CN110358736A - A kind of K562 cell of modification, preparation method and NK cell culture compositions - Google Patents

A kind of K562 cell of modification, preparation method and NK cell culture compositions Download PDF

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CN110358736A
CN110358736A CN201910620164.7A CN201910620164A CN110358736A CN 110358736 A CN110358736 A CN 110358736A CN 201910620164 A CN201910620164 A CN 201910620164A CN 110358736 A CN110358736 A CN 110358736A
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cell
modification
fusion
mbil21
human source
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卓朗
王晖
王柯
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Hangzhou Shuoxi Biopharmaceutical Co ltd
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Hangzhou Excellent Biotechnology Co Ltd
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Abstract

The present invention provides the K562 cell, preparation method and NK cell culture compositions of a kind of modification.The K562 cell of the modification is as obtained from fusion is carried out AAVS1 transposons of the genetic recombination to K562 cell as plasmid；Wherein, the fusion includes human source gene, wherein the human source gene includes mbIL15,4-1BBL and mbIL21.The K562 cell of the modification is the cell line developed by using the integration technology of site-specific genetic, NK cell cell activity with higher, purity, cytotoxicity and the amplification efficiency expanded by it；With great market prospects and economic value.

Description

A kind of K562 cell of modification, preparation method and NK cell culture compositions
It is on May 20th, 2016 that present patent application, which is application No. is the 201610340117.3, applying date, entitled A kind of divisional application of the patent application of " method and its application of efficient amplification freezen protective NK cell ".
Technical field
The invention belongs to cell engineering fields, and in particular to a kind of K562 cell of modification, preparation method and comprising The NK cell culture compositions of the K562 cell of the modification.
Background technique
People's natural kill (NK) cell is the lymph by expression CD56 and the natural immune system for lacking CD3 (CD-CD56+) Cell composition.NK cell has the function of the killing tumor cell of wide spectrum, can pass through cytotoxicity particle such as perforin And granzyme, and interacted by such as death receptor, Fas-Fas ligand binding and the relevant apoptosis-inducing ligand of TNF In conjunction with equal mechanisms plays antitumor action.NK cell can also be thin by membrane receptor FcR γ III (CD16) mediate antibody dependence Cellular toxicity (ADCC).The targets identification of NK cell and activation are by inhibiting and activating NK cell surface receptor and expression in target cell On associated ligands this balance to play a role, allow to pathogen invade and canceration make an immediate response.This unique target It is different from the T cell of major histocompatibility complex (MHC) restricted T cell receptor (TCR) to the mechanism of identification NK cell Dependent mechanism.Therefore, graft versus host disease(GVH disease) (GVHD) may not be caused by carrying out treating cancer using allogeneic NK cell.
In health volunteer, NK cell accounts about the 5-20% of peripheral blood mononuclear cells (PBMC).In adoptive immunity In treatment, large dosage of NK cell context is from 5 × 10^6-5 × 10^7/kg body weight per dose, most 4 dosage.In the prior art, Can from a large amount of peripheral blood Enrichment Amplification NK cell method, majority is by means of using invasive leucocyte separation and big Type magnetic separating device, this mode is sufficiently expensive, and causes certain risk to patient.Another popular NK cell therapy is to make NK cell is expanded with feeder cells, such as the film binding molecule of K562 cell modification, such as interleukin (IL) -15 and 4-1BB ligand (K562-mb15-41BBL).These feeder cells promptly can expand NK cell from peripheral blood mononuclear cells, make NK cell 21.6 times were averagely expanded at the 7th day, expanded 277 times within the 21st day.But the Telomerase as caused by aging shortens, so can limit System continues to expand.
Obtain large dosage NK cell usually require amplification several weeks, the NK cell of feedback must be it is fresh, cell culture with Coordination between multi-dose cell infusion has extremely challenging.Therefore, freezing for the NK cell after amplification is very It is necessary to.However, freezing for traditional NK cell has harmful influence to cell, NK cell viability, cell toxicant are such as reduced Property and the expression to the vital NK cell receptor of function.It is thin that usual NK cell can not crack immediately cancer after freezing defrosting Born of the same parents, the NK cell of recovery need to stimulate cytotoxicity (Denman, the Senyukov to restore the NK cell frozen overnight with IL-2 et al.,2012；Lapteva N et al.,2012).Therefore, in the prior art, cells viability is still a problem, And the NK cell survival after recovering cannot be continued above one week.Meanwhile the NK cells containing sequences of recovery further expand still not It is bright.Therefore, this field is highly desirable to one kind new holding NK cell activity and original function at present and has amplification well The cryopreservation methods of property.
Summary of the invention
For this purpose, technical problem to be solved by the present invention lies in propose a kind of holding NK cell activity, function and amplification The cryopreservation methods of property.
In order to solve the above technical problems, the invention discloses a kind of method of efficient amplification freezen protective NK cell, it is described Method is by introducing fusion by plasmid progress genetic recombination to AAVS1 transposons, and in the cell amplification intermediate stage NK cell expansion step based on freezing/thawing step to modification K562 cell line；Finally carry out amplification and the guarantor of NK cell It deposits.
Preferably, the fusion is that human chromosome 19 encodes mbIL15,4-1BBL and mbIL21.
Preferably, the gene expression plasmid skeleton of the fusion is pFastBac1.
Preferably, the promoter of the plasmid backbone is cytomegalovirus.
Preferably, the recombination of the plasmid uses Zinc finger nuclease-mediation homologous recombination technique.
Preferably, the step of genetic recombination specifically:
K562 cell is subjected to suspension processing；
PFastBac-ZFN and PFB- fusion host's electricity is gone into cell；
The cell of electroporation is subsequently transferred to K562 growth medium to cultivate.
After culture, carries out expanding unicellular, clone progress pcr gene parting, flow cytometry, be made and stablize expression The cell of fusion.
Preferably, the amplification specific steps of the NK cell are as follows:
Peripheral blood mononuclear cells is first taken, line density of going forward side by side gradient method analysis processing；
Then the NK cell is expanded using serum-free stem cell growth culture medium, and with PBMC and gamma-radiation K562 feeder cells co-incubation in NK cell growth medium；
By the obtained NK cell of amplification in the freezing culture medium containing SCGM culture medium and 10%FBS and 10%DMSO It is frozen in cryovial；
More preferably, cell is placed in after freezing processing and is thawed and washed again；Again by the NK cell of defrosting finally It is secondary that stimulation culture is carried out with the ratio of 1:1 with K562 feeder cells.
The invention also discloses method the answering in cell preservation field of the efficient amplification freezen protective NK cell With.
The above technical solution of the present invention has the following advantages over the prior art, by using site-specific genetic The K562-mbIL15-41BBL-mbIL21 cell line of integration technology exploitation, by with high NK cell activity, purity and thin Cellular toxicity realizes high NK cell amplification efficiency to improve NK amplification.
Detailed description of the invention
In order to make the content of the present invention more clearly understood, it below according to specific embodiments of the present invention and combines Attached drawing, the present invention is described in further detail, wherein
Fig. 1 is the fusion of the site-specific integration in embodiment to the process schematic of K562 cell；
Fig. 2 is flow cytometry analysis mbIL15,4-1BBL and mbIL21 in embodiment in 562 cell of K of modification Protein expression level；
Fig. 3 is NK cells expanded in embodiment；
Fig. 4 is to be compared in embodiment using the purity of NK cell after K562mbil15-41BBL-mbil21；
Fig. 5 is NK cell-stimulating receptor after expanding in embodiment, inhibits the expression of receptor and NK cell marking；
Fig. 6 is the killing activity schematic diagram of the NK cell that expands in embodiment to K562 human leukemia cell line；
Fig. 7 is that NK cell fights RaJi B- cell lymphocyte system in the presence of anti-CD20 human antibody in embodiment ADCC acts on schematic diagram；
Fig. 8 is killing of the initial NK cell of EpCAM specific C AR modification in embodiment to PCRC7 colorectal cancer cell Active schematic diagram；
Fig. 9 is that the initial NK cell that EpCAM specific C AR is modified in embodiment is living to the killing of MCF7 breast cancer cell Property.
Specific embodiment
Present embodiment discloses a kind of methods of efficient amplification freezen protective NK cell for embodiment 1, the specific steps are as follows:
1, the culture of human archeocyte system
Wild type K562 and Raji cell line is obtained from American type culture collection (ATCC).And by these cells It is placed in the RPMI-1640 culture medium (GIBCO) containing 10% fetal calf serum (GIBCO), and is put into 37 DEG C, 5%CO2Incubator In cultivated.
2, the building of plasmid
By pFastBac1 (Invitrogen, Carlsbad, CA) as plasmid backbone for expressing ZFN and coding The fusion of mbil15,4-1BBL and mbIL21, wherein promoter is cytomegalovirus (CMV).Wherein, pFastBac-ZFN It is specific building before be reported (Tay, Tan et al.2013).
Building for pFB- fusion host, the DNA fragmentation of the coding fusion is for the first time by AIT biotechnology root According to the sequence gene synthesis reported in the prior art, the construct of synthesis is cloned into pUC57 (ammonia benzyl resistance), then by the piece Section passes through PCR amplification.
In addition, CMV promoter, internal ribosome entry site (IRES), neomycin resistance gene (new) and marmot liver Controlling element (WPRE) is also by PCR amplification after scorching virus transcription.All these segments use the seamless clone of GENEART and assembling Kit (Invitrogen) is cloned into the plasmid of a pUC19.Segment excision will then be merged, cloned using Asc/ClaI I To pFastBac1 backbone, wherein the left homology arm comprising a 810bp belonging to AAVS1 transposons and 837bp it is right together Source arm (Tay, Tan et al.2013).
3, the fusion of site-specific integration is to K562 cell AAVS1 locus
By 2 × 106A K562 cell line is resuspended in the Opti-MEM of 100 μ l respectively.Use 21 electroporation of NE PA By 3 μ g, each pFastBac-ZFN and PFB- fusion host's electricity goes to cell with Bio-Rad company test tube.Electricity is worn The cell in hole is subsequently transferred to the fresh K562 growth medium in 6 orifice plates.It is dense in 200 μ g/mL the 5th day after electroporation G418 selection in 1 month by a definite date is carried out under degree.It replaces within culture medium every 2 days.After selection in 1 month, cell sorter, BD are used FACSAria (BD Biosciences company) is sorted into 96 orifice plates for unicellular.It expands unicellular and clone and carries out pcr gene Parting and flow cytometry, to confirm the expression of the fusion of the site-specific integration and stablize expression.Pcr gene Sorting is with the integration of identification of cell clone and the site AAVS1.The genomic DNA of cell is usedBlood and tissue reagent Box (Qiagen, Xi Erdeng, Germany) is separated.Pcr gene parting be used to detect the site-specific integration of OKSM box. KAPA high-fidelity thermal starting premixing (KAPA Biosystem, Woburn, MA) is used together, forward primer: ATATTGCTGAAGAGCTTGGCGGCGAATGGG and reverse primer: CGGGGATGCAGGGGAACGGGGCTCAGTCTG.Amplification Product is analyzed on 1% Ago-Gel.
4, it the amplification of NK cell and freezes
20 healthy donor's peripheral blood mononuclear cells are taken, and use Ficoll-Paque PREMIUM (GE medical treatment collection Group's life science), density gradient method analysis processing is carried out from the fresh buffycoat of peripheral blood mononuclear cells.Using being supplemented with The serum-free of the GMP of the IL-2 (PeproTech, Rocky.Hill, NJ, USA) of 10%FBS (GIBCO) and 10 or 50IU/ml is dry Cell growth medium (SCGM) (CellGro/CellGenix) expands NK cell, and with PBMC and gamma-radiation K562 Feeder cells (quantity ratio 1:2) co-culture in NK cell growth medium.2 × 10 in T75 culture bottle6A PBMC and 4 × 106K562 feeder cells co-cultured in the NK culture medium of 10ml.Half amount changes liquid and adds 100IU/ml IL-2 within every 2-3 days.
After NK cell expands 7 days, the NK cell expanded is with 2 × 107Cell/mL is containing SCGM culture medium+10% It is frozen in cryovial (NUNC) in the freezing culture medium+10%DMSO (Invitrogen) of FBS.Cell is placed in 1 DEG C of freezing Refrigerated container (Thermo Fisher Scientific, Rochester, NY) freeze 24 hours in -80 DEG C of refrigerators, then It transfers and stores in liquid nitrogen vapor storage tank.The NK cell of freezing is thawed in 37 DEG C of water-baths until being left a small amount of frozen matter, so It is washed afterwards in NK cell growth medium.Defrosting NK cell every 7 days again with K562 feeder cells with the proportional stimulation of 1:1.Freeze Depositing NK cell can at least expand 3 months.
5, flow cytometry analysis
Flow cytometer used be BD Accuri C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ)。
The antibody of used APC label is purchased from U.S. day Ni (German Bergisch Gladbach), BD Biosciences Company or Beckman Coulter (Brea, CA).
6, cytotoxicity analysis
It is describedThe cytotoxic reagent box (Perkin Elmer) of cell is used to test to wild type K562 With the cytotoxicity of Raji cell.
NK cell and target cell at 1:1,1:5 and 1:10 ratio at 37 DEG C, 5%CO2Incubator in co-culture it is 2 small When.
7, experimental result
The fusion of site-specific integration is to K562 feeder cells
Using established Zinc finger nuclease (BV-ZFN) (Tay, Tan et al.2013), two plasmid construction techniques: One coding ZFN, targeting and cracking AAVS1 transposons (pFastBac-ZFN) and other it is used containing CMV promoter to drive The host mbil15,4-1BBL and mbil21 and neomycin gene of the expression of fusion coding.It is specifically shown in Fig. 1.In figure, The fusion of site-specific integration is to K562 cell.The building of pFastBac- fusion host, AAVS1 is extremely The AAVS1 of PPP1R12C gene, shearing site BV-ZFN, and modification is after homologous recombination.E1, E2 and E3: first three The exon of PPP1R12C gene.Arrow indicates that the front PCR and reverse starting (FP and RP) combine the 2.4kb piece formed The AAVS1 of 3 ' modifications of section.
This expression cassette is by the left homology arm flank of 810-bp and the right homology arm of a 837-bp about described AAVS1 transposons (pFastBac-mbIL21- host) (Fig. 1).Two kinds of plasmids are realized that site is special by electroporation by K562 cell Specific integration.After electroporation, K562 cell is subjected to G418 selection, after 5 days, carries out unicellular sorting.It is slender from G418 patience The genome of born of the same parents clone extracts DNA, and is present in the pFastBac- by using the neomycin for being specific to No. 19 chromosomes MbIL21- host gene (downstreams of 3 ' ends of right homology arm) primer pair carries out pcr gene parting.The expansion of the segment of 2.5-kb Increase the homologous recombination success AAVS1 modification for showing to mediate by ZFN.It is being cloned with proving that successfully modification will lead to fusion Stable protein expression, the clone carries out flow cytometry analysis.One is cloned the high-level table for showing protein Up to the feeder cells for being chosen to be the amplification of NK cell.It is specifically shown in Fig. 2.
Freeze the amplification of rear NK cell
Freezing to cell nocuousness, such as cell viability, the decline of cytotoxicity, NK cell receptor table for NK cell is known The reduction reached.In order to overcome this problem, we have developed a kind of scheme, by the NK cell of fresh separated and we obtain K562 cell is 7 days short with the ratio culture of 1:2, the cell that amplification obtains then is frozen, then with the ratio of 1:1 after recovery It is co-cultured with K562 cell.Then NK cell is stimulated again with K562 feeder cells every 7 days, the amplification of NK cell is up to 3 Month.It was found that this method can overcome the problems, such as that cell viability caused by freezen protective declines.As shown in figure 3, freezing is protected The NK cell deposited can be proliferated at least 35 days after recovery.It being shown in figure, average total amplification times of NK cell are 78,880, 152, range is at 280,354 to 226,456,888 times.Every a line represents a PBMC patient (n=3).NK cell is used It is to freeze after -80 DEG C 7 days before 1:2 that K562mbil15-41BBL-mbil21 feeder cells, which expand PBMC:K562 ratio,.Freeze The NK cell deposited is recovered after a week, is expanded again after being stimulated with K562.
For the optimal culture condition for determining NK cell Proliferation, PBMC to K562-mbIL15-41BBL-mbIL21 is evaluated respectively The difference of ratio, 1:2,1:1.5 and 1:1.It observes, may cause the amplification times of NK cell in the reduction of dispensing initial p BMC stimulation Several reductions, the ratio that PBMC:K562 is 1:2 generate highest NK cells expanded.The increase of ratio will also keep higher NK cell purity, in PBMC:K562 be 1:2 can reach after 21 days (Fig. 4) of culture 94.9 ± 2.8% highest purity.
Experimental example
1, the Phenotypic examination experiment of the NK cell expanded
One extensive array of NK cell adhesion molecule and receptor that amplification obtains is tested.Most of high expression Receptor and adhesion molecule, there is a few exceptions-activated receptor, NKp44 and an Inhibitory receptor, surface C D158a, h, CD158i and CD158e1/e2 (Fig. 5).
The expression of adhesion molecule is also compared after 7 days NK cell recoveries of culture simultaneously and with K562-mbIL15- 41BBL-mbIL21 stimulates the expression of adhesion molecule and receptor after 1 week again.Inhibitory receptor, surface C D158a, h and CD158e1/e2's and activated receptor, the surface expression of NKG2D and NKp44 dramatically increase.Surface markers as the result is shown The expression of CD16 can be lowered because of the freezen protective of NK cell (Sakamoto, Ishikawa et al.2015).With these results Consistent, there is significant decline (up to 86%) in the expression of the surface markers CD16 of NK cell after freezing and recovering. But, this problem is improved after stimulating 1 week again using K562-mbIL15-41BBL-mbIL21.CD16 surface expression is aobvious Show and increases to average 90.6 ± 1.8%.
2, the lethal test of NK cells on cancer cells expanded
The NK cell of amplification can directly kill the K562 Leukaemia of NK sensitivity, average 16.1%, and (range is from 0.9% To 31.2%), 69.8% (range from 58.5% to 98.9%) and 86.3% (from 74.9% to 98.7%) imitate target ratio with 1 Respectively 1:1,5:1 and 10:1 (Fig. 6).Wherein NK cell and K562 cell are co-cultured 2 small with the ratio of 1:1,5:1 and 10:1 When.Every a line represents a PBMC healthy volunteer (n=6).But Raji lymphoma cell is less sensitive to NK cell.It is logical The ADCC function using these NK cells is crossed, can obviously be observed to Raji in the presence of antibody CD20-hIgG1 (Fig. 7) The cytotoxicity of cell.Therefore, the ADCC function of the NK cell expanded can be significantly expanded the spectrum of the killing to malignant cell.
Their killing activities to cancer cell are can be improved into immunocyte Chimeric antigen receptor (CAR).After freezen protective The cancer cell killing-efficiency for further increasing the NK cell of amplification redirects whether NK cell shows by test anti-EpCAM CAR- Show the positive colorectal cancer of anti-EpCAM and the cytotoxicity of breast cancer cell.It, will after electroporation encodes the mRNA of anti-EpCAM The NK cell and pCRC7 Human Large Intestine Carcinoma Cells (Fig. 8) and MCF-7 breast cancer cell (Fig. 9) of modification co-culture.Fig. 8, EpCAM in 9 For the initial NK cell of specific C AR modification；CAR is the initial NK cell of control modification；Wt is initial NK cell.
Obtain following result: anti-EpCAM CARNK cell shows strong cellular cytoxicity activity, and can be in E:T ratio 10:1 When 100% kill tumour cell, this more unmodified initial NK cell and mGFP CAR modification control NK cell have more Effective killing activity.
In view of the foregoing it is apparent that adoptive NK cell therapy is a kind of effective cancer treatment method, it is shown that high Antitumor potentiality, while with the risk of extremely low graft versus host disease(GVH disease) (GVHD) in clinical test.However, obtaining enough The NK cell of quantity be used to the adopt difficulty of transplanting is one of NK cell therapy limitation.The amplification in vitro of NK cell is usually and multiple Miscellaneous scheme and expensive cost are associated.The K562-mbIL15- developed by using the integration technology of site-specific genetic 41BBL-mbIL21 cell line, by realizing high NK cell amplification efficiency with high NK cell activity, purity and cytotoxicity To improve NK amplification scheme.The gene of the retroviral vector of random integration coding NK stimulation molecule dye K562 cell is used to produce Raw genetically modified K562 cell, the expression that this may cause stimulation molecule is different, therefore this will be challenging Duplication cell line may express transgenic identical expression.By overcoming the integration by site-specific genetic Problem introduces AAVS1 transposons.AAVS1 is located at protein phosphatase 1, adjusts (inhibitor) subunit 12C (PPP1R12C) gene Within No. 19 chromosomes (19q13.3-qter) of people.This site is used as specific integration locus AAV serotype 2 (AAV2) Non-pathogenic human parvovirus.AAVS1 has transcriptional capability, including an open chromatin Structure and includes natural insulation Body makes the gene integration resistance to transgene silencing.Due to there is the transgenosis box from PPP1R12C gene and insertion point Transcriptional capability destruction caused by be maintained at different types of cell, AAVS1 quilt without known adverse effect on cell It is considered that one " safe port " is addition transgenosis into human genome.
The NK cell of amplification has better efficiency after being frozen preservation, and has improvement in subsequent amplification procedure NK cell function.
Currently, due to freezing the influence expressed CD16, the NK cell recovered immediately cannot be used for ADCC antineoplaston. This therapy will become feasible by being stimulated again with K562-mbIL15-41BBL-mbIl21.NK cytotherapeutic treatment is white at present Blood disease is more successful compared with solid tumor.This is because the display inhibit signal with the solid tumor microenvironment of the lethal effect of NK cell because The influence of element.Therefore, the NK cell that there is the NK cell of the gene modification of CAR targeting solid tumor to have drug resistance opens wider A possibility that general NK cell therapy various cancer types.Therefore, it recovers after the NK cell frozen, anti-EpCAM CAR- is redirected NK cell can effectively kill EpCAM positive colorectal cancer and breast cancer cell.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or It changes still within the protection scope of the invention.

Claims (9)

1. a kind of K562 cell of modification, which is characterized in that the K562 cell of the modification is by the way that fusion is passed through matter Grain carries out obtained from AAVS1 transposons of the genetic recombination to K562 cell；Wherein, the fusion includes human source gene, Wherein the human source gene includes mbIL15,4-1BBL and mbIL21.
2. the K562 cell modified as described in claim 1, which is characterized in that the skeleton of the plasmid is pFastBac1.
3. the K562 cell modified as claimed in claim 2, which is characterized in that the promoter of the skeleton of the plasmid is big and small Cellular virus promoter.
4. the K562 cell modified as described in claim 1, which is characterized in that the genetic recombination uses Zinc finger nuclease The homologous recombination technique that ZFN is mediated.
5. the K562 cell modified as described in claim 1, which is characterized in that the human source gene is by mbIL15,4-1BBL It is formed with mbIL21.
6. a kind of NK cell culture compositions, which is characterized in that the NK cell culture compositions include as claim 1-5 is any K562 cell, IL-2 and the serum-free stem cell growth culture medium of modification described in.
7. NK cell culture compositions as claimed in claim 6, which is characterized in that the NK cell culture compositions further include FBS。
8. a kind of method for the K562 cell for preparing modification according to any one of claims 1-4, comprising:
Construct pFastBac- fusion；Wherein, the fusion includes human source gene, wherein the human source gene includes MbIL15,4-1BBL and mbIL21；
K562 cell is subjected to suspension processing；
PFastBac-ZFN and the pFastBac- fusion electricity are gone into the K562 cell；
The K562 cell turned through the electricity is transferred in K562 growth medium and is cultivated；
After culture, single cell clone amplification is carried out, pcr gene parting, flow cytometry are carried out to single cell clone, be made Stablize the K562 cell for expressing the modification of the fusion.
9. method according to claim 8, which is characterized in that the human source gene is by mbIL15,4-1BBL and mbIL21 group At.
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