CA2328463A1 - Matrix binding factor - Google Patents
Matrix binding factor Download PDFInfo
- Publication number
- CA2328463A1 CA2328463A1 CA002328463A CA2328463A CA2328463A1 CA 2328463 A1 CA2328463 A1 CA 2328463A1 CA 002328463 A CA002328463 A CA 002328463A CA 2328463 A CA2328463 A CA 2328463A CA 2328463 A1 CA2328463 A1 CA 2328463A1
- Authority
- CA
- Canada
- Prior art keywords
- mbf
- factor
- igfs
- igf
- binding
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 239000011159 matrix material Substances 0.000 title claims abstract description 16
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 60
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 54
- 229920001184 polypeptide Polymers 0.000 claims abstract description 53
- 230000000975 bioactive effect Effects 0.000 claims abstract description 49
- 238000000034 method Methods 0.000 claims abstract description 49
- 230000000694 effects Effects 0.000 claims abstract description 26
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 24
- 238000013508 migration Methods 0.000 claims abstract description 23
- 239000000203 mixture Substances 0.000 claims abstract description 19
- 238000004519 manufacturing process Methods 0.000 claims abstract description 17
- 230000005012 migration Effects 0.000 claims abstract description 17
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 15
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 15
- 239000003102 growth factor Substances 0.000 claims abstract description 12
- 230000008685 targeting Effects 0.000 claims abstract description 8
- 230000004071 biological effect Effects 0.000 claims abstract description 7
- 238000006467 substitution reaction Methods 0.000 claims abstract description 6
- 238000007792 addition Methods 0.000 claims abstract description 5
- 239000007943 implant Substances 0.000 claims abstract description 5
- 238000012217 deletion Methods 0.000 claims abstract description 4
- 230000037430 deletion Effects 0.000 claims abstract description 4
- 210000004027 cell Anatomy 0.000 claims description 71
- 210000001519 tissue Anatomy 0.000 claims description 52
- 108020001507 fusion proteins Proteins 0.000 claims description 33
- 102000037865 fusion proteins Human genes 0.000 claims description 30
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims description 25
- 229960002897 heparin Drugs 0.000 claims description 25
- 229920000669 heparin Polymers 0.000 claims description 25
- 150000001413 amino acids Chemical group 0.000 claims description 24
- 108091034117 Oligonucleotide Proteins 0.000 claims description 19
- 239000004033 plastic Substances 0.000 claims description 18
- 229920003023 plastic Polymers 0.000 claims description 18
- 230000012010 growth Effects 0.000 claims description 17
- 239000002299 complementary DNA Substances 0.000 claims description 15
- 239000002773 nucleotide Substances 0.000 claims description 15
- 125000003729 nucleotide group Chemical group 0.000 claims description 15
- 239000013615 primer Substances 0.000 claims description 15
- 239000013604 expression vector Substances 0.000 claims description 14
- 210000003000 inclusion body Anatomy 0.000 claims description 14
- 102000004169 proteins and genes Human genes 0.000 claims description 14
- 108090000623 proteins and genes Proteins 0.000 claims description 14
- 239000003155 DNA primer Substances 0.000 claims description 11
- 108090000386 Fibroblast Growth Factor 1 Proteins 0.000 claims description 11
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 11
- 239000003814 drug Substances 0.000 claims description 11
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 9
- 241000251539 Vertebrata <Metazoa> Species 0.000 claims description 9
- 230000001580 bacterial effect Effects 0.000 claims description 9
- 230000004807 localization Effects 0.000 claims description 9
- 238000002703 mutagenesis Methods 0.000 claims description 9
- 231100000350 mutagenesis Toxicity 0.000 claims description 9
- 230000002265 prevention Effects 0.000 claims description 9
- 108010035532 Collagen Proteins 0.000 claims description 8
- 102000008186 Collagen Human genes 0.000 claims description 8
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 claims description 8
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 claims description 8
- 230000010261 cell growth Effects 0.000 claims description 8
- 229920001436 collagen Polymers 0.000 claims description 8
- 210000002744 extracellular matrix Anatomy 0.000 claims description 8
- 230000001771 impaired effect Effects 0.000 claims description 8
- 238000001727 in vivo Methods 0.000 claims description 8
- 230000003871 intestinal function Effects 0.000 claims description 8
- -1 medicated body wash Substances 0.000 claims description 8
- 102100037362 Fibronectin Human genes 0.000 claims description 7
- 108010067306 Fibronectins Proteins 0.000 claims description 7
- 230000012292 cell migration Effects 0.000 claims description 7
- 239000013599 cloning vector Substances 0.000 claims description 7
- 208000014674 injury Diseases 0.000 claims description 7
- 102000028416 insulin-like growth factor binding Human genes 0.000 claims description 7
- 108091022911 insulin-like growth factor binding Proteins 0.000 claims description 7
- 230000029663 wound healing Effects 0.000 claims description 7
- 101100076157 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) mbf-1 gene Proteins 0.000 claims description 6
- 230000021164 cell adhesion Effects 0.000 claims description 6
- 239000013612 plasmid Substances 0.000 claims description 6
- BBNQQADTFFCFGB-UHFFFAOYSA-N purpurin Chemical compound C1=CC=C2C(=O)C3=C(O)C(O)=CC(O)=C3C(=O)C2=C1 BBNQQADTFFCFGB-UHFFFAOYSA-N 0.000 claims description 6
- 230000008733 trauma Effects 0.000 claims description 6
- 239000004471 Glycine Substances 0.000 claims description 5
- 102000018997 Growth Hormone Human genes 0.000 claims description 5
- 108010051696 Growth Hormone Proteins 0.000 claims description 5
- 108010031318 Vitronectin Proteins 0.000 claims description 5
- 102100035140 Vitronectin Human genes 0.000 claims description 5
- 239000012634 fragment Substances 0.000 claims description 5
- 239000000122 growth hormone Substances 0.000 claims description 5
- 229910052751 metal Inorganic materials 0.000 claims description 5
- 239000002184 metal Substances 0.000 claims description 5
- 150000002739 metals Chemical class 0.000 claims description 5
- 230000017423 tissue regeneration Effects 0.000 claims description 5
- 230000001131 transforming effect Effects 0.000 claims description 5
- 229920001287 Chondroitin sulfate Polymers 0.000 claims description 4
- 229920002307 Dextran Polymers 0.000 claims description 4
- 229920002971 Heparan sulfate Polymers 0.000 claims description 4
- 101000871708 Homo sapiens Proheparin-binding EGF-like growth factor Proteins 0.000 claims description 4
- 102000007547 Laminin Human genes 0.000 claims description 4
- 108010085895 Laminin Proteins 0.000 claims description 4
- 241001465754 Metazoa Species 0.000 claims description 4
- 102100033762 Proheparin-binding EGF-like growth factor Human genes 0.000 claims description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 4
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 4
- 238000003780 insertion Methods 0.000 claims description 4
- 230000037431 insertion Effects 0.000 claims description 4
- 230000004048 modification Effects 0.000 claims description 4
- 238000012986 modification Methods 0.000 claims description 4
- 238000002741 site-directed mutagenesis Methods 0.000 claims description 4
- 229910021653 sulphate ion Inorganic materials 0.000 claims description 4
- 101000846393 Bos taurus Fibroblast growth factor 1 Proteins 0.000 claims description 3
- 102000018233 Fibroblast Growth Factor Human genes 0.000 claims description 3
- 108050007372 Fibroblast Growth Factor Proteins 0.000 claims description 3
- 241000238631 Hexapoda Species 0.000 claims description 3
- 102100027619 Histidine-rich glycoprotein Human genes 0.000 claims description 3
- 210000002469 basement membrane Anatomy 0.000 claims description 3
- 239000000919 ceramic Substances 0.000 claims description 3
- 238000009472 formulation Methods 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 108010044853 histidine-rich proteins Proteins 0.000 claims description 3
- 229910052588 hydroxylapatite Inorganic materials 0.000 claims 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 claims description 3
- 230000001737 promoting effect Effects 0.000 claims description 3
- FPJHWYCPAOPVIV-VOZMEZHOSA-N (2R,3S,4R,5R,6R)-6-[(2R,3R,4R,5R,6R)-5-acetamido-2-(hydroxymethyl)-6-methoxy-3-sulfooxyoxan-4-yl]oxy-4,5-dihydroxy-3-methoxyoxane-2-carboxylic acid Chemical compound CO[C@@H]1O[C@H](CO)[C@H](OS(O)(=O)=O)[C@H](O[C@@H]2O[C@H]([C@@H](OC)[C@H](O)[C@H]2O)C(O)=O)[C@H]1NC(C)=O FPJHWYCPAOPVIV-VOZMEZHOSA-N 0.000 claims description 2
- 229920000045 Dermatan sulfate Polymers 0.000 claims description 2
- 102100031706 Fibroblast growth factor 1 Human genes 0.000 claims description 2
- 239000004472 Lysine Substances 0.000 claims description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 2
- 108020004511 Recombinant DNA Proteins 0.000 claims description 2
- 230000032683 aging Effects 0.000 claims description 2
- 125000000129 anionic group Chemical group 0.000 claims description 2
- 230000005865 ionizing radiation Effects 0.000 claims description 2
- 239000006210 lotion Substances 0.000 claims description 2
- 229910001092 metal group alloy Inorganic materials 0.000 claims description 2
- 239000002324 mouth wash Substances 0.000 claims description 2
- 229940051866 mouthwash Drugs 0.000 claims description 2
- 229920000307 polymer substrate Polymers 0.000 claims description 2
- 239000000843 powder Substances 0.000 claims description 2
- 150000004760 silicates Chemical class 0.000 claims description 2
- 230000037380 skin damage Effects 0.000 claims description 2
- 230000009885 systemic effect Effects 0.000 claims description 2
- 229940034610 toothpaste Drugs 0.000 claims description 2
- 239000000606 toothpaste Substances 0.000 claims description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims 2
- 208000028169 periodontal disease Diseases 0.000 claims 2
- 230000000699 topical effect Effects 0.000 claims 2
- 102000004877 Insulin Human genes 0.000 claims 1
- 108090001061 Insulin Proteins 0.000 claims 1
- 238000004113 cell culture Methods 0.000 claims 1
- 239000002537 cosmetic Substances 0.000 claims 1
- 239000006071 cream Substances 0.000 claims 1
- GICLSALZHXCILJ-UHFFFAOYSA-N ctk5a5089 Chemical group NCC(O)=O.NCC(O)=O GICLSALZHXCILJ-UHFFFAOYSA-N 0.000 claims 1
- 229940126864 fibroblast growth factor Drugs 0.000 claims 1
- 229940029303 fibroblast growth factor-1 Drugs 0.000 claims 1
- 229940125396 insulin Drugs 0.000 claims 1
- 238000007911 parenteral administration Methods 0.000 claims 1
- 125000006850 spacer group Chemical group 0.000 claims 1
- 239000013589 supplement Substances 0.000 claims 1
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 abstract description 80
- 102000013275 Somatomedins Human genes 0.000 abstract description 16
- 125000003275 alpha amino acid group Chemical group 0.000 abstract description 12
- 239000000758 substrate Substances 0.000 abstract description 11
- 230000001413 cellular effect Effects 0.000 abstract description 8
- 239000000126 substance Substances 0.000 abstract description 6
- 230000004069 differentiation Effects 0.000 abstract description 5
- 230000035755 proliferation Effects 0.000 abstract description 4
- 238000013461 design Methods 0.000 abstract description 2
- 230000001575 pathological effect Effects 0.000 abstract description 2
- 238000002360 preparation method Methods 0.000 abstract description 2
- 102000004218 Insulin-Like Growth Factor I Human genes 0.000 description 63
- 235000001014 amino acid Nutrition 0.000 description 19
- 230000014616 translation Effects 0.000 description 19
- 229940024606 amino acid Drugs 0.000 description 18
- 238000001243 protein synthesis Methods 0.000 description 18
- 230000000638 stimulation Effects 0.000 description 14
- 230000000692 anti-sense effect Effects 0.000 description 13
- 239000002609 medium Substances 0.000 description 13
- 235000018102 proteins Nutrition 0.000 description 13
- 239000000243 solution Substances 0.000 description 13
- 108020004414 DNA Proteins 0.000 description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 10
- 230000008569 process Effects 0.000 description 10
- 102000003971 Fibroblast Growth Factor 1 Human genes 0.000 description 9
- 210000002919 epithelial cell Anatomy 0.000 description 9
- 108010073385 Fibrin Proteins 0.000 description 8
- 102000009123 Fibrin Human genes 0.000 description 8
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 8
- 102000048143 Insulin-Like Growth Factor II Human genes 0.000 description 8
- 108090001117 Insulin-Like Growth Factor II Proteins 0.000 description 8
- 238000007796 conventional method Methods 0.000 description 8
- 229950003499 fibrin Drugs 0.000 description 8
- 230000000717 retained effect Effects 0.000 description 8
- 239000007983 Tris buffer Substances 0.000 description 7
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 7
- 210000003098 myoblast Anatomy 0.000 description 7
- 239000002953 phosphate buffered saline Substances 0.000 description 7
- 150000003839 salts Chemical class 0.000 description 7
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 239000004743 Polypropylene Substances 0.000 description 6
- 241000700159 Rattus Species 0.000 description 6
- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 description 6
- 238000003776 cleavage reaction Methods 0.000 description 6
- 108010016616 cysteinylglycine Proteins 0.000 description 6
- 239000012091 fetal bovine serum Substances 0.000 description 6
- 239000000499 gel Substances 0.000 description 6
- 230000001965 increasing effect Effects 0.000 description 6
- 230000007017 scission Effects 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- 210000002435 tendon Anatomy 0.000 description 6
- 239000010936 titanium Substances 0.000 description 6
- 229910052719 titanium Inorganic materials 0.000 description 6
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 5
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 5
- 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 5
- 108010031794 IGF Type 1 Receptor Proteins 0.000 description 5
- 102100039688 Insulin-like growth factor 1 receptor Human genes 0.000 description 5
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 5
- 230000006907 apoptotic process Effects 0.000 description 5
- 238000001574 biopsy Methods 0.000 description 5
- 239000004202 carbamide Substances 0.000 description 5
- 239000011248 coating agent Substances 0.000 description 5
- 238000000576 coating method Methods 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 231100000673 dose–response relationship Toxicity 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 229920001155 polypropylene Polymers 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 102000005962 receptors Human genes 0.000 description 5
- 108020003175 receptors Proteins 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-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
- 239000012901 Milli-Q water Substances 0.000 description 4
- SEQKRHFRPICQDD-UHFFFAOYSA-N N-tris(hydroxymethyl)methylglycine Chemical compound OCC(CO)(CO)[NH2+]CC([O-])=O SEQKRHFRPICQDD-UHFFFAOYSA-N 0.000 description 4
- YYKZDTVQHTUKDW-RYUDHWBXSA-N Phe-Gly-Gln Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)NCC(=O)N[C@@H](CCC(=O)N)C(=O)O)N YYKZDTVQHTUKDW-RYUDHWBXSA-N 0.000 description 4
- ODXKUIGEPAGKKV-KATARQTJSA-N Thr-Leu-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)O)N)O ODXKUIGEPAGKKV-KATARQTJSA-N 0.000 description 4
- 229920004890 Triton X-100 Polymers 0.000 description 4
- 239000013504 Triton X-100 Substances 0.000 description 4
- 229940098773 bovine serum albumin Drugs 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 229960004407 chorionic gonadotrophin Drugs 0.000 description 4
- 238000004090 dissolution Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- 238000005227 gel permeation chromatography Methods 0.000 description 4
- 230000014759 maintenance of location Effects 0.000 description 4
- 238000010369 molecular cloning Methods 0.000 description 4
- 210000005152 placental membrane Anatomy 0.000 description 4
- 230000002285 radioactive effect Effects 0.000 description 4
- 108010007375 seryl-seryl-seryl-arginine Proteins 0.000 description 4
- 229940124597 therapeutic agent Drugs 0.000 description 4
- SUMYEVXWCAYLLJ-GUBZILKMSA-N Ala-Leu-Gln Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O SUMYEVXWCAYLLJ-GUBZILKMSA-N 0.000 description 3
- OTZMRMHZCMZOJZ-SRVKXCTJSA-N Arg-Leu-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O OTZMRMHZCMZOJZ-SRVKXCTJSA-N 0.000 description 3
- 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 3
- MFVQGXGQRIXBPK-WDSKDSINSA-N Gly-Ala-Glu Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O MFVQGXGQRIXBPK-WDSKDSINSA-N 0.000 description 3
- GWCRIHNSVMOBEQ-BQBZGAKWSA-N Gly-Arg-Ser Chemical compound [H]NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(O)=O GWCRIHNSVMOBEQ-BQBZGAKWSA-N 0.000 description 3
- IWAXHBCACVWNHT-BQBZGAKWSA-N Gly-Asp-Arg Chemical compound NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N IWAXHBCACVWNHT-BQBZGAKWSA-N 0.000 description 3
- JJGBXTYGTKWGAT-YUMQZZPRSA-N Gly-Pro-Glu Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O JJGBXTYGTKWGAT-YUMQZZPRSA-N 0.000 description 3
- 239000012981 Hank's balanced salt solution Substances 0.000 description 3
- CGHXMODRYJISSK-NHCYSSNCSA-N Leu-Val-Asp Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC(O)=O CGHXMODRYJISSK-NHCYSSNCSA-N 0.000 description 3
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 3
- 108010047562 NGR peptide Proteins 0.000 description 3
- DXWNFNOPBYAFRM-IHRRRGAJSA-N Phe-Val-Cys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N DXWNFNOPBYAFRM-IHRRRGAJSA-N 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 210000000988 bone and bone Anatomy 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 230000036755 cellular response Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 3
- 230000002860 competitive effect Effects 0.000 description 3
- 238000006073 displacement reaction Methods 0.000 description 3
- 229960001484 edetic acid Drugs 0.000 description 3
- 210000001035 gastrointestinal tract Anatomy 0.000 description 3
- JYPCXBJRLBHWME-UHFFFAOYSA-N glycyl-L-prolyl-L-arginine Natural products NCC(=O)N1CCCC1C(=O)NC(CCCN=C(N)N)C(O)=O JYPCXBJRLBHWME-UHFFFAOYSA-N 0.000 description 3
- 108010079413 glycyl-prolyl-glutamic acid Proteins 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 238000012423 maintenance Methods 0.000 description 3
- 241001515942 marmosets Species 0.000 description 3
- 210000001074 muscle attachment cell Anatomy 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 229920000729 poly(L-lysine) polymer Polymers 0.000 description 3
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 3
- 238000002203 pretreatment Methods 0.000 description 3
- 230000008439 repair process Effects 0.000 description 3
- 239000012679 serum free medium Substances 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 230000035882 stress Effects 0.000 description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- DBKNLHKEVPZVQC-LPEHRKFASA-N Arg-Ala-Pro Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@@H]1C(O)=O DBKNLHKEVPZVQC-LPEHRKFASA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- KLWPJMFMVPTNCC-UHFFFAOYSA-N Camptothecin Natural products CCC1(O)C(=O)OCC2=C1C=C3C4Nc5ccccc5C=C4CN3C2=O KLWPJMFMVPTNCC-UHFFFAOYSA-N 0.000 description 2
- UDDITVWSXPEAIQ-IHRRRGAJSA-N Cys-Phe-Arg Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O UDDITVWSXPEAIQ-IHRRRGAJSA-N 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- QAMMIGULQSIRCD-IRXDYDNUSA-N Gly-Phe-Tyr Chemical compound C([C@H](NC(=O)C[NH3+])C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C([O-])=O)C1=CC=CC=C1 QAMMIGULQSIRCD-IRXDYDNUSA-N 0.000 description 2
- WCORRBXVISTKQL-WHFBIAKZSA-N Gly-Ser-Ser Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O WCORRBXVISTKQL-WHFBIAKZSA-N 0.000 description 2
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 2
- 108010065920 Insulin Lispro Proteins 0.000 description 2
- RIJCHEVHFWMDKD-SRVKXCTJSA-N Lys-Lys-Asn Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O RIJCHEVHFWMDKD-SRVKXCTJSA-N 0.000 description 2
- YJNDFEWPGLNLNH-IHRRRGAJSA-N Met-Tyr-Cys Chemical compound CSCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CS)C(O)=O)CC1=CC=C(O)C=C1 YJNDFEWPGLNLNH-IHRRRGAJSA-N 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- KAHUBGWSIQNZQQ-KKUMJFAQSA-N Phe-Asn-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 KAHUBGWSIQNZQQ-KKUMJFAQSA-N 0.000 description 2
- FRMKIPSIZSFTTE-HJOGWXRNSA-N Phe-Tyr-Phe Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O FRMKIPSIZSFTTE-HJOGWXRNSA-N 0.000 description 2
- DCHQYSOGURGJST-FJXKBIBVSA-N Pro-Thr-Gly Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O DCHQYSOGURGJST-FJXKBIBVSA-N 0.000 description 2
- 102100033237 Pro-epidermal growth factor Human genes 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 108010067787 Proteoglycans Proteins 0.000 description 2
- 102000016611 Proteoglycans Human genes 0.000 description 2
- 101710090749 Purpurin Proteins 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- NLQUOHDCLSFABG-GUBZILKMSA-N Ser-Arg-Arg Chemical compound NC(N)=NCCC[C@H](NC(=O)[C@H](CO)N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O NLQUOHDCLSFABG-GUBZILKMSA-N 0.000 description 2
- KNCJWSPMTFFJII-ZLUOBGJFSA-N Ser-Cys-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(O)=O)C(O)=O KNCJWSPMTFFJII-ZLUOBGJFSA-N 0.000 description 2
- UZMAPBJVXOGOFT-UHFFFAOYSA-N Syringetin Natural products COC1=C(O)C(OC)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 UZMAPBJVXOGOFT-UHFFFAOYSA-N 0.000 description 2
- IMULJHHGAUZZFE-MBLNEYKQSA-N Thr-Gly-Ile Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(O)=O IMULJHHGAUZZFE-MBLNEYKQSA-N 0.000 description 2
- 108090000190 Thrombin Proteins 0.000 description 2
- 239000007997 Tricine buffer Substances 0.000 description 2
- AZGZDDNKFFUDEH-QWRGUYRKSA-N Tyr-Gly-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC1=CC=C(O)C=C1 AZGZDDNKFFUDEH-QWRGUYRKSA-N 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 108010069020 alanyl-prolyl-glycine Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 210000002805 bone matrix Anatomy 0.000 description 2
- VSJKWCGYPAHWDS-FQEVSTJZSA-N camptothecin Chemical compound C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-FQEVSTJZSA-N 0.000 description 2
- 229940127093 camptothecin Drugs 0.000 description 2
- 210000000845 cartilage Anatomy 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 230000033077 cellular process Effects 0.000 description 2
- 238000011210 chromatographic step Methods 0.000 description 2
- 210000002808 connective tissue Anatomy 0.000 description 2
- 239000000356 contaminant Substances 0.000 description 2
- 108010004073 cysteinylcysteine Proteins 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000011033 desalting Methods 0.000 description 2
- KCFYHBSOLOXZIF-UHFFFAOYSA-N dihydrochrysin Natural products COC1=C(O)C(OC)=CC(C2OC3=CC(O)=CC(O)=C3C(=O)C2)=C1 KCFYHBSOLOXZIF-UHFFFAOYSA-N 0.000 description 2
- VSJKWCGYPAHWDS-UHFFFAOYSA-N dl-camptothecin Natural products C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)C5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-UHFFFAOYSA-N 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 108010079547 glutamylmethionine Proteins 0.000 description 2
- XBGGUPMXALFZOT-UHFFFAOYSA-N glycyl-L-tyrosine hemihydrate Natural products NCC(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-UHFFFAOYSA-N 0.000 description 2
- 108010025801 glycyl-prolyl-arginine Proteins 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 210000003041 ligament Anatomy 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 231100000219 mutagenic Toxicity 0.000 description 2
- 230000003505 mutagenic effect Effects 0.000 description 2
- 230000001590 oxidative effect Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 238000007828 protein synthesis assay Methods 0.000 description 2
- 230000003362 replicative effect Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 108010048397 seryl-lysyl-leucine Proteins 0.000 description 2
- 238000001542 size-exclusion chromatography Methods 0.000 description 2
- 210000003491 skin Anatomy 0.000 description 2
- FRGKKTITADJNOE-UHFFFAOYSA-N sulfanyloxyethane Chemical compound CCOS FRGKKTITADJNOE-UHFFFAOYSA-N 0.000 description 2
- 229960004072 thrombin Drugs 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 239000011592 zinc chloride Substances 0.000 description 2
- 235000005074 zinc chloride Nutrition 0.000 description 2
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- HXNNRBHASOSVPG-GUBZILKMSA-N Ala-Glu-Leu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O HXNNRBHASOSVPG-GUBZILKMSA-N 0.000 description 1
- FEGOCLZUJUFCHP-CIUDSAMLSA-N Ala-Pro-Gln Chemical compound [H]N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(O)=O FEGOCLZUJUFCHP-CIUDSAMLSA-N 0.000 description 1
- ADSGHMXEAZJJNF-DCAQKATOSA-N Ala-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](C)N ADSGHMXEAZJJNF-DCAQKATOSA-N 0.000 description 1
- BTRULDJUUVGRNE-DCAQKATOSA-N Ala-Pro-Lys Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(O)=O BTRULDJUUVGRNE-DCAQKATOSA-N 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- 102000000412 Annexin Human genes 0.000 description 1
- 108050008874 Annexin Proteins 0.000 description 1
- 102000004411 Antithrombin III Human genes 0.000 description 1
- 108090000935 Antithrombin III Proteins 0.000 description 1
- VNFWDYWTSHFRRG-SRVKXCTJSA-N Arg-Gln-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O VNFWDYWTSHFRRG-SRVKXCTJSA-N 0.000 description 1
- IRRMIGDCPOPZJW-ULQDDVLXSA-N Arg-His-Phe Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O IRRMIGDCPOPZJW-ULQDDVLXSA-N 0.000 description 1
- AUIJUTGLPVHIRT-FXQIFTODSA-N Arg-Ser-Cys Chemical compound C(C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)O)N)CN=C(N)N AUIJUTGLPVHIRT-FXQIFTODSA-N 0.000 description 1
- JOTRDIXZHNQYGP-DCAQKATOSA-N Arg-Ser-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CCCN=C(N)N)N JOTRDIXZHNQYGP-DCAQKATOSA-N 0.000 description 1
- KSHJMDSNSKDJPU-QTKMDUPCSA-N Arg-Thr-His Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 KSHJMDSNSKDJPU-QTKMDUPCSA-N 0.000 description 1
- GIQCDTKOIPUDSG-GARJFASQSA-N Asn-Lys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CC(=O)N)N)C(=O)O GIQCDTKOIPUDSG-GARJFASQSA-N 0.000 description 1
- IDUUACUJKUXKKD-VEVYYDQMSA-N Asn-Pro-Thr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O IDUUACUJKUXKKD-VEVYYDQMSA-N 0.000 description 1
- RATOMFTUDRYMKX-ACZMJKKPSA-N Asp-Glu-Cys Chemical compound C(CC(=O)O)[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC(=O)O)N RATOMFTUDRYMKX-ACZMJKKPSA-N 0.000 description 1
- SCQIQCWLOMOEFP-DCAQKATOSA-N Asp-Leu-Arg Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O SCQIQCWLOMOEFP-DCAQKATOSA-N 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 101800003265 Beta-thromboglobulin Proteins 0.000 description 1
- 102400001362 Beta-thromboglobulin Human genes 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 101800004419 Cleaved form Proteins 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- PKNIZMPLMSKROD-BIIVOSGPSA-N Cys-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CS)N PKNIZMPLMSKROD-BIIVOSGPSA-N 0.000 description 1
- YMBAVNPKBWHDAW-CIUDSAMLSA-N Cys-Asp-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CS)N YMBAVNPKBWHDAW-CIUDSAMLSA-N 0.000 description 1
- QJUDRFBUWAGUSG-SRVKXCTJSA-N Cys-Cys-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CS)N QJUDRFBUWAGUSG-SRVKXCTJSA-N 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 101001124059 Drosophila melanogaster Vesicle-fusing ATPase 2 Proteins 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 101800003838 Epidermal growth factor Proteins 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 102100033299 Glia-derived nexin Human genes 0.000 description 1
- MLZRSFQRBDNJON-GUBZILKMSA-N Gln-Ala-Lys Chemical compound C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)N)N MLZRSFQRBDNJON-GUBZILKMSA-N 0.000 description 1
- ININBLZFFVOQIO-JHEQGTHGSA-N Gln-Thr-Gly Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CCC(=O)N)N)O ININBLZFFVOQIO-JHEQGTHGSA-N 0.000 description 1
- MXPBQDFWIMBACQ-ACZMJKKPSA-N Glu-Cys-Cys Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CS)C(O)=O MXPBQDFWIMBACQ-ACZMJKKPSA-N 0.000 description 1
- AQNYKMCFCCZEEL-JYJNAYRXSA-N Glu-Lys-Tyr Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 AQNYKMCFCCZEEL-JYJNAYRXSA-N 0.000 description 1
- CLNSYANKYVMZNM-UWVGGRQHSA-N Gly-Lys-Arg Chemical compound NCCCC[C@H](NC(=O)CN)C(=O)N[C@H](C(O)=O)CCCN=C(N)N CLNSYANKYVMZNM-UWVGGRQHSA-N 0.000 description 1
- DNVDEMWIYLVIQU-RCOVLWMOSA-N Gly-Val-Asp Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O DNVDEMWIYLVIQU-RCOVLWMOSA-N 0.000 description 1
- 108090000481 Heparin Cofactor II Proteins 0.000 description 1
- 102100030500 Heparin cofactor 2 Human genes 0.000 description 1
- BSVLMPMIXPQNKC-KBPBESRZSA-N His-Phe-Gly Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)NCC(O)=O BSVLMPMIXPQNKC-KBPBESRZSA-N 0.000 description 1
- 101000997803 Homo sapiens Glia-derived nexin Proteins 0.000 description 1
- 101000599951 Homo sapiens Insulin-like growth factor I Proteins 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- 208000019468 Iatrogenic Disease Diseases 0.000 description 1
- QRTVJGKXFSYJGW-KBIXCLLPSA-N Ile-Glu-Cys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CS)C(=O)O)N QRTVJGKXFSYJGW-KBIXCLLPSA-N 0.000 description 1
- BCISUQVFDGYZBO-QSFUFRPTSA-N Ile-Val-Asp Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC(O)=O BCISUQVFDGYZBO-QSFUFRPTSA-N 0.000 description 1
- 208000022559 Inflammatory bowel disease Diseases 0.000 description 1
- 102000008070 Interferon-gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- UGTHTQWIQKEDEH-BQBZGAKWSA-N L-alanyl-L-prolylglycine zwitterion Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O UGTHTQWIQKEDEH-BQBZGAKWSA-N 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- GLBNEGIOFRVRHO-JYJNAYRXSA-N Leu-Gln-Phe Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O GLBNEGIOFRVRHO-JYJNAYRXSA-N 0.000 description 1
- YFBBUHJJUXXZOF-UWVGGRQHSA-N Leu-Gly-Pro Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N1CCC[C@H]1C(O)=O YFBBUHJJUXXZOF-UWVGGRQHSA-N 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 108010013563 Lipoprotein Lipase Proteins 0.000 description 1
- 102100022119 Lipoprotein lipase Human genes 0.000 description 1
- HQVDJTYKCMIWJP-YUMQZZPRSA-N Lys-Asn-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O HQVDJTYKCMIWJP-YUMQZZPRSA-N 0.000 description 1
- RFQATBGBLDAKGI-VHSXEESVSA-N Lys-Gly-Pro Chemical compound C1C[C@@H](N(C1)C(=O)CNC(=O)[C@H](CCCCN)N)C(=O)O RFQATBGBLDAKGI-VHSXEESVSA-N 0.000 description 1
- NNKLKUUGESXCBS-KBPBESRZSA-N Lys-Gly-Tyr Chemical compound [H]N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O NNKLKUUGESXCBS-KBPBESRZSA-N 0.000 description 1
- VMTYLUGCXIEDMV-QWRGUYRKSA-N Lys-Leu-Gly Chemical compound OC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCCCN VMTYLUGCXIEDMV-QWRGUYRKSA-N 0.000 description 1
- XZFYRXDAULDNFX-UHFFFAOYSA-N N-L-cysteinyl-L-phenylalanine Natural products SCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XZFYRXDAULDNFX-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108010069196 Neural Cell Adhesion Molecules Proteins 0.000 description 1
- 102100023616 Neural cell adhesion molecule L1-like protein Human genes 0.000 description 1
- JEGFCFLCRSJCMA-IHRRRGAJSA-N Phe-Arg-Ser Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CO)C(=O)O)N JEGFCFLCRSJCMA-IHRRRGAJSA-N 0.000 description 1
- 102100024078 Plasma serine protease inhibitor Human genes 0.000 description 1
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 description 1
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 description 1
- ZSKJPKFTPQCPIH-RCWTZXSCSA-N Pro-Arg-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O ZSKJPKFTPQCPIH-RCWTZXSCSA-N 0.000 description 1
- XZONQWUEBAFQPO-HJGDQZAQSA-N Pro-Gln-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XZONQWUEBAFQPO-HJGDQZAQSA-N 0.000 description 1
- 108010001953 Protein C Inhibitor Proteins 0.000 description 1
- 229940122929 Protein C inhibitor Drugs 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 240000003186 Stachytarpheta cayennensis Species 0.000 description 1
- 235000009233 Stachytarpheta cayennensis Nutrition 0.000 description 1
- 208000002847 Surgical Wound Diseases 0.000 description 1
- 208000000491 Tendinopathy Diseases 0.000 description 1
- 206010043255 Tendonitis Diseases 0.000 description 1
- 201000008754 Tenosynovial giant cell tumor Diseases 0.000 description 1
- VYEHBMMAJFVTOI-JHEQGTHGSA-N Thr-Gly-Gln Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(O)=O VYEHBMMAJFVTOI-JHEQGTHGSA-N 0.000 description 1
- JQAWYCUUFIMTHE-WLTAIBSBSA-N Thr-Gly-Tyr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O JQAWYCUUFIMTHE-WLTAIBSBSA-N 0.000 description 1
- 102000002938 Thrombospondin Human genes 0.000 description 1
- 108060008245 Thrombospondin Proteins 0.000 description 1
- RGYDQHBLMMAYNZ-IHRRRGAJSA-N Tyr-Cys-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CC1=CC=C(C=C1)O)N RGYDQHBLMMAYNZ-IHRRRGAJSA-N 0.000 description 1
- ISERLACIZUGCDX-ZKWXMUAHSA-N Val-Asp-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C(C)C)N ISERLACIZUGCDX-ZKWXMUAHSA-N 0.000 description 1
- VLOYGOZDPGYWFO-LAEOZQHASA-N Val-Asp-Glu Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O VLOYGOZDPGYWFO-LAEOZQHASA-N 0.000 description 1
- FPCIBLUVDNXPJO-XPUUQOCRSA-N Val-Cys-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CS)C(=O)NCC(O)=O FPCIBLUVDNXPJO-XPUUQOCRSA-N 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 1
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 1
- 102100039037 Vascular endothelial growth factor A Human genes 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 229960005348 antithrombin iii Drugs 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 108010038633 aspartylglutamate Proteins 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000005341 cation exchange Methods 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 239000002801 charged material Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 239000003636 conditioned culture medium Substances 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 208000035647 diffuse type tenosynovial giant cell tumor Diseases 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 229940116977 epidermal growth factor Drugs 0.000 description 1
- 230000008378 epithelial damage Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 229940012952 fibrinogen Drugs 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 229940044627 gamma-interferon Drugs 0.000 description 1
- 230000002518 glial effect Effects 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 150000002308 glutamine derivatives Chemical class 0.000 description 1
- 108010010147 glycylglutamine Proteins 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 108010092114 histidylphenylalanine Proteins 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 102000044162 human IGF1 Human genes 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000010565 inoculated fermentation Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 108010078274 isoleucylvaline Proteins 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 210000002510 keratinocyte Anatomy 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 108010034529 leucyl-lysine Proteins 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 229940040145 liniment Drugs 0.000 description 1
- 239000000865 liniment Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000003141 lower extremity Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 108010054155 lysyllysine Proteins 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 238000013433 optimization analysis Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 230000002980 postoperative effect Effects 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 238000011533 pre-incubation Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 1
- 238000012342 propidium iodide staining Methods 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000000700 radioactive tracer Substances 0.000 description 1
- 238000002278 reconstructive surgery Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000001223 reverse osmosis Methods 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 238000013341 scale-up Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000013222 sprague-dawley male rat Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000007910 systemic administration Methods 0.000 description 1
- 238000010809 targeting technique Methods 0.000 description 1
- 201000004415 tendinitis Diseases 0.000 description 1
- 208000002918 testicular germ cell tumor Diseases 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 239000010409 thin film Substances 0.000 description 1
- 108010061238 threonyl-glycine Proteins 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 230000008736 traumatic injury Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000007306 turnover Effects 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/65—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0068—General culture methods using substrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Dermatology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Biophysics (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Transplantation (AREA)
- Epidemiology (AREA)
- Cell Biology (AREA)
- Endocrinology (AREA)
- Diabetes (AREA)
- General Engineering & Computer Science (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The present invention relates to the design, manufacture and use of novel polypeptide bioactive factors. More particularly, the invention relates to the targeting of polypeptide bioactive factors to biological or chemical substrates via engineered amino acid motifs, while maintaining or extending the biological activities attributed to that polypeptide bioactive factor. The invention provides a recombinant polypeptide bioactive factor, referred to as a matrix binding factor (MBF), comprising a polypeptide bioactive factor in which the naturally-occurring amino acid sequence of the factor has been modified to introduce one or more amino acid substitutions, deletions and/or additions which increase the affinity of the polypeptide bioactive factor for a negatively-charged surface. Preferably the polypeptide bioactive factor is a growth factor which stimulates proliferation, differentiation, migration or cellular activity, and most preferably is a member of the insulin-like growth factor (IGF) family of growth factors. The invention also provides nucleic acid molecules encoding polypeptide bioactive factors of the invention, methods for production of the factors, and compositions and methods for use of the factors in treatment of a variety of pathological conditions, for use in tissue culture and for the preparation and use of surgical implants or prostheses.
Description
MATRIX BINDING FACTOR
The present invention relates to the design, manufacture and use of novel polypeptide bioactive factors.
More particularly, the invention relates to the targeting of polypeptide bioactive factors to biological or chemical substrates via engineered amino acid motifs, while maintaining or extending the biological activities attributed to that polypeptide :bioactive factor.
l0 BACKGROUND OF THE INVENTION
Polypeptide bioactive factors, particularly growth factors, are crucial for many cellular processes.
Consequently, much research is undertaken in relation to the presence and mechanism of action of such factors in cellular processes, with the objective of identifying novel polypeptide bioactive factors, identifying growth factor synergies, and understanding the mechanisms of processes such as cellular maintenance, growth, development, and apoptosis. The potential usefulness of polypeptide bioactive factors, particularly growth factors, in diagnostics, pharmaceuticals and therapeutics is well recognised.
Considerable interest has been directed to a family of polypeptide bioactive factors, the insulin-like growth factors (IGFs). It is well established in the art that these proteins are structurally related, and that their expression patterns, localisation and bioactivity are important for the differentiation and proliferation of various cellular types. This has led to the suggestion that IGFs are pivotal for cellular migration and proliferation, for connective tissue production, and for turnover processes associated with tissue remodelling, repair and wound healing (Clemmons, D.R., British Medical Bulletin, 1989 45 465-480; Gartner et a1, J. Surg. Res., 1989 52 389-394).
The present invention relates to the design, manufacture and use of novel polypeptide bioactive factors.
More particularly, the invention relates to the targeting of polypeptide bioactive factors to biological or chemical substrates via engineered amino acid motifs, while maintaining or extending the biological activities attributed to that polypeptide :bioactive factor.
l0 BACKGROUND OF THE INVENTION
Polypeptide bioactive factors, particularly growth factors, are crucial for many cellular processes.
Consequently, much research is undertaken in relation to the presence and mechanism of action of such factors in cellular processes, with the objective of identifying novel polypeptide bioactive factors, identifying growth factor synergies, and understanding the mechanisms of processes such as cellular maintenance, growth, development, and apoptosis. The potential usefulness of polypeptide bioactive factors, particularly growth factors, in diagnostics, pharmaceuticals and therapeutics is well recognised.
Considerable interest has been directed to a family of polypeptide bioactive factors, the insulin-like growth factors (IGFs). It is well established in the art that these proteins are structurally related, and that their expression patterns, localisation and bioactivity are important for the differentiation and proliferation of various cellular types. This has led to the suggestion that IGFs are pivotal for cellular migration and proliferation, for connective tissue production, and for turnover processes associated with tissue remodelling, repair and wound healing (Clemmons, D.R., British Medical Bulletin, 1989 45 465-480; Gartner et a1, J. Surg. Res., 1989 52 389-394).
Despite the demonstrated effects of IGFs in cultured vertebrate cells and tissues, as yet there have been no observations in vivo which support any unequivocal role in tissue remodelling or wound healing for exogenously-administered IGFs, whether given systemically, locally or topically. It is therefore accepted in the art that there must be other moieties which influence the bioavailability of IGFs.
The bioavailability of a polypeptide bioactive l0 factor is a measure of that factor's ability to remain active at a site where it can effect a desired cellular response. Bioavailability is modulated by the stability, protease susceptibility and rate of clearance of a factor from the site where it interacts with its cellular receptors.
The bioavailability of IGFs can be modulated by one or more of the six insulin-like growth factor binding proteins (IGFBPs) which are so far known. Furthermore, the bioavailability of an IGF may also be modified by structural changes to the amino acid sequence of the polypeptide. As a hypothetical example, introducing an affinity for one or more biological or chemical substrates may serve to slow or prevent IGF clearance from the local environment, or may even localise an IGF in a biologically active and accessible form. Moreover, this general concept may be extended to polypeptide bioactive factors other than IGFs.
The nature of many biological substrates, such as basement membrane, extracellular matrix (ECM), bone matrix 3o and other connective tissue components, is such that localised and more general patterns of negative charge are created. For example, both heparan sulphate proteoglycans present in the ECM and hydroxyapatite in bone have net negative charges. These charged moieties provide sites of attachment for factors with the appropriate affinities, as determined by their amino acid sequences. The potential of factors able to elicit cell growth effects at the site of such localisation offers opportunities to create a significant improvement in the delivery of therapeutic agents to their preferred site of action. Similarly, therapeutic agents that are retained at the preferred site of action when administered locally may prove useful for the treatment of many conditions, such as bone or ligament grafts and tendonitis, or they may improve the post-operative recovery associated with tissue manipulations, cartilage repair and reconstructive surgery.
Gastrointestinal tract impairment associated with either iatrogenic disorders such as chemotherapy-induced epithelial damage or pathologies such as inflammatory bowel disease are significant causes of morbidity. Accordingly, therapeutic agents directed to the healing of the epithelial lining of the gastrointestinal tract are useful in the prevention and/or treatment of conditions associated with impaired gut function.
The ability of negatively-charged substrates, such as heparan sulphate proteoglycans, to protect bound polypeptides from degradation due to physical stresses such as temperature, chemical stresses such as low pH, and/or biological stresses such as protease degradation, as described in the prior art, provides potential mechanisms for the prevention and treatment of pathological states associated with impaired gut function.
Accordingly, there is a need in the medical and veterinary fields for novel delivery and targeting technologies which create local concentrations of bioactive factors .
SUN~IARY OF THE INVENTION
In a first aspect, the present invention provides a polypeptide bioactive factor, hereinafter referred to as a matrix binding factor (MBF), comprising a polypeptide bioactive factor in which the naturally-occurring amino acid sequence of the factor has been modified to introduce one or more amino acid substitutions, deletions and/or additions which increase the affinity of the polypeptide bioactive factor for a negatively-charged site or surface.
The term "polypeptide bioactive factor" means a polypeptide or small protein which either modulates cellular responses directly, as is the case with growth factors, or acts indirectly by its association with other components, including the extracellular matrix, such that cellular responses are potentiated. This term is also to be understood to encompass mutants, fragments and analogues of such a factor which retain at least one of the biological activities of the factor.
The term "negatively-charged surface" means any surface which displays an array of negative charge that provides association sites for positively-charged polypeptide motifs. Such negatively-charged surfaces may be of natural or artificial origin, and may be in an animal body. They include, but are not limited to, extracellular matrix, dextran sulphate, chondroitin sulphate, dermatan sulphate, collagen, fibronectin, vitronectin, laminin, heparan sulphates, heparin, hydroxyapatite, anionic plastics, silicates, and physiologically-compatible metals and other materials used in surgical implants or prostheses, such as stainless steel, titanium, metal alloys, ceramics, polymers and plastic coated metals.
Metals and ceramics are widely used in orthopaedic applications.
As an integral part of their biological activity, a number of polypeptide bioactive factors have affinities for sites other than their high affinity cellular receptors. For example, they may bind to cell attachment factors, basement membrane moieties, extracellular matrix components, or soluble circulating proteins.
Accordingly, the amino acid modifications include introduction of sequences which are deduced from motifs in the amino acid sequences of polypeptide bioactive factors which have been identified by their particular affinity for specific substrates. Such motifs include, but are not limited to, the heparin-binding amino acid motif characteristic of fibroblast growth factors (FGFs), heparin-binding epidermal growth factor, vitronectin, fibronectin, histidine-rich glycoprotein or purpurin.
Preferably the polypeptide bioactive factor into which changes are introduced in order to create an MBF is a polypeptide bioactive factor which does not already include similar sequence motifs which confer an ability to bind to negatively-charged surfaces.
More preferably the polypeptide bioactive factor into which changes are introduced in order to create an MBF
is a growth factor which stimulates proliferation, differentiation, migration or cellular activity, including, but not limited to, members of the insulin-like growth factor (IGF), transforming growth factor-~3, platelet-derived growth factor, vascular endothelial growth factor or epidermal growth factor families of growth factors.
The present invention also includes within its scope biologically-active mutants, analogues and derivatives of the MBF. Preferably such modified MBFs are in a biologically pure form.
The term "biologicall.y pure" as used herein means a product essentially devoid of unavoidable biologically active impurities or contaminants.
According to a second aspect, the invention provides a nucleic acid molecule whose sequence encodes a polypeptide bioactive factor of the invention. The nucleic acid molecule may be a cDNA, a genomic DNA, or an RNA, and may be in the sense or anti-sense orientation. Preferably the nucleic acid molecule is a cDNA, more preferably a sense cDNA.
In a third aspect, the invention provides a method for producing a recombinant MBF, comprising the steps of subjecting a cloning vector comprising a nucleic acid sequence encoding the polypeptide growth factor to mutagenesis to generate a nucleotide sequence encoding an MBF.
The bioavailability of a polypeptide bioactive l0 factor is a measure of that factor's ability to remain active at a site where it can effect a desired cellular response. Bioavailability is modulated by the stability, protease susceptibility and rate of clearance of a factor from the site where it interacts with its cellular receptors.
The bioavailability of IGFs can be modulated by one or more of the six insulin-like growth factor binding proteins (IGFBPs) which are so far known. Furthermore, the bioavailability of an IGF may also be modified by structural changes to the amino acid sequence of the polypeptide. As a hypothetical example, introducing an affinity for one or more biological or chemical substrates may serve to slow or prevent IGF clearance from the local environment, or may even localise an IGF in a biologically active and accessible form. Moreover, this general concept may be extended to polypeptide bioactive factors other than IGFs.
The nature of many biological substrates, such as basement membrane, extracellular matrix (ECM), bone matrix 3o and other connective tissue components, is such that localised and more general patterns of negative charge are created. For example, both heparan sulphate proteoglycans present in the ECM and hydroxyapatite in bone have net negative charges. These charged moieties provide sites of attachment for factors with the appropriate affinities, as determined by their amino acid sequences. The potential of factors able to elicit cell growth effects at the site of such localisation offers opportunities to create a significant improvement in the delivery of therapeutic agents to their preferred site of action. Similarly, therapeutic agents that are retained at the preferred site of action when administered locally may prove useful for the treatment of many conditions, such as bone or ligament grafts and tendonitis, or they may improve the post-operative recovery associated with tissue manipulations, cartilage repair and reconstructive surgery.
Gastrointestinal tract impairment associated with either iatrogenic disorders such as chemotherapy-induced epithelial damage or pathologies such as inflammatory bowel disease are significant causes of morbidity. Accordingly, therapeutic agents directed to the healing of the epithelial lining of the gastrointestinal tract are useful in the prevention and/or treatment of conditions associated with impaired gut function.
The ability of negatively-charged substrates, such as heparan sulphate proteoglycans, to protect bound polypeptides from degradation due to physical stresses such as temperature, chemical stresses such as low pH, and/or biological stresses such as protease degradation, as described in the prior art, provides potential mechanisms for the prevention and treatment of pathological states associated with impaired gut function.
Accordingly, there is a need in the medical and veterinary fields for novel delivery and targeting technologies which create local concentrations of bioactive factors .
SUN~IARY OF THE INVENTION
In a first aspect, the present invention provides a polypeptide bioactive factor, hereinafter referred to as a matrix binding factor (MBF), comprising a polypeptide bioactive factor in which the naturally-occurring amino acid sequence of the factor has been modified to introduce one or more amino acid substitutions, deletions and/or additions which increase the affinity of the polypeptide bioactive factor for a negatively-charged site or surface.
The term "polypeptide bioactive factor" means a polypeptide or small protein which either modulates cellular responses directly, as is the case with growth factors, or acts indirectly by its association with other components, including the extracellular matrix, such that cellular responses are potentiated. This term is also to be understood to encompass mutants, fragments and analogues of such a factor which retain at least one of the biological activities of the factor.
The term "negatively-charged surface" means any surface which displays an array of negative charge that provides association sites for positively-charged polypeptide motifs. Such negatively-charged surfaces may be of natural or artificial origin, and may be in an animal body. They include, but are not limited to, extracellular matrix, dextran sulphate, chondroitin sulphate, dermatan sulphate, collagen, fibronectin, vitronectin, laminin, heparan sulphates, heparin, hydroxyapatite, anionic plastics, silicates, and physiologically-compatible metals and other materials used in surgical implants or prostheses, such as stainless steel, titanium, metal alloys, ceramics, polymers and plastic coated metals.
Metals and ceramics are widely used in orthopaedic applications.
As an integral part of their biological activity, a number of polypeptide bioactive factors have affinities for sites other than their high affinity cellular receptors. For example, they may bind to cell attachment factors, basement membrane moieties, extracellular matrix components, or soluble circulating proteins.
Accordingly, the amino acid modifications include introduction of sequences which are deduced from motifs in the amino acid sequences of polypeptide bioactive factors which have been identified by their particular affinity for specific substrates. Such motifs include, but are not limited to, the heparin-binding amino acid motif characteristic of fibroblast growth factors (FGFs), heparin-binding epidermal growth factor, vitronectin, fibronectin, histidine-rich glycoprotein or purpurin.
Preferably the polypeptide bioactive factor into which changes are introduced in order to create an MBF is a polypeptide bioactive factor which does not already include similar sequence motifs which confer an ability to bind to negatively-charged surfaces.
More preferably the polypeptide bioactive factor into which changes are introduced in order to create an MBF
is a growth factor which stimulates proliferation, differentiation, migration or cellular activity, including, but not limited to, members of the insulin-like growth factor (IGF), transforming growth factor-~3, platelet-derived growth factor, vascular endothelial growth factor or epidermal growth factor families of growth factors.
The present invention also includes within its scope biologically-active mutants, analogues and derivatives of the MBF. Preferably such modified MBFs are in a biologically pure form.
The term "biologicall.y pure" as used herein means a product essentially devoid of unavoidable biologically active impurities or contaminants.
According to a second aspect, the invention provides a nucleic acid molecule whose sequence encodes a polypeptide bioactive factor of the invention. The nucleic acid molecule may be a cDNA, a genomic DNA, or an RNA, and may be in the sense or anti-sense orientation. Preferably the nucleic acid molecule is a cDNA, more preferably a sense cDNA.
In a third aspect, the invention provides a method for producing a recombinant MBF, comprising the steps of subjecting a cloning vector comprising a nucleic acid sequence encoding the polypeptide growth factor to mutagenesis to generate a nucleotide sequence encoding an MBF.
The nucleic acid sequence of the polypeptide bioactive factor must be subcloned into an appropriate cloning vector or plasmid. This permits mutagenesis of the DNA encoding a polypeptide growth factor to generate a sequence encoding a MBF, by means well known to those in the art.
Preferably the mutagenesis is achieved using site-directed mutagenesis, more preferably using oligonucleotide primers which are based on binding sites able to bind to negatively-charged surfaces, which binding sites are present in other polypeptide bioactive factors, or sequences substantially homologous thereto. Examples of polypeptide bioactive factors comprising such binding sites include vitronectin, 10 kilodalton gamma-interferon inducible protein, histidine-rich glycoprotein, purpurin, beta-thromboglobulin, antithrombin III, heparin cofactor II, FGF-1, FGF-2, heparin-binding epidermal growth factor, lipocortin, protein C inhibitor, fibronectin, thrombospondin, lipoprotein lipase, hepatic triglyceride lipase, vascular endothelial cell growth factor, thrombin, neural cell adhesion molecule and glial-derived nexin.
The altered nucleic acid sequence encoding an MBF
may be subcloned into a suitable expression vector, which may be introduced into host cells by conventional means familiar to those skilled in the art.
Thus the method of the invention preferably also comprises the steps of subcloning the nucleic acid sequence encoding the MBF into a suitable expression vector;
transforming the expression vector into a suitable bacterial, yeast or tissue culture host cell;
cultivating the host cell under conditions suitable to express the MBF; and isolating the MBF.
It will be appreciated that host cells comprising selected constructs so formed may express the MBF as a fusion protein within inclusion bodies (IB). By the term _ 7 _ "MBF fusion protein" we mean a polypeptide consisting of two linked protein components, one of which is selected so as to be expressed in the host cell under the control of a suitable promoter, and the other of which comprises the polypeptide bioactive factor incorporating the motif that confers MBF activity. The fusion protein is produced in order to facilitate the expression and/or processing of the amino acid sequence of the MBF activity. Preferably the MBF fusion protein is produced by an appropriate host cell in a fermenter by conventional means understood by those skilled in the art.
Preferably, the MBF is isolated from the host cell following disruption of the host cell by homogenisation, and processed to its biologically pure form using conventional methods of protein purification well recognised by those skilled in the art. These include oxidative refolding to achieve correct disulphide bonding, chemical cleavage of the fusion partner (if used) from the MBF, and various chromatographic steps. The MBF may be isolated as a biologically pure form of the fusion protein, and may then be cleaved from its fusion partner, yielding a peptide that is not extended.
In a preferred embodiment an MBF is prepared as a fusion protein using the methods described in our Australian Patent No. 633099, the entire disclosure of which is incorporated herein by reference. In this method a fragment of porcine growth hormone is linked to the N-terminal sequence of an MBF, optionally via a cleavable sequence.
In another preferred embodiment of the invention there is provided a process for the production of a cleavable MBF fusion protein, comprising the step of transforming a susceptible bacterial, yeast or tissue culture cell hosts with one or more recombinant DNA
plasmids which include DNA sequences capable of facilitating the expression of an MBF fusion protein.
_ g _ In a further preferred embodiment, the MBF fusion protein is expressed as an insoluble aggregate or inclusion body within the host cell, and isolated by cell disruption and centrifugation. The conventional methodologies which may be employed in the isolation of the MBF include fusion protein dissolution, oxidative refolding, hydroxylamine or proteolytic cleavage, and various chromatographic processes, including size exclusion chromatography, ion exchange chromatography, reversed-phase high performance liquid chromatography and affinity chromatography. Sample fractions may be collected from each purification step, with those exhibiting biological activity in an appropriate assay being pooled and carried forward to the next purification step. This may result in the isolation of biologically pure MBF with or without its fusion partner.
Preferably, the presence of biologically pure MBF
is detected by one or more of a) migration as a single band of the appropriate size on SDS-PAGE gel chromatography;
b) N-terminal sequence analysis, or c) mass spectroscopy.
The biologically pure MBF of the present invention is useful for a wide variety of purposes, including but not limited to maintenance, growth or differentiation of animal or human cells in culture;
maintenance, growth or differentiation of more organised cellular structures, for example, skin, cartilage, tendon, ligament or bone;
coating a negatively-charged surface to promote cell adhesion, growth, migration, or activity, for example culture vessels for use in keratinocyte expansion to provide partial thickness skin grafts for burns patients, or coating of a surgical implant or a prosthesis;
enhancement of tissue remodelling and repair associated with trauma or manipulation, for example in the _ g _ treatment of wounds of all types, including burns, traumatic injuries, or surgical wounds;
as an orally active product for the prevention and/or treatment of impaired gut function;
facilitating tissue targeting following systemic administration, for example by localisation of the factor to bone matrix following intravenous injection; and maintaining higher bioactive factor concentrations at the site of administration in order to effect a prolonged pharmacological action.
The MBF may be used in conjunction with or in combination with one or more other growth factors or therapeutic agents.
Accordingly, in a fourth aspect the present invention provides a composition for promoting adhesion, growth, migration, the prevention of apoptosis, or activity of cells in culture, comprising an effective amount of an MBF together with a pharmaceutically- or veterinarily-acceptable carrier.
In a preferred embodiment, this aspect of the invention provides a composition for the coating of a negatively-charged surface with an effective amount of MBF, thereby to promote adhesion, growth, migration or activity of cells.
In a fifth aspect the invention provides a method for promoting adhesion, growth, migration or activity of cells on a negatively-charged surface coated with an MBF, comprising the step of growing vertebrate cells in a culture medium over or on an appropriate surface pretreated with an MBF.
In both the fourth and fifth aspect of the invention the cells may be of vertebrate, preferably mammalian, or of insect origin.
In a sixth aspect, the invention provides a composition for the enhancement of tissue remodelling or tissue repair associated with tissue trauma or wound healing, comprising an effective amount of an MBF
formulated with a carrier such as an injectible, excipient, carrier, lotion, medicated body wash, dressing, liniment, toothpaste, mouthwash or powder.
In an alternative embodiment, this aspect of the invention provides a composition for alleviation of skin damage associated with ageing or with exposure to ultraviolet or ionizing radiation, comprising an effective amount of an MBF together with a cosmetically-acceptable carrier.
In a seventh aspect, the invention provides a composition for the prevention or treatment of a condition associated with impaired gut function, comprising an effective amount of an MBF formulated with a carrier suitable to produce an orally stable, bioactive enteral formulation.
In an eighth aspect, the invention provides a composition for the targeting or localisation of an MBF to cells or tissues, thereby to promote cell adhesion, growth, migration or activity in vivo, comprising an effective amount of MBF formulated in a pharmaceutically-acceptable carrier.
In a ninth aspect, the invention provides a method for the enhancement of tissue remodelling or tissue repair associated with tissue trauma or wound healing, comprising the step of administering an effective amount of an MBF to a subject in need of such treatment.
In a preferred embodiment, this aspect of the invention provides a method for the prevention or treatment of impaired gut function, comprising the step of administering an effective amount of an MBF to a subject in need of such treatment.
In a second preferred embodiment there is provided a method for the targeting and localisation of an MBF to cells or tissues, thereby to promote cell adhesion, growth, migration or activity in vivo, comprising the step of systemic or local administration of an MBF to a subject in need of such treatment:
It will be appreciated that in the therapeutic methods of the invention, the subject to be treated may be a human, or may be a domestic, companion or zoo animal.
The carrier to be used and the dose and route of administration will depend on the nature of the condition to be treated and the age and general health of the subject, and will be at the discretion of the attending physician or veterinarian. Suitable carriers and formulations are known in the art, for example by reference to Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Company, Easton, Pennsylvania (1995). Suitable dosing regimens are established using methods standard in the art.
For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the results of SDS/PAGE analysis of the biologically pure MBF, as visualised by Novex Tricine gel chromatography.
Figure 2a shows dose-response curves of the extended forms of MBFs, L-MBF-1, L-MBF-2, L-MBF-3, L-MBF-4 and the extended form of IGF-I (L-IGF-I) for the competitive displacement of iodinated IGF-I from the IGF-I
receptor isolated from human placental membranes.
Figure 2b shows dose-response curves of the cleaved forms of MBFs, MBF-1, MBF-2, MBF-3, MBF-4 and IGF-I
for the competitive displacement of iodinated IGF-I from the IGF-I receptor isolated from human placental membranes.
Figure 3a shows dose-response curves of four extended forms of MBFs, L-MBF-1, L-MBF-2, L-MBF-3, L-MBF-4, and L-IGF-I in a protein synthesis assay using a myoblast cell line.
Figure 3b shows dose-response curves of four cleaved forms of MBFs, MBF-1, MBF-2, MBF-3, MBF-4 and IGF-I
in a protein synthesis assay using a myoblast cell line.
DETAILED DESCRIPTION OF THE INVENTION
The present invention will now be more fully described with reference to the accompanying non-limiting examples. It should be understood that the following description is illustrative only, and should not be taken in any way as a restriction on the generality of the invention. In particular, while the invention is specifically exemplified with reference to MBFs derived from IGF-I, it will be clearly understood that the methods described herein are applicable generally to the modification of polypeptide bioactive factors to generate MBFs, and to evaluation of the MBFs thus produced for biological activity. In particular, once the MBF is produced its testing for suitability for the purposes of the invention is a matter of routine.
Representative MBFs according to the invention were produced by introduction of heparin-binding motifs of other polypeptide bioactive factors into the sequence of human IGF-1. The methods used are described in detail below. It will be clearly understood that while the examples utilize the heparin-binding motif from bovine FGF-1, FGF-1 sequences from the human protein or from other species may also be used.
Sequence MBF-1 involves the deletion of the IGF-1 D-domain post Pro63 and its substitution with the heparin-binding motif Lys127 to G1n142 from bovine FGF-1, represented by the single letter code for amino acids as shown below. The introduced heparin-binding motif is shown in bold.
GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEM
YCAPKKNGRSKLGPRTHFGQ
(SEQ ID NO. 1) Sequence MBF-2 contains all of the amino acids of native IGF-1, and Lys127 to G1n142 of FGF-1, through the insertion of the FGF-1 fragment Lys128 to G1y135 in between the residues Lys65 and Pro66 of IGF-1. This is followed by a second insertion of the FGF-1 segment Arg137 to G1n142 in between residues Pro66 and A1a67 of IGF-1. Overall, this results in the insertion of the entire FGF-1 fragment Lys128 to G1y142 which includes by Pro66 of IGF-land is represented below by the single letter code for amino acids.
GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEM
YCAPLKKNGRSKLGPRTHFGQAKSA
(SEQ ID NO. 2) Sequence MBF-3 was constructed using a helical wheel optimised sequence element of FGF-1 (Lys127 to G1n142) that maximised the polarity of a theoretical helical model of the proposed IGF-1 variant. The optimisation analysis resulted in one glycine spacer amino acid being inserted in front of the FGF-1 sequence element and the substitution of glutamine for Lys133 and lysine for Leu134. The IGF-1 D-domain post Pro63 was deleted and this new FGF-1 segment was added and is represented below by the single letter code for amino acids.
GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEM
YCAPGKKNGRSQKGPRTHFGQ
(SEQ ID N0. 4) Sequence MBF-4 most closely represents the native structure of the parent IGF-1 peptide. This variant has been designed utilising substitutions of amino acids and creates two recognised heparin-binding sequences; the octapeptide XBBBXXBX (Asp-Lys-Arg-Gln-Leu-Glu-Lys-Tyr) and the hexapeptide XBBXB (Gly-Lys-Arg-Gly-Arg-Ser). These two structures are positioned either side of Cys61 and collectively constitute 7 changes in the A- and D-domains.
This sequence represents an attempt to mimic heparin-binding structures seen in insulin-like growth factor binding proteins (IGFBPs) and heparin-binding EGF where the sequences surround a cysteine residue. The changes described are represented below by the single letter code for amino acids.
GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDKRQLEK
YCAPGKRGRSA
(SEQ ID NO. 5) IS
In every case, MBF cDNA constructs encode a fusion protein that results in a polypeptide containing the first 11 amino acids of porcine growth hormone, a linker of valine and asparagine (MFPAMPLSSLFVN) and the mutagenised hIGF-I sequence (MBF) and results in the expression of an extended form of MBF or Long MBF (L-MBF). These components permit the restriction digestion of the hIGF-I mutagenised sequence, the bacterial expression of the fusion protein and the hydroxylamine chemical cleavage of the leader sequence from the MBF sequence.
Example 1 Mutagenesis of a Nucleotide Sequence Encoding a Polypeptide Bioactive Factor (IGF-I) to Generate a Nucleotide Sequence Encoding an MBF
In order to perform site-directed mutagenesis on a nucleotide sequence encoding a polypeptide bioactive factor, a suitable plasmid cloning vector PTZ18 was obtained. The cDNA nucleotide sequence pMpGH(11)VN/IGF-I
according to Example 5 of Australian Patent No. 633099, encoding pGH(1-11) joined via a potential hydroxylamine-cleavable linkage N-terminal to IGF-I, was optimised for codon usage in bacteria, and subcloned into the PTZ18 plasmid cloning vector EcoR1/HindIII restriction site in 5'-3' orientation. The construct is hereinafter referred S to as PTZ18/pGH(11)/hIGF-I. The optimisation involved generation, extraction and precipitation of PTZ18 plasmid cloning vector DNA and pMpGH(11)VN/IGF-I DNA, restriction enzyme digestions and legations using conventional methods, for example as described in Molecular Cloning: A Laboratory Manual, Eds Sambrook, Fritsch and Maniatis (second edition), 1989; Pages 1.23-1.24, 1.62-1.68 respectively.
The correct nucleotide sequence was confirmed using the dideoxy-mediated chain termination (Sanger) method as described in Molecular Cloning, Pages 13.3-13.6.
1S Transformation of 200 x..1.1 of competent MV1190 bacterial cell suspension with 5 ~tl of PTZ18/pGH(11)/hIGF-I
legation reaction for the generation of quantities of PTZ18/pGH(11)/hIGF-I double stranded (ds) DNA to be used for restriction digests was carried out as described in Molecular Cloning, Pages 1.74-1.84. The remaining 5ul of legation reaction was used to transform 200 ~1 of CJ236 bacterial cell suspension for the production of single stranded (ss) uracil containing DNA to be used for the production of replicative mutagenised ds DNA. Replicative 2S ds mutagenised DNA was generated using site-directed mutagenesis. In each case two oligonucleotide primers were employed to insert the changes necessary to create a nucleotide sequence encoding an MBF. The complete mutagenesis in each case employed the prime (') oligonucleotide for the first reaction, while the second reaction employed the double prime(") oligonucleotide and used the methods described in Molecular Cloning, Pages 15.74-15.79 and 15.63-15.65. For example, to complete the mutagenesis of MBF-2, IGFS-2' was employed to achieve 3S the first round of mutagenic changes and IGFS-2 " was employed to achieve the second round of mutagenic changes.
Oligonucleotide IGFS-2' (54 mer) 5'-TGCGCTCCGCTGF~i~AAAAAACGGTCGTTCTAAACTGGGCCCGGCTAAATCTGCT-3' (SEQ ID NO. 6) Oligonucleotide primer IGFS-2 " (48 mer) 5'-TCTAAACTGGGTCCGCGTACCCACTTCGGCCAGGCTAAATCTGCTTGA-3' (SEQ ID N0. 7) Transformants carrying the correct ds MBF-2 DNA, hereinafter referred to as PTZ18/pGH(11)/MBF-2, were determined by the dideoxy-mediated chain termination (Sanger) method, and used to generate quantities of PTZ18/pGH(11)/MBF-2 DNA.
A number of other PTZ18/pGH(11)/MBF analogue constructs were generated using different oligonucleotide primers and identical molecular biology techniques (see examples).
Oligonucleotide primers IGFS-1' and IGFS-1" encoding MBF-1:
Oligonucleotide primer IGFS-1' (54 mer) 5'-ATGTACTGCGCTCCGF~~AAAAAACGGTCGTTCTAAACTGCTGAAACCGGCTAAA-3' (SEQ ID NO. 8) Oligonucleotide primer IGFS-1 " (54 mer) 5'-GGTCGTTCTAAACTGGGCCCGCGTACCCACTTCGGTCAGTGATGATGC AAGCTT-3' (SEQ ID NO. 9) Oligonucleotide primers IGFS-3' and IGFS-3 " encoding MBF-3:
Oligonucleotide primer IGFS-3' (57 mer) 5'-ATGTACTGCGCTCCGGGTP~~AAAAAACGGCCGTTCTCAGAAACTGAAACCGGCTAAA-3' (SEQ ID N0. 10) Oligonucleotide primer IGFS-3 " (54 mer) 5'-GGTCGTTCTCAGAAAGGCCCGCGTACCCACTTCGGTCAGTGATGATGCAAGCTT-3' (SEQ ID N0. 11) Oligonucleotide primers IGFS-4' and IGFS-4 " encoding MBF-4:
Oligonucleotide primer IGFS-4' (48 mer) 5'-TTCCGTTCTTGCGACAAACGTCAGCTGGAAAAATACTGCGCTCCGCTG-3' (SEQ ID NO. 12) Oligonucleotide primer IGFS-4 " (45 mer) 5'-AAATACTGCGCTCCGGGTAAACGTGGCCGTTCTGCTTGATGATGC-3' 1~> ( SEQ ID NO . 13 ) Example 2 Subcloning the Nucleotide Sequence Encoding an MBF from PTZ18/pGH(11)/MBF into Expression Vector pGHXSC.4 pGHXSC.4 already contains within its DNA the nucleotide sequence encoding pGH(11). The nucleotide sequence encoding only the MBF was excised from PTZ18/pGH(11)/MBF DNA using restriction enzymes HpaI/HindIII. This MBF nucleotide sequence was subcloned into expression vector pGHXSC.4, using the techniques described in Example 1. The thus-formed expression vector construct is hereinafter referred to as pGHXSC.4/MBF.
ds DNA sequencing as described in Example 1 was again employed to confirm the expected nucleotide sequence of the MBF fusion protein in the expression vector.
Example 3 Transformation of JM101 Bacterial Cells with the Expression Vector pGHXSC.4/MBF Containing the Nucleotide Sequence Encoding an MBF
To facilitate the expression of the MBF fusion protein, pGHXSC.4/MBF (Example 2) was transformed by methods outlined in Example 2 into a suitable host cell, in this case IacIq JM101 cells.
Transformants containing pGHXSC.4/MBF
successfully grew on agarose plates containing ampicillin.
Example 4 Induction of ,IM101 Bacterial Cells Transformed with pGHXSC.4/MBF to Determine Cell Clones Expressing MBF Fusion Proteins as IBs To confirm that the cells were expressing the fusion protein as inclusion bodies (IBs}, inductions of single clones were undertaken. Single pGHXSC.4/MBF
transformed colonies (Example 3) were inoculated into Luria Bertani (LB) medium and cultured overnight. An aliquot from each of these cultures was transferred to a fresh sample of LB medium and incubated at 37°C until an absorbance at 600 nm (A6o~) of 0.8-2.0 was reached. At this time an aliquot was taken from each culture and reserved.
To the remainder of the culture was added isopropyl ~3-D-thiogalactopyranoside (IPTG) to a final concentration of 0.2 mM, to induce the cells into the production of the MBF fusion protein encoded within pGHXSC.4/MBF. Following further incubation, both the reserved culture and the induced culture were centrifuged to pellet the cell, and following removal of the supernatant the cells were treated with 2% ~3-mercaptoethanol/10o SDS to lyse the cells and denature the protein.
Pre-induced cultures were compared directly with post-induced cultures, using SDS/PAGE gel chromatography, 8-25o gradient Phast gel (Pharmacia) to determine MBF
fusion protein expressing clones and to confirm the estimated size of the MBF.
Example 5 Production of MBF Fusion Protein Using Cells Expressing MBF Fusion Protein as Inclusion Bodies (IBs) Clones identified as expressing the fusion protein in Example 4 were used in a scale-up process to produce appropriate quantities of the fusion protein for in vi tro and in vivo experiments .
Four 2 litre Applicon fermenters were employed, each containing 1L of minimal medium and inoculated with an aliquot of a culture, established with clones expressing MBF fusion proteins, growing in log phase. Inoculated fermentation cultures were incubated at 37°C overnight. At an absorbance at 600 nm (A6oo) of between 4-6, IPTG was added to the cultures to a final concentration of 0.2 mM
and the cells further incubated until an A6oo of approximately 15-20 was reached, after which the fermentation suspension was subjected to a number of passes through a homogeniser. This process disrupted the cells, facilitating the further isolation and purification of IBs by three centrifugation and washing steps, using 30 mM
NaCl/10 mM KHZP04 washing buffer.
For pGHXSC.4/MBF-2 this process yielded a wet IB
pellet of 8.2 grams, which was stored at -20°C.
Example 6 Dissolution, Refolding, and Cleavage of MBF
Fusion Protein Produced in Inclusion Bodies, Purification of Biologically Pure MBF
The wet IB pellet containing the MBF-2 fusion protein from Example 5 was solubilised, desalted and refolded by conventional methods. The MBF fusion protein was then isolated and cleaved, followed by chromatographic steps to yield a biologically pure MBF, employing known methods. These processes included, in sequence:
1) dissolution of IBs in buffer (8 M urea, 0.1 M Tris, 40 mM glycine, 0.5 mM ZnCl2 and 40 mM
dithiolthreitol (DTT) pH 9.1), centrifugation and filtration (1 ~.m gradient Whatman filter) to remove particulate contaminants and desalting into 8 M urea, 0.1 M Tris, 40 mM glycine, 0.5 mM ZnCl2 and 1.6 mM dithiothreitol pH 9.1 by size exclusion chromatography on Cellufine GCL-1000m;
2) a 330 ml pool of buffer containing 104.1 mg of fusion protein was reconstituted to 1.320 L in 2.5 M urea, 40 mM glycine, 0.1 M Tris, 0.4 mM DTT and 10 mM ethylenediaminetetra-acetic acid (EDTA) pH 9.0;
refolded over 120 minutes with the addition of 0.12m1.L-1 of oxidised (3-mercaptoethanol and re-acidified to pH 2.5 with concentrated HC1;
3) cation exchange on a SP Sepharose Fast Flow (FFS) matrix, eluting the protein with 8 M urea, 50 mM
Ammonium acetate and 1M NaCl pH 4.8;
4) a 160 ml (FFS) pool of protein containing 97.3 mg was divided with 20% reserved, after which 80% was reconstituted to 2 M urea, 0.1 M Tris, 1 M NH?OH and 1 mM
EDTA pH 8.65 followed by cleavage (see below) for 24 hrs at to 40°C;
5) buffer exchange of both the reserved material and cleaved material was achieved by C18 matrix fast performance liquid chromatography (FPLC) using an XK50/20 column (Pharmacia), washing with 0.1% trifluroacetic acid (TFA) and eluting with 80% acetonitrile/0.08% TFA, followed by 6) final desalting and purification by high performance liquid chromatography (HPLC) on a C4 matrix PrepPak column (Waters) washing with 0.1% TFA and eluting with an 80% acetonitrile/0.08% TFA gradient at 0.1% per minute.
Cleavage of the MBF fusion protein yields a fusion partner and MBF, and may be employed to further potentiate the bioactivity of the MBF if necessary. Thus two MBFs may be derived from the one MBF fusion protein, an extended form of MBF having the first 11 amino acids from porcine growth hormone (pGH(1-11)) N-terminally linked to the MBF amino acid sequence (L-MBF), and a cleaved form not having pGH(1-11).
Dissolution, refolding and cleavage (if used) of MBF fusion protein derived from the pGHXSC.4/MBF expression vector construct in the foregoing manner yielded material that ran as a single band following SDS/PAGE Novex Tricine gel chromatography, as shown in Figure 1. This material was represented as the major species following electrospray mass analysis, and was observed at the calculated theoretical mass.
Example 7 The Affinity of Biologically Pure MBF for Heparin as Measured by Heparin Affinity Chromatography 10 ~,g aliquots of biologically pure MBF-2 and L-MBF-2 from Example 6 were reconstituted in 10 ~,l of mM HCl, taken up into a final volume of 100 ~l and loaded in 10 mM Tris pH 7.0 on to a Pharmacia heparin-Sepharose CL6B affinity column connected to a FPLC and 10 eluted with a linear gradient (0 M NaCl-1M NaCl) of 10 mM Tris/1 M NaCl pH 7.0 over 50 minutes. A
10 ~g aliquot of authentic IGF-I and pGH(1-11) IGF-I
(L-IGF-I) was also loaded and eluted using the same conditions.
The affinity of cleaved MBFs was such that they required salt concentrations of between 0.26 M and 0.33 M
to elute them from the heparin matrix. L-MBFs required salt concentrations of between 0.24 M and 0.32 M, whereas IGF-I and L-IGF-I eluted at salt concentrations of 0.12 M
and 0.11 M respectively.
Similarly, 10 ~g aliquots of MBFs, L-MBFs, IGF-I
and L-IGF-I were prepared as described and loaded in 10 mM Tris pH 7.0 on to a Progel TSK Heparin affinity column connected to a HPLC. Proteins were again eluted with the previously described salt gradient.
MBFs required salt concentrations of between 0.53 M and 0.59 M, while L-MBFs required salt concentration of between 0.56 M and 0.89 M to elute them from this column. IGF-I and L-IGF-I eluted at salt concentrations of 0.28 M and 0.29 M respectively.
This example shows that MBFs do indeed bind more avidly to negatively charged materials as exemplified by two types of heparin-affinity columns.
Example 8 In vitro Binding Affinity of Biologically Pure MBF for the IGF-I Receptor Isolated from Human Placental Membranes The biologically pure MBFs derived in Example 6 had affinities for the IGF-I receptor which ranged from equipotent to three fold lower than authentic IGF-I or uncleaved long (L-IGF-I}. IGF type I receptors isolated from human placental membranes (Cuatrecasas, P., J. Biol.
Chem., 1972 247 1980-1991) were incubated with iodinated hIGF-I or L-hIGF-I in the presence of increasing concentrations of MBFs or L-MBFs (0.01 pmol to 100 pmol).
The affinity of the MBFs or L-MBFs was measured by the competitive displacement of iodinated hIGF-I or L-hIGF-I
from the receptors and the results expressed as percentages IS of iodinated IGF-I remaining bound to the IGF-I receptor.
The results are shown in Figures 2a and 2b.
Example 9 In vitro Stimulation of Protein Synthesis by Biologically Pure MBF in a Myoblast Cell Line The biologically pure MBFs from Example 6 stimulated the production of proteins by rat L6 myoblasts in serum-free medium (Francis et a1, Biochem. J., 1985 233 207). The ability of increasing concentrations of MBFs, ranging from 1 ng/ml to 1 Eig/ml, to stimulate protein synthesis in rat L6 myoblasts was measured, and compared with the ability of both commercially-derived IGF-I and L-IGF-I (GroPep) to stimulate protein synthesis in rat L5 myoblasts. Results are expressed as the percentage stimulation of protein synthesis above that observed in growth factor-free or serum free medium, and shown in Figures 3a and 3b.
Example 10 Characteristics of Binding of Iodinated Biologically Pure MBF to Negatively-Charged ~mrfa~a~
The biologically pure MBFs from Example 6 were iodinated by conventional methods, and shown to have the ability to bind to two negatively-charged surfaces.
Iodinated L-MBFs and reference peptides (10,000 counts per minute cpm/well), when incubated overnight at 4°C in 24-well polyanionic tissue culture plastic plates in the presence of 1 ml of l.Oo bovine serum albumin (BSA) dissolved in phosphate-buffered saline (PBS) and then washed twice using 1 ml of l.Oo BSA/PBS, demonstrated an increase of approximately 6 to 15-fold in their ability to remain bound to the substrate, compared to that of t0 iodinated L-IGF-I (10,000 cpm/well) incubated under the same conditions.
The results, shown in Table 1, are expressed as the number of counts per minute (cpm) retained by the iodinated polypeptide bioactive factor following the washing steps.
Table 1 Radioactive counts per minute {cpm) retained on tissue culture plastic Iodinated polypeptide Counts per minute retained bioactive factor (means sem) FGF-2 1911.6 16.7 IGF-II 967.6 29.3 L-IGF-I 291.3 34.6 L-MBF-1 4758.3 107.4 L-MBF-2 4523.1 89.6 L-MBF-3 4976.3 122.5 L-MBF-4 1970.2 77.4 Similarly, biologically pure, iodinated L-MBFs (10,000 cpm/well) when incubated overnight at 4°C in l.Oo BSA/PBS on HaCat epithelial cell-derived matrix in 24-well plates (Jones et a1, J. Cell Biol., 1993 121 679) and then washed twice with l.Oo BSA/PBS exhibited increases of approximately up to 5-fold in their ability to remain bound to the matrix when compared with iodinated L-IGF-I
(10,000 cpm/ml), as shown in Table 2.
Table 2 Radioactive counts per minute (cpm) retained on HaCaT cell derived matrix Iodinated polypeptide Counts per minute retained bioactive factor (means sem) FGF-2 1697.4 73.6 IGF-II 2168.6 201.3 L-IGF-I gp_g 4,7 L-MBF-1 370.6 29.7 L-MBF-2 406.2 16.8 L-MBF-3 398.7 14.1 L-MBF-4 78.8 7.9 Example 11 In vitro Stimulation of Protein Synthesis in an Epithelial Cell Line by Biologically Pure MBF Bound to a Negatively-Charged Surface The four extended forms of biologically pure MBFs from Example 6, which were shown to stimulate protein synthesis in a myoblast cell line (Example 9) and to have the ability to bind to negatively-charged surfaces (Example 10), were compared with authentic IGF-II, IGF-I
and L-IGF-I for their ability to stimulate protein synthesis in an epithelial cell line following pre-incubation and retention on two negatively-charged surfaces. MBFs, IGF-II, IGF-I and L-IGF-I were incubated overnight at 4°C in 0.5 ml of 1.0% BSA/PBS at concentrations of 2 ng/ml, 20 ng/ml and 200 ng/ml in 24-well tissue culture plates which were either untreated or coated with HaCat epithelial cell-derived matrix, and then washed twice using 1 ml of 1.0°s BSA/PBS, as described in Example 10.
HaCat epithelial cells were serum starved for 2 hours, harvested and resuspended in serum-free medium containing 1 ~Ci/ml H3 leucine, after which they were seeded on to MBF, IGF-II, IGF-I or L-IGF-I pre-incubated and washed wells at a density of 2.85 x 105 cells/well, and incubated for a further 18 hrs at 37°C. Wells were washed twice with 1 ml of cold Hanks balanced salt solution, followed by a single wash in 0.5 ml of cold 5%
trichloroacetic acid , after which wells were washed with 0.5 ml of cold reverse osmosis quality water. Finally 0.25 ml of 0.1o Triton X-100/0.5 M NaOH was added to each well and shaken for 30 mins. The Triton X-100/NaOH
solution from each well was then assayed for beta-emitting radiation, indicative of 3H-leucine which had been incorporated into proteins produced by the cells during the 18 hr incubation at 37°C. The results are shown in Tables 3 and 4.
Bound MBFs showed dose-dependent stimulation of protein synthesis in HaCat epithelial cells, between 1.5 and 2-fold greater than that exhibited by IGF-II, IGF-I or L-IGF-I in the untreated negatively-charged plastic tissue culture vessel. For matrix-bound MBFs, stimulation of protein synthesis in HaCat epithelial cells was approximately 30o above that induced by IGF-I and L-IGF-I, and was observed only at the highest concentration of 200 ng/ml (Table 3 and 4).
' WO 99/54359 PCT/AU99/00292 Table 3 Stimulation of protein synthesis in HaCaT cells seeded onto polypeptide bioactive factor pre-treated tissue culture plastic.
Polypeptide 2 ng/ml 20 200 bioactive factor ng/ml ng/ml FGF-2 112.3 7.5 102.6 4.7 130.4 7.0 IGF-II 117 13 109.8 5.1 134.1 2.7 L-IGF-I 114 6.3 109.8 13.6 132.3 6.3 L-MBF-1 131.7 3.7 175.4 9.4 213.0 10.6 L-MBF-2 131.2 5.2 159.9 10.2 220.5 9.6 L-MBF-3 133.2 10.2 153.7 13.2 226.8 5.6 ~L-MBF-4 143.3 5.6 165.6 6.6 214.6 5.2 Table 4 Stimulation of protein synthesis in HaCaT cells seeded onto polypeptide bioactive factor pre-treated HaCaT cell derived matrix Polypeptide 2 ng/ml 20 200 bioactive ng/ml ng/ml factor FGF-2 82.4 3.3 110.5 13.0 120.9 8.3 IGF-II 96.1 6.1 98.6 10.0 107.3 2.2 L-IGF-I 102,3 3.8 103.3 8.7 113.1 7.3 L-MBF-1 101.3 4.9 100.4 7.3 130.9 6.4 L-MBF-2 98.3 4.2 102 10.5 133.2 3.6 L-MBF-3 103.8 7.9 102.5 9.9 135.2 7.2 L-MBF-4 103.7 5.9 114.1 17.4 124.1 11.8 Example 12 In vitro Binding of Pure MBF-2 and L-MBF-2 to Titanium Screws Biologically pure MBF-2 and L-MBF-2 from Example 6 were iodinated by conventional methods and shown to have an increased ability to bind to titanium screws, compared to iodinated IGF-I. Iodinated MBF-2 and L-MBF-2 were diluted into Dulbecco's modified minimal medium (DMEM) (10,000 counts per minute/ml).Titanium screws were incubated in the presence of 1ml of iodinated MBF or IGF-I
solution (10,000 cpm/ml) overnight at 4°-C in 24-well tissue culture plastic plates. The medium was removed, and the screws were each washed twice with 1 ml of cold DMEM. The washing medium and the screws were analysed for the presence of the iodinated MBF or IGF-I species.
The MBFs demonstrated between 2.5 and 4.5-fold increases in their ability to remain bound to the titanium screws when compared to iodinated IGF-I. The results, shown in Table 5, are expressed as the number of counts per minute (cpm) retained on the screws following the washing steps .
Table 5 Radioactive Counts Remaining Associated with Titanium Screws Following Two DMEM Washes (n=3) Treatment cpm/SCREW cpm/SCREW
(mean sem) IGF-I 97.9 IGF-I 87.3 83.8 11.4 IGF-I 66.1 MBF-2 337.4 MBF-2 416.9 373.4 28.5 MBF-2 365.8 L-MBF-2 211.5 IL-MBF-2 189.7 209.3 13.1 L-MBF-2 226.7 Example 13 In vitro Retention of MBF-2, L-MBF-2 and IGF-I in Fibrin Gels Biologically pure MBF-2 and L-MBF-2 from Example 6 were iodinated by conventional methods, and shown to have an increased ability to remain bound within fibrin gels or clots, compared to iodinated IGF-I. 50 ~1 of 0.4o fibrinogen containing 10,000 cpm of iodinated MBF or IGF-I was combined with 5 ~.l of 0.020 thrombin in 24-well tissue culture plastic plates to form a fibrin clot or gel.
1 ml of DMEM was added to each well to cover the clots and incubated at 4°C for 24 hours, after which the medium was collected and replaced with fresh DMEM. This process of medium collection and replacement continued for 48 hours.
Collected medium was analysed for the presence of iodinated MBF or IGF-I.
The MBFs demonstrated up to 50o increases in retention within the fibrin gels as compared to IGF-1 at all time points, as shown in Table 6.
Table 6 Retention of Radioactive Counts (Iodinated Peptide) Within Fibrin Gels over 48 hours Sample o Counts Retained 24 hours 48 hours IGF-I 19.4 14.9 MBF-2 24.5 19.4 L-MBF-2 26.2 20.9 Example 14 Adsorption of MBF-2, L-MBF-2 and IGF-I on to Polyanionic Tissue Culture Plastic Solutions of MBF-2, L-MBF-2 and IGF-I were prepared to a final concentration of 100 ng/ml in DMEM.
1 ml of each solution was applied to the first well of separate 24-well tissue culture plastic plates, and incubated at room temperature for 15 minutes. Following this incubation period the 1 ml solutions were transferred to the second well of each 24-well plate, and incubated at room temperature for another 15 minutes. This process was repeated for a total of 18 out of the total 24-wells in each plate.
Following the sequential coating of 18 wells by either a 1 ml solution of MBF-2, L-MBF-2 or IGF-I, the wells were washed twice with 1 ml of DMEM, and air dried within a laminar flow cabinet. 1 m1 of DMEM containing 2.5 x 105 HaCat epithelial cells and 1 ~Ci of tritiated leucine was added to each well and incubated at 37°C for 18 hours. Wells were washed twice with 1 ml each of Hank's balanced salt solution, twice with 1 ml each of 5~
trichloroacetic acid (TCA), and once with 2 ml of Milli-Q
water. 1 ml of 0.5M sodium hydroxide/0.1o triton X-100 was added to each well and incubated at room temperature for at least 30 minutes, with shaking. 100 ~,1 samples from each well were transferred to scintillation vials, 2 ml of scintillation fluid was added to each vial, and mixed well with shaking. Samples were analysed for the presence of ~3-emitting tritiated leucine incorporated into newly-synthesized protein in response to the MBF or IGF-I bound to the plastic surface.
Results are expressed as a percentage of protein synthesis compared to a growth factor-free control, and are shown in Table 7. 1 ml solutions of biologically pure MBF-2 and L-MBF-2 (100 ng/ml) from Example 6 were able to be used repeatedly (at least 18 applications) to coat tissue culture plastic surfaces, with 5-8 fold stimulation of protein synthesis in HaCat cells grown on these surfaces.
In contrast, the repeated coating of tissue culture plastic surfaces with IGF-1 solution resulted in a lower stimulation of protein synthesis by the HaCat cells, which returned to baseline after 13 applications.
Table 7 Stimulation of Protein Synthesis in HaCat Cells Grown on to 24-well Tissue Culture Plates Treated with MBF-2, L-MBF-2 or IGF-I, Expressed as a Percentage of a Growth Factor-Free Control Diluted Series IGF-i NSF-2 L-NSF-2 1 334.4 _ 876.8 611.9 2 442.3 578.7 751.3 3 400.'7 560 705.5 4 267 608.2 708.9 293.3 592.6 742 6 243.7 660.1 814.3 7 234.4 643.1 758.7 8 203.5 642.6 830.1 192.9 613.4 706.9 202.3 603.5 712.8 11 230.1 549.1 655.9 12 178.1 539.3 706.2 13 86.4 550.9 632.4 14 128.1 533.7 676.2 184.7 566.5 686 16 122.2 572.7 675.1 17 106 615.3 774 18 97.9 628.2 717.3 Example 15 In vivo Tissue Distribution of Systemically 10 Administered Iodinated MBF-2, L-MBF-2 Compared to IGF-I
Biologically pure MBF-2 and L-MBF-2 from Example 6 iodinated by conventional methods and injected via jugular catheter into male rats appeared to localise 15 preferentially to a variety of tissues, when compared to similarly iodinated and administered IGF-I.
Preferably the mutagenesis is achieved using site-directed mutagenesis, more preferably using oligonucleotide primers which are based on binding sites able to bind to negatively-charged surfaces, which binding sites are present in other polypeptide bioactive factors, or sequences substantially homologous thereto. Examples of polypeptide bioactive factors comprising such binding sites include vitronectin, 10 kilodalton gamma-interferon inducible protein, histidine-rich glycoprotein, purpurin, beta-thromboglobulin, antithrombin III, heparin cofactor II, FGF-1, FGF-2, heparin-binding epidermal growth factor, lipocortin, protein C inhibitor, fibronectin, thrombospondin, lipoprotein lipase, hepatic triglyceride lipase, vascular endothelial cell growth factor, thrombin, neural cell adhesion molecule and glial-derived nexin.
The altered nucleic acid sequence encoding an MBF
may be subcloned into a suitable expression vector, which may be introduced into host cells by conventional means familiar to those skilled in the art.
Thus the method of the invention preferably also comprises the steps of subcloning the nucleic acid sequence encoding the MBF into a suitable expression vector;
transforming the expression vector into a suitable bacterial, yeast or tissue culture host cell;
cultivating the host cell under conditions suitable to express the MBF; and isolating the MBF.
It will be appreciated that host cells comprising selected constructs so formed may express the MBF as a fusion protein within inclusion bodies (IB). By the term _ 7 _ "MBF fusion protein" we mean a polypeptide consisting of two linked protein components, one of which is selected so as to be expressed in the host cell under the control of a suitable promoter, and the other of which comprises the polypeptide bioactive factor incorporating the motif that confers MBF activity. The fusion protein is produced in order to facilitate the expression and/or processing of the amino acid sequence of the MBF activity. Preferably the MBF fusion protein is produced by an appropriate host cell in a fermenter by conventional means understood by those skilled in the art.
Preferably, the MBF is isolated from the host cell following disruption of the host cell by homogenisation, and processed to its biologically pure form using conventional methods of protein purification well recognised by those skilled in the art. These include oxidative refolding to achieve correct disulphide bonding, chemical cleavage of the fusion partner (if used) from the MBF, and various chromatographic steps. The MBF may be isolated as a biologically pure form of the fusion protein, and may then be cleaved from its fusion partner, yielding a peptide that is not extended.
In a preferred embodiment an MBF is prepared as a fusion protein using the methods described in our Australian Patent No. 633099, the entire disclosure of which is incorporated herein by reference. In this method a fragment of porcine growth hormone is linked to the N-terminal sequence of an MBF, optionally via a cleavable sequence.
In another preferred embodiment of the invention there is provided a process for the production of a cleavable MBF fusion protein, comprising the step of transforming a susceptible bacterial, yeast or tissue culture cell hosts with one or more recombinant DNA
plasmids which include DNA sequences capable of facilitating the expression of an MBF fusion protein.
_ g _ In a further preferred embodiment, the MBF fusion protein is expressed as an insoluble aggregate or inclusion body within the host cell, and isolated by cell disruption and centrifugation. The conventional methodologies which may be employed in the isolation of the MBF include fusion protein dissolution, oxidative refolding, hydroxylamine or proteolytic cleavage, and various chromatographic processes, including size exclusion chromatography, ion exchange chromatography, reversed-phase high performance liquid chromatography and affinity chromatography. Sample fractions may be collected from each purification step, with those exhibiting biological activity in an appropriate assay being pooled and carried forward to the next purification step. This may result in the isolation of biologically pure MBF with or without its fusion partner.
Preferably, the presence of biologically pure MBF
is detected by one or more of a) migration as a single band of the appropriate size on SDS-PAGE gel chromatography;
b) N-terminal sequence analysis, or c) mass spectroscopy.
The biologically pure MBF of the present invention is useful for a wide variety of purposes, including but not limited to maintenance, growth or differentiation of animal or human cells in culture;
maintenance, growth or differentiation of more organised cellular structures, for example, skin, cartilage, tendon, ligament or bone;
coating a negatively-charged surface to promote cell adhesion, growth, migration, or activity, for example culture vessels for use in keratinocyte expansion to provide partial thickness skin grafts for burns patients, or coating of a surgical implant or a prosthesis;
enhancement of tissue remodelling and repair associated with trauma or manipulation, for example in the _ g _ treatment of wounds of all types, including burns, traumatic injuries, or surgical wounds;
as an orally active product for the prevention and/or treatment of impaired gut function;
facilitating tissue targeting following systemic administration, for example by localisation of the factor to bone matrix following intravenous injection; and maintaining higher bioactive factor concentrations at the site of administration in order to effect a prolonged pharmacological action.
The MBF may be used in conjunction with or in combination with one or more other growth factors or therapeutic agents.
Accordingly, in a fourth aspect the present invention provides a composition for promoting adhesion, growth, migration, the prevention of apoptosis, or activity of cells in culture, comprising an effective amount of an MBF together with a pharmaceutically- or veterinarily-acceptable carrier.
In a preferred embodiment, this aspect of the invention provides a composition for the coating of a negatively-charged surface with an effective amount of MBF, thereby to promote adhesion, growth, migration or activity of cells.
In a fifth aspect the invention provides a method for promoting adhesion, growth, migration or activity of cells on a negatively-charged surface coated with an MBF, comprising the step of growing vertebrate cells in a culture medium over or on an appropriate surface pretreated with an MBF.
In both the fourth and fifth aspect of the invention the cells may be of vertebrate, preferably mammalian, or of insect origin.
In a sixth aspect, the invention provides a composition for the enhancement of tissue remodelling or tissue repair associated with tissue trauma or wound healing, comprising an effective amount of an MBF
formulated with a carrier such as an injectible, excipient, carrier, lotion, medicated body wash, dressing, liniment, toothpaste, mouthwash or powder.
In an alternative embodiment, this aspect of the invention provides a composition for alleviation of skin damage associated with ageing or with exposure to ultraviolet or ionizing radiation, comprising an effective amount of an MBF together with a cosmetically-acceptable carrier.
In a seventh aspect, the invention provides a composition for the prevention or treatment of a condition associated with impaired gut function, comprising an effective amount of an MBF formulated with a carrier suitable to produce an orally stable, bioactive enteral formulation.
In an eighth aspect, the invention provides a composition for the targeting or localisation of an MBF to cells or tissues, thereby to promote cell adhesion, growth, migration or activity in vivo, comprising an effective amount of MBF formulated in a pharmaceutically-acceptable carrier.
In a ninth aspect, the invention provides a method for the enhancement of tissue remodelling or tissue repair associated with tissue trauma or wound healing, comprising the step of administering an effective amount of an MBF to a subject in need of such treatment.
In a preferred embodiment, this aspect of the invention provides a method for the prevention or treatment of impaired gut function, comprising the step of administering an effective amount of an MBF to a subject in need of such treatment.
In a second preferred embodiment there is provided a method for the targeting and localisation of an MBF to cells or tissues, thereby to promote cell adhesion, growth, migration or activity in vivo, comprising the step of systemic or local administration of an MBF to a subject in need of such treatment:
It will be appreciated that in the therapeutic methods of the invention, the subject to be treated may be a human, or may be a domestic, companion or zoo animal.
The carrier to be used and the dose and route of administration will depend on the nature of the condition to be treated and the age and general health of the subject, and will be at the discretion of the attending physician or veterinarian. Suitable carriers and formulations are known in the art, for example by reference to Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Company, Easton, Pennsylvania (1995). Suitable dosing regimens are established using methods standard in the art.
For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the results of SDS/PAGE analysis of the biologically pure MBF, as visualised by Novex Tricine gel chromatography.
Figure 2a shows dose-response curves of the extended forms of MBFs, L-MBF-1, L-MBF-2, L-MBF-3, L-MBF-4 and the extended form of IGF-I (L-IGF-I) for the competitive displacement of iodinated IGF-I from the IGF-I
receptor isolated from human placental membranes.
Figure 2b shows dose-response curves of the cleaved forms of MBFs, MBF-1, MBF-2, MBF-3, MBF-4 and IGF-I
for the competitive displacement of iodinated IGF-I from the IGF-I receptor isolated from human placental membranes.
Figure 3a shows dose-response curves of four extended forms of MBFs, L-MBF-1, L-MBF-2, L-MBF-3, L-MBF-4, and L-IGF-I in a protein synthesis assay using a myoblast cell line.
Figure 3b shows dose-response curves of four cleaved forms of MBFs, MBF-1, MBF-2, MBF-3, MBF-4 and IGF-I
in a protein synthesis assay using a myoblast cell line.
DETAILED DESCRIPTION OF THE INVENTION
The present invention will now be more fully described with reference to the accompanying non-limiting examples. It should be understood that the following description is illustrative only, and should not be taken in any way as a restriction on the generality of the invention. In particular, while the invention is specifically exemplified with reference to MBFs derived from IGF-I, it will be clearly understood that the methods described herein are applicable generally to the modification of polypeptide bioactive factors to generate MBFs, and to evaluation of the MBFs thus produced for biological activity. In particular, once the MBF is produced its testing for suitability for the purposes of the invention is a matter of routine.
Representative MBFs according to the invention were produced by introduction of heparin-binding motifs of other polypeptide bioactive factors into the sequence of human IGF-1. The methods used are described in detail below. It will be clearly understood that while the examples utilize the heparin-binding motif from bovine FGF-1, FGF-1 sequences from the human protein or from other species may also be used.
Sequence MBF-1 involves the deletion of the IGF-1 D-domain post Pro63 and its substitution with the heparin-binding motif Lys127 to G1n142 from bovine FGF-1, represented by the single letter code for amino acids as shown below. The introduced heparin-binding motif is shown in bold.
GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEM
YCAPKKNGRSKLGPRTHFGQ
(SEQ ID NO. 1) Sequence MBF-2 contains all of the amino acids of native IGF-1, and Lys127 to G1n142 of FGF-1, through the insertion of the FGF-1 fragment Lys128 to G1y135 in between the residues Lys65 and Pro66 of IGF-1. This is followed by a second insertion of the FGF-1 segment Arg137 to G1n142 in between residues Pro66 and A1a67 of IGF-1. Overall, this results in the insertion of the entire FGF-1 fragment Lys128 to G1y142 which includes by Pro66 of IGF-land is represented below by the single letter code for amino acids.
GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEM
YCAPLKKNGRSKLGPRTHFGQAKSA
(SEQ ID NO. 2) Sequence MBF-3 was constructed using a helical wheel optimised sequence element of FGF-1 (Lys127 to G1n142) that maximised the polarity of a theoretical helical model of the proposed IGF-1 variant. The optimisation analysis resulted in one glycine spacer amino acid being inserted in front of the FGF-1 sequence element and the substitution of glutamine for Lys133 and lysine for Leu134. The IGF-1 D-domain post Pro63 was deleted and this new FGF-1 segment was added and is represented below by the single letter code for amino acids.
GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEM
YCAPGKKNGRSQKGPRTHFGQ
(SEQ ID N0. 4) Sequence MBF-4 most closely represents the native structure of the parent IGF-1 peptide. This variant has been designed utilising substitutions of amino acids and creates two recognised heparin-binding sequences; the octapeptide XBBBXXBX (Asp-Lys-Arg-Gln-Leu-Glu-Lys-Tyr) and the hexapeptide XBBXB (Gly-Lys-Arg-Gly-Arg-Ser). These two structures are positioned either side of Cys61 and collectively constitute 7 changes in the A- and D-domains.
This sequence represents an attempt to mimic heparin-binding structures seen in insulin-like growth factor binding proteins (IGFBPs) and heparin-binding EGF where the sequences surround a cysteine residue. The changes described are represented below by the single letter code for amino acids.
GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDKRQLEK
YCAPGKRGRSA
(SEQ ID NO. 5) IS
In every case, MBF cDNA constructs encode a fusion protein that results in a polypeptide containing the first 11 amino acids of porcine growth hormone, a linker of valine and asparagine (MFPAMPLSSLFVN) and the mutagenised hIGF-I sequence (MBF) and results in the expression of an extended form of MBF or Long MBF (L-MBF). These components permit the restriction digestion of the hIGF-I mutagenised sequence, the bacterial expression of the fusion protein and the hydroxylamine chemical cleavage of the leader sequence from the MBF sequence.
Example 1 Mutagenesis of a Nucleotide Sequence Encoding a Polypeptide Bioactive Factor (IGF-I) to Generate a Nucleotide Sequence Encoding an MBF
In order to perform site-directed mutagenesis on a nucleotide sequence encoding a polypeptide bioactive factor, a suitable plasmid cloning vector PTZ18 was obtained. The cDNA nucleotide sequence pMpGH(11)VN/IGF-I
according to Example 5 of Australian Patent No. 633099, encoding pGH(1-11) joined via a potential hydroxylamine-cleavable linkage N-terminal to IGF-I, was optimised for codon usage in bacteria, and subcloned into the PTZ18 plasmid cloning vector EcoR1/HindIII restriction site in 5'-3' orientation. The construct is hereinafter referred S to as PTZ18/pGH(11)/hIGF-I. The optimisation involved generation, extraction and precipitation of PTZ18 plasmid cloning vector DNA and pMpGH(11)VN/IGF-I DNA, restriction enzyme digestions and legations using conventional methods, for example as described in Molecular Cloning: A Laboratory Manual, Eds Sambrook, Fritsch and Maniatis (second edition), 1989; Pages 1.23-1.24, 1.62-1.68 respectively.
The correct nucleotide sequence was confirmed using the dideoxy-mediated chain termination (Sanger) method as described in Molecular Cloning, Pages 13.3-13.6.
1S Transformation of 200 x..1.1 of competent MV1190 bacterial cell suspension with 5 ~tl of PTZ18/pGH(11)/hIGF-I
legation reaction for the generation of quantities of PTZ18/pGH(11)/hIGF-I double stranded (ds) DNA to be used for restriction digests was carried out as described in Molecular Cloning, Pages 1.74-1.84. The remaining 5ul of legation reaction was used to transform 200 ~1 of CJ236 bacterial cell suspension for the production of single stranded (ss) uracil containing DNA to be used for the production of replicative mutagenised ds DNA. Replicative 2S ds mutagenised DNA was generated using site-directed mutagenesis. In each case two oligonucleotide primers were employed to insert the changes necessary to create a nucleotide sequence encoding an MBF. The complete mutagenesis in each case employed the prime (') oligonucleotide for the first reaction, while the second reaction employed the double prime(") oligonucleotide and used the methods described in Molecular Cloning, Pages 15.74-15.79 and 15.63-15.65. For example, to complete the mutagenesis of MBF-2, IGFS-2' was employed to achieve 3S the first round of mutagenic changes and IGFS-2 " was employed to achieve the second round of mutagenic changes.
Oligonucleotide IGFS-2' (54 mer) 5'-TGCGCTCCGCTGF~i~AAAAAACGGTCGTTCTAAACTGGGCCCGGCTAAATCTGCT-3' (SEQ ID NO. 6) Oligonucleotide primer IGFS-2 " (48 mer) 5'-TCTAAACTGGGTCCGCGTACCCACTTCGGCCAGGCTAAATCTGCTTGA-3' (SEQ ID N0. 7) Transformants carrying the correct ds MBF-2 DNA, hereinafter referred to as PTZ18/pGH(11)/MBF-2, were determined by the dideoxy-mediated chain termination (Sanger) method, and used to generate quantities of PTZ18/pGH(11)/MBF-2 DNA.
A number of other PTZ18/pGH(11)/MBF analogue constructs were generated using different oligonucleotide primers and identical molecular biology techniques (see examples).
Oligonucleotide primers IGFS-1' and IGFS-1" encoding MBF-1:
Oligonucleotide primer IGFS-1' (54 mer) 5'-ATGTACTGCGCTCCGF~~AAAAAACGGTCGTTCTAAACTGCTGAAACCGGCTAAA-3' (SEQ ID NO. 8) Oligonucleotide primer IGFS-1 " (54 mer) 5'-GGTCGTTCTAAACTGGGCCCGCGTACCCACTTCGGTCAGTGATGATGC AAGCTT-3' (SEQ ID NO. 9) Oligonucleotide primers IGFS-3' and IGFS-3 " encoding MBF-3:
Oligonucleotide primer IGFS-3' (57 mer) 5'-ATGTACTGCGCTCCGGGTP~~AAAAAACGGCCGTTCTCAGAAACTGAAACCGGCTAAA-3' (SEQ ID N0. 10) Oligonucleotide primer IGFS-3 " (54 mer) 5'-GGTCGTTCTCAGAAAGGCCCGCGTACCCACTTCGGTCAGTGATGATGCAAGCTT-3' (SEQ ID N0. 11) Oligonucleotide primers IGFS-4' and IGFS-4 " encoding MBF-4:
Oligonucleotide primer IGFS-4' (48 mer) 5'-TTCCGTTCTTGCGACAAACGTCAGCTGGAAAAATACTGCGCTCCGCTG-3' (SEQ ID NO. 12) Oligonucleotide primer IGFS-4 " (45 mer) 5'-AAATACTGCGCTCCGGGTAAACGTGGCCGTTCTGCTTGATGATGC-3' 1~> ( SEQ ID NO . 13 ) Example 2 Subcloning the Nucleotide Sequence Encoding an MBF from PTZ18/pGH(11)/MBF into Expression Vector pGHXSC.4 pGHXSC.4 already contains within its DNA the nucleotide sequence encoding pGH(11). The nucleotide sequence encoding only the MBF was excised from PTZ18/pGH(11)/MBF DNA using restriction enzymes HpaI/HindIII. This MBF nucleotide sequence was subcloned into expression vector pGHXSC.4, using the techniques described in Example 1. The thus-formed expression vector construct is hereinafter referred to as pGHXSC.4/MBF.
ds DNA sequencing as described in Example 1 was again employed to confirm the expected nucleotide sequence of the MBF fusion protein in the expression vector.
Example 3 Transformation of JM101 Bacterial Cells with the Expression Vector pGHXSC.4/MBF Containing the Nucleotide Sequence Encoding an MBF
To facilitate the expression of the MBF fusion protein, pGHXSC.4/MBF (Example 2) was transformed by methods outlined in Example 2 into a suitable host cell, in this case IacIq JM101 cells.
Transformants containing pGHXSC.4/MBF
successfully grew on agarose plates containing ampicillin.
Example 4 Induction of ,IM101 Bacterial Cells Transformed with pGHXSC.4/MBF to Determine Cell Clones Expressing MBF Fusion Proteins as IBs To confirm that the cells were expressing the fusion protein as inclusion bodies (IBs}, inductions of single clones were undertaken. Single pGHXSC.4/MBF
transformed colonies (Example 3) were inoculated into Luria Bertani (LB) medium and cultured overnight. An aliquot from each of these cultures was transferred to a fresh sample of LB medium and incubated at 37°C until an absorbance at 600 nm (A6o~) of 0.8-2.0 was reached. At this time an aliquot was taken from each culture and reserved.
To the remainder of the culture was added isopropyl ~3-D-thiogalactopyranoside (IPTG) to a final concentration of 0.2 mM, to induce the cells into the production of the MBF fusion protein encoded within pGHXSC.4/MBF. Following further incubation, both the reserved culture and the induced culture were centrifuged to pellet the cell, and following removal of the supernatant the cells were treated with 2% ~3-mercaptoethanol/10o SDS to lyse the cells and denature the protein.
Pre-induced cultures were compared directly with post-induced cultures, using SDS/PAGE gel chromatography, 8-25o gradient Phast gel (Pharmacia) to determine MBF
fusion protein expressing clones and to confirm the estimated size of the MBF.
Example 5 Production of MBF Fusion Protein Using Cells Expressing MBF Fusion Protein as Inclusion Bodies (IBs) Clones identified as expressing the fusion protein in Example 4 were used in a scale-up process to produce appropriate quantities of the fusion protein for in vi tro and in vivo experiments .
Four 2 litre Applicon fermenters were employed, each containing 1L of minimal medium and inoculated with an aliquot of a culture, established with clones expressing MBF fusion proteins, growing in log phase. Inoculated fermentation cultures were incubated at 37°C overnight. At an absorbance at 600 nm (A6oo) of between 4-6, IPTG was added to the cultures to a final concentration of 0.2 mM
and the cells further incubated until an A6oo of approximately 15-20 was reached, after which the fermentation suspension was subjected to a number of passes through a homogeniser. This process disrupted the cells, facilitating the further isolation and purification of IBs by three centrifugation and washing steps, using 30 mM
NaCl/10 mM KHZP04 washing buffer.
For pGHXSC.4/MBF-2 this process yielded a wet IB
pellet of 8.2 grams, which was stored at -20°C.
Example 6 Dissolution, Refolding, and Cleavage of MBF
Fusion Protein Produced in Inclusion Bodies, Purification of Biologically Pure MBF
The wet IB pellet containing the MBF-2 fusion protein from Example 5 was solubilised, desalted and refolded by conventional methods. The MBF fusion protein was then isolated and cleaved, followed by chromatographic steps to yield a biologically pure MBF, employing known methods. These processes included, in sequence:
1) dissolution of IBs in buffer (8 M urea, 0.1 M Tris, 40 mM glycine, 0.5 mM ZnCl2 and 40 mM
dithiolthreitol (DTT) pH 9.1), centrifugation and filtration (1 ~.m gradient Whatman filter) to remove particulate contaminants and desalting into 8 M urea, 0.1 M Tris, 40 mM glycine, 0.5 mM ZnCl2 and 1.6 mM dithiothreitol pH 9.1 by size exclusion chromatography on Cellufine GCL-1000m;
2) a 330 ml pool of buffer containing 104.1 mg of fusion protein was reconstituted to 1.320 L in 2.5 M urea, 40 mM glycine, 0.1 M Tris, 0.4 mM DTT and 10 mM ethylenediaminetetra-acetic acid (EDTA) pH 9.0;
refolded over 120 minutes with the addition of 0.12m1.L-1 of oxidised (3-mercaptoethanol and re-acidified to pH 2.5 with concentrated HC1;
3) cation exchange on a SP Sepharose Fast Flow (FFS) matrix, eluting the protein with 8 M urea, 50 mM
Ammonium acetate and 1M NaCl pH 4.8;
4) a 160 ml (FFS) pool of protein containing 97.3 mg was divided with 20% reserved, after which 80% was reconstituted to 2 M urea, 0.1 M Tris, 1 M NH?OH and 1 mM
EDTA pH 8.65 followed by cleavage (see below) for 24 hrs at to 40°C;
5) buffer exchange of both the reserved material and cleaved material was achieved by C18 matrix fast performance liquid chromatography (FPLC) using an XK50/20 column (Pharmacia), washing with 0.1% trifluroacetic acid (TFA) and eluting with 80% acetonitrile/0.08% TFA, followed by 6) final desalting and purification by high performance liquid chromatography (HPLC) on a C4 matrix PrepPak column (Waters) washing with 0.1% TFA and eluting with an 80% acetonitrile/0.08% TFA gradient at 0.1% per minute.
Cleavage of the MBF fusion protein yields a fusion partner and MBF, and may be employed to further potentiate the bioactivity of the MBF if necessary. Thus two MBFs may be derived from the one MBF fusion protein, an extended form of MBF having the first 11 amino acids from porcine growth hormone (pGH(1-11)) N-terminally linked to the MBF amino acid sequence (L-MBF), and a cleaved form not having pGH(1-11).
Dissolution, refolding and cleavage (if used) of MBF fusion protein derived from the pGHXSC.4/MBF expression vector construct in the foregoing manner yielded material that ran as a single band following SDS/PAGE Novex Tricine gel chromatography, as shown in Figure 1. This material was represented as the major species following electrospray mass analysis, and was observed at the calculated theoretical mass.
Example 7 The Affinity of Biologically Pure MBF for Heparin as Measured by Heparin Affinity Chromatography 10 ~,g aliquots of biologically pure MBF-2 and L-MBF-2 from Example 6 were reconstituted in 10 ~,l of mM HCl, taken up into a final volume of 100 ~l and loaded in 10 mM Tris pH 7.0 on to a Pharmacia heparin-Sepharose CL6B affinity column connected to a FPLC and 10 eluted with a linear gradient (0 M NaCl-1M NaCl) of 10 mM Tris/1 M NaCl pH 7.0 over 50 minutes. A
10 ~g aliquot of authentic IGF-I and pGH(1-11) IGF-I
(L-IGF-I) was also loaded and eluted using the same conditions.
The affinity of cleaved MBFs was such that they required salt concentrations of between 0.26 M and 0.33 M
to elute them from the heparin matrix. L-MBFs required salt concentrations of between 0.24 M and 0.32 M, whereas IGF-I and L-IGF-I eluted at salt concentrations of 0.12 M
and 0.11 M respectively.
Similarly, 10 ~g aliquots of MBFs, L-MBFs, IGF-I
and L-IGF-I were prepared as described and loaded in 10 mM Tris pH 7.0 on to a Progel TSK Heparin affinity column connected to a HPLC. Proteins were again eluted with the previously described salt gradient.
MBFs required salt concentrations of between 0.53 M and 0.59 M, while L-MBFs required salt concentration of between 0.56 M and 0.89 M to elute them from this column. IGF-I and L-IGF-I eluted at salt concentrations of 0.28 M and 0.29 M respectively.
This example shows that MBFs do indeed bind more avidly to negatively charged materials as exemplified by two types of heparin-affinity columns.
Example 8 In vitro Binding Affinity of Biologically Pure MBF for the IGF-I Receptor Isolated from Human Placental Membranes The biologically pure MBFs derived in Example 6 had affinities for the IGF-I receptor which ranged from equipotent to three fold lower than authentic IGF-I or uncleaved long (L-IGF-I}. IGF type I receptors isolated from human placental membranes (Cuatrecasas, P., J. Biol.
Chem., 1972 247 1980-1991) were incubated with iodinated hIGF-I or L-hIGF-I in the presence of increasing concentrations of MBFs or L-MBFs (0.01 pmol to 100 pmol).
The affinity of the MBFs or L-MBFs was measured by the competitive displacement of iodinated hIGF-I or L-hIGF-I
from the receptors and the results expressed as percentages IS of iodinated IGF-I remaining bound to the IGF-I receptor.
The results are shown in Figures 2a and 2b.
Example 9 In vitro Stimulation of Protein Synthesis by Biologically Pure MBF in a Myoblast Cell Line The biologically pure MBFs from Example 6 stimulated the production of proteins by rat L6 myoblasts in serum-free medium (Francis et a1, Biochem. J., 1985 233 207). The ability of increasing concentrations of MBFs, ranging from 1 ng/ml to 1 Eig/ml, to stimulate protein synthesis in rat L6 myoblasts was measured, and compared with the ability of both commercially-derived IGF-I and L-IGF-I (GroPep) to stimulate protein synthesis in rat L5 myoblasts. Results are expressed as the percentage stimulation of protein synthesis above that observed in growth factor-free or serum free medium, and shown in Figures 3a and 3b.
Example 10 Characteristics of Binding of Iodinated Biologically Pure MBF to Negatively-Charged ~mrfa~a~
The biologically pure MBFs from Example 6 were iodinated by conventional methods, and shown to have the ability to bind to two negatively-charged surfaces.
Iodinated L-MBFs and reference peptides (10,000 counts per minute cpm/well), when incubated overnight at 4°C in 24-well polyanionic tissue culture plastic plates in the presence of 1 ml of l.Oo bovine serum albumin (BSA) dissolved in phosphate-buffered saline (PBS) and then washed twice using 1 ml of l.Oo BSA/PBS, demonstrated an increase of approximately 6 to 15-fold in their ability to remain bound to the substrate, compared to that of t0 iodinated L-IGF-I (10,000 cpm/well) incubated under the same conditions.
The results, shown in Table 1, are expressed as the number of counts per minute (cpm) retained by the iodinated polypeptide bioactive factor following the washing steps.
Table 1 Radioactive counts per minute {cpm) retained on tissue culture plastic Iodinated polypeptide Counts per minute retained bioactive factor (means sem) FGF-2 1911.6 16.7 IGF-II 967.6 29.3 L-IGF-I 291.3 34.6 L-MBF-1 4758.3 107.4 L-MBF-2 4523.1 89.6 L-MBF-3 4976.3 122.5 L-MBF-4 1970.2 77.4 Similarly, biologically pure, iodinated L-MBFs (10,000 cpm/well) when incubated overnight at 4°C in l.Oo BSA/PBS on HaCat epithelial cell-derived matrix in 24-well plates (Jones et a1, J. Cell Biol., 1993 121 679) and then washed twice with l.Oo BSA/PBS exhibited increases of approximately up to 5-fold in their ability to remain bound to the matrix when compared with iodinated L-IGF-I
(10,000 cpm/ml), as shown in Table 2.
Table 2 Radioactive counts per minute (cpm) retained on HaCaT cell derived matrix Iodinated polypeptide Counts per minute retained bioactive factor (means sem) FGF-2 1697.4 73.6 IGF-II 2168.6 201.3 L-IGF-I gp_g 4,7 L-MBF-1 370.6 29.7 L-MBF-2 406.2 16.8 L-MBF-3 398.7 14.1 L-MBF-4 78.8 7.9 Example 11 In vitro Stimulation of Protein Synthesis in an Epithelial Cell Line by Biologically Pure MBF Bound to a Negatively-Charged Surface The four extended forms of biologically pure MBFs from Example 6, which were shown to stimulate protein synthesis in a myoblast cell line (Example 9) and to have the ability to bind to negatively-charged surfaces (Example 10), were compared with authentic IGF-II, IGF-I
and L-IGF-I for their ability to stimulate protein synthesis in an epithelial cell line following pre-incubation and retention on two negatively-charged surfaces. MBFs, IGF-II, IGF-I and L-IGF-I were incubated overnight at 4°C in 0.5 ml of 1.0% BSA/PBS at concentrations of 2 ng/ml, 20 ng/ml and 200 ng/ml in 24-well tissue culture plates which were either untreated or coated with HaCat epithelial cell-derived matrix, and then washed twice using 1 ml of 1.0°s BSA/PBS, as described in Example 10.
HaCat epithelial cells were serum starved for 2 hours, harvested and resuspended in serum-free medium containing 1 ~Ci/ml H3 leucine, after which they were seeded on to MBF, IGF-II, IGF-I or L-IGF-I pre-incubated and washed wells at a density of 2.85 x 105 cells/well, and incubated for a further 18 hrs at 37°C. Wells were washed twice with 1 ml of cold Hanks balanced salt solution, followed by a single wash in 0.5 ml of cold 5%
trichloroacetic acid , after which wells were washed with 0.5 ml of cold reverse osmosis quality water. Finally 0.25 ml of 0.1o Triton X-100/0.5 M NaOH was added to each well and shaken for 30 mins. The Triton X-100/NaOH
solution from each well was then assayed for beta-emitting radiation, indicative of 3H-leucine which had been incorporated into proteins produced by the cells during the 18 hr incubation at 37°C. The results are shown in Tables 3 and 4.
Bound MBFs showed dose-dependent stimulation of protein synthesis in HaCat epithelial cells, between 1.5 and 2-fold greater than that exhibited by IGF-II, IGF-I or L-IGF-I in the untreated negatively-charged plastic tissue culture vessel. For matrix-bound MBFs, stimulation of protein synthesis in HaCat epithelial cells was approximately 30o above that induced by IGF-I and L-IGF-I, and was observed only at the highest concentration of 200 ng/ml (Table 3 and 4).
' WO 99/54359 PCT/AU99/00292 Table 3 Stimulation of protein synthesis in HaCaT cells seeded onto polypeptide bioactive factor pre-treated tissue culture plastic.
Polypeptide 2 ng/ml 20 200 bioactive factor ng/ml ng/ml FGF-2 112.3 7.5 102.6 4.7 130.4 7.0 IGF-II 117 13 109.8 5.1 134.1 2.7 L-IGF-I 114 6.3 109.8 13.6 132.3 6.3 L-MBF-1 131.7 3.7 175.4 9.4 213.0 10.6 L-MBF-2 131.2 5.2 159.9 10.2 220.5 9.6 L-MBF-3 133.2 10.2 153.7 13.2 226.8 5.6 ~L-MBF-4 143.3 5.6 165.6 6.6 214.6 5.2 Table 4 Stimulation of protein synthesis in HaCaT cells seeded onto polypeptide bioactive factor pre-treated HaCaT cell derived matrix Polypeptide 2 ng/ml 20 200 bioactive ng/ml ng/ml factor FGF-2 82.4 3.3 110.5 13.0 120.9 8.3 IGF-II 96.1 6.1 98.6 10.0 107.3 2.2 L-IGF-I 102,3 3.8 103.3 8.7 113.1 7.3 L-MBF-1 101.3 4.9 100.4 7.3 130.9 6.4 L-MBF-2 98.3 4.2 102 10.5 133.2 3.6 L-MBF-3 103.8 7.9 102.5 9.9 135.2 7.2 L-MBF-4 103.7 5.9 114.1 17.4 124.1 11.8 Example 12 In vitro Binding of Pure MBF-2 and L-MBF-2 to Titanium Screws Biologically pure MBF-2 and L-MBF-2 from Example 6 were iodinated by conventional methods and shown to have an increased ability to bind to titanium screws, compared to iodinated IGF-I. Iodinated MBF-2 and L-MBF-2 were diluted into Dulbecco's modified minimal medium (DMEM) (10,000 counts per minute/ml).Titanium screws were incubated in the presence of 1ml of iodinated MBF or IGF-I
solution (10,000 cpm/ml) overnight at 4°-C in 24-well tissue culture plastic plates. The medium was removed, and the screws were each washed twice with 1 ml of cold DMEM. The washing medium and the screws were analysed for the presence of the iodinated MBF or IGF-I species.
The MBFs demonstrated between 2.5 and 4.5-fold increases in their ability to remain bound to the titanium screws when compared to iodinated IGF-I. The results, shown in Table 5, are expressed as the number of counts per minute (cpm) retained on the screws following the washing steps .
Table 5 Radioactive Counts Remaining Associated with Titanium Screws Following Two DMEM Washes (n=3) Treatment cpm/SCREW cpm/SCREW
(mean sem) IGF-I 97.9 IGF-I 87.3 83.8 11.4 IGF-I 66.1 MBF-2 337.4 MBF-2 416.9 373.4 28.5 MBF-2 365.8 L-MBF-2 211.5 IL-MBF-2 189.7 209.3 13.1 L-MBF-2 226.7 Example 13 In vitro Retention of MBF-2, L-MBF-2 and IGF-I in Fibrin Gels Biologically pure MBF-2 and L-MBF-2 from Example 6 were iodinated by conventional methods, and shown to have an increased ability to remain bound within fibrin gels or clots, compared to iodinated IGF-I. 50 ~1 of 0.4o fibrinogen containing 10,000 cpm of iodinated MBF or IGF-I was combined with 5 ~.l of 0.020 thrombin in 24-well tissue culture plastic plates to form a fibrin clot or gel.
1 ml of DMEM was added to each well to cover the clots and incubated at 4°C for 24 hours, after which the medium was collected and replaced with fresh DMEM. This process of medium collection and replacement continued for 48 hours.
Collected medium was analysed for the presence of iodinated MBF or IGF-I.
The MBFs demonstrated up to 50o increases in retention within the fibrin gels as compared to IGF-1 at all time points, as shown in Table 6.
Table 6 Retention of Radioactive Counts (Iodinated Peptide) Within Fibrin Gels over 48 hours Sample o Counts Retained 24 hours 48 hours IGF-I 19.4 14.9 MBF-2 24.5 19.4 L-MBF-2 26.2 20.9 Example 14 Adsorption of MBF-2, L-MBF-2 and IGF-I on to Polyanionic Tissue Culture Plastic Solutions of MBF-2, L-MBF-2 and IGF-I were prepared to a final concentration of 100 ng/ml in DMEM.
1 ml of each solution was applied to the first well of separate 24-well tissue culture plastic plates, and incubated at room temperature for 15 minutes. Following this incubation period the 1 ml solutions were transferred to the second well of each 24-well plate, and incubated at room temperature for another 15 minutes. This process was repeated for a total of 18 out of the total 24-wells in each plate.
Following the sequential coating of 18 wells by either a 1 ml solution of MBF-2, L-MBF-2 or IGF-I, the wells were washed twice with 1 ml of DMEM, and air dried within a laminar flow cabinet. 1 m1 of DMEM containing 2.5 x 105 HaCat epithelial cells and 1 ~Ci of tritiated leucine was added to each well and incubated at 37°C for 18 hours. Wells were washed twice with 1 ml each of Hank's balanced salt solution, twice with 1 ml each of 5~
trichloroacetic acid (TCA), and once with 2 ml of Milli-Q
water. 1 ml of 0.5M sodium hydroxide/0.1o triton X-100 was added to each well and incubated at room temperature for at least 30 minutes, with shaking. 100 ~,1 samples from each well were transferred to scintillation vials, 2 ml of scintillation fluid was added to each vial, and mixed well with shaking. Samples were analysed for the presence of ~3-emitting tritiated leucine incorporated into newly-synthesized protein in response to the MBF or IGF-I bound to the plastic surface.
Results are expressed as a percentage of protein synthesis compared to a growth factor-free control, and are shown in Table 7. 1 ml solutions of biologically pure MBF-2 and L-MBF-2 (100 ng/ml) from Example 6 were able to be used repeatedly (at least 18 applications) to coat tissue culture plastic surfaces, with 5-8 fold stimulation of protein synthesis in HaCat cells grown on these surfaces.
In contrast, the repeated coating of tissue culture plastic surfaces with IGF-1 solution resulted in a lower stimulation of protein synthesis by the HaCat cells, which returned to baseline after 13 applications.
Table 7 Stimulation of Protein Synthesis in HaCat Cells Grown on to 24-well Tissue Culture Plates Treated with MBF-2, L-MBF-2 or IGF-I, Expressed as a Percentage of a Growth Factor-Free Control Diluted Series IGF-i NSF-2 L-NSF-2 1 334.4 _ 876.8 611.9 2 442.3 578.7 751.3 3 400.'7 560 705.5 4 267 608.2 708.9 293.3 592.6 742 6 243.7 660.1 814.3 7 234.4 643.1 758.7 8 203.5 642.6 830.1 192.9 613.4 706.9 202.3 603.5 712.8 11 230.1 549.1 655.9 12 178.1 539.3 706.2 13 86.4 550.9 632.4 14 128.1 533.7 676.2 184.7 566.5 686 16 122.2 572.7 675.1 17 106 615.3 774 18 97.9 628.2 717.3 Example 15 In vivo Tissue Distribution of Systemically 10 Administered Iodinated MBF-2, L-MBF-2 Compared to IGF-I
Biologically pure MBF-2 and L-MBF-2 from Example 6 iodinated by conventional methods and injected via jugular catheter into male rats appeared to localise 15 preferentially to a variety of tissues, when compared to similarly iodinated and administered IGF-I.
Male Sprague Dawley rats (118-130 grams) were administered 1 x 10' cpm of either MBF-2, L-MBF-2 or IGF-I, and decapitated at either 1 minute or 15 minutes, after which samples of blood and the gut, left hind limb, pelt, heart, liver, spleen, lungs, adrenals, kidneys and thymus were frozen in liquid nitrogen. Samples were thawed before being homogenised in 5 volumes (w/v) of 10% TCA and analysed for the presence of TCA precipitable iodinated MBF-2, L-MBF-2 or IGF-I.
Results for each tissue were expressed as cpm/gram of tissue and normalised compared to cpm in the plasma of each respective rat, to produce a ratio of between 0 and 1 correlating to cpm/gram of tissue. Table 8 shows the tissues where preferential localisation of MBFs occurs compared to IGF-I.
m m H
I
w C7 r~l H r101 CO N O LClt11r-iLfl O O r-Iw-1N r--IN N
U~O O O O O O O O
rrr a U
b f~ ~ ~-It1 a~o M ~ m o O [-1c-1c-iN ('1N N N
O ~
U U~0 0 0 0 0 0 0 0 I ~
[z,~,' m f~ L N N ~ d'O rl O O O rlr W ~--IN N
-I
I U pqo O o O o o O o f~ U7 O l~ O O1C~ tI1O LI~N
b r'IN M M M Ll) ~,O O O O O O O O
N ~ O
I .-, H O O O O O O O O
f~ O N
~, H o m ~-I~ O O M M N N
E-'~ ~ .,i ~ .~I a o c~ o 0 0 0 0 0 s~ ~
.~,~ m 'b ~-I rll0 l0 N CO h I~I'~l0 O O U ri c-IN c-iM M c'~1N
O O O O O O O O
4-I((j ,~,"O O O O O O O O
O J~
!~
~1 d~ O LC1M M 00 M
W rrN N l0tI1111l00~ l0 1J rlO O O O O O O O
x ~ . .
U7 ClaO O O O O O O O
r-i m m N
V
O r1a) .t-'-~ [Ci~s ~ G
~
N rl(.," -I
.
r rl~ .~, LCl 'rl~ ri c--I
x N N
~, I I
1.1H H [x, [z, m w H H H
SUBSTITUTE SHEET (Rule 26) (RO/AU) Example 16 Effect of L-MBF-2 on Growth and Cellular Activity of CHO Cells Grown on Polypropylene Discs 30 mls of 0.9o saline, 2 grams of polypropylene discs were placed in four 100 ml spinner flasks and autoclaved. The saline in two of the spinner flasks was removed, replaced with 30 mls of DMEM containing 100 ng/ml L-MBF-2, and all four flasks stored overnight at 4°-C. The polypropylene discs in all four-spinner flasks were washed twice with 50 mls of DMEM and seeded with 1 x 10' CHO cells expressing marmoset chorionic gonadotrophin in 50 mls of PF-CHO protein-free medium. The spinner flasks were allowed to stand for 1 hour, after which they were incubated at 5o COZ, 37°C on a multiple magnetic stirrer.
1 ml sub-samples of medium were collected from each spinner flask at 2 hrs, 4 hrs, 16 hrs, 20 hrs, 24 hrs, 48 hrs, 72 hrs and frozen immediately. The sub-samples were analysed for glucose concentration and marmoset chorionic gonadotrophin (ELISA) as indicators of cellular growth and/or activity.
Results at each sub-sample time are expressed as glucose concentrations (mM), as shown in Table 9, and as protein (chorionic gonadotrophin) production, as measured by colorimetric ELISA assay at 650 nm, shown in Table 10.
These results showed that CHO cells grown on polypropylene discs treated with biologically pure L-MBF-2 from example 6 contained within 200 ml spinner flasks were more active, as indicated by glucose consumption and marginally increased protein production, than identical cells grown on untreated polypropylene discs.
Tahla ~
Glucose Consumption over the 72 hour Period Sample Glucose Concentration (mM) Treated Untreated Discs Discs 2 hours 21.8 21.8 21.8 21.8 4 hours 20.8 20.7 21.6 21.6 16 hours 19.3 19.2 21.1 21.0 20 hours 18.7 18.9 20.9 20.7 24 hours 15.9 15.7 20.7 20.3 48 hours 13.9 13.6 16.6 16.5 72 hours 9.8 8.5 11.5 12.1 Ta~-,~ ~~ ~ n Marmoset Chorionic Gonadotrophin in Conditioned Medium as Measured by ELISA
Sample Absorbance (650 nm) Treated Untreated Discs Discs 2 hours 0.43 0.4 0.36 0.4 (1:4 dilution}
4 hours 0.44 0.4 0.37 0.4 (1:4 dilution) 16 hours 0.41 0.41 0.38 0.37 (1:8 dilution) hours 0.46 0.44 0.39 0.39 (1:8 dilution) '24 hours 0.48 0.51 0.45 0.47 (1:8 dilution) WO 99/54359 PCTlAU99/00292 Example 17 The Protective Effects of MBF Coated Tissue Culture Plastic Against Apoptosis induced by Serum Deprivation or Camptothecin The tissue culture wells were each incubated with 1 ml of DMEM containing 100 ng/ml of MBF-2 or L-MBF-2 for 18 hours at 4°C. Following the 18 hour incubation all wells were washed twice with 1 ml of DMEM, and allowed to air dry in a laminar flow cabinet. An identical number (0.2 x 10') of MCF7 mammary tumour cells was seeded on to i0 and grown on the MBF-treated and untreated 24-well tissue culture plastic plates. The number of cells on the respective plates induced into apoptosis by either serum deprivation or the addition of camptothecin was indicated by the detection of propidium iodide staining.
Results are expressed as the number of cells per field staining positive for propidium iodide. Four fields in each of two wells were assessed for each treatment (n=8), and the results are shown in Table 11. This experiment showed that biologically pure MBF-2 and L-MBF-2 from Example 6 coated on 24-well tissue culture plastic plates had a protective effect against serum deprivation or drug-induced apoptosis in a mammary tumour cell-line.
r-i,..
I
m 3 a N ro 1 ~ w M
w ~
m +i +~
a ~ --~ M
.~, m ~
s~
I
~ m +I
~
o ~ o ~r a ~- ~ co U
ro m b r' m a v w M d~
W ~ m +~ +~
r l ~ o0 3 n .-, a~
U
r~ 00 'I-a m 11 0 3 ~
' y~ ~ .
N
~I Id +~
+~
N
U o b N
s~
S-a U
!~ N
O
b m ~ O
U
SUBSTITUTE SHEET (Rule 26) (RO/AU) Example 18 The Stimulation of Protein Synthesis in HaCat Epithelial Cells Seeded on to Various Biological Substrates Pre-Treated with MBF-2, L-MBF-2 or IGF-I
Preparation of substrates 24-well tissue culture plastic plates were coated with 1 ml of poly-L-lysine solution (O. lo) for 10 minutes, after which each well was washed twice with sterile Milli-Q
water and allowed to air dry for at least 2 hours.
to Heparin, dextran sulphate and chondroitin sulphate A were dissolved in sterile water (100 ~,g/ml), 1 ml of each solution added to respective wells and incubated for 18 hours at 4°C. The wells were washed twice with 1 ml of sterile Milli-Q water, and allowed to air dry inside a t5 laminar flow cabinet for at least 1 hour. These plates were stored at 4°C until pre-treatment with MBFs or IGF-I.
Rat tail collagen was prepared according to a conventional method and 250 x..1,1 of the stock collagen solution added to respective poly-L-lysine coated wells.
20 After a 5 minute incubation inside a laminar flow cabinet the collagen solution was aspirated, leaving only a thin film of collagen remaining, and the collagen-coated wells air dried for at least 1 hour, after which they were stored at 4°C until pre-treatment with MBFs or IGF-I.
25 Fibronectin and laminin coated plates were purchased from Falcon, and pre-treated with MBFs or IGF-I
as supplied.
Pre-treatment of substrates 30 Biologically pure MBF-2 and L-MBF-2 from Example 6 and receptor grade IGF-I were dissolved in DMEM
at 100 ng/m1. 1 ml of these solutions was added to the respective pre-prepared wells and incubated for 4 hours at 4°-C. All wells were washed twice with 1 ml of cold DMEM
35 and stored at 4°C prior to inoculation with cells.
Seeding with cells HaCat cells were grown in the absence of serum for 4 hours and harvested. Cells were counted and resuspended into DMEM containing 1 ~Ci/ml of tritiated leucine at 2 x 105 cells/ml. 1 ml of cell suspension containing radioactive tracer was added to each of the pre-prepared and pre-treated wells, and incubated at 37°C for 18 hours.
Harvesting The wells were washed twice with 2 ml of cold Hank's balanced salt solution, twice with 1 ml of cold 50 trichloroacetic acid, and once with 2 ml of cold Milli-Q
water. 1 ml of 0.1o Triton X-100/0.5M NaOH was added to each well and incubated at room temperature for 30 minutes with shaking. 100 ~l sub-samples from each well were transferred to scintillation vials, 2 ml of scintillation fluid added to each vial and mixed well with shaking. Sub-samples were assayed for the presence of newly synthesized tritiated-leucine containing protein.
Results are expressed as the percentage of protein synthesis stimulation compared to a growth factor-free control, and are shown in Table 12. This study showed that HaCat cells grown on 24-well tissue culture plastic plates coated with heparin, dextran sulphate, chondroitin sulphate A, fibronectin, laminin and rat-tail collagen respectively that had been pre-treated with MBF-2 or L-MBF-2 all exhibited increased stimulations of protein synthesis, compared with the same substrates pre-treated with IGF-I. No effect was found with poly-L-lysine coated plates. Similar results were obtained in a second experiment.
' WO 99/54359 PCT/AU99/00292 M
I M
I
I
I
~
~, ri .,i ~ I' O ~.,'' ood~ ,--~ ,. W D
.~
' . . M O M o~.
~ ~
N O I (~ lD[
m o +~ a ~ r-,o, O a ,-1-,o, U
U
v .~ v v ~ v U
w m t O M O ~ O 01 l0N
O y,~
pp O
L~ O N 1.J .~ d~ C~M
U , tolO o1 U rl lD lDO
r1 IH c--Ic-io1 (jj W '-ir-1a-1 I ~ I
+-~
-u o , . . . O ~ ,~
U o ? o o ~ ~ ? o ~ 0 I ~ ~ 0 w ~ ~ ,-~~, w ~
v a fO I'~M CO
ri M . . N 1J ~"i ' ' O lI1 G' O m o M , cn ~ '~' m v 5-11 U N N H ~ U
L
. ~ ~ N N ~-i -~ p, ~ ID v rd ~ ~ ~ ~
r-1rl . 1-l U7 Ul Ai rl ~
td~ 'Q .,..~~D m (d .~ -rim E'~, ''[SC!1 ~ ~ N CD v Ul b fHM c-1Lfl Li , ~
v O rlO N d~
.~iW -I~-iO
U ~ N N ~-1 ~ V ~ N N ~-1 Gl W
1~
~1 a r-1lD M '~ ~1 .~iN LnO
M ;
x ~ ~ ~; a~ x A N N N ,--1 ~ C~ UlN N ~-1 >~
Cn r1 r~
M (~ L~ ~ O
~
r',o ~ v p, oo ~ c~, Q) .-i~--IO .1.~ ~ ~ c--IO
O x N N r-1 O ,~, N N r-1 W W
U U
r1 u-r r1 O ~.1 1f1[~ 01 O 1~ c~ N M
.~, f~ CItM O1 ~i Id 01 O rl O O O O rl r1 d~c-1 W M M ~ r1 W M M H
l~
(>~ N r..l N
d.aO N CL~H ~ .L!O N fsaH
~
C9 W ~:~7 H C9 W ~ ~7H
'n O_ SUBSTITUTE SHEET (Rule 26) (RO/AU) Example 19 The Stimulation of Tenocyte Cell Migration from Tendon Biopsies by MBF-2 The migration of fibroblast type cells (tenocytes) from circular tendon biopsies into fibrin clots was stimulated by biologically pure MBF-2 from Example 6. 2 mm diameter tendon biopsies were taken from chicken toe flexor tendons and embedded into fibrin clots. The fibrin clots containing tendon biopsies were incubated in medium containing MBF-2 (500 ng/ml) + 5% fetal bovine serum (FBS), IGF-I (500 ng/ml) + 5o FBS or in 5% FBS alone and incubated at 5o COz, 37QC for 4 days. The migration distances of cells were measured four times each day at three-hourly intervals using phase-contrast light microscopy (4X magnification) and the mean calculated for each treatment.
Results were expressed as the migration distance in millimetres at each of the four days, and are shown in Table 13. This experiment demonstrated greater migration distances with MBF-2 and IGF-I above control (5oFBS) cultures. MBF-2 was slightly more active than IGF-I at the earlier time points.
m ~ ~.,, ~ , ~
Migration Distances ~(mm) of Tenocytes from their Biopsy Interface IGF-1 I~F-2 (500 5% Fetal bovine (500 ng/ml) ser~un ng/ml) + 5% FBS
+ 5 %
FBS
Day 1 0.8 0.1 1.0 0.1 0.4 0.1 Day 2 2.7 0.4 3.4 0.3 1.5 0.2 Day 3 5.3 0.6 5.7 0.5 3.5 0.4 Day 4 7.8 0.8 7.8 0.4 5.7 0.6 It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: GROPEP PTY LTD
(B) STREET: GATE 11, VICTORIA DRIVE
(C) CITY: ADELAIDE
(D) STATE: SOUTH AUSTRALIA
(E) COUNTRY: AUSTRALIA
(F) POSTAL CODE (ZIP): 5000 (ii) TITLE OF INVENTION: MATRIX BINDING FACTOR
(iii) NUMBER OF SEQUENCES: 12 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO) (v) CURRENT APPLICATION DATA:
APPLICATION NUMBER: AU PP 2984 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 79 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
G1y Pro G1u Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe Val Cys Gly Asp Arg G1y Phe Tyr Phe Asn Pro Thr Lys Gly Tyr Gly Ser Ser Ser Arg Arg A1a Pro Gln Thr Gly Val Asp Ile Glu Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Tyr Cys Met Ala Pro Lys Lys Asn Gly Arg Ser Lys Leu Gly Pro Arg His Phe Thr Gly Gln (2) INFO RMATION FOR SEQ ID NO:
2:
(i) SEQUENCE
CHARACTERISTICS:
(A) LENGTH: 84 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE
TYPE:
peptide ' WO 99/54359 PCT/AU99/00292 (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Gly Pro Glu Thr Leu Cys G1y Ala Glu Leu Val Asp Ala Leu Gln Phe l0 15 Val Cys Gly Asp Arg G1y Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu Lys Lys Asn Gly Arg Ser Lys Leu Gly Pro Arg Thr His Phe Gly Gln Ala Lys Ser Ala (2) INFORMATION FOR SEQ ID N0: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 80 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (iii)HYPOTHETICAL: NO
(iv)ANTI-SENSE: NO
(xi)SEQUENCE DESCRIPTION: NO: 3:
SEQ ID
t0 Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly Ser Ser Ser Arg Arg Ala Pro G1n Thr Gly Ile Va1 Asp Glu Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Gly Lys Lys Asn Gly Arg Ser G1n Lys Gly Pro Arg Thr His Phe Gly Gln (2) INFORMATION FOR SEQ ID NO: 4:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 70 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys Phe Arg Ser Cys Asp Lys Arg Gln Leu Glu Lys Tyr Cys Ala Pro Gly Lys Arg Gly Arg Ser Ala (2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii)HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 5:
TGC GCTCCGC
TGP.,~~~1AAAAA
CGGTCGTTCT
AAACTGGGCC
CGGCTAAATC
TGCT
15(2) INFORMATION
FOR
SEQ
ID
NO:
6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii)HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
TCT AAACTGG
GTCCGCGTAC
CCACTTCGGC
CAGGCTAAAT
CTGCTTGA
(2) INFORMATION
FOR
SEQ
ID
NO:
7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETTCAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
ATGTACTGCG
CTCCGAA.AAA
AAACGGTCGT
TCTAAACTGC
TGAAACCGGC
TAAA
(2) INFORMATION
FOR SEQ
ID NO:
8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
GGTCGTTCTA
AACTGGGCCC
GCGTACCCAC
TTCGGTCAGT
GATGATGCAA
GCTT
(2) INFORMATION
FOR SEQ
ID NO:
9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
ATGTACTGCG CTCCGGGTAA AAAAAACGGC CGTTCTCAGA AACTGAAACC
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A} LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
GGTCGTTCTC AGAAAGGCCC GCGTACCCAC TTCGGTCAGT GATGATGCAA GCTT
_ g _ (2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
IS (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
TTCCGTTCTT GCGACAA.ACG TCAGCTGGAA AAATACTGCG CTCCGCTG
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
AAATACTGCG CTCCGGGTAA ACGTGGCCGT TCTGCTTGAT GATGC
Results for each tissue were expressed as cpm/gram of tissue and normalised compared to cpm in the plasma of each respective rat, to produce a ratio of between 0 and 1 correlating to cpm/gram of tissue. Table 8 shows the tissues where preferential localisation of MBFs occurs compared to IGF-I.
m m H
I
w C7 r~l H r101 CO N O LClt11r-iLfl O O r-Iw-1N r--IN N
U~O O O O O O O O
rrr a U
b f~ ~ ~-It1 a~o M ~ m o O [-1c-1c-iN ('1N N N
O ~
U U~0 0 0 0 0 0 0 0 I ~
[z,~,' m f~ L N N ~ d'O rl O O O rlr W ~--IN N
-I
I U pqo O o O o o O o f~ U7 O l~ O O1C~ tI1O LI~N
b r'IN M M M Ll) ~,O O O O O O O O
N ~ O
I .-, H O O O O O O O O
f~ O N
~, H o m ~-I~ O O M M N N
E-'~ ~ .,i ~ .~I a o c~ o 0 0 0 0 0 s~ ~
.~,~ m 'b ~-I rll0 l0 N CO h I~I'~l0 O O U ri c-IN c-iM M c'~1N
O O O O O O O O
4-I((j ,~,"O O O O O O O O
O J~
!~
~1 d~ O LC1M M 00 M
W rrN N l0tI1111l00~ l0 1J rlO O O O O O O O
x ~ . .
U7 ClaO O O O O O O O
r-i m m N
V
O r1a) .t-'-~ [Ci~s ~ G
~
N rl(.," -I
.
r rl~ .~, LCl 'rl~ ri c--I
x N N
~, I I
1.1H H [x, [z, m w H H H
SUBSTITUTE SHEET (Rule 26) (RO/AU) Example 16 Effect of L-MBF-2 on Growth and Cellular Activity of CHO Cells Grown on Polypropylene Discs 30 mls of 0.9o saline, 2 grams of polypropylene discs were placed in four 100 ml spinner flasks and autoclaved. The saline in two of the spinner flasks was removed, replaced with 30 mls of DMEM containing 100 ng/ml L-MBF-2, and all four flasks stored overnight at 4°-C. The polypropylene discs in all four-spinner flasks were washed twice with 50 mls of DMEM and seeded with 1 x 10' CHO cells expressing marmoset chorionic gonadotrophin in 50 mls of PF-CHO protein-free medium. The spinner flasks were allowed to stand for 1 hour, after which they were incubated at 5o COZ, 37°C on a multiple magnetic stirrer.
1 ml sub-samples of medium were collected from each spinner flask at 2 hrs, 4 hrs, 16 hrs, 20 hrs, 24 hrs, 48 hrs, 72 hrs and frozen immediately. The sub-samples were analysed for glucose concentration and marmoset chorionic gonadotrophin (ELISA) as indicators of cellular growth and/or activity.
Results at each sub-sample time are expressed as glucose concentrations (mM), as shown in Table 9, and as protein (chorionic gonadotrophin) production, as measured by colorimetric ELISA assay at 650 nm, shown in Table 10.
These results showed that CHO cells grown on polypropylene discs treated with biologically pure L-MBF-2 from example 6 contained within 200 ml spinner flasks were more active, as indicated by glucose consumption and marginally increased protein production, than identical cells grown on untreated polypropylene discs.
Tahla ~
Glucose Consumption over the 72 hour Period Sample Glucose Concentration (mM) Treated Untreated Discs Discs 2 hours 21.8 21.8 21.8 21.8 4 hours 20.8 20.7 21.6 21.6 16 hours 19.3 19.2 21.1 21.0 20 hours 18.7 18.9 20.9 20.7 24 hours 15.9 15.7 20.7 20.3 48 hours 13.9 13.6 16.6 16.5 72 hours 9.8 8.5 11.5 12.1 Ta~-,~ ~~ ~ n Marmoset Chorionic Gonadotrophin in Conditioned Medium as Measured by ELISA
Sample Absorbance (650 nm) Treated Untreated Discs Discs 2 hours 0.43 0.4 0.36 0.4 (1:4 dilution}
4 hours 0.44 0.4 0.37 0.4 (1:4 dilution) 16 hours 0.41 0.41 0.38 0.37 (1:8 dilution) hours 0.46 0.44 0.39 0.39 (1:8 dilution) '24 hours 0.48 0.51 0.45 0.47 (1:8 dilution) WO 99/54359 PCTlAU99/00292 Example 17 The Protective Effects of MBF Coated Tissue Culture Plastic Against Apoptosis induced by Serum Deprivation or Camptothecin The tissue culture wells were each incubated with 1 ml of DMEM containing 100 ng/ml of MBF-2 or L-MBF-2 for 18 hours at 4°C. Following the 18 hour incubation all wells were washed twice with 1 ml of DMEM, and allowed to air dry in a laminar flow cabinet. An identical number (0.2 x 10') of MCF7 mammary tumour cells was seeded on to i0 and grown on the MBF-treated and untreated 24-well tissue culture plastic plates. The number of cells on the respective plates induced into apoptosis by either serum deprivation or the addition of camptothecin was indicated by the detection of propidium iodide staining.
Results are expressed as the number of cells per field staining positive for propidium iodide. Four fields in each of two wells were assessed for each treatment (n=8), and the results are shown in Table 11. This experiment showed that biologically pure MBF-2 and L-MBF-2 from Example 6 coated on 24-well tissue culture plastic plates had a protective effect against serum deprivation or drug-induced apoptosis in a mammary tumour cell-line.
r-i,..
I
m 3 a N ro 1 ~ w M
w ~
m +i +~
a ~ --~ M
.~, m ~
s~
I
~ m +I
~
o ~ o ~r a ~- ~ co U
ro m b r' m a v w M d~
W ~ m +~ +~
r l ~ o0 3 n .-, a~
U
r~ 00 'I-a m 11 0 3 ~
' y~ ~ .
N
~I Id +~
+~
N
U o b N
s~
S-a U
!~ N
O
b m ~ O
U
SUBSTITUTE SHEET (Rule 26) (RO/AU) Example 18 The Stimulation of Protein Synthesis in HaCat Epithelial Cells Seeded on to Various Biological Substrates Pre-Treated with MBF-2, L-MBF-2 or IGF-I
Preparation of substrates 24-well tissue culture plastic plates were coated with 1 ml of poly-L-lysine solution (O. lo) for 10 minutes, after which each well was washed twice with sterile Milli-Q
water and allowed to air dry for at least 2 hours.
to Heparin, dextran sulphate and chondroitin sulphate A were dissolved in sterile water (100 ~,g/ml), 1 ml of each solution added to respective wells and incubated for 18 hours at 4°C. The wells were washed twice with 1 ml of sterile Milli-Q water, and allowed to air dry inside a t5 laminar flow cabinet for at least 1 hour. These plates were stored at 4°C until pre-treatment with MBFs or IGF-I.
Rat tail collagen was prepared according to a conventional method and 250 x..1,1 of the stock collagen solution added to respective poly-L-lysine coated wells.
20 After a 5 minute incubation inside a laminar flow cabinet the collagen solution was aspirated, leaving only a thin film of collagen remaining, and the collagen-coated wells air dried for at least 1 hour, after which they were stored at 4°C until pre-treatment with MBFs or IGF-I.
25 Fibronectin and laminin coated plates were purchased from Falcon, and pre-treated with MBFs or IGF-I
as supplied.
Pre-treatment of substrates 30 Biologically pure MBF-2 and L-MBF-2 from Example 6 and receptor grade IGF-I were dissolved in DMEM
at 100 ng/m1. 1 ml of these solutions was added to the respective pre-prepared wells and incubated for 4 hours at 4°-C. All wells were washed twice with 1 ml of cold DMEM
35 and stored at 4°C prior to inoculation with cells.
Seeding with cells HaCat cells were grown in the absence of serum for 4 hours and harvested. Cells were counted and resuspended into DMEM containing 1 ~Ci/ml of tritiated leucine at 2 x 105 cells/ml. 1 ml of cell suspension containing radioactive tracer was added to each of the pre-prepared and pre-treated wells, and incubated at 37°C for 18 hours.
Harvesting The wells were washed twice with 2 ml of cold Hank's balanced salt solution, twice with 1 ml of cold 50 trichloroacetic acid, and once with 2 ml of cold Milli-Q
water. 1 ml of 0.1o Triton X-100/0.5M NaOH was added to each well and incubated at room temperature for 30 minutes with shaking. 100 ~l sub-samples from each well were transferred to scintillation vials, 2 ml of scintillation fluid added to each vial and mixed well with shaking. Sub-samples were assayed for the presence of newly synthesized tritiated-leucine containing protein.
Results are expressed as the percentage of protein synthesis stimulation compared to a growth factor-free control, and are shown in Table 12. This study showed that HaCat cells grown on 24-well tissue culture plastic plates coated with heparin, dextran sulphate, chondroitin sulphate A, fibronectin, laminin and rat-tail collagen respectively that had been pre-treated with MBF-2 or L-MBF-2 all exhibited increased stimulations of protein synthesis, compared with the same substrates pre-treated with IGF-I. No effect was found with poly-L-lysine coated plates. Similar results were obtained in a second experiment.
' WO 99/54359 PCT/AU99/00292 M
I M
I
I
I
~
~, ri .,i ~ I' O ~.,'' ood~ ,--~ ,. W D
.~
' . . M O M o~.
~ ~
N O I (~ lD[
m o +~ a ~ r-,o, O a ,-1-,o, U
U
v .~ v v ~ v U
w m t O M O ~ O 01 l0N
O y,~
pp O
L~ O N 1.J .~ d~ C~M
U , tolO o1 U rl lD lDO
r1 IH c--Ic-io1 (jj W '-ir-1a-1 I ~ I
+-~
-u o , . . . O ~ ,~
U o ? o o ~ ~ ? o ~ 0 I ~ ~ 0 w ~ ~ ,-~~, w ~
v a fO I'~M CO
ri M . . N 1J ~"i ' ' O lI1 G' O m o M , cn ~ '~' m v 5-11 U N N H ~ U
L
. ~ ~ N N ~-i -~ p, ~ ID v rd ~ ~ ~ ~
r-1rl . 1-l U7 Ul Ai rl ~
td~ 'Q .,..~~D m (d .~ -rim E'~, ''[SC!1 ~ ~ N CD v Ul b fHM c-1Lfl Li , ~
v O rlO N d~
.~iW -I~-iO
U ~ N N ~-1 ~ V ~ N N ~-1 Gl W
1~
~1 a r-1lD M '~ ~1 .~iN LnO
M ;
x ~ ~ ~; a~ x A N N N ,--1 ~ C~ UlN N ~-1 >~
Cn r1 r~
M (~ L~ ~ O
~
r',o ~ v p, oo ~ c~, Q) .-i~--IO .1.~ ~ ~ c--IO
O x N N r-1 O ,~, N N r-1 W W
U U
r1 u-r r1 O ~.1 1f1[~ 01 O 1~ c~ N M
.~, f~ CItM O1 ~i Id 01 O rl O O O O rl r1 d~c-1 W M M ~ r1 W M M H
l~
(>~ N r..l N
d.aO N CL~H ~ .L!O N fsaH
~
C9 W ~:~7 H C9 W ~ ~7H
'n O_ SUBSTITUTE SHEET (Rule 26) (RO/AU) Example 19 The Stimulation of Tenocyte Cell Migration from Tendon Biopsies by MBF-2 The migration of fibroblast type cells (tenocytes) from circular tendon biopsies into fibrin clots was stimulated by biologically pure MBF-2 from Example 6. 2 mm diameter tendon biopsies were taken from chicken toe flexor tendons and embedded into fibrin clots. The fibrin clots containing tendon biopsies were incubated in medium containing MBF-2 (500 ng/ml) + 5% fetal bovine serum (FBS), IGF-I (500 ng/ml) + 5o FBS or in 5% FBS alone and incubated at 5o COz, 37QC for 4 days. The migration distances of cells were measured four times each day at three-hourly intervals using phase-contrast light microscopy (4X magnification) and the mean calculated for each treatment.
Results were expressed as the migration distance in millimetres at each of the four days, and are shown in Table 13. This experiment demonstrated greater migration distances with MBF-2 and IGF-I above control (5oFBS) cultures. MBF-2 was slightly more active than IGF-I at the earlier time points.
m ~ ~.,, ~ , ~
Migration Distances ~(mm) of Tenocytes from their Biopsy Interface IGF-1 I~F-2 (500 5% Fetal bovine (500 ng/ml) ser~un ng/ml) + 5% FBS
+ 5 %
FBS
Day 1 0.8 0.1 1.0 0.1 0.4 0.1 Day 2 2.7 0.4 3.4 0.3 1.5 0.2 Day 3 5.3 0.6 5.7 0.5 3.5 0.4 Day 4 7.8 0.8 7.8 0.4 5.7 0.6 It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: GROPEP PTY LTD
(B) STREET: GATE 11, VICTORIA DRIVE
(C) CITY: ADELAIDE
(D) STATE: SOUTH AUSTRALIA
(E) COUNTRY: AUSTRALIA
(F) POSTAL CODE (ZIP): 5000 (ii) TITLE OF INVENTION: MATRIX BINDING FACTOR
(iii) NUMBER OF SEQUENCES: 12 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO) (v) CURRENT APPLICATION DATA:
APPLICATION NUMBER: AU PP 2984 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 79 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
G1y Pro G1u Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe Val Cys Gly Asp Arg G1y Phe Tyr Phe Asn Pro Thr Lys Gly Tyr Gly Ser Ser Ser Arg Arg A1a Pro Gln Thr Gly Val Asp Ile Glu Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Tyr Cys Met Ala Pro Lys Lys Asn Gly Arg Ser Lys Leu Gly Pro Arg His Phe Thr Gly Gln (2) INFO RMATION FOR SEQ ID NO:
2:
(i) SEQUENCE
CHARACTERISTICS:
(A) LENGTH: 84 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE
TYPE:
peptide ' WO 99/54359 PCT/AU99/00292 (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Gly Pro Glu Thr Leu Cys G1y Ala Glu Leu Val Asp Ala Leu Gln Phe l0 15 Val Cys Gly Asp Arg G1y Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu Lys Lys Asn Gly Arg Ser Lys Leu Gly Pro Arg Thr His Phe Gly Gln Ala Lys Ser Ala (2) INFORMATION FOR SEQ ID N0: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 80 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (iii)HYPOTHETICAL: NO
(iv)ANTI-SENSE: NO
(xi)SEQUENCE DESCRIPTION: NO: 3:
SEQ ID
t0 Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly Ser Ser Ser Arg Arg Ala Pro G1n Thr Gly Ile Va1 Asp Glu Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Gly Lys Lys Asn Gly Arg Ser G1n Lys Gly Pro Arg Thr His Phe Gly Gln (2) INFORMATION FOR SEQ ID NO: 4:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 70 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys Phe Arg Ser Cys Asp Lys Arg Gln Leu Glu Lys Tyr Cys Ala Pro Gly Lys Arg Gly Arg Ser Ala (2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii)HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 5:
TGC GCTCCGC
TGP.,~~~1AAAAA
CGGTCGTTCT
AAACTGGGCC
CGGCTAAATC
TGCT
15(2) INFORMATION
FOR
SEQ
ID
NO:
6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii)HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
TCT AAACTGG
GTCCGCGTAC
CCACTTCGGC
CAGGCTAAAT
CTGCTTGA
(2) INFORMATION
FOR
SEQ
ID
NO:
7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETTCAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
ATGTACTGCG
CTCCGAA.AAA
AAACGGTCGT
TCTAAACTGC
TGAAACCGGC
TAAA
(2) INFORMATION
FOR SEQ
ID NO:
8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
GGTCGTTCTA
AACTGGGCCC
GCGTACCCAC
TTCGGTCAGT
GATGATGCAA
GCTT
(2) INFORMATION
FOR SEQ
ID NO:
9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
ATGTACTGCG CTCCGGGTAA AAAAAACGGC CGTTCTCAGA AACTGAAACC
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A} LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
GGTCGTTCTC AGAAAGGCCC GCGTACCCAC TTCGGTCAGT GATGATGCAA GCTT
_ g _ (2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
IS (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
TTCCGTTCTT GCGACAA.ACG TCAGCTGGAA AAATACTGCG CTCCGCTG
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
AAATACTGCG CTCCGGGTAA ACGTGGCCGT TCTGCTTGAT GATGC
Claims (57)
1. A recombinant matrix binding factor (MBF), comprising an insulin-binding growth factor (IGF), or a biologically-active analogue, mutant or derivative thereof, in which the naturally-occurring amino acid sequence of the factor has been modified to introduce one or more amino acid substitutions, deletions and/or additions in the A
and/or D domains thereof which increase the affinity of the factor for a negatively-charged surface, and in which the factor retains its biological activity.
and/or D domains thereof which increase the affinity of the factor for a negatively-charged surface, and in which the factor retains its biological activity.
2. An MBF according to Claim 1, in which the MBF is able to bind to one or more components selected from the group consisting of cell attachment factors, basement membrane moieties, extracellular matrix components, and soluble circulating proteins.
3. An MBF according to Claim 1 or Claim 2, in which the modification is insertion of a heparin-binding amino acid motif.
4. An MBF according to any one of Claims 1 to 3, in which the heparin-binding motif is one which is present in a fibroblast growth factor, heparin-binding epidermal growth factor, vitronectin, fibronectin, histidine-rich glycoprotein, insulin-like growth factor binding protein (IGFBP), or purpurin.
5. An MBF according to Claim 4, in which the heparin-binding motif is derived from a heparin-binding motif of fibroblast growth factor-1.
6. An MBF according to Claim 5, in which the heparin-binding amino acid motif is derived from the region of bovine fibroblast growth factor-1 from lysine 127 to glycine 141.
7. An MBF according to claim 3, in which the heparin-binding amino acid motif is a consensus heparin-binding sequence based on heparin-binding epidermal growth factor and IGF-binding protein.
8. An MBF according to any one of Claims 3 to 7, in which the heparin-binding amino acid motif is present in a non-contiguous form.
9. An MBF according to any one of Claims 3 to 7, in which the heparin-binding amino acid motif is modified to maximise its polarity.
10. An MBF according to any one of Claims 3 to 9, which additionally comprises an amino acid spacer sequence.
11. An MBF according to Claim 10, in which the spacer sequence is glycine-glycine.
12. An MBF according to any one of Claim 1 to 11, selected from the group consisting of MBF-1, MBF-2, MBF-3 and MBF-4, as herein defined.
13. An MBF according to any one of Claims 1 to 11, selected from the group consisting of L-MBF-1, L-MBF-2, L-MBF-3 and L-MBF-4, as herein defined.
14. An MBF according to any one of Claims 1 to 13, in which the negatively-charged surface is selected from the group consisting of extracellular matrix, dextran sulphate, chondroitin sulphate, dermatan sulphate, heparan sulphates, heparin, collagen, fibronectin, vitronectin, laminin, hydroxyapatite, anionic plastics, silicates and physiologically-compatible metals, metal alloys, ceramics, polymers and plastic-coated metals.
15. An isolated nucleic acid molecule whose sequence encodes an ME3F according to any one of Claims 1 to 14.
16. An isolated nucleic acid molecule according to Claim 15, which is a cDNA.
17. An isolated nucleic acid molecule according to Claim 16, which is a sense cDNA.
18. An expression vector comprising a nucleic acid sequence according to any one of Claims 15 to 17.
19. An expression vector according to Claim 23, further comprising a nucleic acid sequence encoding a portion of porcine growth hormone (pGH) linked to the 5' nucleotide of the sequence encoding the MBF.
20. An expression vector according to Claim 19, in which the nucleic acid sequence encoding a portion of pGH
includes a cleavable sequence.
includes a cleavable sequence.
21. A composition comprising an MBF according to any one of Claims 1 to 14, together with a pharmaceutically or veterinarily acceptable carrier.
22. A composition according to Claim 21, in which the carrier is suitable for topical application.
23. A composition according to Claim 21, in which the carrier is suitable for parenteral administration.
24. A composition comprising an MBF according to any one of Claims 1 to 14, together with a cosmetically acceptable carrier.
25. A composition according to Claim 24, in which the carrier is a cream, lotion, medicated body wash, powder, toothpaste, or mouthwash.
26. A composition for the enhancement of tissue remodelling or tissue repair associated with tissue trauma or wound healing, comprising an effective amount of an MBF
according to any one of Claims 1 to 14, formulated with a carrier suitable for topical application.
according to any one of Claims 1 to 14, formulated with a carrier suitable for topical application.
27. A composition for alleviation of skin damage associated with ageing or with exposure to ultraviolet or ionizing radiation, comprising an effective amount of an MBF according to any one of Claims 1 to 14, together with a cosmetically-acceptable carrier.
28. A composition for the prevention or treatment of a condition associated with impaired gut function, comprising an effective amount of an MBF according to any one of Claims 1 to 14, formulated with a carrier suitable to produce an orally stable, bioactive enteral formulation.
29. A composition for the targeting and localisation of an MBF to cells or tissues, thereby to promote cell adhesion, growth, migration or activity in vivo, comprising an effective amount of MBF according to any one of Claims 1 to 14, formulated in a sterile injectible carrier.
30. A cell or tissue culture supplement comprising an MBF according to any one of Claims 1 to 14, together with a physiologically compatible carrier.
31. A tissue culture vessel or insert, comprising a negatively-charged surface pre-treated with an amount of an MBF according to any one of Claims 1 to 14, effective to promote adhesion, growth, migration or activity of vertebrate cells.
32. A surgical implant or prosthesis, comprising a negatively-charged surface pretreated with an amount of an MBF according to any one of claims 1 to 14 effective to promote adhesion, growth, migration or activity of vertebrate cells.
33. A method of producing a recombinant MBF as defined in any one of Claims 1 to 14, comprising the steps of (a) subcloning the nucleic acid sequence encoding the polypeptide bioactive factor into a cloning vector; and (b) subjecting the cloning vector to mutagenesis to generate a nucleotide sequence encoding an MBF, thereby to increase the affinity of the encoded polypeptide bioactive factor for a negatively-charged surface.
34. A method according to Claim 33, in which the mutagenesis is achieved using site-directed mutagenesis.
35. A method according to Claim 33 or Claim 34, in which the mutagenesis is achieved using one or more oligonucleotide primers which are based on binding sites for negatively-charged surfaces found in other polypeptide bioactive factors, or on sequences substantially homologous thereto.
36. A method according to any one of Claims 33 to 35, further comprising the steps of (a) subcloning the nucleic acid sequence encoding the MBF into an expression vector;
(b) transforming the expression vector into a bacterial, yeast or tissue culture host cell;
(c) cultivating the host cell under conditions suitable to express the MBF; and (d) isolating the MBF.
(b) transforming the expression vector into a bacterial, yeast or tissue culture host cell;
(c) cultivating the host cell under conditions suitable to express the MBF; and (d) isolating the MBF.
37. A method according to any one of Claims 33 to 36, in which the MBF is expressed as a fusion protein.
38. A method according to Claim 37, in which the fusion protein is expressed within inclusion bodies.
39. A method according to Claim 37 or Claim 38, in which a fragment of porcine growth hormone is linked to the N-terminal sequence of an MBF, optionally via a cleavable sequence.
40. A method according to any one of Claims 33 to 39, comprising the step of transforming a susceptible bacterial, yeast or tissue culture cell host with a recombinant DNA plasmid which includes one or more DNA
sequences capable of facilitating the expression of an MBF
fusion protein.
sequences capable of facilitating the expression of an MBF
fusion protein.
41. A method according to any one of Claims 35 to 41, in which the oligonucleotide primer is selected from the group consisting of Oligonucleotide primers IGFS-2' and IGFS-2'' encoding MBF-2:
Oligonucleotide IGFS-2' (54 mer) TGCGCTCCGCTGAAAAAAAACGGTCGTTCTAAACTGGGCCCGGCTAAATCTGCT
(SEQ ID NO. 6]
Oligonucleotide primer IGFS-2'' (48 mer) TCTAAACTGGGTCCGCGTACCCACTTCGGCCAGGCTAAATCTGCTTGA
(SEQ ID NO. 7) Oligonucleotide primers IGFS-1' and IGFS-1 " encoding MBF-1:
Oligonucleotide primer IGFS-1' (54 mer) 5' ATGTACTGCGCTCCGAAAAAAAACGGTCGTTCTAAACTGCTGAAACCGGCTAAA 3' (SEQ ID NO. 8) Oligonucleotide primer IGFS-1" (54 mer) 5' GGTCGTTCTAAACTGGGCCCGCGTACCCACTTCGGTCAGTGATGATGCAAGCTT 3' (SEQ ID NO. 9) Oligonucleotide primers IGFS-3' and IGFS-3" encoding MBF-3:
Oligonucleotide primer IGFS-3' (57 mer) 5' ATGTACTGCGCTCCGGGTAAAAAAAACGGCCGTTCTCAGAAACTGAAACCGGCTAAA 3' (SEQ ID NO. 10) Oligonucleotide primer IGFS-3" (54 mer) 5' GGTCGTTCTCAGAAAGGCCCGCGTACCCACTTCGGTCAGTGATGATGCAAGCTT 3' (SEQ ID NO. 11) Oligonucleotide primers IGFS-4' and IGFS-4" encoding MBF-4:
Oligonucleotide primer IGFS-4' (48 mer) 5' TTCCGTTCTTGCGACAAACGTCAGCTGGAAAAATACTGCGCTCCGCTG 3' (SEQ ID NO. 12) Oligonucleotide primer IGFS-4 " (45 mer) 5' AAATACTGCGCTCCGGGTAAACGTGGCCGTTCTGCTTGATGATGC 3' (SEQ ID NO. 13)
Oligonucleotide IGFS-2' (54 mer) TGCGCTCCGCTGAAAAAAAACGGTCGTTCTAAACTGGGCCCGGCTAAATCTGCT
(SEQ ID NO. 6]
Oligonucleotide primer IGFS-2'' (48 mer) TCTAAACTGGGTCCGCGTACCCACTTCGGCCAGGCTAAATCTGCTTGA
(SEQ ID NO. 7) Oligonucleotide primers IGFS-1' and IGFS-1 " encoding MBF-1:
Oligonucleotide primer IGFS-1' (54 mer) 5' ATGTACTGCGCTCCGAAAAAAAACGGTCGTTCTAAACTGCTGAAACCGGCTAAA 3' (SEQ ID NO. 8) Oligonucleotide primer IGFS-1" (54 mer) 5' GGTCGTTCTAAACTGGGCCCGCGTACCCACTTCGGTCAGTGATGATGCAAGCTT 3' (SEQ ID NO. 9) Oligonucleotide primers IGFS-3' and IGFS-3" encoding MBF-3:
Oligonucleotide primer IGFS-3' (57 mer) 5' ATGTACTGCGCTCCGGGTAAAAAAAACGGCCGTTCTCAGAAACTGAAACCGGCTAAA 3' (SEQ ID NO. 10) Oligonucleotide primer IGFS-3" (54 mer) 5' GGTCGTTCTCAGAAAGGCCCGCGTACCCACTTCGGTCAGTGATGATGCAAGCTT 3' (SEQ ID NO. 11) Oligonucleotide primers IGFS-4' and IGFS-4" encoding MBF-4:
Oligonucleotide primer IGFS-4' (48 mer) 5' TTCCGTTCTTGCGACAAACGTCAGCTGGAAAAATACTGCGCTCCGCTG 3' (SEQ ID NO. 12) Oligonucleotide primer IGFS-4 " (45 mer) 5' AAATACTGCGCTCCGGGTAAACGTGGCCGTTCTGCTTGATGATGC 3' (SEQ ID NO. 13)
42. A method for promoting adhesion, growth, migration or activity of cells on a negatively-charged surface, comprising the step of growing cells in a culture medium in the presence of a negatively-charged surface pretreated with an MBF according to any one of Claims 1 to 14.
43. A method according to Claim 42, in which the cells are of vertebrate or of insect origin.
44. A method of cell culture, comprising the step of growing cells in a culture medium comprising an MBF
according to any one of Claims 1 to 14.
according to any one of Claims 1 to 14.
45. A method according to Claim 44, in which the cells are of vertebrate or of insect origin.
46. A method for the enhancement of tissue remodelling or tissue repair associated with tissue trauma or wound healing, comprising the step of administering an effective amount of an MBF according to any one of Claims 1 to 14, to a subject in need of such treatment.
47. A method for the prevention or treatment of a condition associated with impaired gut function, comprising the step of administering an effective amount of an MBF
according to any one of Claims 1 to 14, to a subject in need of such treatment.
according to any one of Claims 1 to 14, to a subject in need of such treatment.
48. A method for the targeting or localisation of an MBF to cells or tissues, thereby to promote cell adhesion, growth, migration or activity in vivo, comprising the step of systemic or local administration of an MBF according to any one of Claims 1 to 14, to a subject in need of such treatment.
49. A method for the prevention or treatment of periodontal disease, comprising the step of administering an effective amount of an MBF according to any one of Claims 1 to 14, to a subject in need of such treatment.
50. A method of cosmetic treatment, comprising the step of administering an effective amount of an MBF
according to any one of Claims 1 to 14, to the skin or hair of a subject in need of such treatment.
according to any one of Claims 1 to 14, to the skin or hair of a subject in need of such treatment.
51. A method according to any one of Claims 46 to 50, in which the subject to be treated is a human, or is a domestic, companion or zoo animal.
52. Use of an MBF according to any one of Claims 1 to 14, in medicine or in veterinary treatment.
53. Use of an MBF according to any one of Claims 1 to 14, in the manufacture of a medicament for the treatment of a condition associated with impaired gut function or periodontal disease.
54. Use of an MBF according to any one of Claims 1 to 14, in the manufacture of a medicament for enhancement of tissue remodelling or tissue repair associated with tissue trauma or wound healing.
55. Use of an MBF according to any one of Claims 1 to 14, in the manufacture of a medicament for the targeting and localisation of an MBF to cells or tissues, thereby to promote cell adhesion, growth, migration or activity in vivo.
56. Use of an MBF according to any one of claims 1 to 14 in the manufacture of a tissue culture vessel or insert, thereby to promote adhesion, growth, migration or activity of vertebrate cells.
57. Use of an MBF according to any one of claims 1 to 14 in the manufacture of a surgical implant or prosthesis, thereby to promote adhesion, growth, migration or activity of vertebrate cells.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AUPP2984 | 1998-04-17 | ||
AUPP2984A AUPP298498A0 (en) | 1998-04-17 | 1998-04-17 | Matrix binding factor |
PCT/AU1999/000292 WO1999054359A1 (en) | 1998-04-17 | 1999-04-19 | Matrix binding factor |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2328463A1 true CA2328463A1 (en) | 1999-10-28 |
Family
ID=3807243
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002328463A Abandoned CA2328463A1 (en) | 1998-04-17 | 1999-04-19 | Matrix binding factor |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1071718A4 (en) |
JP (1) | JP2002512018A (en) |
AU (1) | AUPP298498A0 (en) |
CA (1) | CA2328463A1 (en) |
WO (1) | WO1999054359A1 (en) |
Families Citing this family (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000064481A1 (en) * | 1999-04-22 | 2000-11-02 | Eidgenössische Technische Hochschule (ETH) | Controlled release of growth factors from heparin containing matrices |
CA2417207C (en) | 2000-08-16 | 2010-04-20 | Toshiyuki Yoneda | Dental products comprising bone growth enhancing peptide |
AUPR030900A0 (en) | 2000-09-22 | 2000-10-12 | Queensland University Of Technology | Growth factor complex |
US20100143442A1 (en) * | 2003-02-05 | 2010-06-10 | Queensland University Of Technology | Growth factor complexes and modulation of cell migration and growth |
US8871709B2 (en) * | 2003-02-05 | 2014-10-28 | Queensland University of Technolgy | Synthetic chimeric proteins comprising epidermal growth factor and vitronectin |
AU2006203882B2 (en) | 2005-01-07 | 2011-05-12 | Regeneron Pharmaceuticals, Inc. | IGF-1 fusion polypeptides and therapeutic uses thereof |
EP1928491A4 (en) | 2005-07-18 | 2009-08-26 | Acologix Inc | Protein formulation for promoting hard tissue formation |
US9187517B2 (en) * | 2006-11-13 | 2015-11-17 | The Brigham And Women's Hospital, Inc. | Methods of promoting cardiac repair using growth factors fused to heparin binding sequences |
NZ600454A (en) | 2009-11-30 | 2014-10-31 | Univ Queensland | Fibronectin: growth factor chimeras |
US9790264B2 (en) | 2012-06-25 | 2017-10-17 | The Brigham And Women's Hospital, Inc. | Compounds and methods for modulating pharmacokinetics |
HUE035607T2 (en) | 2012-06-25 | 2018-05-28 | Brigham & Womens Hospital Inc | Targeted therapeutics |
US9637531B2 (en) | 2012-06-25 | 2017-05-02 | The Brigham And Women's Hospital, Inc | Selective cartilage therapy |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE69021335T2 (en) * | 1989-06-09 | 1996-04-11 | Gropep Pty. Ltd., Adelaide | GROWTH HORMON FUSION PROTEINS. |
-
1998
- 1998-04-17 AU AUPP2984A patent/AUPP298498A0/en not_active Abandoned
-
1999
- 1999-04-19 CA CA002328463A patent/CA2328463A1/en not_active Abandoned
- 1999-04-19 WO PCT/AU1999/000292 patent/WO1999054359A1/en not_active Application Discontinuation
- 1999-04-19 JP JP2000544697A patent/JP2002512018A/en active Pending
- 1999-04-19 EP EP99915364A patent/EP1071718A4/en not_active Withdrawn
Also Published As
Publication number | Publication date |
---|---|
JP2002512018A (en) | 2002-04-23 |
AUPP298498A0 (en) | 1998-05-07 |
EP1071718A1 (en) | 2001-01-31 |
EP1071718A4 (en) | 2003-07-02 |
WO1999054359A1 (en) | 1999-10-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111944057B (en) | Recombinant human collagen peptide and application thereof | |
CN108794639B (en) | Recombinant fibronectin and application thereof | |
CN110845603B (en) | Human collagen 17-type polypeptide, production method and use thereof | |
CA2017379C (en) | Purification and characterization of a glioma-derived growth factor | |
US8691944B2 (en) | Fibronectin polypeptides and methods of use | |
JPH06507304A (en) | Synthetic bioadhesive polypeptide | |
JPS6136223A (en) | Polypeptide cartilage inductive factor in bone | |
US9572869B2 (en) | Chimeric fibronectin matrix mimetics and uses thereof | |
JPH05501655A (en) | Enzyme preparation method for basic fibroblast growth factor | |
CA2328463A1 (en) | Matrix binding factor | |
CA2690734A1 (en) | Polypeptides and methods of use | |
JPH0662677B2 (en) | Purified protein having angiogenin activity and method for producing the same | |
US5302701A (en) | Polypeptide having human fibronectin-like cell adhesive activity | |
JPH07149797A (en) | Structural analogue of heparin binding part of fibroblast growth factor | |
US5498698A (en) | Megakaryocyte potentiator | |
Veis et al. | Collagen Biosynthesi | |
JP2002542824A (en) | Recombinant laminin 5 | |
AU744514B2 (en) | Matrix binding factor | |
JP2003137899A (en) | Fibroblast proliferation-promoting peptide | |
JPH07103156B2 (en) | Osteogenic growth polypeptides identified from regenerated bone marrow | |
JP2002058485A (en) | Osteogenesis-promoting fusion protein having collagen- binding property | |
CA2135123A1 (en) | Medicine for preventing and curing bone fracture | |
EP2516622A2 (en) | Enamel matrix derivative-proteins having tissue generating activity | |
KR101972988B1 (en) | Composition for promoting differentiation of stem cell into osteoblasts containing fibulin-1 | |
DE60108326T2 (en) | Snake proteins with antithrombotic effect |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
FZDE | Discontinued |