Application of an epigenetic drug combined with adoptive monocyte / macrophage immunotherapy in the preparation of biopharmaceuticals for tumor diseases

Abstract

The invention discloses an application of an epigenetic drug combined with adoptive monocyte / macrophage immunotherapy in the preparation of a biopharmaceutical for tumor diseases, belonging to the technical field of biopharmaceuticals. The present invention provides an application of an epigenetic drug combined with adoptive monocyte / macrophage immunotherapy in the preparation of a biopharmaceutical for tumor diseases, wherein the epigenetic drug removes the methylation of tumor cells, increases the susceptibility of tumors to targeted therapy and regulates tumor heterogeneity, and the chimeric antigen receptor extracellular domain of the CAR monocyte / macrophage targets and recognizes and blocks the signal pathway of targeted tumor antigens, and performs cellular immune killing. The epigenetic modification of the present invention and CAR monocyte / macrophage anti-solid tumor treatment have a synergistic therapeutic effect, and the epigenetic modification treatment makes tumor antigens more sensitive to CAR monocyte / macrophage therapy, promotes the expression of tumor suppressor genes, promotes the conversion of tumor cell apoptosis to pyroptosis, and solves the problem of immune escape tolerance caused by tumor heterogeneity.

Description

Technical Field

[0001] The invention belongs to the technical field of biological medicine manufacturing, and specifically relates to the preparation of a biological medicine for treating tumor diseases. Background Art

[0002] CAR-macrophages (CAR-M) are considered to be one of the most promising immunotherapies for conquering malignant solid tumors. Macrophages mainly activate lymphoid immune cells by phagocytosing cell debris and pathogens in body fluids and tissues and presenting antigens to make them respond to pathogens. In the tumor microenvironment, macrophages are innate immune cells with the highest infiltration rate. The development of CAR-macrophages (CAR-M) for tumor immunotherapy has aroused great interest among researchers. The emergence of CAR-M has opened up a new development direction for conquering solid tumors by modifying macrophages with specific CARs to improve the phagocytic activity and antigen presentation of macrophages to tumors. However, the single implementation of CAR-M requires a high cell transfusion dose during clinical treatment, which brings difficulties to production and preparation, generates high costs for patient treatment, and affects the clinical popularization and application of cell therapy. It is particularly important that even if CAR-M has the potential to comprehensively regulate the anti-tumor effect of the tumor microenvironment, in the face of the complex pathogenesis of malignant solid tumors, more effective combination methods are still needed in the clinic to improve the cure effect.

[0003] Tumor heterogeneity and its dynamic development form specific target immune escape tolerance, which is the main reason for tumor recurrence after cell therapy. Tumor heterogeneity is one of the important characteristics of malignant tumors. Tumor heterogeneity can be manifested in the level of tumor differentiation and tumor function, with heterogeneous antigen expression or cell subpopulations with different biological characteristics. This tumor heterogeneity often brings great difficulties to the study and treatment of tumors. Tumor-associated antigen targets in solid tumors show heterogeneity in intensity and distribution. Due to the high heterogeneity of tumor cells, the expression of target antigens may be insufficient or even lost. Currently, CAR-M is hailed as one of the most promising options for the treatment of malignant solid tumors. Even so, problems such as tumor antigen escape, tumor heterogeneity, and insufficient or even lost expression of target antigens will also pose challenges to CAR-M cell therapy. At the same time, the high cost of CAR-M cell transfusion dose limits the clinical application of cell therapy. Although the current immunotherapy for solid tumors has made different progress in various technical fields, the clinical treatment of solid tumors is still very challenging. Tumors start from a single cell and undergo many changes, developing into tumor tissues with tumor heterogeneity and immune heterogeneity, forming complex anti-tumor mechanisms, making it difficult for a single therapeutic measure to effectively treat tumors. Although tumor development is driven by a variety of important genes, changes in many key genes are due to epigenetic changes including DNA methylation. Current studies have shown that changes in DNA methylation status are common in tumors, characterized by a decrease in overall methylation levels and an increase in local methylation levels. In tumor cells, oncogenes are activated in a low methylation state, and tumor suppressor genes are inhibited in a high methylation state. Tumor methylation characteristics already exist to some extent in the initial cells before they are transformed into tumor cells, and the risk of cancer in normal tissues is directly related to the degree of abnormal methylation variation in the tissue. The overall impact of epigenetic events is to change gene expression patterns, allow oncogene expression, or prevent the activation of genes involved in inhibiting growth differentiation or DNA damage response. Anti-tumor immune responses require specific responses to tumor antigens, and the latest high-throughput sequencing studies support that tumors conceal new antigen presentation through methylation of key mechanisms such as promoters, mediate cancer cell immune editing to form immune escape, and promote the development of tumors towards an immunosuppressive microenvironment.

[0004] Tumor cell immunotherapy is based on a long-lasting and efficient living cell therapy mechanism, bringing new hope to cancer patients. Chimeric antigen monocyte / macrophage adoptive cell immunotherapy deeply explores the advantages and potential of macrophages as myeloid cells to fight solid tumors, and is highly anticipated in the treatment of solid tumors. Despite this, it is foreseeable that complex mechanisms such as antigen escape and high heterogeneity of solid tumors will still pose great challenges to cell therapy. Clinical studies have found that most tumor cells that respond to targeted therapy have high levels of target antigen expression. Epigenetic modification will innovatively solve this problem and is extremely suitable for long-term in vivo interactions of cell therapy.

[0005] Simply discovering or replacing more targets cannot fundamentally solve the problem of immune escape tolerance caused by tumor heterogeneity. In this regard, this patent designs an innovative solution to enhance the robustness of CAR macrophage immunotherapy through epigenetic modification, enhance the response sensitivity of cell therapy, and regulate the signal pathway response to effectively fight tumors, thereby reducing the amount of clinical CAR-M cell infusion, overcoming the problem of poor efficacy caused by the loss or low expression of tumor antigens, and ultimately systematically enhancing the robustness of CAR-M efficacy. Summary of the invention

[0006] In view of the problems existing in the above-mentioned prior art, the purpose of the present invention is to design and provide an epigenetic drug combined with adoptive monocyte / macrophage immunotherapy for the preparation of tumor disease biological drugs. The epigenetic drug of the present invention removes the methylation of tumor cells, increases the susceptibility of tumors to targeted therapy and regulates tumor heterogeneity. The chimeric antigen receptor extracellular domain of the CAR monocyte / macrophage targets and recognizes and blocks the signal pathway of the targeted tumor antigen, and performs cellular immune killing.

[0007] The present invention uses epigenetic modification and CAR monocyte / macrophage anti-tumor treatment to achieve a synergistic therapeutic effect. The epigenetic modification treatment makes tumor antigens more sensitive to CAR monocyte / macrophage treatment, promotes the expression of tumor suppressor genes, promotes tumor cell apoptosis to pyroptosis, and solves the problem of immune escape tolerance caused by tumor heterogeneity; it has the anti-tumor treatment effect of reducing the clinical infusion volume of CAR monocyte / macrophage cells, reducing costs, and enhancing efficacy.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] On the one hand, the present invention provides an application of an epigenetic drug combined with adoptive monocyte / macrophage immunotherapy in the preparation of a biopharmaceutical for tumor diseases, wherein the epigenetic drug is used to modify tumor cells, the adoptive monocyte / macrophage is a CAR monocyte / macrophage, and the chimeric antigen receptor extracellular domain of the CAR monocyte / macrophage targets and recognizes a targeted tumor antigen and blocks a signal pathway of the targeted tumor antigen;

[0010] Preferably, the method of modifying tumor cells is selected from the group consisting of removing methylation of tumor cells and histone modification of tumor cells.

[0011] The application, wherein the epigenetic drug is selected from one of decitabine, IDH inhibitors, DNA methyltransferase inhibitors, and histone modification enzyme target inhibitors;

[0012] Preferably, the IDH inhibitor is selected from one of ensidipine and ivosidenib;

[0013] The DNA methyltransferase inhibitor is selected from one of nucleoside DNMTs, non-nucleoside DNMTs, and antisense oligonucleotide DNMTs;

[0014] More preferably, the nucleoside DNMTs is selected from one of decitabine, Zebularine, and azacitidine; the non-nucleoside DNMTs is selected from one of procainamide, SGI-110, and RG108; the antisense oligonucleotide DNMT is MG98;

[0015] The histone modification enzyme target inhibitor is selected from one of a histone methyltransferase inhibitor, a histone deacetylase inhibitor or a histone acetyltransferase inhibitor;

[0016] More preferably, the histone methyltransferase inhibitor is selected from one of Tazemmetostat, EZH2 inhibitor (tazemstat), GSK126 or Pinometostat; the histone deacetylase inhibitor is selected from one of Trichostatin, Vorinostat, Belinostat, Panobinostat, Ouisinostat, Ricolinostat, Entinostat, Mocetinostat, Romidepsin, Plitidepsin, Ethinib, CUDC-907, CUDC-101, Abexinostat, RG2833, Pracinostat or Domatinostat; the histone acetyltransferase inhibitor is selected from one of A-485, C646, Remodelin.

[0017] The application, the CAR monocyte / macrophage comprises a signal peptide domain, a first linker domain, a second linker domain and a transmembrane domain;

[0018] Preferably, the transmembrane domain is selected from one of CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, Glycophorin C, NECL2, GPRCSD, CD179a, ERG, TMPRSS2, PAX3, RhoC, TRP-2, CYP1B1, SART3, PAX5, OY-TES1, AKAP-4, SSX2, MYD88, MAL, IRAK1, FCGR2A, FCGR3A, and FCER1G. In the application, the tumor is a solid tumor or a non-solid tumor;

[0019] Preferably, the solid tumor is selected from ovarian cancer, breast cancer, lymphoma, gastric cancer, brain tumor, intestinal cancer, thyroid cancer, leukemia, adrenal cancer, adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain cancer / CNS cancer, bronchial tumor, heart tumor, cervical cancer, bile duct cancer, chondrosarcoma, chordoma, colon cancer, colorectal cancer, craniopharyngioma, breast duct carcinoma in situ, endometrial cancer Cancer, ependymoma, esophageal cancer, nasal glioma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, fibrohistiocytic sarcoma, fibrosarcoma, gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germ cell tumor, glioma, glioblastoma, head and neck cancer, hemangioblastoma, hepatocellular carcinoma, hypopharyngeal cancer, intraocular melanoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, lip cancer, liposarcoma, liver cancer, lung cancer, non-small cell lung cancer, lung carcinoid tumor, malignant mesothelioma, medullary carcinoma, medulloblastoma, meningioma, melanoma, Merkel cell carcinoma, midline digestive tract cancer, mouth cancer, myxosarcoma, myelodysplastic syndrome, myeloproliferative neoplasm, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oligodendroglioma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic islet cell tumor, papillary carcinoma, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, chromaffin cells one of the following: ovarian cancer, pinealoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, prostate cancer, colorectal cancer, retinoblastoma, renal cell carcinoma, renal pelvis and ureter cancer, rhabdomyosarcoma, salivary gland cancer, sebaceous gland cancer, skin cancer, soft tissue sarcoma, squamous cell carcinoma, small cell lung cancer, small intestine cancer, stomach cancer, sweat gland cancer, synovioma, testicular cancer, laryngeal cancer, thymus cancer, thyroid cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vascular cancer, vulvar cancer, and Wilms tumor;

[0020] The non-solid tumor is a blood tumor;

[0021] In the application, the tumor cells are selected from HER2-positive tumor cells; preferably, the HER2-positive tumor cells are SKOV3 or MKN45.

[0022] The application is characterized in that the target transmembrane domain targeting tumor antigens can be derived from any membrane-bound protein or transmembrane protein, selected from HER2 target, GPC3, CD47, TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, TnAg, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, Folate receptor alpha, meothelin, MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-Ireceptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folatereceptorbeta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialicacid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-AL legumain, HPV E6,E7, MAGEA1, ETV6-AML, sperm protein 17, breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intratestinal carboxyl esterase,mut One of hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, FCRL5, and IGLL1. The application, the blocking of the signal pathway targeting tumor antigens can upregulate the expression of caspase-3 in tumor cells and induce apoptosis of tumor cells.

[0023] The application removes the methylation of tumor cells, causing GSDME to be highly expressed, and caspase-3 cuts GSDME, causing tumor cells to change from apoptosis to pyroptosis, thereby enhancing tumor cell death.

[0024] In a second aspect, the present invention provides a pharmaceutical composition for treating tumor diseases, characterized in that it comprises epigenetic drugs and CAR monocytes / macrophages.

[0025] The pharmaceutical composition for treating tumor diseases is characterized in that the epigenetic drug is decitabine, the solid tumor is HER2-positive ovarian cancer or gastric cancer, and the pharmaceutical composition contains one or more additional active ingredients and / or medically or pharmaceutically acceptable excipients.

[0026] In the present invention, it should be understood by those skilled in the art that the CAR monocyte / macrophage represents a CAR monocyte or a CAR macrophage. The CAR monocyte / macrophage is derived from a monocyte or macrophage having a chimeric antigen receptor (CAR) structure. Monocytes can be induced into macrophages or dendritic cells, and monocytes are common precursor cells of macrophages and dendritic cells, with specific identical or similar properties and functions.

[0027] The nucleic acid sequence encoding the bioactive protein or polypeptide that can be expressed across the membrane is introduced into the macrophage for expression, thereby achieving the functional modification of the macrophage. The cell is a macrophage, a monocyte or a dendritic cell.

[0028] The present invention includes a method of treating a disease or condition associated with a tumor or cancer in a subject, comprising administering to the subject a therapeutically effective amount of a composition of the present invention.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] Epigenetic modification can "expose" tumor antigens, making CAR monocyte / macrophage therapy more sensitive. The combination of epigenetic modification anti-tumor mechanism and CAR monocyte / macrophage anti-tumor mechanism can produce a therapeutic effect of "1+1 is greater than 2", that is, a synergistic therapeutic effect. For example, in an embodiment of the present invention, decitabine is used to remove the methylation of HER2-positive tumor cells, so that they highly express GSDME. In addition, by blocking the HER2 signaling pathway, the expression of caspase-3 in tumor cells is upregulated to induce apoptosis of tumor cells. At this time, high GSDME expression can convert tumor cells from apoptosis to pyroptosis, and can expand the activation of caspase-3, further enhancing cell death, thereby reducing the clinical infusion amount of CAR monocytes / macrophages, overcoming the problem of poor efficacy caused by loss or low expression of tumor antigens, and finally systematically enhancing the robustness of the efficacy of CAR monocytes / macrophages.

[0031] The present invention establishes a "low-cost and high-efficiency" treatment strategy for the clinical application of CAR monocyte / macrophage immunotherapy. It is expected to open up new space for the clinical application of CAR monocyte / macrophages, benefit more patients in the future, and further provide scientific data for the theoretical system of cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the structure of anti-HER2 CAR-M;

[0033] Figure 2Figure 1 is a graph of the safe dose range of DAC and the incubation time, where A is the cytotoxicity of different low doses of DAC to HER2+-4T1; B is the cytotoxicity of different low doses of DAC to SKOV3; C is the change in DFNA5 gene expression after HER2+-4T1 was treated with different low doses of DAC and different incubation times; D is the change in DFNA5 gene expression after SKOV3 was treated with different low doses of DAC and different incubation times;

[0034] Figure 3 The results of anti-HER2 CAR-M combined with chemical drugs inducing apoptosis to pyroptosis are shown in Figure 1, where A shows that anti-HER2 CAR-M upregulates the expression of CAS3; B shows the flow cytometry quantitative results of pyroptosis caused by anti-HER2 CAR-M combined with chemical drugs; and C shows the flow cytometry verification of pyroptosis caused by anti-HER2 CAR-M combined with chemical drugs.

[0035] Figure 4 The results of combined treatment with chemical drugs to kill tumor cells are shown in Figure 1, where A is the cytotoxicity of low-dose DAC combined with anti-HER2 CAR-M to HER2+-4T1; B is the cytotoxicity of low-dose DAC combined with anti-HER2 CAR-M to SKOV3;

[0036] Figure 5 The results of anti-HER2 CAR-M combined with chemical drugs killing tumor cells; A is a schematic diagram of mouse tumor model construction and treatment process; B is a tumor image of anti-HER2 CAR-M combined with DAC treatment; C is the tumor weight after anti-HER2 CAR-M combined with DAC treatment; D is the tumor growth curve after anti-HER2 CAR-M combined with DAC treatment; E is the weight of mice after anti-HER2 CAR-M combined with DAC treatment;

[0037] Figure 6 It is the therapeutic effect of the combination of anti-HER2 CAR-M and DAC; A is the mouse SKOV3 abdominal tumor model; B is the survival curve of the mouse SKOV3 abdominal tumor model; C is the safety assessment of the mouse SKOV3 abdominal tumor treatment model - weight monitoring. DETAILED DESCRIPTION

[0038] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0039] Example 1: Construction of anti-HER2 CAR-M cells (ACT CAR-M)

[0040] 1.1 Construction and packaging of lentivirus (taking lentivirus as an example, not limited to lentivirus)

[0041] Based on the lentiviral vector, the chimeric antigen receptor HER2 scFv (sequence as shown in SEQ ID NO.1), the transmembrane region of α1β1 integrin (sequence as shown in SEQ ID NO.2), the intracellular α1β1 integrin (sequence as shown in SEQ ID NO.3), the intracellular FcγR1 transmembrane signaling domain (sequence as shown in SEQ ID NO.4) and GFP (GFP sequence as shown in SEQ ID NO.5) were connected using a linker (linker sequence as shown in SEQ ID NO.6) to synthesize the chimeric antigen receptor HER2 scFv (sequence synthesis performed by Sangon Biotech (Shanghai) Co., Ltd.), which was transfected into macrophages to construct macrophage-specific CAR, and the synthesized HER2 scFv-CAR lentiviral plasmid was co-transfected with three auxiliary plasmids of Rev, Gag and VSV to package it into a complete HER2 scFv-CAR lentivirus.

[0042] The specific steps are as follows:

[0043] (1) 4×10 5 HEK293T cells in logarithmic growth phase were evenly inoculated in 6-well plates and cultured in a 37°C, 5% CO2 cell culture incubator until the cell confluence reached 60%-70%. Plasmids pCDH-HER2scFv, Rev, Gag and VSV were added to a centrifuge tube containing Opti-MEM at a ratio of 3:1:1:1, mixed and incubated at room temperature, and then TurboFect Transfection Reagent was added, and the mixture was gently pipetted and mixed, and incubated at room temperature.

[0044] (2) The incubated mixture was slowly added dropwise to a 6-well plate inoculated with 293T cells, and the culture plate was gently shaken while adding. After culturing at 37°C and 5% CO2 for 16-24 hours, the transfection mixture in each well of the 6-well plate was discarded, and 2-3 mL of Advanced DMEM complete medium containing 10% fetal bovine serum (FBS) was added. The cell culture plate was reset at 37°C and 5% CO2 for 48 hours to be synthesized into HER2 scFv-CAR lentivirus;

[0045] (3) To remove cell debris, centrifuge the culture medium containing lentivirus at 4°C and 4000g for 10 min. Filter the supernatant with a 0.22μm filter and collect it, and centrifuge it at 4°C and 15000g for 2h. Discard the supernatant, resuspend the virus particles in virus preservation solution, centrifuge it at 10000g for 5min, and then take the supernatant and store it at -80°C for later use;

[0046] (4) Subsequently, the titer of the packaged lentivirus was tested. 90 μL of DMEM complete medium was added to 9 sterile 1.5 mL centrifuge tubes. 10 μL of the obtained recombinant lentiviral particles HER2 scFv-CAR were added to the first centrifuge tube and pipetted evenly. 10 μL of the mixture was aspirated and added to the second centrifuge tube. This process was repeated until the last tube. Three replicates were set for each group. 5×10 4 HEK293T cells were evenly inoculated in a 96-well plate. When the cell confluence reached 60%-70%, the above-mentioned gradient lentiviral dilutions were added in sequence. At the same time, 293T cells without virus dilution were set as negative controls. After 24 hours of culture, fresh DMEM complete medium was replaced. After 48 hours, the conditions of each well were observed under an inverted fluorescence microscope. The formula for calculating the lentiviral titer is: Virus titer (TU / mL) = average number of green fluorescent cells × virus dilution multiple / volume of inoculated virus solution (mL), unit is TU / mL.

[0047] 1.2 Preparation of anti-HER2 CAR-M cell lines

[0048] After packaging into a complete lentivirus and detecting its titer, HER2 scFv-CAR lentivirus was used to infect macrophages to obtain anti-HER2 CAR-M CAR-M stable cell lines. Macrophages were inoculated in 6-well plates. When the cells grew to a confluence of 60-70%, HER2 scFv-CAR lentiviral particles were added to the culture medium of the macrophages at a ratio of 9:1. Fresh DMEM complete culture medium was replaced around 24 hours. Lentiviral infection of macrophages was completed after 48 hours. The cell growth and morphological changes were observed, and the cells were passaged to new culture flasks or culture dishes for amplification or cryopreservation.

[0049] 1.3 Confocal characterization of anti-HER2 CAR-M cell lines

[0050] The anti-HER2 CAR-M cell line was statically cultured in a cell culture incubator at 37° C. and 5% CO 2 in DMEM high-glucose medium (hyclone) containing 10% FBS.

[0051] (1) The cultured macrophages were washed 1-2 times with PBS, digested with trypsin for 2 min, and resuspended in complete culture medium at a cell concentration of 10 5 cells / mL, add 1 mL of cell resuspension to the confocal dish, spread evenly, and culture in a cell culture incubator at 37°C and 5% CO2 for 12 h until the cells adhere to the wall;

[0052] (2) Remove the confocal dish and wash the cells with PBS for 5 min each time for 3 times. Then add 1 mL of 4% paraformaldehyde to the confocal dish and fix for 20-30 min. Aspirate the fixative and wash with PBS for 3 times for 5 min each time.

[0053] (3) DiI treatment: The anti-HER2 CAR-M cell membrane was stained with the DiI cell membrane red fluorescent probe. First, the DiI powder particles were diluted with DMSO to a storage solution with a concentration of 1-5 mM, and then diluted with PBS to a concentration of 1-5 μM to form a working solution. 100 μL of the working solution was added from the edge of the chamber of the confocal dish and gently shaken to cover all cells. After 15-20 minutes, the staining solution was aspirated and washed with PBS three times, each time for 5 minutes;

[0054] (4) DAPI staining: DAPI blue fluorescent dye was used to stain the CAR-M cell nuclei. First, the DAPI storage solution with a concentration of 5 mg / mL was diluted to 0.5-10 μg / mL with PBS to form a working solution. Then 100 μL of the working solution was added to the confocal dish to cover all cells. After 10-15 minutes, the staining solution was aspirated and washed with PBS three times, each time for 5 minutes.

[0055] (5) Sealing and imaging: The sample is sealed with an anti-fluorescence attenuation sealing agent to completely cover the sample cells and prevent bubbles from forming. Finally, the sample is placed on the stage of a confocal scanning microscope for fluorescence monitoring. Figure 1 As shown in the figure, the confocal microscopy results show that the anti-HER2 CAR-M fusion protein is successfully expressed across the membrane. Among them, DAPI represents the successful staining of the CAR-M cell nucleus by DAPI blue fluorescent dye, DiI represents the successful staining of the cell membrane by DiI cell membrane red fluorescent probe, anti-HER2 CAR-M represents the expression of CAR-M, and Merge is the superimposed picture of DAPI, DiI, and anti-HER2 CAR-M, which reflects the co-localization of anti-HER2 CAR-M transmembrane expression and DiI cell membrane staining.

[0056] Example 2: Decitabine safe dose range and incubation time

[0057] 1. Low-dose DAC is non-toxic to tumor cells

[0058] The CCK cell proliferation assay was used to verify the toxicity of low-dose DAC on tumor cells. The specific operation is as follows:

[0059] (1) 100 μL of tumor cell suspension was added to each well of a 96-well plate. The cell suspensions were 4T1 and SKOV3 at a density of 1×10 4 cells / mL, placed in a 37°C, 5% CO2 incubator for static culture;

[0060] (2) After culturing the cells for 24-48 hours, the medium in the 96-well plate was removed, the cells were washed 2-3 times with PBS, and different doses of DAC were added to the 96-well plate to treat the cells for 0 hours, 12 hours, 24 hours, 48 ​​hours, and 72 hours respectively;

[0061] (3) After the treatment is completed, the culture medium in the 96-well plate is aspirated, the cells are washed 2-3 times with PBS, and then 10 μL of CCK-8 detection solution is added to the 96-well plate, and cultured in an incubator at 37°C and 5% CO2 for 3-4 hours;

[0062] The absorbance of each well at 450 nm was measured by an enzyme-labeled instrument. The target cell activity = (absorbance of the effector-target ratio group - absorbance of the effector cell control group) / (absorbance of the target cell positive control group - absorbance of the blank well).

[0063] Different doses of DAC were used to treat tumor cells. DAC did not significantly affect cell viability. Figure 2 In the results of A and C, the cell viability did not decrease significantly after treatment with 0μM-5μM DAC, indicating that low doses of DAC have no obvious toxicity to tumor cells.

[0064] 2. DAC dosage and incubation time

[0065] In order to determine the optimal incubation dose and incubation time for DAC to demethylate tumor cells, qPCR was used to quantify the demethylated DFNA5 gene in 4T1 / skov3 tumor cells. The experimental procedure was as follows: 2×10 5 4T1 / skov3 tumor cells were cultured in 10% FBS 1640 / DMEM medium at 37°C and 5% CO2 incubator for 24 hours, and then treated with different concentrations of DAC and incubated for different time periods for detection. Total RNA of each group of cells was extracted, and the expression level of demethylated DFNA5 gene was detected by qPCR. The expression of related cytokines in the above cells was detected by SuperReal PreMix Plus kit, and the experiment was carried out according to the kit instructions. The qPCR experimental steps are as follows:

[0066] (1) Dissolve 2× SuperReal Premix Plus, 50× ROX Reference Dye, template, primers, and RNase-free ddH2O as usual, and equilibrate all reagents at room temperature and mix thoroughly;

[0067] (2) On ice, mix the cDNA template obtained by reverse transcription with a certain system of forward primer, reverse primer, Mix, ROX, and ddH2O. Use a pipette to add the mixture into an eight-tube strip. The 20 μL system includes: 10 μL 2×SuperRealPremix Plus, 0.6 μL forward primer (10 μM), 0.6 μL reverse primer (10 μM), 0.1-2 μL cDNA template, 0.4 μL 50×ROX Reference Dye. Use RNase-free ddH2O to make up the total volume to 20 μL.

[0068] (3) Cover the tube, pipette and mix, and centrifuge for 5-10 seconds to ensure that all components are at the bottom of the tube;

[0069] (4) The reaction system was placed in the RT-qPCR instrument ABI 7300, and the instrument parameters were set: 95°C pre-denaturation for 15 min, 95°C denaturation for 10 s, 60°C±1°C annealing for 20 s, 72°C extension for 31 s, 40 cycles, and the program was run. After the program was completed, the Ct value was exported and the 2 -ΔΔCt (Livak) method was used to calculate the final result.

[0070] like Figure 2 After cells were treated with different concentrations of DAC, the expression of the demethylation gene DFNA5 would change to a certain extent. Generally speaking, incubation within 0-4μm for 1-2 days produced a better demethylation effect.

[0071] Example 3: Verification of the mechanism of apoptosis-to-pyroptosis in decitabine combined with anti-HER2 CAR-M cells

[0072] Anti-HER2 CAR-M upregulates cas3 expression, and tumor cells treated with DAC upregulate GSDME expression. Cas3 cleaves GSDME to cause pyroptosis. The specific experimental steps are as follows:

[0073] 1. Inoculate 2×10 5 The UTD-M / ACT CAR-M cells were cultured in 10% FBS DMEM medium at 37°C and 5% CO2 incubator for 24 hours, and then the total RNA of each group of cells was extracted, and the expression level of cas3 gene was detected by qPCR. The expression of related cytokines in the above cells was detected by SuperReal PreMix Plus kit, and the experiment was carried out according to the kit instructions. The qPCR experimental steps were the same as above.

[0074] The results showed that anti-HER2 CAR-M indeed upregulated cas3 expression and induced apoptosis.

[0075] 2. (1) The mouse triple-negative breast cancer cell line 4T1 was cultured in a 1640 medium in a 37°C, 5% CO2 cell culture incubator;

[0076] (2) Through preliminary experiments, a HER2+-4T1 tumor model was successfully constructed in vitro

[0077] (3) Add 1 mL of tumor target cell suspension HER2+-4T1 to each well of a 6-well plate at a density of 2×10 5 cells / mL, placed in a 37°C, 5% CO2 incubator for static culture;

[0078] (4) After the tumor target cells adhere to the wall, the culture medium in the well plate is aspirated, the cells are washed 2-3 times with PBS, and then 1 μM DAC (fixed concentration) is added to some of the cells in the 6-well plate for 24 hours.

[0079] (5) After the tumor cells were treated for 24 hours, 1 mL of anti-HER2 CAR-M with a 1:1 effector-target ratio was added to each 6-well plate, and 3 parallel wells were set up for each group of experiments. After the cells were co-cultured for 24 hours, the culture medium in the 6-well plate was aspirated, the cells were washed with PBS 2-3 times, the cells were scraped off with a cell scraper, and the cells were collected and resuspended after centrifugation. The Annexin V-FITC kit was mainly used for cell apoptosis detection. The Annexin V-FITC fluorescent probe was added to the cell suspension and blown, and then PI staining was added and gently stirred. The cells were incubated at room temperature (20-25°C) in the dark for 15 minutes, and finally detected by flow cytometry.

[0080] like Figure 3 The results showed that the cells in the drug-untreated group (PBS) had no PS eversion phenomenon, and PI could not break through the normal cell membrane, so 90% of the cells were in the lower left quadrant, that is, the probe was double negative. The upper right quadrant, that is, the probe was double positive, accounted for only 0.60%. The DAC group had no obvious effect. The ACT CAR-M group had a significant apoptosis effect due to the influence of cas3, and the pyroptosis effect was not obvious, accounting for only 4.80% in the upper right corner. Due to the apoptosis-to-pyroptosis conversion in the DAC+ACT CAR-M group, a large number of cell pyroptosis occurred compared with the single ACT CAR-M group, and the double positive accounted for 60.3%. This shows that only the cells that have been treated with the combination have obvious cell pyroptosis, and the proportion is more than 40%.

[0081] Example 4: Improving the anti-tumor activity of anti-HER2 CAR-M cells in vitro

[0082] The present invention is different from the previous strategy of simply relying on the development of new targets to tackle solid tumors, and is a new paradigm of cell therapy targeting the mature target of HER2. The present invention initially explored the ability of anti-HER2 CAR-M to treat tumors in vivo, but due to the methylation of genes in tumor cells, it will lead to poor tumor treatment effects, and the difficulty in preparing CAR-M in clinical practice is also a problem. Considering the combination of decitabine (DAC) to remove the methylation of tumor cells, DAC combined with anti-HER2 CAR-M treatment can reduce the number of anti-HER2 CAR-M treatments and improve treatment efficiency. The specific operation of low-dose DAC combined with anti-HER2 CAR-M to kill tumor cells is as follows:

[0083] (1) Add 100 μL of tumor target cell suspension 4T1, SKOV3 to each well of a 96-well plate at a density of 1×10 4 cells / mL, placed in a 37°C, 5% CO2 incubator for static culture;

[0084] (2) After the tumor target cells adhere to the wall, the culture medium in the well plate is aspirated, the cells are washed 2-3 times with PBS, and then 1 μM DAC (fixed concentration) is added to some of the cells in the 96-well plate for 24 hours.

[0085] (3) After the tumor cells were treated for 24 hours, 100 μL of UTD-M and anti-HER2 CAR-M with a 1:1 effector-target ratio were added to the 96-well plate, and 3 parallel wells were set up for each group of experiments; after the cells were co-cultured for 24-48 hours, the culture medium in the 96-well plate was aspirated, the cells were washed 2-3 times with PBS, and 10 μL of CCK8 detection solution was added to the 96-well plate, and cultured in an incubator at 37°C and 5% CO2 for 3-4 hours;

[0086] (4) Use an ELISA reader to measure the absorbance of each well at 450 nm. The target cell activity = (absorbance of the effector-target ratio group - absorbance of the effector cell control group) / (absorbance of the target cell positive control group - absorbance of the blank well).

[0087] like Figure 4 , using low doses of DAC to treat tumor cells, and using anti-HER2 CAR-M to kill HER2+ tumor cells. After anti-HER2 CAR-M combined with DAC, its effect of killing tumor cells was significantly improved. This has laid an excellent research foundation for clinical research.

[0088] In summary, the combination of anti-HER2 CAR-M and the specific chemical drug DAC has a significant killing effect at the cell level, providing a favorable basis for clinical research.

[0089] Example 5: Decitabine improves the anti-tumor activity of anti-HER2 CAR-M cells in vivo

[0090] In the early stage of this invention, the anti-HER2 CAR-M and DAC were used to verify the ability of killing tumors in vitro. The mouse HER2+-4T1 tumor model was used to predict the anti-tumor effect of the combination of anti-HER2 CAR-M and DAC. After the HER2+-4T1 tumor model was successfully established in each group of mice, drugs and cells were injected into the tail vein to closely observe the tumor treatment effect.

[0091] 1. Anti-HER2 CAR-M combined with DAC inhibits tumor growth in vivo

[0092] The present invention uses a mouse HER2+-4T1 tumor model to verify the anti-tumor effect of anti-HER2 CAR-M HER2+-4T1. The establishment of the HER2+-4T1 tumor model and the tumor treatment process include the following steps:

[0093] (1) Mouse triple-negative breast cancer cell line 4T1 was cultured in 1640 medium in a cell culture incubator at 37°C and 5% CO2;

[0094] (2) Female Balb / c mice aged 4-6 weeks should be purchased and kept in a constant temperature, sterile and ventilated mouse breeding room for more than one week before conducting mouse experiments;

[0095] (3) The 4T1 cells were expanded and cultured, and a HER2+-4T1 tumor model was successfully constructed in vitro through preliminary experiments. After the mice were fed for about a week, the HER2+-4T1 cells were digested, washed 2-3 times with PBS, and resuspended in PBS at a dilution density of 10 7 cells / mL, 100 μL of the cell resuspension was injected subcutaneously into the upper right limb of the mouse;

[0096] (4) Anti-HER2 CAR-M cells were cultured in DMEM high-glucose medium in a cell culture incubator at 37° C. and 5% CO 2 ;

[0097] (5) After HER2+-4T1 cells formed tumors, six mice were set up in parallel for each group. 100 μL PBS or cell resuspension was injected through the tail vein. 3×10 6 The second injection was performed on the 5th day after the first injection. The injection volume for each group was 3×10 6 cells, injected twice in total.

[0098] (6) The length and width of the mouse tumor were measured using an electronic vernier caliper, and the mouse weight was measured using an electronic balance. The mouse tumor volume = (length * width 2 ) / 2, measured every two days, and finally calculated the mouse survival curve.

[0099] Depend on Figure 5 As shown in B and D, both the anti-HER2 CAR-M and DAC combination group and the anti-HER2 CAR-M group significantly inhibited tumor growth. However, compared with the anti-HER2 CAR-M group without combined treatment, the tumor in the anti-HER2 CAR-M and DAC combination group was significantly inhibited, indicating that the anti-HER2 CAR-M and DAC combination group has a stronger ability to inhibit tumors than the anti-HER2 CAR-M group without combined treatment.

[0100] 2. Anti-HER2 CAR-M combined with DAC reduces tumor weight in mice

[0101] Depend on Figure 3 As shown in C, compared with the anti-HER2 CAR-M group without combined treatment, the tumor weight of mice in the anti-HER2 CAR-M and DAC combined treatment group was significantly lighter, which laid an excellent research foundation for clinical research.

[0102] 3. The combination of anti-HER2 CAR-M and DAC is non-toxic in vivo

[0103] The combination of anti-HER2 CAR-M and DAC therapy did not significantly affect the body weight of mice. Figure 5 As shown in E, the final body weight of mice in the anti-HER2CAR-M and DAC combined treatment group increased without significant decrease, indicating that the anti-HER2CAR-M and DAC combined treatment had no obvious toxicity in vivo.

[0104] In summary, the combined treatment of anti-HER2 CAR-M and DAC has a better tumor inhibition effect than the anti-HER2 CAR-M group, which provides a favorable basis for clinical research.

[0105] Embodiment 6:

[0106] The present invention also uses the SKOV3-luc abdominal metastasis model to further predict the anti-tumor effect of combined treatment. After the SKOV3-luc tumor model is established by intraperitoneal injection in each group of nude mice, drugs and cells are injected into the tail vein respectively, and the tumor treatment effect is closely observed.

[0107] 1. Anti-HER2 CAR-M combined with DAC inhibits metastatic tumor growth

[0108] Depend on Figure 4 As shown in A, 1.5×10 6 Anti-HER2 CAR-M combined with DAC group and 0.75×10 6The anti-HER2 CAR-M and DAC combination groups significantly inhibited tumor growth. Compared with the anti-HER2 CAR-M group without combined treatment, the tumors in the anti-HER2 CAR-M and DAC combination groups at different doses were significantly inhibited, indicating that the anti-HER2 CAR-M and DAC combination group has a good ability to inhibit tumors, and 1.5×10 6 Almost all patients in the anti-HER2 CAR-M and DAC combination group were cured.

[0109] 2. The combination of anti-HER2 CAR-M and DAC is non-toxic in vivo

[0110] The combination of anti-HER2 CAR-M and DAC therapy did not significantly affect the body weight of mice. Figure 6 As shown in C.

[0111] In summary, the combination of anti-HER2 CAR-M and DAC therapy still has a good tumor inhibitory effect in the abdominal metastasis model, providing possibilities for the treatment of more different types of tumors.

Claims

1. An application of an epigenetic drug combined with adoptive macrophages in the preparation of a biological drug for treating tumor diseases, characterized in that: The epigenetic drug is used to modify tumor cells, the adoptive macrophages are anti-HER2 CAR macrophages, and the chimeric antigen receptor extracellular domain of the anti-HER2 CAR macrophages targets and recognizes the targeted tumor antigen and blocks the signal pathway of the targeted tumor antigen; The method of modifying tumor cells is to remove the methylation of tumor cells; The epigenetic drug is decitabine; The target of the targeted tumor antigen is HER2; The nucleic acid sequence of the single-chain antibody against HER2 in HER2 CAR macrophages is shown in SEQ ID NO.1; The tumor is ovarian cancer or breast cancer.

2. The use according to claim 1, characterized in that The anti-HER2 CAR macrophage comprises a signal peptide domain, a first linker domain, a second linker domain and a transmembrane domain.

3. The use according to claim 2, characterized in that The transmembrane domain is selected from one of CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, Toll-like receptor 1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, GlycophorinC, NECL2, GPRCSD, CD179a, ERG, TMPRSS2, PAX3, RhoC, TRP-2, CYP1B1, SART3, PAX5, OY-TES1, AKAP-4, SSX2, MYD88, MAL, IRAK1, FCGR2A, FCGR3A and FCER1G.

4. The use according to claim 1, characterized in that The tumor cells are selected from HER2-positive tumor cells; The HER2-positive tumor cell is SKOV3.

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