CN112522203B - Cell vesicle for expressing chimeric antigen receptor, and preparation method and application thereof - Google Patents
Cell vesicle for expressing chimeric antigen receptor, and preparation method and application thereof Download PDFInfo
- Publication number
- CN112522203B CN112522203B CN202011324758.2A CN202011324758A CN112522203B CN 112522203 B CN112522203 B CN 112522203B CN 202011324758 A CN202011324758 A CN 202011324758A CN 112522203 B CN112522203 B CN 112522203B
- Authority
- CN
- China
- Prior art keywords
- cell
- cells
- sars
- cov
- expressing
- 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
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 title claims abstract description 23
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 210000004027 cell Anatomy 0.000 claims abstract description 152
- 239000003814 drug Substances 0.000 claims abstract description 31
- 238000000034 method Methods 0.000 claims abstract description 19
- 239000012634 fragment Substances 0.000 claims abstract description 16
- 210000000170 cell membrane Anatomy 0.000 claims abstract description 8
- 241001678559 COVID-19 virus Species 0.000 claims description 40
- 239000013612 plasmid Substances 0.000 claims description 31
- 241000700605 Viruses Species 0.000 claims description 27
- 229940079593 drug Drugs 0.000 claims description 12
- 108090000623 proteins and genes Proteins 0.000 claims description 12
- 238000004806 packaging method and process Methods 0.000 claims description 9
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 6
- 238000004520 electroporation Methods 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 5
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 claims description 3
- 208000001528 Coronaviridae Infections Diseases 0.000 claims description 3
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 238000010367 cloning Methods 0.000 claims description 2
- 238000001125 extrusion Methods 0.000 claims description 2
- 210000000822 natural killer cell Anatomy 0.000 claims description 2
- 241000711573 Coronaviridae Species 0.000 abstract description 23
- 230000000694 effects Effects 0.000 abstract description 9
- 231100000331 toxic Toxicity 0.000 abstract description 5
- 230000002588 toxic effect Effects 0.000 abstract description 5
- 238000005516 engineering process Methods 0.000 abstract description 3
- 230000002829 reductive effect Effects 0.000 abstract description 3
- 230000009385 viral infection Effects 0.000 abstract description 3
- 208000036142 Viral infection Diseases 0.000 abstract description 2
- 230000002776 aggregation Effects 0.000 abstract description 2
- 238000004220 aggregation Methods 0.000 abstract description 2
- 230000009545 invasion Effects 0.000 abstract description 2
- 239000002609 medium Substances 0.000 description 16
- 241000713666 Lentivirus Species 0.000 description 14
- 241001112090 Pseudovirus Species 0.000 description 12
- 230000003472 neutralizing effect Effects 0.000 description 12
- 102100031673 Corneodesmosin Human genes 0.000 description 11
- 101710139375 Corneodesmosin Proteins 0.000 description 11
- 239000000203 mixture Substances 0.000 description 11
- 238000010790 dilution Methods 0.000 description 9
- 239000012895 dilution Substances 0.000 description 9
- 210000001808 exosome Anatomy 0.000 description 9
- 108060001084 Luciferase Proteins 0.000 description 8
- 238000003306 harvesting Methods 0.000 description 8
- 239000005089 Luciferase Substances 0.000 description 7
- 239000004698 Polyethylene Substances 0.000 description 7
- 229960001997 adefovir Drugs 0.000 description 7
- WOZSCQDILHKSGG-UHFFFAOYSA-N adefovir depivoxil Chemical compound N1=CN=C2N(CCOCP(=O)(OCOC(=O)C(C)(C)C)OCOC(=O)C(C)(C)C)C=NC2=C1N WOZSCQDILHKSGG-UHFFFAOYSA-N 0.000 description 7
- -1 polyethylene Polymers 0.000 description 7
- 229920000573 polyethylene Polymers 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 108090000975 Angiotensin-converting enzyme 2 Proteins 0.000 description 6
- 239000008188 pellet Substances 0.000 description 6
- 102000053723 Angiotensin-converting enzyme 2 Human genes 0.000 description 5
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 238000006386 neutralization reaction Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000003860 storage Methods 0.000 description 5
- 208000015181 infectious disease Diseases 0.000 description 4
- 230000002147 killing effect Effects 0.000 description 4
- 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 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 239000012124 Opti-MEM Substances 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- XXSMGPRMXLTPCZ-UHFFFAOYSA-N hydroxychloroquine Chemical compound ClC1=CC=C2C(NC(C)CCCN(CCO)CC)=CC=NC2=C1 XXSMGPRMXLTPCZ-UHFFFAOYSA-N 0.000 description 3
- 229960004171 hydroxychloroquine Drugs 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- 238000004448 titration Methods 0.000 description 3
- 238000010361 transduction Methods 0.000 description 3
- 230000026683 transduction Effects 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- WHTVZRBIWZFKQO-AWEZNQCLSA-N (S)-chloroquine Chemical compound ClC1=CC=C2C(N[C@@H](C)CCCN(CC)CC)=CC=NC2=C1 WHTVZRBIWZFKQO-AWEZNQCLSA-N 0.000 description 2
- 208000025721 COVID-19 Diseases 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- OFFWOVJBSQMVPI-RMLGOCCBSA-N Kaletra Chemical compound N1([C@@H](C(C)C)C(=O)N[C@H](C[C@H](O)[C@H](CC=2C=CC=CC=2)NC(=O)COC=2C(=CC=CC=2C)C)CC=2C=CC=CC=2)CCCNC1=O.N([C@@H](C(C)C)C(=O)N[C@H](C[C@H](O)[C@H](CC=1C=CC=CC=1)NC(=O)OCC=1SC=NC=1)CC=1C=CC=CC=1)C(=O)N(C)CC1=CSC(C(C)C)=N1 OFFWOVJBSQMVPI-RMLGOCCBSA-N 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- 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 2
- 201000003176 Severe Acute Respiratory Syndrome Diseases 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 229960003677 chloroquine Drugs 0.000 description 2
- WHTVZRBIWZFKQO-UHFFFAOYSA-N chloroquine Natural products ClC1=CC=C2C(NC(C)CCCN(CC)CC)=CC=NC2=C1 WHTVZRBIWZFKQO-UHFFFAOYSA-N 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 239000012499 inoculation medium Substances 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 229940113983 lopinavir / ritonavir Drugs 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 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 2
- 238000000926 separation method Methods 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
- 238000005199 ultracentrifugation Methods 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 206010001052 Acute respiratory distress syndrome Diseases 0.000 description 1
- 108010083359 Antigen Receptors Proteins 0.000 description 1
- 102000006306 Antigen Receptors Human genes 0.000 description 1
- 238000003737 Bright-Glo Luciferase Assay System Methods 0.000 description 1
- 229940022962 COVID-19 vaccine Drugs 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 208000000059 Dyspnea Diseases 0.000 description 1
- 206010013975 Dyspnoeas Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 102000003996 Interferon-beta Human genes 0.000 description 1
- 108090000467 Interferon-beta Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 208000034486 Multi-organ failure Diseases 0.000 description 1
- 208000010718 Multiple Organ Failure Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- 239000004695 Polyether sulfone Substances 0.000 description 1
- 229940096437 Protein S Drugs 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 241000315672 SARS coronavirus Species 0.000 description 1
- 206010040070 Septic Shock Diseases 0.000 description 1
- 101710198474 Spike protein Proteins 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 206010052015 cytokine release syndrome Diseases 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000008260 defense mechanism Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007865 diluting Methods 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
- 238000001493 electron microscopy Methods 0.000 description 1
- 238000013504 emergency use authorization Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 229960004396 famciclovir Drugs 0.000 description 1
- GGXKWVWZWMLJEH-UHFFFAOYSA-N famcyclovir Chemical compound N1=C(N)N=C2N(CCC(COC(=O)C)COC(C)=O)C=NC2=C1 GGXKWVWZWMLJEH-UHFFFAOYSA-N 0.000 description 1
- 206010016256 fatigue Diseases 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 229960001388 interferon-beta Drugs 0.000 description 1
- 208000021601 lentivirus infection Diseases 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 238000003801 milling Methods 0.000 description 1
- 208000029744 multiple organ dysfunction syndrome Diseases 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 229920000729 poly(L-lysine) polymer Polymers 0.000 description 1
- 229920006393 polyether sulfone Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 229950010131 puromycin Drugs 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000008786 sensory perception of smell Effects 0.000 description 1
- 230000014860 sensory perception of taste Effects 0.000 description 1
- 230000036303 septic shock Effects 0.000 description 1
- 208000013220 shortness of breath Diseases 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
-
- 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
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4706—4-Aminoquinolines; 8-Aminoquinolines, e.g. chloroquine, primaquine
-
- 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
- A61K31/66—Phosphorus compounds
- A61K31/683—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
- A61K31/685—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols one of the hydroxy compounds having nitrogen atoms, e.g. phosphatidylserine, lecithin
-
- 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
- A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/7051—T-cell receptor (TcR)-CD3 complex
-
- 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
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- 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
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
-
- 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
- C12N2740/15021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Epidemiology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Mycology (AREA)
- Botany (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Toxicology (AREA)
- Plant Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Pulmonology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of biological medicine, and discloses a cell vesicle for expressing chimeric antigen receptor, a preparation method and application thereof. The single-chain variable fragment aiming at the novel coronavirus is expressed on a cell membrane, then cell vesicles containing the chimeric antigen receptor are directly collected or squeezed, and finally the anti-novel coronavirus such as the Ruidexivir and the like is loaded on the cell vesicles for treating patients infected by the novel coronavirus. The cell vesicles prepared by the invention can specifically neutralize the new coronavirus and block the invasion of the new coronavirus to cells with high expression of ACE 2; meanwhile, the anti-novel coronavirus medicaments such as the Ruidexivir and the like can be transported to the aggregation part of the novel coronavirus in a targeted manner, so that the toxic and side effects of the medicaments are reduced. Moreover, the treatment method of the invention is universal and can be used as a platform technology for treating other viral infections.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to a cell vesicle for expressing a chimeric antigen receptor, a preparation method and application thereof.
Background
Covd-19 is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Common symptoms include fever, cough, fatigue, shortness of breath, and loss of sense of smell and taste. While most people are less symptomatic, some people may develop Acute Respiratory Distress Syndrome (ARDS) due to cytokine storms, multiple organ failure, septic shock and blood clots. By 8 months and 18 days in 2020, over 2180 thousands of cases were reported in 188 countries and regions, resulting in 773,000 deaths.
In response to the outbreak of covd-19, global researchers have conducted active research on covd-19, but no drugs or vaccines with specific indications have been approved to treat the disease. Vaccines are considered to be the best method for completely eliminating SARS-COV-2, but there is currently no very reliable SARS-COV-2 vaccine, so it is more current to use some drugs to reduce the pain of patients as much as possible.
Drugs for use in the treatment of covd-19 include famciclovir, chloroquine, hydroxychloroquine, lopinavir/ritonavir and lopinavir/ritonavir in combination with interferon beta. Ramexivir was initially shown to be therapeutic and the United states Food and Drug Administration (FDA) granted emergency use authorization for severely COVID-19 hospitalized patients at day 5 and 1 of 2020. By 3 months 4 of 2020, results on hydroxychloroquine as a therapeutic effect of covd-19 are good or bad, and some studies indicate little or no improvement.
Neutralizing antibodies are currently considered one of the most promising therapeutic approaches. The main defense mechanism of neutralizing antibodies is to make it difficult for new coronaviruses to invade human cells by neutralization. The spike protein of SARS-CoV-2 is the primary target of neutralizing antibodies. It has been proposed that the selection of broadly neutralizing antibodies against SARS-CoV-2 and SARS-CoV can be used not only to treat COVID-19, but also to treat future SARS-associated viral infections. However, in the case of treatment with monoclonal antibodies, some side effects may also be caused by the cytotoxicity and/or phagocytosis of antibody-dependent cells.
Disclosure of Invention
In order to overcome the possible antibody enhancement effect and escape caused by virus mutation when SARS-CoV-2 monoclonal antibody is used and the toxic and side effects of medicine such as Ruidexivir for SARS-CoV-2 is used, the primary purpose of the invention is to provide a preparation method of cell vesicles expressing chimeric antigen receptor.
It is a second object of the present invention to provide a cell vesicle obtainable by the above method.
A third object of the present invention is to provide the use of the above cell vesicles for the preparation of a medicament for combating a novel coronavirus pneumonic infection.
The aim of the invention is achieved by the following technical scheme:
a method of preparing a cell vesicle expressing a chimeric antigen receptor, comprising the steps of:
s1, preparing a plasmid containing a single-chain variable fragment gene sequence aiming at SARS-CoV-2:
cloning a nucleotide sequence capable of expressing a single-stranded variable fragment for SARS-CoV-2 into a plasmid containing pCDH-CMV-MCS-EF1-copGFP of CD19-CAR, replacing the anti-CD 19scFv in CD19 CAR to obtain a plasmid containing a single-stranded variable fragment gene sequence for SARS-CoV-2;
s2, packaging the slow virus containing the single-chain variable fragment gene sequence aiming at SARS-CoV-2 by using a three-plasmid system or a four-plasmid system;
s3, infecting target cells by slow viruses, wherein the cell surface expression of the target cells contains single-chain variable fragments aiming at SARS-CoV-2;
s4, separating target cell membranes;
s5, carrying out ultrasonic centrifugation or extrusion filtration on the cell membrane prepared in the step S4 to obtain nano vesicles;
s6, mixing the medicine for SARS-CoV-2 with the cell vesicle, and preparing the cell vesicle for expressing the chimeric antigen receptor by adopting an electroporation mode.
Preferably, the single-stranded variable fragment gene sequence for SARS-CoV-2 includes, but is not limited to, 4A8, B38, H4, CB6, S309, n3130 or n3088.
Preferably, the drug for SARS-CoV-2 includes, but is not limited to, redeSivir, chloroquine, hydroxychloroquine.
Preferably, the mixing ratio of the drug to the cell vesicles of S6 is 1. Mu.g: 9. Mu.L.
Previous studies have shown that cell vesicles from CAR-T cells or 293T cells expressing single chain variable fragments have good targeting to specific tumor antigens and drug carrying functions. Thus, preferably, the target cells include, but are not limited to 293T cells, T cells of healthy humans, or NK cells.
The inventor expresses monoclonal antibody sequence aiming at SARS-CoV-2 on cells, then squeezes or separates cell vesicles expressing the monoclonal antibody sequence of SARS-CoV-2, finally loads medicaments such as Ruidexivir and the like into the cell vesicles through electrotransformation for neutralizing and killing SARS-CoV-2 in a patient. By loading medicaments such as Rede-Sivir and the like into cell vesicles to kill SARS-CoV-2, on one hand, the SARS-CoV-2 can be neutralized and the generation of antibody enhancement effect can be avoided; on the other hand, the medicaments such as the Ruidexivir and the like can be better delivered to the aggregation part of SARS-CoV-2, and the toxic and side effects of the medicaments are reduced. The method can be used for killing SARS-CoV-2 and other viruses as a platform technology.
The invention also provides cell vesicles expressing chimeric antigen receptors obtained by the method.
Preferably, the average diameter of the cell vesicles is 30-300nm.
The invention also provides application of the cell vesicles expressing the chimeric antigen receptor in preparation of medicines for resisting new coronavirus infection.
Compared with the prior art, the invention has the following beneficial effects:
(1) The present invention overcomes the antibody enhancing effect that may occur when monoclonal antibodies are used to treat new coronaries by expressing scFv fragments of monoclonal antibodies directed against SARS-CoV-2 on cellular vesicles.
(2) The technological means of the present invention can express two or more kinds of monoclonal antibody scFv segment against SARS-CoV-2 in cell vesicle to reach the effect of "cocktail treatment" and avoid SARS-CoV-2 escape.
(3) In the method, the anti-novel coronavirus medicaments such as the Ruidexivir and the like are loaded in the cell vesicles, so that the targeting of the cell vesicles to the novel coronavirus can be utilized to transport the medicaments to the aggregated part of SARS-CoV-2, and the toxic and side effects of the medicaments are reduced.
(4) Because the invention combines the neutralizing ability of monoclonal antibody and the killing ability of medicine effectively, not only can prevent SARS-CoV-2 from outside cell, but also can kill SARS-CoV-2 which has entered into cell, so that it has better therapeutic effect on SARS-CoV-2 theoretically.
(5) The invention has universal value as a platform technology, and can be applied to the treatment of other virus infections, thereby having wide application prospect.
Drawings
FIG. 1 is a schematic diagram of the preparation of cell vesicles expressing chimeric antigen receptors and their neutralization of a novel coronapneumovirus;
FIG. 2 is a graph of electron microscopy results for cell vesicles expressing chimeric antigen receptors;
FIG. 3 is a graph of NTA characterization results of vesicles expressing a cellular chimeric antigen receptor;
FIG. 4 is a graph of the WB characterization of cell vesicles expressing chimeric antigen receptors;
FIG. 5 is a graph of absorbance results of Rede Wei Jiazai after cell vesicles;
FIG. 6 is a graph showing the results of neutralization of new coronapneumoviruses by antigen receptor-expressing cell vesicles; the neutralization capacity of the pseudovirus by the cell vesicles is reflected by the half inhibitory concentration (IC 50), with smaller IC50 indicating less cell vesicles are required to inhibit the entry of a certain amount of pseudovirus into ACE2 expressing 293T cells, indicating a greater capacity of the cell vesicles to neutralize new coronavirus. In this example, the IC50 of the cell vesicles expressing both CB5 and B38 was 16.05 μg/mL.
Detailed Description
The following describes the invention in more detail. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
The test methods used in the following experimental examples are all conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
The basic principle of the invention is shown in figure 1, the invention is used for the treatment of patients suffering from a new coronavirus infection by loading a drug on cell vesicles expressing a chimeric antigen receptor against the new coronavirus. Firstly constructing lentiviral plasmids containing nucleotide sequences of novel crown monoclonal antibodies such as CB6, B38 and the like, and then packaging corresponding lentiviruses through a three-plasmid system; by infecting cells such as T cells or 293T cells with a lentivirus comprising a novel coronavirus monoclonal antibody gene, the surface expression of the cells such as T cells or 293T cells can be made to comprise scFv against the novel coronavirus; further, the prepared cells can be obtained by simply collecting the exosomes secreted by the prepared cells or squeezing the cells by using a cell squeezer to obtain cell vesicles expressing scFv against the novel coronavirus. The prepared cell vesicles can be loaded with medicaments such as adefovir and the like by electroporation and the like. Because the surfaces of the cell vesicles express more than one scFv such as CB6, B38, H4 and the like, the novel coronavirus can be well neutralized in human bodies, and the novel coronavirus is prevented from invading cells which express high ACE 2; on the other hand, because the cell vesicles have good targeting on the new coronavirus, the Ruidexi Wei Ba can be transported to the place where the virus is gathered, so that the Ruidexi has higher drug concentration at the place where the virus is gathered, thereby better killing the new coronavirus and reducing the toxic and side effects of the virus.
1. Construction of plasmid containing SARS-CoV-2 monoclonal antibody scFv nucleotide sequence
The reported CB6 and B38 novel crown monoclonal antibodies are used. B38 and CB6 are linked together by a (GGGGS) 3 linker peptide, and their nucleotide sequence synthesis is completed by Huada gene company.
After completion of the synthesis of B38-CB6-scFv, subcloning was performed on the plasmid containing pCDH-CMV-MCS-EF 1-copGGFP of CD19-CAR-T, and the anti-CD 19scFv in CD19 CAR was replaced, thereby obtaining the plasmid containing pCDH-CMV-MCS-EF 1-copGGFP of CB 6-B38-scFv. Wherein the nucleotide sequences of the CD19-CAR are CD19scFv-CD8hinge-CD8TM-4-1BB-CD3 zeta, respectively.
2. Preparation of cells expressing SARS-CoV-2 monoclonal antibody scFv
The plasmid containing pCDH-CMV-MCS-EF1-copGFP of CB6-B38-scFv can be used to further prepare cells expressing SARS-CoV-2 monoclonal antibody scFv.
Lentiviruses were packaged using a three plasmid system for subsequent experiments. The three plasmid systems were the pCDH-CMV-MCS-EF1-copGFP plasmid, the psPAX2 plasmid and the pMD2.G plasmid, respectively, containing CB 6-B38-scFv. In the invention, 293T cells are used for packaging lentiviruses. The virus in the supernatant of 293T medium was collected and then used for infection of target cells by ultracentrifugation, resuspension, and subsequent lentiviral titer titration. The infected target cells were 293T cells: 293T cells were plated in 6-well plates until they were grown to a density of 70% -80%.
The specific operation of the lentivirus package in this example is as follows:
(1) 293T packaging cells were packed at 1.3-1.5X 10 5 The concentration of cells/ml (6 ml per plate) was inoculated into the inoculation medium (dmem+10% fbs without penicillin/streptomycin) in the culture plate. Cells were incubated for 24 hours (37 ℃,5% co) 2 ) Or until the next afternoon. After about 24 hours, the cells should be about 70% confluent.
(2) Transfecting packaging cells: a mixture of 3 transfected plasmids was prepared: 250ng pMD2.G plasmid, 750ng psPAX2 plasmid, 1. Mu.g recombinant plasmid; 10-30. Mu.l OptiMEM medium.
(3) Lipofectamine 2,000 was diluted with OptiMEM: 10 μl Lipofectamine+90 μl OptiMEM. Lipofectamine reagent is added dropwise and mixed by rotating the tip or flicking the tube (without pipetting or vortexing); incubate for 5 minutes at room temperature.
(4) The 3 plasmid mixtures were added drop-wise to diluted Lipofectamine reagent and mixed by rotating the tip or flicking the tube.
(6) The transfection mixture was incubated at room temperature for 20-30 minutes.
(6) Carefully transferring the transfection mixture into packaging cells in the inoculation medium, which may be susceptible to disturbance; care was taken not to remove cells from the dish. The total volume of transfection mixture per plate should be 100 to 125. Mu.l.
(7) Cells were incubated for 18 hours (37 ℃,5% co) 2 ) Or until the next morning, the medium was changed to remove the transfection reagent and virus harvest was performed with 6ml harvest medium (dmemd+30% fbs+1x penicillin/streptomycin).
(8) Cells were incubated for 24 hours (37 ℃,5% co) 2 )。
(9) The lentivirus-containing medium was harvested approximately 40 hours after transfection. The medium is transferred to a storage tube. Replace with 6ml harborest medium.
(10) Repeating the virus harvest every 12-24 hours and replacing with 6ml harvest medium, the virus titer tends to decrease at the later harvest stage; a total of 2-3 time points are typically collected; after the last harvest, the packaging cells are discarded; the virus harvests may be pooled as desired.
(11) The virus-containing medium was spun at 1,250rpm for 5 minutes to pellet all packaging cells collected during harvest. The supernatant was passed through a 45 μm filter and transferred to a sterile storage tube.
(12) Viruses can be stored at 4℃for short periods of time (hours to days), but should be frozen at-20℃or-80℃for long-term storage. To reduce the number of freeze/thaw cycles, large-scale virus preparations are dispensed into smaller storage tubes prior to long-term storage.
By the above-described lentiviral package we obtained a lentivirus containing the gene of interest. Due to the different multiplicity of organelle infection, it is necessary to further determine the titer of lentiviral vectors. Since lentiviruses carry fluorescent markers, microscopy can be used to determine the percentage of fluorescent cells and thus lentivirus titer. The specific procedure for lentiviral titer titration in this example is as follows:
(1) 75,000 cells were seeded into each well of a 6-well petri dish.
(2) Cells were incubated overnight.
(3) If freshly collected virus is used, filtration can be performed through a 0.45 μm polyethersulfone filter to remove cells and debris.
(4) Lentivirus titers decreased during freeze-thaw cycles. If the virus is to be frozen and sub-packaged, the titer must be determined from the frozen stock to account for the titer lost associated with freeze thawing.
(5) If frozen virus is used, it is necessary to stir in warm water and rapidly thaw an aliquot of lentivirus at 37 ℃.
(6) Lentiviral dilutions were prepared in DMEM containing 10 μg/mL polyethylene. The dilutions were mixed thoroughly.
(7) The medium was gently aspirated from the cells.
(8) 1.5mL of virus dilution was added to each well (one well diluted per well, one remaining well).
(9) Counting cells in the remaining wells requires cell counting to calculate the titer.
(10) Incubating for 48-72 hours.
(11) The medium was gently aspirated and replaced with 1mL PBS.
(12) The percentage of fluorescence positive cells in each well was calculated.
When calculating titers, only wells with less than 40% fluorescence positive cells were considered. The titration method assumes 1 integration event per cell. When the percentage exceeds 40%, cells with multiple integration events may be counted, which may lead to underestimation of the true titer.
The dilution factor (method 1) or viral volume (method 2) was used to calculate the transduction units per mL (TU/mL):
method 1: TU/mL= (number of transduced cells x percent fluorescence x dilution factor)/(transduction volume (mL)).
Example of method 1: if 1:100 wells have 25% fluorescent cells in them and initially transduce 150,000 cells, there is (150,000 x 0.25 x 100)/(1.5 mL) =2.5x10 6 TU/mL。
Method 2: TU/mL= (number of transduced cells x percent fluorescence)/(viral volume (mL)).
Example of method 2: if the addition of 15 μl of virus to 150,000 cells resulted in 25% fluorescent cells, there was (150,000×0.25)/(0.015 mL) =2.5x10 6 TU/mL。
After determining the titer of the lentivirus by the above procedure, the target cells (e.g., 293 cells or T cells, etc.) can be infected with the lentivirus, thereby obtaining 293 cells or T cells stably expressing the chimeric antigen receptor. The specific steps for lentivirus infection of cells are as follows:
(1) A batch of DMEM complete+10. Mu.g/mL polyethylene was prepared by diluting 20. Mu.L of 10mg/mL polyethylene into 20mL medium.
(2) Lentiviral aliquots were rapidly thawed at 37 ℃.
(3) A series of lentiviral dilutions were prepared in DMEM complete+10. Mu.g/mL polyethylene.
(4) The dilutions were mixed thoroughly.
(5) 0.5mL of a single viral dilution was added to each well.
(6) "reverse transduction" was performed by seeding 50,000 cells into each well of a 6-well petri dish, and adding these cells to wells already containing 0.5mL of various dilutions of the virus solution ensured that the polyethylene-containing medium was used to make the cell solution in this step.
(7) Mix well with a pipette or inverted tube.
(8) 1mL of the cell suspension (i.e., 50,000 cells) was aliquoted into each well of a 6-well petri dish. This allows the total volume of each well to be 1.5mL. Since all media in these wells were made using DMEM complete solution+10. Mu.g/mL of polyethylene, the final polyethylene concentration in each well should be 10. Mu.g/mL.
(9) Cells were incubated with virus for 48-72 hours.
(10) The medium in the cells was gently aspirated.
(11) 1.5mL of DMEM solution containing the appropriate antibiotic is added, which is the beginning of the selection process, which will begin selecting stable cell pools.
(12) The dishes were observed daily to ensure that the cells in the untransduced wells (0. Mu.L lentivirus described above) were about to die. Liquid changes were performed periodically to monitor cell growth.
(13) As the polyclonal population of drug resistant cells begins to pass through and the individual wells merge, it can be expanded into a larger container. The fusion wells of a 6-well culture dish can be expanded to a 10 cm culture dish. The fused 10 cm dish can be expanded into two 75 cm 2 flasks.
(14) Once the polyclonal population grows well and expands sufficiently, cell reserves are prepared and/or harvested for testing protein expression.
3. Preparation of cell vesicles expressing chimeric antigen receptors
1. The separation of cell membranes is carried out as follows:
(1) Cells were collected and centrifuged at 700 Xg for 5 min;
(2) After centrifugation, the supernatant was discarded and the pellet was washed 3 times with 1×pbs;
(3) The cell pellet was then dispersed in a solution consisting of 1mM NaHCO 3 In a separation buffer consisting of 0.2mM EDTA and 1mM PMSF, overnight at 4 ℃;
(4) The mixture was then loaded into a dunus homogenizer and cells were destroyed by repeated 20 times of milling;
(5) After the break, the mixture was rotated at 800×g for 5 minutes to clear large fragments;
(6) The supernatant was collected and centrifuged again at 10000×g for 25 minutes, and the precipitate was discarded; the supernatant was centrifuged at 100000 Xg for 30 minutes, the supernatant was discarded, and the plasma membrane was collected as an off-white precipitate.
(7) The membrane pellet was then dispersed in PBS at ph=7.4 and resuspended with gentle sonication for subsequent experiments.
(8) The membrane protein content was quantified using the coomassie brilliant blue assay.
4. Rede ciclovir loading into cell vesicles
The specific operation is as follows:
(1) Electroporation cuvettes (0.4 cm cuvettes) were electroporated by irradiation with ultraviolet light for 1 minute, then they were placed on ice (70% ethanol if the cuvettes were dirty, then rinsed in double distilled water).
(2) The sample tube is marked.
(3) When the exosomes are isolated for electroporation, the exosome pellet is preferably resuspended in CytoMix buffer. After resuspension of the exosome pellet, the exosome concentration was read to give a final concentration of 0.25-1. Mu.g/. Mu.l.
(4) Mu.l of exosomes and 10ug of adefovir were added to each sample. Both were mixed and transferred to a 0.4cm electroporation cuvette and then electroporated at 150V and 100 μf.
(5) The mixture was incubated at 37 ℃ for 30 minutes to ensure complete recovery of the plasma membrane of the exosomes.
(6) Washing and centrifuging: all exosomes were resuspended in 20ml of PBS containing 1% (wt/vol) BSA. Ultracentrifugation at 120,000g for 70 min at 4 ℃.
(7) The quality of the adefovir loaded in the cell vesicles can be calculated by measuring the absorbance of adefovir at 247nm using a spectrophotometer to quantify the exosomes loaded with adefovir, as shown in fig. 5, since adefovir has a specific absorption peak at 247 nm. The absorbance of adefovir in 20 μg cell vesicles is shown to be 0.45.
5. Neutralizing ability of cell vesicles expressing chimeric antigen receptors to novel coronaviruses
To verify the neutralizing capacity of cell vesicles expressing scFv against SARS-CoV-2 against new coronaviruses, we co-incubated the prepared cell vesicles with an S protein pseudovirus and examined whether the cell vesicles could block invasion of the S protein pseudovirus into 293T cells that highly express ACE 2. Since the S protein pseudovirus prepared in this example contains a luciferase gene, when the S protein pseudovirus enters 293T cells which highly express ACE2, luciferase is expressed; therefore, the amount of S protein pseudovirus entering the cells can be reacted by detecting the expression amount of luciferase in 293T cells which highly express ACE 2. The neutralizing ability of the cell vesicles to the novel coronavirus is represented by the value of the half inhibitory concentration (IC 50), and the smaller the value of the IC50, the smaller the amount of cell vesicles required for neutralizing a certain amount of S protein pseudovirus, the stronger the neutralizing ability of the cell vesicles.
In this example, 293T cells highly expressing ACE2 were obtained by conventional methods of stable cell line construction, namely: firstly, co-transfecting a PCDH-ACE2 plasmid and a lentiviral helper plasmid (psPAX 2, pMD2. G) into 239T cells to obtain lentiviral particles containing ACE2 genes, then infecting 293T cells with the virus, starting to add puromycin after 48 hours for screening, and obtaining 293T cells with stable and high expression of ACE2 after about 2 weeks.
In this example, the S protein pseudovirus was prepared by co-transfecting a PCDH plasmid containing GFP and luciferase genes, a psPAX2 plasmid, and a pcDNA3.1 plasmid expressing the S protein into 293T cells, collecting the virus supernatant after 48 hours, and concentrating the virus by an ultracentrifuge.
After preparing 293T cells and S protein pseudoviruses for stably expressing ACE2, the neutralization capacity of cell vesicles can be detected, and the specific operation steps are as follows:
(1) On the first day, 1.25X10 4 Individual 293T-ACE2 cells were seeded into poly-L-lysine coated 96-well plates and incubated overnight in an incubator at 37 ℃.
(2) After overnight incubation, 50. Mu.L of the cells were titrated to 1X10 7 TU/mL S protein pseudovirus was incubated with the double diluted cell vesicles at 4℃for 10min and the mixture was then added to a 96-well plate.
(3) 48h later, the intensity of luciferase expression in 293T-ACE2 cells was measured using the Bright-Glo luciferase assay system (E2610, promega, madison, wis., USA) with reference to the manufacturer's instructions. That is, after thawing the luciferase reagent, a portion of the medium is removed, leaving about 30ul of medium; then, 30ul of luciferase reagent was added, 60ul of the mixture was transferred to a black-matrix 96-well plate after mixing, and after incubation in the dark for 2min, luminosity was measured. Luciferase was detected by EnVision Multilabel Plate Reader (Perkin Elmer).
In this example, cell vesicles simultaneously expressing scFv of B38 and CB6 monoclonal antibodies are used, and the detection result shows that the vesicles have good capability of neutralizing S protein pseudovirus, and the IC50 is 16.05 mug/mL.
Claims (5)
1. A method of preparing a cell vesicle expressing a chimeric antigen receptor, comprising the steps of:
s1, preparing a plasmid containing a single-chain variable fragment gene sequence aiming at SARS-CoV-2:
cloning a nucleotide sequence capable of expressing a single-stranded variable fragment for SARS-CoV-2 into a plasmid containing pCDH-CMV-MCS-EF1-copGFP of CD19-CAR, replacing the anti-CD 19scFv in CD19 CAR to obtain a plasmid containing a single-stranded variable fragment gene sequence for SARS-CoV-2;
s2, packaging the slow virus containing the single-chain variable fragment gene sequence aiming at SARS-CoV-2 by using a three-plasmid system or a four-plasmid system;
s3, infecting target cells by slow viruses, wherein the cell surface expression of the target cells contains single-chain variable fragments aiming at SARS-CoV-2;
s4, separating target cell membranes;
s5, carrying out ultrasonic centrifugation or extrusion filtration on the cell membrane prepared in the step S4 to obtain nano vesicles;
s6, mixing a medicine for SARS-CoV-2 with the cell vesicle, and preparing the cell vesicle for expressing the chimeric antigen receptor by adopting an electroporation mode; the gene sequence of the single-chain variable fragment aiming at SARS-CoV-2 is B38 (GGGGS) 3-CB6; the medicine for SARS-CoV-2 is Ruidexivir; the mixing ratio of the drug to the cell vesicles described in S6 was 1. Mu.g: 9. Mu.L.
2. The method of claim 1, wherein the target cell is selected from 293T cells, healthy human T cells, or NK cells.
3. A cell vesicle expressing a chimeric antigen receptor obtained by the method of any one of claims 1 to 2.
4. A cell vesicle expressing a chimeric antigen receptor according to claim 3, wherein the average diameter of the cell vesicle is 30-300nm.
5. Use of a cell vesicle expressing a chimeric antigen receptor according to claim 4 for the preparation of a medicament against a new coronavirus infection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011324758.2A CN112522203B (en) | 2020-11-23 | 2020-11-23 | Cell vesicle for expressing chimeric antigen receptor, and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011324758.2A CN112522203B (en) | 2020-11-23 | 2020-11-23 | Cell vesicle for expressing chimeric antigen receptor, and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112522203A CN112522203A (en) | 2021-03-19 |
| CN112522203B true CN112522203B (en) | 2023-08-11 |
Family
ID=74992956
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011324758.2A Active CN112522203B (en) | 2020-11-23 | 2020-11-23 | Cell vesicle for expressing chimeric antigen receptor, and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112522203B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| MX2021004130A (en) | 2020-04-02 | 2021-06-15 | Regeneron Pharma | Anti-sars-cov-2-spike glycoprotein antibodies and antigen-binding fragments. |
| CR20220660A (en) | 2020-06-03 | 2023-02-17 | Regeneron Pharma | METHODS FOR TREATING OR PREVENTING SARS-CoV-2 INFECTIONS AND COVID-19 WITH ANTI-SARS-CoV-2 SPIKE GLYCOPROTEIN ANTIBODIES |
| CN115337404A (en) * | 2021-05-12 | 2022-11-15 | 中国医学科学院基础医学研究所 | Application of cell microparticles in treatment of respiratory viral pneumonia |
| CN115252760B (en) * | 2022-03-30 | 2024-05-31 | 厦门大学 | Broad-spectrum anti-coronavirus preparation and preparation method thereof |
| CN115058344B (en) * | 2022-08-05 | 2022-11-18 | 深圳湾实验室 | Bait micro-robot for removing SARS-CoV-2 and its variant strain in waste water, its preparation method and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109468282A (en) * | 2018-11-22 | 2019-03-15 | 青岛协和华美医学诊断技术有限公司 | A kind of preparation method and application for the Chimeric antigen receptor T cell targeting CD19 |
| CN110734931A (en) * | 2019-11-18 | 2020-01-31 | 山东省齐鲁细胞治疗工程技术有限公司 | humanized scFv chimeric antigen receptor T cells targeting CD19, and preparation method and application thereof |
| CN111518773A (en) * | 2020-05-09 | 2020-08-11 | 山东兴瑞生物科技有限公司 | CAR-T cell for resisting novel coronavirus S protein, preparation method and application thereof |
-
2020
- 2020-11-23 CN CN202011324758.2A patent/CN112522203B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109468282A (en) * | 2018-11-22 | 2019-03-15 | 青岛协和华美医学诊断技术有限公司 | A kind of preparation method and application for the Chimeric antigen receptor T cell targeting CD19 |
| CN110734931A (en) * | 2019-11-18 | 2020-01-31 | 山东省齐鲁细胞治疗工程技术有限公司 | humanized scFv chimeric antigen receptor T cells targeting CD19, and preparation method and application thereof |
| CN111518773A (en) * | 2020-05-09 | 2020-08-11 | 山东兴瑞生物科技有限公司 | CAR-T cell for resisting novel coronavirus S protein, preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 王晓良.《应用分子药理学》.中国协和医科大学出版社,2015,第527-528页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112522203A (en) | 2021-03-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112522203B (en) | Cell vesicle for expressing chimeric antigen receptor, and preparation method and application thereof | |
| Veillette et al. | The HIV-1 gp120 CD4-bound conformation is preferentially targeted by antibody-dependent cellular cytotoxicity-mediating antibodies in sera from HIV-1-infected individuals | |
| JP4434478B2 (en) | Rhabdovirus with reengineered envelope | |
| CN111349150B (en) | Polypeptide for inhibiting novel coronavirus and application thereof | |
| US20190117691A1 (en) | Pd-1 car-t cell, preparation method therefor, and application thereof | |
| WO2019000620A1 (en) | Construction of recombined gene of chimeric antigen receptor for treatment of hiv infection, and application thereof | |
| CN111978395A (en) | Monoclonal antibody against novel coronavirus RBD domain antigen | |
| CN112386684A (en) | COVID-19 vaccine and preparation method and application thereof | |
| CN110964697A (en) | Anti-tumor NK cell and preparation method and application thereof | |
| CN111518773A (en) | CAR-T cell for resisting novel coronavirus S protein, preparation method and application thereof | |
| CN114763380B (en) | Construction body of nano antibody S43 and application thereof | |
| WO2018028157A1 (en) | Vc-car molecule and application thereof to hiv-1 infection cell clearing | |
| EP2477658A2 (en) | Hiv-1 antibodies | |
| CN104004092B (en) | Two or the multivalence specificity AntiHIV1 RT activity immunoadhesin of single-gene coding | |
| CN111704674B (en) | Chimeric antigen receptor targeting c-Met and autocrine PD-L1 scFv and application thereof | |
| Gustchina et al. | Synergistic inhibition of HIV-1 envelope-mediated membrane fusion by inhibitors targeting the N and C-terminal heptad repeats of gp41 | |
| CN110483636B (en) | Humanized anti-HIVgp 120 specific antibody Z166 and application method thereof | |
| CN115252760B (en) | Broad-spectrum anti-coronavirus preparation and preparation method thereof | |
| CN111704675B (en) | Bispecific chimeric antigen receptor for treating HIV infection, gene, construction method and application thereof | |
| CN114344472A (en) | A method for treating tumor by combination of exogenous antigen and therapeutic agent | |
| Zhang et al. | Down-regulation of CXCR4 expression by SDF-KDEL in CD34+ hematopoietic stem cells: An anti-human immunodeficiency virus strategy | |
| CN116536363A (en) | Cell vesicle for expressing neutralizing antibody and DC-SIGN antibody, and preparation method and application thereof | |
| WO2017088623A1 (en) | Anti-placenta-chondroitin-sulfate chimeric antigen receptor and application thereof | |
| Davis et al. | Antibodies raised to short synthetic peptides with sequences derived from HIV‐1 SF2 gp120 can both neutralize and enhance HIV‐1 SF13: A later variant isolated from the same host | |
| WO2023030315A1 (en) | Gene sequence construct for gene therapy of human immunodeficiency virus infection |
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 |