CN114989310A

CN114989310A - Chimeric antigen receptor, macrophage for expressing chimeric antigen receptor and application

Info

Publication number: CN114989310A
Application number: CN202210698985.4A
Authority: CN
Inventors: 万晓春; 陈有海; 刘茂玄; 戴昆; 刘骏晨
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2022-06-20
Filing date: 2022-06-20
Publication date: 2022-09-02
Also published as: WO2023246004A1

Abstract

The invention discloses a chimeric antigen receptor, macrophage expressing the chimeric antigen receptor and application, wherein the primary protein structure of the chimeric antigen receptor sequentially comprises from an amino terminal to a carboxyl terminal: a signal peptide, NKG2D extracellular, hinge, transmembrane and intracellular signal regions; the NKG2D extracellular region is the extracellular domain of human NKG2D, and the amino acid sequence thereof is shown in SEQ ID NO. 2. The chimeric antigen receptor macrophage of the invention comprises the coding gene of the chimeric antigen receptor or an expression vector of the coding gene of the chimeric antigen receptor. Besides the capability of directly killing tumor cells, the chimeric antigen receptor macrophage disclosed by the invention has the following advantages: can improve the immune microenvironment of solid tumor; can present antigen as antigen presenting cells after phagocytosis of tumor cells; the tumor can be infiltrated into the tumor more easily, and the tumor can be infiltrated by other immune cells in a synergistic manner.

Description

Chimeric antigen receptor, macrophage for expressing chimeric antigen receptor and application

Technical Field

The invention relates to the field of tumor treatment, in particular to cell immunotherapy for tumors, especially to NKG2DCAR-M cell immunotherapy, and more specifically to a chimeric antigen receptor, a macrophage expressing the chimeric antigen receptor and an application of the macrophage.

Background

The global tumor statistical result in 2018 shows that 1,810 ten thousands of newly-added patients and 960 ten thousands of dead patients are added in the world in only one year, wherein 380.4 thousands of newly-added patients and 29.6 thousands of dead patients exist in China. Such a severe situation seriously threatens the health of the national people and causes huge burden for social economy. Although more and more methods are used for treating tumors with the rapid development of medical science and technology, the methods are far from sufficient. Therefore, it is still important to develop new methods for studying tumor therapy for a long time in the future.

In the past, malignant tumor has insufficient curative effect and low cure rate, so that the malignant tumor becomes a serious disease seriously threatening the health of human beings. Traditional radiochemical therapy aims at directly eliminating tumors, but the tumors frequently recur and transfer and can kill normal tissue cells. The goal of cellular immunotherapy of tumors is to promote or modify the attack of immune cells on cancer cells while maintaining the integrity of normal cells. Compared with traditional chemoradiotherapy, adoptive therapy of cells has good specificity, safety, effectiveness and durability, and ranks the first in the ten technological advances in 2013.

In recent years, adoptive cell therapy based on DCs, T cells, NK cells, and the like has achieved a superior effect in tumor therapy. In particular, immunocytotherapy techniques, represented by Chimeric Antigen Receptor T-cells (CAR-T), have shown strong therapeutic effects and great potential for development in the treatment of hematologic tumors, and have been promising for human cancer. Chimeric Antigen Receptor (CAR) T cell immunotherapy is an adoptive cellular immunotherapy for tumors, and the basic principle is to use genetic engineering to modify T lymphocytes to express chimeric antigen receptors, killing tumor cells in a non-Major Histocompatibility Complex (MHC) restricted manner. Although CART cells have achieved good results in clinical and clinical trials, serious side effects including cytokine release syndrome, neurotoxicity, off-target effects, etc. have also been observed. Moreover, CAR-T cell therapy is very powerful for hematological malignancies, especially against Acute Lymphoblastic Leukemia (ALL), but is not ideal for solid tumors treated with CAR-T cells. CAR-T cell therapy has not made major breakthroughs in the treatment of solid tumors for several reasons: first, CAR-T cells must be transported and infiltrate into the tumor, a process that requires extravasation, chemotaxis, and infiltration of stromal tissue. CAR-T cells must cross abnormal tumor vessels with reduced adhesion molecules, undergo chemokine/chemokine receptor mismatch, and must migrate through dense cellular and matrix barriers. Upon entering the Tumor Microenvironment (TME), the effector Cells encounter adverse conditions such as hypoxic and acidic environments, expression of immune checkpoint ligands, and a number of immunosuppressive Cells, such as Tumor Associated Macrophages (TAMs), Myeloid Derived Suppressor Cells (MDSCs), and Regulatory T Cells (T-Cells). Furthermore, chronic antigen exposure can lead to T cell depletion, thereby reducing effector function of CAR T cells. Even if CAR-T cells survive in TME, solid tumors often have heterogeneous surface antigen expression, which may lead to escape CAR T cell detection, incomplete tumor clearance, and eventual growth of antigen-negative tumor cells. This is clearly demonstrated in the treatment of glioblastoma using CAR T cells targeting EGFRvIII, with reduced expression of EGFRvIII in 5/7 patients post-treatment. Finally, CAR-T cells may recognize tumor associated antigens in normal tissues and cells, leading to on-target/off tumor toxicity, which is another obstacle to solid tumor cell therapy. Therefore, the development of novel anti-tumor immune cell types can possibly make up the deficiency of CAR-T in this aspect, and resist the immunosuppressive effect of the local tumor microenvironment of solid tumors, thereby achieving the synergistic anti-tumor effect.

NKG2D is a member of the C-type lectin superfamily, an important activating receptor in the innate immune system, NK cells, CD8 ⁺ T cells, activated macrophages, and tumor-infiltrating γ δ T cell surface are all expressed. There are two main classes of NKG2D ligands: one is MHC class I chain associated A/B molecules (MIC-A/B), and the other is UL16 binding protein 1-6(ULBP 1-6). NKG2D ligand (NKG2DL) is mainly expressed on most upper tumor cells, such as pancreatic cancer, ovarian cancer, prostate cancer, liver cancer, etc., and hardly expressed on normal cells. NKG2D/NKG2DL plays a very important role in the immune regulation of tumors, and the innate immune monitoring and clearing function of the body is started in the early stage of tumorigenesis. NKG2D on immune cells exerts a killing effect on tumor cells by recognizing NKG2D ligands induced on the surface of tumor cells to transmit activation signals and activate the immune system. However, the immune system in cancer patients is often weak, the number of immune cells in the body is small, and the ability of killing tumor cells is poor.

In recent years, the potential of macrophages to kill tumors has received increasing attention. Macrophages possess their powerful phagocytic function, recognize tumor cells via cell surface receptors, and then phagocytose them. Although macrophages are also negatively affected by immunosuppressive factors in the tumor microenvironment, these negative regulatory factors are currently being studied relatively extensively and have been controlled by corresponding means. Therefore, by using the concept that CAR-T targeting enhances the killing function of effector T cell tumor, it is worth discussing the possibility of using macrophage as a cell carrier for immune cell therapy.

Disclosure of Invention

In order to solve the problems existing in the prior art that the CAR-T treats the solid tumor, including tumor heterogeneity, CAR-T is difficult to infiltrate into the tumor, and the influence of the immunosuppressive microenvironment of the solid tumor, the invention aims to provide a chimeric antigen receptor, a macrophage expressing the chimeric antigen receptor and application.

The technical scheme of the invention is as follows:

the first aspect of the present invention provides a chimeric antigen receptor, wherein the primary protein structure of the chimeric antigen receptor sequentially comprises from amino terminal to carboxyl terminal: a signal peptide, NKG2D extracellular, hinge, transmembrane and intracellular signal regions;

the NKG2D extracellular region is the extracellular domain of human NKG2D, and the amino acid sequence thereof is shown in SEQ ID NO. 2.

Further, the signal peptide is selected from a CD8 a signal peptide, a CD28 signal peptide, a CD4 signal peptide or a GM-CSF signal peptide;

said hinge region is selected from a CD8 a hinge region or a CD28 hinge region;

the transmembrane region is selected from the CD8 alpha transmembrane region or the CD28 transmembrane region;

the intracellular signaling region is selected from CD3 ζ or FcR γ.

Further, the signal peptide is derived from human CD8 alpha, and the amino acid sequence of the signal peptide is shown as SEQ ID NO. 1;

the hinge region is derived from human CD8 alpha, and the amino acid sequence of the hinge region is shown in SEQ ID NO. 3;

the transmembrane region is derived from human CD8 alpha, and the amino acid sequence of the transmembrane region is shown as SEQ ID NO. 4;

the intracellular signal region is CD3 zeta, and the amino acid sequence is shown in SEQ ID NO. 5.

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

In a third aspect, the present invention provides an expression vector comprising a gene encoding the chimeric antigen receptor.

Further, the expression vector is selected from a lentivirus expression vector, a retrovirus expression vector or an adenovirus expression vector.

The fourth aspect of the present invention provides a macrophage expressing a chimeric antigen receptor, which comprises a gene encoding the chimeric antigen receptor or an expression vector of the gene encoding the chimeric antigen receptor.

Further, the macrophages are derived from a healthy human or a cancer patient;

the macrophage is selected from autologous macrophage, allogeneic macrophage or iPSC induced macrophage;

preferably, the macrophage is a primary macrophage.

The fifth aspect of the invention provides the application of the macrophage expressing the chimeric antigen receptor in preparing a tumor treatment medicament.

Further, the tumor is a NKG2D ligand-expressing tumor;

the tumor is a hematological tumor or a solid tumor;

preferably, the tumor is leukemia, multiple myeloma, malignant lymphoma, brain glioma, liver cancer, lung cancer, stomach cancer, colon cancer, pancreatic cancer or breast cancer.

The invention has the beneficial effects that:

1. according to the invention, the NKG2D extracellular segment natural sequence is used as a CAR recognition region, so that the chimeric antigen receptor has the advantages of low immunogenicity, easy expression and the like, can be normally expressed on macrophages, and provides a basis for preparing the chimeric antigen receptor macrophage (CAR-M) for specifically killing/phagocytizing tumor cells. CD3 zeta is preferably used as an intracellular signaling region, and the ITAM structure contained in CD3 zeta confers the CAR-M a better phagocytosis/tumor killing effect.

2. The chimeric antigen receptor macrophage (CAR-M) is formed by loading the CAR designed by the invention on macrophage, and the CAR can target NKG2D ligand so as to specifically kill/phagocytize tumor cells, and the CAR-primary macrophage in the embodiment proves the therapeutic effect of the CAR-M. CAR-M has advantages over CAR-T, in addition to killing tumor cells directly: can improve the immune microenvironment of solid tumor; can present antigen as antigen presenting cells after phagocytosis of tumor cells; the tumor can be infiltrated into the tumor more easily, and the tumor can be infiltrated by other immune cells in a synergistic manner. Therefore, the CAR-M cell targeting the NKG2D ligand has important application value in the aspect of treating tumors (particularly solid tumors).

Drawings

FIG. 1: schematic representation of NKG2D chimeric antigen receptor (NKG2D CAR).

FIG. 2 is a schematic diagram: NKG2D CAR-M cell CAR positivity rate.

FIG. 3: the killing rate was counted for each group after 48 hours of killing (effector cells: target cells ═ 1).

Detailed Description

In order that the invention may be more clearly understood, it will now be further described with reference to the following examples and the accompanying drawings. The examples are for illustration only and do not limit the invention in any way. In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.

Example 1

This example provides a NKG2D chimeric antigen receptor (NKG2D CAR), the primary protein structure of NKG2D CAR being, in order from amino terminus to carboxy terminus: signal peptide (Signal pep), NKG2D extracellular region (NKG2D ECD), Hinge region (Hinge), transmembrane region (TM) and intracellular Signal region. Wherein, the signal peptide can be selected from signal peptides of human CD8 alpha, CD28, CD4, GM-CSF and the like; the hinge region and transmembrane region may be selected from those of human CD8 α, CD28, and the like; the intracellular signaling region may be selected from CD3 ζ, FcR γ, and the like.

In a particular embodiment (FIG. 1), the signal peptide is derived from human CD8 α and has the amino acid sequence shown in SEQ ID NO. 1; the NKG2D extracellular region is the extracellular domain of human NKG2D (NM-007360.4), and the amino acid sequence is shown in SEQ ID NO. 2; the hinge region and the transmembrane region are derived from human CD8 alpha, the amino acid sequence of the CD8 alpha hinge region is shown as SEQ ID NO.3, and the amino acid sequence of the CD8 alpha transmembrane region is shown as SEQ ID NO. 4; the intracellular signal region provides an activation signal for CD3 zeta and CD3 zeta, and the amino acid sequence of the intracellular signal region is shown as SEQ ID NO. 5. The specific nucleotide sequence is as follows: the sequence of the NKG2D chimeric antigen receptor (NKG2D CAR) is obtained by artificially synthesizing a CD8 alpha signal peptide (SEQ ID NO.6), an NKG2D extracellular region (SEQ ID NO.7), a CD8 alpha hinge region (SEQ ID NO.8), a CD8 alpha transmembrane region (SEQ ID NO.9) and a CD3 zeta intracellular signal region (SEQ ID NO. 10).

The amino acid and nucleotide sequences in this example are as follows:

CD8 alpha signal peptide amino acid sequence (SEQ ID NO. 1):

MALPVTALLLPLALLLHAARP

NKG2D extracellular region amino acid sequence (SEQ ID NO. 2):

IWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV

CD8 a hinge region amino acid sequence (SEQ ID NO. 3):

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

CD8 alpha transmembrane region amino acid sequence (SEQ ID NO. 4):

IYIWAPLAGTCGVLLLSLVITLYC

CD3 ζ intracellular signaling region amino acid sequence (SEQ ID NO. 5):

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

CD8 a signal peptide nucleotide sequence (SEQ ID NO. 6):

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG

NKG2D extracellular region nucleotide sequence (SEQ ID NO. 7):

ATATGGAGTGCTGTATTCCTAAACTCATTATTCAACCAAGAAGTTCAAATTCCCTTGACCGAAAGTTACTGTGGCCCATGTCCTAAAAACTGGATATGTTACAAAAATAACTGCTACCAATTTTTTGATGAGAGTAAAAACTGGTATGAGAGCCAGGCTTCTTGTATGTCTCAAAATGCCAGCCTTCTGAAAGTATACAGCAAAGAGGACCAGGATTTACTTAAACTGGTGAAGTCATATCATTGGATGGGACTAGTACACATTCCAACAAATGGATCTTGGCAGTGGGAAGATGGCTCCATTCTCTCACCCAACCTACTAACAATAATTGAAATGCAGAAGGGAGACTGTGCACTCTATGCCTCGAGCTTTAAAGGCTATATAGAAAACTGTTCAACTCCAAATACGTACATCTGCATGCAAAGGACTGTG

CD8 a hinge region nucleotide sequence (SEQ ID NO. 8):

ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT

CD8 a transmembrane region nucleotide sequence (SEQ ID NO. 9):

ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC

CD3 ζ intracellular signaling region nucleotide sequence (SEQ ID NO. 10):

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

example 2

This example (one) provides the preparation of NKG2D CAR-M cells by the following steps:

NKG2D CAR adenovirus construction

NKG2D CAR adenovirus is packaged by using a packaging system of pAdEasy system recombinant adenovirus, and the framework vector used for packaging Ad5F35 adenovirus is Ad5F35 Helper. Transfecting Ad5F35 related packaging plasmids in HEK 293 cells by using PEI, uniformly mixing, and placing in CO ₂ Culturing in an incubator. After 14 days of culture, 3500g of cells are collected by centrifugation, adenovirus frozen stock solution is added, then heavy suspension and precipitation are carried out, the suspension is repeatedly frozen and thawed for four times at minus 80 ℃ and 37 ℃, after freezing and thawing are finished, 12000g of cells are centrifuged for 2min, and supernatant is collected. The collected virus supernatant is evenly dripped into 10 culture dishes of 10cm, and the mixture is placed in CO after being evenly mixed ₂ Culturing in an incubator. Centrifuging after 2-3 days to collect supernatant and cells, adding PEG8000 and NaCl into the virus supernatant, mixing, standing at 4 deg.C for overnight, centrifuging the next day to collect supernatant; and (3) storing the cell sediment at-80 ℃, adding an adenovirus freezing solution subsequently, repeatedly freezing and thawing for 4 times after resuspension, and collecting the supernatant after high-speed centrifugation. The remaining cells were sonicated and centrifuged to collect the sample supernatant. And mixing all collected samples, purifying the virus by an iodixanol density gradient centrifugation method, and finally filtering the purified virus solution through a 0.22 micron filter membrane, and subpackaging for low-temperature storage.

2. Whole blood isolation of human Peripheral Blood Mononuclear Cells (PBMC)

Adding 25mL of whole blood into a 50mL centrifuge tube, adding 25mL of PBS, and mixing uniformly; adding 15mL of FICOLL reagent into a 50mL centrifuge tube, and slowly adding 30mL of the diluted whole blood along the wall; centrifuging at 20 deg.C for 30min at 2110r/min with 2 rising and 2 falling steps; dividing the liquid into four layers after centrifugation, removing the uppermost yellow liquid, absorbing the second white membrane layer by a circle, dividing the obtained white membrane layer liquid into two tubes, adding PBS to 40-45mL, mixing uniformly, performing centrifugation at 20 ℃ and 1800r/min, rising 9 and falling 9, and centrifuging for 8 min; removing supernatant, adding 40mL PBS to resuspend the two tubes into one tube, centrifuging at 20 deg.C for 1200r/min, rising 9 and falling 9, and centrifuging for 8 min; removing supernatant, adding 40mL PBS, re-suspending and counting, keeping the remaining cells at 20 ℃, 1200r/min, rising 9 and falling 9, and centrifuging for 8 min; removing supernatant, and freezing with the freezing medium for later use.

3. Isolation of human CD14 ⁺ Cells

Placing the recovered PBMC cells in a centrifuge for 5min at 300g, and discarding supernatant; about 4-5mL of MojoStort ^TM The buffer was resuspended. The cells were filtered through a 70 μ M cell screen, centrifuged at 300g for 5min and the supernatant removed, followed by a MojoStor of appropriate volume ^TM Buffer resuspend and adjust cell concentration to 1X 10 ⁸ and/mL. To 100. mu.L of cell suspension (10) ⁷ Individual cells) was added 5. mu.L of Human TruStain FcX ^TM (Fc receptor blocking solution), mixing evenly, and incubating for 10-15min at room temperature. If more cells are isolated, the reagent dosage is increased proportionally. 10-20. mu.L of Biotin-Antibody Cocktail was added, mixed well and incubated on ice for 15 min. If more cells are isolated, the reagent dosage is increased proportionally. Add 10-15 μ L Streptavidin Nanobeads resuspended by maximum speed vortex, mix well and incubate for 15min on ice. If more cells are isolated, the reagent dosage is increased proportionally. Add 4mL of MojoStort ^TM The buffer solution is used for washing cells, and the cells are placed into a centrifuge for centrifugation at 300g for 5-10min, and then the supernatant is discarded. Adding 2.5-5mL of MojoStort ^TM The buffer resuspended cells in a clean flow tube and placed in a magnetic rack for 5 min. Pour out and collect the liquid in a new 15mL centrifuge tube for use. Then 2.5mL of MojoStort was used ^TM Buffer resuspended the remaining pellet. Combining the collected liquidsAfter centrifugation at 300g for 5min in a centrifuge, the cells were resuspended and counted in prepared human primary macrophage medium (RPMI-1640 medium supplemented with 10% FBS, 1% PS, and human GM-CSF at a final concentration of 10-50 ng/mL), and plated in non-TC-treated 6-well plates for culture. A small portion of the cells can be additionally used for flow staining to check their purity.

4. Culture of human primary macrophages

Culturing the separated human CD14 with prepared human primary macrophage culture medium ⁺ Cells in non-TC treated 6-well plates (3-10X 10) ⁵ /mL), 37 ℃, 5% carbon dioxide incubator. And slightly sucking half amount of supernatant for liquid change on the third day, and obtaining adherent cells as human primary macrophages after the liquid change on the fifth day.

5. Adenovirus infection of human primary macrophage

Isolated human CD14 ⁺ Cells were counted and cultured in non-TC-treated 6-well plates (3-8X 10) ⁵ /mL); adenovirus (control virus and NKG2D CAR adenovirus) was added after 5 days at a MOI of 200-; changing into culture medium without virus after 24-48 h; positive rate after 48-60h with test CAR-M: and (3) carrying out flow-type staining on the digested CAR-M to detect the expression of the CAR.

Flow results show (fig. 2): the positive rate of NKG2D CAR-M was > 50%. Indicating that the human primary CAR-M cell targeting NKG2D is successfully prepared.

(II) the killing effect of human primary CAR-M on tumor cells was examined by the following method

The cell killing experiment is carried out by collecting and analyzing the fluorescence intensity by using a SPECTROStar Omega enzyme-labeling instrument. NKG2D CAR-M, empty M (infected with adenovirus) and UTD-M (not treated with virus) cells were first plated at 2-5X 10 ⁴ And/well inoculating to a lightproof 96-well cell culture plate, each group comprises 5-10 multiple wells, and standing for 24-48 h. After 24h according to effector cell: target cells ═ 1: 1, PC-3 cells stably expressing luciferase (serum-free, M-CSF-free DMEM medium) were added and co-cultured with BMDM of each group. The experiments were divided into 4 groups: BLANK group; UTD-M group (no viral infection); empty M cell group (added to adenovirus control group) and NKG2D CAR-M group (added to adenovirus control group)In NKG2D CAR adenovirus group).

After culturing for 24-48h in a 5% carbon dioxide incubator at 37 ℃, adding D-luciferin and potassiumsalt substrates with the concentration of 100-:

% cell Lysis (lysine%) [ 1- (fluorescence signal of co-cultured cells-background fluorescence signal) — based on blood glucose level in blood

(culture of PC-3 cells alone-background fluorescence Signal) ]100

The killing results of PC-3 cells are shown in FIG. 3: the killing effect of the cells in the UTD-M group and the empty group on the tumor cells is weaker, the killing effect of the NKG2D CAR-M group on the tumor cells is obviously enhanced, and the targeted killing function of NKG2D CAR-T is proved.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Obvious variations or modifications of this invention will be apparent to those skilled in the art and are within the scope of the invention.

SEQUENCE LISTING

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Claims

1. A chimeric antigen receptor, wherein the primary protein structure of said chimeric antigen receptor, in order from amino terminus to carboxy terminus, is: a signal peptide, NKG2D extracellular, hinge, transmembrane and intracellular signal regions;

2. The chimeric antigen receptor according to claim 1, wherein the signal peptide is selected from the group consisting of a CD8 a signal peptide, a CD28 signal peptide, a CD4 signal peptide, or a GM-CSF signal peptide;

said hinge region is selected from a CD8 a hinge region or a CD28 hinge region;

the intracellular signaling region is selected from CD3 ζ or FcR γ.

3. The chimeric antigen receptor according to claim 1, wherein the signal peptide is derived from human CD8 α, and has an amino acid sequence shown in SEQ ID No. 1;

4. A gene encoding the chimeric antigen receptor of any one of claims 1 to 3.

5. An expression vector comprising a gene encoding the chimeric antigen receptor of claim 4.

6. The expression vector of claim 5, wherein the expression vector is selected from the group consisting of a lentiviral expression vector, a retroviral expression vector, and an adenoviral expression vector.

7. A macrophage cell expressing a chimeric antigen receptor, which comprises an expression vector containing a gene encoding the chimeric antigen receptor according to claim 4 or a gene encoding the chimeric antigen receptor according to claim 5.

8. The chimeric antigen receptor-expressing macrophage according to claim 7, wherein said macrophage is derived from a healthy human or a cancer patient;

preferably, the macrophage is a primary macrophage.

9. Use of the chimeric antigen receptor-expressing macrophage of claim 7 in the preparation of a medicament for the treatment of a tumor.

10. The use of claim 9, wherein the tumor is a NKG2D ligand-expressing tumor;

the tumor is a hematological tumor or a solid tumor;