US20130171130A1 - Soluble interleukin-20 receptor - Google Patents
Soluble interleukin-20 receptor Download PDFInfo
- Publication number
- US20130171130A1 US20130171130A1 US13/525,466 US201213525466A US2013171130A1 US 20130171130 A1 US20130171130 A1 US 20130171130A1 US 201213525466 A US201213525466 A US 201213525466A US 2013171130 A1 US2013171130 A1 US 2013171130A1
- Authority
- US
- United States
- Prior art keywords
- seq
- receptor
- polypeptide
- cells
- amino acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 108010001618 interleukin-20 receptor Proteins 0.000 title claims description 28
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 152
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 142
- 229920001184 polypeptide Polymers 0.000 claims abstract description 139
- 101001044895 Homo sapiens Interleukin-20 receptor subunit beta Proteins 0.000 claims abstract description 96
- 102100022705 Interleukin-20 receptor subunit beta Human genes 0.000 claims abstract description 96
- 102100022706 Interleukin-20 receptor subunit alpha Human genes 0.000 claims abstract description 83
- 101710174006 Interleukin-20 receptor subunit alpha Proteins 0.000 claims abstract description 80
- 238000000034 method Methods 0.000 claims description 66
- 201000004681 Psoriasis Diseases 0.000 claims description 37
- 230000027455 binding Effects 0.000 claims description 26
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 21
- 239000012634 fragment Substances 0.000 claims description 15
- 208000027866 inflammatory disease Diseases 0.000 claims description 8
- 230000002757 inflammatory effect Effects 0.000 claims description 7
- 201000004624 Dermatitis Diseases 0.000 claims description 5
- 206010039073 rheumatoid arthritis Diseases 0.000 claims description 5
- 208000019693 Lung disease Diseases 0.000 claims description 4
- 239000000427 antigen Substances 0.000 claims description 3
- 108091007433 antigens Proteins 0.000 claims description 3
- 102000036639 antigens Human genes 0.000 claims description 3
- 208000006673 asthma Diseases 0.000 claims description 3
- 206010006451 bronchitis Diseases 0.000 claims description 3
- 201000003883 Cystic fibrosis Diseases 0.000 claims description 2
- 206010012438 Dermatitis atopic Diseases 0.000 claims description 2
- 206010012442 Dermatitis contact Diseases 0.000 claims description 2
- 208000022559 Inflammatory bowel disease Diseases 0.000 claims description 2
- 201000008937 atopic dermatitis Diseases 0.000 claims description 2
- 208000010668 atopic eczema Diseases 0.000 claims description 2
- 208000010247 contact dermatitis Diseases 0.000 claims description 2
- 102000004114 interleukin 20 Human genes 0.000 abstract description 133
- 108090000681 interleukin 20 Proteins 0.000 abstract description 133
- 102000005962 receptors Human genes 0.000 abstract description 71
- 108020003175 receptors Proteins 0.000 abstract description 71
- 101001044893 Homo sapiens Interleukin-20 receptor subunit alpha Proteins 0.000 abstract description 14
- 108060003951 Immunoglobulin Proteins 0.000 abstract description 12
- 102000018358 immunoglobulin Human genes 0.000 abstract description 12
- 210000004027 cell Anatomy 0.000 description 134
- 108090000623 proteins and genes Proteins 0.000 description 62
- 108020004414 DNA Proteins 0.000 description 44
- 235000018102 proteins Nutrition 0.000 description 36
- 102000004169 proteins and genes Human genes 0.000 description 36
- 125000000539 amino acid group Chemical group 0.000 description 34
- 239000013598 vector Substances 0.000 description 32
- 108010076504 Protein Sorting Signals Proteins 0.000 description 26
- 230000014509 gene expression Effects 0.000 description 26
- 102000040430 polynucleotide Human genes 0.000 description 26
- 108091033319 polynucleotide Proteins 0.000 description 26
- 239000002157 polynucleotide Substances 0.000 description 26
- 230000003248 secreting effect Effects 0.000 description 26
- 108091028043 Nucleic acid sequence Proteins 0.000 description 20
- 230000001105 regulatory effect Effects 0.000 description 19
- 210000003491 skin Anatomy 0.000 description 19
- 108020004705 Codon Proteins 0.000 description 18
- 230000004927 fusion Effects 0.000 description 18
- 210000002510 keratinocyte Anatomy 0.000 description 18
- 102000000589 Interleukin-1 Human genes 0.000 description 17
- 108010002352 Interleukin-1 Proteins 0.000 description 17
- 235000001014 amino acid Nutrition 0.000 description 17
- 102000004127 Cytokines Human genes 0.000 description 16
- 108090000695 Cytokines Proteins 0.000 description 16
- 239000005557 antagonist Substances 0.000 description 15
- 239000002299 complementary DNA Substances 0.000 description 15
- 229940024606 amino acid Drugs 0.000 description 14
- 150000001413 amino acids Chemical class 0.000 description 14
- 239000013604 expression vector Substances 0.000 description 14
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 13
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 13
- 241001452677 Ogataea methanolica Species 0.000 description 13
- 108020004999 messenger RNA Proteins 0.000 description 13
- 239000013612 plasmid Substances 0.000 description 13
- 108010017324 STAT3 Transcription Factor Proteins 0.000 description 12
- 102100024040 Signal transducer and activator of transcription 3 Human genes 0.000 description 12
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 12
- 241000701447 unidentified baculovirus Species 0.000 description 12
- 230000004913 activation Effects 0.000 description 11
- 239000003446 ligand Substances 0.000 description 11
- 230000004044 response Effects 0.000 description 11
- 241000699666 Mus <mouse, genus> Species 0.000 description 10
- 238000003556 assay Methods 0.000 description 10
- 230000000295 complement effect Effects 0.000 description 10
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 10
- 230000000694 effects Effects 0.000 description 10
- 239000000047 product Substances 0.000 description 10
- 238000012408 PCR amplification Methods 0.000 description 9
- 108020001507 fusion proteins Proteins 0.000 description 9
- 102000037865 fusion proteins Human genes 0.000 description 9
- 238000004519 manufacturing process Methods 0.000 description 9
- 241000201370 Autographa californica nucleopolyhedrovirus Species 0.000 description 8
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 8
- 241000588724 Escherichia coli Species 0.000 description 8
- 102100029567 Immunoglobulin kappa light chain Human genes 0.000 description 8
- 101710189008 Immunoglobulin kappa light chain Proteins 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- 239000003814 drug Substances 0.000 description 8
- 239000000499 gel Substances 0.000 description 8
- 238000000746 purification Methods 0.000 description 8
- 241000894007 species Species 0.000 description 8
- 238000013518 transcription Methods 0.000 description 8
- 230000035897 transcription Effects 0.000 description 8
- 238000012546 transfer Methods 0.000 description 8
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 7
- 241000699660 Mus musculus Species 0.000 description 7
- 108091034117 Oligonucleotide Proteins 0.000 description 7
- 201000010099 disease Diseases 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 210000004185 liver Anatomy 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- 239000002773 nucleotide Substances 0.000 description 7
- 125000003729 nucleotide group Chemical group 0.000 description 7
- 230000037361 pathway Effects 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- 238000011830 transgenic mouse model Methods 0.000 description 7
- 239000003155 DNA primer Substances 0.000 description 6
- 239000004471 Glycine Substances 0.000 description 6
- 241000238631 Hexapoda Species 0.000 description 6
- 102000006496 Immunoglobulin Heavy Chains Human genes 0.000 description 6
- 108010019476 Immunoglobulin Heavy Chains Proteins 0.000 description 6
- 108090001007 Interleukin-8 Proteins 0.000 description 6
- 102000004890 Interleukin-8 Human genes 0.000 description 6
- 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 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 239000011324 bead Substances 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 6
- 108010030074 endodeoxyribonuclease MluI Proteins 0.000 description 6
- 208000015181 infectious disease Diseases 0.000 description 6
- 230000003993 interaction Effects 0.000 description 6
- 210000004962 mammalian cell Anatomy 0.000 description 6
- 239000003550 marker Substances 0.000 description 6
- 229960000485 methotrexate Drugs 0.000 description 6
- 239000002953 phosphate buffered saline Substances 0.000 description 6
- 239000013615 primer Substances 0.000 description 6
- 230000001185 psoriatic effect Effects 0.000 description 6
- 230000010076 replication Effects 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 230000019491 signal transduction Effects 0.000 description 6
- 238000006467 substitution reaction Methods 0.000 description 6
- 230000014616 translation Effects 0.000 description 6
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 5
- 108091026890 Coding region Proteins 0.000 description 5
- 108091029865 Exogenous DNA Proteins 0.000 description 5
- 102100026215 Immunoglobulin gamma-1 heavy chain Human genes 0.000 description 5
- 101710111858 Immunoglobulin gamma-1 heavy chain Proteins 0.000 description 5
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 5
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 5
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 5
- 101001044894 Mus musculus Interleukin-20 receptor subunit alpha Proteins 0.000 description 5
- 101710182846 Polyhedrin Proteins 0.000 description 5
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 5
- 108090000373 Tissue Plasminogen Activator Proteins 0.000 description 5
- 102000003978 Tissue Plasminogen Activator Human genes 0.000 description 5
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 5
- 235000004279 alanine Nutrition 0.000 description 5
- 230000002391 anti-complement effect Effects 0.000 description 5
- 108010008730 anticomplement Proteins 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 238000010367 cloning Methods 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 102000003675 cytokine receptors Human genes 0.000 description 5
- 108010057085 cytokine receptors Proteins 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 238000000605 extraction Methods 0.000 description 5
- 239000000833 heterodimer Substances 0.000 description 5
- 210000002865 immune cell Anatomy 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 210000004072 lung Anatomy 0.000 description 5
- 230000001404 mediated effect Effects 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 230000035772 mutation Effects 0.000 description 5
- 230000028327 secretion Effects 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 229960000187 tissue plasminogen activator Drugs 0.000 description 5
- 230000001131 transforming effect Effects 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 102000016938 Catalase Human genes 0.000 description 4
- 108010053835 Catalase Proteins 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 4
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 102100032420 Protein S100-A9 Human genes 0.000 description 4
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 4
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 4
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 4
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- 210000004899 c-terminal region Anatomy 0.000 description 4
- 235000018417 cysteine Nutrition 0.000 description 4
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 4
- 230000001086 cytosolic effect Effects 0.000 description 4
- 125000002228 disulfide group Chemical group 0.000 description 4
- 210000002889 endothelial cell Anatomy 0.000 description 4
- 210000002615 epidermis Anatomy 0.000 description 4
- 239000012091 fetal bovine serum Substances 0.000 description 4
- 238000009396 hybridization Methods 0.000 description 4
- 238000003780 insertion Methods 0.000 description 4
- 230000037431 insertion Effects 0.000 description 4
- 230000003902 lesion Effects 0.000 description 4
- 238000002703 mutagenesis Methods 0.000 description 4
- 231100000350 mutagenesis Toxicity 0.000 description 4
- 210000001672 ovary Anatomy 0.000 description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 4
- 229920005989 resin Polymers 0.000 description 4
- 239000011347 resin Substances 0.000 description 4
- 208000017520 skin disease Diseases 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 210000001550 testis Anatomy 0.000 description 4
- 230000002103 transcriptional effect Effects 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 230000009261 transgenic effect Effects 0.000 description 4
- 241000701161 unidentified adenovirus Species 0.000 description 4
- 241001515965 unidentified phage Species 0.000 description 4
- 239000004474 valine Substances 0.000 description 4
- 229920000936 Agarose Polymers 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 102000005701 Calcium-Binding Proteins Human genes 0.000 description 3
- 108010045403 Calcium-Binding Proteins Proteins 0.000 description 3
- 241000701022 Cytomegalovirus Species 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 101001010591 Homo sapiens Interleukin-20 Proteins 0.000 description 3
- 206010020649 Hyperkeratosis Diseases 0.000 description 3
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 3
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 3
- 102000014150 Interferons Human genes 0.000 description 3
- 108010050904 Interferons Proteins 0.000 description 3
- 102100020881 Interleukin-1 alpha Human genes 0.000 description 3
- 108010082786 Interleukin-1alpha Proteins 0.000 description 3
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 3
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 3
- 239000005089 Luciferase Substances 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 3
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 3
- 108010044012 STAT1 Transcription Factor Proteins 0.000 description 3
- 108700025832 Serum Response Element Proteins 0.000 description 3
- 241000256251 Spodoptera frugiperda Species 0.000 description 3
- 108700019146 Transgenes Proteins 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000000692 anti-sense effect Effects 0.000 description 3
- 238000003491 array Methods 0.000 description 3
- 229940098773 bovine serum albumin Drugs 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 230000004069 differentiation Effects 0.000 description 3
- 231100000673 dose–response relationship Toxicity 0.000 description 3
- 238000004520 electroporation Methods 0.000 description 3
- 239000003623 enhancer Substances 0.000 description 3
- 210000003527 eukaryotic cell Anatomy 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 235000013922 glutamic acid Nutrition 0.000 description 3
- 239000004220 glutamic acid Substances 0.000 description 3
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 3
- 239000008187 granular material Substances 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 239000003102 growth factor Substances 0.000 description 3
- 210000002216 heart Anatomy 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000004054 inflammatory process Effects 0.000 description 3
- 229940047124 interferons Drugs 0.000 description 3
- 238000001990 intravenous administration Methods 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- 229960003136 leucine Drugs 0.000 description 3
- 210000002540 macrophage Anatomy 0.000 description 3
- 229910021645 metal ion Inorganic materials 0.000 description 3
- 230000005937 nuclear translocation Effects 0.000 description 3
- 210000004940 nucleus Anatomy 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 210000001322 periplasm Anatomy 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 238000000159 protein binding assay Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 210000000813 small intestine Anatomy 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 210000002784 stomach Anatomy 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 230000000699 topical effect Effects 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 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 2
- 229930024421 Adenine Natural products 0.000 description 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 2
- 101100437118 Arabidopsis thaliana AUG1 gene Proteins 0.000 description 2
- 101100437119 Arabidopsis thaliana AUG2 gene Proteins 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 2
- 102000004219 Brain-derived neurotrophic factor Human genes 0.000 description 2
- 108090000715 Brain-derived neurotrophic factor Proteins 0.000 description 2
- 108010052500 Calgranulin A Proteins 0.000 description 2
- 108010052495 Calgranulin B Proteins 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 108700010070 Codon Usage Proteins 0.000 description 2
- 241000699802 Cricetulus griseus Species 0.000 description 2
- 208000011231 Crohn disease Diseases 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 108010067193 Formaldehyde transketolase Proteins 0.000 description 2
- 108090000698 Formate Dehydrogenases Proteins 0.000 description 2
- KOSRFJWDECSPRO-WDSKDSINSA-N Glu-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(O)=O KOSRFJWDECSPRO-WDSKDSINSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 102000004457 Granulocyte-Macrophage Colony-Stimulating Factor Human genes 0.000 description 2
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 2
- 101001054732 Homo sapiens Inhibin beta A chain Proteins 0.000 description 2
- 102000013463 Immunoglobulin Light Chains Human genes 0.000 description 2
- 108010065825 Immunoglobulin Light Chains Proteins 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 102100027004 Inhibin beta A chain Human genes 0.000 description 2
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 description 2
- 102100040445 Keratin, type I cytoskeletal 14 Human genes 0.000 description 2
- 241000235058 Komagataella pastoris Species 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 2
- 108060001084 Luciferase Proteins 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 102000018697 Membrane Proteins Human genes 0.000 description 2
- 108010052285 Membrane Proteins Proteins 0.000 description 2
- 101100072406 Mus musculus Il20 gene Proteins 0.000 description 2
- 101001010594 Mus musculus Interleukin-20 Proteins 0.000 description 2
- 101000969137 Mus musculus Metallothionein-1 Proteins 0.000 description 2
- 238000000636 Northern blotting Methods 0.000 description 2
- 206010033733 Papule Diseases 0.000 description 2
- 102100027330 Phosphoribosylaminoimidazole carboxylase Human genes 0.000 description 2
- 101710093543 Probable non-specific lipid-transfer protein Proteins 0.000 description 2
- 102100032442 Protein S100-A8 Human genes 0.000 description 2
- 239000013614 RNA sample Substances 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 206010039509 Scab Diseases 0.000 description 2
- 102100029904 Signal transducer and activator of transcription 1-alpha/beta Human genes 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 101001095983 Staphylococcus aureus Protein rep Proteins 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 2
- 239000004473 Threonine Substances 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 2
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 2
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 2
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 2
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 2
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 2
- 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 2
- 230000005856 abnormality Effects 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 229960000643 adenine Drugs 0.000 description 2
- 210000004100 adrenal gland Anatomy 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 229940077737 brain-derived neurotrophic factor Drugs 0.000 description 2
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 125000000837 carbohydrate group Chemical group 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000007385 chemical modification Methods 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 239000012501 chromatography medium Substances 0.000 description 2
- 230000001684 chronic effect Effects 0.000 description 2
- 239000013599 cloning vector Substances 0.000 description 2
- 239000003636 conditioned culture medium Substances 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 239000006071 cream Substances 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 239000003797 essential amino acid Substances 0.000 description 2
- 235000020776 essential amino acid Nutrition 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 210000004905 finger nail Anatomy 0.000 description 2
- 238000005194 fractionation Methods 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 2
- 206010018797 guttate psoriasis Diseases 0.000 description 2
- 108010044853 histidine-rich proteins Proteins 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 239000002955 immunomodulating agent Substances 0.000 description 2
- 229940121354 immunomodulator Drugs 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 210000003963 intermediate filament Anatomy 0.000 description 2
- 108020001756 ligand binding domains Proteins 0.000 description 2
- 229920002521 macromolecule Polymers 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 238000002483 medication Methods 0.000 description 2
- 238000001823 molecular biology technique Methods 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 230000037311 normal skin Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- 108010035774 phosphoribosylaminoimidazole carboxylase Proteins 0.000 description 2
- 230000004962 physiological condition Effects 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 210000002307 prostate Anatomy 0.000 description 2
- 108020001580 protein domains Proteins 0.000 description 2
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 210000003079 salivary gland Anatomy 0.000 description 2
- 210000004761 scalp Anatomy 0.000 description 2
- 239000004017 serum-free culture medium Substances 0.000 description 2
- 238000002741 site-directed mutagenesis Methods 0.000 description 2
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 125000001424 substituent group Chemical group 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 229940124597 therapeutic agent Drugs 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 125000003396 thiol group Chemical group [H]S* 0.000 description 2
- 239000003053 toxin Substances 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 230000005945 translocation Effects 0.000 description 2
- 102000003390 tumor necrosis factor Human genes 0.000 description 2
- 230000003827 upregulation Effects 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- PDRJLZDUOULRHE-ZETCQYMHSA-N (2s)-2-amino-3-pyridin-2-ylpropanoic acid Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=N1 PDRJLZDUOULRHE-ZETCQYMHSA-N 0.000 description 1
- DFZVZEMNPGABKO-ZETCQYMHSA-N (2s)-2-amino-3-pyridin-3-ylpropanoic acid Chemical compound OC(=O)[C@@H](N)CC1=CC=CN=C1 DFZVZEMNPGABKO-ZETCQYMHSA-N 0.000 description 1
- FQFVANSXYKWQOT-ZETCQYMHSA-N (2s)-2-azaniumyl-3-pyridin-4-ylpropanoate Chemical compound OC(=O)[C@@H](N)CC1=CC=NC=C1 FQFVANSXYKWQOT-ZETCQYMHSA-N 0.000 description 1
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- 108020005345 3' Untranslated Regions Proteins 0.000 description 1
- GUPXYSSGJWIURR-UHFFFAOYSA-N 3-octoxypropane-1,2-diol Chemical compound CCCCCCCCOCC(O)CO GUPXYSSGJWIURR-UHFFFAOYSA-N 0.000 description 1
- XWHHYOYVRVGJJY-QMMMGPOBSA-N 4-fluoro-L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(F)C=C1 XWHHYOYVRVGJJY-QMMMGPOBSA-N 0.000 description 1
- 108020003589 5' Untranslated Regions Proteins 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
- 101150096273 ADE2 gene Proteins 0.000 description 1
- 241000228431 Acremonium chrysogenum Species 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 241000589156 Agrobacterium rhizogenes Species 0.000 description 1
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 1
- 102100024321 Alkaline phosphatase, placental type Human genes 0.000 description 1
- 208000006820 Arthralgia Diseases 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 201000001178 Bacterial Pneumonia Diseases 0.000 description 1
- 102100029516 Basic salivary proline-rich protein 1 Human genes 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 102100021943 C-C motif chemokine 2 Human genes 0.000 description 1
- 102100021935 C-C motif chemokine 26 Human genes 0.000 description 1
- 108010001789 Calcitonin Receptors Proteins 0.000 description 1
- 102100038520 Calcitonin receptor Human genes 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000222128 Candida maltosa Species 0.000 description 1
- 101100007328 Cocos nucifera COS-1 gene Proteins 0.000 description 1
- 206010009900 Colitis ulcerative Diseases 0.000 description 1
- 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 1
- PMATZTZNYRCHOR-CGLBZJNRSA-N Cyclosporin A Chemical compound CC[C@@H]1NC(=O)[C@H]([C@H](O)[C@H](C)C\C=C\C)N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-CGLBZJNRSA-N 0.000 description 1
- 108010036949 Cyclosporine Proteins 0.000 description 1
- 101150074155 DHFR gene Proteins 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 238000012270 DNA recombination Methods 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 108090000204 Dipeptidase 1 Proteins 0.000 description 1
- 241000710188 Encephalomyocarditis virus Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 102000009024 Epidermal Growth Factor Human genes 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 108010075944 Erythropoietin Receptors Proteins 0.000 description 1
- 102100036509 Erythropoietin receptor Human genes 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 108090000331 Firefly luciferases Proteins 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 1
- 102000000340 Glucosyltransferases Human genes 0.000 description 1
- 108010055629 Glucosyltransferases Proteins 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 108010053070 Glutathione Disulfide Proteins 0.000 description 1
- 108010054017 Granulocyte Colony-Stimulating Factor Receptors Proteins 0.000 description 1
- 102100039622 Granulocyte colony-stimulating factor receptor Human genes 0.000 description 1
- 108010092372 Granulocyte-Macrophage Colony-Stimulating Factor Receptors Proteins 0.000 description 1
- 102000016355 Granulocyte-Macrophage Colony-Stimulating Factor Receptors Human genes 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 102100020948 Growth hormone receptor Human genes 0.000 description 1
- 101800001649 Heparin-binding EGF-like growth factor Proteins 0.000 description 1
- 102000008949 Histocompatibility Antigens Class I Human genes 0.000 description 1
- 108010088652 Histocompatibility Antigens Class I Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000690301 Homo sapiens Aldo-keto reductase family 1 member C4 Proteins 0.000 description 1
- 101001125486 Homo sapiens Basic salivary proline-rich protein 1 Proteins 0.000 description 1
- 101000897480 Homo sapiens C-C motif chemokine 2 Proteins 0.000 description 1
- 101000897493 Homo sapiens C-C motif chemokine 26 Proteins 0.000 description 1
- 101000746373 Homo sapiens Granulocyte-macrophage colony-stimulating factor Proteins 0.000 description 1
- 101001055222 Homo sapiens Interleukin-8 Proteins 0.000 description 1
- 101001091088 Homo sapiens Prorelaxin H2 Proteins 0.000 description 1
- 101001116548 Homo sapiens Protein CBFA2T1 Proteins 0.000 description 1
- 101000617830 Homo sapiens Sterol O-acyltransferase 1 Proteins 0.000 description 1
- 101000716102 Homo sapiens T-cell surface glycoprotein CD4 Proteins 0.000 description 1
- 101000946843 Homo sapiens T-cell surface glycoprotein CD8 alpha chain Proteins 0.000 description 1
- 101000801481 Homo sapiens Tissue-type plasminogen activator Proteins 0.000 description 1
- 101000847156 Homo sapiens Tumor necrosis factor-inducible gene 6 protein Proteins 0.000 description 1
- 102000002265 Human Growth Hormone Human genes 0.000 description 1
- 108010000521 Human Growth Hormone Proteins 0.000 description 1
- 239000000854 Human Growth Hormone Substances 0.000 description 1
- 241000709704 Human poliovirus 2 Species 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- 235000003332 Ilex aquifolium Nutrition 0.000 description 1
- 241000209027 Ilex aquifolium Species 0.000 description 1
- 102000009786 Immunoglobulin Constant Regions Human genes 0.000 description 1
- 108010009817 Immunoglobulin Constant Regions Proteins 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 108010060231 Insect Proteins Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 108010038452 Interleukin-3 Receptors Proteins 0.000 description 1
- 102000010790 Interleukin-3 Receptors Human genes 0.000 description 1
- 108010038501 Interleukin-6 Receptors Proteins 0.000 description 1
- 102000010781 Interleukin-6 Receptors Human genes 0.000 description 1
- 102000015696 Interleukins Human genes 0.000 description 1
- 108010063738 Interleukins Proteins 0.000 description 1
- SHGAZHPCJJPHSC-NUEINMDLSA-N Isotretinoin Chemical compound OC(=O)C=C(C)/C=C/C=C(C)C=CC1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-NUEINMDLSA-N 0.000 description 1
- 208000012659 Joint disease Diseases 0.000 description 1
- 108010066321 Keratin-14 Proteins 0.000 description 1
- 229940124091 Keratolytic Drugs 0.000 description 1
- 235000014663 Kluyveromyces fragilis Nutrition 0.000 description 1
- 241001138401 Kluyveromyces lactis Species 0.000 description 1
- 241000235650 Kluyveromyces marxianus Species 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- 239000004395 L-leucine Substances 0.000 description 1
- 235000019454 L-leucine Nutrition 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 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
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 1
- 208000004852 Lung Injury Diseases 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 101710141347 Major envelope glycoprotein Proteins 0.000 description 1
- 101710175625 Maltose/maltodextrin-binding periplasmic protein Proteins 0.000 description 1
- 108090000157 Metallothionein Proteins 0.000 description 1
- 208000034486 Multi-organ failure Diseases 0.000 description 1
- 208000010718 Multiple Organ Failure Diseases 0.000 description 1
- 101000930477 Mus musculus Albumin Proteins 0.000 description 1
- 101000966481 Mus musculus Dihydrofolate reductase Proteins 0.000 description 1
- 101100170937 Mus musculus Dnmt1 gene Proteins 0.000 description 1
- 101100243377 Mus musculus Pepd gene Proteins 0.000 description 1
- 101500006448 Mycobacterium bovis (strain ATCC BAA-935 / AF2122/97) Endonuclease PI-MboI Proteins 0.000 description 1
- 102100025243 Myeloid cell surface antigen CD33 Human genes 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 241000221960 Neurospora Species 0.000 description 1
- 108020004485 Nonsense Codon Proteins 0.000 description 1
- 108091060545 Nonsense suppressor Proteins 0.000 description 1
- 241000320412 Ogataea angusta Species 0.000 description 1
- 238000010222 PCR analysis Methods 0.000 description 1
- 108091008606 PDGF receptors Proteins 0.000 description 1
- 101150029183 PEP4 gene Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241000235648 Pichia Species 0.000 description 1
- 102000011653 Platelet-Derived Growth Factor Receptors Human genes 0.000 description 1
- 206010036030 Polyarthritis Diseases 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 101710098940 Pro-epidermal growth factor Proteins 0.000 description 1
- 102100033762 Proheparin-binding EGF-like growth factor Human genes 0.000 description 1
- 102100034949 Prorelaxin H2 Human genes 0.000 description 1
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 1
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 1
- 108010094028 Prothrombin Proteins 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- ODHCTXKNWHHXJC-GSVOUGTGSA-N Pyroglutamic acid Natural products OC(=O)[C@H]1CCC(=O)N1 ODHCTXKNWHHXJC-GSVOUGTGSA-N 0.000 description 1
- 238000012181 QIAquick gel extraction kit Methods 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 230000004570 RNA-binding Effects 0.000 description 1
- 238000010240 RT-PCR analysis Methods 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 206010057190 Respiratory tract infections Diseases 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- -1 SEQ ID NO: 40 Proteins 0.000 description 1
- 241000235347 Schizosaccharomyces pombe Species 0.000 description 1
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 1
- 108091081021 Sense strand Proteins 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 206010040070 Septic Shock Diseases 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 208000006981 Skin Abnormalities Diseases 0.000 description 1
- 108010068542 Somatotropin Receptors Proteins 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 102100021993 Sterol O-acyltransferase 1 Human genes 0.000 description 1
- 101000697584 Streptomyces lavendulae Streptothricin acetyltransferase Proteins 0.000 description 1
- 229940100514 Syk tyrosine kinase inhibitor Drugs 0.000 description 1
- 108010008038 Synthetic Vaccines Proteins 0.000 description 1
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 description 1
- 102100034922 T-cell surface glycoprotein CD8 alpha chain Human genes 0.000 description 1
- ZMZDMBWJUHKJPS-UHFFFAOYSA-M Thiocyanate anion Chemical compound [S-]C#N ZMZDMBWJUHKJPS-UHFFFAOYSA-M 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 108010022394 Threonine synthase Proteins 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 108090000253 Thyrotropin Receptors Proteins 0.000 description 1
- 102100029337 Thyrotropin receptor Human genes 0.000 description 1
- 108050006955 Tissue-type plasminogen activator Proteins 0.000 description 1
- 102000000887 Transcription factor STAT Human genes 0.000 description 1
- 108050007918 Transcription factor STAT Proteins 0.000 description 1
- 108020004566 Transfer RNA Proteins 0.000 description 1
- 102000004338 Transferrin Human genes 0.000 description 1
- 108090000901 Transferrin Proteins 0.000 description 1
- 206010069363 Traumatic lung injury Diseases 0.000 description 1
- 241000255993 Trichoplusia ni Species 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 102100032807 Tumor necrosis factor-inducible gene 6 protein Human genes 0.000 description 1
- IVOMOUWHDPKRLL-UHFFFAOYSA-N UNPD107823 Natural products O1C2COP(O)(=O)OC2C(O)C1N1C(N=CN=C2N)=C2N=C1 IVOMOUWHDPKRLL-UHFFFAOYSA-N 0.000 description 1
- 201000006704 Ulcerative Colitis Diseases 0.000 description 1
- 206010046306 Upper respiratory tract infection Diseases 0.000 description 1
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical class O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 1
- 244000301083 Ustilago maydis Species 0.000 description 1
- 235000015919 Ustilago maydis Nutrition 0.000 description 1
- 108010053096 Vascular Endothelial Growth Factor Receptor-1 Proteins 0.000 description 1
- 102100033178 Vascular endothelial growth factor receptor 1 Human genes 0.000 description 1
- 108020005202 Viral DNA Proteins 0.000 description 1
- 241000269370 Xenopus <genus> Species 0.000 description 1
- PCBMGUSDYHYVBQ-SOOFDHNKSA-N [4-amino-2-[(3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1H-imidazol-5-yl]phosphonic acid Chemical compound P(=O)(O)(O)C=1N=C(NC1N)C1[C@H](O)[C@H](O)[C@H](O1)CO PCBMGUSDYHYVBQ-SOOFDHNKSA-N 0.000 description 1
- 230000035508 accumulation Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 108020002494 acetyltransferase Proteins 0.000 description 1
- 102000005421 acetyltransferase Human genes 0.000 description 1
- ODHCTXKNWHHXJC-UHFFFAOYSA-N acide pyroglutamique Natural products OC(=O)C1CCC(=O)N1 ODHCTXKNWHHXJC-UHFFFAOYSA-N 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 238000012867 alanine scanning Methods 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000000689 aminoacylating effect Effects 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- NUZWLKWWNNJHPT-UHFFFAOYSA-N anthralin Chemical compound C1C2=CC=CC(O)=C2C(=O)C2=C1C=CC=C2O NUZWLKWWNNJHPT-UHFFFAOYSA-N 0.000 description 1
- 230000000078 anti-malarial effect Effects 0.000 description 1
- 230000000340 anti-metabolite Effects 0.000 description 1
- 239000003430 antimalarial agent Substances 0.000 description 1
- 229940033495 antimalarials Drugs 0.000 description 1
- 229940100197 antimetabolite Drugs 0.000 description 1
- 239000002256 antimetabolite Substances 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 210000001099 axilla Anatomy 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 102000012740 beta Adrenergic Receptors Human genes 0.000 description 1
- 108010079452 beta Adrenergic Receptors Proteins 0.000 description 1
- 239000002876 beta blocker Substances 0.000 description 1
- 229940097320 beta blocking agent Drugs 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 description 1
- LWQQLNNNIPYSNX-UROSTWAQSA-N calcipotriol Chemical compound C1([C@H](O)/C=C/[C@@H](C)[C@@H]2[C@]3(CCCC(/[C@@H]3CC2)=C\C=C\2C([C@@H](O)C[C@H](O)C/2)=C)C)CC1 LWQQLNNNIPYSNX-UROSTWAQSA-N 0.000 description 1
- 229960002882 calcipotriol Drugs 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 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
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 210000004671 cell-free system Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 229960001265 ciclosporin Drugs 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000004186 co-expression Effects 0.000 description 1
- 230000001112 coagulating effect Effects 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000003246 corticosteroid Substances 0.000 description 1
- 229960001334 corticosteroids Drugs 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- 229940095074 cyclic amp Drugs 0.000 description 1
- 229930182912 cyclosporin Natural products 0.000 description 1
- 230000016396 cytokine production Effects 0.000 description 1
- 210000000172 cytosol Anatomy 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 239000003398 denaturant Substances 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000030609 dephosphorylation Effects 0.000 description 1
- 238000006209 dephosphorylation reaction Methods 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 238000002050 diffraction method Methods 0.000 description 1
- 102000004419 dihydrofolate reductase Human genes 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000013024 dilution buffer Substances 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 229960002311 dithranol Drugs 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 239000013583 drug formulation Substances 0.000 description 1
- 230000008482 dysregulation Effects 0.000 description 1
- 150000002061 ecdysteroids Chemical class 0.000 description 1
- 230000005684 electric field Effects 0.000 description 1
- 238000002003 electron diffraction Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 150000002118 epoxides Chemical class 0.000 description 1
- 235000020774 essential nutrients Nutrition 0.000 description 1
- HQMNCQVAMBCHCO-DJRRULDNSA-N etretinate Chemical compound CCOC(=O)\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)C=C(OC)C(C)=C1C HQMNCQVAMBCHCO-DJRRULDNSA-N 0.000 description 1
- 229960002199 etretinate Drugs 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 210000003414 extremity Anatomy 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 102000013361 fetuin Human genes 0.000 description 1
- 108060002885 fetuin Proteins 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 1
- YPZRWBKMTBYPTK-BJDJZHNGSA-N glutathione disulfide Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@H](C(=O)NCC(O)=O)CSSC[C@@H](C(=O)NCC(O)=O)NC(=O)CC[C@H](N)C(O)=O YPZRWBKMTBYPTK-BJDJZHNGSA-N 0.000 description 1
- 230000002414 glycolytic effect Effects 0.000 description 1
- 102000035122 glycosylated proteins Human genes 0.000 description 1
- 108091005608 glycosylated proteins Proteins 0.000 description 1
- 239000005090 green fluorescent protein Substances 0.000 description 1
- 210000004013 groin Anatomy 0.000 description 1
- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical compound N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 238000010231 histologic analysis Methods 0.000 description 1
- 238000007489 histopathology method Methods 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 102000046157 human CSF2 Human genes 0.000 description 1
- 102000054751 human RUNX1T1 Human genes 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- ZMZDMBWJUHKJPS-UHFFFAOYSA-N hydrogen thiocyanate Natural products SC#N ZMZDMBWJUHKJPS-UHFFFAOYSA-N 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000002390 hyperplastic effect Effects 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 238000000760 immunoelectrophoresis Methods 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 239000012678 infectious agent Substances 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 230000003960 inflammatory cascade Effects 0.000 description 1
- 230000004968 inflammatory condition Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 150000004001 inositols Chemical class 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 108040001834 interleukin-20 receptor activity proteins Proteins 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000004068 intracellular signaling Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229960005280 isotretinoin Drugs 0.000 description 1
- 230000003780 keratinization Effects 0.000 description 1
- 230000001530 keratinolytic effect Effects 0.000 description 1
- 239000003410 keratolytic agent Substances 0.000 description 1
- 210000003127 knee Anatomy 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 101150066555 lacZ gene Proteins 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 231100000225 lethality Toxicity 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- 229960001078 lithium Drugs 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 238000003468 luciferase reporter gene assay Methods 0.000 description 1
- 231100000515 lung injury Toxicity 0.000 description 1
- 239000006249 magnetic particle Substances 0.000 description 1
- 206010025482 malaise Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- VDXZNPDIRNWWCW-JFTDCZMZSA-N melittin Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(N)=O)CC1=CNC2=CC=CC=C12 VDXZNPDIRNWWCW-JFTDCZMZSA-N 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 230000004898 mitochondrial function Effects 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
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 230000000921 morphogenic effect Effects 0.000 description 1
- 239000012120 mounting media Substances 0.000 description 1
- 230000036457 multidrug resistance Effects 0.000 description 1
- 208000029744 multiple organ dysfunction syndrome Diseases 0.000 description 1
- 210000000282 nail Anatomy 0.000 description 1
- 239000006199 nebulizer Substances 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 230000037434 nonsense mutation Effects 0.000 description 1
- 230000004942 nuclear accumulation Effects 0.000 description 1
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 210000000287 oocyte Anatomy 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- YPZRWBKMTBYPTK-UHFFFAOYSA-N oxidized gamma-L-glutamyl-L-cysteinylglycine Natural products OC(=O)C(N)CCC(=O)NC(C(=O)NCC(O)=O)CSSCC(C(=O)NCC(O)=O)NC(=O)CCC(N)C(O)=O YPZRWBKMTBYPTK-UHFFFAOYSA-N 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 238000005222 photoaffinity labeling Methods 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 108010031345 placental alkaline phosphatase Proteins 0.000 description 1
- 230000003169 placental effect Effects 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 229920002704 polyhistidine Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000009696 proliferative response Effects 0.000 description 1
- 238000000164 protein isolation Methods 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 230000001823 pruritic effect Effects 0.000 description 1
- 229950010131 puromycin Drugs 0.000 description 1
- 239000002510 pyrogen Substances 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 238000003156 radioimmunoprecipitation Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 229910052711 selenium Inorganic materials 0.000 description 1
- 239000011669 selenium Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000036303 septic shock Effects 0.000 description 1
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 206010040882 skin lesion Diseases 0.000 description 1
- 231100000444 skin lesion Toxicity 0.000 description 1
- 210000002460 smooth muscle Anatomy 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 210000000434 stratum corneum Anatomy 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
- 230000008719 thickening Effects 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 210000004367 thymic lymphocyte Anatomy 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical class CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 210000001685 thyroid gland Anatomy 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 210000003437 trachea Anatomy 0.000 description 1
- 230000002463 transducing effect Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 239000012581 transferrin Substances 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
- 239000012588 trypsin Substances 0.000 description 1
- 102000042287 type II cytokine receptor family Human genes 0.000 description 1
- 108091052254 type II cytokine receptor family Proteins 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 238000009192 ultraviolet light therapy Methods 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 150000003710 vitamin D derivatives Chemical class 0.000 description 1
- 150000003704 vitamin D3 derivatives Chemical class 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
- C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- Cytokines are soluble proteins that influence the growth and differentiation of many cell types. Their receptors are composed of one or more integral membrane proteins that bind the cytokine with high affinity and transduce this binding event to the cell through the cytoplasmic portions of the certain receptor subunits. Cytokine receptors have been grouped into several classes on the basis of similarities in their extracellular ligand binding domains. For example, the receptor chains responsible for binding and/or transducing the effect of interferons (IFNs) are members of the type II cytokine receptor family (CRF2), based upon a characteristic 200 residue extracellular domain.
- IFNs interferons
- FIGS. 1-8 illustrate a representative number of embodiments of the present invention. Common elements in each of the drawings are given the same number.
- FIG. 1 depicts the heterotetramer produced by example 5.
- the soluble receptor construct, designated 10 is comprised of two IL-20 binding site polypeptide chains designated 12 and 14. Each binding site is comprised of the extracellular domain of IL-20RA, designated 16, and the extracellular domain of IL-20RB designated 18.
- the extracellular domain, 16, of IL-20RA is linked to the constant heavy one (CH1) domain, 20, of the human immunoglobulin gamma 1 heavy chain constant region via linker 22, which is SEQ ID NO:72.
- the CH1 domain, 20, is then linked to the CH2 domain, 24, via hinge region 23.
- the CH2 domain, 24, is linked to the CH3 domain, 26, via hinge region 25.
- Chains 12 and 14 are disulfide-bonded together by means of disulfide bonds 28 and 30.
- Extracellular domain, 18, of IL-20RB is linked to the constant region of the human kappa light chain (CL), 34 of FIG. 1 via polypeptide linker 32.
- the constant light chain 34 forms a
- FIG. 2 depicts a construct of the present invention where the two IL-20 binding polypeptides, 12 and 14, are not disulfide bonded together, having hinge region, 27.
- FIG. 3 shows a very simple soluble receptor 38 of the present invention wherein extracellular domain, 16, of IL-20RA is connected to the extracellular domain, 18, of IL-20RB by means of a polypeptide linker, 40.
- the polypeptide linker extends from the amino terminus of extracellular domain, 16, of IL-20RA and is connected to the carboxyl terminus of the extracellular domain, 18, of IL-20RB.
- FIG. 4 shows an embodiment that has the extracellular domain, 16, of IL-20RA linked to the extracellular domain, 18, of IL-20RB by means of linker 40, as in FIG. 3 . While the extracellular domain, 16, of IL-20RA is linked to the CH1 domain, 20, as in FIG. 1 by means of polypeptide linker 42.
- FIG. 5 shows another possible embodiment of the present invention.
- a polypeptide linker 44 links the carboxyl terminus of the extracellular domain, 18, of IL-20RB with the amino terminus of the extracellular domain, 16, of IL-20RA.
- a polypeptide linker 46 extends from the carboxy terminus of the extracellular domain, 16, of IL-20RA to the CH2 domain 24.
- FIG. 6 shows another possible embodiment of the present invention.
- the soluble IL-20 receptor of FIG. 6 is identical to that of FIG. 1 except for the CH3 domain, 26 of FIG. 1 , is not present on the embodiment of FIG. 6 .
- FIG. 7 shows a soluble IL-20 receptor construct that is identical to the construct of FIG. 1 except both the CH2, and CH3 domains are absent.
- FIG. 8 shows a construct wherein both IL-20RA, 16, and IL-20RB have a polypeptide linker, 48, fused to their respective carboxyl termini.
- Each polypeptide linker has two cysteine residues such that when they are expressed the cysteines form two disulfide bonds, 50 and 52.
- the present invention fills this need by providing a newly discovered soluble receptor to Interleukin-20 (IL-20).
- IL-20 Interleukin-20
- the soluble receptor can be used to down-regulate IL-20 and thus treat inflammatory diseases such as psoriasis and inflammatory lung diseases.
- IL-20 was formally called ‘Zcyto10’, (International Patent Publication No. WO 99/27103) and has the amino acid sequences of SEQ ID NOs: 1-9.
- the receptor to IL-20 is comprised of two chains, an alpha chain and a beta chain.
- the alpha chain, hereinafter referred to as IL-20RA was formally called ZcytoR7. See U.S. Pat. No. 5,945,511.
- the beta chain, hereinafter referred to as IL-20RB was formally called DIRS1. See International Patent Application No. PCT/US99/03735.
- the present invention is a soluble receptor comprised of the extracellular domain of IL-20RA and the extracellular domain of IL-20RB.
- the present invention encompasses an isolated soluble receptor comprised of an ‘IL-20RA’ subunit and an ‘IL-20RB’ subunit, wherein the IL-20A subunit is comprised of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 38, 55, 63 and 65, and the IL-20RB subunit is comprised of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 59, 61, 67, 68 and 69.
- the IL-20RA and IL-20RB subunits are generally linked together by a polypeptide linker.
- the linking can be by any means but generally by a peptide bond or a disulfide bond between a polypeptide connected to the IL20RA subunit and a polypeptide connected to the IL-20RB subunit.
- the present invention is also directed towards isolated polynucleotides that encode the novel IL-20RA and IL-20RB polypeptides of the present invention.
- the IL-20RA subunit is fused to the constant region of the heavy chain of an immunoglobulin (Ig) molecule or a portion thereof and the IL-20RB subunit is fused to the constant region of the light chain of an Ig molecule such that the constant region of the light chain is disulfide bonded to the constant region of the heavy chain, generally to a cysteine residue on the hinge region of the heavy chain.
- the IL-20RA subunit can be fused to the constant region of the light chain of an Ig molecule and the IL-20RB subunit can be fused to the constant region of the heavy chain of an Ig molecule.
- the IL-20RA subunit fused to the constant region of the heavy chain is comprised of an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 53, 54 and 62
- the IL-20RB subunit fused to the constant region of the light chain of the Ig molecule is comprised of an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 57, 58, and 60.
- a protein having a first polypeptide and a second polypeptide wherein the first polypeptide is comprised of an amino acid sequence of SEQ ID NO: 66 and the second polypeptide is comprised of an amino acid sequence selected from the group consisting of SEQ ID NOs: 70 and 71.
- the resultant protein can be used to generate antibodies to the IL-20RA subunit and the IL-20RB subunit.
- amino-terminal and “carboxyl-terminal” are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.
- antibody fusion protein refers to a recombinant molecule that comprises an antibody component and a therapeutic agent.
- therapeutic agents suitable for such fusion proteins include immunomodulators (“antibody-immunomodulator fusion protein”) and toxins (“antibody-toxin fusion protein”).
- complement/anti-complement pair denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions.
- biotin and avidin are prototypical members of a complement/anti-complement pair.
- Other exemplary complement/anti-complement pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like.
- the complement/anti-complement pair preferably has a binding affinity of ⁇ 10 9 M ⁇ 1 .
- polynucleotide molecule is a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.
- contig denotes a polynucleotide that has a contiguous stretch of identical or complementary sequence to another polynucleotide. Contiguous sequences are said to “overlap” a given stretch of polynucleotide sequence either in their entirety or along a partial stretch of the polynucleotide. For example, representative contigs to the polynucleotide sequence 5′-ATGGCTTAGCTT-3′ are 5′-TAGCTTgagtct-3′ and 3′-gtcgacTACCGA-5′.
- degenerate nucleotide sequence denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide).
- Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp).
- expression vector is used to denote a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription.
- additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc.
- Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
- isolated when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems.
- isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones.
- Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5′ and 3′ untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78 (1985).
- an “isolated” polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue.
- the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure.
- the term “isolated” does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
- operably linked when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.
- polynucleotide is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′ end.
- Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. Sizes of polynucleotides are expressed as base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases (“kb”). Where the context allows, the latter two terms may describe polynucleotides that are single-stranded or double-stranded.
- double-stranded molecules When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term “base pairs”. It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; thus all nucleotides within a double-stranded polynucleotide molecule may not be paired. Such unpaired ends will in general not exceed 20 nucleotides in length.
- polypeptide is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as “peptides”.
- promoter is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5′ non-coding regions of genes.
- a “protein” is a macromolecule comprising one or more polypeptide chains.
- a protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
- receptor denotes a cell-associated protein that binds to a bioactive molecule (i.e., a ligand) and mediates the effect of the ligand on the cell.
- a bioactive molecule i.e., a ligand
- Membrane-bound receptors are characterized by a multi-domain structure comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal transduction. Binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule(s) in the cell. This interaction in turn leads to an alteration in the metabolism of the cell.
- Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids.
- receptors can be membrane bound, cytosolic or nuclear, monomeric (e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor).
- secretory signal sequence denotes a DNA sequence that encodes a polypeptide (a “secretory peptide”) that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized.
- secretory peptide a polypeptide that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized.
- the larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
- splice variant is used herein to denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene.
- IL-20 (formally called Zcyto10) is defined and methods for producing it and antibodies to IL-20 are contained in International Patent Application No. PCT/US98/25228, publication no. WO 99/27103,published Nov. 25, 1998 and U.S. patent application Ser. No. 09/313,458 filed May 17, 1999.
- the polynucleotide and polypeptide of human IL-20 are represented by SEQ ID NOs: 1 - 4, and mouse IL-20 by SEQ ID NOs: 5-9.
- the receptor to IL-20 has been discovered and is a heterodimer comprised of the polypeptide termed ‘IL-20RA’ (formally called Zcytor7) and a polypeptide termed ‘IL-20RB’.
- IL-20RA polypeptide
- the IL-20RA polypeptide, nucleic acid that encodes it, antibodies to IL-20RA and methods for producing it are disclosed in U.S. Pat. No. 5,945,511 issued Aug. 31, 1999.
- SEQ ID NOs: 10 - 12 are the IL-20RA polynucleotides and polypeptides.
- the extracellular domain of the human IL-20RA is comprised of a polypeptide selected from the group consisting of SEQ ID NOs: 12, 55, 63 and 65, the full-length receptor subunit being comprised of SEQ ID NO: 11.
- the extracellular domain of mouse IL-20RA is SEQ ID NO: 38, SEQ ID NO: 37 being the entire mouse IL-20RA.
- the extracellular domain of IL-20RB is comprised of a polypeptide selected from the group consisting of SEQ ID NOs: 15, 59, 61, 67, 68 and 69.
- the extracellular domain of the IL-20RA polypeptide and the extracellular domain of the IL-20RB polypeptide are covalently linked together.
- one extracellular subunit polypeptide has a constant region of a heavy chain of an immunoglobulin fused to its carboxy terminus and the other extracellular subunit has a constant light chain of an immunoglobulin (Ig) fused to its carboxy terminus such that the two polypeptides come together to form a soluble receptor and a disulfide bond is formed between the heavy and the light Ig chains.
- Ig immunoglobulin
- a peptide linker could be fused to the two carboxy-termini of the polypeptides to form a covalently bonded soluble receptor.
- SEQ ID NOs: 22 and 23 are constructs of the extracellular domain of IL-20RA fused to a mutated human immunoglobulin gamma 1 constant region produced according to the procedure set forth in example 5.
- SEQ ID NO: 62 is the predicted mature sequence without the signal sequence.
- SEQ ID NOs: 20 and 21 are constructs of the extracellular domain of IL-20RB fused to wild type human immunoglobulin kappa light chain constant region produced according to the procedure of example 5.
- SEQ ID NO: 60 is the predicted mature sequence without the signal sequence.
- FIG. 1 depicts the heterotetramer produced by example 5.
- SEQ ID NOs: 52 and 53 are constructs of the extracellular domain of IL-20RA fused to a mutated human immunoglobulin gamma 1 constant region produced according to the procedure set forth in example 12.
- SEQ ID NO: 54 is the predicted mature sequence without the signal sequence.
- SEQ ID NOs: 56 and 57 are constructs of the extracellular domain of IL-20RB fused to wild type human immunoglobulin kappa light chain constant region produced according to the procedure of example 12.
- SEQ ID NO: 58 is the predicted mature sequence without the signal sequence.
- the resultant heterotetramer is almost identical to that produced by example 5, the primary difference being the absence of a polypeptide linker between the extracellular domains and the beginning of the Ig constant regions, 22 in FIG. 1 .
- the term “extracellular domain of a receptor” means the extracellular domain of the receptor or a portion of the extracellular domain that is necessary for binding to its ligand, in this case the ligand being IL-20.
- FIGS. 1-8 illustrate a representative number of embodiments of the present invention. Common elements in each of the drawings are given the same number.
- FIG. 1 represents the embodiment of the present invention produced according to example 5 below.
- the soluble receptor construct, designated 10 is comprised of two IL-20 binding site polypeptide chains designated 12 and 14. Each binding site is comprised of the extracellular domain of IL-20RA, designated 16, and the extracellular domain of IL-20RB designated 18.
- the extracellular domain, 16, of IL-20RA is linked to the constant heavy one (CH1) domain, 20, of the human immunoglobulin gamma 1 heavy chain constant region via linker 22, which is SEQ ID NO:72.
- the CH1 domain, 20, is then linked to the CH2 domain, 24, via hinge region 23.
- the CH2 domain, 24, is linked to the CH3 domain, 26, via hinge region 25.
- the extracellular domain, 16, of IL-20RA extends from amino acid residues 36, a valine, to and including amino acid residue 249, a glutamine of SEQ ID NO:22.
- Polypeptide linker, 22, extends from amino acid residue 250, a glycine to and including amino acid residue 264, a serine, of SEQ ID NO:22.
- the CH1 domain, 22 of FIG. 1 extends from amino acid residue 265, an alanine, to and including amino acid residue 362, a valine, of SEQ ID NO:22. Hinge region 23 of FIG.
- Chains 12 and 14 are disulfide-bonded together by means of disulfide bonds 28 and 30.
- the disulfide bonds are formed between the heavy chains by the cysteine residues at positions 373 and 376 of SEQ ID NO: 22 of each of the two heavy chains.
- Extracellular domain, 18, of IL-20RB is linked to the constant region of the human kappa light chain (CL), 34 of FIG. 1 via polypeptide linker 32, which is the polypeptide SEQ ID NO: 72.
- the extracellular domain, 18, of IL-20RB extends from amino acid residue 30, a valine, to and including amino acid residue 230, an alanine, of SEQ ID NO: 20.
- Polypeptide linker, 32 extends from amino acid residue 231, a glycine, to and including amino acid residue 245, a serine, of SEQ ID NO:20.
- the kappa constant light region, 34 extends from amino acid residue 246, an arginine, to and including the final amino acid residue 352, a cysteine, of SEQ ID NO:20.
- the cysteine at position 352 of SEQ ID NO: 20 forms a disulfide bond, 36 in FIG. 1 , with the cysteine at position 367 of SEQ ID NO: 22.
- the constant light chain 34 is thus linked to the hinge region, 23, by disulfide bond, 36. In this way, the extracellular domain, 16, of IL-20RA is linked to the extracellular domain, 18, of IL-20RB to form a soluble receptor.
- the two IL-20 binding polypeptides, 12 and 14 would not be disulfide bonded together and would form a construct shown in FIG. 2 . having hinge region, 27.
- FIG. 3 shows a very simple soluble receptor 38 of the present invention wherein extracellular domain, 16, of IL-20RA is connected to the extracellular domain, 18, of IL-20RB by means of a polypeptide linker, 40.
- the polypeptide linker extends from the amino terminus of extracellular domain, 16, of IL-20RA and is connected to the carboxyl terminus of the extracellular domain, 18, of IL-20RB.
- the polypeptide linker should be between 100-240 amino acids in length, preferably about 170 amino acid residues in length.
- a suitable linker would be comprised of glycine and serine residues.
- a possible linker would be multiple units of SEQ ID NO: 72, preferably about 12.
- FIG. 4 shows an embodiment that has the extracellular domain, 16, of IL-20RA linked to the extracellular domain, 18, of IL-20RB by means of linker 40, as in FIG. 3 . While the extracellular domain, 16, of IL-20RA is linked to the CH1 domain, 20, as in FIG. 1 by means of polypeptide linker 42, which should be about 30 amino acid residues in length.
- An ideal linker would be comprised of glycine and serine as in SEQ ID NO: 72, and the hinge sequence, 23 of FIG. 1 .
- FIG. 5 shows another possible embodiment of the present invention.
- a polypeptide linker 44 of about 15 amino acid residue e.g. SEQ ID NO: 72
- a polypeptide linker 46 of about 30 amino acid residues extends from the carboxy terminus of the extracellular domain, 16, of IL-20RA to the CH2 domain.
- the carboxyl terminus of linker 46 would preferably be comprised of the hinge region extending from amino acid residue 363, a glutamic acid to and including amino acid residue 377, a proline, of SEQ ID NO: 22. Nonetheless, polypeptide linker 46 would ideally have at least one cysteine residue at its carboxyl terminus so a disulfide bond could be formed.
- the soluble IL-20 receptor of FIG. 6 is identical to that of FIG. 1 except for the CH3 domain, 26 of FIG. 1 , is not present on the embodiment of FIG. 6 .
- the CH3 region begins at amino acid residue 488, a glycine, and extends to the last residue 594 of SEQ ID NO: 22.
- FIG. 7 shows a soluble IL-20 receptor construct that is identical to the construct of FIG. 1 except both the CH2, and CH3 domains are absent.
- the CH2 and CH3 domains run from amino acid residue 378, an alanine, to the end of the polypeptide sequence of SEQ ID NO: 22.
- FIG. 8 shows a construct wherein both IL-20RA, 16, and IL-20RB have a polypeptide linker, 48, fused to their respective carboxyl termini.
- Each polypeptide linker has two cysteine residues such that when they are expressed the cysteines form two disulfide bonds, 50 and 52.
- the polypeptide linker is comprised of the hinge region, 23 in FIG. 1 .
- the hinge region is comprised of amino acid residues 363, a glutamine, to and including amino acid residue 377 of SEQ ID NO: 22.
- a method for producing a soluble receptor comprised of extracellular domains of IL-20RA and IL-20RB comprising (a) introducing into a host cell a first DNA sequence comprised of a transcriptional promoter operatively linked to a first secretory signal sequence followed downstream by and in proper reading frame the DNA that encodes the extracellular portion of IL-20RA and the DNA that encodes an immunoglobulin light chain constant region;(b) introducing into the host cell a second DNA construct comprised of a transcriptional promoter operatively linked to a second secretory signal followed downstream by and in proper reading frame a DNA sequence that encodes the extracellular portion of IL-20RB and a DNA sequence that encodes an immunoglobulin heavy chain constant region domain selected from the group consisting of C H 1, C H 2, C H 3 and C H 4; (c) growing the host cell in an appropriate growth medium under physiological conditions to allow the secretion of a fusion protein comprised of the extracellular domain of IL-20RA and IL-20RB
- the second DNA sequence further encodes an immunoglobulin heavy chain hinge region wherein the hinge region is joined to the heavy chain constant region domain. In another embodiment, the second DNA sequence further encodes an immunoglobulin variable region joined upstream of and in proper reading frame with the immunoglobulin heavy chain constant region.
- a method for producing a soluble receptor comprised of the extracellular domains of IL-20RA and IL-20RB comprising (a) introducing into a host cell a first DNA sequence comprised of a transcriptional promoter operatively linked to a first secretory signal sequence followed downstream by and in proper reading frame the DNA that encodes the extracellular portion of IL-20RB and the DNA that encodes an immunoglobulin light chain constant region;(b) introducing into the host cell a second DNA construct comprised of a transcriptional promoter operatively linked to a second secretory signal followed downstream by and in proper reading frame a DNA sequence that encodes the extracellular portion of IL-20RA and a DNA sequence that encodes an immunoglobulin heavy chain constant region domain selected from the group consisting of C H 1, C H 2, C H 3 and C H 4; (c) growing the host cell in an appropriate growth medium under physiological conditions to allow the secretion of a dimerized heterodimeric fusion protein comprised of the extracellular domain of IL-20
- the second DNA sequence further encodes an immunoglobulin heavy chain hinge region wherein the hinge region is joined to the heavy chain constant region domain. In another embodiment, the second DNA sequence further encodes an immunoglobulin variable region joined upstream of and in proper reading frame with the immunoglobulin heavy chain constant region. (See U.S. Pat. No. 5,843,725.)
- a polynucleotide generally a cDNA sequence, encodes the described polypeptides herein.
- a cDNA sequence that encodes a polypeptide of the present invention is comprised of a series of codons, each amino acid residue of the polypeptide being encoded by a codon and each codon being comprised of three nucleotides. The amino acid residues are encoded by their respective codons as follows.
- Alanine (Ala) is encoded by GCA, GCC, GCG or GCT.
- Cysteine (Cys) is encoded by TGC or TGT.
- Aspartic acid is encoded by GAC or GAT.
- Glutamic acid (Glu) is encoded by GAA or GAG.
- Phenylalanine (Phe) is encoded by TTC or TTT.
- Glycine is encoded by GGA, GGC, GGG or GGT.
- Histidine is encoded by CAC or CAT.
- Isoleucine (Ile) is encoded by ATA, ATC or ATT.
- Lysine is encoded by AAA, or AAG.
- Leucine (Leu) is encoded by TTA, TTG, CTA, CTC, CTG or CTT.
- Methionine (Met) is encoded by ATG.
- Asparagine is encoded by AAC or AAT.
- Proline is encoded by CCA, CCC, CCG or CCT.
- Glutamine (Gln) is encoded by CAA or CAG.
- Arginine (Arg) is encoded by AGA, AGG, CGA, CGC, CGG or CGT.
- Serine (Ser) is encoded by AGC, AGT, TCA, TCC, TCG or TCT.
- Threonine (Thr) is encoded by ACA, ACC, ACG or ACT.
- Valine (Val) is encoded by GTA, GTC, GTG or GTT.
- Trp Tryptophan
- Tyrosine (Tyr) is encoded by TAC or TAT.
- mRNA messenger RNA
- T thymine nucleotide
- U uracil nucleotide
- preferential codon usage or “preferential codons” is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid.
- the amino acid Threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential.
- Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art.
- preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein.
- E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).
- a natural amino acid that is to be replaced e.g., phenylalanine
- desired non-naturally occurring amino acid(s) e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine.
- non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-7476 (1994).
- Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions, Wynn and Richards, Protein Sci. 2:395-403 (1993).
- a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues.
- Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis, Cunningham and Wells, Science 244: 1081-1085 (1989); Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-502 (1991).
- site-directed mutagenesis or alanine-scanning mutagenesis Cunningham and Wells, Science 244: 1081-1085 (1989); Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-502 (1991).
- single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity as disclosed below to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., J. Biol. Chem. 271:4699-708, 1996.
- Sites of ligand-receptor interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-312 (1992); Smith et al., J. Mol. Biol. 224:899-904 (1992); Wlodaver et al., FEBS Lett. 309:59-64 (1992).
- variants of the disclosed IL-20, IL-20RA and IL-20RB DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389-391, (1994), Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-10751 (1994) and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated by in vitro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by using a family of parent DNAs, such as allelic variants or DNAs from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid “evolution” of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.
- Mutagenesis methods as disclosed herein can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides in host cells.
- Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.
- Polypeptides can be produced in genetically engineered host cells according to conventional techniques.
- Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells.
- Eukaryotic cells particularly cultured cells of multicellular organisms, are preferred.
- a DNA sequence encoding a polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector.
- the vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.
- a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector.
- the secretory signal sequence may be that of the native polypeptides, or may be derived from another secreted protein (e.g., t-PA) or synthesized de novo.
- the secretory signal sequence is operably linked to the DNA sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell.
- Secretory signal sequences are commonly positioned 5′ to the DNA sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830).
- the secretory signal sequence contained in the polypeptides of the present invention is used to direct other polypeptides into the secretory pathway.
- the present invention provides for such fusion polypeptides.
- the secretory signal sequence contained in the fusion polypeptides of the present invention is preferably fused amino-terminally to an additional peptide to direct the additional peptide into the secretory pathway.
- Such constructs have numerous applications known in the art.
- these novel secretory signal sequence fusion constructs can direct the secretion of an active component of a normally non-secreted protein, such as a receptor. Such fusions may be used in vivo or in vitro to direct peptides through the secretory pathway.
- Cultured mammalian cells are suitable hosts within the present invention.
- Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection, Wigler et al., Cell 14:725 (1978), Corsaro and Pearson, Somatic Cell Genetics 7:603 (1981); Graham and Van der Eb, Virology 52:456 (1973), electroporation, Neumann et al., EMBO J.
- Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59 (1977) and Chinese hamster ovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines.
- COS-1 ATCC No. CRL 1650
- COS-7 ATCC No. CRL 1651
- BHK ATCC No. CRL 1632
- BHK 570 ATCC No. CRL 10314
- 293 ATCC No. CRL 1573
- Graham et al. J. Gen. Virol. 36:59 (1977)
- Chinese hamster ovary e.g. CHO-K1; ATCC No. CCL 61
- Suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Md.
- strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288.
- Other suitable promoters include those from metallothionein genes (U.S. Pat. Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.
- Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as “transfectants”. Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as “stable transfectants.”
- a preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like.
- Selection systems can also be used to increase the expression level of the gene of interest, a process referred to as “amplification.” Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes.
- a preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate.
- Other drug resistance genes e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
- hygromycin resistance e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
- Alternative markers that introduce an altered phenotype such as green fluorescent protein, or cell surface proteins such as CD4, CD8, Class I MHC, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.
- eukaryotic cells can also be used as hosts, including plant cells, insect cells and avian cells.
- Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. ( Bangalore ) 11:47 (1987). Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al., U.S. Pat. No. 5,162,222 and WIPO publication WO 94/06463.
- Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV).
- DNA encoding a polypeptide is inserted into the baculoviral genome in place of the AcNPV polyhedrin gene coding sequence by one of two methods.
- the first is the traditional method of homologous DNA recombination between wild-type AcNPV and a transfer vector containing the gene flanked by AcNPV sequences.
- Suitable insect cells e.g. SF9 cells, are infected with wild-type AcNPV and transfected with a transfer vector comprising a polynucleotide operably linked to an AcNPV polyhedrin gene promoter, terminator, and flanking sequences. See, King, L. A. and Possee, R.
- the second method of making recombinant baculovirus utilizes a transposon-based system described by Luckow, V. A, et al., J Virol 67:4566 (1993). This system is sold in the Bac-to-Bac kit (Life Technologies, Rockville, Md.). This system utilizes a transfer vector, pFastBac1TM (Life Technologies) containing a Tn7 transposon to move the DNA encoding the polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a “bacmid.” The pFastBac1TM transfer vector utilizes the AcNPV polyhedrin promoter to drive the expression of the gene of interest.
- pFastBac1TM can be modified to a considerable degree.
- the polyhedrin promoter can be removed and substituted with the baculovirus basic protein promoter (also known as Pcor, p6.9 or MP promoter), which is expressed earlier in the baculovirus infection, and has been shown to be advantageous for expressing secreted proteins.
- Pcor baculovirus basic protein promoter
- MP promoter baculovirus basic protein promoter
- transfer vector constructs a short or long version of the basic protein promoter can be used.
- transfer vectors can be constructed that replace the native secretory signal sequences with secretory signal sequences derived from insect proteins.
- a secretory signal sequence from Ecdysteroid Glucosyltransferase (EGT), honey bee Melittin (Invitrogen, Carlsbad, Calif.), or baculovirus gp67 (PharMingen, San Diego, Calif.) can be used in constructs to replace the native secretory signal sequence.
- transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed polypeptide, for example, a Glu-Glu epitope tag, Grussenmeyer, T. et al., Proc Natl Acad Sci. 82:7952 (1985).
- a transfer vector containing a recombinant gene is transformed into E. coli, and screened for bacmids that contain an interrupted lacZ gene indicative of recombinant baculovirus.
- the bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, e.g. Sf9 cells.
- Recombinant virus that expresses the polypeptide is subsequently produced.
- Recombinant viral stocks are made by methods commonly used the art.
- the recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda. See, in general, Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA (ASM Press, Washington, D.C., 1994).
- host cells typically a cell line derived from the fall armyworm, Spodoptera frugiperda.
- Another suitable cell line is the High FiveOTM cell line (Invitrogen) derived from Trichoplusia ni (U.S. Pat. No. 5,300,435).
- Commercially available serum-free media are used to grow and maintain the cells.
- Suitable media are Sf900 IITM (Life Technologies) or ESF 921TM (Expression Systems) for the Sf9 cells; and Ex-cellO405TM (JRH Biosciences, Lenexa, Kans.) or Express FiveOTM (Life Technologies) for the T. ni cells.
- the cells are grown up from an inoculation density of approximately 2 ⁇ 5 ⁇ 10 5 cells to a density of 1 ⁇ 2 ⁇ 10 6 cells at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3.
- MOI multiplicity of infection
- the recombinant virus-infected cells typically produce the recombinant polypeptide at 12-72 hours post-infection and secrete it with varying efficiency into the medium.
- the culture is usually harvested 48 hours post-infection. Centrifugation is used to separate the cells from the medium (supernatant). The supernatant containing the polypeptide is filtered through micropore filters, usually 0.45 ⁇ m pore size. Procedures used are generally described in available laboratory manuals (King, L. A. and Possee, R. D., ibid., O'Reilly, D. R. et al., ibid.; Richardson, C. D., ibid.). Subsequent purification of the polypeptide from the supernatant can be achieved using methods described herein.
- Fungal cells including yeast cells, can also be used within the present invention.
- Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris , and Pichia methanolica.
- Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat. No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat. No. 5,037,743; and Murray et al., U.S. Pat. No.
- Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine).
- a preferred vector system for use in Saccharomyces cerevisiae is the POTJ vector system disclosed by Kawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media.
- Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman et al., U.S. Pat. No. 4,615,974; and Bitter, U.S.
- Aspergillus cells may be utilized according to the methods of McKnight et al., U.S. Pat. No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al., U.S. Pat. No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Pat. No. 4,486,533.
- Pichia methanolica as host for the production of recombinant proteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565.
- DNA molecules for use in transforming P. methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation.
- the promoter and terminator in the plasmid be that of a P. methanolica gene, such as a P. methanolica alcohol utilization gene (AUG1 or AUG2).
- DHAS dihydroxyacetone synthase
- FMD formate dehydrogenase
- CAT catalase
- a preferred selectable marker for use in Pichia methanolica is a P. methanolica ADE2 gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), which allows ade2 host cells to grow in the absence of adenine.
- host cells For large-scale, industrial processes where it is desirable to minimize the use of methanol, it is preferred to use host cells in which both methanol utilization genes (AUG1 and AUG2) are deleted. For production of secreted proteins, host cells deficient in vacuolar protease genes (PEP4 and PRB1) are preferred. Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells. It is preferred to transform P.
- methanolica cells by electroporation using an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40 milliseconds, most preferably about 20 milliseconds.
- Prokaryotic host cells including strains of the bacteria Escherichia coli, Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art; see, e.g., Sambrook et al., ibid.).
- the polypeptide When expressing a polypeptide in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea.
- the denatured polypeptide can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution.
- the polypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.
- Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells.
- suitable media including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required.
- the growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient, which is complemented by the selectable marker carried on the expression vector or co-transfected into the host cell.
- P. methanolica cells are cultured in a medium comprising adequate sources of carbon, nitrogen and trace nutrients at a temperature of about 25° C. to 35° C.
- Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors.
- a preferred culture medium for P. methanolica is YEPD (2% D-glucose, 2% BactoTM Peptone (Difco Laboratories, Detroit, Much.), 1% BactoTM yeast extract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).
- polypeptides of the present invention it is preferred to purify the polypeptides of the present invention to ⁇ 80% purity, more preferably to ⁇ 90% purity, even more preferably ⁇ 95% purity, and particularly preferred is a pharmaceutically pure state, that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents.
- a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.
- Expressed recombinant polypeptides can be purified using fractionation and/or conventional purification methods and media Ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples.
- Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography.
- Suitable chromatographic media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred.
- Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.
- Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties. Examples of coupling chemistries include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries.
- Polypeptides can be isolated by exploitation of their properties. For example, immobilized metal ion adsorption (IMAC) chromatography can be used to purify histidine-rich proteins, including those comprising polyhistidine tags. Briefly, a gel is first charged with divalent metal ions to form a chelate, Sulkowski, Trends in Biochem. 3:1 (1985). Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents. Other methods of purification include purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography.
- a protein fused to the Fc portion of an immunoglobulin can be purified using a ‘Protein A column’. Methods in Enzymol., Vol. 182, “Guide to Protein Purification”, M. Deutscher, (ed.),page 529-539 (Acad. Press, San Diego, 1990).
- a fusion of the polypeptide of interest and an affinity tag e.g., maltose-binding protein, an immunoglobulin domain
- an affinity tag e.g., maltose-binding protein, an immunoglobulin domain
- antibodies includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments, such as F(ab′) 2 and Fab proteolytic fragments. Genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as synthetic antigen-binding peptides and polypeptides, are also included.
- Non-human antibodies may be humanized by grafting non-human CDRs onto human framework and constant regions, or by incorporating the entire non-human variable domains (optionally “cloaking” them with a human-like surface by replacement of exposed residues, wherein the result is a “veneered” antibody). In some instances, humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced.
- assays known to those skilled in the art can be utilized to detect antibodies that bind to protein or peptide. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.) (Cold Spring Harbor Laboratory Press, 1988). Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmuno-precipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western blot assay, inhibition or competition assay, and sandwich assay.
- the soluble receptors of the present invention can be used to down-regulate IL-20, which has been shown to be involved in a number of inflammatory processes. Specifically, IL-20 has been shown to up-regulate IL-8. Inflammatory diseases in which IL-8 plays a significant role, and for which a decrease in IL-8 would be beneficial are, adult respiratory disease (ARD), septic shock, multiple organ failure, inflammatory lung injury such as asthma or bronchitis, bacterial pneumonia, psoriasis, eczema, atopic and contact dermatitis, and inflammatory bowel disease such as ulcerative colitis and Crohn's disease.
- ARD adult respiratory disease
- septic shock septic shock
- multiple organ failure inflammatory lung injury
- bacterial pneumonia bacterial pneumonia
- psoriasis eczema
- atopic and contact dermatitis atopic and contact dermatitis
- inflammatory bowel disease such as ulcerative colitis and Crohn's disease.
- IL-20 receptor subunits Two orphan class II cytokine receptors, both of which are expressed in skin, were identified as IL-20 receptor subunits. Both IL-20 receptor subunits are required for ligand binding, distinguishing their role from that of subunits in the four other known class II cytokine receptors.
- IL-20RA and IL-20RB are also coexpressed in a number of human tissues besides skin, including ovary, adrenal gland, testis, salivary gland, muscle, lung, kidney, heart and to a lesser degree the small intestine suggesting additional target tissues for IL-20 action.
- the IL-20 heterodimeric receptor is structurally similar to other class II cytokine receptors and is expressed in skin where we have demonstrated activity of the IL-20 ligand.
- IL-20RA and IL-20RB mRNA are markedly upregulated in human psoriatic skin compared to normal skin. Both IL-20 receptor subunits are expressed in keratinocytes throughout the epidermis and are also expressed in a subset of immune and endothelial cells. We propose that increased expression of an activated IL-20 receptor may alter the interactions between endothelial cells, immune cells and keratinocytes, leading to dysregulation of keratinocyte proliferation and differentiation.
- IL-20 stimulates signal transduction in the human keratinocyte HaCaT cell line, supporting a direct action of this novel ligand in skin.
- IL-1 ⁇ , EGF and TNF- ⁇ proteins known to be active in keratinocytes and to be involved with proliferative and pro-inflammatory signals in skin, enhance the response to IL-20.
- IL-20 signals through STAT3.
- IL-20 binds its receptor on keratinocytes and stimulates a STAT3-containing signal transduction pathway.
- IL-20 is involved in the pathology of psoriasis.
- the soluble receptors of the present invention can be administered to an individual to down-regulate IL-20 and thus treat psoriasis.
- Psoriasis is one of the most common dermatologic diseases, affecting up to 1 to 2 percent of the world's population. It is a chronic inflammatory skin disorder characterized by erythematous, sharply demarcated papules and rounded plaques, covered by silvery micaceous scale. The skin lesions of psoriasis are variably pruritic. Traumatized areas often develop lesions of psoriasis. Additionally, other external factors may exacerbate psoriasis including infections, stress, and medications, e.g. lithium, beta blockers, and anti-malarials.
- infections, stress, and medications e.g. lithium, beta blockers, and anti-malarials.
- plaque type The most common variety of psoriasis is called plaque type. Patients with plaque-type psoriasis will have stable, slowly growing plaques, which remain basically unchanged for long periods of time. The most common areas for plaque psoriasis to occur are the elbows knees, gluteal cleft, and the scalp. Involvement tends to be symmetrical. Inverse psoriasis affects the intertriginous regions including the axilla, groin, submammary region, and navel, and it also tends to affect the scalp, palms, and soles. The individual lesions are sharply demarcated plaques but may be moist due to their location. Plaque-type psoriasis generally develops slowly and runs an indolent course. It rarely spontaneously remits.
- Eruptive psoriasis is most common in children and young adults. It develops acutely in individuals without psoriasis or in those with chronic plaque psoriasis. Patients present with many small erythematous, scaling papules, frequently after upper respiratory tract infection with beta-hemolytic streptococci. Patients with psoriasis may also develop pustular lesions. These may be localized to the palms and soles or may be generalized and associated with fever, malaise, diarrhea, and arthralgias.
- Psoriasis can be treated by administering antagonists to IL-20.
- the preferred antagonists are either a soluble receptor to IL-20 or antibodies, antibody fragments or single chain antibodies that bind to either the IL-20 receptor or to IL-20.
- the antagonists to IL-20 can be administered alone or in combination with other established therapies such as lubricants, keratolytics, topical corticosteroids, topical vitamin D derivatives, anthralin, systemic antimetabolites such as methotrexate, psoralen-ultraviolet-light therapy (PUVA), etretinate, isotretinoin, cyclosporine, and the topical vitamin D3 derivative calcipotriol.
- PUVA psoralen-ultraviolet-light therapy
- etretinate isotretinoin
- cyclosporine and the topical vitamin D3 derivative calcipotriol.
- the antagonists in particularly the soluble receptor or the antibodies that bind to IL-20 or the IL-20 receptor can be administered to individual subcutaneously, intravenously, or transdermally using a cream or transdermal patch that contains the antagonist of IL-20. If administered subcutaneously, the antagonist can be injected into one or more psoriatic plaques. If administered transdermally, the antagonists can be administered directly on the plaques using a cream containing the antagonist to IL-20.
- the soluble receptor of IL-20 of the present invention can be administered to a person who has asthma, bronchitis or cystic fibrosis or other inflammatory lung disease to treat the disease.
- the antagonists can be administered by any suitable method including intravenous, subcutaneous, bronchial lavage, and the use of inhalant containing an antagonist to IL-20.
- the quantities of the IL-20 soluble necessary for effective therapy will depend upon many different factors, including means of administration, target site, physiological state of the patient, and other medications administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in vivo administration of these reagents Animal testing of effective doses for treatment of particular disorders will provide further predictive indication of human dosage. Methods for administration include oral, intravenous, peritoneal, intramuscular, transdermal or administration into the lung or trachea in spray form by means or a nebulizer or atomizer. Pharmaceutically acceptable carriers will include water, saline, buffers to name just a few.
- Dosage ranges would ordinarily be expected from 1 ⁇ g to 1000 ⁇ g per kilogram of body weight per day.
- a dosage for an average adult of the IL-20 soluble receptor would be about 25 mg given twice weekly as a subcutaneous injection. Injections could be given at the site of psoriatic lesions for the treatment of psoriasis.
- the antibody or soluble receptor can be in phosphate buffered saline.
- the antagonist to IL-20 can be administered via an ointment or transdermal patch. The doses by may be higher or lower as can be determined by a medical doctor with ordinary skill in the art.
- KGM Keratinocyte growth media
- KBM keratinocyte basal media
- IL-20 was made up in KBM media and added at varying concentrations, from 2.5 ⁇ g/ml down to 618 ng/mL in a first experiment and from 2.5 ⁇ g/mL down to 3 ng/mL in a second experiment.
- SEQ ID NO: 16 contains the ATG (Met1) codon with an EcoRI restriction site
- SEQ ID NO: 17 contains the stop codon (TAG) with an XhoI restriction site.
- the PCR amplification was carried out using a human keratinocyte (HaCaT) cDNA library DNA as a template and SEQ ID NO: 16 and SEQ ID NO: 17 as primers.
- the PCR reaction was performed as follows: incubation at 94° C. for 1 min followed by 30 cycles of 94° C. for 30 sec and 68° C.
- PCR products were run on 1% Agarose gel, and a 1 kb DNA band was observed.
- the PCR products were cut from the gel and the DNA was purified using a QIAquick Gel Extraction Kit (Qiagen). The purified DNA was digested with EcoRI and XhoI, and cloned into a pZP vector that was called pZP7N.
- a pZP plasmid is a mammalian expression vector containing an expression cassette having the mouse metallothionein-1 promoter, human tPA leader peptide, multiple restriction sites for insertion of coding sequences, a Glu-Glu tag, and a human growth hormone terminator.
- the plasmid also has an E. coli origin of replication, a mammalian selectable marker expression unit having an SV40 promoter, an enhancer and an origin of replication, as well as a DHFR gene, and the SV40 terminator.
- Several IL-20RB-pZP7N clones were sequenced.
- PCR amplification was carried out using three difference cDNA sources—fetal skin marathon cDNA, HaCaT cDNA library DNA, and prostate smooth muscle cDNA library DNA—as templates.
- the PCR products were gel purified and sequenced. The sequence of each of the three PCR products was consistent with that of the IL-20RB-pZP7N clone.
- IL-20RB is SEQ ID NO: 13 and 14, and the mature extracellular domain is SEQ ID NO: 15.
- a cell-based binding assay was used to verify IL-20 binds to IL-20RA- IL-20RB heterodimer.
- IL-20RB- IL-20RA heterodimer is a receptor for IL-20. The procedure used is described below.
- the COS cell transfection was performed in a 12-well tissue culture plate as follows: 0.5 ⁇ g DNA was mixed with medium containing 5 ⁇ l lipofectamine in 92 ⁇ l serum free Dulbecco's modified Eagle's medium (DMEM) (55 mg sodium pyruvate, 146 mg L-glutamine, 5 mg transferrin, 2.5 mg insulin, 1 ⁇ g selenium and 5 mg fetuin in 500 ml DMEM), incubated at room temperature for 30 minutes and then added to 400 ⁇ l serum free DMEM media. This 500 ⁇ l mixture was then added to 1.5 ⁇ 10 5 COS cells/well and incubated for 5 hours at 37° C. 500 ⁇ l 20% fetal bovine serum (FBS) DMEM media was added and incubated overnight.
- DMEM Dulbecco's modified Eagle's medium
- the assay a modification of the “secretion trap” (Davis, S., et al., Cell 87: 1161-1169 (1996), was performed as follows: cells were rinsed with PBS/1% bovine serum albumin (BSA) and blocked for 1 hour with TNB (0.1 M Tris-HCl, 0.15 M NaCl and 0.5% Blocking Reagent (NEN Renaissance TSA-Direct Kit Cat #NEL701) in water). This was followed by a one-hour incubation with 3 ⁇ g/ml biotinylated IL-20 protein in TNB. Cells were washed with PBS/1% BSA and incubated for another hour with 1:300 diluted streptavidin-HRP (NEN kit) in TNB.
- BSA bovine serum albumin
- TNB 0.1 M Tris-HCl, 0.15 M NaCl and 0.5% Blocking Reagent (NEN Renaissance TSA-Direct Kit Cat #NEL701) in water. This was followed by a one-hour incubation with
- TNT phosphate-buffered saline
- the human keratinocyte cell line, HaCaT was grown at 37° C. to several days post-confluence in T-75 tissue culture flasks. At this point, normal growth media (DMEM+10% FBS) was removed and replaced with serum-free media. Cells were then incubated for two days at 37° C.
- DMEM fetal calf serum
- rh recombinant human (rh) IL-1 alpha at 5 ng/mL
- rh IL-1 alpha at 20 ng/mL
- rh IL-1 alpha at 5 ng/mL +IL-20 at 1 ⁇ g/mL
- IL-20 at 1 ⁇ g/mL
- rh IL-10 at 10 ng/mL.
- AtlasTM arrays were pre-hybridized with Clontech ExpressHyb plus 100 mg/mL heat denatured salmon sperm DNA for at least thirty minutes at 68° C. with continuous agitation. Membranes were then hybridized with 1.9 ⁇ 10 6 CPM/mL (a total of 1.14 ⁇ 10 7 CPM) overnight at 68° C. with continuous agitation. The following day, membranes were washed for thirty minutes ⁇ 4 in 2 ⁇ SSC, 1% SDS at 68° C., plus for thirty minutes ⁇ 1 in 0.1 ⁇ SSC, 0.5% SDS at 68° C., followed by one final room temperature wash for five minutes in 2 ⁇ SSC.
- Array membranes were then placed in Kodak plastic pouches sealed and exposed to a phosphor imager screen overnight at room temperature. The next day, phosphor screens were scanned on a phosphor imager and analyzed using Clontech's AtlaslmageTM 1.0 software.
- the expression vector pEZE3 was used to express the recombinant IL-20 receptor-Ig fusion protein.
- the plasmid pEZE3 is derived from pDC312.
- pDC312 was obtained through license from Immunex Corporation.
- the plasmids pDC312 and pEZE3 contain an EASE segment as described in WO 97/25420. The presence of the EASE segment in an expression vector can improve expression of recombinant proteins two to eight fold in stable cell pools.
- the plasmid pEZE3 is a tricistronic expression vector that may be used to express up to three different proteins in mammalian cells, preferably Chinese Hamster Ovary (CHO) cells.
- the pEZE3 expression unit contains the cytomegalovirus (CMV) enhancer/promoter, the adenovirus tripartite leader sequence, a multiple cloning site for insertion of the coding region for the first recombinant protein, the poliovirus type 2 internal ribosome entry site, a second multiple cloning site for insertion of the coding region for the second recombinant protein, an encephalomyocarditis virus internal ribosome entry site, a coding segment for mouse dihydrofolate reductase, and the SV40 transcription terminator.
- pEZE3 contains an E. coli origin of replication and the bacterial beta lactamase gene.
- the IL-20 receptor-Ig fusion protein is a disulfide linked heterotetramer consisting of two chains of the extracellular domain of the human IL-20RB fused to the wild type human immunoglobulin kappa light chain constant region and two chains of the human IL-20RA protein extracellular domain fused to a mutated human immunoglobulin gamma 1 constant region.
- the human immunoglobulin gamma 1 constant region contains amino acid substitutions to reduce FcyRI binding and Clq complement fixation.
- the human IL-20RB extracellular domain human immunoglobulin kappa light chain constant region fusion construct was generated by overlap PCR.
- the IL-20RB coding segment consists of amino acids 1 to 230.
- the template used for the PCR amplification of the IL-20R segment was generated IL-20RB human kappa light chain constant region expression construct as described below in Example 12.
- Oligonucleotide primers SEQ ID NO: 24 and SEQ ID NO: 25 were used to amplify the IL-20RB segment.
- the entire wild type human immunoglobulin kappa light chain constant region was used.
- the template used for the PCR amplification of the wild type human immunoglobulin kappa light chain constant region segment was generated IL-20RB human kappa light chain constant region expression construct as described in Example 12.
- Oligonucleotide primers SEQ ID NO: 26 and SEQ ID NO: 27 were used to amplify the wild type human immunoglobulin kappa light chain constant region.
- the two protein coding domains were linked by overlap PCR using oligonucleotides SEQ ID NO: 24 and SEQ ID NO: 27.
- a (Gly 4 Ser) 3 (SEQ ID NO: 72) peptide linker was inserted between the two protein domains.
- the (Gly 4 Ser) 3 peptide linker was encoded on the PCR primers SEQ ID NO: 26 and SEQ ID NO:25.
- the resultant IL-20RB extracellular domain/kappa light chain constant region fusion construct is shown by SEQ ID NOs: 20 and 21.
- the predicted mature polypeptide, minus the signal sequence, is SEQ ID NO: 60.
- the portion of the extracellular domain of IL-20RB that was actually used was comprised of the amino acid sequence of SEQ ID NO: 61. N-terminal sequencing resulted in the predicted amino acid sequence.
- the human IL-20RA extracellular domain human immunoglobulin gamma 1 heavy chain constant region fusion construct was generated by overlap PCR of four separate DNA fragments, each generated by separate PCR amplification reactions.
- the first fragment contained an optimized tPA (tissue plasminogen activator) signal sequence.
- the tPA signal sequence was amplified using oligonucleotide primers SEQ ID NO: 28 and SEQ ID NO: 29 using an in-house previously generated expression vector as the template.
- the second fragment contained the IL-20RA extracellular domain-coding region consisting of amino acids 30 to 243 of SEQ ID NO: 11. Oligonucleotide primers SEQ ID NO: 30 and SEQ ID NO: 31 were used to amplify this IL-20RA segment using a previously generated clone of IL-20RA as the template.
- the human gamma 1 heavy chain constant region was generated from 2 segments.
- the first segment containing the C H 1 domain was amplified using oligonucleotide primers SEQ ID NO: 32 and SEQ ID NO: 33 using a clone of the wild type human gamma 1 heavy chain constant region as the template.
- the second segment containing the remaining hinge, C H 2, and C H 3 domains of the human immunoglobulin gamma 1 heavy chain constant region was generated by PCR amplification using oligonucleotide primers SEQ ID NO: 34 and SEQ ID NO: 35.
- the template used for this PCR amplification was from a previously generated human gamma 1 Fc construct that contained codons for amino acid substitutions to reduce FcyRI binding and Clq complement fixation as described in Example 12.
- the four protein coding domains were linked by overlap PCR using oligonucleotides SEQ ID NO: 28 and SEQ ID NO: 35.
- a (Gly 4 Ser) 3 peptide linker was inserted between the IL-20RA and CH1 protein domains.
- the (Gly 4 Ser) 3 peptide linker was encoded on the PCR primers SEQ ID NO: 32 and SEQ ID NO: 31.
- the IL-20RA extracellular domain/ domain human immunoglobulin gamma 1 heavy constant region fusion protein and DNA sequence are shown in SEQ ID NOs: 22 and 23.
- the predicted mature polypeptide sequence, minus the signal sequence, is SEQ ID NO: 62.
- the portion of extracellular domain of IL-20RA that was actually used was comprised of SEQ ID NO: 63.
- the IL-20RB extracellular domain human immunoglobulin kappa light chain constant region fusion coding segment was cloned into the second MCS while the human IL-20RA extracellular domain human immunoglobulin gamma 1 heavy chain constant region fusion coding segment was cloned into the first MCS of pEZE3.
- the plasmid was used to transfect CHO cells. The cells were selected in medium without hypoxanthine or thymidine and the transgene was amplified using methotrexate. The presence of protein was assayed by Western blotting using anti human gamma 1 heavy chain constant region and anti human kappa light chain antibodies. N-terminal sequencing revealed that the optimized tPA leader was not completely cleaved.
- Both human and mouse IL-20 were overexpressed in transgenic mice using a variety of promoters.
- the liver-specific mouse albumin promoter directing expression of human IL-20, was used initially in an attempt to achieve circulating levels of protein.
- Subsequent studies were conducted using the keratin 14 (K14) promoter, which primarily targets expression to the epidermis and other stratified squamous epithelia; the mouse metallothionein-1 promoter, which gives a broad expression pattern; and the E LCK promoter, which drives expression in cells of the lymphoid lineage. Similar results were obtained in all four cases, possibly because these promoters all give rise to circulating levels of IL-20.
- transgenic pups expressing the IL-20 transgene were smaller than non-transgenic littermates, had a shiny appearance with tight, wrinkled skin and died within the first few days after birth. Pups had milk in their stomachs indicating that they were able to suckle. These mice had swollen extremities, tail, nostril and mouth regions and had difficulty moving. In addition, the mice were frail, lacked visible adipose tissue and had delayed ear and toe development. Low expression levels in liver (less than 100 mRNA molecules/cell) were sufficient for both the neonatal lethality and skin abnormalities. Transgenic mice without a visible phenotype either did not express the transgene, did not express it at detectable levels, or were mosaic.
- IL-20 The specificity and affinity of IL-20 for its receptor was determined using BHK cells stably transfected with IL-20RA, IL-20RB or both receptor subunits. Binding assays using radiolabeled ligand demonstrated that IL-20 bound to BHK transfectants expressing both IL-20RA and IL-20RB but not to untransfected cells nor to transfectants expressing either receptor subunit alone. Binding of 125 I-labeled IL-20 was eliminated in the presence of 100-fold excess of unlabeled IL-20 but not with 100-fold excess of the unrelated cytokine, IL-21. The binding affinity (k D ) of IL-20 to the IL-20RA/IL-20RB heterodimeric receptor was determined to be approximately 1.5 nM.
- the factor-dependent pre-B cell line BaF3 was co-transfected with IL-20RA and IL-20RB and treated with IL-20 at various concentrations.
- IL-20 stimulated proliferation in a dose-dependent manner and gave a detectable signal at 1.1 pM, with a half maximal response at 3.4 pM.
- the IL-20 concentration for the half maximal proliferative response in BaF3 cells is 1000 ⁇ lower than that for half maximal binding affinity in BHK cells. Possible explanations for this large difference include the use of different cell lines, different receptor expression levels and different assay outputs.
- IL-20 also stimulated signal transduction in the biologically relevant human keratinocyte cell line HaCaT, which naturally expresses IL-20RA and IL-20RB. Therefore, IL-20 binds and activates the heterodimeric IL-20RA/IL-20RB receptor at concentrations expected for a cytokine. While the negative controls containing untransfected BaF3
- RT-PCR analysis was performed on a variety of human tissues to determine the expression pattern of IL-20RA and IL-20RB. Both receptor subunits are most highly expressed in skin and testis. The significant result is that IL-20RA and IL-20RB are both expressed in skin, where they have been shown to mediate the IL-20-induced response. Both IL-20RA and IL-20RB are also both expressed in monocytes, lung, ovary, muscle, testis, adrenal gland, heart, salivary gland and placenta. IL-20RA is also in brain, kidney, liver, colon, small intestine, stomach, thyroid, pancreas, uterus and prostate while IL-20RB is not.
- IL-20RA and IL-20RB mRNA are Up-regulated in Psoriasis
- In situ hybridization was used to determine whether IL-20 receptor expression is altered in psoriasis. Skin samples from four psoriasis patients and three unaffected patients were assayed with probes specific for the two-receptor subunit mRNAs. All four psoriatic skin samples had high levels of IL-20RA and IL-20RB mRNA in keratinocytes whereas normal skin samples did not have detectable levels of either receptor subunit mRNA. Positive signals in psoriatic skin were also observed in mononuclear immune cells and in endothelial cells in a subset of vessels. Therefore, both IL-20RA and IL-20RB are expressed in keratinocytes, immune cells and endothelial cells, the major cell types thought to interact in psoriasis.
- a cross-species hybridization probe was generated which contained the full-length cDNA fragment encoding human IL-20RA.
- a Southern blot of mouse genomic DNA and Northern blots of mouse RNA were performed to demonstrate that the human IL-20RA cDNA could specifically hybridize to mouse sequences.
- the human IL-20RA full length DNA hybridization probe was used to screen a mouse genomic library.
- the library which was obtained from Clontech (Palo Alto, Calif.), was generated from an MboI partial digest of mouse genomic DNA and cloned into the BamHI site of Lambda bacteriophage EMBL3 SP6/T7. Positive bacteriophage was plaque purified and bacteriophage DNA was prepared using Promega's Wizard Lambda Preps DNA Purification System. Two genomic restriction enzyme fragments, a 5.7 kb EcoRI fragment and an 8.0 kb SacI fragment, were generated from the positive bacteriophage and subcloned into pBluescript. DNA sequence analysis revealed the presence of 3 exons from the mouse ortholog to human IL-20RA.
- PCR primers from the 5′ UTR, SEQ ID NO: 40, and 3′ UTR, SEQ ID NO: 41 were designed to generate a full-length mouse IL-20RA sequence by PCR amplification.
- Mouse embryo 15day plus 17 day cDNA was used as the template for the PCR amplification.
- PCR products were subcloned and sequenced for confirmation.
- the mouse sequences are SEQ ID NOs: 36 and 37.
- the mature extracellular domain is comprised of SEQ ID NO: 38.
- a vector expressing a secreted hIL-20RA/hIL-20B heterodimer was constructed.
- the extracellular domain of hIL-20RA was fused to the heavy chain of IgG gamma 1 (IgG ⁇ 1), while the extracellular portion of IL-20RB was fused to human kappa light chain (human ⁇ light chain).
- the heavy chain of IgG ⁇ 1 was cloned into the Zem229R mammalian expression vector (ATCC deposit No. 69447) such that any extracellular portion of a receptor having a 5′ EcoRI and 3′ NheI site can be cloned in, resulting in an N-terminal extracellular domain-C-terminal IgG ⁇ 1 fusion.
- the IgG ⁇ 1 fragment used in this construct was made by using PCR to isolate the IgG ⁇ 1 sequence from a Clontech human fetal liver cDNA library as template. A PCR reaction using oligos SEQ ID NO: 42 and SEQ ID NO: 43 was run as follows: 40 cycles of 94° for 60 sec., 53° C.
- PCR products were separated by agarose gel electrophoresis and purified using a QiaQuickTM (Qiagen Inc., Valencia, Calif.) gel extraction kit.
- QiaQuickTM Qiagen Inc., Valencia, Calif.
- the isolated, 990 bp, DNA fragment was digested with MluI and EcoRI (Boerhinger-Mannheim), extracted with QiaQuickTM gel extraction kit and ligated with oligos SEQ ID NO: 44 and SEQ ID NO: 45, which comprise an MluI/EcoRI linker, into Zem229R previously digested with MluI and EcoRI using standard molecular biology techniques disclosed herein.
- This generic cloning vector was called Vector#76 hIgGgammal w/Ch1 #786 Zem229R (Vector #76).
- the polynucleotide sequence of the extracellular domain of hIL-20RA fused to the heavy chain of IgG gamma 1 is show in SEQ ID NO: 52 and the corresponding polypeptide sequence shown in SEQ ID NO: 53, the mature polypeptide, minus the signal sequence being comprised of SEQ ID NO: 54.
- the portion of the extracellular domain of IL-20RA used was comprised of SEQ ID NO: 55.
- the human ⁇ light chain was cloned in the Zem228R mammalian expression vector (ATCC deposit No. 69446) such that any extracellular portion of a receptor having a 5′ EcoRI site and a 3′ KpnI site can be cloned in, resulting in an N-terminal extracellular domain-C-terminal human ⁇ light chain fusion.
- the human ⁇ light chain fragment used in this construct was made by using PCR to isolate the human ⁇ light chain sequence from the same Clontech hFetal Liver cDNA library used above. A PCR reaction was run using oligos SEQ ID NO: 46 and SEQ ID NO: 47.
- PCR products were separated by agarose gel electrophoresis and purified using a QiaQuickTM (Qiagen) gel extraction kit.
- the isolated, 315 bp, DNA fragment was digested with MluI and EcoRI (Boerhinger-Mannheim), extracted with QiaQuickTM gel extraction kit and ligated with the MluI/EcoRI linker described above, into Zem228R previously digested with MluI and EcoRI using standard molecular biology techniques disclosed herein.
- This generic cloning vector was called Vector #77 h ⁇ light #774 Zem228R (Vector #77).
- polynucleotide sequence of the extracellular portion of IL-20RB fused to human kappa light chain is shown in SEQ ID NO: 56 and the corresponding polypeptide sequence shown in SEQ ID NO: 57, the mature polypeptide, minus the signal sequence, is comprised of SEQ ID NO: 58.
- the portion of the extracellular domain of IL-20RB actually used was comprised of SEQ ID NO: 59.
- a construct having human IL-20RA fused to IgG ⁇ 1 was made. This construction was done by using PCR to obtain human IL-20RA receptor from hIL-20RA/IgG Vector #102 with oligos SEQ ID NO: 48 and SEQ ID NO: 49 under conditions described as follows: 30 cycles of 94° C. for 60 sec., 57° C. for 60 sec., and 72° C. for 120 sec.; and 72° C. for 7 min. The resulting PCR product was digested with EcoRI and NheI, gel purified, as described herein, and ligated into a previously EcoRI and NheI digested and band-purified Vector #76 (above).
- the resulting vector was sequenced to confirm that the human IL-20R ⁇ /IgG gamma 1 fusion (hIL-20RA/Ch1 IgG) was correct.
- the hIL-20RA/Ch1 IgG gamma 1 #1825 Zem229R vector was called vector #195.
- the IL-20RA/Ch1 IgG ⁇ 1 sequence thus obtained is depicted by SEQ ID NOs: 52 and 53. N-terminal sequencing indicated the presence of the predicted mature polypeptide sequence of SEQ ID NO: 54.
- IL-20RB/human ⁇ light chain construction was performed as above by PCRing from DR1/7N-4 with oligos SEQ ID NO: 50 and SEQ ID NO: 51, digesting the resulting band with EcoRI and KpnI and then ligating this product into a previously EcoRI and KpnI digested and band-purified Vec#77 (above).
- the resulting vector was sequenced to confirm that the IL-20RB/ human ⁇ light chain fusion (IL-20RB/ ⁇ light) was correct.
- This IL-20RB// ⁇ light construct is shown by SEQ ID NOs: 56 and 57. N-terminal sequencing of the resultant polypeptide indicated the presence of the predicted mature amino acid sequence comprised of SEQ ID NO: 58.
- SEQ ID NO:59 is the mature portion of the extracellular domain of IL-20RB used.
- each of vectors #194 and #195, above were co-transfected into BHK-570 cells (ATCC No. CRL-10314) using LipofectamineTM reagent (Gibco/BRL), as per manufacturer's instructions.
- the transfected cells were selected for 10 days in DMEM+5%FBS (Gibco/BRL) containing 1 ⁇ M of methotrexate (MTX) (Sigma, St. Louis, Mo.) and 0.5 mg/ml G418 (Gibco/BRL) for 10 days.
- the resulting pool of transfectants was selected again in 10 ⁇ M MTX and 0.5 mg/ml G418 for 10 days.
- the resulting pool of doubly selected cells was used to generate protein.
- Three factories (Nunc, Denmark) of this pool were used to generate 8 L of serum free conditioned medium. This conditioned media was passed over a 1 ml protein-A column and eluted in (10) 750 microliter fractions. 4 of these fractions found to have the highest concentration were pooled and dialyzed (10 kD MW cutoff) against PBS. Finally, the dialyzed material was analyzed by BCA (Pierce) and found to have a concentration of 317 ⁇ g/ml. A total of 951 ⁇ g was obtained from this 8 L purification.
- IL-20 binds cell lines transfected with both subunits of its receptor. However, these cell lines overexpress the IL-20 receptor relative to its normal level and their relevance to the physiological role of IL-20 is unclear.
- the human HaCaT keratinocyte cell line which expresses endogenous IL-20RA and IL-20RB was used to examine IL-20 signal transduction in a biologically relevant cell type.
- HaCaT cells were infected with recombinant adenovirus containing a reporter construct to allow detection of intracellular signaling.
- the construct consists of the firefly luciferase gene driven by promoter/enhancer sequences comprised of the serum response element (SRE) and signal transducers and activators of transduction elements (STATs).
- SRE serum response element
- STATs signal transducers and activators of transduction elements
- This assay system detects productive ligand-receptor interactions and indicates possible downstream signal transduction components involved in receptor activation.
- Treatment with IL-20 alone resulted in a dose-dependent increase in luciferase activity with a half maximal response occurring at approximately 2.3 nM.
- Subsequent luciferase reporter assays using adenovirus vectors containing only the SRE element or only the STAT elements produced detectable reporter activation only through STATs.
- HaCaT cells were treated with IL-20 alone or in combination with a single submaximal dose of EGF, IL-1 ⁇ , or TNF ⁇ .
- IL-20 treatment resulted in a dose-dependent increase in luciferase activity.
- IL-20 in combination with IL-1 ⁇ results in a half-maximal response at approximately 0.5 nM, about five-fold lower than with IL-20 alone.
- activation of the reporter gene is detectable at 0.1 nM IL-20, a dose that is at least tenfold lower than the IL-20 dose required alone.
- BHK cells transfected with IL-20RA, IL-20RB or both receptor subunits were used to determine whether receptor pairing was required for IL-20 stimulation of STAT-luciferase. As was the case with binding assays, only cells transfected with both receptor subunits responded to IL-20 and did so with a half-maximal response of 5.7 pM.
- the IL-20 concentration for the half-maximal response in BHK cells is 400-fold lower than that for half-maximal response in HaCaT cells. It is likely that a lower concentration of IL-20 is needed for half-maximal response in BHK cells, as compared to HaCaT cells, due to higher receptor levels in the BHK IL-20 receptor transfectants.
- a nuclear translocation assay was used to identify STAT proteins involved in IL-20 action. Both HaCaT cells, with endogenous IL-20 receptors, and BHK cells transfected with IL-20RA and IL-20RB, were treated with IL-20 protein and translocation of STAT3 and STAT1 transcription factors from the cytoplasm to the nucleus was assayed by immunofluorescence.
- BHK cells transfected with IL-20RA and IL-20RB were used to confirm that the IL-20 receptor was required for IL-20 stimulation of STAT3 nuclear translocation.
- STAT3 remained cytosolic following treatment with IL-20.
- STAT1 remained cytosolic regardless of IL-20 treatment or IL-20 receptor expression.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Animal Behavior & Ethology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Cell Biology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Transplantation (AREA)
- Rheumatology (AREA)
- Dermatology (AREA)
- Pain & Pain Management (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A soluble receptor to IL-20 having two polypeptide subunits, IL-20RA (formerly called ZcytoR7) and IL-20RB (formerly called DIRS1). The two subunits are preferably linked together. In one embodiment one subunit is fused to the constant region of the light chain of an immunoglobulin, and the other subunit is fused to the constant region of the heavy chain of the immunoglobulin. The light chain and the heavy chain are connected via a disulfide bond.
Description
- This application is a continuation of U.S. application Ser. No. 12/899,700 filed Oct. 7, 2010 which is a continuation of Ser. No. 12/509,667, filed Jul. 27, 2009, which is a continuation of U.S. application Ser. No. 11/458,367, filed Jul. 18, 2006, now issued as U.S. Pat. No. 7,585,948, which is a divisional of U.S. application Ser. No. 09/745,792, filed Dec. 22, 2000, now issued as U.S. Pat. No. 7,122,632, which claims the benefit of U.S. Provisional Application Ser. No. 60/171,966, filed Dec. 23, 1999, and U.S. Provisional Application Ser. No. 60/213,416, filed Jun. 22, 2000, all of which applications are herein incorporated by reference in their entirety.
- The teachings of all of the references cited herein are incorporated in their entirety herein by reference.
- Cytokines are soluble proteins that influence the growth and differentiation of many cell types. Their receptors are composed of one or more integral membrane proteins that bind the cytokine with high affinity and transduce this binding event to the cell through the cytoplasmic portions of the certain receptor subunits. Cytokine receptors have been grouped into several classes on the basis of similarities in their extracellular ligand binding domains. For example, the receptor chains responsible for binding and/or transducing the effect of interferons (IFNs) are members of the type II cytokine receptor family (CRF2), based upon a characteristic 200 residue extracellular domain. The demonstrated in vivo activities of these interferons illustrate the enormous clinical potential of, and need for, other cytokines, cytokine agonists, and cytokine antagonists. Some cytokines are involved in the inflammatory cascade and can promote such diseases as rheumatoid arthritis, Crohn's disease, psoriasis, heart disease etc. Thus, there is a need to discover cytokines and their receptors that are involved in inflammation. One can then use the isolated soluble receptors of the cytokine to inhibit the cytokine-mediated inflammation.
-
FIGS. 1-8 illustrate a representative number of embodiments of the present invention. Common elements in each of the drawings are given the same number. -
FIG. 1 depicts the heterotetramer produced by example 5. The soluble receptor construct, designated 10, is comprised of two IL-20 binding site polypeptide chains designated 12 and 14. Each binding site is comprised of the extracellular domain of IL-20RA, designated 16, and the extracellular domain of IL-20RB designated 18. The extracellular domain, 16, of IL-20RA is linked to the constant heavy one (CH1) domain, 20, of the human immunoglobulin gamma 1 heavy chain constant region via linker 22, which is SEQ ID NO:72. The CH1 domain, 20, is then linked to the CH2 domain, 24, viahinge region 23. The CH2 domain, 24, is linked to the CH3 domain, 26, viahinge region 25.Chains disulfide bonds FIG. 1 viapolypeptide linker 32. Theconstant light chain 34 forms a disulfide bonded, 36, withhinge region 23. -
FIG. 2 depicts a construct of the present invention where the two IL-20 binding polypeptides, 12 and 14, are not disulfide bonded together, having hinge region, 27. -
FIG. 3 shows a very simplesoluble receptor 38 of the present invention wherein extracellular domain, 16, of IL-20RA is connected to the extracellular domain, 18, of IL-20RB by means of a polypeptide linker, 40. The polypeptide linker extends from the amino terminus of extracellular domain, 16, of IL-20RA and is connected to the carboxyl terminus of the extracellular domain, 18, of IL-20RB. -
FIG. 4 shows an embodiment that has the extracellular domain, 16, of IL-20RA linked to the extracellular domain, 18, of IL-20RB by means of linker 40, as inFIG. 3 . While the extracellular domain, 16, of IL-20RA is linked to the CH1 domain, 20, as inFIG. 1 by means ofpolypeptide linker 42. -
FIG. 5 shows another possible embodiment of the present invention. In this embodiment, apolypeptide linker 44, links the carboxyl terminus of the extracellular domain, 18, of IL-20RB with the amino terminus of the extracellular domain, 16, of IL-20RA. Apolypeptide linker 46, extends from the carboxy terminus of the extracellular domain, 16, of IL-20RA to theCH2 domain 24. -
FIG. 6 shows another possible embodiment of the present invention. The soluble IL-20 receptor ofFIG. 6 is identical to that ofFIG. 1 except for the CH3 domain, 26 ofFIG. 1 , is not present on the embodiment ofFIG. 6 . -
FIG. 7 shows a soluble IL-20 receptor construct that is identical to the construct ofFIG. 1 except both the CH2, and CH3 domains are absent. -
FIG. 8 shows a construct wherein both IL-20RA, 16, and IL-20RB have a polypeptide linker, 48, fused to their respective carboxyl termini. Each polypeptide linker has two cysteine residues such that when they are expressed the cysteines form two disulfide bonds, 50 and 52. - The present invention fills this need by providing a newly discovered soluble receptor to Interleukin-20 (IL-20). The soluble receptor can be used to down-regulate IL-20 and thus treat inflammatory diseases such as psoriasis and inflammatory lung diseases.
- IL-20 was formally called ‘Zcyto10’, (International Patent Publication No. WO 99/27103) and has the amino acid sequences of SEQ ID NOs: 1-9. The receptor to IL-20 is comprised of two chains, an alpha chain and a beta chain. The alpha chain, hereinafter referred to as IL-20RA, was formally called ZcytoR7. See U.S. Pat. No. 5,945,511. The beta chain, hereinafter referred to as IL-20RB, was formally called DIRS1. See International Patent Application No. PCT/US99/03735. The present invention is a soluble receptor comprised of the extracellular domain of IL-20RA and the extracellular domain of IL-20RB.
- The present invention encompasses an isolated soluble receptor comprised of an ‘IL-20RA’ subunit and an ‘IL-20RB’ subunit, wherein the IL-20A subunit is comprised of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 38, 55, 63 and 65, and the IL-20RB subunit is comprised of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 59, 61, 67, 68 and 69. The IL-20RA and IL-20RB subunits are generally linked together by a polypeptide linker. The linking can be by any means but generally by a peptide bond or a disulfide bond between a polypeptide connected to the IL20RA subunit and a polypeptide connected to the IL-20RB subunit. The present invention is also directed towards isolated polynucleotides that encode the novel IL-20RA and IL-20RB polypeptides of the present invention.
- In one embodiment the IL-20RA subunit is fused to the constant region of the heavy chain of an immunoglobulin (Ig) molecule or a portion thereof and the IL-20RB subunit is fused to the constant region of the light chain of an Ig molecule such that the constant region of the light chain is disulfide bonded to the constant region of the heavy chain, generally to a cysteine residue on the hinge region of the heavy chain. Also the opposite can occur, the IL-20RA subunit can be fused to the constant region of the light chain of an Ig molecule and the IL-20RB subunit can be fused to the constant region of the heavy chain of an Ig molecule.
- In one embodiment of the soluble receptor of the present invention, the IL-20RA subunit fused to the constant region of the heavy chain is comprised of an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 53, 54 and 62, and the IL-20RB subunit fused to the constant region of the light chain of the Ig molecule is comprised of an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 57, 58, and 60.
- Also claimed is a protein having a first polypeptide and a second polypeptide wherein the first polypeptide is comprised of an amino acid sequence of SEQ ID NO: 66 and the second polypeptide is comprised of an amino acid sequence selected from the group consisting of SEQ ID NOs: 70 and 71. The resultant protein can be used to generate antibodies to the IL-20RA subunit and the IL-20RB subunit.
- Prior to setting forth the invention in more detail, it may be helpful to the understanding thereof to define the following terms.
- The terms “amino-terminal” and “carboxyl-terminal” are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.
- As used herein, the term “antibody fusion protein” refers to a recombinant molecule that comprises an antibody component and a therapeutic agent. Examples of therapeutic agents suitable for such fusion proteins include immunomodulators (“antibody-immunomodulator fusion protein”) and toxins (“antibody-toxin fusion protein”).
- The term “complement/anti-complement pair” denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions. For instance, biotin and avidin (or streptavidin) are prototypical members of a complement/anti-complement pair. Other exemplary complement/anti-complement pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like. Where subsequent dissociation of the complement/anti-complement pair is desirable, the complement/anti-complement pair preferably has a binding affinity of <109 M−1.
- The term “complements of a polynucleotide molecule” is a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.
- The term “contig” denotes a polynucleotide that has a contiguous stretch of identical or complementary sequence to another polynucleotide. Contiguous sequences are said to “overlap” a given stretch of polynucleotide sequence either in their entirety or along a partial stretch of the polynucleotide. For example, representative contigs to the polynucleotide sequence 5′-ATGGCTTAGCTT-3′ are 5′-TAGCTTgagtct-3′ and 3′-gtcgacTACCGA-5′.
- The term “degenerate nucleotide sequence” denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide). Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp).
- The term “expression vector” is used to denote a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
- The term “isolated”, when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5′ and 3′ untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78 (1985).
- An “isolated” polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure. When used in this context, the term “isolated” does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
- The term “operably linked”, when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.
- A “polynucleotide” is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′ end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. Sizes of polynucleotides are expressed as base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases (“kb”). Where the context allows, the latter two terms may describe polynucleotides that are single-stranded or double-stranded. When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term “base pairs”. It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; thus all nucleotides within a double-stranded polynucleotide molecule may not be paired. Such unpaired ends will in general not exceed 20 nucleotides in length.
- A “polypeptide” is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as “peptides”.
- The term “promoter” is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5′ non-coding regions of genes.
- A “protein” is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
- The term “receptor” denotes a cell-associated protein that binds to a bioactive molecule (i.e., a ligand) and mediates the effect of the ligand on the cell. Membrane-bound receptors are characterized by a multi-domain structure comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal transduction. Binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule(s) in the cell. This interaction in turn leads to an alteration in the metabolism of the cell. Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids. In general, receptors can be membrane bound, cytosolic or nuclear, monomeric (e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor).
- The term “secretory signal sequence” denotes a DNA sequence that encodes a polypeptide (a “secretory peptide”) that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized. The larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
- The term “splice variant” is used herein to denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene.
- Molecular weights and lengths of polymers determined by imprecise analytical methods (e.g., gel electrophoresis) will be understood to be approximate values. When such a value is expressed as “about” X or “approximately” X, the stated value of X will be understood to be accurate to ±10%.
- As was stated above, IL-20 (formally called Zcyto10) is defined and methods for producing it and antibodies to IL-20 are contained in International Patent Application No. PCT/US98/25228, publication no. WO 99/27103,published Nov. 25, 1998 and U.S. patent application Ser. No. 09/313,458 filed May 17, 1999. The polynucleotide and polypeptide of human IL-20 are represented by SEQ ID NOs: 1 - 4, and mouse IL-20 by SEQ ID NOs: 5-9.
- The receptor to IL-20 has been discovered and is a heterodimer comprised of the polypeptide termed ‘IL-20RA’ (formally called Zcytor7) and a polypeptide termed ‘IL-20RB’. The IL-20RA polypeptide, nucleic acid that encodes it, antibodies to IL-20RA and methods for producing it are disclosed in U.S. Pat. No. 5,945,511 issued Aug. 31, 1999. SEQ ID NOs: 10 - 12 are the IL-20RA polynucleotides and polypeptides. The extracellular domain of the human IL-20RA is comprised of a polypeptide selected from the group consisting of SEQ ID NOs: 12, 55, 63 and 65, the full-length receptor subunit being comprised of SEQ ID NO: 11. The extracellular domain of mouse IL-20RA is SEQ ID NO: 38, SEQ ID NO: 37 being the entire mouse IL-20RA.
- The extracellular domain of IL-20RB. SEQ ID NOs: 13-14, and a variant SEQ ID NOs: 18 and 19) is comprised of a polypeptide selected from the group consisting of SEQ ID NOs: 15, 59, 61, 67, 68 and 69. Preferably, the extracellular domain of the IL-20RA polypeptide and the extracellular domain of the IL-20RB polypeptide are covalently linked together. In a preferred embodiment one extracellular subunit polypeptide has a constant region of a heavy chain of an immunoglobulin fused to its carboxy terminus and the other extracellular subunit has a constant light chain of an immunoglobulin (Ig) fused to its carboxy terminus such that the two polypeptides come together to form a soluble receptor and a disulfide bond is formed between the heavy and the light Ig chains. In another method, a peptide linker could be fused to the two carboxy-termini of the polypeptides to form a covalently bonded soluble receptor.
- SEQ ID NOs: 22 and 23 are constructs of the extracellular domain of IL-20RA fused to a mutated human immunoglobulin gamma 1 constant region produced according to the procedure set forth in example 5. SEQ ID NO: 62 is the predicted mature sequence without the signal sequence. SEQ ID NOs: 20 and 21 are constructs of the extracellular domain of IL-20RB fused to wild type human immunoglobulin kappa light chain constant region produced according to the procedure of example 5. SEQ ID NO: 60 is the predicted mature sequence without the signal sequence.
FIG. 1 depicts the heterotetramer produced by example 5. - SEQ ID NOs: 52 and 53 are constructs of the extracellular domain of IL-20RA fused to a mutated human immunoglobulin gamma 1 constant region produced according to the procedure set forth in example 12. SEQ ID NO: 54 is the predicted mature sequence without the signal sequence. SEQ ID NOs: 56 and 57 are constructs of the extracellular domain of IL-20RB fused to wild type human immunoglobulin kappa light chain constant region produced according to the procedure of example 12. SEQ ID NO: 58 is the predicted mature sequence without the signal sequence. The resultant heterotetramer is almost identical to that produced by example 5, the primary difference being the absence of a polypeptide linker between the extracellular domains and the beginning of the Ig constant regions, 22 in
FIG. 1 . Hereinafter, the term “extracellular domain of a receptor” means the extracellular domain of the receptor or a portion of the extracellular domain that is necessary for binding to its ligand, in this case the ligand being IL-20. - One can link together the extracellular domains of IL-20RA and IL-20RB in a number of ways such that the resultant soluble receptor can bind to IL-20.
FIGS. 1-8 illustrate a representative number of embodiments of the present invention. Common elements in each of the drawings are given the same number.FIG. 1 represents the embodiment of the present invention produced according to example 5 below. The soluble receptor construct, designated 10, is comprised of two IL-20 binding site polypeptide chains designated 12 and 14. Each binding site is comprised of the extracellular domain of IL-20RA, designated 16, and the extracellular domain of IL-20RB designated 18. - The extracellular domain, 16, of IL-20RA is linked to the constant heavy one (CH1) domain, 20, of the human immunoglobulin gamma 1 heavy chain constant region via
linker 22, which is SEQ ID NO:72. The CH1 domain, 20, is then linked to the CH2 domain, 24, viahinge region 23. The CH2 domain, 24, is linked to the CH3 domain, 26, viahinge region 25. - Comparing the construct of
FIG. 1 with SEQ ID NO:22, the extracellular domain, 16, of IL-20RA extends fromamino acid residues 36, a valine, to and including amino acid residue 249, a glutamine of SEQ ID NO:22. Polypeptide linker, 22, extends from amino acid residue 250, a glycine to and including amino acid residue 264, a serine, of SEQ ID NO:22. The CH1 domain, 22 ofFIG. 1 , extends from amino acid residue 265, an alanine, to and including amino acid residue 362, a valine, of SEQ ID NO:22.Hinge region 23 ofFIG. 1 extends from amino acid residue 363, a glutamic acid to and including amino acid residue 377, a proline, of SEQ ID NO: 22.Chains disulfide bonds - Extracellular domain, 18, of IL-20RB is linked to the constant region of the human kappa light chain (CL), 34 of
FIG. 1 viapolypeptide linker 32, which is the polypeptide SEQ ID NO: 72. The extracellular domain, 18, of IL-20RB extends fromamino acid residue 30, a valine, to and including amino acid residue 230, an alanine, of SEQ ID NO: 20. Polypeptide linker, 32, extends from amino acid residue 231, a glycine, to and including amino acid residue 245, a serine, of SEQ ID NO:20. The kappa constant light region, 34, extends from amino acid residue 246, an arginine, to and including the final amino acid residue 352, a cysteine, of SEQ ID NO:20. The cysteine at position 352 of SEQ ID NO: 20 forms a disulfide bond, 36 inFIG. 1 , with the cysteine at position 367 of SEQ ID NO: 22. Theconstant light chain 34 is thus linked to the hinge region, 23, by disulfide bond, 36. In this way, the extracellular domain, 16, of IL-20RA is linked to the extracellular domain, 18, of IL-20RB to form a soluble receptor. - If the cysteine residues at positions 373 and 376 of SEQ ID NO:22 were changed to different amino acid residues, the two IL-20 binding polypeptides, 12 and 14, would not be disulfide bonded together and would form a construct shown in
FIG. 2 . having hinge region, 27. -
FIG. 3 shows a very simplesoluble receptor 38 of the present invention wherein extracellular domain, 16, of IL-20RA is connected to the extracellular domain, 18, of IL-20RB by means of a polypeptide linker, 40. The polypeptide linker extends from the amino terminus of extracellular domain, 16, of IL-20RA and is connected to the carboxyl terminus of the extracellular domain, 18, of IL-20RB. The polypeptide linker should be between 100-240 amino acids in length, preferably about 170 amino acid residues in length. A suitable linker would be comprised of glycine and serine residues. A possible linker would be multiple units of SEQ ID NO: 72, preferably about 12. -
FIG. 4 shows an embodiment that has the extracellular domain, 16, of IL-20RA linked to the extracellular domain, 18, of IL-20RB by means oflinker 40, as inFIG. 3 . While the extracellular domain, 16, of IL-20RA is linked to the CH1 domain, 20, as inFIG. 1 by means ofpolypeptide linker 42, which should be about 30 amino acid residues in length. An ideal linker would be comprised of glycine and serine as in SEQ ID NO: 72, and the hinge sequence, 23 ofFIG. 1 . -
FIG. 5 shows another possible embodiment of the present invention. In this embodiment, apolypeptide linker 44 of about 15 amino acid residue, e.g. SEQ ID NO: 72, links the carboxyl terminus of the extracellular domain, 18, of IL-20RB with the amino terminus of the extracellular domain, 16, of IL-20RA. Apolypeptide linker 46 of about 30 amino acid residues extends from the carboxy terminus of the extracellular domain, 16, of IL-20RA to the CH2 domain. The carboxyl terminus oflinker 46 would preferably be comprised of the hinge region extending from amino acid residue 363, a glutamic acid to and including amino acid residue 377, a proline, of SEQ ID NO: 22. Nonetheless,polypeptide linker 46 would ideally have at least one cysteine residue at its carboxyl terminus so a disulfide bond could be formed. - The soluble IL-20 receptor of
FIG. 6 is identical to that ofFIG. 1 except for the CH3 domain, 26 ofFIG. 1 , is not present on the embodiment ofFIG. 6 . The CH3 region begins at amino acid residue 488, a glycine, and extends to the last residue 594 of SEQ ID NO: 22. -
FIG. 7 shows a soluble IL-20 receptor construct that is identical to the construct ofFIG. 1 except both the CH2, and CH3 domains are absent. The CH2 and CH3 domains run from amino acid residue 378, an alanine, to the end of the polypeptide sequence of SEQ ID NO: 22. -
FIG. 8 shows a construct wherein both IL-20RA, 16, and IL-20RB have a polypeptide linker, 48, fused to their respective carboxyl termini. Each polypeptide linker has two cysteine residues such that when they are expressed the cysteines form two disulfide bonds, 50 and 52. In this case the polypeptide linker is comprised of the hinge region, 23 inFIG. 1 . The hinge region is comprised of amino acid residues 363, a glutamine, to and including amino acid residue 377 of SEQ ID NO: 22. - In another aspect of the invention, a method is provided for producing a soluble receptor comprised of extracellular domains of IL-20RA and IL-20RB comprising (a) introducing into a host cell a first DNA sequence comprised of a transcriptional promoter operatively linked to a first secretory signal sequence followed downstream by and in proper reading frame the DNA that encodes the extracellular portion of IL-20RA and the DNA that encodes an immunoglobulin light chain constant region;(b) introducing into the host cell a second DNA construct comprised of a transcriptional promoter operatively linked to a second secretory signal followed downstream by and in proper reading frame a DNA sequence that encodes the extracellular portion of IL-20RB and a DNA sequence that encodes an immunoglobulin heavy chain constant region domain selected from the group consisting of CH1, CH2, CH3 and CH4; (c) growing the host cell in an appropriate growth medium under physiological conditions to allow the secretion of a fusion protein comprised of the extracellular domain of IL-20RA and IL-20RB; and (d) isolating the polypeptide from the host cell. In one embodiment, the second DNA sequence further encodes an immunoglobulin heavy chain hinge region wherein the hinge region is joined to the heavy chain constant region domain. In another embodiment, the second DNA sequence further encodes an immunoglobulin variable region joined upstream of and in proper reading frame with the immunoglobulin heavy chain constant region.
- In an alternative embodiment, a method is provided for producing a soluble receptor comprised of the extracellular domains of IL-20RA and IL-20RB comprising (a) introducing into a host cell a first DNA sequence comprised of a transcriptional promoter operatively linked to a first secretory signal sequence followed downstream by and in proper reading frame the DNA that encodes the extracellular portion of IL-20RB and the DNA that encodes an immunoglobulin light chain constant region;(b) introducing into the host cell a second DNA construct comprised of a transcriptional promoter operatively linked to a second secretory signal followed downstream by and in proper reading frame a DNA sequence that encodes the extracellular portion of IL-20RA and a DNA sequence that encodes an immunoglobulin heavy chain constant region domain selected from the group consisting of CH1, CH2, CH3 and CH4; (c) growing the host cell in an appropriate growth medium under physiological conditions to allow the secretion of a dimerized heterodimeric fusion protein comprised of the extracellular domain of IL-20RA and IL-20RB; and (d) isolating the dimerized polypeptide from the host cell. In one embodiment, the second DNA sequence further encodes an immunoglobulin heavy chain hinge region wherein the hinge region is joined to the heavy chain constant region domain. In another embodiment, the second DNA sequence further encodes an immunoglobulin variable region joined upstream of and in proper reading frame with the immunoglobulin heavy chain constant region. (See U.S. Pat. No. 5,843,725.)
- A polynucleotide, generally a cDNA sequence, encodes the described polypeptides herein. A cDNA sequence that encodes a polypeptide of the present invention is comprised of a series of codons, each amino acid residue of the polypeptide being encoded by a codon and each codon being comprised of three nucleotides. The amino acid residues are encoded by their respective codons as follows.
- Alanine (Ala) is encoded by GCA, GCC, GCG or GCT.
- Cysteine (Cys) is encoded by TGC or TGT.
- Aspartic acid (Asp) is encoded by GAC or GAT.
- Glutamic acid (Glu) is encoded by GAA or GAG.
- Phenylalanine (Phe) is encoded by TTC or TTT.
- Glycine (Gly) is encoded by GGA, GGC, GGG or GGT.
- Histidine (His) is encoded by CAC or CAT.
- Isoleucine (Ile) is encoded by ATA, ATC or ATT.
- Lysine (Lys) is encoded by AAA, or AAG.
- Leucine (Leu) is encoded by TTA, TTG, CTA, CTC, CTG or CTT.
- Methionine (Met) is encoded by ATG.
- Asparagine (Asn) is encoded by AAC or AAT.
- Proline (Pro) is encoded by CCA, CCC, CCG or CCT.
- Glutamine (Gln) is encoded by CAA or CAG.
- Arginine (Arg) is encoded by AGA, AGG, CGA, CGC, CGG or CGT.
- Serine (Ser) is encoded by AGC, AGT, TCA, TCC, TCG or TCT.
- Threonine (Thr) is encoded by ACA, ACC, ACG or ACT.
- Valine (Val) is encoded by GTA, GTC, GTG or GTT.
- Tryptophan (Trp) is encoded by TGG.
- Tyrosine (Tyr) is encoded by TAC or TAT.
- It is to be recognized that according to the present invention, when a polynucleotide is claimed as described herein, it is understood that what is claimed are both the sense strand, the anti-sense strand, and the DNA as double-stranded having both the sense and anti-sense strand annealed together by their respective hydrogen bonds. Also claimed is the messenger RNA (mRNA) that encodes the polypeptides of the president invention, and which mRNA is encoded by the cDNA described herein. Messenger RNA (mRNA) will encode a polypeptide using the same codons as those defined herein, with the exception that each thymine nucleotide (T) is replaced by a uracil nucleotide (U).
- One of ordinary skill in the art will also appreciate that different species can exhibit “preferential codon usage.” In general, see, Grantham, et al., Nuc. Acids Res. 8:1893-1912 (1980); Haas, et al. Curr. Biol. 6:315-324 (1996); Wain-Hobson, et al., Gene 13:355-364 (1981); Grosjean and Fiers, Gene 18:199-209 (1982); Holm, Nuc. Acids Res. 14:3075-3087 (1986); Ikemura, J. Mol. Biol. 158:573-597 (1982). As used herein, the term “preferential codon usage” or “preferential codons” is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid. For example, the amino acid Threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential. Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. Introduction of preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein.
- Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell-free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722 (1991); Ellman et al., Methods Enzymol. 202:301 (1991; Chung et al., Science 259:806-809 (1993); and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-1019 (1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs, Turcatti et al., J. Biol. Chem. 271:19991-19998 (1996). Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-7476 (1994). Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions, Wynn and Richards, Protein Sci. 2:395-403 (1993).
- A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues.
- Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis, Cunningham and Wells, Science 244: 1081-1085 (1989); Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-502 (1991). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity as disclosed below to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., J. Biol. Chem. 271:4699-708, 1996. Sites of ligand-receptor interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-312 (1992); Smith et al., J. Mol. Biol. 224:899-904 (1992); Wlodaver et al., FEBS Lett. 309:59-64 (1992).
- Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer, Science 241:53-57 (1988) or Bowie and Sauer, Proc. Natl. Acad. Sci. USA 86:2152-2156 (1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display, e.g., Lowman et al., Biochem. 30:10832-10837 (1991); Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis, Derbyshire et al., Gene 46:145 (1986); Ner et al., DNA 7:127 (1988).
- Variants of the disclosed IL-20, IL-20RA and IL-20RB DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389-391, (1994), Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-10751 (1994) and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated by in vitro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by using a family of parent DNAs, such as allelic variants or DNAs from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid “evolution” of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.
- Mutagenesis methods as disclosed herein can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides in host cells. Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.
- Protein Production
- Polypeptides can be produced in genetically engineered host cells according to conventional techniques. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and Ausubel et al., eds., Current Protocols in Molecular Biology (John Wiley and Sons, Inc., NY, 1987).
- In general, a DNA sequence encoding a polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector. The vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.
- To direct a polypeptide into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector. The secretory signal sequence may be that of the native polypeptides, or may be derived from another secreted protein (e.g., t-PA) or synthesized de novo. The secretory signal sequence is operably linked to the DNA sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. Secretory signal sequences are commonly positioned 5′ to the DNA sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830).
- Alternatively, the secretory signal sequence contained in the polypeptides of the present invention is used to direct other polypeptides into the secretory pathway. The present invention provides for such fusion polypeptides. The secretory signal sequence contained in the fusion polypeptides of the present invention is preferably fused amino-terminally to an additional peptide to direct the additional peptide into the secretory pathway. Such constructs have numerous applications known in the art. For example, these novel secretory signal sequence fusion constructs can direct the secretion of an active component of a normally non-secreted protein, such as a receptor. Such fusions may be used in vivo or in vitro to direct peptides through the secretory pathway.
- Cultured mammalian cells are suitable hosts within the present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection, Wigler et al., Cell 14:725 (1978), Corsaro and Pearson, Somatic Cell Genetics 7:603 (1981); Graham and Van der Eb, Virology 52:456 (1973), electroporation, Neumann et al., EMBO J. 1:841-845 (1982), DEAE-dextran mediated transfection (Ausubel et al., ibid., and liposome-mediated transfection, Hawley-Nelson et al., Focus 15:73 (1993); Ciccarone et al., Focus 15:80 (1993), and viral vectors, Miller and Rosman, BioTechniques 7:980(1989); Wang and Finer, Nature Med. 2:714 (1996). The production of recombinant polypeptides in cultured mammalian cells is disclosed, for example, by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59 (1977) and Chinese hamster ovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Md. In general, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Other suitable promoters include those from metallothionein genes (U.S. Pat. Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.
- Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as “transfectants”. Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as “stable transfectants.” A preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like. Selection systems can also be used to increase the expression level of the gene of interest, a process referred to as “amplification.” Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes. A preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase) can also be used. Alternative markers that introduce an altered phenotype, such as green fluorescent protein, or cell surface proteins such as CD4, CD8, Class I MHC, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.
- Other higher eukaryotic cells can also be used as hosts, including plant cells, insect cells and avian cells. The use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47 (1987). Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al., U.S. Pat. No. 5,162,222 and WIPO publication WO 94/06463. Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV). DNA encoding a polypeptide is inserted into the baculoviral genome in place of the AcNPV polyhedrin gene coding sequence by one of two methods. The first is the traditional method of homologous DNA recombination between wild-type AcNPV and a transfer vector containing the gene flanked by AcNPV sequences. Suitable insect cells, e.g. SF9 cells, are infected with wild-type AcNPV and transfected with a transfer vector comprising a polynucleotide operably linked to an AcNPV polyhedrin gene promoter, terminator, and flanking sequences. See, King, L. A. and Possee, R. D., The Baculovirus Expression System: A Laboratory Guide, (Chapman & Hall, London); O'Reilly, D. R. et al., Baculovirus Expression Vectors: A Laboratory Manual (Oxford University Press, New York, N.Y., 1994); and, Richardson, C. D., Ed., Baculovirus Expression Protocols. Methods in Molecular Biology, (Humana Press, Totowa, N.J. 1995). Natural recombination within an insect cell will result in a recombinant baculovirus that contains coding sequences driven by the polyhedrin promoter. Recombinant viral stocks are made by methods commonly used in the art.
- The second method of making recombinant baculovirus utilizes a transposon-based system described by Luckow, V. A, et al., J Virol 67:4566 (1993). This system is sold in the Bac-to-Bac kit (Life Technologies, Rockville, Md.). This system utilizes a transfer vector, pFastBac1™ (Life Technologies) containing a Tn7 transposon to move the DNA encoding the polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a “bacmid.” The pFastBac1™ transfer vector utilizes the AcNPV polyhedrin promoter to drive the expression of the gene of interest. However, pFastBac1™ can be modified to a considerable degree. The polyhedrin promoter can be removed and substituted with the baculovirus basic protein promoter (also known as Pcor, p6.9 or MP promoter), which is expressed earlier in the baculovirus infection, and has been shown to be advantageous for expressing secreted proteins. See, Hill-Perkins, M. S. and Possee, R. D., J Gen Virol 71:971 (1990); Bonning, B. C. et al., J Gen Virol 75:1551 (1994); and, Chazenbalk, G. D., and Rapoport, B., J Biol Chem 270:1543 (1995). In such transfer vector constructs, a short or long version of the basic protein promoter can be used. Moreover, transfer vectors can be constructed that replace the native secretory signal sequences with secretory signal sequences derived from insect proteins. For example, a secretory signal sequence from Ecdysteroid Glucosyltransferase (EGT), honey bee Melittin (Invitrogen, Carlsbad, Calif.), or baculovirus gp67 (PharMingen, San Diego, Calif.) can be used in constructs to replace the native secretory signal sequence. In addition, transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed polypeptide, for example, a Glu-Glu epitope tag, Grussenmeyer, T. et al., Proc Natl Acad Sci. 82:7952 (1985). Using a technique known in the art, a transfer vector containing a recombinant gene is transformed into E. coli, and screened for bacmids that contain an interrupted lacZ gene indicative of recombinant baculovirus. The bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, e.g. Sf9 cells. Recombinant virus that expresses the polypeptide is subsequently produced. Recombinant viral stocks are made by methods commonly used the art.
- The recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda. See, in general, Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA (ASM Press, Washington, D.C., 1994). Another suitable cell line is the High FiveO™ cell line (Invitrogen) derived from Trichoplusia ni (U.S. Pat. No. 5,300,435). Commercially available serum-free media are used to grow and maintain the cells. Suitable media are Sf900 II™ (Life Technologies) or ESF 921™ (Expression Systems) for the Sf9 cells; and Ex-cellO405™ (JRH Biosciences, Lenexa, Kans.) or Express FiveO™ (Life Technologies) for the T. ni cells. The cells are grown up from an inoculation density of approximately 2−5×105 cells to a density of 1−2×106 cells at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3. The recombinant virus-infected cells typically produce the recombinant polypeptide at 12-72 hours post-infection and secrete it with varying efficiency into the medium. The culture is usually harvested 48 hours post-infection. Centrifugation is used to separate the cells from the medium (supernatant). The supernatant containing the polypeptide is filtered through micropore filters, usually 0.45 μm pore size. Procedures used are generally described in available laboratory manuals (King, L. A. and Possee, R. D., ibid., O'Reilly, D. R. et al., ibid.; Richardson, C. D., ibid.). Subsequent purification of the polypeptide from the supernatant can be achieved using methods described herein.
- Fungal cells, including yeast cells, can also be used within the present invention. Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica. Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat. No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat. No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine). A preferred vector system for use in Saccharomyces cerevisiae is the POTJ vector system disclosed by Kawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media. Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman et al., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) and alcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltosa are known in the art. See, for example, Gleeson et al., J. Gen. Microbiol. 132:3459 (1986) and Cregg, U.S. Pat. No. 4,882,279. Aspergillus cells may be utilized according to the methods of McKnight et al., U.S. Pat. No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al., U.S. Pat. No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Pat. No. 4,486,533.
- The use of Pichia methanolica as host for the production of recombinant proteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use in transforming P. methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation. For polypeptide production in P. methanolica, it is preferred that the promoter and terminator in the plasmid be that of a P. methanolica gene, such as a P. methanolica alcohol utilization gene (AUG1 or AUG2). Other useful promoters include those of the dihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), and catalase (CAT) genes. To facilitate integration of the DNA into the host chromosome, it is preferred to have the entire expression segment of the plasmid flanked at both ends by host DNA sequences. A preferred selectable marker for use in Pichia methanolica is a P. methanolica ADE2 gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), which allows ade2 host cells to grow in the absence of adenine. For large-scale, industrial processes where it is desirable to minimize the use of methanol, it is preferred to use host cells in which both methanol utilization genes (AUG1 and AUG2) are deleted. For production of secreted proteins, host cells deficient in vacuolar protease genes (PEP4 and PRB1) are preferred. Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells. It is preferred to transform P. methanolica cells by electroporation using an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40 milliseconds, most preferably about 20 milliseconds.
- Prokaryotic host cells, including strains of the bacteria Escherichia coli, Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art; see, e.g., Sambrook et al., ibid.). When expressing a polypeptide in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea. The denatured polypeptide can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution. In the latter case, the polypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.
- Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells. A variety of suitable media, including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required. The growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient, which is complemented by the selectable marker carried on the expression vector or co-transfected into the host cell. P. methanolica cells are cultured in a medium comprising adequate sources of carbon, nitrogen and trace nutrients at a temperature of about 25° C. to 35° C. Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors. A preferred culture medium for P. methanolica is YEPD (2% D-glucose, 2% Bacto™ Peptone (Difco Laboratories, Detroit, Much.), 1% Bacto™ yeast extract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).
- Protein Isolation
- It is preferred to purify the polypeptides of the present invention to ≧80% purity, more preferably to ≧90% purity, even more preferably ≧95% purity, and particularly preferred is a pharmaceutically pure state, that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. Preferably, a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.
- Expressed recombinant polypeptides (or chimeric polypeptides) can be purified using fractionation and/or conventional purification methods and media Ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples. Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography. Suitable chromatographic media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred. Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like. Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties. Examples of coupling chemistries include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Methods for binding receptor polypeptides to support media are well known in the art. Selection of a particular method is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affinity Chromatography: Principles & Methods (Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988).
- Polypeptides can be isolated by exploitation of their properties. For example, immobilized metal ion adsorption (IMAC) chromatography can be used to purify histidine-rich proteins, including those comprising polyhistidine tags. Briefly, a gel is first charged with divalent metal ions to form a chelate, Sulkowski, Trends in Biochem. 3:1 (1985). Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents. Other methods of purification include purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography. A protein fused to the Fc portion of an immunoglobulin can be purified using a ‘Protein A column’. Methods in Enzymol., Vol. 182, “Guide to Protein Purification”, M. Deutscher, (ed.),page 529-539 (Acad. Press, San Diego, 1990). Within additional embodiments of the invention, a fusion of the polypeptide of interest and an affinity tag (e.g., maltose-binding protein, an immunoglobulin domain) may be constructed to facilitate purification.
- As used herein, the term “antibodies” includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments, such as F(ab′)2 and Fab proteolytic fragments. Genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as synthetic antigen-binding peptides and polypeptides, are also included. Non-human antibodies may be humanized by grafting non-human CDRs onto human framework and constant regions, or by incorporating the entire non-human variable domains (optionally “cloaking” them with a human-like surface by replacement of exposed residues, wherein the result is a “veneered” antibody). In some instances, humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced.
- A variety of assays known to those skilled in the art can be utilized to detect antibodies that bind to protein or peptide. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.) (Cold Spring Harbor Laboratory Press, 1988). Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmuno-precipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western blot assay, inhibition or competition assay, and sandwich assay.
- The soluble receptors of the present invention can be used to down-regulate IL-20, which has been shown to be involved in a number of inflammatory processes. Specifically, IL-20 has been shown to up-regulate IL-8. Inflammatory diseases in which IL-8 plays a significant role, and for which a decrease in IL-8 would be beneficial are, adult respiratory disease (ARD), septic shock, multiple organ failure, inflammatory lung injury such as asthma or bronchitis, bacterial pneumonia, psoriasis, eczema, atopic and contact dermatitis, and inflammatory bowel disease such as ulcerative colitis and Crohn's disease. Thus, the soluble receptor to IL-20 of the present invention can be administered to a patient to treat these diseases.
- Two orphan class II cytokine receptors, both of which are expressed in skin, were identified as IL-20 receptor subunits. Both IL-20 receptor subunits are required for ligand binding, distinguishing their role from that of subunits in the four other known class II cytokine receptors. IL-20RA and IL-20RB are also coexpressed in a number of human tissues besides skin, including ovary, adrenal gland, testis, salivary gland, muscle, lung, kidney, heart and to a lesser degree the small intestine suggesting additional target tissues for IL-20 action. We conclude that the IL-20 heterodimeric receptor is structurally similar to other class II cytokine receptors and is expressed in skin where we have demonstrated activity of the IL-20 ligand.
- Two lines of evidence indicate that a role IL-20 and its receptor are involved in psoriasis. This multigenic skin disease is characterized by increased keratinocyte proliferation, altered keratinocyte differentiation, and infiltration of immune cells into the skin. The first line of evidence for a role of IL-20 in psoriasis is that the observed hyperkeratosis and thickened epidermis in the transgenic mice that resemble human psoriatic abnormalities. Decreased numbers of tonofilaments, thought to be related to defective keratinization, are a striking feature of human psoriasis. Intramitochondrial inclusions have been found in both chemically induced and naturally occurring hyperplastic skin conditions in mice. The cause of the inclusions and their effects on mitochondrial function, if any, are unknown. We conclude that IL-20 transgenic mice exhibit many of the characteristics observed in human psoriasis.
- A second line of evidence that implicates the IL-20 receptor in psoriasis is that both IL-20RA and IL-20RB mRNA are markedly upregulated in human psoriatic skin compared to normal skin. Both IL-20 receptor subunits are expressed in keratinocytes throughout the epidermis and are also expressed in a subset of immune and endothelial cells. We propose that increased expression of an activated IL-20 receptor may alter the interactions between endothelial cells, immune cells and keratinocytes, leading to dysregulation of keratinocyte proliferation and differentiation.
- A crucial step in understanding the function of a novel cytokine is the identification and characterization of its cognate receptor. We have successfully used a structure-based approach to isolate a novel interleukin that ultimately led to the isolation of its receptor. IL-20 stimulates signal transduction in the human keratinocyte HaCaT cell line, supporting a direct action of this novel ligand in skin. In addition, IL-1β, EGF and TNF-α, proteins known to be active in keratinocytes and to be involved with proliferative and pro-inflammatory signals in skin, enhance the response to IL-20. In both HaCaT and BHK cells expressing the IL-20 receptor, IL-20 signals through STAT3. Thus, IL-20 binds its receptor on keratinocytes and stimulates a STAT3-containing signal transduction pathway.
- As indicated in the discussion above and the examples below, IL-20 is involved in the pathology of psoriasis. Thus, the soluble receptors of the present invention can be administered to an individual to down-regulate IL-20 and thus treat psoriasis.
- Psoriasis is one of the most common dermatologic diseases, affecting up to 1 to 2 percent of the world's population. It is a chronic inflammatory skin disorder characterized by erythematous, sharply demarcated papules and rounded plaques, covered by silvery micaceous scale. The skin lesions of psoriasis are variably pruritic. Traumatized areas often develop lesions of psoriasis. Additionally, other external factors may exacerbate psoriasis including infections, stress, and medications, e.g. lithium, beta blockers, and anti-malarials.
- The most common variety of psoriasis is called plaque type. Patients with plaque-type psoriasis will have stable, slowly growing plaques, which remain basically unchanged for long periods of time. The most common areas for plaque psoriasis to occur are the elbows knees, gluteal cleft, and the scalp. Involvement tends to be symmetrical. Inverse psoriasis affects the intertriginous regions including the axilla, groin, submammary region, and navel, and it also tends to affect the scalp, palms, and soles. The individual lesions are sharply demarcated plaques but may be moist due to their location. Plaque-type psoriasis generally develops slowly and runs an indolent course. It rarely spontaneously remits.
- Eruptive psoriasis (guttate psoriasis) is most common in children and young adults. It develops acutely in individuals without psoriasis or in those with chronic plaque psoriasis. Patients present with many small erythematous, scaling papules, frequently after upper respiratory tract infection with beta-hemolytic streptococci. Patients with psoriasis may also develop pustular lesions. These may be localized to the palms and soles or may be generalized and associated with fever, malaise, diarrhea, and arthralgias.
- About half of all patients with psoriasis have fingernail involvement, appearing as punctate pitting, nail thickening or subungual hyperkeratosis. About 5 to 10 percent of patients with psoriasis have associated joint complaints, and these are most often found in patients with fingernail involvement. Although some have the coincident occurrence of classic Although some have the coincident occurrence of classic rheumatoid arthritis, many have joint disease that falls into one of five type associated with psoriasis: (1) disease limited to a single or a few small joints (70 percent of cases); (2) a seronegative rheumatoid arthritis-like disease; (3) involvement of the distal interphalangeal joints; (4) severe destructive arthritis with the development of “arthritis mutilans”; and (5) disease limited to the spine.
- Psoriasis can be treated by administering antagonists to IL-20. The preferred antagonists are either a soluble receptor to IL-20 or antibodies, antibody fragments or single chain antibodies that bind to either the IL-20 receptor or to IL-20. The antagonists to IL-20 can be administered alone or in combination with other established therapies such as lubricants, keratolytics, topical corticosteroids, topical vitamin D derivatives, anthralin, systemic antimetabolites such as methotrexate, psoralen-ultraviolet-light therapy (PUVA), etretinate, isotretinoin, cyclosporine, and the topical vitamin D3 derivative calcipotriol. The antagonists, in particularly the soluble receptor or the antibodies that bind to IL-20 or the IL-20 receptor can be administered to individual subcutaneously, intravenously, or transdermally using a cream or transdermal patch that contains the antagonist of IL-20. If administered subcutaneously, the antagonist can be injected into one or more psoriatic plaques. If administered transdermally, the antagonists can be administered directly on the plaques using a cream containing the antagonist to IL-20.
- The soluble receptor of IL-20 of the present invention can be administered to a person who has asthma, bronchitis or cystic fibrosis or other inflammatory lung disease to treat the disease. The antagonists can be administered by any suitable method including intravenous, subcutaneous, bronchial lavage, and the use of inhalant containing an antagonist to IL-20.
- The quantities of the IL-20 soluble necessary for effective therapy will depend upon many different factors, including means of administration, target site, physiological state of the patient, and other medications administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in vivo administration of these reagents Animal testing of effective doses for treatment of particular disorders will provide further predictive indication of human dosage. Methods for administration include oral, intravenous, peritoneal, intramuscular, transdermal or administration into the lung or trachea in spray form by means or a nebulizer or atomizer. Pharmaceutically acceptable carriers will include water, saline, buffers to name just a few. Dosage ranges would ordinarily be expected from 1 μg to 1000 μg per kilogram of body weight per day. A dosage for an average adult of the IL-20 soluble receptor would be about 25 mg given twice weekly as a subcutaneous injection. Injections could be given at the site of psoriatic lesions for the treatment of psoriasis. For subcutaneous or intravenous administration of the antagonist to IL-20, the antibody or soluble receptor can be in phosphate buffered saline. Also in skin diseases such as psoriasis, the antagonist to IL-20 can be administered via an ointment or transdermal patch. The doses by may be higher or lower as can be determined by a medical doctor with ordinary skill in the art. For a complete discussion of drug formulations and dosage ranges see Remington's Pharmaceutical Sciences, 18th Ed., (Mack Publishing Co., Easton, Pa., 1996), and Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 9th Ed. (Pergamon Press 1996).
- The invention is further illustrated by the following non-limiting examples.
- Normal Human Epidermal neonatal keratinocytes (NHEK) (from Clonetics) at passage 2 were plated and grown to confluency in 12 well tissue culture plates. KGM (Keratinocyte growth media) was purchased from Clonetics. When cells reached confluency, they were washed with KGM media minus growth factors=KBM (keratinocyte basal media). Cells were serum starved in KBM for 72 hours prior to the addition of test compounds. Thrombin at 1 I.U./mL and trypsin at 25 nM were used as positive controls. One mL of media/well was added. KBM only was used as the negative control.
- IL-20 was made up in KBM media and added at varying concentrations, from 2.5 μg/ml down to 618 ng/mL in a first experiment and from 2.5 μg/mL down to 3 ng/mL in a second experiment.
- Cells were incubated at 37° C., 5% CO2 for 48 hours. Supernatants were removed and frozen at −80° C. for several days prior to assaying for IL-8 and GM-CSF levels. Human IL-8 Immunoassay kit #D8050 (RandD Systems, Inc.) and human GM-CSF Immunoassay kit #HSGMO (RandD Systems, Inc.) were used to determine cytokine production following manufacturer's instructions.
- The results indicated that the expression of IL-8 and GM-CSF were induced by IL-20.
- Two PCR primers were designed based on the sequence from International Patent Application No. PCT/US99/03735 (publication no. WO 99/46379) filed on Mar. 8, 1999. SEQ ID NO: 16 contains the ATG (Met1) codon with an EcoRI restriction site, SEQ ID NO: 17 contains the stop codon (TAG) with an XhoI restriction site. The PCR amplification was carried out using a human keratinocyte (HaCaT) cDNA library DNA as a template and SEQ ID NO: 16 and SEQ ID NO: 17 as primers. The PCR reaction was performed as follows: incubation at 94° C. for 1 min followed by 30 cycles of 94° C. for 30 sec and 68° C. for 2 min, after additional 68° C. for 4 min, the reaction was stored at 4° C. The PCR products were run on 1% Agarose gel, and a 1 kb DNA band was observed. The PCR products were cut from the gel and the DNA was purified using a QIAquick Gel Extraction Kit (Qiagen). The purified DNA was digested with EcoRI and XhoI, and cloned into a pZP vector that was called pZP7N. A pZP plasmid is a mammalian expression vector containing an expression cassette having the mouse metallothionein-1 promoter, human tPA leader peptide, multiple restriction sites for insertion of coding sequences, a Glu-Glu tag, and a human growth hormone terminator. The plasmid also has an E. coli origin of replication, a mammalian selectable marker expression unit having an SV40 promoter, an enhancer and an origin of replication, as well as a DHFR gene, and the SV40 terminator. Several IL-20RB-pZP7N clones were sequenced. They all contain three non-conservative mutations compared with the sequence of IL-20RB in PCT/US99/03735: (sequence IL-20RB-pZP7N), 146 Pro (CCC)—Thr (ACC), 148 His (CAT)—Asp (GAT), and 171 Thr (ACG)—Arg (AGG).
- To verify the three substitutions in IL-20RB-pZP7N clone, PCR amplification was carried out using three difference cDNA sources—fetal skin marathon cDNA, HaCaT cDNA library DNA, and prostate smooth muscle cDNA library DNA—as templates. The PCR products were gel purified and sequenced. The sequence of each of the three PCR products was consistent with that of the IL-20RB-pZP7N clone. IL-20RB is SEQ ID NO: 13 and 14, and the mature extracellular domain is SEQ ID NO: 15.
- A cell-based binding assay was used to verify IL-20 binds to IL-20RA- IL-20RB heterodimer.
- Expression vectors containing known and orphan Class II cytokine receptors (including IL-20RA and IL-20RB) were transiently transfected into COS cells in various combinations, which were then assayed for their ability to bind biotin-labeled IL-20 protein. The results show IL-20RB- IL-20RA heterodimer is a receptor for IL-20. The procedure used is described below.
- The COS cell transfection was performed in a 12-well tissue culture plate as follows: 0.5 μg DNA was mixed with medium containing 5 μl lipofectamine in 92 μl serum free Dulbecco's modified Eagle's medium (DMEM) (55 mg sodium pyruvate, 146 mg L-glutamine, 5 mg transferrin, 2.5 mg insulin, 1 μg selenium and 5 mg fetuin in 500 ml DMEM), incubated at room temperature for 30 minutes and then added to 400 μl serum free DMEM media. This 500 μl mixture was then added to 1.5×105 COS cells/well and incubated for 5 hours at 37° C. 500
μl 20% fetal bovine serum (FBS) DMEM media was added and incubated overnight. - The assay, a modification of the “secretion trap” (Davis, S., et al., Cell 87: 1161-1169 (1996), was performed as follows: cells were rinsed with PBS/1% bovine serum albumin (BSA) and blocked for 1 hour with TNB (0.1 M Tris-HCl, 0.15 M NaCl and 0.5% Blocking Reagent (NEN Renaissance TSA-Direct Kit Cat #NEL701) in water). This was followed by a one-hour incubation with 3 μg/ml biotinylated IL-20 protein in TNB. Cells were washed with PBS/1% BSA and incubated for another hour with 1:300 diluted streptavidin-HRP (NEN kit) in TNB. Following another wash, cells were fixed for 15 minutes with 1.8% Formaldehyde in phosphate-buffered saline (PBS). Cells were then washed with TNT (0.1 M Tris-HCL, 0.15 M NaCl, and 0.05% Tween-20 in water). Positive binding signals were detected following a five-minute incubation with fluorescein tyramide reagent diluted 1:50 in dilution buffer (NEN kit). Cells were washed with TNT, preserved with Vectashield Mounting Media (Vector Labs) diluted 1:5 in TNT, and visualized using an FITC filter on an inverted fluorescent microscope.
- The human keratinocyte cell line, HaCaT was grown at 37° C. to several days post-confluence in T-75 tissue culture flasks. At this point, normal growth media (DMEM+10% FBS) was removed and replaced with serum-free media. Cells were then incubated for two days at 37° C. DMEM was then removed and four flasks of cells per treatment were treated with one of each of the following conditions for four hours at 37° C.: recombinant human (rh) IL-1 alpha at 5 ng/mL, rh IL-1 alpha at 20 ng/mL, rh IL-1 alpha at 5 ng/mL +IL-20 at 1 μg/mL, IL-20 at 1 μg/mL, or rh IL-10 at 10 ng/mL.
- Following cytokine treatment, media was removed and cells were lysed using a guanidium thiocyanate solution. Total RNA was isolated from the cell lysate by an overnight spin on a cesium chloride gradient. The following day, the RNA pellet was resuspended in a TE/SDS solution and ethanol precipitated. RNA was then quantitated using a spectrophotometer, followed by a DNase treatment as per Section V.B. of Clontech's Atlas™ cDNA Expression Arrays User Manual (version PT3140-1/PR9X390, published Nov. 5, 1999). Quality of RNA samples was verified by purity calculations based on spec readings, and by visualization on agarose gel. Genomic contamination of the RNA samples was ruled out by PCR analysis of the beta-actin gene.
- Clontech's protocols for polyA+ enrichment, probe synthesis and hybridization to Atlas™ arrays were followed (see above, plus Atlas™ Pure Total RNA Labeling System User Manual, PT3231-1/PR96157, published 6/22/99). Briefly, polyA+ RNA was isolated from 50 mg of total RNA using streptavidin coated magnetic beads (by Clontech, Paolo Alto, Calif.) and a magnetic particle separator. PolyA+ RNA was then labeled with alpha32P-dATP via RT-PCR. Clontech CDS primers specific to the 268 genes on the Atlas™ human cytokine/receptor array (Cat. #7744-1) were used in the reaction. Labeled probe was isolated using column chromatography and counted in scintillation fluid.
- Atlas™ arrays were pre-hybridized with Clontech ExpressHyb plus 100 mg/mL heat denatured salmon sperm DNA for at least thirty minutes at 68° C. with continuous agitation. Membranes were then hybridized with 1.9×106 CPM/mL (a total of 1.14×107 CPM) overnight at 68° C. with continuous agitation. The following day, membranes were washed for thirty minutes ×4 in 2×SSC, 1% SDS at 68° C., plus for thirty minutes ×1 in 0.1×SSC, 0.5% SDS at 68° C., followed by one final room temperature wash for five minutes in 2×SSC. Array membranes were then placed in Kodak plastic pouches sealed and exposed to a phosphor imager screen overnight at room temperature. The next day, phosphor screens were scanned on a phosphor imager and analyzed using Clontech's Atlaslmage™ 1.0 software.
-
- 1. Tumor necrosis factor (TNF) was up-regulated 1.9-2.4 fold by IL-20.
- 2. Placental growth factors 1 & 2 (PLGF) were up-regulated 1.9-2.0 fold by IL-20.
- 3. Coagulating factor II receptor was up-regulated 2.0-2.5 fold by IL-20.
- 4. Calcitonin receptor was up-regulated 2.2-2.3 fold by IL-20.
- 5. TNF-inducible hyaluronate-binding protein TSG-6 was up-regulated 2.1-2.2 fold by IL-20.
- 6. Vascular endothelial growth factor (VEGF) receptor-1 precursor, tyrosine-protein kinase receptor (FLT-1) (SFLT) was up-regulated 2.1-2.7 fold by IL-20.
- 7. MRP-8 (calcium binding protein in macrophages MIF- related) was up-regulated 2.9-4.1 fold by IL-20.
- 8. MRP-14 (calcium binding protein in macrophages MIF-related) was up-regulated 3.0-3.8 fold by IL-20.
- 9. Relaxin H2 was up-regulated 3.14 fold by IL-20.
- 10. Transforming growth factor beta (TGFβ) receptor III 300 kDa was up-regulated 2.4-3.6 fold by IL-20.
Genes Showing Synergy with IL-20 +IL-1 Treatment - 1. Bone morphogenic protein 2a was up-regulated 1.8 fold with IL-20 treatment alone, 2.5 fold with IL-1 treatment alone, and 8.2 fold with both IL-20 and IL-1 treatment together.
- 2. MRP-8 was up-regulated 2.9 fold with IL-20 treatment alone, 10.7 fold with IL-1 treatment alone and 18.0 fold with both IL-20 and IL-1 treatment together.
- 3. Erythroid differentiation protein (EDF) was up-regulated 1.9 fold with IL-20 treatment alone, 9.7 fold with IL-1 treatment alone and 19.0 fold with both IL-20 and IL-1 treatment together.
- 4. MRP-14 (calcium binding protein in macrophages, MIF related) was up-regulated 3.0 fold with IL-20 treatment alone, 12.2 fold with IL-1 treatment alone and 20.3 fold with both IL-20 and IL-1 treatment together.
- 5. Heparin-binding EGF-like growth factor was up-regulated 2.0 fold with IL-20 treatment alone, 14 fold with IL-1 treatment alone and 25.0 fold with both IL-20 and IL-1 treatment together.
- 6. Beta-thromboglobulin-like protein was up-regulated 1.5 fold with IL-20 treatment alone, 15 fold with IL-1 treatment alone and 27 fold with both IL-20 and IL-1 treatment together.
- 7. Brain-derived neurotrophic factor (BDNF) was up-regulated 1.7 fold with IL-20 treatment alone, 25 fold with IL-1 treatment alone and 48 fold with both IL-20 and IL-1 treatment together.
- 8. Monocyte chemotactic and activating factor MCAF was up-regulated 1.3 fold with IL-20 treatment alone, 32 fold with IL-1 treatment alone and 56 fold with both IL-20 and IL-1 treatment together.
- The expression vector pEZE3 was used to express the recombinant IL-20 receptor-Ig fusion protein. The plasmid pEZE3 is derived from pDC312. pDC312 was obtained through license from Immunex Corporation. The plasmids pDC312 and pEZE3 contain an EASE segment as described in WO 97/25420. The presence of the EASE segment in an expression vector can improve expression of recombinant proteins two to eight fold in stable cell pools.
- The plasmid pEZE3 is a tricistronic expression vector that may be used to express up to three different proteins in mammalian cells, preferably Chinese Hamster Ovary (CHO) cells. The pEZE3 expression unit contains the cytomegalovirus (CMV) enhancer/promoter, the adenovirus tripartite leader sequence, a multiple cloning site for insertion of the coding region for the first recombinant protein, the poliovirus type 2 internal ribosome entry site, a second multiple cloning site for insertion of the coding region for the second recombinant protein, an encephalomyocarditis virus internal ribosome entry site, a coding segment for mouse dihydrofolate reductase, and the SV40 transcription terminator. In addition, pEZE3 contains an E. coli origin of replication and the bacterial beta lactamase gene.
- The IL-20 receptor-Ig fusion protein is a disulfide linked heterotetramer consisting of two chains of the extracellular domain of the human IL-20RB fused to the wild type human immunoglobulin kappa light chain constant region and two chains of the human IL-20RA protein extracellular domain fused to a mutated human immunoglobulin gamma 1 constant region. The human immunoglobulin gamma 1 constant region contains amino acid substitutions to reduce FcyRI binding and Clq complement fixation.
- The human IL-20RB extracellular domain human immunoglobulin kappa light chain constant region fusion construct was generated by overlap PCR. The IL-20RB coding segment consists of amino acids 1 to 230. The template used for the PCR amplification of the IL-20R segment was generated IL-20RB human kappa light chain constant region expression construct as described below in Example 12. Oligonucleotide primers SEQ ID NO: 24 and SEQ ID NO: 25 were used to amplify the IL-20RB segment. The entire wild type human immunoglobulin kappa light chain constant region was used. The template used for the PCR amplification of the wild type human immunoglobulin kappa light chain constant region segment was generated IL-20RB human kappa light chain constant region expression construct as described in Example 12. Oligonucleotide primers SEQ ID NO: 26 and SEQ ID NO: 27 were used to amplify the wild type human immunoglobulin kappa light chain constant region. The two protein coding domains were linked by overlap PCR using oligonucleotides SEQ ID NO: 24 and SEQ ID NO: 27. A (Gly4Ser)3 (SEQ ID NO: 72) peptide linker was inserted between the two protein domains. The (Gly4Ser)3 peptide linker was encoded on the PCR primers SEQ ID NO: 26 and SEQ ID NO:25. The resultant IL-20RB extracellular domain/kappa light chain constant region fusion construct is shown by SEQ ID NOs: 20 and 21. The predicted mature polypeptide, minus the signal sequence, is SEQ ID NO: 60. The portion of the extracellular domain of IL-20RB that was actually used was comprised of the amino acid sequence of SEQ ID NO: 61. N-terminal sequencing resulted in the predicted amino acid sequence.
- The human IL-20RA extracellular domain human immunoglobulin gamma 1 heavy chain constant region fusion construct was generated by overlap PCR of four separate DNA fragments, each generated by separate PCR amplification reactions. The first fragment contained an optimized tPA (tissue plasminogen activator) signal sequence. The tPA signal sequence was amplified using oligonucleotide primers SEQ ID NO: 28 and SEQ ID NO: 29 using an in-house previously generated expression vector as the template. The second fragment contained the IL-20RA extracellular domain-coding region consisting of
amino acids 30 to 243 of SEQ ID NO: 11. Oligonucleotide primers SEQ ID NO: 30 and SEQ ID NO: 31 were used to amplify this IL-20RA segment using a previously generated clone of IL-20RA as the template. - The human gamma 1 heavy chain constant region was generated from 2 segments. The first segment containing the CH1 domain was amplified using oligonucleotide primers SEQ ID NO: 32 and SEQ ID NO: 33 using a clone of the wild type human gamma 1 heavy chain constant region as the template. The second segment containing the remaining hinge, CH2, and CH3 domains of the human immunoglobulin gamma 1 heavy chain constant region was generated by PCR amplification using oligonucleotide primers SEQ ID NO: 34 and SEQ ID NO: 35. The template used for this PCR amplification was from a previously generated human gamma 1 Fc construct that contained codons for amino acid substitutions to reduce FcyRI binding and Clq complement fixation as described in Example 12.
- The four protein coding domains were linked by overlap PCR using oligonucleotides SEQ ID NO: 28 and SEQ ID NO: 35. A (Gly4Ser)3 peptide linker was inserted between the IL-20RA and CH1 protein domains. The (Gly4Ser)3 peptide linker was encoded on the PCR primers SEQ ID NO: 32 and SEQ ID NO: 31. The IL-20RA extracellular domain/ domain human immunoglobulin gamma 1 heavy constant region fusion protein and DNA sequence are shown in SEQ ID NOs: 22 and 23. The predicted mature polypeptide sequence, minus the signal sequence, is SEQ ID NO: 62. The portion of extracellular domain of IL-20RA that was actually used was comprised of SEQ ID NO: 63.
- The IL-20RB extracellular domain human immunoglobulin kappa light chain constant region fusion coding segment was cloned into the second MCS while the human IL-20RA extracellular domain human immunoglobulin gamma 1 heavy chain constant region fusion coding segment was cloned into the first MCS of pEZE3. The plasmid was used to transfect CHO cells. The cells were selected in medium without hypoxanthine or thymidine and the transgene was amplified using methotrexate. The presence of protein was assayed by Western blotting using anti human gamma 1 heavy chain constant region and anti human kappa light chain antibodies. N-terminal sequencing revealed that the optimized tPA leader was not completely cleaved. The observed mass indicated that the first residue of the polypeptide sequence to be pyroglutamic acid, and the N-terminal sequence appears to be pyroEEIHAELRRFRRVPCVSGG (SEQ ID NO: 64), the underlined portion being remnants of the tPA leader.
- Both human and mouse IL-20 were overexpressed in transgenic mice using a variety of promoters. The liver-specific mouse albumin promoter, directing expression of human IL-20, was used initially in an attempt to achieve circulating levels of protein. Subsequent studies were conducted using the keratin 14 (K14) promoter, which primarily targets expression to the epidermis and other stratified squamous epithelia; the mouse metallothionein-1 promoter, which gives a broad expression pattern; and the E LCK promoter, which drives expression in cells of the lymphoid lineage. Similar results were obtained in all four cases, possibly because these promoters all give rise to circulating levels of IL-20.
- In all cases, transgenic pups expressing the IL-20 transgene were smaller than non-transgenic littermates, had a shiny appearance with tight, wrinkled skin and died within the first few days after birth. Pups had milk in their stomachs indicating that they were able to suckle. These mice had swollen extremities, tail, nostril and mouth regions and had difficulty moving. In addition, the mice were frail, lacked visible adipose tissue and had delayed ear and toe development. Low expression levels in liver (less than 100 mRNA molecules/cell) were sufficient for both the neonatal lethality and skin abnormalities. Transgenic mice without a visible phenotype either did not express the transgene, did not express it at detectable levels, or were mosaic.
- Histologic analysis of the skin of the IL-20 transgenic mice showed a thickened epidermis, hyperkeratosis and a compact stratum corneum compared to non-transgenic littermates. Serocellular crusts (scabs) were observed occasionally. Electron microscopic (EM) analysis of skin from transgenic mice showed intramitochondrial lipoid inclusions, mottled keratohyaline granules, and relatively few tonofilaments similar to that observed in human psoriatic skin and in mouse skin disease models. In addition, many of the transgenic mice had apoptotic thymic lymphocytes. No other abnormalities were detected by histopathological analysis. These histological and EM results support and extend the observed gross skin alterations.
- The specificity and affinity of IL-20 for its receptor was determined using BHK cells stably transfected with IL-20RA, IL-20RB or both receptor subunits. Binding assays using radiolabeled ligand demonstrated that IL-20 bound to BHK transfectants expressing both IL-20RA and IL-20RB but not to untransfected cells nor to transfectants expressing either receptor subunit alone. Binding of 125I-labeled IL-20 was eliminated in the presence of 100-fold excess of unlabeled IL-20 but not with 100-fold excess of the unrelated cytokine, IL-21. The binding affinity (kD) of IL-20 to the IL-20RA/IL-20RB heterodimeric receptor was determined to be approximately 1.5 nM.
- To determine if IL-20 binding leads to receptor activation, the factor-dependent pre-B cell line BaF3 was co-transfected with IL-20RA and IL-20RB and treated with IL-20 at various concentrations. IL-20 stimulated proliferation in a dose-dependent manner and gave a detectable signal at 1.1 pM, with a half maximal response at 3.4 pM. We note that the IL-20 concentration for the half maximal proliferative response in BaF3 cells is 1000× lower than that for half maximal binding affinity in BHK cells. Possible explanations for this large difference include the use of different cell lines, different receptor expression levels and different assay outputs. IL-20 also stimulated signal transduction in the biologically relevant human keratinocyte cell line HaCaT, which naturally expresses IL-20RA and IL-20RB. Therefore, IL-20 binds and activates the heterodimeric IL-20RA/IL-20RB receptor at concentrations expected for a cytokine. While the negative controls containing untransfected BaF3
- RT-PCR analysis was performed on a variety of human tissues to determine the expression pattern of IL-20RA and IL-20RB. Both receptor subunits are most highly expressed in skin and testis. The significant result is that IL-20RA and IL-20RB are both expressed in skin, where they have been shown to mediate the IL-20-induced response. Both IL-20RA and IL-20RB are also both expressed in monocytes, lung, ovary, muscle, testis, adrenal gland, heart, salivary gland and placenta. IL-20RA is also in brain, kidney, liver, colon, small intestine, stomach, thyroid, pancreas, uterus and prostate while IL-20RB is not.
- In situ hybridization was used to determine whether IL-20 receptor expression is altered in psoriasis. Skin samples from four psoriasis patients and three unaffected patients were assayed with probes specific for the two-receptor subunit mRNAs. All four psoriatic skin samples had high levels of IL-20RA and IL-20RB mRNA in keratinocytes whereas normal skin samples did not have detectable levels of either receptor subunit mRNA. Positive signals in psoriatic skin were also observed in mononuclear immune cells and in endothelial cells in a subset of vessels. Therefore, both IL-20RA and IL-20RB are expressed in keratinocytes, immune cells and endothelial cells, the major cell types thought to interact in psoriasis.
- A cross-species hybridization probe was generated which contained the full-length cDNA fragment encoding human IL-20RA. A Southern blot of mouse genomic DNA and Northern blots of mouse RNA were performed to demonstrate that the human IL-20RA cDNA could specifically hybridize to mouse sequences. The Northern blot results indicated that mouse IL-20RA RNA was present in
mouse embryo day 15 and 17 as well as heart, brain, lung, liver, kidney, testes, spleen, thymus, liver, stomach, and small intestine. - The human IL-20RA full length DNA hybridization probe was used to screen a mouse genomic library. The library, which was obtained from Clontech (Palo Alto, Calif.), was generated from an MboI partial digest of mouse genomic DNA and cloned into the BamHI site of Lambda bacteriophage EMBL3 SP6/T7. Positive bacteriophage was plaque purified and bacteriophage DNA was prepared using Promega's Wizard Lambda Preps DNA Purification System. Two genomic restriction enzyme fragments, a 5.7 kb EcoRI fragment and an 8.0 kb SacI fragment, were generated from the positive bacteriophage and subcloned into pBluescript. DNA sequence analysis revealed the presence of 3 exons from the mouse ortholog to human IL-20RA.
- PCR primers from the 5′ UTR, SEQ ID NO: 40, and 3′ UTR, SEQ ID NO: 41, were designed to generate a full-length mouse IL-20RA sequence by PCR amplification. Mouse embryo 15day plus 17 day cDNA was used as the template for the PCR amplification. PCR products were subcloned and sequenced for confirmation. The mouse sequences are SEQ ID NOs: 36 and 37. The mature extracellular domain is comprised of SEQ ID NO: 38.
- A vector expressing a secreted hIL-20RA/hIL-20B heterodimer was constructed. In this construct, the extracellular domain of hIL-20RA was fused to the heavy chain of IgG gamma 1 (IgGγ1), while the extracellular portion of IL-20RB was fused to human kappa light chain (human κ light chain).
- The heavy chain of IgGγ1 was cloned into the Zem229R mammalian expression vector (ATCC deposit No. 69447) such that any extracellular portion of a receptor having a 5′ EcoRI and 3′ NheI site can be cloned in, resulting in an N-terminal extracellular domain-C-terminal IgGγ1 fusion. The IgGγ1 fragment used in this construct was made by using PCR to isolate the IgGγ1 sequence from a Clontech human fetal liver cDNA library as template. A PCR reaction using oligos SEQ ID NO: 42 and SEQ ID NO: 43 was run as follows: 40 cycles of 94° for 60 sec., 53° C. for 60 sec., and 72° for 120 sec.; and 72° C. for 7 minutes. PCR products were separated by agarose gel electrophoresis and purified using a QiaQuick™ (Qiagen Inc., Valencia, Calif.) gel extraction kit. The isolated, 990 bp, DNA fragment was digested with MluI and EcoRI (Boerhinger-Mannheim), extracted with QiaQuick™ gel extraction kit and ligated with oligos SEQ ID NO: 44 and SEQ ID NO: 45, which comprise an MluI/EcoRI linker, into Zem229R previously digested with MluI and EcoRI using standard molecular biology techniques disclosed herein. This generic cloning vector was called Vector#76 hIgGgammal w/Ch1 #786 Zem229R (Vector #76). The polynucleotide sequence of the extracellular domain of hIL-20RA fused to the heavy chain of IgG gamma 1 is show in SEQ ID NO: 52 and the corresponding polypeptide sequence shown in SEQ ID NO: 53, the mature polypeptide, minus the signal sequence being comprised of SEQ ID NO: 54. The portion of the extracellular domain of IL-20RA used was comprised of SEQ ID NO: 55.
- The human κ light chain was cloned in the Zem228R mammalian expression vector (ATCC deposit No. 69446) such that any extracellular portion of a receptor having a 5′ EcoRI site and a 3′ KpnI site can be cloned in, resulting in an N-terminal extracellular domain-C-terminal human κ light chain fusion. The human κ light chain fragment used in this construct was made by using PCR to isolate the human κ light chain sequence from the same Clontech hFetal Liver cDNA library used above. A PCR reaction was run using oligos SEQ ID NO: 46 and SEQ ID NO: 47. PCR products were separated by agarose gel electrophoresis and purified using a QiaQuick™ (Qiagen) gel extraction kit. The isolated, 315 bp, DNA fragment was digested with MluI and EcoRI (Boerhinger-Mannheim), extracted with QiaQuick™ gel extraction kit and ligated with the MluI/EcoRI linker described above, into Zem228R previously digested with MluI and EcoRI using standard molecular biology techniques disclosed herein. This generic cloning vector was called Vector #77 hκlight #774 Zem228R (Vector #77). The polynucleotide sequence of the extracellular portion of IL-20RB fused to human kappa light chain is shown in SEQ ID NO: 56 and the corresponding polypeptide sequence shown in SEQ ID NO: 57, the mature polypeptide, minus the signal sequence, is comprised of SEQ ID NO: 58. The portion of the extracellular domain of IL-20RB actually used was comprised of SEQ ID NO: 59.
- Insertion of hIL-20RA and IL-20RB Extracellular Domains into Fusion Vector Constructs
- Using the construction vectors above, a construct having human IL-20RA fused to IgGγ1 was made. This construction was done by using PCR to obtain human IL-20RA receptor from hIL-20RA/IgG Vector #102 with oligos SEQ ID NO: 48 and SEQ ID NO: 49 under conditions described as follows: 30 cycles of 94° C. for 60 sec., 57° C. for 60 sec., and 72° C. for 120 sec.; and 72° C. for 7 min. The resulting PCR product was digested with EcoRI and NheI, gel purified, as described herein, and ligated into a previously EcoRI and NheI digested and band-purified Vector #76 (above). The resulting vector was sequenced to confirm that the human IL-20Rα/IgG gamma 1 fusion (hIL-20RA/Ch1 IgG) was correct. The hIL-20RA/Ch1 IgG gamma 1 #1825 Zem229R vector was called vector #195. The IL-20RA/Ch1 IgGγ1 sequence thus obtained is depicted by SEQ ID NOs: 52 and 53. N-terminal sequencing indicated the presence of the predicted mature polypeptide sequence of SEQ ID NO: 54.
- A separate construct having IL-20RB fused to x light was also constructed. The IL-20RB/human κ light chain construction was performed as above by PCRing from DR1/7N-4 with oligos SEQ ID NO: 50 and SEQ ID NO: 51, digesting the resulting band with EcoRI and KpnI and then ligating this product into a previously EcoRI and KpnI digested and band-purified Vec#77 (above). The resulting vector was sequenced to confirm that the IL-20RB/ human κ light chain fusion (IL-20RB/κlight) was correct. This IL-20RB//κlight construct is shown by SEQ ID NOs: 56 and 57. N-terminal sequencing of the resultant polypeptide indicated the presence of the predicted mature amino acid sequence comprised of SEQ ID NO: 58. SEQ ID NO:59 is the mature portion of the extracellular domain of IL-20RB used.
- Approximately 16 μg of each of vectors #194 and #195, above, were co-transfected into BHK-570 cells (ATCC No. CRL-10314) using Lipofectamine™ reagent (Gibco/BRL), as per manufacturer's instructions. The transfected cells were selected for 10 days in DMEM+5%FBS (Gibco/BRL) containing 1 μM of methotrexate (MTX) (Sigma, St. Louis, Mo.) and 0.5 mg/ml G418 (Gibco/BRL) for 10 days. The resulting pool of transfectants was selected again in 10 μM MTX and 0.5 mg/ml G418 for 10 days.
- The resulting pool of doubly selected cells was used to generate protein. Three factories (Nunc, Denmark) of this pool were used to generate 8 L of serum free conditioned medium. This conditioned media was passed over a 1 ml protein-A column and eluted in (10) 750 microliter fractions. 4 of these fractions found to have the highest concentration were pooled and dialyzed (10 kD MW cutoff) against PBS. Finally, the dialyzed material was analyzed by BCA (Pierce) and found to have a concentration of 317 μg/ml. A total of 951 μg was obtained from this 8 L purification.
- IL-20 binds cell lines transfected with both subunits of its receptor. However, these cell lines overexpress the IL-20 receptor relative to its normal level and their relevance to the physiological role of IL-20 is unclear. The human HaCaT keratinocyte cell line, which expresses endogenous IL-20RA and IL-20RB was used to examine IL-20 signal transduction in a biologically relevant cell type. HaCaT cells were infected with recombinant adenovirus containing a reporter construct to allow detection of intracellular signaling. The construct consists of the firefly luciferase gene driven by promoter/enhancer sequences comprised of the serum response element (SRE) and signal transducers and activators of transduction elements (STATs). This assay system detects productive ligand-receptor interactions and indicates possible downstream signal transduction components involved in receptor activation. Treatment with IL-20 alone resulted in a dose-dependent increase in luciferase activity with a half maximal response occurring at approximately 2.3 nM. Subsequent luciferase reporter assays using adenovirus vectors containing only the SRE element or only the STAT elements produced detectable reporter activation only through STATs.
- To determine if other cytokines act in concert with IL-20, HaCaT cells were treated with IL-20 alone or in combination with a single submaximal dose of EGF, IL-1β, or TNFα. In the presence of each of these three proteins, IL-20 treatment resulted in a dose-dependent increase in luciferase activity. IL-20 in combination with IL-1β results in a half-maximal response at approximately 0.5 nM, about five-fold lower than with IL-20 alone. In addition, activation of the reporter gene is detectable at 0.1 nM IL-20, a dose that is at least tenfold lower than the IL-20 dose required alone.
- BHK cells transfected with IL-20RA, IL-20RB or both receptor subunits were used to determine whether receptor pairing was required for IL-20 stimulation of STAT-luciferase. As was the case with binding assays, only cells transfected with both receptor subunits responded to IL-20 and did so with a half-maximal response of 5.7 pM. We note that the IL-20 concentration for the half-maximal response in BHK cells is 400-fold lower than that for half-maximal response in HaCaT cells. It is likely that a lower concentration of IL-20 is needed for half-maximal response in BHK cells, as compared to HaCaT cells, due to higher receptor levels in the BHK IL-20 receptor transfectants.
- A nuclear translocation assay was used to identify STAT proteins involved in IL-20 action. Both HaCaT cells, with endogenous IL-20 receptors, and BHK cells transfected with IL-20RA and IL-20RB, were treated with IL-20 protein and translocation of STAT3 and STAT1 transcription factors from the cytoplasm to the nucleus was assayed by immunofluorescence.
- In unstimulated HaCaT cells, STAT3 staining was predominantly in the cytosol. Treatment of HaCaT cells with IL-20 resulted in a distinct accumulation of STAT3 in the nucleus. Nuclear translocation of STAT3 in response to increasing concentrations of IL-20 occurred with a half-maximal IL-20 concentration of 7 nM. In contrast to STAT3 translocation, HaCaT cells treated with IL-20 did not show any detectable nuclear accumulation of STAT1.
- BHK cells transfected with IL-20RA and IL-20RB were used to confirm that the IL-20 receptor was required for IL-20 stimulation of STAT3 nuclear translocation. In BHK cells lacking the IL-20 receptor, STAT3 remained cytosolic following treatment with IL-20. In contrast, in BHK cells transfected with the IL-20 receptor, STAT3 translocated to the nucleus in response to IL-20. Again, STAT1 remained cytosolic regardless of IL-20 treatment or IL-20 receptor expression. We conclude that the IL-20 receptor is required for IL-20-mediated STAT3 activation.
Claims (13)
1. A method of treating an inflammatory disease in a subject in need thereof, comprising:
administering to the subject an effective amount of an antibody that specifically binds to an IL-20 receptor comprising an IL-20RA subunit and an IL-20RB subunit, wherein the IL-20A subunit comprises a polypeptide having the amino acid sequence of SEQ ID NO: 11 and the IL-20RB subunit comprises a polypeptide having the amino acid sequence of SEQ ID NO: 14.
2. The method of claim 1 , wherein the antibody is a monoclonal antibody.
3. The method of claim 1 , wherein the antibody is a human antibody.
4. The method of claim 1 , wherein the antibody is a humanized antibody.
5. The method of claim 1 , wherein the antibody is an antigen-binding fragment.
6. The method of claim 1 , wherein the inflammatory disease is an inflammatory skin disease.
7. The method of claim 6 , wherein the inflammatory skin disease is chosen from psoriasis, eczema, atopic dermatitis, and contact dermatitis.
8. The method of claim 7 , wherein the inflammatory skin disease is psoriasis.
9. The method of claim 1 , wherein the inflammatory disease is chosen from an inflammatory lung disease, rheumatoid arthritis and inflammatory bowel disease.
10. The method of claim 9 , wherein the inflammatory lung disease is chosen from asthma, bronchitis, and cystic fibrosis.
11. The method of claim 9 , wherein the inflammatory disease is rheumatoid arthritis.
12. A method of treating an inflammatory disease in a subject in need thereof, comprising:
administering to the subject an effective amount of an antibody that specifically binds to an IL-20 receptor comprising an IL-20RA subunit and an IL-20RB subunit, wherein the IL-20A subunit comprises a polypeptide having the amino acid sequence of SEQ ID NO: 12 and the IL-20RB subunit comprises a polypeptide having the amino acid sequence of SEQ ID NO: 15.
13. A method of treating an inflammatory disease in a subject in need thereof, comprising:
administering to the subject an effective amount of an antibody that specifically binds to an IL-20 receptor comprising an IL-20RA subunit and an IL-20RB subunit, wherein the IL-20A subunit comprises a polypeptide having the amino acid sequence of SEQ ID NO: 55 and the IL-20RB subunit comprises a polypeptide having the amino acid sequence of SEQ ID NO: 59.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US13/525,466 US20130171130A1 (en) | 1999-12-23 | 2012-06-18 | Soluble interleukin-20 receptor |
Applications Claiming Priority (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17196699P | 1999-12-23 | 1999-12-23 | |
US21341600P | 2000-06-22 | 2000-06-22 | |
US09/745,792 US7122632B2 (en) | 1999-12-23 | 2000-12-22 | Soluble Interleukin-20 receptor |
US11/458,367 US7585948B2 (en) | 1999-12-23 | 2006-07-18 | Soluble interleukin-20 receptor |
US12/509,667 US20100197575A1 (en) | 1999-12-23 | 2009-07-27 | Soluble interleukin-20 receptor |
US12/899,700 US8217008B2 (en) | 1999-12-23 | 2010-10-07 | Methods of treating inflammatory disease using a soluble IL-20 receptor |
US13/525,466 US20130171130A1 (en) | 1999-12-23 | 2012-06-18 | Soluble interleukin-20 receptor |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/899,700 Continuation US8217008B2 (en) | 1999-12-23 | 2010-10-07 | Methods of treating inflammatory disease using a soluble IL-20 receptor |
Publications (1)
Publication Number | Publication Date |
---|---|
US20130171130A1 true US20130171130A1 (en) | 2013-07-04 |
Family
ID=33556250
Family Applications (6)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/745,792 Expired - Lifetime US7122632B2 (en) | 1999-12-23 | 2000-12-22 | Soluble Interleukin-20 receptor |
US11/458,367 Expired - Fee Related US7585948B2 (en) | 1999-12-23 | 2006-07-18 | Soluble interleukin-20 receptor |
US11/458,360 Expired - Lifetime US7387780B2 (en) | 1999-12-23 | 2006-07-18 | Soluble interleukin-20 receptor |
US12/509,667 Abandoned US20100197575A1 (en) | 1999-12-23 | 2009-07-27 | Soluble interleukin-20 receptor |
US12/899,700 Expired - Fee Related US8217008B2 (en) | 1999-12-23 | 2010-10-07 | Methods of treating inflammatory disease using a soluble IL-20 receptor |
US13/525,466 Abandoned US20130171130A1 (en) | 1999-12-23 | 2012-06-18 | Soluble interleukin-20 receptor |
Family Applications Before (5)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/745,792 Expired - Lifetime US7122632B2 (en) | 1999-12-23 | 2000-12-22 | Soluble Interleukin-20 receptor |
US11/458,367 Expired - Fee Related US7585948B2 (en) | 1999-12-23 | 2006-07-18 | Soluble interleukin-20 receptor |
US11/458,360 Expired - Lifetime US7387780B2 (en) | 1999-12-23 | 2006-07-18 | Soluble interleukin-20 receptor |
US12/509,667 Abandoned US20100197575A1 (en) | 1999-12-23 | 2009-07-27 | Soluble interleukin-20 receptor |
US12/899,700 Expired - Fee Related US8217008B2 (en) | 1999-12-23 | 2010-10-07 | Methods of treating inflammatory disease using a soluble IL-20 receptor |
Country Status (1)
Country | Link |
---|---|
US (6) | US7122632B2 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016083304A1 (en) * | 2014-11-24 | 2016-06-02 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Treatment of acute exacerbations of chronic obstructive pulmonary disease by antagonism of the il-20r. |
Families Citing this family (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1803733B1 (en) * | 2000-09-15 | 2010-03-10 | ZymoGenetics, Inc. | Polypeptides comprising the extracellular domains of IL-20RA and/or IL-20RB |
US20070299246A1 (en) * | 2006-06-21 | 2007-12-27 | Taussig Michael J | IN VITRO PROTEIN EXPRESSION PROCESS COMPRISING Ckappa FUSION MOLECULES |
US7611705B2 (en) * | 2007-06-15 | 2009-11-03 | National Cheng Kung University | Anti-IL-20 antibody and its use in treating IL-20 associated inflammatory diseases |
DE102007039124A1 (en) * | 2007-08-18 | 2009-02-19 | Ulrich Dr. Jerichow | Device and method for controlling and / or regulating a training and / or rehabilitation unit |
US7837994B2 (en) * | 2008-10-07 | 2010-11-23 | National Cheng Kung University | Use of anti-IL-20 antibody for treating osteoporosis |
US8012478B2 (en) | 2008-10-07 | 2011-09-06 | National Cheng Kung University | Use of anti-IL-20 antibody for treating stroke |
CA2739794A1 (en) * | 2008-10-07 | 2010-04-15 | National Cheng Kung University | Use of il-20 antagonists for treating rheumatoid arthritis and osteoporosis |
US8454956B2 (en) | 2009-08-31 | 2013-06-04 | National Cheng Kung University | Methods for treating rheumatoid arthritis and osteoporosis with anti-IL-20 antibodies |
US9221904B2 (en) | 2012-07-19 | 2015-12-29 | National Cheng Kung University | Treatment of osteoarthritis using IL-20 antagonists |
US8603470B1 (en) | 2012-08-07 | 2013-12-10 | National Cheng Kung University | Use of IL-20 antagonists for treating liver diseases |
US8852588B2 (en) | 2012-08-07 | 2014-10-07 | National Cheng Kung University | Treating allergic airway disorders using anti-IL-20 receptor antibodies |
WO2017202813A1 (en) | 2016-05-24 | 2017-11-30 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and pharmaceutical compositions for the treatment of pulmonary bacterial infections |
Family Cites Families (31)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5567584A (en) | 1988-01-22 | 1996-10-22 | Zymogenetics, Inc. | Methods of using biologically active dimerized polypeptide fusions to detect PDGF |
US5116964A (en) | 1989-02-23 | 1992-05-26 | Genentech, Inc. | Hybrid immunoglobulins |
US5436155A (en) | 1991-12-31 | 1995-07-25 | Arch Development Corporation | Isolated DNA encoding a somatostatin receptor |
US5789192A (en) | 1992-12-10 | 1998-08-04 | Schering Corporation | Mammalian receptors for interleukin-10 (IL-10) |
DE69637856D1 (en) | 1996-08-30 | 2009-04-16 | Human Genome Sciences Inc | Interleukin-19. |
US5985614A (en) | 1996-08-30 | 1999-11-16 | Human Genome Sciences, Inc. | Polynucleotides encoding interleukin-19 |
US5945511A (en) | 1997-02-20 | 1999-08-31 | Zymogenetics, Inc. | Class II cytokine receptor |
EP0979102A4 (en) | 1997-04-30 | 2005-11-23 | Enzon Inc | Polyalkylene oxide-modified single chain polypeptides |
JP2001510035A (en) | 1997-07-16 | 2001-07-31 | ヒューマン ジノーム サイエンシーズ, インコーポレイテッド | Interleukin-20 |
US6486301B1 (en) | 1997-07-16 | 2002-11-26 | Human Genome Sciences, Inc. | Interleukin-20 |
WO1999007740A2 (en) | 1997-08-06 | 1999-02-18 | Zymogenetics, Inc. | Lipocalin homologs |
US6576743B1 (en) | 1997-11-26 | 2003-06-10 | Zymogenetics, Inc. | Mammalian cytokine-like polypeptide-10 |
IL137178A0 (en) | 1998-01-23 | 2001-07-24 | Immunex Corp | Il-18 receptors |
AU2871899A (en) | 1998-03-09 | 1999-09-27 | Schering Corporation | Human receptor proteins; related reagents and methods |
ES2312205T3 (en) | 1998-03-10 | 2009-02-16 | Genentech, Inc. | NEW POLYPEPTIDE AND NUCLEIC ACIDS THAT CODE IT. |
US7198789B2 (en) | 1998-03-17 | 2007-04-03 | Genetics Institute, Llc | Methods and compositions for modulating interleukin-21 receptor activity |
CN1233476A (en) | 1998-04-24 | 1999-11-03 | 陆道培 | Medicine for treating acute leukemia, and method for preparing same |
WO1999061630A2 (en) | 1998-05-26 | 1999-12-02 | Regeneron Pharmaceuticals, Inc. | Chimeric molecules comprising an extracellular ligand binding domain of a receptor and an ige fc or constant region, and their use in an assay system |
AU4411699A (en) | 1998-06-05 | 1999-12-20 | Human Genome Sciences, Inc. | Interferon receptor hkaef92 |
US7034123B2 (en) | 1998-09-01 | 2006-04-25 | Genetech, Inc. | Anti-PRO1347 antibodies |
WO2000039161A1 (en) | 1998-12-31 | 2000-07-06 | Millennium Pharmaceuticals, Inc. | Class ii cytokine receptor-like proteins and nucleic acids encoding them |
CA2383254A1 (en) | 1999-06-02 | 2000-12-07 | Genentech, Inc. | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
DE60030769T2 (en) | 1999-12-23 | 2007-10-25 | Zymogenetics, Inc., Seattle | PROCESS FOR TREATMENT OF IGNITION |
US6610286B2 (en) | 1999-12-23 | 2003-08-26 | Zymogenetics, Inc. | Method for treating inflammation using soluble receptors to interleukin-20 |
JP4741139B2 (en) | 1999-12-23 | 2011-08-03 | ザイモジェネティクス, インコーポレイテッド | Soluble interleukin-20 receptor |
EP1399472B1 (en) | 2001-03-09 | 2009-06-03 | ZymoGenetics, Inc. | Soluble heterodimeric cytokine receptor |
EP1432431B1 (en) | 2001-10-04 | 2017-05-10 | Genetics Institute LLC | Methods and compositions for modulating interleukin-21 activity |
US7582287B2 (en) | 2001-12-17 | 2009-09-01 | Zymogenetics, Inc. | Method for treating cervical cancer |
WO2003082212A2 (en) | 2002-03-27 | 2003-10-09 | The Governement Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services | Method for treating cancer in humans |
AU2003243415A1 (en) | 2002-06-07 | 2003-12-22 | Zymogenetics, Inc. | Use of il-21 in cancer and other therapeutic applications |
WO2005052001A2 (en) | 2003-11-21 | 2005-06-09 | Zymogenetics, Inc. | Anti-il-20 receptor antibodies and binding partners and methods of using in inflammation |
-
2000
- 2000-12-22 US US09/745,792 patent/US7122632B2/en not_active Expired - Lifetime
-
2006
- 2006-07-18 US US11/458,367 patent/US7585948B2/en not_active Expired - Fee Related
- 2006-07-18 US US11/458,360 patent/US7387780B2/en not_active Expired - Lifetime
-
2009
- 2009-07-27 US US12/509,667 patent/US20100197575A1/en not_active Abandoned
-
2010
- 2010-10-07 US US12/899,700 patent/US8217008B2/en not_active Expired - Fee Related
-
2012
- 2012-06-18 US US13/525,466 patent/US20130171130A1/en not_active Abandoned
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016083304A1 (en) * | 2014-11-24 | 2016-06-02 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Treatment of acute exacerbations of chronic obstructive pulmonary disease by antagonism of the il-20r. |
CN107249630A (en) * | 2014-11-24 | 2017-10-13 | 国家健康与医学研究院 | The acute exacerbation of COPD is treated by IL 20R antagonism |
Also Published As
Publication number | Publication date |
---|---|
US20110027263A1 (en) | 2011-02-03 |
US20050003475A1 (en) | 2005-01-06 |
US7585948B2 (en) | 2009-09-08 |
US8217008B2 (en) | 2012-07-10 |
US20060263850A1 (en) | 2006-11-23 |
US20100197575A1 (en) | 2010-08-05 |
US20060263851A1 (en) | 2006-11-23 |
US7122632B2 (en) | 2006-10-17 |
US7387780B2 (en) | 2008-06-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP1246846B1 (en) | Soluble interleukin-20 receptor | |
US7585948B2 (en) | Soluble interleukin-20 receptor | |
EP1399472B1 (en) | Soluble heterodimeric cytokine receptor | |
US7855269B2 (en) | Method for treating inflammation | |
US7189394B2 (en) | Method for treating inflammation | |
EP1743648B1 (en) | Method for treating inflammation | |
US8133487B2 (en) | Soluble heterodimeric cytokine receptor | |
CA2733610A1 (en) | Soluble heterodimeric cytokine receptor |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |