Application of histone variant macroH2A in regulation of macrophage function in improvement of tumor immune microenvironment

Abstract

The invention discloses an application of a histone variant macroH2A for regulating and controlling a macrophage function in improvement of a tumor immune microenvironment. Specifically, the invention provides a macrophage with deletion of a macroH2A gene and application of the macrophage in tumor resistance, and further provides application of a reagent for detecting the expression quantity of macroH2A in the macrophage in preparation of a product for predicting prognosis of a patient with skin T cell lymphoma and / or liver cancer. The invention finds that the immunosuppression function of macrophages with deletion of the macroH2A gene is weakened, and the anti-tumor function is enhanced. The macrophage provided by the invention has an application prospect in the anti-tumor field.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology. Specifically, the present invention relates to the application of histone variant macroH2A in regulating macrophage function in improving tumor immune microenvironment. Background Art

[0002] At present, CAR-T therapy is widely used in clinical treatment of tumors. It has significant therapeutic effects on blood tumors, but for solid tumors, T cells cannot infiltrate into the corresponding tumor tissues, thus affecting the therapeutic effect. Macrophages are a type of natural immune cells that are ubiquitous in tumor tissues and play a key role in regulating tumor growth and anti-tumor immune response. A large number of studies have found that tumor-associated macrophages are in an immunosuppressive state, with characteristics similar to bypass-activated macrophages (M2). They have weak antigen presentation ability and can secrete a variety of cytokines to inhibit the activation of T cells, NK cells, etc., thereby promoting tumor development and metastasis. In clinical practice, the large enrichment of tumor-associated macrophages is highly correlated with reduced T cell infiltration and poor prognosis. By studying and developing small molecule compounds, new nanomaterials and other methods to reprogram tumor-associated macrophages into macrophages that promote immune response, it is expected to reverse the inhibitory tumor microenvironment and improve patient prognosis.

[0003] The histone variant macroH2A is a variant of the conventional histone H2A, which is widely present in vertebrates and is highly conserved. MacroH2A was first discovered to be closely associated with heterochromatin, including the inactive X chromosome of female mammalian cells and aging-related heterochromatin. However, the function of macroH2A in macrophages is still unclear. Summary of the invention

[0004] In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide an application of histone variant macroH2A in regulating macrophage function in improving tumor immune microenvironment.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] The first aspect of the present invention provides a genetically engineered macrophage.

[0007] Furthermore, the macroH2A gene in the macrophages is knocked out or knocked down.

[0008] Furthermore, the macrophages include commercial macrophages, primary macrophages, immortalized macrophages, tumor-associated macrophages (TAMs), and chimeric antigen receptor macrophages (CAR-Ms).

[0009] Preferably, the macrophages include Raw264.7 macrophages, THP-1 macrophages, immortalized bone marrow-derived mouse macrophages BMDM, and macrophages iMac differentiated from induced pluripotent stem cells (iPSCs).

[0010] More preferably, the macrophages are Raw264.7 macrophages.

[0011] Furthermore, compared with macrophages without macroH2A gene knockout or knockdown, the genetically engineered macrophages have one or more of the following characteristics:

[0012] (1) Low expression of genes related to immunosuppressive macrophages

[0013] (2) High expression of chemokine receptor protein or its mRNA;

[0014] (3) High expression of chemokine protein or its mRNA.

[0015] Furthermore, the immunosuppressive macrophage-related gene is selected from ARG1, IL10, IL4, Mrc1, TGFbeta or a combination thereof.

[0016] Furthermore, the chemokine receptor is selected from CCR1, CXCR3 or a combination thereof.

[0017] Furthermore, the chemokine is selected from CCL2, CCL3, CCL4, CCL5, CCL9, CXCL9, CXCL16 or a combination thereof.

[0018] In the present invention, the terms "gene inactivation" and "gene knockout" are used interchangeably, referring to a genetic operation such as interruption or knockout of a target gene, thereby significantly reducing or even completely losing the expression and / or activity of the target gene.

[0019] In the present invention, gene knockout can be achieved using a gene editor. The gene editor includes a DNA gene editor and an RNA gene editor. In a preferred embodiment, the gene editor of the present invention includes a gene editing protein and an optional gRNA.

[0020] In the present invention, the nucleotides of the gene editing protein can be obtained by genetic engineering techniques, such as genome sequencing, polymerase chain reaction (PCR), etc., and its amino acid sequence can be derived from the nucleotide sequence. In a preferred embodiment of the present invention, the gene editing protein includes but is not limited to Cas13 (such as CasRx), Cpf1, SaCas9, Cas13a, Cas13b, and Cas13c.

[0021] In the present invention, the "high expression" refers to: the ratio (F1 / F2) of the expression level of the protein or its mRNA in genetically engineered macrophages (F1) to the expression level (F2) in macrophages without gene knockdown is ≥2, preferably ≥3, and more preferably ≥4.

[0022] A second aspect of the present invention provides a pharmaceutical composition.

[0023] Furthermore, the pharmaceutical composition comprises the macrophages described in the first aspect of the present invention.

[0024] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier, diluent or excipient.

[0025] In some embodiments, the preparation is a liquid preparation. Preferably, the preparation is an injection. Preferably, the concentration of the macrophages in the preparation is 1×10 3 -1×10 8 cells / ml, more preferably, the concentration of macrophages in the preparation is 1×10 4 -1×10 7 cells / ml.

[0026] In some embodiments, the formulation may include a buffer such as neutral buffered saline, sulfate buffered saline, etc.; a carbohydrate such as glucose, mannose, sucrose or dextran, mannitol; a protein; a polypeptide or an amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The formulation of the present invention is preferably formulated for intravenous administration.

[0027] The dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The dosage ratio for human administration can be implemented according to practices accepted in the art. Typically, 1×10 6 to 1×10 10 The macrophages of the present invention are administered to a patient, for example, by intravenous infusion.

[0028] In the present invention, the term "therapeutically effective amount" refers to an amount that produces a function or activity in humans and / or animals and is acceptable to humans and / or animals. It should be understood by those skilled in the art that the "therapeutically effective amount" may vary depending on the form of the pharmaceutical composition, the route of administration, the adjuvants of the drug used, the severity of the disease, and the combination with other drugs.

[0029] In the present invention, the term "pharmaceutically acceptable" and its grammatical variants refer to compositions, carriers, diluents and agents that can be applied to a subject or applied to a subject without producing undesirable physiological effects to the extent that prevents the application of the composition. The term "excipient" or "carrier" refers to an inert substance added to a pharmaceutical composition to further promote the administration of a compound. The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" covers any of the agents approved by the regulatory agencies of the U.S. federal government or listed in the U.S. Animal (including human) Pharmacopoeia, as well as any carrier or diluent that does not cause considerable irritation to the subject and does not eliminate the biological activity and properties of the administered compound. Includes excipients and carriers that are suitable for preparing pharmaceutical compositions and are generally safe, non-toxic and desirable.

[0030] The third aspect of the present invention provides the use of a biomaterial that inhibits the expression of macroH2A in reversing the immunosuppressive function of macrophages, promoting the anti-tumor effect of macrophages and / or preparing macrophages with high killing effect.

[0031] Furthermore, the biological material includes coding genes, expression cassettes, recombinant vectors, cells, and proteins.

[0032] Furthermore, the macrophage immunosuppressive function refers to its ability to inhibit the activation of other immune cells and promote tumor growth.

[0033] Furthermore, the anti-tumor function of macrophages refers to having a strong killing effect on cancer cells and promoting the activation of other immune cells in the tumor microenvironment. The anti-tumor macrophages and highly killing macrophages described in the present invention have the same meaning and can be used interchangeably.

[0034] In the present invention, in vitro induction of macrophage polarization toward M2 is initiated by treatment with IL-4 and IL-13 (see, e.g., Liu, YC, Zou, XB, Chai, YF, & Yao, YM (2014). Macrophage polarization in inflammatory diseases. International Journal of Biological Sciences, 10(5), 520-529, incorporated by reference for all purposes).

[0035] In the present invention, the biological material for inhibiting the expression of macroH2A is sgRNA targeting macroH2A, and its specific sequence is GCGGGGGCGTGTTGCCGAAT.

[0036] A fourth aspect of the present invention provides a method for reducing the immunosuppressive function of macrophages and inducing the transformation of macrophages into anti-tumor macrophages.

[0037] In the present invention, the term "macrophage transformation" refers to a change that reduces the immunosuppressive ability of macrophages and induces the transformation of macrophages into anti-tumor macrophages.

[0038] Further, the method includes treating the macrophages with a biological material that inhibits the expression of macroH2A.

[0039] Furthermore, the biological material includes coding genes, expression cassettes, recombinant vectors, cells, and proteins.

[0040] A fifth aspect of the present invention provides the use of a reagent for detecting the expression level of macroH2A in macrophages in the preparation of a product for predicting the prognosis of patients with cutaneous T-cell lymphoma and / or liver cancer.

[0041] Furthermore, the expression level includes mRNA expression level and protein expression level.

[0042] Furthermore, the reagents for detecting the mRNA expression amount include reagents used in the following methods: PCR-based detection method, Southern hybridization method, Northern hybridization method, dot hybridization method, fluorescence in situ hybridization method, DNA microarray method, ASO method, and high-throughput sequencing platform method.

[0043] Furthermore, the reagents for detecting protein expression include reagents used in the following methods: hematoxylin-eosin staining, safranin O-fast green staining, protein blotting, enzyme-linked immunosorbent assay, radioimmunoassay, sandwich assay, immunohistochemical staining method, mass spectrometry, immunoprecipitation analysis, complement fixation analysis, flow cytometry fluorescence resolution technology and protein chip method.

[0044] Furthermore, the product includes a chip, a kit or a nucleic acid membrane strip.

[0045] In the present invention, prognosis refers to the expectation about medical development (e.g., long-term survival possibility, disease-free survival rate, etc.), including positive prognosis or negative prognosis, wherein the negative prognosis includes disease progression such as recurrence, growth, metastasis and drug-resistant mortality, and the positive prognosis includes disease remission such as disease-free state, disease improvement such as regression or stabilization of cutaneous T-cell lymphoma / hepatocarcinoma.

[0046] In some embodiments, the quantitative detection method based on PCR includes the step of reverse transcribing mRNA into cDNA and / or the step of measuring the content of cDNA. Preferably, the method for measuring the content of cDNA includes but is not limited to PCR, NASBA, RPA, SDA, LAMP, HAD, NEAR, MDA, RCA, LCR, RAM.

[0047] In the present invention, the chip includes a gene chip and a protein chip. The gene chip includes an oligonucleotide probe for the macroH2A gene for detecting the transcription level of the macroH2A gene, and the protein chip includes a specific binder for the macroH2A protein; the kit includes a gene detection kit and a protein detection kit. The gene detection kit includes a reagent or chip for detecting the transcription level of the macroH2A gene, and the protein detection kit includes a reagent or chip for detecting the expression level of the macroH2A protein.

[0048] The sixth aspect of the present invention provides use of the macrophages described in the first aspect of the present invention in the preparation of a drug for treating malignant tumors.

[0049] Furthermore, the cancer includes gastroesophageal adenocarcinoma, breast cancer, ovarian cancer, endometrial cancer, bladder cancer, lung cancer, colon cancer, rectal cancer, prostate cancer, blood cancer, lymphoma, cervical cancer, osteosarcoma, glioblastoma, melanoma, pancreatic cancer, liver cancer, kidney cancer, gallbladder cancer, bile duct cancer, esophageal cancer or neuroblastoma.

[0050] Preferably, the cancer is melanoma.

[0051] Advantages and beneficial effects of the present invention:

[0052] (1) This invention is the first to demonstrate that macrophages lacking macroH2A play a key role in anti-tumor immunity;

[0053] (2) This invention is the first to clarify the effect of macroH2A on macrophage transformation and found that macroH2A deficiency can promote the transformation of macrophages to an anti-tumor phenotype;

[0054] (3) The present invention demonstrates that the expression level of macroH2A in macrophages is associated with the survival rate of patients with cutaneous T-cell lymphoma and the recurrence of patients with liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Single-cell sequencing results for cutaneous T-cell lymphoma tumor tissue;

[0056] Figure 2 This is the survival curve of patients with cutaneous T-cell lymphoma;

[0057] Figure 3 Single-cell sequencing results from liver cancer patient tissue;

[0058] Figure 4 is the expression level of H2AFY gene in various cells in the liver cancer microenvironment;

[0059] Figure 5 The relationship between H2AFY gene expression in macrophages and recurrence of liver cancer patients;

[0060] Figure 6 The images of macrophages phagocytizing tumors and the statistical graph of phagocytic ability before and after knocking out macroH2A;

[0061] Figure 7 In vivo imaging of control mice and macrophage macroH2A knockout mice after tumor inoculation;

[0062] Figure 8 This is the expression analysis of macrophage immunosuppression-related genes before and after knocking out macroH2A;

[0063] Fig. 9 This is the gene expression analysis diagram of macrophage chemokines and their receptors before and after knocking out macroH2A;

[0064] Fig.10 This is the expression analysis of genes related to the positive immune response of macrophages before and after knocking out macroH2A. DETAILED DESCRIPTION

[0065] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes based on the technical solution and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

[0066] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0067] Example 1 Expression of macroH2A in macrophages

[0068] 1. Experimental Methods

[0069] Source of single-cell sequencing of patients with cutaneous T-cell lymphoma: 15 informed consenting patients with cutaneous T-cell lymphoma were recruited from the cutaneous lymphoma clinic of Peking University First Hospital, and tissues were cut from the tumor site of the patients. Cut into small pieces on ice, add collagenase, and digest in a 37°C water bath for 45 minutes. After digestion, filter using a 40μm cell filter. Cells were incubated and labeled with antibodies for CD45-PE and CD3-BV421, and single-cell sequencing was performed after cells were sorted by flow cytometry.

[0070] Source of single-cell sequencing of liver cancer patients: from published literature, CNSA: CNP0000650.

[0071] 2. Experimental Results

[0072] Figure 1 The results of single-cell sequencing of cutaneous T-cell lymphoma tumor tissue show that the histone variant macroH2A (gene name: H2AFY) is highly expressed in cutaneous T-cell lymphoma tumor-associated macrophages.

[0073] Figure 2 This is the survival curve of patients with cutaneous T-cell lymphoma. The ordinate represents the survival rate of the patients, and the abscissa represents the survival time of the patients. It can be seen that the survival rate of patients in the group with high gene expression levels of the histone variant macroH2A (H2AFY) in tumor-associated macrophages is lower.

[0074] Figure 3 The single-cell sequencing results of liver cancer patient tissues show that tumor-associated macrophages are infiltrating in large numbers.

[0075] Figure 4 This is the expression level of macroH2A (H2AFY) gene in various cells in the liver cancer microenvironment. It can be seen that the histone variant macroH2A (H2AFY) is highly expressed in tumor-associated macrophages of liver cancer.

[0076] Figure 5 The figure shows the relationship between the expression of macroH2A (H2AFY) gene in macrophages and the recurrence of liver cancer patients. The vertical axis represents the expression level of macroH2A (H2AFY) in tumor-associated macrophages, and the horizontal axis represents whether the disease has recurred. Primary represents the liver cancer tissue of patients who underwent the first surgery, and Relapsed represents the liver cancer tissue of patients who underwent the second surgery after the liver cancer recurred. It can be seen that the gene expression of macroH2A (H2AFY) in macrophages is higher in the liver cancer tissue after recurrence.

[0077] Example 2 Macrophages deficient in macroH2A play a key role in anti-tumor immunity

[0078] 1. Experimental Methods

[0079] 1. Establishment of macroH2A knockout cell line: Construct the sgRNA sequence 5'-GCGGGGGCGTGTTGCCGAAT-3' for macroH2A knockout. Subculture RAW264.7 cells in 10 cm cell culture dishes in advance, and perform transfection after the cells grow well and have reached a density of about 50% of the cell culture dish. Prepare 15 μg of plasmid, 15 μL of Lipo3000 reagent, 30 μL of P3000 reagent, and 75 μL of Opti-MEM medium for each 10 cm cell culture dish. Mix the plasmid and Lipo3000, P3000, and Opti-MEM medium in a 1.5 mL EP tube, respectively. After standing at room temperature for 5 minutes, mix the above transfection system in a new 1.5 mL EP tube and stand for another 5 minutes.

[0080] Take out the cultured cells, discard the original culture medium completely with a pipette, add 5 mL of new culture medium (without double antibody and serum), and then add the above-configured transfection system, mix thoroughly, and continue to culture in the incubator for 8 hours. Use a pipette to completely discard the above culture medium, add 10 mL of culture medium containing 10% serum, and continue to culture in the incubator for 48 hours.

[0081] Screening for the lowest drug resistance concentration of cells: The cells were subcultured and inoculated in 6 plates, with 5×10 5 cells. Add screening medium containing different concentrations of bleomycin to each well of the 6-well plate, and select at least 5 gradients. At the same time, set up wells without bleomycin as negative control wells. Culture in a cell culture incubator, replace the new screening medium every day, and observe the cell survival rate under a microscope for 4 consecutive days. The standard for screening bleomycin concentration: the lowest drug concentration that kills all cells within 4 days from the addition of the screening medium.

[0082] After culturing the cells transfected with the knockout plasmid for 48 hours, they are passaged. During the passage, the cells need to be cultured as single clones. First, count the cells and passage 1,000 cells to a 10 cm cell culture dish for culture, and passage a total of 3 10 cm culture dishes. Add culture medium with a bleomycin screening concentration for culture. Replace the screening culture medium every 2 days. After about 14 days of screening, resistant cell clones can be obtained.

[0083] 2. Macrophage phagocytosis of tumor experiment: The macrophage cell line RAW264.7 was inoculated into a 6-well culture plate. After the cells adhered to the wall, melanoma cells (B16F10) were added and then co-cultured in a cell culture incubator for 6 hours.

[0084] The cells were washed twice with PBS, and after collecting the cells, the macrophages were labeled with a flow cytometry antibody (PerCP / Cyanine5.5 anti-human CD11b, purchased from Biolegend), and then analyzed by flow cytometry (FlowSight multidimensional panoramic flow cytometer, 3Laser-12channel).

[0085] 3. In vivo experiment in mice: Cultivate melanoma B16F10 cells. When the number of cells meets the experimental requirements and the cells are in good condition, collect the cells and resuspend them in high-pressure sterile PBS. Count the cells and adjust the cell density to: 100 μL PBS contains 100,000 B16F10 cells. Draw them into a 1 mL syringe.

[0086] Using macrophage-specific macroH2A knockout mice (macroH2A fl / fl Lyz2-Cre), the subcutaneous part of the right upper limb of the mouse was selected and 100 μL of the above cell suspension was injected. After 14 days, the small animal in vivo imaging device was used for imaging.

[0087] 2. Experimental Results

[0088] 1. Figure 6 Results of the macrophage phagocytosis experiment: WT cells and macroH2A-KO cells were co-cultured with melanoma cells (B16F10) to observe the ability of macrophages to phagocytize tumors. Statistical results showed that macrophages with macroH2A knockout had enhanced phagocytosis of tumor cells (B16F10).

[0089] 2. Figure 7 The results of in vivo experiments in mice: wild-type mice and macroH2A-KO mice were subcutaneously injected with melanoma B16F10. Two weeks later, the tumor growth in the mice was detected by live animal imaging. It was found that the tumors in macrophage-specific macroH2A knockout mice were smaller than those in wild-type mice, indicating that the body's anti-tumor function is enhanced after macroH2A knockout in macrophages.

[0090] Example 3 Effect of macroH2A on macrophage functional transformation

[0091] 1. Experimental Methods

[0092] A commercial kit (purchased from Novazonics) was used to extract intracellular mRNA. The detailed information of the product components is shown in Table 1.

[0093] Table 1 Kit component information

[0094] Extract RNA:

[0095] Every 5×10 6 Add 500 μL Buffer RL to each cell and vortex until there are no obvious cell clusters. Transfer the lysed sample to FastPure gDNA-Filter Columns III (FastPure gDNA-Filter Columns III has been placed in the collection tube) and centrifuge at 12,000 rpm for 30 seconds. Discard FastPure gDNA-Filter Columns III and collect the filtrate. Add 0.5 times the volume of the filtrate to the filtrate with anhydrous ethanol and mix thoroughly.

[0096] Transfer all the above mixed solutions to FastPure RNA Columns III (FastPure RNA Columns III has been placed in the collection tube), centrifuge at 12000 rpm for 30 seconds, and discard the filtrate.

[0097] Add 700 μL Buffer RW1 to FastPure RNA Columns III, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate.

[0098] Add 700 μL Buffer RW2 (with anhydrous ethanol) to FastPure RNA Columns III, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate.

[0099] Add 500 μL Buffer RW2 to FastPure RNA Columns III and centrifuge at 12,000 rpm for 2 minutes. Carefully remove the adsorption column from the collection tube to avoid contact with the filtrate, which may cause contamination.

[0100] If there is liquid residue in the adsorption column or it comes into contact with the filtrate, discard the filtrate, put the FastPure RNA Columns III back into the collection tube, and spin at 12,000 rpm for 1 minute to prevent ethanol contamination.

[0101] Carefully transfer the adsorption column to a new RNase-free Collection Tubes 1.5mL centrifuge tube, add 50-200μL of RNase-free ddH2O to the center of the adsorption column, let it stand at room temperature for 1 minute, and centrifuge at 12000 rpm for 1 minute to elute the RNA.

[0102] The obtained RNA was sequenced on the Illumina platform. The differential expression of RNA-seq was analyzed by DESeq2 (https: / / bioconductor.org / packages / 3.17 / bioc / html / DESeq2.html).

[0103] 2. Experimental Results

[0104] like Figure 8 As shown, the R language programming technology was used to analyze the expression of genes related to immunosuppression in macrophages under polarization-induced conditions (20ng / mL IL-13, 20ng / mL IL-4, 48 hours) using the Pheatmap package. The icon changes from light to dark from bottom to top, indicating downregulation and upregulation of differential gene expression. From left to right, every three vertical boxes represent a group of samples. It can be seen that under polarization-induced conditions, genes related to immunosuppression in macrophages are significantly upregulated (WT-M0 vs WT-M2), but in the case of macroH2A-KO, the expression of these genes cannot be upregulated (macroH2A-KO-M2 vs WT-M2), that is, the knockout of macroH2A inhibits the expression of genes related to intracellular immunosuppression under polarization-induced conditions.

[0105] like Fig. 9 As shown, R language programming technology was used to use the Pheatmap package to perform mapping analysis on chemotaxis-related genes. After macroH2A knockout, macrophages significantly upregulated the expression of chemokine receptors (CCR1, CXCR3), which helped promote their migration. At the same time, it also significantly upregulated the expression of multiple chemokines (CCL2 / 3 / 4 / 5 / 9, CXCL9, CXCL16), which can chemotactic monocytes / macrophages, T cells, immature dendritic cells, NK cells, etc., and promote the infiltration of immune cells in tumors. In particular, CCL5 and CXCL9 have been reported to be closely related to T cell infiltration in the tumor microenvironment.

[0106] like Fig.10As shown, the R language programming technology was used to use the Pheatmap package to plot and analyze the anti-tumor genes and "positive immune response" genes expressed in macrophages. Under polarization-inducing conditions (20ng / mL IL-13, 20ng / mL IL-4, 48 hours), the "positive immune response" genes of macrophages did not change (WT-M0 vs WT-M2), but in the case of macroH2A-KO, the expression of these genes was significantly upregulated.

[0107] Therefore, the knockout of macroH2A not only prevents the expression of immunosuppressive genes in macrophages under M2-induced conditions, but also promotes their transformation to "immune activation" function, thereby improving the activity of its own and other related immune cells in the tumor immune microenvironment.

[0108] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims

1. A genetically engineered macrophage, characterized in that: The macroH2A gene in the macrophages is knocked out or knocked down.

2. The macrophage according to claim 1, characterized in that The macrophages include commercial macrophages, primary macrophages, immortalized macrophages, tumor-associated macrophages (TAMs), and chimeric antigen receptor macrophages (CAR-Ms); Preferably, the macrophages include Raw264.7 macrophages, THP-1 macrophages, immortalized bone marrow-derived mouse macrophages BMDM, and macrophages iMac differentiated from induced pluripotent stem cells (iPSCs); More preferably, the macrophages are Raw264.7 macrophages.

3. The macrophage according to claim 1, characterized in that Compared with macrophages without macroH2A gene knockout or knockdown, the genetically engineered macrophages have one or more of the following characteristics: (1) Low expression of genes related to immunosuppressive macrophages; (2) High expression of chemokine receptor protein or its mRNA; (3) High expression of chemokine protein or its mRNA; Preferably, the immunosuppressive macrophage-related gene is selected from ARG1, IL10, IL4, Mrc1, TGFbeta or a combination thereof; Preferably, the chemokine receptor is selected from CCR1, CXCR3 or a combination thereof; Preferably, the chemokine is selected from CCL2, CCL3, CCL4, CCL5, CCL9, CXCL9, CXCL16 or a combination thereof.

4. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the macrophage according to any one of claims 1 to 3; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or excipient.

5. Application of biomaterials that inhibit macroH2A expression in reversing the immunosuppressive function of macrophages, promoting the anti-tumor effect of macrophages and / or preparing macrophages with high cytotoxicity; Preferably, the biological material includes coding genes, expression cassettes, recombinant vectors, cells, proteins.

6. A method for reducing the immunosuppressive function of macrophages, inducing the transformation of macrophages into anti-tumor macrophages and / or preparing macrophages with high killing effect, characterized in that: The method comprises treating macrophages with a biological material that inhibits expression of macroH2A; Preferably, the biological material includes coding genes, expression cassettes, recombinant vectors, cells, proteins.

7. Use of a reagent for detecting the expression of macroH2A in macrophages in the preparation of a product for predicting the prognosis of patients with cutaneous T-cell lymphoma and / or liver cancer; Preferably, the expression level includes mRNA expression level and protein expression level.

8. The use according to claim 7, characterized in that: The reagents for detecting the mRNA expression amount include reagents used in the following methods: PCR-based detection method, Southern hybridization method, Northern hybridization method, dot hybridization method, fluorescence in situ hybridization method, DNA microarray method, ASO method, high-throughput sequencing platform method; Preferably, the reagents for detecting protein expression include reagents used in the following methods: hematoxylin-eosin staining, safranin O-fast green staining, protein blotting, enzyme-linked immunosorbent assay, radioimmunoassay, sandwich assay, immunohistochemical staining method, mass spectrometry, immunoprecipitation analysis, complement fixation analysis, flow cytometry fluorescence resolution technology and protein chip method.

9. Use of the macrophage according to any one of claims 1 to 3 in the preparation of a drug for treating malignant tumors.

10. The use according to claim 9, characterized in that: The cancer includes gastroesophageal adenocarcinoma, breast cancer, ovarian cancer, bladder cancer, lung cancer, colon cancer, rectal cancer, prostate cancer, blood cancer, lymphoma, cervical cancer, osteosarcoma, glioblastoma, melanoma, pancreatic cancer, liver cancer, kidney cancer, gallbladder cancer, bile duct cancer, esophageal cancer or neuroblastoma; Preferably, the cancer is melanoma.

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