Use of ptprt as biomarker and target for predicting efficacy of immune checkpoint therapy in lung cancer

Abstract

The present application relates to the technical field of medicine, and provides a use of PTPRT as a biomarker and a target for predicting the efficacy of immune checkpoint therapy in lung cancer. Further provided is a use of the expression quantity of PTPRT in the prediction or evaluation of the efficacy of immune checkpoint therapy in cancer. Experiments show that the low expression of PTPRT, as a marker for predicting the efficacy of immune checkpoint therapy in lung cancer, can accurately predict progression-free survival (PFS) of lung cancer patients. Inhibition of PTPRT can increase the proportion of tumor-infiltrating CD8+T cells and synergize with immune checkpoint therapy to enhance the anti-tumor effect of an immune checkpoint inhibitor.

Description

Application of PTPRT as a biomarker and target for predicting the efficacy of immune checkpoint therapy in lung cancer Technical Field

[0001] The present application relates to the field of medical technology, and specifically to the application of PTPRT as a biomarker and target for predicting the efficacy of immune checkpoint therapy for lung cancer. Background Art

[0002] Cancer is the leading cause of death worldwide, and the advent of immune checkpoint therapy has transformed cancer treatment. However, only approximately 20% of patients experience sustained benefit from immune checkpoint inhibitors, and 90% of treated patients experience adverse events. Therefore, identifying patients most likely to benefit from immune checkpoint therapy and enabling precision medicine is crucial.

[0003] Currently, the only US Food and Drug Administration-approved and most widely used biomarker for immune checkpoint inhibitor efficacy is tumor PD-L1 expression. However, PD-L1 has limited sensitivity and specificity. A subset of patients with high PD-L1 expression fail to benefit from immune checkpoint inhibitor therapy, while approximately 20% of patients with negative PD-L1 expression respond to treatment. Tumor mutation burden (TMB), a biomarker for immune checkpoint inhibitor efficacy, lacks standardized detection methods and thresholds. Therefore, the search for more precise biomarkers with clinical translational potential to guide immune checkpoint therapy in lung cancer patients is crucial, identifying those who will benefit from immune checkpoint therapy and expanding the response population through multi-target combination therapy, thereby providing survival benefits to more patients. Summary of the Invention

[0004] In view of the shortcomings of the prior art described above, the purpose of this application is to provide the application of PTPRT as a biomarker and target for predicting the efficacy of immune checkpoint therapy for lung cancer, so as to solve the problems in the prior art.

[0005] The inventors of this application have discovered that the accuracy of patient screening based on tumor PD-L1 expression needs to be improved. This innovative technology addresses the shortcomings of existing technologies by discovering that PTPRT expression can predict the efficacy of immune checkpoints in non-small cell lung cancer and is independent of tumor PD-L1 expression. It can be used as a separate indicator to improve patient screening accuracy.

[0006] To achieve the above-mentioned objectives and other related objectives, the first aspect of the present application provides the application of PTPRT expression level in predicting or evaluating the efficacy of immune checkpoint therapy for cancer.

[0007] The second aspect of the present application provides the use of a substance for detecting the expression level of PTPRT in the preparation of a product for evaluating or predicting the efficacy of immune checkpoint therapy for cancer.

[0008] In a third aspect, the present application provides a kit comprising a substance for detecting the expression level of PTPRT, and the kit has at least one of the following uses:

[0009] 1) Predicting the efficacy of immune checkpoint therapy in lung cancer;

[0010] 2) Evaluate the efficacy of immune checkpoint therapy in lung cancer.

[0011] In a fourth aspect, the present application provides a use of a PTPRT inhibitor in preparing a product, wherein the product has at least one of the following effects:

[0012] 1) Increase the infiltration of CD8+ T cells in lung or colorectal cancer tumors;

[0013] 2) Improve the survival of lung cancer patients;

[0014] 3) Improve the tumor immune microenvironment of lung cancer or colorectal cancer;

[0015] 4) Improve the efficacy of immune checkpoint-related drugs in the treatment of lung cancer or colorectal cancer.

[0016] The fifth aspect of the present application provides a pharmaceutical composition comprising an effective amount of an immune checkpoint-related drug and the PTPRT inhibitor for the aforementioned use.

[0017] The sixth aspect of the present application provides the use of the aforementioned pharmaceutical composition in the preparation of a product for treating lung cancer or colorectal cancer.

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

[0019] 1. This application discloses for the first time the relationship between low PTPRT expression and the efficacy of immune checkpoint therapy for lung cancer. Specifically, low PTPRT expression significantly improves the progression-free survival (PFS) of lung cancer patients receiving immune checkpoint therapy; low PTPRT expression significantly improves the objective response rate (ORR) of lung cancer patients receiving immune checkpoint therapy; and low PTPRT expression significantly increases the infiltration of CD8+ T cells in tumor samples from lung cancer patients. Inhibiting PTPRT improves the tumor immune microenvironment of lung cancer patients. This shows that low PTPRT expression is a new marker for predicting the efficacy of immune checkpoint therapy in lung cancer patients.

[0020] 2. This application found that the low expression of PTPRT is independent of the expression of PD-L1. The low expression of PTPRT does not affect the expression of PD-L1 and can serve as a supplement to the expression of PD-L1.

[0021] 3. This application constructed a mouse model (preclinical model) and found that knocking out PTPRT combined with immune checkpoint therapy significantly inhibited tumor growth and tumor size; knocking out PTPRT significantly improved the tumor immune microenvironment of mice and increased the infiltration of CD8+T cells; knocking out PTPRT combined with immune checkpoint therapy significantly improved the survival of mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 shows the correlation between low PTPRT expression and the efficacy of immune checkpoint therapy in lung cancer. Figure 1A shows immunohistochemical staining of PTPRT (-, +, 2+, 3+) in tumor biopsy specimens. Scale bar, 100 μm. Figure 1B shows the correlation between PTPRT expression levels and progression-free survival in lung cancer patients receiving immunotherapy. Figure 1C shows the receiver operating characteristic (ROC) curve for low PTPRT expression in predicting immune checkpoint efficacy. Figure 1D shows the correlation between PTPRT expression levels and objective response rate in lung cancer patients receiving immunotherapy. Figure 1E shows the correlation between PTPRT expression levels and PD-L1 expression in lung cancer patients receiving immunotherapy. Figure 1F shows the correlation between low and high PTPRT expression groups and CD8+ T cell infiltration.

[0023] Figure 2 shows the external validation data for 37 patients. Figure 2A shows the analysis of PTPRT expression levels and progression-free survival in lung cancer patients receiving immunotherapy; Figure 2B shows the receiver operating characteristic (ROC) curve for predicting immune checkpoint efficacy using low PTPRT expression.

[0024] Figure 3 shows the synergistic enhancement of immunotherapy efficacy by combined inhibition of PTPRT expression in a subcutaneous lung cancer animal model. Figure 3A shows changes in related proteins after LLC knockout of PTPRT; Figure 3B shows changes in PD-L1 fluorescence intensity in cells after LLC knockout of PTPRT by flow cytometry; Figure 3C shows changes in tumor volume curves; Figure 3D shows changes in tumor size at endpoint; and Figure 3E shows the proportion of tumor-infiltrating CD8+ T cells.

[0025] Figure 4 shows the synergistic enhancement of immunotherapy efficacy by combined inhibition of PTPRT expression in an orthotopic lung cancer model. Figure 4A shows in vivo fluorescence imaging of the tumor; Figure 4B shows the change in tumor fluorescence at the endpoint; Figure 4C shows the survival rate of orthotopic tumor-bearing mice; and Figure 4D shows the proportion of tumor-infiltrating CD8+ T cells.

[0026] Figure 5 shows the synergistic enhancement of immunotherapy efficacy by combined inhibition of PTPRT expression in a subcutaneous colorectal cancer animal model. Figure 5A shows the changes in related proteins after PTPRT knockout in MC38; Figure 5B shows the changes in tumor volume curve; Figure 5C shows the change in tumor size at the endpoint; and Figure 5D shows the proportion of tumor-infiltrating CD8+ T cells. DETAILED DESCRIPTION

[0027] In order to make the invention purpose, technical scheme and beneficial effect of the present application clearer, the present application is further described below in conjunction with embodiment.It should be understood that the embodiment is only used to explain the present application and is not used to limit the scope of the application.The test method used in the following examples is conventional method unless otherwise specified, according to the technology or conditions described in the literature in this area or according to the product instructions.People familiar with this technology can easily understand other advantages and effects of the present application from the content disclosed in this description.The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0028] This study, conducted in a clinical cohort of immune checkpoint therapy patients at Shanghai Chest Hospital, found that expression of receptor-type protein tyrosine phosphatase T (PTPRT) was highly correlated with progression-free survival (PFS) in lung cancer patients receiving immune checkpoint therapy. Low PTPRT expression can be used as a marker to predict the efficacy of immune checkpoint therapy in lung cancer. PTPRT is a target for predicting the efficacy of immune checkpoint therapy in lung cancer. Inhibiting PTPRT can effectively improve the tumor's immunosuppressive microenvironment by increasing CD8+ T cell infiltration in the tumor.

[0029] On the one hand, the present application provides the application of PTPRT expression in predicting or evaluating the efficacy of immune checkpoint therapy for cancer; the cancer is selected from lung cancer, head and neck squamous cell carcinoma, esophageal cancer, pleural mesothelioma, breast cancer, gastric cancer, hepatocellular carcinoma, colorectal cancer, renal cancer, urothelial carcinoma, cervical cancer, endometrial cancer, ovarian cancer, malignant melanoma, malignant lymphoma, or skin cancer. In a specific embodiment of the present application, the cancer is lung cancer or colorectal cancer.

[0030] In the application provided in the present application, when the expression level of PTPRT in the sample is less than 1% in the staining intensity of immunohistochemical staining, it is predicted that the efficacy of immune checkpoint therapy for cancer is good; when the expression level of PTPRT in the sample is greater than 1% in the staining intensity of immunohistochemical staining, it is predicted that the efficacy of immune checkpoint therapy for cancer is poor.

[0031] PTPRT stands for receptor protein tyrosine phosphatase T. Prior art indicates that PTPRT gene mutations are associated with the prognosis of immunotherapy for lung cancer. This application, however, discloses for the first time the relationship between low PTPRT protein expression and the efficacy evaluation of immune checkpoint inhibitors in treating lung cancer. Specifically, low PTPRT expression significantly increases the infiltration of CD8+ T cells in lung cancer tumors; low PTPRT expression significantly increases the progression-free survival (PFS) of lung cancer patients; low PTPRT expression significantly increases the objective response rate (ORR) of lung cancer patients; and low PTPRT expression significantly increases the infiltration of CD8+ T cells in tumor samples from lung cancer patients. This indicates that low PTPRT expression is a new marker for predicting the efficacy of immune checkpoint inhibitors in lung cancer patients.

[0032] In the application provided herein, lung cancer includes non-small cell lung cancer. The expression level of PTPRT is the expression level of wild-type PTPRT.

[0033] In the application provided herein, the immune checkpoint treatment of lung cancer includes the use of immune checkpoint-related drugs; further, immune checkpoint-related drugs include immune checkpoint inhibitors and / or immune checkpoint activators; further, immune checkpoint inhibitors include a combination of one or more of PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, TIM-3 inhibitors, LAG3 inhibitors, and TIGIT inhibitors. Immune checkpoint activators include one or a combination of two or more of CD27 activators, CD40 activators, OX40 activators, GITR activators, CD137 activators, CD28 activators, and ICOS activators. In a specific embodiment of the present application, immune checkpoint-related drugs are selected from PD-1 inhibitors. Immune checkpoints are expressed on immune cells and can regulate the degree of immune activation. They play an important role in preventing the occurrence of autoimmune effects. Immunotherapy through immune checkpoints is a treatment method that kills tumor cells by regulating T cell activity through a series of pathways such as co-inhibition or co-stimulation signals.

[0034] In the applications provided in this application, the lung cancer tumor microenvironment includes but is not limited to the lung cancer tumor immune microenvironment.

[0035] In the application provided in this application, the low expression of PTPRT is independent of the expression of PD-L1. The low expression of PTPRT does not affect the expression of PD-L1 and can serve as a supplement to the expression of PD-L1.

[0036] In the application provided in this application, low expression of PTPRT refers to a staining intensity of <1% in immunohistochemical staining, which is considered to be low expression.

[0037] The second aspect of the present application provides the use of a substance for detecting the expression level of PTPRT in the preparation of a product for evaluating or predicting the efficacy of immune checkpoint therapy for cancer.

[0038] In the use provided herein, the cancer is selected from lung cancer, head and neck squamous cell carcinoma, esophageal cancer, pleural mesothelioma, breast cancer, gastric cancer, hepatocellular carcinoma, colorectal cancer, renal cancer, urothelial carcinoma, cervical cancer, endometrial cancer, ovarian cancer, malignant melanoma, malignant lymphoma, or skin cancer. Preferably, the cancer is lung cancer or colorectal cancer.

[0039] In the applications provided herein, the substances for detecting PTPRT expression include reagents for detecting PTPRT protein expression, detecting PTPRT protein content, or detecting PTPRT RNA expression. Specifically, the reagents include antibodies, polypeptides, proteins, or nucleic acid molecules that bind to PTPRT proteins.

[0040] The reagent for detecting PTPRT can specifically bind to a specific site on PTPRT, but not to other genes outside of PTPRT, and the reagent can optionally carry a detectable signal. Methods for detecting PTPRT expression in an analyte using antibodies that specifically bind to PTPRT are also well known in the art.

[0041] In a specific embodiment of the present application, the substance for detecting the expression level of PTPRT is an antibody, specifically a PTPRT monoclonal antibody.

[0042] In a third aspect, the present application provides a kit comprising a substance for detecting the expression level of PTPRT, wherein the kit has at least one of the following uses:

[0043] 1) Predicting the efficacy of immune checkpoint therapy in lung cancer;

[0044] 2) Evaluate the efficacy of immune checkpoint therapy in lung cancer.

[0045] The kit includes primers that specifically amplify the PTPRT gene, a probe that specifically recognizes the PTPRT gene, and an antibody or ligand that specifically binds to the RNA or protein encoded by the PTPRT gene. Furthermore, the kit may include various reagents required for DNA extraction, RNA, PCR, hybridization, and color development, including but not limited to extraction solutions, amplification solutions, hybridization solutions, enzymes, control solutions, color development solutions, and washing solutions.

[0046] In a specific embodiment of the present application, the kit includes a PTPRT monoclonal antibody. By performing an immunohistochemical staining experiment on the sample, the staining intensity of PTPRT is determined to predict or evaluate the efficacy of immune checkpoint therapy for lung cancer. Specifically, when the staining intensity of PTPRT in the immunohistochemical staining is less than 1% in the sample, it is determined to be low expression, and the efficacy of immune checkpoint therapy for lung cancer is predicted to be good; when the staining intensity of PTPRT in the immunohistochemical staining is greater than 1%, it is determined to be high expression, and the efficacy of immune checkpoint therapy for lung cancer is predicted to be poor.

[0047] In a fourth aspect, the present application provides a use of a PTPRT inhibitor in preparing a product, wherein the product has at least one of the following effects:

[0048] 1) Increase the infiltration of CD8+ T cells in lung or colorectal cancer tumors;

[0049] 2) Improved the survival of lung cancer patients;

[0050] 3) Improved the tumor immune microenvironment of lung cancer or colorectal cancer;

[0051] 4) Improve the efficacy of immune checkpoint-related drugs in the treatment of lung cancer or colorectal cancer.

[0052] PTPRT inhibitors include inhibitors, antagonists, blockers, and inhibitors, and these terms are used interchangeably.

[0053] In the uses provided in the present application, PTPRT inhibitors include substances that reduce the expression level of PTPRT protein or the content of PTPRT protein, or substances that inhibit PTPRT gene expression. Furthermore, substances that inhibit PTPRT gene expression include substances that knock out or silence PTPRT; further, substances that knock out or silence PTPRT include: CRISPR gene editing systems for PTPRT, interfering molecules that specifically interfere with the expression of PTPRT coding genes, and homologous recombination substances for PTPRT loss-of-function mutations.

[0054] In some embodiments, the CRISPR gene editing system for PTPRT can use the CRISPR / Cas (such as Cas9) system to perform targeted gene editing, thereby knocking out the PTPRT gene in the area of ​​the targeted disease. Common methods for knocking out PTPRT include: co-transferring sgRNA or a nucleic acid that can form sgRNA, Cas9 mRNA or a nucleic acid that can form Cas9 mRNA into the targeted area or targeted cell. After determining the target site, known methods can be used to introduce sgRNA and Cas9 into the cell. The nucleic acid that can form sgRNA is a nucleic acid construct or an expression vector, or the nucleic acid that can form the Cas9 mRNA is a nucleic acid construct or an expression vector, and these expression vectors are introduced into the cell, thereby forming active sgRNA and Cas9 mRNA in the cell. As a particularly preferred embodiment of the present invention, the CRISPR gene editing system for PTPRT includes sgRNA, and the encoding DNA sequence of sgRNA is as shown in SEQ ID NO:1.

[0055] In the applications provided herein, immune checkpoint-related drugs include immune checkpoint inhibitors and / or immune checkpoint activators. Further, immune checkpoint inhibitors include a combination of one or more of PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, TIM-3 inhibitors, LAG3 inhibitors, and TIGIT inhibitors; immune checkpoint activators include one or a combination of two or more of CD27 activators, CD40 activators, OX40 activators, GITR activators, CD137 activators, CD28 activators, and ICOS activators. In a specific embodiment of the present application, immune checkpoint-related drugs are selected from PD-1 inhibitors.

[0056] In the application provided in this application, it was found that knocking out PTPRT combined with immune checkpoint inhibitors significantly inhibited tumor growth and tumor size; knocking out PTPRT significantly improved the tumor immune microenvironment of mice and increased the infiltration of CD8+T cells; knocking out PTPRT combined with immune checkpoint inhibitors significantly improved the survival of mice.

[0057] The fifth aspect of the present application provides a pharmaceutical composition, comprising an effective amount of an immune checkpoint-related drug and a PTPRT inhibitor in the aforementioned use. Immune checkpoint-related drugs include immune checkpoint inhibitors and / or immune checkpoint activators. Further, immune checkpoint inhibitors include a combination of one or more of PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, TIM-3 inhibitors, LAG3 inhibitors, and TIGIT inhibitors; immune checkpoint activators include CD27 activators, CD40 activators, OX40 activators, GITR activators, CD137 activators, CD28 activators, and ICOS activators. One or a combination of two or more of these. In a specific embodiment of the present application, the immune checkpoint-related drug is selected from PD-1 inhibitors.

[0058] The pharmaceutical composition provided in this application also includes a pharmaceutically acceptable carrier or excipient.

[0059] "Pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when appropriately administered to an animal or a human.

[0060] "Pharmaceutically acceptable carriers or excipients" should be compatible with immune checkpoint inhibitors and PTPRT inhibitors, meaning they can be blended with them without significantly reducing the drug's efficacy under normal circumstances. Specific examples of substances that can serve as pharmaceutically acceptable carriers or excipients include sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth powder; 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 butter; polyols, such as propylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; colorants; flavorings; tableting agents, stabilizers, antioxidants; preservatives; pyrogen-free water; isotonic saline solution; and phosphate buffer. These materials are used as needed to aid in the stability of the formulation or to help increase the activity or its bioavailability or to produce an acceptable taste or flavor in the case of oral administration.

[0061] The pharmaceutical composition provided herein can be adapted to any form of administration, which can be oral or parenteral administration, for example, it can be pulmonary, nasal, rectal and / or intravenous injection, more specifically, it can be intradermal, subcutaneous, intramuscular, intraarticular, intraperitoneal, pulmonary, oral, sublingual, nasal, transdermal, vaginal, oral or parenteral administration.

[0062] Those skilled in the art can select a suitable formulation according to the mode of administration. For example, formulations suitable for oral administration may include but are not limited to pills, tablets, chewable tablets, capsules, granules, drops or syrups, etc. For another example, formulations suitable for parenteral administration may include but are not limited to solutions, suspensions, reconstituted dry preparations or sprays, etc. For another example, formulations suitable for rectal administration may generally be suppositories.

[0063] The sixth aspect of the present application provides the use of the aforementioned pharmaceutical composition in the preparation of a product for treating lung cancer or colorectal cancer.

[0064] In a seventh aspect, the present application provides a method for treating lung cancer or colorectal cancer, characterized in that the method comprises administering the aforementioned PTPRT inhibitor or the aforementioned pharmaceutical composition to a subject.

[0065] In the method for treating lung cancer provided herein, the subject is a mammal. Mammals include, for example, rodents, artiodactyls, perissodactyls, lagomorphs, primates, etc. Primates include, for example, monkeys, apes, or Homo sapiens.

[0066] The present application is further described below by way of examples, but the scope of the present application is not limited thereby.

[0067] Example 1

[0068] Application of PTPRT in predicting the efficacy of immune checkpoints in lung cancer

[0069] Case Selection in This Example: This example is a retrospective clinical study. It was conducted in accordance with the principles of the Declaration of Helsinki, and informed consent was obtained from all subjects. This example primarily included patients with stage IIIC / IV lung cancer who received immune checkpoint inhibitor therapy at Shanghai Chest Hospital from January 2020 to June 2022. Follow-up ended on December 31, 2022. The treatment received was either anti-PD-1 therapy or anti-PD-1 therapy plus chemotherapy.

[0070] The efficacy of patients during treatment was evaluated according to the Response Valuation Criteria in Solid Tumors (RECIST version 1.1). Efficacy evaluation indicators included complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD). Progression-free survival (PFS) was defined as the time from the start of immunosuppressant treatment to disease progression or death. The objective response rate (ORR) is the proportion of patients who achieved a 30% reduction in tumor volume and maintained this reduction for the minimum required time. It is the sum of the rates of complete response (CR, i.e., complete disappearance of the tumor) and partial response (PR, i.e., a tumor reduction of 30% or more), excluding stable disease (SD).

[0071] The samples were stained with immunohistochemistry to detect the expression percentage of PTPRT. The IHC staining intensity was divided into: -, <1%; +, 1%-10%; ++, 10%–50%; +++, >50%.

[0072] Specific operations:

[0073] a. Baking: Place the paraffin sections on a 60°C baking machine for 30 minutes.

[0074] b. Dewax and rehydrate the sections: Prepare three cylinders of xylene (pass through three times), quickly place the baked paraffin sections into the glass cylinders, and soak for 5 minutes at each concentration. Then, rinse the sections in anhydrous ethanol twice, 2 minutes each time, and then rinse in 95%, 90%, 80%, and 70% ethanol in sequence, staying at each concentration for 5 minutes. Finally, soak the sections in PBS buffer three times, 3 minutes each time.

[0075] c. Slice fixation: Soak the dewaxed and rehydrated slices in 4% PFA solution for 10 minutes, and then soak the slices in PBS buffer three times, each time for 3 minutes.

[0076] d. Membrane disruption: Soak the fixed sections in 0.3% Triton-100 for 10 minutes, then soak the sections in PBS buffer three times, each time for 3 minutes.

[0077] e. Removal of oxidoreductases: Soak the perforated sections in 3% hydrogen peroxide solution for 10 minutes to remove endogenous oxidoreductases. Then soak the sections in PBS buffer three times for 3 minutes each time.

[0078] f. Blocking: After removing oxidoreductase from the sections, wipe away any liquid surrounding the tissue on the slide. Use an immunohistochemistry pen to draw a circle around the tissue to prevent the liquid applied to the sections in subsequent steps from running out. Place the sections in a light-proof humidified chamber. Apply a blocking solution containing 10% goat serum to the sections. After incubating at room temperature for 1 hour, aspirate the blocking solution and soak the sections in PBS buffer three times for 3 minutes each.

[0079] g. Primary Antibody Incubation: Dilute the primary antibody (R&D Product No. AF3697) with antibody diluent according to the instructions provided in the package insert. Apply the primary antibody to the blocked tissue surface and incubate overnight at 4°C.

[0080] h. Secondary Antibody Incubation: Aspirate the primary antibody from the surface of the sections and soak the sections in PBS buffer three times for 3 minutes each. Apply the secondary antibody (Jackson ImmunoResearch, Cat. No. 705-005-003) corresponding to the species of the primary antibody to the sections. Incubate at room temperature for 1 hour, then soak the sections in PBS buffer three times for 3 minutes each.

[0081] i. DAB staining: Add DAB staining solution to the tissue slice. Preliminary experiments can be performed under a microscope to confirm the appropriate reaction time before proceeding with the formal experiment. The staining time is generally 1-5 minutes. Finally, gently rinse the slice with slow-flowing tap water to terminate the staining.

[0082] j. Staining Cell Nuclei: Place the DAB-stained sections in a hematoxylin solution to counterstain the nuclei of all cells (blue). Staining is generally done at room temperature for 2-5 minutes. Finally, gently rinse the sections with running tap water to terminate the color development.

[0083] k. Mounting: After completing the above steps, the slides need to be mounted for long-term preservation. Dehydrate the slides in 70%, 80%, 90%, 95%, and 100% ethanol in a gradient of 2 minutes. Repeat the dehydration in 100% ethanol once, then soak in xylene twice for 5 minutes each. Add a small amount of neutral resin to the sliced ​​tissue and cover with a coverslip to prevent air bubbles. Finally, allow the slides to dry thoroughly in a fume hood and place in a slide box for subsequent observation and photography.

[0084] 1. Survival curve

[0085] Results, as shown in Figure 1A, were categorized into patients with low PTPRT expression (-) and those with high PTPRT expression (+, ++, +++). Results, as shown in Figures 1B and 1D, were plotted against progression-free survival (PFS) and objective response rate (ORR). The analysis showed that, at the same follow-up time, patients with low PTPRT expression had a significantly increased PFS (16.3 vs. 5.9 m, P = 0.037) and an increased ORR (66.7% vs. 27.3%, P = 0.036). The receiver operating characteristic (ROC) curve in Figure 1C demonstrated that low PTPRT expression had high specificity and sensitivity for predicting immune checkpoint response (AUC = 0.742, P = 0.0367).

[0086] External validation was performed using data from 37 other cases. Case selection in this example: This example is a retrospective clinical study. This study was conducted in accordance with the principles of the Declaration of Helsinki, and informed consent was obtained from all subjects. This example primarily included patients with stage IIIC / IV lung cancer who received PD-1 inhibitor treatment at Shanghai Chest Hospital from March 2019 to June 2022, with follow-up ending on March 31, 2023. The treatment received was either anti-PD-1 therapy or anti-PD-1 therapy plus chemotherapy.

[0087] Treatment efficacy was assessed according to the Response Valuation Criteria in Solid Tumors (RECIST version 1.1). Progression-free survival (PFS) was defined as the time from the start of immunosuppressive therapy to disease progression or death. Immunohistochemical staining was performed on samples to determine the percentage of PTPRT expression. IHC staining intensity was categorized as: -, <1%; +, 1% to 100%.

[0088] The results are shown in Figure 2. At the same follow-up time, the progression-free survival rate of patients with low PTPRT expression was significantly increased compared with that of patients with high PTPRT expression (15.9 m vs. 4.2 m, P = 0.008). The appropriate ROC curve showed that low PTPRT expression had higher specificity and sensitivity.

[0089] The ROC curve of Figure 2B shows that low PTPRT expression has high specificity and sensitivity in predicting the efficacy of immune checkpoints (AUC = 0.776, P = 0.0091).

[0090] 2. Relationship between PTPRT expression and tumor immune microenvironment

[0091] Tumor PD-L1 ratio and CD8+ T cell infiltration were assessed based on immunohistochemical staining. PD-L1 was assessed using the universal Tumor Proportion Score (TPS); samples with CD8+ T cell infiltration were defined as positive (+) and those without CD8+ T cell infiltration were defined as negative (-). As shown in Figure 1 (D-E), there was no statistically significant difference between low PTPRT expression and PD-L1 expression (P = 0.516). Furthermore, patients with low PTPRT expression had an increased proportion of CD8+ T cell infiltration (P = 0.0002).

[0092] In summary, the above results indicate that the PTPR expression level in the tumor tissue of the patient can be used to predict or assist in predicting the efficacy of immune checkpoint inhibitor treatment in the patient. The judgment criteria are as follows:

[0093] Patients in the low PTPRT expression group had better or potentially better immune checkpoint inhibitor treatment efficacy than patients in the high PTPRT expression group. Therefore, PTPRT can be used as a biomarker to evaluate or assist in evaluating the efficacy of immune checkpoint inhibitor treatment.

[0094] Example 2

[0095] PTPRT inhibitors combined with immune checkpoint inhibitors can synergistically enhance the anti-lung cancer effect of immunotherapy

[0096] The PTPRT inhibitor can be a substance that inhibits the expression of the PTPRT gene, silences or knocks out the PTPRT gene, or can be a substance that inhibits or reduces the content and / or activity of the PTPRT protein.

[0097] 1. Selection of cell lines and animals

[0098] The mouse LLC cell line used in this study was obtained from the ATCC Cell Resource Center in the United States.

[0099] This study used 6-week-old female C57BL / 6 mice. All mice were housed in a SPF-grade environment. All procedures performed in this study strictly adhered to the regulations established by the Animal Ethics Committee.

[0100] 2. Construction of LLC cells with PTPRT knockout

[0101] Based on the PTPRT gene sequence and CRISPR-Cas9 principles, the PTPRT gene was knocked out in LLC cells. The sgRNA encoding the PTPRT knockout gene was designed to have the following DNA sequence: 5'-CAGCAACTGCGGGTATAGCG-3' (SEQ ID NO: 1), and the control sgRNA encoding the same DNA sequence: 5'-GAACAGTCGCGTTTGCGACT-3' (SEQ ID NO: 2). LLC cells were transfected with lentivirus encoding the PTPRT knockout gene and a blank control to generate PTPRT knockout and control LLC cells. Western blotting was used to examine changes in PTPRT and PD-L1 expression in the cells, and flow cytometry was used to detect changes in surface PD-L1 expression.

[0102] 3. Mouse lung cancer subcutaneous transplant tumor model

[0103] One million LLC cells (PTPRT knockout and control) were subcutaneously injected into the right side of the back of wild-type C57BL / 6 female mice (6 weeks old). Tumor volume was calculated using the following formula: (width x width x length) / 2. Anti-PD-1 therapy (BE0273, BioXcell) and an IgG isotype control (BE0089, BioXcell) were administered intraperitoneally at a dose of 150 μg each time, starting on day 9 and continuing every three days. Tumors were harvested on day 21 for flow cytometry analysis. Tumors were collected by autopsy after sacrifice under carbon dioxide anesthesia to minimize animal suffering.

[0104] The results are as follows: The protein expression of PTPRT in LLC cells with PTPRT knockout was significantly decreased, while the protein level of PD-L1 did not change significantly (Figure 3A). Flow cytometry showed that the fluorescence intensity of PD-L1 on the cell membrane surface of LLC cells with PTPRT knockout did not change significantly (Figure 3B). In a mouse subcutaneous lung cancer xenograft model, after PTPRT knockout combined with anti-PD-1 monoclonal antibody treatment, the tumor growth rate and size were significantly reduced compared with the control treatment group (anti-PD-1 monoclonal antibody group) and the knockdown group (PTPRT knockout group) (Figure 3C and D), and the proportion of CD8+ T cell infiltration increased (Figure 3E).

[0105] These results indicate that knocking out PTPRT or inhibiting PTPRT expression does not affect PD-L1 expression in tumor cells. Knocking out PTPRT or inhibiting PTPRT expression in combination with anti-PD-1 monoclonal antibodies can significantly inhibit tumor growth, improve the efficacy of immune checkpoint inhibitors, increase CD8+ T cell infiltration in tumors, and enhance the anti-tumor effects of immunotherapy.

[0106] Example 3

[0107] PTPRT inhibitors combined with immune checkpoint inhibitors can synergistically enhance the anti-lung cancer effect of immunotherapy

[0108] 1. Selection of cell lines and animals

[0109] The mouse LLC-luc cell line used in this study was obtained from the ATCC Cell Resource Center in the United States.

[0110] This study used 6-week-old female C57BL / 6 mice. All mice were housed in a SPF-grade environment. All procedures performed in this study strictly adhered to the regulations established by the Animal Ethics Committee.

[0111] 2. Construction of LLC-luc cells with PTPRT knockout

[0112] Based on the PTPRT gene sequence and CRISPR-Cas9 principles, the PTPRT gene was knocked out in LLC cells. The encoding DNA sequence of the sgRNA for the PTPRT knockout gene was designed to be: 5'-CAGCAACTGCGGGTATAGCG-3' (SEQ ID NO: 1), and the encoding DNA sequence of the control sgRNA was: 5'-GAACAGTCGCGTTTGCGACT-3' (SEQ ID NO: 2). LLC cells were transfected with lentivirus containing the PTPRT knockout gene and a blank control to obtain LLC cells with the PTPRT knockout gene and a control.

[0113] 3. Mouse Lung Cancer Orthotopic Xenograft Tumor Model

[0114] Mice were anesthetized with isopentane using an animal gas anesthesia machine. A 3 mm incision was made on the dorsal side of the left lung, 0.5 cm below the scapula. After separating the subcutaneous tissue and muscle, lung movement could be observed. A total volume of 50 μl of a cell suspension (Matrigel:PBS = 1:4) of stably transfected cells, i.e., the PTPRT gene knockout cells obtained in step 2, and control LLC cells (800,000) obtained in step 2, was injected directly into the left lung using an insulin syringe (BD), and the incision was sutured. In vivo fluorescence imaging was performed on day 5 to observe the modeling. Anti-PD-1 treatment (BE0273, BioXcell) and IgG isotype control (BE0089, BioXcell) were intraperitoneally administered at 150 μg each time, starting on day 9 and administered every 3 days. In vivo fluorescence imaging was performed at the end of the experiment on day 21. For survival experiments, anti-PD-1 therapy (BE0273, BioXcell) and IgG isotype control (BE0089, BioXcell) were intraperitoneally administered at 150 μg each time starting on day 7 of modeling until the mice died. Anti-CD8α antibody (BE0061, BioXCell) was intraperitoneally injected twice weekly at 200 μg, starting 7 days before modeling.

[0115] The results are as follows: In a mouse lung cancer orthotopic xenograft model, PTPRT knockout combined with anti-PD-1 monoclonal antibody treatment significantly reduced tumor growth rate and size compared with the control treatment group (anti-PD-1 monoclonal antibody group) and the knockdown group (PTPRT knockout group) (Figure 4A and B), while the proportion of CD8+ T cell infiltration increased (Figure 4C). Furthermore, PTPRT knockout combined with anti-PD-1 monoclonal antibody treatment significantly prolonged the overall survival of the mice (Figure 4D).

[0116] The above results indicate that knocking out PTPRT or inhibiting PTPRT expression in combination with anti-PD-1 monoclonal antibody can significantly inhibit tumor growth, prolong survival, increase the infiltration of CD8+T cells in tumors, and enhance the anti-tumor effect of immunotherapy.

[0117] Example 4

[0118] PTPRT inhibitors combined with immune checkpoint inhibitors can synergistically enhance the anti-colorectal cancer effect of immunotherapy

[0119] The PTPRT inhibitor can be a substance that inhibits the expression of the PTPRT gene, silences or knocks out the PTPRT gene, or can be a substance that inhibits or reduces the content and / or activity of the PTPRT protein.

[0120] 1. Selection of cell lines and animals

[0121] The mouse MC38 cell line used in this study was obtained from the ATCC Cell Resource Center in the United States.

[0122] This study used 6-week-old female C57BL / 6 mice. All mice were housed in a SPF-grade environment. All procedures performed in this study strictly adhered to the regulations established by the Animal Ethics Committee.

[0123] 2. Construction of MC38 cells with PTPRT knockout

[0124] Based on the PTPRT gene sequence and CRISPR-Cas9 principles, the PTPRT gene was knocked out in MC38 cells. The encoding DNA sequence of the sgRNA for the PTPRT knockout gene was designed to be: 5'-CAGCAACTGCGGGTATAGCG-3' (SEQ ID NO: 1), and the encoding DNA sequence of the control sgRNA was: 5'-GAACAGTCGCGTTTGCGACT-3' (SEQ ID NO: 2). MC38 cells were transfected with lentivirus containing the PTPRT knockout gene and a blank control to obtain MC38 cells with the PTPRT knockout gene and a control.

[0125] 3. Mouse colorectal cancer subcutaneous transplant tumor model

[0126] One million PTPRT knockout and control MC38 cells were subcutaneously injected into the right side of the back of wild-type C57BL / 6 female mice (6 weeks old). Tumor volume was calculated using the following formula: (width x width x length) / 2. Anti-PD-1 therapy (BE0273, BioXcell) and an IgG isotype control (BE0089, BioXcell) were administered intraperitoneally at a dose of 150 μg each time, starting on day 9 and continuing every three days. Tumors were harvested on day 21 for flow cytometric analysis. Tumors were collected by autopsy after sacrifice under carbon dioxide anesthesia to minimize animal suffering.

[0127] The results are as follows: PTPRT protein expression in MC38 cells was significantly decreased after PTPRT knockout (Figure 5A). In a mouse subcutaneous colorectal cancer xenograft model, PTPRT knockout combined with anti-PD-1 monoclonal antibody treatment significantly reduced tumor growth rate and size compared with both the control treatment group (anti-PD-1 monoclonal antibody group) and the knockdown group (PTPRT knockout group) (Figure 5B and C), while the proportion of CD8+ T cell infiltration increased (Figure 5D).

[0128] Knocking out PTPRT or inhibiting PTPRT expression in combination with anti-PD-1 monoclonal antibodies can significantly inhibit tumor growth, improve the therapeutic efficacy of immune checkpoint inhibitors, increase the infiltration of CD8+T cells in tumors, and enhance the anti-tumor effect of immunotherapy.

[0129] In summary, the predictive biomarkers developed in the present invention can maximize the efficacy of immune checkpoint inhibitors and screen out people who can benefit from immune checkpoint therapy. In addition, inhibiting PTPRT can expand the response population to immune checkpoint therapy and bring survival benefits to more patients.

[0130] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit this application. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of this application. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical concepts disclosed herein shall be covered by the claims of this application.

Claims

1. Application of PTPRT expression in the prediction or evaluation of the efficacy of immune checkpoint therapy for cancer.

2. The use according to claim 1, characterized in that The cancer is selected from lung cancer, head and neck squamous cell carcinoma, esophageal cancer, pleural mesothelioma, breast cancer, gastric cancer, hepatocellular carcinoma, colorectal cancer, kidney cancer, urothelial carcinoma, cervical cancer, endometrial cancer, ovarian cancer, malignant melanoma, malignant lymphoma, or skin cancer; preferably, the cancer is lung cancer or colorectal cancer.

3. The use according to claim 1, characterized in that When the staining intensity of PTPRT in the sample in immunohistochemical staining is less than 1%, it is judged as low expression, which predicts a good efficacy of immune checkpoint therapy for cancer; when the staining intensity of PTPRT in the sample in immunohistochemical staining is greater than 1%, it is judged as high expression, which predicts a poor efficacy of immune checkpoint therapy for cancer.

4. The use according to claim 1, characterized in that The expression level of the PTPRT is the expression level of the wild-type PTPRT; And / or, immune checkpoint treatment of lung cancer includes the use of immune checkpoint-related drugs; the immune checkpoint-related drugs include immune checkpoint inhibitors and / or immune checkpoint activators; preferably, the immune checkpoint inhibitors include a combination of one or more of PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, TIM-3 inhibitors, LAG3 inhibitors, and TIGIT inhibitors; the immune checkpoint activators include one or a combination of two or more of CD27 activators, CD40 activators, OX40 activators, GITR activators, CD137 activators, CD28 activators, and ICOS activators; more preferably, the immune checkpoint-related drugs are selected from PD-1 inhibitors.

5. Use of substances for detecting the expression level of PTPRT in the preparation of products for evaluating or predicting the efficacy of immune checkpoint therapy for cancer.

6. The use according to claim 5, characterized in that The cancer is selected from lung cancer, head and neck squamous cell carcinoma, esophageal cancer, pleural mesothelioma, breast cancer, gastric cancer, hepatocellular carcinoma, colorectal cancer, renal cancer, urothelial carcinoma, cervical cancer, endometrial cancer, ovarian cancer, malignant melanoma, malignant lymphoma, or skin cancer; preferably, the cancer is lung cancer or colorectal cancer; And / or, the substance for detecting the expression amount of PTPRT includes a reagent for detecting the expression amount of PTPRT protein, detecting the content of PTPRT protein, or detecting the expression amount of PTPRT RNA.

7. The use according to claim 6, characterized in that The reagent includes an antibody, a polypeptide, a protein or a nucleic acid molecule that binds to the PTPRT protein; preferably, the antibody is a PTPRT monoclonal antibody.

8. A kit comprising a substance for detecting the expression of PTPRT, wherein the kit has at least one of the following uses: 1) Predict the efficacy of immune checkpoint therapy for lung cancer; 2) Evaluate the efficacy of immune checkpoint therapy in lung cancer.

9. Use of a PTPRT inhibitor in the preparation of a product, wherein the product has at least one of the following effects: 1) Increase the infiltration of CD8+T cells in lung or colorectal cancer tumors; 2) Improve the survival of lung cancer patients; 3) Improve the tumor immune microenvironment of lung cancer or colorectal cancer; 4) Improve the efficacy of immune checkpoint-related drugs in the treatment of lung cancer or colorectal cancer.

10. The use according to claim 9, characterized in that The PTPRT inhibitor includes a substance that reduces the expression amount of PTPRT protein or the content of PTPRT protein, or a substance that inhibits the expression of PTPRT gene. Further, the substance that inhibits the expression of PTPRT gene includes a substance that knocks out or silences PTPRT. Furthermore, the substance for knocking out or silencing PTPRT includes: a CRISPR gene editing system for PTPRT, an interfering molecule that specifically interferes with the expression of the coding gene of PTPRT, and a homologous recombination substance for a loss-of-function mutation of PTPRT; And / or, the immune checkpoint-related drugs include immune checkpoint inhibitors and / or immune checkpoint activators; preferably, the immune checkpoint inhibitors include a combination of one or more of PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, TIM-3 inhibitors, LAG3 inhibitors, and TIGIT inhibitors; the immune checkpoint activators include one or a combination of two or more of CD27 activators, CD40 activators, OX40 activators, GITR activators, CD137 activators, CD28 activators, and ICOS activators; more preferably, the immune checkpoint-related drugs are selected from PD-1 inhibitors.

11. The use according to claim 10, characterized in that The CRISPR gene editing system for PTPRT includes sgRNA, and the encoding DNA sequence of the sgRNA is shown in SEQ ID NO:

1.

12. A pharmaceutical composition comprising an effective amount of an immune checkpoint-related drug and a PTPRT inhibitor for use according to any one of claims 9 to 11.

13. The pharmaceutical composition according to claim 12, characterized in that The immune checkpoint-related drugs include immune checkpoint inhibitors and / or immune checkpoint activators; preferably, the immune checkpoint inhibitors include a combination of one or more of PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, TIM-3 inhibitors, LAG3 inhibitors, and TIGIT inhibitors; the immune checkpoint activators include one or a combination of two or more of CD27 activators, CD40 activators, OX40 activators, GITR activators, CD137 activators, CD28 activators, and ICOS activators; more preferably, the immune checkpoint-related drugs are selected from PD-1 inhibitors.

14. Use of the pharmaceutical composition according to any one of claims 12 to 13 in the preparation of a product for treating lung cancer or colorectal cancer.

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