Recombinant human acid alpha glucosidase and uses thereof

Abstract

Provided herein is a method of treating Pompe disease comprising administering a population of recombinant human acid α-glucosidase molecules, or a pharmaceutical composition or formulation thereof, and a pharmacological chaperone.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 162,683, filed March 18, 2021, and U.S. Provisional Patent Application No. 63 / 148,596, filed February 11, 2021, the disclosures of each of which are hereby incorporated by reference in their entireties.

[0002] Statement regarding electronically submitted text files The contents of the text file submitted electronically herewith are incorporated by reference in their entirety into this specification: Sequence Listing, a computer-readable copy (file name: AMCS_013_02WO_SeqList_ST25.txt, date of recording: February 11, 2022, file size approximately 45,560 bytes).

[0003] The present disclosure relates to recombinant human alpha-glucosidase (rhGAA) and the treatment of Pompe disease. [Background technology]

[0004] Pompe disease is an inherited lysosomal storage disease caused by a deficiency in acid alpha-glucosidase (GAA) activity. People with Pompe disease lack or have low levels of acid alpha-glucosidase (GAA), an enzyme that breaks down glycogen into glucose, the main source of energy for muscles. This enzyme deficiency causes excess glycogen to accumulate in lysosomes, intracellular organelles that normally contain enzymes that break down glycogen and other cellular debris or waste products. The accumulation of glycogen in certain tissues, especially muscles, of subjects with Pompe disease impairs the cells' ability to function normally. In Pompe disease, glycogen is not metabolized properly and gradually accumulates in the lysosomes, especially in skeletal muscle cells and, in the infantile-onset form of the disease, cardiac muscle cells. The accumulation of glycogen damages muscle and nerve cells as well as other affected tissue cells.

[0005] Traditionally, Pompe disease is recognized clinically as either early infantile or late onset, depending on the age of onset. The age of onset tends to parallel the severity of the genetic mutation causing Pompe disease. The most severe genetic mutations cause a complete loss of GAA activity and manifest as early onset disease in infancy. Genetic mutations that reduce, but do not eliminate, GAA activity are associated with forms of Pompe disease that exhibit delayed onset and progression. Infantile onset Pompe disease manifests shortly after birth and is characterized by muscle weakness, respiratory failure, and cardiac failure. If untreated, death usually occurs within two years. Juvenile and adult onset Pompe disease manifests later in life and usually progresses more slowly than the infantile onset disease. This form of the disease can also be fatal, although it does not generally affect the heart, due to weakness of the skeletal and respiratory muscles.

[0006] Current non-palliative treatments for Pompe disease involve enzyme replacement therapy (ERT) using recombinant alglucosidase alfa preparations sold under the trademarks LUMIZYME® and MYOZYME®. This conventional enzyme replacement therapy seeks to treat Pompe disease by administering rhGAA to replenish the missing GAA in lysosomes, thus restoring the cell's ability to break down lysosomal glycogen. LUMIZYME® and MYOZYME® are conventional rhGAAs approved by the US Food and Drug Administration that are produced or sold as biologics by Genzyme and are listed by reference in the Physician's Desk Reference (2014), which is hereby incorporated by reference. Alglucosidase alfa has the chemical name [199-arginine, 223-histidine] prepro-α-glucosidase (human); molecular formula: C 4758 H 7262 N 1274 O 1369 S 35; identified as CAS number 420794-05-0. These formulations are administered to subjects with Pompe disease, also known as glycogen storage disease type II (GSD-II) or acid maltase deficiency.

[0007] However, current ERT, at best, produces limited improvements in measures of muscle function, strength, and respiratory function for a limited period of time, after which these parameters slowly deteriorate (Toscano and Schoser 2013; Wyatt et al. 2012).

[0008] In 2012, a systematic review of all studies performed in subjects with late-onset Pompe disease (LOPD) was performed by Toscano and Schoser 2013. This review included data on 368 subjects with LOPD from published studies, including 27 adolescent subjects (age range: 2-17 years) and 251 adult subjects who had previously received alglucosidase alfa for at least 2 years. Results indicated that more than 30% of subjects did not achieve initial improvement upon treatment with alglucosidase alfa and continued to experience deterioration in muscle and respiratory function despite treatment. In the group of subjects who initially responded to alglucosidase alfa treatment, several additional long-term studies revealed that improvement usually only lasted for about 2 years. Subjects then generally reached a plateau and then began to progressively worsen.

[0009] In 2012, the United Kingdom Health Technology Assessment programme (Wyatt et al 2012), part of the UK National Institute for Health Research, issued recommendations derived from a review of longitudinal data from 81 patients with Pompe disease (including infantile-onset and late-onset (children and adults)) who received the currently approved standard of care ERT, alglucosidase alfa. Key markers of Pompe disease progression (forced vital capacity, ventilator dependency, mobility, 6-minute walk test, muscle strength, and body mass index) were assessed and modelled by time on treatment receiving alglucosidase alfa therapy. Results of this assessment indicated that improvements in FVC, 6-minute walk distance, and muscle strength in patients with LOPD occurred during the first 2 years after initiation of ERT with alglucosidase alfa, with deterioration occurring alongside continued treatment beyond this time frame. Additionally, a 3-year study in 38 subjects with LOPD receiving alglucosidase alfa found that subjects demonstrated improvement in motor function during the first year of treatment, which remained generally stable during the second year, before beginning to deteriorate during the third year (Regnery et al 2012).

[0010] Moreover, a report providing 10 years of follow-up of the Phase 3 LUMIZYME® (Genzyme) trial found that after some improvements in motor and pulmonary function during the first few years of treatment, subjects slowly began to deteriorate while continuing treatment (van der Ploeg et al. 2017). In this study, there was a mean deterioration of about 10% in baseline 6-minute walk distance percent vs. predicted between the third and sixth years of therapy, with about 80% of subjects experiencing deterioration.

[0011] The most significant tolerability issue with alglucosidase alfa is the occurrence of infusion-related reactions (IARs), which in some cases can include life-threatening anaphylaxis or other severe allergic reactions (MYOZYME® Summary of Product Characteristics, December 2018). Management of these events includes dose reduction, reduction in infusion rate and duration of infusion, and interruption or cessation of administration. Premedication with antihistamines and steroids (before infusion) is also routinely used to prevent and reduce the occurrence and severity of IARs and hypersensitivity reactions associated with alglucosidase alfa infusions. Despite these measures, IARs can still occur in Pompe disease patients, some of whom are unable to tolerate regular infusions of currently approved ERTs.

[0012] In 2017, the European Pompe Consortium, a network of experts in the field of Pompe disease from 11 European countries, conducted a systematic review of the literature (van der Ploeg et al. 2017). The Consortium assessed the evidence of the efficacy of ERT at the group level based on data from one clinical trial and 43 observational studies, covering a total of 586 individual adult subjects. The current consensus of the European Pompe Consortium is that the occurrence of severe IAR or progressive clinical deterioration of disease symptoms, as well as the development of high neutralizing antibody (Ab) titers, which effectively render existing ERT treatment ineffective, should lead to the discontinuation of ERT therapy. The European Pompe Consortium's recommendations also include the consideration of resuming ERT treatment if disease progression and clinical deterioration reoccur after stopping ERT.

[0013] Thus, there remains a need to identify improved rhGAA therapies that are effective in treating Pompe disease with reduced adverse events.

[0014] The cellular uptake of rhGAA molecules is facilitated by a specialized carbohydrate, mannose-6-phosphate (M6P), which binds to the cation-independent mannose-6-phosphate receptor (CIMPR) present on target cells, such as muscle cells. Upon binding, the rhGAA molecules are taken up by the target cells and subsequently transported to the lysosomes within the cells. However, many conventional rhGAA formulations lack a high total content of mono-M6P- and bis-M6P-bearing N-glycans (i.e., N-glycans bearing one M6P residue or N-glycans bearing two M6P residues, respectively), limiting their cellular uptake and lysosomal delivery via CIMPR, which in turn reduces the efficacy of conventional enzyme replacement therapy. For example, conventional rhGAA formulations at doses of 20 mg / kg or greater, while indeed reversing some aspects of Pompe disease, do not adequately reverse disease progression, particularly in terms of (i) treating the underlying cellular dysfunction, (ii) repairing muscle structure, or (iii) reducing accumulated glycogen in many target tissues, such as skeletal muscle. Furthermore, high doses can impose additional burdens on the subject and the medical professionals treating the subject, such as the longer infusion times required for intravenous administration of rhGAA.

[0015] Enzymatic modification of the glycosylation of GAA or rhGAA in vitro with phosphotransferases and decoating enzymes as described by Canfield, et al., U.S. Pat. No. 6,534,300, can create M6P groups. However, enzymatic glycosylation cannot be adequately controlled, and can produce rhGAA with undesirable immunological and pharmacological properties. Enzymatically modified rhGAA may contain only high mannose oligosaccharides, all of which may be enzymatically phosphorylated in vitro by phosphotransferases or decoating enzymes. The glycosylation pattern produced by enzymatic treatment of GAA in vitro is problematic, since additional terminal mannose residues, especially those that are not phosphorylated, adversely affect the pharmacokinetics of the modified rhGAA. When such enzymatically modified preparations are administered in vivo, their mannose groups increase the non-productive clearance of GAA, increase the uptake of enzymatically modified GAA by immune cells, and reduce the therapeutic efficacy of rhGAA by allowing less GAA to reach target tissues such as skeletal muscle cells. For example, terminal non-phosphorylated mannose residues are known ligands for mannose receptors in the liver and spleen, leading to rapid clearance of enzymatically modified rhGAA and less rhGAA targeted to target tissues. Moreover, the glycosylation pattern of enzymatically modified GAA, with high mannose type N-glycans with terminal non-phosphorylated mannose residues, is similar to that found in glycoproteins produced by yeast and mold, increasing the risk of eliciting immune or allergic reactions, such as life-threatening severe allergic (anaphylactic) or hypersensitivity reactions, to enzymatically modified rhGAA.

[0016] Compared with conventional recombinant rhGAA formulations and in vitro phosphorylated rhGAA, the rhGAA used in the binary therapy of the present disclosure has an optimized N-glycan profile for enhanced biodistribution and lysosomal uptake, thereby minimizing non-productive clearance of rhGAA after administration. The present disclosure provides stable or deteriorating Pompe disease patients with an effective therapy that reverses disease progression at the cellular level - including clearing lysosomal glycogen more efficiently than the current standard of care. Patients treated with the binary therapy of the present disclosure, including rhGAA and a pharmaceutical chaperone (e.g., miglustat), exhibit significant health improvements, including improvements in muscle strength, motor function, and / or lung function, and / or reverse disease progression, as demonstrated by various efficacy results (e.g., Examples 8 and 9) from clinical trials. Summary of the Invention

[0017] Provided herein are methods for treating a disease or disorder, such as Pompe disease, in a subject, comprising administering a population of recombinant human acid alpha-glucosidase (rhGAA) molecules and a pharmacological chaperone (e.g., miglustat).

[0018] The rhGAA molecules described herein may be expressed in Chinese Hamster Ovary (CHO) cells and may contain seven potential N-glycosylation sites. In some embodiments, the N-glycosylation profile of a population of rhGAA molecules as described herein is determined using liquid chromatography-tandem mass spectrometry (LC-MS / MS). In some embodiments, the rhGAA molecules contain, on average, 3-4 moles of mannose-6-phosphate (M6P) residues per mole of rhGAA. In some embodiments, the rhGAA molecules contain, on average, at least about 0.5 moles of bisphosphorylated N-glycan groups (bis-M6P) per mole of rhGAA at the first potential N-glycosylation site. In some embodiments, the rhGAA comprises an amino acid sequence at least 95% identical to SEQ ID NO:4 or SEQ ID NO:6. In some embodiments, the rhGAA comprises an amino acid sequence identical to SEQ ID NO:4 or SEQ ID NO:6. In some embodiments, at least 30% of the rhGAA molecules contain one or more N-glycan units carrying one or two M6P residues. In some embodiments, the rhGAA molecules contain, on average, about 0.5 mol to about 7.0 mol of N-glycan units carrying one or two M6P residues per mole of rhGAA. In some embodiments, the rhGAA molecules contain, on average, 2.0 to 8.0 mol of sialic acid per mole of rhGAA. In some embodiments, the rhGAA molecules contain, on average, at least 2.5 mol of M6P residues per mole of rhGAA and at least 4 mol of sialic acid residues per mole of rhGAA.In some embodiments, rhGAA molecules comprising an average of 3 to 4 moles of M6P residues per mole of rhGAA and an average of at least about 0.5 moles of bis-M6P per mole of rhGAA at the first potential N-glycosylation site further comprise, on average, 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 bis-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 mol to about 7.3 mol of sialic acid residues per mole of rhGAA, including about 0.9 to about 1.2 mol of sialic acid per mole of rhGAA at the third potential N-glycosylation site, about 0.8 to about 0.9 mol of sialic acid per mole of rhGAA at the fifth potential N-glycosylation site, and about 1.5 to about 4.2 mol of sialic acid per mole of rhGAA at the sixth potential N-glycosylation site. In some embodiments, the rhGAA molecule population is formulated into a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprising the rhGAA molecule population further comprises at least one buffer selected from the group consisting of citrate, phosphate, and combinations thereof, and at least one excipient selected from the group consisting of mannitol, polysorbate 80, and combinations thereof. In some embodiments, the pH of the pharmaceutical composition is about 5.0 to about 7.0, about 5.0 to about 6.0, or about 6.0. In some embodiments, the pharmaceutical composition further comprises water, an acidifying agent, an alkalizing agent, or a combination thereof. In some embodiments, the pharmaceutical composition has a pH of 6.0 and comprises about 5 to 50 mg / mL of rhGAA molecule population, about 10 to 100 mM sodium citrate buffer, about 10 to 50 mg / mL mannitol, about 0.1 to 1 mg / mL polysorbate 80, and water, and optionally comprises an acidifying agent and / or an alkalizing agent.In some embodiments, the pharmaceutical composition has a pH of 6.0 and comprises about 15 mg / mL of rhGAA molecule population, about 25 mM sodium citrate buffer, about 20 mg / mL mannitol, about 0.5 mg / mL polysorbate 80, and water, and optionally an acidifying agent and / or an alkalizing agent.

[0019] In some embodiments, the rhGAA molecule population is administered at a dose of about 1 mg / kg to about 100 mg / kg or about 5 mg / kg to about 20 mg / kg. In some embodiments, the rhGAA molecule population is administered at a dose of about 20 mg / kg. In some embodiments, the rhGAA molecule population is administered bimonthly, monthly, biweekly, weekly, twice weekly, or daily, e.g., every other week. In some embodiments, the rhGAA molecule population is administered intravenously.

[0020] In some embodiments, the rhGAA molecular population is co-administered or sequentially administered with a pharmacological chaperone such as miglustat (also known as AT2221) or a pharma- ceutically acceptable salt thereof. In some embodiments, miglustat or a pharma- ceutically acceptable salt thereof is orally administered at a dose of, for example, about 50 mg to about 200 mg or about 200 mg to about 600 mg, and optionally about 130 mg, about 195 mg, or about 260 mg. In some embodiments, the rhGAA molecular population is intravenously administered at a dose of about 5 mg / kg to about 20 mg / kg, and miglustat or a pharma- ceutically acceptable salt thereof is orally administered at a dose of about 233 mg to about 500 mg. In some embodiments, the rhGAA molecular population is intravenously administered at a dose of about 5 mg / kg to about 20 mg / kg, and miglustat or a pharma- ceutically acceptable salt thereof is orally administered at a dose of about 50 mg to about 200 mg. In one embodiment, the rhGAA molecular population is administered intravenously at a dose of about 20 mg / kg, and miglustat or a pharma- ceutically acceptable salt thereof is administered orally at a dose of about 260 mg. In one embodiment, the rhGAA molecular population is administered intravenously at a dose of about 20 mg / kg, and miglustat or a pharma- ceutically acceptable salt thereof is administered orally at a dose of about 195 mg. In some embodiments, miglustat or a pharma- ceutically acceptable salt thereof is administered prior to (e.g., about 1 hour prior to) administration of the rhGAA molecular population. In at least one embodiment, the subject fasts for at least 2 hours before and at least 2 hours after administration of miglustat or a pharma- ceutically acceptable salt thereof.

[0021] The embodiments of the present disclosure demonstrate the efficacy of the two-component therapy described herein in treating and reversing the progression of Pompe disease in subjects. In some embodiments, the subject is an ERT-experienced patient. In some embodiments, the subject is an ERT-naive patient.

[0022] In some embodiments, a binary therapy according to the present disclosure improves one or more disease symptoms in a subject with Pompe disease compared to (1) baseline, or (2) a control treatment comprising administering alglucosidase alfa and a pharmacological chaperone placebo, in which a placebo is administered in place of the pharmacological chaperone.

[0023] In some embodiments, the dual-component therapy according to the present disclosure improves the motor function of the subject as measured by 6-minute walk test (6MWT).In some embodiments, compared to baseline, the 6-minute walk distance (6MWD) of the subject increases by at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or 50 meters or at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% after 12, 26, 38, or 52 weeks of treatment.In some embodiments, the 6MWD of the subject increases by at least 20 meters or at least 5% after 52 weeks of treatment. In some embodiments, the subject's 6MWD improves by at least 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, or 50 meters after 12, 26, 38, or 52 weeks of treatment, compared to a control treatment. In some embodiments, the subject's 6MWD improves by at least 13 meters after 52 weeks of treatment, compared to a control treatment. In some embodiments, the subject has a baseline 6MWD of less than 300 meters. In some embodiments, the subject has a baseline 6MWD of 300 meters or greater.

[0024] In some embodiments, the dual-component therapy according to the present disclosure stabilizes the subject's lung function as measured by forced vital capacity (FVC) test. In some embodiments, after 12, 26, 38, or 52 weeks of treatment, the subject's percent-to-predicted FVC either increases compared to baseline or decreases by less than 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% compared to baseline. In some embodiments, after 52 weeks of treatment, the subject's percent-to-predicted FVC decreases by less than 1% compared to baseline. In some embodiments, the subject's percent-to-predicted FVC improves significantly after treatment when compared to control treatment. In some embodiments, the subject's percent predicted FVC is significantly improved by at least 0.5%, 1%, 2%, 3%, 4%, 5%, or 6% after 12, 26, 38, or 52 weeks of treatment when compared to a control treatment. In some embodiments, the subject's percent predicted FVC is significantly improved by at least 3% after 52 weeks of treatment when compared to a control treatment. In some embodiments, the subject has a baseline FVC of less than 55%. In some embodiments, the subject has a baseline FVC of 55% or greater.

[0025] In some embodiments, the dual-component therapy of the present disclosure improves the motor function of the subject as measured by the Walk-Stair-Gowers-Chair (GSGC) test. In some embodiments, the subject's GSGC score improves as indicated by a decrease of at least 0.1, 0.3, 0.5, 0.7, 1.0, 1.5, or 2.5 points after 12, 26, 38, or 52 weeks of treatment, as compared to baseline. In some embodiments, the subject's GSGC score improves as indicated by a decrease of at least 0.5 points after 52 weeks of treatment, as compared to baseline. In some embodiments, the subject's GSGC score significantly improves after treatment, as compared to a control treatment. In some embodiments, the subject's GSGC score significantly improves as indicated by a decrease of at least 0.3, 0.5, 0.7, 1.0, 1.5, 2.5, or 5 points after 12, 26, 38, or 52 weeks of treatment, as compared to a control treatment. In some embodiments, the subject's GSGC score significantly improves as indicated by a reduction of at least 1.0 points after 52 weeks of treatment, when compared to the control treatment.

[0026] In some embodiments, the dual component therapy according to the present disclosure reduces the level of at least one muscle damage marker after treatment. In some embodiments, the at least one muscle damage marker includes creatine kinase (CK). In some embodiments, compared to baseline, the subject's CK level is reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, or 50% after 12, 26, 38, or 52 weeks of treatment. In some embodiments, compared to baseline, the subject's CK level is reduced by at least 20% after 52 weeks of treatment. In some embodiments, compared to a control treatment, the subject's CK level is significantly reduced after treatment. In some embodiments, compared to a control treatment, the subject's CK level is significantly reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, or 50% after 12, 26, 38, or 52 weeks of treatment. In some embodiments, compared to a control treatment, the subject's CK level is significantly reduced by at least 30% after 52 weeks of treatment.

[0027] In some embodiments, the dual component therapy of the present disclosure reduces the level of at least one glycogen accumulation marker after treatment. In some embodiments, the at least one glycogen accumulation marker comprises urinary hexose tetrasaccharide (Hex4). In some embodiments, the subject's urinary Hex4 level is reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60% after 12, 26, 38, or 52 weeks of treatment, as compared to baseline. In some embodiments, the subject's urinary Hex4 level is reduced by at least 30% after 52 weeks of treatment, as compared to baseline. In some embodiments, the subject's urinary Hex4 level is significantly reduced after treatment, as compared to a control treatment. In some embodiments, the subject's urinary Hex4 level is significantly reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60% after 12, 26, 38, or 52 weeks of treatment, as compared to a control treatment. In some embodiments, the subject's urinary Hex4 levels are significantly reduced by at least 40% after 52 weeks of treatment compared to the control treatment.

[0028] In some embodiments, a binary therapy according to the present disclosure improves one or more disease symptoms in a Pompe disease patient subject with a history of ERT compared to (1) baseline, or (2) a control treatment comprising administering alglucosidase alfa and a pharmacological chaperone placebo.

[0029] In some embodiments, the dual-component therapy for the subject with Pompe disease who has a history of ERT improves the motor function of the subject as measured by 6MWT.In some embodiments, compared to baseline, the 6MWD of the subject increases by at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or 50 meters or at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% after 12, 26, 38, or 52 weeks of treatment.In some embodiments, the 6MWD of the subject increases by at least 15 meters or at least 5% after 52 weeks of treatment.In some embodiments, compared to control treatment, the 6MWD of the subject improves significantly after treatment. In some embodiments, the subject's 6MWD is significantly improved by at least 10, 12, 14, 15, 16, 18, 20, 30, 40, or 50 meters after 12, 26, 38, or 52 weeks of treatment, compared to a control treatment. In some embodiments, the subject's 6MWD is significantly improved by at least 15 meters after 52 weeks of treatment, compared to a control treatment. In some embodiments, the subject has a baseline 6MWD of less than 300 meters. In some embodiments, the subject has a baseline 6MWD of 300 meters or greater.

[0030] In some embodiments, the bicomponent therapy for a subject with Pompe disease who has a history of ERT improves the subject's lung function as measured by FVC test. In some embodiments, after 12, 26, 38, or 52 weeks of treatment, the subject's percent predicted FVC increases by at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, or 5% compared to baseline. In some embodiments, after 52 weeks of treatment, the subject's percent predicted FVC increases by at least 0.1% compared to baseline. In some embodiments, the subject's percent predicted FVC improves significantly after treatment when compared to a control treatment. In some embodiments, the subject's percent predicted FVC improves significantly by at least 1%, 2%, 3%, 4%, 5%, 6%, 8%, or 10% after 12, 26, 38, or 52 weeks of treatment when compared to a control treatment. In some embodiments, the subject's percent predicted FVC is significantly improved by at least 4% after 52 weeks of treatment when compared to a control treatment. In some embodiments, the subject has a baseline FVC of less than 55%. In some embodiments, the subject has a baseline FVC of 55% or greater.

[0031] In some embodiments, the dual-component therapy for a subject with Pompe disease who has undergone ERT improves the motor function of the subject as measured by GSGC test. In some embodiments, compared to baseline, the subject's GSGC score improves as indicated by a decrease of at least 0.1, 0.3, 0.5, 0.7, 1.0, 1.5, or 2.5 points after 12, 26, 38, or 52 weeks of treatment. In some embodiments, compared to baseline, the subject's GSGC score improves as indicated by a decrease of at least 0.5 points after 52 weeks of treatment. In some embodiments, compared to control treatment, the subject's GSGC score improves significantly after treatment. In some embodiments, compared to control treatment, the subject's GSGC score improves significantly as indicated by a decrease of at least 0.3, 0.5, 0.7, 1.0, 1.5, 2.5, or 5 points after 12, 26, 38, or 52 weeks of treatment. In some embodiments, the subject's GSGC score significantly improves as indicated by a reduction of at least 1.0 points after 52 weeks of treatment, when compared to the control treatment.

[0032] In some embodiments, the dual component therapy for a subject with Pompe disease who has a history of ERT reduces the level of at least one muscle damage marker after treatment. In some embodiments, the at least one muscle damage marker includes CK. In some embodiments, the subject's CK level is reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, or 50% after 12, 26, 38, or 52 weeks of treatment, compared to baseline. In some embodiments, the subject's CK level is reduced by at least 15% after 52 weeks of treatment, compared to baseline. In some embodiments, the subject's CK level is significantly reduced after treatment, compared to a control treatment. In some embodiments, the subject's CK level is significantly reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, or 50% after 12, 26, 38, or 52 weeks of treatment, compared to a control treatment. In some embodiments, the subject's CK level is significantly reduced by at least 30% after 52 weeks of treatment, compared to a control treatment.

[0033] In some embodiments, the dual component therapy for a subject with Pompe disease who has a history of ERT reduces the level of at least one glycogen storage marker after treatment. In some embodiments, the at least one glycogen storage marker comprises urinary Hex4. In some embodiments, the subject's urinary Hex4 level is reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60% after 12, 26, 38, or 52 weeks of treatment, as compared to baseline. In some embodiments, the subject's urinary Hex4 level is reduced by at least 25% after 52 weeks of treatment, as compared to baseline. In some embodiments, the subject's urinary Hex4 level is significantly reduced after treatment, as compared to a control treatment. In some embodiments, the subject's urinary Hex4 level is significantly reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60% after 12, 26, 38, or 52 weeks of treatment, as compared to a control treatment. In some embodiments, the subject's urinary Hex4 levels are significantly reduced by at least 40% after 52 weeks of treatment compared to the control treatment. [Brief description of the drawings]

[0034] [Figure 1A] Non-phosphorylated high mannose N-glycans, mono-M6P N-glycans, and bis-M6P N-glycans are shown, where each box represents N-acetylglucosamine (GlcNAc), each circle represents mannose, and each P represents phosphate. [Figure 1B] The chemical structure of the M6P group is shown. [Figure 2A] Productive targeting of rhGAA to target tissues (eg, muscle tissue in subjects with Pompe disease) by M6P-bearing N-glycans is described. [Figure 2B] This accounts for non-productive drug clearance to non-target tissues (eg, liver and spleen) or due to binding of non-M6P N-glycans to non-target tissues. [Diagram 3] FIG. 1 is a schematic diagram of an exemplary production, capture and purification process for recombinant lysosomal proteins. [Figure 4] 1 shows a DNA construct for transformation of CHO cells with DNA encoding rhGAA. [Diagram 5] 1 is a graph showing the results of CIMPR affinity chromatography of ATB200 rhGAA with (embodiment 2) and without (embodiment 1) capture on an anion exchange (AEX) column. [Figure 6A] Figures 6A to 6H show the results of site-specific N-glycosylation analysis of ATB200 rhGAA using two different LC-MS / MS analytical techniques. Figure 6A shows the site occupancy of seven potential N-glycosylation sites for ATB200. [Figure 6B] Two analyses of the N-glycosylation profile of the first potential N-glycosylation site for ATB200 are shown. [Figure 6C] Two analyses of the N-glycosylation profile of the second potential N-glycosylation site for ATB200 are shown. [Figure 6D] Two analyses of the N-glycosylation profile of the third potential N-glycosylation site for ATB200 are shown. [Figure 6E] Two analyses of the N-glycosylation profile of the fourth potential N-glycosylation site for ATB200 are shown. [Figure 6F] Two analyses of the N-glycosylation profile of the fifth potential N-glycosylation site for ATB200 are shown. [Figure 6G] Two analyses of the N-glycosylation profile of the sixth potential N-glycosylation site for ATB200 are shown. [Figure 6H] The relative percentages of mono- and bis-phosphorylated species for the first, second, third, fourth, fifth, and sixth potential N-glycosylation sites are summarized. [Figure 7] 1 is a graph showing the Polywax dissolution profiles of LUMIZYME® (thin line, eluting on the left) and ATB200 (thick line, eluting on the right). [Figure 8] 1 is a table showing a summary of the N-glycan structures of LUMIZYME® compared to three different formulations of ATB200 rhGAA identified as BP-rhGAA, ATB200-1 and ATB200-2. [Figure 9A] 1 is a graph showing the results of CIMPR affinity chromatography of LUMIZYME®. [Figure 9B] 1 is a graph showing the results of CIMPR affinity chromatography of MYOZYME®. [Figure 10A] 1 is a graph comparing ATB200 rhGAA (left line) with LUMIZYME® (right line) for CIMPR binding affinity. [Figure 10B] 1 is a table comparing bis-M6P content between LUMIZYME® and ATB200 rhGAA. [Figure 11A] 1 is a graph comparing ATB200 rhGAA activity (left trace) with LUMIZYME® rhGAA activity (right trace) inside normal fibroblasts at various GAA concentrations. [Figure 11B] 1 is a table comparing ATB200 rhGAA activity (left trace) with LUMIZYME® rhGAA activity (right trace) within fibroblasts from a subject with Pompe disease at various GAA concentrations. [Figure 11C] 1 is a table comparing Kuptake in fibroblasts from normal and Pompe disease subjects. [Figure 12] FIG. 1 illustrates the stability of ATB200 in acidic or neutral pH buffers as determined in a thermal stability assay using SYPRO orange, following an increase in dye fluorescence upon protein denaturation. [Figure 13]Tissue glycogen content of WT mice or Gaa KO mice treated with vehicle, alglucosidase alpha, or ATB200 / AT2221 was determined using amyloglucosidase digestion. Bars represent mean ± SEM of 7 mice / group. *p<0.05 compared to alglucosidase alpha in multiple comparisons using Dunnett's method under one-way ANOVA analysis. [Figure 14] Illustrates LAMP1-positive vesicles in muscle fibers from Gaa KO or WT mice treated with vehicle, alglucosidase alpha, or ATB200 / AT2221. Images were taken from the vastus lateralis muscle and were representative of seven mice from each group. Magnification = 200x (inset 1,000x). [Figure 15A] LC3-positive aggregates in muscle fibers from Gaa KO or WT mice treated with vehicle, alglucosidase alpha, or ATB200 / AT2221. Images were taken from the vastus lateralis muscle and were representative of seven mice from each group. Magnification = 400x. [Figure 15B] Western blot analysis of LC3 II protein is shown. A total of 30 mg of protein was loaded in each lane. [Figure 16] Figure 2 shows dysferlin expression in muscle fibers from Gaa KO or WT mice treated with vehicle, alglucosidase alpha, or ATB200 / AT2221. Images were taken from the vastus lateralis muscle and were representative of seven mice from each group. Magnification = 200x. [Figure 17] Co-immunofluorescence staining of LAMP1 (green) (see, e.g., "B") and LC3 (red) (see, e.g., "A") in single fibers isolated from white gastrocnemius muscles of Gaa KO mice treated with vehicle, alglucosidase alpha, or ATB200 is illustrated. "C" illustrates the clearance of autophagy debris and the absence of enlarged lysosomes. A minimum of 30 fibers from each animal were examined. [Figure 18]Illustrates the stabilization of ATB200 by 17 μM and 170 μM AT2221, respectively, as compared to ATB200 alone. [Figure 19A] 19A-19H show the results of site-specific N-glycosylation analysis of ATB200 rhGAA, including the N-glycosylation profile for the seventh potential N-glycosylation site, using LC-MS / MS analysis of protease-digested ATB200. FIG. 19A-19H provide average data for 10 lots of ATB200 made at different scales. FIG. 19A shows the average site occupancy of the seven potential N-glycosylation sites for ATB200. N-glycosylation sites are provided according to SEQ ID NO:1. CV=coefficient of variation. [Figure 19B] Figures 19B-19H show site-specific N-glycosylation analysis of all seven potential N-glycosylation sites for ATB200, with site numbers provided according to SEQ ID NO:5. Bars represent the maximum and minimum percentages of N-glycan species identified as specific N-glycan groups for the 10 lots of ATB200 analyzed. Figure 19B shows the N-glycosylation profile of the first potential N-glycosylation site for ATB200. [Figure 19C] 1 shows the N-glycosylation profile of the second potential N-glycosylation site for ATB200. [Figure 19D] 1 shows the N-glycosylation profile of the third potential N-glycosylation site for ATB200. [Figure 19E] 1 shows the N-glycosylation profile of the fourth potential N-glycosylation site for ATB200. [Figure 19F] 1 shows the N-glycosylation profile of the fifth potential N-glycosylation site for ATB200. [Figure 19G] 1 shows the N-glycosylation profile of the sixth potential N-glycosylation site for ATB200. [Figure 19H] 1 shows the N-glycosylation profile of the seventh potential N-glycosylation site for ATB200. [Figure 20A] Figures 20A-20B further characterize and summarize the N-glycosylation profile of ATB200 as also shown in Figures 19A-19H. Figure 20A shows 2-anthranilic acid (2-AA) glycan mapping and LC / MS-MS analysis of ATB200, summarizing the N-glycan species identified in ATB200 as a percentage of total fluorescence. The 2-AA glycan mapping and LC-MS / MS analysis data are also illustrated in Table 5. [Figure 20B] The average site occupancy and average N-glycan profile including total phosphorylation, monophosphorylation, bisphosphorylation, and sialylation for all seven potential N-glycosylation sites of ATB200 are summarized. ND = not detected. [Figure 21] A schematic diagram of the ATB200-03 study design is shown. [Figure 22] Baseline 6-minute walk distance (6MWD) and seated forced vital capacity (FVC) characteristics of 122 subjects enrolled in the ATB200-03 study: AT-GAA group: ATB200 / AT2221-treated subjects; alglucosidase alfa group: alglucosidase alfa / placebo-treated subjects. [Figure 23A] 6MWD and FVC data are illustrated for the entire population (n=122), showing baseline, change from baseline at week 52 ("CFBL"), difference, and P-values. AT-GAA group: ATB200 / AT2221 treated subjects; Alglucosidase alfa group: Alglucosidase alfa / placebo treated subjects. [Figure 23B] 6MWD and FVC data showing change from baseline over time for the total population are shown (n=122). Sipaglucosidase alfa / miglustat group: ATB200 / AT2221 treated subjects; Alglucosidase alfa / placebo: Alglucosidase alfa / placebo treated subjects. [Figure 24]6MWD and FVC data are illustrated for the ERT-experienced population (n=95), showing CFBL at baseline, week 52, differences, and P values. AT-GAA group: ATB200 / AT2221 treated subjects; Alglucosidase alfa group: Alglucosidase alfa / placebo treated subjects. [Diagram 25] Illustrates relative change versus baseline in 6MWD and FVC at weeks 12, 26, 38, and 52 for the ERT-experienced population (n=95). [Figure 26A] 6MWD and FVC data are illustrated for the ERT-naive population (n=27), showing CFBL at baseline, week 52, differences, and P values. AT-GAA group: ATB200 / AT2221 treated subjects; Alglucosidase alfa group: Alglucosidase alfa / placebo treated subjects. [Figure 26B] 6MWD and FVC data are illustrated for the ERT-naive population (n=27) showing change from baseline over time. Sipaglucosidase alfa / miglustat group: ATB200 / AT2221 treated subjects; Alglucosidase alfa / placebo: Alglucosidase alfa / placebo treated subjects. [Figure 27] Baseline characteristics for key secondary endpoints and biomarkers for the total population and the ERT-experienced population are illustrated. AT-GAA group: ATB200 / AT2221-treated subjects; Alglucosidase alfa group: Alglucosidase alfa / placebo-treated subjects. [Figure 28] Relative change versus baseline in lower extremity manual muscle testing (MMT) at weeks 12, 26, 38, and 52 for the total population (left) and the ERT-experienced population (right) is depicted. [Figure 29]Relative change from baseline in Walk-Stair-Gowers-Chair (GSGC) is illustrated at weeks 12, 26, 38, and 52 for the total population (left) and the ERT-experienced population (right). Sipaglucosidase alfa / miglustat: ATB200 / AT2221-treated subjects; Alglucosidase alfa / placebo: alglucosidase alfa / placebo-treated subjects. [Diagram 30] Patient-Reported Outcomes Measurement Information System (PROMIS) data for relative change from baseline in physical function at weeks 12, 26, 38, and 52 for the total population (left) and the ERT-experienced population (right) are illustrated. [Diagram 31] PROMIS graphical depictions of relative change versus baseline in fatigue at weeks 12, 26, 38, and 52 for the total population (left) and the ERT-experienced population (right). [Diagram 32] Relative change versus baseline in creatine kinase (CK) biomarkers at weeks 12, 26, 38, and 52 are depicted for the total population (left) and the ERT-experienced population (right). [Diagram 33] Relative change versus baseline in urinary hexose tetrasaccharide (Hex4) biomarker at weeks 12, 26, 38, and 52 for the total population (left) and the ERT-experienced population (right) is illustrated. [Diagram 34] Primary, secondary and biomarker endpoint heat maps for the total population (left) and the ERT-experienced population (right) are shown. AT-GAA group: ATB200 / AT2221-treated subjects; alglucosidase alfa group: alglucosidase alfa / placebo-treated subjects. [Diagram 35] Safety data from the ATB200-03 study are summarized. AT-GAA group: ATB200 / AT2221-treated subjects; alglucosidase alfa group: alglucosidase alfa / placebo-treated subjects. TEAE: treatment-emergent adverse events; IAR: infusion-related reactions. [Diagram 36]To summarize the results of the ATB200-03 study. [Figure 37] Explain the test objectives and statistical methods for the ATB200-03 test. [Figure 38] The primary and secondary endpoints of the ATB200-03 study are described below. [Figure 39] Summarize patient allocation for the ATB200-03 study. [Diagram 40] Summarize baseline demographic characteristics for the ATB200-03 study. [Figure 41A] Subgroup analyses of change from baseline in 6MWD and FVC by baseline status for the entire population (n=122) in the ATB200-03 study are shown. [Figure 41B] Subgroup analyses of changes from baseline in 6MWD and FVC by baseline status in ERT-experienced patients (n=95) in the ATB200-03 study are shown. [Diagram 42] Treatment-emergent adverse events (TEAEs) occurring in ≥10% of patients in any arm of the ATB200-03 study are listed below. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] Before describing certain example embodiments of the present disclosure, it is to be understood that the disclosure is not limited to the details of structure or method steps set forth in the following description, as the disclosure is capable of other embodiments and of being practiced or carried out in various ways.

[0036] Provided herein is a method for treating Pompe disease, comprising administering to an individual recombinant human α-glucosidase (rhGAA) and a pharmacological chaperone. rhGAA has a higher total content of mannose-6-phosphate-bearing N-glycans compared to conventional rhGAA formulations, exhibits superior uptake into muscle cells and subsequent delivery to lysosomes, and has other pharmacokinetic properties that make it particularly effective for enzyme replacement therapy in subjects with Pompe disease. Accordingly, the binary therapy according to the present disclosure exhibits superior efficacy in treating and reversing the progression of the disease in subjects suffering from Pompe disease compared to conventional therapies.

[0037] I. Definition The terms used herein generally have their ordinary meaning in the art, within the context of this disclosure and in the specific context in which each term is used. Certain terms are discussed below or elsewhere in this specification to provide additional guidance to the practitioner in describing the compositions and methods of the present disclosure and how to make and use them. The articles "a" and "an" refer to one or more than one (i.e., at least one) of the grammatical referent of the article. The term "or" means, and is used synonymously with, the term "and / or," unless the context clearly dictates otherwise. In this application, the use of the singular includes the plural, unless specifically stated otherwise. Furthermore, the use of the term "including" as well as other forms, such as "includes" and "included in," is not intended to be limiting. It will be understood that any range described herein includes its endpoints and all values ​​between the endpoints. In this specification, unless the context otherwise requires by express language or necessary implication, the word "comprises" or variations such as "comprising" are used in an inclusive sense, i.e., to specify the presence of the indicated features but not to exclude the presence or addition of further features in various embodiments of the present disclosure.

[0038] The term "GAA" refers to the human acid alpha-glucosidase (GAA) enzyme, which catalyzes the hydrolysis of alpha-1,4- and alpha-1,6-glycosidic bonds of lysosomal glycogen, as well as insertion, relational, or substitution variants of the GAA amino acid sequence and fragments of the long GAA sequence that exhibit 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 (location 17q25.2-q25.3). An exemplary amino acid sequence of GAA is NP 000143.2, which is incorporated by reference. The present disclosure also encompasses DNA sequences that encode the amino acid sequence of NP 000143.2. Currently, over 500 mutations have been identified in the human GAA gene, many of which are associated with Pompe disease. Mutations that result in misfolding or misprocessing of the acid α-glucosidase enzyme include T1064C (Leu355Pro) and C2104T (Arg702Cys). In addition, GAA mutations that affect maturation and processing of the enzyme include Leu405Pro and Met519Thr. The conserved hexapeptide WIDMNE (SEQ ID NO: 7) at amino acid residues 516-521 is required for activity of the acid α-glucosidase protein. As used herein, the abbreviation "GAA" is intended to refer to the human acid α-glucosidase enzyme, while the abbreviation "GAA" written in italics is intended to refer to the human gene encoding the human acid α-glucosidase enzyme. The abbreviation "Gaa" written in italics is intended to refer to a non-human gene encoding a non-human acid alpha-glucosidase enzyme, including but not limited to a rat or mouse gene, and the abbreviation "Gaa" is intended to refer to a non-human acid alpha-glucosidase enzyme.

[0039] The term "rhGAA" is intended to refer to recombinant human acid alpha-glucosidase enzyme and is used to distinguish endogenous GAA from synthetic or recombinantly produced GAA (e.g., GAA produced from CHO cells or other host cells transformed with DNA encoding GAA). The term "rhGAA" encompasses populations of individual rhGAA molecules. Properties of rhGAA molecule populations are provided herein. The term "conventional rhGAA formulation" is intended to refer to formulations containing alglucosidase alpha, such as LUMIZYME® or MYOZYME®.

[0040] The term "genetically modified" or "recombinant" refers to cells, such as CHO cells, that express a particular gene product, such as rhGAA, following introduction of a nucleic acid that includes a coding sequence that encodes the gene product together with regulatory elements that control expression of the coding sequence. Introduction of the nucleic acid may 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, for example, by gene activation techniques, to express or overexpress an endogenous gene or a gene product that such cells do not normally express.

[0041] As used herein, the term "alglucosidase alfa" is intended to refer to recombinant human acid alpha-glucosidase identified as [199-Arginine, 223-Histidine] prepro-alpha-glucosidase (human); Chemical Abstracts Registry Number 420794-05-0. Alglucosidase alfa is approved for sale in the United States by Genzyme under the formulations LUMIZYME® and MYOZYME®.

[0042] As used herein, the term "ATB200" is intended to refer to recombinant human acid α-glucosidase as described in U.S. Patent No. 10,961,522, the disclosure of which is incorporated herein by reference. ATB200 is also referred to as "sipa glucosidase alpha."

[0043] As used herein, the term "glycan" is intended to refer to an oligosaccharide covalently attached to an amino acid residue on a protein or polypeptide. As used herein, the term "N-glycan" or "N-linked glycan" is intended to refer to a polysaccharide chain that is attached to an asparagine residue on a protein or polypeptide via a covalent bond with the nitrogen atom of the asparagine residue. In some embodiments, the N-glycan unit attached to rhGAA is determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS) using an instrument such as a Thermo Scientific™ Orbitrap Velos Pro™ mass spectrometer, a Thermo Scientific™ Orbitrap Fusion™ Lumos Tribid™ mass spectrometer, or a Waters Xevo® G2-XS QTof mass spectrometer.

[0044] As used herein, forced vital capacity, or "FVC," is the volume of air that can be forcefully exhaled from a subject's lungs after the subject takes in the deepest possible breath.

[0045] As used herein, the "6-minute walk test" (6MWT) is a test that measures the distance an individual can walk on a hard, flat surface in a total of six minutes. The test is conducted by having an individual walk as far as possible in six minutes.

[0046] As used herein, the "10 Meter Walk Test" (10MWT) is a test that measures the time it takes an individual wearing walking shoes to walk 10 meters on a flat surface.

[0047] 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: [ka]

[0048] One formulation of miglustat is marketed under the trade name ZAVESCA® as monotherapy for Gaucher disease type 1. In some embodiments, miglustat is referred to as AT2221.

[0049] As discussed below, pharma- ceutically acceptable salts of miglustat may also be used in the present disclosure. When a salt of miglustat is used, the dosage of the salt will be adjusted so that the dose of miglustat the patient receives is equivalent to the amount they would have received if miglustat free base had been used.

[0050] 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: [ka]

[0051] 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 α-glucosidase and has one or more of the following effects: Enhances the formation of stable molecular conformations of proteins; · enhancing proper transport of proteins from the endoplasmic reticulum to another cellular site, preferably the native cellular site, thereby preventing endoplasmic reticulum-associated degradation of proteins; · Prevents aggregation of conformationally unstable or misfolded proteins; restore and / or enhance wild-type function, stability, and / or activity of at least a portion of a protein; and / or Improving the phenotype or function of cells harboring acid α-glucosidase.

[0052] Thus, a pharmacological chaperone for acid α-glucosidase is a molecule that binds to acid α-glucosidase, resulting in proper folding, trafficking, non-aggregation, and 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 duvoglustat.

[0053] As used herein, the term "pharmaceutical 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 "pharmaceutical acceptable" means approved by a federal or state regulatory agency for use in animals, more particularly in humans, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias. As used herein, the term "carrier" is intended to refer to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Suitable pharmaceutical carriers are known in the art and, in at least one embodiment, are described in "Remington's Pharmaceutical Sciences" by E.W. Martin, 18th Edition or other editions.

[0054] The term "pharmaceutically acceptable salts" as used herein is intended to mean salts that are generally water- or oil-soluble or dispersible and effective for their intended use, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic reaction, etc., commensurate with a reasonable risk-to-benefit ratio. The term includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. For a list of suitable salts, see, for example, S. M. Berge et al., J. Pharm. Sci., 1977, 66, pp. 1-19, which is incorporated herein by reference. The term "pharmaceutically acceptable acid addition salts" as used herein is intended to mean salts formed with inorganic acids which retain the biological effectiveness and properties of the free bases and which are not biologically or otherwise undesirable. The term "pharmaceutically acceptable base addition salts", as used herein, is intended to mean salts formed with inorganic bases which retain the biological effectiveness and properties of the free acids and which are not biologically or otherwise undesirable.

[0055] As used herein, the term "buffer" refers to a solution containing a weak acid and its conjugate base or a weak base and its conjugate acid that helps prevent changes in pH.

[0056] As used herein, the terms "therapeutically effective dose" and "effective amount" are intended to refer to the amount of acid α-glucosidase and / or the amount of miglustat and / or the amount of a binary therapy thereof sufficient to produce a therapeutic response in a subject.

[0057] Therapeutic response may also include molecular responses, such as glycogen accumulation, lysosomal proliferation, and the formation of autophagy regions. Therapeutic response may be determined by comparing the physiological and molecular responses of muscle biopsies before and after treatment with rhGAA as described herein. For example, the amount of glycogen present in the biopsy sample can be used as a marker in determining therapeutic response. Another example includes biomarkers such as LAMP-1, LC3, and dysferlin, which can be used as indicators of lysosomal storage deficiency. For example, muscle biopsies taken before and after treatment with rhGAA as described herein may be stained with an antibody that recognizes one of these biomarkers. Therapeutic response may also include reduced fatigue or improved other patient-reported outcomes (e.g., activities of daily living, well-being, etc.).

[0058] As used herein, the term "enzyme replacement therapy" or "ERT" is intended to refer to the introduction of a non-naturally occurring purified enzyme into an individual who is deficient in such an enzyme. The administered protein can be obtained from a natural source or by recombinant expression. The term also refers to the introduction of a purified enzyme in an individual who would otherwise require or benefit from the administration of the purified enzyme. In at least one embodiment, such an individual suffers from an enzyme deficiency. The introduced enzyme may be a purified recombinant enzyme made in vitro, or a protein purified from an isolated tissue or body fluid, such as the placenta or milk of an animal, or from a plant.

[0059] As used herein, the term "binary therapy" is intended to refer to any therapy in which two or more individual therapies are administered simultaneously or sequentially. In some embodiments, the results of the binary therapy are enhanced when compared to the effects of each therapy when administered individually. Enhancement may include any improvement in the effects of the various therapies that may result in favorable results compared to the results achieved by those therapies when administered alone. Enhancement of effects or results may include synergistic enhancement, where the enhanced effect is greater than the additive effect of each therapy when administered alone; additive enhancement, where the enhanced effect is substantially equal to the additive effect of each therapy when administered alone; or subadditive effect, where the enhanced effect is less than the additive effect of each therapy when administered alone, but still better than the effect of each therapy when administered alone. Enhanced effects may be measured by any means known in the art to be able to measure therapeutic efficacy or outcome.

[0060] "Pompe disease" refers to an autosomal recessive LSD characterized by impaired lysosomal glycogen metabolism due to deficiency in acid alpha-glucosidase (GAA) activity. This enzymatic deficiency leads to lysosomal glycogen accumulation and progressive skeletal muscle weakness, cardiac decline, respiratory failure, and / or CNS dysfunction in end-stage disease. Genetic mutations in the GAA gene either result in reduced expression or give rise to mutant forms of the enzyme with altered stability and / or biological activity, ultimately leading to disease (for review see Hirschhorn R, 1995, Glycogen Storage Disease Type II: Acid a-Glucosidase(Acid Maltase) Deficiency, The Metabolic and Molecular Bases of Inherited Disease, Scriver et al., eds., McGraw-Hill, New York, 7th ed., pages 2443-2464). The three recognized clinical forms of Pompe disease (infantile, juvenile, and adult) correlate with the level of residual α-glucosidase activity (Reuser AJ et al., 1995, "Glycogenosis Type II (Acid Maltase Deficiency)", Muscle & Nerve Supplement 3, S61-S69). Infantile Pompe disease (type I or A) is the most common and most severe, characterized by failure to thrive within the first 2 years of life, generalized hypotonia, cardiac hypertrophy, and cardiopulmonary insufficiency. Juvenile Pompe disease (type II or B) is of intermediate severity and is characterized by a predominance of muscular symptoms and the absence of cardiac hypertrophy. Individuals with juvenile Pompe disease usually die of respiratory failure before the age of 20 years.Adult Pompe disease (type III or type C) often presents as a slowly progressive myopathy in the teens or as late as the sixth decade (Felicia KJ et al., 1995, Clinical Variability in Adult-Onset Acid Maltase Deficiency: Report of Affected Sibs and Review of the Literature, Medicine 74, 131-135). In Pompe disease, α-glucosidase has been shown to undergo extensive post-translational modification by glycosylation, phosphorylation, and proteolytic processing. Optimal glycogen catalysis requires the conversion of the 110 kilodalton (kDa) precursor to the 76 and 70 KDa mature forms by lysosomal proteolysis. As used herein, the term "Pompe disease" refers to any form of Pompe disease. The formulations and administration regimens disclosed herein may be used, for example, to treat Type I, Type II, or Type III Pompe disease.

[0061] As used herein, "significant" refers to statistical significance.This term refers to the statistical evidence that there is a difference between two treatment groups.It can be defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true.This decision is often made using a p-value < 0.05 derived from suitable statistical analysis of the comparison.See, for example, Example 9.

[0062] A "subject" or "patient" is preferably a human, although other mammals and non-human animals with disorders involving glycogen storage may also be treated. A subject may be a fetus, neonate, child, juvenile, or adult with Pompe disease or other glycogen storage or accumulation disorder. One example of an individual to be treated is an individual (fetal, neonatal, child, juvenile, adolescent, or adult human) with GSD-II (e.g., infantile GSD-II, juvenile GSD-II, or adult-onset GSD-II). The individual may have residual GAA activity or no measurable activity. For example, an individual with GSD-II may have GAA activity less than about 1% of normal GAA activity (infantile GSD-II), about 1-10% of normal GAA activity (juvenile GSD-II), or about 10-40% of normal GAA activity (adult GSD-II). In some embodiments, the subject or patient is an "ERT-historic" or "ERT-switched" patient, which refers to a Pompe disease patient who has previously undergone enzyme replacement therapy. In some embodiments, an "ERT-historic" or "ERT-switched" patient is a Pompe disease patient who has previously undergone or is currently undergoing administration of alglucosidase alfa for 24 months or more. In some embodiments, the subject or patient is an "ERT-naive" patient, which refers to a Pompe disease patient who has not previously undergone enzyme replacement therapy. In certain embodiments, the subject or patient is ambulatory (e.g., an ambulatory ERT-switched patient or an ambulatory ERT-naive patient). In certain embodiments, the subject or patient is non-ambulatory (e.g., a non-ambulatory ERT-switched patient). Ambulatory or non-ambulatory status may be determined by a 6-minute walk test (6MWT). In some embodiments, an ambulatory patient is a Pompe disease patient who is able to walk at least 200 meters in the 6MWT. In some embodiments, a non-ambulatory patient is a Pompe disease patient who is unable to walk unaided or who is wheelchair bound.

[0063] The terms "treat" and "treatment" as used herein refer to improving one or more symptoms associated with a disease, delaying the onset of one or more symptoms of a disease, and / or reducing the severity or frequency of one or more symptoms of a disease. For example, treatment can refer to improving cardiac status (e.g., increasing end-diastolic and / or end-systolic volume, or reducing or improving progressive cardiomyopathy typically seen in GSD-II) or improving pulmonary function (e.g., increasing crying vital capacity compared to baseline volume, and / or normalizing oxygen desaturation during crying); improving neurodevelopment and / or motor skills (e.g., increasing AIMS score); reducing glycogen levels in tissues of individuals affected by the disease; or any combination of these effects. In a preferred embodiment, treatment includes improving cardiac status, particularly reducing GSD-II associated cardiomyopathy.

[0064] The terms "improve," "increase," and "decrease," as used herein, refer to a relative value relative to a baseline measurement or a corresponding value from a control treatment, such as a measurement in the same individual before the initiation of a treatment described herein, a measurement in a control individual (or control individuals) in the absence of a treatment described herein, or a measurement after a control treatment. A control individual is an individual who is afflicted with the same type of GSD-II (either infantile, juvenile, or adult-onset) as the individual under treatment and is of approximately the same age as the individual under treatment (to ensure that the stage of the disease in the treated individual and the control individual or individuals is comparable). In some embodiments, the control treatment comprises administering alglucosidase alfa and a pharmacological chaperone placebo (see Example 9).

[0065] As used herein, the terms "about" and "approximately" are intended to refer to an acceptable degree of error for the quantity being measured given the nature or precision of the measurement. For example, the degree of error may be indicated by the number of significant digits provided for the measurement, as understood in the art, including but not limited to a variation of ±1 to the most accurate significant digit 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. Numerical quantities given herein are approximate unless otherwise specified, meaning that the term "about" or "approximately" can be inferred even when not specified.

[0066] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, the mention of any reference, article, publication, patent, patent publication, or patent application cited herein is not, and should not be construed as, an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0067] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0068] II. Recombinant human acid α-glucosidase (rhGAA) In some embodiments, the 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, SEQ ID NO:5, or SEQ ID NO:6. In some embodiments, the rhGAA is encoded by a nucleotide sequence as set forth in SEQ ID NO:2.

[0069] [Table 1]

[0070] [Table 2]

[0071] [Table 3]

[0072] In some embodiments, rhGAA has the GAA amino acid sequence as set forth in SEQ ID NO: 1, as described in U.S. Patent No. 8,592,362, and has GenBank Accession No. AHE24104.1 (GI:568760974). In some embodiments, rhGAA has the GAA amino acid sequence as encoded by SEQ ID NO: 2, an mRNA sequence with GenBank Accession No. Y00839.1. In some embodiments, rhGAA has the GAA amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, rhGAA has the GAA amino acid sequence as set forth in SEQ ID NO: 4, and has National Center for Biotechnology Information (NCBI) Accession No. NP_000143.2 or UniProtKB Accession No. P10253.

[0073] In some embodiments, rhGAA is initially expressed as having the full-length 952 amino acid sequence of wild-type GAA as set forth in SEQ ID NO:1 or SEQ ID NO:4, and as rhGAA undergoes intracellular processing, some of the amino acids, for example the first 56 amino acids, are removed. Accordingly, rhGAA secreted from a host cell may have a shorter amino acid sequence than rhGAA initially expressed within the cell. In some embodiments, this shorter protein has the amino acid sequence set forth in SEQ ID NO:5, which differs from SEQ ID NO:1 only in that the first 56 amino acids, including the signal peptide and precursor peptide, have been removed, resulting in a protein with 896 amino acids. In some embodiments, this shorter protein has the amino acid sequence set forth in SEQ ID NO:6, which differs from SEQ ID NO:4 only in that the first 56 amino acids, including the signal peptide and precursor peptide, have been removed, resulting in a protein with 896 amino acids. Other variations in the number of amino acids are also possible, such as having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more deletions, substitutions and / or insertions relative to the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. In some embodiments, the rhGAA product includes a mixture of recombinant human acid α-glucosidase molecules having different amino acid lengths.

[0074] In some embodiments, rhGAA comprises an amino acid sequence at least 90%, 95%, 98% or 99% identical to SEQ ID NO:4 or SEQ ID NO:6. A variety of alignment algorithms and / or programs may be used to calculate the identity between two sequences, including FASTA, or BLAST available as part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), which may be used, for example, with default settings. For example, polypeptides having at least 90%, 95%, 98% or 99% identity to a particular polypeptide described herein, and preferably exhibiting substantially the same function, as well as 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 will be based on the BLASTP positive score, and the percentage of sequence identity will be based on the BLASTP identity score. BLASTP "identity" indicates the number and percentage of total residues in the high-scoring sequence pair that are identical; and BLASTP "positive value" indicates the number and percentage of residues for which the alignment score has a positive value and that are similar to each other. Amino acid sequences having these degrees of identity or similarity, or any intermediate degree of identity or similarity, with the amino acid sequences disclosed herein are contemplated and are encompassed by the present disclosure. Polynucleotide sequences of similar polypeptides are inferred using the genetic code and may be obtained by conventional means, in particular by back-translating the amino acid sequences using the genetic code.

[0075] In some embodiments, rhGAA undergoes post-translational and / or chemical modifications to one or more amino acid residues in the protein. For example, methionine and tryptophan residues can undergo oxidation. As another example, the N-terminal glutamine of SEQ ID NO:6 can be further modified to form pyroglutamic acid. As another example, an asparagine residue can undergo deamidation to aspartic acid. As yet another example, an aspartic acid residue can undergo isomerization to isoaspartic acid. As yet another example, an unpaired cysteine ​​residue in the protein can form a disulfide bond with free glutathione and / or cysteine. Accordingly, in some embodiments, the enzyme is initially expressed as having an amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, or an amino acid sequence encoded by SEQ ID NO:2, and the enzyme undergoes one or more of these post-translational and / or chemical modifications. Such modifications are also within the scope of the present disclosure.

[0076] III. N-Linked Glycosylation of rhGAA There are seven potential N-linked glycosylation sites on a single rhGAA molecule. These potential glycosylation sites are at the following positions of SEQ ID NO:6: N84, N177, N334, N414, N596, N826, and N869. Similarly, for the full-length amino acid sequence of SEQ ID NO:4, these potential glycosylation sites are at the following positions: N140, N233, N390, N470, N652, N882, and N925. Other variants of rhGAA may have similar glycosylation sites depending on the position of the asparagine residue. Generally, the sequence Asn-X-Ser or Asn-X-Thr in the protein amino acid sequence indicates a potential glycosylation site, with the proviso that X cannot be His or Pro.

[0077] The rhGAA molecules described herein may have, on average, one, two, three, or four 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 (bisphosphorylated or bis-M6P), or two different N-glycans on the same rhGAA molecule may each carry a single M6P group. In some embodiments, the rhGAA molecules described herein have, on average, 3-4 moles of M6P groups on their N-glycans per mole of rhGAA. The recombinant human acid α-glucosidase molecule may also have N-glycans that do not carry M6P groups. In another embodiment, on average, rhGAA contains 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, on average, rhGAA contains about 3-3.5 moles of M6P per mole of rhGAA. In some embodiments, on average, rhGAA contains 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 have 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 bis-M6P N-glycans, and on average, less than 25% of the total rhGAA does not contain phosphorylated N-glycans that bind to CIMPR. In some embodiments, on average, about 10% to about 14% of the total N-glycans on rhGAA are monophosphorylated. In some embodiments, on average, about 7% to about 25% of the total N-glycans on rhGAA are bisphosphorylated. In some embodiments, on average, rhGAA contains about 1.3 moles of bis-M6P per mole of rhGAA.

[0078] The rhGAA described herein may have an average of 0.5-7.0 moles of M6P per mole of rhGAA, or any intermediate or subrange thereof, including 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, or 7.0 moles of M6P per mole of rhGAA. The rhGAA may be fractionated to provide rhGAA preparations with various average numbers of mono- or bis-M6P-bearing N-glycans by selecting specific fractions or by selectively combining different fractions, thus allowing further customization of rhGAA targeting to the lysosomes of target tissues.

[0079] 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, 50% or less of the N-glycans on rhGAA are fully sialylated. In some embodiments, between 4% and 20% of the total N-glycans are fully sialylated. In other embodiments, up to 5%, 10%, 20% or 30% of the N-glycans on rhGAA have sialic acid and terminal galactose residues (Gal). This range includes all intermediate values ​​and subranges, for example, between 7% and 30% of the total N-glycans on rhGAA may have sialic acid and terminal galactose. In yet other embodiments, no more than 5%, 10%, 15%, 16%, 17%, 18%, 19%, or 20% of the N-glycans on rhGAA have only terminal galactose and no sialic acid, including all intermediate values ​​and subranges within this range, for example, between 8% and 19% of the total N-glycans on rhGAA in the composition have only terminal galactose and no sialic acid.

[0080] In some embodiments, between 40% and 60%, between 45% and 60%, between 50% and 60%, or between 55% and 60% of the total N-glycans on rhGAA are complex type N-glycans; or less than 1%, 2%, 3%, 4%, 5%, 6%, or 7% of the total N-glycans on rhGAA are hybrid type N-glycans; less than 5%, 10%, 15%, 20%, or 25% of the high mannose type N-glycans on rhGAA are non-phosphorylated; or at least 5% or 10% of the high mannose type N-glycans on rhGAA are monophosphorylated. and / or at least 1% or 2% of the high mannose N-glycans on rhGAA are bisphosphorylated. These values ​​include all intermediate values ​​and subranges. rhGAA can meet one or more of the content ranges described above.

[0081] In some embodiments, rhGAA may carry, on average, 2.0 to 8.0 moles of sialic acid residues per mole of rhGAA, 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, and all intermediate values ​​and subranges therein. Without being bound by theory, it is believed that the presence of N-glycan units carrying sialic acid residues may prevent non-productive clearance of rhGAA by the asialoglycoprotein receptor.

[0082] In one or more embodiments, rhGAA has a certain N-glycosylation profile at a certain potential N-glycosylation site. In some embodiments, rhGAA has seven potential N-glycosylation sites. In some embodiments, at least 20% of rhGAA is phosphorylated at the first potential N-glycosylation site (e.g., N84 for SEQ ID NO:6 and N140 for SEQ ID NO:4). For example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of rhGAA may be phosphorylated at the first potential N-glycosylation site. This phosphorylation may be the result of mono-M6P and / or bis-M6P units. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA bears a mono-M6P unit at the first potential N-glycosylation site. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA bears a bis-M6P unit at the first potential N-glycosylation site. In some embodiments, rhGAA contains, on average, about 1.4 moles of M6P (mono-M6P and bis-M6P) per mole of rhGAA at the first potential N-glycosylation site. In some embodiments, rhGAA contains, on average, at least about 0.5 moles of bis-M6P per mole of rhGAA at the first potential N-glycosylation site. In some embodiments, rhGAA contains, on average, about 0.25 moles of mono-M6P per mole of rhGAA at the first potential N-glycosylation site. In some embodiments, rhGAA contains, on average, 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, the rhGAA comprises a first potential N-glycosylation site occupancy as depicted in Figure 6A and an N-glycosylation profile as depicted in Figure 6B. In at least one embodiment, the rhGAA comprises a first potential N-glycosylation site occupancy as depicted in Figure 19A and an N-glycosylation profile as depicted in Figure 19B or Figure 20B.

[0083] In some embodiments, at least 20% of the rhGAA is phosphorylated at the second potential N-glycosylation site (e.g., N177 for SEQ ID NO: 6 and N223 for SEQ ID NO: 4). For example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA may be phosphorylated at the second N-glycosylation site. This phosphorylation may be the result of mono-M6P and / or bis-M6P units. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA bears mono-M6P units at the second N-glycosylation site. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA bears bis-M6P units at the second N-glycosylation site. In some embodiments, rhGAA comprises, on average, about 0.5 moles of M6P (mono-M6P and bis-M6P) per mole of rhGAA at the second potential N-glycosylation site. In some embodiments, rhGAA comprises, on average, about 0.4 to about 0.6 moles of mono-M6P per mole of rhGAA at the second potential N-glycosylation site. In at least one embodiment, rhGAA comprises a second potential N-glycosylation site occupancy as depicted in FIG. 6A and an N-glycosylation profile as depicted in FIG. 6C. In at least one embodiment, rhGAA comprises a second potential N-glycosylation site occupancy as depicted in FIG. 19A and an N-glycosylation profile as depicted in FIG. 19C or FIG. 20B.

[0084] 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:6 and N390 for SEQ ID NO:4). 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 can have a mixture of non-phosphorylated high mannose type N-glycans, diantennary, triantennary, and tetraantennary complex type N-glycans, and hybrid type N-glycans as the predominant species. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the rhGAA is sialylated at the third potential N-glycosylation site. In some embodiments, the rhGAA comprises on average 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, the rhGAA comprises a third potential N-glycosylation site occupancy as depicted in Figure 6A and an N-glycosylation profile as depicted in Figure 6D. In at least one embodiment, the rhGAA comprises a third potential N-glycosylation site occupancy as depicted in Figure 19A and an N-glycosylation profile as depicted in Figure 19D or Figure 20B.

[0085] In some embodiments, at least 20% of the rhGAA is phosphorylated at the fourth potential N-glycosylation site (e.g., N414 for SEQ ID NO: 6 and N470 for SEQ ID NO: 4). For example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA may be phosphorylated at the fourth potential N-glycosylation site. This phosphorylation may be the result of mono-M6P and / or bis-M6P units. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA bears a mono-M6P unit at the fourth potential N-glycosylation site. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA bears a bis-M6P unit at the fourth potential N-glycosylation site. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, or 25% of the rhGAA is sialylated at the fourth potential N-glycosylation site. In some embodiments, the rhGAA contains, on average, about 1.4 moles of M6P (mono-M6P and bis-M6P) per mole of rhGAA at the fourth potential N-glycosylation site. In some embodiments, the rhGAA contains, on average, about 0.4 to about 0.6 moles of bis-M6P per mole of rhGAA at the fourth potential N-glycosylation site. In some embodiments, the rhGAA contains, on average, 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 profile as depicted in FIG. 6E.In at least one embodiment, rhGAA comprises a fourth potential N-glycosylation site occupancy as depicted in FIG. 19A and an N-glycosylation profile as depicted in FIG. 19E or FIG. 20B.

[0086] In some embodiments, at least 5% of the rhGAA is phosphorylated at the fifth potential N-glycosylation site (e.g., N596 for SEQ ID NO:6 and N692 for SEQ ID NO:4). In other embodiments, less than 5%, 10%, 15%, 20%, or 25% of the rhGAA is phosphorylated at the fifth potential N-glycosylation site. For example, the fifth potential N-glycosylation site can have fucosylated diantennary complex-type 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 the rhGAA is sialylated at the fifth potential N-glycosylation site. In some embodiments, rhGAA comprises on average 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 Figure 6A and an N-glycosylation profile as depicted in Figure 6F. In at least one embodiment, rhGAA comprises a fifth potential N-glycosylation site occupancy as depicted in Figure 19A and an N-glycosylation profile as depicted in Figure 19F or Figure 20B.

[0087] In some embodiments, at least 5% of the rhGAA is phosphorylated at the sixth N-glycosylation site (e.g., N826 for SEQ ID NO:6 and N882 for SEQ ID NO:4). In other embodiments, less than 5%, 10%, 15%, 20%, or 25% of the rhGAA is phosphorylated at the sixth N-glycosylation site. For example, the sixth N-glycosylation site can have a mixture of diantennary, triantennary, and tetraantennary complex type 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 the rhGAA is sialylated at the sixth N-glycosylation site. In some embodiments, rhGAA comprises, on average, 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, on average, about 0.9 moles of acetylated sialic acid per mole of rhGAA at the sixth potential N-glycosylation site. In some embodiments, rhGAA comprises a glycan species having an average of at least 0.05 moles of poly-N-acetyl-D-lactosamine (poly-LacNAc) residues per mole of rhGAA at the sixth potential N-glycosylation site. In some embodiments, greater than 10% of rhGAA comprises a glycan bearing a poly-LacNAc residue at the sixth potential N-glycosylation site. In at least one embodiment, rhGAA comprises a sixth potential N-glycosylation site occupancy as depicted in FIG. 6A and an N-glycosylation profile as depicted in FIG. 6G. In at least one embodiment, rhGAA comprises a sixth potential N-glycosylation site occupancy as depicted in FIG. 19A and an N-glycosylation profile as depicted in FIG. 19G or FIG. 20B.

[0088] In some embodiments, at least 5% of the rhGAA is phosphorylated at the seventh potential N-glycosylation site (e.g., N869 for SEQ ID NO:6 and N925 for SEQ ID NO:4). In other embodiments, less than 5%, 10%, 15%, 20%, or 25% of the rhGAA is phosphorylated at the seventh potential N-glycosylation site. In some embodiments, less than 40%, 45%, 50%, 55%, 60%, or 65% of the rhGAA has any N-glycan at the seventh potential N-glycosylation site. In some embodiments, at least 30%, 35%, or 40% of the rhGAA has an N-glycan at the seventh potential N-glycosylation site. In some embodiments, the rhGAA comprises, on average, at least 0.5 moles of sialic acid per mole of rhGAA at the seventh potential N-glycosylation site. In some embodiments, rhGAA contains an average of at least 0.8 moles of sialic acid per mole of rhGAA at the seventh potential N-glycosylation site. In some embodiments, rhGAA contains an average of about 0.86 moles of sialic acid per mole of rhGAA at the seventh potential N-glycosylation site. In some embodiments, rhGAA contains an average of at least 0.3 moles of glycan species bearing poly-LacNAc residues per mole of rhGAA at the seventh potential N-glycosylation site. In some embodiments, nearly half of rhGAA contains glycans bearing poly-LacNAc residues at the seventh potential N-glycosylation site. In at least one embodiment, all N-glycans identified at the seventh potential N-glycosylation site are complex N-glycans. In at least one embodiment, rhGAA comprises a seventh potential N-glycosylation site occupancy as depicted in FIG. 6A, or as depicted in FIG. 19A, and an N-glycosylation profile as depicted in FIG. 19H or FIG. 20B.

[0089] In some embodiments, rhGAA contains, on average, 3-4 moles of M6P residues per mole of rhGAA and about 4 to about 7.3 moles of sialic acid per mole of rhGAA. In some embodiments, the rhGAA contains, on average, at least 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 0.8 to about 0.9 moles of sialic acid per mole of rhGAA at the sixth potential N-glycosylation site. rhGAA further comprises about 1.5 to about 4.2 moles of sialic acid per mole of rhGAA. In some embodiments, rhGAA further comprises an average of at least 0.5 moles of sialic acid per mole of rhGAA at the seventh potential N-glycosylation site. In some embodiments, rhGAA further comprises an average of at least 0.8 moles of sialic acid per mole of rhGAA at the seventh potential N-glycosylation site. In at least one embodiment, rhGAA further comprises an average of about 0.86 moles of sialic acid per mole of rhGAA at the seventh potential N-glycosylation site. In at least one embodiment, rhGAA comprises seven potential N-glycosylation sites with occupancy and N-glycosylation profiles as depicted in Figures 6A-6H. In at least one embodiment, rhGAA contains seven potential N-glycosylation sites with occupancy and N-glycosylation profiles as depicted in Figures 19A-H and 20A-B.

[0090] Methods for making rhGAA are disclosed in U.S. Provisional Patent Application No. 62 / 057,842, filed Sep. 30, 2014, the entire contents of which are incorporated herein by reference.

[0091] Once inside the lysosome, rhGAA can enzymatically degrade accumulated glycogen. However, conventional rhGAA formulations have low total levels of mono-M6P and bis-M6P-bearing N-glycans, which in turn results in poor targeting of muscle cells, resulting in poor delivery of rhGAA to lysosomes. The majority of rhGAA molecules in these conventional formulations do not have phosphorylated N-glycans and therefore lack affinity for CIMPR. Non-phosphorylated high mannose type N-glycans can also be removed by the mannose receptor, resulting in non-productive clearance of ERT (Figure 2B). In contrast, as shown in Figure 2A, the rhGAA described herein can contain higher amounts of mono-M6P and bis-M6P-bearing N-glycans, leading to productive uptake of rhGAA into specific tissues such as muscle.

[0092] IV. Preparation and purification of N-linked glycosylated rhGAA As described in U.S. Patent No. 10,961,522 (which is incorporated herein by reference in its entirety), cells such as Chinese hamster ovary (CHO) cells can be used to produce the rhGAA described herein. Expressing high M6P rhGAA in CHO cells is advantageous, at least in part, because compared to post-translationally modifying the glycan profile of rhGAA, only the former can be converted by glycan degradation to the optimal glycogen hydrolysis form of rhGAA, which can then enhance therapeutic efficacy.

[0093] In some embodiments, rhGAA is preferably produced by one or more CHO cell lines transformed with a DNA construct encoding rhGAA as described herein. Such CHO cell lines may contain multiple copies of the gene, such as 5, 10, 15, or 20 or more copies of a polynucleotide encoding GAA. DNA constructs expressing allelic variants of acid alpha-glucosidase or other variant acid alpha-glucosidase amino acid sequences, such as those at least 90%, 95%, 98%, or 99% identical to SEQ ID NO: 4 or SEQ ID NO: 6, may be constructed and expressed in CHO cells. Those skilled in the art may select alternative vectors suitable for transformation of CHO cells to produce such DNA constructs.

[0094] Methods for making such CHO cell lines are described in U.S. Patent No. 10,961,522, which is incorporated herein by reference in its entirety. Briefly, these methods involve transforming CHO cells with DNA encoding GAA or a GAA variant, selecting CHO cells that stably integrate the DNA encoding GAA into one or more of their chromosomes and stably express GAA, and selecting CHO cells that express GAA with a high content of N-glycans carrying mono-M6P or bis-M6P, and optionally selecting CHO cells with high sialic acid content N-glycans and / or low non-phosphorylated high mannose content N-glycans. The selected CHO cell lines may be used to make rhGAA and rhGAA compositions by culturing the CHO cell lines and recovering the compositions from the culture of the CHO cells. In some embodiments, the rhGAA produced from the selected CHO cell line contains high content of N-glycans carrying mono-M6P or bis-M6P targeting CIMPR. In some embodiments, the rhGAA produced as described herein has low levels of complex type N-glycans with terminal galactose. In some embodiments, the selected CHO cell line is referred to as GA-ATB200 or ATB200-X5-14. In some embodiments, the selected CHO cell line includes subcultures or derivatives of such CHO cell cultures. In some embodiments, the rhGAA produced from the selected CHO cell line is referred to as ATB200.

[0095] rhGAA produced as described herein may be purified by following the methods described in U.S. Patent No. 10,227,577 and U.S. Provisional Patent Application No. 62 / 506,569, both of which are incorporated herein by reference in their entireties. An exemplary process for the production, capture, and purification of rhGAA produced from a CHO cell line is shown in FIG.

[0096] 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 bioreactor suitable for culturing cells, such as a perfusion, batch, or fed-batch bioreactor. Culture medium is removed from the bioreactor after sufficient time for the cells to produce rhGAA. Such medium removal may be continuous in a perfusion bioreactor, or batch-by-batch in a batch or fed-batch reactor. Cells may be removed by filtering the medium through a filtration system 603. Filtration system 603 may be any suitable filtration system, including an alternating tangential flow filtration (ATF) system, a tangential flow filtration (TFF) system, and / or a centrifugal filtration system. In various embodiments, the filtration system utilizes filters having a pore size of about 10 nanometers to about 2 micrometers.

[0097] After filtration, the filtrate is loaded into a protein capture system 605. The protein capture system 605 may comprise one or more chromatography columns. If more than one chromatography column is used, the columns may be arranged in series such that the loading of the next column can begin once the first column has been loaded. Alternatively, the media removal process may be stopped while switching columns.

[0098] In various embodiments, protein capture system 605 comprises one or more anion exchange (AEX) columns for direct product capture of rhGAA, particularly rhGAA with high M6P content. The rhGAA captured by protein capture system 605 is eluted from the column or columns by altering the pH and / or salt content of the column. Exemplary conditions for the AEX columns are provided in Table 2.

[0099] [Table 4]

[0100] The eluted rhGAA may be subjected to further purification and / or quality assurance steps. For example, the eluted rhGAA may be subjected to a virus 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 fed directly to the second chromatography system 609. In various embodiments, the second chromatography system 609 includes one or more immobilized metal affinity chromatography (IMAC) columns to further remove impurities. Exemplary conditions for the IMAC columns are provided in Table 3 below.

[0101] [Table 5]

[0102] After loading the rhGAA into a second chromatography system 609, the recombinant protein is eluted from the column or columns. The eluted rhGAA can be subjected to a virus killing step 611. Similar to virus killing 607, virus killing 611 can also 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 virus killing is performed at the same stage in the purification process.

[0103] The rhGAA from the virus killing step 611 may be introduced into a third chromatography system 613 to further purify the recombinant protein product. Alternatively, the eluted recombinant protein from the second chromatography system 609 may be fed directly into the third chromatography system 613. In various embodiments, the third chromatography system 613 includes one or more cation exchange chromatography (CEX) and / or size exclusion chromatography (SEC) columns to further remove impurities. The rhGAA product is then eluted from the third chromatography system 613. Exemplary conditions for the CEX column are provided below in Table 4.

[0104] [Table 6]

[0105] The rhGAA formulation may also be subjected to further processing steps. For example, a separate filtration system 615 may be used to remove viruses. In some embodiments, such filtration may utilize a filter with a pore size of 5-50 μm. Other product processing steps may include a product conditioning step 617, in which the recombinant protein product may be sterilized, filtered, concentrated, stored, and / or additional components added for final product formulation.

[0106] As used herein, the term "ATB200" refers to rhGAA having a high content of N-glycans carrying mono-M6P and bis-M6P, which is produced and purified from the GA-ATB200 cell line using the methods described herein.

[0107] V. Pharmaceutical Compositions In various embodiments, pharmaceutical compositions are provided that include rhGAA as described herein, either alone or in combination with other therapeutic agents, and / or a pharma- ceutically acceptable carrier.

[0108] In one or more embodiments, the pharmaceutical compositions described herein include pharma- ceutically acceptable salts.

[0109] In some embodiments, the pharma- ceutically acceptable salts used herein are pharma- ceutically acceptable acid addition salts, including but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, nitric acid, phosphoric acid, and organic acids, including but not limited to, acetic acid, trifluoroacetic acid, adipic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, butyric acid, camphoric acid, camphorsulfonic acid, cinnamic acid, citric acid, digluconic acid, ethanesulfonic acid, glutamic acid, glycolic acid, glycerophosphoric acid, hemisulfic acid, acid), hexanoic acid, formic acid, fumaric acid, 2-hydroxyethanesulfonic acid (isethionic acid), lactic acid, hydroxymaleic acid, malic acid, malonic acid, mandelic acid, mesitylenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, nicotinic acid, 2-naphthalenesulfonic acid, oxalic acid, pamoic acid, pectinic acid, phenylacetic acid, 3-phenylpropionic acid, pivalic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, p-toluenesulfonic acid, and undecanoic acid.

[0110] In some embodiments, the pharma- ceutically acceptable salt used herein is a pharma- ceutically acceptable base addition salt. Pharmaceutically acceptable base addition salts can include, but are not limited to, hydroxides, carbonates, or bicarbonates of ammonia or ammonium or metal cations, such as sodium, potassium, lithium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, etc. Salts derived from pharma- ceutically acceptable organic non-toxic bases can include, but are not limited to, primary, secondary, and tertiary amines, quaternary amine compounds, substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, isopropylamine, tripropylamine, tributylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, Examples of salts include salts of arginine, histidine, caffeine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, tetramethylammonium compounds, tetraethylammonium compounds, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine, N,N'-dibenzylethylenediamine, and polyamine resins.

[0111] In some embodiments, rhGAA or a pharma- ceutically acceptable salt thereof may 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 solution. If necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of injection. The components of the pharmaceutical composition may be supplied separately or mixed together in a unit dosage form, for example, as a dry lyophilized powder or a water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the amount of active agent. If the composition is to be administered by injection, it may be dispensed in an infusion bottle containing sterile pharmaceutical grade water, saline, or dextrose / water. In some embodiments, the injection may be administered in a hospital or clinic. In some embodiments, the injection may be administered outside of a hospital or clinic setting, for example, at the subject's home. If the composition is administered by injection, an ampoule of sterile water for injection or saline may be provided to allow the components to be mixed prior to administration.

[0112] In some embodiments, rhGAA or its pharma- ceutically acceptable salts may be formulated for oral administration. The composition to be administered orally may be in the form of tablets, capsules, suppositories, elixirs, solutions or suspensions, gels, syrups, mouthwashes, or dry powders to be reconstituted with water or other suitable medium before use, optionally with flavorings and colorings, and may be formulated for immediate release, delayed release, modulated release, sustained release, pulsed release, or controlled release applications. Solid compositions such as tablets, capsules, lozenges, troches, pills, boluses, powders, pastes, granules, bullets, dragees, or premixed preparations may also be used. Solid and liquid compositions for oral use may be prepared according to methods well known in the art. Such compositions may also contain one or more pharma- ceutically acceptable carriers and excipients, which may be in solid or liquid form. Tablets or capsules can be prepared by conventional means with pharma- ceutically acceptable excipients, including but not limited to binders, fillers, lubricants, disintegrants, or wetting agents.Suitable pharma-ceutically acceptable excipients are known in the art, and include but are not limited to pregelatinized starch, polyvinylpyrrolidone, povidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylethylcellulose (HPEC), hydroxypropylcellulose (HPC), sucrose, gelatin, acacia, lactose, microcrystalline cellulose, calcium hydrogen phosphate, magnesium stearate, stearic acid, glyceryl behenate, talc, silica, corn, potato or tapioca starch, sodium starch glycolate, sodium lauryl sulfate, sodium citrate, calcium carbonate, calcium hydrogen phosphate, glycine croscarmellose sodium, and complex silicates.Tablets can be coated by methods known in the art.

[0113] In some embodiments, the pharmaceutical compositions described herein may be formulated as described in U.S. Pat. No. 10,512,676 and U.S. Provisional Patent Application No. 62 / 506,574 (both of which are incorporated herein by reference in their entirety). For example, in some embodiments, the pH of the pharmaceutical compositions described herein is about 5.0 to about 7.0 or about 5.0 to about 6.0. In some embodiments, the pH ranges from about 5.5 to about 6.0. In some embodiments, the pH of the pharmaceutical composition is 6.0. In some embodiments, the pH may be adjusted to a target pH by using a pH adjuster (e.g., an alkalizing agent and an acidifying agent), such as sodium hydroxide and / or hydrochloric acid.

[0114] The pharmaceutical compositions described herein may include a buffer system, such as a citrate system, a phosphate system, and combinations thereof. The citrate and / or phosphate may be sodium citrate or sodium phosphate. Other salts include potassium and ammonium salts. In one or more embodiments, the buffer comprises a citrate. In further embodiments, the buffer comprises sodium citrate (e.g., a mixture of anhydrous sodium citrate and citric acid monohydrate). In one or more embodiments, the citrate-containing buffer solution may include sodium citrate and citric acid. In some embodiments, both citrate and phosphate buffers are present.

[0115] In some embodiments, the pharmaceutical compositions described herein include at least one excipient. The excipient may function as a tonicity agent, bulking agent, and / or stabilizer. A tonicity agent is an ingredient that helps ensure that the formulation has a similar or the same osmotic pressure as human blood. A bulking agent is an ingredient that adds bulk to the formulation (e.g., lyophilized form) and gives the cake the proper structure. A stabilizer is a compound that can prevent or minimize aggregate formation at the hydrophobic air-liquid interface. An excipient may function as a tonicity agent and bulking agent simultaneously. For example, mannitol may function as a tonicity agent and also provide benefits as a bulking agent.

[0116] 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 the one or more tonicity agents ranges from an amount of about 10 mg / mL to about 50 mg / mL. In further embodiments, the total amount of the one or more tonicity agents ranges from an amount of about 10, 11, 12, 13, 14, or 15 mg / mL to about 16, 20, 25, 30, 35, 40, 45, or 50 mg / mL.

[0117] In some embodiments, the excipient comprises a stabilizer. In some embodiments, the stabilizer is a surfactant. In some embodiments, the stabilizer is polysorbate 80. In one or more embodiments, the total amount of stabilizer ranges from about 0.1 mg / mL to about 1.0 mg / mL. In further embodiments, the total amount of stabilizer ranges from about 0.1, 0.2, 0.3, 0.4, or 0.5 mg / mL to about 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mg / mL. In yet other embodiments, the total amount of stabilizer is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mg / mL.

[0118] In some embodiments, the pharmaceutical composition comprises (a) rhGAA (such as ATB200), (b) at least one buffer selected from the group consisting of citrate, phosphate, and combinations thereof, and (c) at least one excipient selected from the group consisting of mannitol, polysorbate 80, and combinations thereof, and has a pH of (i) about 5.0 to about 6.0, or (ii) about 5.0 to about 7.0. In some embodiments, the composition further comprises water. In some embodiments, the composition may further comprise an acidifier and / or an alkalizer.

[0119] 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.

[0120] In some embodiments, a pharmaceutical composition comprising rhGAA is diluted prior to administration to a subject in need thereof.

[0121] In some embodiments, the pharmaceutical compositions described herein comprise a chaperone. In some embodiments, the chaperone is miglustat or a pharma- ceutically acceptable salt thereof. In other embodiments, the chaperone is duvoglustat or a pharma- ceutically acceptable salt thereof.

[0122] In some embodiments, the rhGAA described herein is formulated in one pharmaceutical composition, while the chaperone, such as miglustat, is formulated in another pharmaceutical composition. In some embodiments, the pharmaceutical composition comprising miglustat is based on the formulation commercially available as ZAVESCA® (Actelion Pharmaceuticals).

[0123] In some embodiments, the pharmaceutical compositions described herein may undergo a lyophilization (freeze-drying) process to provide a cake or powder. Accordingly, in some embodiments, the pharmaceutical compositions described herein relate to rhGAA compositions after lyophilization. The lyophilized mixture may include rhGAA (e.g., ATB200) described herein, a buffer selected from the group consisting of citrate, phosphate, and combinations thereof, and at least one excipient selected from the group consisting of trehalose, mannitol, polysorbate 80, and combinations thereof. In some embodiments, other components (e.g., other excipients) may be added to the lyophilized mixture. The pharmaceutical composition comprising the lyophilized formulation may be provided in a vial, which can then be stored, shipped, reconstituted, and / or administered to a patient.

[0124] VI. Treatment method A. Treatment of Disease Another aspect of the present disclosure relates to a method for treating a disease or disorder associated with glycogen storage dysregulation 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. Also provided herein is the use of rhGAA or ATB200 to treat Pompe disease.

[0125] In some embodiments, the subject treated by the methods disclosed herein is an ERT-experienced patient. In some embodiments, the subject treated by the methods disclosed herein is an ERT-naive patient.

[0126] The rhGAA or pharmaceutical composition described herein is administered by a suitable 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 a target tissue, such as the heart or skeletal muscle (e.g., intramuscularly), or the nervous system (e.g., by direct injection into the brain; intracerebroventricularly; intrathecally). In some embodiments, the rhGAA or pharmaceutical composition is administered orally. If necessary, two or more routes can be used simultaneously.

[0127] In some embodiments, the therapeutic effect of rhGAA or a pharmaceutical composition described herein may be assessed based on one or more of the following criteria: (1) cardiac status (e.g., increased end-diastolic and / or end-systolic volumes, or reduction, improvement, or prevention of progressive cardiomyopathy typically seen in GSD-II); (2) pulmonary function (e.g., increased crying vital capacity compared to baseline volumes and / or normalization of oxygen desaturation during crying); (3) neurodevelopment and / or motor skills (e.g., increased AIMS scores); and (4) reduction in glycogen levels in tissues of individuals affected by the disease.

[0128] In some embodiments, after administration of one or more dosages of rhGAA or pharmaceutical compositions described herein, the subject's cardiac condition improves by 10%, 20%, 30%, 40%, or 50% (or any percentage in between) compared to that of a vehicle-treated subject or that of a subject before treatment. The subject's cardiac condition may be evaluated by measuring end-diastolic and / or end-systolic volumes and / or by clinically assessing cardiomyopathy. In some embodiments, the subject's pulmonary function improves by 10%, 20%, 30%, 40%, or 50% (or any percentage in between) compared to that of a vehicle-treated subject or that of a subject before treatment after administration of one or more dosages of ATB200 or pharmaceutical compositions comprising ATB200. In certain embodiments, the improvement is achieved after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or more (or any period in between) after administration. In certain embodiments, ATB200 or a pharmaceutical composition comprising ATB200 improves pulmonary function in a subject after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or more (or any period in between) of administration.

[0129] In some embodiments, after administration of one or more dosages of rhGAA or pharmaceutical compositions described herein, the subject's pulmonary function is improved by 10%, 20%, 30%, 40%, or 50% (or any percentage in between) compared to that of a vehicle-treated subject or that of a subject before treatment. The subject's pulmonary function may be assessed by normalization of crying lung capacity and / or oxygen desaturation during crying compared to baseline volume. In some embodiments, after administration of one or more dosages of ATB200 or pharmaceutical compositions comprising ATB200, the subject's pulmonary function is improved by 10%, 20%, 30%, 40%, or 50% (or any percentage in between) compared to that of a vehicle-treated subject or that of a subject before treatment. In certain embodiments, the improvement is achieved after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or more (or any period in between) from administration. In certain embodiments, ATB200 or a pharmaceutical composition comprising ATB200 improves pulmonary function in a subject after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or more (or any period in between) of administration.

[0130] In some embodiments, after administration of one or more dosages of rhGAA or pharmaceutical compositions described herein, the subject's neurodevelopment and / or motor skills improve by 10%, 20%, 30%, 40%, or 50% (or any percentage in between) compared to vehicle-treated subjects or to pre-treatment subjects. The subject's neurodevelopment and / or motor skills can be assessed by determining the AIMS score. The AIMS is a 12-item anchored scale administered and scored by a clinician (see Rush JA Jr., Handbook of Psychiatric Measures, American Psychiatric Association, 2000, 166-168). Items 1-10 are scored on a 5-point anchored scale. Items 1-4 assess orofacial movements. Items 5-7 address limb and truncal dyskinesias. Items 8-10 address the overall severity as judged by the examiner, and the patient's awareness of movement and associated pain. Items 11-12 are yes / no questions regarding dental and / or denture problems (such problems may lead to misdiagnosis of dyskinesia). In some embodiments, after administration of one or more dosages of ATB200 or a pharmaceutical composition comprising ATB200, the subject's neurodevelopment and / or motor skills improve by 10%, 20%, 30%, 40%, or 50% (or any percentage in between) compared to those of vehicle-treated subjects or those of subjects prior to treatment. In certain embodiments, the improvement is realized after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or more (or any period in between) after administration. In certain embodiments, ATB200 or a pharmaceutical composition comprising ATB200 improves neurodevelopment and / or motor skills in a subject after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or more (or any period in between) of administration.

[0131] In some embodiments, after administration of one or more dosages of rhGAA or pharmaceutical compositions described herein, the glycogen level of a particular tissue of a subject is reduced by 10%, 20%, 30%, 40%, or 50% (or any percentage between) compared to that of a vehicle-treated subject or that of a subject before treatment.In some embodiments, the tissue is muscle, such as quadriceps, triceps, and gastrocnemius.The glycogen level of tissue can be analyzed using methods known in the art. Determination of glycogen levels based on amyloglucosidase digestion is well known and described in publications such as Amalfitano et al. (1999), "Systemic correction of the muscle disorder glycogen storage disease type II after hepatic targeting of a modified adenovirus vector encoding human acid-alphaglucosidase", Proc Natl Acad Sci USA, 96:8861-8866. In some embodiments, after administration of one or more dosages of ATB200 or a pharmaceutical composition comprising ATB200, the glycogen level in the muscle of the subject is reduced by 10%, 20%, 30%, 40%, or 50% (or any percentage therebetween) compared to that of a vehicle-treated subject or that of the subject before treatment. In certain embodiments, the reduction is achieved after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or more (or any period in between) of administration. In certain embodiments, ATB200 or a pharmaceutical composition comprising ATB200 reduces glycogen levels in muscle of a subject after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or more (or any period in between) of administration.

[0132] B. Biomarkers A biomarker of glycogen accumulation in a subject, such as urinary hexose tetrasaccharide (Hex4), may be used to assess and compare the therapeutic effect of enzyme replacement therapy in a subject 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 urinary Hex4 level in the subject.

[0133] Biomarkers of muscle injury or damage, such as creatine kinase (CK), alanine aminotransferase (ALT), and aspartate aminotransferase (AST), may 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 subject's CK, ALT, and / or AST levels. In at least one embodiment, the therapeutic effect of rhGAA or a pharmaceutical composition comprising rhGAA on muscle damage is assessed by measuring the subject's CK levels.

[0134] Biomarkers such as LAMP-1, LC3, and dysferlin may also be used to evaluate and compare the therapeutic effects of rhGAA or pharmaceutical compositions described herein. In Pompe disease, GAA is unable to hydrolyze lysosomal glycogen, leading to the abnormal accumulation of large lysosomes filled with glycogen in some tissues (Raben et al., JBC 273:19086-19092, 1998). Mouse model studies of Pompe disease have shown that enlarged lysosomes in skeletal muscle cannot adequately explain the reduced mechanical performance, and the presence of large inclusions containing degraded myofibrils (i.e., autophagy buildup) contributes to muscle dysfunction (Raben et al., Human Mol Genet 17:3897-3908, 2008). Reports also suggest that impaired autophagic flux is associated with poor treatment outcomes in patients with Pompe disease (Nascimbeni et al., Neuropathology and Applied Neurobiology doi:10.1111 / nan.12214, 2015; Fukuda et al., Mol Ther 14:831-839, 2006). In addition, late-onset Pompe disease is commonly found in unclassified limb-girdle muscular dystrophies (LGMDs) (Preisler et al., Mol Genet Metab 110:287-289, 2013), a group of genetically heterogeneous neuromuscular disorders with more than 30 genetically defined subtypes of varying severity. IHC studies revealed substantially increased sarcoplasmic abundance of dysferlin in skeletal muscle fibers from Gaa KO mice.

[0135] A variety of known methods can be used to measure gene expression levels and / or protein levels of such biomarkers. For example, a sample, such as a tissue, particularly a muscle biopsy, can be obtained from a subject treated with rhGAA or a pharmaceutical composition described herein. In some embodiments, the sample is a muscle biopsy from the subject. In some embodiments, the muscle is selected from the quadriceps, triceps, and gastrocnemius. The sample obtained from the subject may be stained with one or more antibodies or other detection agents that detect such biomarkers, or may be identified and quantified by mass spectrometry. The sample may also, or alternatively, be processed to detect the presence of nucleic acids, such as mRNAs, that encode the biomarkers, for example, by RT-qPCR.

[0136] In some embodiments, gene expression levels and / or protein levels of one or more biomarkers are measured in muscle biopsies obtained from individuals before and after treatment with rhGAA or pharmaceutical compositions described herein. In some embodiments, gene expression levels and / or protein levels of one or more biomarkers are measured in muscle biopsies obtained from vehicle-treated individuals. In some embodiments, after administration of one or more dosages of rhGAA or pharmaceutical compositions described herein, gene expression levels and / or protein levels of one or more biomarkers are reduced by 10%, 20%, 30%, 40%, or 50% (or any percentage in between) compared to those of vehicle-treated subjects or those of subjects before treatment. In some embodiments, after administration of one or more dosages of ATB200 or a pharmaceutical composition comprising ATB200, 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 that of a vehicle-treated subject or that 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 period in between) after administration. In certain embodiments, ATB200 or a pharmaceutical composition comprising ATB200 reduces the gene expression level and / or protein level of one or more biomarkers after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or more (or any period in between) after administration.

[0137] C. rhGAA Dosage The pharmaceutical formulation or reconstituted composition is administered in a therapeutically effective amount (e.g., a dosage that, when administered at regular intervals, is sufficient to treat the disease, such as by ameliorating symptoms associated with the disease, delaying the onset of the disease, and / or reducing the severity or frequency of symptoms of the disease). The therapeutically effective amount in treating a disease may depend on the nature and extent of the disease's effects and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be used to help identify optimal dosage ranges. In at least one embodiment, rhGAA or pharmaceutical compositions comprising rhGAA described herein are administered at 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, 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 pharmaceutical composition is administered at a dose of about 20 mg / kg. In some embodiments, rhGAA or pharmaceutical composition is administered simultaneously or sequentially with a pharmacological chaperone. In some embodiments, the pharmacological chaperone is miglustat. In at least one embodiment, miglustat is administered as an oral dose of about 260 mg. In at least one embodiment, miglustat is administered as an oral dose of about 195 mg. The effective dose for a particular individual may vary (e.g., may increase or decrease) over time depending on the needs of the individual.For example, the amounts of rhGAA and / or miglustat can be adjusted in the presence of physical illness or stress, or if anti-acid alpha-glucosidase antibodies become present or increase, or if disease symptoms worsen.

[0138] In some embodiments, the therapeutically effective dose of the rhGAA or pharmaceutical composition described herein is lower than that of a conventional rhGAA formulation. For example, if the therapeutically effective dose of a conventional rhGAA formulation is 20 mg / kg, the dose required for the rhGAA or pharmaceutical composition described herein to produce the same or better therapeutic effect as that of the conventional rhGAA formulation may be lower than 20 mg / kg. The therapeutic effect may be evaluated based on one or more of the criteria discussed above (e.g., cardiac status, glycogen level, or biomarker expression). In some embodiments, the therapeutically effective dose of the rhGAA or pharmaceutical composition described herein is at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more lower than that of a conventional rhGAA formulation.

[0139] In some embodiments, the therapeutic effect of rhGAA or a pharmaceutical composition described herein includes improved motor function, improved muscle strength (upper body, lower body, or total body), improved pulmonary function, reduced fatigue, reduced levels of at least one biomarker for muscle injury, reduced levels of at least one biomarker for glycogen accumulation, or a combination thereof. In some embodiments, the therapeutic effect of the rhGAA or pharmaceutical compositions described herein includes reversal of lysosomal lesions in muscle fibers, a faster and / or more effective reduction in glycogen content in muscle fibers, an increase in 6-minute walk test distance, a decrease in timed up and go test time, a decrease in 4-step stair climbing test time, a decrease in 10-meter walk test time, a decrease in walk-stairs-Gowers-chair score, an increase in upper limb strength, an improvement in shoulder adduction, an improvement in shoulder abduction, an improvement in elbow flexion, an improvement in elbow extension, an improvement in upper body strength, an improvement in lower body strength, an improvement in total body strength, an improvement in standing (sitting) forced vital capacity, an improvement in maximum expiratory pressure, an improvement in maximum inspiratory pressure, a decrease in fatigue severity scale score, a decrease in urinary hexose tetrasaccharide levels, a decrease in creatine kinase levels, a decrease in alanine aminotransferase levels, a decrease in aspartate aminotransferase levels, or any combination thereof.

[0140] In some embodiments, the rhGAA or pharmaceutical composition described herein achieves the desired therapeutic effect faster than conventional rhGAA formulations when administered at the same dose. The therapeutic effect may be evaluated based on one or more of the criteria discussed above (e.g., cardiac status, glycogen level, or biomarker expression). For example, if a single dose of a conventional rhGAA formulation reduces glycogen levels in tissues of a treated individual by 10% in one week, administration of the same dose of the rhGAA or pharmaceutical composition described herein may achieve the same degree of reduction in less than one week. In some embodiments, the rhGAA or pharmaceutical composition described herein may achieve the desired therapeutic effect at least about 1.25, 1.5, 1.75, 2.0, 3.0, or more faster than conventional rhGAA formulations when administered at the same dose.

[0141] In some embodiments, a therapeutically effective amount of rhGAA (or a composition or medicament comprising rhGAA) is administered two or more times. In some embodiments, rhGAA or a pharmaceutical composition described herein is administered continuously at regular intervals depending on the nature and extent of the disease effect. Administration at "regular intervals", as used herein, indicates that the therapeutically effective amount is administered periodically (as distinguished from a one-time dose). The intervals can be determined by standard clinical techniques. In certain embodiments, rhGAA is administered bimonthly, monthly, biweekly, weekly, twice weekly, or daily. In some embodiments, rhGAA is administered intravenously twice weekly, weekly, or biweekly. The administration interval for a single individual need not be a fixed interval, but can vary over time depending on the needs of the individual. For example, the interval between doses can be reduced when anti-rhGAA antibodies become present or increase during physical illness or stress, or when disease symptoms worsen.

[0142] In some embodiments, when used at the same dose, the rhGAA or pharmaceutical composition as described herein may be administered less frequently than a conventional rhGAA formulation and still be capable of producing the same or better therapeutic effect than the conventional rhGAA formulation. For example, if a conventional rhGAA formulation is administered at 20 mg / kg every week, the rhGAA or pharmaceutical composition as described herein, when administered at 20 mg / kg, may produce the same or better therapeutic effect than the conventional rhGAA formulation, even if the rhGAA or pharmaceutical composition is administered less frequently, for example, every other week or every month. The therapeutic effect may be evaluated based on one or more of the criteria discussed above (e.g., cardiac status, glycogen level, or biomarker expression). In some embodiments, the interval between two doses of the rhGAA or pharmaceutical composition described herein is longer than the conventional rhGAA formulation. In some embodiments, the interval between two doses of the rhGAA or pharmaceutical composition is at least about 1.25, 1.5, 1.75, 2.0, 3.0, or more longer than the conventional rhGAA formulation.

[0143] In some embodiments, under the same therapeutic conditions (e.g., the same dose administered at the same intervals), the rhGAA or pharmaceutical composition described herein provides a therapeutic effect of a degree greater than that provided by conventional rhGAA formulations. The therapeutic effect may be evaluated based on one or more criteria discussed above (e.g., cardiac status, glycogen level, or biomarker expression). For example, compared to a conventional rhGAA formulation administered at 20 mg / kg weekly, the glycogen level in tissues of a treated individual may be reduced to a greater extent by rhGAA or pharmaceutical composition administered at 20 mg / kg weekly. In some embodiments, when administered under the same therapeutic conditions, the rhGAA or pharmaceutical composition described herein provides a therapeutic effect of at least about 1.25, 1.5, 1.75, 2.0, 3.0, or more than that of conventional rhGAA formulations.

[0144] D. Two-component therapy In one or more embodiments, the rhGAA or pharmaceutical composition comprising rhGAA described herein is administered simultaneously or sequentially with a pharmacological chaperone. In some embodiments, the rhGAA or pharmaceutical composition is administered by a different route compared to the pharmacological chaperone. For example, the pharmacological chaperone may be administered orally, while the rhGAA or pharmaceutical composition is administered intravenously.

[0145] In various embodiments, the pharmacological chaperone is miglustat. Without wishing to be bound by any theory, it is believed that when co-administered, miglustat stabilizes ATB200 from denaturation in systemic circulation, thereby enhancing the delivery of the active ingredient ATB200 to lysosomes.

[0146] In some embodiments, miglustat is administered in an oral dose of about 50 mg to about 600 mg. In at least one embodiment, miglustat is administered in an oral dose of about 200 mg to about 600 mg, or in 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 in an oral dose of about 233 mg to about 500 mg. In at least one embodiment, miglustat is administered in an oral dose of about 250 to about 270 mg, or in 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 as an oral dose of about 260 mg.

[0147] Those skilled in the art will appreciate that oral doses of miglustat in the range of about 200 mg to 600 mg or any smaller range therein may be suitable for adult patients depending on their body weight. For example, lower doses may be deemed suitable by physicians for patients weighing significantly less than about 70 kg, including but not limited to infants, children, or low body weight adults. Thus, in at least one embodiment, miglustat is administered as an oral dose of about 50 mg to about 200 mg, or as an oral dose of about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 130 mg, about 150 mg, about 175 mg, about 195 mg, about 200 mg, or about 260 mg. In at least one embodiment, miglustat is administered as an oral dose of about 65 mg to about 195 mg, or as an oral dose of about 65 mg, about 130 mg, or about 195 mg.

[0148] In some embodiments, rhGAA is administered intravenously at a dose of about 5 mg / kg to about 20 mg / kg, and miglustat is administered orally at a dose of about 50 mg to about 600 mg. In some embodiments, rhGAA is administered intravenously at a dose of about 5 mg / kg to about 20 mg / kg, and miglustat is administered orally at a dose of about 50 mg to about 200 mg. In some embodiments, rhGAA is administered intravenously at a dose of about 5 mg / kg to about 20 mg / kg, and miglustat is administered orally at a dose of about 200 mg to about 600 mg. In some embodiments, rhGAA is administered intravenously at a dose of about 5 mg / kg to about 20 mg / kg, and miglustat is administered orally at a dose of about 200 mg to about 500 mg. In one embodiment, rhGAA is administered intravenously at a dose of about 20 mg / kg, and miglustat is administered orally at a dose of about 260 mg. In some embodiments, rhGAA is administered intravenously at a dose of about 5 mg / kg to about 20 mg / kg, and miglustat is administered orally at a dose of about 130 mg to about 200 mg. In one embodiment, rhGAA is administered intravenously at a dose of about 20 mg / kg, and miglustat is administered orally at a dose of about 195 mg.

[0149] In some embodiments, miglustat and rhGAA are administered simultaneously. For example, miglustat may be administered within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minutes before or after administration of rhGAA. In some embodiments, miglustat is administered within 5, 4, 3, 2, or 1 minutes before or after administration of rhGAA.

[0150] In some embodiments, miglustat and rhGAA are administered sequentially. In at least one embodiment, miglustat is administered before administration of rhGAA. In at least one embodiment, miglustat is administered less than 3 hours before administration of rhGAA. In at least one embodiment, miglustat is administered about 2 hours before administration of rhGAA. For example, miglustat can be administered about 1.5 hours, about 1 hour, about 50 minutes, about 30 minutes, or about 20 minutes before administration of rhGAA. In at least one embodiment, miglustat is administered about 1 hour before administration of rhGAA.

[0151] In some embodiments, miglustat is administered after administration of rhGAA. In at least one embodiment, miglustat is administered within 3 hours after administration of rhGAA. In at least one embodiment, miglustat is administered within 2 hours after administration of rhGAA. For example, miglustat can be administered within about 1.5 hours, about 1 hour, about 50 minutes, about 30 minutes, or about 20 minutes after administration of rhGAA.

[0152] In some embodiments, the subject fasts for at least 2 hours prior to and at least 2 hours after administration of miglustat.

[0153] In some embodiments, the binary therapy disclosed herein improves one or more disease symptoms in a subject with Pompe disease compared to (1) baseline, or (2) a control treatment comprising administering alglucosidase alfa and a pharmacological chaperone placebo, where a placebo is administered instead of the pharmacological chaperone. In some embodiments, the subject treated with the binary therapy is an ERT-experienced patient. In some embodiments, the subject treated with the binary therapy is an ERT-naive patient.

[0154] In some embodiments, the dual-component therapy according to the present disclosure improves the motor function of the subject as measured by 6-minute walk test (6MWT).In some embodiments, compared to baseline, the 6-minute walk distance (6MWD) of the subject increases by at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or 50 meters or at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% after 12, 26, 38, or 52 weeks of treatment.In some embodiments, the 6MWD of the subject increases by at least 20 meters or at least 5% after 52 weeks of treatment. In some embodiments, the subject's 6MWD improves by at least 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, or 50 meters after 12, 26, 38, or 52 weeks of treatment, compared to a control treatment. In some embodiments, the subject's 6MWD improves by at least 13 meters after 52 weeks of treatment, compared to a control treatment. In some embodiments, the subject has a baseline 6MWD of less than 300 meters. In some embodiments, the subject has a baseline 6MWD of 300 meters or greater.

[0155] In some embodiments, the dual-component therapy according to the present disclosure stabilizes the subject's lung function as measured by forced vital capacity (FVC) test. In some embodiments, after 12, 26, 38, or 52 weeks of treatment, the subject's percent-to-predicted FVC either increases compared to baseline or decreases by less than 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% compared to baseline. In some embodiments, after 52 weeks of treatment, the subject's percent-to-predicted FVC decreases by less than 1% compared to baseline. In some embodiments, the subject's percent-to-predicted FVC improves significantly after treatment when compared to control treatment. In some embodiments, the subject's percent predicted FVC is significantly improved by at least 0.5%, 1%, 2%, 3%, 4%, 5%, or 6% after 12, 26, 38, or 52 weeks of treatment when compared to a control treatment. In some embodiments, the subject's percent predicted FVC is significantly improved by at least 3% after 52 weeks of treatment when compared to a control treatment. In some embodiments, the subject has a baseline FVC of less than 55%. In some embodiments, the subject has a baseline FVC of 55% or greater.

[0156] In some embodiments, the dual-component therapy of the present disclosure improves the motor function of the subject as measured by the Walk-Stair-Gowers-Chair (GSGC) test. In some embodiments, the subject's GSGC score improves as indicated by a decrease of at least 0.1, 0.3, 0.5, 0.7, 1.0, 1.5, or 2.5 points after 12, 26, 38, or 52 weeks of treatment, as compared to baseline. In some embodiments, the subject's GSGC score improves as indicated by a decrease of at least 0.5 points after 52 weeks of treatment, as compared to baseline. In some embodiments, the subject's GSGC score significantly improves after treatment, as compared to a control treatment. In some embodiments, the subject's GSGC score significantly improves as indicated by a decrease of at least 0.3, 0.5, 0.7, 1.0, 1.5, 2.5, or 5 points after 12, 26, 38, or 52 weeks of treatment, as compared to a control treatment. In some embodiments, the subject's GSGC score significantly improves as indicated by a reduction of at least 1.0 points after 52 weeks of treatment, when compared to the control treatment.

[0157] In some embodiments, the dual component therapy according to the present disclosure reduces the level of at least one muscle damage marker after treatment. In some embodiments, the at least one muscle damage marker includes creatine kinase (CK). In some embodiments, compared to baseline, the subject's CK level is reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, or 50% after 12, 26, 38, or 52 weeks of treatment. In some embodiments, compared to baseline, the subject's CK level is reduced by at least 20% after 52 weeks of treatment. In some embodiments, compared to a control treatment, the subject's CK level is significantly reduced after treatment. In some embodiments, compared to a control treatment, the subject's CK level is significantly reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, or 50% after 12, 26, 38, or 52 weeks of treatment. In some embodiments, compared to a control treatment, the subject's CK level is significantly reduced by at least 30% after 52 weeks of treatment.

[0158] In some embodiments, the dual component therapy of the present disclosure reduces the level of at least one glycogen accumulation marker after treatment. In some embodiments, the at least one glycogen accumulation marker comprises urinary hexose tetrasaccharide (Hex4). In some embodiments, the subject's urinary Hex4 level is reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60% after 12, 26, 38, or 52 weeks of treatment, as compared to baseline. In some embodiments, the subject's urinary Hex4 level is reduced by at least 30% after 52 weeks of treatment, as compared to baseline. In some embodiments, the subject's urinary Hex4 level is significantly reduced after treatment, as compared to a control treatment. In some embodiments, the subject's urinary Hex4 level is significantly reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60% after 12, 26, 38, or 52 weeks of treatment, as compared to a control treatment. In some embodiments, the subject's urinary Hex4 levels are significantly reduced by at least 40% after 52 weeks of treatment compared to the control treatment.

[0159] In some embodiments, a binary therapy according to the present disclosure improves one or more disease symptoms in a Pompe disease patient subject with a history of ERT compared to (1) baseline, or (2) a control treatment comprising administering alglucosidase alfa and a pharmacological chaperone placebo.

[0160] In some embodiments, the dual-component therapy for the subject with Pompe disease who has a history of ERT improves the motor function of the subject as measured by 6MWT.In some embodiments, compared to baseline, the 6MWD of the subject increases by at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or 50 meters or at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% after 12, 26, 38, or 52 weeks of treatment.In some embodiments, the 6MWD of the subject increases by at least 15 meters or at least 5% after 52 weeks of treatment.In some embodiments, compared to control treatment, the 6MWD of the subject improves significantly after treatment. In some embodiments, the subject's 6MWD is significantly improved by at least 10, 12, 14, 15, 16, 18, 20, 30, 40, or 50 meters after 12, 26, 38, or 52 weeks of treatment, compared to a control treatment. In some embodiments, the subject's 6MWD is significantly improved by at least 15 meters after 52 weeks of treatment, compared to a control treatment. In some embodiments, the subject has a baseline 6MWD of less than 300 meters. In some embodiments, the subject has a baseline 6MWD of 300 meters or greater.

[0161] In some embodiments, the bicomponent therapy for a subject with Pompe disease who has a history of ERT improves the subject's lung function as measured by FVC test. In some embodiments, after 12, 26, 38, or 52 weeks of treatment, the subject's percent predicted FVC increases by at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, or 5% compared to baseline. In some embodiments, after 52 weeks of treatment, the subject's percent predicted FVC increases by at least 0.1% compared to baseline. In some embodiments, the subject's percent predicted FVC improves significantly after treatment when compared to a control treatment. In some embodiments, the subject's percent predicted FVC improves significantly by at least 1%, 2%, 3%, 4%, 5%, 6%, 8%, or 10% after 12, 26, 38, or 52 weeks of treatment when compared to a control treatment. In some embodiments, the subject's percent predicted FVC is significantly improved by at least 4% after 52 weeks of treatment when compared to a control treatment. In some embodiments, the subject has a baseline FVC of less than 55%. In some embodiments, the subject has a baseline FVC of 55% or greater.

[0162] In some embodiments, the dual-component therapy for a subject with Pompe disease who has undergone ERT improves the motor function of the subject as measured by GSGC test. In some embodiments, compared to baseline, the subject's GSGC score improves as indicated by a decrease of at least 0.1, 0.3, 0.5, 0.7, 1.0, 1.5, or 2.5 points after 12, 26, 38, or 52 weeks of treatment. In some embodiments, compared to baseline, the subject's GSGC score improves as indicated by a decrease of at least 0.5 points after 52 weeks of treatment. In some embodiments, compared to control treatment, the subject's GSGC score improves significantly after treatment. In some embodiments, compared to control treatment, the subject's GSGC score improves significantly as indicated by a decrease of at least 0.3, 0.5, 0.7, 1.0, 1.5, 2.5, or 5 points after 12, 26, 38, or 52 weeks of treatment. In some embodiments, the subject's GSGC score significantly improves as indicated by a reduction of at least 1.0 points after 52 weeks of treatment, when compared to the control treatment.

[0163] In some embodiments, the dual component therapy for a subject with Pompe disease who has a history of ERT reduces the level of at least one muscle damage marker after treatment. In some embodiments, the at least one muscle damage marker includes CK. In some embodiments, the subject's CK level is reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, or 50% after 12, 26, 38, or 52 weeks of treatment, compared to baseline. In some embodiments, the subject's CK level is reduced by at least 15% after 52 weeks of treatment, compared to baseline. In some embodiments, the subject's CK level is significantly reduced after treatment, compared to a control treatment. In some embodiments, the subject's CK level is significantly reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, or 50% after 12, 26, 38, or 52 weeks of treatment, compared to a control treatment. In some embodiments, the subject's CK level is significantly reduced by at least 30% after 52 weeks of treatment, compared to a control treatment.

[0164] In some embodiments, the dual component therapy for a subject with Pompe disease who has a history of ERT reduces the level of at least one glycogen storage marker after treatment. In some embodiments, the at least one glycogen storage marker comprises urinary Hex4. In some embodiments, the subject's urinary Hex4 level is reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60% after 12, 26, 38, or 52 weeks of treatment, as compared to baseline. In some embodiments, the subject's urinary Hex4 level is reduced by at least 25% after 52 weeks of treatment, as compared to baseline. In some embodiments, the subject's urinary Hex4 level is significantly reduced after treatment, as compared to a control treatment. In some embodiments, the subject's urinary Hex4 level is significantly reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60% after 12, 26, 38, or 52 weeks of treatment, as compared to a control treatment. In some embodiments, the subject's urinary Hex4 levels are significantly reduced by at least 40% after 52 weeks of treatment compared to the control treatment.

[0165] E.Kit Another aspect of the disclosure relates to kits suitable for carrying out the rhGAA therapy described herein. In one or more embodiments, the kits include a container (e.g., a vial, a tube, a bag, etc.) containing rhGAA or a pharmaceutical composition (either pre- or post-lyophilization) and instructions for reconstitution, dilution, and administration. In one or more embodiments, the kits include a container (e.g., a vial, a tube, a bag, etc.) containing a pharmaceutical composition (either pre- or post-lyophilization) comprising a pharmacological chaperone (e.g., miglustat) and rhGAA, and instructions for reconstitution, dilution, and administration of rhGAA with the pharmacological chaperone. EXAMPLES

[0166] Example 1: Preparation of CHO cells producing rhGAA with high mono- or bis-M6P-bearing N-glycan content. DG44 CHO (DHFR-) cells were transfected with a DNA construct expressing rhGAA, which is shown in Figure 4. After transfection, CHO cells with stably integrated GAA genes were selected in hypoxanthine / thymidine-free (-HT) medium. GAA expression in these cells was induced by methotrexate treatment (MTX, 500 nM).

[0167] Cell pools with high GAA expression were identified by GAA enzyme activity assay and used to establish individual clones producing rhGAA. Individual clones were generated on semi-solid media plates, picked by ClonePix system, and transferred to 24 deep-well plates. These individual clones were assayed for GAA enzyme activity to identify clones with high GAA expression. Conditioned media for determining GAA activity used 4-MU-α-glucosidase substrate. Clones with high GAA production as measured by GAA enzyme assay were further determined for viability, growth potential, GAA productivity, N-glycan structure, and stable protein expression. CHO cell lines expressing rhGAA with enhanced mono-M6P or bis-M6P N-glycans were isolated using this procedure, including the CHO cell line GA-ATB200.

[0168] Example 2: Purification of rhGAA Multiple batches of rhGAA according to the present disclosure were produced in shake flasks and perfusion bioreactors using the CHO cell line GA-ATB200. This product is referred to as "ATB200". ATB200 rhGAA was fractionated based on terminal phosphate and sialic acid using weak anion exchange ("WAX") liquid chromatography. ERT was eluted with increasing amounts of salt to generate elution profiles. These profiles were monitored by UV (A280 nm). Similar CIMPR receptor binding (at least about 70%) profiles were observed for purified ATB200 rhGAA from different production batches (Figure 5), indicating that ATB200 rhGAA can be produced consistently.

[0169] Example 3: Characterization of oligosaccharides of ATB200 rhGAA ATB200 rhGAA was analyzed for site-specific N-glycan profiles using different LC-MS / MS analytical techniques. Results from the first two LC-MS / MS methods are shown in Figures 6A-H. Results from the third LC-MS / MS method with 2-AA glycan mapping are shown in Figures 19A-H, 20A-B, and Table 5.

[0170] In the first LC-MS / MS analysis, the protein was denatured, reduced, alkylated, and digested, and then subjected to LC-MS / MS analysis. For 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 Milli-Q® water were added to a 1.5 mL tube to provide a total volume of 100 μL. The sample was mixed and incubated at 56° C. for 30 minutes in a dry bath. For alkylation, the denatured and reduced protein sample was mixed with 5 μL of 1 mol / L iodoacetamide (IAM, final concentration 50 mM) and then incubated at 10-30° C. for 30 minutes in the dark. After alkylation, 400 μL of pre-chilled acetone was added to the samples and the mixture was frozen under -80°C freezer for 4 hours. The samples were then centrifuged at 13000 rpm for 5 minutes at 4°C and the supernatant was removed. 400 μL of pre-chilled acetone was added to the pellet, which was centrifuged at 13000 rpm for 5 minutes at 4°C and the supernatant was removed. The samples were then air-dried on ice in the dark to remove acetone residues. Forty microliters of 8M urea and 160 μL of 100 mM NH4HCO3 were added to the samples to dissolve the proteins. For trypsin digestion, 50 μg of protein was then added with trypsin digestion buffer to a final volume of 100 μL and 5 μL of 0.5 mg / mL trypsin (protein to enzyme ratio 20 / 1 w / w) was added. The solution was mixed thoroughly and incubated at 37°C overnight (16 ± 2 hours). The reaction was quenched by adding 2.5 microliters of 20% TFA (final concentration 0.5%).The samples were then analyzed using a Thermo Scientific™ Orbitrap Velos Pro™ mass spectrometer.

[0171] In a second LC-MS / MS analysis, ATB200 samples were prepared by a similar denaturation, reduction, alkylation, and digestion procedure, except that iodoacetic acid (IAA) was used instead of IAM as the alkylation reagent, and then analyzed using a Thermo Scientific™ Orbitrap Fusion™ Lumos Tribid™ mass spectrometer.

[0172] The results of the first and second analyses are shown in Figures 6A-6H. In Figures 6A-6H, the results of the first analysis are shown in the left bar (dark gray) and the results of the second analysis are shown in the right bar (light gray). The symbolic nomenclature of the glycan representation follows Varki, A., Cummings, RD, Esko JD, et al., Essentials of Glycobiology, 2nd edition (2009).

[0173] As can be seen from Figures 6A-6H, these two analyses provided similar results, but there was some variability between the results. This variability can be attributed to a number of factors, including the instrumentation used and the completeness of the N-glycan analysis. For example, if some species of phosphorylated N-glycans were not identified and / or quantified, then the total number of phosphorylated N-glycans may be underestimated and the percentage of rhGAA bearing phosphorylated N-glycans at that site may be underestimated. As another example, if some species of non-phosphorylated N-glycans were not identified and / or quantified, then the total number of non-phosphorylated N-glycans may be underestimated and the percentage of rhGAA bearing phosphorylated N-glycans at that site may be overestimated.

[0174] Figure 6A shows the N-glycosylation site occupancy of ATB200. As can be seen from Figure 6A, the first, second, third, fourth, fifth and sixth N-glycosylation sites were nearly occupied, and both analyses detected around 90% or more, up to about 100%, of the ATB200 enzyme having detected N-glycans at each of the potential N-glycosylation sites. However, for the seventh potential N-glycosylation site, about half were N-glycosylated.

[0175] Figure 6B shows the N-glycosylation profile of the first potential N-glycosylation site, N84. As can be seen in Figure 6B, the major N-glycan species is bis-M6P N-glycan. Both the first and second analyses detected that more than 75% of ATB200 had bis-M6P at the first site, which corresponded to an average of about 0.8 mol of bis-M6P per mol of ATB200 at the first site.

[0176] Figure 6C shows the N-glycosylation profile of the second potential N-glycosylation site, N177. As can be seen from Figure 6C, the major N-glycan species are mono-M6P N-glycan and non-phosphorylated high mannose type N-glycan. Both the first and second analyses detected that more than 40% of ATB200 had mono-M6P at the second site, which corresponded to an average of about 0.4 to about 0.6 mol of mono-M6P per mol of ATB200 at the second site.

[0177] Figure 6D shows the N-glycosylation profile of the third potential N-glycosylation site, N334. As can be seen from Figure 6D, the major N-glycan species are non-phosphorylated high mannose type N-glycans, diantennary type, triantennary type, and tetraantennary type complex type N-glycans, and hybrid type N-glycans. Both the first and second analyses detected that more than 20% of ATB200 had sialic acid residues at the third site, which corresponded to an average of about 0.9 to about 1.2 mol of sialic acid per mol of ATB200 at the third site.

[0178] FIG. 6E shows the N-glycosylation profile of the fourth potential N-glycosylation site, N414. As can be seen from FIG. 6E, the major N-glycan species are bis-M6P and mono-M6P N-glycans. Both the first and second analyses detected that more than 40% of ATB200 had bis-M6P at the fourth site, which corresponded to an average of about 0.4 to about 0.6 mol of bis-M6P per mol of ATB200 at the fourth site. Both the first and second analyses also detected that more than 25% of ATB200 had mono-M6P at the fourth site, which corresponded to an average of about 0.3 to about 0.4 mol of mono-M6P per mol of ATB200 at the fourth site.

[0179] Figure 6F shows the N-glycosylation profile of the fifth potential N-glycosylation site, N596. As can be seen from Figure 6F, the major N-glycan species is a fucosylated diantennary complex type N-glycan. Both the first and second analyses detected that more than 70% of ATB200 had a sialic acid residue at the fifth site, which corresponded to an average of about 0.8 to about 0.9 mol of sialic acid per mol of ATB200 at the fifth site.

[0180] Figure 6G shows the N-glycosylation profile of the sixth potential N-glycosylation site, N826. As can be seen from Figure 6G, the major N-glycan species are diantennary, triantennary, and tetraantennary complex type N-glycans. Both the first and second analyses detected that more than 80% of ATB200 had sialic acid residues at the sixth site, which corresponded to an average of about 1.5 to about 1.8 mol of sialic acid per mol of ATB200 at the sixth site.

[0181] Analysis of N-glycosylation at the seventh site, N869, revealed approximately 40% N-glycosylation, with the most prevalent N-glycans being A4S3S3GF (12%), A5S3G2F (10%), A4S2G2F (8%) and A6S3G3F (8%).

[0182] Figure 6H shows a summary of phosphorylation at each of the seven potential N-glycosylation sites. As can be seen in Figure 6H, both the first and second analyses detected high phosphorylation levels at the first, second, and fourth potential N-glycosylation sites. Both analyses detected more than 80% of ATB200 being mono- or bis-phosphorylated at the first site, more than 40% of ATB200 being mono-phosphorylated at the second site, and more than 80% of ATB200 being mono- or bis-phosphorylated at the fourth site.

[0183] Another N-glycosylation analysis of ATB200 was performed by LC-MS / MS method as described below. From this analysis, an average N-glycosylation profile of ATB200 across 10 lots was generated (Figures 19A-H, 20A-B).

[0184] N-linked glycans from ATB200 were enzymatically released with PNGase-F and labeled with 2-anthranilic acid (2-AA). The 2-AA labeled N-glycans were further processed by solid phase extraction (SPE) to remove excess salts and other contaminants. The purified 2-AA N-glycans were dissolved in acetonitrile / water (20 / 80; v / v) and 10 micrograms were loaded onto an aminopolymer analytical column (apHera™, Supelco) for high performance liquid chromatography with fluorescence detection (HPLC-FLD) and high resolution mass spectrometry (HRMS) analysis.

[0185] Liquid chromatographic (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 starting 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) operated in independent data acquisition (IDA) mode. The acquired data files were converted to mzML files using MSConvert in ProteoWizard and then subjected to glycan database search utilizing GRITS toolbox 1.2 Morning Blend software (UGA), followed by annotation of the identified N-glycans. N-glycans were identified using both precursor monoisotopic mass (m / z) and product ion m / z. Experimental product ions and fragmentation patterns were confirmed in silico using the GlycoWorkbench 2 application.

[0186] To determine the relative quantification of N-linked glycans from ATB200, the data obtained from the HPLC-FLD-QTOF MS / MS experiment was processed as follows: All N-glycan peaks in the FLD chromatogram were integrated and each peak was assigned a percentage of the total area of ​​all peaks in the FLD chromatogram. The fluorescence signal expressed as the peak area is a quantitative measure of the amount of each N-glycan in the sample. However, in most cases, multiple N-glycan species were contained in the same FLD peak. Therefore, mass spectrometry data was also required to obtain the relative quantification of each N-glycan species (Table 5). The ion intensity signal of each N-glycan was "extracted" from the data to create a chromatographic peak called an extracted ion chromatogram (XIC). The XIC aligned with the FLD chromatographic peak was specific for only one N-glycan species. The XIC peak created from the ion intensity signal was then integrated. This peak area is a relative quantitative measure of the amount of glycan present. Using both the FLD peak areas and the mass spectrometer XIC peak areas allowed for relative quantification of all N-linked glycan species of ATB200 reported herein.

[0187] The results of this LC-MS / MS analysis are provided in Table 5 below. Symbolic nomenclature for glycan representation is given in Wopereis W,et al.2006.Abnormal glycosylation with hypersialylated O-glycans in patients with Sialuria.Biochimica et Biophysica Acta.1762:598-607;Gornik O,et al.2007.Changes of serum glycans during sepsis and acute pancreatitis.Glycobiology.17:1321-1332;Kattla JJ,et al.2011.Biologic protein glycosylation.In:Murray Moo-Young(ed.),Comprehensive Biotechnology,Second Edition,3:467-486;Tharmalingam-Jaikaran T,et al. "N-glycan profiling of bovine follicular fluid at key dominant follicle developmental stages". 2014. Reproduction. 148:569-580; Clerc F,et al. "Human plasma protein N-glycosylation". 2015. Glycoconj J. DOI 10.1007 / s10719-015-9626-2; and Blackler RJ,et al. 2016."Single-chain antibody-fragment M6P-1 possesses a mannose 6-phosphate monosaccharide-specific binding pocket that distinguishes N-glycan phosphorylation in a branch-specific manner." Glycobiology. 26-2:181-192.

[0188] [Table 7]

[0189] Based on this 2-AA and LC-MS / MS analysis, and as further summarized, the ATB200 tested has an average M6P content (accounting for both mono-M6P and bis-M6P) of 3-5 mol per mol of ATB200 and a sialic acid content of 4-7 mol per mol of ATB200.

[0190] As shown in Figures 19A-19H and summarized in Figure 20B, the first potential N-glycosylation site of ATB200 has an average M6P content of about 1.4 mol M6P / mol ATB200, which is accounted for by an average mono-M6P content of about 0.25 mol mono-M6P / mol ATB200 and an average bis-M6P content of about 0.56 mol bis-M6P / mol ATB200; the second potential N-glycosylation site of ATB200 has an average M6P content of about 0.5 mol M6P / mol ATB200, where the major phosphorylated N-glycan species is mono-M6P N-glycan; and the third potential N-glycosylation site of ATB200 has an average M6P content of about 1 mol sialic acid / mol the fourth potential N-glycosylation site of ATB200 has an average M6P content of about 1.4 mol M6P / mol ATB200, accounted for by an average mono-M6P content of about 0.35 mol mono-M6P / mol ATB200 and an average bis-M6P content of about 0.52 mol bis-M6P / mol ATB200; the fifth potential N-glycosylation site of ATB200 has an average sialic acid content of about 0.86 mol sialic acid / mol ATB200; the sixth potential N-glycosylation site of ATB200 has an average sialic acid content of about 4.2 mol sialic acid / mol ATB200; and the seventh potential N-glycosylation site of ATB200 has an average sialic acid content of about 0.86 mol sialic acid / mol ATB200.

[0191] Also, according to the 2-AA and LC-MS / MS analytical techniques, on average about 65% of the N-glycans at the first potential N-glycosylation site of ATB200 are high mannose type N-glycans, about 89% of the N-glycans at the second potential N-glycosylation site of ATB200 are high mannose type N-glycans, more than half of the N-glycans at the third potential N-glycosylation site of ATB200 are sialylated (nearly 20% are fully sialylated), and about 85% of the N-glycans at the third potential N-glycosylation site of ATB200 are complex type N-glycans, about 84% of the N-glycans at the fourth potential N-glycosylation site of ATB200 are high mannose type N-glycans, and about 85% of the N-glycans at the fifth potential N-glycosylation site of ATB200 are complex type N-glycans. About 70% of the N-glycans at the 5th potential N-glycosylation site of ATB200 are sialylated (about 26% are fully sialylated), and about 100% of the N-glycans at the 5th potential N-glycosylation site of ATB200 are complex type N-glycans, about 85% of the N-glycans at the 6th potential N-glycosylation site of ATB200 are sialylated (nearly 27% are fully sialylated), and about 98% of the N-glycans at the 6th potential N-glycosylation site of ATB200 are complex type N-glycans, and about 87% of the N-glycans at the 7th potential N-glycosylation site of ATB200 are sialylated (nearly 8% are fully sialylated), and about 100% of the N-glycans at the 7th potential N-glycosylation site of ATB200 are complex type N-glycans.

[0192] Example 4: Analytical Comparison of ATB200 and MYOZYME® / LUMIZYME® Purified ATB200 and LUMIZYME® N-glycans were assessed by MALDI-TOF to determine the distinct N-glycan structures found in each ERT. LUMIZYME® was obtained from a commercial source. As shown in FIG. 7, ATB200 exhibited four prominent peaks that eluted to the right of LUMIZYME®. This confirms that ATB200 was more highly phosphorylated than LUMIZYME®, as this determination is due to terminal charge rather than CIMPR affinity. As summarized in FIG. 8, ATB200 samples were found to have a lower content of non-phosphorylated high mannose N-glycans than LUMIZYME®.

[0193] To determine the ability of conventional rhGAA in MYOZYME® and LUMIZYME® to interact with CIMPR, two conventional rhGAA formulations were injected onto a CIMPR affinity column (which binds rhGAA with M6P groups) and the flow-through was collected. Bound material was eluted with a free M6 gradient. Fractions were collected into a 96-well plate and assayed for GAA activity with 4MU-α-glucosidase substrate. The relative amounts of unbound rhGAA (flow-through) and bound rhGAA (eluted M6P) were determined based on GAA activity and reported as a percentage of total enzyme. Figures 9A and 9B show the binding profiles of rhGAA in MYOZYME® and LUMIZYME®: 73% of rhGAA in MYOZYME® (Figure 9B) and 78% of rhGAA in LUMIZYME® (Figure 9A) did not bind to CIMPR. In fact, only 27% of rhGAA in MYOZYME® and 22% of rhGAA in LUMIZYME® contained M6P, which may be productive in targeting it to CIMPR on muscle cells. In contrast, as shown in Figure 5, under the same conditions, more than 70% of rhGAA in ATB200 was found to bind to CIMPR.

[0194] In addition to the large percentage of rhGAA that can bind to CIMPR, it is important to understand the quality of the interaction. LUMIZYME® and ATB200 receptor binding was determined using a CIMPR plate binding assay. Briefly, GAA was captured using a plate coated with CIMPR. Various concentrations of rhGAA were applied to the immobilized receptor, and unbound rhGAA was washed away. The amount of remaining rhGAA was determined by GAA activity. As shown in Figure 10A, ATB200 bound to CIMPR significantly better than LUMIZYME®. Figure 10B shows the relative content of bis-M6P N-glycans in LUMIZYME® (a conventional rhGAA formulation) and ATB200 according to the present invention. For LUMIZYME®, on average, only 10% of the molecules have bis-phosphorylated N-glycans. In contrast, for ATB200, on average, every rhGAA molecule has at least one bis-phosphorylated N-glycan.

[0195] Overall, the higher content of M6P N-glycans in ATB200 compared to LUMIZYME® indicates that a higher proportion of rhGAA molecules can target muscle cells with ATB200. As shown above, the high proportion of mono- and bis-phosphorylated structures determined by MALDI is consistent with the CIMPR profile, which explains the significantly greater binding of ATB200 to the CIMPR receptor. N-glycan analysis by MALDI-TOF mass spectrometry confirmed that, on average, each ATB200 molecule had at least one natural bis-M6P N-glycan structure. This higher bis-M6P N-glycan content in ATB200 was consistent with the higher affinity binding (K ) to CIMPR in the M6P receptor plate binding assay. D This directly correlates with the α-terminal agonist concentration (approximately 2-4 nM).

[0196] The relative cellular uptake of ATB200 and LUMIZYME® rhGAA was compared using normal and Pompe fibroblast cell lines. Comparisons involved 5-100 nM of the disclosed ATB200 and 10-500 nM of the conventional rhGAA formulation LUMIZYME®. After 16 hours of incubation, external rhGAA was inactivated with Tris base and cells were washed three times with PBS prior to harvesting. Internalized GAA was measured by 4MU-α-glucoside hydrolysis and graphed against total cellular protein. Results can be seen in Figures 11A-C.

[0197] ATB200 was also shown to be efficiently internalized by cells. As illustrated in Figures 11A-B, ATB200 was internalized by both normal and Pompe fibroblasts to a greater extent than the conventional rhGAA formulation, LUMIZYME®. ATB200 saturates cell receptors at approximately 20 nM, whereas approximately 250 nM of LUMIZYME® is required to saturate cell receptors. The uptake efficiency constant (K uptake ) is 2-3 nm for ATB200 and 56 nM for LUMIZYME®, as shown in Figure 11C. These results suggest that ATB200 is a well-targeted treatment for Pompe disease.

[0198] Example 5: ATB200 and pharmacological chaperones The stability of ATB200 in acidic or neutral pH buffers was determined in a thermal stability assay using SYPRO orange, which increases the fluorescence of the dye as the protein unfolds. As shown in FIG. 12, the addition of AT2221 stabilized ATB200 at pH 7.4 in a concentration-dependent manner, which was comparable to the stability of ATB200 at pH 5.2, a condition that mimics the acidic environment of the lysosome. As summarized in Table 6, the addition of AT2221 increased the melting temperature (T m ) rose by almost 10°C.

[0199] [Table 8]

[0200] Example 6: Co-administration of ATB200 and AT2221 in Gaa KO mice The therapeutic effects of ATB200 and AT2221 were determined and compared to alglucosidase alfa in Gaa KO mice. Male Gaa KO (3-4 months old) and age-matched wild-type (WT) mice were used in this study. Alglucosidase alfa was administered by bolus tail vein intravenous (IV) injection. In the co-administration regimen, AT2221 was administered by oral gavage (PO) 30 minutes prior to the intravenous injection of ATB200. Treatments were given every other week. 14 days after the final dose, treated mice were sacrificed and various tissues were harvested for further analysis. Table 7 summarizes the study design.

[0201] [Table 9]

[0202] Tissue glycogen content of 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, triceps, gastrocnemius, and heart) when compared to the same dosage of alglucosidase alfa.

[0203] Tissue samples were also analyzed by the methods described in Khanna R, et al. (2012), "The pharmacological chaperone AT2220 increases recombinant human acid α-glucosidase uptake and glycogen reduction in a mouse model of Pompe disease," Plos One 7(7):e40776; and Khanna, R et al. (2014), "The Pharmacological Chaperone AT2220 Increases the Specific Activity and Lysosomal Delivery of Mutant Acid α-Glucosidase,and Promotes Glycogen Reduction in a Transgenic Mouse Model of Pompe Disease." Biomarker changes were also analyzed following the methods discussed in "Amyloidosis and Lysosomal Disease," PLoS ONE 9(7):e102092. As shown in Figure 14, muscle fibers from Gaa KO animals showed a marked increase in LAMP1-positive vesicles, indicative of lysosomal proliferation, and their hypertrophy, compared to WT. Co-administration of ATB200 / AT2221 led to more fibers with normalized LAMP1 levels, while the remaining LAMP1-positive vesicles were also reduced in size (inset).

[0204] Similarly, prominent LC3-positive aggregates in muscle fibers of untreated Gaa KO mice are indicative of the presence of autophagic regions and autophagic build-up. LC3-positive aggregates (red) were preferentially reduced in mice treated with ATB200 / AT2221 co-administration when compared to mice treated with alglucosidase alpha (Figure 15A). Similar observations were made when LC3 expression was assessed using Western blot. As shown in Figure 15B, the majority of animals treated with ATB200 / AT2221 exhibited significantly reduced levels of LC3 II, a lipidated form associated with autophagosomes, suggesting improved autophagic flux. In comparison, alglucosidase alpha had a lesser effect on autophagy.

[0205] Dysferlin, a protein involved in membrane repair whose deficiency / mistransportation is associated with several muscular dystrophies, was also evaluated. As shown in Figure 16, dysferlin (brown) was severely accumulated in the sarcoplasm of Gaa KO mice. Compared with alglucosidase alpha, ATB200 / AT2221 was able to restore dysferlin to the sarcolemma of a greater number of muscle fibers.

[0206] These data are consistent with the cellular improvements demonstrated in human Pompe disease patients treated with ATB200 and miglustat (e.g., these patients exhibit reduced levels of biomarkers of glycogen storage and muscle damage), and lead not only to an effective treatment of Pompe disease, but also to reversal of disease progression. Clinical data in human Pompe disease patients are summarized in Examples 8 and 9 below.

[0207] Example 7: Single Fiber Analysis As shown in FIG. 17, the majority of vehicle-treated mice showed significantly enlarged lysosomes (green) (see, e.g., “B”) and the presence of clumped autophagy buildup (red) (see, e.g., “A”). MYOZYME®-treated mice did not show any significant differences when compared to vehicle-treated mice. In contrast, most fibers isolated from ATB200-treated mice showed a dramatic reduction in lysosome size (see, e.g., “C”). Furthermore, there was also a variable degree of reduction in the extent of autophagy buildup (see, e.g., “C”). As a result, a large proportion (36-60%) of analyzed muscle fibers from ATB200-treated mice appeared normal or near normal. Table 8 below summarizes the single fiber analysis shown in FIG. 17.

[0208] [Table 10]

[0209] Overall, this data indicates that ATB200, with its high M6P content, both alone and further stabilized by the pharmacological chaperone AT2221 at neutral pH of blood, has higher efficiency of tissue targeting and lysosomal transport when administered to Gaa KO mice compared to alglucosidase alfa, consistent with the stabilization of ATB200 by AT2221 as illustrated in FIG. 18. As a result, administration of ATB200 and co-administration of ATB200 / AT2221 were more effective than alglucosidase alfa in correcting some of the disease-related pathologies, such as glycogen accumulation, lysosomal proliferation, and the formation of autophagy areas. Due to these positive therapeutic effects, administration of ATB200 and co-administration of ATB200 / AT2221 may help muscle fibers recover from injury and even reverse injury by removing glycogen accumulated in cells due to lack of optimal GAA activity. As in Example 6, these data are also consistent with the improvements at the cellular level demonstrated in human Pompe disease patients, which lead to both effective treatment of Pompe disease and reversal of disease progression after administration of ATB200 and miglustat. Clinical data of human Pompe disease patients are summarized in Examples 8 and 9 below.

[0210] Example 8: ATB200-02 Clinical Trial A Phase 1 / 2 (ATB200-02, NCT-02675465) open-label, fixed-sequence, dose-escalation clinical trial was conducted to evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics, and preliminary efficacy of intravenous infusions of ATB200 and AT2221 in adult subjects with Pompe disease. The data were reported in WO 2020 / 163480, the disclosure of which is incorporated herein by reference.

[0211] Example 9: ATB200-03 Clinical Trial: Phase 3 In-Human Study of ATB200 / AT2221 in Patients with Pompe Disease The ATB200-03 trial was a phase 3, double-blind, randomized, multicenter, international study of ATB200 / AT2221 compared with alglucosidase alfa / placebo in adult subjects with late-onset Pompe disease (LOPD) who had received enzyme replacement therapy with alglucosidase alfa (i.e., ERT-experienced) or had never received ERT (i.e., ERT-naive).

[0212] Study design As shown in Figure 21, the trial consisted of a screening period of up to 30 days, a treatment period of 12 months, and a safety follow-up period of 30 days. Eligible subjects were randomized in a 2:1 ratio to receive ATB200 / AT2221 or alglucosidase alfa / placebo and stratified by ERT status (ERT-experienced, ERT-naïve) and baseline 6-minute walk distance (6MWD) (≥75m<150m, ≥150m<400m, ≥400m).

[0213] Efficacy assessments (i.e., functional assessments) included assessment of gait function (6MWT), motor function tests (Walk-Stair-Gowers-Chair Movement (GSGC) test and Timed Up-and-Go (TUG) test), muscle strength (manual muscle testing and quantitative muscle testing), and pulmonary function tests (FVC, SVC, MIP, MEP, and SNIP). Patient-reported outcomes (Rasch-built Pompe-specific Activity (R Pact) scale, EuroQol 5 Dimensions 5 Level instrument (EQ-5D-5L), Patient-Reported Outcomes Measurement Information System (PROMIS®) instrument for physical function, fatigue, dyspnea, and upper extremities, and Subject's Global Impression of Change) were recorded. Physician's Global Impression of Change was also performed.

[0214] Pharmacodynamic evaluation included measurement of biomarkers for muscle injury (creatine kinase (CK) and disease substrates (urinary hexose tetrasaccharide (Hex4)). Low-density blood samples for population PK analysis were collected in ERT-experienced subjects to determine plasma total GAA protein levels and AT2221 concentrations. In ERT-naive subjects, time-series blood sampling was performed to characterize the PK profile of total GAA protein and AT2221.

[0215] Safety assessments included monitoring for adverse events (AEs) including infusion-related reactions (IARs), clinical laboratory tests (chemistry, hematology, and urinalysis), vital signs, physical examination including weight, electrocardiogram (ECG), and immunogenicity. Concomitant medications and non-pharmacological therapies were also recorded.

[0216] Selecting a target The subjects who participated in the study met all the following inclusion criteria and did not meet any exclusion criteria. In total, 122 subjects participated in this ATB200-03 trial. Of these, 85 subjects (ERT-experienced: 65 cases; ERT-naïve: 20 cases) received ATB200 / AT2221 treatment, and 37 subjects (ERT-experienced: 30 cases; ERT-naïve: 7 cases) received alglucosidase alfa / placebo treatment. As shown in Figure 22, baseline 6MWD and FVC data were representative of the population and generally similar in the two treatment groups.

[0217] Inclusion Criteria: 1. Subjects provided signed informed consent prior to undergoing any study-related procedures. 2. Male and female subjects were aged 18 years or older and weighed 40 kg or more at the time of screening. 3. Female and male subjects of childbearing potential agreed to use medically accepted methods of contraception during this study and for 90 days after the final dose of study drug. 4. Subject had a documented diagnosis of LOPD based on one of the following: A deficiency of the GAA enzyme b.GAA genotyping 5. Subjects were classified into one of the following regarding ERT status: a. ERT-previous, defined as having received standard of care ERT (alglucosidase alfa) at the recommended dose and regimen (i.e., 20 mg / kg dose every 2 weeks) for ≥24 months Specifically for Australia, ERT-experienced is defined as having received standard of care ERT (alglucosidase alfa) at a dose of 20 mg / kg of lean or ideal body weight every 2 weeks at the recommended dose and regimen. b. ERT naive, defined as never having received any investigational or commercially available ERT 6. Participants had a sitting FVC ≥ 30% of predicted for healthy adults (National Health and Nutrition Examination Survey III) at screening. 7. Subject performed two 6MWTs at screening that were valid as determined by a clinical assessor and met all of the following criteria: a. Both screening values ​​for 6MWD were above 75 meters. b. Both 6MWD screening values ​​were below 90% of the predicted value for healthy adults c. The lower 6MWD was within 20% of the higher 6MWD.

[0218] Exclusion criteria: 1. Subjects had received, or were due to receive during the study, any investigational therapy or pharmacologic treatment for Pompe disease other than alglucosidase alfa within the 30 days prior to Day 1 or 5 half-lives of that therapy or treatment, whichever was longer. 2. Subject had previously received gene therapy for Pompe disease. 3. Subject was taking any of the following prohibited drugs within the 30 days prior to Day 1: Miglitol Miglustat Acarbose Voglibose 4. Subject required the use of invasive or noninvasive ventilatory support for more than 6 hours per day while awake. 5. The subject had a hypersensitivity to ATB200, alglucosidase alfa, or any of the excipients in AT2221. 6. Subject had a medical condition or any other extenuating circumstances that, in the opinion of the Investigator or Medical Monitor, posed an undue safety risk to the subject or impair or adversely affect the subject's ability to comply with the protocol requirements. This included clinical depression with uncontrolled or poorly controlled symptoms (as diagnosed by a psychiatrist or other mental health professional). 7. Subject, if female, was pregnant or lactating at the time of screening. 8. Subjects, regardless of gender, were planning to become pregnant during the study. 9. Subject refused to undergo genetic testing.

[0219] Investigational drug, dosage, and method of administration Subjects were randomized in at least a 2:1 randomization ratio to receive either ATB200 / AT2221 or alglucosidase alfa / placebo. Table 9 below summarizes the treatment of enrolled subjects.

[0220] [Table 11]

[0221] Data evaluation and statistical considerations The primary efficacy outcome was change in 6MWD from baseline to week 52. The primary outcome was tested for superiority of ATB200 / AT2221 versus alglucosidase alfa / placebo using a mixed-effects model for repeated measures (MMRM) and prespecified nonparametric tests when normality was violated.

[0222] Key secondary efficacy outcomes, in prespecified order of hierarchical importance, were as follows: These secondary outcomes were analyzed using analysis of covariance (ANCOVA) models and missing value imputation methods using the most recent observation imputed (ITT LOCF). Change from baseline to week 52 in sitting FVC (% predicted) Change in lower limb manual muscle test score from baseline to week 52 Change from baseline to week 52 in the PROMIS-Physical Function total score Change from baseline to week 52 in the PROMIS-Fatigue total score Change in GSGC total score from baseline to week 52

[0223] Other secondary efficacy outcomes were: Change from baseline to week 52 in the following motor function variables: - Time taken to complete the 10 meter walk of the GSGC test (i.e., gait assessment) - Time taken to complete the GSGC test of climbing four stairs - Time taken to complete the Gowers movement in the GSGC test - Time taken to rise from a chair as part of the GSGC test - Time taken to complete the TUG test Change from baseline to week 52 in the following muscle strength variables: - Upper limb manual muscle test score - Total manual muscle test score - Upper limb quantitative muscle strength test value (kg) - Lower limb quantitative muscle strength test value (kg) - Quantitative Muscle Strength Test Total Value (kg) Change from baseline to week 52 in the following variables from patient-reported outcome measures: - PROMIS-dyspnea total score - PROMIS-Upper Extremity Total Score - R-PAct scale total score - EQ-5D-5L health status Actual subject functional status (improved, stable, or worsening) regarding the effect of the study drug in the following life domains at Week 52, as measured by the Subject's Global Impression of Change: - General physical health - breathing effort - Strength - Muscle function - Ability to move around - Daily life activities - Vitality level - Muscle pain level Actual subject functional status (improved, stable, or worsening) at Week 52 as measured by the Physician's Global Impression of Change Change from baseline to week 52 in the following lung function measures: - Sitting FVC (% predicted) - MIP (cmH2O) - MIP (% of predicted value) - MEP (cmH2O) - MEP (% of predicted value) - SNIP (cmH2O)

[0224] Pharmacodynamic endpoints were as follows: Change in serum CK levels from baseline to week 52 Change in urinary Hex4 levels from baseline to week 52

[0225] For ERT-experienced subjects, pharmacokinetic endpoints from population PK analysis of total GAA protein levels and AT2221 concentrations were collected. For ERT-naive subjects, plasma total GAA protein concentrations and PK parameters of AT2221 were calculated.

[0226] The safety profile of ATB200 / AT2221 was characterized using the incidence of treatment-emergent adverse events (TEAEs), serious adverse events (SAEs), and AEs leading to discontinuation of study drug, the frequency and severity of immediate and delayed IARs, and any abnormalities observed in other safety assessments. The impact of immunogenicity to ATB200 and alglucosidase alfa on safety and efficacy was also evaluated.

[0227] Statistical methods included the following considerations for sample randomization, sample size calculations, efficacy analyses, and safety analyses.

[0228] Randomization. The following two factors were identified as design stratification variables: 1. Baseline 6MWD (≥75 <150 meters, ≥150 <400 meters, ≥400 meters); and 2. ERT status (ERT-experienced, ERT-naive). These two factors formed six factor combinations (i.e., levels, strata). The above risk factors were balanced using a centralized block randomization procedure to 1) reduce bias and increase the precision of statistical inferences, and 2) allow for various planned and unplanned subset analyses. A block randomization scheme was performed for each of the six strata. The randomization ratio was fixed, 2:1, ATB200 / AT2221 vs. alglucosidase alfa / placebo.

[0229] Sample size calculation. A two-sample t-test with a two-sided significance level of 0.05 and a 2:1 randomization scheme (66 subjects in the ATB200 / AT2221 group and 33 subjects in the alglucosidase alfa / placebo group for a total sample size of 99 subjects) was determined to have approximately 90% power to detect a standardized effect size of 0.7 between these two samples in a superiority test. This calculation was performed using Nquery 8©. Assuming a 10% dropout rate, the sample size was approximately 110 subjects.

[0230] Efficacy Analysis. A parametric analysis of covariance (ANCOVA) model was used to analyze the primary efficacy outcome (i.e., change from baseline to week 52 in 6MWD) and compare new treatment with control. The model typically adjusted for baseline 6MWD (as a continuous covariate) and would stratify randomization using two factors: ERT status (ERT-naive vs. ERT-experienced) and baseline 6MWD (75 to <150 meters, 150 to <400 meters, 400 meters or more). However, baseline 6MWD could not be used twice in the model (as both a continuous and categorical variable) because of the expected high point-biserial correlation between them. Thus, the 6MWD continuous variable was left in the model, but the categorical 6MWD was removed. The ANCOVA model then had tops for treatment, baseline 6MWD (continuous), and ERT status (categorical).

[0231] In addition, possible treatment × covariate interactions (i.e., treatment × ERT status and treatment × baseline 6MWD continuous) were examined. If an interaction term was statistically significant (e.g., p<0.10, two-sided) and had a logical biological interpretation, it could then be added to the final ANCOVA model, which would then be used for the primary endpoint analysis. Data were then analyzed based on the ANCOVA model, providing all relevant estimates (e.g., LS means for each treatment group, LS means of the differences, 95% confidence intervals (CIs) for the LS mean differences, and p values ​​for the comparison between the two treatment groups).

[0232] To support interpretation of clinical benefit, a composite subject-level response was defined based on the aggregate treatment outcome data. Subjects were classified according to an ordinal response variable consisting of significant improvement, moderate improvement, or slight improvement / no improvement based on treatment outcome.

[0233] Key secondary outcomes were analyzed in a hierarchical order, using a stepwise closed testing procedure to control for type I error rates. Key secondary and other secondary outcomes were analyzed separately using methods similar to those used for the primary outcome analysis.

[0234] Safety Analyses. Safety data were summarized using counts and percentages for categorical data and descriptive statistics (mean, standard deviation, median, minimum, maximum) for continuous data.

[0235] Efficacy results of the ATB200-03 clinical trial In the entire population, ATB200 / AT2221 treatment showed improvement in 6MWD and stabilization of percent predicted FVC compared to baseline at week 52 (FIG. 23A) and over time (FIG. 23B). Compared to alglucosidase alfa / placebo, ATB200 / AT2221 treatment showed greater improvement in 6MWD in the entire population at week 52 (FIG. 23A). Furthermore, as shown in FIG. 23A, ATB200 / AT2221 treatment showed clinically significant improvement in percent predicted FVC in the entire population at week 52 compared to alglucosidase alfa / placebo.

[0236] In the ERT-experienced population, ATB200 / AT2221 treatment demonstrated improvement in 6MWD and stabilization of percent predicted FVC at 52 weeks compared to baseline ( FIG. 24 ). Compared to alglucosidase alfa / placebo, ATB200 / AT2221 treatment demonstrated improvement in 6MWD over time and stabilization of percent predicted FVC over time in the ERT-experienced population ( FIG. 25 ). Furthermore, as shown in FIG. 24 , ATB200 / AT2221 treatment demonstrated clinically significant improvement in both 6MWD and percent predicted FVC at 52 weeks in the ERT-experienced population compared to alglucosidase alfa / placebo.

[0237] As shown in Figures 26A and 26B, in a smaller ERT-naive population (n=27), ATB200 / AT2221 treatment demonstrated improvements in 6MWD and stabilization of percent predicted FVC at week 52 compared to baseline (Figure 26A) and over time (Figure 26B). There was more variability between the two treatment groups, with no clinically meaningful improvements in 6MWD or percent predicted FVC (Figure 26A).

[0238] As shown in FIG. 28, in the entire population and the population with a history of ERT, the lower limb MMT was more favorable in the ATB200 / AT2221 treatment compared to alglucosidase alfa / placebo.

[0239] As shown in FIG. 29, in the total population and in the ERT-experienced population, ATB200 / AT2221 treatment demonstrated clinically significant improvements in GSGC at 52 weeks compared to alglucosidase alfa / placebo.

[0240] As shown in FIG. 30, PROMIS physical function was more favorable with ATB200 / AT2221 treatment compared to alglucosidase alfa / placebo in the overall population and in the ERT-experienced population.

[0241] As shown in Figure 31, PROMIS Fatigue improved to a similar extent between the two treatment groups in the overall population and in the ERT-experienced population.

[0242] Biomarker results from the ATB200-03 clinical trial In the total population and the ERT-experienced population, ATB200 / AT2221 treatment showed improvement in biomarkers of muscle damage (CK) and disease substrate (Hex4) over time (Figures 32 and 33). Furthermore, as shown in Figures 32 and 33, in the total population and the ERT-experienced population, the reduction in CK and urinary Hex4 was significantly greater with ATB200 / AT2221 treatment at week 52 compared to alglucosidase alfa / placebo.

[0243] As summarized in Figure 34, in the overall population and in the ERT-experienced population, exercise function, pulmonary function, muscle strength, patient-reported outcomes (PROs), and biomarker endpoints were consistently favored with ATB200 / AT2221 treatment compared to alglucosidase alfa / placebo. Furthermore, of the 17 efficacy and biomarker endpoints evaluated, 16 were favored with ATB200 / AT2221 treatment compared to alglucosidase alfa / placebo.

[0244] Safety results of the ATB200-03 clinical trial As shown in FIG. 35, the overall safety profile of the ATB200 / AT2221 treatment group was similar to the alglucosidase alfa / placebo group.

[0245] 36-40 describe additional aspects of the ATB200-03 clinical trial.

[0246] Example 10: Results of the PROPEL Phase 3 Clinical Trial AT-GAA demonstrated clinically meaningful and significant improvements in both musculoskeletal and respiratory measures of late-onset Pompe disease compared to standard of care in the pivotal Phase 3 PROPEL trial. PROPEL is also referred to as "ATB200-03." See Example 9.

[0247] Patients who switched from the approved standard of care ERT (alglucosidase alfa) to AT-GAA walked an average of 17 metres more (p=0.046).

[0248] Patients who switched to AT-GAA also showed improvement in percent-predicted forced vital capacity (FVC), the most important measure of respiratory function in Pompe disease, compared with deterioration in patients treated with alglucosidase alfa (FVC difference 4.1%; p=0.006).

[0249] AT-GAA demonstrated a nominally statistically significant and clinically meaningful difference in superiority in the first key secondary endpoint of FVC compared with patients treated with alglucosidase alfa (FVC difference 3.0%; p=0.023).

[0250] In the combined study population of ERT-converted and ERT-naïve patients, AT-GAA outperformed alglucosidase alfa by 14 m on the primary endpoint (21 m vs. 7 m), with the superiority not being statistically significant (p=0.072).

[0251] Improvement in two key biomarkers of Pompe disease (Hex-4 and CK) in the combination study population was significantly better with AT-GAA compared to alglucosidase alfa (p<0.001).

[0252] PROPEL was a 52-week, double-blind, randomized, international trial designed to evaluate the efficacy, safety, and tolerability of AT-GAA compared with the current standard of care, enzyme replacement therapy (ERT), alglucosidase alfa. The trial enrolled 123 adult Pompe disease patients who were still able to walk and breathe without mechanical ventilation and was conducted at 62 clinical sites in 24 countries across five continents. This was the largest controlled clinical trial ever conducted in a lysosomal disorder.

[0253] Patients enrolled in PROPEL were randomized 2:1, so that for every 2 patients randomized to treatment with AT-GAA, 1 was randomized to treatment with alglucosidase alfa. Of the Pompe disease patients enrolled in PROPEL, 77% had been treated with alglucosidase alfa immediately prior to enrollment (n=95), and 23% had never been treated with any ERT (n=28). 117 patients completed the PROPEL trial, and all 117 willingly enrolled in the long-term extension study and are currently being treated for their Pompe disease exclusively with AT-GAA.

[0254] Pre-specified analyses of 6-minute walk distance (6MWD) and percent predicted forced vital capacity (FVC) in a combined ERT conversion and ERT-naive study population: The primary endpoint of this study was the mean change in 6-minute walk distance compared to baseline measurements at 52 weeks in the ERT-switched and ERT-naive combination patient population. In this combination population, patients taking AT-GAA (n=85) walked an average of 21 meters more at 52 weeks compared to 7 meters in patients treated with alglucosidase alfa (n=37) (Table 10). This primary endpoint in the combination population was evaluated for superiority, and although numerically large, statistical significance of superiority in this combination population was not achieved for the AT-GAA arm when compared to the alglucosidase alfa arm (p=0.072).

[0255] According to the hierarchy of the statistical analysis plan, the first key secondary endpoint of the study was the mean change in percent predicted FVC at 52 weeks in the combination population. In this combination population, patients taking AT-GAA demonstrated a nominally statistically significant and clinically meaningful difference in superiority over patients treated with alglucosidase alfa. AT-GAA significantly slowed the rate of respiratory deterioration in patients after 52 weeks. Patients treated with AT-GAA showed an absolute worsening of 0.9% in percent predicted FVC compared to an absolute worsening of 4.0% in the alglucosidase alfa arm (p=0.023) (Table 11). Percent predicted FVC is the most important measure of respiratory muscle function in Pompe disease and was the basis for the approval of alglucosidase alfa.

[0256] [Table 12]

[0257] [Table 13]

[0258] Pre-specified analyses of 6-minute walk distance (6MWD) and percent predicted forced vital capacity (FVC) in the ERT conversion trial population (n=95): PROPEL-switched patients who had been treated with alglucosidase alfa for at least 2 years were enrolled in the study. More than two-thirds (67%+) of these patients had been on ERT treatment for more than 5 years (mean 7.4 years) prior to enrollment in the PROPEL study.

[0259] A prespecified analysis of 6-minute walk distance in patients who switched from alglucosidase alfa showed that AT-GAA-treated patients (n=65) walked 16.9 meters more than their baseline 52 weeks after switching, compared with 0.0 meters in patients randomized to remain on alglucosidase alfa (n=30) (p=0.046) (Table 12).

[0260] A prespecified analysis of percent predicted FVC in patients who switched from alglucosidase alfa showed that AT-GAA-treated patients stabilized and slightly improved their respiratory function on this important measure, while patients who remained on alglucosidase alfa continued to experience a significant deterioration in respiratory muscle function. AT-GAA patients showed an absolute increase of 0.1% in percent predicted FVC over 1 year, while alglucosidase alfa patients showed an absolute deterioration of 4.0% (p=0.006) (Table 13).

[0261] [Table 14]

[0262] [Table 15]

[0263] Pre-specified analyses of 6-minute walk distance (6MWD) and percent predicted forced vital capacity (FVC) in an ERT-naive population (n=28): A prespecified analysis of 6-minute walk distance in patients never previously treated with any ERT showed that after 52 weeks, AT-GAA-treated patients (n=20) walked 33 meters more than their baseline. Alglucosidase alfa-treated patients (n=7) walked 38 meters more than their baseline. The difference between the two groups was not statistically significant (p=0.60) (Table 14).

[0264] A prespecified analysis of patients never previously treated with any ERT showed a similar deterioration in percent predicted forced vital capacity (FVC) at 52 weeks, -4.1% for AT-GAA-treated patients and -3.6% for alglucosidase alfa-treated patients (Table 15). The difference between the two groups was not statistically significant (p=0.57).

[0265] [Table 16]

[0266] [Table 17]

[0267] Pre-specified analyses of other key secondary and biomarker endpoints in ERT switching and ERT-naive overall study populations: Musculoskeletal and other key secondary endpoints: GSGC (Walking·Stairs·Gowers·Chair): GSGC is a widely used outcome measure capturing muscle strength, coordination and mobility important in Pompe disease. AT-GAA-treated patients demonstrated statistically significant improvement in scores on this important assessment compared with worsening in alglucosidase alfa-treated patients in the overall population (p<0.05).

[0268] Lower limb MMT (manual muscle testing), PROMIS physical function: for both of these validated measures of muscle strength and patient-reported outcomes, AT-GAA-treated patients improved numerically more than alglucosidase alfa-treated patients, but the results were not statistically significant.

[0269] PROMIS Fatigue: Fatigue as measured by this scale was slightly better in AT-GAA-treated patients than in alglucosidase alfa-treated patients.

[0270] Biomarkers of disease response to treatment: Urinary Hex-4: For the combined study population of both ERT-modified and ERT-naive patients, patients receiving AT-GAA showed substantial improvement for this biomarker, with a mean decrease in Hex-4 of -31.5% after 52 weeks compared with a +11.0% increase (i.e., worsening) in Hex-4 in alglucosidase alfa-treated patients (p=<0.001). Urinary Hex-4 is a commonly used biomarker in Pompe disease and is used as an indirect measure of the extent of skeletal glycogen clearance in Pompe disease patients receiving ERT. Glycogen is the substrate that accumulates in the lysosomes of muscle in Pompe disease patients.

[0271] CK (creatine kinase): After 52 weeks, AT-GAA-treated patients showed a substantial improvement in this biomarker as well, with a mean CK reduction of -22.4% compared with an increase (i.e., worsening) of +15.6% in alglucosidase alfa-treated patients (p<0.001). CK is an enzyme that leaks out of damaged muscle cells and is elevated in Pompe disease patients.

[0272] AT-GAA demonstrated a similar safety profile to alglucosidase alfa. Two patients (2.4%) receiving AT-GAA discontinued treatment due to adverse events compared with one (2.6%) unrelated to treatment for alglucosidase alfa. Infusion-related reactions (IARs) were reported in 25% of AT-GAA participants and 26% of alglucosidase alfa patients.

[0273] Post-hoc subgroup analyses: Baseline 6MWD and FVC categories: ERT-naive population (n=27): 3 patients had a baseline 6MWD <300m and 3 had a baseline FVC <55%; CFBL analyses were not performed in these subgroups due to small patient numbers.Baseline 6MWD ≥300m: Both the AT-GAA (n=18) and alglucosidase alfa / placebo (n=6) groups showed similar improvements over time (mean [SE] CFBL by week 52: +34.4[12.1]m and +30.8[9.6]m, respectively). Baseline FVC ≥ 55% in both the sipa glucosidase alfa / miglustat (n = 19) and sipa glucosidase alfa / placebo (n = 5) groups worsened over time (CFBL mean [SE] by week 52: -3.7 [1.5]% and -3.3 [2.6]%, respectively). As shown in Figures 41A and 41B, in the total and previously ERT populations, outcomes were consistently better with sipa glucosidase alfa / miglustat in patients with baseline 6MWD < 300m and ≥ 300m and FVC < 55% and ≥ 55%.

[0274] In the full study population, including ERT-naïve and ERT-experienced patients, sipaglucosidase alfa / miglustat demonstrated positive trends or clinically meaningful improvements compared to approved ERTs in terms of exercise and respiratory function, independent of baseline 6MWD and %FVC assessments, and in both pre-specified and post-hoc subgroup analyses.

[0275] Sipaglucosidase alfa / miglustat demonstrated a safety profile similar to that of alglucosidase alfa / placebo (Figure 42).

[0276] About AT-GAA AT-GAA is an investigational two-component therapy consisting of sipa glucosidase alfa (ATB200), a unique recombinant human acid alpha glucosidase (rhGAA) enzyme with an optimized carbohydrate structure, specifically bisphosphorylated mannose-6-phosphate (bis-M6P) glycan, for enhanced cellular uptake, administered in conjunction with miglustat (AT2221), a stabilizer of sipa glucosidase alfa. In preclinical studies, AT-GA was associated with increased levels of the mature lysosomal form of GAA and decreased levels of glycogen in muscle, reduced autophagy defects, and improved muscle strength.

[0277] About Pompe Disease Pompe disease is an inherited lysosomal disorder caused by a deficiency in the enzyme acid alpha-glucosidase (GAA). It is believed that reduced or absent levels of GAA lead to the accumulation of glycogen in cells, which results in the clinical symptoms of Pompe disease. The disease can be debilitating and is characterized by severe muscle weakness that worsens over time. Pompe disease ranges from a rapidly fatal infantile form with significant effects on cardiac function to a more slowly progressing late-onset form that primarily affects skeletal muscles. It is estimated that approximately 5,000-10,000 people worldwide suffer from Pompe disease.

[0278] Numbered embodiments Notwithstanding the appended claims, the present disclosure presents the following numbered embodiments.

[0279] 1. A method of treating Pompe disease in a subject in need thereof, comprising administering to the subject a population of recombinant human acid alpha-glucosidase (rhGAA) molecules, either simultaneously or sequentially, with a pharmacological chaperone; The rhGAA molecule contains seven potential N-glycosylation sites; Between 40% and 60% of the N-glycans on the rhGAA molecule are complex-type N-glycans; the rhGAA molecule contains at least 0.5 moles of bis-mannose-6-phosphate (bis-M6P) per mole of rhGAA at the first potential N-glycosylation site as determined using liquid chromatography tandem mass spectrometry (LC-MS / MS); and The method improves one or more disease outcomes in a subject compared to 1) baseline, or (2) a control treatment comprising administering alglucosidase alfa and a pharmacological chaperone placebo.

[0280] 2. The method of embodiment 1, which improves the subject's motor function as measured by the 6-minute walk test.

[0281] 3. The method of embodiment 2, wherein the change from baseline in 6-minute walking distance (6MWD) is at least 20 meters.

[0282] The method of embodiment 3, wherein the change from baseline in 4.6MWD is at least 20 meters after 52 weeks of treatment.

[0283] 5. The method of embodiment 2, wherein the subject's 6MWD is increased by at least 10 compared to a control treatment.

[0284] 6. The method of embodiment 5, wherein the subject's 6MWD improves by at least 13 meters after 52 weeks of treatment, compared to a control treatment.

[0285] 7. The method of any one of embodiments 2-6, wherein the subject has a baseline 6MWD of less than 300 meters.

[0286] 8. The method of any one of embodiments 2-6, wherein the subject has a baseline 6MWD of 300 meters or greater.

[0287] 9. The method of embodiment 1, which improves the subject's lung function as measured by a forced vital capacity (FVC) test.

[0288] 10. The method of embodiment 9, wherein after treatment, the subject's percent-predicted FVC is either increased compared to baseline or is decreased by less than 3% compared to baseline.

[0289] 11. The method of embodiment 10, wherein following treatment, the subject's percent-predicted FVC declines by less than 1% compared to baseline.

[0290] 12. The method of embodiment 10 or 11, wherein the subject's percent-predicted FVC declines by less than 1% compared to baseline after 52 weeks of treatment.

[0291] 13. The method of embodiment 9, wherein the subject's percent-predicted FVC significantly improves or stabilizes after treatment compared to a control treatment.

[0292] 14. The method of embodiment 13, wherein the subject's percent-predicted FVC improves by at least 3% after treatment compared to the control treatment.

[0293] 15. The method of embodiment 12 or 13, wherein the subject's percent-predicted FVC improves by at least 3% after 52 weeks of treatment compared to the control treatment.

[0294] 16. The method of any one of embodiments 9-15, wherein the subject has a baseline FVC of less than 55%.

[0295] 17. The method of any one of embodiments 9-15, wherein the subject has a baseline FVC of 55% or greater.

[0296] 18. The method of embodiment 1, which improves the subject's motor function as measured by the Walk-Stair-Gowers-Chair (GSGC) test.

[0297] 19. The method of embodiment 18, wherein the subject's GSGC score improves after treatment as indicated by a decrease of at least 0.5 points compared to baseline.

[0298] 20. The method of embodiment 19, wherein the subject's GSGC score improves as indicated by a decrease of at least 0.5 points after 52 weeks of treatment compared to baseline.

[0299] 21. The method of embodiment 18, wherein the subject's GSGC score is significantly improved after treatment compared to a control treatment.

[0300] 22. The method of embodiment 21, wherein the subject's GSGC score improves after treatment as indicated by a decrease of at least 1 point compared to the control treatment.

[0301] 23. The method of embodiment 21 or 22, wherein the subject's GSGC score improves as indicated by a reduction of at least 1 point after 52 weeks of treatment compared to the control treatment.

[0302] 24. The method of embodiment 1, wherein the level of at least one marker of muscle damage and / or at least one marker of glycogen accumulation is reduced.

[0303] 25. The method of embodiment 24, wherein at least one muscle damage marker comprises creatine kinase (CK) and / or at least one glycogen accumulation marker comprises urinary hexose tetrasaccharide (Hex4).

[0304] 26. The method of embodiment 25, wherein the subject's CK levels are reduced by at least 20% after treatment and / or the subject's urinary Hex4 levels are reduced by at least 30% after treatment compared to baseline.

[0305] 27. The method of embodiment 26, wherein the subject's CK levels are reduced by at least 20% after 52 weeks of treatment and / or the subject's urinary Hex4 levels are reduced by at least 30% after 52 weeks of treatment, compared to baseline.

[0306] 28. The method of embodiment 25, wherein the subject's CK and / or urinary Hex4 levels are significantly reduced after treatment compared to a control treatment.

[0307] 29. The method of embodiment 28, wherein the subject's CK levels are reduced by at least 30% after treatment and / or the subject's urinary Hex4 levels are reduced by at least 40% after treatment compared to a control treatment.

[0308] 30. The method of embodiment 28 or 29, wherein the subject's CK levels are reduced by at least 30% after 52 weeks of treatment and / or the subject's urinary Hex4 levels are reduced by at least 40% after 52 weeks of treatment compared to a control treatment.

[0309] 31. The method of any one of embodiments 1-30, wherein the subject is a patient with a history of ERT.

[0310] 32. The method of any one of embodiments 1-30, wherein the subject is an ERT-naive patient.

[0311] 33. A method of treating Pompe disease in a subject in need thereof, comprising administering to the subject a population of recombinant human acid alpha-glucosidase (rhGAA) molecules, either simultaneously or sequentially, with a pharmacological chaperone; The rhGAA molecule contains seven potential N-glycosylation sites; Between 40% and 60% of the N-glycans on the rhGAA molecule are complex-type N-glycans; the rhGAA molecule contains at least 0.5 moles of bis-mannose-6-phosphate (bis-M6P) per mole of rhGAA at the first potential N-glycosylation site as determined using liquid chromatography tandem mass spectrometry (LC-MS / MS); The method improves one or more disease symptoms in a subject as compared to 1) baseline, or (2) a control treatment comprising administering alglucosidase alfa and a pharmacological chaperone placebo; and Methods: The subjects are patients with a history of ERT.

[0312] 34. The method of embodiment 33, wherein the method improves the subject's motor function as measured by the 6-minute walk test.

[0313] 35. The method of embodiment 34, wherein the subject's 6-minute walking distance (6MWD) is increased by at least 15 meters or at least 5% after treatment compared to baseline.

[0314] 36. The method of embodiment 35, wherein the subject's 6-minute walking distance (6MWD) is increased by at least 15 meters or at least 4% after 52 weeks of treatment compared to baseline.

[0315] 37. The method of embodiment 33, wherein the subject's 6MWD is significantly improved after treatment compared to a control treatment.

[0316] 38. The method of embodiment 37, wherein the subject's 6MWD improves by at least 15 meters after treatment compared to a control treatment.

[0317] 39. The method of embodiment 37 or 38, wherein the subject's 6MWD improves by at least 15 meters after 52 weeks of treatment, compared to a control treatment.

[0318] 40. The method of any one of embodiments 34-39, wherein the subject has a baseline 6MWD of less than 300 meters.

[0319] 41. The method of any one of embodiments 34-39, wherein the subject has a baseline 6MWD of 300 meters or greater.

[0320] 42. The method of embodiment 33, which improves the subject's lung function as measured by a forced vital capacity (FVC) test.

[0321] 43. The method of embodiment 42, wherein after treatment, the subject's percent-to-predicted FVC is increased by at least 0.1% compared to baseline.

[0322] 44. The method of embodiment 43, wherein the subject's percent-predicted FVC is increased by at least 0.1% compared to baseline after 52 weeks of treatment.

[0323] 45. The method of embodiment 42, wherein the subject's percent-predicted FVC significantly improves after treatment compared to the control treatment.

[0324] 46. ​​The method of embodiment 45, wherein the subject's percent-predicted FVC improves by at least 4% after treatment compared to the control treatment.

[0325] 47. The method of embodiment 45 or 46, wherein the subject's percent-predicted FVC improves by at least 4% after 52 weeks of treatment compared to the control treatment.

[0326] 48. The method of any one of embodiments 42-47, wherein the subject has a baseline FVC of less than 55%.

[0327] 49. The method of any one of embodiments 42-47, wherein the subject has a baseline FVC of 55% or greater.

[0328] 50. The method of embodiment 33, which improves the subject's motor function as measured by the Walk-Stair-Gowers-Chair (GSGC) test.

[0329] 51. The method of embodiment 50, wherein the subject's GSGC score improves after treatment as indicated by a decrease of at least 0.5 points compared to baseline.

[0330] 52. The method of embodiment 51, wherein the subject's GSGC score improves as indicated by a decrease of at least 0.5 points after 52 weeks of treatment compared to baseline.

[0331] 53. The method of embodiment 50, wherein the subject's GSGC score is significantly improved after treatment compared to a control treatment.

[0332] 54. The method of embodiment 53, wherein the subject's GSGC score improves after treatment as indicated by a decrease of at least 1 point compared to the control treatment.

[0333] 55. The method of embodiment 53 or 54, wherein the subject's GSGC score improves as indicated by a reduction of at least 1 point after 52 weeks of treatment compared to the control treatment.

[0334] 56. The method of embodiment 33, wherein the level of at least one muscle damage marker and / or at least one glycogen accumulation marker is reduced.

[0335] 57. The method of embodiment 56, wherein at least one muscle damage marker comprises creatine kinase (CK) and / or at least one glycogen accumulation marker comprises urinary hexose tetrasaccharide (Hex4).

[0336] 58. The method of embodiment 57, wherein the subject's CK levels are reduced by at least 15% after treatment and / or the subject's urinary Hex4 levels are reduced by at least 25% after treatment compared to baseline.

[0337] 59. The method of embodiment 58, wherein the subject's CK levels are reduced by at least 15% after treatment and / or the subject's urinary Hex4 levels are reduced by at least 25% after 52 weeks of treatment compared to baseline.

[0338] 60. The method of embodiment 57, wherein the subject's CK and / or urinary Hex4 levels are significantly reduced after treatment compared to a control treatment.

[0339] 61. The method of embodiment 60, wherein the subject's CK levels are reduced by at least 30% after treatment and / or the subject's urinary Hex4 levels are reduced by at least 40% after treatment compared to a control treatment.

[0340] 62. The method of embodiment 60 or 61, wherein the subject's CK levels are reduced by at least 30% after treatment and / or the subject's urinary Hex4 levels are reduced by at least 40% after 52 weeks of treatment compared to a control treatment.

[0341] 63. The method of any one of embodiments 1 to 62, wherein the rhGAA molecule population is administered at a dose of 5 mg / kg to 20 mg / kg, optionally 20 mg / kg.

[0342] 64. The method of any one of embodiments 1 to 63, wherein the population of rhGAA molecules is administered every two weeks.

[0343] 65. The method of any one of embodiments 1 to 64, wherein the population of rhGAA molecules is administered intravenously.

[0344] 66. The method of any one of embodiments 1-65, wherein the pharmacological chaperone is miglustat or a pharma- ceutically acceptable salt thereof, and optionally wherein the miglustat or a pharma- ceutically acceptable salt thereof is administered orally.

[0345] 67. The method of embodiment 66, wherein miglustat or a pharma- ceutically acceptable salt thereof is administered in a dose of 195 mg or 260 mg.

[0346] 68. The method of embodiment 66 or 67, wherein miglustat or a pharma- ceutically acceptable salt thereof is administered prior to administration of the rhGAA molecule population, optionally 1 hour prior to administration of the rhGAA molecule population.

[0347] 69. The method of embodiment 68, wherein the subject fasts for at least 2 hours prior to and at least 2 hours after administration of miglustat or a pharma- ceutically acceptable salt thereof.

[0348] 70. The method of any one of embodiments 1 to 69, wherein the rhGAA molecule comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:4 or SEQ ID NO:6.

[0349] 71. The method of any one of embodiments 1 to 70, wherein the rhGAA molecule comprises the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 6.

[0350] 72. A method according to any one of embodiments 1 to 71, wherein at least 30% of the rhGAA molecules contain one or more N-glycan units carrying one mannose-6-phosphate residue (mono-M6P) or bis-M6P, as determined using LC-MS / MS.

[0351] 73. The method of any one of embodiments 1 to 72, wherein the rhGAA molecules contain, on average, 0.5 mol to 7.0 mol of mono-M6P or bis-M6P per mol of rhGAA as determined using LC-MS / MS.

[0352] 74. A method according to any one of embodiments 1 to 73, wherein the rhGAA molecules contain, on average, 2.0 to 8.0 moles of sialic acid per mole of rhGAA, as determined using LC-MS / MS.

[0353] 75. A method according to any one of embodiments 1 to 73, wherein the rhGAA molecules contain, on average, at least 2.5 mol of M6P per mol of rhGAA and at least 4 mol of sialic acid per mol of rhGAA, as determined using LC-MS / MS.

[0354] On average, rhGAA molecules are (a) 0.4–0.6 mol of mono-M6P at the second potential N-glycosylation site; (b) 0.4 to 0.6 moles of bis-M6P at the fourth potential N-glycosylation site; or (c) 0.3–0.4 mol of mono-M6P at the fourth potential N-glycosylation site Includes; The method of any one of embodiments 1-75, wherein (a)-(c) are determined using LC-MS / MS.

[0355] per 77.1 moles of rhGAA, the rhGAA molecule further comprises 4 moles to 7.3 moles of sialic acid; and On average, 1 mole of rhGAA has rhGAA molecules (a) 0.9–1.2 mol of sialic acid at the third potential N-glycosylation site; (b) 0.8 to 0.9 moles of sialic acid at the fifth potential N-glycosylation site; or (c) 1.5–4.2 mol of sialic acid at the sixth potential N-glycosylation site Includes; 77. The method of embodiment 76, wherein (a)-(c) are determined using LC-MS / MS.

[0356] 78. The method of any one of embodiments 1 to 77, wherein the population of rhGAA molecules is formulated into a pharmaceutical composition.

[0357] 79. The method of embodiment 78, wherein the pharmaceutical composition further comprises at least one buffer selected from the group consisting of citrate, phosphate, and combinations thereof, and at least one excipient selected from the group consisting of mannitol, polysorbate 80, and combinations thereof; and the pharmaceutical composition has a pH of 5.0 to 7.0.

[0358] 80. The method according to embodiment 79, wherein the pharmaceutical composition has a pH of 5.0 to 6.0.

[0359] 81. The method of embodiment 78 or 79, wherein the pharmaceutical composition further comprises water, an acidifying agent, an alkalizing agent, or a combination thereof.

[0360] 82. The method of embodiment 81, wherein in the pharmaceutical composition, the rhGAA molecule population is present at a concentration of 5-50 mg / mL, the at least one buffer is a sodium citrate buffer present at a concentration of 10-100 mM, the at least one excipient is mannitol present at a concentration of 10-50 mg / mL and polysorbate 80 present at a concentration of 0.1-1 mg / mL, and the pharmaceutical composition further comprises water, and optionally an acidifying agent and / or an alkalizing agent; and the pharmaceutical composition has a pH of 6.0.

[0361] 83. The method of embodiment 82, wherein in the pharmaceutical composition, the rhGAA molecule population is present at a concentration of 15 mg / mL, the sodium citrate buffer is present at a concentration of 25 mM, the mannitol is present at a concentration of 20 mg / mL, and the polysorbate 80 is present at a concentration of 0.5 mg / mL.

[0362] 84. The method of any one of embodiments 1 to 83, wherein rhGAA is produced from Chinese hamster ovary cells.

Claims

1. A pharmaceutical for use in a method of treating Pompe disease in a subject in need thereof comprising a population of recombinant human acid alpha-glucosidase (rhGAA) molecules, said method comprising administering to said subject a population of recombinant human acid alpha-glucosidase (rhGAA) molecules simultaneously or sequentially with a pharmacological chaperone; the rhGAA molecule contains seven potential N-glycosylation sites; at least 3% of the total N-glycans on the rhGAA are bis-mannose-6-phosphate (bis-M6P); The method improves one or more disease outcomes in the subject compared to 1) baseline, or (2) a control treatment comprising administering alglucosidase alfa and a placebo of the pharmacological chaperone.

2. The method improves motor function of the subject as measured by a 6-minute walk test; Optionally, (a) a change from baseline in 6-minute walk distance (6MWD) of at least 20 meters; More optionally, said change from baseline in 6MWD is at least 20 meters after 52 weeks of treatment; or (b) the subject's 6MWD increases by at least 10 compared to the control treatment; Further optionally, the subject's 6MWD improves by at least 13 meters after 52 weeks of treatment, compared to the control treatment. The pharmaceutical composition according to claim 1.

3. The method of claim 2, wherein the subject has a baseline 6MWD of less than 300 meters.

4. The method of claim 2, wherein the subject has a baseline 6MWD of 300 meters or greater.

5. The method of claim 1, wherein the method improves pulmonary function of the subject as measured by a forced vital capacity (FVC) test; Optionally, (a) following treatment, the subject's percent-predicted FVC is either increased compared to baseline or is decreased by less than 3% compared to baseline; Further optionally, following treatment, the subject's percent-predicted FVC is reduced by less than 1% compared to baseline; and / or Further optionally, the subject's percent-predicted FVC declines by less than 1% compared to baseline after 52 weeks of treatment; or (b) the subject's percent-predicted FVC significantly improves or stabilizes after treatment compared to the control treatment; Further optionally, the subject's percent-predicted FVC improves by at least 3% after treatment compared to the control treatment; Further optionally, the subject's percent-predicted FVC improves by at least 3% after 52 weeks of treatment, compared to the control treatment. The pharmaceutical composition according to claim 1.

6. The method of claim 5, wherein the subject has a baseline FVC of less than 55%.

7. The method of claim 5, wherein the subject has a baseline FVC of 55% or greater.

8. The method of claim 7, further comprising: improving motor function of the subject as measured by the Walking Stairs and Gowers Chair (GSGC) test. Optionally, (a) the subject's GSGC score improves as indicated by a reduction of at least 0.5 points after treatment compared to baseline; Further optionally, the subject's GSGC score improves as indicated by a reduction of at least 0.5 points after 52 weeks of treatment, compared to baseline; or (b) the subject's GSGC score is significantly improved after treatment compared to the control treatment; Further optionally, the subject's GSGC score improves as indicated by a decrease of at least one point after treatment compared to the control treatment, and / or Further optionally, the subject's GSGC score improves as indicated by a reduction of at least one point after 52 weeks of treatment, as compared to the control treatment. The pharmaceutical composition according to claim 1.

9. The pharmaceutical described in claim 1, wherein the method reduces the level of at least one muscle damage marker and / or at least one glycogen accumulation marker.

10. the at least one muscle damage marker comprises creatine kinase (CK) and / or the at least one glycogen accumulation marker comprises urinary hexose tetrasaccharide (Hex4); Optionally, (a) the subject's CK levels are decreased by at least 20% after treatment, and / or the subject's urinary Hex4 levels are decreased by at least 30% after treatment, compared to baseline; Further optionally, the subject's CK levels are reduced by at least 20% after 52 weeks of treatment, and / or the subject's urinary Hex4 levels are reduced by at least 30% after 52 weeks of treatment, compared to baseline; or (b) the subject's CK and / or urinary Hex4 levels are significantly decreased after treatment compared to the control treatment; Further optionally, the subject's CK levels are reduced by at least 30% after treatment, and / or the subject's urinary Hex4 levels are reduced by at least 40% after treatment, compared to the control treatment, and / or Further optionally, the subject's CK levels are reduced by at least 30% after 52 weeks of treatment, and / or the subject's urinary Hex4 levels are reduced by at least 40% after 52 weeks of treatment, compared to the control treatment. The pharmaceutical composition according to claim 9.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the subject is a patient with a history of ERT.

12. The pharmaceutical composition according to any one of claims 1 to 10, wherein the subject is an ERT-naive patient.

13. (a) the population of rhGAA molecules is administered at a dose of 5 mg / kg to 20 mg / kg, optionally 20 mg / kg; and / or (b) the population of rhGAA molecules is administered every other week; and / or (c) the population of rhGAA molecules is administered intravenously; The pharmaceutical composition according to any one of claims 1 to 12.

14. the pharmacological chaperone is miglustat or a pharma- ceutically acceptable salt thereof, and optionally, the miglustat or a pharma- ceutically acceptable salt thereof is administered orally; Optionally, the miglustat or a pharma- ceutically acceptable salt thereof is administered in a dose of 195 mg or 260 mg. The pharmaceutical composition according to any one of claims 1 to 13.

15. 15. The pharmaceutical composition of claim 14, wherein the miglustat or a pharma- ceutically acceptable salt thereof is administered prior to administration of the population of rhGAA molecules, optionally 1 hour prior to administration of the population of rhGAA molecules, and optionally the subject fasts for at least 2 hours prior to and at least 2 hours after administration of miglustat or a pharma- ceutically acceptable salt thereof.

16. the rhGAA molecule comprises an amino acid sequence at least 95% identical to SEQ ID NO:4 or SEQ ID NO:6; and / or The rhGAA molecule comprises the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:

6. The pharmaceutical composition according to any one of claims 1 to 15. (a) at least 30% of the rhGAA molecules contain one or more N-glycan units bearing one mannose-6-phosphate residue (mono-M6P) or bis-M6P, as determined by LC-MS / MS; and / or (b) the rhGAA molecules contain, on average, between 0.5 mol and 7.0 mol of mono-M6P or bis-M6P per mol of rhGAA as determined using LC-MS / MS; and / or (c) the rhGAA molecules contain, on average, 2.0-8.0 mol of sialic acid per mol of rhGAA as determined using LC-MS / MS, or the rhGAA molecules contain, on average, at least 2.5 mol of M6P per mol of rhGAA and at least 4 mol of sialic acid per mol of rhGAA as determined using LC-MS / MS; and / or (d) per mole of rhGAA, the rhGAA molecules are (i) 0.4-0.6 mol of mono-M6P at the second potential N-glycosylation site; (ii) 0.4 to 0.6 mol of bis-M6P at the fourth potential N-glycosylation site; or (iii) 0.3 to 0.4 mol of mono-M6P at the fourth potential N-glycosylation site Including; (i) to (iii) are determined using LC-MS / MS; Optionally, per mol of rhGAA, the rhGAA molecule further comprises 4 mol to 7.3 mol of sialic acid; and Per 1 mol of rhGAA, the rhGAA molecules are (iv) 0.9 to 1.2 mol of sialic acid at the third potential N-glycosylation site; (v) 0.8 to 0.9 mol of sialic acid at the fifth potential N-glycosylation site; or (vi) 1.5 to 4.2 mol of sialic acid at the sixth potential N-glycosylation site Including; (iv) to (vi) are determined using LC-MS / MS; The pharmaceutical composition according to any one of claims 1 to 16.

18. (a) the population of rhGAA molecules is formulated into a pharmaceutical composition; (b) optionally, 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; the pharmaceutical composition has a pH of 5.0 to 7.0; (c) further optionally, the pharmaceutical composition has a pH of 5.0 to 6.

0. The pharmaceutical composition according to any one of claims 1 to 17.

19. 19. The pharmaceutical composition according to claim 18, wherein in (a) or (b), the pharmaceutical composition further comprises water, an acidifying agent, an alkalizing agent, or a combination thereof; Optionally, in the pharmaceutical composition, the population of rhGAA molecules is present at a concentration of 5-50 mg / mL, the at least one buffer is a sodium citrate buffer present at a concentration of 10-100 mM, the at least one excipient is mannitol present at a concentration of 10-50 mg / mL and polysorbate 80 present at a concentration of 0.1-1 mg / mL, the pharmaceutical composition further comprises water, and optionally an acidifying agent and / or an alkalizing agent; the pharmaceutical composition has a pH of 6.0; Further optionally, the population of rhGAA molecules is present in a concentration of 15 mg / mL, the sodium citrate buffer is present in a concentration of 25 mM, the mannitol is present in a concentration of 20 mg / mL, and the polysorbate 80 is present in a concentration of 0.5 mg / mL in the pharmaceutical composition. Medicine.

20. The pharmaceutical of any one of claims 1 to 19, wherein the rhGAA is produced from Chinese hamster ovary cells.

21. The pharmaceutical of any one of claims 1 to 20, wherein the rhGAA molecule contains at least 0.5 mol of bis-mannose-6-phosphate (bis-M6P) per mol of rhGAA at the first potential N-glycosylation site as determined using liquid chromatography tandem mass spectrometry (LC-MS / MS).

22. The pharmaceutical composition of any one of claims 1 to 21, wherein the rhGAA contains, on average, at least 1 mol of bis-M6P per mol of rhGAA.

23. The pharmaceutical composition of any one of claims 1 to 22, wherein the rhGAA contains, on average, at least 1.3 mol of bis-M6P per mol of rhGAA.

24. The pharmaceutical composition of any one of claims 1 to 23, wherein the rhGAA contains at least 3 mol to at least 81 mol of sialic acid residues per mol of rhGAA.

25. The pharmaceutical composition of any one of claims 1 to 24, wherein the rhGAA contains at least 4 mol of sialic acid residues per mol of rhGAA.

26. The pharmaceutical composition of claim 1, wherein at least 17% of the total N-glycans on the rhGAA are bis-M6P.

27. The pharmaceutical composition of any one of claims 1 to 26, wherein at least 3% to at least 17% of the total N-glycans on the rhGAA are bis-M6P.

28. The pharmaceutical composition of any one of claims 1 to 27, wherein at least 3% to at least 25% of the total N-glycans on the rhGAA are bis-M6P.

29. The pharmaceutical composition of any one of claims 1 to 28, wherein at least 17% to at least 25% of the total N-glycans on the rhGAA are bis-M6P.

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