WO2007113488A1 - Fibronectin type iii domain containing protein - Google Patents
Fibronectin type iii domain containing protein Download PDFInfo
- Publication number
- WO2007113488A1 WO2007113488A1 PCT/GB2007/001109 GB2007001109W WO2007113488A1 WO 2007113488 A1 WO2007113488 A1 WO 2007113488A1 GB 2007001109 W GB2007001109 W GB 2007001109W WO 2007113488 A1 WO2007113488 A1 WO 2007113488A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- polypeptide
- disease
- nucleic acid
- acid molecule
- seq
- Prior art date
Links
- 101710162849 Fibronectin type III domain-containing protein Proteins 0.000 title description 3
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 199
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 124
- 230000004850 protein–protein interaction Effects 0.000 claims abstract description 6
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 462
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 444
- 229920001184 polypeptide Polymers 0.000 claims description 442
- 238000000034 method Methods 0.000 claims description 172
- 150000007523 nucleic acids Chemical class 0.000 claims description 160
- 102000039446 nucleic acids Human genes 0.000 claims description 151
- 108020004707 nucleic acids Proteins 0.000 claims description 151
- 150000001875 compounds Chemical class 0.000 claims description 129
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 124
- 201000010099 disease Diseases 0.000 claims description 92
- 230000014509 gene expression Effects 0.000 claims description 82
- 230000000694 effects Effects 0.000 claims description 61
- 239000003446 ligand Substances 0.000 claims description 57
- 241000282414 Homo sapiens Species 0.000 claims description 53
- 239000013598 vector Substances 0.000 claims description 51
- 239000012634 fragment Substances 0.000 claims description 49
- 239000000523 sample Substances 0.000 claims description 49
- 238000009739 binding Methods 0.000 claims description 47
- 239000000203 mixture Substances 0.000 claims description 47
- 230000027455 binding Effects 0.000 claims description 46
- 210000001519 tissue Anatomy 0.000 claims description 38
- 206010028980 Neoplasm Diseases 0.000 claims description 37
- 238000004519 manufacturing process Methods 0.000 claims description 31
- 208000035475 disorder Diseases 0.000 claims description 29
- 238000012216 screening Methods 0.000 claims description 25
- 238000011282 treatment Methods 0.000 claims description 25
- 239000005557 antagonist Substances 0.000 claims description 24
- 239000000556 agonist Substances 0.000 claims description 23
- 108020001507 fusion proteins Proteins 0.000 claims description 21
- 102000037865 fusion proteins Human genes 0.000 claims description 21
- 230000035772 mutation Effects 0.000 claims description 21
- 230000001225 therapeutic effect Effects 0.000 claims description 21
- 230000015572 biosynthetic process Effects 0.000 claims description 18
- 102000004190 Enzymes Human genes 0.000 claims description 17
- 108090000790 Enzymes Proteins 0.000 claims description 17
- 238000003745 diagnosis Methods 0.000 claims description 17
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 16
- 239000003814 drug Substances 0.000 claims description 16
- 102000005962 receptors Human genes 0.000 claims description 16
- 108020003175 receptors Proteins 0.000 claims description 16
- 206010016654 Fibrosis Diseases 0.000 claims description 15
- 238000001514 detection method Methods 0.000 claims description 15
- 230000004761 fibrosis Effects 0.000 claims description 15
- 238000000338 in vitro Methods 0.000 claims description 15
- 125000000539 amino acid group Chemical group 0.000 claims description 14
- 201000011510 cancer Diseases 0.000 claims description 14
- 241001465754 Metazoa Species 0.000 claims description 13
- 230000000890 antigenic effect Effects 0.000 claims description 13
- 239000000758 substrate Substances 0.000 claims description 13
- 230000004071 biological effect Effects 0.000 claims description 12
- 239000003153 chemical reaction reagent Substances 0.000 claims description 12
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 11
- 239000003795 chemical substances by application Substances 0.000 claims description 11
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 11
- 239000008194 pharmaceutical composition Substances 0.000 claims description 11
- 229960005486 vaccine Drugs 0.000 claims description 11
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 10
- 201000009273 Endometriosis Diseases 0.000 claims description 9
- -1 host cell Substances 0.000 claims description 9
- 108020004711 Nucleic Acid Probes Proteins 0.000 claims description 8
- 229940079593 drug Drugs 0.000 claims description 8
- 208000032839 leukemia Diseases 0.000 claims description 8
- 239000002853 nucleic acid probe Substances 0.000 claims description 8
- 210000002307 prostate Anatomy 0.000 claims description 7
- 238000002560 therapeutic procedure Methods 0.000 claims description 7
- 230000009261 transgenic effect Effects 0.000 claims description 7
- 210000004556 brain Anatomy 0.000 claims description 6
- 239000013068 control sample Substances 0.000 claims description 6
- 201000011461 pre-eclampsia Diseases 0.000 claims description 6
- 208000030507 AIDS Diseases 0.000 claims description 5
- 208000024827 Alzheimer disease Diseases 0.000 claims description 5
- 206010002556 Ankylosing Spondylitis Diseases 0.000 claims description 5
- 208000023275 Autoimmune disease Diseases 0.000 claims description 5
- 208000020084 Bone disease Diseases 0.000 claims description 5
- 208000018084 Bone neoplasm Diseases 0.000 claims description 5
- 208000003174 Brain Neoplasms Diseases 0.000 claims description 5
- 208000006545 Chronic Obstructive Pulmonary Disease Diseases 0.000 claims description 5
- 206010009900 Colitis ulcerative Diseases 0.000 claims description 5
- 208000011231 Crohn disease Diseases 0.000 claims description 5
- 206010011831 Cytomegalovirus infection Diseases 0.000 claims description 5
- 206010012689 Diabetic retinopathy Diseases 0.000 claims description 5
- 208000005171 Dysmenorrhea Diseases 0.000 claims description 5
- 206010013935 Dysmenorrhoea Diseases 0.000 claims description 5
- 208000005189 Embolism Diseases 0.000 claims description 5
- 206010014989 Epidermolysis bullosa Diseases 0.000 claims description 5
- 206010017533 Fungal infection Diseases 0.000 claims description 5
- 208000007882 Gastritis Diseases 0.000 claims description 5
- 208000010412 Glaucoma Diseases 0.000 claims description 5
- 208000032612 Glial tumor Diseases 0.000 claims description 5
- 206010018338 Glioma Diseases 0.000 claims description 5
- 206010018364 Glomerulonephritis Diseases 0.000 claims description 5
- 208000019693 Lung disease Diseases 0.000 claims description 5
- 208000016604 Lyme disease Diseases 0.000 claims description 5
- 208000026072 Motor neurone disease Diseases 0.000 claims description 5
- 208000031888 Mycoses Diseases 0.000 claims description 5
- 208000014767 Myeloproliferative disease Diseases 0.000 claims description 5
- 201000007224 Myeloproliferative neoplasm Diseases 0.000 claims description 5
- 208000022873 Ocular disease Diseases 0.000 claims description 5
- 206010030043 Ocular hypertension Diseases 0.000 claims description 5
- 206010030113 Oedema Diseases 0.000 claims description 5
- 208000018737 Parkinson disease Diseases 0.000 claims description 5
- 208000018262 Peripheral vascular disease Diseases 0.000 claims description 5
- 201000004681 Psoriasis Diseases 0.000 claims description 5
- 208000001435 Thromboembolism Diseases 0.000 claims description 5
- 208000025865 Ulcer Diseases 0.000 claims description 5
- 201000006704 Ulcerative Colitis Diseases 0.000 claims description 5
- 206010052428 Wound Diseases 0.000 claims description 5
- 208000027418 Wounds and injury Diseases 0.000 claims description 5
- 230000032683 aging Effects 0.000 claims description 5
- 230000033115 angiogenesis Effects 0.000 claims description 5
- 206010003246 arthritis Diseases 0.000 claims description 5
- 208000006673 asthma Diseases 0.000 claims description 5
- 208000029028 brain injury Diseases 0.000 claims description 5
- 210000000481 breast Anatomy 0.000 claims description 5
- 208000035269 cancer or benign tumor Diseases 0.000 claims description 5
- 210000001072 colon Anatomy 0.000 claims description 5
- 208000029078 coronary artery disease Diseases 0.000 claims description 5
- 230000007423 decrease Effects 0.000 claims description 5
- 208000019622 heart disease Diseases 0.000 claims description 5
- 201000011066 hemangioma Diseases 0.000 claims description 5
- 208000014951 hematologic disease Diseases 0.000 claims description 5
- 208000018706 hematopoietic system disease Diseases 0.000 claims description 5
- 208000026278 immune system disease Diseases 0.000 claims description 5
- 230000001939 inductive effect Effects 0.000 claims description 5
- 208000017169 kidney disease Diseases 0.000 claims description 5
- 208000019423 liver disease Diseases 0.000 claims description 5
- 208000020816 lung neoplasm Diseases 0.000 claims description 5
- 208000037841 lung tumor Diseases 0.000 claims description 5
- 208000002780 macular degeneration Diseases 0.000 claims description 5
- 201000006417 multiple sclerosis Diseases 0.000 claims description 5
- 230000004770 neurodegeneration Effects 0.000 claims description 5
- 208000018360 neuromuscular disease Diseases 0.000 claims description 5
- 208000004235 neutropenia Diseases 0.000 claims description 5
- 206010039073 rheumatoid arthritis Diseases 0.000 claims description 5
- 208000020431 spinal cord injury Diseases 0.000 claims description 5
- 206010043554 thrombocytopenia Diseases 0.000 claims description 5
- 231100000397 ulcer Toxicity 0.000 claims description 5
- 208000024172 Cardiovascular disease Diseases 0.000 claims description 4
- 239000012472 biological sample Substances 0.000 claims description 4
- 238000012544 monitoring process Methods 0.000 claims description 4
- 201000000596 systemic lupus erythematosus Diseases 0.000 claims description 4
- 239000013610 patient sample Substances 0.000 claims description 3
- 102000002090 Fibronectin type III Human genes 0.000 claims description 2
- 108050009401 Fibronectin type III Proteins 0.000 claims description 2
- 230000002526 effect on cardiovascular system Effects 0.000 claims 1
- 206010025135 lupus erythematosus Diseases 0.000 claims 1
- 230000009885 systemic effect Effects 0.000 claims 1
- 108010067306 Fibronectins Proteins 0.000 abstract description 15
- 102000016359 Fibronectins Human genes 0.000 abstract description 15
- 108090000144 Human Proteins Proteins 0.000 abstract description 2
- 102000003839 Human Proteins Human genes 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 153
- 235000018102 proteins Nutrition 0.000 description 116
- 238000003556 assay Methods 0.000 description 76
- 108020004414 DNA Proteins 0.000 description 59
- 238000003752 polymerase chain reaction Methods 0.000 description 42
- 235000001014 amino acid Nutrition 0.000 description 38
- 150000001413 amino acids Chemical class 0.000 description 35
- 238000012360 testing method Methods 0.000 description 31
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 30
- 239000013612 plasmid Substances 0.000 description 27
- 239000002299 complementary DNA Substances 0.000 description 26
- 238000006243 chemical reaction Methods 0.000 description 25
- 238000000746 purification Methods 0.000 description 25
- 239000000872 buffer Substances 0.000 description 23
- 230000006870 function Effects 0.000 description 23
- 210000002950 fibroblast Anatomy 0.000 description 22
- 125000003729 nucleotide group Chemical group 0.000 description 20
- 239000002773 nucleotide Substances 0.000 description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 19
- 230000001105 regulatory effect Effects 0.000 description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 17
- 230000004048 modification Effects 0.000 description 17
- 238000012986 modification Methods 0.000 description 17
- 239000000047 product Substances 0.000 description 17
- 238000010367 cloning Methods 0.000 description 16
- 239000012528 membrane Substances 0.000 description 16
- 108091026890 Coding region Proteins 0.000 description 15
- 210000000170 cell membrane Anatomy 0.000 description 15
- 238000005516 engineering process Methods 0.000 description 15
- 239000013604 expression vector Substances 0.000 description 15
- 238000009396 hybridization Methods 0.000 description 15
- 210000004379 membrane Anatomy 0.000 description 15
- 108020004999 messenger RNA Proteins 0.000 description 14
- 108091034117 Oligonucleotide Proteins 0.000 description 13
- 230000002068 genetic effect Effects 0.000 description 13
- 238000001727 in vivo Methods 0.000 description 13
- 239000000126 substance Substances 0.000 description 13
- 241000196324 Embryophyta Species 0.000 description 12
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 12
- 238000013459 approach Methods 0.000 description 12
- 230000000295 complement effect Effects 0.000 description 12
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 238000006467 substitution reaction Methods 0.000 description 12
- 238000001890 transfection Methods 0.000 description 12
- 102000003972 Fibroblast growth factor 7 Human genes 0.000 description 11
- 108090000385 Fibroblast growth factor 7 Proteins 0.000 description 11
- 230000003321 amplification Effects 0.000 description 11
- 238000005755 formation reaction Methods 0.000 description 11
- 238000003199 nucleic acid amplification method Methods 0.000 description 11
- 239000007787 solid Substances 0.000 description 11
- 239000003550 marker Substances 0.000 description 10
- 230000008569 process Effects 0.000 description 10
- 238000013519 translation Methods 0.000 description 10
- 238000002965 ELISA Methods 0.000 description 9
- 125000000174 L-prolyl group Chemical group [H]N1C([H])([H])C([H])([H])C([H])([H])[C@@]1([H])C(*)=O 0.000 description 9
- 241000700605 Viruses Species 0.000 description 9
- 230000000692 anti-sense effect Effects 0.000 description 9
- 210000004962 mammalian cell Anatomy 0.000 description 9
- 230000009933 reproductive health Effects 0.000 description 9
- 238000012163 sequencing technique Methods 0.000 description 9
- 239000011780 sodium chloride Substances 0.000 description 9
- 238000013518 transcription Methods 0.000 description 9
- 230000035897 transcription Effects 0.000 description 9
- 238000001712 DNA sequencing Methods 0.000 description 8
- 241000588724 Escherichia coli Species 0.000 description 8
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 8
- 230000004913 activation Effects 0.000 description 8
- 238000004113 cell culture Methods 0.000 description 8
- 239000003937 drug carrier Substances 0.000 description 8
- 239000000499 gel Substances 0.000 description 8
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 8
- 230000004044 response Effects 0.000 description 8
- 108020004635 Complementary DNA Proteins 0.000 description 7
- 238000007792 addition Methods 0.000 description 7
- 230000001580 bacterial effect Effects 0.000 description 7
- 230000001413 cellular effect Effects 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 235000014304 histidine Nutrition 0.000 description 7
- 230000001965 increasing effect Effects 0.000 description 7
- 238000003780 insertion Methods 0.000 description 7
- 230000037431 insertion Effects 0.000 description 7
- 210000004072 lung Anatomy 0.000 description 7
- 230000035755 proliferation Effects 0.000 description 7
- 230000001850 reproductive effect Effects 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 108090000994 Catalytic RNA Proteins 0.000 description 6
- 102000053642 Catalytic RNA Human genes 0.000 description 6
- 108010035532 Collagen Proteins 0.000 description 6
- 102000008186 Collagen Human genes 0.000 description 6
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 6
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 6
- 208000007984 Female Infertility Diseases 0.000 description 6
- 101000777550 Homo sapiens CCN family member 2 Proteins 0.000 description 6
- 206010021928 Infertility female Diseases 0.000 description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 6
- 208000007466 Male Infertility Diseases 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 108010035042 Osteoprotegerin Proteins 0.000 description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 6
- 102100032236 Tumor necrosis factor receptor superfamily member 11B Human genes 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 206010003883 azoospermia Diseases 0.000 description 6
- 239000011324 bead Substances 0.000 description 6
- 238000001516 cell proliferation assay Methods 0.000 description 6
- 230000002759 chromosomal effect Effects 0.000 description 6
- 229920001436 collagen Polymers 0.000 description 6
- 230000000875 corresponding effect Effects 0.000 description 6
- 230000007812 deficiency Effects 0.000 description 6
- 238000012217 deletion Methods 0.000 description 6
- 230000037430 deletion Effects 0.000 description 6
- 238000011161 development Methods 0.000 description 6
- 230000018109 developmental process Effects 0.000 description 6
- 238000001415 gene therapy Methods 0.000 description 6
- 230000002163 immunogen Effects 0.000 description 6
- 208000015181 infectious disease Diseases 0.000 description 6
- 239000003112 inhibitor Substances 0.000 description 6
- 201000010260 leiomyoma Diseases 0.000 description 6
- XXUPLYBCNPLTIW-UHFFFAOYSA-N octadec-7-ynoic acid Chemical compound CCCCCCCCCCC#CCCCCCC(O)=O XXUPLYBCNPLTIW-UHFFFAOYSA-N 0.000 description 6
- 210000001672 ovary Anatomy 0.000 description 6
- 230000001817 pituitary effect Effects 0.000 description 6
- 102000040430 polynucleotide Human genes 0.000 description 6
- 108091033319 polynucleotide Proteins 0.000 description 6
- 239000002157 polynucleotide Substances 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 238000003127 radioimmunoassay Methods 0.000 description 6
- 239000011541 reaction mixture Substances 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 108091092562 ribozyme Proteins 0.000 description 6
- 238000010561 standard procedure Methods 0.000 description 6
- 239000008223 sterile water Substances 0.000 description 6
- 239000000725 suspension Substances 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- 102100031168 CCN family member 2 Human genes 0.000 description 5
- 241000238631 Hexapoda Species 0.000 description 5
- 102100034343 Integrase Human genes 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 5
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 5
- 238000001042 affinity chromatography Methods 0.000 description 5
- 239000000427 antigen Substances 0.000 description 5
- 108091007433 antigens Proteins 0.000 description 5
- 102000036639 antigens Human genes 0.000 description 5
- 230000008827 biological function Effects 0.000 description 5
- 210000001124 body fluid Anatomy 0.000 description 5
- 239000000969 carrier Substances 0.000 description 5
- 238000003776 cleavage reaction Methods 0.000 description 5
- 239000003623 enhancer Substances 0.000 description 5
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 210000003734 kidney Anatomy 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 229910052751 metal Inorganic materials 0.000 description 5
- 239000002184 metal Substances 0.000 description 5
- 238000002493 microarray Methods 0.000 description 5
- 238000012545 processing Methods 0.000 description 5
- 230000007017 scission Effects 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- 238000001262 western blot Methods 0.000 description 5
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 description 4
- 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 4
- 102000053602 DNA Human genes 0.000 description 4
- 208000012239 Developmental disease Diseases 0.000 description 4
- 229920002307 Dextran Polymers 0.000 description 4
- 238000009007 Diagnostic Kit Methods 0.000 description 4
- 108700024394 Exon Proteins 0.000 description 4
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 4
- 102100028967 HLA class I histocompatibility antigen, alpha chain G Human genes 0.000 description 4
- 108010024164 HLA-G Antigens Proteins 0.000 description 4
- 102000003945 NF-kappa B Human genes 0.000 description 4
- 108010057466 NF-kappa B Proteins 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- 108091093037 Peptide nucleic acid Proteins 0.000 description 4
- 102000006382 Ribonucleases Human genes 0.000 description 4
- 108010083644 Ribonucleases Proteins 0.000 description 4
- 230000002159 abnormal effect Effects 0.000 description 4
- 208000009956 adenocarcinoma Diseases 0.000 description 4
- 150000003862 amino acid derivatives Chemical class 0.000 description 4
- 238000010171 animal model Methods 0.000 description 4
- 238000013528 artificial neural network Methods 0.000 description 4
- 239000010839 body fluid Substances 0.000 description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 210000002919 epithelial cell Anatomy 0.000 description 4
- 229960005309 estradiol Drugs 0.000 description 4
- 229930182833 estradiol Natural products 0.000 description 4
- 210000002744 extracellular matrix Anatomy 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 4
- 238000002513 implantation Methods 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 150000002632 lipids Chemical class 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 230000001404 mediated effect Effects 0.000 description 4
- 201000001441 melanoma Diseases 0.000 description 4
- 230000002018 overexpression Effects 0.000 description 4
- 229910052697 platinum Inorganic materials 0.000 description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 4
- 238000000159 protein binding assay Methods 0.000 description 4
- 238000007423 screening assay Methods 0.000 description 4
- 210000004739 secretory vesicle Anatomy 0.000 description 4
- 229910000162 sodium phosphate Inorganic materials 0.000 description 4
- 241000701447 unidentified baculovirus Species 0.000 description 4
- 210000004325 uterine smooth muscle cell Anatomy 0.000 description 4
- 210000004291 uterus Anatomy 0.000 description 4
- 230000003612 virological effect Effects 0.000 description 4
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 3
- FUOOLUPWFVMBKG-UHFFFAOYSA-N 2-Aminoisobutyric acid Chemical compound CC(C)(N)C(O)=O FUOOLUPWFVMBKG-UHFFFAOYSA-N 0.000 description 3
- 102000005606 Activins Human genes 0.000 description 3
- 108010059616 Activins Proteins 0.000 description 3
- 201000009030 Carcinoma Diseases 0.000 description 3
- 102000012422 Collagen Type I Human genes 0.000 description 3
- 108010022452 Collagen Type I Proteins 0.000 description 3
- 102000004127 Cytokines Human genes 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 3
- 102100029727 Enteropeptidase Human genes 0.000 description 3
- 108010013369 Enteropeptidase Proteins 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 108050001049 Extracellular proteins Proteins 0.000 description 3
- NMJREATYWWNIKX-UHFFFAOYSA-N GnRH Chemical compound C1CCC(C(=O)NCC(N)=O)N1C(=O)C(CC(C)C)NC(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)CNC(=O)C(NC(=O)C(CO)NC(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)C(CC=1NC=NC=1)NC(=O)C1NC(=O)CC1)CC1=CC=C(O)C=C1 NMJREATYWWNIKX-UHFFFAOYSA-N 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 201000009794 Idiopathic Pulmonary Fibrosis Diseases 0.000 description 3
- 108060003951 Immunoglobulin Proteins 0.000 description 3
- 239000007836 KH2PO4 Substances 0.000 description 3
- 108060001084 Luciferase Proteins 0.000 description 3
- 206010025323 Lymphomas Diseases 0.000 description 3
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 206010033128 Ovarian cancer Diseases 0.000 description 3
- 206010061535 Ovarian neoplasm Diseases 0.000 description 3
- 206010060862 Prostate cancer Diseases 0.000 description 3
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 3
- 108010029485 Protein Isoforms Proteins 0.000 description 3
- 102000001708 Protein Isoforms Human genes 0.000 description 3
- 241000700159 Rattus Species 0.000 description 3
- 108700008625 Reporter Genes Proteins 0.000 description 3
- 206010039491 Sarcoma Diseases 0.000 description 3
- 229920005654 Sephadex Polymers 0.000 description 3
- 239000012507 Sephadex™ Substances 0.000 description 3
- 101000857870 Squalus acanthias Gonadoliberin Proteins 0.000 description 3
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 3
- 239000008049 TAE buffer Substances 0.000 description 3
- 108700012920 TNF Proteins 0.000 description 3
- 208000024313 Testicular Neoplasms Diseases 0.000 description 3
- 206010057644 Testis cancer Diseases 0.000 description 3
- 102100024598 Tumor necrosis factor ligand superfamily member 10 Human genes 0.000 description 3
- 101710097160 Tumor necrosis factor ligand superfamily member 10 Proteins 0.000 description 3
- 206010046798 Uterine leiomyoma Diseases 0.000 description 3
- HGEVZDLYZYVYHD-UHFFFAOYSA-N acetic acid;2-amino-2-(hydroxymethyl)propane-1,3-diol;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid Chemical compound CC(O)=O.OCC(N)(CO)CO.OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O HGEVZDLYZYVYHD-UHFFFAOYSA-N 0.000 description 3
- 239000000488 activin Substances 0.000 description 3
- 239000011543 agarose gel Substances 0.000 description 3
- 230000004075 alteration Effects 0.000 description 3
- 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 3
- 229960000723 ampicillin Drugs 0.000 description 3
- 230000003510 anti-fibrotic effect Effects 0.000 description 3
- 201000000053 blastoma Diseases 0.000 description 3
- 238000000423 cell based assay Methods 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 230000037319 collagen production Effects 0.000 description 3
- 230000016117 decidualization Effects 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 230000008021 deposition Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 3
- 229910000397 disodium phosphate Inorganic materials 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 231100000673 dose–response relationship Toxicity 0.000 description 3
- 238000007877 drug screening Methods 0.000 description 3
- 201000008184 embryoma Diseases 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 230000013595 glycosylation Effects 0.000 description 3
- 238000006206 glycosylation reaction Methods 0.000 description 3
- 210000002503 granulosa cell Anatomy 0.000 description 3
- 210000000688 human artificial chromosome Anatomy 0.000 description 3
- 238000003018 immunoassay Methods 0.000 description 3
- 102000018358 immunoglobulin Human genes 0.000 description 3
- 238000001114 immunoprecipitation Methods 0.000 description 3
- 238000007901 in situ hybridization Methods 0.000 description 3
- 208000036971 interstitial lung disease 2 Diseases 0.000 description 3
- 239000002502 liposome Substances 0.000 description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 3
- 235000013336 milk Nutrition 0.000 description 3
- 239000008267 milk Substances 0.000 description 3
- 210000004080 milk Anatomy 0.000 description 3
- 238000010369 molecular cloning Methods 0.000 description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 3
- 208000008634 oligospermia Diseases 0.000 description 3
- 230000036616 oligospermia Effects 0.000 description 3
- 231100000528 oligospermia Toxicity 0.000 description 3
- 210000000287 oocyte Anatomy 0.000 description 3
- 230000002611 ovarian Effects 0.000 description 3
- 210000000496 pancreas Anatomy 0.000 description 3
- 229920001223 polyethylene glycol Polymers 0.000 description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 230000006798 recombination Effects 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 230000003248 secreting effect Effects 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 210000003491 skin Anatomy 0.000 description 3
- 150000003384 small molecules Chemical class 0.000 description 3
- 230000021595 spermatogenesis Effects 0.000 description 3
- 230000010009 steroidogenesis Effects 0.000 description 3
- 230000000638 stimulation Effects 0.000 description 3
- 210000002536 stromal cell Anatomy 0.000 description 3
- 229910021653 sulphate ion Inorganic materials 0.000 description 3
- 201000003120 testicular cancer Diseases 0.000 description 3
- 229940124597 therapeutic agent Drugs 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 210000002993 trophoblast Anatomy 0.000 description 3
- 241001515965 unidentified phage Species 0.000 description 3
- 208000010579 uterine corpus leiomyoma Diseases 0.000 description 3
- 201000007954 uterine fibroid Diseases 0.000 description 3
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 101800002812 Anastellin Proteins 0.000 description 2
- 102400001212 Anastellin Human genes 0.000 description 2
- 241000972773 Aulopiformes Species 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 108091033380 Coding strand Proteins 0.000 description 2
- IVOMOUWHDPKRLL-KQYNXXCUSA-N Cyclic adenosine monophosphate Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=CN=C2N)=C2N=C1 IVOMOUWHDPKRLL-KQYNXXCUSA-N 0.000 description 2
- 108010017826 DNA Polymerase I Proteins 0.000 description 2
- 102000004594 DNA Polymerase I Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 2
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 2
- 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 2
- 108010067770 Endopeptidase K Proteins 0.000 description 2
- 201000008808 Fibrosarcoma Diseases 0.000 description 2
- 102100039556 Galectin-4 Human genes 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- 101000608765 Homo sapiens Galectin-4 Proteins 0.000 description 2
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 101710203526 Integrase Proteins 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- 241000829100 Macaca mulatta polyomavirus 1 Species 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 241001529936 Murinae Species 0.000 description 2
- 230000004988 N-glycosylation Effects 0.000 description 2
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 2
- 102000004264 Osteopontin Human genes 0.000 description 2
- 108010081689 Osteopontin Proteins 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 239000013614 RNA sample Substances 0.000 description 2
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 241000283984 Rodentia Species 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 2
- 108020004682 Single-Stranded DNA Proteins 0.000 description 2
- 108091008874 T cell receptors Proteins 0.000 description 2
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 2
- 108010006785 Taq Polymerase Proteins 0.000 description 2
- 102000002933 Thioredoxin Human genes 0.000 description 2
- 108010022394 Threonine synthase Proteins 0.000 description 2
- 241000723873 Tobacco mosaic virus Species 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- 102000040945 Transcription factor Human genes 0.000 description 2
- IVOMOUWHDPKRLL-UHFFFAOYSA-N UNPD107823 Natural products O1C2COP(O)(=O)OC2C(O)C1N1C(N=CN=C2N)=C2N=C1 IVOMOUWHDPKRLL-UHFFFAOYSA-N 0.000 description 2
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 230000002730 additional effect Effects 0.000 description 2
- 210000002588 alveolar type II cell Anatomy 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 210000002459 blastocyst Anatomy 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 230000003491 cAMP production Effects 0.000 description 2
- 238000010804 cDNA synthesis Methods 0.000 description 2
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 230000015861 cell surface binding Effects 0.000 description 2
- 210000003679 cervix uteri Anatomy 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000011210 chromatographic step Methods 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 230000009918 complex formation Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 229940095074 cyclic amp Drugs 0.000 description 2
- 230000001351 cycling effect Effects 0.000 description 2
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 2
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 2
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 2
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 2
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000002405 diagnostic procedure Methods 0.000 description 2
- 102000004419 dihydrofolate reductase Human genes 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000007876 drug discovery Methods 0.000 description 2
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 2
- 210000000981 epithelium Anatomy 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000035558 fertility Effects 0.000 description 2
- 230000003176 fibrotic effect Effects 0.000 description 2
- 238000002866 fluorescence resonance energy transfer Methods 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 238000002523 gelfiltration Methods 0.000 description 2
- 230000030279 gene silencing Effects 0.000 description 2
- 230000009368 gene silencing by RNA Effects 0.000 description 2
- 238000012226 gene silencing method Methods 0.000 description 2
- ZEKANFGSDXODPD-UHFFFAOYSA-N glyphosate-isopropylammonium Chemical compound CC(C)N.OC(=O)CNCP(O)(O)=O ZEKANFGSDXODPD-UHFFFAOYSA-N 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 210000004408 hybridoma Anatomy 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 230000009545 invasion Effects 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 238000005304 joining Methods 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 238000007834 ligase chain reaction Methods 0.000 description 2
- 230000004807 localization Effects 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 210000005075 mammary gland Anatomy 0.000 description 2
- 238000013507 mapping Methods 0.000 description 2
- 108010082117 matrigel Proteins 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 210000000651 myofibroblast Anatomy 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 230000003472 neutralizing effect Effects 0.000 description 2
- 238000007899 nucleic acid hybridization Methods 0.000 description 2
- 239000002751 oligonucleotide probe Substances 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 230000004792 oxidative damage Effects 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 150000008300 phosphoramidites Chemical class 0.000 description 2
- 230000035790 physiological processes and functions Effects 0.000 description 2
- 201000010065 polycystic ovary syndrome Diseases 0.000 description 2
- 102000054765 polymorphisms of proteins Human genes 0.000 description 2
- 230000001323 posttranslational effect Effects 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 230000002206 pro-fibrotic effect Effects 0.000 description 2
- 230000000770 proinflammatory effect Effects 0.000 description 2
- 230000000069 prophylactic effect Effects 0.000 description 2
- 210000001095 prostate stromal cell Anatomy 0.000 description 2
- 230000006337 proteolytic cleavage Effects 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 230000033458 reproduction Effects 0.000 description 2
- 208000017443 reproductive system disease Diseases 0.000 description 2
- 230000011506 response to oxidative stress Effects 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 230000001177 retroviral effect Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 238000012552 review Methods 0.000 description 2
- 235000019515 salmon Nutrition 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- 210000002460 smooth muscle Anatomy 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 2
- 239000001488 sodium phosphate Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 230000009870 specific binding Effects 0.000 description 2
- 210000000278 spinal cord Anatomy 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000002123 temporal effect Effects 0.000 description 2
- 210000001550 testis Anatomy 0.000 description 2
- 108060008226 thioredoxin Proteins 0.000 description 2
- 229940094937 thioredoxin Drugs 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 210000001685 thyroid gland Anatomy 0.000 description 2
- 230000005945 translocation Effects 0.000 description 2
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 2
- 229940038773 trisodium citrate Drugs 0.000 description 2
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 2
- 230000009452 underexpressoin Effects 0.000 description 2
- 241000701161 unidentified adenovirus Species 0.000 description 2
- 239000003981 vehicle Substances 0.000 description 2
- 210000005253 yeast cell Anatomy 0.000 description 2
- WAMWSIDTKSNDCU-ZETCQYMHSA-N (2s)-2-azaniumyl-2-cyclohexylacetate Chemical compound OC(=O)[C@@H](N)C1CCCCC1 WAMWSIDTKSNDCU-ZETCQYMHSA-N 0.000 description 1
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 description 1
- OMGHIGVFLOPEHJ-UHFFFAOYSA-N 2,5-dihydro-1h-pyrrol-1-ium-2-carboxylate Chemical compound OC(=O)C1NCC=C1 OMGHIGVFLOPEHJ-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- ODHCTXKNWHHXJC-VKHMYHEASA-N 5-oxo-L-proline Chemical compound OC(=O)[C@@H]1CCC(=O)N1 ODHCTXKNWHHXJC-VKHMYHEASA-N 0.000 description 1
- 230000005730 ADP ribosylation Effects 0.000 description 1
- 101150094949 APRT gene Proteins 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 102100029457 Adenine phosphoribosyltransferase Human genes 0.000 description 1
- 108010024223 Adenine phosphoribosyltransferase Proteins 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 244000153158 Ammi visnaga Species 0.000 description 1
- 235000010585 Ammi visnaga Nutrition 0.000 description 1
- 108020004491 Antisense DNA Proteins 0.000 description 1
- 101100519158 Arabidopsis thaliana PCR2 gene Proteins 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 238000012935 Averaging Methods 0.000 description 1
- 238000009020 BCA Protein Assay Kit Methods 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 206010004446 Benign prostatic hyperplasia Diseases 0.000 description 1
- 241000219310 Beta vulgaris subsp. vulgaris Species 0.000 description 1
- 101000800130 Bos taurus Thyroglobulin Proteins 0.000 description 1
- 102100021935 C-C motif chemokine 26 Human genes 0.000 description 1
- 102100032367 C-C motif chemokine 5 Human genes 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 241000701489 Cauliflower mosaic virus Species 0.000 description 1
- 102000000844 Cell Surface Receptors Human genes 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 108010055166 Chemokine CCL5 Proteins 0.000 description 1
- 239000005496 Chlorsulfuron Substances 0.000 description 1
- 206010008631 Cholera Diseases 0.000 description 1
- 208000006332 Choriocarcinoma Diseases 0.000 description 1
- 102000008169 Co-Repressor Proteins Human genes 0.000 description 1
- 108010060434 Co-Repressor Proteins Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- 108010039419 Connective Tissue Growth Factor Proteins 0.000 description 1
- 102000015225 Connective Tissue Growth Factor Human genes 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 description 1
- 102000003915 DNA Topoisomerases Human genes 0.000 description 1
- 108090000323 DNA Topoisomerases Proteins 0.000 description 1
- 239000003298 DNA probe Substances 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 108010008655 Epstein-Barr Virus Nuclear Antigens Proteins 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- 108010074860 Factor Xa Proteins 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 108010017707 Fibronectin Receptors Proteins 0.000 description 1
- 241000700662 Fowlpox virus Species 0.000 description 1
- CHDWDBPJOZVZSE-KKUMJFAQSA-N Glu-Phe-Met Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCSC)C(O)=O CHDWDBPJOZVZSE-KKUMJFAQSA-N 0.000 description 1
- 102000006771 Gonadotropins Human genes 0.000 description 1
- 108010086677 Gonadotropins Proteins 0.000 description 1
- 241000219146 Gossypium Species 0.000 description 1
- 241000201858 Granulicatella adiacens Species 0.000 description 1
- 102100020948 Growth hormone receptor Human genes 0.000 description 1
- 101710154606 Hemagglutinin Proteins 0.000 description 1
- 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 description 1
- 108010093488 His-His-His-His-His-His Proteins 0.000 description 1
- 108091006054 His-tagged proteins Proteins 0.000 description 1
- 101000897493 Homo sapiens C-C motif chemokine 26 Proteins 0.000 description 1
- 101001027128 Homo sapiens Fibronectin Proteins 0.000 description 1
- 101000862396 Homo sapiens Follicle-stimulating hormone receptor Proteins 0.000 description 1
- 101000881168 Homo sapiens SPARC Proteins 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-N Hydrogen bromide Chemical class Br CPELXLSAUQHCOX-UHFFFAOYSA-N 0.000 description 1
- 102000009786 Immunoglobulin Constant Regions Human genes 0.000 description 1
- 108010009817 Immunoglobulin Constant Regions Proteins 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 229930010555 Inosine Natural products 0.000 description 1
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 1
- 108010005716 Interferon beta-1a Proteins 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- 241000235649 Kluyveromyces Species 0.000 description 1
- 241000235058 Komagataella pastoris Species 0.000 description 1
- WTDRDQBEARUVNC-LURJTMIESA-N L-DOPA Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-LURJTMIESA-N 0.000 description 1
- WTDRDQBEARUVNC-UHFFFAOYSA-N L-Dopa Natural products OC(=O)C(N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-UHFFFAOYSA-N 0.000 description 1
- ZGUNAGUHMKGQNY-ZETCQYMHSA-N L-alpha-phenylglycine zwitterion Chemical compound OC(=O)[C@@H](N)C1=CC=CC=C1 ZGUNAGUHMKGQNY-ZETCQYMHSA-N 0.000 description 1
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- HXEACLLIILLPRG-YFKPBYRVSA-N L-pipecolic acid Chemical compound [O-]C(=O)[C@@H]1CCCC[NH2+]1 HXEACLLIILLPRG-YFKPBYRVSA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 108010011942 LH Receptors Proteins 0.000 description 1
- 102000023108 LH Receptors Human genes 0.000 description 1
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 108091061960 Naked DNA Proteins 0.000 description 1
- 229920002274 Nalgene Polymers 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- 108700020497 Nucleopolyhedrovirus polyhedrin Proteins 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 241000320412 Ogataea angusta Species 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 101710093908 Outer capsid protein VP4 Proteins 0.000 description 1
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108010067902 Peptide Library Proteins 0.000 description 1
- 102000004861 Phosphoric Diester Hydrolases Human genes 0.000 description 1
- 108090001050 Phosphoric Diester Hydrolases Proteins 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 229920000954 Polyglycolide Polymers 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 101000621511 Potato virus M (strain German) RNA silencing suppressor Proteins 0.000 description 1
- XBDQKXXYIPTUBI-UHFFFAOYSA-N Propionic acid Chemical class CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 1
- 208000004403 Prostatic Hyperplasia Diseases 0.000 description 1
- 101710176177 Protein A56 Proteins 0.000 description 1
- 101710130181 Protochlorophyllide reductase A, chloroplastic Proteins 0.000 description 1
- 108020004518 RNA Probes Proteins 0.000 description 1
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 1
- 239000003391 RNA probe Substances 0.000 description 1
- 108091030071 RNAI Proteins 0.000 description 1
- 230000010799 Receptor Interactions Effects 0.000 description 1
- 102000004278 Receptor Protein-Tyrosine Kinases Human genes 0.000 description 1
- 108090000873 Receptor Protein-Tyrosine Kinases Proteins 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108700005075 Regulator Genes Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 1
- 101710141795 Ribonuclease inhibitor Proteins 0.000 description 1
- 229940122208 Ribonuclease inhibitor Drugs 0.000 description 1
- 102100037968 Ribonuclease inhibitor Human genes 0.000 description 1
- 108010003581 Ribulose-bisphosphate carboxylase Proteins 0.000 description 1
- 102100037599 SPARC Human genes 0.000 description 1
- 240000000111 Saccharum officinarum Species 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 0.000 description 1
- 108091081021 Sense strand Proteins 0.000 description 1
- 241000700584 Simplexvirus Species 0.000 description 1
- 108010068542 Somatotropin Receptors Proteins 0.000 description 1
- 241000256248 Spodoptera Species 0.000 description 1
- 241000295644 Staphylococcaceae Species 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 241000193996 Streptococcus pyogenes Species 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 235000021536 Sugar beet Nutrition 0.000 description 1
- 241000701093 Suid alphaherpesvirus 1 Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 201000009594 Systemic Scleroderma Diseases 0.000 description 1
- 206010042953 Systemic sclerosis Diseases 0.000 description 1
- 206010043376 Tetanus Diseases 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 102000006601 Thymidine Kinase Human genes 0.000 description 1
- 108020004440 Thymidine kinase Proteins 0.000 description 1
- 102000009843 Thyroglobulin Human genes 0.000 description 1
- 239000007984 Tris EDTA buffer Substances 0.000 description 1
- 102100028787 Tumor necrosis factor receptor superfamily member 11A Human genes 0.000 description 1
- 101710178436 Tumor necrosis factor receptor superfamily member 11A Proteins 0.000 description 1
- 241000700618 Vaccinia virus Species 0.000 description 1
- 230000001594 aberrant effect Effects 0.000 description 1
- 238000002679 ablation Methods 0.000 description 1
- 150000001242 acetic acid derivatives Chemical class 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 102000005421 acetyltransferase Human genes 0.000 description 1
- 108020002494 acetyltransferase Proteins 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000010933 acylation Effects 0.000 description 1
- 238000005917 acylation reaction Methods 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 210000001789 adipocyte Anatomy 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- PPQRONHOSHZGFQ-LMVFSUKVSA-N aldehydo-D-ribose 5-phosphate Chemical group OP(=O)(O)OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PPQRONHOSHZGFQ-LMVFSUKVSA-N 0.000 description 1
- 230000009435 amidation Effects 0.000 description 1
- 238000007112 amidation reaction Methods 0.000 description 1
- 229940126575 aminoglycoside Drugs 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 239000001166 ammonium sulphate Substances 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000012197 amplification kit Methods 0.000 description 1
- 238000004873 anchoring Methods 0.000 description 1
- 239000003098 androgen Substances 0.000 description 1
- 208000036878 aneuploidy Diseases 0.000 description 1
- 231100001075 aneuploidy Toxicity 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 230000001772 anti-angiogenic effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000340 anti-metabolite Effects 0.000 description 1
- 230000002001 anti-metastasis Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 229940100197 antimetabolite Drugs 0.000 description 1
- 239000002256 antimetabolite Substances 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 239000003816 antisense DNA Substances 0.000 description 1
- 230000010516 arginylation Effects 0.000 description 1
- 210000001106 artificial yeast chromosome Anatomy 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 210000003567 ascitic fluid Anatomy 0.000 description 1
- 238000011888 autopsy Methods 0.000 description 1
- 108010058966 bacteriophage T7 induced DNA polymerase Proteins 0.000 description 1
- 210000002469 basement membrane Anatomy 0.000 description 1
- 150000001558 benzoic acid derivatives Chemical class 0.000 description 1
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 238000010364 biochemical engineering Methods 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000005277 cation exchange chromatography Methods 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 230000008568 cell cell communication Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 230000005859 cell recognition Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000012412 chemical coupling Methods 0.000 description 1
- 150000005829 chemical entities Chemical class 0.000 description 1
- 125000003636 chemical group Chemical group 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 108700010039 chimeric receptor Proteins 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- VJYIFXVZLXQVHO-UHFFFAOYSA-N chlorsulfuron Chemical compound COC1=NC(C)=NC(NC(=O)NS(=O)(=O)C=2C(=CC=CC=2)Cl)=N1 VJYIFXVZLXQVHO-UHFFFAOYSA-N 0.000 description 1
- 230000014107 chromosome localization Effects 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000012875 competitive assay Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 239000003636 conditioned culture medium Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 description 1
- 108010057085 cytokine receptors Proteins 0.000 description 1
- 102000003675 cytokine receptors Human genes 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 210000004292 cytoskeleton Anatomy 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000017858 demethylation Effects 0.000 description 1
- 238000010520 demethylation reaction Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013400 design of experiment Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- XEYBRNLFEZDVAW-ARSRFYASSA-N dinoprostone Chemical compound CCCCC[C@H](O)\C=C\[C@H]1[C@H](O)CC(=O)[C@@H]1C\C=C/CCCC(O)=O XEYBRNLFEZDVAW-ARSRFYASSA-N 0.000 description 1
- 229960002986 dinoprostone Drugs 0.000 description 1
- 206010013023 diphtheria Diseases 0.000 description 1
- 208000037765 diseases and disorders Diseases 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 1
- 239000008298 dragée Substances 0.000 description 1
- 229940000406 drug candidate Drugs 0.000 description 1
- 239000000890 drug combination Substances 0.000 description 1
- 238000009510 drug design Methods 0.000 description 1
- 238000007878 drug screening assay Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 210000002308 embryonic cell Anatomy 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000006274 endogenous ligand Substances 0.000 description 1
- 210000005168 endometrial cell Anatomy 0.000 description 1
- 210000004696 endometrium Anatomy 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- PHTXVQQRWJXYPP-UHFFFAOYSA-N ethyltrifluoromethylaminoindane Chemical compound C1=C(C(F)(F)F)C=C2CC(NCC)CC2=C1 PHTXVQQRWJXYPP-UHFFFAOYSA-N 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 108010020275 fibrin receptor Proteins 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 230000022244 formylation Effects 0.000 description 1
- 238000006170 formylation reaction Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 238000002825 functional assay Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 230000005021 gait Effects 0.000 description 1
- 230000006251 gamma-carboxylation Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000001476 gene delivery Methods 0.000 description 1
- 238000003197 gene knockdown Methods 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 238000009650 gentamicin protection assay Methods 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 150000002337 glycosamines Chemical group 0.000 description 1
- 210000004368 gonadotroph Anatomy 0.000 description 1
- 239000002622 gonadotropin Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 1
- 230000002363 herbicidal effect Effects 0.000 description 1
- 239000004009 herbicide Substances 0.000 description 1
- 125000005842 heteroatom Chemical group 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 150000002411 histidines Chemical class 0.000 description 1
- 102000046113 human FSHR Human genes 0.000 description 1
- 210000003917 human chromosome Anatomy 0.000 description 1
- 150000003840 hydrochlorides Chemical class 0.000 description 1
- 238000004191 hydrophobic interaction chromatography Methods 0.000 description 1
- 238000012872 hydroxylapatite chromatography Methods 0.000 description 1
- 230000033444 hydroxylation Effects 0.000 description 1
- 238000005805 hydroxylation reaction Methods 0.000 description 1
- 229960002591 hydroxyproline Drugs 0.000 description 1
- 210000001822 immobilized cell Anatomy 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 239000003022 immunostimulating agent Substances 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000012405 in silico analysis Methods 0.000 description 1
- 238000010249 in-situ analysis Methods 0.000 description 1
- QNRXNRGSOJZINA-UHFFFAOYSA-N indoline-2-carboxylic acid Chemical compound C1=CC=C2NC(C(=O)O)CC2=C1 QNRXNRGSOJZINA-UHFFFAOYSA-N 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 208000000509 infertility Diseases 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 208000021267 infertility disease Diseases 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 206010022000 influenza Diseases 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 229960003786 inosine Drugs 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 238000007914 intraventricular administration Methods 0.000 description 1
- 238000007852 inverse PCR Methods 0.000 description 1
- 230000026045 iodination Effects 0.000 description 1
- 238000006192 iodination reaction Methods 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 description 1
- 101150066555 lacZ gene Proteins 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 235000021374 legumes Nutrition 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- 239000006194 liquid suspension Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000006249 magnetic particle Substances 0.000 description 1
- 150000002690 malonic acid derivatives Chemical class 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000010232 migration assay Methods 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- ZAHQPTJLOCWVPG-UHFFFAOYSA-N mitoxantrone dihydrochloride Chemical compound Cl.Cl.O=C1C2=C(O)C=CC(O)=C2C(=O)C2=C1C(NCCNCCO)=CC=C2NCCNCCO ZAHQPTJLOCWVPG-UHFFFAOYSA-N 0.000 description 1
- 230000037230 mobility Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 230000007498 myristoylation Effects 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000010807 negative regulation of binding Effects 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 210000005170 neoplastic cell Anatomy 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- LGQLOGILCSXPEA-UHFFFAOYSA-L nickel sulfate Chemical compound [Ni+2].[O-]S([O-])(=O)=O LGQLOGILCSXPEA-UHFFFAOYSA-L 0.000 description 1
- 229910000363 nickel(II) sulfate Inorganic materials 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 230000031787 nutrient reservoir activity Effects 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 238000002515 oligonucleotide synthesis Methods 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 238000012261 overproduction Methods 0.000 description 1
- 210000003101 oviduct Anatomy 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000006179 pH buffering agent Substances 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000009589 pathological growth Effects 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000001991 pathophysiological effect Effects 0.000 description 1
- 230000006320 pegylation Effects 0.000 description 1
- 239000000816 peptidomimetic Substances 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 210000001322 periplasm Anatomy 0.000 description 1
- 210000003200 peritoneal cavity Anatomy 0.000 description 1
- 210000003245 peritoneal mesothelial cell Anatomy 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 150000003905 phosphatidylinositols Chemical class 0.000 description 1
- 229940080469 phosphocellulose Drugs 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229920000747 poly(lactic acid) Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 230000000379 polymerizing effect Effects 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000034190 positive regulation of NF-kappaB transcription factor activity Effects 0.000 description 1
- 230000029279 positive regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 230000032361 posttranscriptional gene silencing Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 230000013823 prenylation Effects 0.000 description 1
- 230000037452 priming Effects 0.000 description 1
- 238000007639 printing Methods 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000009696 proliferative response Effects 0.000 description 1
- 230000001915 proofreading effect Effects 0.000 description 1
- XEYBRNLFEZDVAW-UHFFFAOYSA-N prostaglandin E2 Natural products CCCCCC(O)C=CC1C(O)CC(=O)C1CC=CCCCC(O)=O XEYBRNLFEZDVAW-UHFFFAOYSA-N 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 230000004952 protein activity Effects 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 229940043131 pyroglutamate Drugs 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000006340 racemization Effects 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 229940038850 rebif Drugs 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 239000003161 ribonuclease inhibitor Substances 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 238000013341 scale-up Methods 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- YZHUMGUJCQRKBT-UHFFFAOYSA-M sodium chlorate Chemical compound [Na+].[O-]Cl(=O)=O YZHUMGUJCQRKBT-UHFFFAOYSA-M 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 230000034005 thiol-disulfide exchange Effects 0.000 description 1
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 229960002175 thyroglobulin Drugs 0.000 description 1
- 238000012090 tissue culture technique Methods 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 1
- 230000005758 transcription activity Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 238000010798 ubiquitination Methods 0.000 description 1
- 230000034512 ubiquitination Effects 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 1
- 229940045145 uridine Drugs 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 210000003501 vero cell Anatomy 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 230000029663 wound healing Effects 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
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
Definitions
- bioinformatics tools increase in potency and in accuracy, these tools are rapidly replacing the conventional techniques of biochemical characterisation. Indeed, the advanced bioinformatics tools used in identifying the present invention are now capable of outputting results in which a high degree of confidence can be placed.
- This tool is a database system, termed the Biopendium search database, that is the subject of WO 01/69507.
- This database system consists of an integrated data resource created using proprietary technology and containing information generated from an all-by-all comparison of all available protein or nucleic acid sequences.
- sequence data from separate data resources is to combine as much data as possible, relating both to the sequences themselves and to information relevant to each sequence, into one integrated resource.
- available data relating to each sequence including data on the three-dimensional structure of the encoded protein, if this is available, are integrated together to make best use of the information that is known about each sequence and thus to allow the most educated predictions to be made from comparisons of these sequences.
- the annotation that is generated in the database and which accompanies each sequence entry imparts a biologically relevant context to the sequence information.
- Fibronectins are multi-domain glycoproteins found in a soluble form in plasma, and in an insoluble form in loose connective tissue and basement membranes. They contain multiple copies of 3 repeat regions (types I, II and III), which bind to a variety of substances including heparin, collagen, DNA, actin, fibrin and fibronectin receptors on cell surfaces. The wide variety of these substances means that fibronectins are involved in a number of important functions: e.g., wound healing; cell adhesion; blood coagulation; cell differentiation and migration; maintenance of the cellular cytoskeleton; and tumour metastasis.
- Proteins containing fibronectin type 3 domains have been shown to play a role in diverse physiological functions, many of which can play a role in disease processes. Alteration of their activity is a means to alter the disease phenotype and as such identification of novel adhesion molecules is highly relevant as they may play a role in many diseases. Their identification will allow the development of new methods for the treatment and diagnosis of diseases and disorders. Accordingly, there remains a need for the identification of such proteins to enable new drugs to be developed for the treatment and prevention of human disease.
- the invention provides a polypeptide, which polypeptide:
- FN3 domain containing protein refers to a molecule containing at least one FN3 domain.
- the "FN3 domain containing protein” may be a molecule containing an FN3 domain detected with an e-value lower than 0.1, 0.01, 0.001, 0.0001, 0.0002, 0.00001, 0.000001 or 0.0000001.
- FN3 domain containing protein may be a molecule matching the HMM build of the Pfam entry detected with an e-value lower than 0.1, 0.01, 0.001, 0.0001, 0.0002, 0.00001, 0.000001 or 0.0000001.
- a polypeptide according to any one of the above-described aspects of the invention functions as a FN3 domain containing protein.
- purified nucleic acid molecule preferably refers to a nucleic acid molecule of the invention that (1) has been separated from at least about 50 percent of proteins, lipids, carbohydrates, or other materials with which it is naturally found when total nucleic acid is isolated from the source cells, (2) is not linked to all or a portion of a polynucleotide to which the "purified nucleic acid molecule" is linked in nature, (3) is operably linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature as part of a larger polynucleotide sequence.
- Ligands to a polypeptide according to the invention may come in various forms, including natural or modified substrates, enzymes, receptors, small organic molecules such as small natural or synthetic organic molecules of up to 2000Da, preferably 800Da or less, peptidomimetics, inorganic molecules, peptides, polypeptides, antibodies, structural or functional mimetics of the aforementioned.
- the present invention provides a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, for use in the manufacture of a medicament for the diagnosis or treatment of a disease including, but not limited to, neoplasm, cancer, brain tumour, glioma, bone tumor, lung tumor, breast tumour, prostate tumour, colon tumour, hemangioma, myeloproliferative disorder, leukemia, hematological disease, neutropenia, thrombocytopenia, angiogenesis disorders, dermatological disease, ageing, wounds, burns, fibrosis, cardiovascular disease, restensosis, heart disease, peripheral vascular disease, coronary artery disease, oedema, thromboembolism, dysmenor
- Such mutants also include polypeptides in which one or more of the amino acid residues includes a substituent group.
- nucleic acid molecules encoding a polypeptide of the present invention into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et ah, Basic Methods in Molecular Biology (1986) and Sambrook et ah, [supra]. Particularly suitable methods include calcium phosphate transfection, DEAE-dextran mediated transfection, trans vection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection (see Sambrook et ah, 1989 [supra]; Ausubel et al., 1991 [supra]; Spector, Goldman & Leinwald, 1998). In eukaryotic cells, expression systems may either be transient (for example, episomal) or permanent (chromosomal integration) according to the needs of the system.
- any number of selection systems are known in the art that may be used to recover transformed cell lines. Examples include the herpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell 11 :223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) genes that can be employed in tk- or aprt ⁇ cells, respectively.
- Compounds that are most likely to be good antagonists are molecules that bind to the polypeptide of the invention without inducing the biological effects of the polypeptide upon binding to it.
- Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to the polypeptide of the invention and thereby inhibit or extinguish its activity. In this fashion, binding of the polypeptide to normal cellular binding molecules may be inhibited, such that the normal biological activity of the polypeptide is prevented.
- the polypeptide of the invention that is employed in such a screening technique may be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly.
- simple binding assays may be used, in which the adherence of a test compound to a surface bearing the polypeptide is detected by means of a label directly or indirectly associated with the test compound or in an assay involving competition with a labelled competitor.
- competitive drug screening assays may be used, in which neutralising antibodies that are capable of binding the polypeptide specifically compete with a test compound for binding. In this manner, the antibodies can be used to detect the presence of any test compound that possesses specific binding affinity for the polypeptide.
- Binding assays may be used for the purification and cloning of the receptor, but may also identify agonists and antagonists of the polypeptide, that compete with the binding of the polypeptide to its receptor. Standard methods for conducting screening assays are well understood in the art.
- this invention relates to the use of a INSP 172 polypeptide or fragment thereof, whereby the fragment is preferably a INSP 172 gene-specific fragment, for isolating or generating an agonist or stimulator of the INSP 172 polypeptide for the treatment of an immune related disorder, wherein said agonist or stimulator is selected from the group consisting of: 1. a specific antibody or fragment thereof including: a) a chimeric, b) a humanized or c) a fully human antibody, as well as;
- Gene therapy of the present invention can occur in vivo or ex vivo.
- Ex vivo gene therapy requires the isolation and purification of patient cells, the introduction of a therapeutic gene and introduction of the genetically altered cells back into the patient.
- in vivo gene therapy does not require isolation and purification of a patient's cells.
- DNA sequence differences may also be detected by alterations in the electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (for example, Myers et al, Science (1985) 230:1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and Sl protection or the chemical cleavage method (see Cotton et al., Proc. Natl. Acad. Sci. USA (1985) 85: 4397-4401).
- This assay uses primary human prostate stromal cells as a model for proliferation of these cells during BPH. The goal will be to identify proteins that inhibit proliferation of these cells.
- Example 4 cDNA cloning of INSP 172 First strand cDNA was prepared from a variety of human tissue total RNA samples (Clontech, Stratagene, Ambion, Biochain Institute and in-house preparations) using Superscript II or Superscript III RNase H " Reverse Transcriptase (Invitrogen) according to the manufacturer's protocol.
- the final 21 ⁇ l reaction mix was diluted by adding 179 ⁇ l sterile water to give a total volume of 200 ⁇ l. This represented approximately 20 ng/ ⁇ l of each individual cDNA template.
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention is based on the discovery that the human protein referred to herein as INSP172 protein is a fibronectin type (3) domain containing protein believed to be involved in protein-protein interactions.
Description
Fibronectin type III domain containing protein
This invention relates to a novel protein (termed INSP 172) herein identified as a fibronectin type 3 domain containing protein and to the use of this protein and nucleic acid sequences from the encoding gene in the diagnosis, prevention and treatment of disease. All publications, patents and patent applications cited herein are incorporated in full by reference.
BACKGROUND
The process of drug discovery is presently undergoing a fundamental revolution as the era of functional genomics comes of age. The term "functional genomics" applies to an approach utilising bioinformatics tools to ascribe function to protein sequences of interest. Such tools are becoming increasingly necessary as the speed of generation of sequence data is rapidly outpacing the ability of research laboratories to assign functions to these protein sequences.
As bioinformatics tools increase in potency and in accuracy, these tools are rapidly replacing the conventional techniques of biochemical characterisation. Indeed, the advanced bioinformatics tools used in identifying the present invention are now capable of outputting results in which a high degree of confidence can be placed.
Various institutions and commercial organisations are examining sequence data as they become available and significant discoveries are being made on an on-going basis. However, there remains a continuing need to identify and characterise further genes and the polypeptides that they encode, as targets for research and for drug discovery.
Recently, a remarkable tool for the evaluation of sequences of unknown function has been developed by the Applicant for the present invention. This tool is a database system, termed the Biopendium search database, that is the subject of WO 01/69507. This database system consists of an integrated data resource created using proprietary technology and containing information generated from an all-by-all comparison of all available protein or nucleic acid sequences.
The aim behind the integration of these sequence data from separate data resources is to combine as much data as possible, relating both to the sequences themselves and to information relevant to each sequence, into one integrated resource. AU the available data relating to each sequence, including data on the three-dimensional structure of the encoded
protein, if this is available, are integrated together to make best use of the information that is known about each sequence and thus to allow the most educated predictions to be made from comparisons of these sequences. The annotation that is generated in the database and which accompanies each sequence entry imparts a biologically relevant context to the sequence information.
This data resource has made possible the accurate prediction of protein function from sequence alone. Using conventional technology, this is only possible for proteins that exhibit a high degree of sequence identity (above about 20%-30% identity) to other proteins in the same functional family. Accurate predictions are not possible for proteins that exhibit a very low degree of sequence homology to other related proteins of known function.
Secreted Proteins
The ability for cells to make and secrete extracellular proteins is central to many biological processes. Enzymes, growth factors, extracellular matrix proteins and signalling molecules are all secreted by cells. This is through fusion of a secretory vesicle with the plasma membrane. In most cases, but not all, proteins are directed to the endoplasmic reticulum and into secretory vesicles by a signal peptide. Signal peptides are cis-acting sequences that affect the transport of polypeptide chains from the cytoplasm to a membrane bound compartment such as a secretory vesicle. Polypeptides that are targeted to the secretory vesicles are either secreted into the extracellular matrix or are retained in the plasma membrane. The polypeptides that are retained in the plasma membrane will have one or more transmembrane domains. Examples of secreted proteins that play a central role in the functioning of a cell are cytokines, hormones, extracellular matrix proteins (adhesion molecules), proteases, and growth and differentiation factors. Introduction to the fibronectin type 3 domain
Fibronectins are multi-domain glycoproteins found in a soluble form in plasma, and in an insoluble form in loose connective tissue and basement membranes. They contain multiple copies of 3 repeat regions (types I, II and III), which bind to a variety of substances including heparin, collagen, DNA, actin, fibrin and fibronectin receptors on cell surfaces. The wide variety of these substances means that fibronectins are involved in a number of important functions: e.g., wound healing; cell adhesion; blood coagulation; cell
differentiation and migration; maintenance of the cellular cytoskeleton; and tumour metastasis.
The fibronectin type 3 domain is one of the three types of internal repeat found within fibronectin and is approximately 100 amino acids long. Fibronectin type 3 domains are found in both extracellular and intracellular proteins. The majority of proteins containing fibronectin type 3 repeats are involved in cell surface binding or are cell-surface receptors. Many are receptor protein tyrosine kinases, cytokine receptors or growth hormone receptors.
Proteins containing fibronectin type 3 domains, have been shown to play a role in diverse physiological functions, many of which can play a role in disease processes. Alteration of their activity is a means to alter the disease phenotype and as such identification of novel adhesion molecules is highly relevant as they may play a role in many diseases. Their identification will allow the development of new methods for the treatment and diagnosis of diseases and disorders. Accordingly, there remains a need for the identification of such proteins to enable new drugs to be developed for the treatment and prevention of human disease.
THE INVENTION
The invention is based on the discovery that the human protein referred to herein as INSP 172 protein is a secreted protein. Further, INSP 172 possesses at least seven FN3 domains. As such, it is believed that INSP 172 is involved in protein-protein interactions, and possibly interacts with a receptor. EST evidence indicates that INSP 172 is predominantly expressed in embryonic tissues in addition to being expressed in the testes and the oviduct. Screening of pools of cDNA templates indicated INSP 172 to be present in the following tissues: pancreas, pituitary, Y79 cell line, uterus, bone marrow, thyroid, spinal cord, cervix, mammary gland, and ovary. Hence, it is believed that INSP 172 may play an important role in development or reproduction.
In a first aspect, the invention provides a polypeptide, which polypeptide:
(i) comprises the amino acid sequence as recited in SEQ ID NO:2;
(ii) is a fragment thereof that contains a fibronectin type III domain and/or has an antigenic determinant in common with the polypeptides of (i); or
(iii) is a functional equivalent of (i) or (ii).
According to a second embodiment of this first aspect of the invention, there is provided a polypeptide which consists of the amino acid sequence as recited in SEQ ID NO: 2.
The polypeptide having the sequence recited in SEQ ID NO: 2 is referred to hereafter as "the cloned INSP 172 mature polypeptide".
Although the Applicant does not wish to be bound by this theory, it is postulated that the the cloned INSP 172 full length polypeptide and the cloned INSP 172 his tag full length polypeptide may further comprise a signal peptide at the N-terminus that is 29 amino acids in length. The cloned INSP 172 full length polypeptide sequence with this postulated signal sequence is recited in SEQ ID NO:6. The cloned INSP172 his tag full length polypeptide sequence with this postulated signal sequence is recited in SEQ ID NO: 8.
The polypeptide having the sequence recited in SEQ ID NO: 6 is hereafter referred to as "the cloned INSP 172 full length polypeptide". The polypeptides of the first aspect of the invention may further comprise a histidine tag. Preferably the histidine tag is found at the C-terminal of the polypeptide. Preferably the histidine tag comprises 1-10 histidine residues (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues). More preferably, the histidine tag comprises 6 histidine residues. Preferred polypeptides are therefore those comprising the sequence recited in SEQ ID NO: 4 and/or SEQ ID NO: 8. The polypeptide having the sequence recited in SEQ ID NO:4 is hereafter referred to as "the cloned INSP 172 his tag mature polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 8 is hereafter referred to as "the cloned INSP 172 his tag full length polypeptide". Preferably polypeptides of the invention containing his tags consist of the sequence recited in SEQ ID NO: 4 and/or SEQ ID NO:8. The term "INSP 172 polypeptides" as used herein includes polypeptides comprising the cloned INSP 172 mature polypeptide, the cloned INSP 172 his tag mature polypeptide, the cloned INSP 172 full length polypeptide and the cloned INSP 172 his tag full length polypeptide.
The term "FN3 domain containing protein" refers to a molecule containing at least one FN3 domain.
Preferably, the "FN3 domain containing protein" may be a molecule containing an FN3
domain detected with an e-value lower than 0.1, 0.01, 0.001, 0.0001, 0.0002, 0.00001, 0.000001 or 0.0000001.
Preferably, the term "FN3 domain containing protein" may be a molecule matching the HMM build of the Pfam entry detected with an e-value lower than 0.1, 0.01, 0.001, 0.0001, 0.0002, 0.00001, 0.000001 or 0.0000001.
Preferably, a polypeptide according to any one of the above-described aspects of the invention functions as a FN3 domain containing protein.
By "functions as a FN3 domain containing protein" we refer to the activity of the polypeptide in participating in protein-protein interactions; receptor interactions; cell recognition; communication between cells; cell surface binding; and/or playing a role in development, reproduction or fibrosis. There are many methods for detecting protein- protein interactions which will be known to those skilled in the art and which include the Yeast 2 hybrid system, immuno-precipitation techniques, and methods using Biacore and FRET. Preferably, a polypeptide according to the first aspect of the invention possesses seven or more FN3 domains.
Briknarova et al. discloses anastellin, a carboxy-terminal fragment of the first fϊbronectin- type III (FN3) domain from human fibronectin (Briknarova et al. J MoI Biol. 2003 Sep 5;332(l):205-15.). Anastellin was capable of polymerizing fibronectin in vitro, and it displayed anti-tumour, anti-metastatic and anti-angiogenic properties in vivo.
Hiro-o Ito et al. found that the epitope of an inhibiting selected mAb, that specifically interferes with the interaction between Fn and bacterial cells, was mapped to the central region of Fn, which consists of the type III repeat domain or cell-binding region known to be responsible for interaction with integrins (Hiro-o Ito et al. Biochem Biophys Res Commun. 2004 JuI 23;320(2):347-53). The central cell-binding region of Fn was found to interact not only with Granulicatella adiacens (a causative agent of infective endocarditis) but also with different species of microorganisms (Staphylococcus aureus, Streptococcus pyogenes and Escherichia coli).
As such, INSP 172, splice variants thereof and fragments containing the FN3 domain (soluble FN3 domain of INSP 172 or fusion proteins containing the FN3 domain of
INSP 172) might be useful for the treatment of tumour and of microbial infections {e.g.
infections from Granulicatella adiacens, Staphylococcus aureus, Streptococcus pyogenes or Escherichia coli).
An "antigenic determinant" of the present invention may be a part of a polypeptide of the present invention, which binds to an antibody-combining site or to a T-cell receptor (TCR). Alternatively, an "antigenic determinant" may be a site on the surface of a polypeptide of the present invention to which a single antibody molecule binds. Generally an antigen has several or many different antigenic determinants and reacts with antibodies of many different specificities. Preferably, the antibody is immunospecific to a polypeptide of the invention. Preferably, the antibody is immunospecific to a polypeptide of the invention, which is not part of a fusion protein. Preferably, the antibody is irmnunospecific to INSP 172 or a fragment thereof. Antigenic determinants usually consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three dimensional structural characteristics, as well as specific charge characteristics. Preferably, the "antigenic determinant" refers to a particular chemical group on a polypeptide of the present invention that is antigenic, i.e. that elicit a specific immune response.
In a second aspect, the invention provides a purified nucleic acid molecule which encodes a polypeptide of the first aspect of the invention. Preferably, the purified nucleic acid molecule has the nucleic acid sequence as recited in SEQ ID NO:1 (encoding the cloned INSP172 mature polypeptide), SEQ ID NO:3 (encoding the cloned INSP172 his tag mature polypeptide), SEQ ID NO: 5 (encoding the cloned INSP 172 full length polypeptide), SEQ ID NO:7 (encoding the cloned INSP172 his tag full length polypeptide).
The term "purified nucleic acid molecule" preferably refers to a nucleic acid molecule of the invention that (1) has been separated from at least about 50 percent of proteins, lipids, carbohydrates, or other materials with which it is naturally found when total nucleic acid is isolated from the source cells, (2) is not linked to all or a portion of a polynucleotide to which the "purified nucleic acid molecule" is linked in nature, (3) is operably linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature as part of a larger polynucleotide sequence. Preferably, the isolated nucleic acid molecule of the present invention is substantially free from any other contaminating nucleic acid molecule(s) or other contaminants that are found in its natural environment that would
interfere with its use in polypeptide production or its therapeutic, diagnostic, prophylactic or research use. In a preferred embodiment, genomic DNA are specifically excluded from the scope of the invention. Preferably, genomic DNA larger than 10 kbp (kilo base pairs), 50 kbp, 100 kbp, 150 kbp, 200 kbp, 250 kbp or 300 kbp are specifically excluded from the scope of the invention. Preferably, the "purified nucleic acid molecule" consists of cDNA only.
In a third aspect, the invention provides a purified nucleic acid molecule which hydridizes under high stringency conditions with a nucleic acid molecule of the second aspect of the invention. High stringency hybridisation conditions are defined as overnight incubation at 42°C in a solution comprising 50% formamide, 5XSSC (15OmM NaCl, 15mM trisodium citrate), 5OmM sodium phosphate (pH7.6), 5x Denhardts solution, 10% dextran sulphate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1X SSC at approximately 65°C. Preferably, the nucleic acid molecules hybridizes with a nucleic acid sequence set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7.
In a fourth aspect, the invention provides a vector, such as an expression vector, that contains a nucleic acid molecule of the second or third aspect of the invention.
In a fifth aspect, the invention provides a host cell transformed with a vector of the fourth aspect of the invention. In a sixth aspect, the invention provides a ligand which binds specifically to, and which preferably inhibits the activity of a polypeptide of the first aspect of the invention.
Ligands to a polypeptide according to the invention may come in various forms, including natural or modified substrates, enzymes, receptors, small organic molecules such as small natural or synthetic organic molecules of up to 2000Da, preferably 800Da or less, peptidomimetics, inorganic molecules, peptides, polypeptides, antibodies, structural or functional mimetics of the aforementioned.
Such compounds may be identified using the assays and screening methods disclosed herein.
In a seventh aspect, the invention provides a compound that is effective to alter the expression of a natural gene which encodes a polypeptide of the first aspect of the invention or to regulate the activity of a polypeptide of the first aspect of the invention.
Such compounds may be identified using the assays and screening methods disclosed herein.
A compound of the seventh aspect of the invention may either increase (agonise) or decrease (antagonise) the level of expression of the gene or the activity of the polypeptide. Importantly, the identification of the function of the INSP 172 polypeptides allows for the design of screening methods capable of identifying compounds that are effective in the treatment and/or diagnosis of disease. Ligands and compounds according to the sixth and seventh aspects of the invention may be identified using such methods. These methods are included as aspects of the present invention. Using these methods, it will now be possible to identify inhibitors or antagonists of INSP 172 polypeptides, such as, for example, monoclonal antibodies, which may be of use in modulating activity in vivo in clinical applications.
Another aspect of this invention resides in the use of an INSP 172 gene or polypeptide as a target for the screening of candidate drug modulators, particularly candidate drugs active against FN3 domain containing protein related disorders.
A further aspect of this invention resides in methods of screening of compounds for therapy of FN3 domain containing protein related disorders, comprising determining the ability of a compound to bind to an INSP 172 gene or polypeptide, or a fragment thereof.
A further aspect of this invention resides in methods of screening of compounds for therapy of FN3 domain containing protein related disorders, comprising testing for modulation of the activity of an INSP 172 gene or polypeptide, or a fragment thereof.
In an eighth aspect, the invention provides a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, for use in therapy or diagnosis.
These molecules may also be used in the manufacture of a medicament for the treatment of diseases including, but not limited to, neoplasm, cancer, brain tumour, glioma, bone tumor, lung tumor, breast tumour, prostate tumour, colon tumour, hemangioma, myeloproliferative disorder, leukemia, hematological disease, neutropenia, thrombocytopenia, angiogenesis disorders, dermatological disease, ageing, wounds, burns,
fibrosis, cardiovascular disease, restensosis, heart disease, peripheral vascular disease, coronary artery disease, oedema, thromboembolism, dysmenorrhea, endometriosis, preeclampsia, lung disease, COPD, asthma bone disease, renal disease, glomerulonephritis, liver disease, Crohn's disease, gastritis, ulcerative colitis, ulcer, immune disorder, autoimmune disease, arthritis, rheumatoid arthritis, psoriasis, epidermolysis bullosa, systemic lupus erythematosus, ankylosing spondylitis, Lyme disease, multiple sclerosis, neurodegeneration, stroke, brain/spinal cord injury, Alzheimer's disease, Parkinson's disease, motor neurone disease, neuromuscular disease, HIV, AIDS, cytomegalovirus infection, fungal infection, ocular disorder, macular degeneration, glaucoma, diabetic retinopathy and ocular hypertension. In view of the EST evidence which indicates that INSP 172 is predominantly expressed in embryonic and reproductive tissues, the moeities of the invention may be particularly useful in the treatment or diagnosis of reproductive disorders (e.g. the moieities of the invention may be used to increase or decrease fertility) or developmental disorders. Thus, the embodiments of the invention provide for the use of the polypeptides of the invention in the treatment of reproductive health disorders (in particular male or female reproductive disorders) or cancer. In one embodiment of the invention, the reproductive health disorder is selected from male or female infertility. In one embodiment of the invention, the male infertility is associated to spermatogenesis deficiencies with oligospermia or azoospermia. In one embodiment of the invention, the female infertility is associated with follicle-genesis deficiency. In one embodiment of the invention, the cancer is selected from carcinoma, including, but not limited to adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma or leukemia. In one embodiment of the invention, the cancer is selected from ovarian cancer, prostate cancer or testicular cancer. The various moieties of the invention (i.e. the polypeptides of the first aspect of the invention, a nucleic acid molecule of the second or third aspect of the invention, a vector of the fourth aspect of the invention, a host cell of the fifth aspect of the invention, a ligand of the sixth aspect of the invention, a compound of the seventh aspect of the invention) are envisaged as being particularly useful in the treatment and diagnosis of developmental disorders, reprodutive health disorders or fibrosis. Such moieities may be identified using an assay described herein, for example in Example 2 or 3.
In a ninth aspect, the invention provides a method of diagnosing a disease in a patient,
comprising assessing the level of expression of a natural gene encoding a polypeptide of the first aspect of the invention or the activity of a polypeptide of the first aspect of the invention in tissue from said patient and comparing said level of expression or activity to a control level, wherein a level that is different to said control level is indicative of disease. Such a method will preferably be carried out in vitro. Similar methods may be used for monitoring the therapeutic treatment of disease in a patient, wherein altering the level of expression or activity of a polypeptide or nucleic acid molecule over a period of time towards a control level is indicative of regression of disease.
One possible method for detecting polypeptides of the first aspect of the invention comprises the steps of: (a) contacting a ligand, such as an antibody, of the sixth aspect of the invention with a biological sample under conditions suitable for the formation of a ligand-polypeptide complex; and (b) detecting said complex.
A number of different such methods according to the ninth aspect of the invention exist, as the skilled reader will be aware, such as methods of nucleic acid hybridization with short probes, point mutation analysis, polymerase chain reaction (PCR) amplification and methods using antibodies to detect aberrant protein levels. Similar methods may be used on a short or long term basis to allow therapeutic treatment of a disease to be monitored in a patient. The invention also provides kits that are useful in these methods for diagnosing disease. In a tenth aspect, the invention provides for the use of a polypeptide of the first aspect of the invention as a protein involved in protein-protein interactions.
One suitable use of the INSP 172 polypeptides (i.e. SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 6 or SEQ ID NO: 8) is in the screening of drug compounds that are effective against the diseases and conditions in which an INSP 172 polypeptide is implicated. In an eleventh aspect, the invention provides a pharmaceutical composition comprising a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, in conjunction with a pharmaceutically- acceptable carrier.
In a twelfth aspect, the present invention provides a polypeptide of the first aspect of the
invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, for use in the manufacture of a medicament for the diagnosis or treatment of a disease including, but not limited to, neoplasm, cancer, brain tumour, glioma, bone tumor, lung tumor, breast tumour, prostate tumour, colon tumour, hemangioma, myeloproliferative disorder, leukemia, hematological disease, neutropenia, thrombocytopenia, angiogenesis disorders, dermatological disease, ageing, wounds, burns, fibrosis, cardiovascular disease, restensosis, heart disease, peripheral vascular disease, coronary artery disease, oedema, thromboembolism, dysmenorrhea, endometriosis, preeclampsia, lung disease, COPD, asthma bone disease, renal disease, glomerulonephritis, liver disease, Crohn's disease, gastritis, ulcerative colitis, ulcer, immune disorder, autoimmune disease, arthritis, rheumatoid arthritis, psoriasis, epidermolysis bullosa, systemic lupus erythematosus, ankylosing spondylitis, Lyme disease, multiple sclerosis, neurodegeneration, stroke, brain/spinal cord injury, Alzheimer's disease, Parkinson's disease, motor neurone disease, neuromuscular disease, HIV, AIDS, cytomegalovirus infection, fungal infection, ocular disorder, macular degeneration, glaucoma, diabetic retinopathy and ocular hypertension. Preferably, the disease is a reproductive or developmental disease or disorder. Thus, the embodiments of the invention provide for the use of the polypeptides of the invention in the manufacture of a medicament for the treatment of reproductive health disorders (in particular male or female reproductive disorders) or cancer. In one embodiment of the invention, the reproductive health disorder is selected from male or female infertility. In one embodiment of the invention, the male infertility is associated to spermatogenesis deficiencies with oligospermia or azoospermia. In one embodiment of the invention, the female infertility is associated with follicle-genesis deficiency. In one embodiment of the invention, the cancer is selected from carcinoma, including, but not limited to adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma or leukemia. In one embodiment of the invention, the cancer is selected from ovarian cancer, prostate cancer or testicular cancer.
In a thirteenth aspect, the invention provides a method of treating a disease in a patient comprising administering to the patient a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the
fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention.
For diseases in which the expression of a natural gene encoding a polypeptide of the first aspect of the invention, or in which the activity of a polypeptide of the first aspect of the invention, is lower in a diseased patient when compared to the level of expression or activity in a healthy patient, the polypeptide, nucleic acid molecule, ligand or compound administered to the patient should be an agonist. Conversely, for diseases in which the expression of the natural gene or activity of the polypeptide is higher in a diseased patient when compared to the level of expression or activity in a healthy patient, the polypeptide, nucleic acid molecule, ligand or compound administered to the patient should be an antagonist. Examples of such antagonists include antisense nucleic acid molecules, ribozymes and ligands, such as antibodies.
The INSP 172 polypeptides are FN3 domain containing proteins and thus have roles in many disease states. Antagonists of the INSP 172 polypeptides are of particular interest as they provide a way of modulating these disease states.
In a fourteenth aspect, the invention provides transgenic or knockout non-human animals that have been transformed to express higher, lower or absent levels of a polypeptide of the first aspect of the invention. Such transgenic animals are very useful models for the study of disease and may also be using in screening regimes for the identification of compounds that are effective in the treatment or diagnosis of such a disease.
As used herein, "functional equivalent" refers to a protein or nucleic acid molecule that possesses functional or structural characteristics that are substantially similar to a polypeptide or nucleic acid molecule of the present invention. A functional equivalent of a protein may contain modifications depending on the necessity of such modifications for the performance of a specific function. The term "functional equivalent" is intended to include the fragments, mutants, hybrids, variants, analogs, or chemical derivatives of a molecule.
Preferably, the "•functional equivalent" may be a protein or nucleic acid molecule that exhibits any one or more of the functional activities of the polypeptides of the present invention.
Preferably, the "functional equivalent" may be a protein or nucleic acid molecule that
displays substantially similar activity compared with INSP 172 or fragments thereof in a suitable assay for the measurement of biological activity or function. Preferably, the "functional equivalent" may be a protein or nucleic acid molecule that displays identical or higher activity compared with INSPl 72 or fragments thereof in a suitable assay for the measurement of biological activity or function. Preferably, the "functional equivalent" may be a protein or nucleic acid molecule that displays 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 100% or more activity compared with INSP 172 or fragments thereof in a suitable assay for the measurement of biological activity or function.
Preferably, the "functional equivalent" may be a protein or polypeptide capable of exhibiting a substantially similar in vivo or in vitro activity as the polypeptides of the invention. Preferably, the "functional equivalent" may be a protein or polypeptide capable of interacting with other cellular or extracellular molecules in a manner substantially similar to the way in which the corresponding portion of the polypeptides of the invention would. For example, a "functional equivalent" would be able, in an immunoassay, to diminish the binding of an antibody to the corresponding peptide (i.e., the peptide the amino acid sequence of which was modified to achieve the "functional equivalent") of the polypeptide of the invention, or to the polypeptide of the invention itself, where the antibody was raised against the corresponding peptide of the polypeptide of the invention.
An equimolar concentration of the functional equivalent will diminish the aforesaid binding of the corresponding peptide by at least about 5%, preferably between about 5% and 10%, more preferably between about 10% and 25%, even more preferably between about 25% and 50%, and most preferably between about 40% and 50%.
For example, functional equivalents can be fully functional or can lack function in one or more activities. Thus, in the present invention, variations can affect the function, for example, of the activities of the polypeptide that reflect its possession of a FN3 domain domain.
A summary of standard techniques and procedures which may be employed in order to utilise the invention are given below. It will be understood that this invention is not limited to the particular methodology, protocols, cell lines, vectors and reagents described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and it is not intended that this terminology should limit the scope of the present invention. The extent of the invention is limited only by the terms of
the appended claims.
Standard abbreviations for nucleotides and amino acids are used in this specification.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology and immunology, which are within the skill of those working in the art.
Such techniques are explained fully in the literature. Examples of particularly suitable texts for consultation include the following: Sambrook Molecular Cloning; A Laboratory Manual, Second Edition (1989); DNA Cloning, Volumes I and II (D.N Glover ed. 1985); Oligonucleotide Synthesis (MJ. Gait ed. 1984); Nucleic Acid Hybridization (B.D. Hames & SJ. Higgins eds. 1984); Transcription and Translation (B.D. Hames & SJ. Higgins eds. 1984); Animal Cell Culture (R.I. Freshney ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the Methods in Enzymology series (Academic Press, Inc.), especially volumes 154 & 155; Gene Transfer Vectors for Mammalian Cells (J.H. Miller and M.P. Calos eds. 1987, Cold Spring Harbor Laboratory); Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds. 1987, Academic Press, London); Scopes, (1987) Protein Purification: Principles and Practice, Second Edition (Springer Verlag, N. Y.); and Handbook of Experimental Immunology, Volumes I-IV (D.M. Weir and C. C. Blackwell eds. 1986).
As used herein, the term "polypeptide" includes any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e. peptide isosteres. This term refers both to short chains (peptides and oligopeptides) and to longer chains (proteins).
The polypeptide of the present invention may be in the form of a mature protein or may be a pre-, pro- or prepro- protein that can be activated by cleavage of the pre-, pro- or prepro- portion to produce an active mature polypeptide. In such polypeptides, the pre-, pro- or prepro- sequence may be a leader or secretory sequence or may be a sequence that is employed for purification of the mature polypeptide sequence.
The polypeptide of the first aspect of the invention may form part of a fusion protein. For example, it is often advantageous to include one or more additional amino acid sequences which may contain secretory or leader sequences, pro-sequences, sequences which aid in purification, or sequences that confer higher protein stability, for example during
recombinant production. Alternatively or additionally, the mature polypeptide may be fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol).
In a further preferred embodiment, the polypeptide of the invention comprising a sequence having at least 85% of homology with INSP172 may be a fusion protein.
These fusion proteins can be obtained by cloning a polynucleotide encoding a polypeptide comprising a sequence having at least 85% of homology with INSP 172 in frame to the coding sequences for a heterologous protein sequence.
The term "heterologous", when used herein, is intended to designate any polypeptide other than a human INSP 172 polypeptide .
Example of heterologous sequences, that can be comprised in the soluble fusion proteins either at N- or at C-terminus, are the following: extracellular domains of membrane-bound protein, immunoglobulin constant regions (Fc region), multimerization domains, domains of extracellular proteins, signal sequences, export sequences, or sequences allowing purification by affinity chromatography.
Many of these heterologous sequences are commercially available in expression plasmids since these sequences are commonly included in the fusion proteins in order to provide additional properties without significantly impairing the specific biological activity of the protein fused to them (Terpe K, Appl Microbiol Biotechnol, 60: 523-33, 2003). Examples of such additional properties are a longer lasting half-life in body fluids, the extracellular localization, or an easier purification procedure as allowed by the a stretch of Histidines forming the so-called "histidine tag" (Gentz et al., Proc Natl Acad Sci USA, 86: 821-4, 1989) or by the "HA" tag, an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell, 37: 767-78, 1994). If needed, the heterologous sequence can be eliminated by a proteolytic cleavage, for example by inserting a proteolytic cleavage site between the protein and the heterologous sequence, and exposing the purified fusion protein to the appropriate protease. These features are of particular importance for the fusion proteins since they facilitate their production and use in the preparation of pharmaceutical compositions. For example, the protein used in the examples (INSP 172) can be purified by means of a hexa-histidine peptide fused at the C-terminus of INSP 172. When the fusion protein comprises an immunoglobulin region, the fusion may be direct, or via a short linker peptide which can be as short as 1 to 3 amino acid residues in length or
longer, for example, 13 amino acid residues in length. Said linker may be a tripeptide of the sequence E-F-M (Glu-Phe-Met), for example, or a 13 -amino acid linker sequence comprising Glu-Phe-Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln-Phe-Met (SEQ ID NO:9) introduced between the sequence of the substances of the invention and the immunoglobulin sequence. The resulting fusion protein has improved properties, such as an extended residence time in body fluids (half-life), increased specific activity, increased expression level, or the purification of the fusion protein is facilitated.
In a preferred embodiment, the protein is fused to the constant region of an Ig molecule. Preferably, it is fused to heavy chain regions, like the CH2 and CH3 domains of human IgGl, for example. Other isoforms of Ig molecules are also suitable for the generation of fusion proteins according to the present invention, such as isoforms IgG2 or IgG4, or other Ig classes, like IgM or IgA, for example. Fusion proteins may be monomeric or multimeric, hetero- or homomultimeric.
In a further preferred embodiment, the functional derivative comprises at least one moiety attached to one or more functional groups, which occur as one or more side chains on the amino acid residues. Preferably, the moiety is a polyethylene (PEG) moiety. PEGylation may be carried out by known methods, such as the ones described in WO99/55377, for example.
Polypeptides may contain amino acids other than the 20 gene-encoded amino acids, modified either by natural processes, such as by post-translational processing or by chemical modification techniques which are well known in the art. Among the known modifications which may commonly be present in polypeptides of the present invention are glycosylation, lipid attachment, sulphation, gamma-carboxylation, for instance of glutamic acid residues, hydroxylation and ADP-ribosylation. Other potential modifications include acetylation, acylation, amidation, covalent attachment of flavin, covalent attachment of a haeme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulphide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, GPI anchor formation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, transfer-
RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. In fact, blockage of the amino or carboxyl terminus in a polypeptide, or both, by a covalent modification is common in naturally-occurring and synthetic polypeptides and such modifications may be present in polypeptides of the present invention.
The modifications that occur in a polypeptide often will be a function of how the polypeptide is made. For polypeptides that are made recombinantly, the nature and extent of the modifications in large part will be determined by the post-translational modification capacity of the particular host cell and the modification signals that are present in the amino acid sequence of the polypeptide in question. For instance, glycosylation patterns vary between different types of host cell.
The polypeptides of the present invention can be prepared in any suitable manner. Such polypeptides include isolated naturally-occurring polypeptides (for example purified from cell culture), recombinantly-produced polypeptides (including fusion proteins), synthetically-produced polypeptides or polypeptides that are produced by a combination of these methods.
The functionally-equivalent polypeptides of the first aspect of the invention may be polypeptides that are homologous to an INSP 172 polypeptide, preferably to a polypeptide sequence as set forth in SEQ ID NO:2. Two polypeptides are said to be "homologous", as the term is used herein, if the sequence of one of the polypeptides has a high enough degree of identity or similarity to the sequence of the other polypeptide. "Identity" indicates that at any particular position in the aligned sequences, the amino acid residue is identical between the sequences. "Similarity" indicates that, at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences. Degrees of identity and similarity can be readily calculated (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I5 Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).
Homologous polypeptides therefore include natural biological variants (for example,
allelic variants or geographical variations within the species from which the polypeptides are derived) and mutants (such as mutants containing amino acid substitutions, insertions or deletions) of the INSP 172 polypeptides. Such mutants may include polypeptides in which one or more of the amino acid residues are substituted with a conserved or non- conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code. Typical such substitutions are among Ala, VaI, Leu and He; among Ser and Thr; among the acidic residues Asp and GIu; among Asn and GIn; among the basic residues Lys and Arg; or among the aromatic residues Phe and Tyr. Particularly preferred are variants in which several, i.e. between 5 and 10, 1 and 5, 1 and 3, 1 and 2 or just 1 amino acids are substituted, deleted or added in any combination. Especially preferred are silent substitutions, additions and deletions, which do not alter the properties and activities of the protein. Also especially preferred in this regard are conservative substitutions.
Such mutants also include polypeptides in which one or more of the amino acid residues includes a substituent group.
In accordance with the present invention, any substitution should be preferably a "conservative" or "safe" substitution, which is commonly defined a substitution introducing an amino acids having sufficiently similar chemical properties (e.g. a basic, positively charged amino acid should be replaced by another basic, positively charged amino acid), in order to preserve the structure and the biological function of the molecule.
The literature provide many models on which the selection of conservative amino acids substitutions can be performed on the basis of statistical and physico-chemical studies on the sequence and/or the structure of proteins (Rogov SI and Nekrasov AN, 2001). Protein design experiments have shown that the use of specific subsets of amino acids can produce foldable and active proteins, helping in the classification of amino acid "synonymous" substitutions which can be more easily accommodated in protein structure, and which can be used to detect functional and structural homologs and paralogs (Murphy LR et ah, 2000). The groups of synonymous amino acids and the groups of more preferred synonymous amino acids are shown in Table 1. Specific, non-conservative mutations can be also introduced in the polypeptides of the invention with different purposes. Mutations reducing the affinity of the FN3 domain containing protein may increase its ability to be reused and recycled, potentially increasing
its therapeutic potency (Robinson CR, 2002). Immunogenic epitopes eventually present in the polypeptides of the invention can be exploited for developing vaccines (Stevanovic S, 2002), or eliminated by modifying their sequence following known methods for selecting mutations for increasing protein stability, and correcting them (van den Burg B and Eijsink 5 V, 2002; WO 02/05146, WO 00/34317, WO 98/52976).
Preferred alternative, synonymous groups for amino acids derivatives included in peptide mimetics are those defined in Table 2. A non-exhaustive list of amino acid derivatives also include aminoisobutyric acid (Aib), hydroxyproline (Hyp), 1,2,3,4-tetrahydro- isoquinoline-3-COOH, indoline-2carboxylic acid, 4-difluoro-proline, L- thiazolidine-4- 10 carboxylic acid, L-homoproline, 3,4-dehydro-proline, 3,4-dihydroxy-phenylalanine, cyclohexyl-glycine, and phenylglycine.
By "amino acid derivative" is intended an amino acid or amino acid-like chemical entity other than one of the 20 genetically encoded naturally occurring amino acids. In particular, the amino acid derivative may contain substituted or non-substituted, linear, branched, or 15 cyclic alkyl moieties, and may include one or more heteroatoms. The amino acid derivatives can be made de novo or obtained from commercial sources (Calbiochem- Novabiochem AG, Switzerland; Bachem, USA).
Various methodologies for incorporating unnatural amino acids derivatives into proteins, using both in vitro and in vivo translation systems, to probe and/or improve protein 20 structure and function are disclosed in the literature (Dougherty DA, 2000). Techniques for the synthesis and the development of peptide mimetics, as well as non-peptide mimetics, are also well known in the art (Golebiowski A et ah, 2001; Hruby VJ and Balse PM, 2000; Sawyer TK, in "Structure Based Drug Design", edited by Veerapandian P, Marcel Dekker Inc., pg. 557-663, 1997).
25 Typically, greater than 30% identity between two polypeptides is considered to be an indication of functional equivalence. Preferably, functionally equivalent polypeptides of the first aspect of the invention have a degree of sequence identity with the INSP 172 polypeptides, or with active fragments thereof, of greater than 70% or 80%. More preferred polypeptides have degrees of identity of greater than 85%, 90%, 95%, 98%,
30 98.5%, 99% or 99.5% respectively.
The functionally-equivalent polypeptides of the first aspect of the invention may also be polypeptides which have been identified using one or more techniques of structural
alignment. For example, the Inpharmatica Genome Threader technology that forms one aspect of the search tools used to generate the Biopendium search database may be used (see WO 01/67507) to identify polypeptides of presently-unknown function which, while having low sequence identity as compared to the INSP 172 polypeptides are predicted to possess FN3 domains by virtue of sharing significant structural homology with the INSP 172 polypeptides.
By "significant structural homology" is meant that the Inpharmatica Genome Threader™ predicts two proteins, or protein regions, to share structural homology with a certainty of at least 10% more preferably, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and above. The certainty value of the Inpharmatica Genome Threader™ is calculated as follows. A set of comparisons was initially performed using the Inpharmatica Genome Threader™ exclusively using sequences of known structure. Some of the comparisons were between proteins that were known to be related (on the basis of structure). A neural network was then trained on the basis that it needed to best distinguish between the known relationships and known not-relationships taken from the CATH structure classification (www.biochem.ucl.ac.uk/bsm/cath). This resulted in a neural network score between 0 and 1. However, again as the number of proteins that are related and the number that are unrelated were known, it was possible to partition the neural network results into packets and calculate empirically the percentage of the results that were correct. In this manner, any genuine prediction in the Biopendium search database has an attached neural network score and the percentage confidence is a reflection of how successful the Inpharmatica Genome Threader™ was in the training/testing set.
The polypeptides of the first aspect of the invention also include fragments of the INSP 172 polypeptides and fragments of the functional equivalents of the INSP 172 polypeptides, provided that those fragments have the activity of an INSP 172 polypeptide (preferably a polypeptide as set forth in SEQ ID NO:2) or an antigenic determinant in common with an INSP172 polypeptide (preferably SEQ ID NO: 2).
As used herein, the term "fragment" refers to a polypeptide having an amino acid sequence that is the same as part, but not all, of the amino acid sequence of the INSP 172 polypeptides or one of its functional equivalents. The fragments should comprise at least n consecutive amino acids from the sequence and, depending on the particular sequence, n preferably is 7 or more (for example, 8, 10, 12, 14, 16, 18, 20 or more). Small fragments
may form an antigenic determinant. Fragments according to the invention may be 1-700 amino acids in length, preferably, 5-600, more preferably 10-500, more preferably 20-400, more preferably 30-300, more preferably 50-200, more preferable 100-150 amino acids.
Nucleic acids according to the invention are preferably 10-2220 nucleotides in length, preferably 20-1500, preferably 30-1000, preferably 50-800 nucleotides, preferably 100- 600, preferably 200-550, preferably 300-500 nucleotides in length. Polypeptides according to the invention are preferably 5-740 amino acids in length, preferably 10-600, preferably 20-500, preferably 50-400, preferably 100-300, preferably 150-250 amino acids in length.
Fragments of the full length INSP 172 polypeptides may consist of combinations of 1 or 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 neighbouring exon sequences in the INSP 172 polypeptide sequences, respectively. These exons may be combined with further mature fragments according to the invention. Accordingly, fragments may include combinations of exon coding sequences made up of exons 1+2, 2+3, 1+3, 2+4, 2+3+4 and so on. Alternatively, fragments may comprise one or more of the FN3 domains of INSP 172 as identified in example 1. Such fragments are included in the present invention.
Such fragments may be "free-standing", i.e. not part of or fused to other amino acids or polypeptides, or they may be comprised within a larger polypeptide of which they form a part or region. When comprised within a larger polypeptide, the fragment of the invention most preferably forms a single continuous region. For instance, certain preferred embodiments relate to a fragment having a pre- and/or pro- polypeptide region fused to the amino terminus of the fragment and/or an additional region fused to the carboxyl terminus of the fragment. However, several fragments may be comprised within a single larger polypeptide.
The polypeptides of the present invention or their immunogenic fragments (comprising at least one antigenic determinant) can be used to generate ligands, such as polyclonal or monoclonal antibodies, that are immunospecific for the polypeptides. Such antibodies may be employed to isolate or to identify clones expressing the polypeptides of the invention or to purify the polypeptides by affinity chromatography. The antibodies may also be employed as diagnostic or therapeutic aids, amongst other applications, as will be apparent to the skilled reader.
The term "immunospecific" means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the
prior art. As used herein, the term "antibody" refers to intact molecules as well as to fragments thereof, such as Fab, F(ab')2 and Fv, which are capable of binding to the antigenic determinant in question. Such antibodies thus bind to the polypeptides of the first aspect of the invention. By "substantially greater affinity" we mean that there is a measurable increase in the affinity for a polypeptide of the invention as compared with the affinity for other related polypeptides in the prior art.
Preferably, the affinity is at least 1.5-fold, 2-fold, 5-fold 10-fold, 100-fold, 103-fold, 104- fold, 105-fold, 106-fold or greater for a polypeptide of the invention than for other related polypeptides in the prior art.
Preferably, there is a measurable increase in the affinity for a polypeptide of the invention as compared with known FN3 domain containing proteins.
If polyclonal antibodies are desired, a selected mammal, such as a mouse, rabbit, goat or horse, may be immunised with a polypeptide of the first aspect of the invention. The polypeptide used to immunise the animal can be derived by recombinant DNA technology or can be synthesized chemically. If desired, the polypeptide can be conjugated to a carrier protein. Commonly used carriers to which the polypeptides may be chemically coupled include bovine serum albumin, thyroglobulin and keyhole limpet haemocyanin. The coupled polypeptide is then used to immunise the animal. Serum from the immunised animal is collected and treated according to known procedures, for example by immunoaffϊnity chromatography.
Monoclonal antibodies to the polypeptides of the first aspect of the invention can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies using hybridoma technology is well known (see, for example, Kohler, G. and Milstein, C, Nature 256: 495-497 (1975); Kozbor et al, Immunology Today 4: 72 (1983); Cole et al., 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985).
Panels of monoclonal antibodies produced against the polypeptides of the first aspect of the invention can be screened for various properties, i.e., for isotype, epitope, affinity, etc. Monoclonal antibodies are particularly useful in purification of the individual polypeptides against which they are directed. Alternatively, genes encoding the monoclonal antibodies
of interest may be isolated from hybridomas, for instance by PCR techniques known in the art, and cloned and expressed in appropriate vectors.
Chimeric antibodies, in which non-human variable regions are joined or fused to human constant regions (see, for example, Liu et al, Proc. Natl. Acad. Sci. USA, 84, 3439 (1987)), may also be of use.
The antibody may be modified to make it less immunogenic in an individual, for example by humanisation (see Jones et al, Nature, 321, 522 (1986); Verhoeyen et al, Science, 239, 1534 (1988); Kabat et al., J. Immunol., 147, 1709 (1991); Queen et al, Proc. Natl Acad. Sci. USA, 86, 10029 (1989); Gorman et al, Proc. Natl Acad. Sci. USA, 88, 34181 (1991); and Hodgson et al, Bio/Technology, 9, 421 (1991)). The term "humanised antibody", as used herein, refers to antibody molecules in which the CDR amino acids and selected other amino acids in the variable domains of the heavy and/or light chains of a non-human donor antibody have been substituted in place of the equivalent amino acids in a human antibody. The humanised antibody thus closely resembles a human antibody but has the binding ability of the donor antibody.
In a further alternative, the antibody may be a "bispecifϊc" antibody, that is an antibody having two different antigen binding domains, each domain being directed against a different epitope.
Phage display technology may be utilised to select genes which encode antibodies with binding activities towards the polypeptides of the invention either from repertoires of PCR amplified V-genes of lymphocytes from humans screened for possessing the relevant antibodies, or from naive libraries (McCafferty, J. et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992) Biotechnology 10, 779-783). The affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al, (1991) Nature 352, 624-628). Antibodies generated by the above techniques, whether polyclonal or monoclonal, have additional utility in that they may be employed as reagents in immunoassays, radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA). In these applications, the antibodies can be labelled with an analytically-detectable reagent such as a radioisotope, a fluorescent molecule or an enzyme. Preferred nucleic acid molecules of the second and third aspects of the invention are those which encode a polypeptide sequence as recited in SEQ ID NO:2, SEQ ID NO:4, SEQ ID
NO:6, or SEQ ID NO:8 and functionally equivalent polypeptides. These nucleic acid molecules may be used in the methods and applications described herein. The nucleic acid molecules of the invention preferably comprise at least n consecutive nucleotides from the sequences disclosed herein where, depending on the particular sequence, n is 10 or more (for example, 12, 14, 15, 18, 20, 25, 30, 35, 40 or more).
The nucleic acid molecules of the invention also include sequences that are complementary to nucleic acid molecules described above (for example, for antisense or probing purposes).
Nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance cDNA, synthetic DNA or genomic
DNA. Such nucleic acid molecules may be obtained by cloning, by chemical synthetic techniques or by a combination thereof. The nucleic acid molecules can be prepared, for example, by chemical synthesis using techniques such as solid phase phosphoramidite chemical synthesis, from genomic or cDNA libraries or by separation from an organism. RNA molecules may generally be generated by the in vitro or in vivo transcription of DNA sequences.
The nucleic acid molecules may be double-stranded or single-stranded. Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non- coding strand, also referred to as the anti-sense strand. The term "nucleic acid molecule" also includes analogues of DNA and RNA, such as those containing modified backbones, and peptide nucleic acids (PNA). The term "PNA", as used herein, refers to an antisense molecule or an anti-gene agent which comprises an oligonucleotide of at least five nucleotides in length linked to a peptide backbone of amino acid residues, which preferably ends in lysine. The terminal lysine confers solubility to the composition. PNAs may be pegylated to extend their lifespan in a cell, where they preferentially bind complementary single stranded DNA and RNA and stop transcript elongation (Nielsen, P.E. et al. (1993) Anticancer Drug Des. 8:53-63).
A nucleic acid molecule which encodes the polypeptide of SEQ ID NO:2 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:1. A nucleic acid molecule which encodes the polypeptide of SEQ ID NO:4 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:3. A nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 6 may be identical to the coding
sequence of the nucleic acid molecule shown in SEQ ID NO:5. A nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 8 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:7.
These molecules also may have a different sequence which, as a result of the degeneracy of the genetic code, encodes a polypeptide of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 6 or SEQ ID NO:8. Such nucleic acid molecules may include, but are not limited to, the coding sequence for the mature polypeptide by itself; the coding sequence for the mature polypeptide and additional coding sequences, such as those encoding a leader or secretory sequence, such as a pro-, pre- or prepro- polypeptide sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with further additional, non-coding sequences, including non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription (including termination signals), ribosome binding and mRNA stability. The nucleic acid molecules may also include additional sequences which encode additional amino acids, such as those which provide additional functionalities.
The nucleic acid molecules of the second and third aspects of the invention may also encode the fragments or the functional equivalents of the polypeptides and fragments of the first aspect of the invention. Such a nucleic acid molecule may be a naturally occurring variant such as a naturally occurring allelic variant, or the molecule may be a variant that is not known to occur naturally. Such non-naturally occurring variants of the nucleic acid molecule may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells or organisms.
Among variants in this regard are variants that differ from the aforementioned nucleic acid molecules by nucleotide substitutions, deletions or insertions. The substitutions, deletions or insertions may involve one or more nucleotides. The variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or insertions.
The nucleic acid molecules of the invention can also be engineered, using methods generally known in the art, for a variety of reasons, including modifying the cloning, processing, and/or expression of the gene product (the polypeptide). DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides are included as techniques which may be used to engineer the nucleotide
sequences. Site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations and so forth.
Nucleic acid molecules which encode a polypeptide of the first aspect of the invention may be ligated to a heterologous sequence so that the combined nucleic acid molecule encodes a fusion protein. Such combined nucleic acid molecules are included within the second or third aspects of the invention. For example, to screen peptide libraries for inhibitors of the activity of the polypeptide, it may be useful to express, using such a combined nucleic acid molecule, a fusion protein that can be recognised by a commercially-available antibody. A fusion protein may also be engineered to contain a cleavage site located between the sequence of the polypeptide of the invention and the sequence of a heterologous protein so that the polypeptide may be cleaved and purified away from the heterologous protein.
The nucleic acid molecules of the invention also include antisense molecules that are partially complementary to nucleic acid molecules encoding polypeptides of the present invention and that therefore hybridize to the encoding nucleic acid molecules (hybridization). Such antisense molecules, such as oligonucleotides, can be designed to recognise, specifically bind to and prevent transcription of a target nucleic acid encoding a polypeptide of the invention, as will be known by those of ordinary skill in the art (see, for example, Cohen, J.S., Trends in Pharm. ScL, 10, 435 (1989), Okano, J. Neurochem. 56, 560 (1991); O'Connor, J. Neurochem 56, 560 (1991); Lee et al., Nucleic Acids Res 6, 3073 (1979); Cooney et al, Science 241, 456 (1988); Dervan et al, Science 251, 1360 (1991).
The term "hybridization" as used here refers to the association of two nucleic acid molecules with one another by hydrogen bonding. Typically, one molecule will be fixed to a solid support and the other will be free in solution. Then, the two molecules may be placed in contact with one another under conditions that favour hydrogen bonding. Factors that affect this bonding include: the type and volume of solvent; reaction temperature; time of hybridization; agitation; agents to block the non-specific attachment of the liquid phase molecule to the solid support (Denhardt's reagent or BLOTTO); the concentration of the molecules; use of compounds to increase the rate of association of molecules (dextran sulphate or polyethylene glycol); and the stringency of the washing conditions following hybridization (see Sambrook et al [supra]).
The inhibition of hybridization of a completely complementary molecule to a target
molecule may be examined using a hybridization assay, as known in the art (see, for example, Sambrook et al [supra]). A substantially homologous molecule will then compete for and inhibit the binding of a completely homologous molecule to the target molecule under various conditions of stringency, as taught in Wahl, G.M. and S. L. Berger (1987; Methods Enzymol. 152:399-407) and Kimmel, A.R. (1987; Methods Enzymol. 152:507- 511).
"Stringency" refers to conditions in a hybridization reaction that favour the association of very similar molecules over association of molecules that differ. High stringency hybridisation conditions are defined as overnight incubation at 42°C in a solution comprising 50% formamide, 5XSSC (15OmM NaCl, 15mM trisodium citrate), 5OmM sodium phosphate (pH7.6), 5x Denhardts solution, 10% dextran sulphate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1X SSC at approximately 65°C. Low stringency conditions involve the hybridisation reaction being carried out at 35°C (see Sambrook et al. [supra]). Preferably, the conditions used for hybridization are those of high stringency.
Preferred embodiments of this aspect of the invention are nucleic acid molecules that are at least 70% identical over their entire length to a nucleic acid molecule encoding an INSP172 polypeptide (i.e. SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO: 8), and nucleic acid molecules that are substantially complementary to these nucleic acid molecules. Preferably, a nucleic acid molecule according to this aspect of the invention comprises a region that is at least 80% identical over its entire length to an INSP72 polypeptide (preferably to SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO: 8) or a nucleic acid molecule that is complementary thereto. In this regard, nucleic acid molecules at least 90%, preferably at least 95%, more preferably at least 98%, 98.5%, 99% or 99% identical over their entire length to the same are particularly preferred. Preferred embodiments in this respect are nucleic acid molecules that encode polypeptides which retain substantially the same biological function or activity as the INSP 172 polypeptide or the INSP 172 mature polypeptide.
The invention also provides a process for detecting a nucleic acid molecule of the invention, comprising the steps of: (a) contacting a nucleic probe according to the invention with a biological sample under hybridizing conditions to form duplexes; and (b) detecting any such duplexes that are formed.
As discussed additionally below in connection with assays that may be utilised according to the invention, a nucleic acid molecule as described above may be used as a hybridization probe for RNA, cDNA or genomic DNA, in order to isolate full-length cDNAs and genomic clones encoding the INSP 172 polypeptides and to isolate cDNA and genomic clones of homologous or orthologous genes that have a high sequence similarity to the gene encoding these polypeptides.
In this regard, the following techniques, among others known in the art, may be utilised and are discussed below for purposes of illustration. Methods for DNA sequencing and analysis are well known and are generally available in the art and may, indeed, be used to practice many of the embodiments of the invention discussed herein. Such methods may employ such enzymes as the Klenow fragment of DNA polymerase I, Sequenase (US Biochemical Corp, Cleveland, OH), Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago, IL), or combinations of polymerases and proof-reading exonucleases such as those found in the ELONGASE Amplification System marketed by Gibco/BRL (Gaithersburg, MD). Preferably, the sequencing process may be automated using machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, NV), the Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).
One method for isolating a nucleic acid molecule encoding a polypeptide with an equivalent function to that of the INSP 172 polypeptides is to probe a genomic or cDNA library with a natural or artificially-designed probe using standard procedures that are recognised in the art (see, for example, "Current Protocols in Molecular Biology", Ausubel et al. (eds). Greene Publishing Association and John Wiley Interscience, New York, 1989,1992). Probes comprising at least 15, preferably at least 30, and more preferably at least 50, contiguous bases that correspond to, or are complementary to, nucleic acid sequences from the appropriate encoding sequence (SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7) are particularly useful probes. Such probes may be labelled with an analytically-detectable reagent to facilitate their identification. Useful reagents include, but are not limited to, radioisotopes, fluorescent dyes and enzymes that are capable of catalysing the formation of a detectable product. Using these probes, the ordinarily skilled artisan will be capable of isolating complementary copies of genomic DNA, cDNA or RNA polynucleotides encoding proteins of interest from human, mammalian or other animal sources and screening such sources for related sequences, for example, for
additional members of the family, type and/or subtype.
In many cases, isolated cDNA sequences will be incomplete, in that the region encoding the polypeptide will be cut short, normally at the 5' end. Several methods are available to obtain full length cDNAs, or to extend short cDNAs. Such sequences may be extended utilising a partial nucleotide sequence and employing various methods known in the art to detect upstream sequences such as promoters and regulatory elements. For example, one method which may be employed is based on the method of Rapid Amplification of cDNA Ends (RACE; see, for example, Frohman et al., PNAS USA 85, 8998-9002, 1988). Recent modifications of this technique, exemplified by the Marathon™ technology (Clontech Laboratories Inc.), for example, have significantly simplified the search for longer cDNAs. A slightly different technique, termed "restriction-site" PCR, uses universal primers to retrieve unknown nucleic acid sequence adjacent a known locus (Sarkar, G. (1993) PCR Methods Applic. 2:318-322). Inverse PCR may also be used to amplify or to extend sequences using divergent primers based on a known region (Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186). Another method which may be used is capture PCR which involves PCR amplification of DNA fragments adjacent a known sequence in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic, 1, 111-119). Another method which may be used to retrieve unknown sequences is that of Parker, J.D. et al. (1991); Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR, nested primers, and PromoterFinder™ libraries to walk genomic DNA (Clontech, Palo Alto, CA). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.
When screening for full-length cDNAs, it is preferable to use libraries that have been size- selected to include larger cDNAs. Also, random-primed libraries are preferable, in that they will contain more sequences that contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
In one embodiment of the invention, the nucleic acid molecules of the present invention may be used for chromosome localisation. In this technique, a nucleic acid molecule is specifically targeted to, and can hybridize with, a particular location on an individual human chromosome. The mapping of relevant sequences to chromosomes according to the
present invention is an important step in the confirmatory correlation of those sequences with the gene-associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found in, for example, V. McKusick, Mendelian Inheritance in Man (available on-line through Johns Hopkins University Welch Medical Library). The relationships between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes). This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localised by genetic linkage to a particular genomic region, any sequences mapping to that area may represent associated or regulatory genes for further investigation. The nucleic acid molecule may also be used to detect differences in the chromosomal location due to translocation, inversion, etc. among normal, carrier, or affected individuals. The nucleic acid molecules of the present invention are also valuable for tissue localisation. Such techniques allow the determination of expression patterns of the polypeptide in tissues by detection of the mRNAs that encode them. These techniques include in situ hybridization techniques and nucleotide amplification techniques, such as PCR. Results from these studies provide an indication of the normal functions of the polypeptide in the organism. In addition, comparative studies of the normal expression pattern of mRNAs with that of mRNAs encoded by a mutant gene provide valuable insights into the role of mutant polypeptides in disease. Such inappropriate expression may be of a temporal, spatial or quantitative nature.
Gene silencing approaches may also be undertaken to down-regulate endogenous expression of a gene encoding a polypeptide of the invention. RNA interference (RNAi)
(Elbashir, SM et ah, Nature 2001, 411, 494-498) is one method of sequence specific post- transcriptional gene silencing that may be employed. Short dsRNA oligonucleotides are synthesised in vitro and introduced into a cell. The sequence specific binding of these dsRNA oligonucleotides triggers the degradation of target mRNA, reducing or ablating target protein expression.
Efficacy of the gene silencing approaches assessed above may be assessed through the measurement of polypeptide expression (for example, by Western blotting), and at the
RNA level using TaqMan-based methodologies.
The vectors of the present invention comprise nucleic acid molecules of the invention and may be cloning or expression vectors. The host cells of the invention, which may be transformed, transfested or transduced with the vectors of the invention may be prokaryotic or eukaryotic.
The polypeptides of the invention may be prepared in recombinant form by expression of their encoding nucleic acid molecules in vectors contained within a host cell. Such expression methods are well known to those of skill in the art and many are described in detail by Sambrook et al {supra) and Fernandez & Hoeffler (1998, eds. "Gene expression systems. Using nature for the art of expression". Academic Press, San Diego, London, Boston, New York, Sydney, Tokyo, Toronto).
Generally, any system or vector that is suitable to maintain, propagate or express nucleic acid molecules to produce a polypeptide in the required host may be used. The appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well- known and routine techniques, such as, for example, those described in Sambrook et ah, {supra). Generally, the encoding gene can be placed under the control of a control element such as a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator, so that the DNA sequence encoding the desired polypeptide is transcribed into RNA in the transformed host cell. Examples of suitable expression systems include, for example, chromosomal, episomal and virus-derived systems, including, for example, vectors derived from: bacterial plasmids, bacteriophage, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as baculoviruses, papova viruses such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, or combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, including cosmids and phagemids. Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained and expressed in a plasmid. The vectors pCR4-TOPO, pCR4-TOPO-INSP172, pEAK12d, pDEST12.2, pDONR221, pEAK12d, pEAK12F, pEAK12R, are preferred examples of suitable vectors for use in accordance with the aspects of this invention relating to INSP172.
Particularly suitable expression systems include microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors;
yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (for example, baculo virus); plant cell systems transformed with virus expression vectors (for example, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (for example, Ti or pBR322 plasmids); or animal cell systems. Cell-free translation systems can also be employed to produce the polypeptides of the invention.
Introduction of nucleic acid molecules encoding a polypeptide of the present invention into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et ah, Basic Methods in Molecular Biology (1986) and Sambrook et ah, [supra]. Particularly suitable methods include calcium phosphate transfection, DEAE-dextran mediated transfection, trans vection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection (see Sambrook et ah, 1989 [supra]; Ausubel et al., 1991 [supra]; Spector, Goldman & Leinwald, 1998). In eukaryotic cells, expression systems may either be transient (for example, episomal) or permanent (chromosomal integration) according to the needs of the system.
The encoding nucleic acid molecule may or may not include a sequence encoding a control sequence, such as a signal peptide or leader sequence, as desired, for example, for secretion of the translated polypeptide into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment. These signals may be endogenous to the polypeptide or they may be heterologous signals. Leader sequences can be removed by the bacterial host in post-translational processing.
In addition to control sequences, it may be desirable to add regulatory sequences that allow for regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory sequences are those which cause the expression of a gene to be increased or decreased in response to a chemical or physical stimulus, including the presence of a regulatory compound or to various temperature or metabolic conditions. Regulatory sequences are those non-translated regions of the vector, such as enhancers, promoters and 5' and 3' untranslated regions. These interact with host cellular proteins to carry out transcription and translation. Such regulatory sequences may vary in their strength and specificity. Depending on the vector system and host utilised, any number of suitable transcription and translation elements, including constitutive and inducible
promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the Bluescript phagemid (Stratagene, LaJolla, CA) or pSportlTM plasmid (Gibco BRL) and the like may be used. The baculo virus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (for example, heat shock, RUBISCO and storage protein genes) or from plant viruses (for example, viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of the sequence, vectors based on SV40 or EBV may be used with an appropriate selectable marker.
An expression vector is constructed so that the particular nucleic acid coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the regulatory sequences being such that the coding sequence is transcribed under the "control" of the regulatory sequences, i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence. In some cases it may be necessary to modify the sequence so that it may be attached to the control sequences with the appropriate orientation; i.e., to maintain the reading frame.
The control sequences and other regulatory sequences may be ligated to the nucleic acid coding sequence prior to insertion into a vector. Alternatively, the coding sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site.
For long-term, high-yield production of a recombinant polypeptide, stable expression is preferred. For example, cell lines which stably express the polypeptide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.
Mammalian cell lines available as hosts for expression are known in the art and include many immortalised cell lines available from the American Type Culture Collection (ATCC) including, but not limited to, Chinese hamster ovary (CHO), HeLa, baby hamster kidney (BHK), monkey kidney (COS), C127, 3T3, BHK, HEK 293, Bowes melanoma and human hepatocellular carcinoma (for example Hep G2) cells and a number of other cell lines.
In the baculovirus system, the materials for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego CA (the "MaxBac" kit). These techniques are generally known to those skilled in the art and are described fully in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Particularly suitable host cells for use in this system include insect cells such as Drosophila S2 and Spodoptera Sf9 cells.
There are many plant cell culture and whole plant genetic expression systems known in the art. Examples of suitable plant cellular genetic expression systems include those described in US 5,693,506; US 5,659,122; and US 5,608,143. Additional examples of genetic expression in plant cell culture has been described by Zenk, Phytochemistry 30, 3861-3863 (1991).
In particular, all plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be utilised, so that whole plants are recovered which contain the transferred gene. Practically all plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugar cane, sugar beet, cotton, fruit and other trees, legumes and vegetables.
Examples of particularly preferred bacterial host cells include streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells. Examples of particularly suitable host cells for fungal expression include yeast cells (for example, S. cerevisiae) and Aspergillus cells.
Any number of selection systems are known in the art that may be used to recover transformed cell lines. Examples include the herpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell 11 :223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) genes that can be employed in tk- or aprt± cells, respectively.
Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for
selection; for example, dihydrofolate reductase (DHFR) that confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt, which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al "(1981) J. MoI. Biol. 150:1-14) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. Additional selectable genes have been described, examples of which will be clear to those of skill in the art.
Although the presence or absence of marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed. For example, if the relevant sequence is inserted within a marker gene sequence, transformed cells containing the appropriate sequences can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding a polypeptide of the invention under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well. Alternatively, host cells that contain a nucleic acid sequence encoding a polypeptide of the invention and which express said polypeptide may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA- DNA or DNA-RNA hybridizations and protein bioassays, for example, fluorescence activated cell sorting (FACS) or immunoassay techniques (such as the enzyme-linked immunosorbent assay [ELISA] and radioimmunoassay [RIA]), that include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein (see Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, St Paul, MN) and Maddox, D.E. et al. (1983) J. Exp. Med, 158, 1211-1216).
A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labelled hybridization or PCR probes for detecting sequences related to nucleic acid molecules encoding polypeptides of the present invention include oligolabelling, nick translation, end-labelling or PCR amplification using a labelled polynucleotide.
Alternatively, the sequences encoding the polypeptide of the invention may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesise RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3 or SP6 and labelled nucleotides. These
procedures may be conducted using a variety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo, MI); Promega (Madison WI); and U.S. Biochemical Corp., Cleveland, OH)).
Suitable reporter molecules or labels, which may be used for ease of detection, include radionuclides, enzymes and fluorescent, chemiluminescent or chromo genie agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
Nucleic acid molecules according to the present invention may also be used to create transgenic animals, particularly rodent animals. Such transgenic animals form a further aspect of the present invention. This may be done locally by modification of somatic cells, or by germ line therapy to incorporate heritable modifications. Such transgenic animals may be particularly useful in the generation of animal models for drug molecules effective as modulators of the polypeptides of the present invention.
The polypeptide can be recovered and purified from recombinant cell cultures by well- known methods including ammonium sulphate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography is particularly useful for purification. Well known techniques for refolding proteins may be employed to regenerate an active conformation when the polypeptide is denatured during isolation and or purification.
Specialised vector constructions may also be used to facilitate purification of proteins, as desired, by joining sequences encoding the polypeptides of the invention to a nucleotide sequence encoding a polypeptide domain that will facilitate purification of soluble proteins. Examples of such purification-facilitating domains include metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilised metals, protein A domains that allow purification on immobilised immunoglobulin, and the domain utilised in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, WA). The inclusion of cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the polypeptide of the invention may be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing the polypeptide of the invention fused to several histidine residues preceding a thioredoxin or an enterokinase
cleavage site. The histidine residues facilitate purification by IMAC (immobilised metal ion affinity chromatography as described in Porath, J. et a (1992), Prot. Exp. Purif. 3: 263-281) while the thioredoxin or enterokinase cleavage site provides a means for purifying the polypeptide from the fusion protein. A discussion of vectors which contain fusion proteins is provided in Kroll, DJ. et a (1993; DNA Cell Biol. 12:441-453).
If the polypeptide is to be expressed for use in screening assays, generally it is preferred that it be produced at the surface of the host cell in which it is expressed. In this event, the host cells may be harvested prior to use in the screening assay, for example using techniques such as fluorescence activated cell sorting (FACS) or immunoaffinity techniques. If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the expressed polypeptide. If polypeptide is produced intracellularly, the cells must first be lysed before the polypeptide is recovered.
As indicated above, the present invention also provides novel targets and methods for the screening of drug candidates or leads. These screening methods include binding assays and/or functional assays, and may be performed in vitro, in cell systems or in animals.
In this regard, a particular object of this invention resides in the use of an INSP 172 polypeptide as a target for screening candidate drugs for treating or preventing FN3 domain containing protein related disorders.
Another object of this invention resides in methods of selecting biologically active compounds, said methods comprising contacting a candidate compound with a INSP 172 gene or polypeptide, and selecting compounds that bind said gene or polypeptide.
A further other object of this invention resides in methods of selecting biologically active compounds, said method comprising contacting a candidate compound with recombinant host cell expressing a INSP 172 polypeptide with a candidate compound, and selecting compounds that bind said INSP 172 polypeptide at the surface of said cells and/or that modulate the activity of the INSP 172 polypeptide.
A "biologically active" compound denotes any compound having biological activity in a subject, preferably therapeutic activity, more preferably a compound having FN3 domain containing protein activity, and further preferably a compound that can be used for treating INSP 172 related disorders, or as a lead to develop drugs for treating FN3 domain containing protein related disorders. A "biologically active" compound preferably is a
compound that modulates the activity of INSP 172.
The above methods may be conducted in vitro, using various devices and conditions, including with immobilized reagents, and may further comprise an additional step of assaying the activity of the selected compounds in a model of FN3 domain containing protein related disorder, such as an animal model.
Preferred selected compounds are agonists of INSP 172, i.e., compounds that can bind to INSP 172 and mimic the activity of an endogenous ligand thereof.
A further object of this invention resides in a method of selecting biologically active compounds, said method comprising contacting in vitro a test compound with a INSP 172 polypeptide according to the present invention and determining the ability of said test compound to modulate the activity of said INSP 172 polypeptide.
A further object of this invention resides in a method of selecting biologically active compounds, said method comprising contacting in vitro a test compound with a INSP 172 gene according to the present invention and determining the ability of said test compound to modulate the expression of said INSP 172 gene, preferably to stimulate expression thereof.
In another embodiment, this invention relates to a method of screening, selecting or identifying active compounds, the method comprising contacting a test compound with a recombinant host cell comprising a reporter construct, said reporter construct comprising a reporter gene under the control of a INSP 172 gene promoter, and selecting the test compounds that modulate (e.g. stimulate or reduce, preferably stimulate) expression of the reporter gene.
The polypeptide of the invention can be used to screen libraries of compounds in any of a variety of drug screening techniques. Such compounds may activate (agonise) or inhibit (antagonise) the level of expression of the gene or the activity of the polypeptide of the invention and form a further aspect of the present invention. Preferred compounds are effective to alter the expression of a natural gene which encodes a polypeptide of the first aspect of the invention or to regulate the activity of a polypeptide of the first aspect of the invention. Agonist or antagonist compounds may be isolated from, for example, cells, cell-free preparations, chemical libraries or natural product mixtures. These agonists or antagonists
may be natural or modified substrates, ligands, enzymes, receptors or structural or functional mimetics. For a suitable review of such screening techniques, see Coligan et al., Current Protocols in Immunology 1(2): Chapter 5 (1991).
Binding to a target gene or polypeptide provides an indication as to the ability of the compound to modulate the activity of said target, and thus to affect a pathway leading to a
FN3 domain containing protein related disorder in a subject. The determination of binding may be performed by various techniques, such as by labelling of the candidate compound, by competition with a labelled reference ligand, etc. For in vitro binding assays, the polypeptides may be used in essentially pure form, in suspension, immobilized on a support, or expressed in a membrane (intact cell, membrane preparation, liposome, etc.).
Modulation of activity includes, without limitation, stimulation of the surface expression of the INSP172 receptor, modulation of multimerization of said receptor {e.g., the formation of multimeric complexes with other sub-units), etc. The cells used in the assays may be any recombinant cell (i.e., any cell comprising a recombinant nucleic acid encoding a INSP 172 polypeptide) or any cell that expresses an endogenous INSP 172 polypeptide. Examples of such cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E.coli, Pichia pastoris, Hansenula polymorpha, Schi∑osaccharomyces pombe, Kluyveromyces or Saccharomyces yeasts, mammalian cell lines {e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary or established mammalian cell cultures {e.g., produced from fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.).
Compounds that are most likely to be good antagonists are molecules that bind to the polypeptide of the invention without inducing the biological effects of the polypeptide upon binding to it. Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to the polypeptide of the invention and thereby inhibit or extinguish its activity. In this fashion, binding of the polypeptide to normal cellular binding molecules may be inhibited, such that the normal biological activity of the polypeptide is prevented. The polypeptide of the invention that is employed in such a screening technique may be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly. In general, such screening procedures may involve using appropriate cells or cell
membranes that express the polypeptide that are contacted with a test compound to observe binding, or stimulation or inhibition of a functional response. The functional response of the cells contacted with the test compound is then compared with control cells that were not contacted with the test compound. Such an assay may assess whether the test compound results in a signal generated by activation of the polypeptide, using an appropriate detection system. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist in the presence of the test compound is observed.
A preferred method for identifying an agonist or antagonist compound of a polypeptide of the present invention comprises:
(a) contacting a cell expressing on the surface thereof the polypeptide according to the first aspect of the invention, the polypeptide being associated with a second component capable of providing a detectable signal in response to the binding of a compound to the polypeptide, with a compound to be screened under conditions to permit binding to the polypeptide; and
(b) determining whether the compound binds to and activates or inhibits the polypeptide by measuring the level of a signal generated from the interaction of the compound with the polypeptide.
Methods for generating detectable signals in the types of assays described herein will be known to those of skill in the art. A particular example is cotransfecting a construct expressing a polypeptide according to the invention, or a fragment such as the LBD, in fusion with the GAL4 DNA binding domain, into a cell together with a reporter plasmid, an example of which is pFR-Luc (Stratagene Europe, Amsterdam, The Netherlands). This particular plasmid contains a synthetic promoter with five tandem repeats of GAL4 binding sites that control the expression of the luciferase gene. When a potential ligand is added to the cells, it will bind the GAL4-polypeptide fusion and induce transcription of the luciferase gene. The level of the luciferase expression can be monitored by its activity using a luminescence reader (see, for example, Lehman et al JBC 270, 12953, 1995; Pawar et al JBC, 277, 39243, 2002). A further preferred method for identifying an agonist or antagonist of a polypeptide of the invention comprises:
(a) contacting a labelled or unlabeled compound with the polypeptide immobilized on any solid support (for example beads, plates, matrix support, chip) and detection of the compound by measuring the label or the presence of the compound itself; or
(b) contacting a cell expressing on the surface thereof the polypeptide, by means of artificially anchoring it to the cell membrane, or by constructing a chimeric receptor being associated with a second component capable of providing a detectable signal in response to the binding of a compound to the polypeptide, with a compound to be screened under conditions to permit binding to the polypeptide; and
(c) determining whether the compound binds to and activates or inhibits the polypeptide by comparing the level of a signal generated from the interaction of the compound with the polypeptide with the level of a signal in the absence of the compound.
For example, a method such as FRET detection of ligand bound to the polypeptide in the presence of peptide co-activators (Norris et al, Science 285, 744, 1999) might be used.
A further preferred method for identifying an agonist or antagonist of a polypeptide of the invention comprises :
(a) contacting a cell expressing on the surface thereof the polypeptide, the polypeptide being associated with a second component capable of providing a detectable signal in response to the binding of a compound to the polypeptide, with a compound to be screened under conditions to permit binding to the polypeptide; and (b) determining whether the compound binds to and activates or inhibits the polypeptide by comparing the level of a signal generated from the interaction of the compound with the polypeptide with the level of a signal in the absence of the compound.
In further preferred embodiments, the general methods that are described above may further comprise conducting the identification of agonist or antagonist in the presence of labelled or unlabelled ligand for the polypeptide.
In another embodiment of the method for identifying agonist or antagonist of a polypeptide of the present invention comprises: determining the inhibition of binding of a ligand to cells which have a polypeptide of the invention on the surface thereof, or to cell membranes containing such a polypeptide, in the presence of a candidate compound under conditions to permit binding to the polypeptide, and determining the amount of ligand bound to the polypeptide. A compound
capable of causing reduction of binding of a ligand is considered to be an agonist or antagonist. Preferably the ligand is labelled.
More particularly, a method of screening for a polypeptide antagonist or agonist compound comprises the steps of: (a) incubating a labelled ligand with a whole cell expressing a polypeptide according to the invention on the cell surface, or a cell membrane containing a polypeptide of the invention,
(b) measuring the amount of labelled ligand bound to the whole cell or the cell membrane;
(c) adding a candidate compound to a mixture of labelled ligand and the whole cell or the cell membrane of step (a) and allowing the mixture to attain equilibrium; (d) measuring the amount of labelled ligand bound to the whole cell or the cell membrane after step (c); and
(e) comparing the difference in the labelled ligand bound in step (b) and (d), such that the compound which causes the reduction in binding in step (d) is considered to be an agonist or antagonist. Similarly, there is provided a method of screening for a polypeptide antagonist or agonist compound which comprises the steps of:
(a) incubating a labelled ligand with a polypeptide according to the invention on any solid support or the cell surface, or a cell membrane containing a polypeptide of the invention.
(b) measuring the amount of labelled ligand bound to the polypeptide on the solid support, whole cell or the cell membrane;
(c) adding a candidate compound to a mixture of labelled ligand and immobilized polypeptide on the solid support, the whole cell or the cell membrane of step (a) and allowing the mixture to attain equilibrium;
(d) measuring the amount of labelled ligand bound to the immobilized polypeptide or the whole cell or the cell membrane after step (c); and
(e) comparing the difference in the labelled ligand bound in step (b) and (d), such that the compound which causes the reduction in binding in step (d) is considered to be an agonist or antagonist.
The polypeptides may be found to modulate a variety of physiological and pathological
processes in a dose-dependent manner in the above-described assays. Thus, the "functional equivalents" of the polypeptides of the invention include polypeptides that exhibit any of the same modulatory activities in the above-described assays in a dose-dependent manner. Although the degree of dose-dependent activity need not be identical to that of the polypeptides of the invention, preferably the "functional equivalents" will exhibit substantially similar dose-dependence in a given activity assay compared to the polypeptides of the invention.
In certain of the embodiments described above, simple binding assays may be used, in which the adherence of a test compound to a surface bearing the polypeptide is detected by means of a label directly or indirectly associated with the test compound or in an assay involving competition with a labelled competitor. In another embodiment, competitive drug screening assays may be used, in which neutralising antibodies that are capable of binding the polypeptide specifically compete with a test compound for binding. In this manner, the antibodies can be used to detect the presence of any test compound that possesses specific binding affinity for the polypeptide.
Assays may also be designed to detect the effect of added test compounds on the production of mRNA encoding the polypeptide in cells. For example, an ELISA may be constructed that measures secreted or cell-associated levels of polypeptide using monoclonal or polyclonal antibodies by standard methods known in the art, and this can be used to search for compounds that may inhibit or enhance the production of the polypeptide from suitably manipulated cells or tissues. The formation of binding complexes between the polypeptide and the compound being tested may then be measured.
Assays may also be designed to detect the effect of added test compounds on the production of mRNA encoding the polypeptide in cells. For example, an ELISA may be constructed that measures secreted or cell-associated levels of polypeptide using monoclonal or polyclonal antibodies by standard methods known in the art, and this can be used to search for compounds that may inhibit or enhance the production of the polypeptide from suitably manipulated cells or tissues. The formation of binding complexes between the polypeptide and the compound being tested may then be measured. Assay methods that are also included within the terms of the present invention are those that involve the use of the genes and polypeptides of the invention in overexpression or ablation assays. Such assays involve the manipulation of levels of these genes/polypeptides
in cells and assessment of the impact of this manipulation event on the physiology of the manipulated cells. For example, such experiments reveal details of signalling and metabolic pathways in which the particular genes/polypeptides are implicated, generate information regarding the identities of polypeptides with which the studied polypeptides interact and provide clues as to methods by which related genes and proteins are regulated.
Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the polypeptide of interest (see International patent application WO84/03564). In this method, large numbers of different small test compounds are synthesised on a solid substrate, which may then be reacted with the polypeptide of the invention and washed. One way of immobilising the polypeptide is to use non-neutralising antibodies. Bound polypeptide may then be detected using methods that are well known in the art. Purified polypeptide can also be coated directly onto plates for use in the aforementioned drug screening techniques.
The polypeptide of the invention may be used to identify membrane-bound or soluble receptors, through standard receptor binding techniques that are known in the art, such as ligand binding and crosslinking assays in which the polypeptide is labelled with a radioactive isotope, is chemically modified, or is fused to a peptide sequence that facilitates its detection or purification, and incubated with a source of the putative receptor (for example, a composition of cells, cell membranes, cell supernatants, tissue extracts, or bodily fluids). The efficacy of binding may be measured using biophysical techniques such as surface plasmon resonance and spectroscopy. Binding assays may be used for the purification and cloning of the receptor, but may also identify agonists and antagonists of the polypeptide, that compete with the binding of the polypeptide to its receptor. Standard methods for conducting screening assays are well understood in the art. In another embodiment, this invention relates to the use of a INSP 172 polypeptide or fragment thereof, whereby the fragment is preferably a INSP 172 gene-specific fragment, for isolating or generating an agonist or stimulator of the INSP 172 polypeptide for the treatment of an immune related disorder, wherein said agonist or stimulator is selected from the group consisting of: 1. a specific antibody or fragment thereof including: a) a chimeric, b) a humanized or c) a fully human antibody, as well as;
2. a bispecific or multispecific antibody,
3. a single chain (e.g. scFv) or
4. single domain antibody, or
5. a peptide- or non-peptide mimetic derived from said antibodies or
6. an antibody-mimetic such as a) an anticalin or b) a fibronectin-based binding molecule (e.g. trinectin or adnectin).
The generation of peptide- or non-peptide minietics from antibodies is known in the art (Saragovi etai, 1991 and Saragovi etal, 1992).
Anticalins are also known in the art (Vogt et ah, 2004). Fibronectin-based binding molecules are described in US6818418 and WO2004029224. Furthermore, the test compound may be of various origin, nature and composition, such as any small molecule, nucleic acid, lipid, peptide, polypeptide including an antibody such as a chimeric, humanized or fully human antibody or an antibody fragment, peptide- or non- peptide mimetic derived therefrom as well as a bispecific or multispecific antibody, a single chain (e.g. scFv) or single domain antibody or an antibody-mimetic such as an anticalin or fibronectin-based binding molecule (e.g. trinectin or adnectin), etc., in isolated form or in mixture or combinations.
The invention also includes a screening kit useful in the methods for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, that are described above.
The invention includes the agonists, antagonists, ligands, receptors, substrates and enzymes, and other compounds which modulate the activity or antigenicity of the polypeptide of the invention discovered by the methods that are described above.
As mentioned above, it is envisaged that the various moieties of the invention (i.e. the polypeptides of the first aspect of the invention, a nucleic acid molecule of the second or third aspect of the invention, a vector of the fourth aspect of the invention, a host cell of the fifth aspect of the invention, a ligand of the sixth aspect of the invention, a compound of the seventh aspect of the invention) may be useful in the therapy or diagnosis of diseases. To assess the utility of the moieties of the invention for treating or diagnosing a disease one or more of the following assays may be carried out. Note that although some of the following assays refer to the test compound as being a protein/polypeptide, a person skilled in the art will readily be able to adapt the following assays so that the other moieties of the invention may also be used as the "test compound".
The invention also provides pharmaceutical compositions comprising a polypeptide, nucleic acid, ligand or compound of the invention in combination with a suitable pharmaceutical carrier. These compositions may be suitable as therapeutic or diagnostic reagents, as vaccines, or as other immunogenic compositions, as outlined in detail below. According to the terminology used herein, a composition containing a polypeptide, nucleic acid, ligand or compound [X] is "substantially free of impurities [herein, Y] when at least 85% by weight of the total X+Y in the composition is X. Preferably, X comprises at least about 90% by weight of the total of X+Y in the composition, more preferably at least about 95%, 98%. 98.5% or even 99% by weight. The pharmaceutical compositions should preferably comprise a therapeutically effective amount of the polypeptide, nucleic acid molecule, ligand, or compound of the invention. The term "therapeutically effective amount" as used herein refers to an amount of a therapeutic agent needed to treat, ameliorate, or prevent a targetted disease or condition, or to exhibit a detectable therapeutic or preventative effect. For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, for example, of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. The precise effective amount for a human subject will depend upon the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the judgement of the clinician. Generally, an effective dose will be from 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg. Compositions may be administered individually to a patient or may be administered in combination with other agents, drugs or hormones.
A pharmaceutical composition may also contain a pharmaceutically acceptable carrier, for administration of a therapeutic agent. Such carriers include antibodies and other polypeptides, genes and other therapeutic agents such as liposomes, provided that the carrier does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.
Suitable carriers may be large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles.
Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).
Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient. Once formulated, the compositions of the invention can be administered directly to the subject. The subjects to be treated can be animals; in particular, human subjects can be treated.
The pharmaceutical compositions utilised in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra- arterial, intramedullary, intrathecal, intraventricular, transdermal or transcutaneous applications (for example, see WO98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal means. Gene guns or hyposprays may also be used to administer the pharmaceutical compositions of the invention. Typically, the therapeutic compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
Direct delivery of the compositions will generally be accomplished by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion. Dosage treatment may be a single dose schedule or a multiple dose schedule.
If the activity of the polypeptide of the invention is in excess in a particular disease state,
several approaches are available. One approach comprises administering to a subject an inhibitor compound (antagonist) as described above, along with a pharmaceutically acceptable carrier in an amount effective to inhibit the function of the polypeptide, such as by blocking the binding of ligands, substrates, enzymes, receptors, or by inhibiting a second signal, and thereby alleviating the abnormal condition. Preferably, such antagonists are antibodies. Most preferably, such antibodies are chimeric and/or humanised to minimise their immunogenicity, as described previously.
In another approach, soluble forms of the polypeptide that retain binding affinity for the ligand, substrate, enzyme, receptor, in question, may be administered. Typically, the polypeptide may be administered in the form of fragments that retain the relevant portions.
In an alternative approach, expression of the gene encoding the polypeptide can be inhibited using expression blocking techniques, such as the use of antisense nucleic acid molecules (as described above), either internally generated or separately administered. Modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5' or regulatory regions (signal sequence, promoters, enhancers and introns) of the gene encoding the polypeptide. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature (Gee, J.E. et al. (1994) In: Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, NY). The complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes. Such oligonucleotides may be administered or may be generated in situ from expression in vivo.
In addition, expression of the polypeptide of the invention may be prevented by using ribozymes specific to its encoding mRNA sequence. Ribozymes are catalytically active RNAs that can be natural or synthetic (see for example Usman, N, et ah, Curr. Opin. Struct. Biol (1996) 6(4), 527-33). Synthetic ribozymes can be designed to specifically cleave mRNAs at selected positions thereby preventing translation of the mRNAs into functional polypeptide. Ribozymes may be synthesised with a natural ribose phosphate backbone and natural bases, as normally found in RNA molecules. Alternatively the
ribozymes may be synthesised with non-natural backbones, for example, 2'-O-methyl RNA, to provide protection from ribonuclease degradation and may contain modified bases.
RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of non-traditional bases such as inosine, queosine and butosine, as well as acetyl-, methyl-, thio- and similarly modified forms of adenine, cytidine, guanine, thymine and uridine which are not as easily recognised by endogenous endonucleases.
For treating abnormal conditions related to an under-expression of the polypeptide of the invention and its activity, several approaches are also available. One approach comprises administering to a subject a therapeutically effective amount of a compound that activates the polypeptide, i.e., an agonist as described above, to alleviate the abnormal condition. Alternatively, a therapeutic amount of the polypeptide in combination with a suitable pharmaceutical carrier may be administered to restore the relevant physiological balance of polypeptide.
Gene therapy may be employed to effect the endogenous production of the polypeptide by the relevant cells in the subject. Gene therapy is used to treat permanently the inappropriate production of the polypeptide by replacing a defective gene with a corrected therapeutic gene.
Gene therapy of the present invention can occur in vivo or ex vivo. Ex vivo gene therapy requires the isolation and purification of patient cells, the introduction of a therapeutic gene and introduction of the genetically altered cells back into the patient. In contrast, in vivo gene therapy does not require isolation and purification of a patient's cells.
The therapeutic gene is typically "packaged" for administration to a patient. Gene delivery vehicles may be non-viral, such as liposomes, or replication-deficient viruses, such as adenovirus as described by Berkner, K.L., in Curr. Top. Microbiol. Immunol., 158, 39-66 (1992) or adeno-associated virus (AAV) vectors as described by Muzyczka, N., in Curr. Top. Microbiol. Immunol., 158, 97-129 (1992) and U.S. Patent No. 5,252,479. For example, a nucleic acid molecule encoding a polypeptide of the invention may be
engineered for expression in a replication-defective retroviral vector. This expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding the polypeptide, such that the packaging cell now produces infectious viral particles containing the gene of interest. These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo (see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics (1996), T Strachan and A P Read, BIOS Scientific Publishers Ltd).
Another approach is the administration of "naked DNA" in which the therapeutic gene is directly injected into the bloodstream or muscle tissue.
In situations in which the polypeptides or nucleic acid molecules of the invention are disease-causing agents, the invention provides that they can be used in vaccines to raise antibodies against the disease causing agent.
Vaccines according to the invention may either be prophylactic (ie. to prevent infection) or therapeutic (ie. to treat disease after infection). Such vaccines comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid, usually in combination with pharmaceutically-acceptable carriers as described above, which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Additionally, these carriers may function as immunostimulating agents ("adjuvants"). Furthermore, the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, and other pathogens.
Since polypeptides may be broken down in the stomach, vaccines comprising polypeptides are preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intradermal injection). Formulations suitable for parenteral admim'stration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The vaccine formulations of the invention may be presented in unit-dose or multi-dose containers. For example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
Genetic delivery of antibodies that bind to polypeptides according to the invention may also be effected, for example, as described in International patent application WO98/55607.
The technology referred to as jet injection (see, for example, www.powderject.com) may also be useful in the formulation of vaccine compositions.
A number of suitable methods for vaccination and vaccine delivery systems are described in International patent application WO00/29428. This invention also relates to the use of nucleic acid molecules according to the present invention as diagnostic reagents. Detection of a mutated form of the gene characterised by the nucleic acid molecules of the invention which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over-expression or altered spatial or temporal expression of the gene. Individuals carrying mutations in the gene may be detected at the DNA level by a variety of techniques.
Nucleic acid molecules for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR, ligase chain reaction (LCR), strand displacement amplification (SDA), or other amplification techniques (see Saiki et ah, Nature, 324, 163-166 (1986); Bej, et ah, Crit. Rev. Biochem. Molec. Biol., 26, 301-334 (1991); Birkenmeyer et ah, J. Virol. Meth., 35, 117-126 (1991); Van Brunt, J., Bio/Technology, 8, 291-294 (1990)) prior to analysis.
In one embodiment, this aspect of the invention provides a method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide according to the invention and comparing said level of expression to a control level, wherein a level that is different to said control level is indicative of disease. The method may comprise the steps of: a)contacting a sample of tissue from the patient with a nucleic acid probe under stringent conditions that allow the formation of a hybrid complex between a nucleic acid molecule of the invention and the probe;
b)contacting a control sample with said probe under the same conditions used in step a); c)and detecting the presence of hybrid complexes in said samples; wherein detection of levels of the hybrid complex in the patient sample that differ from levels of the hybrid complex in the control sample is indicative of disease. A further aspect of the invention comprises a diagnostic method comprising the steps of: a)obtaining a tissue sample from a patient being tested for disease; b)isolating a nucleic acid molecule according to the invention from said tissue sample; and c)diagnosing the patient for disease by detecting the presence of a mutation in the nucleic acid molecule which is associated with disease. To aid the detection of nucleic acid molecules in the above-described methods, an amplification step, for example using PCR, may be included.
Deletions and insertions can be detected by a change in the size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labelled RNA of the invention or alternatively, labelled antisense DNA sequences of the invention. Perfectly-matched sequences can be distinguished from mismatched duplexes by RNase digestion or by assessing differences in melting temperatures. The presence or absence of the mutation in the patient may be detected by contacting DNA with a nucleic acid probe that hybridises to the DNA under stringent conditions to form a hybrid double-stranded molecule, the hybrid double-stranded molecule having an unhybridised portion of the nucleic acid probe strand at any portion corresponding to a mutation associated with disease; and detecting the presence or absence of an unhybridised portion of the probe strand as an indication of the presence or absence of a disease-associated mutation in the corresponding portion of the DNA strand.
Such diagnostics are particularly useful for prenatal and even neonatal testing. Point mutations and other sequence differences between the reference gene and "mutant" genes can be identified by other well-known techniques, such as direct DNA sequencing or single-strand conformational polymorphism, (see Orita et ah, Genomics, 5, 874-879 (1989)). For example, a sequencing primer may be used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures with radiolabeled nucleotides or
by automatic sequencing procedures with fluorescent-tags. Cloned DNA segments may also be used as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR. Further, point mutations and other sequence variations, such as polymorphisms, can be detected as described above, for example, through the use of allele-specific oligonucleotides for PCR amplification of sequences that differ by single nucleotides.
DNA sequence differences may also be detected by alterations in the electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (for example, Myers et al, Science (1985) 230:1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and Sl protection or the chemical cleavage method (see Cotton et al., Proc. Natl. Acad. Sci. USA (1985) 85: 4397-4401).
In addition to conventional gel electrophoresis and DNA sequencing, mutations such as microdeletions, aneuploidies, translocations, inversions, can also be detected by in situ analysis (see, for example, Keller et ah, DNA Probes, 2nd Ed., Stockton Press, New York, N. Y., USA (1993)), that is, DNA or RNA sequences in cells can be analysed for mutations without need for their isolation and/or immobilisation onto a membrane. Fluorescence in situ hybridization (FISH) is presently the most commonly applied method and numerous reviews of FISH have appeared (see, for example, Trachuck et al, Science, 250, 559-562 (1990), and Trask et al, Trends, Genet., 7, 149-154 (1991)).
In another embodiment of the invention, an array of oligonucleotide probes comprising a nucleic acid molecule according to the invention can be constructed to conduct efficient screening of genetic variants, mutations and polymorphisms. Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see for example: M.Chee et al, Science (1996), VoI 274, pp 610-613).
In one embodiment, the array is prepared and used according to the methods described in PCT application WO95/11995 (Chee et at); Lockhart, D. J. et al. (1996) Nat. Biotech. 14: 1675-1680); and Schena, M. et al (1996) Proc. Natl. Acad. Sci. 93: 10614-10619). Oligonucleotide pairs may range from two to over one million. The oligomers are synthesized at designated areas on a substrate using a light-directed chemical process. The substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any
other suitable solid support. In another aspect, an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application W095/25116 (Baldeschweiler et aϊ). In another aspect, a "gridded" array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures. An array, such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any other number between two and over one million which lends itself to the efficient use of commercially-available instrumentation.
In addition to the methods discussed above, diseases may be diagnosed by methods comprising determining, from a sample derived from a subject, an abnormally decreased or increased level of polypeptide or mRNA. Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
Assay techniques that can be used to determine levels of a polypeptide of the present invention in a sample derived from a host are well-known to those of skill in the art and are discussed in some detail above (including radioimmunoassays, competitive-binding assays,
Western Blot analysis and ELISA assays). This aspect of the invention provides a diagnostic method which comprises the steps of: (a) contacting a ligand as described above with a biological sample under conditions suitable for the formation of a ligand- polypeptide complex; and (b) detecting said complex.
Protocols such as ELISA, RIA, and FACS for measuring polypeptide levels may additionally provide a basis for diagnosing altered or abnormal levels of polypeptide expression. Normal or standard values for polypeptide expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably humans, with antibody to the polypeptide under conditions suitable for complex formation The amount of standard complex formation may be quantified by various methods, such as by photometric means.
Antibodies which specifically bind to a polypeptide of the invention may be used for the diagnosis of conditions or diseases characterised by expression of the polypeptide, or in assays to monitor patients being treated with the polypeptides, nucleic acid molecules, ligands and other compounds of the invention. Antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics. Diagnostic assays for the polypeptide include methods that utilise the antibody and a label to detect the polypeptide in human body fluids or extracts of cells or tissues. The antibodies may be used with or without modification, and may be labelled by joining them, either covalently or non-covalently, with a reporter molecule. A wide variety of reporter molecules known in the art may be used, several of which are described above.
Quantities of polypeptide expressed in subject, control and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease. Diagnostic assays may be used to distinguish between absence, presence, and excess expression of polypeptide and to monitor regulation of polypeptide levels during therapeutic intervention. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials or in monitoring the treatment of an individual patient.
A diagnostic kit of the present invention may comprise: (a) a nucleic acid molecule of the present invention; (b) a polypeptide of the present invention; or (c) a ligand of the present invention.
In one aspect of the invention, a diagnostic kit may comprise a first container containing a nucleic acid probe that hybridises under stringent conditions with a nucleic acid molecule according to the invention; a second container containing primers useful for amplifying the nucleic acid molecule; and instructions for using the probe and primers for facilitating the diagnosis of disease. The kit may further comprise a third container holding an agent for digesting unhybridised RNA.
In an alternative aspect of the invention, a diagnostic kit may comprise an array of nucleic acid molecules, at least one of which may be a nucleic acid molecule according to the invention.
To detect polypeptide according to the invention, a diagnostic kit may comprise one or
more antibodies that bind to a polypeptide according to the invention; and a reagent useful for the detection of a binding reaction between the antibody and the polypeptide.
Such kits will be of use in diagnosing a disease or susceptibility to disease, particularly a disease including, but not limited to, neoplasm, cancer, brain tumour, glioma, bone tumor, lung tumor, breast tumour, prostate tumour, colon tumour, hemangioma, myeloproliferative disorder, leukemia, hematological disease, neutropenia, thrombocytopenia, angiogenesis disorders, dermatological disease, ageing, wounds, burns, fibrosis, cardiovascular disease, restensosis, heart disease, peripheral vascular disease, coronary artery disease, oedema, thromboembolism, dysmenorrhea, endometriosis, pre- eclampsia, lung disease, COPD, asthma bone disease, renal disease, glomerulonephritis, liver disease, Crohn's disease, gastritis, ulcerative colitis, ulcer, immune disorder, autoimmune disease, arthritis, rheumatoid arthritis, psoriasis, epidermolysis bullosa, systemic lupus erythematosus, ankylosing spondylitis, Lyme disease, multiple sclerosis, neurodegeneration, stroke, brain/spinal cord injury, Alzheimer's disease, Parkinson's disease, motor neurone disease, neuromuscular disease, HIV, AIDS, cytomegalovirus infection, fungal infection, ocular disorder, macular degeneration, glaucoma, diabetic retinopathy and ocular hypertension. Preferably, the disease is a reproductive or developmental disease or disorder.
Thus, the embodiments of the invention provide kits for use in diagnosing a disease or susceptibility to disease such as of reproductive health disorders (in particular male or female reproductive disorders) or cancer. In one embodiment of the invention, the reproductive health disorder is selected from male or female infertility. In one embodiment of the invention, the male infertility is associated to spermatogenesis deficiencies with oligospermia or azoospermia. In one embodiment of the invention, the female infertility is associated with follicle-genesis deficiency. In one embodiment of the invention, the cancer is selected from carcinoma, including, but not limited to adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma or leukemia. In one embodiment of the invention, the cancer is selected from ovarian cancer, prostate cancer or testicular cancer.
Various aspects and embodiments of the present invention will now be described in more detail by way of example, with particular reference to INSP 172 polypeptides.
It will be appreciated that modification of detail may be made without departing from the scope of the invention.
Brief description of the Figures
Figure 1: DNA and protein sequence of INSP 172. The position and sense of PCR primers INSPl 72-CP 1 /INSP 172-CP2 are indicated by arrows.
Figure 2: Nucleotide sequence with translation of the INSP 172 PCR product cloned using primers INSP 172-CP 1 and INSP 172-CP2. The position and sense of PCR primers INSPl 72-CP 1 /INSP 172-CP2 are indicated by arrows.
Figure 3: Clustal w alignment of the amino acid sequence of the INSP 172 cloned sequence with mouse orthologue (SwissProt Ace. Code Q8BW83).
Figure 4: Sequence features of INSP 172. The signal peptide is in italics. Potential N- glycosylation sites are highlighted in black. Predicted FNIII domains are underlined.
Figure 5: Alignment of INSP 172 full length protein sequence to human genomic sequence.
TABLE 1
TABLE 2
Example 1: Identification and in silico analysis of INSP 172
INSP 172 is a novel fibronectin type III domain containing protein. The sequence consists of 733 amino acids encoded in 11 exons. A signal peptide spans from amino acid residues 1 to 25.
A pair of PCR primers, INSPl 72-CP 1 /INSP 172-CP2 (Figure 1), were designed to amplify the full length cds of INSP 172. The primers were used to screen a proprietary panel of cDNA templates. Bands migrating at the predicted size were identified in pools PS22 (pancreas, pituitary and Y79 cell line), PS 17 (uterus, bone marrow, thyroid, spinal cord and cervix); and PS25 (mammary gland, ovary, pituitary). The resultant bands were subcloned into the pCR4 TOPO vector and several clones from each pool were sequenced. The nucleotide sequence and translation of the cloned INSP 172 is shown in Figure 2. The plasmid of one of the resultant clones is ρCR4 TOPO-INSP 172-PS22-3.
The cloned INSP 172 sequence was analysed with Prosite. Prosite indicates that INSP 172 contains eight FN3 domains between the following amino acids residues: 25-112, 117-199, 201-285, 288-370, 375-455, 456-540, 546-625 and 631-715. Polypeptides comprising these discrete domains form one aspect of the present invention, along with polypeptides that include these domains and combinations of domains.
The INSPl 72 polypeptide sequence was also analysed using NetNGlyc 1.0. INSP 172 is predicted to contain various N-linked glycosylation sites as indicated by the shading in Figure 4. Amino acid residues 159, 162, 230, 331, 367, 415, 433, 446, 465, 561, 602 and 656 are predicted to be N-glycosylated.
EST evidence indicates that INSP 172 is expressed in the following cell lines/tissue (Table 3):
Table 3
The recombinant INSP 172 may be used in a wide variety of screening assays, including those described above, and those described in Examples 2 and 3 below.
Example 2 - Assays Suitable for Exploration of the Biological Relevance of INSP 172 Function - reproductive health
It is believed that the moieities of the invention will be particularly useful for the treatment or diagnosis of disorders/diseases relating to reproductive health. It is believed that the following assays will be useful to test for moieties have useful biological effects. Note that although some of the following assays refer to the test compound as being a protein/polypeptide, a person skilled in the art will readily be able to adapt the following assays so that the other moieties of the invention may also be used as the "test compound".
A Reproductive health assays JEG-3 Implantation assay:
In this assay, a 2-chamber system is used where fluorescently labeled JEG-3 cells invade through a Matrigel-coated porous membrane from an upper chamber into a lower chamber when Ishikawa cells or Ishikawa-conditioned medium are placed into the lower chamber. The cells that migrate are quantified in a plate reader. The goal is to identify proteins that increase invasion of JEG-3 cells for use in aiding implantation in vivo.
This implantation assay using JEG-3 choriocarcinoma cell line has been described for example by Hohn et al. (MoI. Reprod. Dev. 2000, 57:135-145).
Osteopontin bead assay (Ishikawa cells)
In this assay, osteopontin-coated fluorescent beads represent the blastocyst, and the Ishikawa cells are primed to accept them for binding by treating them with estradiol. The goal is to identify proteins that increase the ability of the Ishikawa cells to bind the osteopontin-beads as an aid to increase receptivity of the uterine endometrium at the time of implantation.
HuF6 assay: In this assay the goal is to identify proteins that increase production of PGE2 (a marker for decidualization) by the HuF6 cells as a way of enhancing decidualization during early pregnancy.
This decidualization assay using HuF6 uterine fibroblasts is described by Tran et al. (Fertility and Sterility 2005, 84, Supplement 1, S436-437). Endometriosis assay:
Peritoneal TNFa plays a role in endometriosis by inducing the sloughed endometrial cells from the uterus to adhere to and proliferate on peritoneal mesothelial cells. In this assay, BEND cells are treated with TNFa, which increases their ability to bind fibronectin-coated fluorescent beads as an assay for adherence during endometriosis. The goal is to identify proteins that decrease or inhibit the ability of TNFa to stimulate bead-binding capacity of the cells.
Cyclic AMP assay using JC-410 porcine granulose cells stably transfected with hLHR.
In Polycystic Ovary Syndrome, LH from the pituitary is relatively high, and induces androgen output from the ovarian thecal cells. In this assay, we are looking for an inhibitor of LH signaling which could be used to decrease the action of LH at the ovary
during PCOS. The JC-410 porcine granulosa cell line was stably transfected with the human LH receptor. Treatment with LH results in cAMP production.
Cyclic AMP assay using JC-410 porcine granulose cells stably transfected with hFSHR.
The JC-410 porcine granulosa cell line was stably transfected with the human FSHR. Treatment with FSH stimulates cAMP production, which is measured in this assay. The goal is to identify proteins that enhance FSH action in the granulosa cells.
LbetaT2 (mouse) pituitary cells assay.
The LbT2 is an immortalized murine pituitary gonadotroph cell line. Stimulation with Activin alone or with GnRH + Activin results in secretion of FSH. The cells can either be treated with GnRH + Bioscreen proteins to find proteins that act in concert with GnRH to stimulate FSH production, or they can be treated with Bioscreen proteins alone to find a protein that can stimulate FSH secretion like activin alone.
Cumulus expansion assay.
Using murine cumulus-oocyte complexes an assay can be developed to identify moieties which promote expansion.
Such a cumulus expansion assay (oocyte maturation assay) is described in Hizaki H et al. (Proc. Natl. Acad. Sci. 1999, 96:10501-6).
RWPE Proliferation assay
Benign prostatic hyperplasia is characterized by growth of prostatic epithelium and stroma that is not balanced by apoptosis, resulting in enlargement of the organ. RWPE is a regular human prostatic epithelial cell line that was immortalized with the HPV- 18, and is used in place of primary human prostatic epithelial cells, which are not always available.
HT-1080 fibrosarcoma invasion assay
Fluorescently-labeled HT- 1080 human fibrosarcoma cells are cultured in the upper chamber of a 2-chamber system, and can be stimulated to invade through the porous Matrigel-coated membrane into the bottom chamber where they are quantified. The goal would be to identify a moiety that would inhibit the invasion.
Primary human uterine smooth muscle assay
One of the hallmarks of uterine fibroid disease is collagen deposition by the uterine smooth muscle cells that have become leioymyomas. Primary human uterine smooth muscle cells are stimulated to produce collagen by treatment with TGFb, which is blocked with Rebif. The goal is to discover proteins that inhibit this fibrotic phenotype. Human leiomyoma cells proliferation assay
Human leiomyoma cells may be used as a model for uterine fibroid disease in a proliferation assay. The cells grow very slowly but may be stimulated with estradiol and growth factors. The goal is to identify proteins that inhibit estradiol-dependent growth of leiomyoma cells. U937 Migration assay.
Endometriotic lesions secrete cytokines that recruit immune cells to the peritoneal cavity which then mediate inflammatory symptoms that are common to endometriosis. RANTES has been shown to be produced by endometriotic stromal cells and is present in the peritoneal fluid. In this assay, U937, a monocytic cell line used as a model for activated macrophages, can be induced by treating the lower level of a 2-chamber culture system to migrate from the upper chamber. If the cells are pre-loaded with fluorescent dye, they can be quantified in the lower chamber. The goal will be to identify proteins that inhibit the migration of the U937 cells.
JEG3 human trophoblast assay The trophoblast of the blastocyst produces HLA-G, a class I HLA molecule that is believed to be important in preventing immunological rejection of the embryo by the mother. During pre-eclampsia, HLA-G levels are low or non-existent. The JEG-3 human trophoblast cell line produces HLA-G and may be utilised to identify moieties that can increase HLA-G production. Primary rat ovarian dispersate assay
The amount of estradiol production from cultures of cells from whole ovaries taken from immature rats or other rodents may be measured after treatment with FSH and/or LH. The goal will be to identify proteins that enhance gonadotropin-stimulated steroidogenesis, or proteins that work alone to increase steroidogenesis by these cultures.
Activity of the polypeptides of this invention may also be confirmed using the assay of Adashi et ah (Biol. Reprod. 1982, 26:270-80). In this assay, enhancement of gonadotrophin stimulated steroidogenesis in primary rat ovarian dispersate is measured.
Mouse IVF assay In this assay, sperm function, measured by ability to fertilize oocytes, will be assayed with the goal of finding proteins that stimulate fertilizing potential of sperm. Such an assay may be run with, for example, mouse sperm and oocytes.
Primary human prostate stromal cells proliferation assay.
An assay for the epithelial component of BPH has already been developed (see RWPE above). This assay uses primary human prostate stromal cells as a model for proliferation of these cells during BPH. The goal will be to identify proteins that inhibit proliferation of these cells.
Primary human uterine smooth muscle proliferation assay
Proteins and other moieties may be tested to thereby identify moieties capable of inhibiting the proliferation of primary human uterine smooth muscle cells. Proliferation of uterine smooth muscle cells is a precursor for development of tumours in uterine fibroid disease.
Example 3 - Assays Suitable for Exploration of the Biological Relevance of INSP 172 Function — fibrosis
Activation and pathological proliferation of fibroblasts are the key steps leading to a phenotype known as fibrosis. Fibrosis is characterized by the excessive deposition of extracellular matrix, especially collagen. Stromal cells, including fibroblasts, express specific pro- and anti-fibrotic proteins. Keratinocyte growth factor (KGF) is well- characterized anti-fibrotic molecule. Additionally, oxidative damage and pro-inflammatory stimuli have been proposed to be among major events leading to myofibroblast phenotype and eventually to fibrosis. NF-kB is a mediator of oxidative stress and inflammatory reactions. Based on fibroblast biology, we have developed four cell-based assays, namely fibroblast proliferation, collagen production, NF-kB activation and KGF production assays. Human fibroblast proliferation assay. An activation and pathological proliferation of fibroblasts are the key steps leading to a phenotype known as fibrosis. We have developed a simple assay to measure the
proliferative responses of human skin-derived fibroblasts to novel proteins and small molecules. The assay is based on a Fluorescence enhancement mediated by CyQUANT GR dye bound to cellular nucleic acids.
Type I collagen production by human fibroblasts. Fibrosis is characterized by the excessive deposition of extracellular matrix, especially collagen. Over production of type I collagen is the main manifestation of systemic sclerosis. TGFβ is known to up-regulate production of collagen in vitro and in vivo. A cell- based assay has been developed in order to test the ability of novel pro-or anti-fibrotic molecules to modulate basal or TGFβ 1 -stimulated levels of type I collagen production by human skin-derived fibroblasts.
Keratinocyte growth factor (KGF) production by human fibroblasts.
KGF is an important mediator of stroma-to epithelium interactions in many organs (lung, pancreas, kidney, prostate, mammary, gland, uterus) during normal and pathological growth and development. KGF is specifically produced by stromal cells and its receptor is specifically expressed by epithelial cells. We propose that KGF might be an important player during pathophysiological reactions in fibrosis and can be used as a marker of these reactions. We have developed KGF ELISA and using human lung-derived fibroblasts shown that the KGF production can be significantly up-regulated by IL- lβ and TNFβ and down-regulated by TGFβ. These cytokines will be used as reference molecules in screening for novel proteins capable to induce KGF production.
NF-κB transcription activation in Fibroblasts.
Oxidative damage and pro-inflammatory stimuli have been proposed to be among major events leading to myofibroblast phenotype and eventually to fibrosis. NF-κB is a mediator of oxidative stress and inflammatory reactions. We generated Swiss 3T3 fibroblasts with stably integrated NFKB-SEAP (secreted alkaline phosphatase) construct. NFDB-SEAP is designed to measure the binding of transcription factors to K enhancer allowing a direct measurement of activation of NF-κB pathway. The SEAP enzyme is secreted into the culture medium, so samples can be collected at various time points to assay for transcription activity without harvesting cells. The Swiss 3T3-NFκB-SEAP cell line is being used as a cell-based assay to test novel Functional Genomics proteins and is very
promising to test small molecules, especially those with predicted pro-/anti-inflammatory activity.
Further assays which may be of use include the following:
Connective tissue growth factor f CTGF) promoter activation/repression in fibroblasts. CTGF, a 38-kD cysteine-rich protein, stimulates the production of extracellular matrix elements by fibroblasts. CTGF overexpression has been reportedly found in many fibrotic human tissues, including lung, skin, liver, kidney and blood vessels. In vitro, TGFβ activates CTGF gene transcription in human lung fibroblasts. We constructed CTGF promoter-reporter with secreted alkaline phosphatase (SEAP) as a reporter and generated Swiss 3T3 fibroblasts with stably integrated CTGF-SEAP construct. Using these fibroblasts we have shown that CTGF promoter is down-regulated by SARP-I, OPG and FSH and up-regulated by TGFβ
KL-6 production.
KL-6, originally discovered as a pulmonary adenocarcinoma-related protein and later referred to as MUC-I, is a high-molecular- weight glycoprotein, now classified as Cluster 9 antigen. KL-6 is elevated in both sera and BALF of patients with idiopathic pulmonary fibrosis (IPF) and other lung interstitial diseases. In lung tissue from patients suffering from IPF, the majority of cells labeled with KL-6 antibodies are regenerating type II pneumocytes. We designed two peptides to produce polyclonal antibodies against KL-6 and currently developing KL-6 ELISA to measure KL-6 production by human lung- derived type II pneumocytes.
Neutralization of apoptosis of L-929 fibroblasts treated with soluble recombinant TRAIL (TNF-related apoptosis-inducing ligand).
TRAIL has been shown to be one of the cellular ligands for osteoprotegerin (OPG) and this assay measures the biological activity of OPG.
RANKL (receptor activator of NF-kB ligand) production by human fibroblasts.
RANKL is another ligand for OPG and this assay will be used to measure the biological activity of OPG.
Example 4 : cDNA cloning of INSP 172
First strand cDNA was prepared from a variety of human tissue total RNA samples (Clontech, Stratagene, Ambion, Biochain Institute and in-house preparations) using Superscript II or Superscript III RNase H" Reverse Transcriptase (Invitrogen) according to the manufacturer's protocol. For Superscript II: Oligo (dT)15 primer (lμl at 500 μg/ml) (Promega), 2 μg human total RNA5 1 μl 10 mM dNTP mix (10 mM each of dATP, dGTP, dCTP and dTTP at neutral pH) and sterile distilled water to a final volume of 12 μl were combined in a 1.5 ml Eppendorf tube, heated to 65 0C for 5 min and chilled on ice. The contents were collected by brief centrifugation and 4 μl of 5X First-Strand Buffer, 2 μl 0.1 M DTT, and 1 μl RnaseOUT™ Recombinant Ribonuclease Inhibitor (40 units/μl, Invitrogen) were added. The contents of the tube were mixed gently and incubated at 42 0C for 2 min, then 1 μl (200 units) of Superscript II™ enzyme was added and mixed gently by pipetting. The mixture was incubated at 42 0C for 50 min and then inactivated by heating at 70 °C for 15 min. To remove RNA complementary to the cDNA, 1 μl (2 units) of E. coli RNase H (Invitrogen) was added and the reaction mixture incubated at 37 °C for 20 min.
For Superscript III: 1 μl 01igo(dT)2o primer (50μM, Invitrogen), 2 μg human total RNA, 1 μl 10 mM dNTP mix (10 mM each of dATP, dGTP, dCTP and dTTP at neutral pH) and sterile distilled water to a final volume of 10 μl were combined in a 1.5 ml Eppendorf tube, heated to 65 0C for 5 min and then chilled on ice. For each RT reaction a cDNA synthesis mix was prepared as follows: 2 μl 1 OX RT buffer, 4 μl 25mM MgCl2, 2 μl 0. IM DTT, 1 μl RNaseOUT™ (40 U/μl) and 1 μl Superscript III™ RT enzyme were combined in a separate tube and then 10 μl of this mix added to the tube containing the RNA/primer mixture. The contents of the tube were mixed gently, collected by brief centrifugation, and incubated at 50 0C for 50 min. The reaction was terminated by incubating at 80 0C for 5 min and the reaction mixture then chilled on ice and collected by brief centrifugation. To remove RNA complementary to the cDNA, lμl (2 units) of E. coli RNase H (Invitrogen) was added and the reaction mixture incubated at 37 0C for 20 min.
The final 21 μl reaction mix was diluted by adding 179 μl sterile water to give a total volume of 200 μl. This represented approximately 20 ng/μl of each individual cDNA template.
Gene specific cloning primers for PCR
A pair of PCR primers having a length of between 18 and 30 bases were designed to amplify the predicted INSP 172 cds using Primer Designer Software (Scientific & Educational Software, PO Box 72045, Durham, NC 27722-2045, USA). PCR primers were optimized to have a Tm close to 55 + 10 0C and a GC content of 40-60%. Primers were selected which had high selectivity for the target sequence (INSP 172) with little or no none specific priming.
PCR amplification of INSP 172 from human cDNA templates
Gene-specific cloning primers INSPl 72-CP 1 /INSP 172-CP2 were designed to amplify a cDNA fragment of 2202 bp spanning the entire INSP 172 cds. (Table 4, Figure 1). The primer pair was tested on pools of cDNA containing between 3-5 different cDNAs. PCR was performed in a final volume of 50 μl containing IX Platinum® Tag High Fidelity (HiFi) buffer, 2 mM MgSO4, 200 μM dNTPs, 0.2 μM of each cloning primer, 1 unit of Platinum® Taq DNA Polymerase High Fidelity (HiFi) (Invitrogen), 1 μl of each cDNA pool, and either OX, IX or 2X PCRx Enhancer solution (Invitrogen). Cycling was performed using an MJ Research DNA Engine, programmed as follows: 94 °C, 2 min; 40 cycles of 94 °C, 30 sec, 58 0C, 30 sec, and 68 0C, 3 min; followed by 1 cycle at 68 °C for 8 min and a holding cycle at 4 °C.
All 40 μl of each amplification reaction was visualized on a 0.8 % agarose gel in 1 X TAE buffer (Invitrogen). Products of approximately the expected molecular mass (2202 bp) were identified in pools PS 17, PS22 and PS25. Bands were purified from the gel using the MinElute gel extraction kit (Qiagen), eluted in 10 μil of water, and subcloned directly.
Subcloning of PCR Products
The PCR products were subcloned into the topoisomerase I modified cloning vector (pCR4-TOPO) using the TA cloning kit purchased from the Invitrogen Corporation using the conditions specified by the manufacturer. Briefly, 4 μl of gel purified PCR product was incubated for 15 min at room temperature with 1 μl of TOPO vector and 1 μl salt solution. The reaction mixture was then transformed into E. coli strain TOPlO (Invitrogen) as follows: a 50 μl aliquot of One Shot TOPlO cells was thawed on ice and 2 μl of TOPO reaction was added. The mixture was incubated for 15 min on ice and then heat shocked by incubation at 42 0C for exactly 30 s. Samples were returned to ice and 250 μl of warm (room temperature) SOC media was added. Samples were incubated with shaking (220
rpm) for 1 h at 37 0C. The transformation mixture was then plated on L-broth (LB) plates containing ampicillin (100 μg/ml) and incubated overnight at 37 °C.
Colony PCR
Colonies were inoculated into 50 μl sterile water using a sterile toothpick. A 10 μl aliquot of the inoculum was then subjected to PCR in a total reaction volume of 20 μl containing
IX AmpliTaq™ buffer, 200 μM dNTPs, 20 pmoles of T7 primer, 20 pmoles of T3 primer, and 1 unit of AmpliTaq™ (Applied Biosystems) using an MJ Research DNA Engine. The cycling conditions were as follows: 94 0C, 2 min; 30 cycles of 94 0C, 30 sec, 48 °C, 30 sec and 72 °C for 3 min. Samples were maintained at 4 0C (holding cycle) before further analysis.
PCR reaction products were analyzed on 1 % agarose gels in 1 X TAE buffer. 4 colonies from each of the positive pools which gave PCR products of approximately the expected molecular weight (2202 bp + 105 bp due to the multiple cloning site (MCS) were grown up overnight at 37 0C in 5 ml L-Broth (LB) containing ampicillin (100 μg /ml), with shaking at 220 rpm.
Plasmid DNA preparation and sequencing
Miniprep plasmid DNA was prepared from 5 ml cultures using a Biorobot 8000 robotic system (Qiagen) or Wizard Plus SV Minipreps kit (Promega cat. no. 1460) according to the manufacturer's instructions. Plasmid DNA was eluted in 80 μl of sterile water. The DNA concentration was measured using an Eppendorf BO photometer or Spectramax 190 photometer (Molecular Devices). Plasmid DNA (200-500 ng) was subjected to DNA sequencing with the sequencing primers T7 and T3, and the gene- specific primers INSPl 72-SP 1 and -SP2 (Table 4) using the BigDye Terminator system (Applied Biosystems cat. no. 4390246) according to the manufacturer's instructions. Sequencing reactions were purified using Dye-Ex columns (Qiagen) or Montage SEQ 96 cleanup plates (Millipore cat. no. LSKS09624) then analyzed on an Applied Biosystems 3700 sequencer.
AU clones sequenced contained the INSP 172 cds. A plasmid containing the cloned PCR product is pCR4-TOPO-INSP172.
Table 4
Example 5: Construction of mammalian cell expression vectors for INSP172 Generation of Gateway compatible INSP 172 ORF fused to an in frame 6HIS tag sequence.
Further experiments can be perfomed in order to construct mammalian cell expression vectors for INSP172. The first stage of the Gateway cloning process involves a two step PCR reaction which generates the ORF of INSP 172 flanked at the 5' end by an attBl recombination site and Kozak sequence, and flanked at the 3' end by a sequence encoding an in-frame 6 histidine (6HIS) tag, a stop codon and the attB2 recombination site (Gateway compatible cDNA). Plasmid ρCR4-TOPO-INSP172 is used as a template for the PCR.
The first PCR reaction (in a final volume of 50 μl) typically contains respectively: 1 μl (30 ng) of plasmid pCR4-TOPO-INSP172, 1.5 μl dNTPs (10 niM), 10 μl of 1OX Pfx polymerase buffer, 1 μl MgSO4 (50 mM), 0.5 μl each of gene specific primer (100 μM), and 0.5 μl Platinum Pfx DNA polymerase (Invitrogen). The amplification product is directly purified using the Wizard PCR Preps DNA Purification System (Promega) and recovered in 50μl sterile water according to the manufacturer's instructions.
The second PCR reaction (in a final volume of 50 μl) typically contains 10 μl purified PCRl product, 1.5 μl dNTPs (10 mM), 5 μl of 1OX Pfx polymerase buffer, 1 μl MgSO4 (50 mM), 0.5 μl of each Gateway conversion primer (100 μM) (GCP forward and GCP reverse) and 0.5 μl of Platinum Pfx DNA polymerase. The PCR mixture is cleaned up directly using the Wizard PCR Preps DNA Purification System (Promega) and recovered in 50 μl sterile water according to the manufacturer's instructions. A 10 μl aliquot is visualized on 0.8 % agarose gel in 1 X TAE buffer (Invitrogen) in order to verify that the product is of the expected molecular weight.
Subcloning of Gateway compatible INSP 172 ORF into Gateway entry vector pDONR221 and expression vectors pEAK12d and pDEST12.2
The second stage of the Gateway cloning process involves subcloning of the Gateway modified PCR product into the Gateway entry vector pDONR221 (Invitrogen) as follows: 5 μl of purified product from PCR2 are incubated with 1.5 μl pDONR221 vector (0.1 μg/μl), 2 μl BP buffer and 1.5 μl of BP clonase enzyme mix (Invitrogen) in a final volume of 10 μl at RT for 1.5 h. The reaction is stopped by addition of 1 μl proteinase K (2 μg/μl) and incubated at 37 0C for a further 10 min. An aliquot of this reaction (2 μl) is used to transform E. coli strain TOPlO (Invitrogen) as follows: a 50 μl aliquot of One Shot TOPlO cells is thawed on ice and 2 μl of reaction mixture added. The mixture is incubated for 30 min on ice and then heat shocked by incubation at 42 °C for exactly 30 s. Samples are returned to ice and 250 μl of warm SOC media (room temperature) is added. Samples are incubated with shaking (220 rpm) for 1 h at 37 °C. The transformation mixture is then plated on L-broth (LB) plates containing kanamycin (40 μg/ml) and incubated overnight at 37 0C. Resultant colonies are then inoculated into 1.3 ml of T-broth (TB) using a Qpix2 colony picking robot (Genetix), grown up overnight at 37 0C with shaking (220 rpm), and plasmid miniprep DNA is prepared using a Qiaprep BioRobot 8000 system (Qiagen) as described above. Plasmid DNA (150-200 ng) is subjected to DNA sequencing with typically 21M13 and M13Rev primers using the BigDyeTerminator system (Applied Biosystems cat. no. 4336919) according to the manufacturer's instructions. Sequencing reactions are purified using Montage SEQ 96 cleanup plates (Millipore cat. no. LSKS09624) then analyzed on an Applied Biosystems 3700 sequencer.
Typically, plasmid eluate (2 μl or approx. 150 ng) from one of the clones which contains the correct sequence is then used in a recombination reaction containing 1.5 μl of either pEAK12d vector or pDEST12.2 vector (0.1 μg / μl), 2 μl LR buffer and 1.5 μl of LR clonase (Invitrogen) in a final volume of 10 μl. The mixture is incubated at RT for 1 h, stopped by addition of proteinase K (2 μg) and incubated at 37 °C for a further 10 min. An aliquot of this reaction (2 ul) is used to transform E. coli strain TOPlO (Invitrogen) as described above. The transformation mixture is then plated on L-broth (LB) plates containing ampicillin (100 μg/ml) and incubated overnight at 37 0C.
Plasmid miniprep DNA is prepared from 5ml cultures from the resultant colonies subcloned into each vector using a Qiaprep BioRobot 8000 system (Qiagen). Plasmid
DNA (200-500 ng) in the pEAK12d vector is subjected to DNA sequencing with the sequencing primers pEAK12F and pEAK12R, and gene-specific primers INSP 172-SP 1,
INSP172-SP2, as described above. Plasmid DNA (200-500 ng) in the pDEST12.2 vector is subjected to DNA sequencing typically with the sequencing primers 21M13 and M13Rev, and gene-specific primers INSP172-SP1, INSP172-SP2, as described above.
DNA maxipreps CsCl gradient purified maxi-prep DNA is prepared from 500 ml cultures of sequence verified pEAK12d clones of INSP 172 using the method described by Sambrook J. et al., 1989 (in Molecular Cloning, a Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press). Plasmid DNA is resuspended at a concentration of 1 μg/μl in sterile water (or 10 mM Tris-HCl pH 8.5) and stored at -20 0C. Typically, endotoxin-free maxi-prep DNA is prepared from 500ml cultures of sequence verified pDEST12.2 clones of INSP172 using the EndoFree Plasmid Mega kit (Qiagen) according to the manufacturer's instructions. Purified plasmid DNA is resuspended in endotoxin free TE buffer at a final concentration of at least 3 μg/μl and stored at -20 0C.
Example 6: Expression and purification of INSP 172
Further experiments may now be performed to determine the tissue distribution and expression levels of the INSP 172 polypeptide in vivo, on the basis of the nucleotide and amino acid sequence disclosed herein.
The presence of the transcripts for INSP 172 may be investigated by PCR of cDNA from different human tissues. The INSP 172 transcripts may be present at very low levels in the samples tested. Therefore, extreme care is needed in the design of experiments to establish the presence of a transcript in various human tissues as a small amount of genomic contamination in the RNA preparation will provide a false positive result. Thus, all RNA should be treated with DNAse prior to use for reverse transcription. In addition, for each tissue a control reaction may be set up in which reverse transcription was not undertaken (a
-ve RT control).
For example, 1 μg of total RNA from each tissue may be used to generate cDNA using Multiscript reverse transcriptase (ABI) and random hexamer primers. For each tissue, a control reaction is set up in which all the constituents are added except the reverse transcriptase (-ve RT control). PCR reactions are set up for each tissue on the reverse transcribed RNA samples and the minus RT controls. INSP 172 specific primers may
readily be designed on the basis of the sequence information provided herein. The presence of a product of the correct molecular weight in the reverse transcribed sample together with the absence of a product in the minus RT control may be taken as evidence for the presence of a transcript in that tissue. Any suitable cDNA libraries may be used to screen for the INSP 172 transcripts, not only those generated as described above.
The tissue distribution pattern of the INSP 172 polypeptides will provide further useful information in relation to the function of those polypeptides.
In addition, further experiments may now be performed using expression vectors. Transfection of mammalian cell lines with these vectors may enable the high level expression of the INSP 172 proteins and thus enable the continued investigation of the functional characteristics of the INSP 172 polypeptides. The following material and methods are an example of those suitable in such experiments:
Cell Culture
Human Embryonic Kidney 293 cells expressing the Epstein-Barr virus Nuclear Antigen (HEK293-EBNA, Invitrogen) are maintained in suspension in Ex-cell VPRO serum-free medium (seed stock, maintenance medium, JRH). Sixteen to 20 hours prior to transfection (Day-1), cells are seeded in 2x T225 flasks (50ml per flask in DMEM / F12 (1:1) containing 2% FBS seeding medium (JRH) at a density of 2x105 cells/ml). The next day (transfection day 0) transfection takes place using the JetPEITM reagent (2μl/μg of plasmid DNA, PolyPlus-transfection). For each flask, plasmid DNA is co-transfected with GFP (fluorescent reporter gene) DNA. The transfection mix is then added to the 2xT225 flasks and incubated at 37°C (5%CO2) for 6 days. Confirmation of positive transfection may be carried out by qualitative fluorescence examination at day 1 and day 6 (Axiovert 10 Zeiss). On day 6 (harvest day), supernatants from the two flasks are pooled and centrifuged (e.g. 4°C, 40Og) and placed into a pot bearing a unique identifier. One aliquot (500μl) is kept for QC of the 6His-tagged protein (internal bioprocessing QC).
Scale-up batches may be produced by following the protocol called "PEI transfection of suspension cells", referenced BP/PEI/HH/02/04, with PolyEthylenelmine from Polysciences as transfection agent.
Purification process
The culture medium sample containing the recombinant protein with a C-terminal 6His tag is diluted with cold buffer A (5OmM NaH2PO4; 60OmM NaCl; 8.7 % (w/v) glycerol, pH 7.5). The sample is filtered then through a sterile filter (Millipore) and kept at 40C in a sterile square media bottle (Nalgene). The purification is performed at 40C on the VISION workstation (Applied Biosystems) connected to an automatic sample loader (Labomatic). The purification procedure is composed of two sequential steps, metal affinity chromatography on a Poros 20 MC (Applied Biosystems) column charged with Ni ions (4.6 x 50 mm, 0.83ml), followed by gel filtration on a Sephadex G-25 medium (Amersham Pharmacia) column (1,0 x 10cm). For the first chromatography step the metal affinity column is regenerated with 30 column volumes of EDTA solution (10OmM EDTA; IM NaCl; pH 8.0), recharged with Ni ions through washing with 15 column volumes of a 10OmM NiSO4 solution, washed with 10 column volumes of buffer A, followed by 7 column volumes of buffer B (5OmM NaH2PO4; 60OmM NaCl; 8.7 % (w/v) glycerol, 40OmM; imidazole, pH 7.5), and finally equilibrated with 15 column volumes of buffer A containing 15mM imidazole. The sample is transferred, by the Labomatic sample loader, into a 200ml sample loop and subsequently charged onto the Ni metal affinity column at a flow rate of 10ml/min. The column is washed with 12 column volumes of buffer A, followed by 28 column volumes of buffer A containing 2OmM imidazole. During the 2OmM imidazole wash loosely attached contaminating proteins are eluted from the column. The recombinant His-tagged protein is finally eluted with 10 column volumes of buffer B at a flow rate of 2ml/min, and the eluted protein is collected.
For the second chromatography step, the Sephadex G-25 gel-filtration column is regenerated with 2ml of buffer D (1.137M NaCl; 2.7mM KCl; 1.5mM KH2PO4; 8mM Na2HPO4; pH 7.2), and subsequently equilibrated with 4 column volumes of buffer C (137mM NaCl; 2.7mM KCl; 1.5mM KH2PO4; 8mM Na2HPO4; 20% (w/v) glycerol; pH 7.4). The peak fraction eluted from the Ni-column is automatically loaded onto the Sephadex G-25 column through the integrated sample loader on the VISION and the protein is eluted with buffer C at a flow rate of 2 ml/min. The fraction was filtered through a sterile centrifugation filter (Millipore), frozen and stored at -800C. An aliquot of the sample is analyzed on SDS-PAGE (4-12% NuPAGE gel; Novex) Western blot with anti- His antibodies. The NuPAGE gel may be stained in a 0.1 % Coomassie blue R250 staining
solution (30% methanol, 10% acetic acid) at room temperature for Ih and subsequently destained in 20% methanol, 7.5% acetic acid until the background is clear and the protein bands clearly visible.
Following the electrophoresis the proteins are electrotransferred from the gel to a nitrocellulose membrane. The membrane is blocked with 5% milk powder in buffer E (137mM NaCl; 2.7mM KCl; 1.5mM KH2PO4; 8mM Na2HPO4; 0.1 % Tween 20, pH 7.4) for Ih at room temperature, and subsequently incubated with a mixture of 2 rabbit polyclonal anti-His antibodies (G-18 and H-15, 0.2μg/ml each; Santa Cruz) in 2.5% milk powder in buffer E overnight at 40C. After a further 1 hour incubation at room temperature, the membrane is washed with buffer E (3 x lOmin), and then incubated with a secondary HRP-conjugated anti-rabbit antibody (DAKO, HRP 0399) diluted 1/3000 in buffer E containing 2.5% milk powder for 2 hours at room temperature. After washing with buffer E (3 x 10 minutes), the membrane is developed with the ECL kit (Amersham Pharmacia) for 1 min. The membrane is subsequently exposed to a Hyperfilm (Amersham Pharmacia), the film developed and the western blot image visually analysed.
For samples that showed detectable protein bands by Coomassie staining, the protein concentration may be determined using the BCA protein assay kit (Pierce) with bovine serum albumin as standard.
Furthermore, overexpression or knock-down of the expression of the polypeptides in cell lines may be used to determine the effect on transcriptional activation of the host cell genome. Dimerisation partners, co-activators and co-repressors of the INSP 172 polypeptides may be identified by immunoprecipitation combined with Western blotting and immunoprecipitation combined with mass spectroscopy.
Example 7: Microarray studies
Custom microarrays have been manufactured using Agilent Technologies' (Agilent Technologies Inc, Palo Alto, CA) non-contact in situ synthesis process of printing 60-mer length oligonucleotide probes, base-by-base, from digital sequence files. This is achieved with an inkjet process which delivers extremely small, accurate volumes (picoliters) of the chemicals to be spotted. Standard phosphoramidite chemistry used in the reactions allows for very high coupling efficiencies to be maintained at each step in the synthesis of the full-length oligonucleotide. Precise quantities are reproducibly deposited "on the fly."
This engineering feat is achieved without stopping to make contact with the slide surface and without introducing surface-contact feature anomalies, resulting in consistent spot uniformity and traceability. (Hughes et ah (2001) Nat. Biotech. Apr; 19(4): 342-7. Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer). Probe Synthesis
Methodologies were carried out according to Agilent instructions. Essentially, cDNA synthesis and subsequent T7 polymerase amplification of Cyanine 3(5)-CTP labeled cRNA probe was carried out using Agilent's low RNA input fluorescent linear amplification kit from a template of 5μιg of total RNA according to the kit protocol (version 2 August 2003, Agilent, Palo Alto, CA). cRNA is then fragmented using Agilent's In Situ hybridization kit-plus and hybridized both according to Agilent's protocol (Agilent 60-mer oligo microarray processing protocol version 4.1 April 2004, Agilent, Palo Alto, CA). Microarray Chip Design • 10,536 probes are on the array
• 5557 of the probes designed specifically to detect secreted sequences of primary interest
• 1000 probes designed as negative controls
• 500 probes designed as positive controls • Remainder of the probes were designed to public domain sequences which are known to be either secreted soluble extracellular proteins or membrane bound proteins with an extracellular domain in contact with the extracellular milieu. Studies specific for INSPl 72
INSP 172 is formed from separate component exons. We intend to profile the chips using probe synthesized from 10 normal tissues, bone marrow, brain, lung, ovary, PBMCs, placenta, prostate, spleen and testis. Expression reports are obtainable on an exon by exon basis.
Averaging is performed for the data, using the One-step Tukey Bi- Weight Algorithm (Data Analysis and Regression: A Second Course in Statistics", Mosteller and Tukey, Addison- Wesley, 1977, pp. 203-209; see also Affymetrix MAS5.0 algorithm). The purpose of this is to define a robust estimate of the average value of a dataset. In this case our datasets will comprise multiple probe expression values for a single exon.
This custom array is useful for a number of reasons. First, it allows the existence and sequence of the transcript to be confirmed. Second, the tissue distribution of the INSP 172 polypeptide sequence can be evaluated and thus the role of this polypeptide in disease can be clarified. The array can also be used as a diagnostic tool, to diagnose disease incidence in patients with disease conditions with which this polypeptide is correlated. The use of exon-specifϊc probes allows any variance in expression of splice variants of this polypeptide sequence to be evaluated, in general, in specific tissues and in specific disease states.
Claims
1. A polypeptide, which polypeptide:
(i) comprises the amino acid sequence as recited in SEQ ID NO:2;
(ii) is a fragment thereof that contains a fibronectin type III domain and/or has an antigenic determinant in common with the polypeptides of (i); or
(iii) is a functional equivalent of (i) or (ii).
2. A polypeptide which is a functional equivalent according to part (iii) of claim 1, characterised in that it is homologous to the amino acid sequence as recited in SEQ ID NO:2.
3. A fragment or functional equivalent according to part (ii) of claim 1, which has greater than 50% sequence identity with the amino acid sequence recited SEQ ID NO:2 or with active fragments thereof, preferably greater than 60%, 70%, 80%, 90%, 95%, 98% or 99% sequence identity.
4. A functional equivalent according to any one of the preceding claims, which exhibits significant structural homology with a polypeptide having the amino acid sequence given in SEQ IDNO:2.
5. A fragment as recited in any one of the preceding claims, having an antigenic determinant in common with the polypeptide of part (i) of claim 1 which consists of 7 or more (for example, 8, 10, 12, 14, 16, 18, 20 or more) amino acid residues from the sequence of SEQ ID NO:2.
6. A fusion protein comprising the polypeptide according to any one of the preceding claims.
7. The polypeptide of claim 6, wherein said polypeptide comprises a histidine tag.
8. The polypeptide of claim 7, whose sequence is recited in SEQ ID NO: 4.
9. The polypeptide of any one of the preceding claims, wherein said polypeptide comprises a signal peptide.
10. The polypeptide of claim 9, whose sequence is recited in SEQ ID NO:6 or SEQ ID NO:8.
11. A purified nucleic acid molecule which encodes a polypeptide according to any one of the preceding claims.
12. A purified nucleic acid molecule according to claim 11, which comprises or consists of the nucleic acid sequence as recited in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7.
5 13. A purified nucleic acid molecule according to claims 11 or 12 which consists of the nucleic acid sequence as recited in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO: 7, or is a redundant equivalent or fragment thereof.
14. A purified nucleic acid molecule which hybridizes under high stringency conditions with a nucleic acid molecule according any one of claims 11 to 13.
10 15. A vector comprising a nucleic acid molecule as recited in any one of claims 11 to 14.
16. A host cell transformed with a vector according to claim 15.
17. A ligand which binds specifically to, and preferably modulates the activity of, a polypeptide according to any one of claims 1 to 5.
18. A ligand according to claim 17, which is an antibody.
15 19. A compound that either increases or decreases the level of expression or activity of a polypeptide according to any one of claims 1 to 10.
20. A compound according to claim 19 that binds to a polypeptide according to any one of claims 1 to 10 without inducing any of the biological effects of the polypeptide.
21. A compound according to claim 20, which is a natural or modified substrate, ligand, 20 enzyme, receptor or structural or functional mimetic.
22. A polypeptide according to any one of claims 1 to 10, a nucleic acid molecule according to any one of claims 11 to 14, a vector according to claim 15, a host cell according to claim 16, a ligand according to claim 17 or claim 18, or a compound according to any one of claims 19 to 21, for use in therapy or diagnosis of disease.
25 23. A method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide according to any one of claims 1 to 10, or assessing the activity of a polypeptide according to any one of claims 1 to 10, in tissue from said patient and comparing said level of expression or activity to a control level, wherein a level that is different to said control level is indicative of
30 disease.
24. A method according to claim 23 that is carried out in vitro.
25. A method according to claim 23 or claim 24, which comprises the steps of: (a) contacting a ligand according to claim 17 or claim 18 with a biological sample under conditions suitable for the formation of a ligand-polypeptide complex; and (b) detecting said complex.
26. A method according to claim 23 or claim 24, comprising the steps of: a) contacting a sample of tissue from the patient with a nucleic acid probe under stringent conditions that allow the formation of a hybrid complex between a nucleic acid molecule according to any one of claims 11 to 14 and the probe; b) contacting a control sample with said probe under the same conditions used in step a); and c) detecting the presence of hybrid complexes in said samples; wherein detection of levels of the hybrid complex in the patient sample that differ from levels of the hybrid complex in the control sample is indicative of disease.
27. A method according to claim 23 or claim 24, comprising: a) contacting a sample of nucleic acid from tissue of the patient with a nucleic acid primer under stringent conditions that allow the formation of a hybrid complex between a nucleic acid molecule according to any one of claims 11 to 14 and the primer; b) contacting a control sample with said primer under the same conditions used in step a); and c) amplifying the sampled nucleic acid; and d) detecting the level of amplified nucleic acid from both patient and control samples; wherein detection of levels of the amplified nucleic acid in the patient sample that differ significantly from levels of the amplified nucleic acid in the control sample is indicative of disease.
28. A method according to claim 23 or claim 24 comprising: a) obtaining a tissue sample from a patient being tested for disease; b) isolating a nucleic acid molecule according to any one of claims 11 to 14 from said tissue sample; and c) diagnosing the patient for disease by detecting the presence of a mutation which is associated with disease in the nucleic acid molecule as an indication of the disease.
29. The method of claim 28, further comprising amplifying the nucleic acid molecule to 5 form an amplified product and detecting the presence or absence of a mutation in the amplified product.
30. The method of either claim 28 or 29, wherein the presence or absence of the mutation in the patient is detected by contacting said nucleic acid molecule with a nucleic acid probe that hybridises to said nucleic acid molecule under stringent conditions to form a
10 hybrid double-stranded molecule, the hybrid double-stranded molecule having an unhybridised portion of the nucleic acid probe strand at any portion corresponding to a mutation associated with disease; and detecting the presence or absence of an unhybridised portion of the probe strand as an indication of the presence or absence of a disease-associated mutation.
15 31. A method according to any one of claims 23-30, wherein said disease includes, but is not limited to, neoplasm, cancer, brain tumour, glioma, bone tumor, lung tumor, breast tumour, prostate tumour, colon tumour, hemangioma, myeloproliferative disorder, leukemia, hematological disease, neutropenia, thrombocytopenia, angiogenesis disorders, dermatological disease, ageing, wounds, burns, fibrosis, cardiovascular 0 disease, restensosis, heart disease, peripheral vascular disease, coronary artery disease, oedema, thromboembolism, dysmenorrhea, endometriosis, pre-eclampsia, lung disease, COPD, asthma bone disease, renal disease, glomerulonephritis, liver disease, Crohn's disease, gastritis, ulcerative colitis, ulcer, immune disorder, autoimmune disease, arthritis, rheumatoid arthritis, psoriasis, epidermolysis bullosa, systemic lupus 5 erythematosus, ankylosing spondylitis, Lyme disease, multiple sclerosis, neurodegeneration, stroke, brain/spinal cord injury, Alzheimer's disease, Parkinson's disease, motor neurone disease, neuromuscular disease, HIV, AIDS, cytomegalovirus infection, fungal infection, ocular disorder, macular degeneration, glaucoma, diabetic retinopathy and ocular hypertension. 0 32. Use of a polypeptide according to any one of claims 1 to 10 as a protein involved in protein-protein interactions.
33. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 10, a nucleic acid molecule according to any one of claims 11 to 14, a vector according to claim 15, a host cell according to claim 10, a ligand according to claim 17 or claim 18, or a compound according to any one of claims 19 to 21.
34. A vaccine composition comprising a polypeptide according to any one of claims 1 to 10 or a nucleic acid molecule according to any one of claims 11 to 14.
35. A polypeptide according to any one of claims 1 to 10, a nucleic acid molecule according to any one of claims 11 to 14, a vector according to claim 15, a host cell according to claim 16, a ligand according to claim 17 or claim 18, a compound according to any one of claims 19 to 21, or a pharmaceutical composition according to claim 33, for use in the manufacture of a medicament for the treatment of a certain disease including, but not limited to, neoplasm, cancer, brain tumour, glioma, bone tumor, lung tumor, breast tumour, prostate tumour, colon tumour, hemangioma, myeloproliferative disorder, leukemia, hematological disease, neutropenia, thrombocytopenia, angiogenesis disorders, dermatological disease, ageing, wounds, burns, fibrosis, cardiovascular disease, restensosis, heart disease, peripheral vascular disease, coronary artery disease, oedema, thromboembolism, dysmenorrhea, endometriosis, pre-eclampsia, lung disease, COPD, asthma bone disease, renal disease, glomerulonephritis, liver disease, Crohn's disease, gastritis, ulcerative colitis, ulcer, immune disorder, autoimmune disease, arthritis, rheumatoid arthritis, psoriasis, epidermolysis bullosa, systemic lupus erythematosus, ankylosing spondylitis, Lyme disease, multiple sclerosis, neurodegeneration, stroke, brain/spinal cord injury, Alzheimer's disease, Parkinson's disease, motor neurone disease, neuromuscular disease, HIV, AIDS, cytomegalovirus infection, fungal infection, ocular disorder, macular degeneration, glaucoma, diabetic retinopathy and ocular hypertension.
36. A method of treating a disease in a patient, comprising administering to the patient a polypeptide according to any one of claims 1 to 10, a nucleic acid molecule according to any one of claims 11 to 14, a vector according to claim 15, a host cell according to claim 16, a ligand according to claim 17 or claim 18, a compound according to any one of claims 19 to 21, or a pharmaceutical composition according to claim 33.
37. A method according to claim 36, wherein, for diseases in which the expression of the natural gene or the activity of the polypeptide is lower in a diseased patient when compared to the level of expression or activity in a healthy patient, the polypeptide, nucleic acid molecule, vector, host cell, ligand, compound or composition administered to the patient is an agonist.
38. A method according to claim 36, wherein, for diseases in which the expression of the 5 natural gene or activity of the polypeptide is higher in a diseased patient when compared to the level of expression or activity in a healthy patient, the polypeptide, nucleic acid molecule, vector, host cell, ligand, compound or composition administered to the patient is an antagonist.
39. A method of monitoring the therapeutic treatment of disease in a patient, comprising 10 monitoring over a period of time the level of expression or activity of a polypeptide according to any one of claims 1 to 10, or the level of expression of a nucleic acid molecule according to any one of claims 11 to 14 in tissue from said patient, wherein altering said level of expression or activity over the period of time towards a control level is indicative of regression of said disease.
15 40. A method for the identification of a compound that is effective in the treatment and/or diagnosis of disease, comprising contacting a polypeptide according to any one of claims 1 to 10, or a nucleic acid molecule according to any one of claims 11 to 14 with one or more compounds suspected of possessing binding affinity for said polypeptide or nucleic acid molecule, and selecting a compound that binds specifically to said
20 nucleic acid molecule or polypeptide.
41. A kit useful for diagnosing disease comprising a first container containing a nucleic acid probe that hybridises under stringent conditions with a nucleic acid molecule according to any one of claims 11 to 14; a second container containing primers useful for amplifying said nucleic acid molecule; and instructions for using the probe and
25 primers for facilitating the diagnosis of disease.
42. The kit of claim 41, further comprising a third container holding an agent for digesting unhybridised RNA.
43. A kit comprising an array of nucleic acid molecules, at least one of which is a nucleic acid molecule according to any one of claims 11 to 14.
30 44. A kit comprising one or more antibodies that bind to a polypeptide as recited in any one of claims 1 to 10; and a reagent useful for the detection of a binding reaction between said antibody and said polypeptide.
45. A transgenic or knockout non-human animal that has been transformed to express higher, lower or absent levels of a polypeptide according to any one of claims 1 to 10.
46. A method for screening for a compound effective to treat disease, by contacting a non- human transgenic animal according to claim 45 with a candidate compound and determining the effect of the compound on the disease of the animal.
47. The use of an INSP 172 polypeptide as a target for screening candidate drugs for treating or preventing a FN3 domain containing protein related disorder.
48. Method of selecting biologically active compounds comprising: (i) contacting a candidate compound with recombinant host cells expressing an
INSP172 polypeptide;
(ii) selecting compounds that bind said INSP 172 polypeptide at the surface of said cells and/or that modulate the activity of the INSP 172 polypeptide.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB0606545.2 | 2006-03-31 | ||
GB0606545A GB0606545D0 (en) | 2006-03-31 | 2006-03-31 | Fibronectin type 111 domain containing protein |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2007113488A1 true WO2007113488A1 (en) | 2007-10-11 |
Family
ID=36425030
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB2007/001109 WO2007113488A1 (en) | 2006-03-31 | 2007-03-28 | Fibronectin type iii domain containing protein |
Country Status (2)
Country | Link |
---|---|
GB (1) | GB0606545D0 (en) |
WO (1) | WO2007113488A1 (en) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001069507A2 (en) * | 2000-03-14 | 2001-09-20 | Inpharmatica Limited | Proteomics database |
-
2006
- 2006-03-31 GB GB0606545A patent/GB0606545D0/en not_active Ceased
-
2007
- 2007-03-28 WO PCT/GB2007/001109 patent/WO2007113488A1/en active Application Filing
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001069507A2 (en) * | 2000-03-14 | 2001-09-20 | Inpharmatica Limited | Proteomics database |
Non-Patent Citations (3)
Title |
---|
DATABASE EMBL [online] 18 December 2002 (2002-12-18), "Mus musculus 2 days pregnant adult female oviduct cDNA, RIKEN full-length enriched library, clone:E230011A21 product:hypothetical Fibronectin type III domain/ATP/GTP-binding site motif A (P-loop) containing protein, full insert sequence.", XP002435032, retrieved from EBI accession no. EMBL:AK053989 Database accession no. AK053989 * |
DATABASE UniProt [online] 7 December 2004 (2004-12-07), "Fibronectin type III domain-containing protein 7.", XP002435031, retrieved from EBI accession no. UNIPROT:Q5VTL7 Database accession no. Q5VTL7 * |
LITJENS SANDY H M ET AL: "Modeling and experimental validation of the binary complex of the plectin actin-binding domain and the first pair of fibronectin type III (FNIII) domains of the beta 4 integrin", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 280, no. 23, June 2005 (2005-06-01), pages 22270 - 22277, XP002435028, ISSN: 0021-9258 * |
Also Published As
Publication number | Publication date |
---|---|
GB0606545D0 (en) | 2006-05-10 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU2006221859B2 (en) | Lipocalin protein | |
US20080196113A1 (en) | Lipocalin Protein | |
EP1499640A2 (en) | Immunoglobulin-domain containing cell surface recognition molecules | |
WO2007007116A1 (en) | Defensin proteins | |
WO2003054004A2 (en) | Secreted proteins | |
WO2007072012A1 (en) | Novel members of the kazal family of serine protease inhibitors | |
WO2008047111A1 (en) | Immunoglobulin domain-containing cell surface recognition molecules | |
AU2004203966B2 (en) | Defensin proteins | |
WO2006043060A2 (en) | Mam domain containing protein | |
US20090215672A1 (en) | Cys-Rich, Cell Surface Glycoproteins | |
EP1824881A1 (en) | Tgr3-like protein receptor | |
EP1973936A1 (en) | Reeler domain containing protein | |
WO2007049065A2 (en) | Vwfa, collagen and kunitz domain containing protein | |
WO2003099865A1 (en) | Cation channel proteins | |
WO2007148063A1 (en) | Cd24-like protein | |
WO2007068913A2 (en) | SRCR-B Domain Containing Proteins | |
WO2007113488A1 (en) | Fibronectin type iii domain containing protein | |
WO2007060425A1 (en) | Thrombospondin domain-containing cell surface recognition molecules | |
WO2008001045A1 (en) | Members of the glycoside hydrolase family 31 family of proteins | |
WO2007049062A2 (en) | Vwfa-domain containing proteins | |
WO2007060423A1 (en) | Signal anchored protein | |
WO2007060431A1 (en) | Netrin-like (ntr) domain containing protein | |
WO2007045911A1 (en) | Integral membrane protein | |
WO2008017831A1 (en) | Secreted vit domain containing proteins | |
WO2004009624A2 (en) | Three finger toxin fold protein |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 07732167 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 07732167 Country of ref document: EP Kind code of ref document: A1 |