NZ729507B2 - Highly potent acid alpha-glucosidase with enhanced carbohydrates - Google Patents
Highly potent acid alpha-glucosidase with enhanced carbohydrates Download PDFInfo
- Publication number
- NZ729507B2 NZ729507B2 NZ729507A NZ72950715A NZ729507B2 NZ 729507 B2 NZ729507 B2 NZ 729507B2 NZ 729507 A NZ729507 A NZ 729507A NZ 72950715 A NZ72950715 A NZ 72950715A NZ 729507 B2 NZ729507 B2 NZ 729507B2
- Authority
- NZ
- New Zealand
- Prior art keywords
- rhgaa
- gaa
- atb
- glycans
- composition
- Prior art date
Links
- 108010028144 alpha-Glucosidases Proteins 0.000 title abstract description 12
- 235000014633 carbohydrates Nutrition 0.000 title description 12
- 102100008175 MGAM Human genes 0.000 title description 11
- 150000001720 carbohydrates Chemical class 0.000 title description 10
- 239000002253 acid Substances 0.000 title description 7
- 230000003389 potentiating Effects 0.000 title description 3
- 239000000203 mixture Substances 0.000 claims abstract description 84
- 206010053185 Glycogen storage disease type II Diseases 0.000 claims description 56
- 201000004502 glycogen storage disease II Diseases 0.000 claims description 56
- 101710010383 GAA Proteins 0.000 claims description 55
- 102100008255 GAA Human genes 0.000 claims description 55
- UQRORFVVSGFNRO-UTINFBMNSA-N Miglustat Chemical compound CCCCN1C[C@H](O)[C@@H](O)[C@H](O)[C@H]1CO UQRORFVVSGFNRO-UTINFBMNSA-N 0.000 claims description 21
- 229960001512 Miglustat Drugs 0.000 claims description 21
- 239000003814 drug Substances 0.000 claims description 18
- 238000004519 manufacturing process Methods 0.000 claims description 9
- 239000011780 sodium chloride Substances 0.000 claims description 9
- 230000000144 pharmacologic effect Effects 0.000 claims description 8
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 claims description 8
- 150000003839 salts Chemical class 0.000 claims description 6
- NBSCHQHZLSJFNQ-RWOPYEJCSA-N β-D-mannose 6-phosphate Chemical compound O[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H](O)[C@@H]1O NBSCHQHZLSJFNQ-RWOPYEJCSA-N 0.000 claims description 6
- 125000005629 sialic acid group Chemical group 0.000 claims description 4
- 101000525742 GAA Proteins 0.000 claims description 2
- 102000027592 human GAA protein Human genes 0.000 claims description 2
- NBSCHQHZLSJFNQ-QTVWNMPRSA-N Mannose-6-phosphate Chemical compound OC1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H](O)[C@@H]1O NBSCHQHZLSJFNQ-QTVWNMPRSA-N 0.000 abstract description 50
- 241000282414 Homo sapiens Species 0.000 abstract description 15
- 150000004676 glycans Chemical class 0.000 abstract description 9
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 abstract description 6
- 229920001542 oligosaccharide Polymers 0.000 abstract description 4
- 150000002482 oligosaccharides Polymers 0.000 abstract description 4
- GZCGUPFRVQAUEE-KCDKBNATSA-N D-(+)-Galactose Natural products OC[C@@H](O)[C@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-KCDKBNATSA-N 0.000 abstract description 3
- 102000016679 alpha-Glucosidases Human genes 0.000 abstract 1
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 abstract 1
- 229940091827 Lumizyme Drugs 0.000 description 62
- 229920002527 Glycogen Polymers 0.000 description 58
- BYSGBSNPRWKUQH-UJDJLXLFSA-N Glycogen Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)[C@H](O)[C@@H](O)[C@@H](O[C@@H]2[C@H](O[C@H](O)[C@H](O)[C@H]2O)CO)O1 BYSGBSNPRWKUQH-UJDJLXLFSA-N 0.000 description 58
- 229940096919 Glycogen Drugs 0.000 description 58
- 210000004027 cells Anatomy 0.000 description 56
- 102100013307 IGF2R Human genes 0.000 description 51
- 101710032496 IGF2R Proteins 0.000 description 51
- 230000000694 effects Effects 0.000 description 35
- 210000003712 Lysosomes Anatomy 0.000 description 33
- 230000001868 lysosomic Effects 0.000 description 33
- 210000003205 Muscles Anatomy 0.000 description 31
- 201000010099 disease Diseases 0.000 description 31
- 210000001519 tissues Anatomy 0.000 description 26
- 210000002027 Muscle, Skeletal Anatomy 0.000 description 22
- 102000004190 Enzymes Human genes 0.000 description 21
- 108090000790 Enzymes Proteins 0.000 description 21
- 210000000663 muscle cells Anatomy 0.000 description 21
- 230000001603 reducing Effects 0.000 description 21
- 230000037250 Clearance Effects 0.000 description 20
- 230000035512 clearance Effects 0.000 description 20
- 229940088598 Enzyme Drugs 0.000 description 19
- 229940103023 Myozyme Drugs 0.000 description 19
- 238000002641 enzyme replacement therapy Methods 0.000 description 19
- 230000035492 administration Effects 0.000 description 18
- 238000006722 reduction reaction Methods 0.000 description 18
- 108020003175 receptors Proteins 0.000 description 16
- 102000005962 receptors Human genes 0.000 description 16
- 230000027455 binding Effects 0.000 description 15
- 230000003899 glycosylation Effects 0.000 description 15
- 238000006206 glycosylation reaction Methods 0.000 description 15
- 125000003275 alpha amino acid group Chemical group 0.000 description 14
- 102000004169 proteins and genes Human genes 0.000 description 14
- 108090000623 proteins and genes Proteins 0.000 description 14
- 229920003013 deoxyribonucleic acid Polymers 0.000 description 13
- 238000001990 intravenous administration Methods 0.000 description 13
- 230000002132 lysosomal Effects 0.000 description 11
- 229940079593 drugs Drugs 0.000 description 9
- 230000000366 juvenile Effects 0.000 description 9
- 239000000758 substrate Substances 0.000 description 9
- 230000001413 cellular Effects 0.000 description 8
- 210000002950 fibroblast Anatomy 0.000 description 8
- 230000001976 improved Effects 0.000 description 8
- 241000699802 Cricetulus griseus Species 0.000 description 7
- 241000508725 Elymus repens Species 0.000 description 7
- 102100005918 LAMP1 Human genes 0.000 description 7
- 210000003314 Quadriceps Muscle Anatomy 0.000 description 7
- 230000036461 convulsion Effects 0.000 description 7
- 239000007924 injection Substances 0.000 description 7
- -1 mannose glycan Chemical class 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 230000035755 proliferation Effects 0.000 description 7
- 102000005431 Molecular Chaperones Human genes 0.000 description 6
- 108010006519 Molecular Chaperones Proteins 0.000 description 6
- 210000004165 Myocardium Anatomy 0.000 description 6
- 238000009825 accumulation Methods 0.000 description 6
- 230000001058 adult Effects 0.000 description 6
- 239000000835 fiber Substances 0.000 description 6
- 230000001131 transforming Effects 0.000 description 6
- 108010009254 Lysosomal-Associated Membrane Protein 1 Proteins 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 229960004593 alglucosidase alfa Drugs 0.000 description 5
- 230000037396 body weight Effects 0.000 description 5
- 239000000969 carrier Substances 0.000 description 5
- 238000010828 elution Methods 0.000 description 5
- 230000001965 increased Effects 0.000 description 5
- 238000001802 infusion Methods 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 125000000311 mannosyl group Chemical group C1([C@@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 150000007523 nucleic acids Chemical class 0.000 description 5
- 230000000275 pharmacokinetic Effects 0.000 description 5
- 230000002829 reduced Effects 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- HAPOVYFOVVWLRS-UHFFFAOYSA-N Ethosuximide Chemical compound CCC1(C)CC(=O)NC1=O HAPOVYFOVVWLRS-UHFFFAOYSA-N 0.000 description 4
- 101700048185 LAMP1 Proteins 0.000 description 4
- 210000000587 Skeletal Muscle Fibers Anatomy 0.000 description 4
- 238000004166 bioassay Methods 0.000 description 4
- 230000000747 cardiac effect Effects 0.000 description 4
- 230000004700 cellular uptake Effects 0.000 description 4
- 230000002950 deficient Effects 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 230000001264 neutralization Effects 0.000 description 4
- 230000036231 pharmacokinetics Effects 0.000 description 4
- 229920000023 polynucleotide Polymers 0.000 description 4
- 239000002157 polynucleotide Substances 0.000 description 4
- 229920001184 polypeptide Polymers 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- ZJIHMALTJRDNQI-VFQQELCFSA-N (2R,3R,4R,5S)-2-(hydroxymethyl)piperidine-3,4,5-triol;hydrochloride Chemical compound Cl.OC[C@H]1NC[C@H](O)[C@@H](O)[C@@H]1O ZJIHMALTJRDNQI-VFQQELCFSA-N 0.000 description 3
- 210000002351 Embryonic Muscle Cell Anatomy 0.000 description 3
- 206010020751 Hypersensitivity Diseases 0.000 description 3
- 210000004185 Liver Anatomy 0.000 description 3
- 229940037201 Oris Drugs 0.000 description 3
- 229920002472 Starch Polymers 0.000 description 3
- 238000001042 affinity chromatography Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 102000004965 antibodies Human genes 0.000 description 3
- 108090001123 antibodies Proteins 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 239000000356 contaminant Substances 0.000 description 3
- 230000003247 decreasing Effects 0.000 description 3
- 230000002255 enzymatic Effects 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 230000002349 favourable Effects 0.000 description 3
- 230000002068 genetic Effects 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 230000003601 intercostal Effects 0.000 description 3
- 108010031099 mannose receptor Proteins 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 230000000750 progressive Effects 0.000 description 3
- 238000009256 replacement therapy Methods 0.000 description 3
- 201000004193 respiratory failure Diseases 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 210000002363 skeletal muscle cell Anatomy 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 239000008107 starch Substances 0.000 description 3
- 235000019698 starch Nutrition 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- 239000003981 vehicle Substances 0.000 description 3
- 230000003442 weekly Effects 0.000 description 3
- LXBIFEVIBLOUGU-FSIIMWSLSA-N 1,5-Dideoxy-1,5-Imino-D-Mannitol Chemical compound OC[C@@H]1NC[C@@H](O)[C@H](O)[C@H]1O LXBIFEVIBLOUGU-FSIIMWSLSA-N 0.000 description 2
- LXBIFEVIBLOUGU-JGWLITMVSA-N 1-Deoxynojirimycin Natural products OC[C@H]1NC[C@H](O)[C@@H](O)[C@@H]1O LXBIFEVIBLOUGU-JGWLITMVSA-N 0.000 description 2
- 108009000409 Autophagy Proteins 0.000 description 2
- 210000004369 Blood Anatomy 0.000 description 2
- 208000005846 Cardiomyopathy Diseases 0.000 description 2
- 229920001405 Coding region Polymers 0.000 description 2
- 206010011469 Crying Diseases 0.000 description 2
- 210000000805 Cytoplasm Anatomy 0.000 description 2
- FBPFZTCFMRRESA-KAZBKCHUSA-N D-Mannitol Natural products OC[C@@H](O)[C@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KAZBKCHUSA-N 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N DEOXYTHYMIDINE Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 229940093738 Enzymes for ALIMENTARY TRACT AND METABOLISM Drugs 0.000 description 2
- 210000003754 Fetus Anatomy 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N Hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 102100005117 IGF2 Human genes 0.000 description 2
- 101700070236 IGF2 Proteins 0.000 description 2
- 210000000987 Immune System Anatomy 0.000 description 2
- GUBGYTABKSRVRQ-UUNJERMWSA-N Lactose Natural products O([C@@H]1[C@H](O)[C@H](O)[C@H](O)O[C@@H]1CO)[C@H]1[C@@H](O)[C@@H](O)[C@H](O)[C@H](CO)O1 GUBGYTABKSRVRQ-UUNJERMWSA-N 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- 210000003470 Mitochondria Anatomy 0.000 description 2
- 208000010428 Muscle Weakness Diseases 0.000 description 2
- 210000003699 Muscle, Striated Anatomy 0.000 description 2
- 206010028372 Muscular weakness Diseases 0.000 description 2
- 210000004413 Myocytes, Cardiac Anatomy 0.000 description 2
- 102000036913 Myoglobin Human genes 0.000 description 2
- 108010062374 Myoglobin Proteins 0.000 description 2
- 210000003463 Organelles Anatomy 0.000 description 2
- 210000001672 Ovary Anatomy 0.000 description 2
- 210000001184 Pharyngeal Muscles Anatomy 0.000 description 2
- 102000030951 Phosphotransferases Human genes 0.000 description 2
- 108091000081 Phosphotransferases Proteins 0.000 description 2
- 241000411545 Punargentus Species 0.000 description 2
- 208000004756 Respiratory Insufficiency Diseases 0.000 description 2
- 210000000952 Spleen Anatomy 0.000 description 2
- 230000036462 Unbound Effects 0.000 description 2
- 230000000172 allergic Effects 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 239000003708 ampul Substances 0.000 description 2
- 108010006523 asialoglycoprotein receptor family Proteins 0.000 description 2
- 102000005427 asialoglycoprotein receptor family Human genes 0.000 description 2
- 201000008937 atopic dermatitis Diseases 0.000 description 2
- 230000006877 autophagy Effects 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M buffer Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 201000008031 cardiomyopathy Diseases 0.000 description 2
- 230000024881 catalytic activity Effects 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 230000002596 correlated Effects 0.000 description 2
- 238000010192 crystallographic characterization Methods 0.000 description 2
- 230000000994 depressed Effects 0.000 description 2
- 230000001627 detrimental Effects 0.000 description 2
- 239000008121 dextrose Substances 0.000 description 2
- 239000003995 emulsifying agent Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 2
- 230000000670 limiting Effects 0.000 description 2
- 235000019359 magnesium stearate Nutrition 0.000 description 2
- 239000000594 mannitol Substances 0.000 description 2
- 235000010355 mannitol Nutrition 0.000 description 2
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 2
- 239000002609 media Substances 0.000 description 2
- 230000000051 modifying Effects 0.000 description 2
- 210000000107 myocyte Anatomy 0.000 description 2
- MYMOFIZGZYHOMD-UHFFFAOYSA-N oxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 239000008194 pharmaceutical composition Substances 0.000 description 2
- 239000000825 pharmaceutical preparation Substances 0.000 description 2
- 238000000159 protein binding assay Methods 0.000 description 2
- 230000002797 proteolythic Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000001105 regulatory Effects 0.000 description 2
- 210000001189 slow twitch fiber Anatomy 0.000 description 2
- 230000001225 therapeutic Effects 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- ZMANZCXQSJIPKH-UHFFFAOYSA-N triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 2
- 229920002554 vinyl polymer Polymers 0.000 description 2
- 239000000080 wetting agent Substances 0.000 description 2
- WQZGKKKJIJFFOK-PHYPRBDBSA-N α-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N β-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- MIJDSYMOBYNHOT-UHFFFAOYSA-N 2-(ethylamino)ethanol Chemical compound CCNCCO MIJDSYMOBYNHOT-UHFFFAOYSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 244000215068 Acacia senegal Species 0.000 description 1
- 229920000856 Amylose Polymers 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- WVDDGKGOMKODPV-UHFFFAOYSA-N Benzyl alcohol Natural products OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 description 1
- 230000037177 Biodistribution Effects 0.000 description 1
- 210000004204 Blood Vessels Anatomy 0.000 description 1
- 210000004556 Brain Anatomy 0.000 description 1
- 210000001736 Capillaries Anatomy 0.000 description 1
- 206010007554 Cardiac failure Diseases 0.000 description 1
- 206010007572 Cardiac hypertrophy Diseases 0.000 description 1
- 208000006029 Cardiomegaly Diseases 0.000 description 1
- 206010051093 Cardiopulmonary failure Diseases 0.000 description 1
- 210000000349 Chromosomes Anatomy 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N D-sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 206010016165 Failure to thrive Diseases 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 102000004366 Glucosidases Human genes 0.000 description 1
- 108010056771 Glucosidases Proteins 0.000 description 1
- 206010053250 Glycogen storage disease type III Diseases 0.000 description 1
- 229920000084 Gum arabic Polymers 0.000 description 1
- 229940031574 HYDROXYMETHYL CELLULOSE Drugs 0.000 description 1
- 206010019280 Heart failure Diseases 0.000 description 1
- 210000003494 Hepatocytes Anatomy 0.000 description 1
- 102000038585 IGF Type 2 Receptor Human genes 0.000 description 1
- 108010031792 IGF Type 2 Receptor Proteins 0.000 description 1
- JJWLVOIRVHMVIS-UHFFFAOYSA-N Isopropylamine Chemical compound CC(C)N JJWLVOIRVHMVIS-UHFFFAOYSA-N 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- VAYOSLLFUXYJDT-RDTXWAMCSA-N LSD Chemical compound C1=CC(C=2[C@H](N(C)C[C@@H](C=2)C(=O)N(CC)CC)C2)=C3C2=CNC3=C1 VAYOSLLFUXYJDT-RDTXWAMCSA-N 0.000 description 1
- 229950002454 Lysergide Drugs 0.000 description 1
- 206010024579 Lysosomal storage disease Diseases 0.000 description 1
- ZLNQQNXFFQJAID-UHFFFAOYSA-L Magnesium carbonate Chemical compound [Mg+2].[O-]C([O-])=O ZLNQQNXFFQJAID-UHFFFAOYSA-L 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241000489861 Maximus Species 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 230000036740 Metabolism Effects 0.000 description 1
- 230000035633 Metabolized Effects 0.000 description 1
- 210000002464 Muscle, Smooth, Vascular Anatomy 0.000 description 1
- 210000000329 Myocytes, Smooth Muscle Anatomy 0.000 description 1
- 206010028640 Myopathy Diseases 0.000 description 1
- 108010040066 N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase Proteins 0.000 description 1
- 241000158526 Nasalis Species 0.000 description 1
- ODUCDPQEXGNKDN-UHFFFAOYSA-N Nitroxyl Chemical compound O=N ODUCDPQEXGNKDN-UHFFFAOYSA-N 0.000 description 1
- 229920001850 Nucleic acid sequence Polymers 0.000 description 1
- 210000000062 Pectoralis major Anatomy 0.000 description 1
- 210000000989 Pectoralis minor Anatomy 0.000 description 1
- 229940023488 Pill Drugs 0.000 description 1
- 210000002381 Plasma Anatomy 0.000 description 1
- 230000037289 Plasma half life Effects 0.000 description 1
- 230000037240 Plasma half-life Effects 0.000 description 1
- 241000223503 Platysma Species 0.000 description 1
- 229940068968 Polysorbate 80 Drugs 0.000 description 1
- 229940068965 Polysorbates Drugs 0.000 description 1
- MFDFERRIHVXMIY-UHFFFAOYSA-N Procaine Chemical compound CCN(CC)CCOC(=O)C1=CC=C(N)C=C1 MFDFERRIHVXMIY-UHFFFAOYSA-N 0.000 description 1
- 241000225674 Procerus Species 0.000 description 1
- 208000008425 Protein Deficiency Diseases 0.000 description 1
- 102000007312 Recombinant Proteins Human genes 0.000 description 1
- 108010033725 Recombinant Proteins Proteins 0.000 description 1
- 210000001139 Rectus Abdominis Anatomy 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- CVHZOJJKTDOEJC-UHFFFAOYSA-N Saccharin Chemical compound C1=CC=C2C(=O)NS(=O)(=O)C2=C1 CVHZOJJKTDOEJC-UHFFFAOYSA-N 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- RMAQACBXLXPBSY-UHFFFAOYSA-N Silicic acid Chemical compound O[Si](O)(O)O RMAQACBXLXPBSY-UHFFFAOYSA-N 0.000 description 1
- 210000001088 Stapedius Anatomy 0.000 description 1
- CZMRCDWAGMRECN-GDQSFJPYSA-N Sucrose Natural products O([C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O1)[C@@]1(CO)[C@H](O)[C@@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-GDQSFJPYSA-N 0.000 description 1
- 210000000516 Tensor Tympani Anatomy 0.000 description 1
- 229940104230 Thymidine Drugs 0.000 description 1
- 206010066901 Treatment failure Diseases 0.000 description 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O Chemical class [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- 239000000205 acacia gum Substances 0.000 description 1
- 235000010489 acacia gum Nutrition 0.000 description 1
- 230000002378 acidificating Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000002730 additional Effects 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000002052 anaphylactic Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 239000012062 aqueous buffer Substances 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 235000019445 benzyl alcohol Nutrition 0.000 description 1
- 150000003938 benzyl alcohols Chemical class 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 229960000074 biopharmaceuticals Drugs 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 210000002317 cardiac myocyte Anatomy 0.000 description 1
- 230000001925 catabolic Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 230000000536 complexating Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000003636 conditioned culture media Substances 0.000 description 1
- 230000000875 corresponding Effects 0.000 description 1
- 230000003316 cricothyroid Effects 0.000 description 1
- 230000002354 daily Effects 0.000 description 1
- 230000000593 degrading Effects 0.000 description 1
- 230000003111 delayed Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000002708 enhancing Effects 0.000 description 1
- 238000007824 enzymatic assay Methods 0.000 description 1
- 230000001747 exhibiting Effects 0.000 description 1
- 230000028023 exocytosis Effects 0.000 description 1
- 239000003925 fat Substances 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000010363 gene targeting Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 201000004543 glycogen storage disease III Diseases 0.000 description 1
- 230000002414 glycolytic Effects 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000004217 heart function Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 150000004679 hydroxides Chemical class 0.000 description 1
- 229920003063 hydroxymethyl cellulose Polymers 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000010874 in vitro model Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000000977 initiatory Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000003834 intracellular Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000009114 investigational therapy Methods 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- 239000003589 local anesthetic agent Substances 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 239000001095 magnesium carbonate Substances 0.000 description 1
- 239000011776 magnesium carbonate Substances 0.000 description 1
- 229910000021 magnesium carbonate Inorganic materials 0.000 description 1
- 101700009216 malA Proteins 0.000 description 1
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000002503 metabolic Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000035786 metabolism Effects 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- 230000004118 muscle contraction Effects 0.000 description 1
- 230000003387 muscular Effects 0.000 description 1
- 201000010770 muscular disease Diseases 0.000 description 1
- 210000003098 myoblast Anatomy 0.000 description 1
- 201000009623 myopathy Diseases 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 210000002569 neurons Anatomy 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000003204 osmotic Effects 0.000 description 1
- 239000006174 pH buffer Substances 0.000 description 1
- 239000006179 pH buffering agent Substances 0.000 description 1
- 239000005022 packaging material Substances 0.000 description 1
- 230000003285 pharmacodynamic Effects 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000000865 phosphorylative Effects 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000000244 polyoxyethylene sorbitan monooleate Substances 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- OZAIFHULBGXAKX-UHFFFAOYSA-N precursor Substances N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 description 1
- 230000002335 preservative Effects 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 229960004919 procaine Drugs 0.000 description 1
- 230000012846 protein folding Effects 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 230000002685 pulmonary Effects 0.000 description 1
- 230000035812 respiration Effects 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- KEAYESYHFKHZAL-UHFFFAOYSA-N sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000008227 sterile water for injection Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002459 sustained Effects 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 235000012222 talc Nutrition 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- 231100000730 tolerability Toxicity 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 210000003854 type 2 muscle cell Anatomy 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 230000003313 weakening Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
- C12N2510/02—Cells for production
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2465—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/0102—Alpha-glucosidase (3.2.1.20)
Abstract
Recombinant human alpha glucosidase (rhGAA) composition derived from CHO cells that contains a more optimized glycan composition consisting of a higher amount of rhGAA containing N-glycans carrying mannose-6-phosphate (M6P) or bis-M6P than conventional rhGAAs, along with low amount of non-phosphorylated high mannose glycans, and low amount of terminal galactose on complex oligosaccharides. Compositions containing the rhGAA, and methods of use are described. ated high mannose glycans, and low amount of terminal galactose on complex oligosaccharides. Compositions containing the rhGAA, and methods of use are described.
Description
TITLE OF THE INVENTION
HIGHLY POTENT ACID ALPHA-GLUCOSIDASE WITH
ENHANCED CARBOHYDRATES
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of priority to U.S. Provisional 62/057,842, filed
September 30, 2014, U.S. Provisional 62/057,847, filed September 30, 2014, U.S. Provisional
62/112,463, filed February 5, 2015, and to U.S. Provisional 62/135,345, filed March 19, 2015
each of which is incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention involves the fields of medicine, genetics and recombinant
glycoprotein biochemistry, and, specifically, relates to recombinant human alpha glucosidase
(rhGAA) compositions that have a higher total content of mannose 6-phosphate-bearing
glycans that efficiently target CIMPR on muscle cells and subsequently deliver rhGAA to the
lysosomes where it can break down abnormally high levels of accumulated glycogen. The
rhGAA of the invention exhibits superior targeting to muscle cells and subsequent delivery to
lysosomes compared to conventional rhGAA products and exhibits other pharmacokinetic
properties that make it particularly effective for enzyme replacement therapy of subjects
having Pompe disease.
Description of the Related Art
Existing enzyme replacement therapies for Pompe Disease use conventional rhGAA
products that have a low total content of M6P and bis-M6P bearing glycans. Conventional
products are known under the names Lumizyme®, Myozyme® and Alglucosidase alfa.
“Lumizyme” and “Myozyme” are conventional forms of rhGAA produced or marketed as
biologics by Genzyme and approved by the U.S. Food and Drug Administration and are
described by reference to the Physician’s Desk Reference (2014)(which is hereby
incorporated by reference) or by the products named “Lumizyme®” or “Myozyme®”
approved for use in the United States by the FDA as of October 1, 2014. Alglucosidase Alfa is
identified as chemical name [199-arginine,223-histidine]prepro-α-glucosidase (human);
molecular formula, C H N O S CAS number 4207940. These products are
4758 7262 1274 1369 35;
administered to subjects with Pompe Disease, also known as glycogen storage disease type II
(GSD-II) or acid maltase deficiency disease. Enzyme replacement therapy seeks to treat
Pompe Disease by replacing the missing GAA in lysosomes by administering rhGAA thus
restoring the ability of cell to break down lysosomal glycogen
Pompe disease is an inherited lysosomal storage disease that results from a deficiency
in acid α-glucosidase (GAA) activity. A person having Pompe Disease lacks or has reduced
levels of acid alpha-glucosidase (GAA), the enzyme which breaks down glycogen, and a
substance the body uses as an energy source. This enzyme deficiency causes excess glycogen
accumulation in the lysosomes, which are intra-cellular organelles containing enzymes that
ordinarily break down glycogen and other cellular debris or waste products. Glycogen
accumulation in certain tissues of a subject having Pompe Disease, especially muscles,
impairs the ability of cells to function normally. In Pompe Disease, glycogen is not properly
metabolized and progressively accumulates in the lysosomes, especially in skeletal muscle
cells and, in the infant onset form of the disease, in cardiac muscle cells. The accumulation
of glycogen damages the muscle and nerve cells as well as those in other affected tissues
Traditionally, depending on the age of onset, Pompe disease is clinically recognized
as either an early infantile form or as a late onset form. The age of onset tends to parallel the
severity of the genetic mutation causing Pompe Disease. The most severe genetic mutations
cause complete loss of GAA activity manifest as early onset disease during infancy. Genetic
mutations that diminish GAA activity but do not completely eliminate it are associated with
forms of Pompe disease having delayed onset and progression. Infantile onset Pompe disease
manifests shortly after birth and is characterized by muscular weakness, respiratory
insufficiency and cardiac failure. Untreated, it is usually fatal within two years. Juvenile and
adult onset Pompe disease manifest later in life and usually progress more slowly than
infantile onset disease. This form of the disease, while it generally does not affect the heart,
may also result in death, due to weakening of skeletal muscles and those involved in
respiration.
Current non-palliative treatment of Pompe disease involves enzyme replacement
therapy (ERT) using recombinant human GAA (rhGAA) such as Lumizyme® or
Myozyme®. The rhGAA is administered in an attempt to replace or supplement the missing
or defective GAA in a subject having Pompe Disease. However, since most of the rhGAA in
conventional rhGAA products does not target muscle tissue it is non-productively eliminated
after administration.
This occurs because conventional rhGAAs lack a high total content of M6P- and bis-
M6P-bearing glycans which target a rhGAA molecule to the CIMPR on target muscle cells
where it is subsequently transported into the cell’s lysosomes. This cellular uptake of rhGAA
for enzyme replacement therapy is facilitated by the specialized carbohydrate, mannose
phosphate (M6P), which binds to the cation-independent mannose 6-phosphate receptor
(CIMPR) present on cell surfaces for subsequent delivery of the exogenous enzyme to
lysosomes.
There are seven potential N-linked glycosylation sites on rhGAA. Since each
glycosylation site is heterogeneous in the type of N-linked oligosaccharides (N-glycans)
present, rhGAA consist of a complex mixture of proteins with N-glycans having varying
binding affinities for M6P receptor and other carbohydrate receptors. rhGAA that contains a
high mannose N-glycans having one M6P group (mono-M6P) binds to CIMPR with low
(~6,000 nM) affinity while rhGAA that contains two M6P groups on same N-glycan (bis-
M6P) bind with high (~2 nM) affinity. Representative structures for non-phosphorylated,
mono-M6P, and bis-M6P glycans are shown by Fig. 1A. The mannoseP group is shown
by Fig. 1B. Once inside the lysosome, rhGAA can enzymatically degrade accumulated
glycogen. However, conventional rhGAAs have low total levels of M6P- and bis-M6P-
bearing glycans and, thus, target muscle cells poorly resulting in inferior delivery of rhGAA
to the lysosomes. The majority of rhGAA molecules in these conventional products do not
have phosphorylated N-glycans, thereby lacking affinity for the CIMPR. Non-phosphorylated
high mannose glycans can also be cleared by the mannose receptor which results in non-
productive clearance of the ERT (Fig. 2).
The other type of N-glycans, complex carbohydrates, which contain, galactose and
sialic acids are also present on rhGAA. Since complex N-glycans are not phosphorylated they
have no affinity for CIMPR. However, complex -type N-glycans with exposed galactose
residues have moderate to high affinity for the asialoglycoprotein receptor on liver
hepatocytes which leads to rapid non-productive clearance of rhGAA (Fig 2).
The glycosylation of GAA or rhGAA can be enzymatically modified in vitro by the
phosphotransferase and uncovering enzymes described by Canfield, et al., U.S. Patent No.
6,534,300, to generate M6P groups. Enzymatic glycosylation cannot be adequately
controlled and produces rhGAA having undesirable immunological and pharmacological
properties. Enzymatically modified rhGAA may contain only high-mannose N-glycans
which all could be potentially enzymatically phosphorylated in vitro with a
phosphotransferase/uncovering enzyme and may contain on average 5-6 M6P groups per
GAA. The glycosylation patterns produced by in vitro enzymatic treatment of GAA are
problematic because the additional terminal mannose residues, particularly non-
phosphorylated terminal mannose residues, negatively affect the pharmacokinetics of the
modified rhGAA. When such an enzymatically modified product is administered in vivo,
these mannose groups increase non-productive clearance of the GAA, increase the uptake of
the enzymatically-modified GAA by immune cells, and reduce rhGAA therapeutic efficacy
due to less of the GAA reaching targeted tissues, such as cardiac or skeletal muscle
myocytes. For example, terminal non-phosphorylated mannose residues are known ligands
for mannose receptors in the liver and spleen which leads to rapid clearance of the
enzymatically-modified rhGAA and reduced targeting of rhGAA to target tissue. Moreover,
the glycosylation pattern of enzymatically-modified GAA having high mannose N-glycans
with terminal non-phosphorylated mannose residues resembles that on glycoproteins
produced in yeasts, molds and function increasing the risk of triggering immune or allergic
responses, such as life-threatening severe allergic (anaphylactic) or hypersensitivity reactions,
to the enzymatically modified rhGAA.
As explained above, conventional rhGAA products like Lumizyme have low levels
of mono-phosphorylated glycans and even lower bis-phosphorylated glycans. In order for a
Pompe disease therapy to be efficacious rhGAA must be delivered to the lysosomes in
muscle cells. The low total amount of mono-M6P and bis-M6P targeting groups on
conventional rhGAA limits cellular uptake via CIMPR and lysosomal delivery, thus making
conventional enzyme replacement therapy inefficient. For example, while conventional
rhGAA products at 20 mg/kg or higher doses do ameliorate some aspects of Pompe disease,
they are not able to adequately reduce accumulated glycogen in many target tissues,
particularly skeletal muscles to reverse disease progression.
Due to the inefficiency of delivering conventional enzyme replacement therapies to
lysosomes, such therapies are often associated with other problems, including generation of
immune responses to GAA. A large portion of the GAA in a conventional rhGAA does not
contain glycans bearing mono- or bis-M6P, which target the rhGAA to muscle cells. A
subject’s immune system is exposed to this excess non-phosphorylated GAA and can
generate detrimental immune responses that recognize GAA. Induction of an immune
responses to the non-phosphorylated GAA that does not enter the target tissues and deliver to
the lysosomes increase the risk of treatment failure due to immunological inactivation of the
administered rhGAA and increases the risk of the patient experiencing detrimental
autoimmune or allergic reactions to the rhGAA treatment. The rhGAA according to the
invention contains significantly less of this non-targeted, non-phosphorylated rhGAA, thus
reducing exposure of a patient’s immune system to it.
Logistically, larger doses impose additional burdens on the subject as well as medical
professionals treating the subject, such as lengthening the infusion time needed to administer
rhGAA intravenously. This is because conventional rhGAA’s contain a higher content of
non-phosphorylated rhGAA which does not target the CIMPR on muscle cells. rhGAA that
does not bind to CIMPR on muscle cells and then enter the lysosome does not enzymatically
degrade glycogen there. When equivalent doses of a conventional rhGAA and the rhGAA
according to the invention are administered, more rhGAA in the composition according to the
invention binds CIMPR on muscle cells and then delivers to the lysosome. The rhGAA of
the invention provides a doctor with the option of administering a lower amount of rhGAA
while delivering the same or more rhGAA to the lysosome.
Current manufacturing processes used to make conventional rhGAA, such as
Myozyme®, Lumizyme® or Alglucosidase Alfa, have not significantly increased the content of M6P or
bis-M6P because cellular carbohydrate processing is naturally complex and extremely difficult
to manipulate. In view of these deficiencies of conventional rhGAA products, the inventors
diligently sought and identified ways to efficiently target rhGAA to muscle cells and deliver
it to the lysosome, minimize non-productive clearance of rhGAA once administered, and thus
more productively target rhGAA to muscle tissue.
BRIEF SUMMARY OF THE INVENTION
In response to the problems associated with targeting and administering conventional
forms of rhGAA and to the difficulties associated with producing such well-targeted forms of
rhGAA, the inventors have investigated and developed procedures for making rhGAA that
more efficiently targets the CIMPR and deliver it to lysosomes in muscle tissues because it
has a higher content of M6P- and bis-M6P glycan than conventional rhGAA compositions.
Moreover, rhGAA of the invention has well-processed complex-type N-glycans which
minimize non-productive clearance of the rhGAA by non-target tissues.
Taking into account the problems associated with current enzyme replacement
treatments using conventional rhGAA products such as Lumizyme®, through diligent study
and investigation the inventors have developed a method for producing rhGAA in CHO cells
having significantly higher total content of mono-M6P and bis-M6P glycans which target
CIMPR on muscle cells and then deliver the rhGAA to the lysosomes.
The rhGAA produced by this method also has advantageous pharmacokinetic
properties by virtue of its overall glycosylation pattern that increases target tissue uptake and
decreases non-productive clearance following administration to a subject having Pompe
Disease. The inventors show that the rhGAA of the invention, as exemplified by rhGAA
designated as ATB-200, is more potent in and more efficient at targeting skeletal muscle
tissues than conventional rhGAA such as Lumizyme®. The rhGAA according to the
invention has a superior ability to productively target muscle tissues in patients having
Pompe Disease and reduce non-productive clearance of rhGAA as illustrated by Fig. 2.
The superior rhGAA according to the invention may be further completed or
combined with chaperones or conjugated to other groups that target the CIMPR in muscle
tissue, such as portions of IGF2 that bind to this receptor. The Examples below show that the
rhGAA of the invention, exemplified by ATB-200 rhGAA, exceeds the existing standard of
care for enzyme replacement therapy by providing significantly better glycogen clearance in
skeletal muscle as compared to existing regimen using the conventional rhGAA product
Lumizyme®.
BRIEF DESCRIPTION OF THE DRAWINGS
This application file contains at least one drawing executed in color.
Fig. 1 Fig. 1A shows a non-phosphorylated high mannose glycan, a mono-M6P glycan,
and a bis-M6P glycan. Fig. 1B shows the chemical structure of the M6P group.
Fig. 2 Fig. 2A describes productive targeting of rhGAA via glycans bearing M6P to target
tissues (e.g., muscle tissues of subject with Pompe Disease). Fig. 2B describes
non-productive drug clearance to non-target tissues (e.g., liver and spleen) or by
binding of non-M6P glycans to non-target tissues.
Fig. 3 Fig. 3A graphically depicts a CIMPR receptor (also known as an IGF2 receptor)
and domains of this receptor. Fig. 3B is a table showing binding affinity (nMolar)
of glycans bearing bis- and mono-M6P for CIMPR, the binding affinity of high
mannose-type glycans to mannose receptors, and the binding affinity of de-
sialyated complex glycan for asialyoglycoprotein receptors. RhGAA that has
glycans bearing M6P and bis-M6P can productively bind to CIMPR on muscle
target cells. RhGAA that has high mannose glycans and de-sialylated glycans can
non-productively bind to non-target cells bearing the corresponding receptors.
Fig. 4 Figs. 4A and 4B respectively, show the results of CIMPR affinity chromatography
of Lumizyme® and Myozyme®. The dashed lines refer to the M6P elution
gradient. Elution with M6P displaces GAA molecules bound via an M6P-
containing glycan to CIMPR. As shown in Fig. 4A, 78% of the GAA activity in
Lumizyme® eluted prior to addition of M6P. Fig. 4B shows that 73% of the GAA
Myozyme® activity eluted prior to addition of M6P. Only 22% or 27% of the
rhGAA in Lumizyme® or Myozyme, respectively, was eluted with M6P. These
figures show that most of the rhGAA in these two conventional rhGAA products
lack glycans having M6P needed to target CIMPR in target muscle tissues.
Fig. 5 A DNA construct for transforming CHO cells with DNA encoding rhGAA. CHO
cells were transformed with a DNA construct encoding rhGAA (SEQ ID NO: 4).
Fig. 6 Figs. 6A and 6B show the results of CIMPR affinity chromatography of Myozyme
and ATB-200 rhGAA. As apparent from Fig. 6B, about 70% of the rhGAA in
ATB-200 rhGAA contained M6P.
Fig. 7 ATB-200 rhGAA purification, Embodiment 1 & 2.
Fig. 8 Polywax elution profiles of Lumizyme® and ATB-200.rhGAAs.
Fig. 9 Summary of N-glycan structures of Lumizyme® compared to three different
preparations of ATB200 rhGAA, identified as BP-rhGAA, ATB200-1 and
ATB200-2.
Fig. 10 Fig. 10A compares the CIMPR binding affinity of ATB-200 rhGAA (left trace)
with that of Lumizyme® (right trace). Fig. 10B describes the Bis-M6P content of
Lumizyme® and ATB-200 rhGAA.
Fig. 11 Fig. 11A compares ATB-200 rhGAA activity (left trace) with Lumizyme® rhGAA
activity (right trace) inside normal fibroblasts at various GAA concentrations. Fig.
11B compares ATB-200 rhGAA activity (left trace) with Lumizyme® rhGAA
activity (right trace) inside fibroblasts from a subject having Pompe Disease at
various GAA concentrations. Fig. 11C compares (K ) of fibroblasts from
uptake
normal subjects and subjects with Pompe Disease.
Fig. 12 Fig. 12A shows the amount of glycogen relative to protein in heart muscle after
contact with vehicle (negative control), with 20 mg/ml Alglucosidase alfa, or with
, 10 or 20 mg/kg ATB-200 rhGAA. Fig. 12B shows the amount of glycogen
relative to protein in quadriceps muscle after contact with vehicle (negative
control), with 20 mg/ml Lumizyme®, or with 5, 10 or 20 mg/kg ATB-200 rhGAA.
Fig. 12C shows the amount of glycogen relative to protein in triceps muscle after
contact with vehicle (negative control), with 20 mg/ml Lumizyme®, or with 5, 10
or 20 mg/kg ATB-200 rhGAA. ATB-200 rhGAA produced significant glycogen
reductions in quadriceps and triceps muscle compared to the negative control and
compared to Lumizyme®.
Fig. 13 ATB-200 rhGAA stability is improved in the presence of chaperone AT2221. The
first, left trace in Fig. 13A shows percentage of unfolded ATB-200 rhGAA protein
at various temperatures at pH 7.4 (blood pH). The last, right trace shows
percentage of unfolded ATB-200 rhGAA protein at various temperatures at pH 5.2
(lysosome pH). The three intermediate traces show the effects of 10µg, 30 µg , or
100 µg of AT2221 chaperone on protein folding. These data show that AT2221
prevents unfolding of ATB-200 rhGAA at blood pH compared to the control
sample. The improvement of Tm at neutral pH by AT2221 is summarized in
Figure 13B.
Fig. 14 This table shows that the combination of ATB-200 rhGAA and chaperone AT2221
provided significantly better glycogen clearance in GAA knock-out mice than
treatments with Lumizyme® and AT2221 or controls of either Lumizyme® or
ATB200 rhGAAs without the AT2221 chaperone.
Fig. 15 Residual glycogen in quadriceps muscle after treatment with Lumizyme, ATB-200
rhGAA, or ATB-200 rhGAA and various concentrations of the AT2221 chaperone.
Fig. 16 Improvement of Skeletal Muscle Pathology in Mice treated with ATB200 +
Miglustat (AT2221) over those treated with ERT alone. PAS glycogen staining
(Fig. 16A) and EM (Fig. 16 B) of muscle tissue from GAA KO mice treated with
conventional rhGAA or ATB-200 rhGAA and miglustat (AT-2221). Fig. 16C;
Evaluation of lysosomal proliferation by LAMP-1 marker. Fig. 16D Identification
of Type I and Type II muscle fibers.
Fig. 17. Improvement of Skeletal Muscle Pathology in Mice treated with ATB-200 +
Miglustat (AT2221) over those treated with ERT alone. PAS glycogen staining
(Fig. 17A) of muscle tissue from GAA KO mice treated with conventional rhGAA
or ATB-200 rhGAA and miglustat (AT-2221). Fig. 17B; Evaluation of lysosomal
proliferation by LAMP-1 marker.
DETAILED DESCRIPTION OF THE INVENTION
Definitions: The terms used in this specification generally have their ordinary
meanings in the art, within the context of this invention and in the specific context where
each term is used. Certain terms are discussed below, or elsewhere in the specification, to
provide additional guidance to the practitioner in describing the compositions and methods of
the invention and how to make and use them.
The term “GAA” refers to human acid α-glucosidase (GAA) an enzyme that catalyzes
the hydrolysis of α-1,4- and α-1,6-glycosidic linkages of lysosomal glycogen as well as to
insertional, relational or substitution variants of the GAA amino acid sequence and fragments
of a longer GAA sequence that exert enzymatic activity. The term “rhGAA” is used to
distinguish endogenous GAA from synthetic or recombinant-produced GAA, such as that
produced by transformation of CHO cells with DNA encoding GAA. An exemplary DNA
sequence encoding GAA is NP_000143.2 (SEQ ID NO: 4) which is incorporated by
reference. GAA and rhGAA may be present in a composition containing a mixture of GAA
molecules having different glycosylation patterns, such as a mixture of rhGAA molecules
bearing mono-M6P or bis-M6P groups on their glycans and GAA molecules that do not bear
M6P or bis-M6P. GAA and rhGAA may also be completed with other compounds, such as
chaperones, or may be bound to other moieties in a GAA or rhGAA conjugate, such as bound
to an IGF2 moiety that targets the conjugate to CIMPR and subsequently delivers it to the
lysosomes.
A "subject" or "patient" is preferably a human, though other mammals and non-
human animals having disorders involving accumulation of glycogen may also be treated. A
subject may be a fetus, a neonate, child, juvenile or an adult with Pompe disease or other
glycogen storage or accumulation disorder. One example of an individual being treated is an
individual (fetus, neonate, child, juvenile, adolescent, or adult human) having GSD-II (e.g.,
infantile GSD-II, juvenile GSD-II, or adult-onset GSD-II). The individual can have residual
GAA activity, or no measurable activity. For example, the individual having GSD-II can have
GAA activity that is less than about 1% of normal GAA activity (infantile GSD-II), GAA
activity that is about 1-10% of normal GAA activity (juvenile GSD-II), or GAA activity that
is about 10-40% of normal GAA activity (adult GSD-II).
The terms, "treat" and "treatment," as used herein, refer to amelioration of one or
more symptoms associated with the disease, prevention or delay of the onset of one or more
symptoms of the disease, and/or lessening of the severity or frequency of one or more
symptoms of the disease. For example, treatment can refer to improvement of cardiac status
(e.g., increase of end-diastolic and/or end-systolic volumes, or reduction, amelioration or
prevention of the progressive cardiomyopathy that is typically found in GSD-II) or of
pulmonary function (e.g., increase in crying vital capacity over baseline capacity, and/or
normalization of oxygen desaturation during crying); improvement in neurodevelopment
and/or motor skills (e.g., increase in AIMS score); reduction of glycogen levels in tissue of
the individual affected by the disease; or any combination of these effects. In one preferred
embodiment, treatment includes improvement of cardiac status, particularly in reduction or
prevention of GSD-II-associated cardiomyopathy.
The terms, "improve," "increase" or "reduce," as used herein, indicate values that are
relative to a baseline measurement, such as a measurement in the same individual prior to
initiation of the treatment described herein, or a measurement in a control individual (or
multiple control individuals) in the absence of the treatment described herein. A control
individual is an individual afflicted with the same form of GSD-II (either infantile, juvenile
or adult-onset) as the individual being treated, who is about the same age as the individual
being treated (to ensure that the stages of the disease in the treated individual and the control
individual(s) are comparable).
The term "purified" as used herein refers to material that has been isolated under
conditions that reduce or eliminate the presence of unrelated materials, i.e., contaminants,
including native materials from which the material is obtained. For example, a purified
protein is preferably substantially free of other proteins or nucleic acids with which it is
associated in a cell; a purified nucleic acid molecule is preferably substantially free of
proteins or other unrelated nucleic acid molecules with which it can be found within a cell.
As used herein, the term "substantially free" is used operationally, in the context of analytical
testing of the material. Preferably, purified material substantially free of contaminants is at
least 95% pure; more preferably, at least 97% pure, and more preferably still at least 99%
pure. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay,
composition analysis, biological assay, enzymatic assay and other methods known in the art.
In a specific embodiment, purified means that the level of contaminants is below a level
acceptable to regulatory authorities for safe administration to a human or non-human animal.
Recombinant proteins may be isolated or purified from CHO cells using methods known in
the art including by chromatographic size separation, affinity chromatography or anionic
exchange chromatography.
The term "genetically modified" or “recombinant” refers to cells, such as CHO cells,
that express a particular gene product, such as rhGAA or ATB-200 rhGAA, following
introduction of a nucleic acid comprising a coding sequence which encodes the gene product,
along with regulatory elements that control expression of the coding sequence. Introduction
of the nucleic acid may be accomplished by any method known in the art including gene
targeting and homologous recombination. As used herein, the term also includes cells that
have been engineered to express or overexpress an endogenous gene or gene product not
normally expressed by such cell, e.g., by gene activation technology.
"Pompe Disease" refers to an autosomal recessive LSD characterized by deficient acid
alpha glucosidase (GAA) activity which impairs lysosomal glycogen metabolism. The
enzyme deficiency leads to lysosomal glycogen accumulation and results in progressive
skeletal muscle weakness, reduced cardiac function, respiratory insufficiency, and/or CNS
impairment at late stages of disease. Genetic mutations in the GAA gene result in either lower
expression or produce mutant forms of the enzyme with altered stability, and/or biological
activity ultimately leading to disease. (see generally Hirschhorn R, 1995, Glycogen Storage
Disease Type II: Acid α-Glucosidase (Acid Maltase) Deficiency, The Metabolic and
Molecular Bases of Inherited Disease, Scriver et al., eds., McGraw-Hill, New York, 7th ed.,
pages 2443-2464). The three recognized clinical forms of Pompe Disease (infantile, juvenile
and adult) are correlated with the level of residual α-glucosidase activity (Reuser A J et al.,
1995, Glycogenosis Type II (Acid Maltase Deficiency), Muscle & Nerve Supplement 3, S61-
S69). Infantile Pompe disease (type I or A) is most common and most severe, characterized
by failure to thrive, generalized hypotonic, cardiac hypertrophy, and cardiorespiratory failure
within the second year of life. Juvenile Pompe disease (type II or B) is intermediate in
severity and is characterized by a predominance of muscular symptoms without
cardiomegaly. Juvenile Pompe individuals usually die before reaching 20 years of age due to
respiratory failure. Adult Pompe disease (type III or C) often presents as a slowly progressive
myopathy in the teenage years or as late as the sixth decade (Felicia K J et al., 1995, Clinical
Variability in Adult-Onset Acid Maltase Deficiency: Report of Affected Sibs and Review of
the Literature, Medicine 74, 131-135). In Pompe, it has been shown that α-glucosidase is
extensively modified post-translationally by glycosylation, phosphorylation, and proteolytic
processing. Conversion of the 110 kilo Dalton (kids) precursor to 76 and 70 kids mature
forms by proteolysis in the lysosome is required for optimum glycogen catalysis. As used
herein, the term "Pompe Disease" refers to all types of Pompe Disease. The formulations and
dosing regimens disclosed in this application may be used to treat, for example, Type I, Type
II or Type III Pompe Disease.
Non-limiting embodiments of the invention
A rhGAA composition derived from CHO cells that contains a higher amount of
rhGAA containing N-glycans carrying mono-mannosephosphate (M6P) or bis-M6P than
conventional rhGAA as exemplified by Lumizyme® (Alglucosidase Alfa; CAS 4207940).
An exemplary rhGAA composition according to the invention is ATB-200 (sometimes
designated ATB-200, ATB-200 or CBP-rhGAA) which is described in the Examples. The
rhGAA of the invention (ATB-200) has been shown to bind the CIMPR with high affinity
(K ~ 2-4 nM) and to be efficiently internalized by Pompe fibroblasts and skeletal muscle
myoblasts (K ~ 7-14 nM). ATB-200 was characterized in vivo and shown to have a
uptake
shorter apparent plasma half-life (t ~ 45 min) than the current rhGAA ERT (t ~ 60 min).
1/2 1/2
The amino acid sequence of the rhGAA can be at least 70%, 75%, 80%, 85%, 95% or
99% identical, or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more deletions, substitutions or
additions to the amino acid sequence described by SEQ ID NO: 1, 3 or 4. In some
embodiments of the GAA or rhGAA of the invention, such as in ATB-200 rhGAA, the GAA
or rhGAA will comprise a wild-type GAA amino acid sequence such as that of SEQ ID NO:
1 or 3. In other non-limiting embodiments, the rhGAA comprises a subset of the amino acid
residues present in a wild-type GAA, wherein the subset includes the amino acid residues of
the wild-type GAA that form the active site for substrate binding and/or substrate reduction.
In one embodiment, the rhGAA is glucosidase alfa, which is the human enzyme acid α-
glucosidase (GAA), encoded by the most predominant of nine observed haplotypes of this
gene. The rhGAA of the invention, including ATB-200 rhGAA, may comprise an amino
acid sequence that is 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid
sequence of human alpha glucosidase, such as that given by accession number AHE24104.1
(GI:568760974)(SEQ ID NO: 1) and which is incorporated by reference to U.S. Patent No.
8,592,362 or to the amino acid sequence of NP_000143.2 (SEQ ID NO: 4). A nucleotide and
amino acid sequence for GAA is also given by SEQ ID NOS: 2 and 3, respectively. Variants
of this amino acid sequence also include those with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more
amino acid deletions, insertions or substitutions to the GAA amino acid sequence below.
Polynucleotide sequences encoding GAA and such variant human GAAs are also
contemplated and may be used to recombinantly express rhGAAs according to the invention.
Various alignment algorithms and/or programs may be used to calculate the identity
between two sequences, including FASTA, or BLAST which are available as a part of the
GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used
with, e.g., default setting. For example, polypeptides having at least 70%, 85%, 90%, 95%,
98% or 99% identity to specific polypeptides described herein and preferably exhibiting
substantially the same functions, as well as polynucleotide encoding such polypeptides, are
contemplated. Unless otherwise indicated a similarity score will be based on use of
BLOSUM62. When BLASTP is used, the percent similarity is based on the BLASTP
positives score and the percent sequence identity is based on the BLASTP identities score.
BLASTP “Identities” shows the number and fraction of total residues in the high scoring
sequence pairs which are identical; and BLASTP “Positives” shows the number and fraction
of residues for which the alignment scores have positive values and which are similar to each
other. Amino acid sequences having these degrees of identity or similarity or any
intermediate degree of identity of similarity to the amino acid sequences disclosed herein are
contemplated and encompassed by this disclosure. The polynucleotide sequences of similar
polypeptides are deduced using the genetic code and may be obtained by conventional means,
in particular by reverse translating its amino acid sequence using the genetic code.
Preferably, no more than 70, 65, 60, 55, 45, 40, 35, 30, 25, 20, 15, 10, or 5% of the
total rhGAA in the composition according to the invention lacks an N-glycan bearing M6P or
bis-M6P or lacks a capacity to bind to the cationic independent manosephosphate receptor
(CIMPR). Alternatively, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99%, <100% or
more of the rhGAA in the composition comprises at least one N-glycan bearing M6P and/or
bis-M6P or has the capacity to bind to CIMPR.
The rhGAA molecules in the rhGAA composition of the invention may have 1, 2, 3 or
4 M6P groups on their glycans. For example, only one N-glycan on an rhGAA molecule may
bear M6P (mono-phosphorylated), a single N-glycan may bear two M6P groups (bis-
phosphorylated), or two different N-glycans on the same rhGAA molecule may bear single
M6P groups. rhGAA molecules in the rhGAA composition may also have N-glycans bearing
no M6P groups. In another embodiment, on average the N-glycans contain greater than 3
mol/mol of M6P and greater than 4 mol/mol sialic acid. On average at least about 3, 4, 5, 6,
7, 8, 9, or 10% of the total glycans on the rhGAA may be in the form of a mono-M6P glycan,
for example, about 6.25% of the total glycans may carry a single M6P group and on average,
at least about 0.5, 1, 1.5, 2.0, 2.5, 3.0% of the total glycans on the rhGAA are in the form of a
bis-M6P glycan and on average less than 25% of total rhGAA of the invention contains no
phosphorylated glycan binding to CIMPR.
The rhGAA composition according to the invention may have an average content of
N-glycans carrying M6P ranging from 0.5 to 7.0 mol/mol rhGAA or any intermediate value
of subrange including 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, or 7.0
mol/mol rhGAA. As shown in the Examples, the rhGAA of the invention can be fractionated
to provide rhGAA compositions with different average numbers of M6P-bearing or bis-M6P-
bearing glycans on the rhGAA thus permitting further customization of rhGAA targeting to
the lysosomes in target tissues by selecting a particular fraction or by selectively combining
different fractions.
Up to 60% of the N-glycans on the rhGAA may be fully sialyated, for example, up to
%, 20%, 30%, 40%, 50% or 60% of the N-glycans may be fully sialyated. In some
embodiments from 4 to 20% of the total N-glycans in the rhGAA composition are fully
sialylated.
In other embodiments no more than 5%, 10%, 20% or 30% of N-glycans on the
rhGAA carry sialic acid and a terminal Gal. This ranges includes all intermediate values and
subranges, for example, 7 to 30% of the total N-glycans on the rhGAA in the composition
can carry sialic acid and terminal Gal.
In yet other embodiments, no more than 5, 10, 15, 16, 17, 18, 19 or 20% of the N-
glycans on the rhGAA have a terminal Gal only and do not contain sialic acid. This range
includes all intermediate values and subranges, for example, from 8 to 19% of the total N-
glycans on the rhGAA in the composition may have terminal Gal only and do not contain
sialic acid.
In other embodiments of the invention 40, 45, 50, 55 to 60% of the total N-glycans on
the rhGAA in the composition are complex type N-glycans; or no more than 1, 2, 3, 4, 5, 6,
7% of total N-glycans on the rhGAA in the composition are hybrid-type N-glycans; no more
than 5, 10, or 15% of the high mannose-type N-glycans on the rhGAA in the composition are
non-phosphorylated; at least 5% or 10% of the high mannose-type N-glycans on the rhGAA
in the composition are mono-M6P phosphorylated; and/or at least 1 or 2% of the high
mannose-type N-glycans on the rhGAA in the composition are bis-M6P phosphorylated.
These values include all intermediate values and subranges. An rhGAA composition
according to the invention may meet one or more of the content ranges described above.
In some embodiments, the rhGAA composition of the invention will bear, on average,
2.0 to 8.0 sialic acid residues per mol of rhGAA. This range includes all intermediate values
and subranges including 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0
residues/mol rhGAA. Sialic acid residues may prevent non-productive clearance by
asialoglycoprotein receptors.
The rhGAA composition of the invention is preferably produced by CHO cells, such
as CHO cell line GA-ATB-200, or by a subculture or derivative of such a CHO cell culture.
DNA constructs, which express allelic variants of GAA or other variant GAA amino acid
sequences such as those that are at least 90%, 95% or 99% identical to SEQ ID NO: 1, may
be constructed and expressed in CHO cells. Those of skill in the art can select alternative
vectors suitable for transforming CHO cells for production of such DNA constructs.
The inventors have found that rhGAA having superior ability to target the CIMPR
and cellular lysosomes as well as glycosylation patterns that reduce its non-productive
clearance in vivo can be produced using Chinese hamster ovary (CHO) cells. These cells can
be induced to express rhGAA with significantly higher levels of total M6P and bis-M6P than
conventional rhGAA products. The recombinant human GAA produced by these cells, for
example, as exemplified by rhGAA ATB-200 described in the Examples, has significantly
more muscle cell-targeting M6P and bis-M6P groups than conventional GAA, such as
Lumizyme® and has been shown to efficiently bind to CIMPR and be efficiently taken up by
skeletal muscle and cardiac muscle. It has also been shown to have a glycosylation pattern
that provides a favorable pharmacokinetic profile and reduces non-productive clearance in
vivo.
The rhGAA according to the invention may be formulated as a pharmaceutical
composition or used in the manufacture of a medicament for treatment of Pompe Disease or
other conditions associated with a deficient of GAA. The compositions can be formulated
with a physiologically acceptable carrier or excipient. The carrier and composition can be
sterile and otherwise suit the mode of administration.
Suitable pharmaceutically acceptable carriers include but are not limited to water, salt
solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic,
vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose,
amylose or starch, sugars such as mannitol, sucrose, or others, dextrose, magnesium stearate,
talc, silicic acid, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well
as combinations thereof. The pharmaceutical preparations can, if desired, be mixed with
auxiliary agents, e.g., surfactants, such as polysorbates like polysorbate 80, lubricants,
preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure,
buffers, coloring, flavoring and/or aromatic substances and the like which do not
deleteriously react with the active compounds. In a preferred embodiment, a water-soluble
carrier suitable for intravenous administration is used.
The composition or medicament, if desired, can also contain minor amounts of
wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid
solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
The composition can also be formulated as a suppository, with traditional binders and carriers
such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical
grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidone, sodium
saccharine, cellulose, magnesium carbonate, etc. In a preferred embodiment the rhGAA is
administered by IV infusion.
The composition or medicament can be formulated in accordance with the routine
procedures as a pharmaceutical composition adapted for administration to human beings. For
example, in a preferred embodiment, a composition for intravenous administration is a
solution in sterile isotonic aqueous buffer. Where necessary, the composition may also
include a solubilizing agent and a local anesthetic to ease pain at the site of the injection.
Generally, the ingredients are supplied either separately or mixed together in unit dosage
faun, for example, as a dry lyophilized powder or water free concentrate in a hermetically
sealed container such as an ampule or sachet indicating the quantity of active agent. Where
the composition is to be administered by infusion, it can be dispensed with an infusion bottle
containing sterile pharmaceutical grade water, saline or dextrose/water. Where the
composition is administered by injection, an ampule of sterile water for injection or saline
can be provided so that the ingredients may be mixed prior to administration.
The rhGAA can be formulated as neutral or salt forms. Pharmaceutically acceptable
salts include those formed with free amino groups such as those derived from hydrochloric,
phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups
such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides,
isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
rhGAA (or a composition or medicament containing GAA) is administered by an
appropriate route. In one embodiment, the GAA is administered intravenously. In other
embodiments, GAA is administered by direct administration to a target tissue, such as to
heart or skeletal muscle (e.g., intramuscular), or nervous system (e.g., direct injection into the
brain; intraventricularly; intrathecally). More than one route can be used concurrently, if
desired.
The rhGAA (or a composition or medicament containing GAA) is administered in a
therapeutically effective amount (e.g., a dosage amount that, when administered at regular
intervals, is sufficient to treat the disease, such as by ameliorating symptoms associated with
the disease, preventing or delaying the onset of the disease, and/or also lessening the severity
or frequency of symptoms of the disease, as described above). The amount which will be
therapeutically effective in the treatment of the disease will depend on the nature and extent
of the disease's effects, and can be determined by standard clinical techniques. In addition, in
vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges.
The precise dose to be employed will also depend on the route of administration, and the
seriousness of the disease, and should be decided according to the judgment of a practitioner
and each patient's circumstances. Effective doses may be extrapolated from dose-response
curves derived from in vitro or animal model test systems. In a preferred embodiment, the
therapeutically effective amount is equal of less than 20 mg enzyme/kg body weight of the
individual, preferably in the range of about 1-10 mg enzyme/kg body weight, and even more
preferably about 10 mg enzyme/kg body weight or about 5 mg enzyme/kg body weight. The
effective dose for a particular individual can be varied (e.g., increased or decreased) over
time, depending on the needs of the individual. For example, in times of physical illness or
stress, or if anti-GAA antibodies become present or increase, or if disease symptoms worsen,
the amount can be increased.
The therapeutically effective amount of GAA (or composition or medicament
containing GAA) is administered at regular intervals, depending on the nature and extent of
the disease's effects, and on an ongoing basis. Administration at a "regular interval," as used
herein, indicates that the therapeutically effective amount is administered periodically (as
distinguished from a one-time dose). The interval can be determined by standard clinical
techniques. In preferred embodiments, GAA is administered monthly, bimonthly; weekly;
twice weekly; or daily. The administration interval for a single individual need not be a fixed
interval, but can be varied over time, depending on the needs of the individual. For example,
in times of physical illness or stress, if anti-GAA antibodies become present or increase, or if
disease symptoms worsen, the interval between doses can be decreased. In some
embodiments, a therapeutically effective amount of 5, 10, 20, 50, 100, or 200 mg enzyme/kg
body weight is administered twice a week, weekly or every other week with or without a
chaperone.
The GAA or rhGAA of the invention may be prepared for later use, such as in a unit
dose vial or syringe, or in a bottle or bag for intravenous administration. Kits containing the
GAA or rhGAA, as well as optional excipients or other active ingredients, such as chaperones
or other drugs, may be enclosed in packaging material and accompanied by instructions for
reconstitution, dilution or dosing for treating a subject in need of treatment, such as a patient
having Pompe disease.
GAA (or a composition or medicament containing GAA) can be administered alone,
or in conjunction with other agents, such as a chaperone. rhGAA with different degrees of
glycosylation with mono-M6P or bis-M6P may be administered or combinations of rhGAAs
with different degrees of M6P or bisM6P glycosylate administered.
In some embodiments the rhGAA composition of the invention will be complexed or
admixed with a chaperone, such as AT-2220 or AT-2221. Chaperones, sometimes referred to
as “pharmacological chaperones,” are compounds that when complexed or coadministered
with rhGAA modify its pharmacokinetics and other pharmacological properties.
Representative chaperones exemplified herein include AT2221 (miglustat, N-butyl-
deoxynojirimycin) and AT2220 (duvoglustat HCl, 1-deoxynojirimycin). Such complexing or
admixing may occur outside the body or inside the body, for example, where separate
dosages of the rhGAA and chaperone are administered. For example, targeting of active
rhGAA, its fractions, or derivatives of the invention to CIMPR and subsequently to cellular
lysosomes may be improved by combining it duvoglustat-HCl (AT2220, deoxynojirimycine,
AT2220) or miglustat (AT2221, N-butyl-deoxynojirimycin). The Examples below show
significant glycogen substrate reductions in key skeletal muscles of GAA-knock-out mice
receiving the well-targeted rhGAA of the invention in combination with a chaperone.
Another aspect of the invention pertains to CHO cells or their derivatives or other
equivalents that produce the rhGAA according to the invention. One example of such a CHO
cell line is GA-ATB-200 or a subculture thereof that produces a rhGAA composition as
described herein. Such CHO cell lines may contain multiple copies of a gene, such as 5, 10,
, or 20 or more copies, of a polynucleotide encoding GAA.
The high M6P and bis-M6P rhGAA of the invention, such as ATB-200 rhGAA, can
be produced by transforming CHO cells (Chinese hamster ovary cells) with a DNA construct
that encodes GAA. While CHO cells have been previously used to make rhGAA, it was not
appreciated that transformed CHO cells could be cultured and selected in a way that would
produce rhGAA having a high content of M6P and bis-M6P glycans which target the
CIMPR.
Surprisingly, the inventors found that it was possible to transform CHO cell lines, select
transformants that produce rhGAA containing a high content of glycans bearing M6P or bis-
M6P that target the CIMPR, and to stably express this high-M6P rhGAA. Thus, a related
aspect of the invention is directed to method for making these CHO cell lines. This method
involves transforming a CHO cell with DNA encoding GAA or a GAA variant, selecting a
CHO cell that stably integrates the DNA encoding GAA into its chromosome(s) and that
stably expresses GAA, and selecting a CHO cell that expresses GAA having a high content of
glycans bearing M6P or bis-M6P, and, optionally, selecting a CHO cell having N-glycans
with high sialic acid content and/or having N-glycans with a low non-phosphorylated high-
mannose content.
These CHO cell lines may be used to produce rhGAA and rhGAA compositions according to
the invention by culturing the CHO cell line and recovering said composition from the culture
of CHO cells.
The rhGAA composition of the invention or its fractions or derivatives is
advantageously used to treat subjects having a condition, disorder or disease associated with
insufficient lysosomal GAA by administering the rhGAA composition. A subject in need of
treatment includes those having Glycogen Storage Disease Type II (Pompe Disease) as well
as other conditions, disorders or diseases which would benefit from the administration of the
rhGAA.
The Examples below show that the rhGAA of the invention (ATB-200) is taken up by
skeletal muscle cells, binds to CIMPR and effectively removes glycogen from skeletal
muscle cells when administered at a significantly lower dosage than conventional rhGAA
products. A reduction of up to 75% of glycogen in skeletal muscle myoblast was attained in
GAA-knockout mice using a biweekly regimen of intravenous administration of ATB-200.
These reductions exceeded those provided by the same amount of Lumizyme® showing that
the rhGAA of the invention, which has an enhanced content of N-glycans bearing M6P and
bis-M6P, provided superior reductions in glycogen substrate. Due to the improved targeting,
pharmacodynamics and pharmacokinetics of the rhGAA composition of the invention may be
administered in a lower dosage than conventional rhGAA products such as Lumizyme® or
Myozyme®.
It may be used to degrade, decrease or remove glycogen from cardiac muscle, smooth
muscle, or striated muscle. Examples of skeletal or striated muscles subject to treatment
include at least one muscle selected from the group consisting of abductor digiti minimi
(foot), abductor digiti minimi (hand), abductor halluces, abductor pollicis brevis, abductor
pollicis longus, adductor brevis, adductor halluces, adductor longus, adductor magnus,
adductor pollicis, anconeus, articularis cubiti, articularis genu, aryepiglotticus,
aryjordanicus, auricularis, biceps brachii, biceps femoris, brachialis, brachioradialis,
buccinators, bulbospongiosus, constrictor of pharynx–inferior, constrictor of pharynx–
middle, constrictor of pharynx–superior, coracobrachialis, corrugator supercilii,
cremaster, cricothyroid, dartos, deep transverse perinei, deltoid, depressor anguli oris,
depressor labii inferioris, diaphragm, digastric, digastric (anterior view), erector spinae–
spinalis, erector spinae–iliocostalis, erector spinae–longissimus, extensor carpi radialis
brevis, extensor carpi radialis longus, extensor carpi ulnaris, extensor digiti minimi
(hand), extensor digitorum (hand), extensor digitorum brevis (foot), extensor digitorum
longus (foot), extensor hallucis longus, extensor indicis, extensor pollicis brevis,
extensor pollicis longus, external oblique abdominis, flexor carpi radialis, flexor carpi
ulnaris, flexor digiti minimi brevis (foot), flexor digiti minimi brevis (hand), flexor
digitorum brevis, flexor digitorum longus (foot), flexor digitorum profundus, flexor
digitorum superficialis, flexor hallucis brevis, flexor hallucis longus, flexor pollicis
brevis, flexor pollicis longus, frontalis, gastrocnemius, gemellus inferior, gemellus
superior, genioglossus, geniohyoid, gluteus maximus, gluteus medius, gluteus minimus,
gracilis, hyoglossus, iliacus, inferior oblique, inferior rectus, infraspinatus, intercostals
external, intercostals innermost, intercostals internal, internal oblique abdominis,
interossei-dorsal of hand, interossei-dorsal of foot, interossei-palmar of hand, interossei-
plantar of foot, interspinales, intertransversarii, intrinsic muscles of tongue,
ishiocavernosus, lateral cricoarytenoid, lateral pterygoid, lateral rectus, latissimus dorsi,
levator anguli oris, levator ani-coccygeus, levator ani – iliococcygeus, levator ani-
pubococcygeus, levator ani-puborectalis, levator ani-pubovaginalis, levator labii
superioris, levator labii superioris, alaeque nasi, levator palpebrae superioris, levator
scapulae, levator veli palatine, levatores costarum, longus capitis, longus colli,
lumbricals of foot (4),lumbricals of hand, masseter, medial pterygoid, medial rectus,
mentalis, m. uvulae, mylohyoid, nasalis, oblique arytenoid, obliquus capitis inferior,
obliquus capitis superior, obturator externus, obturator internus (A), obturator internus
(B), omohyoid, opponens digiti minimi (hand), opponens pollicis, orbicularis oculi,
orbicularis oris, palatoglossus, palatopharyngeus, palmaris brevis, palmaris longus,
pectineus, pectoralis major, pectoralis minor, peroneus brevis, peroneus longus,
peroneus tertius, piriformis (A), piriformis (B), plantaris, platysma, popliteus, posterior
cricoarytenoid, procerus, pronator quadratus, pronator teres, psoas major, psoas minor,
pyramidalis, quadratus femoris, quadratus lumborum, quadratus plantae, rectus
abdominis, rectus capitus anterior, rectus capitus lateralis, rectus capitus posterior
major, rectus capitus posterior minor, rectus femoris, rhomboid major, rhomboid
minor, risorius, salpingopharyngeus, sartorius, scalenus anterior, scalenus medius,
scalenus minimus, scalenus posterior, semimembranosus, semitendinosus, serratus
anterior, serratus posterior inferior, serratus posterior superior, soleus, sphincter ani,
sphincter urethrae, splenius capitis, splenius cervicis, stapedius, sternocleidomastoid,
sternohyoid, sternothyroid, styloglossus, stylohyoid, stylohyoid (anterior view),
stylopharyngeus, subclavius, subcostalis, subscapularis, superficial transverse, perinei,
superior oblique, superior rectus, supinator, supraspinatus, temporalis, temporoparietalis,
tensor fasciae lata, tensor tympani, tensor veli palatine, teres major, teres minor,
thyro-arytenoid & vocalis, thyro-epiglotticus, thyrohyoid, tibialis anterior, tibialis
posterior, transverse arytenoid, transversospinalis–multifidus, transversospinalis–rotatores,
transversospinalis –semispinalis, transversus abdominis, transversus thoracis, trapezius,
triceps, vastus intermedius, vastus lateralis, vastus medialis, zygomaticus major, and
zygomaticus minor.
The GAA composition of the invention may also be administered to, or used to treat,
type 1 (slow twitch) muscle fiber or type 2 (fast twitch) muscle fiber or subjects accumulating
glycogen in such muscle fibers. Type I, slow twitch, or "red" muscle, is dense with
capillaries and is rich in mitochondria and myoglobin, giving the muscle tissue its
characteristic red color. It can carry more oxygen and sustain aerobic activity using fats or
carbohydrates as fuel. Slow twitch fibers contract for long periods of time but with little
force. Type II, fast twitch muscle, has three major subtypes (IIa, IIx, and IIb) that vary in
both contractile speed and force generated. Fast twitch fibers contract quickly and
powerfully but fatigue very rapidly, sustaining only short, anaerobic bursts of activity before
muscle contraction becomes painful. They contribute most to muscle strength and have
greater potential for increase in mass. Type IIb is anaerobic, glycolytic, "white" muscle that is
least dense in mitochondria and myoglobin. In small animals (e.g., rodents) this is the major
fast muscle type, explaining the pale color of their flesh.
The rhGAA composition of the invention, its fractions or derivatives may be administered
systemically, for example, by intravenous (IV) infusion, or administered directly into a
desired site, such as into cardiac or skeletal muscle, such as quadriceps, triceps, or
diaphragm. It may be administered to myocytes, particular muscle tissues, muscles, or
muscle groups. For example, such a treatment may administer intramuscularly the rhGAA
composition directly into a subject’s quadriceps or triceps or diaphragm.
As mentioned above, the rhGAA composition of the invention, its fractions or
derivatives can be complexed or admixed with a chaperone, such as AT-2220 (Duvoglustat
HCl, 1-Deoxynojirimycin) or AT-2221(Miglustat, N-butyl-deoxynojirimycin) or their salts to
improve the pharmacokinetics of the rhGAA administration. The rhGAA and the chaperone
may be administered together or separately. When administered simultaneously the GAA in
the composition may be preloaded with the chaperone. Alternatively, the GAA and the
chaperone may be administered separately either at the same time or at different times.
Representative dosages of AT2221 range from 0.25 to 400 mg/kg, preferably from
0.5-200 mg/kg, and most preferably from 2 to 50 mg/kg. Specific dosages of AT2221
include 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 mg/kg. These dosages may be
combined with rhGAA, such as ATB-200 rhGAA, at a molar ratio of AT2221 to rhGAA
ranging from 15:1 to 150: 1. Specific ratios include 15:1, 20:1, 25:1, 50:1, 60: 1, 65:1, 70:1,
75:1, 80:1, 85:1, 90: 1, 100:1, 125:1, and 150:1. rhGAA and AT2221 may be coadministered
in these amounts or molar ratios either concurrently, sequentially or separately. The ranges
above include all intermediate subranges and values, such as all integer values between the
range endpoints.
Representative dosages of AT2220 range from 0.1 to 120 mg/kg, preferably 0.25 to
60, and most preferably from 0.6 to 15 mg/kg. Specific dosages of AT2220 include 1, 2, 3, 4,
, 6, 7, 8, 9, 10, 15, 20, 25 and 30 mg/kg. These dosages may be combined with rhGAA,
such as ATB-200 rhGAA, at a molar ratio of AT2220 to rhGAA ranging from 15:1 to 150: 1.
Specific ratios include 15:1, 20: 125:1, 50:1, 60: 1, 65:1, 70:1, 75:1, 80:1, 85:1, 90: 1, 100:1,
125:1, and 150:1. rhGAA and AT2220 may be coadministered in these amounts or molar
ratios either concurrently, sequentially or separately. The ranges above include all
intermediate subranges and values, such as all integer values between the range endpoints.
The rhGAA composition of the invention, its fractions or derivatives may also be used
for metabolizing, degrading, removing or otherwise decreasing glycogen in tissue, muscle,
muscle fiber, muscle cells, lysosomes, organelles, cellular compartments, or cytoplasm. By
administering the rhGAA composition to a subject, optionally along with a chaperone or a
drug that reduces immunological responses to rhGAA.
In another embodiment of its method of use, the rhGAA of the invention may be used
for modulating lysosomal proliferation, autophagy, or exocytosis in a cell by administering it,
its fractions, or derivatives to cells, tissues, or subjects in need of such modulation, optionally
in combination with a chaperone or optionally as a conjugate with another targeting moiety.
Autophagy is a catabolic mechanism that allows a cell to degrade glycogen or other
unnecessary or dysfunctional cellular components through the actions of it lysosomes. This
method can also involve systemically or locally administering the GAA composition to a
subject in need of treatment.
The rhGAA according to the invention, which is enriched for mono-M6P and bis-
M6P, compared to Lumizyme® and Myozyme, and which has favorable pharmacokinetic
properties conferred by its glycosylation pattern may also be used for treatment of other
conditions requiring the breakdown of complex carbohydrates, such as other disorders in
which glycogen or other carbohydrates degraded by rhGAA accumulate in the lysosomes or
other parts of the cell, such as in the cytoplasm accessible to rhGAA, such as Glycogen
storage disease III. It may also be used non-therapeutic purposes, such as for the production
of foods, beverages, chemicals and pharmaceutical products which require breaking down
complex carbohydrates such as starch and glycogen into their monomers.
EXAMPLES
The following non-limiting Examples exemplify aspects of the invention.
Section I: ATB-200 rhGAA and its properties
Limitations of existing Myozyme® and Lumizyme® rhGAA products
To evaluate the ability of the rhGAA in Myozyme® and Lumizyme®, the only
currently approved treatments for Pompe disease, these rhGAA preparations were injected
onto a CIMPR column (which binds rhGAA having M6P groups) and subsequently eluted
with a free M6 gradient. Fractions were collected in 96-well plate and GAA activity assayed
by 4MU- -glucose substrate. The relative amounts of bound and unbound rhGAA were
determined based on GAA activity and reported as the fraction of total enzyme.
Fig. 5 describes the problems associated with conventional ERTs (Myozyme® and
Lumizyme®): 73% of the rhGAA in Myozyme® (Fig. 5B) and 78% of the rhGAA in
Lumizyme® (Fig. 5A) did not bind to the CIMPR, see the left-most peaks in each figure.
Only 27% of the rhGAA in Myozyme® and 22% of the rhGAA in Lumizyme® contained
M6P that can productive target it to the CIMPR on muscle cells, see Fig. 2 which describes
productive drug targeting and non-productive drug clearance.
An effective dose of Myozyme® and Lumizyme® corresponds to the amount of
rhGAA containing M6P which targets the CIMPR on muscle cells. However, most of the
rhGAA in these two conventional products does not target the CIMPR receptor on target
muscle cells. The administration of a conventional rhGAA where most of the rhGAA is not
targeted to muscle cells increases the risk of allergic reaction or induction of immunity to the
non-targeted rhGAA.
Preparation of CHO Cells Producing ATB-200 rhGAA having a high content of mono- or
bis-M6P-bearing N-glycans.
CHO cells were transfected with DNA that expresses rh-GAA followed by selection
of transformants producing rhGAA. A DNA construct for transforming CHO cells with
DNA encoding rh-GAA is shown in Fig. 5. CHO cells were transfected with DNA that
expresses rh-GAA followed by selection of transformants producing rhGAA.
After transfection, DG44 CHO (DHFR-) cells containing a stably integrated GAA
gene were selected with hypoxanthine/thymidine deficient (-HT) medium). Amplification of
GAA expression in these cells was induced by methotrexate treatment (MTX, 500 nM). Cell
pools that expressed high amounts of GAA were identified by GAA enzyme activity assays
and were used to establish individual clones producing rhGAA. Individual clones were
generated on semisolid media plates, picked by ClonePix system, and were transferred to 24-
deep well plates. The individual clones were assayed for GAA enzyme activity to identify
clones expressing a high level of GAA. Conditioned media for determining GAA activity
used a 4-MU-α-Glucosidase substrate. Clones producing higher levels of GAA as measured
by GAA enzyme assays were further evaluated for viability, ability to grow, GAA
productivity, N-glycan structure and stable protein expression. CHO cell lines, including
CHO cell line GA-ATB-200, expressing rhGAA with enhanced mono-M6P or bis-M6P N-
glycans were isolated using this procedure.
Purification of rhGAA ATB-200 rhGAA
Multiple batches of the rhGAA according to the invention were produced in shake
flasks and in perfusion bioreactors using CHO cell line GA-ATB-200 and CIMPR binding
was measured. Similar CIMPR receptor binding (~70%) to that shown in Fig. 6B and Fig. 7
was observed for purified ATB-200 rhGAA from different production batches indicating that
ATB-200 rhGAA can be consistently produced. As shown by Figs. 6A and 6B, Myozyme®
and Lumizyme® rhGAAs exhibited significantly less CIMPR binding than ATB-200 rhGAA.
Analytical Comparison of ATB-200 to Lumizyme
Weak anion exchange (“WAX”) liquid chromatography was used to fractionate ATB-
200 rhGAA according to terminal phosphate. Elution profiles were generated by eluting the
ERT with increasing amount of salt. The profiles were monitored by UV (A280nm). ATB-
200 rhGAA was obtained from CHO cells and purified. Lumizyme® was obtained from a
commercial source. Lumizyme® exhibited a high peak on the left of its elution profile. ATB-
200 rhGAA exhibited four prominent peaks eluting to the right of Lumizyme® (Fig. 8). This
confirms that ATB-200 rhGAA was phosphorylated to a greater extent than Lumizyme®
since this evaluation is by terminal charge rather than CIMPR affinity.
Oligosaccharide Characterization of ATB-200 rhGAA
Purified ATB-200 rhGAA and Lumizyme® glycans were evaluated by MALDI-TOF
to determine the individual glycan structures found on each ERT (Fig. 9). ATB-200 samples
were found to contain slightly lower amounts of non-phosphorylated high-mannose type N-
glycans than Lumizyme®. The higher content of M6P glycans in ATB-200 than in
Lumizyme, targets ATB-200 rhGAA to muscle cells more effectively. The high percentage
of mono-phosphorylated and bis-phosphorylated structures determined by MALDI agree with
the CIMPR profiles which illustrated significantly greater binding of ATB-200 to the CIMPR
receptor. N-glycan analysis via MALDI-TOF mass spectrometry confirmed that on average
each ATB200 molecule contains at least one natural bis-M6P N-glycan structure. This higher
bis-M6P N-glycan content on ATB-200 rhGAA directly correlated with high-affinity binding
to CIMPR in M6P receptor plate binding assays (K about 2-4 nM) Figure 10A.
Characterization of CIMPR Affinity of ATB-200
In addition to having a greater percentage of rhGAA that can bind to the CIMPR, it is
important to understand the quality of that interaction. Lumizyme® and ATB200 rhGAA
receptor binding was determined using a CIMPR plate binding assay. Briefly, CIMPR-
coated plates were used to capture GAA. Varying concentrations of rhGAA were applied to
the immobilized receptor and unbound rhGAA was washed off. The amount of remaining
rhGAA was determined by GAA activity. As shown by Fig. 10A, ATB-200 rhGAA bound to
CIMPR significantly better than Lumizyme.
Fig. 10B shows the relative content of bis-M6P glycans in Lumizyme, a conventional
rhGAA, and ATB-200 according to the invention. For Lumizyme® there is on average only
% of molecules have a bis-phosphorylated glycan. Contrast this with ATB-200 where on
average every rhGAA molecule has at least one bis-phosphorylated glycan.
ATB-200 rhGAA was more efficiently internalized by fibroblast than Lumizyme.
The relative cellular uptake of ATB-200 and Lumizyme® rhGAA were compared
using normal and Pompe fibroblast cell lines. Comparisons involved 5-100 nM of ATB-200
rhGAA according to the invention with 10-500 nM conventional rhGAA Lumizyme®. After
16-hr incubation, external rhGAA was inactivated with TRIS base and cells were washed 3-
times with PBS prior to harvest. Internalized GAA measured by 4MU-α-Glucoside
hydrolysis and was graphed relative to total cellular protein and the results appear in Fig. 11.
ATB-200 rhGAA was also shown to be efficiently internalized into cells (Figure 11A
and 11B), respectively, show that ATB-200 rhGAA is internalized into both normal and
Pompe fibroblast cells and that it is internalized to a greater degree than conventional
Lumizyme® rhGAA. ATB-200 rhGAA saturates cellular receptors at about 20 nM, while
about 250 nM of Lumizyme® is needed. The uptake efficiency constant (K )
uptake
extrapolated from these results is 2-3 nm for ATB-200 and 56 nM for Lumizyme® as shown
by Fig. 11C. These results suggest that ATB-200 rhGAA is a well-targeted treatment for
Pompe disease.
Section II: Preclinical Studies
ATB-200 rhGAA with Superior Glycosylation was Significantly Better than Standard
of Care ERT for Glycogen Clearance in Skeletal Muscles of GAA KO Mice
As explained above, enzyme replacement therapy (ERT) using recombinant human
GAA (rhGAA) is the only approved treatment available for Pompe disease. This ERT
requires the specialized carbohydrate mannose 6-phosphate (M6P) for cellular uptake and
subsequent delivery to lysosomes via cell surface cation-independent M6P receptors
(CIMPRs). However, the current rhGAA ERT contains low amounts of M6P that limit drug
targeting and efficacy in disease-relevant tissues. The inventors developed a production cell
line and manufacturing process that yield rhGAA (designated as ATB-200 rhGAA) with
superior glycosylation and higher M6P content than conventional rhGAA, particularly the
high-affinity bis-M6P N-glycan structure, for improved drug targeting. ATB-200 rhGAA
binds the CI-MPR with high affinity (KD~ 2-4 nM) and was efficiently internalized by
Pompe fibroblasts and skeletal muscle myoblasts (K ~7-14 nM).
uptake
ATB-200 rhGAA clears glycogen significantly better than Lumizyme® in skeletal
muscle. The effects of administering Lumizyme® and ATB-200 rhGAA for glycogen
clearance in GAA KO mice were evaluated. Animals were given two IV bolus
administrations (every other week); tissues were harvested two weeks after the last dose and
analyzed for GAA activity and glycogen content (Fig 12). ATB-200 rhGAA and Lumizyme®
rhGAA were equally effective for clearing glycogen in heart (Fig 12A). As show in in Figs.
12B and 12C, ATB-200 rhGAA at 5 mg/kg was equivalent to Lumizyme® rhGAA at 20
mg/kg for reducing glycogen in skeletal muscles; ATB-200 dosed at 10 and 20 mg/kg was
significantly better than Lumizyme® for clearing glycogen in skeletal muscles.
Rationale for Co-administration of ATB-200 rhGAA with AT2221 (CHART
Technology)
A chaperone binds to and stabilizes rhGAA ERT, increases uptake of active enzyme
into tissues, improves tolerability and potentially mitigates immunogenicity. As shown
above, the protein stability of ERT under unfavorable conditions was substantially improved
using CHART™. CHART: chaperone-advanced replacement therapy, see
http://_www.amicusrx.com/chaperone.aspx (last accessed September 22, 2015) which is
incorporated by reference. As shown by Figs. 13A and 13B, the stability of ATB-200 was
significantly improved by AT2221 (Miglustat, N-butyl-deoxynojirimycin). Folding of
rhGAA protein was monitored at 37 C by thermal denaturation in neutral (pH 7.4 – plasma
environment) or acidic (pH 5.2 – lysosomal environment) buffers. AT2220 stabilized rhGAA
protein in neutral pH buffer over 24 hours.
Co-administration of Myozyme® with AT2221 (Miglustat) Compared to Co-
administration of ATB-200 rhGAA with Miglustat
Twelve week old GAA KO mice treated with Lumizyme® or ATB200, 20 mg/kg IV
every other week 4 injections; Miglustat was co-administered at 10 mg/kg PO, 30 min prior
to rhGAA as indicated. Tissues were collected 14 days after last enzyme dose for glycogen
measurement. Fig. 14 shows the relative reduction of glycogen in quadriceps and triceps
skeletal muscle.
Reduction of tissue glycogen with ATB-200 rhGAA coadministered with
pharmacological chaperone AT2221 (Miglustat).
The combination of a pharmacological chaperone and ATB-200 rhGAA was found to
enhance glycogen clearance in vivo. GAA KO mice were given two IV bolus administrations
of rhGAA at 20 mg/kg every other week. The pharmacological chaperone AT2221 was
orally administered 30 mins prior to rhGAA at dosages of 0, 1, 2 and 10 mg/kg. Tissues were
harvested two weeks after the last dose of ERT and analyzed for GAA activity, glycogen
content cell specific glycogen and lysosome proliferation.
As shown by Fig. 15, the animals receiving ATB200+ chaperone AT2221 exhibited
enhanced glycogen clearance from quadriceps muscle. ATB-200 rhGAA (20 mg/kg)
reduced glycogen more than the same dose of Lumizyme® and when ATB-200 rhGAA was
combined with 10 mg/kg of AT2220 near normal levels of glycogen in muscle were attained.
As shown by Figs. 16A and 16B, unlike conventional rhGAA, which showed limited
glycogen reduction (indicated by abundant punctate PAS signal), ATB-200 rhGAA alone
showed a significant decrease in PAS signals. Co-administration with 10 mg/kg miglustat
resulted in a substantial further reduction in substrate. TEM revealed that the majority of
glycogen in the lysosomes as membrane-bound, electron-dense material, which correspond to
the punctate PAS signals. Co-administration of ATB-200 rhGAA with miglustat, reduced the
number, size and density of substrate-containing lysosomes suggesting targeted delivery of
ATB-200 rhGAA to the muscle cells and subsequent delivery to the lysosomes.
From the study (2 IV bolus every other week injection) shown above, tissues were
processed for lysosomal proliferation using a LAMP 1 marker, the up-regulation is another
hallmark of Pompe disease. LAMP: lysosome-associated membrane protein. From the study
(2 IV bolus EOW injection) shown above, soleus tissue was processed for LAMP 1 staining
in adjacent sections and type I fiber-specific antibody (NOQ7.5.4D) in adjacent sections Fig
16C and 16D) ATB-200 rhGAA results in a more substantial LAMP1 reduction compared to
conventional rhGAA, with reductions leading to levels seen in WT animals Figure 16C).
In addition, unlike rhGAA, where the effect is mostly restricted to type I fibers (slow
twitch, marked with asterisks), ATB-200 rhGAA also led to significant reduction in LAMP1
signals in a fraction of type II (fast twitch) fibers (red arrow heads) (Figure 16D).
Importantly, co-administration with miglustat further improved ATBmediated reduction
of LAMP1 proliferation in the majority of type II fibers (Fig 16C and 16D). As a result, there
did not appear to be a significant fiber type-specific difference in the level of LAMP1 signals.
Similar conclusions were drawn from quadriceps and diaphragm (data not shown).
In a separate and similarly designed study, the effect of ATB-200 ± AT2221 was
examined over a longer term with 4 biweekly IV bolus injections. In heart, the main glycogen
store in the cardiomyocytes was readily cleared by repeat administration of either rhGAA or
ATB-200 to levels seen in wild-type (WT) animals (Fig 17A). However, the substrate in
cardiac smooth muscle cells seems to be cleared preferably by ATB-200 rhGAA, suggesting
a potentially broader bio-distribution of ATB-200 compared to rhGAA (asterisks mark the
lumen of cardiac blood vessels). Importantly, co-administration with miglustat further
improved ATBmediated reduction of LAMP1 proliferation.
These results show that ATB-200 rhGAA, which has higher levels of M6P and bis-
M6P on its N-glycans efficiently targets CIMPR in skeletal muscle. ATB-200 rhGAA also
has well-processed complex-type N-glycans that minimize non-productive clearance in vivo,
has pharmacokinetic properties favorable for its use in vivo and exhibits good targeting to key
muscle tissues in vivo. They also show that ATB-200 rhGAA is better than the conventional
standard of care, Lumizyme, for reducing glycogen in muscle tissue and that a combination
of ATB-200 rhGAA and chaperone AT2221 further improve removal of glycogen from target
tissues and improves muscle pathology.
Claims (10)
1. A composition comprising recombinant human acid alpha-glucosidase (rhGAA), wherein 40%-60% of the N-glycans on the rhGAA are complex type N-glycans; the rhGAA comprises 2.0 to 8.0 mol sialic acid residues per mol of rhGAA; the rhGAA 5 comprises at least one mol bis-phosphorylated N-glycan per mol rhGAA; and the rhGAA comprises 3.0-5.0 mol mannosephosphate (M6P) residues per mol rhGAA.
2. The composition of claim 1, wherein the rhGAA comprises 3.0 to 4.0 mol M6P per mol rhGAA.
3. The composition of claim 1, wherein the rhGAA comprises 4.0 to 5.0 mol 10 M6P per mol rhGAA.
4. The composition of any one of claims 1-3, wherein the rhGAA comprises at least 4 mol sialic acid per mol rhGAA.
5. The composition of any one of claims 1-4, wherein the composition further comprises a pharmacological chaperone. 15
6. The composition of any one of claims 1-5, wherein about 45%-55% of the N- glycans on the rhGAA are complex type N-glycans.
7. The composition of claim 6, wherein 50% of the N-glycans on the rhGAA are complex type N-glycans.
8. The composition of claim 1, wherein the rhGAA comprises 4 mol M6P and 20 4.5 mol sialic acid per mol rhGAA.
9. The composition of claim 8, wherein the composition further comprises the pharmacological chaperone N-butyl-deoxynojirimycin or a pharmaceutically acceptable salt thereof.
10. Use of the composition of any one of claims 1-9 for the manufacture of a 25 medicament for the treatment of Pompe disease.
Applications Claiming Priority (9)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201462057842P | 2014-09-30 | 2014-09-30 | |
US201462057847P | 2014-09-30 | 2014-09-30 | |
US62/057,842 | 2014-09-30 | ||
US62/057,847 | 2014-09-30 | ||
US201562112463P | 2015-02-05 | 2015-02-05 | |
US62/112,463 | 2015-02-05 | ||
US201562135345P | 2015-03-19 | 2015-03-19 | |
US62/135,345 | 2015-03-19 | ||
PCT/US2015/053252 WO2016054231A1 (en) | 2014-09-30 | 2015-09-30 | Highly potent acid alpha-glucosidase with enhanced carbohydrates |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ729507A NZ729507A (en) | 2021-10-29 |
NZ729507B2 true NZ729507B2 (en) | 2022-02-01 |
Family
ID=
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11753632B2 (en) | Highly potent acid alpha-glucosidase with enhanced carbohydrates | |
JP7046003B2 (en) | Method for selecting high M6P recombinant protein | |
NZ729507B2 (en) | Highly potent acid alpha-glucosidase with enhanced carbohydrates | |
TWI789758B (en) | Highly potent modified acid alpha-glucosidase and method of making and using it | |
TWI843172B (en) | Highly potent modified acid alpha-glucosidase and method of making and using it | |
TW202417620A (en) | Highly potent modified acid alpha-glucosidase and method of making and using it |