Bladder cancer combined treatment method based on CTSE enhancement

Abstract

The invention discloses a bladder cancer combined treatment method based on CTSE enhancement. According to the application, research finds that CTSE is a key factor related to ICB reaction and patient survival rate improvement. Functional studies find that CTSE improves the curative effect of ICB by enhancing MHC expression, so that antigen presentation, T cell activation and anti-tumor immunity are promoted, and the anti-inflammatory phenotype of macrophages is adjusted at the same time. Importantly, overexpression of the CTSE can further enhance the immunoreaction for blocking the PD-1, has the effect of synergistically treating the bladder cancer, and provides a new idea for effective treatment of the bladder cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a combined treatment method for bladder cancer based on CTSE enhancement. Background Technology

[0002] Bladder cancer (BC) is a malignant tumor that occurs on the bladder mucosa and is the most common malignant tumor of the urinary system. Bladder cancer can occur at any age, even in children. Its incidence increases with age, with a peak incidence between 50 and 70 years of age. The incidence of bladder cancer in men is 3 to 4 times that in women.

[0003] Currently, the molecular mechanisms underlying the development and progression of bladder cancer remain unclear, and significant progress has been made in its treatment and diagnosis over the past few decades. Existing treatments primarily rely on surgery, especially for muscle-invasive bladder cancer, which requires radical cystectomy and urinary diversion, causing considerable suffering for patients. To explore better and more effective methods for the diagnosis and treatment of bladder cancer, further research is necessary to investigate its pathogenesis and identify more sensitive biomarkers and therapeutic targets to modify existing treatment approaches. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, this invention provides a combined treatment method for bladder cancer based on CTSE enhancement.

[0005] To achieve the above objectives, the present invention adopts the following technical solution.

[0006] A first aspect of the invention provides the use of CTSE promoters and / or PD-1 blockers in the preparation of pharmaceutical compositions for treating bladder cancer.

[0007] Furthermore, the promoter includes a carrier or compound that overexpresses CTSE.

[0008] Furthermore, the PD-1 blocker is selected from PD-1 antibodies.

[0009] Furthermore, the CTSE is applied via a delivery carrier.

[0010] Furthermore, the delivery vector is a CAR-T cell.

[0011] Furthermore, the CAR-T cells are CD19 CAR-T cells.

[0012] Furthermore, the pharmaceutical composition also includes other drugs for treating bladder cancer.

[0013] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0014] A second aspect of the invention provides the use of a CTSE promoter in the preparation of a pharmaceutical composition that enhances the efficacy of PD-1 blockade agents in the treatment of bladder cancer.

[0015] Furthermore, the promoter includes a carrier or compound that overexpresses CTSE.

[0016] Furthermore, the PD-1 blocker is selected from PD-1 antibodies.

[0017] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0018] A third aspect of the invention provides the use of a reagent for detecting CTSE expression levels in the preparation of products for predicting the efficacy of drug treatment in bladder cancer patients / predicting the prognosis of bladder cancer.

[0019] Furthermore, the reagent is selected from probes that specifically recognize the CTSE gene, primers that specifically amplify the CTSE gene, or binding agents that specifically bind to the protein encoded by the CTSE gene.

[0020] Furthermore, the drug is a drug used in immunotherapy.

[0021] Furthermore, the reagent also includes a detectable marker.

[0022] Furthermore, the bladder cancer in question is MIBC.

[0023] A fourth aspect of the invention provides the use of CTSE promoters in the preparation of products that promote T cell activation in tumor cells.

[0024] Furthermore, the tumor cells are bladder cancer cells.

[0025] Furthermore, the T cells include CD4. + T cells and / or CD8 + T cells.

[0026] The fifth aspect of the invention provides the use of CTSE promoters in the preparation of products that promote the expression of MHC molecules in tumor cells.

[0027] Furthermore, the tumor cells are bladder cancer cells.

[0028] Furthermore, the MHC molecules include MHC class I molecules and / or MHC class II molecules.

[0029] A sixth aspect of the invention provides the use of a CTSE promoter in the preparation of a product that inhibits TGF-β in tumor cells.

[0030] Furthermore, the tumor cells are bladder cancer cells.

[0031] A seventh aspect of the invention provides the use of CTSE promoters in the preparation of products that regulate macrophage phenotypes in tumor cells.

[0032] Furthermore, the tumor cells are bladder cancer cells.

[0033] Furthermore, the regulation of macrophage phenotype in tumor cells is to promote macrophage polarization toward a pro-inflammatory phenotype.

[0034] An eighth aspect of the present invention provides a pharmaceutical composition for treating bladder cancer, the pharmaceutical composition comprising a CTSE promoter and / or a PD-1 blocker.

[0035] Furthermore, the CTSE also includes a delivery carrier.

[0036] Furthermore, the delivery vector is a CAR-T cell.

[0037] Furthermore, the CAR-T cells are CD19 CAR-T cells.

[0038] Furthermore, the PD-1 blocker is selected from PD-1 antibodies.

[0039] Furthermore, the dosage forms of the pharmaceutical composition include gastrointestinal dosage forms and non-gastrointestinal dosage forms.

[0040] Furthermore, the gastrointestinal dosage forms include tablets, granules, capsules, solutions, dry suspensions, powders, sustained-release preparations, effervescent tablets, emulsions, suspensions, syrups, drops, and chewable tablets.

[0041] Furthermore, the non-gastrointestinal dosage forms include injectable dosage forms, respiratory dosage forms, cavity dosage forms, mucosal dosage forms, and skin dosage forms.

[0042] The ninth aspect of the present invention provides the use of CTSE as a target in screening candidate drugs for the treatment of bladder cancer.

[0043] Furthermore, the method for screening candidate drugs for treating bladder cancer includes: treating a culture system expressing or containing the CTSE gene or its encoded protein with a substance to be screened; and detecting the expression or activity of the CTSE gene or its encoded protein in the system; wherein, when the substance to be screened promotes the expression level or activity of the CTSE gene or its encoded protein, the substance to be screened is a candidate drug for treating bladder cancer.

[0044] The tenth aspect of the present invention provides any of the following methods:

[0045] 1) A method for promoting T cell activation in tumor cells in vitro, the method comprising administering a promoter of CTSE;

[0046] 2) A method for promoting the expression of MHC molecules in tumor cells in vitro, the method comprising administering a promoter of CTSE;

[0047] 3) A method for inhibiting TGF-β in tumor cells in vitro, the method comprising administering a promoter of CTSE;

[0048] 4) A method for promoting macrophage polarization in tumor cells in vitro, the method comprising administering a promoter of CTSE;

[0049] 5) A method for screening candidate drugs for treating bladder cancer, the method comprising: treating a culture system expressing or containing a CTSE gene or its encoded protein with a substance to be screened; and detecting the expression or activity of the CTSE gene or its encoded protein in the system; wherein, when the substance to be screened promotes the expression level or activity of the CTSE gene or its encoded protein, the substance to be screened is a candidate drug for treating bladder cancer.

[0050] Furthermore, the tumor cells are bladder cancer cells.

[0051] Furthermore, the T cells include CD4. + T cells and / or CD8 + T cells.

[0052] Furthermore, the MHC molecules include MHC class I molecules and / or MHC class II molecules.

[0053] Furthermore, the regulation of macrophage phenotype in tumor cells is to promote macrophage polarization toward a pro-inflammatory phenotype.

[0054] Furthermore, the promoter includes a carrier or compound that overexpresses CTSE.

[0055] Furthermore, the method described is not for therapeutic purposes.

[0056] Advantages and beneficial effects of the present invention:

[0057] This application reveals that CTSE is a key factor associated with improved ICB response and patient survival. Functional studies show that CTSE enhances ICB efficacy by increasing MHC expression, thereby promoting antigen presentation, T cell activation, and anti-tumor immunity, while also regulating the anti-inflammatory phenotype of macrophages. Importantly, overexpression of CTSE further enhances the immune response to PD-1 blockade, exhibiting a synergistic therapeutic effect on bladder cancer and providing a new approach for the effective treatment of bladder cancer. Attached Figure Description

[0058] Figure 1This is a single-cell atlas of immunotherapy responsiveness in muscle-invasive bladder cancer. 1A is a schematic diagram of a single-arm exploratory clinical trial of bladder cancer patients receiving anti-PD-1 therapy and GC chemotherapy. 1B is a diagram of clinical response results. 1C is a CT image of tumors before and after ICB treatment with and without response (NR). 1D is a schematic diagram of scRNA-seq and animal model experiments. 1E is a demographic diagram of 36 patients. 1F is a clustering diagram of captured cells. 1G is a diagram of the expression of marker genes in 12 major cell types. 1H is a representative diagram of the proportion of immune cells and non-immune cells in the TME of different patients.

[0059] Figure 2 This is a CTSE prognostic map for bladder cancer immunotherapy. 2A shows differentially expressed genes in tumor cells; 2B shows pathway enrichment in responding tumor cells compared to non-responding tumor cells; 2C shows pathway enrichment in responding tumor cells compared to non-responding tumor cells; 2D shows survival analysis of response characteristics in the TCGA-BLCA cohort; 2E shows survival analysis of genes positively correlated with survival in the TCGA-BLCA cohort; 2F shows AUC of genes positively correlated with survival in the TCGA-BLCA cohort; 2G shows survival analysis of response characteristics in the IMvigor210 cohort; and 2H shows survival analysis of genes positively correlated with survival in the IMvigor210 cohort. 1 is the AUC plot of genes predicting survival in the IMvigor210 cohort; 2J is the survival plot of genes positively correlated with survival in the GSE176307 cohort; 2K is the AUC plot of genes predicting survival in the GSE176307 cohort; 2L is the t-SNE visualization plot of tumor cells; 2M is the t-SNE visualization plot of CTSE expression in tumor cells; 2N is the statistical analysis plot of CTSE expression in the responsive and non-responsive groups; 2O is the IHC plot of CTSE expression in tumors after treatment in the responsive and non-responsive groups; 2P is the quantitative plot of CTSE score in tumor IHC staining after treatment in the responsive and non-responsive groups; 2Q is the CTSE overexpression plot in the MB49 cell line; 2R is the experimental timeline of MB49 EV / OE cells seeded into C57BL / 6 mice, anti-PD-1 treatment, and tumor monitoring; 2S is the tumor growth curve.

[0060] Figure 3This section presents data from single-cell sequencing cohorts. 3A shows a single-cell copy number variation (CNV) graph based on InferCNV analysis; 3B shows survival curves based on gene characteristics in the non-responder group in the TCGA-BLCA and IMvigor210 cohorts; 3C shows survival curves for differentially expressed genes in the TCGA-BLCA cohort; 3D shows survival curves for differentially expressed genes in the IMvigor210 cohort; 3E shows survival curves for differentially expressed genes in the GSE176307 cohort; 3F shows the expression levels of CTS family genes in tumor cells of the responder and non-responder groups; and 3G shows survival curves related to CTS family genes in the TCGA-BLCA cohort.

[0061] Figure 4 This is an in vitro validation of mouse CTSE-overexpressing bladder cancer cell lines and IHC staining characteristics of CTSE in bladder cancer sections from patients. Among them, 4A is a statistical graph of CTSE expression level, 4B is an immunohistochemical image before and after treatment, 4C is a statistical graph of qPCR analysis of CTSE expression in MB49 EV and MB49 OE cells, 4D is a Western blot image of CTSE expression in the culture supernatant of MB49 EV / MB49 OE cells, 4E is a graph of proliferation rate of MB49 EV and MB49 OE cells, 4F is a graph of apoptosis level of MB49 EV and MB49 OE cells, and 4G is a graph of body weight of mice during anti-PD-1 treatment.

[0062] Figure 5 CTSE upregulates MHC molecules and promotes CD4. + T cell expansion and exhaustion precursor phenotypic differentiation diagrams, where 5A is the GSEA diagram including CTSE in tumor cells of the response group, 5B is the gene expression level diagram of MHC class II molecules in tumor cells of the response and non-responder groups, and 5C is the CD4+ phenotype differentiation diagram. + GSEA diagram of MHC-related pathways in T cells, 5D is CD4 + Annotated diagram of T cell subsets; 5E represents different CD4 groups. + Enrichment levels of T cell subset characteristic genes in the responding and non-responding groups, with 5F being CD4+. + Expression map of CCR7, IFNG, CXCL13 and MKI67 in T cells, 5G is CD4 + TCR clonal abundance analysis in T cells, 5H represents CD4. + TCR clonal abundance statistics of T cell subsets, 5I is CD4 + The graph shows the diversity of TCR clones in T cells; 5J is a graph showing the differential expression analysis of MHC class II molecules in MB49 EV / MB49OE; 5K is a graph showing the expression level analysis and statistical analysis of MHC class II molecules in mouse tumor cells; 5L is a graph showing the expression level of CD4+.+ Analysis and statistical graph of Ki67 expression level in tumor-infiltrating lymphocytes (TILs), 5M is tumor CD4 + Analysis and statistical graph of IFN-γ expression level in TILs; 5N is a tumor CD4+ expression level. + Expression level analysis and statistical graphs of PD-1 and Ly108 on TILs, 5O is CD4 + The diagram shows the TCR clonal diversity of T cells. 5P is the flowchart for the OT II cell killing experiment using MB49 EV / MB49 OE loaded with OVA peptides. 5Q is the OT II cell cytotoxicity diagram. 5R is the OT II cell IFN-γ... + Proportional statistics chart;

[0063] Figure 6 It is CD4 + Phenotypic diagrams of T cells in ICB therapy, where 6A shows the expression of MHC class II molecules on antigen-presenting cells in the responding and non-responding groups, and 6B shows the expression of different CD4 subsets. + The expression analysis diagram of T cell-specific genes shows that 6C represents different CD4 expression levels before and after anti-PD-1 treatment. + T cell subset ratio changes, 6D represents CD4 counts in the responding and non-responding groups. + The graph shows changes in T cell subsets. 6E is a comparison of glycolysis and pentose phosphate pathway levels. 6F is a graph showing CD4+ changes. + Flow cytometry plot (left) and statistical plot (right) of CD69 and Ly108 expression on tumor-infiltrating lymphocytes (TILs). 6G is a t-SNE visualization of all cells captured in mouse tumor scRNA-seq. 6H is a dot plot of marker gene expression for each major cell type.

[0064] Figure 7 CTSE promotes CD8 + T cells maintain the exhausted precursor phenotype and enhance IFN-γ secretion. (Graph showing 7A being CD8+). + T cell subset annotation diagram, 7B is CD8 + Expression maps of key markers in T cells; 7C is the TCR clonal abundance map, and 7D is the different CD8 subsets. + T cell TCR clone frequency analysis plot, 7E is the IFNG expression plot in CD8 Tprex, 7F is the comparison plot of IFNG expression in the IMvigor210 cohort, 7G is the correlation plot of CD8A and IFNG expression in the IMvigor210 cohort, 7H is the survival curve plot comparing different IFNG expression levels in the IMvigor210 cohort, 7I is the survival curve plot of different IFNG expression levels in the TCGA dataset, 7J is the tumor infiltration CD8A clone frequency analysis plot. +Expression map of IFN-γ in T cells, 7K is tumor-infiltrating CD8 + Statistical graph of IFN-γ expression in T cells, 7L is CD8 expression in tumor-infiltrating T cells. + The percentage of T cells, 7M is Ki-67. + CD8 + The percentage of T cells, 7N represents tumor-infiltrating CD8 cells. + Expression map of PD-1 and Ly108 on T cells, 7O represents tumor-infiltrating CD45. + CD8 cells + T cell subtype diagram, 7P is CD8 + A graph showing the ratio of observed to expected T cell numbers for different T cell subsets; 7Q represents CD8. + Expression map of Mki67 in T cells, 7R is CD8 + RNA velocity analysis diagram of the transition between T cell Tprex and Tex, 7S is CD8 + TCR clonal abundance map in T cells, 7T is CD8 + TCR clone frequency map in T cells, 7U is CD8 + Expression of Ifng in T cells;

[0065] Figure 8 CD8 in human scRNA-seq and mouse CTSE overexpression bladder cancer models + T cell analysis diagrams are shown, where 8A is the cell communication analysis diagram, 8B is the ligand-receptor (LR) pair analysis diagram, 8C is the expression diagram of major MHC class I related genes in tumor cells, 8D is the expression diagram of MHC class I genes in mouse tumor cells, and 8E is the CD8 expression diagram of 21 patients. + T cell percentage diagram, 8F is CD8 + The plot shows the differential expression of characteristic genes in T cells. 8G is the correlation plot between CD8A and IFNG expression in the TCGA-BLCA dataset, and 8H is the plot between CD8A and IFNG expression. + TCR clonal diversity map in T cells, 8I is CD8 + A diagram of carbon metabolism in the T cell metabolic pathway, where 8J is CD8. + A diagram of the citric acid cycle in T cell metabolic pathways, where 8K is CD8. + Gene enrichment analysis of T-cell glucose metabolism pathways; 8L is the tumor-infiltrating CD8+ pathway in the mouse MB49 tumor model. + CD69 and Ly108 expression patterns in T cells;

[0066] Figure 9This is a map showing the pro-inflammatory phenotype of macrophages promoted by CTSE. Specifically, 9A is the expression level map of IFNGR1 and IFNGR2; 9B is the cell-cell communication analysis map; 9C is the UMAP visualization of bone marrow cell subsets; 9D is the GSEA map of cellular type II interferon response in human scRNA-seq data; 9E is the GSEA map of cellular type II interferon response in mouse scRNA-seq data; 9F is the expression map of pro-inflammatory markers in human scRNA-seq data; 9G is the CCL5 expression map in the IMvigor210 cohort; and 9H... 9I is a correlation plot between CD14 and CCL5 expression in the IMvigor210 cohort; 9J is a survival analysis plot related to CCL5 in the IMvigor210 cohort; 9K is a correlation plot between CTSE and CD163 expression in the IMvigor210 cohort; 9L is a correlation plot between CTSE and TREM2 expression in the IMvigor210 cohort; 9M is a plot of macrophage CD86 expression; 9N is a plot of macrophage CD206 expression; and 9O is a plot of tumor-infiltrating CD11b expression. + F4 / 80 + MFI image of CD86 in macrophages, 9P represents tumor-infiltrating CD11b. + F4 / 80 + MFI image of CD206 in cells (macrophages);

[0067] Figure 10 These are pro-inflammatory phenotype maps of TAMs obtained by scRNA-seq. 10A shows the frequency distribution of DC, pDC, and TAM; 10B shows the correlation between CD14 and CCL5 expression in the TCGABLCA dataset; 10C shows the correlation between CTSE and MRC1 expression in the IMvigor210 cohort; 10D shows the correlation between CTSE and TLR4 in the IMvigor210 cohort; 10E shows the enrichment analysis of the NF-κB signaling pathway in the scRNA-seq data; 10F shows the enrichment analysis of the PI3K-Akt signaling pathway in the scRNA-seq data; and 10G shows the CD8+ signaling pathway. + LT signal transduction map between T cell subsets and TAM, 10H is CD8 + TNF signaling pathway between T cell subsets and TAM, 10I is CD8 + LTA cell communication diagram of T cell subsets, 10J is CD8 + TNF-IL-1β cell communication diagram of T cell subsets;

[0068] Figure 11This diagram illustrates how CTSE inhibits the production of active TGF-β and attenuates its immunosuppressive effect. Specifically, 11A shows the expression level of TGFB1 in the IMvigor210 cohort; 11B shows the expression of TGFB1 in tumor cells; 11C shows the interaction between tumor cell-derived TGF-β and other cellular signaling pathways; 11D shows the level of active TGF-β in the tumor cell supernatant (Sup); and 11E shows the activation of naive CD4+ co-cultured in the tumor supernatant. + Flow cytometry plot of IFN-γ cells after T cell activation; 11F represents the activation of naïve CD4 cells co-cultured with tumor supernatant. + Flow cytometry image of T cell granzyme B; 11G is activated by co-culturing naïve CD8 cells in tumor supernatant. + Flow cytometry image of T cells after IFN-γ activation; 11H represents the activation of tumor supernatant CD8 cells co-cultured. + Flow cytometry image of T cell granzyme B, 11I is activated by co-culturing naïve CD4 cells in tumor supernatant. + IFN-γ expression level after T cell activation, 11J is the expression level of naive CD4 cells co-cultured with tumor supernatant. + T cell granzyme B expression level diagram; 11K activates naïve CD8 cells co-cultured in tumor supernatant. + IFN-γ expression level after T cell activation; 11L is activated tumor supernatant CD8 co-cultured. + The graph shows the expression levels of granzyme B after T cell transplantation, and 11M represents the expression levels of MHC I and MHC II in MB49 EV / OE tumor cells.

[0069] Figure 12 This is a diagram illustrating the immunosuppressive effect of CTSE on TGF-β. 12A shows the TGFB1 expression level in Treg cells; 12B shows cell communication analysis of TGF-β signaling among different cell subsets; 12C shows the TGF-β receptor expression level in different cell subsets; and 12D shows CD4+ expression. + GSEA results of TGF-β receptor signal transduction in T cells, 12E is CD8 + GSEA results of TGF-β receptor signal transduction in T cells, 12F is an naive CD4 cell that activates CFSE-labeled receptors. + CFSE fluorescence intensity map of T cells, 12G represents the activation of CFSE-labeled naïve CD4 at different time points. + IFN-γ flow cytometry image of T cells; 12H is the GSEA result of TGF-β receptor signal transduction in macrophages.

[0070] Figure 13This diagram illustrates the enhanced response of endogenous T cells to PD-1 blockade therapy via CAR-T cell delivery of CTSE. 13A is a schematic diagram of the structure of CD19 CAR and CTSE-armored CD19 CAR. 13B is a diagram confirming CTSE overexpression in CAR-T cells using qPCR results. 13C is a flowchart of the C57BL / 6 mouse animal experiment, including the time points of MB49CD19 tumor cell implantation, cyclophosphamide (CTX) pretreatment, adoptive cell infusion (ACT, containing CTSE-armored CAR-T or control CAR-T cells), and PD-1 blockade therapy. 13D is a curve showing the differences in tumor progression among the groups (PBS group, n=10; CD19 CAR-T group, n=11; CTSE CD19 CAR-T group, n=11; CD19 CAR-T combined with PD-1 group, n=6; CTSE CD19 CAR-T combined with PD-1 group, n=6). Detailed Implementation

[0071] The following provides definitions for some of the terms used in this specification. Unless otherwise stated, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0072] This invention provides the use of CTSE promoters and / or PD-1 blockers in the preparation of pharmaceutical compositions for the treatment of bladder cancer.

[0073] In some implementations, CTSE includes wild-type, mutant, or fragments thereof. The term encompasses full-length, unprocessed CTSE, any form of CTSE derived from cell-processed CTSE, and naturally occurring variants of CTSE (e.g., splice variants or allelic variants). The term covers, for example, human CTSE and CTSE from any other vertebrate source, including mammalian CTSEs such as primates and rodents (e.g., mice and rats), with gene IDs 1510 and / or 13034.

[0074] In some embodiments, a CTSE promoter refers to any substance that can enhance the activity of the CTSE protein, enhance the stability of the CTSE gene or protein, promote the expression level of CTSE, and increase the effective duration of the CTSE protein. This includes, but is not limited to, CTSE overexpression vectors, naturally purified substances that can promote CTSE expression, modified naturally purified substances, semi-synthetic substances, chemically synthesized substances, and / or any combination thereof. Any substance that can promote CTSE expression is within the scope of protection of this application.

[0075] The CTSE is applied via a delivery carrier.

[0076] In some embodiments, the delivery vector includes, but is not limited to, CAR-T cells, nanocarriers, and oncolytic viruses.

[0077] In a preferred embodiment, the delivery vector is a CAR-T cell. The CAR-T cell can be any CAR-T cell capable of drug delivery.

[0078] In a specific implementation, the CAR-T cells are CD19 CAR-T cells.

[0079] The pharmaceutical composition also includes other drugs for treating bladder cancer.

[0080] In some implementations, other drugs for treating bladder cancer include, but are not limited to, chemotherapy drugs (common drugs include pirarubicin, epirubicin, hydroxycamptothecin, etc.), immunotherapy drugs (such as pembrolizumab, nivolumab, durvalumab, etc.), and targeted drugs (anti-angiogenic drugs: such as bevacizumab, ramucirumab, pan-FGFR inhibitors: such as erdatinib, antibody-drug conjugates (ADCs: such as vedictetumab (RC48), veentumumab (EV)).

[0081] The pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0082] In some embodiments, pharmaceutically acceptable excipients include any and all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic and absorption-retarding agents. The use of these media and solvents for pharmaceutically active substances is well known in the art. Its use in therapeutic compositions is considered unless any conventional media or agents are incompatible with the active ingredient to date. Furthermore, various adjuvants, such as those commonly used in the art, may be included.

[0083] Examples of pharmaceutically acceptable excipients include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powdered yarrow; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa soybean oil; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as TWEENS; wetting agents such as sodium dodecyl sulfate; colorants; flavoring agents; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline; and phosphate buffer solutions.

[0084] This invention provides the application of reagents for detecting CTSE expression levels in the preparation of products for predicting the efficacy of drug treatment in bladder cancer patients / predicting the prognosis of bladder cancer.

[0085] The reagent also includes a detectable marker.

[0086] In some embodiments, a detectable marker refers to a composition capable of generating a detectable signal indicating the presence of a target polynucleotide in a sample. Suitable markers include, but are not limited to, radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent components, magnetic particles, and bioluminescent components. Therefore, a marker is any composition detectable by a device or method, including but not limited to spectroscopic, photochemical, biochemical, immunochemical, electrochemical, optical, chemical detection devices, or any other suitable device. In some embodiments, the marker can be detected visually without the aid of a device.

[0087] Among them, radioactive isotopes include but are not limited to 3 H, 14 C 35 S, 125 I, 131 I.

[0088] Enzymes include, but are not limited to, horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and acetylcholinesterase.

[0089] Fluorescent molecules include, but are not limited to, FITC, rhodamine, and lanthanide phosphors.

[0090] The invention is further illustrated below with reference to specific embodiments. It should be understood that the specific embodiments described herein are by way of example and are not intended to limit the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention.

[0091] Example

[0092] 1. Experimental Materials and Methods

[0093] Laboratory animals: C57BL / 6 mice were purchased from the Laboratory Animal Center of Southern Medical University and the Guangdong Provincial Laboratory Animal Center. All animal experiments were conducted in accordance with the operating procedures approved by the Animal Ethics Committee of Southern Medical University.

[0094] Cell lines: Mouse bladder cancer cell line MB49 and its derivatives MB49-MigR1 EV, MB49-CTSE OE, and MB49-CD19 were cultured in DMEM medium containing 10% heat-inactivated fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. The NIH3T3-TGF-β reporter cell line was cultured under the same conditions. All cell lines were cultured at 37°C in a humidified environment of 5% CO2 and were confirmed to be free of mycoplasma contamination.

[0095] Experimental methods

[0096] Western Blot: Cells were washed twice with phosphate-buffered saline (PBS), lysed on ice in lysis buffer containing protease inhibitors, and lysed for 15–25 minutes. After lysis, fragments were removed by centrifugation at 12,000 × g for 30 minutes at 4°C. After protein concentration determination, the sample was mixed with SDS-PAGE buffer and denatured at 95°C for 5 minutes. 10–20 μg of protein was separated by 10% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with TBST containing 5% skim milk for 1 hour, followed by loading with primary antibody (CTSE).

[0097] Incubate overnight at 4°C with 1:1000 (α-actinin 1:1000). After washing with TBST, incubate with HRP-labeled secondary antibody at room temperature for 1 hour, develop with ECL, and analyze band intensity using ImageJ software.

[0098] Apoptosis and necrosis analysis: An APC-labeled Annexin V apoptosis detection kit (containing PI) was used. Cells were collected by centrifugation at 300×g for 5 minutes, washed with cold PBS, resuspended in binding buffer, and incubated with APC-Annexin V (1:100) at room temperature for 15 minutes in the dark. After washing with PBS, PI (1:100) was added for staining, and apoptosis (Annexin V) was detected by flow cytometry. + PI - ), necrosis (Annexin V) + PI + ) and survival (Annexin V) - PI - Cells. The experiment was repeated three times.

[0099] Animal experiment: PD-1 blockade therapy: On day 0, 5 × 10⁵ PD-1 was subcutaneously injected into the flank of male C57BL / 6 rats. 5 MB49 EV or CTSE OE tumor cells. Anti-PD-1 antibody (100 μg / mouse) or isotype control was injected intraperitoneally on days 6, 9, 20, and 23. Anti-PD-1 antibody (200 μg / mouse) or control was injected on days 12 and 16. Tumor volume was measured every 3 days (formula: volume = width² × length / 2). Mice were sacrificed on day 40, and tumor tissue was collected for analysis (10 mice each in the EV and OE groups, and 7 mice each in the EV anti-PD-1 and OE anti-PD-1 groups). Tumor tissue was digested with collagenase IV (1 mg / mL) and DNase I (200 μg / mL), then filtered through a 70 μm screen to prepare a single-cell suspension for subsequent staining.

[0100] Adoptive cell transplantation therapy: C57BL / 6 mice were inoculated with 1×10 6MB49-CD19 tumor cells were randomly divided into three groups after 14 days: CTSE-CD19 CAR-T group, CD19 CAR-T control group, and PBS control group. 1.5 × 10⁻⁶ cells were injected intravenously. 6 CAR-T cells were transduced with retroviruses and expanded in vitro for 6 days. Mice were sacrificed 30 days after treatment, and single-cell suspensions were prepared from tumor tissues, spleen, and other tissues. Flow cytometry analysis was performed to identify the phenotypes of tumor-infiltrating lymphocytes and systemic immune cells.

[0101] CAR-T combined with PD-1 blockade therapy: Mice were randomly divided into five groups after inoculation with tumor cells: PBS control group, CD19CAR-T group, CTSE-CD19 CAR-T group, CD19 CAR-T + anti-PD-1 group, and CTSE-CD19 CAR-T + anti-PD-1 group. CAR-T treatment was the same as before, and the PD-1 blockade regimen was as follows: anti-PD-1 antibody (100 μg / mouse) was injected on days 6, 9, 20, and 23 after ACT, and 200 μg / mouse was injected on days 12 and 16. Tumor progression and survival were monitored for 30 days, and tissue samples were collected for flow cytometry analysis.

[0102] Proliferation assay: The proliferation of MB49 EV and OE cells was detected using the CCK-8 assay. Cells were sputtered at a rate of 5 × 10⁶ cells / year. 4 / wells were seeded in 96-well plates, and after 12 hours, CCK-8 solution (2 μL / well) was added. The plates were incubated at 37°C for 2 hours, and the OD value was measured at 450 nm. The proliferation rate was calculated as (OD at time point - baseline OD) / baseline OD. Each group had four replicates, and results are expressed as mean ± standard error (sem).

[0103] Real-time quantitative PCR: Total RNA was extracted from T cells and cDNA was synthesized. qPCR was performed using the SYBR Green method, with GAPDH as the internal reference gene.

[0104] Plasmid construction: The CD19 coding sequence was cloned into the pUltra vector via the XbaI restriction site. When constructing CD19 CAR and CTSE-CD19 CAR plasmids in the MIGR1 vector, XhoI / HpaI and MluI restriction sites were introduced, respectively. After restriction digestion, the fragments were ligated and sequenced for verification.

[0105] T cell activation and co-culture: Initial CD4 cells were isolated from the spleen of C57BL / 6J or CD45.1 mice. + and CD8 + T cells, CFSE-labeled, were used at a concentration of 5 × 10⁻⁶. 5 / wells were inoculated into 48-well plates. + T cells were activated by plate-loaded anti-CD3 (5 μg / mL) and soluble anti-CD28 (1 μg / mL), CD8 +T cells were additionally supplemented with IL-2 (100 U / mL). MB49 EV or OE cell supernatant (1:1 dilution) was added to the T cell culture system, and TGF-β neutralizing antibody (10 μg / mL) was added to some groups.

[0106] Flow cytometry: Cells were surface-stained (anti-CD25, IFN-γ, granzyme B, Foxp3, etc.) or intracellularly stained, and then detected using a BD LSR Fortessa X-20 flow cytometer. Tumor-infiltrating immune cells were filtered through a 40μm filter, and red blood cells were lysed before analysis. Data were processed using FlowJo software, and live cell CD45 levels were analyzed. + Sorting purity > 98%.

[0107] Virus production and transduction: The CD19 CAR sequence was cloned into the reverse transcription MIGR1 vector, and the viral supernatant was collected after packaging in 293T cells. Mouse CD3 + T cells, activated with anti-CD3 / CD28, were co-incubated with viral supernatant and polybrene (10 μg / mL), centrifuged for infection, and expanded to day 10, where GFP was sorted. + CAR-T cells are used in experiments.

[0108] MB49 cell MHC expression analysis: MB49 EV and OE cells expressed at 4 × 10⁻⁶ mcg / mL. 5 / wells were seeded, and TGF-β (10 ng / mL), anti-TGF-β antibody (10 μg / mL), or blank control were added, while IFN-γ (1 ng / mL) was stimulated. After incubation at 37°C for 24 hours, the samples were analyzed.

[0109] 2. Experimental Results

[0110] Immune patterns and variability in response to PD-1 blockers in muscle-invasive bladder cancer

[0111] To evaluate the efficacy and safety of gemcitabine, cisplatin, and tolipalimab in patients with T2-4aN0M0 muscle-invasive bladder cancer (MIBC), this application initiated an open-label, single-arm exploratory clinical trial (GZZJU-2021NB) with 36 participants. All patients received four 21-day cycles of gemcitabine and cisplatin concurrently with tolipalimab at the recommended phase II dose. Tumor resection was performed within six weeks of completion of neoadjuvant therapy. Figure 1 A). Imaging evaluations (CT and MRI) were performed during treatment to monitor tumor size and morphology and to detect signs of metastasis or pelvic recurrence.

[0112] The response to anti-PD-1 therapy varied widely among patient cohorts, ranging from disease progression to significant tumor regression. Figure 1B). Clinical response is assessed based on the reduction in tumor size. Figure 1 C). The mean age of the cohort was 61.1 years (range: 27–87 years), predominantly male (31 males, 5 females). Of these, 16.7% experienced disease progression (PD), 11.9% had stable disease (SD), 33.3% achieved partial response (PR), and 38.1% achieved complete response (CR). The overall mean response rate was 0.65, ranging from 0 (complete tumor regression) to 2.25 (worst response). Figure 1 E). No major treatment-related adverse events occurred, indicating that the therapy has a good safety profile.

[0113] To investigate systemic immunological changes in the tumor microenvironment (TME), this application performed single-cell RNA sequencing (scRNA-seq) on nine tumor samples from seven patients. The responsive / reactive (CR / PR) group included three patients, one of whom had samples from both pre- and post-treatment studies. The non-responsive / progressive (SD / PD) group included four patients, one of whom had samples from both pre- and post-treatment studies. These findings were further validated using animal models. Figure 1 D). Following rigorous quality control, a total of 40,094 high-quality single cells were analyzed. Unsupervised clustering identified 12 primitive cell types, including tumor-associated macrophages (TAMs), endothelial cells, plasmacytic dendritic cells (pDCs), dendritic cells (DCs), T cells, proliferating T cells, B cells, plasma cells, mesenchymal CAFs (mCAFs), inflammatory CAFs (iCAFs), epithelial cells, and mast cells. Figure 1 F, G). Significant differences in immune cell and non-immune cell populations were observed between reactive and non-reactive samples, and the composition of the TME varied greatly among patients. Figure 1 H).

[0114] CTSE can enhance the efficacy of bladder cancer immunotherapy in human patients and mouse models.

[0115] To investigate how tumor cells respond to PD-1 blockade, this application performed copy number variation (CNV) analysis to identify malignant epithelial cells. CNV was present in most epithelial cells; therefore, all epithelial cells were classified as malignant cells. Figure 3 A). Differential expression analysis ( Figure 2 A) showed that the upregulated genes in responders were significantly enriched in the antigen presentation pathway. Figure 2 B), while downregulated genes are associated with epithelial cell proliferation and chemotaxis. Figure 2 C). These findings suggest that in responders, the immune response is enhanced and tumor growth is reduced.

[0116] To further explore the relationship between tumor characteristics and prognosis in bladder cancer patients, this application used responsive and non-responsive gene markers in the TCGA-BLCA cohort, including patients who had not received PD-1 therapy. Analysis showed that patients with higher response scores had better prognoses compared to those with lower scores. Figure 2 D), while patients with no response score have a poorer prognosis ( Figure 3 B). In the reaction group ( Figure 2 A) Of the top 10 upregulated genes, 4 were positively correlated with improved survival rates. Figure 2 E, 3C). It is worth noting that CTSE is the gene with the strongest predictive power for survival. Figure 2 F). The prognostic value of these genetic traits was further validated in the Imvigor210 immunotherapy cohort. Figure 2 Of the top 10 upregulated genes, only two were positively correlated with survival rate (G). Figure 2 H, 3D). Importantly, CTSE has a stronger prognostic value than LEAP2 (H, 3D). Figure 2 I). This was further confirmed in the GSE176307 immunotherapy cohort, reinforcing CTSE's status as a potentially unique predictive biomarker for response to immune checkpoint blockade therapy (ICB) in MIBC patients. Figure 2 J, 2K, 3E).

[0117] Other analyses have shown that CTSE is primarily expressed in tumor cells of responders. Figure 2 The difference (L, M) remains statistically significant at the sample level. Figure 2 N). Immunohistochemistry (IHC) further confirmed these findings, showing that CTSE expression was significantly higher in responders compared to non-responders. Figure 2 O, P). Within the cathepsin family, only CTSE possesses such strong predictive potential ( Figure 3 F and G) emphasize their unique role as biomarkers.

[0118] These results suggest that high expression of CTSE may enhance tumor sensitivity to immunotherapy. To test this hypothesis, this application overexpressed CTSE in the mouse bladder cancer cell line MB49. In vitro analysis showed no significant difference in proliferation or apoptosis between cells overexpressing CTSE (OE) and control cells (EV). Figure 2 Q, Figure 4 Subsequently, MB49 OE or control cells were implanted subcutaneously into C57BL / 6 mice, and the animals were treated with anti-PD-1 or isotype control antibodies. Figure 2 R, S). Among the four treatment groups, the OE group treated with anti-PD-1 therapy had the slowest tumor growth (R, S). Figure 2 This demonstrates that CTSE overexpression can enhance the antitumor effect of PD-1 blockers in bladder cancer.

[0119] To investigate the synergistic effect of CTSE and PD-1 inhibitors, this application first successfully constructed CTSECD19 CAR-T cells and verified the overexpression of CTSE in CAR-T cells using qPCR. Figure 13 A, B). To evaluate whether CTSECD19 CAR-T can deliver CTSE to bladder tumors and activate endogenous immune cells to counteract PD-1 therapy, this application conducted animal experiments. The specific procedure is as follows: C57BL / 6 mice were inoculated with MB49 CD19 tumor cells and then pretreated with cyclophosphamide (CTX), followed by infusion of two groups of CAR-T cells for adoptive cell therapy (ACT), and finally given PD-1 blockade therapy (…). Figure 13 C). The results showed that, compared with conventional CD19 CAR-T, CTSE CD19CAR-T significantly delayed tumor progression and exhibited a stronger anti-tumor effect. Notably, CD19CAR-T alone and PD-1 therapy did not show a synergistic effect, while CTSE CD19CAR-T combined with PD-1 therapy demonstrated the most significant advantage in tumor control. Figure 13 D).

[0120] According to the King's formula (Q-value method), the anti-PD-1 antibody (800 micrograms / animal) and CTSE CD19 CAR-T (2×10⁻⁶) were compared. 6 The Q value for the combination of cells / animals was 1.57 (Q > 1.15), indicating that the two have a synergistic effect.

[0121] CTSE-mediated MHC II upregulation promotes CD4 + T cell activation drives the pre-exhaustion phenotype in bladder cancer immunotherapy.

[0122] To elucidate the mechanism by which CTSE enhances the therapeutic effect of MIBC, this application performed a gene set enrichment analysis (GSEA) on tumor cells from both the responsive and non-responsive groups. The analysis results showed that "Antigen Processing and Presentation of Peptide Antigen" was the only pathway enriched in the responsive group, and it was particularly correlated with CTSE expression. Figure 5 A). This indicates that CTSE plays a role in regulating antigen processing and presentation. Further research revealed that, compared to the non-responsive group, the expression levels of MHC class II in tumor cells and antigen-presenting cells (APCs) were significantly higher in the responsive group. Figure 5 B and Figure 6 A) This is positively correlated with the expression level of CTSE, indicating that there is a broader regulatory relationship between CTSE and MHC molecules.

[0123] MHC class II molecules are CD4 + T cell activation is key because they mediate antigen presentation to CD4. + T cells trigger an immune response. CD4 + GSEA analysis of T cells showed that the MHC protein binding pathway was significantly enriched in the responding group, indicating that CD4+ in this group was significantly enriched. + T cells have enhanced ability to recognize and activate tumor cells and APCs. Figure 5 C). By integrating existing data and data from this application, CD4 + Dimensionality reduction and clustering of T cell subsets revealed eight distinct subsets. Figure 5 D, Figure 6 B). Notably, in the responsive group, anti-PD-1 treatment led to TOX. + CD4 dysfunction + T cell count decreased, while CD4 precursors were depleted. + T cell counts increased. These exhausted progenitor cells co-expressed CXCL13 and IFNG, indicating that they possess effector functions. Figure 5 E, F and Figure 6 C). The response group also showed stem cell-like CD4 expression of markers such as CCR7 and SELL. + The enrichment of T cells indicates that their proliferative capacity has been preserved. Figure 5 E, F). Similarly, after anti-PD-1 treatment, the CD4+ response group... + T cells showed a higher progenitor cell exhaustion score. Figure 6 D). These findings suggest that CTSE-mediated MHC II upregulation promotes an immune environment that encourages CD4 activation. + T cells evolve into exhaustion precursors and proliferative phenotypes.

[0124] For CD4 + Further analysis of T cells revealed a greater degree of metabolic reprogramming following anti-PD-1 treatment, including increased activity in the glycolysis and pentose phosphate pathways. These pathways are associated with the expansion of exhausted precursor T cells and enhanced anti-tumor function. Most importantly, the increase in the pentose phosphate pathway also corresponded to an increase in exhausted precursor cells in the response group. Figure 6 E). These findings suggest that, under the influence of external cellular signals, CD4 in the responder group... + T cells underwent metabolic reprogramming, resulting in an increased proportion of exhausted precursor T cells.

[0125] Increased MHC II expression promoted enhanced antigen presentation, which was related to CD4 expression in the responder group. + The increased TCR clonal expansion and clonal diversity in T cells correspond to this, as evidenced by the elevated expression of the proliferation marker MKI67 and the increase in TCR clones in both the Treg subset and the exhausted precursor T cell subset. Figure 5 GI).

[0126] To directly assess the role of CTSE in regulating MHC II expression, RNA-seq analysis was performed on MB49 cells with and without CTSE overexpression. CTSE overexpression significantly upregulated MHC II molecules, a finding confirmed by flow cytometry. Figure 5 J). In vivo, compared with the control group, the expression level of MHC II on tumor cells in the CTSE overexpression group (OE) was consistently higher (J). Figure 5 K), independent of isotype or anti-PD-1 therapy. Importantly, in the CTSE-OE group, tumor-infiltrating CD4 + T cells showed higher expression of KI67 and IFN-γ. Figure 5 LM) highlights the enhancement of activation and functional states.

[0127] To explore the CD4 observed in patient samples + Whether the T cell exhaustion precursor state can be reproduced in a mouse model, this application analyzes tumor-infiltrating CD4. + Exhaustion markers Ly108 and PD-1 in T cells. In the CTSE-OE anti-PD-1 group, PD-1... + Ly108 + and CD69 + Ly108 + CD4 + The significantly increased proportion of T cells indicates a marked exhaustion precursor state. Figure 5 N, Figure 6 F). CD45 + Further scRNA-seq and scTCR-seq of immune cells confirmed that CD4 in the CTSE-OE anti-PD-1 group... + T cells exhibited higher TCR clonal diversity, enhancing CTSE-mediated CD4 activation. + Hypotheses on T cell expansion and diversity ( Figure 6 GH and Figure 5 O).

[0128] To verify the functional relevance of CTSE-mediated MHC II upregulation, OTII Th1 cells were co-cultured with MB49 cells loaded with OVA peptide, with or without CTSE overexpression. CD4+ in the CTSE-OE group... +T cells produced significantly higher levels of IFN-γ and exhibited stronger cytotoxicity. Figure 5 PR, Figure 7 This highlights that CTSE enhances the recognition of the TCR-MHC II peptide complex and CD4. + The ability to activate T cells.

[0129] CTSE can enhance MHC class I expression, thereby activating CD8. + T cells and maintaining the exhausted precursor phenotype in tumors.

[0130] This study observed that some respondents exhibited CD4+. + and CD8 + Enhanced intercellular communication between T cells, characterized by increased expression of co-stimulatory molecules and increased MHC class I signaling, suggests that CD4+ + T cells may be CD8 + T cells provided the necessary help ( Figure 8 A, B). Sc-RNA-seq analysis of human tumor samples and flow cytometry analysis of a CTSE-overexpressing mouse model consistently showed increased MHC class I expression in tumor cells. Figure 8 (C, D). This upregulation may promote CD8. + T cells play a crucial role in activation and subsequent immune responses.

[0131] To further explore the effects of CTSE on CD8 + The influence of T cell phenotype on CD8 surface marker expression + T cells are divided into four subsets: memory CD8+ + T cells (CD8 Tm), effector CD8 + T cells (CD8 Teff), terminally depleted CD8 + T cells (CD8Tex) and depleted precursor CD8 + T cells (CD8 Tprex) Figure 7 A, B, Figure 8 E, F). Cloning frequency analysis revealed a unique pattern of T cell expansion, with the CD8 Tprex subset exhibiting the highest cloning frequency, although there was no significant difference in the cloning frequency of these four subsets between the responder and non-responder groups. Figure 7 C, D, Figure 8 H). Previous studies have identified CD8 Tprex cells as the primary responder population for immune checkpoint blockade therapy (ICB), and this study focused on this subgroup. In the responder group, CD8 Tprex cells showed significantly higher IFN-γ (IFNG) expression levels. Figure 7E), this was confirmed in the IMvigor210 bladder cancer ICB treatment cohort ( Figure 7 F). A positive correlation was also observed between IFNG and CD8A expression. Figure 7 G, Figure 8 G). Patients in the IMvigor210 and TCGA BLCA cohorts were further divided into high IFNG expression and low IFNG expression groups. The results showed that patients with higher IFNG expression had significantly better survival prognosis. Figure 7 H, I). In addition, there is a reaction group with CD8. + T cells exhibited enhanced carbon metabolism, upregulation of the citric acid cycle and glycolysis pathways, indicating a more activated immune state. Figure 8 IK).

[0132] To determine CD8 + To investigate whether T cell phenotype is driven by CTSE overexpression in tumor cells, mouse experiments were conducted using MB49 cells from both the control and CTSE-overexpressing groups. The CTSE-overexpressing group showed tumor-infiltrating CD8+ cells. + T cells showed significantly higher levels of IFNG ( Figure 7 J, K). In addition, CD8 in the CTSE group + A higher proportion of T cells, KI67 + CD8 proliferation + A higher proportion of T cells ( Figure 7 L, M). Importantly, tumors with CTSE overexpression showed a higher proportion of depleted precursor CD8. + T cells are a hallmark of effective ICB treatment response. Figure 7 N, Figure 8 L).

[0133] CD45 sorted in mouse models + ScRNA sequence analysis performed on immune cells confirmed these findings, showing depletion and depletion precursor CD8 in the CTSE group. + The proportion of T cells was higher ( Figure 7 O). Major KI67 expression was observed in the CTSE group, consistent with flow cytometry results. RNA velocity analysis further indicated that CD8+ expression was present. + The T cell population may transition from a pre-exhaustion state (Tprex) to a terminal exhaustion state (Tex). Figure 7 R). T-cell receptor (TCR) clonal expansion analysis showed that the CTSE group exhibited larger clonal sizes, primarily in the CD8 Tprex and Tex subsets, with higher IFNG expression associated with larger clonal sizes, while the control group showed lower IFNG expression. Figure 7 S, 7T, 7U).

[0134] These results indicate that CTSE expression in tumor cells can enhance CD8 expression by upregulating MHC class I expression. + T cell activation, thereby promoting CD8 + T cell recognition and activation. To further verify this hypothesis, OTI CD8 was used... + T cells were co-cultured with MB49 cells with or without CTSE overexpression. CD8+ cells in the CTSE-overexpressing group... + T cells exhibited significantly higher IFNG levels and stronger cytotoxic activity. These findings highlight the role of CTSE in promoting TCR-MHC class I peptide complex recognition and enhancing CD8. + T cell plays a key role in tumor cytotoxicity.

[0135] CTSE-induced T cell-derived IFN-γ drives pro-inflammatory macrophage polarization in the tumor microenvironment. In responders, single-cell RNA sequencing revealed increased IFN-γ expression in T cells, suggesting that T cell-mediated signaling plays a role in shaping the tumor microenvironment. The expression of IFN-γ receptors IFNGR1 and IFNGR2 is primarily localized to TAMs, indicating that macrophages are the main targets of T cell-derived IFN-γ in the tumor microenvironment. Figure 9 A). Intercellular communication analysis showed that responders had CD8 + Enhanced IFN-γ signaling between T cells and TAMs highlights the potential link between T cell activation and macrophage polarization. Figure 9 B).

[0136] Although the overall distribution of TAMs in the myeloid population did not differ significantly between groups ( Figure 10 A), but gene set enrichment analysis (GSEA) showed enhanced type II interferon signaling in responders and TAMs with CTSE-overexpressing tumors. Figure 9 (D, E). These findings are consistent with observations that the key macrophage activation cytokine IFN-γ promotes macrophage polarization towards a pro-inflammatory phenotype. In responders, TAMs showed increased expression of pro-inflammatory markers (such as CCL5), while TAMs in non-responders primarily expressed anti-inflammatory markers (…). Figure 9 F). CCL5 is a key driver of the pro-inflammatory TAM phenotype, and it is consistently upregulated in human single-cell RNA sequencing data and the IMvigor210 cohort. Figure 9 F, G). In the IMvigor210 population and TCGA BLCA dataset, the TAM marker CD14 was positively correlated with CCL5 expression, which strengthened the association between CCL5 and pro-inflammatory macrophage phenotype. Figure 9H, 10B).

[0137] Dividing patients into high CCL5 expression and low CCL5 expression groups revealed that, in both the IMvigor210 and TCGA-BLCA cohorts, elevated CCL5 levels were significantly associated with improved survival outcomes. Figure 9 I, J). Furthermore, CTSE expression was negatively correlated with the expression of anti-inflammatory markers, further demonstrating its role in driving TAMs into a pro-inflammatory state. Figure 9 K, L, Figure 10 C, D).

[0138] Pathway analysis highlighted these observations, showing that responders' TAMs exhibited increased nuclear factor-κB (NF-κB) signaling, decreased PI3K-Akt pathway activation, and increased pro-inflammatory cytokine signaling, including lymphotoxin (LT), tumor necrosis factor (TNF), and interleukin-1 (IL-1) pathways. Figure 10 Consistent with these findings, TAMs derived from tumors with overexpressing CTSE in vivo exhibited enhanced expression of pro-inflammatory markers and decreased expression of anti-inflammatory markers such as CD206. Figure 9 O, P).

[0139] To directly assess the role of T cell-derived IFN-γ in TAM programming, macrophages were cultured in the supernatant generated from the co-culture of CTSE-overexpressing MB49 tumor cells and T cells. Macrophages exposed to the CTSE co-culture supernatant exhibited a marked pro-inflammatory phenotype, with increased CD86 expression and decreased CD206 expression. Figure 9 Neutralizing IFN-γ in these supernatants reduced CD86 levels while increasing CD206 expression, confirming that T cell-derived IFN-γ is crucial for inducing a pro-inflammatory state in TAMs.

[0140] CTSE can mitigate TGF-β activation and enhance anti-tumor immunity by modulating MHC expression and T cell function. Previous studies have highlighted that TGF-β expression is elevated in bladder cancer samples with an immune desert phenotype and is associated with resistance to immunotherapy. In the analysis of this application, the non-responder group in the bladder cancer immunotherapy cohort showed higher TGF-β expression levels compared to the responder group. Figure 11 A). These observations suggest that TGF-β plays a crucial role in immune evasion, particularly in immune checkpoint blockade therapy.

[0141] To investigate whether CTSE overexpression in tumor cells regulates MHC expression and T cell activation through TGF-β signaling, this application analyzed human single-cell data and found that, compared with the non-responder group, the levels of TGF-β expressed in tumor cells and Treg cells in the responder group were significantly lower. Figure 11 B, Figure 12 A). Furthermore, cell communication analysis showed stronger TGF-β signaling interaction in the non-responder group, suggesting that TGF-β signaling may suppress the immune response in these tumors. Figure 12 B). These results suggest that tumor cells in the unresponsive group may utilize TGF-β to suppress anti-tumor immune responses, thereby leading to resistance to immunotherapy.

[0142] To assess whether CTSE affects TGF-β signaling, this application used a TGF-β reporter system to measure the level of active TGF-β in the supernatant of CTSE-overexpressing (OE) and control (EV) tumor cells. Figure 11 D). Data showed that the level of active TGF-β was significantly reduced in the CTSE overexpression group, suggesting that CTSE may reduce the inhibitory effect of active TGF-β in the tumor microenvironment.

[0143] Although T cells themselves express low levels of TGF-β receptors ( Figure 12 C), but GSEA analysis showed that CD4 levels were higher in the responding group compared to the non-responding group. + and CD8 + T cells have low levels of TGF-β receptor signaling. Figure 12 This indicates that although T cells express very few TGF-β receptors, TGF-β in the tumor microenvironment still has an inhibitory effect on T cells in the unresponsive group.

[0144] To validate these findings, CFSE-labeled CD4 was activated using anti-CD3 and anti-CD28 antibodies. + and CD8 + T cells were incubated with the supernatant of MB49 cells overexpressing CTSE (OE Sup) or control cells (EV Sup) for 72 hours. Flow cytometry analysis showed that, compared with the EV group, the supernatant of tumor cells overexpressing CTSE had a significantly weaker inhibitory effect on the production of IFN-γ and granzyme B (GzmB) and on T cell proliferation. Figure 11 (EJ). Importantly, neutralizing TGF-β in these supernatants alleviated the inhibitory effect, confirming the role of TGF-β in inhibiting T cell activation.

[0145] The role of TGF-β in inducing regulatory T cells (Tregs) has been well-established. In this study, lower CD25 expression was observed in the EV group, consistent with previous findings, while Foxp3 expression was lower. + The frequency of Tregs was significantly higher. Figure 11 K). Neutralizing TGF-β reversed the induction of Tregs in the EV group, which further confirms the role of TGF-β in promoting Treg differentiation. Figure 11 L).

[0146] Next, this application investigated whether TGF-β could also regulate the antigen presentation capacity of tumor cells. To simulate a cytokine environment that promotes MHC expression, MB49 cells (EV and OE) were cultured with IFN-γ for 24 hours. Compared with the EV group, the expression levels of MHC class I and II molecules were significantly increased in tumor cells with high CTSE expression. Figure 11 The results showed that CTSE enhanced the antigen-presenting ability of tumor cells. However, when TGF-β was added to the culture medium, MHC expression in both EV and OE cells was significantly reduced. Notably, although TGF-β still reduced MHC expression in both groups of cells, the MHC expression level in CTSE-high expression cells was significantly higher than that in the control group. Figure 11 These findings suggest that TGF-β not only impairs the immune response by regulating T cell activation and promoting Treg differentiation, but also directly inhibits MHC expression on tumor cells, thereby limiting T cell recognition and clearance of tumor cells.

[0147] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. The use of CTSE promoters and / or PD-1 blockers in the preparation of pharmaceutical compositions for the treatment of bladder cancer; Preferably, the promoter includes a carrier or compound that overexpresses CTSE; Preferably, the PD-1 blocker is selected from PD-1 antibodies; Preferably, the CTSE is applied via a delivery carrier; Preferably, the delivery vector is a CAR-T cell; Preferably, the CAR-T cells are CD19 CAR-T cells; Preferably, the pharmaceutical composition further includes other drugs for treating bladder cancer; Preferably, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

2. The application of CTSE promoters in the preparation of pharmaceutical compositions that enhance the efficacy of PD-1 blockade agents in the treatment of bladder cancer; Preferably, the promoter includes a carrier or compound that overexpresses CTSE; Preferably, the PD-1 blocker is selected from PD-1 antibodies; Preferably, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

3. Application of reagents for detecting CTSE expression levels in the preparation of products for predicting the efficacy of drug treatment in bladder cancer patients / predicting the prognosis of bladder cancer; Preferably, the reagent is selected from probes that specifically recognize the CTSE gene, primers that specifically amplify the CTSE gene, or binding agents that specifically bind to the protein encoded by the CTSE gene. Preferably, the drug is a drug used in immunotherapy; Preferably, the reagent further includes a detectable marker; Preferably, the bladder cancer is MIBC.

4. The application of CTSE promoters in the preparation of products that promote T cell activation in tumor cells; preferably, the tumor cells are bladder cancer cells; Preferably, the T cells include CD4. + T cells and / or CD8 + T cells.

5. The application of CTSE promoters in the preparation of products that promote the expression of MHC molecules in tumor cells; preferably, the tumor cells are bladder cancer cells; Preferably, the MHC molecules include MHC class I molecules and / or MHC class II molecules.

6. Application of CTSE promoters in the preparation of products that inhibit TGF-β in tumor cells; Preferably, the tumor cells are bladder cancer cells.

7. The use of CTSE promoters in the preparation of products that regulate macrophage phenotype in tumor cells; preferably, the tumor cells are bladder cancer cells; Preferably, the regulation of macrophage phenotype in tumor cells is to promote macrophage polarization toward a pro-inflammatory phenotype.

8. A pharmaceutical composition for treating bladder cancer, characterized in that, The pharmaceutical composition includes a CTSE promoter and / or a PD-1 blocker; Preferably, the CTSE further includes a delivery carrier; Preferably, the delivery vector is a CAR-T cell; Preferably, the CAR-T cells are CD19 CAR-T cells; Preferably, the PD-1 blocker is selected from PD-1 antibodies; Preferably, the dosage form of the pharmaceutical composition includes a gastrointestinal dosage form and a non-gastrointestinal dosage form; preferably, the gastrointestinal dosage form includes tablets, granules, capsules, solutions, dry suspensions, powders, sustained-release preparations, effervescent tablets, emulsions, suspensions, syrups, drops, and chewable tablets; Preferably, the non-gastrointestinal dosage forms include injectable dosage forms, respiratory dosage forms, cavity dosage forms, mucosal dosage forms, and skin dosage forms.

9. Application of CTSE as a target in screening candidate drugs for the treatment of bladder cancer; Preferably, the method for screening candidate drugs for treating bladder cancer includes: Treat culture systems that express or contain the CTSE gene or its encoded protein with the substance to be screened; The system is used to detect the expression or activity of the CTSE gene or its encoded protein; wherein, when the substance to be screened promotes the expression level or activity of the CTSE gene or its encoded protein, the substance to be screened is a candidate drug for the treatment of bladder cancer.

10. Any one of the following methods: 1) A method for promoting T cell activation in tumor cells in vitro, characterized in that, The method includes administering a CTSE promoter; 2) A method for promoting the expression of MHC molecules in tumor cells in vitro, characterized in that the method includes administering a promoter of CTSE; 3) A method for inhibiting TGF-β in tumor cells in vitro, characterized in that the method includes administering a promoter of CTSE; 4) A method for regulating macrophage phenotype in tumor cells in vitro, characterized in that the method includes administering a promoter of CTSE; 5) A method for screening candidate drugs for treating bladder cancer, characterized in that the method comprises: treating a culture system expressing or containing a CTSE gene or its encoded protein with a substance to be screened; and detecting the expression or activity of the CTSE gene or its encoded protein in the system; wherein, when the substance to be screened promotes the expression level or activity of the CTSE gene or its encoded protein, the substance to be screened is a candidate drug for treating bladder cancer. Preferably, the tumor cells are bladder cancer cells; Preferably, the T cells include CD4. + T cells and / or CD8 + T cells; Preferably, the MHC molecules include MHC class I molecules and / or MHC class II molecules; Preferably, the regulation of macrophage phenotype in tumor cells is to promote macrophage polarization toward a pro-inflammatory phenotype; Preferably, the promoter includes a carrier or compound that overexpresses CTSE.

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