CN107034237A - A kind of CAR NK cells and preparation method and application - Google Patents
A kind of CAR NK cells and preparation method and application Download PDFInfo
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Abstract
The present invention relates to a kind of CAR NK cells and preparation method and application.Methods described includes：S1, specific antibody encoding gene is connected in the cloning site area of engineered CAR skeletons to acquisition CAR genes, the engineered CAR skeletons include film signaling zone, cloning site area, the extracellular hinge areas of CD244, CD244 transmembrane segments area and CD244 intracellular parts area on the CD244 that is sequentially connected, S2, the CAR genes are passed through into slow-virus infection NK cells, obtain expressing the NK cells of the CAR albumen, i.e., described CAR NK cells.Immunotherapy of tumors cell CAR NK cells prepared by the present invention can also be applied to allosome and feed back in addition to it can be applied to cancer patient.Therefore, the immunotherapy of tumors cell prepared by the present invention and existing CAR T-phase ratio, wider using scope, prepares cost lower.
Description
Technical field
It is specifically a kind of to be used to clinical treatment class and prepare CAR-NK (or other exempting from the present invention relates to biological technical field
Epidemic disease cell) etc., the cellular immunotherapy for tumour.
Background technology
Cell biological treatment is the novel cancer therapies with self immune system, is a kind of to have significant curative effect
Therapeutic mode.It uses Protocols in Molecular Biology and cellular engineering technology, with biological agent to exempting from for gathering from the patient
Epidemic disease cell is cultivated and expanded in vitro, then is fed back in patient body, so that excite, strengthen the immunologic function of body itself,
Reach the purpose for monitoring and killing sick cell or repair damaged cell.
NK (natural killer cell, NK) belongs to granular lymphocytic, and being that body is important is immunized
Cell.It is generally acknowledged that NK cell deriveds are in marrow lymphoid stem cells, it breaks up, development depends on marrow or thymus microenvironment,
Peripheral blood and spleen are distributed mainly on, also has a small amount of presence in lymph node and its hetero-organization.Expression specificity does not resist NK cells
Former identification receptor, is to be different from T, the 3rd quasi-lymphocyte of bone-marrow-derived lymphocyte.Compared with other lymphocytes, NK cells are killed
Wound activity is without MHC limitations, without antigen presensitization, therefore referred to as Nk Cell Activity.NK cell cytosols enrich, containing larger
Azurophilic granule, and the content of azurophilic granule and the killing activity of NK cells are proportionate, therefore NK cytosiies are thin in target
There is early, 1 hour in vitro, internal 4 hours i.e. visible lethal effect in lethal effect after born of the same parents.The target cell of NK cells mainly has
Some tumour cells (including part cell line), virus infected cell etc., therefore NK cells can some tumour cells of direct killing
And virus infected cell.It can play important body is antitumor, in viral infection resisting and immunological regulation this means NK cells
Effect.
It, by the human protein of CD244 gene codes, also known as NK cell receptors 2B4, is that a NK cell is important that CD244, which is,
Surface receptor.NK cells pass through CD244 receptor activation cytotoxicity functions.
CAR means Chimeric antigen receptor (Chimeric Antigen Receptor), when CAR structures are related to tumour anti-
Original antibody amalgamation and expression can assign the energy of NK cell-specifics identification correspondence tumour cell in the cell membrane surface of NK cells
Power, NK cells now are CAR-NK cells.One complete CAR structure generally includes film signaling zone, antibody district (generally
From correspondence monoclonal antibody scFV sections), extracellular hinge area, transmembrane region, intracellular signal area.The NK crossed by genetic modification
Cell (CAR-NK) can produce the stronger killing functions of immunocytes of specificity to the cell with its antigen being directed to, so as to
Reach the effect to treatments such as tumours.
Slow virus carrier refer to human immunodeficiency virus-1 (HIV-1) originate a kind of viral vector, can will outside
Source gene is effectively incorporated on host chromosome, is that foreign gene is normal so as to reach that persistence expresses the effect of aim sequence
With one of carrier format.Its basic process is exactly the slow virus carrier for carrying foreign gene in slow virus packaging plasmid, cell
Under the auxiliary of system, turn into infectious virion by virus packaging, by infection cell or biological tissue, realize external source
Gene is expressed in cell or biological tissue.Slow virus carrier can effectively infect neuronal cell in terms of infection ability, swell
Polytype cell such as oncocyte, stem cell, cardiac muscle cell.More convenient mesh can be quickly realized using slow virus carrier
Gene long-term, stable expression.
At present, method and its answering in terms of immunotherapy of tumors prepared by a kind of effective CAR-NK cells are not yet set up
Use precedent.
The content of the invention
For defect present in prior art, it is an object of the invention to provide a kind of CAR-NK cells and its preparation side
Method and application.
The purpose of the present invention and the technical problem to be solved are：People source CD244 sequences are transformed, are enabled thin in NK etc.
Cellular surface expression specificity albumen, the specific proteins can recognize specific tumors cell, while NK cells can be activated, be allowed to
With the killing ability to specific tumors cell.Slow virus can be coated with by including the slow virus carrier of the CD244 sequences of transformation,
The CD244 protein sequences of NK cells expression transformation can be made after slow-virus infection NK cells.
To achieve the above objectives, the present invention is adopted the technical scheme that：
The present invention provides a kind of preparation method of CAR-NK cells, comprises the following steps：
S1, by specific antibody (the specific antibody CD19 antibody or PSMA antibody of such as tumour cell target antigen) encode
Gene (chain moiety, the coupling part of heavy chain and light chain and the heavy chain moiety that include specific antibody) is connected to engineered
CAR genes are obtained in the cloning site area of CAR skeletons,
It is extracellular that the engineered CAR skeletons include film signaling zone, cloning site area, CD244 on the CD244 that is sequentially connected
Hinge area, CD244 transmembrane segments area and CD244 intracellular parts area,
S2, by the CAR genes by slow-virus infection NK cells, obtain expressing the CAR albumen (i.e. described CAR bases
CAR-CD244-antiCD19 protein structures and CAR-CD244-antiPSMA albumen in the expressing protein of cause, such as embodiment 4
Structure) NK cells (such as NK92MI cells), i.e., described CAR-NK cells.
In the preparation method of above-mentioned CAR-NK cells, the DNA sequence dna of film signaling zone such as sequence on the CD244
Shown in the 1st to the 63rd in 1；
And/or, the DNA sequence dna in the cloning site area is as shown in the 64th to the 95th in sequence 1；
And/or, the extracellular hinge areas of CD244, CD244 transmembrane segments area and the DNA sequence dna in CD244 intracellular parts area are such as
Shown in the 96th to the 560th in sequence 1.
In the preparation method of above-mentioned CAR-NK cells, the DNA sequence dna such as sequence of the engineered CAR skeletons
Shown in 1.
In the preparation method of above-mentioned CAR-NK cells, the step S2 is carried out according to the method comprised the following steps：
S21, the CAR genes are connected on the cloning site of slow virus carrier to acquisition recombined lentivirus vector A, will recombinated
Slow virus carrier A transfection aim cells (such as 293T cells) are cultivated, and obtain slow virus；
S22, by slow-virus infection NK cells, obtain the CAR-NK cells.
In the preparation method of above-mentioned CAR-NK cells, in step S21, the slow virus carrier carries for Plenti slow virus
Body；
The transfection is carried out using slow virus packaging plasmid；
The slow virus packaging plasmid is specially psPAX2 and PMD2.G, during transfection, according to by the slow virus carrier,
PsPAX2 and PMD2.G is using quality ratio ratio as 5ug:3.2ug:1.8ug—7.5ug:4.8ug:2.7ug transfects 3X106Individual purpose
Cell；
It is described to include slow-virus infection NK cells the slow virus of concentration is added in complete medium in step S22,
The step of being mixed with NK cells.
It is described also to include slow-virus infection NK cells to add in step S22 in the preparation method of above-mentioned CAR-NK cells
The step of entering polybrene；The final concentration of 4ug/mL of addition of the polybrene；
The slow virus of concentration is after recombined lentivirus vector A transfections aim cell is cultivated, by the supernatant of culture
After being centrifuged 1 hour under the conditions of 20000g, take slow virus to be suspended with DPBS buffer solutions and obtain.
It is following any of (1) or (2) present invention also offers a kind of DNA molecular：
(1) film signaling zone, cloning site area, the extracellular hinge areas of CD244, CD244 cross-films on the CD244 that is sequentially connected are included
Part area and CD244 intracellular parts area；
(2) it is that specific antibody is inserted in the cloning site area of (1) described DNA molecular (such as tumour cell target antigen
Specific antibody) DNA molecular that is formed after encoding gene.
In above-mentioned DNA molecular, the 1st on the CD244 in the DNA sequence dna of film signaling zone such as sequence 1 is extremely
Shown in 63rd；
And/or, the DNA sequence dna in the cloning site area is as shown in the 64th to the 95th in sequence 1；
And/or, the extracellular hinge areas of CD244, CD244 transmembrane segments area and the DNA sequence dna in CD244 intracellular parts area are such as
Shown in the 96th to the 560th in sequence 1；
The sequence of the DNA molecular of (1) concretely sequence shown in sequence 1.
Recombinant vector (such as recombinant expression carrier, recombined lentivirus vector), again of the present invention protection containing the DNA molecular
Group cell (such as immunocyte NK cells or T cell, or 293T cells) or recombinant cell lines.
The present invention protection DNA molecular, the recombinant vector, recombinant cell or recombinant cell lines, any above method
Prepare CAR-NK cells and prepare oncotherapy (as suppressed tumour growth or killing tumor cell) medicine (such as tumour immunity
Treat cell), the application in reagent or kit.
Described prepare contains following liquid in anti-tumor medicine, reagent or kit：It is 5X10 containing concentration6It is individual described
CAR-NK cells/100ul physiological saline.
The present invention has the advantages that：
The cell material that the present invention is used to build novel tumor immunization therapy cell is NK (NK cells), NK
Cell immunogenicity is low, can carry out allosome feedback, cell derived is solved the problems, such as compared to T cell；
Tumor vaccine prepared by the present invention can induce the target cell lysis of antigentic specificity；
Engineered CAR skeletons of the present invention are equally applicable to other immunocytes；Preparation method institute through the present invention
The NK cells of modification can produce the stronger cell killing of specificity to such as tumour cell of the cell with its antigen being directed to and make
With so as to reach the effect of the treatments such as tumour.
Immunotherapy of tumors cell prepared by the present invention can also be applied to different in addition to it can be applied to cancer patient
Body is fed back.Therefore, the immunotherapy of tumors cell prepared by the present invention is wider using scope compared with existing CAR-T, prepares
Cost is lower.
Brief description of the drawings
The present invention has drawings described below：
Fig. 1 is the engineered CD244 skeleton schematic diagrames for being connected to specific antibody encoding gene.
Fig. 2 is recombined lentivirus vector Plenti-CAR-CD244-antiCD19 schematic diagrames.
Fig. 3 is expression figures of the CD244-antiCD19 and CD244-antiPSMA in NK92MI cells.
Fig. 4 is CAR structure representation results in Flow cytometry NK cells, wherein, ordinate is counts, and abscissa is
CD19-FITC (GRN-Hlog) green fluorescence intensity (logarithm value).
Fig. 5 is killing experiments of the NK92MI-CD244-antiCD19 to Daudi tumour cells.
Fig. 6 is killing experiments of the NK92MI-CD244-antiPSMA to PC3 tumour cells.
Fig. 7 is that NK92MI-CD244-antiCD19 is incubated after Daudi tumour cells 20h, the detection of IFN γ Interferon level.
Fig. 8 is that NK92MI-CD244-antiPSMA is incubated after PC3 tumour cells 20h, the detection of IFN γ Interferon level.
Fig. 9 is that the tumour growth situation result after living body fluorescent imaging is carried out to mouse.
Embodiment
The present invention is described in further detail below in conjunction with accompanying drawing.
It is prepared by embodiment 1, recombinant vector CAR-CD244-antiCD19 and recombinant vector CAR-CD244-antiPSMA
1. specific antibody encoding gene is carried out using primer of the end with BfuAI restriction enzyme enzyme recognition sites
PCR is expanded；
The specific antibody coding gene sequence used in the present embodiment is respectively CD19 antibody (antiCD19)
The partial sequence (sequence 3) of partial sequence (sequence 2) and PSMA antibody (antiPSMA)；
2. the PCR primer of step 1 is purified respectively；
3. the PCR primer that step 2 is purified carries out single endonuclease digestion (BfuAI), and simultaneously by the CD244 skeletons containing transformation
Cloning vector carries out single endonuclease digestion (BfuAI)；
4. product after digestion is purified；
5. it is 3 by the cloning vector mol ratio of CD244 skeleton of the product after purification by antibody fragment and containing transformation:1
After proportioning, it is attached, converted using T4 ligases, coated plate, picking monoclonal extracts plasmid order-checking；Correct plasmid will be connected
(being connected into antiCD19 or antiPSMA in the cloning site area of the CD244 skeletons of the transformation) is respectively designated as：Restructuring
Support C AR-CD244-antiCD19 and recombinant vector CAR-CD244-antiPSMA.
The CD244 frame sequences of the transformation as shown in sequence 1, wherein, the 1st in sequence 1 is extremely
63rd shown sequence is the DNA sequence dna of film signaling zone on CD244；The 64th to the 95th shown sequence in sequence 1
It is classified as the DNA sequence dna in cloning site area；The 96th to the 560th shown sequence in sequence 1 is the extracellular hinges of CD244
(extracellular hinge area is included in transmembrane segment area and intracellular to the DNA sequence dna in area, CD244 transmembrane segments area and CD244 intracellular parts area
Part area) in；The 561st to the 575th shown sequence in sequence 1 marks for DDDDK-tag；Sequence 1
In the 576th to the 578th shown sequence be terminator codon.
The CD244 skeleton schematic diagrames for connecting the transformation of specific antibody encoding gene are as shown in Figure 1：In Fig. 1,
CD244leader represents film signaling zone on CD244, VLThe chain moiety of specific antibody is represented, GSlinker represents specificity
The coupling part of heavy chain of antibody and light chain, VHThe heavy chain moiety of specific antibody is represented, CD244 represents CD244 extracellular hinge
Area, transmembrane segment area and intracellular part area, DDDDK represent DDDDK-tag marks.
The preparation of embodiment 2, recombined lentivirus vector
1. the recombinant vector CAR-CD244-antiCD19 and recombinant vector CAR-CD244- that are prepared respectively with embodiment 1
AntiPSMA is expanded as template using the primer PCR with BamHI and XbaI restriction enzyme digestion sites sequences；
2. a pair PCR primer is purified respectively, digestion (BamHI and XbaI), purifying；
3. the product of step 2 is connected into Plenti slow virus carriers (entitled pLenti respectively respectively using T4 ligases
CMV/TO eGFP Puro (w159-1), purchased from addgene, network address：http://www.addgene.org/17481/)
Between BamHI and XbaI sites；
4. conversion, coated plate, picking monoclonal extract plasmid order-checking, correct recombined lentivirus vector will be sequenced and names respectively
For recombined lentivirus vector Plenti-CAR-CD244-antiCD19 and Plenti-CAR-CD244-antiPSMA.
Recombined lentivirus vector Plenti-CAR-CD244-antiCD19 schematic diagram is as shown in Fig. 2 wherein, CMV-
Promoter represents CMV promoter, and CD244peptide is film signaling zone on CD244, and antiCD19 is CD19 antibody, CD244
For the extracellular hinge areas of CD244, transmembrane segment area and intracellular part area, DDDDK marks for DDDDK-tag, and WPRE is that function is similar
In polyA 3 ' UTR region.
Embodiment 3, the preparation and concentration of slow virus
1st, the preparation of slow virus
The recombined lentivirus vector Plenti-CAR-CD244-antiCD19 and psPAX2 (purchases prepared using embodiment 2
From addgene, network address is https://www.addgene.org/12260/), pMD2.G (be purchased from addgene, network address is
https://www.addgene.org/12259/) carrier in mass ratio be 5ug:3.2ug:1.8ug transfection 293T cells are (about
3X106Individual 293T cells are laid on 10cm wares), fresh culture (DMEM culture mediums add 10% hyclone) is replaced by after 8h,
A supernatant being collected every 24h afterwards and adding fresh culture (DMEM culture mediums add 10% hyclone), 3 are collected altogether
It is secondary, obtain slow virus supernatant A.
Using embodiment 2 prepare by recombined lentivirus vector Plenti-CAR-CD244-antiPSMA and psPAX2,
PMD2.G carriers are 5ug in mass ratio:3.2ug:1.8ug transfection 293T cells (about 3X106Individual 293T cells are laid on 10cm wares),
Fresh culture (DMEM culture mediums add 10% hyclone) is replaced by after 8h, a supernatant is collected every 24h afterwards and adds
Enter fresh culture (DMEM culture mediums add 10% hyclone), collect 3 times altogether, obtain slow virus supernatant B.
2nd, the concentration of slow virus
The slow virus supernatant A and B that step 1 is obtained respectively loads high speed centrifugation pipe by 30ml/ pipes, uses supercentrifuge
Centrifugation, 20000g is centrifuged 1 hour, collects slow virus on tube wall, is suspended using 100ul DPBS buffer solutions, and concentration is obtained respectively
Slow virus A and B, -80 DEG C of preservations.
Embodiment 4, the slow-virus infection NK cells of concentration prepare CAR-NK cells and detection
1st, the slow-virus infection NK cells of concentration
The slow virus A and B of the concentration respectively prepared by embodiment 3, add 1ml complete mediums, with 105Individual NK92MI is thin
Born of the same parents (are purchased from ATCC, network address：https://www.atcc.org/) mix (addition final concentration 4ug/ml polybrene
Polybrene), recombinant cell NK92MI-CD244-antiCD19 and recombinant cell NK92MI-CD244-antiPSMA are obtained
(recombinant cell obtained herein is cell mixing, and wherein CAR-NK cells refer to NK92MI recombinant cell NK92MI-CD244-
AntiCD19 and NK92MI-CD244-antiPSMA).
2nd, detection of expression of the CAR structures in NK cells
Step 1 is obtained into the 293T transfection carriers that recombinant cell NK92MI-CD244-antiCD19 and embodiment 3 are obtained
Plenti-CAR-CD244-antiCD19 cell carries out Western blot detection surface antibody expressions:With NK92MI
It is control to transfect the cell after Plenti empty carriers, and Plenti empty carriers are transfected respectively at NK92MI using CD19-FITC antigens
After cell, recombinant cell NK92MI-CD244-antiCD19 afterwards is incubated, Western blot detections are carried out, as a result such as
Shown in Fig. 3.
Fig. 3 result shows, NK92MI-CD244-antiCD19 and 293T transfection carriers Plenti-CAR-CD244-
AntiCD19 cell being capable of specifically expressing CAR-CD244-antiCD19 protein structures.
The 293T transfection carriers Plenti-CAR- that recombinant cell NK92MI-CD244-antiPSMA and embodiment 3 are obtained
The detection of expression of CD244-antiPSMA cell is carried out according to the method described above, as a result, NK92MI-CD244-antiPSMA and
293T transfection carriers Plenti-CAR-CD244-antiPSMA cell being capable of specific expressed CAR-CD244-antiPSMA eggs
White structure.
3rd, in Flow cytometry CAR-NK cells CAR protein structures expression
Concrete operations are carried out by streaming antigenic label operation instruction, specific as follows：
Cell to be detected (the recombinant cell NK92MI-CD244-antiCD19 in step 2) is taken about 105Individual cell centrifugation
After remove supernatant；
Using 200ul DPBS suspensions cell to be detected, streaming antigenic label is added, is gently mixed, in 37 DEG C of shaking tables
(100 turns) are incubated 30 minutes, and supernatant is removed in centrifugation；
Using 500ul complete medium suspension cells, 37 DEG C of incubator cultures 5 minutes, supernatant is abandoned in centrifugation；
It is resuspended using 200ul DPBS after cell, upper machine testing.
As a result：As shown in figure 4, Flow cytometry recombinant cell NK92MI-CD244-antiCD19 cell surfaces CAR
The expression of protein structure, darker non-flanged solid line grey parts are that shallower has reality without any modification NK92MI cell controls
Line edge grey parts are that recombinant cell NK92MI-CD244-antiCD19 is incubated CD19-FITC antigens, and displacement explanation restructuring is thin
Born of the same parents' NK92MI-CD244-antiCD19 cell membrane surface has CD19 antibody expressions；Boundary line is correspondence abscissa fluorescence signal position
Put corresponding cell count.
The killing experiments of embodiment 5, CAR-NK cells against tumor cells
Take 104Individual NK92MI-CD244-antiCD19 cells and 104Individual bone-marrow-derived lymphocyte knurl (Daudi) cell incubation, with
NK92MI is control, is designated as 1:1；
5,000 NK92MI-CD244-antiCD1 cells and 104Individual Daudi is incubated, and using NK92MI as control, is designated as
0.5:1；
2,500 NK92MI-CD244-antiCD19 cells and 104Individual Daudi is incubated, and using NK92MI as control, is designated as
0.25:1；
1,250 NK92MI-CD244-antiCD19 cells and 104Individual Daudi is incubated, and using NK92MI as control, is designated as
0.125:1；
By NK92MI-CD244-antiPSMA and prostate cancer (PC3) cell incubation, using NK92MI as control, ratio is pressed
The above method is carried out.
If three groups of parallel controls, detected after being incubated 20 hours using CellTox-Green (promega G8471) kit
Cell viability (universal method), drawing.
As a result as shown in Figure 5 and Figure 6, wherein, Diamond spot be unmodified NK92MI cells under the conditions of oncolysis
Degree；Square points are that (NK92MI-antiCD19 represents NK92MI-CD244- to the NK92MI cells through the modification of the method for embodiment 4
AntiCD19, NK92MI-antiPSMA represent NK92MI-CD244-antiPSMA) under the conditions of tumor lysis degree.As a result demonstrate,prove
The NK cells of bright recombinant C D244 modifieds, i.e. CAR-NK recombinant cells are compared to the NK cells against tumor cells of unmodified mistake
Fragmentation effect is more preferable.
IFN γ Interferon level detection after embodiment 6, CAR-NK cell incubation Daudi, PC3 cells 20h
Take 105Individual NK92MI-CD244-antiCD19 and 105Individual Daudi cells are incubated 20h (notes in 500ul culture mediums
For NK92MI-antiCD19+Daudi)；Using unmodified NK92MI cells as control (being designated as NK92MI+Daudi)；
Take 105Individual NK92MI-CD244-antiPSMA cells and 105Individual PC3 cells are incubated 20h in 500ul culture mediums
(being designated as NK92MI-antiPSMA+PC3)；Using unmodified NK92MI cells as control (being designated as NK92MI+PC3)；
Cell centrifuging and taking supernatant after incubation is subjected to IFN γ Interferon level detection, specific method is used by kit
Illustrate to carry out (different kit methods are slightly different).
As a result as shown in Figure 7 and Figure 8, wherein, NK92MI is the result that is individually incubated of unmodified NK92MI cells,
NK92MI-antiCD19 is the result that NK92MI-CD244-antiCD19 is individually incubated, and NK92MI-antiPSMA is
The result that NK92MI-CD244-antiPSMA is individually incubated.
As a result show：The NK cells of recombinant C D244 modifieds be CAR-NK cells NK92MI-CD244-antiCD19 and
NK92MI-CD244-antiPSMA can discharge more IFN γs after being incubated corresponding tumour cell, illustrate CAR-NK activity ratio
Unmodified common NK cytoactives are stronger, higher to tumor cytotoxicity effect.
Embodiment 7, the tumor model experiment in vivo of experimental animal
The nude mice (Nude Mouse) of health is randomly divided into 3 groups for totally 9, every group 3,3 groups are respectively blank control group,
NK cell controls groups, CAR-NK groups of cells.5X10 is injected near the oxter of all mouse shirtfronts6It is individual to have GFP fluorescent protein labelings
PC3 tumour cells/only, and normally feed 14 days.After 14 days, living body fluorescent imaging, record are carried out to the mouse of all groups
The growth situation of mouse interior tumor.After fluorescence imaging, respectively to entering respectively near each group mouse PC3 tumor cell injection points
The following administration of row：
Give blank control group mice received saline injection 100ul；
Common NK92MI cell suspending liquids 100ul is injected to NK cell controls groups mouse, sum is 5X106Individual NK cells/
100ul physiological saline；
CAR-NK (the NK92MI- being made to CAR-NK groups of cells mouse injection needles for the embodiment 4 of PC3 tumour cells
CD244-antiPSMA) (concentration is 5X10 to cell suspending liquid6Individual CAR-NK (NK92MI-CD244-antiPSMA) cell/
100ul physiological saline).
Injection finishes rear and normal feed 7 days and carries out living body fluorescent imaging, record to the mouse of all groups respectively afterwards
Mouse interior tumor situation.
As a result as shown in figure 9, the row of left, center, right three are respectively in Fig. 9：Blank control group, NK cell controls group, CAR-NK are thin
Born of the same parents' group, the first behavior injection finish after result, the second behavior normally feed 7 days after result, Fig. 9 results show, compared to
Mean tumour volume in blank control group and NK cell controls groups, CAR-NK groups of cells Mice Bodies is significantly smaller, tumour growth
Substantially it is inhibited.
The content not being described in detail in this specification belongs to prior art known to professional and technical personnel in the field.
<110>Beijing is in promise medical science and technology Co., Ltd
<120>A kind of CAR-NK cells and preparation method and application
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 578
<212> DNA
<213>Artificial sequence
<400> 1
atgctggggc aagtggtcac cctcatactc ctcctgctcc tcaaggtgta tcagggcaaa 60
ggattttgca ggtaaatttc ccgggacctg caaaacagga ctgtcagaat gcccatcagg 120
aattcagatt ttggccgttt ttggtgatca tcgtgattct aagcgcactg ttccttggca 180
cccttgcctg cttctgtgtg tggaggagaa agaggaagga gaagcagtca gagaccagtc 240
ccaaggaatt tttgacaatt tacgaagatg tcaaggatct gaaaaccagg agaaatcacg 300
agcaggagca gacttttcct ggagggggga gcaccatcta ctctatgatc cagtcccagt 360
cttctgctcc cacgtcacaa gaaccagcat atacattata ttcattaatt cagccttcca 420
ggaagtctgg atccaggaag aggaaccaca gcccttcctt caatagcact atctatgaag 480
tgattggaaa gagtcaacct aaagcccaga accctgctcg attgagccgc aaagagctgg 540
agaactttga tgtttattcc gatgacgatg acaagtga 578
<210> 2
<211> 774
<212> DNA
<213>Artificial sequence
<400> 2
gaggtgaaac tgcaggagtc aggacctggc ctggtggcgc cctcacagag cctgtccgtc 60
acatgcactg tctcaggggt gtcattaccc gactatggtg taagctggat tcgccagcct 120
ccacgaaagg gtctggagtg gctgggagta atatggggta gtgaaaccac atactataat 180
tcagctctca aatccagact gaccatcatc aaggacaact ccaagagcca agttttctta 240
aaaatgaaca gtctgcaaac tgatgacaca gccatttact actgtgccaa acattattac 300
tacggtggta gctatgctat ggactactgg ggtcaaggaa cctcagtcac cgtctcctca 360
ggtggtggtg gttccggtgg tggtggttcc ggtggtggtg gttccgacat ccagatgaca 420
cagactacat cctccctgtc tgcctctctg ggagacagag tcaccatcag ttgcagggca 480
agtcaggaca ttagtaaata tttaaattgg tatcagcaga aaccagatgg aactgttaaa 540
ctcctgatct accatacatc aagattacac tcaggagtcc catcaaggtt cagtggcagt 600
gggtctggaa cagattattc tctcaccatt agcaacctgg agcaagaaga tattgccact 660
tacttttgcc aacagggtaa tacgcttccg tacacgttcg gaggggggac taagttggaa 720
ataacacggg ctgatgctgc accaactgta tccatcttcc caccatccag taat 774
<210> 3
<211> 834
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<213>Artificial sequence
<400> 3
agtcctgccc agttcctgtt tctgttagtg ctctggattc aggaaaccaa cggtgatgtt 60
gtaatgaccc agactccact cactttgtcg gttaccattg gacaaccagc ctctatctct 120
tgcaagtcaa gtcagagcct cttatatagt aatggaaaaa cctatttgaa ttggttatta 180
cagaggccag gccagtctcc aaagcgccta atctatctgg tgtctaaact ggactctgga 240
gtccctgaca ggttcactgg cagtggatca ggaacagatt ttacactgaa aatcagcaga 300
gtggaggctg aggatttggg agtttattac tgcgtgcaag gtacacattt tcctcacacg 360
ttcggagggg ggaccaagct ggaaataaaa cggggtggct caggatcggg tggctctggc 420
tctggtggct caggatcgaa cttcgggctc agcttgattt tccttgtcct tgttttaaaa 480
ggtgtccagt gtgaagtgaa ggtggtggag tctgggggag gcttagtgaa gcctggagcg 540
tctctgaaac tctcctgtgc agcctctgga ttcactttca gtaactatgg catgtcttgg 600
gttcgccaga cttcagacaa gaggctggag tgggtcgcat ccattagtag tggtggtgat 660
agcaccttct atgcagacaa tgtaaagggc cgattcacca tctccagaga gaatgccaag 720
aacaccctgt acctgcaaat gagtagtctg aagtctgagg acacggcctt gtattactgt 780
gcaagagacg atctatttaa ctggggccaa ggcaccactc tcacagtctc atca 834
Claims (10)
1. a kind of preparation method of CAR-NK cells, comprises the following steps：
S1, specific antibody encoding gene is connected in the cloning site area of engineered CAR skeletons to acquisition CAR genes,
The engineered CAR skeletons include film signaling zone, cloning site area, the extracellular hinges of CD244 on the CD244 that is sequentially connected
Area, CD244 transmembrane segments area and CD244 intracellular parts area,
S2, by the CAR genes by slow-virus infection NK cells, obtain expressing the NK cells of the CAR albumen, i.e., it is described
CAR-NK cells.
2. the preparation method of CAR-NK cells as claimed in claim 1, it is characterised in that：
The DNA sequence dna of film signaling zone is as shown in the 1st to the 63rd in sequence 1 on the CD244；
And/or, the DNA sequence dna in the cloning site area is as shown in the 64th to the 95th in sequence 1；
And/or, the extracellular hinge areas of CD244, CD244 transmembrane segments area and the DNA sequence dna such as sequence in CD244 intracellular parts area
Shown in the 96th to the 560th in table sequence 1.
3. the preparation method of CAR-NK cells as claimed in claim 1, it is characterised in that：The engineered CAR skeletons
DNA sequence dna is as shown in sequence 1.
4. the preparation method of CAR-NK cells as claimed in claim 1, it is characterised in that：The step S2 is according to including as follows
The method of step is carried out：
S21, the CAR genes are connected on the cloning site of slow virus carrier to acquisition recombined lentivirus vector A, by recombinant lentiviral disease
Poisonous carrier A transfections aim cell is cultivated, and obtains slow virus；
S22, by slow-virus infection NK cells, obtain the CAR-NK cells.
5. the preparation method of CAR-NK cells as claimed in claim 4, it is characterised in that：In step S21, the slow virus carries
Body is Plenti slow virus carriers；
The transfection is carried out using slow virus packaging plasmid；
The slow virus packaging plasmid is specially psPAX2 and PMD2.G, during transfection, according to by the slow virus carrier, psPAX2
With PMD2.G using quality ratio ratio as 5ug:3.2ug:1.8ug—7.5ug:4.8ug:2.7ug transfects 3X106Individual aim cell；
It is described to include slow-virus infection NK cells the slow virus of concentration is added in complete medium in step S22, with NK
The step of cell is mixed.
6. the preparation method of CAR-NK cells as claimed in claim 5, it is characterised in that：It is described by slow virus in step S22
Infect the step of NK cells also include adding polybrene；The final concentration of 4ug/mL of addition of the polybrene；
The slow virus of concentration is that after recombined lentivirus vector A transfections aim cell is cultivated, the supernatant of culture is existed
After being centrifuged 1 hour under the conditions of 20000g, take slow virus to be suspended with DPBS buffer solutions and obtain.
7. a kind of DNA molecular, is following any of (1) or (2)：
(1) film signaling zone, cloning site area, the extracellular hinge areas of CD244, CD244 transmembrane segments on the CD244 that is sequentially connected are included
Area and CD244 intracellular parts area；
(2) it is that the DNA molecular formed after specific antibody encoding gene is inserted in the cloning site area of (1) described DNA molecular.
8. DNA molecular as claimed in claim 7, it is characterised in that：
The DNA sequence dna of film signaling zone is as shown in the 1st to the 63rd in sequence 1 on the CD244；
And/or, the DNA sequence dna in the cloning site area is as shown in the 64th to the 95th in sequence 1；
And/or, the extracellular hinge areas of CD244, CD244 transmembrane segments area and the DNA sequence dna such as sequence in CD244 intracellular parts area
Shown in the 96th to the 560th in table sequence 1.
9. recombinant vector, recombinant cell or recombinant cell lines containing DNA molecular described in claim 7 or 8.
10. DNA molecular described in claim 7 or 8, recombinant vector, recombinant cell or recombinant cell lines, power described in claim 9
Profit requires that any methods described prepares CAR-NK cells in medicine, reagent or the kit for preparing oncotherapy in 1-6
Application.
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| CN201810984124.6A CN109097401B (en) | 2017-03-31 | 2017-03-31 | Preparation method of recombinant vector CAR-CD 244-anti-SMA |
| CN201810984136.9A CN109097402B (en) | 2017-03-31 | 2017-03-31 | Preparation method of recombinant vector CAR-CD244-anti CD19 |
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| CN107267463A (en) * | 2017-08-23 | 2017-10-20 | 安徽惠恩生物科技股份有限公司 | A kind of Car NK cell preparation methods for breast cancer |
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| CN109825528A (en) * | 2019-02-21 | 2019-05-31 | 北京呈诺医学科技有限公司 | A kind of CAR-NK cell preparation method for Huppert's disease |
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| CN114835820A (en) * | 2022-04-14 | 2022-08-02 | 呈诺再生医学科技(珠海横琴新区)有限公司 | Chimeric Fc receptor for genetically modified pluripotent stem cells and natural killer cells |
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| US11738051B2 (en) | 2017-12-06 | 2023-08-29 | Asclepius (Suzhou) Technology Company Group Co., Ltd. | Nucleotide sequences for encoding CAR, ROBO1 CAR-NK cells of expressing the CAR, and preparation and application thereof |
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| CN114835820A (en) * | 2022-04-14 | 2022-08-02 | 呈诺再生医学科技(珠海横琴新区)有限公司 | Chimeric Fc receptor for genetically modified pluripotent stem cells and natural killer cells |
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| CN117384858B (en) * | 2023-12-12 | 2024-03-08 | 山东丽山生物科技有限公司 | Preparation method and application of chimeric antigen receptor T cells |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107034237B (en) | 2018-10-16 |
| CN109097401B (en) | 2021-09-07 |
| CN109097401A (en) | 2018-12-28 |
| CN109097402A (en) | 2018-12-28 |
| CN109097402B (en) | 2021-09-07 |
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