US 2009019 1178A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0191178 A1 Zankel et al. (43) Pub. Date: Jul. 30, 2009

(54) MANUFACTURE OF HIGHLY Related U.S. Application Data PHOSPHORYLATED LYSOSOMAL (63) Continuation-in-part of application No. 10/588,425, ENZYMES AND USES THEREOF filed on Jun. 6, 2007, filed as application No. PCT/ US05/04345 on Feb. 7, 2005. (75) Inventors: Todd C. Zankel, San Francisco, (60) Provisional application No. 60/542,586, filed on Feb. CA (US); Christopher M. Starr, 6, 2004. Sonoma, CA (US) Publication Classification Correspondence Address: (51) Int. C. A638/47 (2006.01) MARSHALL, GERSTEIN & BORUN LLP CI2N 9/24 (2006.01) 233 SOUTH WACKER DRIVE, 6300 SEARS CI2P 2L/04 (2006.01) TOWER CI2N 5/06 (2006.01) CHICAGO, IL 60606–6357 (US) (52) U.S. Cl...... 424/94.61; 435/200; 435/69.1; 435/358 (73) Assignee: BOMARIN ABSTRACT PHARMACEUTICAL INC., (57) Novato, CA (US) This invention provides compositions of highly phosphory lated lysosomal enzymes, their pharmaceutical composi tions, methods of producing and purifying Such lysosomal (21) Appl. No.: 12/182,818 enzymes and compositions and their use in the diagnosis, prophylaxis, or treatment of diseases and conditions, includ (22) Filed: Jul. 30, 2008 ing particularly lysosomal storage diseases.

Construct: pCINt (5952 bp)

HindIII (2141) neo WPRE HSV-tk BGHpA XhoI (2153) Patent Application Publication Jul. 30, 2009 Sheet 1 of 19 US 2009/01911 78A1

FIGURE 1

GAAF: 5’-GCGATAGGTACCGCCATGGGAGTGAGGCACCCGCCCTGCTCCC-3 (SEQ ID NO:3) GAAR: 5’-GCGATACTCGAGTCAACACCAGCTGACGAGAAACTGCTCTCCC-3 (SEQ ID NO:4) Patent Application Publication Jul. 30, 2009 Sheet 2 of 19 US 2009/0191178 A1

FIGURE 2A

Construct: pCINt (5952 bp) Patent Application Publication Jul. 30, 2009 Sheet 3 of 19 US 2009/0191178 A1

FIGURE 2 B

pBioMP 14 EIN

Patent Application Publication Jul. 30, 2009 Sheet 6 of 19 US 2009/0191178 A1

FIGURE 4

AMINO ACID SEQUENCE ALIGNMENT OF HUMAN ACID ALPHA GLUCOSIDASE ISOFORM 1 (GAA. SEQ ID NO: 1) AND 2 (GAA2. SEQ ID NO:12)

7 y G R H P C S R L id-- As c A. L j S i:A. AAA I G H 1. L H i. l W R E l S {G S S P -Yi E. T P A H C Q(G A. S R

ERISSSEf RTEFETRIAEERLEFERRYE

SSSEAGYLETTLEFPK). RLDW-NETERRIERPARRYEWL

GAA1 ETERVESRAPSPLYSWEFSEEPEG, VEROCGRVT, LTTWAFL FRADCFLLSTSLPSYITGI. 2 t GAA2 FRHSRASLSEFSEEE LLETTWAPLFSACFLQLSTSLPSYITG

GAA1 AELSPLMS SWR ANRA GANGSHEY AIEDGGSAHGVFLENSNAA DVWL 2 GAA2 AHSELMISTSWRITLEJNRELATP CANLY GSHPFY L.A.T.EIGCSAHG, FITNS NAMDVIPS 3 5.

GAA PALSWRSTGGLD, IFT,GPE PKSV WCYT, DJ, CYPFM

FLSRS (GLEWIGPEEK

GAA1 MITRA - FELDV CWND LOY-IS VEI, CGGRRYSEWAISSSGPAGS G3. --- 2 T A. H f p - FFAM7CE, HCGRRY: IM WDPAISSS PAGSY

GAA R YDEGLRR. VFITNEGOPLICKVWPGSTAF PET. TALAWWE DrivAEFEDQWPFOSISNIDR1) 5 2. g YDEGLRRGVFITNETGQPLIGKVWPGSTAFPDFTNPTALAWWEDMVAEFHDQ7 PFDGL IDMN 5, 2 (AA ESNFIRGSEDGCPNNEL ENPPY WGWWGGTLAATICASSHCFLSTHYNLHNLYGLTEAIASR 8 5. (GAA2 EPSNFIRGSEDGCPNNELENPPY WPG, VGSTLCAAFICASS HQFISTHYNLHNLYGLTEPIASHR 5 8 5

w , L R T R F f S R SSFAGGRYAGHWTGD, -- -- SSNEOASSVPE LLGWPL, GADVCGF s . 2 Aij KARSTRFWSRST. FASHGRYAGE. WTGD, WSS ELASS, PEI, CEELLGVLWGAD.CG

G A. i.

Patent Application Publication Jul. 30, 2009 Sheet 11 of 19 US 2009/0191178 A1

FIGURE 8

A) Blue Eluate B) Q Eluate C) Phe Eluate

75 75

Ag+ 5ug BM 103/lane Western (1:5000) Primary Commassie 5ug BM 103/lane 0.5ug BM 103 f lane Patent Application Publication Jul. 30, 2009 Sheet 12 of 19 US 2009/0191178 A1

FIGURE 9

2 3 4 3

Markers GAA, 23 FBS PS 83005 PSO9205 Load FT Wash : :::::::3& :::::::: Eluate-1 (1-5) 28 Eluate-II (#14-43). Main 9 4 COOmmassie Anti-BM103 (1:5000) Patent Application Publication Jul. 30, 2009 Sheet 13 of 19 US 2009/01911 78A1

FIGURE 10

parkers S05906 Eluate - | OSO-52306 Eluate SEHCapoS2506 Eluate SO-2508 Eate.

Coomassie Silver stain Western (1:5000) primary 2.5 lug BM103/lane 2.5 ug BM103/lane 0.5 pug BM103/lane Patent Application Publication Jul. 30, 2009 Sheet 14 of 19 US 2009/0191178 A1

FIGURE 11

1 2 3 4 5 6 7 1 2 3 4 5 6 7

COOmmassie anti-BM103 (1:5000)

rakers ESO 27 Lad BSO 27 Eiate SOF 5. Eat SEH Capo7O607 FT-w-E) S. Eigte C3 FBDS (PSO 70907+PSO 1007) Patent Application Publication Jul. 30, 2009 Sheet 15 of 19 US 2009/0191178 A1

FIGURE 12

FACE profiles for GAA from G715 and DUXBll

G4 standard ...:------. G4 standard

O7P 19% of total oligosaccharide profile O7P 6.7% of total oligosaccharide profile Patent Application Publication Jul. 30, 2009 Sheet 16 of 19 US 2009/0191178 A1

FIGURE 13

BSA Patent Application Publication Jul. 30, 2009 Sheet 17 of 19 US 2009/0191178 A1

FIGURE 14

Kuptake for DUX rhGAA = 2.95 nM, Kuptake for G71 rhGAA = 1.31 nM

1OOOO

9000 8000 7000

6000 5000

4000 3000 2000 1000

enzyme applied, nM Patent Application Publication Jul. 30, 2009 Sheet 18 of 19 US 2009/01911 78A1

FIGURE 15A

53. - . Heart, 40x (Vehicle) Heart, 40x (20 mg/kg rhGAA) Patent Application Publication Jul. 30, 2009 Sheet 19 of 19 US 2009/0191178 A1

FIGURE 15B

Diaphragm, 40x (Vehicle) Diaphragm, 40x (20 mg/kg rhGAA) US 2009/019 1178 A1 Jul. 30, 2009

MANUFACTURE OF HIGHLY ing site. A second enzyme, phosphodiester C-GlcNAcase, PHOSPHORYLATED LYSOSOMAL then cleaves the GlcNAc-phosphate bond to remove ENZYMES AND USES THEREOF N-acetylglucosamine to give a mannose 6-phosphate termi nal oligosaccharide. The purpose of the mannose 6-phosphate CROSS-REFERENCE TO RELATED modification is to divert lysosomal enzymes from the secre APPLICATIONS tory pathway to the lysosomal pathway within the cell. Phos phate-bearing enzyme is bound by the MPR in the trans Golgi 0001. This application is a continuation-in-part of U.S. and routed to the lysosome instead of the cell surface. application Ser. No. 10/588,425, filed Jun. 6, 2007, which is 0006 Large-scale production of lysosomal enzymes the National Stage of International Application No. PCT/ involves expression in mammalian cell lines. The goal is the US2005/004345, filed Feb. 7, 2005, which claims the benefit predominant secretion of recombinant enzyme into the Sur of and priority to U.S. Provisional Application No. 60/542, rounding growth medium for harvest and processing down 586, filed Feb. 6, 2004, the disclosures of which are herein stream. In an ideal system for the large-scale production of incorporated by reference in their entirety. lysosomal enzymes, enzyme would be efficiently phospho rylated and then directed primarily toward the cell surface FIELD OF THE INVENTION (secretion) rather than primarily to the lysosome. As 0002 The present invention relates to the technical fields described above, this proportionation of phosphorylated of cellular and molecular biology and medicine, particularly enzymes is the exact opposite of what occurs in normal cells. to the manufacture of highly phosphorylated lysosomal Manufacturing lines often used for lysosomal enzyme pro enzymes and their use in the management of lysosomal Stor duction focus on maximizing the level of mannose 6-phos age diseases. phate per mole of enzyme and are characterized by low spe cific productivity. In vitro attempts at producing lysosomal BACKGROUND OF THE INVENTION enzymes containing high levels of mannose-6 phosphate 0003) Lysosomal storage diseases (LSDs) result from the moieties have resulted in mixed success (Canfield, U.S. Pat. deficiency of specific lysosomal enzymes within the cell that No. 6,537,785). The in vitro enzyme exhibits high levels of are essential for the degradation of cellular waste in the lyso mannose-6-phosphate, as well as high levels of unmodified some. A deficiency of such lysosomal enzymes leads to accu terminal mannose. Competition between the mannose mulation of undegraded “storage material' within the lyso 6-phosphate and mannose receptors for enzyme results in the some, which causes swelling and malfunction of the necessity for high doses of enzyme for effectiveness, and lysosomes, and ultimately cellular and tissue damage. A large could lead to greater immunogenicity to the detriment of the number of lysosomal enzymes have been identified and cor Subject being treated. related with their related diseases. Once a missing enzyme 0007 Thus, there exists a need in the art for an efficient has been identified, treatment can be reduced to the sole and productive system for the large-scale manufacture of problem of efficiently delivering replacement enzyme to the therapeutically effective lysosomal enzymes for management affected tissues of patients. of lysosomal storage disorders. 0004 One way to treat lysosomal storage diseases is by intravenous enzyme replacement therapy (ERT) (Kakkis. SUMMARY OF INVENTION Expert Opin. Investig. Drugs 11(5):675-685, 2002). ERT 0008. The present invention relates to the discovery that a takes advantage of the vasculature to carry enzyme from a CHO-K1 derivative, designated G71, which is defective in single site of administration to most tissues. Once the enzyme endosomal acidification, produces high yields of phosphory has been widely distributed, it must be taken up into cells. The lated, recombinant enzyme by preventing loss of material to basis for uptake into cells is found in a unique feature of the lysosomal compartment of the manufacturing cell line lysosomal enzymes, which constitute a separate class of gly itself. Such enzymes also preferably have a low level of coproteins defined by phosphate at the 6-position of terminal unphosphorylated high-mannose oligosaccharides. In one mannose residues. Mannose-6-phosphate (M6P) is bound embodiment, the invention provides an END3 complementa with high affinity and specificity by a receptor found on the tion group cell line that overexpresses recombinant lysosomal surface of most cells (Munier-Lehmann et al., Biochem. Soc. enzymes, resulting in high yields of highly phosphorylated Trans. 24(1):133-136, 1996; Marnell et al., J. Cell. Biol. enzyme. Exemplary cell lines are G71 or a derivative thereof 99(6): 1907-1916, 1984). The mannose-6-phosphate receptor that retains the desired property of G71, i.e., the ability to (MPR) directs uptake of enzyme from blood to tissue and then produce high yields of highly phosphorylated recombinant mediates intracellular routing to the lysosome. enzyme preferably with a low level of unphosphorylated high 0005. The therapeutic effectiveness of a lysosomal mannose oligosaccharides. This application of an END3 enzyme preparation depends crucially on the level of man complementation group modified CHO-K1 line would be nose 6-phosphate in that preparation. Phosphate is added to especially useful for the manufacture of lysosomal enzymes the glycoprotein by a post-translational modification in the to be used for management of lysosomal storage diseases by endoplasmic reticulum and early Golgi. Folded lysosomal enzyme replacement therapy (ERT). In one embodiment, the enzymes display a unique tertiary determinant that is recog lysosomal enzyme can be human acid alpha-glucosidase nized by an oligosaccharide modification enzyme. The deter (rhGAA). minant is composed of a set of specifically spaced lysines and 0009. In one aspect, the present invention features a novel is found on most lysosomal enzymes despite absence of pri method of producing highly phosphorylated lysosomal mary sequence homology. The modification enzyme, UDP enzymes in amounts that enable their therapeutic use. In a GlcNAc phosphotransferase, binds to the protein determinant broad embodiment, the method comprises the step of trans and adds GlcNAc-1-phosphate to the 6-position of terminal fecting a cDNA encoding for all or part of the lysosomal mannose residues on oligosaccharides proximate to the bind enzyme into a cell suitable for the expression thereof. In some US 2009/019 1178 A1 Jul. 30, 2009 embodiments, a cDNA encoding for a full-length lysosomal thereof and comprises an expression vector for recombinant enzyme is used, whereas in other embodiments a cDNA human acid alpha-glucosidase (rhGAA). In a preferred encoding for a biologically active fragment, variant, deriva embodiment, the END3 complementation group CHO cell tive or mutant thereof may be used. In other preferred line expresses and secretes rhGAA. In another preferred embodiments, an expression vector is used to transfer the embodiment, the END3 complementation group cell line is cDNA into a suitable cell line for expression thereof. In a selected from the group consisting of 1B8, C3, D7 and F5. In preferred embodiment, the method comprises the step of pro a more preferred embodiment, the END3 complementation ducing highly phosphorylated lysosomal enzymes from cell group cell line is C3. In another preferred embodiment, the lines with defects in endosomal trafficking. In a particularly END3 complementation group cell line comprises G71 or a preferred embodiment, the method comprises the step of pro derivative thereof that is adapted to growth in Suspension. ducing highly phosphorylated recombinant lysosomal 0013. In a third aspect, the invention provides novel lyso enzymes from the END3 complementation group CHO cell Somal enzymes produced in accordance with the methods of line G71. In one embodiment, the lysosomal enzyme can be the present invention and thereby present in amounts that human acid alpha-glucosidase (rhGAA). enable using the enzyme therapeutically. The enzymes may 0010. In a preferred embodiment, the invention provides a be full-length proteins, or fragments, variants, derivatives or method for producing a recombinant human acid alpha-glu mutants thereof. In some embodiments, the full-length pro cosidase (rhGAA) enzyme or variant thereof, or derivative of tein, or fragment, variant, derivative or mutant thereof accord the rhGAA enzyme or variant thereof, which has a high level ing to the invention may be modified as desired to enhance its of phosphorylation and a low level of unphosphorylated high stability or pharmacokinetic properties (e.g., PEGylation, mannose oligosaccharides, exceeding at least about 0.7 moles mutagenesis, fusion, conjugation). In preferred embodi bis-phosphorylated oligomannose chains per mole of rhGAA ments, the enzyme is a human enzyme, a fragment of the enzyme or variant thereof, comprising the steps of: (a) cul human protein or enzyme having a biological activity of a turing Chinese Hamster Ovary (CHO)-derived END3 native protein or enzyme, or a polypeptide that has substantial complementation group cells, preferably G71 cells or a amino acid sequence homology with the human protein or derivative thereof; (b) preparing a mammalian expression enzyme. In some embodiments, the enzyme agent is a protein vector encoding the rhGAA enzyme or variant thereof suit of human or mammalian sequence, origin or derivation. In able for the END3 complementation group cells; (c) trans other embodiments, the enzyme or protein is such that its fecting the END3 complementation group cells with the deficiency causes a human disease Such as Pompe disease expression vector; (d) selecting and cloning a END3 comple (e.g. acid alpha-glucosidase). In other embodiments, the mentation group cell transfectant; and (e) optimizing cell enzyme is selected for its beneficial effect. culture process methods for manufacturing the END3 0014. The enzyme or protein can also be of human or complementation group cell transfectant. In a more preferred mammalian sequence origin or derivation. In yet other embodiment, the rhGAA enzyme comprises an amino acid embodiments of the invention, in each of its aspects, the sequence selected from the group consisting of SEQ ID enzyme or protein is identical in amino acid sequence to the NO:11 (GAA1-M), SEQID NO:12 (GAA2), SEQID NO:14 corresponding portion of a human or mammalian polypeptide (GAA1-L) and SEQID NO:15 (GAA4). In a most preferred amino acid sequence. In other embodiments, the polypeptide embodiment, the rhGAA enzyme comprises the amino acid moiety is the native protein from the human or mammal. In sequence of SEQID NO:11 (GAA1-M). In another preferred other embodiments, the polypeptide is Substantially homolo embodiment, the rhGAA enzyme has high levels of phospho gous (i.e., at least 80%, 85%, 90%. 95%, 96%,97%.98%, or rylation (greater than about 0.7 moles oligomannose bis 99% identical in amino acid sequence) over a length of at least phosphate per mole of enzyme, preferably greater than about 25, 50, 100, 150, or 200 amino acids, or the entire length of the 0.85 moles oligomannose bis-phosphate per mole of enzyme, polypeptide, to the native enzyme sequence of human or and more preferably greater than about 1.0 mole oligoman mammalian enzyme. In other embodiments, the Subject to nose bis-phosphate per mole of enzyme) and low levels of which the enzyme is to be administered is human. high-mannose oligosaccharide. 0015. In a preferred embodiment, the lysosomal enzyme 0011. In a second aspect, the present invention provides an of the invention is a recombinant human acid alpha-glucosi endosomal acidification-deficient cell line characterized by dase (rhGAA) enzyme or variant thereof, or derivative of the its ability to produce lysosomal enzymes in amounts that rhGAA enzyme or variant thereof, which is produced in enable use of the enzyme therapeutically. In preferred END3 complementation group CHO cells, preferably a G71 embodiments, the invention provides CHO-K1-derived cell line or derivative thereof, and which has a high level of END3 complementation group cell lines, designated G71 and phosphorylation and a low level of unphosphorylated high derivatives thereof, that are capable of producing high yields mannose oligosaccharides, exceeding at least about 0.7 moles of highly phosphorylated lysosomal enzymes, thereby bis-phosphorylated oligomannose chains per mole of rhGAA enabling the large scale production of therapeutic lysosomal enzyme or variant thereof. In a more preferred embodiment, enzymes. In most preferred embodiments, the cell line the rhGAA enzyme comprises an amino acid sequence expresses and secretes recombinant lysosomal enzymes in selected from the group consisting of SEQID NO:11 (GAA1 amounts of at least about 0.25 picogram/cell/day, preferably M), SEQID NO:12 (GAA2), SEQID NO:14 (GAA1-L) and at least about 0.5 picogram/cell/day, more preferably at least SEQID NO:15 (GAA4). In a most preferredembodiment, the about 1.0 picogram/cell/day, or more. rhGAA enzyme comprises the amino acid sequence of SEQ 0012. An END3 complementation group cell line is any ID NO:11 (GAA1-M). modified CHO cell line that retains the properties of an END3 0016. In another preferred embodiment, the lysosomal complementation group cell. Such as defective endosomal enzyme of the invention is a recombinant human acid alpha acidification. In one embodiment, the END3 complementa glucosidase (rhGAA) enzyme or variant thereof, or derivative tion group CHO cell line is derived from G71 or a derivative of said rhGAA enzyme or variant thereof, which has a high US 2009/019 1178 A1 Jul. 30, 2009 level of phosphorylation and a low level of unphosphorylated needs of the subjects to be treated, preferably mammals and high-mannose oligosaccharides, exceeding at least about 0.7 most preferably humans, to most effectively ameliorate the moles bis-phosphorylated oligomannose chains per mole of disease symptoms. rhGAA enzyme or variant thereof, which is produced by the 0020 Such therapeutic enzymes are particularly useful, methods of the invention. In a more preferred embodiment, for example, in the treatment of lysosomal storage diseases the rhGAA enzyme comprises an amino acid sequence such as MPSI, MPS II, MPS IIIA, MPS IIIB, Metachromatic selected from the group consisting of SEQID NO:11 (GAA1 Leukodystrophy, Gaucher, Krabbe, Pompe, Neuronal Ceroid M), SEQID NO:12 (GAA2), SEQID NO:14 (GAA1-L) and Lipofuscinosis CLN2 Batten disease, Niemann-Pick and SEQID NO:15 (GAA4). In a most preferredembodiment, the Tay-Sachs disease wherein a lysosomal protein deficiency rhGAA enzyme comprises the amino acid sequence of SEQ contributes to the disease state. In yet other embodiments, the invention also provides a pharmaceutical composition com ID NO:11 (GAA1-M). prised of the deficient protein or enzyme causing a lysosomal 0017. In preferred embodiments, the enzyme is a recom storage disease. binant human lysosomal enzyme produced by an endosomal 0021. In some embodiments, the lysosomal enzymes, acidification-deficient cell line. In more preferred embodi compositions, and methods of the invention can be used to ments, the recombinant human lysosomal enzyme has a high treat Such lysosomal storage diseases as Aspartylglu level of phosphorylated oligosaccharides, exceeding at least cosaminuria, Cholesterol ester storage diseasef Wolman dis about 0.7 moles bis-phosphorylated oligomannose chains per ease, Cystinosis, Danon disease, Fabry disease, Farber mole of protein, preferably exceeding at least about 0.85 Lipogranulomatosis/Farber disease, Fucosidosis, Galacto moles bis-phosphorylated oligomannose chains per mole of sialidosis types I/II, Gaucher disease types I/II/III Gaucher protein, and more preferably exceeding at least about 1.0 disease, Globoid cell leukodystrophy/Krabbe disease, Glyco mole bis-phosphorylated oligomannose chains per mole of gen storage disease II/Pompe disease, GM1-Gangliosidosis protein, and low level of unphosphorylated high-mannose types I/II/III, GM2-Gangliosidosis type I/Tay-Sachs disease, oligosaccharides. In most preferred embodiments, the GM2-Gangliosidosis type II Sandhoff disease, GM2-Gangli enzyme is a highly phosphorylated recombinant human acid osidosis, alpha-Mannosidosis types I/II, alpha-Mannosido alpha-glucosidase (rhGAA). sis, Metachromatic leukodystrophy, Mucolipidosis type 0018. In a fourth aspect, the invention provides a method I/Sialidosis types I/II Mucolipidosis types II/IIII-cell disease, to purify the lysosomal enzymes produced by the methods of Mucolipidosis type IIIC pseudo-Hurler polydystrophy, the present invention. In preferred embodiments, the inven Mucopolysaccharidosis type I, Mucopolysaccharidosis type tion provides a method for purifying a lysosomal enzyme II Hunter syndrome, Mucopolysaccharidosis type IIIA San comprising the steps of: (a) dia-filtering a cell culture harvest filippo syndrome. Mucopolysaccharidosis type IIIB Sanfil containing the lysosomal enzyme; (b) adjusting the pH of the ippo syndrome, Mucopolysaccharidosis type IIIC Sanfilippo dia-filtered harvest to induce precipitation of contaminating syndrome, Mucopolysaccharidosis type IIID Sanfilippo Syn proteins; and (c) isolating the lysosomal enzyme by sequen drome. Mucopolysaccharidosis type IVA Morquio syn tial chromatography on dye-ligand, anion exchange and drome, Mucopolysaccharidosis type IVB Morquio syn hydrophobic resins. In other preferred embodiments, the drome, Mucopolysaccharidosis type VI, invention provides a method for purifying a lysosomal Mucopolysaccharidosis type VII Sly syndrome. Muco enzyme comprising the steps of: (a) ultra-filtering a cell cul polysaccharidosis type IX, Multiple sulfatase deficiency, ture harvest containing the lysosomal enzyme; (b) adjusting Pompe, Neuronal Ceroid Lipofuscinosis CLN1 Batten dis the pH of the ultra-filtered harvest to induce precipitation of ease, Neuronal Ceroid Lipofuscinosis CLN2 Batten disease, contaminating proteins; and (c) isolating the lysosomal Niemann-Pick disease types A/B Niemann-Pick disease, enzyme by sequential chromatography on dye-ligand, anion Niemann-Pick disease type C1 Niemann-Pick disease, exchange, cation exchange and hydrophobic resins. In some Niemann-Pick disease type C2 Niemann-Pick disease, Pyc embodiments, the dye-ligand resin is Blue-Sepharose. In nodysostosis, Schindler disease types I/II Schindler disease, Some embodiments, the anion exchange resin is and Sialic acid storage disease. In particularly preferred Q-Sepharose. In some embodiments, the cation exchange embodiments, the lysosomal storage disease is MPS III, resin is SE Hi-Cap. In some embodiments, the hydrophobic MLD, or GM1. In a most preferred embodiment, the lysoso resin is Phenyl-Sepharose. mal storage disease is Pompe. 0019. In a fifth aspect, the present invention provides a 0022. In still another embodiment, the present invention method of treating diseases caused all or in part by deficiency provides for a method of enzyme replacement therapy by of lysosomal enzyme. In most preferred embodiments, the administering a therapeutically effective amount of a fusion method comprises administering the therapeutic enzyme pro or conjugate to a Subject in need of the enzyme replacement duced by the methods of the present invention, wherein the therapy, wherein the cells of the patient have lysosomes enzyme binds to an MPR receptor and is transported across which contain insufficient amounts of the enzyme to prevent the cell membrane, enters the cell and is delivered to the or reduce damage to the cells, whereby Sufficient amounts of lysosomes within the cell. In one embodiment, the method the enzyme enter the lysosomes to prevent or reduce damage comprises administering a therapeutic recombinant enzyme, to the cells. The cells may be within or without the CNS or or a biologically active fragment, variant, derivative or mutant need not be set off from the blood by capillary walls whose thereof, alone or in combination with a pharmaceutically endothelial cells are closely sealed to diffusion of an active acceptable carrier. In other embodiments, this method fea agent by tight junctions. tures transfer of a nucleic acid sequence encoding the full 0023. In a particular embodiment, the invention provides length lysosomal enzyme or a fragment, variant, derivative or lysosomal enzymes comprising an active agent having a bio mutant thereof into one or more of the host cells in vivo. logical activity which is reduced, deficient, or absent in the Preferred embodiments include optimizing the dosage to the target lysosome and which is administered to the Subject. US 2009/019 1178 A1 Jul. 30, 2009

Preferred active agents include, but are not limited to aspar rhGAA enzyme or variant thereof, wherein the rhGAA tylglucosaminidase, acid lipase, cysteine transporter, Lamp enzyme or variant thereof has at least about 0.7 bis-phospho 2, alpha-galactosidase A, acid ceramidase, alpha-L-fucosi rylated oligomannose chains per mole of rhGAA enzyme, dase, beta-hexosaminidase A, GM2-activator deficiency, which is produced comprising the steps of: (a) culturing Chi alpha-D-mannosidase, beta-D-mannosidase, arylsulfatase A, nese Hamster Ovary (CHO)-derived END3 complementation saposin B, neuraminidase, alpha-N-acetylglucosaminidase group cells; (b) preparing a mammalian expression vector phosphotransferase, phosphotransferase Y-Subunit, alpha-L- encoding the rhGAA enzyme or variant thereof suitable for iduronidase, iduronate-2-sulfatase, heparan-N-sulfatase, the END3 complementation group cells; (c) transfecting the alpha-N-acetylglucosaminidase, acetylCoA:N-acetyltrans END3 complementation group cells with the expression vec ferase, N-acetylglucosamine 6-sulfatase, galactose 6-sulfa tor; (d) selecting and cloning a END3 complementation tase, alpha-galactosidase, N-acetylgalactosamine 4-Sulfa group cell transfectant; and (e) optimizing cell culture pro tase, hyaluronoglucosaminidase, palmitoyl protein cess methods for manufacturing the END3 complementation thioesterase, tripeptidyl peptidase I, acid sphingomyelinase, group cell transfectant, wherein the END3 complementation cholesterol trafficking, cathepsin K, beta-galactosidase B. group cell is a G71 cell line or a derivative thereof, and a C-glucosidase, and sialic acid transporter. In a preferred pharmaceutically acceptable carrier, diluent or excipient. In a embodiment, alpha-L-iduronidase, C-glucosidase or more preferred embodiment, the rhGAA enzyme comprises N-acetylgalactosamine 4-sulfatase is the enzyme. In a most an amino acid sequence selected from the group consisting of preferred embodiment, C-glucosidase is the enzyme. SEQID NO:11 (GAA1-M), SEQID NO:12 (GAA2), SEQ 0024. In a preferred embodiment, the invention provides a ID NO:14 (GAA1-L) and SEQID NO:15 (GAA4). In an even method of treating Pompe disease by administering a lysoso more preferred embodiment, the rhGAA comprises the mal enzyme of the invention useful to treat Pompe disease, amino acid sequence of SEQ ID NO:11 (GAA1-M). In wherein the lysosomal enzyme is a recombinant human acid another more preferred embodiment, the rhGAA enzyme has alpha-glucosidase (rhGAA) enzyme or variant thereof, or at least about 0.85 bis-phosphorylated oligomannose chains derivative of said rhGAA enzyme or variant thereof, which is per mole of rhGAA enzyme. In an even more preferred produced by END3 complementation group cells, preferably embodiment, the rhGAA enzyme has at least about 1.0 bis G71 cells or a derivative thereof, and which has a high level of phosphorylated oligomannose chains per mole of rhGAA phosphorylation and a low level of unphosphorylated high enzyme. mannose oligosaccharides, exceeding at least about 0.7 moles 0028. Other features and advantages of the invention will bis-phosphorylated oligomannose chains per mole of rhGAA become apparent from the following detailed description. It enzyme or variant thereof. In a more preferred embodiment, should be understood, however, that the detailed description the rhGAA enzyme comprises an amino acid sequence and the specific examples, while indicating preferred embodi selected from the group consisting of SEQID NO:11 (GAA1 ments of the invention, are given by way of illustration only, M), SEQID NO:12 (GAA2), SEQID NO:14 (GAA1-L) and because various changes and modifications within the spirit SEQID NO:15 (GAA4). In a most preferredembodiment, the and scope of the invention will become apparent to those rhGAA enzyme comprises the amino acid sequence of SEQ skilled in the art from this detailed description. ID NO:11 (GAA1-M). In another preferred embodiment, the rhGAA enzyme has high levels of phosphorylation (greater BRIEF DESCRIPTION OF THE DRAWINGS than about 0.7 moles oligomannose bis-phosphate per mole 0029 FIG. 1 describes the nucleic acid sequences of the of enzyme, preferably greater than about 0.85 moles oligom primers used to amplify human acid alpha-glucosidase annose bis-phosphate per mole of enzyme, and more prefer (hGAA) from human liver mRNA by high-stringency PCR ably greater than about 1.0 mole oligomannose bis-phosphate (SEQ ID NOs: 3 and 4). per mole of enzyme) and low levels of high-mannose oli 0030 FIG. 2 describes (A) the CINt vector and (B) the gosaccharide. pBMP14 vector. 0025 Corresponding use of highly phosphorylated lyso 0031 FIG.3 describes the nucleotide (top) and amino acid Somal enzymes of the invention, which are preferably pro (bottom) sequences (SEQID NOs: 1 and 2, respectively) of duced by methods of the invention, in preparation of a medi human acid alpha-glucosidase (hGAA) inserted into the CINt cament for the treatment of the diseases described above is Vector. also contemplated. 0032 FIG. 4 shows an amino acid sequence alignment of 0026. In a sixth aspect, the present invention provides human acid alpha-glucosidase isoform 1 (GAA1, top, SEQ pharmaceutical compositions comprising recombinant thera ID NO:11) and 2 (GAA2, bottom, SEQID NO:12). peutic lysosomal enzymes useful for treating a disease caused 0033 FIG. 5 describes the nucleotide sequence of human all or in part by the deficiency in Such lysosomal enzyme. acid alpha-glucosidase isoform 1-M (GAA1-M) (SEQ ID Such compositions may be suitable for administration by NO:13). several routes such as intrathecal, parenteral, topical, intrana 0034 FIG. 6 describes the amino sequence of human acid sal, inhalational or oral administration. Within the scope of alpha-glucosidase isoform 1-L (GAA1-L) (SEQID NO:14). this aspectare embodiments featuring nucleic acid sequences 0035 FIG. 7 describes the amino sequence of human acid encoding the full-length enzymes or fragments, variants, alpha-glucosidase isoform 4 (GAA4) (SEQID NO:15). derivatives or mutants thereof, which may be administered in 0036 FIG. 8 describes a method for purifying highly vivo into cells affected with a lysosomal enzyme deficiency. phosphorylated recombinant human acid alpha-glucosidase 0027. The lysosomal enzyme of the invention may be for (rhGAA) from a G71 transfectant (CIN11) grown on micro mulated as a pharmaceutical composition for therapeutic use. carrier beads following a three-column purification proce In a preferred embodiment, the pharmaceutical composition dure. comprises a recombinant human acid alpha-glucosidase 0037 FIG. 9 describes a method for purifying highly (rhGAA) enzyme or variant thereof, or derivative of said phosphorylated recombinant human acid alpha-glucosidase US 2009/019 1178 A1 Jul. 30, 2009

(rhGAA) from a G71 transfectant (1B8) grown in suspension resistant to toxins at elevated temperatures. The lesion in G71 following a three-column purification procedure. was not due to the uptake or mechanism of action of the two 0038 FIG. 10 describes a method for purifying highly toxins, but resulted from an inability of the clone to acidify phosphorylated recombinant human acid alpha-glucosidase endoSomes at elevated temperatures. This inability was also (rhGAA) from a G71 transfectant (F5) grown on microcarrier evident at permissive temperatures (34° C.), although to a beads following a four-column purification procedure. lesser extent. G71 cells were also found to be auxotrophic for 0039 FIG. 11 describes a method for purifying highly iron at elevated temperatures, despite normal uptake of trans phosphorylated recombinant human acid alpha-glucosidase ferrin from the medium (Timchak et al., J. Biol. Chem. 261 (rhGAA) from a G71 transfectant (C3) grown in suspension (30): 14154-14159, 1986). Since iron was released from following a four-column purification procedure. transferrin only at low pH, auxotrophy for iron despite normal 0040 FIG. 12 shows FACE analysis of recombinant transferrin uptake indicated a failure in endosomal acidifica human acid alpha-glucosidase (rhGAA) expressed by from tion. Another study demonstrated that the acidification defect G715 (G71) and 3.1.36 (DUXB11) cells. was manifested primarily in endoSomes rather than lysos 0041 FIG. 13 demonstrates binding of G71 produced omes (Stone et al., J. Biol. Chem. 262(20):9883-9886, 1987). recombinant human acid alpha-glucosidase (rhGAA) to a The data on G71 were consistent with the conclusion that a mannose 6-phosphate receptor column. mutation resulted in the destabilization of the vacuolar 0042 FIG. 14 compares the uptake of G71 recombinant ATPase responsible for endosomal acidification. Destabiliza human acid alpha-glucosidase (rhGAA) and DUX rhGAA tion was most evident at elevated temperatures (39.5°C.), but into GM244 Pompe fibroblasts. was partially expressed even at lower temperatures (34° C.). 0043 FIGS. 15A and 15B demonstrate glycogen clear A study of the trafficking of two endogenous lysosomal ance in the heart and diaphragm, respectively, in a mouse enzymes, cathepsin D and acid alpha-glucosidase, in G71 model of Pompe disease upon administration of G71 pro cells (Park et al., (1991), supra) showed that both enzymes duced recombinant human acid alpha-glucosidase (rhGAA). were quantitatively secreted at elevated temperatures, and glycosylation of the enzymes was unaffected. The Secretion DETAILED DESCRIPTION OF THE INVENTION of phosphorylated acid alpha-glucosidase was significantly enhanced at non-permissive temperatures. 0044) The present invention relates to the discovery of a 0047 Thus, the ability of G71 cells, mutant CHO cells that method that reconciles the need for large-scale manufacture are defective in endosomal acidification, to overexpress a of lysosomal enzymes with the requirement of a highly phos human lysosomal enzyme could provide a mechanism for the phorylated lysosomal enzyme product that is efficient in tar large-scale production of highly phosphorylated recombinant geting lysosomes and hence is therapeutically effective. human lysosomal enzymes. 0045. In addition to the presence of the mannose 6-phos phate marker on lysosomal enzyme oligosaccharide, lysoso mal routing of enzymes depends on the acidification of traf I. DEFINITIONS ficking endoSomes emerging from the end of the trans Golgi 0048. Unless otherwise defined, all technical and scien stack. Chemical quenching of the acidic environment within tific terms used herein have the same meaning as commonly these endosomes with diffusible basic molecules results in understood by one of ordinary skill in the art to which this disgorgement of the vesicular contents, including lysosomal invention belongs. The following references provide one of enzymes, into the extracellular milieu (Braulke et al., Eur: J. skill with a general definition of many of the terms used in this Cell Biol. 43(3):316-321, 1987). Acidification requires a spe invention: Singleton et al., DICTIONARY OF MICROBIOL cific vacuolar ATPase embedded within the membrane of the OGY AND MOLECULAR BIOLOGY (2d ed. 1994): THE endosome (Nishi et al., Nat. Rev. Mol. Cell Biol. 3(2):94-103. CAMBRIDGE DICTIONARY OF SCIENCE AND TECH 2002). Failure of this ATPase is expected to enhance the NOLOGY (Walker ed., 1988): THE GLOSSARY OF secretion of lysosomal enzymes at the expense of lysosomal GENETICS, 5THED., R. Riegeretal. (eds.), Springer Verlag routing. Manufacturing cell lines which carry defects in the (1991); and Hale & Marham, THE HARPER COLLINS vacuolar ATPase would be expected to prevent non-produc DICTIONARY OF BIOLOGY (1991). tive diversion of phosphorylated recombinant enzyme to the 0049. Each publication, patent application, patent, and intracellular lysosomal compartment. other reference cited herein is incorporated by reference in its 0046. In 1984, Chinese hamster ovary (CHO) cell mutants entirety to the extent that it is not inconsistent with the present specifically defective in endosomal acidification were gener disclosure. ated and characterized (Park et al., Somat. Cell Mol. Genet. 0050. It is noted here that as used in this specification and 17(2):137-150, 1991). CHO-K1 cells were chemically the appended claims, the singular forms “a,” “an and “the mutagenized and selected for Survival at elevated tempera include plural reference unless the context clearly dictates tures in the presence of toxins. These toxins required endo otherwise. somal acidification for the full expression of their lethality 0051. As used herein, the following terms have the mean (Marnell et al., (1984), supra). In the former study, a cocktail ings ascribed to them unless specified otherwise. of two toxins with different mechanisms of action was chosen 0.052 Allelic variant” refers to any of two or more poly to avoid selection of toxin-specific resistance. The principle is morphic forms of a gene occupying the same genetic locus. that while the probability of serendipitous mutations that Allelic variations arise naturally through mutation, and may result in resistance to one particular toxin is Small, the prob result in phenotypic polymorphism within populations. Gene ability of two simultaneous serendipitous mutations specific mutations can be silent (no change in the encoded polypep for two entirely different toxins is non-existent. Selections tide) or may encode polypeptides having altered amino acid were carried out at elevated temperature to allow for tempera sequences. Allelic variants' also refer to cDNAs derived ture-sensitive mutations. This genetic screen resulted in two from mRNA transcripts of geneticallelic variants, as well as mutants, one of which was designated G.7.1 (G71), that were the proteins encoded by them. US 2009/019 1178 A1 Jul. 30, 2009

0053 Amplification” refers to any means by which a 0066. The term “derivative' when used in reference to cell polynucleotide sequence is copied and thus expanded into a lines refers to cell lines that are descendants of the parent cell larger number of polynucleotide molecules, e.g., by reverse line; for example, this term includes cells that have been transcription, polymerase chain reaction, and ligase chain passaged or Subcloned from parent cells and retain the desired reaction. property, descendants of the parent cell line that have been 0054. A first sequence is an “antisense sequence' with mutated and selected for retention of the desired property, and respect to a second sequence if a polynucleotide whose descendants of the parent cell line which have been altered to sequence is the first sequence specifically hybridizes with a contain different expression vectors or different exogenously polynucleotide whose sequence is the second sequence. added nucleic acids. 0055 “cDNA refers to a DNA that is complementary or 0067 "Detecting refers to determining the presence, identical to an mRNA, in either single stranded or double absence, or amount of an analyte in a sample, and can include stranded form. quantifying the amount of the analyte in a sample or per cell 0056 Conventional notation is used herein to describe in a sample. polynucleotide sequences: the left-hand end of a single 0068 “Detectable moiety” or a “label” refers to a compo stranded polynucleotide sequence is the 5'-end; the left-hand sition detectable by spectroscopic, photochemical, biochemi direction of a double-stranded polynucleotide sequence is cal, immunochemical, or chemical means. For example, use referred to as the 5'-direction. The direction of 5' to 3' addition ful labels include “P, S, fluorescent dyes, electron-dense of nucleotides to nascent RNA transcripts is referred to as the reagents, enzymes (e.g., as commonly used in an ELISA), transcription direction. The DNA strand having the same biotin-streptavadin, dioxigenin, haptens and proteins for sequence as an mRNA is referred to as the “coding strand’’: which antisera or monoclonal antibodies are available, or sequences on the DNA strand having the same sequence as an nucleic acid molecules with a sequence complementary to a mRNA transcribed from that DNA and which are located 5' to target. The detectable moiety often generates a measurable the 5'-end of the RNA transcript are referred to as “upstream signal. Such as a radioactive, chromogenic, or fluorescent sequences'; sequences on the DNA strand having the same signal, that can be used to quantitate the amount of bound sequence as the RNA and which are 3' to the 3' end of the detectable moiety in a sample. The detectable moiety can be coding RNA transcript are referred to as “downstream incorporated in or attached to a primer or probe either sequences.” covalently, or through ionic, van der Waals or hydrogen 0057 "Complementary” refers to the topological compat bonds, e.g., incorporation of radioactive nucleotides, or bioti ibility or matching together of interacting surfaces of two nylated nucleotides that are recognized by streptavadin. The polynucleotides. Thus, the two molecules can be described as detectable moiety may be directly or indirectly detectable. complementary, and furthermore, the contact surface charac Indirect detection can involve the binding of a second directly teristics are complementary to each other. A first polynucle or indirectly detectable moiety to the detectable moiety. For otide is complementary to a second polynucleotide if the example, the detectable moiety can be the ligand of a binding nucleotide sequence of the first polynucleotide is identical to partner, Such as biotin, which is a binding partner for strepta the nucleotide sequence of the polynucleotide binding partner Vadin, or a nucleotide sequence, which is the binding partner of the second polynucleotide. Thus, the polynucleotide for a complementary sequence, to which it can specifically whose sequence 5'-TATAC-3' is complementary to a poly hybridize. The binding partner may itself be directly detect nucleotide whose sequence is 5'-GTATA-3'. A nucleotide able, for example, an antibody may be itself labeled with a sequence is “substantially complementary to a reference fluorescent molecule. The binding partner also may be indi nucleotide sequence if the sequence complementary to the rectly detectable, for example, a nucleic acid having a Subject nucleotide sequence is Substantially identical to the complementary nucleotide sequence can be a part of a reference nucleotide sequence. branched DNA molecule that is in turn detectable through 0058 “Conservative substitution” refers to the substitu hybridization with other labeled nucleic acid molecules. (See, tion in a polypeptide of an amino acid with a functionally e.g., Fahrlander et al., Bio/Technology 6:1165, 1988). Quan similar amino acid. The following six groups each contain titation of the signal is achieved by, e.g., Scintillation count amino acids that are conservative Substitutions for one ing, densitometry, or flow cytometry. another: 0069. “Diagnostic’ means identifying the presence or nature of apathologic condition. Diagnostic methods differin 0059) 1) Alanine (A), Serine (S), Threonine (T): their specificity and selectivity. While a particular diagnostic 0060 2) Aspartic acid (D), Glutamic acid (E): method may not provide a definitive diagnosis of a condition, 0061 3) Asparagine (N), Glutamine (Q); it suffices if the method provides a positive indication that 0062 4) Arginine (R), Lysine (K); aids in diagnosis. 0063 5) Isoleucine (1), Leucine (L), Methionine (M), 0070 The term “effective amount’ means a dosage suffi Valine (V); and cient to produce a desired result on a health condition, pathol 0064 6) Phenylalanine (F), Tyrosine (Y), Tryptophan ogy, and disease of a Subject or for a diagnostic purpose. The (W). desired result may comprise a subjective or objective 0065. The term “derivative' when used in reference to improvement in the recipient of the dosage. “Therapeutically polypeptides refers to polypeptides chemically modified by effective amount” refers to that amount of an agent effective techniques such as, for example, ubiquitination, labeling to produce the intended beneficial effect on health. (e.g., with radionuclides or various enzymes), covalent poly 0071. “Encoding refers to the inherent property of spe merattachment Such as pegylation (derivatization with poly cific sequences of nucleotides in a polynucleotide. Such as a ethylene glycol) and insertion or Substitution by chemical gene, a cDNA, or an mRNA, to serve as templates for Syn synthesis of amino acids such as ornithine, which do not thesis of other polymers and macromolecules in biological normally occur in human proteins. processes having either a defined sequence of nucleotides US 2009/019 1178 A1 Jul. 30, 2009

(i.e., rRNA, tRNA and mRNA) or a defined sequence of greater than 10 nM or the fraction of enzyme that binds a man amino acids and the biological properties resulting therefrom. 6-P receptor column is less than 30-50%. Thus, a gene encodes a protein if transcription and translation 0079 “Low level of unphosphorylated high-mannose oli of mRNA produced by that gene produces the protein in a cell gosaccharide” refers to a preparation of protein in which each or other biological system. Both the coding Strand, the nucle molecule of protein has at least one molecule of complex otide sequence of which is identical to the mRNA sequence oligosaccharide in place of a high-mannose oligosaccharide. and is usually provided in sequence listings, and non-coding Complex oligosaccharide contains galactose, acetylgluc Strand, used as the template for transcription, of a gene or samine (GlcNAc) and sialic acid, in addition to other Sugars. cDNA can be referred to as encoding the protein or other 0080 “Naturally-occurring as applied to an object refers product of that gene or cDNA. Unless otherwise specified, a to the fact that the object can be found in nature. For example, “nucleotide sequence encoding an amino acid sequence' a polypeptide or polynucleotide sequence that is present in an includes all nucleotide sequences that are degenerate versions organism (including viruses) that can be isolated from a of each other and that encode the same amino acid sequence. Source in nature and which has not been intentionally modi Nucleotide sequences that encode proteins and RNA may fied by man in the laboratory is naturally-occurring. include introns. I0081 "Pharmaceutical composition” refers to a composi 0072 “Equivalent dose' refers to a dose, which contains tion Suitable for pharmaceutical use in Subject animal, includ the same amount of active agent. ing humans and mammals. A pharmaceutical composition 0073. “Expression control sequence” refers to a nucle comprises a pharmacologically effective amount of a thera otide sequence in a polynucleotide that regulates the expres peutic lysosomal enzyme and also comprises a pharmaceuti sion (transcription and/or translation) of a nucleotide cally acceptable carrier. A pharmaceutical composition sequence operatively linked thereto. “Operatively linked' encompasses a composition comprising the active ingredient refers to a functional relationship between two parts in which (S), and the inertingredient(s) that make up the carrier, as well the activity of one part (e.g., the ability to regulate transcrip as any product which results, directly or indirectly, from tion) results in an action on the other part (e.g., transcription combination, complexation or aggregation of any two or of the sequence). Expression control sequences can include, more of the ingredients, or from dissociation of one or more for example and without limitation, sequences of promoters of the ingredients, or from other types of reactions or inter (e.g., inducible or constitutive), enhancers, transcription ter actions of one or more of the ingredients. Accordingly, the minators, a start codon (i.e., ATG), splicing signals for pharmaceutical compositions of the present invention introns, and stop codons. encompass any composition made by admixing a therapeutic 0074 "Expression vector” refers to a vector comprising a lysosomal enzyme of the present invention and a pharmaceu recombinant polynucleotide comprising expression control tically acceptable carrier. sequences operatively linked to a nucleotide sequence to be I0082 “Pharmaceutically acceptable carrier refers to any expressed. An expression vector comprises sufficient cis-act of the standard pharmaceutical carriers, buffers, and excipi ing elements for expression; other elements for expression ents, such as a phosphate buffered Saline solution, 5% aque can be supplied by the host cell or in vitro expression system. ous solution of dextrose, and emulsions, such as an oil/water Expression vectors include all those known in the art, such as or water/oil emulsion, and various types of wetting agents cosmids, plasmids (e.g., naked or contained in liposomes) and/or adjuvants. Suitable pharmaceutical carriers and for and viruses that incorporate the recombinant polynucleotide. mulations are described in Remington's Pharmaceutical Sci 0075) “Highly phosphorylated”, “high level of phospho ences, 19th Ed. (Mack Publishing Co., Easton, 1995). Pre rylation' and “high level of phosphorylated oligosaccha ferred pharmaceutical carriers depend upon the intended rides’ refers to preparations of protein in which at least 70% mode of administration of the active agent. Typical modes of of the protein binds to the cation-independent mannose administration include enteral (e.g., oral) or parenteral (e.g., 6-phosphate receptor through phosphorylated oligosaccha Subcutaneous, intramuscular, intravenous or intraperitoneal rides. Binding is further characterized by sensitivity to com injection; or topical, transdermal, or transmucosal adminis petition with mannose 6-phosphate. A highly phosphorylated tration). A "pharmaceutically acceptable salt' is a salt that can enzyme may also refer to an enzyme with at least 0.7 bis be formulated into a compound for pharmaceutical use phosphorylated oligomannose chains per mole of protein. including, e.g., metal salts (Sodium, potassium, magnesium, 0076. The terms “identical” or percent “identity,” in the calcium, etc.) and salts of ammonia or organic amines. context of two or more polynucleotide or polypeptide I0083. “Polynucleotide' refers to a polymer composed of sequences, refer to two or more sequences or Subsequences nucleotide units. Polynucleotides include naturally occurring that are the same or have a specified percentage of nucleotides nucleic acids, such as deoxyribonucleic acid (“DNA) and oramino acid residues that are the same, when compared and ribonucleic acid (“RNA) as well as nucleic acid analogs. aligned for maximum correspondence, as measured using Nucleic acid analogs include those which include non-natu one of the following sequence comparison algorithms or by rally occurring bases, nucleotides that engage in linkages visual inspection. with other nucleotides other than the naturally occurring 0077 “Linker” refers to a molecule that joins two other phosphodiester bond or which include bases attached through molecules, either covalently, or through ionic, van der Waals linkages other than phosphodiester bonds. Thus, nucleotide or hydrogen bonds, e.g., a nucleic acid molecule that hybrid analogs include, for example and without limitation, phos izes to one complementary sequence at the 5' end and to phorothioates, phosphorodithioates, phosphorotriesters, another complementary sequence at the 3' end, thus joining phosphoramidates, boranophosphates, methylphosphonates, two non-complementary sequences. chiral-methyl phosphonates, 2-O-methyl ribonucleotides, 0078 “Low level of phosphorylation' or “low phosphory peptide-nucleic acids (PNAS), and the like. Such polynucle lation” refers to a preparation of protein in which the uptake otides can be synthesized, for example, using an automated into fibroblast cells has a half maximal concentration of DNA synthesizer. The term “nucleic acid' typically refers to US 2009/019 1178 A1 Jul. 30, 2009 large polynucleotides. The term "oligonucleotide' typically non-coding function (e.g., promoter, origin of replication, refers to short polynucleotides, generally no greater than ribosome-binding site, etc.) as well. about 50 nucleotides. It will be understood that when a nucle I0089) Hybridizing specifically to” or “specific hybridiza otide sequence is represented by a DNA sequence (i.e., A, T, tion” or “selectively hybridize to’, refers to the binding, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in duplexing, or hybridizing of a nucleic acid molecule prefer which “U” replaces “T” entially to a particular nucleotide sequence under stringent 0084) “Polypeptide' refers to a polymer composed of conditions when that sequence is present in a complex mix amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs ture (e.g., total cellular) DNA or RNA. thereof linked via peptide bonds, related naturally occurring 0090 The term “stringent conditions' refers to conditions structural variants, and synthetic non-naturally occurring under which a probe will hybridize preferentially to its target analogs thereof. Synthetic polypeptides can be synthesized, Subsequence, and to a lesser extent to, or not at all to, other for example, using an automated polypeptide synthesizer. sequences. “Stringent hybridization' and “stringent hybrid The term “protein' typically refers to large polypeptides. The ization wash conditions” in the context of nucleic acid hybrid term "peptide' typically refers to short polypeptides. Con ization experiments such as Southern and Northern hybrid ventional notation is used herein to portray polypeptide izations are sequence dependent, and are different under sequences: the left-hand end of a polypeptide sequence is the different environmental parameters. An extensive guide to the amino-terminus; the right-hand end of a polypeptide hybridization of nucleic acids is found in Tijssen (1993) sequence is the carboxyl-terminus. Laboratory Techniques in Biochemistry and Molecular Biol I0085 “Primer' refers to a polynucleotide that is capable of ogy—Hybridization with Nucleic Acid Probes part I chapter specifically hybridizing to a designated polynucleotide tem 2 “Overview of principles of hybridization and the strategy of plate and providing a point of initiation for synthesis of a nucleic acid probe assays, Elsevier, New York. Generally, complementary polynucleotide. Such synthesis occurs when highly stringent hybridization and wash conditions are the polynucleotide primer is placed under conditions in which selected to be about 5°C. lower than the thermal melting point synthesis is induced, i.e., in the presence of nucleotides, a (Tm) for the specific sequence at a defined ionic strength and complementary polynucleotide template, and an agent for pH. The Tm is the temperature (under defined ionic strength polymerization Such as DNA polymerase. A primer is typi and pH) at which 50% of the target sequence hybridizes to a cally single-stranded, but may be double-stranded. Primers perfectly matched probe. Very stringent conditions are are typically deoxyribonucleic acids, but a wide variety of selected to be equal to the Tm for a particular probe. synthetic and naturally occurring primers are useful for many 0091 An example of stringent hybridization conditions applications. A primer is complementary to the template to for hybridization of complementary nucleic acids which have which it is designed to hybridize to serve as a site for the more than 100 complementary residues on a filter in a South initiation of synthesis, but need not reflect the exact sequence ern or northern blot is 50% formalin with 1 mg of heparin at of the template. In such a case, specific hybridization of the 42°C., with the hybridization being carried out overnight. An primer to the template depends on the stringency of the example of highly stringent wash conditions is 0.15 MNaCl hybridization conditions. Primers can be labeled with, e.g., at 72°C. for about 15 minutes. An example of stringent wash chromogenic, radioactive, or fluorescent moieties and used as conditions is a 0.2xSSC wash at 65° C. for 15 minutes (see, detectable moieties. Sambrook, et al. for a description of SSC buffer). Often, a I0086) “Probe,” when used in reference to a polynucle high Stringency wash is preceded by a low stringency wash to otide, refers to a polynucleotide that is capable of specifically remove background probe signal. An example medium strin hybridizing to a designated sequence of another polynucle gency wash for a duplex of e.g., more than 100 nucleotides, otide. A probe specifically hybridizes to a target complemen is 1xSSC at 45° C. for 15 minutes. An example low stringency tary polynucleotide, but need not reflect the exact comple wash for a duplex of, e.g., more than 100 nucleotides, is mentary sequence of the template. In Such a case, specific 4-6xSSC at 40°C. for 15 minutes. In general, a signal to noise hybridization of the probe to the target depends on the strin ratio of 2x (or higher) than that observed for an unrelated gency of the hybridization conditions. Probes can be labeled probe in the particular hybridization assay indicates detection with, e.g., chromogenic, radioactive, or fluorescent moieties of a specific hybridization. and used as detectable moieties. 0092. A “subject of diagnosis or treatment is a human or 0087. A "prophylactic' treatment is a treatment adminis non-human animal, including a mammal or a primate. tered to a subject who does not exhibit signs of a disease or 0093. The phrase “substantially homologous' or “sub exhibits only early signs for the purpose of decreasing the risk stantially identical” in the context of two nucleic acids or of developing pathology. The lysosomal enzymes of the polypeptides, generally refers to two or more sequences or invention may be given as a prophylactic treatment to reduce subsequences that have at least 40%, 60%, 80%, 90%, 95%, the likelihood of developing a pathology or to minimize the 96%, 97%, 98% or 99% nucleotide or amino acid residue severity of the pathology, if developed. identity, when compared and aligned for maximum corre 0088 “Recombinant polynucleotide' refers to a poly spondence, as measured using one of the following sequence nucleotide having sequences that are not naturally joined comparison algorithms or by visual inspection. Preferably, together. An amplified or assembled recombinant polynucle the Substantial identity exists over a region of the sequences otide may be included in a suitable vector, and the vector can that is at least about 50 residues in length, more preferably be used to transform a suitable host cell. A host cell that over a region of at least about 100 residues, and most prefer comprises the recombinant polynucleotide is referred to as a ably the sequences are substantially identical over at least “recombinant host cell. The gene is then expressed in the about 150 residues. In a most preferred embodiment, the recombinant host cell to produce, e.g., a “recombinant sequences are Substantially identical over the entire length of polypeptide. A recombinant polynucleotide may serve a either or both comparison biopolymers. US 2009/019 1178 A1 Jul. 30, 2009

0094 For sequence comparison, typically one sequence to as the neighborhood word score threshold (Altschul et al. acts as a reference sequence, to which test sequences are (1990), supra). These initial neighborhood word hits act as compared. When using a sequence comparison algorithm, seeds for initiating searches to find longer HSPs containing test and reference sequences are input into a computer, Sub them. The word hits are then extended in both directions sequence coordinates are designated, if necessary, and along each sequence for as far as the cumulative alignment sequence algorithm program parameters are designated. The score can be increased. Cumulative scores are calculated sequence comparison algorithm then calculates the percent using, for nucleotide sequences, the parameters M (reward sequence identity for the test sequence(s) relative to the ref score for a pair of matching residues; always >0) and N erence sequence, based on the designated program param (penalty score for mismatching residues; always <0). For eters. amino acid sequences, a scoring matrix is used to calculate 0095 Optimal alignment of sequences for comparison can the cumulative score. Extension of the word hits in each be conducted, e.g., by the local homology algorithm of Smith direction are halted when: the cumulative alignment score & Waterman, Adv. Appl Math. 2:482, 1981, by the homology falls off by the quantity X from its maximum achieved value: alignment algorithm of Needleman & Wunsch, J. Mol. Biol. the cumulative score goes to Zero or below, due to the accu 48:443, 1970, by the search for similarity method of Pearson mulation of one or more negative-scoring residue alignments; & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988, by or the end of either sequence is reached. The BLAST algo computerized implementations of these algorithms (GAP, rithm parameters W. T. and X determine the sensitivity and BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics speed of the alignment. The BLASTN program (for nucle Software Package, Genetics Computer Group, 575 Science otide sequences) uses as defaults a wordlength (W) of 11, an Dr. Madison, Wis.), or by visual inspection (see generally expectation (E) of 10, M-5, N=-4, and a comparison of both Ausubel, et al., Supra). Strands. For amino acid sequences, the BLASTP program 0096. One example of a useful algorithm is PILEUP. uses as defaults a wordlength (W) of 3, an expectation (E) of PILEUP creates a multiple sequence alignment from a group 10, and the BLOSUM62 scoring matrix (see Henikoff & of related sequences using progressive, pairwise alignments Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989). to show relationship and percent sequence identity. It also 0098. In addition to calculating percent sequence identity, plots a tree or dendogram showing the clustering relation the BLAST algorithm also performs a statistical analysis of ships used to create the alignment. PILEUP uses a simplifi the similarity between two sequences (see, e.g., Karlin & cation of the progressive alignment method of Feng & Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). Doolittle, J. Mol. Evol. 35:351-360, 1987. The method used is One measure of similarity provided by the BLAST algorithm similar to the method described by Higgins & Sharp, CABIOS is the smallest sum probability (P(N)), which provides an 5:151-153, 1989. The program can align up to 300 sequences, indication of the probability by which a match between two each of a maximum length of 5,000 nucleotides or amino nucleotide or amino acid sequences would occur by chance. acids. The multiple alignment procedure begins with the pair For example, a nucleic acid is considered similar to a refer wise alignment of the two most similar sequences, producing ence sequence if the Smallest Sum probability in a comparison a cluster of two aligned sequences. This clusteris then aligned of the test nucleic acid to the reference nucleic acid is less than to the next most related sequence or cluster of aligned about 0.1, more preferably less than about 0.01, and most sequences. Two clusters of sequences are aligned by a simple preferably less than about 0.001. extension of the pairwise alignment of two individual 0099. A further indication that two nucleic acid sequences sequences. The final alignment is achieved by a series of or polypeptides are Substantially identical is that the polypep progressive, pairwise alignments. The program is run by des tide encoded by the first nucleic acid is immunologically ignating specific sequences and their amino acid or nucle cross reactive with the polypeptide encoded by the second otide coordinates for regions of sequence comparison and by nucleic acid, as described below. Thus, a polypeptide is typi designating the program parameters. For example, a refer cally substantially identical to a second polypeptide, for ence sequence can be compared to other test sequences to example, where the two peptides differ only by conservative determine the percent sequence identity relationship using Substitutions. Another indication that two nucleic acid the following parameters: default gap weight (3.00), default sequences are substantially identical is that the two molecules gap length weight (0.10), and weighted end gaps. Another hybridize to each other under stringent conditions, as algorithm that is useful for generating multiple alignments of described herein. sequences is Clustal W (Thompson, et al. CLUSTAL W: 0100 “Substantially pure' or "isolated” means an object improving the sensitivity of progressive multiple sequence species is the predominant species present (i.e., on a molar alignment through sequence weighting, positions-specific basis, more abundant than any other individual macromo gap penalties and weight matrix choice, Nucleic Acids lecular species in the composition), and a Substantially puri Research 22:4673-4680, 1994). fied fraction is a composition wherein the object species 0097 Another example of algorithm that is suitable for comprises at least about 50% (on a molar basis) of all mac determining percent sequence identity and sequence similar romolecular species present. Generally, a Substantially pure ity is the BLAST algorithm, which is described in Altschulet composition means that about 80% to 90% or more of the al., J. Mol. Biol. 215:403-410, 1990. Software for performing macromolecular species present in the composition is the BLAST analyses is publicly available through the National purified species of interest. The object species is purified to Center for Biotechnology Information (http://www.ncbi.nlm. essential homogeneity (contaminant species cannot be nih.gov/). This algorithm involves first identifying high scor detected in the composition by conventional detection meth ing sequence pairs (HSPs) by identifying short words of ods) if the composition consists essentially of a single mac length W in the query sequence, which either match or satisfy romolecular species. Solvent species, small molecules (<500 some positive-valued threshold score T when aligned with a Daltons), stabilizers (e.g., BSA), and elemental ion species word of the same length in a database sequence. T is referred are not considered macromolecular species for purposes of US 2009/019 1178 A1 Jul. 30, 2009

this definition. In some embodiments, the conjugates of the 0107 Cells that contain and express DNA or RNA encod invention are Substantially pure or isolated. In some embodi ing the chimeric protein are referred to herein as genetically ments, the conjugates of the invention are substantially pure modified cells. Mammalian cells that contain and express or isolated with respect to the macromolecular starting mate DNA or RNA encoding the chimeric protein are referred to as rials used in their synthesis. In some embodiments, the phar genetically modified mammalian cells. Introduction of the maceutical composition of the invention comprises a Substan DNA or RNA into cells is by a known transfection method, tially purified or isolated therapeutic enzyme admixed with Such as electroporation, microinjection, microprojectile one or more pharmaceutically acceptable excipient. bombardment, calcium phosphate precipitation, modified 0101. A “therapeutic' treatment is a treatment adminis calcium phosphate precipitation, cationic lipid treatment, tered to a Subject who exhibits signs or symptoms of pathol photoporation, fusion methodologies, receptor mediated ogy for the purpose of diminishing or eliminating those signs transfer, or polybrene precipitation. Alternatively, the DNA or or symptoms. The signs or symptoms may be biochemical, RNA can be introduced by infection with a viral vector. cellular, histological, functional, subjective or objective. The Methods of production for cells, including mammalian cells, lysosmal enzymes of the invention may be given as a thera which express DNA or RNA encoding a chimeric protein are peutic treatment or for diagnosis. described in co-pending patent applications U.S. Ser. No. 0102 “Therapeutic index” refers to the dose range 08/334,797, entitled “In Vivo Protein Production and Deliv (amount and/or timing) above the minimum therapeutic ery System for Gene Therapy”, by Richard F Selden, Douglas amount and below an unacceptably toxic amount. A. Treco and Michael W. Heartlein (filed Nov. 4, 1994); U.S. Ser. No. 08/334,455, entitled “In Vivo Production and Deliv 0103 “Treatment” refers to prophylactic treatment or ery of Erythropoietin or Insulinotropin for Gene Therapy”, by therapeutic treatment or diagnostic treatment. Richard F Selden, Douglas A. Treco and Michael W. 0104. The term “unit dosage form,” as used herein, refers Heartlein (filed Nov. 4, 1994) and U.S. Ser. No. 08/231,439, to physically discrete units suitable as unitary dosages for entitled “Targeted Introduction of DNA Into Primary or Sec human and animal Subjects, each unit containing a predeter ondary Cells and Their Use for Gene Therapy”, by Douglas A. mined quantity of lysosomal enzymes of the present inven Treco, Michael W. Heartlein and Richard F Selden (filed Apr. tion calculated in an amount Sufficient to produce the desired 20, 1994). The teachings of each of these applications are effect in association with a pharmaceutically acceptable dilu expressly incorporated herein by reference in their entirety. ent, carrier or vehicle. The specifications for the novel unit 0108. In preferred embodiments, the host cell used to pro dosage forms of the present invention depend on the particu duce proteins is an endosomal acidification-deficient cell line lar lysosomal enzyme employed and the effect to be achieved, characterized by its ability to produce lysosomal enzymes in and the pharmacodynamics associated with each lysosomal amounts that enable use of the enzyme therapeutically. In enzyme in the host. preferred embodiments, the invention provides a CHO-K1 derived, END3 complementation group cell line, designated II. PRODUCTION OF LYSOSOMAL ENZYMES G71, that is capable of producing high yields of highly phos phorylated lysosomal enzymes, as specified in “DEFINI 0105. In one aspect, the present invention features a novel TIONS'', thereby enabling the large scale production of thera method of producing lysosomal enzymes in amounts that peutic lysosomal enzymes. In most preferred embodiments, enable therapeutic use of Such enzymes. In general, the the cell line expresses and secretes recombinant lysosomal method features transformation of a suitable cell line with the enzymes in amounts of at least about 0.25 picogram/cell/day, cDNA encoding for full-length lysosomal enzymes or a bio preferably at least about 0.5 picogram/cell/day, more prefer logically active fragment, variant, or mutant thereof. Those of ably at least about 1.0 picogram/cell/day, or more. skill in the art may prepare expression constructs other than those expressly described herein for optimal production of Vectors and Nucleic Acid Constructs Such lysosomal enzymes in Suitable transfected cell lines therewith. Moreover, skilled artisans may easily design frag 0109. A nucleic acid construct used to express the chi ments of cDNA encoding biologically active fragments, Vari meric protein can be one which is expressed extrachromo ants, or mutants of the naturally occurring lysosomal Somally (episomally) in the transfected mammalian cell or enzymes that possess the same or similar biological activity to one which integrates, either randomly or at a pre-selected the naturally occurring full-length enzyme. targeted site through homologous recombination, into the recipient cell's genome. A construct which is expressed extra chromosomally comprises, in addition to chimeric protein Host Cells encoding sequences, sequences Sufficient for expression of 0106 Host cells used to produce proteins are endosomal the protein in the cells and, optionally, for replication of the acidification-deficient cell lines characterized by their ability construct. It typically includes a promoter, chimeric protein to produce lysosomal enzymes in amounts that enable use of encoding DNA and a polyadenylation site. The DNA encod the enzyme therapeutically. The invention provides a CHO ing the chimeric protein is positioned in the construct in Such K1-derived, END3 complementation group cell line, desig a manner that its expression is under the control of the pro nated G71. The invention also provides G71 cell lines which moter. Optionally, the construct may contain additional com have been subcloned further or which contain different ponents such as one or more of the following: a splice site, an expression plasmids, designated G715 and G71 GAA2, enhancer sequence, a selectable marker gene under the con respectively. The invention also provides G71 cell lines which trol of an appropriate promoter, and an amplifiable marker contain different expression plasmids, designated 1B8, C3, gene under the control of an appropriate promoter. D7 and F5. The invention also provides G71 cell lines and 0110. In those embodiments in which the DNA construct derivatives thereof which have been adapted for growth in integrates into the cell's genome, it need include only the suspension, designated G71-S and derivatives thereof. chimeric protein-encoding nucleic acid sequences. Option US 2009/019 1178 A1 Jul. 30, 2009 ally, it can include a promoter and an enhancer sequence, a activity, of the lysosomal enzyme. Other preferred variants polyadenylation site or sites, a splice site or sites, nucleic acid include variants of a polypeptide of acid alpha-glucosidase sequences which encode a selectable marker or markers, which retain at least about 50%, preferably at least about nucleic acid sequences which encode an amplifiable marker 75%, more preferably at least about 90%, of the catalytic and/or DNA homologous to genomic DNA in the recipient activity of the acid alpha-glucosidase. cell, to target integration of the DNA to a selected site in the 0115 Substitutional variants typically exchange one genome (to target DNA or DNA sequences). amino acid of the wild-type for another at one or more sites within the protein, and may be designed to modulate one or Cell Culture Methods more properties of the polypeptide. Such as stability against 0111 Mammalian cells containing the chimeric protein proteolytic cleavage, without the loss of other functions or encoding DNA or RNA are cultured under conditions appro properties. Substitutions of this kind preferably are conser priate for growth of the cells and expression of the DNA or Vative, that is, one amino acid is replaced with one of similar RNA. Those cells which express the chimeric protein can be shape and charge. Conservative Substitutions are well known identified, using known methods and methods described in the art and include, for example, the changes of alanine to herein, and the chimeric protein can be isolated and purified, serine; arginine to lysine; asparagine to glutamine or histi using known methods and methods also described herein, dine; aspartate to glutamate; cysteine to serine; glutamine to either with or without amplification of chimeric protein pro asparagine; glutamate to aspartate; glycine to proline; histi duction. Identification can be carried out, for example, dine to asparagine or glutamine; isoleucine to leucine or through screening genetically modified mammaliancells that Valine; leucine to valine or isoleucine; lysine to arginine; display a phenotype indicative of the presence of DNA or methionine to leucine or isoleucine; phenylalanine to RNA encoding the chimeric protein, such as PCR screening, tyrosine, leucine or methionine; serine to threonine; threo screening by Southern blot analysis, or screening for the nine to serine; tryptophan to tyrosine; tyrosine to tryptophan expression of the chimeric protein. Selection of cells which or phenylalanine; and valine to isoleucine or leucine. contain incorporated chimeric protein-encoding DNA may 0116. One aspect of the present invention contemplates be accomplished by including a selectable marker in the DNA generating glycosylation site mutants in which the O- or construct, with Subsequent culturing of transfected or N-linked glycosylation site of the lysosomal enzyme protein infected cells containing a selectable marker gene, under has been mutated. Such mutants will yield important infor conditions appropriate for survival of only those cells that mation pertaining to the biological activity, physical structure express the selectable marker gene. Further amplification of and Substrate binding potential of the highly phosphorylated the introduced DNA construct can be affected by culturing lysosomal enzyme. In particular aspects it is contemplated genetically modified mammaliancells under appropriate con that other mutants of the highly phosphorylated lysosomal ditions (e.g., culturing genetically modified mammalian cells enzyme polypeptide may be generated that retain the biologi containing an amplifiable marker gene in the presence of a cal activity but have increased or decreased substrate binding concentration of a drug at which only cells containing mul activity. As such, mutations of the active site or catalytic tiple copies of the amplifiable marker gene can Survive). region are particularly contemplated in order to generate pro tein variants with altered substrate binding activity. In such 0112 Genetically modified mammalian cells expressing embodiments, the sequence of the highly phosphorylated the chimeric protein can be identified, as described herein, by lysosomal enzyme is compared to that of the other related detection of the expression product. For example, mamma enzymes and selected residues are specifically mutated. lian cells expressing highly phosphorylated enzymes can be 0117 Numbering the amino acids of the mature protein identified by a sandwich enzyme immunoassay. The antibod (e.g., recombinant human acid alpha-glucosidase or rhGAA) ies can be directed toward the active agent portion. from the putative amino terminus as amino acid number 1, exemplary mutations that may be useful include, for example, Variants of Lysosomal Enzymes deletion of all or some of glycosylated asparagines, including 0113. In certain embodiments, highly phosphorylated N140, N233, N390, N470, N652, N882 and N925 (Hermans lysosomal enzyme analogs and variants may be prepared and et al., Biochem. J. 289 (Pt3):681-686, 1993). Substrate bind will be useful in a variety of applications in which highly ing can be modified by mutations at D91 (the amino acid that phosphorylated lysosomal enzymes may be used. Amino acid differs between alleles GAA1 and GAA2 (Swallow et al., sequence variants of the polypeptide can be substitutional, Ann. Hum. Genet. 53 (Pt 2): 177-178, 1989). Taking into insertional or deletion variants. Deletion variants lack one or consideration Such mutations are exemplary, those of skill in more residues of the native protein which are not essential for the art will recognize that other mutations of the enzyme function or immunogenic activity. A common type of deletion sequence can be made to provide additional structural and variant is one lacking secretory signal sequences or signal functional information about this protein and its activity. sequences directing a protein to bind to a particular part of a 0118. In order to construct mutants such as those cell. Insertional mutants typically involve the addition of described above, one of skill in the art may employ well material at a non-terminal point in the polypeptide. This may known standard technologies. Specifically contemplated are include the insertion of an immunoreactive epitope or simply N-terminal deletions, C-terminal deletions, internal dele a single residue. Terminal additions, also called fusion pro tions, as well as random and point mutagenesis. teins, are discussed below. 0119 N-terminal and C-terminal deletions are forms of 0114 Variants may be substantially homologous or sub deletion mutagenesis that take advantage for example, of the stantially identical to the unmodified lysosomal enzyme as set presence of a suitable single restriction site near the end of the out above. Preferred variants are those which are variants of a C- or N-terminal region. The DNA is cleaved at the site and highly phosphorylated lysosomal enzyme polypeptide which the cut ends are degraded by nucleases such as BAL31, exo retain at least Some of the biological activity, e.g. catalytic nuclease III, DNase I, and S1 nuclease. Rejoining the two US 2009/019 1178 A1 Jul. 30, 2009 ends produces a series of DNAs with deletions of varying size 0.126 Exemplary amino acid substitutions that may be around the restriction site. Proteins expressed from such used in this context of the invention includebutare not limited mutant can be assayed for appropriate biological function, to exchanging arginine and lysine; glutamate and aspartate; e.g. enzymatic activity, using techniques standard in the art, serine and threonine; glutamine and asparagine; and valine, and described in the specification. Similar techniques may be leucine and isoleucine. Other such substitutions that take into employed for internal deletion mutants by using two Suitably account the need for retention of some or all of the biological placed restriction sites, thereby allowing a precisely defined activity whilst altering the secondary structure of the protein deletion to be made, and the ends to be religated as above. will be well known to those of skill in the art. 0120 Also contemplated are partial digestion mutants. In I0127. Another type of variant that is contemplated for the such instances, one of skill in the art would employ a “fre preparation of polypeptides according to the invention is the quent cutter that cuts the DNA in numerous places depend use of peptide mimetics. Mimetics are peptide-containing ing on the length of reaction time. Thus, by varying the molecules that mimic elements of protein secondary struc reaction conditions it will be possible to generate a series of ture. See, for example, Johnson et al., “Peptide Turn Mimet mutants of varying size, which may then be screened for ics’ in BIOTECHNOLOGY AND PHARMACY, Pezzuto et activity. al., Eds. Chapman and Hall, New York (1993). The underly 0121. A random insertional mutation may also be per ing rationale behind the use of peptide mimetics is that the formed by cutting the DNA sequence with a DNase I, for peptide backbone of proteins exists chiefly to orient amino example, and inserting a stretch of nucleotides that encode, 3. acid side chains in Such a way as to facilitate molecular 6, 9, 12 etc., amino acids and religating the end. Once Such a interactions, such as those of antibody and antigen. A peptide mutation is made the mutants can be screened for various mimetic is expected to permit molecular interactions similar activities presented by the wild-type protein. to the natural molecule. These principles may be used, in 0122) Point mutagenesis also may be employed to identify conjunction with the principles described above, to engineer with particularity which amino acid residues are important in second generation molecules having many of the natural particular activities associated with lysosomal enzyme bio properties of lysosomal enzymes, but with altered and even logical activity. Thus, one of skill in the art will be able to improved characteristics. generate single base changes in the DNA strand to result in an altered codon and a missense mutation. Modified Glycosylation 0123. The amino acids of a particular protein can be I0128 Variants of a highly phosphorylated lysosomal altered to create an equivalent, or even an improved, second enzyme can also be produced that have a modified glycosy generation molecule. Such alterations contemplate Substitu lation pattern relative to the parent polypeptide, for example, tion of a given amino acid of the protein without appreciable deleting one or more carbohydrate moieties, and/or adding loss of interactive binding capacity with structures such as, one or more glycosylation sites that are not present in the for example, antigen-binding regions of antibodies orbinding native polypeptide. sites on substrate molecules or receptors. Since it is the inter I0129 Glycosylation is typically either N-linked or active capacity and nature of a protein that defines that pro O-linked. N-linked refers to the attachment of the carbohy tein's biological functional activity, certain amino acid Sub drate moiety to the side chain of an asparagine residue. The stitutions can be made in a protein sequence, and its tripeptide sequences asparagine-X-Serine and asparagine-X- underlying DNA coding sequence, and nevertheless obtain a threonine, where X is any amino acid except proline, are the protein with like properties. Thus, various changes can be recognition sequences for enzymatic attachment of the car made in the DNA sequences of genes without appreciable bohydrate moiety to the asparagine side chain. The presence loss of their biological utility or activity, as discussed below. of either of these tripeptide sequences in a polypeptide creates 0124. In making Such changes, the hydropathic index of a potential glycosylation site. Thus, N-linked glycosylation amino acids may be considered. It is accepted that the relative sites may be added to a polypeptide by altering the amino acid hydropathic character of the amino acid contributes to the sequence Such that it contains one or more of these tripeptide secondary structure of the resultant protein, which in turn sequences. O-linked glycosylation refers to the attachment of defines the interaction of the protein with other molecules, for one of the Sugars N-aceylgalactosamine, galactose, or xylose example, enzymes, Substrates, receptors, DNA, antibodies, to a hydroxyamino acid, most commonly serine or threonine, antigens, and the like. Each amino acid has been assigned a although 5-hydroxyproline or 5-hydroxylysine may also be hydropathic index on the basis of their hydrophobicity and used. O-linked glycosylation sites may be added by inserting charge characteristics (Kyte & Doolittle, J. Mol. Biol. 157(1): or Substituting one or more serine or threonine residues to the 105-132, 1982, incorporated herein by reference). Generally, sequence of the original polypeptide. amino acids may be substituted by other amino acids that have a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a Domain Switching biological functionally equivalent protein. 0.130 Various portions of lysosomal enzyme proteins pos 0125. In addition, the substitution of like amino acids can sess a great deal of sequence homology. Mutations may be be made effectively on the basis of hydrophilicity. U.S. Pat. identified in lysosomal enzyme polypeptides which may alter No. 4,554,101, incorporated herein by reference, states that its function. These studies are potentially important for at the greatest local average hydrophilicity of a protein, as gov least two reasons. First, they provide areasonable expectation erned by the hydrophilicity of its adjacent amino acids, cor that still other homologs, allelic variants and mutants of this relates with a biological property of the protein. As such, an gene may exist in related species, such as rat, rabbit, monkey, amino acid can be substituted for another having a similar gibbon, chimp, ape, baboon, cow, pig, horse, sheep and cat. hydrophilicity value and still obtain a biologically equivalent Upon isolation of these homologs, variants and mutants, and and immunologically equivalent protein. in conjunction with other analyses, certain active or func US 2009/019 1178 A1 Jul. 30, 2009

tional domains can be identified. Second, this will provide a immunogenicity of a lysosomal enzyme fusion construct. starting point for further mutational analysis of the molecule This may be useful in the production of antibodies to the as described above. One way in which this information can be highly phosphorylated lysosomal enzyme to enable detection exploited is in “domain switching.” of the protein. In other embodiments, fusion construct can be 0131 Domain switching involves the generation of chi made which will enhance the targeting of the lysosomal meric molecules using different but related polypeptides. For enzyme-related compositions to a specific site or cell. example, by comparing the sequence of a lysosomal enzyme, 0.136. Other fusion constructs including a heterologous e.g. acid alpha-glucosidase, with that of a similar lysosomal polypeptide with desired properties, e.g., an Ig constant enzyme from another source and with mutants and allelic region to prolong serum half life or an antibody or fragment variants of these polypeptides, one can make predictions as to thereof for targeting also are contemplated. Other fusion sys the functionally significant regions of these molecules. It is tems produce polypeptide hybrids where it is desirable to possible, then, to switch related domains of these molecules excise the fusion partner from the desired polypeptide. In one in an effort to determine the criticality of these regions to embodiment, the fusion partner is linked to the recombinant enzyme function and effects in lysosomal storage disorders. highly phosphorylated lysosomal enzyme polypeptide by a These molecules may have additional value in that these peptide sequence containing a specific recognition sequence "chimeras' can be distinguished from natural molecules, for a protease. Examples of Suitable sequences are those while possibly providing the same or even enhanced function. recognized by the Tobacco Etch Virus protease (Life Tech 0132 Based on the numerous lysosomal enzymes now nologies, Gaithersburg, Md.) or Factor Xa (New England being identified, further analysis of mutations and their pre Biolabs, Beverley, Mass.). dicted effect on secondary structure will add to this under standing. It is contemplated that the mutants that Switch Derivatives domains between the lysosomal enzymes will provide useful information about the structure/function relationships of 0.137 As stated above, derivative refers to polypeptides these molecules and the polypeptides with which they inter chemically modified by Such techniques as ubiquitination, act. labeling (e.g., with radionuclides or various enzymes), cova lent polymer attachment Such as pegylation (derivatization Fusion Proteins with polyethylene glycol) and insertion or substitution by chemical synthesis of amino acids such as ornithine. Deriva 0133. In addition to the mutations described above, the tives of the lysosomal enzyme are also useful as therapeutic present invention further contemplates the generation of a agents and may be produced by the method of the invention. specialized kind of insertional variant known as a fusion I0138 Polyethylene glycol (PEG) may be attached to the protein. This molecule generally has all or a Substantial por lysosomal enzyme produced by the method of the invention tion of the native molecule, linked at the N- or C-terminus, to to provide a longer half-life in vivo. The PEG group may be of all or a portion of a second polypeptide. For example, fusions any convenient molecular weight and may be linear or typically employ leader sequences from other species to per branched. The average molecular weight of the PEG will mit the recombinant expression of a protein in a heterologous preferably range from about 2 kiloDaltons (“kDa) to about host. Another useful fusion includes the addition of a immu 100 kDa, more preferably from about 5kDa to about 50 kDa, nologically active domain, Such as an antibody epitope, to most preferably from about 5 kDa to about 10 kDa. The PEG facilitate purification of the fusion protein. Inclusion of a groups will generally be attached to the lysosomal enzymes cleavage site at or near the fusion junction will facilitate of the invention via acylation or reductive alkylation through removal of the extraneous polypeptide after purification. a reactive group on the PEG moiety (e.g., an aldehyde, amino, Other useful fusions include linking of functional domains, thiol, or ester group) to a reactive group on the inventive Such as active sites from enzymes, glycosylation domains, lysosomal enzyme (e.g., an aldehyde, amino, or ester group). cellular targeting signals or transmembrane regions. Addition of PEG moieties to polypeptide of interest can be 0134. There are various commercially available fusion carried out using techniques well-known in the art. See, e.g., protein expression systems that may be used in the present International Publication No. WO96/11953 and U.S. Pat. No. invention. Particularly useful systems include but are not 4,179,337. limited to the glutathione S-transferase (GST) system (Phar 0.139 Ligation of the enzyme polypeptide with PEG usu macia, Piscataway, N.J.), the maltose binding protein system ally takes place in aqueous phase and can be easily monitored (NEB, Beverley, Mass.), the FLAG system (IBI, New Haven, by reverse phase analytical HPLC. The PEGylated peptides Conn.), and the 6xHis system (Qiagen, Chatsworth, Calif.). can be easily purified by preparative HPLC and characterized These systems are capable of producing recombinant by analytical HPLC, amino acid analysis and laser desorption polypeptides bearing only a small number of additional mass spectrometry. amino acids, which are unlikely to affect the antigenic ability of the recombinant polypeptide. For example, both the FLAG system and the 6xHis system add only short sequences, both Labels of which are known to be poorly antigenic and which do not 0140. In some embodiments, the therapeutic enzyme is adversely affect folding of the polypeptide to its native con labeled to facilitate its detection. A "label' or a “detectable formation. Another N-terminal fusion that is contemplated to moiety' is a composition detectable by spectroscopic, pho be useful is the fusion of a Met-Lys dipeptide at the N-termi tochemical, biochemical, immunochemical, chemical, or nal region of the protein or peptides. Such a fusion may other physical means. For example, labels suitable for use in produce beneficial increases in protein expression or activity. the present invention include, radioactive labels (e.g., “P), 0135 A particularly useful fusion construct may be one in fluorophores (e.g., fluorescein), electron-dense reagents, which a highly phosphorylated lysosomal enzyme polypep enzymes (e.g., as commonly used in an ELISA), biotin, tide or fragment thereof is fused to a hapten to enhance digoxigenin, or haptens as well as proteins which can be made US 2009/019 1178 A1 Jul. 30, 2009 detectable, e.g., by incorporating a radiolabel into the hapten detecting the resulting reaction product. Colorimetric or or peptide, or used to detect antibodies specifically reactive chemiluminescent labels may be detected simply by observ with the hapten or peptide. ing the color associated with the label. Other labeling and 01.41 Examples of labels suitable for use in the present detection systems suitable for use in the methods of the invention include, but are not limited to, fluorescent dyes present invention will be readily apparent to those of skill in (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the art. Such labeled modulators and ligands can be used in the like), radiolabels (e.g., H, I, S, ''C, or 'P), enzymes the diagnosis of a disease or health condition. (e.g., horse radish peroxidase, alkaline phosphatase and oth 0145. In a preferred embodiment, the method comprises ers commonly used in an ELISA), and colorimetric labels the step of producing highly phosphorylated lysosomal Such as colloidal gold, colored glass or plastic beads (e.g., enzymes from cell lines with defects in endosomal traffick polystyrene, polypropylene, latex, etc.). ing. In a particularly preferred embodiment, the method com 0142. The label may be coupled directly or indirectly to prises the step of producing highly phosphorylated recombi the desired component of the assay according to methods well nant human acid alpha-glucosidase (rhGAA) from the CHO known in the art. Preferably, the label in one embodiment is cell line, G71. Production of lysosomal enzymes comprises covalently bound to the biopolymer using an isocyanate the steps of: (a) development of recombinant G71 expressing reagent for conjugation of an active agent according to the acid alpha-glucosidase (GAA); (b) culture of the cells; and (c) invention. In one aspect of the invention, the bifunctional Scaling up of cell line to bioreactor for production of lysoso isocyanate reagents of the invention can be used to conjugate mal enzymes. In preferred embodiments, human GAA is a label to a biopolymer to form a label biopolymer conjugate amplified from human liver mRNA (Clontech 6510-1) and without an active agent attached thereto. The label biopoly subcloned into the mammalian expression vector pCINt mer conjugate may be used as an intermediate for the synthe (BioMarin). The vector pCINt comprises the human CMV sis of a labeled conjugate according to the invention or may be enhancer-promoter, rabbit B-globin IVS2 intron, multiple used to detect the biopolymer conjugate. As indicated above, cloning site from pcDNA3.1 (+) (Invitrogen), bovine growth a wide variety of labels can be used, with the choice of label hormone poly-adenylation signal for efficient transcript ter depending on sensitivity required, ease of conjugation with mination, and selection marker neomycin phosphotrans the desired component of the assay, Stability requirements, ferase gene with a point mutation to decrease enzyme effi available instrumentation, and disposal provisions. Non-ra ciency. The attenuated marker is further handicapped with the dioactive labels are often attached by indirect means. Gener weak HSV-tkpromoter. In another preferred embodiment, the ally, a ligand molecule (e.g., biotin) is covalently bound to the human GAA cDNA is amplified and subcloned into the mam molecule. The ligand then binds to another molecules (e.g., malian expression vector pBMP14 EIN (BioMarin). streptavidin) molecule, which is either inherently detectable pBMP14 EIN is similar to vector pCINt, except that the or covalently bound to a signal system, such as a detectable human elongation factor-1C. promoter replaces the human enzyme, a fluorescent compound, or a chemiluminescent CMV enhancer-promoter. compound. 0146 For cell line development, in a first series of trans 0143. The lysosomal enzymes of the invention can also be fections, G71 was transfected with linearized expression conjugated directly to signal-generating compounds, e.g., by plasmids derived from pCINt (BioMarin) and recombinants conjugation with an enzyme or fluorophore. Enzymes Suit were selected. After a first round of subcloning of transfec able for use as labels include, but are not limited to, hydro tants, four cell lines were selected using the fluorescent sub lases, particularly phosphatases, esterases and glycosidases, strate and specifically designated. CIN cell lines were ana or oxidotases, particularly peroxidases. Fluorescent com lyzed for cell-specific productivity (pg of product/cell) in pounds, i.e., fluorophores, Suitable for use as labels include, spinners with microcarriers. Cell lines were cultured in JRH but are not limited to, fluorescein and its derivatives, Excell 302 medium supplemented with 2 mM glutamine and rhodamine and its derivatives, dansyl, umbelliferone, etc. 5% fetal calf serum, seeded onto Cytopore microcarriers and Further examples of suitable fluorophores include, but are not grown in 200 mL spinner flasks. Serum was removed by limited to, eosin, TRITC-amine, quinine, fluorescein W. acri dilution over the course of a week until BSA was undetectable dine yellow, lissamine rhodamine, B sulfonyl chloride eryth by ELISA. The best producer was identified and scaled-up to roscein, ruthenium (tris, bipyridinium), Texas Red, nicotina bioreactor for production of pre-clinical material. mide adenine dinucleotide, flavin adenine dinucleotide, etc. 0.147. In a second series of transfections, G.71 was trans Chemiluminescent compounds Suitable for use as labels fected with linearized expression plasmids derived from include, but are not limited to, luciferin and 2,3-dihydro pBMP 14 EIN and recombinants were selected. rhGAA pro phthalazinediones, e.g., luminol. For a review of various ducing G71 cell lines were selected, analyzed, cultured and labeling or signal producing systems that can be used in the scaled-up for production as described supra. G71 transfec methods of the present invention, see U.S. Pat. No. 4.391.904. tants were also adapted to Suspension as described infra. 0144. Means for detecting labels are well known to those III. PURIFICATION OF LYSOSOMAL ENZYMES of skill in the art. Thus, for example, where the label is radioactive, means for detection include a Scintillation 0.148. The invention provides methods to purify lysosomal counter or photographic film, as in autoradiography. Where enzymes, e.g., recombinant human acid alpha-glucosidase the label is a fluorescent label, it may be detected by exciting (rhGAA), produced by the methods described herein. In one the fluorochrome with the appropriate wavelength of light embodiment, the invention provides a method for purifying a and detecting the resulting fluorescence. The fluorescence lysosomal enzyme comprising the steps of: (a) dia-filtering a may be detected visually, by the use of electronic detectors cell harvest medium containing the lysosomal enzyme, (b) Such as charge coupled devices (CCDS) or photomultipliers adjusting the pH of the dia-filtered harvest to 5.5 and then to and the like. Similarly, enzymatic labels may be detected by 4.5 to induce precipitation of contaminating proteins, (c) providing the appropriate Substrates for the enzyme and re-filtering the pH adjusted dia-filtered harvest to remove US 2009/019 1178 A1 Jul. 30, 2009 precipitate; and (d) isolating the lysosomal enzyme by and the like. The Q-Sepharose resin, an anion exchange resin, sequential chromatography comprising the steps of: (i) load may be replaced with otheranion exchange resins, including ing the filtrate from step (c) onto Blue-Sepharose, washing for example and not for limitation DEAE-Sepharose, and the with 20 mM acetate/phosphate, 50 mM NaCl, pH 4.5 and like. The SE Hi-CAP resin, a cation exchange resin, may be eluting with 20 mMacetate/phosphate, 50 mMNaCl, pH 5.9; replaced with other cation exchange resins, including for (ii) loading the eluate from step (i) onto Q-Sepharose, wash example and not for limitation Capto S, Giga Cap S, SP ing with 10 mM histidine, pH 6.0, 70 mM NaCl and eluting Sepharose, and the like. The Phenyl-Sepharose resin, a with 10 mM histidine, pH 6.0, 165 mM NaCl; (iii) adjusting hydrophobic interaction chemistry resin, may be replaced the salt concentration and pH of the eluate from step (ii) to 1.3M NaCl and 5.0, respectively: (and iv) loading the salt and with other hydrophobic interaction chemistry resins, includ pH adjusted eluate from step (iii) onto Phenyl-Sepharose and ing for example and not for limitation ToyoPearl Butyl 650M, gradient eluting with 1.3M to 0.5 M NaCl. In a preferred Phenyl-Sepharose Hi-Sub, ToyoPearl PPG 600M, Fractogel embodiment, the lysosomal enzyme is recombinant human EMD, and the like. Alternatively, multi-modal resins can acid alpha-glucosidase (rhGAA). replace the anion exchange and/or hydrophobic interaction 0149. In another embodiment, the invention provides a chemistry resins, including for example and not for limitation method for purifying a lysosomal enzyme comprising the Capto MMC, Capto Adhere, PPA Hypercel, and the like. steps of: (a) ultrafiltering a cell harvest medium containing the lysosomal enzyme, (b) adjusting the pH of the ultrafiltered IV. LYSOSOMAL ENZYMES AND LYSOSOMAL harvest to 4.5 to induce precipitation of contaminating pro STORAGE DISEASES teins; (c) re-filtering the pH adjusted ultrafiltered harvest to remove precipitate; and (d) isolating the lysosomal enzyme 0151. The lysosomal enzyme is a full-length enzyme or by sequential chromatography comprising the steps of: (i) any fragment, variant, derivative or mutant thereof that still loading the filtrate from step (c) onto Blue-Sepharose, wash retains some, Substantially all, or all of the therapeutic or ing with 20 mMacetate/phosphate, 50 mM NaCl, pH 4.5 and eluting with 20 mMacetate/phosphate, 50 mMNaCl, pH 5.9; biological activity of the enzyme. In some embodiments, the (ii) loading the eluate from step (i) onto Q-Sepharose, wash enzyme is one that, if not expressed or produced, or if Sub ing with 10 mM histidine, pH 6.0, 70 mM NaCl and eluting stantially reduced in expression or production, would give with 10 mMhistidine, pH 6.0, 165 mMNaCl; (iii) loading the rise to a disease, including but not limited to, lysosomal eluate from step (ii) onto SE Hi-CAP, washing with 10 mM storage diseases. In some embodiments, the enzyme is one acetate, 1 mM phosphate, 5 mM NaCl, pH 5.0 and eluting that is not expressed or produced, or is substantially reduced with a gradient of 5-300 mM. NaCl in 10 mM acetate, 1 mM in expression or production, in a lysosomal storage disease phosphate, pH 5.0, wherein the lysosomal enzyme, i.e., acid (i.e., the reduction or absence of the enzyme is associated alpha-glucosidase, is present in the flow-through, wash and with a lysosomal storage disease). Preferably, the enzyme is early part of the salt elution gradient; (iv) adjusting the salt derived or obtained from a human. concentration of pooled fractions containing lysosomal 0152 Preferably, the enzyme is a lysosomal storage enzyme to 2 MNaCl, (v) loading the salt adjusted eluate from enzyme, such as alpha-L-iduronidase, iduronate-2-sulfatase, step (iv) onto Phenyl-Sepharose and gradient eluting with heparan N-sulfatase, alpha-N-acetylglucosaminidase, aryl 1.3M to 0.5 MNaCl; and (vi) formulating the eluate from step Sulfatase A, galactosylceramidase, acid-alpha-glucosidase, (v) in pH 5.8 buffer. In a preferred embodiment, the lysosomal thioesterase, hexosaminidase A, acid sphingomyelinase, enzyme is recombinant human acid alpha-glucosidase alpha-galactosidase, or any other lysosomal storage enzyme. (rhGAA). Table 1 lists lysosomal storage diseases and the proteins 0150. The chromatography resins listed above may be deficient therein, which are useful as a full-length enzyme or Substituted with resins belonging to the same or similar class. any fragment, variant, derivative or mutant thereof that still For example, the Blue-Sepharose resin (i.e., Blue Sepharose retains some, Substantially all, or all of the therapeutic or 6 FF) may be replaced with other Blue resins, including for biological activity of the enzyme for the treatment of a lyso example and not for limitation Capto Blue, ToyoPearl Blue, Somal storage disease.

TABLE 1 Proteins Deficient in Lysosomal Storage Diseases

Lysosomal Storage Disease Protein Deficiency Mucopolysaccharidosis ty be I L-Iduronidase Mucopolysaccharidosis ty pe II Hunter syndrome Iduronate-2-sulfatase Mucopolysaccharidosis ty pe IIIA Sanfilippo Heparan-N-sulfatase syndrome Mucopolysaccharidosis ty pe IIIB Sanfilippo C-N-Acetylglucosaminidase syndrome Mucopolysaccharidosis ty pe IIIC Sanfilippo AcetylCoA:N-acetyltransferase syndrome Mucopolysaccharidosis ty pe IIID Sanfilippo N-Acetylglucosamine 6-sulfatase syndrome Mucopolysaccharidosis ty pe IVA Morquio syndrome Galactose 6-sulfatase Mucopolysaccharidosis ty pe IVB Morquio syndrome B-Galactosidase Mucopolysaccharidosis ty be VI N-Acetylgalactosamine 4-sulfatase US 2009/019 1178 A1 Jul. 30, 2009 16

TABLE 1-continued Proteins Deficient in Lysosomal Storage Diseases Lysosomal Storage Disease Protein Deficiency Mucopolysaccharidosis type VII Sly Syndrome B-Glucuronidase Mucopolysaccharidosis type IX Hyaluronoglucosaminidase Aspartylglucosaminuria Aspartylglucosaminidase Cholesterol ester storage disease? Wolman disease Acid lipase Cystinosis Cystine transporter Danon disease Lamp-2 Fabry disease C-Galactosidase A Farber Lipogranulomatosis/Farber disease Acid ceramidase Fucosidosis C-L-Fucosidase Galactosialidosis types III Protective protein Gaucher disease types I/IIIII Gaucher disease Glucocerebrosidase (3-glucosidase) Globoid cell leukodystrophy/Krabbe disease Galactocerebrosidase Glycogen storage disease II Pompe disease C-Glucosidase GM1-Gangliosidosis types III/III B-Galactosidase GM2-Gangliosidosis type I/Tay Sachs disease 3-Hexosaminidase A GM2-Gangliosidosis type II Sandhoff disease 3-Hexosaminidase A GM2-Gangliosidosis GM2-activator deficiency C-Mannosidosis types III C-D-Mannosidase 3-Mannosidosis B-D-Mannosidase Metachromatic leukodystrophy Arylsulfatase A Metachromatic leukodystrophy Saposin B Mucolipidosis type I Sialidosis types III Neuraminidase Mucolipidosis types II/IIII-cell disease Phosphotransferase Mucolipidosis type IIIC pseudo-Hurler polydystrophy Phosphotransferase Y-subunit Multiple sulfatase deficiency Multiple sulfatases Neuronal Ceroid Lipofuscinosis, CLN1 Batten Palmitoyl protein thioesterase disease Neuronal Ceroid Lipofuscinosis, CLN2 Batten Tripeptidyl peptidase I disease Niemann-Pick disease types A/B Niemann-Pick Acid sphingomyelinase disease Niemann-Pick disease type C1 Niemann-Pick disease Cholesterol trafficking Niemann-Pick disease type C2 Niemann-Pick disease Cholesterol trafficking Pycnodysostosis Cathepsin K Schindler disease types III Schindler disease C-Galactosidase B Sialic acid storage disease Sialic acid transporter

0153 In preferred embodiments, the enzyme is a recom involving Krabbe, the preferred enzyme is galactosylcerami binant human lysosomal enzyme produced by an endosomal dase. For methods involving Pompe, the preferred enzyme is acidification-deficient cell line. In more preferred embodi acid C-glucosidase. For methods involving Neuronal Ceroid ments, the recombinant human lysosomal enzyme has a high Lipofuscinosis CLN2 Batten disease, the preferred enzyme is level of phosphorylated oligosaccharides and low level of tripeptidyl peptidase I. For methods involving Tay-Sachs, the unphosphorylated high-mannose oligosaccharides as speci preferred enzyme is hexosaminidase alpha. For methods fied under “DEFINITIONS'. In most preferred embodi involving Niemann-Pick A and B the preferred enzyme is ments, the enzyme is a highly phosphorylated recombinant acid sphingomyelinase. human acid alpha-glucosidase (rhGAA). 0154 Thus, the lysosomal storage diseases that can be V. PHARMACEUTICAL COMPOSITIONS AND treated or prevented using the methods of the present inven ADMINISTRATION tion include, but are not limited to. Mucopolysaccharidosis I 0156 The lysosomal enzymes of the invention may be (MPSI), MPS II, MPS IIIA, MPS IIIB, Metachromatic Leu administered by a variety of routes. For oral preparations, the kodystrophy (MLD), Krabbe, Pompe, Neuronal Ceroid Lipo lysosomal enzymes can be used alone or in combination with fuscinosis CLN2 Batten Disease, Tay-Sachs, Niemann-Pick appropriate additives to make tablets, powders, granules or A and B, and other lysosomal storage diseases. capsules, for example, with conventional additives, such as 0155 Thus, per the above table, for each disease the lactose, mannitol, corn starch or potato starch; with binders, enzyme would preferably comprise a specific lysosomal Such as crystalline cellulose, cellulose derivatives, acacia, enzyme deficient or reduced in expression or production in corn starch or gelatins; with disintegrators, such as corn the lysosomal storage disease. For instance, for methods starch, potato starch or sodium carboxymethylcellulose; with involving MPS I, the enzyme is C.-L-iduronidase. For meth lubricants, such as talc or magnesium Stearate; and if desired, ods involving MPS II, the preferred enzyme is iduronate-2- with diluents, buffering agents, moistening agents, preserva sulfatase. For methods involving MPS IIIA, the preferred tives and flavoring agents. enzyme is heparan N-sulfatase. For methods involving MPS 0157. The lysosomal enzymes of the invention can be IIIB, the preferred enzyme is C.-N-acetylglucosaminidase. formulated into preparations for injection by dissolving, Sus For methods involving Metachromatic Leukodystropy pending or emulsifying them in an aqueous or nonaqueous (MLD), the preferred enzyme is arylsulfatase A. For methods Solvent, such as Vegetable or other similar oils, synthetic US 2009/019 1178 A1 Jul. 30, 2009

aliphatic acid glycerides, esters of higher aliphatic acids or and intravenous), pulmonary (nasal or buccal inhalation), or propylene glycol; and if desired, with conventional additives nasal administration, although the most Suitable route in any Such as solubilizers, isotonic agents, Suspending agents, given case will depend in part on the nature and severity of the emulsifying agents, stabilizers and preservatives. conditions being treated and on the nature of the active ingre 0158. The lysosomal enzymes of the invention can be dient. Exemplary routes of administration are the oral and utilized in aerosol formulation to be administered via inhala intravenous routes. The compositions may be conveniently tion. The lysosomal enzymes of the present invention can be presented in unit dosage form and prepared by any of the formulated into pressurized acceptable propellants such as methods well-known in the art of pharmacy. dichlorodifluoromethane, propane, nitrogen and the like. 0.165. In practical use, the lysosomal enzymes according 0159 Furthermore, the lysosomal enzymes of the inven to the invention can be combined as the active ingredient in tion can be made into Suppositories by mixing with a variety intimate admixture with a pharmaceutical carrier according of bases Such as emulsifying bases or water-soluble bases. to conventional pharmaceutical compounding techniques. The lysosomal enzymes of the present invention can be The carrier may take a wide variety of forms depending on the administered rectally via a Suppository. The Suppository can form of preparation desired for administration, e.g., oral or include vehicles Such as cocoa butter, carbowaxes and poly parenteral (including intravenous). In preparing the compo ethylene glycols, which melt at body temperature, yet are sitions for oral dosage form, any of the usual pharmaceutical solidified at room temperature. media may be employed, such as, for example, water, glycols, 0160 Unit dosage forms of the lysosomal enzymes for oils, alcohols, flavoring agents, preservatives, coloring agents oral or rectal administration Such as syrups, elixirs, and Sus and the like; in the case of oral liquid preparations. Such as, for pensions may be provided wherein each dosage unit, for example, Suspensions, elixirs and Solutions; or carriers such example, teaspoonful, tablespoonful, tablet or Suppository, as starches, Sugars, microcrystalline cellulose, diluents, contains a predetermined amount of the composition contain granulating agents, lubricants, binders, disintegrating agents ing a lysosomal enzyme. Similarly, unit dosage forms for and the like; in the case of oral Solid preparations such as, for injection or intravenous administration may comprise the example, powders, hard and Soft capsules and tablets, with the lysosomal enzyme in a composition as a solution in sterile Solid oral preparations being preferred over the liquid prepa water, normal saline or another pharmaceutically acceptable rations. carrier. (0166 Because of their ease of administration, tablets and 0161 In practical use, the lysosomal enzymes of the capsules represent the most advantageous oral dosage unit invention can be combined as the active ingredient in intimate form in which case solid pharmaceutical carriers are obvi admixture with a pharmaceutical carrier according to conven ously employed. If desired, tablets may be coated by standard tional pharmaceutical compounding techniques. The carrier aqueous or non-aqueous techniques. The percentage of an may take a wide variety of forms depending on the preferable active lysosomal enzyme in these compositions may, of form of preparation desired for administration, e.g., oral or course, be varied and may conveniently be between about 2 parenteral (including intravenous). In preparing the compo percent to about 60 percent of the weight of the unit. sitions for oral dosage form, any of the usual pharmaceutical 0167. The lysosomal enzymes of the invention are useful media may be employed. Such as, for example, water, glycols, for therapeutic, prophylactic and diagnostic intervention in oils, alcohols, flavoring agents, preservatives, coloring agents animals, and particularly in humans. As described herein, the and the like in the case of oral liquid preparations, for lysosomal enzymes show preferential accumulation and/or example, Suspensions, elixirs and solutions; or carriers such release of the lysosomal enzyme in any target organ, com as starches, Sugars, microcrystalline cellulose, diluents, partment, or site depending upon the lysosomal enzyme used. granulating agents, lubricants, binders, disintegrating agents 0168 Lysosomal enzyme compositions of the present and the like in the case of oral Solid preparations, for example, invention may be administered encapsulated in or attached to powders, hard and soft capsules and tablets, with the solid viral envelopes or vesicles, or incorporated into cells. Vesicles oral preparations being preferred over the liquid preparations. are micellular particles which are usually spherical and which 0162. With respect to transdermal routes of administra are frequently lipidic. Liposomes are vesicles formed from a tion, methods for transdermal administration of drugs are bilayer membrane. Suitable vesicles include, but are not lim disclosed in Remington's Pharmaceutical Sciences, 17th Edi ited to, unilamellar vesicles and multilamellar lipid vesicles tion, (Gennaro et al., Eds. Mack Publishing Co., 1985). Der or liposomes. Such vesicles and liposomes may be made from mal or skin patches are a preferred means for transdermal a wide range of lipid or phospholipid compounds, such as delivery of the lysosomal enzymes of the invention. Patches phosphatidylcholine, phosphatidic acid, phosphatidylserine, preferably provide an absorption enhancer such as DMSO to phosphatidylethanolamine, Sphingomyelin, glycolipids, gan increase the absorption of the lysosomal enzymes. Other gliosides, etc. using standard techniques, such as those methods for transdermal drug delivery are disclosed in U.S. described in, e.g., U.S. Pat. No. 4.394,448. Such vesicles or Pat. Nos. 5,962,012, 6,261,595, and 6,261,595, each of which liposomes may be used to administer lysosomal enzymes is incorporated by reference in its entirety. intracellularly and to deliver the lysosomal enzymes to the 0163 Pharmaceutically acceptable excipients, such as target organs. Controlled release of a p97-composition of vehicles, adjuvants, carriers or diluents, are commercially interest may also be achieved using encapsulation (see, e.g., available. Moreover, pharmaceutically acceptable auxiliary U.S. Pat. No. 5,186,941). Substances, such as pH adjusting and buffering agents, tonic 0169. Any route of administration that dilutes the lysoso ity adjusting agents, stabilizers, wetting agents and the like, mal enzyme composition into the blood stream, or preferably, are also commercially available. at least outside of the blood-brain barrier, may be used. Pref 0164. In each of these aspects, the compositions include, erably, the lysosomal enzyme composition is administered but are not limited to, compositions suitable for oral, rectal, peripherally, most preferably intravenously or by cardiac topical, parenteral (including Subcutaneous, intramuscular, catheter. Intrajugular and intracarotid injections are also use US 2009/019 1178 A1 Jul. 30, 2009 ful. Lysosomal enzyme compositions may be administered e.g., symptom, associated with the pathological condition locally or regionally, Such as intraperitoneally, Subcutane being treated. Such as inflammation and pain associated there ously or intramuscularly. In one aspect, lysosomal enzyme with. As such, treatment also includes situations where the compositions are administered with a suitable pharmaceuti pathological condition, or at least symptoms associated there cal diluent or carrier. with, are completely inhibited, e.g., prevented from happen 0170 Lysosomal enzyme compositions may be adminis ing, or stopped, e.g., terminated. Such that the host no longer tered to bypass the blood-brain barrier, such as, for example Suffers from the pathological condition, or at least no longer and not for limitation, by direct intra-cranial injection, by Suffers from the symptoms that characterize the pathological transient permeabilization of the blood-brain barrier, and by condition. modification of the lysosomal enzyme to alter its tissue dis 0176 A variety of hosts or subjects are treatable according tribution. Lysosomal enzyme compositions may be adminis to the subject methods. Generally such hosts are “mammals' tered by intrathecal injection (i.e., directly into the cere or “mammalian.” where these terms are used broadly to brospinal fluid). See, e.g., Kakkis, U.S. Patent Application describe organisms which are within the class mammalia, Publication No. 2005/0048047, the disclosure of which is including the orders carnivore (e.g., dogs and cats), rodentia herein incorporated by reference in its entirety. (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, 0171 Those of skill will readily appreciate that dose levels chimpanzees, and monkeys). In many embodiments, the can vary as a function of the specific lysosomal enzyme, the hosts will be humans. severity of the symptoms and the susceptibility of the subject 0177. Having now generally described the invention, the to side effects. Preferred dosages for a given lysosomal same may be more readily understood through the following enzyme are readily determinable by those of skill in the art by reference to the following examples which provide exem a variety of means including, but not limited to, dose response plary protocols for the production, and purification of highly and pharmacokinetic assessments conducted in patients, test phosphorylated lysosomal enzymes and their use in the treat animals, and in vitro. ment of lysosomal storage diseases. The examples are offered 0172 Dosages to be administered may also depend on for illustrative purposes only, and are not intended to limit the individual needs, on the desired effect, the lysosomal enzyme scope of the present invention in any way. Efforts have been used and on the chosen route of administration. Preferred made to ensure accuracy with respect to numbers used (e.g., dosages of a lysosomal enzyme range from about 0.2 pmol/kg amounts, temperatures, etc.), but some experimental error to about 25 nmol/kg, more preferred dosages range from and deviation should, of course, be allowed for. about 2 to 2500 pmol/kg, and particularly preferred dosages range from about 2 to 250 pmol/kg; alternatively, preferred EXAMPLES doses of the lysosomal enzyme range from about 0.02 to 2000 Example I mg/kg, more preferred dosages range from about 0.2 to 200 mg/kg, and particularly preferred dosages range from 1 to 40 G71 Cell Line Development mg/kg. These dosages will be influenced by the number of lysosomal enzymes associated with the biopolymer. Alterna 0.178 The objective was to produce a recombinant, highly tively, dosages may be calculated based on the lysosomal phosphorylated lysosomal enzyme that is useful therapeuti enzyme administered. cally at low doses. A cell line was developed that provides 0173 The lysosomal enzymes of the invention are useful improved phosphorylation levels of recombinantly expressed for therapeutic, prophylactic and diagnostic intervention in lysosomal enzymes. animals, and in particular in humans. Lysosomal enzymes (0179 G71 cells (from Rockford K. Draper) were derived may show preferential accumulation in particular tissues. directly from CHO-K1 (ATCC CCL-61). Preferred medical indications for diagnostic uses include, for 0180. The adherent G71 cells were maintained at 34°C. in example, any condition associated with a target organ (e.g., BioWhittaker UltraCHO medium supplemented with 2.5% lung, liver, kidney, spleen) or target tissue (e.g., bone, muscle) fetal calf serum, 2 mM glutamine, gentamycin and amphot of interest. ericin. 0.174. The subject methods find use in the treatment of a 0181. To allow easier use of cell lines for protein produc variety of different disease conditions. In certain embodi tion, the adherent G71 cells may be pre-adapted to serum-free ments, of particular interest is the use of the subject methods growth medium using a protocol for adapting anchorage in disease conditions where a lysosomal enzyme having dependent, serum-dependent mammalian cells to high den desired activity has been previously identified, but in which sity serum-free Suspension culture (Sinacore et al., Mol. Bio the lysosomal enzyme is not adequately delivered to the target technol. 15(3):249-257, 2000), resulting in the serum-free site, area or compartment to produce a fully satisfactory suspension culture adapted cell line, G71S. Alternatively, therapeutic result. With such lysosomal enzymes, the subject adherent G71 cells, after being stably transfected as described methods of producing highly phosphorylated lysosomal infra, may be adapted to serum-free Suspension culture fol enzymes can be used to enhance the therapeutic efficacy and lowing procedures known in the art and basically as outlined therapeutic index of active lysosomal enzyme. in Sinacore et al., (2000), supra. 0175 Treatment is meant to encompass any beneficial outcome to a Subject associated with administration of a Example II lysosomal enzyme including a reduced likelihood of acquir Human Acid Alpha-Glucosidase (GAA) Expression ing a disease, prevention of a disease, slowing, stopping or Vectors reversing, the progression of a disease or an amelioration of the symptoms associated with the disease condition afflicting 0182. The objective was to produce a recombinant, human the host, where amelioration or benefit is used in abroad sense acid alpha-glucosidase (hGAA). Mammalian expression vec to refer to at least a reduction in the magnitude of a parameter, tors were generated encoding hCAA. US 2009/019 1178 A1 Jul. 30, 2009

0183 Human acid alpha-glucosidase (hGAA) cDNA was FIG. 5 (SEQ ID NO:13). The amino acid sequence of the amplified from human liver mRNA (Clontech 6510-1) by recombinant hCAA isoform GAA1 or GAA1-M, which is high-stringency PCR using the primers designated GAAF encoded by the expression vectors of the invention, is shown and GAAR (SEQ ID NO:3 and SEQID NO:4, respectively) in FIG. 4 (SEQID NO:11). (FIG. 1). 0191 The GAA1 or GAA1-M isoform was mutagenized 0184 The amplified hCAA clNA sequence was sub to a GAA1-L isoform (amino acid at position 515 changed cloned using flanking KpnI and XhoI sites into mammalian from methionine to leucine) using the Stratagene expression vector pCINt (BioMarin) (FIG. 2). This expres QuikChange kit and appropriate primers. The amino acid sion vector contained the human CMV enhancer-promoter sequence of the recombinanthCAA isoform GAA 1-L, which linked to the rabbit B-globin IVS2 intron and the multiple was inserted into the expression vectors of the invention, is cloning site from pcDNA3.1 (+) (Invitrogen, Carlsbad, depicted in FIG. 6 (SEQID NO:14). Calif.). Efficient transcript termination was ensured by the 0.192 The GAA1 or GAA1-M isoform was mutagenized bovine growth hormone poly-adenylation signal. The selec to a GAA4 isoform (amino acid at position 689 changed from tion marker was a neomycin phosphotransferase gene that glutamic acid to lysine) using the Stratagene QuikChange kit carries a point mutation to decrease enzyme efficiency. The and appropriate primers. The amino acid sequence of the attenuated marker was further handicapped with the weak recombinant hCAA isoform GAA4, which was inserted into HSV-tk promoter. The resultant rhGAA expression plasmid the expression vectors of the invention, is depicted in FIG. 7 was named pCINt-GAA. (SEQ ID NO:15). 0185. Alternatively, amplified hCAA clNA sequence 0193 Table 2 shows the amino acid residues at the indi was subcloned into the multiple cloning site of the mamma cated positions in different human acid alpha-glucosidase lian expression vector pBMP 14 EIN (BioMarin) (FIG. 2). (GAA) isoforms. This expression vector contained the human elongation fac tor-1C. promoter linked to the rabbit B-globin IVS2 intron and TABLE 2 the multiple cloning site from pcDNA3.1 (+) (Invitrogen, Carlsbad, Calif.). Efficient transcript termination was ensured Amino Acid Sequences in Human Acid by the bovine growth hormone poly-adenylation signal. The Alpha-Glucosidase (GAA) Isoforms selection marker was a neomycin phosphotransferase gene SEQID that carries a point mutation to decrease enzyme efficiency. Isoform NO: AA91 AA515 AA580 AA 689 The attenuated marker was further handicapped with the GAA1 (GAA1-M) 11 D M A. E weak HSV-tk promoter. The resultant rhGAA expression GAA1-L 14 D L A. E plasmid was named pBMP14-GAA. GAA2 12 N L P E 0186. In certain constructs, the hCAA cDNA was modi GAA4 15 D M A. K fied to replace the pre-prosequence of hCGAA (MGVRHPPC SHRLLAVCALVSLATAALLG) (SEQ ID NO:5) with the presequence of human arylsulfatase B (MGPRGAASLPRG Example III PGPRRLLLPVVPLLLLLLLAPPG) (SEQ ID NO:6). The resultant plasmid was named pBMP14-asbGAA. Development of G71 Cell Lines Expressing Recom 0187. In certain constructs, the hCAA cDNA was modi binant Human Acid Alpha-Glucosidase (GAA) fied to replace the pre-prosequence of hCGAA with the prese 0194 The objective was to obtain highly phosphorylated quence of human prolactin (MKGSLLLLLVSNLLLCQS) recombinant human acid alpha-glucosidase (rhGAA). (SEQID NO:7) followed by a portion of human arylsulfatase Expression vectors encoding rhGAA were transfected into B (GSGAGA) (SEQ ID NO:8). The nucleotide and amino G71 cells. acid sequences of this recombinant hCAA inserted into the (0195 In a first series of transfections, G71 cells were expression vector plasmids is shown in FIG. 3 (SEQID NO:9 transfected with linearized pCINt-based rhGAA expression and SEQID NO:10, respectively). The resultant plasmid was plasmids. Transfections were performed by lipofection with named pBMP14-proGAA. Cytofectene (BioRad) using manufacturer's protocols. Stably 0188 Human GAA exists in at least three different iso transfected cells were selected on UltraCHO (BioWhittaker) forms, the preponderant allele GAA1, GAA2 and GAA4. Supplemented with glutamine, gentamycin, amphotericinand There are no known functional differences between these 200 lug/mL G418 (Mediatech). Individual clones were GAA isoforms. The GAA1 isoform is identical to the human obtained by limiting dilution and screened for activity by acid alpha-glucosidase sequence in GenBank (Accession enzyme assay (see below). Following selection, stably trans Number CAA687631), whereas the GAA2 isoform has fected cells were transferred to JRH302 medium supple amino acid substitutions at positions 91 (D91N), 515 (M515 mented with glutamine, gentamycin, amphotericin and G418 L) and 580 (A580P). An amino acid sequence alignment of for expression studies. G71 cells are grown at 34°C. in 5% GAA1 (top, SEQ ID NO:11) and GAA2 (bottom, SEQ ID CO, and induced at 39°C. in 5% CO, in the presence of 3 uM NO:12) is shown in FIG. 4. FeSO. Control BHK cells are grown at 37° C. in 5% CO. (0189 The GAA2 isoform was inserted into the pCINt 0196. After a first round of subcloning of transfectants, GAA and p3MP14-GAA plasmids. The GAA2 isoform was four rhGAA positive cell lines were selected using the fluo mutagenized to the GAA1 isoform using the Stratagene rescent Substrate, 4MU-alpha-glucoside, and enzyme pro QuikChange kit and appropriate primers. duced by the cells (Reuser et al., Am. J. Hum. Genet. 30:132 0190. The nucleotide sequence of the recombinant hCAA 43, 1978). This substrate yields 4-methylumbelliferone isoform GAA1, also known as GAA1-M (amino acid (4MU) after hydrolysis, which is detectable by a characteris methionine at position 515 of hCGAA), which was inserted tic blue fluorescence when illuminated with UV-light (ap into the expression vectors of the invention, is depicted in proximately 366 mm). These positive G71 clones were desig US 2009/019 1178 A1 Jul. 30, 2009 20 nated CIN4, 5, 6 and 11. Cell-specific productivity ranged C3-S, D7-S and F5-S were scaled-up to bioreactor for pro from 1.8 and 4.6 pg cell of product. The four CIN cell lines duction of pre-clinical material and analyzed for rhGAA pro were analyzed forenzyme production in spinners with micro duction. carriers. 0.197 For comparison, dihydrofolate reductase deficient Example V CHO cells, DUXB11, overexpressing rhGAA were prepared Purification of Recombinant Human Acid Alpha by similar means. The highest producing DUXB11 clone, Glucosidase 3.1.36, was selected for further studies. 0205 The objective was to obtain a large quantity of 0198 In a second series of transfections, G71 cells were recombinant human GAA (rhGAA). Transfected G71 cells transfected with linearized p3MP14-based rhGAA expres expressing rhGAA were grown under bioreactor culture con sion plasmids. Transfections were performed in adherent G71 ditions on microcarrier beads or in Suspension and rhGAA and CHOK1 cell lines as described above. Pools of transfec enzyme was purified from the culture medium. tants were subcloned by limiting dilution cloning at 0.5, 1.0 0206 For enzymes from the first series of transfections, and 1.5 cells/well in 96-well format. The activity was deter including rhGAA from CIN4, 5, 6 and 11, or from the second mined by the 4MU alpha-glucoside hydrolysis assay for series of transfections, from 1B8, dia-filtered cell harvest approximately 300 clones. The clones with the highest activ medium was pH adjusted to 5.5, stored, adjusted to pH 4.5 ity were subsequently transferred and expanded. and stored for 4 days at 4°C. Material was then re-filtered to 0199 Twenty clones from each pool of transfectants were remove precipitate. Yield was >90% for this step. Filtrate was analyzed at the T-25 flask level for activity and cell growth. then loaded onto Blue-Sepharose (Pharmacia/Amersham), CHOK1 clones transfected with GAA1 were B6 and B7. A washed with 20 mMacetate/phosphate, 50 mMNaCl, pH 4.5 G71 clone transfected with GAA1-L was 1B8. G71 clones and eluted with 20 mMacetate/phosphate, 50 mM NaCl, pH transfected with GAA1-M were C3, D7 and F5. These clones 5.9. Yield for this step was >70%. Eluate was then loaded to were further evaluated for cell growth and production. Q-Sepharose (Pharmacia/Amersham), washed with 10 mM histidine, pH 6.0, 70 mM. NaCl and eluted with 10 mM Example IV histidine, pH 6.0, 165 mM NaCl. Yield for this step was >50%. Eluate was salt and pH adjusted to 1.3M NaCl and 5.0, Culture of Recombinant Human Acid Alpha-Glu respectively, loaded to Phenyl-Sepharose (Pharmacia Amer cosidase Expressing G71 Cells sham) and gradient eluted with 1.3M to 0.5 M NaCl. Final purity of the rhGAA from a G71 transfectant grown on micro 0200. The objective was to measure recombinant human carrier beads (CIN11) was greater than 98% as assessed by acid alpha-glucosidase (rhGAA) production from the G71 SDS-PAGE followed by Coomassie stain, silver stain and transfectants. Cell lines were further assessed for rhGAA Western blot (FIG. 8). Final purity of the rhGAA from a G71 enzyme production in cell culture. transfectant grown in Suspension (e.g., 1B8) was at least 95% 0201 G71 transfected cell lines were cultured in JRH as assessed by SDS-PAGE followed by Coomassie stain and Excell 302 medium supplemented with 2 mM glutamine and Western blot (FIG. 9). Similar results were obtained for 5% fetal calf serum. Cells were seeded onto Cytopore micro rhGAA from other G71 or G71-S transfectants using this carriers (Pharmacia/Amersham) and grown in 200 mL spin three-column purification procedure. ner flasks. Serum was removed by dilution over the course of 0207 Forenzymes from the second series of transfections, a week until BSA was undetectable by ELISA. The four CIN including 1B8, C3, D7, F5 and F6, ultrafiltered cell harvest lines from the first series of transfections were analyzed for medium was pH adjusted to pH 4.5 and stored at 4°C. Mate rhGAA production. CIN11 titer was the best producer at rial was then re-filtered to remove precipitate. Yield was approximately 4 mg/L/day. DUXB11 3.1.36 titer was >90% for this step. Filtrate was then loaded onto Blue approximately 1 mg/L/day. Sepharose, washed with 20 mM acetate/phosphate, 50 mM NaCl, pH 4.5 and eluted with 20 mM acetate/phosphate, 50 0202 The best candidate from this screen, CIN11 (also mM NaCl, pH 5.9. Yield for this step was >70%. Eluate was known as G71 GAA2) was scaled-up to bioreactor for pro then loaded onto Q-Sepharose, washed with 10 mM histidine, duction of pre-clinical material and analyzed for rhGAA pro pH 6.0, 70 mM. NaCl and eluted with 10 mM histidine, pH duction. 6.0, 165 mM NaCl. Yield for this step was >50%. Eluate was 0203 The GAA1-expressing G71 and CHOK1 cell lines then loaded onto SE Hi-CAP washed with 10 mMacetate, 1 from the second series of transfections were cultured as mM phosphate, 5 mM NaCl, pH 5.0 and eluted with a gradi described above and analyzed for rhGAA production. Clones ent of 5-300 mM NaCl in 10 mM acetate, 1 mM phosphate, 1B8, B6, B7, C3, D7 and F5 were scaled-up to bioreactor for pH 5.0. Yield for this step was -85%. The lysosomal enzyme, production of pre-clinical material and analyzed for rhGAA i.e., acid-alpha-glucosidase, was present in the flow-through, production. wash and early part of the salt elution gradient. Fractions 0204 The GAA1-expressing G71 cell lines were adapted containing lysosomal enzyme were pooled, adjusted to 2 M for growth in suspension. Cells were suspended in JRH NaCl and then loaded onto Phenyl-Sepharose and gradient Excell 302 medium containing 5% FBS, and then sequen eluted with 1.3M to 0.5MNaCl. Yield for this step was -80%. tially through 2.5%, 1% or no FBS in the presence of G418, Eluate was formulated in pH 5.8 buffer. Final purity of the followed by removal of G418. The G71 suspension clones rhGAA from a G71 transfectant grown on microcarrier beads were adapted to growth in CD-CHO medium (Invitrogen) by (e.g., F5) at least 95% as assessed by SDS-PAGE followed by resuspending cells in a 50:50 mixture of JRH Excell 302:CD Coomassie stain, silver stain and Western blot (FIG. 10). CHO medium, and after two high viability passages, cells Final purity of the rhGAA from a G71 transfectant grown in were resuspended into 100% CD-CHO medium. Clones suspension (e.g., C3) at least 95% as assessed by SDS-PAGE US 2009/019 1178 A1 Jul. 30, 2009

followed by Coomassie stain, silver stain and Western blot 0214. The level of mannose 6-phosphorylation on lysoso (FIG. 11). Similar results were obtained for rhGAA from mal enzymes from G71 cells expressing rhGAA was also other G71 or G71-S transfectants using this four-column determined by N-linked carbohydrate profiling by capillary purification procedure. electrophoresis as described in Ma et al., Anal. Chem. 71 (22): 0208. These assays indicate that the protocols described 5185-51.92, 1999. The method used PNGase F to cleave above for making recombinant lysosomal enzyme provide N-linked oligosaccharides. The cleaved oligosaccharides efficient methods for production of large quantities of highly were isolated and derivatized with fluorescent dye, and purified enzyme, in particular, rhGAA. applied to a G10 spin column to remove excess dye. The purified, fluorescently labeled oligosaccharides were sepa Example VI rated electrophoretically and peaks Subsequently quantified Analysis of Recombinant Human Acid Alpha-Glu using the MDQ-CE software (32 Karat Ver, 7.0). 0215 Using this method, between 15% and 18% of the cosidase total oligosaccharides contained bis-phosphorylated man 0209. The G71 cell line produces lysosomal enzymes with nose7 (BispMan7) in rhGAA derived from G71 derivatives, greater levels of high mannose phosphorylation than is noted including C3 grown in suspension in JRH-302 or CD-CHO in an average mammalian cell line, and a low level of unphos medium, and F5 grown on microcarrier culture. This percent phorylated high-mannose oligosaccharides. A molecule com age of BispMan7 oligosaccharides, assuming that all of the 7 prising a low level of unphosphorylated high-mannose oli N-linked glycosylation sites were glycosylated, corre gosaccharides, as defined herein, is compared to molecules sponded to between 1.05 to 1.26 moles BispMan7 per mol of obtained in U.S. Pat. No. 6,537,785 (Canfield), which do not enzyme. The results are shown in Table 3. comprise complex oligosaccharides, and exhibit only high mannose oligosaccharides. TABLE 3 0210. To determine levels of unphosphorylated high-man nose, one of skill in the art can use exoglycosidase sequencing Characterization of rhGAA Produced in G71 Cells of released oligosaccharides ("FACE sequencing), to pin Sp. Sialic Kuptake point the percentages of unphosphorylated high-mannose oli G71 Titer Activity BispMan7 Acid GM248 gosaccharide chains. On a normal lot-release FACE profiling Clone Media (mg/mL) (U/mg) 9% by CE (Dionex)" (nM) gel, unphosphorylated high mannose co-migrates with par B7 JRH- 2.7 6.1 14 9.8 1.6 ticular complex oligosaccharides (for example, oligoman 302 nose 6 and fully sialylated biantennary complex). Unphos C3-S CD- 8.0 5.2 17 5.5 2.1 CHO phorylated high mannose is then differentiated from the other C3-S JRH- 1O.O 8.0 18 5.9 1.9 oligosaccharides by enzymatic sequencing. 3O2 0211 To determine if purified, recombinant human acid FS JRH- 1S.O 7.2 15 3.5 2.2 alpha-glucosidase (rhGAA) exhibits increased phosphoryla 3O2 tion, the level of mannose-6-phosphate on the protein and *S signifies G71 transfectant adapted to suspension culture. enzyme binding to the mannose 6-phosphate receptor were "Data is shown in mol of sialic acid/mol of protein using a Dionex instru determined. ment. 0212 Purified, recombinant enzyme from the two trans fected cell lines, G71 CIN11 and DUXB11, was analyzed by 0216. As shown in Table 3, the rhGAA from the G71 fluorescence assisted carbohydrate electrophoresis (FACE) transfectant C3-S, which was adapted to growth in Suspen and by chromatography on MPR-Sepharose resin. The rela sion in JRH-302 medium, was produced at a higher titer, with tive intensity of the oligomannose 7 bis-phosphate (O7P) higher specific activity, higher level of phosphorylation, and band on FACE (Hague et al., Electrophoresis 19(15):2612 lower level of sialic acid content than the rhGAA from the 2620, 1998) and the percent activity retained on the MPR CHOK1 transfectant B7. column (Cacia et al., Biochemistry 37(43): 15154-15161, 0217. The ability of rhGAA from G71 cells in the second 1998) gave reliable measures of phosphorylation level per series of transfections to bind to the MPR was determined. mole of protein. A FACE comparison of material prepared The soluble extracellular domain of the bovine cation inde from the G71 and DUXB11 lines showed that approximately pendent mannose-6-phosphate receptor (CI-MPR) was 19.6% of the total G71 rhGAA oligosaccharide is O7P while coupled to an Affigel-15 column using N-hydroxysuccinim only 6.7% of DUXB11 rhGAA is O7P (FIG. 12). This assay ide chemistry. Samples of rhGAA (6 ug) were bound to the also demonstrated that approximately 75% of total binding CI-MPR column in a buffer of PBS pH 6.0/5 mM B-glycero activity to mannose 6 phosphate receptor column was attrib phosphate/0.05% Tween-20/5 mM glucose-1-phosphate/0. uted to G71 rhGAA (FIG. 12). Relative retention of enzyme 02% sodium azide. Bound enzyme was subsequently eluted analyzed by MPR column also demonstrated that approxi using a linear gradient of 0.5 mM mannose-6-phosphate in mately 75% of rhGAA bound to the receptor whereas binding the same buffer without glucose-1-phosphate. Fractions were of control protein was negligible (FIG. 13). collected and assayed for GAA enzyme activity using the 0213. These results showed that the levels of mannose 4MU-alpha-glucoside assay or by an ELISA procedure. 6-phosphorylation was approximately 3-times higher in Using this assay, approximately 70% of rhGAA from the G71 rhGAA enzyme produced by G71 cells than other CHO cell transfectant F5 was able to bind to the immobilized MPR. lines. Thus, G71 cells transfected with lysosomal enzyme Similar data was obtained for other rhGAA enzymes from efficiently produce highly phosphorylated enzyme, leading to G71 cells in the second series of transfections. an increased level of high mannose residues on these 0218. The percentage of rhGAA from the G71 transfectant enzymes, which may lead to increased uptake by MPR on F5 that was able to bind to the immobilized MPR in this assay, cells. 70%, compared favorably to the 30%-40% of rhGAA pro US 2009/019 1178 A1 Jul. 30, 2009 22 duced in CHO cells that was able to bind to a similar CI-MPR 0226 Fibroblasts from patients symptomatic of a glyco column, as reported in Zhu et al., J. Biol. Chem. 279:50336 gen storage disorder are seeded and grown to confluence in 50341, 2004 and Zhu et al., Biochem. J. 389:619-628, 2005. 12-well plates. On the day of the experiment, cells are fed 0219. In summary, rhGAA has been produced in G71 cells with fresh medium containing 4 mM glucose. Cells are also with a high titer and having a high specific activity, high level supplemented with rhGAA in the presence or absence of 10 of phosphorylation and ability to bind to the MPR in vitro. mM mannose 6-phosphate. Cells are harvested each day for 4 days. After rinsing with PBS, cells are lysed by freeze-thaw. Example VII Stored glycogen is assayed by boiling the lysate, precipitation Uptake of Recombinant Human Acid Alpha-Glucosi with 80% ethanol, digestion with Aspergillis niger glucosi dase into Pompe Fibroblasts dase and glucose assay (Van Hove et al., Proc. Natl. Acad. Sci. USA 93:65-70, 1996). Stored glycogen values are normalized 0220. The objective was to determine if the purified to the protein content of the cell lysates. recombinant human acid alpha-glucosidase (rhGAA) protein 0227. It is expected that cells receiving G71 rhGAA clear binds efficiently to the MPR on cells. Cells obtained from stored glycogen more efficiently than cells which are treated patients with the lysosomal storage disorder Pompe disease with enzyme produced by other recombinant methods or were assessed for their ability to bind recombinant, highly control protein. Ability of G71 rhGAA treated cells to clear phosphorylated rhGAA. glycogen at levels comparable to cells from normal donors 0221) The uptake of rhGAA enzyme from the first series of indicates that the G71 produced lysosomal enzyme is as G71 transfections was investigated in GM244 Pompe patient effective as native rhGAA enzyme in relieving symptoms of fibroblasts. Cells were seeded and grown to confluence in Pompe disease. 12-well plates. On the day of the experiment, cells were fed with fresh medium containing 4 mM glucose and varying Example IX concentrations of either G71 rhGAA or DUXB11 rhGAA. Cells were incubated for 4 hours, rinsed with PBS and lysed Effect of Recombinant Human Acid-Alpha-Glucosi by freeze-thaw. GAA enzyme activity was then measured dase in a Mouse Model of Pompe Disease using 4MU-alpha-glucoside using published methods. The 0228. The effects of the highly phosphorylated human 4MU-alpha-glucoside assay demonstrated that the rate of lysosomal enzymes of the invention, e.g., recombinant enzyme uptake (K) for DUXB11 rhGAA was 2.95 nM human acid alpha-glucosidase (rhGAA), in mice deficient in and the K for G71 rhGAA was 1.31 nM (approximately GAA (i.e., mouse model of Pompe disease) were studied. 2.25 times more efficient that the DXB11 rhGAA) (FIG. 14). 0229. The rhGAA enzyme from G71 clone C3 was 0222. This result demonstrated that phosphorylated high expressed in G71 cells, purified, and administered into GAA mannose oligosaccharide on the G71-derived acid alpha-glu deficient (Pompe) mice. cosidase bound to the MPR with an affinity similar to that 0230. At least three mouse models of GAA deficiency seen for other properly phosphorylated lysosomal enzymes (i.e., Pompe disease) have been described (Raben et al., J. (Sando et al., Cell 12:619-27, 1977). This affinity for the MPR Biol. Chem. 273(30): 19086-92, 1998: Bijvoet et al., Hum. exceeded that for acid alpha-glucosidase made in DUXB11. Mol. Genet. 7(1):53-62, 1998). The 6"/6' mice, which 0223) The uptake of rhGAA enzyme from the second have markedly reduced mobility and strength by 3.5 weeks of series of G71 transfections, either from clone F5 or C3-S, was age, recapitulate the critical features of the infantile and adult investigated in confluent GM248 Pompe fibroblasts. Cells forms of Pompe disease, whereas the A6/A6 and Gaa(-/-) were exposed to a dilution series of each enzyme in serum mice, despite displaying similar biochemical and pathologi free uptake medium (DMEM/25 mH HEPES, pH 7.5/2 mM cal features as the 6"/6' mice, have unimpaired strength glutamine/0.5 mg/mL BSA) for 4 hours. After exposure, cells and mobility up to 6.5 months of age. were washed with PBS and released from the plate with 0231. The effect of administration of rhGAA into 6"/ trypsin/EDTA digestion. Cells were collected, washed with 6" mice, also referred to as GAA"' mice, on tissue PBS and lysed by freezing overnight in phosphate-citrate glycogen clearance was investigated. GAA"*" mice or buffer pH4.0/0.1% Triton-X100. Crude extracts were thawed wild-type mice of the background strain of about three and centrifuged to separate the soluble and insoluble phases, months of age were administered vehicle or various doses of and then the soluble lysate was analyzed for enzyme activity rhGAA by intravenous infusions in the tail vein over 30 min using the 4MU-alpha-glucoside Substrate assay. twice weekly for two weeks. Mice were sacrificed two days 0224. As shown in Table 3 above, this assay demonstrated after the last injection. Details of this study are shown in Table that the rate of enzyme uptake (K.) for rhGAA from G71 4. cells was 1.9-2.2 nM. These results suggested that the highly phosphorylated rhGAA produced in G71 cells bound to the TABLE 4 MPR on cells with high affinity and was taken up into the cells in an active form. Design of Study to Determine Effect of rhGAA in Mouse Model of Pompe Disease Example VIII No. Adminis- Dose Measurement of Specific Uptake of Recombinant Group Animals Strain tered (mg/kg) Route Frequency Human Acid-Alpha-Glucosidase into Enzyme-Defi 1 10 Wild-type Vehicle O IV 2x week, cient Patient Fibroblasts with Concomitant Clear 2 wks ance of Stored Glycogen 2 10 GAAt Rob Vehicle O IV 2x week, 2 wks 0225. The ability of the recombinant human acid alpha 3 10 GAA 6' rhGAA 5 IV 2x week, glucosidase (rhGAA) enzyme to clear glycogen stored in 2 wks cells is assessed. US 2009/019 1178 A1 Jul. 30, 2009 23

determine the percent area occupied by PAS stained glycogen TABLE 4-continued in three fields for each tissue. The results for heart and dia phragm, comparing vehicle (left panels) and 20 mg/kg Design of Study to Determine Effect of rhGAA (right panels) are shown in FIGS. 15A and 15B, rhGAA in Mouse Model of Pompe Disease respectively. No. Adminis- Dose 0238. The results showed that rhGAA was able to signifi Group Animals Strain tered (mg/kg) Route Frequency cantly reduce the glycogen storage that accumulated in the 4 10 GAAt Rabi' rhGAA 10 IV 2x week, heart and diaphragm in Pompe mice. 2 wks 0239 Quantitative measurement of glycogen in tissue 5 10 GAA or rhGAA 2O IV 2x week, homogenates. A quantitative glycogen determination method 2 wks was used with a limit of detection of 0.02 mg glycogen/g tissue. Tissue was homogenized in 0.2 M sodium acetate, 0232. The following tissues were collected and analyzed 0.5% NP-40 (pH 4.8) using the FastPrep homogenizer (MP for rhGAA activity and for glycogen clearance: heart (left Bio). Glycogen was detected by using two enzymatic steps as Ventricle); diaphragm; and skeletal muscle (quadriceps, tri described in Zhu et al., J. Biol. Chem. 279:50336-50341, ceps, psoas. Soleus, plantaris). 2004, and reported as mg glycogen/g tissue. The results are 0233 Sera was collected from the mice at day 0 prior to the shown in Table 6 and expressed as glycogen levels in indi first injection, at day 7 before the third injection, and two days cated tissues relative to those in the same tissues in after the last injection when the mice were sacrificed. GAA"''''' mice treated with vehicle. 0234 Quantitative GAA enzyme activity assay in tissue homogenates. A method was used to measure GAA enzyme TABLE 6 activity in Pompe mouse tissue with a limit of detection of 0.2 mu GAA/g tissue. Tissue was homogenized in 0.2 M sodium rhGAA Reduces Tissue Glycogen in phosphate, 0.1 M citrate (pH 4.3) using the FastPrep homog Mouse Model of Pompe Disease" enizer (MP Bio). GAA was captured on a plate coated with Normal 5 mg/kg 10 mg/kg 20 mg/kg anti-GAA as described in Umapathysivam et al., Clin. Chem. Tissue Mice rhGAA rhGAA rhGAA 47:1378-1383, 2001. Enzyme activity was measured using a Heart 21** 51 14** 3113** 14 7:8: fluorometric assay as described in Kallwasset al., Mol. Genet. Diaphragm 64** 87 - 19 72 14 51 - 12:8: Metab. 90:449-452, 2007, and reported as mu GAA activity Quadriceps 7 2** 106 16 10016 78 12** Triceps 53** 86 16* 90 - 15 74.13** per gram of tissue. The results are shown in Table 5 and Psoas 83** 92.13 91 - 24 71 - 13:8: expressed as GAA activity in indicated tissues relative to Plantaris 143** 91 - 18 82 15* 76. 12.** those in the same tissues in normal mice treated with vehicle. Soleus 10+ 4** 79 16** 82 18* 54 - 15**

TABLE 5 "Glycogen levels in GAA"'mice treated with vehicle were taken as Tissue rhGAA Enzyme Activity in Mouse Model of Pompe Disease Normal 5 mg/kg 10 mg/kg 20 mg/kg Pompe 0240. The results showed that rhGAA was able, in a dose Tissue Miceti rhGAA rhGAA rhGAA Mice dependent fashion, to reduce glycogen accumulation in all of the tissues examined, with the most striking effect being Diaphragm 1OO 15 114 - 77 165 75 386- 145 6 - O Psoas 1OO 11 63 35 175 102 251 86 8 - O observed in the heart, as expected (Zhu et al., Biochem. J. Quadriceps 1OO 6 36 - 18 12677 1668O 7 - O 389:619-628, 2005). Thus, rhGAA produced in G71 cells was Triceps 1OO 9 42 - 18 139 65 218 106 7 - O able to exert beneficial effects in vivo in a mouse model of Pompe disease. "GAA activity in normal mice treated with vehicle was taken as 100. 0241. Detection of antibodies to GAA in sera of Pompe 0235. The results showed a dose-dependent increase in mice after injection with rhGAA. A qualitative electrochemi GAA enzyme activity in Pompe mouse tissues upon admin luminescent assay (ECLA) was used to detect antibodies istration of rhGAA. The levels of rhGAA enzyme activity against rhGAA in mouse serum (White et al., U.S. application achieved were greater than the levels of endogenous GAA Ser. No. 1 1/724,983, filed Mar. 16, 2007). To determine the enzyme activity in Pompe and normal mice. titer, serial 3-fold dilutions were prepared and the highest 0236 Light microscopic analysis of tissue glycogen. High dilution factor yielding a positive result obtained (three times resolution light microscopy method was used to quantitate the serum dilution). The logo of this dilution factor was glycogen content in tissue as described in Lynch et al., J. reported as the titer. The lowest reported titer was 1.5, which Histochem. Cytochem. 53(1): 63-73, 2005. Tissues were cut corresponded to logo (30). Antibodies against rhGAA into 1 mm pieces immediately after collection, immersion present in mouse serum were measured at three timepoints: fixed in 3% gluteraldehyde in 0.2 M cacodylic buffer, pH 7.3, predose (day 0), prior to the third dose (day 7), and at post-fixed in 1% osmium tetroxide in 0.2M cacodylate buffer, necropsy (day 13). pH 7.3, and then embedded in Epon-Araldite. Polymerized 0242 All predose serum samples were below the limit of blocks were cut to 1 um thick sections and mounted on detection, and only three of the day 7 serum samples gener charged slides. Slides were stained for Epon-Araldite, and ated a positive anti-GAA antibody titer. Less than half the then dried completely in a 60°C. oven and coverslipped with mice had a detectable serum anti-GAA antibody titer at Acrytol without the use of xylene. necropsy (day 13). 0237 Stained sections were evaluated and photographed 0243 A second study was performed to further investigate at the 40x magnification. ImagePro Software was used to the effect of administration of rhGAA on tissue glycogen US 2009/019 1178 A1 Jul. 30, 2009 24 clearance and on anti-rhGAA antibody formation in 0247 This second study showed that rhGAA caused a GAA"' mice. As in the first study, the rhGAA was dose-dependent reduction in glycogen accumulation in all expressed from the G71 clone C3 in G71 cells as described tissues examined, with the most striking effect in the heart, above. GAA"' mice or wild-type mice of the back consistent with the first study. ground strain (normal) of about five months of age were 0248. It was also apparent that glycogen clearance in this administered vehicle or various doses of rhGAA by bolus second (five week) study was improved compared to the first injection in the tail vein weekly for four weeks. Mice were (three week) study. Thus, rhGAA produced in G71 cells is sacrificed one week after the last injection. This study was able to exert beneficial effects in vivo in a mouse model of modeled after the studies described in Zhu et al., J. Biol. Pompe disease. 0249 Detection of antibodies to GAA in sera of Pompe Chem. 279:50336-50341, 2004 and Zhu et al., Biochem. J. mice after injection with rhGAA. The qualitative ECLA 389:619–628, 2005, the disclosures of which are herein incor described above was used to detectantibodies against rhGAA porated by reference in their entirety. Details of this study are in mouse serum. The highest dilution factor that provided a shown in Table 7. positive result in the assay was the reported value (logo reported as titer). TABLE 7 0250) No pre-dose serum samples were positive, whereas Design of Study to Determine Effect of most serum samples obtained post-dose showed a detectable rhGAA in Mouse Model of Pompe Disease anti-GAA antibody titer. The median anti-GAA titer No. Adminis- Dose increased for all treatment groups (5, 10 and 20 mg/kg Group Animals Strain tered (mg/kg) Route Frequency rhGAA) between day 14 and day 29. This increased anti GAA titer, however, was not rhGAA dose-dependent, and no 1 10 GAA or rhGAA 5 Bolus 1x week, correlation was observed between the anti-GAA titers and 4 wks 2 10 GAAt Rabi' rhGAA 10 Bolus 1x week, pharmacodynamic activity (i.e., glycogen clearance). These 4 wks findings showed that rhGAA induced an antibody response in 3 10 GAA or rhGAA 20 Bolus 1x week, Pompe mice, but that the presence of anti-GAA antibodies 4 wks did not preclude efficacy in this model of Pompe disease. 4 10 GAAt Rabi' Vehicle — Bolus 1Xiweek, 4 wks 5 10 Normal Vehicle — Bolus 1Xiweek, Example X 4 wks Treatment of Patients with Pompe Disease with Recombinant Human Acid-Alpha-Glucosidase 0244. The following tissues were collected and analyzed 0251 Human patients manifesting a clinical phenotype of for rhGAA activity and for glycogen clearance: heart (left lysosomal enzyme deficiency. Such as in patients with Pompe Ventricle); diaphragm; and skeletal muscle (quadriceps, tri Disease with an acid alpha-glucosidase (GAA) level of less ceps, psoas. Soleus, plantaris). than 1% of normal in leukocytes and fibroblasts are contem 0245 Sera was collected from the mice at day 0 prior to the plated for enzyme replacement therapy with the recombinant first injection, at day 7 before the third injection, at day 14 human acid alpha-glucosidase (rhGAA) enzyme produced before the fourth injection, and at day 29, after the last injec according to the methods described herein. All Pompe Dis tion when the mice were sacrificed. ease patients manifest some clinical evidence of muscular 0246 Quantitative measurement of glycogen in tissue accumulation of glycogen with varying degrees of functional homogenates. The quantitative glycogen determination impairment. Efficacy is evaluated by measuring enhance method described above was used to measure the amount of ments or stabilization in cardiac, pulmonary and motor func glycogen per gram of tissue. The results are shown in Table 8 tion and/or determining the percentage of patients who die and expressed as glycogen levels in indicated tissues relative during the study or require ventilatory Support. Assessment of to those in the same tissues in GAA"'mice treated with liversize is also performed as this is the most widely accepted vehicle. means for evaluating successful ERT in Pompe disease (Hoogerbrugge et al., Lancet 345:1398, 1995). TABLE 8 0252. A randomized, double-blinded clinical trial of puri fied, highly phosphorylated rhGAA produced according to rhGAA Reduces Tissue Glycogen in the methods described herein is conducted in patients with Mouse Model of Pompe Disease" Pompe Disease, in particular the infantile-onset form of the Normal 5 mg/kg 10 mg/kg 20 mg/kg disease, but may also be conducted in patients with the juve Tissue Mice rhGAA rhGAA rhGAA nile- and late-onset forms of the disease. Heart 11 ** 4010** 32 14** 10. 5** 0253 A pharmaceutical composition consisting of an Diaphragm 4 + 2** 101 - 23 74.13** 42 + 15** enzyme composition comprising rhGAA is administered Quadriceps 21** 42 21** 45 15** 31, 12.** intravenously. The final dosage form of the fluid comprises Triceps 54** 91 68 9236 41 14** Psoas 5 2** 84 - 15 94 - 18 57 25** rhGAA, normal saline, phosphate buffer at pH 5.8 and human Plantaris 93** 8222* 77 - 12.** 46 20** albumin at 1 mg/ml, prepared in a bag of normal saline. A Soleus 53** 45 16** 54 - 21** 21, 12.** preferred composition comprises rhGAA in an amount rang ing from 0.05-0.5 mg/mL or 12,500-50,000 units per mL: "Glycogen levels in GAA"' mice treated with vehicle were taken as sodium chloride solution 150 mM; sodium phosphate buffer 100. 10-50 mM, pH 5.8: human albumin 1 mg/mL. The composi tion may be in an intravenous bag of 50 to 250 mL. When administered to patients, the rhGAA is prepared by diluting US 2009/019 1178 A1 Jul. 30, 2009

the appropriate amount of drug Solution in the intravenous joint range of motion (flexion, extension, rotation), functional bag with normal saline at room temperature. Patients may be status (e.g., Childhood Health Assessment Questionnaire, pre-medicated with an anti-histamine prior to infusion to including assessment of pain and stiffness), levels of urinary reduce the potential for infusion-related reactions. glycogen and glycosaminoglycans, and changes in 0254 The objectives of this study are to evaluate the hepatomegaly, assessment of grip and pinch strength, visual safety, efficacy and pharmacokinetic profile of various doses acuity, cardiac function (e.g., ECG) and sleep apnea. Efficacy of rhGAA as ERT in patients with Pompe Disease. Patients can be assessed by measuring cardiac, pulmonary and/or are randomized in a double-blind fashion to various dose motor function according to established guidelines and com groups, which may range from about 1 to 40 mg/kg rhGAA. paring the results among patient groups administered differ Drug is administered at a frequency which may range from ent doses of rhGAA. Efficacy can also be assessed by com about once per week to once per month as an infusion over a paring the percentage of patients who die or require period of time which may range from about 1-hour to 6-hours, ventilatory Support during the study with an historical cohort depending on the dose of enzyme. The length of treatment of untreated Pompe patients of similar age and disease sever may range from about 26 to 104 weeks or more. Enrollment ity. in this study may be limited to those Pompe Disease patients 0257 Pharmacokinetic evaluations are conducted during having clinical signs of the disease, but not requiring venti infusions periodically to measure rhGAA enzyme levels dur latory Support at the time of entry into the study. ing and after infusions. 0255 Measures of safety include, for example, complete 0258 Based on the invention and examples disclosed chemistry panel, urinalysis, complete blood count with dif herein, those skilled in the art will be able to develop other ferential, ECG and tracking of adverse events. Immune embodiments of the invention. The examples are not intended response and infusion-associated reactions, complementacti to limit the scope of the claims set out below in any way. Vation and antibody formation are assessed for all patients. Although the foregoing invention has been described in some Safety can be assessed by comparing the adverse safety detail by way of illustration and example for purposes of events among patient groups administered different doses of clarity of understanding, it will be readily apparent to those of rhGAA. ordinary skill in the art, in light of the teachings of this 0256 Efficacy parameters include, for example, exercise invention, that numerous modifications and variations in the tolerance/endurance (e.g., 6-minute or 12-minute walk tests, invention as set forth above illustrative examples are expected an expanded timed get up and go test, a 3-minute stair climb to occur to those skilled in the art and may be made thereto test and physical activity), respiratory capacity (e.g., pulmo without departing from the spirit or scope of the appended nary function tests such as forced vital capacity, forced expi claims. Consequently only such limitations as appear in the ratory time and forced expiratory Volume for one second), appended claims should be placed on the invention.

SEQUENCE LISTING

<16 Oc NUMBER OF SEO ID NOS : 15

<210 SEQ ID NO 1 <211 LENGTH: 2847 &212> TYPE: DNA <213> ORGANISM: Mus musculus

<4 OO SEQUENCE: 1 atgaaagggit Coctoctgct gctgctggtg tcaaacctgc tic ctgtgcca gagcgggit Co 60

ggagc.cgggg cc cacatcct actic catgat tt Cotgctgg titcc cc.gaga gctgagtggc 12O toctic cc cag to Ctggagga gactic accca gct caccagc agggagc.cag Cagaccaggg 18O

cc.ccgggatg ccCaggcaca ccc.cggc.cgt CC cagagcag toccacaca gtgcgacgtc 24 O

cc.ccc.caac a gcc.gctt.cga ttgcgc.ccct gacaaggcca to acccagga acagtgcgag 3 OO gcc.cgcggct gctgctacat Coctgcaaag caggggotgc agggagcc.ca gatggggcag 360

ccctggtgct tctt.cccacc cagot acco c agctacaagc tiggaga acct gagctic ct ct 42O

gaaatgggct acacggccac cctdaccc.gt accaccc.cca ccttct tcc c caagga catc 48O

Ctgaccctgc ggctggacgt gatgatggag actgaga acc gcct coactt cacgatcaaa 54 O

gatccagcta acaggcgct a cdaggtgc.cc ttggaga.ccc cycgtgtc.ca cago.cgggca 6 OO

ccgt.ccc.cac totacagcgt ggagttct cc gaggagc cct tcggggtgat cqtgcaccgg 660

cagctggacg gC cqcgtgct gctgaac acg acggtggcgc cc ctgttctt toggaccag 72O

US 2009/019 1178 A1 Jul. 30, 2009 27

- Continued

<213> ORGANISM: Mus musculus

<4 OO SEQUENCE: 2 Met Lys Gly Ser Lieu Lleu Lleu Lieu. Lieu Val Ser Asn Lieu. Lieu. Lieu. Cys 1. 5 1O 15 Glin Ser Gly Ser Gly Ala Gly Ala His Ile Lieu. Lieu. His Asp Phe Lieu. 2O 25 3 O Lieu Val Pro Arg Glu Lieu Ser Gly Ser Ser Pro Val Lieu. Glu Glu Thr 35 4 O 45 His Pro Ala His Glin Glin Gly Ala Ser Arg Pro Gly Pro Arg Asp Ala SO 55 6 O Glin Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr Glin Cys Asp Wall 65 70 7s 8O Pro Pro Asn. Ser Arg Phe Asp Cys Ala Pro Asp Lys Ala Ile Thr Glin 85 9 O 95 Glu Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro Ala Lys Glin Gly OO OS 1O Lieu. Glin Gly Ala Gln Met Gly Glin Pro Trp Cys Phe Phe Pro Pro Ser 15 2O 25 yr Pro Ser Tyr Lys Lieu. Glu Asn Lieu Ser Ser Ser Glu Met Gly Tyr 3O 35 4 O Thr Ala Thr Lieu. Thr Arg Thr Thr Pro Thr Phe Phe Pro Lys Asp Ile 45 SO 55 160 Lieu. Thir Lieu. Arg Lieu. Asp Wal Met Met Glu Thr Glu Asn Arg Lieu. His 65 70 7s Phe Thir Ile Lys Asp Pro Ala Asn Arg Arg Tyr Glu Val Pro Lieu. Glu 8O 85 90 Thr Pro Arg Val His Ser Arg Ala Pro Ser Pro Leu Tyr Ser Val Glu 95 2 OO 2O5 Phe Ser Glu Glu Pro Phe Gly Val Ile Val His Arg Gln Lieu. Asp Gly 210 215 22O Arg Val Lieu. Lieu. Asn. Thir Thr Val Ala Pro Lieu. Phe Phe Ala Asp Glin 225 23 O 235 24 O Phe Leu Gln Leu Ser Thr Ser Leu Pro Ser Glin Tyr Ile Thr Gly Lieu. 245 250 255 Ala Glu. His Leu Ser Pro Leu Met Leu Ser Thr Ser Trp Thr Arg Ile 26 O 265 27 O Thir Lieu. Trp Asn Arg Asp Lieu Ala Pro Thr Pro Gly Ala Asn Lieu. Tyr 27s 28O 285 Gly Ser His Pro Phe Tyr Lieu Ala Lieu. Glu Asp Gly Gly Ser Ala His 290 295 3 OO Gly Val Phe Lieu. Lieu. Asn. Ser Asn Ala Met Asp Val Val Lieu. Glin Pro 3. OS 310 315 32O Ser Pro Ala Lieu. Ser Trp Arg Ser Thr Gly Gly Ile Lieu. Asp Val Tyr 3.25 330 335 Ile Phe Leu Gly Pro Glu Pro Llys Ser Val Val Glin Glin Tyr Lieu. Asp 34 O 345 350 Val Val Gly Tyr Pro Phe Met Pro Pro Tyr Trp Gly Lieu. Gly Phe His 355 360 365 Lieu. Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr Arg Glin Val Val 37O 375 38O US 2009/019 1178 A1 Jul. 30, 2009 28

- Continued Glu Asn Met Thr Arg Ala His Phe Pro Lieu. Asp Val Glin Trp Asn Asp 385 390 395 4 OO Lieu. Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe Asn Lys Asp Gly 4 OS 410 415 Phe Arg Asp Phe Pro Ala Met Val Glin Glu Lieu. His Glin Gly Gly Arg 42O 425 43 O Arg Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser Ser Gly Pro Ala 435 4 4 O 445 Gly Ser Tyr Arg Pro Tyr Asp Glu Gly Lieu. Arg Arg Gly Val Phe Ile 450 45.5 460 Thr Asn Glu Thr Gly Glin Pro Leu. Ile Gly Llys Val Trp Pro Gly Ser 465 470 47s 48O Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu Ala Trp Trp Glu 485 490 495 Asp Met Val Ala Glu Phe His Asp Glin Val Pro Phe Asp Gly Lieu. Trip 5 OO 5 OS 510 Ile Asp Met Asn. Glu Pro Ser Asn. Phe Ile Arg Gly Ser Glu Asp Gly 515 52O 525 Cys Pro Asn Asn Glu Lieu. Glu Asn Pro Pro Tyr Val Pro Gly Val Val 53 O 535 54 O Gly Gly Thr Lieu. Glin Ala Ala Thr Ile Cys Ala Ser Ser His Glin Phe 5.45 550 555 560 Lieu. Ser Thr His Tyr Asn Lieu. His Asn Lieu. Tyr Gly Lieu. Thr Glu Pro 565 st O sfs Ile Ala Ser His Arg Ala Lieu Val Lys Ala Arg Gly Thr Arg Pro Phe 58O 585 590 Val Ile Ser Arg Ser Thr Phe Ala Gly His Gly Arg Tyr Ala Gly His 595 6 OO 605 Trp Thr Gly Asp Val Trp Ser Ser Trp Glu Gln Leu Ala Ser Ser Val 610 615 62O Pro Glu Ile Lieu. Glin Phe Asn Lieu. Lieu. Gly Val Pro Lieu Val Gly Ala 625 630 635 64 O Asp Val Cys Gly Phe Lieu. Gly Asn. Thir Ser Glu Glu Lieu. Cys Val Arg 645 650 655 Trp Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg Asn His Asn Ser 660 665 670 Lieu. Leu Ser Leu Pro Glin Glu Pro Tyr Ser Phe Ser Glu Pro Ala Glin 675 68O 685 Glin Ala Met Arg Lys Ala Lieu. Thir Lieu. Arg Tyr Ala Lieu. Lieu Pro His 690 695 7 OO Lieu. Tyr Thr Lieu Phe His Glin Ala His Val Ala Gly Glu Thr Val Ala 7 Os 71O 71s 72O Arg Pro Leu Phe Lieu. Glu Phe Pro Lys Asp Ser Ser Thr Trp Thr Val 72 73 O 73 Asp His Glin Lieu Lleu Trp Gly Glu Ala Lieu. Lieu. Ile Thr Pro Val Lieu 740 74. 75O Glin Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro Leu Gly. Thir Trp 75s 760 765 Tyr Asp Leu Gln Thr Val Pro Ile Glu Ala Leu Gly Ser Leu Pro Pro 770 775 78O Pro Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser Glu Gly Gln Trp US 2009/019 1178 A1 Jul. 30, 2009 29

- Continued

78s 79 O 79. 8OO Val Thir Lieu Pro Ala Pro Lieu. Asp Thir Ile Asn. Wal His Lieu. Arg Ala 805 810 815 Gly Tyr Ile Ile Pro Leu Gln Gly Pro Gly Lieu. Thir Thr Thr Glu Ser 82O 825 830 Arg Glin Glin Pro Met Ala Lieu Ala Val Ala Lieu. Thir Lys Gly Gly Glu 835 84 O 845 Ala Arg Gly Glu Lieu. Phe Trp Asp Asp Gly Glu Ser Lieu. Glu Val Lieu 850 855 860 Glu Arg Gly Ala Tyr Thr Glin Val Ile Phe Lieu Ala Arg Asn. Asn. Thir 865 87O 87s 88O Ile Val Asn. Glu Lieu Val Arg Val Thir Ser Glu Gly Ala Gly Lieu. Glin 885 890 895 Lieu Gln Llys Val Thr Val Lieu. Gly Val Ala Thr Ala Pro Glin Glin Val 9 OO 9 OS 910 Lieu. Ser Asn Gly Val Pro Val Ser Asn Phe Thr Tyr Ser Pro Asp Thr 915 92O 925 Llys Val Lieu. Asp Ile Cys Val Ser Lieu. Lieu Met Gly Glu Glin Phe Lieu. 930 935 94 O Val Ser Trp Cys 945

<210 SEQ ID NO 3 <211 LENGTH: 43 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OO SEQUENCE: 3 gcgataggta CCC catggg agtgaggcac cc.gc.cctgct CCC 43

<210 SEQ ID NO 4 <211 LENGTH: 43 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OO SEQUENCE: 4 gcgatact cq agt caacacic agctgacgag aaactgctict CCC 43

<210 SEQ ID NO 5 <211 LENGTH: 28 &212> TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OO SEQUENCE: 5 Met Gly Val Arg His Pro Pro Cys Ser His Arg Lieu. Lieu Ala Val Cys 1. 5 1O 15 Ala Lieu Val Ser Lieu Ala Thr Ala Ala Lieu. Lieu. Gly 2O 25

<210 SEQ ID NO 6 <211 LENGTH: 35 &212> TYPE: PRT <213> ORGANISM: Homo sapiens

US 2009/019 1178 A1 Jul. 30, 2009 32

- Continued

Lieu Val Pro Arg Glu Lieu Ser Gly Ser Ser Pro Val Lieu. Glu Glu Thr 35 4 O 45 His Pro Ala His Glin Glin Gly Ala Ser Arg Pro Gly Pro Arg Asp Ala SO 55 6 O Glin Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr Glin Cys Asp Wall 65 70 7s 8O Pro Pro Asn. Ser Arg Phe Asp Cys Ala Pro Asp Lys Ala Ile Thr Glin 85 9 O 95 Glu Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro Ala Lys Glin Gly OO OS 1O Lieu. Glin Gly Ala Gln Met Gly Glin Pro Trp Cys Phe Phe Pro Pro Ser 15 2O 25 yr Pro Ser Tyr Lys Lieu. Glu Asn Lieu Ser Ser Ser Glu Met Gly Tyr 3O 35 4 O Thr Ala Thr Lieu. Thr Arg Thr Thr Pro Thr Phe Phe Pro Lys Asp Ile 45 SO 55 160 Lieu. Thir Lieu. Arg Lieu. Asp Wal Met Met Glu Thr Glu Asn Arg Lieu. His 65 70 7s Phe Thir Ile Lys Asp Pro Ala Asn Arg Arg Tyr Glu Val Pro Lieu. Glu 8O 85 90 Thr Pro Arg Val His Ser Arg Ala Pro Ser Pro Leu Tyr Ser Val Glu 95 2 OO 2O5 Phe Ser Glu Glu Pro Phe Gly Val Ile Val His Arg Gln Lieu. Asp Gly 210 215 22O Arg Val Lieu. Lieu. Asn. Thir Thr Val Ala Pro Lieu. Phe Phe Ala Asp Glin 225 23 O 235 24 O Phe Leu Gln Leu Ser Thr Ser Leu Pro Ser Glin Tyr Ile Thr Gly Lieu. 245 250 255 Ala Glu. His Leu Pro Thr Pro Gly Ala Asn Lieu. Tyr Gly Ser His Pro 26 O 265 27 O Phe Tyr Lieu Ala Lieu. Glu Asp Gly Ser Pro Leu Met Leu Ser Thr Ser 27s 28O 285 Trp Thr Arg Ile Thr Lieu. Trp Asin Arg Asp Lieu Ala Gly Ser Ala His 290 295 3 OO Gly Val Phe Lieu. Lieu. Asn. Ser Asn Ala Met Asp Val Val Lieu. Glin Pro 3. OS 310 315 32O Ser Pro Ala Lieu. Ser Trp Arg Ser Thr Gly Gly Ile Lieu. Asp Val Tyr 3.25 330 335 Ile Phe Leu Gly Pro Glu Pro Llys Ser Val Val Glin Glin Tyr Lieu. Asp 34 O 345 350 Val Val Gly Tyr Pro Phe Met Pro Pro Tyr Trp Gly Lieu. Gly Phe His 355 360 365 Lieu. Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr Arg Glin Val Val 37O 375 38O Glu Asn Met Thr Arg Ala His Phe Pro Lieu. Asp Val Glin Trp Asn Asp 385 390 395 4 OO Lieu. Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe Asn Lys Asp Gly 4 OS 410 415 Phe Arg Asp Phe Pro Ala Met Val Glin Glu Lieu. His Glin Gly Gly Arg 42O 425 43 O US 2009/019 1178 A1 Jul. 30, 2009 33

- Continued Arg Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser Ser Gly Pro Ala 435 4 4 O 445 Gly Ser Tyr Arg Pro Tyr Asp Glu Gly Lieu. Arg Arg Gly Val Phe Ile 450 45.5 460 Thr Asn Glu Thr Gly Glin Pro Leu. Ile Gly Llys Val Trp Pro Gly Ser 465 470 47s 48O Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu Ala Trp Trp Glu 485 490 495 Asp Met Val Ala Glu Phe His Asp Glin Val Pro Phe Asp Gly Lieu. Trip 5 OO 5 OS 510 Ile Asp Met Asn. Glu Pro Ser Asn. Phe Ile Arg Gly Ser Glu Asp Gly 515 52O 525 Cys Pro Asn Asn Glu Lieu. Glu Asn Pro Pro Tyr Val Pro Gly Val Val 53 O 535 54 O Gly Gly Thr Lieu. Glin Ala Ala Thr Ile Cys Ala Ser Ser His Glin Phe 5.45 550 555 560 Lieu. Ser Thr His Tyr Asn Lieu. His Asn Lieu. Tyr Gly Lieu. Thr Glu Pro 565 st O sfs Ile Ala Ser His Arg Ala Lieu Val Lys Ala Arg Gly Thr Arg Pro Phe 58O 585 590 Val Ile Ser Arg Ser Thr Phe Ala Gly His Gly Arg Tyr Ala Gly His 595 6 OO 605 Trp Thr Gly Asp Val Trp Ser Ser Trp Glu Gln Leu Ala Ser Ser Val 610 615 62O Pro Glu Ile Lieu. Glin Phe Asn Lieu. Lieu. Gly Val Pro Lieu Val Gly Ala 625 630 635 64 O Asp Val Cys Gly Phe Lieu. Gly Asn. Thir Ser Glu Glu Lieu. Cys Val Arg 645 650 655 Trp Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg Asn His Asn Ser 660 665 670 Lieu. Leu Ser Leu Pro Glin Glu Pro Tyr Ser Phe Ser Glu Pro Ala Glin 675 68O 685 Glin Ala Met Arg Lys Ala Lieu. Thir Lieu. Arg Tyr Ala Lieu. Lieu Pro His 690 695 7 OO Lieu. Tyr Thr Lieu Phe His Glin Ala His Val Ala Gly Glu Thr Val Ala 7 Os 71O 71s 72O Arg Pro Leu Phe Lieu. Glu Phe Pro Lys Asp Ser Ser Thr Trp Thr Val 72 73 O 73 Asp His Glin Lieu Lleu Trp Gly Glu Ala Lieu. Lieu. Ile Thr Pro Val Lieu 740 74. 75O Glin Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro Leu Gly. Thir Trp 75s 760 765 Tyr Asp Leu Gln Thr Val Pro Ile Glu Ala Leu Gly Ser Leu Pro Pro 770 775 78O Pro Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser Glu Gly Gln Trp 78s 79 O 79. 8OO Val Thir Lieu Pro Ala Pro Lieu. Asp Thir Ile Asn. Wal His Lieu. Arg Ala 805 810 815 Gly Tyr Ile Ile Pro Leu Gln Gly Pro Gly Lieu. Thir Thr Thr Glu Ser 82O 825 830 Arg Glin Glin Pro Met Ala Lieu Ala Val Ala Lieu. Thir Lys Gly Gly Glu US 2009/019 1178 A1 Jul. 30, 2009 34

- Continued

835 84 O 845 Ala Arg Gly Glu Lieu. Phe Trp Asp Asp Gly Glu Ser Lieu. Glu Val Lieu 850 855 860 Glu Arg Gly Ala Tyr Thr Glin Val Ile Phe Lieu Ala Arg Asn. Asn. Thir 865 87O 87s 88O Ile Val Asn. Glu Lieu Val Arg Val Thir Ser Glu Gly Ala Gly Lieu. Glin 885 890 895 Lieu Gln Llys Val Thr Val Lieu. Gly Val Ala Thr Ala Pro Glin Glin Val 9 OO 9 OS 910 Lieu. Ser Asn Gly Val Pro Val Ser Asn Phe Thr Tyr Ser Pro Asp Thr 915 92O 925 Llys Val Lieu. Asp Ile Cys Val Ser Lieu. Lieu Met Gly Glu Glin Phe Lieu. 930 935 94 O Val Ser Trp Cys 945

<210 SEQ ID NO 11 <211 LENGTH: 952 &212> TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO SEQUENCE: 11 Met Gly Val Arg His Pro Pro Cys Ser His Arg Lieu. Lieu Ala Val Cys 1. 5 1O 15 Ala Lieu Val Ser Lieu Ala Thr Ala Ala Lieu. Lieu. Gly. His Ile Lieu. Lieu 2O 25 3 O His Asp Phe Lieu. Lieu Val Pro Arg Glu Lieu. Ser Gly Ser Ser Pro Val 35 4 O 45 Lieu. Glu Glu Thr His Pro Ala His Glin Glin Gly Ala Ser Arg Pro Gly SO 55 6 O Pro Arg Asp Ala Glin Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr 65 70 7s 8O Glin Cys Asp Val Pro Pro Asn. Ser Arg Phe Asp Cys Ala Pro Asp Llys 85 9 O 95 Ala Ile Thr Glin Glu Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro OO OS 1O Ala Lys Glin Gly Lieu. Glin Gly Ala Gln Met Gly Glin Pro Trp Cys Phe 15 2O 25 Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Lieu. Glu Asn Lieu Ser Ser Ser 3O 35 4 O Glu Met Gly Tyr Thr Ala Thr Lieu. Thr Arg Thr Thr Pro Thr Phe Phe 45 SO 55 160 Pro Lys Asp Ile Lieu. Thir Lieu. Arg Lieu. Asp Wal Met Met Glu Thr Glu 65 70 7s Asn Arg Lieu. His Phe Thir Ile Lys Asp Pro Ala Asn Arg Arg Tyr Glu 8O 85 90 Val Pro Leu Glu Thr Pro Arg Val His Ser Arg Ala Pro Ser Pro Leu 95 2 OO 2O5 yr Ser Val Glu Phe Ser Glu Glu Pro Phe Gly Val Ile Val His Arg 210 215 22O Glin Lieu. Asp Gly Arg Val Lieu. Lieu. Asn. Thir Thr Val Ala Pro Lieu. Phe 225 23 O 235 24 O US 2009/019 1178 A1 Jul. 30, 2009 35

- Continued Phe Ala Asp Glin Phe Leu Gln Leu Ser Thr Ser Leu Pro Ser Glin Tyr 245 250 255 Ile Thr Gly Lieu Ala Glu. His Leu Ser Pro Leu Met Leu Ser Thr Ser 26 O 265 27 O Trp Thr Arg Ile Thr Lieu. Trp Asin Arg Asp Lieu Ala Pro Thr Pro Gly 27s 28O 285 Ala Asn Lieu. Tyr Gly Ser His Pro Phe Tyr Lieu Ala Lieu. Glu Asp Gly 290 295 3 OO Gly Ser Ala His Gly Val Phe Lieu. Lieu. Asn. Ser Asn Ala Met Asp Wall 3. OS 310 315 32O Val Lieu Gln Pro Ser Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile 3.25 330 335 Lieu. Asp Val Tyr Ile Phe Leu Gly Pro Glu Pro Llys Ser Val Val Glin 34 O 345 350 Gln Tyr Lieu. Asp Val Val Gly Tyr Pro Phe Met Pro Pro Tyr Trp Gly 355 360 365 Lieu. Gly Phe His Lieu. Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr 37O 375 38O Arg Glin Val Val Glu Asn Met Thr Arg Ala His Phe Pro Lieu. Asp Wall 385 390 395 4 OO Glin Trp Asn Asp Lieu. Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe 4 OS 410 415 Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala Met Val Glin Glu Lieu. His 42O 425 43 O Gln Gly Gly Arg Arg Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser 435 4 4 O 445 Ser Gly Pro Ala Gly Ser Tyr Arg Pro Tyr Asp Glu Gly Lieu. Arg Arg 450 45.5 460 Gly Val Phe Ile Thr Asn Glu Thr Gly Glin Pro Leu. Ile Gly Llys Val 465 470 47s 48O Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu 485 490 495 Ala Trp Trp Glu Asp Met Val Ala Glu Phe His Asp Glin Val Pro Phe 5 OO 5 OS 510 Asp Gly Met Trp Ile Asp Met Asn. Glu Pro Ser Asn. Phe Ile Arg Gly 515 52O 525 Ser Glu Asp Gly Cys Pro Asn. Asn. Glu Lieu. Glu ASn Pro Pro Tyr Val 53 O 535 54 O Pro Gly Val Val Gly Gly Thr Lieu. Glin Ala Ala Thr Ile Cys Ala Ser 5.45 550 555 560 Ser His Glin Phe Leu Ser Thr His Tyr Asn Lieu. His Asn Lieu. Tyr Gly 565 st O sfs Lieu. Thr Glu Ala Ile Ala Ser His Arg Ala Lieu Val Lys Ala Arg Gly 58O 585 590 Thr Arg Pro Phe Val Ile Ser Arg Ser Thr Phe Ala Gly His Gly Arg 595 6 OO 605 Tyr Ala Gly His Trp Thr Gly Asp Val Trp Ser Ser Trp Glu Gln Leu 610 615 62O Ala Ser Ser Val Pro Glu Ile Leu Glin Phe Asn Lieu. Leu Gly Val Pro 625 630 635 64 O Lieu Val Gly Ala Asp Val Cys Gly Phe Lieu. Gly Asn. Thir Ser Glu Glu US 2009/019 1178 A1 Jul. 30, 2009 36

- Continued

645 650 655

Lell Wall Arg Trp Thir Glin Luell Gly Ala Phe Pro Phe Met Arg 660 665 670

Asn His Asn Ser Lell Lell Ser Luell Pro Glin Glu Pro Ser Phe Ser 675 68O 685

Glu Pro Ala Glin Glin Ala Met Arg Ala Luell Thir Lell Arg Ala 690 695 7 OO

Lell Luell Pro His Lell Thir Luell Phe His Glin Ala His Wall Ala Gly 7 Os 71s

Glu Thir Wall Ala Arg Pro Lell Phe Luell Glu Phe Pro Asp Ser Ser 72 73 O 73

Thir Trp Thir Wall Asp His Glin Luell Luell Trp Gly Glu Ala Luell Luell Ile 740 74. 75O

Thir Pro Wall Luell Glin Ala Gly Ala Glu Wall Thir Gly Phe Pro 75s 760 765

Lell Gly Thir Trp Tyr Asp Lell Glin Thir Wall Pro Ile Glu Luell Gly 770 775 78O

Ser Luell Pro Pro Pro Pro Ala Ala Pro Arg Glu Pro Ala His Ser 78s 79 O 79.

Glu Gly Glin Trp Wall Thir Lell Pro Ala Pro Luell Asp Thir Asn Wall 805 810 815

His Luell Arg Ala Gly Tyr Ile Ile Pro Luell Glin Gly Pro Luell Thir 82O 825 830

Thir Thir Glu Ser Arg Glin Glin Pro Met Ala Luell Ala Wall Luell Thir 835 84 O 845

Lys Gly Gly Glu Ala Arg Gly Glu Luell Phe Trp Asp Asp Glu Ser 850 855 860

Lell Glu Wall Luell Glu Arg Gly Ala Thir Glin Wall Ile Phe Luell Ala 865 87O 87s

Arg Asn Asn Thir Ile Wall Asn Glu Luell Wall Arg Wall Thir Ser Glu Gly 885 890 895

Ala Gly Luell Glin Lell Glin Wall Thir Wall Luell Gly Wall Ala Thir Ala 9 OO 9 OS 910

Pro Glin Glin Wall Lell Ser Asn Gly Wall Pro Wall Ser Asn Phe Thir Tyr 915 92O 925

Ser Pro Asp Thir Lys Wall Lell Asp Ile Cys Wall Ser Lell Luell Met Gly 930 935 94 O

Glu Glin Phe Luell Wall Ser Trp 945 950

<210 SEQ ID NO 12 <211 LENGTH: 952 &212> TYPE : PRT <213> ORGANISM: Homo sapiens

<4 OO SEQUENCE: 12 Met Gly Val Arg His Pro Pro Cys Ser His Arg Lieu. Lieu Ala Val Cys 1. 5 1O 15

Ala Lieu Wal Ser Lieu. Ala Thir Ala Ala Lieu. Lieu. Gly His Ile Lieu. Lieu. 25 3 O His Asp Phe Lieu. Lieu. Val Pro Arg Glu Lieu. Ser Gly Ser Ser Pro Val 35 4 O 45 US 2009/019 1178 A1 Jul. 30, 2009 37

- Continued Lieu. Glu Glu Thr His Pro Ala His Glin Glin Gly Ala Ser Arg Pro Gly SO 55 6 O Pro Arg Asp Ala Glin Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr 65 70 7s 8O Glin Cys Asp Val Pro Pro Asn. Ser Arg Phe Asn. Cys Ala Pro Asp Llys 85 9 O 95 Ala Ile Thr Glin Glu Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro OO OS 1O Ala Lys Glin Gly Lieu. Glin Gly Ala Gln Met Gly Glin Pro Trp Cys Phe 15 2O 25 Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Lieu. Glu Asn Lieu Ser Ser Ser 3O 35 4 O Glu Met Gly Tyr Thr Ala Thr Lieu. Thr Arg Thr Thr Pro Thr Phe Phe 45 SO 55 160 Pro Lys Asp Ile Lieu. Thir Lieu. Arg Lieu. Asp Wal Met Met Glu Thr Glu 65 70 7s Asn Arg Lieu. His Phe Thir Ile Lys Asp Pro Ala Asn Arg Arg Tyr Glu 8O 85 90 Val Pro Leu Glu Thr Pro Arg Val His Ser Arg Ala Pro Ser Pro Leu 95 2 OO 2O5 Tyr Ser Val Glu Phe Ser Glu Glu Pro Phe Gly Val Ile Val His Arg 210 215 22O Glin Lieu. Asp Gly Arg Val Lieu. Lieu. Asn. Thir Thr Val Ala Pro Lieu. Phe 225 23 O 235 24 O Phe Ala Asp Glin Phe Leu Gln Leu Ser Thr Ser Leu Pro Ser Glin Tyr 245 250 255 Ile Thr Gly Lieu Ala Glu. His Leu Ser Pro Leu Met Leu Ser Thr Ser 26 O 265 27 O Trp Thr Arg Ile Thr Lieu. Trp Asin Arg Asp Lieu Ala Pro Thr Pro Gly 27s 28O 285 Ala Asn Lieu. Tyr Gly Ser His Pro Phe Tyr Lieu Ala Lieu. Glu Asp Gly 290 295 3 OO Gly Ser Ala His Gly Val Phe Lieu. Lieu. Asn. Ser Asn Ala Met Asp Wall 3. OS 310 315 32O Val Lieu Gln Pro Ser Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile 3.25 330 335 Lieu. Asp Val Tyr Ile Phe Leu Gly Pro Glu Pro Llys Ser Val Val Glin 34 O 345 350 Gln Tyr Lieu. Asp Val Val Gly Tyr Pro Phe Met Pro Pro Tyr Trp Gly 355 360 365 Lieu. Gly Phe His Lieu. Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr 37O 375 38O Arg Glin Val Val Glu Asn Met Thr Arg Ala His Phe Pro Lieu. Asp Wall 385 390 395 4 OO Glin Trp Asn Asp Lieu. Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe 4 OS 410 415 Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala Met Val Glin Glu Lieu. His 42O 425 43 O Gln Gly Gly Arg Arg Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser 435 4 4 O 445 Ser Gly Pro Ala Gly Ser Tyr Arg Pro Tyr Asp Glu Gly Lieu. Arg Arg US 2009/019 1178 A1 Jul. 30, 2009 38

- Continued

450 45.5 460 Gly Val Phe Ile Thr Asn Glu Thr Gly Glin Pro Leu. Ile Gly Llys Val 465 470 47s 48O Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu 485 490 495 Ala Trp Trp Glu Asp Met Val Ala Glu Phe His Asp Glin Val Pro Phe 5 OO 5 OS 510 Asp Gly Lieu. Trp Ile Asp Met Asn. Glu Pro Ser Asn. Phe Ile Arg Gly 515 52O 525 Ser Glu Asp Gly Cys Pro Asn. Asn. Glu Lieu. Glu ASn Pro Pro Tyr Val 53 O 535 54 O Pro Gly Val Val Gly Gly Thr Lieu. Glin Ala Ala Thr Ile Cys Ala Ser 5.45 550 555 560 Ser His Glin Phe Leu Ser Thr His Tyr Asn Lieu. His Asn Lieu. Tyr Gly 565 st O sfs Lieu. Thr Glu Pro Ile Ala Ser His Arg Ala Lieu Val Lys Ala Arg Gly 58O 585 590 Thr Arg Pro Phe Val Ile Ser Arg Ser Thr Phe Ala Gly His Gly Arg 595 6 OO 605 Tyr Ala Gly His Trp Thr Gly Asp Val Trp Ser Ser Trp Glu Gln Leu 610 615 62O Ala Ser Ser Val Pro Glu. Ile Lieu. Glin Phe ASn Lieu. Lieu. Gly Val Pro 625 630 635 64 O Lieu Val Gly Ala Asp Val Cys Gly Phe Lieu. Gly Asn. Thir Ser Glu Glu 645 650 655 Lieu. Cys Val Arg Trp Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg 660 665 670 Asn His Asn Ser Lieu Lleu Ser Leu Pro Glin Glu Pro Tyr Ser Phe Ser 675 68O 685 Glu Pro Ala Glin Glin Ala Met Arg Lys Ala Lieu. Thir Lieu. Arg Tyr Ala 690 695 7 OO Lieu. Leu Pro His Lieu. Tyr Thr Lieu Phe His Glin Ala His Val Ala Gly 7 Os 71O 71s 72O Glu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser 72 73 O 73 Thir Trp Thr Val Asp His Glin Lieu. Lieu. Trp Gly Glu Ala Lieu. Lieu. Ile 740 74. 75O Thr Pro Val Lieu. Glin Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro 75s 760 765 Lieu. Gly. Thir Trp Tyr Asp Leu Gln Thr Val Pro Ile Glu Ala Leu Gly 770 775 78O Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser 78s 79 O 79. 8OO Glu Gly Gln Trp Val Thr Lieu Pro Ala Pro Leu Asp Thr Ile Asin Val 805 810 815 His Lieu. Arg Ala Gly Tyr Ile Ile Pro Lieu. Glin Gly Pro Gly Lieu. Thir 82O 825 830 Thir Thr Glu Ser Arg Glin Gln Pro Met Ala Leu Ala Val Ala Lieu. Thr 835 84 O 845 Lys Gly Gly Glu Ala Arg Gly Glu Lieu. Phe Trp Asp Asp Gly Glu Ser 850 855 860

US 2009/019 1178 A1 Jul. 30, 2009 41

- Continued

Ala Glin Gly Lieu. Glin Gly Ala Glin Met Gly Glin Pro Trp Cys Phe 15 12O 125

Phe Pro Pro Ser Tyr Pro Ser Luell Glu Asn Lell Ser Ser Ser 3O 135 14 O

Glu Met Gly Tyr Thr Ala Thir Luell Thir Arg Thir Thir Pro Thir Phe Phe 45 150 155 160

Pro Asp Ile Lieu. Thir Lell Arg Luell Asp Wall Met Met Glu Thir Glu 65 17O 17s

Arg Luell His Phe Thir Ile Asp Pro Ala Asn Arg Arg Glu 185 190

Wall Pro Luell Glu Thir Pro Arg Wall His Ser Arg Ala Pro Ser Pro Luell 95 2 OO 2O5

yr Ser Wall Glu Phe Ser Glu Glu Pro Phe Gly Wall Ile Wall His Arg 210 215 22O

Glin Luell Asp Gly Arg Wall Lell Luell Asn Thir Thir Wall Ala Pro Luell Phe 225 23 O 235 24 O

Phe Ala Asp Glin Phe Lell Glin Luell Ser Thir Ser Lell Pro Ser Glin 245 250 255

Ile Thir Gly Lieu Ala Glu His Luell Ser Pro Luell Met Lell Ser Thir Ser 26 O 265 27 O

Trp Thir Arg Ile Thr Lell Trp Asn Arg Asp Luell Ala Pro Thir Pro Gly 27s 28O 285

Ala Asn Luell Ser His Pro Phe Luell Ala Lell Glu Asp Gly 290 295 3 OO

Gly Ser Ala His Gly Wall Phe Luell Luell Asn Ser Asn Ala Met Asp Wall 3. OS 310 315

Wall Luell Glin Pro Ser Pro Ala Luell Ser Trp Arg Ser Thir Gly Gly Ile 3.25 330 335

Lell Asp Wall Tyr Ile Phe Lell Gly Pro Glu Pro Ser Wall Wall Glin 34 O 345 350

Glin Luell Asp Val Wall Gly Pro Phe Met Pro Pro Trp Gly 355 360 365

Lell Gly Phe His Lieu. Cys Arg Trp Gly Ser Ser Thir Ala Ile Thir 37O 375 38O

Arg Glin Wall Wall Glu Asn Met Thir Arg Ala His Phe Pro Luell Asp Wall 385 390 395 4 OO

Glin Trp Asn Asp Lieu. Asp Met Asp Ser Arg Arg Asp Phe Thir Phe 4 OS 410 415

Asn Asp Gly Phe Arg Asp Phe Pro Ala Met Wall Glin Glu Luell His 42O 425 43 O

Glin Gly Gly Arg Arg Tyr Met Met Ile Wall Asp Pro Ala Ile Ser Ser 435 4 4 O 445

Ser Gly Pro Ala Gly Ser Arg Pro Asp Glu Gly Luell Arg Arg 450 45.5 460

Gly Wall Phe Ile Thr Asn Glu Thir Gly Glin Pro Lell Ile Gly Wall 465 470 47s

Trp Pro Gly Ser Thir Ala Phe Pro Asp Phe Thir Asn Pro Thir Ala Luell 485 490 495

Ala Trp Trp Glu Asp Met Wall Ala Glu Phe His Asp Glin Wall Pro Phe 5 OO 5 OS 510

Asp Gly Luell Trp Ile Asp Met Asn Glu Pro Ser Asn Phe Ile Arg Gly US 2009/019 1178 A1 Jul. 30, 2009 42

- Continued

515 52O 525 Ser Glu Asp Gly Cys Pro Asn. Asn. Glu Lieu. Glu ASn Pro Pro Tyr Val 53 O 535 54 O Pro Gly Val Val Gly Gly Thr Lieu. Glin Ala Ala Thr Ile Cys Ala Ser 5.45 550 555 560 Ser His Glin Phe Leu Ser Thr His Tyr Asn Lieu. His Asn Lieu. Tyr Gly 565 st O sfs Lieu. Thr Glu Ala Ile Ala Ser His Arg Ala Lieu Val Lys Ala Arg Gly 58O 585 590 Thr Arg Pro Phe Val Ile Ser Arg Ser Thr Phe Ala Gly His Gly Arg 595 6 OO 605 Tyr Ala Gly His Trp Thr Gly Asp Val Trp Ser Ser Trp Glu Gln Leu 610 615 62O Ala Ser Ser Val Pro Glu Ile Leu Glin Phe Asn Lieu. Leu Gly Val Pro 625 630 635 64 O Lieu Val Gly Ala Asp Val Cys Gly Phe Lieu. Gly Asn. Thir Ser Glu Glu 645 650 655 Lieu. Cys Val Arg Trp Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg 660 665 670 Asn His Asn Ser Lieu Lleu Ser Leu Pro Glin Glu Pro Tyr Ser Phe Ser 675 68O 685 Glu Pro Ala Glin Glin Ala Met Arg Lys Ala Lieu. Thr Lieu. Arg Tyr Ala 690 695 7 OO Lieu. Leu Pro His Lieu. Tyr Thr Lieu Phe His Glin Ala His Val Ala Gly 7 Os 71O 71s 72O Glu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser 72 73 O 73 Thir Trp Thr Val Asp His Glin Lieu. Lieu. Trp Gly Glu Ala Lieu. Lieu. Ile 740 74. 75O Thr Pro Val Lieu. Glin Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro 75s 760 765 Lieu. Gly. Thir Trp Tyr Asp Leu Gln Thr Val Pro Ile Glu Ala Leu Gly 770 775 78O Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser 78s 79 O 79. 8OO Glu Gly Gln Trp Val Thr Lieu Pro Ala Pro Leu Asp Thr Ile Asin Val 805 810 815 His Lieu. Arg Ala Gly Tyr Ile Ile Pro Lieu. Glin Gly Pro Gly Lieu. Thir 82O 825 830 Thir Thr Glu Ser Arg Glin Gln Pro Met Ala Leu Ala Val Ala Lieu. Thr 835 84 O 845 Lys Gly Gly Glu Ala Arg Gly Glu Lieu. Phe Trp Asp Asp Gly Glu Ser 850 855 860 Lieu. Glu Val Lieu. Glu Arg Gly Ala Tyr Thr Glin Val Ile Phe Lieu Ala 865 87O 87s 88O Arg Asn. Asn. Thir Ile Val Asn. Glu Lieu Val Arg Val Thir Ser Glu Gly 885 890 895 Ala Gly Lieu. Glin Lieu. Glin Llys Val Thr Val Lieu. Gly Val Ala Thir Ala 9 OO 9 OS 910 Pro Glin Glin Val Lieu. Ser Asn Gly Val Pro Val Ser Asn Phe Thr Tyr 915 92O 925 US 2009/019 1178 A1 Jul. 30, 2009 43

- Continued

Ser Pro Asp Thir Lys Val Lieu. Asp Ile Cys Val Ser Lieu. Lieu Met Gly 930 935 94 O Glu Glin Phe Leu Val Ser Trp Cys 945 950

<210 SEQ ID NO 15 <211 LENGTH: 952 &212> TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO SEQUENCE: 15 Met Gly Val Arg His Pro Pro Cys Ser His Arg Lieu. Lieu Ala Val Cys 1. 5 1O 15 Ala Lieu Val Ser Lieu Ala Thr Ala Ala Lieu. Lieu. Gly. His Ile Lieu. Lieu 2O 25 3 O His Asp Phe Lieu. Lieu Val Pro Arg Glu Lieu. Ser Gly Ser Ser Pro Val 35 4 O 45 Lieu. Glu Glu Thr His Pro Ala His Glin Glin Gly Ala Ser Arg Pro Gly SO 55 6 O Pro Arg Asp Ala Glin Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr 65 70 7s 8O Glin Cys Asp Val Pro Pro Asn. Ser Arg Phe Asp Cys Ala Pro Asp Llys 85 9 O 95 Ala Ile Thr Glin Glu Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro OO OS 1O Ala Lys Glin Gly Lieu. Glin Gly Ala Gln Met Gly Glin Pro Trp Cys Phe 15 2O 25 Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Lieu. Glu Asn Lieu Ser Ser Ser 3O 35 4 O Glu Met Gly Tyr Thr Ala Thr Lieu. Thr Arg Thr Thr Pro Thr Phe Phe 45 SO 55 160 Pro Lys Asp Ile Lieu. Thir Lieu. Arg Lieu. Asp Wal Met Met Glu Thr Glu 65 70 7s Asn Arg Lieu. His Phe Thir Ile Lys Asp Pro Ala Asn Arg Arg Tyr Glu 8O 85 90 Val Pro Leu Glu Thr Pro Arg Val His Ser Arg Ala Pro Ser Pro Leu 95 2 OO 2O5 Tyr Ser Val Glu Phe Ser Glu Glu Pro Phe Gly Val Ile Val His Arg 210 215 22O Glin Lieu. Asp Gly Arg Val Lieu. Lieu. Asn. Thir Thr Val Ala Pro Lieu. Phe 225 23 O 235 24 O Phe Ala Asp Glin Phe Leu Gln Leu Ser Thr Ser Leu Pro Ser Glin Tyr 245 250 255 Ile Thr Gly Lieu Ala Glu. His Leu Ser Pro Leu Met Leu Ser Thr Ser 26 O 265 27 O Trp Thr Arg Ile Thr Lieu. Trp Asin Arg Asp Lieu Ala Pro Thr Pro Gly 27s 28O 285 Ala Asn Lieu. Tyr Gly Ser His Pro Phe Tyr Lieu Ala Lieu. Glu Asp Gly 290 295 3 OO Gly Ser Ala His Gly Val Phe Lieu. Lieu. Asn. Ser Asn Ala Met Asp Wall 3. OS 310 315 32O Val Lieu Gln Pro Ser Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile US 2009/019 1178 A1 Jul. 30, 2009 44

- Continued

3.25 330 335 Lieu. Asp Val Tyr Ile Phe Leu Gly Pro Glu Pro Llys Ser Val Val Glin 34 O 345 350 Gln Tyr Lieu. Asp Val Val Gly Tyr Pro Phe Met Pro Pro Tyr Trp Gly 355 360 365 Lieu. Gly Phe His Lieu. Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr 37O 375 38O Arg Glin Val Val Glu Asn Met Thr Arg Ala His Phe Pro Lieu. Asp Wall 385 390 395 4 OO Glin Trp Asn Asp Lieu. Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe 4 OS 410 415 Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala Met Val Glin Glu Lieu. His 42O 425 43 O Gln Gly Gly Arg Arg Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser 435 4 4 O 445 Ser Gly Pro Ala Gly Ser Tyr Arg Pro Tyr Asp Glu Gly Lieu. Arg Arg 450 45.5 460 Gly Val Phe Ile Thr Asn Glu Thr Gly Glin Pro Leu. Ile Gly Llys Val 465 470 47s 48O Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu 485 490 495 Ala Trp Trp Glu Asp Met Val Ala Glu Phe His Asp Glin Val Pro Phe 5 OO 5 OS 510 Asp Gly Met Trp Ile Asp Met Asn. Glu Pro Ser Asn. Phe Ile Arg Gly 515 52O 525 Ser Glu Asp Gly Cys Pro Asn. Asn. Glu Lieu. Glu ASn Pro Pro Tyr Val 53 O 535 54 O Pro Gly Val Val Gly Gly Thr Lieu. Glin Ala Ala Thr Ile Cys Ala Ser 5.45 550 555 560 Ser His Glin Phe Leu Ser Thr His Tyr Asn Lieu. His Asn Lieu. Tyr Gly 565 st O sfs Lieu. Thr Glu Ala Ile Ala Ser His Arg Ala Lieu Val Lys Ala Arg Gly 58O 585 590 Thr Arg Pro Phe Val Ile Ser Arg Ser Thr Phe Ala Gly His Gly Arg 595 6 OO 605 Tyr Ala Gly His Trp Thr Gly Asp Val Trp Ser Ser Trp Glu Gln Leu 610 615 62O Ala Ser Ser Val Pro Glu Ile Leu Glin Phe Asn Lieu. Leu Gly Val Pro 625 630 635 64 O Lieu Val Gly Ala Asp Val Cys Gly Phe Lieu. Gly Asn. Thir Ser Glu Glu 645 650 655 Lieu. Cys Val Arg Trp Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg 660 665 670 Asn His Asn Ser Lieu Lleu Ser Leu Pro Glin Glu Pro Tyr Ser Phe Ser 675 68O 685 Llys Pro Ala Glin Glin Ala Met Arg Lys Ala Lieu. Thir Lieu. Arg Tyr Ala 690 695 7 OO Lieu. Leu Pro His Lieu. Tyr Thr Lieu Phe His Glin Ala His Val Ala Gly 7 Os 71O 71s 72O Glu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser 72 73 O 73 US 2009/019 1178 A1 Jul. 30, 2009 45

- Continued

Thir Trp Thr Val Asp His Glin Lieu. Lieu. Trp Gly Glu Ala Lieu. Lieu. Ile 740 74. 75O Thr Pro Val Lieu. Glin Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro 75s 760 765 Lieu. Gly. Thir Trp Tyr Asp Leu Gln Thr Val Pro Ile Glu Ala Leu Gly 770 775 78O Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser 78s 79 O 79. 8OO Glu Gly Gln Trp Val Thr Lieu Pro Ala Pro Leu Asp Thr Ile Asin Val 805 810 815 His Lieu. Arg Ala Gly Tyr Ile Ile Pro Lieu. Glin Gly Pro Gly Lieu. Thir 82O 825 830 Thir Thr Glu Ser Arg Glin Gln Pro Met Ala Leu Ala Val Ala Lieu. Thr 835 84 O 845 Lys Gly Gly Glu Ala Arg Gly Glu Lieu. Phe Trp Asp Asp Gly Glu Ser 850 855 860 Lieu. Glu Val Lieu. Glu Arg Gly Ala Tyr Thr Glin Val Ile Phe Lieu Ala 865 87O 87s 88O Arg Asn. Asn. Thir Ile Val Asn. Glu Lieu Val Arg Val Thir Ser Glu Gly 885 890 895 Ala Gly Lieu. Glin Lieu. Glin Llys Val Thr Val Lieu. Gly Val Ala Thir Ala 9 OO 9 OS 910 Pro Glin Glin Val Lieu. Ser Asn Gly Val Pro Val Ser Asn Phe Thr Tyr 915 92O 925 Ser Pro Asp Thir Lys Val Lieu. Asp Ile Cys Val Ser Lieu. Lieu Met Gly 930 935 94 O Glu Glin Phe Leu Val Ser Trp Cys 945 950

What is claimed: (c) transfecting said END3 complementation group cells 1. A recombinant human acid alpha-glucosidase (rhGAA) with said expression vector, enzyme comprising an amino acid sequence at least 90% (d) selecting and cloning a END3 complementation group identical to the GAA amino acid sequence set out in SEQID cell transfectant; and NO: 2, produced by END3 complementation group CHO cells, wherein said rhGAA enzyme has at least about 0.7 (e) optimizing cell culture process methods for manufac bis-phosphorylated oligomannose chains per mole of rhGAA turing said END3 complementation group cell transfec enzyme, and wherein said END3 complementation group tant, CHO cell is a G71 cell line or derivative thereof. wherein said END3 complementation group cell is a G71 2. The rhGAA enzyme of claim 1, wherein said rhGAA cell line or derivative thereof. enzyme comprises an amino acid sequence at least 95% iden 5. The method of claim 4, wherein said rhGAA enzyme tical to the GAA amino acid sequence set out in SEQID NO: comprises an amino acid sequence at least 95% identical to 2 the GAA amino acid sequence set out in SEQID NO: 2. 3. A method for producing the rhGAA enzyme of claim 1, 6. A recombinant human acid alpha-glucosidase (rhGAA) comprising culturing Chinese Hamster Ovary (CHO)-de enzyme produced by the method of any one of claims 3 to 5. rived END3 complementation group cells under conditions 7. A pharmaceutical composition comprising the rhGAA appropriate for growth of the cells and expression of the GAA enzyme of claim 1 or 2, and a pharmaceutically acceptable enzyme. carrier, diluent or excipient. 4. A method for producing the rhGAA enzyme of claim 1, 8. A pharmaceutical composition comprising the rhGAA comprising the steps of enzyme of claim 6, and a pharmaceutically acceptable carrier, (a) culturing Chinese Hamster Ovary (CHO)-derived diluent or excipient. END3 complementation group cells; 9. A method of treating a deficiency of acid alpha-glucosi (b) preparing a mammalian expression vector encoding dase comprising administering to a subject in need a thera said rhGAA enzyme or variant thereof suitable for said peutically effective amount of the rhGAA enzyme of claim 1 END3 complementation group cells; or 2. US 2009/019 1178 A1 Jul. 30, 2009 46

10. The method of claim 9, wherein said subject has Pompe 13. The END3 complementation group CHO cell line of disease. claim 12, wherein the END3 complementation group cell line 11. The method of claim 9, wherein the subject is human. expresses and secretes rhGAA or a variant thereof. 12. An END3 complementation group CHO cell line com 14. The END3 complementation group CHO cell line of prising an expression vector for recombinant human acid claim 13, wherein said rhGAA enzyme comprises an amino alpha-glucosidase (rhGAA) enzyme, wherein said rhGAA acid sequence at least 95% identical to the GAA amino acid enzyme comprises an amino acid sequence at least 90% iden sequence set out in SEQID NO: 2. tical to the GAA amino acid sequence set out in SEQID NO: 2. c c c c c