CN105647873A - Preparation method and kit of bispecific chimeric antigen receptor gene modified natural killer cells - Google Patents
Preparation method and kit of bispecific chimeric antigen receptor gene modified natural killer cells Download PDFInfo
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Abstract
The invention provides a preparation method of bispecific chimeric antigen receptor gene modified natural killer cells, comprising the features: constructing a bispecific chimeric antigen receptor gene containing specific-binding signaling lymphocyte activation molecule family member 7 and fibronectin mutants, recombining the bispecific chimeric antigen receptor gene to a viral vector, transfecting with human natural killer cells, and highly expressing the bispecific chimeric antigen receptor gene, thus specifically binding tumor cells that express the signaling lymphocyte activation molecule family member 7 and fibronectin mutants, inhibiting expression of natural killer cell inhibitory receptors, and preventing immune escape of tumor cells; meanwhile, activating a first signal and a co-stimulatory signal to trigger toxic activity of the tumor cells, and great anti-tumor killing toxicity is shown in in-vivo and in-vitro experiments; the invention also provides a kit for the preparation of autologous natural killer cells through the above method, and with the kit, it is possible to highly express the bispecific chimeric antigen receptor gene and trigger great anti-tumor effect.
Description
Technical field
The present invention relates to the preparation method of the natural killer cell of a kind of bispecific chimeric antigen receptor genetic modification and test kit thereof, including following feature: be built into containing specific bond signal lymphocyte activator molecule families member 7 (SignalingLymphocyticActivationMoleculeFamily, Member7, and fibronectin variant (fibronectinvariant SLAMF7), FNv) double, two distinctive embedment antigen receptor (doublechimericantigenreceptor, Di-CAR) gene recombinaton is to viral vector and transfected with human natural killer cell (Naturalkillercells, NK), this bispecific chimeric antigen receptor gene of high expressed, can the tumor cell of specific bond expression signal lymphocyte activator molecule families member 7 and fibronectin variant, and the cell inhibiting expression of receptor of suppression of natural killer, avoid tumor cell immune evasion, activating the first signal and costimulatory signal simultaneously and cause antitumor cell toxic activity, vivo and vitro test antitumor has and kills very by force toxicity, present invention also offers a kind of test kit prepared containing the autologous natural killer cell obtained by said method, this bispecific chimeric antigen receptor gene of high expressed can be obtained and cause powerful Graft Versus Tumor.
Background technology
Natural killer cell is to carry out the immunocyte technology that clinical treatment is common at home at present. Current NK cell obtains mainly through trophocyte or antibody activation-inducing effect, expresses CD16+CD56+ positive cell. Its antitumor main mechanism is express activation receptor part on tumor cell membrane by it to be combined, release particles enzyme and perforin directly kill tumor cell, and the CD16 molecule of high expressed causes antibody-dependent cellular cytoxicity activity that tumor cell is produced apoptotic effect.
But, it is also limited that NK cell treats malignant tumor aspect curative effect clinically, include the effective percentage of the complete incidence graph of cases of complete remission and the partial rcsponse of tumor section reduction between 40-50% according to clinical treatment data statistics, also can not meet far away the needs of clinical treatment malignant tumor.The key issue affecting the effect of NK cell therapy cancer is NK cell-targeting tumor cytotoxicity poor specificity, and its expression inhibiting receptor so that tumor cell can pass through to produce immunologic escape in conjunction with it. Improving NK cell quantity, CD16+CD56+ phenotype at present and reduce Inhibitory receptor expression, all existing research is carried out, to be effectively improved the effective percentage of its treatment cancer.
The invention provides double; two distinctive embedment antigen genes of SLAMF7 and FNv in conjunction with tumor cell specifically expressing and recombinate on viral vector and transfected with human natural killer cell, this bispecific chimeric antigen receptor gene of high expressed, can the tumor cell of specific bond expression signal lymphocyte activator molecule families member 7 and fibronectin variant, such as all two tumor antigens of specifically expressing SLAMF7 and FNv such as melanoma, uveal and melanoma of choroid; And suppression of natural killer cell inhibiting expression of receptor, it is to avoid tumor cell immune evasion; Activating the first signal and costimulatory signal simultaneously and cause antitumor cell toxic activity, vivo and vitro test antitumor has and kills very by force toxicity, will significantly improve clinical efficacy and prolongation survival of patients phase.
Summary of the invention
The present invention is directed to the deficiency of existing clinical practice NK cell technology, particularly because of NK cell-targeting tumor cytotoxicity poor specificity, NK cell expression inhibiting receptor so that tumor cell can pass through to produce immunologic escape in conjunction with it, and it is not enough to activate patient's autoimmunization antitumor in vivo, thus results in that to kill the low and most for the treatment of malignant tumor of tumor activity limited. the present invention finds melanoma in previous research work, uveal and melanoma of choroid etc. be two tumor antigen SLAMF7 and FNv of specifically expressing all, extracellular region according to the two tumor antigen obtains the monoclonal cell strain with both specific binding reactions and monoclonal antibody by hybridoma technology, protein sequencing determines the heavy chain of monoclonal antibody and the variable region of light chain, utilize gene PCR technology that by linking son (linker), heavy chain and light chain are connected generation single-chain antibody (singlechainantibodyfragment, scFv), at euzymelinked immunosorbent assay (ELISA) and these two scFv energy two tumor antigens of specific bond SLAMF7 and FNv of cell flow cytometer detection, tumor cell is melanoma, uveal and melanoma of choroid etc., the two scFv can as the antibody in conjunction with two tumor antigens of SLAMF7 and FNv.
Present inventor finds that transcription factor-proto-oncogene c-Myc is in conjunction with the cell inhibiting receptor (killercellimmunoglobulin-likereceptor of NK, KIR) express promoter, start the immunologic escape function of the expression of NK cell KIR and mediate tumor cell. We have found that and 362 site leucines in c-Myc are sported the c-Myc mutant that valine produces, can be combined with KIR gene expression promoter, but KIR gene is not expressed, show that this c-Myc mutant can suppress KIR gene expression, thus preventing the receptor-mediated immunologic escape effect of NK cell expression inhibiting. Thus we are according to these scientific research results, it is built into the double; two distinctive embedment antigen receptor genes containing specific bond SLAMF7 and FNv, and introduce c-Myc mutant and the genetic fragment in CD28, CD137 and CD3 �� intracellular signal district, recombinate to viral vector and transfected with human natural killer cell, this bispecific chimeric antigen receptor gene of high expressed, vivo and vitro test antitumor has and kills very by force toxicity.
The preparation method that the present invention relates to the natural killer cell of a kind of bispecific chimeric antigen receptor genetic modification, it is characterized in that, it is built into the double; two distinctive embedment antigen receptor gene recombinaton containing specific bond SLAMF7 and FNv to viral vector and transfected with human natural killer cell, this bispecific chimeric antigen receptor gene of high expressed, can the tumor cell of specific bond expression signal lymphocyte activator molecule families member 7 and fibronectin variant, and suppression of natural killer cell inhibiting expression of receptor, it is to avoid tumor cell immune evasion; Activate the first signal and costimulatory signal simultaneously and cause antitumor cell toxic activity, vivo and vitro test have activated antitumor response consumingly.
The cDNA (Di-CAR) of described bispecific chimeric antigen receptor gene builds in viral vector, and transfected NK cells, NK cell is cultivated to the 21st day again, obtain Di-CAR-NK cell, being detected by fluorescent quantitative PCR technique, its expression Di-CAR gene of NKs having transfected Di-CAR viral vector is more than 50 times higher than the NKs of untransfected;
Obtained natural killer cell, its high expressed its specific markers CD16+/CD56+, positive rate is higher than more than 80%, and activation receptor positive rate is more than more than 80%, and Inhibitory receptor positive rate is lower than less than 10%.
SLAMF7 and the FNv antigen that in of the present invention pair of distinctive embedment antigen receptor gene, SLAMF7 and FNv single-chain antibody specific bond malignant cell is expressed, NK cell is made to produce antineoplastic targeting specificity, preferably, for targeting melanoma, uveal and melanoma of choroid etc.
The distinctive embedment antigen receptor gene of fibronectin variant of the present invention, including the single-chain antibody of fibronectin variant and linked the genetic fragment of transcription factor mutant (c-Myc mutant) of specific bond Inhibitory receptor gene promoter, suppression of natural killer cell expression inhibiting receptor, avoid tumor cell immune evasion, its high expressed activation receptor specific bond tumor cell surface part and produce cytotoxic effect. Described transcription factor c-Myc mutant, it is preferable that be g by this gene c1641 site mutation, namely leucine (Leu362) sports valine, in combinations with KIR upstream region of gene promoter and inhibit it to transcribe.
Distinctive embedment antigen receptor gene containing specific bond signal lymphocyte activator molecule families member 7 of the present invention, genetic fragment including the single-chain antibody of specific bond signal lymphocyte activator molecule families member 7 connected two costimulatory moleculeses born of the same parents' internal area (CD28 and CD137) and CD3 �� intracellular signal district, the first signal and costimulatory signal can be activated, namely activate immune response and cause antitumor cell toxic activity and tumor death effect.
Having linked furin cleavage site between described bispecific chimeric antigen receptor gene, it can produce the Chimeric antigen receptor of two independent functions after intracellular protein is translated simultaneously.
In the method for the invention, natural killer cell derives from the mononuclearcell of autologous vein blood, autologous bone marrow, Cord blood and placental blood etc. Preferably, derive from cancer patient perform the operation one month after, the fresh peripheral blood that gathers after one month of chemicotherapy or bone marrow.
Of the present invention by people's IFN ��-signal peptide-scFv (SLAMF7)-CD8a hinge region-CD28-CD137-CD3 �� intracellular region, and scFv (FNv)-IgD hinge region-c-Myc mutant is linked together by Furin cleavage site and catenation sequence, form recombination sequence, gene order is obtained by chemosynthesis, form the cDNA, i.e. Di-CAR of complete bispecific chimeric antigen receptor gene.
Di-CAR is built in viral vector, and transfects autologous patient NK cell, then NK cell is cultivated to the 21st day, it is thus achieved that Di-CAR-NK cell. Being detected by fluorescent quantitative PCR technique, its expression Di-CAR gene of NKs having transfected Di-CAR viral vector is more than 50 times higher than the NKs of untransfected. Western blotting technique detects its Di-CAR expressing fusion protein level, can express the fusion protein of scFv (SLAMF7)-CAR53kDa and scFv (FNv)-CAR42kDa.
30ml PERIPHERAL BLOOD MONONUCLEAR CELL inducing culture of originating in the method for the invention obtains natural killer cell, and proliferation times reaches more than 1000 times, cultivates and reaches 3 �� 10 to 21 days natural killer cell numbers9Above.
The method of the invention is cultivated the natural killer cell of acquisition, high expressed its specific markers CD16+/CD56+, positive rate is higher than more than 80%, activation receptor positive rate is more than more than 80%, and Inhibitory receptor positive rate is lower than less than 10%, described natural killer cell causes the cellular cytoxicity activity of antibody dependent, and vivo and vitro test has very powerful antitumor and kills toxicity.
The NK cell-stimulating incubator that the present invention uses is for using the coated culture plate of mouse anti human CD16 monoclonal antibody, including 48 orifice plates, 24 orifice plates, 12 orifice plates, 6 orifice plates and culture bottle, it is therefore preferable to 24 orifice plates.
The cell culture medium that the present invention uses is that lymphocyte serum or RPMI1640 culture medium add containing 1-500ng/mlIL-15,1-500ng/mlIL-12,1-500ng/mlIL-18,10-2000 active unit (IU)/mlIL-2 and 10% (volume ratio) autoserum or hyclone, preferably, cell culture medium is lymphocyte serum, such as CellGroSCGM, AIM-V, X-VIVO and GT-T551 etc., containing 1-500ng/mlIL-15,1-500ng/mlIL-12,1-500ng/mlIL-18 and 10-2000IU/mlIL-2.
Present invention also offers the test kit of a kind of natural killer cell preparing bispecific chimeric antigen receptor gene, it is characterised in that described test kit includes:
(1) viral vector of stably express Di-CAR as above is obtained;
(2) NK cytokine, including IL-15, IL-12, IL-18 and IL-2;
(3) mouse-anti people CD16 is coated 24 orifice plates;
(4) lymphocytes culture medium, including RPMI1640 or serum-free medium;
(5) lymphocyte separation medium;
(6) normal saline;
(7) operation instructions;
Wherein, described operation instructions include method as above.
Present invention also offers said method and test kit prepares NK cell, can be used for the immunization therapy of melanoma, uveal and melanoma of choroid.
Accompanying drawing explanation
Fig. 1 is expressed as the bispecific chimeric antigen receptor fusion protein schematic diagram built;
Fig. 2 cultivate 21 days after being expressed as the Di-CAR slow-virus transfection NK cell of embodiment two preparation after its expression;
Fig. 3 is expressed as Di-CAR fusion protein expression in Di-CAR slow-virus transfection NK cell of western blotting technique detection embodiment two preparation;
Fig. 4 is expressed as the melanoma patients Di-CAR slow-virus transfection NK cell FCM analysis of embodiment two preparation;
Fig. 5 is expressed as the advanced melanoma of embodiment two preparation and uveal patient originates the Di-CAR slow-virus transfection NK cell killing activity to A875;
Fig. 6 is expressed as the advanced melanoma of embodiment two preparation and uveal patient originates the Di-CAR slow-virus transfection NK cell killing activity to MUM-2B;
Fig. 7 is expressed as the complete tumor size change curve afterwards of lotus uveal MUM-2BSCID mouse model NK cell therapy.
Detailed description of the invention
Present invention discover that double; two distinctive embedment antigen receptor gene recombinaton of specific bond SLAMF7 and FNv are to viral vector and transfected with human natural killer cell, this bispecific chimeric antigen receptor gene of high expressed, can the tumor cell of specific bond expression signal lymphocyte activator molecule families member 7 and fibronectin variant, and suppression of natural killer cell inhibiting expression of receptor, it is to avoid tumor cell immune evasion; Activate the first signal and costimulatory signal simultaneously and cause antitumor cell toxic activity, vivo and vitro test have activated antitumor response consumingly.
The preparation method of the natural killer cell of a kind of bispecific chimeric antigen receptor genetic modification that the present invention relates to is specifically described.
The preparation method that the present invention relates to the natural killer cell of a kind of bispecific chimeric antigen receptor genetic modification, it is characterized in that, it is built into the double; two distinctive embedment antigen receptor gene recombinaton containing specific bond SLAMF7 and FNv to viral vector and transfected with human natural killer cell, this bispecific chimeric antigen receptor gene of high expressed, can the tumor cell of specific bond expression signal lymphocyte activator molecule families member 7 and fibronectin variant, and suppression of natural killer cell inhibiting expression of receptor, it is to avoid tumor cell immune evasion; Activate the first signal and costimulatory signal simultaneously and cause antitumor cell toxic activity, vivo and vitro test have activated antitumor response consumingly.
SLAMF7 and the FNv antigen that in of the present invention pair of distinctive embedment antigen receptor gene, SLAMF7 and FNv single-chain antibody specific bond malignant cell is expressed, NK cell is made to produce antineoplastic targeting specificity, preferably, for targeting melanoma, uveal and melanoma of choroid etc.
The distinctive embedment antigen receptor gene of fibronectin variant of the present invention, including the single-chain antibody of fibronectin variant and linked the genetic fragment of transcription factor mutant (c-Myc mutant) of specific bond Inhibitory receptor gene promoter, suppression of natural killer cell expression inhibiting receptor, avoid tumor cell immune evasion, its high expressed activation receptor specific bond tumor cell surface part and produce cytotoxic effect. Described transcription factor c-Myc mutant, it is preferable that be g by this gene c1641 site mutation, namely leucine (Leu362) sports valine, in combinations with KIR upstream region of gene promoter and inhibit it to transcribe.
Distinctive embedment antigen receptor gene containing specific bond signal lymphocyte activator molecule families member 7 of the present invention, genetic fragment including the single-chain antibody of specific bond signal lymphocyte activator molecule families member 7 connected two costimulatory moleculeses born of the same parents' internal area (CD28 and CD137) and CD3 �� intracellular signal district, the first signal and costimulatory signal can be activated, namely activate immune response and cause antitumor cell toxic activity and tumor death effect.
Having linked furin cleavage site between described bispecific chimeric antigen receptor gene, it can produce the Chimeric antigen receptor of two independent functions after intracellular protein is translated simultaneously.
Of the present invention by people's IFN ��-signal peptide-scFv (SLAMF7)-CD8a hinge region-CD28-CD137-CD3 �� intracellular region, and scFv (FNv)-IgD hinge region-c-Myc mutant is linked together by Furin cleavage site and catenation sequence (Linker), form recombination sequence, gene order is obtained by chemosynthesis, form the cDNA, i.e. Di-CAR of complete bispecific chimeric antigen receptor gene.
By two specific chimeric antigen receptor genes, i.e. people's IFN ��-signal peptide-scFv (SLAMF7)-CD8a hinge region-CD28-CD137-CD3 �� intracellular region, and scFv (FNv)-IgD hinge region-c-Myc mutant is linked together by Furin cleavage site and catenation sequence, form recombination sequence, sequence is obtained by chemosynthesis, form the cDNA, i.e. Di-CAR of complete bispecific chimeric antigen receptor gene;
After the target DNA fragment of Di-CAR and pUC229 carrier HindIII/BamHI double digestion, under T4DNA ligase effect, both are connected liquid in coupled reaction in 4 DEG C, 12 hours preparation clone, carries out positive colony PCR qualification after converting competent escherichia coli cell DH5a and order-checking is identified; After PCR primer detected through gel electrophoresis and order-checking qualification meet Di-CAR size and sequence, by checking order, correct bacterium solution is transferred in 10ml containing in corresponding antibiotic LB fluid medium, 37 DEG C of overnight incubation, carrying middle amount test kit with sky, Beijing root biology carry out plasmid extraction without endotoxin plasmid is little, the qualified recombiant plasmid of extracting places-80 DEG C of medium-term and long-term preservations of ultra cold storage freezer;
After the target DNA fragment of Di-CAR and pFLAG-CMV-2 carrier HindIII/BamHI double digestion, under T4DNA ligase effect, both are connected liquid in coupled reaction in 37 DEG C, 12 hours preparation clone, carries out positive colony PCR qualification after converting competent escherichia coli cell DH5a and order-checking is identified;
Take cell state good, be in the 293FT cell of exponential phase, after cell counting, according to the culture dish 6 �� 10 of each 10cm6Individual cell number is inoculated in culture dish, 37 DEG C, 5%CO2Incubator in overnight incubation; Remove culture fluid before transfection in second day, change 5mlOpti-MEM culture fluid; Taking 9 �� g to pack in mixed liquor and 3 �� g slow virus expression plasmids addition 1.5mlOpti-MEM, mix gently, take 36 �� llipofectamine2000 and add in 1.5mlOpti-MEM, mix gently, room temperature places 5min; Mixing plasmid solution and lipofectamine2000 diluent, put room temperature 20min; Mixed liquor is slowly added dropwise to the culture fluid of 293FT cell, and mixing, in 37 DEG C, 5%CO2Cell culture incubator is cultivated; Discarding the culture medium containing transfection mixture after cultivating 6h, the PBS liquid adding 10ml cleans once, softly rocks culture dish to abandon after washing remaining transfection mixture; It is slowly added to the cell culture medium 20ml containing 10% serum, in 37 DEG C, containing 5%CO2Continue in incubator to cultivate 48-72h;
According to cell state, collect the 293FT cell conditioned medium liquid of 48h after transfecting; In 4 DEG C, 4000g is centrifuged 10min, removes cell debris; With 0.45 ��m of frit supernatant in 40ml ultracentrifugation pipe; Trim sample, puts into Beckman supercentrifuge one by one by the ultracentrifugation pipe with vial supernatant respectively, and arranging parameter of noncentricity is 25000rpm, and centrifugation time is 2h, and centrifuging temperature controls at 4 DEG C; After centrifugal end, supernatant discarded, remove the liquid remaining on tube wall as far as possible, add virus preservation liquid, repeatedly blow and beat resuspended gently; After fully dissolving, high speed centrifugation 10000rpm, after centrifugal 5min, take supernatant fluorescence spectrometry titre, virus, according to 50 �� l2E+8TU/ml subpackages, is stored in-80 DEG C of ultra cold storage freezers.
In the method for the invention, natural killer cell derives from the mononuclearcell of autologous vein blood, autologous bone marrow, Cord blood and placental blood etc. Preferably, derive from cancer patient perform the operation one month after, the fresh peripheral blood that gathers after one month of chemicotherapy or bone marrow.
Collection derives from autologous patient peripheral blood, after lymphocyte separation medium separation purification obtains mononuclearcell, resuspended by RPMI1640 culture medium and be diluted to 2-5 �� 106Cell/ml, and supplementary 1-500ng/mlIL-15,1-500ng/mlIL-12,1-500ng/mlIL-18,10-2000 active unit (IU)/mlIL-2 and 10% autoserum, every hole 1.2ml joins in coated 24 orifice plates of mouse anti human CD16, in 37 DEG C, containing 5%CO2Continue in incubator to cultivate by the 5th day.5th day harvested by centrifugation NK cell, and expect blue counting by tongue.
5th day with 3 �� 106Cell/mlNK cell joins six well culture plates, and every hole is 2ml, in 37 DEG C, containing 5%CO2Overnight incubation in incubator. Within 6th day, being joined in six well culture plates of people NK by 20 �� l1E+7TU/ml restructuring Di-CAR slow viruss, system is 2ml, mixing, 37 DEG C, 5%CO2Incubator in hatch 8-12 hour after, changing complete culture solution RPMI16404ml, described complete culture solution is containing 1-500ng/mlIL-15,1-500ng/mlIL-12,1-500ng/mlIL-18,10-2000 active unit (IU)/mlIL-2 and 10% autoserum; When NK cell density reaches 6 �� 106During cell/ml, proceed to 75cm2Culture bottle grows, corresponding 1 culture bottle in 6 holes, supplement complete culture solution RPMI1640 to 50ml cultivating system; In 37 DEG C, containing 5%CO2Cultivating by the 15-21 days in incubator, period changes according to NK cell density and culture medium color supplements the fresh complete culture solution RPMI1640 of the autoserum containing 10-2000IU/mlIL-2 and 10%, and expands bottle to 175cm2In culture bottle or transfer in 1.5L culture bag grow.
The NK cell cultivated the 21st day is detected by fluorescent quantitative PCR technique, and its expression Di-CAR gene of NKs having transfected Di-CAR viral vector is more than 50 times higher than the NKs of untransfected. Western blotting technique detects its Di-CAR expressing fusion protein level, can express the fusion protein of scFv (SLAMF7)-CAR53kDa and scFv (FNv)-CAR42kDa.
30ml PERIPHERAL BLOOD MONONUCLEAR CELL inducing culture of originating in the method for the invention obtains natural killer cell, and proliferation times reaches more than 1000 times, cultivates and reaches 3 �� 10 to 21 days natural killer cell numbers9Above.
The method of the invention is cultivated the natural killer cell of acquisition, high expressed its specific markers CD16+/CD56+, positive rate is higher than more than 80%, activation receptor positive rate is more than more than 80%, and Inhibitory receptor positive rate is lower than less than 10%, described natural killer cell causes the cellular cytoxicity activity of antibody dependent, and vivo and vitro test has very powerful antitumor and kills toxicity.
The NK cell-stimulating incubator that the present invention uses is for using the coated culture plate of mouse anti human CD16 monoclonal antibody, including 48 orifice plates, 24 orifice plates, 12 orifice plates, 6 orifice plates and culture bottle, it is therefore preferable to 24 orifice plates.
The cell culture medium that the present invention uses is that lymphocyte serum or RPMI1640 culture medium add containing 1-500ng/mlIL-15,1-500ng/mlIL-12,1-500ng/mlIL-18,10-2000 active unit (IU)/mlIL-2 and 10% (volume ratio) autoserum or hyclone, preferably, cell culture medium is lymphocyte serum, such as CellGroSCGM, AIM-V, X-VIVO and GT-T551 etc., containing containing 1-500ng/mlIL-15,1-500ng/mlIL-12,1-500ng/mlIL-18 and 10-2000IU/mlIL-2.
Present invention also offers the test kit of a kind of natural killer cell preparing bispecific chimeric antigen receptor gene, it is characterised in that described test kit includes:
(1) viral vector of stably express Di-CAR as above is obtained;
(2) NK cytokine, including IL-15, IL-12, IL-18 and IL-2;
(3) mouse-anti people CD16 is coated 24 orifice plates;
(4) lymphocytes culture medium, including RPMI1640 or serum-free medium;
(5) lymphocyte separation medium;
(6) normal saline;
(7) operation instructions;
Wherein, described operation instructions include method as above.
In cultivating as NK cell proliferation of the present invention, the Tissue Culture Plate of use, Tissue Culture Dish, culture bottle, cell culture bags, can be exemplified by 75cm2Tissue Culture Flask, 175cm2Tissue Culture Flask, 225cm2The cells such as Tissue Culture Flask, 1L or 2L cell culture bags are cultivated with equipment (container), are used equally to the present invention, it is preferable that cell culture bags.
Carrying out frozen to NK cell in the manufacture method of the present invention, to frozen stock solution without particular restriction, but be preferably such as 50% calf serum, 40% cell culture fluid and 10% dimethyl sulfoxide, wherein cell culture fluid is more preferably NK cell culture fluid.
Serum can be added in the medium or blood plasma is cultivated. They additions in the medium are not particularly limited, and as more than 0 capacity % to 20 capacity %, and can change the consumption of serum or blood plasma according to different cultivation stages, it is preferred to 5% (volume ratio). For example, it is possible to interim minimizing serum or plasma concentration use. Additionally, source as serum or blood plasma, any one that can be oneself in (meaning identical from institute's cultured cells source) or non-oneself (meaning different with the source of institute cultured cells), set out from a security point, it is preferable that the serum in oneself source or blood plasma. Alternatively, it is also possible to add the serum composition of purification as separated in human serum albumin etc.
The preparation of the autologous NK cells propagation of the present invention uses above-mentioned various compositions and culture medium to implement. The cultivation condition of culture used in the present invention is also without particular restriction, it is possible to use the condition that common cell uses in cultivating. Such as, can at 37 DEG C, 5%CO2Cultivate etc. under condition. Can also implement such as inferior operation: interval reasonable time adds fresh culture and carrys out diluting cells culture fluid, or changes culture medium, or changes cell cultivation equipment etc.
The present invention also provides for NK cell antineoplaston repeatedly in clinical practice, plays anti-tumor immune response better in vivo, reaches good therapeutic effect. In addition, above-mentioned NK cell also has the advantage that, repeatedly NK cell therapy will cause NK cell to kill tumor activity better, prevent immunosurveillance escape, activate body autoimmune response, produce efficiently, antineoplastic immune effect longer, be therefore especially advantageous for improving and the prolongation of life cycle of patient's curative effect.
Hereinafter, the present invention is done in conjunction with the embodiments and describe more specifically, but the invention is not restricted to this.
Embodiment one
Di-CAR is gene constructed and the preparation of slow virus in restructuring
By people's IFN ��-signal peptide-scFv (SLAMF7)-CD8a hinge region-CD28-CD137-CD3 �� intracellular region, and scFv (FNv)-IgD hinge region-c-Myc mutant is consisted of the genetic fragment of Furin cleavage site and catenation sequence, 362nd leucine of c-Myc mutant sports valine, i.e. c-Myc-L/V, see Fig. 1, i.e. Di-CAR fusion protein schematic diagram.
By people's IFN ��-signal peptide-scFv (SLAMF7)-CD8a hinge region-CD28-CD137-CD3 �� intracellular region, and scFv (FNv)-IgD hinge region-c-Myc mutant is linked together by Furin cleavage site and catenation sequence (Linker), form recombination sequence, gene order is obtained by chemosynthesis, form the cDNA, i.e. Di-CAR of complete bispecific chimeric antigen receptor gene;
After the target DNA fragment of Di-CAR and pUC229 carrier HindIII/BamHI double digestion, under T4DNA ligase effect, both are connected liquid in coupled reaction in 4 DEG C, 12 hours preparation clone, carries out positive colony PCR qualification after converting competent escherichia coli cell DH5a and order-checking is identified; After PCR primer detected through gel electrophoresis and order-checking qualification meet Di-CAR size and sequence, by checking order, correct bacterium solution is transferred in 10ml containing in corresponding antibiotic LB fluid medium, 37 DEG C of overnight incubation, carrying middle amount test kit with sky, Beijing root biology carry out plasmid extraction without endotoxin plasmid is little, the qualified recombiant plasmid of extracting places-80 DEG C of medium-term and long-term preservations of ultra cold storage freezer; Described Di-CAR size is about 2607bp;
After the target DNA fragment of Di-CAR and pFLAG-CMV-2 carrier HindIII/BamHI double digestion, under T4DNA ligase effect, both are connected liquid in coupled reaction in 37 DEG C, 12 hours preparation clone, carries out positive colony PCR qualification after converting competent escherichia coli cell DH5a and order-checking is identified;
Take cell state good, be in the 293FT cell of exponential phase, after cell counting, according to the culture dish 6 �� 10 of each 10cm6Individual cell number is inoculated in culture dish, 37 DEG C, 5%CO2Incubator in overnight incubation; Remove culture fluid before transfection in second day, change 5mlOpti-MEM culture fluid; Taking 9 �� g to pack in mixed liquor and 3 �� g slow virus expression plasmids addition 1.5mlOpti-MEM, mix gently, take 36 �� llipofectamine2000 and add in 1.5mlOpti-MEM, mix gently, room temperature places 5min; Mixing plasmid solution and lipofectamine2000 diluent, put room temperature 20min; Mixed liquor is slowly added dropwise to the culture fluid of 293FT cell, and mixing, in 37 DEG C, 5%CO2Cell culture incubator is cultivated; Discarding the culture medium containing transfection mixture after cultivating 6h, the PBS liquid adding 10ml cleans once, softly rocks culture dish to abandon after washing remaining transfection mixture; It is slowly added to the cell culture medium 20ml containing 10% serum, in 37 DEG C, containing 5%CO2Continue in incubator to cultivate 72h.
According to cell state, collect the 293FT cell conditioned medium liquid of 48h after transfecting; In 4 DEG C, 4000g is centrifuged 10min, removes cell debris; With 0.45 ��m of frit supernatant in 40ml ultracentrifugation pipe; Trim sample, puts into Beckman supercentrifuge one by one by the ultracentrifugation pipe with vial supernatant respectively, and arranging parameter of noncentricity is 25000rpm, and centrifugation time is 2h, and centrifuging temperature controls at 4 DEG C; After centrifugal end, supernatant discarded, remove the liquid remaining on tube wall as far as possible, add virus preservation liquid, repeatedly blow and beat resuspended gently; After fully dissolving, high speed centrifugation 10000rpm, after centrifugal 5min, take supernatant fluorescence spectrometry titre, virus, according to 50 �� l2E+8TU/ml subpackages, is stored in-80 DEG C of ultra cold storage freezers, has namely prepared into Di-CAR slow virus.
Embodiment two
The preparation of the natural killer cell of bispecific chimeric antigen receptor genetic modification
Gather advanced melanoma and uveal patient 30ml (signing Informed Consent Form with it), add equivalent 1 �� PBS dilution, it is superimposed on gently on lymphocyte separation medium along centrifuge tube tube wall, form sharp interface, the centrifugal 20min of 2000rpm under room temperature, draw the PBMC of boundary layer, wash twice with 1 �� PBS (pH value is 7.4). After tongue dish orchid counting, resuspended by RPMI1640 culture medium and be diluted to 3 �� 106Cell/ml, resuspended by RPMI1640 culture medium and be diluted to 3 �� 106Cell/ml, and supplementary 50ng/mlIL-15,20ng/mlIL-12,20ng/mlIL-18,100IU/mlIL-2 and 10% autoserum, every hole 1.2ml joins in coated 24 orifice plates of mouse anti human CD16, in 37 DEG C, containing 5%CO2Continue in incubator to cultivate by the 5th day.5th day harvested by centrifugation NK cell, and expect blue counting by tongue.
5th day with 3 �� 106Cell/mlNK cell joins six well culture plates, and every hole is 2mlRPMI1640 culture medium (containing 50ng/mlIL-15,20ng/mlIL-12,20ng/mlIL-18,100IU/mlIL-2 and 10% autoserum), in 37 DEG C, containing 5%CO2Overnight incubation in incubator. Within 6th day, being joined in six well culture plates of people NK by 20 �� l1E+7TU/ml restructuring Di-CAR slow viruss, system is 2ml, mixing, 37 DEG C, 5%CO2Incubator in hatch 8-12 hour after, change complete culture solution RPMI16404ml, described complete culture solution is containing 50ng/mlIL-15,20ng/mlIL-12,20ng/mlIL-18,100IU/mlIL-2 and 10% autoserum; When NK cell density reaches 6 �� 106During cell/ml, NK cell is transferred to growth 75cm in 1.5L culture bag2Culture bottle, supplements complete culture solution RPMI1640 to 100ml cultivating system; In 37 DEG C, containing 5%CO2Cultivating by the 21st day in incubator, period changes according to NK cell density and culture medium color supplements complete medium RPMI1640 (containing 50ng/mlIL-15,20ng/mlIL-12,20ng/mlIL-18,100IU/mlIL-2 and 10% autoserum).
Fig. 2 is its expression after cultivating 21 days after Di-CAR slow-virus transfection NK cell. Detected according to operation instructions (American I nvitrogen company) by PCR kit for fluorescence quantitative, the NK cell cultivated the 21st day is detected by fluorescent quantitative PCR technique, and the NKs that its expression Di-CAR gene of NKs having transfected Di-CAR virus transfection advanced melanoma and uveal patient is respectively higher than untransfected is 68.9 and 59.5 times. CAR-mNK is the melanoma patients source NK cell of Di-CAR slow-virus transfection, CAR-uNK is that the uveal patient of Di-CAR slow-virus transfection originates NK cell, Blank-mNK is the melanoma patients source NK cell of blank slow-virus transfection, Blank-uNK is that the uveal patient of blank slow-virus transfection originates NK cell, mNK is melanoma patients source NK cell, and uNK is that uveal patient originates NK cell.
Embodiment three
The natural killer cell Western blot of bispecific chimeric antigen receptor genetic modification and FCM analysis
The advanced melanoma of Example two preparation, uveal patient and normal healthy people are originated the 15th day and are cultivated the NK cell each 1 �� 10 obtained6, by carrying out proteins gel electrophoresis after ultrasonication. Molecular weight according to destination protein, prepares 10% separation gel, and concentration gum concentration is 5%. Protein sample applied sample amount to be detected: 20 �� g/ holes. Deposition condition: concentration glue constant voltage 90V, about 20 minutes; Separation gel constant voltage 120V, determines electrophoresis dwell time by standard molecular weight pre-dyed albumen. Wet robin, transferring film condition: 300mA constant current; 0.45 ��m of aperture pvdf membrane, 80 minutes transferring film time. After transferring film completes, film is dyeed by Ponceaux staining reagent, observes transferring film effect. Close: being totally submerged by film in the transferring film buffer (containing volume ratio 3% tween 20, TBST) in 5% (mg/mL) bovine serum albumin (BSA), under room temperature, horizontal shaker hatches 1 hour. Primary antibodie is hatched: 5% (mg/mL) BSA-TBST dilutes the goat anti-mouse IgG (H+L) 1: 2000 of marked by streptavidin, purchased from the green skies Bioisystech Co., Ltd in Shanghai; 4 DEG C of horizontal shaker overnight incubation.Next day, wash film: TBST washes 3 times, each 10 minutes. Chemical luminous substrate ECL is added drop-wise to the protein powder of film, reacts 3-5 minute; Exposure: 10 seconds-5 minutes (time of exposure adjusts with different light intensities), develops 2 minutes, fixing.
Film scanner scanning is the TIF format picture more than 300DPI, use QuantityOne to analyze software (Bio-Rad company) and carry out gray analysis, Fig. 3 is the NK cell that western blotting technique detection Di-CAR fusion protein is originated at Di-CAR slow-virus transfection advanced melanoma and uveal patient, and Di-CAR fusion protein expression in escherichia coli level (positive control), i.e. westernblotting result figure, all there are two bands in two example patients, wherein one is SLAMF7-CAR, it is about 482 amino acid residues, molecular weight is about 53kD, another is FNv-CAR, about 383 amino acid residue, and molecular weight is about 42kD. 1st band is Di-CAR slow-virus transfection advanced melanoma NK cell, 2nd band is Di-CAR slow-virus transfection grape in late period film melanoma NK cell, 3rd band is Di-CAR fusion protein expression in escherichia coli, band molecular weight is about 95kD, i.e. SLAMF7-CAR and FNv-CAR molecular weight sum.
The advanced melanoma of Example two preparation, uveal patient and normal healthy people are originated the 15th day and are cultivated obtain 1 �� 106NK cell, carries out the detection of streaming antibody staining and analyzes. First group of NK cell respectively 20 �� L mouse-anti people CD16-FITC, 20 �� L mouse-anti people CD56-PE and 20 �� L mouse-anti people CD3-PerCP; Second component is not 20 �� L mouse-anti people NKG2D-FITC, 20 �� L mouse-anti people NKp46-PE and 20 �� L mouse-anti people KIR3DL1-PerCP; Isotype control group is 20 �� LmIgG1-FITC, 20 �� LmIgG1-PE and 20 �� LmIgG1-PerCP; Place and wash 3 times with PBS after 4 DEG C of refrigerators dye 30 minutes, then in the upper machine testing of FACSCalibur instrument (U.S. company BD) and analysis.
Fig. 4 is the melanoma patients Di-CAR slow-virus transfection NK cell FCM analysis of embodiment two preparation, and this NK cell CD16 and CD56 positive rate are 94.44%, and CD3 positive rate is 3.4%; Activation receptor NKG2D and NKp46 positive rate respectively 93.04% and 75.84%; Inhibitory receptor KIR3DL1 positive rate is 8.13%. The uveal patient of embodiment two preparation and normal person Di-CAR slow-virus transfection NK cell CD16 and CD56 positive rate are all more than more than 85%, and CD3 positive rate is lower than 10%; Activation receptor NKG2D and NKp46 positive rate are respectively higher than more than 75%; Inhibitory receptor KIR3DL1 positive rate is lower than less than 10%. And the NK cell CD16 of untransfected and CD56 positive rate are all also above more than 85%, CD3 positive rate is also below 10%; Activation receptor NKG2D and NKp46 positive rate are also respectively higher than more than 65%; But Inhibitory receptor KIR3DL1 high expressed, positive rate is more than more than 50%.
Embodiment four
The natural killer cell of bispecific chimeric antigen receptor genetic modification is external kills tumor test
Respectively with K-1735 A875 and uveal melanoma cells system MUM-2B for target cell, with the Di-CAR slow-virus transfection NK cell action effect cell of preparation in embodiment two, it is separately added in 96 well culture plates by effect target than 40: 1,20: 1,10: 1,5: 1 and 2.5: 1, be subsequently placed in 37 DEG C, 5%CO2 when cultivate 4h, be all provided with 3 multiple holes.Adopting LDH cellulotoxic experiment method to measure NK activity: Aspirate supernatant 50 �� L, be placed in 96 orifice plates, add LDH base fluid 50 �� L, room temperature lucifuge 30min, add 50 �� L stop buffers and terminate reaction, 490nm wavelength place surveys absorbance (OD) value. The killing rate of PC-3 and MCF-7 cell is calculated respectively: killing rate=(experimental group OD value-effector lymphocyte's Spontaneous release group OD value-target cell Spontaneous release group OD value)/(the maximum release group OD value of target cell-target cell Spontaneous release group OD value) �� 100% by following equation.
Fig. 5 is advanced melanoma and uveal patient the originates Di-CAR slow-virus transfection NK cell killing activity to A875, along with effect target ratio improves, cellular cytoxicity activity also improves constantly, Di-CAR slow-virus transfection melanoma patients NK cell killing A875 kills toxicity and is significantly higher than the NK cell of untransfected, p value is 0.016, and compared with Di-CAR slow-virus transfection uveal patient's NK cell zero difference. Fig. 6 is advanced melanoma and uveal patient the originates Di-CAR slow-virus transfection NK cell killing activity to MUM-2B, along with effect target ratio improves, cellular cytoxicity activity also improves constantly, Di-CAR slow-virus transfection uveal patient NK cell killing MUM-2B kills toxicity and is significantly higher than the NK cell of untransfected, p value is 0.021, and compared with Di-CAR slow-virus transfection melanoma patients NK cell zero difference.
Embodiment five
The effect in tumor experiment is killed in the natural killer cell body of bispecific chimeric antigen receptor genetic modification
24 8 weeks SCID nude mice abdominal part flank subcutaneous injections 5 �� 106Uveal system MUM-2B, after raising 7 days, is randomly divided into A, B, C and D4 group, and often group 6: A group is Di-CAR slow-virus transfection uveal patient's NK cell therapy group of embodiment two preparation; B group is blank slow-virus transfection uveal patient's NK cell therapy group of embodiment two preparation; C group is uveal patient's NK cell therapy group of the untransfected of embodiment two preparation; D group is normal saline placebo group. A group cultivates the 14th day and the 16th day tail vein injection 2 �� 10 at Di-CAR slow-virus transfection uveal patient's NK cell4Cell/gram, B group cultivates the 14th day and the 16th day tail vein injection 2 �� 10 at blank slow-virus transfection uveal patient's NK cell4Cell/gram, C group cultivates the 14th day and the 16th day tail vein injection 2 �� 10 at untransfected uveal patient's NK cell4Cell/gram, D group and A, B and C treatment group same time, the normal saline of tail vein injection same volume, each 1mL. Draw neck to put to death respectively at 7d, 14d, 21d, 28d and 35d after treatment, take lotus carcinoma of prostate MUM-2BSCID mouse model tumor tissues, calculate mouse model lotus tumor size.
Tumor size change curve after Fig. 7 lotus uveal MUM-2BSCID mouse model NK cell therapy is complete, compared with normal saline group D and CAR-uNK treatment group A, Blank-uNK treatment group B and uNK treatment group C, treatment group A, treatment group B reduce with uveal tumor size in treatment group C, but treatment group A and treatment group B uveal tumor size compared with treatment group C reduces and becomes apparent from, p value respectively 0.004 and 0.006, it was shown that Di-CAR slow-virus transfection uveal patient's NK cell has caused and powerful internal kills tumor activity.
Claims (10)
1. the preparation method of the natural killer cell of a bispecific chimeric antigen receptor genetic modification, it is characterized in that, it is built into containing specific bond signal lymphocyte activator molecule families member 7 (SignalingLymphocyticActivationMoleculeFamily, Member7, and fibronectin variant (fibronectinvariant SLAMF7), FNv) bispecific chimeric antigen receptor (doublechimericantigenreceptor, Di-CAR) gene recombinaton is to viral vector and transfected with human natural killer cell (Naturalkillercells, NK),
The cDNA of described bispecific chimeric antigen receptor (Di-CAR) gene builds in viral vector, and transfected NK cells, then NK cell is cultivated the 21st day, it is thus achieved that Di-CAR-NK cell; Being detected by fluorescent quantitative PCR technique, its expression Di-CAR gene of NKs having transfected Di-CAR viral vector is more than 50 times higher than the NKs of untransfected;
The natural killer cell obtained, this bispecific chimeric antigen receptor gene of high expressed, can the tumor cell of specific bond expression signal lymphocyte activator molecule families member 7 and fibronectin variant, and the cell inhibiting expression of receptor of suppression of natural killer, prevent tumor cell immune evasion, activating the first signal and costimulatory signal simultaneously and cause antitumor cell toxic activity, vivo and vitro test antitumor has and kills very by force toxicity; Its high expressed its specific markers CD16+/CD56+, positive rate is higher than more than 80%, and activation receptor positive rate is more than more than 80%, and Inhibitory receptor positive rate is lower than less than 10%.
2. method according to claim 1, it is characterized in that, the distinctive embedment antigen receptor gene of described fibronectin variant, including the single-chain antibody of fibronectin variant and linked the genetic fragment of transcription factor mutant (c-Myc mutant) of specific bond Inhibitory receptor gene promoter.
3. the method according to any one of claim 1-2, it is characterized in that, the described distinctive embedment antigen receptor gene containing specific bond signal lymphocyte activator molecule families member 7, including the genetic fragment of the single-chain antibody of specific bond signal lymphocyte activator molecule families member 7 connected two costimulatory molecules born of the same parents' internal areas and CD3 �� intracellular signal district; Said two costimulatory molecules is CD28 and CD137.
4. according to the method in any one of claims 1 to 3, it is characterized in that, having linked furin cleavage site between described bispecific chimeric antigen receptor gene, it can produce the Chimeric antigen receptor of two independent functions after intracellular protein is translated.
5. method according to any one of claim 1 to 4, it is characterised in that described natural killer cell derives from the mononuclearcell of autologous vein blood, autologous bone marrow, Cord blood and placental blood etc.
6. the preparation method of the natural killer cell of a bispecific chimeric antigen receptor genetic modification, it is characterised in that
By two specific chimeric antigen receptor genes, i.e. people's IFN ��-signal peptide-scFv (SLAMF7)-CD8a hinge region-CD28-CD137-CD3 �� intracellular region, and scFv (FNv)-IgD hinge region-c-Myc mutant is linked together by Furin cleavage site and catenation sequence, form recombination sequence, sequence is obtained by chemosynthesis, form the cDNA, i.e. Di-CAR of complete bispecific chimeric antigen receptor gene;
After the target DNA fragment of Di-CAR and pUC229 carrier HindIII/BamHI double digestion, under T4DNA ligase effect, in coupled reaction in 4 DEG C, 12 hours preparation, both are cloned connection liquid, and the PCR carrying out positive colony after converting competent escherichia coli cell DH5a identifies and order-checking qualification; After PCR primer detected through gel electrophoresis and order-checking qualification meet Di-CAR size and sequence, by checking order, correct bacterium solution is transferred in 10ml containing in corresponding antibiotic LB fluid medium, 37 DEG C of overnight incubation, carrying middle amount test kit with sky, Beijing root biology carry out plasmid extraction without endotoxin plasmid is little, the qualified recombiant plasmid of extracting places-80 DEG C of medium-term and long-term preservations of ultra cold storage freezer;
After the target DNA fragment of Di-CAR and pFLAG-CMV-2 carrier HindIII/BamHI double digestion, under T4DNA ligase effect, in coupled reaction in 37 DEG C, 12 hours preparation, both are cloned connection liquid, and the PCR carrying out positive colony after converting competent escherichia coli cell DH5a identifies and order-checking qualification;
Take cell state good, be in the 293FT cell of exponential phase, after cell counting, according to the culture dish 6 �� 10 of each 10cm6Individual cell number is inoculated in culture dish, 37 DEG C, 5%CO2Incubator in overnight incubation; Remove culture fluid before transfection in second day, change 5mlOpti-MEM culture fluid; Taking 9 �� g to pack in mixed liquor and 3 �� g slow virus expression plasmids addition 1.5mlOpti-MEM, mix gently, take 36 �� llipofectamine2000 and add in 1.5mlOpti-MEM, mix gently, room temperature places 5min; Mixing plasmid solution and lipofectamine2000 diluent, put room temperature 20min; Mixed liquor is slowly added dropwise to the culture fluid of 293FT cell, and mixing, in 37 DEG C, 5%CO2Cell culture incubator is cultivated; Discarding the culture medium containing transfection mixture after cultivating 6h, the PBS liquid adding 10ml cleans once, softly rocks culture dish to abandon after washing remaining transfection mixture; It is slowly added to the cell culture medium 20ml containing 10% serum, in 37 DEG C, containing 5%CO2Continue in incubator to cultivate 48-72h;
According to cell state, collect the 293FT cell conditioned medium liquid of 48h after transfecting; In 4 DEG C, 4000g is centrifuged 10min, removes cell debris; With 0.45 ��m of frit supernatant in 40ml ultracentrifugation pipe; Trim sample, puts into Beckman supercentrifuge one by one by the ultracentrifugation pipe with vial supernatant respectively, and arranging parameter of noncentricity is 25000rpm, and centrifugation time is 2h, and centrifuging temperature controls at 4 DEG C; After centrifugal end, supernatant discarded, remove the liquid remaining on tube wall as far as possible, add virus preservation liquid, repeatedly blow and beat resuspended; After fully dissolving, high speed centrifugation 10000rpm, after centrifugal 5min, take supernatant fluorescence spectrometry titre, virus, according to 50 �� l2E+8TU/ml subpackages, is stored in-80 DEG C of ultra cold storage freezers;
The cDNA (Di-CAR) of described bispecific chimeric antigen receptor gene builds in viral vector, and transfected NK cells, then NK cell is cultivated the 21st day, it is thus achieved that Di-CAR-NK cell; Being detected by fluorescent quantitative PCR technique, its expression Di-CAR gene of NKs having transfected Di-CAR viral vector is more than 50 times higher than the NKs of untransfected;
Obtained natural killer cell, its high expressed its specific markers CD16+/CD56+, positive rate is higher than more than 80%, and activation receptor positive rate is more than more than 80%, and Inhibitory receptor positive rate is lower than less than 10%.
7. method according to any one of claim 1 to 6, it is characterised in that
Collection derives from autologous patient peripheral blood, after lymphocyte separation medium separation purification obtains mononuclearcell, resuspended by RPMI1640 culture medium and be diluted to 2-5 �� 106Cell/ml, and supplementary 1-500ng/mlIL-15,1-500ng/mlIL-12,1-500ng/mlIL-18,10-2000IU/mlIL-2 and 10% autoserum, every hole 1.2ml joins in coated 24 orifice plates of mouse anti human CD16, in 37 DEG C, containing 5%CO2Continue in incubator to cultivate by the 5th day; 5th day harvested by centrifugation NK cell, and expect blue counting by tongue.
5th day with 3 �� 106Cell/mlNK cell joins six well culture plates, and every hole is 2ml, in 37 DEG C, containing 5%CO2Overnight incubation in incubator; Within 6th day, being joined in six well culture plates of people NK by 20 �� l1E+7TU/ml restructuring Di-CAR slow viruss, system is 2ml, mixing, 37 DEG C, 5%CO2Incubator in hatch 8-12 hour after, changing complete culture solution RPMI16404ml, described complete culture solution is preferably containing 1-500ng/mlIL-15,1-500ng/mlIL-12,1-500ng/mlIL-18,10-2000 active unit (IU)/mlIL-2 and 10% autoserum; When NK cell density reaches 6 �� 106During cell/ml, proceed to 75cm2Culture bottle grows, corresponding 1 culture bottle in 6 holes, supplement complete culture solution RPMI1640 to 50ml cultivating system; In 37 DEG C, containing 5%CO2Cultivating by the 15-21 days in incubator, period changes according to NK cell density and culture medium color supplements the fresh complete culture solution RPMI1640 of the autoserum containing 10-2000IU/mlIL-2 and 10%, and expands bottle to 175cm2In culture bottle or transfer in 1.5L culture bag grow;
The NK cell cultivated the 21st day is detected by fluorescent quantitative PCR technique, and its expression Di-CAR gene of NKs having transfected Di-CAR viral vector is more than 50 times higher than the NKs of untransfected; Western blotting technique detects its Di-CAR expressing fusion protein level, can express the fusion protein of scFv (SLAMF7)-CAR53kDa and scFv (FNv)-CAR42kDa.
8. method according to any one of claim 1 to 7, it is characterized in that, described method is cultivated and obtains natural killer cell, high expressed its specific markers CD16+/CD56+ positive rate is higher than more than 85%, activation receptor positive rate is more than more than 80%, and Inhibitory receptor positive rate is lower than less than 10%, described natural killer cell causes the cellular cytoxicity activity of antibody dependent, and vivo and vitro test has very powerful antitumor and kills toxicity.
9. the test kit of the natural killer cell preparing bispecific chimeric antigen receptor gene, it is characterised in that described test kit includes;
(1) viral vector of acquisition stably express Di-CAR described in any one of claim 1-8;
(2) NK cytokine, including IL-15, IL-12, IL-18 and IL-2;
(3) mouse-anti people CD16 is coated 24 orifice plates;
(4) lymphocytes culture medium, including RPMI1640 or serum-free medium;
(5) lymphocyte separation medium;
(6) normal saline;
(7) operation instructions;
Wherein, described operation instructions include the method according to any one of claim 1-8.
10. the purposes of the natural killer cell of bispecific chimeric antigen receptor genetic modification according to any one of claim 1-9.
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