Application of PTPRT as biomarker and target spot for predicting lung cancer immune checkpoint treatment effect

Abstract

The invention relates to the technical field of medicine, in particular to application of PTPRT as a biomarker and a target spot for predicting the treatment effect of a lung cancer immune checkpoint. The invention provides application of the expression quantity of PTPRT in prediction or evaluation of the curative effect of immune checkpoint treatment on cancer. Experiments show that low expression of PTPRT is used as the marker for predicting the curative effect of the immune checkpoint on the lung cancer, and the progression-free lifetime (PFS) of a lung cancer patient after the immune checkpoint treatment can be accurately predicted. The proportion of CD8 + T cells infiltrated by tumors can be increased by inhibiting PTPRT, and the anti-tumor effect of the immune checkpoint inhibitor is enhanced in cooperation with immune checkpoint treatment.

Description

Technical Field

[0001] This application relates to the field of medical technology, and specifically relates 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 globally, and the emergence of immune checkpoint therapy has changed the way cancer is treated. However, only about 20% of patients continuously benefit from immune checkpoint inhibitors, and 90% of the patients receiving treatment suffer from adverse reaction events. Therefore, it is of great significance to identify patients who are more likely to benefit from immune checkpoint therapy and achieve precision treatment.

[0003] Currently, the only biomarker for the efficacy of immune checkpoint inhibitor therapy approved by the US Food and Drug Administration and most widely used is the expression of tumor PD-L1. However, the sensitivity and specificity of PD-L1 are limited. Some patients with high PD-L1 expression do not benefit from immune checkpoint inhibitor therapy, while about 20% of patients with negative PD-L1 expression respond to immune checkpoint inhibitor therapy. As a biomarker for the efficacy of immune checkpoint inhibitor therapy, there is no unified standard for the selection of detection methods and thresholds for tumor mutation burden (TMB). Therefore, finding more accurate and clinically translatable biomarkers to guide lung cancer patients to receive immune checkpoint therapy, screening out the population that can benefit from immune checkpoint therapy, and expanding the responsive population of immune checkpoint therapy through multi-target combination therapy can bring survival benefits to more patients. Summary of the Invention

[0004] In view of the above-mentioned disadvantages of the prior art, 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 found through research that: the accuracy of screening patients by detecting the expression of tumor PD-L1 needs to be improved. The innovation of this technology lies in solving the deficiencies of the prior art, discovering that the expression of PTPRT can predict the efficacy of immune checkpoint in non-small cell lung cancer, and is independent of the method of detecting the expression of tumor PD-L1. It can be detected as a separate indicator to improve the accuracy of screening patients.

[0006] To achieve the above purpose and other related purposes, the first aspect of this application provides the application of the expression level of PTPRT 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] The third aspect of the present application provides a kit, the kit includes a substance for detecting the expression level of PTPRT, and the kit has at least one of the following uses:

[0009] 1) Predict 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 the present application provides the use of a PTPRT inhibitor in the preparation of a product, and the product has at least one of the following effects:

[0013] 1) Increase the infiltration of CD8+ T cells in lung cancer or colorectal cancer tumors;

[0014] 2) Increase the survival period of lung cancer patients;

[0015] 3) Improve the immune microenvironment of lung cancer or colorectal cancer tumors;

[0016] 4) Improve the efficacy of immune checkpoint-related drugs in treating lung cancer or colorectal cancer.

[0017] The fifth aspect of the present application provides a pharmaceutical composition, including an effective amount of an immune checkpoint-related drug and a PTPRT inhibitor in the foregoing use.

[0018] The sixth aspect of the present application provides the use of the foregoing pharmaceutical composition in the preparation of a product for treating lung cancer or colorectal cancer.

[0019] Compared with the prior art, the beneficial effects of the present application are:

[0020] 1. The present application first discloses the relationship between low PTPRT expression and the efficacy of immune checkpoint therapy for lung cancer. Specifically, low PTPRT expression significantly increases the progression-free survival (PFS) of lung cancer patients receiving immune checkpoint therapy; low PTPRT expression significantly increases the objective response rate (ORR) of lung cancer patients receiving immune checkpoint therapy; low PTPRT expression significantly increases the infiltration of CD8+ T cells in tumor samples of lung cancer patients. Inhibiting PTPRT improves the tumor immune microenvironment of lung cancer patients. This indicates that low PTPRT expression is a new biomarker for predicting the efficacy of immune checkpoint therapy for lung cancer patients.

[0021] 2. The present application finds that low PTPRT expression is independent of PD-L1 expression, and low PTPRT expression does not affect PD-L1 expression, which can be used as a supplement to PD-L1 expression.

[0022] 3. In this application, by constructing a mouse model (preclinical model), it was 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 prolonged the survival of mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Low expression of PTPRT is related to the efficacy of lung cancer immune checkpoint therapy. Figure 1 In A, immunohistochemical staining pictures of PTPRT (-、+、2+、3+) in tumor biopsy samples. The scale bar is 100μm; Figure 1 In B, analysis of progression-free survival of lung cancer patients with PTPRT expression levels and immunotherapy; Figure 1 In C, ROC curve of PTPRT low expression predicting the efficacy of immune checkpoint; Figure 1 In D, analysis of objective response rate of lung cancer patients with PTPRT expression levels and immunotherapy; Figure 1 In E, analysis of PD-L1 expression levels of lung cancer patients with PTPRT expression levels and immunotherapy; Figure 1 In F, analysis of the correlation between PTPRT low and high expression groups and CD8+ T cell infiltration.

[0024] Figure 2 It is 37 cases of external validation data. Figure 2 In A, analysis of progression-free survival of lung cancer patients with PTPRT expression levels and immunotherapy; Figure 2 In B, ROC curve of PTPRT low expression predicting the efficacy of immune checkpoint.

[0025] Figure 3 Combined inhibition of PTPRT expression synergistically enhances the effect of immunotherapy in a subcutaneous tumor model of lung cancer. Figure 3 In A, changes in related proteins after knocking out PTPRT in LLC; Figure 3 In B, changes in the fluorescence intensity of PD-L1 in cells after knocking out PTPRT in LLC detected by flow cytometry; Figure 3 In C, changes in the tumor volume curve; Figure 3 In D, changes in tumor size at the end point; Figure 3 In E, the proportion of tumor-infiltrating CD8+ T cells.

[0026] Figure 4 Combined inhibition of PTPRT expression synergistically enhances the effect of immunotherapy in an orthotopic tumor model of lung cancer. Figure 4 In A, results of in vivo fluorescence imaging of tumors; Figure 4 In B, changes in tumor fluorescence at the end point; Figure 4 In C, survival rate of mice with orthotopic tumorsFigure 4 In which, D is the proportion of tumor-infiltrating CD8+ T cells.

[0027] Figure 5 It is for synergistically enhancing the effect of immunotherapy by jointly inhibiting the expression of PTPRT in a subcutaneous tumor model of colorectal cancer in animals. Figure 5 In which, A is the change of related proteins after knocking out PTPRT in MC38; Figure 5 In which, B is the change of the tumor volume curve; Figure 5 In which, C is the change of tumor size at the end point; Figure 5 In which, D is the proportion of tumor-infiltrating CD8+ T cells. Detailed implementation manners

[0028] In order to make the invention purpose, technical solutions and beneficial effects of this application clearer, the following further illustrates this application in combination with embodiments. It should be understood that the embodiments are only used to explain this application and are not used to limit the scope of the application. The test methods used in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the technologies or conditions described in the literature in this field or according to the product instructions. Those familiar with this technology can easily understand other advantages and effects of this application from the content disclosed in this description. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0029] This invention studied the clinical cohort of immune checkpoint therapy in Shanghai Chest Hospital and found that the expression of receptor-type protein tyrosine phosphatase T (PTPRT) is highly correlated with the progression-free survival (PFS) of lung cancer patients after receiving immune checkpoint therapy. The low expression of PTPRT can be used as a biomarker for predicting the efficacy of immune checkpoint therapy for lung cancer. As a target for predicting the efficacy of immune checkpoint therapy for lung cancer, inhibiting PTPRT can effectively improve the tumor immunosuppressive microenvironment by increasing the infiltration of tumor CD8+ T cells.

[0030] This application provides an application of the expression level of PTPRT 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.

[0031] In the application provided by this application, when the staining intensity of the expression level of PTPRT in the sample in immunohistochemical staining < 1%, it is predicted that the efficacy of immune checkpoint therapy for cancer is good; when the staining intensity of the expression level of PTPRT in the sample in immunohistochemical staining > 1%, it is predicted that the efficacy of immune checkpoint therapy for cancer is poor.

[0032] PTPRT is Receptor protein tyrosine phosphatase T. It is known in the prior art that PTPRT gene mutations are somewhat correlated with the prognosis of immunotherapy for lung cancer. This application for the first time discloses the relationship between low expression of PTPRT protein and the efficacy evaluation of immune checkpoint inhibitors in the treatment of lung cancer, specifically manifested as follows: low expression of PTPRT significantly increases the infiltration of CD8+ T cells in lung cancer tumors; low expression of PTPRT significantly increases the progression-free survival (PFS) of lung cancer patients; low expression of PTPRT significantly increases the objective response rate (ORR) of lung cancer patients; low expression of PTPRT significantly increases the infiltration of CD8+ T cells in tumor samples of lung cancer patients. This indicates that low expression of PTPRT is a brand-new biomarker for predicting the efficacy of immune checkpoint inhibitors in lung cancer patients.

[0033] In the application provided by this application, lung cancer includes non-small cell lung cancer. The expression level of PTPRT is the expression level of wild-type PTPRT.

[0034] In the application provided by this application, immune checkpoint treatment of lung cancer includes using immune checkpoint-related drugs; further, immune checkpoint-related drugs include immune checkpoint inhibitors and / or immune checkpoint activators; furthermore, immune checkpoint inhibitors include one or a combination of more than one 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 this application, the immune checkpoint-related drug is selected from PD-1 inhibitors. Immune Checkpoint is expressed on immune cells and can regulate the degree of immune activation, and they play an important role in preventing the occurrence of autoimmune effects. Immune checkpoint immunotherapy is a treatment method that regulates the activity of T cells to kill tumor cells through a series of pathways such as co-inhibitory or co-stimulatory signals.

[0035] In the application provided by this application, the lung cancer tumor microenvironment includes, but is not limited to, the lung cancer tumor immune microenvironment.

[0036] In the application provided by 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.

[0037] In the application provided by this application, low expression of PTPRT means that in immunohistochemical staining, a staining intensity < 1% is considered low expression.

[0038] 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.

[0039] In the use provided by the present 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, renal cancer, urothelial cancer, cervical cancer, endometrial cancer, ovarian cancer, malignant melanoma, malignant lymphoma, or skin cancer. Preferably, the cancer is lung cancer or colorectal cancer.

[0040] In the use provided by the present application, the substance for detecting the expression level of PTPRT includes a reagent for detecting the expression level of PTPRT protein, detecting the content of PTPRT protein, or detecting the expression level of PTPRT RNA. Specifically, the reagent includes an antibody, polypeptide, protein, or nucleic acid molecule that binds to the PTPRT protein.

[0041] The reagent for detecting PTPRT can specifically bind to a specific site on PTPRT, and does not specifically bind to other genes other than PTPRT, and the reagent may optionally carry a detectable signal. The method of using an antibody that specifically binds to PTPRT to detect the expression of PTPRT in an analyte is also a technique well known to those skilled in the art.

[0042] In a specific embodiment of the present application, the substance for detecting the expression level of PTPRT is an antibody, specifically a monoclonal antibody against PTPRT.

[0043] The third aspect of the present application provides a kit, including a substance for detecting the expression level of PTPRT, and the kit has at least one of the following uses:

[0044] 1) Predict the efficacy of immune checkpoint therapy for lung cancer;

[0045] 2) Evaluate the efficacy of immune checkpoint therapy for lung cancer.

[0046] The kit includes: primers for specifically amplifying the PTPRT gene, probes for specifically recognizing the PTPRT gene, antibodies or ligands that specifically bind to the RNA encoded by the PTPRT gene, or antibodies or ligands that specifically bind to the protein encoded by the PTPRT gene. In addition, the kit may also include various reagents required for DNA extraction, RNA extraction, PCR, hybridization, color development, etc., including but not limited to: extraction solution, amplification solution, hybridization solution, enzyme, control solution, color development solution, washing solution, etc.

[0047] 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 judged to predict or evaluate the efficacy of immunotherapy for lung cancer. Specifically, when the staining intensity of PTPRT in the immunohistochemical staining of the sample is <1%, it is determined as low expression, predicting good efficacy of immunotherapy for lung cancer; when the staining intensity of PTPRT in the immunohistochemical staining of the sample is >1%, it is determined as high expression, predicting poor efficacy of immunotherapy for lung cancer.

[0048] The fourth aspect of the present application provides the use of a PTPRT inhibitor in the preparation of a product, and the product has at least one of the following effects:

[0049] 1) Increase the infiltration of CD8+ T cells in lung cancer or colorectal cancer tumors;

[0050] 2) Increase the survival period of lung cancer patients;

[0051] 3) Improve the immune microenvironment of lung cancer or colorectal cancer tumors;

[0052] 4) Improve the efficacy of immunotherapy-related drugs in the treatment of lung cancer or colorectal cancer.

[0053] PTPRT inhibitors include inhibitors, antagonists, blockers, blocking agents, etc., and these terms can be used interchangeably.

[0054] In the use provided by the present application, the PTPRT inhibitor includes a substance that reduces the expression level or content of PTPRT protein, or a substance that inhibits the expression of the PTPRT gene. Further, the substance that inhibits the expression of the PTPRT gene includes a substance that knocks out or silences PTPRT; furthermore, the substance that knocks out or silences PTPRT includes: a CRISPR gene editing system targeting PTPRT, an interfering molecule that specifically interferes with the expression of the coding gene of PTPRT, and a homologous recombination substance targeting loss-of-function mutations of PTPRT.

[0055] In some embodiments, the CRISPR gene editing system targeting PTPRT can use the CRISPR / Cas (such as Cas9) system for targeted gene editing to knockout the PTPRT gene in the targeted disease region. Common methods for knocking out the PTPRT gene include: co-transfecting the sgRNA or nucleic acid capable of forming sgRNA, Cas9 mRNA or nucleic acid capable of forming Cas9 mRNA into the targeted region or targeted cells. After determining the target site, known methods can be used to introduce the 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 the Cas9 mRNA is a nucleic acid construct or expression vector. These expression vectors are introduced into the cell to form active sgRNA and Cas9 mRNA in the cell. As a particularly preferred embodiment of the present invention, the CRISPR gene editing system targeting PTPRT includes sgRNA, and the coding DNA sequence of the sgRNA is as shown in SEQ ID NO:1.

[0056] In the application provided by this application, the immune checkpoint-related drugs include immune checkpoint inhibitors and / or immune checkpoint activators. Further, the 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; 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. In a specific embodiment of this application, the immune checkpoint-related drug is selected from PD-1 inhibitors.

[0057] In the application provided by this application, it is found that knocking out PTPRT in combination with immune checkpoint inhibitors significantly inhibits tumor growth and tumor size; knocking out PTPRT significantly improves the tumor immune microenvironment of mice and increases the infiltration of CD8+ T cells; knocking out PTPRT in combination with immune checkpoint inhibitors significantly increases the survival period of mice.

[0058] 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 foregoing use. The immune checkpoint-related drug includes an immune checkpoint inhibitor and / or an immune checkpoint activator. Further, the immune checkpoint inhibitor includes one or a combination of more than one of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIM-3 inhibitor, a LAG3 inhibitor, and a TIGIT inhibitor; the immune checkpoint activator includes one or a combination of two or more of a CD27 activator, a CD40 activator, an OX40 activator, a GITR activator, a CD137 activator, a CD28 activator, and an ICOS activator. In a specific embodiment of the present application, the immune checkpoint-related drug is selected from a PD-1 inhibitor.

[0059] The pharmaceutical composition provided by the present application further includes a pharmaceutically acceptable carrier and / or excipient.

[0060] "Pharmaceutically acceptable" means that when the molecular entity and the composition are appropriately administered to an animal or a human, they do not produce adverse, allergic, or other untoward reactions.

[0061] "Pharmaceutically acceptable carrier or excipient" should be compatible with the immune checkpoint inhibitor and the PTPRT inhibitor, that is, it can be blended with them without significantly reducing the efficacy of the drug under normal circumstances. Specific examples of some substances that can be used as pharmaceutically acceptable carriers or excipients are sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium methylcellulose, ethyl cellulose, and methyl cellulose; 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; coloring agents; flavoring agents; tabletting agents, stabilizers, antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; and phosphate buffer solutions, etc. These substances are used as needed to help the stability of the formulation or to enhance the activity or its bioavailability or to produce an acceptable taste or odor in the case of oral administration.

[0062] The pharmaceutical composition provided by the present application can be adapted to any form of administration, which can be oral or parenteral administration. For example, it can be administered by inhalation, intranasally, rectally, and / or by intravenous injection. More specifically, it can be administered intradermally, subcutaneously, intramuscularly, intra-articularly, intraperitoneally, by inhalation, orally, sublingually, intranasally, transdermally, vaginally, orally, or parenterally.

[0063] Those skilled in the art can select a suitable dosage form according to the administration method. For example, the dosage forms suitable for oral administration can include, but are not limited to, pills, tablets, chewable tablets, capsules, granules, drops or syrups, etc. For another example, the dosage forms suitable for parenteral administration can include, but are not limited to, solutions, suspensions, reconstitutable dry preparations or sprays, etc. For still another example, those usually suitable for rectal administration can be suppositories.

[0064] 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.

[0065] The seventh aspect of the present application provides a method for treating lung cancer or colorectal cancer, which is characterized in that the aforementioned PTPRT inhibitor or the aforementioned pharmaceutical composition is administered to an object.

[0066] In the method for treating lung cancer provided by the present application, the object is a mammal. Mammals are, for example, rodents, artiodactyls, perissodactyls, lagomorphs, primates, etc. Primates are, for example, monkeys, apes or Homo sapiens.

[0067] The present application will be further illustrated by the following examples, but the scope of the present application is not limited thereby.

[0068] Example 1

[0069] Application of PTPRT in predicting the efficacy of immune checkpoint in lung cancer

[0070] Case selection in this example: This example is a retrospective clinical study. It is carried out in accordance with the principles of the Helsinki Declaration and the informed consent of all subjects has been obtained. This example mainly included lung cancer patients in stages IIIC / IV who received immune checkpoint inhibitor treatment at the Shanghai Chest Hospital from January 2020 to June 2022, and the follow-up date ended on December 31, 2022. The treatment methods received were anti-PD-1 treatment or anti-PD-1 treatment plus chemotherapy.

[0071] The efficacy evaluation of patients during the treatment was carried out according to the Response Evaluation Criteria in Solid Tumors, version 1.1 (RECIST version 1.1). The 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 immunosuppressive therapy to disease progression or death in patients. The objective response rate was the proportion of patients whose tumor volume shrank by 30% and could maintain the minimum time limit requirement, which was the sum of the proportions of complete response (CR, that is, the tumor completely disappeared) and partial response (PR, that is, the tumor shrank by 30% or more), excluding stable disease (SD).

[0072] The samples were subjected to immunohistochemical staining to detect the expression percentage of PTPRT. The IHC staining intensity was divided into: -, <1%; +, 1% - 10%; ++, 10% - 50%; +++, >50%.

[0073] Specific operations:

[0074] a. Baking the slices: Place the paraffin sections on a 60°C baking machine for 30 min.

[0075] b. Dewaxing and rehydrating the sections: Prepare 3 cylinders of xylene (pass through 3 times), quickly place the baked paraffin sections into the glass cylinders, soak in each concentration for 5 min, then pass the sections through absolute ethanol twice, 2 min each time, and then successively through 95%, 90%, 80%, 70% ethanol, stay in each concentration for 5 min, and finally soak the sections in PBS buffer 3 times, 3 min each time.

[0076] c. Fixing the sections: Soak the sections that have completed dewaxing and rehydrating in 4% PFA solution for 10 min, and then soak the sections in PBS buffer 3 times, 3 min each time.

[0077] d. Membrane permeabilization: Soak the fixed sections in 0.3% Triton-100 for 10 min, and then soak the sections in PBS buffer 3 times, 3 min each time.

[0078] e. Removing redox enzymes: Soak the membrane-permeabilized sections in 3% hydrogen peroxide solution for 10 min, aiming to remove endogenous redox enzymes in the tissue. Then soak the sections in PBS buffer 3 times, 3 min each time.

[0079] f. Blocking: After removing the oxidoreductase from the sections, dry the liquid around the tissue on the glass slide. Use an immunohistochemistry pen to draw a circle around the tissue to prevent the liquid added in subsequent steps from flowing out of the section tissue. Place the section in a light-proof wet box, and then add a blocking solution containing 10% goat serum to the section tissue. After leaving it at room temperature for 1 h, aspirate the blocking solution on the section tissue, and then soak the section in PBS buffer three times, 3 min each time.

[0080] g. Primary antibody incubation: Dilute the primary antibody (product number AF3697 from R&D) according to the instructions of the antibody diluent, and then drop the primary antibody on the surface of the tissue that has been blocked. Incubate overnight in a 4°C refrigerator.

[0081] h. Secondary antibody incubation: Aspirate the primary antibody liquid on the surface of the section, and then soak the section in PBS buffer three times, 3 min each time. Drop the secondary antibody corresponding to the species of the primary antibody (product number 705 - 005 - 003 from Jackson ImmunoResearch) on the section tissue. After incubating at room temperature for 1 h, soak the section in PBS buffer three times, 3 min each time.

[0082] i. DAB color development: Drop DAB color development solution on the section tissue. It is advisable to first conduct a preliminary experiment to confirm the appropriate reaction time under a microscope and then carry out the formal experiment. Generally, the staining time is 1 - 5 min. Finally, gently rinse the section with slow running tap water to terminate the color development.

[0083] j. Staining the cell nuclei: Put the section after DAB color development into hematoxylin solution for counterstaining to make the cell nuclei of all cells stained (blue). Generally, staining at room temperature for 2 - 5 min is sufficient. Finally, gently rinse the section with slow running tap water to terminate the color development.

[0084] k. Sealing the slide: After completing the above steps, the slide needs to be sealed for long - term preservation. Gradient dehydrate the slide in 70%, 80%, 90%, 95%, 100% ethanol for 2 min each, repeat dehydration in 100% ethanol once, and then soak it in xylene twice, 5 min each time. Drop a small amount of neutral resin on the section tissue, cover with a coverslip, and avoid generating air bubbles. Finally, let the slide dry thoroughly in a fume hood, place it in a slide box for subsequent observation and photography.

[0085] 1. Survival curve

[0086] The results are as Figure 1 shown in A below. According to the expression of PTPRT in patients, they were divided into patients with low PTPRT expression (-) and patients with high PTPRT expression (+, ++, +++). The results are as Figure 1As shown in B and D, the progression-free survival time of the patients was plotted to draw the survival curve and the objective response rate. The analysis results indicated that at the same follow-up time, the progression-free survival rate of patients with low PTPRT expression was significantly increased (16.3 m vs. 5.9 m, P = 0.037), and at the same time, the objective response rate was also significantly increased (66.7% vs. 27.3%, P = 0.036). Figure 1 The ROC curve in C showed that low PTPRT expression had high specificity and sensitivity in predicting the efficacy of immune checkpoint (AUC = 0.742, P = 0.0367).

[0087] Another 37 cases of other data were used for external validation: Case selection in this example: This example was a retrospective clinical study. This study was conducted in accordance with the principles of the Declaration of Helsinki and informed consent of all subjects had been obtained. This example mainly included lung cancer patients in stage IIIC / IV who received PD-1 inhibitor treatment at Shanghai Chest Hospital from March 2019 to June 2022, and the follow-up date ended on March 31, 2023. The treatment methods received were anti-PD-1 treatment or anti-PD-1 treatment plus chemotherapy.

[0088] The efficacy evaluation of the patients during the treatment was carried out according to the Response Evaluation Criteria in Solid Tumors, version 1.1 (RECIST version 1.1). Progression-free survival (PFS) was defined as the time from the start of immunosuppressive treatment to disease progression or death of the patients. Immunohistochemical staining was performed on the samples to detect the expression percentage of PTPRT, and the IHC staining intensity was divided into: -, <1%; +, 1% - 100%.

[0089] The results were as Figure 2 shown. 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 high specificity and sensitivity.

[0090] Figure 2 The ROC curve in B showed that low PTPRT expression had high specificity and sensitivity in predicting the efficacy of immune checkpoint (AUC = 0.776, P = 0.0091).

[0091] 2. Relationship between PTPRT expression and tumor immune microenvironment

[0092] The proportion of tumor PD-L1 and CD8+ T cell infiltration were evaluated according to the results of immunohistochemical staining. PD-L1 was scored using the common 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 as Figure 1 shown in D-E of Figure 1 . There was no statistical difference in the expression level of PD-L1 between the low-expression of PTPRT group and the high-expression of PTPRT group (P = 0.516), while the proportion of CD8+ T cell infiltration in patients with low-expression of PTPRT increased (P = 0.0002).

[0093] Based on the above results, it can be known that the efficacy of immune checkpoint inhibitors in patients with lung cancer to be tested can be predicted or assisted in prediction by detecting the expression level of PTPR in the tumor tissue of the patients with lung cancer to be tested. The judgment criteria are as follows:

[0094] The efficacy of immune checkpoint inhibitors in the low-expression of PTPRT group was better than or potentially better than that in the high-expression of PTPRT group. Therefore, PTPRT can be used as a biomarker to evaluate or assist in evaluating the efficacy of immune checkpoint inhibitors.

[0095] Example 2

[0096] The combination of PTPRT inhibitor and immune checkpoint inhibitor can synergistically enhance the anti-lung cancer effect of immunotherapy

[0097] The PTPRT inhibitor can be a substance that inhibits the expression of the PTPRT gene, silences or knocks out the PTPRT gene, or a substance that inhibits or reduces the content and / or activity of the PTPRT protein.

[0098] 1. Selection of cell lines and animals

[0099] The mouse LLC cell line used in this study was derived from the American Type Culture Collection (ATCC).

[0100] Six-week-old female C57BL / 6 mice were used in this study. All the mice used in the experiment were housed in a specific pathogen-free (SPF) environment. All the operations in the study were strictly in accordance with the regulations formulated by the Animal Ethics Committee.

[0101] 2. Construction of LLC cells with knocked-out PTPRT

[0102] According to the PTPRT gene sequence and the CRISPR-Cas9 principle, the PTPRT gene was knocked out in LLC cells. The coding DNA sequence of the sgRNA designed to knock out the PTPRT gene was: 5’-CAGCAACTGCGGGTATAGCG-3’ (SEQ ID NO:1), and the coding DNA sequence of the control sgRNA was: 5’-GAACAGTCGCGTTTGCGACT-3’ (SEQ ID NO:2). After lentivirus transfection of LLC cells with PTPRT knockout and blank control, LLC cells with PTPRT knockout and control were obtained. The changes in cell PTPRT and PD-L1 were detected by immunoblotting (Western blotting), and the changes in cell membrane surface PD-L1 were detected by flow cytometry.

[0103] 3. Subcutaneous xenograft tumor model of mouse lung cancer

[0104] LLC cells with PTPRT knockout and control (one million) were subcutaneously injected into the right back of wild-type C57BL / 6 female mice (6 weeks old). The tumor volume was calculated using the following formula: (width diameter * width diameter * length diameter) / 2. Anti-PD-1 treatment (BE0273, BioXcell) and IgG isotype control (BE0089, BioXcell) were administered intraperitoneally at 150 μg each time, starting from day 9, once every 3 days, and tumors were collected on day 21 for flow analysis. When collecting tumors, they were dissected after anesthesia with carbon dioxide to reduce animal suffering.

[0105] The results were as follows: The protein expression level of PTPRT in LLC cells with PTPRT knockout decreased significantly, while the protein level of PD-L1 did not change significantly ( Figure 3 in A). Flow cytometry detection found that the fluorescence intensity of PD-L1 on the cell membrane surface of LLC cells with PTPRT knockout did not change significantly ( Figure 3 in B). In the subcutaneous xenograft tumor model of mouse lung cancer, after treatment with PTPRT knockout combined with anti-PD-1 monoclonal antibody, the tumor growth rate and size were significantly lower than those in the control treatment group (anti-PD-1 monoclonal antibody group) and the knockdown group (PTPRT knockout group) ( Figure 3 in C and D), and at the same time, the infiltration ratio of CD8+ T cells increased ( Figure 3 in E).

[0106] The above results indicate that knocking out PTPRT or inhibiting PTPRT expression does not affect the expression of PD-L1 in tumor cells. Knocking out PTPRT or inhibiting PTPRT expression synergistically with anti-PD-1 monoclonal antibody 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.

[0107] Example 3

[0108] The combination therapy of PTPRT inhibitor and immune checkpoint inhibitor can synergistically enhance the anti-lung cancer effect of immunotherapy

[0109] 1. Selection of cell lines and animals

[0110] The mouse LLC-luc cell line used in this study was derived from the American Type Culture Collection (ATCC) cell resource center

[0111] Six-week-old female C57BL / 6 mice were used in this study. All the mice used in the experiments were housed in a specific pathogen-free (SPF) environment. All the operations in the study were strictly in accordance with the regulations established by the Animal Ethics Committee

[0112] 2. Construction of LLC-luc cells with PTPRT knockout

[0113] According to the PTPRT gene sequence and the CRISPR-Cas9 principle, the PTPRT gene was knocked out in LLC cells. The coding DNA sequence of the sgRNA designed to knock out the PTPRT gene was: 5’-CAGCAACTGCGGGTATAGCG-3’ (SEQ ID NO:1), and the coding DNA sequence of the control sgRNA was: 5’-GAACAGTCGCGTTTGCGACT-3’ (SEQ ID NO:2). After lentiviral transfection of LLC cells with PTPRT knockout and blank control, LLC cells with PTPRT gene knockout and control were obtained

[0114] 3. Mouse orthotopic transplantation tumor model of lung cancer

[0115] Anesthetize mice with isopentane using an animal gas anesthetic machine. Make a 3-mm incision at the dorsal side of the left lung of the mouse, 0.5 cm below the scapula. After separating the subcutaneous tissue and muscle, the movement of the lungs can be observed. Use an insulin syringe (BD) to directly inject a cell suspension (Matrigel:PBS = 1:4) of 50 μl in total volume of stably transfected cells, namely the LLC cells (eight hundred thousand) with the PTPRT gene knocked out and the control obtained in step 2, into the left lung, and suture the incision. Perform in vivo fluorescence imaging on the 5th day to observe the modeling situation. Anti-PD-1 treatment (BE0273, BioXcell) and IgG isotype control (BE0089, BioXcell) are administered intraperitoneally at 150 μg each time. Administration starts on the 9th day, once every 3 days, and in vivo fluorescence imaging is performed at the end of the experiment on the 21st day. For the survival experiment, anti-PD-1 treatment (BE0273, BioXcell) and IgG isotype control (BE0089, BioXcell) are administered intraperitoneally at 150 μg each time starting from the 7th day after modeling until the mice die. Anti-CD8α antibody (BE0061, BioXCell) at 200 μg is injected intraperitoneally twice a week starting from 7 days before modeling.

[0116] The results are as follows: In the mouse orthotopic transplantation tumor model of lung cancer, after knocking out PTPRT in combination 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 4 in A and B), and at the same time, the infiltration ratio of CD8+ T cells increased ( Figure 4 in C). At the same time, knocking out PTPRT in combination with anti-PD-1 monoclonal antibody significantly prolonged the overall survival period of the mice ( Figure 4 in D).

[0117] The above results indicate that knocking out PTPRT or inhibiting PTPRT expression synergistically with anti-PD-1 monoclonal antibody can significantly inhibit tumor growth, prolong the survival period, increase the infiltration of CD8+ T cells in tumors, and enhance the anti-tumor effect of immunotherapy.

[0118] Example 4

[0119] The combination treatment of PTPRT inhibitor and immune checkpoint inhibitor can synergistically enhance the anti-colorectal cancer effect of immunotherapy

[0120] The PTPRT inhibitor can be a substance that inhibits PTPRT gene expression, silences or knocks out the PTPRT gene, or a substance that inhibits or reduces the content and / or activity of the PTPRT protein.

[0121] 1. Selection of cell lines and animals

[0122] The mouse MC38 cell line used in this study was derived from the American Type Culture Collection (ATCC).

[0123] Six-week-old female C57BL / 6 mice were used in this study. All the mice used in the experiments were housed in a specific pathogen-free (SPF) environment. All procedures in this study were strictly in accordance with the regulations established by the Animal Ethics Committee.

[0124] 2. Construction of MC38 cells with PTPRT knockout

[0125] According to the PTPRT gene sequence and the CRISPR-Cas9 principle, the PTPRT gene was knocked out in MC38 cells. The coding DNA sequence of the sgRNA designed to knock out the PTPRT gene was: 5’-CAGCAACTGCGGGTATAGCG-3’ (SEQ ID NO:1), and the coding DNA sequence of the control sgRNA was: 5’-GAACAGTCGCGTTTGCGACT-3’ (SEQ ID NO:2). After lentivirus transfection of MC38 cells with PTPRT knockout and blank control, MC38 cells with PTPRT knockout and control were obtained.

[0126] 3. Subcutaneous xenograft tumor model of mouse colorectal cancer

[0127] MC38 cells with PTPRT knockout and control (one million) were subcutaneously injected into the right back of wild-type C57BL / 6 female mice (6 weeks old). The tumor volume was calculated using the following formula: (width diameter * width diameter * length diameter) / 2. Anti-PD-1 treatment (BE0273, BioXcell) and IgG isotype control (BE0089, BioXcell) were administered intraperitoneally at a dose of 150 μg each time. Administration started on the 9th day, once every 3 days, and tumors were collected on the 21st day for flow cytometry analysis. When collecting tumors, the animals were sacrificed by carbon dioxide anesthesia and then dissected for sampling to reduce animal suffering.

[0128] The results were as follows: The protein expression level of PTPRT in MC38 cells with PTPRT knockout decreased significantly ( Figure 5 in A). In the subcutaneous xenograft tumor model of mouse colorectal cancer, after treatment with PTPRT knockout combined with anti-PD-1 monoclonal antibody, the tumor growth rate and size were significantly lower than those in the control treatment group (anti-PD-1 monoclonal antibody group) and the knockdown group (PTPRT knockout group) ( Figure 5 in B and C), and at the same time, the infiltration ratio of CD8+ T cells increased ( Figure 5 in D).

[0129] Knocking out PTPRT or inhibiting PTPRT expression synergistically with anti-PD-1 monoclonal antibody 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.

[0130] In summary, the predictive biomarker developed in the present invention can maximize the efficacy of immune checkpoint inhibitors, screen out the population that can benefit from immune checkpoint therapy, and inhibiting PTPRT can expand the responsive population of immune checkpoint therapy, bringing survival benefits to more patients.

[0131] The above embodiments are only illustrative of the principles and effects of the present invention and are not intended to limit this application. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of this application. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of this application.

Claims

1. Use of the expression level of PTPRT in predicting or evaluating the efficacy of cancer immunotherapy checkpoint.

2. The use according to claim 1, wherein, 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.

3. The use according to claim 1, wherein, when the staining intensity of PTPRT in immunohistochemical staining in the sample is <1%, it is determined as low expression, predicting good efficacy of cancer immunotherapy checkpoint; when the staining intensity of PTPRT in immunohistochemical staining in the sample is >1%, it is determined as high expression, predicting poor efficacy of cancer immunotherapy checkpoint.

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

5. Use of a substance for detecting the expression level of PTPRT in the preparation of a product for evaluating or predicting the efficacy of cancer immunotherapy checkpoint.

6. The use according to claim 5, wherein, 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 level of PTPRT includes a reagent for detecting the expression level of PTPRT protein, detecting the content of PTPRT protein, or detecting the expression level of PTPRT RNA.

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

8. A kit, the kit includes a substance for detecting the expression level of PTPRT, and the kit has at least one of the following uses: 1) Predicting the efficacy of lung cancer immunotherapy checkpoint; 2) Evaluating the efficacy of lung cancer immunotherapy checkpoint.

9. Use of a PTPRT inhibitor in the preparation of a product, the product having at least one of the following effects: 1) Increase the infiltration of CD8+ T cells in lung cancer or colorectal cancer tumors; 2) Increase the survival period of lung cancer patients; 3) Improve the immune microenvironment of lung cancer or colorectal cancer tumors; 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, wherein, the PTPRT inhibitor includes a substance that reduces the expression level or content of the PTPRT protein, or a substance that inhibits the expression of the PTPRT gene. Further, the substance that inhibits the expression of the PTPRT gene includes a substance that knocks out or silences PTPRT; Still further, the substance that knocks out or silences PTPRT includes: a CRISPR gene editing system targeting PTPRT, an interfering molecule that specifically interferes with the expression of the coding gene of PTPRT, and a homologous recombination substance for loss-of-function mutations of PTPRT; and / or, the immune checkpoint-related drug includes an immune checkpoint inhibitor and / or an immune checkpoint activator; preferably, the immune checkpoint inhibitor includes one or a combination of more of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIM-3 inhibitor, a LAG3 inhibitor, and a TIGIT inhibitor; the immune checkpoint activator includes one or a combination of two or more of a CD27 activator, a CD40 activator, an OX40 activator, a GITR activator, a CD137 activator, a CD28 activator, and an ICOS activator; more preferably, the immune checkpoint-related drug is selected from PD-1 inhibitors.

11. The use according to claim 10, wherein, the CRISPR gene editing system targeting PTPRT includes sgRNA, and the coding DNA sequence of the sgRNA is as shown in SEQ ID NO:

1.

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

13. The pharmaceutical composition according to claim 12, wherein, the immune checkpoint-related drug includes an immune checkpoint inhibitor and / or an immune checkpoint activator; preferably, the immune checkpoint inhibitor includes one or a combination of more of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIM-3 inhibitor, a LAG3 inhibitor, and a TIGIT inhibitor; the immune checkpoint activator includes one or a combination of two or more of a CD27 activator, a CD40 activator, an OX40 activator, a GITR activator, a CD137 activator, a CD28 activator, and an ICOS activator; more preferably, the immune checkpoint-related drug is selected from PD-1 inhibitors.

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

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