Application of inhibitor for reducing O-GlcNAc modification level in medicine or medicine composition for treating bladder cancer

Abstract

The invention belongs to the field of biological medicine, and relates to application of an inhibitor for reducing the O-GlcNAc modification level in a medicine or a medicine composition for treating bladder cancer, and the antigen presentation capacity of a TAP1 or HLA-A system is enhanced by reducing the O-GlcNAc modification level. The invention further provides application of the medicine or the medicine composition for enhancing the antigen presentation capacity of the TAP1 or HLA-A system in bladder cancer treatment. The OGT inhibitor is used for reducing the modification level of O-GlcNAc on TAP1, so that the capacity of the TAP1 for transmitting antigen peptide fragments to HLA-A and the capacity of the TAP1 for combining with the HLA-A are improved, degradation of the HLA-A through an autophagy-lysosome pathway is inhibited, and immune escape is reduced.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to the application of an inhibitor for reducing the level of O-GlcNAc modification in a drug or a drug composition for treating bladder cancer. Background Art

[0002] Bladder cancer (BC) is one of the most common malignant tumors of the urinary system in humans and the ninth most common malignant tumor in the world. Bladder cancer can be divided into bladder urothelial cell carcinoma, bladder squamous cell carcinoma, and bladder adenocarcinoma, among which urothelial carcinoma is the most common type of bladder cancer. According to its invasive state, bladder urothelial carcinoma can be divided into muscle invasive bladder urothelial carcinoma (MIBC) and non-muscle invasive bladder urothelial carcinoma (NMIBC). Due to the different characteristics of NMBC and MIBC, there are significant differences in the prognosis of patients; MIBC accounts for about 75% of bladder cancer, is more malignant, more metastatic, and has a worse clinical prognosis than NMIBC. In the process of bladder cancer development, bladder cancer cells go through three stages: immune surveillance, immune balance, and immune escape, and immune escape makes the treatment of bladder cancer more complicated. Therefore, understanding the mechanism of immune escape in bladder cancer is particularly important for the treatment of bladder cancer.

[0003] The major histocompatibility complex (MHC) is a multi-gene family located on human chromosome 6, also known as human leukocyte antigen (HLA), which is closely related to human immune response and immune escape. MHC is mainly divided into two categories, namely MHC Class I and MHC Class II. MHC-I class molecules are mainly responsible for the recognition and transmission of endogenous antigen peptides: after the endogenous protein is cleaved by the proteasome, it is hydrolyzed to produce a peptide of 9-12 amino acids, which is recognized by the transporter associated with antigen processing 1 (TAP1) located on the endoplasmic reticulum membrane. With the help of Tapasin and Calnexin proteins, TAP1 protein forms a quaternary complex with MHC-I, presents the antigen to MHC-I class molecules, and is transported to the cell membrane surface by MHC-I for CD8+T cells to recognize and kill, thereby triggering an immune response. HLA-I class genes mainly include classic HLA-A, HLA-B and HLA-C. Studies have reported that 80% of human tumors downregulate or lose the expression of MHC-I class molecules, resulting in dysregulation of antigen presentation mediated by them.

[0004] O-GlcNAcylation (O-GlcNAc) glycosylation modification is an O-linked glycosylation modification, which only connects one N-acetylglucosamine to the serine and threonine of the protein. O-GlcNAc modification is dynamically regulated by two enzymes, O-GlcNAc glycosyltransferase (O-GlcNAc transferase, OGT) and O-GlcNAc glycosidase (O-GlcNAcase, OGA). O-GlcNAc modification plays an important role in the occurrence and development of tumors, and also has the function of maintaining and regulating the homeostasis of the immune system. In tumor cells, the dysregulation of O-GlcNAc modification often leads to damage to immune checkpoints and tumor immune escape. More and more studies have shown that the level of O-GlcNAc modification has a certain regulatory effect on the immune response of mammalian organisms.

[0005] Therefore, the related applications of reducing the O-GlcNAc modification level of tumor cells, increasing the expression of the antigen presenting molecule MHC-I, and enhancing the ability of the TAP1 / MHC-I system to present tumor-associated antigens urgently need to become a new means to eliminate the immune escape of tumor cells. Summary of the invention

[0006] The purpose of the present invention is to overcome the disadvantages of the prior art mentioned above. The present invention proposes the use of an inhibitor that reduces the level of O-GlcNAc modification in the treatment of bladder cancer.

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

[0008] The present invention provides an application of an inhibitor for reducing the O-GlcNAc modification level in a drug or a pharmaceutical composition for treating bladder cancer, and enhances the antigen presenting ability of TAP1 or HLA-A system by reducing the O-GlcNAc modification level.

[0009] The present invention provides the use of a synergist for enhancing the antigen presenting ability of TAP1 or HLA-A system in a medicine or a medicine composition for treating bladder cancer.

[0010] Furthermore, the medicine or pharmaceutical composition also includes pharmaceutical excipients.

[0011] Furthermore, the pharmaceutical excipients include 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 or release retardants.

[0012] Furthermore, the drug or pharmaceutical composition comprises an inhibitor for reducing the level of O-GlcNAc modification and a pharmaceutically acceptable carrier.

[0013] Furthermore, the carrier includes microcapsules, microspheres, nanoparticles or liposomes.

[0014] Furthermore, the drug or drug composition includes a compound inhibitor, a small protein inhibitor or a monoclonal antibody.

[0015] 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.

[0016] The present invention provides a drug or a drug composition for enhancing the antigen presenting ability of TAP1 or HLA-A system, wherein the drug or the drug composition comprises an OGT inhibitor.

[0017] The present invention provides an application of an inhibitor for reducing the level of O-GlcNAc modification in the preparation of food or health products for preventing or inhibiting tumors.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. The present invention uses an OGT inhibitor to reduce the O-GlcNAc modification level on TAP1, thereby improving its ability to transmit antigenic peptides to HLA-A and its ability to bind to HLA-A, thereby improving the antigen presentation ability of tumor cells and avoiding immune escape.

[0020] 2. The present invention uses an OGT inhibitor to reduce the level of autophagy in bladder cancer cells, resulting in an increase in the level of HLA-A protein.

[0021] 3. The present invention uses an OGT inhibitor to increase the proportion of infiltrating CD8+T cells in the tumor microenvironment and improve the body's immune response level.

[0022] 4. The present invention further confirmed in a BALB / c (nude) mouse subcutaneous tumor-bearing model that reducing the level of O-GlcNAc on TAP1 can effectively inhibit the proliferation of subcutaneous tumors, increase the proportion of tissue-infiltrating CD8+T cells, and improve the survival rate and survival time of mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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.

[0024] 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.

[0025] Figure 1 This is a schematic diagram of flow cytometry detection of reducing the O-GlcNAc level of bladder cancer cells to enhance the killing ability of CD8+T cells against bladder cancer cells in Example 1 of the present invention;

[0026] Figure 2 The results of ELISA in Example 1 of the present invention for detecting bladder cancer cells with low O-GlcNAc levels stimulating CD8+T cells to release TNF-α and IFN-γ;

[0027] FIG3 (A) is the result of enzyme-linked immunosorbent assay in Example 2 of the present invention showing that the O-GlcNAc modification level in bladder cancer tissue is negatively correlated with HLA-A;

[0028] FIG3 (B) is the result of Western blotting detection in Example 2 of the present invention showing that reducing the level of O-GlcNAc in bladder cancer cells promotes the protein expression of HLA-A;

[0029] FIG4 (A) shows the results of establishing a primary mouse bladder cancer model;

[0030] FIG4 (B) is the HE staining result of bladder cancer tissue in the primary mouse bladder cancer model;

[0031] FIG4(C) is a schematic diagram of the primary mouse bladder cancer model treated with OGT inhibitor OSMI-1;

[0032] Figure 4 (D) shows the immunohistochemical results of mouse bladder cancer cells;

[0033] FIG4(E) is the flow cytometry results of mouse bladder cancer cells;

[0034] FIG5 (A) shows the results of the inhibition of HLA-A degradation through the autophagy-lysosome pathway by reduced O-GlcNAc levels in bladder cancer cells;

[0035] FIG5(B) is the result of Western blotting to detect the inhibition of cell autophagy by reducing the level of O-GlcNAc in bladder cancer cells;

[0036] FIG5 (C) shows the results of HLA-A degradation through the autophagy-lysosome pathway;

[0037] FIG6 (A) is the result of immunoprecipitation detection that the decreased O-GlcNAc level in bladder cancer cells promotes the interaction between HLA-A and TAP1;

[0038] FIG6 (B) is the result of isothermal titration calorimetry to determine whether the reduction of O-GlcNAc level on TAP1 promotes the affinity of TAP1 to antigenic peptides;

[0039] Figure 6 (C) is a schematic diagram of in vitro detection of TAP1 antigen presentation ability;

[0040] FIG6 (D) shows that the reduction of O-GlcNAc levels in bladder cancer cells enhances the ability of HLA-A / TAP1 to bind antigenic peptides;

[0041] FIG7 (A) shows the result of constructing the pLVX-Ser63-Flag plasmid using the fusion PCR method;

[0042] FIG7 (B) is the result of verifying the construction of TAP1 overexpression and Ser63 mutation cell lines;

[0043] FIG7 (C) is the result of immunoprecipitation detection of the ability of Ser63 mutation on TAP1 to promote the antigen presentation ability of TAP1 / HLA-A;

[0044] FIG7 (D) is the result of flow cytometry detection of the effect of O-GlcNAc modification at Ser63 on TAP1 on immune escape of bladder cancer;

[0045] Figure 7 (E) is a schematic diagram of the results of the CD8+ T cell in vivo attack experiment;

[0046] FIG7(F) is the result of small animal imaging detection of the effect of O-GlcNAc on bladder cancer immune escape;

[0047] FIG. 7(G) shows the survival curve of Balb / C nude mice after CD8+ T cell treatment. DETAILED DESCRIPTION

[0048] 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.

[0049] The present invention provides an application of an inhibitor for reducing the level of O-GlcNAc modification in a drug or pharmaceutical composition for treating bladder cancer, and enhances the antigen presentation ability of TAP1 or HLA-A system by reducing the level of O-GlcNAc modification. The inhibitor is an OGT inhibitor, and preferably, the OGT inhibitor is OSMI-1.

[0050] The present invention provides the use of a synergist for enhancing the antigen presenting ability of TAP1 or HLA-A system in a medicine or a medicine composition for treating bladder cancer.

[0051] Furthermore, the medicine or pharmaceutical composition includes pharmaceutical excipients.

[0052] Furthermore, the pharmaceutical excipients include 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 or release retardants.

[0053] Furthermore, the drug or pharmaceutical composition comprises an inhibitor for reducing the level of O-GlcNAc modification and a pharmaceutically acceptable carrier.

[0054] Furthermore, the carrier includes microcapsules, microspheres, nanoparticles or liposomes.

[0055] Furthermore, the drug or drug composition includes a compound inhibitor, a small protein inhibitor or a monoclonal antibody.

[0056] 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.

[0057] The present invention provides a drug or a drug composition for enhancing the antigen presenting ability of TAP1 or HLA-A system, wherein the drug or the drug composition comprises an OGT inhibitor.

[0058] The present invention provides an application of an inhibitor for reducing the level of O-GlcNAc modification in the preparation of food or health products for preventing or inhibiting tumors.

[0059] The drug or pharmaceutical composition can also be used in combination with other drugs for treating bladder cancer.

[0060] In one embodiment of the present invention, the medicament or pharmaceutical composition may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.

[0061] In one embodiment of the present invention, the drug includes substances that reduce O-GlcNAc modification on TAP1, promote TAP1 antigen presentation ability, HLA-A binding ability and promote HLA-A expression.

[0062] In one embodiment of the present invention, the drug includes but is not limited to compound inhibitors, small protein inhibitors, and monoclonal antibodies.

[0063] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the present technical field.

[0064] Unless otherwise specified, the reagents and materials used in the following examples are commercially available or can be prepared by known methods.

[0065] The cell names in the following examples are explained as follows:

[0066] YTS-1 cells: human bladder invasive tumor cell line;

[0067] HEK-293T cells: human embryonic kidney cells.

[0068] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0069] Example 1 Reduced O-GlcNAc levels in bladder cancer inhibit immune escape

[0070] (1) T cell killing experiment

[0071] When the target cells (bladder cancer cell YTS-1) grew to a confluency of about 80%, CFSE dye with a final concentration of 100 μM was added to the culture medium to label the target cells, incubated at 37°C in the dark for 2 h, and then washed three times with saline. After digesting the cells with trypsin, 3×10 5 YTS-1 cells were inoculated into six-well plates containing complete culture medium (RPMI-1640 containing 10% FBS) and cultured for 12 h until the target cells adhered to the wall. + T cells were co-cultured at a ratio of 1:1, 1:3 and 1:9; after 4 hours, the culture medium was discarded, the cells were washed three times with normal saline, digested with EDTA-free trypsin, transferred to the corresponding flow tubes, centrifuged at 250g for 5 minutes, and the supernatant was discarded; 200μL of 1×Binding buffer was added to each flow tube to resuspend the cells, and 5μL of APC-labeled Annexin V and erCP-labeled 7-AAD were added and incubated at 4°C for 30 minutes to stain the apoptotic cells; at the same time, unstained cells, cells stained with Annexin V alone, and cells stained with 7-AAD alone were set as negative controls; 400μL of 1×Binding buffer was added to resuspend the cells, and samples were collected using a flow cytometer for detection.

[0072] The experimental results are as follows Figure 1 As shown, the O-GlcNAc modification level of YTS-1 was reduced after treatment with OGT inhibitors and was blocked by CD8 + The proportion of T cells killing increased, showing CD8 + T cells are more sensitive; and with co-culture with CD8 + As the proportion of T cells increases, the apoptosis rate of YTS-1 cells increases synchronously. Preferably, the OGT inhibitor is OSMI-1.

[0073] (2) ELISA detection of bladder cancer cells with low O-GlcNAc levels stimulating CD8 + T cells release TNF-α and IFN-γ

[0074] CD8 + CD8 T cells were transferred to serum-free RPMI 1640 medium, and 50 μM phorbol ester and 100 μM ionomycin were added. The cells were cultured at 37°C and 5% CO2 for 24 h to activate CD8 + T cells.

[0075] YTS-1 cells were treated with OSMI-1 for 48 h and then incubated with activated CD8 +T cells were co-cultured for 48 h. The culture medium was then collected and centrifuged at 1000 rpm for 5 min to collect the supernatant. 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% 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 + The levels of TNF-α and IFN-γ released by T cells. Figure 2 As shown, CD8 + After T cells were co-cultured with YTS-1 cells treated with OSMI-1, the levels of TNF-α and IFN-γ released increased, indicating that reducing the level of O-GlcNAc modification of tumor cells helps activate CD8 + T cells.

[0076] Example 2 Reduced O-GlcNAc levels in bladder cancer cells promote HLA-A expression

[0077] (1) Enzyme-linked immunosorbent assay detected that the O-GlcNAc modification level in bladder cancer tissue was negatively correlated with HLA-A.

[0078] Take appropriate amount of tissue blocks from 30 bladder cancer tissue samples, wash in pre-cooled PBS to remove blood, weigh and set aside (large tissue blocks need to be cut into pieces before homogenization). Then move the tissue blocks into a glass homogenizer, add 5-10ml pre-cooled PBS (the mass volume ratio of tissue to PBS is 1:5) for full grinding. This process needs to be done on ice. Centrifuge the prepared homogenate at 5000×g for 5 minutes, and keep the supernatant for enzyme-linked immunosorbent assay of O-GlcNAc, MHC-I and its subtype proteins HLA-A, HLA-B and HLA-C levels.

[0079] The experimental results are shown in Figure 3(A), and the O-GlcNAc modification level is negatively correlated with MHC-I, HLA-A and HLA-C.

[0080] (2) Western blotting showed that reducing the level of O-GlcNAc in bladder cancer cells promoted the protein expression of HLA-A

[0081] 2×10 5 YTS-1 cells were inoculated into a 6-well plate containing complete culture medium. When the cells grew to a confluence of about 30%, the cells were treated with OSMI-1 at final concentrations of 0 μg / mL, 10 μg / mL, 20 μg / mL, and 40 μg / mL for 48 h; the culture medium was discarded, the cells were washed three times with PBS, 200 μL of protein lysis buffer (with 1% protease inhibitor, PMSF) was added, the cells were scraped with a cell scraper, lysed on ice for 30 min, the lysate was aspirated into a 500 μL centrifuge tube, centrifuged at 14000g for 15 min at 4°C, the supernatant was taken, and protein immunoblotting was performed.

[0082] 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 PVDF 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, incubated at 4°C overnight, washed 5 times with 1×TBST, and then the horseradish peroxide-labeled secondary antibody (1:5000) was added, incubated at room temperature for 1 h, washed 5 times with 1×TBST, and developed with a chemiluminescent detection reagent. Finally, the gel was scanned and imaged using a gel imaging system.

[0083] The experimental results are shown in FIG3(B) , and the reduction of O-GlcNAc level in bladder cancer cell YTS-1 can significantly increase the expression of HLA-A protein in YTS-1 cells.

[0084] Example 3 Reduction of O-GlcNAc levels in mouse bladder cancer cells can treat mouse bladder cancer

[0085] (1) Construction of BBN-induced primary bladder cancer mouse model

[0086] Four-week-old BALB / c (nude) mice were selected, and N-butyl-N(4-hydroxybutyl)nitrosamine (BBN) with a final concentration of 0.05% (V / V) was added to the drinking water of the BALB / c (nude) mice for the model group mice to drink; and 300 μL of drinking water containing 0.05% BBN was gavaged to the model group mice every 3 days for 60 days.

[0087] The experimental results are shown in Figure 4 (A). After 60 days, the urine was collected and observed under a microscope. A large number of suspended red blood cells were found in the urine. At the same time, 20 μL of fresh mouse urine was titrated on a blood urine test strip for detection. By comparing the color of the blood urine test strip, it was observed that the density of red blood cells in the mouse urine was greater than or equal to 200 / μL, indicating that the mouse had initially developed hematuria.

[0088] Three representative mice were euthanized, and their bladder tissues were dissected and stained with hematoxylin and eosin (HE). The experimental results are shown in Figure 4 (B), and it was found that there were multiple cell nuclei in the bladder tissue that had aberrations such as enlarged nuclei and irregular nuclei.

[0089] The above experimental results prove that the primary bladder cancer mouse model was successfully established.

[0090] (2) Reducing the level of O-GlcNAc in mouse bladder cancer cells can treat mouse bladder cancer

[0091] The BBN-induced primary bladder cancer model was randomly divided into three groups. The control group: the model mice were injected with 0.9% saline through the tail vein; the experimental group: the model mice were injected with OSMI-1 at a final concentration of 40 μg / mL through the tail vein for treatment; and a group of healthy wild-type mice without BBN induction were set up and injected with 0.9% saline through the tail vein as a negative control; the experimental scheme is shown in Figure 4 (C).

[0092] After four weeks of treatment with the OGT inhibitor OSMI-1, mice were euthanized, and the bladders were removed and embedded in paraffin for immunohistochemical staining. The obtained sections were incubated at 63°C for 1 hour; dewaxed in xylene and gradient ethanol, and washed with pure water; sodium citrate repair solution with a final concentration of 0.01M at a temperature of 95°C was added, and the water bath was 15 minutes; after the sections were cooled naturally, the sections were washed 3 times with PBS; 5% BSA was added and incubated at 37°C for 2 hours; the primary antibody to be stained was added, incubated overnight at 4°C, and washed 3 times with PBS; HRP-labeled secondary antibody was added and incubated at 37°C for 1 hour; DAB color developer was added for color development, and photos were taken using a microscope. Data statistics were calculated using ImagePro Plus software based on the average optical density.

[0093] The experimental results are shown in Figure 4 (D) and Figure 4 (E). In the bladder cancer model mice successfully induced by BBN, the MHC-I level was significantly decreased; after treatment with OSMI-1, the MHC-I level in the bladder tissue recovered to a certain extent, and the infiltrating CD8 + The level of T cells was significantly increased; the experimental results are shown in Figure 4 (E). After the bladder tissue was digested into single cells with collagenase and hyaluronic acid, flow cytometry was used to detect it. It was also found that after OSMI-1 treatment, the infiltrating CD8 + The number of T cells increased significantly, indicating that reducing the O-GlcNAc level in mouse bladder cancer cells can inhibit the immune escape of bladder cancer, thereby treating primary bladder cancer in mice.

[0094] Example 4 Reduced O-GlcNAc levels in bladder cancer cells inhibit HLA-A degradation via the autophagy-lysosome pathway

[0095] (1) Lysosomal immunoprecipitation detected that decreased O-GlcNAc levels in bladder cancer cells inhibited the degradation of HLA-A through the lysosomal pathway

[0096] 2×10 5 YTS-1 cells were inoculated into a 6-well plate containing complete medium. When the cells grew to a confluency of about 30%, the cells were treated with OSMI-1 at a final concentration of 40 μg / mL for 48 h; the medium was discarded and the cells were washed 3 times with PBS buffer. 1 mL of non-denaturing lysis buffer was added to the culture dish and lysed on ice for 20 min. The cells were gently scraped with a scraper and transferred to a 1.5 mL centrifuge tube; the cells were broken using a pre-cooled homogenizer. Subsequently, the cell lysate was centrifuged for 15 min at 4°C and 1000 rpm, and the supernatant was taken. IP Buffer was added to the supernatant at a ratio of 1:3 to dilute the sample. 1 μg of Lamp-2 monoclonal antibody was added to the diluted supernatant, and incubated for 2 h at 4°C using a flip shaker. 20 μL of Protein A / G Plus-Agarose was added to the lysate, and incubated overnight at 4°C on a flip shaker. Centrifuge at 1000 rpm for 5 min at 4°C, discard the supernatant, add 1 mL of PBS buffer to resuspend the precipitate at the bottom of the centrifuge tube, and repeat 5 times. Add 100 μL of 1× Loading buffer to the precipitate, place in a boiling water bath for 10 min, then centrifuge at 1000 rpm for 5 min, take the supernatant, obtain lysosomal proteins, and perform protein immunoblotting detection.

[0097] The experimental results are shown in Figure 5(A). OSMI-1 treatment reduced the level of intracellular O-GlcNAc modification, reduced HLA-A enriched in lysosomes, and reduced HLA-A degraded by lysosomes.

[0098] (2) Western blotting to detect the inhibition of cell autophagy by reducing the level of O-GlcNAc in bladder cancer cells

[0099] 2×10 5YTS-1 cells were inoculated into a 6-well plate containing complete culture medium. When the cells grew to a confluence of about 30%, the cells were treated with OSMI-1 at final concentrations of 0 μg / mL, 10 μg / mL, 20 μg / mL, and 40 μg / mL for 48 h; the culture medium was discarded, the cells were washed three times with PBS, 200 μL of protein lysis buffer (with 1% protease inhibitor, PMSF) was added, the cells were scraped with a cell scraper, lysed on ice for 30 min, the lysate was aspirated into a 500 μL centrifuge tube, centrifuged at 14000g at 4°C for 15 min, the supernatant was taken, and the levels of autophagy markers SQSTM1 / p62, Beclin 1, and LC3B-II / LC3B-I were detected by western blotting. The experimental results are shown in Figure 5(B). The decrease in intracellular O-GlcNAc levels caused the expression of SQSTM1 / p62, which inhibits autophagy, to increase in a concentration-dependent manner, while the expression of Beclin 1, which promotes autophagy, decreased in a concentration-dependent manner, and the ratio of LC3B-II / LC3B-I decreased, indicating that the decrease in O-GlcNAc levels in bladder cancer cells can inhibit cell autophagy.

[0100] (1) HLA-A degradation via the autophagy-lysosome pathway

[0101] Synthetic shRNA was designed to interfere with or inhibit the expression of ATG5 in YTS-1 cells. shRNA was designed and synthesized for the sequence of ATG5 (NCBI reference sequence: NM_004849.4), and the synthesized shRNA-ATG5-A, shRNA-ATG5-B and shRNA-ATG5-C sequences were constructed into the PLKO.1 plasmid (Addgene number #: 8453), respectively, to obtain silencing plasmids ATG5-A, ATG5-B and ATG5-C. When the 293T cells grow to a confluency of 40%, replace the original culture medium with DMEM empty culture medium (without serum and double antibody) and store at 37°C for use; take two 1.5mL centrifuge tubes A and B, add 250μl of DMEM empty culture medium (without serum and double antibody) respectively; and add 5μg of silencing plasmid, 3.75μg of psPAX2 coating plasmid (Addgene number #: 12260), 1.25μg of pMD2.G coating plasmid (Addgene number #: 12259) to centrifuge tube A, and add 30μl of 10μM PEI transfection reagent, gently mix; incubate at room temperature for 5 minutes; transfer the culture medium in centrifuge tube B to centrifuge tube A, gently mix, incubate at room temperature for 30 minutes, add the mixture to 293T cells, gently mix, place in the incubator and culture for 24 hours, then replace with 10mL of fresh complete culture medium; after 48 hours, collect the culture medium into a 15mL centrifuge tube, centrifuge at 1000rpm, take the supernatant, filter the supernatant with a 2μm filter membrane to obtain the virus liquid.

[0102] 1×10 5 YTS-1 cells were inoculated into a 12-well plate containing complete medium. When the cells grew to a confluency of about 20%, the original medium was removed, and the above-mentioned coated virus solution was added. After incubation at 37°C for 4 hours, a complete medium equal to the volume of the virus solution was added, and the cells were cultured for 24 hours; all the liquid in the well plate was removed, and the cells were gently washed 3 times with DMEM empty medium (without serum and double antibody) to wash away the residual virus solution, and fresh complete medium was added, and the cells were cultured for 24 hours. 0.5ug / mL of puromycin was added to screen the cells, and finally the ATG5-silenced stable transfected cell lines ATG5-A, ATG5-B and ATG5-C were obtained.

[0103] YTS-1 cells were used to detect the effect of shRNA on HLA-A. As shown in Figure 5(C), when ATG5 was interfered with and the intracellular autophagy level was inhibited, the HLA-A protein level increased significantly, indicating that HLA-A can be degraded through the autophagy-lysosome pathway.

[0104] Example 5 Reduced O-GlcNAc levels in bladder cancer cells promote TAP1 antigen presentation ability

[0105] (1) Immunoprecipitation detection: decreased O-GlcNAc levels in bladder cancer cells promote the interaction between HLA-A and TAP1

[0106] 2×105 YTS-1 cells were inoculated into a 6-well plate containing complete medium. When the cells grew to a confluency of about 30%, the cells were treated with an OGT inhibitor (OSMI-1) at a final concentration of 40μg / mL and an OGA inhibitor (TMG) at 20mM for 48h. The protein was then extracted using IP lysis buffer, and the concentration of the extracted protein was determined using a BCA kit. Take a cell lysate containing 1mg of protein, add 1μg of the corresponding primary antibody to the lysate, incubate at 4℃ for 2h, then continue to add 20μL of protein A / GAgrose, and incubate at 4℃ overnight. Then, centrifuge at 1000×g for 5min at 4℃, discard the supernatant, add 1mL of PBS buffer to resuspend the precipitate, repeat this step 5 times, and discard the supernatant; add 100μL of 1×Loading buffer, mix well, and boil in a water bath for 10min. Then centrifuge at 1000×g for 5 min, transfer the supernatant to a new 1.5 mL centrifuge tube, and perform protein immunoblotting. The experimental results are shown in Figure 6 (A). After YTS-1 cells were treated with OSMI-1, the O-GlcNAc modification on TAP1 decreased, and the interaction between TAP1 and HLA-A was enhanced.

[0107] (2) Isothermal titration calorimetry determination of the affinity of TAP1 for antigenic peptides by reducing the level of O-GlcNAc on TAP1

[0108] TAP1 protein was purified from Escherichia coli, and 100 μg of purified protein was prepared into the following system: substrate protein: 100 μg; OGT: 200 μg; MgCl2: 12.5 μM; UDP-GlcNAc: 5 mM. The mixture was incubated on a shaker at 37°C for 4 h to obtain a protein modified with O-GlcNAc.

[0109] Subsequently, the biotin-labeled OVA256-264 peptide was used to titrate wild-type TAP1 (TAP1-WT) and O-GlcNAc-modified TAP1 (TAP1-Glc), and Nano-ITC was used to measure the heat released when the peptides bind to the proteins. The loading needle, titration needle, sample cell and reference cell of Nano-ITC were rinsed with degassed ultrapure water. 200 μL of ultrapure water was added to the reference cell as a reference for heat release during titration. 50 μL of 100 μM OVA256-264 peptide was added to the titration needle as a titration solution for standby use; 300 μL of 1 mM TAP1-WT or TAP1-Glc protein was added to the sample cell. At a constant temperature of 25 °C, 2 μL of titration solution was added to the titration needle each time, and titration was performed every 150 s for a total of 25 times. During titration, the sample pool was stirred continuously at a rate of 150 rpm. The baseline was automatically generated by Microcal origin, and data collection began after the baseline was balanced. The experimental results are shown in Figure 6 (B). When there is no O-GlcNAc modification on TAP1, TAP1 binds more tightly to OVA256-264.

[0110] (3) Reduced O-GlcNAc levels in bladder cancer cells enhance the ability of HLA-A / TAP1 to bind antigenic peptides

[0111] The biotin-labeled OVA256-264 peptide was incubated with YTS-1 cell lysate treated with OSMI-1 or TMG at 4°C to allow OVA256-264 and TAP1 / HLA-A to fully bind. Subsequently, the OVA256-264 peptide was enriched using streptavidin magnetic beads and separated and detected using Tricine-SDS Page. The schematic diagram of the in vitro detection of TAP1 antigen presentation ability is shown in Figure 6(C).

[0112] The experimental results are shown in Figure 6(D). After YTS-1 cells were treated with OSMI-1, the O-GlcNAc level decreased, and the binding of biotin-labeled OVA256-264 to TAP1 and HLA-A increased significantly, indicating that the reduction of O-GlcNAc levels in bladder cancer cells enhanced the ability of HLA-A / TAP1 to bind to antigenic peptides.

[0113] Example 6 Mutation at Ser63 on TAP1 inhibits immune escape

[0114] (1) Construction of pLVX-Ser63-Flag plasmid using fusion PCR

[0115] It has been reported that the serine at position 63 (Ser63) of TAP1 has O-GlcNAc modification. The Ser at position 63 of TAP1 was mutated to Ala by fusion PCR and then inserted into the MCS region of the pLVX-Flag vector to form a new recombinant plasmid. The scheme is shown in Figure 7 (A).

[0116] Using the pLVX-TAP1-Flag vector as a template, Ser63 mutation-specific primers were designed. First, the partial sequence of TAP1 was amplified using primers TAP1-BamH IF and TAP1-Ser63-R; the partial sequence of TAP1 was amplified using TAP1-Ser63-F and TAP1-Age IR; finally, the full-length TAP1 containing the mutation site was amplified using sequence 1 and sequence 2 as templates and TAP1-BamH IF and TAP1-Age IR as primers. The pLVX-Flag vector and the full-length TAP1 containing the mutation site were double-digested with BamH I and Age I, and then connected to form a new recombinant plasmid.

[0117] (2)YTS-1 TAP1 With YTS-1 Ser63 Cell line construction

[0118] Packaging of lentivirus: When HEK-293T cells grow to 50% confluence in T25 cell culture flasks, replace with empty DMEM medium and continue to culture at 37°C, 5% (CO2 for 30 min. Take two sterile centrifuge tubes A and B with a specification of 1.5 mL, and add 250 μL of empty DMEM medium; add 30 μL of PEI to centrifuge tube A, add 3.75 μg of psPAX2 plasmid, 1.25 μg of pMD2.G plasmid and 5 μg of pLVX-TAP1-Flag or pLVX-Ser63-Flag plasmid to centrifuge tube B, and let stand at room temperature for 5 min. Then gently transfer the solution in centrifuge tube A to centrifuge tube B, incubate at room temperature for 30 min, and add to HEK-293T cells, shake gently, and incubate at 37°C, 5% Continue to culture under CO2 conditions for 24 hours. After 24 hours, replace with fresh complete medium and continue to culture for 48 hours. Collect HEK-293T cell culture medium, centrifuge at 1000 rpm for 5 minutes at room temperature, collect supernatant, and filter the collected supernatant with a 0.22 μm filter membrane to obtain pLVX-TAP1-Flag or pLVXSer63-Flag virus liquid, which can be used for subsequent experiments.

[0119] Infect YTS-1 cells with viruses: Subculture YTS-1 cells into 12-well plates. When the cells grow to a confluency of about 10%, remove the culture medium and add 1 mL of the above-obtained virus solution; 12 hours later, add 1 mL of fresh complete culture medium to the cells and continue culturing for 12 hours. After replacing the fresh complete culture medium and continuing culturing for 24 hours; add hygromycin B at a final concentration of 500 μg / mL to the cells and continue culturing for 7 days to screen the successfully infected cells until all uninfected cells are killed.

[0120] Overexpression of TAP1 (YTS-1 TAP1 ) and the mutant Ser63 of TAP1 (YTS-1 Ser63 ) YTS-1 cells were detected by western blotting for TAP1 level. The results are shown in Figure 7(B). The intracellular TAP1 protein level increased, indicating that the YTS-1TAP1 and YTS-1Ser63 stable cell lines were successfully constructed; and as the TAP1 expression increased, the MHC-I and HLA-A expressions in the cells also increased.

[0121] (3) O-GlcNAc modification of Ser63 on TAP1 affects immune escape of bladder cancer cells

[0122] In order to explore the effect of O-GlcNAc modification at Ser63 on the ability of TAP1 to bind and deliver antigenic peptides, this study injected YTS-1 and YTS-1 TAP1 With YTS-1 Ser63 Add OVA to the cell culture medium 256-264 After the peptides were co-incubated for 12 h, the peptides were detected using Tricine-SDS Page. The results are shown in Figure 7(C). Ser63 TAP1 of cell lines binds to OVA on HLA-A 256-264 The number of peptides increased significantly, indicating that O-GlcNAc modification at Ser63 had a significant effect on TAP1 binding to antigenic peptides.

[0123] YTS-1, YTS-1 TAP1 With YTS-1 Ser63 cells, according to tumor cells and CD8 + T cells were co-cultured at a ratio of 1:1, 1:3, and 1:9 for 48 h, and flow cytometry was used to detect the apoptosis of tumor cells regulated by YTS-1. The experimental results are shown in Figure 7(D). Ser63 CD8 + The proportion of T cell killing was the highest, YTS-1 TAP1 cells, indicating that TAP1 is highly glycosylated at Ser63, which can lead to the immune escape of bladder cancer cells.

[0124] (4) Mouse model verifies that O-GlcNAc modification on TAP1 reduces and inhibits tumor immune escape

[0125] In order to verify the effect of mutation of Ser63 O-GlcNAc modification site on TAP1's ability to present HLA-A antigen in vivo, 2×10 5 YTS-1 Luc / OVA / WT 、YTS-1 Luc / OVA / TAP1 With YTS-1 Luc / OVA / Ser63 The cells were injected into 4-week-old male Balb / C nude mice through the tail vein; 7 days later, the mice were observed and photographed using a small animal imaging system. After confirming that the tumor cells were colonized in the mice, 4×10 5 CD8 + T cells, once every 5 days; at the same time, some mice were injected with CD8a neutralizing antibodies to block CD8 + After three injections, the Balb / C nude mice were photographed again using small animal imaging to observe the changes in the tumor area in their bodies. + A schematic diagram of the T cell in vivo attack experiment is shown in Figure 7(E).

[0126] The small animal imaging results are shown in Figure 7(F), and it was found that YTS-1 Luc / OVA / WT 、YTS-1 Luc / OVA / TAP1 With YTS-1 Luc / OVA / Ser63 All three types of cells mainly colonized the lungs of mice. + Before T cell therapy, the areas of tumors formed by the three types of cells were basically the same, indicating that TAP1 overexpression and Ser63 mutation did not affect the tumorigenicity of bladder cancer cells; however, after CD8 + After T cell therapy, YTS-1 Luc / OVA / Ser63 The tumor area formed by cells was significantly reduced, and YTS-1 Luc / OVA / TAP1 The tumor area was also significantly reduced; YTS-1 Luc / OVA / WT Although the tumor volume was reduced to a certain extent, it was not significant. Luc / OVA / WT 、YTS-1 Luc / OVA / TAP1 Still YTS-1 Luc / OVA / Ser63 In mice with CD8a cells, the tumor area did not decrease significantly after injection of CD8a neutralizing antibodies, and the tumor area also showed an increasing trend, indicating that the reduction in tumor area was caused by the injected CD8 + Produced by T cell killing.

[0127] The survival curve was drawn based on the survival time of the Balb / C nude mice in the above experimental groups. The results are shown in Figure 7(G). It was found that the YTS-1 Luc / OVA / Ser63 CD8 + After T cell therapy, the survival rate was the highest; loading YTS-1 Luc / OVA / TAP1 Mouse cells were second; loaded with YTS-1 Luc / OVA / WT The survival time of mice with tumors from the three types of cells was relatively short. + After T cell treatment, their survival time was rapidly shortened: loading YTS-1 Luc / OVA / WT The mice with tumor cells died first, and the YTS-1-loaded Luc / OVA / TAP1 and YTS-1 Luc / OVA / Ser63 The death time of mice with tumor cells was basically the same, and there was no significant difference in the survival curves of mice injected with CD8a neutralizing antibodies.

[0128] In summary, it is shown that the O-GlcNAc modification at Ser63 on TAP1 is crucial for TAP1 to exert its antigen presenting function. When the O-GlcNAc modification at Ser63 is mutated, YTS-1 Luc / OVA / Ser63 Increased levels of HLA-A in cells are suppressed by CD8 + The T cell killing rate is higher and the level of immune escape is significantly reduced.

[0129] 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.

[0130] 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 an inhibitor for reducing the level of O-GlcNAc modification in a drug or pharmaceutical composition for treating bladder cancer, characterized in that: Enhancement of TAP1 or HLA-A system antigen presentation ability by reducing O-GlcNAc modification levels.

2. Use of a synergist that enhances the antigen presentation ability of TAP1 or HLA-A system in a drug or pharmaceutical composition for treating bladder cancer.

3. The use according to claim 1 or 2, wherein the medicine or pharmaceutical composition comprises pharmaceutical excipients.

4. The use according to claim 3, wherein the pharmaceutical excipients include 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, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids or release retardants.

5. The use according to claim 1 or 2, wherein the medicine or pharmaceutical composition comprises an inhibitor for reducing the level of O-GlcNAc modification and a pharmaceutically acceptable carrier.

6. The use according to claim 5, wherein the carrier comprises a microcapsule, a microsphere, a nanoparticle or a liposome.

7. The use according to claim 1 or 2, wherein the medicine or pharmaceutical composition comprises a compound inhibitor, a small protein inhibitor or a monoclonal antibody.

8. The use according to claim 1 or 2, wherein the dosage form of the drug or pharmaceutical composition comprises injection, lyophilized powder for injection, controlled release injection, liposome injection, suspension, implant, embolic agent, capsule, tablet, pill or oral solution.

9. A drug or a drug composition for enhancing the antigen presentation ability of TAP1 or HLA-A system, characterized in that: The medicament or pharmaceutical composition comprises an OGT inhibitor.

10. Use of an inhibitor for reducing the level of O-GlcNAc modification in the preparation of food or health products for preventing or inhibiting tumors.

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