CN111808200A

CN111808200A - CD19 and CD22 double-target chimeric antigen receptor and application thereof

Info

Publication number: CN111808200A
Application number: CN202010687675.3A
Authority: CN
Inventors: 秦乐; 汤朝阳; 邓殷建; 魏志辉; 其他发明人请求不公开姓名
Original assignee: Guangdong Zhaotai In Vivo Biomedical Technology Co ltd
Current assignee: Guangdong Zhaotai In Vivo Biomedical Technology Co ltd
Priority date: 2020-07-16
Filing date: 2020-07-16
Publication date: 2020-10-23
Anticipated expiration: 2040-07-16
Also published as: CN111808200B; WO2022011846A1

Abstract

The invention provides a CD19 and CD22 dual-target chimeric antigen receptor and application thereof, wherein the chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain and a signal transduction domain; the antigen binding domain includes the light chain variable region of an anti-CD 19 single chain antibody, the heavy chain variable region of an anti-CD 19 single chain antibody, the light chain variable region of an anti-CD 22 single chain antibody, and the heavy chain variable region of an anti-CD 22 single chain antibody. The anti-CD 19 and CD22 double-target chimeric antigen receptor has targeting activity on CD19 positive and/or CD22 positive cells, and T cells expressing the anti-CD 19 and CD22 double-target chimeric antigen receptor have killing effects on tumor cells with low or no expression of CD19 antigen and tumor cells with low or no expression of CD22 antigen, so that the immune escape phenomenon is avoided, and the possibility of disease relapse is reduced.

Description

CD19 and CD22 double-target chimeric antigen receptor and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and relates to a CD19 and CD22 double-target chimeric antigen receptor and application thereof.

Background

A Chimeric Antigen Receptor (CAR) consists of a tumor-associated antigen binding region, an extracellular hinge region, a transmembrane region, and an intracellular signaling region. Generally, CAR molecules comprise an antibody single chain fragment variable (scFv) with specific binding for a Tumor Associated Antigen (TAA), which is coupled to the cytoplasmic domain of a signaling molecule via a hinge and a transmembrane region.

In recent years, with the development of tumor immunology theory and clinical technology, Chimeric antigen receptor T-cell immunotherapy (CAR-T) has become one of the most promising tumor immunotherapy. At present, CAR-T cell therapy has been widely applied to the treatment of B cell malignant tumors, and CAR-T cells targeting CD19 are precursors for the treatment of B cell malignant tumors by CAR-T therapy, and provide an effective scheme for the treatment of B cell malignant tumors. However, medical diagnosis shows that tumor cells in some blood tumors do not express CD19 molecules, but express CD22 molecules, and the CAR-T cells only targeting CD19 molecules have poor treatment effect, and the tumor recurrence phenomenon appears in some patients after a period of time.

Therefore, there is a need to develop chimeric antigen receptors that target both CD19 and CD22 molecules, improving the targeting and clearance of CAR-T cells to tumor cells.

Disclosure of Invention

Aiming at the defects and practical requirements of the prior art, the invention provides the CD19 and CD22 double-target chimeric antigen receptor and the application thereof, wherein the chimeric antigen receptor can simultaneously target CD19 and CD22 molecules and has wide prospects in the aspect of treating hematological malignant diseases.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a chimeric antigen receptor comprising an antigen binding domain, a transmembrane domain, and a signaling domain;

the antigen binding domain includes the light chain variable region of an anti-CD 19 single chain antibody, the heavy chain variable region of an anti-CD 19 single chain antibody, the light chain variable region of an anti-CD 22 single chain antibody, and the heavy chain variable region of an anti-CD 22 single chain antibody.

Compared with a single-target chimeric antigen receptor, the anti-CD 19 and anti-CD 22 double-target chimeric antigen receptor has stronger targeting activity on CD19 positive and/or CD22 positive cells, has efficient targeting effect on tumor cells with little or no expression of CD19 antigen and tumor cells with little or no expression of CD22 antigen, and is favorable for avoiding the immune escape phenomenon.

Preferably, the antigen binding domain comprises a light chain variable region of an anti-CD 19 single chain antibody, a heavy chain variable region of an anti-CD 19 single chain antibody, a light chain variable region of an anti-CD 22 single chain antibody and a heavy chain variable region of an anti-CD 22 single chain antibody, connected in series in that order.

Preferably, the antigen binding domain comprises a light chain variable region of an anti-CD 19 single chain antibody, a heavy chain variable region of an anti-CD 19 single chain antibody, a heavy chain variable region of an anti-CD 22 single chain antibody and a light chain variable region of an anti-CD 22 single chain antibody, connected in series in that order.

Preferably, the antigen binding domain comprises a light chain variable region of an anti-CD 19 single chain antibody, a light chain variable region of an anti-CD 22 single chain antibody, a heavy chain variable region of an anti-CD 19 single chain antibody and a heavy chain variable region of an anti-CD 22 single chain antibody, connected in series in that order.

Preferably, the antigen binding domain comprises a light chain variable region of an anti-CD 19 single chain antibody, a light chain variable region of an anti-CD 22 single chain antibody, a heavy chain variable region of an anti-CD 22 single chain antibody and a heavy chain variable region of an anti-CD 19 single chain antibody, connected in series in that order.

Preferably, the antigen binding domain comprises a light chain variable region of an anti-CD 19 single chain antibody, a heavy chain variable region of an anti-CD 22 single chain antibody, a heavy chain variable region of an anti-CD 19 single chain antibody and a light chain variable region of an anti-CD 22 single chain antibody, connected in series in that order.

Preferably, the antigen binding domain comprises a light chain variable region of an anti-CD 19 single chain antibody, a heavy chain variable region of an anti-CD 22 single chain antibody, a light chain variable region of an anti-CD 22 single chain antibody and a heavy chain variable region of an anti-CD 19 single chain antibody, connected in series in that order.

Preferably, the antigen binding domain comprises a heavy chain variable region of an anti-CD 19 single chain antibody, a light chain variable region of an anti-CD 19 single chain antibody, a light chain variable region of an anti-CD 22 single chain antibody and a heavy chain variable region of an anti-CD 22 single chain antibody, connected in series in that order.

Preferably, the antigen binding domain comprises a heavy chain variable region of an anti-CD 19 single chain antibody, a light chain variable region of an anti-CD 19 single chain antibody, a heavy chain variable region of an anti-CD 22 single chain antibody and a light chain variable region of an anti-CD 22 single chain antibody, connected in series in that order.

Preferably, the antigen binding domain comprises a heavy chain variable region of an anti-CD 19 single chain antibody, a light chain variable region of an anti-CD 22 single chain antibody, a light chain variable region of an anti-CD 19 single chain antibody and a heavy chain variable region of an anti-CD 22 single chain antibody, connected in series in that order.

Preferably, the antigen binding domain comprises a heavy chain variable region of an anti-CD 19 single chain antibody, a light chain variable region of an anti-CD 22 single chain antibody, a heavy chain variable region of an anti-CD 22 single chain antibody and a light chain variable region of an anti-CD 19 single chain antibody, connected in series in that order.

Preferably, the antigen binding domain comprises a heavy chain variable region of an anti-CD 19 single chain antibody, a heavy chain variable region of an anti-CD 22 single chain antibody, a light chain variable region of an anti-CD 19 single chain antibody and a light chain variable region of an anti-CD 22 single chain antibody, connected in series in that order.

Preferably, the antigen binding domain comprises a heavy chain variable region of an anti-CD 19 single chain antibody, a heavy chain variable region of an anti-CD 22 single chain antibody, a light chain variable region of an anti-CD 22 single chain antibody and a light chain variable region of an anti-CD 19 single chain antibody, connected in series in that order.

Preferably, the antigen binding domain comprises a light chain variable region of an anti-CD 22 single chain antibody, a light chain variable region of an anti-CD 19 single chain antibody, a heavy chain variable region of an anti-CD 19 single chain antibody and a heavy chain variable region of an anti-CD 22 single chain antibody, connected in series in that order.

Preferably, the antigen binding domain comprises a light chain variable region of an anti-CD 22 single chain antibody, a light chain variable region of an anti-CD 19 single chain antibody, a heavy chain variable region of an anti-CD 22 single chain antibody and a heavy chain variable region of an anti-CD 19 single chain antibody, connected in series in that order.

Preferably, the antigen binding domain comprises a light chain variable region of an anti-CD 22 single chain antibody, a heavy chain variable region of an anti-CD 19 single chain antibody, a light chain variable region of an anti-CD 19 single chain antibody and a heavy chain variable region of an anti-CD 22 single chain antibody, connected in series in that order.

Preferably, the antigen binding domain comprises a light chain variable region of an anti-CD 22 single chain antibody, a heavy chain variable region of an anti-CD 19 single chain antibody, a heavy chain variable region of an anti-CD 22 single chain antibody and a light chain variable region of an anti-CD 19 single chain antibody, connected in series in that order.

Preferably, the antigen binding domain comprises a light chain variable region of an anti-CD 22 single chain antibody, a heavy chain variable region of an anti-CD 22 single chain antibody, a light chain variable region of an anti-CD 19 single chain antibody and a heavy chain variable region of an anti-CD 19 single chain antibody, connected in series in that order.

Preferably, the antigen binding domain comprises a light chain variable region of an anti-CD 22 single chain antibody, a heavy chain variable region of an anti-CD 22 single chain antibody, a heavy chain variable region of an anti-CD 19 single chain antibody and a light chain variable region of an anti-CD 19 single chain antibody, connected in series in that order.

Preferably, the antigen binding domain comprises a heavy chain variable region of an anti-CD 22 single chain antibody, a light chain variable region of an anti-CD 19 single chain antibody, a heavy chain variable region of an anti-CD 19 single chain antibody and a light chain variable region of an anti-CD 22 single chain antibody, connected in series in that order.

Preferably, the antigen binding domain comprises a heavy chain variable region of an anti-CD 22 single chain antibody, a light chain variable region of an anti-CD 19 single chain antibody, a light chain variable region of an anti-CD 22 single chain antibody and a heavy chain variable region of an anti-CD 19 single chain antibody, connected in series in that order.

Preferably, the antigen binding domain comprises a heavy chain variable region of an anti-CD 22 single chain antibody, a heavy chain variable region of an anti-CD 19 single chain antibody, a light chain variable region of an anti-CD 19 single chain antibody and a light chain variable region of an anti-CD 22 single chain antibody, connected in series in that order.

Preferably, the antigen binding domain comprises a heavy chain variable region of an anti-CD 22 single chain antibody, a heavy chain variable region of an anti-CD 19 single chain antibody, a light chain variable region of an anti-CD 22 single chain antibody and a light chain variable region of an anti-CD 19 single chain antibody, connected in series in that order.

Preferably, the antigen binding domain comprises a heavy chain variable region of an anti-CD 22 single chain antibody, a light chain variable region of an anti-CD 22 single chain antibody, a light chain variable region of an anti-CD 19 single chain antibody and a heavy chain variable region of an anti-CD 19 single chain antibody, connected in series in that order.

Preferably, the antigen binding domain comprises a heavy chain variable region of an anti-CD 22 single chain antibody, a light chain variable region of an anti-CD 22 single chain antibody, a heavy chain variable region of an anti-CD 19 single chain antibody and a light chain variable region of an anti-CD 19 single chain antibody, connected in series in that order.

Preferably, the light chain variable region of the anti-CD 19 single chain antibody comprises the amino acid sequence shown in SEQ ID NO. 1;

SEQ ID NO:1：

DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPPRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSAYTFGQGTKLEIK。

preferably, the heavy chain variable region of the anti-CD 19 single-chain antibody comprises the amino acid sequence shown in SEQ ID NO. 2;

SEQ ID NO:2：

QVQLVESGGGVVQPGRSLRLSCAASGFTFSRHGMHWVRQAPGKGLEWVAVIWYDGSNQYYVDSVKGRFTISRDNSKNTLDLQMNSLRVEDTAVYYCARRSITWYGGFDIWGQGTMVTVSSAQTTAPSVYPLAP。

preferably, the light chain variable region of the anti-CD 22 single chain antibody comprises the amino acid sequence shown in SEQ ID NO. 3;

SEQ ID NO:3：

DIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIK。

preferably, the heavy chain variable region of the anti-CD 22 single chain antibody comprises the amino acid sequence shown in SEQ ID NO. 4;

SEQ ID NO:4：

QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTVSS。

preferably, the light chain variable region of the anti-CD 19 single-chain antibody, the heavy chain variable region of the anti-CD 19 single-chain antibody, the light chain variable region of the anti-CD 22 single-chain antibody and the heavy chain variable region of the anti-CD 22 single-chain antibody are connected through a connecting peptide.

Preferably, the connecting peptide comprises an amino acid sequence shown in SEQ ID NO. 5, SEQ ID NO. 6 or SEQ ID NO. 7;

SEQ ID NO:5：GSGGGGSGGGGSGGGGS；

SEQ ID NO:6：GGGGSGGGGSGGGGSGGGGS；

SEQ ID NO:7：GSTSGSGKPGSGEGSTKG。

preferably, the transmembrane domain comprises CD28 and/or CD8 a.

Preferably, the signalling domain comprises any one of CD3 ζ, 4-1BB, CD28, TLR1, TLR2, CD27, OX40 or DAP10, or a combination of at least two.

Preferably, the chimeric antigen receptor further comprises a signal peptide.

Preferably, the signal peptide comprises a GM-CSF signal peptide.

Preferably, the GM-CSF signal peptide comprises the amino acid sequence shown in SEQ ID NO. 8;

SEQ ID NO:8：MLLLVTSLLLCELPHPAFLL。

preferably, the chimeric antigen receptor is composed of a GM-CSF signal peptide, a light chain variable region of an anti-CD 19 single chain antibody, a first linker peptide, a heavy chain variable region of an anti-CD 19 single chain antibody, a second linker peptide, a heavy chain variable region of an anti-CD 22 single chain antibody, a third linker peptide, a light chain variable region of an anti-CD 22 single chain antibody, CD28, and CD3 ζ in tandem.

In a second aspect, the present invention provides a coding gene encoding the chimeric antigen receptor of the first aspect.

Preferably, the coding genes comprise the light chain variable region coding sequence of an anti-CD 19 single chain antibody, the heavy chain variable region coding sequence of an anti-CD 19 single chain antibody, the heavy chain variable region coding sequence of an anti-CD 22 single chain antibody and the light chain variable region coding sequence of an anti-CD 22 single chain antibody.

Preferably, the coding gene further comprises a linker peptide coding sequence.

Preferably, the coding gene further comprises a coding sequence for GM-CSF signal peptide, a coding sequence for CD28, and a coding sequence for CD3 zeta.

Preferably, the light chain variable region encoding sequence of the anti-CD 19 single chain antibody comprises the nucleic acid sequence shown in SEQ ID NO. 9;

SEQ ID NO:9：

gacatccagatgacccagagccccagcaccctgagcgccagcgtgggcgaccgcgtgaccatcacctgccgcgccagccagagcatcagcagctggctggcctggtaccagcagaagcccggcaaggcccccaagctgctgatctacaaggccagcagcctggagagcggcgtgcccccccgcttcagcggcagcggcagcggcaccgagttcaccctgaccatcagcagcctgcagcccgacgacttcgccacctactactgccagcagtacaacagcgcctacaccttcggccagggcaccaagctggagatcaag。

preferably, the heavy chain variable region encoding sequence of the anti-CD 19 single chain antibody comprises the nucleic acid sequence shown in SEQ ID NO. 10;

SEQ ID NO:10：

caggtgcagctggtggagagcggcggcggcgtggtgcagcccggccgcagcctgcgcctgagctgcgccgccagcggcttcaccttcagccgccacggcatgcactgggtgcgccaggcccccggcaagggcctggagtgggtggccgtgatctggtacgacggcagcaaccagtactacgtggacagcgtgaagggccgcttcaccatcagccgcgacaacagcaagaacaccctggacctgcagatgaacagcctgcgcgtggaggacaccgccgtgtactactgcgcccgccgcagcatcacctggtacggcggcttcgacatctggggccagggcaccatggtgaccgtgagcagcgcccagaccaccgcccccagcgtgtaccccctggccccc。

preferably, the heavy chain variable region encoding sequence of the anti-CD 22 single chain antibody comprises the nucleic acid sequence shown in SEQ ID NO. 11;

SEQ ID NO:11：

caggtgcagctgcagcagtctggccctggcctcgtgaagcctagccagaccctgagcctgacctgtgccatcagcggcgatagcgtgtccagcaatagcgccgcctggaactggatcagacagagccctagcagaggcctggaatggctgggccggacctactaccggtccaagtggtacaacgactacgccgtgtccgtgaagtcccggatcaccatcaaccccgacaccagcaagaaccagttctccctgcagctgaacagcgtgacccccgaggataccgccgtgtactactgcgccagagaagtgaccggcgacctggaagatgccttcgacatctggggccagggcacaatggtcaccgtgtctagc。

preferably, the light chain variable region encoding sequence of the anti-CD 22 single chain antibody comprises the nucleic acid sequence shown in SEQ ID NO. 12;

SEQ ID NO:12：

gacatccagatgacacagagccccagctccctgagcgccagcgtgggagacagagtgaccatcacctgtcgggccagccagaccatctggtcctacctgaactggtatcagcagcggcctggcaaggcccccaacctgctgatctatgccgccagctcactgcagagcggcgtgcccagcagattttccggcagaggcagcggcaccgacttcaccctgacaatcagttccctgcaggccgaggacttcgccacctactactgccagcagagctacagcatcccccagaccttcggccaggggaccaagctggaaatcaaa。

preferably, the linker peptide coding sequence comprises the nucleic acid sequence shown in SEQ ID NO 13, SEQ ID NO 14 or SEQ ID NO 15;

SEQ ID NO:13：

ggatccggtggcggtggcagcggcggtggtggttccggaggcggcggttct；

SEQ ID NO:14：

ggtggaggcggcagtggcggaggtgggagcggagggggcggttccggtggcgggggatct；

SEQ ID NO:15：

ggctccacctctggatccggcaagcccggatctggcgagggatccaccaagggc。

preferably, the GM-CSF signal peptide coding sequence comprises the nucleic acid sequence shown in SEQ ID NO. 16;

SEQ ID NO:16：

atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctg。

in a third aspect, the present invention provides an expression vector comprising the coding gene of the second aspect.

Preferably, the expression vector is a viral vector comprising the encoding gene of the second aspect.

Preferably, the expression vector is any one of a lentiviral vector, a retroviral vector or an adeno-associated viral vector containing the coding gene of the second aspect.

In a fourth aspect, the present invention provides a recombinant lentivirus which is a mammalian cell transfected with an expression vector and a helper plasmid as described in the third aspect.

In a fifth aspect, the invention provides a CAR-T cell expressing the chimeric antigen receptor of the first aspect.

In the invention, the T cells expressing anti-CD 19 and CD22 double-target chimeric antigen receptors have high-efficiency targeting activity and killing efficacy on CD19 positive and/or CD22 positive cells, and have killing effects on tumor cells with little or no expression of CD19 antigen and tumor cells with little or no expression of CD22 antigen, thereby being beneficial to avoiding the immune escape phenomenon and reducing the possibility of disease relapse.

Preferably, the CAR-T cell has integrated into its genome the gene encoding the second aspect.

Preferably, the CAR-T cell comprises the expression vector of the third aspect and/or the recombinant lentivirus of the fourth aspect.

In a sixth aspect, the present invention provides a method of producing a CAR-T cell according to the fifth aspect, the method comprising the step of introducing into a T cell a gene encoding the chimeric antigen receptor according to the first aspect.

In a seventh aspect, the present invention provides a chimeric antigen receptor of the first aspect, a coding gene of the second aspect, an expression vector of the third aspect, a recombinant lentivirus of the fourth aspect, or a CAR-T cell of the fifth aspect, for use in the preparation of a medicament for the treatment of a disease.

Preferably, the disease comprises a hematological tumor.

Preferably, the disease comprises a CD19 positive and/or CD22 positive disease.

Compared with the prior art, the invention has the following beneficial effects:

(1) compared with a single-target chimeric antigen receptor, the anti-CD 19 and anti-CD 22 double-target chimeric antigen receptor has stronger targeting activity on CD19 positive and/or CD22 positive cells, has efficient targeting effect on tumor cells with little or no expression of CD19 antigen and tumor cells with little or no expression of CD22 antigen, and is favorable for avoiding the immune escape phenomenon;

(2) the T cell expressing the anti-CD 19 and CD22 double-target chimeric antigen receptor has high-efficiency targeting activity and killing effect on CD19 positive cells and/or CD22 positive cells, has killing effect on tumor cells with small or non-expressed CD19 antigen expression quantity and tumor cells with small or non-expressed CD22 antigen expression quantity, is favorable for avoiding immune escape phenomenon, and reduces the possibility of disease relapse.

Drawings

FIG. 1 is a lentiviral vector map containing the genes encoding a CAR targeting both CD19 and CD22 targets;

FIG. 2A is a graph of the flow results for WT and 2228zT2, and FIG. 2B is a graph of the flow results for 1928zT2, 31A2 and 11H8.22.28. zGFP;

FIG. 3 shows the killing efficiency of WT, 2228zT2 and 11H8.22.28.zGFP on tumor cells K52-CD22-GL at different E: T ratios;

FIG. 4 shows the killing efficiency of WT, 31A2, 1928zT2 and 11H8.22.28.zGFP on tumor cells K52-CD19-GL at different E: T ratios.

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

Example 1 construction of CAR molecular vectors

In the embodiment, firstly, a coding gene of anti-CD 19 and CD22 double-target chimeric antigen receptor is synthesized by gene, and a restriction enzyme Pme1 restriction site and a protective base thereof, and a restriction enzyme Spe1 restriction enzyme site and a protective base thereof are respectively added at the C end and the N end of the coding gene;

the coding gene is subjected to double enzyme digestion by restriction enzymes Pme1 and Spe1, an enzyme digestion product containing a sticky end is obtained by agar gel electrophoresis recovery, the enzyme digestion product is connected into a linearized pWPXld-eGFP plasmid (containing the sticky end) which is also subjected to double enzyme digestion by Pme1 and Spe1, a connection reaction is carried out in the presence of T4 DNA polymerase (Invitrogen company), and a lentiviral vector containing the coding gene of the CAR targeting CD19 and CD22 double targets is obtained, and the map is shown in FIG. 1.

In this example, the antigen binding domains of CAR (anti CD19scFv-CD28-CD3 zeta) and CAR (anti CD22 scFv-CD28-CD3 zeta) against CD19scFv and CD22 scFv were constructed simultaneously, and corresponding lentiviral vectors were constructed.

Example 2 Lentiviral packaging

In order to introduce CAR molecules into T cells, recombinant lentiviruses were prepared using 293T cells, and lentivirus packaging was performed when 293T cells were plated at 80-90% on 100mm plates:

2h before virus packaging, the culture medium is replaced by DMEM containing 1% fetal calf serum, and the addition amount is 6mL/100mm culture dish;

preparing a plasmid mixed solution as shown in table 1, wherein the pWPXld-expression plasmid comprises a lentiviral vector containing a coding gene of a CAR targeting CD19 and CD22 double targets, a lentiviral vector containing a coding gene of a CAR targeting CD19 single target, and a lentiviral vector containing a coding gene of a CAR targeting CD22 single target, and the pWPXld-eGFP plasmid is an empty vector containing no CAR coding gene;

TABLE 1

Adding 36 μ g PEI into another 500 μ L opti-MEM medium, mixing, and standing at room temperature for 5 min;

mixing the pWPXld-expression plasmid or pWPXld-eGFP plasmid with PEI, blowing and uniformly mixing, and standing for 25-30 min at room temperature;

dropwise adding the mixed solution to 293T cells cultured in a 100mm culture dish;

after culturing for 6h, changing the culture medium into DMEM containing 1% fetal calf serum, and adding the DMEM into a culture dish with the volume of 7mL/100 mm;

collecting virus supernatant 24h, 48h and 72h after packaging, and simultaneously supplementing a culture medium to 293T cells, wherein the addition amount is 7mL/100mm culture dish;

centrifugation at 1000g for 10min, filtration through a 0.45 μm filter, to obtain recombinant lentivirus expressing CAR or a blank control eGFP lentivirus, and storage at 4 ℃ for future use.

Example 3T cell activation and lentivirus transfection

Separating Peripheral Blood Mononuclear Cells (PBMC) from whole blood by using a Ficoll density gradient centrifugation kit (GE company), removing red blood cells, and then separating T cells by using MACS Pan-T magnetic beads;

the separated T cells were diluted with a medium (AIM-V medium + 5% FBS + penicillin 100U/mL + streptomycin 0.1mg/mL) to a cell concentration of 2.5X 10⁶Per mL for standby;

activating T cells by using CD2/CD3/CD 28T cell activation and expansion kit (Meitian whirlwind company), namely mixing coated magnetic beads with T cells at a ratio of 1:2, and finally, the density of the T cells is 5 x 10⁶Per mL/cm²Mixing, and standing at 37 deg.C and 5% CO₂Culturing and stimulating for 48h in an incubator;

after 48h of T cell activation, beads were removed, 300g was centrifuged for 5min, supernatant was removed, the T cells were resuspended in fresh medium, CAR-expressing recombinant lentivirus or blank control eGFP lentivirus (MOI 10) was added, and 8. mu.g/mL polybrene and 300IU/mL IL-2 were added, placed at 37 ℃ and 5% CO₂Culturing in an incubator;

after 24h, centrifuging for 5min at 300g, removing supernatant, and resuspending T cells in fresh culture medium containing 300IU/mL IL-2 to obtain CAR-T cells;

maintenance of CAR-T cell density at 1X 10⁶About one/mL, half the amount is carried out every 2-3 daysTwo weeks after fluid change, CAR-T cell numbers expanded 100-fold.

CAR-T cells constructed in this example were 11h8.22.28 zgfp (expressing anti-CD 19 and CD22 dual-target CARs), 31a2 (expressing anti-murine CD19 single-target CAR), 1928zT2 (expressing anti-murine CD19 single-target CAR), 2228zT2 (expressing anti-murine CD22 single-target CAR), and a WT control group (transfection blank control eGFP lentivirus) was set.

Lentiviral transfection conditions are shown in Table 1.

TABLE 1

Example 4 expression of CAR molecules by T cells

Since the lentiviral vector expressing the CAR molecule carries the eGFP gene, the expression of the CAR molecule on the T cell can be indicated by eGFP, and the eGFP on the T cell is detected in this example by flow cytometry, acearocyte.

As shown in fig. 2A and 2B, which are flow-through results of the experimental group and the different control groups, it can be seen that the transfection efficiency of lentiviruses to WT cells is 0.72%, the transfection efficiency to 2228zT2 is 3.71%, the transfection efficiency to 1928zT2 is 18.5%, the transfection efficiency to 31a2 is 7.77%, and the transfection efficiency to 11h8.22.28 zgfp is 9.79%.

Example 5 in vitro testing of the killing function of CAR-T cells against tumor cells K52-CD22-GL

WT prepared in example 3, 2228zT2 and 11H8.22.28.zGFP were mixed with 1X 10 of each⁴Mixing tumor cells K52-CD22-GL at E: T ratio of 4:1, 2:1, 1:2, 1:4, 1:8, 1:16, adding into 96-well plate, setting 3 multiple wells in each group, centrifuging for 5min at 250g, standing at 37 deg.C and 5% CO₂Co-culturing for 18h in an incubator;

after 18h, 100. mu.L/well Luciferase substrate (1X) was added to a 96-well plate, the cells were resuspended and mixed well, RLU (relative light unit) was immediately measured by a multifunctional microplate reader for 1 second, and the killing effect of WT, 2228zT2 and 11H8.22.28.zGFP on K52-CD22-GL was compared in vitro by the Luciferase (Luciferase) quantitative killing efficiency assessment method, and the killing ratio was calculated as follows:

100% × (control well reading-experimental well reading)/control well reading (blank reading without cells negligible)

As shown in FIG. 3, the in vitro killing efficiency of 11H8.22.28.zGFP on K52-CD22-GL is significantly higher than that of WT and 2228zT2, and under the condition of small E: T, namely the tumor target cells are far larger than effector T cells, the 11H8.22.28.zGFP can also show strong tumor killing activity.

Example 6 in vitro testing of the killing function of CAR-T cells against tumor cells K52-CD19-GL

WT prepared in example 3, 31A2, 1928zT2 and 11H8.22.28.zGFP were mixed with 1X 10⁴Mixing tumor cells K52-CD19-GL at E: T ratio of 8:1, 4:1, 2:1, 1:2, 1:4, 1:8, adding into 96-well plate, setting 3 multiple wells in each group, centrifuging for 5min at 250g, standing at 37 deg.C and 5% CO₂Co-culturing for 18h in an incubator;

after 18h, 100. mu.L/well Luciferase substrate (1X) was added to the 96-well plate, the cells were resuspended and mixed well, RLU (relative light unit) was immediately measured by a multifunctional microplate reader for 1 second, and the killing effect of WT, 31A2, 1928zT2 and 11H8.22.28.zGFP on K52-CD19-GL was compared in vitro by the Luciferase (Luciferase) quantitative killing efficiency assessment method, and the killing ratio was calculated as follows:

The results are shown in FIG. 4, where killing efficiency of 1928zT2 and 11H8.22.28.zGFP on K52-CD19-GL in vitro is significantly higher than that of WT and 31A 2.

In conclusion, the anti-CD 19 and CD22 double-target chimeric antigen receptor constructed by the invention has targeting activity on CD19 positive and/or CD22 positive cells, and T cells expressing the anti-CD 19 and CD22 double-target chimeric antigen receptor have killing effects on tumor cells with low or no expression of CD19 antigen and tumor cells with low or no expression of CD22 antigen, so that the immune escape phenomenon is avoided, and the possibility of disease relapse is reduced.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

SEQUENCE LISTING

<110> Guangdong Shoutai biomedical science and technology Co., Ltd

<120> CD19 and CD22 dual-target chimeric antigen receptor and application thereof

<130>20200714

<160>16

<170>PatentIn version 3.3

<210>1

<211>106

<212>PRT

<213> Artificial sequence

<400>1

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Pro Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Ala Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>2

<211>133

<212>PRT

<213> Artificial sequence

<400>2

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg His

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Gln Tyr Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Asp

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ser Ile Thr Trp Tyr Gly Gly Phe Asp Ile Trp Gly Gln

100 105 110

Gly Thr Met Val Thr Val Ser Ser Ala Gln Thr Thr Ala Pro Ser Val

115 120 125

Tyr Pro Leu Ala Pro

130

<210>3

<211>107

<212>PRT

<213> Artificial sequence

<400>3

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Trp Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Asn Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Arg Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ile Pro Gln

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>4

<211>124

<212>PRT

<213> Artificial sequence

<400>4

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp ThrSer Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Glu Val Thr Gly Asp Leu Glu Asp Ala Phe Asp

100 105 110

Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210>5

<211>17

<212>PRT

<213> Artificial sequence

<400>5

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

1 5 10 15

Ser

<210>6

<211>20

<212>PRT

<213> Artificial sequence

<400>6

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210>7

<211>18

<212>PRT

<213> Artificial sequence

<400>7

Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr

1 5 10 15

Lys Gly

<210>8

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<212>PRT

<213> Artificial sequence

<400>8

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu

20

<210>9

<211>318

<212>DNA

<213> Artificial sequence

<400>9

gacatccaga tgacccagag ccccagcacc ctgagcgcca gcgtgggcga ccgcgtgacc 60

atcacctgcc gcgccagcca gagcatcagc agctggctgg cctggtacca gcagaagccc 120

ggcaaggccc ccaagctgct gatctacaag gccagcagcc tggagagcgg cgtgcccccc 180

cgcttcagcg gcagcggcag cggcaccgag ttcaccctga ccatcagcag cctgcagccc 240

gacgacttcg ccacctacta ctgccagcag tacaacagcg cctacacctt cggccagggc 300

accaagctgg agatcaag 318

<210>10

<211>399

<212>DNA

<213> Artificial sequence

<400>10

caggtgcagc tggtggagag cggcggcggc gtggtgcagc ccggccgcag cctgcgcctg 60

agctgcgccg ccagcggctt caccttcagc cgccacggca tgcactgggt gcgccaggcc 120

cccggcaagg gcctggagtg ggtggccgtg atctggtacg acggcagcaa ccagtactac 180

gtggacagcg tgaagggccg cttcaccatc agccgcgaca acagcaagaa caccctggac 240

ctgcagatga acagcctgcg cgtggaggac accgccgtgt actactgcgc ccgccgcagc 300

atcacctggt acggcggctt cgacatctgg ggccagggca ccatggtgac cgtgagcagc 360

gcccagacca ccgcccccag cgtgtacccc ctggccccc 399

<210>11

<211>372

<212>DNA

<213> Artificial sequence

<400>11

caggtgcagc tgcagcagtc tggccctggc ctcgtgaagc ctagccagac cctgagcctg 60

acctgtgcca tcagcggcga tagcgtgtcc agcaatagcg ccgcctggaa ctggatcaga 120

cagagcccta gcagaggcct ggaatggctg ggccggacct actaccggtc caagtggtac 180

aacgactacg ccgtgtccgt gaagtcccgg atcaccatca accccgacac cagcaagaac 240

cagttctccc tgcagctgaa cagcgtgacc cccgaggata ccgccgtgta ctactgcgcc 300

agagaagtga ccggcgacct ggaagatgcc ttcgacatct ggggccaggg cacaatggtc 360

accgtgtcta gc 372

<210>12

<211>321

<212>DNA

<213> Artificial sequence

<400>12

gacatccaga tgacacagag ccccagctcc ctgagcgcca gcgtgggaga cagagtgacc 60

atcacctgtc gggccagcca gaccatctgg tcctacctga actggtatca gcagcggcct 120

ggcaaggccc ccaacctgct gatctatgcc gccagctcac tgcagagcgg cgtgcccagc 180

agattttccg gcagaggcag cggcaccgac ttcaccctga caatcagttc cctgcaggcc 240

gaggacttcg ccacctacta ctgccagcag agctacagca tcccccagac cttcggccag 300

gggaccaagc tggaaatcaa a 321

<210>13

<211>51

<212>DNA

<213> Artificial sequence

<400>13

ggatccggtg gcggtggcag cggcggtggt ggttccggag gcggcggttc t 51

<210>14

<211>60

<212>DNA

<213> Artificial sequence

<400>14

ggtggaggcg gcagtggcgg aggtgggagc ggagggggcg gttccggtgg cgggggatct 60

<210>15

<211>54

<212>DNA

<213> Artificial sequence

<400>15

ggctccacct ctggatccgg caagcccgga tctggcgagg gatccaccaa gggc 54

<210>16

<211>60

<212>DNA

<213> Artificial sequence

<400>16

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

Claims

1. A chimeric antigen receptor, comprising an antigen binding domain, a transmembrane domain, and a signaling domain;

2. The chimeric antigen receptor according to claim 1, wherein the antigen-binding domain comprises, in series, the light chain variable region of an anti-CD 19 single-chain antibody, the heavy chain variable region of an anti-CD 19 single-chain antibody, the light chain variable region of an anti-CD 22 single-chain antibody, and the heavy chain variable region of an anti-CD 22 single-chain antibody;

preferably, the antigen binding domain comprises a light chain variable region of an anti-CD 19 single chain antibody, a heavy chain variable region of an anti-CD 19 single chain antibody, a heavy chain variable region of an anti-CD 22 single chain antibody and a light chain variable region of an anti-CD 22 single chain antibody, connected in series in this order;

preferably, the antigen binding domain comprises a light chain variable region of an anti-CD 19 single chain antibody, a light chain variable region of an anti-CD 22 single chain antibody, a heavy chain variable region of an anti-CD 19 single chain antibody and a heavy chain variable region of an anti-CD 22 single chain antibody, connected in series in that order;

preferably, the antigen binding domain comprises a light chain variable region of an anti-CD 19 single chain antibody, a light chain variable region of an anti-CD 22 single chain antibody, a heavy chain variable region of an anti-CD 22 single chain antibody and a heavy chain variable region of an anti-CD 19 single chain antibody, connected in series in that order;

preferably, the antigen binding domain comprises a light chain variable region of an anti-CD 19 single chain antibody, a heavy chain variable region of an anti-CD 22 single chain antibody, a heavy chain variable region of an anti-CD 19 single chain antibody and a light chain variable region of an anti-CD 22 single chain antibody, connected in series in this order;

preferably, the antigen binding domain comprises a light chain variable region of an anti-CD 19 single chain antibody, a heavy chain variable region of an anti-CD 22 single chain antibody, a light chain variable region of an anti-CD 22 single chain antibody and a heavy chain variable region of an anti-CD 19 single chain antibody, connected in series in this order;

preferably, the antigen binding domain comprises a heavy chain variable region of an anti-CD 19 single chain antibody, a light chain variable region of an anti-CD 19 single chain antibody, a light chain variable region of an anti-CD 22 single chain antibody and a heavy chain variable region of an anti-CD 22 single chain antibody, connected in series in that order;

preferably, the antigen binding domain comprises a heavy chain variable region of an anti-CD 19 single chain antibody, a light chain variable region of an anti-CD 19 single chain antibody, a heavy chain variable region of an anti-CD 22 single chain antibody and a light chain variable region of an anti-CD 22 single chain antibody, connected in series in this order;

preferably, the antigen binding domain comprises a heavy chain variable region of an anti-CD 19 single chain antibody, a light chain variable region of an anti-CD 22 single chain antibody, a light chain variable region of an anti-CD 19 single chain antibody and a heavy chain variable region of an anti-CD 22 single chain antibody, connected in series in that order;

preferably, the antigen binding domain comprises a heavy chain variable region of an anti-CD 19 single chain antibody, a light chain variable region of an anti-CD 22 single chain antibody, a heavy chain variable region of an anti-CD 22 single chain antibody and a light chain variable region of an anti-CD 19 single chain antibody, connected in series in this order;

preferably, the antigen binding domain comprises a heavy chain variable region of an anti-CD 19 single chain antibody, a heavy chain variable region of an anti-CD 22 single chain antibody, a light chain variable region of an anti-CD 19 single chain antibody and a light chain variable region of an anti-CD 22 single chain antibody, connected in series in that order;

preferably, the antigen binding domain comprises a heavy chain variable region of an anti-CD 19 single chain antibody, a heavy chain variable region of an anti-CD 22 single chain antibody, a light chain variable region of an anti-CD 22 single chain antibody and a light chain variable region of an anti-CD 19 single chain antibody, connected in series in that order;

preferably, the antigen binding domain comprises a light chain variable region of an anti-CD 22 single chain antibody, a light chain variable region of an anti-CD 19 single chain antibody, a heavy chain variable region of an anti-CD 19 single chain antibody and a heavy chain variable region of an anti-CD 22 single chain antibody, connected in series in that order;

preferably, the antigen binding domain comprises a light chain variable region of an anti-CD 22 single chain antibody, a light chain variable region of an anti-CD 19 single chain antibody, a heavy chain variable region of an anti-CD 22 single chain antibody and a heavy chain variable region of an anti-CD 19 single chain antibody, connected in series in that order;

preferably, the antigen binding domain comprises a light chain variable region of an anti-CD 22 single chain antibody, a heavy chain variable region of an anti-CD 19 single chain antibody, a light chain variable region of an anti-CD 19 single chain antibody and a heavy chain variable region of an anti-CD 22 single chain antibody, connected in series in this order;

preferably, the antigen binding domain comprises a light chain variable region of an anti-CD 22 single chain antibody, a heavy chain variable region of an anti-CD 19 single chain antibody, a heavy chain variable region of an anti-CD 22 single chain antibody and a light chain variable region of an anti-CD 19 single chain antibody, connected in series in this order;

preferably, the antigen binding domain comprises a light chain variable region of an anti-CD 22 single chain antibody, a heavy chain variable region of an anti-CD 22 single chain antibody, a light chain variable region of an anti-CD 19 single chain antibody and a heavy chain variable region of an anti-CD 19 single chain antibody, connected in series in this order;

preferably, the antigen binding domain comprises a light chain variable region of an anti-CD 22 single chain antibody, a heavy chain variable region of an anti-CD 22 single chain antibody, a heavy chain variable region of an anti-CD 19 single chain antibody and a light chain variable region of an anti-CD 19 single chain antibody, connected in series in this order;

preferably, the antigen binding domain comprises a heavy chain variable region of an anti-CD 22 single chain antibody, a light chain variable region of an anti-CD 19 single chain antibody, a heavy chain variable region of an anti-CD 19 single chain antibody and a light chain variable region of an anti-CD 22 single chain antibody, connected in series in this order;

preferably, the antigen binding domain comprises a heavy chain variable region of an anti-CD 22 single chain antibody, a light chain variable region of an anti-CD 19 single chain antibody, a light chain variable region of an anti-CD 22 single chain antibody and a heavy chain variable region of an anti-CD 19 single chain antibody, connected in series in that order;

preferably, the antigen binding domain comprises a heavy chain variable region of an anti-CD 22 single chain antibody, a heavy chain variable region of an anti-CD 19 single chain antibody, a light chain variable region of an anti-CD 19 single chain antibody and a light chain variable region of an anti-CD 22 single chain antibody, connected in series in that order;

preferably, the antigen binding domain comprises a heavy chain variable region of an anti-CD 22 single chain antibody, a heavy chain variable region of an anti-CD 19 single chain antibody, a light chain variable region of an anti-CD 22 single chain antibody and a light chain variable region of an anti-CD 19 single chain antibody, connected in series in that order;

preferably, the antigen binding domain comprises a heavy chain variable region of an anti-CD 22 single chain antibody, a light chain variable region of an anti-CD 22 single chain antibody, a light chain variable region of an anti-CD 19 single chain antibody and a heavy chain variable region of an anti-CD 19 single chain antibody, connected in series in that order;

preferably, the antigen binding domain comprises a heavy chain variable region of an anti-CD 22 single chain antibody, a light chain variable region of an anti-CD 22 single chain antibody, a heavy chain variable region of an anti-CD 19 single chain antibody and a light chain variable region of an anti-CD 19 single chain antibody, connected in series in this order;

preferably, the heavy chain variable region of the anti-CD 22 single chain antibody comprises the amino acid sequence shown in SEQ ID NO. 4.

3. The chimeric antigen receptor according to claim 1 or 2, wherein the light chain variable region of the anti-CD 19 single-chain antibody, the heavy chain variable region of the anti-CD 19 single-chain antibody, the light chain variable region of the anti-CD 22 single-chain antibody and the heavy chain variable region of the anti-CD 22 single-chain antibody are linked by a connecting peptide;

preferably, the connecting peptide comprises an amino acid sequence shown in SEQ ID NO. 5, SEQ ID NO. 6 or SEQ ID NO. 7.

4. The chimeric antigen receptor according to any one of claims 1-3, wherein the transmembrane domain comprises CD28 and/or CD8 a;

preferably, the signalling domain comprises any one of CD3 ζ, 4-1BB, CD28, TLR1, TLR2, CD27, OX40 or DAP10, or a combination of at least two;

preferably, the chimeric antigen receptor further comprises a signal peptide;

preferably, the signal peptide comprises a GM-CSF signal peptide;

5. A coding gene encoding the chimeric antigen receptor of any one of claims 1 to 4;

preferably, the encoding genes comprise the light chain variable region encoding sequence of an anti-CD 19 single chain antibody, the heavy chain variable region encoding sequence of an anti-CD 19 single chain antibody, the heavy chain variable region encoding sequence of an anti-CD 22 single chain antibody, and the light chain variable region encoding sequence of an anti-CD 22 single chain antibody;

preferably, the coding gene further comprises a linker peptide coding sequence;

preferably, the coding gene further comprises a coding sequence for GM-CSF signal peptide, a coding sequence for CD28, and a coding sequence for CD3 ζ;

preferably, the coding sequence for the GM-CSF signal peptide comprises the nucleic acid sequence shown in SEQ ID NO 16.

6. An expression vector comprising the coding gene of claim 5;

preferably, the expression vector is a viral vector containing the encoding gene of claim 5;

preferably, the expression vector is any one of a lentiviral vector, a retroviral vector or an adeno-associated viral vector containing the coding gene of claim 5.

7. A recombinant lentivirus, wherein the recombinant lentivirus is a mammalian cell transfected with the expression vector of claim 6 and a helper plasmid.

8. A CAR-T cell expressing the chimeric antigen receptor of any one of claims 1-4;

preferably, the CAR-T cell has integrated into its genome the encoding gene of claim 5;

preferably, the CAR-T cell comprises the expression vector of claim 6 and/or the recombinant lentivirus of claim 7.

9. A method of producing a CAR-T cell according to claim 8, which comprises the step of introducing into a T cell a gene encoding a chimeric antigen receptor according to any one of claims 1 to 4.

10. Use of the chimeric antigen receptor of any one of claims 1 to 4, the coding gene of claim 5, the expression vector of claim 6, the recombinant lentivirus of claim 7 or the CAR-T cell of claim 8 for the preparation of a medicament for the treatment of a disease;

preferably, the disease comprises a hematological tumor;

preferably, the disease comprises a CD19 positive and/or CD22 positive disease.