CN109536455A - A kind of CAR-NK cell and its preparation method and application - Google Patents

A kind of CAR-NK cell and its preparation method and application Download PDF

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CN109536455A
CN109536455A CN201811509243.2A CN201811509243A CN109536455A CN 109536455 A CN109536455 A CN 109536455A CN 201811509243 A CN201811509243 A CN 201811509243A CN 109536455 A CN109536455 A CN 109536455A
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CN109536455B (en
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张同存
顾潮江
周勇
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Wuhan Ruida Biotechnology Co Ltd
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Wuhan Ruida Biotechnology Co Ltd
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    • 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
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2510/00—Genetically modified cells

Abstract

The invention discloses a kind of CAR-NK cells and its preparation method and application, belong to cell therapy field, field of biological pharmacy, the present invention is using bleeding of the umbilicus as NK cell origin, solving the problems, such as the CAR-NK cell of NK cell line, peripheral blood and self blood source, there are some, such as it is complicated for operation, there is tumor formation risk, cell activity is inadequate, the problems such as cell quantity is few, and the cell of part xenogenic origin can draw GVHD.

Description

A kind of CAR-NK cell and its preparation method and application
Technical field
The invention belongs to cell therapy fields, field of biological pharmacy, and in particular to using bleeding of the umbilicus as variant cell source CAR-NK cell, and modified using source of people Chimeric antigen receptor to carry out the treatment of tumour and infectious diseases.
Background technique
The T cell (CAR-T) of Chimeric antigen receptor (chimeric antigen receptor, CAR) modification is in leukaemia Important breakthrough is achieved in research, brings new hope for patients with hematological tumor.But CAR-T treatment is asked there is also some Topic, such as undershooting-effect, cytokine storm, insertion mutation etc., and significant curative effect is not yet obtained to solid tumor.It researches and develops newly Effector cell with powerful antitumor action has important theory significance and clinical value.NK cell (natural kill Cell, natural killer cell) because of mechanism, of short duration physiological period, the extensive tumour of its special identification target cell The advantages such as killing ability are considered as the same potential effector cell that its anti-tumor capacity of enhancing is modified by CAR.
NK cell be it is a kind of there is strength lethal effect and the non-dependent lymphocyte of MHC to tumour cell, to tumour The identification of cell depends on the regulation that intersects of its surface active receptor and Inhibitory receptor.Work as tumor cell Later, NK cell makes target cell apoptosis, expression film TNF family molecule induce target by release killing medium perforin and granzyme The number of ways killing tumor cell such as cytotoxicity of Apoptosis and antibody-dependant.NK cell is modified by CAR to be expected to increase The ability of its strong target killing tumor cell simultaneously develops the effector cell with powerful antitumor action.It is existing at present largely to grind Study carefully and designs corresponding CAR-NK for different tumor targets, the significant effect obtained in experimental study, and it is clinical Its feasibility is verified in research.Such as number of patent application be CN201810069667 text in elaborate that a kind of targeting CSF1R is embedding Close the NK92MI cell and T cell and its preparation method and application that antigen receptor is modified.It is illustrated in application number CN201810036254 text It is a kind of using CD33 as the specific antibody of target spot, CAR-NK cell and its preparation and application.Application number CN201810036245 It is elaborated in text a kind of using CD19 as the specific antibody of target spot, CAR-NK cell and its preparation and application.
The NK cell origin for making CAR-NK in presently relevant patent mainly has 3 kinds of approach.(1) NK cell line, including NK- 92, NKG, YT, NK-YS, HANK-1 and NKL etc., wherein NK-92 is studied the most extensively in CAR-NK, and NK-92 has powerful Cytotoxicity, height express a series of molecules relevant to cell dissolution, such as perforin and granzyme B.Lack surface simultaneously to kill The surfaces such as killer cell immunoglobulin receptor (killer-cell immunoglobulin-like receptors, KIRs) Inhibitory receptor, the missing of Inhibitory receptor signal is so that it is better than primary NK cells to the killing ability of kinds of tumors.But It is that there is also some obvious disadvantages, such as oncogenicity and potential Epstein-Barr virus neurological susceptibility etc. by NK-92.Therefore, as safety Consider, NK-92 can be used after having to pass through irradiation.(2) autologous peripheral blood source NK cell, self CAR-NK cell return Defeated, Inhibitory receptor generates in conjunction with the HLA- class Ⅰmolecule that self normal cell is expressed inhibits signal, can inhibit NK cell Lethal effect.Although tumour cell is lost classical HLA- class Ⅰmolecule, non-classical HLA- class Ⅰmolecule HLA-G, The expression of HLA-E etc. is equally able to suppress the activation of NK cell.Simultaneously because NK cell quantity in tumor patient body, quality The presence of decline and tumor escape mechanism, anti-tumor function in vivo fail to be not fully exerted.(3) allosome peripheral blood comes The NK cell in source, the KIRs of allosome NK cell due to patient HLA- class Ⅰmolecule and mismatch, inhibition letter can't be generated Number cause to interfere.But there is other lymphocytes based on T cell in allosome NK cell donor blood, these other The presence of immunocyte can cause graft versus host disease(GVH disease) (graft versus host disease, GVHD).Therefore it is being used for Before treatment, it is necessary to remove T cell, but can also lose some other lymphs for playing miscellaneous function during killing simultaneously Cell.
Summary of the invention
For above-mentioned deficiency in the prior art, the present invention provides a kind of CAR-NK cells, and the NK cell origin is in navel Blood mononuclear cell.
Bleeding of the umbilicus has a many advantages as another alloimmune cell origin, no ethics problem, and Biohazard Waste is sharp again With abundance is easy the optimal donor of screening, bleeding of the umbilicus medium size lymphocyte developmental immaturity, mostly juvenile cell, immunogenicity Low, the probability low degree that acute chronic GVHD occurs after transplanting is light.NK cell in bleeding of the umbilicus by in-vitro separation it is amplifying activated after Can obtain largely has the NK cell for killing tumor activity to freeze after CAR gene modification as patent medicine, and recovery makes when needing With becoming the equally ready-made therapeutic modality being easy to get of drug, the i.e. concept of " off-the-shelf ".Therefore, bleeding of the umbilicus can be at Pharmaceutical requirements can also be met by experiment for the source that allosome CAR-NK cell is more satisfactory.
Umbilical cord and placenta of the bleeding of the umbilicus of the present invention from the healthy babies of term birth, no family's heredity medication history produce Woman's free from infection history, foetus health stages of labor are smooth.
NK optional or preferred, that NK cell induces for candidate stem cell in the NK cell and/or bleeding of the umbilicus in bleeding of the umbilicus Cell.
Optional or preferred, the antigen of CAR specific recognition is tumour specific antigen, viral antigen, bacterial antigens, posts The glycoprotein antigen of infested proteantigen or helminth；
The tumour specific antigen are as follows: CAIX, CD19, CD20, CD22, CD30, CD44v7/8, CEA, EGP-2, EGP- 40、erb-B2、erb-B 2,3,4、FBP、Fetal acetylcholine receptor、GD2、GD3、Her2/neu、IL- 13R-a2、KDR、LeY、MAGE-A1、Mesothelin、MUC1、NKG2D ligands、Oncofetal antigen、PSCA、 PSMA, TAG-72 or VEGF-R2.
It is optional or preferred, the structure of the CAR include sequentially connected SP, scFv, CD8 hinge area, CD28TM+ICD, 4-1BB and CD3 ζ.
The CAR be the third generation, not only include CD3 ζ signal domain, further include two costimulatory signal molecule CD28TM+ICD, 4-1BB, costimulatory signal molecule solves the problems such as CAR-NK cytosis is not lasting, and CD8 hinge area, scFv are (extracellular anti- Original identification domain, usually single-chain antibody, is also possible to polypeptide or other albumen), SP (signal peptide) same to first generation.Wherein SP and ScFv be located at it is extracellular, CD28TM+ICD, 4-1BB and CD3 ζ be located at it is intracellular.
Optional or preferred, in above-mentioned CAR-NK cell, as shown in SEQ ID NO.1, scFv is the amino acid sequence of CAR EGFRvIII。
SEQ ID NO.1EGFRvIII CAR sequence
MALPVTALLLPLALLLHAARPEIQLVQSGAEVKKPGESLRISCKGSGFNIEDYYIHWVRQMPGKGLEW MGRIDPENDETKYGPIFQGHVTISADTSINTVYLQWSSLKASDTAMYYCAFRGGVYWGQGTTVTVSSGGGGSGGGG SGGGGSGGGGSDVVMTQSPDSLAVSLGERATINCKSSQSLLDSDGKTYLNWLQQKPGQPPKRLISLVSKLDSGVPD RFSGSGSGTDFTLTISSLQAEDVAVYYCWQGTHFPGTFGGGTKVEIKNWSHPQFEKGGGGSTTTPAPRPPTPAPTI ASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRR PGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPA YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDG LYQGLSTATKDTYDALHMQALPPR*
Wherein, SP amino acid sequence is as shown in SEQ ID NO.2: MALPVTALLLPLALLLHAA.
Scfv amino acid sequence is as shown in SEQ ID NO:3: (EGFRvIII)
DVVMTQSPDSLAVSLGERATINCKSSQSLLDSDGKTYLNWLQQKPGQPPKRLISLVSKLDSGVPDRFS GSGSGTDFTLTISSLQAEDVAVYYCWQGTHFPGTFGGGTKVEIK。
CD8hinge amino acid sequence is as shown in SEQ ID NO:4:
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD。
CD28 cross-film domain amino acid sequence is as shown in SEQ ID NO:5:
FWVLVVVGGVLACYSLLVTVAFIIFWV。
Intracellular signal structural domain is made of CD28,4-1BB and CD3zeta, and amino acid sequence is as shown in SEQ ID NO:6:
RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDG CSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNE LQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*。
Optional or preferred, CAR nucleotide sequence is as shown in SEQ ID NO.7:
atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgagat tcagctcgtgcaatcgggagcggaagtcaagaagccaggagagtccttgcggatctcatgcaagggtagcggcttt aacatcgaggattactacatccactgggtgaggcagatgccggggaagggactcgaatggatgggacggatcgacc cagaaaacgacgaaactaagtacggtccgatcttccaaggccatgtgactattagcgccgatacttcaatcaatac cgtgtatctgcaatggtcctcattgaaagcctcagataccgcgatgtactactgtgctttcagaggaggggtctac tggggacagggaactaccgtgactgtctcgtccggcggaggcgggtcaggaggtggcggcagcggaggaggagggt ccggcggaggtgggtccgacgtcgtgatgacccagagccctgacagcctggcagtgagcctgggcgaaagagctac cattaactgcaaatcgtcgcagagcctgctggactcggacggaaaaacgtacctcaattggctgcagcaaaagcct ggccagccaccgaagcgccttatctcactggtgtcgaagctggattcgggagtgcccgatcgcttctccggctcgg gatcgggtactgacttcaccctcactatctcctcgcttcaagcagaggacgtggccgtctactactgctggcaggg aacccactttccgggaaccttcggcggagggacgaaagtggagatcaagaactggagccacccccagttcgagaag ggcggtggcggaagcaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgt ccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgattt ttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgg gtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgca agcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccaaacggggcagaaagaaactcctgta tatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaa gaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccaga accagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccc tgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcg gaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctca gtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa。
The present invention also provides the preparation method of any description above CAR-NK cell, be by NK cell with containing cell because The culture medium of son is cultivated together to be activated, then is expressed in CAR in NK cell by gene modification.
Gene modification includes the recombination of gene, overexpression and gene knockout, such as the expression of Chimeric antigen receptor, PD- 1, the knockout of TIM3.The technological means that gene modification uses includes slow virus, retrovirus, electricity turn, sleeping beauty's swivel base etc..Institute The gene knockout method stated, including but not limited to CRISPR technology, ZFN technology, TALEN technology etc..
Optional or preferred, in above-mentioned preparation method, the cell factor includes in IL-2, IL-15, IL-7, IL-21 It is one or more of.
The present invention also provides a kind of above-mentioned CAR preparation method containing SEQ ID NO.1-6 or SEQ ID NO.7, packets Include following steps:
(1) cord blood mononuclear cells separation and Extraction: taking bleeding of the umbilicus, obtains mononuclearcell by density gradient centrifugation.
(2) NK cell activation: the mononuclearcell of step (1) be seeded in containing 1~10% bleeding of the umbilicus autologous plasma, 200~ The IL-21 lymph of the IL-2 of 1500IU/mL, the IL-15 of 10~50ng/mL, the IL-7 of 10~30ng/mL and 50~100ng/mL Cell culture medium culture, and cell density 0.8~1.0 × 10 is pressed in 3d6A/mL fluid infusion；Culture harvests activation to 5d NK cell；
(3) with the NK cell for the lentiviruses transduction activation for being built with CAR, culture；Obtain CAR-NK cell；
(4) CAR-NK cell amplification cultivation: 5d turns bag and by cell density 0.8~1.0 × 106A/mL fluid infusion, later Cell density 0.8~1.0 × 10 is pressed every embedding 2d6A/mL fluid infusion after amplification to 15d, harvests cell.
The present invention also provides the application of any description above CAR-NK cell in medicine preparation, the drug therapy Disease corresponds to the type of chimeric antigen in CAR-NK.
Compared with prior art, the invention has the following advantages:
The present invention solves the CAR-NK of NK cell line, peripheral blood and self blood source using bleeding of the umbilicus as NK cell origin Cell is such as complicated for operation there are some problems, has tumor formation risk, cell activity is inadequate, and cell quantity is few, and part allosome comes The cell in source can draw the problems such as GVHD.
CAR-NK cell of the present invention is significantly better than NK cell in terms of killing tumor.
Detailed description of the invention
Fig. 1 is the transduction efficiency testing result of EGFRvIII-CAR-NK cell in the embodiment of the present invention.
Fig. 2 is EGFRvIII-CAR-NK cell in the embodiment of the present invention and NK cell to positive target cell U87 (EGFRvIII) Cytotoxicity in vitro efficiency.
Fig. 3 is EGFRvIII-CAR-NK cell in the embodiment of the present invention and NK cell to the external of negative targets Raji Killing-efficiency.
Fig. 4 is EGFRvIII CAR structure in the embodiment of the present invention.
Fig. 5 is PLVX-EGFRvIII-strepII plasmid map in the embodiment of the present invention.
Specific embodiment
The present invention is further explained in the light of specific embodiments, so that those skilled in the art can be preferably Understand the present invention and can be practiced, but illustrated embodiment is not as a limitation of the invention.
1 cord blood mononuclear cells of embodiment is extracted
(1) bleeding of the umbilicus 50mL is taken, average mark is loaded in 2 50mL centrifuge tubes, and 650g is centrifuged 15min at room temperature, takes upper layer Faint yellow blood plasma (lower layer's red liquid is for extracting mononuclearcell) into new 50mL centrifuge tube, by blood plasma in 56 DEG C of water-baths 30min is inactivated in pot, then 900g is centrifuged 10min, takes supernatant, is placed in 15min in -20 DEG C of environment；It is centrifuged again, 900g, 10min takes supernatant, is placed in 4 DEG C and saves for use.(centrifuge adjusts raising speed 1, reduction of speed 1).
(2) lower layer's red liquid obtained in the extraction of previous step blood plasma is taken to be diluted in equal volume with physiological saline, total volume is about 20mL is mixed by inversion, spare.The centrifuge tube of 2 new 50mL is separately taken, lymphocyte separation medium 20mL is added in every pipe.By 20mL Blood after dilution is added in the centrifuge tube of the lymphocyte separation medium containing 20mL, and blood and lymphocyte separation medium is made to form one A apparent layering, room temperature 650g are centrifuged 30min.(centrifuge adjusts raising speed 1, drops peripheral blood speed 1).
(3) it gentle aspiration monocyte (tunica albuginea layer) and the lymphocyte separation medium of half and is transferred to new below it In 50mL centrifuge tube；Isometric physiological saline is added, room temperature 260g is centrifuged 10min.Supernatant is abandoned, is cleaned carefully with physiological saline again Supernatant is abandoned in born of the same parents, centrifugation.Repeated washing step is resuspended cell with physiological saline, while taking a small amount of cell suspension Trypan Blue meter Number, 260g is centrifuged 10min after counting, abandons supernatant, spare.
2 Cord Blood Natural Killer Cells: Impact induced activation of embodiment
The mononuclearcell obtained in embodiment 1 is by cell density 2 × 106A/mL is seeded in containing 1~10% bleeding of the umbilicus certainly Body blood plasma, the IL-7 and 50~100ng/ of the IL-15 of the IL-2 of 200~1500IU/mL, 10~50ng/mL, 10~30ng/mL In the IL-21 lymphocytes culture medium of mL, 37 DEG C are put into, 5%CO2Cultivated in incubator, and 3d by cell density 0.8~ 1.0×106A/mL fluid infusion.
The detection of the NK cell surface marker of 3 peripheral blood of embodiment and derived from cord blood
50mL peripheral blood is taken, mononuclearcell is extracted by the method in embodiment 1, is activated by method in embodiment 2 Culture.
The NK cell of cell and the derived from peripheral blood of activation culture in Example 2, is washed 2 times with PBS, rear that mouse IgG is added, 4 DEG C are protected from light 30min；It is separately added into specific antibody CD16, CD161, NKG2A, NKG2D, NKp46,4 DEG C are protected from light incubation 30min. Cell after dyeing is washed 2 times, with the above-mentioned marker of flow cytomery.Statistical method uses SPSS10 statistical software. All data are indicated with ˉ x ± s, are examined using t and Mann-Whitney is examined.It the results are shown in Table 1, the NK cell in two kinds of sources exists There is no notable difference in phenotype.
1 bleeding of the umbilicus of table and the expression of peripheral blood NK cell surface marker
Observation item Cord Blood Natural Killer Cells: Impact Peripheral blood NK cell
CD16 90.71±8.05 93.22±5.44
CD161 83.08±9.18 70.87±12.54
NKG2A 75.34±8.39 25.88±2.31
NKG2D 93.67±5.05 96.54±2.56
NKp46 94.23±5.06 88.98±6.74
Note: compared with peripheral blood NK cell, P < 0.05.
The detection of the NK cell function index of correlation of 4 peripheral blood of embodiment and derived from cord blood
In Example 2 the NK cell of cell and the derived from peripheral blood of activation culture in 37 DEG C with PMA (20ng/ml) and Ionomycin (1 μ g/ml) stimulates 4h, and monensin (10 μ g/ml) is added and blocks cytokine secretion；Cell is washed 2 times, is added Specific antibody CD3, CD56,4 DEG C are protected from light incubation 30min, mark surface marker.By Fix/Perm liquid be added it is labeled after it is thin It is placed at room temperature for 20min in born of the same parents, the internal standards antibody at room temperature such as IFN-γ, TNF-α, granzyme B, perforin are added and place 1h.Carefully Born of the same parents wash 2 times, upper machine testing.Peripheral blood and the NK cell granulations enzyme B of derived from cord blood, perforin, FasL, IFN-γ, TNF-α table It reaches.Statistical method uses 10 statistical software of SPSS.All data are indicated with ˉ x ± s, using t inspection and Mann-Whitney It examines.It the results are shown in Table 2, the NK cell of derived from cord blood is less in addition to the release of granzyme B, and other function albumen comes with peripheral blood The NK in source does not have notable difference.
2 bleeding of the umbilicus of table and the expression of peripheral blood NK cell functional protein
Observation item Cord Blood Natural Killer Cells: Impact Peripheral blood NK cell
IFN-γ 45.43±8.36 34.98±12.88
Granzyme B 2.45±1.98 70.34±4.94
Perforin 40.36±18.53 35.27±13.84
TNF-α 1.97±1.24 1.84±1.38
Note: in addition to granzyme, other indexs are compared with peripheral blood NK cell, P < 0.01.
Embodiment 5 is transfected EGFRvIII-CAR into derived from cord blood NK cell using slow virus
(1) cell is harvested after the NK cell activation culture to 5d in embodiment 2, is 3 addition LVEGFRvIII- by MOI (virus contains expression plasmid PLVX-EGFRvIII-strepII to CAR virus, and structure can be expressed referring to embodiment 7 EGFRvIII-CAR it) transduces, mixing is placed on CO2Incubator is incubated for, and suitable NK cell is added after 4 hours and is cultivated completely Base is cultivated and (obtains EGFRvIII-CAR-NK cell after lentiviruses transduction success).It will transduction after lentiviruses transduction 24 hours EGFRvIII-CAR-NK cell changes to fresh medium afterwards, and adjusting viable cell density is 1.0 × 106/ mL continues culture and expands Increase 10~20 days, observed and counted daily, and fluid infusion is carried out according to the cell quantity counted and expands culture, remains thin Born of the same parents' culture density is 1.0 × 106/mL。
(2) EGFRvIII-CAR-NK transduction efficiency detects
Take 1.0 × 106NK cell after a transduction, with Goat anti-Human IgG Antibody, FITC Conjugate is incubated at room temperature 30 minutes, after physiological saline cleaning twice, by flow cytomery FITC fluorescence signal, is surveyed FITC positive cell ratio is measured, ratio of the CAR-NK cell in total cell is reflected.Testing result is as shown in Fig. 1 and following table. Illustrate to be successfully prepared EGFRvIII-CAR-NK cell.
The transduction efficiency testing result of 3 EGFRvIII-CAR-NK cell of table
Transduction type EGFRvIII-CAR-NK
Transduction efficiency 30.6%
6 EGFRvIII-CAR-NK Cytotoxicity in vitro Function detection of embodiment
EGFRvIII-CAR-NK cell is carried out using calcein detection method to kill tumor Function detection in vitro.Take appropriate U87 Cell is as target cell, 1 × 106Calcein-acetyl methylol is added in the cell suspension (PBS, 5% fetal calf serum) of/mL Ester (Calcein-AM) is incubated for 30min to 25 μM of final concentration in incubator.Under room temperature, cell is resuspended to 1.5 after washing twice × 105/mL.It is centrifuged 30 seconds by different effect targets than EGFRvIII-CAR-NK cell, 200g is added, 37 DEG C are incubated for 2~3 hours.It is incubated for Supernatant is taken after the completion, is measured the fluorescence intensity of wherein calcein, and compare according to spontaneous release control and maximum release, is calculated Target cell lysis percentage.
It kills tumor experimental data: needing to carry out it before application to function such as tumor cell line killings to the NK cell of lentiviruses transduction Energy property detection, uses calcein detection method.As a result referring to figs. 2 and 3 and table 4 and table 5.The results show that EGFRvIII-CAR- NK cell has specific killing activity to the tumour cell of high expression EGFRvIII, and does not kill tumor to EGFRvIII negative cells Activity embodies good targeting.
The Cytotoxicity in vitro efficiency of 4 EGFRvIII-CAR-NK cell of table and NK cell to positive target cell U87
The Cytotoxicity in vitro efficiency of 5 EGFRvIII-CAR-NK cell of table and NK cell to negative targets Raji
Embodiment 7EGFRvIII-CAR expression vector establishment
The DNA fragmentation for being 1647bp by length shown in artificial synthesized SEQ ID NO:7, wherein 1-57 core Thuja acid encodes SP, the SCFV of 58-801 nucleotide coding targeting EGFR vIII, 844-978 nucleotide codings CD8 hinge area, 979-1182 nucleotide coding CD28TM+ICD, 1183-1308 nucleotide coding 4-1BB, 1309-1647 nucleotide coding CD3 zeta, concrete structure schematic diagram are shown in attached drawing 4.Three regions next constitute intracellular Signaling zone.
By the EF1alpha promoter downstream of above-mentioned DNA fragmentation insertion Lentiviral PLVX, targeted The Chimeric antigen receptor expression plasmid PLVX-EGFRvIII-strepII of EGFRvIII, plasmid map are shown in attached drawing 5.
Embodiment described above is only to absolutely prove preferred embodiment that is of the invention and being lifted, protection scope of the present invention It is without being limited thereto.Those skilled in the art's made equivalent substitute or transformation on the basis of the present invention, of the invention Within protection scope.Protection scope of the present invention is subject to claims.
Sequence table
<110>Wuhan wave is farsighted reaches Biotechnology Co., Ltd
<120>a kind of CAR-NK cell and its preparation method and application
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 548
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Glu Ile Gln Leu Val Gln Ser Gly Ala Glu Val
20 25 30
Lys Lys Pro Gly Glu Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Phe
35 40 45
Asn Ile Glu Asp Tyr Tyr Ile His Trp Val Arg Gln Met Pro Gly Lys
50 55 60
Gly Leu Glu Trp Met Gly Arg Ile Asp Pro Glu Asn Asp Glu Thr Lys
65 70 75 80
Tyr Gly Pro Ile Phe Gln Gly His Val Thr Ile Ser Ala Asp Thr Ser
85 90 95
Ile Asn Thr Val Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr
100 105 110
Ala Met Tyr Tyr Cys Ala Phe Arg Gly Gly Val Tyr Trp Gly Gln Gly
115 120 125
Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr
145 150 155 160
Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile
165 170 175
Asn Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr
180 185 190
Leu Asn Trp Leu Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu Ile
195 200 205
Ser Leu Val Ser Lys Leu Asp Ser Gly Val Pro Asp Arg Phe Ser Gly
210 215 220
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala
225 230 235 240
Glu Asp Val Ala Val Tyr Tyr Cys Trp Gln Gly Thr His Phe Pro Gly
245 250 255
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Asn Trp Ser His Pro
260 265 270
Gln Phe Glu Lys Gly Gly Gly Gly Ser Thr Thr Thr Pro Ala Pro Arg
275 280 285
Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg
290 295 300
Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly
305 310 315 320
Leu Asp Phe Ala Cys Asp Phe Trp Val Leu Val Val Val Gly Gly Val
325 330 335
Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp
340 345 350
Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met
355 360 365
Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala
370 375 380
Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys
385 390 395 400
Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr
405 410 415
Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly
420 425 430
Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala
435 440 445
Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg
450 455 460
Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu
465 470 475 480
Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn
485 490 495
Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met
500 505 510
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
515 520 525
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala
530 535 540
Leu Pro Pro Arg
545
<210> 2
<211> 19
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 2
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala
<210> 3
<211> 112
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 3
Asp Val Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser
20 25 30
Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Lys Pro Gly Gln Pro
35 40 45
Pro Lys Arg Leu Ile Ser Leu Val Ser Lys Leu Asp Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
65 70 75 80
Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Trp Gln Gly
85 90 95
Thr His Phe Pro Gly Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 4
<211> 45
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 4
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 45
<210> 5
<211> 27
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 5
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
1 5 10 15
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val
20 25
<210> 6
<211> 195
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 6
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
20 25 30
Pro Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu
35 40 45
Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln
50 55 60
Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly
65 70 75 80
Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr
85 90 95
Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
100 105 110
Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met
115 120 125
Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu
130 135 140
Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys
145 150 155 160
Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu
165 170 175
Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu
180 185 190
Pro Pro Arg
195
<210> 7
<211> 1647
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60
cccgagattc agctcgtgca atcgggagcg gaagtcaaga agccaggaga gtccttgcgg 120
atctcatgca agggtagcgg ctttaacatc gaggattact acatccactg ggtgaggcag 180
atgccgggga agggactcga atggatggga cggatcgacc cagaaaacga cgaaactaag 240
tacggtccga tcttccaagg ccatgtgact attagcgccg atacttcaat caataccgtg 300
tatctgcaat ggtcctcatt gaaagcctca gataccgcga tgtactactg tgctttcaga 360
ggaggggtct actggggaca gggaactacc gtgactgtct cgtccggcgg aggcgggtca 420
ggaggtggcg gcagcggagg aggagggtcc ggcggaggtg ggtccgacgt cgtgatgacc 480
cagagccctg acagcctggc agtgagcctg ggcgaaagag ctaccattaa ctgcaaatcg 540
tcgcagagcc tgctggactc ggacggaaaa acgtacctca attggctgca gcaaaagcct 600
ggccagccac cgaagcgcct tatctcactg gtgtcgaagc tggattcggg agtgcccgat 660
cgcttctccg gctcgggatc gggtactgac ttcaccctca ctatctcctc gcttcaagca 720
gaggacgtgg ccgtctacta ctgctggcag ggaacccact ttccgggaac cttcggcgga 780
gggacgaaag tggagatcaa gaactggagc cacccccagt tcgagaaggg cggtggcgga 840
agcaccacga cgccagcgcc gcgaccacca acaccggcgc ccaccatcgc gtcgcagccc 900
ctgtccctgc gcccagaggc gtgccggcca gcggcggggg gcgcagtgca cacgaggggg 960
ctggacttcg cctgtgattt ttgggtgctg gtggtggttg gtggagtcct ggcttgctat 1020
agcttgctag taacagtggc ctttattatt ttctgggtga ggagtaagag gagcaggctc 1080
ctgcacagtg actacatgaa catgactccc cgccgccccg ggcccacccg caagcattac 1140
cagccctatg ccccaccacg cgacttcgca gcctatcgct ccaaacgggg cagaaagaaa 1200
ctcctgtata tattcaaaca accatttatg agaccagtac aaactactca agaggaagat 1260
ggctgtagct gccgatttcc agaagaagaa gaaggaggat gtgaactgag agtgaagttc 1320
agcaggagcg cagacgcccc cgcgtaccag cagggccaga accagctcta taacgagctc 1380
aatctaggac gaagagagga gtacgatgtt ttggacaaga gacgtggccg ggaccctgag 1440
atggggggaa agccgagaag gaagaaccct caggaaggcc tgtacaatga actgcagaaa 1500
gataagatgg cggaggccta cagtgagatt gggatgaaag gcgagcgccg gaggggcaag 1560
gggcacgatg gcctttacca gggtctcagt acagccacca aggacaccta cgacgccctt 1620
cacatgcagg ccctgccccc tcgctaa 1647

Claims (10)

1. a kind of CAR-NK cell, which is characterized in that the NK cell origin is in cord blood mononuclear cells.
2. CAR-NK cell according to claim 1, which is characterized in that the NK cell be bleeding of the umbilicus in NK cell and/ Or the NK cell that candidate stem cell induces in bleeding of the umbilicus.
3. CAR-NK cell according to claim 1, which is characterized in that the antigen of CAR specific recognition is tomour specific Property antigen, viral antigen, bacterial antigens, the proteantigen of helminth or the glycoprotein antigen of helminth；
The tumour specific antigen are as follows: CAIX, CD19, CD20, CD22, CD30, CD44v7/8, CEA, EGP-2, EGP-40, erb-B2、erb-B 2,3,4、FBP、Fetal acetylcholine receptor、GD2、GD3、Her2/neu、IL-13R- a2、KDR、LeY、MAGE-A1、Mesothelin、MUC1、NKG2D ligands、Oncofetal antigen、PSCA、PSMA、 TAG-72 or VEGF-R2.
4. CAR-NK cell according to claim 1, which is characterized in that the structure of the CAR include sequentially connected SP, ScFv, CD8 hinge area, CD28TM+ICD, 4-1BB and CD3 ζ.
5. CAR-NK cell according to claim 4, which is characterized in that the amino acid sequence of CAR such as SEQ ID NO.1 institute Show.
6. CAR-NK cell according to claim 5, which is characterized in that CAR nucleotide sequence such as SEQ ID NO.7 institute Show.
7. the preparation method of any CAR-NK cell of claim 1~6, which is characterized in that by NK cell and contain cell The culture medium of the factor is cultivated activated together, then is expressed in CAR in NK cell by gene modification.
8. preparation method according to claim 7, which is characterized in that the cell factor include IL-2, IL-15, IL-7, One or more of IL-21.
9. the preparation method of the CAR-NK cell of claim 5 or 6, which comprises the following steps:
(1) cord blood mononuclear cells separation and Extraction: taking bleeding of the umbilicus, obtains mononuclearcell by density gradient centrifugation.
(2) NK cell activation: the mononuclearcell of step (1) be seeded in containing 1~10% bleeding of the umbilicus autologous plasma, 200~ The IL-21 lymph of the IL-2 of 1500IU/mL, the IL-15 of 10~50ng/mL, the IL-7 of 10~30ng/mL and 50~100ng/mL Cell culture medium culture, and cell density 0.8~1.0 × 10 is pressed in 3d6A/mL fluid infusion；Culture harvests activation to 5d NK cell；
(3) with the NK cell for the lentiviruses transduction activation for being built with CAR, culture；Obtain CAR-NK cell；
(4) CAR-NK cell amplification cultivation: 5d turns bag and by cell density 0.8~1.0 × 106A/mL fluid infusion, later every Embedding 2d presses cell density 0.8~1.0 × 106A/mL fluid infusion after amplification to 15d, harvests cell.
10. the application of any CAR-NK cell of claim 1~6 in medicine preparation, the disease pair of the drug therapy Should in CAR-NK chimeric antigen type.
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CN111560076B (en) * 2020-05-15 2021-05-07 广州百暨基因科技有限公司 Chimeric antigen receptor immune cell and preparation method and application thereof
CN111675765A (en) * 2020-06-02 2020-09-18 南京凯地生物科技有限公司 Armed chimeric antigen receptor cell of targeted coronavirus SPIKE, preparation method and application
CN111675765B (en) * 2020-06-02 2024-02-06 南京凯地医疗技术有限公司 Armed chimeric antigen receptor cell targeting coronavirus SPIKE, preparation method and application
CN115505600A (en) * 2022-09-30 2022-12-23 北京奇迈永华生物科技有限公司 Method for efficiently infecting human NK cells by lentiviruses
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