CN116514975B - 一种鼠抗人b7-h3单克隆抗体及其制备方法和应用 - Google Patents
一种鼠抗人b7-h3单克隆抗体及其制备方法和应用 Download PDFInfo
- Publication number
- CN116514975B CN116514975B CN202310242833.8A CN202310242833A CN116514975B CN 116514975 B CN116514975 B CN 116514975B CN 202310242833 A CN202310242833 A CN 202310242833A CN 116514975 B CN116514975 B CN 116514975B
- Authority
- CN
- China
- Prior art keywords
- human
- chain variable
- variable region
- antibody
- monoclonal antibody
- 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.)
- Active
Links
- 238000002360 preparation method Methods 0.000 title description 5
- 241001529936 Murinae Species 0.000 claims abstract description 20
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 4
- 239000013604 expression vector Substances 0.000 claims description 15
- 239000000203 mixture Substances 0.000 claims description 10
- 102000039446 nucleic acids Human genes 0.000 claims description 6
- 108020004707 nucleic acids Proteins 0.000 claims description 6
- 150000007523 nucleic acids Chemical class 0.000 claims description 6
- 238000012360 testing method Methods 0.000 claims description 3
- 239000002671 adjuvant Substances 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 239000008194 pharmaceutical composition Substances 0.000 claims description 2
- 206010028980 Neoplasm Diseases 0.000 abstract description 30
- 238000009739 binding Methods 0.000 abstract description 24
- 241000699670 Mus sp. Species 0.000 abstract description 21
- 241000699666 Mus <mouse, genus> Species 0.000 abstract description 20
- 238000002965 ELISA Methods 0.000 abstract description 11
- 230000004614 tumor growth Effects 0.000 abstract description 9
- 102100022623 Hepatocyte growth factor receptor Human genes 0.000 abstract description 6
- 101710184069 Hepatocyte growth factor receptor Proteins 0.000 abstract description 6
- 238000005516 engineering process Methods 0.000 abstract description 6
- 230000000903 blocking effect Effects 0.000 abstract description 5
- 230000009286 beneficial effect Effects 0.000 abstract description 4
- 230000005847 immunogenicity Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 43
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 26
- 108090000623 proteins and genes Proteins 0.000 description 22
- 238000001514 detection method Methods 0.000 description 15
- 241000894006 Bacteria Species 0.000 description 13
- 239000002609 medium Substances 0.000 description 13
- 102000004169 proteins and genes Human genes 0.000 description 12
- 238000005406 washing Methods 0.000 description 12
- 210000004408 hybridoma Anatomy 0.000 description 11
- 239000007788 liquid Substances 0.000 description 10
- 238000000034 method Methods 0.000 description 9
- 238000003752 polymerase chain reaction Methods 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 239000006228 supernatant Substances 0.000 description 9
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 8
- 102000008022 Proto-Oncogene Proteins c-met Human genes 0.000 description 8
- 108010089836 Proto-Oncogene Proteins c-met Proteins 0.000 description 8
- 229960002685 biotin Drugs 0.000 description 8
- 239000011616 biotin Substances 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 8
- 239000007640 basal medium Substances 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 238000011161 development Methods 0.000 description 7
- 230000018109 developmental process Effects 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- 238000012163 sequencing technique Methods 0.000 description 7
- 239000002299 complementary DNA Substances 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 230000006870 function Effects 0.000 description 6
- 230000005764 inhibitory process Effects 0.000 description 6
- 102000005962 receptors Human genes 0.000 description 6
- 108020003175 receptors Proteins 0.000 description 6
- 210000004881 tumor cell Anatomy 0.000 description 6
- 239000002202 Polyethylene glycol Substances 0.000 description 5
- 239000011248 coating agent Substances 0.000 description 5
- 238000000576 coating method Methods 0.000 description 5
- 239000013642 negative control Substances 0.000 description 5
- 229920001223 polyethylene glycol Polymers 0.000 description 5
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 5
- 238000011729 BALB/c nude mouse Methods 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 4
- 240000000220 Panda oleosa Species 0.000 description 4
- 235000016496 Panda oleosa Nutrition 0.000 description 4
- 150000001413 amino acids Chemical group 0.000 description 4
- 235000020958 biotin Nutrition 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 239000012091 fetal bovine serum Substances 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 230000001131 transforming effect Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000004698 Polyethylene Substances 0.000 description 3
- 229920001213 Polysorbate 20 Polymers 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 238000007792 addition Methods 0.000 description 3
- 229960000723 ampicillin Drugs 0.000 description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 239000013599 cloning vector Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 239000006260 foam Substances 0.000 description 3
- 238000013115 immunohistochemical detection Methods 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 230000019491 signal transduction Effects 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 230000009261 transgenic effect Effects 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 206010009944 Colon cancer Diseases 0.000 description 2
- 238000012286 ELISA Assay Methods 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 102100021866 Hepatocyte growth factor Human genes 0.000 description 2
- 239000002033 PVDF binder Substances 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 description 2
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- 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 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 239000012679 serum free medium Substances 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 210000004989 spleen cell Anatomy 0.000 description 2
- 229910001220 stainless steel Inorganic materials 0.000 description 2
- 239000010935 stainless steel Substances 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 102000010400 1-phosphatidylinositol-3-kinase activity proteins Human genes 0.000 description 1
- TVZGACDUOSZQKY-LBPRGKRZSA-N 4-aminofolic acid Chemical compound C1=NC2=NC(N)=NC(N)=C2N=C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 TVZGACDUOSZQKY-LBPRGKRZSA-N 0.000 description 1
- 102000008096 B7-H1 Antigen Human genes 0.000 description 1
- 108010074708 B7-H1 Antigen Proteins 0.000 description 1
- 238000011725 BALB/c mouse Methods 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
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 101001044893 Homo sapiens Interleukin-20 receptor subunit alpha Proteins 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 208000029462 Immunodeficiency disease Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102100022706 Interleukin-20 receptor subunit alpha Human genes 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- 206010061309 Neoplasm progression Diseases 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 108091007960 PI3Ks Proteins 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 108010039918 Polylysine Proteins 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 239000006180 TBST buffer Substances 0.000 description 1
- 210000000447 Th1 cell Anatomy 0.000 description 1
- 102000005747 Transcription Factor RelA Human genes 0.000 description 1
- 108010031154 Transcription Factor RelA Proteins 0.000 description 1
- 102100032990 Trem-like transcript 2 protein Human genes 0.000 description 1
- 101710149051 Trem-like transcript 2 protein Proteins 0.000 description 1
- 101100311214 Xenopus laevis stat3.1 gene Proteins 0.000 description 1
- 229960003896 aminopterin Drugs 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 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
- 102000023732 binding proteins Human genes 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000012830 cancer therapeutic Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000000139 costimulatory effect Effects 0.000 description 1
- 230000016396 cytokine production Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 230000017188 evasion or tolerance of host immune response Effects 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000012921 fluorescence analysis Methods 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 230000034659 glycolysis Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
- 230000005934 immune activation Effects 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000007813 immunodeficiency Effects 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 238000000643 oven drying Methods 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 108091005981 phosphorylated proteins Proteins 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 229920000656 polylysine Polymers 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000000284 resting effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000004565 tumor cell growth Effects 0.000 description 1
- 230000005751 tumor progression Effects 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
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
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
- G01N2333/70532—B7 molecules, e.g. CD80, CD86
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Hematology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Urology & Nephrology (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pathology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Food Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Veterinary Medicine (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Plant Pathology (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开了一种鼠抗人B7‑H3单克隆抗体,包括重链可变区和轻链可变区,所述重链可变区的氨基酸序列如SEQ ID NO.1所示;所述轻链可变区的氨基酸序列如SEQ ID NO.2所示。本发明的有益效果为:本发明提供了一种鼠抗人B7‑H3单克隆抗体3E8(克隆号/酶标板编号:3E8)是鼠源的抗体通过抗体工程技术进行改造,具有较弱的免疫原性,更有利于未来的人体应用;能够抑制B7‑H3与原癌基因c‑Met结合,使得该抗体具有明确的阻断位点;能够抑制荷瘤小鼠体内肿瘤生长的作用,使得该抗体具有广泛的临床应用前景。
Description
技术领域
本发明属于生物技术领域,具体涉及一种鼠抗人B7-H3单克隆抗体及其制备方法、肿瘤抑制功能以及功能阻断位点。
背景技术
B7-H3于2001年从人树突状细胞cDNA文库中首次克隆出来。早期的B7-H3被认为是一种正性共刺激分子,能够正性调控T细胞产生免疫活化效应。但随着研究的深入,越来越多的研究显示B7-H3更多地展现出负性共刺激分子的效应,包括抑制Th1细胞活性,促进免疫抑制细胞因子产生,促进肿瘤免疫逃逸等。遗憾的是,B7-H3的受体至今尚未发现。曾有研究发现TLT-2可以作为B7-H3的受体发挥功能,但存在争议,并且也应该不是唯一的受体,因为B7-H3具有正性和负性的双重作用,很可能通过不同受体发挥功能。除了受体的研究,B7-H3还被发现可以与一些结合蛋白,如IL20RA,MVP等结合发挥生物学功能。
此外,B7-H3的非免疫学效应最近也越来越多地受到关注。不仅在骨分化、炎症反应以及代谢等多种疾病和功能中B7-H3都扮演非免疫功能的角色,在肿瘤细胞中B7-H3也可以不依赖于免疫效应而独立地通过调控肿瘤细胞生物信号发挥功能。B7-H3可以通过多种信号途径直接参与多种肿瘤细胞的肿瘤进展、化疗药物敏感性以及糖酵解,包括PI3K/Akt、Jak/STAT、NF-κB-p65/MAPK-p38和Ras/Raf/MEK等信号。B7-H3能够在多种癌症类型的肿瘤患者组织中检测到并异常表达,并预测预后不良,因此B7-H3可以作为一个候选的癌症治疗靶点。
到目前为止,针对B7-H3作为肿瘤治疗靶点,已有3种具有ADCC或ADC作用的抗B7-H3的单克隆抗体正在进行至少11项一期临床试验,并取得了相当不错的初步结果。此外,B7-H3已被广泛地用作CAR-T细胞(B7-H3.CAR-Ts)的作用靶点,在体外和小鼠体内有效地控制肿瘤细胞,且无明显毒性。尽管受体尚未阐明,B7-H3由于其独特的功能吸引了越来越多地基础和临床研究。
因此,研制鼠抗人B7-H3单克隆抗体可以为进一步研究该分子在肿瘤中的作用奠定物质基础,阻断位点的阐明也为探明B7-H3信号传导通路及功能机制提供可能的解释,具有肿瘤生长抑制功能的抗人B7-H3单抗有望成为肿瘤生物治疗的关键药物。
发明内容
本申请的主要目的在于提供一种可阻断B7-H3与原癌基因c-Met结合,且能抑制荷瘤小鼠体内肿瘤生长的鼠抗人B7-H3单克隆抗体及其应用。
为了实现上述目的,本发明提供如下技术方案:
一种鼠抗人B7-H3单克隆抗体,包括重链可变区(mVH)和轻链可变区(mVL),所述重链可变区的氨基酸序列如SEQ ID NO.1所示;所述轻链可变区的氨基酸序列如SEQ ID NO.2所示;
所述SEQ ID NO.1的序列为:
QVQLQESGAELVKPGASVKLSCKTSGYTFTNYPIHWVKQSPGQGLEWIARIYWGIDSIYYNEIFRGKVTLTADTSTSTAYMQLSSLKSEDTAVYFCANSGYTDYGTYYLMDYWGQGTTVTVSS;
所述SEQ ID NO.2的序列为:
DIVMTQSALSVSVTPGESVSISCRSTRSLLHSNGCTYLYWFLQRPGQSPQLLIYWMYNLASGVPDRFSGSGSGTTFTLKISRVEAEDVGVYYCMSNLEYLFTFGGGTKLEIK。
上述一种鼠抗人B7-H3单克隆抗体,作为一种优选的实施方案,所述重链可变区具有三个高变区,具体为:GYTFTNYP(SEQ ID NO.3)、IYWGIDSI(SEQ ID NO.4)、ANSGYTDYGTYYLMDY(SEQ ID NO.5)。
上述一种鼠抗人B7-H3单克隆抗体,作为一种优选的实施方案,所述轻链可变区具有三个高变区,具体为:
RSTRSLLHSNGCTYLY(SEQ ID NO.6)、WMYNLA(SEQ ID NO.7)、MSNLEYLFT(SEQ IDNO.8)。
本发明的第二方面,提供一种鼠抗人B7-H3单克隆抗体在重链可变区和轻链可变区的检测方法,包括如下步骤:
(1)制备B7-H3单克隆抗体的杂交瘤;
(2)制备B7-H3鼠/人嵌合抗体:
(2.1)提取杂交瘤细胞的cDNA:从该杂交瘤细胞株中提取RNA,并使用RT-PCR技术,将获得的RNA反转录为cDNA,进一步PCR获得单抗重链可变区和轻链可变区;
(2.2)将步骤2.1所述重链可变区和轻链可变区分别与克隆载体pJET连接,连接产物转化感受态细菌DH5a,由于pJET载体带有氨苄抗性基因,将转化菌液涂在氨苄青霉素(Amp)抗性的LB固体培养基上,37°过夜培养;
(2.3)待涂板细菌长出分散菌落,选择边缘清晰、生长良好的菌落,进一步测序鉴定重链和轻链可变区基因序列;
(2.4)根据测序结果保留候选的重链可变区和轻链可变区序列,再次PCR扩增出与表达载体匹配的重链可变区和轻链可变区序列,将PCR产物与BmtI(酶切位点GCTAG^C),FspI(酶切位点TGC^GCA)限制性内切酶预先处理的线性表达载体连接,连接产物转化感受态细菌DH5a,由于表达载体带有卡那霉素(Kana+)抗性基因,可将转化菌液涂在Kana抗性的LB固体培养基上,37°过夜培养;
(2.5)对比两次测序结果,测试方法参照(2.4),挑选前后两次序列均一致的转化菌,扩大培养后进行质粒抽提;将包含了单抗重链可变区和轻链可变区基因的表达载体共转染真核表达细胞株293;
(2.6)收获上清含有目的抗体,流式检测表达单抗与M435结合良好,阳性率达95%以上,表明已获得正确的抗体重链和轻链可变区序列。
本发明的第三方面,提供一种编码所述的鼠抗人B7-H3单克隆抗体的核酸分子。
本发明的第四方面,提供一种表达载体,包含上述的鼠抗人B7-H3单克隆抗体的核酸分子。
本发明的第五方面,提供一种组合物,包含所述的鼠抗人B7-H3单克隆抗体,和可接受的辅料或载体。
本发明的第六方面,提供一种药用组合物,包含所述的鼠抗人B7-H3单克隆抗体或所述的组合物,和药学上可接受的载体。
本发明的第七方面,提供一种检测试剂盒,包含所述的鼠抗人B7-H3单克隆抗体、所述的核酸分子、所述的表达载体或所述的组合物。
本发明的第八方面,提供一种鼠抗人B7-H3单克隆抗体的应用,所述鼠抗人B7-H3单克隆抗体可阻断B7-H3与原癌基因c-Met结合。
本发明的有益效果为:本发明提供的鼠抗人B7-H3单克隆抗体3E8是鼠源的抗体通过抗体工程技术进行改造,具有较弱的免疫原性,更有利于未来的人体应用;能够抑制B7-H3与原癌基因c-Met结合,使得该抗体具有明确的阻断位点;能够抑制荷瘤小鼠体内肿瘤生长的作用,使得该抗体具有广泛的临床应用前景。
附图说明
图1a为SPR技术检测B7-H3和c-Met结合的检测结果。
图1b为SPR技术检测B7-H3和c-Met结合的实验方法示意图。
图1c为ELISA检测B7-H3和c-Met结合的示意图。
图1d为ELISA检测不同浓度梯度B7-H3与c-Met结合的示意图。
图2a为流式细胞术检测B7-H3单抗3E8能够识别并结合B7-H3的示意图。
图2b为ELISA检测不同浓度梯度的3E8与B7-H3蛋白结合的示意图。
图2c为ELISA检测B7-H3单抗3E8能够阻断B7-H3蛋白与c-Met蛋白的结合,并且存在浓度梯度依赖。
图2d为western blot检测B7-H3单抗3E8能够抑制细胞的c-Met活化信号p-Met以及p-Stat3的表达,且存在浓度依赖。
图3a为HCT116细胞荷瘤小鼠检测3E8抗体对肿瘤生长的抑制作用。
图3b为NCI-N87细胞荷瘤小鼠检测3E8抗体对肿瘤生长的抑制作用
图3c为HCT116细胞荷瘤小鼠用3E8抗体治疗后的生存图。
图3d为HCT116细胞荷瘤小鼠用3E8抗体治疗后的肿瘤大小示意图。
图3e为荷瘤小鼠检测3E8抗体对敲减B7-H3的HCT116肿瘤细胞生长的抑制作用示意图。
图3f为HCT116细胞荷瘤小鼠用3E8抗体治疗后的肿瘤HE染色结果以及免疫组化检测B7-H3表达、CD133表达结果示意图。
具体实施方式
为了使本技术领域的人员更好地理解本申请方案,下面将结合案例对本申请实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本申请一部分的实施例,而不是全部的实施例。基于本申请中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都应当属于本申请保护的范围。
本发明所述一种鼠抗人B7-H3单克隆抗体利用Co-IP、表面等离子共振实验(SPR)以及ELISA技术,检测B7-H3和原癌基因c-Met的相互作用;利用ELISA技术,检测该抗体3E8是否能够阻断B7-H3与c-Met蛋白之间的结合;利用western blot技术以及人结直肠癌细胞株HCT116,检测3E8是否能够抑制c-Met信号活化的标志p-Met以及p-Stat3的表达。
本发明所述一种鼠抗人B7-H3单克隆抗体利用高表达B7-H3的恶性肿瘤细胞株接种免疫缺陷小鼠皮下成瘤,检测该抗体3E8是否能抑制肿瘤生长;利用敲减B7-H3的恶性肿瘤细胞株接种免疫缺陷小鼠皮下成瘤,检测3E8是否抑制肿瘤生长。
实施例中若无特殊说明,均为本领域常规的实验技术。
实施例1
制备B7-H3单克隆抗体的杂交瘤:
1、免疫小鼠:用融合蛋白或转基因细胞免疫小鼠,免疫四次,每次间隔21天,第四次免疫7-10天后测小鼠眼眶血效价,效价测定好则加强免疫。
2、细胞培养
2.1于融合前1d,取6~7周龄BALB/c小鼠1只,置于75%乙醇溶液中2min。
2.2无菌取出小鼠脾脏,置于200目的不锈钢筛网中,研磨获得单个细胞悬液。用1640基础培养基洗涤两遍(1400rpm,5min)备用。用15% FBS(胎牛血清)的1640培养基,调整细胞浓度为2×105个/ml,滴加入96孔培养板中,每孔100μL,37℃、5%CO2培养箱中培养。
2.3过夜培养,第二天在低倍显微镜下观察。并铺亚克隆细胞150ul。
3、融合与筛选
器材:泡老鼠杯;简易解剖台;杂交瘤包;保温水浴杯;温度计;96孔培养板。
试剂:75%乙醇溶液;37℃预热1640基础培养基;37℃预热PEG(聚乙二醇)1ml;37℃预热1640基础培养基14ml,终止PEG反应;37℃预热1640选择培养基(含15%FBS,并计算加入HAT(含次黄嘌呤、氨基喋呤和胸腺嘧啶的培养试剂)的量,使得最终96孔培养板中培养基的HAT终浓度为1%)。
具体步骤:
3.1.将免疫小鼠,用流水冲洗,置于75%乙醇溶液中2min。
3.2.无菌取出小鼠脾脏,置于200目的不锈钢筛网中,研磨获得单个细胞悬液。用预热1640基础培养基洗涤两遍(1400rpm,5min)备用。
3.3.收集生长良好、处于对数生长期的SP2/0细胞,用预热1640基础培养基洗涤两遍(1400rpm,5min)备用。
3.4.将SP2/0细胞或Ag8细胞与脾脏细胞混合于50ml透明塑料离心管中,一般脾细胞:骨髓瘤细胞比例为5:1,预热1640基础培养基洗涤一遍(1400rpm,5min),弃尽上清(以避免对PEG产生不必要的稀释)并用掌心搓管底(或手指轻轻弹击管底),使两种细胞充分混匀成悬浮细胞状。
3.5.将离心管置于37℃保温水浴杯中预热,吸取1ml经37℃预温的50%的PEG溶液,在1min内匀速加完,且边加边轻轻震荡离心管,加完后在37℃水浴中轻晃60s。(3秒滴一滴)
3.6.沿管壁轻柔加入14ml 37℃预温的1640基础培养基终止(第1min加1ml,3min加3ml,最后缓慢加入10ml)。(匀速滴加)
3.7.37℃静止5min后离心(800rpm,5min),弃去上清(将离心管倾斜,吸去上清)。将沉淀细胞轻轻用37℃预热的1640选择培养基重悬(不可吹打),加入到事先准备好的预热培养基中后,混匀后滴加在上述含有滋养细胞的96孔培养板中,100μl/孔,置于37℃、5%CO2培养箱中培养,3-4d后半量换液,10d后改用HT培养基培养,2周后转用含10%FBS的1640培养基培养。
3.8.期间每天观察96孔板中克隆生长情况,一般在杂交瘤细胞布满孔底1/10面积时,即可开始检测特异性抗体,筛选出所需要的杂交瘤细胞系。对于有特异性分泌抗体的细胞应及时克隆培养并冻存。通常需3-5次亚克隆才能获得稳定基因型和稳定分泌表型的细胞,且培养一段时间后需再次亚克隆。
鼠抗人B7-H3单克隆抗体重链可变区和轻链可变区的测序:
1、提取杂交瘤细胞的cDNA:从该杂交瘤细胞株中提取RNA,并使用RT-PCR技术,将获得的RNA反转录为cDNA;以该cDNA为模板PCR扩增该杂交瘤细胞重链可变区(mVH)和轻链可变区(mVL);
2、将所得重链可变区和轻链可变区分别与克隆载体(pJET cloning vector)连接,连接产物转化感受态细菌DH5a,由于pJET载体带有氨苄(Amp+)抗性基因,可将转化菌液涂在Amp抗性的LB固体培养基上,37℃过夜培养;
3、待涂板细菌长出分散菌落,选择边缘清晰、分散的菌落,进一步测序鉴定重链和轻链可变区基因序列;
4、根据测序结果保留候选的重轻链可变区序列,再次PCR扩增出与表达载体匹配的重链、轻链可变区序列,将PCR产物BmtI,FspI限制性内切酶预先处理的线性表达载体连接,连接产物转化感受态细菌DH5a,由于表达载体带有卡那霉素(Kana+)抗性基因,可将转化菌液涂在Kana抗性的LB固体培养基上,37°过夜培养;
5、测序方法参照4对比两次测序结果,挑选前后两次序列均一致的转化菌,扩大培养后进行质粒抽提;
6、将包含了单抗重链可变区和轻链可变区基因的表达载体共转染真核表达细胞株293。293细胞为悬浮培养,SFM4Transfx-293without L-glutamine(liquid)无血清培养基传代扩增,转染时用FreeStyleTM293,Expression Medium无血清培养基替换,通过7天连续培养后收获上清,4000g离心30min,去除上清中细胞等杂质,并用0.45um滤器过滤除菌;收获的上清含有目的抗体,流式检测表达单抗与乳腺癌细胞M435结合良好。
通过上述方法获得的一种鼠抗人B7-H3单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO.1所示:
SEQ ID NO.1的序列为:
QVQLQESGAELVKPGASVKLSCKTSGYTFTNYPIHWVKQSPGQGLEWIARIYWGIDSIYYNEIFRGKVTLTADTSTSTAYMQLSSLKSEDTAVYFCANSGYTDYGTYYLMDYWGQGTTVTVSS;
重链可变区的三个高变区为:
GYTFTNYP、IYWGIDSI、ANSGYTDYGTYYLMDY。
通过上述方法获得的一种鼠抗人B7-H3单克隆抗体的轻链可变区的氨基酸序列如SEQ ID NO.2所示:
SEQ ID NO.2的序列为:
DIVMTQSALSVSVTPGESVSISCRSTRSLLHSNGCTYLYWFLQRPGQSPQLLIYWMYNLASGVPDRFSGSGSGTTFTLKISRVEAEDVGVYYCMSNLEYLFTFGGGTKLEIK
轻链可变区的三个高变区为:
RSTRSLLHSNGCTYLY、WMYNLA、MSNLEYLFT。
本发明从分泌抗人B7-H3单克隆抗体的杂交瘤细胞中提取重链可变区(mVH)和轻链可变区(mVL),根据测序结果保留候选的重轻链可变区序列,PCR扩增出与表达载体匹配的重链、轻链可变区序列,将PCR产物与双酶切预先处理的线性表达载体连接,连接产物转化感受态细菌DH5a。连接有目的单抗重链和轻链可变区基因的表达载体共转染真核表达细胞株293,培养收获的上清含有目的抗体,证明得到的重链和轻链可变区序列正确。
实施例2
为了确定B7-H3和c-Met之间的相互作用,使用Biacore T200仪器进行表面等离子共振实验检测。将c-Met-his蛋白质用10mM乙酸钠(pH 4.5)稀释至200μg/mL,并固定在CM5传感器芯片上。在最大浓度为100μM的连续倍增浓度下分析B7-H3-his。结合分析在25℃以30μL/min的流速进行。最后通过Biacore T200 v3.0软件中全局拟合1:1模型(一对一结合模型)计算动力学速率(KD)常数,结果如图1a所示(实验方法如图1b所示)。
从图1a和图1b可以看出:B7-H3可与原癌基因c-Met结合,且B7-H3的浓度越高,结合越强,结合常数KD值为1.66E-06。
实施例3
为了检测B7-H3与c-Met蛋白的结合,我们做了ELISA实验,具体步骤如下:
1.蛋白包被:将c-Met蛋白用0.01M Tris-CL缓冲液(pH 8.9)调整为5μg/mL,包被96孔酶标板,4℃过夜。PBS(含0.1% Tween 20)洗涤3次后,1%BSA(200μL/well)4℃封闭过夜。
2.结合反应:PBS洗涤3次后,加入带biotin标记的梯度B7-H3蛋白(100μL/well),以及空白对照、阴性对照(PD-L1-biotin或EGFR包板,B7-H3-biotin检测)、阳性对照蛋白(HGF包板,c-Met-biotin检测),37℃反应2h。
3.显色:PBS洗涤3次,加入streptavidin-HRP(1:1000,100μL/well),37℃反应30min,PBS洗涤5次,再加入现配的底物TMB(1:3,100μL/well),37℃反应10min,用2M H2SO4(50μL/well)终止反应。
4.读数:用酶标仪测定酶标板各孔在450nm波长下的读数。根据检测蛋白的浓度绘制OD值曲线。
检测结果如图1c和图1d所示:从图1c和图1d中可以看出:B7-H3可与c-Met结合,且B7-H3的浓度增加,二者结合的能力也逐渐增加。阳性对照组HGF蛋白与c-Met蛋白结合,阴性对照组PD-L1蛋白不与c-Met结合,B7-H3也同样不与EGFR结合。
实施例4
为了验证B7-H3单抗3E8对B7-H3分子的识别,使用流式细胞术进行分析,步骤如下:将L929转染B7-H3的转基因细胞1×105个,分别与PE标记的B7-H3商品化抗体、3E8抗体以及小鼠IgG在室温避光孵育30min,之后PBS洗涤两次,在3E8抗体管和小鼠IgG管中加入PE标记的抗鼠二抗,之后PBS洗涤两次,随后通过流式细胞仪检测上述L929转基因细胞中的PE荧光表达。
测试结果如图2a所示:B7-H3单抗3E8能够识别L929-B7-H3细胞上的B7-H3分子,阳性对照抗体(商品化B7-H3抗体)也可以识别,阴性对照抗体鼠IgG无法识别。
实施例5
为了检测B7-H3单抗3E8是否能与B7-H3蛋白直接结合,我们进行了ELISA检测,具体步骤如下:
1.蛋白包被:将B7-H3蛋白用0.01M Tris-CL缓冲液(pH 8.9)调整为5μg/mL,包被96孔酶标板,4℃过夜。PBS(含0.1% Tween 20)洗涤3次后,1%BSA(200μL/well)4℃封闭过夜。
2.结合反应:PBS洗涤3次后,加入带biotin标记的梯度B7-H3单抗3E8(100μL/well),以及空白对照、阴性对照(鼠IgG-biotin),37℃反应2h。
3.显色:PBS洗涤3次,加入streptavidin-HRP(1:1000,100μL/well),37℃反应30min,PBS洗涤5次,再加入现配的底物TMB(1:3,100μL/well),37℃反应10min,用2M H2SO4(50μL/well)终止反应。
4.读数:用酶标仪测定酶标板各孔在450nm波长下的读数。根据检测蛋白的浓度绘制OD值曲线。
检测结果如图2b所示:从图2b中可以看出抗人B7-H3单抗3E8可以与B7-H3结合,且随着3E8浓度的提升,结合力逐渐提升。
实施例6
为了检测B7-H3单抗3E8是否能阻断B7-H3蛋白与c-Met蛋白的直接结合,我们进行了ELISA检测,具体步骤如下:
1.蛋白包被:将c-Met蛋白用0.01M Tris-CL缓冲液(pH 8.9)调整为5μg/mL,包被96孔酶标板,4℃过夜。PBS(含0.1% Tween 20)洗涤3次后,1%BSA(200μL/well)4℃封闭过夜。
2.结合反应:PBS洗涤3次后,加入带biotin标记的B7-H3蛋白和梯度B7-H3单抗3E8的混合液(100μL/well),以及空白对照、阴性对照(带biotin标记的B7-H3蛋白和梯度鼠IgG的混合液),37℃反应2h。
3.显色:PBS洗涤3次,加入streptavidin-HRP(1:1000,100μL/well),37℃反应30min,PBS洗涤5次,再加入现配的底物TMB(1:3,100μL/well),37℃反应10min,用2M H2SO4(50μL/well)终止反应。
4.读数:用酶标仪测定酶标板各孔在450nm波长下的读数。根据检测蛋白的浓度绘制OD值曲线。
检测结果如图2c所示:从图2c中可以看出抗人B7-H3单抗3E8可以阻止B7-H3与c-Met的结合,且随着3E8抗体浓度的增加,B7-H3与c-Met的结合逐渐减弱。而对照抗体鼠IgG则无法阻断B7-H3与c-Met的结合,随着鼠IgG抗体浓度的增加,B7-H3与c-Met的结合无明显变化。
实施例7
为了检测B7-H3单抗3E8是否能阻断B7-H3蛋白与c-Met蛋白结合后的信号通路,我们通过western blot技术对p-Met以及p-Stat3蛋白进行了检测,具体步骤如下:
1.用双蒸水将玻璃板清洗干净,烘箱烘干,配制分离胶和浓缩胶;
2.放入电泳槽,加入电泳液,安装电泳仪。垂直向上拔出梳子;
3.在胶中加入HCT116细胞经抗体处理的裂解蛋白样品,将电压设置到150V,电泳60min左右;
4.等到蛋白样品跑到分离胶下缘时,关闭电源,取下玻璃板,暂时放到转膜液中保存;
5.将PVDF膜置于无水甲醇中激活1min。将6张滤纸和2张海绵垫放入预冷好的转膜液中充分浸湿;
6.夹板的黑色面朝下,先放好一层海绵垫和三层滤纸,小心地把凝胶铺到滤纸上。再用镊子夹出PVDF膜,放到凝胶的上层。去除凝胶和膜之间的所有气泡。最后再叠加三层滤纸和一层海绵垫。扣紧夹板;
7.将组装好的转膜夹板放入转膜槽。倒入预冷好的转模液,放到冰盒中。将电流大小设置为220mA,时间2小时;
8.将膜放入5%封闭液中。室温缓慢摇床2小时;
9.取出膜,TBST洗去封闭液。将膜按照目的条带大小进行裁剪,然后依次将膜放到装有对应一抗(p-Met、p-Stat3、c-Met、Stat3、GAPDH)的反应盒中。在4℃缓慢摇床过夜;
10.将膜放入装有二抗(分别针对之前一抗来源的二抗)的反应盒中。缓慢摇床,室温下1小时;
11.使用条形荧光分析系统进行显影,用现配的TMB显影剂覆盖每个条带。保存显影结果图。
检测结果如图2d所示:从图2d中可以看出抗人B7-H3单抗3E8可抑制c-Met信号通路中p-Met和p-Stat3的表达,且随着3E8抗体的浓度上升,p-Met和p-Stat3的表达逐渐下降,当3E8浓度增加到10μg/ml时,两个磷酸化蛋白的表达水平最低。
实施例8
荷瘤小鼠抗体治疗
取5-6周龄大小的雄性BALB/C裸鼠10只,皮下注射生长良好的HCT116细胞(人结肠癌细胞),细胞密度为2×105个/只(图3a);或5-6周龄大小的雌性BALB/C裸鼠10只,皮下注射生长良好的NCI-N87细胞(人胃癌细胞),细胞密度为2.5×105个/只(图3b);或5-6周龄大小的雄性BALB/C裸鼠10只,皮下注射生长良好的HCT116细胞,细胞密度为2×106个/只(图3c);或5-6周龄大小的雄性BALB/C裸鼠10只,皮下注射生长良好的B7-H3敲减的HCT116细胞HCT116 shB7-H3,细胞密度为2×105个/只(图3e);成瘤后将小鼠随机分为两组。第一组用10mg/kg的IgG治疗,第二组用10mg/kg的本申请实施例1所述的3E8(B7-H3抗体)治疗。所有药物每三天腹腔注射一次,共注射五次;从治疗开始每三天测量小鼠体重和肿瘤大小,肿瘤大小(mm3)=1/2×长度×宽度2;
进行生存研究时(图3c),当最大的肿瘤超过1500mm3时,即脱颈处死小鼠,结果按死亡记录。同时取出肿瘤组织,拍照记录肿瘤外观(图3d)。按需要将组织切块,用于后续提取组织RNA及蛋白样品。剩下的存放在福尔马林中,择期制成切片,进行免疫组化检测(图3f)。
记录结果如图3a至图3e所示:
从图3a和图3b可以看出:抗人B7-H3单抗3E8能够有效抑制肿瘤的生长。
从图3c可以看出:对照组IgG抗体治疗组33天时所有小鼠都大于1500mm3(记录为死亡),而本申请所述抗人B7-H3单抗3E8治疗组37天时仍有60%的小鼠小于1500mm3(记录为存活)。
从图3d可以看出:本发明所述抗人B7-H3单抗3E8抑制肿瘤的效果明显优于对照组IgG抗体的效果。
从图3e中可以看出:HCT116敲减B7-H3后用抗人B7-H3单抗3E8抗体或对照IgG抗体以10mg/kg的剂量进行治疗后,可见HCT116敲减B7-H3后3E8抗体的治疗效果不明显。
实施例9
取HCT116细胞荷瘤小鼠治疗后的新鲜组织(实施例8保存的组织),修剪为1cm×1cm×0.3cm大小组织块,中性福尔马林液中固定24h,浸蜡(共两次,第一次1h,第二次2h),石蜡包埋后切5μm薄片,贴附于涂布多聚赖氨酸的玻片。
当需要进行免疫组化检测时,将切片经二甲苯脱蜡(共两次,每次各20min),梯度酒精溶液脱水(无水乙醇5min两次、90%酒精5min、80%酒精5min、70%酒精5min各一次)。接着用0.03% H2O2孵育30分钟去除内源性过氧化物酶,PBS清洗后滴加5%BSA,室温孵育30分钟,滴加一抗(抗人CD133或抗人B7-H3),4℃孵育过夜,之后用PBS冲洗,滴加对应的HRP标记二抗,37℃孵育30min,PBS冲洗三遍,DAB显色后经苏木素复染,1%盐酸-酒精分化,梯度乙醇中脱水(75%乙醇3min,95%乙醇I 3min,95%乙醇II 3min,100%无水乙醇I 3min,100%无水乙醇II 3min)后,吹风机吹干无水乙醇,中性树脂封片,显微镜下观察并拍照。
结果如图3f所示:从图3f中可以看出相对于对照IgG抗体组,3E8抗体组的肿瘤区域减少,且肿瘤组织中的B7-H3和CD133的表达显著减弱。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员,在不脱离本发明方法的前提下,还可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。
Claims (6)
1.一种鼠抗人B7-H3单克隆抗体,包括重链可变区和轻链可变区,其特征在于,所述重链可变区的氨基酸序列如SEQ ID NO.1所示;所述轻链可变区的氨基酸序列如SEQ ID NO.2所示;
所述SEQ ID NO.1的序列为:
QVQLQESGAELVKPGASVKLSCKTSGYTFTNYPIHWVKQSPGQGLEWIARIYWGIDS IYYNEIFRGKVTLTADTSTSTAYMQLSSLKSEDTAVYFCANSGYTDYGTYYLMDYWGQG TTVTVSS;
所述SEQ ID NO.2的序列为:
DIVMTQSALSVSVTPGESVSISCRSTRSLLHSNGCTYLYWFLQRPGQSPQLLIYWMYNL ASGVPDRFSGSGSGTTFTLKISRVEAEDVGVYYCMSNLEYLFTFGGGTKLEIK。
2.编码如权利要求1所述的鼠抗人B7-H3单克隆抗体的核酸分子。
3.一种表达载体,其特征在于,包含如权利要求2所述的核酸分子。
4.一种组合物,其特征在于,包含如权利要求1所述的鼠抗人B7-H3单克隆抗体,和可接受的辅料或载体。
5.一种药用组合物,其特征在于,包含权利要求1所述的鼠抗人B7-H3单克隆抗体或权利要求4所述的组合物,和药学上可接受的载体。
6.一种检测试剂盒,其特征在于,包含权利要求1所述的鼠抗人B7-H3单克隆抗体、权利要求2所述的核酸分子、权利要求3所述的表达载体或权利要求4所述的组合物。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310242833.8A CN116514975B (zh) | 2023-03-14 | 2023-03-14 | 一种鼠抗人b7-h3单克隆抗体及其制备方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310242833.8A CN116514975B (zh) | 2023-03-14 | 2023-03-14 | 一种鼠抗人b7-h3单克隆抗体及其制备方法和应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN116514975A CN116514975A (zh) | 2023-08-01 |
CN116514975B true CN116514975B (zh) | 2024-02-09 |
Family
ID=87394733
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310242833.8A Active CN116514975B (zh) | 2023-03-14 | 2023-03-14 | 一种鼠抗人b7-h3单克隆抗体及其制备方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116514975B (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN117603916A (zh) * | 2023-11-17 | 2024-02-27 | 赛业(苏州)生物科技有限公司 | 一种基于全人源化抗体鼠的人b7-h3抗体及其制备方法和应用 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109851673A (zh) * | 2019-01-22 | 2019-06-07 | 苏州旭光科星生物技术有限公司 | 一种抗人b7-h3单克隆抗体的制备方法及其免疫组化检测方法及其应用及其试剂盒 |
CN114380910A (zh) * | 2022-01-07 | 2022-04-22 | 苏州旭光科星抗体生物科技有限公司 | 靶向人b7-h3分子的人源化单克隆抗体及其应用 |
WO2022117040A1 (zh) * | 2020-12-02 | 2022-06-09 | 迈威(上海)生物科技股份有限公司 | 抗人b7-h3抗体及其应用 |
-
2023
- 2023-03-14 CN CN202310242833.8A patent/CN116514975B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109851673A (zh) * | 2019-01-22 | 2019-06-07 | 苏州旭光科星生物技术有限公司 | 一种抗人b7-h3单克隆抗体的制备方法及其免疫组化检测方法及其应用及其试剂盒 |
WO2022117040A1 (zh) * | 2020-12-02 | 2022-06-09 | 迈威(上海)生物科技股份有限公司 | 抗人b7-h3抗体及其应用 |
CN114380910A (zh) * | 2022-01-07 | 2022-04-22 | 苏州旭光科星抗体生物科技有限公司 | 靶向人b7-h3分子的人源化单克隆抗体及其应用 |
Non-Patent Citations (1)
Title |
---|
B7-H3 promotes colorectal cancer angiogenesis through activating the NF-κB pathway to induce VEGFA expression;Ruoqin Wang等;Cell Death & Disease;第11卷;https://doi.org/10.1038/s41419-020-2252-3,第1-15页 * |
Also Published As
Publication number | Publication date |
---|---|
CN116514975A (zh) | 2023-08-01 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN109851673B (zh) | 一种抗人b7-h3单克隆抗体的制备方法及其免疫组化检测方法及其应用及其试剂盒 | |
CN104725509B (zh) | 结合转化生长因子α且对Ras基因突变的癌具有增殖抑制活性的抗体 | |
KR20150030675A (ko) | 항bmp9 항체를 유효 성분으로 하는, 신장성 빈혈, 암성 빈혈 등의 빈혈에 대한 치료제 | |
CN108948204B (zh) | 抗人b7-h4单克隆抗体、鉴定方法、应用和试剂盒 | |
JP5723270B2 (ja) | 抗カドヘリン抗体 | |
CN114560938B (zh) | 抗gdf15中和性单克隆抗体及其应用 | |
EA023679B1 (ru) | АНТИ-EpCAM АНТИТЕЛО И ЕГО ПРИМЕНЕНИЕ | |
CN112111462B (zh) | 烯醇化酶eno1单克隆抗体及其应用 | |
CN110461881B (zh) | 嵌合抗原受体 | |
CN112794909A (zh) | 一种抗tigit单克隆抗体及其应用 | |
CN116514975B (zh) | 一种鼠抗人b7-h3单克隆抗体及其制备方法和应用 | |
WO2021057866A1 (zh) | 一种单域抗体及包含抗体结构的嵌合抗原受体 | |
JPWO2020059772A1 (ja) | 抗her3抗体−薬物コンジュゲート投与によるher3変異がんの治療 | |
CN116847864A (zh) | 治疗剂和诊断剂以及其用途 | |
EP4257617A1 (en) | Antigen-binding protein targeting cd70 and use thereof | |
CN108084263B (zh) | 一种抗人cd25嵌合单克隆抗体及其制备方法和应用 | |
CN115028730A (zh) | 一种成体干细胞衍生的类器官制备方法 | |
CN117050183B (zh) | 一种ptn-ptprz1通路的阻断抗体及其在胶质瘤靶向治疗的应用 | |
CN111793134A (zh) | 一种用于癌症治疗中的药物、肿瘤疫苗及抑制剂 | |
CN109468380A (zh) | Il1r2在乳腺癌预后评估与靶向治疗中的应用 | |
CN117567620A (zh) | 一种Nectin-4单克隆抗体及其CAR-NK细胞 | |
CN116199781A (zh) | 一种靶向cd22的单域抗体、嵌合抗原受体及其应用 | |
CN103361355A (zh) | 编码重组rhHER2-mAb人源化单克隆抗体的基因及其应用 | |
JP2022513321A (ja) | 固形腫瘍を治療するための遺伝子操作しているγδ-T細胞及び遺伝子操作していないγδ-T細胞に関する組成物及び方法 | |
CN114853889B (zh) | 针对人gpr48的单克隆抗体及其应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |