Application of histone variant macroH2A in regulating macrophage function to improve tumor immune microenvironment

Abstract

The present invention discloses the application of histone variant macroH2A in regulating macrophage function to improve the tumor immune microenvironment. Specifically, the present invention provides macrophages lacking the macroH2A gene and the use of such cells in anti-tumor treatment. The present invention also provides the application 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. The present invention discovers that macrophages lacking the macroH2A gene have weakened immunosuppressive function and enhanced anti-tumor function. The macrophages of the present invention have the prospect of application in the field of anti-tumor treatment.

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 murine 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 the protein or its mRNA of chemokine receptors;

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

[0015] Furthermore, the genes related to immunosuppressive macrophages are selected from ARG1, IL10, IL4, Mrc1, TGFbeta, or a combination thereof.

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

[0017] Furthermore, the chemokines are 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 genetic operations such as interruption and knockout of a certain target gene, so that the expression and / or activity of the target gene are significantly decreased or even completely lost.

[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 optionally a gRNA.

[0020] In the present invention, the nucleotide 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 deduced from the nucleotide sequence. In a preferred example of the present invention, the gene editing protein includes but is not limited to Cas13 (such as CasRx), Cpf1, SaCas9, Cas13a, Cas13b, Cas13c.

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

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

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

[0024] Furthermore, the pharmaceutical composition further 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, and more preferably, the concentration of the macrophages in the preparation is 1×10 4 -1×10 7 cells / ml.

[0026] In some embodiments, the preparation may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The preparation of the present invention is preferably formulated for intravenous administration.

[0027] The dose of the above treatment administered to a patient will vary with the precise nature of the disorder being treated and the recipient of the treatment. The dosage ratios for human administration can be practiced according to accepted practice in the art. Generally, for each treatment or each course of treatment, 1×10 6 to 1×10 10 macrophages of the present invention can be administered to a patient, for example, by intravenous reinfusion.

[0028] In the present invention, the term "therapeutically effective amount" refers to an amount that produces a function or activity in a human and / or animal and is acceptable to the human and / or animal. Those of ordinary skill in the art should understand that the "therapeutically effective amount" may vary depending on factors such as the form of the pharmaceutical composition, the route of administration, the excipients used in the drug, the severity of the disease, and co-administration with other drugs.

[0029] In the present invention, the term "pharmaceutically acceptable" and its grammatical variants refer to a composition, carrier, diluent, and reagent that can be administered to a subject or administered on a subject without producing an undesired physiological effect to the extent that it prevents the administration of the composition. The term "excipient" or "carrier" refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of the compound. The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" encompasses any of the agents approved by the regulatory agencies of the federal government of the United States or listed in the pharmacopoeias for use in animals (including humans) in the United States, as well as any carrier or diluent that does not cause substantial irritation to the subject and does not eliminate the biological activity and properties of the administered compound. It includes excipients and carriers that are suitable for the preparation of 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 effects.

[0031] Furthermore, the biomaterial includes a coding gene, an expression cassette, a recombinant vector, a cell, and a protein.

[0032] Furthermore, the immunosuppressive function of the macrophages refers to their function of inhibiting the activation of other immune cells and promoting tumor growth.

[0033] Furthermore, the anti-tumor function of the 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 macrophages with high killing effects as described in the present invention have the same meaning and can be used interchangeably.

[0034] In the present invention, the in vitro induction of macrophage polarization towards the M2 direction is initiated by treatment with IL-4 and IL-13 (see, for example, Liu, Y.C., Zou, X.B., Chai, Y.F., & Yao, Y.M. (2014). Macrophage polarization in inflammatory diseases. International Journal of Biological Sciences, 10(5), 520–529, which is incorporated by reference for all purposes).

[0035] In the present invention, the biomaterial that inhibits the expression of macroH2A is an sgRNA targeting macroH2A, and its specific sequence is GCGGGGGCGTGTTGCCGAAT.

[0036] The 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 the change of reducing the immunosuppressive ability of macrophages and inducing the transformation of macrophages into anti-tumor macrophages.

[0038] Furthermore, the method includes treating macrophages with a biomaterial that inhibits the expression of macroH2A.

[0039] Furthermore, the biomaterial includes a coding gene, an expression cassette, a recombinant vector, a cell, and a protein.

[0040] The 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 the mRNA expression level and the protein expression level.

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

[0043] Furthermore, the reagent for detecting the protein expression level includes the reagents used in the following methods: hematoxylin-eosin staining, safranin O-fast green staining, Western blotting, enzyme-linked immunosorbent assay, radioimmunoassay, sandwich assay, immunohistochemical staining method, mass spectrometry, immunoprecipitation analysis, complement fixation analysis, flow cytometry fluorescence discrimination 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 regarding the medical development (e.g., the possibility of long-term survival, disease-free survival rate, etc.), including positive prognosis or negative prognosis. 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 status, disease improvement such as the regression or stability of cutaneous T-cell lymphoma / liver cancer.

[0046] In some embodiments, the PCR-based quantitative detection method 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 oligonucleotide probes for macroH2A gene for detecting the transcription level of the macroH2A gene, and the protein chip includes specific binders for macroH2A protein; the kit includes a gene detection kit and a protein detection kit. The gene detection kit includes reagents or chips for detecting the transcription level of the macroH2A gene, and the protein detection kit includes reagents or chips for detecting the expression level of macroH2A protein.

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

[0049] Furthermore, the cancers include 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, cholangiocarcinoma, esophageal cancer or neuroblastoma.

[0050] Preferably, the cancer is melanoma.

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

[0052] (1) The present invention for the first time proves that macrophages lacking macroH2A play a key role in anti-tumor immunity;

[0053] (2) The present invention for the first time clarifies the effect of macroH2A on macrophage transformation, and finds that the deletion of macroH2A can promote the transformation of macrophages into an anti-tumor phenotype;

[0054] (3) The present invention proves that the expression level of macroH2A in macrophages is related to the survival rate of patients with cutaneous T-cell lymphoma and the recurrence of patients with liver cancer. Description of the Drawings

[0055] Figure 1 Is the single-cell sequencing result of the tumor tissue of cutaneous T-cell lymphoma;

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

[0057] Figure 3 Single-cell sequencing results of liver cancer patient tissues;

[0058] Figure 4 H2AFY gene expression levels in various cell types in the liver cancer microenvironment;

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

[0060] Figure 6 Images of macrophages phagocytosing tumors and statistical graphs of phagocytic ability before and after knocking out macroH2A;

[0061] Figure 7 In vivo imaging of control mice and mice with macroH2A knocked out in macrophages after tumor inoculation;

[0062] Figure 8 Expression analysis diagrams of macrophage immunosuppression-related genes before and after knocking out macroH2A;

[0063] Figure 9 Expression analysis diagrams of macrophage chemokines and their receptors before and after knocking out macroH2A;

[0064] Figure 10 Expression analysis diagrams of macrophage positive immune response-related genes before and after knocking out macroH2A. Detailed implementation manners

[0065] The following further illustrates the present invention with specific embodiments, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and should be covered by the protection scope of the present invention.

[0066] The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels without special instructions.

[0067] Example 1 Expression of macroH2A in macrophages

[0068] I. Experimental method

[0069] Sources of single-cell sequencing for patients with cutaneous T-cell lymphoma: 15 patients with cutaneous T-cell lymphoma who gave informed consent were recruited from the cutaneous lymphoma clinic of Peking University First Hospital. Tissue was excised from the tumor sites of the patients, placed on ice, cut into small pieces, collagenase was added, and digestion was carried out in a 37°C water bath for 45 minutes. After digestion was completed, filtration was performed using a 40μm cell strainer. Cells were incubated and labeled with antibodies against CD45-PE and CD3-BV421, and after sorting the cells by flow cytometry, single-cell sequencing was carried out.

[0070] Sources of single-cell sequencing for patients with liver cancer: From a published literature, CNSA: CNP0000650.

[0071] II. Experimental results

[0072] Figure 1 This is the result of single-cell sequencing of cutaneous T-cell lymphoma tumor tissue. It can be seen that the histone variant macroH2A (gene name: H2AFY) is highly expressed in tumor-associated macrophages of cutaneous T-cell lymphoma.

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

[0074] Figure 3 This is the result of single-cell sequencing of liver cancer patient tissues. It can be seen that tumor-associated macrophages are massively infiltrated.

[0075] Figure 4 This is the expression level of the macroH2A (H2AFY) gene in various types of 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 This is the relationship between the expression level of the 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 there is recurrence. Primary represents the liver cancer tissue of patients undergoing primary surgery, and Relapsed represents the liver cancer tissue of patients undergoing the second surgery after liver cancer recurrence. It can be seen that in the liver cancer tissue after recurrence, the expression level of the macroH2A (H2AFY) gene in macrophages is higher.

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

[0078] I. Experimental Methods

[0079] 1. Establishment of macroH2A knockout cell line: Construct the sgRNA sequence for macroH2A knockout: 5’-GCGGGGGCGTGTTGCCGAAT-3’. Passage RAW264.7 cells in advance into a 10-cm cell culture dish. When the cells are in good growth condition and reach about 50% density of the cell culture dish, perform the transfection operation. Prepare 15 μg plasmid, 15 μL Lipo3000 reagent, 30 μL P3000 reagent, and 75 μL Opti-MEM medium for each 10-cm cell culture dish. Mix the plasmid with 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 into a new 1.5-mL EP tube and stand for another 5 minutes.

[0080] Take out the cultured cells, use a pipette to completely discard the original medium, add 5 mL of new medium (without double antibiotics and without serum), and then add the above-prepared transfection system. After mixing well, place it in the incubator and continue to culture for 8 hours. Use a pipette to completely discard the above medium, add 10 mL of medium containing 10% serum, and place it in the incubator and continue to culture for 48 hours.

[0081] Screening of the minimum drug resistance concentration of cells: Passage the cells and inoculate them in a 6-well plate, with 5×10 5 cells inoculated in each well. 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 the wells without adding bleomycin as negative control wells. Place them in the cell incubator for culture. It is necessary to change the new screening medium every day and observe the cell survival rate under the microscope. Culture continuously for 4 days. The standard for screening the bleomycin concentration: The lowest drug concentration that can kill all cells within 4 days starting from adding the screening medium.

[0082] After culturing the cells transfected with the knockout plasmid for 48 hours, passage them. When passaging, cell monoclonal culture is required. First, perform cell counting and passage according to the number of 1000 cells into 10-cm cell culture dishes for culture, with a total of 3 10-cm culture dishes passaged. Add the medium with the bleomycin screening concentration for culture. Change the screening medium every 2 days. After screening for about 14 days, resistant cell clones can be obtained.

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

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

[0085] 3. In vivo experiment in mice: Melanoma cells B16F10 were cultured. When the cell number met the experimental requirements and the cell state was good, the cells were collected and resuspended in sterile PBS under high pressure. Cell counting was performed, and the cell density was adjusted to: 100,000 B16F10 cells in 100 μL PBS. It was aspirated into a 1 mL syringe.

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

[0087] II. Experimental results

[0088] 1. Figure 6 This is the result of the macrophage phagocytosis of tumor experiment: WT cells and macroH2A-KO cells were co-cultured with melanoma cells (B16F10) to observe the ability of macrophages to phagocytose tumors. The statistical results showed that the phagocytic ability of macroH2A-knockout macrophages against tumor cells (B16F10) was enhanced.

[0089] 2. Figure 7 This is the result of the in vivo experiment in mice: Wild-type mice and macroH2A-KO mice were subcutaneously injected with melanoma B16F10. After 2 weeks, the tumor growth in the mice was detected by in vivo animal imaging. It was found that the tumors in the macrophage-specific knockout macroH2A mice were smaller than those in the wild-type mice, indicating that the anti-tumor function of the body was enhanced after knocking out macroH2A in macrophages.

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

[0091] I. Experimental methods

[0092] Extract intracellular mRNA using a commercial kit (purchased from Novoprotein). The detailed information of the product components is shown in Table 1.

[0093] Table 1 Information on Kit Components

[0094]

[0095] RNA Extraction:

[0096] Add 500 μL of Buffer RL to every 5×10 6 cells and vortex until there are no obvious cell clumps. 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 anhydrous ethanol with a volume 0.5 times that of the filtrate to the filtrate and mix well.

[0097] Transfer all of the above mixture to FastPure RNA Columns III (FastPure RNA Columns III has been placed in the collection tube) and centrifuge at 12,000 rpm for 30 seconds. Discard the filtrate.

[0098] Add 700 μL of Buffer RW1 to FastPure RNA Columns III and centrifuge at 12,000 rpm for 30 seconds. Discard the filtrate.

[0099] Add 700 μL of Buffer RW2 (anhydrous ethanol has been added) to FastPure RNA Columns III and centrifuge at 12,000 rpm for 30 seconds. Discard the filtrate.

[0100] Add 500 μL of 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, avoiding contact with the filtrate to prevent contamination.

[0101] If there is liquid residue on the adsorption column or it comes into contact with the filtrate, discard the filtrate, place FastPure RNA Columns III back into the collection tube, and centrifuge empty at 12,000 rpm for 1 minute to prevent ethanol contamination.

[0102] Carefully transfer the adsorption column to a new 1.5 mL centrifuge tube of RNase-free Collection Tubes. Drop 50 - 200 μL of RNase-free ddH2O onto the center of the adsorption column in a suspended manner, let it stand at room temperature for 1 minute, and centrifuge at 12,000 rpm for 1 minute to elute the RNA.

[0103] Sequence the obtained RNA on the Illumina platform. The differential expression of RNA-seq was analyzed by DESeq2 (https: / / bioconductor.org / packages / 3.17 / bioc / html / DESeq2.html).

[0104] II. Experimental Results

[0105] As Figure 8 shown, using R language programming technology and the Pheatmap package to analyze the gene expression of immunosuppression-related genes in macrophages under polarization induction conditions (20 ng / mL IL-13, 20 ng / mL IL-4, 48 hours). From bottom to top, the lighter color changing to darker color in the graph indicates the down-regulation and up-regulation of differential gene expression. Every three vertical boxes from left to right represent a group of samples. It can be seen that under polarization induction conditions, the immunosuppression-related genes in macrophages are significantly up-regulated (WT-M0 vs WT-M2), but in the case of macroH2A-KO, the expression of these genes cannot be up-regulated (macroH2A-KO-M2 vs WT-M2), that is, the knockout of macroH2A inhibits the expression of immunosuppression-related genes in cells under polarization induction conditions.

[0106] As Figure 9 shown, using R language programming technology and the Pheatmap package to plot and analyze chemotaxis-related genes. After the knockout of macroH2A, macrophages significantly up-regulate the expression of chemokine receptors (CCR1, CXCR3), which helps to promote their migration. At the same time, it also significantly up-regulates the expression of a variety of chemokines (CCL2 / 3 / 4 / 5 / 9, CXCL9, CXCL16), which can chemotax 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 the infiltration of T cells in the tumor microenvironment.

[0107] As Figure 10As shown, the R language programming technology was adopted, and the Pheatmap package was used to plot and analyze the anti-tumor genes and "positive immune response" genes expressed in macrophages. Under the polarization induction conditions (20 ng / mL IL-13, 20 ng / 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 up-regulated.

[0108] Therefore, the knockout of macroH2A not only prevents the expression of immunosuppressive genes in macrophages under M2 induction conditions, but also promotes their transformation into "immune activation" functions, thereby improving the activity of themselves and other related immune cells in the tumor immune microenvironment.

[0109] The above content is a further detailed description of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention belongs, without departing from the concept of the present invention, several simple deductions or substitutions can be made, and all should be regarded as belonging to 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; The knockout is achieved by sgRNA specifically targeting macroH2A; The sequence of the sgRNA is GCGGGGGCGTGTTGCCGAAT.

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).

3. The macrophage according to claim 1 or 2, characterized in that 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).

4. The macrophage according to claim 3, characterized in that The macrophages are Raw264.7 macrophages.

5. 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; The immunosuppressive macrophage-related gene is selected from ARG1, IL10, IL4, Mrc1, TGFbeta or a combination thereof; The chemokine receptor is selected from CCR1, CXCR3 or a combination thereof; The chemokine is selected from CCL2, CCL3, CCL4, CCL5, CCL9, CXCL9, CXCL16 or a combination thereof.

6. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the macrophage according to any one of claims 1 to 5.

7. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition further includes a pharmaceutically acceptable carrier, diluent or excipient.

8. Use of a biomaterial that inhibits the expression of macroH2A in the preparation of macrophages with high cytotoxicity, characterized in that: The biological material is sgRNA that specifically targets macroH2A; The sequence of the sgRNA is GCGGGGGCGTGTTGCCGAAT.

9. A method for preparing macrophages with high killing effect, characterized in that: The method comprises treating macrophages with a biological material that inhibits expression of macroH2A; The biological material is sgRNA that specifically targets macroH2A; The sequence of the sgRNA is GCGGGGGCGTGTTGCCGAAT.

10. 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.

11. The use according to claim 10, characterized in that: The expression level includes mRNA expression level and protein expression level.

12. The use according to claim 11, 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, and high-throughput sequencing platform method.

13. The use according to claim 11, characterized in that: 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, mass spectrometry, immunoprecipitation analysis, complement fixation analysis, flow cytometry fluorescence resolution technology and protein chip method.

14. Use of the macrophage according to any one of claims 1 to 5 in the preparation of a drug for treating melanoma.

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