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 (GA. 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 G 3. --- 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.