CN114748448B - 一种巨噬细胞膜纳米囊泡的制备方法与应用 - Google Patents
一种巨噬细胞膜纳米囊泡的制备方法与应用 Download PDFInfo
- Publication number
- CN114748448B CN114748448B CN202210460518.8A CN202210460518A CN114748448B CN 114748448 B CN114748448 B CN 114748448B CN 202210460518 A CN202210460518 A CN 202210460518A CN 114748448 B CN114748448 B CN 114748448B
- Authority
- CN
- China
- Prior art keywords
- cells
- macrophage membrane
- macrophage
- application
- tumor
- 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
- 210000002540 macrophage Anatomy 0.000 title claims abstract description 50
- 239000012528 membrane Substances 0.000 title claims abstract description 44
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- KURQKNMKCGYWRJ-HNNXBMFYSA-N 7-(5-methylfuran-2-yl)-3-[[6-[[(3s)-oxolan-3-yl]oxymethyl]pyridin-2-yl]methyl]triazolo[4,5-d]pyrimidin-5-amine Chemical compound O1C(C)=CC=C1C1=NC(N)=NC2=C1N=NN2CC1=CC=CC(CO[C@@H]2COCC2)=N1 KURQKNMKCGYWRJ-HNNXBMFYSA-N 0.000 claims abstract description 17
- 239000012634 fragment Substances 0.000 claims abstract description 13
- 229940121359 adenosine receptor antagonist Drugs 0.000 claims abstract description 11
- 239000000296 purinergic P1 receptor antagonist Substances 0.000 claims abstract description 11
- 210000004027 cell Anatomy 0.000 claims description 69
- 239000013612 plasmid Substances 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 10
- 241000700605 Viruses Species 0.000 claims description 9
- 210000004881 tumor cell Anatomy 0.000 claims description 8
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 claims description 6
- 239000000137 peptide hydrolase inhibitor Substances 0.000 claims description 6
- 238000011068 loading method Methods 0.000 claims description 4
- 201000001441 melanoma Diseases 0.000 claims description 4
- 238000004806 packaging method and process Methods 0.000 claims description 4
- 230000037396 body weight Effects 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 229940076838 Immune checkpoint inhibitor Drugs 0.000 claims description 2
- 102000037984 Inhibitory immune checkpoint proteins Human genes 0.000 claims description 2
- 108091008026 Inhibitory immune checkpoint proteins Proteins 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 239000012274 immune-checkpoint protein inhibitor Substances 0.000 claims description 2
- 229940126585 therapeutic drug Drugs 0.000 claims description 2
- 101150051188 Adora2a gene Proteins 0.000 claims 2
- 210000001744 T-lymphocyte Anatomy 0.000 abstract description 23
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 abstract description 14
- 230000000694 effects Effects 0.000 abstract description 13
- 239000003814 drug Substances 0.000 abstract description 10
- 229940079593 drug Drugs 0.000 abstract description 9
- 239000002126 C01EB10 - Adenosine Substances 0.000 abstract description 7
- 229960005305 adenosine Drugs 0.000 abstract description 7
- 238000002560 therapeutic procedure Methods 0.000 abstract description 6
- 102000009346 Adenosine receptors Human genes 0.000 abstract description 5
- 108050000203 Adenosine receptors Proteins 0.000 abstract description 5
- 230000000259 anti-tumor effect Effects 0.000 abstract description 4
- 206010027476 Metastases Diseases 0.000 abstract description 3
- 239000005557 antagonist Substances 0.000 abstract description 3
- 230000009401 metastasis Effects 0.000 abstract description 3
- 210000003171 tumor-infiltrating lymphocyte Anatomy 0.000 abstract description 3
- 206010061309 Neoplasm progression Diseases 0.000 abstract description 2
- 230000005751 tumor progression Effects 0.000 abstract description 2
- 206010028980 Neoplasm Diseases 0.000 description 37
- 210000000170 cell membrane Anatomy 0.000 description 36
- 241000699670 Mus sp. Species 0.000 description 33
- 102100040678 Programmed cell death protein 1 Human genes 0.000 description 32
- 101710089372 Programmed cell death protein 1 Proteins 0.000 description 32
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 22
- 238000011282 treatment Methods 0.000 description 21
- 238000002474 experimental method Methods 0.000 description 20
- 239000006228 supernatant Substances 0.000 description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 16
- 239000012091 fetal bovine serum Substances 0.000 description 13
- 241000699666 Mus <mouse, genus> Species 0.000 description 11
- 239000000243 solution Substances 0.000 description 11
- 235000019441 ethanol Nutrition 0.000 description 9
- 239000007788 liquid Substances 0.000 description 8
- 238000001514 detection method Methods 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 210000000056 organ Anatomy 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 238000011160 research Methods 0.000 description 7
- 210000001808 exosome Anatomy 0.000 description 6
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 238000000684 flow cytometry Methods 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 210000003289 regulatory T cell Anatomy 0.000 description 5
- 238000007789 sealing Methods 0.000 description 5
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 4
- 102000004127 Cytokines Human genes 0.000 description 4
- 108090000695 Cytokines Proteins 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 229910052802 copper Inorganic materials 0.000 description 4
- 239000010949 copper Substances 0.000 description 4
- 238000009169 immunotherapy Methods 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 3
- 102000037982 Immune checkpoint proteins Human genes 0.000 description 3
- 108091008036 Immune checkpoint proteins Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000002033 PVDF binder Substances 0.000 description 3
- 229930040373 Paraformaldehyde Natural products 0.000 description 3
- KHGNFPUMBJSZSM-UHFFFAOYSA-N Perforine Natural products COC1=C2CCC(O)C(CCC(C)(C)O)(OC)C2=NC2=C1C=CO2 KHGNFPUMBJSZSM-UHFFFAOYSA-N 0.000 description 3
- 210000001015 abdomen Anatomy 0.000 description 3
- 230000006470 autoimmune attack Effects 0.000 description 3
- 230000005540 biological transmission Effects 0.000 description 3
- 238000002619 cancer immunotherapy Methods 0.000 description 3
- 238000004624 confocal microscopy Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 210000002865 immune cell Anatomy 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 210000004072 lung Anatomy 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 229920002866 paraformaldehyde Polymers 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 229930192851 perforin Natural products 0.000 description 3
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 230000001737 promoting effect Effects 0.000 description 3
- 229950010131 puromycin Drugs 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 239000008399 tap water Substances 0.000 description 3
- 235000020679 tap water Nutrition 0.000 description 3
- 230000004614 tumor growth Effects 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- 102000008096 B7-H1 Antigen Human genes 0.000 description 2
- 108010074708 B7-H1 Antigen Proteins 0.000 description 2
- 101710088172 HTH-type transcriptional regulator RipA Proteins 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
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 239000012097 Lipofectamine 2000 Substances 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241001529936 Murinae Species 0.000 description 2
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 2
- 108010019160 Pancreatin Proteins 0.000 description 2
- 239000006180 TBST buffer Substances 0.000 description 2
- ABUBSBSOTTXVPV-UHFFFAOYSA-H [U+6].CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O Chemical compound [U+6].CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O ABUBSBSOTTXVPV-UHFFFAOYSA-H 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000003149 assay kit Methods 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 239000006059 cover glass Substances 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000002296 dynamic light scattering Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 239000012737 fresh medium Substances 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 210000002216 heart Anatomy 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 230000001506 immunosuppresive effect Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 229940055695 pancreatin Drugs 0.000 description 2
- 230000010287 polarization Effects 0.000 description 2
- 238000010791 quenching Methods 0.000 description 2
- 230000000171 quenching effect Effects 0.000 description 2
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 2
- 238000001959 radiotherapy Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 239000012096 transfection reagent Substances 0.000 description 2
- 230000005748 tumor development Effects 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- 239000012224 working solution Substances 0.000 description 2
- 239000008096 xylene Substances 0.000 description 2
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 239000000592 Artificial Cell Substances 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 108700031361 Brachyury Proteins 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 description 1
- 229920000858 Cyclodextrin Polymers 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 239000001116 FEMA 4028 Substances 0.000 description 1
- 102000001398 Granzyme Human genes 0.000 description 1
- 108060005986 Granzyme Proteins 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 238000010867 Hoechst staining Methods 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 210000004322 M2 macrophage Anatomy 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 208000009525 Myocarditis Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 206010041047 Slow virus infection Diseases 0.000 description 1
- 230000006044 T cell activation Effects 0.000 description 1
- 230000006052 T cell proliferation Effects 0.000 description 1
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 1
- 241000700647 Variola virus Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000002390 adhesive tape Substances 0.000 description 1
- 238000011467 adoptive cell therapy Methods 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 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 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 description 1
- 235000011175 beta-cyclodextrine Nutrition 0.000 description 1
- 229960004853 betadex Drugs 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- KQNZDYYTLMIZCT-KQPMLPITSA-N brefeldin A Chemical compound O[C@@H]1\C=C\C(=O)O[C@@H](C)CCC\C=C\[C@@H]2C[C@H](O)C[C@H]21 KQNZDYYTLMIZCT-KQPMLPITSA-N 0.000 description 1
- JUMGSHROWPPKFX-UHFFFAOYSA-N brefeldin-A Natural products CC1CCCC=CC2(C)CC(O)CC2(C)C(O)C=CC(=O)O1 JUMGSHROWPPKFX-UHFFFAOYSA-N 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000007012 clinical effect Effects 0.000 description 1
- 230000008045 co-localization Effects 0.000 description 1
- 206010009887 colitis Diseases 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000000139 costimulatory effect Effects 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000000779 depleting effect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 238000001125 extrusion Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 1
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
- 230000000004 hemodynamic effect Effects 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 230000037451 immune surveillance Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 238000003125 immunofluorescent labeling Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 210000005008 immunosuppressive cell Anatomy 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 230000005917 in vivo anti-tumor Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- PGHMRUGBZOYCAA-UHFFFAOYSA-N ionomycin Natural products O1C(CC(O)C(C)C(O)C(C)C=CCC(C)CC(C)C(O)=CC(=O)C(C)CC(C)CC(CCC(O)=O)C)CCC1(C)C1OC(C)(C(C)O)CC1 PGHMRUGBZOYCAA-UHFFFAOYSA-N 0.000 description 1
- PGHMRUGBZOYCAA-ADZNBVRBSA-N ionomycin Chemical compound O1[C@H](C[C@H](O)[C@H](C)[C@H](O)[C@H](C)/C=C/C[C@@H](C)C[C@@H](C)C(/O)=C/C(=O)[C@@H](C)C[C@@H](C)C[C@@H](CCC(O)=O)C)CC[C@@]1(C)[C@@H]1O[C@](C)([C@@H](C)O)CC1 PGHMRUGBZOYCAA-ADZNBVRBSA-N 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229960003301 nivolumab Drugs 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229960002621 pembrolizumab Drugs 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 238000013379 physicochemical characterization Methods 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- -1 small molecule compound Chemical class 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 206010043778 thyroiditis Diseases 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
Classifications
-
- 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
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
-
- 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
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
- A61K9/5068—Cell membranes or bacterial membranes enclosing drugs
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
-
- 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
- C07K16/2818—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 CD28 or CD152
-
- 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
- C12N15/86—Viral vectors
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
-
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
-
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
-
- 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
- C12N2510/00—Genetically modified cells
-
- 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
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- 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
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15051—Methods of production or purification of viral material
- C12N2740/15052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Epidemiology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Cell Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Virology (AREA)
- Hematology (AREA)
- Endocrinology (AREA)
- Mycology (AREA)
- Gastroenterology & Hepatology (AREA)
- Oncology (AREA)
- Botany (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
本发明公开了一种巨噬细胞膜纳米囊泡的制备方法与应用，涉及生物医药技术领域。本发明的巨噬细胞膜纳米囊泡，所述巨噬细胞膜过表达PD‑1单链可变片段抗体，并负载腺苷受体拮抗剂。本发明的巨噬细胞膜纳米囊泡通过PD‑1阻断增强T细胞的抗肿瘤活性；本发明的巨噬细胞膜纳米囊泡负载了A2a腺苷受体(A2aR)拮抗剂CPI‑444，可协助T细胞拮抗腺苷，增加肿瘤浸润T细胞的密度和活性，直接抑制肿瘤进展和转移，为当前的联合PD‑1抗体疗法提供了新的思路。
Description
技术领域
本发明涉及生物医药技术领域，具体涉及一种巨噬细胞膜纳米囊泡的制备方法与应用。
背景技术
恶性肿瘤是威胁人类生命与健康的最主要疾病之一，对我国人民生命和健康造成了严重威胁。肿瘤的传统治疗方法主要包括手术切除、放疗和化疗等，但这些治疗方法都具有一定的局限性，比如手术治疗无法解决非实体瘤，放疗和化疗具有极大副作用且易复发。近年来，癌症免疫疗法取得了令人瞩目的临床效果，并在2013年被《科学》杂志评为年度最重要的科学突破。癌症免疫治疗是一种通过激活人体内源的免疫系统来杀伤肿瘤细胞的治疗方式。目前肿瘤免疫治疗的研究主要集中于免疫检查点(Checkpoint)阻断剂疗法，肿瘤疫苗，过继性细胞疗法，并在临床上取得了显著的治疗效果。然而，这些免疫疗法都存在其一定的局限性：PD-1单抗药物在临床治疗中的总体响应率低于30％，肿瘤疫苗目前还处于临床研究，而嵌合抗原受体T细胞免疫疗法(CAR-T)细胞疗法仅在血液肿瘤中有较好的疗效。
T细胞在激活后表达PD-1受体，作为一种刹车来限制自身免疫攻击。肿瘤细胞巧妙地利用这种负调控机制，通过表达程序性死亡配体1(programmed death ligand 1,PD-L1)来逃避免疫监视，从而使T细胞衰竭，从而避免免疫消除。因此，免疫检查点阻断疗法(ICB)，一种用Nivolumab和Pembrolizumab等单克隆抗体阻断PD-1/PD-L1轴的策略，在各种类型的癌症中获得了前所未有的应答。但ICB治疗仍有一定的局限性，如临床应答率有限、治疗效果短暂、自身免疫攻击等。这些问题与免疫抑制肿瘤微环境(TME)有所关联。M2巨噬细胞和调节性T细胞(Tregs)等免疫抑制细胞总是被招募到肿瘤组织中，从而抑制T细胞活性。此外，外周核苷酸酶(CD39和CD73)在肿瘤细胞中的高表达导致了TME中腺苷的积累。腺苷与A2a腺苷受体(A2aR)结合，降低T细胞的抗肿瘤活性，促进调节性T细胞(Treg)的极化，进一步限制ICB治疗的疗效。此外，PD-1抗体在临床治疗中可引起多种自身免疫攻击，包括心肌炎、甲状腺炎、1型糖尿病、结肠炎等，甚至危及患者的生命。因此，迫切需要提高应答率，同时减少免疫检查点抗体的毒性作用。除了单克隆抗体，小分子化合物药物也被开发来调节免疫细胞的活性。值得注意的是，化学药物靶向给药不仅能提高药物疗效，而且能降低毒副作用，包括外泌体在内的天然细胞膜泡被认为是药物输送的潜在载体。外泌体表现出良好的生物相容性、优异的载药能力和循环稳定性。然而，外泌体的产量太低，无法满足研究和临床治疗的需要。因此，采用挤压和超声方法制备了人工细胞膜纳米囊泡(NVs)。此外，与外泌体相比，细胞膜NVs的数量和大小更可控。特别是，NVs可以通过基因工程表达功能性蛋白，实现对癌症治疗的特异功能，因为这些NVs中的重组蛋白可以保持其完整的生物活性。此外，NVs可以靶向并浸润到特定的肿瘤中，从而促进其发挥作用。基于目前的免疫疗法存在的局限性，开发新型癌症免疫疗法有着迫切的临床需求。
发明内容
本发明的目的在于克服现有技术的不足，提供一种巨噬细胞膜纳米囊泡的制备方法与应用。
为实现上述目的，本发明采取的技术方案为：一种巨噬细胞膜纳米囊泡，所述巨噬细胞膜过表达PD-1单链可变片段抗体，并负载腺苷受体拮抗剂。
研究表明，细胞膜囊泡可作为多种药物体内递送的载体，细胞膜囊泡由脂质、蛋白和少量糖类组成，表面的蛋白有助于提高其靶向性，具有良好的生物相容性，免疫原性低，有助于降低肝脏和肾脏对药物的清除，在血液中半衰期长，提高药物的生物利用度。本发明利用慢病毒感染的方法构建过表达PD-1单链可变片段抗体的巨噬细胞系，并制得细胞膜纳米囊泡，并系统地研究了该细胞膜囊泡的理化表征、体外活性和体内抗肿瘤效应。
作为本发明所述的巨噬细胞膜纳米囊泡的优选实施方式，所述巨噬细胞为RAW264.7细胞系。
研究表明，单核细胞/巨噬细胞可以被趋化因子(CSF1,CCL2)招募到肿瘤微环境。本发明利用巨噬细胞的肿瘤靶向能力，制备巨噬细胞来源的人工外泌体，以恢复“耗竭”的肿瘤特异性T细胞的功能。本申请发明人经大量的研究筛选验证发现，采用RAW 264.7细胞系制备过表达PD-1单链可变片段抗体的细胞膜纳米囊泡，能够稳定表达aPD-1-scFv。
作为本发明所述的巨噬细胞膜纳米囊泡的优选实施方式，所述腺苷受体拮抗剂为A2a腺苷受体拮抗剂。
腺苷是癌细胞分泌的一种免疫抑制代谢物，腺苷与A2a腺苷受体(A2aR)结合，诱导降低T细胞的抗肿瘤活性，促进Treg的极化，进一步限制ICB治疗的疗效。本申请发明人经大量的研究筛选验证发现，NVs负载A2a腺苷受体(A2aR)拮抗剂CPI-444，以协助T细胞拮抗腺苷，增加肿瘤浸润T细胞的密度和活性，直接抑制肿瘤进展和转移，为当前的联合PD-1抗体疗法提供了新的思路。
作为本发明所述的巨噬细胞膜纳米囊泡的优选实施方式，所述A2a腺苷受体拮抗剂为CPI-444。
作为本发明所述的巨噬细胞膜纳米囊泡的优选实施方式，所述CPI-444的载药率为15.2％。
本申请发明人经研究发现，将0.5mg/mL的CPI-444与aPD-1-scFv NVs孵育，能获得具有良好的载药能力的巨噬细胞膜纳米囊泡。
本发明还提供上述的巨噬细胞膜纳米囊泡的制备方法，包括以下步骤：
(1)构建和扩增PD-1单链可变片段抗体慢病毒载体质粒；
(2)使用步骤(1)的质粒进行慢病毒包装，得PD-1单链抗体病毒；
(3)构建稳定过表达PD-1单链可变片段抗体的巨噬细胞；
(4)扩增培养步骤(3)的巨噬细胞，离心收集细胞，用含蛋白酶抑制剂的PBS溶液重悬细胞，经过滤得过表达PD-1单链可变片段抗体的巨噬细胞膜纳米囊泡；
(5)将腺苷受体拮抗剂负载到过表达PD-1单链可变片段抗体的巨噬细胞膜纳米囊泡，得所述巨噬细胞膜纳米囊泡。
本发明还提供所述的巨噬细胞膜纳米囊泡在制备免疫检查点抑制剂中的应用。
本发明还提供所述的巨噬细胞膜纳米囊泡在制备靶向肿瘤细胞治疗药物中的应用。
本申请发明人经研究发现，本发明的巨噬细胞膜纳米囊泡比未负载CPI-444的纳米囊泡具有更好的抑制肿瘤生长效果。
作为本发明所述应用的优选实施方式，所述肿瘤细胞为黑色素瘤细胞。
作为本发明所述应用的优选实施方式，所述巨噬细胞膜纳米囊泡的浓度为25mg/kg体重。
本发明的有益效果：本发明提供了一种巨噬细胞膜纳米囊泡的制备方法，本发明的巨噬细胞膜纳米囊泡通过PD-1阻断增强T细胞的抗肿瘤活性；本发明的巨噬细胞膜纳米囊泡负载了A2a腺苷受体(A2aR)拮抗剂CPI-444，可协助T细胞拮抗腺苷，增加肿瘤浸润T细胞的密度和活性，直接抑制肿瘤进展和转移，为当前的联合PD-1抗体疗法提供了新的思路。
附图说明
图1为巨噬细胞膜纳米囊泡的制备流程示意图。
图2为aPD-1-scFv-EGFP慢病毒载体质粒的图谱。
图3为巨噬细胞Raw 264.7的激光共聚焦图、流式细胞图和Western Blot图。
图4为NVs的粒径和电位表征图。
图5为细胞膜囊泡蛋白的表达验证图。
图6为细胞膜囊泡与细胞结合的激光共聚焦图。
图7为细胞膜囊泡的血液循环状况图。
图8为细胞膜囊泡在小鼠器官中的分布图。
图9为实施例3PD-1单链抗体细胞膜囊泡治疗小鼠肿瘤模型的给药流程图。
图10为实验小鼠体积增长图、肿瘤重量变化图和生存率变化图。
图11为小鼠成像和单个肿瘤增长曲线图。
图12为小鼠肿瘤免疫细胞和细胞因子流式图。
图13为小鼠脏器HE染色图。
具体实施方式
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
实施例1巨噬细胞膜纳米囊泡的制备
本实施例提供了巨噬细胞膜纳米囊泡的制备，其制备的流程示意图如图1所示。
1.1PD-1单链抗体慢病毒载体质粒构建和扩增
(1)实验材料：aPD-1-scFv-EGFP慢病毒载体质粒购自擎科生物科技有限公司；包装质粒psPAX2和pMD2.G购自擎科生物科技有限公司。
(2)实验方法：a.将TransStbl3感受态细胞置于冰上解冻，在超净台环境条件下加入500ng质粒，轻弹混匀，冰上静置30min；b.42℃，水浴热激50s，冰浴3min；c.在超净台加入200μL LB培养基，37℃，220rpm，摇1.5h；d.吸200μL培养物涂布Amp平板培养基，在细菌培养箱培养12h；e.挑平板培养基上的单菌落至含有60μg/mL氨卞青霉素的LB摇菌管，37℃，220rpm，摇12h；f.每管吸200μL菌液，送到擎科生物科技有限公司进行碱基测序，剩余的菌液暂存4℃冰箱。测序结果与设计序列碱基序列一致，将剩余菌液用天根高纯度质粒小提试剂盒进行质粒提取，步骤按照说明书进行。提取的质粒测浓度和纯度，要求OD260/OD280>1.8，OD260/OD230>2。检测完毕后，质粒保存在-20℃。上述aPD-1-scFv-EGFP慢病毒载体质粒的图谱如图2所示。
1.2包病毒和纯化病毒
具体实验方法：提前一天将HEK 293T细胞铺板到10cm皿，密度为50％。第二天，在生物安全柜内，分别向4管15mL离心管内加入1mL MEM基础培养基，不含血清和抗生素。两管分别加入目的质粒10μg aPD-1、10μg psPAX2质粒、5μg pMD2.G质粒。另外1管分别加入12μLLipofectamine 2000转染试剂。室温静置5min后，分别将一管质粒管与一管Lipofectamine2000管混合，室温静置30min。将提前铺板的HEK 293T细胞从细胞培养箱取出，用基础培养基MEM润洗细胞一次，然后加入4mL MEM培养基，再滴加上述质粒和转染试剂混合液，放回培养箱培养8h，然后换上10％胎牛血清和1％青霉素/链霉素的DMEM培养基正常培养，分别在24h、48h、72h收集细胞上清液，并添加新鲜培养基。
将收集的上清培养基在4℃条件下离心，500g，10min。然后在生物安全柜内，取上清液过0.45μm滤头，加入5×慢病毒浓集液稀释至1×，轻轻摇匀，4℃冰箱过夜。第二天，将混合液4℃离心，3500g，25min。去掉上清液后，4℃条件下离心，3500g，5min，用移液枪吸掉剩余液体，用本来上清液体积的1/100的PBS重悬沉淀，分装成6份，保存于-80℃。
1.3构建稳定过表达PD-1单链抗体的RAW 264.7细胞系
具体实验方法：提前一天将RAW 264.7细胞铺板于六孔板，密度为50％。第二天，从细胞培养箱取出细胞，去掉培养基，每孔加入500μL新鲜培养基。加入终浓度为8μg/mLPolybrene，每孔加入一份PD-1单链抗体病毒。将细胞放回培养箱，72h后，在荧光显微镜下观察荧光。用胰酶消化细胞，传至新的六孔板，分别用嘌呤霉素对单独表达PD-1单链抗体的RAW 264.7细胞进行筛选。经过3-4次传代和筛选后，可筛选出稳定表达PD-1单链抗体的RAW264.7细胞系，大量扩增和冻存。提前一天将稳定表达PD-1单链抗体的RAW 264.7细胞系铺板于六孔板，密度为50％。第二天，换上含有终浓度为8μg/mL Polybrene的500μL新鲜培养基，加入一份PD-1单链抗体的病毒，培养72h。观察荧光后，用胰酶对细胞进行传代处理，同时用嘌呤霉素对细胞进行筛选，反复传代和筛选后，可筛选出稳定过表达PD-1单链抗体的RAW 264.7细胞系，大量扩增细胞和冻存。
1.4激光共聚焦显微镜验证PD-1单链抗体的表达
具体实验方法：用嘌呤霉素对病毒感染后的RAW 264.7细胞系进行筛选后，将细胞铺板于激光共聚焦培养皿，密度约为60％。培养过夜后，吸掉培养基，用PBS洗一次，然后用4％多聚甲醛室温固定15min，然后用PBS洗一次，加入1mL 1μg/mL的DAPI染液，室温避光孵育10min，PBS洗三次，每次5min。最后加20μL抗荧光淬灭封片剂，上机拍片。
1.5Western Blot验证PD-1单链抗体表达
具体实验方法：将空白对照、单独表达EGFP的RAW 264.7细胞、表达PD-1单链抗体的RAW 264.7细胞铺板于六孔板，密度高达90％以上。第二天，在冰上用细胞刮对细胞进行收样，4℃，3500rpm，10min。然后用PBS洗两次，用150μL加了蛋白酶抑制剂的RIPA裂解液重悬细胞，超声波细胞破碎仪进行细胞破碎，冰上，25％功率，超声3s后暂停3s，重复4次。将超声后的内容物进行离心，4℃，12000g，10min，取上清。然后用BCA蛋白浓度测定试剂盒对各组蛋白浓度进行检测，步骤按照说明书进行。测完浓度后，每组取120μg，用RIPA裂解液补充体积至150μL，加入37.5μL 5×SDS-PAGE蛋白上样缓冲液，混匀后，100℃，加热10min，暂时保存在-20℃。然后，进行Western blot步骤：
a.配制浓度为10％的SDS-PAGE凝胶，每孔上样品20μL，电压70V，进行电泳；b.预先用甲醇活化PVDF膜，按黑板-海绵-滤纸-胶-PVDF膜-滤纸-海绵-白板制作转膜“三明治”。将转膜槽置于冰中，恒流转膜，250mA，2h；c.配5％脱脂奶粉，室温封闭PVDF膜1h；d.剪膜，分别孵EGFP抗体和β-actin抗体，4℃，慢速摇床过夜；e.在摇床上用TBST洗膜三次，每次10min。室温孵育二抗，1h；f.在摇床上用TBST洗膜三次，每次10min；g.按1：1比例配置ECL发光液，机器曝光。
实验结果如图3所示，共聚焦显微镜、流式细胞术表明巨噬细胞RAW 264.7中EGFP-aPD-1-scFv融合蛋白正确表达。Western blot结果显示EGFP-aPD-1-scFv融合蛋白约为58kDa，证明抗体正确显示在NVs上。
1.6细胞膜囊泡制作和表征
具体实验方法：用15cm细胞培养皿大量培养细胞，然后用细胞刮收集细胞，4℃离心，800rpm，收集细胞沉淀。用含蛋白酶抑制剂混合物的PBS洗两次细胞，用3-4mL的含蛋白酶抑制剂混合物的PBS重悬细胞。将玻璃匀浆器用酒精和紫外消毒后，置于冰上，加3-4mL细胞重悬液，手动匀浆200次。收集匀浆液后，4℃离心，1000g，5min，取上清液，去沉淀。上清液在4℃条件下，3000g，离心5min。再次取上清液，去沉淀，在14800g的条件下，4℃离心40min。最后去上清，取沉淀，用含蛋白酶抑制剂混合物的PBS重悬沉淀，依次过0.8μm滤头八次和0.22μm滤头八次，即得细胞膜囊泡。用纳米粒度和Zeta分析仪检测细胞膜囊泡的粒径大小和zeta电位。
实验结果如图4所示，动态光散射(DLS)分析表明，NVs的平均直径约为110nm。NVs的zeta电位在-10mV左右。
1.7透射电子显微镜观察样品
具体实验方法：用镊子将铜网夹住后，加燕尾夹固定，防止铜网掉落。将镊子用胶带固定在冰上，滴加10μL细胞膜囊泡溶液，静置5min，用滤纸从铜网边缘吸走液体，反复滴加六次，然后滴加10μL 3％醋酸铀染色5min，用滤纸从边缘吸走液体。最后室温晾干铜网，用120kV透射电子显微镜观察囊泡的形貌和拍照。实验过程中与醋酸铀相关的枪头和滤纸等均要按照安全准则处理。
实验结果如图4所示，透射电子显微镜(TEM)表征了负染NVs的形态，发现NVs具有与外泌体相似的结构。
1.8细胞膜囊泡浓度测定
具体实验方法：采用碧云天的BCA蛋白浓度测定试剂盒对细胞膜囊泡浓度进行测定。将0.5mg/mL的BSA蛋白标准品梯度稀释至0、0.025、0.05、0.1、0.2、0.3、0.4和0.5mg/mL，细胞膜囊泡与PBS 2：18稀释10倍。取各浓度标准品与待测样品20μL，分别与200μL BCA工作液涡旋混匀，然后吸200μL混合物至酶标板，套上PE薄膜手套，在37℃的培养箱孵育30min。用酶标仪的A562吸光度对标准品和样品进行读数检测。根据检测结果，用标准品的读数制作浓度-读数标准曲线，求出公式和R平方值，代入待测样品的读数，从而求出稀释后的样品浓度。
1.9细胞膜囊泡激光共聚焦显微镜观察
具体实验方法：将稳定表达PD-1单链抗体的RAW 264.7细胞制成的细胞膜囊泡，滴加10μL在玻片上，盖上盖玻片，四周加指甲油封片，室温避光晾片。然后在激光共聚焦显微镜下，观察囊泡的荧光情况。
实验结果如图5所示，通过共聚焦显微镜观察到EGFP信号在NVs上显示。
实施例二巨噬细胞膜纳米囊泡的体外生物活性
2.1细胞膜囊泡结合细胞实验
具体实验方法：按照实施例一1.6的方法，将制好的细胞膜囊泡在生物安全柜中经0.22μm滤头过滤，去除杂菌。将Cl.ly1+2-/9细胞用1640培养基重悬在激光共聚焦显微镜专用的培养皿中，密度约为40-50％，加入80μg细胞膜囊泡，水平混匀。在细胞培养箱培养6h后，取出培养皿，然后加入Hoechst染液染色细胞核。在激光共聚焦显微镜下，观察囊泡和细胞的共定位情况。
实验结果如图6所示，在包括CI.ly1+2-/9和CTLL-2在内的两种小鼠T细胞上检测PD-1的表达。PD-1在CI.ly1+2-/9细胞上表达，而CTLL-2细胞为阴性。接下来，进行细胞结合试验，发现aPD-1-scFv NVs可以与CI.ly1+2-/9细胞上的PD-1结合。相比之下，aPD-1-scFvNVs与CTLL-2细胞的结合能力较差，CTLL-2细胞可能缺乏PD-1受体。为了进一步确认结合是否依赖于PD-1受体，CI.ly1+2-/9细胞与游离PD-1单克隆抗体孵育4h，然后与aPD-1-scFvNVs孵育。结果显示，aPD-1-scFv NVs的结合被游离的PD-1抗体明显阻断，而CTLL-2细胞摄取了一些NVs，而不是与它们结合。此外，NHS-Cy5.5标记的无aPD-1-scFv的游离NVs不能与CI.ly1+2-/9细胞结合。
2.2细胞膜囊泡在小鼠血液中的血液动力学研究
具体实验方法：采用BCA试剂盒测细胞膜囊泡蛋白浓度，向细胞膜囊泡加入NHS-Cy5.5溶液，室温在摇床上孵育40min，在14800g的条件下，4℃离心40min，去上清。用PBS洗细胞膜囊泡两次，最后用PBS重悬，每只老鼠尾静脉注射500μg/150μL，分别在2min、30min、1h、2h、4h、12h、24h、48h用抗凝管眼眶取血，然后室温静置2h，3000rpm离心10min，取上清。将上清吸到酶标板，使用多功能酶标仪，在670nm波长处检测荧光度值。
实验结果如图7所示，aPD-1-scFv NVs的血液潴留比游离NVs略高。在注射后24h和48h,aPD-1-scFv NVs的保留率分别为13.2％和6.2％，高于游离NVs的7.2％和2.9％。
2.3细胞膜囊泡在小鼠器官中的分布研究
具体实验方法：小鼠左腹部脱毛，每只小鼠皮下注射106个B16-F10-Luciferase细胞。五天后，给成瘤小鼠尾静脉注射NHS-Cy5.5染料标记的细胞膜囊泡，然后分别在2h、6h、12h、24h和48h颈椎脱臼处死小鼠，解剖小鼠，取心、肝、脾、肺、肾和肿瘤，使用小动物活体成像仪，选择Cy5.5成像体系，检测各脏器细胞膜囊泡含量水平。
实验结果如图8所示，aPD-1-scFv NVs和空白NVs相似，NHS-Cy5.5标记的NVs信号首先在12h检测，最后在48h检测，肺最后得到了很好的Cy5.5信号检测。
实施例三PD-1单链抗体细胞膜囊泡治疗小鼠肿瘤模型
3.1小鼠黑色素瘤模型治疗实验
具体实验方法：提前一天将6-8w龄的C57BL/6小鼠腹部脱毛，第二天皮下注射106个鼠源黑色素瘤细胞B16-F10-Luciferase。成瘤后，将小鼠随机分成7组，接受不同的治疗：1组：PBS(150μl)，2组：Free NVs(25mg/kg)，3组：CPI-444(10mg/kg,溶解于含有20％β-环糊精的生理盐水中)，4组：CPI-444-loaded free NVs(25mg/kg)，5组：aPD-1antibody(Biolegend,114115,2.5mg/kg)，6组：aPD-1-scFv NVs(25mg/kg)，7组：CPI-444-loadedaPD-1-scFv NVs(25mg/kg)。给药频率均为每三天一次，共给药四次。每三天测量小鼠的体重和肿瘤的长与宽，按照公式：体积＝长径*短径*短径*1/2计算肿瘤体积。当小鼠体积超过1500mm3时，根据伦理要求，处死动物，记录生存期。实验给药方案流程图如图9所示。
实验结果如图10、11所示，用aPD-1-scFv NVs治疗的小鼠的肿瘤发育明显延迟。相比之下，分别用游离NVs、CPI-444和CPI-444游离NVs处理的小鼠，肿瘤发展迅速，与PBS处理的小鼠相似。同时，接受aPD-1或CPI-444-aPD-1-scFv NVs治疗的小鼠比接受aPD-1-scFvNVs治疗的小鼠有更好的抑制肿瘤生长的治疗效果。分别用PBS、游离NVs、CPI-444和CPI-444游离NVs处理的小鼠均在肿瘤接种后26天内死亡。相比而言，20％的小鼠接受aPD-1-scFv NVs治疗，30％的小鼠接受aPD-1和CPI-444-aPD-1-scFv NVs治疗，存活超过45天，存活率显著提高。此外，小鼠在治疗期间体重没有明显下降，这表明没有明显的毒性发生。
3.2流式检测小鼠肿瘤免疫细胞和细胞因子
具体实验方法：在3.1小鼠给药结束后2-4天，采用流式细胞术检测肿瘤处的免疫相关细胞含量和细胞因子水平。颈椎脱臼处死小鼠后，剥离小鼠腹部皮下肿瘤，用PBS洗两次，去除血丝。用滤纸吸干后，称重肿瘤，做好记录。称重后，将肿瘤置于70μm细胞筛网上，筛网下接着50mL离心管，全程冰上操作。加入含有2％FBS的PBS润湿后，轻轻研磨，过程不断加入含有2％FBS的PBS冲洗，得到单细胞悬液。将单细胞悬液置在4℃条件下，350g离心5min，去上清，然后加入2％FBS的PBS洗两次，用500μL 2％FBS的PBS重悬，分到1.5mL EP管中，进行下游实验。流式检测步骤如下：(1)细胞表面分子检测：a.用2％FBS的PBS重悬每管细胞体积为30-50μL，加入流式抗体，4℃孵育30min。b.染色结束后，用2％FBS的PBS洗两次，加500μL 2％FBS的PBS重悬细胞，尽快上机检测。(2)细胞胞内分泌细胞因子检测：a.将肿瘤悬液接种于含有500μL 1640培养液的48孔板，加入终浓度为50ng/mL PMA和1μg/mL离子霉素，细胞培养箱培养1h后，加入终浓度为1-50μM brefeldin A，继续培养3-6h。b.去掉培养基后，用PBS洗两次。用2％FBS的PBS重悬每管细胞体积为30-50μL，加入表面分子流式抗体，4℃孵育30min。c.染色结束后，用2％FBS的PBS洗两次，用4％多聚甲醛室温避光孵育固定20min。d.4℃，350g，离心5min，去上清，加2％FBS的PBS洗两次。e.加0.2％Trixton-x100对固定后的细胞进行破膜，室温孵育20min，350g离心5min。f.用30-50μL 0.01％Trixton-x100重悬每管细胞，加入胞内流式抗体，4℃孵育30min。g.4℃，350g，离心5min，去上清，用2％FBS的PBS洗两次。h.500μL 2％FBS的PBS重悬固定染色后的细胞，尽快上机检测。
实验结果如图12所示，我们分析了不同处理小鼠肿瘤浸润CD8+T细胞的数量。接收到aPD-1-scFv NVs和CPI-444-aPD-1-scFv NVs治疗的小鼠的CD8+T细胞比例显著增加。4-1BB被鉴定为共刺激分子，主要表达于NK细胞和活性T细胞上，是T细胞活化和增殖的标志。流式细胞术结果显示，CPI-444-aPD-1-scFv NVs处理小鼠后，激活的4-1BB+CD8+T细胞显著增加。颗粒酶B(Gzm B)和穿孔素(Perforin)是CD8+T细胞产生的杀伤肿瘤细胞的关键分子，采用流式细胞术检测两种细胞因子。结果显示，与PBS组相比，CPI-444-aPD-1-scFv NVs组小鼠的Gzm B+CD8+T细胞和Perforin+CD8+T细胞增加更为密集。结果显示，CPI-444与aPD-1-scFv NVs的联合治疗可以更好地降低调节性T细胞比例。免疫荧光染色检测肿瘤组织中浸润肿瘤的CD8+T细胞数量。我们发现，经aPD-1-scFv NVs处理的小鼠可显著促进CD8+T细胞向肿瘤浸润。
3.3免疫荧光检测小鼠肿瘤T细胞含量
具体实验方法：剥离小鼠肿瘤后，用PBS洗两次，滤纸吸干水分，用OCT包埋后，置于-20℃冰箱冻到OCT凝固。将冷冻切片机预冷，切片厚度调为6μm，进行切片。将切好的片子室温放置1-2h，再进行下游操作，防止脱片。将恢复到室温的切片在PBS中浸泡15min，去除OCT。用滤纸吸走大部分水分，用免疫组化笔离肿瘤组织远一点画圈，避免边缘效应。加入100μL 3％BSA配制的0.2％Trixton-x100室温孵育30min，吸走液体后，加入CD8的一抗，放于湿盒中，4℃过夜。第二天，回收一抗，用PBS浸泡，在摇床上洗三次，每次10min。用滤纸吸走液体后，加入荧光二抗液，室温避光孵育1h。吸走二抗后，用PBS浸泡，在摇床上洗三次，每次10min。用滤纸吸走大部分水分后，加入终浓度为1μg/mL的DAPI染液，室温避光孵育15min。吸走染液后，用PBS浸泡，在摇床上洗三次，每次10min。吸走水分后，滴加防荧光淬灭剂封片，盖上盖玻片，用指甲油固定盖玻片四周。尽快用激光共聚焦显微镜观察和拍片。
3.4小鼠脏器HE染色
具体实验方法：颈椎脱臼处死小鼠后，取小鼠的心、肝、脾、肺和肾，用PBS洗两次，然后用滤纸吸走水分，用4％多聚甲醛在4℃条件下固定24-72h，然后用75％乙醇脱水组织，石蜡包埋。将石蜡切片机调节切片厚度至5μm，进行切片。将切片浸泡在二甲苯中脱蜡5min，重复3次，依次浸泡两次无水乙醇5min，95％乙醇5min,80％乙醇5min，70％乙醇5min，蒸馏水2min，梯度水化。然后将切片放入苏木素工作液中染色5min,再用自来水洗，用1％的盐酸酒精分化数秒钟，自来水中止分化。将切片浸泡伊红染液1min，自来水洗。依次用70％乙醇、80％乙醇、95％乙醇、两次无水乙醇脱水，每次5min，用二甲苯透化5min，重复两次，最后用中性树胶封片，显微镜拍片。
实验结果如图13所示，采集不同处理组小鼠的主要脏器，进行HE染色，显示各组小鼠主要脏器未见损伤。
最后应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
Claims (7)
1.一种巨噬细胞膜纳米囊泡，其特征在于，所述巨噬细胞膜过表达PD-1单链可变片段抗体，并负载腺苷受体拮抗剂；所述腺苷受体拮抗剂为A2a腺苷受体拮抗剂；所述A2a腺苷受体拮抗剂为CPI-444。
2.根据权利要求1所述的巨噬细胞膜纳米囊泡，其特征在于，所述巨噬细胞为RAW264.7细胞系。
3.根据权利要求1所述的巨噬细胞膜纳米囊泡，其特征在于，所述CPI-444的载药率为15.2%。
4.根据权利要求1~3任一项所述的巨噬细胞膜纳米囊泡的制备方法，其特征在于，包括以下步骤：
（1）构建和扩增 PD-1单链可变片段抗体慢病毒载体质粒；
（2）使用步骤（1）的质粒进行慢病毒包装，得PD-1单链抗体病毒；
（3）构建稳定过表达PD-1单链可变片段抗体的巨噬细胞；
（4）扩增培养步骤（3）的巨噬细胞，离心收集细胞，用含蛋白酶抑制剂的PBS溶液重悬细胞，经过滤得过表达PD-1单链可变片段抗体的巨噬细胞膜纳米囊泡；
（5）将腺苷受体拮抗剂负载到过表达PD-1单链可变片段抗体的巨噬细胞膜纳米囊泡，得所述巨噬细胞膜纳米囊泡。
5.根据权利要求1~3任一项所述的巨噬细胞膜纳米囊泡在制备免疫检查点抑制剂中的应用。
6.根据权利要求1~3任一项所述的巨噬细胞膜纳米囊泡在制备靶向肿瘤细胞治疗药物中的应用；所述肿瘤细胞为黑色素瘤细胞。
7.根据权利要求6所述应用，其特征在于，所述巨噬细胞膜纳米囊泡的浓度为25 mg/kg体重。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210460518.8A CN114748448B (zh) | 2022-04-28 | 2022-04-28 | 一种巨噬细胞膜纳米囊泡的制备方法与应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210460518.8A CN114748448B (zh) | 2022-04-28 | 2022-04-28 | 一种巨噬细胞膜纳米囊泡的制备方法与应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114748448A CN114748448A (zh) | 2022-07-15 |
| CN114748448B true CN114748448B (zh) | 2023-10-24 |
Family
ID=82332484
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210460518.8A Active CN114748448B (zh) | 2022-04-28 | 2022-04-28 | 一种巨噬细胞膜纳米囊泡的制备方法与应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114748448B (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115501334B (zh) * | 2022-08-15 | 2023-09-19 | 中山大学附属第五医院 | 一种负载艾拉莫德-铑纳米颗粒的pd-1纳米囊泡及其制备方法和应用 |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018187508A1 (en) * | 2017-04-04 | 2018-10-11 | Corvus Pharmaceuticals, Inc. | Methods of treating cancer |
| CN110215514A (zh) * | 2019-04-22 | 2019-09-10 | 上海瑞可新生物科技有限公司 | 一种基因工程化细胞膜纳米囊泡及其制备与应用 |
| CN110446489A (zh) * | 2017-03-20 | 2019-11-12 | 南加利福尼亚大学 | 具有表面缀合的载药纳米颗粒的car t细胞的过继转移及其用途 |
| CN110974964A (zh) * | 2019-12-25 | 2020-04-10 | 中南大学湘雅医院 | Dpcpx抑制剂在制备pd-l1/pd-1单抗肿瘤免疫治疗药物中的应用 |
| CN111920769A (zh) * | 2020-06-29 | 2020-11-13 | 中山大学·深圳 | 一种包裹免疫抑制剂并过表达pd-l1的细胞膜纳米囊泡及其制备方法和应用 |
| CN113769106A (zh) * | 2021-08-13 | 2021-12-10 | 深圳湾实验室 | 用于肿瘤免疫治疗的融合细胞膜纳米囊泡、其制备方法及应用 |
| CN114366821A (zh) * | 2022-02-09 | 2022-04-19 | 台州学院 | 一种表达受体蛋白的细胞膜纳米囊泡及其制备方法和应用 |
-
2022
- 2022-04-28 CN CN202210460518.8A patent/CN114748448B/zh active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110446489A (zh) * | 2017-03-20 | 2019-11-12 | 南加利福尼亚大学 | 具有表面缀合的载药纳米颗粒的car t细胞的过继转移及其用途 |
| WO2018187508A1 (en) * | 2017-04-04 | 2018-10-11 | Corvus Pharmaceuticals, Inc. | Methods of treating cancer |
| CN110215514A (zh) * | 2019-04-22 | 2019-09-10 | 上海瑞可新生物科技有限公司 | 一种基因工程化细胞膜纳米囊泡及其制备与应用 |
| CN110974964A (zh) * | 2019-12-25 | 2020-04-10 | 中南大学湘雅医院 | Dpcpx抑制剂在制备pd-l1/pd-1单抗肿瘤免疫治疗药物中的应用 |
| CN111920769A (zh) * | 2020-06-29 | 2020-11-13 | 中山大学·深圳 | 一种包裹免疫抑制剂并过表达pd-l1的细胞膜纳米囊泡及其制备方法和应用 |
| CN113769106A (zh) * | 2021-08-13 | 2021-12-10 | 深圳湾实验室 | 用于肿瘤免疫治疗的融合细胞膜纳米囊泡、其制备方法及应用 |
| CN114366821A (zh) * | 2022-02-09 | 2022-04-19 | 台州学院 | 一种表达受体蛋白的细胞膜纳米囊泡及其制备方法和应用 |
Non-Patent Citations (1)
| Title |
|---|
| Roberta Turiello et al.Exosomal CD73 from serum of patients with melanoma suppresses lymphocyte functions and is associated with therapy resistance to anti-PD- 1 agents.《Journal for Immunotherapy of Cancer》.2022,第10卷第e004043页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114748448A (zh) | 2022-07-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Luo et al. | Extracellular vesicles from Akkermansia muciniphila elicit antitumor immunity against prostate cancer via modulation of CD8+ T cells and macrophages | |
| CN111920769B (zh) | 一种包裹免疫抑制剂并过表达pd-l1的细胞膜纳米囊泡及其制备方法和应用 | |
| He et al. | Folate-modified chitosan nanoparticles containing the IP-10 gene enhance melanoma-specific cytotoxic CD8+ CD28+ T lymphocyte responses | |
| CN110172479B (zh) | 能同时表达lmp1和cd30双靶点car的质粒、car-t细胞、构建方法及其应用 | |
| CN114748448B (zh) | 一种巨噬细胞膜纳米囊泡的制备方法与应用 | |
| CN108289942A (zh) | 用生产免疫调节剂的免疫-分离的细胞接种疫苗 | |
| CN114404571A (zh) | 一种装载化疗药物且tigit过表达的工程化载药细胞膜囊泡以及制备方法和应用 | |
| CN111905105B (zh) | 一种用于癌症靶向治疗的蛋白类纳米药物及其制备方法 | |
| CN113621077B (zh) | 一种tim-3/cd28融合蛋白及所述融合蛋白修饰的car-t细胞 | |
| Wang et al. | Genetically Engineered Hematopoietic Stem Cells Deliver TGF‐β Inhibitor to Enhance Bone Metastases Immunotherapy | |
| JP2024500748A (ja) | 腫瘍浸潤リンパ球の処理 | |
| CN109745326B (zh) | 一种包含吉非替尼和组蛋白去乙酰酶抑制剂的药物组合物，其脂质体制剂及其制药用途 | |
| CN114835781A (zh) | Wtn多肽及其在检测、治疗癌症中的用途 | |
| Lyu et al. | Roles of exosomes in immunotherapy for solid cancers | |
| CN114712496B (zh) | 一种展示新抗原的细菌衍生外膜囊泡疫苗及制备方法和在制备癌症免疫治疗试剂盒中的应用 | |
| CN117210411A (zh) | 一种免疫细胞及其表达载体、应用和制备方法 | |
| CN114796483B (zh) | 活化t细胞与阻断性抗体联合制备抗肿瘤药物的用途及抗肿瘤药物 | |
| Sun et al. | Impact of synchronized anti-PD-1 with Ad-CEA vaccination on inhibition of colon cancer growth | |
| CN114908054B (zh) | 一种细胞膜囊泡及其制备方法与应用 | |
| CN114588274A (zh) | 一种负载cRGD和小分子抗肿瘤药物的复合外泌体及其制备方法和应用 | |
| CN112725283B (zh) | 靶向cd30和cd24的双靶点car-t细胞的构建方法及应用 | |
| CN114191556A (zh) | 敲降rbms1的试剂在制备治疗三阴性乳腺癌的药物中的应用 | |
| CN114788877B (zh) | 一种基因工程化造血干细胞给药系统及其制备方法和应用 | |
| KR100569609B1 (ko) | 세포면역치료제의 제조를 위한 림프구 장기 보관 방법 | |
| CN116179606B (zh) | 转录因子tcf1在抵抗car-t细胞耗竭和终末分化中的应用 |
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 |