CN106749677A - Bispecific chimeric antigen receptor gene targeting MLL leukemia and application thereof - Google Patents
Bispecific chimeric antigen receptor gene targeting MLL leukemia and application thereof Download PDFInfo
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Abstract
The invention relates to a bispecific chimeric antigen receptor gene targeting MLL leukemia and application thereof, and the bispecific chimeric antigen receptor can specifically recognize two tumor-associated antigens, namely CD19 and CD133 on the cell surface of MLL leukemia. The TanCAR molecule is recombined on a virus vector and is transfected into a T lymphocyte cell line to express a bispecific chimeric antigen receptor, so that the dual-positive tumor cells of CD19CD133 can be specifically identified and killed, the killing effect on non-tumor cells is reduced, and the escape probability of tumor antigens is reduced. And the TanCAR (1linker) -Jurkat can kill MLL leukemia cells specifically at a low effective target ratio, and has less toxic and side effects such as cytokine storm, neurotoxicity and the like in later clinical use. The bispecific chimeric antigen receptor can be applied to the treatment of MLL leukemia, eliminate tumors in a patient body and prolong the life cycle of the patient.
Description
Technical field
It is a kind of bispecific chimeric of targeting MLL leukaemia specifically the present invention relates to biomedicine technical field
Antigen receptor gene.
Background technology
The immune response of tumor specific T cells mediation is the Main Means that body removes tumour cell, is normally being exempted from
In epidemic disease reaction, T cell activation needs two stimulus signals.Wherein, TCR-CD3 complexs in T cell surface divide with Antigenic Peptide-MHC
Son is combined, there is provided the first signal of T cell activation, determines the killing specificity of T cell；Secondary signal is by T cell and antigen
The costimulatory molecules mediation of presenting cells (APC) or other cell surfaces is produced.Tumour cell participates in T cell identification by lowering
And antigen reactive developed by molecule, reduce the approach such as immunogenicity and produce immunologic escape, so that the immune system of body can not
Tumour is removed well.
Research finds, with the variable region of single-stranded variable region (scFv) the substitution TCR of tumor specific monoclonal antibodies, and will
ScFv is directly connected with costimulatory molecules (such as CD28,4-1BB etc.) and T cell signal transduction region CD3-zata, and formation is embedding
Close antigen receptor (CAR) and be expressed in T cell surface, tumour specific antigen is recognized by scFv, by non-MHC restrictive approaches
With antigen-reactive, there is provided the signal of T cell activation etc. first, second, promote T cell activation, propagation, specificity kill tumour is thin
Born of the same parents.From 1993, since proposing the concept of CAR first by Eshhar and his colleagues, CAR-T had evolved to the third generation.
ScFv fragments of the first generation CAR comprising extracellular specific recognition tumour antigen, intracellular activation signal is by CD3-zata or Fc sections
ITAM (immonoreceptor tyrosine-based activation motifs) signal chains are transmitted, but the first generation
CAR lacks the costimulatory signal of T cell, causes T cell instantaneously to play effect.Second generation CAR is on the basis of first generation CAR
On increased an intracellular domain for costimulatory signal molecule, there is provided two kinds of signals of T cell activation, such as CD28, CD134/
OX40, CD137/4-1BB etc., enhance the propagation of T cell and the ability of cytokine secretion.Third generation CAR molecules are second
The intracellular domain of another costimulatory signal is increased on the basis of generation again, such as son is melted after costimulation structure C CD28
Unification costimulatory molecules such as 4-1BB, produces a CAR acceptor for triple signals, the T cell tool of third generation CAR acceptors transformation
There are more preferable effector function and internal time-to-live.
In recent years, using the Chimeric antigen receptor restructuring T cell treatment B cell ALL of targeting CD19
(B-ALL) major progress is obtained, subversiveness therapeutic effect is clinically achieved.But because CD19 is the surface mark of B cell
Will, has expression in normal B cells and B cell tumour, and it is exactly to make that the therapeutic strategy can bring inevitable side effect
Normal B cells reduce, although therefore patient CD19CAR-T treat after have significantly alleviate or cure completely,
However it is necessary that supplementing gamma globulin for a long time or even all the life by vein to avoid due under immunity caused by B cell reduction
Drop, greatly reduces the life quality of patient.
The content of the invention
MLL leukaemia is the common acutus malignus leukaemia of a class, you can to show as B-ALL, it is also possible to show as
AML, accounts for the 10% of all acute leukaemia.Especially baby's leukaemia and topoisomerase enzyme inhibitor treatment cause after
It is even more in hair property leukaemia and has accounted for more than 70%.MLL is extremely dangerous, poor prognosis, it is considered to be the most refractory white blood of a class
Disease, needs the effective treatment method of searching badly.
CTL19 lays a good foundation in the successful Application for the treatment of leukaemia to treat MLL.But it is normal in order to protect
B cell, it is necessary to the improvement that the CAR-T to targetting CD19 is carried out, makes CAR-T cells in combination with two tumor associated antigens,
So that it can only recognize malignant B cell, nonrecognition normal B cells.In to the research process of MLL leukaemia, Wo Menfa
Now show as surface antigen CD133 expression high in the MLL leukaemia of B-ALL, and other B-ALL cells or normal B cells
Do not express CD133.CD133 is common stem cell labeling thing, and normal stem cell is not express CD19.That is,
Only this class shows as the MLL leukaemia of B-ALL just while having two kinds of surface antigens of CD19 and CD133, therefore target
This quasi-leukemia cell will be specifically killed to the double special CAR-T of CD19/CD133, normal B cells will be protected, will be the white blood of MLL
The healing of disease brings hope.
First purpose of the invention is directed to deficiency of the prior art, there is provided a kind of double spies of targeting MLL leukaemia
Different in nature Chimeric antigen receptor.
Second object of the present invention is to provide the bispecific chimeric antigen that coding targets MLL leukaemia as described above
The gene of acceptor.
Third object of the present invention is to provide the bispecific chimeric antigen receptor of targeting MLL leukaemia as described above
Purposes.
To realize above-mentioned first purpose, the present invention is adopted the technical scheme that：
A kind of bispecific chimeric antigen receptor of targeting MLL leukaemia, the bispecific of the targeting MLL leukaemia is embedding
Close hinge area and transmembrane region, CD28 intracellular of the antigen receptor by CD8leader, CD19scFv, linker, CD133scFv, CD8
Signaling zone, 4-1BB intracellular signals area, CD3-zata intracellular signals area are followed in series to form, and its structure is as follows；
The nucleotide sequence such as SEQ ID of the gene of the bispecific chimeric antigen receptor of the coding targeting MLL leukaemia
Shown in NO.1.
Further, the nucleotide sequence of the gene of the coding CD8 leader is as shown in SEQ ID NO.2；
Further, the nucleotide sequence of the gene of the coding CD19 scFv is as shown in SEQ ID NO.3；
Further, the nucleotide sequence of the gene of the coding linker is as shown in SEQ ID NO.4；
Further, the nucleotide sequence of the gene of the coding CD133 scFv is as shown in SEQ ID NO.5；
Further, the nucleotide sequence of the gene of the hinge area of the coding CD8 is as shown in SEQ ID NO.6；
Further, the nucleotide sequence of the gene of the transmembrane region of the coding CD8 is as shown in SEQ ID NO.7；
Further, the nucleotide sequence of the gene in the coding CD28 intracellular signals area is as shown in SEQ ID NO.8；
Further, the nucleotide sequence of the gene in the coding 4-1BB intracellular signals area is as shown in SEQ ID NO.9；
Further, the nucleotide sequence of the gene of the coding CD3-zata intracellular signals is as shown in SEQ ID NO.10.
Further, bispecific chimeric antigen receptor specificity identification MLL the leukemia cell surfaces CD19 and CD133
Two kinds of tumor associated antigens, specific recognition kills the double positive tumor cells of CD19/CD133.
To realize above-mentioned second purpose, the present invention is adopted the technical scheme that：
Coding targets the gene of the bispecific chimeric antigen receptor of MLL leukaemia, the nucleosides of the gene as described above
As shown in SEQ ID NO.1, wherein 1-63 is CD8leader to acid sequence, and 64-789 is CD19scFv, and 790-804 is linker,
805-1536 is CD133scFv, and 1537-1671 is CD8hinge, and 1672-1743 is CD8transmember, and 1744-1866 is
CD28intracellular domain, 1867-1992 are for 4-1BB intracellular domain, 1993-2328
CD3-zata domain。
To realize above-mentioned 3rd purpose, the present invention is adopted the technical scheme that：
The bispecific chimeric antigen receptor of targeting MLL leukaemia is preparing anti-ALL as described above
Medicine in application.
The present invention also provides a kind of expression vector, and as above any described gene is mounted with the expression vector.
Further, the expression vector is slow virus, retrovirus, adenovirus or plasmid.
Further, the expression vector is pCDH-MCS-T2A-copGFP-MSCV viral vectors.
The invention has the advantages that：
1st, bispecific chimeric antigen receptor TanCAR of the invention can specific recognition MLL leukemia cell surfaces CD19
With two kinds of tumor associated antigens of CD133.In TanCAR molecular recombinations to viral vectors and T lymphocytic series will be transfected, expression is double
Specific chimeric antigen receptor, can specific recognition kill the double positive tumor cells of CD19CD133, alleviate to non-tumor cell
Lethal effect, reduce tumour antigen escape probability.And TanCAR (1linker)-Jurkat are in more poorly efficient target ratio
Can specific killing MLL leukaemia, less toxic and side effect, such as cell factor can occur in Clinical practice that can be after
Storm, neurotoxicity etc..Therefore bispecific chimeric antigen receptor of the invention can be applied in MLL leukemia treatings,
Patient's in-vivo tumour is removed, extends patient survival.
2nd, the double positive MLL leukaemias of TanCAR (1linker) targeting CD19/CD133 of the invention, and TanCAR
(1linker)-Jurkat specific killing MLL leukaemias than by more poorly efficient target, illustrate TanCAR (1linker) points
Sub reasonable in design, effect is significant, for treatment MLL leukaemia lays the foundation.
Brief description of the drawings
Accompanying drawing 1 is third generation TanCAR, (L in combination with CD19 and CD133:Linker the third generation of CD19) is combined
CD19CAR, the third generation CD133CAR ideographs with reference to CD133.
Accompanying drawing 2 is TanCAR expression vector structure charts.
Accompanying drawing 3 be through TanCAR (the 3linker)-Jurkat after different disposal, TanCAR (2linker)-Jurkat,
TanCAR (1linker)-Jurkat, CD19CAR-Jurkat and CD133CAR-Jurkat and target cell REH, after SEM is co-cultured
CD69 expressions.
Accompanying drawing 4 be through TanCAR (the 3linker)-Jurkat after different disposal, TanCAR (2linker)-Jurkat,
TanCAR (1linker)-Jurkat, CD19CAR-Jurkat and CD133CAR-Jurkat and target cell REH, after SEM is co-cultured
The secretory volume of IL-2.In figure per cluster first five root pillar be followed successively by from left to right TanCAR (3linker)-Jurkat,
TanCAR (2linker)-Jurkat, TanCAR (1linker)-Jurkat, CD19CAR-Jurkat and CD133CAR-
Jurkat。
Accompanying drawing 5 be through TanCAR (the 3linker)-Jurkat after different disposal, TanCAR (2linker)-Jurkat,
TanCAR (1linker)-Jurkat, CD19CAR-Jurkat and CD133CAR-Jurkat make to target cell REH, the killing of SEM
With.
Specific embodiment
The invention will be further elucidated with reference to specific embodiments.It should be understood that these embodiments are merely to illustrate this hair
Bright rather than limitation the scope of the present invention.In addition, it is to be understood that after the content for having read record of the present invention, art technology
Personnel can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited
Fixed scope.
Embodiment
The present invention provide targeting MLL leukaemia bispecific chimeric antigen receptor, its by CD8leader,
The hinge area and transmembrane region of CD19scFv, linker, CD133scFv, CD8, CD28 intracellular signals area, 4-1BB intracellular signals area,
CD3-zata intracellular signals area is in series, and its structure is as shown in Figure 1 (TanCAR (1linker)).
The nucleotide sequence such as SEQ ID of the gene of the bispecific chimeric antigen receptor of the coding targeting MLL leukaemia
Shown in NO.1, wherein 1-63 is CD8leader, and 64-789 is CD19scFv, and 790-804 is linker, and 805-1536 is
CD133scFv, 1537-1671 are CD8hinge, and 1672-1743 is CD8transmember, and 1744-1866 is
CD28intracellular domain, 1867-1992 are for 4-1BB intracellular domain, 1993-2328
CD3-zata domain。
(1), pCDH-TanCAR (3linker), pCDH-TanCAR (2linker) and pCDH-TanCAR (1linker)
The structure of plasmid
The present invention is from the hybridoma cell strain HB-12346 of IgG1 monoclonal antibodies AC133 that can secrete specific recognition CD133
The cDNA sequence for encoding the antibody variable heavy (VH) and light chain (VL) is obtained, splicing synthesis can combine CD133 antibody
Single-chain antibody (single chain antibody fragment, scFv) sequence.And according to it has been reported that simultaneously extensive use
CD19 sequences, TanCAR sequence of the design with CD19-scFv and CD133-scFv and introduce CD8 transmembrane regions and CD28,4-
The genetic fragment in 1BB, CD3zata intracellular signal area.CD19-scFv and CD133-scFv is connected with 3 kinds of linker of form, point
It is not：(G4S) x1 is 1linker, the i.e. 2linker of (G4S) x2 and the i.e. 3linker of (G4S) x3.According to TanCAR (3linker)
DNA encoding sequence, commission Sangon Biotech (Shanghai) Co., Ltd. synthesizes whole expression cassette, pCDH- inserted after digestion
Between the EcoRI-BamHI sites of MCS-T2A-copGFP-MSCV viral vectors (Fig. 2), pCDH-TanCAR is obtained
(3linker), obtains pCDH-TanCAR (2linker) and pCDH-TanCAR by BamHI digestions and Insert Fragment respectively
(1linker) plasmid.After through sequencing correctly, promega companies plasmid extraction kit is used to extract plasmid.
(2), slow virus packaging
Using pSPAX2, pMD2G Viral Packaging Systems, Lenti-X cells are transfected with CaCl2 transfection reagents, are harvested
After virus, purified.
(3), the genetic modification of T cell strain
The virus infection Jurkat E6.1 of acquisition, obtain stable cell strain TanCAR (3linker)-Jurkat, TanCAR
(2linker)-Jurkat, TanCAR (1linker)-Jurkat, CD19CAR-Jurkat and CD133CAR-Jurkat.
(4) activation situation of the vitro detection tumour target cell to CAR-T cells
1. the expression of flow cytometer detection CAR-T cell surfaces CD69 molecules
Effector cell TanCAR (3linker)-Jurkat, TanCAR (2linker)-Jurkat, TanCAR
(1linker)-Jurkat, CD19CAR-Jurkat and CD133CAR-Jurkat respectively with target cell REH (CD19+/
), CD133- SEM (CD19+/CD133+) each 1x105/ hole is co-cultured in 24 orifice plates, while set CAR-T cells individually cultivating
Group, as negative control.RPMI1640 culture medium of each hole containing 10%FBS supplies 1ml, 37 degree of incubation 24h.Collect cell in
In 1.5mlEP pipes, room temperature 300g centrifugation 5min, supernatant discarded, every kind of sample adds 100ul antibody labeling systems, per reactant
Antibody containing 1ulCD69-APC (BD) and 99ulPBS in system.Room temperature lucifuge is washed twice after being incubated 30min with PBS, flow cytometer detection
CD69 expressions.Result is shown in Fig. 3.Result above show TanCAR (3linker)-Jurkat, TanCAR (2linker)-
Jurkat, TanCAR (1linker)-Jurkat can be by target cell REH, SEM activation；CD19CAR-Jurkat can be by target cell
REH, SEM are activated；But CD133CAR-Jurkat can only be by SEM cell-stimulatings.Wherein TanCAR (1linker)-Jurkat quilts
SEM activation levels are significantly higher than by REH activation levels, illustrate TanCAR (1linker)-Jurkat have specific recognition CD19+/
The function of the double positive target cells of CD133+.
2.ELISA methods determine the level of cell factor
By TanCAR (3linker)-Jurkat, TanCAR (2linker)-Jurkat, TanCAR (1linker)-
Jurkat, CD19CAR-Jurkat and CD133CAR-Jurkat respectively with target cell REH, each 1x10 of SEM, K5625/ Kong Gongpei
Support in 24 orifice plates, supernatant is collected after 24h, ELISA method determines IL-2 secretory volumes.(IL-2ELISA kits are purchased from BD companies).Knot
Fruit sees Fig. 4.Result shows that TanCAR-Jurkat (1linker) cell can be big after being co-cultured with CD19, CD133 double positive cells
Amount secretion IL-2, secretory volume is significantly higher than CD19CAR-Jurkat and CD133CAR-Jurkat, and mono- positive higher than with CD19
TanCAR-Jurkat (1linker) cell that REH is co-cultured.Result above shows that CD19, CD133 double positive cells compare CD19
Single positive cell can preferably activate TanCAR-Jurkat (1linker) cell.From the result of Fig. 4 can further be seen that with
The secretory volume of TanCAR-Jurkat (1linker) cells IL-2 is significantly higher than after the co-cultivation of CD19, CD133 double positive cells
TanCAR-Jurkat (2linker) cells and TanCAR-Jurkat (3linker) cell, illustrate at identical conditions, this
TanCAR-Jurkat (1linker) cell of invention can be activated preferably by CD19, CD133 double positive cells.
3.Annexin-V labelling methods detect CAR-T cells to REH, the lethal effect of SEM cells
By TanCAR (1linker)-Jurkat respectively with celltrace yellow (be purchased from ThermoFisher companies)
The target cell REH of mark, SEM is with 1:1 co-cultures in 24 orifice plates.After 24h, with 50ulAnnexin-V (being purchased from BD companies) body
System's mark apoptotic cell, adds 200ul Bounding Burrer, upper machine testing after 30min.Result is shown in Fig. 5.Result shows
TanCAR (1linker)-Jurkat with specific recognition and can kill SEM cells, but to REH without obvious effect, explanation
The double positive MLL leukaemias of TanCAR (1linker) targetings CD19/CD133.And TanCAR (1linker)-Jurkat exists
More poorly efficient target specific killing MLL leukaemia than by, illustrates TanCAR (1linker) MOLECULE DESIGN rationally, and effect shows
Write, for treatment MLL leukaemia lays the foundation.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, on the premise of the inventive method is not departed from, can also make some improvement and supplement, and these are improved and supplement also should be regarded as
Protection scope of the present invention.
SEQUENCE LISTING
<110>Ruijin Hospital, Shanghai Jiao Tong University School of Medicine
<120>A kind of bispecific chimeric antigen receptor gene of targeting MLL leukaemia and its application
<130> /
<160> 10
<170> PatentIn version 3.3
<210> 1
<211> 2328
<212> DNA
<213>Artificial sequence
<400> 1
atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60
cccgaaattg tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca 120
accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta tcaacagaag 180
cccggacagg ctcctcgcct tctgatctac cacaccagcc ggctccattc tggaatccct 240
gccaggttca gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag 300
ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta cacctttgga 360
cagggcacca agctggagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt 420
ggaggaagcc aggtccaact ccaagaaagc ggaccgggtc ttgtgaagcc atcagaaact 480
ctttcactga cttgtactgt gagcggagtg tctctccccg attacggggt gtcttggatc 540
agacagccac cggggaaggg tctggaatgg attggagtga tttggggctc tgagactact 600
tactactctt catccctcaa gtcacgcgtc accatctcaa aggacaactc taagaatcag 660
gtgtcactga aactgtcatc tgtgaccgca gccgacaccg ccgtgtacta ttgcgctaag 720
cattactatt atggcgggag ctacgcaatg gattactggg gacagggtac tctggtcacc 780
gtgtccagcg gaggtggtgg atccgatgtt gtgatgactc agtctccact ctccctgccc 840
gtcacccctg gagagccggc ctccatctcc tgcaggtcta gtcagagtct tgcaaacagt 900
tatgggaaca cctatttgtc ttggtacctg cagaagccag ggcagtctcc acagctcctg 960
atctatggga tttccaacag attttctggg gtccctgaca ggttcagtgg cagtggatca 1020
ggcacagatt ttacactgaa aatcagcaga gtggaggctg aggatgttgg ggtttattac 1080
tgcttacaag gtacacatca gccgtacacg tttggccagg ggaccaagct ggagatcaaa 1140
ggcagtacta gcggtggtgg ctccgggggc ggttccggtg ggggcggcag cagccaggtt 1200
cagctggtgc agtctggagc tgaggtgaag aagcctgggg cctcagtgaa ggtctcctgc 1260
aaggcttctg gttacacctt taccgacttt gaaatgcact gggtgcgaca ggcccctgga 1320
caagggcttg agtggatggg agatattgat cctggaactg gtgatactgc ctacaatctg 1380
aagttcaagg gcagagtcac catgaccaca gacacatcca cgagcacagc ctacatggag 1440
ctgaggagcc tgaggtctga cgacacggcc gtgtattact gtgcgttggg ggcctttgtt 1500
tactggggcc agggaaccct ggtcaccgtc tcctcaacca ctaccccagc accgaggcca 1560
cccaccccgg ctcctaccat cgcctcccag cctctgtccc tgcgtccgga ggcatgtaga 1620
cccgcagctg gtggggccgt gcatacccgg ggtcttgact tcgcctgcga tatctacatt 1680
tgggcccctc tggctggtac ttgcggggtc ctgctgcttt cactcgtgat cactctttac 1740
tgtaggagta agaggagcag gctcctgcac agtgactaca tgaacatgac tccccgccgc 1800
cccgggccca cccgcaagca ttaccagccc tatgccccac cacgcgactt cgcagcctat 1860
cgctccaagc gcggtcggaa gaagctgctg tacatcttta agcaaccctt catgaggcct 1920
gtgcagacta ctcaagagga ggacggctgt tcatgccggt tcccagagga ggaggaaggc 1980
ggctgcgaac tgcgcgtgaa attcagccgc agcgcagatg ctccagccta caagcagggg 2040
cagaaccagc tctacaacga actcaatctt ggtcggagag aggagtacga cgtgctggac 2100
aagcggagag gacgggaccc agaaatgggc gggaagccgc gcagaaagaa tccccaagag 2160
ggcctgtaca acgagctcca aaaggataag atggcagaag cctatagcga gattggtatg 2220
aaaggggaac gcagaagagg caaaggccac gacggactgt accagggact cagcaccgcc 2280
accaaggaca cctatgacgc tcttcacatg caggccctgc cgcctcgg 2328
<210> 2
<211> 63
<212> DNA
<213>Artificial sequence
<400> 2
atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60
ccc 63
<210> 3
<211> 726
<212> DNA
<213>Artificial sequence
<400> 3
gaaattgtga tgacccagtc acccgccact cttagccttt cacccggtga gcgcgcaacc 60
ctgtcttgca gagcctccca agacatctca aaatacctta attggtatca acagaagccc 120
ggacaggctc ctcgccttct gatctaccac accagccggc tccattctgg aatccctgcc 180
aggttcagcg gtagcggatc tgggaccgac tacaccctca ctatcagctc actgcagcca 240
gaggacttcg ctgtctattt ctgtcagcaa gggaacaccc tgccctacac ctttggacag 300
ggcaccaagc tggagattaa aggtggaggt ggcagcggag gaggtgggtc cggcggtgga 360
ggaagccagg tccaactcca agaaagcgga ccgggtcttg tgaagccatc agaaactctt 420
tcactgactt gtactgtgag cggagtgtct ctccccgatt acggggtgtc ttggatcaga 480
cagccaccgg ggaagggtct ggaatggatt ggagtgattt ggggctctga gactacttac 540
tactcttcat ccctcaagtc acgcgtcacc atctcaaagg acaactctaa gaatcaggtg 600
tcactgaaac tgtcatctgt gaccgcagcc gacaccgccg tgtactattg cgctaagcat 660
tactattatg gcgggagcta cgcaatggat tactggggac agggtactct ggtcaccgtg 720
tccagc 726
<210> 4
<211> 15
<212> DNA
<213>Artificial sequence
<400> 4
ggaggtggtg gatcc 15
<210> 5
<211> 732
<212> DNA
<213>Artificial sequence
<400> 5
gatgttgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60
atctcctgca ggtctagtca gagtcttgca aacagttatg ggaacaccta tttgtcttgg 120
tacctgcaga agccagggca gtctccacag ctcctgatct atgggatttc caacagattt 180
tctggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240
agcagagtgg aggctgagga tgttggggtt tattactgct tacaaggtac acatcagccg 300
tacacgtttg gccaggggac caagctggag atcaaaggca gtactagcgg tggtggctcc 360
gggggcggtt ccggtggggg cggcagcagc caggttcagc tggtgcagtc tggagctgag 420
gtgaagaagc ctggggcctc agtgaaggtc tcctgcaagg cttctggtta cacctttacc 480
gactttgaaa tgcactgggt gcgacaggcc cctggacaag ggcttgagtg gatgggagat 540
attgatcctg gaactggtga tactgcctac aatctgaagt tcaagggcag agtcaccatg 600
accacagaca catccacgag cacagcctac atggagctga ggagcctgag gtctgacgac 660
acggccgtgt attactgtgc gttgggggcc tttgtttact ggggccaggg aaccctggtc 720
accgtctcct ca 732
<210> 6
<211> 135
<212> DNA
<213>Artificial sequence
<400> 6
accactaccc cagcaccgag gccacccacc ccggctccta ccatcgcctc ccagcctctg 60
tccctgcgtc cggaggcatg tagacccgca gctggtgggg ccgtgcatac ccggggtctt 120
gacttcgcct gcgat 135
<210> 7
<211> 72
<212> DNA
<213>Artificial sequence
<400> 7
atctacattt gggcccctct ggctggtact tgcggggtcc tgctgctttc actcgtgatc 60
actctttact gt 72
<210> 8
<211> 123
<212> DNA
<213>Artificial sequence
<400> 8
aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60
gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120
tcc 123
<210> 9
<211> 126
<212> DNA
<213>Artificial sequence
<400> 9
aagcgcggtc ggaagaagct gctgtacatc tttaagcaac ccttcatgag gcctgtgcag 60
actactcaag aggaggacgg ctgttcatgc cggttcccag aggaggagga aggcggctgc 120
gaactg 126
<210> 10
<211> 336
<212> DNA
<213>Artificial sequence
<400> 10
cgcgtgaaat tcagccgcag cgcagatgct ccagcctaca agcaggggca gaaccagctc 60
tacaacgaac tcaatcttgg tcggagagag gagtacgacg tgctggacaa gcggagagga 120
cgggacccag aaatgggcgg gaagccgcgc agaaagaatc cccaagaggg cctgtacaac 180
gagctccaaa aggataagat ggcagaagcc tatagcgaga ttggtatgaa aggggaacgc 240
agaagaggca aaggccacga cggactgtac cagggactca gcaccgccac caaggacacc 300
tatgacgctc ttcacatgca ggccctgccg cctcgg 336
Claims (7)
1. a kind of bispecific chimeric antigen receptor of targeting MLL leukaemia, it is characterised in that the targeting MLL leukaemia
Bispecific chimeric antigen receptor by CD8leader, CD19scFv, linker, CD133scFv, CD8 hinge area and cross-film
Area, CD28 intracellular signals area, 4-1BB intracellular signals area, CD3-zata intracellular signals area are in series, and its structure is as follows；
The nucleotide sequence such as SEQ ID of the gene of the bispecific chimeric antigen receptor of the coding targeting MLL leukaemia
Shown in NO.1.
2. the bispecific chimeric antigen receptor of MLL leukaemia is targetted according to claim 1, it is characterised in that
The nucleotide sequence of gene of the CD8leader is encoded as shown in SEQ ID NO.2；
The nucleotide sequence of gene of the CD19scFv is encoded as shown in SEQ ID NO.3；
The nucleotide sequence of gene of the linker is encoded as shown in SEQ ID NO.4；
The nucleotide sequence of gene of the CD133scFv is encoded as shown in SEQ ID NO.5；
The nucleotide sequence of gene of the hinge area of the CD8 is encoded as shown in SEQ ID NO.6；
The nucleotide sequence of gene of the transmembrane region of the CD8 is encoded as shown in SEQ ID NO.7；
The nucleotide sequence of gene in the CD28 intracellular signals area is encoded as shown in SEQ ID NO.8；
The nucleotide sequence of gene in the 4-1BB intracellular signals area is encoded as shown in SEQ ID NO.9；
The nucleotide sequence of gene of the CD3-zata intracellular signals is encoded as shown in SEQ ID NO.10.
3. the bispecific chimeric antigen receptor of MLL leukaemia is targetted according to claim 1, it is characterised in that described double
Specific chimeric antigen receptor specificity recognizes MLL leukemia cell surfaces two kinds of tumor associated antigens of CD19 and CD133, specifically
Property identification kill the double positive tumor cells of CD19/CD133.
4. the gene of the bispecific chimeric antigen receptor of MLL leukaemia is targetted described in coding claim 1-3, and its feature exists
In the nucleotide sequence of the gene is as shown in SEQ ID NO.1.
5. the bispecific chimeric antigen receptor of MLL leukaemia is targetted described in claim 1 to prepare anti-acute lymphoblastic white
Application in the medicine of blood disease.
6. a kind of expression vector, it is characterised in that gene as claimed in claim 4 is mounted with the expression vector.
7. expression vector according to claim 6, it is characterised in that the expression vector be slow virus, retrovirus,
Adenovirus or plasmid.
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| Publication number | Publication date |
|---|---|
| CN106749677B (en) | 2020-06-12 |
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