Highly potent modified acid alpha-glucosidase and method of making and using it

Abstract

This invention describes a recombinant human α-glucosidase (rhGAA) composition derived from CHO cells, containing a higher amount of rhGAA containing N-glycans with mannose-6-phosphate (M6P) or bis-M6P than conventional rhGAA; a composition containing the rhGAA; and a method of use.

Description

Technical Field

[0001] This application claims priority to International Application No. PCT / US / 2015 / 039098, filed July 2, 2015, U.S. Provisional Application No. 62 / 057,842, filed September 30, 2014, U.S. Provisional Application No. 62 / 057,847, filed September 30, 2014, U.S. Provisional Application No. 62 / 112,463, filed February 5, 2015, and U.S. Provisional Application No. 62 / 135,345, filed March 19, 2015, each of which is incorporated herein by reference in its entirety. This application is related to U.S. Provisional Application No. 62 / 020,653, filed July 3, 2014, which is incorporated herein by reference in its entirety.

[0002] The present invention relates to the fields of medicine, genetics, and recombinant glycoprotein biochemistry, and more particularly to recombinant human alpha-glucosidase (rhGAA) compositions having a higher total content of mannose-6-phosphate glycans that effectively targets rhGAA to target tissues, such as the muscle cells of individuals with Pompe disease, where it can translocate to cellular lysosomes and break down abnormally high levels of accumulated glycogen. The rhGAA of the present invention exhibits superior lysosomal targeting in muscle cells compared to conventional rhGAA products and other pharmacokinetic properties that make it particularly effective for enzyme replacement therapy in individuals with Pompe disease. Prior Art

[0003] Current enzyme replacement therapies for Pompe disease use conventional rhGAA products with low levels of total glycans containing M6P and bis-M6P. These conventional products are known by the names Lumizyme®, Myozyme®, and Alglucosidase α. "Lumizyme" and "Myozyme" are conventional forms of rhGAA manufactured or marketed as biologics by Genzyme and approved by the US Food and Drug Administration (FDA). These are described in the Physician's Desk Reference (2014), which is incorporated herein by reference, or by products named "Lumizyme®" or "Myozyme®" approved for use in the United States by the FDA as of October 1, 2014. Glucosidase alfa is identified by the chemical name [199-arginine, 223-histidine] prepro-α-glucosidase (human); molecular formula C 4758H 7262N 1274O 1369S 35; CAS number 420794-05-0. These products are administered to individuals with Pompe disease, also known as glycogen storage disease type II (GSD-II) or acid maltase deficiency. Enzyme replacement therapy attempts to treat Pompe disease by replacing the missing GAA with rhGAA, thereby restoring the cells' ability to break down glycogen.

[0004] Pompe disease is an inherited lysosomal storage disorder caused by a deficiency in the activity of acid alpha-glucosidase (GAA). Individuals with Pompe disease lack the enzyme acid alpha-glucosidase (GAA), which breaks down glycogen, a substance the body uses as energy. This enzyme deficiency causes excess glycogen to accumulate in lysosomes, organelles found within cells that contain enzymes that normally break down glycogen, along with other cellular debris or waste products. Glycogen accumulates in certain tissues, particularly muscle, in individuals with Pompe disease, impairing the tissue's ability to function properly. In Pompe disease, glycogen is not properly metabolized and gradually accumulates in lysosomes, particularly in skeletal muscle cells and, in the infantile-onset form of the disease, in the lysosomes of heart muscle cells. Glycogen accumulation damages muscle and nerve cells, as well as other affected tissues.

[0005] Pompe disease has traditionally been clinically recognized as either an early infantile or late-onset form, depending on the age of onset. However, Pompe disease spans a spectrum, with the most severe gene mutations resulting in complete loss of GAA activity and presenting during infancy, while other gene mutations (such as splicing defects and missense mutations) result in some residual GAA activity, slowing the onset and progression of the disease. Infantile-onset Pompe disease manifests shortly after birth and is characterized by muscle weakness, respiratory insufficiency, and heart failure. Untreated, it is usually fatal within two years. Juvenile- and adult-onset Pompe disease manifests later in life and typically progresses more slowly than the infantile-onset form. While these forms of the disease generally do not affect the heart, they can also be fatal due to weakening of skeletal muscles and those involved in breathing.

[0006] Current non-remedial treatments for Pompe disease include enzyme replacement therapy (ERT) using recombinant human GAA (rhGAA), such as Lumizyme® or Myozyme®. rhGAA replaces or supplements the GAA that is deficient or missing in individuals with Pompe disease. However, most rhGAA in conventional rhGAA products is not targeted to lysosomes and is not effectively eliminated after administration.

[0007] This occurs because conventional rhGAA lacks a high total content of glycans with M6P and bis-M6P, which target the rhGAA molecule to cells and subsequently the lysosomes of the cell. Cellular uptake of rhGAA for enzyme replacement therapy requires the specialized carbohydrate mannose-6-phosphate (M6P), which binds to the cation-independent mannose-6-phosphate receptor (CIMPR) present on the cell surface for subsequent delivery of the exogenous enzyme to the lysosome.

[0008] rhGAA typically contains different types of N-linked oligosaccharides (N-glycans) with varying binding affinities for the M6P receptor and other carbohydrate receptors. rhGAA containing N-glycans with one M6P group (mono-M6P) binds to CIMPR with moderate affinity, while rhGAA containing glycans with two M6P groups on the same N-glycan (bis-M6P) binds with high affinity. Once within lysosomes, GAA can enzymatically degrade accumulated glycogen, alleviating disease. However, conventional rhGAA has a low total content of glycans containing M6P and bis-M6P and is therefore poorly targeted to lysosomes. The majority of rhGAA molecules in these conventional products contain N-glycans that are non-phosphorylated, high-mannose N-glycans with no affinity for CIMPR. However, they have a relatively high affinity for mannose receptors on macrophages in the liver, spleen, and other non-target tissues, resulting in rapid and ineffective clearance of rhGAA. Figure 1A shows representative structures of non-phosphorylated mono- and di-M6P glycans. Figure 1B shows the mannose-6-P group.

[0009] Other types of N-glycans, called complex N-glycans, containing complex carbohydrates such as N-acetylglucosamine, galactose, and sialic acid, are also present on rhGAA. Complex N-glycans have no affinity for CIMPR. However, incompletely processed complex N-glycans with exposed galactose residues have moderate to high affinity for the asialoglycoprotein receptor on hepatocytes, leading to rapid and inefficient clearance of rhGAA.

[0010] Glycosylation of GAA or rhGAA can be modified in vitro using enzymes, such as the phosphotransferase / mannosidase described in U.S. Patent No. 6,534,30 by Canfield et al., to introduce M6P groups. Enzymatic glycosylation is not well controlled and produces rhGAA with undesirable immunological and pharmacological properties. Enzymatically modified rhGAA may contain only high-mannose N-glycans, all of which are potentially phosphorylated in vitro by phosphotransferase / mannosidases, and each GAA may contain an average of 5-6 M6P groups. The glycosylation pattern generated by in vitro enzymatic treatment of GAA is problematic because the additional mannose residues, particularly the non-phosphorylated terminal mannose residues, negatively impact the pharmacokinetics of the modified rhGAA. When such enzymatically modified products are administered in vivo, these mannose groups increase the inefficient clearance of GAA, enhance the uptake of enzymatically modified GAA by immune cells, and reduce the therapeutic efficacy of rhGAA due to less GAA reaching target tissues (such as cardiac or skeletal muscle cells). For example, terminal non-phosphorylated mannose residues are known ligands for mannose receptors in the liver and spleen, leading to rapid clearance of enzymatically modified rhGAA and reduced targeting of rhGAA to target tissues. Furthermore, the glycosylation pattern of enzymatically modified GAA with high-mannose N-glycans containing terminal non-phosphorylated mannose residues resembles that of enzymatically modified rhGAA in terms of its function as glycoproteins produced by yeast and mold, increasing the risk of triggering immune or allergic reactions, such as life-threatening allergic / anaphylactic or hypersensitivity reactions.

[0011] As mentioned above, conventional rhGAA products (such as Lumizyme®) have low amounts of mono-M6P and even lower amounts of bis-M6P on their glycans. This is problematic because the efficacy of Pompe disease treatment depends on rhGAA targeting to lysosomes. The low total amount of M6P and bis-M6P targeting groups on conventional rhGAA limits its cellular uptake and delivery to lysosomes, thereby rendering conventional enzyme replacement therapy ineffective. For example, while conventional rhGAA products do improve Pompe disease at doses of 20 mg / kg or higher, they fail to sufficiently reduce accumulated glycogen in many target tissues, particularly skeletal muscle.

[0012] Furthermore, conventional enzyme replacement therapy is associated with several issues. These include the necessity of administering large doses of rhGAA compositions to achieve adequate cellular uptake of rhGAA in target tissues. However, large doses of rhGAA, particularly large amounts of unphosphorylated rhGAA, increase the risk of individuals developing an immune response to rhGAA. Immunity to rhGAA is undesirable because it increases the risk of treatment failure and can induce harmful autoimmune or allergic reactions. Logistically, large doses impose additional burdens on individuals and the medical professionals treating them, such as prolonged infusion times required for intravenous rhGAA administration.

[0013] Conventional forms of rhGAA, such as Myozyme®, Lumizyme®, or AL glucosidase alpha, cannot significantly increase M6P levels because carbohydrate processing is complex and difficult to regulate in cells by current manufacturing methods.

[0014] In view of these deficiencies of conventional rhGAA products, there is a need to develop an rhGAA that is effectively and efficiently targeted to the lysosomes of muscle tissue and in which inefficient clearance to non-target tissues is minimized. Summary of the Invention

[0015] In response to the problems associated with targeting and administering conventional forms of rhGAA and the difficulties associated with producing such better-targeted forms of rhGAA, the present inventors have researched and developed a process for producing rhGAA that is more effectively targeted to lysosomes in muscle tissue because it has higher levels of M6P and bis-M6P glycans than conventional rhGAA compositions. Furthermore, the rhGAA of the present invention has better processed complex N-glycans, which minimizes ineffective clearance of rhGAA from non-target tissues.

[0016] Considering the issues associated with current enzyme replacement therapy using conventional rhGAA products (such as Lumizyme®), the present inventors have developed, through intensive research and investigation, a method for producing rhGAA in CHO cells with significantly higher total levels of M6P and bis-M6P glycans, enabling rhGAA to be targeted to lysosomes in target tissues, such as muscle. The rhGAA produced by this method also possesses favorable pharmacokinetic properties due to its overall glycosylation pattern, increasing its uptake into target tissues and reducing its inefficient clearance following administration to individuals with Pompe disease. The present inventors have demonstrated that the rhGAA of the present invention, as exemplified by the rhGAA designated ATB-200, is more effective and efficient at targeting cardiac and skeletal muscle tissue than conventional rhGAA (such as Lumizyme®). As depicted in Figure 1C , the rhGAA of the present invention, as illustrated in Figure 1D , has superior ability to effectively target muscle tissue in patients with Pompe disease and reduce its inefficient clearance.

[0017] The superior rhGAA according to the present invention may further be combined with or possess a chaperone, or may be conjugated to other moieties that target the CIMPR in muscle tissue, such as the IGF2 moiety that binds to this receptor. The following examples demonstrate that the rhGAA of the present invention (exemplified by ATB-200 rhGAA) surpasses the current standard of care of enzyme replacement therapy by providing significantly better glycogen clearance compared to current therapy using the conventional rhGAA product, Lumizyme®. Simple diagram description

[0018] Figures 1A, 1B, 1C, and 1D. Figure 1A shows non-phosphorylated high-mannose glycans, mono-M6P glycans, and di-M6P glycans. Some conjugates of rhGAA have IGF2 fragments that bind to CIMPR. Figure 1B shows the chemical structure of the M6P group. Figure 1C depicts effective targeting of rhGAA via M6P-bearing glycans. Figure 1D depicts ineffective drug clearance.

[0019] Figures 2A and 2B. Figure 2A depicts the CIMPR receptor (also known as the IGF2 receptor) and its domains. Figure 2B is a table showing the binding affinities (in nanomolar terms) of glycans containing bis-M6P and mono-M6P for CIMPR, the binding affinity of high-mannose glycans for the mannose receptor, and the binding affinity of asialoglycoprotein receptors for desialylated complex glycans. RhGAA with M6P- and bis-M6P-containing glycans effectively binds to CIMPR on muscle target cells. RhGAA with high-mannose glycans and desialylated glycans ineffectively binds to non-target cells bearing the corresponding receptors.

[0020] Figures 3A, 3B, 3C, and 3D. Figures 3A, 3B, and 3C show the results of CIMPR ("IGF2 / CI-MPR") affinity chromatography of Myozyme®, Lumizyme®, and ATB-200 rhGAA, respectively. The dashed line indicates the M6P elution gradient. Figure 3D demonstrates that binding of ATB-200 rhGAA ("CBP rhGAA") is abolished after dephosphorylation.

[0021] Figure 4 compares the binding of Lumizyme® and ATB-200 rhGAA to CIMPR (“IGF2 / CI-MPR”).

[0022] FIG5 depicts the internalization of ATB-200 rhGAA (“CBP rhGAA”) and Lumizyme® rhGAA following uptake by L6 myoblasts.

[0023] FIG6 . Flowchart of a method for producing CHO cells expressing rhGAA with high M6P and bis-M6P glycan contents.

[0024] Figure 7. DNA constructs used to transform CHO cells with DNA encoding rhGAA.

[0025] 8A and 8B depict the survival rate ( FIG. 8A ) and viable cell density ( FIG. 8B ) of GAA-transformed CHO cells over time.

[0026] Figures 9A and 9B depict the selection of clones producing high amounts of rhGAA.

[0027] 10A and 10B show the results of survival testing of rhGAA-producing clones.

[0028] Figures 11A, 11B, and 11C depict the number of GAA gene copies in selected clones 110-011-X5-2 and 110-011-X5-14. Figure 11A (110-011-X5-2), Figure 11B (110-011-X5-14), and Figure 11C (mean and standard deviation of the number of copies).

[0029] Figures 12A, 12B, and 12C. Stability of clones producing high-mannose rhGAA.

[0030] Figures 13A and 13B. Comparative threshold analysis of GAA gene copy number. Figure 13A (clone 110-011-X5-2) and Figure 13B (clone 110-011-X5-14).

[0031] Figure 14. Flowchart of the purification scheme for rhGAA.

[0032] Figures 15A and 15B. Relative purity of rhGAA obtained by the original and improved purification schemes. Figures 15A (initial purification method) and 15B (optimized method).

[0033] Figures 16A, 16B, and 16C. Figures 16A and 16B depict the purification of samples ATB-200-1 and ATB-200-2 using an AEX-based purification scheme. Figure 16C shows an overlay view.

[0034] FIG. 17 summarizes the N-glycan content of three different formulations of ATB-200 rhGAA identified as CBP-rhGAA, ATB-200-1, and ATB-200-2.

[0035] Figures 18A and 18B. Figure 18A: Comparison of isoelectric points between ATB-200 and Lumizyme®. Figure 18B shows the isoelectric point range of the samples.

[0036] Figures 19A and 19B. Analysis of glycans from ATB-200 and Lumizyme®.

[0037] Figures 20A and 20B. Gradient profiles for partial separation of rhGAA using ATB-200. Figure 20A (graph), Figure 20B (table).

[0038] Figure 21 compares the Poleax dissolution curves of Lumizyme® and ATB-200.

[0039] FIG. 22 defines the extent of each of the four Poleax portions of ATB-200 rhGAA.

[0040] FIG. 23 shows the GAA activity of each of the four Poleax fractions of ATB-200 rhGAA.

[0041] Figure 24. Decreased glycogen in quadriceps muscle of GAA knockout (KO) mice following rapid intravenous administration of ATB-200, ATB-200 IGF2 conjugate, or Lumizyme.

[0042] Figure 25. CIMPR receptor binding of Lumizyme®, ATB-200 rhGAA, and vIGF2-ATB-200 rhGAA conjugate.

[0043] Figure 26. Uptake of Lumizyme®, ATB-200 rhGAA, and rhGAA vIGF2 conjugate by L6 myoblasts.

[0044] Figure 27. Graph showing that AT2220 stabilizes rhGAA activity in blood (upper trace) compared to rhGAA alone (lower trace).

[0045] Figure 28. Unfolded rhGAA at pH seen in plasma. At pH 7.4, rhGAA is unfolded over a period of approximately 20 hours.

[0046] Figures 29A and 29B. Figure 29A shows that the half-life of rhGAA is increased when co-administered with the chaperone AT2220. Figure 29B depicts the amount of rhGAA detected by Western blotting at selected time points.

[0047] Figures 30A, 30B, and 30C. Figure 30A shows GAA activity in the heart, quadriceps, and triceps muscles after administration of rhGAA (Lumizyme®) and rhGAA + AT2220 (duvoglustat). Figure 30B shows glycogen levels in the heart, quadriceps, and triceps muscles after administration of rhGAA (Lumizyme®) and rhGAA + AT2220. Figure 30C shows glycogen levels in heart and quadriceps tissue after treatment.

[0048] Figures 31A, 31B, 31C, and 31D. Lumizyme® rhGAA plasma activity with and without the chaperone AT2220. Figures 31A, 31B, 31C, and 31D depict the effects of Lumizyme® rhGAA + 50 mg, 100 mg, 250 mg, and 600 mg oral doses of AT2220 compared to Lumizyme® rhGAA alone.

[0049] Figure 32. Absolute difference in GAA activity compared to Lumizyme® rhGAA ERT alone.

[0050] FIG33 : Flowchart of steps in mouse muscle tissue analysis.

[0051] Figure 34. Analysis of rhGAA ERT muscle tissue for studies involving 4 or 6 rhGAA administrations.

[0052] FIG35. Bar graph showing reduction of glycogen in quadriceps, triceps, and heart tissues of GAA KO mice treated with unlabeled rhGAA and the AT2221 (miglustat) chaperone. V: Vehicle, L: Lumizyme®, COF: Co-formulation with AT2220 (miglustat).

[0053] FIG36 . Decreased hepatic glucose in GAA KO mice treated with unlabeled rhGAA and chaperones. V: Vehicle, L: Lumizyme®, COF: Co-formulation with AT2220 (Dugruct).

[0054] FIG37. Glucose reduction in GAA KO mice treated with IGF2-labeled rhGAA and chaperones. V: Vehicle, L: Lumizyme®, COF: Co-formulation with AT2220 (Dugruct).

[0055] FIG38. Glucose reduction in GAA KO mice treated with IGF2-labeled rhGAA and chaperones. V: Vehicle, L: Lumizyme®, COF: Co-formulation with AT2220 (Dugruct).

[0056] Figure 39. GAA activity in tissues—6 doses. V: Vehicle, L: Lumizyme®, COF: Co-formulation with AT2220 (Dugruct).

[0057] FIG40. Bar graph comparing triceps glycogen levels after treatment with 4 or 6 doses of ATB-200 rhGAA or ATB-200 in combination with the chaperone protein AT2221 (miglustat). V: Vehicle; L: Lumizyme®; COF: Co-formulation with AT2220 (duglustat); U: Unlabeled ATB-200 rhGAA; T: ATB-200 rhGAA labeled with vIGF2.

[0058] Figure 41. Bar graph comparing triceps glycogen levels after treatment with 4 or 6 doses of ATB-200 rhGAA or ATB-200 in combination with the chaperone protein AT2221 (miglustat). V: Vehicle, L: Lumizyme®, COF: Co-formulation with AT2220 (duglustat); U: Unlabeled ATB-200 rhGAA; T: ATB-200 rhGAA labeled with vIGF2.

[0059] FIG42. Bar graph comparing cardiac glycogen levels following treatment with 4 or 6 doses of ATB-200 rhGAA or ATB-200 rhGAA in combination with the chaperone protein AT2221 (miglustat). V: Vehicle; L: Lumizyme®; COF: Co-formulation with AT2220 (duglustat); U: Unlabeled ATB-200 rhGAA; T: ATB-200 rhGAA labeled with vIGF2.

[0060] Figure 43. Comparison of two methods for measuring glycogenotype (6 doses, quadriceps muscle). V: Vehicle, L: Lumizyme®, COF: Co-formulation with AT2220 (Dugruct); U: Unlabeled ATB-200 rhGAA; T: ATB-200 rhGAA labeled with vIGF2.

[0061] Figure 44. Comparison of two methods for measuring glycogenotype (6 doses, quadriceps muscle). V: Vehicle, L: Lumizyme®, COF: Co-formulation with AT2220 (Dugruct); U: Unlabeled ATB-200 rhGAA; T: ATB-200 rhGAA labeled with vIGF2.

[0062] Figure 45. Preclinical study 294A.

[0063] Figure 46. Bar graph showing reduction of glycogen in skeletal muscle from Study 296 (POC2 - twice biweekly dosing) by ATB-200 rhGAA or the combination of ATB-200 rhGAA and the chaperone AT2221.

[0064] Figure 47. Plasma PK from an infusion PK study in GAA KO mice.

[0065] Figure 48. Study 296 (POC2): Bar graph showing relative glycogen reduction in skeletal muscle following ATB-200 rhGAA or vIGF2-ATB-rhGAA treatment.

[0066] Figure 49. Study 296 (POC2): Bar graph showing relative glycogen reduction in skeletal muscle following Lumizyme® rhGAA or vIGF2-ATB-rhGAA treatment.

[0067] Figures 50A, 50B, and 50C. Study 296 (POC2): Bar graphs showing relative glycogen reduction in skeletal muscle following Lumizyme® rhGAA or vIGF2-ATB-rhGAA treatment in combination with AT2220 or AT2221 chaperones. Figure 50A (quadriceps), Figure 50B (gastrocnemius), and Figure 50C (triceps).

[0068] Figures 51A and 51B. PK of ATB-200 rhGAA, vIGF2-ATB-200 rhGAA, and Lumizyme® rhGAA (individual mice) in combination with chaperones AT2220 or AT2221.

[0069] Figure 52. Normalized data for treatment with ATB-200 rhGAA, vIGF2-ATB-200 rhGAA, or combination with chaperones AT2220 or AT2221.

[0070] FIG53 . Comparison of glycogen levels in quadriceps muscle after treatment with ATB-200 or vIGF2-ATB-200.

[0071] Figure 54. Glycogen staining of cardiac and skeletal muscle tissues after treatment with ATB-200 rhGAA, vIGF2-ATB-200 rhGAA, or Lumizyme® rhGAA.

[0072] FIG55. Bar graph showing glycogen levels in muscle tissue treated with ATB-200 rhGAA, vIGF2-ATB-200, or Lumizyme®.

[0073] FIG56 . Glycogen staining of quadriceps muscle tissue treated with ATB-200 rhGAA with co-administration of the chaperone protein AT2221 (miglustat).

[0074] Figure 57. Bar graph showing the reduction of glycogen in quadriceps muscle tissue provided by the combination of chaperone and ATB-200 rhGAA.

[0075] FIG58. Glycogen staining of quadriceps muscle tissue treated with ATB-200 rhGAA and the chaperone protein AT2220 (Dugruct).

[0076] FIG59. Glycogen staining of triceps muscle tissue from Study 296 treated with ATB-200 rhGAA and 1, 3, 10, or 30 mg / kg of the chaperone AT2221 (migludec).

[0077] Figure 60. Bar graph showing the reduction in glycogen in triceps muscle tissue from Study 296 following treatment with ATB-200 rhGAA and 1, 3, 10, or 30 mg / kg AT-2221 (migludec).

[0078] FIG61. Glycogen staining of triceps muscle tissue from Study 296 treated with co-formulations of ATB-200 rhGAA and 3 or 10 mg / kg chaperone protein AT2220 (Dugruct).

[0079] FIG62. Glycogen staining of heart tissue from Study 296 treated with ATB-200 rhGAA and AT2221 chaperones.

[0080] FIG63 . Relative glycogen reduction in cardiac tissue from Study 296 by co-administration of ATB-200 rhGAA with AT2221 chaperone and co-formulation with AT2220.

[0081] FIG64. Glycogen staining of cardiac tissue from Study 296 treated with co-formulations of ATB-200 rhGAA and AT2220 chaperone protein.

[0082] FIG65. Glycogen staining of diaphragm tissue from Study 296 treated with ATB-200 rhGAA and AT2220 chaperones.

[0083] FIG66. Glycogen staining of diaphragm tissue from Study 296 following treatment with a co-formulation of ATB-200 and AT2220 (Dugroit) chaperones.

[0084] Figure 67. Bar graph showing reductions in glycogen in quadriceps, gastrocnemius, triceps, and heart following treatment with rhGAA or a combination of rhGAA and a chaperone from Study 296.

[0085] FIG. 68 . Glycogen staining of treated heart and skeletal muscle tissue from Study 296.

[0086] Figure 69. Bar graph showing relative tissue glycogen levels after treatment from Study 296.

[0087] FIG70 . Glycogen staining of quadriceps muscle tissue from Study 296 treated with ATB-200 rhGAA or vIGF2-ATB-200 rhGAA conjugate.

[0088] FIG71 . Glycogen staining of quadriceps muscle tissue from Study 296 with or without the AT2221 (miglustat) chaperone or co-formulation of ATB-200 rhGAA and AT2220 (duglustat) chaperones.

[0089] FIG72 . Glycogen staining of triceps muscle tissue from Study 296 treated with ATB-200 rhGAA or vIGF2-ATB-200 rhGAA.

[0090] FIG. 73 . Glycogen staining of triceps muscle tissue from Study 296 treated with ATB-200 rhGAA and the chaperones AT2221 or AT2220.

[0091] FIG. 74 . Glycogen staining of diaphragm tissue from Study 296 treated with ATB-200 rhGAA or vIGF2-ATB-200 rhGAA.

[0092] FIG. 75 . Glycogen staining of diaphragm tissue from Study 296 treated with ATB-200 rhGAA and the chaperones AT2221 or AT2220.

[0093] Figure 76. Glycogen staining of heart tissue treated with ATB-200 rhGAA vIGF2-ATB-200 rhGAA.

[0094] FIG. 77 . Glycogen staining of heart tissue from Study 296 treated with ATB-200 rhGAA and the chaperones AT2221 or AT2220.

[0095] FIG78. Effects of repeated doses of rhGAA on glycogen levels in quadriceps muscle as determined by Study 296 (POC2) and Studies POC1a and POC1b.

[0096] Figures 79A and 79B. Figure 79A shows the pharmacokinetics of ATB-200 rhGAA, Lumizyme® rhGAA, and vIGF2-ATB-200 rhGAA, and Figure 79B: the respective half-lives of these products as shown from Study 302.

[0097] Figures 80A and 80B. Study 302: As shown in Study 302, the PK of ATB-200 rhGAA was dose-dependent and lower than that of Lumizyme®. Figure 80A (GAA activity), Figure 80B (half-life).

[0098] Figures 81A and 81B. Study 302: As shown in Study 302, the chaperones AT2220 and AT2221 did not significantly improve the plasma PK of ATB-200 rhGAA. Figure 81A (GAA activity), Figure 81B (half-life).

[0099] FIG82 . Antibody titers in groups treated with enzyme rhGAA alone or IGF2-labeled rhGAA from Study 294A (6 biweekly injections).

[0100] FIG83 . Antibody titers of rhGAA and IGF23 labeled rhGAA enzyme-only treatment groups from Study 294A (six biweekly doses).

[0101] FIG84 . Effects of AT2220 and AT2221 chaperones from Study 294A (six biweekly doses) on rhGAA antibody titers.

[0102] FIG85 . Effects of chaperones AT2220 and AT2221 on antibody titers to unlabeled and labeled rhGAA in the Study 294A treatment group (six biweekly doses).

[0103] FIG. 86 . Antibody titers to rhGAA in the treatment group of Study 294B (POC1b) (four biweekly doses).

[0104] FIG. 87. Antibody titers to ATB-200 rhGAA in Study 296 treatment group (administered twice biweekly).

[0105] Figures 88A, 88B, and 88C. Conventional rhGAA, ATB-200 rhGAA, and the combination of ATB-200 rhGAA and the chaperone AT2221 display reduced glycogen levels, EM micrographs, and bar graphs.

[0106] FIG89. Glycogen staining of cardiac and skeletal muscle tissues treated with conventional rhGAA, ATB-200 rhGAA, and the combination of ATB-200 rhGAA and the chaperone protein AT2221.

[0107] Figures 90A and 90B. LAMP-1 staining of tissues treated with ATB-200 rhGAA and the combination of ATB-200 rhGAA and the chaperone protein AT2221.

[0108] Figures 91A and 91B. Bis-M6P content and CIMPR binding affinity of Lumizyme® and ATB-200 rhGAA. Figure 91A (Bis-M6P analysis), Figure 91B (CIMPR binding affinity).

[0109] Figures 92A, 92B, and 92C. ATB-200 rhGAA internalization into normal and Pompeii fibroblasts. Figure 92A (normal fibroblasts), Figure 92B (Pompeii fibroblasts), and Figure 92C (K uptake)

[0110] Figures 93A and 93B. Comparative pharmacokinetics of Lumizyme® and ATB-200 rhGAA. Figure 93A (plasma PK), Figure 93B (PK profile).

[0111] Figures 94A and 94B. Comparative glycogen clearance of ATB-200 rhGAA and Lumizyme® rhGAA in quadriceps and triceps muscles. Figure 94A (quadriceps) and Figure 94B (triceps).

[0112] Figures 95A and 95B. Effect of temperature on ATB-200 rhGAA folding in the presence of chaperone AT2221. Figure 95A (% unfolded rhGAA) and Figure 95B (Table: Effect of temperature).

[0113] Figure 96. Glycogen clearance of ATB-200 rhGAA and AT-2221 chaperones in quadriceps muscle.

[0114] Figure 97. Comparison of Lumizyme® and ATB-200 rhGAA in myocardial glycogen reduction.

[0115] Figures 98A and 98B. Comparison of glycogen reduction in skeletal muscle by Lumizyme® and ATB-200 rhGAA, Figure 98A (quadriceps) and Figure 98B (triceps).

[0116] Figures 99A, 99B, and 99C. Figure 99A: Lumizyme® and ATB-200 rhGAA plasma PK; Figure 99B: Lumizyme® (rhGAA) and ATB-200 plasma half-life regression; Figure 99C: Lumizyme® and ATB-200 rhGAA PK data.

[0117] Figures 100A, 100B, and 100C. Glycogen reduction in quadriceps (99A), triceps (99B), and myocardium (99C) by the combination of ATB-200 and chaperone protein AT2220.

[0118] Figures 101A, 101B, and 101C. Glycogen is reduced in skeletal (101A, 101B) and cardiac (101C) muscles of GAA KO mice.

[0119] Figures 102A, 102B, and 102C. Relative reduction of glycogen in quadriceps and triceps muscles by Myozyme® rhGAA and chaperone AT-2221. Figures 102(A) and 102(B): Figure, Figure 102C: Table.

[0120] FIG103. Glycogen staining of the heart, diaphragm, and quadriceps muscle after treatment with ATB-200 rhGAA or ATB-200 rhGAA and AT2221 (migludec).

[0121] Figures 104A and 104B. Bar graphs of GAA activity and glycogenolysis in quadriceps muscle after treatment with ATB-200 rhGAA or ATB-200 rhGAA and AT2221 (miglutide). Figure 104A (GAA activity) and Figure 104B (glycogenolysis).

[0122] Figures 105A and 105B. PAS glycogen staining (105A) and EM (105B) of muscle tissue from GAA KO mice treated with conventional rhGAA or ATB-200 rhGAA and miglustat (AT-2221). Figure 105A (PAS glycogen staining) and Figure 105B (EM).

[0123] FIG106 . LAMP-1 labeling signal in tissues from GAA KO mice treated with ATB-200 rhGAA or ATB-200 rhGAA and miglustat (AT2221) compared to untreated or wild-type tissues.

[0124] Figure 107. Assessment of lysosomal proliferation by LAMP-1 labeling.

[0125] Figures 108A and 108B. Comparison of the pharmacokinetics of different batches of ATB-200 rhGAA. Figure 108A (Study 317) and Figure 108B (Study 321).

[0126] Figure 109. Bar graph showing the reduction of glycogen in muscle tissue by different batches of ATB-200 rhGAA.

[0127] Figures 110A and 110B. Half-life of ATB-200 rhGAA and the combination of Lumizyme® rhGAA and miglustat (AT-2221). Figure 110A (half-life) and Figure 110B (PK profile).

[0128] Figure 111. Muscle morphology after ATB-200 rhGAA and miglustat (AT2221) treatment.

[0129] Figures 112A, 112B, and 112C. Pharmacokinetics of chaperones AT2221 and AT2220 in wild-type C57BL / 6 mice. Figure 112A (plasma levels), Figure 112B (quadriceps muscle levels), and Figure 94C (rhGAA inhibition assay).

[0130] Figures 113A and 113B. Pharmacokinetics of migludec (AT2221) after oral administration in mice. Figure 113A (plasma levels of migludec) and Figure 113B (PK profile).

[0131] Figure 114. Pharmacokinetics of Migludec (AT2221) in patients with Gaucher's disease and Pompe's disease.

[0132] FIG. 115 . Effect of chaperone protein AT-2220 on survival of mice administered conventional Myozyme® rhGAA.

[0133] FIG. 116 . Survival rate following administration of 3 mg / kg Myozyme® or Myozyme® in combination with the chaperone protein AT2220 (Dugrostat).

[0134] FIG. 117 . Survival rate following administration of 10 mg / kg Myozyme® or the combination of Myozyme® and the chaperone protein AT2220.

[0135] Figure 118. Myozyme® survival dose-response curve. Implementation Method

[0136] [Definition]: The terms used in this specification generally have their ordinary meanings in the art within the context of the present invention and in the specific context in which each term is used. Certain terms are discussed below or elsewhere in this specification to provide additional guidance to practitioners in describing the compositions and methods of the present invention and how to make and use them.

[0137] The term "GAA" refers to human acid α-glucosidase (GAA), an enzyme that catalyzes the hydrolysis of α-1,4- and α-1,6-glycosidic bonds of lysosomal glycogen; as well as insertional, related, or substitutional variants of the GAA amino acid sequence and enzymatically active fragments of longer GAA sequences. The term "rhGAA" is used to distinguish endogenous GAA from synthetically or recombinantly produced GAA, such as GAA produced by transforming CHO cells with GAA-encoding DNA. GAA and rhGAA can exist as compositions containing a mixture of GAA molecules with different glycosylation patterns, such as a mixture of rhGAA molecules with M6P or bis-M6P groups on their glycans and GAA molecules without M6P or bis-M6P. GAA and rhGAA may also have other compounds, such as chaperones, or may be bound to other moieties in the GAA or rhGAA conjugate, such as to the IGF2 moiety that targets the conjugate to the CIMPR and subsequently the lysosome.

[0138] A "subject" or "patient" is preferably a human, although other mammals and non-human animals with disorders involving glycogen accumulation may also be treated. The subject may be a fetus, neonate, child, adolescent, or adult with Pompe disease or another glycogen storage or accumulation disorder. An example of a treated subject is a subject (fetus, neonate, child, adolescent, young adult, or adult) with GSD-II (e.g., infantile GSD-II, juvenile GSD-II, or adult-onset GSD-II). The subject may have residual GAA activity or no measurable activity. For example, a subject with GSD-II may have GAA activity of less than about 1% of normal (infantile GSD-II), about 1-10% of normal (juvenile GSD-II), or about 10-40% of normal (adult GSD-II).

[0139] As used herein, the terms "treat" and "treatment" refer to ameliorating one or more symptoms associated with a disease, preventing or delaying the onset of one or more symptoms of a disease, and / or reducing the severity or frequency of one or more symptoms of a disease. For example, treatment may refer to improving cardiac status (e.g., increasing end-diastolic and / or end-systolic volumes, or reducing, ameliorating, or preventing the progressive cardiomyopathy typically seen in GSD-II) or pulmonary function (e.g., increasing vital capacity above baseline capacity and / or normalizing oxygen desaturation during crying); improving neurodevelopmental and / or motor skills (e.g., improving AIMS scores); reducing glycogen levels in tissues of individuals affected by the disease; or any combination of these effects. In a preferred embodiment, treatment includes improving cardiac status, particularly reducing or preventing GSD-II-associated cardiomyopathy.

[0140] As used herein, the terms "increase," "increase," or "decrease" refer to values ​​relative to a baseline measurement, such as the measurement in the same individual before starting a treatment described herein, or the measurement in a control individual (or multiple control individuals) in the absence of a treatment described herein. A control individual is an individual with the same form of GSD-II as the treated individual (infantile, juvenile, or adult-onset form) who is approximately the same age as the treated individual (to ensure that the disease stage in the treated and control individuals is comparable).

[0141] As used herein, the term "purified" refers to a substance that has been isolated under conditions that reduce or eliminate the presence of unrelated substances (i.e., contaminants), including the natural substance from which the substance was obtained. For example, a purified protein is preferably substantially free of other proteins or nucleic acids with which it is associated in cells; a purified nucleic acid molecule is preferably substantially free of proteins or other unrelated nucleic acid molecules with which it can be found within cells. As used herein, the term "substantially free" can be applied in the context of analytical testing of a substance. Preferably, a purified substance that is substantially free of contaminants is at least 95% pure; more preferably, at least 97% pure, and even more preferably, at least 99% pure. Purity can be assessed by chromatography, gel electrophoresis, immunoassays, compositional analysis, bioassays, and other methods known in the art. In a specific embodiment, purified means that the level of contaminants is below that acceptable to regulatory authorities for safe administration to humans or non-human animals. Recombinant proteins can be isolated or purified from CHO cells using methods known in the art, including chromatographic size separation, affinity chromatography, or anion exchange chromatography.

[0142] The terms "genetically modified" or "recombinant" refer to cells, such as CHO cells, that express a specific gene product (such as rhGAA or ATB-200 rhGAA) following the introduction of a nucleic acid comprising a coding sequence encoding the gene product and regulatory elements that control the expression of the coding sequence. Introduction of the nucleic acid can be achieved by any method known in the art, including gene targeting and homologous recombination. As used herein, the term also includes cells that have been engineered, for example, by gene activation techniques, to express or overexpress an endogenous gene or gene product that is not normally expressed by the cell.

[0143] "Pompe disease" refers to an autosomal recessive LSD characterized by a deficiency in acid α-glucosidase (GAA) activity, impairing lysosomal glucose metabolism. This enzyme deficiency leads to lysosomal glycogen accumulation and, in the late stages of the disease, progressive skeletal muscle weakness, decreased cardiac function, respiratory insufficiency, and / or central nervous system damage. Mutations in the GAA gene result in lower expression or in the production of mutant forms of the enzyme with altered stability and / or biological activity, ultimately leading to the disease. (See generally Hirschhorn R, 1995, Glycogen Storage Disease Type II: Acid α-Glucosidase (Acid Maltase) Deficiency, in The Metabolic and Molecular Bases of Inherited Disease, Scriver et al., eds., McGraw-Hill, New York, 7th ed., pp. 2443-2464). The three recognized clinical forms of Pompe disease (infantile, juvenile, and adult) are associated with the level of residual α-glucosidase activity (Refuser AJ et al., 1995, Glycogenosis Type II (Acid Maltase Deficiency), Muscle & Nerve Suppl 3, S61-S69). Infantile Pompe disease (Type I or A) is the most common and most severe, characterized by failure to thrive, generalized hypotonicity, cardiomegaly, and cardiopulmonary failure within the second year of life. Juvenile Pompe disease (Type II or B) is of intermediate severity and is characterized by prominent muscle symptoms without cardiomegaly. Juvenile Pompe disease patients typically die before reaching the age of 20 due to respiratory failure. Adult Pompe disease (type III or C) typically presents as a slowly progressive myopathy during adolescence or as late as the sixth decade (Felicia KJ et al., 1995, Clinical Variability in Adult-Onset Acid Maltase Deficiency: Report of Affected Sibs and Review of the Literature, Medicine 74, 131-135). In Pompe disease, α-glucosidase has been shown to be extensively modified post-translationally by glycosylation, phosphorylation, and proteolytic processing. Proteolytic conversion of the 110 kilodalton (kids) precursor to the 76 and 70 kids mature forms is required in the lysosome for optimal glycogen catalysis. As used herein, the term "Pompe disease" refers to all types of Pompe disease. The formulations and dosing regimens disclosed in this application can be used to treat, for example, types I, II, or III Pompe disease. Non-limiting embodiments of the present invention

[0144] A CHO cell-derived rhGAA composition contains a higher amount of rhGAA containing N-glycans with mannose-6-phosphate (M6P) or bis-M6P than conventional rhGAA (alglucosidase alfa; CAS 420794-05-0), such as that exemplified by Lumizyme®. An exemplary rhGAA composition according to the present invention is ATB-200 (sometimes designated ATB-200 or CBP-rhGAA), which is described in the Examples. The rhGAA of the present invention (ATB-200) has been shown to bind to CIMPR with high affinity (KD approximately 2-4 nM) and is efficiently internalized by Pompeii fibroblasts and skeletal myoblasts (KD approximately 7-14 nM). ATB-200 was characterized in vivo and shown to have a shorter apparent plasma half-life (t 1 / 2 -45 min) than the current rhGAA ERT (t 1 / 2 -75 min).

[0145] The amino acid sequence of rhGAA can be at least 70%, 75%, 80%, 85%, 95%, or 99% identical to the amino acid sequence depicted by SEQ ID NO: 1 or 3, or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more deletions, substitutions, or additions. In some embodiments of GAA or rhGAA of the present invention, such as in ATB-200 rhGAA, GAA or rhGAA comprises a wild-type GAA amino acid sequence, such as the amino acid sequence of SEQ ID NO: 1 or 3. In other non-limiting embodiments, rhGAA comprises a subset of the amino acid residues present in wild-type GAA, wherein the subset includes the amino acid residues of wild-type GAA that form the active site for substrate binding and / or substrate reduction. Thus, the present invention encompasses rhGAA, which is a fusion protein comprising the wild-type GAA active site for substrate binding and / or substrate reduction, as well as additional amino acid residues that may or may not be present in wild-type GAA. In one embodiment, the rhGAA is glucosidase alpha, which is the human enzyme acid alpha-glucosidase (GAA), encoded by the most predominant of the nine observed haplotypes of this gene. The rhGAAs of the present invention, including ATB-200 rhGAA, may comprise an amino acid sequence that is 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of human alpha-glucosidase, such as the amino acid sequence set forth in accession number AHE24104.1 (GI:568760974) and incorporated by reference in U.S. Patent No. 8,592,362 (SEQ ID NO: 1). The nucleotide and amino acid sequences of GAA are also set forth in SEQ ID NOs: 2 and 3, respectively. Variants of this amino acid sequence also include amino acid sequences having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more amino acid deletions, insertions or substitutions relative to the following GAA amino acid sequence. Polynucleotide sequences encoding GAA and such variant human GAA are also encompassed and can be used to recombinantly express rhGAA according to the present invention.

[0146] Various alignment algorithms and / or programs can be used to calculate the identity between two sequences, including FASTA or BLAST, which are available as part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.) and can be used, for example, with default settings. For example, polypeptides and polynucleotides encoding such polypeptides that are at least 70%, 85%, 90%, 95%, 98%, or 99% identical to a specific polypeptide described herein and preferably exhibit substantially the same function are contemplated. Unless otherwise indicated, similarity scores are based on the use of BLOSUM62. When BLASTP is used, % similarity is based on BLASTP positive scores and % sequence identity is based on BLASTP identity scores. BLASTP "identity" indicates the number and fraction of total residues in the same high-scoring sequence; and BLASTP "positive" indicates the number and fraction of residues that have a positive alignment score and are similar to each other. Amino acid sequences having this degree of identity or similarity, or any intermediate degree of identity or similarity, to the amino acid sequences disclosed herein are encompassed and encompassed by the present invention. Polynucleotide sequences of similar polypeptides can be inferred using the genetic code and obtained by conventional means, specifically by reverse translating their amino acid sequences using the genetic code. Preferably, no more than 70, 65, 60, 55, 45, 40, 35, 30, 25, 20, 15, 10, or 5% of the total rhGAA in a composition according to the present invention lacks N-glycans with M6P or bis-M6P or lacks the ability to bind to the cation-independent mannose-6-phosphate receptor (CIMPR). Alternatively, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99%, <100%, or more of the rhGAA in the composition comprises at least one N-glycan with M6P and / or bis-M6P or has the ability to bind to CIMPR.

[0147] The rhGAA molecules in the rhGAA compositions of the present invention may have one, two, three, or four M6P groups on their glycans. For example, only one N-glycan on a rhGAA molecule may have an M6P, a single N-glycan may have two M6P groups, or two different N-glycans on the same rhGAA molecule may have a single M6P group. The rhGAA molecules in the rhGAA compositions may also have N-glycans without M6P groups. However, on average, at least about 3, 4, 5, 6, 7, 8, 9, or 10% of the total glycans on rhGAA are in the form of mono-M6P glycans, for example, about 6.25% of the total glycans may carry a single M6P group, and on average, at least about 0.5, 1, 1.5, 2.0, 2.5, or 3.0% of the total glycans on rhGAA are in the form of di-M6P glycans, and on average, less than 25% of the total rhGAA of the present invention does not contain phosphorylated glycans bound to CIMPR.

[0148] The average content of M6P-bearing N-glycans in the rhGAA compositions according to the present invention can be in the range of 0.5 to 2.0 mol / mol rhGAA or 1.0 to 2.0 mol / mol rhGAA, or subranges thereof. As shown in the Examples, the rhGAA of the present invention can be partially fractionated to provide rhGAA compositions having varying average numbers of M6P-bearing or bis-M6P-bearing glycans on rhGAA, thereby allowing for further tailored targeting of rhGAA to lysosomes in target tissues by selecting specific fractions or by selectively combining different fractions.

[0149] In some embodiments, 4-16% of the total N-glycans in the rhGAA composition are fully sialylated. In other embodiments, 15-30% of the total N-glycans on rhGAA in the composition bear sialic acid and terminal Gal. In still other embodiments, 8-19% of the total N-glycans on rhGAA in the composition bear only terminal Gal and no sialic acid.

[0150] In other embodiments of the present invention, 40, 45, 50, 55, or 60% of the total N-glycans on rhGAA in the composition are complex N-glycans; or no more than 6.5% of the total N-glycans on rhGAA in the composition are mixed N-glycans; no more than 15% of the high-mannose N-glycans on rhGAA in the composition are non-phosphorylated; at least 10% of the high-mannose N-glycans on rhGAA in the composition are mono-M6P phosphorylated; and / or at least 2% of the high-mannose N-glycans on rhGAA in the composition are di-M6P phosphorylated. These values ​​include all intermediate values ​​and subranges. The rhGAA composition according to the present invention may meet one or more of the above-described ranges.

[0151] In some embodiments, the rhGAA compositions of the present invention will have an average of 2.0 to 6.0 moles of sialic acid residues per mole of rhGAA. This range includes all intermediate values ​​and subranges, including 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, and 6.0 moles of residues per mole of rhGAA. Sialic acid residues may prevent inefficient clearance by asialoprotein receptors.

[0152] The rhGAA compositions of the present invention are preferably produced using CHO cells (such as the CHO cell line GA-ATB-200) or subcultures or derivatives of such CHO cell cultures. The present inventors have discovered that Chinese hamster ovary (CHO) cells can be used to produce rhGAA with superior targeting to CIMPR and lysosomes, as well as a glycosylation pattern that reduces inefficient clearance in vivo. These cells can be induced to express rhGAA with significantly higher levels of total M6P and bis-M6P than conventional rhGAA products. Recombinant human GAA produced by these cells, as exemplified by the rhGAA ATB-200 described in the Examples, contains significantly more lysosomal-targeting M6P and bis-M6P groups than conventional GAAs (such as Lumizyme®), has been shown to effectively bind to CIMPR, and is efficiently taken up by skeletal and cardiac muscle. It has also been shown to have a glycosylation pattern that provides a favorable pharmacokinetic profile and reduces inefficient clearance in vivo.

[0153] The rhGAA according to the present invention can be formulated into pharmaceutical compositions or used in the manufacture of medicaments for the treatment of Pompe disease or other conditions associated with GAA deficiency. These compositions can be formulated with physiologically acceptable carriers or excipients. The carriers and compositions can be sterile and otherwise suitable for the mode of administration.

[0154] Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline solutions (e.g., NaCl), saline, buffered saline, alcohol, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohol, polyethylene glycol, gelatin, carbohydrates (such as lactose, amylose, or starch), sugars (such as mannitol, sucrose, or others), dextrose, magnesium stearate, talc, silicic acid, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, and the like, and combinations thereof. If desired, pharmaceutical preparations may be mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavorings, and / or aromatic substances that do not deleteriously react with the active compound, and the like. In a preferred embodiment, a water-soluble carrier suitable for intravenous administration is used.

[0155] If desired, the composition or medicament may also contain a small amount of a wetting agent, emulsifier, or pH buffer. The composition may be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained-release formulation, or powder. The composition may also be formulated as a suppository using conventional binders and carriers (such as triglycerides). Oral formulations may include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, and the like. In a preferred embodiment, rhGAA is administered by intravenous infusion.

[0156] The composition or medicament can be formulated according to conventional procedures into a pharmaceutical composition suitable for human administration. For example, in a preferred embodiment, the composition for intravenous administration is a sterile isotonic aqueous buffer solution. If necessary, the composition may also include a cosolvent and a local anesthetic to relieve pain at the injection site. Generally, the ingredients are provided separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container (such as an ampoule or sachet) indicating the amount of active agent. When the composition is administered by infusion, the composition can be dispensed from an infusion bottle containing sterile pharmaceutical-grade water, saline, or dextrose / water. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0157] rhGAA can be formulated in neutral or salt form. Pharmaceutically acceptable salts include those formed with free amine groups, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, and tartaric acid, and those formed with free carboxyl groups, such as those derived from sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, and procaine.

[0158] rhGAA (or a composition or medicament containing GAA) is administered via an appropriate route. In one embodiment, GAA is administered intravenously. In other embodiments, GAA is administered by direct administration to target tissues, such as cardiac or skeletal muscle (e.g., intramuscularly) or the nervous system (e.g., direct injection into the brain; intraventricularly; intrathecally). If desired, more than one route may be used concurrently.

[0159] rhGAA (or a composition or medicament containing GAA) is administered in a therapeutically effective amount (e.g., a dose sufficient, when administered at regular intervals, to treat the disease, such as by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and / or reducing the severity or frequency of disease symptoms, as described above). The amount that will be therapeutically effective in treating the disease will depend on the nature and extent of the disease and can be determined by standard clinical techniques. Additionally, in vitro or in vivo assays may be employed, as appropriate, to help identify the optimal dosage range. The precise dosage employed will also depend on the route of administration and the severity of the disease and should be determined according to the physician's judgment and individual patient circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems. In a preferred embodiment, the therapeutically effective amount is less than about 15 mg enzyme per kg body weight of the subject, preferably in the range of about 1 to 10 mg enzyme per kg body weight, and even more preferably about 10 mg enzyme per kg body weight or about 5 mg enzyme per kg body weight. The effective dosage for a particular individual may be varied (e.g., increased or decreased) over time depending on the individual's needs. For example, the dosage may be increased during times of physical illness or stress, or when anti-GAA antibodies become present or increase, or when disease symptoms worsen.

[0160] A therapeutically effective amount of GAA (or a composition or medicament containing GAA) is administered at regular intervals, depending on the nature and severity of the disease and on an ongoing basis. As used herein, administration at "regular intervals" indicates periodic administration of a therapeutically effective amount (as distinguished from a single dose). The intervals can be determined by standard clinical techniques. In preferred embodiments, GAA is administered monthly, bimonthly, weekly, twice weekly, or daily. The dosing interval for an individual need not be fixed but can vary over time depending on individual needs. For example, the interval between doses may be reduced during times of physical illness or stress, if anti-GAA antibodies become present or increase, or if disease symptoms worsen. In some embodiments, a therapeutically effective amount of 5, 10, 15, or 20 mg of enzyme per kilogram of body weight is administered twice weekly, weekly, or every other week, with or without a chaperone.

[0161] GAA or rhGAA of the present invention can be prepared for later use, such as in unit-dose vials or syringes, or bottles or bags for intravenous administration. Kits containing GAA or rhGAA and, optionally, excipients or other active ingredients (such as companion proteins or other drugs) can be enclosed in packaging material and accompanied by instructions for reconstitution, dilution, or administration for treatment of a subject in need thereof (such as a patient with Pompe disease).

[0162] GAA (or a composition or drug containing GAA) can be administered alone or in combination with other agents (such as chaperones). rhGAA with M6P or bis-M6P of varying degrees of glycosylation can be administered, or a combination of rhGAA and M6P or bis-M6P of varying degrees of glycosylation can be administered.

[0163] In some embodiments, the rhGAA compositions of the present invention are complexed or blended with a chaperone protein, such as AT-2220 or AT-2221. Chaperone proteins, sometimes referred to as "pharmacological chaperones," are compounds whose pharmacokinetic and other pharmacological properties are altered when complexed or co-administered with rhGAA. Representative chaperone proteins exemplified herein include AT2221 (miglustat, N-butyl-deoxynojirimycin) and AT2220 (duglustat hydrochloride, 1-deoxynojirimycin). Such complexation or blending can be performed in vitro or in vivo, for example, where separate doses of rhGAA and the chaperone protein are administered. For example, targeting of rhGAA, portions, or derivatives thereof to the CIMPR and subsequently to the cellular lysosome can be customized by combining them with duglustat hydrochloride (AT2220, deoxynojirimycin) or miglustat (AT2221, N-butyl-deoxynojirimycin). The following example demonstrates a significant reduction in glycogen substrates in key skeletal muscles of GAA knockout mice receiving the preferred combination of the present invention targeting rhGAA and a chaperone protein.

[0164] In other embodiments, the rhGAA in the composition will be modified to contain an additional targeting ligand, such as a ligand that binds to CIMPR, such as the IGF2 portion that binds to CIMPR. Such conjugates can be produced by introducing chimeric DNA encoding the conjugate into CHO cells and recovering the chimeric rhGAA, or by chemically modifying the rhGAA contained in the rhGAA composition according to the present invention. A representative conjugate is rhGAA bound to the IGF2 portion that binds to CIMPR. Such conjugates may contain one or more of the amino acid sequence modifications set forth in SEQ ID NO: 4, which depicts [(del 1-4)-Arg6-Leu27-Arg65], an IGF-2 peptide with an N-terminal extension linker and a TEV protease recognition site.

[0165] Another aspect of the present invention relates to CHO cells or derivatives thereof or other equivalents that produce rhGAA according to the present invention. One example of such a CHO cell line is GA-ATB-200 or a subculture thereof that produces the rhGAA composition described herein. Other exemplary CHO cell lines are the clones 110-001-X5-2 and 110-001-X5-14 described in the Examples below. Such CHO cell lines may contain multiple gene copies, such as 5, 10, 15, or 20 or more copies of a polynucleotide encoding GAA.

[0166] An aspect of the present invention relating to CHO cells expressing the rhGAA compositions of the present invention is a method for producing such CHO cell lines. Such methods include transforming CHO cells with DNA encoding GAA or a GAA variant, selecting CHO cells that have stably integrated the GAA-encoding DNA into their chromosomes and stably express GAA, selecting CHO cells that express GAA with high levels of glycans bearing M6P or bis-M6P, and optionally selecting CHO cells that have N-glycans with high sialic acid content and / or N-glycans with low non-phosphorylated mannose content. Representative recombinant DNA and screening procedures are described in the Examples. These CHO cell lines can be used to produce rhGAA and rhGAA compositions of the present invention by culturing the CHO cell lines and recovering the rhGAA compositions of the present invention from the CHO cell cultures. The recovered rhGAA compositions can be further purified or partially isolated, for example, by chromatography or the fractionation procedures described in the Examples. The rhGAA compositions of the present invention, or portions or derivatives thereof, are preferably used to treat subjects suffering from conditions, disorders, or diseases associated with lysosomal GAA deficiency by administering the rhGAA compositions. Subjects in need of treatment include those suffering from type II glycogen storage disease (Pompe disease) and other conditions, disorders, or diseases that would benefit from administration of rhGAA.

[0167] The following examples demonstrate that the rhGAA of the present invention (ATB-200) is taken up by cardiac and skeletal muscle cells, binds to CIMPR, and effectively removes glycogen from skeletal and cardiac muscle cells when administered at significantly lower doses than conventional rhGAA products. A biweekly regimen of intravenously administered ATB-200 achieved up to 75% reductions in skeletal myoblasts in GAA knockout mice. These reductions exceeded those achieved by the same amount of Lumizyme®, demonstrating that the rhGAA of the present invention, with its enhanced content of N-glycans bearing M6P and bis-M6P, superiorly reduces glycogen substrates. Due to its improved targeting, pharmacodynamics, and pharmacokinetics, the rhGAA compositions of the present invention can be administered at lower doses than conventional rhGAA products (such as Lumizyme® or Myozyme®).

[0168] It can be used to degrade, reduce or remove glycogen from cardiac muscle, smooth muscle or striated muscle. Examples of skeletal or striated muscles to be treated include at least one muscle selected from the group consisting of: abductor digiti minimi (foot), abductor digiti minimi (hand), abductor hallucis, abductor hallucis brevis, abductor hallucis longus, adductor brevis, adductor hallucis magnus, adductor hallucis, adductor hallucis longus, adductor hallucis magnus, adductor hallucis, anconeus, anconeus, knee joint, aryepiglottis, iliopsoas, auricle, biceps brachii, biceps femoris, brachialis, brachioradialis, buccinator, bulbospongiosus, inferior pharyngeal constrictor, middle pharyngeal constrictor, superior pharyngeal constrictor, coracobrachialis, corrugator supercilii, cremaster muscle, cricothyroid muscle, tunica carnosus, deep transverse perineal muscle, deltoid muscle, depressor anguli oris, depressor labii inferioris, diaphragm, digastric muscle, digastric muscle (anterior view), erector spinae-spinalis, erector spinae-iliocostalis, erector spinae- Longissimus, extensor carpi radialis brevis, extensor carpi radialis longus, extensor carpi ulnaris, extensor digiti minimi (hand), extensor digitorum brevis (hand), extensor digitorum brevis (foot), extensor digitorum longus (foot), extensor digitorum indica longus, extensor digitorum indexis longus, extensor pollicis brevis, extensor pollicis longus, external obliques, flexor carpi radialis, flexor carpi ulnaris, flexor digiti minimi brevis (foot), flexor digitorum minimi brevis (hand), flexor digitorum brevis, flexor digitorum longus (foot), flexor digitorum profundus, flexor digitorum superficialis, flexor digitorum brevis, flexor pollicis longus, flexor pollicis brevis, flexor pollicis longus, frontalis, gastrocnemius, inferior gemellus, superior gemellus, genioglossus, geniohyoid, gluteus maximus, gluteus medius, gluteus minimus, gracilis, osteoglossus, iliacus, inferior oblique, inferior rectus, infraspinatus, external intercostal muscles, innermost intercostal muscles, internal intercostal muscles, internal obliques, dorsal interosseous muscles of the hand, dorsal interosseous muscles of the foot, Palmar interosseous muscles, plantar interosseous muscles, interspinous muscles, intertransverse processes, internal tongue, ischiocavernosus, cricoarytenoids, lateral pterygoids, lateral rectus, latissimus dorsi, levator anguli oris, levator ani-coccygeus, levator ani-iliococcygeus, levator ani-pubococcygeus, levator ani-puborectalis, levator ani-pubovaginal, levator labii superioris, levator labii superioris, alar nasalis, levator palpebrae superioris, levator scapulae, levator veli palatini, levator costalis, longus capitis, longus colli, lumbricalis (4), lumbricalis hand, masseter, medial pterygoid, medial rectus, mentalis, uvula, mylohyoid, nasal muscles, oblique arytenoids, oblique capitis inferioris, oblique capitis capitis, obturator externus, obturator internus (A), obturator internus (B), omohyoid, opponents of the digiti minimi (hand), opponents of the pollicis, orbicularis oculi, orbicularis oris Muscles, palatoglossal muscles, palatopharyngeus, palmaris brevis, palmaris longus, pectineus, pectoralis major, pectoralis minor, peroneus brevis, peroneus longus, peroneus tertius, piriformis (A), piriformis (B), plantaris, platysma, popliteus, cricoarytenoids, procerus, pronator quadratus, pronator teres, psoas major, psoas minor, pyramidalis, quadratus femoris, quadratus lumborum, quadratus plantaris, rectus abdominis, rectus capitis anterior muscle, rectus capitis lateralis, rectus capitis major, rectus capitis minor, rectus femoris, rhomboid major, rhomboid minor, risorius, tympanipharyngeal, sartorius, scalene anterior, scalene middle, scalene minor, scalene posterior, semimembranosus, semitendinosus, serratus anterior, serratus posterior inferior, serratus posterior superior, soleus, anal sphincter, urethral sphincter, splenius capitis, splenius cervicis, stapedius, sternocleidomastoid,Sternohyoid, sternothyroid, styloglossus, stylohyoid, stylohyoid (anterior view), stylopharyngeus, subclavius, subcostalis, subscapularis, transverse superficialis, perineum, superior oblique, superior rectus, supinator, supraspinatus, temporalis, temporaloparis, tensor fasciae latae, tensor tympani, tensor veli palatini, teres major, teres minor, thyroarytenoid and vocal muscles, thyroepiglottis, thyrohyoid, tibialis anterior, tibialis posterior, transverse arytenoid, transverse spinae-multifidus, transverse spinae-rotators, transverse spinae-semispinalis, transverse abdominis, transverse thoracis, trapezius, triceps, vastus intermedius, vastus lateralis, vastus medialis, zygomaticus major, and zygomaticus minor.

[0169] The GAA compositions of the present invention can also be administered or used to treat type 1 (slow-twitch) or type 2 (fast-twitch) muscle fibers, or individuals with glycogen accumulation in these types of muscle fibers. Type I slow-twitch, or "red," muscle has dense capillaries and is rich in mitochondria and myoglobin, giving the muscle tissue its characteristic red color. It can carry more oxygen and uses fat or carbohydrates as fuel to sustain aerobic activity. Slow-twitch fibers contract for extended periods but with minimal force. Type II fast-twitch muscle has three major subtypes (IIa, IIx, and IIb), which differ in contraction speed and force generated. Fast-twitch fibers contract quickly and forcefully but fatigue very quickly, lasting only briefly, with bursts of anaerobic activity before the contraction becomes painful. They contribute most to muscle strength and have greater potential for mass gain. Type IIb anaerobic glycolytic "white" muscle has the least dense mitochondria and myoglobin. In small animals (e.g., rodents), this is the predominant fast-twitch muscle type, explaining the pale color of their meat.

[0170] The rhGAA compositions, portions, or derivatives thereof of the present invention can be administered systemically, for example, by intravenous (IV) infusion, or directly to a desired site, such as the heart or skeletal muscle, such as the quadriceps, triceps, or diaphragm. Administration can be to muscle cells, specific muscle tissue, muscle, or muscle group. For example, such treatments can involve intramuscular administration of the rhGAA composition directly to the quadriceps or triceps muscle, or diaphragm, of a subject.

[0171] As mentioned above, the rhGAA compositions, portions, or derivatives thereof of the present invention can be complexed or blended with a chaperone protein, such as AT-2220 (duglut hydrochloride, 1-deoxynojirimycin) or AT-2221 (miglut, N-butyl-deoxynojirimycin), or their salts, to improve the pharmacokinetics of rhGAA administration. rhGAA and the chaperone protein can be administered together or separately. For simultaneous administration, the GAA in the composition can be preloaded with the chaperone protein. Alternatively, GAA and the chaperone protein can be administered simultaneously or separately at different times.

[0172] Representative dosages of AT2221 range from 0.25 to 400 mg / kg, preferably from 0.5 to 200 mg / kg, and most preferably from 2 to 50 mg / kg. Specific dosages of AT2221 include 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 mg / kg. These dosages can be combined with rhGAA (such as ATB-200 rhGAA) at a molar ratio of AT2221 to rhGAA ranging from 15:1 to 150:1. Specific ratios include 15:1, 20:1, 25:1, 50:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 100:1, 125:1, and 150:1. rhGAA and AT2221 can be co-administered in equal amounts or molar ratios, concurrently, sequentially or separately. The above ranges include all intermediate subranges and values, such as all integer values ​​between the range endpoints.

[0173] Representative dosages of AT2220 range from 0.1 to 120 mg / kg, preferably 0.25 to 60, and most preferably 0.6 to 15 mg / kg. Specific dosages of AT2220 include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, and 30 mg / kg. These dosages can be combined with rhGAA (such as ATB-200 rhGAA) at a molar ratio of AT2220 to rhGAA ranging from 15:1 to 150:1. Specific ratios include 15:1, 20:1, 25:1, 50:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 100:1, 125:1, and 150:1. rhGAA and AT2220 can be co-administered in equal amounts or molar ratios, concurrently, sequentially or separately. The above ranges include all intermediate subranges and values, such as all integer values ​​between the range endpoints.

[0174] The rhGAA compositions, portions, or derivatives thereof of the present invention can also be used to metabolize, degrade, remove, or otherwise reduce glycogen in tissues, muscles, myofibers, myocytes, lysosomes, organelles, cellular compartments, or cytoplasm. The rhGAA compositions are administered to a subject, optionally along with a companion protein or drug that reduces the immune response to rhGAA.

[0175] In another embodiment of its method of use, the rhGAA of the present invention can be used to modulate lysosomal proliferation, autophagy, or exocytosis in cells by administering it, a portion, or derivative thereof, optionally in combination with a chaperone protein or conjugated with another targeting moiety, to cells, tissues, or subjects in need of such modulation. Autophagy is a mechanism that allows cells to degrade glycogen or other unnecessary or abnormal cellular components through their lysosomal function. This method may also include systemically or locally administering a GAA composition to a subject in need of treatment.

[0176] The rhGAA according to the present invention, which is enriched in M6P and bis-M6P compared to Lumizyme® and Myozyme® and has favorable pharmacokinetic properties conferred by its glycosylation pattern, can also be used to treat other conditions requiring the breakdown of complex carbohydrates, such as other conditions where accumulation of rhGAA in lysosomes or other parts of cells (such as the cytoplasm where rhGAA can reach) leads to the degradation of glycogen or other carbohydrates, such as glycogen storage disease III. It can also be used for non-therapeutic purposes, such as in the production of food, beverages, chemicals, and pharmaceutical products, where the breakdown of complex carbohydrates (such as starch and glycogen) into their monomers is required. Examples

[0177] The following non-limiting examples illustrate aspects of the present invention. Part I: ATB-200 rhGAA and its characteristics

[0178] Limitations of existing Myozyme® and Lumizyme® rhGAA products

[0179] The only currently approved treatment for Pompe disease uses existing rhGAA enzyme replacement therapy products, Lumizyme® and Myozyme. These products poorly target and deliver rhGAA to tissues containing the CIMPR receptor that binds to M6P.

[0180] To evaluate the activity of rhGAA in Myozyme® and Lumizyme®, these rhGAA preparations were injected onto a CIMPR column (which binds rhGAA with an M6P group) and subsequently eluted using a free M6 gradient. Fractions were collected in a 96-well plate and analyzed for GAA activity using a 4MU-α-glucose matrix. The relative amounts of bound and unbound rhGAA were determined based on GAA activity and reported as a fraction of total enzyme. Elution curves for Myozyme® and Lumizyme® are shown in Figures 3A and 3B. Figure 3C shows the elution curve for high-M6P rhGAA (ATB-200) according to the present invention.

[0181] Dephosphorylation of rhGAA of the present invention using purified purple acid phosphatase from sweet potato completely prevented GAA from binding to CIMPR, and the receptor on the lysosome took up rhGAA, see Figure 3D.

[0182] Figures 3A and 3B illustrate a problem associated with conventional Myozyme® and Lumizyme® ERTs: 73% of rhGAA in Myozyme® and 79% of rhGAA in Lumizyme® do not bind to CIMPR, as seen in the leftmost peak in each figure. Only 27% and 22% of Myozyme® and Lumizyme® rhGAA, respectively, contain M6P. As shown in Figure 3D, M6P is required for rhGAA to target CIMPR in lysosomes. The effective doses of Myozyme® and Lumizyme® correspond to the amount of M6P-containing rhGAA targeted to lysosomes. However, the majority of rhGAA in these two conventional products does not target CIMPR receptors on lysosomes. This requires administration of large doses of rhGAA, which is largely non-lysosomal. This increases the risk of allergic reactions to rhGAA or induction of immunity to rhGAA.

[0183] ATB-200 rhGAA has a higher affinity for CIMPR than Lumizyme

[0184] Lumizyme® and ATB-200 rhGAA receptor binding were compared using a receptor disk binding assay. CIMPR-coated disks were used to capture GAA with M6P. Various concentrations of rhGAA were applied, and unbound rhGAA was washed away. As shown in Figure 4, ATB-200 rhGAA bound significantly better to CIMPR than to Lumizyme.

[0185] [ATB-200 rhGAA] [Compared to Lumizyme] [quilt] [More Efficient Internalization by Muscle Cells.] The relative cellular uptake of ATB-200 and Lumizyme® rhGAA was compared using L6 myoblasts. The comparison involved 5-100 nM ATB-200 rh-GAA according to the present invention and 10-500 nM conventional rh-GAA (Lumizyme®). After 16 hours of incubation, external rhGAA was inactivated with TRIS base, and the cells were washed three times with PBS before harvesting. Internalized GAA, measured by 4-MUG, was plotted relative to total cellular protein, and the results are presented in Figure 5.

[0186] Figure 5 shows that substantially lower concentrations of ATB-200 rhGAA are required to achieve the same cellular uptake as Lumizyme® rhGAA. These results also show that ATB-200 rhGAA, which has a greater concentration of M6P glycans, is internalized by myoblasts approximately 10-fold more efficiently than Lumizyme.

[0187] As further shown below (in the section also describing ATB-200 conjugates), ATB-200 significantly reduced glycogen levels compared to Lumizyme when administered to myocytes.

[0188] Preparation of CHO cells producing ATB-200 rhGAA and high-M6P rhGAA

[0189] The high-M6P and bis-M6P rhGAAs of the present invention, such as ATB-200 rhGAA, are produced using CHO cells (Chinese Hamster Ovary cells) by transforming the cells with a DNA construct encoding GAA. CHO cells have previously been used to produce rhGAA, however, it was not known that CHO cells could be cultured and selected in a manner that produces rhGAA with high levels of M6P and bis-M6P glycans that target CIMPR in lysosomes.

[0190] Surprisingly, the present inventors have discovered that it is possible to transform CHO cell lines, select transformants that produce rhGAA with a high M6P content and stably express high-mannose rhGAA.

[0191] CHO cells are transfected with DNA expressing rh-GAA, followed by selection of transformants producing rhGAA. Figure 6 shows a scheme of such a method. Figure 6 summarizes a method for producing CHO cells expressing high amounts of M6P and bis-M6P glycans.

[0192] FIG7 shows a representative DNA construct used for transformation of CHO cells with DNA encoding rh-GAA. Other DNA constructs expressing allelic variants of GAA or other variant GAA amino acid sequences (such as an amino acid sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 1) can also be constructed and expressed in CHO cells. Those skilled in the art can also select alternative vectors suitable for transforming CHO cells to produce such DNA constructs. FIG7 depicts a DNA construct used to transform CHO cells with DNA encoding rhGAA.

[0193] After transfection, DG44 CHO (DHFR-) cells containing stably integrated GAA genes were selected using hypoxanthine / thymidine (-HT)-deficient culture medium. Amplified GAA expression in these cells was induced by methotrexate (MTX, 500 nM). Cell pools expressing high levels of GAA were identified by GAA enzyme activity analysis and used to establish individual rhGAA-producing clones. Figures 8A and 8B depict the survival rate and viable cell density of GAA-transformed CHO cells over time.

[0194] Individual clones were generated using two pools of high-expressing cells on semi-solid culture plates. 926 individual clones selected using the ClonePix system were transferred to 24 deep-well plates. These clones were analyzed for GAA enzyme activity to identify clones expressing high GAA levels. The modified culture medium used to determine GAA activity used a 4-MU-α-glucose substrate.

[0195] Figures 9A and 9B depict 42 different clones that exhibited high GAA activity (>105 fluorescent units). These top 42 clones were expanded in shake flask cultures to assess the stability of rhGAA expression in each clone. Six of the 42 clones were identified using a GAA enzyme activity assay (see Table 1).

[0196] Table 1: rhGAA potency Potency: Unit: mg / L [sample] [D6] [D7] [D8] [D9] [D10] [D]

[11] [D12] [D13] [D14] 110-001-X5-2-1 111.0 194.9 326.0 333.7 308.6 415.1 479.0 448.6 477.1 110-001-X5-2-2 111.3 185.1 321.5 327.3 305.3 401.7 562.8 479.7 526.0 110-001-X5-1 87.5 155.5 279.7 339.5 236.8 393.1 544.6 562.2 528.7 110-001-X5-4 85.6 141.5 241.1 330.1 211.3 311.6 370.8 368.0 375.6 110-001-X5-5 87.8 135.0 224.0 292.2 195.8 272.1 331.8 369.5 356.4 110-001-X5-14 87.7 166.9 265.3 314.1 270.0 584.1 620.3 609.5 488.8 110-001-X5-2 77.8 133.6 244.8 352.7 268.2 361.9 443.6 439.2 396.8

[0197] The top six rhGAA-expressing clones, identified by GAA enzyme activity assay, were further evaluated using both batch-fed and perfusion bioreactor systems, using other predetermined criteria, including cell growth and viability, and N-glycan structure (e.g., high sialic acid content and low non-phosphorylated high-mannose N-glycans). Sample 110-001-X5-14 corresponds to cells producing ATB-200. rhGAA purified from these individual clones was characterized by enzyme activity, isoelectric focusing, N-glycan analysis, and sialic acid content.

[0198] As shown in Figures 10A and 10B, the best three clones out of these six were identified by predetermined criteria (e.g., productivity, enzyme quality, cell viability, N-glycan structure, stable protein expression in bioreactor, etc.), see Figures 10A and 10B.

[0199] Two clones were selected: 110-001-X5-2 and 110-001-X5-14. The number of integrated copies of the GAA gene in these two clones was determined by quantitative PCR (qPCR), as shown in Figures 11A, 11B, and 11C. As shown in Figures 11A, 11B, and 11C, clone 110-001-X5-2 was found to have 34 integrated copies of the GAA gene, while clone 110-001-X5-14 contained 22 copies.

[0200] As shown in Figures 12A, 12B and 12C, these two clones showed stable expression of GAA over a nine-week period for more than 60 passages.

[0201] These results demonstrate that clones generated according to the selection method of the present invention are genetically stable as tested over a 9 week period and >60 passages, and are suitable for larger scale production of rhGAA.

[0202] GAA expression was tested in shake flask cultures at t = 0 and after 6 and 9 weeks. These data demonstrate that the best clones maintained very similar GAA protein expression throughout the study and that these two best clones were genetically stable for >60 passages and suitable for use as production cell lines. Comparative threshold analysis further demonstrated the genetic stability of these two clones.

[0203] Quantitative PCR (qPCR) was used to measure the number of integrated GAA gene copies in the best clones at t = 0 and after 9 weeks of cell culture. These data indicate that the best two clones maintained the number of integrated GAA gene copies throughout the 9-week study (see comparative threshold analysis in Figures 13A and 13B).

[0204] ATB-200 rhGAA is produced by cells derived from 110-001-X5-14. However, both of the above clones produce rhGAA with high M6P content relative to conventional rhGAA.

[0205] Consistent with the CHO strain stability described above, the inventors discovered that different batches of ATB-200 rhGAA had similar protein and carbohydrate profiles. Multiple batches of rhGAA according to the present invention were produced in both shaker flasks and perfusion bioreactors. Similar CIMPR receptor binding (approximately 70%) was observed for purified ATB-200 (CBP rhGAA) from different production batches. These data indicate that CBP rhGAA can be consistently produced and demonstrate that rhGAA produced from the CHO cell line according to the present invention contains significantly more M6P than Lumizyme® rhGAA.

[0206] Purified High-Mannose rhGAA ATB-200 (CBP-rhGAA)

[0207] ATB-200 rhGAA was purified by chromatography to obtain a composition containing 70% to 96% rhGAA with M6P glycans, which can be targeted to CIMPR on lysosomes. As shown in Figure 14, two different purification schemes were used.

[0208] In the original purification protocol, immobilized metal ion affinity chromatography (IMAC) was used to remove CHO cell-derived impurities from the crude rhGAA (CBP-rhGAA). Subsequently, the IMAC-purified rhGAA was run through a phenyl-agarose column. 70% of the rhGAA was determined to contain M6P glycans. See Figure 15A.

[0209] To minimize oligosaccharide dephosphorylation, an additional purification scheme was developed using direct AEX product capture, followed by IMAC impurity removal and size exclusion chromatography as shown in ATB-200-1 and ATB-200-2 (see Figure 14 ). This resulted in an rhGAA composition containing 92% of rhGAA containing M6P glycans. This purification scheme retains rhGAA with a high M6P content by removing contaminating phosphatases. See Figure 15B .

[0210] Figures 16A and 16B also demonstrate the advantages of the AEX-based purification scheme for rhGAA. ATB-200-1 eluted with less free M6P than ATB-200-1. The leftward shift in GAA activity in ATB-200-2 indicates a higher bis-M6P content than ATB-200-1.

[0211] Although the improved method of AT B200-2 required more M6P to elute rhGAA compared to the non-improved method AT B200-1, it resulted in a higher percentage of rhGAA bound to CIMPR. The leftward shift in the overlay in Figure 9 indicates that optimizing the new method resulted in ATB-200 rhGAA with a higher bis-M6P content. Compared to rhGAA containing a single M6P residue (mono-M6P rhGAA), bis-M6P rhGAA had significantly higher binding affinity for CIMPR on lysozyme.

[0212] FIG. 17 shows the different glycan contents of CBP-rhGAA, ATB-200-1, and ATB-200-2, and shows that each of these rhGAAs has higher M6P and bis-M6P contents than Lumizyme.

[0213] In addition to improving glycan phosphorylation via M6P, the improved AEX-based method reduces the amount of unphosphorylated rhGAA, increases sialic acid levels, and provides a product with improved targeting to the IGF2 / CIMP receptor on lysosomes. Table 2 below compares the sialic acid and M6P levels (mol / mol) of ATB-200 compositions produced by the old method (AT B200 ATF2; CBP rhGAA) and the new method using direct capture (AT B200-1, AT B200-2). This table shows that the sialic acid levels of rhGAA according to the present invention ranged from 2.6 to 5.4 mol / mol protein, and the M6P levels were 2.0 to 3.1 mol / mol protein, with 70% to 86% to 96% of the rhGAA in these samples bound to CIMPR. Table 2 sample Sialic acid (mol / mol protein) M6P (mol / mol protein) M6P combination ATB-200 ATF2 2.6 2.0 70% ATB-200-1 4.8 2.3 86% ATB-200-2 5.4 3.1 96%

[0214] Isoelectric point of ATB-200 (CBP-rhGAA).

[0215] Lumizyme® and ATB-200 rhGAA have different isoelectric points. Although both rhGAAs are produced in CHO cells, ATB-200, as described above, exhibits differences in glycosylation, most likely reflected in its different isoelectric points. The present inventors used capillary isoelectric focusing to determine the slightly different isoelectric points of different ATB-200 rhGAA (CBP-rhGAA) samples compared to Lumizyme®. As shown in Figure 18, the isoelectric point range of Lumizyme® (sample WBP110-STD) is slightly lower than that of the ATB-200 sample.

[0216] [ATB-200 rhGAA] [Compared to Lumizyme®] [Contains lower amounts of non-phosphorylated glycans.] The amounts of non-phosphorylated glycans in ATB-200 samples and Lumizyme® were analyzed and compared. The ATB-200 sample was found to contain lower amounts of non-phosphorylated high-mannose N-glycans than Lumizyme® (WBP110-STD), but similar amounts. The lower amounts of non-phosphorylated glycans inhibit the clearance of rhGAA from the circulation. The higher content of M6P glycans in ATB-200 compared to Lumizyme® may enable more effective lysosomal targeting of ATB-200. Figures 19A and 19B show the results.

[0217] ATB-200 rhGAA administered to muscles

[0218] As is apparent from Figure 1C, pharmacokinetic data cannot adequately distinguish between efficient and inefficient drug clearance, as rhGAA can be efficiently removed from the circulation and enter target tissues (such as lysosomes in muscle cells), or inefficiently removed through non-target tissues (such as the liver or spleen) or other types of inefficient binding that are not located in target tissues. Although ATB-200 rhGAA has a shorter biological half-life than Lumizyme®, the results demonstrate that ATB-200 rhGAA produces a significant reduction in glycogen compared to Lumizyme® in this animal model—comparing glycogen clearance of ATB-200 and Lumizyme®.

[0219] Partially isolated ATB-200 rhGAA

[0220] ATB-200 rhGAA was partially isolated using weak anion exchange (WAX) liquid chromatography. The ATB-200 fraction was detected using UV light and the content of M6P and other carbohydrates was determined. ATB-200 was obtained from CHO cells and purified. Lumizyme® was obtained from a commercial source.

[0221] Prior to fraction separation and analysis, ATB-200 samples were prepared by buffer exchange at 4°C using MWCO filters (30KD) with a gradient starting mobile composition containing 95% mobile phase A: 20 mM HEPES, pH 6; and 5% mobile phase B: 20 ​​mM HEPES (pH 6.5) + 1.5 M NaCl.

[0222] The ATB-200 fraction was separated by WAX chromatography using a PolyWax LP analytical column (guard column: PolyWAX LP Javelin 10 × 2.1 mm item #J22GCWX0503; analytical column 100 × 2.1 mm; 5 μm 1000A item #102WX0510; PolyWAX LP PolyLC Inc., Columbia, MD) and an Alliance Waters 2695 separation module (Empower Pro software). The column flow rate was 0.400 mL / min, and the total run time was 75 minutes. This method separated ATB-200 protein species based on their phosphorylation status.

[0223] The protein content of each fraction was measured by UV absorbance at 280 nM, and the mannose-6-phosphate (M6P) content was determined using high performance anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD).

[0224] After analysis and collection, the collected ATB-200 fractions were buffer exchanged and concentrated using MWCO filters (30 KD) in 50 mM sodium phosphate (pH 6.2), 50 mM sodium chloride at 4°C.

[0225] Figures 20A and 20B below depict gradient curves. Figure 21 compares the PolyWax elution curves of Lumizyme® and ATB-200. Lumizyme® exhibits a peak on the left side of its elution curve. The ATB-200 rhGAA curve shows four main peaks.

[0226] Based on the elution curve of ATB-200, as shown in FIG22 , four different fractions were obtained and analyzed.

[0227] Each of the four fractions was collected and desalted, then dried and reconstituted in deionized water. One portion of each fraction was hydrolyzed with 4N TFA to determine the M6P content by HPAEC-PAD. The results are shown in Table 3 below. Table 3 ATB-200-P039 Peak area (%) Concentration (µg) Concentration (µM) M6P (µM) M6P / GAA (Mol) Part 1 5.60 29.6 2.7 ND - Part 2 11.8 62.3 5.7 0.385 0.0680 Part 3 32.4 171 15.5 15.0 0.965 Part 4 49.8 263 23.9 47.3 1.98

[0228] The average M6P content per mole of rhGAA ranged from 0.0680 to 1.98. The average concentration of M6P-containing rhGAA can be adjusted by selecting a specific fraction or a mixture of different ATB-200 fractions. For example, the average M6P content of ATB-200 rhGAA can be adjusted to 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, or 2.0, or any subrange or intermediate value within these ranges. The glycans in each fraction of ATB-200 rhGAA were analyzed as shown in Table 4. Table 4 [N-] [Glycan type] ATB-200 Part 1 Part 2 Part 3 Part 4 [Compound] 40% 58% 47% 37% 40% Completely sialylated 5% 16% 10% 5% 4% SA and terminal Gal 16% 30% 25% 15% 15% Only terminal Gal 17% 8% 10% 14% 19% [High mannose] 56% 40% 48% 58% 57% Dual-M6P 17% 1% 8% 14% twenty four% Single M6P 15% 14% 20% twenty one% 10% Non-phosphorylated 28% 27% 26% 28% 26% [Hybrid] 4% 2% 5% 5% 3%

[0229] Each of these four fractions (Fractions 1-4) was then run through a PolyWax column and subfractions were tested for GAA activity. The GAA activity for each fraction is shown in Figure 23. The average concentration of specific N-glycans can be adjusted to within the ranges described in Table 4 by selecting specific fractions or combining and blending different fractions. For example, the content of di-M6P glycans can be selected or adjusted to within a range of 1% to 24%, the content of mono-M6P glycans can be selected or adjusted to within a range of 10% to 20%, the content of fully sialylated glycans can be selected or adjusted to within a range of 4% to 16%, the content of SA and terminal Gal can be selected or adjusted to within a range of 15% to 25%, and the content of N-glycans with only terminal Gal can be selected or adjusted to within a range of 8% to 19%. These ranges include any subranges or intermediate values, such as all integer values ​​within each range. Part II: In vivo efficacy of ATB-200 rhGAA and ATB-200 rhGAA conjugated to IGF2 targeting moieties.

[0230] ATB-200 rhGAA can be modified by conjugating it to an IGF2 peptide to further enhance the conjugate's ability to target the IGF2 / CI-MP receptor on lysosomes. Methods for producing such conjugates and targeting moieties are known and are also disclosed in WO 2012 / 166653, incorporated by reference. The rhGAA of the present invention (such as ATB-200 rhGAA) can be conjugated to a moiety that binds to the IGF2 receptor / CI-MPR (such as a fragment or modified fragment of the IGF2 amino acid sequence). This moiety may contain one or more of the amino acid sequence modifications shown in SEQ ID NO: 4, which depicts [(del 1-4)-Arg6-Leu27-Arg65], an IGF-2 peptide with an N-terminal extension linker and a TEV protease recognition site.

[0231] This IGF2 peptide lacks residues 1-4, leaving the N-terminal serine residue corresponding to residue 5 of wild-type IGF2. Substitution of arginine for glutamine at position 6 is known to substantially reduce the binding affinity of IGF2 peptides for serum IGF-binding protein (IGFBP). Substitution of leucine for tyrosine at position 27 is known to substantially reduce the binding affinity of IGF2 peptides for insulin and IGF1 receptors. A conservative substitution of arginine for lysine at position 65 allows for chemical modification of only the N-terminal extension linker for binding to lysosomal enzymes. The N-terminal extension, represented by GGGGSGGG (SEQ ID NO: 5), can be chemically modified for coupling to lysosomal enzymes such as rhGAA. Figure 2 shows the relative affinities of IGF2 and bi-M6P for the IGF2 / CI-MP receptor.

[0232] To further enhance rhGAA uptake, the present inventors developed a method for linking a variant IGF2 (vIGF2) peptide to ATB-200 rhGAA to further enhance binding to the IGF2 / CI-MPR receptor. As described in WO 2012 / 166653, the peptide portion targeting vIGF2 contains several amino acid substitutions to provide a significantly improved selectivity profile. The vIGF2 peptides according to the present invention can contain various combinations of these substitutions, and include a substitution at residue 65 (Lys to Arg) to eliminate a chemically reactive group, enabling chemical crosslinking only at the N-terminus. This allows for the production of a single chemically modified IGF2 peptide species for subsequent ERT coupling. Without this substitution, the reaction produces multiple (3-4) species of IGF2 peptide that are difficult to separate, resulting in low yields of the desired species and significantly reducing the overall efficiency of the method.

[0233] [Vote to ATB-200] [or ATB-200 IGF2] [GAA of the conjugate] [Gene knockout mice (GAA KO)] [Mouse] ATB-200 rhGAA was conjugated to the targeting moiety of the vIGF2 peptide, and the binding affinity of the IGF2-ATB-200 conjugate was compared with that of unconjugated ATB-200 rhGAA and Lumizyme®.

[0234] Glycogen clearance was further assessed after 4 weeks of intravenous rapid administration to KO mice. Mouse tissues were collected one week after the last dose and analyzed for GAA activity and glycogen content. Figure 24 shows glycogen content in quadriceps muscle for each sample tested. As is apparent from these results, ATB-200 and its combination with vIGF2 produced significantly reduced glycogen compared to Lumizyme® or the negative control (vehicle alone).

[0235] CIMPR Binding of ATB-200 IGF2 Conjugate and Uptake of the Conjugate by Myoblasts

[0236] CIMPR Binding Assay. Binding assays of ATB-200 and ATB-200 vIGF2 conjugates were performed to determine whether binding of the IGF2 moiety enhances rhGAA binding to receptors involved in lysosomal uptake of rhGAA. The discs were coated with CIMPR (cation-independent M6P receptor, IGF2 receptor) to capture rhGAA. The vIGF2 peptide was chemically coupled to Lumizyme® rhGAA and ATB-200 rhGAA. Chemical coupling of vIGF2 did not impair rhGAA enzymatic activity. Binding of the samples described in Figure 25 is shown in that figure.

[0237] Conjugation of the vIGF2 peptide was found to further enhance rhGAA binding to the IGF2 / CI-MPR, consistent with vIGF2 binding enhancing GAA affinity for the IGF2 / CI-MPR. Chemical conjugation of vIGF2 did not impair GAA enzymatic activity. As shown in Figure 25 , ATB-200-vIGF2 had the highest affinity for the CI-MPR, followed by Lumizyme-vIGF2, unconjugated ATB-200, and finally unconjugated Lumizyme (see Figure 25 ).

[0238] [Myocyte Uptake Analysis.] Myocyte (skeletal muscle model) uptake of Lumizyme® rhGAA, ATB-200 rhGAA, and vIGF2-bound ATB-200 was directly compared as described above. Results showed that under identical conditions, ATB-200 was internalized >10-fold better than Lumizyme® in L6 myocytes, while vIGF2-CBP GAA internalization exceeded Lumizyme® by approximately 50-fold. The data demonstrate that the increased receptor affinity of ATB-200 and vIGF2-bound ATB-200 directly translates into improved cellular uptake in target skeletal muscle cells (see Figure 26). Internalized vIGF2-ATB-200 was also properly processed into intermediate and mature GAA forms in L6 myoblasts (after 16 hours). IGF2 peptides are known to be naturally degraded in lysosomes, and the results demonstrate that bound vIGF2 peptides are removed from GAA in lysosomes, enabling proper GAA processing. Part III: Co-administration of ATB-200 rhGAA and chaperone proteins

[0239] The only approved treatment for Pompe disease is enzyme replacement therapy with Lumizyme® rhGAA via intravenous (IV) infusion. The present inventors investigated the use of the small molecule AT2220 (deoxynojirimycin; dugruent hydrochloride) to improve the pharmacokinetics of rhGAA therapy, for example, by physically stabilizing rhGAA.

[0240] AT2220 is an orally available pharmacological chaperone protein that reversibly binds and stabilizes endogenous and exogenous forms of GAA. When co-administered with rhGAA ERT, AT2220 is intended to bind to the infused enzyme and stabilize it in its properly folded, active form, leading to greater GAA uptake and glycogen reduction in disease-related tissues. The present inventors investigated the effects of administering AT2220 in combination with conventional rhGAA (Lumizyme) and high-M6P rhGAA (ATB-200).

[0241] AT2220 stabilizes rhGAA in human blood.

[0242] The activity of rhGAA (0.5 μM) ± 50 μM AT2220 was monitored during incubation in human blood (pH 7.4) at 37°C. As shown in Figure 27, AT2220 reduced the loss of rhGAA activity. In the absence of AT2220, at least 90% of rhGAA activity was lost over 24 hours (see Figure 27).

[0243] As shown in Figure 28, unfolded rhGAA loses activity, while folded rhGAA retains most of its activity. The folding of the rhGAA protein was monitored by heat denaturation at 37°C in either neutral (pH 7.4 - plasma environment) or acidic (pH 5.2 - lysosomal environment) buffer. AT2220 stabilized the rhGAA protein in neutral pH buffer for over 24 hours.

[0244] In vivo rhGAA activity following a single intravenous bolus administration of rhGAA.

[0245] Eight-week-old male Sprague-Dawley rats received a single intravenous (IV) dose of rhGAA (10 mg / kg) via rapid tail vein injection, with or without oral co-administration of AT2220 (3 or 30 mg / kg, free base equivalent) 30 minutes prior to the IV injection. Plasma GAA enzyme activity and protein levels were analyzed using a 4-MUG activity assay and Western blotting at selected time points. Co-administration of AT2220 significantly and dose-dependently increased the circulating half-life and exposure of rhGAA. Figure 29A shows that two AT2220 doses increased rhGAA half-life. Figure 29B depicts the amount of rhGAA detected by Western blotting at selected time points. These results demonstrate that AT2220 increases the biological half-life of rhGAA in vivo.

[0246] Co-administration of AT2220 increases rhGAA uptake and ameliorates rhGAA-mediated glycogen reduction in GAA KO mouse tissues

[0247] Twelve-week-old male GAA knockout (KO) mice were administered rhGAA (20 mg / kg) intravenously via rapid tail vein injection every other week for 8 weeks, with or without oral co-administration of AT2220 30 minutes prior to each intravenous injection.

[0248] FIG30A. GAA activity was assessed in disease-relevant tissues (n=5) 7 days after the last intravenous injection. Oral co-administration of 30 mg / kg AT2220 significantly increased rhGAA uptake in disease-relevant tissues compared to rhGAA alone. FIG30B and FIG30C. Tissue glycogen content was assessed using amyloglucosidase digestion ( FIG30B ) and histology ( FIG30C ) 21 days after the last intravenous injection (n=7). Co-administration of AT2220 resulted in significantly greater glycogen reduction in key disease-relevant tissues compared to rhGAA alone. Histological data also supported greater glycogen reduction in cardiac and quadriceps muscle fibers with co-administration of AT2220.

[0249] Effect of co-administration of AT2220 on Lumizyme® rhGAA activity in plasma and muscle of Pompe patients

[0250] Study Design: AT2220-010 was a two-period, open-label, non-randomized, Phase 2a drug-drug interaction study. During Period 1, patients received Lumizyme® rhGAA ERT alone according to their standard of care. During Period 2, single ascending oral doses of AT2220 were administered, followed one hour later by the next intravenous ERT infusion at the same dose and schedule as in Period 1. Plasma and muscle total GAA activity was assessed in Period 2 relative to Period 1. AT2220 groups: 50 mg (N=6), 100 mg (N=6), 250 mg (N=6), and 600 mg (N=7). Patient Demographics: Thirteen males and 12 females, aged 32-65 years. Weights ranged from 55.8 to 147 kg. Figures 31A, 31B, 31C, and 31D compare rhGAA activity in plasma when rhGAA is administered with or without the chaperone protein AT-2220.

[0251] Figures 31A, 31B, 31C, and 31D show active plasma rhGAA levels (presented as mean [SD], as determined by the 4-MUG GAA activity assay) during treatment with ERT alone (blue squares, Period 1) and two weeks later when ERT and AT2220 were co-administered (referred to in the figures as ERT + AT2220; red circles, Period 2). These data demonstrate an increase in active rhGAA in plasma when co-administered with AT2220. Figure 32 shows the absolute difference in GAA activity.

[0252] FIG32 shows the absolute difference in total GAA activity in muscle on day 3, calculated from individuals with matched biopsies after ERT alone (Period 1) and co-administration of AT2220 (Period 2). Each individual is represented by a unique symbol. The mean GAA muscle activity for each treatment group is indicated by the black horizontal bar. These data demonstrate a dose-related increase in muscle GAA activity when AT2220 is co-administered with ERT. The greatest increase in muscle GAA activity was observed in the 600 mg treatment group. Part IV: Preclinical Studies (POC1, Studies 294A and 294B)

[0253] Preclinical Studies POC 294A and 294B. Lumizyme, ATB-200, ATB-200 vIGF2, with or without co-administration of the chaperones AT2221 and AT2220 were administered intravenously.

[0254] The GAA KO mouse model was used to investigate the effects of different rhGAA treatment regimens. ATB-200 rhGAA or ATB-200 rhGAA vIGF conjugate was administered without or with the chaperones AT2221 (miglustat, N-butyl-deoxynojirimycin) or AT2220 (duglustat hydrochloride, 1-deoxynojirimycin).

[0255] Study 294a (POC 1a) involved six biweekly intravenous bolus doses of ATB-200 over three months. Study 294b involved four biweekly intravenous bolus doses of ATB-200 over two months. GAA KO mice were divided into treatment groups: 10 and 20 mg / kg unlabeled ATB-200; 5, 10, and 20 mg / kg vIGF2-labeled ATB-200; oral co-administration of AT2221 at 10 and 30 mg / kg, testing each ATB-200 enzyme; 30 mg / kg AT2220 co-formulated with 20 mg / kg unlabeled and labeled ATB-200; a 20 mg / kg Lumizyme® group; and a vehicle (negative control) group.

[0256] At treatment endpoint, glycogenometry was used to determine glycogenometry levels in primary disease-related tissues (e.g., heart, quadriceps, and triceps) using biochemical or HPLC-based methods. Glycogenometry levels were also measured in relevant secondary tissues (gastrocnemius, tongue, soleus, biceps, and liver). Glycogenometry and tissue enzyme uptake measured by 4-MUC assay were analyzed in tissue samples of heart, quadriceps, triceps, and diaphragm and then stained for PAS staining. Plasma anti-GAA IgG levels were measured. Tables 6 and 7 below describe the treatment groups for each study.

[0257] Table 6. Treatment Groups for Study 294a (POC1a). [Group] [Combined treatment] [ERT] [dose] [(mg / kg)] [AT2221] [dose] [(mg / kg)] [Deaths] [(] [total] [= 11)] 1 [Vehicle] [Vehicle] -- 1 (not directly related to drug administration) 2 Lumizyme® alone 20 -- 2 3 ATB-200 10 -- 0 4 Oral AT2221; followed by ATB-200 10 10 2 5 Oral AT2221; followed by ATB-200 10 30 1 6 ATB-200 20 0 7 Oral AT2221; followed by ATB-200 20 10 1 8 Oral AT2221; followed by ATB-200 20 30 0 9 COF AT2220 + ATB-200 20 AT2220 = 30 0 10 Mark ATB-200 5 -- 2 11 Oral administration of AT2221 followed by vIGF2-labeled ATB-200 5 10 0 12 Oral administration of AT2221 followed by vIGF2-labeled ATB-200 5 30 2 13 Mark ATB-200 10 -- 0 14 Oral administration of AT2221 followed by vIGF2-labeled ATB-200 10 10 0 15 Oral administration of AT2221 followed by vIGF2-labeled ATB-200 10 30 0 16 Mark ATB-200 20 0 17 Oral administration of AT2221 followed by vIGF2-labeled ATB-200 20 10 0 18 Oral administration of AT2221 followed by vIGF2-labeled ATB-200 20 30 0 19 COF AT2220 + vIGF2-labeled ATB-200 20 AT2220 = 30 0 [Group] [Number of animals] [Last dose date] [Note] 2 2 February 26, 2014 Found dead on 3 / 1 / 14, not drug related 4 5 3 / 12 / 2014 Euthanasia 1 hour after the third dose 7 4 3 / 12 / 2014 Death was discovered 45 minutes after the third dose 12 2 3 / 12 / 2014 Found dead 30 minutes after the third dose 12 1 3 / 26 / 2014 Found dead 50 minutes after the fourth dose 5 4 3 / 12 / 2014 Euthanasia on 3 / 26 / 14 before medication, unrelated to medication 4 6 3 / 26 / 2014 Found dead on 4 / 1 / 2014 10 2 3 / 26 / 2014 Found dead on 4 / 8 / 2014 1 4 4 / 9 / 2014 Found dead on 4 / 17 / 2014 10 1 4 / 23 / 2014 Found dead on 5 / 1 / 2014 2 1 4 / 23 / 2014 Found dead on 5 / 7 / 2014

[0258] Table 7: Treatment Groups Study 294b (POC1b). [Group] [Combined treatment] [ERT] [dose] [(mg / kg)] [AT2221] [dose] [(mg / kg)] [Deaths] [(] [total] [= 6)] 1 [Vehicle] [Vehicle] -- 2 Lumizyme® alone 20 -- 0 3 ATB-200 10 -- 1 4 Oral AT2221; followed by ATB-200 10 10 1 5 Oral AT2221; followed by ATB-200 10 30 0 6 ATB-200 20 0 7 Oral AT2221; followed by ATB-200 20 10 0 8 Oral AT2221; followed by ATB-200 20 30 0 9 COF AT2220 + ATB-200 20 AT2220 = 30 0 10 Mark ATB-200 5 -- 1 11 Oral administration of AT2221 followed by vIGF2-labeled ATB-200 5 10 0 12 Oral administration of AT2221 followed by vIGF2-labeled ATB-200 5 30 0 13 Mark ATB-200 10 -- 0 14 Oral administration of AT2221 followed by vIGF2-labeled ATB-200 10 10 1 15 Oral administration of AT2221 followed by vIGF2-labeled ATB-200 10 30 2 16 Mark ATB-200 20 0 17 Oral administration of AT2221 followed by vIGF2-labeled ATB-200 20 10 0 18 Oral administration of AT2221 followed by vIGF2-labeled ATB-200 20 30 0 19 COF AT2220 + vIGF2-labeled ATB-200 20 AT2220 = 30 0 [Group] [Number of animals] [Last dose date] [Note] 15 2 March 19, 2014 Found dead by vivarium staff on 3 / 27 / 2014 14 5 March 19, 2014 Found dead on 4 / 2 / 2014 before taking the medicine 4 2 4 / 2 / 2014 Found dead by vivarium staff on 4 / 5 / 2014 3 6 4 / 16 / 2014 Found dead after intraperitoneal administration on 4 / 16 / 2014 15 5 4 / 16 / 2014 Found dead by vivarium staff on 4 / 25 / 2014 10 4 4 / 16 / 2014 Found dead by vivarium staff on 4 / 24 / 2014

[0259] Figure 33 illustrates how mouse muscle tissue was analyzed for glycogen, GAA, and protein. Figure 34 illustrates analysis of muscle tissue from mice that received either rhGAA alone or the rhGAA-IGF2 conjugate without the chaperone protein. Dose-dependent glycogen reduction was observed in tissue from mice treated with ATB-200 (particularly with four doses). Both unlabeled ATB-200 rhGAA and IGF2-labeled ATB-200 rhGAA provided glycogen reduction in GAA KO mouse muscle tissue, generally better than the glycogen reduction provided by the same dose of Lumizyme®. Limited improvement was observed in the group that received six, rather than four, intravenous boluses.

[0260] FIG35 depicts analysis of muscle tissue from mice that received unlabeled ATB-200 rhGAA and chaperones (four intravenous boluses). At the doses tested (10 and 30 mg / kg), oral co-administration of AT2221 with 10 mg / kg of unlabeled ATB-200 showed little additional benefit over either enzyme alone. Oral co-administration of 10 mg / kg AT2221 with 20 mg / kg of unlabeled ATB-200 showed a modest trend toward further glycogen reduction; 30 mg / kg AT2221 was not as effective. Co-formulation of 30 mg / kg AT2220 with 20 mg / kg of unlabeled ATB-200 showed no clear benefit or harm over either enzyme alone.

[0261] Figure 36 depicts analysis of muscle tissue from mice that received unlabeled ATB-200 rhGAA and chaperones for the group that received six intravenous boluses. The benefit of 10 mg / kg AT2221 when co-administered orally with 20 mg / kg unlabeled ATB-200 appears less pronounced after six doses than after four doses. A co-formulation of 30 mg / kg AT2220 and 20 mg / kg unlabeled ATB-200 demonstrated significantly worse glucose reduction than either enzyme alone after six doses, potentially indicating PC inhibition of the enzyme. Figure 37 depicts analysis of muscle tissue from mice that received IGF2-labeled ATB-200 rhGAA and chaperones for the group that received four intravenous boluses.

[0262] In general, oral co-administration of AT2221 with 5, 10, or 20 mg / kg of vIGF2-labeled ATB-200 showed little additional benefit over either enzyme alone at the doses tested (10 and 30 mg / kg). Co-formulation of 30 mg / kg AT2220 with 20 mg / kg of vIGF2-labeled ATB-200 showed no significant effect over either enzyme alone. Figure 38 depicts analysis of muscle tissue from mice that received six intravenous bolus doses of IGF2-labeled ATB-200 rhGAA and chaperone.

[0263] In general, at the doses tested (10 and 30 mg / kg), oral co-administration of AT2221 with 5 and 10 mg / kg vIGF2-labeled ATB-200 showed little additional benefit over the enzymes alone. Oral co-administration of 10 mg / kg AT2221 with 20 mg / kg vIGF2-labeled ATB-200 showed a modest trend toward further glycogen reduction, while co-formulation of 30 mg / kg AT2220 with 20 mg / kg vIGF2-labeled ATB-200 did not demonstrate a significant effect over the enzymes alone. Figure 39 depicts GAA activity in muscle tissue from mice that received unlabeled and IGF2-labeled ATB-200 rhGAA (with or without a chaperone) for groups that received six intravenous boluses over three months. Tissue activity was measured 14 days after the last intravenous dose. At these later time points, activity appeared to have trended back toward baseline. For reliable absorption, FIG43 compares HPLC- and biochemical-based methods for determining glycogenolysis.

[0264] Figure 44 depicts the glycogen levels of various muscle tissues in groups of GAA KO mice treated with six IC boluses of either labeled (T) or unlabeled (U) ATB-200 rhGAA, as measured using two different assays. Figure 45 depicts the glycogen levels of quadriceps muscle tissue following treatment with six intravenous boluses of unlabeled (ATB-200) or IGF2-labeled ATB-200 (vIGF2-ATB-200).

[0265] The above data demonstrate the significant improvements provided by using ATB-200 rhGAA alone as a conjugate to the IGF2 targeting portion of CIMPR or in combination with a chaperone. Part V. Preclinical Studies 296 (POC 2)

[0266] Effects of different doses of AT2221 (migludec) and AT2220 (dugludec) on ATB-200 rhGAA uptake and glycogen reduction in GAA KO mice.

[0267] The two POC1 studies 294A and 294B described above evaluated the effects of biweekly administration of rh-GAA for 6 or 4 times. The goal of POC2 study 296 (2 biweekly injections) was to test the effects of different doses of AT2221-HCl (1, 3, and 10 mg / kg free base equivalent) on rhGAA absorption and rhGAA-mediated glycogen reduction in disease-relevant tissues of GAA KO mice under 2 repeated biweekly intravenous bolus dosing schedules when co-administered orally with unlabeled ATB-200 (5, 10, and 20 mg / kg). Table 8. Comparison of study designs between POC1 and POC2 [Research] [POC 1] [(294A] [and] [294B)] [POC 2] [(296)] Dosage 4 or 6 bolus injections every two weeks 2 rapid intravenous injections every two weeks Lumizyme® 20 mg / kg 20 mg / kg ATB-200* 10 and 20 mg / kg 5, 10, and 20 mg / kg AT2221 joint investment (ATB-200 administered orally 30 minutes before intravenous administration) 10 and 30 mg / kg (with 10 and 20 mg / kg ATB-200) 1, 3, and 10 mg / kg (Tested with 5, 10, and 20 mg / kg ATB-200; 30 mg / kg also tested with 20 mg / kg ATB-200) AT2220 co-formulation (ATB-200 + AT2220 intravenous) 30 mg / kg 3 and 10 mg / kg vIGF2-ATB-200 5, 10, and 20 mg / kg 20 mg / kg end Glycogen measurement, 14 days after the last (4th or 6th) injection Glycogen measurement, 14 days after the last (second injection) *The batch material used for POC 2 is different from that for POC 1

[0268] Additionally, the effect of AT2220-HCl (3 and 10 mg / kg) co-formulated with unlabeled ATB-200 (20 mg / kg) was evaluated by POC 2. The bridging group of Study 294 (POC 1) was also tested, including 30 mg / kg AT2221-HCl co-administered with 20 mg / kg unlabeled ATB-200 and 20 mg / kg vIGF2-labeled ATB-200 alone. Lumizyme® alone at 20 mg / kg was also included as a reference. For these purposes, Lumizyme® (20 mg / kg), unlabeled ATB-200 (5, 10, and 20 mg / kg), and labeled ATB-200 (20 mg / kg) were administered to GAA KO mice via rapid tail vein intravenous (IV) injection (a total of six injections) every other week. In addition, at each dose of unlabeled ATB200, 1.17, 3.5, or 11.7 mg / kg AT2221-HCl (1, 3, or 10 mg / kg free base equivalent, respectively) was administered via oral gavage (oral) 30 minutes prior to each intravenous administration of the enzyme; and 35 mg / kg AT2221-HCl (30 mg / kg free base equivalent) was administered orally 30 minutes prior to each intravenous injection of 20 mg / kg unlabeled ATB-200. 3.66 or 12.2 mg / kg AT2220-HCl (1, 3, or 10 mg / kg free base equivalent, respectively) was co-formulated with 20 mg / kg unlabeled ATB-200 and administered via intravenous bolus injection.

[0269] Study 296 involved only two biweekly intravenous dosings. No DPH was administered. However, animals were closely observed after each dosing. In Study 296, plasma and tissues—heart, diaphragm, tongue, liver, kidney, hindlimb, and forelimb—were collected 14 days after the second dosing. Antibody titers / IgG levels were measured against Lumizyme and unlabeled and labeled ATB-200.

[0270] Study Design: Animals: A total of 124 male GAA KO mice (approximately 30 g) with an average age of 16 weeks were used in this study. Six age-matched C57BL16 male mice served as controls. The treatment regimen is described in Table 5 below. Table 5. Drug treatment: [Group] [Number of mice] [ERT / ] [Vehicle] [Intravenous] [(mg / kg)] [AT2221] [Oral] [(mg / kg)] [AT2220] [+ERT] [Intravenous] [(mg / kg)] [ERT] [concentration] [(mg / mL)] [Weekly Requirement] [ERT / ] [Vehicle Volume] [(mL)] [AT2221-HCl] [concentration] [(mg / mL)] [Number of doses] [(] [Every other week] [)] 1 5 medium - - 1.9 - 2 2 7 Lumizyme (20) - - 2 2.1 - 2 3 7 Unmarked ATB-200 (5) - - 0.5 2.1 - 2 4 7 Unmarked ATB-200 (5) AT2221 (1) - 0.5 2.1 0.12 2 5 7 Unmarked ATB-200 (5) AT2221 (3) - 0.5 2.1 0.35 2 6 7 Unmarked ATB-200 (5) AT2221 (10) - 0.5 2.1 1.17 2 7 7 Unlabeled ATB-200 (10) - - 1 2.1 - 2 8 7 Unlabeled ATB-200 (10) AT2221 (1) - 1 2.1 0.12 2 9 7 Unlabeled ATB-200 (10) AT2221 (3) - 1 2.1 0.35 2 10 7 Unlabeled ATB-200 (10) AT2221 (10) - 1 2.1 1.17 2 11 7 Unmarked ATB-200 (20) - - 2 2.1 - 2 12 7 Unmarked ATB-200 (20) AT2221 (1) - 2 2.1 0.12 2 13 7 Unmarked ATB-200 (20) AT2221 (3) - 2 2.1 0.35 2 14 7 Unmarked ATB-200 (20) AT2221 (10) - 2 2.1 1.17 2 15 7 Unmarked ATB-200 (20) AT2221 (30) - 2 2.1 3.5 2 16 7 - Unmarked ATB-200 + AT2220 (20 + 3) 2 + 0.3 1.89 (On the day of administration, add a 10-fold stock solution of AT2220 hydrochloride (36.6 mg / mL) to the enzyme) - 2 17 7 - Unmarked ATB-200 + AT2220 (20 + 10) 2 + 1 1.89 (On the day of administration, add a 10-fold stock solution of AT2220 hydrochloride (12.2 mg / mL) to the enzyme) - 2 18 7 Mark ATB-200 (20) - 2 2.1 - 2 19 6 WT - - - - -

[0271] Dosing solution.

[0272] Formulation buffer. The formulation buffer consists of 50 mM sodium phosphate (pH 6.0) / 100 mM NaCl / 2% (wt / v) mannitol 0.02% (v / v) polysorbate 80. This buffer is sterile, endotoxin-free, and suitable for administering test samples to animals. The formulation buffer will be used to dilute the rhGAA enzyme preparation and will also serve as the "vehicle" sample for negative control mice without ERT.

[0273] Lumizyme® alone was administered intravenously. Lumizyme® was diluted to 2 mg / mL with formulation buffer and aliquoted into sterile, pyrogen-free vials. Each 2.1 mL aliquot was used for a 10 mL / kg (based on mouse weight) intravenous injection. The formulated test article vials were stored at 4°C until use. Each vial contained sufficient test article for weekly dosing of seven mice.

[0274] Unlabeled ATB-200 was administered intravenously. Unlabeled ATB-200 was diluted with formulation buffer to achieve the appropriate concentrations listed in Table 5 above. Unlabeled ATB-200 was diluted to 0.5 mg / ml (for the 5 mg rhGAA / kg group), 1 mg / ml (for the 10 mg rhGAA / kg group), or 2 mg / ml (for the 20 mg rhGAA / kg group) and aliquoted into sterile, pyrogen-free vials. 2.1 mL per vial was used for a 10 mL / kg (based on mouse weight) intravenous injection. The formulated test article vials were stored at 4°C until use. Each vial contained sufficient test article for weekly dosing of seven mice.

[0275] vIGF2-labeled ATB-200 was administered intravenously. vIGF2-labeled ATB-200 was diluted with formulation buffer to achieve the appropriate concentrations listed in Table 5 above. vIGF2-ATB-200 was diluted to 0.5 mg / ml (for the 5 mg rhGAA / kg group), 1 mg / ml (for the 10 mg rhGAA / kg group), or 2 mg / ml (for the 20 mg rhGAA / kg group) and aliquoted into sterile, pyrogen-free vials, 2.1 mL per vial, for intravenous injections of 10 mL / kg (based on mouse body weight). Vials of formulated test article were stored at 4°C until use. Each vial contained sufficient test article for weekly dosing of seven mice. Please note: Group 1 (untreated mice) received a bolus intravenous injection of vehicle alone (10 mL / kg) every other week for a total of two injections.

[0276] All animals received two 10 mL / kg intravenous injections via the lateral tail vein. Care should be taken to administer the dose slightly slower than the typical 5 mL / kg intravenous bolus injection to avoid any tail vein rupture / burst. The dose volume for each animal, along with any other findings from the live observations, was recorded on the Dosing and Sample Collection Sheet.

[0277] Oral Dosing of AT2221-HCl: A stock solution of 11.7 mg / mL AT2221-HCl was prepared in deionized water. The stock solution was then diluted with deionized water to achieve final concentrations of 0.12, 0.35, 1.17, and 3.5 mg / mL, respectively, for PO doses of 1, 3, 10, and 30 mg / kg. All solutions were vortexed for several seconds to ensure complete dissolution of the powder. Approximately 100 µl aliquots of each solution were collected for analysis. 10 ml / kg AT2221-HCl was administered by oral gavage 30 minutes prior to the intravenous injection of unlabeled ATB-200.

[0278] Coformulation Intravenous Drug Preparation: Unlabeled ATB-200 and vIGF2-labeled ATB-200 in the coformulation group were diluted to 2.2 mg / mL in formulation buffer and aliquoted into sterile, pyrogen-free vials at 1.89 mL per vial. Vials of the formulated test article were stored at 4°C until use. Each vial contained sufficient test article for weekly dosing of seven mice. Stock solutions of AT2220-HCl at 36.6 mg / mL and 12.2 mg / mL, respectively, were prepared in formulation buffer. On the day of dosing, 0.21 mL of the 36.6 mg / mL and 12.2 mg / mL AT2220-HCl stock solutions, respectively, were added to each 1.89 mL enzyme vial to prepare the final co-formulation solution (total volume 2.1 mL). This solution, for intravenous injection based on mouse body weight, contains either 0.366 mg / mL AT2220-HCl (equivalent to 0.3 mg / mL free base and a 3 mg / kg dose) or 1.22 mg / mL AT2220-HCl (equivalent to 1 mg / mL free base and a 10 mg / kg dose) and 2 mg / mL enzyme. The solution should be mixed gently to avoid foaming and should appear clear and colorless.

[0279] Experimental: At necropsy, 14 days after the last intravenous administration, whole blood was collected via cardiac puncture. The collected blood was placed in lithium heparinized vials and centrifuged at 2700 × g for 10 minutes at 4°C. Approximately 200 µl of plasma was collected and immediately snap-frozen on dry ice. Following sacrifice, the following tissues were collected: heart, diaphragm, tongue, liver, kidney, hind limbs, and forelimbs.

[0280] Results from the POC2 study demonstrated that ATB-200 was more effective than Lumizyme® when administered intravenously twice (once every two weeks), with ATB-200 demonstrating a clear dose-dependent and significantly greater reduction in glycogen compared to Lumizyme. The following comparative glycogen reduction levels were observed: 20 mg / kg ATB-200 > 10 mg / kg ATB-200 > 5 mg / kg ATB-200 ≈ 20 mg / kg Lumizyme. The glycogen reduction achieved with vIGF2-ATB-200 was comparable to that achieved with ATB-200 rhGAA: 20 mg / kg ATB-200 ≈ 20 mg / kg vIGF2-ATB-200. Co-administration of 10 mg / kg AT2221 and 3 mg / kg AT2220 demonstrated modestly improved glycogen reduction compared to ATB-200 rhGAA alone. While it's difficult to compare two different routes, AT2220 co-formulations performed better than AT2221 co-administration. A dose-dependent effect was observed with either chaperone at the "optimal" dose that demonstrated improvement (1, 3, 10, and 30 mg / kg AT2221 and 3 and 10 mg / kg AT2220 tested to date). There was inhibition at higher doses, and no effect at lower doses. Representative data are shown in Figure 46 et seq.

[0281] At 20 mg / kg ATB-200, both AT2221 and AT2220 showed a trend toward improved glycogen reduction (see Figure 46). Co-administration of 10 mg / kg AT2221 showed a trend toward a beneficial effect; 1, 3, or 30 mg / kg were not effective. 3 and 10 mg / kg AT2220 co-formulations showed a trend toward improved glycogen reduction in the quadriceps and triceps muscles; in particular, 3 mg / kg AT2220 co-formulation resulted in significantly greater glycogen reduction than either enzyme alone. Combining AT2221 or AT2220 with ATB-200 significantly outperformed Lumizyme® at the same dose. As shown in Figure 47, while ATB-200's higher M6P content allows for better lysosomal targeting, as evident from the improved substrate reduction, its half-life is shorter than Lumizyme®. Table 6 describes the pharmacokinetic parameters of ATB-200 rhGAA.

[0282] Table 6: ATB-200 pharmacokinetics Cmax (nmol / mL / hr) 21316 45547 88799 104702 AUC (nmol / mL) 58350 101290 247068 514743 Half-life (hr) 0.62 0.61 0.79 1.32

[0283] The PK of ATB-200 rhGAA was dose-dependent and linear based on Cmax and AUC. The half-life of ATB-200 rhGAA was similar at 5, 10, and 20 mg / kg. At 20 mg / kg, the half-life of ATB-200 was approximately 47 minutes, while that of Lumizyme® was approximately 1.3 hours; studies on the impact of chaperones on ATB-200 PK are ongoing. Overall, these mouse infusion PK data were similar to the intravenous bolus PK in mice (data not shown), indicating that despite ATB-200's shorter half-life (compared to Lumizyme), it is well targeted due to its high M6P content and results in improved glycogen reduction in skeletal muscle.

[0284] As shown in Figure 48, dose-dependent glycogen reduction was observed with ATB-200 rhGAA. 5 mg / kg ATB-200 rhGAA ≈ 20 mg / kg Lumizyme®. 10 and 20 mg / kg ATB-200 rhGAA demonstrated significantly greater glycogen reduction than 20 mg / kg Lumizyme®. The vIGF2-ATB-200 rhGAA conjugate showed a slight trend toward greater glycogen reduction compared to the same dose of ATB-200, but overall, the two forms of ATB-200 were comparable.

[0285] At 20 mg / kg of ATB-200 rhGAA, both AT2221 and AT2220 showed a trend toward improved glycogen reduction (see Figure 49). Co-administration of 10 mg / kg AT2221 showed a trend toward a beneficial effect in the quadriceps, but not the triceps; 1, 3, or 30 mg / kg AT2221 was not effective. 3 and 10 mg / kg AT2220 co-formulations showed a trend toward improved glycogen reduction in both the quadriceps and triceps; in particular, 3 mg / kg AT2220 co-formulation resulted in significantly greater glycogen reduction than either enzyme alone. Combining AT2221 or AT2220 with ATB-200 significantly outperformed Lumizyme at the same dose.

[0286] Results from the POC1 (4 and 6 biweekly doses) and POC2 (2 biweekly doses) studies showed significantly better glycogen reduction with ATB-200 than with Lumizyme. ATB-200 10 mg / kg > 20 mg / kg Lumizyme; ATB-200 20 mg / kg > 20 mg / kg Lumizyme; ATB-200 alone demonstrated significant dose-dependent glycogen reduction; 20 mg / kg vIGF2-ATB-200 was comparable to ATB-200; an equivalent dose of 20 mg / kg ATB-200 ≈ vIGF2-ATB-200 > Lumizyme; and co-administration of AT2221 (10 mg / kg) with 20 mg / kg ATB-200 showed a trend toward improved glycogen reduction compared to 20 mg / kg ATB-200 alone.

[0287] The POC2 study also demonstrated that administration of both enzymes was sufficient for glycogen reduction in GAA KO mice. While 20 mg / kg Lumizyme® showed only minimal, if any, glycogen reduction, 10 and 20 mg / kg ATB-200 substantially reduced glycogen (approximately 60% and 40% in the quadriceps and triceps, respectively). ATB-200 rhGAA 5 mg / kg ≈ 20 mg / kg Lumizyme. At 20 mg / kg ATB-200, both AT2221 and AT2220 showed a trend toward improved glycogen reduction (compared to 20 mg / kg ATB-200 alone). AT2221 10 mg / kg and AT2220 3 mg / kg were effective in reducing glycogen. The beneficial effects of AT2221 were evident in the quadriceps and gastrocnemius muscles, but not as profound in the triceps. AT2221 at 1 and 3 mg / kg and ATB-200 at 5, 10, or 20 mg / kg were not effective in reducing glycogen. The beneficial effects of AT2220 were particularly evident in the quadriceps, gastrocnemius, and triceps muscles (only the high dose of 30 mg / kg AT2220 was tested in the POC1 study and demonstrated an inhibitory effect). Combining AT2221 or AT2220 with ATB-200 significantly outperformed Lumizyme at the same dose. See Figures 50A, 50B, and 50C, which show glycogen levels in selected muscles after treatment. Table 7. Study 96 (POC): Glycogen in Muscle. Observations in Daily Life [Group] [Combined treatment] [Deaths] [(] [total] [= 7)] 1 [Vehicle] 1 2 Lumizyme® 20 mg / kg 0 3 ATB-200 5 mg / kg 0 4 AT2221 1 mg / kg orally; followed by ATB-200 5 mg / kg 1 5 3 mg / kg oral AT2221; followed by 5 mg / kg ATB-200 0 6 AT2221 10 mg / kg orally; followed by ATB-200 5 mg / kg 0 7 ATB-200 10 mg / kg 0 8 AT2221 1 mg / kg orally; followed by ATB-200 10 mg / kg 0 9 3 mg / kg oral AT2221; followed by ATB-200 10 mg / kg 0 10 AT2221 10 mg / kg orally; followed by ATB-200 10 mg / kg 0 11 ATB-200 20 mg / kg 1 12 1 mg / kg oral AT2221; followed by ATB-200 20 mg / kg 0 13 3 mg / kg oral AT2221; followed by ATB-200 20 mg / kg 0 14 AT2221 10 mg / kg orally; followed by ATB-200 20 mg / kg 1 15 30 mg / kg oral AT2221; followed by 20 mg / kg ATB-200 1 16 Co-formulation: 3 mg / kg ATB220 + ATB-200 20 mg / kg 1 17 Co-formulation: 10 mg / kg ATB220 + ATB-200 20 mg / kg 0 18 vIGF2-labeled ATB-200 20 mg / kg 1 19 WT 0 [Group] [Number of animals] [Last dose] [DOD] [Note] 15 7 July 10, 2014 July 16, 2014 Found dead on 7 / 16 / 14; not drug related 5 1 July 24, 2014 July 24, 2014 Death occurred 30 minutes after the second intravenous dose 14 6 July 24, 2014 July 24, 2014 Euthanasia was performed 20 min after the second intravenous administration. 16 4 July 24, 2014 July 24, 2014 Death occurred 35 minutes after the second intravenous dose 18 6 July 24, 2014 July 30, 2014 Found dead on 7 / 30 / 14; may not be drug related 1 1 July 24, 2014 8 / 7 / 2014 Death discovered immediately before autopsy 11 6 July 24, 2014 ?? Lost from cage, may have died before necropsy

[0288] Overall, 7 deaths occurred (among 124 GAA KO mice)

[0289] Animals received two biweekly intravenous bolus injections. No DPH was administered during this study. After the second dose, all groups receiving concomitant protein administration (AT2221 co-administered or AT2220 co-formulation) exhibited signs of an acute immune response; three animals in three different groups died shortly after dosing. The enzyme-alone group appeared normal and healthy immediately after each injection. Antibody titers were measured in treated animals to illustrate these observations. Figure 51 shows the pharmacokinetics of ATB-200, vIGF2-ATB-200, and Lumizyme® (individual mice). Figure 52 shows normalized pharmacokinetic data. Table 8: rhGAA half-life [half life] [(hr)] [animal] [1] [animal] [2] [animal] [3] [animal] [4] [animal] [5] [average value] Lumizyme® 1.3 1.6 1.2 1.4 1.1 1.32 ATB-200 5 mg / kg 0.53 0.60 0.64 0.73 0.59 0.62 ATB-200 10 mg / kg 0.69 0.55 0.60 0.67 0.55 0.61 ATB-200 20 mg / kg 0.81 0.83 0.83 0.78 0.70 0.79 ATB-200 20 mg / kg + AT2221 10 mg / kg (PO) 1.04 0.69 0.72 1.18 0.70 0.87 ATB-200 20 mg / kg + AT2220 10 mg / kg (COF) 0.96 0.75 0.94 0.82 0.82 0.86 vIGF2-ATB-200 0.46 0.44 0.48 0.53 1.47 0.68 (0.48)*

[0290] * Animal 5 in the vIGF2-ATB-200 group exhibited a significantly longer enzyme half-life compared to the other animals in the group, substantially affecting the mean half-life for the group. Excluding this animal, the mean half-life was 0.48 hr.

[0291] POC2 Study (Study 296). Effects of ATB-200 Administration with and without a Chaperone on Glycogen Reduction. Table 9. Summary of some POC1 and POC2 study results: [treat] [dose] [(mg / kg)] [POC1] [(4] [and] [6] [Injection] [)] ATB-200 (single) 10, 20 + AT2221 (co-administered) + 10. + 30 + AT2220 (co-formulation) + 30 vIGF2-ATB-200 (alone) 5, 10, 20 + AT2221 (co-administered) + 10. + 30 + AT2220 (co-formulation) + 30 End point: Glycogen 14 days after the last injection Amyloglucosidase digestion; acid hydrolysis; PAS staining

[0292] As shown in Figure 53, ATB-200 and vIGF2-ATB-200 produced significant reductions in glycogen compared to Lumizyme® treatment in quadriceps muscle tissue. Dose-dependent glycogen reductions were observed with ATB-200 and vIGF-2-ATB-200. 20 mg / kg (vIGF2-)ATB-200 > 10 mg / kg (vIGF2-)ATB-200 > 5 mg / kg vIFG2-ATB-200 = 20 mg / kg Lumizyme. Glycogen reduction was improved when 20 mg / kg (vIGF2-)ATB-200 was co-administered with 10 mg / kg AT2221. Potential inhibition was observed at the higher AT2221 dose (30 mg / kg).

[0293] Study 296 (POC 2, 2 injections): ERT alone: ​​ATB-200 and vIGF2-ATB-200 were more effective than Lumizyme.

[0294] Figure 54 shows the histology of heart, diaphragm, triceps, and quadriceps tissues treated with rhGAA. One section from each animal was analyzed. Each image (20×) represents n=6-7 per group. Images of WT tissue are shown in the inset.

[0295] [Research] [296 (POC2-2] [Injection] [)] [: , Amyloglucosidase digestion , ]

[0296] As shown in Figure 55 , dose-dependent reductions in hepatic glucose were observed with ATB-200 rhGAA. 5 mg / kg ATB-200 ≈ 20 mg / kg Lumizyme. 10 and 20 mg / kg ATB-200 demonstrated significantly greater reductions in hepatic glucose than 20 mg / kg Lumizyme. In general, vlFG2-ATB-200 was comparable to ATB-200. This was consistent with histological assessment by PAS.

[0297] [Study 296.] [Quadriceps: ATB-200 rhGAA] [with pharmacological chaperone protein (PC)] [The combination resulted in less glycogen than ATB-200 and rhGAA alone] [Bigger].

[0298] As shown in Figures 56, 57, and 58, the combination of ATB-200 rhGAA and PC AT2221 or AT2220 provided greater reductions in quadriceps glycogen compared to ATB-200 rhGAA alone. Co-administration of PC AT2221 was most effective at 10 mg / kg, with possible inhibition observed at 30 mg / kg. Co-formulations of AT2220 were more effective at 3 mg / kg compared to the higher dose of 10 mg / kg.

[0299] [Study 296. ATB-200 rhGAA] [with chaperone protein AT2221 (] [Megrute)] [Combination compared to ATB-200 rhGAA alone] [Provides triceps glycogen reduction].

[0300] As described for Study 296, GAA KO mice were given two intravenous doses of ATB-200 rhGAA. Figure 59 shows the effects on muscle (triceps) glycogen levels following treatment with Lumizyme® rhGAA, ATB-200 rhGAA alone, or a combination of ATB-200 rhGAA and 1, 3, 10, or 30 mg / kg AT-2221. Figure 60 shows the reduction in glycogen in triceps following treatment with ATB-200 rhGAA and 1, 3, 10, or 30 mg / kg AT-2221. Co-administration: No significant additional benefit was observed with the combination of AT2221 by enzymatic analysis. A slight further reduction was observed with 3 and 10 mg / kg AT2221 by PAS. Co-formulation: AT2220 was more effective at the 3 mg / kg dose than the 10 mg / kg dose.

[0301] [Study 296. ATB-200 rhGAA] [with chaperone protein AT2221 (] [Megrute)] [Combination compared to ATB-200 rhGAA alone] [Provide myocardial glycogen reduction].

[0302] Figure 62 shows glycogen staining of cardiac tissue following treatment with ATB-200 rhGAA or with a combination of ATB-200 rhGAA and 1, 3, 10, or 30 mg / kg AT-2221. Figure 63 is a bar graph showing the relative reduction in glycogen in cardiac tissue. Figure 64 shows glycogen staining of cardiac tissue treated with ATB-200 rhGAA alone or with a combination of ATB-200 rhGAA and the chaperone AT-2200 (dugruct) at 3 or 10 mg / kg. Co-administration: Significantly further glycogen reduction was observed in the group treated with AT-2221 (30 mg / kg). This effect was further evident by PAS staining. Co-formulation: AT2220 was more effective at 3 mg / kg than at 10 mg / kg.

[0303] [Study 296. ATB-200 rhGAA] [with chaperone protein AT2221 (] [Megrute)] [Combination compared to ATB-200 rhGAA alone] [Provides diaphragmatic glycogen reduction].

[0304] Figure 65 shows glycogen staining of diaphragm tissue treated with ATB-200 rhGAA and 1, 3, 10, or 30 mg / kg of the chaperone protein AT2221 (miglutide). Figure 66 shows glycogen staining of diaphragm tissue treated with ATB-200 rhGAA and 3 or 10 mg / kg of the chaperone protein AT2220 (duglutide). Co-administration: A consistent further reduction in glycogen levels was observed when AT2221 (30 mg / kg) was co-administered with 20 mg / kg ATB-200. Co-formulation: ATB-200 (20 mg / kg) further improved glycogen reduction with co-formulation compared to ERT alone. The effects of co-formulations of ATB-200 rhGAA with 3 and 10 mg / kg AT2220 were comparable to the effects of co-administration (30 mg / kg AT2221).

[0305] [Study 296.] [with PC] [Combination compared to ATB-200 rhGAA alone] [Leads to Further Glucose Reduction]. Consistent with POC1, the effects of PC were seen with 20 mg / kg ATB-200. PC provided no additional benefit when combined with lower doses of ATB-200 (5 and 10 mg / kg, see Appendix). Co-administration: AT2221 at a suitable dose of 10 mg / kg was observed in most tissues. This demonstrated the greatest reduction in glycogen in the quadriceps and gastrocnemius muscles, with improved reduction in glycogen in the triceps, heart, and diaphragm, without signs of suppression, compared to Lumizyme® rhGAA alone. Co-formulation: 3 mg / kg AT2220 was a suitable dose in most tissues and resulted in greater glycogen reduction in all analyzed tissues compared to ATB-200 alone. This effect was similar to or superior to co-administration with 10 mg / kg AT2221 in most analyzed tissues. With only 2 injections (POC 2), the effect was greater than 4 or 6 injections of 30 mg / kg AT2220 co-formulated with ATB-200 (POC 1). Figure 67 is a bar graph summarizing the relative glycogen reduction in heart and different skeletal muscle tissues after treatment with ATB-200 rhGAA and the combination of ATB-200 rhGAA and chaperones.

[0306] [Study 296.] [Using ATB-200 rhGAA] [or ATB-200 rhGAA] [with AT2221] [Glycogen staining of cardiac and skeletal muscle tissues following combined protein treatment].

[0307] As described above for Study 296, animals were treated with Lumizyme® rhGAA, ATB-200 rhGAA alone, or ATB-220 in combination with 1, 3, or 10 mg / kg of the AT2221 (miglutide) chaperone. Figure 68 shows glycogen staining of representative tissue sections. Figure 69 is a bar graph showing relative tissue glycogen levels. No additional benefit was observed with coadministration of ATB-200 (10 mg / kg) compared to rhGAA ERT alone. Coadministration of ATB-200 (5 and 10 mg / kg) provided no additional benefit compared to rhGAA ERT alone.

[0308] [Study 296.] [Effects of treatment on quadriceps muscle tissue].

[0309] Quadriceps ERT Alone: ​​A dose response was observed with ATB-200 rhGAA. ATB-200 at 5 mg / kg was comparable in efficacy to Lumizyme® (20 mg / kg). ATB-200 at 20 mg / kg was significantly more effective than Lumizyme® (20 mg / kg). vIGF2-ATB-200 (20 mg / kg) was comparable in efficacy to ATB-200 (20 mg / kg). vIGF2-ATB-200 rhGAA was more effective than Lumizyme rhGAA. Coadministration: Glucose reduction was not significantly improved when AT2221 was coadministered with lower doses of ATB-200 (5 or 10 mg / kg). Glucose reduction was slightly increased when AT2221 (10 mg / kg) was coadministered with 20 mg / kg ATB-200. Less inhibition was observed with co-administration of ATB-200 (20 mg / kg) and AT2221 (30 mg / kg). Co-formulation: A greater reduction in glycogen was observed with ATB-200 rhGAA (20 mg / kg) co-formulation with AT2220 compared to ATB-200 rhGAA alone. AT2220 was more effective at 3 mg / kg than at 10 mg / kg (see Figures 70 and 71).

[0310] [Study 296.] [Effects of treatment on triceps muscle tissue].

[0311] As shown in Figure 72, ATB-200 and vIGF2-ATB-200 were more effective than Lumizyme® in reducing glycogen. Figure 73 shows the effects of treating triceps muscle tissue with ATB-200 rhGAA co-administered with the chaperone protein AT2221 and co-formulated with the chaperone protein AT2220. For triceps ERT alone, a dose response was observed with ATB-200; 5 mg / kg ATB-200 was comparable in efficacy to Lumizyme® (20 mg / kg); 20 mg / kg ATB-200 was significantly more effective than Lumizyme® (20 mg / kg); vIGF2-ATB-200 (20 mg / kg) was more effective than Lumizyme® (20 mg / kg); and vIGF2-ATB-200 was slightly less effective than ATB-200 (both at 20 mg / kg). Co-administration of triceps: A small increase in glycogen reduction was observed when AT2221 was co-administered with lower doses of ATB-200 (5 or 10 mg / kg). Moderate improvement in glycogen reduction was observed when AT2221 (3 and 10 mg / kg) was co-administered with 20 mg / kg of ATB-200. Co-formulation of triceps: ATB-200 efficacy was significantly improved with co-formulation compared to co-administration. AT2220 was more effective at 3 mg / kg than at 10 mg / kg.

[0312] [Study 296.] [Effects of treatment on diaphragmatic tissue].

[0313] As shown in Figure 74, ATB-200 and vIGF2-ATB-200 were more effective than Lumizyme® in reducing glycogen in diaphragm tissue. Figure 75 shows the effects of treating diaphragm tissue with ATB-200 rhGAA co-administered with the chaperone protein AT2221 and co-formulated with the chaperone protein AT2220. Diaphragm ERT alone: ​​A dose response was observed with ATB-200 rhGAA. 5 mg / kg ATB-200 was comparable in efficacy to Lumizyme® (20 mg / kg). 20 mg / kg ATB-200 rhGAA was significantly more effective than Lumizyme® (20 mg / kg). At the same dose, vIGF2-ATB-200 (20 mg / kg) was comparable to ATB-200 and more effective than Lumizyme®. Co-administration of diaphragm: There was no significant improvement in glycogen reduction when AT2221 was co-administered with lower doses of ATB-200 (5 or 10 mg / kg). Glycogen levels were consistent and modestly further reduced when AT2221 (30 mg / kg) was co-administered with 20 mg / kg ATB-200. The AT2221 dose response was subtle. Co-formulation treatment of diaphragm showed less improvement in the efficacy of ATB-200 (20 mg / kg) compared to rhGAA ERT alone. The effects of the co-formulation (3 and 10 mg / kg AT2220) were comparable to those of ATB-200 (20 mg / kg) co-administered with 30 mg / kg AT2221.

[0314] [Study 296] [:Effects of treatment on cardiac tissue].

[0315] As shown in Figure 76, ATB-200 and vIGF2-ATB-200 were more effective than Lumizyme® in reducing glycogen in cardiac tissue. Figure 77 shows the effects of treating cardiac tissue with ATB-200 rhGAA co-administered with the chaperone protein AT2221 and co-formulated with the chaperone protein AT2220. ERT in cardiac tissue alone: ​​A dose response was observed with ATB-200 rhGAA. 5 mg / kg ATB-200 was comparable in efficacy to Lumizyme® (20 mg / kg). 20 mg / kg ATB-200 rhGAA was significantly more effective than Lumizyme® (20 mg / kg). vIGF2-ATB-200 was slightly more effective than Lumizyme®. vIGF2-ATB-200 (20 mg / kg) was comparable to a moderate dose (10 mg / kg) of ATB-200 rhGAA. Co-administration of cardiac tissue showed no significant improvement in glycogen reduction when AT2221 was co-administered with lower doses of ATB-200 (5 or 10 mg / kg). A substantial further reduction in glycogen levels was observed when AT2221 (30 mg / kg) was co-administered with 20 mg / kg ATB-200. A dose response was observed for AT2221. Co-administration of cardiac tissue showed a modest improvement in the efficacy of ATB-200 (20 mg / kg) compared to ERT alone. The effects of the co-administration (3 and 10 mg / kg AT2220) were similar to those of the co-administration (3 and 10 mg AT2221).

[0316] [Research] [296. ATB-200 rhGAA] [Effects of drug administration.]

[0317] In Study 296 (POC2), knockout mice were dosed with ATB-200 rhGAA twice. In previous studies POC1a and POC1b, animals were dosed with ATB-200 rhGAA six or four times. Figure 78 compares the effects of these different dosing regimens on glycogen levels in quadriceps muscle. For both Lumizyme® rhGAA and ATB-200 rhGAA alone, lower glycogen levels correlated with the number of doses. [VI] [Part. ATB-200] [Pharmacology Overview]

[0318] [Study 302] [:ATB-200 rhGAA] [, vIGF2-ATB-200 rhGAA] [and Lumizyme® rhGAA] [In GAA] [Pharmacokinetics in knockout mice(] [Intravenous Infusion)]

[0319] The present inventors sought to evaluate the pharmacokinetic (PK) profiles of ATB-200 rhGAA and vIGF2-labeled ATB-200 rhGAA when administered via intravenous infusion to GAA knockout (GAA) knockout (KO) mice and compare their pharmacokinetic (PK) profiles with those of Lumizyme. Study design: 40 GAA knockout (GAA) mice approximately 14-16 weeks old (n=5 mice / group) were administered a single 30-minute intravenous infusion of the test composition. The following doses were used: Lumizyme at 20 mg / kg; ATB-200 rhGAA at 5, 10, and 20 mg / kg; vIGF2-labeled ATB-200 rhGAA at 20 mg / kg. Chaperone protein AT2221-HCl was co-administered orally at 10 mg / kg over 30 minutes, followed by an intravenous infusion of ATB-200 rhGAA. The chaperone AT2220-HCl was co-formulated with ATB-200 rhGAA at a dose of 10 mg / kg and administered via intravenous infusion. At the study endpoint, plasma was collected before infusion and at 30 minutes, 1, 1.5, 2, 3, and 24 hours (final bleed) from the start of the infusion; tissues were collected at 24 hours. Plasma PK of Lumizyme® rhGAA, ATB-200 rhGAA, and vIGF2-labeled ATB-200 rhGAA was analyzed as a measure of GAA activity. Tissue uptake was measured at 24 hours as a measure of GAA activity in the heart, quadriceps, and triceps muscles. Table 10. Comparison of the pharmacokinetics of rhGAA products Group n = 5 Lumizyme (20 mg / kg) ATB-200 (20 mg / kg) vIGF2-ATB-200 (20 mg / kg) C max (nmol / mL / hr) 104702 88799 77547 AUC (nmol / mL) 514743 247068 120945 Half-life (hr) 1.32 0.79 0.48* (0.68)

[0320] As can be seen, the half-lives of ATB-200 rhGAA (approximately 47 minutes) and vIGF2-ATB-200 rhGAA (approximately 29 minutes) are shorter than those of Lumizyme® (1.3 hours). Overall, Lumizyme® PK > ATB-200 > vIGF2-ATB-200 (see Figures 79A and 79B). The PK of ATB-200 rhGAA is dose-dependent and lower than that of Lumizyme®, as shown in Figures 80A and 80B. Addition of the chaperones AT2220 or AT2221 did not significantly improve the PK of ATB-200 rhGAA, as shown in Figures 81A and 81B. Table 11. Comparative pharmacokinetics of ATB-200 rhGAA alone and in combination with chaperones. Group ATB-200 alone (20 mg / kg) ATB-200 (20 mg / kg) + AT2221 (10 mg / kg) (PO) ATB-200 (20 mg / kg) + AT2220 (10 mg / kg) (co-formulation) C max (nmol / mL / hr) 88799 74987 96699 AUC (nmol / mL) 247068 239390 286042 Half-life (hr) 0.79 0.81 0.85

[0321] Neither AT2220 nor AT2221 demonstrated any significant improvement in ATB-200 PK at the doses tested. Study 302 demonstrated that ATB-200 rhGAA and vIGF2 exhibited lower PK than Lumizyme® in GAA KO mice, but was more effective than Lumizyme in reducing tissue glycogen. The PK of ATB-200 was dose-dependent (at the doses tested of 5, 10, and 20 mg / kg) and linear with similar half-lives. At equivalent doses, the half-life of ATB-200 rhGAA was approximately 47 minutes, compared to vIGF2-ATB-200 (approximately 29 minutes) and Lumizyme® (1.3 hours). Neither AT2220 nor AT2221 chaperones showed any significant improvement in ATB-200 PK at the 10 mg / kg dose (10 mg / kg AT2221 + 20 mg / kg ATB-200 showed improved glycogen reduction in GAA KO mice). [VII] [part.] [Anti-GAA] [Antibody titer]

[0322] [In Study 294A (POC1a)] [, 294B (POC1b)] [and 29 (POC2)] [Evaluation of anti-rhGAA IgG] [Titer]. The protocol described above for Study 296 generally followed that for Studies 294A and 294B, with the exception of the biweekly dosing schedule. Generally, all animals receiving rhGAA produced IgG titers against rhGAA. Lumizyme® rhGAA IgG titers were generally higher than those for ATB-200 rhGAA; and IgG titers against IGF2-labeled ATB-200 were substantially equivalent to those for unlabeled ATB-200 rhGAA.

[0323] [Study 294A] In this study, animals received six biweekly injections. Anti-rhGAA IgG titers - Enzyme-only group (Test 1). Plasma samples were incubated on Imulon 2HB plates coated with the same rhGAA as the animals were treated with; Test 1 included only the group receiving enzyme alone. All animals receiving rhGAA developed anti-rhGAA titers (20,000-160,000). No substantial differences in anti-rhGAA titers were observed between unlabeled and labeled ATB-200. The titers against unlabeled and IGF2-labeled rhGAA were similar. No significant differences in antibody titers were observed between the different doses of rhGAA; see Figure 82.

[0324] [Anti-rhGAA IgG] [Titer] - Enzyme Only Groups (Test 1 vs. 2). Plasma samples were incubated on Immulon 2HB plates coated with the same rhGAA as the animals were treated with. Test 1 included only the group receiving enzyme alone, while Test 2 included all groups receiving unlabeled or labeled ATB-200 (see Figure 83). All animals receiving rhGAA developed anti-rhGAA titers. Two independent dilutions and measurements yielded very comparable results. No substantial differences in anti-rhGAA titers were observed between unlabeled and labeled ATB-200; titers against labeled ATB-200 did not appear to be higher than those against unlabeled ATB-200. No substantial differences in antibody titers were observed between the different enzyme doses.

[0325] [Anti-rhGAA IgG] [Titer] - All unlabeled and IGF2-labeled ATB-200 groups were tested with or without a chaperone. Plasma samples were incubated on Immulon 2HB plates coated with the same rhGAA as the animals were treated with; all groups receiving unlabeled or labeled ATB-200 were tested for anti-rhGAA titers. Figure 84 shows the results. All animals receiving ATB-200 developed anti-rhGAA titers ranging from 12,500 to 400,000. There was no substantial difference in anti-rhGAA titers between unlabeled and labeled ATB-200. No significant differences in antibody titers were observed between the different enzyme doses. In general, no reduction in antibody titer was observed with co-administration of AT2221 or AT2220 co-formulation.

[0326] [Anti-rhGAA IgG] [Titer] - All unlabeled and labeled ATB-200 groups. Plasma samples were incubated on Immulon 2HB plates coated with the same rhGAA as the animals received; anti-rhGAA titers were tested for all groups receiving unlabeled or labeled ATB-200. Results are presented in Figure 85. All animals receiving ATB-200 rhGAA developed anti-rhGAA titers ranging from 12,500 to 400,000. No substantial differences in anti-rhGAA titers were observed between unlabeled and labeled ATB-200 rhGAA products. No significant differences in antibody titers were observed between different enzyme doses. In general, no reduction in antibody titer was observed with co-administration of AT2221 or AT2220 co-formulation.

[0327] [From Accept Four Times(] [Biweekly) rhGAA] [Administration Study 294B (POC1b)] [Antibody titers of animals] FIG86 shows the antibody titers from this group.

[0328] [From Accept Twice(] [Biweekly) rhGAA] [Product Administration Study 296 (POC2)] Figure 87 shows the antibody titer for this panel. The titer was determined by incubating antibody samples on Immunlon 4HBX plates coated with ATB-200 rhGAA. The higher potency of ATB-200 rhGAA compared to Lumizyme® allows for the administration of lower doses of ATB-200 rhGAA to achieve the same or similar glycogen reduction as higher doses of Lumizyme®. For example, biweekly administration of 5 mg / kg ATB-200 is equivalent to 20 mg / kg rhGAA Lumizyme® in reducing glycogen. As shown in Figure 87, these lower doses induce less anti-rhGAA IgG responses than higher doses of Lumizyme®, for example, comparing the data in row 1 (Lumizyme, 20 mg / kg) with the data in row 2 (unlabeled ATB-200, 5 mg / kg). Unlabeled ATB-200 also showed a gradual decrease in IgG titer when administered with increasing amounts of AT2221, see rows 3, 4, and 5. [Chapter VIII] [part.] [Vote to ATB-200] [with chaperone protein AT-2221] [Impact]

[0329] [rhGAA in ATB-200] [with pharmacological chaperone protein AT2221 (] [Megrute)] [Reduction in tissue glycogen with co-administration].

[0330] Twelve-week-old male GAA KO mice were administered a total of two biweekly bolus intravenous (IV) injections of 20 mg / kg rhGAA or ATB-200 via tail vein injection (n=5 per group). Additionally, 10 mg / kg AT2221 was orally administered 30 minutes prior to each IV administration of ATB-200 (hereafter referred to as "co-administration" or "ATB-200 + AT2221"). A group consisting of vehicle-injected (i.e., "untreated") GAA KO and age-matched wild-type (WT) mice served as baseline / controls. Fourteen days after the last dose, animals were euthanized, and quadriceps muscles were harvested for glycogenolysis.

[0331] Figure 88A shows PAS staining of paraffin sections of quadriceps muscle from untreated mice, demonstrating the presence of glycogen accumulation as numerous dense magenta dots (inset) per myofiber. Unlike the standard of care rhGAA, which exhibited limited effect, ATB-200 rhGAA alone resulted in a significant reduction in PAS signal. Furthermore, co-administration with AT2221 resulted in a substantial further reduction in matrix, as evidenced by the elimination of PAS signal in most myofibers (asterisks).

[0332] Figure 88B shows TEM examination of Epon-embedded quadriceps sections, which show that in untreated GAA KO mice, most glycogen is stored in lysosomes as membrane-bound, electron-dense materials (arrows and insets), which likely correspond to punctate PAS signals (see Figure 88A). In animals co-administered with ATB-200 and AT2221, not only was the number and size of matrix-containing lysosomes reduced, but the electron density of the remaining lysosomes was also reduced. Furthermore, the reduction in lysosomal glycogen indicates that ATB-200 is delivered to lysosomes.

[0333] Glycogen levels in quadriceps muscle were also measured using amyloglucosidase digestion. The results support the conclusions from histological examination: ATB-200 rhGAA is superior to conventional rhGAA in reducing glycogen, and its effect can be further improved by co-administration with AT2221. Similar results were obtained in gastrocnemius muscle and heart. Figure 88C shows quadriceps glycogen levels in different rhGAA-treated groups.

[0334] [Long-term repeated administration of ATB-200] [± AT2221] [Leading to GAA KO] [Glycogen reduction was greater in extramuscular tissue of mice].

[0335] In a separate, similarly designed study, the effects of ATB-200 ± AT2221 were examined over a longer period using four biweekly intravenous boluses. Following rhGAA treatment, modest overall reductions in glycogen levels were observed in quadriceps (data not shown), triceps (asterisks mark myofibers with significantly reduced PAS signal), and diaphragm. A more pronounced reduction in glycogen levels was observed in animals treated with ATB-200, and this effect was even greater with co-administration of AT2221. Notably, the diaphragm, a tissue refractory to rhGAA treatment, was more responsive to ATB-200 alone or co-administered with AT2221. In the heart, glycogen, primarily stored in cardiomyocytes, was readily cleared by repeated administration of rhGAA or ATB-200 to levels comparable to those seen in wild-type (WT) animals. However, substrates in cardiac smooth muscle cells appeared to be better cleared by ATB-200, suggesting a potentially broader biodistribution of ATB-200 compared to rhGAA (asterisks mark the lumen of cardiac vessels), see Figure 89, which shows glycogen staining of cardiac and skeletal muscle tissues treated with conventional rhGAA, ATB-200 rhGAA, and a combination of ATB-200 rhGAA and the chaperone protein AT2221.

[0336] [exist, GAA KO , ] [AT2221 in multiple muscle tissues of mice] [Co-administered with substantially improved ATB-200] [Mediated reduction in lysosomal proliferation]

[0337] Samples from the above study (see Figure 89) were also analyzed by IHC to examine the lysosomal marker LAMP 1. Upregulation of LAMP 1 indicates lysosomal proliferation, which is one of the hallmarks of Pompe disease.

[0338] Figure 90A. In quadriceps muscle, the number and size of LAMP1-positive vesicles (i.e., lysosomes) were greatly increased in untreated GAA KO mice compared to age-matched WT animals. Notably, a significant reduction in LAMP1 IHC signal was generally observed following administration of ATB-200, but not standard-of-care rhGAA. Co-administration with AT2221 resulted in a further significant reduction in lysosomal proliferation, with LAMP1 levels and patterns in most myofibers returning to those seen in WT animals. Similar conclusions were inferred in heart, diaphragm, and soleus muscles (see Panel B). In general, the effects of ATB-200 rhGAA ± AT2221 on LAMP1 appeared to closely mimic the positive effects seen on glycogen levels (see Figure 90).

[0339] Figure 90B. IHC of adjacent sections in soleus muscle using the specific antibody NOQ7.5.4D in type I (slow-twitch) fibers demonstrates that ATB-200 leads to a more substantial reduction in LAMP1 (asterisks) compared to standard-of-care rhGAA, with the reduction reaching levels seen in WT animals. Unlike rhGAA, whose effects are primarily restricted to type I fibers, ATB-200 also significantly reduces LAMP1 signaling in a subset of type II fibers (arrows). Furthermore, co-administration of ATB-200 in the majority of type II fibers further enhances ATB-200 efficacy. Thus, there appear to be no significant fiber-type-specific differences in LAMP1 signaling levels. Similar conclusions were reached in quadriceps and diaphragm (data not shown).

[0340] Histological data based on PAS staining demonstrated that ATB-200 rhGAA was more effective than standard-of-care rhGAA in reducing glycogen in multiple disease-relevant muscle tissues of GAA KO mice, including the quadriceps, triceps, diaphragm, and heart. PAS staining also demonstrated that co-administration with AT2221 further improved ATB-200-mediated glycogen reduction. EM examination revealed a reduction in substrates in lysosomes, suggesting trafficking of ATB-200 to target organelles. Generally, histological data were consistent with biochemical assessments of glycogen reduction using amyloglucosidase digestion. IHC analysis of LAMP1 demonstrated that ATB-200 ± AT2221 exhibited beneficial effects in reducing lysosomal proliferation, similar to the effects on glycogen substrate reduction. Fiber type analysis demonstrated that, unlike rhGAA, ATB-200 ± AT2221 was effective not only in type I fibers but also in type II fibers. This is supported by the superiority of ATB-200 ± AT2221 in glycogen reduction in quadriceps, triceps, and diaphragm, as these tissues are primarily composed of type II fibers.

[0341] Overall, these data suggest that ATB-200 is more readily absorbed into disease-relevant muscle tissue than standard-of-care rhGAA, as its higher M6P content leads to greater reductions in lysosomal glycogen and proliferation. Co-administration with AT2221 potentially further enhances ATB-200 efficacy by binding and stabilizing ATB-200 in the blood, maintaining the enzyme in a properly folded, active form that is more amenable to tissue uptake and lysosomal delivery. As a result, ATB-200 exhibits a broader biodistribution than standard-of-care rhGAA and achieves greater glycogen reduction in disease-relevant tissues / cell types that do not respond well to standard-of-care rhGAA, such as type II skeletal myofibers and cardiac vascular smooth muscle cells. Taken together, these preclinical data support the enhanced efficacy of ATB-200 rhGAA ± AT2221.

[0342] [For GAA KO] [Glycogen clearance in mouse skeletal muscle with ATB-200 rhGAA with excellent glycosylation] [ERT than standard of care] [Significantly better]

[0343] As mentioned above, enzyme replacement therapy (ERT) using recombinant human GAA (rhGAA) is the only approved treatment for Pompe disease. This ERT requires the specific carbohydrate mannose 6-phosphate (M6P) for cellular uptake and subsequent delivery to lysosomes via the cell surface cation-independent M6P receptor (CIMPR). However, current rhGAA ERTs contain low amounts of M6P, limiting drug targeting and efficacy in disease-relevant tissues. The present inventors have developed a production cell line and manufacturing method that produces rhGAA (designated ATB-200 rhGAA) that is superior in glycosylation and has a higher M6P content than conventional rhGAA. Specifically, it possesses a high-affinity bi-M6P N-glycan structure to improve drug targeting. ATB-200 rhGAA binds to CI-MPR with high affinity (KD approximately 2-4 nM) and is efficiently internalized by Pompe fibroblasts and skeletal myoblasts (KD approximately 7-14 nM). ATB-200 was evaluated in vivo and demonstrated to be more effective than current rhGAA ERT in clearing accumulated glycogen in skeletal muscle of GAA KO mice. Results indicate that biweekly administration of 5 mg / kg ATB-200 was equivalent to 20 mg / kg rhGAA in reducing glycogen, with substantially greater glycogen clearance observed in key skeletal muscles with 20 mg / kg ATB-200 compared to 20 mg / kg rhGAA. These results were confirmed by histological examination. Addition of a pharmacological chaperone (PC) enhanced the glycogen reduction effect of ATB-200. In combination, these data demonstrate that ATB-200 rhGAA with a higher M6P content leads to better lysosomal targeting and matrix reduction, which can be further improved by combination with PC, thus warranting further investigation of this next-generation therapy for Pompe disease. Figure 91A shows the relative levels of bis-M6P glycans in Lumizyme according to the present invention, conventional rhGAA, and ATB-200.

[0344] Figure 91A shows the bis-M6P glycan content of Lumizyme® and ATB-200 rhGAA. ATB-200 rhGAA has over ten times more bis-M6P glycans than Lumizyme® rhGAA. Figure 94B shows the relative binding affinities of Lumizyme® and ATB-200 rhGAA for CIMPR. Figure 91B shows the significantly superior binding affinity of ATB-200 rhGAA for CIMPR, a receptor that binds to rhGAA prior to internalization into lysosomes.

[0345] N-glycan analysis by MALDI-TOF mass spectrometry confirmed that, on average, each ATB-200 molecule contained native bis-M6P N-glycan structures. The higher bis-M6P N-glycan content of ATB-200 rhGAA was directly correlated with its high affinity for binding to CIMPR in an M6P receptor disk binding assay (KD approximately 2-4 nM).

[0346] ATB-200 rhGAA also demonstrates efficient cellular internalization. Figures 92A and 92B demonstrate internalization of ATB-200 rhGAA into normal and Pompe disease fibroblasts, respectively, to a greater extent than conventional Lumizyme® rhGAA. ATB-200 rhGAA saturates cellular receptors at approximately 20 nM, while requiring approximately 250 nM Lumizyme®. As shown in Figure 92C, the uptake efficiency constant (Kuptake) extrapolated from these results is 2-3 nM for ATB-200 and 56 nM for Lumizyme®. Cellular uptake of rhGAA is dependent on M6P, and free excess M6P blocks rhGAA uptake. These results suggest that ATB-200 rhGAA is a well-characterized targeted therapy for Pompe disease.

[0347] As shown in Figures 93A, 93B, and 93C, ATB-200 rhGAA has a favorable pharmacokinetic profile. The pharmacokinetics of ATB200 rhGAA and Lumizyme® rhGAA were compared after a single dose of rhGAA was administered to rats via a 30-minute infusion. Plasma GAA levels were measured by GAA enzyme activity. ATB200 rhGAA was cleared more rapidly than Lumizyme®, which can be attributed to its faster absorption via the M6P pathway. ATB-200 exhibited similar half-lives at the different doses tested. Cmax and AUC were dose-proportional. As is evident from these results, ATB200 has a favorable pharmacokinetic profile and produces effective ERT for Pompe disease.

[0348] ATB-200 rhGAA significantly clears glycogen in skeletal muscle compared to Lumizyme®. The effects of Lumizyme® and ATB200 rhGAA on glycogen clearance were evaluated in GAA KO mice. Animals were given two intravenous boluses (one week apart); tissues were collected two weeks after the last dose and analyzed for GAA activity and glycogen content. As shown in Figures 94A and 94B, 5 mg / kg of ATB200 rhGAA was equivalent to 20 mg / kg of Lumizyme® rhGAA in reducing glycogen in skeletal muscle. ATB200, administered at 10 and 20 mg / kg, was significantly better than Lumizyme® at clearing glycogen in skeletal muscle. Furthermore, ATB200 rhGAA and Lumizyme® rhGAA were equally effective at clearing glycogen in the heart.

[0349] [CHART] [Technology greatly improves rhGAA] [Stability]. Chaperones bind to and stabilize rhGAA ERT, increasing the uptake of the active enzyme into tissues, improving tolerability, and potentially reducing immunogenicity. As demonstrated above, CHART™ significantly improves ERT protein stability under adverse conditions. CHART: Chaperone Advanced Replacement Therapies, see http: / / www.amicusrx.com / chaperone.aspx (last accessed June 21, 2015), incorporated by reference. As shown in Figures 95A and 95B, ATB-200 stability is significantly improved by AT2221 (migludec, N-butyl-deoxynojirimycin).

[0350] The combination of a pharmacological chaperone and ATB-200 rhGAA was found to enhance glycogen clearance in vivo. GAA KO mice were given two intravenous boluses of 20 mg / kg rhGAA every other week. The pharmacological chaperone AT2221 was orally administered 30 minutes prior to doses of 0, 1, 2, and 10 mg / kg rhGAA. Tissues were collected two weeks after the last ERT dose and analyzed for GAA activity and glycogen levels. As shown in Figure 96, animals receiving rhGAA plus the chaperone AT2221 exhibited enhanced glycogen clearance from quadriceps muscle. ATB-200 rhGAA (20 mg / kg) reduced glycogen more than the same dose of Lumizyme®, and nearly normal glycogen levels in muscle were achieved when ATB-200 rhGAA was combined with 10 mg / kg AT2220.

[0351] These results demonstrate that ATB-200 rhGAA, with its high content of M6P and bis-M6P on its N-glycans, effectively targets CIMPR on lysosomes in skeletal muscle. ATB-200 rhGAA also possesses well-processed complex N-glycans, minimizing inefficient clearance in vivo; possesses pharmacokinetic properties favorable for in vivo use; and exhibits good targeting of key muscle tissues in vivo. They also demonstrate that ATB-200 rhGAA is superior to the conventional standard of care, Lumizyme, in reducing glycogen in muscle tissue, and that the combination of ATB-200 rhGAA with the chaperone protein AT2221 further improves glycogen removal from target tissues.

[0352] [ATB-200 rhGAA] [Compared to Lumizyme® rhGAA (] in removing glycogen from skeletal and cardiac muscle] [Glucosidase α) [Significantly better]

[0353] The ability of ATB-200 rhGAA and Lumizyme® (alglucosidase α) to clear myocardial glycogen was evaluated in GAA KO mice. Two bolus intravenous doses of rhGAA were administered weekly, and tissues were collected two weeks after the last dose and analyzed for GAA activity and glycogen content. 5 mg / kg of ATB-200 rhGAA was equivalent to 20 mg / kg of alglucosidase α in reducing myocardial glycogen. As shown in Figure 97, 5 mg / kg of ATB-200 was equivalent to 10 and 20 mg / kg of ATB-200 in reducing myocardial glycogen.

[0354] The ability of ATB-200 rhGAA and Lumizyme® (alglucosidase α) to clear glycogen from skeletal muscle (quadriceps and triceps) and cardiac muscle was evaluated in GAA KO mice. Two bolus doses of rhGAA were administered intravenously at weekly intervals, and tissues were collected two weeks after the last dose and analyzed for GAA activity and glycogen content. As shown in Figures 98A and 98B, ATB-200 rhGAA significantly reduced glycogen in skeletal muscle (quadriceps and triceps) compared to Lumizyme® (alglucosidase α). 5 mg / kg of ATB-200 was equivalent to 20 mg / kg of alglucosidase α in reducing glycogen in skeletal muscle.

[0355] These results are consistent with the properties of ATB-200 rhGAA described herein. ATB-200 rhGAA contains well-processed, complex N-glycans to minimize ineffective drug clearance. This superior carbohydrate structure is maintained during scale-up manufacturing processes, for example, to volumes of 250 L or greater. rhGAA (ATB-200) exhibits favorable PK and excellent drug targeting to key muscle tissues in vivo. rhGAA (ATB-200) is significantly more effective than alglucosidase α in reducing glycogen in skeletal muscle of GAA KO mice. Furthermore, the addition of the pharmacological chaperone protein AT2221 to rhGAA (ATB-200) further improved glycogen reduction and reduced skeletal muscle pathology in GAA KO mice. rhGAA (ATB-200) reduces glycogen in both type I and type II myofibers and decreases autophagic vesicles in treated cells. [IX] [part.] [In GAA KO] [Intravenous Infusion of ATB-200 in Mice] Pharmacokinetics

[0356] [In GAA] [Intravenous Infusion of ATB-200 in Knockout Mice] Pharmacokinetics

[0357] GAA KO mice were infused with 5, 10, or 20 mg / kg ATB-200 or 20 mg / kg Lumizyme® (rhGAA). As shown in Figure 99A, ATB-200 rhGAA was cleared from serum more rapidly than Lumizyme® rhGAA, indicating faster tissue uptake (top trace = Lumizyme; bottom trace = ATB-200, at concentrations of 5, 10, and 20 mg / kg). As shown in Figures 99B and 99C, no substantial differences in the circulating half-life were observed for the different doses of ATB-200 tested (5, 10, and 20 mg / kg), and the AUC (area under the curve) was approximately dose proportional.

[0358] [In GAA KO] [Effects of repeated rapid intravenous injections on hepatic glucose levels in mice]

[0359] Twelve- to fourteen-week-old GAA-KO mice (N = 5-6 male GAA mice) were administered bolus injections of 10 or 20 mg / kg ATB-200 or 20 mg / kg Lumizyme® (rhGAA) at weeks 0, 2, 4, and 6 (every other week). DPH (phenytoin) was administered after the second dose of ATB-200 or Lumizyme®, and tissue glycogen was measured in quadriceps, triceps, and heart tissue 14 days after the final (fourth) injection. As shown in Figures 100A, 100B, and 100C, ATB-200 rhGAA produced a greater decrease in glycogen compared to Lumizyme® (rhGAA) in GAA-KO mice. Similar results were obtained in a parallel study with six intravenous bolus injections of ATB-200 or Lumizyme®.

[0360] [Study 294B] [:ATB-200] [with and without oral co-administration of AT2221 (] [Megrute)] [In case of GAA KO] [Effects on tissue glycogen content in mice]

[0361] Twelve- to fourteen-week-old GAA KO mice were administered four biweekly (every other week) bolus injections of the indicated doses of ATB-200 rhGAA and either 10 mg / kg AT2221 (migludec) or 20 mg / kg Lumizyme® (rhGAA) alone. DPH was administered after the second dose; N = 5-6 male GAA KO mice. Tissue glycogen was measured 14 days after the final (fourth) injection. Co-administration of migludec (10 mg / kg) improved glycogen reduction in GAA KO mice compared to ATB-200 (20 mg / kg) or rhGAA (20 mg / kg) alone. Figures 101A, 101B, and 101C are bar graphs showing glycogen reduction in skeletal and cardiac muscle by different rhGAA products. The data were similar to the six-injection study (Study 294A); 30 mg / kg migludec was also tested in this study and did not demonstrate any improvement in glycogen clearance compared to ATB-200 rhGAA alone.

[0362] About Myozyme® [Compared with ATB-200 after co-administration with miglustat] [Historical data on co-administration with Miglustat]

[0363] Twelve-week-old GAA KO mice were treated with four weekly intravenous injections of 20 mg / kg rhGAA (Myozyme). Migludec (10 mg / kg) was co-administered orally 30 minutes before Myozyme. Tissues were collected for glycogenometry 14 days after the last Myozyme dose. Figures 102A, 102B, and 102C show the relative decrease in muscle glycogenometry.

[0364] [ATB-200] [GAA KO with and without oral co-administration of migludec] [Effects of Cell Type-Specific Glycogen Levels in Mice]

[0365] GAA KO mice were treated with conventional rhGAA, ATB-200 rhGAA, or a combination of ATB-200 rhGAA and miglustat (AT-2221) as indicated in Figure 103. Tissues were obtained and stained for glycogen as shown in Figure 103.

[0366] Tissues were processed for histological PAS staining based on the study shown in Figure 103 (four rapid intravenous EOW injections). Glycogen is represented by magenta spots (arrows in untreated sections). Co-administration of meglucagon (10 mg / kg) improved glycogen reduction in GAA KO mice compared to either ATB-200 (20 mg / kg) or rhGAA (20 mg / kg) alone. Data show representative micrographs from 5 to 6 mice per group (one section per animal).

[0367] Dose-response data for miglustat were generated. Different doses of each rhGAA formulation were administered intravenously via the tail vein twice weekly in GAA KO mice. Tissues were collected 14 days after the last (second) dose and analyzed for tissue GAA enzyme activity and residual glycogen. Tissue GAA activity and muscle glycogen content are shown in Figures 104A and 104B.

[0368] Significantly higher tissue GAA levels were observed with 20 mg / kg ATB-200 compared to 20 mg / kg rhGAA, which correlated with better glycogen reduction. Co-administration of 10 mg / kg migludec with 20 mg / kg ATB-200 further improved enzyme absorption and glycogen clearance. As shown in Figure 104, glycogen reduction in GAA KO mice was greatest with co-administration of 20 mg / kg ATB-200 and 10 mg / kg migludec (POC 2, Study 296).

[0369] [Study 311] [:at GAA KO] [PAS in mice] [and TEM] [Staining assessed in ATB-200 + / -] [Glycogen reduction in the case of Migludec]

[0370] Twelve-week-old GAA KO mice were given two intravenous bolus injections of rhGAA and ATB-200 + / - 10 mg / kg migludec. Tissues were harvested 14 days after the final (second) dose for cell-type-specific glycogenotyping using PAS and TEM (transmission electron microscopy). The results are presented in Figures 105A and 105B.

[0371] As shown in Figures 105A and 105B, unlike conventional rhGAA, which exhibited limited glycogen reduction (indicated by numerous punctate PAS signals), ATB-200 rhGAA alone exhibited a significant reduction in PAS signals. Co-administration with 10 mg / kg miglustat resulted in a substantial further reduction in matrix. TEM revealed that the majority of glycogen in lysosomes was membrane-bound, electron-dense, corresponding to the punctate PAS signals. Co-administration of ATB-200 rhGAA with miglustat reduced the number, size, and density of matrix-containing lysosomes, demonstrating targeted delivery of ATB-200 rhGAA to lysosomes.

[0372] [Study 311] [:at GAA KO] [PAS in mice] [and TEM] [Staining assessed in ATB-200 + / -] [Lysosomal proliferation in the presence of migludec]

[0373] Based on the studies described above (two rapid intravenous EOW injections), tissues were treated with LAMP 1 to label lysosomal proliferation, upregulation of which is another hallmark of Pompe disease. LAMP: lysosomal associated membrane protein. Figure 106 shows the effects of conventional rhGAA, ATB-200 rhGAA, and ATB-200 + miglustat (AT-2221) treatment on muscle tissue.

[0374] LAMP1 IHC revealed widespread signaling in untreated GAA KO mice, which was significantly reduced with ATB-200 compared to rhGAA. Notably, co-administration with miglustat resulted in a further significant reduction. Overall, the effects of ATB-200 ± miglustat on LAMP1 closely mimicked glycogen depletion.

[0375] [Study 311] [:at GAA KO] [Assessment of ATB-200 rhGAA in mice using fiber type-specific staining] [Lysosomal proliferation in the presence of migludec]

[0376] Following the studies described above (two rapid intravenous EOW injections), soleus muscle tissue was processed for type I fiber-specific antibody (NOQ7.5.4D) and LAMP 1 staining in adjacent sections, see FIG107 .

[0377] ATB-200 rhGAA resulted in a more substantial reduction in LAMP1 expression than conventional rhGAA, with the reduction reaching levels seen in WT animals. Furthermore, unlike rhGAA, whose effects were primarily restricted to type I fibers (slow-twitch, marked with an asterisk), ATB-200 rhGAA also significantly reduced LAMP1 signaling in a subset of type II (fast-twitch) fibers (red arrows). Importantly, co-administration with miglustat further ameliorated the ATB-200-mediated reduction in LAMP1 proliferation in the majority of type II fibers. Therefore, there appear to be no significant fiber-type-specific differences in LAMP1 signaling levels. Similar conclusions were obtained from the quadriceps and diaphragm (data not shown).

[0378] [Study 317] [and 321] [:In Engineering Batch (EB)] [ATB-200] [Preclinical Animal Studies in Context]

[0379] ATB-200 (EB) pharmacokinetics were evaluated in GAA KO mice by intravenous infusion. GAA KO mice were administered an engineered batch of ATB-200 rhGAA by intravenous infusion. Plasma PK was compared with mice administered conventional rhGAA (Lumizyme), as shown in Figures 108A and 108B. Fourteen-week-old GAA KO mice received a single intravenous bolus injection of 10 mg / kg EB1, EB2, or Lumizyme. Plasma samples were collected from each mouse prior to dosing and 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and 24 hours after intravenous submandibular bleeding.

[0380] The half-life of ATB-200 EB was determined to be shorter than that of Lumizyme®, which can be attributed to the presence of more M6P N-glycans on ATB-200 rhGAA. At 10 mg / kg intravenous administration in mice, EB2 ATB-200 exhibited a slightly longer half-life (approximately 36 minutes) compared to EB1 ATB-200 (approximately 30 minutes based on studies 307 and 321).

[0381] [Study 327B.] [Assessing GAA KO] [By repeated intravenous rapid injection of EB1 in mice] [and EB2 ATB-200] [Reduction in glycogenolysis]

[0382] Two different batches of ATB-200 rhGAA demonstrated efficacy in reducing glycogen in muscle tissue. Overall, ATB-200 EB1 did not demonstrate a clear dose-dependent effect on glycogen reduction. EB1 and EB2 demonstrated similar glycogen reduction in key muscles at a 20 mg / kg dose. EB1 and EB2 (20 mg / kg) demonstrated significantly better glycogen reduction than Lumizyme® (similar to the POC batch) (see Figure 109).

[0383] [Study #304] [:As shown in Figure 112] [Shown, ATB-200 rhGAA] [Half-life longer than Lumizyme®] [Short and suitably improved with Megroot].

[0384] The half-life of ATB-200 rhGAA was compared with that of conventional Lumizyme® rhGAA, as shown in Figures 110A and 110B. Addition of a chaperone (co-administered with AT2221) modestly increased the circulating half-life, and the AUC of ATB-200 was similar to that observed for Lumizyme.

[0385] [ATB-200 rhGAA +] [Chaperone protein significantly improves muscle morphology]

[0386] As described above, GAA KO mice were treated with two rapid intravenous injections of Lumizyme® (20 mg / kg), ATB-200 (20 mg / kg) and / or miglustat (10 mg / kg). Plastic sections of quadriceps muscle samples were stained with methylene blue and examined for HRLM. One section each from the WT and untreated GAA KO groups; two sections each from the Lumizyme, ATB-200, and co-administered groups were obtained. Figure 111 shows the muscle morphology of these sections.

[0387] A significant reduction in the area of ​​autophagy (arrows) was observed in both samples from the co-administered groups compared to the untreated GAA KO sample. No significant differences were observed among the untreated, Lumizyme®, and ATB-200 samples examined. H&E staining of paraffin sections from all animals also showed improved morphology (i.e., fewer vacuoles) in the presence of ATB-200 rhGAA and chaperones.

[0388] Migludec is well-characterized as a pharmacological chaperone for Pompe disease. The pharmacokinetics of the pharmacological chaperones AT2221 (migludec) and AT-2220 were evaluated in eight-week-old wild-type C57BL / B mice following a 100 mg / kg PO dose. Figures 112A and 112B show the results. Figure 112C depicts the results of an rhGAA inhibition assay. As is apparent from the above data, migludec is rapidly cleared from muscle. At 24 hours, tissue concentrations of migludec were approximately 30 ng / gm tissue, corresponding to a molar concentration of 0.14 µM (>10-fold below the IC50 at pH 5.2).

[0389] Figures 113A and 113B show the pharmacokinetics of different doses of migludec after oral administration to mice. Mice were orally administered the doses described in Figure 113A, and plasma migludec levels were measured over time.

[0390] Preclinical data indicate that co-administration of 10 mg / kg migludec enhances the effectiveness of 20 mg / kg ATB-200 rhGAA in clearing glycogen from key skeletal muscles in mice. Oral administration of 10 mg / kg migludec results in a plasma Cmax of approximately 30 µM in mice. Similar plasma concentrations can be achieved in human patients with a 300 mg dose of migludec. Therefore, in the POC study, a 300 mg dose of migludec was co-administered with 20 mg / kg ATB-200 rhGAA.

[0391] [AT2220] [About Myozyme®] [In GAA KO] [Effects on immunogenicity in mice(] [Study 49)]

[0392] GAA KO mice (5-6 mice / group) were administered Myozyme® intravenously once weekly in the drinking water for up to 5 weeks in the absence or presence of AT2220 (1, 10, or 100 mg / kg daily). The effects of these administrations are shown in Figure 115 . Myozyme® alone caused an allergic reaction (manifested by mortality) after 4-5 injections. Co-administration with AT2220 modestly prolonged survival, but this was not dose-dependent. The study was terminated at week 5 because no mice survived in the Myozyme® alone group.

[0393] [GAA KO] [Myozyme / AT2220 in mice] [Survival Study: Study Design] [Study 83)]

[0394] The present inventors investigated the effect of AT2220 on the survival of GAA knockout (GABA)-KO (GABA-KO) mice treated with Myozyme. Twelve-week-old GAA knockout (GABA-KO) mice (7 mice / group) were administered weekly for 8 weeks via tail vein injections of: saline alone; Myozyme® alone; premixed Myozyme® + AT2220; AT2220 followed by premixed Myozyme® + AT2220; or AT2220 followed by Myozyme®. Myozyme® was administered weekly at 3, 10, and 30 mg / kg. The groups treated with 3 and 10 mg / kg Myozyme® alone were also given 100 µM AT2220. Mice were observed for adverse reactions and survival immediately following Myozyme® injection. The results are presented in Figures 116, 117, and 118.

[0395] No deaths were observed at any time point in mice receiving saline. No adverse events were observed with the first two Myozyme® injections. Hypersensitivity reactions (manifested as deaths) were observed in some mice after the third week of Myozyme® injection. Survival rates were appropriately prolonged in the groups co-administered with AT2220, followed by pre-mixed Myozyme® + AT2220, or Myozyme® alone. Pre-mixed Myozyme® + AT2220 exhibited the highest incidence of adverse events (see Figure 116).

[0396] No deaths were observed at any time point in mice administered saline. No adverse events were observed with the first two Myozyme® injections. Hypersensitivity reactions (manifested as deaths) were observed in some mice after the 3-week Myozyme® injection. Survival rates in those groups co-administered with AT2220 were appropriately prolonged with pre-administration of AT2220 followed by pre-mixed Myozyme® + AT2220 or Myozyme® alone. Pre-mixed Myozyme® + AT2220 showed a similar incidence of adverse events as the Myozyme® only group, see Figure 117

[0397] No deaths were observed at any time point in mice administered saline. No adverse events were observed with the first three Myozyme® injections. Hypersensitivity reactions (manifested as deaths) were observed in some mice after Myozyme® injection at week 4. An overall dose-dependent hypersensitivity reaction was observed after Myozyme® injection: 3 mg / kg Myozyme® resulted in fewer deaths over 8 weeks than 10 and 30 mg / kg Myozyme® (all deaths by week 6), see Figure 118.

[0398] none

[0399] Domestic storage information (please note the order of storage institution, date, and number) none Overseas deposit information (please note the order of deposit country, institution, date, and number) none

[0400]

Claims

1. A composition comprising recombinant human acid α-glucosidase (rhGAA), wherein the rhGAA is produced by Chinese hamster ovary (CHO) cells expressing the rhGAA, wherein at least 2% of the total polysaccharides on the rhGAA are diphosphorylated mannose-6-phosphate (bis-M6P) polysaccharides, and wherein the rhGAA carries an average of 2.0 to 6.0 moles of sialic acid residues per mole of rhGAA.

2. The composition as claimed in claim 1, wherein at least 2.5% of the total polysaccharide on the rhGAA is bis-M6P polysaccharide.

3. The composition as claimed in claim 1, wherein at least 3.0% of the total polysaccharide on the rhGAA is bis-M6P polysaccharide.

4. The composition as claimed in claim 1, wherein at least 17% of the total polysaccharide on the rhGAA is bis-M6P polysaccharide.

5. The composition as claimed in any one of claims 1 to 4, wherein 4% to 16% of the total N-glycans on the rhGAA are completely sialylated.

6. The composition as claimed in any one of claims 1 to 4, wherein 15% to 30% of the total N-glycans on the rhGAA contain sialic acid and terminal galactose.

7. The composition as claimed in any one of claims 1 to 4, wherein 8% to 19% of the total N-glycans on the rhGAA have only terminal galactose and do not contain sialic acid.

8. The composition as claimed in any one of claims 1 to 4, wherein no more than 15% of the high-mannose N-glycan on the rhGAA in the composition is non-phosphorylated.

9. The composition as claimed in any one of claims 1 to 4, wherein less than 25% of the total rhGAA on average is free of phosphorylated polysaccharides bound to CIMPR.

10. The composition as claimed in any one of claims 1 to 4, wherein at least 3% of the total polysaccharide on the rhGAA is in the form of monophosphorylated mannose-6-phosphate (mono-M6P) polysaccharide.

11. The composition of claim 1, wherein at least 3% of the total polysaccharide on the rhGAA is in the form of monophosphorylated mannose-6-phosphate (mono-M6P) polysaccharide, at least 3.0% of the total polysaccharide on the rhGAA is in the form of diphosphorylated mannose-6-phosphate (bis-M6P) polysaccharide, and less than 25% of the total rhGAA on average is free of phosphorylated polysaccharides bound to CIMPR.

12. The composition as claimed in claim 1, wherein at least 3.0% of the total polysaccharides on the rhGAA are in the form of bisphosphorylated mannose-6-phosphate (bis-M6P) polysaccharides, and less than 25% of the total rhGAA on average is free of phosphorylated polysaccharides bound to CIMPR.

13. The composition as claimed in claim 1, wherein at least 3.0% of the total polysaccharides on the rhGAA are in the form of diphosphorylated mannose-6-phosphate (bis-M6P) polysaccharides, and no more than 15% of the high-mannose N-polysaccharides on the rhGAA in the composition are nonphosphorylated.

14. The composition according to any one of claims 1 to 4, wherein the rhGAA comprises 2.3 to 3.1 mol of mannose-6-phosphate (M6P) residues per mol of rhGAA.

15. The composition as claimed in any one of claims 11 to 13, wherein at least 17% of the total polysaccharides on the rhGAA are bis-M6P polysaccharides.

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