AU2016256663A1 - Treatment of clycogen storage disease type II - Google Patents

Abstract

Methods of treating glycogen storage disease type i, by administering acid o-glucosidase, are described, as are compositions for use in treatment of glycogen storage disease type I

Description

2016256663 07 Nov 2016 -1-

TREATMENT OF GLYCOGEN STORAGE MEASE TYPE II

This application is a divisional of Australian Application No. 2015202140, the entire contents of which are incorporated herein by reference.

lELATBD AJ^IICAXION

This application claims the benefit of iJ.S, Provisional Application No, 60/219,232 filed My 18,2000, the entire teachings of which are incorporated herein

BACKGROUND OF THE INVENTION

Glycogen storage disease type H (GSD-Π) (also known as Pompe disease or acidmaltase deficiency) is a latal geiietic muscle disorder caused by a deficiency of acid a-ghieosidase (GAA), a glycogen degrading lysosomal enzyme (Hirsehhorn, R., 10 “Glycogen Storage disease type If: acid a-ghicosidase (acid maitase) deficiency^, in

Scriver, C.R, et al, (eds) The Metabolic and Molecular Basis oflrJieriteddisease, 7,s Ed., McGraw-Hill, New York, 1995, pp. 2443-2464). Tile deficiency results in lysosomal glycogm aecuimfiaiion In almost alltissu.es of the body, with cardiac and skdetfinmsele being the most senously affected. The combined incidence of all 15 forms of GSD-Π is estimated to be 1:40,000, and the disease affects all groups without an ethnic predilection (Martimuk, F, etal,Amer. X Med Genet 79:69-72 (1998); Ausems, M.GE.M. et«/,, Bur, J. Hum. Genet. 7:713-716 (1999)).

Clinically, GSD-Π encompasses a range of phenotypes differing as to age of onset, organs involved and clinical severity, generally correlating with the residual 20 amount of GAA activity, In its most severe presentation (infantile GSD-U, or Pong» disease, in which less than 1% of normal GAA activity is present), infants are affected by a hypertrophic cardiomyopathy, generalized muscle weakness and. hypotonia secondary' to massive glycogen accmnulation m cardiac and skeletal muscles (for review, see Hirschhom, supra). The disease progresses rapidly, with. 25 death from cardiac failure usually occurring by 1 year of age. juvenile (1-1034 of normal GAA activity) and adult-onset (10-40% of normal GAA activity) forms of 2016256663 07 Nov 2016 -2- die disease are characterized by lack of severe cardiac involvement, later age of onset, and slower progression, but eventual respiratory or limb muscle involvement results in significant morbidity and mortality for the affected individuals,

Drug treatment strategies, dietary manipulations, and bone marrow 5 transplantation have been employed as means for treatment for GSD-ΪΙ, without significant success (Hug, G, et at, Birth Defects Org Sen 5:160-183 (1967)1 Slonim, A.E. et at, Neurology 33/34 (1983); Watson, IG. ei at, N EnglJ. Med. 314:385 (1986)). Early attempts at enzyme replacement were also unsuccessful (Hug, G. and Schubert, W.K,, J. Clin. Invest, 46:1073 (1967); deBarsy, T. ei al, 10 Birth Defects Orig. Art. Ser. 9:84-190 (1973); WilHams, J.C. and Murray, A.K., “Enzyme replacement in Pompe disease with aft alpha glucosidase low-density lipoprotein complex”, in Desnick, R J. (ed), Enzyme Therapy in Genetic Diseases: 2, New York, Alan R. Liss 1980; pp. 415-423)). A need remains for effective treatment of GSD-H, 15

SUMMARY OF THE INVENTION

The present invention is drawn to methods of treating glycogen storage disease type Π (infantile, juvenile or adult-onset) in anindiyidual, by administering to the individual a therapeutically effective amount of acid α-glucosidase (e.g., less 20 than about 15 mg enzyme per kilogram of body weight, preferably about 1-10 mg enzyme per kilogram of body weight, more preferably about 10 enzyme per kilogram of body weight or about 5 mg enzyme per kilogram of body weight), at a regular- interval (e.g,, monthly*, bimonthly, weekly, twice weekly, daily). The acid «-glucosidase is human acid α-glucosidase, preferably recombinant human acid «-25 glucosidase, more preferably, precursor form of human acid α-glucosidase, and even more preferably precursor form of human acid α-glucosidase produced in Chinese hamster ovary'· cells, The acid u-glucosidase is administered periodically (e,g., monthly, bimonthly, weekly, twice weekly, daily). In preferred embodiments, the acid α-glucosidase is administered intravenously; intramuscularly; intrathecally; pr 30 intraventriculaxly. 2016256663 07 Nov 2016 -a- means to treat an

Fig. 1A-1C are a seriesof graphic representations depleting longitudinal 5 data (for the first 16 months of age) on motor development as assessed by Alberta Infant Motor Scale (AIMS) (closed diamonds), and dter of antibodies to recombinant human acid α-glucosidase (thGAA) (open diamonds) in three patients (patient 1, Fig. 1A; patient 2, Fig, IB; patient 3, Fig, 1C) with infantile Pompe disuse receiving enzyme replacement therapy. The arrow radicals when the 10 enzyme therapy was initiated. AIMS scores in normal patients are plotted as dotted curves against age (5th, 10th, 25* 5#, 75* and 95* percentile, from bottom to top).

Fig. 2A-2F are a series of graphic representations depicting longitudinal two-dimensional echocardiographic measurements of left ventricular volume pug. 2A-2C) and mass (Fig. 2D-2F) in the three infantile Pompe disease patients 15 receiving enzyme replacement therapy (patient 1, Fig. 2A and 2D; patient 2, Fig. 2B and 2E; patient 3, Fig, 2C and 2F). Week 0 depicts the measurements at the time of enzyme therapy initiation. Open diamonds, end-diastolic volume measurement; closed diamonds, end-systolic volume measurement. DETAILED DESCRIPTION OF THE INVENTION 20 The present invention is drawn to methods of treating glycogen storage disease type H (GSD-H) in an individual, by administering the enzyme, acid «-giucosidase (GAA) to the individual, as Well as the use of the enzyme, GAA,in the manufacture of a medicament for the treatment of glycogen storagedisease type H. As described herein, Applicants have successfully treated infants suffering from 25 GSD-H by administering GAA to the infants on a regular basis; the infants demonstrated improvement ofcardiac- status, pulmonary function, and neurodevelopmeni, as well as reduction of glycogen levels in tissue.

As a result of these findings, it is now possible for the first time to treat GSD-Π, including infantile, juvenile and adult-onset GSD-DL Although the results 2016256663 07 Nov 2016 -4- described herein discuss individuals with the most severe form of GSD-Π (infantile GSD-HT it is expected that fixe methods will be equally effociive in individuals affected by juvenile or adult-onset GSD-Π, and may, in fact, be even more effective, as individuals with juvenile or adult-onset GSD-Π have higher levels of residual 5 GAA activity (1-10%, or 10-40%, respectively), and therefore are likelyfo he more immunologically tolerant of the administered GAA (e.g., they me generally cross-reactive immunoreactive material (CRIM)-positive for endogenous GAA, so that their immune systems do not perceive the GAA as a “foreign” protein, and they do not develop ann-GAA antibodies). Hie enhanced efficacy in such individuals can : 10 be seen ill patient 3, who was CRM-positive and did not develop anti-GAA antibodies, and who demonstrated a normal progression of developmental milestones, in contrast with the variable course that was seen in CRIM-negative patients 1 and 2 (Who did develop anti-GAA antibodies).

The terms, “treat” and “treatment,” as used herein, refer to amelioration of 15 one or more symptoms associated with the disease, prevention or delay of the onset of one or more symptoms of the disease, and/or lessening of the severity or frequency of one or more symptoms of tire disease. For example, feeatmeni can refer to improvement of cardiac status (e.g,, increase of end-diastolic and/Or end-systolic volumes, or reduction, amelioration or prevention ofthe progressive cmdiomyopathy 20 that xs typically found in GSD-B) or of pulmonary function (e.g., increase in crying vital capacity over baseline capacity, and/or normalzation of oxygen desaiuration during crying); improvement in neurodevelopment and/or motor skills (e.g., increase in AIMS score); reduction of glycogen levelsin tissue of fixemdividualaffecied by the disease; or any combination of these effects. In one preferred embodiment, 25 treatment includes improvement of cardiac status, particularly in reduction or prevention of GSD-i-associated cardiomyopathy. The terms, *%spmve ” “inmeaseP or “reduce,” as used herein, indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control individual (or multiple 30 control individuals) in the absence of the treatment described herein. A control individual is an individual afflicted with the same form of GSD-Π 2016256663 07 Nov 2016 30 -5- juveniie or adult-onset) as the individual being treated, who is about the same age as the individual being treated (to ensure that the stages of the disease in the heated individual and the control mdividual(s) are comparable).

The individual being treated is an individual (fetus, child, adolescent, or 5 adult human) having GSD-H (i.e., either infantile GSD-Π, juvenile GSD-I1, or adult-onset GSD-H). The individual can have residual GAA activity, or no measurable activity. For example, the mdividual having GSjD-S can have GAA activity that is less than about 1% of normal GAA activity (infantile GSD-H), GAA activity that is about 1*10% of normal GAA activity (juvenile GSD-Π), or GAA activity that is 10 about 10-40% of normal GAA activity (adult GSD-H), The individual can be CElM-positive or CRIM-negative for endogenous GAA., ha a preferred embodiment, the individual is CRM-positive for endogenous GAA. hi another preferred embodiment die mdividual is an individual who has been recently diagnosed with the disease. Early treatment (treatment commencing as soon as 15 possible after diagnosis) is important for to minimize the effects of die disease and to maximize the benefits of treatment.

In the methods of the invention, human acid α-glucosidase (GAA) is administered to the mdividual. The GAA is in a form that, when administered, targets tissues such as the tissues affected by the disease (e.g,, heart, muscle). In one 20 preferred embodiment, the human GAA is administered in Its precursor form, as the precursor contains motifs which allow efficient receptor-mediated uptake of GAA. Alternatively, a mature form of human GAA that has been modified to contain motifs to allow efficient uptake of GAA, can be administered, in a particularly preferred embodiment, the GAA is the precursor form of recombinant human GAA. 25 GAA is obtainable horn a variety of sources, in a particularly preferred embodiment, recombinant human add α-glucosidase (rhGAA) has been produced in. Chinese hamster ovary (CHO) cell cultures is used (see, e,g>, Fuller, M, et a!.. Bur, J. Biochem, 254:903-909 (1995); Van Hove, J.L,K. et al, Proc, Natl Aaad Set. USA 93:65-70 (1994); the entire teachings of these references are incorporated herein by reference). Production of GAA in CHO cells appears to yield a product 2016256663 07 Nov 2016 desired tissues (hean and inuscle); it is assessed-that the glycosylat-ion differs from that of GAA that is produced in transgenic mouse and rabbit milk (see, e.g., Bijvoet, A.G.A. et al, Hum, Mol Genet 7:18154824 (1998); Bijvoet, A.G.A. et al., Hum. 5 Mol Genet £:2145-2153 (1999)).

The GAA has a specific enzyme activity in the range of about 1.0-3.5 pmoi/min/mg protein, preferably in the range of about 2-3 ,5 pmoLmtin/mg protein. In one preferred embodiment, the GAA has a specific enzyme activity of at least about 1.0 pmohmin/mg protein; more preferably, a specific enzyme activity of at 1.0 least about 2,0 pmol/min/mg protein; even more preferably, a specific enzyme activity of at least about 2.5 umol/min/mg protein; and still more preferably, a specific enzyme activity of at least about 2.75 pmol/min/mg protein. GAA can be administered alone, or in. compositions or medicaments comprising the GAA (e.g., in the manufacture of a medicament for the treatment of 15 the disease), as described herein. The compositions can be formulated with a physiologically acceptable carrier or excipient to prepare a pharmaceutical composition. The carrier and composition can be sterile. The formulation should suit die mode of administration.

Suitable pharmaceutically acceptable carfim include but are not limited to 20 water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabie, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amyloseor starch, sugars such as mannitol, sucrose, or others, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxynethylceliulose, polyvinyl pyrolidone, etc., as well as 25 combinations thereof. The pharmaceutical preparations can, if desired, he mixed with anxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffer, coloring, flavoring and/or aromatic substances and the like Which do not deleteriously react with the active compounds. Jn a preferred embodiment, a w'ater-soluble carrier suitable for 30 intravenous administration is used. 2016256663 07 Nov 2016

The composition or medicament, if desired, can also containminor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition Gan also he formulated as a suppository, 5 with traditional binders and earners such as triglycerides, Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidono, sodium saccharine, cellulose, magnesium carbonate, etc.

The composition or medicament can be formulated in accordance with the 1Θ routine procedures as a pharmaceutical composition adapted for adminisiration to human beings. For example, in a preferred embodiment, a composition for intravenous administration typically is a solution in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection. Generally, the ingredients are 15 supplied either 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 ampule or sachette indicating the quantity Of active agent. Where the composition is to be administered by infusion, it cm be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water. 20 Where the composition is administered by injection, an ampule of sterile water for hfection or saline can be provided so that the ingmdi^ts may he mixed priorto administration.

The GAA can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free ammo groups such as those derived 25 from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed With free carboxyl groups such as those derived from sodium, potassium, ammonium, ctieium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino eilianol, histidirie, procaine, etc, GAA (or composition or medicament containing GAA) is administered by an 30 appropriate route. In one embodiment, the GAA is adinmistered intravenously. M other embodiments, GAA is administered by direct administration to a target tissue. 2016256663 07 Nov 2016 ~8~ such as heart or muscle (e.g., intramuscular), or .nervous system (e.g., direct injection, into the brain; intraventricularly; inirafiiecally}. More than one route can be used concurrently, if desired. GAA (or composition or medicament containing GAA) can be administered 5 alone, or in conjunction with other agents, such as antihistamines (e.g., diphenhydramine) or immunosuppressants or other immunotherapeutic agents which counteract anti-GAA antibodies. The term,.:¾ conjunction with,” indicates that the agent is administered at about the same time as tire GAA (or compositioncontaining GAA). For example, the agent can be mixed into a composition containing GAA, 10 and thereby administered contemporaneously with the GAA; alternatively, die agent can be administered contemporaneously, without mixing (e.g., by “piggybacking*5 delivery of the agent on the intravenous line by which the GAA is also administered, or vice versa), ha another example, the agent can be administered separately (e.g., not admixed), but within a short time Same (e.g., within 24 hours) of administration 15 of the GAA, fix one preferred embodiment» :if the Individual is CSJM-negative for endogenous GAA, GAA (or composition containing GAA) is administered in conjunction with an immunosuppressive or immunotherapeutic regimen designed to reduce amounts of, or prevent production of, anti-GAA antibodies. For example, a protocol similar to those used in hemophilia patients (Nilsson, LM. ei αί., Ν. Engl. J, 20 Med. 575:947-50 (1988)) can be used to reduce anti-GAA antibodies^ Such a regimen can also be used in individuals who are CRIM-positive for endogenous GAA but who have, or are at risk of having, anti-GAA antibodies. In a particularly' preferred embodiment, the immunosuppressive or immtmoiherapeutic regimen is begun prior to the first administration of GAA, in order to minimize the possibility 25 of production· of anti-GAA antibodies, GAA (or eompositioh or medicament containing GAA) is administered in a therapeutically effective amount (i,e., a dosage amount that, when administered at regular intervals, is sufficient to treat the disease, such as by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and/or 30 also lessening the severity or frequency of symptoms of the disease, as described above). The ainourit which will be therapeutically effective in the treatment the 2016256663 07 Nov 2016 -9- disease will depend on the nature and extent of fee disease’s effects, and can be determined by standard clinical techniques, ία addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges, The precise dose to be employed 'will also depend on the route of administration, and the 5 seriousness of the disease, and should be decided according to the judgment of a practitioner and each patient's circumstaices. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems, hi a preferred embodiment, the therapeutically effective amotmt is less than about 15 xng enzyme/kg bodyweight of the individual, preferably in the range of about 1-10 mg 10 enzyme/kg body weight, and even mom preferably about 10 mg enzyme/kg body weight or about 5 mg enzyme/kg body wei^at The effective dosefor aparticular individual can be varied (e.g., increased or decreased) over time, depending on the needs of the individnal. For example, in times of physical illness or Stress, or if anti-GAA antibodies become present or increase, or if disease symptoms worsen, the 15 amount can be increased.

The therapeutically effective amount of GAA (or composition or medicament containing GAA) is administered at regular intervals, depending on the nature and extent of the disease’s effects, and on an ongoing basis. Administration at a ‘-regular interval,” as used herein, indicates that the therapeitiically effective 20 amount is administered periodically (as distinguished from a one-time dose). The interval can be determined by standard clinical techniques. In preferred embodiments, GAA is administered monthly, bimonthly; weekly; twice weekly; or daily. The administration interval for a single individual need not be a fixed interval, but can be varied over time, depending on the needs of the individual. For 25 example, in times of physical illness or stress, if anti-GAA antibodies become present or increase, or if disease symptoms worsen, the interval between doses can be decreased. frt one preferred embodiment, a therapeutically effective amount of 10 nag enzyme/kg body weight is administered weekly. In another preferred embodiment, a 30 therapeutically effective amount of 5 mg enzyme/kg body weight is administered twice weekly. 2016256663 07 Nov 2016 -ιο-

The invention additionally pertains to a pharmaceutical composition comprising human acid cc-glucosidase, as described herein, in a container (e.g,, a vial, bottle., bag for intravenous administration, syringe, etc,) with a label containing instructions for adrmrdstration of the composition for treatment of glycogen storage 5 disease type E, such as by the methods described herein, the invention will be further and more specifically described by the following examples. EXEMPLIFICATION; Phase W Trial of Use of Recombinant Human Acid K-glucosidase

Patients: Inclusion criteria were infants affected with infeutile GSD-H having virtually' absent GAA activity' (<1% of normal in skin fibroblasts and/or muscle-biopsy) and less than one year of age. Exclusion criteria included severe cardiorespiratory failure at baseline and/or other medical conditions likely to 15 decrease survival. Because of the limited life expectancy of the disease foUowing «riagaosiSj no placebo control was used. Historical control data indicated that virtually all patients died before 1 y'ear of age (Table 1). 2016256663 07 Nov 2016 -u- Table 1 Historical control data of infantile glycogen storage disease, type H Onset (months) Death (months) Length of disease course (months) Duke IMvemty Medical Center (a- 30)* Mean + SB 5,1 + 1.8 8.6 ±2.4 3,5 + 2.3 Range 2.4 -10.3 3.3-12.4 0.0-9,0 Slonimeia/. (a-10)** Meant SB 2.5 ±1.0 7.2 + 2.8 4.7+2.4 Range 1,0-4.0 4.0 -12.0 2.0 - 9.0 * Data from Duke University Pompe Disease Registry ** Data from Slonim etal.X Pediatr. 137:283-285 (2000), 10 Three infants affected with infantile GSD-H as evidenced by reduced acid a- ghicosidase activity to less than 1% of normal in skis fibroblasts and/or muscle biopsy were enrolled in the study. At the protein level, both patients 1 and 2 had no detectable GAA protein while patient 3 had. reduced levels of GAA protein detected by bnrnunoblot analysis, The baseline clinical data before the initiation of tie 15 therapy are summarized in Table 2. 2016256663 07 Nov 2016

Table 2. Baseline Clinical Data on 3 infanlik *.

Patient Number /Sex Ethnic Background Age JiiGAA Started Cardiac Status Pulmonary Function Motor Development (AIMS Score) GAAAe&amp;vftjrin Skin Fibroblasts CR1M* Status Current Patient 1/male Caucasian 4 months Severe cardiomyopathy ; status post cardiac arrest Borderline normal, left main bronchus compression due to markedly enlarged heart, Oa desaturation «5lh% 0.84% Negative 29 mouths Patient 2/male African^ Ameriean 3 months Moderate cardiomyopathy Oa desaturation during crying 0.57% Negative 25 months Patient 3/male Caucasian ......... 2½ months Borderline cardiomyopathy Normal «5α'% 0.69% Positive 23 months *ϊ» 2016256663 07 Nov 2016 -13-

Patient 1 presented at 2 months of age with cardiac arrest during elective surgical repair of an inguinal hernia. Subsequent evaluation when hewas 4 monthsofage demonstrated evidence of severe hypotonia, with a motor development age estimated to be equivalent to that of a 3 week old fie also had profound 5 cardiomyopathy and severe cardiomegaly with compression of the left main bx-onchus resulting iapartial atelectasis of the left lung, and feeding difficulties and failure to thrive. Patients 2 and 3 were prenatally diagnosed with Pompe disease; importantly, each had a previous sibling that had died of symptoms typically attributable to the infantile GSD-E Both patients had evidence of motor delays; in 10 addition patient 2 had feeding difficulty, failure to thrive and severe cardiomyqpaSiy.

Basic Design: The study was designed as a Phase Idl, open-label, singledose, safety and efficacy study of rhGAA administered twice weekly in the 3 patients with infantile Pornpe disease, The study was approved by the institutional 15 review board, and parental written informed consent was obtained.

The study consisted of an initial Screening Phase, a 13-week Treatment Phase, and a Follow-up Treatment Phase. During the Screening Phase the initial clinical status of the patients was assessed; in addition, GAA and glycogen levels were determined in skeletal muscle biopsy samples. During the Treatment Phase, 20 patients received intravenous infusions of rhGAA (5mg/kg) twice weekly. Patients were closely monitored, for any adverse responses to the enzyme infhsions, as well as tor any impact the rhGAA administrations had on the clinical progression of infantile GSD-Π. General clinical assessments included routine physical examinations, supplemented by complete urine, hematological, and clinical 25 chemistry' analyses (electrolytes, glucose, creatinine, BUN, CO,, protein, albumin, AIT, AST, bilirubin, alkaline phosphatase, CK aiidisozyme, uric add). Exhausti ve neurologic and motor function evaluations ineluded raanual muscle strength testing, Denver development testing, and AIMS (Alberta infant Motor Seale; see Piper, M.C. andDarrah,1., Motor Assessment of the Dm^I&amp;pmglnfant, WB Sanders 30 Company, Philadelphia, 1994). Two-dimensional, M-mode and Doppler echocardiography were used to assess left ventricular mass, wall thickness and 2016256663 07 Nov 2016 *14- systolic as well as diastolic Amotions, Adfetionally, a variety bf pulmonary functions (crying vital capacity* trend pulse-oximetry and end tidal carbon dioxide measurement, as well as negative inspiratory force maneuver) weremonitored throughout die study. At the conclusion of the 13-week treatment phase, GAA 5 activity, glycogen levels mid histopathology of muscle biopsies obtained fiom the quadriceps muscles of the contra-lateral thigh of the pre-treatment biopsies-were determined. The muscle biopsies were taken 3 days after the rhGAA infusion.

Enzyme source: rhGAA purified from the culture medium of rhGAA secreting CHO cells (Van Hove, J.L.K. etal, Proc, Natl Acad, Set USA 93:65-10 70 (1996)) Was provided as a GMP-grade, sterile and colorless solution by Synpac (North Carolina), Inc,, 99 Alexander Drive, Suite NW20, Research Triangle Park, North Carolina 27709. rhGAA was purified primarily as the 110~kD precursor protein with specific enzyme activity of2,77-3.02 umohmin/mg protein. ELISA for anti-rkGAi antibodies: The ELISA for anti-rhGAA antibodies 15 was a standard sandwich assay performed by Phoenix International Life Sciences, Inc, (Saint-Laurent, Quebec), Briefly, microtiter plates were coated with rhGAA at 2.0 pg/ml overnight and then blocked with bovine IgG. Patient serum, diluted to 1:100 and then serially diluted at 1:2, was reacted with the rhGAA on the plate. The amount of bound antibody was detected with a horseradish peroxidase conjugated 20 goat anti-human secondary antibody and tehamethylbenzidine substrate by measuring the absorbances at 450 run. Positive samples were defined as having an absorbancethat was higher than the negative cutoff. This was defined as twice the A450 value of the normal human serum negative control Titer was defined as the dilution of the serum feat still had an A450 reading above the negative cutoff value, 25 GAA activity, glycogen content and Western Mot analysis: GAA acti vity was

assessed by measurement of 4-methyl-umbellifeiyl-ci-D-glucoside cleavage at pH 4.3 as previously described (Reuser, AM, et al, Am. J Hum. Genet. 50:132-143 (1978)). As an internal standard, acid-p-galactosidase activity was similarly assayed with the 4-niefcyl~tnabilliferyl derivative as the substrate (Wenger, DA. and 2016256663 07 Nov 2016 -15-

Williams, C., “Screening for lysosomal disorders” in Homines, P.A. (ed.%

Tecimiqim in diagnostic human biochemical genetics: a laboratory manual, Wiley-Liss, New York, '1991,. pp. 587-617). Glycogen content was determined by treatment, of tissue extracts with A. niger amyloglncosidase and measurement of 5 glucose released (Van Hove, J.L.K. et at, Proc. Natl Acad. Set, USA 93:65-70 (1996)). Western blot analysis was performed withautibody raised in rabbits against purified placenta GAA (Van Hove, J.L.K. et al, supra).

Histology: One specimen of muscle was mounted on a chuck with gum tragacanrb and quick-ftozen in isopentane cooled by lipid nitrogen. Five micron 10 sections were obtained Pd stained with hematoxylin and eosin, modified Goniori tiichrome, ATPase at pH 4.35 and 9.4, mcotmamide dehydrogenase tetrazolium bine reductase, and phosphorylase. A second specimen was clamped in stm Md placed in 2.5% glntaraldehyde. The tissue was processed without en bloc staining with many] acetate in order to avoid loss of glycogen, Ssmithin sections (0.5 micron) were 15 stained with tolui dine bine and thin sections stained with uranyl acetate and lead citrate and mounted on a copper grid for electron microscopy.

Patient Reaction to Treatment. Thethree patients with infantile Pompe disease received twice weekly intravenous inftisions of daGAAfor 21-25 months. 20 No serious allergic reactions occurred during enzyme therapy. However, three episodes of skin rash, accompanied by a mild fever and increased irritability occurred in two of the patients (patient 1 two episodes, patient 2 single episode). These symptoms resolved promptly after intravenous administration of diphenhydramine. After a second episode of skin rash, patient 1 was premedieated 25 with oral diphenhydramine just prior to all subsequent ihGAA infusions, without further episodes. Patient 2 was similarly premedieated with oral diphenhydramine just prior to all subsequent infusions, without further episodes. Multiple hematological parameters, liver functions, renal functions, and urinalyses have all been in the normal range throughout the therapy period in all trotted patients. 2016256663 07 Nov 2016 -16-

Anti-rhGAA antibodies of IgG class were detected in patients! and 2 as early as 3 weeks after ike initiation of ft» enzyme therapy (Figs. 1A4C). Anti-ihGAA antibody titers increased to 1:1600 by w-eek 16 inpatient 1 pig. 1A) and 1:6400-1;12,800 between weelcs 11-19 in patient 2 (Fi g. IB). As anti-rhGAA 5 antibody titers increased, we noted that climcal improvements {noted early during therapy - see below) were no longer advancing. Neither untoward effects nor anti-rhGAA antibodies have been detected in patient 3 (Fig. 1C).

Cardiac status: Prior to the initiation of the enzyme therapy, patients 1 and 2 had severe hypertrophic cardiomyopathy associated with an increased left ventricular-10 (LV) mass, concentric thickening of the ventricular wall and a decrease in size of the ventricular cavity (Fig. 2B, the cavity in patient 2 was almost obliterated at the end of systole). All of these features are typically seen in the untreated patient with the infantile form of Pompe disease. Additionally patient 2 was noted to have an increased LV ejection fraction (shortening fraction, 84%) reflective of a 15 hyperdynamic shortening. None of the patients, however, had any evidence of obstruction of the ventricular outflow tract. The longitudinal echocafdiographic data assessed in the patients during the first 3 months of rhGAA therapy are shown in Fig. 2A-2C. During thetreatmeni period, in both patients 1 and 2, tire LV end-diastolic and end-systolic volumes (2-D measurements) progressively increased, and 20 Up to almost 2-3 fold by the end of 3 months of tiierapy as compared to those measured dnring the pre-treatment phase (Fig. 2A and 2B, respectively). Similar increases were noted by M-mode analysis (data not shown). The two-dimensional LV mass measurements (Fig 2D-2F) initially increased as the LV volumes increased, but then steadily decreased during therapy, to a value that was less than 25 the pre-treatment LV mass (reduced to 60-70% of the baseline pretreatment levels). The initial increase in mass was most likely due to an increase in the LV volume, without any changes in the LV wall thickness. These overall improvements in cardiac parameters, were sustained through the latest follow-up evaluation, although patient 1 required an intensive daily enzyme infusion for 10 days when LV mass was 30 further increased and cardiac Junction compromised at the time of viral pneumonia, Otherwise the ventricular ftmction in both patients had been normal and remained 2016256663 07 Nov 2016 -17- normal at the latest fokow-up. Thus, the progressive cardiac morbidity normally noted in untreated infantile Pompe disease was clearly averted.

Patient 3 had aLv mass of 64 g/β2 (upper norma! limits 65) but otherwise of normal baseline cardiac evaluation at the initiation of therapy, and has continued to 5 be normal (with LV mass now of 3 3 g/β2) since 7 months post-therapy.

Pulmonary fimction: 3h the first 2 months of therapy, improvement of pulmonary funeiion was evident by increases in crying vital capacity (improvements of greater than 28% and 70%, in patients 1 and 2, respectively) over baseline capacities, and normalization of 02 desaturation dming crying (02 saturation of 70% 10 in patient 1 and 81% in patient 2 during maximal crying) . Decreased respiratory' muscle strength was also evidenced in patient 1 before the therapy by a negative inspiratory force maneuver (NIEM) of -45 cm H20, With treatment, theMFM increased to -5 5 cm H20. The initial improvements noted in the pulmonary functions of both patients, however, plateaued over the next 2-3 months and 15 declined subsequently, concomitant with die.np^;a3^rhi3A^.t®^0dies* Both pneumonia precipitated respiratory insufficiency.

Patient 3 had a normal pulmonary function at initiation of therapy and has continued to demonstrate normal pulmonary inaction testing at the latest follow-up. 20 Neurodevelopmeni and motor assessment'. Alberta JhfantMotor Seale (AIMS) was used to evaluate the motor development in these infants, AIMS scores for all 3 patients started below the 5th percentile for age (Fig. IA-1C). Patient 1 remained below die 5th percentile but showed increases within that range before beginning to decline at week 13 of the therapy (Fig. 1 A). Patient 2 rose to the 25 th 25 percentile by week 5, dropped back to remain below the 5th percentile after week 7 despite increasing skills, then showed a rapid decline and loss of skills between weeks 13 and 17 (Fig. IB), The onset of clinical declines, again was concomitant with the rising anti-rhGAA antibodies (Fig. 1 A, IB).

Goncuirently administered neurologic and Denver Developmental 30 evaluations showed in patient 1, normal personal-social, language, and fine motor 2016256663 07 Nov 2016 5 18- motor skills had shown significant progress mill week 10 hot never reached normal. Patient 2 showed mild developmental delay in the gross motor sphere only with attainment of normal developmental skills in the fee motor, personal-social, and language domains until weeks 14-16 when regression occurred. Currently, both patients have normal personal-social development for age but delayin all other domains,

Patient 3 showed a steady increase of AIMS score, rising oyer the 10,h 10 percentile by week 11 of the therapy and rising above the 25 - percentile by week 20 (Fig, 1C), and 90f!l percentile at latest follow-up. At age 9 months, he maintained independent sitting, belly crawled reciprocally for mobility, and maintained standing with hands held. Remarkably, he has been walking independently since 12 months of age and has been able to move betwemsquatting and standing without hand use 15 since 14 months of age. He currently also has normal for age neurologic and Denver development evaluations in all domains.

Muscle GAA activity and glycogen content: Muscle biopsies were performed at baseline 1 week prior to the start of the rhGAA therapy except in patient 1 who had a biopsy done at the time of diagnosis which was 2 months prior to imitation of 20 rhGAA. therapy. After 4 months of rhGAA therapy, muscle biopsies were obtained from the contra-lateral quadriceps 3 days after the enzyme infusion (trough level). With rhGAA treatment GAA activity increased 2-3 fold over baseline pre-treatment levels in both patients i and 2, and IB fold in patient 3 (Table 3). 2016256663 07 Nov 2016 25 -19-

Table 3. Muscle Acid α-glucosidase Activity and Glycogen Content in Infantile

Disease Patients Treated with xhGAA GAA Activity nmole/ltr/mg Protein Glycogen Content % WetWght Patient 1 Pre-therapy 0.41 5.903¾ . Post-therapy 1 0.95 7.50% Patient 2 Pre-therapy 0.67 5.68% Post-therapy 1.97 443% Patient 3 Pre-therapy 0.1 5.13% Post-therapy 1.84 1.43% Control 23.92 +/- 8.63 0.94+/- 0.55% (upper normal limit; 1,5%} 15 The absolute level of GAA activity approached 8% of the GAA activity seen in nonnal muscles. There were no appreciable changes in the muscle glycogen content in patients 1 and 2, but glycogen levels were reduced to within normal range in patients.

Histelogyi The pre-treatment biopsies of all the patients showed marked 20 vacuolization of the muscle fibers in the frozen sections. Evaluation of the semithin sections demonstrated the fibers to be expanded by glycogen with the formation of glycogen lakes, in some fibers faint outlines of residual membranes eould be discerned. Electron microscopy confirmed the presence of glycogen boil in expanded lysosomes and lying free in the cytoplasm* The biopsy from patient 3 had more glycogen remaining within lysosomes than did fire other two patients (data not shown). 2016256663 07 Nov 2016 -20-

The 4-month post-treatment biopsies of patients I and 2 were similar to the pre-treatment biopsies in terms of glycogen accumulation. The post-treatment biopsy of patient 3, however, had a marked decrease Iq visible glycogen and essentially normal histology' in most of the muscle fibers. Electron mieroseppy 5 showed many remaining distended lysosomes were depleted of glycogen. Some glycogen lakes and glycogen-rich lysosomes remained.

Western Mot analysis

To investigate why anti-rhGAA antibodies developed in patients 1 and 2, but not 3, we performed a Western blot analysis specific for detection of expressed (but 10 nonfunctional) GAA protein in fibroblasts derived from each of the patients, No GAA protein was detected in the fibroblasts of patients 1 and2, whereas a readily detectable precursor form of GAA protein (110 kD) was found in patient 3. These pafiems were previously seen in other patients with infantile GSD-If (Van der Floegj A.T. st at, Am. J Hum. Genet 44:787-793 (1989)). Normal fibroblasts as expected 15 have GAA protein predominantly of 95 kD mid 76 kD.

FURTHEB. STUDIES

Three more patients have been enrolled in an additional study. All three are CRM positive. After treatment (10 mg/ldlogram body weight, weekly intravenous infusions of rhGAA) for 3-6 weeks, improvement of heart function, muscle strength, 20 and motor development have been seen.

The teachings of all publications cited herein are incorporated herein by reference in their entirety. 25 the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. 2016256663 07 Nov 2016 -20a-

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises” or "comprising", will be understood to imply the inclusion of a stated element or integer or method step or group of elements or integers or method steps but not the exclusion of any other element or integer or method steps or group of elements or integers or method steps.

Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

Claims (18)

1. CLAIMS:

   1. A method of treating glycogen storage disease type II in an individual, comprising administering to the individual a therapeutically effective amount of human acid α-glucosidase at a regular interval.
2. 2. A method of treating cardiomyopathy associated with glycogen storage disease type II in an individual, comprising administering to the individual a therapeutically effective amount of human acid α-glucosidase at a regular interval.
3. 3. The method of Claim 1 or Claim 2, wherein the glycogen storage disease type II is infantile glycogen storage disease type II, juvenile glycogen storage disease type II, or adult-onset glycogen storage disease type II.
4. 4. The method of Claim 1 or Claim 2, wherein the therapeutically effective amount of human acid α-glucosidase is less than about 15 mg of acid α-glucosidase per kilogram of body weight of the individual.
5. 5. The method of Claim 1 or Claim 2, wherein the human acid α-glucosidase is recombinant human acid α-glucosidase and/or a precursor of recombinant human acid a-glucosidase.
6. 6. The method of Claim 5, wherein the recombinant human acid α-glucosidase is produced in Chinese hamster ovary cells.
7. 7. The method of Claim 1 or Claim 2, wherein the regular interval is monthly, bimonthly, weekly, twice weekly or daily.
8. 8. The method of Claim 1 or Claim 2, wherein the human acid α-glucosidase is administered by a route selected from the group consisting of: intravenously, intramuscularly, intrathecally and intraventricularly.
9. 9. The method of Claim 1 or Claim 2, wherein the human acid α-glucosidase is administered in conjunction with, or subsequent to, administration of an immunosuppressant.
10. 10. A pharmaceutical composition comprising human acid α-glucosidase in a container with a label containing instructions for administration of the composition for treatment of glycogen storage disease type II.
11. 11. Use of a therapeutically effective amount of human acid α-glucosidase for the manufacture of a medicament for the treatment of glycogen storage disease II in an individual.
12. 12. Use of a therapeutically effective amount of human acid α-glucosidase for the manufacture of a medicament for the treatment of cardiomyopathy associated glycogen storage disease type II in an individual.
13. 13. The use of Claim 11 or Claim 12, wherein the therapeutically effective amount of human acid α-glucosidase is less than about 15 mg of acid α-glucosidase per kilogram of body weight of the individual.
14. 14. The use of Claim 11 or Claim 12, wherein the human acid α-glucosidase is recombinant human acid α-glucosidase and/or a precursor of recombinant human acid a-glucosidase.
15. 15. The use of Claim 14, wherein the recombinant human acid α-glucosidase is produced in Chinese hamster ovary cells.
16. 16. The use of Claim 11 or Claim 12, wherein the medicament is prepared to be administered at a regular interval that is monthly, bimonthly, weekly, twice weekly or daily.
17. 17. The use of Claim 11 or Claim 12, wherein the medicament is prepared to be administered by a route selected from the group consisting of: intravenously, intramuscularly, intrathecally and intraventricularly.
18. 18. The use of Claim 11 or Claim 12, wherein the medicament further comprises an immunosuppressant.