Use of car-t and car-m combination in preparation of anti-tumor drugs

Abstract

The application discloses application of combination of CAR-T and CAR-M in preparation of antitumor drugs, wherein the CAR-T is a T cell modified by a chimeric antigen receptor, and the primary protein structure of the chimeric antigen receptor sequentially comprises a signal peptide, an NKG2D extracellular region, a hinge region, a transmembrane region, an intracellular costimulatory signal region and an intracellular signal region from an amino terminus to a carboxyl terminus; the CAR-M is a macrophage modified by a chimeric antigen receptor, and the primary protein structure of the chimeric antigen receptor sequentially comprises a signal peptide, an NKG2D extracellular region, a hinge region, a transmembrane region and an intracellular signal region from an amino terminus to a carboxyl terminus; and the NKG2D extracellular region is a human NKG2D extracellular domain, and the amino acid sequence is shown as SEQ ID NO. 2. The combination of the CAR-T and the CAR-M combines the ability of the CAR-M to improve a tumor microenvironment with the ability of the CAR-T to directly kill tumor cells, so that strong and efficient killing of tumor cells can be realized, and the curative effect is greatly improved.

Description

Technical Field

[0001] This invention relates to the field of tumor treatment, specifically to cell immunotherapy for tumors, particularly to chimeric antigen receptor T cell (CAR-T) and chimeric antigen receptor macrophage (CAR-M) cell immunotherapy, and more specifically to the application of CAR-T and CAR-M in combination in the preparation of anti-tumor drugs. Background Technology

[0002] Despite the rapid development of medical technology and the increasing number of methods and techniques used in cancer treatment, it is far from sufficient, and there remains a huge unmet clinical need. Therefore, it is crucial to accumulate experience in developing new cancer treatments.

[0003] For a long time, the inadequate treatment efficacy and low cure rate of malignant tumors have made them a major disease that seriously threatens human health. Traditional radiotherapy and chemotherapy aim to directly eliminate tumors, but tumors often recur and metastasize, and they also kill normal tissue cells. The goal of cell immunotherapy for tumors is to promote or modify immune cells to attack cancer cells while maintaining the integrity of normal cells. Compared with traditional radiotherapy and chemotherapy, adoptive cell therapy has good specificity, safety, efficacy and durability, ranking first among the top ten scientific and technological advances in 2013.

[0004] In recent years, adoptive cell therapy based on DCs, T cells, and NK cells has achieved good results in cancer treatment. In particular, immunotherapy technologies represented by chimeric antigen receptor T-cells (CAR-T) have demonstrated powerful efficacy and enormous development potential in the treatment of hematological malignancies, becoming a new hope for humanity in the fight against cancer. Chimeric antigen receptor (CAR) T-cell immunotherapy is a type of adoptive cell immunotherapy for tumors. Its basic principle is to use genetic engineering to modify T lymphocytes to express chimeric antigen receptors, thereby killing tumor cells in a manner restricted by the major histocompatibility complex (MHC). Although CAR T-cells have achieved good results in clinical trials, they have also shown serious side effects, including cytokine release syndrome, neurotoxicity, and off-target effects. Moreover, the effectiveness of CAR-T cell therapy for other tumors besides some hematological malignancies (such as solid tumors) in clinical trials is not satisfactory and urgently needs improvement. Several factors have hindered the significant breakthrough of CAR-T cell therapy in the treatment of solid tumors. First, CAR-T cells must be transported and infiltrated into the tumor, a process requiring extravasation, chemotaxis, and stromal infiltration. CAR-T cells must traverse abnormal tumor blood vessels with reduced adhesion molecules, overcome chemokine / chemokine receptor mismatch, and migrate through dense cellular and stromal barriers. Once inside the tumor microenvironment (TME), effector cells encounter adverse conditions such as hypoxia and acidity, expression of immune checkpoint ligands, and a large number of immunosuppressive cells, including tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and regulatory T cells (T-regs). Furthermore, prolonged antigen exposure can lead to T cell exhaustion, thereby reducing the effector function of CAR T cells. Even when CAR-T cells survive in the TME, solid tumors often exhibit heterogeneous surface antigen expression, which can lead to evasion of CAR T cell detection, incomplete tumor clearance, and the eventual growth of antigen-negative tumor cells. This has been clearly demonstrated in the use of CAR T cell therapy targeting EGFRvIII to treat glioblastoma, where EGFRvIII expression decreased in 5 out of 7 patients after treatment.Finally, CAR-T cells may recognize tumor-associated antigens in normal tissues and cells, leading to on-target / offtumor toxicity, which is another obstacle in cell therapy for solid tumors. Therefore, combining CAR-T with other therapies may compensate for CAR-T's shortcomings in this regard, counteract the immunosuppressive effects of the local tumor microenvironment in solid tumors, and thus achieve a synergistic anti-tumor effect.

[0005] In recent years, the tumor-killing potential of macrophages has gradually attracted attention. CAR-M modified macrophages are considered a promising cell type. Macrophages primarily activate lymphocytes or other immune cells by phagocytosing cellular debris and pathogens in body fluids and tissues and presenting antigens, thus inducing a response against pathogens. In the tumor microenvironment, macrophages are the innate immune cells with the highest infiltration rate and can interact with almost all cellular components of the TME, stimulating angiogenesis, increasing tumor invasion, and mediating immunosuppression. However, the direct tumor-killing effect of CAR-M is limited. Summary of the Invention

[0006] In order to address the shortcomings of existing technologies in (1) CAR-T therapy for solid tumors, including tumor heterogeneity leading to the escape of CAR target-negative cells; CAR-T's difficulty in infiltrating the tumor and insufficient killing effect; the influence of the immunosuppressive microenvironment of solid tumors leading to CAR-T depletion and loss of killing function; and the side effects of CAR-T itself, such as cytokine release syndrome, neurotoxicity and off-target effects; and (2) the low efficiency of CAR-M cells in killing tumor cells by phagocytosis or cytophagy, this invention aims to provide the application of CAR-T and CAR-M in the preparation of anti-tumor drugs.

[0007] Based on the comparative advantages of CAR-M and CAR-T: (1) CAR-M can improve the tumor immune microenvironment of solid tumors; after phagocytizing tumor cells, it can act as an antigen-presenting cell to present antigens; it is easier to infiltrate the tumor and cooperate with other immune cells to infiltrate the tumor, etc.; (2) CAR-T has the ability to directly kill tumor cells. This invention proposes a scheme for tumor treatment using a combination of CAR-T and CAR-M. The specific scheme is as follows:

[0008] The first aspect of this invention provides the application of CAR-T and macrophages in the preparation of antitumor drugs, wherein the CAR-T is a T cell modified with a chimeric antigen receptor, and the primary protein structure of the chimeric antigen receptor, from the amino terminus to the carboxyl terminus, consists of: a signal peptide, an extracellular NKG2D region, a hinge region, a transmembrane region, an intracellular co-stimulatory signal region, and an intracellular signal region.

[0009] The extracellular region of NKG2D is the extracellular domain of human NKG2D, and its amino acid sequence is shown in SEQ ID NO.2.

[0010] The second aspect of the present invention provides the application of CAR-M and T cells in the preparation of antitumor drugs, wherein the CAR-M is a macrophage modified with a chimeric antigen receptor, and the primary protein structure of the chimeric antigen receptor, from the amino terminus to the carboxyl terminus, consists of: a signal peptide, an extracellular region of NKG2D, a hinge region, a transmembrane region, and an intracellular signal region.

[0011] The extracellular region of NKG2D is the extracellular domain of human NKG2D, and its amino acid sequence is shown in SEQ ID NO.2.

[0012] The third aspect of the present invention provides the application of CAR-T and CAR-M in combination in the preparation of antitumor drugs, wherein the CAR-T is a T cell modified with a chimeric antigen receptor, and the primary protein structure of the chimeric antigen receptor, from the amino terminus to the carboxyl terminus, is as follows: signal peptide, extracellular NKG2D region, hinge region, transmembrane region, intracellular co-stimulatory signal region, and intracellular signal region.

[0013] The CAR-M is a macrophage modified with a chimeric antigen receptor. The primary protein structure of the chimeric antigen receptor, from the amino terminus to the carboxyl terminus, consists of: signal peptide, extracellular NKG2D region, hinge region, transmembrane region, and intracellular signal region.

[0014] The extracellular region of the chimeric antigen receptor NKG2D in the CAR-T and CAR-M is the extracellular domain of human NKG2D, and its amino acid sequence is shown in SEQ ID NO.2.

[0015] In the above applications, the signal peptide is selected from CD8α signal peptide, CD28 signal peptide, CD4 signal peptide or GM-CSF signal peptide;

[0016] The hinge area is selected from the CD8α hinge area or the CD28 hinge area;

[0017] The transmembrane region is selected from the CD8α transmembrane region or the CD28 transmembrane region;

[0018] The intracellular signaling region is selected from CD3ζ or FcRγ.

[0019] In the above applications, the signal peptide is derived from human CD8α, and its amino acid sequence is shown in SEQ ID NO.1;

[0020] The hinge region is derived from human CD8α, and its amino acid sequence is shown in SEQ ID NO.3;

[0021] The transmembrane region is derived from human CD8α, and its amino acid sequence is shown in SEQ ID NO.4;

[0022] The intracellular signaling region is CD3ζ, and its amino acid sequence is shown in SEQ ID NO.5.

[0023] In the above applications, the intracellular co-stimulatory signal region is selected from 4-1BB, CD28, CD27, OX40, or ICOS;

[0024] Preferably, the intracellular co-stimulatory signal region is selected from 4-1BB, and its amino acid sequence is shown in SEQ ID NO.11.

[0025] In the above applications, the CAR-T construction method is as follows: introducing the nucleic acid or vector encoding the CAR-T chimeric antigen receptor gene into T cells;

[0026] The methods of introduction include electroporation, transduction, or transfection;

[0027] Preferably, the nucleic acid is located on different viral vectors; the viral vector is a lentiviral vector, an adenovirus vector, or a retroviral vector;

[0028] Preferably, the vector is a transposon or mRNA vector;

[0029] Preferably, a lentiviral vector containing the CAR-T chimeric antigen receptor encoding gene is introduced into T cells via transfection.

[0030] In the above applications, the CAR-M construction method is as follows: introducing the nucleic acid or vector of the CAR-M chimeric antigen receptor encoding gene into macrophage cells;

[0031] The methods of introduction include electroporation, transduction, or transfection;

[0032] Preferably, the nucleic acid is located on different viral vectors; the viral vector is a lentiviral vector, an adenovirus vector, or a retroviral vector;

[0033] Preferably, the vector is a transposon or mRNA vector;

[0034] Preferably, an adenovirus vector containing the CAR-M chimeric antigen receptor encoding gene is introduced into macrophage cells via transfection.

[0035] In the above applications, the macrophages are derived from healthy individuals or cancer patients;

[0036] The macrophages are selected from autologous macrophages, allogeneic macrophages, or iPSC-induced macrophages;

[0037] Preferably, the macrophages are primary macrophages;

[0038] The T cells are derived from healthy individuals or cancer patients;

[0039] The T cells are selected from autologous T cells, allogeneic T cells, or iPSC-induced T cells.

[0040] In the above applications, the tumor is a tumor expressing NKG2D ligand;

[0041] The tumor is either a hematologic tumor or a solid tumor;

[0042] Preferably, the tumor is leukemia, multiple myeloma, malignant lymphoma, glioma, liver cancer, lung cancer, stomach cancer, colon cancer, pancreatic cancer, or breast cancer.

[0043] The beneficial effects of this invention are as follows:

[0044] 1. This invention uses the natural extracellular sequence of NKG2D as the CAR recognition region, giving the chimeric antigen receptor advantages such as low immunogenicity and easy expression. It can be normally expressed on macrophages and T cells, providing a basis for the preparation of chimeric antigen receptor macrophages (CAR-M) and chimeric antigen receptor T cells (CAR-T) that specifically kill / phagocytose tumor cells. Preferably, CD3ζ is used as the intracellular signaling region, and the ITAM structure contained in CD3ζ endows CAR-M with better phagocytic / killing effects on tumors.

[0045] 2. This invention achieves synergistic effects in anti-tumor therapy by combining CAR-T and CAR-M, CAR-T and M cells, or CAR-M and T cells, thereby improving the efficacy of tumor treatment. In particular, the combination of CAR-T and CAR-M cells in tumor treatment combines the ability of CAR-M to improve the tumor microenvironment with the ability of CAR-T to directly kill tumor cells, resulting in a synergistic effect. This allows for powerful and efficient killing of tumor cells (especially solid tumors), greatly improving efficacy and addressing the shortcomings of CAR-T therapy alone in treating solid tumors, as well as the low efficiency of CAR-M alone in killing tumor cells. Furthermore, the combination regimen can reduce the amount of CAR-T cells used, thereby reducing the side effects of CAR-T therapy. Attached Figure Description

[0046] Figure 1 : A schematic diagram of the NKG2D CAR in NKG2D CAR-M.

[0047] Figure 2 : Schematic diagram of NKG2D CAR in NKG2D CAR-T.

[0048] Figure 3 : CAR positivity rate of NKG2D CAR-M cells.

[0049] Figure 4: CAR positivity rate of NKG2D CAR-T cells.

[0050] Figure 5 Experiments on the killing effect of human primary CAR-M and CAR-T cells on tumor cells, and statistical analysis of the killing rate of each group 48 hours after killing (effective cells: target cells = 1).

[0051] Figure 6 Experiments on the killing effect of human primary macrophages (M) and CAR-T cells on tumor cells, and statistical analysis of the killing rate of each group 48 hours after killing (effective cells: target cells = 1).

[0052] Figure 7 Experiments on the killing effect of human primary CAR-M and primary T cells on tumor cells, and statistical analysis of the killing rate of each group 48 hours after killing (effective cells: target cells = 1). Detailed Implementation

[0053] To better understand the present invention, it is now further described with reference to the following embodiments and accompanying drawings. The embodiments are for illustrative purposes only and do not limit the invention in any way. In the embodiments, all original reagents and materials are commercially available, and experimental methods not specifically specified are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0054] Example 1

[0055] 1. The NKG2D chimeric antigen receptor used in NKG2D CAR-M

[0056] This embodiment provides the NKG2D chimeric antigen receptor used in NKG2D CAR-M, whose primary protein structure, from the amino terminus to the carboxyl terminus, consists of: a signal peptide (Signal pep), an NKG2D extracellular region (NKG2D ECD), a hinge region, a transmembrane region (TM), and an intracellular signaling region. The signal peptide can be selected from human CD8α, CD28, CD4, GM-CSF, etc.; the hinge region and transmembrane region can be selected from the hinge region and transmembrane region of human CD8α, CD28, etc.; and the intracellular signaling region can be selected from CD3ζ, FcRγ, etc. In one specific embodiment (… Figure 1The signal peptide is derived from human CD8α, and its amino acid sequence is shown in SEQ ID NO.1; the extracellular region of NKG2D is the extracellular domain of human NKG2D (NM_007360.4), and its amino acid sequence is shown in SEQ ID NO.2; the hinge region and transmembrane region are derived from human CD8α, and the amino acid sequence of the CD8α hinge region is shown in SEQ ID NO.3, and the amino acid sequence of the CD8α transmembrane region is shown in SEQ ID NO.4; the intracellular signal region is CD3ζ, which provides the activation signal, and its amino acid sequence is shown in SEQ ID NO.5. Specifically, the CD8α signal peptide (SEQ ID NO.6), NKG2D extracellular region (SEQ ID NO.7), CD8α hinge region (SEQ ID NO.8), CD8α transmembrane region (SEQ ID NO.9), and CD3ζ intracellular signal region (SEQ ID NO.10) were artificially synthesized to obtain the NKG2D chimeric antigen receptor sequence used in NKG2D CAR-M.

[0057] 2. The NKG2D chimeric antigen receptor used in NKG2D CAR-T

[0058] This embodiment provides the NKG2D chimeric antigen receptor used in NKG2D CAR-T, whose primary protein structure, from the amino terminus to the carboxyl terminus, is as follows: signal peptide, NKG2D extracellular region (NKG2D ECD), hinge region, transmembrane region (TM), intracellular co-stimulatory signal region, and intracellular signal region. The signal peptide can be selected from human CD8α, CD28, CD4, GM-CSF, etc.; the hinge region and transmembrane region can be selected from the hinge region and transmembrane region of human CD8α, CD28, etc.; the intracellular co-stimulatory signal region can be selected from 41-BB, CD28, CD27, OX40, or ICOS; and the intracellular signal region can be selected from CD3ζ, FcRγ, etc. In one specific embodiment ( Figure 2The signal peptide is derived from human CD8α, and its amino acid sequence is shown in SEQ ID NO.1; the extracellular region of NKG2D is the extracellular domain of human NKG2D (NM_007360.4), and its amino acid sequence is shown in SEQ ID NO.2; the hinge region and transmembrane region are derived from human CD8α, and the amino acid sequence of the CD8α hinge region is shown in SEQ ID NO.3, and the amino acid sequence of the CD8α transmembrane region is shown in SEQ ID NO.4; the intracellular co-stimulatory signal region is selected from 41BB, and its amino acid sequence is shown in SEQ ID NO.11; the intracellular signal region is CD3ζ, which provides the activation signal, and its amino acid sequence is shown in SEQ ID NO.5. The specific nucleotide sequences—CD8α signal peptide (SEQ ID NO.6), NKG2D extracellular region (SEQ ID NO.7), CD8α hinge region (SEQ ID NO.8), CD8α transmembrane region (SEQ ID NO.9), 41BB intracellular co-stimulatory signal region (SEQ ID NO.12), and CD3ζ intracellular signal region (SEQ ID NO.10)—were artificially synthesized to obtain the NKG2D chimeric antigen receptor sequence used in NKG2D CAR-M.

[0059] 3. Amino acid and nucleotide sequences in this embodiment

[0060] CD8α signal peptide amino acid sequence (SEQ ID NO.1):

[0061] MALPVTALLLPLALLLHAARP

[0062] NKG2D extracellular amino acid sequence (SEQ ID NO.2):

[0063] IWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV

[0064] The amino acid sequence of the CD8α hinge region (SEQ ID NO.3):

[0065] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0066] CD8α transmembrane region amino acid sequence (SEQ ID NO.4):

[0067] IYIWAPLAGTCGVLLLSLVITLYC

[0068] The amino acid sequence of the CD3ζ intracellular signaling region (SEQ ID NO.5):

[0069] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0070] CD8α signal peptide nucleotide sequence (SEQ ID NO.6):

[0071] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG

[0072] NKG2D extracellular nucleotide sequence (SEQ ID NO.7):

[0073] ATATGGAGTGCTGTATTCCTAAACTCATTATTCAACCAAGAAGTTCAAATTCCCTTGACCGAAAGTTACTGTGGCCCATGTCCTAAAAACTGGATATGTTACAAAAATAACTGCTACCAATTTTTTGATGAGAGTAAAAACTGGTATGAGAGCCAGGCTTCTTGTATGTCTCAAAATGCCAGCCTTCTGAAAGTATACAGCAAAGAGGACCAGGAT TTACTTAAACTGGTGAAGTCATATCATTGGATGGGACTAGTACACATTCCAACAAATGGATCTTGGCAGTGGGAAGATGGCTCCATTCTCTCACCCAACCTACTAACAATAATTGAAATGCAGAAGGGAGACTGTGCACTCTATGCCTCGAGCTTTAAAGGCTATATAGAAAACTGTTCAACTCCAAATACGTACATCTGCATGCAAAGGACTGTG

[0074] CD8α hinge region nucleotide sequence (SEQ ID NO.8):

[0075] ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT

[0076] CD8α transmembrane nucleotide sequence (SEQ ID NO.9):

[0077] ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC

[0078] The nucleotide sequence of the CD3ζ intracellular signal region (SEQ ID NO.10):

[0079] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAA GGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

[0080] 41BB intracellular co-stimulatory signal region amino acid sequence (SEQ ID NO.11):

[0081] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0082] nucleotide sequence of the 4-1BB intracellular co-stimulatory signal region (SEQ ID NO.12):

[0083] AAACGGGGCAGAAAGAAACTCCTGTATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAA

[0084] Example 2

[0085] 1. Preparation of NKG2D CAR-M

[0086] 1.1. Construction of recombinant adenovirus

[0087] NKG2D CAR adenovirus was packaged using the pAdEasy system recombinant adenovirus packaging system, with the Ad5F35 Helper serving as the backbone vector for Ad5F35 adenovirus packaging. HEK 293 cells were transfected with PEI-related packaging plasmids and cultured in a CO2 incubator after mixing. After 14 days of culture, cells were collected by centrifugation at 3500g, resuspended in adenovirus cryopreservation solution, and subjected to four freeze-thaw cycles at -80℃ and 37℃. After each freeze-thaw cycle, the cells were centrifuged at 12000g for 2 min, and the supernatant was collected. The collected viral supernatant was evenly added to ten 10cm culture dishes, mixed, and then cultured in a CO2 incubator. Two to three days later, the supernatant and cells were collected by centrifugation. PEG8000 and NaCl were added to the viral supernatant, mixed, and incubated upright at 4°C overnight. The supernatant was collected by centrifugation the next day. The cell pellet was stored at -80°C, subsequently resuspended in adenovirus cryopreservation solution, and subjected to four freeze-thaw cycles. The supernatant was collected after high-speed centrifugation. The remaining cells were sonicated, and the supernatant was collected after centrifugation. All collected samples were mixed, and the virus was purified using iodixanol density gradient centrifugation. Finally, the purified virus solution was filtered through a 0.22-micron filter and aliquoted for low-temperature storage.

[0088] 1.2 Preparation of chimeric antigen receptor M cells targeting NKG2D

[0089] a) Isolation of human peripheral blood mononuclear cells (PBMCs) from whole blood

[0090] Add 25 mL of whole blood to a 50 mL centrifuge tube, then add 25 mL of PBS and mix well. Add 15 mL of FICOLL reagent to the 50 mL centrifuge tube, then slowly add 30 mL of the diluted blood along the wall. Centrifuge at 20°C, 2110 rpm, 2 dips, for 30 min. After centrifugation, the liquid will separate into four layers. Remove the top yellow layer, and aspirate the second white film layer in a circular motion. Divide the resulting white film layer into two tubes, add PBS to 40-45 mL and mix well. Centrifuge at 20°C, 1800 rpm, 9 dips, for 8 min. Discard the supernatant, add 40 mL of PBS to resuspend the two tubes into one tube, and centrifuge at 20°C, 1200 rpm, 9 dips, for 8 min. Discard the supernatant, add 40 mL of PBS to resuspend and count the cells. Centrifuge the remaining cells at 20°C, 1200 rpm, 9 dips, for 8 min. Discard the supernatant and freeze in cryopreservation solution for later use.

[0091] b) Separation of human CD14 + cell

[0092] Centrifuge the revived PBMC cells at 300g for 5 minutes, discard the supernatant; add approximately 4-5 mL of MojoSort. TM Resuspend in buffer. Filter cells through a 70 μM cell strainer, centrifuge at 300 g for 5 min, discard the supernatant, and then resuspend in an appropriate volume of MojoSort. TM Resuspend in buffer and adjust cell concentration to 1×10⁻⁶ 8 Cells / mL. Add to 100 μL of cell suspension (10 7 Add 5 μL of Human TruStain FCX ​​to each cell. TM (Fc receptor blocking solution), mix well and incubate at room temperature for 10 min. If more cells are isolated, increase the reagent volume proportionally. Add 10 μL of Biotin-Antibody Cocktail, mix well and incubate on ice for 15 min. If more cells are isolated, increase the reagent volume proportionally. Add 10 μL of Streptavidin Nanobeads resuspended by vortexing at maximum speed, mix well and incubate on ice for 15 min. If more cells are isolated, increase the reagent volume proportionally. Add 4 mL of MojoSort. TM Cells were washed with buffer, centrifuged at 300g for 5 minutes, and the supernatant was discarded. 2.5 mL of MojoSort was added. TM Resuspend the cells in buffer solution in a clean flow cytometry tube and incubate on a magnetic rack for 5 minutes. Pour out and collect the liquid in a new 15 mL centrifuge tube for later use. Then, use 2.5 mL of MojoSort... TMResuspend the remaining precipitate in buffer. Combine the collected liquids, centrifuge at 300g for 5 min, resuspend in prepared human primary macrophage culture medium (RPMI-1640 medium with 10% FBS, 1% PS, and a final concentration of 20 ng / mL human GM-CSF), count the cells, and seed them into non-TC treated 6-well plates. A small fraction of cells can be used for flow cytometry staining to determine their purity.

[0093] c) Culture of primary human macrophages

[0094] Human CD14 cells were cultured and isolated using a prepared human primary macrophage culture medium. + Cells were stored in non-TC-treated 6-well plates (3-8 × 10⁻⁶). 5 Cells were cultured at 37°C in a 5% CO2 incubator (cells / mL). On the third day, half of the supernatant was gently aspirated and the medium was replaced. On the fifth day, the medium was completely replaced, and the resulting adherent cells were human primary macrophages.

[0095] d) Adenovirus infection of human primary macrophages

[0096] Good separation of CD14 + Cells were counted and cultured in non-TC-treated 6-well plates (3-8 × 10⁶ cells / well). 5 (number / mL); after 5 days, adenovirus (control virus and NKG2D CAR adenovirus) was added at a viral titer of MOI = 200-1000; after 24-48 hours, the medium was replaced with a virus-free medium; after 48 hours, the CAR-M positivity rate was detected by flow cytometry staining of the digested CAR-M.

[0097] Flow cytometry results (attached) Figure 3 The positive rate of NKG2D CAR-M was >50%, indicating that primary human CAR-M cells targeting NKG2D were successfully prepared.

[0098] 2. Preparation of NKG2D CAR-T

[0099] 2.1. Construction of pWPXLd-CAR-NKG2D recombinant plasmid

[0100] The CAR-NKG2D coding gene was inserted between the BamHI and EcoRI restriction sites of the pWPXLD vector, following the extension factor 1α (EF1α) of the pWPXLD vector, with EF1α as the promoter. When the CAR-NKG2D coding gene was inserted into the pWPXLD vector, a start codon (e.g., ATG) could be added to the 5' end of the CAR-NKG2D coding gene, linking it to the BamHI restriction site in the pWPXLD vector, and a stop codon (e.g., TAA) could be added to the 3' end, linking it to the EcoRI restriction site in the pWPXLD vector. The resulting plasmid was then transformed into *E. coli* competent cells DH5α, and positive clones were identified by PCR and sequencing. After PCR product gel electrophoresis and sequencing confirmation, the plasmid met the target fragment size and sequence, thus obtaining the pWPXLd-CAR-NKG2D recombinant plasmid.

[0101] 2.2. Construction of Recombinant Lentiviral Virus

[0102] The recombinant plasmid pWPXLd-CAR-NKG2D obtained above, along with the packaging plasmid psPAX2 and the envelope plasmid pMD2G, were co-transfected into cultured HEK293T cells using the liposome transfection reagent Lipofectamine 3000. At 48 hours, the virus-containing supernatant was harvested, filtered through a 0.45 μm filter, and stored at -80°C. At 72 hours, the virus-containing supernatant was harvested again, filtered through a 0.45 μm filter, and combined with the virus supernatant harvested at 48 hours. These were then added to ultracentrifuge tubes and placed one by one into a Beckman ultracentrifuge. The centrifugation parameters were set to 25,000 rpm for 2 hours at 4°C. After centrifugation, the supernatant was discarded, and as much residual liquid as possible was removed from the tube walls. Virus preservation solution was added, and the cells were gently resuspended by repeated pipetting. After thorough dissolution, the cells were centrifuged at 10,000 rpm for 5 minutes. The supernatant titer was determined by fluorescence method. The virus was administered at a concentration of 100 μl, 2 × 10⁻⁶. 8 The cells were aliquoted per mL and stored at -80°C to obtain recombinant lentiviruses carrying the CAR-NKG2D encoding gene.

[0103] 2.3 Preparation of chimeric antigen receptor T cells targeting NKG2D

[0104] a) Isolation of PBMCs (peripheral blood mononuclear cells)

[0105] PBMCs are derived from autologous venous blood, autologous bone marrow, umbilical cord blood, and placental blood. Ideally, they should be obtained from fresh peripheral blood or bone marrow collected from cancer patients one month after surgery or one month after radiotherapy and chemotherapy.

[0106] Patient blood was drawn and sent to the blood separation room; peripheral blood mononuclear cells were collected, and the intermediate layer cells were obtained after Ficoll centrifugation; after washing with PBS, PBMCs were obtained.

[0107] b) Immunomagnetic bead method for isolating antigen-specific T lymphocytes

[0108] Take the above PBMCs and add them to serum-free basal culture medium to prepare a cell suspension; add CD3 / CD28 immunomagnetic beads at a ratio of 3:1 of magnetic beads to cells and incubate at room temperature for 1-2 hours; use a magnet to screen the cells after incubation of magnetic beads; wash with PBS to remove the immunomagnetic beads and obtain CD3 positive T lymphocytes.

[0109] c) Preparation of antigen-specific T lymphocytes via viral transfection

[0110] Take the CD3-positive T lymphocytes obtained by immunomagnetic bead separation in b), add the recombinant lentivirus described in 2.2 with a viral titer corresponding to the number of CD3-positive cells, and culture them.

[0111] On day 3 of culture, cell counting and medium change were performed, and the cell concentration was adjusted to 1×10⁻⁶. 6 Inoculate at 100 cells / mL and culture. On day 5 of culture, observe the cell status. If the cell density increases, dilute the cell concentration to 1×10⁻⁶. 6 Cell viability was assessed by measuring cells / mL, and the cells were cultured further. Cells were expanded and cultured for 9-11 days, at which point they were collected, and the expression of the chimeric antigen receptor CAR-NKG2D targeting NKG2D was detected by flow cytometry. The results are as follows: Figure 4 As shown in the figure. The positive rate of CAR-NKG2D in T cells infected with the above recombinant lentivirus was approximately 70%, indicating that CAR-T cells targeting NKG2D were successfully prepared.

[0112] 3. Killing effect of primary human CAR-M and CAR-T cells on tumor cells

[0113] The experiment was divided into four groups: Control group; CAR-M group; CAR-T group; CAR-T and CAR-M combination group; and 50% CAR-T and 50% CAR-M group. Fluorescence intensity was collected and analyzed using a SPECTROstar Omega microplate reader to perform cell killing experiments. First, NKG2D CAR-M cells were cultured at a rate of 2 × 10⁻⁶ cells / year. 4 1 unit / well (CAR-M group; CAR-T and CAR-M combination group) or 1×10 410 cells / well (50% CAR-T and 50% CAR-M groups) were seeded into opaque 96-well cell culture plates, with 10 replicates per group, and incubated for 24 h. After 24 h, PC-3 cells stably expressing luciferase and 2 × 10⁶ cells / well were added at an effector cell:target cell ratio of 1:1. 4 1 unit / well (CAR-T group; CAR-T and CAR-M combination group) or 1×10 4 Number of CAR-T cells per well (50% CAR-T and 50% CAR-M groups).

[0114] After co-culturing for 48 hours, the cells were incubated at 37°C in a 5% CO2 incubator for another 48 hours. Then, 100-200 μg / mL of D-luciferin and potassium salt substrate were added to opaque 96-well plates. After incubation at 37°C in the dark for 10 minutes, fluorescence intensity was collected and analyzed using a SPECTROstar Omega microplate reader. The killing effect of each immune cell group on PC-3 target cells was calculated using the following formula:

[0115] % cell lysis (Lysis%) = [1 - (fluorescence signal of co-cultured cells - background fluorescence signal) /

[0116] (Fluorescence signal of PC-3 cells cultured alone - background fluorescence signal) *100

[0117] The results of PC-3 cell killing are attached. Figure 5 As shown, the CAR-T group was superior to the CAR-M group in killing cells, and the 50% CAR-T and 50% CAR-M groups were significantly superior to the CAR-T group. The CAR-T+CAR-M combination exhibited the strongest killing effect. Given that CAR-T cells are more effective at killing cells than CAR-M cells, the total number of cells in the 50% CAR-T and 50% CAR-M groups was the same as that in the CAR-T group, containing 50% CAR-T and 50% CAR-M cells. However, the killing effect of the 50% CAR-T and 50% CAR-M groups was significantly superior to that of the CAR-T group. This indicates that CAR-T and CAR-M have a synergistic effect (1+1>2).

[0118] 4. Killing effect of human primary macrophages (M) and CAR-T cells on tumor cells

[0119] The experiment was divided into four groups: Control group; macrophage group without CAR conversion (M group); CAR-T group; CAR-T and M combination group; and 50% CAR-T and 50% M group. Fluorescence intensity was collected and analyzed using a SPECTROstar Omega microplate reader to perform cell killing experiments. First, macrophages (M) were cultured at 2 × 10⁻⁶ cells / cells. 4pcs / well (Group M; CAR-T and M combination group) or 1×10 4 50% CAR-T and 50% M groups were seeded per well into opaque 96-well cell culture plates, with 10 replicates per group, and incubated for 24 h. After 24 h, PC-3 cells stably expressing luciferase and 2 × 10⁶ cells were added at an effector cell:target cell ratio of 1:1. 4 One per well (CAR-T group; CAR-T and M combination group) or 1×10 4 Number of CAR-T cells per well (50% CAR-T and 50% M group).

[0120] After co-culturing for 48 hours, the cells were incubated at 37°C in a 5% CO2 incubator for another 48 hours. Then, 100-200 μg / mL of D-luciferin and potassium salt substrate were added to opaque 96-well plates. After incubation at 37°C in the dark for 10 minutes, fluorescence intensity was collected and analyzed using a SPECTROstar Omega microplate reader. The killing effect of each immune cell group on PC-3 target cells was calculated using the following formula:

[0121] % cell lysis (Lysis%) = [1 - (fluorescence signal of co-cultured cells - background fluorescence signal) /

[0122] (Fluorescence signal of PC-3 cells cultured alone - background fluorescence signal) *100

[0123] The results of PC-3 cell killing are attached. Figure 6 As shown, the CAR-T group was superior to the M group in killing cells, and the 50% CAR-T and 50% M groups were significantly superior to the CAR-T group. The CAR-T+M combination showed the strongest killing effect. Given that CAR-T cells are more effective at killing M cells than M cells, the total number of cells in the 50% CAR-T and 50% M groups was the same as that in the CAR-T group, containing 50% CAR-T and 50% M cells. However, the killing effect of the 50% CAR-T and 50% M groups was significantly superior to that of the CAR-T group, indicating that CAR-T and M cells have a synergistic effect (1+1>2).

[0124] 5. Killing effects of primary human CAR-M and primary T cells on tumor cells

[0125] The experiment was divided into four groups: Control group; CAR-M group; T cell group; T cell and CAR-M combination group; and 50% T and 50% CAR-M group. Fluorescence intensity was collected and analyzed using a SPECTROstar Omega microplate reader to perform cell killing experiments. First, NKG2D CAR-M cells were cultured at a rate of 2 × 10⁻⁶ cells / year. 4 1 unit / well (CAR-M group; T and CAR-M combination group) or 1×104 Seeds were planted per well (50% T and 50% CAR-M groups) into opaque 96-well cell culture plates, with 10 replicates per group, and incubated for 24 h. After 24 h, PC-3 cells stably expressing luciferase and 2 × 10⁶ cells were added at an effector cell:target cell ratio of 1:1. 4 pcs / well (T group; T and CAR-M combined group) or 1×10 4 Number of Ts per well (50% T and 50% CAR-M group).

[0126] After co-culturing for 48 hours, the cells were incubated at 37°C in a 5% CO2 incubator for another 48 hours. Then, 100-200 μg / mL of D-luciferin and potassium salt substrate were added to opaque 96-well plates. After incubation at 37°C in the dark for 10 minutes, fluorescence intensity was collected and analyzed using a SPECTROstar Omega microplate reader. The killing effect of each immune cell group on PC-3 target cells was calculated using the following formula:

[0127] % cell lysis (Lysis%) = [1 - (fluorescence signal of co-cultured cells - background fluorescence signal) /

[0128] (Fluorescence signal of PC-3 cells cultured alone - background fluorescence signal) *100

[0129] The results of PC-3 cell killing are attached. Figure 7 As shown, the 50% T and 50% CAR-M groups were significantly superior to the T and CAR-M groups, with T+CAR-M exhibiting the strongest killing effect. In the 50% T and 50% CAR-M combination group, the total cell count was the same as that of the T and CAR-M groups, containing 50% T and 50% CAR-M cells. However, the killing effect of the 50% T and 50% CAR-M groups was significantly better than that of the T and CAR-M groups, indicating that CAR-T and CAR-M have a synergistic effect (1+1>2).

[0130] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention. SEQUENCE LISTING <110> Shenzhen Institutes of Advanced Technology <120> Application of CAR-T and CAR-M combination in the preparation of antitumor drugs <130> CP122010529C <160> 12 <170> PatentIn version 3.3 <210> 1 <211> 21 <212> PRT <213> artificial sequence <400> 1 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 2 <211> 144 <212> PRT <213> artificial sequence <400> 2 Ile Trp Ser Ala Val Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln 1 5 10 15 Ile Pro Leu Thr Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile 20 25 30 Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp 35 40 45 Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys 50 55 60 Val Tyr Ser Lys Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr 65 70 75 80 His Trp Met Gly Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp 85 90 95 Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met 100 105 110 Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile 115 120 125 Glu Asn Cys Ser Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val 130 135 140 <210> 3 <211> 45 <212> PRT <213> Artificial Sequence <400> 3 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 4 <211> 24 <212> PRT <213> Artificial Sequence <400> 4 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys 20 <210> 5 <211> 112 <212> PRT <213> Artificial sequence <400> 5 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 6 <211> 63 <212> DNA <213> Artificial sequence <400> 6 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccg 63 <210> 7 <211> 432 <212> DNA <213> artificial sequence <400> 7 atatggagtg ctgtattcct aaactcatta ttcaaccaag aagttcaaat tcccttgacc 60 gaaagttact gtggcccatg tcctaaaaac tggatatgtt acaaaaataa ctgctaccaa 120 ttttttgatg agagtaaaa ctggtatgag agccaggctt cttgtatgtc tcaaaatgcc 180 agccttctga aagtatacag caaagaggac caggatttac ttaaactggt gaagtcatat 240 cattggatgg gactagtaca cattccaaca aatggatctt ggcagtggga agatggctcc 300 attctctcac ccaacctact aacaataatt gaaatgcaga agggagactg tgcactctat 360 gcctcgagct ttaaaggcta tatagaaaac tgttcaactc caaatacgta catctgcatg 420 caaaggactg tg 432 <210> 8 <211> 135 <212> DNA <213> artificial sequence <400> 8 accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60 tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120 gacttcgcct gtgat 135 <210> 9 <211> 72 <212> DNA <213> Artificial sequence <400> 9 atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60 accctttact gc 72 <210> 10 <211> 336 <212> DNA <213> Artificial sequence <400> 10 agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180 gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240 cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300 tacgacgccc ttcacatgca ggccctgccc cctcgc 336 <210> 11 <211> 42 <212> PRT <213> Artificial sequence <400> 11 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Gly Cys Glu Leu 35 40 <210> 12 <211> 123 <212> DNA <213> The snowstorm <400> 12 60. aaacggggca cctgtatata ttcaaacaac catttatgag accagtacaa actactcaag aggagatgg ctgtagctgc cgatttccag aagaagaga aggagatgt gaa 123

Claims

1. The application of CAR-T and macrophage combined in the preparation of antitumor drugs, characterized in that, The CAR-T is a T cell modified with a chimeric antigen receptor. The primary protein structure of the chimeric antigen receptor, from the amino terminus to the carboxyl terminus, consists of: signal peptide, extracellular NKG2D region, hinge region, transmembrane region, intracellular co-stimulatory signal region, and intracellular signal region. The signal peptide is derived from human CD8α, and its amino acid sequence is shown in SEQ ID NO.1; The extracellular region of NKG2D is the extracellular domain of human NKG2D, and its amino acid sequence is shown in SEQ ID NO.2; The hinge region is derived from human CD8α, and its amino acid sequence is shown in SEQ ID NO.3; The transmembrane region is derived from human CD8α, and its amino acid sequence is shown in SEQ ID NO.4; The intracellular co-stimulatory signal region is selected from 4-1BB, and its amino acid sequence is shown in SEQ ID NO.11; The intracellular signaling region is CD3ζ, and its amino acid sequence is shown in SEQ ID NO.5; The ratio of CAR-T cells to macrophage cells is 1:1; The tumor is prostate cancer.

2. The application of CAR-M and T cell combination in the preparation of antitumor drugs, characterized in that, The CAR-M is a macrophage modified with a chimeric antigen receptor. The primary protein structure of the chimeric antigen receptor, from the amino terminus to the carboxyl terminus, consists of: signal peptide, extracellular NKG2D region, hinge region, transmembrane region, and intracellular signal region. The signal peptide is derived from human CD8α, and its amino acid sequence is shown in SEQ ID NO.1; The extracellular region of NKG2D is the extracellular domain of human NKG2D, and its amino acid sequence is shown in SEQ ID NO.2; The hinge region is derived from human CD8α, and its amino acid sequence is shown in SEQ ID NO.3; The transmembrane region is derived from human CD8α, and its amino acid sequence is shown in SEQ ID NO.4; The intracellular co-stimulatory signal region is selected from 4-1BB, and its amino acid sequence is shown in SEQ ID NO.11; The intracellular signaling region is CD3ζ, and its amino acid sequence is shown in SEQ ID NO.5; The ratio of CAR-M cells to T cells is 1:1; The tumor is prostate cancer.

3. The application of the combined use of CAR-T and CAR-M in the preparation of antitumor drugs, characterized in that, The CAR-T is a T cell modified with a chimeric antigen receptor. The primary protein structure of the chimeric antigen receptor, from the amino terminus to the carboxyl terminus, consists of: signal peptide, extracellular NKG2D region, hinge region, transmembrane region, intracellular co-stimulatory signal region, and intracellular signal region. The CAR-M is a macrophage modified with a chimeric antigen receptor. The primary protein structure of the chimeric antigen receptor, from the amino terminus to the carboxyl terminus, consists of: signal peptide, extracellular NKG2D region, hinge region, transmembrane region, and intracellular signal region. The signal peptide is derived from human CD8α, and its amino acid sequence is shown in SEQ ID NO.1; The extracellular region of the chimeric antigen receptor NKG2D in the CAR-T and CAR-M is the extracellular domain of human NKG2D, and its amino acid sequence is shown in SEQ ID NO.2; The hinge region is derived from human CD8α, and its amino acid sequence is shown in SEQ ID NO.3; The transmembrane region is derived from human CD8α, and its amino acid sequence is shown in SEQ ID NO.4; The intracellular co-stimulatory signal region is selected from 4-1BB, and its amino acid sequence is shown in SEQ ID NO.11; The intracellular signaling region is CD3ζ, and its amino acid sequence is shown in SEQ ID NO.5; The ratio of CAR-T to CAR-M cell numbers is 1:1; The tumor is prostate cancer.

4. The application according to claim 1 or 3, characterized in that, The CAR-T construction method is as follows: introducing the nucleic acid or vector encoding the CAR-T chimeric antigen receptor gene into T cells; The methods of introduction include electroporation, transduction, or transfection.

5. The application according to claim 4, characterized in that, The nucleic acids are located on different viral vectors; the viral vectors are lentiviral vectors, adenovirus vectors, or retroviral vectors.

6. The application according to claim 4, characterized in that, The vector is a transposon or mRNA vector.

7. The application according to claim 4, characterized in that, A lentiviral vector containing the CAR-T chimeric antigen receptor encoding gene was introduced into T cells via transfection.

8. The application according to claim 2 or 3, characterized in that, The CAR-M construction method is as follows: introducing the nucleic acid or vector of the CAR-M chimeric antigen receptor encoding gene into macrophage cells; The methods of introduction include electroporation, transduction, or transfection.

9. The application according to claim 8, characterized in that, The nucleic acids are located on different viral vectors; the viral vectors are lentiviral vectors, adenovirus vectors, or retroviral vectors.

10. The application according to claim 8, characterized in that, The vector is a transposon or mRNA vector.

11. The application according to claim 8, characterized in that, An adenovirus vector containing the CAR-M chimeric antigen receptor encoding gene was introduced into macrophage cells via transfection.

12. The application according to any one of claims 1-3, characterized in that, The macrophages are derived from healthy individuals or cancer patients; The macrophages are selected from autologous macrophages, allogeneic macrophages, or iPSC-induced macrophages; The T cells are derived from healthy individuals or cancer patients; The T cells are selected from autologous T cells, allogeneic T cells, or iPSC-induced T cells.

13. The application according to any one of claims 1-3, characterized in that, The macrophages mentioned are primary macrophages.

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