CN107699547A - The targeting CD133 of the gene silencings of PD 1 CAR T cells and its application - Google Patents

The targeting CD133 of the gene silencings of PD 1 CAR T cells and its application Download PDF

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CN107699547A
CN107699547A CN201710793243.9A CN201710793243A CN107699547A CN 107699547 A CN107699547 A CN 107699547A CN 201710793243 A CN201710793243 A CN 201710793243A CN 107699547 A CN107699547 A CN 107699547A
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朱学锴
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ShanghaiTech University
University of Shanghai for Science and Technology
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells

Abstract

The invention provides the targeting CD133 of the gene silencings of PD 1 CAR T cells and its application.The targeting CD133 of the described gene silencings of PD 1 CAR T cells, it is characterised in that its preparation method includes：CRISPR Cas9 and Transposon System are imported in PBMC simultaneously first with core transfection system, T cell is then activated, carries out T cell amplification, obtain the targeting CD133 of the gene silencings of PD 1 CAR T cells.The targeting CD133 of the gene silencings of PD 1 provided by the invention CAR T cells can solve the immunosupress of the paths of PD 1 mediation, prevent metastases and recurrence.And the therapeutic alliance based on PD 1 or PD L1 antibody is compared, the targeting CD133 of the gene silencings of PD 1 CAR T cells can effectively avoid systemic toxicity problem, precisely the immune microenvironment of regulation.

Description

The targeting CD133 of PD-1 gene silencings CAR T cells and its application
Technical field
The invention belongs to the immune cell therapy field of tumour, and in particular to a kind of targeting CD133 of PD-1 gene silencings CART cells and its application.
Background technology
Tumor stem cell hypothesis thinks a small set of cell in tumour be present, it possess self duplication, start tumour occur and It is divided into the ability of other tumour cells.Substantial amounts of experimental data also reveal that the presence of this group cell.Importantly, grind Study carefully and show that tumor stem cell has the resistance to the action of a drug, it is related to the failure of the traditional treatment such as chemotherapy, radiotherapy, it is to cause metastases and multiple An important factor for hair.Therefore targeting tumor stem cells will be that oncotherapy is successfully crucial [1].
CD133 is one of molecular marked compound of the most frequently used identification tumor stem cell at present, the equal table in a variety of solid tumors Reach, including glioma, colorectal cancer, liver cancer, cancer of pancreas, breast cancer and oophoroma etc., therefore can be by identifying CD133 targets To killing related neoplasms stem cell [2].The method for the targeting CD133 positive cells reported includes anti-CD133 immunotoxin [3,4], bispecific antibody [5,6] etc., show certain application prospect.In addition, targeting tumor stem cells target spot CD133's is embedding It can be CAR T cell targeting therapy on tumor with specific killing human brain glioma stem cells to close antigen receptor (CAR) modification T cell Lay a good foundation [7].
Tumour-specific Chimeric antigen receptor (CAR) is thin by the land of tumor cell surface antigen and multiple T The recombinant molecule that the fusion of born of the same parents' signaling zone is formed, wherein land is usually the native ligand of single-chain antibody variable region or antigen, And signaling zone then comes from the intracellular part [8] of tcr complex and costimulation acceptor.Therefore, express chimeric antigen by The T cell (CAR T) of body can cause t cell activation, and then killing tumor cell [9,10] by tumor cell.
The treatment of CAR T cells shows extraordinary curative effect [11] in the positive hematologic malignancies of CD19.But The effect of CAR T cells treatment solid tumor is still limited.CAR T cells facing challenges in the treatment of entity tumor mainly wrap Include：Lack the antigen of tumour-specific, so as to there is the risk that attack normal cell causes serious side reaction；T cell is difficult to have Effect ground reaches tumor locus；Tumor microenvironment is often immunosuppressive so that T cell loses function and apoptosis [12].Swollen In knurl immune response, the apoptosis-induced and anergy of specific for tumour antigen T cell is the master in immunosurveillance escape Want mechanism, and Programmed death 1 (Programmed death 1, PD-1)/programmed death molecule ligand 1 (Programmed death-ligand 1, PD-L1) signal is exactly the important channel that many tumour cells realize immunologic escape. Main mechanism is to be combined by the PD-1 albumen of T cell with the PD-L1 of tumor cell surface, causes PD-1 path sustained activations, T Cell function is suppressed, so that it cannot attack and killing tumor cell [13].
It is applied to PD-1 the or PD-L1 antibody of clinical research at present, by blocking PD-1/PD-L1 paths, part is recovered The function of T cell, killing tumor cell can be continued, the potentiality with treatment polytype tumour.It is thin in melanoma, kidney Born of the same parents' cancer, carcinoma of urinary bladder, malignant hematologic disease and non-small cell lung cancer etc. show good clinical efficacy [14] so that PD-1 or PD-L1 antibody drugs get the Green Light listing in multiple countries.Research shows CD133-CAR T cells in animal model only Possess the suppression tumor growth ability [7] of short time, thus it is speculated that CAR T cells in vivo perform function when also by PD-1/PD-L1 Path immunosupress.In fact, John etc. research shows, it is combined PD-1 antibody in mouse and CAR T cells can be notable Strengthen the suppression tumour function [15] of CAR T cells, disclose PD-1/PD-L1 paths to a certain extent to CAR T cell functions Influence.
But carry out immunization therapy using immunologic test point blocking antibody and 2 at least be present：1st, immunologic test point hinders Disconnected antibody can also break the normal immune tolerance of body while tumour is treated so that body normal structure and organ are lived The T cell attack of change, long-term use can have systemic toxicity [16]；2nd, immunologic test point includes multiple members, only blocks PD-1/PD-L1 paths are probably inadequate.Nearest Koyama etc. research shows, tolerance of the tumour to PD-1 Antybody therapies with The up-regulation of other checkpoint molecules is related, such as T cell immunoglobulin and the (T-cell of mucin domain albumen 3 Immunoglobulin mucin-3, TIM-3) [17].And developing multigroup efficient antibody needs longer time and input, because The research and development difficulty that multiple antibody are used in combination in this is big.
The mode of any blocking immunity checkpoint, is all likely to be breached same therapeutic effect.Programmable endonuclease Zymotechnic, including Zinc finger nuclease (zinc-finger nucleases, ZFNs) technology and activating transcription factor sample effector core Sour enzyme (transcription activator-like effectors nucleases, TALENs) technology, and rule into The short palindrome in cluster interval repeats system (clustered regularly interspaced short palindromic repeat；CRISPR-associated, CRISPR-Cas9), specific DNA sequence dna can be identified, and carry out cutting cause it is double Chain DNA is broken, and under conditions of no template, non-homologous end joining reparation occurs, causes frameshift mutation to cause gene knockout [18], thus for the operation of immunologic test point gene good instrument is provided.
Bibliography
1.Pattabiraman, D.R.and R.A.Weinberg, Tackling the cancer stem cells- what challenges do they poseNat Rev Drug Discov, 2014.13 (7)：p.497-512.
2.Grosse-Gehling, P., et al., CD133 as a biomarker for putative cancer stem cells in solid tumours：Limitations, problems and challenges.J Pathol, 2013.229(3)：p.355-78.
3.Bostad, M., et al., Light-controlled endosomal escape of the novel CD133-targeting immunotoxin AC133-saporin by photochemicalinternalization-A Minimally invasive cancer stem cell-targeting strategy.J Control Release, 2015.206：p.37-48.
4.Ohlfest, J.R., et al., Immunotoxin targeting CD133 (+) breast carcinoma Cells.Drug DelivTransl Res, 2013.3 (2)：p.195-204.
5.Prasad, S., et al., Effective Eradication of Glioblastoma Stem Cells by Local Application of an AC133/CD133-Specific T-cell-Engaging Antibody and CD8 T Cells.Cancer Res, 2015.75 (11)：p.2166-76.
6.Zhao, L., et al., Targeting CD 133high Colorectal Cancer Cells In Vitro and In Vivo With an Asymmetric Bispecific Antibody.J Immunother, 2015.38 (6)：p.217-28.
7.Zhu, X., et al., Patient-derived glioblastoma stem cells are killed by CD133-specific CAR T cells but induce the T cell aging marker CD57.Oncotarget, 2015.6 (1)：p.171-84.
8.Sadelain, M., R.Brentjens, and I.Riviere, The basic principles of Chimeric antigen receptor design.Cancer Discov, 2013.3 (4)：p.388-98.
9.Dai, H., et al., Chimeric Antigen Receptors Modified T-Cells for Cancer Therapy.J Natl Cancer Inst, 2016.108 (7)
10.Rosenberg, S.A.and N.P.Restifo, Adoptive cell transfer as Personalized immunotherapy for human cancer.Science, 2015.348 (6230)：p.62-8.
11.Sadelain, M., CAR therapy：The CD19 paradigm.J Clin Invest, 2015.125 (9)：p.3392-400.
12.Morello, A., M.Sadelain, and P.S.Adusumilli, Mesothelin-Targeted CARs： Driving T Cells to Solid Tumors.Cancer Discov, 2016.6 (2)：p.133-46.
13.Baumeister, S.H., et al., Coinhibitory Pathways in Immunotherapy for Cancer.Annu Rev Immunol, 2016.
14.Page, D.B., et al., Immune modulation in cancer with antibodies.Annu Rev Med, 2014.65：p.185-202.
15.John, L.B., et al., Anti-PD-1 antibody therapy potently enhances the Eradication of established tumors by gene-modified T cells.Clin Cancer Res, 2013.19(20)：p.5636-46.
16.Naidoo, J., et al., Toxicities of the anti-PD-1 and anti-PD-L1 immune Checkpoint antibodies.Ann Oncol, 2015.26 (12)：p.2375-91.
17.Koyama, S., et al., Adaptive resistance to therapeutic PD-1 blockade Is associated with upregulation of alternative immune checkpoints.Nat Commun, 2016.7：p.10501.
18.Kim, H.and J.S.Kim, A guide to genome engineering with programmable Nucleases.Nat Rev Genet, 2014.15 (5)：p.321-34.
The content of the invention
The technical problems to be solved by the invention be to provide the targeting CD133 of PD-1 gene silencings CAR T cells and its Methods for making and using same.
In order to solve the above-mentioned technical problem, present invention employs following technical scheme：
A kind of targeting CD133 of PD-1 gene silencings CAR T cells, it is characterised in that by by CRISPR-Cas9 and The PD-1 genes that piggyBac transposon system imports in PBMC in the CAR T cells for knocking out targeting CD133 simultaneously obtain.
Present invention also offers the preparation method of the targeting CD133 of above-mentioned PD-1 gene silencings CAR T cells, and it is special Sign is, including：CRISPR-Cas9 and piggyBac transposon system are imported in PBMC simultaneously first with core transfection system The PD-1 genes in targeting CD133 CAR T cells are knocked out, then activate T cell, T cell amplification is carried out, obtains PD-1 genes The targeting CD133 of silence CAR T cells.
Preferably, described CRISPR-Cas9 and piggyBac transposon system include plasmid pGL3-U6-hPD1- SgRNA (1+2), pST1374-NLS-flag-Cas9-ZF, AC133-CARpiggyBac transposon and Super piggyBactransposase。
Preferably, the CD3/CD28 antibody that described activation T cell is presented using soluble CD3 antibody or magnetic bead is realized.
Preferably, described T cell amplification is realized in the presence of IL2 or feeder cells.
Preferably, described preparation method also includes PD-1 gene knockout efficiency analysis in T cell.
It is highly preferred that the efficiency analysis of PD-1 gene knockouts is included by T7EN1 cuttings and gene sequencing in described T cell The ratio that detection PD-1 genes are edited and the knockout by inducing PD-1 on CAR T cells expression PD-1 detection protein levels At least one of efficiency.
Present invention also offers the targeting CD133 of above-mentioned PD-1 gene silencings CAR T cells to prepare tumour immunity Application in therapeutic reagent or prevention metastases and recurrence reagent.
Preferably, the targeting CD133 of described PD-1 gene silencings CAR T cells can target the swollen of the CD133 positives Knurl stem cell.
Preferably, the targeting CD133 of described PD-1 gene silencings CAR T cells can solve the problem that the mediation of PD-1 paths Immune-suppression problems.
A kind of immunotherapy of tumors reagent, it is characterised in that the targeting CD133's containing above-mentioned PD-1 gene silencings CAR T cells.
One kind prevention metastases and recurrence reagent, it is characterised in that the targeting containing above-mentioned PD-1 gene silencings CD133 CAR T cells.
The present invention is established based on the method that CRISPR-Cas9 and CAR T technology genetic modification T cells are used in combination；This Invention provides the method that the electricity based on DNA turns to realize the genetic engineering transformation of T cell.Firstly the need of for PD-1 genes Design, synthesize, verifying different sgRNA and combination.SgRNA last selection depends on different sequences and combination knocks out efficiency Comparative result.The present invention verifies by early stage, finally have selected a pair of sgRNA combination targeting PD-1 genes.On CAR genes Delivering, the present invention have selected suitable Transposon System, realize stable expression of the CAR genes in T cell.
The invention provides the method for improving CAR T cell anti-tumor functions based on PD-1 gene silencings.By target cell and T After cell co-cultures 24 hours, culture supernatant is taken quantitatively to can detect the secretory volume of various cell factors with AlphaLISA.And by table Target cell up to luciferase co-cultures with T cell, after 16-24 hours, collects all cells, adds the substrate of luciferase, T cell killing target cell situation can be analyzed.Invention further provides the internal analysis for suppressing tumour function.In immune deficiency The lotus knurl model in situ of glioma is established in mouse, by 3 in-situ injection CAR T cells, analysis T cell suppresses tumour growth Situation；Finally compare the life cycle of mouse.
The present invention knocks out the PD-1 genes in CD133-CAR T cells with CRISPR-Cas9 systems.On the one hand, CAR T are thin The anti-tumor function of born of the same parents is recovered；On the other hand, due to being only that the T cell of adoptive input loses PD-1 expression, thus The xicity related T cell that would be limited to input of PD-1 blocking treatments, is more susceptible to monitoring.
Compared with prior art, the beneficial effects of the invention are as follows：
The scheme of the CAR T cells of PD-1 genes is knocked out the invention provides the preparation of optimization.Relatively conventional CAR T are thin Born of the same parents, the targeting CD133 of PD-1 gene silencings provided by the invention CAR T cells can solve the immune suppression of PD-1 paths mediation System, prevent metastases and recurrence.And the therapeutic alliance based on PD-1 or PD-L1 antibody is compared, the targeting of PD-1 gene silencings CD133 CAR T cells can effectively avoid systemic toxicity problem, precisely the immune microenvironment of regulation.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing There is the required accompanying drawing used in technology description to be briefly described.
Fig. 1 is the AC133-CAR piggyBac transposon expression vector structure charts used in the present invention；
Fig. 2 is pGL3-U6-hPD1-sgRNA (1+2) expression of the specific knockdown people's PD-1 genes used in the present invention Carrier structure figure；
Fig. 3 is the CD133-CAR T cells of obtained PD-1 gene silencings and traditional CD133- in the embodiment of the present invention 2 The comparing result figure of CAR expression rates in CAR T cells；
Fig. 4 is the CD133-CAR T cells of obtained PD-1 gene silencings and traditional CD133- in the embodiment of the present invention 2 PD-1 knockout result figure in CAR T cells；
Fig. 5 is the U251 CD133-OE surface antigen analysis used in the present invention, and flow cytometry is shown while high table Up to antigen molecule CD133 and immunoregulatory molecules PD-L1；
Fig. 6 is the CD133-CAR T cells of obtained PD-1 gene silencings and traditional CD133- in the embodiment of the present invention 2 CAR T cells, after being co-cultured respectively with overexpression CD133 human glioma cells U251 (U251CD133-OE), cell factor IL-2, IFN-γ, TNF-α and GM-CSF secretory volume column diagram；
Fig. 7 is the CD133-CAR T cells of obtained PD-1 gene silencings and traditional CD133- in the embodiment of the present invention 2 CAR T cells, after being co-cultured respectively with overexpression CD133 human glioma cells U251 (U251 CD133-OE), cytotoxicity The comparison diagram of detection.
Fig. 8 is the CD133-CAR T cells of obtained PD-1 gene silencings and traditional CD133- in the embodiment of the present invention 2 CAR T cells, there is Immune deficient mice NSG of the U251 CD133-OE cells into knurl to intracerebral transplantation respectively, carry out 3 wheels note in situ After penetrating treatment, survivorship curve comparison diagram and the uciferase activity change of mouse.
Embodiment
Clear, complete description is carried out to embodiment of the present invention below in conjunction with embodiment, it is clear that described reality Apply example and be merely to illustrate the part of the embodiment of the present invention, and be not construed as limiting the scope of the present invention.It is unreceipted in embodiment Actual conditions person, the condition suggested according to normal condition or manufacturer are carried out.Agents useful for same or the unreceipted production firm person of instrument, It is considered as the conventional products that can be obtained by commercially available purchase.
Embodiment 1
It is prepared by PBMC separation：
1. gather Healthy People (or patient) peripheral blood with anticoagulant tube.It is slowly added to after diluting anticoagulation (1: 1) with PBS It is slow to rise slow drop centrifugation with 450g centrifugal force in 50ml centrifuge tubes equipped with isometric lymphocyte separation medium (Ficoll) 25min；
After 2. centrifugation terminates, the careful tunica albuginea layer drawn above lymphocyte separation medium, it is transferred to a new 50ml centrifugation Guan Zhong, PBS is added, it is slow to rise slow drop centrifugation 10min with 300g centrifugal force, supernatant is abandoned, the cell for retaining centrifugation bottom of the tube sinks Form sediment；PBS is added again, it is slow to rise slow drop centrifugation 15min with 160g centrifugal force, abandon supernatant；Be eventually adding PBS, with 300g from Mental and physical efforts, it is slow to rise slow drop centrifugation 10min, supernatant is abandoned, that is, obtains PBMC.
Embodiment 2
Prepare CAR T cells：
1. after PBMC slightly is cultivated into 1 to 2 hour with T cell culture medium (culture mediums of RPMI 1640 containing 10%FBS) Carry out electricity to turn, transfection is following two groups respectively：
(1) by pGL3-U6-hPD1-sgRNA (1+2) (structure chart is as shown in Figure 2), pST1374-NLS-flag-Cas9- ZF, AC133-CAR piggyBac transposon (structure chart is as shown in Figure 1) and Super piggyBactransposase Each 5 μ g of four plasmids turn buffer solution mixing with the electricity in human T-cell's consideration convey transfection reagent box (Lonza, VPA-1002), comprising About 110 μ l electricity of four kinds of plasmids turn mixed liquor；
(2) to include original plasmid pGL3-U6-sgRNA, pST1374-NLS-flag-Cas9-ZF, AC133-CAR The electricity of piggyBac transposon and Super tetra- plasmids of piggyBactransposase turns mixed liquor as control；Separately A part of cell is stayed to turn without electricity；
Plasmid pGL3-U6-hPD1-sgRNA (1+2), the pST1374-NLS-flag- used in two above step Cas9-ZF and pGL3-U6-sgRNA preparation method is referring to document Su, S., et al., CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients.Sci Rep, 2016.6：20070.doi：10.1038/srep20070 with patent CN201410077474.6.
Plasmid AC133-CAR piggyBac transposon and Super piggyBactransposase preparation side Method is referring to document Zhu, X., et al., Patient-derived glioblastoma stem cells are killed by CD133-specific CAR T cells but induce the T cell aging marker CD57.Oncotarget, 2015.6 (1)：p.171-84.
2. with reference to the specification of human T-cell's consideration convey transfection reagent box (Lonza, VPA-1002), turned respectively with described two groups electricity Mixed liquor has hanged 1-2 × 107Individual PBMC cells carry out electricity and turned, and electricity turns that cell is transferred to the T cell culture of preheating at once after terminating Base；A subculture is changed again after 2 hours；
3. adding the T cell culture medium of the IL-2 containing 100IU/ml after 16-18 hours, there is CD3/CD28 antibody with coupling Magnetic bead (ThermoFisher SCIENTIFIC, 11141D) stimulate activation CD3 positive T cells；
4. the IL-2 containing 100IU/ml more renewed for every 2 to 3 days T cell culture medium, while observe cell amplification situation. Determine that it stimulates duration according to its proliferative conditions, but stimulation time is no more than one week；
5. separate except after magnetic bead, 1 μ g/ml puromycins of addition continue culture 5 to 7 days.A small amount of cell is collected to be used to examine Survey, remaining cell can be continuing with rapid amplification and further largely expand, and obtain the targeting CD133's of PD-1 gene silencings CAR T cells (the CD133- of CAR T cells (i.e. the CD133-CAR T cells that PD-1 is knocked out) and traditional targeting CD133 CAR T cells).
Embodiment 3
Utilize Flow cytometry CAR expression
By 105Expand obtained CAR-T cells and be resuspended in 100 microlitres of FACS buffer solutions (EDTA containing 2mM and 0.5%BSA PBS) in, added in cell suspension Myc-Tag (9B11) Mouse mAb (1: 500 dilution, Cell Signaling, 2276S), 4 DEG C are incubated 15 minutes；Cleaning cell is once resuspended in 100 microlitres of FACS buffer solutions afterwards, adds the sheep anti mouse of PE marks Secondary antibody (1: 50 dilution, Jackson, 115-116-072), 4 DEG C are incubated 10 minutes；CytoFLEX (Beckman Coulter) flows Formula cell instrument is used to obtain staining cell, and FlowJo is used for analysis result.As shown in figure 3, flow cytometry display tradition CD133-CAR T (the PD1-KO that CD133-CAR T, PD-1 are knocked out；High CD133-CAR points of the expression of cell such as CD133-CAR) Son.
Embodiment 4
Cellular genome is extracted to be analyzed with cutting effect
Use cell pyrolysis liquid vitellophag (0.1-1 × 10 containing Proteinase K6), and it is thin with phenol chloroform method extraction CAR-T Born of the same parents' genomic DNA.With detection on-target primer hPDltest For and hPD1 test Rev, (sequence is referring to patent SEQ ID NO.12 and SEQ ID NO.13 in CN201410077474.6) PCR amplification purpose fragments.Reaction system is：12μl 10 × buffer, 12 μ l dNTP (0.5mM), 500ng genomic DNAs, 0.6 μ l hPD1 test For (0.5 μM), 0.6 μ l HPD1 test Rev (0.5 μM), 3U Super-Taq, moisturizing to 120 μ l systems.Response procedures are：95℃5min；95℃ 30s, 60 DEG C of 30s, 72 DEG C of 1min, 30cycles；72℃10min；4℃for ever.Reclaimed and tried using Axygen PCR primers The purifying recovery of agent box.Using T7EN1 cleavage PCR annealed products, 3% agarose gel electrophoresis tests and analyzes digestion products, As a result as shown in Fig. 4 gel electrophoresis figures.
PCR primer is connected into pMD19-T carriers (TAKARA, 3271), connection method is said with reference to pMD19-T carriers Bright book and patent CN201410077474.6.Connection product is converted into DH5 α competence bacteriums (TransGen, CD201), The single bacterium colony that random picking is more than 20, is sequenced using universal primer M13-47, assesses target fragment and be cut base afterwards Change and cutting efficiency.As shown in figure 4, the CD133-CAR T (PD1-KO knocked out in PD-1；CD133-CAR) in cell, PD-1 There is missing and insertion mutation in gene, the efficiency of gene editing is up to more than 57%.Further, the CD133-CAR that PD-1 is knocked out T cell is in the case where target cell stimulates 3 days, a large amount of up-regulations of the Surface testing less than PD-1 acceptors.
Embodiment 5：
The CD133-CAR T cell cytokine release outcome evaluations that PD-1 is knocked out
The present invention is used as target cell, U251 using the human glioma cells system U251 (U251 CD133-OE) for being overexpressed CD133 CD133-OE construction method bibliography Prasad, S., et al., Effective Eradication of Glioblastoma Stem Cells by Local Application of am AC133/CD133-Specific T- Cell-Engaging Antibody and CD8 T Cells.Cancer Res, 2015.75 (11)：p.2166-76.Respectively Cultivate the CD133-CAR T cells that U251 CD133-OE cells and effector cell CD133-CAR T and PD-1 are knocked out.Such as Fig. 5 Shown, flow cytometry shows U251 CD133-OE overexpression protection original molecule CD133 and immunoregulatory molecules PD-L1 simultaneously.
By target cell U251 CD133-OE with every hole 1x105Kind in 96 orifice plates, by effector cell CD133-CAR T and The CD133-CAR T that PD-1 is knocked out are respectively with 2x 105Mixed with target cell, with the μ l of cumulative volume 200 nutrient solution (RPMI 1640 Culture medium+10%FBS) in 37 DEG C of 5%CO224 hours are co-cultured in incubator.Every group of 3 multiple holes.Centrifuging and taking supernatant afterwards 10 μ l are detected respectively with PerkinElmer AlphaLISAassay reagents series box (AL208, AL216, AL217, AL221) TNF-α, GM-CSF, IFN-γ, and IL-2 cytokine concentrations are horizontal.As shown in fig. 6, IFN-γ, IL-2, TNF-α and The cytokine levels such as GM-CSF are in knockout group and do not knock out and have no difference between group, illustrate the CD133- of PD-1 gene silencings CAR T cells can be activated effectively.
Embodiment 6
The CD133-CAR T cells fragmentation effect that PD-1 is knocked out is assessed
With reference first to document Prasad, S., et al., Effective Eradication of Glioblastoma Stem Cells by Local Application of an AC133/CD 133-Specific T-cell-Engaging Antibody and CD8 T Cells.Cancer Res, 2015.75 (11)：P.2166-76 stable expressing luciferase is built Target cell (U251 CD133-OE-luc).U251 CD133-OE-luc and effector cell are collected (in embodiment 2 CAR-T cells), 300g, 10min is centrifuged, abandons supernatant；Target cell and effector cell are resuspended respectively with 1ml PBS solutions, 300g, 10min is centrifuged, abandons supernatant；Repeated washing is once；Target cell is resuspended to dense with nutrient solution (culture medium+10%FBS of RPMI 1640) Spend for 2x106/ ml, resuspension effector cell to concentration is 3.2x107/ml。
Use the method for doubling dilution to set and imitate target ratio as 1: 1,2: 1,4: 1,8: 1 experimental port, and control group is set, it is empty White group, every group of 3 multiple holes, 37 DEG C of 5%CO216 to 24h are cultivated in incubator；All cells, 500g centrifugations 5min are drawn in digestion； Cell is resuspended with 100 μ l PBS, adds in 96 hole detection plates of white, and adds 100 μ l D- in every hole rapidly Luciferin substrate solutions (lucifuge operation), ELIASA detection 560nm absorbances.As a result as shown in fig. 7, what PD1- was knocked out To U251 CD133-OE target cells, the killing-efficiency under the conditions of different effect target ratios is above not knocking out group CD133-CAR T cells.
Embodiment 7：
Assessed in Mice Body into knurl and Experiment on therapy
2x10 is implanted into advance in immunodeficient mouse NSG encephalic5The target cell U251 of stable expressing luciferase CD133-OE-luc (bibliography Prasad, S., et al., Effective Eradication of Glioblastoma Stem Cells by Local Application of an AC133/CD133-Specific T-cell-Engaging Antibody and CD8 T Cells.Cancer Res, 2015.75 (11)：P.2166-76 build), it is every with living imaging instrument Its stable situation into knurl was observed every 2-3 days.It is implanted into latter week, in-situ injection 2x106CD133-CAR T or PD-1 are knocked out CD133-CAR T cells, and set electricity turn group NT for compare.Every injection in 3 days once, co-injection 3 times.Biology is utilized afterwards Fluorescence signal (BLI) detection uciferase activity change, observes mouse survival state and records survivorship curve.As shown in figure 8, strike Except group can be obviously prolonged the mouse survival life-span, the suppression for having the longer time to tumour growth.
Present pre-ferred embodiments are the foregoing is only, are not intended to limit the invention, all spirit in the present invention Within principle, any modification, equivalent substitution and improvements made etc., it should be included in the scope of the protection.

Claims (10)

  1. A kind of 1. targeting CD133 of PD-1 gene silencings CAR T cells, it is characterised in that by by CRISPR-Cas9 and The PD-1 genes that piggyBac transposon system imports in PBMC in the CAR T cells for knocking out targeting CD133 simultaneously obtain.
  2. 2. the preparation method of the targeting CD133 of the PD-1 gene silencings described in claim 1 CAR T cells, it is characterised in that Including：CRISPR-Cas9 and piggyBac transposon system are imported simultaneously first with core transfection system target is knocked out in PBMC PD-1 genes into CD133 CAR T cells, T cell is then activated, carry out T cell amplification, obtain PD-1 gene silencings Target CD133 CAR T cells.
  3. 3. the preparation method of the targeting CD133 of PD-1 gene silencings as claimed in claim 2 CAR T cells, its feature exist In, described CRISPR-Cas9 and piggyBac transposon system include plasmid pGL3-U6-hPD1-sgRNA (1+2), PST1374-NLS-flag-Cas9-ZF, AC133-CARpiggyBac transposon and Super piggyBactransposase。
  4. 4. the preparation method of the targeting CD133 of PD-1 gene silencings as claimed in claim 2 CAR T cells, its feature exist In the CD3/CD28 antibody that described activation T cell is presented using soluble CD3 antibody or magnetic bead is realized.
  5. 5. the preparation method of the targeting CD133 of PD-1 gene silencings as claimed in claim 2 CAR T cells, its feature exist In the amplification of described T cell is realized in the presence of IL2 or feeder cells.
  6. 6. the preparation method of the targeting CD133 of PD-1 gene silencings as claimed in claim 2 CAR T cells, its feature exist In described preparation method also includes PD-1 gene knockout efficiency analysis in T cell.
  7. 7. the preparation method of the targeting CD133 of PD-1 gene silencings as claimed in claim 6 CAR T cells, its feature exist In the efficiency analysis of PD-1 gene knockouts includes cutting by T7EN1 in described T cell and gene sequencing detects PD-1 gene quilts The ratio of editor and by induce CAR T cells express PD-1 detect protein level on PD-1 knockout efficiency at least one Kind.
  8. 8. the targeting CD133 of the PD-1 gene silencings described in claim 1 CAR T cells are preparing immunotherapy of tumors reagent Or the application in prevention metastases and recurrence reagent.
  9. 9. application as claimed in claim 8, it is characterised in that the targeting CD133 of described PD-1 gene silencings CAR T are thin Born of the same parents can target the positive tumor stem cells of CD133, and solve the immune-suppression problems of PD-1 paths mediation.
  10. A kind of 10. immunotherapy of tumors reagent, it is characterised in that the targeting containing the PD-1 gene silencings described in claim 1 CD133 CAR T cells.
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CN109136190A (en) * 2018-04-11 2019-01-04 北京中诚华科生物科技有限公司 A kind of BTLA for resisting tumour immunity and inhibiting environment-/-The preparation method and application of T lymphocyte
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CN109082442A (en) * 2018-07-17 2018-12-25 杭州观梓健康科技有限公司 A kind of preparation method for the mescenchymal stem cell for releasing immunosupress and enhancing tumor-targeting killing
CN109082442B (en) * 2018-07-17 2019-09-20 杭州观梓健康科技有限公司 A kind of preparation method for the mescenchymal stem cell for releasing immunosupress and enhancing tumor-targeting killing
CN109652381A (en) * 2019-01-25 2019-04-19 苏州茂行生物科技有限公司 The CAR-T cell preparation method and application of CD133 is targeted based on base editor
CN111549062A (en) * 2020-05-07 2020-08-18 西南大学 Whole genome knockout vector library of silkworm based on CRISPR/Cas9 system and construction method

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