CN111808821B - Construction and preparation of FLT3-NKG2D double-target CAR-T - Google Patents

Construction and preparation of FLT3-NKG2D double-target CAR-T Download PDF

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CN111808821B
CN111808821B CN202010587905.9A CN202010587905A CN111808821B CN 111808821 B CN111808821 B CN 111808821B CN 202010587905 A CN202010587905 A CN 202010587905A CN 111808821 B CN111808821 B CN 111808821B
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黄宇贤
李可昕
吴惠阳
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Southern Medical University Zhujiang Hospital
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Abstract

The invention discloses construction and preparation of FLT3-NKG2D double-target CAR-T. The CAR belongs to a double-target second-generation CAR and has the functions of overcoming tumor immune escape and enhancing specific recognition and killing of tumor cells. It can be used together with Gilitinib to effectively solve FLT3mut+The treatment dilemma of intractable acute myeloid leukemia, and provides a new treatment strategy for the treatment of acute myeloid leukemia.

Description

Construction and preparation of FLT3-NKG2D double-target CAR-T
Technical Field
The invention relates to the field of biological immunocyte treatment, in particular to construction and preparation of FLT3-NKG2D double-target CAR-T.
Background
The international cell therapy association (interna) in 2012 indicates that biological immune cell therapy has become a fourth means for treating tumors besides surgery, radiotherapy and chemotherapy, and will become a necessary means for treating tumors in the future. The immune cell therapy is to collect peripheral venous blood of a patient, separate peripheral blood mononuclear cells in a GMP laboratory, greatly expand immune effector cells with high-efficiency antitumor activity under the induction of various cytokines, and then return the cells into the body of the patient through intravenous injection, intradermal injection, intervention and the like so as to achieve the purposes of enhancing the immune function of the patient and killing tumor cells. However, when a tumor is found clinically, the tumor is generally in the middle-late stage, at the moment, tumor cells in a patient are dominant, the immune function of an organism is seriously damaged, the DC cell function is damaged under the microenvironment of the immune system, the T cell activating efficiency is low, the ability of attacking cancer cells is insufficient, and the accuracy is not high enough; in addition, tumor cells escape from immune cell attack by escape mechanisms that either underexpress or do not express MHC molecules. There is a need to create a precisely guided, precisely targeted immune cell weapon that overcomes the MHC-mediated oncocidal mechanism. Thus, targeted anti-tumor cell CAR-T technology is in force.
A Chimeric Antigen Receptor (CAR) mainly includes an extracellular antigen-binding region, a transmembrane region, and an intracellular region: the extracellular region is mainly an antigen-specific monoclonal antibody single-chain variable region sequence and comprises a heavy-chain variable region and a light-chain variable region which are connected by a hinge region; the transmembrane region is the transmembrane region of protein molecules such as CD3, CD4, CD8, CD28 and the like; the intracellular domains are mainly the CD3 zeta chain or the immunoglobulin Fc receptor fcepsilon RI gamma chain of the T cell receptor TCR/CD3 complex, usually with Immunoreceptor Tyrosine Activation Motifs (ITAMs), responsible for signal transduction; the CAR extracellular region is a ligand, a Tumor Associated Antigen (TAA) is a receptor per se, the CAR can activate T cells to play an effector function through the intracellular region of a CD3 zeta or a high affinity receptor FceRI gamma once the CAR is combined with the TAA, CAR-T cells are induced to be activated after being combined with the TAA, the CAR-T cells are infused in a body and show CAR-dependent killing, proliferation and cytokine release, and the continuous existence of the infused CAR-T cells in a patient is related to potential curative effect.
Currently, most CAR-T cell designs are mainly directed to lymphocytic leukemias such as CD19-CAR T cells, where single target CAR-T cells are not sufficiently potent for tumor killing, while overdose CAR-T cells are prone to severe complications such as Cytokine Release Syndrome (CRS). Moreover, single-target CAR-T cell therapy is prone to target antigen loss, resulting in recurrence of the tumor after CAR-T cell therapy.
Second, most current monotherapies of FLT3 inhibitors and single CAR-T cell therapies did not significantly improve acute myeloid leukemia OS (overall survival time) and were targeted to refractory FLT3mut+AML also did not produce significant effects, and a new anti-leukemia immune mechanism with clinical therapeutic transformation significance is generally lacking, for FLT3mut+The treatment of AML patients brings new hopes.
Disclosure of Invention
The invention aims to provide a double-target FLT3-NKG2D-CAR-T cell combined with gelitinib in preparation of a medicine for treating FLT3mut+Application in the medicine for treating acute myelogenous leukemia.
The technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided:
a double-target CAR-T cell can simultaneously express FLT3 single-chain antibody and NKG2D receptor gene sequences.
Further, the gene sequence of the FLT3 single-chain antibody is FLT3(EB10) SCFV gene sequence:
GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGAAACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCTGGGGTCCCAGACAGATTCAGCGGCAGTGGGTCAGACACTGATTTCACACTGCAAATCAGTAGGGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTACACACCCCGCCATCTCCTTCGGCCAAGGGACACGACTGGAGATTAAAGGTGGCGGAGGATCTGGCGGAGGTGGAAGCGGCGGAGGCGGATCTGAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTAGTGGTGGTAGCACAAGCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGGGGAGTGGGAGCGCATGATGCTTTTGATATCTGGGGCCAAGGGACCACGGTCACCGTCTCAAGC(SEQ ID NO.1)。
furthermore, the FLT3 single-chain antibody and the NKG2D receptor gene sequence vector are lentiviral vectors.
Further, the CAR-T cell structure described above includes a signal peptide, FLT3(EB10) single chain antibody, spacer region, CD8 hinge region, transmembrane region, and intracellular signal region.
Further, the signal peptide is selected from the group consisting of CD8SP signal peptide.
Furthermore, the spacer is selected from T2A.
Furthermore, the intracellular signaling region is an intracellular activation signaling region, and CD137(4-1BB) and CD3 zeta are used as co-stimulatory molecules.
In a second aspect of the present invention, there is provided:
the CAR-T cell is applied to the preparation of drugs for treating leukemia.
In a third aspect of the present invention, there is provided:
the CAR-T cell is combined with an FLT3 inhibitor in the preparation of FLT3mut+Application in the medicine for treating acute myelogenous leukemia.
In acute myeloid leukemia, the expression rates of various ligands of NKG2D such as MICA/B, ULBP1/2 and ULBP3 were 0-75%, 16-63% and 16-100%, respectively, while they were hardly expressed in normal tissue cells. In addition, the dual-target CAR-T cells also express anti-FLT3 recognizes FLT3 target antigen on AML cells, and the double target point formed by the ti-FLT3 and NKG2D can mutually promote CAR-T to AML cells, particularly to FLT3mut+Specific killing of AML cells.
Further, the FLT3 inhibitor is selected from the group consisting of gillitinib (Gilteritinib).
On one hand, the gelitinib serving as an FLT3 inhibitor has good killing capacity on leukemia cells in rescue chemotherapy of patients with acute myelogenous leukemia after relapse; on the other hand, the gilitinib can induce the expression of tumor cells FLT3 and NKG2D, and when the gilitinib is combined with the FLT3-NKG2D-CAR-T cells, the gilitinib can effectively promote the killing of the CAR-T cells on target cells, reduce off-target effects and form the efficacy of '1 +1> 2'.
The beneficial effects of the invention are:
1. compared with the first generation CAR, the second generation CAR introduces a co-stimulation receptor domain on the basis of the first generation CAR, improves the initial T cell to obtain durable in vitro proliferation and stronger cytokine secretion, obviously improves the problem of CAR-T immune activity activation in clinical tests, and improves the action durability of the CAR-T immune activity. Compared with the current third-generation CAR, the CAR is more stable and has higher transfection efficiency.
2. The invention is a dual-target FLT3-NKG2D-CAR-T that, on the one hand, helps to overcome tumor immune escape and enhance CAR-T cell specific recognition and killing function on tumor cells compared to single-target CAR-T cells. On the other hand, NKG2D receptor expressed on this dual-targeted CAR-T cell can specifically target killing of AML cells carrying NKG2D ligand without causing normal tissue damage.
3. The invention provides a novel treatment mode of combining double-target FLT3-NKG2D-CAR-T and gillitinib, on one hand, the deficiency and off-target phenomena of killing tumor cells by the single-target CAR-T are compensated, on the other hand, the drug can kill tumors by cytotoxicity due to the combination of the double-target FLT3-NKG2D-CAR-T and the gillitinib, on the other hand, the drug can induce the tumor cells to express FLT3 and up-regulate NKG2DLs, and the defect of insufficient killing caused by the low expression of CAR-T surface recognition sites due to the low large transfection rate of a constructed vector of the double-target CAR is compensated. The treatment modeBlock FLT3mut+The treatment dilemma of the refractory acute myeloid leukemia provides a new treatment strategy for the treatment of the acute myeloid leukemia.
Drawings
FIG. 1 is a schematic structural diagram of the chimeric antigen receptor FLT3-NKG 2D-CAR;
FIG. 2 is an enzyme digestion identification map of FLT3-NKG2D-CAR vector;
FIG. 3 is a FACS analysis of GFP expression by 293T cells transfected with FLT3-NKG2D-CAR lentiviral expression vector and NKG 2D;
FIG. 4 shows that FLT3-NKG2D-CAR lentivirus infects human CD3+T cell fluorescence microscopy images; magnification of X200
FIG. 5 is a bar graph of the sensitivity of Gilitinib in promoting the killing of leukemic cells by FLT3-NKG2D-CAR T cells;
FIG. 6 shows FLT3-NKG2D-CAR-T cells in combination with Gelitinib killing CD33+Effect of tumor cells versus line graph; t ratio is the ratio of effector cells, namely FLT3-NKG2D-CAR T cells to target cells, namely MV 4-11;
FIG. 7 is a bar graph of variable concentrations of gelitinib induced leukemia cell FLT3 expression and upregulation of NKG2 DLs;
FIG. 8 is a schematic diagram of the combined treatment pattern of FLT3-NKG2D-CAR-T cells and gelitinib.
Detailed Description
In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
Firstly, construction and identification of FLT3-NKG2D-CAR lentiviral vector
1. Lentiviral vector preparation and identification
Taking the gene sequences of the FLT3(EB10) SCFV and NKG2D extracellular region, the specific sequence of the FLT3(EB10) SCFV is GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGAAACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCTGGGGTCCCAGACAGATTCAGCGGCAGTGGGTCAGACACTGATTTCACACTGCAAATCAGTAGGGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTACACACCCCGCCATCTCCTTCGGCCAAGGGACACGACTGGAGATTAAAGGTGGCGGAGGATCTGGCGGAGGTGGAAGCGGCGGAGGCGGATCTGAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTAGTGGTGGTAGCACAAGCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGGGGAGTGGGAGCGCATGATGCTTTTGATATCTGGGGCCAAGGGACCACGGTCACCGTCTCAAGC (SEQ ID NO. 1).
CD137 and CD3 zeta are used as activation signals and are subcloned into a lentivirus expression vector Lenti-EF1a-AT-Free to construct a double-CAR lentivirus expression vector as shown in figure 1, the structure of the chimeric antigen receptor FLT3-NKG2D-CAR comprises an SP signal peptide, a spacer region is T2A, a hinge region is CD8 hinge, a FLT3(EB10) single-chain antibody, a transmembrane region is NKG2D, and an intracellular signal region is CD137(4-1BB) and CD3 zeta used as intracellular activation signal regions of co-stimulatory molecules. After Sanger sequencing confirmed the correct sequence inserted into the vector, the endotoxin-free plasmid was prepared using the Qiagen Maxi prep kit. The enzyme cutting identification map of the FLT3-NKG2D-CAR vector is shown in figure 2.
2. Lentiviral expression vector validation
Transiently transfecting the prepared CAR lentiviral expression vector into 293T cells, and detecting the expression of EGFP by FACS after successful transfection; after collecting cell samples and incubating the cell samples by Anti-NKG2D antibody, FACS is carried out to detect the expression of NKG2D so as to confirm that the constructed lentivirus expression vector can correctly express the target protein.
FIG. 3 is a FACS analysis of the expression of EGFP and NKG2D, which further shows that the CAR lentiviral expression vector can successfully transfect cells and that transfected cells can significantly express the NKG2D receptor.
Preparation and identification of FLT3-NKG2D-CAR-T cell
1. Pre-blood collection and peripheral blood lymphocyte pre-separation
(1) The method comprises the steps of collecting heparin anticoagulation peripheral blood of a volunteer by using a disposable sterile syringe according to a clinical standard process, and then separating peripheral blood mononuclear cells.
(2) The anticoagulated blood sample in the syringe was transferred to a sterile centrifuge tube, the cap was tightened, the centrifuge force was adjusted to 800Xg, the centrifuge deceleration was set to the lowest, and the blood sample was centrifuged at room temperature for 20 minutes.
(3) After the centrifugation is finished, taking out the centrifuge tube from the centrifuge, avoiding violent shaking or turning the centrifuge tube upside down, opening a centrifuge tube cover on an operation table top of the biosafety cabinet, and transferring the upper light yellow serum layer into a new sterile centrifuge tube by using a disposable pipettor; then adding physiological saline with the same volume to the lower red peripheral blood cell layer, screwing the centrifugal tube cover, and slightly reversing and mixing.
(4) The lymphocyte separation medium was removed and turned upside down several times, and mixed well.
(5) In the biological safety cabinet, the lymphocyte separation solution is firstly added into a centrifuge tube by a disposable sterile pipette, and then the blood sample diluted by the physiological saline in the step (2) is carefully and slowly added to the upper layer of the lymphocyte separation reagent along the tube wall by using the pipette, so that the separation reagent and the blood sample are prevented from being mixed.
(6) The centrifugal force of the centrifuge was set at 800Xg, the rotational speed reduction rate was set at the lowest, the temperature was set at 20 ℃ and the centrifuge was performed for 20 minutes.
(7) After centrifugation is finished, a disposable sterile pipettor is used for sucking the middle white mononuclear cell layer into a new sterile centrifuge tube in the biological safety cabinet, after isometric normal saline is added, a centrifuge tube cover is screwed, the centrifuge tube cover is turned upside down and mixed evenly, the centrifugal force of the centrifuge is set to be 800xg, centrifugation is carried out for 5 minutes, after centrifugation is finished, the sterile pipettor is used for sucking out all upper liquid, then normal saline is added, after the centrifugation is carried out gently and mixed evenly, partial cell suspension is taken for counting, the total number of cells is calculated, the centrifugal force of the centrifuge is set to be 800xg, and centrifugation is carried out for 5 minutes.
(8) And (3) after the centrifugation is finished, sucking out all the upper layer liquid by using a sterile pipettor, calculating the volume of the cryopreservation liquid required to be added according to the cell density according to the cell counting result in the step (7), adding the cell cryopreservation liquid CryoStor CS10 by using the sterile pipettor, slightly blowing the cell mass at the bottom of the centrifuge tube by using the pipettor, re-suspending the cells to prepare cell suspension, and transferring the cell suspension to a new sterile cell cryopreservation tube. Immediately transferring the frozen tube to a gradient cooling box at 2-8 ℃, and incubating for 10 minutes in a refrigerator. After the incubation is finished, the gradient cooling box is immediately transferred to an ultra-low temperature refrigerator with the temperature of minus 80 ℃, and the incubation is continued for 4 hours.
CAR T cell preparation and culture
(1) The required virus amount was calculated according to the instructions. The calculation formula is as follows:
Figure GDA0003613641760000061
(2) the lentivirus was removed from an ultra-low temperature freezer at-80 deg.C and rapidly thawed in a 37 deg.C water bath.
(3) The outer surface of the cryopreservation tube for storing viruses was sterilized with 75% medical alcohol.
(4) Taking out the T cells prepared in the cell culture experiment from the incubator, adding polybrene into the culture vessel, adding the calculated virus amount, gently blowing and beating by using a pipette, fully and uniformly mixing to blow out all cell clusters/beads, sealing the culture vessel by using a sealing film, and centrifuging at the room temperature of 800Xg for 1 hour.
(5) After the centrifugation is finished, the sealing film is torn off, and the culture vessel is placed in an incubator with 37 ℃ and 5% CO2 for further culture for 24 hours.
(6) Centrifugation at 250Xg removed the virus-containing medium supernatant, resuspension of the cell pellet with fresh medium, transfer of the cells to a new culture vessel and culture for an additional 5 days. Gently blowing and beating beads/cell clusters in the culture system every day until the beads/cell clusters are completely separated; the cells were counted once a day (50. mu.L of cell suspension was taken and counted under a microscope using a hemocytometer), and the cell density was adjusted by adding fresh medium according to the cell density.
(7) A portion of the cells were taken and FACS was used to detect expression of the CAR molecules on the surface of the T cells.
Identification of FLT3-NKG2D-CAR-T cells and detection of killing Effect
(1) T cells transfected with FLT3-NKG2D-CAR lentiviral vector were observed under a fluorescence microscope, and successfully transfected CAR-T cells expressed GFP (green fluorescent protein) due to the carried lentiviral vector and fluorescence was observed under a fluorescence microscope. T cell fluorescence observations of FLT3-NKG2D-CAR lentiviral vector are shown in FIG. 4.
(2) The LDH release assay detects the killing activity of FLT3-NKG2D-CAR-T cells on leukemia tumor cells MV4-11, MOLM-13 and K562 before and after FLT3 inhibition drug treatment. The target cells of each treatment group were collected and divided into untreated group, drug-treated group and positive control group. The cell density of each group of target cells was adjusted by using a culture medium, 50. mu.L of the cells were added to each well in a 96-well plate, and 3 wells were provided for each group. FLT3-NKG2D-CAR-T cells are used as effector cells, and different amounts of effector cells are respectively added according to different effective target ratios (10:1 and 20:1), wherein the amount of the effector cells is 50 mu l. After the cells are incubated for 4 hours under the conventional culture condition, 50 mu L of supernatant is absorbed and added into a 96-hole flat-bottom enzyme label plate, and LDH substrate reaction solution and stop solution are added according to the kit specification. LDH values were measured using a fully automated biochemical analyzer, and the killing activity of each group of FLT3-NKG2D-CAR-T cells was calculated according to the formula in the description of the LDH assay kit, and plotted in FIGS. 5 and 6.
According to FIGS. 5 and 6, the killing sensitivity of the leukemia cells FLT3-NKG2D-CAR-T cells treated by the FLT3 inhibitor is found to be far better than that of the untreated group; for the percentage of cells remaining on CD33+ cells (i.e., leukemic tumor cells), the greater the killing, the fewer CD33+ cells remaining; FIG. 6 further demonstrates that the FLT3 inhibitor in combination with CAR T cells kills significantly better than CAR T cells alone (E: T ratio is the ratio of effector cells, FLT3-NKG2D-CAR T cells, to target cells, MV 4-11).
According to the figure 7, the Gilitinib (Giltertinib) can remarkably induce leukemia cell strains MOLM-13 and MV4-11 to express FLT3 and MICA, and can be beneficial to improving FLT3-NKG2D-CAR-T recognition target cells and reducing off-target effects of the FLT3-NKG2D-CAR-T recognition target cells. The method lays a foundation and a theoretical basis for the combination of the Gilitinib and the FLT3-NKG2D-CAR-T cells.
FIG. 8 is a diagram of the combined application of Gelitinib and FLT3-NKG2D-CAR-T, and according to the diagram in FIG. 8, Gelitinib can inhibit pathways such as PI3K/AKT/mTOR, RAS/RAF/MEK/ERK, induce apoptosis and toxicity to leukemia cells, enhance the expression of FLT3 on the surface of AML cells, induce the leukemia cells to express immune activator NKG2DLs, and promote the recognition and killing of target cells by FLT3-NKG2D-CAR-T cells.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> Zhujiang Hospital of southern medical university
<120> Dual-target FLT3-NKG2D-CAR-T in combination with Gilletinib for treating FLT3mut + acute leukemia
<130>
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 738
<212> DNA
<213> Artificial sequence
<400> 1
gatgttgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60
atctcctgca ggtctagtca gagcctcctg catagtaatg gaaacaacta tttggattgg 120
tacctgcaga agccagggca gtctccacag ctcctgatct atttgggttc taatcgggcc 180
tctggggtcc cagacagatt cagcggcagt gggtcagaca ctgatttcac actgcaaatc 240
agtagggtgg aggctgagga tgttggggtt tattactgca tgcaaggtac acaccccgcc 300
atctccttcg gccaagggac acgactggag attaaaggtg gcggaggatc tggcggaggt 360
ggaagcggcg gaggcggatc tgaggtccag ctggtgcagt ctggggctga ggtgaagaag 420
cctggggcct cagtgaaggt ttcctgcaag gcatctggat acaccttcac cagctactat 480
atgcactggg tgcgacaggc ccctggacaa gggcttgagt ggatgggaat aatcaaccct 540
agtggtggta gcacaagcta cgcacagaag ttccagggca gagtcaccat gaccagggac 600
acgtccacga gcacagtcta catggagctg agcagcctga gatctgagga cacggccgtg 660
tattactgtg cgaggggagt gggagcgcat gatgcttttg atatctgggg ccaagggacc 720
acggtcaccg tctcaagc 738

Claims (1)

1. Preparation of FLT3 by combining double-target CAR-T cells with FLT3 inhibitormut+The application of the medicine for treating acute myeloid leukemia; the FLT3 inhibitor is gillitinib,
the double-target CAR-T cell expresses FLT3 single-chain antibody and NKG2D receptor gene sequences at the same time,
the CAR-T cell structure comprises a signal peptide, an antigen recognition domain, a spacer region, a CD8 hinge region, a transmembrane region and an intracellular signal region, wherein the antigen recognition domain is a FLT3 single-chain antibody and an NKG2D extracellular region,
the signal peptide is selected from a CD8SP signal peptide; the spacer is T2A; the intracellular signal area is an intracellular activation signal area, CD137(4-1BB) and CD3 zeta are used as costimulatory molecules,
the FLT3 single-chain antibody has the gene sequence of FLT3 EB10 SCFV, and the gene sequence is as follows:
GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGAAACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCTGGGGTCCCAGACAGATTCAGCGGCAGTGGGTCAGACACTGATTTCACACTGCAAATCAGTAGGGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTACACACCCCGCCATCTCCTTCGGCCAAGGGACACGACTGGAGATTAAAGGTGGCGGAGGATCTGGCGGAGGTGGAAGCGGCGGAGGCGGATCTGAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTAGTGGTGGTAGCACAAGCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGGGGAGTGGGAGCGCATGATGCTTTTGATATCTGGGGCCAAGGGACCACGGTCACCGTCTCAAGC。
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