CN107858352A - A kind of shRNA cluster sequences and its expression vector and purposes - Google Patents

A kind of shRNA cluster sequences and its expression vector and purposes Download PDF

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CN107858352A
CN107858352A CN201711096268.XA CN201711096268A CN107858352A CN 107858352 A CN107858352 A CN 107858352A CN 201711096268 A CN201711096268 A CN 201711096268A CN 107858352 A CN107858352 A CN 107858352A
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cell
shrna
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CN107858352B (en
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张辉
邹帆
卢丽娟
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Guangzhou Qianyang Bio Pharmaceutical Technology Co Ltd
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Abstract

The present invention provides a kind of shRNA cluster sequences, and present invention also offers a kind of expression vector, the expression vector expresses shRNA cluster sequences described above and CAR molecules, and present invention also offers their purposes.The present invention lowers PD 1, the expression of 3 three acceptors of Tim 3, Lag or the downward expression of receptor of PD 1 by the sequence-specific in miRNA cluster sources.The present invention suppresses the expression of the multiple Inhibitory receptors in its surface while the T cell after using CAR molecular modifications, so as to strengthen the inhibition of its antagonism entity tumor microenvironment, and then the killing to tumour is forcefully mediated, it can preferably be used for anti entity tumour immunization therapy.

Description

A kind of shRNA-cluster sequences and its expression vector and purposes
Technical field
The present invention relates to a kind of shRNA-cluster sequences and its expression vector and purposes.
Background technology
From CAR-T immunological techniques 2011 for CD19 antigens acute lymphoblastic type leukaemia clinical test obtain into Fruit simultaneously was chosen as starting first of ten big technological breakthroughs then in 2013 by Science magazines, the technology obtain from scientific circles The favorable comment consistent to financial quarters.
The new hair tumor patient that diagnoses every year of China more than 3,000,000 people, the probability of most tumors postoperative recurrence more than 50%, Median survival interval less than 5 years, catch the flap and see by current traditional treatment means curative effect when in face of most of refractory, recurrence tumours Elbow, research and development specific can infiltrate simultaneously effectively antagonism entity tumor microenvironment and then mediate the CAR-T cells of effective killing to it Possesses important meaning.The great achievement obtained relative to CAR-T cells in blood type tumour, CAR-T cells are for entity The Phase I clinical trial individual difference of tumour is very big, only a small amount of successful story, solves infiltrations of the CAR-T to entity tumor It is to capture the key of solid tumor cell immunotherapy with avoiding premature depletion.
The development of immunotherapy based on entity tumor, scientist have developed largely can specific recognition entity tumor surface The monoclonal antibody class medicine of antigen, these protein medicaments provide for the research and development of the CAR-T immune cell therapies for entity tumor Ensure, for example, for treat Her-2 positives colon cancer, oophoroma monoclonal antibody class medicine Trastuzumab (Herceptin) from It is developed within 2010 specific recognition and the CAR-T immunocytes of Her-2 positive tumors can be killed, for treating EGFR sun Property kinds of tumors Buddhist nun's trastuzumab, Cetuximab, Victibix be developed to related scFv be film foreign lands CAR-T Immunocyte has simultaneously carried out substantial amounts of clinical test.However, except minority achieves good outcome, it is most of for real The clinical test results of body tumour are unsatisfactory, and the solid tumor for removing patient's body using CAR-T immune cell therapies is present extensively General dispute.In addition to lymthoma, using to IL13R α 2 antigentic specificity CAR-T cell therapies, one brain colloid of 50 years old In the clinical test of the patient of knurl, clinician was carrying out 16 altogether more than 220 days to the tumor locus and encephalic of patient Secondary cell observes the disappearance of tumour finally after feeding back.Multiple team are removing pancreas using current CAR-T immune cell therapies The clinical test of the entity tumors such as gland cancer, prostate cancer, it was confirmed that the security of related CAR-T cells and have to tumour cell The killing of limit.To find out its cause, the inhibition of tumor microenvironment causes CAR-T cells equally to press down without the soluble monoclonal antibody medicine of the image of Buddha Simultaneously killing tumor cell is made, therefore should be partial to strengthen CAR-T cells for the emphasis of the research and development of the CAR-T technologies of entity tumor Wetting capacity to entity tumor and the tolerance to hypoxia condition in tumor microenvironment.Therefore it is designed to fully infiltrate Tumor microenvironment, it is long-term to maintain self-renewing to be resistant to low-oxygen environment simultaneously, further enhance killing to entity tumor and in body It is interior continue in the presence of exercise immune surveillance function CAR-T immunocytes technology of future generation be captured using immune cell therapy it is various The key of entity tumor.
The content of the invention
It is an object of the present invention to provide a kind of shRNA-cluster sequences, and present invention also offers one kind expression to carry Body, the expression vector express shRNA-cluster sequences described above and CAR molecules, present invention also offers they Purposes.
To achieve the above object, the technical solution used in the present invention：A kind of shRNA-cluster sequences, it is described The nucleotide sequence of shRNA-cluster sequences such as SEQ ID NO:1 or such as SEQ ID NO:Shown in 2.The present invention passes through The sequence-specific in miRNA-cluster sources lowers PD-1, the expression of tri- acceptors of Tim-3, Lag-3 or downward PD-1 acceptors Expression.
The invention provides a kind of expression vector, the expression vector is by such as SEQ ID NO:1 or such as SEQ ID NO:2 Shown sequence, and the coded sequence of CAR molecules are connected to obtained by carrier is carrier；The expression vector expresses above-mentioned institute The shRNA-cluster sequences and CAR molecules stated.Will be such as SEQ ID NO:Sequence shown in 1, and the code sequence of CAR molecules Row are connected to expression vector obtained by carrier is carrier and are referred to as PTL-CAR molecules；Will be such as SEQ ID NO:Sequence shown in 2, with It is described such as SEQ ID and the coded sequence of CAR molecules is connected to expression vector obtained by carrier is carrier and is referred to as P-CAR molecules NO:1 or such as SEQ ID NO:Sequence and CAR molecules shown in 2 are driven respectively by two different promoters, and CAR molecules can be special The antigen of opposite sex identification targeting.The structure of P-CAR molecules is as shown in the upper figure in Fig. 1；In the structure such as Fig. 1 of PTL-CAR molecules Figure below shown in.
Preferably, the carrier is carrier is slow virus carrier.
The invention provides a kind of CAR-T cells of transformation, the T cell is to be transferred to expression vector described above Obtained by CD8+T cells.The CD8+T cells of PTL-CAR molecular modifications are referred to as PTL-CAR-T cells, P-CAR molecular modifications CD8+T cells are referred to as P-CAR-T cells.Cultivated for a long time through external, the cell-mediated expression Her2 of CD8+T of PTL-CAR transformations Cell line SK-OV3 killing and the effect of secrete cytokines are more obvious.
The invention provides a kind of preparation method of the CAR-T cells of transformation described above, methods described includes following Step：
(1) PMNC and separation and concentration CD8+T cells therein are collected, using anti-CD3, anti- CD28 and IL-2 activates the CD8+T cells；
(2) CD8+T cell-stimulatings are after 48 hours, with 1ml/1 × 106The ratio of cell adds as claimed in claim 3 Lentiviral is infected, while adds polybrene solution, continues to cultivate after centrifugation；After 8~12 hours, second is carried out Wheel infection, the CAR-T cells transformed.
Preferably, the concentration of the anti-CD3 is that 1 μ g/mL, anti-CD28 concentration is 1 μ g/ ML, the IL-2 concentration are 10ng/mL.
Preferably, the concentration of the polybrene solution is 8 μ g/mL.
The invention provides a kind of method for the CAR-T cells for expanding the transformation described in claim 4, including following step Suddenly：(1) cell is resuspended with fresh culture in the CAR-T cells of transformation, and adds IL-2 and IL-7 and keep cell state；(2) Improved 3rd day and the 5th day, according to cell state and proliferative conditions, complete RMPI1640 culture mediums, cell are added to cell Concentration maintains 2 × 106/ ml, and IL-2 and IL-7 are supplemented, continue to cultivate and pass in time, further amplifying cells.
Preferably, it is characterised in that the concentration of the IL-2 is 10ng/mL, and IL-7 concentration is 10ng/mL.
The invention provides shRNA-cluster sequences described above in the preparation for removing solid tumor cell is prepared Application.
The invention provides expression vector described above or the CAR-T cells transformed as claimed in claim 4 to make Application in the standby preparation for removing solid tumor cell.
The invention provides a kind of method of improvement CAR-T cell functions, methods described is to lower CAR-T by shRNA Tri- expression of receptor of PD-1, Tim-3 and Lag-3 of cell, or lower the PD-1 expression of receptor of CAR-T cells.
Improving the invention provides tri- acceptor joints of PD-1 acceptors or PD-1, Tim-3 and Lag-3 as target spot The application of CAR-T cell functions.
The invention provides lower CAR-T cells tri- expression of receptor of PD-1, Tim-3 and Lag-3 shRNA or under Adjusting the shRNA of the PD-1 expression of receptor of CAR-T cells is improving the application of CAR-T cell functions.
The beneficial effects of the present invention are：
The PTL-CAR-T cells completely newly transformed in the present invention are complete in the present invention in the NSG mouse for the treatment of SK-OV3 lotus knurls The PTL-CAR-T cells newly transformed compare the CAR-T cells reported, the size of PTL-CAR-T cell therapy group mouse tumors It is significantly less than CAR-T cells, and PTL-CAR-T cell therapy group mouse survival rates are substantially better than CAR-T cells.PTL-CAR-T Cell can be swollen by specific activation and the cell factor IFN-γ of a large amount of secretory cell toxicity correlations and then forcefully mediation The necrosis of knurl.
The present invention is in the NSG mouse for the treatment of CD19 positive Raji cell lotus knurls, compared to the CAR-T cells reported, The downright bad effect of the entity tumor positive cell-mediated expression CD19 of PTL-CAR-T is more obvious, associated treatment group mouse tumor Size be significantly less than CAR-T cells.
Brief description of the drawings
Fig. 1 is the structural representation of the PTL-CAR molecules of structure.
Fig. 2 is pLKO-IRES-GFP slow virus carrier structural representations.
Fig. 3 is the expression for the Inhibitory receptor that PTL-Her2-CAR suppress longterm culture in vitro CD8+CAR-T cells.
Fig. 4 is that the CD8+T cells that PTL-Her2-CAR and P-Her2-CAR is transformed mix with Her2 positive target cells system respectively The comparison of secretion of gamma-IFN after conjunction.
Fig. 5 is to be killed after the CD8+T cells that PTL-Her2-CAR and P-CAR is transformed mix with Her2 positive target cells system respectively Hinder the comparison of effect.
Fig. 6 is SK-OV3 tumor-bearing mice tumours after the CD8+T cell therapies that PTL-Her2-CAR-T and Her2-CAR is transformed Size and mouse survival rate.
Fig. 7 is SK-OV3 tumor-bearing mice tumours after the CD8+T cell therapies that PTL-Her2-CAR-T and Her2-CAR is transformed The transformation CD8+T cells of middle infiltration and the ability for discharging IFN-γ.
After the CD8+T cell therapies that Fig. 8 transforms for PTL-Her2-CAR-T and Her2-CAR, SK-OV3 tumor-bearing mices are normal Organ infiltration's immunocyte.
After the CD8+T cell therapies that Fig. 9 transforms for PTL-CD19-CAR-T and CD19-CAR, Raji tumor-bearing mice tumours The change of size.
Embodiment
The present invention is made with reference to Figure of description and specific embodiment and further being elaborated, the embodiment It is served only for explaining the present invention, is not intended to limit the scope of the present invention.Test method used in following embodiments is such as without spy Different explanation, is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent commercially obtained And material.
Embodiment 1
A kind of shRNA-cluster sequences transcribed jointly with CAR molecules, pass through the sequence in miRNA-cluster sources The expression of tri- acceptors of specific downregulation PD-1, Tim-3, Lag-3 or the technology for lowering PD-1 expression of receptor, its nucleic acid nucleotides Sequence such as SEQ ID NO：1 or SEQ ID NO：Shown in 2.SEQ ID NO：The structure of shRNA-cluster molecules shown in 1 is such as Shown in figure below in Fig. 1；SEQ ID NO：The structure of shRNA-cluster molecules shown in 2 is as shown in the upper figure in Fig. 1.
Embodiment 2
First, shRNA-cluster-CAR molecular recombinations pLKO.3G slow virus carriers：According to SEQ ID NO：1 or SEQ ID NO：Sequent synthesis shRNA-cluster shown in 2, the shRNA-cluster of synthesis pass through EcoR I and Pac I double digestions, insertion (see Fig. 2) in pLKO.3G slow virus carriers, digestion, the specific steps of connection refer to the conventional method of this area；By homologous heavy The molecular cloning means of group replace GFP albumen in plasmid to obtain CAR molecules, and corotation record shRNA-cluster is obtained after restructuring With the pLKO.3G slow virus carriers of CAR sequences.
The HEK293T cells of one growth conditions are averagely laid in the 10cm culture dishes handled with poly-D-lysine (thin Born of the same parents' density is about 6.5 × 104/cm2), it is desirable to cell is uniformly distributed in single.After culture about 24 hours, cell confluency degree should approach 80%, now each ware changes liquid with 12ml complete mediums, while adds 3.75ul chloroquines (100mM, 4000 ×).Change after liquid by Table 1 prepares calcium phosphate-DNA mixed liquors, stands 1 minute after reverse mixing for several times, is then rapidly added each ware cell with 3ml/ wares In nutrient solution, side edged shakes up, and rapidly joins dropwise.
Table 1 is calcium phosphate-DNA mixture system formulas
12 hours after transfection, cell confluency degree should be close to 100%, and now each ware changes liquid with 12ml fresh cultures, together When add 90 μ l sodium butyrates (1M, 100 ×).About 48 hours after transfection, whole 34ml HEK293T cell supernatants are collected, with Load 50ml centrifuge tubes after 0.45 μm of filter filtering, the ratio of according to the form below 2 adds PEG-NaCl-PBS mixed liquors, overturns and mixes Placed after 4 DEG C 1.5 hours, mix within during which every 20~30 minutes once or reverse mix stands overnight after 4 DEG C.
Table 2
By mixed liquor in 4 DEG C, 7000g is centrifuged 10 minutes, and visible white precipitates on tube wall, carefully removes whole supernatants, The fresh culture of appropriate volume (300ul~3ml) is added, jiggling dissolves precipitation, that is, obtains shRNA-cluster- The viral concentration liquid of CAR molecular recombinations, use or dispense immediately after -80 DEG C of preservations.
2nd, shRNA-cluster-CAR is in CD8+Expression in T cell：Peripheral blood is separated from peripheral blood sample Mononuclearcell, then pass through the CD8 antibody separation and concentrations CD8 of couple biotin+T lymphocytes, count, centrifugation.With RPMI1640 complete mediums are resuspended, then by 2 × 106/ ml cell concentration, uniformly paving is into Tissue Culture Plate.Utilize Anti-CD3 (final concentration of 1 μ g/ml), anti-CD28 (final concentration of 1 μ g/ml) antibody and IL2 (final concentration of 10ng/ml) Cell is stimulated, after acting on 48 hours, collects cell, the infection for pseudovirus.
The target cell suspension that appropriate growth conditions are good is taken, 300g in centrifuge tube is put into and centrifuges 5 minutes.Supernatant discarding Liquid, with 1ml/1 × 106The ratio of cell adds pseudovirus concentrate, while adds the μ g/ml of final concentration 8 Polybrene, gently Mixing is played in featheriness.Cell suspension is moved into culture dish, 37 DEG C, 350 × g centrifuge 90 minutes, after centrifugation terminates, put back in incubator Continue to cultivate.After about 8~12 hours, the second wheel infection is carried out, step is same as above, and after about 12 hours, liquid is changed in centrifugation, is washed away with PBS Virus in culture medium, cell is resuspended with fresh culture, and adds the IL-2 and IL-7 that final concentration is 10ng/ml and keep thin Born of the same parents' state.Continue to cultivate and pass in time, further amplifying cells.Metainfective 3rd day, according to cell state and propagation feelings Condition, complete RMPI1640 culture mediums are added to cell, cell concentration maintains 2 × 106/ml.And IL-2 and IL-7 are supplemented, it is dense eventually Degree is 10ng/ml.Metainfective 5th day, continue amplifying cells according to the flow of the 3rd day.And utilize flow cytometer detection fluorescence egg White mouse source Fab expression, determine the CD8 of shRNA-cluster-CAR downward longterm culture in vitro+T cell Inhibitory receptor The efficiency (Fig. 3) of expression.Metainfective 5th day, cell was used for subsequent experimental and detects or continue to cultivate and freeze.Hereafter, it is For the sake of convenience, by SEQ ID NO：Tested below the shRNA-cluster-CAR plasmids of 1 structure and be called PTL-CAR, by SEQ ID NO：Tested below the shRNA-cluster-CAR-T plasmids of 2 structures and be called P-CAR.
Embodiment 3
First, by SEQ ID NO：1 builds and targets to test below Her2 shRNA-cluster-CAR plasmids and is called PTL-Her2-CAR, by SEQ ID NO：2 build and target to test below Her2 shRNA-cluster-CAR-T plasmids and claim It is P-Her2-CAR.
The CD8 of PTL-Her2-CAR and P-Her2CAR transformations+After T cell amplification in vitro culture, mixed respectively with target cell system The comparison of IFN-γ effect is discharged after conjunction：With same condition transduction of CD 8+T lymphocytes, then with express Her2 albumen target Cell line (SK-OV3) is mixed in the ELIspot plates of pre-coated IFN-γ antibody, by detecting it to target cell secretion of gamma-IFN Ability probe into Inhibitory receptor expression Her2-CAR-T cell functions between relation.As a result PTL-Her2- is shown The ability of the stimulation secretion of gamma-IFN of CAR-T cellular response target cells is better than P-CAR-T cells, i.e., the downward of more Inhibitory receptors Lifting to Her2-CAR-T cell functions is better than the lifting that independent Inhibitory receptor is lowered to Her2-CAR-T cell functions and (schemed 4)。
2nd, the comparison of fragmentation effect：We will transduce unloaded and PTL-Her2-CAR, P-Her2-CAR molecule CD8+T effector cell's amplification cultivation in vitro, the 5th day, 10 days, 15 days, 20 days, 25 days five time points difference are cultivated in vitro The cell of equivalent is frozen, recover after the 5th batch of cell cryopreservation simultaneously after and cultivates 24 hours, then three kinds of effector cells' difference With target cell system with 8：1 ratio is mixed 24 hours, by detect lactic dehydrogenase (lactate dehydrogenase, LDH the method for release), the killing activity of Her2-CAR-T lymphocytes is detected.Experimental result is shown：During in vitro culture five Between put control effector cell Mock (zero load transduction CD8+T lymphocytes) there is no fragmentation effect to target cell system, In vitro culture three time points of 5 days to 15 days, killing and P-Her2-CAR-T of the PTL-Her2-CAR-T cells to target cell There was no significant difference for cell, and the killing to target cell gradually reduces with the enhancing of incubation time.In vitro cultivate 20 days, 25 Its two time points, killing effect of two kinds of effector cells to target cell continue to reduce, and P-Her2-CAR-T cells are to target cell Killing effect almost disappear, PTL-Her2-CAR-T cells to target cell still possesses limited killing ability, both are to target Significant difference (Fig. 5) be present in the killing of cell.
Embodiment 4
First, the survival rate of the oncolytic effect of PTL-Her2-CAR-T cell therapies and mouse：The NSG mouse of 6~8 weeks 21 Subcutaneous vaccination contains the SK-OV3 cells 1 × 10 of 40%Corning companies high concentration basement membrane matrix glue6Individual/only, by lotus knurl NSG mouse are randomly divided into three groups：PTL-Her2-CAR-T cell therapies group, Her2-CAR-T cell therapies group, Mock-T cells Treatment control group, every group 7.Treat that tumour reaches 500mm3Afterwards, by way of tail vein feedback, every mouse adoptive immunity is thin Born of the same parents 5 × 105It is individual.Experimental result is shown, after adoptive immunity cell, tail vein has been adopted the experiments of PTL-Her2-CAR-T cells Group tumor-bearing mice tumour size be significantly less than Her2-CAR-T cells of adopting experimental mice and the Mock-T that adopted it is thin The control group mice of born of the same parents, while the survival rate of PTL-Her2-CAR-T cell therapy group mouse is also thin apparently higher than Her2-CAR-T Born of the same parents treatment group mouse and Mock-T cell therapy group mouse, experimental result confirm PTL-Her2-CAR-T to Her2 positive tumors There is notable and specific killing, and the survival rate (Fig. 6) of mouse can be effectively improved.
2nd, infiltration of the PTL-Her2-CAR-T cells to entity tumor and effectively killing：The frost for preparing tumor tissues is cut Piece simultaneously carries out experiment by indirect immunofluorescence.We are used as the mark for assessing CAR-T cell functions using IFN-γ.Frost is cut Piece result shows that the quantity of the CAR-T cells infiltrated in the tumour of PTL-Her2-CAR-T cell therapy group mouse is significantly more than The experimental mice of Her2-CART cells and the tumor tissues of control group mice, and in the border in neoplasm necrosis region, PTL- Her2-CAR-T secretes a large amount of further mediate tumor tissues of IFN-γ more towards the borderline region enrichment in neoplasm necrosis Downright bad (Fig. 7).
3rd, PTL-Her2-CAR-T safety evaluation of cell：Immunocyte is due to the expression of a series of " brake " gene It is suppressed, related CAR-T cells, which once miss the target, to bring serious toxicity.In order to assess the security of clinical test, The main organs that we have collected mouse carry out HE dyeing, and experimental result show, PTL-Her2-CAR-T cells do not infiltrate with The main organs of mouse are killed, its security has obtained preliminary demonstration (Fig. 8).
4th, the CAR-T cells for suppressing three Inhibitory receptor expression to probe into specificity infiltrate and killed to entity tumor Whether the enhancing of ability is confined to the positive SK-OV3 ovarian cancer cell lines of Her-2, and we establish can selectively targeted CD19 CAR-T cells, further assess the feasibility that CD19-CAR-T cells are used to remove entity lymthoma.We establish notch graft The lymthoma NSG mouse models of kind Raji cells, reach 200mm in mouse tumor3When, tail vein is adopted three kinds of same battens The CAR-T cells 5 × 10 transduceed under part5Individual/only.Experimental result shows, adopts PTL-CD19-CAR-T cells through tail vein The mouse hypodermic tumour for the treatment of group is significantly less than the treatment group of CD19-CAR-T cells and the control group (figure of VC-CAR-T cells 9)。
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected The limitation of scope is protected, although being explained in detail with reference to preferred embodiment to the present invention, one of ordinary skill in the art should Understand, technical scheme can be modified or equivalent substitution, without departing from the essence of technical solution of the present invention And scope.
Sequence table
<110>Raise biological medicine technology Co., Ltd in Guangzhou thousand
<120>A kind of shRNA-cluster sequences and its expression vector and purposes
<130> 2017
<160> 2
<170> PatentIn version 3.3
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<211> 690
<212> DNA
<213>Artificial sequence（Artificial)
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tggtaagtgc ccaaattgct ggagggccat ctgttttgac ccttaaaggg gtagctcctt 60
accgtgctct cattgccgcc tccccacctc ccgctccagc cctgccgggg cgcttcgtgc 120
taaactggta gctttgtgta ggctaccagt ttagcacgaa gcgctccagc agggcacgca 180
cagcgtccgt ggagggaaag gccttttccc cacttcttaa ccttcactga gagggtggtt 240
ggggtctgtt tcactccatg tgtcctagat cctgtgctac agaccttcct ttctgtcctc 300
ccgtcttgga cctcagtcct gggggctcac tctagattgg ccaatgagct ttgtgtaggc 360
tcattggcca atctagagtg agcccccggg acacgttctc tctgccaatt gtcttcttgg 420
ctgagctccc caagctccat ctgtcatgct ggggagccca gtggcgttca aaagggtctg 480
gtctccctca caggacagct gaactccggg actggccagt gttgagtggc gactttaccc 540
ttcgagcttt gtgtaggctc gaagggtaaa gtcgccactc agtgccggcc caacactgcg 600
gatgctgggg ggagggggga ttccactcct gttttgtgag taggcgaccc atgggctgcc 660
cagccttaaa gccagaacaa gggtgtcccc 690
<210> 2
<211> 690
<212> DNA
<213>Artificial sequence（Artificial)
<400> 2
tggtaagtgc ccaaattgct ggagggccat ctgttttgac ccttaaaggg gtagctcctt 60
accgtgctct cattgccgcc tccccacctc ccgctccagc cctgccgggg cgcttcgtgc 120
taaactggta gctttgtgta ggctaccagt ttagcacgaa gcgctccagc agggcacgca 180
cagcgtccgt ggagggaaag gccttttccc cacttcttaa ccttcactga gagggtggtt 240
ggggtctgtt tcactccatg tgtcctagat cctgtgctac agaccttcct ttctgtcctc 300
ccgtcttgga cctcagtcct gggggctcta aggctatgaa gagatacgct ttgtgtaggc 360
tagcgactaa acacatcaag agcccccggg acacgttctc tctgccaatt gtcttcttgg 420
ctgagctccc caagctccat ctgtcatgct ggggagccca gtggcgttca aaagggtctg 480
gtctccctca caggacagct gaactccggg actggccagt gttgagtaag gctatgaaga 540
gatacgcttt gtgtaggcta gcgactaaac acatcaactc agtgccggcc caacactgcg 600
gatgctgggg ggagggggga ttccactcct gttttgtgag taggcgaccc atgggctgcc 660
cagccttaaa gccagaacaa gggtgtcccc 690

Claims (10)

  1. A kind of 1. shRNA-cluster sequences, it is characterised in that the nucleotide sequence such as SEQ of the shRNA-cluster sequences ID NO:1 or such as SEQ ID NO:Shown in 2.
  2. 2. a kind of expression vector, it is characterised in that the expression vector is by such as SEQ ID NO:1 or such as SEQ ID NO:2 institutes The sequence shown, and the coded sequence of CAR molecules are connected to obtained by carrier is carrier；Expression vector expression such as the right will Seek the shRNA-cluster sequences and CAR molecules described in 1.
  3. 3. expression vector according to claim 1, it is characterised in that the carrier is carrier is slow virus carrier.
  4. 4. the CAR-T cells of a kind of transformation, it is characterised in that the T cell is to carry expression as claimed in claim 2 or claim 3 Body is transferred to obtained by CD8+T cells.
  5. 5. a kind of preparation method for the CAR-T cells transformed as claimed in claim 4, it is characterised in that methods described includes Following steps：
    (1) PMNC and separation and concentration CD8+T cells therein are collected, using anti-CD3, anti-CD28 and IL-2 activates the CD8+T cells；
    (2) CD8+T cell-stimulatings are after 48 hours, with 1ml/1 × 106The ratio of cell adds slow virus as claimed in claim 3 Expression vector is infected, while adds polybrene solution, continues to cultivate after centrifugation；After 8~12 hours, the second wheel sense is carried out Dye, the CAR-T cells transformed.
  6. A kind of 6. method for the CAR-T cells for expanding the transformation described in claim 4, it is characterised in that comprise the following steps： (1) cell is resuspended with fresh culture in the CAR-T cells of transformation, and adds IL-2 and IL-7 and keep cell state；(2) transform The 3rd day afterwards and the 5th day, according to cell state and proliferative conditions, complete RMPI1640 culture mediums, cell concentration are added to cell Maintain 2 × 106/ ml, and IL-2 and IL-7 are supplemented, continue to cultivate and pass in time, further amplifying cells.
  7. 7. shRNA-cluster sequences as claimed in claim 1, expression vector as claimed in claim 2 or claim 3 or such as right It is required that application of the CAR-T cells of the transformation described in 4 in the preparation for removing solid tumor cell is prepared.
  8. A kind of 8. method of improvement CAR-T cell functions, it is characterised in that methods described is to lower CAR-T cells by shRNA Tri- acceptors of PD-1, Tim-3 and Lag-3 expression, or lower CAR-T cells PD-1 expression of receptor.
  9. Tri- acceptor joints of 9.PD-1 acceptors or PD-1, Tim-3 and Lag-3 are improving CAR-T cell functions as target spot Using.
  10. 10. lower the shRNA of tri- expression of receptor of PD-1, Tim-3 and Lag-3 of CAR-T cells or lower CAR-T cells The shRNA of PD-1 expression of receptor is improving the application of CAR-T cell functions.
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CN112239759A (en) * 2020-10-28 2021-01-19 重庆斯德姆生物技术有限公司 MicroRNA for knocking down PD1 gene expression and construction of chimeric antigen receptor-T/NK cell thereof

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WO2020191336A1 (en) * 2019-03-21 2020-09-24 Yamaguchi, Yukiko Composition and method for reducing expression of checkpoint inhibitors in t cells expressing a car or ctl
CN112239759A (en) * 2020-10-28 2021-01-19 重庆斯德姆生物技术有限公司 MicroRNA for knocking down PD1 gene expression and construction of chimeric antigen receptor-T/NK cell thereof

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