CN101687927A - 针对g蛋白偶联的受体(gpcr)的抗体 - Google Patents
针对g蛋白偶联的受体(gpcr)的抗体 Download PDFInfo
- Publication number
- CN101687927A CN101687927A CN200880015679A CN200880015679A CN101687927A CN 101687927 A CN101687927 A CN 101687927A CN 200880015679 A CN200880015679 A CN 200880015679A CN 200880015679 A CN200880015679 A CN 200880015679A CN 101687927 A CN101687927 A CN 101687927A
- Authority
- CN
- China
- Prior art keywords
- antibody
- acceptor
- receptor
- cell
- igg
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 102000003688 G-Protein-Coupled Receptors Human genes 0.000 title claims abstract 6
- 108090000045 G-Protein-Coupled Receptors Proteins 0.000 title claims abstract 6
- 210000004027 cell Anatomy 0.000 claims abstract description 61
- 102000005962 receptors Human genes 0.000 claims abstract description 10
- 108020003175 receptors Proteins 0.000 claims abstract description 10
- 230000033300 receptor internalization Effects 0.000 claims abstract description 9
- 102100038294 Metabotropic glutamate receptor 7 Human genes 0.000 claims description 37
- 238000000034 method Methods 0.000 claims description 26
- 108020004459 Small interfering RNA Proteins 0.000 claims description 24
- 230000008878 coupling Effects 0.000 claims description 24
- 238000010168 coupling process Methods 0.000 claims description 24
- 238000005859 coupling reaction Methods 0.000 claims description 24
- 102000043136 MAP kinase family Human genes 0.000 claims description 18
- 108091054455 MAP kinase family Proteins 0.000 claims description 18
- 108010038449 metabotropic glutamate receptor 7 Proteins 0.000 claims description 17
- 150000001875 compounds Chemical class 0.000 claims description 16
- 238000002360 preparation method Methods 0.000 claims description 16
- 239000003053 toxin Substances 0.000 claims description 11
- 231100000765 toxin Toxicity 0.000 claims description 11
- 230000019491 signal transduction Effects 0.000 claims description 10
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 9
- 201000010099 disease Diseases 0.000 claims description 7
- 108010010914 Metabotropic glutamate receptors Proteins 0.000 claims description 6
- 102000016193 Metabotropic glutamate receptors Human genes 0.000 claims description 6
- 230000004913 activation Effects 0.000 claims description 6
- 230000006698 induction Effects 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- 239000008194 pharmaceutical composition Substances 0.000 claims description 4
- 206010012601 diabetes mellitus Diseases 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 3
- 239000003937 drug carrier Substances 0.000 claims description 3
- 230000003993 interaction Effects 0.000 claims description 3
- 230000001404 mediated effect Effects 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 3
- 210000004408 hybridoma Anatomy 0.000 claims description 2
- 102000027411 intracellular receptors Human genes 0.000 claims 1
- 108091008582 intracellular receptors Proteins 0.000 claims 1
- 210000004897 n-terminal region Anatomy 0.000 abstract 1
- 239000000370 acceptor Substances 0.000 description 39
- 239000000243 solution Substances 0.000 description 32
- 238000011534 incubation Methods 0.000 description 24
- 101001032841 Homo sapiens Metabotropic glutamate receptor 7 Proteins 0.000 description 21
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 19
- OHCQJHSOBUTRHG-KGGHGJDLSA-N FORSKOLIN Chemical compound O=C([C@@]12O)C[C@](C)(C=C)O[C@]1(C)[C@@H](OC(=O)C)[C@@H](O)[C@@H]1[C@]2(C)[C@@H](O)CCC1(C)C OHCQJHSOBUTRHG-KGGHGJDLSA-N 0.000 description 18
- 238000004043 dyeing Methods 0.000 description 18
- 230000000694 effects Effects 0.000 description 15
- 101100230376 Acetivibrio thermocellus (strain ATCC 27405 / DSM 1237 / JCM 9322 / NBRC 103400 / NCIMB 10682 / NRRL B-4536 / VPI 7372) celI gene Proteins 0.000 description 14
- 241000699802 Cricetulus griseus Species 0.000 description 14
- 210000001672 ovary Anatomy 0.000 description 14
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 238000001914 filtration Methods 0.000 description 12
- WFZFMHDDZRBTFH-CZEFNJPISA-N 2-[(e)-2-(5-carbamimidoyl-1-benzofuran-2-yl)ethenyl]-1-benzofuran-5-carboximidamide;dihydrochloride Chemical compound Cl.Cl.NC(=N)C1=CC=C2OC(/C=C/C=3OC4=CC=C(C=C4C=3)C(=N)N)=CC2=C1 WFZFMHDDZRBTFH-CZEFNJPISA-N 0.000 description 11
- 239000003153 chemical reaction reagent Substances 0.000 description 11
- DDOQBQRIEWHWBT-VKHMYHEASA-N (2S)-2-amino-4-phosphonobutanoic acid Chemical compound OC(=O)[C@@H](N)CCP(O)(O)=O DDOQBQRIEWHWBT-VKHMYHEASA-N 0.000 description 10
- CWHJIJJSDGEHNS-MYLFLSLOSA-N Senegenin Chemical compound C1[C@H](O)[C@H](O)[C@@](C)(C(O)=O)[C@@H]2CC[C@@]3(C)C(CC[C@]4(CCC(C[C@H]44)(C)C)C(O)=O)=C4[C@@H](CCl)C[C@@H]3[C@]21C CWHJIJJSDGEHNS-MYLFLSLOSA-N 0.000 description 10
- 239000000872 buffer Substances 0.000 description 10
- 239000009871 tenuigenin Substances 0.000 description 10
- SUZLHDUTVMZSEV-UHFFFAOYSA-N Deoxycoleonol Natural products C12C(=O)CC(C)(C=C)OC2(C)C(OC(=O)C)C(O)C2C1(C)C(O)CCC2(C)C SUZLHDUTVMZSEV-UHFFFAOYSA-N 0.000 description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- OHCQJHSOBUTRHG-UHFFFAOYSA-N colforsin Natural products OC12C(=O)CC(C)(C=C)OC1(C)C(OC(=O)C)C(O)C1C2(C)C(O)CCC1(C)C OHCQJHSOBUTRHG-UHFFFAOYSA-N 0.000 description 9
- 239000012634 fragment Substances 0.000 description 9
- 230000001939 inductive effect Effects 0.000 description 9
- 230000008569 process Effects 0.000 description 9
- 108700012359 toxins Proteins 0.000 description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 8
- 101100174574 Mus musculus Pikfyve gene Proteins 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 238000012744 immunostaining Methods 0.000 description 8
- 201000005702 Pertussis Diseases 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 150000008300 phosphoramidites Chemical class 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- LLTDOAPVRPZLCM-UHFFFAOYSA-O 4-(7,8,8,16,16,17-hexamethyl-4,20-disulfo-2-oxa-18-aza-6-azoniapentacyclo[11.7.0.03,11.05,9.015,19]icosa-1(20),3,5,9,11,13,15(19)-heptaen-12-yl)benzoic acid Chemical compound CC1(C)C(C)NC(C(=C2OC3=C(C=4C(C(C(C)[NH+]=4)(C)C)=CC3=3)S(O)(=O)=O)S(O)(=O)=O)=C1C=C2C=3C1=CC=C(C(O)=O)C=C1 LLTDOAPVRPZLCM-UHFFFAOYSA-O 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 239000000556 agonist Substances 0.000 description 6
- 239000004744 fabric Substances 0.000 description 6
- 238000012545 processing Methods 0.000 description 6
- 108091027075 5S-rRNA precursor Proteins 0.000 description 5
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 5
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 5
- 238000013016 damping Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 239000012530 fluid Substances 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 230000007794 irritation Effects 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 4
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 4
- BAVYZALUXZFZLV-UHFFFAOYSA-N Methylamine Chemical compound NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 description 4
- 101001032835 Rattus norvegicus Metabotropic glutamate receptor 7 Proteins 0.000 description 4
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 4
- 238000000137 annealing Methods 0.000 description 4
- 230000001086 cytosolic effect Effects 0.000 description 4
- -1 for example Substances 0.000 description 4
- 125000000524 functional group Chemical group 0.000 description 4
- 235000011187 glycerol Nutrition 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 238000003118 sandwich ELISA Methods 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 125000004213 tert-butoxy group Chemical group [H]C([H])([H])C(O*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 4
- 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 3
- YRQCDCNQANSUPB-UHFFFAOYSA-N AMN082 dihydrochloride Chemical compound Cl.Cl.C=1C=CC=CC=1C(C=1C=CC=CC=1)NCCNC(C=1C=CC=CC=1)C1=CC=CC=C1 YRQCDCNQANSUPB-UHFFFAOYSA-N 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 101150060240 FAB1 gene Proteins 0.000 description 3
- 101001032837 Homo sapiens Metabotropic glutamate receptor 6 Proteins 0.000 description 3
- 102100038300 Metabotropic glutamate receptor 6 Human genes 0.000 description 3
- 241001416149 Ovis ammon Species 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 229920004890 Triton X-100 Polymers 0.000 description 3
- 239000013504 Triton X-100 Substances 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 231100000673 dose–response relationship Toxicity 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 229940127121 immunoconjugate Drugs 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000208199 Buxus sempervirens Species 0.000 description 2
- 102000020313 Cell-Penetrating Peptides Human genes 0.000 description 2
- 108010051109 Cell-Penetrating Peptides Proteins 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 239000012981 Hank's balanced salt solution Substances 0.000 description 2
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- GHAZCVNUKKZTLG-UHFFFAOYSA-N N-ethyl-succinimide Natural products CCN1C(=O)CCC1=O GHAZCVNUKKZTLG-UHFFFAOYSA-N 0.000 description 2
- HDFGOPSGAURCEO-UHFFFAOYSA-N N-ethylmaleimide Chemical compound CCN1C(=O)C=CC1=O HDFGOPSGAURCEO-UHFFFAOYSA-N 0.000 description 2
- 239000012124 Opti-MEM Substances 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 230000003281 allosteric effect Effects 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000005660 chlorination reaction Methods 0.000 description 2
- 238000010612 desalination reaction Methods 0.000 description 2
- 238000002866 fluorescence resonance energy transfer Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 239000012139 lysis buffer Substances 0.000 description 2
- 230000002101 lytic effect Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000003068 molecular probe Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- INAAIJLSXJJHOZ-UHFFFAOYSA-N pibenzimol Chemical compound C1CN(C)CCN1C1=CC=C(N=C(N2)C=3C=C4NC(=NC4=CC=3)C=3C=CC(O)=CC=3)C2=C1 INAAIJLSXJJHOZ-UHFFFAOYSA-N 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 238000001542 size-exclusion chromatography Methods 0.000 description 2
- 239000001488 sodium phosphate Substances 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 125000003396 thiol group Chemical group [H]S* 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 238000003146 transient transfection Methods 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
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 2
- QGVLYPPODPLXMB-UBTYZVCOSA-N (1aR,1bS,4aR,7aS,7bS,8R,9R,9aS)-4a,7b,9,9a-tetrahydroxy-3-(hydroxymethyl)-1,1,6,8-tetramethyl-1,1a,1b,4,4a,7a,7b,8,9,9a-decahydro-5H-cyclopropa[3,4]benzo[1,2-e]azulen-5-one Chemical compound C1=C(CO)C[C@]2(O)C(=O)C(C)=C[C@H]2[C@@]2(O)[C@H](C)[C@@H](O)[C@@]3(O)C(C)(C)[C@H]3[C@@H]21 QGVLYPPODPLXMB-UBTYZVCOSA-N 0.000 description 1
- BLSQLHNBWJLIBQ-OZXSUGGESA-N (2R,4S)-terconazole Chemical compound C1CN(C(C)C)CCN1C(C=C1)=CC=C1OC[C@@H]1O[C@@](CN2N=CN=C2)(C=2C(=CC(Cl)=CC=2)Cl)OC1 BLSQLHNBWJLIBQ-OZXSUGGESA-N 0.000 description 1
- PRDFBSVERLRRMY-UHFFFAOYSA-N 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bibenzimidazole Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-UHFFFAOYSA-N 0.000 description 1
- RFCQJGFZUQFYRF-ZOQUXTDFSA-N 2'-O-methylcytidine Chemical compound CO[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)N=C(N)C=C1 RFCQJGFZUQFYRF-ZOQUXTDFSA-N 0.000 description 1
- BLSAPDZWVFWUTL-UHFFFAOYSA-N 2,5-dioxopyrrolidine-3-sulfonic acid Chemical compound OS(=O)(=O)C1CC(=O)NC1=O BLSAPDZWVFWUTL-UHFFFAOYSA-N 0.000 description 1
- AXAVXPMQTGXXJZ-UHFFFAOYSA-N 2-aminoacetic acid;2-amino-2-(hydroxymethyl)propane-1,3-diol Chemical compound NCC(O)=O.OCC(N)(CO)CO AXAVXPMQTGXXJZ-UHFFFAOYSA-N 0.000 description 1
- 125000000143 2-carboxyethyl group Chemical group [H]OC(=O)C([H])([H])C([H])([H])* 0.000 description 1
- VKIGAWAEXPTIOL-UHFFFAOYSA-N 2-hydroxyhexanenitrile Chemical compound CCCCC(O)C#N VKIGAWAEXPTIOL-UHFFFAOYSA-N 0.000 description 1
- 206010001557 Albinism Diseases 0.000 description 1
- 208000019901 Anxiety disease Diseases 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 208000036693 Color-vision disease Diseases 0.000 description 1
- FCKYPQBAHLOOJQ-UHFFFAOYSA-N Cyclohexane-1,2-diaminetetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)C1CCCCC1N(CC(O)=O)CC(O)=O FCKYPQBAHLOOJQ-UHFFFAOYSA-N 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 206010013883 Dwarfism Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- KRHYYFGTRYWZRS-UHFFFAOYSA-N Fluorane Chemical class F KRHYYFGTRYWZRS-UHFFFAOYSA-N 0.000 description 1
- 208000031886 HIV Infections Diseases 0.000 description 1
- 208000037357 HIV infectious disease Diseases 0.000 description 1
- 101000945318 Homo sapiens Calponin-1 Proteins 0.000 description 1
- 101000652736 Homo sapiens Transgelin Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 208000018737 Parkinson disease Diseases 0.000 description 1
- 101001032842 Rattus norvegicus Metabotropic glutamate receptor 6 Proteins 0.000 description 1
- 208000001647 Renal Insufficiency Diseases 0.000 description 1
- 108091027981 Response element Proteins 0.000 description 1
- 208000007014 Retinitis pigmentosa Diseases 0.000 description 1
- KJTLSVCANCCWHF-UHFFFAOYSA-N Ruthenium Chemical compound [Ru] KJTLSVCANCCWHF-UHFFFAOYSA-N 0.000 description 1
- 108091081021 Sense strand Proteins 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 208000013738 Sleep Initiation and Maintenance disease Diseases 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 239000012505 Superdex™ Substances 0.000 description 1
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 description 1
- 108700007696 Tetrahydrofolate Dehydrogenase Proteins 0.000 description 1
- 102100031013 Transgelin Human genes 0.000 description 1
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical class CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000002223 anti-pathogen Effects 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000000337 buffer salt Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 201000007254 color blindness Diseases 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000006059 cover glass Substances 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 102000004419 dihydrofolate reductase Human genes 0.000 description 1
- KZNICNPSHKQLFF-UHFFFAOYSA-N dihydromaleimide Natural products O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 1
- 238000007865 diluting Methods 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
- 208000035475 disorder Diseases 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 208000024732 dysthymic disease Diseases 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 239000006274 endogenous ligand Substances 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- RSIHJDGMBDPTIM-UHFFFAOYSA-N ethoxy(trimethyl)silane Chemical compound CCO[Si](C)(C)C RSIHJDGMBDPTIM-UHFFFAOYSA-N 0.000 description 1
- XEKOWRVHYACXOJ-UHFFFAOYSA-N ethyl acetate Substances CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 1
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000008098 formaldehyde solution Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 229940089256 fungistat Drugs 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 235000015220 hamburgers Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 208000033519 human immunodeficiency virus infectious disease Diseases 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 238000003703 image analysis method Methods 0.000 description 1
- 150000002460 imidazoles Chemical class 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 239000002955 immunomodulating agent Substances 0.000 description 1
- 230000002584 immunomodulator Effects 0.000 description 1
- 229940121354 immunomodulator Drugs 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 206010022437 insomnia Diseases 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 201000006370 kidney failure Diseases 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- SMBBZHGTZJNSRQ-UHFFFAOYSA-N n'-(6,6-dichlorohexyl)methanediimine Chemical compound ClC(Cl)CCCCCN=C=N SMBBZHGTZJNSRQ-UHFFFAOYSA-N 0.000 description 1
- HOGDNTQCSIKEEV-UHFFFAOYSA-N n'-hydroxybutanediamide Chemical compound NC(=O)CCC(=O)NO HOGDNTQCSIKEEV-UHFFFAOYSA-N 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- QGVLYPPODPLXMB-QXYKVGAMSA-N phorbol Natural products C[C@@H]1[C@@H](O)[C@]2(O)[C@H]([C@H]3C=C(CO)C[C@@]4(O)[C@H](C=C(C)C4=O)[C@@]13O)C2(C)C QGVLYPPODPLXMB-QXYKVGAMSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Substances OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000002207 retinal effect Effects 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 229910052707 ruthenium Inorganic materials 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 201000000980 schizophrenia Diseases 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 210000000278 spinal cord Anatomy 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229960002317 succinimide Drugs 0.000 description 1
- JJAHTWIKCUJRDK-UHFFFAOYSA-N succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate Chemical compound C1CC(CN2C(C=CC2=O)=O)CCC1C(=O)ON1C(=O)CCC1=O JJAHTWIKCUJRDK-UHFFFAOYSA-N 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- FBWNMEQMRUMQSO-UHFFFAOYSA-N tergitol NP-9 Chemical compound CCCCCCCCCC1=CC=C(OCCOCCOCCOCCOCCOCCOCCOCCOCCO)C=C1 FBWNMEQMRUMQSO-UHFFFAOYSA-N 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 230000001256 tonic effect Effects 0.000 description 1
- 238000005829 trimerization reaction Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/286—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against neuromediator receptors, e.g. serotonin receptor, dopamine receptor
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/111—General methods applicable to biologically active non-coding nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/77—Internalization into the cell
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/31—Chemical structure of the backbone
- C12N2310/315—Phosphorothioates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/32—Chemical structure of the sugar
- C12N2310/321—2'-O-R Modification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/35—Nature of the modification
- C12N2310/351—Conjugate
- C12N2310/3513—Protein; Peptide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/35—Nature of the modification
- C12N2310/351—Conjugate
- C12N2310/3517—Marker; Tag
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2320/00—Applications; Uses
- C12N2320/30—Special therapeutic applications
- C12N2320/32—Special delivery means, e.g. tissue-specific
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Pharmacology & Pharmacy (AREA)
- Molecular Biology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Epidemiology (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Neurology (AREA)
- Diabetes (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Plant Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- General Chemical & Material Sciences (AREA)
- Emergency Medicine (AREA)
- Endocrinology (AREA)
- Obesity (AREA)
- Neurosurgery (AREA)
- Hematology (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
本发明提供针对G蛋白偶联受体的表位的抗体,其中所述抗体结合该受体的胞外N-端区域并且该抗体与G蛋白偶联受体的结合诱导细胞内的受体内在化。
Description
本发明涉及针对G蛋白偶联的受体(GPCR)的抗体,具体涉及代谢型谷氨酸受体。
GPCR是已知的最大的受体超家族之一。这些受体在生物学上非常重要,且这些受体的故障引起疾病,诸如阿尔茨海默氏病、帕金森氏病、糖尿病、侏儒症、色盲症、色素性视网膜炎(retinal pigmentosa)和哮喘。GPCR还参与抑郁症、精神分裂症、失眠症、高血压、焦虑症、压力、肾衰竭和若干其他心血管的、代谢的、神经系统的、肿瘤学的和免疫病症(F.Horn和G.Vriend,J.Mol.25 Med.(分子25医学杂志),76:464-468(1998)。它们还显示在HIV感染中起作用(Y.Feng等,Science(科学),272:872-877(1996))。GPCR的结构由7个由环相连接的跨膜螺旋组成。N-端总在胞外且C-膜在胞内。GPCR参与信号转导。在胞外N-端侧接收信号。信号可以是内源配体、化学部分或光。该信号然后通过膜转导到胞质侧,在其中激活异源三聚G蛋白,随之激发反应(F.Horn等,Recept.and Chann.(受体和通道),5:305-314(1998))。
本发明的一个目的是提供针对G蛋白偶联的受体的表位的抗体,其中所述抗体结合该受体的胞外N-端区域并且抗体与G蛋白偶联的受体的结合诱导细胞中的受体内在化。
在优选的实施方案中,所述抗体针对GPCR C类受体,优选代谢型谷氨酸受体,具体针对代谢型谷氨酸受体mGluR7。
在另一个实施方案中,所述抗体是单克隆抗体。
在另一个优选的实施方案中,所述抗体诱导的受体内在化不依赖与该受体有关的G蛋白介导的信号传导途径,优选与该受体有关的Gi偶联的cAMP信号传导途径的活化。在另一个优选的实施方案中,所述抗体诱导的受体内在化涉及促分裂原活化的蛋白激酶(MAPK)信号传导途径。
在另一个实施方案中,所述抗体是通过用表达目的G蛋白偶联的受体,优选mGluR7的全细胞免疫合适动物制备的。
在另一个实施方案中,所述抗体通过在2007年8月8日保藏在DSMZ(German Collection of Microorganisms and Cell Cultures(德国微生物和细胞培养物保藏中心))并获得保藏号DSM ACC2855的杂交瘤细胞系mGluR7-CHO-1/28制备。
在第二个目的中,本发明涉及本发明的抗体在制备用于治疗疾病的药物中的用途,所述疾病涉及G蛋白偶联的受体信号传导途径的调节。所述疾病优选是神经学病症或糖尿病。
在另一个实施方案中,本发明的抗体用作用于胞内递送活性化合物的工具。所述活性化合物优选与所述抗体共价偶联。“活性化合物”可以是任何合适的分子,包括DNA、RNA、siRNA、蛋白质、肽、或药物活性试剂,诸如,例如,毒素、抗生素、抗病原体试剂、抗原、抗体、抗体片段、免疫调节剂、酶、或治疗剂。本发明的抗体适合于胞内递送活性化合物,原因在于该抗体容许通过与GPCR的结合靶向胞内递送活性化合物。
在第三个目的中,本发明涉及用于筛选G蛋白偶联的受体的配体的方法。所述方法包括:
a)使表达G蛋白偶联的受体的细胞或包含G蛋白偶联的受体的细胞制剂与待筛选的化合物和本发明的抗体相接触,和
b)测量抗体与G蛋白偶联的受体的相互作用,其中抗体结合或内在化的水平是配体/G蛋白偶联的受体内在化的指示。
在优选的实施方案中,细胞稳定地表达G蛋白偶联的受体。
在进一步优选的实施方案中,G蛋白偶联的受体是代谢型谷氨酸受体,优选mGluR7。
术语“抗体”用于本文中时包括抗体或抗体片段,包括但不仅限于诸如Fv、Fab、F(ab’)2、单链抗体的抗体片段。
在另一个目的中,本发明提供缀合物,所述缀合物包括本发明的抗体和与该抗体共价连接的活性化合物。
在优选的实施方案中,所述活性化合物是毒素或siRNA分子,优选siRNA分子。
通过将本发明的抗体与siRNA分子的末端基团共价键合来制备处于A-X-Y形式的siRNA-抗体缀合物的方法,所述方法包括:选择预定的siRNA分子;和使该siRNA分子与本发明的抗体共价键合,其中A是本发明的抗体,X是连接体-介导的共价键,且Y是siRNA分子。
制备siRNA-抗体缀合物的方法可以包括活化siRNA官能团,和使活化的官能团与抗体共价键合。待活化的官能团可以包括,但不仅限于,胺基、硫醇基、磷酸基、或其组合。在一些实施方案中,活化siRNA官能团的物质包括1-乙基-3,3-二乙基氨基丙基碳二亚胺,咪唑,N-羟基琥珀酰亚胺,二氯己基-碳二亚胺,N-β-马来酰亚胺丙酸,N-β-马来酰亚胺丙-氧基琥珀酰亚胺酯,N-琥珀酰亚胺基吡啶基二硫代丙酸酯,或其组合。用于制备本发明的siRNA抗体缀合物的另一种方法可以参见Handbook of CellPenetrating Peptides(细胞穿透肽手册),第18章,第二版,2006年4月,编辑:
在另一个目的中,本发明提供药物组合物,其包括本发明的抗体或缀合物和药用载体。
为了更好的施用,所述组合物除上述活性成分外还可以包含至少一种药用载体。所述载体的实例包括盐溶液、无菌水、Ringer’s溶液、缓冲盐溶液、葡萄糖溶液、麦芽糖糊精(水性)溶液、甘油、乙醇及其混合物。如果需要,可以添加典型的添加剂,诸如抗氧化剂、缓冲剂、抑菌剂等。而且,该组合物可以通过添加更多的添加剂,诸如稀释剂、分散剂、表面活性剂、结合试剂和润滑剂而在制药学上被制备以用于以水溶液、混悬液、乳剂等形式注射。
可以通过不同的施药途径,包括静脉内施药、肌肉内施药、动脉内施药、脊髓内施药、鞘内施药、心脏内施药、经皮施药、皮下施药、腹膜内施药、舌下施药、和局部施药使本发明的药物组合物与身体相接触。
为了这样的临床施药,可以利用常规技术将本发明的药物组合物制备为适当的产品。
附图简述
图1A-I显示IgG和Fab1诱导的mGluR7内在化的比较;
图2显示分别由IgG和Fab1片段诱导的mGluR7内在化的量化表面染色和细胞质斑点强度;
图3显示IgG和Fab诱导的mGluR7内在化的动力学;
图4显示百日咳毒素对IgG诱导的mGluR7内在化的影响;4A:细胞表面染色中的平均像素强度。4B:细胞质斑点像素强度/细胞质面积。
图5显示荧光siRNA在与IgG共价连接时的共内在化。图5A-D使用由siRNA-Cy5标记的初级抗体,图5E-H使用与siRNA-Cy5混合的初级抗体;
图6显示IgG(实心圆)和FAB(实心三角)片段的浓度-反应曲线。用3μM弗司扣林,L-AP4的EC80和多种浓度的IgG或FAB刺激细胞。测量cAMP的细胞含量并将其表述为cAMP含量占仅用3μM弗司扣林处理的细胞的百分比。所有的测量重复进行三次,并且数值表示平均值±SE;
图7显示瞬间表达野生型或嵌合mGlu6或mGlu7受体的活CHO细胞的免疫染色,如所示。用IgG-Alexa488染色细胞。只有具有mGlu7受体N-端结构域的受体被IgG识别,这提示IgG结合mGlu7受体的N-端结构域。所示的是在40x放大倍数,很短暴露时间的代表性图像。
图8A显示IgG对表达mGlu7受体的CHO细胞中Thr202/Tyr204处的p44/42MAPK水平的影响。p44/42MAPK水平通过PathScanTM夹心ELISA试剂盒,按照制造商的说明确定。IgG-诱导的p44/42MAPK水平的增高在处理后5分钟时瞬间达到最高水平,并随后迅速下降。
图8B显示增高剂量的IgG在处理表达mGlu7受体的CHO细胞后5分钟时,诱导可饱和水平的p44/42 MAPK。IgG触发p44/42 MAPK活性的潜力处于与如图4中所示抑制L-AP4-诱导的弗司扣林活性抑制的潜力相同的数量级。
图8C显示处理表达mGlu7受体的CHO细胞后IgG-诱导的p44/42MAPK活性水平的特异性。在与IgG的比较中,抗6xHis IgG、orthostericmGlu7激动剂L-AP4和变构mGlu7激动剂AMN082不触发p44/42 MAPK活性的增加。p44/42 MAPK活性的功效与用阳性对照PMA获得的水平相当。
图8D显示与用阳性对照PMA获得的活性相比,用IgG处理未转染的CHO细胞后p44/42 MAPK活性的缺少,这提示IgG的mGlu7-特异性活性。
实验部分
实施例1:使用抗体量化受体由膜到细胞质的迁移
样品制备
第1天:在实验前24小时接种稳定表达大鼠mGlu7受体的中国仓鼠卵巢细胞:
用Trypsin/EDTA分离细胞。加入生长培养基并通过流经10ml吸移管10-20次重新混悬细胞。确定细胞浓度并将细胞混悬液稀释到合适的浓度。为了在96孔板中进行实验,以处于100μl培养基中25.000个细胞/孔接种细胞。在具有5%CO2的湿润细胞培养温育器中37℃温育细胞,以容许细胞粘附在孔上。
第2天:制备I-缓冲剂(1x HBSS,20mM Hepes,0.1%BSA,用三次蒸馏的水制备,未调节pH)。用37℃的温暖的I-缓冲剂制备Hoechst 33258和氯化TrueBlue溶液(TrueBlue Chloride solution)。在以下添加溶液的过程中,在孔中保持37℃的温度。轻轻吸出细胞培养基。在每个孔中,加入100μl Hoechst和Trueblue溶液,并在湿润的温育器中37℃温育板30分钟。
制备抗体/Fab1溶液并在37℃平衡5分钟。用100μl在37℃预平衡的I-缓冲剂/孔小心并快速地清洗细胞一次。然后将60μl抗体溶液/孔加入到孔中,并以37℃的温度温育板30分钟。
Alexa532山羊-抗-鼠二级抗体的1∶400溶液用冰冷的PBS制备。在室温下用100μl PBS/孔清洗孔三次。将该板转移到冰浴中,将60μl冰冷的抗体溶液/孔加入到孔中,并温育该板60分钟。用100μl冰冷的PBS/孔清洗孔三次。每孔中加入温度为-20℃的150μl甲醇/孔,并在冰上温育该板10分钟。用100μl PBS/孔在室温下清洗孔,向孔中加入100μl甲醛溶液/孔,并在室温下温育板15分钟。在室温下用PBS清洗孔一次。通过用0.25% Triton X-100溶液在RT温育5分钟渗透化(permeabilize)细胞膜。用PBS清洗孔一次,并用在PBS中稀释的10%体积/体积山羊血清溶液温育30分钟。用新鲜制备的稀释在PBS中的1∶400Alexa647山羊-抗-鼠二级抗体替换山羊血清。在RT温育细胞30分钟,并用100μl PBS/孔在室温下小心并快速清洗三次。然后加入100μl 4%甲醛/孔,并在室温下温育孔15分钟。将溶液替换为150μl PBS/孔。
量化免疫染色亚细胞水平
利用来自德国汉堡的Evotec Technologies(Evotec技术)的Opera QEHSHCS读数器量化抗体(IgG或Fab1片段)的内在化。该机器装配有反向共聚焦荧光显微镜,并设置为从在透明底部微滴定板中制备样品自动获取图像。在该读数器中,引入用于图像分析的软件“Accapella”,其中可以制备图像分析方法(原稿(script)),其确定预定类型目标的定位。
开发在该实施例中用于量化的原稿,以确定免疫染色分别在细胞表面上和位于胞内腔内(公认为是斑点样结构,其不与细胞表面免疫染色共-定位)的强度。该分析基于对用DNA-特异性荧光团、同源完整细胞染色TrueBlue、和两种具有不同缀合的荧光团的二抗染色的样品平行获得的三幅图像。由每个图像,确定目标。由第一幅特异于DNA染色和TrueBlue染色的图像,细胞核的数量、位置、尺寸和形状由更亮的hoechst染色确定,且细胞质的轮廓由TrueBlue染色确定。由第二幅对Alexa532二级抗体具有选择性的图像,确定细胞表面免疫染色的面积,并量化强度。由第三幅,确定细胞胞质中斑点样结构并量化荧光强度。
比较IgG和Fab1片段诱导的mGluR7内在化
图1显示表达大鼠-mGluR7的细胞的图像,所述细胞在清洗和转移到冰上进行如流程中所述的第二次染色和固定前已经与67nM初级抗体、fab1片段或缓冲剂一起在37℃温育。每排包含在孔的相同视野中平行获得的图像。图A-C使用33nM初级抗体,图D-F使用67nM fab1片段和图G-I不使用初级抗体或fab1片段。图A、D和G显示以对trueblue和hoechst染色具有选择性的过滤设置获得的图像:激光405nm,由Longpath650过滤器反射,经过Shortpath 568过滤器和Bandpath 455/70过滤器过滤的发射光。图B、E和H显示以对使用Alexa532二级抗体的细胞表面染色具有选择性的过滤设置获得的图像:激光532nm,由LP650反射和经过LP568和BP586/40过滤的发射光。图C、F和I显示以对在膜渗透化后使用Alexa647二级抗体的全细胞染色具有选择性的过滤设置获得的图像:激光635nm,通过LP650并经BP690/50过滤的发射光。像素强度从黑到白的灰度变化范围显示在每幅图像的左下角。
完整的IgG和Fab1片段在与表达mGluR7的CHO细胞一起在37℃温育时表现不同。IgG很大程度地内在化,而Fab1片段几乎专门位于细胞表面上。
图2显示关于各自IgG和Fab1片段量化的表面染色和细胞质斑点强度。图A:细胞表面染色中的平均像素强度。图B:细胞质斑点像素强度/细胞质面积。
IgG和Fab片段诱导的mGluR7内在化的动力学
实验按照具有以下改进的流程进行:用初级抗体或Fab片段在37℃温育细胞至多60分钟,然后转移到冰上,并在用二级抗体染色前将溶液替换为新鲜冰冷的初级抗体或Fab片段溶液,1小时。用遵从甲醛固定的染色步骤代替细胞质的Trueblue和hoechst染色,其中样品在与处于PBS中的3uM Hoechst和2μg/ml CellmaskBlue一起在室温下温育15分钟,随后用4%甲醛在室温下进行二次固定15分钟。
图3显示IgG和Fab结合和吸收的动力学。黑圈:22nM IgG,白圈:33nM Fab。曲线是两个不同实验的平均值+/-标准偏差,每个实验重复进行三次。图A:细胞表面染色中的平均像素强度。图B:细胞质斑点像素强度/细胞质面积。IgG显示胞内斑点结构增加的累积,其曲线在开始的20-30分钟后平化。在比较中,关于Fab片段,可以检测到非常微小的增加。
百日咳毒素对IgG诱导的内在化的影响
实验按照具有以下改进的流程进行,即在实验日,在用抗体温育前5小时,将百日咳毒素在PBS中的20x稀释液,最终为500ng/ml加入到孔中。
图4显示使用不同浓度IgG的剂量反应曲线。黑圈:百日咳毒素处理的细胞,白圈:无百日咳毒素。每个点是三个孔的平均值+/-标准偏差。图A:细胞表面染色中的平均像素强度。图B:细胞质斑点像素强度/细胞质面积。百日咳毒素预处理不影响IgG诱导的mGluR7内在化,即IgG诱导的受体内在化不依赖于受体的cAMP信号转导级联。
抗体介导的siRNA的细胞吸收
实验按照具有以下改进的流程进行:将细胞与67nm与siRNA-Cy5缀合的初级抗体(摩尔比:1∶0.3)或67nm初级抗体与20nM siRNA-Cy5的混合物在一起37℃温育40分钟。用于检测细胞表面第一次免疫染色的二级抗体用Alexa488标记,且用于检测全细胞第一次免疫染色的二级抗体用Alexa532标记。用遵从甲醛固定的染色步骤代替细胞质的Trueblue和hoechst染色,其中样品与处于PBS中的3uM Hoechst和2μg/mlCellmaskBlue一起在室温下温育15分钟,随后用4%甲醛在室温下进行二次固定15分钟。
图5显示表达大鼠-mGluR7的细胞的图像,所述细胞在如上所述染色和固定之前已经与siRNA-Cy5缀合的初级抗体或初级抗体和siRNA-Cy5的混合物一起在37℃温育。每排包含在孔的相同视野中平行获得的图像。图A-D是用siRNA-Cy5标记的初级抗体,图E-H是与siRNA-Cy5混合的初级抗体。
图A和E显示以对trueblue和hoechst染色具有选择性的过滤设置获得的图像:激光405nm激发,由Longpath 650过滤器反射、经过Shortpath568过滤器和Bandpath 455/70过滤器过滤的发射光。
图B和F显示以对Cy5具有选择性的过滤设置获得的图像:用激光635nm激发,通过LP650并经BP690/50过滤的发射光。
图C和G显示以对初级抗体和Alexa532二级抗体的全细胞染色具有选择性的过滤设置获得的图像:用激光532nm激发,由LP650和shortpath568反射和经过BP586/40过滤的发射光。
图D和H显示以对在膜渗透化后初级抗体和Alexa488二级抗体的细胞表面特异性染色具有选择性的过滤设置获得的图像:激光488nm激发,由LP650反射和经过Shortpass 568过滤器和Bandpath532/60过滤器的发射光。
像素强度从黑到白的灰度变化范围显示在每幅图像的下部。
IgG可以介导siRNA的细胞吸收。当与siRNA-Cy5共价连接的IgG与表达mGluR7的CHO细胞一起在37℃温育时,IgG和siRNA-Cy5均内在化,并共定位在胞内腔中。在无IgG和siRNA之间的共价连接的条件下,只有IgG内在化。
实施例2:产生表达大鼠mGlu7的细胞系
将大鼠mGlu7a受体剪接变体的cDNA(Genbank:D16817)插入到真核表达载体pcDNA3.1(+)中。按照制造商的说明,利用Lipofectamine加,将该质粒转染到缺乏二氢叶酸还原酶活性的CHO细胞(CHO-dhfr-)中,所述细胞具有在5个cAMP-反应元件(CRE)的控制下的荧光素酶受体基因。通过有限的稀释分离并通过在受体基因测定中的活性识别克隆。用处于测定缓冲液中的1μM弗司扣林和0.5mM L-AP4刺激细胞。4小时后,将上清液更换为裂解缓冲液并测量荧光素酶活性。反应范围从无减弱到约83%减弱。选择表现出最大抑制弗司扣林-介导的荧光素酶活性的克隆细胞系并将其确定为高达至少20代始终提供良好反应。
对于全细胞免疫化,利用Lipofectamine加使用mGlu7表达质粒再次瞬间转染稳定的细胞系并冷冻以在液氮中进行免疫化。
实施例3:全细胞免疫化
使用稳定表达mGluR7转染的CHO细胞,通过重复注射活细胞进行对瑞士白化病小鼠的免疫化。按照G.和C.Milstein(1975)″Continuous cultures of fused cells secreting antibody of predefined specificity(连续培养分泌预定特异性抗体的融合细胞)″.Nature(自然)256:495-497,一旦动物表现出针对mGluR7的特异性免疫反应,摘除脾细胞并与Ag8融合。
实施例4:cAMP测定
在实验前17-24小时,将稳定表达大鼠mGluR7受体的细胞接种到具有透明底部的黑色96孔板中((Corning Costar # 3904)的具有7.5%透析胎牛血清的生长培养基中,并在湿润的温育器中以5% CO2和37℃温育。将生长培养基更换为具有1mM IBMX的Krebs Ringer碳酸氢盐缓冲液,并在30℃温育30分钟。将0.3mM L-AP4和3μM弗司扣林加入到最终测定体积100μl中之前15分钟时添加化合物,并在30℃温育30分钟。该测定通过添加50μl裂解试剂和50μl检测溶液并在室温下摇动2小时终止。
时间-分辨的能量转移通过装备有ND:YAG激光作为激发源的板::视觉TRF读数器(Evotec Technologies GmbH,德国汉堡)测量。用355nm处的激发和具有100ns延迟和100ns门的发射光测量该板2次,在730(带宽30nm)或645nm(带宽75nm)的总曝光时间分别是10s。在730nm处测量的信号必须针对钌背景,Alexa的直接发射光和缓冲液对照进行校正。FRET信号计算如下:FRET=T730-Alexa730-P(T645-B645),P=Ru730-B730/Ru645-B645,其中T730是在730nm处测量的测试孔,T645是在645nm处测量的测试孔,B730和B645分别是在730nm和645nm处的缓冲液对照。由跨度为10μM-0.13nM cAMP的标准曲线的函数确定cAMP的含量。
图6显示IgG(实心圆)和FAB(实心三角)片段的浓度-反应曲线。用3μM弗司扣林,L-AP4的EC80和不同浓度的IgG或FAB刺激细胞。测量cAMP的细胞含量并将其表述为cAMP含量占仅用3μM弗司扣林处理的细胞的百分比。所有的测量重复进行三次,并且数值表示平均值±SE。orthosteric mGlu7激动剂L-AP4的活性受到添加IgG的完全抑制。IC50计算为2.4nM。FAB部分对抗激动剂L-AP4(IC50:486nM),这提示为了有效对抗L-AP4活性需要IgG的二价结合模式。
实施例5:构建嵌合受体
利用交换PCR构建编码嵌合mGlu6和mGlu7受体的cDNA。mGlu6/7受体构建体含有565个源自大鼠mGluR6的N-端胞外区的氨基酸和剩余的大鼠mGluR7a受体C-端部分,其包括完整的跨膜区;mGluR7/6构建体基本是在氨基酸576处具有融合点的反向嵌合物。将cDNA克隆到pcDNA3.1中并利用Lipofectamine加瞬间转染到CHO-dhfr-细胞中。转染24小时后,将细胞接种在培养基中由聚-D-赖氨酸包被的盖玻片上并在第二天进行染色。将在24孔板中生长的细胞置于冰上,加入IgG或IgG-Alexa488(1∶100)并在DMEM中温育30分钟,在添加兔-抗鼠FITC抗体(1∶100)前用DMEM清洗2次,静置于冰上温育30分钟,并且在进行如前的两次清洗步骤后用甘油/PBS(1∶1)封固。
图7显示瞬间表达野生型或嵌合mGlu6或mGlu7受体的活CHO细胞的免疫染色,如所示。用IgG-Alexa488染色细胞。只有具有mGlu7受体N-端结构域的受体被IgG识别,这提示IgG结合mGlu7受体的N-端结构域。所示的是在40x放大倍数,很短暴露时间的代表性图像。
实施例6:IgG-诱导的p44/42MAPK活性
将稳定表达大鼠mGluR7受体的细胞接种到6孔板的生长培养基中。第二天,将培养基替换为2ml饥饿培养基(含有0.1%无脂肪酸BSA的Opti-MEM)。下一天,用处于150μl饥饿培养基中的化合物在37℃刺激细胞指定的时间,否则刺激细胞5分钟。用冰冷PBS清洗细胞并将其溶解中150μl含有1mM β-甘油磷酸、1mM EDTA、1mM EGTA、1μg/ml抑酶醛肽、150mM氯化钠、2.5mM磷酸钠、20mM Tris-CI、1mM PMSF和1%Triton X-100的缓冲液中,刮掉并在冰上进行超声波降级。离心后,利用PathScanTM夹心ELISA试剂盒,按照制造商的说明分析上清液的p44/42 MAPK活性。利用EnVision读数器(Perkin Elmer)阅读吸光度(450nM)。
图8A显示IgG对表达mGlu7受体的CHO细胞中Thr202/Tyr204处的p44/42 MAPK水平的影响。p44/42 MAPK水平通过PathScanTM夹心ELISA试剂盒,按照制造商的说明确定。IgG-诱导的p44/42 MAPK水平的增高在处理后5分钟时瞬间达到最高水平,并随后迅速下降。
图8B显示增高剂量的IgG在处理表达mGlu7受体的CHO细胞后5分钟时,诱导可饱和水平的p44/42 MAPK。IgG触发p44/42 MAPK活性的潜力处于与如图4中所示抑制L-AP4-诱导的弗司扣林活性抑制的潜力相同的数量级。
图8C显示处理表达mGlu7受体的CHO细胞后IgG-诱导的p44/42MAPK活性水平的特异性。在与IgG的比较中,抗6xHis IgG、orthostericmGlu7激动剂L-AP4和变构mGlu7激动剂AMN082不触发p44/42 MAPK活性的增加。p44/42 MAPK活性的功效与用阳性对照PMA获得的水平相当。
图8D显示与用阳性对照PMA获得的活性相比,用IgG处理未转染的CHO细胞后p44/42 MAPK活性的缺少,这提示IgG的mGlu7-特异性活性。
实施例7:siRNA制备
寡核糖核苷酸合成
寡核糖核苷酸按照10μmol规模使用ABI 394合成器(AppliedBiosystems(应用生物系统))的固相上的亚磷酰胺技术合成。关于RNA序列信息参见表1。合成在由受控的孔玻璃(CPG,具有75μmol/g的负荷,获自Prime Synthesis,Aston,PA,美国)制成的固体支持物上进行。常规RNA亚磷酰胺,2’-O-甲基亚磷酰胺以及辅剂购自Proligo(汉堡,德国)。具体地,使用下列amidites:(5’-O-二甲氧基三苯甲基-N6-(苯甲酰)-2’-O-t-丁基二甲基甲硅烷基-腺苷-3’-O-(2-氰基乙基-N,N-二异丙基氨基)亚磷酰胺,5’-O-二甲氧基三苯甲基-N4-(乙酰基)-2’-O-t-丁基二甲基甲硅烷基-胞苷-3’-O-(2-氰基乙基-N,N-二异丙基氨基)亚磷酰胺,(5’-O-二甲氧基三苯甲基-N2-(异丁酰)-2’-O-t-丁基二甲基甲硅烷基-鸟苷-3’-O-(2-氰基乙基-N,N-二异丙基氨基)亚磷酰胺,和5’-O-二甲氧基三苯甲基-2’-O-t-丁基二甲基甲硅烷基-尿苷-3’-O-(2-氰基乙基-N,N-二异丙基氨基)亚磷酰胺。2’-O-甲基亚磷酰胺与常规RNA amidites携带相同的保护基团,除了N4-(t-丁基苯氧基乙酰基)保护的2’-O-甲基-胞苷。将全部的amidites溶于无水乙腈(100mM)中并加入分子筛()。为了在该低聚物的3’-末端产生巯基连接体,使用来自Glen Research(Glen研究)(Sterling,弗吉尼亚,美国)的1-O-二甲氧基三苯甲基-己基-二硫化物,1′-[(2-氰基乙基)-(N,N-二异丙基)]-亚磷酰胺连接体。在不改变该合成周期的条件下,利用相应的亚磷酰胺(获自GEHealthcare,德国慕尼黑)使Cy5荧光染料附着于5’-末端。5-乙基硫代四唑(ETT,500mM处于乙腈中)用作活化剂溶液。偶联时间是6分钟。为了引入硫代磷酸酯键合,使用100mM处于无水乙腈中的3-乙氧基-1,2,4-二噻唑啉-5-酮(EDITH,获自Link Technologies(连接技术),Lanarkshire,苏格兰)溶液。
支持物结合的低聚物的分裂和去保护
在固相合成完成后,将干燥的固体支持物转移到15mL管中并用甲胺的甲醇溶液(2M,Aldrich)45℃处理180分钟。离心后,将上清液转移到新的15mL管中并用1200μL N-甲基吡咯烷-2-酮(N-methylpyrolidine-2-one)(NMP,Fluka,Buchs,瑞士)清洗CPG。清洗物与甲醇甲胺溶液合并并加入450μL三乙胺三氢氟化物(TEA·3HF,Alfa Aesar,Karlsruhe,德国)。将该混合物置于65℃ 150分钟。冷却至室温后,加入0.75mL NMP和1.5mL of乙氧基三甲基硅烷(Fluka,Buchs,瑞士)。10分钟后,通过离心收集沉淀的寡核糖核苷酸,弃去上清液并将固体重构到1mL缓冲液A(见下)中。
纯化寡核糖核苷酸
粗低聚物通过在AKTA Explorer系统(GE Helthcare)上使用XTerra PrepMS C8 10×50mm柱(Waters,Eschborn,德国)的RP HPLC纯化。缓冲液A是100mM三乙基醋酸铵(Biosolve,Valkenswaard,荷兰)且缓冲液B含有处于缓冲液A中的50%乙腈。使用5mL/min的流速。记录260nm,280nm和643nm处的UV踪迹。使用58柱体积(CV)中5%B到60%B的梯度。汇集合适的馏分,并用3M NaOAc,pH=5.2和70%乙醇进行沉淀。
最后,纯化的低聚物通过在含有Sephadex G-25(GE Healthcare)的柱上进行尺寸排阻层析法脱盐。溶液的浓度通过在UV光度计(Beckman Coulter,Krefeld,德国)中在260nm处的吸光度测量确定。直到进行退火,将各个链作为冷冻溶液保存在-20℃。
退火寡核糖核苷酸以产生siRNA
互补链通过结合等摩尔RNA溶液退火。将混合物冷冻干燥并用适当体积的退火缓冲液(100mM NaCl,20mM磷酸钠,pH 6.8)重构以获得理想的浓度。将该溶液置于95℃水浴中,其在3h内冷却至室温。
siRNA序列信息
有义链(5′--3′):(Cy5)cuuAcGcuGAGuAcuucGAdTdT(C6SSC6)dT
反义链(5′--3′):UCGAAGuACUcAGCGuAAGdTsdT
小写字体:2′OMe核苷酸;s:硫代磷酸酯键合;dT:脱氧胸苷
实施例8:抗体-siRNA缀合物的制备
抗体的马来酰亚胺活化:单克隆抗体mGluR7-CHO-1/28与10倍摩尔过量SMCC(磺基琥珀酰亚胺基4-[N马来酰亚胺甲基]环己胺-1-羧酸酯)反应,随后通过脱盐除去过量的(未反应的)试剂。
siRNA活化:用TCEP(Tris[2-羧基乙基]磷化氢)还原Cy5标记的具有单脱氧胸苷的C6SSC6-连接体的siRNA,从而选择性还原二硫键。
然后加入含有巯基的siRNA,以与已经与该单克隆抗体附着的马来酰亚胺基团反应。然后利用NEM(N-乙基马来酰亚胺)封闭siRNA上的未反应的游离巯基,并通过尺寸排阻层析法在Superdex 200HR 10/30柱上纯化终产物。
最终的标记-比通过nanodrop(在280nm处的IgG吸附,646nm处的Cy5,消光系数Cy5:250’000)确定。
塑料器皿,溶液和试剂
测试板Costar 96孔特殊视觉板(Special Optics plate),目录号3614
10x HBSS GIBCO目录号14065-049
Hepes 1M溶液,GIBCO目录号15630-056
BSA 牛血清白蛋白级分V,Sigma,目录号A-3059
Hoechst 33258 Sigma # B-2261(bisBenzimide),在DMSO中溶解到
10mM.
氯化TrueBlue Molecular Probes(分子探针)目录号T1323,在1∶2
MeOH∶DMSO中溶解到2mM
CellMask Blue DMSO中5mg/ml,Invitrogen目录号H34558
甲醛 4%,在PBS中由36.5%储液(Fluka,目录号47629)稀释
山羊抗鼠Alexa 488抗体:1μg/μl溶液Invitrogen目录号A11029
山羊抗鼠Alexa 532抗体:1μg/μl溶液Invitrogen目录号A11200A
山羊抗鼠Alexa 647抗体:1μg/μl溶液Invitrogen目录号A21235A
Triton X-100: Fluka目录号93426,在PBS中稀释到4%体积/体积
山羊血清: Sigma目录号G9023
百日咳毒素: Sigma目录号P-2980.储液:在50%甘油,0.5M NaCl,
50mM Tris-甘氨酸中0.2mg/ml
Krebs Ringer Sigma # K-4002,
L-AP4 Tocris # 0103
弗司扣林 Sigma # F 3917
IBMX Sigma
裂解试剂 Tris,NaCl,1.5%Triton X100,2.5%NP40,10%NaN3
检测溶液 20μM mAb Alexa700-cAMP 1∶1,和48μM钌
-2-AHA-cAMP
生长培养基 DMEM(Invitrogen No 31331),1x HT补剂,10%FCS
Opti-MEM Invitrogen # 11058-021
Lipofectamine加 Invitrogen
pcDNA3.1(+) Invitrogen
CHO-dhfr- ATCC No CRL-9096
测定缓冲液 5mM KCL,154mM NaCl,2.3mM CaCl2,5mM
NaHCO3,1mM MgCl2,5.5mM葡萄糖,5mM Hepes,10
μM IBMX,pH 7.4
裂解缓冲液 25mM Tris,0.4mM DTT,0.4mM CDTA,0.2%甘油,
0.2%Triton X-100,pH 7.8
PMA 佛波醇12-肉豆蔻酸酯13-乙酸酯(Sigma # P8139)
AMN082 Dibenzhydrylethane-1,2-二胺二氢氯化物,机构内部合成
PathScanTM 夹心ELISA试剂盒#7315,Cell Signaling,Beverly,MA
无脂肪酸BSA Sigma # A-6003-25G
尽管显示并描述了本发明目前优选的实施方案,但是显然应该理解为本发明不受其限制,而是可以另外在以下权利要求的范围内不同的实施和实践。
Claims (20)
1.针对G蛋白偶联受体的表位的抗体,其中所述抗体结合所述受体的胞外N-端区域并且所述抗体与G蛋白偶联受体的结合诱导细胞内的受体内在化。
2.权利要求1的抗体,其中所述受体属于GPCR C类,优选所述受体是代谢型谷氨酸受体。
3.权利要求2的抗体,其中所述代谢型谷氨酸受体是mGluR7。
4.权利要求1-3的抗体,其中所述抗体诱导的受体内在化涉及促分裂原活化的蛋白激酶(MAPK)信号传导途径。
5.权利要求1-4的抗体,其是单克隆抗体。
6.权利要求1-5的抗体,其是IgG。
7.权利要求1-6的抗体,其中所述抗体通过杂交瘤细胞系mGluR7-CHO-1/28制备。
8.权利要求1-7的抗体,其中所述抗体诱导的受体内在化不依赖于与所述受体有关的G蛋白介导的信号传导途径,优选与所述受体有关的Gi偶联的cAMP信号传导途径的活化。
9.权利要求1-8的抗体,其作为药物。
10.一种缀合物,其包括权利要求1-8的抗体和与所述抗体共价连接的活性化合物。
11.权利要求10的缀合物,其中所述活性化合物是毒素或siRNA分子,优选siRNA分子。
12.一种药物组合物,其包括权利要求1-8的抗体或权利要求10或11的缀合物和药用载体。
13.权利要求1-8的抗体或权利要求10或11的缀合物用于胞内递送活性化合物的应用。
14.权利要求13的应用,其中所述活性化合物是与所述抗体共价偶联的毒素或siRNA分子。
15.权利要求1-8的抗体在制备用于治疗疾病的药物中的应用,所述疾病涉及G蛋白偶联的受体信号传导途径的调节。
16.权利要求15的应用,其中所述疾病是神经学病症或糖尿病。
17.一种用于筛选G蛋白偶联的受体的配体的方法,所述方法包括以下步骤:
使表达所述G蛋白偶联的受体的细胞或包含所述G蛋白偶联的受体的细胞制剂与待筛选的化合物和权利要求1-8的抗体相接触,和
测量抗体与所述G蛋白偶联的受体的相互作用,其中抗体结合或内在化的水平是配体/G蛋白偶联的受体相互作用的指示。
18.权利要求17的方法,其中使用稳定表达所述G蛋白偶联的受体的细胞。
19.权利要求17或18的方法,其中所述G蛋白偶联的受体是代谢型谷氨酸受体,优选mGluR7。
20.基本如本文中所述的化合物、方法和应用,尤其参考前述实施例。
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP07108213 | 2007-05-15 | ||
EP071082135 | 2007-05-15 | ||
EP07108213.5 | 2007-05-15 | ||
PCT/EP2008/003689 WO2008138536A2 (en) | 2007-05-15 | 2008-05-08 | Antibody directed to g protein coupled receptors (gpcr) |
Publications (2)
Publication Number | Publication Date |
---|---|
CN101687927A true CN101687927A (zh) | 2010-03-31 |
CN101687927B CN101687927B (zh) | 2013-10-30 |
Family
ID=39817060
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN2008800156794A Expired - Fee Related CN101687927B (zh) | 2007-05-15 | 2008-05-08 | 针对g蛋白偶联的受体(gpcr)的抗体 |
Country Status (6)
Country | Link |
---|---|
US (1) | US8354102B2 (zh) |
EP (1) | EP2152752B1 (zh) |
JP (1) | JP5425056B2 (zh) |
CN (1) | CN101687927B (zh) |
CA (1) | CA2686238C (zh) |
WO (1) | WO2008138536A2 (zh) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105907759A (zh) * | 2016-05-30 | 2016-08-31 | 东北师范大学 | 靶向沉默Gβ2的shRNA |
CN109883932A (zh) * | 2019-02-18 | 2019-06-14 | 武汉伊莱瑞特生物科技股份有限公司 | 流式抗体及其制备方法和滴定方法 |
Families Citing this family (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JO3672B1 (ar) | 2008-12-15 | 2020-08-27 | Regeneron Pharma | أجسام مضادة بشرية عالية التفاعل الكيماوي بالنسبة لإنزيم سبتيليسين كنفرتيز بروبروتين / كيكسين نوع 9 (pcsk9). |
GB201014715D0 (en) * | 2010-09-06 | 2010-10-20 | Vib Vzw | Nanobodies stabilizing functional conformational states of GPCRS |
AR087305A1 (es) | 2011-07-28 | 2014-03-12 | Regeneron Pharma | Formulaciones estabilizadas que contienen anticuerpos anti-pcsk9, metodo de preparacion y kit |
CN103930444B (zh) | 2011-09-16 | 2020-08-04 | 瑞泽恩制药公司 | 用前蛋白转化酶枯草溶菌素-9(PCSK9)抑制剂降低脂蛋白(a)水平的方法 |
WO2014078306A1 (en) | 2012-11-13 | 2014-05-22 | Regeneron Pharmaceuticals, Inc. | Anti-prokineticin receptor (prokr) antibodies and uses thereof |
US10111953B2 (en) | 2013-05-30 | 2018-10-30 | Regeneron Pharmaceuticals, Inc. | Methods for reducing remnant cholesterol and other lipoprotein fractions by administering an inhibitor of proprotein convertase subtilisin kexin-9 (PCSK9) |
KR20180034672A (ko) | 2015-08-18 | 2018-04-04 | 리제너론 파아마슈티컬스, 인크. | 지단백질 분리반출술을 경험하고 있는 고지혈증을 갖는 환자를 치료하기 위한 항-pcsk9 억제성 항체 |
EP3473270B1 (en) * | 2016-06-20 | 2022-11-30 | Genahead Bio, Inc. | Antibody-drug conjugate |
CA3050668C (en) * | 2017-01-17 | 2023-08-15 | Daiichi Sankyo Company, Limited | Anti-gpr20 antibody and anti-gpr20 antibody-drug conjugate |
TWI782000B (zh) * | 2017-03-30 | 2022-11-01 | 日商第一三共股份有限公司 | 抗gpr20抗體、其製造方法及其應用 |
IL272964B2 (en) | 2017-08-31 | 2024-02-01 | Daiichi Sankyo Co Ltd | Production method for antibody-drug conjugates |
KR20200041993A (ko) | 2017-08-31 | 2020-04-22 | 다이이찌 산쿄 가부시키가이샤 | 항체-약물 콘주게이트의 개량 제조 방법 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5738999A (en) * | 1993-12-30 | 1998-04-14 | Zymogenetics, Inc. | L-AP4 sensitive glutamate receptors |
JP2004121212A (ja) * | 2002-06-26 | 2004-04-22 | Japan Science & Technology Agency | 抗mGluR1モノクローナル抗体の製造方法 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1995008627A1 (en) * | 1993-09-20 | 1995-03-30 | Ciba-Geigy Ag | Human metabotropic glutamate receptor subtypes (hmr4, hmr6, hmr7) and related dna compounds |
WO2005075988A1 (en) * | 2004-01-28 | 2005-08-18 | Bayer Healthcare Ag | Diagnostics and therapeutics for diseases associated with human metabotropic glutamate receptor 7 (mglur7) |
-
2008
- 2008-05-08 CN CN2008800156794A patent/CN101687927B/zh not_active Expired - Fee Related
- 2008-05-08 JP JP2010507829A patent/JP5425056B2/ja not_active Expired - Fee Related
- 2008-05-08 CA CA2686238A patent/CA2686238C/en active Active
- 2008-05-08 EP EP08749387.0A patent/EP2152752B1/en not_active Not-in-force
- 2008-05-08 WO PCT/EP2008/003689 patent/WO2008138536A2/en active Application Filing
- 2008-05-08 US US12/599,698 patent/US8354102B2/en active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5738999A (en) * | 1993-12-30 | 1998-04-14 | Zymogenetics, Inc. | L-AP4 sensitive glutamate receptors |
JP2004121212A (ja) * | 2002-06-26 | 2004-04-22 | Japan Science & Technology Agency | 抗mGluR1モノクローナル抗体の製造方法 |
Non-Patent Citations (1)
Title |
---|
DOROTHEA HAASEN ET AL.: "G Protein-Coupled Receptor Internalization Assays in the High-Content Screening Format", 《METHODS IN ENZYMOLOGY》 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105907759A (zh) * | 2016-05-30 | 2016-08-31 | 东北师范大学 | 靶向沉默Gβ2的shRNA |
CN109883932A (zh) * | 2019-02-18 | 2019-06-14 | 武汉伊莱瑞特生物科技股份有限公司 | 流式抗体及其制备方法和滴定方法 |
CN109883932B (zh) * | 2019-02-18 | 2021-08-10 | 武汉伊莱瑞特生物科技股份有限公司 | 流式抗体及其制备方法和滴定方法 |
Also Published As
Publication number | Publication date |
---|---|
WO2008138536A2 (en) | 2008-11-20 |
CN101687927B (zh) | 2013-10-30 |
WO2008138536A3 (en) | 2009-01-29 |
EP2152752A2 (en) | 2010-02-17 |
US8354102B2 (en) | 2013-01-15 |
CA2686238C (en) | 2018-09-25 |
US20100303802A1 (en) | 2010-12-02 |
EP2152752B1 (en) | 2017-11-08 |
JP2010527921A (ja) | 2010-08-19 |
CA2686238A1 (en) | 2008-11-20 |
JP5425056B2 (ja) | 2014-02-26 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN101687927B (zh) | 针对g蛋白偶联的受体(gpcr)的抗体 | |
Barr et al. | Subcellular localization and internalization of the four human leptin receptor isoforms | |
Zhang et al. | Quantification of epidermal growth factor receptor expression level and binding kinetics on cell surfaces by surface plasmon resonance imaging | |
Zen et al. | JAM-C is a component of desmosomes and a ligand for CD11b/CD18-mediated neutrophil transepithelial migration | |
Matthews et al. | Ultra-rapid activation of TRPV4 ion channels by mechanical forces applied to cell surface β1 integrins | |
Sun et al. | Regulation of MMP-1 and MMP-2 production through CD147/extracellular matrix metalloproteinase inducer interactions | |
Kast et al. | Epitope insertion favors a six transmembrane domain model for the carboxy-terminal portion of the multidrug resistance-associated protein | |
Caiolfa et al. | Monomer–dimer dynamics and distribution of GPI-anchored uPAR are determined by cell surface protein assemblies | |
Nishi et al. | Fc domain mediated self-association of an IgG1 monoclonal antibody under a low ionic strength condition | |
CN1862258B (zh) | 核仁素辅助的癌症诊断与治疗方法 | |
Schröder et al. | The tetraspanin network modulates MT1-MMP cell surface trafficking | |
Knutti et al. | Soluble extracellular matrix metalloproteinase inducer (EMMPRIN, EMN) regulates cancer‐related cellular functions by homotypic interactions with surface CD 147 | |
Pratt et al. | Fluorogenic green-inside red-outside (GIRO) labeling approach reveals adenylyl cyclase-dependent control of BKα surface expression | |
Yano et al. | Expression and localization of ecto-nucleotide pyrophosphatase/phosphodiesterase I-1 (E-NPP1/PC-1) and-3 (E-NPP3/CD203c/PD-Iβ/B10/gp130RB13-6) in inflammatory and neoplastic bile duct diseases | |
Mihai et al. | Discoidin domain receptor 2 inhibits fibrillogenesis of collagen type 1 | |
Selby et al. | Quantifying cellular internalization with a fluorescent click sensor | |
Nakamura et al. | Ubiquitin-like protein MNSFβ/endophilin II complex regulates Dectin-1-mediated phagocytosis and inflammatory responses in macrophages | |
CN111704669A (zh) | 一种用于治疗晚期胃癌的抗cldn18全人源化抗体 | |
Lofgren et al. | Anti-tumor efficacy of an MMAE-conjugated antibody targeting cell surface TACE/ADAM17-cleaved Amphiregulin in breast cancer | |
Morrison et al. | Standardized Preclinical In Vitro Blood–Brain Barrier Mouse Assay Validates Endocytosis-Dependent Antibody Transcytosis Using Transferrin-Receptor-Mediated Pathways | |
Mousavi et al. | Affinity and kinetics of proprotein convertase subtilisin/kexin type 9 binding to low‐density lipoprotein receptors on HepG2 cells | |
Ingle et al. | DropArray™, a wall‐less 96‐well plate for uptake and immunofluorescence microscopy, confirms CD22 recycles | |
Szilágyi et al. | Complex formation among TGF-β receptors in live cell membranes measured by patch-FRAP | |
JP2023549761A (ja) | 核タンパク質ターゲティング操作脱ユビキチン化酵素およびその使用方法 | |
Li et al. | The antiangiogenic activity of a soluble fragment of the VEGFR extracellular domain |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20131030 |
|
CF01 | Termination of patent right due to non-payment of annual fee |