A kind of NK cell expansion ex vivo compositions and NK methods for cell expansionTechnical field
The present invention relates to a kind of NK cell amplification compositions and amplification method.
Background technology
The general status of 28 kinds of cancers of " report of world's cancer " of latest edition country individual to the whole world more than 180 and fashion trendHaving carried out comprehensive description and analysis, address prediction whole world cases of cancer will present swift and violent growing trend, the whole world in 2012Increase 14,000,000 cases of cancers altogether newly and have 8,200,000 people dead.Wherein, China increases 3,070,000 cancer patients newly and causes about 220Ten thousand people are dead, account for the 21.9% and 26.8% of whole world total amount respectively.Wherein, pulmonary carcinoma, gastric cancer, hepatocarcinoma are that sickness rate is the highestCancer, and breast carcinoma, colorectal cancer, cervical cancer make women's health be on the hazard.
Immunotherapy of tumors is known as the fourth-largest tumor therapy by domestic and international medical circle, wherein autoimmune cell (T cell,NK cell) treatment technology belongs to health ministry and allows the 3rd class medical skill of clinical practice in the first batch.In recent years, naturally killHinder cell (natural killer cell, NK) immunotherapy techniques and the most become a kind of reliable anticancer treatmentMethod, it is adaptable to clinical treatment and the side effect of Several Kinds of Malignancy are little, do not damage normal structure.NK was in 70 years 20th centuryIn generation, enters the sight line of people, the so far developing history of existing nearly 40 years.NK cell is defined as large granular lymphocyte(LGL), it is to be differentiated by lymphoid progenitor cell, and constitutes three major types immunity carefully with bone-marrow-derived lymphocyte and T lymphocyteBorn of the same parents, only account for the 5-10% of peripheral blood lymphocyte.NK cell can kill target cell (such as virus without contacting antigen in advanceThe host cell infected and tumor cell), owing to the killing activity of NK cell limits without MHC, because the most naturally killingWound activity, it plays an important role during immune surveillance and early stage anti-infectious immunity.The tumor-killing mechanism bag of NK cellInclude: (1) FasL Yu Fas phase separation, finally start target cell Apoptosis System.(2) perforin/granzyme effect, swashsSend out apoptosis relevant enzyme system and cause apoptosis.(3) the CD16 molecule of NK cell surface expression and tumour-specific IgGAntibody combines, thus identifies, kills the tumor cell specific binding with IgG antibody generation.(4)TNF-α、IFN-γDeng the combination of receptor corresponding to target cells, start the Apoptosis System of target cell.NK cell is except target cell is had dissolvingOutside lethal effect, NK cell also has critical function in organism immune response.NK cell can be by thin to M Ф, grainBorn of the same parents, the regulation effect of dendritic cell and control natural immunity.NK cell can act on CD4+T cell and CD8+TCell is to strengthen acquired immunity.
At present, external acquisition NK cells of human beings is mainly carried out by following approach: one, use the method for immunomagnetic beads from PBMCMiddle separation；Two, the method stimulating amplification cultivation is used to expand NK cell from PBMC.The former can quickly obtain NKCell, but a small amount of NK cell can only be obtained and study use altogether.The latter is then with PBMC as original material, passes through certain detailThe stimulation of intracellular cytokine, makes NK cell obtain special amplification in vitro, is cultivated by external stimulation and can carry out relatively large rulePrepared by the NK cell of mould, thus be preferably applied for clinic.But existing in vitro method can only make NK cell in vitroExpand tens of to Radix Achyranthis Bidentatae, and CD3-CD56+ cell proportion is relatively low.The cytokines such as IL-2, IL-15, IL-18 and IL-21Being very important to the growth of NK cell with amplification, meanwhile, 4-1BBL with NK cell surface receptor can be situated between after being combinedThe costimulatory signal led promotes NK cell proliferation, the secretion etc. of cytokine.Research confirms that the cytokines such as IL-2 are NKThe essential condition of cell amplification, but only cytokine does not ensures that NK cell continues and efficient amplification, on a large scaleThe amplification of NK cell also needs to the support of more signaling system.K562 cell line is to suffer from from chronic granulocytic leukemiaExtract the cell line set up in person's body, the amplification of NK cell can be activated.
At present, the main method of NK amplification is that the simple cell factor stimulates TRAP, and its amplification efficiency and purity are relatively low, withTime there is poor repeatability shortcoming.In recent years, occur that some stimulate with the trophocyte that have expressed IL-15 and 4-1BBLNK methods for cell expansion, the method expand 3 weeks after cell number up to 277 times, purity more than 90%, killing activity up to80% (E:T=10:1).But needed for the NK cell quantity that this amplification method is cultivated needs the long period to can be only achieved clinical treatmentQuantity, the most corresponding cost is the most of a relatively high.To sum up, existing NK cell injuring model method need to improve, simultaneouslyThe most more be necessary to design the specification being consistent therewith reagent set incompatible guarantee cultural method timely, accurate, be smoothed out.
Summary of the invention
The present invention is to solve that current NK cells expanded is low, purity is low, amplification cycle length, problem that cost is high, carryFor a kind of NK cell expansion ex vivo compositions and NK methods for cell expansion.
NK cell expansion ex vivo compositions of the present invention include NK amplification trophocyte, IL-2, the autologous plasma of inactivation andGT-T551 H3 culture medium.Every milliliter of GT-T551 H3 culture medium wherein adds 0.65 × 106Individual～1 × 106Individual NK expandsIncrease trophocyte, every milliliter of GT-T551 H3 culture medium adds the IL-2 of 200-500IU, the body of the autologous plasma of inactivationAmassing 5% for GT-T551 H3 culture volume, described NK amplification trophocyte is K562-4-1BBL-IL-15-IL-21Trophocyte.
The present invention is by first the most frozen for the NK amplification trophocyte of inactivation, with when carry out recovery process again.
The preparation method of described K562-4-1BBL-IL-15-IL-21 trophocyte is:
A, utilize method for synthesizing gene, it is thus achieved that the full length coding region sequence of 4-1BBL-IL-15 fusion gene and IL-21 gene,Article two, upstream region of gene all adds CD8 alpha signal peptide, and downstream all adds CD8 α cross-film district；Meanwhile, on CD8 alpha signal peptideTrip introduces Nhe I restriction enzyme site, and CD8 α cross-film district introduces Not I restriction enzyme site.
B, build recombinant slow virus expression plasmid pCDH-CMV-MCS-4-1BBL-IL-15 andPCDH-CMV-MCS-IL-21:
Gene carrier pCDH-CMV-MCS-Vector and step A obtained is respectively with restricted enzyme Nhe IWith Not I double digestion, 1% agarose gel reclaims after purification, is utilized respectively T4DNALigase and is attached, it is thus achieved thatConnect product and be respectively designated as pCDH-CMV-MCS-4-1BBL-IL-15 and pCDH-CMV-MCS-IL-21, through enzyme actionAfter identifying correctly ,-20 DEG C save backup.Wherein said pCDH-CMV-MCS-Vector buys the most biological lucky section from ShanghaiSkill company limited, container name is pCDH-CMV-MCS-EF1-Puro (article No.: CD510B-1).
C, slow virus packaging:
A. dilute, with 235 μ L Opti-MEM, the connection product that 2.7 μ g steps B obtainPCDH-CMV-MCS-4-1BBL-IL-15, adds 1.76 μ g pLp1 (Gal/Pol), 0.95 μ g pLpVSVG and 0.68 μ gPLp2 (pREV), obtains a1 solution；The connection product that 2.7 μ g steps B obtain is diluted with 235 μ L Opti-MEMPCDH-CMV-MCS-IL-21, adds 1.76 μ g pLp1 (Gal/Pol), 0.95 μ g pLpVSVG and 0.68 μ gPLp2 (pREV), obtains a2 solution；
B. dilute 15 μ L lipofectamine2000 with 235 μ L Opti-MEM, obtain b solution；
C.5min, after, a1 solution, a2 solution are mixed with b solution respectively, is added drop-wise to respectively connect after the static 30min of room temperaturePlant in 6 orifice plates of 293-T cell；
D.6-10h after, remove culture medium, add the DMED culture medium containing 10% (v/v) FBS, in 48h and 72hEach receive virus once, final obtain pCDH-CMV-MCS-4-1BBL-IL-15 slow virus andPCDH-CMV-MCS-IL-21 slow virus.
D., after two kinds of slow viruss of results being concentrated to 3E+8 TU/ml, virus mixes than 1:1 with titre, with 1000MOI value carries out the infection of K562 cell line, and being cloned by flow cytometry and limiting dilution assay after infecting 72h can be steadyThe K562-4-1BBL-IL-15-IL-21 engineering cell of fixed heredity.
E. engineering cell strain is carried out 100Gy lonizing radiation and carry out lethal exposure, after irradiation, mix extraction 1 × 106Individual trainSupport, within every 2 days, calculate a Cell viability, find no survivaling cell after adding up 15 days and exist.
F. the K562-4-1BBL-IL-15-IL-21 engineering cell strain frozen stock solution of inactivation is carried out frozen, often pipe 1mL is denseDegree is 1 × 107Individual/mL, removes frozen stock solution with front 37 DEG C of recoveries.The formula of described frozen stock solution is 90% (v/v) FBS+10%(v/v)DMSO。
The method utilizing above-mentioned NK cell expansion ex vivo compositions to carry out NK cell amplification, sequentially includes the following steps:
By 30mL GT-T551 H3 culture medium by 2 × 107Individual～3 × 107After individual PBMCs is resuspended, accesses T75 cell and cultivateBottle, IL-2, NK amplification trophocyte being subsequently adding and the autologous plasma of the inactivation accounting for culture volume 5%, the most oftenMilliliter GT-T551 H3 culture medium adds the IL-2 of 200-500IU, and every milliliter of GT-T551 H3 culture medium adds 0.65 × 106Individual～1 × 106Individual NK expands trophocyte, in 37 DEG C of 5%CO2Under the conditions of co culture system in vitro 15 days, wherein cultivate the 7thIt time add NK and expand trophocyte, before adding, the NK cell in culture fluid is counted, the NK added expandsIncrease the 1/5-1/4 that quantity is NK cell quantity of trophocyte.In incubation, NK cell observation is counted by every day, whenNK cell density is more than 2.5 × 106/ mL must be supplemented with culture medium, and the culture medium added is for containing 200-500IU/mL IL-2GT-T551 H3 culture medium, after fluid infusion, NK cell density maintains 0.8 × 106Individual/mL～1.0 × 106Individual/mL, cultivatesWithin 13 and 15 days, collect cell to detect.
Described NK amplification trophocyte is K562-4-1BBL-IL-15-IL-21 trophocyte.
Beneficial effects of the present invention:
(1) present invention is that the method being packed and infecting K562 cell by slow virus carries out 4-1BBL, IL-15, IL-21The transduction of gene, the blood cell of suspension and tumor cell belong to difficult transfectional cell, and liposome transfection method effect is extremely low, and electricity is wornHole method efficiency is higher, but relatively big to cell injury, and dead cell is very difficult to remove, the screening operation after impact transfection.Slow sickPoison infection method is a kind of a kind of gentleer to cell and that transfection efficiency is higher method, solves suspension cell transfection and is facedThe problems referred to above.
(2) present invention first carries out selected by flow cytometry apoptosis with the purplish red element IL-21 antibody of labelling and IL-15 antibody, removesNegative K562 cell, then double positive cell groups of screening are carried out limiting dilution assay clone can stablize hereditaryK562-4-1BBL-IL-15-IL-21 engineering cell, two kinds of methods combine and can shorten the screening cycle, make whole process 1I.e. can complete within individual month, save R&D costs simultaneously.
(3) engineering cell strain is carried out 100Gy lonizing radiation and carries out lethal exposure by the present invention, mixes extraction 1 × 10 after irradiation6Individual cultivating, within every 2 days, calculate a Cell viability, find no survivaling cell existence after add up 15 days, the method canTo ensure the engineering cell strain safety for NK amplification.
(4) present invention uses autologous plasma to promote the propagation of cell, and exogenous factor only adds the IL-2 of relatively low-dose, keeps awayExempt from the probability of the pollution of extrinsic protein and virus, and greatly reduce production cost；
(5) NK cell culture compositions of the present invention can promote a large amount of propagation of NK cell, and purity is higher, can improveThe killing activity of its cell.Its killing of interior tumor cell that can be not only used for tumor patient and immune regulation,Can apply to the health care of sub-health population simultaneously.
IL-21, while K562 surface of cell membrane expresses IL-15 Yu 4-1BBL, is expressed in K562 by the present invention the most simultaneouslyCell surface, the most solvable state adds IL-2, IL-15 Yu IL-21 is combined by the present invention, the two synergism, significantlyImprove invention effect.Said composition can remarkably promote a large amount of amplifications of NK cell, meets clinical practice demand, and K562 is thinCellular expression is also embedded in the albumen on its surface NK cell aggregation can be made fully to be stimulated in its surface, it is thus achieved that NKCell purity is higher, and the method it also avoid the probability of pollution of extrinsic protein and virus simultaneously, saves NK cellPreparation cost, and improve the killing activity of tumor cell.
Figure of description
Fig. 1 is the cell proliferation number change in test 1 amplification procedure；
Cell purity when Fig. 2 is that in test 1, experimental group is cultivated 13 days；
Cell purity when Fig. 3 is that in test 1, experimental group is cultivated 15 days；
Cell purity when Fig. 4 is that in test 1, matched group 1 is cultivated 13 days；
Cell purity when Fig. 5 is that in test 1, matched group 1 is cultivated 15 days；
Cell purity when Fig. 6 is that in test 1, matched group 2 is cultivated 13 days；
Cell purity when Fig. 7 is that in test 1, matched group 2 is cultivated 15 days；
Fig. 8 is the killing activity contrast after test 1 is cultivated 15 days to NK cell A549.
Detailed description of the invention
Technical solution of the present invention is not limited to act detailed description of the invention set forth below, also includes appointing between each detailed description of the inventionMeaning combination.
Detailed description of the invention one: present embodiment NK cell expansion ex vivo compositions include NK amplification trophocyte, IL-2,The autologous plasma of inactivation and GT-T551 H3 culture medium, wherein add 0.65 × 10 in every milliliter of GT-T551 H3 culture medium6Individual～1 × 106Individual NK expands trophocyte, adds the IL-2 of 200-500IU, go out in every milliliter of GT-T551 H3 culture mediumVolume is GT-T551 H3 culture volume the 5% of the autologous plasma lived, described NK amplification trophocyte isK562-4-1BBL-IL-15-IL-21 trophocyte.
The factor promoting NK cell activation propagation is mainly embedded in this by the amplification in vitro method of NK cell of the present inventionBody has the K562 cell surface of inducing action to NK cell, makes NK cell can preferably activate and expand so that it isThere is the killing activity of more preferable tumor cell；The method takes a short time, and safety is high, can apply to clinical cytology rawThing is treated, including renal carcinoma, malignant melanoma, leukemia, breast carcinoma, rectal cancer, gastric cancer, pulmonary carcinoma, the esophageal carcinoma, palaceThe malignancy diseases such as neck cancer, ovarian cancer, multiple myeloma, malignant lymphoma (non-T cell lymphoma), especiallyCan apply to immune system research and the research of tumor-killing effect.
Detailed description of the invention two: present embodiment is unlike detailed description of the invention one: every milliliter of GT-T551 H3 cultivatesBase adds 0.7 × 106Individual～0.9 × 106Individual NK expands trophocyte.Other is identical with detailed description of the invention one.
Detailed description of the invention three: present embodiment is unlike detailed description of the invention one or two: every milliliter of GT-T551 H3Culture medium adds 0.8 × 106Individual NK expands trophocyte.Other is identical with detailed description of the invention one or two.
Detailed description of the invention four: present embodiment is unlike one of detailed description of the invention one to three: every milliliter of GT-T551H3 culture medium adds the IL-2 of 300-400IU.Other is identical with one of detailed description of the invention one to three.
Detailed description of the invention five: present embodiment is unlike one of detailed description of the invention one to three: every milliliter of GT-T551H3 culture medium adds the IL-2 of 350IU.Other is identical with one of detailed description of the invention one to three.
Detailed description of the invention six: present embodiment is unlike one of detailed description of the invention one to five: describedThe preparation method of K562-4-1BBL-IL-15-IL-21 trophocyte is:
A, utilize method for synthesizing gene, it is thus achieved that the fusion gene 4-1BBL-IL-15 of 4-1BBL gene and IL-15 gene andThe full length coding region sequence of IL-21 gene, two upstream region of gene all add CD8 alpha signal peptide, and downstream all adds CD8 α cross-filmDistrict；Meanwhile, CD8 alpha signal peptide upstream introduces Nhe I restriction enzyme site, and CD8 α cross-film district introduces Not I restriction enzyme site；
B, build recombinant slow virus expression plasmid pCDH-CMV-MCS-4-1BBL-IL-15 andPCDH-CMV-MCS-IL-21:
Gene pCDH-CMV-MCS-Vector and step A obtained is respectively with restricted enzyme Nhe I and NotI double digestion, 1% agarose gel reclaim after purification, be utilized respectively T4DNALigase and be attached, it is thus achieved that connectionProduct is respectively designated as pCDH-CMV-MCS-4-1BBL-IL-15 and pCDH-CMV-MCS-IL-21, identifies through enzyme actionAfter Zheng Que ,-20 DEG C save backup；
C, slow virus packaging:
A. dilute, with 235 μ L Opti-MEM, the connection product that 2.7 μ g steps B obtainPCDH-CMV-MCS-4-1BBL-IL-15, adds 1.76 μ g pLp1 (Gal/Pol), 0.95 μ g pLpVSVG and 0.68 μ gPLp2 (pREV), obtains a1 solution；The connection product that 2.7 μ g steps B obtain is diluted with 235 μ L Opti-MEMPCDH-CMV-MCS-IL-21, adds 1.76 μ g pLp1 (Gal/Pol), 0.95 μ g pLpVSVG and 0.68 μ gPLp2 (pREV), obtains a2 solution；
B. dilute 15 μ L lipofectamine2000 with 235 μ L Opti-MEM, obtain b solution；
C.5min, after, a1 solution, a2 solution are mixed with b solution respectively, is added drop-wise to respectively connect after the static 30min of room temperaturePlant in 6 orifice plates of 293-T cell；
D.6-10h after, remove culture medium, add the DMED culture medium containing 10% (v/v) FBS, in 48h and 72hEach receive virus once, final obtain pCDH-CMV-MCS-4-1BBL-IL-15 slow virus andPCDH-CMV-MCS-IL-21 slow virus.
D., after two kinds of slow viruss of results being concentrated to 3E+8 TU/ml, virus mixes than 1:1 with titre, with 1000MOI value carries out the infection of K562 cell line, and being cloned by flow cytometry and limiting dilution assay after infecting 72h can be steadyThe K562-4-1BBL-IL-15-IL-21 engineering cell of fixed heredity.
E. engineering cell strain is carried out 100Gy lonizing radiation and carry out lethal exposure, after irradiation, mix extraction 1 × 106Individual trainSupport, within every 2 days, calculate a Cell viability, find no survivaling cell after adding up 15 days and exist.
F. the K562-4-1BBL-IL-15-IL-21 engineering cell strain frozen stock solution of inactivation is carried out frozen, often pipe 1mL is denseDegree is 1 × 107Individual/mL, removes frozen stock solution with front 37 DEG C of recoveries.Other is identical with one of detailed description of the invention one to five.
Detailed description of the invention seven: present embodiment is unlike detailed description of the invention six: the formula of described frozen stock solution is 90%(v/v) FBS+10% (v/v) DMSO.Other is identical with detailed description of the invention six.
Detailed description of the invention eight: present embodiment NK methods for cell expansion, sequentially includes the following steps:
By 30mL GT-T551 H3 culture medium by 2 × 107Individual～3 × 107After individual PBMCs is resuspended, accesses T75 cell and cultivateBottle, IL-2, NK amplification trophocyte being subsequently adding and the autologous plasma of the inactivation accounting for culture volume 5%, the most oftenMilliliter GT-T551 H3 culture medium adds the IL-2 of 200-500IU, and every milliliter of GT-T551 H3 culture medium adds 0.65 × 106Individual～1 × 106Individual NK expands trophocyte, in 37 DEG C of 5%CO2Under the conditions of co culture system in vitro 15 days, wherein cultivate the 7thIt time add NK and expand trophocyte, before adding, the NK cell in culture fluid is counted, the NK added expandsIncrease the 1/5-1/4 that quantity is NK cell quantity of trophocyte.In incubation, NK cell observation is counted by every day, whenNK cell density is more than 2.5 × 106/ mL must be supplemented with culture medium, and the culture medium added is for containing 200-500IU/mL IL-2GT-T551 H3 culture medium, after fluid infusion, NK cell density maintains 0.8 × 106Individual/mL～1.0 × 106Individual/mL, cultivatesWithin 13 and 15 days, collect cell to detect.
Detailed description of the invention nine: present embodiment is unlike detailed description of the invention eight: train with 30mL GT-T551 H3Support base by 2.5 × 107Individual PBMCs is resuspended.Other is identical with detailed description of the invention eight.
Detailed description of the invention ten: present embodiment is unlike detailed description of the invention eight: every milliliter of GT-T551 H3 cultivatesBase adds the IL-2 of 300-400IU.Other is identical with detailed description of the invention eight.
Detailed description of the invention ten: present embodiment is unlike detailed description of the invention eight: every milliliter of GT-T551 H3 cultivatesBase adds the IL-2 of 240IU.Other is identical with detailed description of the invention eight.
Detailed description of the invention 11: present embodiment is unlike detailed description of the invention eight: every milliliter of GT-T551 H3 trainingSupport base and add 0.7 × 106Individual～0.9 × 106Individual NK expands trophocyte.Other is identical with detailed description of the invention eight.
Detailed description of the invention 12: present embodiment is unlike detailed description of the invention eight: every milliliter of GT-T551 H3 trainingSupport base and add 0.8 × 106Individual NK expands trophocyte.Other is identical with detailed description of the invention eight.
Detailed description of the invention 13: present embodiment is unlike detailed description of the invention eight: described NK expands trophocyteFor K562-4-1BBL-IL-15-IL-21 trophocyte, the acquisition of described K562-4-1BBL-IL-15-IL-21 trophocyteMethod is:
A, utilize method for synthesizing gene, it is thus achieved that the fusion gene 4-1BBL-IL-15 of 4-1BBL gene and IL-15 gene andThe full length coding region sequence of IL-21 gene, two upstream region of gene all add CD8 alpha signal peptide, and downstream all adds CD8 α cross-filmDistrict；Meanwhile, CD8 alpha signal peptide upstream introduces Nhe I restriction enzyme site, and CD8 α cross-film district introduces Not I restriction enzyme site；
B, build recombinant slow virus expression plasmid pCDH-CMV-MCS-4-1BBL-IL-15 andPCDH-CMV-MCS-IL-21:
Gene pCDH-CMV-MCS-Vector and step A obtained is respectively with restricted enzyme Nhe I and NotI double digestion, 1% agarose gel reclaim after purification, be utilized respectively T4DNALigase and be attached, it is thus achieved that connectionProduct is respectively designated as pCDH-CMV-MCS-4-1BBL-IL-15 and pCDH-CMV-MCS-IL-21, identifies through enzyme actionAfter Zheng Que ,-20 DEG C save backup；
C, slow virus packaging:
A. dilute, with 235 μ L Opti-MEM, the connection product that 2.7 μ g steps B obtainPCDH-CMV-MCS-4-1BBL-IL-15, adds 1.76 μ g pLp1 (Gal/Pol), 0.95 μ g pLpVSVG and 0.68 μ gPLp2 (pREV), obtains a1 solution；The connection product that 2.7 μ g steps B obtain is diluted with 235 μ L Opti-MEMPCDH-CMV-MCS-IL-21, adds 1.76 μ g pLp1 (Gal/Pol), 0.95 μ g pLpVSVG and 0.68 μ gPLp2 (pREV), obtains a2 solution；
B. dilute 15 μ L lipofectamine2000 with 235 μ L Opti-MEM, obtain b solution；
C.5min, after, a1 solution, a2 solution are mixed with b solution respectively, is added drop-wise to respectively connect after the static 30min of room temperaturePlant in 6 orifice plates of 293-T cell；
D.6-10h after, remove culture medium, add the DMED culture medium containing 10% (v/v) FBS, in 48h and 72hEach receive virus once, final obtain pCDH-CMV-MCS-4-1BBL-IL-15 slow virus andPCDH-CMV-MCS-IL-21 slow virus.
D., after two kinds of slow viruss of results being concentrated to 3E+8 TU/ml, virus mixes than 1:1 with titre, with 1000MOI value carries out the infection of K562 cell line, and being cloned by flow cytometry and limiting dilution assay after infecting 72h can be steadyThe K562-4-1BBL-IL-15-IL-21 engineering cell of fixed heredity.
E. engineering cell strain is carried out 100Gy lonizing radiation and carry out lethal exposure, after irradiation, mix extraction 1 × 106Individual trainSupport, within every 2 days, calculate a Cell viability, find no survivaling cell after adding up 15 days and exist.
F. the K562-4-1BBL-IL-15-IL-21 engineering cell strain frozen stock solution of inactivation is carried out frozen, often pipe 1mL is denseDegree is 1 × 107Individual/mL, goes frozen stock solution, the formula of described frozen stock solution to be 90% (v/v) FBS+10% with front 37 DEG C of recoveries(v/v)DMSO.Other is identical with detailed description of the invention eight.
For checking beneficial effects of the present invention, carry out tests below:
Test 1: this test NK cell expansion ex vivo compositions and NK methods for cell expansion
One, preparation K562-4-1BBL-IL-15-IL-21 trophocyte
A, utilize method for synthesizing gene, it is thus achieved that the fusion gene 4-1BBL-IL-15 of 4-1BBL gene and IL-15 gene,The fusion gene 4-1BBL-IL-21 of 4-1BBL gene and IL-21 gene and the full length coding region sequence of IL-21 gene, twoBar upstream region of gene all adds CD8 alpha signal peptide, and downstream all adds CD8 α cross-film district；Meanwhile, CD8 alpha signal peptide upstreamIntroducing Nhe I restriction enzyme site, CD8 α cross-film district introduces Not I restriction enzyme site；
B, build recombinant slow virus expression plasmid pCDH-CMV-MCS-4-1BBL-IL-15,PCDH-CMV-MCS-4-1BBL-IL-21 and pCDH-CMV-MCS-IL-21:
Gene pCDH-CMV-MCS-Vector and step A obtained is respectively with restricted enzyme Nhe I and NotI double digestion, 1% agarose gel reclaim after purification, be utilized respectively T4DNALigase and be attached, it is thus achieved that connectionProduct be respectively designated as pCDH-CMV-MCS-4-1BBL-IL-15, pCDH-CMV-MCS-4-1BBL-IL-21 andPCDH-CMV-MCS-IL-21, after enzyme action is identified correctly ,-20 DEG C save backup；
C, slow virus packaging:
A. dilute, with 235 μ L Opti-MEM, the connection product that 2.7 μ g steps B obtainPCDH-CMV-MCS-4-1BBL-IL-15, adds 1.76 μ g pLp1 (Gal/Pol), 0.95 μ g pLpVSVG and 0.68 μ gPLp2 (pREV), obtains a1 solution；The connection product that 2.7 μ g steps B obtain is diluted with 235 μ L Opti-MEMPCDH-CMV-MCS-IL-21, adds 1.76 μ g pLp1 (Gal/Pol), 0.95 μ g pLpVSVG and 0.68 μ gPLp2 (pREV), obtains a2 solution；The connection product that 2.7 μ g steps B obtain is diluted with 235 μ L Opti-MEMPCDH-CMV-MCS-4-1BBL-IL-21, adds 1.76 μ g pLp1 (Gal/Pol), 0.95 μ g pLpVSVG and 0.68 μ gPLp2 (pREV), obtains a3 solution；
B. dilute 15 μ L lipofectamine2000 with 235 μ L Opti-MEM, obtain b solution；
C.5min, after, a1 solution, a2 solution, a3 solution are mixed with b solution respectively, after the static 30min of room temperature respectivelyIt is added drop-wise in 6 orifice plates of inoculation 293-T cell；
D.6-10h after, remove culture medium, add the DMED culture medium containing 10% (v/v) FBS, in 48h and 72hRespectively receive virus once, final acquisition pCDH-CMV-MCS-4-1BBL-IL-15 slow virus,PCDH-CMV-MCS-4-1BBL-IL-21 slow virus and pCDH-CMV-MCS-IL-21 slow virus.
D. after two kinds of slow viruss of results are concentrated to 3E+8 TU/ml by experimental group, willPCDH-CMV-MCS-4-1BBL-IL-15 slow virus and pCDH-CMV-MCS-IL-21 slow virus compare 1:1 with titreMixing, carries out the infection of K562 cell line with the MOI value of 1000, and matched group 1 willPCDH-CMV-MCS-4-1BBL-IL-15 slow virus carries out K562 cell infection, matched group 2 with the MOI value of 500PCDH-CMV-MCS-4-1BBL-IL-21 slow virus is carried out K562 cell infection with the MOI value of 500, infects 72hCloned by flow cytometry and limiting dilution assay afterwards can stablize heredity K562-4-1BBL-IL-15 (matched group 1),K562-4-1BBL-IL-21 (matched group 2) and K562-4-1BBL-IL-15-IL-21 (experimental group) engineering cell.
The present invention first carries out selected by flow cytometry apoptosis with the purplish red element IL-21 antibody of labelling and IL-15 antibody, removes feminine genderK562 cell, then double positive cell groups of screening are carried out limiting dilution assay clone can stablize hereditaryK562-4-1BBL-IL-15-IL-21 engineering cell, two kinds of methods combine and can shorten the screening cycle, make whole process 1I.e. can complete within individual month, save R&D costs simultaneously.
E. engineering cell strain is carried out 100Gy lonizing radiation and carry out lethal exposure, after irradiation, mix extraction 1 × 106Individual trainSupport, within every 2 days, calculate a Cell viability, find no survivaling cell after adding up 15 days and exist.
F. the K562-4-1BBL-IL-15-IL-21 engineering cell strain of inactivation is carried out frozen (frozen stock solution formula is90%FBS+10%DMSO), every pipe 1 × 107Individual/ml, removes frozen stock solution with front 37 DEG C of recoveries.
Two, experimental group by 30mL GT-T551 H3 culture medium by 2 × 107Individual～3 × 107After individual PBMCs is resuspended, accessT75 Tissue Culture Flask, IL-2, the NK being subsequently adding amplification trophocyte and account for inactivation autologous of culture volume 5%Blood plasma, the wherein IL-2 of every milliliter of GT-T551 H3 culture medium addition 200-500IU, every milliliter of GT-T551 H3 cultivatesBase adds 0.65 × 106Individual～1 × 106Individual NK expands trophocyte, in 37 DEG C of 5%CO2Under the conditions of co culture system in vitro 15 days,Add a NK when wherein cultivating the 7th day and expand trophocyte, before adding, the NK cell in culture fluid is counted,Quantity is NK cell quantity the 1/4 of the NK amplification trophocyte added.In incubation, every day is to NK cell observationCounting, when NK cell density is more than 2.5 × 106/ mL must be supplemented with culture medium, and the culture medium added is for containing 240IU/mLThe GT-T551 H3 culture medium of IL-2, after fluid infusion, NK cell density maintains 1.0 × 106Individual/mL, respectively at cultivating the 5thMy god, 7 days, 9 days, 11 days, 13 and 15 days collect cell detect.
Matched group 1, matched group 2 are identical with the operation of experimental group, and simply trophocyte is replaced by by matched group 1K562-4-1BBL-IL-15, trophocyte is replaced by K562-4-1BBL-IL-21 by matched group 2.
Testing result is as follows:
(1) the NK cell quantity (hundred million) expanded
The PBMCs quantity of each group initial inoculation is 3 × 107Cells, within i.e. the 0th day, cell number is 0.30 ± 0.00 hundred million
Table 1
Fig. 1 is the cell proliferation number change in amplification procedure, in Fig. 1 ● representing experimental group, ■ represents matched group 1, ▲Represent matched group 2.During cell amplification 15d, experimental group cell quantity is 114.00 ± 12.37, and matched group 1 cell quantity is68.4 ± 9.75, matched group 2 cell quantity is 59.6 ± 8.67.The cell quantity of experimental group amplification is significantly higher than matched group 1And matched group 2 (P1=0.0037 < 0.01, P2=0.0034 < 0.01), the cell quantity of matched group 1 and matched group 2 amplification withoutSignificant difference (P=0.1538 > 0.05), illustrates that 4-1BBL, IL-15 and IL-21 are expressed in cell surface simultaneously thin to NKThe expanding effect of born of the same parents is better than containing only 4-1BBL, IL-15 or 4-1BBL, the effect of IL-21.
(2) motility rate, purity:
Table 2
Fig. 2 be experimental group cultivate 13 days time cell purity；Fig. 3 be experimental group cultivate 15 days time cell purity；Fig. 4Cell purity when cultivating 13 days for matched group 1；Fig. 5 is the matched group 1 cell purity when cultivating 15 days；Fig. 6 isMatched group 2 cultivates cell purity when 13 days；Fig. 7 is the matched group 2 cell purity when cultivating 15 days.
As seen from the figure, in experimental group cell, CD3-, CD56+ ratio is significantly higher than experimental group 1 and experimental group 2, and matched group1 and matched group 2 without significant difference, illustrate that 4-1BBL, IL-15 and IL-21 are expressed in cell surface simultaneously and more only expressThe amplification impurities affect effect of NK cell is become apparent from by 4-1BBL, IL-15 or 4-1BBL, IL-21.Each group cell expandsIncreasing 13 days and the CD3-of 15 days, the change of CD56+ cell purity is inconspicuous, illustrates that when 13 days, NK cell is the most ripe.
(3) the NK cell the gathered in the crops killing activity to A549 cell
A549 cell inoculum concentration is 10000cells/ hole, and effect target ratio for 10:1, observes NK cell constantly to A549 cellFragmentation effect.Add NK cell when inoculating about 19h, about during 90h, reach maximum fragmentation effect, experimental group, matched group1 and matched group 2NK killing-efficiency be respectively as follows: 90.64%, 82.61%, 73.33%.
Fig. 8 is to cultivate after 15 days the killing activity to NK cell A549 to contrast, and in Fig. 8, curve 1,2 represents blank rightAccording to group (i.e. without immunocyte), curve 3,4 represents matched group 1, and curve 5,6 represents matched group 2, curve 7,8 represent experimental group.A549 cell inoculum concentration is 10000cells/ hole, and effect target ratio for 10:1, observes NK cell pair constantlyThe fragmentation effect of A549 cell.Add NK cell when inoculating about 19h, about during 90h, reach maximum fragmentation effect, experimentGroup, matched group 1 and matched group 2NK killing-efficiency average are respectively as follows: 90.64%, 82.61%, 73.33, and each group all hasThere is good tumors inhibition activity, and the tumor-killing effect of experimental group becomes apparent from.