Compositions and methods for treating pompe disease

Abstract

To provide compositions and methods for treating Pompe disease.SOLUTION: A method for treating Pompe disease such as infantile-onset Pompe disease (IOPD) using a pharmaceutical composition comprising an oligosaccharide-acid α-glucosidase (GAA) conjugate such as avalglucosidase alpha is provided. Also provided are formulations of the oligosaccharide-GAA conjugates.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 971,930, filed February 8, 2020, and U.S. Provisional Patent Application No. 63 / 115,975, filed November 19, 2020, both of which are incorporated by reference in their entireties.

[0002] The present application relates to compositions and methods for treating Pompe disease, such as infantile-onset Pompe disease (IOPD). [Background technology]

[0003] Pompe disease (also known as glycogen storage disease type II, or GSD-II) is a genetically inherited lysosomal storage disorder in which deficiency of the glycogen-degrading enzyme acid α-glucosidase (GAA) leads to glycogen accumulation in lysosomes, resulting in muscle weakness and cardiac hypertrophy (Non-Patent Document 1, Non-Patent Document 2). The most severe form of Pompe disease is infantile-onset Pompe disease (IOPD), in which symptoms begin early in life and the disease progresses very rapidly: IOPD symptoms begin at a median age of 2 months, and if left untreated, patients die at a median age of 8.7 months (Non-Patent Document 3, Non-Patent Document 1).

[0004] Enzyme replacement therapy (ERT) using recombinant GAA has been developed to treat IOPD (e.g., Non-Patent Document 4). Nevertheless, IOPD is a difficult disease to treat, and there is still a need to improve the long-term prognosis and alleviate symptoms of IOPD patients (Non-Patent Document 5, Non-Patent Document 3). Therefore, there is a need in the art for methods and compositions for treating Pompe disease, particularly for treating IOPD. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Chien, Y. and Hwu, W. Biologics 2007 1:3 [Non-patent document 2] Kishnani, PS et al. Genetics in Medicine 2006 8 [Non-patent document 3] Chien, Y. et al., The Journal of Pediatrics 2015, 166:4 [Non-patent document 4] Kishnani, PS et al., Neurology 2007, 68:2 [Non-patent document 5] Prater, SN et al. Genet. Med. 2012, 14:9 Summary of the Invention [Problem to be solved by the invention]

[0006] The present application provides methods and compositions for treating Pompe disease, such as IOPD, in an individual in need thereof. Also provided are compositions and formulations for use in treating Pompe disease, such as IOPD, and the use of the compositions and formulations in the manufacture of a medicament for treating Pompe disease, such as IOPD. [Means for solving the problem]

[0007] One aspect of the present application is a method for treating infantile-onset Pompe disease (IWD) comprising administering to a human individual in need thereof a pharmaceutical composition comprising an oligosaccharide-protein conjugate and a pharmaceutically acceptable carrier. The present invention provides a method for treating IOPD, wherein the oligosaccharide-protein conjugate has the structure of Formula I: [ka] wherein GAA is acid α-glucosidase, L is a chemical linker connecting the oligosaccharide and GAA, and n is 1 to 10, and the pharmaceutical composition is administered at a dose of about 20 mg / kg to about 40 mg / kg. In some embodiments, the pharmaceutical composition is administered at a dose of about 20 mg / kg. In some embodiments, the pharmaceutical composition is administered at a dose of about 40 mg / kg. In some embodiments, the pharmaceutical composition is administered to an individual once every two weeks. In some embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the pharmaceutical composition is administered to an individual for at least about 25 weeks.

[0008] In some embodiments of any one of the above methods, the pharmaceutical composition is reconstituted from a lyophilized formulation comprising an oligosaccharide-protein conjugate. In some embodiments, the pharmaceutically acceptable carrier comprises a sugar that is not degraded by GAA. In some embodiments, the sugar that is not degraded by GAA is mannitol. In some embodiments, the pharmaceutically acceptable carrier further comprises glycine. In some embodiments, the pharmaceutically acceptable carrier comprises histidine. In some embodiments, the pharmaceutical composition has a pH of about 6.2. In some embodiments, the pharmaceutically acceptable carrier comprises about 10-50 mM histidine, about 0.25-2% glycine, about 1-4% mannitol, and about 0.005-0.05% polysorbate 80. In some embodiments, the pharmaceutically acceptable carrier comprises about 10 mM histidine, about 2% glycine, about 2% mannitol, and about 0.01% polysorbate 80.

[0009] In some embodiments according to any one of the above methods, the individual has cardiomyopathy at the time of diagnosis within the first year of life. In some embodiments, the individual has arrhythmia. In some embodiments, the individual has cardiac hypertrophy. In some embodiments, the individual is 18 years of age or younger. In some embodiments, the individual is about 6 months of age or younger.

[0010] In some embodiments of any one of the above methods, the individual has been treated with recombinant GAA for at least 6 months. In some embodiments, the individual shows clinical attenuation after treatment with recombinant GAA, wherein the clinical attenuation is measured by measuring respiratory function, exercise capacity, and cardiac parameters. In some embodiments, the individual has a suboptimal clinical response in response to treatment with recombinant GAA, and the clinical response is determined by assessing one or more parameters selected from the group consisting of respiratory function, exercise capacity, and cardiac parameters. In some embodiments, the individual has not received treatment with recombinant GAA. In some embodiments, the recombinant GAA is alglucosidase alfa.

[0011] In some embodiments according to any one of the above methods, the individual is cross-reactive immune material (CRIM) negative. In some embodiments, the individual is CRIM positive.

[0012] In some embodiments of any one of the above methods, the method further comprises administering to the individual an effective amount of methotrexate. In some embodiments, the effective amount of methotrexate is administered in a single cycle or three cycles. In some embodiments, methotrexate is administered in a single cycle. In some embodiments, methotrexate is administered in two, three, four, five, or more cycles. In some embodiments, one cycle of methotrexate consists of one day of methotrexate administration or two, three, four, five, six, seven, eight, nine, ten, or eleven consecutive days of methotrexate administration. In some embodiments, methotrexate is administered to the individual at a time selected from one or more of before, during, and after administration of the oligosaccharide-protein conjugate. In some embodiments, methotrexate is administered between 48 hours before and 48 hours after administration of the oligosaccharide-protein conjugate. In some embodiments, methotrexate is administered simultaneously with administration of the oligosaccharide-protein conjugate, and about 24 hours and about 48 hours after administration of the oligosaccharide-protein conjugate. In some embodiments, methotrexate is administered at about 0.1 mg / kg to about 5 mg / kg. In some embodiments, the method further comprises administering to the individual an additional immune tolerance induction therapy. In some embodiments, the additional immune tolerance induction therapy comprises rituximab and intravenous immunoglobulin (IVIG).

[0013] In some embodiments according to any one of the above methods, the individual has a decreased level of anti-drug antibodies (ADA) against the oligosaccharide-protein conjugate over time.

[0014] In some embodiments of any one of the aforementioned methods, the individual's creatine kinase (CK) levels are reduced by at least about 100 IU / L when measured after at least about 25 weeks of treatment.

[0015] In some embodiments of any one of the aforementioned methods, the individual's urinary hexose tetrasaccharide (Hex4) levels are reduced by at least about 10 mmol / mol when measured after at least about 25 weeks of treatment.

[0016] In some embodiments of any one of the aforementioned methods, the individual's Gross Motor Function Scale (GMFM-88) score is increased by at least 5% when measured after at least about 25 weeks of treatment.

[0017] In some embodiments of any one of the above-described methods, the individual exhibits improvement or stabilization of one or more parameters selected from the group consisting of respiratory function, motor capacity, cardiac parameters, and eyelid position. In some embodiments, the individual exhibits improvement or stabilization of one or more parameters selected from the group consisting of Alberta Infant Motor Development Scale (AIMS) score, Pompe-Pediatric Assessment of Disability (PEDI) Functional Capacity Scale, Echocardiogram (ECHO)-Left Ventricular Mass (LVM) Z-score, ECHO LVMI score, Gross Motor Function Classification System-Extended and Revised (GMFCS-E&R) score, Brief Motor Function Test, 6-Minute Walk Test (6MWT), Interpalpebral Fissure Diameter (IPFD), Eyelid Margin Corneal Reflection Distance-1 (MRD-1), Eyelid Margin Pupillary Distance (MPD), development of ptosis, and use of respiratory support. In some embodiments, improvement or stabilization is determined based on one or more parameters selected from the group consisting of: (a) a score for the Pompe-PEDI Functional Capacity Scale; (b) a score for the ECHO-LVM Z-score; (c) a score for the ECHO-LVM Z-score;

[0018] In some embodiments according to any one of the methods described above, the oligosaccharide-protein conjugate has the structure of Formula II: [ka] wherein GAA is acid α-glucosidase, L′ is a chemical linker, and n is 1-10.

[0019] In some embodiments according to any one of the methods described above, the oligosaccharide-protein conjugate has the structure of Formula III: [ka] wherein GAA is acid α-glucosidase, n is 1 to 10, and m and p are independently selected from integers ranging from 1 to 10. In some embodiments, m is 3 and p is 1. In some embodiments, n is 5 to 7.

[0020] In some embodiments according to any one of the methods described above, the GAA is In some embodiments, the human GAA is produced in human ovarian (CHO) cells. In some embodiments, the human GAA has glycoform alpha. In some embodiments, the oligosaccharide-protein conjugate is avalglucosidase alpha.

[0021] Another aspect of the present application provides a formulation comprising: (a) an oligosaccharide-protein conjugate; and (b) one or more cryoprotectants comprising sugars that are not degraded by acid-alpha glucosidase; the oligosaccharide-protein conjugate has the structure of Formula I: [ka] wherein GAA is acid α-glucosidase, L is a chemical linker, and n is 1 to 10. In some embodiments, the sugar is mannitol. In some embodiments, the formulation includes about 1 to 4% (w / w) mannitol, such as about 2% mannitol.

[0022] In some embodiments of any one of the above formulations, the one or more cryoprotectants further comprise an amino acid. In some embodiments, the amino acid is glycine. In some embodiments, the formulation comprises about 0.25-2% (w / w) glycine, such as about 2% glycine.

[0023] In some embodiments of any one of the above formulations, the one or more cryoprotectants further comprise a surfactant. In some embodiments, the surfactant is polysorbate 80. In some embodiments, the formulation comprises about 0.005-0.05% (w / w) polysorbate 80, such as about 0.01% polysorbate 80.

[0024] In some embodiments of any one of the above formulations, the formulation further comprises a buffering agent. In some embodiments, the buffering agent is histidine. In some embodiments, the formulation comprises about 10-50 mM histidine, such as about 10 mM histidine.

[0025] In some embodiments of any one of the above formulations, the formulation comprises about 10 mM histidine, about 2% glycine, about 2% mannitol, and about 0.01% polysorbate 80.

[0026] In some embodiments of any one of the above formulations, the formulation is a lyophilized formulation. In some embodiments, the formulation is a pre-lyophilized (i.e., lyophilizable) formulation. In some embodiments, the formulation is a reconstituted liquid formulation. In some embodiments, the pH of the formulation is about 5.5 to about 6.5. In some embodiments, the pH of the formulation is about 6.2.

[0027] In some embodiments of any one of the above formulations, the formulation comprises about 5-10 mg / mL (eg, about 5 mg / mL) of oligosaccharide-protein conjugate.

[0028] In some embodiments according to any one of the above formulations, the oligosaccharide-protein conjugate has the structure of Formula II: [ka] wherein GAA is acid α-glucosidase, L′ is a chemical linker, and n is 1-10.

[0029] In some embodiments according to any one of the above formulations, the oligosaccharide-protein conjugate has the structure of Formula III: [ka] wherein GAA is acid α-glucosidase, n is 1 to 10, and m and p are independently selected from integers ranging from 1 to 10. In some embodiments, m is 3 and p is 1. In some embodiments, n is 5 to 7.

[0030] In some embodiments of any one of the above formulations, the GAA is human GAA produced in Chinese hamster ovary (CHO) cells. AA has the glycoform alpha. In some embodiments, the oligosaccharide-protein conjugate is avalglucosidase alpha.

[0031] Another aspect of the present application provides an article of manufacture comprising a container containing a formulation according to any one of the formulations described above. In some embodiments, the container is a vial. In some embodiments, the formulation is a lyophilized formulation.

[0032] Further provided is a method of treating Pompe disease, comprising administering to a human individual in need thereof an effective amount of a pharmaceutical composition comprising a formulation according to any one of the above-described formulations. In some embodiments, the Pompe disease is IOPD.

[0033] Also provided are kits containing a formulation according to any one of the above formulations. In some embodiments, the kit further comprises instructions for use in treating Pompe disease (e.g., IOPD). [Brief explanation of the drawings]

[0034] [Figure 1]

[0023] Figure 1 provides a schematic overview of the study design. Cohort 1 is shown in blue, Cohort 2 in orange, and the two arms of Cohort 3 in pink and green. Time from baseline is shown from left to right. [Figure 2]Figures 2A-2B show the change in the Gross Motor Function Scale-88 (GMFM-88) total percent score compared to baseline. Figure 2A shows the GMFM-88 scores for Cohort 1 (blue) and Cohort 2 (orange) during the primary analysis period. Figure 2B shows the GMFM-88 scores for Cohort 3, who received alglucosidase alfa followed by either avalglucosidase alfa (pink) or alglucosidase alfa (green) at 25 weeks during the primary analysis period. In Figure 2B, * indicates that GMFM-88 was not measured for one patient with GMFCS level V. In Figures 2A-2B, the x-axis shows the amount of time in weeks from baseline, and the y-axis shows the change from baseline (mean ± standard deviation). [Figure 3] 1 shows the Pompe Pediatric Disability Assessment Inventory (Pompe-PEDI) Functional Disability Scale: Scaled scores over time for the mobility domain by patient age. Patient age at visit is shown on the x-axis and scaled scores are shown on the y-axis. Individuals in Cohort 1 are shown in blue, individuals in Cohort 2 are shown in orange, individuals in Cohort 3 receiving alglucosidase alfa followed by avalglucosidase alfa at 25 weeks are shown in pink, and individuals in Cohort 3 receiving alglucosidase alfa are shown in green. [Figure 4]Figures 4A-4B show the change in creatine kinase (CK) levels over time compared to baseline. Figure 4A shows the change in CK levels for Cohort 1 (blue) and Cohort 2 (orange) during the primary analysis period. Figure 4B shows the change in CK levels for Cohort 3 receiving alglucosidase alfa followed by either avalglucosidase alfa (pink) or alglucosidase alfa (green) at 25 weeks during the primary analysis period. In Figures 4A-4B, the x-axis shows the amount of time in weeks from baseline, and the y-axis shows the change from baseline (median). The CK levels at baseline in IU / L for the different cohorts were as follows: Cohort 1 mean: 1102.17, standard deviation: 932.43, median: 750, minimum: 318.0, and maximum: 2607.0; Cohort 2 mean: 1444.80, standard deviation: 164.17, median: 1472, minimum: 1188.0, maximum: 1645.0; Cohort 3 receiving avalglucosidase alfa mean: 1211.40, standard deviation: 597.45, median: 1528, minimum: 347.0, and maximum: 1704.0; and Cohort 3 receiving avalglucosidase alfa followed by avalglucosidase alfa at 25 weeks. Mean: 1136.17, standard deviation: 672.61, median: 1179, minimum: 273.0, and maximum: 1830.0. [Figure 5]Figures 5A-5B show the change in HEX4 levels over time compared to baseline. Figure 5A shows the change in HEX4 levels for Cohort 1 (blue) and Cohort 2 (orange) during the primary analysis period. Figure 5B shows the change in HEX4 levels for Cohort 3, who received alglucosidase alfa followed by either avalglucosidase alfa (pink) or alglucosidase alfa (green) at 25 weeks during the primary analysis period. In Figures 5A-5B, the x-axis shows the amount of time in weeks from baseline, and the y-axis shows the change from baseline (median). The HEX4 levels at baseline in mmol / mol for the different cohorts were as follows: Cohort 1, mean: 80.25, standard deviation: 48.38, median: 73.12, minimum: 16.1, and maximum: 143.3; Cohort 2, mean: 63.43, standard deviation: 30.71, median: 71.31, minimum: 20.0, maximum: 97.3; Cohort 3, which received avalglucosidase alfa, mean: 54.81, standard deviation: 50.41, median: 42.54, minimum: 11.9, and maximum: 141.0; and Cohort 3, which received avalglucosidase alfa followed by avalglucosidase alfa at 25 weeks, mean: 52.16, standard deviation: 33.93, median: 69.17, minimum: 4.2, and maximum: 80.6. [Figure 6] 6A to 6D are diagrams showing measurement of eyelid position. [Figure 7A] These figures show the mean ± standard deviation (SD) from baseline at 13 and 25 weeks for the interpalpebral fissure diameter (IPFD; Figure 7A: right eye; Figure 7B: left eye), lid margin corneal reflex distance-1 (MRD-1; Figure 7C: right eye; Figure 7D: left eye), and lid margin pupillary distance (MPD; Figure 7E: right eye; Figure 7F: left eye). [Figure 7B] These figures show the mean ± standard deviation (SD) from baseline at 13 and 25 weeks for the interpalpebral fissure diameter (IPFD; Figure 7A: right eye; Figure 7B: left eye), lid margin corneal reflex distance-1 (MRD-1; Figure 7C: right eye; Figure 7D: left eye), and lid margin pupillary distance (MPD; Figure 7E: right eye; Figure 7F: left eye). [Figure 7C]These figures show the mean ± standard deviation (SD) from baseline at 13 and 25 weeks for the interpalpebral fissure diameter (IPFD; Figure 7A: right eye; Figure 7B: left eye), lid margin corneal reflex distance-1 (MRD-1; Figure 7C: right eye; Figure 7D: left eye), and lid margin pupillary distance (MPD; Figure 7E: right eye; Figure 7F: left eye). [Figure 7D] These figures show the mean ± standard deviation (SD) from baseline at 13 and 25 weeks for the interpalpebral fissure diameter (IPFD; Figure 7A: right eye; Figure 7B: left eye), lid margin corneal reflex distance-1 (MRD-1; Figure 7C: right eye; Figure 7D: left eye), and lid margin pupillary distance (MPD; Figure 7E: right eye; Figure 7F: left eye). [Figure 7E] These figures show the mean ± standard deviation (SD) from baseline at 13 and 25 weeks for the interpalpebral fissure diameter (IPFD; Figure 7A: right eye; Figure 7B: left eye), lid margin corneal reflex distance-1 (MRD-1; Figure 7C: right eye; Figure 7D: left eye), and lid margin pupillary distance (MPD; Figure 7E: right eye; Figure 7F: left eye). [Figure 7F] These figures show the mean ± standard deviation (SD) from baseline at 13 and 25 weeks for the interpalpebral fissure diameter (IPFD; Figure 7A: right eye; Figure 7B: left eye), lid margin corneal reflex distance-1 (MRD-1; Figure 7C: right eye; Figure 7D: left eye), and lid margin pupillary distance (MPD; Figure 7E: right eye; Figure 7F: left eye). [Figure 8A] Figure 8A shows the change from baseline to week 25 in hexose tetrasaccharide (Hex4) ordered by cohort (Figure 8A) and by baseline values ​​(Figure 8B), and in creatine kinase (CK) ordered by cohort (Figure 8C) and by baseline values ​​(Figure 8D). 1: Cohort 1 (avalglucosidase alfa 20 mg / kg every other week); 2: Cohort 2 (avalglucosidase alfa 40 mg / kg every other week); 3: Cohort 3 (avalglucosidase alfa 40 mg / kg every other week); and 4: Cohort 3 (avalglucosidase alfa 20 mg / kg every other week to 40 mg / kg every week). [Figure 8B]Figure 8A shows the change from baseline to week 25 in hexose tetrasaccharide (Hex4) ordered by cohort (Figure 8A) and by baseline values ​​(Figure 8B), and in creatine kinase (CK) ordered by cohort (Figure 8C) and by baseline values ​​(Figure 8D). 1: Cohort 1 (avalglucosidase alfa 20 mg / kg every other week); 2: Cohort 2 (avalglucosidase alfa 40 mg / kg every other week); 3: Cohort 3 (avalglucosidase alfa 40 mg / kg every other week); and 4: Cohort 3 (avalglucosidase alfa 20 mg / kg every other week to 40 mg / kg every week). [Figure 8C] Figure 8A shows the change from baseline to week 25 in hexose tetrasaccharide (Hex4) ordered by cohort (Figure 8A) and by baseline values ​​(Figure 8B), and in creatine kinase (CK) ordered by cohort (Figure 8C) and by baseline values ​​(Figure 8D). 1: Cohort 1 (avalglucosidase alfa 20 mg / kg every other week); 2: Cohort 2 (avalglucosidase alfa 40 mg / kg every other week); 3: Cohort 3 (avalglucosidase alfa 40 mg / kg every other week); and 4: Cohort 3 (avalglucosidase alfa 20 mg / kg every other week to 40 mg / kg every week). [Figure 8D] Figure 8A shows the change from baseline to week 25 in hexose tetrasaccharide (Hex4) ordered by cohort (Figure 8A) and by baseline values ​​(Figure 8B), and in creatine kinase (CK) ordered by cohort (Figure 8C) and by baseline values ​​(Figure 8D). 1: Cohort 1 (avalglucosidase alfa 20 mg / kg every other week); 2: Cohort 2 (avalglucosidase alfa 40 mg / kg every other week); 3: Cohort 3 (avalglucosidase alfa 40 mg / kg every other week); and 4: Cohort 3 (avalglucosidase alfa 20 mg / kg every other week to 40 mg / kg every week). [Figure 9A]Figure 9 shows the change from baseline to week 25 in the Gross Motor Function Scale-88 (GMFM-88) ordered by cohort (Figure 9A) and by baseline values ​​(Figure 9B), and the Quick Motor Function Test (QMFT) ordered by cohort (Figure 9C) and by baseline values ​​(Figure 9D). 1: Cohort 1 (avalglucosidase alfa 20 mg / kg every other week); 2: Cohort 2 (avalglucosidase alfa 40 mg / kg every other week); 3: Cohort 3 (avalglucosidase alfa 40 mg / kg every other week); and 4: Cohort 3 (avalglucosidase alfa 20 mg / kg every other week to 40 mg / kg every week). [Figure 9B] Figure 9 shows the change from baseline to week 25 in the Gross Motor Function Scale-88 (GMFM-88) ordered by cohort (Figure 9A) and by baseline values ​​(Figure 9B), and the Quick Motor Function Test (QMFT) ordered by cohort (Figure 9C) and by baseline values ​​(Figure 9D). 1: Cohort 1 (avalglucosidase alfa 20 mg / kg every other week); 2: Cohort 2 (avalglucosidase alfa 40 mg / kg every other week); 3: Cohort 3 (avalglucosidase alfa 40 mg / kg every other week); and 4: Cohort 3 (avalglucosidase alfa 20 mg / kg every other week to 40 mg / kg every week). [Figure 9C] Figure 9 shows the change from baseline to week 25 in the Gross Motor Function Scale-88 (GMFM-88) ordered by cohort (Figure 9A) and by baseline values ​​(Figure 9B), and the Quick Motor Function Test (QMFT) ordered by cohort (Figure 9C) and by baseline values ​​(Figure 9D). 1: Cohort 1 (avalglucosidase alfa 20 mg / kg every other week); 2: Cohort 2 (avalglucosidase alfa 40 mg / kg every other week); 3: Cohort 3 (avalglucosidase alfa 40 mg / kg every other week); and 4: Cohort 3 (avalglucosidase alfa 20 mg / kg every other week to 40 mg / kg every week). [Figure 9D] Figure 9 shows the change from baseline to week 25 in the Gross Motor Function Scale-88 (GMFM-88) ordered by cohort (Figure 9A) and by baseline values ​​(Figure 9B), and the Quick Motor Function Test (QMFT) ordered by cohort (Figure 9C) and by baseline values ​​(Figure 9D). 1: Cohort 1 (avalglucosidase alfa 20 mg / kg every other week); 2: Cohort 2 (avalglucosidase alfa 40 mg / kg every other week); 3: Cohort 3 (avalglucosidase alfa 40 mg / kg every other week); and 4: Cohort 3 (avalglucosidase alfa 20 mg / kg every other week to 40 mg / kg every week). [Figure 10A] FIG. 1 shows Gross Motor Function Scale-88 item (GMFM-88) performance demonstrated after initiation of avalglucosidase alfa treatment for retrospective chart data and individual participants from a 3-case study. [Figure 10B] FIG. 1 shows Gross Motor Function Scale-88 item (GMFM-88) performance demonstrated after initiation of avalglucosidase alfa treatment for retrospective chart data and individual participants from a 3-case study. [Figure 10C] FIG. 1 shows Gross Motor Function Scale-88 item (GMFM-88) performance demonstrated after initiation of avalglucosidase alfa treatment for retrospective chart data and individual participants from a 3-case study. [Figure 11A] Figure 11A shows 6-minute walk test (6MWT) distance at baseline and at weeks 13 and 25 in ambulatory participants older than 6 years at baseline. Figure 11A shows results in cohort 2 (avalglucosidase alfa 40 mg / kg every other week) and cohort 3 (avalglucosidase alfa 40 mg / kg every other week). [Figure 11B]Figure 11B shows 6-minute walk test (6MWT) distance at baseline and at weeks 13 and 25 in ambulatory participants older than 6 years at baseline. Figure 11B shows results in cohort 1 (avalglucosidase alfa 20 mg / kg every other week) and cohort 3 (alglucosidase alfa 20 mg / kg every other week and 40 mg / kg every week). [Figure 12] Figure 1 shows an exemplary dosing regimen for CRIM-negative patients: ERT: enzyme replacement therapy; ITI: immune tolerance induction therapy, i.e., methotrexate, rituximab, and IVIG in this dosing regimen. DETAILED DESCRIPTION OF THE INVENTION

[0035] The present application provides compositions and methods for treating Pompe disease, including infantile-onset Pompe disease (IOPD), the most severe form of Pompe disease, using an oligosaccharide-acid alpha-glucosidase (GAA) conjugate. In some embodiments, the compositions described herein are lyophilized formulations of oligosaccharide-GAA conjugates, which have high stability after storage and / or reconstitution. In some embodiments, the oligosaccharide-GAA conjugate is avalglucosidase alfa. Clinical trials of avalglucosidase alfa in human patients have demonstrated the efficacy of the compositions described herein for treating Pompe disease, such as IOPD, even in patients who show clinical attenuation or a suboptimal clinical response after treatment with recombinant GAA.

[0036] I. Definition Terms are used herein as commonly used in the art unless otherwise defined as follows.

[0037] The terms "acid alpha-glucosidase" and "GAA" are used interchangeably herein and refer to the protein acid alpha-glucosidase. In some embodiments, the GAA is recombinant GAA. In some embodiments, the GAA is human GAA.

[0038] As used herein, "treatment" or "treating" is an approach for obtaining beneficial or desired results, including clinical results. For purposes of this application, beneficial or desired clinical results include the following: reducing one or more symptoms resulting from a disease, lessening the severity of the disease, stabilizing the disease (e.g., preventing or slowing the progression of the disease), or reducing the severity of the disease. "Treatment" includes, but is not limited to, one or more of: reducing or delaying the progression of the disease (prolonging the progression of the disease), preventing or delaying the spread of the disease, preventing or delaying the onset or recurrence of the disease, delaying or slowing the progression of the disease, ameliorating the disease state, providing remission (partial or complete) of the disease, reducing the dose of one or more other medications required to treat the disease, slowing the progression of the disease, improving quality of life, and / or extending survival. Also encompassed by "treatment" is the reduction of the pathological consequences of Pompe disease (such as IOPD). The methods of the present application contemplate any one or more of these aspects of treatment.

[0039] The terms "individual," "subject," and "patient" are used interchangeably herein to refer to a mammal, including a human. In some embodiments, the individual is a human. In some embodiments, the individual is suffering from a disease, such as IOPD. In some embodiments, the individual is in need of treatment.

[0040] As understood in the art, an "effective amount" refers to an amount of a therapeutic agent or composition (e.g., a composition comprising avalglucosidase alfa) sufficient to produce a desired therapeutic outcome (e.g., reducing the severity or duration of, stabilizing the severity of, or eliminating one or more symptoms of IOPD) or achieve a desired prophylactic result (e.g., inducing immune tolerance to the therapeutic agent). An effective amount herein may vary according to factors such as the patient's disease state, age, sex, and weight, and the ability of the antibody to elicit a desired response in the individual. An effective amount is also one in which the therapeutically beneficial effects outweigh any toxic or detrimental effects of the treatment. For prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk of, reducing the severity of, or delaying the onset of, disease, including its biochemical, histological, and / or behavioral symptoms, its complications, and intermediate pathological phenotypes present during disease development. Beneficial or desired results for therapeutic use include, for example, reducing one or more symptoms resulting from a disease (biochemical, histological, and / or behavioral), including its complications and intermediate pathological phenotypes present during the development of the disease; improving the quality of life of those afflicted with the disease; reducing the dose of other medications required to treat the disease; enhancing the effectiveness of other medications; slowing disease progression; and / or extending patient survival. In some embodiments, an effective amount of a therapeutic agent can extend a subject's survival (including overall survival and progression-free survival); produce an objective response (including a complete or partial response); alleviate to some extent one or more signs or symptoms of a disease or condition; and / or improve quality of life. For purposes of this application, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment, either directly or indirectly.

[0041] As used herein, "baseline value" refers to the value of an individual's biomarker before or at the start of treatment.

[0042] The term "pharmaceutical composition" refers to a manufacture that is in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0043] A "pharmaceutically acceptable carrier" refers to one or more ingredients in a pharmaceutical formulation, other than an active ingredient, that are non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, cryoprotectants, tonicity agents, preservatives, and combinations thereof.

[0044] The terms "lyophilization," "lyophilized," and "freeze-dried" refer to a process in which the material to be dried is first frozen, and then the ice or freezing solvent is removed by sublimation in a vacuum environment. Excipients are included in pre-lyophilized formulations to enhance the stability of the lyophilized product during storage.

[0045] The terms "pre-lyophilized formulation" and "lyophilizable formulation" are used interchangeably herein and refer to a formulation that has been subjected to lyophilization to produce a lyophilized formulation.

[0046] The term "package insert" is used to refer to instructions customarily included on commercial packaging of a therapeutic product that contain information about the directions, usage, dosage, administration, concomitant therapy, contraindications and / or warnings surrounding the use of such therapeutic product.

[0047] An "article of manufacture" is any product (e.g., package or container) or kit that includes at least one reagent, e.g., an agent for treating a disease or condition (e.g., IOPD), or a probe for specifically detecting a biomarker described herein. In certain embodiments, the product or kit is promoted, delivered, or sold as a unit for performing the methods described herein.

[0048] As used herein, reference to "about" a value or parameter includes (and describes) a variation about the value or parameter itself. For example, a statement expressed as "about X" includes the statement of "X."

[0049] As used herein, a reference to "not being" a value or parameter generally means and refers to "other than" a value or parameter. For example, "this method is not used to treat disease type X" means that this method is used to treat diseases other than type X.

[0050] As used herein, the term "about X to Y" has the same meaning as "about X to about Y."

[0051] As used in this specification and the appended claims, the singular forms "a," "an," or "the" include plural referents unless the context clearly dictates otherwise.

[0052] For clarity, it will be understood that certain features of the invention that are described in the context of separate embodiments can also be provided in combination in one embodiment. Conversely, for brevity, various features of the invention that are described in the context of one embodiment can also be provided separately or in any suitable subcombination. All combinations of the embodiments relating to the compositions and methods described herein are expressly embraced by the present invention and disclosed herein, just as if each and every combination were individually and explicitly disclosed herein. In addition, all subcombinations of the features and characteristics of the compositions and methods in the embodiments that describe such variability are also expressly embraced by the present invention and disclosed herein, just as if each and every such subcombination were individually and explicitly disclosed herein.

[0053] II. Treatment Methods The present application provides methods of treating Pompe disease, such as infantile-onset Pompe disease (IOPD), in an individual in need thereof using any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa).

[0054] In some embodiments, the present disclosure provides a method for the preparation of oligosaccharide-protein conjugates and pharmaceutically acceptable carriers. and a carrier comprising: an oligosaccharide-protein conjugate having the structure of Formula I: [ka] wherein GAA is acid α-glucosidase, L is a chemical linker connecting the oligosaccharide and GAA, and n is 1 to 10. In some embodiments, the pharmaceutical composition is administered at a dose of about 20 mg / kg to about 40 mg / kg (e.g., about 20 mg / kg or about 40 mg / kg). In some embodiments, the pharmaceutically acceptable carrier comprises histidine, glycine, mannitol, and / or polysorbate 80. In some embodiments, the pharmaceutically acceptable carrier comprises about 10 to 50 mM (e.g., about 10 mM) histidine, about 0.25 to 2% (e.g., about 2%) glycine, about 1 to 4% (e.g., about 2%) mannitol, and about 0.005 to 0.05% (e.g., about 0.01%) polysorbate 80. In some embodiments, the pharmaceutical composition has a pH of about 5.5 to about 6.5, such as about 6.2. In some embodiments, the Pompe disease is IOPD. In some embodiments, the oligosaccharide-protein conjugate has the structure of formula (II): [ka] wherein GAA is acid α-glucosidase, L' is a chemical linker, and n is 1 to 10. In some embodiments, the oligosaccharide-protein conjugate has the structure of formula (III): [ka] wherein GAA is acid α-glucosidase, n is 1 to 10, and m and p are independently selected from integers ranging from 1 to 10. In some embodiments, m is 3 and p is 1. In some embodiments, n is 5 to 7. In some embodiments, the oligosaccharide-protein conjugate is avaruglucosidase alfa.

[0055] In some embodiments, there is provided a method of treating IOPD comprising administering to a human individual in need thereof a pharmaceutical composition comprising an oligosaccharide-protein conjugate and a pharmaceutically acceptable carrier, wherein the oligosaccharide-protein conjugate has the structure of Formula I: [ka] wherein GAA is acid-glucosidase, L is a chemical linker connecting the oligosaccharide and GAA, and n is 1 to 10, and the pharmaceutical composition is administered at a dose of about 20 mg / kg to about 40 mg / kg (e.g., about 20 mg / kg or about 40 mg / kg). In some embodiments, the oligosaccharide-protein conjugate has the structure of Formula II: [ka] wherein GAA is acid α-glucosidase, L' is a chemical linker, and n is 1 to 10. In some embodiments, the oligosaccharide-protein conjugate has the structure of Formula III: [ka] wherein GAA is acid α-glucosidase, n is 1 to 10, and m and p are independently selected from integers ranging from 1 to 10. In some embodiments, m is 3 and p is 1. In some embodiments, n is 5 to 7. In some embodiments, the oligosaccharide-protein conjugate is avaruglucosidase alfa.

[0056] In some embodiments, a method of treating IOPD is provided, comprising administering a pharmaceutical composition comprising any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa) and a pharmaceutically acceptable carrier to a human individual in need thereof, wherein the individual is 6 months of age or younger. In some embodiments, a method of treating IOPD is provided, comprising administering a pharmaceutical composition comprising any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa) and a pharmaceutically acceptable carrier to a human individual in need thereof, wherein the individual begins treatment at 6 months of age or younger. In some embodiments, the individual has not been treated with recombinant GAA prior to receiving the oligosaccharide-GAA conjugate. In some embodiments, the pharmaceutical composition is administered at a dose of about 20 mg / kg to about 40 mg / kg (e.g., about 20 mg / kg or about 40 mg / kg). In some embodiments, the oligosaccharide-protein conjugate is It is avalglucosidase alfa.

[0057] Creatine kinase (CK, also known as creatine phosphokinase) is a serum enzyme biomarker for Pompe disease that indicates muscle damage; CK levels are elevated in patients with classic IOPD (Burton, BK et al., Pediatrics 2017 140:s1). A CK blood test is performed to measure the amount of CK enzyme in an individual's blood.

[0058] Thus, some embodiments of the present application provide a method for treating IOPD, comprising administering to a human individual in need thereof a pharmaceutical composition comprising any one of the oligosaccharide-GAA conjugates described herein (e.g., of Formulas I-III, or avalglucosidase alfa) and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered at a dose of about 20 mg / kg to about 40 mg / kg (e.g., about 20 mg / kg or about 40 mg / kg), and the individual's creatine kinase (CK) levels are reduced by at least about 100 IU / L as measured at least about 25 weeks after treatment. Some embodiments provide a method for reducing creatine kinase levels in a human individual with IOPD, comprising administering to the individual a pharmaceutical composition comprising any one of the oligosaccharide-GAA conjugates described herein (e.g., of Formulas I-III, or avalglucosidase alfa) and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered at a dose of about 20 mg / kg to about 40 mg / kg (e.g., about 20 mg / kg or about 40 mg / kg). In some embodiments, the oligosaccharide-protein conjugate is avalglucosidase alfa.

[0059] In embodiments, CK levels are reduced by at least about 50 IU / L, 60 IU / L, 70 IU / L, 80 IU / L, 90 IU / L, 100 IU / L, 110 IU / L, 120 IU / L, 130 IU / L, 140 IU / L, 150 IU / L, 160 IU / L, 170 IU / L, 180 IU / L, 190 IU / L, 200 IU / L, 250 IU / L, 300 IU / L, 400 IU / L, 500 IU / L, 600 IU / L, 700 IU / L, 800 IU / L or more when measured after a particular duration of treatment. In some embodiments, CK levels, when measured after a particular treatment duration, are reduced by about 50 IU / L, 60 IU / L, 70 IU / L, 80 IU / L, 90 IU / L, 100 IU / L, 110 IU / L, 120 IU / L, 130 IU / L, 140 IU / L, 150 IU / L, 160 IU / L, 170 IU / L, 180 IU / L, 190 IU / L, 200 IU / L, 250 IU / L, 300 IU / L, 400 IU / L, 500 IU / L, 600 IU / L, 700 IU / L, 800 IU / L or more, including any value or range therebetween. In some embodiments, CK levels are reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to baseline when measured after a specific treatment duration. In some embodiments, CK levels are reduced by about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% or less compared to baseline when measured after a specific treatment duration. In some embodiments, CK levels are reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or any value or range therebetween. In some embodiments, CK levels are measured at about 1 month, 3 months, 6 months, 25 weeks, 7 months, 8 months, 9 months, 12 months, 2 years, 5 years, 10 years or more after treatment, or any value or range therebetween.

[0060] Another biomarker of IOPD is glucose tetrasaccharide, which is elevated in the urine and plasma of patients with Pompe disease (Burton, BK et al., Pediatrics 2017 Glucose tetrasaccharides can be measured as urinary hexose tetrasaccharide (Hex4) levels, as Hex4 is a breakdown product of glycogen. Therefore, urinary Hex4 serves as a biomarker of glycogen stores (Burton, BK et al., Pediatrics 2017 140:s1).

[0061] Accordingly, some embodiments of the methods herein provide a method of treating IOPD, comprising administering to a human individual in need thereof a pharmaceutical composition comprising any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa) and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered at a dose of about 20 mg / kg to about 40 mg / kg (e.g., about 20 mg / kg or about 40 mg / kg), and the individual's urinary hexose tetrasaccharide (Hex4) levels are reduced by at least about 10 mmol / mol when measured at least about 25 weeks after treatment. In some embodiments, provided are methods for reducing urinary Hex4 levels in a human individual with IOPD, comprising administering to the individual a pharmaceutical composition comprising any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa) and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered at a dose of about 20 mg / kg to about 40 mg / kg (e.g., about 20 mg / kg or about 40 mg / kg). In some embodiments, the oligosaccharide-protein conjugate is avalglucosidase alfa.

[0062] In some embodiments, the individual's urinary Hex4 levels, when measured after a particular duration of treatment, are reduced by at least about any of 5 mmol / mol, 6 mmol / mol, 7 mmol / mol, 8 mmol / mol, 9 mmol / mol, 10 mmol / mol, 11 mmol / mol, 12 mmol / mol, 13 mmol / mol, 14 mmol / mol, 15 mmol / mol, 20 mmol / mol, 25 mmol / mol, 30 mmol / mol, 35 mmol / mol, 40 mmol / mol, or 50 mmol / mol. In some embodiments, an individual's urinary Hex4 levels, when measured after a particular duration of treatment, are reduced by about 5 mmol / mol, 6 mmol / mol, 7 mmol / mol, 8 mmol / mol, 9 mmol / mol, 10 mmol / mol, 11 mmol / mol, 12 mmol / mol, 13 mmol / mol, 14 mmol / mol, 15 mmol / mol, 20 mmol / mol, 25 mmol / mol, 30 mmol / mol, 35 mmol / mol, 40 mmol / mol, or 50 mmol / mol, including any value or range therebetween. In some embodiments, urinary Hex4 levels, when measured after a particular duration of treatment, are reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to baseline. In some embodiments, urinary Hex4 levels are reduced by about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% or less compared to baseline when measured after a particular duration of treatment. In some embodiments, Hex4 levels are reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, including any value or range therebetween. In some embodiments, Hex4 levels are measured at about 1 month, 3 months, 6 months, 25 weeks, 7 months, 8 months, 9 months, 12 months, 2 years, 5 years, 10 years, or more after treatment, including any value or range therebetween.

[0063] IOPD-related muscle weakness is assessed using the Gross Motor Function Scale-88 (GMFM-88) score. The GMFM-88 is an 88-item measure that considers an individual's gross motor activity in five domains: (1) lying down and rolling over, (2) sitting, (3) crawling and kneeling, (4) standing, and (5) walking, running, and jumping. The GMFM-88 items were selected to represent motor functions typically performed by children up to 5 years of age without motor dysfunction. Each item is scored on a 4-point Likert scale (i.e., 0 = unable; 1 = initiated [less than 10% of the task]; 2 = partially completed [10% to less than 100% of the task]; 3 = completed the task). The score for each domain is expressed as a percentage of the maximum score for that domain. The total score is obtained by adding the percentage scores for each domain and dividing the sum by the total number of domains. Thus, each region contributes equally to the total score.

[0064] The GMFM-88 test is intended for use in children and can be used on infants under 1 year of age. There is no age restriction for the GMFM-88. The GMFM-88 is used in accordance with published manuals in the art (Russell, DJ, et al., Gross Motor Function Measure (GMFM-66 & GMFM-88) User Manual, 2nd Edition 2013). The GMFM-88 was not originally validated for children with diagnoses other than cerebral palsy, but has been used for other children with motor difficulties, including those with osteogenesis imperfecta (Russell, DJ, et al., Gross Motor Function Measure (GMFM-66 & GMFM-88) User Manual, 2nd Edition 2013) and acute lymphoblastic leukemia (Wright, MJ, et al., Med Pediatr Oncol. 1998;31:2). The GMFM-88 is used to evaluate children and adults with Pompe disease (Winkel, LP et al., Ann Neurol. 2004;55:4) and can be used to measure changes in motor performance over time secondary to improvements or declines in muscle strength.

[0065] Accordingly, some embodiments of the methods herein provide a method of treating IOPD, comprising administering to a human individual in need thereof a pharmaceutical composition comprising any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa) and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered at a dose of about 20 mg / kg to about 40 mg / kg (e.g., about 20 mg / kg or about 40 mg / kg), and wherein the individual's Gross Motor Performance Scale-88 (GMFM-88) score increases by at least 5% when measured at least about 25 weeks after treatment. In some embodiments, methods are provided for improving and / or increasing the GMFM-88 score in a human individual with IOPD, comprising administering to the individual a pharmaceutical composition comprising any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa) and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered at a dose of about 20 mg / kg to about 40 mg / kg (e.g., about 20 mg / kg or about 40 mg / kg). In some embodiments, the oligosaccharide-protein conjugate is avalglucosidase alfa.

[0066] In some embodiments, the GMFM-88 score increases by at least about 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% or more, including any value or range therebetween. In some embodiments, the GMFM-88 score increases by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to baseline when measured after a specific treatment duration. In some embodiments, the GMFM-88 score increases by no more than about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% compared to baseline when measured after a specific treatment duration. In some embodiments, the GMFM-88 score increases by about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, including any value or range therebetween. In some embodiments, the GMFM-88 score is determined at about any of 1 month, 3 months, 6 months, 25 weeks, 7 months, 8 months, 9 months, 12 months, 2 years, 5 years, 10 years or more after treatment, including any value or range therebetween.

[0067] In some embodiments, individuals show improvement or stabilization as measured by Gross Motor Function Classification System-Extended and Revised (GMFCS-E&R) parameters. The GMFCS-E&R emphasizes concepts from the World Health Organization's International Classification of Functioning, Disability and Health. It emphasizes normal performance in home, work, and community settings without making prognostic judgments about the quality of movement or improvement. The GMFCS-E&R is a standardized system originally developed to classify gross motor skills in children with cerebral palsy (Palisano, R., et al., D Dev. Med. Child Neurol. 1997; 39:4). The GMFCS-E&R has also been applied to the assessment of Pompe disease (e.g., Spiridigliozzi, GA, et al., Mol. Genet. Metab. 2017; 121:2). The GMFCS-E&R is a five-level classification system (Levels I-V) based on self-initiated activities for specific age ranges, emphasizing sitting, transfers, and mobility. The divisions between levels are based on functional limitations, the need for assistive mobility devices, and are designed to be meaningful in daily life, regardless of the quality of the activities (Palisano RJ, et al., Dev. Med. Child Neurol. 2008; 50:10).

[0068] The general headings for the five levels are: Level I: Walk without restrictions Level II: Walking with limitations Level III: Walking with a hand-held mobility device Level IV: Mobility with limitations; may use motorized transportation; Level V: Transported in a manual wheelchair is.

[0069] Thus, some embodiments of the methods herein provide a method of treating IOPD, comprising administering to a human individual in need thereof a pharmaceutical composition comprising any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa) and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered at a dose of about 20 mg / kg to about 40 mg / kg (e.g., about 20 mg / kg or about 40 mg / kg), and the individual's GMFCS-E&R score is increased as measured at least about 25 weeks after treatment. Some embodiments provide a method of improving the GMFCS-E&R score in a human individual with IOPD, comprising administering to the individual a pharmaceutical composition comprising any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa) and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered at a dose of about 20 mg / kg to about 40 mg / kg (e.g., about 20 mg / kg or about 40 mg / kg). In some embodiments, the oligosaccharide-protein conjugate is avalglucosidase alfa.

[0070] In some embodiments, the GMFCS-E&R score, when measured after a specific treatment duration, increases by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to baseline. In some embodiments, the GMFCS-E&R score, when measured after a specific treatment duration, increases by about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% or less compared to baseline. In some embodiments, the GMFCS-E&R score, when measured after a specific treatment duration, increases by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, including any value or range therebetween. In some embodiments, the GMFCS-E&R score, when measured after a specific treatment duration, increases by 1, 2, 3, or 4 levels compared to baseline. In some embodiments, the GMFCS-E&R score is determined at about 1 month, 3 months, 6 months, 25 weeks, 7 months, 8 months, 9 months, 12 months, 2 years, 5 years, 10 years or more after treatment, including any value or range therebetween.

[0071] In some embodiments, the individual shows improvement or stabilization of one or more parameters selected from the group consisting of respiratory function, exercise capacity, cardiac parameters, and eyelid position. Exemplary parameters include the Pompe-Pediatric Disability Assessment (PEDI) Functional Capacity Scale, Echocardiogram (ECHO)-Left Ventricular Mass (LVM) Z-score, ECHO LVMI score, Gross Motor Function Classification System-Extended and Revised (GMFCS-E&R) score, Simplified Motor Function Classification (SFM) score, and the like. These include, but are not limited to, a visual acuity test, a 6-minute walk test (6MWT), interpalpebral fissure diameter (IPFD), lid margin corneal reflex distance-1 (MRD-1), lid margin pupillary distance (MPD), the development of ptosis, and the use of respiratory support. In some embodiments, an individual shows improvement or stabilization of one or more parameters compared to baseline after a specific treatment duration. The treatment duration may be at least about 10 weeks, 13 weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks, 49 weeks, 61 weeks, 73 weeks, 97 weeks, or 100 weeks, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, or more.

[0072] In some embodiments, the individual demonstrates improvement or stabilization of one or more motor skills as assessed by the Pompe Pediatric Disability Assessment Inventory (Pompe-PEDI) Functional Capacity Scale: Mobility Domain (Haley, SM, et al., Pediatr Rehabil 2003 6:2; Haley, SM, et al., Pediatr. Neurol. 2004 31). The Pompe-PEDI is a modified version of the Pediatric Disability Assessment Inventory specific to Pompe disease, and includes both self-care and mobility items (e.g., head control, floor activity, sitting, standing, etc.) (Haley, SM, et al., Pediatr. Neurol. 2004 31). The Pompe-PEDI raw score is the sum of the items that the child is able to perform. The standard and scaled scores, as well as the associated standard deviations, were generated from a statistical program created as part of the research study described in SM, et al., Pediatr. Neurol. 2004 31; this program requires input of the subject's age and score and generates the corresponding standard and scaled score data.

[0073] In some embodiments, provided are methods for improving or stabilizing Pompe-PEDI scores in a human individual with IOPD, comprising administering to the individual a pharmaceutical composition comprising any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa) and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered at a dose of about 20 mg / kg to about 40 mg / kg (e.g., about 20 mg / kg or about 40 mg / kg). In some embodiments, the oligosaccharide-protein conjugate is avalglucosidase alfa.

[0074] In some embodiments, the Pompe-PEDI Functional Ability Scale: Mobility Domain scaled score is increased by at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 25, 30, 40, or 50 points. In some embodiments, the Pompe-PEDI Functional Ability Scale: Mobility Domain scaled score is increased by at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more points compared to baseline when measured after a specified treatment duration. In some embodiments, the Pompe-PEDI Functional Capacity Scale: Mobility Domain scaled score, when measured after a particular treatment duration, increases by no more than about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% compared to baseline. In some embodiments, the Pompe-PEDI Functional Capacity Scale: Mobility Domain scaled score increases by no more than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, including any value or range therebetween. In some embodiments, the Pompe-PEDI Functional Capacity Scale: Mobility Domain scaled score is determined at about 1 month, 3 months, 6 months, 25 weeks, 7 months, 8 months, 9 months, 12 months, 2 years, 5 years, 10 years, or more after treatment, including any value or range therebetween.

[0075] In some embodiments, any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa) and a pharmaceutically acceptable carrier are used.

[0010] Also provided are methods for improving or stabilizing an Alberta Infant Motor Development Scale (AIMS) score in a human individual with IOPD, comprising administering to the individual a pharmaceutical composition comprising the oligosaccharide-protein complex, wherein the pharmaceutical composition is administered at a dose of about 20 mg / kg to about 40 mg / kg (e.g., about 20 mg / kg or about 40 mg / kg). In some embodiments, the oligosaccharide-protein complex is avaruglucosidase alfa.

[0076] The Alberta Infant Motor Scale (AIMS) assesses gross infant motor skills from 0 to 18 months of age. This scale evaluates an infant's weight support, posture, and antigravity movements. See, e.g., Piper MC et al., Construction and validation of the Alberta Infant Motor Scale (AIMS). Can J Public Health. 1992 Jul-Aug;83 Suppl 2:S46-50. PMID:1468050.

[0077] In some embodiments, the individual demonstrates improvement or stabilization of one or more motor functions as assessed by the Quick Motor Function Test (QMFT) (van Capelle, CI, et al., J Inherit Metab Dis. 2012 35:2). The QMFT is a functional motor measure specific to Pompe disease in which an assessor observes the patient's performance and scores each of 16 items (e.g., sitting up, flexing the neck, picking up an object, etc.) on a 5-point scale.

[0078] In some embodiments, there is provided a method for improving or stabilizing QMFT scores in a human individual with IOPD, comprising administering to the individual a pharmaceutical composition comprising any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa) and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered at a dose of about 20 mg / kg to about 40 mg / kg (e.g., about 20 mg / kg or about 40 mg / kg). In some embodiments, the oligosaccharide-protein conjugate is avalglucosidase alfa.

[0079] In some embodiments, the individual shows improvement in QMFT scale. For example, the individual may show improvement in QMFT scale of about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% change from baseline after a specific treatment duration. In some embodiments, the QMFT score increases by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to baseline when measured after a specific treatment duration. In some embodiments, the QMFT score increases by about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% or less compared to baseline when measured after a specific treatment duration. In some embodiments, the QMFT score increases by about any of the following: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, including any value or range therebetween. In some embodiments, the QMFT score is determined at about any of the following: 1 month, 3 months, 6 months, 25 weeks, 7 months, 8 months, 9 months, 12 months, 2 years, 5 years, 10 years or more after treatment, including any value or range therebetween.

[0080] In some embodiments, the individual exhibits improvement or stabilization of one or more cardiac parameters as determined by echocardiographic (ECHO) endpoints, such as left ventricular mass index (LVMI) and / or left ventricular mass (LVM) Z-score (Barker, PAC, et al., Mol. Genet. Metab. 2010 101:4). Heart failure is the leading cause of death in Pompe disease patients, and therefore echocardiography (also known as echo) is used in the art to assess cardiac response to ERT (van Cap (Chubb, H. and J.M. Simpson, Ann. Pediatr. Cardiol. 2012; 5:2). The echo Z-score indicates how many standard deviations a given measurement is above or below a size- or age-specific population mean.

[0081] In some embodiments, there is provided a method for reducing LVMI in a human individual with IOPD, comprising administering to the individual a pharmaceutical composition comprising any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa) and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered at a dose of about 20 mg / kg to about 40 mg / kg (e.g., about 20 mg / kg or about 40 mg / kg). In some embodiments, the oligosaccharide-protein conjugate is avalglucosidase alfa.

[0082] In some embodiments, the LVMI is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to baseline when measured after a specific treatment duration. In some embodiments, the LVMI is reduced by about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% or less compared to baseline when measured after a specific treatment duration. In some embodiments, the LVMI is reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or any value or range therebetween. In some embodiments, the LVMI is determined at about 1 month, 3 months, 6 months, 25 weeks, 7 months, 8 months, 9 months, 12 months, 2 years, 5 years, 10 years or more after treatment, or any value or range therebetween.

[0083] In some embodiments, a method for treating LVM in a human individual with IOPD includes administering to the individual a pharmaceutical composition comprising any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa) and a pharmaceutically acceptable carrier. A method for reducing a Z-score is provided, wherein the pharmaceutical composition is administered at a dose of about 20 mg / kg to about 40 mg / kg (e.g., about 20 mg / kg or about 40 mg / kg). In some embodiments, the oligosaccharide-protein complex is avalglucosidase alfa.

[0084] In some embodiments, the LVM Z-score is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to baseline when measured after a particular duration of treatment. The Z-score, when measured after a particular treatment duration, is reduced by no more than about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% compared to baseline. In some embodiments, the LVM Z-score is reduced by no more than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, including any value or range therebetween. In some embodiments, the LVM Z-score is determined at about 1 month, 3 months, 6 months, 25 weeks, 7 months, 8 months, 9 months, 12 months, 2 years, 5 years, 10 years, or more after treatment, including any value or range therebetween.

[0085] In some embodiments, an individual exhibits improvement or stabilization of one or more respiratory function, exercise capacity, and / or cardiac parameters as determined by a 6-minute walk test (6MWT). As used herein, the 6MWT determines the distance walked in 6 minutes by a patient who is able to walk at least 40 meters without stopping or using an assistive device. For example, if an individual is able to walk a greater distance in 6 minutes after a particular treatment duration compared to baseline, the 6MWT is improved. In some embodiments, an oligosaccharide-GAA conjugate described herein (e.g., a compound of Formulas I-III, or avalglucosamine) is administered.

[0013] The present invention provides a method for improving 6MWT scores (e.g., increasing distance walked) in a human individual with IOPD, comprising administering to the individual a pharmaceutical composition comprising one of the oligosaccharide-protein complexes avaruglucosidase alfa and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered at a dose of about 20 mg / kg to about 40 mg / kg (e.g., about 20 mg / kg or about 40 mg / kg). In some embodiments, the oligosaccharide-protein complex is avaruglucosidase alfa.

[0086] In some embodiments, the individual exhibits improvement or stabilization of one or more motor functions, as determined by the onset of ptosis. Ptosis is the drooping of the upper eyelid above the eye. In some embodiments, ptosis is delayed compared to the average onset of ptosis in untreated IOPD patients, as measured after a specific duration of treatment. In some embodiments, a method is provided for delaying the onset of ptosis in a human individual with IOPD, comprising administering to the individual a pharmaceutical composition comprising any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa) and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered at a dose of about 20 mg / kg to about 40 mg / kg (e.g., about 20 mg / kg or about 40 mg / kg). In some embodiments, the oligosaccharide-protein conjugate is avalglucosidase alfa.

[0087] Ptosis is determined by measuring one or more eyelid positions using methods known in the art, such as the measuring tool shown in FIG. 6A. Exemplary eyelid position measurements include, but are not limited to, the interpalpebral fissure diameter (IPFD), the lid margin corneal reflex distance-1 (MRD-1), and the lid margin pupillary distance (MPD), as shown in FIGS. 6B-6D. Ptosis (drooping of the eyelid) has been shown to occur in long-term survivors of infantile-onset Pompe disease (IOPD) who received alglucosidase alfa and is attributed to glycogen storage in the levator palpebrae superioris muscle. Ptosis can lead to amblyopia and, in severe cases, may require surgical intervention. See, for example, De Wilde F. et al., Surgical treatment of myogenic blepharoptosis. Bull Soc Belge Ophtalmol. 1995;255:139-46; Slingerland NW, et al., Ptosis, extraocular motility disorder, and myopia as features of Pompe disease. Orbit. 2011;30(2):111-3; and Prakalapakorn SG, et al., Ocular and histologic findings in a series of children with infantile Pompe disease treated with enzyme replacement therapy. J Pediatr Ophthalmol Strabismus. 2014;51(6):355-9, which are incorporated herein by reference. Eyelid position is measured using flash or non-flash photography. In some embodiments, eyelid position (e.g., IPDF and MPD) is measured using non-flash photography. In some embodiments, the method reduces one or more of IPFD, MRD-1, and MPD in an individual. In some embodiments, the method reduces IPFD, MRD-1, and MPD in an individual. In some embodiments, the reduction is in both eyes. In some embodiments, the reduction is in one eye.

[0088] In some embodiments, provided are methods for reducing interocular fissure diameter (IPFD) in a human individual with IOPD, comprising administering to the individual a pharmaceutical composition comprising any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa) and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered at a dose of about 20 mg / kg to about 40 mg / kg (e.g., about 20 mg / kg or about 40 mg / kg). In some embodiments, the pharmaceutical composition is administered every other week or every week. In some embodiments, the oligosaccharide-protein complex is avalglucosidase alfa. In some embodiments, the reduction is in both eyes. In some embodiments, the reduction is in one eye.

[0089] In some embodiments, provided are methods for reducing palpebral corneal reflex distance-1 (MRD-1) in a human individual with IOPD, comprising administering to the individual a pharmaceutical composition comprising any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa) and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered at a dose of about 20 mg / kg to about 40 mg / kg (e.g., about 20 mg / kg or about 40 mg / kg). In some embodiments, the pharmaceutical composition is administered every other week or every week. In some embodiments, the oligosaccharide-protein conjugate is avalglucosidase alfa. In some embodiments, the reduction is in both eyes. In some embodiments, the reduction is in one eye.

[0090] In some embodiments, provided is a method for reducing eyelid margin pupillary distance (MPD) in a human individual with IOPD, comprising administering to the individual a pharmaceutical composition comprising any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa) and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered at a dose of about 20 mg / kg to about 40 mg / kg (e.g., about 20 mg / kg or about 40 mg / kg). In some embodiments, the pharmaceutical composition is administered biweekly or weekly. In some embodiments, the oligosaccharide-protein conjugate is avalglucosidase alfa. In some embodiments, the reduction is in both eyes. In some embodiments, the reduction is in one eye.

[0091] In some embodiments, the individual exhibits improvement or stabilization of one or more respiratory functions as determined by the use of a ventilator. IOPD patients may require the use of a ventilator. The ventilator may be invasive or non-invasive, and may be used only during the day or at night, or both during the day and at night. In some embodiments, the individual uses a ventilator less frequently during a day, week, or month when measured after a specific treatment duration compared to baseline. In some embodiments, the individual uses a less invasive ventilator when measured after a specific treatment duration compared to baseline, such as about 1 month, 3 months, 6 months, 25 weeks, 7 months, 8 months, 9 months, 12 months, 2 years, 5 years, 10 years, or more, including any value or range between these values.

[0092] In some embodiments, methods are provided for delaying the use of mechanical ventilation in a human individual with IOPD, comprising administering to the individual a pharmaceutical composition comprising any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa) and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered at a dose of about 20 mg / kg to about 40 mg / kg (e.g., about 20 mg / kg or about 40 mg / kg). In some embodiments, the oligosaccharide-protein conjugate is avalglucosidase alfa.

[0093] In some embodiments, methods are provided for extending invasive ventilator-free survival and / or overall survival in an individual with IOPD, comprising administering to the individual a pharmaceutical composition comprising any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa) and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered at a dose of about 20 mg / kg to about 40 mg / kg (e.g., about 20 mg / kg or about 40 mg / kg). In some embodiments, the oligosaccharide-protein conjugate is avalglucosidase alfa. In some embodiments, the invasive ventilator-free survival is extended to about 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, 15 years, 20 years, 25 years, 30 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 21 years, 22 years, 23 years, 24 years, 25 years, 26 years, 27 years, 28 years, 29 years, 30 years, 31 years, 32 years, 33 years, 34 years, 35 years, 36 years, 37 years, 38 years, 39 years, 40 years, 41 years, 42 years, 43 years, 44 years, 45 years, 46 years, 47 years, 48 ​​years, 49 years, 50 years, 51 years, 52 years, 53 years, 54 years, 55 years, 56 years, 57 years, 58 years, 59 years, 60 years, 61 years, 62 years, 63 years, In some embodiments, the overall survival time is increased by about 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, 15 years, 20 years, 25 years, 30 years, 40 years, or more, including any value or range therebetween.

[0094] The methods described herein are also associated with fewer associated adverse effects. In some embodiments, a method of treating IOPD is provided, comprising administering to a human individual in need thereof a pharmaceutical composition comprising any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa) and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered at a dose of about 20 mg / kg to about 40 mg / kg (e.g., about 20 mg / kg or about 40 mg / kg), and the individual has a decrease in the level of anti-drug antibodies (ADA) against the oligosaccharide-protein conjugate over time. ADA can form in response to drugs such as oligosaccharide-protein conjugates. ADA can neutralize the effects of the drug, thus presenting a challenge in ERT.

[0095] In some embodiments, the methods described herein further comprise preparing a pharmaceutical composition. In some embodiments, the pharmaceutical composition is reconstituted from a lyophilized formulation. In some embodiments, the pharmaceutical composition is reconstituted from a lyophilized formulation comprising an oligosaccharide-protein conjugate.

[0096] In some embodiments, there is provided a use of any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa) in the manufacture of a medicament for treating IOPD in a human individual in need thereof. In some embodiments, there is provided a pharmaceutical composition comprising any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa) for treating IOPD in a human individual in need thereof.

[0097] The pharmaceutical compositions described herein are administered to an individual intravenously or by another suitable route. A suitable dosage of the pharmaceutical composition is about 20 mg / kg to about 40 mg / kg, including about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 mg / kg, including any value or range therebetween. In some embodiments, the pharmaceutical composition is administered at a dose of about 20 mg / kg. In some embodiments, the pharmaceutical composition is administered at a dose of about 40 mg / kg.

[0098] The pharmaceutical compositions described herein are administered at a suitable frequency, including, for example, every other week, once a week, once every two weeks, once every three weeks, or once a month. In some embodiments, the pharmaceutical composition is administered to an individual once every two weeks.

[0099] The pharmaceutical compositions of the present application may be administered to an individual in a single dose or multiple doses. For example, the pharmaceutical compositions described herein may be administered by intravenous infusion at a dose of about 20 mg / kg to about 40 mg / kg of body weight every two weeks for approximately, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours. In some embodiments, the administration rate may be initiated at, for example, 1 mg / kg / hour, and then increased by, for example, 2 mg / kg / hour every 30 minutes, until the patient's tolerance to the infusion rate is established, at which point the rate may be increased to a maximum value, for example, 7 mg / kg / hour.

[0100] The pharmaceutical compositions described herein may be administered for at least about 25 weeks, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 12 years, 15 years, 20 years, 25 years, 30 years or more, for example. The dose may be administered to an individual for an extended period of time, including any value or range between these values.

[0101] A. Patient Population Pompe disease (also known as acid maltase deficiency or glycogen storage disease (GSD) type II) is a rare, autosomal recessive disorder caused by a deficiency of lysosomal acid alpha-glucosidase (GAA), an enzyme that breaks down glycogen (see OMIM 232300). Pompe disease has been classified into infantile-onset and late-onset forms. Classic infantile-onset Pompe disease (IOPD) is characterized by fatality by 1 year of age if untreated. Late-onset Pompe disease (LOPD) has a more variable course.

[0102] Specifically, IOPD is characterized by rapidly progressive hypertrophic cardiomyopathy, left ventricular outflow tract obstruction, hypotonia and weakness, respiratory distress, and progressive lack of independent ventilation (Kohler, L. et al., Neurotherapeutics 2018 15:4). Respiratory distress, feeding problems, and macroglossia are common signs of IOPD, and motor development is typically significantly delayed (Kohler, L. et al., Neurotherapeutics 2018 15:4). IOPD is characterized by rapidly progressive hypertrophic cardiomyopathy, left ventricular outflow tract obstruction, hypotonia and weakness, respiratory distress, and progressive lack of independent ventilation (Kohler, L. et al., Neurotherapeutics 2018 15:4). It is diagnosed by newborn screening as described in 140:s1.

[0103] Alglucosidase alfa contains the active ingredient recombinant human acid α-glucosidase (rhGAA) and can be used as long-term enzyme replacement therapy (ERT) for patients with a confirmed diagnosis of Pompe disease. Alglucosidase alfa treatment has been approved worldwide for the treatment of Pompe disease (trade names MYOZYME® and LUMIZYME®) based on its efficacy in extending invasive ventilator-free survival and overall survival in infants, and its ability to improve mobility and stabilize respiratory function in children and adults with the disease. Despite the availability of rhGAA, there remains an unmet need in Pompe disease, particularly for IOPD patients, who often experience long-term motor decline despite early treatment. Without wishing to be bound by theory, this is thought to be due, at least in part, to the relatively low levels of bis-mannose-6-phosphate (bis-M6P) on alglucosidase alfa. Increasing the levels of bis-M6P on alglucosidase alfa may provide a mechanism to drive uptake into skeletal muscle, thereby improving outcomes in IOPD and LOPD.

[0104] The methods described herein are suitable for treating human individuals with IOPD. In some embodiments, the individual has cardiomyopathy at the time of diagnosis within the first year of life. In some embodiments, the individual has hypertrophic cardiomyopathy at the time of diagnosis within the first year of life. In some embodiments, the individual has arrhythmia at the time of diagnosis within the first year of life. In some embodiments, the individual has cardiac hypertrophy at the time of diagnosis within the first year of life. In some embodiments, the individual has severe hypotonia at the time of diagnosis within the first year of life. In some embodiments, the individual has severe myopathy at the time of diagnosis within the first year of life.

[0105] In some embodiments, the individual is 18 years of age or younger. In specific embodiments, the individual is 17 years of age or younger, 16 years of age or younger, 15 years of age or younger, 14 years of age or younger, 13 years of age or younger, 12 years of age or younger, 11 years of age or younger, 10 years of age or younger, 9 years of age or younger, 8 years of age or younger, 7 years of age or younger, 6 years of age or younger, 5 years of age or younger, 4 years of age or younger, 3 years of age or younger, 2 years of age or younger, or 1 year of age or younger. In some embodiments, the individual is older than 18 years of age. In some embodiments, the individual is 11 months of age or younger, 10 months of age or younger, 9 months of age or younger, 8 months of age or younger, 7 months of age or younger, 6 months of age or younger, 5 months of age or younger, 4 months of age or younger, 3 months of age or younger, 2 months of age or younger, or younger than 1 month. In some embodiments, the individual is a newborn. In some embodiments, the individual is about 0-1 month, 0-2 months, 0-3 months, 0-4 months, 0-5 months, 0-6 months, 1-6 months, 2-6 months, 3-6 months, 4-6 months, 5-6 months, 2-4 months, 3-6 months, In some embodiments, the individual is first administered an oligosaccharide-GAA conjugate within the first month of life. In some embodiments, the individual is administered an immune induction therapy (e.g., methotrexate, rituximab, and IVIG) within the first month of life.

[0106] In some embodiments, the individual has been treated with recombinant GAA for at least 6 months. In some embodiments, the individual has been treated with recombinant GAA for at least about 8 months, 10 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years or more, including any value or range therebetween. In other embodiments, the individual has not been treated with recombinant GAA. Exemplary recombinant GAAs include, but are not limited to, alglucosidase alfa, leveglucosidase alfa (BioMarin), and ATB200 (Amicus).

[0107] In some embodiments, the individual shows clinical decline after treatment with recombinant GAA, and the clinical decline is determined by assessing one or more parameters selected from the group consisting of respiratory function, exercise capacity, and cardiac parameters. In some embodiments, the individual has a suboptimal clinical response to treatment with recombinant GAA. The suboptimal clinical response is determined by assessing respiratory function, exercise capacity, or cardiac parameters. Respiratory function, exercise capacity, and / or cardiac parameters can be determined by CK level, urinary Hex4 level, GMFM-88, Alberta Infant Motor Development Scale (AIMS) score, Pompe-Pediatric Assessment of Disability (PEDI) Functional Capacity Scale, Echocardiogram (ECHO)-Left Ventricular Mass (LVM) Z-score, ECHO LVMI score, Gross Motor Function Classification System-Extended and Revised (GMFCS-E&R) score, Brief Motor Function Test, 6-Minute Walk Test (6MWT), onset of ptosis, interpalpebral fissure diameter (IPFD), eyelid margin corneal reflex distance-1 (MRD-1), and eyelid margin pupillary distance (MPD), and / or use of respiratory support.

[0108] In some embodiments, the individual is cross-reactive immunoglobulin (CRIM) negative. CRIM-negative patients do not produce the GAA enzyme and, as a result, can produce high levels of neutralizing antibodies against GAA ERT (Dasouki, M. et al., Neurol Clin. 2014; 32:3). In other embodiments, the individual is CRIM-positive. CRIM-positive patients have some residual GAA activity. CRIM status is determined by Western blot analysis of cultured skin fibroblasts, predicted from GAA gene mutations, or determined from a blood test (Burton, BK et al., Pediatrics 2017; 140:s1; Bali, DS et al., Am J Med Genet C Semin Med Genet. 2012; 15:160C; Wang, Z. et al., Mol Genet Metab. 2014; 111:2).

[0109] B. Oligosaccharide-protein complexes The methods described herein use an oligosaccharide-protein conjugate (also referred to herein as an "oligosaccharide-GAA conjugate") that includes an oligosaccharide, GAA (also known as acid α-glucosidase), and a chemical linker that connects the oligosaccharide and GAA, for example, via an oxime group. In some embodiments, the oligosaccharide is a hexasaccharide. In some embodiments, the oligosaccharide has two mannose-6-phosphate (M6P) units. In some embodiments, the oligosaccharide-protein conjugate has about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or more M6P units, including any value or range therebetween. In some embodiments, the oligosaccharide-protein conjugate is avaruglucosidase alpha.

[0110] In some embodiments, an oligosaccharide-protein conjugate comprising (1) a GAA protein, (2) an oligosaccharide, and (3) an oxime group linking the GAA protein and the oligosaccharide is provided herein. It shall be recorded in the book.

[0111] In some embodiments, the oligosaccharide-protein conjugate has the structure of Formula (I): [ka] wherein GAA is acid α-glucosidase, L is a chemical linker connecting the oligosaccharide and GAA, and n is 1 to 10. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8. In some embodiments, n is 9. In some embodiments, n is 10. In some embodiments, n is at least 1, 2, 3, 4, 5, 6, 7, 8, or 9. In some embodiments, n is no greater than 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, n is selected from the following ranges: 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 1 to 3, 3 to 5, 5 to 7, 7 to 9, 1 to 4, 4 to 7, 7 to 10, 1 to 5, 5 to 10, 2 to 10, 3 to 9, 4 to 8, 2 to 9, 3 to 8, 4 to 7, 5 to 8, 5 to 9, 5 to 10, 4 to 7, 3 to 7, 2 to 7, and 1 to 7. In some embodiments, n is 5 to 9.

[0112] In some embodiments, the oligosaccharide-protein conjugate has the structure of formula (II): [ka] wherein GAA is acid α-glucosidase, L' is a chemical linker, and n is 1-10. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8. In some embodiments, n is 9. In some embodiments, n is 10. In some embodiments, n is at least 1, 2, 3, 4, 5, 6, 7, 8, or 9. In some embodiments, n is no greater than 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, n is selected from the following ranges: 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 1 to 3, 3 to 5, 5 to 7, 7 to 9, 1 to 4, 4 to 7, 7 to 10, 1 to 5, 5 to 10, 2 to 10, 3 to 9, 4 to 8, 2 to 9, 3 to 8, 4 to 7, 5 to 8, 5 to 9, 5 to 10, 4 to 7, 3 to 7, 2 to 7, and 1 to 7. In some embodiments, n is 5 to 9.

[0113] In some embodiments, the oligosaccharide-protein conjugate has the structure of formula (III): [ka] wherein GAA is acid α-glucosidase, n is 1 to 10, and m and p are independently selected from integers ranging from 1 to 10. In some embodiments, n is 1. In embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8. In some embodiments, n is 9. In some embodiments, n is 10. In some embodiments, n is at least 1, 2, 3, 4, 5, 6, 7, 8, or 9. In some embodiments, n is no greater than 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, n is selected from the following ranges: 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 1 to 3, 3 to 5, 5 to 7, 7 to 9, 1 to 4, 4 to 7, 7 to 10, 1 to 5, 5 to 10, 2 to 10, 3 to 9, 4 to 8, 2 to 9, 3 to 8, 4 to 7, 5 to 8, 5 to 9, 5 to 10, 4 to 7, 3 to 7, 2 to 7, and 1 to 7. In some embodiments, n is 5 to 9. In some embodiments, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, m and p are independently selected from integers selected from the following ranges: 1 to 4, 4 to 8, 8 to 10, 2 to 4, 2 to 6, 2 to 8, 2 to 10, 1 to 3, 3 to 6, 6 to 9, and 4 to 10. In some embodiments, m and p are independently selected from integers in the range of 1 to 5. In some embodiments, m is 1, 2, 3, 4, or 5, and p is 1, 2, 3, 4, or 5. In some embodiments, m is 1, 2, or 3, and p is 1, 2, or 3. In some embodiments, m is 5 and p is 1. In some embodiments, m is 4 and p is 1. In some embodiments, m is 3 and p is 1. In some embodiments, m is 2 and p is 1. In some embodiments, m is 1 and p is 1. In some embodiments, m is 5 or less, and p is 3 or less. In some embodiments, m is 4 or less, and p is 2 or less. In some embodiments, m is less than or equal to 3 and p is less than or equal to 1. In some embodiments, m is at least 1 and p is at least 1. In some embodiments, m is at least 2 and p is at least 1.In some embodiments, m is at least 3 and p is at least 1.

[0114] In some embodiments, the number of phosphate groups per oligosaccharide-protein conjugate (e.g., a conjugate of Formula (I), (II), or (III)) is about 2-20. In some embodiments, the number of phosphate groups per oligosaccharide-protein conjugate is 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20. In some embodiments, the number of phosphate groups per oligosaccharide-protein conjugate is at least 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20. In some embodiments, the number of phosphate groups per oligosaccharide-protein conjugate is at least 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20. , 8, 10, 12, 14, 16, 18, or 20 or less. In some embodiments, the number of phosphate groups per oligosaccharide-protein conjugate is an even integer selected from the following ranges: 2-4, 4-6, 6-8, 8-10, 10-12, 12-14, 14-16, 16-18, 18-20, 2-6, 6-10, 10-14, 14-18, 2-8, 8-14, 14-20, 2-10, 10-20, 4-20, 6-18, 8-16, 4-18, 6-16, 8-14, 10-16, 10-18, 10-20, 8-14, 6-14, 4-14, and 2-14.

[0115] In some embodiments, the oligosaccharide-protein conjugate is avalglucosidase alfa. Avalglucosidase alfa is also known as neoGAA or GZ402666. Avalglucosidase alfa is an oligosaccharide-modified form of human GAA containing multiple hexamannose structures containing two terminal mannose-6-phosphate (M6P) moieties conjugated to GAA.

[0116] Methods for producing the oligosaccharide-protein conjugates described herein are known in the art, see, e.g., US7723296, WO2008 / 089403 and WO2010 / 075010, the entire contents of which are incorporated herein by reference.

[0117] Oligosaccharides are isolated from natural sources or produced by chemical or enzymatic synthesis. Oligosaccharides isolated from natural sources can be homogeneous or a heterogeneous mixture of related oligosaccharides. In some embodiments, oligosaccharides are produced by chemical or enzymatic modification of oligosaccharides isolated from natural sources ("semi-synthetic"). In some embodiments, oligosaccharides can be synthetic oligosaccharides having the chemical structure of naturally occurring oligosaccharides.

[0118] The sequence of GAA is well known (see, e.g., Martiniuk et al., Proc. Natl. Acad. Sci. USA 83:9641-9644 (1986); Hoefsloot et al., Biochem. J. 272:493-497 (1990); Moreland et al., J. Biol. Chem. 280:6780-6791 (2005)). See also GenBank accession number NM_000152. GAA is wild-type GAA or a sequence variant thereof.

[0119] GAA can be obtained from natural sources or recombinantly. In some embodiments, GAA is a glycoprotein. In some embodiments, GAA is human GAA produced in Chinese hamster ovary (CHO) cells. In some embodiments, human GAA has glycoform alpha. In some embodiments, GAA has at least one carbonyl group. For example, GAA having at least one carbonyl group can be obtained by oxidation of GAA by any means known to those skilled in the art. In some embodiments, for example, GAA having at least one carbonyl group can be obtained by oxidation of GAA using periodate (e.g., sodium periodate) or galactose oxidase. In some embodiments, GAA having at least one carbonyl group can be chemically conjugated to an oligosaccharide functionalized at the reducing end with a carbonyl-reactive group (e.g., hydrazine, hydrazide, aminooxy, thiosemicarbazide, semicarbazide, or amine group) to obtain an oligosaccharide-GAA conjugate. In some embodiments, GAA is oxidized with about 1, 2, 3, 4, 5, 7.5, 10, or 22.5 mM periodate. In some embodiments, GAA is oxidized under conditions sufficient to oxidize the sialic acid residues on the glycan of GAA and minimize the oxidation of fucose and mannose. In some embodiments, the periodate concentration used is less than about 2, 3, 4, or 5 mM. In some embodiments, the periodate is sodium periodate.

[0120] Other methods of conjugating oligosaccharides to GAA proteins can be used. In some embodiments, oligosaccharide-protein conjugates are prepared by reacting an oligosaccharide containing a first reactive group with a GAA protein having a second reactive group. In some embodiments, oligosaccharide-protein conjugates are prepared by reacting an oligosaccharide containing an aminooxy group with GAA having at least one carbonyl group.

[0121] In some embodiments, the oligosaccharide is conjugated to an amino acid of the GAA protein, such as cysteine ​​or lysine.

[0122] In some embodiments, oligosaccharides are conjugated to glycans on GAA. In some embodiments, oligosaccharides are conjugated to sialic acid residues on the glycan. In other embodiments, oligosaccharides are conjugated to mannose, fucose, galactose, and / or sialic acid residues on the glycan. For conjugation via galactose, the glycoprotein is first treated with sialidase to remove sialic acid residues, and then treated with galactose oxidase prior to reaction with the oligosaccharide.

[0123] For example, oligosaccharide-protein conjugates may be any oligosaccharides present on proteins (e.g., containing amines, thiols, carboxylic acids, hydroxyls) and / or introduced into proteins. The glycoprotein is prepared by reacting a functional group of the carboxyl group with a suitable second functional group on the oligosaccharide. Methods for introducing functional groups are well known in the art. For example, a glycoprotein having at least one carbonyl group can be obtained by oxidizing the glycoprotein using, for example, periodate (e.g., sodium periodate) or galactose oxidase. In another example, the carbonyl group is introduced by using an expression system with an extended genetic code, as described, for example, in Wang et al., Proc. Natl. Acad. Sci. USA 100:56-61 (2003). See also, for example, U.S. Patent Application Publication No. 2006 / 0228348, which describes the introduction of reactive groups into glycoproteins.

[0124] In certain embodiments, the method further comprises adding a reducing agent to the oligosaccharide-protein conjugate, which can be any reducing agent known to those of skill in the art, such as, for example, sodium cyanoborohydride or sodium triacetoxyborohydride (STAB).

[0125] In certain embodiments, protein aggregates that form during conjugation can be removed using various chromatographic methods. In one embodiment, hydrophobic interaction chromatography (HIC) is utilized. Examples of HIC columns include Butyl 650C and 650M, Hexyl 650C, Phenyl 6FF, Capto Octyl, and Capto Phenyl. In other embodiments, aggregates are removed by metal chelation chromatography, such as copper, nickel, cobalt, or mercury. In one embodiment, a copper column can be used in a bind-and-elute or flow-through mode.

[0126] In some embodiments, the oligosaccharide comprising a first reactive group is an oligosaccharide comprising an aminooxy group and has the structure of formula (IV): [ka] where L' is a chemical linker.

[0127] In some embodiments, the oligosaccharide of formula (III) is an oligosaccharide of formula (V): [ka] wherein m and p are independently selected from integers in the range of 1 to 10. In some embodiments, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, p is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, m and p are independently selected from integers selected from the following ranges: 1 to 4, 4 to 8, 8 to 10, 2 to 4, 2 to 6, 2 to 8, 2 to 10, 1 to 3, 3 to 6, 6 to 9, and 4 to 10. In some embodiments, m and p are independently selected from integers in the range of 1 to 5. In some embodiments, m is 1, 2, 3, 4, or 5 and p is 1, 2, 3, 4, or 5. In some embodiments, m is 1, 2, or 3, and p is 1, 2, or 3. In some embodiments, m is 5 and p is 1. In some embodiments, m is 4 and p is 1. In some embodiments, m is 3 and p is 1. In some embodiments, m is 2 and p is 1. In some embodiments, m is 1 and p is 1. In some embodiments, m is 5 or less, and p is 3 or less. In some embodiments, m is 4 or less, and p is 2 or less. In some embodiments, m is 3 or less, and p is 1 or less. In some embodiments, m is at least 1, and p is at least 1. In some embodiments, m is at least 2, and p is at least 1. In some embodiments, m is at least 3, and p is at least 1.

[0128] C. Immune Tolerance Induction Therapy Any one of the methods described herein may further comprise administering an immune tolerance induction therapy. In some embodiments, the immune tolerance induction therapy comprises methotrexate. In some embodiments, the immune tolerance induction therapy comprises methotrexate and an additional immune tolerance induction therapy. In some embodiments, the immune tolerance induction therapy comprises methotrexate, rituximab, and IVIG. In some embodiments, the immune tolerance induction therapy is for prophylactic use.

[0129] In some embodiments, the method further comprises administering to the individual an effective amount of methotrexate. In some embodiments, methotrexate is used to induce immune tolerance in the individual to the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa). Any of the available dosing regimens can be used to induce immune tolerance, including continuous weekly repeat dosing regimens (Garman et al., Clin Exp Immunol, 137(3):496-502 ( 2004); Joseph et al., Clin Exp Immunol, 152(1):138-46 (2008); Mendelsohn et al., N Engl J Med, 360(2):194-5 (2009)), and temporary low-dose regimens (see U.S. Patent Publication No. 20140135337). In some embodiments, the effective amount of methotrexate is administered in three cycles. In some embodiments, the methotrexate is administered in a single cycle. In some embodiments, the methotrexate is administered in 1, 2, 3, 4, 5, or more cycles.

[0130] As used herein, a single cycle refers to a treatment regimen or treatment unit of consecutive or non-consecutive days, preferably starting within 5 days (e.g., within 3 days) after administration of the oligosaccharide-GAA complex. A single cycle of methotrexate can consist of a single dose of methotrexate, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 consecutive daily doses of methotrexate. When the oligosaccharide-GAA complex is administered in multiple periods, a single cycle of treatment with methotrexate preferably does not extend beyond the first period of oligosaccharide-GAA complex administration. As an example, in the administration of an oligosaccharide-GAA complex once a week, once a month, or once a year, a single cycle of methotrexate consists of taking methotrexate (e.g., orally) for three consecutive days, starting on day 0, the day the oligosaccharide-GAA complex is first administered to the patient. The patient then receives a single dose of methotrexate on day 1 (about 24 hours later) and day 2 (about 48 hours later). In some embodiments, a single cycle of methotrexate lasts no longer than about 8 days.

[0131] Methotrexate and oligosaccharide-GAA complexes can be administered in any order deemed appropriate. For example, methotrexate is administered to the subject before, during, and / or after the administration of the oligosaccharide-GAA complexes. In some embodiments, methotrexate is administered between about 48 hours before and about 48 hours after the administration of the oligosaccharide-GAA complexes. For example, methotrexate is administered about 48 hours, 36 hours, 24 hours, 12 hours before, simultaneously with, 12 hours, 24 hours, 36 hours, or 48 hours after the administration of the oligosaccharide-GAA complex treatment. In some embodiments, methotrexate is administered simultaneously with the oligosaccharide-GAA complexes. In some embodiments, methotrexate is administered simultaneously with the administration of the oligosaccharide-GAA complexes, and about 24 hours and about 48 hours after the administration of the oligosaccharide-GAA complexes. In some embodiments, the oligosaccharide-GAA conjugate is alglucosidase alfa, and methotrexate is administered simultaneously with administration of the oligosaccharide-GAA conjugate, and about 24 hours and about 48 hours after administration of the oligosaccharide-GAA conjugate.

[0132] Methotrexate is administered at any dose effective to reduce undesired immunological responses, such as antibody or cellular responses. Effective amounts of methotrexate in human patients can range from about 0.05 mg / kg to about 10 mg / kg, such as about 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, 6 mg / kg, 7.5 mg / kg, or up to 10 mg / kg. In some embodiments, the effective amount is about 0.1 mg / kg to about 1.5 mg / kg, about 0.12 mg / kg to about 1.28 mg / kg, about 1 mg / kg to about 2.5 mg / kg, or about 2.5 mg / kg to about 5 mg / kg, or about 0.1 mg / kg to about 5 mg / kg. In some embodiments, methotrexate administration may have minimal safety risks because the administration regimen involves only a short course of methotrexate at a dose level more similar to rheumatoid arthritis doses than low neoplastic doses. Patients with rheumatoid arthritis can receive up to 25 mg of methotrexate per week without suffering significant toxicity. Methotrexate The low neoplastic dose is 30 mg / m 2 It is thought that this is the case.

[0133] In some embodiments, methotrexate is administered for two or more cycles but at a low total dosage, for example, methotrexate is administered for two or more cycles (e.g., 3, 4, 5, 6, etc.), but at a combined total dosage of 5 mg / kg or less per patient.

[0134] In some embodiments, in addition to methotrexate, the subject is administered an additional immune tolerance induction therapy simultaneously with the administration of any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa). In some embodiments, methotrexate and / or the additional immune tolerance induction therapy is administered to the individual at the initiation of the oligosaccharide-GAA conjugate, as opposed to the immune tolerance induction therapy being administered in additional cycles of treatment with the oligosaccharide-GAA conjugate. In some embodiments, methotrexate and / or the additional immune tolerance induction therapy is administered to the individual at the initiation of the oligosaccharide-GAA conjugate and in one or more additional cycles of treatment with the oligosaccharide-GAA conjugate. In some embodiments, the additional immune tolerance induction therapy is administered simultaneously with methotrexate. In some embodiments, the additional immune tolerance induction therapy is administered after the administration of methotrexate, such as any of 1, 2, 3, 4, 5, 6, 7 or more days after the administration of methotrexate. In some embodiments, the additional immune tolerance induction therapy is administered prior to the administration of methotrexate, such as any of 1, 2, 3, 4, 5, 6, 7 or more days prior to the administration of methotrexate. In some embodiments, the subject has CRIM-negative IOPD. In some embodiments, the subject has CRIM-positive IOPD. In some embodiments, the methotrexate is administered enterally. In some embodiments, the methotrexate is administered at a dose of about 0.5 mg / kg. In some embodiments, the methotrexate is administered weekly. In some embodiments, the methotrexate is administered subcutaneously. In some embodiments, the methotrexate is administered at a dose of about 0.4 mg / kg. In some embodiments, the methotrexate is administered three times per week for three weeks.

[0135] In some embodiments, the additional immune tolerance induction therapy comprises rituximab, intravenous immunoglobulin (IVIG), or a combination thereof. In some embodiments, the additional immune tolerance induction therapy comprises administration of rituximab. In some embodiments, rituximab is administered at a dose of about 375 mg / m 2In some embodiments, the subject's body surface area (BSA) is administered at a dose of 0.5 m 2 In some embodiments, the rituximab is administered at a dose of about 12.5 mg / kg. In some embodiments, the rituximab is administered intravenously. In some embodiments, the rituximab is administered weekly. In some embodiments, the additional immune tolerance induction therapy includes administration of IVIG. In some embodiments, the IVIG is administered at a dose of about 0.5 g / kg. In some embodiments, the IVIG is administered at a dose of about 400-500 mg / kg. In some embodiments, the IVIG is administered about every four weeks. In some embodiments, the IVIG is administered monthly. In some embodiments, the additional immune tolerance induction therapy includes administration of both rituximab and IVIG.

[0136] In some embodiments, the method comprises: (a) administering to the individual an effective amount of any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa); and (b) administering to the individual an effective amount of methotrexate, an effective amount of rituximab, and an effective amount of IVIG. In some embodiments, the oligosaccharide-GAA conjugate is administered at about 20 mg / kg every other week. In some embodiments, the methotrexate is administered subcutaneously. In some embodiments, the methotrexate is administered at about 0.4 mg / kg. In some embodiments, the methotrexate is administered three times per week for three weeks. In some embodiments, the rituximab is administered at about 375 mg / m 2 In some embodiments, the subject's body surface area (BSA) is 0.5 m 2 Less than In such cases, rituximab is administered at a dose of about 12.5 mg / kg. In some embodiments, rituximab is administered intravenously. In some embodiments, rituximab is administered weekly. In some embodiments, IVIG is administered at a dose of about 400-500 mg / kg. In some embodiments, IVIG is administered about every 4 weeks. In some embodiments, the dosing regimen for oligosaccharide-GAA conjugate, methotrexate, rituximab, and IVIG is as shown in FIG. 12.

[0137] The effectiveness of methotrexate and additional immune tolerance induction therapy in managing unwanted antibody responses in patients can be monitored by well-known methods, including the patient's clinical examination, symptoms, blood tests to determine anti-drug antibody titers, and immunohistochemical assays (e.g., C4 deposition assays and other solid-phase antibody detection methods such as enzyme-linked immunosorbent assays (ELISAs) and bead-based fluorometric assays). This effectiveness can also be monitored by measuring the levels of biomarkers such as MCP-1, IL-13, IL-6, and IL-12, whose levels have been shown to decrease with methotrexate treatment, and by measuring transitional 2 B cells, transitional 3 B cells, follicular B cells, marginal zone B cells, B10 B cells, and B1 B cells, whose numbers have been shown to increase with methotrexate treatment. Additionally, TGF-beta, FoxP3, IL-5, IL-10, IL-15, and GM-CSF are used as biomarkers to monitor the effect of methotrexate on unwanted immune responses, if necessary. Biomarker levels are also used to monitor the effect of methotrexate on T cell responsiveness to oligosaccharide-GAA conjugates. Biomarkers related to T cell activation, such as IL-2, interferon-γ, and TNF-α, are also monitored as readouts for the effect of methotrexate on T cell responses. In some embodiments, CD19 levels are further detected to monitor unwanted immune responses. In some embodiments, individuals are monitored throughout treatment for one or more anti-drug antibody levels (such as anti-rhGAA IgG antibody levels), CD19 levels, and disease progression.

[0138] III. Formulations and Pharmaceutical Compositions Further provided are formulations and pharmaceutical compositions comprising any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa). In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the formulation is a lyophilizable formulation. In some embodiments, the pharmaceutical composition is reconstituted from a lyophilized formulation. Also provided are methods and uses of the formulations and pharmaceutical compositions described herein for treating Pompe disease, such as IOPD.

[0139] The oligosaccharide-GAA complexes described herein are unstable in liquid formulations when stored for extended periods of time. Without being bound by theory or hypothesis, the chemical bond between the oligosaccharide moiety and the GAA protein may be subject to degradation in liquid formulations. Lyophilized formulations have been developed to allow storage of the oligosaccharide-GAA complexes in dry powder form, which are then reconstituted into a liquid formulation before administration to a patient. Previous attempts to formulate recombinant GAA as a lyophilized formulation have been unsuccessful, resulting in aggregates upon reconstitution, because GAA is an enzyme capable of degrading the sugar moiety commonly used as a cryoprotectant in lyophilized or lyophilizable formulations. The lyophilized formulations described herein contain one or more cryoprotectants, such as mannitol, which are not subject to degradation by GAA.

[0140] In some embodiments, a formulation is provided that includes: (a) any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa); and (b) a sugar that is not degraded by GAA. Formulations are provided that include any one of the described oligosaccharide-GAA conjugates (e.g., Formulas I-III, or avalglucosidase alfa); (b) a sugar that is not degraded by GAA; and (c) a buffering agent. In some embodiments, the sugar is mannitol. In some embodiments, the buffering agent is histidine. In some embodiments, the formulation further includes one or more stabilizers, such as glycine or arginine. In some embodiments, the formulation further includes a surfactant, such as a polysorbate, e.g., polysorbate 80. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the formulation is a lyophilizable formulation. In some embodiments, a pharmaceutical composition reconstituted from a lyophilized formulation is provided.

[0141] In some embodiments, lyophilized or lyophilizable formulations are provided, comprising: (a) any one of the oligosaccharide-GAA conjugates described herein (e.g., any one of Formulas I-III, or avalglucosidase alfa); and (b) one or more cryoprotectants comprising a sugar that is not degraded by GAA. In some embodiments, lyophilized or lyophilizable formulations are provided, comprising: (a) any one of the oligosaccharide-GAA conjugates described herein (e.g., any one of Formulas I-III, or avalglucosidase alfa); (b) one or more cryoprotectants comprising a sugar that is not degraded by GAA; and (c) a buffer. In some embodiments, the one or more cryoprotectants comprise mannitol. In some embodiments, the one or more cryoprotectants comprise glycine and mannitol. In some embodiments, the one or more cryoprotectants comprise arginine and mannitol. In some embodiments, the one or more cryoprotectants further comprise a surfactant, such as a polysorbate, e.g., polysorbate 80. In some embodiments, the buffer is histidine. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the formulation is a lyophilizable formulation. In some embodiments, a pharmaceutical composition reconstituted from a lyophilized formulation is provided.

[0142] In some embodiments, a formulation is provided that includes: (a) any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa); (b) mannitol; and (c) a buffering agent, such as histidine. In some embodiments, the formulation further includes a surfactant, such as a polysorbate, e.g., polysorbate 80. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the formulation is a lyophilizable formulation. In some embodiments, a pharmaceutical composition reconstituted from a lyophilized formulation is provided.

[0143] In some embodiments, a formulation is provided that includes: (a) any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa); (b) mannitol; and (c) a buffering agent, such as histidine. In some embodiments, the formulation further includes a surfactant, such as a polysorbate, e.g., polysorbate 80. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the formulation is a lyophilizable formulation. In some embodiments, a pharmaceutical composition reconstituted from a lyophilized formulation is provided.

[0144] In some embodiments, a formulation is provided that includes: (a) any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa); (b) mannitol; (c) glycine; and (d) a buffering agent, such as histidine. In some embodiments, the formulation further includes a surfactant, such as a polysorbate, e.g., polysorbate 80. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the formulation is a lyophilizable formulation. In some embodiments, a pharmaceutical composition reconstituted from a lyophilized formulation is provided.

[0145] In some embodiments, a formulation is provided that includes: (a) any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa); (b) mannitol; (c) arginine; and (d) a buffering agent, such as histidine. In some embodiments, the formulation further includes a surfactant, such as a polysorbate, e.g., polysorbate 80. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the formulation is a lyophilizable formulation. In some embodiments, a pharmaceutical composition reconstituted from a lyophilized formulation is provided.

[0146] In some embodiments, a formulation is provided comprising: (a) any one of the oligosaccharide-GAA conjugates described herein (e.g., any one of Formulas I-III, or avalglucosidase alfa); (b) mannitol; (c) glycine; and (d) histidine. In some embodiments, a formulation is provided comprising: (a) any one of the oligosaccharide-GAA conjugates described herein (e.g., any one of Formulas I-III, or avalglucosidase alfa); (b) mannitol; (c) glycine; (d) histidine; and (e) a polysorbate (e.g., polysorbate 80). In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the formulation is a lyophilizable formulation. In some embodiments, a pharmaceutical composition reconstituted from a lyophilized formulation is provided.

[0147] Unless otherwise indicated, the percentage concentrations described herein are weight-to-weight percentages.When expressing the concentration of oligosaccharide-GAA complexes or various excipients in lyophilized formulations, the concentration is determined in the composition before lyophilization (i.e., in lyophilizable formulations) or after reconstitution of the lyophilized formulation.The pH of the lyophilized formulation also refers to the pH of the composition before lyophilization (i.e., in lyophilizable formulations) or after reconstitution of the lyophilized formulation.

[0148] In some embodiments, the formulation contains about 1% to about 5% sugars that are not degraded by GAA, for example, about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5% sugars, including any value or range therebetween. In some embodiments, the formulation contains about 1%-2%, 2%-3%, 3%-4%, 4%-5%, 1%-3%, 2%-4%, 1%-4%, or 1%-5% sugars. In some embodiments, the formulation contains about 2% sugars.

[0149] In some embodiments, the formulation comprises about 1% to about 5% mannitol, for example, about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5% mannitol, including any value or range therebetween. In some embodiments, the formulation comprises about 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 1% to 3%, 2% to 4%, 1% to 4%, or 1% to 5% mannitol. In some embodiments, the formulation comprises about 1% to 4% mannitol. In some embodiments, the formulation comprises about 2% mannitol.

[0150] In some embodiments, the formulation comprises between about 0.5% and about 4% of an amino acid (e.g., arginine), for example, between about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, or 4% of an amino acid, including any value or range therebetween. In some embodiments, the formulation comprises between about 0.5% and 1%, 1% and 2%, 2% and 3%, 3% and 4%, 1% and 3%, 2% and 4%, or 1% and 4% of an amino acid.

[0151] In some embodiments, the formulation comprises about 0.25% to about 4% glycine, for example, about 0.25%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, or 4% glycine, including any value or range therebetween. In some embodiments, the formulation includes glycine at about 0.25%-0.5%, 0.5%-1%, 1%-2%, 2%-3%, 3%-4%, 1%-3%, 2%-4%, 0.25%-1%, 0.25%-1.5%, or 0.25%-2%. In some embodiments, the formulation includes glycine at about 0.25%-2%. In some embodiments, the formulation includes glycine at about 2%.

[0152] In some embodiments, the formulation comprises mannitol and an amino acid (e.g., arginine or glycine) in a weight ratio of about 16:1, 8:1, 4:1, 2:1, 1:1, 1:2, 1:3, 1:4, or 1:5, including any value or range therebetween, hi some embodiments, the formulation comprises mannitol and glycine in a weight ratio of about 1:1.

[0153] In some embodiments, the formulation comprises about 5 mM to about 50 mM of buffering agent, for example, about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM, including any value or range therebetween. In some embodiments, the formulation comprises about 5-10 mM, 10-20 mM, 20-30 mM, 30-40 mM, 40-50 mM, 5-20 mM, 10-40 mM, 10-50 mM, 5-25 mM, or 25-50 mM of buffering agent.

[0154] In some embodiments, the formulation comprises about 5 mM to about 50 mM histidine, e.g., about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM, including any value or range therebetween. In some embodiments, the formulation comprises about 5-10 mM, 10-20 mM, 20-30 mM, 30-40 mM, 40-50 mM, 5-20 mM, 10-40 mM, 10-50 mM, 5-25 mM, or 25-50 mM histidine. In some embodiments, the formulation comprises about 10-50 mM histidine. In some embodiments, the formulation comprises about 10 mM histidine.

[0155] In some embodiments, the formulation has a pH of about 5.5 to about 6.5, including, for example, about 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5, including any value or range therebetween. In some embodiments, the formulation has a pH of about 5.5 to 6, 6 to 6.5, 5.5 to 5.75, 5.75 to 6, 6 to 6.25, 5.75 to 6.25, or 6.25 to 6.5. In some embodiments, the formulation has a pH of about 6.2.

[0156] In some embodiments, the formulation comprises a surfactant (e.g., polysorbate) at about 0.005-0.05%, for example, about 0.005%, 0.0075%, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, or 0.05%, including any value or range therebetween. In some embodiments, the formulation comprises a surfactant (e.g., polysorbate) at about 0.005-0.01%, 0.01-0.02%, 0.02-0.03%, 0.03-0.04%, 0.04-0.05%, 0.005-0.02%, 0.01-0.04%, or 0.005-0.05%.

[0157] In some embodiments, the formulation comprises about 0.005-0.05% polysorbate 80, such as about 0.005%, 0.0075%, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, or 0.05%, including any value or range therebetween. In some embodiments, the formulation comprises about 0.005-0.01%, 0.01-0.02%, 0.02-0.03%, 0.03-0.04%, 0.04-0.05%, 0.005-0.02%, 0.01-0.04%, or 0.005-0.05% polysorbate 80. In some embodiments, the formulation comprises about 0.01% polysorbate 80.

[0158] In some embodiments, the formulation comprises about 1-10 mg / mL of oligosaccharide-GAA complex, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / mL of oligosaccharide-GAA complex. In some embodiments, the formulation comprises about 1-5, 2-8, 3-7, 4-6, 1-2.5, 2.5-5, 5-7.5, 7.5-10, or 5-10 mg / mL of oligosaccharide-GAA complex. In some embodiments, the formulation comprises about 5-10 mg / mL of oligosaccharide-GAA complex. In some embodiments, the formulation comprises about 5 mg / mL of oligosaccharide-GAA complex.

[0159] In some embodiments, a formulation is provided that includes: (a) any one of the oligosaccharide-GAA complexes described herein (e.g., Formulas I-III, or avalglucosidase alfa); and (b) about 1-4% mannitol. In some embodiments, a formulation is provided that includes: (a) any one of the oligosaccharide-GAA complexes described herein (e.g., Formulas I-III, or avalglucosidase alfa); (b) about 1-4% mannitol; and (c) about 0.25-2% glycine. In some embodiments, a formulation is provided that includes: (a) any one of the oligosaccharide-GAA complexes described herein (e.g., Formulas I-III, or avalglucosidase alfa); (b) about 1-4% mannitol; (c) about 0.25-2% glycine; and (d) about 10-50 mM histidine. In some embodiments, a formulation is provided that includes: (a) any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa); (b) about 1-4% mannitol; (c) about 0.25-2% glycine; (d) about 10-50 mM histidine; and (e) about 0.005-0.05% polysorbate (e.g., polysorbate 80). In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the formulation is a lyophilizable formulation. In some embodiments, a pharmaceutical composition reconstituted from a lyophilized formulation is provided. In some embodiments, the formulation includes about 5 mg / mL to about 10 mg / mL of the oligosaccharide-GAA conjugate. In some embodiments, the formulation has a pH of about 5.5 to about 6.5.

[0160] In some embodiments, a formulation is provided that includes: (a) any one of the oligosaccharide-GAA complexes described herein (e.g., Formulas I-III, or avalglucosidase alfa); and (b) about 2% mannitol. In some embodiments, a formulation is provided that includes: (a) any one of the oligosaccharide-GAA complexes described herein (e.g., Formulas I-III, or avalglucosidase alfa); (b) about 2% mannitol; and (c) about 2% glycine. In some embodiments, a formulation is provided that includes: (a) any one of the oligosaccharide-GAA complexes described herein (e.g., Formulas I-III, or avalglucosidase alfa); (b) about 2% mannitol; (c) about 2% glycine; and (d) about 10 mM histidine. In some embodiments, a formulation is provided comprising: (a) any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa); (b) about 2% mannitol; (c) about 2% glycine; (d) about 10 mM histidine; and (e) about 0.01% polysorbate 80. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the formulation is a lyophilizable formulation. In some embodiments, a pharmaceutical composition reconstituted from a lyophilized formulation is provided. In some embodiments, the formulation comprises about 5 mg / mL of the oligosaccharide-GAA conjugate. In some embodiments, the formulation has a pH of about 6.2.

[0161] In some embodiments, a lyophilized or lyophilizable formulation is provided, comprising: (a) avalglucosidase alfa; (b) about 1-4% mannitol; (c) about 0.25-2% glycine; (d) about 10-50 mM histidine; and (e) about 0.005-0.05% polysorbate 80. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the formulation is a lyophilizable formulation. In some embodiments, the formulation is a lyophilizable formulation. provides a pharmaceutical composition reconstituted from a lyophilized formulation. In some embodiments, the formulation contains about 5 mg / mL of avalglucosidase alfa. In some embodiments, the formulation has a pH of about 6.2.

[0162] In some embodiments, a lyophilized or lyophilizable formulation is provided, comprising: (a) avalglucosidase alfa; (b) about 2% mannitol; (c) about 2% glycine; (d) about 10 mM histidine; and (e) about 0.01% polysorbate 80. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the formulation is a lyophilizable formulation. In some embodiments, a pharmaceutical composition reconstituted from a lyophilized formulation is provided. In some embodiments, the formulation comprises about 5 mg / mL of avalglucosidase alfa. In some embodiments, the formulation has a pH of about 6.2.

[0163] The term "buffer" encompasses agents that maintain the solution pH in an acceptable range prior to lyophilization and may include succinate (sodium or potassium), histidine, phosphate (sodium or potassium), Tris (tris(hydroxymethyl)aminomethane), diethanolamine, citrate (sodium), etc. Examples of buffers that control pH in this range include succinate (such as sodium succinate), gluconate, histidine, citrate, and other organic acid buffers.

[0164] The term "cryoprotectant" generally includes agents that provide protein stability against freezing-induced stress, presumably by preferential exclusion from the protein surface. Cryoprotectants may also provide protection during primary and secondary drying and long-term product storage. Examples include polymers such as dextran and polyethylene glycol; sugars such as mannitol; surfactants such as polysorbates; and amino acids such as glycine, arginine, and serine.

[0165] The formulations described herein may further comprise one or more pharmaceutically acceptable excipients, such as bulking agents, tonicity modifiers, antioxidants, and preservatives. Cryoprotectants described herein, such as mannitol and glycine, can serve multiple roles, including being bulking agents and / or tonicity modifiers. The term "bulking agent" includes agents that provide a lyophilized product with structure. In addition to providing a pharmaceutically high-quality cake, bulking agents can also impart useful qualities such as modifying the collapse temperature, providing freeze-thaw protection, and enhancing protein stability over long-term storage. The term "tonicity modifier" includes salts (such as NaCl, KCl, MgCl2, CaCl2) that are used as tonicity modifiers to control osmotic pressure.

[0166] In some embodiments, the formulations described herein include an antioxidant, such as methionine. In some embodiments, the formulations described herein do not include an antioxidant. In some embodiments, the formulations include about 1-20 mM of an antioxidant (e.g., methionine), for example, about 1, 2, 5, 10, 15, or 20 mM of antioxidant, including any value or range therebetween.

[0167] The formulations described herein are stable. A "stable" formulation is one in which the protein therein essentially retains its physical stability and / or chemical stability and / or biological activity upon storage. Various analytical techniques for measuring protein stability are available in the art, including those described in Peptide and Protein Drug Delivery, pp. 247-301, Vincent Lee, ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). ) Stability can be measured at a selected temperature for a selected period of time.

[0168] A "stable" lyophilized formulation is one in which no significant changes are observed for at least 12 months, preferably 2 years, and more preferably 3 years, at refrigerated temperatures (2-8°C); or at least 3 months, preferably 6 months, and more preferably 1 year, at room temperature (23-27°C). The criteria for stability are as follows: 10% or less, preferably 5%, 3%, 2%, or 1% or less of the oligosaccharide-GAA conjugate monomers are degraded as measured by size exclusion chromatography (SEC). The rehydrated solution is colorless or clear to slightly opalescent by visual analysis. The concentration, pH, and osmolality of the formulation vary by no more than + / -10%. Potency is controlled within 70-130%, preferably 80-120%. 10% or less, preferably 5%, 3%, 2%, or 1% or less of clipping is observed. 10% or less, preferably 5%, 3%, 2%, or 1% or less of aggregates are formed.

[0169] The following criteria are considered in developing a stable lyophilized formulation: Protein unfolding during lyophilization must be minimized. Various degradation pathways, including breakdown of the chemical bond between GAA and oligosaccharides and degradation of the GAA protein, must be minimized. The glass transition temperature (Tg) must be higher than the product storage temperature. Residual moisture must be low (<1% by weight). A strong, high-quality cake structure must be obtained. The preferred shelf life should be at least 3 months, preferably 6 months, and more preferably 1 year at room temperature (22-28°C). The reconstitution time should be short, e.g., less than 5 minutes, preferably less than 2 minutes, and more preferably less than 1 minute. If the lyophilized product is reconstituted, the reconstituted sample should be stable for at least 48 hours at 2-8°C.

[0170] The formulations described herein minimize aggregate and particulate formation in pharmaceutical compositions containing oligosaccharide-GAA complexes and ensure that the oligosaccharide-GAA complexes maintain their activity after reconstitution from the lyophilized formulation. The pharmaceutical compositions described herein may include sterile, pharmaceutically acceptable lyophilized formulations made from aqueous pre-lyophilized (i.e., lyophilizable) formulations.

[0171] Lyophilization is a freezing-drying process often used in the manufacture of pharmaceutical products to preserve their biological activity. A liquid composition is produced and then lyophilized to form a dry cake-like product. This process typically involves drying a previously frozen sample in a vacuum to remove the ice, leaving the anhydrous components unchanged in the form of a powder or cake-like substance. Lyophilized products can be stored for extended periods, can be easily reconstituted at elevated temperatures without loss of biological activity, and can be easily reconstituted into a particle-free solution with the addition of a suitable diluent. A suitable diluent is any liquid that is biologically acceptable and in which the lyophilized powder dissolves completely. Water, particularly sterile, pyrogen-free water, is the preferred diluent because it does not contain salts or other compounds that may affect the stability of the oligosaccharide-GAA complex. An advantage of lyophilization is that it reduces the water content to a level that significantly reduces various molecular events that could lead to product instability during long-term storage. Lyophilized products can also more easily withstand the physical stresses of transportation. Because the reconstituted product is particle-free, it can be administered without prior filtration.

[0172] The liquid pre-lyophilized (i.e., lyophilizable) formulation can be lyophilized using appropriate drying parameters. For example, the following drying parameters can be used: a primary drying stage temperature of about -20°C to -50°C and a pressure of about 80 mTorr to about 120 mTorr; and a secondary drying stage at ambient temperature and a pressure of about 80 mTorr. ~120mTorr pressure.

[0173] The lyophilized formulation is rehydrated in a diluent (e.g., sterile water or saline) at the time of use to obtain a particle-free pharmaceutical composition. The reconstituted pharmaceutical composition is particle-free even after long-term storage of the lyophilized cake at ambient temperature. The reconstituted pharmaceutical composition is administered to a subject parenterally, preferably intravenously or subcutaneously.

[0174] An important characteristic of lyophilized formulations is the reconstitution time, or the time it takes to rehydrate the dried powder. It is important that the cake have a highly porous structure to allow for very fast and complete rehydration. The cake structure is a function of several parameters, including the concentration of the oligosaccharide-GAA complex, the type and concentration of excipients, and the process parameters of the lyophilization cycle. Generally, the reconstitution time increases with increasing concentration; therefore, a short reconstitution time is an important goal in developing lyophilized formulations with high concentrations of oligosaccharide-GAA complex.

[0175] Further provided is the use of any one of the combinations or pharmaceutical compositions described herein in the manufacture of a medicament for treating Pompe disease (such as IOPD).Also provided is any one of the combinations or pharmaceutical compositions described herein for use in treating Pompe disease (such as IOPD).

[0176] IV. Kits and Articles of Manufacture The present application further provides kits and articles of manufacture for use in any of the embodiments of the treatment methods described herein. The kits and articles of manufacture may include any one of the formulations and pharmaceutical compositions described herein. For example, a kit is provided that includes a lyophilized formulation of avalglucosidase alfa.

[0177] In some embodiments, provided herein are kits comprising a formulation comprising any one of the oligosaccharide-GAA complexes described herein (e.g., of Formulas I-III, or avaruglucosidase alfa), one or more cryoprotectants, one or more buffering agents (e.g., histidine), and one or more cryoprotectants comprising a sugar that is not degraded by GAA (e.g., mannitol). In some embodiments, the one or more cryoprotectants comprise mannitol and glycine. In some embodiments, the one or more cryoprotectants further comprise a surfactant (e.g., polysorbate 80). In some embodiments, the kit comprises a formulation comprising any one of the oligosaccharide-GAA complexes described herein (e.g., of Formulas I-III, or avaruglucosidase alfa), histidine, glycine, mannitol, and polysorbate 80. In some embodiments, the formulation comprises about 10-50 mM (e.g., about 10 mM) histidine, about 0.25-2% (e.g., about 2%) glycine, about 1-4% (e.g., about 2%) mannitol, and about 0.005-0.05% (e.g., about 0.01%) polysorbate 80. In some embodiments, the formulation is a lyophilized formulation, a lyophilizable formulation, or a reconstituted liquid formulation. In some embodiments, the pH of the formulation is about 5.5 to about 6.5 (e.g., about 6.2). In some embodiments, the formulation comprises about 5 mg / mL to about 10 mg / mL of an oligosaccharide-GAA complex. In some embodiments, the formulation comprises avaruglucosidase alfa.

[0178] In some embodiments, provided herein are articles of manufacture that include a container containing a formulation that includes any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa), one or more cryoprotectants, one or more buffering agents (e.g., histidine), and one or more cryoprotectants that include a sugar that is not degraded by GAA (e.g., mannitol). The cryoprotectant further comprises mannitol and glycine. In some embodiments, the one or more cryoprotectants further comprise a surfactant (e.g., polysorbate 80). In some embodiments, an article of manufacture comprises a container containing a formulation comprising any one of the oligosaccharide-GAA conjugates described herein (e.g., Formulas I-III, or avalglucosidase alfa), about 10-50 mM histidine (e.g., about 10 mM), about 0.25-2% glycine (e.g., about 2%), about 1-4% mannitol (e.g., about 2%), and about 0.005-0.05% polysorbate 80 (e.g., about 0.01%). In some embodiments, the formulation is a lyophilized formulation, a lyophilizable formulation, or a reconstituted liquid formulation. In some embodiments, the pH of the formulation is about 5.5 to about 6.5 (e.g., about 6.2). In some embodiments, the formulation comprises about 5 mg / mL to about 10 mg / mL of an oligosaccharide-GAA complex. In some embodiments, the formulation comprises avalglucosidase alfa. In some embodiments, the container is a vial. In some embodiments, the container is a 20 cc vial.

[0179] The kits and articles of manufacture of the present application are suitably packaged. Suitable packaging includes, but is not limited to, vials, bottles, jars, lightweight packaging (e.g., Mylar or plastic bags), and the like. The kits may optionally provide additional components such as buffers and instructional information. Thus, the present application also provides articles of manufacture that include vials (such as sealed vials), bottles, jars, lightweight packaging, and the like.

[0180] The kit further comprises instructions for use of the oligosaccharide-GAA conjugate in the treatment of Pompe disease (such as IOPD). In some embodiments, the kit further comprises a guide, such as a manual, describing the protocol for any embodiment of the treatment method described herein. The instructions may include information regarding the dosage, administration schedule, and route of administration of the oligosaccharide-GAA conjugate (e.g., avalglucosidase alfa). In some embodiments, the kit further comprises instructions for selecting an individual for a treatment method. In some embodiments, the kit further comprises instructions for monitoring an individual after receiving treatment.

[0181] The containers can be unit doses, bulk packages (e.g., multi-dose packages), or small unit doses. Kits can be provided that contain sufficient oligosaccharide-GAA conjugates (e.g., avalglucosidase alfa) described herein to provide effective treatment to an individual for an extended period of time, such as 3 weeks, 6 weeks, 9 weeks, 3 months, 4 months, 5 months, 6 months, 8 months, 9 months, 1 year, or more.

[0182] All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features. [Example]

[0183] The following examples are intended to be purely illustrative of the present invention and, as such, should not be considered to impose any limitations on the present invention. The following examples and detailed description are offered by way of illustration and not by way of limitation. [Example]

[0184] Clinical trial of avalglucosidase alfa in infantile-onset Pompe disease The following example describes an open-label, ascending-dose cohort study to determine the safety, pharmacokinetics, and preliminary efficacy of avalglucosidase alfa in patients with infantile-onset Pompe disease who were treated with avalglucosidase alfa and showed clinical attenuation or a suboptimal clinical response.

[0185] A. Research design the purpose The primary objective of this study was to evaluate the safety profile of avalglucosidase alfa in patients with infantile-onset Pompe disease (IOPD) previously treated with alglucosidase alfa. Secondary objectives were to characterize the pharmacokinetic profile of avalglucosidase alfa and evaluate the preliminary efficacy of avalglucosidase alfa compared with alglucosidase alfa.

[0186] methodology We conducted a multistage, phase 2, open-label, multicenter, multinational, ascending-dose cohort study involving repeated intravenous infusions of avalglucosidase alfa in pediatric IOPD patients who had previously been treated with avalglucosidase alfa and who had demonstrated clinical attenuation (Stage 1) or suboptimal clinical response (Stage 2) in specific respiratory function, exercise capacity, cardiac parameters, and / or new-onset ptosis (including eyelid position measurements such as interpalpebral fissure diameter (IPFD), eyelid margin corneal reflex distance-1 (MRD-1), and eyelid margin pupillary distance (MPD)). A schematic overview of the study design is provided in Figure 1.

[0187] Patients in cohorts 1 and 2 had demonstrated evidence of clinical decline in at least one of the following parameters associated with Pompe disease: respiratory function, exercise capacity, and / or cardiac parameters. Patients in cohort 3 had demonstrated evidence of a suboptimal clinical response in at least one of the following parameters associated with Pompe disease: respiratory function, exercise capacity, and / or new-onset ptosis (confirmed by at least two consecutive assessments) while receiving alglucosidase alfa.

[0188] In Stage 1, patients in Cohort 1 received avalglucosidase alfa 20 mg / kg throughout the study, and patients in Cohort 2 received avalglucosidase alfa 40 mg / kg throughout the study (Figure 1). In Stage 2, patients enrolled in Cohort 3 were randomized to receive either the maximum tolerated dose of avalglucosidase alfa (i.e., 40 mg / kg) from Cohorts 1 and 2, or avalglucosidase alfa (a stable dose for at least 6 months prior to enrollment in the study, the labeled dose, or higher) for the first 6 months of the study before switching to avalglucosidase alfa 40 mg / kg. All patients received avalglucosidase alfa for the remainder of the study.

[0189] Diagnosis and inclusion and exclusion criteria Included patients had documented GAA deficiency, were under 18 years of age, had cardiac complications at the time of Pompe disease diagnosis, had been receiving a stable dose of alglucosidase alfa regularly for a minimum of 6 months immediately prior to study entry, and had documented evidence of clinical attenuation (stage 1 patients) or suboptimal clinical response (stage 2 patients).

[0190] The inclusion criteria for this study were as follows: Patients have confirmed acid alpha-glucosidase (GAA) enzyme deficiency from any tissue source (e.g., blood, skin, or muscle tissue). The patient, who had reached the legal age of majority as defined by local regulations, or the patient's legal guardian(s) provided signed informed consent before any study-related procedures were performed. If the patient was a legal minor according to local regulations, consent was obtained from multiple patients, if applicable. · The patient (or the patient's legal guardian, if the patient is a legal minor as defined by local regulations) had to be competent to comply with the clinical protocol. The patient was under 18 years old. Patients, if female and of childbearing potential, had to have a negative serum pregnancy test (beta-human chorionic gonadotropin) and not be lactating at screening / baseline. Patients had cardiomyopathy at diagnosis within the first year of life: i.e., a left ventricular mass index (LVMI) equivalent to the mean age-specific LVMI plus two standard deviations. Patients had been receiving a stable dose of alglucosidase alfa regularly for at least 6 months immediately prior to study entry. For participants in Stage 1: Patients had demonstrated evidence of clinical decline in at least one of the following parameters related to Pompe disease and not related to intercurrent illness: respiratory function, exercise capacity, and / or cardiac parameters. For participants in Stage 2: Patients had demonstrated evidence of a suboptimal clinical response in at least one of the following parameters related to Pompe disease and not related to intercurrent illness: respiratory function, exercise capacity, and / or new-onset ptosis.

[0191] Excluded patients had high antibody titers (i.e., anti-alglucosidase alfa antibody titers ≥ 1:25,600 at two consecutive time points separated by more than one month); clinically significant organic disease (other than Pompe disease) that could pose a high risk for abnormal laboratory parameters or reduced survival; or severe allergic reaction to alglucosidase alfa (i.e., history of previous moderate / severe anaphylactic reaction, IgE antibodies, or high IgG antibodies to alglucosidase alfa).

[0192] The exclusion criteria for this study were as follows: The patient had high antibody titers against alglucosidase alfa. The patient was at high risk of a severe allergic reaction to avalglucosidase alfa. Patients required any prohibited concomitant medication (e.g., immunomodulatory treatment) for the duration of the study. Patients had previously participated in any ACT14132 study cohort. Female patients of childbearing potential who have not been protected with a highly effective method of birth control and / or who are unwilling or unable to undergo a pregnancy test.

[0193] Primary and key secondary endpoints The primary safety endpoints included: (1) adverse events / treatment-emergent adverse events (TEAEs), including infusion-related reactions (IARs) (Table 5); (2) clinical laboratory analyses, including standard biochemistry, hematology, and urinalysis, as narrative summaries; and (3) immunogenicity assessment, as narrative summaries.

[0194] Secondary pharmacokinetic endpoints included C max , t max , area under the curve (AUC 0-last ), elimination phase half-life (t 1 / 2z Plasma parameters including ), clearance (CL), and volume of distribution (Vd) were included.

[0195] Secondary efficacy endpoints included changes in the following parameters at 6 months: Gross Motor Function Scale-88 (GMFM-88, see Figures 2A-2B) and Gross Motor Function Classification System-Extended and Revised (GMFCS-E&R); Pompe Pediatric Evaluation of Disability Indicator (Pompe-PEDI) Functional Capacity Scale: Mobility domain (Figure 3); Quick Motor Function Test (QMFT); echocardiography (ECHO) endpoints (Table 6): left ventricular mass index (LVMI) and left ventricular mass (LVM) Z-score; and creatine kinase levels (Figures 4A-4B).

[0196] Other secondary endpoints included the 6-minute walk test (6MWT), which measures the distance walked without stopping and using assistive devices by patients who are able to ambulate at least 40 meters (approximately 130 feet); ventilator use, and urinary HEX-4 levels (Figures 5A-5B).

[0197] Another secondary outcome was eyelid position measurement. Assessments were performed at screening / baseline, day 1, and every 2 weeks from week 3 through week 25 (inclusive). Diagnosis of ptosis, defined as an inclusion criterion for suboptimal improvement, was based on the assessment of the treating investigator; in addition, site readers assessed the presence of ptosis based on photographs taken at baseline and each visit, including week 25. Images of participants' eyes were taken while wearing a novel measurement device consisting of a pair of empty eyeglass frames with an attached ruler as a standardized measurement tool (Figure 6A). The following eyelid position measurements were performed by readers at an independent institution blinded to treatment group and dose: (1) interpalpebral fissure diameter (IPFD): the vertical distance between the upper and lower eyelid margins (Figure 6B); (2) lid margin corneal reflex distance-1 (MRD-1): the distance between the corneal light reflex and the upper eyelid margin (Figure 6C); (3) lid margin pupillary distance (MPD): the distance between the center of the pupil and the upper eyelid margin (Figure 6D).

[0198] statistical methods A narrative summary of safety results was provided based on the safety population, defined as all patients (partial or total) who received at least one infusion during the study. Data on adverse events, laboratory safety variables, and immunogenicity were summarized.

[0199] Narrative summaries and / or plots of efficacy endpoints, pharmacokinetic and pharmacodynamic endpoints were provided for all cohorts.

[0200] Avalglucosidase alfa formulation and administration Avalglucosidase alfa was formulated in vials, each containing approximately 105 mg of avalglucosidase alfa in 10 mM histidine, 2% glycine, 2% mannitol, and 0.01% polysorbate 80, pH 6.2. This was reconstituted with sterile water to a final concentration of approximately 10 mg / mL for injection prior to administration. The reconstituted solution was diluted with 5% dextrose in water to a final concentration of 0.5 mg / mL to 4 mg / mL.

[0201] Infusions should be administered gradually over approximately 6 hours for IOPD patients, as determined by the patient's response and comfort. For IOPD patients, it is recommended that the infusion be initiated at an initial rate of 1 mg / kg / h and gradually increased by 2 mg / kg / h every 30 minutes in the absence of signs of an infusion-related reaction (IAR) until a maximum rate of 7 mg / kg / h (for LOPD patients) and 10 mg / kg / h (for IOPD patients) is reached. Vital signs should be obtained before each stepwise increase in infusion rate. Patients are pretreated with antihistamines, antipyretics, and / or corticosteroids to prevent or reduce allergic reactions.

[0202] B. Results Population characteristics: Six patients were enrolled in Cohort 1, 5 in Cohort 2, and 11 in Cohort 3 (Table 1). Patients were enrolled at two centers in France, two in Japan, two in the UK, three in the US, and one each in China and Taiwan. Of the patients in Cohort 3, six were randomized to alglucosidase alfa and five to avalglucosidase alfa. Patients were between 1 and 12 years of age.

[0203] Baseline patient demographics and characteristics were comparable across the three cohorts and treatment arms, except for age: patients in Cohort 3 randomized to alglucosidase alfa were younger (Table 1). Table 2 provides a summary of patients' medical histories from the time of diagnosis prior to study enrollment. Baseline functional levels were heterogeneous across all patients, with motor dysfunction less severe in Cohort 3 (Table 3). There were two cross-reactive immunoglobulin (CRIM)-negative patients and 20 CRIM-positive patients (Table 3).

[0204] Participants in the alglucosidase alfa treatment arm of Cohort 3 were younger than participants in the other cohorts at baseline and received dose regimens ranging from 20 mg / kg every other week to 40 mg / kg weekly. Participants in Cohort 1 were predominantly male. Overall, participants' growth parameters were generally within normal ranges.

[0205] [Table 1]

[0206] [Table 2]

[0207] [Table 3]

[0208] No patients discontinued during the primary analysis period (25-week treatment period) or by the data cutoff date after the last patient's last visit (LPLV) of the primary analysis period on September 30, 2019. All patients had at least 6 months of available data.

[0209] Study drug exposure during the primary analysis period There were no missed infusions or dose reductions, and all 22 enrolled patients completed the primary analysis period (through week 25; Table 4). Overall treatment compliance was good; no participants missed infusions or had dose reductions.

[0210] In the alglucosidase alfa treatment arm of Cohort 3, each patient had their own regimen based on their previous stable dose, ranging from 20 mg / kg every 2 weeks to 40 mg / kg weekly. Two patients in Cohort 1 had their dose increased after the primary analysis period, per protocol.

[0211] [Table 4]

[0212] safety results There was a similar incidence of treatment-emergent adverse events (TEAEs) across all cohorts and between the two treatment arms in Cohort 3 (Table 5). Safety was consistent with the known safety profiles of both products and underlying conditions at both dose levels tested (20 and 40 mg / kg every other week) in severely affected, previously treated IOPD patients. The most common TEAEs were vomiting and fever (6 patients each), upper respiratory tract infection (5 patients), cough and rash (4 patients each).

[0213] [Table 5]

[0214] There were no treatment-related serious adverse events. There were no deaths or withdrawals due to adverse events in either cohort.

[0215] Four patients experienced at least one infusion-related reaction (IAR) during the primary analysis period, and four patients experienced at least one IAR during the extended treatment period (ETP) (Table 5).

[0216] There were no adverse events related to potentially clinically significant laboratory or echocardiographic abnormalities.

[0217] Six patients developed anti-drug antibodies (ADA) to avalglucosidase alfa with a median peak titer of 6400, and ADA increased, i.e., after administration of the study drug (first This included one patient with a prior ADA that increased by at least two titer steps from baseline (i.e., a four-fold increase in titer) at any time after drug administration. All but one patient had a decrease in ADA over time; this patient had a peak titer of 6400 at the last available time point.

[0218] Of note, very few patients were CRIM-negative. Patients with previous high antibody titers (anti-alglucosidase alfa titers ≥ 1:25600), including those who had previously undergone alglucosidase alfa treatment, were excluded.

[0219] Efficacy results The majority of patients (14 / 16 treated with avalglucosidase alfa and 6 / 6 treated with alglucosidase alfa) experienced symptomatic stabilization or improvement (i.e., entry criteria of clinical attenuation / suboptimal response). Two worsening patients had their avalglucosidase alfa dose increased from 20 to 40 mg / kg per protocol after the primary analysis period.

[0220] With avalglucosidase alfa treatment, there were positive trends (stabilization or improvement) for the exercise scale GMFM-88 (Figures 2A-2B), Pompe-PEDI (Figure 3), and echocardiographic assessments (e.g., echocardiographic left ventricular mass (LVM) Z-score) (Table 6). Individual patients across all cohorts showed improvement or stabilization for the Pompe-PEDI functional capacity scale (Figure 3). During the primary analysis period, no decline from baseline in ECHO-LVM Z-score was observed, and the only patient with an abnormal LVM Z-score at baseline improved to the normal range at week 25 (a patient in CRIM-negative cohort 2) (Table 6).

[0221] [Table 6]

[0222] At the group level, no clear differences were observed in cohort 3 at week 25 between avalglucosidase alfa (40 mg / kg every other week) and alglucosidase alfa (previous stable dose; range 20–40 mg / kg every other week / week), except for an imbalance between the treatment arms regarding patient age (i.e., there were younger patients in the alglucosidase alfa arm).

[0223] Pharmacokinetic results Creatine kinase (Figures 4A-4B) and urinary HEX4 (Figures 5A-5B) parameters showed high interindividual variability at all dose levels, with a trend toward more pronounced decreases in the avalglucosidase alfa 40 mg / kg group. Levels of pharmacodynamic disease biomarkers for muscle damage (creatine kinase [CK]) and glycogen loading (hexose tetrasaccharide [Hex4]) were also reduced with avalglucosidase alfa compared with stable levels with alglucosidase alfa.

[0224] ptosis The results of eyelid position measurements are shown in Table 7 below. At baseline, seven of the 22 participants enrolled in Mini-COMET had a clinical diagnosis of ptosis, including three bilateral and four unilateral cases. At week 25, seven participants had ptosis, including three bilateral and four unilateral cases. In cohort 1 (avalglucosidase alfa 20 mg / kg every other week), one participant had ptosis in the left eye, which resolved, and two additional participants developed bilateral ptosis by week 25. In cohort 2 (avalglucosidase alfa 40 mg / kg every other week), one participant had ptosis in the right eye at baseline, and none had ptosis in either eye by week 25. In cohort 3 (alglucosidase alfa 40 mg / kg every other week), one additional participant developed right ptosis by week 25, while one participant had bilateral ptosis at baseline that was absent by week 25. In cohort 3 (alglucosidase alfa 20 mg / kg every other week to 40 mg / kg every week), two participants had right ptosis at baseline that was still present in one participant by week 25 and had resolved in one participant, while one participant had left ptosis at baseline that was still present at week 25.

[0225] [Table 7]

[0226] Figures 7A–7F compare the results of various eyelid position measurements across different cohorts. From baseline to week 25, there was a trend toward improvement in eyelid position measurements in the 40 mg / kg every other week avalglucosidase alfa group (Cohort 2 and the avalglucosidase alfa arm of Cohort 3). In contrast, the 20 mg / kg every other week avalglucosidase alfa group (Cohort 1) and the alglucosidase alfa group (Cohort 3, the alglucosidase alfa arm) showed stabilization or deterioration in these measurements. Differences between treatment groups were more pronounced for vertical palpebral fissure diameter (both eyes, with and without flash) than for lid margin corneal reflex distance (both eyes) or lid margin pupillary distance (both eyes). Of note, non-flash images, especially those measuring vertical palpebral fissure diameter and lid margin-pupillary distance, allowed for more accurate measurements because there was no squinting caused by flash photography.

[0227] Individual participant responses Individual participant responses in the Mini-COMET study were analyzed by plotting baseline and week 25 data in individual spaghetti plots.

[0228] Figures 8A-8D show Pompe disease burden biomarkers: (1) hexose tetrasaccharide (Hex4) Figure 8 shows individual responses in different cohorts, as measured by (1) creatine kinase (CK) levels and (2) creatine kinase (CK) levels. The decrease in Hex4 from baseline to week 25 was greatest in participants treated with avalglucosidase alfa 40 mg / kg every other week with baseline values ​​>40 mmol / mol, whereas changes were more variable for participants treated with alglucosidase alfa and avalglucosidase alfa 20 mg / kg every other week (Figures 8A-8B). The decrease in CK from baseline to week 25 appeared to be primarily observed in participants with baseline levels >900 IU / L, decreasing in participants treated with avalglucosidase alfa and tending to remain stable in participants treated with alglucosidase alfa (Figures 8C-8D).

[0229] Figures 9A-9D show individual responses in different cohorts as measured by motor function parameters, including GMFM-88 and QMFT. The GMFM-88 and QMFT are considered to have maximal improvement with baseline scores of 20-70% (on a 0-100% scale) and 10-45 (on a 0-64 scale), respectively. Participants with lower or higher functioning remained stable or trended toward decline (Figures 9A-9D).

[0230] Participants with GMFM-88 scores prior to initiating avalglucosidase alfa treatment demonstrated consistent improvements in motor function after switching from weekly high avalglucosidase alfa treatment to avalglucosidase alfa 40 mg / kg every other week. Case studies of three individuals are shown in Figures 10A-10C and Table 8 below.

[0231] [Table 8]

[0232] Six-minute walk test (6MWT) distance in ambulatory participants older than 6 years at baseline was significantly improved with avalglucosidase alfa 40 mg / kg every other week in cohorts 2 and 3. improved for all participants receiving avalglucosidase alfa (20 mg / kg every other week) (Figure 11A), while participants in the alglucosidase alfa arm of Cohort 1 and Cohort 3 who received a lower dose of avalglucosidase alfa (20 mg / kg every other week) (dose range, 20 mg / kg every other week to 40 mg / kg every week) remained stable or declined during the first 25 weeks of treatment (Figure 11B).

[0233] Limited data were available on pulmonary function testing based on age-related limitations in the ability to reliably undergo testing. At baseline, five participants were ventilated, and few changes without respiratory attenuation were observed.

[0234] conclusion Baseline patient demographics and characteristics were comparable across the three cohorts and treatment arms, except for age: (Mean age was younger in Cohort 3 patients randomized to alglucosidase alfa.) Baseline functional levels were heterogeneous across all patients, with less severe motor dysfunction in Cohort 3.

[0235] No patients discontinued during the primary analysis period or by the data cutoff date. Additionally, there were no deaths or treatment-related serious adverse events.

[0236] The safety and immunogenicity profile was favorable. In patients with severely affected IOPD who had previously been treated with dose regimens ranging from 20 mg / kg every 2 weeks to 40 mg / kg every week and had an incomplete treatment response to alglucosidase alfa, avalglucosidase alfa was generally safe and well tolerated at 20 and 40 mg / kg every 2 weeks. Disease biomarkers and exploratory efficacy measures showed a trend toward improvement in most study participants.

[0237] There was a trend toward more pronounced creatine kinase (and HEX4) declines in patients receiving avalglucosidase alfa 40 mg / kg. Interindividual variability was high.

[0238] Additionally, a trend toward improved eyelid position was observed in participants receiving avalglucosidase alfa 40 mg / kg every other week, compared with stabilization or attenuation in participants receiving avalglucosidase alfa 20 mg / kg every other week or avalglucosidase alfa at doses ranging from 20 mg / kg every other week to 40 mg / kg every week. This effect was more pronounced for vertical palpebral fissure diameter than for either the lid margin corneal reflex distance-1 or the lid margin pupillary distance. These data further support the stronger effect of avalglucosidase alfa 40 mg / kg every other week on clinically meaningful outcome measures (i.e., prevention of worsening ptosis, ultimately reducing the risk of amblyopia or the need for surgical intervention) in long-term survivors of IOPD.

[0239] The majority of patients (14 / 16 patients treated with avalglucosidase alfa and 6 / 6 patients treated with alglucosidase alfa) experienced stabilization or improvement of the entry criterion of clinical attenuation / suboptimal response.

[0240] Patient-level analyses clarify the effect of avalglucosidase alfa due to heterogeneous baseline functional levels across patient cohorts. Analyses were performed to account for heterogeneous baseline functional levels across patients in the three cohorts and treatment arms, as well as differences in age at diagnosis and study entry, previous avalglucosidase alfa treatment, and gender balance.

[0241] Avalglucosidase alfa at 20 and 40 mg / kg every other week was Participant-level analyses confirmed that avalglucosidase alfa (20 mg / kg every two weeks to 40 mg / kg every week) appears to improve or better stabilize Pompe disease symptoms with respect to pharmacodynamic disease biomarkers, motor outcomes, respiratory parameters, and eyelid measures. The highest dose of avalglucosidase alfa tested, 40 mg / kg every two weeks, appears to provide additional benefit in meaningful outcome measures while maintaining a favorable safety profile and acceptable immunogenicity.

[0242] These data support the promising clinical impact associated with the use of avalglucosidase alfa in patients with IOPD, the most severely affected population of Pompe disease patients. [Example]

[0243] Clinical trial of avalglucosidase alfa in treatment-naive infantile-onset Pompe disease The following example describes a phase 3, open-label, multinational, multicenter, intravenous infusion study of the efficacy, safety, pharmacokinetics, and pharmacodynamics of avalglucosidase alfa in treatment-naive pediatric patients aged 6 months or younger with infantile-onset Pompe disease.

[0244] A. Research design the purpose The primary objective of this study was to determine the effect of avalglucosidase alfa treatment on survival and invasive ventilation-free survival in patients with infantile-onset Pompe disease (IOPD) aged 6 months or younger after 52 weeks of treatment. Secondary objectives of the study were to determine the effect of avalglucosidase alfa treatment on survival and invasive ventilation-free survival at 12 and 18 months of age, as well as on changes in left ventricular mass Z-score (LVM Z-score), Alberta Infant Motor Development Scale (AIMS) score, body length, weight, and head circumference percentiles, and urinary Hex4 levels at 52 weeks of study. Other secondary objectives of the study were to determine the safety, tolerability, and immunogenicity of avalglucosidase alfa and to determine its pharmacokinetic (PK) profile at 12 and 52 weeks.

[0245] methodology Patients were enrolled in a single, experimental arm and received intravenous (IV) infusions of avalglucosidase alfa every 2 weeks. Primary endpoint data were collected at week 52, prior to study completion.

[0246] Diagnosis and inclusion and exclusion criteria Included patients had to have a confirmed diagnosis of infantile-onset Pompe disease and be aged 6 months or younger.

[0247] The inclusion criteria for this study were as follows: Patients had a confirmed diagnosis of infantile-onset Pompe disease, defined as: (1) the presence of two lysosomal acid alpha-glucosidase (GAA) pathogenic variants and documented GAA deficiency from blood, skin, or muscle tissue, or (2) the presence of one GAA pathogenic variant and documented GAA deficiency in two separate samples from blood, skin, or muscle tissue (either from two different tissues or the same tissue but from two different sampling dates). Patients had established cross-reactive immunomaterial (CRIM) status available prior to enrollment, as determined by previous CRIM test results, prediction of CRIM based on genotyping, or testing at screening by a Clinical Laboratory Improvement Amendments (CLIA) or other appropriately certified laboratory. was provided. Patients had cardiomyopathy at the time of diagnosis; i.e., a left ventricular mass index (LVMI) equivalent to the mean age-specific LVMI plus one standard deviation for participants diagnosed by newborn screening or sibling screening, or equivalent to two standard deviations for participants diagnosed by clinical evaluation. The patient's parents or legally authorized representative(s) were able to provide signed informed consent.

[0248] The exclusion criteria for this study were as follows: Patients had symptoms of respiratory failure or were receiving any type of mechanical ventilation (invasive or non-invasive) at the time of enrollment. The patient had a severe congenital anomaly. Patients had clinically significant organic disease (excluding conditions related to Pompe disease). Patients were receiving enzyme replacement therapy (ERT) with recombinant human acid alpha-glucosidase (rhGAA) from any source. Patients were previously treated in any clinical trial of avalglucosidase alfa. The patient was deemed by the investigator to be unsuitable for participation for any reason, including a medical or clinical condition, or the patient was at risk of non-compliance with study procedures.

[0249] Primary and key secondary endpoints The primary endpoint of the study was the proportion of patients alive and invasively ventilator-free at 52 weeks.

[0250] Secondary endpoints of the study included assessment of the following at 52 weeks: proportion of patients alive and free of invasive ventilation at 12 and 18 months of age; proportion of participants alive at 52 weeks; proportion of patients alive at 12 and 18 months of age; and proportion of patients ventilator-free and not using supplemental oxygen at 52 weeks.

[0251] Secondary endpoints also included assessment of the following at week 52: assessment of treatment-emergent adverse events (TEAEs), including infusion-related reactions; physical examination; clinical laboratory assessments; vital sign measurements; 12-lead electrocardiogram (ECG); and immunogenicity assessment.

[0252] Other secondary endpoints included changes from baseline to week 52 in the following parameters: left ventricular mass (LMV) Z-score, Alberta Infant Motor Development Scale (AIMS) score, physical growth Z-score, urinary Hex4, and percentiles of body length, weight, and head circumference.

[0253] Additionally, plasma concentrations of acid alpha-glucosidase (GAA) were determined at day 1, week 12, and week 52.

[0254] Duration of the study period Patients were treated for 52 weeks during the primary analysis period and continued treatment in a 52-week extension treatment period. This was followed by an extended treatment period of up to 104 weeks plus a 4-week follow-up period, for a total study duration of up to 4.08 years. This duration may vary by country, but patients continued until avalglucosidase alfa was approved in their country or until 4.08 years, whichever occurred first.

[0255] Avalglucosidase alfa combination Avalglucosidase alfa was formulated as a sterile, lyophilized powder that was reconstituted prior to administration by IV infusion.

Claims

1. A pharmaceutical composition for treating infant-onset Pompe disease (IOPD) in a human individual requiring such treatment, comprising an oligosaccharide-protein complex and a pharmaceutically acceptable carrier, wherein the oligosaccharide-protein complex is aggregate glucosidase alpha, the pharmaceutical composition contains a dose of 40 mg / kg of aggregate glucosidase alpha, the human individual is 6 months of age or younger, and the human individual has not received enzyme replacement therapy with recombinant human acid alpha-glucosidase (GAA).

2. For the aforementioned individual: (i) Once every two weeks; (ii) intravenously; and / or (iii) At least about 25 weeks The pharmaceutical composition according to claim 1, which is administered.

3. The pharmaceutical composition according to claim 1 or 2, which is reconstituted from a freeze-dried formulation containing the oligosaccharide-protein complex.

4. The pharmaceutical composition according to claim 3, wherein the pharmaceutically acceptable carrier comprises a sugar that is not decomposed by GAA.

5. The pharmaceutical composition according to claim 4, wherein the sugar that is not broken down by the GAA is mannitol.

6. The pharmaceutical composition according to any one of claims 3 to 5, wherein the pharmaceutically acceptable carrier further comprises glycine or histidine.

7. A pharmaceutical composition according to any one of claims 3 to 6, having a pH of approximately 6.

2.

8. The aforementioned pharmaceutically acceptable carrier is approximately 10-50 mM histidine, approximately 0.25-2% A pharmaceutical composition according to any one of claims 3 to 7, comprising glycine, about 1 to 4% mannitol, and about 0.005 to 0.05% polysorbate 80.

9. The pharmaceutical composition according to claim 8, wherein the pharmaceutically acceptable carrier comprises about 10 mM histidine, about 2% glycine, about 2% mannitol, and about 0.01% polysorbate 80.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the individual has cardiomyopathy at the time of diagnosis within one year of birth.

11. The pharmaceutical composition according to claim 10, wherein the individual has arrhythmia or cardiac hypertrophy.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the recombinant GAA is alglucosidase alpha.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the individual is negative for cross-reactive immunosorbents (CRIM).

14. The pharmaceutical composition according to any one of claims 1 to 12, wherein the individual is CRIM positive.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the individual exhibits a decrease in the level of anti-drug antibodies (ADA) against oligosaccharide-protein complexes over time.

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein the individual exhibits improvement or stabilization of one or more respiratory functions as evaluated by the use of a ventilator.

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein the individual uses a ventilator less frequently per day, week, or month compared to baseline, as measured after 52 weeks of treatment.

18. The pharmaceutical composition according to any one of claims 1 to 17, wherein administration of the pharmaceutical composition extends the invasive ventilator-free survival period and / or overall survival period of an individual having IOPD.

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