Application of PTPRT as biomarker and target for predicting efficacy of lung cancer immunotherapy

Abstract

The application relates to the field of medical technology, in particular to application of PTPRT as a biomarker and target point for predicting the curative effect of lung cancer immunological checkpoint treatment. The application provides application of the expression amount of PTPRT in curative effect prediction or evaluation of immunological checkpoint treatment of cancer. The experiment of the application shows that low expression of PTPRT can accurately predict the progression-free survival (PFS) of lung cancer patients after receiving immunological checkpoint treatment as a marker for predicting the curative effect of lung cancer immunological checkpoint treatment. Inhibition of PTPRT can increase the proportion of CD8+ T cells infiltrating tumors, and synergistically enhances the anti-tumor effect of an immunological 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] This application relates to the field of medical technology, specifically to the application of PTPRT as a biomarker and target for predicting the efficacy of immune checkpoint therapy for lung cancer. Background Technology

[0002] Cancer is a leading cause of death worldwide, and the advent of immune checkpoint therapy has transformed cancer treatment. However, only about 20% of patients consistently benefit from immune checkpoint inhibitors, and 90% of those treated experience adverse events. Therefore, identifying patients more likely to benefit from immune checkpoint therapy and achieving precision medicine is of great significance.

[0003] However, PD-L1 has limited sensitivity and specificity. A portion of patients with high PD-L1 expression do not benefit from immune checkpoint inhibitor therapy, while approximately 20% of patients with negative PD-L1 expression do respond to immune checkpoint inhibitor therapy. Tumor mutation burden (TMB), as a biomarker for the efficacy of immune checkpoint inhibitor therapy, lacks standardized detection methods and threshold selection. Therefore, finding more precise biomarkers with clinical translational potential to guide lung cancer patients in receiving immune checkpoint therapy, identifying patients who can benefit from immune checkpoint therapy, and expanding the response population through multi-target combination therapy, ultimately bringing 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, in order to solve the problems in the prior art.

[0005] The inventors of this application discovered that the accuracy of screening patients based on detecting tumor PD-L1 expression needs improvement. The innovative achievement of this technology lies in overcoming the shortcomings of existing techniques by discovering that PTPRT expression can predict the efficacy of immune checkpoint therapy in non-small cell lung cancer, and that this method is independent of the method used to detect tumor PD-L1 expression. It can be used as a separate indicator to improve the accuracy of patient screening.

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

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

[0008] A third aspect of this application provides a kit comprising a substance for detecting PTPRT expression levels, the kit having at least one of the following uses:

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

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

[0011] The fourth aspect of this application provides the use of a PTPRT inhibitor in the preparation of a product, said product having at least one of the following effects:

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

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

[0014] 3) Improve the tumor immune microenvironment in 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 this application provides a pharmaceutical composition comprising an effective amount of an immune checkpoint-related drug and a PTPRT inhibitor used in the aforementioned uses.

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

[0018] Compared with the prior art, the beneficial effects of this application are as follows:

[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 improved progression-free survival (PFS) in lung cancer patients receiving immune checkpoint therapy; low PTPRT expression significantly improved the objective response rate (ORR) in lung cancer patients receiving immune checkpoint therapy; and low PTPRT expression significantly increased CD8+ T cell infiltration in tumor samples from lung cancer patients. Inhibiting PTPRT improved the tumor immune microenvironment in lung cancer patients. This indicates that low PTPRT expression is a novel biomarker 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, and the low expression of PTPRT does not affect the expression of PD-L1, and can be used as a supplement to the expression of PD-L1.

[0021] 3. This application, through the construction of a mouse model (preclinical model), found that PTPRT knockout combined with immune checkpoint therapy significantly inhibited tumor growth and tumor size; PTPRT knockout significantly improved the tumor immune microenvironment in mice and increased CD8+ T cell infiltration; PTPRT knockout combined with immune checkpoint therapy significantly improved the survival of mice. Attached Figure Description

[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 images of PTPRT (-, +, 2+, 3+) in tumor biopsy samples. Scale bar is 100 μm. Figure 1B shows the correlation between PTPRT expression level and progression-free survival in lung cancer patients receiving immunotherapy. Figure 1C shows the ROC curve of low PTPRT expression predicting the efficacy of immune checkpoint therapy. Figure 1D shows the correlation between PTPRT expression level and objective response rate in lung cancer patients receiving immunotherapy. Figure 1E shows the correlation between PTPRT expression level and PD-L1 expression level 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 external validation data from 37 cases. In Figure 2, A represents the correlation between PTPRT expression levels and progression-free survival in lung cancer patients receiving immunotherapy; Figure 2, B represents the ROC curve of low PTPRT expression predicting the efficacy of immune checkpoint therapy.

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

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

[0026] Figure 5 shows the synergistic effect of combined inhibition of PTPRT expression in a subcutaneous tumor model of colorectal cancer, enhancing the immunotherapy effect. In Figure 5, A shows the changes in related proteins after MC38 PTPRT knockout; B shows the changes in tumor volume curve; C shows the changes in tumor size at the endpoint; and D shows the proportion of tumor-infiltrating CD8+ T cells. Detailed Implementation

[0027] To make the inventive purpose, technical solution, and beneficial effects of this application clearer, the following description, in conjunction with embodiments, further illustrates this application. It should be understood that the embodiments described are for illustrative purposes only and are not intended to limit the scope of the application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this description. Unless otherwise specified, the materials, reagents, etc., used in the following embodiments are commercially available.

[0028] This invention investigated a clinical cohort of immune checkpoint therapy at Shanghai Chest Hospital and found that the expression of receptor-type protein tyrosine phosphatase T (PTPRT) was highly correlated with progression-free survival (PFS) in lung cancer patients after immune checkpoint therapy. Low PTPRT expression can serve as a biomarker for predicting the efficacy of immune checkpoint therapy in lung cancer. As a target for predicting the efficacy of immune checkpoint therapy in lung cancer, inhibiting PTPRT can effectively improve the tumor immunosuppressive microenvironment by increasing tumor CD8+ T cell infiltration.

[0029] This application provides, in one aspect, the application of PTPRT expression levels 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 this application, the cancer is lung cancer or colorectal cancer.

[0030] In the application provided in this application, when the expression level of PTPRT in the sample is <1% in immunohistochemical staining, the predicted efficacy of immune checkpoint therapy for cancer is good; when the expression level of PTPRT in the sample is >1% in immunohistochemical staining, the predicted efficacy of immune checkpoint therapy for cancer is poor.

[0031] PTPRT stands for receptor protein tyrosine phosphatase T. Existing technologies indicate that PTPRT gene mutations are associated with prognostic outcomes in lung cancer immunotherapy. This application, for the first time, discloses the relationship between low PTPRT protein expression and the evaluation of the efficacy of immune checkpoint inhibitor therapy in lung cancer. Specifically, low PTPRT expression significantly increases CD8+ T cell infiltration in lung cancer tumors; low PTPRT expression significantly improves progression-free survival (PFS) in lung cancer patients; low PTPRT expression significantly improves objective response rate (ORR) in lung cancer patients; and low PTPRT expression significantly increases CD8+ T cell infiltration in tumor samples from lung cancer patients. This demonstrates that low PTPRT expression is a novel biomarker for predicting the efficacy of immune checkpoint inhibitor therapy in lung cancer patients.

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

[0033] In the applications provided in this application, immune checkpoint therapy for lung cancer includes the use of immune checkpoint-related drugs; further, immune checkpoint-related drugs include immune checkpoint inhibitors and / or immune checkpoint activators; even further, immune checkpoint inhibitors include one or more combinations of PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, TIM-3 inhibitors, LAG3 inhibitors, and TIGIT inhibitors. Immune checkpoint activators include one or more combinations of CD27 activators, CD40 activators, OX40 activators, GITR activators, CD137 activators, CD28 activators, and ICOS activators. In a specific embodiment of this application, the immune checkpoint-related drug is 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 autoimmune reactions. Immune checkpoint immunotherapy is a treatment method that uses a series of pathways, such as co-inhibition or co-stimulation signals, to regulate T cell activity and kill tumor cells.

[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 be used as a supplement to the expression of PD-L1.

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

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

[0038] In the uses provided in this application, 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.

[0039] In the uses provided in this application, the substances for detecting PTPRT expression levels include reagents for detecting PTPRT protein expression levels, detecting PTPRT protein content, or detecting PTPRT RNA expression levels. Specifically, the reagents include antibodies, peptides, proteins, or nucleic acid molecules that bind to the PTPRT protein.

[0040] The reagents for detecting PTPRT can specifically bind to specific sites on PTPRT, without specifically binding to other genes besides PTPRT, and optionally carry a detectable signal. The method of using antibodies that specifically bind to PTPRT to detect PTPRT expression in an analyte is also a well-known technique in the art.

[0041] In a specific embodiment of this application, the substance used to detect the expression level of PTPRT is an antibody, specifically a PTPRT monoclonal antibody.

[0042] A third aspect of this application provides a kit comprising a substance for detecting PTPRT expression levels, the kit having at least one of the following uses:

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

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

[0045] The kit includes: primers for specifically amplifying the PTPRT gene, probes for specifically recognizing the PTPRT gene, and antibodies or ligands that specifically bind to the RNA encoded by the PTPRT gene or the protein encoded by the PTPRT gene. Furthermore, the kit may also include various reagents required for DNA and RNA extraction, PCR, hybridization, and color development, including but not limited to: extraction buffer, amplification buffer, hybridization buffer, enzymes, control solutions, color development solutions, and washing buffers.

[0046] In one specific embodiment of this application, the kit includes a PTPRT monoclonal antibody. The efficacy of immune checkpoint therapy for lung cancer is predicted or evaluated by determining the staining intensity of PTPRT in the sample through immunohistochemical staining. Specifically, when the staining intensity of PTPRT in the sample is <1% in immunohistochemical staining, it is considered low expression, predicting good efficacy of immune checkpoint therapy for lung cancer; when the staining intensity of PTPRT in the sample is >1% in immunohistochemical staining, it is considered high expression, predicting poor efficacy of immune checkpoint therapy for lung cancer.

[0047] The fourth aspect of this application provides the use of a PTPRT inhibitor in the preparation of a product, the product having at least one of the following effects:

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

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

[0050] 3) Improved the tumor immune microenvironment in 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 blocking agents; these terms are used interchangeably.

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

[0054] In some embodiments, the CRISPR gene editing system for PTPRT can employ a CRISPR / Cas (e.g., Cas9) system for targeted gene editing, thereby knocking out the PTPRT gene in the target disease region. Common methods for knocking out the PTPRT gene include co-transferring sgRNA or a nucleic acid capable of forming sgRNA, Cas9 mRNA or a nucleic acid capable of forming Cas9 mRNA to the target region or target cells. After identifying the target site, known methods can be used to introduce sgRNA and Cas9 into the cell. The nucleic acid capable of forming sgRNA is a nucleic acid construct or expression vector, or the nucleic acid capable of forming said Cas9 mRNA is a nucleic acid construct or expression vector. These expression vectors are introduced into the cell, thereby forming active sgRNA and Cas9 mRNA within the cell. As a particularly preferred embodiment of the present invention, the CRISPR gene editing system for PTPRT includes sgRNA, the encoding DNA sequence of which is shown in SEQ ID NO:1.

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

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

[0057] A fifth aspect of this application provides a pharmaceutical composition comprising an effective amount of an immune checkpoint-related drug and a PTPRT inhibitor as described above. The immune checkpoint-related drug includes immune checkpoint inhibitors and / or immune checkpoint activators. Further, the immune checkpoint inhibitor includes one or more combinations of PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, TIM-3 inhibitors, LAG3 inhibitors, and TIGIT inhibitors; the immune checkpoint activator includes one or more combinations of CD27 activators, CD40 activators, OX40 activators, GITR activators, CD137 activators, CD28 activators, and ICOS activators. In a specific embodiment of this 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] "Pharmaceutical acceptable" means that when the molecular basis and the composition are properly administered to animals or humans, they do not produce adverse, allergic, or other adverse reactions.

[0060] "Pharmaceutically acceptable carriers or excipients" should be compatible with immune checkpoint inhibitors and PTPRT inhibitors, meaning they can be mixed 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 gum 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, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavoring agents; tableting agents, stabilizers, antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffers, etc. These substances are used as needed to help stabilize the formulation or to improve its activity or bioavailability or to produce an acceptable taste or smell when taken orally.

[0061] The pharmaceutical composition provided in this application can be adapted to any form of administration, including oral or parenteral administration, for example, via pulmonary, nasal, rectal and / or intravenous injection, and more specifically via intradermal, subcutaneous, intramuscular, intra-articular, intraperitoneal, pulmonary, oral, sublingual, nasal, percutaneous, vaginal, oral or parenteral administration.

[0062] Those skilled in the art can select appropriate formulations based on the route 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. For another example, formulations suitable for parenteral administration may include, but are not limited to, solutions, suspensions, rehydrated dry formulations, or sprays. For yet another example, suppositories are typically suitable for rectal administration.

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

[0064] The seventh aspect of this application provides a method for treating lung cancer or colorectal cancer, characterized in that it involves administering the aforementioned PTPRT inhibitor or the aforementioned pharmaceutical composition to a subject.

[0065] The method for treating lung cancer provided in this application targets mammals. Examples of mammals include rodents, even-toed ungulates, perissodactyls, lagomorphs, and primates. Examples of primates include monkeys, apes, or Homo sapiens.

[0066] The present application will be further illustrated by the following examples, but these examples do not limit the scope of the present application.

[0067] Example 1

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

[0069] Case selection in this embodiment: This embodiment 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 participants. This embodiment primarily includes stage IIIC / IV lung cancer patients who received immune checkpoint inhibitor therapy at Shanghai Chest Hospital from January 2020 to June 2022, with follow-up ending on December 31, 2022. The treatment received was anti-PD-1 therapy or anti-PD-1 therapy plus chemotherapy.

[0070] The efficacy of treatment during the patient's treatment period was assessed according to the Response Evaluation 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 therapy to disease progression or death. The objective response rate (ORR) was the proportion of patients whose tumor volume shrank by 30% and maintained this reduction for the minimum required duration; it was the sum of the proportions of complete response (CR, i.e., complete disappearance of the tumor) and partial response (PR, i.e., tumor shrinkage of 30% or more), excluding stable disease (SD).

[0071] Immunohistochemical staining was performed on the samples to detect the expression percentage of PTPRT. IHC staining intensity was divided into: -, <1%; +, 1%-10%; ++, 10%-50%; +++, >50%.

[0072] Specific operations:

[0073] a. Baking: Place the paraffin slices on a baking machine at 60℃ for 30 minutes.

[0074] b. Dewaxing and rehydration of sections: Prepare 3 tanks of xylene (pass through 3 times), quickly put the baked paraffin sections into the glass tank, soak for 5 minutes at each concentration, then pass the sections through anhydrous ethanol twice, 2 minutes each time, then pass them through 95%, 90%, 80%, and 70% ethanol in sequence, 5 minutes at each concentration, and finally soak the sections in PBS buffer 3 times, 3 minutes each time.

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

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

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

[0078] f. Blocking: After removing oxidoreductase from the sections, wipe the fluid around the tissue on the slide dry. Use an immunohistochemical pen to draw a circle around the tissue to prevent the fluid added to the sections in subsequent steps from flowing out. Place the sections in a humidified chamber protected from light, and then add blocking solution containing 10% goat serum to the sections. After incubating at room temperature for 1 hour, aspirate the blocking solution from the sections, and then soak the sections in PBS buffer 3 times, 3 minutes each time.

[0079] g. Primary antibody incubation: Dilute the antibody with antibody diluent according to the instructions of the primary antibody (R&D Company product number AF3697), then drop the primary antibody onto the blocked tissue surface and incubate overnight at 4°C.

[0080] h. Secondary antibody incubation: Aspirate the primary antibody liquid from the surface of the sections, then soak the sections in PBS buffer three times, 3 min each time. Place the prepared secondary antibody (Jackson ImmunoResearch product catalog number 705-005-003) corresponding to the primary antibody species onto the tissue sections. Incubate at room temperature for 1 h, then soak the sections in PBS buffer three more times, 3 min each time.

[0081] i. DAB staining: Add DAB staining solution to the tissue section. A preliminary experiment can be conducted 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 section with slow-flowing tap water to stop the staining process.

[0082] j. Staining cell nuclei: After DAB staining, the sections are counterstained in hematoxylin solution to make the nuclei of all cells appear blue. Generally, staining at room temperature for 2-5 minutes is sufficient. Finally, gently rinse the sections with slow-flowing tap water to stop the staining process.

[0083] k. Mounting: After completing the above steps, the slides need to be mounted for long-term preservation. Immerse the slides sequentially in 70%, 80%, 90%, 95%, and 100% ethanol for 2 minutes each, repeating the dehydration process once in 100% ethanol. Then, soak the slides twice in xylene for 5 minutes each time. Add a small amount of neutral resin to the tissue section, cover with a coverslip, and avoid air bubbles. Finally, allow the slides to dry completely in a fume hood, then place them in a slide box for subsequent observation and photography.

[0084] 1. Survival Curve

[0085] The results are shown in Figure 1A. Patients were divided into those with low PTPRT expression (-) and those with high PTPRT expression (+, ++, +++). The results are shown in Figures 1B and D. Progression-free survival (PFS) curves and objective response rates were plotted. Analysis showed that, at the same follow-up time, patients with low PTPRT expression had a significantly increased PFS (16.3 months vs. 5.9 months, P = 0.037) and a significantly increased objective response rate (66.7% vs. 27.3%, P = 0.036). The ROC curve in Figure 1C shows that low PTPRT expression has high specificity and sensitivity in predicting immune checkpoint therapy efficacy (AUC = 0.742, P = 0.0367).

[0086] External validation was performed using data from 37 other cases: Case selection in this embodiment: This embodiment 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 participants. This embodiment primarily included stage IIIC / IV lung cancer patients who received PD-1 inhibitor therapy 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] The efficacy of treatment was assessed according to the Response Evaluation Criteria in Solid Tumors (RECIST version 1.1). Progression-free survival (PFS) was defined as the time from the start of immunosuppressant therapy to disease progression or death. Immunohistochemical staining was performed on samples to detect the percentage of PTPRT expression; IHC staining intensity was categorized as: - (<1%); + (1%–100%).

[0088] As 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 months vs. 4.2 months, P = 0.008). Appropriate ROC curves showed that low PTPRT expression had high specificity and sensitivity.

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

[0090] 2. The relationship between PTPRT expression and the tumor immune microenvironment

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

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

[0093] Patients in the PTPRT low expression group showed better or better efficacy with immune checkpoint inhibitor therapy than patients in the PTPRT high expression group. Therefore, PTPRT can be used as a biomarker to assess or assist in the assessment of the efficacy of immune checkpoint inhibitor therapy.

[0094] Example 2

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

[0096] PTPRT inhibitors can be substances that inhibit PTPRT gene expression, silence or knock out the PTPRT gene, or substances that inhibit or reduce the content and / or activity of 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] Six-week-old female C57BL / 6 mice were used in this study. All mice were housed in an SPF-protected environment. All procedures in the study were strictly performed in accordance with the regulations established by the animal ethics committee.

[0100] 2. Construct LLC cells with PTPRT knockout

[0101] Based on the PTPRT gene sequence and the CRISPR-Cas9 principle, the PTPRT gene was knocked out in LLC cells. The encoding DNA sequence of the sgRNA for PTPRT gene knockout was designed as: 5'-CAGCAACTGCGGGTATAGCG-3' (SEQ ID NO:1), and the encoding DNA sequence of the control sgRNA was: 5'-GAACAGTCGCGTTTGCGACT-3' (SEQ ID NO:2). After transfecting LLC cells with lentiviruses that knocked out PTPRT and those that were blank controls, PTPRT gene knockout and control LLC cells were obtained. Changes in PTPRT and PD-L1 levels in cells were detected by Western blotting, and changes in PD-L1 levels on the cell membrane surface were detected by flow cytometry.

[0102] 3. Mouse subcutaneous xenograft model of lung cancer

[0103] One million LLC cells with the PTPRT gene knocked out and those used as a control were subcutaneously injected into the right back of wild-type C57BL / 6 female mice (6 weeks old). Tumor volume was calculated using the following formula: (width * width * length) / 2. Anti-PD-1 treatment (BE0273, BioXcell) and IgG isotype control (BE0089, BioXcell) were administered intraperitoneally at 150 μg each time, starting on day 9 and every 3 days. Tumors were collected on day 21 for flow cytometry analysis. Animals were euthanized under carbon dioxide anesthesia and dissected after tumor collection to minimize suffering.

[0104] The results are as follows: PTPRT protein expression was significantly decreased in LLC cells with PTPRT knockout, while PD-L1 protein levels did not change significantly (Figure 3A). Flow cytometry analysis revealed no significant change in the fluorescence intensity of PD-L1 on the cell membrane surface of LLC cells with PTPRT knockout (Figure 3B). In a mouse subcutaneous xenograft model of lung cancer, PTPRT knockout combined with anti-PD-1 monoclonal antibody treatment significantly reduced tumor growth rate and size compared to the control treatment group (anti-PD-1 monoclonal antibody group) and the knockdown group (PTPRT knockout group) (Figure 3C and D), while increasing the proportion of CD8+ T cell infiltration (Figure 3E).

[0105] The above results indicate that knocking out or inhibiting PTPRT expression does not affect PD-L1 expression in tumor cells. Knocking out or inhibiting PTPRT expression, in combination with anti-PD-1 monoclonal antibodies, can significantly inhibit tumor growth, improve the efficacy of immune checkpoint inhibitor therapy, increase CD8+ T cell infiltration in tumors, and enhance the anti-tumor effect 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] Six-week-old female C57BL / 6 mice were used in this study. All mice were housed in an SPF-protected environment. All procedures in the study were strictly performed in accordance with the regulations established by the animal ethics committee.

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

[0112] Based on the PTPRT gene sequence and the CRISPR-Cas9 principle, the PTPRT gene was knocked out in LLC cells. The encoding DNA sequence of the sgRNA for PTPRT gene knockout was designed as: 5'-CAGCAACTGCGGGTATAGCG-3' (SEQ ID NO:1), and the encoding DNA sequence of the control sgRNA was: 5'-GAACAGTCGCGTTTGCGACT-3' (SEQ ID NO:2). After transfecting LLC cells with lentiviruses that knocked out PTPRT and those that were blank controls, LLC cells with PTPRT gene knockout and those that were controls were obtained.

[0113] 3. Mouse orthotopic xenograft model of lung cancer

[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 suspension of stable transfected cells (800,000 LLC cells with the PTPRT gene knocked out in step 2 and control cells) (Matrigel:PBS = 1:4) was directly injected into the left lung using an insulin injector (BD), and the incision was sutured. In vivo fluorescence imaging was performed on day 5 to observe the model establishment. Anti-PD-1 treatment (BE0273, BioXcell) and IgG isotype control (BE0089, BioXcell) were administered intraperitoneally at 150 μg each time, starting on day 9, every 3 days, until the experimental endpoint was reached on day 21, at which point in vivo fluorescence imaging was performed. For the survival experiment, anti-PD-1 treatment (BE0273, BioXcell) and IgG isotype control (BE0089, BioXcell) were administered intraperitoneally at 150 μg per dose, starting from day 7 of modeling, until the mice died. Anti-CD8α 200 μg antibody (BE0061, BioXCell) was injected intraperitoneally twice a week, starting 7 days before modeling.

[0115] The results are as follows: In a mouse orthotopic xenograft model of lung cancer, the tumor growth rate and size were significantly reduced after PTPRT knockout combined with anti-PD-1 monoclonal antibody treatment 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 significantly prolonged the overall survival of mice (Figure 4D).

[0116] The above results indicate that knocking out PTPRT or inhibiting PTPRT expression in combination with anti-PD-1 monoclonal antibodies 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] PTPRT inhibitors can be substances that inhibit PTPRT gene expression, silence or knock out the PTPRT gene, or substances that inhibit or reduce the content and / or activity of 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] Six-week-old female C57BL / 6 mice were used in this study. All mice were housed in an SPF-protected environment. All procedures in the study were strictly performed in accordance with the regulations established by the animal ethics committee.

[0123] 2. Constructing MC38 cells with PTPRT knockout

[0124] Based on the PTPRT gene sequence and the CRISPR-Cas9 principle, the PTPRT gene was knocked out in MC38 cells. The encoding DNA sequence of the sgRNA for PTPRT gene knockout was designed as: 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 lentiviruses that knocked out the PTPRT gene and those that served as a blank control to obtain MC38 cells with the PTPRT gene knocked out and those that served as a control.

[0125] 3. Mouse subcutaneous xenograft model of colorectal cancer

[0126] One million PTPRT-knockout and control MC38 cells were subcutaneously injected into the right back of wild-type C57BL / 6 female mice (6 weeks old). Tumor volume was calculated using the following formula: (width * width * length) / 2. Anti-PD-1 treatment (BE0273, BioXcell) and IgG isotype control (BE0089, BioXcell) were administered intraperitoneally at 150 μg each time, starting on day 9 and every 3 days. Tumors were collected on day 21 for flow cytometry analysis. Animals were euthanized under carbon dioxide anesthesia and dissected to minimize suffering during tumor collection.

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

[0128] Knocking out or inhibiting PTPRT expression in combination with anti-PD-1 monoclonal antibodies can significantly inhibit tumor growth, improve the efficacy of immune checkpoint inhibitor therapy, 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 this invention can maximize the efficacy of immune checkpoint inhibitors, screen out individuals who can benefit from immune checkpoint therapy, and inhibit PTPRT can expand the response population of immune checkpoint therapy, bringing 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. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of this application. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in this invention should still be covered by the claims of this application.

Claims

1. The use of a substance for detecting PTPRT expression levels in the preparation of products for evaluating or predicting the efficacy of immune checkpoint therapy for cancer; wherein the cancer is lung cancer, and the immune checkpoint therapy for cancer includes the use of immune checkpoint-related drugs selected from PD-1 inhibitors; the substance for detecting PTPRT expression levels includes reagents for detecting PTPRT protein expression levels or detecting PTPRT protein content.

2. The use as described in claim 1, characterized in that, The reagents include antibodies, peptides, proteins, or nucleic acid molecules that bind to the PTPRT protein.

3. The use as described in claim 2, characterized in that, The antibody is a PTPRT monoclonal antibody.

4. The use as described in claim 1, characterized in that, When the staining intensity of PTPRT in the sample is <1% in immunohistochemical staining, it is considered low expression, which predicts good efficacy of immune checkpoint therapy for cancer; when the staining intensity of PTPRT in the sample is >1% in immunohistochemical staining, it is considered high expression, which predicts poor efficacy of immune checkpoint therapy for cancer.

5. The use as described in claim 1, characterized in that, The expression level of PTPRT is the expression level of wild-type PTPRT.

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