Recombinant human acidic alpha-glucosidase

Abstract

The invention provides a recombinant acidic alpha-glucosidase and a pharmaceutical composition containing the recombinant acidic alpha-glucosidase. Wherein the recombinant acidic [alpha]-glucosidase is expressed in Chinese hamster ovary (CHO) cells and comprises an increased amount of N-glycan units bearing one or two mannose-6-phosphate residues when compared to the amount of N-glycan units bearing one or two mannose-6-phosphate residues of aglucosidase [alpha]. Also provided herein are methods of generating, purifying, and formulating the recombinant acidic alpha-glucosidase or pharmaceutical composition for administration to a subject and methods of using the recombinant acidic alpha-glucosidase or pharmaceutical composition to treat a disease or condition, such as Poincare Disease.

Description

Technical Field

[0001] This application claims U.S. Provisional Application Serial No. 62 / 506,561 filed on May 15, 2017, U.S. Provisional Application Serial No. 62 / 506,569 filed on May 15, 2017, U.S. Provisional Application Serial No. 62 / 506,574 filed on May 15, 2017, U.S. Provisional Application Serial No. 62 / 564,083 filed on September 27, 2017, U.S. Provisional Application Serial No. 62 / 564,083 filed on October 3, 2017 No. 62 / 567,334, U.S. Provisional Application Serial No. 62 / 618,021 filed on January 16, 2018, U.S. Provisional Application Serial No. 62 / 624,638 filed on January 31, 2018, and U.S. Provisional Application Serial No. 62 / 660,758 filed on April 20, 2018, each of which claims priority and is incorporated herein by reference in its entirety.

[0002] The present invention relates to the fields of medicine, genetics and recombinant glycoprotein biochemistry, and in particular, to recombinant human α-glucosidase (rhGAA) compositions having a high total content of N-glycans with mannose-6-phosphate, thereby effectively targeting CIMPR on cells and subsequently delivering rhGAA to lysosomes, where the rhGAA can break down abnormally high levels of accumulated glycogen. The rhGAA of the present invention exhibits superior targeting to muscle cells and subsequent delivery to lysosomes compared to traditional rhGAA products and exhibits other pharmacokinetic properties, making it particularly effective for enzyme replacement therapy in subjects with Pompe disease.

[0003] The present invention also provides methods for treating Pompe disease, comprising administering to an individual a combination of rhGAA and a pharmacological chaperone. For example, in some embodiments, the present invention provides methods for treating Pompe disease, comprising administering to an individual a combination of rhGAA and miglustat. The rhGAA of the present invention exhibits surprising efficacy in treating and reversing the disease process in subjects suffering from Pompe disease. Background Art

[0004] Pompe disease is a hereditary lysosomal storage disease resulting from a lack of acid alpha-glucosidase (GAA) activity. 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 causes excess glycogen to accumulate in lysosomes, which are intracellular organelles that contain enzymes that generally break down glycogen and other cellular debris or waste products. Glycogen accumulation in certain tissues, especially upper muscles, 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, especially in skeletal muscle cells and, in the infantile onset form of the disease, in cardiac muscle cells. The accumulation of glycogenolysis damages muscle and nerve cells, as well as those cells in other affected tissues.

[0005] Traditionally, Pompe disease is clinically considered as an early infantile form or a late-onset form, depending on the age of onset. The age of onset tends to parallel the severity of the genetic mutation that causes Pompe disease. The most serious genetic mutation causes a complete loss of GAA activity and manifests as an early-onset disease during infancy. Genetic mutations that reduce GAA activity but do not completely eliminate it are associated with a form of Pompe disease with a delayed onset and progression. Infantile onset Pompe disease does not manifest after birth and is characterized by muscle weakness, respiratory insufficiency, and heart failure. When not treated, it is usually fatal within two years. Pompe disease in adolescents and adults manifests later in life and usually progresses more slowly than infantile onset diseases. Although this form of disease does not usually affect the heart, it can also cause death, because of the weakening of skeletal muscles and muscles involved in breathing.

[0006] Current non-painful treatments for Pompe disease involve enzyme replacement therapy (ERT) using recombinant human GAA (rhGAA), which is known as Or alglucosidase alfa. This traditional enzyme replacement therapy seeks to treat Pompe disease by administering rhGAA to replace the missing GAA in the lysosomes, thereby restoring the cell's ability to break down lysosomal glycogen. and It is a traditional form of rhGAA produced or sold by Genzyme as a biological agent and approved by the U.S. Food and Drug Administration and described with reference to the Physician's Desk Reference (2014) (which is hereby incorporated by reference). Alpha-glucosidase alfa is identified by the chemical name [199-arginine, 223-histidine] precursor l-α-glucosidase (human); the molecular formula is C 4758 H 7262 N1274 O 1369 S 35 ; CAS number 420794-05-0. These products are administered to subjects with Pompe disease, also known as glycogen storage disease type II (GSD-II) or acid maltase deficiency.

[0007] The cellular uptake of rhGAA molecules is facilitated by a specific carbohydrate, mannose-6-phosphate (M6P), which binds to cation-independent mannose-6-phosphate receptors (CIMPRs) present on target cells such as muscle cells. After binding, rhGAA molecules are taken up by target cells and subsequently transported to lysosomes within the cells. However, most conventional rhGAA products lack a high total content of mono- and di-M6P-bearing N-glycans (i.e., N-glycans with one M6P residue or N-glycans with two M6P residues, respectively), thereby limiting their cellular uptake and lysosomal delivery through CIMPRs, thus making conventional enzyme replacement therapy inefficient. For example, while conventional rhGAA products at doses of 20 mg / kg or higher do improve some aspects of Pompe disease, they are particularly inadequate to (i) treat underlying cellular dysfunction, (ii) restore muscle structure, or (iii) reduce accumulated glycogen in many target tissues such as skeletal muscle to reverse disease progression. In addition, higher doses can impose additional burdens on subjects and medical professionals treating subjects, such as lengthening the infusion time required for intravenous administration of rhGAA. There remains a need for further improved enzyme replacement therapies for the treatment of Pompe disease, such as rhGAA with improved tissue uptake, improved enzyme activity, improved stability, or reduced immunogenicity.

[0008] Glycosylation of GAA or rhGAA can be enzymatically modified in vitro by phosphotransferases and unmasking enzymes as described in U.S. Pat. No. 6,534,300 to Canfield et al. to produce M6P groups. Enzymatic glycosylation cannot be adequately controlled and produces rhGAA with undesirable immunological and pharmacological properties. Enzymatically modified rhGAA may contain only high mannose N-glycans, all of which may potentially be enzymatically phosphorylated in vitro by phosphotransferases / unmasking enzymes and may contain an average of 5-6 M6P groups per GAA. The glycosylation pattern produced by in vitro enzymatic treatment of GAA is problematic because additional terminal mannose residues, especially non-phosphorylated terminal mannose residues, negatively affect the pharmacokinetics of the modified rhGAA. When such enzymatically modified products are administered in vivo, these mannose groups increase the non-bulk 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 cardiac or skeletal muscle myocytes. For example, terminal non-phosphorylated mannose residues are known ligands for mannose receptors in the liver and spleen, which results in rapid clearance of enzymatically modified rhGAA and reduced targeting of rhGAA to target tissues. In addition, the glycosylation pattern of enzymatically modified GAA having high mannose N-glycans with 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.

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

[0010] The present invention is directed to a method of treating a disease or condition, such as Pompe disease, in a subject comprising administering a population of recombinant human acid alpha-glucosidase (rhGAA) molecules.

[0011] The rhGAA molecules described herein can be expressed in Chinese hamster ovary (CHO) cells and include seven potential N-glycosylation sites. In some embodiments, the N-glycosylation distribution of the rhGAA molecule population described herein is determined using liquid chromatography-tandem mass spectrometry (LC-MS / MS). In some embodiments, the rhGAA molecules contain an average of 3-4 mannose-6-phosphate (M6P) residues. In some embodiments, the rhGAA molecules contain an average of about 0.5 moles of double phosphorylated N-glycan groups (double M6P) per mole of rhGAA at the first potential N-glycosylation site. In some embodiments, rhGAA comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:1 or SEQ ID NO:5. In some embodiments, rhGAA comprises an amino acid sequence that is consistent with SEQ ID NO:1 or SEQ ID NO:5. In some embodiments, at least 30% of the rhGAA molecules contain one or more N-glycan units with one or two M6P residues. In some embodiments, the rhGAA molecule comprises an average of about 0.5 to about 7.0 moles of N-glycan units with one or two M6P residues per mole of rhGAA. In some embodiments, the rhGAA molecule comprises an average of at least 2.5 moles of M6P residues per mole of rhGAA and at least 4 moles of sialic acid residues per mole of rhGAA. In some embodiments, the rhGAA molecule comprising an average of 3-4 M6P residues per molecule and an average of about 0.5 moles of di-M6P per mole of rhGAA at the first potential N-glycosylation site further comprises an average of about 0.4 to about 0.6 moles of monophosphorylated N-glycans (mono-M6P) per mole of rhGAA at the second potential N-glycosylation site, about 0.4 to about 0.6 moles of di-M6P per mole of rhGAA at the fourth potential N-glycosylation site, and about 0.3 to about 0.4 moles of mono-M6P per mole of rhGAA at the fourth potential N-glycosylation site. In some embodiments, the rhGAA molecules further comprise an average of about 4 to about 5.4 moles of sialic acid residues per mole of rhGAA, including about 0.9 to about 1.2 moles of sialic acid per mole of rhGAA at the third potential N-glycosylation site, about 0.8 to about 0.9 moles of sialic acid per mole of rhGAA at the fifth potential N-glycosylation site, and about 1.5 to about 4.2 moles of sialic acid per mole of rhGAA at the sixth potential N-glycosylation site. In some embodiments, the population of rhGAA molecules is formulated in a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprising the population of rhGAA molecules further comprises at least one buffer selected from the group consisting of citrate, phosphate, and combinations thereof, and at least one excipient selected from the group consisting of mannitol, polysorbate 80, and combinations thereof.In some embodiments, the pH of the pharmaceutical composition is about 5.0 to about 7.0, about 5.0 to about 6.0, or about 6.0. In some embodiments, the pharmaceutical composition further comprises water, an acidulant, an alkalizer, or a combination of the foregoing. In some embodiments, the pharmaceutical composition has a pH of 6.0 and comprises a population of about 5-50 mg / mL of rhGAA molecules, about 10-100 mM sodium citrate buffer, about 10-50 mg / mL mannitol, about 0.1-1 mg / mL polysorbate 80, and water, and optionally comprises an acidulant and / or an alkalizer. In some embodiments, the pharmaceutical composition has a pH of 6.0 and comprises a population of about 15 mg / mL of rhGAA molecules, about 25 mM sodium citrate buffer, about 20 mg / mL mannitol, about 0.5 mg / mL polysorbate 80, and water, and optionally comprises an acidulant and / or an alkalizer.

[0012] In some embodiments, the population of rhGAA molecules is administered at a dose of about 1 mg / kg to about 100 mg / kg. In some embodiments, the population of rhGAA molecules is administered at a dose of about 20 mg / kg. In some embodiments, the population of rhGAA molecules is administered bimonthly, monthly, biweekly, weekly, twice weekly, or daily, for example, biweekly. In some embodiments, the population of rhGAA molecules is administered intravenously.

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

[0014] The present examples demonstrate the efficacy of rhGAA described herein to treat and reverse disease progression in subjects with Pompe disease.

[0015] In some embodiments, the population of rhGAA molecules is administered at a dose capable of reversing cellular damage in the subject's muscle fibers. For example, after treatment, the subject's muscles or muscle fibers exhibit normalized lysosomal size and / or relief of autophagic accumulation. In some embodiments, after treatment, less than 65% of the muscle fibers analyzed in the subject have autophagic accumulation. In some embodiments, after treatment, at least 36% of the muscle fibers analyzed in the subject have a normal or near-normal appearance. In some embodiments, the subject who experiences a reversal of disease progression after treatment is an ERT conversion patient, such as an ERT conversion patient who has previously been treated with alglucosidase alfa for at least two years.

[0016] In some embodiments, the population of rhGAA molecules is administered at a dose that is capable of reducing glycogen content in the muscle of a subject faster than the same dose of alglucosidase alpha. rhGAA can reduce glycogen content at least about 1.25, 1.5, 1.75, 2.0, or 3.0 times faster than the same dose of alglucosidase alpha. In some embodiments, the population of rhGAA molecules is administered at a dose that is further capable of reducing glycogen content in the muscle of a subject more effectively than alglucosidase alpha administered at the same dose when assessed after one, two, three, four, five, or six administrations. In some embodiments, the population of rhGAA molecules is at least about 10%, 20%, 30%, 50%, 75%, or 90% more effective than alglucosidase alpha administered at the same dose to reduce glycogen content. In some embodiments, after treatment, the subject exhibits a lower level of the glycogen accumulation biomarker urinary hexotetraose (Hex4). In at least one embodiment, the Hex4 level in the subject is reduced by at least 30% relative to baseline six months after treatment. For example, an active or inactive subject previously treated with enzyme replacement therapy (ERT switch patient) may exhibit at least a 35% reduction in Hex4 level six months after treatment relative to baseline. In another case, an active subject who did not previously receive enzyme replacement therapy (ERT naive patient) may exhibit at least a 45% reduction in Hex4 level six months after treatment relative to baseline.

[0017] In some embodiments, the population of rhGAA molecules is administered at a dose that can improve the motor function of the subject. The improvement in motor function can be measured by a motor function test, such as a six-minute walk test (six-minute walk test; 6MWT), a timed stand and walk test, a four-stair climbing test, a ten-meter walk test, a Gowers test, a gait-stair-gower-chair (gait-stair-gower-chair; GSGC) test, or a combination of the foregoing. In some embodiments, six months after treatment, the subject (when compared to baseline) shows an increase in 6MWT distance of at least 20 meters, a decrease in the timed stand and walk test time of at least 1 second, a decrease in the four-stair climbing test time of at least 0.6 seconds, a decrease in the ten-meter walk test time of at least 0.7 seconds, a decrease in the Gowers test time of at least 1 second, and / or a decrease in the GCSC score of at least 1. For example, an ERT-switch patient who is mobile may demonstrate an increase in 6MWT of at least 20 meters, a decrease in timed up and go test time of at least 1.5 seconds, a decrease in four-stair climb test time of at least 0.6 seconds, and / or a decrease in Gowers' test time of at least 1 second six months after treatment (compared to baseline). In another case, an ERT-naive patient who is mobile may demonstrate an increase in 6MWT distance of at least 40 meters, a decrease in timed up and go test time of at least 1 second, a decrease in four-stair climb test time of at least 0.6 seconds, a decrease in ten-meter walk test time of at least 0.7 seconds, and / or a decrease in GSGC score of at least 1 six months after treatment (compared to baseline). In some embodiments, an ERT-switch patient exhibits improvement in the motor function test results of a patient following ERT prior to treatment with alglucosidase alfa in at least one motor function test.

[0018] In some embodiments, the population of rhGAA molecules is administered at a dose that improves upper body strength in a subject. In some embodiments, the population of rhGAA molecules is administered to an active subject and further improves lower body strength and / or total body strength in the subject.

[0019] In some embodiments, the improvement in upper body strength is measured using a manual muscle strength score. The subject's manual muscle strength score may improve by at least 1 (for ERT-switching patients with mobility) or at least 5.5 (for ERT-switching patients without mobility) relative to baseline six months after treatment. In some embodiments, ERT-switching patients show improvement in upper body strength after treatment relative to the upper body strength of patients following ERT prior to alglucosidase alfa.

[0020] In some embodiments, the population of rhGAA molecules is administered at a dose that improves upper extremity strength as measured by quantitative muscle testing or manual muscle testing of shoulder adduction, shoulder abduction, elbow flexion, and / or elbow extension. For example, six months after treatment, an amobile ERT switch patient may demonstrate at least an 8-pound-force improvement in shoulder adduction, at least a 1-pound-force improvement in shoulder abduction, at least a 2-pound-force improvement in elbow flexion, and / or at least a 5-pound-force improvement in elbow extension relative to baseline.

[0021] In some embodiments, the population of rhGAA molecules is administered at a dose capable of improving lung function in a subject. Improvements in motor function may be measured by a lung function test, such as an upright (sitting) forced spirometry test, a maximal expiratory pressure (MEP) test, a maximal inspiratory pressure (MIP) test, or a combination of the foregoing. In some embodiments, the subject shows at least a 4% improvement in FVC, at least a 16 cmH2O improvement in MEP, and / or at least a 0.3 cmH2O improvement in MIP six months after treatment (when compared to baseline). For example, an ERT switch patient who is mobile may demonstrate an improvement in MEP of at least 16 cmH2O six months after treatment (compared to baseline). In another instance, an ERT original patient who is mobile may demonstrate at least a 4% improvement in FVC and / or at least a 11 cmH2O improvement in MIP six months after treatment (compared to baseline). In some embodiments, the ERT switch patient exhibits improvement in at least one lung function test after treatment relative to the lung function test results of the patient following prior ERT with alglucosidase alfa.

[0022] In some embodiments, the population of rhGAA molecules is administered at a dose capable of reducing fatigue in a subject as measured by a fatigue severity scale (FSS) score. For example, the subject may be an ERT switch patient who is not capable of mobility and exhibits a reduction in FSS score of at least 3.5 relative to baseline at six months after treatment. In another example, the subject may be an ERT switch patient who is capable of mobility and exhibits a reduction in FSS score of at least 8 relative to baseline at six months after treatment. In yet another example, the subject may be an ERT naive patient who is capable of mobility and exhibits a reduction in FSS score of at least 5 relative to baseline at six months after treatment. In some embodiments, the ERT switch patient exhibits a lower FSS score after treatment relative to the FSS score of the patient after ERT prior to utilizing alglucosidase alfa.

[0023] In some embodiments, the population of rhGAA molecules is administered at a dose capable of reducing the level of at least one biomarker of muscle damage, such as creatine kinase (CK), alanine aminotransferase (ALT), aspartate aminotransferase (AST), or a combination of the foregoing. In some embodiments, the subject's CK level is reduced by at least 15% relative to baseline six months after treatment, the subject's ALT level is reduced by at least 5% relative to baseline six months after treatment, and / or the subject's AST level is reduced by at least 5% relative to baseline six months after treatment. For example, the subject may be an ERT-switching patient who is capable of mobility and exhibits at least a 15% reduction in CK level, at least a 15% reduction in ALT level, and / or at least a 10% reduction in AST level six months after treatment relative to baseline. In another example, the subject may be an ERT-switching patient who is incapable of mobility and exhibits at least a 20% reduction in CK level, at least a 5% reduction in ALT level, and / or at least a 5% reduction in AST level six months after treatment relative to baseline. In yet another example, the subject may be an active ERT naive patient and exhibit at least a 35% reduction in CK levels, at least a 35% reduction in ALT levels, and / or at least a 30% reduction in AST levels relative to baseline six months after treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1A shows non-phosphorylated high mannose N-glycans, mono-M6P N-glycans, and di-M6P N-glycans. Figure 1B shows the chemical structure of the M6P group. Each square represents N-acetylglucosamine (GlcNAc), each circle represents mannose, and each P represents phosphate.

[0025] Figure 2A depicts the extensive targeting of rhGAA to target tissues (e.g., muscle tissue in subjects with Pompe disease) via M6P-bearing N-glycans. Figure 2B depicts the non-extensive drug clearance to non-target tissues (e.g., liver and spleen) or binding to non-target tissues via non-M6P N-glycans.

[0026] FIG. 3 is a schematic diagram of an exemplary method for producing, capturing, and purifying recombinant lysosomal proteins.

[0027] FIG. 4 shows the DNA construct used to transform CHO cells with DNA encoding rhGAA.

[0028] FIG. 5 is a graph showing the results of CIMPR affinity chromatography of ATB200 rhGAA with (Example 2) and without (Example 1) capture on an anion exchange (AEX) column.

[0029] Figure 6A-Figure 6H show the results of site-specific N-glycosylation analysis of ATB200 rhGAA using two different LC-MS / MS analysis techniques. Figure 6A shows the site occupancy of seven potential N-glycosylation sites for ATB200. Figure 6B shows two analyses of the N-glycosylation distribution for the first potential N-glycosylation site of ATB200. Figure 6C shows two analyses of the N-glycosylation distribution for the second potential N-glycosylation site of ATB200. Figure 6D shows two analyses of the N-glycosylation distribution for the third potential N-glycosylation site of ATB200. Figure 6E shows two analyses of the N-glycosylation distribution for the fourth potential N-glycosylation site of ATB200. Figure 6F shows two analyses of the N-glycosylation distribution for the fifth potential N-glycosylation site of ATB200. Figure 6G shows two analyses of the N-glycosylation distribution for the sixth potential N-glycosylation site of ATB200. Figure 6H summarizes the relative percentages of mono- and di-phosphorylated species for the first, second, third, fourth, fifth, and sixth potential N-glycosylation sites.

[0030] Figure 7 shows Graph of the Polywax elution profiles for alglucosidase alfa (thinner line, eluted to the left) and ATB200 (thicker line, eluted to the right).

[0031] Figure 8 shows Table showing a summary of the N-glycan structures of three different preparations of ATB200 rhGAA compared to BP-rhGAA, ATB200-1 and ATB200-2.

[0032] Figures 9A and 9B show and Graph of the results of CIMPR affinity chromatography.

[0033] Figure 10A shows the CIMPR binding affinity of ATB200 rhGAA (left trace) compared to Figure 10B is a graph comparing the binding affinity of CIMPR (right trace). And the table of double M6P content of ATB200rhGAA.

[0034] Figure 11A compares the activity of ATB200 rhGAA in normal fibroblasts (left trace) with that in Figure 11B is a graph comparing ATB200 rhGAA activity (left trace) in fibroblasts from a subject with Pompe disease at various GAA concentrations to Figure 11C is a table comparing the K of fibroblasts from normal subjects and subjects with Pompe disease. 吸收 of table.

[0035] Figure 12 depicts the stability of ATB200 in acidic or neutral pH buffers as assessed in a thermal stability assay using SYPRO Orange as the fluorescence of the dye increases upon protein denaturation.

[0036] FIG. 13 shows tissue glycogen content of WT mice or Gaa KO mice treated with vehicle, alglucosidase alfa, or ATB200 / AT2221 as determined using amyloglucosidase digestion. Bars represent mean ± SEM of 7 mice / group. *p<0.05 compared to alglucosidase alfa in one-way ANOVA analysis using Dunnett's multiple comparisons.

[0037] FIG. 14 depicts LAMP1-positive vesicles in muscle fibers of Gaa KO mice or WT mice treated with vehicle, alglucosidase alfa, or ATB200 / AT2221. Images were obtained from the vastus lateralis muscle and are representative of 7 mice per group. Magnification = 200x (inset is 1,000x).

[0038] Figure 15A shows LC3 positive aggregates in muscle fibers of Gaa KO mice or WT mice treated with vehicle, alglucosidase alpha, or ATB200 / AT2221. Images were taken from the vastus lateralis muscle and represent 7 mice per group. Magnification = 400x. Figure 15B shows Western blot analysis of LC3 II protein. A total of 30 mg protein was loaded per lane.

[0039] Figure 16 shows Dysferlin expression in muscle fibers of Gaa KO mice or WT mice treated with vehicle, alglucosidase alfa, or ATB200 / AT2221. Images were obtained from the vastus lateralis muscle and represent 7 mice per group. Magnification = 200x.

[0040] FIG. 17 depicts co-immunofluorescence staining of LAMP1 (green) and LC3 (red) in single fibers isolated from white gastrocnemius muscle of Gaa KO mice treated with vehicle, alglucosidase alpha, or ATB200. "C" depicts clearance of autophagic debris and absence of enlarged lysosomes. A minimum of 30 fibers were examined from each animal.

[0041] FIG. 18 depicts the stabilization of ATB200 by AT2221 at 17 μM and 170 μM AT2221, respectively, compared to ATB200 alone.

[0042] Figures 19A and 19B show the ATB200-02 study design. Low dose = 130 mg. High dose = 260 mg. In Figure 26A, "6MWT" = 6-minute walk test; "FVC" = forced vital capacity; "QOW" = every other week. "a" = safety data of 2 sentinel patients from Group 1 were reviewed at each dose level before dosing in Groups 2 and 3; "b" = AT2221 was administered orally before starting ATB200 intravenous infusion during Phases 2 and 3. ATB200 was an intravenous infusion for 4 hours in duration for all doses. "c" = The first 2 patients in Groups 2 and 3 served as sentinel patients for their respective groups. Figure 19C summarizes the baseline characteristics of patients enrolled across Groups 1, 2, and 3. "NA" = Not applicable. "SD" = Standard deviation. "a" = Group 1 patients were required to take alglucosidase alfa for 2-6 years at baseline. LOPD = late-onset Pompe disease.

[0043] FIG. 20 depicts pharmacokinetic data for AT2221. “AUC” = area under the curve; “CL / F” = plasma clearance adjusted for oral bioavailability of AT2221; “C max ” = maximum drug concentration; “CV” = coefficient of variation; “t 1 / 2 " = half-life; "t max ” = time to reach maximum drug concentration; “V Z / F" = apparent terminal phase distribution volume adjusted for oral bioavailability of AT2221. "a" = geometric mean (CV%); "b" = median (min-max); "c" = arithmetic mean (CV%).

[0044] FIG. 21 depicts total GAA protein by signature peptide T09 for Groups 1 and 3. “AUC” = Area Under the Curve; “CL T ”=systemic clearance; “C max ” = maximum drug concentration; “CV” = coefficient of variation; “MD” = multiple dose; “t 1 / 2 " = half-life; "t max " = time to reach maximum drug concentration; "F rel" = AUC ratios of 20 mg / kg ATB200 alone and 10 mg / kg ATB200 alone versus 5 mg / kg ATB200 alone, and 20 mg / kg ATB200 + low or high dose AT2221 versus 20 mg / kg ATB200 alone. "a" = geometric mean (CV%); "b" = median (min-max); "c" = arithmetic mean (CV%); "d" = n = 11; "e" = n = 5. Low dose = 130 mg. High dose = 260 mg.

[0045] Figures 22A, 22B, 22C, 22D, 22E, and 22F depict total GAA protein by group. Low dose = 130 mg. High dose = 260 mg. Figure 22A shows the average total GAA protein concentration-time profile for Group 1 (single dose). Figure 22B shows the average total GAA protein concentration-time profile for Group 1 (multiple doses). Figure 22C shows the average total GAA protein concentration-time profile for Group 1 vs. Group 3 (single dose). Figure 22D shows the average total GAA protein concentration-time profile for Group 1 vs. Group 3 (multiple doses). Figure 22E shows a comparison of total GAA protein with 20 mg / kg ATB200 at 12 hours post-dose; *=p<0.05; **=p<0.01; ***p<0.001. Figure 22F shows total GAA protein comparison with 20 mg / kg ATB200 at 24 hours post-dose; * = p < 0.05; ** = p < 0.01; "ns" = not significant.

[0046] Figure 23 shows the analysis of variance (ANOVA) of total GAA protein by signature peptide T09. Area under the curve (AUC) is provided in μg-h / mL; "CI" = confidence interval.

[0047] FIG. 24A depicts a summary of the analysis and available interim data from the 6-Minute Walk Test (“6MWT”), showing the change from baseline (“CFBL”) at Months 6, 9, and 12 for patients in Cohorts 1 and 3.

[0048] FIG. 24B depicts 6MWT data for individual Cohort 1 and Cohort 3 patients.

[0049] Figure 24C depicts an analysis of other motor function tests: timed stand-up and walk motor function test; 4-stair climbing test; 10-meter (10M) walk test; Gowers' test; and gait-stair-gower-chair (gait-stair-gower-chair; "GSGC") motor function test and a summary of available interim data, showing the change from baseline ("CFBL") at 6 months, 9 months, and 12 months for patients in Group 1 and Group 3. GSGC is an observer-graded composite score for the following four motor function assessments: gait (10-meter walk), 4-stair climbing, Gowers' (standing from the floor), and sitting up from a chair. Each test is scored from 1 (normal) to 7 (unable to perform; the maximum score for the test of sitting up from a chair is 6). The total score range is 4 to 27. "a" = n = 9, missing values ​​not obtained because patients refused to perform the test; "b" = median change from baseline was -1.5, and 7 / 9 patients had a reduction; "c" = median change from baseline was -0.8, and 4 / 5 patients had a reduction.

[0050] FIG. 25 depicts a summary of the analysis and available interim data from the muscle strength test (QMT), showing the change from baseline ("CFBL") at Months 6 and 9 for patients in Cohort 2. QMT = Quantitative Muscle Testing. Values ​​shown represent pounds of force for right and left sides combined. "a" = shoulder adduction not available for one subject; "b" = score: (1) visible muscle movement, but no movement at the joint; (2) movement at the joint, but not against gravity; (3) movement against gravity, but not against added resistance; (4) movement against resistance, but less than normal; (5) normal strength.

[0051] FIG. 26A depicts a summary of the analysis and available interim data from the manual muscle test (MMT) in patients in Cohort 1. MMT scores were calculated for the upper body (maximum score: 40), lower body (maximum score: 40), and total body (maximum score: 80). In patients in Cohort 1, increases in manual muscle strength were observed at 6, 9, and 12 months. "SD" = standard deviation.

[0052] FIG. 26B depicts a summary of the analysis and available interim data from the manual muscle test (MMT) in patients in Cohort 2. MMT scores were calculated for the upper body (maximum score: 40). In patients in Cohort 2, increases in manual muscle strength were observed at 6 and 9 months. "SD" = standard deviation. The MMT results were generally consistent with the QMT results (shown in FIG. 28).

[0053] FIG. 26C depicts a summary of the analysis and available interim data from the manual muscle test (MMT) in patients in Cohort 3. MMT scores were calculated for upper body (maximum score: 40), lower body (maximum score: 40), and total body (maximum score: 80). In patients in Cohort 3, increases in manual muscle strength were observed at each of Months 6, 9, and 12. "SD" = standard deviation.

[0054] FIG. 27 depicts a summary of the analysis and available interim data from forced vital capacity ("FVC"), maximum inspiratory pressure ("MIP"), and maximum expiratory pressure ("MEP"), showing the change from baseline ("CFBL") at Months 6, 9, and 12 for patients in Cohorts 1 and 3. "a" = FVC was not available for one subject. MEP and MIP are measured in centimeters of water.

[0055] FIG. 28 depicts a summary of the analysis and available interim data from the Fatigue Severity Scale ("FSS"), a nine-question self-rating questionnaire, each scored on a scale of 1 to 7. Total scores range from 9 to 63, with higher values ​​indicating higher levels of fatigue due to disease conditions. Normal values ​​in a healthy population are about 21. FIG. 28 shows the change from baseline ("CFBL") at 6, 9, and 12 months for patients in Cohort 1, Cohort 2, and Cohort 3.

[0056] Figures 29A-29C depict the mean percent change from baseline for markers of marker damage (alanine transaminase, aspartate transaminase, and creatine kinase) in all patient groups. Figure 29A depicts data from patients in Group 1 over 58 weeks, Figure 29B depicts data from patients in Group 2 over 24 weeks, and Figure 29C depicts data from patients in Group 3 over 36 weeks. Figure 29D depicts the mean percent change from baseline for markers of muscle damage (CK=creatine kinase) and markers of disease substrates (Hex4=urinary hexotetraose) for patients in Groups 1, 2, and 3 up to 12 months. "BL" = baseline. "SE" = standard error. "WK" = week. "M" = month.

[0057] Figure 30 summarizes safety data from the ATB200-02 study. "AE" = adverse event. "IAR" = infusion-related reaction; "a" = reported via interim data analysis (up to 20+ months); "b" = includes upper and lower abdominal pain.

[0058] Figure 31 summarizes the available efficacy and safety data from the ATB200-02 study.

[0059] Figures 32A-32H show the results of site-specific N-glycosylation analysis of ATB200 rhGAA using LC-MS / MS analysis of protease-digested ATB200, including the N-glycosylation distribution of the seventh potential N-glycosylation site. Figures 32A-32H provide average data for ten batches of ATB200 produced at different scales.

[0060] Figure 32A shows the average site occupancy of seven potential N-glycosylation sites of ATB200. The N-glycosylation sites are provided according to SEQ ID NO: 1. CV = coefficient of variation.

[0061] Figures 32B-32H show site-specific N-glycosylation analysis of all seven potential N-glycosylation sites of ATB200, with site numbers provided according to SEQ ID NO: 5. Each bar represents the maximum and minimum percentages of N-glycan species identified as specific N-glycan groups of the ten batches of ATB200 analyzed. Figure 32B shows the N-glycosylation distribution of the first potential N-glycosylation site of ATB200. Figure 32C shows the N-glycosylation distribution of the second potential N-glycosylation site of ATB200. Figure 32D shows the N-glycosylation distribution of the third potential N-glycosylation site of ATB200. Figure 32E shows the N-glycosylation distribution of the fourth potential N-glycosylation site of ATB200. Figure 32F shows the N-glycosylation distribution of the fifth potential N-glycosylation site of ATB200. Figure 32G shows the N-glycosylation distribution of the sixth potential N-glycosylation site of ATB200. Figure 32H shows the N-glycosylation distribution of the seventh potential N-glycosylation site of ATB200.

[0062] As also shown in Figures 32A-32H, Figures 33A-33B further characterize and summarize the N-glycosylation distribution of ATB200. Figure 33A shows the 2-anthranilic acid (2-AA) glycan map distribution and LC / MS-MS analysis of ATB200, and summarizes the percentage of N-glycan species identified in ATB200 as a percentage of total fluorescence. Table 5 also lists the data of 2-AA glycan map distribution and LC-MS / MS analysis. Figure 33B summarizes the average site occupancy and average N-glycan distribution for all seven potential N-glycosylation sites of ATB200, including total phosphorylation, monophosphorylation, diphosphorylation and sialylation, ND = not detected. DETAILED DESCRIPTION

[0063] Before describing several exemplary embodiments of the present invention, it should be understood that the present invention is not limited to the details of construction or method steps set forth in the following description. The present invention is capable of other embodiments and of being practiced or carried out in various ways.

[0064] I. Definitions

[0065] The terms used in this specification generally have their ordinary meanings in this technology within the context of the present invention and in the specific circumstances in which each term is used. Certain terms are discussed below or elsewhere in the specification to provide practitioners with additional guidance for describing the compositions and methods of the present invention and how to make and use. The articles "a" and "an" refer to one or more than one (that is, at least one) grammatical objects of the articles. Unless the context clearly indicates otherwise, the term "or" means the term "and / or" and can be used interchangeably with the term "and / or". In this application, unless otherwise specifically stated, the use of the singular includes the plural. In addition, the use of the term "including" and other forms such as "includes" and "included" is not limited. Any range described herein will be understood to include all values ​​between the endpoints and the endpoints. In this specification, unless the context requires otherwise due to express language or necessary implication, the word "comprises" or variations such as "comprising" are used in an inclusive sense, that is, to specify the presence of stated features but not to exclude the presence or addition of other features in various embodiments of the invention.

[0066] The term "GAA" refers to human acid alpha-glucosidase (GAA) that catalyzes the hydrolysis of α-1,4- and α-1,6-glycosidic bonds of lysosomal glycogen, as well as insertion, related, or substitution variants of the GAA amino acid sequence and fragments of longer GAA sequences that exert enzymatic activity. Human acid alpha-glucosidase is encoded by the GAA gene (National Centre for Biotechnology Information; NCBI) gene ID 2548), which has been mapped to the long arm of chromosome 17 (position 17q25.2-q25.3). An exemplary DNA sequence encoding GAA is NP000143.2, which is incorporated by reference. More than 500 mutations have been identified in the human GAA gene, many of which are associated with Pompe disease. Mutations that cause misfolding or misprocessing of acid alpha-glucosidase include T1064C (Leu355Pro) and C2104T (Arg702Cys). In addition, GAA mutations that affect the maturation and processing of the enzyme include Leu405Pro and Met519Thr. The conserved hexapeptide WIDMNE at amino acid residues 516-521 is required for the activity of acid alpha-glucosidase protein. As used herein, the abbreviation "GAA" is intended to refer to human acid alpha-glucosidase, and the italic abbreviation "GAA" is intended to refer to the human gene encoding human acid alpha-glucosidase. The italic abbreviation "Gaa" is intended to refer to non-human genes encoding non-human acid alpha-glucosidase, including but not limited to rat or mouse genes, and the abbreviation "Gaa" is intended to refer to non-human acid alpha-glucosidase.

[0067] The term "rhGAA" is intended to refer to recombinant human acid alpha-glucosidase and is used to distinguish endogenous GAA from synthetically or recombinantly produced GAA (e.g., GAA produced by CHO cells transformed with DNA encoding GAA). The term "rhGAA" encompasses a population of individual rhGAA molecules. The properties of a population of rhGAA molecules are provided herein. The term "traditional rhGAA product" is intended to refer to products containing alglucosidase alpha, such as or

[0068] The term "genetically modified" or "recombinant" refers to cells, such as CHO cells, that express a particular gene product, such as rhGAA, following 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 the nucleic acid can be accomplished by any method known in the art including gene targeting and homologous recombination. As used herein, the term also includes cells that have been engineered to express or overexpress an endogenous gene or gene product that is not normally expressed by such cells, such as by gene activation techniques.

[0069] As used herein, the term "purified" refers to a material that has been isolated under conditions that reduce or eliminate the presence of extraneous materials, that is, contaminants, including the original material from which the material was obtained. For example, a purified protein is preferably substantially free of other proteins or nucleic acids with which it is associated in the cell; a purified nucleic acid molecule is preferably substantially free of proteins or other extraneous nucleic acid molecules that may be found with it in the cell. As used herein, the term "substantially free" is used operationally in the context of analytical testing of a material. Preferably, a purified material that is substantially free of contaminants is at least 95% pure; more preferably, at least 97% pure, and even more preferably at least 99% pure. Purity can be assessed by chromatography, gel electrophoresis, immunoassays, compositional analysis, bioassays, enzymatic assays, and other methods known in the art. In specific embodiments, purified means that the level of contaminants is below a level acceptable to regulatory agencies for safe administration to humans or non-human animals. Recombinant proteins such as rhGAA can be isolated or purified from CHO cells using methods known in the art, including chromatographic size separation, affinity chromatography, or anion exchange chromatography. In some embodiments, rhGAA is purified by a method comprising anion exchange chromatography, followed by immobilized metal affinity chromatography, optionally, followed by purification using a third chromatography column.

[0070] As used herein, the term "alglucosidase alfa" is intended to refer to the recombinant human acid alpha-glucosidase identified as [199-arginine, 223-histidine] precursor l-alpha-glucosidase (human); Chemical Abstract Registration Number 420794-05-0. Alglucosidase alfa is approved in the United States by Genzyme as a product and Listed for sale.

[0071] As used herein, the term "ATB200" is intended to refer to the recombinant human acid alpha-glucosidase described in International Application PCT / US2015 / 053252, the disclosure of which is incorporated herein by reference.

[0072] As used herein, the term "polysaccharide" is intended to refer to a polysaccharide chain covalently bound to an amino acid residue on a protein or polypeptide. As used herein, the term "N-polysaccharide" or "N-linked polysaccharide" is intended to refer to a polysaccharide chain connected to an amino acid residue on a protein or polypeptide by covalent bonding to the nitrogen atom of the amino acid residue on the protein or polypeptide. For example, an N-polysaccharide can be covalently bound to a side chain nitrogen atom of an asparagine residue. A polysaccharide can contain one or more monosaccharide units, and the monosaccharide units can be covalently linked to form a straight chain or a branched chain. In at least one embodiment, the N-polysaccharide unit connected to rhGAA can include one or more monosaccharide units independently selected from N-acetylglucosamine, mannose, galactose, trehalose, mannose-6-phosphate or sialic acid. The N-polysaccharide unit on the protein can be determined by any appropriate analytical technique such as mass spectrometry. In some embodiments, the N-glycan units attached to rhGAA are determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS) using instruments such as Thermo Scientific TM Orbitrap Velos Pro TM Mass spectrometer, Thermo Scientific TM OrbitrapFusion TM Lumos Tribid TM Mass spectrometer or Waters G2-XS QTof mass spectrometer.

[0073] As used herein, the term "high mannose N-glycan" is intended to refer to N-glycans having one to six or more mannose units. In some embodiments, the high mannose N-glycan unit may contain a bis(N-acetylglucosamine) chain bonded to an asparagine residue and further bonded to a branched polymannose chain. As used interchangeably herein, the term "M6P" or "mannose-6-phosphate" is intended to refer to a mannose unit phosphorylated at the 6 position, that is, having a phosphate group bonded to a hydroxyl group at the 6 position. In some embodiments, one or more mannose units of one or more N-glycan units are phosphorylated at the 6 position to form a mannose-6-phosphate unit. In some embodiments, the term "M6P" or "mannose-6-phosphate" refers to a mannose phosphodiester having N-acetylglucosamine (GlcNAc) as a "cap" on the phosphate group, as well as a mannose unit having an exposed phosphate group that lacks a GlcNAc cap. In at least one embodiment, the N-glycan of a protein may have multiple M6P groups, wherein at least one M6P group has a GlcNAc cap and at least one other M6P group lacks a GlcNAc cap.

[0074] As used herein, the term "complex N-glycan" refers to an N-glycan comprising sugar types other than GlcNac and mannose, such as one or more galactose and / or sialic acid units. In at least one embodiment, the complex N-glycan may be a high mannose N-glycan, wherein one or more mannose units are further bonded to one or more monosaccharide units, each of which is independently selected from N-acetylglucosamine, galactose, and sialic acid. As used herein, "hybrid N-glycan" refers to an N-glycan comprising at least one high mannose branch and at least one complex branch. Representative structures of non-phosphorylated, single M6P, and double M6P N-glycans are shown in Figure 1A. The mannose-6-phosphate group is shown in Figure 1B.

[0075] As used herein, "normalization" of lysosomes in muscle fibers refers to the process of restoring the affected muscle fibers to the lysosomal morphology of wild-type muscle fibers by reducing the size and number of accumulated glycogen in the muscle fibers so that the affected muscle fibers will essentially resemble normal lysosomal morphology, ultimately leading to a reversal of the disease process.

[0076] As used herein, "reversal of disease progression" refers, inter alia, to sufficient (i) reduction or elimination of glycogen accumulation, (ii) reduction or elimination of lysosomal swelling and / or dysfunction, and (iii) reduction or elimination of accumulation of autophagic debris. In ERT Pompe disease patients with mobility, reversal of disease progression may be manifested as two or more of the following "clinical improvements"; (a) an average increase of at least 20 meters in the six-minute walk test distance; (b) an average increase of at least 16 cm of water in the maximum expiratory pressure; (c) an average decrease of at least 7 in the fatigue severity score. In non-mobile ERT Pompe disease patients, reversal of disease progression may be manifested as two or more of the following "clinical improvements": (a) an average improvement of at least 8 pounds of force in shoulder adduction; (b) an average improvement of at least 5 pounds of force in elbow extension; and (c) an average decrease of at least 3.5 in the fatigue severity score. Reversal of disease progression in ERT-naive Pompe disease patients may be demonstrated by two or more of the following “clinical improvement”: (a) a mean increase in six-minute walk test distance of at least 40 meters; (b) a mean increase in upright (seated) forced vital capacity of at least 4%; (c) a mean increase in maximum inspiratory pressure of at least 11 cmH2O, and (d) a mean decrease in fatigue severity score of at least 5.

[0077] Compared to the administration of alglucosidase alfa, the advantage of the treatment methods disclosed herein is that Pompe patients treated with the former show prolonged clinical improvement. For example, improvements can be observed two to three years or longer from the start of the first treatment, including, for example, four, five or six years from the start of the first treatment. In contrast, after two years of enzyme replacement therapy with standard care (e.g., alglucosidase alfa), Pompe disease patients either (i) maintain elevated baseline levels before treatment, but do not have significant improvement scores after two or three years or (ii) gradually decline and lose any benefits within two to three years after receiving standard care. "Kuperus et al. 2017, Long-term benefits of enzyme replacement therapy in Pompe disease; a 5-year prospective study, Neurology, 89: 2365-2373". In contrast, the rhGAA described herein clears lysosomal glycogen more effectively than standard care and has been shown to cause improvements in patients (e.g., "Actionable ERT switching", cohort 1 of study ATB200-02), but is not expected to improve after at least two years of taking enzyme replacement therapy. To date, clinical data using rhGAA or pharmaceutical compositions described herein are promising for continued improvement in patient outcomes even two years after treatment. Thus, in some embodiments, patients treated with rhGAA or pharmaceutical compositions described herein continue to show progression of one or more clinical improvements (improvements beyond those achieved prior to or at the two-year mark) more than two years after treatment.

[0078] As used herein, "reversal of lysosomal pathology" means the partial or complete clearance of glycogen that has accumulated in the cell due to lack of optimal GAA activity.

[0079] 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 breath possible.

[0080] As used herein, the "Six Minute Walk Test" (6MWT) is a test used to measure the distance an individual can walk on a hard, flat surface over a total of six minutes. The test is performed by having the individual walk as far as possible in six minutes.

[0081] As used herein, the "Ten Meter Walk Test" (10MWT) is a test used to measure the time it takes an individual to walk ten meters on a flat surface while wearing a walking shoe.

[0082] 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:

[0083]

[0084] One formulation of Miglutec is sold under the trade name It is marketed as a monotherapy for Gaucher disease type 1. In some embodiments, miglustat is referred to as AT2221.

[0085] As discussed below, pharmaceutically acceptable salts of miglustat are also useful in the present invention. When a salt of miglustat is used, the dosage of the salt will be adjusted so that the patient receives a dose of miglustat equal to the amount that would be received when miglustat free base is used.

[0086] 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:

[0087]

[0088] As used herein, the term "pharmacological chaperone" or sometimes simply the term "chaperone" is intended to refer to a molecule that specifically binds to acid alpha-glucosidase and has one or more of the following effects:

[0089] Enhance the formation of stable molecular conformation of proteins;

[0090] Enhancing proper transport of proteins from the endoplasmic reticulum to another cellular location, preferably the original cellular location, in order to prevent endoplasmic reticulum-associated degradation of proteins;

[0091] Prevent aggregation of conformationally unstable or misfolded proteins;

[0092] Restoring and / or enhancing at least part of the wild-type function, stability, and / or activity of a protein; and / or

[0093] Improve the phenotype or function of cells harboring acid α-glucosidase.

[0094] Therefore, the pharmacological chaperone of acid α-glucosidase is a protein that binds to acid α-glucosidase, thereby producing proper folding, transport, non-aggregation, and activity of acid α-glucosidase. As used herein, this term includes, but is not limited to, active site-specific chaperones (ASSCs) that bind to the active site of the enzyme, inhibitors or antagonists, and agonists. In at least one embodiment, the pharmacological chaperone may be an inhibitor or antagonist of acid α-glucosidase. As used herein, the term "antagonist" is intended to refer to any molecule that binds to acid α-glucosidase and partially or completely blocks, inhibits, reduces, or neutralizes the activity of acid α-glucosidase. In at least one embodiment, the pharmacological chaperone is miglustat. Another non-limiting example of a pharmacological chaperone for acid α-glucosidase is duglustat.

[0095] As used herein, the term "active site" is intended to refer to a region in a protein that is associated with and necessary for a specific biological activity of the protein. In at least one embodiment, an active site may be a site that binds a substrate or other binding partner and contributes amino acid residues that are directly involved in forming and breaking chemical bonds. The active site in the present invention may encompass the catalytic site of an enzyme, the antigen binding site of an antibody, the ligand binding domain of a receptor, the binding domain of a regulatory protein, or the receptor binding domain of a secreted protein. The active site may also encompass transcriptional activation, protein-protein interactions, or DNA binding domains of transcription factors and regulatory proteins.

[0096] As used herein, the term "AUC" or "area under the curve" is intended to refer to a mathematical calculation that estimates the body's overall exposure to a given drug over time. In a graph that plots how the concentration of a drug administered to a subject changes over time in the blood after administration, the drug concentration variable is on the y-axis and time is on the x-axis. The area between the drug concentration curve and the x-axis for a specified time interval is the AUC. The AUC is used as a guide for dosing schedules and is used to compare the bioavailability of different drugs available in the body.

[0097] As used herein, the term “C max ” is intended to refer to the maximum plasma concentration of drug achieved following administration to a subject.

[0098] As used herein, the term "distribution volume" or "V" is intended to refer to the theoretical volume necessary to contain the total amount of administered drug at the same concentration observed in blood plasma, and it indicates the extent to which the drug is distributed in body tissues rather than in plasma. Higher V values ​​indicate a greater degree of tissue distribution. "Central distribution volume" or "Vc" is intended to refer to the distribution volume within the blood and tissues that are highly perfused by the blood. "Peripheral distribution volume" or "V2" is intended to refer to the distribution volume within peripheral tissues.

[0099] As used interchangeably herein, the terms "clearance," "systemic clearance," or "CL" are intended to refer to the volume of plasma that completely clears an administered drug per unit time. "Peripheral clearance" is intended to refer to the volume of peripheral tissue that clears an administered drug per unit time.

[0100] As used herein, the term "pharmaceutically acceptable" is intended to refer to molecular entities and compositions that are physiologically tolerable and typically do not produce adverse reactions when administered to humans. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of a federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia, suitable for animals, and more particularly suitable for humans. As used herein, the term "carrier" is intended to refer to a diluent, adjuvant, excipient, or vehicle administered with a compound. Suitable pharmaceutical carriers are known in the art and, in at least one embodiment, are described in "Remington's Pharmaceutical Sciences", EW Martin, 18th edition or other versions.

[0101] As used herein, the term "pharmaceutically acceptable salt" is intended to refer to a salt that is suitable for contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic reaction, and the like, commensurate with a reasonable benefit / risk ratio, and is generally water-soluble or oil-soluble or water-dispersible or oil-dispersible, and effective for its designed use. The term includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. A list of suitable salts is found, for example, in SM Berge et al., J. Pharm. Sci., 1977, 66, pp. 1-19, which is incorporated herein by reference. As used herein, the term "pharmaceutically acceptable acid addition salt" is intended to refer to those salts that retain the biological effectiveness and properties of the free base and are not biologically or otherwise undesirable, and are formed with inorganic acids. As used herein, the term "pharmaceutically acceptable base addition salts" is intended to refer to those salts formed with inorganic bases which retain the biological effectiveness and properties of the free acids and which are not biologically or otherwise undesirable.

[0102] As used herein, the term "buffer" refers to a solution containing a weak acid and its conjugate base to help prevent changes in pH.

[0103] As used herein, the terms "therapeutically effective dose" and "effective amount" are intended to refer to an amount of acid alpha-glucosidase and / or miglustat and / or a combination of the foregoing that is sufficient to produce a therapeutic response in a subject. A therapeutic response can be any response that a user (e.g., a clinician) would consider to be an effective response to therapy, including an effective response to any surrogate clinical marker or symptom described herein and known in the art. Thus, in at least one embodiment, a therapeutic response can be an improvement or inhibition of one or more symptoms or markers of Pompe disease, such as those known in the art. Symptoms or markers of Pompe disease include, but are not limited to, decreased acid alpha-glucosidase tissue activity; cardiomyopathy; cardiac hypertrophy; progressive muscle weakness, especially in the trunk or lower extremities; profound hypotonia; macroglossia (and in some cases, protrusion of the tongue); difficulty swallowing, sucking, and / or eating; respiratory insufficiency; hepatomegaly (moderate); facial muscle flaccidity; areflexia; lack of exercise persistence; exertional dyspnea; orthopnea; sleep apnea; morning headaches; narcolepsy; lordosis and / or scoliosis; decreased deep tendon reflexes; lower back pain; and failure to meet developmental motor milestones. It should be noted that concentrations of miglustat that have an inhibitory effect on acid alpha-glucosidase may constitute an "effective amount" for the purposes of the present invention due to dilution (and subsequent binding changes due to changes in equilibrium), bioavailability, and metabolism of miglustat following in vivo administration.

[0104] The therapeutic response may also include molecular responses, such as glycogen accumulation, lysosomal proliferation, and the formation of autophagic zones. The therapeutic response can be assessed by comparing the physiological and molecular responses of muscle biospecimens before and after treatment with rhGAA as described herein. For example, the amount of glycogen present in the biospecimen sample can be used as a marker for determining the therapeutic response. Another example includes biomarkers such as LAMP-1, LC3, and Dysferlin, which can be used as indicators of lysosomal storage dysfunction. For example, muscle biospecimens collected before and after treatment with rhGAA as described herein can be stained with an antibody that recognizes one of the biomarkers.

[0105] As used herein, the term "enzyme replacement therapy" or "ERT" is intended to refer to the introduction of a non-native, purified enzyme into an individual who lacks such an enzyme. The administered protein may 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 needs or benefits from the administration of a purified enzyme. In at least one embodiment, the individual lacks the enzyme. The introduced enzyme may be a purified, recombinant enzyme produced in vitro, or a protein purified from isolated tissue or fluid such as, for example, the placenta or animal milk, or from a plant.

[0106] As used herein, the term "combination therapy" is intended to refer to any therapy in which two or more individual therapies are administered in parallel or sequentially. In some embodiments, the result of the combination therapy is an enhancement compared to the effect of each therapy when each therapy is performed individually. Enhancement can include any improvement in the effect of the various therapies, which can produce a favorable result compared to the result achieved by the therapy when performed alone. The enhanced effect or result may include synergistic enhancement, where the enhanced effect is greater than the additive effect of each therapy when performed by itself; additive enhancement, where the enhanced effect is substantially equal to the additive response of each therapy when performed by itself; or less than a synergistic effect, where the enhanced effect is less than the additive effect of each therapy when performed by itself, but better than the effect of each therapy when performed by itself. The enhanced effect can be measured by any means known in the art, by which the efficacy or result of the treatment can be measured.

[0107] As used herein, the term "concurrently" is intended to mean simultaneously or within a reasonably short period of time before or after, as will be understood by those skilled in the art. For example, if two treatments are administered in parallel with each other, one treatment may be administered before or after the other treatment to allow for the time required to prepare the latter of the two treatments. Thus, "concurrent administration" of two treatments includes, but is not limited to, one treatment being administered 30 minutes or less, about 30 minutes, 20 minutes or less, about 20 minutes, about 15 minutes, about 10 minutes, about 9 minutes, about 8 minutes, about 7 minutes, about 6 minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, about 1 minute, or less than 1 minute after the other treatment.

[0108] "Pompe disease" refers to an autosomal recessive LSD characterized by a deficiency in acid α-glucosidase (GAA) activity, thereby impairing lysosomal glycogen metabolism. Enzyme deficiency leads to lysosomal glycogen accumulation and causes progressive skeletal muscle weakness, reduced cardiac function, respiratory insufficiency, and / or CNS damage in the late stages of the disease. Inherited mutations in the GAA gene result in lower expression of the enzyme or produce mutant forms of the enzyme with altered stability and / or biological activity that ultimately leads to disease (see generally Hirschhorn R, 1995, Glycogen Storage Disease Type II: Acid α-glucosidase (Acid Maltase) Deficiency, The Metabolic and Molecular Bases of Inherited Disease, Scriver et al., eds., McGraw-Hill, New York, 7th ed., pp. 2443-2464). The three recognized clinical forms of Pompe disease (infantile, juvenile, and adult) are associated with the level of residual α-glucosidase activity (Reuser AJ et al., 1995, Glycogenosis Type II (Acid Maltase Deficiency), Muscle & Nerve Supplement 3, S61-S69). Infantile Pompe disease (Type I or A) is the most common and most severe, characterized by developmental failure, systemic hypotonicity, cardiomegaly, and cardiopulmonary failure in the second year of life. Juvenile Pompe disease (Type II or B) is moderately severe and is characterized by prominent muscle symptoms without cardiac hypertrophy. Juvenile Pompe disease individuals usually die before reaching the age of 20 due to respiratory failure. Adult-onset Pompe disease (type III or C) often presents as a slowly progressive myopathy in adolescence or as late as the sixth decade of life (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 post-translationally modified by glycosylation, phosphorylation, and proteolytic processing. Optimal glycogen catalysis requires the conversion of a 110 kilodalton (kDa) precursor to a 76 and 70 KDa mature form by proteolysis in the lysosome. As used herein, the term "Pompe disease" refers to all types of Pompe disease. The formulations and dosing regimens disclosed in the present application can be used to treat, for example, type I, type II, or type III Pompe disease.

[0109] A "subject" or "patient" is preferably a human, but other mammals and non-human animals with disorders involving the accumulation of glycogen may also be treated. The subject may be a fetus, neonate, child, adolescent, or adult with Pompe disease or other glycogen storage or accumulation disorders. An example of an individual treated is an individual (fetus, neonate, child, adolescent, young adult, or adult human) with GSD-II (e.g., infantile-onset GSD-II, juvenile-onset 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 (infantile GSD-II), a GAA activity of about 1-10% of normal GAA activity (juvenile 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 "ERT-switched" patient, which refers to a Pompe disease patient who has previously received enzyme replacement therapy. In some embodiments, the subject or patient is an "ERT-original" patient, which refers to a Pompe disease patient who has not previously received enzyme replacement therapy. In certain embodiments, the subject or patient is mobile (e.g., a mobile ERT-switched patient or a mobile ERT-original patient). In certain embodiments, the subject or patient is incapable of mobility (e.g., an incapable ERT-switched patient). The state of mobility or incapacity can be determined by a six-minute walk test (six-minute walk test; 6MWT). In some embodiments, a mobile patient is a Pompe disease patient who is able to walk at least 200 meters in a 6MWT. In some embodiments, an incapable patient is a Pompe disease patient who cannot walk or sit in a wheelchair without assistance.

[0110] As used herein, the terms "treat" and "treatment" refer to the improvement of one or more symptoms associated with a disease, the prevention or delay of the onset of one or more symptoms of a disease, and / or the reduction in the severity or frequency of one or more symptoms of a disease. For example, treatment may refer to an improvement in cardiac status (e.g., an increase in end-diastolic and / or end-systolic volume, or a reduction, improvement, or prevention of progressive cardiomyopathy typically seen in GSD-II) or an improvement in lung function (e.g., an increase in crying vital capacity relative to baseline capacity, and / or normalization of oxygen desaturation during crying); improvement in neurodevelopment and / or motor skills (e.g., an increase in AIMS scores); a reduction in glycogen levels in disease-affected tissues of an individual; or any combination of these effects. In a preferred embodiment, treatment includes an improvement in cardiac status, particularly a reduction or prevention of cardiomyopathy associated with GSD-II.

[0111] As used herein, the terms "improvement," "increase," and "decrease" refer to values ​​relative to baseline measurements, such as measurements in the same individual prior to initiation of a treatment described herein, or measurements in a control individual (or multiple control individuals) in the absence of a treatment described herein. A control individual is an individual suffering from the same form of GSD-II (infantile onset, juvenile onset, or adult onset) as the individual being treated, who is about the same age as the individual being treated (to ensure that the stage of the disease in the treated individual and the control individual is comparable).

[0112] As used herein, the terms "about" and "approximately" are intended to refer to an acceptable degree of error in a measured quantity given the nature or precision of the measurement. For example, the degree of error may be indicated by the number of significant figures provided for a measurement as understood in the art, and includes, but is not limited to, a variation of ±1 of the most accurate significant figure reported for the measurement. Typical exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Unless otherwise indicated, numerical quantities given herein are approximate, meaning that the term "about" or "approximately" can be inferred when not explicitly stated.

[0113] II Recombinant human acid alpha-glucosidase (rhGAA)

[0114] In some embodiments, recombinant human acid alpha-glucosidase (rhGAA) is an enzyme having an amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In some embodiments, rhGAA is encoded by a nucleotide sequence as set forth in SEQ ID NO: 2.

[0115] Table 1. Nucleotide sequences and protein sequences

[0116]

[0117]

[0118]

[0119] In some embodiments, rhGAA has a wild-type GAA amino acid sequence as set forth in SEQ ID NO: 1, as described in U.S. Pat. No. 8,592,362 and having GenBank Accession No. AHE24104.1 (GI: 568760974). In some embodiments, rhGAA has a wild-type GAA amino acid sequence as encoded in SEQ ID NO: 2, and the mRNA sequence has GenBank Accession No. Y00839.1. In some embodiments, rhGAA has a wild-type GAA amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, rhGAA has a GAA amino acid sequence as set forth in SEQ ID NO: 4, and has National Centre for Biotechnology Information (NCBI) Accession No. NP_000143.2. In some embodiments, rhGAA is alglucosidase alpha, which is a human acid alpha-glucosidase encoded by the nine most predominant observed haplotypes of the GAA gene.

[0120] In some embodiments, rhGAA is initially expressed as having a full-length 952 amino acid sequence of wild-type GAA as set forth in SEQ ID NO: 1, and rhGAA undergoes intracellular processing to remove a portion of the amino acids, such as the first 56 amino acids. Therefore, the rhGAA secreted by the host cell may have an amino acid sequence shorter than the rhGAA initially expressed in the cell. In one embodiment, the shorter protein has an amino acid sequence set forth in SEQ ID NO: 5, which differs from SEQ ID NO: 1 only in that the first 56 amino acids including the signal peptide and the precursor peptide have been removed, thereby producing a protein with 896 amino acids. Other changes in the number of amino acids are also possible, such as having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more deletions, substitutions, and / or insertions relative to the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 5. In some embodiments, the rhGAA product includes a mixture of recombinant human acid alpha-glucosidase molecules having different amino acid lengths.

[0121] In some embodiments, rhGAA comprises an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to SEQ ID NO: 1 or SEQ ID NO: 5. Various alignment algorithms and / or programs can be used to calculate the identity between two sequences, including FASTA or BLAST, which can be obtained as part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.) and can be used, for example, with default settings. For example, polypeptides having at least 90%, 95%, 98% or 99% identity to the specific polypeptides described herein and preferably exhibiting substantially the same function, and polynucleotides encoding such polypeptides are contemplated. Unless otherwise indicated, similarity scores will be based on the use of BLOSUM62. When BLASTP is used, the percentage of similarity is based on the BLASTP positive score and the percentage of sequence identity is based on the BLASTP consensus score. BLASTP "consistency" shows the total number and fraction of residues that are consistent in high-scoring sequence pairs; and BLASTP "positive value" shows the number and fraction of residues for which the alignment score has a positive value and is similar to each other. The disclosure contemplates and encompasses amino acid sequences with these degrees of identity or similarity or any intermediate degrees of identity or similarity to the amino acid sequences disclosed herein. The polynucleotide sequence of similar polypeptides is inferred using the genetic code and can be obtained by conventional means, particularly by reversely translating its amino acid sequence using the genetic code.

[0122] In some embodiments, rhGAA undergoes post-translational modification and / or chemical modification at one or more amino acid residues in the protein. For example, methionine and tryptophan residues may undergo oxidation. As another example, N-terminal glutamine may form pyroglutamate. As another example, asparagine residues may undergo deamidation to aspartic acid. As yet another example, aspartic acid residues may undergo isomerization to isoaspartic acid. As another example, unpaired cysteine ​​residues in the protein may form disulfide bonds with free glutathione and / or cysteine. Thus, in some embodiments, the enzyme is initially represented as having an amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, or 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.

[0123] III. N-linked glycosylation of rhGAA

[0124] There are seven potential N-linked glycosylation sites on a single rhGAA molecule. These potential glycosylation sites are at the following positions of SEQ ID NO:5: N84, N177, N334, N414, N596, N826, and N869. Similarly, for the full-length amino acid sequence of SEQ ID NO:1, these potential glycosylation sites are at the following positions: N140, N233, N390, N470, N652, N882, and N925. Other variants of rhGAA may have similar glycosylation sites, depending on the position of the asparagine residue. In general, the sequence of Asn-X-Ser or Asn-X-Thr in the protein amino acid sequence indicates a potential glycosylation site, with the exception that X cannot be His or Pro.

[0125] The rhGAA molecules described herein may have an average of 1, 2, 3, or 4 mannose-6-phosphate (M6P) groups on their N-glycans. For example, only one N-glycan on a rhGAA molecule may carry M6P (monophosphorylated or mono-M6P), a single N-glycan may carry two M6P groups (diphosphorylated 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 M6P groups 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 moles of M6P per mole of rhGAA and greater than 4 moles of sialic acid per mole of rhGAA. In some embodiments, rhGAA comprises, on average, about 3-3.5 moles of M6P per mole of rhGAA. In some embodiments, rhGAA comprises, on average, about 4-5.4 moles of sialic acid per mole of rhGAA. On average, at least about 3, 4, 5, 6, 7, 8, 9, 10%, or 20% of the total N-glycans on rhGAA may be in the form of mono-M6P N-glycans, for example, about 6.25% of the total N-glycans may carry a single M6P group and on average at least about 0.5, 1, 1.5, 2.0, 2.5, 3.0% of the total N-glycans on rhGAA are in the form of di-M6P N-glycans and on average less than 25% of the total rhGAA does not contain phosphorylated N-glycans that bind to CIMPR. In some embodiments, on average, about 10% to about 14% of the total N-glycans on rhGAA are mono-phosphorylated. In some embodiments, on average about 7% to about 25% of the total N-glycans on rhGAA are diphosphorylated. In some embodiments, the rhGAA comprises, on average, about 1.3 moles of bis-M6P per mole of rhGAA.

[0126] The rhGAA described herein can have an average content of M6P-carrying N-glycans ranging from 0.5 to 7.0 moles of M6P per mole of 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, 7.0 moles of M6P per mole of rhGAA. The rhGAA can be fractionated to provide rhGAA preparations having different average amounts of single-M6P or double-M6P-carrying N-glycans, thus allowing further customization of rhGAA targeted to lysosomes in target tissues by selecting specific fractions or by selectively combining different fractions.

[0127] In some embodiments, up to 60% of the N-glycans on rhGAA may be fully sialylated, for example, up to 10%, 20%, 30%, 40%, 50%, or 60% of the N-glycans may be fully sialylated. In some embodiments, no more than 50% of the N-glycans 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 terminal galactose residues (Gal). This range includes all intermediate values ​​and subranges, for example, 7% to 30% of the total N-glycans on rhGAA may carry sialic acid and terminal galactose. In 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 no sialic acid. This range includes all intermediate values ​​and subranges, for example, 8% to 19% of the total N-glycans on rhGAA in the composition may have only terminal galactose and no sialic acid.

[0128] In some embodiments, 40%, 45%, 50%, 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 mixed N-glycans; no more than 5%, 10%, or 15% of the high mannose N-glycans on rhGAA are not phosphorylated; at least 5% or 10% of the high mannose N-glycans on rhGAA are monophosphorylated; and / or at least 1% or 2% of the high mannose N-glycans on rhGAA are diphosphorylated. These values ​​include all intermediate values ​​and subranges. rhGAA can meet one or more of the content ranges described above.

[0129] In some embodiments, rhGAA may have 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 with sialic acid residues prevents the non-substantial clearance of rhGAA by the asialoglycoprotein receptor.

[0130] In one or more embodiments, rhGAA has a specific N-glycosylation distribution 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 for SEQ ID NO:5 and N140 for SEQ ID NO:1). For example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of rhGAA may be phosphorylated at the first potential N-glycosylation site. This phosphorylation may be the result of a single M6P and / or double M6P unit. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA carries a single M6P unit at the first potential N-glycosylation site. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA carries a double M6P unit at the first potential N-glycosylation site. In some embodiments, the rhGAA comprises an average of about 1.4 moles of M6P (mono- and double-M6P) per mole of rhGAA at the first potential N-glycosylation site. In some embodiments, rhGAA comprises an average of about at least 0.5 moles of di-M6P per mole of rhGAA at the first potential N-glycosylation site. In some embodiments, rhGAA comprises an average of about 0.25 moles of mono-M6P per mole of rhGAA at the first potential N-glycosylation site. In some embodiments, rhGAA comprises an average of about 0.2 moles to about 0.3 moles of sialic acid per mole of rhGAA at the first potential N-glycosylation site. In at least one embodiment, rhGAA comprises a first potential N-glycosylation site occupancy as depicted in Figure 6A and an N-glycosylation distribution as depicted in Figure 6B. In at least one embodiment, rhGAA comprises a first potential N-glycosylation site occupancy as depicted in Figure 32A and an N-glycosylation distribution as depicted in Figure 32B or Figure 33B.

[0131] In some embodiments, at least 20% of rhGAA is phosphorylated at the second potential N-glycosylation site (e.g., N177 for SEQ ID NO: 5 and N223 for SEQ ID NO: 1). For example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of rhGAA may be phosphorylated at the second N-glycosylation site. Such phosphorylation may be the result of a single M6P and / or double M6P unit. 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 carries a single 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 carries a double M6P unit at the second N-glycosylation site. In some embodiments, rhGAA comprises an average of about 0.5 moles of M6P (single and double M6P) per mole of rhGAA at the second potential N-glycosylation site. In some embodiments, the rhGAA comprises an average of about 0.4 to about 0.6 moles of monoM6P per mole of rhGAA at the second potential N-glycosylation site. In at least one embodiment, the rhGAA comprises a second potential N-glycosylation site occupancy as depicted in FIG. 6A and an N-glycosylation distribution as depicted in FIG. 6C. In at least one embodiment, the rhGAA comprises a second potential N-glycosylation site occupancy as depicted in FIG. 32A and an N-glycosylation distribution as depicted in FIG. 32C or FIG. 33B.

[0132] In one or more embodiments, at least 5% of the rhGAA is phosphorylated at the third potential N-glycosylation site (e.g., N334 for SEQ ID NO:5 and N390 for SEQ ID NO:1). In other embodiments, less than 5%, 10%, 15%, 20%, or 25% of the 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, diantennary, triantennary, and tetraantennary complex N-glycans, and mixed N-glycans as the main species. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the rhGAA is sialylated at the third potential N-glycosylation site. In some embodiments, rhGAA comprises an average of about 0.9 to about 1.2 moles of sialic acid per mole of rhGAA at the third potential N-glycosylation site. In at least one embodiment, rhGAA comprises a third potential N-glycosylation site occupancy as depicted in FIG. 6A and an N-glycosylation distribution as depicted in FIG. 6D. In at least one embodiment, rhGAA comprises a third potential N-glycosylation site occupancy as depicted in FIG. 32A and an N-glycosylation distribution as depicted in FIG. 32D or FIG. 33B.

[0133] In some embodiments, at least 20% of rhGAA is phosphorylated at the fourth potential N-glycosylation site (e.g., N414 for SEQ ID NO: 5 and N470 for SEQ ID NO: 1). For example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of rhGAA may be phosphorylated at the fourth potential N-glycosylation site. Such phosphorylation may be the result of a single M6P and / or double M6P unit. 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 has a single 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 has a double 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 moles of M6P (mono-M6P and di-M6P) per mole of rhGAA at the fourth potential N-glycosylation site. In some embodiments, rhGAA comprises an average of about 0.4 to about 0.6 moles of di-M6P per mole of rhGAA at the fourth potential N-glycosylation site. In some embodiments, rhGAA comprises an average of about 0.3 to about 0.4 moles of mono-M6P per mole of rhGAA at the fourth potential N-glycosylation site. In at least one embodiment, rhGAA comprises a fourth potential N-glycosylation site occupancy as depicted in FIG. 6A and an N-glycosylation distribution as depicted in FIG. 6E. In at least one embodiment, the rhGAA comprises a fourth potential N-glycosylation site occupancy as depicted in FIG. 32A and an N-glycosylation distribution as depicted in FIG. 32E or FIG. 33B.

[0134] In some embodiments, at least 5% of rhGAA is phosphorylated at the fifth potential N-glycosylation site (e.g., N596 for SEQ ID NO:5 and N692 for SEQ ID NO:1). In other embodiments, less than 5%, 10%, 15%, 20%, or 25% of rhGAA is phosphorylated at the fifth potential N-glycosylation site. For example, the fifth potential N-glycosylation site may have a fucosylated biantennary complex N-glycan 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 moles of sialic acid per mole of rhGAA at the fifth potential N-glycosylation site. In at least one embodiment, rhGAA comprises a fifth potential N-glycosylation site occupancy as depicted in FIG. 6A and an N-glycosylation distribution as depicted in FIG. 6F. In at least one embodiment, rhGAA comprises a fifth potential N-glycosylation site occupancy as depicted in FIG. 32A and an N-glycosylation distribution as depicted in FIG. 32F and FIG. 33B.

[0135] In some embodiments, at least 5% of rhGAA is phosphorylated at the sixth N-glycosylation site (e.g., N826 for SEQ ID NO:5 and N882 for SEQ ID NO:1). In other embodiments, less than 5%, 10%, 15%, 20%, or 25% of rhGAA is phosphorylated at the sixth N-glycosylation site. For example, the sixth N-glycosylation site may have a mixture of diantennary, triantennary, and tetraantennary 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 moles of sialic acid per mole of rhGAA at the sixth potential N-glycosylation site. In some embodiments, rhGAA comprises an average of about 0.9 moles of acetylated sialic acid per mole of rhGAA at the sixth potential N-glycosylation site. In at least one embodiment, rhGAA comprises the sixth potential N-glycosylation site occupancy as depicted in Figure 6A and the N-glycosylation distribution as depicted in Figure 6G. In at least one embodiment, rhGAA comprises the sixth potential N-glycosylation site occupancy as depicted in Figure 32A and the N-glycosylation distribution as depicted in Figures 32G and 33B.

[0136] In some embodiments, at least 5% of rhGAA is phosphorylated at the seventh potential N-glycosylation site (e.g., N869 for SEQ ID NO:5 and N925 for SEQ ID NO:1). In other embodiments, less than 5%, 10%, 15%, 20%, or 25% of rhGAA is phosphorylated at the seventh potential N-glycosylation site. In some embodiments, less than 40%, 45%, 50%, 55%, 60%, or 65% of rhGAA has any N-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 has an average of 0.86 moles of sialic acid per mole of rhGAA at the seventh potential N-glycosylation site. In at least one embodiment, the rhGAA comprises a seventh potential N-glycosylation site occupancy as depicted in FIG. 6A and an N-glycosylation distribution as depicted in FIG. 32H or FIG. 33B.

[0137] In some embodiments, rhGAA comprises an average of 3-4 M6P residues per rhGAA molecule and about 4 to about 7.3 moles of sialic acid per mole of rhGAA. In some embodiments, the rhGAA further comprises an average of about 0.5 moles of bis-M6P per mole of rhGAA at the first potential N-glycosylation site, about 0.4 to about 0.6 moles of mono-M6P per mole of rhGAA at the second potential N-glycosylation site, about 0.9 to about 1.2 moles of sialic acid per mole of rhGAA at the third potential N-glycosylation site, about 0.4 to about 0.6 moles of bis-M6P per mole of rhGAA at the fourth potential N-glycosylation site, about 0.3 to about 0.4 moles of mono-M6P per mole of rhGAA at the fourth potential N-glycosylation site, about 0.8 to about 0.9 moles of sialic acid per mole of rhGAA at the fifth potential N-glycosylation site, and about 1.5 to about 4.2 moles of sialic acid per mole of rhGAA at the sixth potential N-glycosylation site. In at least one embodiment, rhGAA comprises a seventh potential N-glycosylation site occupancy and N-glycosylation distribution as depicted in Figures 6A-6H. In at least one embodiment, rhGAA comprises seven potential N-glycosylation sites, wherein the occupancy and N-glycosylation distribution are as depicted in Figures 32A-32H and Figures 33A-33B.

[0138] Methods for preparing rhGAA are disclosed in US Provisional Patent Application No. 62 / 057,842, filed September 30, 2014, the entire contents of which are incorporated herein by reference.

[0139] Once inside the lysosome, rhGAA can enzymatically degrade the accumulated glycogen. However, traditional rhGAA products have an overall low level of N-glycans with single M6P and double M6P, and therefore poorly target muscle cells, resulting in poor delivery of rhGAA to lysosomes. Most rhGAA molecules in these traditional 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, resulting in non-mass clearance of ERT (Figure 2B). In contrast, as shown in Figure 2A, the rhGAA described herein may contain a higher amount of N-glycans with single M6P and double M6P, resulting in a large amount of rhGAA absorbed into specific tissues such as muscle.

[0140] IV. Production and purification of N-linked glycosylated rhGAA

[0141] As described in International Application PCT / US2015 / 053252 (the entire contents of which are incorporated herein by reference), cells such as Chinese hamster ovary (CHO) cells can be used to produce rhGAA described herein. Expressing high M6P rhGAA in CHO cells is superior to at least partially post-translationally modifying the glycan distribution of rhGAA, because only the former can be converted to a form of rhGAA with optimal glycogen hydrolysis ability through glycan degradation, thereby improving the therapeutic effect.

[0142] In some embodiments, rhGAA is preferably produced by one or more CHO cell lines transformed with a DNA construct encoding rhGAA described herein. Such a CHO cell line may contain multiple copies of the gene, such as 5, 10, 15, or 20 or more copies of a polynucleotide encoding GAA. DNA constructs may be constructed and expressed in CHO cells that express allele variants of acid α-glucosidase or other variant acid α-glucosidase amino acid sequences, such as those that are at least 90%, 95%, 98%, or 99% identical to SEQ ID NO: 1 or SEQ ID NO: 5. Those skilled in the art may select an alternative vector suitable for transforming CHO cells for use in producing such a DNA construct.

[0143] Methods for preparing such CHO cell lines are described in international application PCT / US2015 / 053252, the entire contents of which are incorporated herein by reference. Briefly, these methods involve transforming CHO cells with DNA encoding GAA or GAA variants, selecting CHO cells that stably integrate the DNA encoding GAA into their chromosomes and stably express GAA, and selecting CHO cells that express GAA with high levels of N-glycans with single M6P or double M6P, and optionally, selecting CHO cells with N-glycans containing high sialic acid content and / or with N-glycans containing low non-phosphorylated high mannose 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 culture of the CHO cell. In some embodiments, the rhGAA produced by the selected CHO cell line contains a high content of N-glycans with single M6P or double M6P targeting CIMPR. In some embodiments, the rhGAA produced as described herein has low levels of complex N-glycans with terminal galactose. In some embodiments, the selected CHO cell line is referred to as GA-ATB200 or ATB200-X5-14. In some embodiments, the selected CHO cell line encompasses a subculture or derivative of such a CHO cell culture. In some embodiments, the rhGAA produced by the selected CHO cell line is referred to as ATB200.

[0144] The rhGAA produced as described herein can be purified by following the methods described in International Application PCT / US2017 / 024981 and U.S. Provisional Application No. 62 / 506,569, both of which are incorporated herein by reference in their entirety. An exemplary method for producing, capturing, and purifying rhGAA produced by a CHO cell line is shown in FIG. 3 .

[0145] Briefly, bioreactor 601 contains a culture of cells, such as CHO cells, that express and secrete rhGAA into the surrounding liquid culture medium. Bioreactor 601 may be any suitable bioreactor for culturing cells, such as a perfusion, batch, or batch-fed bioreactor. After the cells have been through a sufficient period of time to produce rhGAA, the culture medium is removed from the bioreactor. This culture medium removal may be continuous for a perfusion bioreactor or may be performed batch by batch for a batch or fed-batch reactor. The culture medium may be filtered through a filtration system 603 to remove cells. Filtration system 603 may be any suitable filtration system, including an alternating tangential flow filtration (ATF) system, a tangential flow filtration (TFF) system, and / or a centrifugal filtration system. In various embodiments, the filtration system utilizes a filter having a pore size between about 10 nanometers and about 2 microns.

[0146] After filtration, the filtrate is loaded onto a protein capture system 605. The protein capture system 605 may include one or more chromatography columns. If more than one chromatography column is used, the columns may be placed in series so that loading of the next column can begin as soon as the first column is loaded. Alternatively, the media removal process may be stopped during the time it takes to switch columns.

[0147] In various embodiments, the protein capture system 605 includes one or more anion exchange (AEX) columns for direct product capture of rhGAA, particularly rhGAA with a high M6P content. The rhGAA captured by the protein capture system 605 is eluted from the column by changing the pH and / or salt content in the column. Exemplary conditions for the AEX column are provided in Table 2.

[0148] Table 2. Exemplary conditions for AEX columns

[0149]

[0150] The eluted rhGAA may be subjected to further purification steps and / or quality assurance steps. For example, the eluted rhGAA may be subjected to a virus killing step 607. Such virus killing 607 may include one or more of: low pH killing, detergent killing, or other techniques known in the art. The rhGAA from the virus killing step 607 may be introduced into a second chromatography system 609 to further purify the rhGAA product. Alternatively, the eluted rhGAA from the protein capture system 605 may be directly supplied to the second chromatography system 609. In various embodiments, the second chromatography system 609 includes one or more immobilized metal affinity chromatography (IMAC) columns for further removal of impurities. Exemplary conditions for an IMAC column are provided in Table 3 below.

[0151] Table 3. Exemplary conditions for IMAC columns

[0152]

[0153] After rhGAA is loaded onto the second chromatography system 609, the recombinant protein is eluted from the column. The eluted rhGAA may be subjected to a virus killing step 611. As with virus killing 607, virus killing 611 may include one or more of: low pH killing, detergent killing, or other techniques known in the art. In some embodiments, only one of virus killing 607 or 611 is used, or these virus killing steps are performed at the same stage in the purification process.

[0154] The rhGAA from the virus killing step 611 can be introduced into the third chromatography system 613 to further purify the recombinant protein product. Alternatively, the eluted recombinant protein from the second chromatography system 609 can be directly supplied to 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 removal of impurities. The rhGAA product is then eluted from the third chromatography system 613. Exemplary conditions for the CEX column are provided in Table 4 below.

[0155] Table 4. Exemplary conditions for CEX columns

[0156]

[0157] The rhGAA product may also be further processed. 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 μm and 50 μm. Other product processing may include a product conditioning step 617, wherein the recombinant protein product may be sterilized, filtered, concentrated, stored, and / or have additional components added to the final product formulation.

[0158] As used herein, the term "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.

[0159] V. Pharmaceutical Compositions

[0160] 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.

[0161] In one or more embodiments, the pharmaceutical compositions described herein comprise a pharmaceutically acceptable salt.

[0162] In some embodiments, the pharmaceutically acceptable salts used herein are pharmaceutically acceptable acid addition salts. Pharmaceutically acceptable acid addition salts may 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, asparagine, benzenesulfonic acid, benzoic acid, butyric acid, camphoric acid, camphorsulfonic acid, cinnamic acid, citric acid, digluconic acid, ethanesulfonic acid, glutamic acid, glycolic acid, glycerophosphoric acid, hemisulfonic acid, hexanoic acid, formic acid, fumaric acid, 2-hydroxyethanesulfonic acid (2-hydroxyethanesulfonic acid), lactic acid, hydroxymaleic acid, malic acid, malonic acid, mandelic acid, trimethylolbenzenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, nicotinic acid, 2-naphthalenesulfonic acid, oxalic acid, pamoic acid, pectinic acid, phenylacetic acid, 3-phenylpropionic acid, trimethylolacetic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, p-toluenesulfonic acid, undecanoic acid, and the like.

[0163] In some embodiments, the pharmaceutically acceptable salt used herein is a pharmaceutically acceptable base addition salt. Pharmaceutically acceptable base addition salts may include, but are not limited to, ammonia, or hydroxides, carbonates, or bicarbonates of 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 amines, secondary amines, and tertiary amines, quaternary amine compounds, including naturally occurring substituted amines, cyclic amines, and alkaline ion exchange resins, such as methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, isopropylamine, tripropylamine, tributylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicycloamine, lysine, spermine Acid, histidine, caffeine, hydrazine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, tetramethylammonium compounds, tetraethylammonium compounds, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicycloamine, dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine, N,N'-dibenzylethylenediamine, polyamine resins, and the like.

[0164] In some embodiments, rhGAA or a pharmaceutically acceptable salt thereof can be formulated as 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 ingredients of the pharmaceutical composition may be supplied separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or anhydrous concentrate in an airtight sealed container such as an ampoule or capsule indicating the amount of active agent. In the case where the composition is to be administered by infusion, an infusion bottle containing sterile pharmaceutical grade water, saline or glucose / water may be used to distribute the composition. In some embodiments, the infusion may be performed in a hospital or clinic. In some embodiments, the infusion may be performed outside a hospital or clinic facility, for example, at the subject's residence. In the case where the composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients can be mixed before administration.

[0165] In some embodiments, rhGAA or a pharmaceutically acceptable salt thereof can be formulated for oral administration. Orally administrable compositions can be formulated in the following forms: tablets, capsules, ovules, elixirs, solutions or suspensions, gels, syrups, mouthwashes, or dry powders for rehydration with water or other suitable vehicles before use, optionally with flavoring agents and coloring agents for immediate release, delayed release, modified release, sustained release, pulsed release, or controlled release applications. Solid compositions such as tablets, capsules, lozenges, pellets, pills, boluses, powders, pastes, granules, pellets, dragees, or premixed formulations can also be used. Solid and liquid compositions for oral use can be prepared according to methods well known in the art. Such compositions can also contain one or more pharmaceutically acceptable carriers and excipients, which can be in solid or liquid form. Tablets or capsules can be prepared by conventional means using 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), hydroxypropyl cellulose (HPC), sucrose, gelatin, gum arabic, lactose, microcrystalline cellulose, calcium hydrogen phosphate, magnesium stearate, stearic acid, glyceryl behenate, talc, silica, corn, potato or tapioca starch, sodium starch glycolate, sodium lauryl sulfate, sodium citrate, calcium carbonate, dicalcium hydrogen phosphate, sodium croscarmellose, and complex silicates. Tablets may be coated by methods well known in the art.

[0166] In some embodiments, the pharmaceutical compositions described herein may be formulated according to International Application PCT / US2017 / 024982 and U.S. Provisional Application No. 62 / 506,574, both of which are incorporated herein by reference in their entirety. For example, in some embodiments, the pH of the pharmaceutical compositions described herein is about 5.0 to about 7.0 or about 5.0 to about 6.0. In some embodiments, the pH range is 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 alkalizer and an acidulant) such as sodium hydroxide and / or hydrochloric acid.

[0167] The pharmaceutical compositions described herein may comprise a buffer system, such as a citrate system, a phosphate system, and combinations of the foregoing. The citrate and / or phosphate may be sodium citrate or sodium phosphate. Other salts include potassium salts and ammonium salts. In one or more embodiments, the buffer comprises citrate. In other embodiments, the buffer comprises sodium citrate (e.g., a mixture of sodium citrate dehydrate and citric acid monohydrate). In one or more embodiments, a buffer solution comprising citrate may comprise sodium citrate and citric acid. In some embodiments, there is a citrate and phosphate buffer.

[0168] In some embodiments, the pharmaceutical compositions described herein include at least one excipient. An excipient can act as a tonicity agent, a bulking agent, and / or a stabilizer. A tonicity agent is a component that helps ensure that the formulation has an osmotic pressure similar to or the same as human blood. A bulking agent is a component that adds mass to the formulation (e.g., freeze drying) and provides sufficient structure to the cake. A stabilizer is a compound that can prevent or minimize the formation of aggregates at the hydrophobic air-water interface surface. An excipient can act as a tonicity agent and a bulking agent at the same time. For example, mannitol can act as a tonicity agent and also provide benefits as a bulking agent.

[0169] Examples of tonicity agents include sodium chloride, mannitol, sucrose, and trehalose. In some embodiments, the tonicity agent comprises mannitol. In some embodiments, the total amount of tonicity agent ranges from about 10 mg / mL to about 50 mg / mL. In other embodiments, the total amount of tonicity agent 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.

[0170] In some embodiments, the excipient comprises a stabilizer. In some embodiments, the stabilizer is a surfactant. In some embodiments, the stabilizer is polysorbate 80. In one or more embodiments, the total amount of the stabilizer ranges from about 0.1 mg / mL to about 1.0 mg / mL. In other embodiments, the total amount of the stabilizer ranges from about 0.1, 0.2, 0.3, 0.4 or 0.5 mg / mL to about 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 mg / mL. In other embodiments, the total amount of the stabilizer is about 1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 mg / mL.

[0171] In some embodiments, the pharmaceutical composition comprises (a) rhGAA (such as ATB200), (b) at least one buffer selected from the group consisting of citrate, phosphate, and a combination thereof, and (c) at least one excipient selected from the group consisting of mannitol, polysorbate 80, and a combination thereof, and has a pH of (i) about 5.0 to about 6.0 or (ii) about 5.0 to about 7.0. In some embodiments, the composition further comprises water. In some embodiments, the composition may further comprise an acidifier and / or an alkalizer.

[0172] In some embodiments, the pharmaceutical composition comprises (a) rhGAA (such as ATB200) at a concentration of about 5-50 mg / mL, about 5-30 mg / mL, or about 15 mg / mL, (b) sodium citrate buffer at a concentration of about 10-100 mM or about 25 mM, (c) mannitol at a concentration of about 10-50 mg / mL, or about 20 mg / mL, (d) polysorbate 80 present at a concentration of about 0.1-1 mg / mL, about 0.2-0.5 mg / mL, or about 0.5 mg / mL, and (e) water, and has a pH of about 6.0. In at least one embodiment, the pharmaceutical composition comprises (a) 15 mg / mL rhGAA (such as ATB200), (b) 25 mM sodium citrate buffer, (c) 20 mg / mL mannitol, (d) 0.5 mg / mL polysorbate 80, and (e) water, and has a pH of about 6.0. In some embodiments, the composition may further comprise an acidifying agent and / or an alkalizing agent.

[0173] In some embodiments, the pharmaceutical composition comprising rhGAA is diluted prior to administration to a subject in need thereof.

[0174] In some embodiments, the pharmaceutical compositions described herein comprise a chaperone. In some embodiments, the chaperone is miglustat or a pharmaceutically acceptable salt thereof. In another embodiment, the chaperone is duglustat or a pharmaceutically acceptable salt thereof.

[0175] In some embodiments, rhGAA described herein is formulated in one pharmaceutical composition and a chaperone such as miglustat is formulated in another pharmaceutical composition. In some embodiments, a pharmaceutical composition comprising miglustat is based on a (Actelion Pharmaceuticals) commercially available formulation.

[0176] In some embodiments, the pharmaceutical compositions described herein may be subjected to a freeze-drying (freeze-drying) process to provide a cake or powder. Therefore, another aspect of the present invention is to provide a pharmaceutical composition after freeze-drying. The freeze-dried mixture may include rhGAA (e.g., ATB200) described herein, a buffer selected from a group consisting of citrate, phosphate, and a combination of the foregoing, and at least one excipient selected from a group consisting of trehalose, mannitol, polysorbate 80, and a combination of the foregoing. 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 may be provided in a vial, which may then be stored, transported, rehydrated, and / or administered to a patient.

[0177] VI. Treatment Methods

[0178] A. Treatment of diseases

[0179] Another aspect of the invention is a method of treating a disease or condition associated with poorly regulated glycogen storage by administering rhGAA or a pharmaceutical composition 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. In some embodiments, the pharmaceutical composition comprises ATB200.

[0180] The rhGAA or pharmaceutical compositions described herein are administered by an appropriate route. In one embodiment, the rhGAA or pharmaceutical composition is administered intravenously. In other embodiments, the rhGAA or pharmaceutical composition is administered by direct administration to the target tissue to reach the heart or skeletal muscle (e.g., intramuscularly), or the nervous system (e.g., directly injected 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 in parallel.

[0181] 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 seen in GSD-II), (2) lung function (e.g., an increase in crying vital capacity relative to baseline volume, and / or normalization of oxygen desaturation during crying), (3) neurodevelopment and / or motor skills (e.g., an increase in AIMS scores), and (4) a reduction in glycogen levels in disease-affected tissues of the subject.

[0182] In some embodiments, the cardiac state of the subject improves by 10%, 20%, 30%, 40%, or 50% (or any percentage in between) after administering one or more doses of rhGAA or a pharmaceutical composition described herein compared to the cardiac state of a subject treated with a vehicle or the cardiac state of a subject before treatment. The cardiac state of the subject can be assessed by measuring the end-diastolic and / or end-systolic volumes and / or by clinically evaluating cardiomyopathy. In some embodiments, the pulmonary function of the subject improves by 10%, 20%, 30%, 40%, or 50% (or any percentage in between) after administering one or more doses of ATB200 or a pharmaceutical composition comprising ATB200 compared to the pulmonary function of a subject treated with a vehicle or the pulmonary function of a subject before treatment. In certain embodiments, improvement is achieved after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or longer (or any time period in between) from administration. In certain embodiments, ATB200 or a pharmaceutical composition comprising ATB200 improves lung function in a subject after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or more (or any time period in between) from administration.

[0183] In some embodiments, the lung function of the subject is improved by 10%, 20%, 30%, 40%, or 50% (or any percentage in between) after administering one or more doses of rhGAA or a pharmaceutical composition described herein compared to the lung function of a subject treated with a vehicle or the lung function of a subject before treatment. The lung function of the subject can be assessed by normalization of the cry vital capacity and / or oxygen desaturation during cry relative to the baseline capacity. In some embodiments, the lung function of the subject is improved by 10%, 20%, 30%, 40%, or 50% (or any percentage in between) after administering one or more doses of ATB200 or a pharmaceutical composition comprising ATB200 compared to the lung function of a subject treated with a vehicle or the lung function of a subject before treatment. In certain embodiments, improvement is achieved after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or longer (or any time period in between) from administration. In certain embodiments, ATB200 or a pharmaceutical composition comprising ATB200 improves lung function in a subject after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or more (or any time period in between) from administration.

[0184] In some embodiments, the neurodevelopment and / or motor skills of the subject are improved by 10%, 20%, 30%, 40%, or 50% (or any percentage in between) after administration of one or more doses of rhGAA or a pharmaceutical composition described herein compared to the neurodevelopment and / or motor skills of the subject treated with a vehicle or the neurodevelopment and / or motor skills of the subject before treatment. The neurodevelopment and / or motor skills of the subject can be assessed by measuring the AIMS score. The AIMS is a 12-item fixed scale that is administered and scored by a clinician (see Rush JA Jr., Handbook of Psychiatric Measures, American Psychiatric Association, 2000, 166-168). Items 1-10 are graded on a 5-point fixed scale. Items 1-4 assess oral-facial movement. Items 5-7 deal with limb and trunk movement difficulties. Items 8-10 assess the overall severity as judged by the examiner, and the patient's awareness of movement and the pain associated with it. Items 11-12 are true / false questions about teeth and / or dentures (such questions can lead to the misdiagnosis of motor difficulties). In some embodiments, the neurodevelopment and / or motor skills of the subject are improved by 10%, 20%, 30%, 40%, or 50% (or any percentage in between) compared to the neurodevelopment and / or motor skills of the subject treated with the vehicle or the neurodevelopment and / or motor skills of the subject before treatment after applying one or more doses of ATB200 or a pharmaceutical composition comprising ATB200. In certain embodiments, improvement is achieved after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or longer (or any time period in between) from the application. In certain embodiments, ATB200 or a pharmaceutical composition comprising ATB200 improves the neurodevelopment and / or motor skills of the subject after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or longer (or any time period in between) from the application.

[0185] In some embodiments, the glycogen level of a tissue of a subject is reduced by 10%, 20%, 30%, 40%, or 50% (or any percentage in between) after administration of one or more doses of rhGAA or a pharmaceutical composition described herein compared to the glycogen level of a subject treated with vehicle or the glycogen level of a subject before treatment. In some embodiments, the tissue is muscle, such as the quadriceps, deltoid, and gastrocnemius. The glycogen level of the tissue can be analyzed using methods known in the art. It is known that the determination of glycogen levels is based on amyloglucosidase digestion and is described in publications such as Amalfitano et al. (1999), "Systemic correction of the muscledisorder glycogenstorage disease type ii after hepatic targeting of a modified adenovirus vector encoding human acid-alphaglucosidase", Proc Natl Acad Sci USA, 96: 8861-8866. In some embodiments, the glycogen level in the muscle of the subject is reduced by 10%, 20%, 30%, 40%, or 50% (or any percentage in between) after administration of one or more doses of ATB200 or a pharmaceutical composition comprising ATB200 compared to the glycogen level of a subject treated with a vehicle or the glycogen level of a subject before treatment. In certain embodiments, the reduction is achieved after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or more (or any time period in between) from administration. In certain embodiments, ATB200 or a pharmaceutical composition comprising ATB200 reduces the glycogen level in the muscle of the subject after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or more (or any time period in between) from administration.

[0186] B. Biomarkers

[0187] Biomarkers of glycogen accumulation in muscle fibers of a subject, such as urinary hexotetrasaccharide (Hex4), can be used to assess and compare the therapeutic effect of enzyme replacement therapy in subjects with Pompe disease. In some embodiments, the therapeutic effect of rhGAA or a pharmaceutical composition comprising rhGAA on glycogen accumulation is assessed by measuring the level of Hex4 in a subject.

[0188] Biomarkers of muscle damage or destruction such as creatine kinase (CK), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) can be used to assess and compare the therapeutic effect of enzyme replacement therapy in subjects with Pompe disease. In some embodiments, the therapeutic effect of rhGAA or a pharmaceutical composition comprising rhGAA on muscle damage is assessed by measuring the levels of CK, 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 levels of CK in the subject.

[0189] Biomarkers such as LAMP-1, LC3, and Dysferlin can also be used to assess and compare the therapeutic effects of rhGAA or pharmaceutical compositions described herein. In Pompe disease, GAA is unable to hydrolyze lysosomal glycogen, resulting in abnormal accumulation of large lysosomes filled with glycogen in some tissues. (Raben et al., JBC 273: 19086-19092, 1998.) Studies in mouse models of Pompe disease have shown that increased lysosomes in skeletal muscle cannot fully explain the reduction in mechanical efficiency, and the presence of large contents containing degraded myofibrils (that is, autophagic accumulation) contributes to the impairment of muscle function. (Raben et al., Human Mol Genet 17: 3897-3908, 2008.) Reports also suggest that impaired autophagic flux is associated with poor treatment 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 common in unclassified limb-girdle muscular dystrophy (LGMD) (Preisler et al., Mol Genet Metab 110: 287-289, 2013), which is caused by the deficiency of various components of the dystrophin-glycoprotein complex (DGC). IHC examination showed a substantially increased sarcoplasmic presence of dysferlin in skeletal muscle fibers of Gaa KO mice.

[0190] Various known methods can be used to measure the gene expression level and / or protein level of such biomarkers. For example, a sample from a subject treated with rhGAA or a pharmaceutical composition described herein, such as a biological specimen of tissue, especially muscle, can be obtained. In some embodiments, the sample is a biological specimen of muscle in a subject. In some embodiments, the muscle is selected from the quadriceps, deltoid, and gastrocnemius. The sample obtained from the subject can be stained with one or more antibodies or other detection agents for detecting such biomarkers. The sample may also or alternatively be processed for detecting the presence of nucleic acids encoding biomarkers such as mRNA by, for example, RT-qPCR methods.

[0191] In some embodiments, the gene expression level and / or protein level of one or more biomarkers is measured in a muscle biopsy obtained from an individual before and after treatment with rhGAA or a pharmaceutical composition described herein. In some embodiments, the gene expression level and / or protein level of one or more biomarkers is measured in a muscle biopsy obtained from an individual treated with a vehicle. 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 in between) after administration of one or more doses of rhGAA or a pharmaceutical composition described herein compared to the gene expression level and / or protein level of a subject treated with a vehicle or the gene expression level and / or protein level 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 in between) compared to the gene expression level and / or protein level of a subject treated with a vehicle or the gene expression level and / or protein level of a subject before treatment after administration of one or more doses of ATB200 or a pharmaceutical composition comprising ATB200. In certain embodiments, the reduction is achieved after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or longer (or any time period in between) from administration. In certain embodiments, ATB200 or a pharmaceutical composition comprising ATB200 reduces the gene expression level and / or protein level of one or more biomarkers after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or longer (or any time period in between) from administration.

[0192] C. Administration of rhGAA

[0193] The pharmaceutical formulation or rehydration composition is administered in a therapeutically effective amount (e.g., an amount sufficient to treat the disease, such as to improve symptoms associated with the disease, prevent or delay the onset of the disease, and / or reduce the severity or frequency of disease symptoms when administered at regular intervals). An amount that is therapeutically effective in treating a disease may depend on the nature of the disease and the extent of the effects of the disease, and may be determined by standard clinical techniques. In addition, in vitro or in vivo assays may be used to help identify optimal dosage ranges as appropriate. In at least one embodiment, rhGAA or a pharmaceutical composition comprising rhGAA described herein is administered in a dose of about 1 mg / kg to about 100 mg / kg, such as about 5 mg / kg to about 30 mg / kg, typically about 5 mg / kg to about 20 mg / kg. In at least one embodiment, the rhGAA or pharmaceutical composition described herein is administered at a dose of about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 50 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg. In some embodiments, 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 concurrently or sequentially with a pharmacological chaperone. In some embodiments, the pharmacological chaperone is miglustat. In at least one embodiment, miglustat is administered at an oral dose of about 260 mg. The effective dose for a particular individual may vary (e.g., increase or decrease) over time, depending on the individual's needs. For example, the amount may be increased when the body is ill or stressed, or if anti-acid α-glucosidase antibodies become present or increase, or if disease symptoms worsen.

[0194] In some embodiments, the therapeutically effective dose of rhGAA or a pharmaceutical composition described herein is lower than that 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 than a conventional rhGAA product may be lower than 20 mg / kg. The therapeutic effect may be assessed based on one or more criteria discussed above (e.g., cardiac status, glycogen levels, or biomarker performance). 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 greater than that of a conventional rhGAA product.

[0195] In some embodiments, the therapeutic effect of rhGAA or a pharmaceutical composition described herein comprises an improvement in motor function, an improvement in muscle strength (upper, lower, or whole body), an improvement in lung function, a reduction in fatigue, a reduction in the level of at least one biomarker of muscle damage, a reduction in the level of at least one biomarker of glycogen accumulation, or a combination of the foregoing. In some embodiments, the therapeutic effects of rhGAA or a pharmaceutical composition described herein comprise reversal of lysosomal pathology in muscle fibers, faster and / or more effective reduction in glycogen content in muscle fibers, increase in six minute walk test distance, decrease in timed up and go test time, decrease in four stair climb test time, decrease in ten meter walk test time, decrease in gait-stair-gower-chair score, increase in upper extremity strength, improvement in shoulder adduction, improvement in shoulder abduction, improvement in elbow flexion, improvement in elbow extension, improvement in upper body strength, improvement in lower body strength, improvement in whole body strength, improvement in upright (sitting) forced vital capacity, improvement in maximum expiratory pressure, improvement in maximum inspiratory pressure, decrease in fatigue severity scale score, decrease in urine hexotetraose levels, decrease in creatine kinase levels, decrease in alanine transaminase levels, decrease in aspartate transaminase levels, or any combination thereof.

[0196] 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 performance). For example, if a single dose of a conventional rhGAA product reduces glycogen levels in the tissues of the 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 times, or faster than conventional rhGAA products when administered at the same dose.

[0197] In some embodiments, a therapeutically effective amount of rhGAA (or a composition or medicament comprising rhGAA) is administered more than once. In some embodiments, the rhGAA or pharmaceutical composition described herein is administered at regular intervals, depending on the nature of the disease and the extent of the effect of the disease, and on the developing condition. Administration at "regular intervals" as used herein indicates that the therapeutically effective amount is administered periodically (as distinguished from a single 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 interval for administration of a single individual does not need to be a fixed interval, but may vary over time, depending on the needs of the individual. For example, when the body is ill or stressed, if anti-rhGAA antibodies are present or increased, or if symptoms of the disease worsen, the interval between doses can be reduced.

[0198] In some embodiments, when used at the same dose, rhGAA or pharmaceutical compositions as described herein may be administered less frequently than traditional rhGAA products and still be able to produce the same or better therapeutic effect than traditional rhGAA products. For example, if a traditional rhGAA product is administered at 20 mg / kg once a week, rhGAA or pharmaceutical compositions as described herein may produce the same or better therapeutic effect when administered at 20 mg / kg compared to traditional rhGAA products, even if the rhGAA or pharmaceutical composition is administered less frequently, for example, once every two weeks or once a month. The therapeutic effect may be assessed based on one or more criteria discussed above (e.g., cardiac status, glycogen levels, or biomarker performance). In some embodiments, the interval between two doses of rhGAA or pharmaceutical compositions described herein is longer than that of traditional rhGAA products. In some embodiments, the interval between two doses of rhGAA or pharmaceutical compositions is at least about 1.25, 1.5, 1.75, 2.0, 3.0 times, or longer than that of traditional rhGAA products.

[0199] In some embodiments, under the same treatment conditions (e.g., the same dose is administered at the same interval), the rhGAA or pharmaceutical composition described herein provides a therapeutic effect to a degree that is superior to that provided by a traditional rhGAA product. The therapeutic effect can be assessed based on one or more criteria discussed above (e.g., cardiac status, glycogen levels, or biomarker performance). For example, when compared to a traditional rhGAA product administered at 20 mg / kg once a week, rhGAA or a pharmaceutical composition administered once a 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 composition described herein provides a therapeutic effect that is at least about 1.25, 1.5, 1.75, 2.0, 3.0 times, or greater than those of a traditional rhGAA product.

[0200] D. Combination therapy

[0201] In one or more embodiments, rhGAA or a pharmaceutical composition comprising rhGAA described herein is administered concurrently or sequentially with a pharmacological chaperone. In some embodiments, rhGAA or a pharmaceutical composition is administered by a different route than the pharmacological chaperone. For example, the pharmacological chaperone may be administered orally while rhGAA or a pharmaceutical composition is administered intravenously.

[0202] In various embodiments, the pharmacological chaperone is miglustat. In some embodiments, miglustat is administered at an oral dose of about 50 mg to about 600 mg. In at least one embodiment, miglustat is administered at an oral dose of about 200 mg to about 600 mg, or at an oral dose of about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, or about 600 mg. In at least one embodiment, miglustat is administered at an oral dose of about 233 mg to about 500 mg. In at least one embodiment, miglustat is administered at an oral dose of about 250 to about 270 mg, or at an oral dose of about 250 mg, about 255 mg, about 260 mg, about 265 mg, or about 270 mg. In at least one embodiment, miglustat is administered at an oral dose of about 260 mg.

[0203] It will be understood by those skilled in the art that an oral dose of Miglut in the range of about 200 mg to 600 mg, or any smaller range therein, may be suitable for adult patients depending on their weight. For example, for patients with a body weight significantly less than about 70 kg, including but not limited to infants, children, or underweight adults, a smaller dose may be appropriately considered by a physician. Thus, in at least one embodiment, Miglut 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, 125 mg, about 150 mg, about 175 mg, or about 200 mg. In at least one embodiment, Miglut is administered at an oral dose of about 65 mg to about 195 mg, or at an oral dose of about 65 mg, about 130 mg, or about 195 mg.

[0204] In some embodiments, rhGAA is administered intravenously at a dose of about 5 mg / kg to about 20 mg / kg and miglustat is administered orally at a dose of about 50 mg to about 600 mg. In some embodiments, rhGAA is administered intravenously at a dose of about 5 mg / kg to about 20 mg / kg and miglustat is administered orally at a dose of about 50 mg to about 200 mg. In some embodiments, rhGAA is administered intravenously at a dose of about 5 mg / kg to about 20 mg / kg and miglustat is administered orally at a dose of about 200 mg to about 600 mg. In some embodiments, rhGAA is administered intravenously at a dose of about 5 mg / kg to about 20 mg / kg and miglustat is administered orally at a dose of about 233 mg to about 500 mg. In one embodiment, rhGAA is administered intravenously at a dose of about 20 mg / kg and miglustat is administered orally at a dose of about 260 mg.

[0205] In some embodiments, miglustat and rhGAA are administered concurrently. For example, miglustat may 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.

[0206] 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 about two hours prior to the administration of rhGAA. For example, miglustat may be administered about 1.5 hours, about 1 hour, about 50 minutes, about 30 minutes, or about 20 minutes prior to the administration of rhGAA. In at least one embodiment, miglustat is administered about one hour prior to the administration of rhGAA.

[0207] 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 may be administered within about 1.5 hours, about 1 hour, about 50 minutes, about 30 minutes, or about 20 minutes after administration of rhGAA.

[0208] In some embodiments, the subject fasts for at least two hours before and at least two hours after administration of miglustat.

[0209] E. Set

[0210] Another aspect of the invention relates to a kit comprising rhGAA or a pharmaceutical composition described herein. In one or more embodiments, the kit comprises a container (e.g., a vial, a tube, a bag, etc.) comprising rhGAA or a pharmaceutical composition (before or after lyophilization) and comprising instructions for reconstitution, dilution, and administration.

[0211] Examples

[0212] Example 1: Preparation of rhGAA-producing CHO cells with high content of N-glycans with mono- or di-M6P.

[0213] DG44 CHO (DHFR-) cells were transfected with a DNA construct expressing rhGAA. The DNA construct is shown in Figure 4. After transfection, CHO cells containing a stably integrated GAA gene were selected using medium lacking hypoxanthine / thymidine (-HT). GAA expression in these cells was induced by methotrexate treatment (MTX, 500 nM).

[0214] The cell pools showing high amounts of GAA are identified by GAA enzyme activity assay and used to establish individual pure lines producing rhGAA. Individual pure lines are produced on semisolid culture plates, picked up by the ClonePix system, and transferred to 24 deep well plates. The GAA enzyme activity of individual pure lines is analyzed to identify the pure lines showing high levels of GAA. The conditioned medium used to measure GAA activity uses 4-MU-α-glucosidase substrate. Further assessment is made of the viability, growth ability, GAA productivity, N-polysaccharide structure and stable protein performance of the pure lines producing higher levels of GAA as measured by GAA enzyme assay. This procedure is used to separate the CHO cell lines showing rhGAA with enhanced single M6P or double M6P N-polysaccharides, including the CHO cell line GA-ATB200.

[0215] Example 2: Purification of rhGAA

[0216] Batches of rhGAA according to the present invention were produced using the CHO cell line GA-ATB200 in shake flasks and in perfusion bioreactors, the product being referred to as "ATB200". Weak anion exchange ("WAX") liquid chromatography was used to fractionate ATB200 rhGAA based on terminal phosphates. Elution profiles were generated by eluting the ERT with increasing amounts of salt. The profiles were monitored by UV (A280nm). Similar CIMPR receptor binding (at least about 70%) profiles were observed for purified ATB200 rhGAA from different production batches (Figure 5), indicating that ATB200 rhGAA can be consistently produced.

[0217] Example 3: Oligosaccharide Characterization of ATB200 rhGAA

[0218] The site-specific N-glycan distribution of ATB200 rhGAA was analyzed using two different LC-MS / MS analytical techniques. The results of the first two LC-MS / MS methods are shown in Figures 6A-6H. The results of the third LC-MS / MS method with the 2-AA glycan profile are shown in Figures 32A-32H, Figures 33A-33B Figure and Table 5.

[0219] In the first analysis, proteins were denatured, reduced, alkylated, and digested prior to LC-MS / MS analysis. During protein denaturation and reduction, 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 to a 1.5 mL tube to provide a total volume of 100 μL. The samples are mixed and incubated at 56° C. in a waterless bath for 30 minutes. During alkylation, the denatured and reduced protein samples are mixed with 5 μL of 1 mol / L iodoacetamide (IAM, final concentration 50 mM), then incubated in the dark at 10-30° C. for 30 minutes. After alkylation, 400 μL of pre-cooled acetone is added to the sample and the mixture is frozen at -80° C. for 4 hours. The sample is then centrifuged at 13000 rpm at 4° C. for 5 min and the supernatant is removed. 400 μL of pre-cooled acetone is added to the mass, which is then centrifuged at 13000 rpm at 4° C. for 5 min and the supernatant is removed. The sample is then air dried on ice in the dark to remove acetone residues. Forty microliters of 8M urea and 160 μL of 100 mM NH4HCO3 are added to the sample to dissolve the protein. During trypsin digestion, 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 (protein to enzyme ratio of 20 / 1 w / w). The solution was mixed well and incubated at 37°C overnight (16 ± 2 hours). 2.5 microliters of 20% TFA (final concentration 0.5%) was added to quench the reaction. Thermo Scientific TM Orbitrap Velos Pro TM A mass spectrometer analyzes the samples.

[0220] 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 as the alkylation agent instead of IAM, and then the ATB200 samples were purified by Thermo Scientific TM Orbitrap Fusion TM Lumos Tribid TM Mass spectrometry analysis.

[0221] The results of the first and second analyses are shown in FIGS. 6A-6H. In FIGS. 6A-6H, the results of the first analysis are represented by the left bars (dark grey) and the results from the second analysis are represented by the right bars (light grey). The symbol nomenclature used for glycan representation is according to Varki, A., Cummings, RD, Esko JD et al., Essentials of Glycobiology, 2nd ed. (2009).

[0222] As can be seen from Figures 6A-6H, the two analyses provide similar results, but there is some variation between the results. This variation may be due to many factors, including the instrument used and the integrity of the N-glycan analysis. For example, if some species of phosphorylated N-glycans are not identified and / or quantified, the total number of phosphorylated N-glycans may be underrepresented, and the percentage of rhGAA with phosphorylated N-glycans at that site may be underrepresented. As another example, if some species of non-phosphorylated N-glycans are not identified and / or quantified, the total number of non-phosphorylated N-glycans may be underrepresented, and the percentage of rhGAA with phosphorylated N-glycans at that site may be overrepresented.

[0223] FIG. 6A shows the N-glycosylation site occupancy of ATB200. As can be seen from FIG. 6A, the first, second, third, fourth, fifth, and sixth N-glycosylation sites are almost all occupied, with two analyses detecting approximately or more than 90% and up to approximately 100% of the ATB200 enzymes having detected N-glycans at each potential N-glycosylation site. However, the seventh potential N-glycosylation site is N-glycosylated about half of the time.

[0224] FIG. 6B shows the N-glycosylation distribution of the first potential N-glycosylation site N84. As can be seen from FIG. 6B, the main N-glycan species is di-M6P N-glycan. The first and second analyses detected that more than 75% of ATB200 at the first site had di-M6P, which corresponds to an average of about 0.8 moles of di-M6P per mole of ATB200 at the first site.

[0225] FIG. 6C shows the N-glycosylation distribution of the second potential N-glycosylation site N177. As can be seen from FIG. 6C, the main N-glycan species are mono-M6P N-glycans and non-phosphorylated high mannose N-glycans. The first and second analyses detected that more than 40% of ATB200 at the second site had mono-M6P, which corresponds to an average of about 0.4 to about 0.6 moles of mono-M6P per mole of ATB200 at the second site.

[0226] FIG. 6D shows the N-glycosylation distribution of the third potential N-glycosylation site N334. As can be seen from FIG. 6D, the main N-glycan species are non-phosphorylated high mannose N-glycans, diantennary, triantennary, and tetraantennary complex N-glycans, and mixed N-glycans. The first analysis and the second analysis detected that more than 20% of ATB200 at the third site had sialic acid residues, which corresponded to an average of about 0.9 to about 1.2 moles of sialic acid per mole of ATB200 at the third site.

[0227] FIG. 6E shows the N-glycosylation distribution of the fourth potential N-glycosylation site N414. As can be seen from FIG. 6E, the main N-glycan species are di-M6P and mono-M6P N-glycans. The first analysis and the second analysis detected that more than 40% of ATB200 at the fourth site had di-M6P, which corresponds to an average of about 0.4 to about 0.6 moles of di-M6P per mole of ATB200 at the fourth site. The first analysis and the second analysis also detected that more than 25% of ATB200 at the fourth site had mono-M6P, which corresponds to an average of about 0.3 to about 0.4 moles of mono-M6P per mole of ATB200 at the fourth site.

[0228] FIG. 6F shows the N-glycosylation distribution of the fifth potential N-glycosylation site N596. As can be seen from FIG. 6F, the main N-glycan species is fucosylated biantennary complex N-glycan. The first and second analyses detected that more than 70% of ATB200 at the fifth site had sialic acid residues, which corresponds to an average of about 0.8 to about 0.9 moles of sialic acid per mole of ATB200 at the fifth site.

[0229] FIG. 6G shows the N-glycosylation distribution of the sixth potential N-glycosylation site N826. As can be seen from FIG. 6G, the main N-glycan species are diantennary, triantennary, and tetraantennary complex N-glycans. The first analysis and the second analysis detected that more than 80% of ATB200 at the sixth site had sialic acid residues, which corresponds to an average of about 1.5 to about 1.8 moles of sialic acid per mole of ATB200 at the sixth site.

[0230] Analysis of N-glycosylation at the seventh position N869 showed approximately 40% N-glycosylation, with the most prevalent N-glycans being A4S3S3GF (12%), A5S3G2F (10%), A4S2G2F (8%), and A6S3G3F (8%).

[0231] Figure 6H shows a summary of phosphorylation at each of the seven potential N-glycosylation sites. As can be seen from Figure 6H, 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 at the first site was single or double phosphorylated, more than 40% of ATB200 at the second site was single phosphorylated, and more than 80% of ATB200 at the fourth site was single or double phosphorylated.

[0232] Another N-glycosylation analysis of ATB200 was performed according to the LC-MS / MS method described below. The analysis yielded an average N-glycosylation profile of ten batches of ATB200 (Figs. 32A-32H, Figs. 33A-33B).

[0233] 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.

[0234] 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 ammonium acetate in water 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 from ProteoWizard, and then the glycan database search and subsequent annotation of the identified N-glycans were performed using GRITS Toolbox 1,2Morning Blend software (UGA). N-glycans were identified using precursor monoisotopic mass number (m / z) and product ion m / z. Experimental product ions and fragmentation patterns were confirmed in silico using the GlycoWorkbench 2 application.

[0235] To determine the relative quantification of N-linked glycans from ATB200, the data acquired 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, expressed as peak area, is a quantitative measure of the amount of each N-glycan in the sample ( Fig.33A ). However, in most cases, multiple N-glycans are contained in the same FLD peak. Therefore, mass spectrometer data are also needed to obtain relative quantification of each N-glycan species (Table 5). 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 specific to only one N-glycan species. The XIC peak generated by the ion intensity signal is then integrated, and the peak area is a relative quantitative measure of the amount of glycan present. Both the FLD peak area and the mass spectrometer XIC peak area were used to perform relative quantification of all N-linked glycan species for ATB200 reported here.

[0236] The results of the LC-MS / MS analysis are provided in Table 5 below. Glycan nomenclature is based on 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 https: / / doi.org / 10.1093. / glycob / cwm.106; Kattla JJ et al., 2011. Biologic protein glycosylation. In: Murray Moo-Young (ed.), Comprehensive Biotechnology, Second Edition, 3: 467-486; Thamalingarn-Jaikarjsn 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., Humanplasma protein N-glycosylation, 2015.Glycoconj J.DOI 10.1007 / sl0719-015-9626-2; and Blackler RJ et al. 2016.Single-chain antibody-fragment M6P-1possesses amannose6-phosphate monosaccharide-specific binding pocket that distinguishes N-glycan phosphorylation in a branch-specific manner. Glycobiology. 26-2:181-192.

[0237] Table 5: Oligosaccharide types and occurrences identified on ATB200 based on 2-AA glycan profile and LC-MS / MS identification

[0238]

[0239] According to this 2-AA and LC-MS / MS analysis, and further as Fig.33B In summary, the average M6P content of the ATB200 tested was 3-5 mol / mol ATB200 (considering mono-M6P and di-M6P) and the sialic acid content per mol ATB200 was 4-7 mol.

[0240] As shown in Figures 32A-32H and Fig.33B As summarized in , the first potential N-glycosylation site of ATB200 has an average M6P content of about 1.4 mol M6P / mol ATB200, including an average mono-M6P content of about 0.25 mol mono-M6P per mol ATB200 and an average di-M6P content of about 0.56 mol di-M6P per mol ATB200; the average M6P content of the second potential N-glycosylation site of ATB200 is about 0.5 mol M6P / mol ATB200, and the major phosphorylated N-glycan species is mono-M6P N-glycans; the average sialic acid content of the third potential N-glycosylation site of ATB200 is about 1 mol sialic acid / mol ATB200; the average M6P content of the fourth potential N-glycosylation site of ATB200 is about 1.4 mol M6P / mol ATB200, of which the average mono M6P content is about 0.35 mol mono M6P / mol ATB200 and the average di-M6P content is about 0.52 mol di-M6P / mol ATB200; the average sialic acid content of the fifth potential N-glycosylation site of ATB200 is about 0.86 mol sialic acid / mol ATB200; the average sialic acid content of the sixth potential N-glycosylation site of ATB200 is about 4.2 mol sialic acid / mol ATB200; and the average sialic acid content of the seventh potential N-glycosylation site of ATB200 is about 0.86 mol sialic acid / mol TB200.

[0241] Also according to this 2-AA and LC-MS / MS analysis technique, on average 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), 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. About 70% of the N-glycans at the glycosylation sites are sialylated (about 26% are fully sialylated), while about 100% of the N-glycans at the fifth potential N-glycosylation site of ATB200 are complex N-glycans, about 85% of the N-glycans at the sixth potential N-glycosylation site of ATB200 are sialylated (nearly 27% are fully sialylated), about 98% of the N-glycans at the sixth potential N-glycosylation site of ATB200 are complex N-glycans, about 87% of the N-glycans at the seventh potential N-glycosylation site of ATB200 are sialylated (nearly 8% are fully sialylated), and about 100% of the N-glycans at the seventh potential N-glycosylation site of ATB200 are complex N-glycans.

[0242] Example 4: ATB200 and Comparison of analysis

[0243] Purified ATB200 and N-glycans were evaluated by MALDI-TOF to determine the individual N-glycan structures found on each ERT. was obtained from a commercial source. As shown in Figure 7, ATB200 exhibits This confirms that ATB200 is phosphorylated to As summarized in Figure 8, it was found that the ATB200 samples were Contains lower amounts of non-phosphorylated high-mannose N-glycans.

[0244] For evaluation and To determine 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 an M6P group) and the flow-through was collected. The bound material was then eluted using a free M6 gradient. The fractions were collected in a 96-well plate and analyzed for GAA activity using a 4MU-α-glucose substrate. The relative amounts of separated (flow-through) and bound (M6P eluted) rhGAA were determined based on GAA activity and reported as a fraction of the total enzyme. Figures 9A and 9B show and Binding distribution of rhGAA in: 73% of rhGAA (Figure 9B) and 78% of rhGAA in (Fig. 9A) did not bind to CIMPR. Only 27% of rhGAA and 22% of rhGAA in ATB200 contained M6P which may be sufficient to target it to CIMPR on myocytes. In contrast, as shown in FIG5 , under the same conditions, more than 70% of rhGAA in ATB200 was found to bind to CIMPR.

[0245] In addition to having a larger percentage of rhGAA available to bind to CIMPR, it is important to understand the quality of that interaction. The ATB200 and ATB200 receptor binding was determined using the CIMPR plate binding assay. Briefly, a CIMPR-coated plate was used to capture GAA. Varying concentrations of rhGAA were applied to the immobilized receptors and unbound rhGAA was washed off. The amount of remaining rhGAA was determined by GAA activity. As shown in Figure 10A, ATB200 vs. Significantly better binding to CIMPR. Figure 10B shows The relative content of double M6P N-glycans in ATB200 (traditional rhGAA product) and the present invention. For ATB200, there were an average of only 10% of the molecules with dual phosphorylated N-glycans. In contrast, on average each rhGAA molecule in ATB200 had at least one dual phosphorylated N-glycan.

[0246] In general, ATB200 The higher content of M6P N-glycans in ATB200 indicates that a higher portion of rhGAA molecules in ATB200 can target myocytes. As shown above, the high percentage of mono- and di-phosphorylated structures determined by MALDI conform to the CIMPR distribution, indicating that ATB200 has significantly greater binding to the CIMPR receptor. N-glycan analysis by MALDI-TOF mass spectrometry confirmed that on average each ATB200 molecule contains at least one native di-M6P N-glycan structure. This higher di-M6P N-glycan content on ATB200 is directly related to the high affinity binding to CIMPR in the M6P receptor plate binding assay (K D about 2-4 nM).

[0247] ATB200 and The relative cellular uptake of rhGAA was compared using normal and Pompe fibroblast cell lines. The comparison involved 5-100 nM of ATB200 according to the invention and 10-500 nM of a conventional rhGAA product. After 16 hours incubation, external rhGAA was inactivated using TRIS base and cells were washed 3 times with PBS before harvesting. Internalized GAA was measured by 4MU-α-glycoside hydrolysis and plotted relative to total cellular protein and the results are presented in Figures 11A-11C.

[0248] ATB200 was also shown to be efficiently internalized in cells. As depicted in FIGS. 11A-11B , ATB200 was internalized in normal and Pompe fibroblasts and was more potent than conventional rhGAA products. ATB200 saturates cell receptors at about 20 nM, while about 250 nM is required. to saturate the cell receptors. The absorption efficiency constant (K 吸收 ) is 2-3nm for ATB200 and The results suggest that ATB200 is a good targeted therapy for Pompe disease.

[0249] Example 5: ATB200 and pharmacological chaperones

[0250] The stability of ATB200 in acidic or neutral pH buffer was evaluated in a thermal stability assay using SYPRO Orange when the fluorescence of the dye increases upon protein denaturation. As shown in Figure 12, the addition of AT2221 at pH 7.4 stabilized ATB200 in a concentration-dependent manner, equivalent to the stability of ATB200 at pH 5.2, which is a condition that simulates the acidic environment of lysosomes. As summarized in Table 6, the addition of AT2221 increased the melting temperature (Tm ) increased by almost 10°C.

[0251] Table 6. Stability of the combination of ATB200 and AT2221

[0252]

[0253] Example 6: Co-administration of ATB200 and AT2221 in Gaa KO mice

[0254] The therapeutic effects of ATB200 and AT2221 were evaluated in Gaa KO mice and compared with those of alglucosidase alpha. For the study, male Gaa KO (3 to 4 months old) and age-matched wild-type (WT) mice were used. Alglucosidase alpha was administered by intravenous (IV) injection through a bolus tail vein. In the co-administration protocol, AT2221 was administered by oral gavage (PO) 30 minutes before the IV injection of ATB200. Treatment was given once every two weeks. The treated mice were sacrificed 14 days after the last administration and various tissues were collected for further analysis. Table 7 summarizes the study design:

[0255] Table 7. Co-administration study design

[0256]

[0257] Tissue glycogen content in tissue samples was determined using amyloglucosidase digestion, as discussed above. As shown in Figure 13, the combination of 20 mg / kg ATB200 and 10 mg / kg AT2221 significantly reduced glycogen content in four different tissues (quadriceps, deltoid, gastrocnemius, and heart) compared to the same dose of alglucosidase alfa.

[0258] Tissue samples were also analyzed for biomarker changes following the methods discussed in Khanna R et al. (2012), "The pharmacological chaperone AT2220 increases recombinant human acid α-glucosidase uptake and glycogen reduction in a mouse model of 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 Acid α-glucosidase, and Promotes Glycogen Reduction in a Transgenic Mouse Model of Pompe Disease", PLoS ONE 9(7): e102092. As shown in FIG. 14 , increased depth and enlargement of LAMP1-positive vesicles were seen in muscle fibers of Gaa KO animals compared to WT, indicating lysosomal proliferation. Co-administration of ATB200 / AT2221 produced more fibers with normalized LAMP1 levels, while the remaining LAMP1-positive vesicles also decreased in size (inset).

[0259] Similarly, the dense LC3 positive aggregates in the muscle fibers of untreated GaaKO mice indicate the presence of autophagic zones and autophagic accumulation. LC3 positive aggregates (red) are preferably reduced in mice treated with ATB200 / AT2221 co-administered for treatment compared to mice treated with alglucosidase α (Figure 15A). When the performance of LC3 was assessed using Western blot, similar observations were obtained. As shown in Figure 15B, most animals treated with ATB200 / AT2221 showed a significant reduction in the level of LC3 II, which is a lipidated form associated with autophagosomes, thereby suggesting improved autophagic flux. Comparatively, the effect of alglucosidase α on autophagy is moderate.

[0260] Dysferlin, a protein involved in membrane repair and whose deficiency / misdelivery is associated with many muscular dystrophies, was also assessed. As shown in Figure 16, dysferlin is abundantly accumulated in the sarcoplasm of Gaa KO mice. ATB200 / AT2221 was able to restore dysferlin to the sarcolemma in a greater number of muscle fibers than alglucosidase alfa.

[0261] These data are consistent with the improvements demonstrated at the cellular level in human Pompe disease patients treated with ATB200 and Miglustat (e.g., patients exhibited reduced levels of biomarkers of glycogen accumulation and muscle damage), resulting in not only effective treatment of Pompe disease but also reversal of disease progression. Clinical data in human Pompe disease patients are summarized in Examples 8-13 below.

[0262] Example 7: Single fiber analysis

[0263] As shown in FIG. 17 , most vehicle-treated mice displayed grossly enlarged lysosomes (green) (see, e.g., “B”) and the presence of large autophagic accumulations (red) (see, e.g., “A”). The treated mice did not show any significant differences compared to the mice treated with the vehicle. In contrast, most of the fibers isolated from mice treated with ATB200 showed a dramatic reduction in lysosome size (see, e.g., "C"). In addition, the areas with autophagy accumulation were also reduced to varying degrees (see, e.g., "C"). As a result, most of the muscle fibers analyzed from mice treated with ATB200 (36-60%) appeared normal or near normal. Table 8 below summarizes the single fiber analysis shown in Figure 17.

[0264] Table 8. Single fiber analysis

[0265]

[0266] *This includes fibers with varying degrees of reduced autophagic accumulation. Overall, the extent of accumulation was less in the ATB200 treated groups compared to the vehicle or alglucosidase alfa treated groups.

[0267] Overall, the data indicate that ATB200 with its higher M6P content, alone and further stabilized by the pharmacological chaperone AT2221 at the neutral pH of blood, is more effective than alglucosidase alfa in tissue targeting and lysosomal trafficking when administered to Gaa KO mice, consistent with the stabilization of ATB200 by AT2221, as depicted in Figure 18. As a result, administration of ATB200 and co-administration of ATB200 / AT2221 are more effective than alglucosidase alfa in correcting some disease-related pathological parameters such as glycogen accumulation, lysosomal proliferation, and autophagic zone formation. Due to these positive therapeutic effects, administration of ATB200 and co-administration of ATB200 / AT2221 are shown to improve the potential for muscle fiber recovery from self-destruction and even reverse the destruction by clearing glycogen that has accumulated in the cells due to the lack of optimal GAA activity. As with Example 6, these data are also consistent with the improvements demonstrated in human Pompe disease patients at the cellular level, which resulted in effective treatment of Pompe disease and reversal of disease progression following administration of ATB200 and Miglustat. Clinical data in human Pompe disease patients are summarized in Examples 8-13 below.

[0268] Example 8: ATB200-02 Trial: Non-Human Study of ATB200 / AT2221 in Patients with Pompe Disease

[0269] Preclinical studies were conducted in Gaa knockout mice to evaluate the pharmacokinetics (PK) and glycogen reduction efficiency at varying doses of ATB200 enzyme replacement therapy (ERT) and AT2221 chaperones. These data were used to estimate comparable AT2221 chaperone doses in humans. The study ATB200-02 (NCT02675465) was subsequently designed as an open-label fixed sequence, ascending dose, first-in-human, Phase 1 / 2 study to evaluate the safety, tolerability, PK, pharmacodynamics (PD), and efficacy of co-administration of ATB200 and AT2221 in patients with Pompe disease. Figures 19A-19B present the ATB200-02 study design. Ambulatory patients who have previously received enzyme replacement therapy with alglucosidase alpha are referred to as ambulatory ERT switching (or ERT switching ambulatory) patients or Group 1 patients. Incapable patients who had previously received enzyme replacement therapy with alglucosidase alfa were referred to as incapable ERT-switched (or ERT-switched incapable) patients or Cohort 2 patients. Capable patients who had not previously received enzyme replacement therapy with alglucosidase alfa were referred to as ERT-naive (or ERT-naive capable) patients or Cohort 3 patients.

[0270] Sixteen clinical sites in five countries participated in the ATB200-02 study. The study used the following key inclusion criteria: men and women aged 18-65 years who were diagnosed with Pompe disease based on documented GAA enzyme activity deficiency or GAA phenotype and had received enzyme replacement therapy with alglucosidase alfa for 2-6 years (Cohort 1) (or >2 years for Cohort 2) prior to trial initiation. Eligible subjects were those who were currently receiving alglucosidase alfa at a frequency of every other week and had completed the last 2 infusions without a drug-related adverse event (AE) resulting in dose interruption (Cohorts 1 and 2). Subjects had to be able to walk between 200 and 500 meters on the 6-Minute Walk Test (6MWT) (Cohorts 1 and 3), have an upright forced vital capacity (FVC) of 30-80% of predicted normal values ​​(Cohorts 1 and 3), or be wheelchair-bound and unable to walk without assistance (Cohort 2). The protocol for 6MWT and FVC testing can be found, for example, in Lachman and Schoser, Journal of Rare Diseases, 2013, 8: 160, and Bittner and Singh, The 6 Minute Walk Test, Cardiology Advisor, 2013. Figure 19C provides baseline characteristics of 20 subjects. Safety, tolerability, and biomarkers of groups 1, 2, and 3 were assessed. The following functional assessment results of groups 1 and 3 were assessed: 6MWT, other motor function tests (time test and gait-stair-gower-chair (gait-stair-gower-chair; GSGC)), manual muscle testing, and lung function (FVC, maximum inspiratory pressure (maximal inspiratory pressure; MIP) / maximal expiratory pressure (maximal expiratory pressure; MEP)). The protocol for time test and GSGC testing can be found, for example, in Lachman and Schoser, Journal of Rare Diseases, 2013, 8: 160. For Cohort 2, functional assessment included muscle strength testing.

[0271] Example 9: Interim PK Results from the ATB200-02 Trial

[0272] A summary of the pharmacokinetic data for AT2221 is provided in Figure 20. Total GAA protein concentrations in plasma for ATB200 at 5 mg / kg, 10 mg / kg, and 20 mg / kg were determined by validated LC-MS / MS quantification of rhGAA-specific "signature" peptides T09 (primary) and T50 (confirming) for 11 Cohort 1 patients who completed Phases 1 and 2, and five Cohort 2 patients who completed Phase 3 of the PK study. For Phase 1, blood samples for plasma total GAA protein concentrations were collected prior to the start of ATB200 infusion and at 1, 2, 3, 3.5, 4, 4.5, 5, 6, 8, 10, 12, and 24 hours after the start of the infusion. For Phases 2 and 3, blood samples for plasma total GAA protein concentrations were collected prior to the start of the infusion and at 1, 2, 3, 3.5, 4, 4.5, 5, 6, 8, 10, 12, and 24 hours after the start of the infusion.

[0273] AT2221 PK analysis was also performed on 11 Cohort 1 patients who completed Phase 1 and 2, and on five Cohort 2 patients who completed the PK study in Phase 3. Blood samples for plasma AT2221 concentrations were obtained just before (time 0) and at 1, 1.5, 2, 2.5, 3, 4, 5, 6, 9, 11, and 25 hours after oral administration of AT2221. Plasma AT2221 was determined by a validated LC-MS / MS assay.

[0274] As shown in Figure 21, when administered alone, the level of ATB200 increased in a slightly greater than dose-proportional manner. Co-administration of ATB200 at 20 mg / kg with a single high dose (260 mg) of AT2221 increased the area under the total GAA protein exposure curve (area under the curve; AUC) by approximately 17% compared to ATB200 administered alone at 20 mg / kg (Figure 21, Figure 22C). Co-administration of ATB200 at 20 mg / kg with multiple high doses (260 mg) of AT2221 increased the area under the total GAA protein exposure curve (area under the curve; AUC) by approximately 29% compared to ATB200 administered alone at 20 mg / kg (Figure 21, Figure 22D). An increase in the distribution half-life and partial AUC-24h was observed on a logarithmic scale during the terminal elimination phase (Figure 21, Figure 22A, Figure 22B). As shown in Figure 21, the distribution half-life (α phase) increased by 40%, consistent with the stabilizing effect of high-dose AT2221 on ATB200 in plasma. The increase in the distribution half-life was accompanied by a partial AUC increase of 42.2% from the time of reaching maximum plasma concentration to 24 hours after administration (Figure 21, Figure 22B). Further evidence of stabilization of ATB200 by AT2221 was observed in the comparison of low-dose AT2221 and high-dose AT2221 versus ATB200 alone 12 hours and 24 hours after administration (Figure 22E and Figure 22F). There was no statistically significant difference in plasma total GAA protein exposure between ERT original (Group 3) patients and ERT switching patients (Group 1) (Figure 23). The PK placement of the signature peptide T50 was no different from that of the signature peptide T09 (AUC ratio: 1.00).

[0275] Example 10: Interim Efficacy Results from the ATB200-02 Trial

[0276] As shown in Figures 24A and 24B, 6MWT improved in both ERT-switch patients and ERT-naive patients who were active at month 6 and sustained benefit was observed up to month 12. 7 / 10, 8 / 10, and 8 / 8 of ERT-switch patients had an increase in 6MWT at month 6, month 9, and month 12, respectively. 5 / 5, 5 / 5, and 2 / 2 of ERT-naive patients had an increase in 6MWT at month 6, month 9, and month 12, respectively.

[0277] As shown in Figures 24C, 26A, and 26C, improvements in motor function tests and manual muscle strength along with the 6MWT were consistent with overall improvements in muscle function in both ERT switch and ERT naive patients over 12 months.

[0278] As shown in Figures 25 and 26B, consistent and significant increases in upper limb strength were observed in all immobile ERT-switched patients at both 6 and 9 months.

[0279] As shown in FIG27 , FVC was stable or increased in 5 / 9, 6 / 9, and 3 / 7 ERT switch patients at 6, 9, and 12 months, respectively, and was stable or increased in 5 / 5, 5 / 5, and 2 / 2 ERT naive patients at 6, 9, and 12 months, respectively. Also as shown in FIG27 , maximal inspiratory pressure (MIP) was stable and maximal expiratory pressure (MEP) increased in ERT switch ambulatory patients, while MIP increased and MEP was stable in ERT naive patients.

[0280] The Fatigue Severity Scale (FSS) is a self-assessment questionnaire consisting of nine questions, each of which is scored on a scale of 1 to 7. The total score range is 9 to 63, with higher values ​​indicating higher fatigue levels due to disease symptoms. Normal values ​​in healthy populations are approximately 21 (Grace J et al. Parkinsonism Relat Disord. 2007; 13: 443-445). As shown in Figure 28, all groups were significantly affected by fatigue at baseline, and all groups demonstrated improvements in their FSS after receiving ATB200 / AT2221.

[0281] Example 11: Interim Results from the ATB200-02 Trial: Markers of Muscle Damage

[0282] The following muscle destruction markers were assessed: creatine kinase (CK), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Results available after nine months of the clinical trial are reported in Figures 29A-29C (from data up to 58 weeks, 24 weeks, and 36 weeks for Cohorts 1, 2, and 3, respectively; lower n values ​​reflect that some data could not be analyzed or had not been analyzed). The mean reductions from baseline observed at these corresponding time points were approximately 30-35% for ambulatory ERT switch patients (n=9), 5-20% for inactive ERT switch patients (n=4), and 40-55% for ERT naive patients (n=5). Results available for CK enzyme after twelve months of the clinical trial are reported in Figure 29D (from data up to 12 months for Cohorts 1, 2, and 3; lower n values ​​reflect that some data could not be analyzed or had not been analyzed).

[0283] Urinary hexotetrasaccharide (Hex4) was assessed as a marker of glycogen accumulation. Results for Hex4 available after twelve months of the clinical trial are reported in Figure 29D (data from up to 12 months for Cohorts 1, 2, and 3; lower n values ​​reflect that some data could not be analyzed or had not been analyzed).

[0284] Example 12: Interim Safety Results from the ATB200-02 Trial

[0285] The maximum duration of treatment was over 20 months. Adverse events (AEs) were generally mild and transient, with a very low rate of infusion-related reactions (less than 1%) across all three groups after 400 total infusions. These events were managed by standard premedication.

[0286] The most common AEs reported as treatment-related through Week 72 were nausea (3 / 20), tremor (3 / 20), headache (3 / 20), fatigue (3 / 20), diarrhea (2 / 20), muscle cramps (2 / 20), and joint swelling (2 / 20).

[0287] The most common AEs reported as treatment-related through 20+ months were abdominal pain (including upper and lower abdominal pain) (8 / 20), diarrhea (8 / 20), nasopharyngitis (6 / 20), nausea (5 / 20), headache (5 / 20), and upper respiratory tract infection (5 / 20) (Figure 30). One serious AE was reported that was not related to study drug (hospitalization for lower respiratory tract infection). No patient discontinued the study due to an AE.

[0288] Three infusion-associated reactions (IARs) occurred in 550+ infusions, which were controlled by standard premedication. One IAR event (skin depigmentation) occurred in an incapacitated ERT switch patient (Group 2). Two IAR events (local pruritus, erythema, and burning) occurred in an ERT naive patient (Group 3) (Figure 30).

[0289] Example 13: Summary and conclusions from interim results of the ATB200-02 trial

[0290] As summarized in Figure 31, there is consistency in the interim data from the ATB200-02 trial, confirming the markers of muscle damage and substrate accumulation, muscle function tests (timed tests and endurance), significant and unexpected parallel improvements in unarmed muscle strength, and stability and / or improvement in respiratory function tests across different groups. Muscle function improved in 16 / 18 and 10 / 10 patients at 6 and 9 months, respectively. The increase in 6MWT distance was consistent and lasting until the 12th month in patients with mobility in ERT switching and original patients with ERT, as was the improvement of other motor function tests in patients with mobility in ERT switching and original patients with ERT. Qualitative and quantitative measurements showed an increase in upper limb strength in patients with no mobility in ERT switching at 6 and 9 months. FVC, MIP, and MEP were generally stable in patients with ERT switching and increased in original patients with ERT. Improvements in fatigue scores were observed in all groups. Biomarker levels (eg, levels of CK and Hex4) were reduced in all groups and ATB200 / AT2221 was generally well tolerated.

[0291] Therefore, the multidimensional impact of therapy suggests that the combination regimen of ATB200 / AT2221 has the potential to become an important treatment option for patients with Pompe disease. These clinical results support the results from the single fiber analysis study described in Example 7, demonstrating that the treatment is effective in clearing pathology from muscle fibers. Further studies in clinical trials are still ongoing.

Claims

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

2. The method of claim 1, wherein reversing disease progression comprises reducing lysosomal size in the subject's muscle.

3. The method of claim 1 or claim 2, wherein reversing the disease process comprises resolving autophagic accumulation in the muscle of the subject.

4. The method of claim 3, wherein after treatment less than 65% of muscle fibers analyzed in the subject have autophagic accumulation.

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

6. The method according to claim 5, wherein: The ERT-switching patients had previously been treated with alglucosidase alfa for at least two years.

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

8. A method of treating Pompe disease in a subject in need thereof, comprising the steps of: administering to the subject a population of recombinant human acid alpha-glucosidase (rhGAA) molecules from Chinese hamster ovary (CHO) cells; wherein the rhGAA molecule comprises seven potential N-glycosylation sites; wherein the rhGAA molecule contains an average of 3-4 mannose-6-phosphate (M6P) residues; wherein the rhGAA molecules comprise, on average, at least about 0.5 moles of dimannose-6-phosphate (bis-M6P) per mole of rhGAA at the first potential N-glycosylation site when measured using LC-MS / MS; and wherein the glycogen content in the subject's muscle decreases faster following treatment than when alglucosidase alfa is administered at the same dose.

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

10. The method of claim 8 or claim 9, wherein after treatment, the glycogen content in the subject's muscle is more effectively reduced than when alglucosidase alfa is administered at the same dose when assessed after one, two, three, four, five, or six administrations.

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

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

13. The method of any one of claims 8-12, wherein the subject exhibits reduced levels of urinary hexotetrasaccharides after treatment.

14. The method of claim 13, wherein the level of urinary hexotetrasaccharide is reduced by at least 30% compared to baseline at six months following treatment.

15. The method of claim 13, wherein the subject is an ambulatory ERT switch patient or an incapable ERT switch patient, and wherein the subject's urinary hexotetrasaccharide level is reduced by at least 35% compared to baseline six months after treatment.

16. The method of claim 13, wherein the subject is an active ERT naive patient and the subject's urinary hexotetrasaccharide levels are reduced by at least 45% from baseline six months after treatment.

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

18. The method of claim 17, wherein the improved motor function in the subject is measured by at least one motor function test selected from the group consisting of a six minute walk test (6MWT), a timed up and go test, a four-stair climb test, a ten-meter walk test, a Gowers test, a gait-stair-gower-chair (GSGC) test, or a combination of the foregoing.

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

20. The method of claim 18, wherein the subject is an ambulatory ERT switching patient, and wherein, Compared to baseline, the subject's 6MWT distance increased by at least 20 meters six months after treatment, the subject's timed up and go test time decreased by at least 1.5 seconds six months after treatment, the subject's four-stair climb test time decreased by at least 0.6 seconds six months after treatment, or the subject's Gowers test time decreased by at least 1 second six months after treatment.

21. The method of claim 18, wherein the subject is an ERT-naive patient who is able to move, and wherein, Compared to baseline, six months after treatment, the subject's 6MWT distance increased by at least 40 meters, six months after treatment, the subject's timed up and go test time decreased by at least 1 second, six months after treatment, the subject's four-stair climbing test decreased by at least 0.6 seconds, six months after treatment, the subject's ten-meter walk test time decreased by at least 0.7 seconds, or six months after treatment, the subject's GSGC score decreased by at least 1.

22. The method of claim 18, wherein the subject previously received alglucosidase alfa enzyme replacement therapy, wherein the subject exhibits an improvement in at least one motor function following the population treatment with rhGAA as compared to the subject's motor function test result following the prior alglucosidase alfa enzyme replacement therapy.

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

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

25. The method of claim 24, wherein the subject is an ambulatory ERT switch patient and demonstrates an improvement in an upper body manual muscle strength score of at least 1 compared to baseline at six months following treatment.

26. The method of claim 24, wherein the subject is an immobile ERT switch patient and demonstrates an improvement in an upper body manual muscle strength score of at least 5.5 compared to baseline at six months following treatment.

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

28. The method of claim 27, wherein the subject is an incapacitated ERT switching patient, and wherein, Compared to baseline, the subject's shoulder adduction is improved by at least 8 pounds of force at six months after treatment, the subject's shoulder abduction is improved by at least 1 pound of force at six months after treatment, the subject's elbow flexion is improved by at least 2 pounds of force at six months after treatment, or the subject's elbow extension is improved by at least 5 pounds of force at six months after treatment.

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

30. The method of claim 23, wherein the subject previously received alglucosidase alfa enzyme replacement therapy, wherein the subject exhibits an improvement in upper body strength following the population treatment with rhGAA compared to the subject's upper body strength following the prior alglucosidase alfa enzyme replacement therapy.

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

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

33. The method of claim 32, wherein the subject's FVC improves by at least 4% at six months after treatment, the subject's MEP improves by at least 16 cmH2O at six months after treatment, or the subject's MIP improves by at least 0.3 cmH2O at six months after treatment compared to baseline.

34. The method of claim 32, wherein the subject is an ambulatory ERT switching patient and wherein, The subject's MEP improved by at least 16 cmH2O at six months following treatment compared to baseline.

35. The method of claim 32, wherein the subject is an ERT-naive patient who is able to move and wherein, The subject's FVC improves by at least 4% at six months after treatment or the subject's MIP improves by at least 11 cmH2O at six months after treatment compared to baseline.

36. The method of claim 32, wherein the subject previously received alglucosidase alfa enzyme replacement therapy, wherein the subject exhibits an improvement in at least one pulmonary function test following the population treatment with rhGAA as compared to the subject's pulmonary function test result following the prior alglucosidase alfa enzyme replacement therapy.

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

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

39. The method of claim 38, wherein the subject is an incapacitated ERT switching patient.

40. The method of claim 37 or claim 38, wherein the subject is an ambulatory ERT switch patient and wherein the subject's FSS score decreases by at least 8 compared to baseline at six months following treatment.

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

42. The method of claim 37, wherein the subject previously received alglucosidase alfa enzyme replacement therapy, wherein the subject has a lower FSS score after the population treatment with rhGAA compared to the subject's FSS score after the previous alglucosidase alfa enzyme replacement therapy.

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

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

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

46. ​​The method of claim 43, wherein the subject is an ambulatory ERT switching patient and wherein, The subject's creatine kinase level is reduced by at least 15% at six months after treatment, the subject's ALT level is reduced by at least 15% at six months after treatment, or the subject's AST level is reduced by at least 10% at six months after treatment, compared to baseline.

47. The method of claim 43, wherein the subject is an incapacitated ERT switching patient and wherein, The subject's creatine kinase level is reduced by at least 20% at six months after treatment, the subject's ALT level is reduced by at least 5% at six months after treatment, or the subject's AST level is reduced by at least 5% at six months after treatment, compared to baseline.

48. The method of claim 43, wherein the subject is an ERT-naive patient who is able to move and wherein, The subject's creatine kinase level is reduced by at least 35% at six months after treatment, the subject's ALT level is reduced by at least 35% at six months after treatment, or the subject's AST level is reduced by at least 30% at six months after treatment, compared to baseline.

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

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

51. The method of any one of claims 1-50, wherein the population of rhGAA molecules is administered bimonthly, monthly, biweekly, weekly, twice weekly, or daily.

52. The method of claim 51, wherein the population of rhGAA molecules is administered biweekly.

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

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

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

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

57. The method of claim 55 or claim 56, wherein the miglustat or a pharmaceutically acceptable salt thereof is administered at a dose of about 200 mg to about 600 mg.

58. The method of claim 57, wherein the miglustat or a pharmaceutically acceptable salt thereof is administered at a dose of about 260 mg.

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

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

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

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

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

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

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

5.

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

5.

67. The method of any one of claims 1-66, 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 when measured using LC-MS / MS.

68. The method of any one of claims 1-67, wherein the rhGAA molecules comprise an average of about 0.5 moles to about 7.0 moles of mono- or di-M6P per mole of rhGAA when measured using LC-MS / MS.

69. The method of any one of claims 1-68, wherein the rhGAA molecules comprise an average of at least 2.5 moles of M6P per mole of rhGAA and at least 4 moles of sialic acid per mole of rhGAA when measured using LC-MS / MS.

70. The method according to any one of claims 1 to 69, wherein: Per mole of rhGAA, the rhGAA molecule contains on average: (a) from about 0.4 to about 0.6 moles of mono M6P at the second potential N-glycosylation site; (b) from about 0.4 to about 0.6 moles of bis-M6P at the fourth potential N-glycosylation site; and (c) about 0.3 to about 0.4 moles of monoM6P at the fourth potential N-glycosylation site, wherein (a)-(c) are determined using LC-MS / MS.

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

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

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

0.

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

0.

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

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

0.

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

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