NZ743635B2 - Biomarker for use in treating anemia - Google Patents
Biomarker for use in treating anemia Download PDFInfo
- Publication number
- NZ743635B2 NZ743635B2 NZ743635A NZ74363513A NZ743635B2 NZ 743635 B2 NZ743635 B2 NZ 743635B2 NZ 743635 A NZ743635 A NZ 743635A NZ 74363513 A NZ74363513 A NZ 74363513A NZ 743635 B2 NZ743635 B2 NZ 743635B2
- Authority
- NZ
- New Zealand
- Prior art keywords
- seq
- gdf11
- level
- activin
- inhibitor
- Prior art date
Links
- 208000007502 anemia Diseases 0.000 title claims abstract description 96
- 239000000090 biomarker Substances 0.000 title abstract description 5
- 230000000694 effects Effects 0.000 claims abstract description 251
- 102100040898 Growth/differentiation factor 11 Human genes 0.000 claims abstract description 242
- 101000893545 Homo sapiens Growth/differentiation factor 11 Proteins 0.000 claims abstract description 242
- 108010059616 Activins Proteins 0.000 claims abstract description 183
- 239000000488 activin Substances 0.000 claims abstract description 181
- 102100026818 Inhibin beta E chain Human genes 0.000 claims abstract description 175
- 239000003112 inhibitor Substances 0.000 claims abstract description 147
- 210000003743 erythrocyte Anatomy 0.000 claims abstract description 79
- 238000011282 treatment Methods 0.000 claims abstract description 74
- 230000001965 increasing effect Effects 0.000 claims abstract description 67
- 102000018918 Activin Receptors Human genes 0.000 claims abstract description 64
- 108010052946 Activin Receptors Proteins 0.000 claims abstract description 64
- 210000003924 normoblast Anatomy 0.000 claims abstract description 52
- 208000005980 beta thalassemia Diseases 0.000 claims abstract description 33
- 208000018240 Bone Marrow Failure disease Diseases 0.000 claims abstract description 4
- 206010065553 Bone marrow failure Diseases 0.000 claims abstract description 4
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 370
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 361
- 229920001184 polypeptide Polymers 0.000 claims description 359
- 108090000623 proteins and genes Proteins 0.000 claims description 165
- 102000004169 proteins and genes Human genes 0.000 claims description 105
- 210000001519 tissue Anatomy 0.000 claims description 98
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 82
- 230000003247 decreasing effect Effects 0.000 claims description 75
- 241000282414 Homo sapiens Species 0.000 claims description 71
- 210000002966 serum Anatomy 0.000 claims description 63
- 102000005962 receptors Human genes 0.000 claims description 61
- 108020003175 receptors Proteins 0.000 claims description 61
- 210000001185 bone marrow Anatomy 0.000 claims description 50
- 210000000952 spleen Anatomy 0.000 claims description 46
- 238000004519 manufacturing process Methods 0.000 claims description 26
- 210000004185 liver Anatomy 0.000 claims description 14
- 230000001684 chronic effect Effects 0.000 claims description 13
- 239000003814 drug Substances 0.000 claims description 11
- 201000003793 Myelodysplastic syndrome Diseases 0.000 claims description 10
- 238000012544 monitoring process Methods 0.000 claims description 9
- 208000007056 sickle cell anemia Diseases 0.000 claims description 9
- 102100021886 Activin receptor type-2A Human genes 0.000 claims description 8
- 108020004999 messenger RNA Proteins 0.000 claims description 7
- 108010057429 activin receptor type II-A Proteins 0.000 claims description 6
- 102100027647 Activin receptor type-2B Human genes 0.000 claims 2
- 108010057453 activin receptor type II-B Proteins 0.000 claims 2
- 230000000505 pernicious effect Effects 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 252
- 108091006082 receptor inhibitors Proteins 0.000 abstract description 79
- 230000010437 erythropoiesis Effects 0.000 abstract description 72
- 239000003446 ligand Substances 0.000 abstract description 40
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 26
- 201000010099 disease Diseases 0.000 abstract description 21
- 230000004043 responsiveness Effects 0.000 abstract description 4
- 208000011580 syndromic disease Diseases 0.000 abstract description 4
- 235000001014 amino acid Nutrition 0.000 description 137
- 210000004027 cell Anatomy 0.000 description 133
- 239000000203 mixture Substances 0.000 description 103
- 235000018102 proteins Nutrition 0.000 description 99
- 229940024606 amino acid Drugs 0.000 description 95
- 150000001413 amino acids Chemical class 0.000 description 95
- 230000014509 gene expression Effects 0.000 description 77
- 241000894007 species Species 0.000 description 73
- 241000699670 Mus sp. Species 0.000 description 69
- 150000007523 nucleic acids Chemical group 0.000 description 66
- 230000027455 binding Effects 0.000 description 64
- 239000000523 sample Substances 0.000 description 63
- 108020004707 nucleic acids Proteins 0.000 description 56
- 102000039446 nucleic acids Human genes 0.000 description 56
- 150000001768 cations Chemical class 0.000 description 50
- 108020001507 fusion proteins Proteins 0.000 description 49
- 102000037865 fusion proteins Human genes 0.000 description 49
- 102000001554 Hemoglobins Human genes 0.000 description 47
- 108010054147 Hemoglobins Proteins 0.000 description 47
- 239000013598 vector Substances 0.000 description 43
- 230000004927 fusion Effects 0.000 description 37
- 208000002903 Thalassemia Diseases 0.000 description 36
- 238000003556 assay Methods 0.000 description 33
- 230000007423 decrease Effects 0.000 description 32
- 230000035772 mutation Effects 0.000 description 32
- -1 Nodal Proteins 0.000 description 30
- 238000005534 hematocrit Methods 0.000 description 30
- 239000002243 precursor Substances 0.000 description 30
- 230000004069 differentiation Effects 0.000 description 27
- 239000012634 fragment Substances 0.000 description 27
- 230000000925 erythroid effect Effects 0.000 description 26
- 108010023082 activin A Proteins 0.000 description 25
- 108010023079 activin B Proteins 0.000 description 24
- 210000004369 blood Anatomy 0.000 description 23
- 239000008280 blood Substances 0.000 description 23
- 150000001875 compounds Chemical class 0.000 description 23
- 239000002773 nucleotide Substances 0.000 description 22
- 125000003729 nucleotide group Chemical group 0.000 description 22
- 239000013612 plasmid Substances 0.000 description 22
- 230000002829 reductive effect Effects 0.000 description 22
- 108020004414 DNA Proteins 0.000 description 20
- 108091028043 Nucleic acid sequence Proteins 0.000 description 20
- 230000013595 glycosylation Effects 0.000 description 19
- 238000006206 glycosylation reaction Methods 0.000 description 19
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 18
- 238000001514 detection method Methods 0.000 description 18
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 17
- 230000006907 apoptotic process Effects 0.000 description 17
- 210000000468 rubriblast Anatomy 0.000 description 17
- 230000011664 signaling Effects 0.000 description 17
- 238000006467 substitution reaction Methods 0.000 description 17
- 238000004458 analytical method Methods 0.000 description 16
- 238000002703 mutagenesis Methods 0.000 description 16
- 231100000350 mutagenesis Toxicity 0.000 description 16
- 239000003795 chemical substances by application Substances 0.000 description 15
- 238000004587 chromatography analysis Methods 0.000 description 15
- 238000004163 cytometry Methods 0.000 description 15
- 210000003527 eukaryotic cell Anatomy 0.000 description 15
- 239000013604 expression vector Substances 0.000 description 15
- 230000001105 regulatory effect Effects 0.000 description 15
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 description 14
- 210000000988 bone and bone Anatomy 0.000 description 14
- 102000016970 Follistatin Human genes 0.000 description 13
- 108010014612 Follistatin Proteins 0.000 description 13
- 239000002253 acid Substances 0.000 description 13
- 230000008859 change Effects 0.000 description 13
- 238000010186 staining Methods 0.000 description 12
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 11
- 239000000427 antigen Substances 0.000 description 11
- 108091007433 antigens Proteins 0.000 description 11
- 102000036639 antigens Human genes 0.000 description 11
- 238000012217 deletion Methods 0.000 description 11
- 230000037430 deletion Effects 0.000 description 11
- 208000014951 hematologic disease Diseases 0.000 description 11
- 210000001236 prokaryotic cell Anatomy 0.000 description 11
- 238000012216 screening Methods 0.000 description 11
- 208000019838 Blood disease Diseases 0.000 description 10
- 206010053138 Congenital aplastic anaemia Diseases 0.000 description 10
- 102000004190 Enzymes Human genes 0.000 description 10
- 108090000790 Enzymes Proteins 0.000 description 10
- 102100039939 Growth/differentiation factor 8 Human genes 0.000 description 10
- 101100437153 Rattus norvegicus Acvr2b gene Proteins 0.000 description 10
- 238000007792 addition Methods 0.000 description 10
- 238000004820 blood count Methods 0.000 description 10
- 150000001720 carbohydrates Chemical group 0.000 description 10
- 229940088598 enzyme Drugs 0.000 description 10
- 208000018706 hematopoietic system disease Diseases 0.000 description 10
- 239000003642 reactive oxygen metabolite Substances 0.000 description 10
- 230000004044 response Effects 0.000 description 10
- 230000001177 retroviral effect Effects 0.000 description 10
- 101000970954 Homo sapiens Activin receptor type-2A Proteins 0.000 description 9
- 101000937269 Homo sapiens Activin receptor type-2B Proteins 0.000 description 9
- 101000886562 Homo sapiens Growth/differentiation factor 8 Proteins 0.000 description 9
- 241001465754 Metazoa Species 0.000 description 9
- 241000700605 Viruses Species 0.000 description 9
- 150000007513 acids Chemical class 0.000 description 9
- 235000004279 alanine Nutrition 0.000 description 9
- 239000005557 antagonist Substances 0.000 description 9
- 230000006870 function Effects 0.000 description 9
- 102000045412 human ACVR2B Human genes 0.000 description 9
- 210000004408 hybridoma Anatomy 0.000 description 9
- 229910052742 iron Inorganic materials 0.000 description 9
- 239000000126 substance Substances 0.000 description 9
- 235000000346 sugar Nutrition 0.000 description 9
- 238000003786 synthesis reaction Methods 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 9
- 230000001225 therapeutic effect Effects 0.000 description 9
- 239000004475 Arginine Substances 0.000 description 8
- 238000002965 ELISA Methods 0.000 description 8
- 102000003951 Erythropoietin Human genes 0.000 description 8
- 108090000394 Erythropoietin Proteins 0.000 description 8
- 241000238631 Hexapoda Species 0.000 description 8
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 8
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 8
- 101150038444 Ment gene Proteins 0.000 description 8
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 8
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 8
- 230000006378 damage Effects 0.000 description 8
- 210000003013 erythroid precursor cell Anatomy 0.000 description 8
- 229940105423 erythropoietin Drugs 0.000 description 8
- 238000001727 in vivo Methods 0.000 description 8
- 239000002502 liposome Substances 0.000 description 8
- 230000001404 mediated effect Effects 0.000 description 8
- 238000010172 mouse model Methods 0.000 description 8
- 210000002381 plasma Anatomy 0.000 description 8
- 238000003118 sandwich ELISA Methods 0.000 description 8
- 239000003381 stabilizer Substances 0.000 description 8
- 239000012581 transferrin Substances 0.000 description 8
- 241000701447 unidentified baculovirus Species 0.000 description 8
- 230000004988 N-glycosylation Effects 0.000 description 7
- 102000043168 TGF-beta family Human genes 0.000 description 7
- 108091085018 TGF-beta family Proteins 0.000 description 7
- 102000004338 Transferrin Human genes 0.000 description 7
- 108090000901 Transferrin Proteins 0.000 description 7
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 7
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 7
- 230000004075 alteration Effects 0.000 description 7
- 238000004113 cell culture Methods 0.000 description 7
- 238000003776 cleavage reaction Methods 0.000 description 7
- 238000000338 in vitro Methods 0.000 description 7
- 210000004005 intermediate erythroblast Anatomy 0.000 description 7
- 239000003550 marker Substances 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 239000008194 pharmaceutical composition Substances 0.000 description 7
- 230000001323 posttranslational effect Effects 0.000 description 7
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 230000007017 scission Effects 0.000 description 7
- 102100033051 40S ribosomal protein S19 Human genes 0.000 description 6
- 208000033932 Blackfan-Diamond anemia Diseases 0.000 description 6
- 201000004449 Diamond-Blackfan anemia Diseases 0.000 description 6
- 102000005720 Glutathione transferase Human genes 0.000 description 6
- 108010070675 Glutathione transferase Proteins 0.000 description 6
- 241001529936 Murinae Species 0.000 description 6
- 108091034117 Oligonucleotide Proteins 0.000 description 6
- 208000031845 Pernicious anaemia Diseases 0.000 description 6
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 6
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 6
- 125000000539 amino acid group Chemical group 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 230000000295 complement effect Effects 0.000 description 6
- 230000008878 coupling Effects 0.000 description 6
- 238000010168 coupling process Methods 0.000 description 6
- 238000005859 coupling reaction Methods 0.000 description 6
- 235000018417 cysteine Nutrition 0.000 description 6
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 6
- 239000003623 enhancer Substances 0.000 description 6
- 230000002255 enzymatic effect Effects 0.000 description 6
- 238000001415 gene therapy Methods 0.000 description 6
- 238000002991 immunohistochemical analysis Methods 0.000 description 6
- 230000003993 interaction Effects 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 239000000816 peptidomimetic Substances 0.000 description 6
- 229920001223 polyethylene glycol Polymers 0.000 description 6
- 230000017854 proteolysis Effects 0.000 description 6
- 150000003839 salts Chemical class 0.000 description 6
- 230000009885 systemic effect Effects 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 5
- 108010076504 Protein Sorting Signals Proteins 0.000 description 5
- 239000004473 Threonine Substances 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- 210000002960 bfu-e Anatomy 0.000 description 5
- 208000020832 chronic kidney disease Diseases 0.000 description 5
- 208000035475 disorder Diseases 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 5
- 150000002500 ions Chemical class 0.000 description 5
- 238000002955 isolation Methods 0.000 description 5
- 150000002632 lipids Chemical class 0.000 description 5
- 210000004962 mammalian cell Anatomy 0.000 description 5
- 238000012545 processing Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 230000000087 stabilizing effect Effects 0.000 description 5
- 230000002103 transcriptional effect Effects 0.000 description 5
- UBWXUGDQUBIEIZ-UHFFFAOYSA-N (13-methyl-3-oxo-2,6,7,8,9,10,11,12,14,15,16,17-dodecahydro-1h-cyclopenta[a]phenanthren-17-yl) 3-phenylpropanoate Chemical compound CC12CCC(C3CCC(=O)C=C3CC3)C3C1CCC2OC(=O)CCC1=CC=CC=C1 UBWXUGDQUBIEIZ-UHFFFAOYSA-N 0.000 description 4
- 102000005606 Activins Human genes 0.000 description 4
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 4
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 4
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 4
- 102000053028 CD36 Antigens Human genes 0.000 description 4
- 108010045374 CD36 Antigens Proteins 0.000 description 4
- 102220479051 CD59 glycoprotein_L79D_mutation Human genes 0.000 description 4
- 108020004705 Codon Proteins 0.000 description 4
- 108010013369 Enteropeptidase Proteins 0.000 description 4
- 102100029727 Enteropeptidase Human genes 0.000 description 4
- 108010075944 Erythropoietin Receptors Proteins 0.000 description 4
- 102100036509 Erythropoietin receptor Human genes 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 102100035716 Glycophorin-A Human genes 0.000 description 4
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 4
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 4
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 4
- 101710154606 Hemagglutinin Proteins 0.000 description 4
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 4
- 241000282412 Homo Species 0.000 description 4
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 4
- 102000008100 Human Serum Albumin Human genes 0.000 description 4
- 108091006905 Human Serum Albumin Proteins 0.000 description 4
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 4
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 4
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 101710093908 Outer capsid protein VP4 Proteins 0.000 description 4
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 description 4
- 229910019142 PO4 Inorganic materials 0.000 description 4
- 108091005804 Peptidases Proteins 0.000 description 4
- 239000004365 Protease Substances 0.000 description 4
- 101710176177 Protein A56 Proteins 0.000 description 4
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 4
- 241000714474 Rous sarcoma virus Species 0.000 description 4
- 101150054830 S100A6 gene Proteins 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 4
- 230000002159 abnormal effect Effects 0.000 description 4
- 230000002378 acidificating effect Effects 0.000 description 4
- 230000009471 action Effects 0.000 description 4
- 230000001154 acute effect Effects 0.000 description 4
- 235000009582 asparagine Nutrition 0.000 description 4
- 229960001230 asparagine Drugs 0.000 description 4
- 235000003704 aspartic acid Nutrition 0.000 description 4
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 4
- 239000011230 binding agent Substances 0.000 description 4
- 230000036772 blood pressure Effects 0.000 description 4
- 230000008468 bone growth Effects 0.000 description 4
- 210000004899 c-terminal region Anatomy 0.000 description 4
- 239000001506 calcium phosphate Substances 0.000 description 4
- 230000001413 cellular effect Effects 0.000 description 4
- 238000007796 conventional method Methods 0.000 description 4
- 230000022811 deglycosylation Effects 0.000 description 4
- 231100000673 dose–response relationship Toxicity 0.000 description 4
- 239000000839 emulsion Substances 0.000 description 4
- 235000011187 glycerol Nutrition 0.000 description 4
- 239000000185 hemagglutinin Substances 0.000 description 4
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 4
- 230000001939 inductive effect Effects 0.000 description 4
- 238000005304 joining Methods 0.000 description 4
- 210000003734 kidney Anatomy 0.000 description 4
- 239000011159 matrix material Substances 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 108091033319 polynucleotide Proteins 0.000 description 4
- 102000040430 polynucleotide Human genes 0.000 description 4
- 239000002157 polynucleotide Substances 0.000 description 4
- 238000000159 protein binding assay Methods 0.000 description 4
- 230000009467 reduction Effects 0.000 description 4
- 239000011347 resin Substances 0.000 description 4
- 229920005989 resin Polymers 0.000 description 4
- 210000001995 reticulocyte Anatomy 0.000 description 4
- 210000004989 spleen cell Anatomy 0.000 description 4
- 210000000130 stem cell Anatomy 0.000 description 4
- 150000008163 sugars Chemical class 0.000 description 4
- 230000008685 targeting Effects 0.000 description 4
- 206010043554 thrombocytopenia Diseases 0.000 description 4
- 238000013518 transcription Methods 0.000 description 4
- 230000035897 transcription Effects 0.000 description 4
- 230000003612 virological effect Effects 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- 210000005253 yeast cell Anatomy 0.000 description 4
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 3
- 208000020084 Bone disease Diseases 0.000 description 3
- 238000011740 C57BL/6 mouse Methods 0.000 description 3
- 108091026890 Coding region Proteins 0.000 description 3
- 102220525561 Coiled-coil domain-containing protein 200_A24N_mutation Human genes 0.000 description 3
- 206010009944 Colon cancer Diseases 0.000 description 3
- 208000018698 Congenital Bone Marrow Failure Syndromes Diseases 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 108091006020 Fc-tagged proteins Proteins 0.000 description 3
- 108010010803 Gelatin Proteins 0.000 description 3
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 3
- 108091005250 Glycophorins Proteins 0.000 description 3
- 206010018910 Haemolysis Diseases 0.000 description 3
- 101000835093 Homo sapiens Transferrin receptor protein 1 Proteins 0.000 description 3
- 206010020772 Hypertension Diseases 0.000 description 3
- 108060003951 Immunoglobulin Proteins 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 3
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 3
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 3
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 3
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 3
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 3
- 240000007472 Leucaena leucocephala Species 0.000 description 3
- 235000010643 Leucaena leucocephala Nutrition 0.000 description 3
- 101710175625 Maltose/maltodextrin-binding periplasmic protein Proteins 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 230000004989 O-glycosylation Effects 0.000 description 3
- 102000013566 Plasminogen Human genes 0.000 description 3
- 108010051456 Plasminogen Proteins 0.000 description 3
- 239000002202 Polyethylene glycol Substances 0.000 description 3
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 3
- 108091030071 RNAI Proteins 0.000 description 3
- 238000011529 RT qPCR Methods 0.000 description 3
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 3
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 3
- 108700008625 Reporter Genes Proteins 0.000 description 3
- 241001000605 Semia Species 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N 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 3
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 3
- 102100026144 Transferrin receptor protein 1 Human genes 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 239000002671 adjuvant Substances 0.000 description 3
- 230000000692 anti-sense effect Effects 0.000 description 3
- 210000000628 antibody-producing cell Anatomy 0.000 description 3
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 239000006227 byproduct Substances 0.000 description 3
- 239000002775 capsule Substances 0.000 description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 3
- 230000033077 cellular process Effects 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 238000001246 colloidal dispersion Methods 0.000 description 3
- 238000010293 colony formation assay Methods 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 230000003111 delayed effect Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 239000006185 dispersion Substances 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 201000000523 end stage renal failure Diseases 0.000 description 3
- 210000000267 erythroid cell Anatomy 0.000 description 3
- 235000019441 ethanol Nutrition 0.000 description 3
- 239000008273 gelatin Substances 0.000 description 3
- 229920000159 gelatin Polymers 0.000 description 3
- 235000019322 gelatine Nutrition 0.000 description 3
- 235000011852 gelatine desserts Nutrition 0.000 description 3
- 230000009368 gene silencing by RNA Effects 0.000 description 3
- 108060003196 globin Proteins 0.000 description 3
- 235000013922 glutamic acid Nutrition 0.000 description 3
- 239000004220 glutamic acid Substances 0.000 description 3
- 108010067216 glycyl-glycyl-glycine Proteins 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 230000002489 hematologic effect Effects 0.000 description 3
- 230000008588 hemolysis Effects 0.000 description 3
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 3
- 238000003018 immunoassay Methods 0.000 description 3
- 230000002163 immunogen Effects 0.000 description 3
- 102000018358 immunoglobulin Human genes 0.000 description 3
- 230000001771 impaired effect Effects 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 238000004255 ion exchange chromatography Methods 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 229920002521 macromolecule Polymers 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 3
- 239000006187 pill Substances 0.000 description 3
- 102000054765 polymorphisms of proteins Human genes 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 230000002028 premature Effects 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 3
- 230000000644 propagated effect Effects 0.000 description 3
- 230000019491 signal transduction Effects 0.000 description 3
- 238000001542 size-exclusion chromatography Methods 0.000 description 3
- 239000001509 sodium citrate Substances 0.000 description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 3
- 230000035882 stress Effects 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 239000000375 suspending agent Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 239000003826 tablet Substances 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 238000011285 therapeutic regimen Methods 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 3
- 241000701161 unidentified adenovirus Species 0.000 description 3
- 239000003981 vehicle Substances 0.000 description 3
- 239000000080 wetting agent Substances 0.000 description 3
- PUPZLCDOIYMWBV-UHFFFAOYSA-N (+/-)-1,3-Butanediol Chemical compound CC(O)CCO PUPZLCDOIYMWBV-UHFFFAOYSA-N 0.000 description 2
- VBICKXHEKHSIBG-UHFFFAOYSA-N 1-monostearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(O)CO VBICKXHEKHSIBG-UHFFFAOYSA-N 0.000 description 2
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 description 2
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 2
- 102000013563 Acid Phosphatase Human genes 0.000 description 2
- 108010051457 Acid Phosphatase Proteins 0.000 description 2
- 101710191686 Activin receptor type-2A Proteins 0.000 description 2
- 206010002536 Anisocytosis Diseases 0.000 description 2
- 102220638160 Arylamine N-acetyltransferase 1_R64K_mutation Human genes 0.000 description 2
- 241000416162 Astragalus gummifer Species 0.000 description 2
- 241000271566 Aves Species 0.000 description 2
- 108010049931 Bone Morphogenetic Protein 2 Proteins 0.000 description 2
- 108010049955 Bone Morphogenetic Protein 4 Proteins 0.000 description 2
- 102100024506 Bone morphogenetic protein 2 Human genes 0.000 description 2
- 102100024505 Bone morphogenetic protein 4 Human genes 0.000 description 2
- 241000701822 Bovine papillomavirus Species 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 2
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 2
- 101710132601 Capsid protein Proteins 0.000 description 2
- 102000053642 Catalytic RNA Human genes 0.000 description 2
- 108090000994 Catalytic RNA Proteins 0.000 description 2
- 102220578645 Chorion-specific transcription factor GCMb_N65A_mutation Human genes 0.000 description 2
- 101710094648 Coat protein Proteins 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 2
- 206010062759 Congenital dyskeratosis Diseases 0.000 description 2
- 241000699802 Cricetulus griseus Species 0.000 description 2
- 241000701022 Cytomegalovirus Species 0.000 description 2
- 238000008789 Direct Bilirubin Methods 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241000701959 Escherichia virus Lambda Species 0.000 description 2
- 108010074860 Factor Xa Proteins 0.000 description 2
- 102000009109 Fc receptors Human genes 0.000 description 2
- 108010087819 Fc receptors Proteins 0.000 description 2
- 101150050927 Fcgrt gene Proteins 0.000 description 2
- 102000008857 Ferritin Human genes 0.000 description 2
- 238000008416 Ferritin Methods 0.000 description 2
- 108050000784 Ferritin Proteins 0.000 description 2
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 2
- KOSRFJWDECSPRO-WDSKDSINSA-N Glu-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(O)=O KOSRFJWDECSPRO-WDSKDSINSA-N 0.000 description 2
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 2
- 241000713858 Harvey murine sarcoma virus Species 0.000 description 2
- 101000777658 Homo sapiens Platelet glycoprotein 4 Proteins 0.000 description 2
- 101001059454 Homo sapiens Serine/threonine-protein kinase MARK2 Proteins 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- 102000006496 Immunoglobulin Heavy Chains Human genes 0.000 description 2
- 108010019476 Immunoglobulin Heavy Chains Proteins 0.000 description 2
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- 101710125418 Major capsid protein Proteins 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 241000713869 Moloney murine leukemia virus Species 0.000 description 2
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 2
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 2
- OVRNDRQMDRJTHS-CBQIKETKSA-N N-Acetyl-D-Galactosamine Chemical compound CC(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-CBQIKETKSA-N 0.000 description 2
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 2
- MBLBDJOUHNCFQT-UHFFFAOYSA-N N-acetyl-D-galactosamine Natural products CC(=O)NC(C=O)C(O)C(O)C(O)CO MBLBDJOUHNCFQT-UHFFFAOYSA-N 0.000 description 2
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 2
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 2
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 2
- 101710141454 Nucleoprotein Proteins 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 208000013234 Pearson syndrome Diseases 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 108091000080 Phosphotransferase Proteins 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- 206010035774 Poikilocytosis Diseases 0.000 description 2
- 101710083689 Probable capsid protein Proteins 0.000 description 2
- 108010014608 Proto-Oncogene Proteins c-kit Proteins 0.000 description 2
- 102000016971 Proto-Oncogene Proteins c-kit Human genes 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 102100028904 Serine/threonine-protein kinase MARK2 Human genes 0.000 description 2
- 108020004459 Small interfering RNA Proteins 0.000 description 2
- 206010041660 Splenomegaly Diseases 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 101710137500 T7 RNA polymerase Proteins 0.000 description 2
- 206010043391 Thalassaemia beta Diseases 0.000 description 2
- 102000002933 Thioredoxin Human genes 0.000 description 2
- 108090000190 Thrombin Proteins 0.000 description 2
- 238000008050 Total Bilirubin Reagent Methods 0.000 description 2
- 101710120037 Toxin CcdB Proteins 0.000 description 2
- 229920001615 Tragacanth Polymers 0.000 description 2
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 2
- 102000000523 Type II Activin Receptors Human genes 0.000 description 2
- 108010041546 Type II Activin Receptors Proteins 0.000 description 2
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 2
- 241000269370 Xenopus <genus> Species 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 230000021736 acetylation Effects 0.000 description 2
- 238000006640 acetylation reaction Methods 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 230000010933 acylation Effects 0.000 description 2
- 238000005917 acylation reaction Methods 0.000 description 2
- 235000010443 alginic acid Nutrition 0.000 description 2
- 229920000615 alginic acid Polymers 0.000 description 2
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 2
- 125000003368 amide group Chemical group 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000001640 apoptogenic effect Effects 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 125000003118 aryl group Chemical group 0.000 description 2
- 229940009098 aspartate Drugs 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 210000003651 basophil Anatomy 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- SESFRYSPDFLNCH-UHFFFAOYSA-N benzyl benzoate Chemical compound C=1C=CC=CC=1C(=O)OCC1=CC=CC=C1 SESFRYSPDFLNCH-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-FPRJBGLDSA-N beta-D-galactose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-FPRJBGLDSA-N 0.000 description 2
- 108010005774 beta-Galactosidase Proteins 0.000 description 2
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 2
- 230000000975 bioactive effect Effects 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 210000000601 blood cell Anatomy 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 230000037182 bone density Effects 0.000 description 2
- 210000002798 bone marrow cell Anatomy 0.000 description 2
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 2
- 230000021523 carboxylation Effects 0.000 description 2
- 238000006473 carboxylation reaction Methods 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 210000000845 cartilage Anatomy 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 238000005277 cation exchange chromatography Methods 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 230000030833 cell death Effects 0.000 description 2
- 230000024245 cell differentiation Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 2
- OSASVXMJTNOKOY-UHFFFAOYSA-N chlorobutanol Chemical compound CC(C)(O)C(Cl)(Cl)Cl OSASVXMJTNOKOY-UHFFFAOYSA-N 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000013375 chromatographic separation Methods 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000003501 co-culture Methods 0.000 description 2
- 229920001436 collagen Polymers 0.000 description 2
- 108010049937 collagen type I trimeric cross-linked peptide Proteins 0.000 description 2
- 230000001332 colony forming effect Effects 0.000 description 2
- 238000004040 coloring Methods 0.000 description 2
- 238000012937 correction Methods 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 2
- 229960003957 dexamethasone Drugs 0.000 description 2
- 230000000447 dimerizing effect Effects 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 208000009356 dyskeratosis congenita Diseases 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 239000003995 emulsifying agent Substances 0.000 description 2
- 208000028208 end stage renal disease Diseases 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000007717 exclusion Effects 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 102000018146 globin Human genes 0.000 description 2
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 2
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 2
- 230000002414 glycolytic effect Effects 0.000 description 2
- 229930182470 glycoside Natural products 0.000 description 2
- 150000002338 glycosides Chemical class 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 102000051522 human CD36 Human genes 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 229960002591 hydroxyproline Drugs 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 238000011532 immunohistochemical staining Methods 0.000 description 2
- 238000001114 immunoprecipitation Methods 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 239000003701 inert diluent Substances 0.000 description 2
- 208000018337 inherited hemoglobinopathy Diseases 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 208000032839 leukemia Diseases 0.000 description 2
- 108020001756 ligand binding domains Proteins 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000008297 liquid dosage form Substances 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 2
- 229920000609 methyl cellulose Polymers 0.000 description 2
- 239000001923 methylcellulose Substances 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 230000037257 muscle growth Effects 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- 229950006780 n-acetylglucosamine Drugs 0.000 description 2
- 108091008104 nucleic acid aptamers Proteins 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 239000004006 olive oil Substances 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 125000001151 peptidyl group Chemical group 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 235000021317 phosphate Nutrition 0.000 description 2
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 2
- 150000003904 phospholipids Chemical class 0.000 description 2
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 2
- 230000026731 phosphorylation Effects 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- 102000020233 phosphotransferase Human genes 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 229920000747 poly(lactic acid) Polymers 0.000 description 2
- 229920002704 polyhistidine Polymers 0.000 description 2
- 239000004626 polylactic acid Substances 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000002708 random mutagenesis Methods 0.000 description 2
- 239000002464 receptor antagonist Substances 0.000 description 2
- 229940044551 receptor antagonist Drugs 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 206010038796 reticulocytosis Diseases 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 206010039073 rheumatoid arthritis Diseases 0.000 description 2
- 108091092562 ribozyme Proteins 0.000 description 2
- 201000006681 severe congenital neutropenia Diseases 0.000 description 2
- 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 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 239000006188 syrup Substances 0.000 description 2
- 235000020357 syrup Nutrition 0.000 description 2
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 2
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- 229940124597 therapeutic agent Drugs 0.000 description 2
- 125000003396 thiol group Chemical group [H]S* 0.000 description 2
- 108060008226 thioredoxin Proteins 0.000 description 2
- 229940094937 thioredoxin Drugs 0.000 description 2
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 description 2
- 229960004072 thrombin Drugs 0.000 description 2
- 239000000196 tragacanth Substances 0.000 description 2
- 235000010487 tragacanth Nutrition 0.000 description 2
- 229940116362 tragacanth Drugs 0.000 description 2
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 230000001052 transient effect Effects 0.000 description 2
- 230000010474 transient expression Effects 0.000 description 2
- 102000035160 transmembrane proteins Human genes 0.000 description 2
- 108091005703 transmembrane proteins Proteins 0.000 description 2
- 229910000391 tricalcium phosphate Inorganic materials 0.000 description 2
- 235000019731 tricalcium phosphate Nutrition 0.000 description 2
- 229940078499 tricalcium phosphate Drugs 0.000 description 2
- 238000013042 tunel staining Methods 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- 230000002485 urinary effect Effects 0.000 description 2
- 239000004474 valine Substances 0.000 description 2
- JNYAEWCLZODPBN-JGWLITMVSA-N (2r,3r,4s)-2-[(1r)-1,2-dihydroxyethyl]oxolane-3,4-diol Chemical class OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O JNYAEWCLZODPBN-JGWLITMVSA-N 0.000 description 1
- XJOTXKZIRSHZQV-RXHOOSIZSA-N (3S)-3-amino-4-[[(2S,3R)-1-[[(2S)-1-[[(2S)-1-[(2S)-2-[[(2S,3S)-1-[[(1R,6R,12R,17R,20S,23S,26R,31R,34R,39R,42S,45S,48S,51S,59S)-51-(4-aminobutyl)-31-[[(2S)-6-amino-1-[[(1S,2R)-1-carboxy-2-hydroxypropyl]amino]-1-oxohexan-2-yl]carbamoyl]-20-benzyl-23-[(2S)-butan-2-yl]-45-(3-carbamimidamidopropyl)-48-(hydroxymethyl)-42-(1H-imidazol-4-ylmethyl)-59-(2-methylsulfanylethyl)-7,10,19,22,25,33,40,43,46,49,52,54,57,60,63,64-hexadecaoxo-3,4,14,15,28,29,36,37-octathia-8,11,18,21,24,32,41,44,47,50,53,55,58,61,62,65-hexadecazatetracyclo[32.19.8.26,17.212,39]pentahexacontan-26-yl]amino]-3-methyl-1-oxopentan-2-yl]carbamoyl]pyrrolidin-1-yl]-1-oxo-3-phenylpropan-2-yl]amino]-3-(1H-imidazol-4-yl)-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxobutan-2-yl]amino]-4-oxobutanoic acid Chemical compound CC[C@H](C)[C@H](NC(=O)[C@@H]1CCCN1C(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](Cc1cnc[nH]1)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(O)=O)[C@@H](C)O)C(=O)N[C@H]1CSSC[C@H](NC(=O)[C@@H]2CSSC[C@@H]3NC(=O)[C@@H]4CSSC[C@H](NC(=O)[C@H](Cc5ccccc5)NC(=O)[C@@H](NC1=O)[C@@H](C)CC)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](Cc1cnc[nH]1)NC3=O)C(=O)NCC(=O)N[C@@H](CCSC)C(=O)N2)C(=O)NCC(=O)N4)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XJOTXKZIRSHZQV-RXHOOSIZSA-N 0.000 description 1
- NRJAVPSFFCBXDT-HUESYALOSA-N 1,2-distearoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCCCC NRJAVPSFFCBXDT-HUESYALOSA-N 0.000 description 1
- TZCPCKNHXULUIY-RGULYWFUSA-N 1,2-distearoyl-sn-glycero-3-phosphoserine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCCCCCCCCCCCC TZCPCKNHXULUIY-RGULYWFUSA-N 0.000 description 1
- 229940058015 1,3-butylene glycol Drugs 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- JNODDICFTDYODH-UHFFFAOYSA-N 2-hydroxytetrahydrofuran Chemical compound OC1CCCO1 JNODDICFTDYODH-UHFFFAOYSA-N 0.000 description 1
- HVCOBJNICQPDBP-UHFFFAOYSA-N 3-[3-[3,5-dihydroxy-6-methyl-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid;hydrate Chemical compound O.OC1C(OC(CC(=O)OC(CCCCCCC)CC(O)=O)CCCCCCC)OC(C)C(O)C1OC1C(O)C(O)C(O)C(C)O1 HVCOBJNICQPDBP-UHFFFAOYSA-N 0.000 description 1
- OSJPPGNTCRNQQC-UWTATZPHSA-N 3-phospho-D-glyceric acid Chemical compound OC(=O)[C@H](O)COP(O)(O)=O OSJPPGNTCRNQQC-UWTATZPHSA-N 0.000 description 1
- 102100034134 Activin receptor type-1B Human genes 0.000 description 1
- 101710173005 Activin receptor type-1C Proteins 0.000 description 1
- 102100034135 Activin receptor type-1C Human genes 0.000 description 1
- 208000030090 Acute Disease Diseases 0.000 description 1
- 102220556625 Acyl-coenzyme A thioesterase 11_K55A_mutation Human genes 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 238000008940 Alkaline Phosphatase assay kit Methods 0.000 description 1
- 239000005995 Aluminium silicate Substances 0.000 description 1
- 235000003276 Apios tuberosa Nutrition 0.000 description 1
- 241000256844 Apis mellifera Species 0.000 description 1
- 102000010565 Apoptosis Regulatory Proteins Human genes 0.000 description 1
- 108010063104 Apoptosis Regulatory Proteins Proteins 0.000 description 1
- 244000105624 Arachis hypogaea Species 0.000 description 1
- 235000010777 Arachis hypogaea Nutrition 0.000 description 1
- 235000010744 Arachis villosulicarpa Nutrition 0.000 description 1
- 240000003291 Armoracia rusticana Species 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 241000714235 Avian retrovirus Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 206010065687 Bone loss Diseases 0.000 description 1
- 102100028726 Bone morphogenetic protein 10 Human genes 0.000 description 1
- 101710118482 Bone morphogenetic protein 10 Proteins 0.000 description 1
- 102100022544 Bone morphogenetic protein 7 Human genes 0.000 description 1
- 101000937267 Bos taurus Activin receptor type-2B Proteins 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 240000001546 Byrsonima crassifolia Species 0.000 description 1
- 235000003197 Byrsonima crassifolia Nutrition 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 241000202252 Cerberus Species 0.000 description 1
- 102220578642 Chorion-specific transcription factor GCMb_K74M_mutation Human genes 0.000 description 1
- 108010022452 Collagen Type I Proteins 0.000 description 1
- 102000012422 Collagen Type I Human genes 0.000 description 1
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 108091035707 Consensus sequence Proteins 0.000 description 1
- 201000006306 Cor pulmonale Diseases 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 208000011231 Crohn disease Diseases 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 102100033215 DNA nucleotidylexotransferase Human genes 0.000 description 1
- 108010008286 DNA nucleotidylexotransferase Proteins 0.000 description 1
- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 description 1
- 235000019739 Dicalciumphosphate Nutrition 0.000 description 1
- HJEINPVZRDJRBY-UHFFFAOYSA-N Disul Chemical group OS(=O)(=O)OCCOC1=CC=C(Cl)C=C1Cl HJEINPVZRDJRBY-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 102220575187 Egl nine homolog 1_F82A_mutation Human genes 0.000 description 1
- LVGKNOAMLMIIKO-UHFFFAOYSA-N Elaidinsaeure-aethylester Natural products CCCCCCCCC=CCCCCCCCC(=O)OCC LVGKNOAMLMIIKO-UHFFFAOYSA-N 0.000 description 1
- 102100040897 Embryonic growth/differentiation factor 1 Human genes 0.000 description 1
- 108010036395 Endoglin Proteins 0.000 description 1
- 102100037241 Endoglin Human genes 0.000 description 1
- 101710088235 Envelope glycoprotein C homolog Proteins 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 201000004939 Fanconi anemia Diseases 0.000 description 1
- 102100027286 Fanconi anemia group C protein Human genes 0.000 description 1
- 239000004606 Fillers/Extenders Substances 0.000 description 1
- 102100029379 Follistatin-related protein 3 Human genes 0.000 description 1
- 241000206672 Gelidium Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 1
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 description 1
- 229930186217 Glycolipid Natural products 0.000 description 1
- 102000006354 HLA-DR Antigens Human genes 0.000 description 1
- 108010058597 HLA-DR Antigens Proteins 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 208000017604 Hodgkin disease Diseases 0.000 description 1
- 208000021519 Hodgkin lymphoma Diseases 0.000 description 1
- 208000010747 Hodgkins lymphoma Diseases 0.000 description 1
- 101000799189 Homo sapiens Activin receptor type-1B Proteins 0.000 description 1
- 101000899361 Homo sapiens Bone morphogenetic protein 7 Proteins 0.000 description 1
- 101000893552 Homo sapiens Embryonic growth/differentiation factor 1 Proteins 0.000 description 1
- 101000931668 Homo sapiens Follistatin Proteins 0.000 description 1
- 101001062529 Homo sapiens Follistatin-related protein 3 Proteins 0.000 description 1
- 101000934338 Homo sapiens Myeloid cell surface antigen CD33 Proteins 0.000 description 1
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 1
- 241000257303 Hymenoptera Species 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 102000009490 IgG Receptors Human genes 0.000 description 1
- 108010073807 IgG Receptors Proteins 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 102100025885 Inhibin alpha chain Human genes 0.000 description 1
- 108010004250 Inhibins Proteins 0.000 description 1
- 206010065973 Iron Overload Diseases 0.000 description 1
- 241000726306 Irus Species 0.000 description 1
- 101710177504 Kit ligand Proteins 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 240000003183 Manihot esculenta Species 0.000 description 1
- 235000016735 Manihot esculenta subsp esculenta Nutrition 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 108010036176 Melitten Proteins 0.000 description 1
- 208000036696 Microcytic anaemia Diseases 0.000 description 1
- 101100060131 Mus musculus Cdk5rap2 gene Proteins 0.000 description 1
- 101000987583 Mus musculus Eosinophil peroxidase Proteins 0.000 description 1
- 101000716728 Mus musculus Kit ligand Proteins 0.000 description 1
- 102100025243 Myeloid cell surface antigen CD33 Human genes 0.000 description 1
- 108010056852 Myostatin Proteins 0.000 description 1
- 208000015914 Non-Hodgkin lymphomas Diseases 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 240000007817 Olea europaea Species 0.000 description 1
- 206010031252 Osteomyelitis Diseases 0.000 description 1
- 238000010222 PCR analysis Methods 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 102220496976 Platelet-activating factor acetylhydrolase 2, cytoplasmic_L79A_mutation Human genes 0.000 description 1
- 241000276498 Pollachius virens Species 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 229920002732 Polyanhydride Polymers 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 102220542870 Presenilins-associated rhomboid-like protein, mitochondrial_L79E_mutation Human genes 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 201000004681 Psoriasis Diseases 0.000 description 1
- 102220495689 Putative uncharacterized protein FLJ43944_R40A_mutation Human genes 0.000 description 1
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
- 235000004443 Ricinus communis Nutrition 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 201000004283 Shwachman-Diamond syndrome Diseases 0.000 description 1
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- SSZBUIDZHHWXNJ-UHFFFAOYSA-N Stearinsaeure-hexadecylester Natural products CCCCCCCCCCCCCCCCCC(=O)OCCCCCCCCCCCCCCCC SSZBUIDZHHWXNJ-UHFFFAOYSA-N 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 102100032120 Toll/interleukin-1 receptor domain-containing adapter protein Human genes 0.000 description 1
- 102000046299 Transforming Growth Factor beta1 Human genes 0.000 description 1
- 101800002279 Transforming growth factor beta-1 Proteins 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 108010015920 Type I Activin Receptors Proteins 0.000 description 1
- 102000002138 Type I Activin Receptors Human genes 0.000 description 1
- 206010046865 Vaccinia virus infection Diseases 0.000 description 1
- 108010003533 Viral Envelope Proteins Proteins 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- ATBOMIWRCZXYSZ-XZBBILGWSA-N [1-[2,3-dihydroxypropoxy(hydroxy)phosphoryl]oxy-3-hexadecanoyloxypropan-2-yl] (9e,12e)-octadeca-9,12-dienoate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCC(O)CO)OC(=O)CCCCCCC\C=C\C\C=C\CCCCC ATBOMIWRCZXYSZ-XZBBILGWSA-N 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 235000010419 agar Nutrition 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 1
- 238000012867 alanine scanning Methods 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- AWUCVROLDVIAJX-UHFFFAOYSA-N alpha-glycerophosphate Natural products OCC(O)COP(O)(O)=O AWUCVROLDVIAJX-UHFFFAOYSA-N 0.000 description 1
- 150000004645 aluminates Chemical class 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 235000012211 aluminium silicate Nutrition 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000002788 anti-peptide Effects 0.000 description 1
- 230000005875 antibody response Effects 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000004872 arterial blood pressure Effects 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 239000000440 bentonite Substances 0.000 description 1
- 229910000278 bentonite Inorganic materials 0.000 description 1
- SVPXDRXYRYOSEX-UHFFFAOYSA-N bentoquatam Chemical compound O.O=[Si]=O.O=[Al]O[Al]=O SVPXDRXYRYOSEX-UHFFFAOYSA-N 0.000 description 1
- 229960002903 benzyl benzoate Drugs 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-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 1
- 239000003462 bioceramic Substances 0.000 description 1
- 239000005312 bioglass Substances 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 208000034158 bleeding Diseases 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 210000001772 blood platelet Anatomy 0.000 description 1
- 238000010322 bone marrow transplantation Methods 0.000 description 1
- 230000018678 bone mineralization Effects 0.000 description 1
- 230000037118 bone strength Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- 235000019437 butane-1,3-diol Nutrition 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- CJZGTCYPCWQAJB-UHFFFAOYSA-L calcium stearate Chemical compound [Ca+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O CJZGTCYPCWQAJB-UHFFFAOYSA-L 0.000 description 1
- 235000013539 calcium stearate Nutrition 0.000 description 1
- 239000008116 calcium stearate Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 239000004359 castor oil Substances 0.000 description 1
- 238000000423 cell based assay Methods 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000008809 cell oxidative stress Effects 0.000 description 1
- 230000007248 cellular mechanism Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 229930183167 cerebroside Natural products 0.000 description 1
- 150000001784 cerebrosides Chemical class 0.000 description 1
- 229960000541 cetyl alcohol Drugs 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 229960004926 chlorobutanol Drugs 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000011210 chromatographic step Methods 0.000 description 1
- 208000022831 chronic renal failure syndrome Diseases 0.000 description 1
- 239000004927 clay Substances 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 206010009887 colitis Diseases 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 230000005757 colony formation Effects 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000002742 combinatorial mutagenesis Methods 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 230000001447 compensatory effect Effects 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 231100001021 decreased hematocrit Toxicity 0.000 description 1
- 210000003074 dental pulp Anatomy 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 229910003460 diamond Inorganic materials 0.000 description 1
- 239000010432 diamond Substances 0.000 description 1
- NEFBYIFKOOEVPA-UHFFFAOYSA-K dicalcium phosphate Chemical compound [Ca+2].[Ca+2].[O-]P([O-])([O-])=O NEFBYIFKOOEVPA-UHFFFAOYSA-K 0.000 description 1
- 229910000390 dicalcium phosphate Inorganic materials 0.000 description 1
- 229940038472 dicalcium phosphate Drugs 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 235000015872 dietary supplement Nutrition 0.000 description 1
- UGMCXQCYOVCMTB-UHFFFAOYSA-K dihydroxy(stearato)aluminium Chemical compound CCCCCCCCCCCCCCCCCC(=O)O[Al](O)O UGMCXQCYOVCMTB-UHFFFAOYSA-K 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 239000008298 dragée Substances 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 238000009547 dual-energy X-ray absorptiometry Methods 0.000 description 1
- 230000004821 effect on bone Effects 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 230000002500 effect on skin Effects 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 230000000913 erythropoietic effect Effects 0.000 description 1
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 description 1
- GWEJFLVSOGNLSS-WPFOTENUSA-N ethyl (2s)-2-amino-5-[[(2r)-3-benzylsulfanyl-1-[[(1r)-2-ethoxy-2-oxo-1-phenylethyl]amino]-1-oxopropan-2-yl]amino]-5-oxopentanoate Chemical compound C([C@H](NC(=O)CC[C@H](N)C(=O)OCC)C(=O)N[C@@H](C(=O)OCC)C=1C=CC=CC=1)SCC1=CC=CC=C1 GWEJFLVSOGNLSS-WPFOTENUSA-N 0.000 description 1
- LVGKNOAMLMIIKO-QXMHVHEDSA-N ethyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC LVGKNOAMLMIIKO-QXMHVHEDSA-N 0.000 description 1
- 229940093471 ethyl oleate Drugs 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 229940014144 folate Drugs 0.000 description 1
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 1
- 235000019152 folic acid Nutrition 0.000 description 1
- 239000011724 folic acid Substances 0.000 description 1
- 108700017752 gamma-Glu-S-BzCys-PhGly diethyl ester Proteins 0.000 description 1
- 150000002270 gangliosides Chemical class 0.000 description 1
- 239000007903 gelatin capsule Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 1
- 150000004676 glycans Polymers 0.000 description 1
- YQEMORVAKMFKLG-UHFFFAOYSA-N glycerine monostearate Natural products CCCCCCCCCCCCCCCCCC(=O)OC(CO)CO YQEMORVAKMFKLG-UHFFFAOYSA-N 0.000 description 1
- SVUQHVRAGMNPLW-UHFFFAOYSA-N glycerol monostearate Natural products CCCCCCCCCCCCCCCCC(=O)OCC(O)CO SVUQHVRAGMNPLW-UHFFFAOYSA-N 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- 210000000777 hematopoietic system Anatomy 0.000 description 1
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
- 208000034737 hemoglobinopathy Diseases 0.000 description 1
- 102000018511 hepcidin Human genes 0.000 description 1
- 108060003558 hepcidin Proteins 0.000 description 1
- 229940066919 hepcidin Drugs 0.000 description 1
- BXWNKGSJHAJOGX-UHFFFAOYSA-N hexadecan-1-ol Chemical compound CCCCCCCCCCCCCCCCO BXWNKGSJHAJOGX-UHFFFAOYSA-N 0.000 description 1
- 239000000710 homodimer Substances 0.000 description 1
- 102000050536 human FST Human genes 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000010874 in vitro model Methods 0.000 description 1
- 238000005462 in vivo assay Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000000893 inhibin Substances 0.000 description 1
- 108010032856 inhibin-alpha subunit Proteins 0.000 description 1
- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
- 150000002614 leucines Chemical class 0.000 description 1
- 230000029226 lipidation Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000007937 lozenge Substances 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 210000004324 lymphatic system Anatomy 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 201000004792 malaria Diseases 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- VDXZNPDIRNWWCW-UHFFFAOYSA-N melitten Chemical compound NCC(=O)NC(C(C)CC)C(=O)NCC(=O)NC(C)C(=O)NC(C(C)C)C(=O)NC(CC(C)C)C(=O)NC(CCCCN)C(=O)NC(C(C)C)C(=O)NC(CC(C)C)C(=O)NC(C(C)O)C(=O)NC(C(C)O)C(=O)NCC(=O)NC(CC(C)C)C(=O)N1CCCC1C(=O)NC(C)C(=O)NC(CC(C)C)C(=O)NC(C(C)CC)C(=O)NC(CO)C(=O)NC(C(=O)NC(C(C)CC)C(=O)NC(CCCCN)C(=O)NC(CCCNC(N)=N)C(=O)NC(CCCCN)C(=O)NC(CCCNC(N)=N)C(=O)NC(CCC(N)=O)C(=O)NC(CCC(N)=O)C(N)=O)CC1=CNC2=CC=CC=C12 VDXZNPDIRNWWCW-UHFFFAOYSA-N 0.000 description 1
- 210000004779 membrane envelope Anatomy 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 239000000693 micelle Substances 0.000 description 1
- 239000004530 micro-emulsion Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- CQDGTJPVBWZJAZ-UHFFFAOYSA-N monoethyl carbonate Chemical compound CCOC(O)=O CQDGTJPVBWZJAZ-UHFFFAOYSA-N 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 239000002324 mouth wash Substances 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 239000002088 nanocapsule Substances 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 239000012457 nonaqueous media Substances 0.000 description 1
- 230000007959 normoxia Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 150000002895 organic esters Chemical class 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 230000005298 paramagnetic effect Effects 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 235000010603 pastilles Nutrition 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 239000002304 perfume Substances 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 102000013415 peroxidase activity proteins Human genes 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 238000009521 phase II clinical trial Methods 0.000 description 1
- 125000001095 phosphatidyl group Chemical group 0.000 description 1
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 150000004804 polysaccharides Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000013105 post hoc analysis Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 229960004063 propylene glycol Drugs 0.000 description 1
- 235000013772 propylene glycol Nutrition 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000003571 reporter gene assay Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 239000003340 retarding agent Substances 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 102220125997 rs141863326 Human genes 0.000 description 1
- 102220288227 rs141863326 Human genes 0.000 description 1
- 102220140258 rs146572379 Human genes 0.000 description 1
- 102200145357 rs1555341957 Human genes 0.000 description 1
- 102220036189 rs273585616 Human genes 0.000 description 1
- 238000007423 screening assay Methods 0.000 description 1
- 239000008159 sesame oil Substances 0.000 description 1
- 235000011803 sesame oil Nutrition 0.000 description 1
- 150000004760 silicates Chemical class 0.000 description 1
- RMAQACBXLXPBSY-UHFFFAOYSA-N silicic acid Chemical compound O[Si](O)(O)O RMAQACBXLXPBSY-UHFFFAOYSA-N 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 210000002027 skeletal muscle Anatomy 0.000 description 1
- 208000017520 skin disease Diseases 0.000 description 1
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000007909 solid dosage form Substances 0.000 description 1
- 239000008247 solid mixture Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 230000035488 systolic blood pressure Effects 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
- 229930101283 tetracycline Natural products 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 230000036964 tight binding Effects 0.000 description 1
- 230000030968 tissue homeostasis Effects 0.000 description 1
- 238000007492 two-way ANOVA Methods 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 208000007089 vaccinia Diseases 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
Abstract
Provided herein are methods of treating diseases associated with anemia or ineffective erythropoiesis by using the level and/or activity of a ligand of an activin receptor, in particular, Growth differentiation factor 11 (GDF11), as an indicator of responsiveness of a patient to the treatment, efficacy of the treatment, or appropriate dosage for the treatment with an activin type II receptor inhibitor. In particular, the present invention relates to the use of an activin receptor type II inhibitor for treating anemia, beta thalassemia, increasing red blood cell levels, increasing orthochromatic erythroblasts (Ery-C), inherited bone marrow failures or myelodsplastic syndromes in patients having elevated GDF11 levels. GDF11 levels are used as a biomarker of treatment efficacy. acy of the treatment, or appropriate dosage for the treatment with an activin type II receptor inhibitor. In particular, the present invention relates to the use of an activin receptor type II inhibitor for treating anemia, beta thalassemia, increasing red blood cell levels, increasing orthochromatic erythroblasts (Ery-C), inherited bone marrow failures or myelodsplastic syndromes in patients having elevated GDF11 levels. GDF11 levels are used as a biomarker of treatment efficacy.
Description
KER FOR USE IN TREATING ANEMIA This ation is a divisional application of New Zealand Patent Application No. 707469 and claims priority to U.S. provisional patent application No. 61/718,126, October 24, 2012, the disclosure of each of which is herein incorporated by reference in its entirety. 1. INTRODUCTION Provided herein are s of treating diseases associated with anemia or ineffective erythropoiesis by using the level and/or activity of a ligand of an n type II receptor, in particular, Growth differentiation factor 11 ), as an indicator of responsiveness of a patient to the treatment, efficacy of the treatment, or appropriate dosage for the treatment with an activin type II receptor inhibitor. For e, provided herein are methods for determining the level and/or activity of GDF11, ing patients having elevated level and/or activity of GDF11, and treating patients having elevated level and/or activity of GDF11 with an activin type II receptor inhibitor. Further provided herein are s comprising determining the level and/or activity of GDF11 in a patient treated with an activin type II receptor inhibitor, and administering the activin type II receptor tor in a dose amount depending on the level and/or ty of GDF11 in the patient. Such activin type II receptor inhibitor can be an inhibitor of ActRIIA and / or ActRIIB signaling (e.g., a humanized fusion-protein consisting of the extracellular domain of activin-receptor type IIA and the human IgG1 Fc domain). 2. BACKGROUND Anemia is a se in number of red blood cells or less than the normal quantity of hemoglobin in the blood. Anemia can also be caused by decreased oxygen-binding ability of the hemoglobin. Anemia is the most common disorder of the blood.
Anemia can be caused by ineffective erythropoiesis. Ineffective erythropoiesis is present if active erythropoiesis takes place but mature red blood cells fail to develop at the proper rate. Progenitor cells undergo apoptosis before the stage of mature red blood cells is reached.
Thalassemia is a form of ineffective erythropoiesis. In thalassemia, ineffective opoiesis is characterized by apoptosis of the maturing nucleated oid cells.
Specifically beta-thalassemia is a disease characterized by defects in Hemoglobin (or Hgb) synthesis leading to impaired erythrocyte maturation and production. The decrease is primarily thought to be due to abnormally accelerated erythroid differentiation and apoptosis at the late basophilic/polychromatic erythroblast stage ofred blood cell differentiation, resulting in an overall decrease in mature red blood cell production. beta-thalassemia is characterized by a hypercellular bone marrow compartment where abnormal erythroblasts accumulate and o apoptosis, resulting in systemic anemia.
The transforming growth factor beta (TGF-beta) family, which include TGF-beta, activins, bone genic proteins (BMPs) and growth and entiation s (GDFs) are secreted proteins known to regulate numerous cellular processes during both development and tissue homeostasis. TGF-beta1, activin A, BMP-2 and BMP-4 have all been associated with the regulation of erythropoiesis in various model systems. TGF—betal both inhibits and promotes erythroid differentiation depending on the context, activin A was shown to be a pro-erythroid differentiation agent, while BMP-4 has been ated in stress erythropoiesis and recovery from acute anemia in murine models. BMP-2 acts on early erythroid cells to increase colony formation in samples obtained from mobilized peripheral blood or bone marrow CD34+ cells. ally high levels of some of these growth factors have been ated with various hematologic diseases. For example, high levels of GDP—15 are not usually a feature of normal erythropoiesis but in conditions of ineffective erythropoiesis, GDF-lS expression is elevated.
The TGF-beta superfamily consists ofmore than 30 proteins and they signal through only a limited number of receptors and signaling pathways, pointing to inherent cuity and redundancy in their actions. Furthermore, in any given tissue, l different ligands may be found and signaling presumably occurs via an overlapping subset of receptors complicating the ability to ate particular ligands to their function. GDFll is a member of the GDP ily and shares about 90% amino acid gy with GDF8, also known as myostatin.
Both can bind the activin type 11A and B ors and activate the Smad 2/3 ing pathway.
GDFll plays a big role in development, taking part in the ion of muscle, cartilage, bone, kidney, and nervous system while in adult tissues, GDFll was detected in the pancreas, intestine, kidney, skeletal muscle, brain and dental pulp. Low amounts of GDF-l] can be also found in the circulation. To this date, however, there is no evidence describing a role for GDP- 11 in erythropoiesis.
Type 11 receptors for activins can also bind GDF11. Two related type 11 receptors, ActRIIa and ActRIIb, have been identi?ed (Mathews and Vale, 1991, Cell 65 :973-982; Attisano et al., 1992, Cell 68: 97-108). Besides GDP] 1, ActRIIa and ActRIIb can biochemically interact with several other TGF-beta family proteins, including BMP7, Nodal, GDF8, and activin (Yamashita et al., 1995, J. Cell Biol. 130:217-226; Lee and McPherron, 2001, Proc. Natl.
Acad. Sci. 98:9306-9311; Yeo and Whitman, 2001, Mol. Cell 7: 949-957; Oh et al., 2002, Genes Dev. 16:2749-54). ALK4 is the primary type I receptor for ns, particularly for activin A, and ALK-7 may serve as a receptor for activins as well, particularly for activin B.
A humanized -protein consisting of the extracellular domain of activinreceptor type IIA IA) and the human IgG1 Fc (ActRIIA-hFc) is tly being evaluated in phase II clinical trials for treatment of patients with anemia and bone disorders associated with end-stage renal disease (ESRD) as well as for those patients with betathalassemia.
In healthy post-menopausal women, ActRIIA-hFc was shown to significantly increase hematocrit (Hct) and hemoglobin (Hgb) as well as bone l density. However, the identity of ActRIIA ligands sequestered by ActRIIA-hFc in the bloodstream s unknown.
SUMMARY In one aspect, provided herein are methods for treating anemia, or a blood disorder associated with anemia or ineffective erythropoiesis, using GDF11 as an indicator of responsiveness of a t to the treatment, cy of the treatment, or riate dosage for the treatment of the patient with an activin type II receptor inhibitor. In particular the present invention relates to the use of an activing receptor type II inhibitor which is a polypeptide comprising an amino acid sequence selected from the group consisting of: a 90% identical to SEQ ID NO:2; b 95% identical to SEQ ID NO:2; c 98% identical to SEQ ID NO:2; d SEQ ID NO:2; e 90% identical to SEQ ID NO:3; f 95% identical to SEQ ID NO:3; g 98% cal to SEQ ID NO:3; h SEQ ID NO:3; i 90% identical to SEQ ID NO:7; j 95% identical to SEQ ID NO:7; k 98% identical to SEQ ID NO:7; l SEQ ID NO:7; m 90% identical to SEQ ID NO:12; n 95% identical to SEQ ID NO:12; o 98% cal to SEQ ID NO:12; p SEQ ID NO:12; q 90% identical to SEQ ID NO:17; r 95% identical to SEQ ID NO:17; s 98% identical to SEQ ID NO:17; t SEQ ID NO:17; u 90% identical to SEQ ID NO:20; v 95% identical to SEQ ID NO:20; w 98% identical to SEQ ID NO:20; x SEQ ID NO:20; y 90% identical to SEQ ID NO:21; z 95% identical to SEQ ID NO:21; aa 98% identical to SEQ ID NO:21; bb SEQ ID NO:21 cc 90% identical to SEQ ID NO:22; dd 95% identical to SEQ ID NO:22; ee 98% identical to SEQ ID NO:22; ff SEQ ID NO:22; gg 90% identical to SEQ ID NO:23; hh 95% identical to SEQ ID NO:23; ii 98% identical to SEQ ID NO:23; jj SEQ ID NO:23; kk 90% cal to SEQ ID NO:24; ll 95% identical to SEQ ID NO:24; mm 98% identical to SEQ ID NO:24; nn SEQ ID NO:24; oo 90% identical to SEQ ID NO:25; pp 95% identical to SEQ ID NO:25; qq 98% identical to SEQ ID NO:25; and rr SEQ ID NO:25; wherein the activin receptor type II tor is a polypeptide comprising the extracellular domain of activin receptor type IIA linked to the Fc portion of an antibody; or wherein the activin receptor type II inhibitor is a polypeptide comprising the extracellular domain of activin or type IIB linked to the Fc portion of an dy, according to one of the embodiments below: (1) Use of an activin receptor type II inhibitor in the manufacture of a medicament for treatment of anemia, beta thalassemia, or for increasing red blood cell levels or orthochromatic erythroblasts (Ery-C) in a human subject in need thereof, n the ent ses: (a) assessing the level of GDF11 in a tissue sample of the human subject, and (b) if the level of GDF11 is ed over the normal level of GDF11, administering an activin receptor type II inhibitor to the human subject. (2) Use of an activin receptor type II tor in the manufacture of a medicament for treating anemia, chronic pernicious anemia, sickle cell anemia, myelodysplastic syndromes, inherited bone marrow failure, or beta-thalassemia, or for increasing red blood cell levels or orthochromatic erythroblasts (Ery-C) in a human subject in need of treatment, wherein the human t treated has an elevated level of GDF11 as compared to the level of GDF11 of one or more healthy human subjects. (3) Use of an activin receptor type II inhibitor in manufacture of a medicament for the treatment of anemia, beta semia, or for increasing red blood cell levels or orthochromatic erythroblasts (Ery-C) in a human subject in need thereof, wherein the treatment comprises: (a) administering an activin receptor type II inhibitor, and (b) monitoring the level of GDF11 in a tissue sample of the human subject. (4) The use of any one of (1)-(3), wherein the treatment comprises: (a) administering a dose amount of an activin receptor type II inhibitor, (b) assessing the level of GDF11 in a tissue sample of the human subject, and (c) adjusting the dose amount of the activin receptor type II tor, wherein, if the level of GDF11 is elevated over the normal level of GDF11, the dose amount of the activin receptor type II inhibitor is increased, and wherein, if the level of GDF11 is decreased over the normal level and/or activity of GDF11, the dose amount of the activin receptor type II inhibitor is sed. (5) The use of any one of (1)-(3), n the treatment comprises: (i) determining a first level of GDF11 in a tissue sample of the human subject; (ii) administering the activin receptor type II inhibitor, and (iii) determining a second level of GDF11 in a tissue sample of the human subject, and (iv) adjusting a subsequent dose amount of the activin receptor type II inhibitor; wherein, if the second level of GDF11 is elevated over the first level of GDF11, the subsequent dose amount of the activin receptor type II inhibitor is increased, and wherein, if the second level of GDF11 is decreased over the level of GDF11 in the same tissue of one or more y human subjects, the subsequent dose amount of the activin receptor type II tor is decreased. (6) The use of (4) or (5), wherein the level of GDF11 is decreased when it is sed at least 25%, at least 50%, at least 75%, or at least 90% over the normal level of GDF11, or over the level of GDF11 in the same tissue of one or more healthy human subjects. (7) The use of (4) or (5), wherein the level and/or activity of GDF11 is decreased when it is decreased at least 2 times over the normal level of GDF11 or over the level of GDF11 in the same tissue of one or more healthy human subjects. (8) The use of any one of (1), (2) and (4) to (7), wherein the level of GDF11 is elevated when it is elevated at least 25%, at least 50%, at least 75%, at least 100% or at least 200% over the normal or first level of GDF11 or the level of GDF11 in the same tissue of one or more healthy human subjects. (9) The use of any one of (1), (2) and (4) to (7), wherein the level of GDF11 is elevated when it is elevated at least 2 times over the normal or first level of GDF11 or the level of GDF11 in the same tissue of one or more y human subjects. (10) The use of any one of (1), (4), and ), wherein the normal level of GDF11 is an average level of GDF11 in a sample from the same tissue of one or more healthy human subjects in the same age category. (11) The use of any one of (1) and (3)-(10), wherein the tissue is obtained from serum, bone marrow, liver, or spleen. (12) The use of any one of (1)-(11), n the level of GDF11 is the protein level of GDF11; or wherein the level of GDF11 is mRNA level of GDF11. (13) The use of any one of (1)-(12), n the Fc portion of an antibody is Fc portion of an IgG1 antibody.
In certain embodiments, provided herein is a method of determining the level and/or activity of GDF11 in a tissue sample of a patient having anemia, or a disorder, e.g., a blood disorder associated with anemia or ineffective erythropoiesis. In some embodiments, provided herein is a method of determining the level and/or activity of GDF11 in a tissue sample of a t having anemia, or a disorder, e.g., a blood disorder associated with anemia or ineffective erythropoiesis, and comparing it to the normal level and/or activity of GDF11 (e.g., the average level and/or activity of GDF11 in a sample from the same tissue of a healthy subject or subjects). In some embodiments, provided herein is a method of determining the level and/or ty of GDF11 in a tissue sample (such as serum/plasma, blood, bone marrow, spleen, or liver) of a patient having anemia, or a disorder, e.g., a blood disorder associated with anemia or ineffective opoiesis, and ing it to the level and/or activity of GDF11 in a sample from a different tissue of the subject (such as a tissue in which the level of GDF11 is not elevated in patients having anemia).
In certain embodiments, ed herein is a method for treating anemia, or a blood disorder associated with anemia or ineffective erythropoiesis, wherein the method ses: (a) assessing the level and/or activity of GDF11 in a tissue sample of the subject, and (b) if the level and/or activity of GDF11 is elevated over the normal level and/or activity of GDF11, administering an activin or type II inhibitor to the subject. In some embodiments, provided herein is a method for treating anemia, or a blood disorder associated with anemia or ineffective erythropoiesis, wherein the method ses: (a) administering an activin receptor type II inhibitor, and (b) monitoring the level and/or activity of GDF11 in a tissue sample of the subject. In certain embodiments, provided herein is a method for treating anemia, or a blood disorder associated with anemia or ctive erythropoiesis, n the method comprises: (a) administering a dose amount of an activin receptor type II inhibitor, (b) assessing the level and/or activity of GDF11 in a tissue sample of the subject, and (c) adjusting the dose amount of the activin receptor type II inhibitor wherein, if the level and/or activity of GDF11 is elevated over the normal level and/or ty of GDF11, the dose amount of the activin receptor type II inhibitor is increased, and wherein, if the level and/or ty of GDF11 is decreased over the normal level and/or activity of GDF11, the dose amount of the activin or type II inhibitor is decreased. The blood disorders associated with anemia or ineffective erythropoiesis treated in accordance with the methods described herein e, without limitation, thalassemia (e.g., beta thalassemia), myelodysplastic (followed by page 7A) me, chronic pernicious anemia, sickle cell anemia, Diamond Blackfan anemia, decreased red blood cell levels, or decreased orthochromatic erythroblasts (Ery-C). In a red embodiment, the subject being treated in accordance with the methods described herein is a human.
In certain embodiments, ed herein is a method for treating anemia in a subject in need of treatment, wherein the method comprises: (a) ing the level and/or ty of GDF11 in a tissue sample of the subject, and (b) if the level and/or activity of GDF11 is elevated over the normal level and/or activity of GDF11, administering an activin receptor type II inhibitor to the subject.
Further, in some embodiments, provided herein is a method for treating thalassemia (e.g., beta thalassemia) in a subject in need of ent, wherein the method ses: (a) assessing the level and/or ty of GDF11 in a tissue sample of the subject, and (b) if the level and/or activity of GDF11 is elevated over the normal level and/or activity of GDF11, administering an activin receptor type II tor to the subject. In particular embodiments, provided herein is a method for treating thalassemia (e.g., beta thalassemia) in a subject in need of treatment, wherein the method comprises: (a) administering a dose amount of an activin receptor type II inhibitor, (b) assessing the level and/or activity of GDF11 in a tissue sample of the subject, and (c) adjusting the dose amount of the activin receptor type II inhibitor n, if the level and/or activity of GDF11 is elevated over the normal level and/or activity of GDF11, the dose amount of the n receptor type II inhibitor is increased, and wherein, if the level and/or activity of GDF11 is decreased over the normal level and/or activity of GDF11, the dose amount of the activin receptor type II inhibitor is decreased.
In some embodiments, provided herein is a method for increasing red blood cell levels or orthochromatic erythroblast (Ery-C) levels in a subject in need thereof, wherein the method comprises: (a) assessing the level and/or activity of GDF11 in a tissue sample of the subject, and (b) if the level and/or activity of GDF11 is elevated over the normal level and/or ty of GDF11, administering an activin receptor type II inhibitor to the subject. In other embodiments, provided herein is a method for increasing red blood cell levels or orthochromatic erythroblast ) levels in a subject in need thereof, wherein the method comprises: (a) administering a dose amount of an activin receptor type II inhibitor, (b) assessing the level and/or activity of GDF11 in a tissue sample of the subject, and (c) adjusting the dose amount of the activin receptor type II inhibitor wherein, if the level and/or (followed by page 7B) activity of GDF11 is elevated over the normal level and/or activity of GDF11, the dose amount of the activin receptor type II inhibitor is increased, and wherein, if the level and/or activity of GDF11 is decreased over the normal level and/or activity of GDF11, the dose amount of the activin receptor type II inhibitor is decreased.
In specific embodiments, administering of an activin receptor type II inhibitor in accordance with the methods described herein results in a decreased cell count of late basophilic and polychromatic erythroblasts (Ery-B) in the patient. In some embodiments, said administering results in a decreased level of bin (e.g., serum level of bilirubin) in the patient. In some embodiments, said administering results in a decreased level of lactate ogenase (e.g., serum level of lactate dehydrogenase) in the patient. In some embodiments, said administering s in decreased bone marrow and/or spleen cellularity in the patient. In some embodiments, said administering results in decreased reticulocyte counts in the patient. In some embodiments, said administering results in decreased erring saturation and/or sed systemic iron levels in the patient. In preferred embodiments, stering of an activin receptor type II inhibitor in accordance with the methods described herein results in increased red blood cell counts, increased hematocrit levels and/or increased hemoglobin levels in the patient. In particular embodiments, administering of an activin receptor type II inhibitor in ance with the methods described herein results in increased mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and/or MCH concentration in the patient. In certain embodiments, such results are assessed in the t in addition to assessment of the level and/or activity of GDF11 in the subject.
In certain embodiments, the ActRII inhibitor that can be used in the methods provided herein is a polypeptide comprising an amino acid sequence selected from the group consisting of: 90% identical to SEQ ID NO:2; 95% cal to SEQ ID NO:2; 98% identical to SEQ ID NO:2; SEQ ID NO:2; 90% identical to SEQ ID NO:3; 95% identical to SEQ ID NO:3; 98% identical to SEQ ID NO:3; SEQ ID NO:3; 90% cal to SEQ ID NO:6; 95% cal to SEQ ID NO:6; 98% identical to SEQ ID NO:6; SEQ ID NO:6; 90% cal to SEQ ID NO:7; 95% cal to SEQ ID NO:7; 98% identical to SEQ ID NO:7; SEQ ID NO:7; 90% identical to SEQ ID NO:12; 95% identical to SEQ ID NO:12; 98% identical to SEQ ID NO:12; SEQ ID NO:12; 90% identical to SEQ ID NO:17; 95% identical to SEQ ID NO:17; 98% identical to SEQ ID NO:17; SEQ ID NO:17; 90% identical to SEQ ID NO:20; 95% identical to SEQ ID NO:20; 98% identical to SEQ ID NO:20; SEQ ID NO:20; 90% (followed by page 7C) identical to SEQ ID NO:21; 95% identical to SEQ ID NO:21; 98% identical to SEQ ID NO:21; and SEQ ID NO:21. In some embodiments, the ActRII inhibitor is a humanized fusion-protein consisting of the extracellular domain of ActRIIA and the human IgG1 Fc domain. In a more ic embodiment, the ActRII inhibitor is a polypeptide comprising the amino acid sequence of SEQ ID NO:7. In one ment, the ActRII inhibitor is administered parenterally.
In certain embodiments, the normal level and/or activity of GDF11 is an average level and/or activity of GDF11 in a tissue sample of one or more healthy subjects (e.g., healthy subjects in the same age category and/or of the same gender). In some embodiments, the level and/or ty of GDF11 in a tissue sample of the treated subject is compared to the level and/or activity of GDF11 in a sample from the same tissue of one or more healthy subjects. In some embodiments, the tissue in which the level and/or activity of GDF11 is assessed is whole blood, blood serum/plasma, bone marrow, liver, or spleen. In one embodiment, the tissue in which the level and/or activity of GDF11 is assessed is serum.
The level and/or activity of GDF11 can be measured by any method known in the art or described herein. In one embodiment, the level and/or activity of GDF11 is the protein level of GDF11. In one embodiment, the level and/or activity of GDF11 is mRNA level of GDF11.
In n embodiments, the level and/or activity of GDF11 is deemed to be elevated over the normal level and/or activity of GDF11 when it is elevated at least 10%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, or at least 500% over the normal level and/or activity of GDF11. In certain embodiments, the level and/or ty of GDF11 is deemed to be ed over the normal level and/or activity of GDF11 when it is elevated at least 25%, at least 50%, at least 75%, at least 100%, or at least 200% over the normal level and/or activity of GDF11. In a particular ment, the level and/or activity of GDF11 is deemed to be elevated over the normal level and/or activity of GDF11 when it is ed at least 50% over the normal level and/or activity of GDF11. In a particular embodiment, the level and/or activity of GDF11 is deemed to be elevated over the normal level and/or activity of GDF11 when it is elevated at least 100% over the normal level and/or activity of GDF11.
In a particular embodiment, the level and/or activity of GDF11 is deemed to be elevated over the normal level and/or activity of GDF11 when it is elevated at least 200% over the normal level and/or ty of GDF11. In one embodiment, the level and/or activity of GDF11 is (followed by page 7D) deemed to be elevated over the normal level and/or ty of GDF11 when it is elevated at least 1.5 or 2 times over the normal level and/or activity of GDF11. In some embodiments, the level and/or activity of GDF11 is deemed to be decreased over the normal level and/or activity of GDF11 when it is decreased at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, or at least 90% over the normal level and/or activity of GDF11. In some embodiments, the level and/or activity of GDF11 is deemed to be decreased over the normal level and/or activity of GDF11 when it is decreased at least 25%, at least 50%, at least 75%, or at least 90% over the normal level and/or activity of GDF11. In one embodiment, the level and/or activity of GDF11 is deemed to be decreased over the normal level and/or activity of GDF11 when it is decreased at least 25% over the normal level and/or activity of GDF11. In one embodiment, the level and/or activity of GDF11 is deemed to be decreased over the normal level and/or activity of GDF11 when it is decreased at least 50% over the normal level and/or activity of GDF11. In one embodiment, the level and/or activity of GDF11 is deemed to be sed over the normal level and/or ty of GDF11 when it is decreased at least 75% over the normal level and/or activity of GDF11. In one ment, the level and/or activity of GDF11 is deemed to be decreased over the normal level and/or activity of GDF11 when it is decreased at least 1.5 or 2 times over the normal level and/or activity of GDF11. (followed by page 8) 4. BRIEF PTION OF THE FIGURES illustrates that the murine counterpart of SEQ ID NO 7 IIA-Fc) ameliorates hematological parameters in B—thalassemic mice. be thl/thl mice (Skow LC et al., Cell 34: 1043-52, 1983) were treated with PBS or mActRIIA-Fc (10 mg/Kg body weight twice a week) for 60 days. Hematological parameters were evaluated on days 5, 10, 30 and 60.
Evaluation of (A) red blood cell counts, (B) hematocrit and (C) hemoglobin was associated with a decreased (D) reticulocytosis. Analysis of circulating red blood cells (RBC) parameters also show that (E) mean corpuscular volume (MCV), (F) mean corpuscular hemoglobin (MCH) and (G) MCH tration (MCHC) increased in mice treated with mActRIIA-Fc. (H) Total antioxidant status. (I) Morphological analysis showed a reduction in anisocytosis, poikilocytosis and target cells. (J) Systemic iron levels, (K) transferrin sis, (L) transferrin and (M) ferritin levels tion were also evaluated on thalassemic mice. (N) In?ammatory cell counts were also ed. Effects of mActRIIA-Fc on splenomegaly in thalassemic mice ted by (O) spleen weight and total cell numbers. (P) Bone marrow erythroblasts numbers and expansion /hematoxylin staining) were also decreased in mice treated with mActRIIA-Fc. (Q) Bone marrow and spleen erythroblasts were quanti?ed by ?ow cytometry by TER119 staining. p<0.05, N=3-5 for each independent experiment. illustrates that IA-Fc decreases ineffective erythropoiesis in thalassemic mice. (A-C) Bone marrow and spleen were harvested and erythroblast differentiation was evaluated by ?ow try by CD7l/TER119 staining and FSC/SSC distribution. (D) Analysis of total and direct bilirubin levels. (E) Reactive oxygen species (ROS) generation on primary pro-erythroblast differentiation was evaluated by ?ow cytometry using rodihydro?uorescein. (F) Analysis of hemoglobin solubility on y thalassemic pro- erythroblasts treated for 48 hours with mActRIIA—Fc or PBS. *p<0.05, **p<0.01, ***p<0.005 , N=3-5 for each independent experiment. illustrates the effect of mActRIIA-Fc on the apoptosis of erythroblasts from thalassemic mice. Expression of Fas-L on the bone marrow (A) and spleen (B) erythroblasts from mice treated with mActRIIA-Fc or PBS. (C) Tunel staining of erythroblasts was increased in mActRIIA-Fc-treated mice. illustrates the expression of ActRIIA s in the spleen of thalassemic mice.
(A) mRNA expression levels of ActRII, activin A, activin B and GDFll were increased in mActRIIA-Fc-treated animals. (B) Western blot analysis of GDFll protein levels, which were decreased in mActRIIA-Fc-treated animals. (C) Immunohistochemical staining of bone marrow for GDFll showed no change between wildtype and mice treated with mActRIIA-Fc. illustrates the effect ofmActRIIA-Fc on GDF11 expression in y thalassemic proerythroblasts. (A) Immunohistochemical analysis of activin/GDF signaling pathway on thalassemic mice treated with PBS or mActRIIA-Fc for 30 days, showing increased levels of GDF1 1, ActRII and 2 on thalassemic mice. (B) Immunohistochemical analysis of activin A, activin B and GDFll on thalassemic mice, compared to other models of anemia.
(C) FACS analysis of y semic pro-erythroblasts treated with PBS or mActRIIA-Fc for 48 hours using speci?c antibodies against activin A, activin B, GDFll propeptide and GDF8/GDF11 d e. ?cation of GDFll staining is demonstrated on the graph bars. (D) Immunohistochemical is of GDFll proform expression in the spleens of thalassemic mice treated with PBS or IA—Fc. * p<0.05, N=4. illustrates that inhibition of GDFll reduces ineffective erythropoiesis in thalassemic mice. (A) Bone marrow and spleen were harvested and primary pro-erythroblast differentiation was evaluated by ?ow cytometry by CD71/TER119 staining and FSC/SSC bution. (B) ROS generation on primary pro-erythroblast differentiation was evaluated by ?ow cytometry using dichlorodihydrofluorescein. * p<0.05, N=4. rates a sandwich ELISA assay to detect GDF11 in serum. (A) Schematic of assay. (B) Plates were coated with 5 ug/mL ofmActRIIA-Fc and increasing doses of recombinant GDF11 (0 ng/ul, 0.1 ng/ul, 0.5 ng/ul, 2.5 ng/ul) or control sera (1/4 to 1/500 dilution) were added to plates coated with mActRIIA-Fc, the plates washed and bound protein detected with anti-GDF8/ 11 antibodies, followed by detection using a horseradish peroxidase- coupled anti-rabbit IgG. GDF11 protein bound the plates in a dose-dependent manner. illustrates the detection of increased levels of GDF11 in the serum of [3- thalassemic patients. Sera were obtained from patients presenting thalassemia and healthy controls. illustrates a sandwich ELISA assay to detect activin A in serum. (A) Schematic of assay. (B) Plates were coated with 5 ug/mL of ActRIIA-Fc (SEQ ID NO. 7) and increasing doses of recombinant activin A (0 ng/ul, 0.1 ng/ul, 0.5 ng/ul, 2.5 ng/ul) or control sera (1/4 to 1/500 dilution) were added to ActRIIA—Fc , the plates washed and bound protein detected with anti-activin A antibodies, followed by detection using a adish peroxidase-coupled anti-rabbit IgG. Activin A protein bound the plates in a dose-dependent manner. (C) Detection of levels of activin A in the serum of B-thalassemic ts. Sera were obtained from ts presenting thalassemia and healthy controls. There was no change in the serum levels of activin A in thalassemic patients.
FIG 10 illustrates a sandwich ELISA assay to detect activin B in serum. (A) Schematic of assay. (B) Plates were coated with 5 ug/mL of ActRIIA—Fc and increasing doses of recombinant activin B (0 ng/ul, 0.1 ng/ul, 0.5 ng/ul, 2.5 ng/ul) or control sera (1/4 to 1/500 dilution) were added to ActRIIA-Fc plates, the plates washed and bound protein detected with ctivin B antibodies, followed by detection using a horseradish peroxidase-coupled anti- rabbit IgG. Activin B protein bound the plates in a dose-dependent manner. (C) Detection of levels of activin B in the serum of B—thalassemic patients. Sera were obtained from patients presenting semia and healthy controls. There was no change in the serum levels of activin B in thalassemic patients. illustrates that stration ofmActRIIA-Fc to C57BL/6 wildtype mice does not change their logical parameters. Evaluation of (A) red blood cell counts, (B) hematocrit, (C) hemoglobin showed no association with mActRIIA-Fc. There was a slight decrease (D) reticulocytosis. mActRIIA-Fc did not change red blood cells (RBC) parameters such as (E) mean corpuscular volume (MCV), (F) mean corpuscular hemoglobin (MCH) or (G) MCH concentration . * p<0.05, N=3-5 for each independent experiment. illustrates that administration ofmActRIIA-Fc to C57BL/6 wildtype mice had no effect on the number of spleen and bone marrow cells of the mice. illustrates that mActRIIA-Fc stimulates erythroid differentiation through inhibition of GDFl l. (A-C) Erythroid differentiation of CD34+/CD36+ cells was performed by ing in media ning EPO, g/mL ofmActRIIA—Fc; (A) erythroid progenitors, (B) cell proliferation and (C) erythoid precursors were analyzed. (D-F) Erythroid differentiation of CD34+/CD36+ cells when co-cultured with bone marrow (BM) cells in media containing EPO, g/mL of mActRIIA-Fc; (D) erythroid progenitors, (E) cell proliferation and (F) erythoid precursors were analyzed. (G-H) Erythroid differentiation of CD36+ cells was performed by culturing in media containing EPO, +/- 200ng/mL of GDP] 1, +/- 100ug/mL of mActRIIA-Fc; (G) cell proliferation and (H) percentage of erythroid precursors GPA+ were analyzed . DETAILED PTION .1 OVERVIEW Provided herein are methods of treating anemia, or diseases or conditions associated with anemia or ineffective erythropoiesis, by using the level and/or activity of GDFll as an tor of responsiveness of a patient to the treatment with an activin type II receptor inhibitor, efficacy of the treatment with an activin type II receptor inhibitor, or appropriate dosage for the treatment with an activin type II receptor tor. The diseases associated with anemia or ineffective erythropoiesis that can be treated in accordance with the methods described herein include, without limitation, thalassemia (e.g., beta thalassemia), myelodysplastic syndrome, chronic ious anemia, sickle cell anemia, and Diamond Blackfan anemia. The conditions associated with anemia or ineffective erythropoiesis that can be treated in ance with the methods described herein e, without limitation, decreased red blood cell levels, decreased hemoglobin levels, decreased hematocrit levels, and decreased hromatic erythroblasts (Ery-C). In one embodiment, provided herein are methods for treating anemia. In one embodiment, provided herein are methods for treating thalassemia (e.g., beta thalassemia). In one embodiment, provided herein are s for treating decreased red blood cell levels or methods for sing red blood cell levels. An activin type II receptor tor used in the methods described herein can be an tor of ActRIIA and / or ActRIIB, such as any of the inhibitors bed herein or known in the art. In a preferred embodiment, an activin type II receptor tor is a humanized fusion-protein consisting of the extracellular domain of ActRIIA and the human IgG1 Fc domain ("ActRIIA-Fc," e.g., SEQ ID NO:7).
The methods provided herein are based, in part, on the discovery that the levels of GDFll are increased in the blood of human patients with beta-thalassemia and in a mouse model of beta-thalassemia, and that ActRIIA-ch IA fused to a murine IgG) reduces increased levels of GDFll in the mouse model of beta—thalassemia (see es). Further, without being limited by theory, ligand trapping by ActRIIA-ch corrects ineffective opoiesis and improves anemia in an experimental mouse model of thalassemia intermedia (bethl/m1 mice).
As shown in the Examples presented herein, treating of thalassemic mice (bethl/thl) with ActRIIA-ch increased red blood cell counts, hematocrit, hemoglobin, mean cell volume (MCV) and mean cell hemoglobin, increased orthochromatic oblasts (Ery-C), reduced bone marrow and spleen cellularity, reduced late basophilic/polychromatic erythroblasts (Ery-B), in the spleen and bone marrow, reduced the levels of bilirubin levels (indicative of a decrease in red blood cell destruction), and reduced apoptotic cells. Furthermore, the methods provided herein are based, in part, on the discovery that in an in vitro culture system GDF-ll inhibits growth of human bone marrow-derived erythroid precursors and ActRIIA-ch rescues this inhibitory effect (see Examples). Taken together, the data ted herein indicate that GDF-ll levels, e.g., blood and/or serum levels of GDP] 1, can identify which patients may respond to ActRIIA-Fc and can be used to monitor clinical response to the drug. The data presented herein also indicate that ActRIIA-Fc (e. g., A-ch, or ActRIIA—hFc such as SEQ ID NO:7) is useful in treating anemia associated with ineffective erythropoiesis.
The ?ndings described herein, illustrated in the Examples, indicate that detection of the levels and/or activity of GDFll can be used (i) as a marker (e. g., serum biomarker) of the extent of ineffective erythropoiesis in a patient, (ii) as a marker to measure the patient’s response to an activin type II or tor (such as ActRIIA—Fc), or (iii) to evaluate the pharmacodynamic effects of an activin type II receptor inhibitor (such as ActRIIA-Fc) in a patient ing treatment, wherein the patient is a patient with anemia or a disease associated with anemia or ineffective erythropoiesis (such as thalassemia, e.g., beta-thalassemia). Thus, in certain embodiments, GDFll can be used in the s bed herein as an indicator of efficacy of ActRIIA-Fc (e.g., ActRIIA-hFc such as SEQ ID NO:7) treatment and/or as an indicator of e of response to the treatment with ActRIIA-Fc (e.g., A-hFc such as SEQ ID NO:7). In addition, as described herein, GDFll can be used as a reliable molecular marker to evaluate time-course treatment ef?cacy of ActRIIA-Fc (e.g., ActRIIA-hFc such as SEQ ID NO:7). Further, in a speci?c embodiment, provided herein is a method which comprises ion of the level and/or activity of GDFll in the blood (e.g., detecting abnormal expression in a disease associated with ineffective erythropoiesis), and administration of an activin type II receptor inhibitor, such as ActRIIA-Fc, in a dose ent on the level and/or activity of GDFl l. .2 DIAGNOSTIC/ PROGNOSTIC/ THERAPEUTIC METHODS In one aspect, the methods provided herein comprise determining the level and/or activity of GDF11 in a tissue sample of a subject (e.g., serum or blood), selecting ts having elevated level and/or activity of GDFll over the normal level and/or activity of GDF11, and treating subjects having ed level and/or activity of GDFll with an activin type II receptor tor. Elevated levels and/or activity of GDP" in a tissue sample of a subject (e. g., serum or blood) can indicate that the patient may be responsive to the treatment with an activin type II receptor inhibitor. In one embodiment of the methods described herein, the activin type II receptor inhibitor is ActRIIA-Fc such as ActRIIA—hFc (e.g., SEQ ID NO:7).
GDFll level and/or activity may be further used to evaluate appropriate dosing for a subject who is a candidate to be treated with an activin type II receptor inhibitor, to evaluate whether to adjust the dosage of the activin type II receptor inhibitor during treatment, and/or to te an appropriate maintenance dose of the activin type II receptor inhibitor. If the GDFll level and/or activity is outside the normal level and/or activity, dosing with an n type II receptor inhibitor may be initiated, increased, reduced, d or terminated depending on the level and/or activity of GDFl 1. For example, if the GDFll level and/or activity is ed over the normal level and/or activity, dosing with an activin type II receptor inhibitor may be initiated or increased, and if the GDFll level and/or activity is decreased over the normal level and/or activity, dosing with an activin type II or inhibitor may be reduced, delayed or terminated.
In one aspect, the methods bed herein comprise administering to a subject an activin type II or inhibitor, and monitoring the level and/or activity of GDFll in the subject. A decrease in the level and/or activity of GDFll after administration of an activin type II receptor inhibitor (relative to the level and/or activity before administration of the activin type II inhibitor) can indicate that the subject is responsive to the treatment with the activin type II receptor inhibitor and/or that the treatment with the activin type II receptor inhibitor is ef?cacious. No change or increase in the level and/or activity of GDFll after administration of an activin type II or tor (relative to the level and/or activity before administration of the activin type II inhibitor) can indicate that the subject is not responsive to the ent with an activin type II receptor inhibitor or that the subject requires a higher dose of the activin type II receptor inhibitor for efficacy.
In other aspects, the degree of change in the level and/or activity of GDFll after administration of an activin type II receptor tor (relative to the level and/or ty before administration of the activin type II inhibitor or relative to the normal levels and/or activity of GDFl 1) can te the appropriate dosage or dosing regimen of activin type II receptor inhibitor. For example, no decrease or a small decrease in the level and/or activity of GDFll (e.g., less than 5%, less than 10%, less than 15%, less than 20%, or less than 25%, or less than % decrease) after administration of a dose of an activin type II receptor inhibitor (relative to the level and/or activity before administration of the activin type 1] inhibitor) can indicate that a higher dose of the activin type II receptor tor is required or desirable for y.
Accordingly, in some embodiments, when assessment of the level and/or activity of GDFll shows no se or an undesirably small decrease in the level and/or activity of GDFll (e.g., less than 5%, less than 10%, less than 15%, less than 20%, or less than 25%, or less than 30% decrease) after administration of a dose of an n type II receptor inhibitor to the patient, the patient is administered a higher dose of an activin type II receptor inhibitor (e.g., 20%, 25%, %, 50%, 75%,100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% higher dose).
In other ments, a decrease in the level and/or activity of GDF11 (for example, slight or average decrease, such as less than 10%, less than 15%, less than 20%, or less than %, less than 30%, less than 50%, or less than 75% decrease, or from 10% to 75% decrease, or from 25% to 50% decrease) after administration of a dose of an activin type II receptor tor (relative to the level and/or activity before administration of the n type II tor) can indicate that the treatment with the activin type II receptor inhibitor is ef?cacious at the given dose and/or dosing regimen. Accordingly, in some embodiments, when assessment of the level and/or activity of GDF11 shows a decrease in the level and/or activity of GDF11 (for example, slight or average decrease, such as less than 10%, less than 15%, less than 20%, or less than %, less than 30%, less than 50%, or less than 75% decrease, or from 10% to 75% decrease, or from 25% to 50% decrease) after stration of a dose of an activin type II receptor inhibitor to the patient, the patient is administered the same dose of an activin type II receptor inhibitor.
In a particular embodiment, when assessment of the level and/or activity of GDF11 shows an average or desirable se in the level and/or activity of GDF11 (for example, between 20% and 75%, between 25% and 75%, or between 30% and 60% decrease) after administration of a dose of an n type II receptor inhibitor to the patient, the patient is administered the same dose of an activin type II receptor inhibitor. A large (or undesirably large) decrease in the level and/or activity of GDF11 (e. g., more than 50%, more than 60%, more than 70%, more than 75%, more than 80%, more than 90%, more than 95% decrease) after administration of a dose of an activin type II receptor inhibitor (relative to the level and/or activity before administration of the activin type II inhibitor) can indicate that a lower dose of the activin type II receptor inhibitor is desirable (e.g., to avoid the side effects of the treatment, such as hypertension). Accordingly, in some embodiments, when assessment of the level and/or activity of GDF11 shows a large (or rably large) se in the level and/or activity of GDF11 (e.g., more than 50%, more than 60%, more than 70%, more than 75%, more than 80%, more than 90%, more than 95% decrease) after administration of a dose of an activin type II receptor inhibitor to the patient, the patient is administered a lower dose of an activin type II receptor inhibitor (e.g., 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, or 95% lower dose).
The monitoring of the level and/or ty of GDFll can be performed by assessing the level and/or activity of GDFll 1 day, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 , 4 months, 5 , 6 months, or 1 year after administration of an activin type II receptor inhibitor to the patient. The level and/or activity of GDFll after stration of an activin type II receptor inhibitor to a patient, can also be used as an indicator of the appropriate or desirable frequency of administration of an activin type II receptor inhibitor to the patient. For example, if a decrease in the level and/or activity of GDFll after administration of an activin receptor type II inhibitor is maintained over a time period (e.g., 1 day, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 10 weeks, 3 , 4 months, 5 months, 6 months, or 1 year), then the activin type II receptor inhibitor can be stered once during the time period (e.g. once a day, once a week, once in 2 weeks, once in 3 weeks, once in 4 weeks, once in 5 weeks, once in 6 weeks, once in 7 weeks, once in 8 weeks, once in 3 , once in 4 months, once in 5 months, once in 6 months, or once a year).
In a speci?c embodiment, if a se in the level and/or activity of GDFll after administration of an activin receptor type II inhibitor is maintained over, e.g., 1 month, 2 months or 3 months, then the activin type II receptor inhibitor can be administered once in 1 month, 2 months, or 3 months, respectively.
In a speci?c embodiment, provided herein are methods comprising (i) assessing the level of GDFll in a tissue (e.g., blood, serum, plasma, liver, bone marrow, and/or spleen) of a subject (e.g., a subject having anemia); (ii) administering an activin type II receptor tor to the subject; and (iii) assessing the level of GDFll in a tissue (e.g., blood, serum, plasma, liver, bone marrow, and/or spleen) of a subject (e.g., a subject having anemia) following the administration of the activin type II receptor inhibitor. The administration of step (ii) can comprise a single administration of an activin type II receptor inhibitor (i.e., a single dose is administered one time to the subject) or multiple administrations of an n type II receptor inhibitor (e. g., the administration may comprise a complete administration regimen). The assessment of step (iii) can be performed at any point ing the administration of step (ii).
For example, the assessment of step (iii) can be performed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 10 weeks, 3 , 4 months, 5 months, 6 months, or 1 year following the administration of step (ii). As another example, the the assessment of step (iii) can be performed 1-3 days, 2-4 days, 3-5 days, -7 days, 1-2 weeks, 2-3 weeks, 3-4 weeks, 1—2 months, 2-3 months, 3-4 months, 4-5 , 5- 6 months, or 6-12 months following the administration of step (ii). The therapeutic regimen can be adjusted based on the outcome of the assessments in steps (i) and (iii). For example, if the levels of GDFll in the tissue of the t determined in the ment of step (i) are decreased relative to the levels of GDFll in the tissue of the subject determined in the assessment of step (iii), then the dose of the activin type II receptor inhibitor administered to the subject can be maintained or sed. Conversely, if the levels of GDFll in the tissue of the subject determined in the assessment of step (i) are sed relative to the levels of GDFll in the tissue of the subject determined in the assessment of step (iii), then the dose of the activin type II receptor inhibitor administered to the t can be increased. The steps of the foregoing method can be repeated/altered as necessary to determine the appropriate dose/therapeutic regimen that is appropriate for the subject undergoing treatment.
In another aspect, provided herein are methods comprising (a) administering a first dose of an activin type II receptor inhibitor to a patient, (b) determining the level and/or activity of GDFll in a tissue sample of the patient (e.g., serum), and (c) stering a second dose of the activin type II or inhibitor, wherein if the level and/or activity of GDFll is elevated over normal, the second dose of the activin or type II inhibitor is higher than the first dose (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% higher), and wherein, if the level and/or activity of GDFll is decreased over normal, the second dose ofthe activin receptor type II inhibitor is lower than the ?rst dose (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% . In these embodiments, the level and/or activity of GDFll in a tissue sample is compared to the normal level and/or activity of GDFll (e. g., the average level and/or activity of GDFll in a sample from the same tissue of healthy subjects, such as subjects in the same age group who do not have anemia). In certain embodiments, the level and/or activity of GDFll is deemed to be elevated over the normal level and/or activity of GDFll when it is ed at least 10%, at least 15%, at least %, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, or at least 500% over the normal level and/or activity of GDFl 1. In certain embodiments, the level and/or activity of GDFll is deemed to be elevated over the normal level and/or activity of GDFll when it is elevated at least %, at least 50%, at least 75%, at least 100%, or at least 200% over the normal level and/or activity of GDF11. In some embodiments, the level and/or activity of GDF11 is deemed to be decreased over the normal level and/or ty of GDF11 when it is decreased at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% over the normal level and/or activity of GDFl 1. In some ments, the level and/or activity of GDF11 is deemed to be decreased over the normal level and/or activity of GDF11 when it is decreased at least 25%, at least 50%, at least 75%, or at least 90% over the normal level and/or activity of GDP] 1.
The methods described herein are based, in part, on a ?nding that GDF11 inhibits growth of precursors of red blood cell, and thus the level of GDF11 can correlate with the level of red blood cells. It is desirable to maintain an optimal level of red blood cells in a t because, while decreases in the levels ofred blood cells, hemoglobin or hematocrit are associated with ineffective erythropoiesis and anemia, excessive increases in the levels of red blood cells, hemoglobin or hematocrit are associated with increase in blood pressure and other undesirable side s (which may be caused by treatment with higher than optimal doses of an activin type II receptor inhibitor). ingly, it can be desirable to administer an activin type II receptor inhibitor in a dosing regimen that maintains the level and/or activity of GDF11 in a patient at or around (e.g., within 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60% or 75% of) the normal level and/or activity of GDF11.
In certain embodiments, if the level and/or activity of GDF11 is determined to be reduced below the normal level and/or activity of GDP] 1, then the administration of an activin type II receptor inhibitor is adjusted ingly, e.g., is delayed, until the level and/or activity of GDF11 is returned to normal or within 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60% or 75% of the normal level and/or activity of GDF11, or terminated either temporarily or permanently.
In other embodiments, wherein the level and/or activity of GDF11 is determined to be reduced below the normal level and/or activity of GDF11, the administration of an activin type II receptor inhibitor is not delayed, but the dosage amount or frequency of dosing of the inhibitor is set at an amount that would reduce the risk of an unacceptable se in red blood cells, hemoglobin or hematocrit, or alternatively, a therapeutic regimen is developed for the patient that combines the use of the tor with an agent that ses the unacceptable increase in red blood cells, hemoglobin or crit (e.g., a blood pressure lowering agent).
In certain ments, ed herein is a method of treating anemia in an individual in need thereof by determining the level and/or activity of GDFll in the individual, and if the level and/or activity of GDF11 is elevated, administering to the individual a therapeutically effective amount of an activin type II or inhibitor, particularly an ActRII polypeptide (e.g., ActRIIa-hFc), and optionally, r monitoring (or determining) the level and/or activity of GDF11, and adjusting the dose of the activin type II receptor inhibitor (wherein, for example, if GDF11 is elevated over the normal level and/or activity the dose of the activin type II receptor inhibitor is sed, and if the level and/or ty of GDF11 is lower than the normal level and/or activity the dose of the activin type II receptor inhibitor is decreased).
In certain embodiments, provided herein is a method of treating beta-thalassemia in an individual in need thereof by determining the level and/or activity of GDF11 in the individual, and if the level and/or activity of GDF11 is elevated, administering to the individual a therapeutically effective amount of an activin type II receptor inhibitor, particularly an ActRII polypeptide (e.g., ActRIIa-hFc), and ally, ?irther monitoring (or determining) the level and/or activity of GDFll and ing the dose of the activin type II or tor (wherein, for example, if the level and/or activity of GDF11 is elevated over normal the dose of the activin type II receptor inhibitor is increased, and if the level and/or activity of GDFll is lower than normal the dose of the activin type II receptor inhibitor is decreased).
In n embodiments, provided herein is a method of increasing red blood cell, hemoglobin, hematocrit or Ery-C levels in an individual in need thereof by determining the level and/or activity of GDFll in the individual, and if the level and/or activity of GDFll is elevated, administering to the individual a therapeutically effective amount of an activin type II receptor tor, particularly an ActRII polypeptide (e.g., ActRIIa-hFc), and optionally, ?irther monitoring (or determining) the level and/or ty of GDF11, and adjusting the dose of the activin type II receptor tor (wherein, for example, if the level and/or activity of GDF11 is elevated over normal the dose of the n receptor antagonist is increased, and if the level and/or activity of GDF11 is lower than normal the dose of the activin type II receptor inhibitor is decreased).
In certain embodiments, the methods provided herein are utilized in connection with a method for treating or ameliorating anemia, ineffective erythropoiesis, decreased red blood cell levels or any other blood disorder described herein. In certain embodiments, the methods provided herein are utilized in connection with a method for increasing red blood cell levels, hemoglobin levels, hematocrit levels, or levels of colony forming units in a patient with anemia, ineffective erythropoiesis, decreased red blood cell levels or any other blood disorder described . In certain embodiments, the red blood cell levels are increased by at least 5%, 10%, %, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, or by at least 500%.
In certain embodiments, the hemoglobin levels are increased by at least 5%, 10%, 15%, 20%, %, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, or by at least 500%. In certain embodiments, the hematocrit levels are increased by at least 5%, 10%, 15%, 20%, 25%, 30%, %, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, or by at least 500%. In certain embodiments, the colony g units levels are sed by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, or by at least 500%. In certain embodiments, the methods ed herein are utilized in connection with a method for decreasing the levels of sis of erythroid progenitors and precursors. .3 T POPULATIONS The subjects treated in accordance with the methods described herein can be any mammals such as rodents and es, and in a preferred embodiment, humans. In n embodiments, the methods described herein can be used to treat anemia or ineffective erythropoiesis, or to monitor and/or increase red blood cell, hemoglobin, hematocrit or Ery-C levels, in any mammals such as s and primates, and in a preferred embodiment, in human patients.
In one aspect, the methods provided herein are utilized in connection with a method of treatment. In some embodiments, provided herein is a method for treating anemia (e.g., anemia caused by ineffective erythropoiesis). In certain embodiments, ed herein is a method for treating a disease associated with ineffective erythropoiesis (e.g., thalassemia, myelodysplastic syndromes, chronic ious anemia, or sickle cell anemia). In certain embodiments, provided herein is a method for treating an inherited bone marrow failure syndrome (such as, but not limited to, Amegakaryocytic Thrombocytopenia, Diamond-Blackfan Anemia, Dyskeratosis Congenita, Fanconi Anemia, Pearson Syndrome, Severe Congenital Neutropenia, Shwachman-Diamond me, Thrombocytopenia Absent Radii). In speci?c embodiments, provided herein is a method for treating an inherited bone marrow failure syndrome that cally affects red blood cells. In certain embodiments, provided herein is a method for treating anemia and/or bone disorders associated with end stage renal disease. In some ments, provided herein is a method for treating thalassemia (e.g., B-thalassemia), myelodysplastic syndromes, chronic pernicious anemia, sickle cell anemia, or Diamond- an Anemia. In one embodiment, provided herein is a method for treating B-thalassemia.
In one ment, provided herein is a method for treating Diamond-Blackfan Anemia.
In certain embodiments, provided herein are methods for treating a patient having anemia (e.g., diagnosed with anemia). In some ments, provided herein are methods for treating a patient having (e.g., diagnosed with) a disease associated with ineffective erythropoiesis, such as thalassemia, myelodysplastic syndromes, chronic pernicious anemia, or sickle cell anemia. In certain embodiments, provided herein are methods for treating a patient having (e.g., diagnosed with) an inherited bone marrow failure syndrome, such as Amegakaryocytic Thrombocytopenia, Diamond—Blackfan Anemia, Dyskeratosis Congenita, Fanconi , Pearson Syndrome, Severe Congenital Neutropenia, Shwachman-Diamond Syndrome, Thrombocytopenia Absent Radii, or a bone marrow e syndrome that speci?cally s red blood cells. In speci?c embodiments, provided herein are methods for treating a patient having (e.g., diagnosed with) anemia and/or bone disorders associated with end stage renal disease. In speci?c embodiments, provided herein are methods for treating a patient having (e.g., diagnosed with) thalassemia (e.g., B-thalassemia), myelodysplastic syndromes, chronic pernicious anemia, sickle cell , or Diamond-Blackfan Anemia. In one embodiment, ed herein are methods for treating a patient having (e.g., diagnosed with) B-thalassemia.
Anemia is ated with a variety of ers and ions that include, without limitation: chronic renal failure, myelodysplastic syndrome, rheumatoid arthritis, bone marrow transplantation, solid tumors (e. g. breast cancer, lung cancer, colon cancer), tumors of the lymphatic system (e. g. chronic lymphocyte leukemia, non-Hodgkins and Hodgkins lymphomas), tumors of the hematopoietic system (e. g. leukemia, myelodysplastic syndrome, multiple a), radiation y, chemotherapy (e.g. platinum containing regimens), in?ammatory and autoimmune diseases (including, but not limited to, rheumatoid arthritis, other in?ammatory arthritides, systemic lupus erythematosus (SLE), acute or chronic skin diseases (e.g. psoriasis), in?ammatory bowel disease (e.g. Crohn's disease and tive colitis)), acute or chronic renal disease or failure including idiopathic or congenital conditions, acute or chronic liver e, acute or chronic bleeding, situations where transfusion of red blood cells is not possible due to patient allo- or auto-antibodies and/or for religious reasons, infections (e.g. malaria, osteomyelitis), hemoglobinopathies (including, for example, sickle cell e, thalassemias), drug use or abuse (e.g. l misuse; pediatric patients with anemia from any cause to avoid transfusion), and situations where elderly patients or patients with underlying cardiopulmonary disease with anemia cannot receive transfusions due to concerns about atory overload. In certain embodiments, the methods described herein are used to treat anemia, or monitor and/or increase red blood cell, hemoglobin, hematocrit or Ery-C , in any t having one or more of the above-listed disorders or conditions.
In certain embodiments, provided herein is a method for ng an anemia wherein the subject is unresponsive to administration of erythropoietin. In certain embodiments, provided herein is a method for ng an anemia wherein the subject is unresponsive to administration of iron, vitamin B-l2, and / or folate. In n embodiments, provided herein is a method for treating an anemia resulting from erythroid progenitors and precursors being highly sensitive to death by apoptosis.
In certain embodiments, the ts treated (e.g., selected for treatment) in accordance with the methods described herein have elevated levels and/or activity of GDF11 relative to the normal levels of GDF11. For example, the subjects treated (e.g., selected for treatment) in accordance with the methods described herein have elevated levels and/or activity of GDF11 is a tissue relative to the average level and/or activity of GDF11 in the same tissue from the healthy subjects in the same age category. The level and/or activity of GDF11 can be assessed by any method known in the art or described herein. In some embodiments, subjects treated in ance with the methods described herein have the level and/or activity of GDF11 which is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, or 1000% over the normal level and/or activity of GDF11 (e.g., the average level in a healthy subject or subjects). In specific embodiments, subjects treated in accordance with the methods described herein have the level and/or activity of GDF11 which is at least 50%, at least 75%, at least 100%, at least 200%, or at least 500% % over the normal level and/or activity of GDF11. In some ments, the methods described herein are used to r, modulate or increase the level of red blood cells, hemoglobin, hematocrit or Ery-C levels in subjects that have been determined to have elevated levels of GDP] 1.
In certain embodiments, the subjects treated (e.g., selected for treatment) in accordance with the methods described herein have (i) elevated levels and/or activity of GDFll ve to the normal levels of GDFII (e.g., in serum, bone marrow, liver, and/or spleen), and (ii) anemia or a disease or condition associated with anemia or ineffective erythropoiesis (e.g., subjects diagnosed with anemia). In certain embodiments, a human patient d (e.g., selected for treatment) in accordance with the methods described herein has elevated serum levels and/or activity of GDFll relative to the normal levels of GDP] 1, and has anemia (e.g., has been diagnosed with anemia).
In some embodiments, the subjects treated in accordance with the methods described herein have an undesirably low red blood cell count, an undesirably low level of hemoglobin, an undesirably low level of hematocrit, and/or an undesirably low level of Ery-C. The methods described herein can be used to monitor, modulate and/or increase red blood cell, hemoglobin, hematocrit or Ery-C levels in selected patient populations. In other embodiments, provided herein is a method for treating a t at risk for developing undesirably low red blood cell or obin , such as those ts that are about to undergo major surgery or other procedures that may result in substantial blood loss. In some embodiments, subjects treated in accordance with the methods described herein are about to undergo major y or another procedure that may result in ntial blood loss.
When determining whether the level of hemoglobin is undesirably low, a level of less than normal for the appropriate age and gender category may be tive of anemia, although individual variations are taken into account. For example, a hemoglobin level of 12 g/dl is generally considered the lower limit ofnormal in adult . Potential causes of low levels of hemoglobin include blood-loss, nutritional de?cits, medication reaction, various problems with the bone marrow and many diseases. Patients may be treated with a closing n intended to restore the patient to a target obin level, usually between about 10 g/dl and about 12.5 g/dl, and typically about 11.0 g/dl (see also Jacobs et a1. (2000) Nephrol Dial lant 15, 15- 19), although lower target levels may cause fewer cardiovascular side effects. Optimally, the target hemoglobin level can be individualized for each patient. In certain embodiments, a patient to be treated with the methods described herein has hemoglobin levels of less than 13 g/dl, less than 12.5 g/dl, less than 12 g/dl, less than 11.5 g/dl, less than 11 g/dl, less than 10.5 g/dl, less than 10 g/dl, less than 9.5 g/dl, or less than 9 g/dl.
When determining whether the level of red blood cells is undesirably low, hematocrit levels (percentage of the volume of a blood sample occupied by the cells) can be used to assess the condition of red blood cells. Hematocrit levels for healthy individuals range from 41 to 51% for adult males and from 35 to 45% for adult females. Target hematocrit levels are usually around , although hematocrit levels vary from person to . Optimally, the target hematocrit level can be individualized for each patient.
In some embodiments, the s described herein are used to treat anemia, or a disease or condition associated with anemia or ineffective erythropoiesis, in patients that are susceptible to adverse effects when stered higher than optimal dose of an activin type II or inhibitor, particularly an ActRII polypeptide (e.g., ActRIIa-hFc). e effects that may be associated with administration of higher than optimal dose of an activin type II receptor inhibitor include, without limitation, an excessive increase in red blood cell , hematocrit levels or hemoglobin levels, an excessive increase in iron stores, and bone marrow or spleen hypercellularity. In turn, excessive increases in red blood cell levels, hemoglobin levels, or hematocrit levels may cause an increase in blood pressure and/or other undesirable side effects.
In speci?c ments, patients treated in accordance with the methods described herein are susceptible to hypertension (e.g., increase in systolic blood pressure, lic blood pressure and/or mean arterial blood pressure) or another condition that may be related to excessive increase in red blood cell levels (e.g., headaches, in?uenza-like syndrome, or vascular thrombosis). In a speci?c embodiment, patients treated in accordance with the methods described herein are susceptible to hypertension or another condition that may be related to excessive increase in red blood cell levels when treated with an activin type II receptor inhibitor (e.g., ActRIIA-hFc such as SEQ ID NO:7).
Subjects of any age can be treated in accordance with the methods described herein.
In some embodiments, subjects treated in ance with the methods described herein are over 55 years of age. In some embodiments, subjects d in accordance with the methods described herein are under 3 or 10 years of age. In other embodiments, subjects treated in ance with the methods described herein are under 18 years of age. In yet other ments, ts treated in accordance with the methods described herein are 18 to 55 years of age. .4 ASSESSING GDFll LEVEL AND/OR ACTIVITY The level or the activity of GDFll can be determined by any method known in the art or described . For example, the level of GDFll in a tissue sample can be ined by assessing (e.g., quantifying) transcribed RNA of GDFll in the sample using, e. g., Northern blotting, PCR analysis, real time PCR analysis, or any other technique known in the art or described herein. In one embodiment, the level of GDFll in a tissue sample can be determined by ing (e.g., quantifying) mRNA of GDFll in the sample.
The level of GDFll in a tissue sample can also be determined by assessing (e.g., quantifying) the level of protein expression ofGDP" in the sample using, e. g., immunohistochemical analysis, Western blotting, ELISA, immunoprecipitation, ?ow cytometry analysis, or any other technique known in the art or described herein. In particular embodiments, the level of GDFll is determined by a method capable of quantifying the amount of GDFll present in a tissue sample of a t (e.g., in human serum), and/or capable of detecting the correction of the level of GDFll following treatment with an activin type II or inhibitor.
In one embodiment, the level of GDFll in a tissue sample is determined by assessing (e.g., fying) protein expression of GDFll in the sample using ELISA. For example, GDFll can be identi?ed and quanti?ed in the human serum using sandwich ELISA method described in the Examples. The sandwich ELISA method for use in determining the level of GDFll in a tissue sample can comprise ActRIIA-Fc coating , e.g., A-ch, or ActRIIA-hFc such as SEQ ID NO:7) of ELISA plates, contacting the plates with the tissue sample (e.g., human serum), and detecting ActRIIA ligand (such as GDFl l) in the tissue sample (e.g., human serum) that bind to ActRIIA-Fc by speci?c antibodies. In some embodiments, the method for use in determining the level and/or activity of GDFll as described herein (e.g., sandwich ELISA) is capable of detecting from 100 pg/ml mbinant GDFl l, ActiVin A and/or ActiVin B bound to ActRIIA.
Antibodies for use in assays that measure the levels of GDFll in a sample (e.g., in a tissue sample, e. g., a sample of blood, serum, plasma, liver, spleen, and/or bone marrow) are known in the art or could be y developed using approaches known to those of skill in the art. Examples of monoclonal antibodies that can be used in assays that measure the levels of GDFll in a sample include antibodies from LifeSpan Biosciences Inc., Seattle, WA, with catalog numbers LS-Cl21127, LS-Cl38772, LS—C105098; antibodies available from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, with catalog number (X-l9): sc-81952; and antibodies available from Sigma-Aldrich Co. LLC, with t number: WH0010220M3.
The activity of GDFll can be measured by any assay known in the art including, without limitation, colony formation assay, a reporter gene assay (e.g., containing GDFl l- responsive reporter gene construct), alkaline phosphatase assay, or any other ivity assay.
Exemplary assays that can be used to measure ty of GDFll are described in Souza et al., 2008, Mol. Endocrinology 22(12):2689-2702; and Bessa et al., 2009, Protein Expression and Puri?cation 63 289-94.
The level and/or activity ofGDP" can be assessed in any tissue sample obtained from a patient d in accordance with the methods described herein. In certain embodiments, GDFll level and/or activity is assessed in a sample obtained from serum, liver, spleen or bone marrow of a patient treated in accordance with the methods described herein. In one embodiment, GDFll level and/or activity is ed in a sample obtained from serum of a patient d in accordance with the methods described herein. In another embodiment, GDFll level and/or activity is assessed in a sample obtained from spleen of a patient treated in accordance with the methods described . In yet another embodiment, GDFll level and/or activity is assessed in a sample obtained from bone marrow of a patient treated in accordance with the methods described herein.
In some embodiments, the level and/or activity of GDFll in a tissue sample of a patient is compared to the average level and/or activity of GDFll in tissue samples (e.g., in samples from the same ) of healthy subjects (e.g., subjects that do not have anemia), such as subjects in the same age group and, optionally, of the same gender. In some embodiments, the level and/or activity of GDFll in a tissue sample of a t is compared to the level and/or activity of GDFll in a tissue sample (e. g., in a sample from the same tissue) of the subject at an earlier time point (e.g., before the onset of disease, before the onset of treatment, or during treatment). In some ments, the level and/or activity of GDFll in a tissue sample (e.g., serum, spleen, liver, or blood marrow) of a patient is compared to the level and/or activity of GDFII in another tissue sample of the t. In some embodiments, the level and/or activity of GDFll in a tissue sample of a patient is compared to the level and/or activity of another gene t in the tissue sample of the patient (e.g., b—actin, n A, Activin B).
In some embodiments, the level and/or activity of GDFll in a tissue sample of a patient is compared to the normal level and/or activity of GDFll in the tissue sample. The normal level and/or activity of GDF11 in a tissue sample can be an average level and/or activity of GDF11 in tissue samples (e. g., in s from the same tissue) of one or more healthy subjects (e.g., subjects that do not have anemia), such as subjects in the same age group and, optionally, of the same gender. In some embodiments, detection of the elevated level and/or activity of GDF11 in comparison to the normal level and/or activity of GDF11 is followed by administration of an activin receptor inhibitor (such as one or more activin receptor inhibitors described herein). In some embodiments, administration of an activin receptor inhibitor (such as one or more activin receptor inhibitors described herein) is followed by monitoring of the level and/or ty of GDF11 and, optionally, comparing the level and/or activity of GDF11 to the normal level and/or activity of GDF11. In some embodiments, administration of a ?rst dose of n type II receptor inhibitor (e. g., ActRIIA-hFc such as SEQ ID NO:7) is followed by determining the level and/or activity of GDFl 1, and if the level and/or activity of GDF11 is elevated over the normal level and/or activity administering a second dose of the activin type II receptor inhibitor which is higher (e.g., 1.25, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 times higher) than the first dose, and if the level and/or activity ofGDF11 is sed over the normal level and/or actiVity administering a second dose of the activin type II receptor inhibitor which is lower (e.g., 1.25, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 times lower) than the ?rst dose.
As used herein, in certain embodiments, the normal level and/or activity of GDF11 is an average level and/or activity of GDF11 in a tissue sample of one or more y subjects (e.g., healthy subjects in the same age category and/or of the same gender). In some embodiments, the level and/or activity of GDF11 in a tissue sample of the treated subject is compared to the level and/or activity of GDF11 in a sample from the same tissue of one or more healthy subjects. In some embodiments, the tissue in which the level and/or activity of GDF11 is assessed is blood serum, bone marrow, liver, or . In one embodiment, the tissue in which the level and/or activity of GDF11 is assessed is serum. .5 THERAPEUTIC USES In certain embodiments, Activin type II receptor inhibitors are used for treatment of patients having anemia, or a disease or ion associated with anemia or ineffective erythropoiesis, in the methods bed herein. In some embodiments, activin type II receptor tors are administered to a patient having anemia, or a disease or condition associated with anemia or ineffective opoiesis. In some embodiments, activin type II receptor inhibitors are administered to a t having anemia, or a disease or condition associated with anemia or ineffective erythropoiesis, and having elevated levels and/or activity of GDFll (e. g., in serum, bone marrow, liver, and/or spleen of the patient). The activin type II receptor inhibitors described in this section and known in the art can be used in the methods provided . In certain embodiments, the activin type II receptor inhibitors described in this section can be used in the methods ed herein.
Inhibitors of ActRII receptors assed herein include ActRIIA inhibitors and ActRIIB inhibitors (see below). In certain embodiments, an ActRII receptor inhibitor is speci?c to ActRIIA. In other embodiments, an ActRII receptor inhibitor is speci?c to ActRIIB. In certain embodiments, an ActRII receptor inhibitor entially inhibits ActRIIA. In other embodiments, an ActRII receptor inhibitor preferentially inhibits ActRIIB. In certain embodiments, an ActRII receptor inhibitor inhibits both A and ActRIIB.
In certain embodiments, inhibitors of ActRII ors can be polypeptides comprising activin-binding domains of ActRII. Without being bound by theory, such activin- binding domain comprising polypeptides sequester activin and y prevent activin ing.
These activin-binding domain comprising polypeptides may comprise all or a portion of the ellular domain of an ActRII or (i.e., all or a portion of the ellular domain of ActRIIA or all or a portion of the extracellular domain of ActRIIB). In specific embodiments, the extracellular domain of an ActRII receptor is soluble.
In certain embodiments, the activin—binding domain comprising polypeptides are linked to an Fc portion of an antibody (i.e., a conjugate comprising an activin-binding domain sing polypeptide of an ActRII receptor and an Fc portion of an antibody is ted).
Without being bound by theory, the antibody portion confers increased ity on the conjugate.
In certain embodiments, the activin-binding domain is linked to an Fc portion of an antibody via a linker, e.g., a peptide linker.
The inhibitors of ActRII ors used in the compositions and methods described herein comprise molecules that t ActRIIA and/or ActRIIB, directly or indirectly, either extracellularly or intracellularly. In some embodiments, the inhibitors of ActRIIA and/or ActRIIB used in the itions and methods described herein inhibit ActRIIA and/or ActRIIB via interactions with the receptor(s) itself. In other embodiments, the inhibitors of ActRIIA and/or ActRIIB used in the compositions and methods described herein inhibit ActRIIA and/or ActRIIB via interactions with an ActRIIA and/or ActRIIB ligand, e.g., Activin. .5.1 INHIBITORS OF ACTRIIA As used herein, the term "ActRIIA" refers to a family of activin receptor type Ila (ActRIIA) ns from any s and variants derived from such ActRIIA proteins by mutagenesis or other modi?cation. Reference to A herein is understood to be a reference to any one of the currently identified forms. Members of the ActRIIA family are lly transmembrane proteins, composed of a ligand-binding extracellular domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with ted serine/threonine kinase activity.
ActRIIA inhibitors to be used in the compositions and s described herein include, without limitation, activin-binding soluble ActRIIA polypeptides; antibodies that bind to activin (particularly the activin A or B subunits, also referred to as BA or BB) and disrupt ActRIIA binding; antibodies that bind to ActRIIA and disrupt n binding; non-antibody proteins selected for activin or ActRIIA binding (see e.g., WO/2002/088l7l, WO/2006/055689, WO/2002/032925, WO/2005/037989, US 2003/0133939, and US 238646, each h is incorporated herein by reference in its entirety, for examples of such proteins and methods for design and selection of same); and randomized peptides selected for activin or ActRIIA binding, which can be conjugated to an Fc domain.
In certain ments, two or more different proteins (or other moieties) with activin or ActRIIA binding activity, especially activin binders that block the type I (e.g., a soluble type I activin or) and type II (e.g., a soluble type II activin receptor) binding sites, respectively, may be linked together to create a bi?inctional or multi?inctional binding molecule that ts ActRIIA and thus can be used in the compositions and methods described herein. In certain ments, Activin-ActRIIA signaling axis antagonists that inhibit ActRIIA include nucleic acid aptamers, small molecules and other agents are used in the itions and methods described herein include. (a) ActRIIA Inhibitors Comprising ActRIIA ptides The term "ActRIIA polypeptide" includes polypeptides comprising any naturally occurring polypeptide of an ActRIIA family member as well as any variants thereof (including mutants, nts, fusions, and peptidomimetic forms) that retain a use?Jl activity. For example, ActRIIA polypeptides include polypeptides derived from the sequence of any known ActRIIA having a sequence at least about 80% identical to the sequence of an ActRIIA ptide, and optionally at least 85%, 90%, 95%, 97%, 98%, 99% or greater identity. For example, an ActRIIA polypeptide may bind to and t the function of an ActRIIA protein and/or activin. An ActRIIB polypeptide may be selected for its ability to promote bone growth and bone mineralization. Examples of ActRIIA polypeptides e human ActRIIA precursor polypeptide (SEQ ID NO: 1) and soluble human ActRIIA polypeptides (e.g., SEQ ID NOS: 2, 3, 7 and 12). With respect to the ActRIIA precursor polypeptide whose amino acid sequence is depicted at SEQ ID NO: 1, the signal peptide of the human ActRIIA precursor polypeptide located at amino acid positions 1 to 20; the extracellular domain is located at amino acid positions 21 to 135 and the N-linked glycosylation sites of the human ActRIIA precursor polypeptide (SEQ ID NO: 1) are located at amino acid positions 43 and 56 of SEQ ID NO:1.
The nucleic acid sequence encoding the human B precursor polypeptide of SEQ ID NO:1 is disclosed as SEQ ID N024 (nucleotides 164—1705 of Genbank entry NM_001616). The nucleic acid sequence encoding the e human ActRIIA polypeptide of SEQ ID NO:2 is disclosed as SEQ ID NO:5. See Table 1 for a description of the sequences.
In c ments, the ActRIIA polypeptides used in the compositions and methods bed herein are soluble ActRIIA polypeptides. An extracellular domain of an ActRIIA protein can bind to activin and is generally e, and thus can be termed a soluble, n-binding ActRIIA ptide. Thus, as used herein, the term "soluble ActRIIA polypeptide" generally refers to polypeptides comprising an extracellular domain of an ActRIIA protein, including any naturally occurring extracellular domain of an ActRIIA protein as well as any variants thereof ding s, fragments and peptidomimetic forms). Soluble ActRIIA polypeptides can bind to activin; however, the wild type ActRIIA protein does not exhibit signi?cant selectivity in binding to activin versus GDF8/11. Native or altered ActRIIA proteins may be given added icity for activin by coupling them with a second, activin-selective binding agent. Examples of soluble, activin-binding ActRIIA polypeptides include the soluble polypeptides illustrated in SEQ ID NOs: 2, 3, 7, 12 and 13. Other examples of soluble, activin- g ActRIIA polypeptides comprise a signal sequence in on to the extracellular domain of an ActRIIA protein, for example, the honey bee mellitin leader sequence (SEQ ID NO: 8), the tissue plasminogen tor (TPA) leader (SEQ ID NO: 9) or the native ActRIIA leader (SEQ ID NO: 10). The ActRIIA-hFc polypeptide illustrated in SEQ ID NO:13 uses a TPA leader.
In certain embodiments, the inhibitors of ActRIIA used in the compositions and methods described herein comprise a conjugate/fusion protein comprising an activin-binding domain ofActRIIA linked to an Fc portion of an antibody. In n ments, the activin- binding domain is linked to an Fc portion of an antibody via a linker, e.g., a peptide linker.
Optionally, the Fc domain has one or more mutations at residues such as Asp-265, lysine 322, and Asn-434. In certain cases, the mutant Fc domain having one or more of these ons (e.g., an Asp-265 mutation) has a reduced ability to bind to the Fcy receptor relative to a wild- type Fc domain. In other cases, the mutant Fc domain having one or more of these ons (e.g., an Asn-434 mutation) has an increased ability to bind to the MHC class I- related Fc- receptor (FcRN) relative to a wild-type Fc . ary fusion proteins comprising a soluble extracellular domain of ActRIIA fused to an EC domain are set forth in SEQ ID NOs: 6, 7, 12, and 13.
In a speci?c embodiment, the ActRIIA inhibitors used in the compositions and methods described herein comprise the extracellular domain of A, or a portion thereof, linked to an Fc portion of an antibody, wherein said ActRIIA tor comprises an amino acid sequence that is at least 75% identical to an amino acid sequence selected from SEQ ID NOS: 6, 7, l2, and 13. In another speci?c embodiment, the ActRIIA inhibitors used in the compositions and methods described herein comprise the extracellular domain of ActRIIA, or a portion thereof, linked to an Fc n of an antibody, wherein said ActRIIA inhibitor comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 6, 7, l2, and 13.
In certain embodiments, the inhibitors of ActRIIA used in the compositions and methods described herein comprise a ted form of an extracellular domain of ActRIIA. The truncation can be at the y us and/or the amino terminus of the ActRIIA polypeptide.
In certain embodiments, the truncation can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, l2, l3, l4, l5, l6, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids long relative to the mature ActRIIB polypeptide ellular domain. In certain embodiments, the truncation can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, or 25 N—terminal amino acids ofthe mature ActRIIA polypeptide extracellular domain. In certain embodiments, the truncation can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 C-terminal amino acids of the mature ActRIIA polypeptide extracellular domain. For example, truncated forms of ActRIIA include polypeptides with amino acids 20—119; 20—128; 20-129; ; 20-131; 20- 13220-133; 20-134; 20-131; 21—131; ;23—131;24—131; and 25-131, wherein the amino acid positions refer to the amino acid positions in SEQ ID NO: 1.
In certain embodiments, the inhibitors of ActRIIA used in the compositions and methods described herein se an extracellular domain of ActRIIA with one or more amino acid substitutions. In certain embodiments, the inhibitors of ActRIIA used in the compositions and methods described herein comprise a truncated form of an ActRIIA extracellular domain that also carries an amino acid substitution.
In a speci?c embodiment, the ActRIIA inhibitor to be used in the itions and methods described herein is a fusion protein between the ellular domain of the human ActRIIA receptor and the Fc portion of IgG1. In another speci?c embodiment, the ActRIIA inhibitor to be used in the compositions and methods described herein is a fusion protein between a truncated extracellular domain of the human A or and the Fc portion of IgG1. In r speci?c embodiment, the ActRIIA inhibitor to be used in the compositions and s described herein is a fusion protein between a ted extracellular domain of the human ActRIIA receptor and the Fc n of IgG1 wherein the truncated extracellular domain of the human ActRIIA receptor possesses one or more amino acid substitutions.
Functionally active fragments ofActRIIA polypeptides can be obtained, for example, by screening polypeptides recombinantly produced from the corresponding fragment of the nucleic acid encoding an ActRIIA polypeptide. In addition, fragments can be chemically synthesized using techniques known in the art such as conventional Merri?eld solid phase f-Moc or t-Boc chemistry. The fragments can be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments that can function as antagonists (inhibitors) of ActRIIA protein or signaling mediated by activin.
In addition, functionally active variants of ActRIIA polypeptides can be obtained, for example, by ing libraries of modi?ed polypeptides recombinantly produced from the corresponding mutagenized nucleic acids encoding an A polypeptide. The variants can be produced and tested to identify those that can function as antagonists (inhibitors) of ActRIIA protein or signaling mediated by activin. In certain embodiments, a ?anctional variant of the ActRIIA polypeptides ses an amino acid sequence that is at least 75% identical to an amino acid sequence selected from SEQ ID NOS: 2 or 3. In certain cases, the functional variant has an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% cal to an amino acid sequence selected from SEQ ID NOS: 2 or 3. onal ts may be generated, for example, by modifying the ure of an ActRIIA polypeptide for such purposes as enhancing therapeutic y, or stability (e.g., ex vivo shelf life and resistance to proteolytic degradation in vivo). Such modi?ed ActRIIA ptides when selected to retain activin binding, can be considered functional equivalents of the naturally-occurring ActRIIA polypeptides. Modi?ed ActRIIA polypeptides can also be produced, for ce, by amino acid substitution, deletion, or addition. For instance, it is reasonable to expect that an isolated replacement of a leucine with an cine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar ement of an amino acid with a structurally related amino acid (e.g., conservative mutations) will not have a major effect on the biological activity of the resulting molecule. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Whether a change in the amino acid sequence of an ActRIIA polypeptide s in a onal homolog can be readily determined by assessing the ability of the variant ActRIIA polypeptide to produce a response in cells in a fashion similar to the wild—type ActRIIA polypeptide.
In certain embodiments, the A inhibitor to be used in the compositions and methods described herein provided herein may comprise an ActRIIA polypeptide having one or more speci?c mutations that can alter the ylation of the ptide. Such mutations may introduce or eliminate one or more glycosylation sites, such as O-linked or N—linked glycosylation sites. Asparagine-linked glycosylation recognition sites lly se a tripeptide sequence, asparagine-X-threonine (or asparagines-X-serine) (where "X" is any amino acid) which is speci?cally recognized by riate ar glycosylation enzymes. The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the wild-type ActRIIA polypeptide (for O-linked glycosylation sites). A variety of amino acid substitutions or deletions at one or both of the ?rst or third amino acid positions of a glycosylation recognition site (and/or amino acid deletion at the second position) results in non-glycosylation at the modi?ed tripeptide sequence. Another means of increasing the number of carbohydrate moieties on an ActRIIA ptide is by chemical or enzymatic coupling of glycosides to the ActRIIA polypeptide. Depending on the coupling mode used, the sugar(s) may be attached to (a) arginine and histidine; (b) free carboxyl ; (c) free sulfhydryl groups such as those of cysteine; ((1) free hydroxyl groups such as those of serine, ine, or hydroxyproline; (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan; or (f) the amide group of glutamine. These methods are described in WO 87/05330 hed Sep. 11, 1987, and in Aplin and Wriston (1981) CRC Crit.
Rev. Biochem., pp. 259-3 06, incorporated by reference herein. Removal of one or more carbohydrate moieties t on an A polypeptide may be accomplished chemically and/or enzymatically. Chemical deglycosylation may involve, for e, re of the ActRIIA polypeptide to the compound tri?uoromethanesulfonic acid, or an equivalent compound. This treatment s in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N—acetylgalactosamine), while leaving the amino acid sequence .
Chemical deglycosylation is further described by Hakimuddin et a1. (1987) Arch. Biochem.
Biophys. 259252 and by Edge et al. (1981) Anal. Biochem. 118: 13 1. Enzymatic cleavage of carbohydrate moieties on ActRlIA polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al. (1987) Meth. Enzymol. 138:350. The sequence of an ActRIIA polypeptide may be adjusted, as appropriate, depending on the type of sion system used, as mammalian, yeast, insect and plant cells may all introduce differing glycosylation patterns that can be affected by the amino acid sequence of the peptide. In general, ActRIIA proteins for use in humans can be expressed in a mammalian cell line that provides proper glycosylation, such as HEK293 or CHO cell lines, although other expression systems, such as other mammalian expression cell lines, yeast cell lines with engineered ylation enzymes and insect cells, are expected to be useful as well.
Further provided herein are methods of generating mutants, particularly sets of atorial mutants of an ActRIIA polypeptide, as well as truncation mutants; pools of combinatorial mutants are especially useful for identifying ?anctional variant sequences. The purpose of screening such atorial libraries may be to generate, for example, ActRIIA ptide variants which can act as either ts or antagonist, or alternatively, which possess novel activities all together. A variety of screening assays are provided below, and such assays may be used to evaluate variants. For example, an ActRIIA polypeptide variant may be screened for ability to bind to an A ligand, to prevent binding of an ActRIIA ligand to an ActRIIA polypeptide or to interfere with signaling caused by an ActRIIA ligand.
Combinatorially-derived variants can be generated which have a selective or generally increased potency relative to a naturally occurring ActRIIA polypeptide. Likewise, mutagenesis can give rise to variants which have intracellular half-lives dramatically different than the corresponding a wild—type ActRIIA polypeptide. For example, the altered protein can be rendered either more stable or less stable to proteolytic degradation or other cellular processes which result in destruction of, or ise inactivation of a native ActRIIA polypeptide. Such variants, and the genes which encode them, can be ed to alter ActRIIA polypeptide levels by modulating the half-life of the ActRIIA polypeptides. For instance, a short half-life can give rise to more transient biological effects and can allow tighter control of recombinant ActRIIA polypeptide levels within the patient. In an Fc fusion protein, mutations may be made in the linker (if any) and/or the Fc portion to alter the half—life of the protein.
A combinatorial library may be produced by way of a degenerate library of genes encoding a library of polypeptides which each e at least a portion of potential ActRIIA polypeptide sequences. For instance, a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential A polypeptide nucleotide sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e. g., for phage y).
There are many ways by which the library of potential homologs can be generated from a degenerate oligonucleotide sequence. Chemical sis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the tic genes then be ligated into an appropriate vector for expression. The synthesis of degenerate oligonucleotides is well known in the art (see for example, Narang, S A (1983) Tetrahedron 39:3; Itakura et al., (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp 273-289; Itakura et al., (1984) Annu. Rev. Biochem. 53:323; Itakura et al., (1984) Science 198: 1056; Ike et al., (1983) Nucleic Acid Res. 11:477). Such ques have been employed in the directed evolution of other ns (see, for e, Scott et al., (1990) Science 249:386-390; Roberts et al., (1992) PNAS USA 89:2429-2433; Devlin et al., (1990) Science 249: 6; Cwirla et al., (1990) PNAS USA 87: 6378-6382; as well as US. Pat. Nos. ,223,409, 5,198,346, and 5,096,815).
Alternatively, other forms ofmutagenesis can be ed to generate a combinatorial library. For example, A polypeptide variants can be generated and ed from a library by screening using, for example, alanine scanning mutagenesis and the like (Ruf et al., (1994) Biochemistry 33:1565-1572; Wang et al., (1994) J. Biol. Chem. 95-3099; Balint et al., (1993) Gene 137: 109-1 18; Grodberg et al., (1993) Eur. J. Biochem. 218:597-601; Nagashima et al., (1993) J. Biol. Chem. 268:2888-2892; Lowman et al., (1991) mistry 30: 10832-10838; and Cunningham et al., (1989) Science 244: 1081-1085), by linker scanning mutagenesis (Gustin et al., (1993) Virology 193:653—660; Brown et al., (1992) Mol. Cell Biol. 12:2644-2652; ht et al., (1982) Science 2321316); by saturation mutagenesis (Meyers et al., (1986) Science 232:613); by PCR mutagenesis (Leung etal., (1989) Method Cell Mol Biol 1:11-19); or by random mutagenesis, including chemical mutagenesis, etc. (Miller et al., (1992) A Short Course in Bacterial Genetics, CSHL Press, Cold Spring , N.Y.; and Greener et al., (1994) Strategies in Mol Biol 7:32-34). Linker scanning mutagenesis, particularly in a combinatorial g, is an attractive method for identifying truncated (bioactive) forms of ActRIIA polypeptides.
A wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations and truncations, and, for that matter, for ing cDNA libraries for gene products having a certain property. Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of ActRIIA polypeptides. The most widely used techniques for ing large gene libraries typically ses cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of s, and sing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector ng the gene whose product was detected. Preferred assays include activin binding assays and activin-mediated cell ing assays.
In certain embodiments, ActRIIA polypeptides used in the inhibitors of the methods and compositions described herein may further comprise post-translational modi?cations in addition to any that are naturally present in the A polypeptides. Such modi?cations may include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, tion, and acylation. As a result, the modi?ed ActRIIA polypeptides may contain non- amino acid elements, such as polyethylene glycols, lipids, poly- or mono-saccharide, and phosphates. Effects of such non-amino acid elements on the ?anctionality of a ActRIIA polypeptide may be tested by any method known to the skilled n. When an ActRIIA ptide is produced in cells by cleaving a nascent form of the ActRIIA polypeptide, post- translational processing may also be important for correct folding and/or function of the protein.
Different cells (such as CHO, HeLa, MDCK, 293, W138, NIH-3T3 or HEK293) have speci?c cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modi?cation and processing of the ActRIIA polypeptides.
In certain aspects, functional variants or modi?ed forms of the ActRIIA ptides used in the inhibitors of the methods and compositions described herein include fusion proteins having at least a portion of the ActRIIA polypeptides and one or more fusion domains. Well known es of such fusion s include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin heavy chain constant region (Fc), maltose binding protein (MBP), or human serum albumin. A ?lSlOI‘l domain may be selected so as to confer a desired property. For example, some fusion domains are particularly useful for isolation of the fusion proteins by af?nity chromatography. For the purpose of af?nity puri?cation, relevant matrices for af?nity chromatography, such as g1utathione-, amylase-, and nickel- or cobalt-conjugated resins are used. Many of such matrices are available in "kit" form, such as the Pharmacia GST puri?cation system and the QIAexpress.TM. system (Qiagen) useful with (H186) fusion partners. As another example, a fusion domain may be selected so as to facilitate detection of the A ptides.
Examples of such detection domains include the various ?uorescent proteins (e.g., GFP) as well as "epitope tags," which are usually short peptide ces for which a speci?c antibody is available. Well known epitope tags for which speci?c monoclonal dies are readily available include FLAG, in?uenza virus hemagglutinin (HA), and c-myc tags. In some cases, the ?lsion domains have a protease cleavage site, such as for Factor Xa or Thrombin, which allows the relevant protease to partially digest the fusion proteins and thereby liberate the recombinant proteins therefrom. The liberated proteins can then be isolated from the ?lSlOI‘l domain by subsequent chromatographic separation. In certain red embodiments, an ActRIIA polypeptide is fused with a domain that izes the ActRIIA polypeptide in vivo (a "stabilizer" domain). By "stabilizing" is meant anything that ses serum half life, regardless of whether this is because of decreased destruction, decreased clearance by the kidney, or other pharmacokinetic effect. Fusions with the Fc portion of an immunoglobulin are known to confer desirable pharmacokinetic properties on a wide range of ns. Likewise, s to human serum albumin can confer desirable properties. Other types of fusion domains that may be selected include multimerizing (e.g., dimerizing, tetramerizing) domains and ?anctional domains (that confer an additional ical function, such as r stimulation of bone growth or muscle growth, as desired).
It is tood that different elements of the ?Jsion proteins may be ed in any manner that is consistent with the desired functionality. For example, an ActRIIA ptide may be placed C-terminal to a heterologous domain, or, alternatively, a logous domain may be placed C-terminal to an ActRIIA polypeptide. The A polypeptide domain and the heterologous domain need not be adjacent in a fusion protein, and additional domains or amino acid sequences may be included C- or inal to either domain or between the domains.
In certain embodiments, the ActRIIA polypeptides used in the inhibitors of the methods and compositions described herein may contain one or more modi?cations that are e of stabilizing the ActRIIA polypeptides. For example, such modi?cations may e the in Vitro half life of the ActRIIA polypeptides, enhance circulatory half life of the ActRIIA polypeptides or reduce proteolytic degradation of the ActRIIA polypeptides. Such stabilizing modi?cations may include, but are not limited to, ?ision proteins (including, for example, fusion proteins comprising an ActRIIA polypeptide and a stabilizer domain), modi?cations of a glycosylation site (including, for e, addition of a glycosylation site to an A polypeptide), and modi?cations of ydrate moiety ding, for e, removal of carbohydrate moieties from an ActRIIA ptide). In the case of ?ision proteins, an ActRIIA polypeptide is fused to a stabilizer domain such as an IgG molecule (e. g., an Fc domain). As used herein, the term "stabilizer domain" not only refers to a fusion domain (e.g., PC) as in the case of fusion proteins, but also includes nonproteinaceous modi?cations such as a carbohydrate moiety, or nonproteinaceous polymer, such as polyethylene glycol.
In certain embodiments, isolated and/or puri?ed forms of ActRIIA polypeptides, which are isolated ?om, or otherwise ntially free of, other proteins can be used with the methods and compositions described herein. ActRIIA polypeptides can generally be produced by expression from recombinant nucleic acids.
In certain aspects, the ActRIIA polypeptides used in the compositions and methods described herein are generated using isolated and/or recombinant nucleic acids encoding any of the ActRIIA polypeptides (e.g., soluble ActRIIA polypeptides), including nts, ?inctional variants and fusion proteins sed herein. For example, SEQ ID NO: 4 encodes the naturally occurring human ActRIIA precursor ptide, while SEQ ID NO: 5 s the processed extracellular domain of ActRIIA. Such nucleic acids may be single—stranded or double ed.
Such nucleic acids may be DNA or RNA molecules. These nucleic acids may be used, for e, in methods for making ActRIIA polypeptides or as direct therapeutic agents (e.g., in a gene therapy ch).
In certain aspects, nucleic acids encoding ActRIIA polypeptides may include nucleic acids that are variants of SEQ ID NO: 4 or 5. Variant nucleotide sequences include sequences that differ by one or more tide substitutions, additions or deletions, such as allelic variants.
In certain embodiments, isolated or recombinant nucleic acid sequences encoding ActRIIA polypeptides may be least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 4 or 5. One of ordinary skill in the art will appreciate that nucleic acid sequences mentary to SEQ ID NO: 4 or 5, and variants of SEQ ID NO: 4 or 5 may be used in the production of ActRIIA polypeptides le for use in the methods and compositions described herein. In further embodiments, such nucleic acid sequences can be isolated, recombinant, and/or fused to a heterologous nucleotide sequence, or be from a DNA y.
In other embodiments, nucleic acids used in the production of ActRIIA polypeptides suitable for use in the methods and compositions described herein may include nucleotide sequences that hybridize under highly stringent conditions to the nucleotide sequence designated in SEQ ID NO: 4 or 5, ment sequence of SEQ ID NO: 4 or 5, or fragments thereof. One of ordinary skill in the art will understand that appropriate stringency conditions which promote DNA ization can be varied. For example, one can m the ization at 6.0 times sodium chloride/sodium citrate (SSC) at about 45 degree Celsius, followed by a wash of 2.0 times SSC at 50 degree Celsius. For e, the salt concentration in the wash step can be selected from a low stringency of about 2.0 times SSC at 50 degree Celsius to a high stringency of about 0.2 times SSC at 50 degree Celsius. In on, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22 degree Celsius, to high stringency conditions at about 65 degree Celsius. Both temperature and salt may be varied, or temperature or salt concentration may be held constant while the other variable is changed. In one embodiment, nucleic acids which ize under low stringency conditions of 6 times SSC at room temperature followed by a wash at 2 times SSC at room temperature can be used with the methods and compositions described herein.
Isolated c acids which differ from the nucleic acids as set forth in SEQ ID NOs: 4 or 5 due to degeneracy in the genetic code also can be used in the production of ActRIIA polypeptides suitable for use in the methods and compositions described . For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or ms (for example, CAU and CAC are synonyms for histidine) may result in "silent" ons which do not affect the amino acid sequence of the protein. However, it is expected that DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the subject proteins will exist among mammalian cells. One skilled in the art will appreciate that these variations in one or more nucleotides (up to about 3-5% of the nucleotides) of the nucleic acids encoding a particular protein may exist among individuals of a given species due to natural allelic variation.
In certain embodiments, the recombinant nucleic acids may be operably linked to one or more regulatory nucleotide sequences in an expression uct. tory nucleotide sequences will generally be appropriate to the host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a y of host cells. lly, said one or more regulatory nucleotide sequences may include, but are not limited to, er sequences, leader or signal sequences, mal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences. tutive or inducible promoters as known in the art are contemplated herein. The promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter. An expression construct may be present in a cell on an episome, such as a plasmid, or the sion construct may be inserted in a chromosome. In a preferred embodiment, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. able marker genes are well known in the art and will vary with the host cell used.
In certain aspects, the a nucleic acid used in the production of ActRIIA polypeptides suitable for use in the methods and compositions described herein can be provided in an expression vector comprising a nucleotide sequence encoding an ActRIIA polypeptide and operably linked to at least one regulatory sequence. Regulatory sequences are art-recognized and are selected to direct expression of the ActRIIA polypeptide. Accordingly, the term regulatory sequence includes promoters, enhancers, and other sion l elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, Calif. (1990). For ce, any of a wide variety of expression control sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express DNA sequences encoding an ActRIIA polypeptide. Such useful expression control ces, include, for example, the early and late promoters of SV40, tet promoter, adenovirus or cytomegalovirus immediate early promoter, RSV promoters, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda, the control regions for fd coat protein, the promoter for phoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., PhoS, the promoters of the yeast .-mating factors, the polyhedron promoter of the baculovirus system and other sequences known to l the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof It should be tood that the design of the expression vector may depend on such factors as the choice of the host cell to be ormed and/or the type of protein desired to be expressed. Moreover, the vector's copy number, the ability to l that copy number and the expression of any other protein encoded by the vector, such as antibiotic markers, should also be considered.
A recombinant nucleic acid used in the production of ActRIIA polypeptides le for use in the methods and compositions described herein can be produced by ligating the cloned gene, or a portion thereof, into a vector suitable for expression in either prokaryotic cells, eukaryotic cells (yeast, avian, insect or mammalian), or both. Expression es for production of a recombinant ActRIIA polypeptide include plasmids and other s. For instance, suitable vectors include plasmids of the types: pBR322-derived plasmids, derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC—derived plasmids for expression in prokaryotic cells, such as E. coli.
Some mammalian expression vectors contain both yotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, ka2, o, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression s suitable for transfection of otic cells. Some of these vectors are modified with sequences from ial plasmids, such as pBR322, to tate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as the bovine papilloma virus (BPV-l), or n-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells. Examples of other viral (including retroviral) expression systems can be found below in the description of gene therapy delivery systems. The various methods employed in the preparation of the plasmids and in transformation of host organisms are well known in the art.
For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as l recombinant procedures, see Molecular Cloning A Laboratory Manual, 3rd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 2001). In some instances, it may be desirable to express the inant polypeptides by the use of a baculovirus expression system. Examples of such baculovirus sion systems include pVL- derived vectors (such as pVLl392, pVLl393 and pVL941), pAcUW—derived vectors (such as pAcUWl), and ac-derived vectors (such as the beta-gal containing pBlueBac III).
Vectors can be designed for production of the subject ActRIIA ptides in CHO cells, such as a Pcmv-Script vector (Stratagene, La Jolla, , pcDNA4 s (Invitrogen, Carlsbad, Calif.) and pCI-neo s (Promega, Madison, Wis.) As will be apparent, the t gene constructs can be used to cause expression of the subject ActRIIA polypeptides in cells propagated in culture, e.g., to e proteins, including ?ision proteins or variant proteins, for puri?cation.
Host cells transfected with a recombinant gene including a coding sequence (e.g., SEQ ID NO: 4 or 5) for one or more of the subject ActRIIA polypeptides can be used in the production of ActRIIA polypeptides suitable for use in the methods and itions described herein. The host cell may be any prokaryotic or eukaryotic cell. For example, an A polypeptide provided herein may be expressed in bacterial cells such as E. coli, insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art.
Accordingly, provided herein are methods of producing the ActRIIA polypeptides.
For example, a host cell transfected with an sion vector encoding an ActRIIA polypeptide can be cultured under appropriate conditions to allow expression of the ActRIIA polypeptide to occur. The ActRIIA polypeptide may be secreted and isolated from a mixture of cells and medium containing the ActRIIA polypeptide. atively, the ActRIIA polypeptide may be ed cytoplasmically or in a membrane on and the cells harvested, lysed and the protein isolated. A cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art. The subject ActRIIA polypeptides can be isolated from cell culture medium, host cells, or both, using techniques known in the art for purifying proteins, including ion-exchange chromatography, gel ion chromatography, ultra?ltration, electrophoresis, immunoaf?nity puri?cation with antibodies speci?c for particular epitopes of the ActRIIA polypeptides and af?nity puri?cation with an agent that binds to a domain ?Jsed to the ActRIIA polypeptide (e.g., a protein A column may be used to purify an ActRIIA-Fc ?Jsion).
In a preferred embodiment, the ActRlIA polypeptide is a fusion protein containing a domain which facilitates its puri?cation. In one embodiment, puri?cation is achieved by a series of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography, and cation exchange chromatography. The puri?cation could be ted with viral ?ltration and buffer exchange. As demonstrated herein, ActRIIA-hFc protein was puri?ed to a purity of>98% as ined by size exclusion tography and >95% as determined by SDS PAGE. This level of purity was suf?cient to achieve ble s on bone in mice and an acceptable safety pro?le in mice, rats and non- human primates.
In another embodiment, a fusion gene coding for a ation leader sequence, such as a poly-(His)/enterokinase ge Site sequence at the N-terminus of the desired portion of a recombinant ActRIIA polypeptide, can allow puri?cation of the expressed ?lSiOIl protein by af?nity chromatography using a Ni2+ metal resin. The puri?cation leader sequence can then be subsequently removed by treatment with enterokinase to provide the puri?ed ActRIIA polypeptide (e.g., see Hochuli et al., (1987) J. Chromatography 411:177; and Janknecht et al., PNAS USA 88:8972).
Techniques for making fusion genes are well known. Essentially, the joining of various DNA fragments coding for different polypeptide ces is performed in accordance with conventional techniques, employing blunt—ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, ?lling-in of cohesive ends as appropriate, alkaline phosphatase ent to avoid undesirable joining, and enzymatic ligation.
In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR ampli?cation of gene fragments can be carried out using anchor primers which give rise to complementary overhangs n two consecutive gene fragments which can subsequently be annealed to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular y, eds. Ausubel et al., John Wiley & Sons: 1992).
ActRIIA-Fc fusion protein can be expressed in stably transfected CHO-DUKX B1 1 cells from a pAID4 vector (SV40 ori/enhancer, CMV promoter), using a tissue plasminogen leader sequence of SEQ ID N029. The Fc portion is a human IgG1 Fc sequence, as shown in SEQ ID NO:7. In certain embodiments, upon sion, the protein ned has, on average, between about 1.5 and 2.5 moles of sialic acid per molecule of ActRIIA-Fc fusion protein.
In certain embodiments, the long serum half-life of an ActRIIA-Fc fusion can be 25- 32 days in human patients. Additionally, the CH0 cell sed al can have a higher af?nity for activin B ligand than that reported for an ActRIIA-hFc fusion protein expressed in human 293 cells (del Re et al., J Biol Chem. 2004 Dec 17;279(51):53l26-35). Additionally, without being bound by theory, the use ofthe TPA leader sequence provided greater production than other leader ces and, unlike ActRIIA-Fc expressed with a native leader, may e a highly pure N—terminal ce. Use of the native leader sequence may result in two major species of ActRIIA-Fc, each having a ent N—terminal sequence. .5.2 INHIBITORS OF ACTRIIB As used herein, the term "ActRIIB" refers to a family of activin or type IIB (ActRIIB) proteins from any species and variants derived from such B proteins by mutagenesis or other modi?cation. Reference to ActRIIB herein is understood to be a reference to any one of the currently identi?ed forms of the receptor. Members of the ActRIIB family are generally transmembrane proteins, composed of a -binding extracellular domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine kinase activity.
B tors to be used in the compositions and methods described herein include, without limitation, activin-binding soluble ActRIIB polypeptides; antibodies that bind to activin (particularly the activin A or B subunits, also ed to as BA or BB) and disrupt ActRIIB binding; antibodies that bind to ActRIIB and disrupt activin binding; non-antibody proteins selected for activin or B binding; and randomized peptides selected for activin or ActRIIB binding, which can be conjugated to an Fc .
In certain embodiments, two or more different proteins (or other moieties) with activin or ActRIIB binding activity, especially activin binders that block the type I (e.g., a soluble type I activin receptor) and type II (e.g., a e type II activin receptor) binding sites, respectively, may be linked together to create a bi?inctional or multi?inctional binding molecule that inhibits ActRIIB and thus can be used in the compositions and methods described herein include. In certain embodiments, Activin—ActRIIB signaling axis nists that inhibit ActRIIB include nucleic acid aptamers, small molecules and other agents are used in the compositions and methods described herein include. (a) ActRIIB tors sing ActRIIB Polypeptides As used herein, the term "ActRIIB polypeptide" refers to polypeptides comprising any naturally occurring polypeptide of an ActRIIB family member as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity.
For example, B polypeptides include polypeptides derived from the sequence of any known ActRIIB receptor having a sequence at least about 80% identical to the sequence of an ActRIIB polypeptide, and optionally at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity. For example, an ActRIIB polypeptide may bind to and inhibit the on of an ActRIIB protein and/or activin. An example of an ActRIIB polypeptide includes the human ActRIIB precursor polypeptide (SEQ ID NO:16 or SEQ ID NO:28). With respect to the ActRIIB precursor polypeptide whose amino acid sequence is depicted as SEQ ID NO: 16 or SEQ ID NO:28 (i.e., the human B precursor polypeptide), the signal peptide of the ActRIIB precursor polypeptide is located at amino acids 1 to 18; the extracellular domain is located at amino acids 19 to 134 and the potential N-linked glycosylation sites are located at amino acid positions 42 and 65. The nucleic acid sequence encoding the human B precursor ptide of SEQ ID NOzl6 is sed as SEQ ID NO: 19 (SEQ ID NO: 19 provides an alanine at the codon corresponding to amino acid on 64, but could be readily modi?ed by one of skill in the art using methods known in the art to provide an arginine at the codon corresponding to amino acid position 64 d). See Table l for a description of the SGQUGIICGS.
The numbering of amino acids for all of the ActRIIB-related polypeptides described herein is based on the amino acid ing for SEQ ID NO: 16 and SEQ ID NO:28 (which only differ in the amino acid expressed at position 64), unless speci?cally designated otherwise. For e, if an ActRIIB polypeptide is described as having a substitution/mutation at amino acid position 79, then it is to be understood that position 79 refers to the 79th amino acid in SEQ ID NO: 16 or SEQ ID NO:28, from which the ActRIIB polypeptide is derived. Likewise, if an ActRIIB polypeptide is bed as having an alanine or an arginine at amino acid position 64, then it is to be understood that position 64 refers to the 64th amino acid in SEQ ID NO:l6 or SEQ ID NO:28, from which the ActRIIB polypeptide is derived.
In certain embodiments, the inhibitors of ActRIIB used in the compositions and methods described herein comprise polypeptides comprising an activin-binding domain of ActRIIB. In some ments, the activin-binding domains of ActRIIB se the extracellular domain of ActRIIB, or a portion thereof. In speci?c embodiments, the extracellular domain or portion thereof of ActRIIB is soluble. Illustrative modi?ed forms of ActRIIB polypeptides are disclosed in US Patent ation Publication Nos. 20090005308 and 20100068215, the disclosures of which are incorporated herein by reference in their entireties.
In speci?c embodiments, the ActRIIB polypeptides used in the compositions and methods described herein are e ActRIIB polypeptides. The term "soluble ActRIIB polypeptide" generally refers to polypeptides comprising an extracellular domain of an ActRIIB protein, ing any naturally occurring extracellular domain of an ActRIIB protein as well as any variants thereof (including mutants, fragments and peptidomimetic forms). e ActRIIB polypeptides can bind to activin; however, the wild type ActRIIB protein does not exhibit signi?cant ivity in binding to activin versus GDF8/11. In certain ments, altered forms of ActRIIB with different binding properties can be used in the methods provided herein.
Such altered forms are sed, e.g., in ational patent application publication Nos. WO 2006/012627 and , the disclosures ofwhich are incorporated herein by reference in their entireties. Native or altered ActRIIB proteins may be given added speci?city for activin by coupling them with a second, activin—selective binding agent. Exemplary soluble ActRIIB polypeptides include the ellular domain of a human ActRIIB polypeptide (e.g., SEQ ID NOs: 17, 18, 23, 26, 27, 29, 30, 31, 32, 33, 36, 37, 42, and 43).
An Fc fusion n having the ActRIIB extracellular sequence disclosed by Hilden et al. (Blood, 1994, 83(8):2163-70), which has an alanine at the position ponding to amino acid 64 of the ActRIIB precursor amino acid sequence, i.e., SEQ ID NO: 16 (herein referred to as "A64"), has been demonstrated to possess a relatively low af?nity for activin and GDF-l 1.
By contrast, an EC fusion protein with an arginine at position 64 of the ActRIIB precursor amino acid ce (herein referred to as "R64") has an af?nity for activin and GDF-ll in the low nanomolar to high picomolar range (see, e.g., US. Patent Application ation No. 20100068215, the disclosure of which is herein incorporated in its entirety). An ActRIIB sor amino acid sequence with an arginine at position 64 is presented in SEQ ID NO:28.
As such, in certain ments, the ActRIIB polypeptides used in accordance with the compositions and methods bed herein may comprise either (i) an alanine at the position corresponding to amino acid 64 of the ActRIIB precursor amino acid sequence, i.e., SEQ ID NO: 16; or (ii) an arginine at position 64 of the ActRIIB precursor amino acid sequence, i.e., SEQ ID NO: 28. In other ments, the ActRIIB polypeptides used in accordance with the compositions and methods described herein may comprise an amino acid that is not alanine or arginine at the position corresponding to amino acid 64 of the ActRIIB precursor amino acid sequence, i.e., SEQ ID NO: 16 or SEQ ID N0228.
It has been shown that a deletion of the proline knot at the C-terminus of the extracellular domain of ActRIIB reduces the af?nity of the receptor for activin (see, e.g., Attisano et al., Cell, 1992, 97-108). An ActRIIB-Fc fusion protein containing amino acids -119 of SEQ ID NO: 28 (i.e., SEQ ID NO:32), "ActRIIB(20—119)-Fc" has reduced binding to GDF-ll and activin relative to an ActRIIB-Fc fusion protein containing amino acids 20-134 of SEQ ID NO: 28 (i.e., SEQ ID NO:31), "ActRIIB(20—134)-Fc", which includes the proline knot region and the complete juxtamembrane . However, an ActRIIB—Fc fusion protein containing amino acids 20-129 of SEQ ID NO: 28, "ActRIIB(20-129)-Fc" retains similar but somewhat reduced activity relative to the non-truncated extracellular domain of B, even though the proline knot region is disrupted. Thus, ActRIIB polypeptides comprising extracellular domains that stop at amino acid 134, 133, 132, 131, 130 and 129 of SEQ ID NO: 28 (or SEQ ID NO: 16) are all expected to be active, but constructs ng at amino acid 134 or 133 may be most . Similarly, mutations at any of residues 4 are not expected to alter ligand binding af?nity by large margins, as indicated by the fact that mutations of P129 and P130 of SEQ ID NO: 28 do not substantially decrease ligand binding. Therefore, the ActRIIB polypeptides used in accordance with the methods and compositions described herein may end as early as amino acid 109 (i.e., the ?nal cysteine) of SEQ ID NO:28 (or SEQ ID NO:16), however, forms ending at or between amino acid ons 109 and 119 of SEQ ID NO:28 (or SEQ ID NO:16) are expected to have reduced ligand binding ability.
Amino acid 29 of SEQ ID NO:16 and SEQ ID NO:28 represents the initial ne in the B precursor ce. It is ed that an ActRIIB polypeptide beginning at amino acid 29 of the N—terminus of SEQ ID NO:16 or SEQ ID NO:28, or before these amino acid positions, will retain ligand g activity. An alanine to gine mutation at position 24 of SEQ ID NO: 16 or SEQ ID NO:28 introduces an N—linked glycosylation sequence without substantially affecting ligand binding. This con?rms that mutations in the region between the signal ge peptide and the cysteine cross-linked region, corresponding to amino acids 20-29 of SEQ ID NO:16 or SEQ ID NO:28, are well tolerated. In particular, ActRIIB polypeptides beginning at amino acid position 20, 21, 22, 23 and 24 of SEQ ID NO:16 or SEQ ID NO:28 will retain activity, and ActRIIB polypeptides beginning at amino acid positions 25, 26, 27, 28 and 29 of SEQ ID NO:16 or SEQ ID NO:28 are also expected to retain activity. An ActRIIB polypeptide beginning at amino acid position 22, 23, 24 or 25 of SEQ ID NO: 16 or SEQ ID NO:28 will have the most activity.
Taken together, the active portions (i.e., ActRIIB polypeptides) of the ActRIIB precursor protein (i.e., SEQ ID NO:16 or SEQ ID NO:28) to be used in ance with the methods and compositions described herein will generally se amino acids 29-109 of SEQ ID NO: 16 or SEQ ID NO:28, and such ActRIIB polypeptides may, for example, begin at a residue corresponding to any one of amino acids 19-29 of SEQ ID NO: 16 or SEQ ID NO:28 and end at a position corresponding to any one of amino acids 109-134 of SEQ ID NO: 16 or SEQ ID NO:28. Speci?c examples of ActRIIB polypeptides encompassed herein include those that begin at an amino acid position from 19-29, 20-29 or 21-29 of SEQ ID NO:16 or SEQ ID NO:28 and end at an amino acid position from 119-134, 119-133 or 129-134, 129-133 of SEQ ID NO: 16 or SEQ ID NO:28. Other speci?c examples of ActRIIB polypeptides encompassed herein e those that begin at an amino acid position from 20-24 (or 21-24, or 22-25) of SEQ ID NO:16 or SEQ ID N028 and end at an amino acid position from 109-134 (or 109-133), 119- 134 (or 119-133) or 129-134 (or 129-133) of SEQ ID NO:16 or SEQ ID NO:28. Variant ActRIIB ptides falling within these ranges are also contemplated, particularly those having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or sequence homology to the corresponding portion of SEQ ID NO: 16 or SEQ ID NO:28.
] In certain embodiments, the inhibitors of ActRIIB used in the compositions and methods described herein comprise a truncated form of an ellular domain of B. The truncation can be at the carboxy terminus and/or the amino terminus of the ActRIIB polypeptide.
In certain embodiments, the truncation can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, l2, l3, l4, l5, l6, l7, l8, 19, 20, 21, 22, 23, 24, or 25 amino acids long relative to the mature ActRIIB polypeptide extracellular domain. In certain ments, the truncation can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, l2, l3, l4, l5, l6, 17, 18, 19, 20, 21, 22, 23, 24, or 25 N-terminal amino acids ofthe mature ActRIIB polypeptide extracellular domain. In n embodiments, the truncation can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 C-terminal amino acids of the mature ActRIIB polypeptide extracellular domain. For example, truncated forms of B include polypeptides with amino acids 20-119; 20-128; ; 20-130; 20-131; 20- 133; 20-134; 20—131; 2l—l31; 22—131;23—131;24—l3l; and 25-131, wherein the amino acid positions refer to the amino acid positions in SEQ ID NO:16 or SEQ ID NO:28.
Additional exemplary truncated forms of ActRIIB include (i) polypeptides ing at amino acids at any of amino acids 21-29 of SEQ ID NO:16 or SEQ ID NO:28 (optionally beginning at 22-25 of SEQ ID NO:16 or SEQ ID NO:28) and ending at any of amino acids 109- 134 of SEQ ID NO:16 or SEQ ID NO:28; (ii) polypeptides beginning at any of amino acids 20- 29 of SEQ ID NO:16 or SEQ ID NO:28 (optionally beginning at 22—25 of SEQ ID NO:16 or SEQ ID NO:28) and ending at any o acids 109—133 of SEQ ID NO:16 or SEQ ID NO:28; (iii) polypeptides beginning at any of amino acids 20-24 of SEQ ID NO:16 or SEQ ID NO:28 (optionally beginning at 22-25 of SEQ ID NO:16 or SEQ ID NO:28) and ending at any of amino acids 109-133 of SEQ ID NO:16 or SEQ ID NO:28; (iv) ptides beginning at any of amino acids 21-24 of SEQ ID NO:16 or SEQ ID NO:28 and ending at any of amino acids 109-134 of SEQ ID NO:16 or SEQ ID NO:28; (V) polypeptides beginning at any of amino acids 20-24 of SEQ ID NO:16 or SEQ ID NO:28 and ending at any ofamino acids 118-133 of SEQ ID NO:16 or SEQ ID NO:28; (Vi) polypeptides beginning at any of amino acids 21-24 of SEQ ID NO:16 or SEQ ID NO:28 and ending at any of amino acids 118—134 of SEQ ID NO:16 or SEQ ID NO:28; (Vii) polypeptides beginning at any of amino acids 20—24 of SEQ ID NO:16 or SEQ ID NO:28 and ending at any of amino acids 128-133 of SEQ ID NO:16 or SEQ ID NO:28; (Viii) polypeptides beginning at any of amino acids 20-24 of SEQ ID NO:16 or SEQ ID NO:28 and ending at any of amino acids 128-133 of SEQ ID NO:16 or SEQ ID NO:28; (ix) polypeptides beginning at any of amino acids 21-29 of SEQ ID NO:16 or SEQ ID NO:28 and ending at any of amino acids 118-134 of SEQ ID NO:16 or SEQ ID NO:28; (x) polypeptides beginning at any of amino acids 20-29 of SEQ ID NO:16 or SEQ ID NO:28 and ending at any of amino acids 118- 133 of SEQ ID NO:16 or SEQ ID NO:28; (Xi) polypeptides beginning at any of amino acids 21- 29 of SEQ ID NO:16 or SEQ ID NO:28 and ending at any of amino acids 128-134 of SEQ ID NO:16 or SEQ ID NO:28; and (xii) polypeptides beginning at any of amino acids 20-29 of SEQ ID NO:16 or SEQ ID NO:28 and ending at any ofamino acids 128—133 of SEQ ID NO:16 or SEQ ID NO:28. In a speci?c ment, an ActRIIB polypeptides ses, consists essentially of, or consists of, an amino acid sequence beginning at amino acid position 25 of SEQ ID NO:16 or SEQ ID NO:28 and ending at amino acid position 131 of SEQ ID NO:16 or SEQ ID NO:28. In another speci?c embodiment, an B polypeptide consists of, or ts essentially of, the amino acid sequence of SEQ ID NO:17, 18, 23, 26, 27, 29, 30, 31, 32, 33, 36, 37, 42, or 43.
Any of the ActRIIB polypeptides used in the compositions and methods described herein may be produced as a homodimer. Any of the ActRIIB polypeptides used in the compositions and methods described herein may be ated as a fusion protein having a heterologous portion that comprises a constant region from an IgG heavy chain, such as an F0 domain. Any of the ActRIIB polypeptides used in the compositions and methods described herein may comprise an acidic amino acid at the position corresponding to position 79 of SEQ ID NO: 16 or SEQ ID NO:28, optionally in combination with one or more additional amino acid substitutions, ons or insertions relative to SEQ ID NO: 16 or SEQ ID NO:28.
In speci?c embodiments, the inhibitors of ActRIIB used in the compositions and methods described herein se an extracellular domain of ActRIIB with one or more amino acid tutions/mutations. Such an amino acid tution/mutation can be, for e, an exchange from the e at amino acid position 79 of SEQ ID NO: 16 or SEQ ID NO:28 to an acidic amino acid, such as aspartic acid or glutamic acid. For example, position L79 of SEQ ID NO: 16 or SEQ ID NO:28 may be d in ActRIIB extracellular domain polypeptides to confer d activin-myostatin (GDF-l 1) binding properties. L79A and L79P mutations reduce GDF- ll g to a greater extent than n g. L79E and L79D mutations retain GDF-ll binding, While demonstrating greatly reduced activin binding.
In certain embodiments, the inhibitors of ActRIIB used in the compositions and methods described herein comprise a truncated form of an ActRIIB extracellular domain that also carries an amino acid substitution, e. g., an exchange from the leucine at amino acid position 79 of SEQ ID NO: 16 or SEQ ID NO:28 to an acidic amino acid, such as aspartic acid or glutamic acid. In a speci?c embodiment, the truncated form of an extracellular domain of ActRIIB polypeptide that also carries an amino acid substitution used in the compositions and methods described herein is SEQ ID NO:23. Forms of ActRIIB that are truncated and/or carry one or more amino acid tutions can be linked to an EC domain of an antibody as discussed above.
Functionally active fragments of ActRIIB polypeptides can be obtained, for example, by screening polypeptides recombinantly produced from the corresponding fragment of the nucleic acid encoding an ActRIIB ptide. In addition, fragments can be chemically synthesized using techniques known in the art such as conventional Merri?eld solid phase f—Moc or t-Boc chemistry. The fragments can be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments that can function as antagonists (inhibitors) of ActRIIB protein or signaling mediated by activin.
In addition, functionally active variants of ActRIIB polypeptides can be obtained, for example, by screening libraries of modi?ed polypeptides recombinantly produced from the corresponding mutagenized nucleic acids encoding an ActRIIB polypeptide. The variants can be produced and tested to identify those that can function as antagonists (inhibitors) of ActRIIB protein or signaling mediated by activin. In certain embodiments, a ?anctional variant of the ActRIIB polypeptides comprises an amino acid sequence that is at least 75% cal to an amino acid ce selected from SEQ ID NO:17, 18, 23, 26, 27, 29, 30, 31, 32, 33, 36, 37, 42, and 43. In certain embodiments, the functional t has an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid ce selected from SEQ ID NO:17, 18, 23, 26, 27, 29, 30, 31, 32, 33, 36, 37, 42, and 43. onal variants may be generated, for example, by modifying the structure of an ActRIIB ptide for such purposes as enhancing therapeutic ef?cacy, or stability (e.g., ex vivo shelf life and resistance to proteolytic degradation in vivo). Such modi?ed ActRIIB polypeptides when selected to retain activin binding, are considered functional equivalents of the naturally-occurring ActRIIB polypeptides. Modi?ed ActRIIB polypeptides can also be produced, for instance, by amino acid tution, deletion, or addition. For instance, it is reasonable to expect that an isolated ement of a leucine with an isoleucine or valine, an aspartate with a ate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (e.g., conservative mutations) will not have a major effect on the biological activity of the resulting molecule. Conservative replacements are those that take place within a family of amino acids that are related in their side . r a change in the amino acid sequence of an ActRIIB polypeptide results in a functional homolog can be readily determined by assessing the ability of the variant B polypeptide to produce a response in cells in a fashion similar to the wild-type ActRIIB ptide.
ActRIIB polypeptide mutants, ularly sets of combinatorial mutants of an ActRIIB polypeptide, as well as truncation mutants; pools of atorial mutants are especially useful for fying functional variant sequences can be used in the methods and compositions described herein. The e of screening such combinatorial libraries may be to generate, for example, ActRIIB polypeptide variants which can act as either agonists or antagonist, or alternatively, which possess novel activities all er.
It has been trated that the ligand binding pocket of ActRIIB is de?ned by residues Y31, N33, N35, L38 through T41, E47, E50, Q53 through K55, L57, H58, Y60, S62, K74, W78 through N83, Y85, R87, A92, and E94 through F101 of SEQ ID NO:16 or SEQ ID NO:28. At these positions, it is expected that conservative mutations will be tolerated, although a K74A mutation is well-tolerated, as are R40A, K55A, F82A and ons at on L79.
R40 is a K in Xenopus, indicating that basic amino acids at this position will be tolerated. Q53 is R in bovine ActRIIB and K in Xenopus ActRIIB, and therefore amino acids including R, K, Q, N and H will be tolerated at this position. Thus, a general formula for an ActRIIB polypeptide for use in the methods and compositions described herein is one that comprises amino acids 29- 109 of SEQ ID NO: 16 or SEQ ID NO:28, but optionally beginning at an amino acid position ranging from 20-24 or 22-25 of SEQ ID NO:16 or SEQ ID NO:28 and ending at an amino acid position g from 129-134 of SEQ ID NO:16 or SEQ ID NO:28, and comprising no more than 1, 2, 5, or 15 vative amino acid changes in the ligand binding pocket, and zero, one or more non-conservative alterations at amino acid positions 40, 53, 55, 74, 79 and/or 82 of SEQ ID NO: 16 or SEQ ID NO:28 in the ligand binding pocket. Such an B polypeptide may retain greater than 80%, 90%, 95% or 99% sequence identity or sequence homology to the sequence of amino acids 29-109 of SEQ ID NO: 16 or SEQ ID NO:28. Sites outside the g pocket, at which variability may be particularly well tolerated, include the amino and carboxy termini of the extracellular domain of ActRIIB, and positions 42—46 and 65-73. An asparagine to alanine tion at position 65 of SEQ ID NO: 16 or SEQ ID NO:28 (N65A) actually improves ligand binding in the A64 background, and is thus expected to have no detrimental effect on ligand binding in the R64 background. This change probably eliminates glycosylation at N65 in the A64 background, thus demonstrating that a signi?cant change in this region is likely to be tolerated. While an R64A change is poorly tolerated, R64K is well-tolerated, and thus another basic residue, such as H may be tolerated at position 64.
] As a speci?c example of an ActRIIB polypeptide with a mutation in the ligand binding domain, the positively-charged amino acid e Asp (D80) of the ligand-binding domain of ActRIIB can be d to a different amino acid residue such that the variant ActRIIB polypeptide preferentially binds to GDF8, but not n. In a speci?c embodiment, the D80 residue is changed to an amino acid residue selected from the group consisting of: an uncharged amino acid residue, a negative amino acid residue, and a hydrophobic amino acid residue. As a r speci?c example, the hydrophobic residue L79 can be altered to the acidic amino acids aspartic acid or glutamic acid to greatly reduce activin binding while retaining GDFll binding. As will be recognized by one of skill in the art, most of the described mutations, variants or modi?cations may be made at the nucleic acid level or, in some cases, by post translational modi?cation or chemical synthesis. Such techniques are well known in the art.
In speci?c embodiments, the inhibitors of ActRIIB used in the compositions and methods described herein comprise a conjugate/fusion protein comprising an extracellular domain (e.g., an n—binding domain) of an ActRIIB receptor linked to an Fc portion of an antibody. Such conjugate/fusion proteins may se any of the ActRIIB polypeptides disclosed herein (e.g., any of SEQ ID NOs217, 18, 23, 26, 27, 29, 30, 31, 32, 33, 36, 37, 42, or 43), any ActRIIB polypeptides known in the art, or any ActRIIB polypeptides generated using methods known in the art and/or provided herein.
In certain embodiments, the extracellular domain is linked to an Fc portion of an antibody via a linker, e.g. a e linker. Exemplary linkers e short polypeptide sequences such as 2-10, 2-5, 2-4, 2-3 amino acid residues (e.g., glycine residues), such as, for example, a Gly-Gly-Gly linker. In a speci?c embodiment, the linker comprises the amino acid sequence Gly-Gly-Gly (GGG). In another speci?c embodiment, the linker comprises the amino acid ce Thr-Gly-Gly-Gly (TGGG). Optionally, the Fc domain has one or more mutations at residues such as Asp-265, lysine 322, and Asn-434. In certain cases, the mutant Fc domain having one or more of these mutations (e.g., an Asp-265 mutation) has a reduced ability to bind to the Fcy or relative to a wild-type Fc domain. In other cases, the mutant Fc domain having one or more of these mutations (e.g., an 4 on) has an increased ability to bind to the MHC class I- related Fc-receptor (FcRN) relative to a wild-type Fc domain. ary fusion proteins comprising a soluble extracellular domain of ActRIIB fused to an EC domain are set forth in SEQ ID NOS:20, 21, 24, 25, 34, 35, 38, 39, 40, 41, 44, 46, and 47.
] In a speci?c embodiment, the ActRIIB inhibitors used in the compositions and methods described herein comprise the extracellular domain of ActRIIB, or a portion thereof, linked to an Fc portion of an antibody, wherein said B inhibitor comprises an amino acid sequence that is at least 75% cal to an amino acid sequence selected from SEQ ID NOs:20, 21, 24, 25, 34, 35, 38, 39, 40, 41, 44, 46, and 47. In another speci?c embodiment, the ActRIIB inhibitors used in the compositions and methods described herein comprise the extracellular domain of ActRIIB, or a portion thereof, linked to an Fc portion of an antibody, wherein said ActRIIB inhibitor ses an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs:20, 21, 24, , 34, 35, 38, 39, 40, 41, 44, 46, and 47.
In a speci?c embodiment, the ActRIIB inhibitor to be used in the compositions and methods described herein is a fusion protein n the extracellular domain of the human ActRIIB receptor and the F0 portion of IgG1. In another speci?c embodiment, the ActRIIB inhibitor to be used in the compositions and methods described herein is a fusion protein between a truncated ellular domain of the human ActRIIB receptor and the Fc n of IgG1. In another speci?c embodiment, the B inhibitor to be used in the compositions and methods described herein is a fusion protein between a truncated extracellular domain of the human ActRIIB or and the Fc portion of IgG1 wherein the truncated extracellular domain of the human ActRIIB receptor possesses an amino acid substitution at the amino acid position corresponding to amino acid 79 of SEQ ID NO:16 or SEQ ID NO:28. In one embodiment, the amino acid substitution at the amino acid position corresponding to amino acid 79 of SEQ ID NO: 16 or SEQ ID NO:28 is substitution of Leucine for Aspartic Acid (i.e., an L79D mutation).
In a c embodiment, the ActRIIB inhibitor to be used in the compositions and methods described herein is SEQ ID NO:24 or 25, which represents a ?ision protein between the extracellular domain of the human ActRIIB receptor and the Fc portion of IgG1, wherein said ActRIIB extracellular domain comprises amino acids 25—131 of SEQ ID NO:28 with an L79D mutation. The nucleic acid sequence encoding the ActRIIB-Fc fusion protein of SEQ ID NO:24 is presented in SEQ ID NO:45.
In another speci?c embodiment, the ActRIIB tor to be used in the compositions and methods described herein is SEQ ID NO:34 or 35, which represents a fusion protein between the extracellular domain of the human ActRIIB receptor and the Fc portion of IgG1, wherein said ActRIIB extracellular domain comprises amino acids 25—131 of SEQ ID NO:16 with an L79D mutation.
] Asparagine-linked glycosylation recognition sites lly se a tripeptide sequence, gine-X-threonine (or asparagine—X—serine) (where "X" is any amino acid) which is speci?cally recognized by riate cellular ylation enzymes. The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the Wild-type ActRIIB polypeptide (for O—linked glycosylation sites). A variety of amino acid substitutions or deletions at one or both of the ?rst or third amino acid positions of a ylation recognition site (and/or amino acid deletion at the second position) s in non-glycosylation at the d tripeptide sequence. Another means of increasing the number of carbohydrate moieties on an ActRIIB polypeptide is by chemical or tic coupling of glycosides to the ActRIIB polypeptide. Depending on the coupling mode used, the sugar(s) may be attached to (a) arginine and histidine; (b) free carboxyl groups; (c) free sulfhydryl groups such as those of cysteine; ((1) free hydroxyl groups such as those of serine, threonine, or hydroxyproline; (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan; or (f) the amide group of glutamine. These methods are described in ational Patent Application No. WO 87/05330 published Sep. 11, 1987, and in Aplin and Wriston (1981) CRC Crit. Rev. Biochem., pp. 25 9-3 06, incorporated by reference herein. Removal of one or more carbohydrate moieties present on an ActRIIB polypeptide may be accomplished chemically and/or enzymatically. Chemical deglycosylation may e, for example, exposure of the ActRIIB polypeptide to the compound tri?uoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage ofmost or all sugars except the linking sugar (N—acetylglucosamine or N—acetylgalactosamine), while leaving the amino acid sequence intact.
Chemical deglycosylation is further described by ddin et al. (1987) Arch. Biochem.
Biophys. 259:52 and by Edge et al. (1981) Anal. Biochem. 118: 13 1. Enzymatic cleavage of ydrate moieties on ActRIIB polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as bed by Thotakura et a1. (1987) Meth. Enzymol. 138:350. The sequence of an B polypeptide may be adjusted, as riate, ing on the type of expression system used, as mammalian, yeast, insect and plant cells may all introduce differing glycosylation patterns that can be affected by the amino acid sequence of the peptide. In general, ActRIIB proteins for use in humans will be expressed in a ian cell line that provides proper ylation, such as HEK293 or CHO cell lines, although other expression systems, such as other mammalian expression cell lines, yeast cell lines with engineered glycosylation s and insect cells, are expected to be useful as well.
In speci?c embodiments, mutated ActRIIB polypeptides comprising the addition of a further N—linked glycosylation site (N-X-S/T) that increases the serum half-life of an ActRIIB-Fc fusion protein, relative to the ActRIIB(R64)—Fc form can be used in the methods and compositions bed herein. In a speci?c embodiment, introduction of an asparagine at on 24 of SEQ ID NO: 16 or SEQ ID NO:28 (A24N) results in the creation of an NXT sequence that confers a longer half-life. Other NX(T/S) ces can be found at 42-44 (NQS) and 65-67 (NSS), although the latter may not be ef?ciently glycosylated with the R at position 64 (i.e., in R64 polypeptides). N—X-S/T sequences may be generally introduced at positions outside the ligand binding pocket of ActRIIB, which is detailed above. Particularly suitable sites for the introduction of non-endogenous N-X-S/T sequences include amino acids 20-29, 20-24, 22-25, 109-134, 120-134 or 129-134 of SEQ ID NO:16 or SEQ ID NO:28. N-X-S/T sequences may also be introduced into the linker between the ActRIIB sequence and the F0 or other fusion component. Such a site may be introduced with minimal effort by introducing an N in the correct on with respect to a pre—existing S or T, or by introducing an S or T at a position corresponding to a pre-existing N. Thus, ble tions that would create an N—linked glycosylation site are: A24N, R64N, S67N (possibly combined with an N65A alteration), E106N, R112N, G120N, E123N, P129N, A132N, R112S and R112T (with all amino acid positions corresponding to the positions they can be found in SEQ ID NO:l6 or SEQ ID NO:28).
Any S that is predicted to be glycosylated may be altered to a T t creating an genic site, because of the tion afforded by the glycosylation. Likewise, any T that is predicted to be glycosylated may be altered to an S. Thus the alterations S67T and S44T are encompassed herein. Likewise, in an A24N variant, an S26T alteration may be used.
Accordingly, an ActRIIB polypeptide may include one or more additional, dogenous N- linked glycosylation consensus sequences.
A variety of ing assays may be used to evaluate ActRIIB polypeptide variants.
For example, an ActRIIB polypeptide variant may be screened for ability to bind to an ActRIIB ligand, to prevent binding of an ActRIIB ligand to an ActRIIB polypeptide or to interfere with signaling caused by an ActRIIB ligand. The activity of an ActRIIB polypeptide or its variants may also be tested in a cell-based or in vivo assay.
Combinatorially—derived variants can be generated which have a selective or generally increased potency relative to a naturally occurring ActRIIB polypeptide. Likewise, mutagenesis can give rise to variants which have ellular half-lives dramatically different than the corresponding wild-type ActRIIB ptide. For e, the altered protein can be rendered either more stable or less stable to proteolytic degradation or other cellular processes which result in destruction of, or otherwise inactivation of a native ActRIIB ptide. Such variants, and the genes which encode them, can be utilized to alter ActRIIB polypeptide levels by modulating the half-life of the ActRIIB polypeptides. For instance, a short half-life can give rise to more transient biological s and can allow r control of recombinant ActRIIB polypeptide levels within the patient. In an Fc fusion protein, mutations may be made in the linker (if any) and/or the Fc portion to alter the half-life of the protein.
A combinatorial library may be produced by way of a degenerate library of genes encoding a y of ptides which each include at least a portion of potential ActRIIB polypeptide sequences. For instance, a mixture of synthetic oligonucleotides can be enzymatically d into gene sequences such that the degenerate set of potential ActRIIB polypeptide tide sequences are expressible as individual ptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display).
] There are many ways by which the library of potential homologs can be generated from a rate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic genes then be ligated into an appropriate vector for expression. The sis of degenerate oligonucleotides is well known in the art (see for example, , S A (1983) Tetrahedron 39:3; Itakura et al., (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp 273-289; a et al., (1984) Annu. Rev. Biochem. 53:323; Itakura et al., (1984) Science 198: 1056; Ike et al., (1983) Nucleic Acid Res. 11:477). Such techniques have been employed in the directed evolution of other proteins (see, for example, Scott et al., (1990) e 249:386-390; Roberts et al., (1992) PNAS USA 89:2429—2433; Devlin et al., (1990) Science 249: 404-406; Cwirla etal., (1990) PNAS USA 87: 6378-6382; as well as US. Pat. Nos. ,223,409, 5,198,346, and 5,096,815).
Alternatively, other forms of mutagenesis can be utilized to generate a combinatorial library. For example, ActRIIB polypeptide variants can be generated and isolated from a library by screening using, for example, alanine ng mutagenesis and the like (Ruf et al., (1994) Biochemistry 33:1565-1572; Wang et al., (1994) J. Biol. Chem. 269:3095-3099; Balint et al., (1993) Gene 137: 109-1 18; Grodberg et al., (1993) Eur. J. Biochem. 218:597-601; Nagashima et al., (1993) J. Biol. Chem. 268:2888-2892; Lowman et al., (1991) Biochemistry 30: 10832-10838; and Cunningham et al., (1989) Science 244:1081-1085), by linker scanning mutagenesis (Gustin et a1., (1993) Virology 3-660; Brown et a1., (1992) M01. Cell Biol. 12:2644-2652; ht et a1., (1982) Science 2322316); by saturation mutagenesis (Meyers et al., (1986) Science 2322613); by PCR mutagenesis (Leung et a1., (1989) Method Cell Mol Biol 1:11-19); or by random mutagenesis, including chemical mutagenesis, etc. (Miller et al., (1992) A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor, NY; and r et a1., (1994) gies in Mol Biol 7:32-34). Linker scanning mutagenesis, particularly in a combinatorial setting, is an attractive method for identifying truncated (bioactive) forms of ActRIIB polypeptides.
A wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations and tions, and, for that matter, for ing cDNA libraries for gene products having a certain property. Such techniques will be generally adaptable for rapid ing of the gene libraries generated by the combinatorial nesis of ActRIIB polypeptides. The most widely used techniques for screening large gene libraries typically comprises cloning the gene library into able expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene whose product was ed. red assays include activin binding assays and activin-mediated cell ing assays.
In certain embodiments, ActRIIB polypeptides used in the methods and compositions described herein may further comprise post—translational modi?cations in on to any that are naturally present in the ActRIIB polypeptides. Such modi?cations include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. As a result, the modi?ed ActRlIB polypeptides may n non-amino acid elements, such as polyethylene s, lipids, poly- or accharide, and phosphates. Effects of such non- amino acid elements on the functionality of a ActRJIB polypeptide may be tested by any method known to the skilled artisan. When an ActRJIB polypeptide is produced in cells by cleaving a nascent form of the ActRIIB polypeptide, post—translational processing may also be important for correct folding and/or function of the protein. Different cells (such as CHO, HeLa, MDCK, 293, W138, NIH-3T3 or HEK293) have speci?c cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modi?cation and processing of the B ptides.
In n aspects, functional ts or modi?ed forms of the ActRIIB polypeptides include fusion proteins having at least a n of the ActRIIB polypeptides and one or more fusion domains. Well known examples of such fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin heavy chain constant region (Fc), maltose g n (MBP), or human serum albumin. A fusion domain may be selected so as to confer a desired property. For example, some fusion domains are particularly useful for isolation of the fusion proteins by af?nity chromatography. For the purpose of af?nity puri?cation, nt matrices for af?nity chromatography, such as glutathione—, amylase—, and nickel— or -conjugated resins are used. Many of such matrices are available in "kit" form, such as the Pharmacia GST ation system and the QIAexpressTM system (Qiagen) useful with (H186) fusion partners. As another example, a fusion domain may be selected so as to facilitate detection of the ActRIIB polypeptides. Examples of such detection domains include the various ?uorescent proteins (e.g., GFP) as well as "epitope tags," which are usually short peptide sequences for which a speci?c antibody is available. Well known epitope tags for which speci?c monoclonal antibodies are readily available include FLAG, in?uenza Virus hemagglutinin (HA), and c-myc tags. In some cases, the fusion domains have a protease cleavage site, such as for Factor Xa or Thrombin, which allows the relevant protease to partially digest the fusion proteins and thereby liberate the recombinant proteins therefrom. The liberated proteins can then be isolated from the ?ision domain by subsequent chromatographic separation. In certain preferred embodiments, an ActRIIB polypeptide is fused with a domain that stabilizes the ActRIIB polypeptide in Vivo (a "stabilizer" domain). By lizing" is meant anything that increases serum half life, regardless of whether this is because of decreased destruction, decreased clearance by the kidney, or other pharmacokinetic effect. Fusions with the Fc portion of an immunoglobulin are known to confer desirable pharmacokinetic ties on a wide range of proteins. Likewise, fusions to human serum albumin can confer desirable properties. Other types of fusion s that may be selected include multimerizing (e.g., dimerizing, tetramerizing) domains and functional s (that confer an onal biological function, such as further stimulation of bone growth or muscle growth, as desired).
It is understood that different elements of the ?ision proteins may be arranged in any manner that is consistent with the desired functionality. For example, an ActRIIB polypeptide may be placed inal to a heterologous domain, or, alternatively, a heterologous domain may be placed C-terminal to an ActRIIB polypeptide. The ActRIIB polypeptide domain and the heterologous domain need not be nt in a ?ision protein, and additional domains or amino acid sequences may be included C- or N—terminal to either domain or between the domains.
In certain ments, the ActRIIB polypeptides used in the methods and compositions described herein contain one or more modi?cations that are capable of stabilizing the ActRIIB polypeptides. For e, such modi?cations enhance the in vitro half life of the ActRIIB polypeptides, enhance atory half life of the ActRIIB polypeptides or reduce proteolytic degradation of the ActRIIB polypeptides. Such stabilizing ations e, but are not limited to, fusion proteins (including, for example, fusion proteins comprising an ActRIIB polypeptide and a stabilizer domain), modi?cations of a glycosylation site (including, for example, addition of a glycosylation site to an ActRIIB polypeptide), and modi?cations of carbohydrate moiety (including, for e, removal of carbohydrate moieties from an ActRIIB polypeptide). In the case of fusion proteins, an B polypeptide is fused to a stabilizer domain such as an IgG molecule (e.g., an F0 domain). As used herein, the term "stabilizer domain" not only refers to a fusion domain (e.g., PC) as in the case of fusion proteins, but also includes nonproteinaceous modi?cations such as a carbohydrate moiety, or nonproteinaceous polymer, such as polyethylene glycol.
In certain embodiments, the methods and compositions described herein use isolated or puri?ed ActRIIB ptides, i.e., ActRIIB polypeptides which are isolated from, or otherwise ntially free of, other proteins can be used with the methods and compositions described herein. ActRIIB polypeptides will lly be produced by expression from recombinant nucleic acids.
In certain aspects, the ActRIIB polypeptides used in the methods and compositions described herein are d by ed and/or recombinant nucleic acids, including fragments, functional variants and fusion proteins disclosed herein. For example, SEQ ID NO: 19 encodes the naturally occurring human ActRIIB sor polypeptide. The subject nucleic acids may be single-stranded or double stranded. Such c acids may be DNA or RNA molecules. These nucleic acids may be used, for example, in methods for making ActRIIB ptides or as direct therapeutic agents (e. g., in a gene therapy approach).
In certain aspects, the c acids that can be used to produce ActRIIB polypeptides suitable for use in the methods and compositions described herein are ?thher understood to include nucleic acids that are variants of SEQ ID NO: 19 as well as variants of those nucleic acid sequences that encode soluble B polypeptides (e.g., nucleic acids that encode SEQ ID NOS: 17, 18, 23, 26, 27, 29, 30, 31, 32, 33, 36, 37, 42, and 43). Variant nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants.
In certain embodiments, the isolated or recombinant nucleic acid sequences that can be used to produce B polypeptides suitable for use in the methods and itions described herein are at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 19 or those nucleic acid sequences that encode soluble ActRIIB polypeptides (e.g., nucleic acids that encode SEQ ID NOS: 17, 18, 23, 26, 27, 29, 30, 31, 32, 33, 36, 37,42, and 43).
One of ordinary skill in the art will appreciate that nucleic acid sequences complementary to SEQ ID NO: 19 or those nucleic acid sequences that encode soluble ActRIIB polypeptides (e.g., nucleic acids that encode SEQ ID NOS: 17, 18, 23, 26, 27, 29, 30, 31, 32, 33, 36, 37,42, and 43), and variants of SEQ ID NO: 19 or those nucleic acid sequences that encode soluble ActRIIB polypeptides (e.g., nucleic acids that encode SEQ ID NOS: 17, 18, 23, 26, 27, 29, 30, 31, 32, 33, 36, 37, 42, and 43) can be used with the methods and compositions described herein. In further embodiments, the nucleic acid sequences can be isolated, recombinant, and/or fused with a heterologous nucleotide ce, or in a DNA library.
In other embodiments, nucleic acids that can be used to produce ActRIIB polypeptides suitable for use in the methods and compositions described herein include nucleotide sequences that hybridize under highly stringent conditions to the nucleotide sequence ated in SEQ ID NO: 19 or those nucleic acid sequences that encode soluble ActRIIB polypeptides (e.g., nucleic acids that encode SEQ ID NOS: l7, 18, 23, 26, 27, 29, 30, 31, 32, 33, 36, 37, 42, and 43), complement ce of SEQ ID NO:19 or those nucleic acid sequences that encode soluble B polypeptides (e.g., c acids that encode SEQ ID NOS: 17, 18, 23, 26, 27, 29, 30, 31, 32, 33, 36, 37, 42, and 43), or nts thereof. One of ordinary skill in the art will understand that appropriate ency conditions Which promote DNA hybridization can be varied. For example, one can perform the hybridization at 6.0 times sodium chloride/sodium citrate (SSC) at about 45 degree Celsius, followed by a wash of 2.0 times SSC at 50 degree s. For example, the salt concentration in the wash step can be ed from a low stringency of about 2.0 times SSC at 50 degree Celsius to a high stringency of about 0.2 times SSC at 50 degree Celsius. In on, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22 degree Celsius, to high stringency conditions at about 65 degree Celsius. Both temperature and salt may be , or temperature or salt concentration may be held nt while the other variable is changed. In one embodiment, nucleic acids which hybridize under low stringency conditions of 6 times SSC at room ature followed by a wash at 2 times SSC at room temperature can be used with the methods and compositions described herein.
Isolated nucleic acids which differ from the c acids as set forth in SEQ ID NO: 19 or those nucleic acid sequences that encode soluble ActRIIB polypeptides (e.g., nucleic acids that encode SEQ ID NOs: 17, 18, 23, 26, 27, 29, 30, 31, 32, 33, 36, 37, 42, and 43) due to degeneracy in the genetic code can also be used to produce ActRIIB polypeptides suitable for use in the methods and compositions described herein. For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) may result in "silent" mutations which do not affect the amino acid ce of the protein. However, it is ed that DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the subject proteins will exist among mammalian cells. One skilled in the art will appreciate that these variations in one or more nucleotides (up to about 3-5% of the nucleotides) of the nucleic acids encoding a particular protein may exist among individuals of a given species due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms can be used with the methods and compositions described herein.
In certain embodiments, the recombinant nucleic acids that can be used to produce ActRIIB polypeptides suitable for use in the methods and compositions described herein may be operably linked to one or more regulatory nucleotide ces in an expression construct.
Regulatory tide sequences will generally be riate to the host cell used for expression. Numerous types of appropriate expression vectors and suitable tory ces are known in the art for a variety of host cells. Typically, said one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences. Constitutive or inducible ers as known in the art can be used with the methods and compositions described herein. The promoters may be either lly occurring promoters, or hybrid promoters that combine elements of more than one promoter. An expression construct may be present in a cell on an episome, such as a plasmid, or the expression construct may be inserted in a chromosome. In a preferred embodiment, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used.
In certain aspects, the nucleic acids that can be used to produce ActRIIB polypeptides suitable for use in the methods and compositions described herein are provided in an expression vector comprising a nucleotide sequence encoding an ActRIIB ptide and ly linked to at least one regulatory sequence. Regulatory ces are art-recognized and are selected to direct expression of the ActRIIB polypeptide. Accordingly, the term regulatory sequence includes promoters, enhancers, and other expression control elements. Exemplary regulatory ces are described in l; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, Calif. (1990). For instance, any of a wide variety of expression control ces that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express DNA ces encoding an B polypeptide. Such useful expression control sequences, e, for example, the early and late promoters of SV40, tet promoter, adenovirus or cytomegalovirus immediate early promoter, RSV promoters, the lac system, the trp system, the TAC or TRC system, T7 promoter whose sion is directed by T7 RNA polymerase, the major operator and er regions of phage lambda, the control regions for fd coat protein, the promoter for 3—phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., PhoS, the promoters of the yeast alpha.- mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the sion of genes of yotic or eukaryotic cells or their viruses, and various combinations f. It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed. Moreover, the vector's copy , the ability to control that copy number and the sion of any other protein encoded by the vector, such as antibiotic markers, should also be considered.
] A recombinant nucleic acid can be produced by ligating the cloned gene, or a portion thereof, into a vector suitable for expression in either prokaryotic cells, eukaryotic cells (yeast, avian, insect or mammalian), or both. Expression vehicles for production of a recombinant ActRIIB polypeptide include plasmids and other vectors. For instance, suitable vectors e plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.
Some mammalian expression vectors contain both prokaryotic sequences to facilitate the ation of the vector in ia, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, V, pSVngt, pSVZneo, pSVZ-dhfr, ka2, pRSVneo, pMSG, pSVT7, pko—neo and pHyg derived vectors are examples of mammalian expression vectors le for transfection of eukaryotic cells. Some of these s are modi?ed with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as the bovine papilloma Virus (BPV-l), or Epstein-Barr Virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells. Examples of other Viral (including retroviral) expression systems can be found below in the description of gene therapy delivery systems. The various methods employed in the preparation of the plasmids and in transformation of host organisms are well known in the art.
For other suitable expression s for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular Cloning A Laboratory Manual, 3rd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 2001). In some instances, it may be desirable to express the recombinant polypeptides by the use of a baculovirus expression system. Examples of such baculovirus expression systems include pVL- d vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived s (such as pAcUWl), and pBlueBac—derived vectors (such as the beta-gal containing ac 111).
] In one embodiment, a vector can be designed for tion of the ActRIIB ptides used in the methods and itions described herein in CHO cells, such as a Pcmv-Script vector (Stratagene, La Jolla, Calif), pcDNA4 vectors (Invitrogen, Carlsbad, Calif.) and pCI-neo vectors (Promega, Madison, Wis.). As will be apparent, the subject gene constructs can be used to cause expression of the subject ActRIIB polypeptides in cells propagated in culture, e.g., to produce ns, including ?lSlOIl proteins or variant proteins, for puri?cation.
Host cells transfected with a recombinant gene including a coding ce (e.g., SEQ ID NO: 19 or those nucleic acid ces that encode soluble ActRIIB polypeptides (e. g., c acids that encode SEQ ID N05: 17, 18, 23, 26, 27, 29, 30, 31, 32, 33, 36, 37, 42, and 43)) for one or more of the subject ActRIIB polypeptides can be used to produce ActRIIB polypeptides suitable for use in the methods and compositions described herein. The host cell may be any prokaryotic or eukaryotic cell. For e, an ActRIIB polypeptide may be expressed in bacterial cells such as E. coli, insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art. ingly, provided herein are methods of producing the B polypeptides used in the methods and compositions described herein. For example, a host cell transfected with an expression vector encoding an ActRIIB polypeptide can be cultured under riate conditions to allow expression of the ActRIIB polypeptide to occur. The ActRIIB polypeptide may be ed and isolated ?om a mixture of cells and medium containing the ActRIIB ptide. Alternatively, the ActRIIB polypeptide may be retained cytoplasmically or in a membrane ?action and the cells harvested, lysed and the protein isolated. A cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art.
The subject ActRIIB polypeptides can be isolated from cell culture medium, host cells, or both, using techniques known in the art for ing proteins, including ion-exchange chromatography, gel ?ltration chromatography, ultra?ltration, electrophoresis, immunoaf?nity puri?cation with antibodies speci?c for particular epitopes of the ActRIIB polypeptides and af?nity ation with an agent that binds to a domain fused to the ActRIIB polypeptide (e.g., a protein A column may be used to purify an ActRIIB-Fc ?Jsion). In a preferred embodiment, the ActRIIB polypeptide is a fusion protein containing a domain which facilitates its puri?cation.
In a preferred embodiment, ation is achieved by a series of colunm chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography, and cation exchange chromatography. The puri?cation could be completed with viral ?ltration and buffer exchange. As demonstrated herein, ActRIIB -hFc protein was puri?ed to a purity of >98% as determined by size exclusion chromatography and >95% as determined by SDS PAGE.
This level of purity was suf?cient to achieve desirable effects on bone in mice and an acceptable safety pro?le in mice, rats and non-human primates.
In another embodiment, a fusion gene coding for a puri?cation leader sequence, such as a poly-(His)/enterokinase cleavage site sequence at the N-terminus of the desired portion of the inant B polypeptide, can allow puri?cation of the expressed fusion protein by af?nity chromatography using a Ni2+ metal resin. The puri?cation leader sequence can then be subsequently d by treatment with enterokinase to provide the puri?ed ActRIIB polypeptide (e.g., see Hochuli et al., (1987) J. Chromatography 411:177; and Janknecht et al., PNAS USA 88:8972).
Techniques for making fusion genes are well known. Essentially, the joining of various DNA nts coding for different polypeptide sequences is performed in accordance with conventional techniques, ing blunt—ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, ?lling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid rable joining, and enzymatic ligation.
In another embodiment, the fusion gene can be synthesized by tional techniques including automated DNA synthesizers. Alternatively, PCR ampli?cation of gene fragments can be carried out using anchor primers which give rise to complementary overhangs n two consecutive gene fragments which can subsequently be annealed to generate a ic gene sequence (see, for example, t Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992).
ActRIIB -Fc ?lSlOIl n can be expressed in stably transfected CHO-DUKX B1 1 cells from a pAID4 vector (SV40 ori/enhancer, CMV promoter), using a tissue plasminogen leader sequence of SEQ ID N028. The Fc portion can comprise a human IgG1 Fc sequence, as shown in SEQ ID N027. In n embodiments, upon expression, the protein contained has, on average, between about 1.5 and 2.5 moles of sialic acid per molecule of ActRIIB-Fc fusion protein.
In certain embodiments, the long serum half—life of an ActRIIB-Fc fusion can be 25- 32 days in human patients. Additionally, the CH0 cell sed material can have a higher y for activin B ligand than that reported for an ActRIIB-hFc fusion protein expressed in human 293 cells (del Re et al., J Biol Chem. 2004 Dec l7;279(51):53l26-35). Additionally, without being bound by theory, the use ofthe TPA leader sequence provided greater production than other leader sequences and, unlike ActRIIB-Fc expressed with a native leader, may provide a highly pure N-terminal sequence. Use of the native leader ce may result in two major species of ActRIIB-Fc, each having a different N—terminal sequence. .5.3 Other ActRII Receptor Inhibitors In n embodiments, the tors of ActRII ors used in the compositions and methods described herein are nucleic acid compounds. es of categories ofnucleic acid compounds that inhibit ActRII receptors include antisense nucleic acids, siRNA or RNAi constructs and catalytic nucleic acid constructs.
A nucleic acid compound may be — or double—stranded. A double-stranded compound may also include regions of overhang or non—complementarity, where one or the other of the strands is single-stranded. A single-stranded compound may include regions of self-complementarity, meaning that the compound may form a so-called "hairpin" or "stem-loop" structure, with a region of double l structure.
In certain embodiments, the nucleic acid compounds that inhibit ActRII receptors may comprise a tide sequence that is complementary to a region consisting of no more than 1000, no more than 500, no more than 250, no more than 100 or no more than 50, 35, 30, , 22, 20 or 18 nucleotides of the full-length ActRII receptor nucleic acid sequence or activin c acid sequence (e.g., the nucleic acid sequence of an activin A or activin B subunit, also ed to as BA or 133). In specific embodiments, the region of complementarity will be at least 8 nucleotides, and optionally at least 10 or at least 15 nucleotides, and optionally between 15 and nucleotides. A region of complementarity may fall within an intron, a coding sequence or a noncoding ce of the target ript, such as the coding sequence portion. Generally, a nucleic acid compound that inhibits an ActRII receptor will have a length of about 8 to about 500 nucleotides or base pairs in , and optionally the length will be about 14 to about 50 nucleotides. A nucleic acid compound that inhibits an ActRII receptor may be a DNA (particularly for use as an antisense), an RNA, or an RNA2DNA hybrid. Any one strand may include a mixture ofDNA and RNA, as well as modi?ed forms that cannot readily be classi?ed as either DNA or RNA. Likewise, a double stranded nucleic acid compound may be DNAzDNA, DNA:RNA, or RNA:RNA, and any one strand may also include a mixture ofDNA and RNA, as well as modi?ed forms that cannot readily be classi?ed as either DNA or RNA.
The nucleic acid compounds that inhibit an ActRII receptor may include any of a variety of modi?cations, ing one or modi?cations to the backbone (the sugar-phosphate portion in a natural nucleic acid, including internucleotide linkages) or the base portion (the purine or pyrimidine portion of a natural nucleic acid). In certain embodiments, an antisense nucleic acid compound will have a length of about 15 to about 30 nucleotides and will often contain one or more modi?cations to improve certain characteristics, such as stability in the serum, stability in a cell, or stability in a place where the compound is likely to be delivered, such as, e.g., the stomach in the case of orally delivered compounds and the lung for inhaled compounds. In the case of an RNAi construct, the strand complementary to the target transcript will generally be RNA or modi?cations f. The other strand may be RNA, DNA, or any other variation. The duplex portion of double ed or single stranded "hairpin" RNAi construct may, in certain embodiments, have a length of 18 to 40 nucleotides in length and ally about 21 to 23 tides in length, so long as it serves as a Dicer substrate.
Catalytic or enzymatic nucleic acids may be ribozymes or DNA enzymes and may also contain modi?ed forms. In certain embodiments, nucleic acid nds that inhibit ActRII receptors may inhibit expression of their target by about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more under physiological conditions and at a tration where a nonsense or sense control has little or no effect. Concentrations for testing the effect of nucleic acid compounds include 1, 5, 10 micromolar, or more.
In other ments, the inhibitors of ActRII receptors used in the compositions and methods described herein are dies. Such dies e antibodies that bind to activin (particularly the activin A or B subunits, also referred to as BA or BB) and disrupt ActRII receptor g; and antibodies that bind to ActRII receptor polypeptides (e.g., a soluble ActRIIA or soluble ActRIIB polypeptide) and disrupt activin binding.
By using immunogens derived from an ActRII receptor polypeptide or an activin polypeptide, anti-protein/anti—peptide antisera or monoclonal antibodies can be made by standard protocols (see, for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)). A , such as a mouse, a hamster or rabbit can be immunized with an genic form ofthe ActRII receptor polypeptide, an antigenic fragment which is e of eliciting an antibody response, or a fusion n. Techniques for conferring immunogenicity on a n or peptide include conjugation to carriers or other techniques well known in the art. An immunogenic portion of an ActRII receptor or activin polypeptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies.
Following immunization of an animal with an antigenic preparation of an ActRII receptor polypeptide, antisera can be obtained and, if desired, onal antibodies can be isolated from the serum. To produce monoclonal antibodies, antibody-producing cells (lymphocytes) can be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells.
Such techniques are well known in the art, and include, for example, the hybridoma technique (originally developed by Kohler and Milstein, (1975) Nature, 256: 495-497), the human B cell oma technique (Kozbar et al., (1983) logy Today, 4: 72), and the EBV-hybridoma que to e human monoclonal antibodies (Cole et al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96). Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with an ActRII receptor ptide and onal antibodies ed from a culture comprising such hybridoma cells.
The term "antibody" as used herein is intended to include fragments thereof which are also speci?cally reactive with a subject polypeptide. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab)2 fragments can be ted by ng antibody with pepsin. The resulting F(ab)2 fragment can be treated to reduce disul?de bridges to produce Fab fragments. An antibody is further intended to include bispeci?c, single-chain, chimeric, humanized and fully human molecules having af?nity for an ActRII receptor or activin polypeptide conferred by at least one CDR region of the antibody. An antibody may further comprise a label ed thereto and able to be detected (e.g., the label can be a radioisotope, cent compound, enzyme or enzyme co-factor).
In certain embodiments, the antibody is a recombinant antibody, which term encompasses any dy generated in part by techniques of molecular biology, including CDR- grafted or chimeric antibodies, human or other antibodies assembled from library-selected antibody domains, single chain antibodies and single domain antibodies (e.g., human VH proteins or camelid VHH proteins). In certain embodiments, an antibody can be a monoclonal antibody, and in certain embodiments. For e, a method for generating a monoclonal antibody that binds speci?cally to an ActRII receptor polypeptide or activin polypeptide may comprise administering to a mouse an amount of an immunogenic composition comprising the antigen polypeptide effective to ate a detectable immune response, obtaining antibody-producing cells (e. g., cells from the spleen) from the mouse and fusing the antibody-producing cells with myeloma cells to obtain dy-producing hybridomas, and testing the antibody-producing hybridomas to identify a hybridoma that produces a monoclonal antibody that binds speci?cally to the antigen. Once ed, a hybridoma can be propagated in a cell culture, optionally in culture conditions where the hybridoma—derived cells e the monoclonal antibody that binds speci?cally to the n. The monoclonal antibody may be puri?ed from the cell culture.
] The adjective "speci?cally reactive wit " as used in nce to an antibody is intended to mean, as is generally understood in the art, that the antibody is suf?ciently selective between the antigen of interest (e.g., an ActRII receptor polypeptide) and other antigens that are not of interest that the dy is useful for, at minimum, detecting the presence of the antigen of interest in a particular type of biological sample. In certain methods employing the antibody, such as therapeutic applications, a higher degree of city in binding may be desirable.
Monoclonal antibodies generally have a greater tendency (as compared to polyclonal antibodies) to discriminate ively between the desired antigens and cross-reacting polypeptides. One characteristic that in?uences the city of an antibodyzantigen interaction is the af?nity of the antibody for the antigen. Although the desired speci?city may be reached with a range of different af?nities, generally red antibodies will have an af?nity (a dissociation constant) of about 106, 107, 10's, 10'9 or less. Given the extraordinarily tight binding between activin and an ActRII or, it is expected that a neutralizing anti-activin or ctRII receptor antibody would generally have a dissociation constant of 10'10 or less.
In addition, the techniques used to screen antibodies in order to identify a desirable antibody may in?uence the properties of the antibody obtained. For example, if an antibody is to be used for binding an n in solution, it may be desirable to test on binding. A variety of different techniques are available for testing interaction between dies and antigens to identify particularly desirable antibodies. Such ques include ELISAs, surface plasmon resonance binding assays (e.g., the Biacore.TM. binding assay, Biacore AB, Uppsala, Sweden), sandwich assays (e.g., the paramagnetic bead system of IGEN International, Inc., Gaithersburg, Md), Western blots, immunoprecipitation assays, and immunohistochemistry.
In certain embodiments, ActRII receptor inhibitors to be used in the compositions and methods described herein include ative forms of activin, particularly those with alterations in the type I receptor g domain can bind to type 11 receptors and fail to form an active ternary complex. In certain embodiments, nucleic acids, such as nse molecules, siRNAs or ribozymes that inhibit activin A, B, C or E, or, particularly, ActRII receptor sion, can be used in the compositions and s described herein. In certain embodiments, the ActRII receptor inhibitors to be used in the compositions and methods described herein exhibit selectivity for inhibiting GDFl l—mediated signaling versus other members of the TGF-beta family, particularly with t to GDF8 and activin.
In other embodiments, the inhibitors of ActRII receptors used in the compositions and methods described herein are non-antibody proteins with ActRII receptor antagonist ty, including inhibin (i.e., inhibin alpha subunit), follistatin (e. g., follistatin-288 and follistatin-315), Cerberus, follistatin related protein "), endoglin, activin C, alpha(2)-macroglobulin, and an MlO8A (methionine to alanine change at position 108) mutant activin A.
In a speci?c embodiment, the ActRII receptor inhibitor to be used in the compositions and methods described herein is a follistatin polypeptide that antagonizes activin bioactivity and/or binds to activin. The term "follistatin polypeptide" includes polypeptides comprising any lly occurring polypeptide of follistatin as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity, and further includes any functional monomer or er of tatin. Variants of follistatin polypeptides that retain activin binding properties can be identi?ed based on previous studies involving tatin and activin interactions. For example, WO2008/030367, which is included by reference herein in its entirety, discloses speci?c follistatin domains ("FSDs") that are shown to be important for activin binding. tatin polypeptides include polypeptides derived from the sequence of any known follistatin having a sequence at least about 80% identical to the sequence of a follistatin polypeptide, and optionally at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity.
Examples of follistatin polypeptides include the mature follistatin ptide or r ms or other ts of the human follistatin precursor polypeptide as described, for e, in W02005/025601, which is ed by reference herein in its entirety.
In a speci?c embodiment, the ActRII receptor inhibitor to be used in the itions and s described herein is a follistatin-like related gene (FLRG) that nizes activin bioactivity and/or binds to activin. The term "FLRG polypeptide" includes polypeptides comprising any naturally occurring polypeptide of FLRG as well as any ts thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity.
Variants of FLRG polypeptides that retain n binding properties can be identi?ed using e methods to assay FLRG and activin interactions. See, for example, US. Pat. No. 6,537,966, which is ed by reference herein in its entirety. FLRG polypeptides include polypeptides derived from the sequence of any known FLRG having a sequence at least about 80% identical to the sequence of an FLRG polypeptide, and optionally at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity.
In certain embodiments, functional variants or modi?ed forms of the follistatin polypeptides and FLRG polypeptides include fusion proteins having at least a portion of the tatin ptides or FLRG polypeptides and one or more fusion domains, such as, for example, domains that facilitate isolation, detection, stabilization or multimerization of the polypeptide. Suitable fusion domains are discussed in detail above with reference to the ActRIIA and ActRIIB polypeptides. In one ment, an ActRII receptor inhibitor is a fusion n comprising an activin binding portion of a follistaton polypeptide fused to an Fc domain.
In another embodiment, an ActRII receptor inhibitor is a fusion protein comprising an activin binding portion of an FLRG polypeptide fused to an Fc domain. .6 ASSAYS Various ActRII polypeptide variants, or soluble ActRII polypeptide variants, may be tested for their ability to inhibit ActRII. In addition, compounds can be tested for their ability to inhibit ActRII. Once inhibitors of ActRII activity are con?rmed, these nds can be used with the methods provided herein. ActRII can be ActRIIa or ActRIIb. The assays below are described for ActRIIa but can be performed analogously for ActRIIb. .6.1 RED BLOOD CELL LEVELS RBC count is a count of the actual number of red blood cells per volume of blood and may be included as part of a standard complete blood count. Normally, males have a RBC count of between 4.7 to 6.1 million cells per microliter and s have a RBC count of between 4.2 to 5.4 million cells per microliter. r, thalassemia patients may have a RBC count lower than that normally seen. Thus, determination of the RBC count in an anemia (e.g., thalassemia) patient being d in ance with the methods provided herein allows for the determination of the ef?cacy of such ent. .6.2 ERYTHROID COLONY-FORMING UNITS (CFU-E) CFU-e can be assayed and identi?ed, e.g., in a colony formation assay by the number and morphology of the cells and by the presence or absence of certain cell surface markers.
Levels of erythroid colony—forming units can be measured using, e.g., dy staining followed by ?ow cytometry analysis (FACs) to evaluate expression ofmarkers, such as entiation state markers, e.g., Epo receptor, c—Kit (Stem cell factor receptor), transferring receptor (CD71+), CD36 and Ter119 (glycophorin—A associated antigen) (CFU-e cells are Ter119 (glycophorin-A associated antigen)—negative (see, e.g., Terszowsky et al., 2005). Cells at the CFU-e stage express erythropoietin receptor (EpoR) and can be induced to terminally differentiate in vitro in 2-3 days in the ce of only erythropoietin in a culture media. CFU-e cells can be plated on methylcellulose and stained with obenzidine reagent for hemoglobin, and then CFU-e colonies can be counted. By day 2 from the time of plating, each CFU-e colony can yield between 8 and 64 hemoglobinized cells most of which are in their end- stage of erythroid differentiation.
Colony-forming unit assays are known in the art (e.g., MesenCultTM medium, Stem Cell Technologies Inc., ver British Columbia; see also Wu et a1. (Wu H, Liu X, Jaenisch R, Lodish HF . "Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor". Cell 83 (1): 59—67; Marley SB, Lewis JL, Goldman JM, Gordon MY (1996)). .6.3 ERYTHROID BURST-FORMING UNITS (BFU-E) Similarly to CFU-e, BFU-e can be assayed and identi?ed, e. g., in a colony formation assay by the number and morphology of the cells and by the presence or absence of certain cell surface markers. Speci?cally, BFU—e can be identi?ed by the expression of several cell surface markers such as CD33, CD34 and HLA—DR, and lack of expression of glycophorin-A. For example, BFU-e assays described in Wu et a1. can be utilized (Wu H, Liu X, Jaenisch R, Lodish HF . "Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor". Cell 83 (1): 59—67). .6.4 HEMATOCRIT A hematocrit es the percentage of red blood cells in a given volume of whole blood and may be included as part of a standard complete blood count. The hematocrit is normally about 45% for men and about 40% for women. However, thalassemia patients typically have a hematocrit lower than that normally seen. Thus, determination of the hematocrit in a thalassemia patient being treated in accordance with the methods provided herein allows for the determination of the ef?cacy of such treatment. .6.5 URINARY N-TELOPEPTIDE (uNTX) Urinary N-telopeptide of type I collagen (NTx) can be measured in using, e.g., an automated immunoassay (Vitros ECi; Ortho Clinical, Rochester, NT, USA). .6.6 SERUM BONE IC ALKALINE PHOSPHATASE (BSAP) Serum bone speci?c alkaline phosphatase (BSAP) levels can be measured using, e.g., an enzyme immunoassay. .6.7 SIS OF OID PROGENITORS Apoptosis of Erythroid progenitors can be determined, e.g., by using Terminal deoxynucleotidyltransferase-mediated dUTP nick end—labeling (TUNEL) ng. TUNEL staining can be performed using an in situ Apoptosis ion Kit (Takara Bio, Otsu, Japan). .6.8 ERYTHROID CO-CULTURE SYSTEM To test the effect of an agent on erythroid entiation in an in Vitro environment more analogous to the in Vivo environment, a co-culture system of bone marrow cells and human CD36+ cells can be used. Human CD36+ cells, which are highly enriched for erythroid progenitor cells, are tured with long-term bone marrow cultures in erythropoietin-(EPO) supplemented media (2U/mL). After 6 days, the cellular output (e.g., cell type) of the culture can be ed by, for example, ?ow cytometry (e.g., FACS) analysis. The numbers of erythroid cells at the various levels of erythroid differentiation (e. g., proerythroblast, basophil, late basophilic/polychromatic, orthochromatic/reticulocytes, glycoprotein A+ cells) indicate the ability of the agent being tested to te erythroid differentiation. .6.9 TRANSCRIPTIONAL RESPONSE ASSAY In certain embodiments, a transcription response assay can be used to test an activin type II or inhibitor or activity of GDP] 1. Upon ActRII and GDFll ing, transcription of certain genes is up- or downregulated. A cell culture system used and the transcriptional response can be measured (e.g., by RT-PCT). The effect of an agent on the transcriptional se is a e of its effectiveness or activity. In certain embodiments, the promoter region that is known to be responsive to ActRII or GDFll signaling can be cloned am of a reporter gene. In this way, the assay can be simpli?ed such that only the activity of the reporter gene need to be assayed. .7 DOSE OF ACTIVIN RECEPTOR TYPE 11 INHIBITOR In certain embodiments, an ActRII inhibitor is an inhibitor of ActRIIa as set forth in Section 5.5. 1. In other embodiments, an ActRII inhibitor is an inhibitor of ActRIIb as set forth in Section 5.5.2 In certain embodiments, an ActRII tor is a combination of an ActRIIa tor and an ActRIIb inhibitor.
In certain embodiments, a therapeutically effective amount of an ActRII inhibitor is sufficient to ameliorate one symptom of anemia. In certain embodiments, a therapeutically effective amount of an ActRII inhibitor is suf?cient to prevent at least one symptom of anemia from ing. In certain embodiments, a therapeutically effective amount of an ActRII inhibitor increases the red blood cell level, hemoglobin levels, hematocrit levels, and/or Ery-C in the patient.
In certain embodiments, the ActRII inhibitor is dosed at intervals and amounts suf?cient to achieve serum trations of 0.2 microgram/kg or greater, and serum levels of 1 ram/kg or 2 microgram/kg or greater are desirable for achieving cant effects on bone density and strength. Dosing ns may be designed to reach serum concentrations of between 0.2 and 15 microgram/kg, and optionally between 1 and 5 microgram/kg. In humans, serum levels of 0.2 microgram/kg may be achieved with a single dose of 0.1 mg/kg or r and serum levels of l microgram/kg may be achieved with a single dose of 0.3 mg/kg or greater.
The observed serum half-life of the molecule is between about 20 and 30 days, substantially longer than most Fc fusion proteins, and thus a sustained effective serum level may be achieved, for example, by dosing with 4 mg/kg on a weekly or biweekly basis, or higher doses may be used with longer intervals between dosings. For example, doses of 1-3 mg/kg might be used on a monthly or bimonthly basis, and the effect on bone may be iently durable that dosing is necessary only once every 3, 4, 5, 6, 9, 12 or more months. Serum levels of the ActRII inhibitor can be measured by any means known to the skilled artisan. For example, antibodies against the ActRII inhibitor can be used to determine the serum levels of the ActRII inhibitor using, e.g., an ELISA.
In certain embodiments, the dose of the ActRII inhibitor ranges from 0.01 to 3.0 mg/kg intravenously or from 0.03 to 0.1 mg/kg subcutaneously. In certain embodiments, the dose of ActRII inhibitor is about 0.01 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 1.5 mg/kg, about 2.0 mg/kg, about 2.5 mg/kg, about 3.0 mg/kg, about 3.5 mg/kg, about 4.0 mg/kg, about 4.5 mg/kg, or about .0 mg/kg. In certain embodiments, the dose of ActRII inhibitor is about 10.0 mg/kg, about 15.0 mg/kg, about 20.0 mg/kg, about 25.0 mg/kg, or about 30.0 mg/kg. In n embodiments, the dose of ActRII inhibitor is between 0.01 mg/kg and 0.1 mg/kg, n 0.1 mg/kg and 0.3 mg/kg, between 0.3 mg/kg and 0.5 mg/kg, between 0.5 mg/kg and 1.0 mg/kg, between 1.0 mg/kg and 2.0 mg/kg, between 1.0 mg/kg and 3.0 mg/kg, n 2.0 mg/kg and 3.0 mg/kg, between 2.0 mg/kg and 4.0 mg/kg, between 3.0 mg/kg and 5.0 mg/kg, between 5.0 mg/kg and 10.0 mg/kg, between 10.0 mg/kg and 15.0 mg/kg, between 10.0 mg/kg and 20.0 mg/kg, between 15.0 mg/kg and 20.0 mg/kg, or between 20.0 mg/kg and 30.0 mg/kg. When used in conjunction with a dose provided herein (e.g., a dose of an ActRII inhibitor or a dose of a second active agent), the word "about" refers to any number within 1, 5 or 10% of the referenced number. .8 PHARMACEUTICAL COMPOSITIONS In certain embodiments, activin-ActRII antagonists (e.g., ActRII polypeptides) are formulated with a pharmaceutically acceptable carrier for use with the methods bed .
For example, an ActRII polypeptide can be administered alone or as a component of a pharmaceutical formulation (therapeutic ition). The subject compounds may be formulated for administration in any convenient way for use in human or veterinary medicine.
ActRII can be a or b.
In certain embodiments, the therapeutic methods provided herein include administering the composition (comprising an ActRII inhibitor) systemically, or locally as an t or device. When administered, the eutic composition for uses provided herein is in a pyrogen-free, physiologically acceptable form. Therapeutically useful agents other than the ActRII antagonists which may also optionally be included in the composition as described above, may be administered simultaneously or sequentially with the t compounds (e.g, ActRII polypeptides, such as ActRIIa and / or ActRIIb polypeptides (see Section 5.2)).
Typically, ActRlI nists will be stered parenterally. Pharmaceutical compositions suitable for parenteral administration may comprise one or more ActRII polypeptides in combination with one or more ceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be tituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, iostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions for use in the methods described herein include water, ethanol, s (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper ?uidity can be maintained, for example, by the use of coating materials, such as in, by the nance of the required particle size in the case of dispersions, and by the use of surfactants.
Further, the composition may be encapsulated or injected in a form for ry to a target tissue site (e.g., bone). In certain embodiments, compositions for use in the methods described herein may include a matrix e of delivering one or more therapeutic compounds (e.g., ActRIIa polypeptides) to a target tissue site (e.g., bone), providing a structure for the developing tissue and optimally capable of being ed into the body. For example, the matrix may provide slow release of the ActRIIa polypeptides. Such matrices may be formed of materials presently in use for other implanted medical applications.
The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and ace properties. The particular application of the subject compositions will define the riate formulation. Potential matrices for the compositions may be biodegradable and ally de?ned calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid and polyanhydrides. Other potential materials are biodegradable and biologically well de?ned, such as bone or dermal collagen.
Further matrices are comprised of pure proteins or extracellular matrix components. Other potential matrices are non-biodegradable and chemically de?ned, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalciumphosphate. The bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, le size, particle shape, and radability.
In certain embodiments, the compositions for use in the methods described herein (comprising ActRII inhibitor) can be administered orally, e.g., in the form of capsules, cachets, pills, tablets, lozenges (using a ?avored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a sion in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of an agent as an active ingredient. An agent may also be administered as a bolus, electuary or paste.
In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, and the like), one or more therapeutic compounds described herein may be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) ?llers or extenders, such as starches, lactose, e, glucose, mannitol, and/or silicic acid; (2) s, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3) ants, such as glycerol; (4) disintegrating agents, such as gar, m carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium ate; (5) solution retarding agents, such as paraf?n; (6) absorption rators, such as quaternary um compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring . In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a r type may also be ed as ?llers in soft and hard—filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight hylene s and the like.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and s. In addition to the active ient, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other ts, solubilizing agents and emulsi?ers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl l, benzyl benzoate, propylene glycol, 1,3- butylene glycol, oils (in particular, seed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of an, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, ng, coloring, perfuming, and preservative agents.
Suspensions, in on to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol, and sorbitan esters, rystalline cellulose, aluminum metahydroxide, ite, agar-agar and tragacanth, and mixtures thereof.
The compositions described herein may also n adjuvants, such as vatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, n, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium de, and the like into the compositions. In addition, prolonged absorption of the injectable ceutical form may be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.
It is understood that the dosage regimen will be determined by the attending ian considering various factors which modify the action of the compounds described herein (e.g., ActRII polypeptides, such as ActRIIa and / or ActRIIb polypeptides (see Section .2)). The various factors include, but are not limited to, amount of bone weight desired to be formed, the degree of bone density loss, the site of bone damage, the condition of the damaged bone, the t’s age, sex, and diet, the severity of any disease that may be contributing to bone loss, time of administration, and other clinical factors. Optionally, the dosage may vary with the type of matrix used in the reconstitution and the types of compounds in the composition. The addition of other known growth factors to the ?nal composition, may also affect the dosage.
Progress can be monitored by periodic assessment of bone growth and/or repair, for example, X- rays (including DEXA), histomorphometric determinations, and tetracycline labeling.
In certain ments, provided herein is gene therapy for the in vivo tion of ActRII polypeptides. Such therapy would achieve its therapeutic effect by introduction of the ActRII cleotide sequences into cells or s having the ers as listed above.
Delivery of ActRII polynucleotide sequences can be achieved using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system. Preferred for therapeutic delivery of ActRII cleotide sequences is the use of targeted liposomes. The ActRII polypeptides can be ActRIIa and / or ActRIIb polypeptides (see n 5.2)).
Various viral vectors which can be utilized for gene therapy as taught herein include adenovirus, herpes Virus, vaccinia, or, preferably, an RNA virus such as a irus. Preferably, the retroviral vector is a tive of a murine or avian retrovirus. Examples of retroviral s in which a single foreign gene can be inserted include, but are not d to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). A number of additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a able marker so that transduced cells can be identified and generated. Retroviral vectors can be made target-specific by attaching, for example, a sugar, a glycolipid, or a protein.
Preferred targeting is accomplished by using an antibody. Those of skill in the art will recognize that speci?c polynucleotide sequences can be inserted into the retroviral genome or attached to a viral envelope to allow target specific delivery of the retroviral vector containing the ActRII polynucleotide. In a preferred embodiment, the vector is targeted to bone or cartilage.
Alternatively, tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, pol and env, by conventional calcium phosphate transfection.
These cells are then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector into the culture medium.
Another targeted delivery system for ActRII polynucleotides is a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed es, and liposomes. The preferred colloidal system for use in the methods described herein is a liposome. Liposomes are ial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. RNA, DNA and intact s can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (see e.g., Fraley, et al., Trends Biochem. Sci., 6:77, 1981). s for ef?cient gene transfer using a me vehicle, are known in the art, see e.g., Mannino, et a1., Biotechniques, 6:682, 1988. The composition of the liposome is usually a combination of phospholipids, usually in ation with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic th, and the presence of divalent cations.
Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, olipids, cerebrosides, and gangliosides. Illustrative olipids include egg phosphatidylcholine, itoylphosphatidylcholine, and distearoylphosphatidylcholine. The targeting of liposomes is also possible based on, for example, organ-speci?city, cell—speci?city, and organelle—speci?city and is known in the art.
In certain embodiments, the ActRII inhibitor is substantially pure in a pharmaceutical composition. Speci?cally, at most 20%, 10%, 5%, 2.5%, 1%, 0.1%, or at most 0.05% of the compounds in the pharmaceutical composition are compounds other than the ActRII tor and the pharmaceutical able carrier. 6. EXAMPLE ] The example presented herein demonstrates that GDFll protein levels are elevated in thalassemia and that inhibition of GDFll can treat anemia in a mouse model of beta-thalassemia.
Accordingly, the example ed herein demonstrates that GDFll can be used as a biomarker for thalassemia, and that methods of treating anemia such as thalassemia can assess 6.1 AN ACTRIIA DECOY TREATS beta-THALASSEMIA Beta-thalassemia is associated with ctive erythropoiesis, accelerated erythoid differentiation and sis, resulting in anemia and iron overload. The molecular mechanism underlying the effects of ineffective erythropoiesis is incompletely understood. Although members of the TGF-beta superfamily are implicated in both proliferation and differentiation of erythroid progenitor cells, the role of the numerous TGF-beta family members in the ineffective erythropoiesis seen in halassemia is unknown.
To assess the role of TGF-beta family members in the ctive erythropoiesis of beta-thalassemia, a recombinant fusion protein that binds a number of TGF-beta superfamily ligands was used in a mouse model of human beta—thalassemia. The ActRIIA-Fc ?tsion protein (i.e., the murine counterpart of SEQ ID N027) consists of the extracellular domain of Activin Receptor IIA IA) linked to a human immunoglobulin 1 (IgGl) Fc domain and the protein acts as a ligand trap for TGF-beta family members like activin A, activin B, growth differentiation factor-11 (GDFl 1) and bone morphogenetic protein-10 (BMP- 1 0). bethl/thl is a mouse model of human beta-thalassemia, the mice having a naturally occurring deletion of the ajor gene (Skow et al., 1983). bethl/thl mice have abnormally low hemoglobin (Hgb), hemocrit (Hct) and mean cell volume (MCV) as well as bone marrow (BM) hypercellularity and ally high levels of bilirubin, a by-product of hemoglobin own that represents extensive red blood cell destruction. 6.2 MATERIALS AND METHODS 6.2.1 Mice C57BL/6 were bred and housed in the pathogen—free facilities of INSERM U699. All protocols were approved by the Animal Care Committee of . The bethl/thl model was ated from a natural occurring deletion of the B—major gene (Skow LC et al; Cell 1983). thl mice constitute a model of B—thalassemia intermedia. These mice have several clinical parameters reproducing human B—thalassemia such as ineffective bone marrow erythropoiesis, precursor apoptosis, parenchymal iron distribution, decreased hepcidin expression and lower bone marrow iron levels while levels in the liver and spleen are increased. 6.2.2 Complete blood counts Blood samples were collected in EDTA—coated tubes and complete blood counts were measured on a MS9-5 Blood Analyzer (Melet Schloesing Laboratories) according to the manufacturer's ctions. The selected parameters were red blood cells (RBC), hematocrit (Ht), mean corpuscular volume (MCV), hemoglobin (Hb). Reticulocyte numbers were determined with retic-count reagent (BD Biosciences Retic-CountTM Kit). 6.2.3 Quantitative real-time RT—PCR RNA was extracted from oid itors using RNeasy Plus Mini Kit (Qiagen).
One microgram of total RNA was used for reverse transcription using iScript reverse transcription ix (Bio—rad) during 30 minutes at 42°C. The enzyme was then inactivated at 85°C for 5 minutes. For qPCR, the cDNA samples were ed in a CFX96 PCR System (Bio-rad). The PCR products were quanti?ed using SsoFast EvaGreen Supermix (Bio-rad). 6.2.4 In vitro erythroblast cultures Cells from each tissue were resuspended in serum-free "erythroid expansion media" consisting in either StemPro34 plus nutrient supplement (Life Technologies Gibco-BRL) supplemented with 2 U/mL human recombinant Epo (Roche), 100 ng/mL SCF Tech), —6 M dexamethasone (D2915; Sigma), 40 and penicillin/streptomycin (Pen/Strep; Invitrogen).
After 5 days of e, the nonadherent cells were transferred to the differentiation medium (StemPro-34 supplemented with lU/ml Epo and 1mg/ml ferro-transferrin (Sigma)) during two to three days supplemented or not with 10ug/ml of mActRIIA-Fc. Live and dead cell numbers were determined daily by Trypan Blue (Gibco/BRL) exclusion, and cell concentration was adjusted to 2 X 106 total cells/mL daily through partial medium changes. 6.2.5 Methylcellulose assays Single cell suspensions of adult mouse BM or spleen were mixed with ult M3434 medium (Stem Cell Technologies), plated into 35 mm dishes and cultured at 37°C under a 5% C02 humidified atmosphere. The BFU—E colonies were scored at day 10 (in some experiments mouse BFU-E were scored from day 7 onward to day 10 and there was no ence between colony s at day 7 and at day 10). 6.2.6 Statistical analyses Statistical analyses were performed with ad Prism (version 5.0; GraphPad Software). The data are expressed as the mean i SEM ofN inations unless noted otherwise. Student's t-test or the Mann-Whitney test were used to compare two groups, whereas multigroup comparisons were made using two-way ANOVA test followed by post-hoc analysis (Bonferroni test). Differences were considered signi?cant at a P value less than 0.05 (*), less than 0.01 (**) or less than 0.001 (***). 6.2.7 Immuno?uorescence analysis by ?ow cytometry For bone marrow (BM) and splenocyte sions from mice, blocking of IgG receptors was performed with anti-chammaR mAb 2.4G2. Cells (1 x 106) were then stained with anti-TER—l 19 dy and anti-mouse Tle antibody. Stained cells were further analyzed by ?ow cytometry (FACScalibur; Becton Dickinson) using FlowJo software (Tree Star). 6.2.8 Tissue collection and histology ] Bone marrow and spleens were collected and ?xed in 10% formalin, paraf?n embedded, and sectioned at 3—6 um for hematoxylin and eosin (H&E) staining. 6.2.9 Iron, in, bilirubin and transferrin quanti?cation.
Blood was drawn into nized tubes and centrifuged (5 min, 4 °C, 1,100g).
Plasma was de novo centrifuged (5 min, 4 OC, 1,100g) to remove contaminating red blood cells.
Biochemical parameters were quanti?ed with an Olympus AU400 automat according to the manufacturer's instructions. 6.3 RESULTS 6.3.1 mActRIIA—Fc ameliorates hematological parameters in thalassemic mice. beta-thalassemia is a e characterized by a defect in obin synthesis leading to impaired erythrocyte tion and production. The se in erythrocytes is primarily thought to be due to abnormally accelerated erythroid differentiation and apoptosis at the late basophilic/polychromatic oblast stage of red blood cell differentiation that results in an overall decrease in mature red blood cell production. The disease is characterized by a hypercellular bone marrow compartment in which abnormal erythroblasts accumulate and undergo apoptosis, resulting in systemic anemia.
] To examine the role of TGF-BETA ligands in the disease mechanism of beta- thalassemia, bethl/thl mice were subcutaneously treated with mActRIIA-Fc (the murine counterpart of SEQ ID N027) or PBS for 0, 5, 10, 30 or 60 days (lOmg/Kg body weight) twice a week (*p <0.05, N=3-5 for each independent experiment). Compared to PBS-treated animals, treatment with mActRIIA-Fc signi?cantly increased red blood cell counts (FIG 1A) and hemocrit (FIG 1B) and hemoglobin (FIG 1C) levels, with a concomitant decrease in reticulocyte counts (FIG ID) (from 10 days reatment until day 60). Analysis of circulating red blood cell (RBC) parameters also showed that mean cular volume (MCV) (FIG 1E), mean corpuscular hemoglobin (MCH) (FIG 1F) and MCH concentration (MCHC) (FIG 1G) all increased in all mice treated with mActRIIA-Fc, suggesting that IA-Fc improved the microcytic anemia of thalassemia and restored hemoglobin content per RBC. In addition, bone marrow and spleen cellularity and late basophilic/polychromatic erythroblasts were reduced signi?cantly following treatment with mActRIIA—Fc. Morphological analysis of erythrocytes was evaluated by May-Griinwald (MGG) staining and showed a reduction in anisocytosis, poikilocytosis and target cells (FIG 11) To determine the effect of mActRIIA-Fc on the anemia associated with beta-thalassemia, systemic iron levels (FIG 1]), transferrin synthesis (FIG 1K), transferrin tion (FIG 1L) and ferritin levels (FIG 1M) were evaluated on thalassemic mice.
Transferrin saturation was reduced with the induction of transferrin synthesis or decreased systemic iron levels (FIG 1L). Platelet, monocyte, lymphocyte and neutrophil levels were also assessed (FIG IN).
The effects of mActRIIA-Fc on splenomegaly in thalassemic mice was ted by spleen weight and ing total spleen cell number. Spleen cell number and spleen weight were reduced in animals treated with mActRIIA-Fc compared to PBS-treated thalassemic mice (FIG 10). Similarly, bone marrow erythroblasts numbers and expansion (as determined by eosin/hematoxylin staining) (FIG 1P) were also decreased in mActRIIA-Fc-treated mice. Bone marrow and spleen were harvested and erythroblasts were quanti?ed by ?ow cytometry by TERl l9 staining. Treatment with mActRIIA—Fc signi?cantly reduced the number of erythrocytes in thalassemic mice (FIG lQ), indicating that it corrected the ineffective erythropoiesis in the mice. 6.3.2 mActRIIA-Fc reduces ineffective erythropoiesis in thalassemic mice.
To r e the role of TGF-BETA superfamily s in ineffective erythropoiesis in beta-thalassemia, spleen (FIG 2A, FIG 2C) and bone marrow (FIG 2B, FIG 2C) were harvested and erythroblast differentiation ted by ?ow cytometry by ER119 staining and forward scatter/side scatter SC) distribution. A time course analysis of the percent of cells at progressive stages in erythropoietic entiation, Proerythroblast (Pro-E), Basophil erythroblasts (Ery-A), late basophilic (Ery—B) and polychromatic erythroblasts and orthochromatic erythroblasts ), showed that mice treated with mActRIIA-Fc presented signi?cantly decreased immature TER119/CD71 cells (late basophilic and polychromatic erythroblasts, Ery-B) in spleen with a itant increase in the percentage of orthochromatic oblasts (Ery-C) (FIG 2A). These results were in accordance with mActRIIA-Fc reduction of ineffective erythropoiesis. Although there was a decrease in TER-199+ erythroblasts and in Ery-B s in bone marrow from mice treated with mActRIIA-Fc (FIG 2B), there was no increase in the amount of mature erythroblasts suggesting that ineffective erythropoiesis in the bone marrow of thalassemic mice is not corrected by treatment of mice with mActRIIA-Fc.
Chronic anemia of thalassemia induces stress erythropoiesis compensatory responses.
However, these responses are unproductive because of ineffective erythropoiesis. Ineffective opoiesis is characterized by a requirement of RBC production which cannot be compensated by an accelerated proliferation and differentiation of immature erythroblasts due to the premature cell death by apoptosis of maturing cells. Therefore, imbalanced re/mature erythroblasts ratio is a feature of ineffective erythropoiesis of thalassemia. To further study the impact of mActRIIA-Fc on erythroid differentiation and ineffective erythropoiesis, cell suspensions from mActRIIA-Fc-treated mice and their respective controls were labeled with antibodies to Tle and TER119. Erythroid precursors differentiation was analyzed by ?ow cytometry in TERl l9high gate as previously described (Liu et al., Blood 2006). Accordingly, mActRIIA-Fc-treated mice presented a decreased ratio of immature/mature erythroblasts ratio in spleen indicating the correction of ineffective stress erythropoiesis. In bone marrow the immature/mature erythroblasts ratio did not differ between control and mActRIIA-Fc-treated mice further suggesting that ineffective erythropoiesis in the bone marrow of thalassemic mice was not corrected by mActRIIA-Fc treatment. These s suggest that ActRIIa ligands contribute to erythroblast differentiation but also to ctive spleen erythropoiesis in B- thalassemia.
Bilirubin is a t of hemoglobin ation and increased plasma bilirubin resulting from hemolysis is a feature of ineffective erythropoiesis in assemia 18. In time- course analysis, serum levels of total and direct bilirubin were decreased in thalassemic mice treated with mActRIIA-Fc from 5 days oftreatment ting that hemolysis ing from ineffective erythropoiesis was affected by mActRIIA-Fc administration (FIG 2D). In ent with bilirubin values, levels of serum lactate dehydrogenase (LDH) were also reduced in s treated with mActRIIA-Fc compared to controls after 60 days of treatment (FIG 2D), further con?rming that tissue hemolysis was reduced in mice d with mActRIIA-Fc. tage erythropoiesis is largely committed to the production of the oxygen carrier hemoglobin (Hb), a eric protein composed of two u-globin and two B-globin subunits. [3- thalassemia is a common inherited hemoglobinopathy characterized by impaired or absent [3- globin gene production with subsequent accumulation of unpaired nits. The excess of unbound free (x-globin in maturing erythroid cells precipitates and leads to the production of reactive oxygen species (ROS) and cellular oxidative stress damage inducing premature death of erythroid precursors. The impact of mActRIIA—Fc on the generation of globin precipitates was further investigated. ROS tion on primary pro-erythroblast differentiation was evaluated by ?ow cytometry using dichlorodihydro?uorescein (FIG 2E). Analysis of hemoglobin solubility on primary thalassemic pro-erythroblasts treated for 48 hours with mActRIIA-Fc or PBS (FIG 2F).
To gain insight into the cellular mechanisms associated with treatment with mActRIIA-Fc, spleen-derived proeryhtoblasts were cultured and red from bethl/thl mice in the presence or in the e of mActRIIA-Fc. A well-established in vitro model of pro-erythroblast differentiation was used (spleen precursors were cultured for 5 days in serum free stem cell expansion medium mented with mouse Stem Cell Factor, Epo and dexamethasone). These pro-erythroblast-enriched cultures were then differentiated in the presence of lU/ml Epo and lmg/ml Fe—Tf for three days. Similar to in vivo observations, treatment with mActRIIA-Fc increased total amounts of hemoglobin in thalassemic erythroblasts. However, those cells presented decreased amounts of membrane-associated precipitated hemoglobin when compared to control treated cells (FIG 2F). Accordingly, the amount of reactive oxygen species (ROS) detected on those cells decreased in cells treated with mActRIIA-Fc (FIG 2E). Therefore, treatment with mActRIIA-Fc resulted in decreased xic globin precipitation and its associated ROS production, contributing to ineffective erythropoiesis.
Altogether, these data ted that targeting ActRIIa signaling modulated oblast differentiation and trapping of ActRIIa ligands by mActRIIA-Fc corrected ineffective erythropoiesis by favoring the formation ofmature erythroblasts and reducing membrane- associated obin precipitates. 6.3.3 mActRIIA-Fc tes apoptosis in thalassemic mice.
The involvement of TGF-beta family members on the apoptotic process in beta- semia-associated ineffective erythropoiesis was investigated by analyzing the expression of pro-apoptotic proteins on erythrocytes using ?ow try. Although there were no signi?cant changes in the expression of the apoptotic ns in bone marrow erythroblasts (FIG 3A), analysis of spleen erythroblasts showed that Fas-L was increased on the Ery-B population of cells (FIG 3B). Multiparametric ?ow cytometry comparative analysis on spleen cells from mice treated with PBS and mActRIIA—Fc showed that Fas—L expression was sed on immature late basophilic and polychromatic erythroblasts (Ery—B) and decreased on orthochromatic erythroblasts (Ery-C) (FIG 3B). In st, Fas—L expression was not modulated on BM oblasts (FIG 3A). Together, these results showed that ActRIIa signaling modulated Fas/Fas-L pathway on maturing oblasts. Collectively, these data suggested that ActRIIa signaling induced premature cell death of maturing erythroblasts committed to ineffective opoiesis. Surprisingly, Fas-L expression was decreased on Ery-A and Ery-C s of cells showing that the impact of ActRIIa signaling was more pronounced on maturing erythroblasts (FIG 3B). The number of tunel ve cells was also increased in animals treated with mActRIIA-Fc (FIG 3C). Ineffective erythropoiesis of thalassemia is characterized by a massive apoptosis of maturing erythroblasts. Studying the impact of ent of mActRIIA-Fc on apoptosis indicated that treatment of mice with mActRIIA-Fc presented decreased numbers of tunnel positive cells compared to their tive controls (FIG 3C), suggesting that ActRIIa signaling trough Smad-2,3 activation could control ineffective erythropoiesis by ting apoptosis levels of maturing erythroblasts. 6.3.4 Activin/GDFll ligands are overexpressed in spleen from thalassemic mice.
RNA (mRNA) expression levels ofActRII, activin A, activin B and GDFll were ted in spleen from ype and thalassemic mice by qPCR. The mRNA levels of ActRII, activin A, activin B and GDFll were all increased suggesting that mActRIIA-Fc could be acting h one of its ligands in its improvement of ineffective erythropoiesis (FIG 4A).
Western blot analysis of protein obtained from the spleen from thalassemic mice treated with mActRIIA-Fc showed a signi?cant decrease in GDF11 protein levels compared to PBS-treated mice (FIG 4B), further implicating GDFll as the ActRIIA ligand responsible for ctive erythropoiesis. Further immunohistochemical analysis revealed that GDF11 protein levels (and to a much lesser extent activin A or activin B) were greatly increased on spleen biopsies from thalassemic mice and were abrogated in animals treated with mActRIIA-Fc (FIG 3A). These results were further con?rmed by blotting (FIG 4B). In contrast to spleen sections, analysis of ActRIIa ligands on BM showed no accumulation of GDF11 in thalassemic mice (FIG 4B). Therefore, GDF11 overexpression in spleen section of thalassemic mice could be associated with ineffective erythropoiesis. 6.3.5 mActRIIA—Fc reduces the increased GDF11 expression levels seen in primary thalassemic pro-erythroblasts.
] To determine which TGF—beta family members might be ated in the treatment of beta-thalassemia with IA-Fc, immunohistochemical analysis of proteins in the activin/GDF ing pathway was performed. Thalassemic mice were treated with PBS or mActRIIA-Fc for 30 days and spleen was harvested, ?xed and stained for activin A, activin B, GDF8, GDFl l, ActRII and p-Smad2 (FIG 5A). Immunohistochemical staining showed increased levels of GDFl l, ActRII and p-Smad2 in thalassemic mice. To explore whether proteins in the activin/GDF signaling pathway were overexpressed in other mouse models of anemia, the expression of activin A, activin B and GDFll in thalassemic mice was compared to normoxia, hypoxia and alphaRBC mice (FIG 5B). FACS analysis of primary thalassemic pro- erythroblasts d with PBS or mActRIIA-Fc for 48 hours then incubated with speci?c antibodies against activin A, activin B, the GDFll tide and the GDF8/GDF11 cleaved peptide indicated that mActRIIA—Fc treatment normalized GDFll expression. Quanti?cation of GDFll staining indicated that treatment ofmice with mActRIIA-Fc signi?cantly reduced GDFll levels (FIG 5C). This reduction in GDFll sion upon treatment of thalassemic mice with IA-Fc was con?rmed by immunohistochemical analysis of the spleens of the mice (). * p<0.05, N=4. Thus, the fact that mActRIIA-Fc decreased GDFll overexpression in thalassemic tissues was additional ce that mActRIIA-Fc corrected ineffective erythropoiesis by targeting GDFl 1. 6.3.6 Neutralization of GDFll restores erythroblast entiation.
To determine if increased GDFll expression was responsible for ineffective erythropoiesis, thalassemic pro-erythroblasts were cultured in the presence of blocking antibodies directed against activins A and B and GDFl l. Anti-GDFll antibodies (but not activin A and B antibodies) promoted erythropoiesis, ?irther con?rming that GDFll negatively regulated erythropoiesis by inducing ctive erythropoiesis as quanti?ed by ?ow try following CD7l/TERl l9 staining (). ROS tion on primary pro-erythroblast differentiation was evaluated by ?ow cytometry using dichlorodihydro?uorescein (). *p<0.05, N=4. Thus, anti-GDFII blocking antibodies restored cell entiation and reduced hemoglobin ates in thalassemic erythroblasts. 6.3.7 An ActRIIA ligand-detecting assay An assay was developed to detect, identify and quantify A s present in blood serum and, speci?cally, their abnormal expression in diseases associated with ctive erythropoiesis (e.g., thalassemia, myelodysplastic syndromes, chronic pernicious anemia and sickle cell anemia). The assay consists of a sandwich ELISA based on an ActRIIA-Fc coating followed by the detection of ActRIIA ligands present in serum. The ActRIIA ELISA assay can be used experimentally (for e.g., s) or clinically (for e.g., human patients) to identify, detect and/or fy ligand or receptor levels in blood serum in order to aid in ent decisions and/or determine the iveness of a treatment designed to modulate TGF-beta ligand or receptor levels. 6.3.8 GDF11 levels are elevated in the serum of thalassemia patients.
The sandwich ELISA assay described above was developed to measure GDF11 levels in serum. ELISA plates were coated with 5 microg/mL of mActRIIA-Fc (FIG 7A). Increasing doses of recombinant GDF11 were added to the plates (0.1 ng/microliter, 0.5 ng/microliter, 2.5 ng/microliter). The plates were washed with PBS 0.1% Tween and bound proteins detected using anti-GDF8/11 antibodies, followed by detection using a horseradish peroxidase-coupled anti-rabbit IgG. GDF11 bound the plates coated with mActRIIA-Fc in a dose-dependent manner indicating that the assay could effectively be used to detect and quantify GDFll levels (FIG 7B).
The ELISA using mActRIIA-Fc detected as little as 100 pg/mL of recombinant GDF11.
Serum from patients haVing thalassemia was tested for GDF11 expression using the ch ELISA with mActRIIA-Fc. As shown in FIG 8, GDF11 levels were elevated 3-fold in patients with thalassemia compared to levels in healthy controls.
To determine if the levels of other A ligands were also elevated in semia patients, activin A and activin B expression levels were also ed in the serum of those patients. An ELISA assay was also ped to detect activin A and activin B. ELISA plates were coated with 5 microgram/mL of ActRIIA-Fc. Increasing doses of recombinant activin A and activin B were added to the plates (0.1 ng/microliter, 0.5 ng/microliter, 2.5 ng/microliter).
The plates were washed with PBS 0.1% Tween, and bound proteins detected with anti-activin A (FIG 9A) and anti-activin B (FIG 10A) antibodies (R&D systems), followed by detection using a horseradish peroxidase-coupled anti-rabbit IgG. Both activin A (FIG 9B) and activin B (FIG 10B) bound the ActRIIA-Fc-coated plates in a dose-dependent manner indicating that the assay could effectively be used to detect and quantify both proteins. Like GDF11, activin A and activin B were also detected at a levels as low as 100 pg/mL.
] The A-Fc ELISA was used to determine the sion levels activin A and actiVin B in the serum of patients with thalassemia. In contrast to GDF11, neither activin A (FIG 9C), nor activin B (FIG 10C) protein levels were elevated in thalassemic patients, indicating that the ActRIIA ligand GDF11 was ly implicated in the thalassemia disease process. 6.3.9 mActRIIA-Fc does not change hematological parameters in wild-type mice.
Wild-type C57BL/6 mice were d for 30 days with mActRIIA-Fc (10mg/Kg BW twice a week) or PBS. Evaluation of red blood cell counts (FIG 11A), hematocrit (FIG 11B), hemoglobin (FIG 11C) indicated no change in the levels of the parameters as a result of treatment of the mice with mActRIIA-Fc. Only a slight decrease in locytosis was observed (FIG 11D). mActRIIA-Fc also did not alter red blood cells (RBC) parameters such as MCV (FIG 11E), MCH (FIG 11F) and MCHC (FIG 12G). *p<0.05, N=3-5 for each independent experiment.
The effect of mActRIIA—Fc on the spleen and bone marrow was also assessed in pe 6 mice. mActRIIA-Fc increased spleen weight in wildtype mice, but had no signi?cant effect on spleen cell number. mActRIIA—Fc also had no effect on bone marrow cell number (FIG 12). 6.3.10 mACTRIIA-FC ATES ERYTHROPOIETIC DIFFERENTIATION THROUGH INHIBITION OF GDFll In order to investigate the cellular mechanism by which mActRIIA-Fc increased red blood cell parameters, a series of in vitro experiments were conducted in which no evidence was found to support direct effects of mActRIIA—Fc on human CD34+ cells as assessed in colony formation assays (A) and in erythroid differentiation in liquid culture (FIGs 13B and 13C). As both clinical and pharmacological ?ndings pointed to a clear role for IA-Fc in stimulating RBC parameters, it was hypothesized that the effects of mActRIIA-Fc could be mediated by accessory cells in the bone marrow (BM) microenvironment. Human CD36+ cells, which are highly enriched for oid progenitors, were co-cultured with long-term BM cultures and then their erythroid differentiation was assessed following 6 days of culture in EPO (2U/mL)- supplemented media. At day 6, the output of the cultures was predominantly characterized as EryA (~basophilic erythroblast) but with the addition of mActRIIA-Fc , a signi?cant on of CD36+ cells d into EryB/C cells (polychromatic/orthochromatic erythroblasts), suggesting that factors produced by BM accessory cells mediated erythropoietic effects of mActRIIA-Fc and that, in contrast to EPO, IA-Fc could play a role in the latter stages or erythroblast maturation (FIGS 13D — 13F). To identify cytokines that might mediate the effects of mActRIIA-Fc, CD36+ cells were treated with several ActRIIA ligands. GDFll treatment signi?cantly decreased proliferation of glycoprotein A positive (GPA+) cells during the differentiation process and mActRIIA-Fc effectively reversed this effect, while having no consequence on untreated cells (FIGS 13G and 13H). These data show that tion of GDFll mediates the erythropoietic stimulatory effects ofmActRIIA-Fc. 6.4 CONCLUSION ] Altogether, the data demonstrate that activin/BMP signaling controls erythroblast differentiation and that targeting BMP type ivin type 11 receptors can decrease ineffective erythropoiesis and e anemia in beta-thalassemia. In particular, the data show the involvement of GDFll in beta—thalassemia—associated anemia and te that GDFll levels are important as a biomarker in subjects having anemia.
Umm>wHOZHMQMMEZmmmQMVm>OQHMQMUZHQFQEUGOM>|EnmwmFZM3HfmM9;HZMMmmMmH>mZmHmOD>mEmm@MmhvHZUZZQHUUULM FOQmMQUQQmmmmmmmeOHm>é>mmw3LMMMZOEmOMFOnmhFm><>MEZFQO¢ME>memw memze?mkmoqngm>>za>mewmmm<6 wmmmmqmqwmamHO7MmmHQmmmH?mmmw?mqmeQmmgw? ?>Q©2¢WZQqummmOOEHQwmmmmmm>>>MOZ?mammmOwHmmmwwq?wmm>mwmdmeo ZH>>H>HQMHHHHZHHmOEOHHmmw>owZH> mwmHZMBB?mUEmmxmxowwomm>OBOZBMDMm324meQUmOHmmquH Smmmwmmxmzozzwmooomw>mmWDMMM>ODBMD%UZHDDQBUOOM>HmH mmMmB>mZmBmOB>m mwmHZMBB?mUEMMMDMDUWUmm>OBOZHMDMm324meHUm0BmmquH 2mmmwmmMmZUZZUMUUUmW>mmmDMMm>UDBMD%UZHDDHBUOOM>HmH UBBOBUUBUB?BHUHHHUHUUUOBHBOUOOBBO???UUBUOBUO?OOOB? 4BBBUBREEDBOBOdUU?UBU???U?UB?w??ww??o????ow?ww?o?? ¢HOQ ?HHmuo? dHHMuod amass swasg Ummmmoonm mocmsvmm c655: Ummmmoonm mocmdem qu?EHmu UmumHmU o?waosm mc?Uoon MOmMSUwHQ 6 29g 0mm DH uOZ H 9292 moqmsvmm UO?A4VD>>>UB>MQHMmHZQBDMmMmmmqm>mmww??mm?mommo?m? meOZHBDOEQ>BQ>m>>mw?mzwommmmMBM¢Zm>m>OD>%BZmM>mmD B?m>OZMBmemmmm?Hw>OmmmmOOM¢MmHBMHHm>m?¢MZm>A?vmom mocmsvmm mHQSHOm QHmEOU c685: qo?um?yommo U?om m mHQSHOm q?muonm m o?waodq mc?vooqw 4HHMu04 AHMHSHHwomHquV mUHuQmQ%HOQ co?mSw mq?m?nmaoo HmHSHamomnuxw 0mm DH uOZ 9393 moqmsvmm MmMD>BQMqummmOQqu>mmBBM%ZmeOOmeBm>4HDmmwmwm>?o *MwmmqmquOmeA?vzm??mmz>momm>zwooz mOmHZMBB?mommmx?x?wwomm>OHUZBMDMmBZ¢meHUmOBmmquH ZmamwmmMmzozzmeUUmw>mmmQMMm>UDBMD%UZHDDHBUOOM>HmH HZQBQMQMQmmdm>mmwwd?mm?mUmmUBmBUOOBmmMmB>mZmBmOB>m zwdmmmmMBM?zm>m>UQ>%BZmM>mmDmmm>m>>>o?>mmBMm meBw>OmmmmOUM?MmHHMmHm>mH?MZm>MUMmeOZHBDOEQ>BQ>m mUQde>QmBBM%ZmeOUmeBm>¢HQmmmeM>qUqu>OZMBmemm MUQmamquOmezm?mUmm>ZUOOBMmMD>BHme?mm ?wmeHw>>Zm>Q4>Z>?mMZ mm>m>¢OOQQQ>OOQOMMZ¢DZ ?wmequm>?m4QM¢¢¢UZ MOBmmMOQH mwmHZMBH?hUEMMmemwwomm>UBOZBMDMm324meQUmOBmmmw?H 2mmmwmmMmZUZZUMUUth>mmmDMMm>UDBmD%UZHDDHBUOOM>HMH mm>m>>>o?>mm?mwHEQHDMmMmmmQm>mmww?qmm?mommo?m?www? DOEQ>HQ>W>>MNHmzwommmmMBM¢Zm>m>OD>%BZmM>mmOm HUBQm>OZMB2mmmwmeHw>OmmmmOOM¢MmHBMHHm>mq¢MZm>MUM% >BQMqummWUDmDQ>mmBHM%ZmeOOme3m>4HDmmwmwm> MOmmamamMOmeZEH4MEZ>mUmm>ZUO BOZBMDMMBZ?meQUmOHmmquH??wmm>m>4OUHHQ>UUHOMM24DZ MMH>UDBMD$UZHDDQEUUOM>HEHmOmHZMBB?mUmmmmmmmwwomm>w mmUBEBOOOBmmMmB>mZmBmOB>mmemwmmMmZUZZOMUUUmw>mmmm %BZmM>mmDmmm>D>>>UH>mmBmmHZQBDMmMmmmqm>mmOO??mm4mo H?MZm>MOmmeOZQEQOmQ>HQ>m>>mw?mzwommmmMBM¢Zm>m>OD> om Op gu?s mv?om am QHMEOU UmmSM mo mo gmvmm? .0 On QHmEOU Ummzw HMHDHHmomHuXm «HHmuom mocmsvmm Q?u?HHmE moqwsvmm ammoq?EmmHm Admhv cam HmQHEHmMIZ q?muoum OQHEm ?HHmuo? om?l?uo¢ w>?umz mocmsvwm om-4HHMuo¢ co?umeU qu?sywu mnu wo q?msov Ummmwoowmmb :uHB mocmswmm 0mm DH uOZ 9494 moqmsvmm 4HQmmmeM>QOHQm>02MBmeMmmm??w>OmmmmOOM¢MmHBMHHm>m Z>m0mm>ZUOOBmmMQ>BQme?mmmUDmmq>mmBBM%ZmeOUme3m> ?mMOmezm??mm O0400BUBUBUUHUOHUUHOHUHUUHUHowwwdwdw?dw?ddow??ww?? UG?OBO??dU?UH?U?HUOHHO?H?HUUUUUUOOUUUOUBBBOUBBUB04 UBU¢¢?mHBOWmZMBm?NUEQMMDOmOmUmm?UmOZBmmqm324Z%%HUHMBM mww?mmQm?mmmzomzwmooomw>0m2mm34>0m0mm%UZmDDDBUOMM mmmmw?>m mm UmmmmUOHQQD 4&5 mHQSHOm m??ummghaog .2 m??om m??om SMHB moqmsvmm ~AMmH5HHwowMquV map quEm q?mEOU map quEm Qu?s qo?um?yommo U?om mHHmU04 m Ucm w O?waosz mQ?Uoosm om?l?HHmuo< wamma c655: memmoonm mocmsvmm Hmc?anmu m?u mo UmumHmU qu??nmu 0mm DH uOZ 9595 _gmzzrmmmmmmw _wmm03mo oqngeHszmqwoeq >ZMmmmo HmnmmamomzH¢omq> >>>moqmmnmmmoon gm>mmm>ooQEFH©mmZ mme>mmwwEQNmmmmEm MQOQmmFm><>MCZZQ m0MQEbmq mm mmO©mHQ© mmmeZNHQ>m©QWH> moqmsvmm 39m©399¢9<>30 0m M23h<9f>kéméwwkm O¢M3>memwmE mmmwmmmm>mqwmm?q UMm>QmBQ>Qw2mMZQ mmMQBEQMHHmmzmmm Qm>QOQmHHDZ>mMMH OmmZ?Bm??UEQMMDOMUMUMMQOwOZHmmqm324zwwHUHMBm?mOMOm mmQm?mmmZUmZUMUUUmN>OmZmHH4>UHOMDNUZmDDHBUOMM>QmHE Bm??mmmmw?>mmww? OmmZ?Bm?womQMMDOmwmommqwmOZHmmqm324Z%%HUHMBm?mOMOm mmzomzwmooomw>0m2mHH4>UHOMD%UZmDDHBUOMM>QmHE UUUUBUBUOUH?UUUUHUHUUHUUUUUBOOUUUBUUUBUUUUUUUOdUB? mU?om AwNuOZ HOmHSUmHQ AwmOmZmMB4>UMOMD%UZmDDHBUOMM>QmHE HZHBDMmMmmmqm>mmww?qmm?mommo?m?www?m??mmmmw?>mmww? >>M%BmzwommmmMBM¢Zm>m>OD>%BZmM>mmDmmm>m>>>o?>mmBMm "Omnsomyg Mo QHmEOU DH qu5: mHQSHOm 0mm qo?um?yommo m 393 c?muonm m uwm?v $5304 Q?muoum co?mSw mc?m?wm?oo momnpxw mQ?Uoonm mHHmp04 0mm DH uOZ ON 9797 moqmsvmm mm??w>0mmmmOUM¢MmHHMmHm>mHdMZm>MommeOZQBDOEQ>BH>m mODmQQ>QmBBM%ZmeOUme3m>4HDmmwmwm>qo??m>OZMBZHHMm MUQm?mde0B%EZEQ4mmZ>mUmm>ZOOOBMmMD>B?me?mm UmmZ?Bm?womdmx?omwmomqumOZHmmqm324Z%%HUMMBm?mOMOm mmQm?mmmzomzwmooomw>0m2mm??>UmOMD%UZmDDHBUOMM>HmHE mm>m>>>OB>MQBmmHEQHQMQMQmmqm>mmww??mm?mommo?m?www? QOEQ>BQ>W>>mw?mzwommmmMHM¢Zm>m>OD>%BZmM>mmOm JOBQm>OZMBmemmmdew>OmmmmOUM¢MmHHMHHQ>QH¢MZm>Momw OBMmMQ>BQMmwdmmmUQmQ?>mmHHM%ZmeOUme3m>4HDmmwmwm> ?mMOmezmq?mmZ>mUmm>ZUO >AmHBUmmzmzm?womqumomwmommqwmOZBmmqm3242>>HOHMBH mww?mmqm?mmmzomzwmooomw>0m2mMB4>OMOMD%UZmDDDBOUMM mmmw?>m >QmHBOWmZMBm?NUEQMMDOmOmUmeOmOZBmm?m324Z%%HUMMBH mww?mmQm?mmmzomzwmvoomw>0m2mHB?>UMOMD%UZmDDDBUUMM Bmmmmw?>m 0m mgu DH om mHQSHOm QHmEOU mHQSHOm mU?ung>HOQ .2 mU?om mv?om .2 gu?s OQHEm am mv?om AwNHOZ m??om .0 mHQSHOm m??om cm ommv .0 Op On DH map q?muoam map amp; map m ma \AHmHSHHwowHuXQV oq?Em Q?mEOU OQHEm UmmSw mHHmuo< Su?B q?maov oq?amv Ummdw HmHDHHmomHuXm Awmmv UmumHmU 0mm m??ugmghaog OQHEm quEm Qu?s q?mEOU q?mEOU qo?um?yommo cm m vgw m mHHMu04 m qu m om om mo om om APHHOZ Q?mEOU Qo?mSM mc?m?HQEoo mHHmuo< HmQ?Ekulo mU?om Ama oz Q?mEOU amass um wocwsvwm st?snwu map mo Umuw?mv wu mg» :u?s w?u map wo UwumHmU mNHImN Una co?umuSE c655: ~AMmH5HHwowMuK®V Ummmmooum wocmdem Hmc?snwu mo UmumHmU Hmc?anmu mo 0mm DH uOZ 9898 moqmsvmm OMMQUmOZBmm4m324zwwHUWMBH¢m>4UUHQQ>UUQUMM24DZ >OmZMMB¢>UMOmQ%UZmQQQBOUMM>QmHHUmmszm?wom?mmmomUm UUQQMQ?mUmmUHEHOUUEmmmmw?>mmww?mmqm?mmmzomzwmooomw 42m>m>0m>%32m¥>mmQmmm>Q>>>UH>mmHmmHZQBDMmMmmmHm>mm mHBMmHm?md?MZm>MUMNmMUZQBQOEH>HQ>m>>M%BmzwommmmMBM mOUme3m>£HDmmmeM>qOHqm>OZMHZHHMmQQQBw>OmmmmOUM¢M mezmd?mmz>m0mm>ZUOOBMMMQ>E?Mmw?mmmUQmQQ>mmBBM%ZZm mquO M>QmHHUMmzmzw?womdeDOMOMOMMQOwOZHmm?m324zwwHUEMHH >mmww?mmthmmmzomzwmooomw>0m2mMH?>UMOMDNUZmDQDBUOM >UH>MQBMmHZQHDMmMmmmQm>wmwwqqmm?mommo?m?www?mmmmw? DOEQ>BQ>m>>memZNOmmmmMHM¢Zm>m>OD>NBZmM>mmDmmm>m>> OZMHZMMMmQQQHw>OQMMQOOM¢MmHHMmHm?mQ¢MZm>MommeOZQB >HQme?mmmUQmDQ>mmBHM%ZmeOOZmMBm>¢HDmmwmwm>?o?qm> *MUmmgmgmMO?wmzmQ4mmZ>mUmm>ZUOOBMmMD OmmZ?Bm?womQMMDOmwmommqwmOZBmmqm324Z%%HUHMBm?mwmw mm?m?mmmzomzwmooomw>0m2mHB4>UHOMD%UZmDDHBUOMM>HmHE Bm??mmmmw?>mmww? AwNuOZ omlmHHmMo? map g?mgov mUHom Awmnoz map mUHom AmHuOZ ?mh mv?om AwNuOZ q?meov .0 m??om DH OQHEm mU?om Su?s QH omlmHHmpo? Qu?3 oq?Em Dana Qu?B quEm mv?om ma?a Q?mEOU quEm q?meov DH m DH om map om Doha OQHEMV 0mm map gm q?muoam map Uqw m oq?Emv 0mm qu m map quEmv 0mm m??ummghaog quEmv 0mm qo?um?yommo Ummmmooamsb gm vgw Gm m MO ma?a HmQ?Ekulz om zo?umuse "0 woqwsvwm Q?muoum om mo mHHMu04 MO UmumHmU Mo mo HMHImN map Qu?a Qu?S mg» Una Qo?mSM mw?om w st?symu so?umuse mo UwumeU Hma-mm Ucm HwawH memwoonm qo?mSw qu?symulz mv?om Umumamv qu?symu wo UmumHmU HMHImN Cam qo?umuSE c685: ~AMmH5HHwowMuN®V Ummmmoong mocmswmm wmalom 0mm DH uOZ 9999 4 4 UmmZ<3m0m2mm??>UmOMD%UZmDDQBUOMM>HmHE MWFGmOZHmWQEEZOEOWQMOZMQ #me<é>BQNBm£Hmm Qd?quw>qmmmmmm© MZQmBmMHmmmwOEmO OHMZBHHHZUMQNQHQ >mHHQmMQQ>ZMmMMQ ngm >>>m0gmmgmmmO©Hm Qm>x??>oo?m?mgmkewmmz mmwe>mmwwmmemmmmm mooqmmHm><>m922q momommmquzqm>mq mm mm<2wmmew>owmaow mmquZwmo>mwomH> moqmsvmm Eémwgéé?é?>3m¢92 m3m0m NZEhdHH>HHmHOQHm O¢ME>UOhmwmm mmmwmmmm>mqwmmmq OMm>Qm3Q>QOZ¢MZQ mmMQEmQMaHLZEMME mHHOZ>mZZa OmmZMBm?NUEQMMDOMUMUmeUmOZHmeHBZ¢waHUHMBm?mOMOm mmQm?mmmZUmZUMUUUmN>OmZmHH4>UHOMD%UZmDDHBUOMM>QmHE Bm??mmmmw?>mmww? m??UEQMMDOmUMUmeOmOZBmm?m324Z%%HUmm?m?mwmwm mm?m?mmmzomzwmooomw>0m2mHB¢>UHOMD%UZmDDQBUOMM>HmHE ®HQSHOm mv?ummmhaom no mUHom m??om AmHUOZ Awmmv AwNuOZ .0 man mUHom mUHom DH mHQSHOm mHQSHOm m??om OQHEm HOmHSUmHQ DH map Su?: OQHEmV 0mm moqmsvmm mUHunghaom OQHEMV quEm 0mm m??ummghaog Su?: OQHEmv qo?um?mommo mHHMUO< ma < mHHMu04 ma MO mHHmUod wO QGEDQ ~AHMHSHH®OQHUX®V Ummmmoowm moswsvmm Hms?EHmu UmumeU mHHION amass Q?muoam c655: ~AMMHSHHmowMquV onm m wmalm? c685: ~AMmH5HHwaMuNwV Ummmmoonm moqmsvmm qu?anwu UmumHmU 0mm DH "02 hm 100100 moqmsvmm Ummzmzm?womdmx?omwmommqwmOZBmmqm324Z%%HUHMBm?mwmw mmQm?mmmzomzwmooomw>0m2mm??>UmOMD%UZmDDHBUOMM>HmHE Bm??mmmmw?>mmww? Ummzmzm?womdmx?omwmomm?wmOZBmmqm324Z%%HUHMBm?mwmw mmQm?mmmzomzwmooomw>0m2mWH¢>UHOMQ%UZmQDHBUUMM>HmHE >AmHmemZ?Bw?womdeDOMOMOMMQOWOZBmmqm324zwwHOHMBm mww?mmdm?mmmzomzwmooomw>0m2mMB¢>OMOMQNOZmDDDBOOMM ?>m UMHHOWOZBMMQMEZ?zwwHUMMBm??wmm>m>4OUQHH>UUQOMM24DZ >OmZmHB¢>UMOMDNUZmQDDBUOMM>QmHBOmm243m?wom?mmmomOm qu?mm?mommUBEBUUUBmmmmw?>mmww?mmHm?mmmzomzwmooomw 42m>m>wm>%BZmM>memmm>D>>>UB>mmHmmHZHBDMmMmmmHm>mm mHBMMHm?mQdMZW>MUM>HMOZHBDOEH>BH>m>>M%BmzwommmmMBM mOOmeBm>¢HDmmmeM>QUBQm>OZMBZMMMmmmq?w>OmmmmOOM¢M mezmq?mm2>m0mm>ZUOOBMMMD>BHMmw?mmmODme>mmBBM%ZZm AwNuOZ mHQSHOm mv?ummmhaom mUHom AwNuOZ mHQSHOm mv?ugmghaog .0 mU?om map mU?om Awmnoz mv?om mv?om AmHUOZ mUHom .0 q?meow .0 DH DH map DH mHQSHOm mU?uQmQ>HOQ .2 map mv?om DH omlmHHMuo? ?u?s ~AHMHSHH®OQHMX®V OQHEmV ~AHmH5HHwomHuX®V OQ?Em OQ?Emv \Anm?s?awownuxwv OQHEm g?maov map OQHEm Doha OQHEm om map 0mm OQHEm m qo?um?yommo mHHMuO< 0mm q?meov q?mEOU mHHmuo< Qu?a 0mm 0mm ma mHHMuo< Qu?B m vgw m OQHEmV Gm q?muoum map Ucm m MO Ummmmooam MO Ummmmooam mo om st?samu om MO om U memwooum moswsvmm woswsvmm wocwsvwm u mg» Umumamv qu?EMwu mg» UwuwHwU ?u?B wmalom HMHImN map U?m SE UmmmmOOMQQD co?mSw ch?symplz wo mHHImH amen: amass mHH-om amass wo wo mw?om UmumeU qu?aymu wo 0mm DH uOZ 101101 moqmsvmm *Mwmmqmqmmo M>QmHBUmm243m?womdmxmomwmomm?wmOZBmmqm324Z%%HUMMBM >mmww?mmdm?mmmzomzwmooomw>0m2mm??>0MOMD%UZmDDDBUUM >OB>mmBMmHZQBQMQMQQmqm>mmwwqqmm?mommo?m?www?mmmm%B QOEd>?d>m>>memZNOmmmmMHMm>UQ>%BZmM>mmDmmm>m>> OZMBZMMMmmmABw>OQMMQOUM¢MmHHMmHm?m?¢M2m>MommeUZQ3 >HAMm>AmmmUQde>mmHBMNZmeOOme3m>4HQmmmeM>QUBQm> *MUmm?mamMOBVEZEQ4MEZ>mUmm>ZOOOBMmMQ Ommzmzm?womdmxmomwmomm?wWOZEMMQWBZ¢Z%%Homm?m?mwmw mmthmmmzomzwmooomw>0m2mMH¢>OMOMD%UZmDDDBUOMM>QmHE mmw?>mmww? OmmZ?Bm?womqumOmOmommqwmOZBmm?m324Z%%HUHMBm?mwmw mmQm?mmmZUmZOMUUUmN>OmZmHH4>UHOMD%UZmDDDBUOMM>HmHE Bm??mmmmw?>mmww? Ommzmgm?womdmmemmeMMQUmOZBmm?m324Z%%HUMMBm?mwmw mmHm?mmmZUmZOMUUUmN>OmZmMB4>UMOMD%UZmDDDBUOMM>QmHE OUQQmm?mommo?m?www?m??mmmmw?>mmww? 42m>m>wm>wzzmM>mmQmmm>m>>>o?>mmBMmHZQBDMmMmmem>mm mHBMHHm?mddMZm>MUM%HMOZHBDOEH>BQ>m>>M%BmzwommmmMBM mUHom AmHuOZ 4&5 Su?s g?mgou mU?om OQHEm mU?om Am?noz mUHom AwNUOZ '0 m??om AmHuOZ m??om AwNuOZ DH Su?s DH wHQSHOm mHQSHOm mHQSHOm Doha DH DH DH OQHEMV 0mm om oq?Em Dara map cm Ucm omlmHHmpo? Q?mEOU map 0mm mv?ugmgh?og m??ugmghaom OQHEmV 0mm GOHuprE quEmv 0mm QOHumu?E mhaog quEmv 0mm MO moqmsvmm q?muomm qo?um?yommo qu m oq?Emv Qu?s Mo mHHMuod "0 MO UmumHmU mHHMu04 < wamma Ummm®UOHm Su?B HMHImN map mo Dmbq m0 Dara Una mo?umuSE Qo?mSM HmQ?Ekulz mw?om UmumeU u mo UwuwHwU Hma-mm USS Go?uwuse :wEsg \AHmHSHHGOwHuXGV ~AMMHSHHmowMuK®V UmmmmOOMQ UmmmmOOMQ mocmsvwm wmalom QMHB c655: mocmdem wmalom QMHB amass ~AMMHSHHmoprN®V UmmmmUOMQ moqm?vwm wmalom 0mm DH uOZ 102102 moqmsvmm m>4HQmmmeM>?UH?m>OZMBZMMMmmm??w>OmmmmOOM¢M mezm??mmz>m0mm>20003mmMQ>H?Mmw?mmmUDmD?>mmBBM%ZZm *Mwmm?m?mmo mmmzom2mme<>omommw02mmmmzowmm>qmHE wwqqmammommoemewwwemdemmmmwe>mmwwd <2m>m>wm>w22mz>mmommm>o>>>oe>mmHmmquemMmMmmmqm>mm mHBMmHm4m4¢M2m>momwmmwzqzoomq>3q>m>>mwemzwommmmmem mOGmezm>¢Hmmmwmwm>qoeqm>02M32mmmmmmqew>ommmmowm¢m mezmq4mmz>momm>ZUOOEMmMQ>quqummm0mmmq>mmeemw22m *Mwmmqmqmmo UmOZBmmAMBZ>HOMMBmdmwmwmdw?m>m>4OOQQA>UUQUMM24DZ Owwzm3w4%OEQMMDOmwmommq mmdmhmmmzomzwmooom>>0m2mMB4>OMOMD%OZMQDDBOOMM>QHHE Owd?mm?mOQmOHEEOOOEm??mmmmw?>mmww? £ZE>M>OD>%BZmM>mmQmmw>m>>>OB>mmHmwHZQHDMmMmmem>mm mHHMmHm?mQ?MZw>MOM>MMOZQBDOEQ>HQ>m>>mNHmZNOmmmmMHM mOUme3m>QOHQw>OZMHZHHMmQQQHw>OmmmmOUM¢M B?EZEQ?mmz>mUmm>ZUOOBMwMD>BQMqummmODmDQ>mmBBM%ZZm *Mwmm?m?mMO OmOZBmmqMBZ¢waHUWMBm4MOMOm40mm>m>4OUHQQ>UUQOMM24DZ UmmZ?Bm?womqmmmomwmommq mmQm?mmmzumzwmooomw>0m2mHH4>Um0mD%UZmDDDBUOMM>HmHE UUQQmm?mommo?m?www?m??mmmmw?>mmww? 42$>M>OD>$BZhM>mmDmmm>m>>>o?>mmBMmHZHBDMmMmmem>mm m?mQdMZm>MUMNHMOZQBDOEQ>BQ>m>>M%BmzwommmmMBM mOOme3m>¢HQmmmeM>QUBQm>OZMBZHMMmmmq?w>OmmmmOOM¢M mezm??mmz>m0mm>ZUOOBMmMD>B?Mmw?mmmODme>mmBBM%ZZm *MOmm?mquO memZMBm?$UEQMMQOMUmUmm?UmOZBmm?m3242%%HUMMBm?mwmw 4mm?m?mmmzomzwmvoomw>0m2mMB4>UMOMD%UZmDDQBUOMM>QmH mm?mUMQUHBQ40mUODU¢¢HOQ om om mm mHHmuo< oq?Emv 0mm QO?UMUDE mU?ung>HOQ mm oq?Emv 0mm SE om qo?um?yommo dmh mUHunghaog Go?umu?E m??ummmhaog am OQHEmV 0mm mm ?mH oq?>ms Doha 000 MO Cu thd mHHMuo< \AHmHSHHwowHuXQV MO Cu Dara On Qu?s mHHMu04 mo Dmbq o» mHHMuo? m :u?s QUHB Umm5m QU?B amass Ummmmooam woswsvmm wmalom QU?B me5w Qwsss memwooum wocwsvwm wmalom Qu?B meSw 6cm mocmsvmm c655: ~AHMHSHHmowMuK®V Ummmmooym mocmsvmm wmalom QMHB Umwa 6cm wocmswmm amass ~AMMHSHHmUMprmV Ummmmoong momma-mm 0mm DH uOZ 103103 B00mzmzm4w0mQMMQOm0m0mm?0mOZHmm?mBZ4Z%%HOMMBm4m0m0 4mmQm?mmm20mz0m000mw>0m2mmH4>0m0mmw0ZmDDDBO0MM>QmH mm4m0m40QBQ40m0OQ0444B4mm00mmHBBm4Bm0mm00 30mmzm3m4W0mQMMQOm0m0MMQOmOZBMMQmzz4zwwHUMMHm4m0m0 4mmdmhmmm20mz0m000mw>0m2mMB4>0m0mow0ZmDQQBO0MM>JmH mm4m0md043?4000000444B4mm00mmHBBm43m0mm00 4ZE>m>0D>?BZmM>memEw>D>>>0H>mmHmwHZQHQMmMmmem>mm mHHMMHm4mQ4MZM>MOM>mMOZQBDOEQ>HQ>m>>MNHmZNOmmmmMHM mO0me3m>4HDwm>m0x>?0??w>OZMHZHHMmQQQBw>OmmmmO0M4M mezm94mmz>m0mm>Z0OOBMwMD>BQMqummm0DmDQ>mmBBM%ZZm 00HO0HO0H0 0444000000 4400H04400 0440000040 0B04400400 04000HO0H0 OHOH 0004000BHO 0004400000 0500004000 B0440B0040 0004444000 BOBO0BOB00 000000000B 0BH44BO0B4 BOB0004400 HO0H000H4H 004440440B 4400400000 BHOHOHHB4B B0040004BB 000000440B 040000B000 000HBOHO0B 00??mm4m0mm0?m?000? 0040404403 BBO0BBOB04 404BB4BBB4 B000000B44 00BB400B00 00B0440BB4 40B4BBOBB4 000B B000440B44 4B0040B004 4000034040 HOB040B000 moqmsvmm *M0mmqmquO 4400B400B4 00400BOBOB BOB4400000 0044004000 00444B4004 00400000B0 40BBB40040 4404044000 BOBOBBB440 0000000000 00B0 4000000B00 SH SH Q? m GUIU UmmoHomHUV mHQSHOm Umm5m m mc?>mg mHQSHOm Qu?s m oz HH®omHuX®V mUHuQmQ>HOQ mq?>m£ HMQHEHmulo m?UV se \AHmHSHHwowHuXwV mU?ung>HOQ mq?>ms Ummo?om?vv moqmsvmm wm mq?>m£ qu?EHmu-o qo?umusa quEOU QH qo?um?yommo mHHmuo4 mHHmuo4 U?o4 0mm UQQ?HMNV mm AmhhmmO\DOONOB om meq?? amass Ummmmooam woswsvmm usm?mm> mosmsvmm Amppmm0\noomoz thqGm Qwsss memwooum Amhhmm0\boomoz wocwsvwm usw?nw> woswsvwm Dawn am cw On wwwe z mc?woocm 0mm DH uOZ 104104 9404090040 4090 0009449400 0049004004 9000904004 0004440440 0444000444 0940000009 0090040900 9040090000 0400404404 9490900990 4000040040 4404009090 4449000000 9000040000 0040440040 9000 0440490400 0040090090 4400 0090940044 0004049090 0409004004 0940400040 9044044040 9900900004 0090040040 0040940094 0909000909 0909409409 0040900400 0040090049 4004000000 0040900900 9044049040 4404009400 0404004404 4044004094 9900 0009 0009040090 4404009000 0900090094 0004044040 moqmsvmm 0004040044 9009009000 0090440990 0044404044 0409009090 9440 0000400090 0000004000 0400400000 0444090090 4400404000 0900009000 4090044004 0909909004 0040490400 UmmZm3m4womAMMQOMUMOMMQOwOZBMMQmBZ4zwwHOMM9m4mOmwm mmzomzwmooomw>0m2mm94>0m0m0902mQQQBUOMK>QHH9 HZQBDMmMmmmam>wm00qgmm4m0mm09390009m49mmmm99>mmww4 290mmmmMHM4ZE>m>OD>932mM>mmDmmm>m>>>09>mm9mw me99>OmmmmOUM4MmHBMmHm>mQ4MZm>MUM9mMUZQBDOEQ>BQ>m mOQmQQ>mm99MWZmeOUZwm3m>4HDmmwmwm>409qm>02¥92mmMm MUmmaQOMOmezm??mmZ>mUmm>ZOOOBMmMD>BQMm9qmm Ommzm3m4903QMMDOmUmvmmqwmOZBmmqm324zwwHUmm?m?mwmwm mmqm?mmmzomzwmooomw>om2mm94>0m0mm902mmm?gowmm>qmH9 mm>m>>>09>mm9mwHEQBDMmMmmmQm>mmwwq?mm?momm09m?www? HMOZQBDOEQ>HQ>W>>MNHmzwommmmMBM4Zm>m>OD>932mM>mmOm HUBHm>OZMBZMMMWQQQBw>OmmmmOOM4MmHBMmHm>m?4MZm>MUM9 OBMmMD>BQMmw?mmmUQmDQ>mm99¥922mm002mm3m>4HDmmwmwm> MOmmamquOmezmq?mmZ>mUmm>ZOO mo wo um map DH om mHQSHOm Q?mEOU DH um am 0mm mHQSHOm QHmEOU gu?s OQHEm Ommv Op On q?wuonm m m ma qo?um?yommo "wmmv mc?m?umsoo UmmSm q?muonm Awmmv Ummdw mHHMuo4 mq?m?nmsoo UmumHmU Go?mSw HmHSHHwownuxw Amm oz q?msov co?mSw MMHSHHwomMuxm mHHMuo4 qu?symul0 mU?om Aomuoz q?mEOU 0mm DH "02 105105 48 full-length, unprocessed RTVLAAPLLLGFLLLALELRPRGEAAEGPAA$MV&&AAA precursor protein GDFl 1, AAAAAGVGGERSSRPAPSVAPEPDGCPVCWNNRQHSREL i.e., GDFll preproprotein RLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLQQIL DLHDFQGDALQPEDFLEEDEYHATTETVEmmAQETDPA VQTDGSHXXHHWHBPKVMFTKVLKAQLWRHIRPVPR PATVYLQHRLKPLTGEGTAGGGGGGRRHHURSJUEL HSRSGHWQSIDFKQVLHSWFRQPQSNWGIEINAFDPSG TDLAVTSLGPGAEGLHPFMELRVLENTKRSRRNLGLDC SRCCRYPLTVDFEAFGWHNVHAPKRYKANYCS GQCEYMFMQKYPHTHLVQQANPRGSAGHXH?TKMSP INMLYFNDKOOHYGKWGMVVDRCGCS Nucleic acid sequence ATGGTGCTCGCGGCCCCGCTGCTGCTGGGCTTCCTGC encoding SEQ ID NO: 48 TCCTCGCCCTGGAGCTGCGGCCCCGGGGGGAGGCGG CCGAGGGCCCCGCGGCGGCGGCGGCGGCGGCGGCG GCGGCGGCAGCGGCGGGGGTCGGGGGGGAGCGCTC GCCAGCCCCGTCCGTGGCGCCCGAGCCGGA CGGCTGCCCCGTGTGCGTTTGGCGGCAGCACAGCCG GCGCCTAGAGAGCATCAAGTCGCAGATCTT GAGCAAACTGCGGCTCAAGGAGGCGCCCAACATCAG CCGCGAGGTGGTGAAGCAGCTGCTGCCCAAGGCGCC GCCGCTGCAGCAGATCCTGGACCTACACGACTTCCA GGGCGACGCGCTGCAGCCCGAGGACTTCCTGGAGGA GGACGAGTACCACGCCACCACCGAGACCGTCATEMS CATGGCCCAGGAGACGGACCCAGCAGTACAGACAGA TGGCAGCCCTCTCTGCTGCCATTTTCACTTCAGCCCC AAGGTGATGTTCACAAAGGTACTGAAGGCCCAGCTG TGGGTGTACCTACGGCCTGTACCCCGCCCAGCCACA GTCTACCTGCAGATCTTGCGACTAAAACCCCTAACTG GGGAAGGGACCGCAGGGGGAGGGGGCGGAGGCCGG CGTCACATCCGTATCCGCTCACTGAAGATTGAGCTGC ACTCACGCTCAGGCCATTGGCAGAGCATCGACTTCA AGCAAGTGCTACACAGCTGGTTCCGCCAGCCACAGA GGGGCATCGAGATCAACGCCTTTGATCCCA GTGGCACAGACCTGGCTGTCACCTCCCTGGGGCCGG GAGCCGAGGGGCTGCATCCATTCATGGAGCTTCGAG TCCTAGAGAACACAAAACGTTCCCGGCGGAACCTGG ACTGCGACGAGCACTCAAGCGAGTCCCGCT GCTGCCGATATCCCCTCACAGTGGACTTTGAGGCTTT CGGCTGGGACTGGATCATCGCACCTAAGCGCTACAA CTACTGCTCCGGCCAGTGCGAGTACATGTTC ATGCAAAAATATCCGCATACCCATTTGGTGCAGCAG GCCAATCCAAGAGGCTCTGCTGGGCCCTGTTGTACCC CCACCAAGATGTCCCCAATCAACATGCTCTACTTCAA TGACAAGCAGCAGATTATCTACGGCAAGATCCCTGG CATGGTGGTGGATCGCTGTGGCTGCTCT GDFll propeptide of AEGPAAJU&&AAA£JU&&AGVGGERSSRPAPSVAPEPDG human GDFll protein CPVCVWRQHSRELRLESIKSQILSKLRLKEAPNISREVV KQLLPKAPHXXMLDLHDFQGDALQPEDFLEEDEYHAT TETVISMAQETDPAVQTDGSPLCCHFHFSPKVMFTKVL KAQL"mndnrvPRPATVYLQHBLKPLTGEGTAGGGG GGRRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQ SNWGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELRVLE 106106 NTKRSRR l?mhmaddmmmmm GCCGAGGGCCCCGCGGCGGCGGCGGCGGCGGCGGC encoding SEQ ID NO: 50 GGCGGCGGCAGCGGCGGGGGTCGGGGGGGAGCGCT CCAGCCGGCCAGCCCCGTCCGTGGCGCCCGAGCCGG ACGGCTGCCCCGTGTGCGTTTGGCGGCAGCACAGCC GCGAGCTGCGCCTAGAGAGCATCAAGTCGCAGATCT TGAGCAAACTGCGGCTCAAGGAGGCGCCCAACATCA GCCGCGAGGTGGTGAAGCAGCTGCTGCCCAAGGCGC CGCCGCTGCAGCAGATCCTGGACCTACACGACTTCC AGGGCGACGCGCTGCAGCCCGAGGACTTCCTGGAGG AGGACGAGTACCACGCCACCACCGAGACCGTCATTA GCATGGCCCAGGAGACGGACCCAGCAGTACAGACAG GCCCTCTCTGCTGCCATTTTCACTTCAGCCC GATGTTCACAAAGGTACTGAAGGCCCAGCT GTGGGTGTACCTACGGCCTGTACCCCGCCCAGCCAC AGTCTACCTGCAGATCTTGCGACTAAAACCCCTAACT GGGACCGCAGGGGGAGGGGGCGGAGGCCG GCGTCACATCCGTATCCGCTCACTGAAGATTGAGCTG CACTCACGCTCAGGCCATTGGCAGAGCATCGACTTC AAGCAAGTGCTACACAGCTGGTTCCGCCAGCCACAG AGCAACTGGGGCATCGAGATCAACGCCTTTGATCCC AGTGGCACAGACCTGGCTGTCACCTCCCTGGGGCCG GGAGCCGAGGGGCTGCATCCATTCATGGAGCTTCGA GTCCTAGAGAACACAAAACGTTCCCGGCGG Mature human GDFl l NLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRY n KANYCSGQCEYMFMQKYPHTHLVQQANPRGSAGPCC TPTKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS Nucleic acid sequence AACCTGGGTCTGGACTGCGACGAGCACTCAAGCGAG encoding SEQ ID NO: 52 TCCCGCTGCTGCCGATATCCCCTCACAGTGGACTTTG AGGCTTTCGGCTGGGACTGGATCATCGCACCTAAGC GCTACAAGGCCAACTACTGCTCCGGCCAGTGCGAGT TCATGCAAAAATATCCGCATACCCATTTGGT GCAGCAGGCCAATCCAAGAGGCTCTGCTGGGCCCTG TTGTACCCCCACCAAGATGTCCCCAATCAACATGCTC TACTTCAATGACAAGCAGCAGATTATCTACGGCAAG ATCCCTGGCATGGTGGTGGATCGCTGTGGCTGCTCT Extracellular domain of Murine counterpart of SEQ ID NO: 7 murine ActRIIA fused to a murine Fc domain "mActRIIA-Fc" 8. EQUIVALENTS Although the invention is described in detail with reference to speci?c embodiments thereof, it will be understood that variations which are functionally lent are within the scope of this invention. Indeed, various modi?cations of the invention in addition to those shown and described herein Will become apparent to those skilled in the art from the foregoing description and accompanying gs. Such modi?cations are intended to fall within the 107107 scope of the appended claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the speci?c embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
All publications, patents and patent applications mentioned in this speci?cation are herein incorporated by reference into the speci?cation to the same extent as if each individual publication, patent or patent application was cally and individually ted to be incorporated herein by reference in their entireties. 108108
Claims (16)
1. Use of an activin receptor type II inhibitor in the cture of a ment for treatment of anemia, beta thalassemia, or for increasing red blood cell levels or orthochromatic oblasts (Ery-C) in a human subject in need thereof, wherein the treatment comprises: (a) assessing the level of GDF11 in a tissue sample of the human subject, and (b) if the level of GDF11 is ed over the normal level of GDF11, administering an activin receptor type II inhibitor to the human subject, wherein the activin receptor type II inhibitor is a polypeptide comprising an amino acid sequence selected from the group consisting of: a 90% identical to SEQ ID NO:2; b 95% identical to SEQ ID NO:2; c 98% cal to SEQ ID NO:2; d SEQ ID NO:2; e 90% identical to SEQ ID NO:3; f 95% identical to SEQ ID NO:3; g 98% identical to SEQ ID NO:3; h SEQ ID NO:3; i 90% identical to SEQ ID NO:7; j 95% identical to SEQ ID NO:7; k 98% identical to SEQ ID NO:7; l SEQ ID NO:7; m 90% identical to SEQ ID NO:12; n 95% identical to SEQ ID NO:12; o 98% identical to SEQ ID NO:12; p SEQ ID NO:12; q 90% identical to SEQ ID NO:17; r 95% identical to SEQ ID NO:17; s 98% identical to SEQ ID NO:17; t SEQ ID NO:17; u 90% identical to SEQ ID NO:20; v 95% identical to SEQ ID NO:20; w 98% identical to SEQ ID NO:20; x SEQ ID NO:20; y 90% identical to SEQ ID NO:21; z 95% identical to SEQ ID NO:21; aa 98% identical to SEQ ID NO:21; bb SEQ ID NO:21; cc 90% identical to SEQ ID NO:22; dd 95% identical to SEQ ID NO:22; ee 98% identical to SEQ ID NO:22; ff SEQ ID NO:22; gg 90% identical to SEQ ID NO:23; hh 95% identical to SEQ ID NO:23; ii 98% identical to SEQ ID NO:23; jj SEQ ID NO:23; kk 90% identical to SEQ ID NO:24; ll 95% identical to SEQ ID NO:24; mm 98% identical to SEQ ID NO:24; nn SEQ ID NO:24; oo 90% identical to SEQ ID NO:25; pp 95% identical to SEQ ID NO:25; qq 98% identical to SEQ ID NO:25; and rr SEQ ID NO:25; n the activin receptor type II inhibitor is a polypeptide comprising the extracellular domain of activin receptor type IIA linked to the Fc portion of an antibody; wherein the activin receptor type II inhibitor is a polypeptide sing the extracellular domain of activin or type IIB linked to the Fc portion of an antibody.
2. Use of an activin receptor type II inhibitor in the cture of a medicament for treating anemia, chronic pernicious , sickle cell anemia, myelodysplastic syndromes, inherited bone marrow failure, or beta-thalassemia, or for increasing red blood cell levels or orthochromatic erythroblasts (Ery-C) in a human subject in need of treatment, wherein the human subject treated has an elevated level of GDF11 as compared to the level of GDF11 of one or more healthy human subjects, and wherein the activin receptor type II inhibitor is a polypeptide comprising an amino acid sequence ed from the group consisting of: a 90% identical to SEQ ID NO:2; b 95% identical to SEQ ID NO:2; c 98% cal to SEQ ID NO:2; d SEQ ID NO:2; e 90% identical to SEQ ID NO:3; f 95% identical to SEQ ID NO:3; g 98% identical to SEQ ID NO:3; h SEQ ID NO:3; i 90% identical to SEQ ID NO:7; j 95% identical to SEQ ID NO:7; k 98% identical to SEQ ID NO:7; l SEQ ID NO:7; m 90% identical to SEQ ID NO:12; n 95% identical to SEQ ID NO:12; o 98% identical to SEQ ID NO:12; p SEQ ID NO:12; q 90% identical to SEQ ID NO:17; r 95% cal to SEQ ID NO:17; s 98% identical to SEQ ID NO:17; t SEQ ID NO:17; u 90% identical to SEQ ID NO:20; v 95% identical to SEQ ID NO:20; w 98% identical to SEQ ID NO:20; x SEQ ID NO:20; y 90% identical to SEQ ID NO:21; z 95% identical to SEQ ID NO:21; aa 98% identical to SEQ ID NO:21; bb SEQ ID NO:21 cc 90% identical to SEQ ID NO:22; dd 95% identical to SEQ ID NO:22; ee 98% identical to SEQ ID NO:22; ff SEQ ID NO:22; gg 90% identical to SEQ ID NO:23; hh 95% identical to SEQ ID NO:23; ii 98% identical to SEQ ID NO:23; jj SEQ ID NO:23; kk 90% identical to SEQ ID NO:24; ll 95% identical to SEQ ID NO:24; mm 98% cal to SEQ ID NO:24; nn SEQ ID NO:24; oo 90% cal to SEQ ID NO:25; pp 95% identical to SEQ ID NO:25; qq 98% identical to SEQ ID NO:25; and rr SEQ ID NO:25; wherein the activin receptor type II inhibitor is a polypeptide comprising the extracellular domain of activin receptor type IIA linked to the Fc portion of an antibody; n the activin receptor type II inhibitor is a polypeptide comprising the extracellular domain of activin receptor type IIB linked to the Fc portion of an antibody.
3. Use of an n receptor type II inhibitor in manufacture of a medicament for the treatment of anemia, beta thalassemia, or for increasing red blood cell levels or orthochromatic erythroblasts (Ery-C) in a human subject in need f, wherein the treatment comprises: (a) administering an activin receptor type II inhibitor, and (b) monitoring the level of GDF11 in a tissue sample of the human subject, wherein the activin or type II inhibitor is a polypeptide comprising an amino acid sequence selected from the group consisting of: a 90% identical to SEQ ID NO:2; b 95% identical to SEQ ID NO:2; c 98% identical to SEQ ID NO:2; d SEQ ID NO:2; e 90% identical to SEQ ID NO:3; f 95% identical to SEQ ID NO:3; g 98% identical to SEQ ID NO:3; h SEQ ID NO:3; i 90% identical to SEQ ID NO:7; j 95% identical to SEQ ID NO:7; k 98% identical to SEQ ID NO:7; l SEQ ID NO:7; m 90% identical to SEQ ID NO:12; n 95% identical to SEQ ID NO:12; o 98% identical to SEQ ID NO:12; p SEQ ID NO:12; q 90% cal to SEQ ID NO:17; r 95% identical to SEQ ID NO:17; s 98% identical to SEQ ID NO:17; t SEQ ID NO:17; u 90% identical to SEQ ID NO:20; v 95% identical to SEQ ID NO:20; w 98% identical to SEQ ID NO:20; x SEQ ID NO:20; y 90% cal to SEQ ID NO:21; z 95% identical to SEQ ID NO:21; aa 98% identical to SEQ ID NO:21; bb SEQ ID NO:21 cc 90% identical to SEQ ID NO:22; dd 95% identical to SEQ ID NO:22; ee 98% cal to SEQ ID NO:22; ff SEQ ID NO:22; gg 90% identical to SEQ ID NO:23; hh 95% identical to SEQ ID NO:23; ii 98% identical to SEQ ID NO:23; jj SEQ ID NO:23; kk 90% cal to SEQ ID NO:24; ll 95% identical to SEQ ID NO:24; mm 98% identical to SEQ ID NO:24; nn SEQ ID NO:24; oo 90% identical to SEQ ID NO:25; pp 95% cal to SEQ ID NO:25; qq 98% identical to SEQ ID NO:25; and rr SEQ ID NO:25; wherein the activin or type II inhibitor is a polypeptide comprising the extracellular domain of activin receptor type IIA linked to the Fc portion of an antibody; wherein the activin receptor type II inhibitor is a polypeptide comprising the extracellular domain of activin receptor type IIB linked to the Fc portion of an antibody.
4. The use of any one of claims 1-3, wherein the treatment comprises: (a) administering a dose amount of an activin receptor type II inhibitor, (b) assessing the level of GDF11 in a tissue sample of the human subject, and (c) adjusting the dose amount of the activin receptor type II inhibitor, wherein, if the level of GDF11 is elevated over the normal level of GDF11, the dose amount of the activin receptor type II inhibitor is increased, and wherein, if the level of GDF11 is decreased over the normal level and/or activity of GDF11, the dose amount of the activin receptor type II inhibitor is decreased.
5. The use of any one of claims 1-3, wherein the treatment comprises: (i) determining a first level of GDF11 in a tissue sample of the human subject; (ii) administering the activin receptor type II inhibitor, and (iii) determining a second level of GDF11 in a tissue sample of the human subject, (iv) adjusting a subsequent dose amount of the activin receptor type II inhibitor; wherein, if the second level of GDF11 is elevated over the first level of GDF11, the subsequent dose amount of the activin receptor type II inhibitor is increased, and wherein, if the second level of GDF11 is decreased over the level of GDF11 in the same tissue of one or more healthy human subjects, the subsequent dose amount of the activin receptor type II tor is decreased.
6. The use of claim 4 or 5, wherein the level of GDF11 is decreased when it is sed at least 25%, at least 50%, at least 75%, or at least 90% over the normal level of GDF11, or over the level of GDF11 in the same tissue of one or more healthy human subjects.
7. The use of claim 4 or 5, wherein the level and/or activity of GDF11 is decreased when it is sed at least 2 times over the normal level of GDF11 or over the level of GDF11 in the same tissue of one or more healthy human subjects.
8. The use of any one of claims 1, 2 and, 4 to 7, wherein the level of GDF11 is elevated when it is elevated at least 25%, at least 50%, at least 75%, at least 100% or at least 200% over the normal or first level of GDF11 or the level of GDF11 in the same tissue of one or more healthy human subjects.
9. The use of any one of claims 1, 2 and 4 to 7, wherein the level of GDF11 is elevated when it is elevated at least 2 times over the normal or first level of GDF11 or the level of GDF11 in the same tissue of one or more y human subjects.
10. The use of any one of claims 1, 4, and 6-9, n the normal level of GDF11 is an average level of GDF11 in a sample from the same tissue of one or more healthy human subjects in the same age category.
11. The use of any one of claims 1 and 3-10, wherein the tissue is obtained from serum, bone marrow, liver, or spleen.
12. The use of any one of claims 1-11, wherein the level of GDF11 is the protein level of GDF11; or wherein the level of GDF11 is mRNA level of GDF11.
13. The use of any one of claims 1-12, wherein the Fc portion of an antibody is Fc n of an IgG1 dy.
14. A use according to claim 1 substantially as herein described or exemplified.
15. A use according to claim 2 substantially as herein described or exemplified.
16. A use according to claim 3 substantially as herein described or exemplified. 1/
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201261718126P | 2012-10-24 | 2012-10-24 | |
US61/718,126 | 2012-10-24 | ||
NZ70746913 | 2013-10-23 |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ743635A NZ743635A (en) | 2020-09-25 |
NZ743635B2 true NZ743635B2 (en) | 2021-01-06 |
Family
ID=
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP7146850B2 (en) | Biomarkers for use in treating anemia | |
EP3227675B1 (en) | Activin-actrii antagonists and uses for treating myelodysplastic syndrome | |
EP2303917B1 (en) | Antagonists of actriib and uses for increasing red blood cell levels | |
JP2020183390A (en) | Methods for treating anemia | |
EP2303918B1 (en) | SOLUBLE ACTRIIa AS ACTIVIN-ACTRIIA ANTAGONIST FOR USE IN TREATING ANEMIA | |
AU2007334333B2 (en) | Activin-ActRII antagonists and uses for increasing red blood cell levels | |
EP3308796A1 (en) | Activin-actrii antagonists and uses for treating bone and other disorders | |
AU2016250354B2 (en) | Activin-ActRII antagonists and uses for increasing red blood cell levels | |
NZ743635B2 (en) | Biomarker for use in treating anemia | |
US20230142386A1 (en) | Activin-actrii antagonists and uses for treating anemia | |
NZ743635A (en) | Biomarker for use in treating anemia | |
NZ747422B2 (en) | Activin-actrii antagonists and uses for treating bone and other disorders |