Application of bisecting GlcNAc level accelerant in enhancing tumor immunotherapy curative effect

Abstract

The invention belongs to the field of biological medicine, and particularly relates to application of a bisecting GlcNAc level accelerant in enhancing tumor immunotherapy curative effect, research shows that the bisecting GlcNAc level is increased, PD-L1 expression is adjusted to enhance the action mechanism of tumor immunity, verification is carried out in a BALB / c (nude) mouse body, the bisecting GlcNAc level accelerant has a good tumor immunotherapy curative effect, and in addition, the bisecting GlcNAc level accelerant has a good application prospect. The expression and secretion of TGF beta 1 (transforming growth factor beta 1) can also be inhibited. According to the present invention, the promoter capable of improving the level of the equally divided GlcNAc is adopted as the main active component for preparing the tumor treatment drug or the drug composition so as to achieve the purpose of improving the immunotherapy response efficiency of the tumor patient, and the drug or the drug composition has characteristics of low toxic-side effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the application of a bisecting GlcNAc level promoter in enhancing the efficacy of tumor immunotherapy. Background Art

[0002] Bisecting GlcNAc (N-acetylglucosamine) modification is catalyzed by β-1,4-N-acetylglucosamine transferase III (MGAT3), which connects GlcNAc to the mannose of the pentasaccharide core with a β-1,4-glycosidic bond and is a special form of N-glycosylation.

[0003] Currently, clinical treatments for tumors include surgical treatment, secretory therapy, and chemotherapy, which have achieved certain therapeutic effects. However, these traditional treatments have some defects. The quality of life caused by surgical trauma has decreased significantly; the side effects of chemotherapy such as bone marrow suppression, nausea and vomiting, hair loss, and cardiotoxicity, as well as skin damage and fibrosis caused by radiotherapy, have caused the treatment of tumors to gradually enter a new era of immunotherapy from traditional treatments to targeted drugs.

[0004] Unlike traditional treatments, immunotherapy uses some cytokines, chemokines and immune cells to reshape the tumor microenvironment, thereby improving anti-tumor efficacy and preventing recurrence. However, due to the influence of primary or acquired drug resistance caused by tumor immune escape, the vast majority of patients cannot benefit from immunotherapy. Therefore, in-depth exploration of the immune escape mechanism and the development of new strategies to overcome immune escape are the only way to improve the efficiency of immunotherapy response in tumor patients and make it more widely used.

[0005] In view of this, the present inventors provide a bisecting GlcNAc level promoter for use in enhancing the efficacy of tumor immunotherapy to solve the above technical problems. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the above-mentioned prior art and propose an application of a bisecting GlcNAc level promoter in enhancing the efficacy of tumor immunotherapy, so as to achieve the purpose of improving the efficiency of immunotherapy response in tumor patients.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In one aspect, the present invention provides a use of a bisecting GlcNAc level enhancer in the preparation of a tumor immunotherapy enhancer, or in the preparation of a tumor treatment drug or pharmaceutical composition.

[0009] Furthermore, the drug or pharmaceutical composition comprises a promoter for increasing the level of bisecting GlcNAc and a pharmaceutically acceptable carrier.

[0010] Furthermore, the carrier includes microcapsules, microspheres, nanoparticles or liposomes.

[0011] Furthermore, the medicine or pharmaceutical composition also contains any commonly used non-toxic pharmaceutical excipients.

[0012] Further optionally, the pharmaceutical excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids and release retardants.

[0013] Furthermore, the dosage form of the drug or pharmaceutical composition includes injection, lyophilized powder for injection, controlled release injection, liposome injection, suspension, implant, embolic agent, capsule, tablet, pill or oral solution.

[0014] Furthermore, the drug or drug composition can also be used in combination with other drugs for treating tumors.

[0015] Preferably, the drug or drug composition can also be used in combination with other drugs for treating breast cancer.

[0016] Furthermore, the drug or pharmaceutical composition may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.

[0017] Furthermore, the tumor immunotherapy includes immune checkpoint inhibitor therapy, and the immune checkpoint inhibitor is a PD-L1 antibody.

[0018] On the other hand, the present invention provides a use of a bisecting GlcNAc level promoter in the preparation of a first inhibitory drug that inhibits PD-L1 expression and activity.

[0019] Furthermore, the first inhibitory drug includes a compound inhibitor, a small protein inhibitor or a monoclonal antibody.

[0020] In another aspect, the present invention provides a use of a promoter of bisecting GlcNAc levels in the preparation of a second inhibitory drug for inhibiting the expression and secretion of transforming growth factor β1 (TGFβ1).

[0021] In addition, the present invention provides a use of a bisecting GlcNAc level promoter in the preparation of food or health products for preventing or inhibiting tumors.

[0022] Furthermore, the promoter includes forskolin or MGAT3.

[0023] Further, the tumor is selected from breast cancer, hematological cancer or a combination thereof. In addition to the above, it can also be selected from other cancers.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1) The present invention innovatively utilizes the promoter of bisecting GlcNAc level in the preparation of tumor immunotherapy synergist, or in the preparation of tumor treatment drugs or pharmaceutical compositions, which can promote the transformation of TAMs (tumor-associated macrophages) to a pro-inflammatory phenotype and reduce their inhibitory effect on the immune system, thereby enhancing CD8 + The anti-tumor immune effect of T cells (T lymphocytes) in tumor cells inhibits the immune escape phenomenon. The present invention provides a method for improving the immune response and therapeutic effect of tumor patients.

[0026] 2) The present invention innovatively utilizes the use of a promoter of bisecting GlcNAc levels in the preparation of a tumor immunotherapy enhancer, or in the preparation of a tumor treatment drug or pharmaceutical composition, and also provides the use of a promoter of bisecting GlcNAc levels in the preparation of a first inhibitory drug that inhibits the expression and activity of PD-L1. The drug or pharmaceutical composition or first inhibitory drug can inhibit the expression of PD-L1 (programmed cell death ligand 1) and promote the degradation of PD-L1 via the lysosomal pathway. It clarifies the molecular mechanism by which increasing the level of bisecting GlcNAc enhances anti-tumor immunity by regulating the expression of PD-L1, which is of great significance for guiding the clinical application of PD-L1-related immune checkpoint inhibitors.

[0027] 3) Use of the promoter of the bisecting GlcNAc level proposed in the present invention in the preparation of a second inhibitory drug that inhibits the expression and secretion of transforming growth factor β1, wherein the second inhibitory drug can significantly inhibit the expression and secretion of transforming growth factor β1 (TGFβ1), and further reduce the immunosuppressive effect of M2 macrophages; specifically, the promoter is forskolin, which can effectively enhance the level of bisecting GlcNAc, significantly inhibit the M2 polarization of tumor-associated macrophages (TAMs), thereby reducing the expression of PD-L1 on its surface, and can ensure that the drug is rapidly delivered to the target tissue through the blood, while significantly reducing the risk of side effects due to its low toxicity and good biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings are incorporated in and constitute a part of this specification and, together with the description, serve to explain the principles of the present invention.

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0030] Figure 1 The results of detecting the level of bisecting GlcNAc in breast cancer cells after forskolin treatment in Example 1 of the present invention;

[0031] Figure 2 (A) and Figure 2 (B) is the flow cytometry analysis of forskolin-treated cells and CD8 + Results of apoptosis rate of 231 cells co-cultured with T cells;

[0032] Figure 3 (A) is the ELISA method used in Example 1 of the present invention to detect CD8 + Results on the level of IFN-γ secretion by T cells;

[0033] Figure 3 (B) is the ELISA method used in Example 1 of the present invention to detect CD8 + Results of the levels of TNF-α secretion by T cells;

[0034] Figure 4 The results of detecting the level of bisecting GlcNAc in breast cancer cells after overexpressing MGAT3 in Example 2 of the present invention;

[0035] Figure 5 This is a tumor growth curve analysis diagram in Example 2 of the present invention;

[0036] Figure 6 The BABL / c nude mice with 4T1 / 4T1-MGAT3 cells as described in Example 2 of the present invention were treated with CD8 + Analysis results of tumors and tumor weights obtained after T cell killing;

[0037] Figure 7 (A) and Figure 7 (B) is the result of detecting the level of bisecting GlcNAc in breast cancer cells after overexpression of MGAT3 in Example 3 of the present invention;

[0038] Figure 8 (A) and Figure 8 (B) is the immunofluorescence experiment in Example 3 of the present invention to detect the expression of PD-L1 protein in 231 and 231-MGAT3;

[0039] Fig. 9The expression of PD-L1 was detected by western blotting after 231 / 231-MGAT3 / 231-Forskolin cells were treated with CHX (20 μM) in Example 4 of the present invention;

[0040] Fig.10 The expression of PD-L1 in 231-MGAT3 / 231-Forskolin treated with MG132 or chloroquine in Example 4 of the present invention;

[0041] Fig.11 The polarization result of macrophages of MDS / AML patients detected by flow cytometry in Example 5 of the present invention;

[0042] Fig.12 The results of qPCR detection of the level of bisecting GlcNAc in bone marrow stromal cells of MDS / AML patients in Example 5 of the present invention;

[0043] Fig.13 The protein immunoblotting in Example 5 of the present invention is used to detect the effect of bone marrow stromal cells of MDS / AML patients on macrophage polarization;

[0044] Fig.14 The flow cytometry analysis of macrophages “tamed” by bone marrow stromal cells of MDS / AML patients for CD8 + Effects on T cell function (TNF-α);

[0045] Fig.15 The flow cytometry analysis of macrophages “tamed” by bone marrow stromal cells of MDS / AML patients for CD8 + Effects on T cell function (IFN-γ);

[0046] Fig.16 The results of Western blotting to detect TGF-β1 levels in bone marrow stromal cells of MDS / AML patients in Example 6 of the present invention;

[0047] Fig.17 The result of enzyme-linked immunosorbent assay for TGF-β1 secretion in bone marrow stromal cells of MDS / AML patients in Example 6 of the present invention;

[0048] Fig.18 The protein immunoblotting in Example 6 of the present invention detects the effect of HS27a of bone marrow stromal cells of MDS / AML patients on macrophage polarization and PD-L1 expression;

[0049] Fig.19 The protein immunoblotting in Example 6 of the present invention detects the effect of HS5, a bone marrow stromal cell of MDS / AML patients, on macrophage polarization and PD-L1 expression;

[0050] Fig. 20 The results of Western blotting to detect TGF-β1 levels in bone marrow stromal cells of MDS / AML patients after forskolin treatment in Example 7 of the present invention;

[0051] Fig.21 To detect the effect of forskolin-treated bone marrow stromal cells from MDS / AML patients on macrophage polarization and PD-L1 expression by flow cytometry;

[0052] Fig. 22 To detect the effect of forskolin-treated bone marrow stromal cells on the "training" of macrophages to CD8 + Effects on T cell function;

[0053] Fig.23 (A) and Fig.23 (B) Flow cytometry analysis of the effects of inhibiting the expression of bisecting GlcNAc in stromal cells and combining with PD-L1 neutralizing antibodies on tumor cell proliferation (D) and CD8 + Effects on T cell function (E). DETAILED DESCRIPTION

[0054] Here, exemplary embodiments will be described in detail, and the embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present invention. Instead, they are only examples consistent with some aspects of the present invention described in detail in the appended claims.

[0055] Unless otherwise specified, the reagents and materials used in the following examples are commercially available or can be prepared by known methods.

[0056] The present invention uses breast cancer, myelodysplastic syndrome (MDS blood tumor) and acute myeloid leukemia (AML blood tumor) as examples for experimental analysis. Among them, myelodysplastic syndrome (MDS) is a complex heterogeneous malignant tumor with significant immunosuppressive properties. Its pathological manifestations are abnormal proliferation and differentiation block of myeloid progenitor cells, accompanied by a high risk of acute myeloid leukemia (AML) transformation.

[0057] Explanation of terms: MDA-MB-231 cells (hereinafter referred to as 231): human breast cancer cell line; 4T1 cells: mouse breast cancer cell line; HEK-293T cells: human embryonic kidney cell line; HS5, HS27a cells: human bone marrow mesenchymal stem cell line; NBS Stroma: primary bone marrow stromal cell line derived from bone marrow.

[0058] Example 1 (Using forskolin to promote the increase of bisecting GlcNAc levels and inhibit immune escape in breast cancer)

[0059] The experimental process is as follows:

[0060] (1) Extraction and culture of CD8 + T cells

[0061] Preparation of PBMC (peripheral blood mononuclear cells): 2 ml of fresh venous blood was diluted with 2 ml of physiological saline and mixed in a 15 ml centrifuge tube, 2 ml of human peripheral blood lymphocyte separation solution was added, the diluted blood sample was slowly and uniformly added to the surface of the separation solution; centrifuged at room temperature at 800 g for 30 min; after centrifugation, the centrifuge tube was divided into four layers from top to bottom: the first layer was the plasma layer, the second layer was the white lymphocyte layer, the third layer was the transparent separation solution layer, and the fourth layer was the red blood cell layer. The lymphocyte layer was carefully aspirated, 3 times the volume of PBS (phosphate buffered saline) buffer was added, and the cells were inverted for washing, and the cells were collected and resuspended for counting at room temperature and 250 g for 10 min. The cell concentration was adjusted to 1×10 8 cells / ml.

[0062] Magnetic bead sorting: 100 μl of cell suspension (10 7 cells) into a new tube, add 10 μl antibody-magnetic beads, and incubate at 4°C for 15 min; after the incubation, place the centrifuge tube on a magnetic rack for 5 min, discard the supernatant (unlabeled cells), and resuspend the magnetic beads (labeled cells) in T cell culture medium.

[0063] (2) T cell killing experiment

[0064] Induce T cell activation: Phorbol 12-myristate 13-acetate (PMA) and ionomycin are commonly used polyclonal stimulators for activating T cells and promoting cytokine secretion. PMA (40ng / μl) and ionomycin (1ng / μl) were added to the T cell culture medium at a ratio of 800:1 and 1000:1 respectively, and induced for 24 hours, then washed twice with saline, and counted on a counting plate for later use.

[0065] Mark target cells: Add CFSE fluorescent dye at a ratio of 1000:1 to the digested cell suspension at 5μM and mix well, then place in an incubator at 37°C, 5% CO2 to mark the target cells for 1 hour. After marking, wash twice with physiological saline, resuspend and count, then inoculate into a six-well plate, and wait for it to adhere to the wall before conducting subsequent experiments.

[0066] T cell killing: 231 cells were treated with 10 μM forskolin for 48 h and then the cells were collected to obtain 231-Forskolin. 5 231 or 231-Forskolin cells were seeded into 6-well plates containing complete medium, and adherent breast cancer cells were expressed according to target cells: CD8 + T cells were cultured in a concentration gradient of 1:0, 1:1, and 1:3 in an incubator for 48 h.

[0067] (3) Cell apoptosis experiment

[0068] Tumor cells via CD8 + After T cell treatment, the culture supernatant was collected, and the adherent cells were digested with 0.25% trypsin. The culture supernatant and the digested cell suspension were mixed and centrifuged at 500g for 5 minutes. The cells were washed 3 times with pre-cooled PBS, and the cell pellet was resuspended in 100μl 1× staining buffer (Binding buffer). 2.5μl Annexin V and 2.5μl 7-AAD were added to each sample, and the staining was carried out on ice in the dark for 15 minutes. 400μl 1×Binding buffer was added to terminate the staining, and the cell apoptosis rate was detected on a flow cytometer.

[0069] (4) Enzyme-linked immunosorbent assay (ELISA) to detect TNF-α and IFN-γ levels

[0070] After the co-culture, the culture medium was collected, centrifuged at 1000 rpm for 5 min, and the supernatant was collected. 100 μl of the supernatant was aspirated and added to the ELISA plate and incubated at 37°C for 2 h. The liquid in the ELISA plate was aspirated, and the plate was washed with PBS buffer for 3 times, each time for 5 min; 100 μl of 3% bovine serum albumin (BSA) blocking solution was added to each well, and the plate was reacted at 37°C for 1 h; the liquid in the well was aspirated, and the plate was washed with PBS buffer for 3 times, each time for 5 min; the primary antibody was added at a ratio of 1:1000, and the plate was reacted at 4°C overnight; the liquid in the well was aspirated, and the plate was washed with PBS buffer for 3 times, each time for 5 min; the secondary antibody was added at a ratio of 1:5000, and the plate was reacted at 37°C for 2 h; the liquid in the well was aspirated, and the plate was washed with PBS buffer for 3 times, each time for 5 min; 100 μl of TMB colorimetric solution was added to each well, and the plate was incubated at room temperature for 30 min in the dark; 50 μl of 2 M H2SO4 was added to terminate the reaction; the absorbance of the sample was measured at a wavelength of 450 nm to detect CD8 + Levels of TNF-α and IFN-γ released by T cells.

[0071] The results are as follows Figure 1 to Figure 3 As shown, forskolin treatment can significantly increase the level of bisecting GlcNAc. Figure 2 (A) and Figure 2 (B) It can be seen that 231-Forskolin cells are more susceptible to CD8 + T cell killing. ELISA was used to detect CD8 + IFN-γ and TNF-α secreted by T cells Figure 3 (A) and Figure 3 (B)) levels also increased accordingly, indicating that increasing the bisecting GlcNAc modification can promote CD8 + T cells exert anti-tumor immune function.

[0072] Example 2 (Increasing the level of bisecting GlcNAc by MGAT3 in mice and promoting CD8 + T cells play an anti-tumor immune function

[0073] The experimental process is as follows:

[0074] (1) Construction of cell lines overexpressing MGAT3

[0075] HEK-293T cells were transfected with lentiviral packaging plasmids pMD.2G, pSPAX2 and transfection reagent polyethyleneimine (PEI) for lentiviral packaging. Cell supernatants were collected 48 hours after transfection and filtered through a 0.22μm filter membrane. The synthesized lentivirus was transfected into 4T1, and the MGAT3 gene was amplified by polymerase chain reaction (PCR) and connected to the lentiviral overexpression vector pLVX-AcGFP-N1 to establish a cell line 4T1-MGAT3 that stably expresses MGAT3. Drug-resistant cell lines were screened and verified by immunoblotting. The primers for MGAT3 real-time fluorescence quantitative PCR (qRT-PCR) were as follows: 5'-ATGAAGATGAGACGCTACAAGC-3' (upstream primer), 5'-CTGGATACGAGGTTAGGGCTG-3' (downstream primer).

[0076] (2) Mouse CD8 + T cell extraction and culture

[0077] The mice were killed by cervical dislocation and soaked in 75% ethanol. The spleen of the mice was removed in a clean bench, and the aseptic operation was noted. 5 ml of mouse lymphocyte separation solution was placed in a 5 cm culture dish with a built-in 40 μm cell mesh. The spleen was placed on the cell mesh and the spleen was gently ground with a syringe piston; the separation solution with spleen cells was immediately transferred to a 15 ml centrifuge tube, covered with 1 ml of RPMI 1640 culture medium developed by the Roswell Park Memorial Institute (keep the liquid surface boundary clear), and centrifuged at room temperature and 800 g for 30 min; the lymphocyte layer was aspirated, and 10 ml of RPMI 1640 culture medium was added, inverted for washing, and centrifuged at room temperature and 250 g for 10 min to collect cells, the supernatant was poured out, and the cells were resuspended and counted with culture medium to adjust the cell concentration to 1×10 8 cells / ml.

[0078] 100 μl of cell suspension (10 7 cells) into a new tube, add 10 μl of biotin antibody mixture, mix well, and incubate at 4°C for 15 min; after thorough vortexing, add 10 μl of streptavidin magnetic beads, mix well, and incubate at 4°C for 15 min; after the incubation, place the centrifuge tube on a magnetic rack for 5 min, aspirate the supernatant, and resuspend in T cell culture medium.

[0079] (3) CD8 in mice + T cell killing assay

[0080] Specific pathogen-free, albino BALB / c nude mice were purchased from Shaanxi Pharmaceutical Biotechnology Co., Ltd. and maintained in a standardized laboratory at 25±2°C, 50±5% relative humidity, and 12h light and 12h dark. The experiment began after one week of adaptive feeding.

[0081] Eight healthy BALB / c nude female mice aged 4 weeks were randomly divided into two groups, with 4 mice in each group: control group and 4T1-MGAT3 group. 4T1 cells were prepared at a concentration of 1×10 6 Each BALB / c nude mouse was subcutaneously injected with 200ul of cell suspension (i.e., 2×10 5 cells) to construct a subcutaneous tumor model, and the tumor grew to about 100 mm 3 When mouse CD8 + T cells were injected into BABL / c nude mice once every 3 days, and the volume of subcutaneous tumors in the mice was recorded. After 22 days, the mice were euthanized, and the tumors were removed and immersed in 4% paraformaldehyde.

[0082] Results from Figures 4 to 6As shown, overexpression of MGAT3 can significantly increase the level of bisecting GlcNAc in 4T1 cells ( Figure 4 ). In vivo experiments in mice showed that increasing the level of bisecting GlcNAc in breast cancer cells can significantly enhance CD8 + The anti-tumor immune effect of T cells significantly inhibits the growth rate of tumors ( Figure 5 ), reduce tumor burden ( Figure 6 ).

[0083] Example 3 (MGAT3 promotes the increase of bisecting GlcNAc levels in breast cancer cells to inhibit the expression of PD-L1)

[0084] The experimental process is as follows:

[0085] (1) Construction of cell lines overexpressing MGAT3

[0086] HEK-293T cells were transfected with lentiviral packaging plasmids pMD.2G, pSPAX2 and transfection reagent polyethyleneimine (PEI) for lentiviral packaging. Cell supernatants were collected 48 hours after transfection and filtered through a 0.22 μm filter membrane. The synthesized lentivirus was transfected into 231, and the MGAT3 gene was amplified by PCR and connected to the lentiviral overexpression vector pLVX-AcGFP-N1 to establish a cell line 231-MGAT3 that stably expresses MGAT3. Drug-resistant cell lines were screened and verified by immunoblotting. The qRT-PCR primers for MGAT3 used were as follows: 5'-ATGAAGATGAGACGCTACAAGC-3' (upstream primer), 5'-CTGGATACGAGGTTAGGGCTG-3' (downstream primer).

[0087] (2) Western blotting assay showed that increasing the level of bisecting GlcNAc in breast cancer cells would reduce the protein expression of PD-L1

[0088] 2×10 5 231 and 231-MGAT3 cells were inoculated into 6-well plates containing complete culture medium. When the cells grew to a confluence of about 90%, the culture medium was discarded, the cells were washed three times with PBS, 200 μl of protein lysis buffer (with 1% protease inhibitor) was added, the cells were scraped with a cell scraper, lysed on ice for 30 min, the lysate was aspirated into a 0.5 ml centrifuge tube, and centrifuged at 14000g for 15 min at 4°C. The supernatant was taken for protein immunoblotting experiment.

[0089] The protein concentration was determined using a BCA kit. According to the measured protein concentration, an equal amount of protein was taken for electrophoresis. After electrophoresis, the protein was transferred to a 0.45 μm polyvinylidene fluoride membrane and blocked with 3% bovine serum albumin (BSA) in TBST at room temperature for 1 h. Then, the primary antibody (1:1000) was added and incubated overnight at 4°C. The membrane was washed 5 times with 1×TBST. Then, the secondary antibody labeled with horseradish peroxide (1:5000) was added and incubated at room temperature for 1 h. The membrane was washed 5 times with 1×TBST. The membrane was developed with a chemiluminescent detection reagent and scanned with a gel imaging system.

[0090] The experimental results are as follows Figure 7-8 As shown, increased levels of bisecting GlcNAc can significantly inhibit the expression of PD-L1 protein in 231 cells.

[0091] (3) Immunofluorescence assay detected that increasing the level of bisecting GlcNAc in breast cancer cells would reduce the protein expression of PD-L1

[0092] 231 and 231-MGAT3 cells were inoculated into confocal dishes, and when the cell confluence reached 50%, the complete medium was discarded, and the cells were washed with PBS three times, and the residual PBS was aspirated. The cells were fixed with 4% paraformaldehyde at room temperature in the dark for 15 minutes, and the cells were washed with PBS three times. 3% BSA solution was added to block at room temperature for 1 hour. After the blocking solution was aspirated, the dilution containing the primary antibody or lectin was added at a dilution ratio of 1:100, and incubated at 4°C overnight. After washing the cells with PBS three times, the secondary antibody or streptavidin conjugated with the corresponding fluorescent group was added at a dilution ratio of 1:500, and incubated at room temperature in the dark for 1 hour, and the cells were washed with PBS three times. 4',6-diamidino-2-phenylindole (DAPI) was used for cell nuclei staining, and the cells were washed with PBS for 15 minutes at room temperature in the dark. The cells were washed with PBS three times, and an appropriate amount of PBS was added, and the cells were placed under a Leica TCSSP8 microscope for photography.

[0093] The results showed that overexpression of MGAT3 could significantly increase the level of bisecting GlcNAc in 231 cells, and immunoblotting ( Figure 7 (A) Figure 7 (B)) and immunofluorescence experiments ( Figure 8 (A) Figure 8 (B)) Detection found that increased levels of bisecting GlcNAc would reduce the protein expression level of PD-L1.

[0094] Example 4 (Increased levels of bisecting GlcNAc in breast cancer cells promote lysosomal degradation of PD-L1)

[0095] The experimental process is as follows:

[0096] (1) Cycloheximide (CHX) tracking assay for protein half-life detection 5 231, 231-Forskolin, and 231-MGAT3 cells were inoculated into 6-well plates containing complete medium. When the cells grew to a confluency of about 60%, the cells were treated with CHX at a final concentration of 20 μM at a time gradient of 0 h, 4 h, 8 h, and 12 h. The cell extract proteins at each time point were collected, and the expression level of PD-L1 protein was detected by western blotting.

[0097] (2) Analysis of PD-L1 degradation pathway

[0098] In order to explore the specific pathway by which bisecting GlcNAc modification affects PD-L1 degradation, this study pre-treated 231, 231-Forskolin and 231-MGAT3 with CHX for 8 h, and then treated them with the proteasome pathway inhibitor MG132 (5 μM) and the lysosomal pathway inhibitor chloroquine (Chloroquine, Chl) (5 μM) for 12 h, respectively, and then collected the cells to extract proteins, and used western blotting to detect the expression level of PD-L1 protein.

[0099] The results are as follows Fig. 9 , Fig.10 As shown in Figure 2, stable transfection of MGAT3 or application of forskolin to increase the level of bisecting GlcNAc can promote the degradation rate of PD-L1 ( Fig. 9 ), and the expression of PD-L1 can be restored after the application of Chl, indicating that increasing the level of bisecting GlcNAc in breast cancer cells can promote the degradation of PD-L1 via the lysosomal pathway ( Fig.10 ).

[0100] Example 5 (Changes in the immune microenvironment of macrophages in patients with MDS / AML (myelodysplastic syndrome / acute myeloid leukemia))

[0101] The specific experimental process is as follows:

[0102] In this example, we systematically analyzed the immune microenvironment of bone marrow macrophages in patients with MDS / AML. First, bone marrow puncture fluid was collected from MDS / AML patients and healthy controls, monocytes were separated by lymphocytes, and red blood cells were removed using red blood cell lysis buffer. Next, the cells were washed with 2% FBS / PBS and blocked with FC blocker (1:200) on ice for 15 minutes. Cell labeling was performed using CD68-PE, CD14-FITC, and CD163-PECy7 antibodies. After labeling, the cells were washed once with 2% FBS / PBS, centrifuged at 800g for 3 minutes at 4°C, and the supernatant was removed. Finally, the cells were resuspended with 400 μL DAPI (1:1000), and 50,000 cells were collected for analysis by flow cytometry. The experimental results are shown in the following figure. Fig.11 As shown, macrophages from MDS / AML patients exhibit characteristics of M2 polarization.

[0103] In the second part of the experiment, bone marrow stromal cells were isolated from MDS / AML patients and healthy controls, RNA was extracted and reverse transcribed into cDNA. The expression level of bisecting GlcNAc was detected by qPCR, and the results were analyzed using GraphPad Prism 7. Fig.12 As shown, the expression of bisecting GlcNAc in stromal cells of MDS / AML patients was significantly downregulated.

[0104] To further study the effect of stromal cells from MDS / AML patients on macrophage polarization, we inoculated THP-1 cells in the lower chamber of a 6-well plate and treated THP-1 with phorbol ester (PMA, 100 ng / mL) for 24 hours to induce polarization into M0 macrophages. Then, primary bone marrow stromal cells and the control group were inoculated in the upper chamber of the co-culture chamber and co-cultured with M0 macrophages for 48 hours. The "tamed" TAMs were collected, and the expression of M2 polarization marker CD163, M1 polarization marker TNFα and PD-L1 was detected by Western blot. The experimental results are as follows Fig.13 As shown in the figure, macrophages from MDS / AML patients were obviously polarized toward M2, and PD-L1 expression was significantly increased.

[0105] In order to evaluate the effect of “tamed” TAMs on CD8 + We used human CD8 + T cell kit to isolate CD8 from healthy peripheral blood + T cells were cultured in serum-free 1640 medium with 50 μM PMA and 100 μM ionomycin for 24 h to activate CD8 +CD8 T cells were stained with 100 μM CFSE. + T cells were stained and co-cultured with the “tamed” TAMs for 48 hours. Leukocyte Activation Cocktail was added for 5 hours to inhibit the secretion of TNF-α and IFN-γ. CFSE-labeled CD8 + The secretion of TNF-α and IFN-γ by T cells. Fig.14 and Fig.15 As shown, CD8 + The ability of T cells to secrete TNFα and IFN-γ was significantly reduced, indicating that their immune function was suppressed.

[0106] Example 6 (Bisecting GlcNAc in stromal cells of MDS / AML patients regulates TGFβ1 secretion)

[0107] In this example, bone marrow stromal cells from MDS / AML patients were first isolated and their proteins were extracted for Western Blot detection. Fig.16 As shown in the results, the expression level of bisecting GlcNAc in the bone marrow stromal cells of MDS / AML patients was significantly reduced, while the amount of TGFβ1 secreted was significantly increased. In order to study the role of bisecting GlcNAc in the function of bone marrow stromal cells, mouse and human bone marrow stromal cells were treated with 10 μmol forskolin for 12 hours. Subsequently, the secretion level of TGFβ1 was detected by enzyme-linked immunosorbent assay (ELISA). The experimental results are shown in Fig.17 As shown, after forskolin treatment, the expression of bisecting GlcNAc in bone marrow stromal cells was significantly enhanced and the secretion of TGFβ1 was significantly reduced.

[0108] In further experiments, stromal cell lines HS5 / HS27a were used to overexpress or silence MGAT3, and these cells and control cells were co-cultured with M0 macrophages to detect TAMs polarization and PD-L1 expression. Fig.18 and Fig.19 As shown. Enhancing the level of bisecting GlcNAc in bone marrow stromal cells significantly promoted macrophage polarization to M1 type, inhibited M2 polarization, and inhibited the expression of PD-L1; while the opposite results were observed after inhibiting the expression of bisecting GlcNAc. The above results indicate that by regulating the expression of bisecting GlcNAc in bone marrow stromal cells, the secretion of TGFβ1 in the bone marrow microenvironment of MDS / AML patients can be changed, and ultimately the immune microenvironment can be regulated by regulating the polarization state of macrophages.

[0109] Example 7 (Regulatory effect of forskolin treatment on the immune microenvironment of MDS / AML)

[0110] In this example, mouse and human bone marrow stromal cells were treated with 10 μmol forskolin for 12 hours. Fig. 20 As shown in the figure, the level of bisecting GlcNAc in bone marrow stromal cells increased significantly after treatment, while the secretion of TGFβ1 decreased significantly. Subsequently, the bone marrow stromal cells treated with forskolin were co-cultured with M0 macrophages to detect the expression of M2 polarization markers CD163 and PD-L1. The results showed that bone marrow stromal cells treated with forskolin significantly downregulated the expression of M2 polarization markers CD163 and PD-L1. In addition, the “tamed” TAMs were co-cultured with CD8 + After T cells, CD8 + The ability of T cells to secrete TNF-α and IFN-γ. Fig.21 and Fig. 22 These results indicate that regulating the expression of bisecting GlcNAc in bone marrow stromal cells can significantly change the immune microenvironment, thereby affecting the polarization state of macrophages and T cell function.

[0111] Example 8 (Therapeutic effect of forskolin treatment combined with PD-L1 neutralizing antibody on AML mice)

[0112] In this example, chlorodronate liposomes were used to remove macrophages in C57 mice, and the combined treatment of stromal cell bisection GlcNAc deficiency and PD-L1 neutralizing antibodies was combined to evaluate its effect on tumor immune response. First, chlorodronate liposomes (10 mg / kg) were injected into the tail vein to remove macrophages in mice. Subsequently, the conditioned medium of HS5-shMGAT3 cells was separated and used to treat mouse RAW264.7 macrophages to induce their conversion into M0 macrophages. These TAMs were injected into mice through the tail vein together with the mouse M1-GFP acute myeloid leukemia cell line. On this basis, mice were treated with PD-L1 neutralizing antibodies (7.5 mg / kg, twice a week for two consecutive weeks) by intraperitoneal injection. The experimental results showed that the combined use of stromal cell bisection GlcNAc deficiency and PD-L1 neutralizing antibodies significantly inhibited the proliferation of M1-GFP cells in mice and significantly enhanced CD8 + The levels of TNF-α and IFN-γ secreted by T cells ( Fig.23 ), indicating that the treatment regimen effectively restored CD8 + Immune function of T cells.

[0113] The above description is only a specific embodiment of the present invention, so that those skilled in the art can understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention.

[0114] It should be understood that the present invention is not limited to what has been described above and that various modifications and changes may be made without departing from its scope. The scope of the present invention is limited only by the appended claims.

Claims

1. Use of a bisecting GlcNAc level enhancer in the preparation of a tumor immunotherapy enhancer, or in the preparation of a tumor therapeutic drug or pharmaceutical composition.

2. The use according to claim 1, characterized in that: The medicine or pharmaceutical composition comprises a promoter for increasing the level of bisecting GlcNAc and a pharmaceutically acceptable carrier.

3. The use according to claim 2, characterized in that: The carrier includes microcapsule, microsphere, nanoparticle or liposome.

4. The use according to claim 1, characterized in that: The tumor immunotherapy includes immune checkpoint inhibitor therapy, and the immune checkpoint inhibitor is a PD-L1 antibody.

5. Use of a bisecting GlcNAc level promoter in the preparation of a first inhibitory drug for inhibiting PD-L1 expression and activity.

6. The use according to claim 5, characterized in that: The first inhibitory drug includes a compound inhibitor, a small protein inhibitor or a monoclonal antibody.

7. Use of a promoter of bisecting GlcNAc levels in the preparation of a second inhibitory drug for inhibiting the expression and secretion of transforming growth factor β1.

8. Use of a promoter of bisecting GlcNAc levels in the preparation of food or health products for preventing or inhibiting tumors.

9. The use according to any one of claims 1 to 8, characterized in that: The promoter includes forskolin or MGAT3.

10. The use according to claim 9, characterized in that: The tumor is selected from breast cancer, hematological cancer, or a combination of both.

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