%. In some embodiments, the subject has a baseline FVC greater than or equal to 55%.In some embodiments, the subject has less than 50% of baseline FVC. In some embodiments, the subject has greater than or equal to 50% of baseline FVC.

[0302] In certain embodiments, the two-component therapy according to the present disclosure improves and / or stabilizes the muscle strength of the subject, as measured by manual muscle test (MMT). In certain embodiments, compared with baseline, the subject's MMT lower limb score is improved, such as after 12, 26, 38 or 52 weeks, or after treatment of 12, 18, 24, 30, 36 or 48 months, an increase of at least 0.1, 0.3, 0.5, 0.7, 1.0, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5 points. In certain embodiments, compared with control treatment, the subject's MMT lower limb score is significantly improved and / or stable after treatment. In some embodiments of the method for treating a subject who has experienced ERT, muscle strength is measured by MMT; and the improvement in MMT lower extremity score relative to baseline is at least 1.9, 2, 2.1, 2.2, or 2.3 points 24 months after starting treatment. In some embodiments of the method for treating a subject who has experienced ERT, muscle strength is measured by MMT; and the improvement in MMT lower extremity score relative to baseline is at least 1.5, 1.6, 1.7, 1.8, or 1.9 points 36 or 48 months after starting treatment. In some embodiments of the method for treating an ERT-naive subject, muscle strength is measured by MMT; and the improvement in MMT lower extremity score relative to baseline is at least 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 points 24 months after starting treatment. In some embodiments of the method for treating ERT-naive subjects, muscle strength is measured by MMT; and the improvement from baseline in the MMT lower extremity score is at least 2.8, 2.9, 3.0, 3.1, 3.2, or 3.3 points 36 or 48 months after initiation of treatment. In some embodiments, the subject has a baseline MMT lower extremity score of less than 25. In some embodiments, the subject has a baseline MMT lower extremity score of greater than or equal to 25.

[0303] In certain embodiments, the two-component therapy according to the present disclosure improves and / or stabilizes the motor function of the subject, as measured by gait, stairs, Gowers, chair (GSGC) test. In certain embodiments, compared with baseline, the GSGC score of the subject improves, as after 12, 26, 38 or 52 weeks, or after treating 18, 24, 30, 36 or 48 months, reducing at least 0.1, 0.3, 0.5, 0.7, 1.0, 1.5 or 2.5 points. In certain embodiments, compared with baseline, the GSGC score of the subject improves, as after treating 52 weeks, at least reducing 0.5 points. In certain embodiments, compared with control treatment, the GSGC score of the subject significantly improves after treatment. In certain embodiments, compared with control treatment, the GSGC score of the subject significantly improves, as after 12, 26, 38 or 52 weeks, or after treating 18, 24, 30, 36 or 48 months, reducing at least 0.3, 0.5, 0.7, 1.0, 1.5, 2.5 or 5 points. In some embodiments, the subject's GSGC score is significantly improved compared to the control treatment, as indicated by a decrease of at least 1.0 point after 52 weeks of treatment.

[0304] In some embodiments, the two-component therapy according to the present disclosure improves cardiac function in a subject. In some embodiments, the therapy improves cardiac function in a subject as measured by left ventricular mass index (LVMi).

[0305] In some embodiments, the two-component therapy according to the present disclosure reduces the level of at least one muscle damage marker after treatment. In some embodiments, muscle damage markers include one or more of creatine kinase (CK), alanine aminotransferase (ALT) and aspartate aminotransferase (AST). In some embodiments, at least one muscle damage marker includes CK. In some embodiments, compared to baseline, the CK level of the subject is reduced by at least 10%, 15%, 20%, 25%, 30%, 40% or 50% after 12, 26, 38 or 52 weeks, or after treatment of 18, 24, 30 or 36 months. In some embodiments, compared to baseline, the CK level of the subject is reduced by at least 20% after treatment of 52 weeks. In some embodiments, compared to control treatment, the CK level of the subject is significantly reduced after treatment. In some embodiments, compared to control treatment, the CK level of the subject is significantly reduced by at least 10%, 15%, 20%, 25%, 30%, 40% or 50% after 12, 26, 38 or 52 weeks, or after treatment of 18, 24, 30 or 36 months. In some embodiments, the subject's CK level is significantly reduced by at least 30% after 52 weeks of treatment compared to a control treatment.

[0306] In some embodiments, the two-component therapy according to the present disclosure reduces the level of at least one glycogen accumulation marker after treatment. In some embodiments, at least one marker of glycogen accumulation includes urinary hexose tetraose (Hex4). In some embodiments, the subject's urinary Hex4 level is reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, 50% or 60% after 12, 26, 38 or 52 weeks, or after 18, 24, 30 or 36 months of treatment compared to baseline. In some embodiments, the subject's urinary Hex4 level is reduced by at least 30% after 52 weeks of treatment compared to baseline. In some embodiments, the subject's urinary Hex4 level is significantly reduced after treatment compared to a control treatment. In some embodiments, the subject's urinary Hex4 level is significantly reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, 50% or 60% after 12, 26, 38 or 52 weeks, or after 18, 24, 30 or 36 months of treatment compared to a control treatment. In some embodiments, the subject's urinary Hex4 levels are significantly reduced by at least 40% after 52 weeks of treatment, compared to a control treatment.

[0307] In some embodiments, a two-component therapy according to the present disclosure improves and / or stabilizes one or more disease symptoms in a patient subject with Pompe disease who has undergone ERT, compared to (1) baseline or (2) a control treatment comprising administration of alglucosidase alfa and an enzyme stabilizer placebo.

[0308] In some embodiments, the dual component therapies according to the present disclosure improve quality of life and / or patient-reported outcome measures, such as by the European Quality of Life - Five Dimensions Five Levels (EQ 5D 5L / EQ-5D-Y) questionnaire and / or the Patient Reported Outcomes Measurement Information System (PROMIS Physical Function, PROMIS Fatigue, PROMIS Dyspnea).

[0309] In some embodiments, a dual component therapy according to the present disclosure reduces, delays, and / or maintains a user's need for mobility or respiratory support devices, such as by monitoring the onset / changes in use of a mobility assistance device and the type of device and / or monitoring the onset / changes in use of respiratory support and the type of support.

[0310] In some embodiments, the dual-component therapy for ERT-experienced subjects with Pompe disease improves and / or stabilizes motor function in the subject as measured by the 6MWT. In some embodiments, the subject's 6MWD increases by at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or 50 meters, or by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, after 12, 26, 38, or 52 weeks, or after 18, 24, 30, 36, or 48 months of treatment compared to baseline. In some embodiments, the subject's 6MWD increases by at least 15 meters or by at least 5% after 52 weeks of treatment. In some embodiments, the subject's 6MWD increases by at least 25 meters or by at least 6% after 24 months of treatment. In some embodiments, the subject's 6MWD increases by at least 10 meters or by at least 2% after 36 months of treatment. In some embodiments, the subject's 6MWD increases by at least 20 meters or at least 5% after 52 weeks of treatment. In some embodiments, the subject's 6MWD is significantly improved after treatment compared to a control treatment. In some embodiments, the subject's 6MWD is significantly improved by at least 10, 12, 14, 15, 16, 18, 20, 30, 40, or 50 meters after 12, 26, 38, or 52 weeks or after 18, 24, 30, 36, or 48 months of treatment compared to a control treatment. In some embodiments, the subject's 6MWD is significantly improved by at least 15 meters after 52 weeks of treatment compared to a control treatment. In some embodiments, the subject has a baseline 6MWD of less than 300 meters. In some embodiments, the subject has a baseline 6MWD of greater than or equal to 300 meters.

[0311] In some embodiments, a two-component therapy for a subject who has undergone ERT with Pompe disease improves and / or stabilizes the subject's lung function as measured by an FVC test. In some embodiments, after 12, 26, 38, or 52 weeks, or after 18, 24, 30, 36, or 48 months of treatment, the subject's predicted FVC percentage increases by at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, or 5% compared to baseline. In some embodiments, after 52 weeks of treatment, the subject's predicted FVC percentage increases by at least 0.1% compared to baseline. In some embodiments, the subject's predicted FVC percentage significantly improves and / or stabilizes after treatment compared to a control treatment. In some embodiments, after 12, 26, 38, or 52 weeks, or after 18, 24, 30, 36, or 48 months of treatment, the subject's predicted FVC percentage significantly improves by at least 1%, 2%, 3%, 4%, 5%, 6%, 8%, or 10% compared to a control treatment. In some embodiments, the subject's predicted FVC percentage is significantly improved by at least 4% after 52 weeks of treatment compared to the control treatment. In some embodiments, the subject has a baseline FVC of less than 55%. In some embodiments, the subject has a baseline FVC greater than or equal to 55%.

[0312] In some embodiments, as measured by the GSGC test, a two-component therapy for a subject who has experienced ERT with Pompe disease improves and / or stabilizes the motor function of the subject. In some embodiments, the GSGC score of the subject is improved compared to baseline, as shown in 12, 26, 38 or 52 weeks, or after treating 18, 24, 30, 36 or 48 months, by reducing at least 0.1, 0.3, 0.5, 0.7, 1.0, 1.5 or 2.5 points. In some embodiments, the GSGC score of the subject is improved compared to baseline, as shown in at least 0.5 points after treating 52 weeks. In some embodiments, the GSGC score of the subject is significantly improved and / or stable after treatment compared to a control treatment. In some embodiments, the GSGC score of the subject is significantly improved compared to a control treatment, as shown in 12, 26, 38 or 52 weeks, or after treating 18, 24, 30, 36 or 48 months, by reducing at least 0.3, 0.5, 0.7, 1.0, 1.5, 2.5 or 5 points. In some embodiments, the subject's GSGC score is significantly improved compared to the control treatment, as indicated by a decrease of at least 1.0 point after 52 weeks of treatment.

[0313] In some embodiments, a two-component therapy for subjects with Pompe disease who have undergone ERT reduces the level of at least one marker of muscle damage after treatment. In some embodiments, at least one marker of muscle damage includes CK. In some embodiments, the subject's CK level is reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, or 50% after 12, 26, 38, or 52 weeks, or after 18, 24, 30, 36, or 48 months of treatment compared to baseline. In some embodiments, the subject's CK level is reduced by at least 15% after 52 weeks of treatment compared to baseline. In some embodiments, the subject's CK level is reduced by at least 25% after 24 months of treatment compared to baseline. In some embodiments, the subject's CK level is reduced by at least 30% after 36 months of treatment compared to baseline. In some embodiments, the subject's CK level is reduced by at least 35% after 48 months of treatment compared to baseline. In some embodiments, the subject's CK level is significantly reduced after treatment compared to a control treatment. In some embodiments, the subject's CK level is significantly reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, or 50% after 12, 26, 38, or 52 weeks, or after 18, 24, 30, 36, or 48 months of treatment, compared to a control treatment. In some embodiments, the subject's CK level is significantly reduced by at least 30% after 52 weeks of treatment, compared to a control treatment.

[0314] In some embodiments, a dual-component therapy for ERT-experienced subjects with Pompe disease reduces the level of at least one marker of glycogen accumulation after treatment. In some embodiments, the at least one marker of glycogen accumulation comprises urinary Hex4. In some embodiments, the subject's urinary Hex4 levels are reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60% after 12, 26, 38, or 52 weeks, or after 18, 24, 30, or 36 months of treatment, compared to baseline. In some embodiments, the subject's urinary Hex4 levels are reduced by at least 25% after 52 weeks of treatment, compared to baseline. In some embodiments, the subject's urinary Hex4 levels are reduced by at least 35% after 36 months of treatment, compared to baseline. In some embodiments, the subject's urinary Hex4 levels are reduced by at least 30% after 48 months of treatment, compared to baseline. In some embodiments, the subject's urinary Hex4 levels are significantly reduced after treatment compared to a control treatment. In some embodiments, the subject's urinary Hex4 levels are significantly reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60% after 12, 26, 38, or 52 weeks, or after 18, 24, 30, or 36 months of treatment, compared to a control treatment. In some embodiments, the subject's urinary Hex4 levels are significantly reduced by at least 40% after 52 weeks of treatment, compared to a control treatment. E. Kit

[0315] Another aspect of the present disclosure relates to a kit suitable for performing the rhGAA therapy described herein. In one or more embodiments, the kit includes a container (e.g., a vial, a tube, a bag, etc.) containing rhGAA or a pharmaceutical composition (before or after lyophilization) and instructions for reconstitution, dilution, and administration. In one or more embodiments, the kit includes a container (e.g., a vial, a tube, a bag, etc.) containing an enzyme stabilizer (e.g., miglustat) and a pharmaceutical composition containing rhGAA (before or after lyophilization), as well as instructions for reconstitution, dilution, and administration of rhGAA and the enzyme stabilizer. Examples

[0316] The present invention is further illustrated by the following non-limiting examples. Example 1: Preparation of CHO cells producing rhGAA with high content of N-glycans with mono-M6P or di-M6P.

[0317] DG44 CHO (DHFR-) cells were transfected with a DNA construct expressing rhGAA. Figure 4 After transfection, CHO cells containing stably integrated GAA genes were selected using hypoxanthine / thymidine-deficient (-HT) medium. GAA expression in these cells was induced by methotrexate treatment (MTX, 500 nM).

[0318] Cell pools expressing large 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 culture plates, selected by the ClonePix system, and transferred to 24 deep-well plates. Individual clones were assayed for GAA enzyme activity to identify clones expressing high levels of GAA. The conditioned medium used to determine GAA activity used a 4-MU-α-glucosidase substrate. Clones producing higher levels of GAA as measured by the GAA enzyme assay were further evaluated for viability, growth ability, GAA productivity, N-glycan structure, and stable protein expression. CHO cell lines (including the CHO cell line GA-ATB200, expressing rhGAA with enhanced single-M6P or double-M6P N-glycans) were isolated using this procedure. Example 2: Purification of rhGAA

[0319] Multiple batches of rhGAA according to the present disclosure were produced in shake flasks and perfusion bioreactors using the CHO cell line GA-ATB200, the product of which is referred to as "ATB200". Weak anion exchange ("WAX") liquid chromatography was used to fractionate ATB200 rhGAA based on terminal phosphates and sialic acids. Elution profiles were generated by eluting the ERT with increasing amounts of salt. The profiles were monitored by UV (A280 nm). Similar CIMPR receptor binding (at least about 70%) distribution profiles were observed for purified ATB200 rhGAA from different production batches ( Figure 5 ), indicating that ATB200 rhGAA can be produced consistently. Example 3: Oligosaccharide Properties of ATB200 rhGAA

[0320] The site-specific N-glycan profiles of ATB200 rhGAA were analyzed using different LC-MS / MS analytical techniques. The results of the first two LC-MS / MS methods are shown in Figure 2. Figures 6A-6H The results of the third LC-MS / MS method with 2-AA glycan profile are shown in Figures 19A-19H 、 Figures 20A-20B and Table 9.

[0321] In the first LC-MS / MS analysis, the protein was denatured, reduced, alkylated, and digested before LC-MS / MS analysis. During the protein denaturation and reduction process, 200 μg of 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 Water is added in a 1.5mL test tube to provide a cumulative volume of 100 μL. In a dry bath, the sample is mixed and incubated at 56°C for 30 minutes. In the alkylation process, the protein sample of denaturation and reduction is mixed with 5 μL of 1mol / L iodoacetamide (IAM, final concentration 50mM), then incubated at 10-30°C in the dark for 30 minutes. After the alkylation, 400 μL of pre-cooled acetone are added to the sample, and the mixture is frozen for 4 hours in -80°C refrigeration. The sample is then centrifuged at 4°C for 5min at 13000rpm, and the supernatant is removed. The sample is then air-dried on ice in the dark to remove the acetone residue. Forty microliters of 8M urea and 160 μL of 100mM NH4HCO3 were added to the sample to dissolve the protein. During the trypsin digestion process, 50 μg of protein was then added to a final volume of 100 μL with trypsin digestion buffer and 5 μL of 0.5 mg / mL trypsin was added (the protein to enzyme ratio was 20 / 1 w / w). The solution was mixed thoroughly and incubated at 37°C overnight (16 ± 2 hours). Two and a half microliters of 20% TFA (final concentration 0.5%) was added to quench the reaction. The reaction was then quenched using a Thermo Scientific TM Orbitrap Velos Pro TM The samples were analyzed by mass spectrometry.

[0322] In the second LC-MS / MS analysis, ATB200 samples were prepared according to a similar denaturation, reduction, alkylation, and digestion procedure, except that iodoacetic acid (IAA) was used instead of IAM as the alkylating agent, and then the ATB200 samples were purified using a Thermo Scientific TM Orbitrap Fusion TM Lumos Tribid TM Mass spectrometer analysis.

[0323] The results of the first and second analyses are shown in Figures 6A-6H In. Figures 6A-6H In the table, the results of the first analysis are represented by the left column (dark gray), and the results of the second analysis are represented by the right column (light gray). The symbol nomenclature for glycan representation is consistent with Varki, A., Cummings, RD, Esko JD, et al., Essentials of Glycobiology, 2nd ed. (2009).

[0324] from Figures 6A-6HAs can be seen, the two analyses provide similar results, although there are some differences between the results. This variation can be attributed to multiple factors, including the instrument used and the integrity of the N-glycan analysis. For example, if certain types of phosphorylated N-glycans are not identified and / or quantified, the total number of phosphorylated N-glycans may be underestimated, and the percentage of rhGAA carrying phosphorylated N-glycans at that site may be underestimated. As another example, if certain types of non-phosphorylated N-glycans are not identified and / or quantified, the total number of non-phosphorylated N-glycans may be underestimated, and the percentage of rhGAA carrying phosphorylated N-glycans at that site may be overestimated.

[0325] Figure 6A The N-glycosylation site occupancy of ATB200 is shown. Figure 6A As can be seen, the first, second, third, fourth, fifth, and sixth N-glycosylation sites were largely occupied, with both assays detecting approximately 90% or more, and up to approximately 100%, of the ATB200 enzymes detecting N-glycans at each potential N-glycosylation site. However, the seventh potential N-glycosylation site was N-glycosylated approximately half of the time.

[0326] Figure 6B The N-glycosylation profile of the first potential N-glycosylation site N84 is shown. Figure 6B As can be seen, the major N-glycan species is bis-M6P N-glycan. Both the first and second analyses detected that more than 75% of ATB200 had bis-M6P at the first site, corresponding to an average of approximately 0.8 mol bis-M6P / mol ATB200 at the first site.

[0327] Figure 6C The N-glycosylation profile at the second potential N-glycosylation site N177 is shown. Figure 6C As can be seen, the major N-glycan species are mono-M6P N-glycans and non-phosphorylated high mannose N-glycans. Both the first and second analyses detected that more than 40% of ATB200 had mono-M6P at the second site, corresponding to an average of about 0.4 to about 0.6 mol mono-M6P / mol ATB200 at the second site.

[0328] Figure 6D The N-glycosylation profile at the third potential N-glycosylation site N334 is shown. Figure 6DAs can be seen, the major N-glycan species are non-phosphorylated high mannose N-glycans, diantennary, triantennary, and tetraantennary complex N-glycans, and hybrid N-glycans. Both the first and second analyses detected that more than 20% of ATB200 had a sialic acid residue at the third position, corresponding to an average of about 0.9 to about 1.2 mol sialic acid / mol ATB200 at the third position.

[0329] Figure 6E The N-glycosylation profile at the fourth potential N-glycosylation site N414 is shown. Figure 6E As can be seen, the major N-glycan species are di-M6P and mono-M6P N-glycans. Both the first and second analyses detected that more than 40% of ATB200 had di-M6P at the fourth position, corresponding to an average of about 0.4 to about 0.6 mol di-M6P / mol ATB200 at the fourth position. Both the first and second analyses also detected that more than 25% of ATB200 had mono-M6P at the fourth position, corresponding to an average of about 0.3 to about 0.4 mol mono-M6P / mol ATB200 at the fourth position.

[0330] Figure 6F The N-glycosylation profile at the fifth potential N-glycosylation site N596 is shown. Figure 6F As can be seen, the major N-glycan species are fucosylated biantennary complex N-glycans. Both the first and second analyses detected that more than 70% of ATB200 had a sialic acid residue at the fifth position, corresponding to an average of about 0.8 to about 0.9 mol sialic acid / mol ATB200 at the fifth position.

[0331] Figure 6G The N-glycosylation profile at the sixth potential N-glycosylation site N826 is shown. Figure 6G As can be seen in Figure 2, the major N-glycan species are diantennary, triantennary, and tetraantennary complex N-glycans. Both the first and second analyses detected that more than 80% of ATB200 had a sialic acid residue at the sixth position, corresponding to an average of about 1.5 to about 1.8 mol sialic acid / mol ATB200 at the sixth position.

[0332] N-glycosylation analysis of 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%).

[0333] Figure 6H A summary of phosphorylation for each of the seven potential N-glycosylation sites is shown. Figure 6HAs can be seen, 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 doubly phosphorylated at the first site, more than 40% of ATB200 was mono- or doubly phosphorylated at the second site, and more than 80% of ATB200 was mono- or doubly phosphorylated at the fourth site.

[0334] Another N-glycosylation analysis of ATB200 was performed according to the LC-MS / MS method described below. This analysis yielded an average N-glycosylation profile of ten batches of ATB200 ( Figures 19A-19H , Figures 20A-20B ).

[0335] N-linked glycans from ATB200 were enzymatically released using PNGase-F and labeled with 2-anthranilic acid (2-AA). 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 μg was loaded onto an amino polymer analytical column (apHera TM , Supelco) for high performance liquid chromatography with fluorescence detection (HPLC-FLD) and high resolution mass spectrometry (HRMS) analysis.

[0336] 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; 20 mM aqueous ammonium acetate adjusted to pH 4.3 with ammonium hydroxide). The initial mobile phase composition was 70% A / 30% B. For fluorescence detection, the detector (RF-20Axs, Shimadzu) parameters were excitation (Ex): 320 nm; emission (Em): 420 nm. HRMS analysis was performed using a quadrupole time-of-flight mass spectrometer (Sciex X500B QTOF) operating in independent data acquisition (IDA) mode. The acquired data files were converted to mzML files using MSConvert of ProteoWizard, and then the glycan database search and subsequent annotation of the identified N-glycans were performed using GRITS Toolbox 1.2 Morning Blend software (UGA). N-glycans were identified using precursor monoisotopic mass (m / z) and product ion m / z. Experimental product ions and fragmentation patterns were confirmed on-the-computer using the GlycoWorkbench 2 application.

[0337] In order to determine the relative quantification of N-linked glycans from ATB200, the data obtained from the HPLC-FLD-QTOF MS / MS experiment were 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 represented by the peak area is a quantitative measurement of the number of each N-glycan in the sample. However, in most cases, multiple N-glycan species are contained in the same FLD peak. Therefore, mass spectrometer data is also needed to obtain the relative quantification of each N-glycan species (Table 9). The ion intensity signal of each N-glycan is "extracted" from the data to create a chromatographic peak called an extracted ion chromatogram (XIC). The XIC is aligned with the FLD chromatographic peak and is only for one N-glycan species. The XIC peak generated from the ion intensity signal is then integrated, and the peak area is a relative quantitative measurement of the amount of glycans present. Both the FLD peak area and the mass spectrometer XIC peak area are used to achieve the relative quantification of all N-linked glycan species of ATB200 reported herein.

[0338] The results of this LC-MS / MS analysis are provided in Table 9 below. Symbolic nomenclature for glycan representation is consistent with the following: 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; Kattla JJ et al. 2011. Biologic protein glycosylation. In: Murray Moo-Young (ed.), Comprehensive Biotechnology, 2nd ed., 3:467-486; Tharmalingam-Jaikaran et al. T et al. N-glycan profiling of bovine follicular fluid at key dominant follicle developmental 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 amannose 6-phosphate monosaccharide-specific binding pocket that distinguishes N-glycan phosphorylation in a branch-specific manner.[Single-chain antibody fragment M6P-1 has a mannose 6-phosphate monosaccharide-specific binding pocket that discriminates N-glycan phosphorylation in a branch-specific manner] Glycobiology. [Glycobiology] 26-2: 181-192. Table 9: Types and prevalence of oligosaccharides identified on ATB200 based on 2-AA glycan profiles and LC-MS / MS identification

[0339] Based on this 2-AA and LC-MS / MS analysis, and as further summarized, the ATB200 tested had an average 3-5 mol M6P content / mol ATB200 (considering mono-M6P and bis-M6P) and 4-7 mol sialic acid content / mol ATB200.

[0340] like Figures 19A-19H and Figure 20B As shown, the first potential N-glycosylation site of ATB200 has an average M6P content of about 1.4 mol M6P / mol ATB200, considering an average mono-M6P content of about 0.25 mol mono-M6P / mol ATB200 and an average di-M6P content of about 0.56 mol di-M6P / mol ATB200; the second potential N-glycosylation site of ATB200 has an average M6P content of about 0.5 mol M6P / mol ATB200, and the major phosphorylated N-glycan species is 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 M6P content of about 1.4 mol M6P / mol ATB200, considering an average mono-M6P content of about 0.35 mol mono-M6P / mol ATB200 has an average mono-M6P content 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 / mol ATB200.

[0341] Also according to this 2-AA and LC-MS / MS analysis technique, an average of about 65% of the N-glycans at the first potential N-glycosylation site of ATB200 were high mannose N-glycans, about 89% of the N-glycans at the second potential N-glycosylation site of ATB200 were high mannose N-glycans, more than half of the N-glycans at the third potential N-glycosylation site of ATB200 were sialylated (nearly 20% were fully sialylated), and about 85% of the N-glycans at the third potential N-glycosylation site of ATB200 were complex N-glycans, about 84% of the N-glycans at the fourth potential N-glycosylation site of ATB200 were high mannose N-glycans, and about 10% of the N-glycans at the fifth potential N-glycosylation site of ATB200 were high mannose N-glycans. Approximately 70% of the N-glycans at the fifth potential N-glycosylation site of ATB200 are sialylated (approximately 26% are fully sialylated) and approximately 100% of the N-glycans at the fifth potential N-glycosylation site of ATB200 are complex N-glycans, approximately 85% of the N-glycans at the sixth potential N-glycosylation site of ATB200 are sialylated (nearly 27% are fully sialylated) and approximately 98% of the N-glycans at the sixth potential N-glycosylation site of ATB200 are complex N-glycans, and approximately 87% of the N-glycans at the seventh potential N-glycosylation site of ATB200 are sialylated (nearly 8% are fully sialylated) and approximately 100% of the N-glycans at the seventh potential N-glycosylation site of ATB200 are complex N-glycans. Example 4: ATB200 and Analysis and comparison

[0342] Purified ATB200 and N-glycans were evaluated by MALDI-TOF to identify the individual N-glycan structures found on each ERT. were obtained from commercial sources. Figure 7 As shown, ATB200 The right side of the graph shows four prominent peaks eluting. This confirms that ATB200 is more The phosphorylation of α is greater because the assessment is made by terminal charge rather than CIMPR affinity. Figure 8 As summarized in the previous section, the ATB200 sample was found to contain Lower amounts of non-phosphorylated high-mannose N-glycans.

[0343] To evaluate and To investigate the ability of conventional rhGAA in CIMPR 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 collected. Bound material was eluted with a gradient of free M6. Fractions were collected in a 96-well plate and assayed for GAA activity using 4MU-α-glucosidase substrate. The relative amounts of unbound (flow-through) and bound (M6P-eluted) rhGAA were determined based on GAA activity and reported as a fraction of total enzyme. Figure 9A and 9B Shown in and Binding spectrum of rhGAA in: ( Figure 9B ) 73% of rhGAA and ( Figure 9A ) does not bind to CIMPR. In fact, Only 27% of rhGAA and Only 22% of rhGAA in the 18 samples contained M6P, which could effectively target it to CIMPR on muscle cells. Figure 5 As shown, under the same conditions, more than 70% of rhGAA in ATB200 was found to be bound to CIMPR.

[0344] In addition to having a greater percentage of rhGAA available to bind CIMPR, it is important to understand the quality of this interaction. Binding of ATB200 to the receptor was determined using the CIMPR plate binding assay. Briefly, CIMPR-coated plates were used to capture GAA. Different concentrations of rhGAA were applied to the immobilized receptors, and unbound rhGAA was washed away. The amount of remaining rhGAA was determined by GAA activity. As shown in Figure 10, ATB200 binds significantly better to CIMPR than to ATB200.

[0345] Overall, the M6P N-glycan content in ATB200 was higher than that in This suggests that a higher proportion of rhGAA molecules in ATB200 can target muscle cells. As shown above, the high percentage of mono- and di-phosphorylated structures determined by MALDI are consistent with the CIMPR spectrum, indicating that ATB200 binding to the CIMPR receptor is significantly enhanced. N-glycan analysis via MALDI-TOF mass spectrometry confirmed that on average each ATB200 molecule contains at least one neutral di-M6P N-glycan structure. This higher di-M6PN-glycan content on ATB200 is directly correlated with the high affinity binding to CIMPR in the M6P receptor plate binding assay (K D about 2-4 nM).

[0346] Comparison of ATB200 and Relative cellular uptake of rhGAA. Comparison involving 5-100 nM ATB200 according to the present disclosure and 10-500 nM conventional rhGAA products After 16-hr 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 plotted relative to total cellular protein. Figures 11A-11C middle.

[0347] ATB200 has also been shown to be efficiently internalized into cells. Figures 11A-11B As shown, ATB200 is internalized into normal and Pompe disease fibroblasts to a greater extent than conventional rhGAA products. ATB200 saturates cell receptors at approximately 20 nM, while approximately 250 nM is required to saturate cell receptors. The uptake efficiency constant (K 摄取 ) for ATB200 is 2-3nm, for is 56nM, such as Figure 11C These results suggest that ATB200 is a targeted therapeutic for Pompe disease. Example 5: ATB200 and enzyme stabilizer

[0348] The stability of ATB200 in acidic or neutral pH buffers was assessed in a thermal stability assay using SYPRO Orange, as the fluorescence of the dye increases when the protein is denatured. Figure 12 As shown in Table 10, the addition of miglustat stabilized ATB200 at pH 7.4 in a concentration-dependent manner, which was comparable to the stability of ATB200 at pH 5.2 (a condition that simulates the acidic environment of lysosomes). m ) increased by nearly 10℃. Table 10. Stability of ATB200 with Miglustat Test conditions Tm(℃) pH 7.4 56.2 pH 7.4 + 10 μM miglustat 61.6 pH 7.4 + 30 μM miglustat 62.9 pH 7.4 + 100 μM miglustat 66.0 pH 5.2 67.3 Example 6: Co-administration of ATB200 and Miglustat in Gaa KO mice

[0349] In Gaa KO mice, the therapeutic effects of ATB200 and meglustat were evaluated and compared with the therapeutic effects of alglucosidase alfa. In this study, male Gaa KO (3 to 4 months old) and age-matched wild-type (WT) mice were used. Alglucosidase alfa was administered by intravenous (IV) injection via tail vein bolus. In the co-administration scheme, meglustat was administered by oral gavage (PO) 30 minutes before IV injection of ATB200. Treatment was performed every two weeks. The treated mice were sacrificed 14 days after the last administration, and various tissues were collected for further analysis. Table 11 summarizes the study design: Table 11. Co-administration study design

[0350] Tissue glycogen content in tissue samples was determined using amyloglucosidase digestion as described above. Figure 13 As shown, the combination of 20 mg / kg ATB200 and 10 mg / kg miglustat significantly reduced glycogen content in four different tissues (quadriceps, triceps, gastrocnemius, and heart) compared to the same dose of alglucosidase alfa.

[0351] Tissue samples were also analyzed for biomarker changes as discussed in Khanna R et al. (2012), “The pharmacological chaperone AT2220 increases recombinant human acidα-glucosidase uptake and glycogen reduction in a mouse model of Pompedisease,” Plos One 7(7):e40776; and Khanna, R et al. (2014), “The Pharmacological Chaperone AT2220 Increases the Specific Activity and Lysosomal Delivery of Mutant Acidα-Glucosidase, and Promotes GlycogenReduction in a Transgenic Mouse Model of Pompe Disease.” Disease [Pharmacological chaperone AT2220 increases specific activity and lysosomal delivery of mutant acid α-glucosidase and promotes glycogen depletion in a transgenic mouse model of Pompe disease]” PLoS ONE [Public Library of Science - One] 9(7):e102092. Figure 14 As shown, a significant increase and enlargement of LAMP1-positive vesicles were observed in myofibers of Gaa KO animals compared to WT, indicating lysosomal proliferation. Co-administration of ATB200 / miglustat resulted in more fibers with normal LAMP1 levels, while the remaining LAMP1-positive vesicles were also reduced in size (inset).

[0352] Similarly, strong LC3-positive aggregates in muscle fibers of untreated Gaa KO mice indicated the presence of autophagic zones and autophagic accumulation. LC3-positive aggregates (red) were preferentially reduced in mice treated with ATB200 / miglustat co-administered compared to mice treated with alglucosidase alfa ( Figure 15A ). Similar observations were made when the expression of LC3 was assessed using Western blotting. Figure 15B As shown, most animals treated with ATB200 / miglustat showed a significant reduction in the levels of LC3 II (a lipidated form associated with autophagosomes), indicating improved autophagic flux. In contrast, alglucosidase alfa had little effect on autophagy.

[0353] Desferlin (a protein involved in membrane repair, whose defects / mis-trafficking are associated with many muscular dystrophies) was also evaluated. Figure 16 As shown, desferlin (brown) accumulates abundantly in the sarcoplasm of Gaa KO mice. Compared with alglucosidase alfa, ATB200 / migestastat can restore desferlin to the sarcolemma in more myofibers.

[0354] These data are consistent with the cellular level improvements shown in human Pompe disease patients treated with ATB200 and Miglustat (e.g., patients showed reduced levels of biomarkers of glycogen accumulation and muscle damage), which not only effectively treat Pompe disease but also reverse disease progression. Clinical data from human Pompe disease patients are summarized in Examples 8 and 9 below. Example 7: Single Fiber Analysis

[0355] like Figure 17 As shown, most of the vehicle-treated mice showed marked enlargement of lysosomes (see, e.g., "B") and the presence of numerous autophagic accumulations (see, e.g., "A"). The treated mice did not show any significant differences compared to the vehicle-treated mice. In contrast, most fibers isolated from mice treated with ATB200 showed a significant decrease in lysosome size (see, e.g., "C"). In addition, areas with autophagic accumulation were also reduced to varying degrees (see, e.g., "C"). As a result, a large portion of muscle fibers analyzed from ATB200-treated mice (36-60%) appeared normal or near normal. Table 12 below summarizes the Figure 17 Single fiber analysis shown in . Table 12. Single fiber analysis *This includes fibers with varying degrees of autophagic accumulation. Overall, ATB200-treated groups showed less accumulation compared to vehicle- or alglucosidase alfa-treated groups.

[0356] exist Figure 78 In another study, 12 biweekly bolus injections of ATB200 (20 mg / kg) administered to GaaKO mice showed significant improvements in muscle fiber size, quantified in histological sections of quadriceps muscles, compared to mice treated with alglucosidase alfa (20 mg / kg). Like Pompe disease patients, Gaa mice exhibit reduced muscle fiber size due to muscle atrophy. The mean minimum Feret diameter was significantly larger in mice treated with ATB200 compared to mice treated with alglucosidase alfa, indicating a clear functional difference in efficacy.

[0357] Overall, the data suggest that ATB200 with a higher M6P content, either alone or further stabilized by the enzyme stabilizer miglustat at neutral blood pH, is more efficient in tissue targeting and lysosomal trafficking compared with alglucosidase alfa when administered to Gaa KO mice, consistent with the findings in the literature. Figure 18 The stabilizing effect of miglustat on ATB200 described in Example 6 is consistent. Therefore, the administration of ATB200 and the co-administration of ATB200 / miglustat are more effective than alglucosidase α in correcting some disease-related pathologies, such as glycogen accumulation, lysosomal proliferation, and the formation of autophagic zones. Due to these positive therapeutic effects, the administration of ATB200 and the co-administration of ATB200 / miglustat have been shown to increase the chances of myofibers recovering from injury, and even reverse the injury by clearing glycogen accumulated in cells due to lack of optimal GAA activity. As with Example 6, these data are also consistent with the improvements at the cellular level demonstrated in human Pompe disease patients, which resulted in effective treatment of Pompe disease and reversal of disease progression after administration of ATB200 and miglustat. Clinical data for human Pompe disease patients are summarized in Examples 8 and 9 below. Example 8: ATB200-02 test

[0358] A Phase 1 / 2 (ATB200-02, NCT-02675465) open-label, fixed-sequence, ascending-dose clinical study was conducted to evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics, and mid-term efficacy of IV infusion of ATB200 and miglustat in adult subjects with Pompe disease. The data are reported in International Publication No. WO 2020 / 163480 (the disclosure of which is incorporated herein by reference). Example 9: ATB200-03 trial: a phase 3 human study of ATB200 / miglustat in patients with Pompe disease

[0359] The ATB200-03 trial was a Phase 3, double-blind, randomized, multicenter, international study of ATB200 / miglustat versus alglucosidase alfa / placebo in adult subjects with late-onset Pompe disease (LOPD) who had either received alglucosidase alfa enzyme replacement therapy (ie, ERT-experienced) or had never received ERT (ie, ERT-naive). Study Design

[0360] like Figure 21The trial included a 30-day screening period, a 12-month treatment period, and a 30-day safety follow-up period. Eligible subjects were randomly assigned in a 2:1 ratio to receive ATB200 / miglustat or alglucosidase alfa / placebo and were stratified by ERT status (ERT-experienced, ERT-naive) and baseline 6-minute walk distance (6MWD) (75 to <150 m, 150 to <400 m, ≥400 m).

[0361] Efficacy assessments (i.e., functional assessments) included dynamic functional assessment (6MWT), motor function tests (gait, stair, Gowers, and chair maneuver (GSGC) test and timed up and go (TUG) test), muscle strength (manual muscle strength test and quantitative muscle testing), and pulmonary function tests (FVC, SVC, MIP, MEP, and SNIP). Patient-reported outcomes (Rasch-developed Pompe-Specific Activity (R Pact) scale, EuroQol 5 Dimensions 5 Levels instrument (EQ-5D-5L), Patient-Reported Outcomes Measurement Information System for physical function, fatigue, dyspnea, and upper limb function) were recorded. instrument, and the subject's global impression of change). A physician's global impression of change was also conducted.

[0362] Pharmacodynamic assessments included measurement of biomarkers of muscle damage (creatine kinase (CK) and disease substrates (urinary hexotetraose (Hex4)). Diluted blood samples were collected to determine total GAA protein levels and miglustat concentrations in plasma for population PK analysis in ERT-experienced subjects. Serial blood sampling was performed in ERT-naive subjects to characterize the PK profiles of total GAA protein and miglustat.

[0363] Safety assessments included monitoring of adverse events (AEs), including infusion-related reactions (IARs), clinical laboratory tests (chemistry, hematology, and urinalysis), vital signs, physical examinations including weight, electrocardiograms (ECGs), and immunogenicity. Concomitant medications and non-pharmacological treatments were also recorded. Subject selection

[0364] The subjects who participated in the study met all the following inclusion criteria but did not meet any of the exclusion criteria. A total of 122 subjects participated in the ATB200-03 trial. Among them, 85 subjects (ERT-experienced: 65; ERT-naive: 20) received ATB200 / miglustat treatment, and 37 subjects (ERT-experienced: 30; ERT-naive: 7) received alglucosidase alfa / placebo treatment. Figure 22 As shown, baseline 6MWD and FVC data were representative of the population and were generally similar in the two treatment groups.

[0365] Inclusion criteria: 1. Subjects provided signed informed consent before any study-related procedures were performed. 2. Male and female subjects must be ≥18 years old and weigh ≥40 kg at the time of screening. 3. Female and male subjects of childbearing potential agree to use medically acceptable contraceptive methods during the study and within 90 days after the last dose of study drug. 4. The subject has been diagnosed with LOPD based on one of the following documents: a. GAA enzyme deficiency b. GAA genotyping 5. The subject is classified as one of the following ERT statuses: a. ERT-experienced, defined as having received standard of care ERT (alglucosidase alfa) at the recommended dose and schedule (i.e., 20 mg / kg every 2 weeks) for ≥ 24 months Specific to Australia, ERT-experienced, defined as having received standard of care ERT (alglucosidase alfa) at the recommended dose and schedule of 20 mg / kg every 2 weeks (based on lean body mass or ideal body weight) b. ERT-naive, defined as never receiving investigational or commercial ERT 6. The subject's sitting FVC at screening is ≥ 30% of the predicted value for healthy adults (National Health and Nutrition Examination Survey III). 7. The subject has performed two valid 6MWTs at screening, as determined by the clinical assessor, and meets all of the following criteria: a. Both screening values ​​of 6MWD are ≥ 75 meters b. Both screening values ​​of 6MWD are ≤ 90% of the predicted value for healthy adults c. The lower limit of 6MWD is within 20% of the upper limit of 6MWD

[0366] Exclusion criteria: 1. The subject has received any investigational therapy or drug for Pompe disease other than alglucosidase alfa before Day 1 or is expected to receive any investigational therapy or drug treatment during the study within 30 days or 5 half-lives of therapy or treatment (whichever is longer). 2. The subject has received gene therapy for Pompe disease. 3. The subject has used any of the following illicit drugs within 30 days prior to Day 1: Miglitol Miglustat Acarbose Voglibose 4. The subject needs to use invasive or non-invasive ventilation support for more than 6 hours per day while awake. 5. The subject is allergic to ATB200, alglucosidase alfa, or any excipients in miglustat. 6. The subject has a medical condition or any other extenuating circumstances that, in the opinion of the Investigator or Medical Monitor, creates an undue safety risk to the subject or impairs his / her ability to comply with or adversely affects the requirements of the protocol. This includes clinical depression (diagnosed by a psychiatrist or other mental health professional) with uncontrolled or poorly controlled symptoms. 7. The subject, if female, is pregnant or breastfeeding at the time of screening. 8. The subject, whether male or female, plans to become pregnant during the study. 9. The subject refuses to undergo genetic testing. Study product, dosage, and route of administration

[0367] Subjects were randomly assigned to receive ATB200 / miglustat or alglucosidase alfa / placebo in a randomization ratio of at least 2:1. Table 13 below summarizes the treatments of the enrolled subjects. Table 13. Treatment designation and regimen Abbreviation: IV = intravenous a Note: Subjects were required to fast for at least 2 hours before administration of miglustat or placebo and for 2 hours after administration of placebo. Data evaluation and statistical considerations

[0368] The primary efficacy endpoint was the change in 6MWD from baseline to week 52. The primary endpoint tested the superiority of ATB200 / miglustat versus alglucosidase alfa / placebo using a mixed-effects model for repeated measures (MMRM) and prespecified nonparametric tests for violations of normality.

[0369] The key secondary efficacy endpoints are listed below in order of prespecified importance. These secondary endpoints were analyzed using an analysis of covariance (ANCOVA) model with the last observation carried forward (ITT LOCF) method. Change in sitting FVC from baseline to week 52 (% predicted) Change in lower limb manual muscle strength test scores from baseline to week 52 Change from baseline to week 52 in PROMIS total score - physical function Change from baseline to week 52 in PROMIS total score - Fatigue Change in GSGC total score from baseline to week 52

[0370] Other secondary efficacy endpoints are as follows: Changes from baseline to week 52 in the following motor function-related variables: - Time to complete the 10-meter walk portion of the GSGC test (i.e., gait assessment) -Complete the GSGC test of time to climb 4 stairs - Time to complete the GSGC test Gowers maneuver - Time to stand up from a chair as part of the GSGC test - Time to complete the TUG test Changes from baseline to week 52 in the following muscle strength-related variables: - Upper limb manual muscle strength test score -Total score of manual muscle strength test -Upper limb muscle quantitative test value (kg) - Lower limb quantitative muscle test value (kg) -Quantitative muscle test total value (kg) Changes from baseline to Week 52 in the following patient-reported outcome measures: - PROMIS Total Score - Dyspnea -PROMIS Total Score - Upper Limb -R-PAct total score -EQ-5D-5L health status The actual value of the subject's functional status (improved, stable, or declined) at Week 52 related to the effects of the study drug in the following life domains, as measured by the subject's global impression of change - Overall physical health - Breathing intensity - Muscle strength -Muscle function -Ability to move around -Activities of daily living -Energy levels - Muscle pain level The actual value of the subject's functional status (improved, stable, or declined) at week 52, as measured by the Physician's Global Impression of Change Changes from baseline to Week 52 in the following lung function measures were as follows: -Seated FVC (% predicted) -MIP (cmH2O) -MIP (% predicted) -MEP (cmH2O) -MEP (% forecast) -SNIP(cmH2O)

[0371] The pharmacodynamic endpoints are as follows: Change in serum CK levels from baseline to week 52 Change in urinary Hex4 levels from baseline to week 52

[0372] For ERT-experienced subjects, pharmacokinetic endpoints from population PK analyses of total GAA protein levels and miglustat concentrations were collected. For ERT-naive subjects, plasma total GAA protein concentrations and miglustat PK parameters were calculated.

[0373] The safety profile of ATB200 / miglustat was characterized using the following: treatment-emergent adverse events (TEAEs), serious adverse events (SAEs), and AEs leading to study drug discontinuation, the frequency and severity of immediate and late IARs, and any abnormalities identified during other safety assessments. The impact of the immunogenicity of ATB200 and alglucosidase alfa on safety and efficacy was also evaluated.

[0374] Statistical methods included the following considerations regarding sample randomization, sample size calculation, power analysis, and safety analysis.

[0375] Randomization. The following two factors were identified as design stratification variables: 1. Baseline 6MWD (75 to <150 m, 150 to <400 m, ≥400 m); and 2. ERT status (ERT experienced, ERT naive). These two factors formed six factor combinations (ie, levels, strata). A centralized block randomization procedure was used to balance the above risk factors, 1) reduce bias and improve the precision of statistical inference, and 2) allow for various planned and unplanned subset analyses. A block randomization scheme was performed for each of the 6 strata. The randomization ratio was fixed at 2:1 ATB200 / migestipagliflozin vs alglucosidase alfa / placebo.

[0376] Sample size calculation. A 2-group t-test with a 2-sided significance level of 0.05 and a 2:1 randomization scheme (66 subjects in the ATB200 / miglustat group and 33 subjects in the alglucosidase alfa / placebo group, for a total sample size of 99 subjects) was determined to have approximately 90% power to detect a standardized effect size of 0.7 between the two groups in a superiority test. This calculation was performed using Nquery Assuming a 10% dropout rate, the sample size was approximately 110 subjects.

[0377] Efficacy analysis. The primary efficacy endpoint (i.e., change in 6MWD from baseline to week 52) was analyzed using a parametric analysis of covariance (ANCOVA) model to compare the new treatment and the control. The model would typically adjust for baseline 6MWD (as a continuous covariate) and two factors used to stratify randomization: ERT status (ERT-naive vs. ERT-experienced) and baseline 6MWD (75 to <150 m, 150 to <400 m, ≥400 m). However, due to the expected high biserial correlation between them, baseline 6MWD could not be used twice in the model (as a continuous variable and a categorical variable). Therefore, the 6MWD continuous variable was retained in the model, but the categorical 6MWD was deleted. The ANCOVA model then had the following terms: treatment, baseline 6MWD (continuous), and ERT status (categorical).

[0378] In addition, potential treatment covariate interactions (i.e., treatment in ERT status and baseline 6MWD continuous treatment) were examined. If the interaction term was statistically significant (e.g., p < 0.10, 2-sided) and there was a logical biological explanation, the interaction term could be added to the final ANCOVA model for the primary endpoint analysis. The data were then analyzed based on the ANCOVA model and all relevant estimates (e.g., LS mean, LS mean difference, 95% confidence interval (CI) of the LS mean difference for each treatment group) and p values ​​for comparisons between the two treatment groups were provided.

[0379] To support the interpretation of clinical benefit, a composite subject-level response was defined based on the overall treatment outcome data. Subjects were categorized according to an ordinal response variable consisting of marked improvement, moderate improvement, or slight / no improvement based on treatment outcome.

[0380] Key secondary end points were analyzed in a hierarchical order, using a stepwise closed-test procedure to control the type I error rate. Key secondary end points and other secondary end points were analyzed separately using methods similar to those used for the primary end point analysis.

[0381] Safety Analysis. Safety data were summarized using counts and percentages for categorical data and descriptive statistics (mean, standard deviation, median, minimum, maximum) for continuous data. Efficacy Results from the ATB200-03 Trial

[0382] In the overall population, the 52-week difference in Figure 23A ) and over time ( Figure 23B ), ATB200 / miglustat treatment showed improvement in 6MWD and stabilization of predicted FVC percentage. Compared with alglucosidase alfa / placebo, ATB200 / miglustat treatment showed greater improvement in 6MWD in the overall population at week 52 ( Figure 23A). In addition, if Figure 23A As shown, ATB200 / miglustat treatment demonstrated clinically significant improvements in percent predicted FVC at week 52 in the overall population compared with alglucosidase alfa / placebo.

[0383] In the ERT-experienced group, the difference in the baseline ( Figure 24 ), ATB200 / miglustat treatment showed improvement in 6MWD and stabilization of predicted FVC percentage over time. Compared with alglucosidase alfa / placebo, ATB200 / miglustat treatment in the ERT-experienced population showed improvement in 6MWD over time and stabilization of predicted FVC percentage over time ( Figure 25 ). In addition, if Figure 24 As shown, ATB200 / miglustat treatment demonstrated clinically significant improvements in both 6MWD and percent predicted FVC at week 52 compared with alglucosidase alfa / placebo in the ERT-experienced population.

[0384] like Figure 26A and 26B As shown, in a smaller ERT-naive population (n=27), the 52-week difference in the mean ... Figure 26A ) and over time ( Figure 26B ), ATB200 / migestastat treatment showed improvement in 6MWD and stabilization of percent predicted FVC. The differences between the two treatment groups were greater, and no clinically significant improvements were observed in 6MWD or percent predicted FVC ( Figure 26A ).

[0385] like Figure 28 As shown, in the overall population who underwent ERT, lower extremity MMT numerically favored ATB200 / miglustat treatment compared with alglucosidase alfa / placebo.

[0386] The mean change (SD) in lower extremity MMT scores from baseline to week 52 showed an improvement of 1.6 (3.78) in subjects co-administered with alglucosidase alfa and miglustat compared to an improvement of 0.9 (2.58) in subjects receiving alglucosidase alfa and placebo (p=0.191).

[0387] like Figure 29 As shown, ATB200 / miglustat treatment demonstrated clinically significant improvements in GSGC at week 52 compared with alglucosidase alfa / placebo in both the overall population and the ERT-experienced population.

[0388] The mean change from baseline to Week 52 in total GSGC score showed an improvement of -0.53 (2.5) for subjects co-administered with alglucosidase alfa and miglustat compared to an improvement of 0.77 (1.8) for subjects receiving alglucosidase alfa and placebo (p=0.009).

[0389] like Figure 30 As shown, PROMIS physical function numerically favored ATB200 / miglustat treatment compared with alglucosidase alfa / placebo in both the overall population and the ERT-experienced population.

[0390] like Figure 31 As shown, PROMIS fatigue improvements were similar between the two treatment groups in the overall population and the population that underwent ERT.

[0391] Biomarker Results from the ATB200-03 Trial

[0392] In both the overall population and those who underwent ERT, ATB200 / miglustat treatment demonstrated improvements over time in biomarkers of muscle damage (CK) and disease substrates (Hex4) ( Figure 32 and 33 ). In addition, if Figure 32 and 33 As shown, at week 52, reductions in CK and urinary Hex4 were significantly greater with treatment with ATB200 / miglustat compared with alglucosidase alfa / placebo in both the overall population and the ERT-experienced population.

[0393] In patients who had undergone ERT, the mean baseline urinary Glc4 concentrations were 4.6 mmol / mol and 7.2 mmol / mol in the ATB200 / miglustat and alglucosidase alfa placebo groups, respectively. At week 52, the mean urinary Glc4 concentrations were 2.9 mmol / mol and 9.1 mmol / mol in the ATB200 / miglustat and alglucosidase alfa placebo groups, respectively.

[0394] like Figure 34 As summarized in the , motor function, lung function, muscle strength, patient-reported outcomes (PROs), and biomarker endpoints consistently favored ATB200 / miglustat over alglucosidase alfa / placebo in both the overall and ERT-experienced populations. Furthermore, 16 of the 17 efficacy and biomarker endpoints evaluated favored ATB200 / miglustat over alglucosidase alfa / placebo.

[0395] Safety Results from the ATB200-03 Trial

[0396] like Figure 35As shown, the overall safety profile of the ATB200 / miglustat treatment group was similar to that of the alglucosidase alfa / placebo group.

[0397] Figures 36-40 Other aspects of the ATB200-03 trial are described. Example 10: Results of the PROPEL Phase 3 Clinical Trial

[0398] AT-GAA demonstrated clinically meaningful and significant improvements in musculoskeletal and respiratory measures in late-onset Pompe disease compared to standard of care in the pivotal Phase 3 PROPEL study, also known as "ATB200-03," see Example 9.

[0399] Patients switched from approved standard of care ERT (alglucosidase alfa) to AT-GAA walked an average of 17 meters more (p=0.046).

[0400] Patients switched to AT-GAA also showed improvement in the percentage of predicted forced vital capacity (FVC), the most important measure of respiratory function in Pompe disease, compared with a decline in patients receiving alglucosidase alfa (FVC Diff. 4.1%; p = 0.006).

[0401] AT-GAA demonstrated a nominally statistically significant and clinically meaningful superior difference in the first key secondary endpoint of FVC compared to patients receiving alglucosidase alfa (FVC Diff. 3.0%; p=0.023).

[0402] In the combined study population of ERT-switched and ERT-naive patients, AT-GAA was 14 m superior to alglucosidase alfa on the primary endpoint (21 m vs. 7 m), and the superiority was not statistically significant (p=0.072).

[0403] Improvements in two important biomarkers of Pompe disease (Hex-4 and CK) in the combined study population significantly favored AT-GAA compared with alglucosidase alfa (p<0.001).

[0404] PROPEL is a 52-week, double-blind, randomized, global study designed to evaluate the efficacy, safety, and tolerability of AT-GAA compared to the current standard of care, alglucosidase alfa, an enzyme replacement therapy (ERT). The study enrolled 123 adult patients with Pompe disease who were able to walk and breathe without mechanical ventilation and was conducted at 62 clinical sites in 24 countries across five continents. This is the largest controlled clinical study ever conducted for a lysosomal disorder.

[0405] Patients enrolled in PROPEL were randomized in a 2:1 ratio, so for every two patients randomized to AT-GAA, one was randomized to alglucosidase alfa. Among the Pompe disease patients enrolled in PROPEL, 77% had received alglucosidase alfa immediately prior to enrollment (n=95), and 23% had never received any ERT (n=28). 117 patients completed the PROPEL study, and all 117 patients volunteered to participate in the long-term extension study and now receive only AT-GAA for their Pompe disease. Prespecified Analyses of 6-Minute Walk Distance (6MWD) and Percentage of Predicted Forced Vital Capacity (FVC) in the Combined ERT-Converted and ERT-Naive Study Populations:

[0406] The primary endpoint of the study was the mean change in 6-minute walk distance at 52 weeks compared to baseline measurements in the combined population of ERT-converted and ERT-naive patients. In this combined population, patients using AT-GAA (n=85) walked an average of 21 meters more at 52 weeks, while patients receiving alglucosidase alfa (n=37) walked an average of 7 meters more (Table 14). This primary endpoint was assessed as superior in the combined population, and although numerically greater, the advantage of the AT-GAA group compared to the alglucosidase alfa group in this combined population did not reach statistical significance (p=0.072).

[0407] According to the hierarchy of the statistical analysis plan, the first key secondary endpoint of the study was the mean change in percent predicted FVC at 52 weeks in the combined population. In this combined population, patients treated with AT-GAA demonstrated a nominally statistically significant and clinically meaningful advantage over patients treated with alglucosidase alfa. After 52 weeks, AT-GAA significantly slowed the rate of respiratory decline in patients. Patients treated with AT-GAA showed an absolute decline in percent predicted FVC of 0.9%, compared to an absolute decline of 4.0% in the alglucosidase alfa group (p = 0.023) (Table 15). Percent predicted FVC is the most important measure of respiratory muscle function in Pompe disease and is the basis for the approval of alglucosidase alfa. Table 14. 6MWD (m) in the overall ERT conversion and ERT-naive study population Table 15. FVC (% predicted) in the overall ERT switching and ERT naive study populations Prespecified Analyses of 6-Minute Walk Distance (6MWD) and Percentage of Predicted Forced Vital Capacity (FVC) in the ERT Conversion Study Population (n=95):

[0408] PROPEL switch patients had received at least two years of alglucosidase alfa treatment at study entry. More than two-thirds (67%+) of patients had received ERT for more than five years (mean, 7.4 years) prior to entering the PROPEL study.

[0409] A prespecified analysis of 6-minute walk distance in patients who switched from alglucosidase alfa showed that at 52 weeks after switching, AT-GAA-treated patients (n=65) walked 16.9 meters farther than baseline, compared with 0.0 meters for those randomized to continue alglucosidase alfa (n=30) (p=0.046) (Table 16).

[0410] A prespecified analysis of the percentage of predicted FVC in patients who switched from alglucosidase alfa showed that patients treated with AT-GAA experienced stabilization and slight improvement in respiratory function on this important measure, while those who continued on alglucosidase alfa continued to experience a significant decline in respiratory muscle function. AT-GAA patients showed an absolute increase of 0.1% in the percentage of predicted FVC, while alglucosidase alfa patients showed an absolute decline of 4.0% over the year (p = 0.006) (Table 17). Table 16. 6MWD (meters) in the ERT conversion study population Table 17. FVC (% predicted) in the ERT conversion study population Prespecified analyses of 6-minute walk distance (6MWD) and percentage of predicted forced vital capacity (FVC) in an ERT-naive population (n=28):

[0411] A pre-specified analysis of 6-minute walk distance in patients who had never received any ERT treatment showed that after 52 weeks, patients treated with AT-GAA (n=20) walked 33 meters more than their baseline. Patients treated with alglucosidase alfa (n=7) walked 38 meters more than their baseline. The difference between the two groups was not statistically significant (p=0.60) (Table 18).

[0412] A prespecified analysis of patients who had never received any ERT treatment showed a similar decline in the percentage of predicted forced vital capacity (FVC) at 52 weeks, at -4.1% for patients treated with AT-GAA and -3.6% for patients treated with alglucosidase alfa (Table 19). The difference between the two groups was not statistically significant (p = 0.57). Table 18. 6MWD (meters) in ERT-naive population Table 19. FVC (% predicted) in an ERT-naive population Note: One patient in the alglucosidase alfa group was excluded from study analysis because of use of an investigational anabolic steroid that affected his baseline performance. Pre-specified analyses of other key secondary and biomarker endpoints in the overall ERT conversion and ERT-naive study population:

[0413] Musculoskeletal and other key secondary endpoints:

[0414] GSGC (Gait, Stairs, Gowers): GSGC is an important and commonly used endpoint in Pompe disease that captures strength, coordination, and mobility. In the overall population, patients treated with AT-GAA demonstrated a statistically significant improvement in scores on this important assessment, compared to a worsening in patients treated with alglucosidase alfa (p<0.05).

[0415] Lower Extremity MMT (Manual Muscle Test), PROMIS Physical Function: On these two validated measures of muscle strength and patient-reported outcomes, AT-GAA-treated patients improved numerically more than alglucosidase alfa-treated patients, although the results were not statistically significant.

[0416] PROMIS Fatigue: Fatigue measured by this scale slightly favored AT-GAA-treated patients over alglucosidase alfa-treated patients. Biomarkers of disease treatment efficacy:

[0417] Urinary Hex-4: For the combined study population of ERT-switched and ERT-naive patients, those receiving AT-GAA demonstrated significant improvements in this biomarker, with a mean decrease of -31.5% in Hex-4 after 52 weeks, compared to an increase of +11.0% (i.e., worsening) in Hex-4 in patients treated with alglucosidase alfa (p = < 0.001). Urinary Hex-4 is a common biomarker in Pompe disease and can be used as an indirect measure of the extent of bone glycogen clearance in Pompe disease patients receiving ERT. Glycogen is a substrate that accumulates in muscle lysosomes in Pompe disease patients.

[0418] Creatine kinase (CK): After 52 weeks, patients treated with AT-GAA also showed significant improvements in this biomarker, with a mean decrease of -22.4% in CK, compared to a +15.6% increase (i.e., worsening) in patients treated with alglucosidase alfa (p<0.001). CK is an enzyme that leaks from damaged muscle cells and is elevated in patients with Pompe disease.

[0419] AT-GAA demonstrated a similar safety profile to alglucosidase alfa. Two patients (2.4%) receiving AT-GAA discontinued treatment due to adverse events, compared with one patient (2.6%) receiving alglucosidase alfa, who discontinued treatment unrelated to treatment. Injection-related reactions (IARs) were reported in 25% of participants receiving AT-GAA and 26% of those receiving alglucosidase alfa. Post hoc subgroup analysis:

[0420] Baseline 6MWD and FVC categories: ERT-naive population (n=27): Three patients had a baseline 6MWD <300 m and three patients had a baseline FVC <55%; CFBL analyses were not performed in these subgroups due to the small number of patients. Baseline 6MWD ≥300 m: Both the AT-GAA (n=18) and alglucosidase alfa / placebo (n=6) groups improved similarly over time (mean [SE] CFBL to Week 52: +34.4 [12.1] m and +30.8 [9.6] m, respectively). Baseline FVC ≥55%: Both the AT-GAA (n=19) and alglucosidase alfa / placebo (n=5) groups declined over time (mean [SE] CFBL to Week 52: -3.7 [1.5]% and -3.3 [2.6]%, respectively). Figure 41 As shown, in patients with baseline 6MWD <300 m and ≥300 m, FVC <55% and ≥55%, the results consistently favored sipagliflozin / miglustat in both the overall and ERT-experienced populations.

[0421] In the overall study population, including both ERT-naive and ERT-experienced patients, sipagliflozin / miglustat demonstrated positive trends or clinically meaningful improvements in motor and respiratory function compared with approved ERT, regardless of baseline 6MWD and %FVC assessments, and this was true in prespecified and post hoc subgroup analyses.

[0422] Sipaglucosidase alfa / miglustat showed a similar safety profile to alglucosidase alfa / placebo ( Figure 42 ). About AT-GAA

[0423] AT-GAA is an investigational two-component therapy consisting of sipa-glucosidase alfa (ATB200), a unique recombinant human acid α-glucosidase (rhGAA) enzyme with an optimized carbohydrate structure, specifically a diphosphorylated mannose-6-phosphate (bis-M6P) glycan, for enhanced cellular uptake, administered in combination with miglustat (AT2221), a stabilizer of sipa-glucosidase alfa. In preclinical studies, AT-GAA was associated with increased levels of the mature lysosomal form of GAA and decreased glycogen levels in muscle, alleviation of autophagy defects, and improved muscle strength. About Pompe Disease

[0424] Pompe disease is an inherited lysosomal disorder caused by a deficiency of the enzyme acid alpha-glucosidase (GAA). Reduced or absent levels of GAA lead to a buildup of glycogen in cells, which is believed to contribute to the clinical manifestations of Pompe disease. The disease can be debilitating, characterized by severe muscle weakness that worsens over time. Pompe disease ranges from a rapidly fatal infantile form with significant effects on heart function to a more slowly progressive late-onset form that primarily affects skeletal muscle. Pompe disease is estimated to affect approximately 5,000 to 10,000 people worldwide. Example 11: Results of an open-label phase I / II study (ATB200-02)

[0425] ATB200-02 (NCT02675465) is an open-label, Phase I / II clinical trial designed to evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics, and efficacy of sipa-glucosidase alfa / miglustat in adult patients with Pompe disease. Sipa-glucosidase alfa / miglustat is an investigational, two-component therapy for the treatment of late-onset Pompe disease (LOPD) consisting of intravenous sipa-glucosidase alfa (rhGAA) and oral miglustat, an enzyme stabilizer.

[0426] Figure 43 The study design of the Phase I / II ATB200-02 study is shown. The study was conducted at 16 centers in five countries. The ATB200-02 study enrolled four groups of patients with Pompe disease: Cohort 1: ERT-experienced patients aged 18-65 years with 2-6 years of prior ERT who received 20 mg / kg alglucosidase alfa every 2 weeks (n=11) Cohort 2: Non-ambulatory, ERT-experienced patients aged 18-65 years with ≥2 years of prior ERT who received 20 mg / kg alglucosidase alfa every 2 weeks (n=6) Cohort 3: ERT-naive patients, aged 18-65 years (n=6) Cohort 4: ERT-experienced patients aged 18-75 years with ≥7 years of prior ERT who received 20 mg / kg alglucosidase alfa every 2 weeks (n=6)

[0427] Eligible ambulatory patients had a 6-minute walk distance (6MWD) of at least 200 m (cohorts 1 and 3) or 75 m (cohort 4) and an upright forced vital capacity (FVC) of 30-80% of predicted normal.

[0428] Changes from baseline (CFBL) in 6MWD, % predicted sitting FVC, manual muscle testing (MMT), and biomarkers—urinary glucose tetrasaccharide (Hex4) and serum creatine kinase (CK)—were assessed regularly. This article presents follow-up data for up to 36 months in ERT-experienced and ERT-naive patients. Figure 44 A summary of the reported endpoints and cohorts is shown. Figure 45 Baseline characteristics and patient disposition are shown. Due to staggered enrollment of patients, the number of patients for whom data are currently available will be reduced at later time points in this ongoing study.

[0429] Patients who underwent ERT showed sustained mean improvement in 6MWD from baseline to 48 months. After 12, 24, 36, and 48 months of follow-up, 13 of 16, 9 of 13, 6 of 12, and 6 of 9 patients who underwent ERT, respectively, had improved 6MWD from baseline ( Figure 46A ). The average increase was 33 meters (m) by the 12th month, 25 meters by the 24th month, 9 meters by the 36th month, and 20 meters by the 48th month.

[0430] For patients who underwent ERT, the change from baseline in FVC (CFBL) generally remained stable up to 48 months of follow-up. After 12, 24, 36, and 48 months of follow-up, the change from baseline in FVC was significantly higher in 9 / 16, 11 / 13, 8 / 10, and 4 / 6 patients, respectively. Figure 47A ) FVC improved (>3%) or remained stable (±3%) relative to baseline in the control group. The mean changes were -1.2% by month 12, +1.0% by month 24, -0.3% by month 36, and +1.0% by month 48.

[0431] In patients who underwent ERT, the mean change from baseline in MMT lower extremity scores improved numerically, and the improvement was maintained up to 48 months of follow-up ( Figure 48AAt 12, 24, 36, and 48 months of follow-up, 14 of 15, 11 of 13, 10 of 10, and 8 of 8 ambulatory patients, respectively, showed improvement in their MMT lower extremity scores relative to baseline. The mean increase was 3.1 points at month 12, 2.1 points at month 24, 2.5 points at month 36, and 3.5 points at month 48.

[0432] ERT-naive patients showed sustained mean improvement in 6MWD from baseline to 48 months. After 12, 24, 36, and 48 months of follow-up, 6 / 6, 6 / 6, 4 / 5, and 4 / 4 of the ERT-naive patients, respectively, had a numerical improvement in 6MWD relative to baseline ( Figure 46B The average increase was 57 meters by the 12th month, 54 meters by the 24th month, 43 meters by the 36th month, and 52 meters by the 48th month.

[0433] In ERT-naive patients, mean CFBL in FVC improved relative to baseline at up to 48 months of follow-up. After 12, 24, 36, and 48 months of follow-up, CFBL in 5 / 6, 6 / 6, 5 / 5, and 4 / 4 patients ( Figure 47B ) FVC improved (>3%) or remained stable (±3%) relative to baseline. Figure 47B As shown by the asterisk in the figure, one patient in the ERT-naive cohort had a large decrease in % predicted FVC at month 21, which returned to its previous level at the next visit (month 24). The mean changes were 3.2% by month 12, 4.7% by month 24, 6.2% by month 36, and 8.3% by month 48.

[0434] In ERT-naive patients, the mean change from baseline in MMT lower extremity scores improved numerically, and the improvement was maintained up to 48 months of follow-up ( Figure 48B At 12, 24, 36, and 48 months of follow-up, 4 / 5, 4 / 4, and 3 / 4 of the ambulatory patients, respectively, had an improvement in their MMT lower extremity scores relative to baseline. The mean increase was 2.8 points at month 12, 3.0 points at month 24, 3.3 points at month 36, and 1.0 point at month 48.

[0435] During the 48-month follow-up period, sipa-glucosidase alfa / miglustat was generally associated with mean reductions in urinary Hex4 from baseline, with greater reductions in ERT-naive patients. After 12, 24, 36, and 48 months of follow-up, Hex4 levels were numerically reduced from baseline in 16 / 16, 11 / 14, 11 / 12, and 6 / 9 ERT-experienced patients and in 5 / 6, 5 / 6, 4 / 5, and 4 / 5 ERT-naive patients, respectively. Figure 49A ).

[0436] During the 48-month follow-up period, sipagliflozin / miglustat was associated with stable levels of plasma CK or mean reductions from baseline, with greater reductions in ERT-naive patients. After 12, 24, 36, and 48 months of follow-up, CK levels were numerically reduced from baseline in 13 / 15, 14 / 15, 9 / 11, and 8 / 9 ERT-experienced patients and in 6 / 6, 6 / 6, 5 / 5, and 4 / 5 ERT-naive patients, respectively. Figure 49B ).

[0437] Figure 50 A summary of treatment-emergent adverse events (TEAEs) occurring on or after the first dose of study drug in the ATB200-02 study is shown. The mean (SD) treatment duration in cohorts 1 (2-6 years prior to ERT), 4 (≥7 years prior to ERT), and 3 (ERT-naive) was 37.2 (14.48), 19.9 (4.13), and 36.9 (12.14) months, respectively. The most common TEAEs included falls, nasopharyngitis, arthralgia, headache, and diarrhea; most TEAEs were mild or moderate in severity and did not lead to study withdrawal.

[0438] Results from the ATB200-02 study of sipaglucosidase alfa plus miglustat in ambulatory patients with up to 48 months of follow-up are as follows. ERT-experienced patients experienced sustained mean improvements from baseline in motor function, persisting through 48 months of follow-up, while respiratory function remained stable over the same period: improvements compared with the expected decline seen in many patients undergoing long-term ERT. ERT-naive patients demonstrated sustained mean improvements from baseline in motor and respiratory function, persisting through 48 months of follow-up. Mean levels of two biomarkers, Hex4 and CK, either remained stable or decreased from baseline through 48 months of follow-up, with the greatest decreases seen in the ERT-naive cohort. The safety profile of sipaglucosidase alfa plus miglustat was similar to that of alglucosidase alfa.

[0439] Figure 55 End points reported for Cohort 2 (ERT-experienced patients who were nonambulatory) and a summary of the cohort are shown. Figure 56 Baseline characteristics and patient disposition of cohort 2 are shown.

[0440] In these nonambulatory ERT-experienced patients, the change from baseline in FVC (CFBL) was numerically improved from baseline and / or remained stable at up to 48 months of follow-up ( Figure 57 The mean change was +2.5% by month 12, +2.0% by month 24, -2.0% by month 36, and -1.0% by month 48.

[0441] In these nonambulatory ERT-experienced patients, the mean change from baseline in the MMT total score (upper body) improved numerically and / or remained stable at up to 36 months of follow-up ( Figure 57 The mean change was +1.3 points by month 12, +2.0 points by month 24, and -0.8 points by month 36.

[0442] During the 48-month follow-up period in these nonambulatory ERT-experienced patients, sipa-glucosidase alfa / miglustat was generally associated with mean decreases from baseline in urinary Hex4 and plasma CK ( Figure 57 After 12, 24, 36, and 48 months of follow-up, Hex4 levels decreased numerically relative to baseline, with mean decreases of -15.6%, -34.1%, -36.5%, and -4.0%, respectively. After 12, 24, 36, and 48 months of follow-up, plasma CK levels decreased numerically relative to baseline, with mean decreases of -20.8%, -25.3%, -27.0%, and -23.7%, respectively.

[0443] Figure 58 A summary of treatment-emergent adverse events (TEAEs) occurring at or after the first dose of study drug in the ATB200-02 study or in Cohort 2 (in nonambulatory ERT-experienced patients) is shown. The mean (SD) treatment duration was 46.3 (22.86) months. The most common TEAEs included nasopharyngitis and diarrhea (both occurring in 3 patients); most TEAEs were mild or moderate in severity and did not lead to study withdrawal.

[0444] Results from the ATB200-02 study of sipa-glucosidase alfa plus miglustat in nonambulatory ERT-experienced patients with up to 48 months of follow-up showed that these patients experienced sustained mean improvements and / or stabilization of motor and lung function from baseline for up to 48 months of follow-up, an improvement compared to the expected decline seen in many patients undergoing long-term ERT. Mean levels of two biomarkers, Hex4 and CK, either remained stable or decreased from baseline over the 48 months of follow-up. Sipa-glucosidase alfa plus miglustat was generally well tolerated in this patient group. Example 12: Long-term effects of ERT on patients with Pompe disease

[0445] Figure 51 Shown is a comparison of the long-term effects of sipagnosidase alfa / miglustat and efavirenz alfa on changes from baseline in 6MWD and percent predicted FVC (sitting) in subjects who had undergone ERT. Figure 52A comparison of the long-term effects of alglucosidase alfa / miglustat and efglustat on the change from baseline in 6MWD and percent predicted FVC (sitting) in ERT-naive subjects is shown. The comparison showed that ERT-experienced patients who switched from alglucosidase alfa to efglustat continued to experience progressive loss of motor function as assessed by 6MWD, similar to the progression shown above for patients who continued long-term alglucosidase alfa, while patients who switched from alglucosidase alfa to alglucosidase alfa / miglustat experienced a categorical change, showing a gradual improvement in motor function as assessed by 6MWD. A similar trend was observed with FVC, where patients who switched to efglustat experienced progressive loss of lung function as assessed by FVC, while patients who switched from alglucosidase alfa to alglucosidase alfa / miglustat had stable lung function as assessed by FVC.

[0446] Figure 53A-Figure 53B Shown are the percentages of predicted 6-minute walk test (6MWT) during treatment with alglucosidase alfa. Figure 53B Shows the Figure 53A Data replotted only from year 2 onwards. Figure 54 The percentage of predicted FVC during treatment with alglucosidase alfa is shown. Data from year 2 show that patients who have undergone ERT and continue on alglucosidase alfa are expected to decline. Example 13: Comparison of Alglu, Aval, and Cipa+mig

[0447] In the absence of head-to-head trials comparing Aval and Cipa+mig, indirect treatment comparisons (ITCs) are an appropriate approach to better understand the clinical differences between the three treatments for LOPD. ITCs are widely requested by health technology assessment agencies (HTAs) to support comparative health economic evaluations.

[0448] An ITC was performed that provided relative efficacy estimates for the target population (ie, the LOPD population, which included a mix of ERT-naive and ERT-experienced subjects, such as in the pivotal Phase III trial comparing Cipa+mig with Alglu [PROPEL]).

[0449] Methods: A systematic literature review (SLR) was conducted to identify relevant published clinical studies of ERT in LOPD. The outcomes assessed were change from baseline in 6-minute walk distance (6MWD) (m) and forced vital capacity (FVC; percent predicted) at week 52, which are considered key LOPD trial endpoints by clinicians, HTA agencies, and payers. Summary results of change from baseline in 6MWD and FVC over time and baseline characteristics (age, sex, race, prior ERT duration, baseline 6MWD, and baseline FVC) were extracted from the included studies.

[0450] Methods: Multilevel network meta-regression (ML-NMR), an extension of standard network meta-analysis (NMA) that accounts for the effects of study-level covariates and can be applied to any connected network with a mixture of individual patient-level data (IPD) and population data, was performed. ML-NMR is a method accepted by the National Institute for Health and Care Excellence (NICE) to support cost-effectiveness analysis. Results from single-group studies were matched to appropriate comparators from comparative studies to allow inclusion in the network. Mean treatment differences and associated 95% confidence intervals (CrIs) for changes from baseline in 6MWD and FVC at week 52 were calculated.

[0451] A base-case scenario was evaluated in which all covariates were set to the target population of the PROPEL trial. To investigate the effect of prior ERT duration on relative efficacy, ERT duration values ​​were varied, keeping the remaining covariates the same as in the base-case scenario. A sensitivity analysis was performed by excluding all matched single-group evidence from the network to assess its impact on the results.

[0452] Fixed-effect (FE) and random-effect (RE) ML-NMR models were applied, and the deviance information criterion (DIC) was used to assess the goodness of fit of the models and determine the appropriate model (FE or RE model) for the data. The models were implemented in a Bayesian framework using Stan with the help of the R package multinma.

[0453] The SLR identified seven clinical studies with baseline characteristics such as Figure 59 These studies include, but are not limited to, three randomized clinical trials (LOTS: Alglu vs. placebo; COMET: Aval vs. Alglu; PROPEL: Cipa+mig vs. Alglu). Each shared 6MWD and FVC as key primary or secondary endpoints (see Figure 60 and 61) but their trial populations were different (PROPEL was the only randomized controlled trial [RCT] that included both ERT-naive and ERT-experienced subjects). Figure 60 shown.

[0454] For both endpoints, the network is identical, e.g. Figure 61 Evidence from single-group studies LOTS OLE, NEO-1 / -EXT, COMET OLE, and ATB200-02 has been included in the network, as shown in blue boxes, by matching the single-group results with the corresponding comparison results from head-to-head trials.

[0455] In the base-case scenario, covariates were set to the baseline characteristics of the target population (ie, the PROPEL trial; see Table 20), and time was set to 52 weeks. Table 20: Base case scenario covariate settings Age (years) %male % Caucasian ERT duration (years) 6MWD (meter) FVC (% predicted) 46.95 45.08 84.43 5.744 355.8 70.42

[0456] Based on DIC, the RE model was selected for 6MWD and the FE model was selected for FVC. Figure 62 and 63 ): Cipa+mig showed statistically significant favorable effects compared with Alglu and Aval; while Cipa+mig showed numerical favorable effects compared with placebo.

[0457] Note that the 95% CrIs for the estimates of relative effect compared to placebo are generally much wider than those for the estimates of relative effect compared to Alglu or compared to Aval. This reflects the greater uncertainty in these estimates, as the placebo data are only available for ERT-naive subjects and the previous base-case scenario had a relatively long duration of ERT (5.7 years).

[0458] Figure 64 (6MWD) and Figure 65 Relative effect estimates for different prior ERT durations (prior ERT duration = 0 years [ie, treatment-naive patients], 2.5 years, 5 years, and 9.2 years) are shown in (FVC).

[0459] Based on DIC, the RE model was selected for 6MWD and FVC. Figure 66 and Figure 67 Relative effect estimates with 95% CrI for the base case scenario using sensitivity analysis are summarized and show that: Incorporating matched single-group evidence into the main analysis network reduces uncertainty in relative effect estimates Cipa+mig: statistically favorable compared to Alglu; numerically unfavorable compared to Aval; numerically favorable compared to placebo (6MWT and FVC) Aval: Numerically favorable compared to Alglu and placebo (6MWT and FVC) Alglu: Numerically favorable compared to placebo

[0460] In summary, the ML-NMR comparisons presented here showed that Cipa+mig was statistically significantly superior to Alglu and Aval for 6MWD and FVC in the base-case scenario of the primary analysis. Cipa+mig was also statistically significantly superior to Alglu and Aval for 6MWD and FVC across different ERT durations, with one exception: for FVC, Cipa+mig was only numerically superior to Aval in the ERT-naive setting.

[0461] Sensitivity analyses (including only RCT data) showed that incorporating matched single-group evidence into the primary analysis network reduced uncertainty in the relative effect estimates. Overall, these results suggest that Cipa+mig may have a different clinical spectrum compared with other ERTs, particularly in individuals who have previously received some degree of ERT. Further analyses are expected to test and refine the findings when more long-term data are released. Example 14: Long-term comparison of Cipa+mig and alglucosidase alfa (Alglu)

[0462] The Phase III double-blind PROPEL study (NCT03729362; ATB200-03) described in Examples 9 and 10 above compared the investigational two-component therapy cipa / mig with alglucosidase alfa / placebo (alglu) over 52 weeks in adult patients with late-onset Pompe disease (LOPD). The ongoing open-label extension (OLE) of PROPEL (NCT04138277; ATB200-07) is evaluating the long-term safety and efficacy of cipa / mig. Outcomes include 6-minute walk distance (6MWD), forced vital capacity (FVC), creatine kinase (CK), and hexotetraose (Hex4) levels and safety. Data are reported as changes from PROPEL baseline to OLE Week 52 (104 weeks after PROPEL baseline). Study design and patient disposition are as follows: Figure 68 and baseline characteristics are shown in Figure 69Results are shown in Figures 70-74 and are described in further detail below. In the OLE (N=119; 91 enzyme replacement therapy [ERT]-experienced and 28 ERT-naive), 82 / 85 (96.5%) patients previously treated with cipa / mig continued with cipa / mig and 37 / 38 (97.4%) switched from alglu to cipa / mig; 90.8% of patients remained in the OLE study through week 52. In ERT-experienced patients, the mean change in % predicted 6MWD was +3.1 (8.07 standard deviations) for cipa / mig-cipa / mig and -0.5 (7.76) for alglu-cipa / mig, and in ERT-naive patients, it was +8.6 (8.57) for cipa / mig-cipa / mig and +8.9 (11.65) for alglu-cipa / mig. In ERT-experienced patients, the mean change in % predicted FVC was -0.6 (7.50) for cipa / mig-cipa / mig and -3.8 (6.23) for alglu-cipa / mig, and -4.8 (6.48) and -3.1 (6.66) in ERT-naive patients. The mean decrease in CK (U / L) was -132.1 (215.74) and -216.9 (243.66) for cipa / mig-cipa / mig in ERT-experienced and ERT-naive patients, respectively, and -161.0 (269.52) and -218.6 (316.47) for alglu-cipa / mig, respectively. The mean reduction in Hex4 (mmol / mol) was -1.9 (3.22) in ERT-experienced patients and -2.9 (2.45) in ERT-naive patients for cipa / mig-cipa / mig, and -2.6 (3.75) and -2.9 (2.22) for alglu-cipa / mig, respectively. During PROPEL through Week 52 of the OLE, 84 (98.8%) patients on cipa / mig-cipa / mig and 36 (97.3%) on alglu-cipa / mig experienced treatment-emergent adverse events. Three patients discontinued the OLE due to infusion-related reactions (urticaria, hives and hypotension, and anaphylaxis, respectively).

[0463] Throughout PROPEL and OLE, ERT-experienced patients treated with cipa / mig experienced improvements from baseline in 6MWD and biomarker levels, and % predicted FVC remained stable throughout PROPEL and through week 104 of the OLE. ERT-experienced patients treated with alg / pla during PROPEL maintained stable 6MWD, worsened % predicted FVC and biomarker levels, and stabilized or improved after switching to cipa / mig in the OLE. For ERT-naive patients treated with cipa / mig throughout PROPEL and OLE, 6MWD and biomarker levels improved throughout PROPEL and remained stable throughout the OLE. % predicted FVC declined throughout PROPEL and stabilized throughout the OLE. ERT-naive patients treated with alg / pla during PROPEL and switched to cipa / mig in the OLE showed similar patterns to those who remained on cipa / mig throughout. No new safety signals were identified. Data showed that cipa / mig treatment for up to 104 weeks was associated with durable effects and was well tolerated, supporting the long-term benefit of treatment in patients with LOPD. Example 15: Comparison of N-glycan profiles in sipa-glucosidase alfa and alglucosidase alfa

[0464] Alphaglucosidase alfa and three preparations of sipa-glucosidase alfa were analyzed using LC-FLD to evaluate their N-glycan profiles, which were classified into 10 different classes based primarily on charge and hydrophilicity. LC-FLD analysis showed that levels of the di-M6P (class 10) N-glycan species were consistently and in absolute terms higher in sipa-glucosidase alfa compared to alglucosidase alfa. Additional differences in the relative abundance of monosialylated (class 2), disialylated (class 4), and monophosphorylated (mono-M6P, class 6) glycan species that distinguished sipa-glucosidase alfa from alglucosidase alfa were observed. The results are shown in Figure 75 and Table 21.

[0465] Included in the analysis and Figure 75 The three formulations of Sipaglucosidase alfa reported in Table 21 are 110C161009a (nonclinical drug substance produced in a 1000-L working volume in the first 1000-L bioreactor engineering run), 110C171011a (clinical drug substance), and 2S1802 (clinical drug product).

[0466] The analysis was performed using a hydrophilic interaction liquid chromatography (HILIC) column coupled with fluorescence detection (LC-FLD) under the control of an ultra-high performance liquid chromatography (UHPLC) system to separate fluorescently labeled 2-AA (2-anthranilic acid) N-glycans released from sipa-glucosidase α by the amidase PNGase F. The classes (1-10) are labeled in Table 21. Table 21: Summary of the percent abundance of 2-AA glycans identified in Sipaglucosidase alfa preparations and alglucosidase alfa based on LC-FLD analysis.

[0467] In LC-FLD analysis, proteins are denatured, reduced, and treated with the enzyme PNGase F to release N-linked glycans. The deglycosylated proteins are analyzed by SDS-PAGE. The released glycans are labeled with 2-AA and analyzed by normal phase chromatography. Briefly, 50 μg of ATB200 is mixed with 15 μL of 10× denaturation buffer containing 5% SDS and 400 mM dithiothreitol (DTT) in a total volume of 150 μL, heated at 100°C for 10 minutes, and then cooled to 20°C. To each denatured ATB200 sample, 20 μL of 10× NP-40 and NEB Glyco Reaction Buffer 2 are added to bring the final reaction volume to 190 μL. 10 μL of PNGase F is added to release N-linked glycans, and the reaction is incubated at 37°C for 16-18 hours. The released glycans are purified using a 10 kDa MWCO ultrafiltration centrifuge device. 50 μL of the deglycosylation reaction was added to a centrifuge apparatus and centrifuged at 14,000 RCF for 10 minutes. A total of 150 μL of ddH2O was added in three 50 μL steps, and glycans were washed from the deglycosylated ATB200 by centrifuging the ultrafiltration apparatus at 14,000 RCF for 10 minutes after each wash step. The purified glycans were dried by speed vacuum and labeled with 2AA. The labeled glycans were precipitated in 95% CAN / 5% HO and pelleted by centrifugation at 14,000 RCF for 10 minutes at room temperature. The precipitate was washed with 95% CAN aqueous solution, centrifuged again, and dried using speed vacuum. The dried labeled glycans were dissolved in 100 μL of 20% CAN aqueous solution and stored at 4°C until chromatographic separation and analysis. During the labeling reaction, 2 μL of each glycosylated and deglycosylated ATB200 sample was separated by 4-12% SDS-PAGE. The gel was stained with Imperial Blue and visualized using the BioRad ChemiDoc MP imaging system. Glycan class assignment was based on established peak shapes and chromatographic retention times, as determined in previous work. Glycan peak integration and quantification were performed using Chromeleon.

[0468] Overall, N-glycan profiling of 2-AA-labeled N-glycans by LC-FLD distinguished Sipaglucosidase α from alglucosidase α. As shown above, Sipaglucosidase α had a higher content of M6P glycans, highlighting the structural differences. In particular, Sipaglucosidase α had a higher content of di-M6P glycans, which was attributed to significantly higher CIMPR binding and uptake into muscle cells. Example 16: Role of M6P in CIMPR Binding and Cellular Uptake of Sipaglucosidase α

[0469] Removal of the phosphate groups on sipa-glucosidase α blocked CIMPR binding and prevented rhGAA uptake into fibroblasts from Pompe disease patients, demonstrating the importance of phosphorylated glycans for therapeutically relevant biological effects. Dephosphorylated sipa-glucosidase α was obtained by removing the phosphate groups on mono- and di-M6P using purple acid phosphatase (PAP).

[0470] The removal of mono- and di-M6P phosphates is accomplished by purple acid phosphatase (PAP). PAP hydrolyzes phosphates and anhydrides. Under acidic conditions, the phosphates on mono- and di-M6P phosphates are removed by PAP, e.g. Figure 76 A and 76B, while retaining the enzymatic activity of sipa-glucosidase α, as shown by the increase in electrophoretic mobility in Figure 76 Hydrolysis of 4MU-α-glucoside is shown in D. Sipa-glucosidase α was treated with PAP for 18 hours, while alglucosidase α was treated under the same reaction conditions but without the addition of PAP as a mock-treated control. PAP treatment resulted in the complete removal of the di-M6P peak and nearly all of the mono-M6P peaks from the Sipa-glucosidase α LC-FLD chromatogram. Additional peaks were clearly observed in the neutral high-mannose glycan region due to the removal of phosphates from mono- and di-phosphorylated high-mannose glycans.

[0471] Figure 76 The results of the CIMPR overlay assay, shown in C, show that Sipaglucosidase α binds to CIMPR with high affinity, as indicated by the intense band, whereas PAP-treated Sipaglucosidase α has no detectable band, indicating no binding to CIMPR. Furthermore, the band intensity of alglucosidase α is much lower than that of Sipaglucosidase α, indicating lower binding affinity for CIMPR. Compared to mock-treated Sipaglucosidase α, alglucosidase α and dephosphorylated Sipaglucosidase α showed significantly reduced CIMPR binding, clearly demonstrating that the N-glycan structures, mono-M6P and di-M6P, are crucial for the properties of Sipaglucosidase α and are important for its therapeutic biological effects.

[0472] The CIMPR overlay assay is a variation of western blotting in which proteins are separated on SDS-PAGE, transferred to a nitrocellulose membrane, and incubated with purified CIMPR instead of the primary antibody in conventional western blotting. Binding of CIMPR to M6P-containing glycans on the rhGAA protein is then visualized by anti-CIMPR antibodies.

[0473] The relative cellular uptake of dephosphorylated sipa-glucosidase alfa by Pompe disease patient fibroblasts was assessed compared to mock-treated sipa-glucosidase alfa and mock-treated alglucosidase alfa. A 20 nM concentration of rhGAA was used to simulate the interstitial concentration of sipa-glucosidase alfa that crosses into tissues, as Figure 77 After 18 hours of enzyme uptake incubation, cells were washed for 2 hours, harvested, lysed, and analyzed for GAA activity and Western blot. Internalized GAA was measured by 4MU-α-glucoside hydrolysis and plotted relative to total cellular protein. Figure 77 B. Figure 77 C, cell lysates (3 μg) were analyzed for CIMPR binding by Western blot analysis.

[0474] Dephosphorylated sipa-glucosidase α was shown to be inefficiently internalized into cells. Figure 77 As shown in Figures B and 77C, mock-treated α-glucosidase α was internalized into Pompe disease fibroblasts to a greater extent than conventional α-glucosidase α via 4MU-α-glucosidase hydrolysis and CIMPR binding. However, cellular uptake of α-glucosidase α was abolished by PAP-treated α-glucosidase α. These results demonstrate the important role of M6P and suggest that α-glucosidase α has superior cellular uptake compared with α-glucosidase α due to the structure and number of mono-M6P and di-M6P N-glycans. Example 17: Clinical Development Plan Summary

[0475] A total of three clinical trials (Studies ATB200-02, ATB200-03, and ATB200-07), including both ERT-experienced and ERT-naive adult patients with Pompe disease (≥18 years) (Studies ATB200-02 and ATB200-03) or only ERT-experienced adult patients with Pompe disease (Study ATB200-07), are included in this Summary of Clinical Efficacy (SCE), as detailed in Table 22. One hundred fifty-two adult subjects were incorporated into the clinical development program for cepaglucosidase alfa / miglustat, including 29 adult subjects exposed to cepaglucosidase alfa / miglustat in the ongoing Phase 1 / 2 study ATB200-02 and 123 adult subjects exposed to cepaglucosidase alfa / miglustat (85 subjects) or alglucosidase alfa / placebo (38 subjects) in the completed Phase 3 study ATB200-03. Of the 123 subjects who participated in Study ATB200-03 (85 receiving alglucosidase alfa / miglustat and 38 receiving alglucosidase alfa / placebo), 117 completed the study and then participated in Study ATB200-07. Two additional subjects (1107-1681 and 2010-1352) did not complete Study ATB200-03 but participated in Study ATB200-07, bringing the total number of subjects who participated in Study ATB200-07 (OLE-ES population) to 119 (91 ERT-experienced and 28 ERT-naive). Table 22: Summary of clinical studies in adult subjects Table 22: Summary of Clinical Studies in Adult Subjects (Continued) Abbreviations: 6MWD = 6-minute walk distance; 6MWT = 6-minute walk test; GSGC = gait, stair, Gowers maneuver, and chair test; IV = intravenous (intravenous); LOPD = late-onset Pompe disease; MMT = manual muscle testing; N = number of subjects; PD = pharmacodynamics; PK = pharmacokinetics; PRO = patient-reported outcomes; PROMIS = Patient-Reported Outcomes Measurement Information System; QOW = every other week a Two subjects discontinued from Study ATB200-03 and participated in Study ATB200-07. A total of 119 subjects were enrolled in Study ATB200-07, and 118 subjects were dosed. NOTE: In Studies ATB200-03 and ATB200-07, the miglustat dose was adjusted to 195 mg in subjects <50 kg. Phase 1 / 2 study ATB200-02

[0476] As described above with respect to Examples 8 and 11, Study ATB200-02 is an ongoing, open-label, fixed-sequence, single- and multiple-ascending-dose, first-in-human study evaluating the safety, tolerability, PK, PD, efficacy, and immunogenicity of IV sipa-glucosidase alfa alone and co-administered with oral miglustat in adult subjects with Pompe disease. The study design includes 4 phases and 4 cohorts, with Phases 1 and 2 for Cohort 1 only and Phases 3 and 4 for all 4 cohorts ( Figure 79 The efficacy data for sipaglucosidase alfa / miglustat summarized in the SCE were from phases 3 and 4.

[0477] The four cohorts in the study are as follows: Cohort 1: Ambulatory subjects who had undergone ERT (2 to 6 years) (11 subjects) Cohort 2: Non-ambulatory subjects who had undergone ERT (≥2 years) (6 subjects) Cohort 3: ERT-naive ambulatory subjects (6 subjects) Cohort 4: Ambulatory subjects who had undergone ERT (≥7 years) (6 subjects)

[0478] According to the treatment assignment in Table 22, subjects were administered either sipa-glucosidase alfa as a single agent or sipa-glucosidase alfa co-administered with miglustat. Table 22: Treatment Allocation for Periods 1, 2, 3, and 4 – Study ATB200-02 Abbreviations: AT2221 = miglustat; ATB200 = sipa-glucosidase alfa; NA = not applicable; n = number of subjects; SSC = safety steering committee NOTE: At least 1 of the 2 sentinel subjects will complete Period 5, Phase 2 dosing and safety data will be reviewed by the SSC before dosing of any new subjects in Cohorts 2 and 3. The first 2 subjects in Cohorts 2 and 3 will also serve as sentinel subjects in their respective cohorts.

[0479] The primary objectives of the Phase 1 / 2 study ATB200-02 were to evaluate the safety, tolerability, PK, PD, and efficacy of sipaglucosidase alfa alone and co-administered with oral miglustat.

[0480] Dose selection for the pivotal Phase 3 study, ATB200-03, was based on PK, PD / biomarker, efficacy, and safety data from Study ATB200-02. The PK of sipa-glucosidase alfa was well characterized in Phase 1 and Phase 2 of Study ATB200-02, which demonstrated an increase in overall exposure to sipa-glucosidase alfa, with an additional increase in exposure with the addition of miglustat. This increase in plasma exposure of sipa-glucosidase alfa with miglustat is consistent with data from nonclinical studies, in which increased exposure was associated with an incremental reduction in glycogen and an increase in muscle strength.

[0481] Study ATB200-02 provided supportive efficacy data from Phases 3 and 4, demonstrating that the increased exposure observed in Phases 1 and 2 resulted in clinically meaningful improvements across a broad range of endpoints with treatment with sipagliflozin / miglustat in Phases 3 and 4. Where data were available, efficacy improvements were observed through 48 months, supporting the long-term efficacy of sipagliflozin / miglustat. Phase 3 Study ATB200-03

[0482] The pivotal Phase 3 study, ATB200-03, has been completed. The ATB200-03 study was a double-blind, randomized, multicenter, global, controlled trial designed to evaluate the efficacy and safety of sipaglucosidase alfa / miglucosidase compared to alglucosidase alfa / placebo (an approved therapy) in adult subjects with LOPD who had previously received alglucosidase alfa (i.e., ERT-experienced) or had never received ERT (i.e., ERT-naive). Myozyme (alglucosidase alfa) was sourced directly from Sanofi Genzyme through a third-party supplier. Amicus conducted a scientific review of Sanofi Genzyme's documentation. The results demonstrated that the U.S. product (Lumizyme) and the non-U.S. product (Myozyme) were comparable. Subjects were randomized in a 2:1 ratio to receive sipaglucosidase alfa / miglucosidase or alglucosidase alfa / placebo every other week for 52 weeks ( Figure 80 ).

[0483] The primary endpoint of Study ATB200-03 was to evaluate the efficacy of co-administered sipa-glucosidase alfa / miglustat compared to alglucosidase alfa / placebo on motor function as measured by the 6MWT. The first key secondary endpoint was to evaluate the efficacy of co-administered sipa-glucosidase alfa / miglustat compared to alglucosidase alfa / placebo on pulmonary function as measured by percent predicted sitting FVC. Other key secondary endpoints included MMT lower extremity score, 6MWD at Week 26, - Physical Function Short Form 20a total score, PROMIS-Fatigue Short Form 8a total score, and GSGC total score. Other secondary endpoints included additional assessments of motor function, lung function, muscle strength, and PROs. Biomarkers of muscle damage (CK) and disease substrates (Hex4) were also assessed. Phase 3 Study ATB200-07

[0484] Study ATB200-07 is an ongoing open-label extension (OLE) study designed to evaluate the long-term safety and efficacy of alglucosidase alfa / miglustat in adult subjects with LOPD who have completed Study ATB200-03. Subjects who received alglucosidase alfa / placebo in Study ATB200-03 were switched to alglucosidase alfa / miglustat in Study ATB200-07. The study dose and schedule remained the same as in Study ATB200-03. Summary of findings

[0485] This section summarizes the results from the ongoing Phase 1 / 2 open-label study ATB200-02, the completed pivotal Phase 3 study ATB200-03, and the Phase 3 long-term extension study ATB200-07. Data from these studies are presented individually and as a pooled data set. Case 18: Study of ATB200-02

[0486] The ATB200-02 study is a Phase 1 / 2 study, and the efficacy of sipa-glucosidase alfa / miglustat is primarily based on results from Phases 3 and 4 of the study.

[0487] As of December 2021, 29 subjects received sipaglucosidase alfa / miglustat, 3 subjects discontinued prematurely, and 26 subjects are currently in Stage 4. Three (10.3%) subjects discontinued the study during Stage 3. The mean (standard deviation [SD]) duration of exposure was 48.4 (18.04) months.

[0488] In study ATB200-02, all ERT-experienced and ERT-naive subjects showed a significant effect of Pompe disease based on baseline characteristics. For cohorts 1 and 4 (16 ERT-experienced ambulatory subjects), the mean (SD) predicted 6MWD% at baseline was 60.2% (16.20%). For cohort 3 (6 ERT-naive ambulatory subjects), the mean (SD) predicted 6MWD% at baseline was 67.8% (12.61%).

[0489] Subjects who had undergone ERT prior to study entry had ERT treatment duration >2 years, with an overall median dose of 20 mg / kg. The mean (SD) duration of ERT treatment ranged from 5.1 (1.27) years in Cohort 1 to 10.6 (2.06) years in Cohort 4.

[0490] The results from Study ATB200-02 referenced below are based on the data cutoff date of December 13, 2021, at which point all subjects, except those in Cohort 4, had completed at least 48 months of follow-up (or had discontinued). Data supporting long-term efficacy up to 48 months are presented here and are included in the pooled analysis below. Data from Study ATB200-02, which evaluated key clinical areas of Pompe disease such as motor function, lung function, and muscle strength, support the results obtained in Study ATB200-03. Motor function

[0491] In Study ATB200-02, motor function was assessed in all ambulatory subjects (i.e., Cohorts 1, 3, and 4) (Table 23, Figures 46A-46D After 48 months of treatment with co-administration of 20 mg / kg IV infusion of sipa-glucosidase alfa and 260 mg of miglustat in Phases 3 and 4, clinically meaningful improvements in motor function, as measured by the 6MWT, were observed in ERT-experienced subjects (Cohorts 1 and 4), ERT-naive subjects (Cohort 3), and overall (Cohorts 1, 3, and 4) (Table 23). Initial improvements were observed within 1-2 years and remained above baseline through Month 48.

[0492] Both the ERT-experienced and ERT-naive cohorts showed a mean improvement in predicted 6MWD% from baseline that was sustained through 48 months ( Figure 46C and 46D ; Table 26B ), where 88.9% of ERT-experienced patients and 100% of ERT-naive patients had an improvement in mean 6MWD % predicted relative to baseline at Month 48. After 12, 24, 36, and 48 months of follow-up, 13 of 16, 9 of 13, 6 of 12, and 6 of 9 ERT-experienced patients, respectively, had a numerical improvement in mean 6MWD in meters relative to baseline. After 12, 24, 36, and 48 months of follow-up, 6 of 6, 6 of 6, 4 of 5, and 4 of 4 ERT-naive patients, respectively, had a numerical improvement in mean 6MWD in meters relative to baseline. Table 23: Summary of 6-MWD (Meters) from Baseline to Month 48—Phase 3 and 4 (Efficacy Population)—Study ATB200-02 Table 23: Summary of 6-MWD (Meters) from Baseline to Month 48—Phase 3 and Phase 4 (Efficacy Population)—Study ATB200-02 (Continued) Abbreviations: 6MWD = 6-minute walk distance; CI = confidence interval; max = maximum value; min = minimum value; N = total number of subjects enrolled; n = subset of subjects; Q1 = first quartile; Q3 = third quartile; SD = standard deviation NOTE: The 6-minute walk distance (6MWD) is the distance walked in meters during the 6-minute walk test. It is performed only by subjects who can walk.

[0493] Co-administration of 20 mg / kg IV infusion of sipa-glucosidase alfa and 260 mg of miglustat (i.e., in periods 2, 5, 3, and 4) also resulted in improvements in other motor function measures (including 10MWT, GSGC, and TUG) in ERT-experienced and ERT-naive ambulatory subjects. Initial improvements were observed within 1 to 2 years and remained above baseline through month 48. lung function

[0494] After 48 months of treatment in Periods 3 and 4, co-administration of 20 mg / kg IV infusion of sipa-glucosidase alfa and 260 mg of miglustat (i.e., in Periods 2, 5, 3, and 4) resulted in stable or improved % predicted FVC in ERT-experienced subjects (Cohorts 1 and 4), ERT-naive subjects (Cohort 3), and overall (Cohorts 1, 3, and 4) ( Table 24 , Table 24 ). Figures 47A-47B These values ​​remained at or above baseline through month 48. For Cohort 2 (nonambulatory subjects), % predicted sitting FVC remained stable in 2 subjects with available data for up to 36 months.

[0495] In the ERT-experienced cohort, mean CFBL expressed as % predicted sitting FVC was generally stable (CFBL ± 3% score) at up to 48 months of follow-up ( Figure 47A , Table 26B), of which 66.7% of ERT-experienced patients and 75% of ERT-naive patients improved at month 48 (CFBL>3% score). After 12, 24, 36, and 48 months of follow-up, predicted sitting FVC% improved (>3% score) or remained stable (±3% score) relative to baseline in 9 / 16, 11 / 13, 8 / 10, and 4 / 6 ERT-experienced patients, respectively. This was further supported by the maximum expiratory pressure (MEP) results, which also improved numerically relative to baseline in ERT-experienced patients at 12, 24, 36, and 48 months of follow-up (Table 26B).

[0496] In the ERT-naive cohort, mean CFBL expressed as % predicted sitting FVC improved at month 3 and then remained generally stable during follow-up up to 48 months ( Figure 47B , Table 26B), except that only one patient had a large decrease in % predicted sitting FVC at month 21, which recovered to the previous level at month 24. At 48 months of follow-up, the mean % predicted sitting FVC improved more significantly in the ERT-naive cohort compared with the ERT-experienced cohort ( Figure 47B , Table 26B). After 12, 24, 36, and 48 months of follow-up, % predicted sitting FVC improved (>3% points) or remained stable (±3% points) relative to baseline in 5 / 6, 6 / 6, 5 / 5, and 4 / 4 ERT-naive patients, respectively. The % predicted sitting FVC data were supported by similar results for other pulmonary measurements, including % predicted maximal inspiratory pressure (MIP) and MEP. MIP improved numerically in ERT-naive patients at 12, 24, 36, and 48 months of follow-up (Table 26B).

[0497] Long-term efficacy data for cohort 2, who were nonambulatory and had undergone ERT, were limited. Percent predicted sitting FVC data were available for two nonambulatory ERT-experienced patients after 36 months and for one patient at 48 months of follow-up. After 36 months of follow-up, one patient had improved and the other had deteriorated relative to baseline. Patients with available data after 48 months of follo...

Claims

1. A method of improving and / or stabilizing one or more of muscle strength, motor function, lung function, or pharmacodynamic measures relative to baseline for greater than 24 months in a subject with Pompe disease, the method comprising administering to the subject a population of recombinant human acid alpha-glucosidase (rhGAA) molecules simultaneously or sequentially with an enzyme stabilizer, Each rhGAA molecule comprises seven potential N-glycosylation sites, and the rhGAA molecule comprises at least 0.5 mol bis-mannose-6-phosphate (bis-M6P) / mol rhGAA at the first potential N-glycosylation site.

2. The method of claim 1, wherein the measure is improved and / or stabilized for greater than 36 months.

3. The method of claim 1 or 2, wherein the measure is improved and / or stabilized for at least 48 months.

4. The method of any one of claims 1-3, wherein the amount of di-M6P at the first potential glycosylation site is determined by LC MS / MS.

5. The method of any one of claims 1-4, wherein the measure improves relative to a baseline for a first time period and is stable for a second time period.

6. The method of any one of claims 1-5, wherein the subject is an enzyme replacement therapy (ERT)-experienced subject.

7. The method of any one of claims 1-5, wherein the subject is an enzyme replacement therapy (ERT) naive subject.

8. The method of any one of claims 1-7, wherein muscle strength is measured by manual muscle testing (MMT).

9. The method of any one of claims 1-8, wherein after greater than 24 months of treatment, the muscle strength score improves by at least 1.5, 2.0, or 2.5 relative to baseline.

10. The method of any one of claims 1-9, wherein motor function is measured by a 6-minute walk test.

11. The method of any one of claims 1-10, wherein the subject has undergone ERT and motor function improves by at least 15, 20, or 25 meters relative to baseline after greater than 24 months of treatment.

12. The method of any one of claims 1-11, wherein the subject is ERT naive and motor function improves by at least 30, 40, or 50 meters relative to baseline after greater than 24 months of treatment.

13. The method of any one of claims 1-12, wherein lung function is measured by a seated forced vital capacity (FVC) test.

14. The method of any one of claims 1-13, wherein after greater than 24 months of treatment, the subject's percent predicted FVC is stable compared to baseline.

15. The method of any one of claims 1-14, wherein the ERT-experienced subject has been previously treated with alglucosidase alfa.

16. The method of any one of claims 1-15, wherein the ERT-experienced subject has been previously treated with alglucosidase alfa for about 2 to about 6 years.

17. The method of any one of claims 1-15, wherein the ERT-experienced subject has been previously treated with alglucosidase alfa for at least about 7 years.

18. The method of any one of claims 1-17, wherein the subject is unable to walk.

19. The method of any one of claims 1-17, wherein the subject is able to walk.

20. The method of any one of claims 1-19, wherein the pharmacodynamic measure is creatine kinase (CK) or urinary hexotetraose (Hex4).

21. The method of any one of claims 1-20, wherein the population of rhGAA molecules is administered at a dose of 5 mg / kg to 40 mg / kg.

22. The method of any one of claims 1-21, wherein the population of rhGAA molecules is administered at a dose of 20 mg / kg.

23. The method of any one of claims 1-22, wherein the population of rhGAA molecules is administered once every two weeks.

24. The method of any one of claims 1-23, wherein the population of rhGAA molecules is administered intravenously.

25. The method of any one of claims 1-24, wherein the enzyme stabilizer is miglustat or a pharmaceutically acceptable salt thereof.

26. The method of claim 25, wherein the miglustat or a pharmaceutically acceptable salt thereof is administered orally.

27. The method of claim 25 or 26, wherein the miglustat or a pharmaceutically acceptable salt thereof is administered at a dose of 195 mg or 260 mg.

28. The method of any one of claims 25-27, wherein the miglustat or a pharmaceutically acceptable salt thereof is administered prior to administering the population of rhGAA molecules.

29. The method of any one of claims 25-28, wherein the miglustat or a pharmaceutically acceptable salt thereof is administered one hour prior to administration of the population of rhGAA molecules.

30. The method of any one of claims 25-29, wherein the subject fasts for at least two hours prior to administration of miglustat or a pharmaceutically acceptable salt thereof and fasts for at least two hours after administration of miglustat or a pharmaceutically acceptable salt thereof.

31. The method of any one of claims 1-30, wherein the rhGAA molecule comprises an amino acid sequence at least 90% identical to SEQ ID NO:

4.

32. The method of any one of claims 1-31, wherein the rhGAA molecule comprises an amino acid sequence at least 98% identical to SEQ ID NO:

6.

33. The method of any one of claims 1-32, wherein the rhGAA molecule comprises the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:

6.

34. The method of any one of claims 1-33, wherein 40%-60% of the N-glycans on the rhGAA molecule are complex N-glycans.

35. The method of any one of claims 1-34, wherein at least 30% of the rhGAA molecules comprise one or more N-glycan units with one mannose-6-phosphate residue (mono-M6P) or di-M6P.

36. The method of any one of claims 1-35, wherein the rhGAA molecules comprise an average of 0.5 mol to 7.0 mol mono-M6P or di-M6P / mol rhGAA.

37. The method of any one of claims 1-36, wherein the rhGAA molecules comprise an average of 2.0 to 8.0 mol sialic acid / mol rhGAA.

38. The method of any one of claims 1-37, wherein the rhGAA molecules comprise an average of at least 2.5 mol M6P / mol rhGAA and at least 4 mol sialic acid / mol rhGAA.

39. The method of any one of claims 1 to 38, wherein: Per mol of rhGAA, the rhGAA molecule comprises: (a) an average of 0.4 to 0.6 mol mono-M6P at the second potential N-glycosylation site; (b) an average of 0.4 to 0.6 mol bis-M6P at the fourth potential N-glycosylation site; or (c) Average 0.3 to 0.4 mol mono-M6P at the fourth potential N-glycosylation site.

40. The method of claim 39, wherein: The rhGAA molecule further comprises 4 mol to 7.3 mol of sialic acid per mol of rhGAA, Wherein, per mol of rhGAA, the rhGAA molecule comprises: (a) an average of 0.9 to 1.2 mol of sialic acid at the third potential N-glycosylation site; (b) an average of 0.8 to 0.9 mol sialic acid at the fifth potential N-glycosylation site; or (c) an average of 1.5 to 4.2 mol of sialic acid at the sixth potential N-glycosylation site.

41. The method of any one of claims 1-40, wherein the population of rhGAA molecules is formulated in a pharmaceutical composition further comprising at least one pharmaceutically acceptable buffer, excipient, or carrier.

42. The method of claim 41, 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; wherein the pharmaceutical composition has a pH of 5.0 to 7.

0.

43. The method of claim 41 or 42, wherein the pharmaceutical composition has a pH of 5.0 to 6.

0.

44. The method of any one of claims 41-43, wherein the pharmaceutical composition further comprises water, an acidifying agent, an alkalizing agent, or a combination thereof.

45. The method of any one of claims 41 to 44, wherein: In the pharmaceutical composition, the rhGAA molecule population is present at a concentration of 5-50 mg / mL, the at least one buffer is a sodium citrate buffer present at a concentration of 10-100 mM, the at least one excipient is mannitol present at a concentration of 10-50 mg / mL and polysorbate 80 present at a concentration of 0.1-1 mg / mL, and the pharmaceutical composition further comprises water and optionally an acidifier and / or an alkalizer; wherein the pharmaceutical composition has a pH of 6.

0.

46. ​​The method of any one of claims 41 to 45, wherein: In the pharmaceutical composition, the rhGAA molecular population is present at a concentration of 15 mg / mL, the sodium citrate buffer is present at a concentration of 25 mM, the mannitol is present at a concentration of 20 mg / mL, and the polysorbate 80 is present at a concentration of 0.5 mg / mL.

47. The method of any one of claims 1-46, wherein the rhGAA is produced by Chinese Hamster Ovary (CHO) cells.

48. The method of any one of claims 1-47, wherein at least 6% of the total N-glycan units on the rhGAA molecule are glycans with a single-M6P residue.

49. The method of any one of claims 1-48, wherein at least 3% of the total N-glycan units on the rhGAA molecule are glycans with di-M6P residues.

50. The method of any one of claims 1-49, wherein up to 55% of the total N-glycans on the rhGAA molecule are sialylated complex glycans.

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