NO863657L - REGULATING FACTORS FOR HUMAN ENDOGENIC CANCER. - Google Patents
REGULATING FACTORS FOR HUMAN ENDOGENIC CANCER.Info
- Publication number
- NO863657L NO863657L NO863657A NO863657A NO863657L NO 863657 L NO863657 L NO 863657L NO 863657 A NO863657 A NO 863657A NO 863657 A NO863657 A NO 863657A NO 863657 L NO863657 L NO 863657L
- Authority
- NO
- Norway
- Prior art keywords
- kbs
- molecular weight
- cancer
- range
- human
- Prior art date
Links
- 206010028980 Neoplasm Diseases 0.000 title claims description 28
- 201000011510 cancer Diseases 0.000 title claims description 17
- 230000001105 regulatory effect Effects 0.000 title claims description 5
- 102000004169 proteins and genes Human genes 0.000 claims description 24
- 108090000623 proteins and genes Proteins 0.000 claims description 24
- 238000000034 method Methods 0.000 claims description 17
- 239000003638 chemical reducing agent Substances 0.000 claims description 14
- 102000037983 regulatory factors Human genes 0.000 claims description 11
- 108091008025 regulatory factors Proteins 0.000 claims description 11
- 239000003398 denaturant Substances 0.000 claims description 10
- 108010033276 Peptide Fragments Proteins 0.000 claims description 7
- 102000007079 Peptide Fragments Human genes 0.000 claims description 7
- 239000003104 tissue culture media Substances 0.000 claims description 7
- 125000000539 amino acid group Chemical group 0.000 claims description 6
- 238000003776 cleavage reaction Methods 0.000 claims description 6
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 239000000825 pharmaceutical preparation Substances 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 210000004027 cell Anatomy 0.000 description 31
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 26
- 235000018102 proteins Nutrition 0.000 description 20
- 230000000694 effects Effects 0.000 description 19
- 239000007853 buffer solution Substances 0.000 description 17
- 150000001413 amino acids Chemical group 0.000 description 15
- 239000011780 sodium chloride Substances 0.000 description 13
- 239000000126 substance Substances 0.000 description 13
- 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 11
- 239000000243 solution Substances 0.000 description 11
- 235000001014 amino acid Nutrition 0.000 description 10
- 229940024606 amino acid Drugs 0.000 description 10
- 239000002158 endotoxin Substances 0.000 description 10
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 10
- 229920005654 Sephadex Polymers 0.000 description 9
- 239000012507 Sephadex™ Substances 0.000 description 9
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 230000000638 stimulation Effects 0.000 description 8
- -1 thiol compounds Chemical class 0.000 description 8
- 206010024305 Leukaemia monocytic Diseases 0.000 description 7
- 241000699670 Mus sp. Species 0.000 description 7
- 238000005119 centrifugation Methods 0.000 description 7
- 201000006894 monocytic leukemia Diseases 0.000 description 7
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 7
- 230000000259 anti-tumor effect Effects 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 229920006008 lipopolysaccharide Polymers 0.000 description 6
- 210000001616 monocyte Anatomy 0.000 description 6
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 5
- 108010062580 Concanavalin A Proteins 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 5
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 5
- 241000282412 Homo Species 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 239000012894 fetal calf serum Substances 0.000 description 5
- 238000005194 fractionation Methods 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 230000007017 scission Effects 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 4
- 229920002684 Sepharose Polymers 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 208000032839 leukemia Diseases 0.000 description 4
- 210000000265 leukocyte Anatomy 0.000 description 4
- 230000014759 maintenance of location Effects 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 241000192125 Firmicutes Species 0.000 description 3
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 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
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 3
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 3
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 3
- 239000013543 active substance Substances 0.000 description 3
- 239000002269 analeptic agent Substances 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 239000012228 culture supernatant Substances 0.000 description 3
- 230000001472 cytotoxic effect Effects 0.000 description 3
- 229960000789 guanidine hydrochloride Drugs 0.000 description 3
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 3
- NBZBKCUXIYYUSX-UHFFFAOYSA-N iminodiacetic acid Chemical compound OC(=O)CNCC(O)=O NBZBKCUXIYYUSX-UHFFFAOYSA-N 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 229930027917 kanamycin Natural products 0.000 description 3
- 229960000318 kanamycin Drugs 0.000 description 3
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 3
- 229930182823 kanamycin A Natural products 0.000 description 3
- 210000002540 macrophage Anatomy 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000002093 peripheral effect Effects 0.000 description 3
- 229920002401 polyacrylamide Polymers 0.000 description 3
- 230000000644 propagated effect Effects 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 229960005322 streptomycin Drugs 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 108010042708 Acetylmuramyl-Alanyl-Isoglutamine Proteins 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 241000186216 Corynebacterium Species 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 229930182566 Gentamicin Natural products 0.000 description 2
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 108091006905 Human Serum Albumin Proteins 0.000 description 2
- 102000008100 Human Serum Albumin Human genes 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 108010047620 Phytohemagglutinins Proteins 0.000 description 2
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 150000001450 anions Chemical class 0.000 description 2
- 235000009582 asparagine Nutrition 0.000 description 2
- 229960001230 asparagine Drugs 0.000 description 2
- 235000003704 aspartic acid Nutrition 0.000 description 2
- 239000012298 atmosphere Substances 0.000 description 2
- 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 2
- 229940088623 biologically active substance Drugs 0.000 description 2
- 229960000074 biopharmaceutical Drugs 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 230000024245 cell differentiation Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000011098 chromatofocusing Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- 238000002523 gelfiltration Methods 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 210000005260 human cell Anatomy 0.000 description 2
- 230000003211 malignant effect Effects 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- BSOQXXWZTUDTEL-ZUYCGGNHSA-N muramyl dipeptide Chemical compound OC(=O)CC[C@H](C(N)=O)NC(=O)[C@H](C)NC(=O)[C@@H](C)O[C@H]1[C@H](O)[C@@H](CO)O[C@@H](O)[C@@H]1NC(C)=O BSOQXXWZTUDTEL-ZUYCGGNHSA-N 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 230000001885 phytohemagglutinin Effects 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N serine Chemical compound OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000001488 sodium phosphate Substances 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 2
- 239000000021 stimulant Substances 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- CWERGRDVMFNCDR-UHFFFAOYSA-N thioglycolic acid Chemical compound OC(=O)CS CWERGRDVMFNCDR-UHFFFAOYSA-N 0.000 description 2
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 2
- JNYAEWCLZODPBN-JGWLITMVSA-N (2r,3r,4s)-2-[(1r)-1,2-dihydroxyethyl]oxolane-3,4-diol Chemical compound OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O JNYAEWCLZODPBN-JGWLITMVSA-N 0.000 description 1
- OIXLLKLZKCBCPS-RZVRUWJTSA-N (2s)-2-azanyl-5-[bis(azanyl)methylideneamino]pentanoic acid Chemical compound OC(=O)[C@@H](N)CCCNC(N)=N.OC(=O)[C@@H](N)CCCNC(N)=N OIXLLKLZKCBCPS-RZVRUWJTSA-N 0.000 description 1
- UNUFVVHXFJOAMX-UHFFFAOYSA-N 1-n,1-n,1-n',1-n',1-n",1-n"-hexaethyl-2-phosphanylethane-1,1,1-triamine Chemical compound CCN(CC)C(CP)(N(CC)CC)N(CC)CC UNUFVVHXFJOAMX-UHFFFAOYSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- YVOOPGWEIRIUOX-UHFFFAOYSA-N 2-azanyl-3-sulfanyl-propanoic acid Chemical compound SCC(N)C(O)=O.SCC(N)C(O)=O YVOOPGWEIRIUOX-UHFFFAOYSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 description 1
- 206010000871 Acute monocytic leukaemia Diseases 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 1
- 208000002109 Argyria Diseases 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 241000186427 Cutibacterium acnes Species 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 229920002271 DEAE-Sepharose Polymers 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 239000006145 Eagle's minimal essential medium Substances 0.000 description 1
- 241000283074 Equus asinus Species 0.000 description 1
- 241000223218 Fusarium Species 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- 101000766306 Homo sapiens Serotransferrin Proteins 0.000 description 1
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- 125000000570 L-alpha-aspartyl group Chemical group [H]OC(=O)C([H])([H])[C@]([H])(N([H])[H])C(*)=O 0.000 description 1
- 125000000899 L-alpha-glutamyl group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C([H])([H])C([H])([H])C(O[H])=O 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
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 208000006552 Lewis Lung Carcinoma Diseases 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 208000035489 Monocytic Acute Leukemia Diseases 0.000 description 1
- 241001467552 Mycobacterium bovis BCG Species 0.000 description 1
- 231100000678 Mycotoxin Toxicity 0.000 description 1
- 241000460117 Neonothopanus gardneri Species 0.000 description 1
- 241000187654 Nocardia Species 0.000 description 1
- 108010058846 Ovalbumin Proteins 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 241000224016 Plasmodium Species 0.000 description 1
- 241000276498 Pollachius virens Species 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 1
- URWAJWIAIPFPJE-UHFFFAOYSA-N Rickamicin Natural products O1CC(O)(C)C(NC)C(O)C1OC1C(O)C(OC2C(CC=C(CN)O2)N)C(N)CC1N URWAJWIAIPFPJE-UHFFFAOYSA-N 0.000 description 1
- 241000235070 Saccharomyces Species 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- 208000006268 Sarcoma 180 Diseases 0.000 description 1
- 239000012506 Sephacryl® Substances 0.000 description 1
- 241001522306 Serinus serinus Species 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-N Sulfurous acid Chemical class OS(O)=O LSNNMFCWUKXFEE-UHFFFAOYSA-N 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical compound OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 241000223996 Toxoplasma Species 0.000 description 1
- 101710162629 Trypsin inhibitor Proteins 0.000 description 1
- 229940122618 Trypsin inhibitor Drugs 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
- 206010054094 Tumour necrosis Diseases 0.000 description 1
- 208000037386 Typhoid Diseases 0.000 description 1
- 229920000392 Zymosan Polymers 0.000 description 1
- 229960004308 acetylcysteine Drugs 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 150000008051 alkyl sulfates Chemical class 0.000 description 1
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 1
- 239000001099 ammonium carbonate Substances 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 239000001166 ammonium sulphate Substances 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 229960000190 bacillus calmette–guérin vaccine Drugs 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- VJLOFJZWUDZJBX-UHFFFAOYSA-N bis(2-hydroxyethyl)azanium;chloride Chemical compound [Cl-].OCC[NH2+]CCO VJLOFJZWUDZJBX-UHFFFAOYSA-N 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 244000309464 bull Species 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000003636 conditioned culture medium Substances 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 229960002433 cysteine Drugs 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- ZZTURJAZCMUWEP-UHFFFAOYSA-N diaminomethylideneazanium;hydrogen sulfate Chemical compound NC(N)=N.OS(O)(=O)=O ZZTURJAZCMUWEP-UHFFFAOYSA-N 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- XJWSAJYUBXQQDR-UHFFFAOYSA-M dodecyltrimethylammonium bromide Chemical compound [Br-].CCCCCCCCCCCC[N+](C)(C)C XJWSAJYUBXQQDR-UHFFFAOYSA-M 0.000 description 1
- 239000008298 dragée Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 150000002170 ethers Chemical class 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 239000007863 gel particle Substances 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 150000002500 ions Chemical class 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
- 239000000644 isotonic solution Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- DLEDLHFNQDHEOJ-UDTOXTEMSA-N mezerein Chemical compound O([C@@H]1[C@H]([C@@]23[C@H]4[C@](C(C(C)=C4)=O)(O)[C@H](O)[C@@]4(CO)O[C@H]4[C@H]3[C@H]3O[C@@](O2)(O[C@]31C(C)=C)C=1C=CC=CC=1)C)C(=O)\C=C\C=C\C1=CC=CC=C1 DLEDLHFNQDHEOJ-UDTOXTEMSA-N 0.000 description 1
- DLEDLHFNQDHEOJ-KVZAMRGJSA-N mezerein Natural products CC1C(OC(=O)C=C/C=C/c2ccccc2)C3(OC4(OC3C5C6OC6(CO)C(O)C7(O)C(C=C(C)C7=O)C15O4)c8ccccc8)C(=C)C DLEDLHFNQDHEOJ-KVZAMRGJSA-N 0.000 description 1
- 210000000865 mononuclear phagocyte system Anatomy 0.000 description 1
- 239000002636 mycotoxin Substances 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229940054441 o-phthalaldehyde Drugs 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229940092253 ovalbumin Drugs 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- PHEDXBVPIONUQT-RGYGYFBISA-N phorbol 13-acetate 12-myristate Chemical compound C([C@]1(O)C(=O)C(C)=C[C@H]1[C@@]1(O)[C@H](C)[C@H]2OC(=O)CCCCCCCCCCCCC)C(CO)=C[C@H]1[C@H]1[C@]2(OC(C)=O)C1(C)C PHEDXBVPIONUQT-RGYGYFBISA-N 0.000 description 1
- 150000003003 phosphines Chemical group 0.000 description 1
- ZWLUXSQADUDCSB-UHFFFAOYSA-N phthalaldehyde Chemical compound O=CC1=CC=CC=C1C=O ZWLUXSQADUDCSB-UHFFFAOYSA-N 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- XNSAINXGIQZQOO-SRVKXCTJSA-N protirelin Chemical compound NC(=O)[C@@H]1CCCN1C(=O)[C@@H](NC(=O)[C@H]1NC(=O)CC1)CC1=CN=CN1 XNSAINXGIQZQOO-SRVKXCTJSA-N 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- URWAJWIAIPFPJE-YFMIWBNJSA-N sisomycin Chemical compound O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H](CC=C(CN)O2)N)[C@@H](N)C[C@H]1N URWAJWIAIPFPJE-YFMIWBNJSA-N 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- 235000010265 sodium sulphite Nutrition 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 239000007940 sugar coated tablet Substances 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- TUQOTMZNTHZOKS-UHFFFAOYSA-N tributylphosphine Chemical compound CCCCP(CCCC)CCCC TUQOTMZNTHZOKS-UHFFFAOYSA-N 0.000 description 1
- 229930013292 trichothecene Natural products 0.000 description 1
- 150000003327 trichothecene derivatives Chemical class 0.000 description 1
- 239000002753 trypsin inhibitor Substances 0.000 description 1
- 201000008297 typhoid fever Diseases 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
- 238000000108 ultra-filtration Methods 0.000 description 1
- 229940045136 urea Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
Anvendelsesområde for op pfinnelsenScope of the invention
Oppfinnelsen vedrører nye, lavmolekylære reguleringsfaktorer for endogen kreft hos mennesker, fremgangsmåte for fremstilling av disse og farmasøytiske preparater som inneholder dem. The invention relates to new, low molecular weight regulatory factors for endogenous cancer in humans, methods for producing these and pharmaceutical preparations containing them.
Bakgrunn for oppfinnelsenBackground for the invention
Oppfinnerne isolerte tidligere reguleringsfaktorer for endogen kreft hos mennesker ("Krebs Statika"; KBS), nye biologisk aktive stoffer, ved å dyrke human-monocyter eller klonede stammer derav i et vevkulturmedium. De derved erholdte KBS var av to typer: en med en relativ molekylvekt på 82.000 The inventors previously isolated regulatory factors for endogenous cancer in humans ("Krebs Statika"; KBS), new biologically active substances, by culturing human monocytes or cloned strains thereof in a tissue culture medium. The KBS thus obtained were of two types: one with a relative molecular weight of 82,000
- 10.000 og et isoelektrisk punkt på pH 6,5 - 0,5, og den andre med en relativ molekylvekt i området 50.000 til 72.000 - 10,000 and an isoelectric point of pH 6.5 - 0.5, and the other with a relative molecular weight in the range of 50,000 to 72,000
og et isoelektrisk punkt i pH-området fra 6,0 til 8,5and an isoelectric point in the pH range from 6.0 to 8.5
(japanske patentsøknad 128893/1983 og nr. 99370/1984). KBS(Japanese Patent Application No. 128893/1983 and No. 99370/1984). KBS
av den sistnevnte type ble videre oppdelt i tre stoffer med forskjellig isoelektrisk punkt: KBS-a (pH omtrent 6,5), KBS-[3 (pH omtrent 7,0) og KBS-y (pH omtrent 8,0). KBS er en gruppe biologisk aktive stoffer avledet fra menneskeceller, og ansees på grunn av den kraftige aktivitet med mange tumorslag, både in vitro og in vivo, å være blant de stoffer som produ-seres i svært små mengder i kroppen til pasienter som spon-tant er blitt friske igjen etter å ha hatt kreft. of the latter type was further divided into three substances with different isoelectric points: KBS-a (pH about 6.5), KBS-[3 (pH about 7.0) and KBS-y (pH about 8.0). KBS is a group of biologically active substances derived from human cells, and due to its powerful activity with many types of tumors, both in vitro and in vivo, is considered to be among the substances that are produced in very small quantities in the bodies of patients who sponsor aunt has recovered after having cancer.
Uventet har videre undersøkelser ført til at vi har funnet at et nytt biologisk aktivt stoff kunne erholdes fra denne KBS ved behandling med reduksjonsmiddel og/eller proteindenaturerende middel. Dette nye biologiske aktive stoff avledet fra KBS har en molekylvekt i området fra 17.000 til 30.000 og har anti-tumorvirkning, idet en fraksjon har en molekylvekt i området fra 20.000 til 30.000, særlig i området 22.500 - 1500, som er det mest foretrukkede område. Vi kalte stoffet lavmolekylært, humant endogent anti-tumorstoff (nedre "Krebs-statika; n-KBS) på grunn av dets lavere molekylvekt sammenlignet med KBS. Unexpectedly, further investigations have led us to find that a new biologically active substance could be obtained from this KBS by treatment with a reducing agent and/or a protein denaturing agent. This new biologically active substance derived from KBS has a molecular weight in the range from 17,000 to 30,000 and has an anti-tumor effect, a fraction having a molecular weight in the range from 20,000 to 30,000, particularly in the range of 22,500 - 1,500, which is the most preferred range. We named the substance low-molecular-weight, human endogenous anti-tumor substance (lower "Krebs statica; n-KBS) because of its lower molecular weight compared to KBS.
Angivelse av oppfinnelsenDisclosure of the invention
n-KBS, de lavmolekylære reguleringsfaktorer for endogen kreft hos mennesker ifølge oppfinnelsen, er nye proteiner med en relativ molekylvekt i området 17.000 til 30.000, og n-KBS, the low molecular weight regulatory factors for endogenous cancer in humans according to the invention, are new proteins with a relative molecular weight in the range of 17,000 to 30,000, and
som har anti-tumoraktivitet, og kan fremstilles ved å behandle KBS (relativ molekylvekt 50.000 til 92.000, isoelektrisk punkt pH 6,0 til 8,5) med reduksjonsmiddel og/eller proteindenaturerende middel. n-KBS ifølge oppfinnelsen kan således ansees for å være en av underenhetene til KBS som er blitt skilt fra KBS som et resultat av behandling med reduksjonsmiddel og/ eller proteindenaturerende middel gjennom i det minste delvis nedbrytning av dets struktur av høyere orden, eller i det minste delvis spalting av dets disulfidbindinger. Som KBS, which has anti-tumor activity, and can be prepared by treating KBS (relative molecular weight 50,000 to 92,000, isoelectric point pH 6.0 to 8.5) with reducing agent and/or protein denaturant. n-KBS according to the invention can thus be considered to be one of the subunits of KBS that has been separated from KBS as a result of treatment with a reducing agent and/or protein denaturant through at least partial degradation of its higher order structure, or in the least partial cleavage of its disulfide bonds. As KBS,
har n-KBS kraftig virkning mot mange tumorslag, både in vitro og in vivo, og har også necrotisk tumoraktivitet in vivo. I tillegg er det god grunn til å tro at n-KBS som er avledet fra humane celler som KBS, ikke gjenkjennes som heterologe i menneskekropper og derfor forårsaker liten, om noen, antigen-antistoff-reaksjon. Det er også mulig å bygge opp igjen KBS fra n-KBS ifølge oppfinnelsen gjennom protein-renaturering eller gjenopprettelse av disulfidbindinger. n-KBS has a powerful effect against many tumor types, both in vitro and in vivo, and also has necrotic tumor activity in vivo. In addition, there is good reason to believe that n-KBS derived from human cells such as KBS are not recognized as heterologous in human bodies and therefore cause little, if any, antigen-antibody reaction. It is also possible to rebuild KBS from n-KBS according to the invention through protein renaturation or restoration of disulphide bonds.
Kort beskrivelse av tegningeneBrief description of the drawings
Fig. 1 er en elueringsprofil for n-KBS som viser KBS-aktivitet, etter at KBS er blitt utsatt for elektroforese (eksempel 1), hvor ordinaten representerer KBS-aktivitet ut-vunnet fra hvert gelstykke og abscissen angir fraksjonsnummer. Pilene indikerer elueringsposisjonene til referanseproteiner (TF, human transferriu (molekylvekt 76.000); HSA, humant serumalbumin (molekylvekt 67.000); OVA, ovalbumin (molekylvekt 43.000); SBTI, trypsin-inhibitor fra soyabønne (molekylvekt 21.500) og HG, hemoglobin (molekylvekt 15.500); og fig. 2 viser en SDS-polyacrylamidgelelektroforese (SDS-PAGE i nærvær av 2-mercaptoethanol) av hovedpeptidfragmentet og n-KBS (eksempel 1). Fig. 1 is an elution profile for n-KBS showing KBS activity, after KBS has been subjected to electrophoresis (Example 1), where the ordinate represents KBS activity recovered from each gel piece and the abscissa indicates fraction number. The arrows indicate the elution positions of reference proteins (TF, human transferrin (molecular weight 76,000); HSA, human serum albumin (molecular weight 67,000); OVA, ovalbumin (molecular weight 43,000); SBTI, soybean trypsin inhibitor (molecular weight 21,500) and HG, hemoglobin (molecular weight 15,500 ); and Fig. 2 shows an SDS-polyacrylamide gel electrophoresis (SDS-PAGE in the presence of 2-mercaptoethanol) of the main peptide fragment and n-KBS (Example 1).
Nærmere beskrivelse av de foretrukkede utførelsesformerDetailed description of the preferred embodiments
n-KBS ifølge oppfinnelsen kan fåes ved å dyrke monocyt-celler fra mennesker som er i stand til å produsere KBS, eller en klonet stamme derav, i et dyrkningsmedium for vev, hvorved det fåes KBS med en relativ molekylvekt i området fra 50.000 til 92.000, og et isoelektrisk punkt i pH-området fra 6,0 til 8,5, etterfulgt av behandling med reduksjonsmiddel og/eller proteindenaturerende middel. Nærmere bestemt dyrkes n-KBS according to the invention can be obtained by culturing human monocyte cells capable of producing KBS, or a cloned strain thereof, in a culture medium for tissue, whereby KBS is obtained with a relative molecular weight in the range from 50,000 to 92,000 , and an isoelectric point in the pH range from 6.0 to 8.5, followed by treatment with reducing agent and/or protein denaturant. Specifically cultivated
humane, normale eller leukemi-celler, som er i stand til å produsere KBS, eller en klonet stamme derav, i et dyrkningsmedium for vev og gjennomgår første og andre stimulering, hvilket gir KBS med en relativ molekylvekt i området fra 50.000 til 92.000 og et isoelektrisk punkt i pH-området fra 6,0 til 8,5, og hvor det således erholdte KBS deretter behandles med reduksjonsmiddel og/eller proteindenaturerende middel. human, normal or leukemic cells, capable of producing KBS, or a cloned strain thereof, in a tissue culture medium and undergoing first and second stimulation, yielding KBS with a relative molecular weight in the range of 50,000 to 92,000 and a isoelectric point in the pH range from 6.0 to 8.5, and where the KBS thus obtained is then treated with a reducing agent and/or protein denaturing agent.
De normale eller leukemiske monocyt-celler som brukes ved oppfinnelsen, omfatter normale monocyter, makrofag-celler og leukemi-celler derav, og de som lett kan formeres i et dyrkningsmedium for vev er mest foretrukket. The normal or leukemic monocyte cells used in the invention include normal monocytes, macrophage cells and leukemia cells thereof, and those which can be easily propagated in a tissue culture medium are most preferred.
Særlig foretrukket er leukemi-celler av monocyt-makrofag-rekken. Typiske eksempler omfatter HL-50 som er leukemi-celler av monocyt/makrofag-rekken; YKBS-7-15, YKBS-7-16 og YKBS-7-17 som er monocytiske leukemi-celler valgt fra klinisk fastslåtte, perifere leukocyter av monocytisk leukemi-opp-rinnelse; og klonede stammer derav, slik som klonene 2A2, 2A6, 2C6, 3A1, 3B3, 3B4, 3C1, 3D5, 4B3 og andre kloner fra YKBS-7-15. Particularly preferred are leukemia cells of the monocyte-macrophage lineage. Typical examples include HL-50 which are leukemia cells of the monocyte/macrophage lineage; YKBS-7-15, YKBS-7-16 and YKBS-7-17 which are monocytic leukemia cells selected from clinically established peripheral leukocytes of monocytic leukemia origin; and cloned strains thereof, such as clones 2A2, 2A6, 2C6, 3A1, 3B3, 3B4, 3C1, 3D5, 4B3 and other clones from YKBS-7-15.
Som et typisk eksempel på kloning, vil den fremgangsmåte som er brukt for utvelgelse av de ønskede kloner fra den etablerte cellelinje YKBS-7-15 (japansk patentsøknad nr. 239355/1983) bli beskrevet nedenunder. As a typical example of cloning, the method used for selecting the desired clones from the established cell line YKBS-7-15 (Japanese Patent Application No. 239355/1983) will be described below.
Dyrkede celler av YKBS-7-15 (monocytiske leukemi-celler) avledet fra humane leukemiske perifere leukocyter, ble plassert på en mikroplate med 9 9 brønner som på forhånd var blitt tilført 1 x 10^ mus-thymocyter som fødeceller, ved en konsentrasjon på 1 til 2 celler/brønn (begrensende for-tynningsmetode), og dyrket ved 37°C under en atmosfære med 5% C02og 95% luft. Etter at koloniene var blitt synlige, ble subdyrking utført ved etterhvert å overføre dem til en mikroplate med 24 brønner, og kolonier med stor evne til KBS-produksjon ble valgt ut. Lignende operasjoner ble gjentatt mens dyrkingen gradvis ble oppskalert, mens etterhvert klonene 2A2, 2A6, 2C6, 3A1, 3B3, 3B4, 3C1, 3D5 og 4B3 ble plukket ut som de stammer som hadde høyest KBS-produktivitet. Cultured cells of YKBS-7-15 (monocytic leukemia cells) derived from human leukemic peripheral leukocytes were plated on a 99-well microplate previously seeded with 1 x 10^ mouse thymocytes as feeder cells, at a concentration of 1 to 2 cells/well (limiting dilution method), and cultured at 37°C under an atmosphere of 5% CO 2 and 95% air. After the colonies had become visible, subculturing was performed by gradually transferring them to a microplate with 24 wells, and colonies with a high capacity for KBS production were selected. Similar operations were repeated while the cultivation was gradually scaled up, while eventually clones 2A2, 2A6, 2C6, 3A1, 3B3, 3B4, 3C1, 3D5 and 4B3 were selected as the strains with the highest KBS productivity.
De etablerte cellelinjer, YKBS-7-15, YKBS-7-16 og They established cell lines, YKBS-7-15, YKBS-7-16 and
YKBS-7-17, er moncytiske leukemi-cellelinjer som oppfinnerne og Institute of Medical Science, University of Tokyo har lykkes i å isolere ved å dyrke leukocytene med brungult belegg fra det perifere blod hos en pasient som led av akutt monocytisk leukemi, på konvensjonell måte (inkludert ammonium-kloridbehandling for fjerning av erythrocyter. Blant disse er YKBS-7-15, og YKBS-4B3, som er en isolert klon fra YKBS-7-15, blitt deponert ved C.N.C.M., Pasteur Institute, Frankrike (Februar 29, 1984) (henholdsvis 1-258 og 1-319 (Juli 25, YKBS-7-17, are monocytic leukemia cell lines that the inventors and the Institute of Medical Science, University of Tokyo have succeeded in isolating by culturing the leukocytes with brown-yellow coating from the peripheral blood of a patient suffering from acute monocytic leukemia, on conventional manner (including ammonium chloride treatment to remove erythrocytes. Among these, YKBS-7-15, and YKBS-4B3, which is an isolated clone from YKBS-7-15, have been deposited at C.N.C.M., Pasteur Institute, France (February 29, 1984) (respectively 1-258 and 1-319 (July 25,
1984) . 1984).
Som andre eksempler på cellelinjer av monocyt/makrofag-rekken, kan nevnes Mono-1 og Mono-1-207 (Virchow Arch. A. Pathol. Anat. Histol. 371'15 (1976) og 379, 269 (1978)). As other examples of cell lines of the monocyte/macrophage series, mention can be made of Mono-1 and Mono-1-207 (Virchow Arch. A. Pathol. Anat. Histol. 371'15 (1976) and 379, 269 (1978)).
I tillegg kan selvfølgelig celler som er i stand tilIn addition, of course, cells that are capable of
å differensieretil monocytiske celler, brukes ved oppfinnelsen. Disse celler oppviser etterhvert egenskapene ved monocytisk leukemi, og maligne myeloma-celler er et eksempel. to differentiate into monocytic cells, is used in the invention. These cells eventually show the characteristics of monocytic leukaemia, and malignant myeloma cells are an example.
De ovenfor angitte celler utsettes for vevsdyrking vanligvis i et dyrkningsmedium som inneholder serum fra kalve-foster (FCS). F.eks. kan YKBS-4B3, en klon av YKBS-7-15 som brukes i eksempel 1, med hell dyrkes i RPMI-1640-medium. The above cells are subjected to tissue culture usually in a culture medium containing fetal calf serum (FCS). E.g. can YKBS-4B3, a clone of YKBS-7-15 used in Example 1, be successfully grown in RPMI-1640 medium.
(Gibco Co.) som inneholder 10% FCS, 15 mM HEPES (Wako Junyaku Co.), 50 mg/l kanamycin og 25 mg/l streptomycin. I denne blandingen kan også penicillin, gentamycin, sisomicin og andre antibiotika brukes i stedet for kanamycin og streptomycin. Det er også mulig å utføre vevsdyrkningen i serumfrie dyrkningsmedier, slik som HB101 TM (Hana Biologics Inc.) eller ISCOVE's medium (Flow laboratories). I tillegg kan cellene også formeres intraperitonealt eller subkutant i andre varm-blodige dyr enn mennesker, som mus uten pels og unge hamstere. Vevkulturmediene brukt ved oppfinnelsen omfatter slike in (Gibco Co.) containing 10% FCS, 15 mM HEPES (Wako Junyaku Co.), 50 mg/l kanamycin and 25 mg/l streptomycin. In this mixture, penicillin, gentamycin, sisomycin and other antibiotics can also be used instead of kanamycin and streptomycin. It is also possible to perform the tissue culture in serum-free culture media, such as HB101 TM (Hana Biologics Inc.) or ISCOVE's medium (Flow laboratories). In addition, the cells can also be propagated intraperitoneally or subcutaneously in warm-blooded animals other than humans, such as mice without fur and young hamsters. The tissue culture media used in the invention include such in
vivo dyrkningsmedier.in vivo culture media.
Eksempler på det første stimulerende middel brukt ved oppfinnelsen omfatter kondisjonerte medier med lymfocyter eller lymfoblaster, kjemiske stoffer eller naturlige ekstrakter som induserer cellulær differensiering, samt blandinger Examples of the first stimulating agent used in the invention include conditioned media with lymphocytes or lymphoblasts, chemical substances or natural extracts that induce cellular differentiation, as well as mixtures
derav. Et annet eksempel er supernatanten som fåes ved dyrking av sensitiviserte celler i dyr administrert med et visst hence. Another example is the supernatant obtained from the cultivation of sensitized cells in animals administered with a certain
kjemisk stoff som f.eks. mycotoxin fra Trichothecene. Ett eksempel på det kjemiske stoff som induserer cellulær differensiering, er fytohemagglutinin (PHA), men makrofag-aktive-rende stoffer, slik som muramyl-dipeptid (MDP), 12-0-tetra-decanoylforbol-13-acetat (TPA), 12,13-forbol-butyrat, dimethyl-sulfoxyd /DMSO) og mezerein er særlig foretrukket. chemical substance such as mycotoxin from Trichothecenes. One example of the chemical that induces cellular differentiation is phytohemagglutinin (PHA), but macrophage-activating substances, such as muramyl dipeptide (MDP), 12-0-tetra-decanoylphorbol-13-acetate (TPA), 12 ,13-phorbol-butyrate, dimethyl sulfoxide (DMSO) and mezerein are particularly preferred.
I tillegg kan også stoffer som er i stand til å akti-vere det reticuloendotheliale system brukes som det første stimulerende middel ved oppfinnelsen. Disse er ordinære gram-positive bakterier, stoffer produsert av sopp, protozoer og gjær, som brukes i form av levende celler, døde celler (f.eks. etter varme- eller formalinbehandling) eller celle-ekstrakter. Som eksempler på gram-positive bakterier kan nevnes Propioni-bakterier, som f.eks. P. acnes (Corynebacterium parvum) og P. Granulocum (Corynebacterium granulocum); Mycobacteria, som f.eks. Bacillus Calmette-Guerin (B.C.G.) In addition, substances capable of activating the reticuloendothelial system can also be used as the first stimulating agent in the invention. These are ordinary gram-positive bacteria, substances produced by fungi, protozoa and yeast, which are used in the form of living cells, dead cells (e.g. after heat or formalin treatment) or cell extracts. Examples of gram-positive bacteria include Propioni bacteria, such as e.g. P. acnes (Corynebacterium parvum) and P. Granulocum (Corynebacterium granulocum); Mycobacteria, such as Bacillus Calmette-Guerin (B.C.G.)
og M. segmentis; og Nocardia, som f.eks. N. erythropolis og N. gardneri. Eksempler på stoffer produsert av sopp er toxiner produsert av Fusarium. Typiske protozoer er Plasmodium og Toxoplasma. En vanlig brukt gjær er Zymosan ekstrahert fra Saccharomyces cereviciae eller lignende. Visse syntetiske polymerer som f.eks. pyran-copolymerer kan også anvendes som det første stimuleringsmiddel. and M. segmentis; and Nocardia, such as N. erythropolis and N. gardneri. Examples of substances produced by fungi are toxins produced by Fusarium. Typical protozoa are Plasmodium and Toxoplasma. A commonly used yeast is Zymosan extracted from Saccharomyces cereviciae or similar. Certain synthetic polymers such as pyran copolymers can also be used as the first stimulant.
Det andre stimuleringsmiddel som brukes ved oppfinnelsen, er et endotoxin produsert av gram-positive eller gram-negative bakterier. Typiske eksempler omfatter lipopolysaccharider avlet fra E. coli, Pseudomonas aeruginosa og tyfoid-bacillus. Lipopolysaccharider fra gram-positive bakterier kan også brukes med gode resultater. The second stimulant used in the invention is an endotoxin produced by gram-positive or gram-negative bacteria. Typical examples include lipopolysaccharides derived from E. coli, Pseudomonas aeruginosa and typhoid bacillus. Lipopolysaccharides from Gram-positive bacteria can also be used with good results.
KBS kan fremstilles ved å dyrke normale humane monocytiske celler eller monocytiske leukemiceller i et dyrkningsmedium eller et vanlig vevkulturmedium som inneholder serum og andre næringsstoffer, og tilsette det første stimuleringsmiddel før, under eller etter dyrking, etterfulgt av tilset-ning av det andre stimuleringsmiddel. Selv om begge stimulerings-midler (første og andre) er vanlig brukt for induksjon, kan KBS fremstilles ved virkningen av ett av stimuleringsmidlene alene eller til og med i fravær av stimuleringsmiddel. Van ligvis formeres imidlertid cellene fullstendig i et vanlig vevkulturmedium, og det første stimuleringsmiddel (f.eks. KBS can be produced by growing normal human monocytic cells or monocytic leukemia cells in a culture medium or a normal tissue culture medium containing serum and other nutrients, and adding the first stimulant before, during or after cultivation, followed by the addition of the second stimulant. Although both stimulants (first and second) are commonly used for induction, KBS can be produced by the action of one of the stimulants alone or even in the absence of stimulant. Usually, however, the cells are propagated completely in a normal tissue culture medium, and the first stimulating agent (e.g.
0,1 til 100 mg/l TPA) tilsettes så for å indusere den første stimulering. Etter dyrking over et tidsrom på 12 timer til 3 dager, tilsettes det andre stimuleringsmiddel (f.eks. 0,1 0.1 to 100 mg/l TPA) is then added to induce the first stimulation. After culturing for a period of 12 hours to 3 days, the second stimulant (e.g., 0.1
til 10 ug/ml lipopolysaccharid avledet fra E. coli) for å indusere den andre stimulering, hvorved det fåes, etter ytterligere dyrking i et visst tidsrom, f.eks. 8 til 24 timer, KBS i supernatant-dyrkingsvæsken. to 10 µg/ml lipopolysaccharide derived from E. coli) to induce the second stimulation, thereby obtaining, after further cultivation for a period of time, e.g. 8 to 24 hours, KBS in the supernatant culture fluid.
Det således fremstilte KBS behandles, etter å væreThe KBS produced in this way is processed, after being
blitt isolert eller uten å være blitt isolert, med reduksjonsmiddel og/eller denatureringsmiddel hvorved det fåes n-KBS ifølge oppfinnelsen. Når KBS skal isoleres før den etter-følgende behandling, kan dette gjøres ved å bruke kjente sepa-rasjons- og rensingsteknikker, som f.eks. dialyse, utsalting, ultrafiltrering, sentrifugering, oppkonsentrering og fryse-tørking. Dersom ytterligere rensing er påkrevet, kan disse teknikker kombineres med slike ytterligere operasjoner som adsorpsjon på ionebyttere etterfulgt av eluering fra disse, gelfiltrering, affinitetskromatografi under anvendelse av concanavalin A eller et egnet antistoff på Sepharose<®>som bærer, isoelektrisk fraksjonering, væskekromatografi med høy yteevne, kromatofokusering ved hjelp av væskekromatografi med høy ytteevne for proteiner, ionebytting (f.eks. på kolon-ner med FPLC/Mono-P og Mono-Q; Pharmacia AB) og plateelektroforese eller andre typer elektroforese på polyacrylamidgel. Vanligvis adskilles supernatanten som inneholder KBS fra dyrkingsmediet ved sentrifugering og behandles deretter med en anionbytter, etterfulgt av rensing ved hjelp av andre teknikker. Egnede anionbyttere omfatter DEAE-Sephadex<®>A-50, DEAE-Sepharose<®>CL-6B,"DEAE-Sephacel", QAE-Sephadex<®>A-50 (produkter fra Pharmacia AB), "AIECDE 52" (Wattman Co.), "Servacel AE" (Serva Co.) og "Cellex QAE" (Bio-Rad Laboratories). has been isolated or without having been isolated, with reducing agent and/or denaturing agent whereby n-KBS according to the invention is obtained. When KBS is to be isolated before the subsequent treatment, this can be done by using known separation and purification techniques, such as e.g. dialysis, salting out, ultrafiltration, centrifugation, concentration and freeze-drying. If further purification is required, these techniques can be combined with such additional operations as adsorption onto ion exchangers followed by elution therefrom, gel filtration, affinity chromatography using concanavalin A or a suitable antibody on Sepharose<®> as a carrier, isoelectric fractionation, high-performance liquid chromatography performance, chromatofocusing using high performance liquid chromatography for proteins, ion exchange (e.g. on columns with FPLC/Mono-P and Mono-Q; Pharmacia AB) and plate electrophoresis or other types of polyacrylamide gel electrophoresis. Typically, the supernatant containing KBS is separated from the culture medium by centrifugation and then treated with an anion exchanger, followed by purification by other techniques. Suitable anion exchangers include DEAE-Sephadex<®>A-50, DEAE-Sepharose<®>CL-6B,"DEAE-Sephacel", QAE-Sephadex<®>A-50 (products of Pharmacia AB), "AIECDE 52" ( Wattman Co.), "Servacel AE" (Serva Co.) and "Cellex QAE" (Bio-Rad Laboratories).
Således fremstilte KBS behandles så med reduksjonsmiddel og/éller denatureringsmiddel hvorved det fåes n-KBS ifølge oppfinnelsen ved å bruke teknikker som er vanlig an-vendt på dette tekniske område. Som eksempler på reduksjons-midlet, kan det nevnes thiolforbindelser, som f.eks. 2-mer captoethanol, dithiothreitol, dithioerythitol, thioglycolsyre, monothiofosforsyre, cystein, N-acetylcystein og glutathion i redusert form; tertiære fosfiner, som f.eks. tri-n-butyl-fosfin og tris-diethylaminoethylfosfin; sulfitter, som f.eks. natriumsulfitt; og natriumborhydrid. Typiske proteindenaturerende midler omfatter urea, guanidinhydroklorid, guanidin-sulfat og overflateaktive midler (anioniske, kationiske, amfo-tære og ikke-ioniske). Illustrerende eksempler på de overflateaktive midler er natriumdodecylsulfat (SDS), estere av sorbitan og fettsyre, estere av glycerol og fettsyre, ethere av poly-oxyethylen og nonylfenyl, natrium-polyoxyethylenalkylsulfater, natrium-di-2-ethylhexylsulfosuccinat, dodecyltrimethylammonium-bromid og deoxylysolecithin. The KBS produced in this way is then treated with a reducing agent and/or denaturing agent whereby n-KBS according to the invention is obtained by using techniques that are commonly used in this technical area. As examples of the reducing agent, mention may be made of thiol compounds, such as e.g. 2-mer captoethanol, dithiothreitol, dithioerythitol, thioglycolic acid, monothiophosphoric acid, cysteine, N-acetylcysteine and glutathione in reduced form; tertiary phosphines, such as tri-n-butyl-phosphine and tris-diethylaminoethylphosphine; sulphites, such as sodium sulfite; and sodium borohydride. Typical protein denaturants include urea, guanidine hydrochloride, guanidine sulfate, and surfactants (anionic, cationic, amphoteric, and nonionic). Illustrative examples of the surfactants are sodium dodecyl sulfate (SDS), esters of sorbitan and fatty acid, esters of glycerol and fatty acid, ethers of polyoxyethylene and nonylphenyl, sodium polyoxyethylene alkyl sulfates, sodium di-2-ethylhexyl sulfosuccinate, dodecyltrimethylammonium bromide and deoxylysolecithin.
Behandlingen utføres under milde betingelser, f.eks. ved å tilsette reduksjonsmiddel og/eller proteindenaturerende middel til en oppløsning av KBS i vann eller i en egnet buffer, eller til en fraksjon som inneholder KBS. Konsentrasjonen av reduksjonsmiddel og/eller proteindenaturerende middel, behand-lingstemperatur (vanligvis værelsetemperatur), behandlingstid, pH og andre behandlingsbetingelser velges på passende måte i overensstemmelse med konsentrasjonen av KBS som brukes, og andre faktorer. Alternativt kan KBS omdannes til n-KBS ved hjelp av polyacrylamidgelelektroforese (PAGE) i nærvær av reduksjonsmiddel og/eller proteindenaturerende middel. Det er også mulig å oppnå n-KBS fra KBS ved å utsette det for gelfiltrering under anvendelse av gelpartikler som f.eks. dextran, polyacrylamid og agarose (f.eks. Sephadex<®>G-50, G-75, Sepha-cryl<®>S-200, "Biogel P-10, P-30, P-60, "Ultrogel Ac44" og "AcA54") i nærvær av reduksjonsmiddel og/eller proteindenaturerende middel. The treatment is carried out under mild conditions, e.g. by adding reducing agent and/or protein denaturant to a solution of KBS in water or in a suitable buffer, or to a fraction containing KBS. The concentration of reducing agent and/or protein denaturant, treatment temperature (usually room temperature), treatment time, pH and other treatment conditions are appropriately selected in accordance with the concentration of KBS used, and other factors. Alternatively, KBS can be converted to n-KBS by polyacrylamide gel electrophoresis (PAGE) in the presence of reducing agent and/or protein denaturant. It is also possible to obtain n-KBS from KBS by subjecting it to gel filtration using gel particles such as e.g. dextran, polyacrylamide and agarose (e.g. Sephadex<®>G-50, G-75, Sepha-cryl<®>S-200, "Biogel P-10, P-30, P-60, "Ultrogel Ac44" and "AcA54") in the presence of reducing agent and/or protein denaturant.
Det således fremstilte n-KBS som deretter utvinnes og renses, har de nedenfor angitte egenskaper: The n-KBS produced in this way, which is then extracted and purified, has the following properties:
(1) Molekylvekt(1) Molecular weight
Den relative molekylvekt for n-KBS målt ved hjelp av polyacrylamidgel-plateelektroforese (PAGE) i nærvær av 2-mercaptoethanol og natriumdodecylsulfat (SDS) er 17.000 til 30.000. The relative molecular weight of n-KBS as measured by polyacrylamide gel plate electrophoresis (PAGE) in the presence of 2-mercaptoethanol and sodium dodecyl sulfate (SDS) is 17,000 to 30,000.
(2) Hoved-peptidfragment(2) Main peptide fragment
Den relative molekylvekt for hoved-peptidfragmentet fra- skilt ved hjelp av cyanbromidspalting av n-KBS er omtrent The relative molecular weight for the main peptide fragment separated by means of cyanobromide cleavage of n-KBS is approximately
18.500. 18,500.
(3) Partiell aminosyresekvens n-KBS har følgende partielle aminosyresekvens: (3) Partial amino acid sequence n-KBS has the following partial amino acid sequence:
(hvor og hver representerer en uidentifisert amino-syrerest). (4) Aminosyresammensetning n-KBS inneholder følgende aminosyrer i tillegg til Cys og Trp: (where and each represents an unidentified amino acid residue). (4) Amino acid composition n-KBS contains the following amino acids in addition to Cys and Trp:
(De ovenfor viste innhold er molprosenter av individuelle aminosyrerester basert på de samlede aminosyre- (The contents shown above are mole percentages of individual amino acid residues based on the total amino acid
rester bortsett fra Cys og Trp. Standardavvik er vanligvis - 15%, selv om dette nivå overskrides i noen tilfeller) . residues except Cys and Trp. Standard deviation is usually - 15%, although this level is exceeded in some cases).
(5) Retensjonstid i væskekromatografi med høy yteevne.(5) Retention time in high performance liquid chromatography.
n-KBS viste seg ved en retensjonstid på 28 minutter som en enkel topp når det ble målt på "TSK-gel G2000 SW-kolonne (7,5 mm ID x 60 cm) ved værelsetemperatur under anvendelse av 0,IM natriumfosfat (pH 6,0)/5,5M guanidinhydroklorid som elueringsmiddel ved en hastiget på 0,5 ml/minutt. n-KBS appeared at a retention time of 28 minutes as a single peak when measured on a TSK-gel G2000 SW column (7.5 mm ID x 60 cm) at room temperature using 0.1 M sodium phosphate (pH 6 .0)/5.5M guanidine hydrochloride as eluent at a rate of 0.5 ml/minute.
Anti-tumor-aktiviteten til n-KBS ifølge oppfinnelsenThe anti-tumor activity of n-KBS according to the invention
er beskrevet nedenunder.are described below.
Celler (5 x 10^) av mus-avledet methylchoranthren-A-indusert sarcoma ("Meth A"), fortløpende opprettholdt i Balb/C-mus, ble transplantert inn i rygghuden til Balb/C-mus (han-kjønn, 7 uker gamle). Etter 7 dager ble størrelsen på utviklede tumorer målt og 0,5 ml seriefortynninger av n-KBS (prøven fremstilt i eksempel 1, molekylvekt 22.500) ble injisert intravenøst. Cells (5 x 10^) of mouse-derived methylchoranthrene-A-induced sarcoma ("Meth A"), continuously maintained in Balb/C mice, were transplanted into the dorsal skin of Balb/C mice (male, 7 weeks old). After 7 days, the size of developed tumors was measured and 0.5 ml serial dilutions of n-KBS (the sample prepared in Example 1, molecular weight 22,500) were injected intravenously.
Tumorstørrelsen målt 9 dager etter administreringTumor size measured 9 days after administration
viste at fullstendig helbredelse kunne iakttas i alle prøve-gruppene. Hos alle musene hvor noen grad av reduksjon i tumor-størrelse kunne sees, fortsatte tumorstørrelsen å avta fra showed that complete healing could be observed in all test groups. In all mice where some degree of reduction in tumor size could be seen, the tumor size continued to decrease
da av, idet den transplanterte "Meth A"-tumor til slutt forsvant fullstendig. Hos kontrollgruppen økte derimot størrelsen^ på den transplanterte tumor til mer enn 1,5 cm etter 19 dager og nesten alle musene var døde 40 dager senere. Den bemerkelses-verdige anti-tumor-aktivitet til n-KBS ifølge oppfinnelsen er åpenbar ut fra resultatet av dette forsøk. then off, with the transplanted "Meth A" tumor finally disappearing completely. In the control group, on the other hand, the size of the transplanted tumor increased to more than 1.5 cm after 19 days and almost all the mice were dead 40 days later. The remarkable anti-tumor activity of n-KBS according to the invention is obvious from the results of this experiment.
Det ble også påvist at n-KBS ifølge oppfinnelsen har bemerkelsesverdig tumornecroseaktivitet når det ble testet på mus uten pels transplantert med humanavledet gastrisk kreftcellelinje MKN-45 (fortløpende opprettholdt i mus uten peis) ifølge fremgangsmåten til Haranaka et al. (The Japanese Journal of Clinical Medicine, 4_0, No. 8, s. 186-193 (1982)). n-KBS viste seg også å være effektiv mot Sarcoma 180, Ehrich fast tumor, P388 fast tumor og Lewis lunge-carcinoma. It was also demonstrated that n-KBS according to the invention has remarkable tumor necrosis activity when tested on mice without fur transplanted with the human-derived gastric cancer cell line MKN-45 (continuously maintained in mice without fur) according to the method of Haranaka et al. (The Japanese Journal of Clinical Medicine, 4_0, No. 8, pp. 186-193 (1982)). n-KBS was also shown to be effective against Sarcoma 180, Ehrich solid tumor, P388 solid tumor and Lewis lung carcinoma.
Den biologiske aktivitet (KBS-aktivitet) til den lavmolekylære humane reguleringsfaktor mot endogen kreft hos menneske (n-KBS) ifølge oppfinnelsen ble evaluert ved hjelp av en in vitro cytotoksisitetstest under anvendelse av normale eller maligne pattedyrceller ifølge fremgangsmåten beskrevet av Carswell et al. [Proe. Nat. Acad. Sei. USA, 7_2/No- 9' 3666-3670 (1975)]. Testen ble utført ved å bruke en mikroplate med 96 brønner (Nunk Inc., Danmark), "Eagle's minimum essential medium" (MEM) som inneholder 10% serum fra kalve-foster, 50 ug/ml kanamycin og 5 u/ml streptomycin eller 50 enheter/ml gentamycin eller sisomicm. 2,5 x 10 4 L-celler ble inkubert med seriefortynnede prøver med likt volum ved 37°C i 48 timer i en atmosfære av luft som inneholdt 5% carbondioxyd, og den cytotoksiske virkning ble iakttatt under et mikroskop. 1 enhet (U) ble bestemt ved en resiprok fortyn-ning av prøven som ga 50% avliving av L-cellene. The biological activity (KBS activity) of the low molecular human regulatory factor against endogenous cancer in humans (n-KBS) according to the invention was evaluated by means of an in vitro cytotoxicity test using normal or malignant mammalian cells according to the method described by Carswell et al. [Pro. Nat. Acad. Pollock. USA, 7_2/No-9' 3666-3670 (1975)]. The test was performed using a 96-well microplate (Nunk Inc., Denmark), "Eagle's minimum essential medium" (MEM) containing 10% fetal calf serum, 50 ug/ml kanamycin and 5 u/ml streptomycin or 50 units/ml gentamycin or sisomicm. 2.5 x 10 4 L cells were incubated with serially diluted samples of equal volume at 37°C for 48 hours in an atmosphere of air containing 5% carbon dioxide, and the cytotoxic effect was observed under a microscope. 1 unit (U) was determined by a reciprocal dilution of the sample which gave 50% killing of the L cells.
Kreftreguleringsfaktoren ifølge oppfinnelsen administreres i en form som gir full virkning av dens anti-tumor-effekt når den brukes som medisin. Selv om den kan administreres direkte, er det også mulig å gi den, ifølge farmasøytisk prak-sis, som en blanding med farmasøytisk akseptable fortynnings-midler og/eller ett eller flere andre farmakologisk effektive stoffer. n-KBS kan således ha hvilken som helst ønsket form som er egnet for oral eller parenteral administrering, slik som pulvere, granulater, tabletter, sukkerbelagte tabletter, kapsler, piller, stikkpiller, oppslemninger, væsker, emul-sjoner, injeksjonsoppløsninger og aerosoler. Typisk er fak-toren på forhånd lyofilisert og administreres intravenøst, subkutant eller intramuskulært til pasienten etter å være The cancer regulatory factor of the invention is administered in a form that gives full effect of its anti-tumor effect when used as medicine. Although it can be administered directly, it is also possible to give it, according to pharmaceutical practice, as a mixture with pharmaceutically acceptable diluents and/or one or more other pharmacologically effective substances. n-KBS can thus have any desired form suitable for oral or parenteral administration, such as powders, granules, tablets, sugar-coated tablets, capsules, pills, suppositories, slurries, liquids, emulsions, injection solutions and aerosols. Typically, the factor is lyophilized in advance and is administered intravenously, subcutaneously or intramuscularly to the patient after being
blitt oppløst før bruk i fysiologisk saltoppløsning, sterilt vann eller aseptisk isoton oppløsning for injeksjon. Det er også mulig å tilsette før lyofilisering egnede stabilisatorer som f.eks. mannitol og humant serumalbumin, samt oppløselig-gjøringsmidler som f.eks. glycin. has been dissolved before use in physiological saline, sterile water or aseptic isotonic solution for injection. It is also possible to add suitable stabilizers such as e.g. mannitol and human serum albumin, as well as solubilizing agents such as e.g. glycine.
Den egnede dose n-KBS ifølge oppfinnelsen kan variere avhengig av pasientsensitivitet, -alder, -kjønn, -kroppsvekt og betingelser for administrasjonsmåte, tidspunkt og hyppig-het, av typen og egenskapene til blandingen, og av andre faktorer. Det nedenfor viste dosenivå bør derfor betraktes som en rettesnor, idet en lavere dose kan tjene formålet i noen tilfeller, og en høyere dose kan være nødvendig for enkelte andre tilfeller. Normalt er imidlertid den minimale daglige dose for voksne mennesker 10.000. The suitable dose of n-KBS according to the invention may vary depending on patient sensitivity, age, sex, body weight and conditions for administration method, timing and frequency, on the type and properties of the mixture, and on other factors. The dose level shown below should therefore be regarded as a guideline, as a lower dose may serve the purpose in some cases, and a higher dose may be necessary in certain other cases. Normally, however, the minimum daily dose for adults is 10,000.
Eksemplene nedenunder vil ytterligere illustrere oppfinnelsen, men skal ikke betraktes som en begrensning av denne. The examples below will further illustrate the invention, but should not be considered as a limitation thereof.
Preparateksempel ( injeksjon)Preparation example (injection)
n-KBS ifølge oppfinnelsen (1.000.000 enheter) ble opp-løst i 100 ml fysiologisk saltoppløsning, oppløsningen ble filtrert fri for sporer og filtratet ble helt over i ampuller (1 ml hver) og frysetørket. n-KBS according to the invention (1,000,000 units) was dissolved in 100 ml of physiological saline solution, the solution was filtered free of spores and the filtrate was poured into ampoules (1 ml each) and freeze-dried.
Aminosyrer kan heretter representeres ved hjelp av forkortelser som er angitt av Commission on Biochemical Nomen-clature (CBN) under IUPAC-IUB, eller ved hjelp av trivial-forkortelser som er vanlig brukt på dette bestemte område. Amino acids can hereafter be represented using abbreviations specified by the Commission on Biochemical Nomenclature (CBN) under IUPAC-IUB, or using trivial abbreviations commonly used in this particular field.
For aminosyrer hvor det kan foreligge optisk isomerisme, er L-isomeren ment med mindre annet er angitt. For amino acids where optical isomerism may exist, the L isomer is intended unless otherwise indicated.
Asp AsparaginsyreAsp Aspartic acid
Asn AsparaginDonkey Asparagine
Thr ThreoninThr Threonine
Ser SerinSee Serin
Glu GlutaminsyreGlu Glutamic acid
Gin GlutaminGin Glutamine
Gly GlycinGly Glycine
Ala AlaninAla Alanine
Val ValinVal Valin
Met MethioninMet Methionine
Ile IsoleucinIle Isoleucine
Leu LeucinLeu Leucine
Tyr TyrosinBull Tyrosine
Phe FenylalaninPhe Phenylalanine
Lys LysinLight Lysine
His Histidin-His Histidine-
Arg ArgininArg Arginine
Pro ProlinPro Proline
Cys CysteinCys Cysteine
Trp TryptofanTrp Tryptophan
Asp/Asn Asparaginsyre og.asparaginAsp/Asn Aspartic acid and.asparagine
Glu/Gln Glutaminsyre og glutaminGlu/Gln Glutamic acid and glutamine
Eksempel 1Example 1
a) YKBS-4B3 - en klonet stamme av YKBS-7-15 (monocytiske leukemiceller) som er en etablert cellelinje avledet a) YKBS-4B3 - a cloned strain of YKBS-7-15 (monocytic leukemia cells) which is an established cell line derived
fra perifere leukocyter med gulbrunt belegg fra en monocy-TM from peripheral leukocytes with yellow-brown coating from a monocy-TM
tisk leukemipasient - ble dyrket i 8 liter serumfritt HB101 medium (Hana Biologics Inc.) ved 37°C i et tilstrekkelig langt tidsrom med omrøring. TPA (5 ng/ml) ble tilsatt for å gi den første stimulering; 1 ug/ml endotoxin {lipopolysaccharid avledet fra E. coli 0111-B4) ble tilsatt 36 timer senere som den andre stimulering, celledyrking ble fortsatt i ytterligere 16 timer og kultursupernatanten ble samlet opp ved sentrifugering. tic leukemia patient - was cultured in 8 liters of serum-free HB101 medium (Hana Biologics Inc.) at 37°C for a sufficiently long period of time with agitation. TPA (5 ng/ml) was added to provide the initial stimulation; 1 µg/ml endotoxin (lipopolysaccharide derived from E. coli 0111-B4) was added 36 hours later as the second stimulation, cell culture was continued for another 16 hours and the culture supernatant was collected by centrifugation.
Denne supernatant ble oppkonsentrert ca. 2 0 ganger ved hjelp av "Pellicon Cassette" og mettet vandig ammoniumsulfat ble tilsatt ved 4°C til en sluttkonsentrasjon på 50% (volum/ volum). Etter nøye lagring ved 4°C ble bunnfallet samlet opp ved sentrifugering, oppløst i en liten mengde 20mM Tris-HCl-bufferoppløsning som inneholdt 0,04M natriumklorid (pH 7,8), og dialysert med den samme buffer som angitt ovenfor. Dialysatet ble satt på DEAE-Sephadex<®>A-50 anion-bytter (fylt på en Buchner-trakt og på forhånd ekvilibrert med den samme buffer som ovenfor) og eluert med 20 mM Tris-HCl-bufferopp-løsnina som inneholdt 0.08M natriumklorid (pH 7,8). Eluatet ble på nytt oppkonsentrert ved hjelp av "Pellicon Cassette", konsentratet ble nøye dialysert mot 50mM vandig oppløsning av ammoniumbicarbonat, deretter ble dialysatet frysetørket. This supernatant was concentrated approx. 20 times using the "Pellicon Cassette" and saturated aqueous ammonium sulfate was added at 4°C to a final concentration of 50% (vol/vol). After careful storage at 4°C, the precipitate was collected by centrifugation, dissolved in a small amount of 20 mM Tris-HCl buffer solution containing 0.04 M sodium chloride (pH 7.8), and dialyzed with the same buffer as indicated above. The dialysate was loaded onto DEAE-Sephadex<®>A-50 anion exchanger (filled on a Buchner funnel and pre-equilibrated with the same buffer as above) and eluted with 20 mM Tris-HCl buffer solution containing 0.08 M sodium chloride (pH 7.8). The eluate was re-concentrated using the "Pellicon Cassette", the concentrate was carefully dialysed against 50mM aqueous solution of ammonium bicarbonate, then the dialysate was freeze-dried.
Det således erholdte tørre pulver ble oppløst i en liten mengde 20mM Tris-HCl-bufferoppløsning som inneholdt 0,04M natriumklorid, og oppløsningen ble utsatt for gelkromatografi på en "Ultrogel AcA44"-kolonne, hvorved man fikk en aktiv fraksjon ved en relativ molekylvekt på 60.000 - 10.000. Det totale utbytte så langt var ca. 25%. (Når denne fraksjon ble utsatt for kromatofokusering ved hjelp av væskekromatografi med høy yteevne med hensyn på proteiner (FPLC, under anvendelse av "Mono P"-kolonne, Pharmacia AB) for å bevirke isoelektrisk fraksjonering ved hjelp av lineærgradientmetoden, ga tre KBS-aktivitetstopper i nærheten av pH 6,5 (KBS-oc) , pH 7,0 (KBS-(3) og 8,0 (KBS-y) • b) den således erholdte aktive fraksjon (relativ molekylvekt 60.000 - 10.000) ble nøye dialysert og utsatt for polyacrylamidgel-plateelektroforese (PAGE) i nærvær av 2 mM 2-mercaptoethanol og 0,1% natriumdodecylsulfat (12,5% T, plate: 8 cm) ifølge metoden til Laemmli [Nature, 227, 680 (1970)]. Etter elektroforese ble gelen kuttet opp i 1 mm store stykker, protein ble eluert og KBS-aktivitet ble bestemt som beskrevet ovenfor. KBS-aktivitet ble gjenvunnet i fraksjoner svarende til det relative molekylvektområde 22.500 - 1500. Resultatet er oppsummert i fig. 1. Utbyttet av det således erholdte n-KBS var ca. 25%. The dry powder thus obtained was dissolved in a small amount of 20mM Tris-HCl buffer solution containing 0.04M sodium chloride, and the solution was subjected to gel chromatography on an "Ultrogel AcA44" column, whereby an active fraction was obtained at a relative molecular weight of 60,000 - 10,000. The total dividend so far was approx. 25%. (When this fraction was subjected to chromatofocusing by protein high performance liquid chromatography (FPLC, using "Mono P" column, Pharmacia AB) to effect isoelectric fractionation by the linear gradient method, three KBS activity peaks in the vicinity of pH 6.5 (KBS-oc), pH 7.0 (KBS-(3) and 8.0 (KBS-y) • b) the thus obtained active fraction (relative molecular weight 60,000 - 10,000) was carefully dialyzed and subjected to polyacrylamide gel plate electrophoresis (PAGE) in the presence of 2 mM 2-mercaptoethanol and 0.1% sodium dodecyl sulfate (12.5% T, plate: 8 cm) according to the method of Laemmli [Nature, 227, 680 (1970)]. After electrophoresis, the gel was cut into 1 mm pieces, protein was eluted and KBS activity was determined as described above. KBS activity was recovered in fractions corresponding to the relative molecular weight range 22,500 - 1,500. The result is summarized in Fig. 1. The yield of the n-KBS thus obtained was approximately 25%.
Denne prøve av n-KBS ble sendt til følgende tester.This sample of n-KBS was sent to the following tests.
(1) Aminosyresammensetning(1) Amino acid composition
n-KBS (0,5 til 1,0 ug) ble hydrolysert i 0,1 ml 6N-HC1 ved 110°C i 24, 48 og 72 timer i et forseglet rør, og salt-syren ble fjernet under redusert trykk, og 0,30 til 0,35 ml 0,02N-HC1 ble tilsatt for å fremstille testprøver. Aminosyre-analyse ble utført ved hjelp av fluorescens-metoden under anvendelse av o-fthalaldehyd på en Hitachi aminosyreanalysator Modell 835 (Hitachi, Ltd.). Aminosyresammensetning bestemt n-KBS (0.5 to 1.0 µg) was hydrolyzed in 0.1 mL of 6N-HCl at 110°C for 24, 48, and 72 h in a sealed tube, and the hydrochloric acid was removed under reduced pressure, and 0.30 to 0.35 mL of 0.02N-HCl was added to prepare test samples. Amino acid analysis was performed by the fluorescence method using o-phthalaldehyde on a Hitachi amino acid analyzer Model 835 (Hitachi, Ltd.). Amino acid composition determined
på grunnlag av to forsøk er vist i tabell 1 nedenunder.on the basis of two trials is shown in Table 1 below.
(De ovenfor viste innhold er mol% av individuelle aminosyrerester basert på de samlede aminosyrerester bortsett fra Cys og Trp. Standardavvik er angitt ved hjelp av verdien som følger etter symbolet -). (The contents shown above are mol% of individual amino acid residues based on the total amino acid residues except for Cys and Trp. Standard deviation is indicated by the value following the symbol -).
(2) Partiell aminosyresekvens(2) Partial amino acid sequence
(a) Cyanbromidspalting og isolering av peptidfragmenter. (a) Cyan bromide cleavage and isolation of peptide fragments.
Cyanbromidspalting ble utført ved å behandle en del av n-KBS med ti deler cyanbromid i 88% maursyre ved værelsetemperatur i 24 timer. Ved slutten av reaksjonen ble blandingen fortynnet med destillert vann, den resulterende opp-løsning ble oppkonsentrert til tørrhet under redusert trykk, en del av den således erholdte tørre prøve ble underkastet plate-SDS/PAGE og de utviklede proteinbånd ble kontrollert ved hjelp av sølvfargingsmetoden. Som et resultat av cyanbromidspalting, ble det funnet at proteinbåndet som svarte til en molekylvekt på 22.500 forsvant og nye bånd kom tilsyne i stedet for dette (et hovedbånd svarende til en molekylvekt på ca. 18.500 og noen andre som var svakere i styrke). Resten av den tørkede prøve ble gjort til gjenstand for SDS/PAGE i nærvær av 2 mM 2-mercaptoethanol (15% T, plate: 15 cm) og hovedbåndet svarende til en molekylvekt på ca. 18.500 ble skåret bort. Proteinet ble eluert fra gelstykket ved hjelp av elektroforetisk eluering. Fig. 2 viser en sølvfarget pro-fil av SDS/PAGE av dette hoved-peptidfragment og n-KBS. Pro-teinmarkører for standardmolekylvekt (Bio-Rad Laboratories Ltd.) er også angitt i figuren, Cyanogen bromide cleavage was performed by treating one part of n-KBS with ten parts of cyanogen bromide in 88% formic acid at room temperature for 24 hours. At the end of the reaction, the mixture was diluted with distilled water, the resulting solution was concentrated to dryness under reduced pressure, part of the thus obtained dry sample was subjected to plate SDS/PAGE and the developed protein bands were checked by means of the silver staining method. As a result of cyanogen bromide cleavage, it was found that the protein band corresponding to a molecular weight of 22,500 disappeared and new bands appeared in its place (a major band corresponding to a molecular weight of about 18,500 and some others that were weaker in strength). The rest of the dried sample was subjected to SDS/PAGE in the presence of 2 mM 2-mercaptoethanol (15% T, plate: 15 cm) and the main band corresponding to a molecular weight of approx. 18,500 were cut away. The protein was eluted from the gel piece by electrophoretic elution. Fig. 2 shows a silver profile of SDS/PAGE of this major peptide fragment and n-KBS. Standard molecular weight protein markers (Bio-Rad Laboratories Ltd.) are also indicated in the figure,
b) Bestemmelse av partiell aminosyresekvens b) Determination of partial amino acid sequence
Aminosyresekvensen til hoved-peptidfragmentet erholdt The amino acid sequence of the main peptide fragment obtained
ovenfor ble undersøkt ved å anvende den automatiske Edman nedbrytningsmetode under bruk av et apparat for bestemmelse av proteinsekvens i gassfase (Modell 470A, Applied Biosystems). Analysen viste.femten aminosyrer ordnet i den nedenfor viste rekkefølge. above was investigated using the automatic Edman degradation method using a gas phase protein sequence determination apparatus (Model 470A, Applied Biosystems). The analysis showed fifteen amino acids arranged in the order shown below.
(hvor X^og X^hver representerer en uidentifisert aminosyre-rest). (3) Retensjonstid ved væskekromatografi med høy yteevne. (where X^ and X^ each represent an unidentified amino acid residue). (3) Retention time in high performance liquid chromatography.
n-KBS kom tilsyne ved en retensjonstid på 28 minutter som en enkel topp når det ble målt på "TSK gel G2000 SW-kolonne (7,5mm indre diameter x 60 cm) ved værelsetemperatur under anvendelse av 0,IM natriumfosfat (pH 6,0)/5,5M guanidinhydroklorid som elueringsmiddel ved en hastighet på 0,5 ml/ minutt. n-KBS appeared at a retention time of 28 minutes as a single peak when measured on a TSK gel G2000 SW column (7.5 mm ID x 60 cm) at room temperature using 0.1 M sodium phosphate (pH 6, 0)/5.5M guanidine hydrochloride as eluent at a rate of 0.5 ml/minute.
Eksempel 2Example 2
a) YKBS-4B3, en klonet stamme fra YKBS-7-15, ble dyrket i 8 liter RPMI 1640 medium (Gibco Co.), som er et vanlig brukt grunnmedium for dyrking som inneholder 10% serum fra kalve-foster, ved 37°C i et tilstrekkelig langt tidsrom under om-røring, 5 ng/ml 12-O-tetradecanoylforbol-13-acetat (TPA) ble tilsatt for å indusere den første stimulering, 1 ug/ml endotoxin (lipopolysaccharid avledet fra E. coli 0111:B4) ble tilsatt som det andre stimuleringsmiddel 36 timer senere, celledyrkingen ble fortsatt i ytterligere 16 timer og kultursupernatanten ble samlet opp ved sentrifugering. a) YKBS-4B3, a cloned strain from YKBS-7-15, was cultured in 8 liters of RPMI 1640 medium (Gibco Co.), which is a commonly used basal culture medium containing 10% fetal calf serum, at 37 °C for a sufficiently long time with stirring, 5 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA) was added to induce the first stimulation, 1 µg/ml endotoxin (lipopolysaccharide derived from E. coli 0111 :B4) was added as the second stimulant 36 hours later, the cell culture was continued for another 16 hours and the culture supernatant was collected by centrifugation.
Denne supernatant ble fortynnet med 20mM Tris-HCl-buf-feroppløsning (pH 7,8) inntil den endelige saltkonsentrasjon falt til under 0,04M, og den fortynnede oppløsning fikk renne satsvis gjennom en Buchner-trakt fylt med DEAE-Sephadex<®>A-50 anion-.bytter, på forhånd ekvilibrert med 20mM Tris-HCl-buffer-oppløsning (pH 7,8) som inneholdt 0,04M natriumklorid, for å utvinne den ikke-adsorberte fraksjon. This supernatant was diluted with 20mM Tris-HCl buffer solution (pH 7.8) until the final salt concentration fell below 0.04M, and the diluted solution was allowed to flow batchwise through a Buchner funnel filled with DEAE-Sephadex<®> A-50 anion exchanger, pre-equilibrated with 20mM Tris-HCl buffer solution (pH 7.8) containing 0.04M sodium chloride, to recover the non-adsorbed fraction.
a-i) Denne ikke-adsorberte fraksjon ble oppkonsentrert ved hjelp av "Pellicon Cassette", saltkonsentrasjonen ble regulert til 0,5M natriumklorid og den resulterende oppløsning ble gjort til gjenstand for affinitetskromatografi under anvendelse av en "Con-A-Sepharose<®>"-kolonne som på forhånd var blitt ekvilibrert med 20mM Tris-HCl-bufferoppløsning (pH 7,8) som inneholdt 0,5M natriumklorid. Kolonnen ble nøye vasket med den samme bufferoppløsning som ovenfor for å utvinne ikke-adsorberte stoffer, fraksjonen adsorbert på Con-A ble eluert med 20mM Tris-HCl-bufferoppløsning (pH 7,8) som inneholdt a-methylmannosid og 0,5M natriumklorid, og eluatet ble oppkonsentrert og dialysert over natten mot 25mM triethanolamin/ iminodieddiksyre-bufferoppløsning (pH 8,1). Dialysatet ble deretter kromatofokusert ved hjelp av væskekromatografi med høy yteevne med hensyn på proteiner (FPLC, "Mono-P"-kolonne, Pharmacia AB) for å bevirke isoelektrisk fraksjonering (lineær-gradientmetode over pH-området fra 8,1 til 5,0), hvilket ga separate KBS-aktivitets-topper - ved pH ca. 7,0 (KBS-(3) og ved pH høyere enn 8,0. a-i) This non-adsorbed fraction was concentrated using the "Pellicon Cassette", the salt concentration was adjusted to 0.5 M sodium chloride and the resulting solution was subjected to affinity chromatography using a "Con-A-Sepharose<®>"- column that had been previously equilibrated with 20mM Tris-HCl buffer solution (pH 7.8) containing 0.5M sodium chloride. The column was carefully washed with the same buffer solution as above to recover unadsorbed substances, the fraction adsorbed on Con-A was eluted with 20mM Tris-HCl buffer solution (pH 7.8) containing α-methylmannoside and 0.5M sodium chloride, and the eluate was concentrated and dialyzed overnight against 25mM triethanolamine/iminodiacetic acid buffer solution (pH 8.1). The dialysate was then chromato-focused by high-performance liquid chromatography with respect to proteins (FPLC, "Mono-P" column, Pharmacia AB) to effect isoelectric fractionation (linear-gradient method over the pH range from 8.1 to 5.0 ), which gave separate KBS activity peaks - at pH approx. 7.0 (KBS-(3) and at pH higher than 8.0.
Hver av disse aktive fraksjoner ble samlet opp og be-handlet hver for seg på følgende måte.KBS-(3-fraksjonen ble dialysert over natten mot 25mM triethanolamin/iminodieddiksyre-buf f eroppløsning (pH 8,1) og dialysatet ble kromatofoku sert på FPLC, "Mono P"-kolonne på samme måte som angitt ovenfor, hvilket ga den aktive fraksjon (KBS-(3) som en enkel skarp topp ved pH ca. 7,0. Den KBS-aktive fraksjon ved pH høyere enn 8,0 ble på den andre side dialysert over natten mot 25mM diethanolamin-HCl-bufferoppløsning og dialysatet ble kromatofokusert på FPLC, "Mono P"-kolonne (lineærgradient-metode over pH-området fra 9,5 til 6,8), hvilket ga den aktive fraksjon (KBS-?) som en en enkel skarp topp ved pH ca. 8,0. Den samlede cytotoksiske aktivitet hos de således utvundne aktive stoffer mot L-celler løp opp i 4,0 x 10 4 enheter for KBS-13 og 1,0 x 10 4 enheter for KBS-?. Each of these active fractions was collected and treated separately in the following manner. The KBS-(3) fraction was dialyzed overnight against 25mM triethanolamine/iminodiacetic acid buffer solution (pH 8.1) and the dialysate was chromatofocused on FPLC, "Mono P" column in the same manner as above, which gave the active fraction (KBS-(3) as a single sharp peak at pH about 7.0. The KBS active fraction at pH higher than 8, 0, on the other hand, was dialyzed overnight against 25mM diethanolamine-HCl buffer solution and the dialysate was chromatofocused on FPLC, "Mono P" column (linear gradient method over the pH range from 9.5 to 6.8), giving the active fraction (KBS-?) as a single sharp peak at pH approx. 8.0. The total cytotoxic activity of the thus extracted active substances against L-cells amounted to 4.0 x 10 4 units for KBS-13 and 1.0 x 10 4 units for KBS-?.
En del av hver av disse to aktive fraksjoner ble tilsatt en kolonne (diameter 5 x 1400 mm) pakket med Sephadex<®>G-200 (Pharmacia AB) for bestemmelse av relativ molekylvekt. A portion of each of these two active fractions was added to a column (diameter 5 x 1400 mm) packed with Sephadex<®>G-200 (Pharmacia AB) for determination of relative molecular weight.
Den erholdte relative molekylvekt var 60.000 - 10.000 for KBS-13 og 62. 000 - 10. 000 for KBS-7. The relative molecular weight obtained was 60,000 - 10,000 for KBS-13 and 62,000 - 10,000 for KBS-7.
a-ii) Fraksjonen adsorbert på DEAE-Sephadex<®>A-50 ble eluert med 20mM Tris-HCl-bufferoppløsning som inneholdt 0,IM natriumklorid (pH 7,8), eluatet ble oppkonsentrert og saltkonsentrasjonen ble regulert til 0,5M natriumklorid. Den resulterende oppløsning ble affinitetskromatografert under anvendelse av en "Con-A Sepharose<®>"-kolonne på samme måte som under a-i) a-ii) The fraction adsorbed on DEAE-Sephadex<®>A-50 was eluted with 20mM Tris-HCl buffer solution containing 0.1M sodium chloride (pH 7.8), the eluate was concentrated and the salt concentration was adjusted to 0.5M sodium chloride . The resulting solution was affinity chromatographed using a "Con-A Sepharose<®>" column in the same manner as under a-i)
og fraksjonen adsorbert på "Con-A" ble oppkonsentrert, dialysert over natten og kromatofokusert på FPLC, "Mono P"-kolonne (lineærgradient-metode over pH-området fra 8,1 til 5,0) for and the fraction adsorbed on "Con-A" was concentrated, dialyzed overnight and chromatofocused on FPLC, "Mono P" column (linear gradient method over the pH range from 8.1 to 5.0) for
å bevirke isoelektrisk fraksjonering, hvilket ga en fraksjon med KBS-aktivitet ved pH ca. 6,5 (KBS-a). Den totale cytotoksiske aktivitet for KBS-a mot L-celler var 2,5 x 10 4 enheter. to effect isoelectric fractionation, which gave a fraction with KBS activity at pH approx. 6.5 (KBS-a). The total cytotoxic activity of KBS-a against L-cells was 2.5 x 10 4 units.
En del av den ovenfor erholdte aktive fraksjon ble tilsatt en kolonne (diameter 5 x 1400 mm) pakket med Sephadex<®>G-200 (Pharmacia AB) for bestemmelse av relativ molekylvekt. Part of the active fraction obtained above was added to a column (diameter 5 x 1400 mm) packed with Sephadex<®>G-200 (Pharmacia AB) for determination of relative molecular weight.
Den relative molekylvekt for KBS-a viste seg å være 60.000 - 10.000. The relative molecular weight for KBS-a was found to be 60,000 - 10,000.
b) n-KBS med en relativ molekylvekt på 22.500 - 1500 ble erholdt fra fraksjonen av KBS-(3 på en lignende måte som ifølge b) n-KBS with a relative molecular weight of 22,500 - 1,500 was obtained from the fraction of KBS-(3) in a similar manner as according to
1-b). Utbyttet var omtrent 28%. Likeledes kunne n-KBS også erholdes fra fraksjonen av KBS-a. 1-b). The yield was approximately 28%. Similarly, n-KBS could also be obtained from the fraction of KBS-a.
Eksempel 3Example 3
a) YKBS-4B3, en klonet stamme av YKBS-7-15, ble dyrket i TM o 8 liter serumfritt HB101 -medium ved 37 C i et tilstrekkelig langt tidsrom under omrøring.TPA (5 ng/ml) ble tilsatt for å bevirke den første stimulering, 1 ug/ml endotoxin (lipopolysaccharid avledet fra E. coli 0111-B4) ble tilsatt 36 timer senere som den andre stimulering, celledyrking ble fortsatt i ytterligere 16 timer og kultursupernatanten ble samlet opp etter sentrifugering. a) YKBS-4B3, a cloned strain of YKBS-7-15, was grown in TM o 8 liters of serum-free HB101 medium at 37 C for a sufficiently long time with agitation. TPA (5 ng/ml) was added to effect the first stimulation, 1 µg/ml endotoxin (lipopolysaccharide derived from E. coli 0111-B4) was added 36 hours later as the second stimulation, cell culture was continued for another 16 hours and the culture supernatant was collected after centrifugation.
Denne supernatant ble oppkonsentrert ca. tyve ganger ved hjelp av "Pellicon Cassette", og mettet vandig ammoniumsulfat ble tilsatt ved 4°C hvorved man fikk en sluttkonsentrasjon på 50% (volum/volum). This supernatant was concentrated approx. twenty times using the "Pellicon Cassette", and saturated aqueous ammonium sulphate was added at 4°C, whereby a final concentration of 50% (vol/vol) was obtained.
Etter nøye lagring ved 4°C, ble bunnfallet samlet opp ved sentrifugering, oppløst i en liten mengde 20mM Tris-HCl-buf f eroppløsning som inneholdt 0,04M natriumklorid (pH 7,8), og dialysert mot den samme buffer som ovenfor. Dialysatet ble adsorbert på DEAE-Sephadex<®>A-50 anionbytter fylt på en Buchner-trakt og eluert med 20mM Tris-HCl-bufferoppløsning som inneholdt 0,08M natriumklorid (pH 7,8). After careful storage at 4°C, the precipitate was collected by centrifugation, dissolved in a small amount of 20 mM Tris-HCl buffer solution containing 0.04 M sodium chloride (pH 7.8), and dialyzed against the same buffer as above. The dialysate was adsorbed onto DEAE-Sephadex<®>A-50 anion exchanger filled onto a Buchner funnel and eluted with 20mM Tris-HCl buffer solution containing 0.08M sodium chloride (pH 7.8).
Eluatet ble på nytt oppkonsentrert ved hjelp av "Pellicon Cassette", saltkonsentrasjonen ble regulert til 0,5M natriumklorid og den resulterende oppløsning ble affinitetskromatografert under anvendelse av en "Con A Sepharose<®>"-kolonne, som på forhånd var ekvilibrert med 20mM Tris-HCl-buf f eroppløsning (pH 7,8) som inneholdt 0,5M natriumklorid. Kolonnen ble vasket med den samme bufferoppløsning som ovenfor for å samle opp den ikke-adsorberte fraksjon som ble oppkonsentrert og dialysert over natten mot 25mM triethanolamin/ iminodieddiksyre-buffer (pH 8,1), hvilket ga fraksjoner som inneholdt KBS-(3 og KBS-Y. Disse ble kromatofokusert (FPLC, "Mono P"-kolonne, Pharmacia) for å bevirke isoelektrisk fraksjonering ved hjelp av metoden med lineær konsentrasjons-gradient, hvilket ga topper med KBS-aktivitet ved pH ca. 7,0 (KBS-13) og ved pH ca. 8,0 (KBS-?) . The eluate was re-concentrated using the "Pellicon Cassette", the salt concentration was adjusted to 0.5M sodium chloride and the resulting solution was affinity chromatographed using a "Con A Sepharose<®>" column pre-equilibrated with 20mM Tris -HCl buffer solution (pH 7.8) containing 0.5M sodium chloride. The column was washed with the same buffer solution as above to collect the non-adsorbed fraction which was concentrated and dialyzed overnight against 25mM triethanolamine/iminodiacetic acid buffer (pH 8.1), yielding fractions containing KBS-(3 and KBS -Y. These were chromatofocused (FPLC, "Mono P" column, Pharmacia) to effect isoelectric fractionation using the linear concentration gradient method, giving peaks of KBS activity at pH about 7.0 (KBS- 13) and at pH approx. 8.0 (KBS-?) .
b) n-KBS med en relativ molekylvekt på 22.500 - 1500 ble erholdt fra fraksjonene som inneholdt KBS-3 og KBS-?, på en b) n-KBS with a relative molecular weight of 22,500 - 1,500 was obtained from the fractions containing KBS-3 and KBS-?, on a
lignende måte som ifølge eksempel 1-b). similar way as according to example 1-b).
Claims (6)
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP472185 | 1985-01-14 | ||
| JP13667085 | 1985-06-21 | ||
| PCT/JP1986/000011 WO1986004069A1 (en) | 1985-01-14 | 1986-01-13 | Human endogenous cancer regulatory factors |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| NO863657L true NO863657L (en) | 1986-09-12 |
| NO863657D0 NO863657D0 (en) | 1986-09-12 |
Family
ID=26338538
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| NO1986863657A NO863657D0 (en) | 1985-01-14 | 1986-09-12 | REGULATING FACTORS FOR HUMAN ENDOGENIC CANCER. |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP0216929A1 (en) |
| KR (1) | KR870700072A (en) |
| DK (1) | DK436786D0 (en) |
| ES (1) | ES8705466A1 (en) |
| NO (1) | NO863657D0 (en) |
| PT (1) | PT81823B (en) |
| WO (1) | WO1986004069A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0193032A3 (en) * | 1985-02-13 | 1988-04-06 | Gesellschaft für Biotechnologische Forschung mbH (GBF) | Composition comprising or containing cytokinin, process for its preparation, dna sequence and its use |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SE8204382L (en) * | 1981-07-21 | 1983-01-22 | Hayashibara Biochem Lab | PUT TO MAKE MALCELLY FACTOR AND USE THEREOF |
| ZA834976B (en) * | 1982-07-30 | 1984-08-29 | Genentech Inc | Human lymphotoxin |
-
1986
- 1986-01-10 PT PT81823A patent/PT81823B/en not_active IP Right Cessation
- 1986-01-13 ES ES550833A patent/ES8705466A1/en not_active Expired
- 1986-01-13 EP EP86900636A patent/EP0216929A1/en not_active Withdrawn
- 1986-01-13 KR KR1019860700634A patent/KR870700072A/en not_active Withdrawn
- 1986-01-13 WO PCT/JP1986/000011 patent/WO1986004069A1/en not_active Ceased
- 1986-09-12 DK DK436786A patent/DK436786D0/en not_active Application Discontinuation
- 1986-09-12 NO NO1986863657A patent/NO863657D0/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| EP0216929A1 (en) | 1987-04-08 |
| DK436786A (en) | 1986-09-12 |
| ES550833A0 (en) | 1987-05-01 |
| DK436786D0 (en) | 1986-09-12 |
| PT81823A (en) | 1986-02-01 |
| KR870700072A (en) | 1987-02-28 |
| WO1986004069A1 (en) | 1986-07-17 |
| NO863657D0 (en) | 1986-09-12 |
| PT81823B (en) | 1987-11-30 |
| ES8705466A1 (en) | 1987-05-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Erlanson et al. | Purification and characterization of two proteins with co-lipase activity from porcine pancreas | |
| JP3375949B2 (en) | Antibodies to cytotoxic lymphocyte maturation factor | |
| US3948875A (en) | Process for the preparation of epidermal growth factor and new derivative using cross-linked polyacrylamide gel at a pH of 1-3 | |
| AU724940B2 (en) | Protein which induces interferon-gamma production by immunocompetent cell | |
| JP2525023B2 (en) | Purification of recombinant interleukin-1 | |
| EP0212501B1 (en) | Therapeutic agent for treating hematopoietic diseases | |
| EP0132125B1 (en) | A protein having antitumor activity | |
| EP0621285B1 (en) | Novel megakaryocyte potentiator | |
| NO830697L (en) | HOMOGENT HUMAN IMMUN INTERFERON AND MANUFACTURING THEREOF | |
| NO863657L (en) | REGULATING FACTORS FOR HUMAN ENDOGENIC CANCER. | |
| JP3030312B2 (en) | Mature hepatocyte growth factor (I) | |
| CN1017626B (en) | Growth resulating factor for new cell | |
| WO1987006591A1 (en) | Immunosuppressive factor | |
| WO1991018925A1 (en) | Novel megakaryocyte colony stimulating factor and production thereof | |
| van der Weyden et al. | Acanthoxin, a toxic phospholipase A2 from the venom of the common death adder (Acanthophis antarcticus) | |
| Shinomiya et al. | Rat liver arginase suppresses mixed lymphocyte reaction | |
| JP4163834B2 (en) | Hematopoietic stem cell proliferating agent containing cyclophilin | |
| US6306823B1 (en) | Purified new epididymal forward motility protein and a process for isolation of the said epididymal forward motility protein useful as a fertility promoter/blocker | |
| NO850152L (en) | PROCEDURE FOR MANUFACTURING HUMAN, EVEN CANCER REGULATORY FACTOR. | |
| US5635356A (en) | Anti-oncoimmunin-M antibodies and uses thereof | |
| JPH05504134A (en) | Novel proteins with Oncostatin M activity and methods for their preparation | |
| JPH0731482A (en) | Recombined growth factor for human monocyte, and dna sequence coding the same | |
| Jaken et al. | Purification and comparison of several catalytic parameters of the γ-glutamyltranspeptidase of rat mammary adenocarcinoma (13762) and of normal rat mammary gland | |
| JPS63500376A (en) | Human endogenous cancer regulators | |
| JP3677054B2 (en) | Human T-cell leukemia virus infection / proliferation inhibitor |