CN116785417B - 芦荟苷与her2-car-t细胞联合制备治疗骨肉瘤的药物 - Google Patents
芦荟苷与her2-car-t细胞联合制备治疗骨肉瘤的药物 Download PDFInfo
- Publication number
- CN116785417B CN116785417B CN202311007658.0A CN202311007658A CN116785417B CN 116785417 B CN116785417 B CN 116785417B CN 202311007658 A CN202311007658 A CN 202311007658A CN 116785417 B CN116785417 B CN 116785417B
- Authority
- CN
- China
- Prior art keywords
- her2
- car
- aloin
- cells
- osteosarcoma
- 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
- AFHJQYHRLPMKHU-XXWVOBANSA-N Aloin Natural products O=C1c2c(O)cc(CO)cc2[C@H]([C@H]2[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O2)c2c1c(O)ccc2 AFHJQYHRLPMKHU-XXWVOBANSA-N 0.000 title claims abstract description 54
- AFHJQYHRLPMKHU-UHFFFAOYSA-N isobarbaloin Natural products OC1C(O)C(O)C(CO)OC1C1C2=CC(CO)=CC(O)=C2C(=O)C2=C(O)C=CC=C21 AFHJQYHRLPMKHU-UHFFFAOYSA-N 0.000 title claims abstract description 54
- CPUHNROBVJNNPW-UHFFFAOYSA-N aloin A Natural products OC1C(O)C(O)C(CO)OC1OC1C2=CC(CO)=CC(O)=C2C(=O)C2=C(O)C=CC=C21 CPUHNROBVJNNPW-UHFFFAOYSA-N 0.000 title claims abstract description 51
- 201000008968 osteosarcoma Diseases 0.000 title claims abstract description 34
- 238000002360 preparation method Methods 0.000 title claims abstract description 22
- 239000003814 drug Substances 0.000 title claims abstract description 21
- AFHJQYHRLPMKHU-WEZNYRQKSA-N aloin B Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1[C@H]1C2=CC(CO)=CC(O)=C2C(=O)C2=C(O)C=CC=C21 AFHJQYHRLPMKHU-WEZNYRQKSA-N 0.000 title 1
- 210000004027 cell Anatomy 0.000 claims abstract description 74
- AFHJQYHRLPMKHU-OSYMLPPYSA-N aloin A Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1[C@@H]1C2=CC(CO)=CC(O)=C2C(=O)C2=C(O)C=CC=C21 AFHJQYHRLPMKHU-OSYMLPPYSA-N 0.000 claims abstract description 59
- 210000004881 tumor cell Anatomy 0.000 claims abstract description 24
- 239000002773 nucleotide Substances 0.000 claims description 24
- 125000003729 nucleotide group Chemical group 0.000 claims description 24
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 claims description 20
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 claims description 20
- 239000000427 antigen Substances 0.000 claims description 20
- 108091007433 antigens Proteins 0.000 claims description 20
- 102000036639 antigens Human genes 0.000 claims description 20
- 230000003834 intracellular effect Effects 0.000 claims description 13
- 201000011510 cancer Diseases 0.000 claims description 12
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 claims description 8
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 claims description 8
- 229940079593 drug Drugs 0.000 claims description 8
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 4
- 101100425901 Rattus norvegicus Tpm1 gene Proteins 0.000 claims description 3
- 108700010039 chimeric receptor Proteins 0.000 claims description 2
- 230000004068 intracellular signaling Effects 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 19
- 230000002147 killing effect Effects 0.000 abstract description 13
- 230000008901 benefit Effects 0.000 abstract description 5
- 238000011357 CAR T-cell therapy Methods 0.000 abstract description 4
- 230000002195 synergetic effect Effects 0.000 abstract description 4
- 231100000331 toxic Toxicity 0.000 abstract description 3
- 230000002588 toxic effect Effects 0.000 abstract description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 17
- 238000000034 method Methods 0.000 description 13
- 238000011282 treatment Methods 0.000 description 13
- 206010028980 Neoplasm Diseases 0.000 description 11
- 239000006228 supernatant Substances 0.000 description 8
- 241001116389 Aloe Species 0.000 description 6
- 206010006187 Breast cancer Diseases 0.000 description 6
- 208000026310 Breast neoplasm Diseases 0.000 description 6
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 description 6
- 235000011399 aloe vera Nutrition 0.000 description 6
- 210000004698 lymphocyte Anatomy 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 5
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 5
- 206010033128 Ovarian cancer Diseases 0.000 description 5
- 206010061535 Ovarian neoplasm Diseases 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 230000008685 targeting Effects 0.000 description 5
- 108090000695 Cytokines Proteins 0.000 description 4
- 102000004127 Cytokines Human genes 0.000 description 4
- 230000000259 anti-tumor effect Effects 0.000 description 4
- 230000022534 cell killing Effects 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- 238000001990 intravenous administration Methods 0.000 description 4
- 238000010253 intravenous injection Methods 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 230000028327 secretion Effects 0.000 description 4
- 230000005909 tumor killing Effects 0.000 description 4
- 101710088194 Dehydrogenase Proteins 0.000 description 3
- 108010002350 Interleukin-2 Proteins 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 2
- 102000001398 Granzyme Human genes 0.000 description 2
- 108060005986 Granzyme Proteins 0.000 description 2
- 241000713666 Lentivirus Species 0.000 description 2
- 206010027476 Metastases Diseases 0.000 description 2
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 2
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 208000005718 Stomach Neoplasms Diseases 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 150000004056 anthraquinones Chemical class 0.000 description 2
- 230000001093 anti-cancer Effects 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 230000005779 cell damage Effects 0.000 description 2
- 208000037887 cell injury Diseases 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 201000004101 esophageal cancer Diseases 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 206010017758 gastric cancer Diseases 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 201000005787 hematologic cancer Diseases 0.000 description 2
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 238000009169 immunotherapy Methods 0.000 description 2
- 238000001802 infusion Methods 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000009401 metastasis Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 201000003733 ovarian melanoma Diseases 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 201000002528 pancreatic cancer Diseases 0.000 description 2
- 208000008443 pancreatic carcinoma Diseases 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 201000011549 stomach cancer Diseases 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 239000012224 working solution Substances 0.000 description 2
- LUEWUZLMQUOBSB-FSKGGBMCSA-N (2s,3s,4s,5s,6r)-2-[(2r,3s,4r,5r,6s)-6-[(2r,3s,4r,5s,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5s,6r)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-4,5-dihydroxy-2-(hydroxymethyl)oxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@@H](O[C@@H]2[C@H](O[C@@H](OC3[C@H](O[C@@H](O)[C@@H](O)[C@H]3O)CO)[C@@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O LUEWUZLMQUOBSB-FSKGGBMCSA-N 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 229930192334 Auxin Natural products 0.000 description 1
- 208000003950 B-cell lymphoma Diseases 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 206010005949 Bone cancer Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 229930182476 C-glycoside Natural products 0.000 description 1
- 150000000700 C-glycosides Chemical class 0.000 description 1
- 238000011523 CAR-T cell immunotherapy Methods 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- SGNBVLSWZMBQTH-FGAXOLDCSA-N Campesterol Natural products O[C@@H]1CC=2[C@@](C)([C@@H]3[C@H]([C@H]4[C@@](C)([C@H]([C@H](CC[C@H](C(C)C)C)C)CC4)CC3)CC=2)CC1 SGNBVLSWZMBQTH-FGAXOLDCSA-N 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- VWDXGKUTGQJJHJ-UHFFFAOYSA-N Catenarin Natural products C1=C(O)C=C2C(=O)C3=C(O)C(C)=CC(O)=C3C(=O)C2=C1O VWDXGKUTGQJJHJ-UHFFFAOYSA-N 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- NBSCHQHZLSJFNQ-QTVWNMPRSA-N D-Mannose-6-phosphate Chemical compound OC1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H](O)[C@@H]1O NBSCHQHZLSJFNQ-QTVWNMPRSA-N 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 239000010282 Emodin Substances 0.000 description 1
- RBLJKYCRSCQLRP-UHFFFAOYSA-N Emodin-dianthron Natural products O=C1C2=CC(C)=CC(O)=C2C(=O)C2=C1CC(=O)C=C2O RBLJKYCRSCQLRP-UHFFFAOYSA-N 0.000 description 1
- 229930191978 Gibberellin Natural products 0.000 description 1
- 229920002581 Glucomannan Polymers 0.000 description 1
- 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 1
- BTEISVKTSQLKST-UHFFFAOYSA-N Haliclonasterol Natural products CC(C=CC(C)C(C)(C)C)C1CCC2C3=CC=C4CC(O)CCC4(C)C3CCC12C BTEISVKTSQLKST-UHFFFAOYSA-N 0.000 description 1
- YOOXNSPYGCZLAX-UHFFFAOYSA-N Helminthosporin Natural products C1=CC(O)=C2C(=O)C3=CC(C)=CC(O)=C3C(=O)C2=C1O YOOXNSPYGCZLAX-UHFFFAOYSA-N 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- NTGIIKCGBNGQAR-UHFFFAOYSA-N Rheoemodin Natural products C1=C(O)C=C2C(=O)C3=CC(O)=CC(O)=C3C(=O)C2=C1O NTGIIKCGBNGQAR-UHFFFAOYSA-N 0.000 description 1
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- GZOSMCIZMLWJML-VJLLXTKPSA-N abiraterone Chemical compound C([C@H]1[C@H]2[C@@H]([C@]3(CC[C@H](O)CC3=CC2)C)CC[C@@]11C)C=C1C1=CC=CN=C1 GZOSMCIZMLWJML-VJLLXTKPSA-N 0.000 description 1
- 229960000853 abiraterone Drugs 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000011226 adjuvant chemotherapy Methods 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- PYKYMHQGRFAEBM-UHFFFAOYSA-N anthraquinone Natural products CCC(=O)c1c(O)c2C(=O)C3C(C=CC=C3O)C(=O)c2cc1CC(=O)OC PYKYMHQGRFAEBM-UHFFFAOYSA-N 0.000 description 1
- RJGDLRCDCYRQOQ-UHFFFAOYSA-N anthrone Chemical compound C1=CC=C2C(=O)C3=CC=CC=C3CC2=C1 RJGDLRCDCYRQOQ-UHFFFAOYSA-N 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000003178 anti-diabetic effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 239000003472 antidiabetic agent Substances 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 239000002363 auxin Substances 0.000 description 1
- 238000009412 basement excavation Methods 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000003181 biological factor Substances 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- SGNBVLSWZMBQTH-PODYLUTMSA-N campesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CC[C@@H](C)C(C)C)[C@@]1(C)CC2 SGNBVLSWZMBQTH-PODYLUTMSA-N 0.000 description 1
- 235000000431 campesterol Nutrition 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 238000005119 centrifugation 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
- 238000003501 co-culture Methods 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 230000000139 costimulatory effect Effects 0.000 description 1
- 206010052015 cytokine release syndrome Diseases 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000008355 dextrose injection Substances 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- RHMXXJGYXNZAPX-UHFFFAOYSA-N emodin Chemical compound C1=C(O)C=C2C(=O)C3=CC(C)=CC(O)=C3C(=O)C2=C1O RHMXXJGYXNZAPX-UHFFFAOYSA-N 0.000 description 1
- VASFLQKDXBAWEL-UHFFFAOYSA-N emodin Natural products OC1=C(OC2=C(C=CC(=C2C1=O)O)O)C1=CC=C(C=C1)O VASFLQKDXBAWEL-UHFFFAOYSA-N 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- IXORZMNAPKEEDV-UHFFFAOYSA-N gibberellic acid GA3 Natural products OC(=O)C1C2(C3)CC(=C)C3(O)CCC2C2(C=CC3O)C1C3(C)C(=O)O2 IXORZMNAPKEEDV-UHFFFAOYSA-N 0.000 description 1
- 239000003448 gibberellin Substances 0.000 description 1
- 229940046240 glucomannan Drugs 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000000055 hyoplipidemic effect Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 238000002843 lactate dehydrogenase assay Methods 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 229920005610 lignin Polymers 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- MQYXUWHLBZFQQO-QGTGJCAVSA-N lupeol Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C)CC[C@@H](C(=C)C)[C@@H]5[C@H]4CC[C@@H]3[C@]21C MQYXUWHLBZFQQO-QGTGJCAVSA-N 0.000 description 1
- PKGKOZOYXQMJNG-UHFFFAOYSA-N lupeol Natural products CC(=C)C1CC2C(C)(CCC3C4(C)CCC5C(C)(C)C(O)CCC5(C)C4CCC23C)C1 PKGKOZOYXQMJNG-UHFFFAOYSA-N 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Chemical class 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 230000009437 off-target effect Effects 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 1
- 108010013617 perforin-granzyme B Proteins 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- PKUBGLYEOAJPEG-UHFFFAOYSA-N physcion Natural products C1=C(C)C=C2C(=O)C3=CC(C)=CC(O)=C3C(=O)C2=C1O PKUBGLYEOAJPEG-UHFFFAOYSA-N 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 210000001778 pluripotent stem cell Anatomy 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 229960004889 salicylic acid Drugs 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- 235000017709 saponins Nutrition 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000011669 selenium Substances 0.000 description 1
- 229910052711 selenium Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- -1 vitamins (vitamins a Chemical class 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Classifications
-
- 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
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
- A61K39/001102—Receptors, cell surface antigens or cell surface determinants
- A61K39/001103—Receptors for growth factors
- A61K39/001106—Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ErbB4
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/351—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom not condensed with another ring
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5156—Animal cells expressing foreign proteins
-
- 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
- A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
- A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Physical Education & Sports Medicine (AREA)
- Mycology (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Rheumatology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Oncology (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Cell Biology (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明公开了一种芦荟苷与HER2‑CAR‑T细胞联合制备治疗骨肉瘤的药物，属于生物医药领域。包括芦荟苷和HER2‑CAR‑T细胞；芦荟苷的CAS号为1415‑73‑2，分子式C21H22O9，分子量：418.394，芦荟苷与HER2‑CAR‑T细胞联合制剂的优点如下：第一，发挥协同作用，增加对骨肉瘤等肿瘤细胞的杀伤作用；第二，降低HER2‑CAR‑T细胞的应用剂量；第三，减少HER2‑CAR‑T细胞治疗产生的毒副作用；第四，由于芦荟苷价格经济，二者联合应用后能够降低医疗费用。
Description
技术领域
本发明涉及生物医药技术领域，包括芦荟苷与靶向HER2抗原的CAR-T淋巴细胞联合在制备骨肉瘤药物中的用途。
背景技术
骨肉瘤是全球范围内常见恶性骨肿瘤之一，好发于儿童和年轻人。骨肉瘤具有恶性程度高、复发率高、早期转移率高和预后差的特点，严重影响患者的生活质量和生存期。目前，手术切除和辅助化疗是治疗骨肉瘤的常用方法，尽管这些方法在一定程度上延长了患者生存期、改善了生活质量，但仍面临治疗效果有限、化疗耐药性、副作用、肿瘤复发和远处转移等问题。因此，骨肉瘤的治疗一直是医学领域的未能攻克的难题也是研究热点，为了应对上述困难，探索新型安全有效的治疗方法至关重要。
嵌合抗原受体T淋巴细胞（Chimeric antigen receptor T cell, CAR-T cell）细胞疗法是一种新型、有前景、可能治愈恶性肿瘤的免疫疗法。简单地说，CAR-T细胞由CAR和T淋巴细胞两部分构成，CAR的作用是特异性识别和结合肿瘤细胞，而T淋巴细胞的作用是杀伤肿瘤细胞，二者组合后完成靶向杀伤肿瘤细胞。目前，我国国家监督管理局已批准了首个CAR-T细胞制剂（阿基仑赛注射液）上市，美国FDA的批准了将抗CD19的CAR-T细胞用于临床治疗复发或难治性大B细胞淋巴瘤成人患者，并已获得了治愈的效果。基于CAR-T细胞疗法在血液肿瘤中取得的巨大进展，CAR-T细胞免疫疗法为骨肉瘤的治疗带来了新思路。由于骨肉瘤属于实体瘤，实体瘤与血液肿瘤有许多不同之处。多项研究发现CAR-T细胞治疗骨肉瘤仍面临许多问题，例如，肿瘤抗原逃逸、脱靶效应、难以归巢和定殖、免疫抑制肿瘤微环境以及细胞因子释放综合征，这些问题直接影响CAR-T细胞的治疗效果。骨肉瘤患者的免疫系统往往比较弱，体内免疫细胞的数量较少、杀伤肿瘤细胞的能力并没有预想的强。因此，有必要进一步研究如何增强CAR-T细胞疗法的抗肿瘤作用和降低其副作用。
芦荟是一种多年生绿色草本植物，有明亮的黄色管状花，广泛分布于北非，亚洲中东，地中海南部和加那利群岛的炎热干燥地区。芦荟源自“Allaeh”（阿拉伯语，意思是“发光的苦味物质”）和“Vera”（拉丁语，意思是“真”）。芦荟中的多种提取物近年来已广泛应用于抗癌，抗氧化剂，抗糖尿病药、降血脂药以及创面处理敷料等。芦荟大约包含超过 75 种不同化合物，包括维生素（维生素 A，C，E 和 B12 为主），酶（淀粉酶，过氧化氢酶和过氧化物酶），矿物质（主要为锌，铜，硒和钙），糖（6 磷酸甘露糖和葡甘露聚糖等多糖），蒽醌（芦荟苷和大黄素），脂肪酸（羽扇豆酚和菜油甾醇），激素（生长素和赤霉素）和其他生物因子（即水杨酸，木质素和皂苷）。芦荟苷（Aloin，Alo）是从芦荟叶分泌物中提取的主要蒽醌类成分之一，即芦荟蒽酮的天 然 C-糖苷：（10-glucopyranosyl-1,8-dihydroxy-3-hydroxymethyl-9(10H)-anthracenone），据报道其具有抗炎、抗菌、抗氧化、抗病毒和抗癌等药理作用。研究表明，芦荟苷能够诱导多种种系肿瘤细胞的凋亡，包括乳腺癌、卵巢癌、B16-F10 小鼠黑色素瘤和人类 Jurkat T 淋巴细胞。另外，与化学制剂相比，其来源广泛，安全易得，在预防和治疗相关癌症方面具有较大的挖掘潜力。目前，业界内尚无芦荟苷联合靶向HER2抗原的CAR-T淋巴细胞来制备抗骨肉瘤药物的报告。
发明内容
本发明提供了芦荟苷与HER2-CAR-T细胞联合制剂治疗骨肉瘤的新用途，并证明了其应用结果。尽管本发明专利以抗骨肉瘤为例，该药物同样适用于表达HER2抗原的其他癌症，如乳腺癌、卵巢癌、黑色素瘤等等恶性肿瘤。芦荟苷与HER2-CAR-T细胞联合制剂的优点如下：第一，发挥协同作用，增加对骨肉瘤等肿瘤细胞的杀伤作用；第二，降低HER2-CAR-T细胞的应用剂量；第三，减少HER2-CAR-T细胞治疗产生的毒副作用；第四，由于芦荟苷价格经济，二者联合应用后能够降低医疗费用。
专利申请人研究发现芦荟苷可通过上调骨肉瘤HER2抗原表达来增强HER2-CAR-T细胞对骨肉瘤、乳腺癌、卵巢癌、黑色素瘤的识别、结合以及杀伤作用。重要的是，芦荟苷可以通过还原、取代等方法化学方法对其骨架上的侧链基团进行编辑、修饰以及去除，保留了能够增强CAR-T细胞杀伤能力的多种衍生物，并且包括药学上可以接受的异构体、盐、金属络等合物等。本发明专利中芦荟苷的CAS 号为1415-73-2，分子式C21H22O9，分子量：418.394，结构式如下：
HER2抗原在骨肉瘤细胞表面呈高表达，但是在正常组织细胞中表达量却很少，这为CAR-T细胞提供了结合靶点。HER2抗原同样高表达于食管癌、胃癌、胰腺癌、乳腺癌和卵巢癌等恶性肿瘤，所以芦荟苷联合HER2-CAR-T细胞制剂同样适用于这些恶性肿瘤，但不限于此。芦荟苷具有较强的抗乳腺癌、膀胱癌、结肠癌和肝癌等癌症的活性，在癌症的预防和治疗中有广泛的应用前景。
本专利构建了靶向HER2抗原的HER2-CAR结构，核苷酸序列为SEQ ID No.1。HER2-CAR结构包括胞外结构域、跨膜区和胞内结构域。进一步，胞外结构域由CD8α Leader、HER2scFv单链抗体以及CD8α铰链区组成；跨膜区由为CD8α构成；胞内结构域由CD28共刺激结构域、CD137共刺激结构域以及CD3ζ胞内信号区共同组成。
CD8α Leader为HER2-CAR中的嵌合受体信号肽，核苷酸序列为SEQ ID No.2。
HER2 scFv为HER2-CAR中的靶向恶性肿瘤细胞表面HER2抗原的人源化单链抗体，具有较高的安全性，避免引起人机体的免疫反应的优点，核苷酸序列为SEQ ID No.3。
CD8α-hinge/CD8α TM为HER2-CAR中的铰链区和跨膜区，核苷酸序列为SEQ IDNo.4。
CD28为HER2-CAR中的胞内的共刺激结构域，具有抗原提呈、促进T淋巴细胞活化和分泌抗肿瘤的细胞因子的作用，核苷酸序列为SEQ ID No.5。
HER2-CAR中的CD137为胞内的另一个共刺激结构域，与CD28共刺激结构域相似，也具有抗原提呈、促进T淋巴细胞活化和分泌抗肿瘤的细胞因子的作用，核苷酸序列为SEQ IDNo.6。
HER2-CAR中的CD3ζ为胞内信号区，具有提呈信号活化T淋巴细胞的作用，核苷酸序列为SEQ ID No.7。
HER2-CAR及其载体的核苷酸序列为SEQ ID No.8。
HER2-CAR-T细胞中的T淋巴细胞可以来源于自体T淋巴细胞、异体T淋巴细胞或多能干细胞诱导的T淋巴细胞。上述来源的多种T淋巴细胞中，来自患者自体T淋巴细胞为首选，这有利于降低免疫反应的发生率和副作用。由于骨肉瘤等恶性肿瘤细胞表面HER2抗原高表达，而正常组织细胞表明HER2抗原表达量很低，因此靶向HER2抗原的CAR-T细胞可以特异性的识别和杀伤恶性肿瘤细胞，从而发挥抗肿瘤作用，而又避免正常组织收到攻击。
本发明提供了一种治疗骨肉瘤、食管癌、胃癌、胰腺癌、乳腺癌和卵巢癌等多种恶性肿瘤的药物，所述药物成分包括芦荟苷和HER2-CAR-T细胞，该药物使用方法为静脉注射。芦荟苷与HER2-CAR-T细胞可以单独应用，也可以混合应用。具体而言，HER2-CAR-T细胞的常见给药方式为注射，包括静脉注射、皮下注射、皮内注射、鞘内注射或肌内注射，常用方法是将CAR-T细胞溶于生理盐水或者葡萄糖注射液中静脉注射。芦荟苷的常见给药方式为静脉注射，芦荟苷可通过静脉注射，通过静脉注射可以药物分布广泛，可以到达心、骨、肺、肝。联合制剂药物中含有药学上可搭配载体物质，如水、生理盐水、葡萄糖水溶液以及其他非水性溶剂等至少一种稀释剂，以及稳定剂中的至少一种，但不限于此。
给药方法一：先静脉输注芦荟苷注射液，再静脉输入HER2-CAR-T细胞；给药方法二：芦荟苷与HER2-CAR-T细胞混合液溶于生理盐水后，同时静脉输注。芦荟苷与HER2-CAR-T细胞联合制剂的好处如下：第一，芦荟苷增加骨肉瘤细胞表面的HER2抗原表达的数量，有利于提高HER2-CAR-T细胞对骨肉瘤细胞的识别、结合以及杀伤效果；第二，减少HER2-CAR-T细胞的应用剂量，避免HER2-CAR-T细胞引起的不良反应；第三，联合制剂的治疗效果优于单一用药的治疗效果；第四，降低患者医疗费用。
总之，芦荟苷与HER2-CAR-T细胞联合制剂能够加强对骨肉瘤等高表达HER2抗原的恶性肿瘤的治疗效果，发挥协同增效作用，在保证肿瘤杀伤效能前提下，降低了HER2-CAR-T细胞的使用剂量、减少毒副作用和减少医疗费用。
附图说明
图1为HER2-CAR的结构示意图；
图2为Alo，HER2-CAR-T以及Alo+HER2-CAR-T对U2OS细胞裂解作用的柱状图；
图3为Alo，HER2-CAR-T以及Alo+HER2-CAR-T为杀伤U2OS细胞过程中产生肿瘤细胞杀伤因子TNF-γ、IL-2的柱状图；
图4为Alo，HER2-CAR-T以及Alo+HER2-CAR-T为杀伤U2OS细胞过程中产生肿瘤细胞杀伤因子穿孔素颗粒酶B的柱状图；
其中，Alo代表芦荟苷，HER2-CAR-T代表靶向HER2抗原的嵌合抗原受体T淋巴细胞，U2OS细胞为人骨肉瘤细胞。
具体实施方式
本发明提供了芦荟苷与HER2-CAR-T细胞联合制剂在制备治疗恶性肿瘤药物中的用途。本专利申请人发现，芦荟苷能够上调恶性肿瘤表达HER2抗原的数量，进而明显增强HER2-CAR-T细胞对肿瘤细胞的识别、结合以及杀伤效果。两者的联用表现出协同效应，可达到良好的治疗肿瘤效果，并可使HER2-CAR-T淋巴细胞的用量降低、降低单独的CAR-T淋巴细胞治疗导致的副作用。此外，芦荟苷为小分子有机化合物，价格便宜，能够降低CAR-T细胞的免疫治疗成本。
下面通过具体的实施例验证芦荟苷与HER2-CAR-T细胞联合制剂用于治疗骨肉瘤的协同作用。如图1所示，HER2-CAR序列的信号肽选择了CD8α，胞外识别区为HER2单链抗体，之后顺次连接了CD8α的胞外区和CD8α的跨膜区作为连接结构，然后顺次连接CD28、CD137、CD3ζ的胞内区。
实施例1
构建质粒：本专利申请构建了第三代嵌合抗原受体质粒，即CD8α-HER2scFv-CD8-CD28-CA137-CD3ζ。其中，CD8α为信号肽，对应的核苷酸序列为5’-atg gcc tta cca gtgacc gcc ttg ctc ctg ccg ctg gcc ttg ctg ctc cac gcc gcc agg ccg-3’（核苷酸序列为SEQ ID No.2），连接HER2 scFv胞外识别区（核苷酸序列为SEQ ID No.3），顺次连接CD8α铰链区和CD8α跨膜区（核苷酸序列为SEQ ID No.4）以及CD28（核苷酸序列为SEQ ID No.5）、CD137（核苷酸序列为SEQ ID No.6）和CD3ζ（核苷酸序列为SEQ ID No.7）的胞内区共刺激信号。本发明专利所述的第三代嵌合抗原受体HER2-CAR的核苷酸序列为SEQ ID No.1。第三代CAR-T细胞相比于第一代和第二代CAR-T细胞，增加了胞内区的共刺激结构域，能够增加肿瘤细胞杀伤因子的分泌数量，提高肿瘤杀伤效果。
构建慢病毒载体和包装质粒：将嵌合抗原受体HER2-CAR的编码基因片段（核苷酸序列为SEQ ID No.1）插入到带有酶切位点AtsI和BstXI的慢病毒真核表达载体pLV[Exp]中，经过酶切、连接、鉴定等步骤确保基因片段的准确性，从而得到HER2-CAR重组质粒pLV[Exp]-HER2-CAR；计算核酸纯度及核酸含量，最后分装保存于-20℃备用。
T淋巴细胞慢病毒转染：采集并分离得到健康人的外周血单个核细胞，加入CD3/CD28免疫磁珠进行孵育，筛选得到CD3阳性T淋巴细胞；向其中加入上述重组慢病毒进行培养，获得HER2-CAR-T淋巴细胞，转染成功后，采用流式细胞仪、免疫荧光等方法检测转染效率≥70％，这表明成功制备得到靶向HER2抗原的CAR-T细胞，保存在回输专用的细胞冻存液中，以供患者回输。
实施例2
芦荟苷和HER2-CAR-T细胞联合制剂对肿瘤细胞的体外杀伤作用，靶细胞选择表达HER2的U2OS细胞（人骨肉瘤细胞），将其在含胎牛血清的DMEM高糖培养基中传代培养。将U2OS细胞接种于96孔细胞培养板，细胞数为1×104/孔，培养24小时。根据不同处理方法为3个实验组，即芦荟苷组（芦荟苷注射剂3mL）、HER2-CAR-T细胞组（1×106/mL细胞悬液）和芦荟苷与HER2-CAR-T细胞联合制剂组（1×106/mL细胞悬液+5%芦荟苷注射剂3mL）。
乳酸脱氢酶释放实验评估药物对肿瘤细胞杀伤效果，乳酸脱氢酶释放法利用乳酸脱氢酶释放存在于细胞质中，当细胞受损细胞膜破裂，乳酸脱氢酶释放释放到细胞外的培养基中，测定乳酸脱氢酶释放的量评估细胞损害的程度；应用乳酸脱氢酶释放细胞毒性检测试剂盒检测细胞培养上清中的乳酸脱氢酶释放活性，判定药物对肿瘤细胞杀伤效果。将不同效应细胞分别加入U2OS细胞中进行共培养。24小时后，在未加淋巴细胞肿瘤细胞组加入试剂盒提供的细胞裂解液20μl，37℃作用1h；用多孔板离心机进行离心，400g，5min。取上清液120μl加入到一个新96孔板中；用乳酸溶液20μl、INT溶液（1X） 20μl及酶溶液 20μl，共计每孔体系60μl，完成乳酸脱氢酶检测工作液配置；将配置好的乳酸脱氢酶检测工作液60μl加至含有细胞培养上清的96孔板中；混匀，室温，约25℃，避光条件下孵育30min；490nm处测吸光度；单纯肿瘤细胞孔上清设为样品对照孔，裂解的肿瘤细胞孔上清设为最大酶活性对照孔；细胞杀伤效率计算：（作用样品孔（OD值）-样本空白孔（OD值）/（最大酶活性对照孔（（OD值）-样品对照孔（（OD值））×100%。不同治疗方法对肿瘤细胞的裂解作用，如图2。
实施例3
通过细胞因子检测揭示芦荟苷与HER2-CAR-T细胞制剂发挥强效肿瘤杀伤作用的药理，将表达HER2抗原的U2OS细胞以每孔1×105种于96孔板种，将不同组别的药物与靶细胞混合，以总体积200μl的培养液（RPMI 1640培养基+10％FBS）在CO2培养箱中共孵育24小时，温度为37℃。每组3个复孔。接下来，离心后取10μl上清，用Perkin Elmer的Alpha LISAassay试剂盒检测TNF-γ以及IL-2的细胞因子浓度水平。如图3所示，芦荟苷与HER2-CAR-T细胞联合制剂中检测出的TNF-γ、IL-2细胞因子含量高于单独HER2-CAR-T细胞组和单独芦荟苷组，这阐明了二者联合应用增强了对肿瘤细胞杀伤作用的原因。
实施例4
通过ELISA法检测颗粒酶B，收集上清后，将其用试剂检测增强剂稀释5倍；向标准品管内加入500μl样本稀释液；配置成浓度为220ng/ml。进行倍比稀释成10000pg/ml，5000pg/ml，2500pg/ml，1250pg/ml，625pg/ml，313pg/ml，156pg/ml，0pg/ml；向检测版内分别加入标准品及样本上清50μl，加入抗体混合物50μl，室温震荡孵育1h；去上清，用350μl洗液，洗三次，每次2分钟。最后一次清洗后，用吸水纸拍板，洗去剩余液体；加入100μl TMB显色液，室温孵育10min，加100μl 终止液，混匀；利用酶标仪的450nm波长，测各孔吸光度值；制标准曲线，利用回归方程式，将各样品吸光度值代入后，得出的结果浓度需乘以稀释倍数（5倍），确定待测样品浓度。图4所示，芦荟苷与HER2-CAR-T细胞联合制剂中检测出的颗粒酶B的含量高于单独HER2-CAR-T细胞组和单独芦荟苷组，这阐明了二者联合应用增强了对肿瘤细胞杀伤作用的原因。
Claims (6)
1.一种芦荟苷与HER2-CAR-T细胞联合在制备骨肉瘤药物中的应用，其特征在于：包括芦荟苷和HER2-CAR-T细胞；芦荟苷的CAS 号为1415-73-2，分子式C21H22O9，分子量：418.394，结构式如下：
；
所述HER2-CAR及其载体的核苷酸序列为SEQ ID No.8；HER2-CAR结构包括胞外结构域、跨膜区和胞内结构域；
胞外结构域由CD8α Leader、HER2 scFv单链抗体以及CD8α铰链区组成；跨膜区由CD8αTM构成；胞内结构域由CD28共刺激结构域、CD137共刺激结构域以及CD3ζ胞内信号区共同组成；
CD8α Leader为HER2-CAR中的嵌合受体信号肽，核苷酸序列为SEQ ID No.2。
2.如权利要求1所述一种芦荟苷与HER2-CAR-T细胞联合在制备骨肉瘤药物中的应用，其特征在于：HER2 scFv为HER2-CAR中的靶向恶性肿瘤细胞表面HER2抗原的人源化单链抗体，核苷酸序列为SEQ ID No.3。
3.如权利要求1所述一种芦荟苷与HER2-CAR-T细胞联合在制备骨肉瘤药物中的应用，其特征在于：CD8α-hinge/CD8α TM为HER2-CAR中的铰链区和跨膜区，核苷酸序列为SEQ IDNo.4。
4.如权利要求1所述一种芦荟苷与HER2-CAR-T细胞联合在制备骨肉瘤药物中的应用，其特征在于：CD28为HER2-CAR中的胞内的共刺激结构域，核苷酸序列为SEQ ID No.5。
5.如权利要求1所述一种芦荟苷与HER2-CAR-T细胞联合在制备骨肉瘤药物中的应用，其特征在于：HER2-CAR中的CD137为胞内的另一个共刺激结构域，核苷酸序列为SEQ IDNo.6。
6.如权利要求1所述一种芦荟苷与HER2-CAR-T细胞联合在制备骨肉瘤药物中的应用，其特征在于：HER2-CAR中的CD3ζ为胞内信号区，核苷酸序列为SEQ ID No.7。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311007658.0A CN116785417B (zh) | 2023-08-11 | 2023-08-11 | 芦荟苷与her2-car-t细胞联合制备治疗骨肉瘤的药物 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311007658.0A CN116785417B (zh) | 2023-08-11 | 2023-08-11 | 芦荟苷与her2-car-t细胞联合制备治疗骨肉瘤的药物 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116785417A CN116785417A (zh) | 2023-09-22 |
| CN116785417B true CN116785417B (zh) | 2023-11-03 |
Family
ID=88049937
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311007658.0A Active CN116785417B (zh) | 2023-08-11 | 2023-08-11 | 芦荟苷与her2-car-t细胞联合制备治疗骨肉瘤的药物 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116785417B (zh) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104758278A (zh) * | 2015-03-24 | 2015-07-08 | 宁夏医科大学 | 芦荟苷作为治疗缺血性脑卒中药物的用途 |
| CN105384824A (zh) * | 2014-08-26 | 2016-03-09 | 中国人民解放军总医院 | 嵌合抗原受体及其基因和重组表达载体、工程化her2靶向性的nkt细胞及其应用 |
| CN108164599A (zh) * | 2016-12-07 | 2018-06-15 | 南开大学 | 芦荟苷的偶联物及其制备方法与应用 |
| CN109824781A (zh) * | 2019-01-21 | 2019-05-31 | 卢英 | 抗人her 2抗原的特异性嵌合抗原受体、编码基因和表达载体以及应用 |
| CN113368181A (zh) * | 2021-04-29 | 2021-09-10 | 严伟琪 | 一种芦荟多糖-纳米金载药材料的制备方法 |
| CN113527514A (zh) * | 2021-06-30 | 2021-10-22 | 徐州医科大学 | Gstp1在制备增效型CAR-T中的应用 |
-
2023
- 2023-08-11 CN CN202311007658.0A patent/CN116785417B/zh active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105384824A (zh) * | 2014-08-26 | 2016-03-09 | 中国人民解放军总医院 | 嵌合抗原受体及其基因和重组表达载体、工程化her2靶向性的nkt细胞及其应用 |
| CN104758278A (zh) * | 2015-03-24 | 2015-07-08 | 宁夏医科大学 | 芦荟苷作为治疗缺血性脑卒中药物的用途 |
| CN108164599A (zh) * | 2016-12-07 | 2018-06-15 | 南开大学 | 芦荟苷的偶联物及其制备方法与应用 |
| CN109824781A (zh) * | 2019-01-21 | 2019-05-31 | 卢英 | 抗人her 2抗原的特异性嵌合抗原受体、编码基因和表达载体以及应用 |
| CN113368181A (zh) * | 2021-04-29 | 2021-09-10 | 严伟琪 | 一种芦荟多糖-纳米金载药材料的制备方法 |
| CN113527514A (zh) * | 2021-06-30 | 2021-10-22 | 徐州医科大学 | Gstp1在制备增效型CAR-T中的应用 |
Non-Patent Citations (2)
| Title |
|---|
| Immunotherapy for Osteosarcoma: Genetic Modification of T cells OvercomesLow Levels of Tumor Antigen Expression;Nabil Ahmed等;Molecular Therapy;第17卷(第10期);摘要，第1779-1786页，图3 * |
| 芦荟苷通过PI3K/AKT/mTOR 途径介导人骨肉瘤自噬与凋亡的研究;何嘉铭;中国博士学位论文全文数据库医药卫生科技辑(第2期);第116-119页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116785417A (zh) | 2023-09-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| TWI747280B (zh) | 余甘子萃取發酵物及其製備與應用 | |
| CN116785417B (zh) | 芦荟苷与her2-car-t细胞联合制备治疗骨肉瘤的药物 | |
| AU2004285803A1 (en) | Plant-origin beta3-adrenoceptor agonist and use of the same | |
| CN114761020A (zh) | (5r)-5-羟基雷公藤内酯醇在制备药物中的应用 | |
| CN111419832B (zh) | 药物组合物及其在制备治疗肿瘤药物中的用途 | |
| CN108685916A (zh) | 噻唑烷二酮类化合物双靶点治疗垂体生长激素腺瘤的用途 | |
| KR102051393B1 (ko) | 아데노부속바이러스 기반 유전자 전달용 재조합 벡터 및 이를 이용한 암질환 예방 또는 치료용 조성물 | |
| CN112755015A (zh) | Pt2385在制备防治肺动脉高压的药物中的应用 | |
| JP6535101B2 (ja) | 抗心筋虚血再灌流傷害の香青蘭活性成分組成物 | |
| JP6306634B2 (ja) | 食品用組成物 | |
| CN104491496B (zh) | 天麻素/天麻粉在制备抗肝纤维化药物中的应用 | |
| CN115068610B (zh) | 抑制乳腺癌细胞中muc1表达的物质在降低抗乳腺癌药物耐药性中的应用 | |
| CN114887080B (zh) | 一种mRNA剂型的免疫抑制剂及其在制备肿瘤治疗药物中的应用 | |
| KR102073759B1 (ko) | Nadh를 포함하는 대사성 질환 예방 또는 치료용 조성물 | |
| CN114642668B (zh) | 拉坦前列素的药物新用途 | |
| KR20010101065A (ko) | 표고버섯 균사체 추출물 유래의 ｌａｋ 활성증강물질 및이를 함유하는 ｌａｋ 활성증강용 제제 | |
| CN118045168A (zh) | 一种同时靶向两种肿瘤抗原的car-t细胞治疗上皮性卵巢癌的药物 | |
| CN116585478A (zh) | Brd7联合全反式维甲酸和pd-l1抗体在制备治疗鼻咽癌制剂中的应用 | |
| KR20200131071A (ko) | 디오스민을 포함하는 피부염을 예방 또는 개선하는데 사용하기 위한 조성물 및 그의 용도 | |
| CN118059234A (zh) | 抗pd-1单抗联合米托蒽醌脂质体在制备治疗肿瘤心脏并发症药物中的应用 | |
| US20210161858A1 (en) | Composition for preventing and treating muscle disease, improving muscle function or enhancing motor performance comprising hydrangenol or hydrangea extract as active ingredient | |
| CN118021848A (zh) | 微生物菌株在制备用于预防或治疗代谢性疾病的药物中的用途 | |
| CN116919935A (zh) | 没食子酸在治疗结直肠癌和促进Anti-PD-1介导抗肿瘤效应的应用 | |
| CN107841501A (zh) | 核酸分子ctl4hsh20、其制备方法及应用 | |
| CN107841500A (zh) | 核酸分子ctl4hsh18、其制备方法及应用 |
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 |