CN108218999A - Secrete Chimeric antigen receptor, viral vectors, expression cell and the preparation method and drug of IL-7 - Google Patents
Secrete Chimeric antigen receptor, viral vectors, expression cell and the preparation method and drug of IL-7 Download PDFInfo
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Abstract
The present invention provides a kind of Chimeric antigen receptor, viral vectors, expression cell and preparation methods and drug for secreting IL 7, the Chimeric antigen receptor of secretion IL 7 includes extracellular domain, 7 cytokine domain of transmembrane domain, intracellular domain and IL that can combine antigen, and 7 cytokine domains of IL are by self cleavage peptide and the non-fused connection of intracellular domain.The present invention adds in self cleavage peptide (2A peptides) and 7 gene orders of IL on original CAR expression vectors, so as to which the CAR T for enabling design express secretion IL 7, enhances Tumor cytotoxicity, improves tumor microenvironment.The Chimeric antigen receptor T cell of the present invention significantly improves the improvement of the inside and outside tumor-killing function of CAR T bodies.
Description
Background technology
Chimeric antigen receptor (CAR, Chimeric Antigen Receptors) T cell is the mileage for being expected to cure tumour
Upright stone tablet event, CAR-T technologies are using genetic modification technology, by single-chain antibody (the single chain of one section of identification tumour antigen
Fragment variable, scFv) and intracellular activation motifs recombination transfect on T lymphocytes to reach better knowledge
Other and killing tumour effect.CAR-T molecules generally include extracellular hinge region, transmembrane region and intracellular signal area.Wherein extracellular strand
Sequence is to connect to be formed by a peptide fragment with light chain variable region by the heavy chain of single-chain antibody;Transmembrane region then mostly come from as
The equimolecular transmembrane region of CD8, CD28 or 4-1BB;Intracellular signal area then mostly come from CD28,4-1BB, CD3zeta, CD27 or
The intracellular section chimera of the T cells conducted signal molecule such as OX-40.High affinity, the scFv of high specific determine that CAR-T targets certain
Kind of antigen, once with reference to antigen, the intracellular section of CAR can transmit activation and costimulatory signal to T cell, activate T cell and then
Efficient targeting killing tumor cell.The activation of T cell needs the TCR-CD3 and MHC-I of two kinds of activation signals, i.e. T cell surface
Molecule is combined into the first signal of activation, it is resolved that T cell is to the killing activity of tumour cell;The costimulatory molecules on T cell surface
The second signal of activation is combined into respective ligand, it is resolved that T cell is proliferated.In brief, CAR-T cells pass through Ag-Ab
The special molecular of tumor cell surface is identified in recognition mode, then by the signal transduction of its intracellular into line activating, increase
It grows and plays cell killing function.
The research and development in the structure design experience mostly generation of CAR molecules.It is thin that the structure of first generation CAR molecules includes identification tumour
The scFv of cellular surface antigen, transmembrane domain and activate T cell TCR compounds CD3zeta intracellular domain.Due to first
Signal domain for CAR is single signal molecule, lacks costimulatory signal, the time-to-live is short in vivo for first generation CAR-T cells, right
The lethality of tumour cell is weak, curative effect is unsatisfactory.(JensenMC, PopplewellL, CooperLJ, et
al.Antitransgene rejection responses contribute to attenuated persistence of
adoptively transferred CD20/CDl9-specific chimeric antigen receptor
Redirected T cells in humane [J] .Biol Blood Marrow Transplant, 2010,16 (9):1245–
1256.) second generation, three generations CAR then introduce one or more costimulatory signal respectively on the basis of the first generation, at present
The costimulatory signal of report include CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2,4-1BB, OX-40, ICOS-L,
ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HEVM, lymphotoxin-beta-receptor, ILT3, ILT4, HVEM,
The proliferative capacity and antitumor action of CAR-T cells are significantly improved, extends the duration of CAR-T cells in vivo.(MaQ,
GomesEM,LoAS,et al.Advanced generation anti-prostate specific membrane
antigen designer T cells for prostate cancer immunotherapy[J].Prostate,2014,
74(3):286-296.) research of the University of Pennsylvania finds defeated to recurrence/refractory patients with chronic lymphocytic
The two generation CD19-CAR-T cells containing 4-1BB costimulatory signals can expand in vivo more than 1000 times, and in blood note self
The time survived in liquid and marrow was also above 6 months.
The CAR-T cells for targeting CD19 molecules obtain on the treatment for recurrence/refractory precursor B cells neoplastic hematologic disorder
Preferable clinical efficacy, part result of study show that complete remission rate reaches 80%, have become in immunotherapy of tumors most
For noticeable field.However, so far, following problems are not yet resolved:CAR-T cells plays immunologic cytotoxicity is made
With dependence CAR-T cells and tumour cell contact with each other, and need to overcome tumor by local immunosupress microenvironment.Due to solid tumor
Knurl often is wrapped up with fibroblast, there are a large amount of immunosuppressant cell, the factor and regulation and control in tumor microenvironment
How molecule makes CAR-T cell migrations that tumour immunity be overcome to inhibit microenvironment, CAR-T cell killings are played and make to tumor locus
With being then significant challenge that CAR-T cell therapy technologies are pushed to clinical practice comprehensively.Therefore, it is thin to prepare more effective CAR-T
Born of the same parents and be used to prepare the CAR-T cells expression vector it is extremely urgent.(Xia AL,Wang XC,Lu YJ,Lu XJ,Sun
B.Chimeric-antigen receptor T(CAR-T)cell therapy for solid tumors:challenges
and opportunities.Oncotarget.2017Jul 18;8(52):90521-90531.)
Invention content
In view of this, it is an object of the present invention to advanced optimizing CAR designs, tumour immunity is overcome to inhibit micro-loop
The problem of border, neoplastic hematologic disorder easily recur is provided comprising the Chimeric antigen receptor that can secrete IL-7.
The second object of the present invention is to provide the slow virus carrier for including the Chimeric antigen receptor that can secrete IL-7.
The third object of the present invention is to provide using described comprising the Chimeric antigen receptor or described that can secrete IL-7
Slow virus carrier prepare expression comprising can secrete IL-7 Chimeric antigen receptor immunocyte.
The present invention four be to provide it is described comprising the Chimeric antigen receptor of IL-7 or the slow virus can be secreted
Carrier is being prepared comprising the application in can secreting drug of the Chimeric antigen receptor of IL-7 in targets neoplastic cells.
Above-mentioned purpose that the invention is realized by the following technical scheme:
In a first aspect, the present invention provides a kind of Chimeric antigen receptor, it includes can combine antigen extracellular domain,
Transmembrane domain, intracellular domain and IL-7 cytokine domains, wherein, IL-7 cytokine domains are to pass through Self cleavage
Peptide (2A peptides) and the non-fused connection of intracellular domain.
For above-mentioned Chimeric antigen receptor molecule, preferably, the antigen can be tumour antigen, the tumour antigen
Such as including:5 β 1- integrins of 5T4, α, 707-AP, AFP, ART-4, B7H4, BAGE, beta-catenin/m, Bcr-abl, MN/C
IX antibody, CA125, CAMEL, CAP-1, CASP-8, CD4, CD19, CD20, CD22, CD25, CDC27/m, CD30, CD33,
CD52、CD56、CD80、CDK4/m、CEA、CT、Cyp-B、DAM、EGFR、ErbB3、ELF2M、EMMPRIN、EpCam、ETV6-
AML1、G250、GAGE、GnTV、Gp100、HAGE、HER-2/new、HLA-A*0201-R170I、HPV-E7、HSP70-2M、
HST-2, hTERT (or hTRT), iCE, IGF-1R, IL-2R, IL-5, KIAA0205, LAGE, LDLR/FUT, MAGE, MART-1/
Melan-A, MART-2/Ski, MC1R, Mesothelin, myosin/m, MUC1, MUM-1, MUM-2, MUM3, NA88-A,
PAP, proteinase-3, p190minor bcr-abl, Pml/RAR α, PRAME, PSA, PSM, PSMA, RAGE, RU1 or RU2,
SAGE, SART-1 or SART-3, survivin protein, TEL/AML1, TGF β, TPI/m, TRP-1, TRP-2, TRP-2/INT2, VEGF,
WT1, NY-Eso-1 or NY-Eso-B etc.;It is further preferred that the tumour antigen is CD19.Patent of the present invention carries antigen
Can also be the inflammatory cell surface molecular occurred in autoimmune disease or the TCR for leading to autoimmunity.
Preferably, the Chimeric antigen receptor is single-stranded variable including antitumor-antigen antibody successively since the N-terminal side
Area as extracellular domain, the transmembrane domain of CD28 molecules and 4-1BB intracellular domain, CD3 ζ intracellular domain, 2A peptides and
IL-7 gene orders.
Second aspect, the present invention provides a kind of slow virus carriers for including Chimeric antigen receptor as described above.
The third aspect, the present invention provides a kind of Chimeric antigen receptor expression cell, wherein introducing such as first aspect institute
The Chimeric antigen receptor stated;Preferably, the cell is T cell or the cell mass containing T cell.
Fourth aspect, the present invention provides a kind of sides of Chimeric antigen receptor expression cell prepared as described in the third aspect
Method, including by as described in relation to the first aspect Chimeric antigen receptor introduce cell the step of;Preferably, the cell is T cell
Or the cell mass containing T cell.
5th aspect, the present invention provides a kind of Chimeric antigen receptor as described in relation to the first aspect, as described in second aspect
Use in the drug for preparing treatment tumour of slow virus carrier or Chimeric antigen receptor expression cell as described in the third aspect
On the way.
Preferably, the tumour is solid tumor or blood tumor.
In the specific embodiment of the purposes provided by the invention, the tumour is B-ALL.It is worth noting that, this hair
Bright CAR is characterized in that it includes secretion IL-7 cytokine domains.
IL-7 is the key factor during cellullar immunologic response, thin for the T of B cell, thymocyte and periphery maturation
Born of the same parents etc. have the activity for promoting to grow, and IL-7 can theoretically improve the micro- inhibition environment of tumour with antagonism TGF-β, activation with
Immunocyte is adjusted, important function is played the part of in mediation T, B cell activation, proliferation and differentiation in oncotherapy.(Ye Lin, Zhao Hongwen
.IL-7 biological characteristics and its potential applicability in clinical practice《World breathing magazine》,2007,27(15):It is 1161-1164) into one
Step optimization CAR designs, overcome the problems, such as that tumour immunity inhibits microenvironment, neoplastic hematologic disorder easily to recur, the present invention is expressed in original CAR
Self cleavage peptide (2A peptides) and IL-7 gene orders are added on carrier, so as to which the CAR T for enabling design express secretion IL-7, enhancing
Tumor cytotoxicity improves tumor microenvironment.The Chimeric antigen receptor T cell of the present invention significantly improves the inside and outside tumour of CAR T bodies
The improvement of killing ability.
Description of the drawings
Fig. 1 a are the Chimeric antigen receptor viral vectors containing secretion IL-7 cytokine gene sequences.
Fig. 1 b are the Chimeric antigen receptor viral vectors for not containing secretion IL-7 cytokine gene sequences.
Fig. 2 shows that GFP T, CAR19 T and IL-7 CAR19T cells kill the external of NALM6-GL cells for expressing CD19
Hinder effect, show that IL-7 CAR19T are better than GFP T, CAR19 T.
Fig. 3 shows that GFP T, CAR19 T and IL-7 CAR19T cells imitate the Cytotoxicity in vitro for expressing the Raji cells of CD19
Should, show that IL-7 CAR19T are better than GFP T, CAR19 T.
Fig. 4 shows GFPT, CAR19-GFP T, IL-7 CAR19-GFP T cells IL-7 secretion situations, as a result shows control
Group GFPT, CAR19-GFP T do not secrete IL-7, and IL-7 CAR19-GFP T can secrete higher IL-7 cell factors.*P<
0.05。
Fig. 5 show CAR19C3aR T can significantly reduce the loads of NALM6 tumour cells in NCG Mice Bodies better than GFP T,
CAR19 T。*P<0.05。
Fig. 6 shows that IL-7 CAR19T more can significantly extend the NCG mouse growth phases compared with GFP T, CAR19 T.*P<0.05.
Has present embodiment
With reference to embodiment, present disclosure is further illustrated.It should be understood that the implementation of the present invention is not limited to
In the following examples, the accommodation in any form and/or change made to the present invention both fall within the scope of the present invention.It is real
Apply the implementation that actual conditions are not specified in example, usually according to normal condition described in condition or built according to manufacturer
The condition of view.
Embodiment 1 contains anti-CD19 ScFv-4-1BB-CD3 ζ (CAR19), anti-CD19 ScFv-4-1BB-CD3 ζ-IL-7
The preparation of (IL-7 CAR19) plasmid
The plasmid that the present invention carries the Chimeric antigen receptor gene of the IL-7 cell factors containing secretion is prepared as follows:
(1) matter containing anti-CD19 ScFv-4-1BB-CD3 ζ (CAR19) is obtained by means such as gene chemical synthesis, molecular clonings
Grain pUC57-CAR19, gene include anti-CD19 monoclonal antibodies ScFV, CD28 transmembrane region and 4-1BB, CD3 ζ intracellular regions, i.e. CD19
ScFv-4-1BB-CD3 ζ, as shown in Figure 1.
(2) it is obtained by means such as gene chemical synthesis, molecular clonings containing anti-CD19 ScFv-4-1BB-CD3 ζ-IL-7 (IL-7
CAR19 plasmid pUC57-IL-7 CAR19), gene include anti-CD19 monoclonal antibodies ScFV, CD28 transmembrane region and 4-1BB, CD3 ζ
Intracellular region and IL-7 cell factor nucleic acid series, i.e. CD19 ScFv-4-1BB-CD3 ζ-IL-7.
(3) digestion is carried out to obtained pUC57-CAR19 plasmids by restriction endonuclease Pmel and Sepl, obtains CAR19 bases
Then CAR19 genes are connected to slow virus carrier pWPXLd-GFP by cause, build pWPXLd-CAR19-GFP.
The slow virus packaging of 2 CAR plasmids of embodiment
The CAR plasmids and related control plasmid of the present invention that are prepared using embodiment 1 are packed by slow virus, are divided
Not Biao Da GFP (blank control), CAR19-GFP (control), IL-7 CAR19-GFP three kinds of slow virus.The present embodiment 2 and 3
In, it is pWPXLd-CAR-GFP plasmids that will contain CAR plasmids Unify legislation, is CAR by the slow virus Unify legislation for being overexpressed CAR
Slow virus.
It is as follows:
(1) 293T cells are cultivated in 10cm culture dishes, culture medium is:DMEM high glucose mediums+10%FBS (tire ox bloods
Clearly)+1% dual anti-(100 × Pen .- Strep mixed solution);
(2) when the 293T cell densities in 150mm culture dishes reach 80-90%, culture medium is replaced:DMEM high glucose mediums
+ 1%FBS+1% is dual anti-;
(3) medium culture is replaced after 2-6 hours, with PEI respectively by bis- kinds of plasmids of pWPXLd-CAR-GFP (that is, respectively
Include CAR19, IL-7 CAR19) or blank control plasmid pWPXLd-GFP respectively with slow virus packaging helper plasmid pMD2.G,
PsPAX2 co-transductions enter 293T cells, add in reagent and dosage such as the following table 1:
Table 1
(4) respectively convert after 24,48 and 72 it is small into, collect culture medium supernatant, and add in fresh culture (DMEM high sugar trains
It is dual anti-to support base+1%FBS+1%);
(5) culture medium supernatant collection finishes, after supernatant 2500g is centrifuged 0.5 hour;
(6) centrifugation supernatant is taken, it is small using Ultracentrifuge 28000rpm centrifugations 1.5 after being filtered with 0.45um filters
When;
(7) after ultracentrifugation, supernatant is gently removed, adds in 200ul PBS, is placed in 4 degree of dissolvings in 12-16 hours to get 2
Kind CAR slow virus or blank control GFP slow virus;
(8) it after virolysis, collects virus and is sub-packed in cryopreservation tube, freeze for use in -80 DEG C.
Embodiment 3 uses the CAR virus-infected human's T cells packed
(1) T cell isolates and purifies:The mononuclearcell in blood is isolated by Ficoll density gradient methods, through red
After cell pyrolysis liquid cracking removal red blood cell, then T cell gone out by MACS Pan-T magnetic bead sortings;
(2) T cell sorted out culture medium (AIM-V culture medium+5%FBS+ penicillin 100U/ml+ streptomysins
It is for use 0.1mg/ml) to be diluted to 2.5 × 106/ml of cell concentration;
(3) by being coated with the magnetic bead (rule of origin of CD2, CD3, CD28 antibody:Germany U.S. day Ni) stimulation T cell, that is, it wraps
It is mixed by magnetic bead and T cell with 1: 2 ratio, T cell final densities should be 5 × 106A/ml/cm2.After mixing, be placed in 37 DEG C,
The culture of 5%CO2 incubators stimulates 48 hours.
(4) slow-virus transfection T cell:Magnetic bead in the T cell of activation-magnetic bead mixed liquor is removed by magnetic fields,
300g centrifuges 5min, removes supernatant, is resuspended with fresh culture, is separately added into expression CAR and GFP (blank control) slow virus (disease
Malicious addition is MOI=10) after, polybrene the and 300IU/ml IL-2 of 8 μ g/ml of addition.37 DEG C are placed in, 5%CO2 cultures
After case culture for 24 hours, 300g centrifugation 5min remove supernatant, are resuspended with the fresh culture of the IL-2 containing 300IU/ml to get overexpression
The T cell of CAR.
(5) CAR T cells expand:CAR T cell density is maintained 1 × 106A/ml or so was carried out primary per 2-3 days
Half amount changes liquid.After two weeks, amplifiable 100 times of CAR T cells number.For the cell of the GFP positives to transfect successful cell, GFP is positive
Ratio is detected by streaming to get to 2 kinds of CAR T cells (abbreviation CAR19-GFP, IL-7 CAR19-GFP respectively) or empty
The ratio of white control T cell (GFP-T).
4 CAR T cells of embodiment identify the effect of killing tumour in vitro
GFP T (blank control) prepared by embodiment 3, CAR19-GFP T (control), IL-7 CAR19-GFP T cells
In varing proportions respectively with 5x104Tumour cell mixing, be added in 96 hole U-shaped boards, every group sets 3 multiple holes, and set individually
Tumour cell group is added after 250g centrifuges 5min, to be placed in 37 degree of 5%CO2 incubators as positive control and co-culture for 24 hours;External ratio
During compared with GFP T, CAR19-GFP T, IL-7 CAR19-GFP T cells to the identification killing ability of neoplastic hematologic disorder, tumour cell choosing
With bis- kinds of leukaemia of NALM6-GL, Raji or lymphoma cell line.The bis- dye detection method for cytotoxicity of gas-liquid assessments of CFSE/PI are quantitatively commented
Estimate killing-efficiency:1. priming effect cell (GFP T (blank control), CAR19-GFP T (control), IL-7 CAR19-GFP T
Cell) and target cell, it counts, the GFP% of CAR T cells with a batch of WT cells is calibrated, makes each group GFP% big
It is small close;With 1640+10%FBS+1%P/S culture mediums dilution T cell 8x106/ml.2. (Nalm6 is thin for the desired amount of target cell
Born of the same parents, Raji cells), 1640 culture mediums of 1ml are resuspended, and add in 5umol CSFE, after 37 DEG C are protected from light incubation 20min, add in 10ml trainings
It supports base and terminates 5min, centrifugation removes supernatant, target cell is diluted to 5x105/ml.3. T cell is subjected to doubling dilution, with the volley of rifle fire in V
100ul fresh cultures are previously added in 96 orifice plate of type bottom, first row is not added with, then first row addition T cell 100ul, and second
Row adds in T cell 100ul, then with volley of rifle fire piping and druming second row 5 times or more, draws 100ul and is put into third row, and so on, by T
Cell carries out doubling dilution along number of rows, adds in 100ul target cells in all well, T cell is made to be mixed in varing proportions with target cell
Close (16:1,8:Isosorbide-5-Nitrae:1,2:1,1:1,1:2,1:4).It is cultivated 24 hours 4. the cell mixed is put in incubator.5. by 96
Cell in orifice plate is drawn, centrifugation.6. washed once with 4 DEG C of PBS, 1 x IP Buffer of 100ul are added in, add 5ul
PI is protected from light after being incubated 15min, adds 1 x IP Buffer of 200ul, upper machine testing in 1 hour.7. killing rate calculates:With
The Target cell wells of T cell are not added with as reference, kill percentage=purpose hole CSFE+PI+%- control wells CSFE+PI+%.Knot
Fruit shows that the Cytotoxicity in vitro efficiency of IL7 CAR19 and the tumour target cell to expressing CD19 is all significantly higher than CAR19 T cells.
(Fig. 2, Fig. 3)
5 CAR T cell IL-7 cytokines measurements of embodiment
Situation is secreted to compare GFPT, CAR19-GFPT, IL-7 CAR19-GFP T cell IL-7, is prepared by embodiment
GFP T (blank control), CAR19-GFP T (control), IL-7 CAR19-GFP T cells culture 3 days recycle supernatant, by city
The ELISA kit (R&D systems companies) sold measures the concentration of IL-7.Testing result shows control group GFPT, CAR19-
IL-7 cell factors are not detected in GFP T, and IL-7 CAR19-GFP T detect that IL-7 cell factors are more than 400pg/ml.
Also further verification IL-7CAR19-GFP T can secrete IL-7 cell factors.(Fig. 4)
Identification killing tumour in 6 IL-7 CAR19 T cells body of embodiment
Effect for the internal identification killing tumour for comparing CAR19 T, IL-7 CAR19 T cells, by equivalent amount (5 ×
105) NALM6 cells pass through 7 NCG (NOD/SCID IL2rg of tail vein respectively-/-) in immunodeficient mouse body;NALM6 is thin
The 2nd day and the 8th day after born of the same parents' transplanting (being the 0th day on the day of tumor cell transplantation), by 5 × 106A (three groups of T cell:GFP T、
CAR19 T, IL-7 CAR19 T, 6 mouse of every group of injection) be injected intravenously it is small into the NSI immune deficiencies for having transplanted NALM6 cells
In mouse body.NCG mouse tail vein blood is taken, with flow cytometer detection CD19 expressions, assesses NALM6 tumour cells in NCG Mice Bodies
Load, analysis data result show that IL-7 CAR19 T can significantly reduce the load of NALM6 tumour cells in NCG Mice Bodies
(Fig. 5), and continue to observe survival time of mice, directly dead, final result IL-7 CAR19 T can significantly extend NCG mouse growths
Phase (Fig. 6).The CAR-T of prompting secretion IL-7 can significantly improve second generation CAR T cells (CAR19 T) and the killing of tumour is imitated
Fruit extends NCG mouse tumor models.
By more than experimental result, contrast experiment's group and control group CAR T are to the power of tumour identification killing ability, verification
The CAR T of secretion IL-7 can improve the inside and outside tumor-killing function of body.
Applicant's statement illustrates product, purposes and its occupation mode of the present invention, but this hair by above-mentioned embodiment
It is bright to be not limited to above-mentioned detailed use of commodity or occupation mode, that is, do not mean that the present invention has to rely on above-mentioned detailed use of commodity and use
Mode could test.The personnel of technical field are it will be clearly understood that any improvement in the present invention, to each raw material of product of the present invention
Equivalence replacement and auxiliary element addition, the selection of concrete mode etc. all falls within protection scope of the present invention and the open scope
Within.
Sequence table
<110>Guangdong People's Hospital(The Guangdong Province Academy of Medical Sciences)
<120>Secrete Chimeric antigen receptor, viral vectors, expression cell and the preparation method and drug of IL-7
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ctaatggtca gcatcgatca attattggac agcatgaaag aaattggtag caattgcctg 180
aataatgaat ttaacttttt taaaagacat atctgtgatg ctaataagga aggtatgttt 240
ttattccgtg ctgctcgcaa gttgaggcaa tttcttaaaa tgaatagcac tggtgatttt 300
gatctccact tattaaaagt ttcagaaggc acaacaatac tgttgaactg cactggccag 360
gttaaaggaa gaaaaccagc tgccctgggt gaagcccaac caacaaagag tttggaagaa 420
aataaatctt taaaggaaca gaaaaaactg aatgacttgt gtttcctaaa gagactatta 480
caagagataa aaacttgttg gaataaaatt ttgatgggca ctaaagaaca c 531
<210> 2
<211> 54
<212> DNA
<213> artificial
<220>
<221> misc_feature
<400> 2
gagggcagag gaagtcttct aacatgcggt gacgtggagg agaatcccgg ccct 54
Claims (10)
1. a kind of Chimeric antigen receptor for secreting IL-7, it is characterised in that:Include extracellular domain, the cross-film that can combine antigen
Structural domain, intracellular domain and IL-7 cytokine domains, IL-7 cytokine domains are by self cleavage peptide and intracellular
The non-fused connection of structural domain.
2. Chimeric antigen receptor according to claim 1, it is characterised in that:Antigen is tumour antigen or exempts from itself
The inflammatory cell surface molecular that occurs in epidemic disease disease or the TCR for leading to autoimmunity, the tumour antigen are 5 β 1- of 5T4 or α
Integrin or 707-AP or AFP or ART-4 or B7H4 or BAGE or beta-catenin/m or Bcr-abl or MN/C
IX antibody or CA125 or CAMEL or CAP-1 or CASP-8 or CD4 or CD19 or CD20 or CD22 or CD25 or
CDC27/m or CD30 or CD33 or CD52 or CD56 or CD80 or CDK4/m or CEA or CT or Cyp-B or DAM,
Or EGFR or ErbB3 or ELF2M or EMMPRIN or EpCam or ETV6-AML1 or G250 or GAGE or GnTV or
Gp100 or HAGE or HER-2/new or HLA-A*0201-R170I or HPV-E7 or HSP70-2M or HST-2 or
HTERT or hTRT or iCE or IGF-1R, I or L-2R or IL-5 or KIAA0205 or LAGE or LDLR/FUT or
MAGE or MART-1/melan-A or MART-2/Ski or MC1R or Mesothelin or myosin/m or MUC1 or
MUM-1 or MUM-2 or MUM3 or NA88-A or PAP or proteinase-3 or p190minor bcr-abl or Pml/RAR
α or PRAME or PSA or PSM or PSMA or RAGE or RU1 or RU2 or SAGE or SART-1 or SART-3 or existence
Albumen or TEL/AML1 or TGF β or TPI/m or TRP-1 or TRP-2 or TRP-2/INT2 or VEGF or WT1 or
NY-Eso-1 or NY-Eso-B.
3. Chimeric antigen receptor according to claim 1, it is characterised in that:The Chimeric antigen receptor is opened from N-terminal side
Begin to include successively the single-stranded variable region of antitumor-antigen antibody as extracellular domain, the transmembrane domain and 4- of CD28 molecules
1BB intracellular domain, CD3 ζ intracellular domain, 2A peptides and IL-7 gene orders.
4. Chimeric antigen receptor according to claim 1, it is characterised in that:IL-7 cytokine domains contain such as sequence
Nucleotide sequence shown in table 1.
5. Chimeric antigen receptor according to claim 1, it is characterised in that:Such as sequence table of the nucleotide sequence of self cleavage peptide
Shown in 2 sequence.
6. a kind of slow virus carrier of the Chimeric antigen receptor comprising as described in Claims 1 to 5 is any.
7. a kind of Chimeric antigen receptor expression cell, it is characterised in that:Introduce the chimeric antigen as described in Claims 1 to 5
Receptor.
8. Chimeric antigen receptor expression cell according to claim 7, it is characterised in that:The Chimeric antigen receptor expression
Cell is T cell or the cell mass containing T cell.
A kind of 9. method for preparing Chimeric antigen receptor expression cell as claimed in claim 7 or 8, it is characterised in that including inciting somebody to action
The step of Chimeric antigen receptor as described in Claims 1 to 5 is any introduces cell.
10. a kind of drug prevented or treat tumour, it is characterised in that include the inosculating antibody as described in Claims 1 to 5 is any
Original receptor or slow virus carrier as claimed in claim 6 or Chimeric antigen receptor as claimed in claim 7 or 8 expression are thin
Born of the same parents.
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109722420A (en) * | 2019-03-15 | 2019-05-07 | 江苏艾洛特医药研究院有限公司 | A kind of preparation and its application improveing Chimeric antigen receptor T cell |
CN109735558A (en) * | 2018-12-12 | 2019-05-10 | 中南大学 | A kind of recombinant C AR19-IL24 gene, slow virus carrier, CAR19-IL24-T cell and application |
CN110922490A (en) * | 2019-12-05 | 2020-03-27 | 浙江启新生物技术有限公司 | CAR expression vector secreting interleukin 7 and chemokine 21 and application thereof |
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WO2022174498A1 (en) * | 2021-02-22 | 2022-08-25 | 广州百暨基因科技有限公司 | Chimeric antigen receptor fusion protein co-expressing il-7 and ccr2b, and application thereof |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104177499A (en) * | 2013-05-27 | 2014-12-03 | 张鸿声 | Chimeric antigen receptor, coding gene, expression vector and application thereof |
WO2015188141A2 (en) * | 2014-06-06 | 2015-12-10 | Memorial Sloan-Kettering Cancer Ceneter | Mesothelin-targeted chimeric antigen receptors and uses thereof |
CN105949325A (en) * | 2016-07-08 | 2016-09-21 | 重庆精准生物技术有限公司 | Chimeric antigen receptor containing CD27 intracellular domain, lentiviral vector and application thereof |
CN107337737A (en) * | 2017-07-18 | 2017-11-10 | 深圳市免疫基因治疗研究院 | A kind of Chimeric antigen receptor and its application |
-
2018
- 2018-01-02 CN CN201810001910.XA patent/CN108218999B/en active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104177499A (en) * | 2013-05-27 | 2014-12-03 | 张鸿声 | Chimeric antigen receptor, coding gene, expression vector and application thereof |
WO2015188141A2 (en) * | 2014-06-06 | 2015-12-10 | Memorial Sloan-Kettering Cancer Ceneter | Mesothelin-targeted chimeric antigen receptors and uses thereof |
CN107106665A (en) * | 2014-06-06 | 2017-08-29 | 纪念斯隆-凯特琳癌症中心 | Target Chimeric antigen receptor of mesothelin and application thereof |
CN105949325A (en) * | 2016-07-08 | 2016-09-21 | 重庆精准生物技术有限公司 | Chimeric antigen receptor containing CD27 intracellular domain, lentiviral vector and application thereof |
CN107337737A (en) * | 2017-07-18 | 2017-11-10 | 深圳市免疫基因治疗研究院 | A kind of Chimeric antigen receptor and its application |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109735558A (en) * | 2018-12-12 | 2019-05-10 | 中南大学 | A kind of recombinant C AR19-IL24 gene, slow virus carrier, CAR19-IL24-T cell and application |
CN109735558B (en) * | 2018-12-12 | 2022-04-15 | 中南大学 | Recombinant CAR19-IL24 gene, lentiviral vector, CAR19-IL24-T cell and application |
CN109722420A (en) * | 2019-03-15 | 2019-05-07 | 江苏艾洛特医药研究院有限公司 | A kind of preparation and its application improveing Chimeric antigen receptor T cell |
CN110922490A (en) * | 2019-12-05 | 2020-03-27 | 浙江启新生物技术有限公司 | CAR expression vector secreting interleukin 7 and chemokine 21 and application thereof |
CN110922490B (en) * | 2019-12-05 | 2023-03-03 | 浙江启新生物技术有限公司 | CAR expression vector secreting interleukin 7 and chemokine 21 and application thereof |
CN111849917A (en) * | 2020-07-22 | 2020-10-30 | 武汉大学 | Fourth generation CAR-T cell for fusion expression of type I IL-10 receptor and preparation method and application thereof |
WO2022174498A1 (en) * | 2021-02-22 | 2022-08-25 | 广州百暨基因科技有限公司 | Chimeric antigen receptor fusion protein co-expressing il-7 and ccr2b, and application thereof |
CN113481167A (en) * | 2021-08-04 | 2021-10-08 | 深圳市先康达生命科学有限公司 | Immune cell exosome and application thereof |
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