Chimeric cytokine receptor for activating lymphocytes by using soluble immunosuppressive molecules and application of chimeric cytokine receptor in tumor treatment

Abstract

The invention relates to the field of biological medicines, in particular to a chimeric cytokine activated receptor for activating lymphocytes by using soluble immunosuppressive molecules and application of the chimeric cytokine activated receptor in tumor treatment. The chimeric cytokine activation receptor and a chimeric antigen receptor for recognizing tumor antigens are connected in parallel to form a co-expressed polycistron structure. Wherein the chimeric cytokine activation receptor comprises an scFv structural domain, a transmembrane structural domain and a cytokine chimeric activation structural domain which specifically recognize and bind soluble immunosuppressive molecules. The CAR-NK cell can specifically recognize soluble inhibitory factors in the tumor microenvironment and improve the in-vivo and in-vitro anti-tumor efficacy of the CAR-NK cell, and can be used for treating solid malignant tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a chimeric cytokine activated receptor for activating lymphocytes using soluble immunosuppressive molecules and its application in tumor treatment. Background Art

[0002] In recent years, chimeric antigen receptor (CAR)-modified T cells have become one of the most popular technologies in the field of tumor immunotherapy. This technology enables T cells to specifically target tumor cell surface antigens by transfecting chimeric antigen receptors into T cells. At the same time, the intracellular domain of the chimeric antigen receptor can provide the first and second signals to activate T cells, making it a new generation of cell therapy technology with precise targeting, which can overcome the lack of immune cell infiltration and maintain sustained killing activity. CAR-T cells have shown excellent efficacy in the treatment of hematological tumors. However, at the same time, safety issues have also become a major problem that plagues the clinical application of CAR-T: cytokine storm, off-target effects, severe graft versus host disease (GvHD) risk and neurotoxicity, as well as safety risks brought by viral vectors, all seriously limit the further promotion of CAR-T.

[0003] Natural killer cells (NK) are a subset of killer cells that constitute the innate immune system and can quickly eliminate malignant transformed cells and virus-infected cells. NK cells do not need to be pre-sensitized to kill tumor cells, are not MHC-restricted, and have no risk of GvHD. Therefore, in recent years, the infusion of NK cells into patients after in vitro expansion and / or genetic modification (CAR-NK) has attracted much attention as a new cell immunotherapy. CAR-NK targeting CD19 has shown good clinical efficacy in the treatment of hematological malignancies, but there is limited evidence of clinical benefit of CAR-NK cell therapy for solid tumors. The tumor microenvironment is an important factor leading to the failure of NK adoptive reinfusion therapy, which seriously damages the phenotype, activation, function and persistence of NK cells.

[0004] Immunosuppressive factors in the tumor microenvironment play important roles in tumor development and immune escape. These factors include various cytokines, metabolites, enzymes, and chemokines. Cytokines such as transforming growth factor-β (TGF-β), interleukin-6 (IL-6), and interleukin-4 (IL-4) weaken the anti-tumor immune response by inhibiting the function of natural killer (NK) cells and inducing the generation of regulatory T cells (Tregs). Metabolites such as lactic acid and adenosine inhibit the function of immune cells by acidifying the tumor microenvironment. Indoleamine 2,3-dioxygenase (IDO) and arginase 1 (ARG1) inhibit T cell activity by degrading key amino acids. Chemokines such as CCL2 and CXCL12 further enhance the immunosuppressive environment by recruiting immunosuppressive cells. These soluble factors significantly inhibit the anti-tumor immunity of endogenous and cellular drugs and promote tumor progression. Therefore, designing targeted cellular drugs that recognize these factors has become an important direction in tumor immunotherapy.

[0005] IL-2 is an activating cytokine for NK cells, and the IL-2 receptor is a multi-subunit receptor complex composed of IL2Rα, IL2Rβ, and IL2Rγ. After activation of the IL-2 receptor, conformational changes occur in the intracellular region of the receptor, thereby activating downstream signaling molecules, which in turn phosphorylate members of the intracellular tyrosine kinase (JAK) family and recruit and phosphorylate members of the signal transducer and activator of transcription (STAT) family, such as STAT5. The phosphorylated STAT proteins form dimers and enter the nucleus, binding to the promoter region of specific genes to promote the activation and proliferation of NK cells. Therefore, artificially designing a chimeric antigen receptor that recognizes soluble immunosuppressive molecules and tandemly connecting the intracellular domains of IL2Rβγ is expected to achieve the activation of CAR-NK cells in the tumor microenvironment of solid tumors, thereby improving the clinical efficacy of CAR-NK ( Figure 1 ). Summary of the Invention

[0006] The object of the present invention is to provide a chimeric cytokine-activated receptor that activates lymphocytes using soluble immunosuppressive molecules and its application in tumor treatment.

[0007] To achieve the above object, in the first aspect of the present invention, a chimeric antigen receptor is provided, which includes a signal peptide, an antigen-binding domain, a hinge region, a transmembrane domain, and an intracellular domain:

[0008] The signal peptide is the signal peptide of a protein selected from the following group: the OSM signal peptide, whose amino acid sequence is as shown in Seq.01 and nucleotide sequence is as shown in Seq.02; the IL-2 signal peptide, whose amino acid sequence is as shown in Seq.03 and nucleotide sequence is as shown in Seq.04; the HSA signal peptide, whose amino acid sequence is as shown in Seq.05 and nucleotide sequence is as shown in Seq.06; the CD8α signal peptide, whose amino acid sequence is as shown in Seq.07 and nucleotide sequence is as shown in Seq.08; the insulin signal peptide, whose amino acid sequence is as shown in Seq.09 and nucleotide sequence is as shown in Seq.10; the trypsinogen 2 signal peptide, whose amino acid sequence is as shown in Seq.11 and nucleotide sequence is as shown in Seq.12.

[0009] Preferably, in an application example of the present invention, the amino acid and nucleotide sequences of the signal peptide are as shown in Seq.07 and Seq.08 respectively.

[0010] The antigen-binding domain is an scFv selected from the following group: αGPC3 scFv, whose corresponding amino acid and nucleotide sequences are as shown in Seq.13 and Seq.14 respectively; αCD19 scFv, whose corresponding amino acid and nucleotide sequences are as shown in Seq.15 and Seq. No. 16 respectively; αMSLN scFv, whose corresponding amino acid and nucleotide sequences are as shown in Seq.17 and Seq.18 respectively; αHER2 scFv, whose corresponding amino acid and nucleotide sequences are as shown in Seq.19 and Seq.20 respectively; αPSMA scFv, whose corresponding amino acid and nucleotide sequences are as shown in Seq.21 and Seq.22 respectively. Preferably, in an application example of the present invention, the amino acid and nucleotide sequences of the antigen-binding domain are as shown in Seq.13 and Seq.14 respectively.

[0011] The hinge region connects the extracellular domain of the chimeric antigen receptor to the transmembrane region, and the hinge region is selected from the following proteins: CD8α, whose amino acid sequence is as shown in Seq.23 and nucleotide sequence is as shown in Seq.24; CD28, whose amino acid sequence is as shown in Seq.25 and nucleotide sequence is as shown in Seq.26; IgG1, whose amino acid sequence is as shown in Seq.27 and nucleotide sequence is as shown in Seq.28; IgG4, whose amino acid sequence is as shown in Seq.29 and nucleotide sequence is as shown in Seq.30.

[0012] Preferably, in an application example of the present invention, the amino acid sequence of the hinge region is as shown in Seq.23.

[0013] The transmembrane domain is selected from the following proteins: CD4, whose amino acid sequence is shown in Seq.31 and nucleotide sequence is shown in Seq.32; CD8α, whose amino acid sequence is shown in Seq.33 and nucleotide sequence is shown in Seq.34; CD28, whose amino acid sequence is shown in Seq.35 and nucleotide sequence is shown in Seq.36; NKG2D, whose amino acid sequence is shown in Seq.37 and nucleotide sequence is shown in Seq.38.

[0014] Preferably, in an application example of the present invention, the amino acid sequence of the transmembrane domain is shown in Seq.31.

[0015] The intracellular domain can be composed of 2 to 4 tandem signal transduction domains or co-stimulatory domains of the following molecules, including: CD28, whose amino acid sequence is shown in Seq.39 and nucleotide sequence is shown in Seq.40; CD137, whose amino acid sequence is shown in Seq.41 and nucleotide sequence is shown in Seq.42; CD3ζ, whose amino acid sequence is shown in Seq.43 and nucleotide sequence is shown in Seq.44; DAP10, whose amino acid sequence is shown in Seq.45 and nucleotide sequence is shown in Seq.46; DAP12, whose amino acid sequence is shown in Seq.47 and nucleotide sequence is shown in Seq.48; IL2Rγ, whose amino acid sequence is shown in Seq.49 and nucleotide sequence is shown in Seq.50; ICOS, whose amino acid sequence is shown in Seq.51 and nucleotide sequence is shown in Seq.52; OX40, whose amino acid sequence is shown in Seq.53 and nucleotide sequence is shown in Seq.54; NCR3, whose amino acid sequence is shown in Seq.55 and nucleotide sequence is shown in Seq.56.

[0016] Preferably, in an application example of the present invention, the intracellular domain is composed of the tandem of Seq.39 and Seq.45.

[0017] In the second aspect of the present invention, a chimeric cytokine-activated receptor that recognizes a soluble inhibitory factor and converts an inhibitory signal into an activation signal is provided, including a signal peptide, a soluble molecule binding domain that recognizes a soluble inhibitory factor, a hinge region, a transmembrane domain, and an intracellular domain of the chimeric cytokine-activated receptor that converts an inhibitory signal into an activation signal; wherein, the signal peptide, hinge region, and transmembrane domain are the same as those in the first aspect of the invention.

[0018] The soluble molecular binding domains for identifying soluble inhibitory factors are scFvs selected from the following group: scFvs that can specifically bind TGF-β, the corresponding amino acid and nucleotide sequences are shown in Seq. 57 and Seq. 58 respectively; scFvs that can specifically bind soluble or membrane-shed PD-L1, the corresponding nucleotide and amino acid sequences are shown in Seq. 59 and Seq. 60 respectively; scFvs that can specifically bind VEGF, the corresponding nucleotide and amino acid sequences are shown in Seq. 61 and Seq. 62 respectively; scFvs that can specifically bind CD73, the corresponding nucleotide and amino acid sequences are shown in Seq. 63 and Seq. 64 respectively; scFvs that can specifically bind IL-10, the corresponding amino acid and nucleotide sequences are shown in Seq. 65 and Seq. 66 respectively.

[0019] Preferably, in an application example of the present invention, Seq. 58 is used as the extracellular soluble molecular binding domain. Preferably, in an application example of the present invention, Seq. 62 is used as the soluble molecular binding domain.

[0020] The intracellular domains of the chimeric cytokine-activated receptors that convert inhibitory signals into activating signals are selected from the following group: the intracellular segment of IL2Rβ, the corresponding nucleotide and amino acid sequences are shown in Seq. 67 and Seq. 68 respectively; the intracellular segment of IL2Rγ, the corresponding nucleotide and amino acid sequences are shown in Seq. 69 and Seq. 70 respectively; the intracellular segment of IFNGR1, the corresponding nucleotide and amino acid sequences are shown in Seq. 71 and Seq. 72 respectively; the intracellular segment of IFNGR2, the corresponding nucleotide and amino acid sequences are shown in Seq. 73 and Seq. 74 respectively; the intracellular segment of IL12Rβ1, the corresponding nucleotide and amino acid sequences are shown in Seq. 75 and Seq. 76 respectively; the intracellular segment of IL12Rβ2, the corresponding nucleotide and amino acid sequences are shown in Seq. 77 and Seq. 78 respectively; the intracellular domain of IL18Rβ, the corresponding nucleotide and amino acid sequences are shown in Seq. 79 and Seq. 80 respectively.

[0021] Preferably, in an application example of the present invention, Seq. 68 and Seq. 70 are used in tandem to form the intracellular domain of the chimeric cytokine-activated receptor.

[0022] In the third aspect of the present invention, a polycistronic structure co-expressing a chimeric antigen receptor targeting a tumor antigen and a chimeric cytokine-activated receptor is provided, and the sequence fragments can be directly linked by P2A, T2A, E2A or F2A peptides; the amino acid sequence of the polycistronic structure is selected from any one of Seq. 81 to 84.

[0023] Preferably, in an application example of the present invention, Seq.82 is used as a polycistronic structure to establish a cell line.

[0024] In a fourth aspect of the present invention, there is provided a method for establishing a cell line using the above-mentioned polycistronic structure co-expressing a chimeric antigen receptor targeting a tumor antigen and a chimeric cytokine-activated receptor, with the NK92 cell line as the host cell, wherein the cytokine is VEGF or TGF-β, comprising the following steps:

[0025] A. Design gene elements

[0026] Design a polycistronic structure co-expressing a chimeric antigen receptor and a chimeric cytokine-activated receptor targeting the inhibitory cytokine VEGF according to the amino acid sequence, and the corresponding amino acid sequence is shown as Seq.85; design a polycistronic structure co-expressing a chimeric antigen receptor and a chimeric cytokine-activated receptor targeting the inhibitory cytokine TGFβ, and the corresponding amino acid sequence is shown as Seq.86.

[0027] B. Lentiviral vector construction

[0028] Clone the gene fragments (Seq.88) of the polycistronic structure co-expressing a chimeric antigen receptor and a chimeric cytokine-activated receptor targeting the inhibitory cytokine VEGF, and the polycistronic structure co-expressing a chimeric antigen receptor and a chimeric cytokine-activated receptor targeting the inhibitory cytokine TGFβ into the pLVX plasmid respectively. The lentiviral packaging plasmids are pMD2.G and pCMVR8.74; transfect the plasmids into Lenti-X-293 cells for culture. This process can be cultured for 48 hours, 60 hours or 72 hours. Preferably, in the present invention, it is cultured for 72 hours. Collect the virus supernatant at 36 hours during the culture, store it at 4°C, supplement fresh medium and culture for another 36 hours, and then collect the virus supernatant; mix the two virus supernatants and centrifuge at 4°C and 20000 rpm for 2.5 hours, then discard the supernatant, collect the virus particle precipitate, and resuspend the virus with 50 μL - 100 μL of serum-free medium;

[0029] C. Establishment of CAR-NK cells targeting VEGF

[0030] To construct CAR-NK cells that recognize VEGF and convert the inhibitory signal into an activation signal, it can be selected to infect low-passage NK92 cells with the lentivirus carrying the polycistronic structure co-expressing a chimeric antigen receptor and the structure recognizing VEGF, or on the basis of CAR-NK cells (hereinafter referred to as BBz), infect low-passage BBz cells with the lentivirus carrying the gene of the inhibitory signal-activated chimeric cytokine receptor recognizing VEGF.

[0031] Preferably, in an application example of the present invention, low-passage BBz cells are infected with lentivirus carrying the chimeric cytokine receptor structural gene that recognizes VEGF. The specific procedure is as follows: Resuscitate low-passage BBz cells and passage them 3 to 4 times; Plate the cells. You can choose to plate 48-well plates at 5×10 4 / well, plate 24-well plates at 1×10 5 / well, and plate 6-well plates at 5×10 5 / well (preferably, in an application example of the present invention, the plating density used is 1×10 5 / well for plating 24-well plates). Incubate at 37°C and 5% CO 2Incubate for 2 to 4 hours (in one application example of the present invention, 4 hours of incubation is selected). Add polybrene to each well, and its final concentration can be selected as 1 μg / mL, 2 μg / mL, 4 μg / mL, 5 μg / mL or 6 μg / mL (preferably, in one application example of the present invention, 2 mg / mL polybrene is used for the BBz cell line). After thorough mixing, add the coated virus according to a certain MOI, and the MOI can be selected as MOI = 10, MOI = 20, MOI = 30, MOI = 40, and MOI = 50 (preferably, in one application example of the present invention, add the coated virus according to MOI = 30). After 12 hours of infection, aspirate all the cell suspension containing cells and virus and transfer it to a culture flask containing complete medium. Culture until the 4th day, pick monoclonal cell clusters into a 24-well plate for continued culture. A total of 24 single cell clusters are picked. After the cells in the 24-well plate are expanded, transfer them to a 6-well plate for further amplification. Add Brefeldin A to each well 12 hours before flow cytometry verification, and the concentration that can be used is 1 to 50 μM (preferably, in one application example of the present invention, add Brefeldin A at a working concentration of 10 μM). Take the BBz cells that recognize the chimeric cytokine-activated receptor structural gene of VEGF and the BBz cells after fixation and permeabilization, and label them with the flow antibody anti-HA-PE (the chimeric cytokine-activated receptor structural gene targeting TGFβ carries an HA-TAG) to identify the expression of the chimeric cytokine-activated receptor; and record the doubling time of each monoclonal. Finally, select the monoclonal with the highest HA-TAG positive ratio and the shortest doubling time, continue to expand the culture and transfer it to a 6-well plate. Take the expanded positive cells expressing the chimeric cytokine-activated receptor that recognizes VEGF and the BBz cells and label them with the flow antibody anti-HA-PE to identify the HA-TAG expression. Again, select the monoclonal with the highest HA-TAG positive ratio and the shortest doubling time, and complete the establishment of the cell line after expanding the culture. According to this construction method, the cell population after the establishment of the cell line is homogeneous, and the chimeric cytokine-activated receptor shows positive expression. The stability of the expression of the chimeric cytokine-activated receptor of the VEGF-recognizing BBz cells after continuous passage is detected by FACS. The CAR-NK cells that recognize VEGF are hereinafter referred to as BBz-αVEGF ( Figure 2 ).

[0032] D. Establishment of the BBz cell line that recognizes TGFβ and converts the inhibitory signal into an activating signal

[0033] To construct BBz cells that recognize TGFβ and convert inhibitory signals into activating signals, one can choose to infect low-passage NK cells with a lentivirus carrying a polycistronic structure that co-expresses a chimeric antigen receptor and recognizes TGFβ. Alternatively, based on BBz cells, low-passage BBz cells can be infected with a lentivirus carrying the structural gene of a chimeric cytokine-activated receptor that recognizes TGFβ.

[0034] Preferably, in an application example of the present invention, low-passage BBz cells are infected with a lentivirus carrying the structural gene of a chimeric cytokine-activated receptor that recognizes TGFβ. The specific procedure is as follows: Resuscitate low-passage BBz cells and passage them 3 to 4 times; for cell plating, one can choose to plate 5×10 4 cells per well in a 48-well plate, 1×10 5 cells per well in a 24-well plate, and 5×10 5 cells per well in a 6-well plate (preferably, in an application example of the present invention, the plating density used is 1×10 5 cells per well in a 24-well plate). Incubate at 37°C in 5% CO 2Incubate for 2 to 4 hours (in an application example of the present invention, 4 hours of incubation is selected). Add polybrene to each well, and its final concentration can be selected as 1 μg / mL, 2 μg / mL, 4 μg / mL, 5 μg / mL or 6 μg / mL (preferably, in an application example of the present invention, 2 mg / mL polybrene is used for the BBz cell line). After thorough mixing, add the coated virus according to a certain MOI, and the MOI can be selected as MOI = 10, MOI = 20, MOI = 30, MOI = 40, and MOI = 50 (preferably, in an application example of the present invention, add the coated virus according to MOI = 30). After 12 hours of infection, aspirate all the cell fluid containing cells and virus and transfer it to a culture flask containing complete medium. Culture until the 4th day, pick monoclonal cell clusters into a 24-well plate for continued culture. A total of 24 single cell clusters are picked. After the cells in the 24-well plate are expanded, transfer them to a 6-well plate for further amplification. Add Brefeldin A to each well 12 hours before flow cytometry verification, and the concentration that can be used is 1 to 50 μM (preferably, in an application example of the present invention, add Brefeldin A at a working concentration of 10 μM). Take the BBz cells expressing the chimeric cytokine-activated receptor structure gene recognizing TGFβ and the BBz cells after fixation and permeabilization, and label them with the flow antibody anti-HA-PE (the chimeric cytokine-activated receptor structure gene targeting TGFβ carries an HA-TAG) to identify the expression of the chimeric cytokine-activated receptor; and record the doubling time of each monoclonal. Finally, select the monoclonal with the highest HA-TAG positive ratio and the shortest doubling time, continue to expand the culture and transfer it to a 6-well plate. Take the expanded positive cells expressing the chimeric cytokine-activated receptor recognizing TGFβ and the BBz cells and label them with the flow antibody anti-HA-PE to identify the HA-TAG expression. Again, select the monoclonal with the highest HA-TAG positive ratio and the shortest doubling time, and complete the establishment of the cell line after expansion culture. According to this construction method, the cell population after cell line establishment is uniform, the chimeric cytokine-activated receptor shows positive expression, and the stability of the expression of the chimeric cytokine-activated receptor of the CAR-NK cells recognizing TGFβ in continuous passage is detected by FACS. The CAR-NK cells recognizing TGFβ are hereinafter referred to as BBz-αTGFβ ( Figure 3 )

[0035] In the fifth aspect of the present invention, there is provided the use of the chimeric antigen receptor, chimeric cytokine-activated receptor, and polycistronic structure co-expressing the chimeric antigen receptor and chimeric cytokine-activated receptor targeting tumor antigens as described above in the preparation of tumor therapeutic drugs.

[0036] Preferably, the tumor is a solid malignant tumor.

[0037] In an application example of the present invention, the tumor is liver cancer, gastric cancer, pancreatic cancer or breast cancer.

[0038] The advantages of the present invention are as follows:

[0039] 1. The stromal cells and immunosuppressive cells in the tumor microenvironment (TME) of solid tumors are rich, and the high expression of VEGF promotes angiogenesis. The inhibitory factors secreted in large amounts by liver macrophages, such as transforming growth factor-β (TGF-β), prostaglandin E2 (PGE2), and adenosine and other soluble inhibitory molecules, are the biggest obstacles that prevent CAR-NK cells from effectively infiltrating tumors and exerting their tumor-suppressing efficacy. The present invention specifically recognizes the soluble inhibitory factors in the tumor microenvironment and improves the in vivo and in vitro anti-tumor efficacy of CAR-NK cells, and can be used for the treatment of solid malignant tumors.

[0040] 2. The present invention enables CAR-NK cells to simultaneously express chimeric cytokine-activated receptors that recognize VEGF or TGFβ through gene modification, promotes the tumor-suppressing activity of CAR-NK cells, realizes the recognition of inhibitory soluble molecules, and converts the inhibitory signal into an IL2Rβγ activation signal, enabling NK cells to be effectively activated and exert their tumor-suppressing function in the tumor microenvironment rich in inhibitory cytokines, significantly improving the clinical efficacy of CAR-NK, and providing a new strategy for promoting the in vivo and in vitro killing efficacy of CAR-NK cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Shows a schematic diagram of the chimeric cytokine receptor structure for activating CAR-NK cells using soluble immunosuppressive molecules. Among them, after recognizing extracellular soluble immunosuppressive molecules, the intracellular sequences of IL2-Rβ and γ dimerize, mediating the activation signal of NK cells ( Figure 1 Left); when there is no chimeric cytokine receptor structure that recognizes soluble immunosuppressive molecules, after the inhibitory molecule recognition receptor on the surface of CAR-NK cells recognizes the inhibitory signal, the cytotoxic activity of CAR-NK cells is inhibited ( Figure 1 Right);

[0042] Figure 2 Shows the detection of the expression of αVEGF in BBz-αVEGF cells by FACS ( Figure 2 A); FACS detection of the stability of the expression of the chimeric cytokine-activated receptor αVEGF in continuously passaged BBz cells ( Figure 2 B);

[0043] Figure 3 Shows the detection of the expression of the chimeric cytokine-activated receptor in BBz-αTGFβ cells by FACS ( Figure 3A); Detection of the expression stability of chimeric cytokine-activated receptors in BBz-αTGFβ cells after continuous passage by FACS ( Figure 3 B);

[0044] Figure 4 Proliferation efficiency of BBz-αTGFβ cells in medium with TGFβ added at gradient concentrations and proliferation efficiency of BBz-αVEGF cells in medium with VEGF added at gradient concentrations ( Figure 4 );

[0045] Figure 5 Lysis efficiency of effector cells NK92, BBz, and BBz-αTGFβ cells against target cell HepG2 at effector-to-target ratios of 1:1, 2:1, and 5:1 ( Figure 5 A); Figure 5 B is the IFN-γ secretion after co-culture of effector cells NK92, BBz, and BBz-αTGFβ cells with HepG2 at an effector-to-target ratio of 1:1 ( Figure 5 B);

[0046] Figure 6 Lysis efficiency of CAR-NK cells that recognize TGFβ and target pancreatic cancer mesothelin (MSLN), gastric cancer Claudin-18.2 (CLDN18.2), and breast cancer human epidermal growth factor receptor 2 (HER2) against target cells AsPC-1, MKN45, and BT-474 at effector-to-target ratios of 1:1, 2:1, and 5:1.

[0047] Figure 7 Activation of the Jak / STAT signaling pathway detected by Western-Blot after 30 min, 60 min, and 120 min of interaction between BBz and BBz-αTGFβ cells and HepG2 cells;

[0048] Figure 8 Apoptosis of cells detected by flow cytometry after 30 min, 60 min, and 120 min of interaction between BBz and BBz-αTGFβ cells and HepG2 cells;

[0049] Figure 9 Inhibitory effects of CAR-NK cells that recognize TGFβ and target MSLN, CLDN18, and HER2 on subcutaneous xenografts of pancreatic cancer AsPC-1, gastric cancer MKN45, and breast cancer BT-474 cells;

[0050] Figure 10The tumor volume indicates the inhibitory effects of NK92, BBz, and BBz-αTGFβ cells on subcutaneous transplanted Hep G2 tumors and their effects on the survival of tumor-bearing mice. Detailed implementation manners

[0051] The following describes in detail the specific implementation manners provided by the present invention in conjunction with the embodiments.

[0052] Example 1: Construction and stability detection of BBz-αVEGF cell line

[0053] Experimental method:

[0054] 1. Lentivirus packaging of αVEGF chimeric cytokine-activated receptor: Clone the αVEGF chimeric cytokine-activated receptor sequence (Seq.86) into the lentiviral vector, co-transfect the Lenti-X-293 cell line with the packaging plasmid, and continuously culture for 72 hours (collect the cell culture medium once during this period, store it at 4°C, and add fresh cell culture medium to the cell culture dish to continue culturing). Collect the virus supernatant and filter it. Concentrate the virus by centrifugation at 20,000 rpm and 4°C for 2.5 hours, resuspend the virus particles, and quantify the titer.

[0055] 2. Establishment of BBz-αVEGF cell line: Infect BBz cells with lentivirus at MOI = 30, expand the culture, pick monoclonal colonies, expand the culture to a 24-well plate, label the cells with the flow antibody anti-HA TAG-PerCP / Cyanine5.5 (add Brefeldin A 12 hours before labeling, then fix and permeabilize the cells), and identify the expression of HA TAG by flow cytometry; record the doubling time of each monoclonal colony. Finally, select the monoclonal colony with the highest positive ratio of BBz cells expressing HA TAG and the shortest doubling time, continue to expand the culture and transfer it to a 6-well plate, pick monoclonal colonies again, verify the expression of HA TAG after amplification, and again select the monoclonal colony with the highest positive ratio of BBz cells expressing HA TAG and the shortest doubling time to complete the establishment of the cell line. According to this construction method, the cell population after establishment is homogeneous, and HA TAG (i.e., αVEGF chimeric cytokine-activated receptor) shows positive expression. Detect the stability of the expression of αVEGF chimeric cytokine-activated receptor in continuously passaged BBz-αVEGF cells by FACS.

[0056] 3. Stability detection of BBz-αVEGF cells: Continuously culture and passage BBz-αVEGF cells in complete medium, and use the anti-HA TAG-PerCP / Cyanine5.5 flow antibody to detect the positive rate of αVEGF chimeric cytokine-activated receptor in the established cells every 7 days.

[0057] Experimental results:

[0058] Detection of the stability of BBz-αVEGF cells after establishing the cell line: The BBz-αVEGF cell line has a uniform cell population, and the HA TAG shows positive expression with a good expression abundance ( Figure 2 A). During the continuous 8-week culture period, the stability of BBz-αVEGF cells after establishing the cell line was detected: The BBz-αVEGF cells have good stability, and the positive rate of the αVEGF chimeric cytokine-activated receptor remains stable above 95% ( Figure 2 B).

[0059] Example 2: Construction and stability detection of the BBz-αTGFβ cell line

[0060] Experimental method:

[0061] 1. Lentivirus packaging of the αTGFβ chimeric cytokine-activated receptor: Clone the αTGFβ chimeric cytokine-activated receptor sequence (Seq.88) into the lentiviral vector, and co-transfect the Lenti-X-293 cell line with the packaging plasmid. Continuously culture for 72 hours (collect the cell culture medium once during this period, store it at 4°C and add fresh cell culture medium to the cell culture dish to continue the culture), collect the virus supernatant and filter it. Concentrate the virus by centrifugation at 20000 rpm and 4°C for 2.5 hours, and quantify the titer after resuspending the virus particles.

[0062] 2. Establishment of the BBz-αTGFβ cell line: Infect BBz cells with lentivirus at MOI = 30, pick monoclonal colonies after expanded culture, expand the culture to a 24-well plate, label the cells with the flow antibody anti-HA TAG-PE (add Brefeldin A 12 hours before labeling, then fix and permeabilize the cells), and identify the expression of HA TAG by flow cytometry; record the doubling time of each monoclonal colony. Finally, select the monoclonal colony with the highest positive proportion of BBz cells expressing HA TAG and the shortest doubling time, continue to expand the culture and transfer it to a 6-well plate, pick monoclonal colonies again, verify the expression of HA TAG after amplification, and select the monoclonal colony with the highest positive proportion of BBz cells expressing HA TAG and the shortest doubling time again to complete the establishment of the cell line. According to this construction method, the cell population of the established cell line is uniform, and HA TAG (i.e., the αTGFβ chimeric cytokine-activated receptor) shows positive expression. Detect the stability of the expression of the αTGFβ chimeric cytokine-activated receptor in continuously passaged BBz-αTGFβ cells by FACS.

[0063] 3. Detection of the stability of BBz-αTGFβ cells: Continuously culture and passage BBz-αTGFβ cells in the complete medium, and use the anti-HA TAG-PE flow antibody to detect the positive rate of the αTGFβ chimeric cytokine-activated receptor in the established cells every 7 days.

[0064] Experimental results:

[0065] Detection of the stability of BBz-αTGFβ cells after establishing the coordinate system: The BBz-αTGFβ cell line has a uniform cell population, and the HA TAG shows positive expression with a good expression abundance ( Figure 3 A). During the continuous 8-week culture period, the stability of BBz-αTGFβ cells after establishing the coordinate system was detected: The stability of BBz-αTGFβ cells is good, and the positive rate of the αTGFβ chimeric cytokine-activated receptor is stable above 95% ( Figure 3 B).

[0066] Example 3: Amplification of BBz-αTGFβ cells under TGFβ culture conditions and amplification of BBz-αVEGF cells under VEGF culture conditions

[0067] Experimental method:

[0068] Cell killing experiment: Set the TGFβ concentration gradients in the NK medium to 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, and 25 ng / mL. The NK cell medium with different TGFβ concentration gradients was used to continuously culture BBz cells and BBz-αTGFβ cells for 21 days, and the amplification multiples of BBz cells and BBz-αTGFβ cells were detected.

[0069] Cell killing experiment: Set the VEGF concentration gradients in the NK medium to 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 3 ng / mL, and 5 ng / mL. The NK cell medium with different VEGF concentration gradients was used to continuously culture BBz cells and BBz-αVEGF cells for 21 days, and the amplification multiples of BBz cells and BBz-αVEGF cells were detected.

[0070] Experimental results:

[0071] The results show that as the TGFβ concentration in the medium increases, BBz cells cannot proliferate and gradually apoptose, while the amplification multiple of BBz-αTGFβ cells increases significantly; as the VEGF concentration in the medium increases, BBz cells cannot proliferate and gradually apoptose, while the amplification multiple of BBz-αVEGF cells increases significantly ( Figure 4 ).

[0072] Example 4: The αTGFβ chimeric cytokine-activated receptor significantly enhances the killing activity of BBz cells

[0073] Experimental method:

[0074] Cytotoxicity assay: The effector-to-target ratios (E:T) of NK92, BBz, and BBz-αTGFβ cells to the GPC3-high-expressing hepatocellular carcinoma cell line Hep G2 target cells were set at 1:1, 2:1, and 5:1, and the killing time was 2 hours. The proportion of dead tumor cells was detected by LDH. After cell fixation and permeabilization, flow antibodies (Biolegend, 308504) were labeled to detect IFN-γ secretion.

[0075] Experimental results:

[0076] The results showed that both BBz and BBz-αTGFβ cells could kill HepG2 target cells; however, compared with BBz cells, the killing activity of BBz-αTGFβ cells against HepG2 target cells was significantly increased ( Figure 5 A), and the IFN-γ expression was significantly increased ( Figure 5 B).

[0077] Example 5: αTGFβ chimeric cytokine-activated receptor significantly enhances the killing activity of CAR-NK cells targeting different antigens

[0078] Experimental method:

[0079] Cytotoxicity assay: The effector-to-target ratios of CAR-NK cells that recognize TGFβ and target mesothelin (MSLN), Claudin-18 (CLDN18), and human epidermal growth factor receptor 2 (HER2) to the corresponding target cells AsPC-1, MKN45, and BT-474 were set at 1:1, 2:1, and 5:1, and the killing time was 2 hours. The proportion of dead tumor cells was detected by LDH.

[0080] Experimental results:

[0081] The results showed that the killing activity of CAR-NK cells targeting MSLN, CLDN18, and HER2 and simultaneously recognizing TGFβ was significantly increased compared with the control group cells ( Figure 6 ).

[0082] Example 6: Study on the signal transduction pathways activated by BBz-αTGFβ cells

[0083] Experimental method:

[0084] Western-blot: After co-culturing BBz cells and BBz-αTGFβ cells with tumor cells at a ratio of 2:1 for 30 min, 5×10 of BBz cells and BBz-αTGFβ cells were collected respectively 6Protein was extracted using a protein extraction reagent (Thermo Scientific, 78503), and protein quantification was performed using a BCA protein quantification kit (Thermo Scientific, 23222). After quantification, the protein concentrations of each group of samples were adjusted to be consistent, and Western Blot was used to verify the activation of the JAK-STAT pathway.

[0085] Experimental results:

[0086] The difference in the activation of the JAK-STAT pathway between BBz cells and BBz-αTGFβ cells was compared. The results showed that the phosphorylation level of the JAK-STAT pathway in BBz-αTGFβ cells was significantly increased compared to BBz ( Figure 7 ); β-actin was used as the internal reference.

[0087] Example 7: Apoptosis of BBz-αTGFβ cells

[0088] HepG2 cells were digested and collected, and 200 μL × 10 5 cell suspension was added to each well of a 24-well plate. The culture plate was placed in a 37°C, 5% CO 2 2 cell incubator for 6 hours until the cells adhered. The culture plate was taken out, and Transwell chambers were placed, and 200 μL × 10 5 NK92 cells, BBz cells, and BBz-αTGFβ cells were added respectively. The culture plate was placed in a 37°C, 5% CO 2 2 cell incubator for 8 h. All NK92 cells, BBz cells, and BBz-αTGFβ cells were collected and centrifuged at 300 G for 5 min at 4°C. Annexin V / PI was used to detect NK cell apoptosis.

[0089] Experimental results:

[0090] After co-culture, a certain degree of apoptosis occurred in both NK92 cells and BBz cells. BBz-αTGFβ cells did not show apoptotic characteristics. This phenomenon suggests that targeted inhibition of the cytokine TGFβ can effectively protect CAR-NK cells from apoptosis, thereby potentially improving their persistence and efficacy in tumor immunotherapy ( Figure 8 ).

[0091] Example 8: In vivo subcutaneous tumor killing effect of CAR-NK cells targeting different tumor cell targets that recognize TGFβ on different tumor cells

[0092] Experimental method: NSG mice were subcutaneously injected with 1 × 10 7Individuals / mice were used to establish tumor-bearing mouse models. After 7 days, NK92, αMSLN CAR-NK, and αMSLN / αTGFβ CAR-NK cells were respectively administered for treatment (intravenous injection via the tail vein at a dose of 1×10 7 , once every 5 days, for a total of 3 times). The therapeutic effects of each group were compared.

[0093] NSG mice were subcutaneously injected with 5×10 6 individuals / mice of CLDN18.2-high-expressing gastric cancer MKN45 cells in the logarithmic growth phase to establish tumor-bearing mouse models. After 7 days, NK92, αCLDN18.2 CAR-NK, and αCLDN18.2 / αTGFβ CAR-NK cells were respectively administered for treatment (intravenous injection via the tail vein at a dose of 1×10 7 , once every 5 days, for a total of 3 times). The therapeutic effects of each group were compared.

[0094] NSG mice were subcutaneously injected with 1×10 7 individuals / mice of HER2-high-expressing breast cancer BT-474 cells in the logarithmic growth phase to establish tumor-bearing mouse models. After 7 days, NK92, αHER2 CAR-NK, and αHER2 / αTGFβ CAR-NK cells were respectively administered for treatment (intravenous injection via the tail vein at a dose of 1×10 7 , once every 5 days, for a total of 3 times). The therapeutic effects of each group were compared.

[0095] Experimental results:

[0096] The therapeutic effects of αMSLN / αTGFβ CAR-NK cells ( Figure 9 A), αCLDN18.2 / αTGFβ CAR-NK cells ( Figure 9 B), and αHER2 / αTGFβ CAR-NK cells ( Figure 9 C) were all higher than those of the control group, and the tumor volumes of the tumor-bearing mice were significantly reduced.

[0097] Example 9: Killing effect of BBz-αTGFβ cells on subcutaneous tumors of HepG2 in vivo

[0098] Experimental method: NSG mice were subcutaneously injected with 1×10 7 individuals / mice of GPC3-high-expressing liver cancer HepG2 cells in the logarithmic growth phase to establish tumor-bearing mouse models. After 7 days, NK92, BBz, and BBz-αTGFβ cells were respectively administered for treatment (intravenous injection via the tail vein at a dose of 1×10 7 , once every 5 days, for a total of 3 times). The therapeutic effects of each group were compared.

[0099] Experimental results:

[0100] The therapeutic effect of BBz-αTGFβ cells is significantly better than that of BBz cells, with a significant reduction in tumor volume and a significant prolongation of the survival period of tumor-bearing mice ( Figure 10 ).

[0101] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art can also make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A chimeric antigen receptor, characterized in that Including signal peptide, antigen binding domain, hinge region, transmembrane domain and intracellular domain: The signal peptide is a signal peptide of a protein selected from the group consisting of an OSM signal peptide, whose amino acid sequence is shown in Seq.01 and whose nucleotide sequence is shown in Seq.02; an IL-2 signal peptide, whose amino acid sequence is shown in Seq.03 and whose nucleotide sequence is shown in Seq.04; an HSA signal peptide, whose amino acid sequence is shown in Seq.05 and whose nucleotide sequence is shown in Seq.06; a CD8α signal peptide, whose amino acid sequence is shown in Seq.07 and whose nucleotide sequence is shown in Seq.08; an insulin signal peptide, whose amino acid sequence is shown in Seq.09 and whose nucleotide sequence is shown in Seq.10; a trypsinogen 2 signal peptide, whose amino acid sequence is shown in Seq.11 and whose nucleotide sequence is shown in Seq.12; The antigen binding domain is selected from the following group of scFvs: αGPC3 scFv, the corresponding amino acid and nucleotide sequences are shown in Seq.13 and Seq.14, respectively; αCD19 scFv, the corresponding amino acid and nucleotide sequences are shown in Seq.15 and Seq.No. 16, respectively; αMSLN scFv, the corresponding amino acid and nucleotide sequences are shown in Seq.17 and Seq.18, respectively; αHER2 scFv, the corresponding amino acid and nucleotide sequences are shown in Seq.19 and Seq.20, respectively; αPSMA scFv, the corresponding amino acid and nucleotide sequences are shown in Seq.21 and Seq.22, respectively; The hinge region connects the extracellular domain of the chimeric antigen receptor to the transmembrane region, and the hinge region is selected from the following proteins: CD8α, whose amino acid sequence is shown in Seq.23 and whose nucleotide sequence is shown in Seq.24; CD28, whose amino acid sequence is shown in Seq.25 and whose nucleotide sequence is shown in Seq.26; IgG1, whose amino acid sequence is shown in Seq.27 and whose nucleotide sequence is shown in Seq.28; IgG4, whose amino acid sequence is shown in Seq.29 and whose nucleotide sequence is shown in Seq.30; The transmembrane domain is selected from the following proteins: CD4, whose amino acid sequence is shown in Seq.31 and whose nucleotide sequence is shown in Seq.32; CD8α, whose amino acid sequence is shown in Seq.33 and whose nucleotide sequence is shown in Seq.34; CD28, whose amino acid sequence is shown in Seq.35 and whose nucleotide sequence is shown in Seq.36; NKG2D, whose amino acid sequence is shown in Seq.37 and whose nucleotide sequence is shown in Seq.38; The intracellular domain is composed of 2 to 4 tandem signal transduction domains or co-stimulatory domains of the following molecules, including: CD28, whose amino acid sequence is shown in Seq.39 and whose nucleotide sequence is shown in Seq.40; CD137, whose amino acid sequence is shown in Seq.41 and whose nucleotide sequence is shown in Seq.42; CD3ζ, whose amino acid sequence is shown in Seq.43 and whose nucleotide sequence is shown in Seq.44; DAP10, whose amino acid sequence is shown in Seq.45 and whose nucleotide sequence is shown in Seq.46 as shown; DAP12, whose amino acid sequence is shown in Seq.47, and whose nucleotide sequence is shown in Seq.48; IL2Rγ, whose amino acid sequence is shown in Seq.49, and whose nucleotide sequence is shown in Seq.50; ICOS, whose amino acid sequence is shown in Seq.51, and whose nucleotide sequence is shown in Seq.52; OX40, whose amino acid sequence is shown in Seq.53, and whose nucleotide sequence is shown in Seq.54; NCR3, whose amino acid sequence is shown in Seq.55, and whose nucleotide sequence is shown in Seq.

56.

2. A chimeric cytokine-activated receptor that recognizes a soluble inhibitory factor and converts the inhibitory signal into an activation signal, characterized in that: It includes a signal peptide, a soluble molecule binding domain that recognizes soluble inhibitory factors, a hinge region, a transmembrane domain, and an intracellular domain of a chimeric cytokine-activated receptor that converts inhibitory signals into activation signals: The signal peptide is a signal peptide of a protein selected from the group consisting of an OSM signal peptide, whose amino acid sequence is shown in Seq.01 and whose nucleotide sequence is shown in Seq.02; an IL-2 signal peptide, whose amino acid sequence is shown in Seq.03 and whose nucleotide sequence is shown in Seq.04; an HSA signal peptide, whose amino acid sequence is shown in Seq.05 and whose nucleotide sequence is shown in Seq.06; a CD8α signal peptide, whose amino acid sequence is shown in Seq.07 and whose nucleotide sequence is shown in Seq.08; an insulin signal peptide, whose amino acid sequence is shown in Seq.09 and whose nucleotide sequence is shown in Seq.10; a trypsinogen 2 signal peptide, whose amino acid sequence is shown in Seq.11 and whose nucleotide sequence is shown in Seq.12; The hinge region connects the extracellular domain of the chimeric antigen receptor to the transmembrane region, and the hinge region is selected from the following proteins: CD8α, whose amino acid sequence is shown in Seq.23 and whose nucleotide sequence is shown in Seq.24; CD28, whose amino acid sequence is shown in Seq.25 and whose nucleotide sequence is shown in Seq.26; IgG1, whose amino acid sequence is shown in Seq.27 and whose nucleotide sequence is shown in Seq.28; IgG4, whose amino acid sequence is shown in Seq.29 and whose nucleotide sequence is shown in Seq.30; The transmembrane domain is selected from the following proteins: CD4, whose amino acid sequence is shown in Seq.31 and whose nucleotide sequence is shown in Seq.32; CD8α, whose amino acid sequence is shown in Seq.33 and whose nucleotide sequence is shown in Seq.34; CD28, whose amino acid sequence is shown in Seq.35 and whose nucleotide sequence is shown in Seq.36; NKG2D, whose amino acid sequence is shown in Seq.37 and whose nucleotide sequence is shown in Seq.38; The soluble molecule binding domain that recognizes the soluble inhibitory factor is selected from the following group of scFv: a scFv that can specifically bind to TGF-β, and the corresponding amino acid and nucleotide sequences are shown in Seq. 57 and Seq. 58, respectively; A scFv that can specifically bind to soluble or membrane-shed PD-L1, the corresponding nucleotide and amino acid sequences are shown in Seq. 59 and Seq. 60, respectively; A scFv that can specifically bind to VEGF, the corresponding nucleotide and amino acid sequences are shown in Seq.61 and Seq.62, respectively; A scFv that can specifically bind to CD73, the corresponding nucleotide and amino acid sequences are shown in Seq.63 and Seq.64, respectively; The scFv that can specifically bind to IL-10, the corresponding amino acid and nucleotide sequences are shown in Seq.65 and Seq.66, respectively; The intracellular domain of the chimeric cytokine-activated receptor that converts an inhibitory signal into an activating signal is selected from the following group: the IL2Rβ intracellular segment, the corresponding nucleotide and amino acid sequences are shown in Seq.67 and Seq. 68, respectively; the IL2Rγ intracellular segment, the corresponding nucleotide and amino acid sequences are shown in Seq.69 and Seq. 70, respectively; the IFNGR1 intracellular segment, the corresponding nucleotide and amino acid sequences are shown in Seq.71 and Seq.72, respectively; the IFNGR2 intracellular segment, the corresponding nucleotide and amino acid sequences are shown in Seq.73 and Seq.74, respectively; the IL12Rβ1 intracellular segment, the corresponding nucleotide and amino acid sequences are shown in Seq.75 and Seq.76, respectively; the IL12Rβ2 intracellular segment, the corresponding nucleotide and amino acid sequences are shown in Seq.77 and Seq.78, respectively; the IL18Rβ intracellular domain, the corresponding nucleotide and amino acid sequences are shown in Seq.79 and Seq.80, respectively.

3. A polycistronic structure co-expressing a chimeric antigen receptor targeting a tumor antigen and a chimeric cytokine-activated receptor, characterized in that: The sequence fragments are directly connected through P2A, T2A, E2A or F2A peptides; the amino acid sequence of the polycistronic structure is selected from any one of Seq.81~84.

4. A method for establishing a cell line using the polycistronic structure of a chimeric antigen receptor co-expressing a tumor-targeting antigen and a chimeric cytokine-activated receptor as claimed in claim 3, with NK92 cell line as a host cell, characterized in that: The cytokine is VEGF or TGF-β, comprising the following steps: A. Designing genetic elements A polycistronic structure for co-expressing a chimeric antigen receptor and a chimeric cytokine-activated receptor targeting an inhibitory factor VEGF is designed according to the amino acid sequence, and the corresponding amino acid sequence and nucleotide sequence are shown in Seq.85 and Seq.86, respectively; a polycistronic structure for co-expressing a chimeric antigen receptor and a chimeric cytokine-activated receptor targeting an inhibitory cytokine TGFβ is designed, and the corresponding amino acid sequence and nucleotide sequence are shown in Seq.87 and Seq.88, respectively; B. Lentiviral vector construction The gene fragments of the polycistronic structure of co-expressing chimeric antigen receptor and chimeric cytokine-activated receptor targeting inhibitory factor VEGF and the polycistronic structure of co-expressing chimeric antigen receptor and chimeric cytokine-activated receptor targeting inhibitory cytokine TGFβ were cloned into pLVX plasmid, and pMD2.G and pCMVR8.74 were used as lentiviral packaging plasmids; the plasmids were transfected into Lenti-X-293 cells and cultured for 48 hours, 60 hours or 72 hours, and the virus supernatant was collected in the middle of the culture and stored at 4°C. After supplementing with fresh culture medium and culturing again, the virus supernatant was collected; the two virus supernatants were mixed and centrifuged at 4°C and 20,000 rpm for 2.5 hours, and the supernatant was discarded, the virus particle precipitate was collected, and the virus was resuspended in 50 μL to 100 μL serum-free culture medium; C. Establishment of CAR-NK cell line targeting VEGF Construction of CAR-NK cells that recognize VEGF and convert inhibitory signals into activation signals: Low-passage NK92 cells are infected with lentiviruses carrying a polycistronic structure that co-expresses a chimeric antigen receptor and recognizes VEGF, or low-passage BBz cells are infected with lentiviruses carrying an inhibitory signal activation-type chimeric cytokine receptor gene that recognizes VEGF based on CAR-NK cells, hereinafter referred to as BBz cells; D. Construction of a BBz cell line that recognizes TGFβ and converts inhibitory signals into activation signals To construct BBz cells that recognize TGFβ and convert inhibitory signals into activation signals, low-passage NK cells are infected with a lentivirus carrying a polycistronic structure that co-expresses a chimeric antigen receptor and recognizes TGFβ, or, based on BBz cells, low-passage BBz cells are infected with a lentivirus carrying a chimeric cytokine activation receptor structural gene that recognizes TGFβ.

5. The method according to claim 4, characterized in that The method of infecting low-passage BBz cells with a lentivirus carrying a chimeric cytokine receptor gene that recognizes VEGF and inhibits signal activation is as follows: revive low-passage BBz cells, passage them 3 to 4 times; plate the cells, select 5×10 4 / well in a 48-well plate, 1×10 5 24-well plate, or 5 × 10 5 / well of a 6-well plate; incubate at 37°C, 5% CO2 for 2-4 hours; add polybrene to each well, and the final concentration is 1 μg / mL, 2 μg / mL, 4 μg / mL, 5μg / mL or 6 μg / mL; after thorough mixing, add the coated virus according to MOI=10, MOI=20, MOI=30, MOI=40 and MOI=50; after 12 hours of infection, aspirate all the cell fluid containing cells and viruses and transfer it to a culture bottle containing complete culture medium, culture until the 4th day, pick up a monoclonal cell cluster to continue culturing in a 24-well plate, pick up a total of 24 single cell clusters, expand the culture of the 24-well plate cells, and transfer them to a 6-well plate for further amplification; add BrefeldinA to each well 12 hours before flow cytometry verification, and the concentration used is 1-50 μM; take BBz cells expressing the chimeric cytokine-activated receptor structural gene that recognizes VEGF and fix and permeabilize BBz cells, label them with flow cytometry antibody anti-HA-PE, and the chimeric cytokine-activated receptor structural gene targeting TGFβ carries HA-TAG, and the expression of the chimeric cytokine-activated receptor is identified; and the doubling time of each monoclonal clone is recorded; finally, the monoclonal clone with the highest HA-TAG positive ratio and the shortest doubling time is selected, and the culture is continued to be expanded and moved to a 6-well plate, and the amplified chimeric cytokine-activated receptor-positive cells expressing and recognizing VEGF and BBz cells are taken and labeled with flow cytometry antibody anti-HA-PE to identify the expression of HA-TAG, and the monoclonal clone with the highest HA-TAG positive ratio and the shortest doubling time is selected again, and the culture is expanded to complete the establishment of the system.

6. The method according to claim 4, characterized in that The method of infecting low-passage BBz cells with a lentivirus carrying a chimeric cytokine receptor gene that recognizes TGFβ is as follows: revive low-passage BBz cells, passage them 3 to 4 times; plate the cells, select 5×10 4 / well in a 48-well plate, 1×10 5 24-well plate, 5×10 5 / well of a 6-well plate; culture at 37°C, 5% CO2 for 2-4 hours; add polybrene to each well, and the final concentration is 1 μg / mL, 2 μg / mL, 4 μg / mL, 5 μg / mL or 6 μg / mL; after thorough mixing, add the coated virus according to MOI=10, MOI=20, MOI=30, MOI=40 and MOI=50; after 12 hours of infection, aspirate all the cell fluid containing cells and viruses and transfer it to a culture bottle containing complete culture medium, culture until the 4th day, pick up monoclonal cell clusters to continue culture in a 24-well plate, pick up a total of 24 single cell clusters, expand the culture of the 24-well plate cells and transfer them to a 6-well plate for further amplification; add Brefeldin A to each well 12 hours before flow cytometry verification, with a concentration of 1-50 μM; take BBz cells expressing the structural gene of chimeric cytokine-activated receptor that recognizes TGFβ and fix and permeabilize BBz cells, label them with flow cytometry antibody anti-HA-PE, the structural gene of chimeric cytokine-activated receptor targeting TGFβ carries HA-TAG, and identify the expression of chimeric cytokine-activated receptor; and record the doubling time of each monoclonal clone; finally, select the monoclonal clone with the highest HA-TAG positive ratio and the shortest doubling time, continue to expand the culture and move to a 6-well plate, take the amplified chimeric cytokine-activated receptor-positive cells expressing and recognizing TGFβ and BBz cells, label them with flow cytometry antibody anti-HA-PE, identify the expression of HA-TAG, select the monoclonal clone with the highest HA-TAG positive ratio and the shortest doubling time again, and expand the culture to complete the establishment of the system.

7. Use of the chimeric antigen receptor according to claim 1, the chimeric cytokine-activated receptor according to claim 2, and the polycistronic structure of the chimeric antigen receptor and the chimeric cytokine-activated receptor co-expressing a tumor-targeting antigen according to claim 3 in the preparation of tumor therapeutic drugs.

8. The use according to claim 7, characterized in that: The tumor is a solid malignant tumor.

9. The use according to claim 8, characterized in that: The tumor is liver cancer, stomach cancer, pancreatic cancer or breast cancer.

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