A method and kit for predicting the efficacy of tumor immunotherapy by DIAPH1 protein

Abstract

The present invention provides a method and a kit for predicting the efficacy of tumor immunotherapy by using DIAPH1 protein. The method includes the following steps: using DIAPH1 protein as a molecular marker, and combining with immunohistochemical reagents by using a DIAPH1 monoclonal antibody or a polyclonal antibody to detect the expression level of DIAPH1 protein in tumor tissues, and predicting the efficacy of immunotherapy for patients according to the expression level of DIAPH1 protein in tumor tissues. The present invention uses DIAPH1 protein as a protein molecular marker for judging the efficacy of immunotherapy for patients. Compared with the expression of PD-L1, the expression of DIAPH1 has a more significant predictive value for immunotherapy, and has important guiding significance for whether patients are suitable for immunotherapy.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly, to a method and kit for predicting the efficacy of tumor immunotherapy by DIAPH1 protein. Background Art

[0002] In the past decade or so, immunotherapy targeting immune checkpoints has opened up a new path for the treatment of malignant tumors. Currently, immunotherapy drugs represented by PD-1 / PD-L1 inhibitors have been widely used in the treatment of various malignant tumors such as lung cancer, breast cancer, malignant melanoma, and Hodgkin lymphoma. The expression of tumor cell PD-L1 is currently considered to be the most reasonable biomarker for the selection of advantageous populations for anti-PD-1 / PD-L1 therapy. The results of multiple clinical trials of advanced lung cancer all support that the positive expression of tumor cell PD-L1 is related to its efficacy and prognosis.

[0003] However, a large amount of clinical practice has also found that there are certain deficiencies in the use of the expression level of tumor cell PD-L1 as a biomarker for predicting the efficacy of immunotherapy. The CheckMate017 and OAK studies have found that patients with negative PD-L1 expression can also benefit from immunotherapy. The Keynote407 study published at ASCO in 2018, which compared Pembrolizumab combined with chemotherapy with chemotherapy alone for metastatic squamous cell carcinoma, showed benefits in all cases of different PD-L1 TPS expression. The HRs for PD-L1 <1%, 1% - 50%, and ≥50% were 0.61, 0.57, and 0.64, respectively.

[0004] The reasons for the defects of PD-L1 as an immunotherapy biomarker may include: (1) The expression of PD-L1 is regulated by multiple mechanisms. On the one hand, it includes the signaling pathways during the carcinogenesis of tumor cells themselves. On the other hand, inflammatory response cytokines can not only lead to the expression of PD-L1 in tumor cells, but also stimulate the expression of PD-L1 in other types of cells in the immune microenvironment; (2) PD-L1 can be expressed in both tumor cells and immune cells, and the detected PD-L1 level may be the expression level of tumor cells, immune cells, or both; (3) Different antibodies for detecting PD-L1 expression, different detection platforms, and different evaluation systems have different positive cut-off values, making it difficult to form a consistent standard to measure the expression of tumor cell PD-L1.

[0005] In actual work, we found that the expression of PD-L1 shows an obvious skewed distribution in the patient population, with more cases of negative expression. However, the proportion of patients who cannot benefit from immunotherapy clinically is significantly less than the proportion of negative expression. In summary, the expression of PD-L1 is still an imperfect biomarker for predicting the efficacy of tumor immunotherapy.

[0006] Formin is a class of cytoskeleton-related proteins that are mainly involved in the process of monomeric actin polymerization into linear filaments. All formin proteins have two characteristic domains: the FH2 domain, which catalyzes actin polymerization; and the FH1 domain, which binds profilin to enable actin to form the FH2 domain. A large number of studies have shown that most formin proteins play the role of oncogenes in malignant tumors and promote tumor metastasis by mediating cytoskeletal remodeling.

[0007] Mammalian diaphanous-related formins homology 1 (DIAPH1) is the most abundant member of the formin protein family in almost all tumor types and is significantly highly expressed in tumor tissues. In the past two decades, scholars have studied the biological functions of DIAPH1 in various tumors. The results have shown that knocking down DIAPH1 significantly inhibits the proliferation, migration, and invasion of tumor cells and promotes their apoptosis. A large number of studies have confirmed that DIAPH1 is an oncogene in a variety of malignant tumors. At the molecular mechanism level, in addition to regulating cytoskeletal remodeling, DIAPH1 is also involved in mediating a variety of key oncogenic signals, including the EGF signaling pathway, TGFβ1 signaling pathway, AGE signaling pathway, etc.

[0008] Currently, there are no reports on any research related to DIAPH1 and tumor immunity, especially the lack of application of DIAPH1 protein in the preparation of a kit for predicting the efficacy of tumor immunotherapy. Summary of the Invention

[0009] Object of the Invention: The object of the present invention is to provide a method and a kit for predicting the efficacy of tumor immunotherapy through DIAPH1 protein.

[0010] Technical Solution: The present invention provides a method for predicting the efficacy of tumor immunotherapy through DIAPH1 protein, including the following steps:

[0011] Using DIAPH1 protein as a molecular marker, and using a DIAPH1 monoclonal antibody or polyclonal antibody, in combination with immunohistochemical reagents, to detect the expression level of DIAPH1 protein in tumor tissues, and predicting the efficacy of immunotherapy for patients based on the expression level of DIAPH1 protein in tumor tissues.

[0012] Preferably, the immunotherapy is anti-PD-1 immunotherapy. The tumor type is not limited. The specific drugs for PD-1 immunotherapy include camrelizumab, pembrolizumab, and sintilimab. The specific drug application methods include single drug and combined chemotherapy.

[0013] Preferably, the method for predicting the efficacy of tumor immunotherapy by DIAPH1 protein comprises the following steps:

[0014] (a) Immunohistochemically stain tumor tissue sections;

[0015] (b) Take digital photos using a microscope and an imaging device;

[0016] (c) Analyze the positive signal intensity and staining range of tumor tissue using biological image processing software;

[0017] (d) Calculate the relative expression level of DIAPH1 protein in tumor tissue according to the scoring rule, set a standard value. When the score is lower than or equal to the standard value, it is low expression of DIAPH1 protein, and the patient is not sensitive to immunotherapy and the clinical benefit is uncertain; when the immunohistochemical score is higher than the standard value, it is high expression of DIAPH1 protein, and the patient is sensitive to immunotherapy and the clinical benefit is significant.

[0018] More preferably, in step (d), the scoring rule is as follows: Staining intensity scoring standard: negative 0 points, weakly positive 1 point, moderately positive 2 points, strongly positive 3 points; Scoring standard for the proportion of positive cells: 0 points: <1%, 1 point: 1%-25%, 2 points: 26%-50%, 3 points: 51%-75%, 4 points: >76%; Multiply the staining intensity score by the scoring for the proportion of positive cells to obtain the immunoreactivity score (IRS), ranging from 0 to 12 points (note: only tumor cells in the field of view are included when predicting cell staining intensity and staining range); The standard value is 2 points.

[0019] The present invention also provides a kit for predicting the efficacy of tumor immunotherapy, which kit comprises the following:

[0020] DIAPH1 monoclonal antibody or polyclonal antibody and immunohistochemical reagents.

[0021] Preferably, the immunohistochemical reagents comprise the following:

[0022] 2-4% H2O2 solution, 0.5-1.5% BSA blocking solution, DAB chromogenic reagent, hematoxylin, horseradish peroxidase-labeled goat anti-mouse IgG, xylene, ethanol.

[0023] The present invention finally also provides the application of the kit in predicting the efficacy of tumor immunotherapy.

[0024] Preferably, the application comprises the following steps:

[0025] Using the DIAPH1 protein as a molecular marker, and using the kit to detect the expression level of the DIAPH1 protein in tumor tissues, and predicting the efficacy of immunotherapy for patients based on the expression level of the DIAPH1 protein in tumor tissues.

[0026] Preferably, the immunotherapy is anti-PD-1 immunotherapy. The tumor type is not limited. The specific drugs for PD-1 immunotherapy include camrelizumab, pembrolizumab, and sintilimab. The specific application methods of the drugs include single-agent and combined chemotherapy.

[0027] Beneficial effects: Through extensive and in-depth research, the applicant has first discovered that detecting the expression level of DIAPH1 in the tissues of tumor patients undergoing immunotherapy in the advanced stage by immunohistochemistry can determine the sensitivity of patients to immunotherapy. Based on this correlation between the expression level of the DIAPH1 protein and tumor immunotherapy, using this protein as a molecular marker and detecting its expression level can be used as a predictive indicator for the efficacy of tumor immunotherapy. Compared with the expression of PD-L1, the expression of DIAPH1 has a more significant predictive value for immunotherapy.

[0028] Based on the above findings, the present invention provides a method and a kit for predicting the efficacy of tumor immunotherapy through the DIAPH1 protein. This method uses the DIAPH1 protein as a molecular marker, and uses monoclonal or polyclonal antibodies against DIAPH1, as well as corresponding immunohistochemical reagents, to analyze the expression level of the DIAPH1 protein in tumor tissues and predict the sensitivity of patients to immunotherapy.

[0029] The discovery of the correlation between the DIAPH1 protein of the present invention and the efficacy of tumor immunotherapy provides a new biomarker for predicting the efficacy of immunotherapy clinically. When the score is less than or equal to 2, it is low expression of the DIAPH1 protein, and the patient is not sensitive to immunotherapy, and the clinical benefit is uncertain. When the immunohistochemical score is higher than 2, it is high expression of the DIAPH1 protein, and the patient is sensitive to immunotherapy, and the clinical benefit is significant. Therefore, detecting the expression of DIAPH1 has important guiding significance for assisting in judging the efficacy of immunotherapy for advanced tumor patients. Description of the Drawings

[0030] Figure 1Correlation of DIAPH1 with immune-related genes, PD-L1 expression, and T cell inflammation score. (A) DIAPH1 is positively correlated with immune regulatory genes in most tumor types, * indicates a statistically significant correlation; (B) DIAPH1 is positively correlated with PD-L1 expression in most tumor types; (C) DIAPH1 is positively correlated with T cell inflammation score in most tumor types; (D, E) In 11 tumor tissues, DIAPH1 is positively correlated with PD-L1 expression. Note: Immunohistochemical detection of DIAPH1 and PD-L1 was performed on the multi-tumor tissue microarray HOrgC120PG04, which was purchased from Shanghai Biochip Co., Ltd. and includes 11 common cancers, with 1 - 6 cases of each type.

[0031] Figure 2 Expression of DIAPH1 and PD-L1 in tumor patients with different treatment responses in three public datasets. (A) DIAPH1 expression was significantly different in the GSE91061 dataset; (B) PD-L1 expression was not significantly different in the GSE91061 dataset; (C) DIAPH1 expression was significantly different in the GSE78220 dataset; (D) PD-L1 expression was not significantly different in the GSE78220 dataset; (E) There was a statistically significant trend in DIAPH1 expression in the GSE165252 dataset; (F) PD-L1 expression was not significantly different in the GSE165252 dataset. Note: * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, ns represents no statistical difference.

[0032] Figure 3 CT example images, DIAPH1 and PD-L1 immunohistochemical staining example images, and expression differences in patients with different treatment responses. (A) CT images of cases represented by PD, SD, PR; (B) DIAPH1 and PD-L1 immunohistochemical staining of cases represented by PD, SD, PR; (C) DIAPH1 expression in PD, SD, PR cases, and the expression of DIAPH1 was significantly different in tumor patients with different treatment responses; (D) PD-L1 expression in PD, SD, PR cases, and the expression of PD-L1 was significantly different in tumor patients with different treatment responses, but not as statistically significant as DIAPH1. Note: * represents P < 0.05, ** represents P < 0.01, ns represents no statistical difference.

[0033] Note: The efficacy was defined according to the RECIST 1.1 standard, specifically as follows: ① Complete remission (CR): All target lesions and non-target lesions disappeared, and tumor markers were normal; ② Partial remission (PR): The sum of the longest diameters of the baseline lesions decreased by ≥ 30%; ③ Stable disease (SD): The reduction did not reach PR or the increase did not reach PD, and one or more non-target lesions and / or marker abnormalities; ④ Progressive disease (PD): The sum of the longest diameters of the baseline lesions increased by ≥ 20% or new lesions appeared, or / and non-target lesions progressed.

[0034] Figure 4 For the predictive value of DIAPH1 and PD-L1 on the efficacy and prognosis of immunotherapy. (A) Relationship between DIAPH1 expression and treatment response, DIAPH1 expression has a significant predictive value for immunotherapy; (B) Correlation between DIAPH1 expression and prognosis, patients with high DIAPH1 expression have a longer overall survival after immunotherapy; (C) Relationship between PD-L1 expression and treatment response, PD-L1 expression has no significant predictive value for immunotherapy; (D) Correlation between PD-L1 expression and prognosis, there is no significant difference in overall survival among patients with different PD-L1 expression status after immunotherapy. Generally speaking, compared with PD-L1 expression, DIAPH1 expression has a more significant predictive value for immunotherapy. Note: *** represents P < 0.001, ns represents no statistical difference; referring to the expression pattern of PD-L1 and the existing scoring criteria, when IRS is 0, PD-L1 is low expression, and the rest are high expression.

[0035] Note: The specific names represented by the abbreviations of the tumors in the figure are shown in the following table.

[0036]

[0037] Specific implementation manners

[0038] The following detailed description of the specific implementation manners provided by the present invention will be given in combination with the embodiments.

[0039] The kit prepared by the method of the present invention has the following formulation: including DIAPH1 monoclonal antibody or polyclonal antibody, 3% H2O2 solution, 1% BSA blocking solution, DAB chromogenic reagent, hematoxylin, horseradish peroxidase-labeled goat anti-mouse IgG, xylene, ethanol.

[0040] The above kit was used to carry out relevant experiments. Implementation examples:

[0041] Thirty-five puncture or previous surgical samples collected from patients with various types of advanced tumors before immunotherapy at Wuxi People's Hospital Affiliated to Nanjing Medical University were used to make and cut paraffin tissue sections with a thickness of 4 mm for immunohistochemical staining. The specific steps are as follows:

[0042] (1) Dewaxing of sections: Xylene I① for 10 min → Xylene II② for 10 min → Xylene III③ for 10 min → 100% ethanol for 5 min → 95% ethanol for 5 min → 85% ethanol for 5 min → 75% ethanol for 5 min → double-distilled water for 5 min

[0043] (2) Inactivation of enzymes: Add 3% H₂O₂ solution dropwise to the pre-treated sections and place at room temperature for 20 min;

[0044] (3) Wash with double-distilled water for 5 min × 3;

[0045] (4) Antigen retrieval: Place the sections in 1× EDTA antigen retrieval solution (pH 9.0) and boil for 30 min;

[0046] (5) Cool naturally to room temperature and wash with double-distilled water for 5 min × 3;

[0047] (6) Block with 1% BSA for 30 min at 37°C;

[0048] (7) Discard the blocking solution without washing, and directly add the primary antibody (mouse DIAPH1 monoclonal antibody, purchased from Santa Cruz Biotechnology, dilution ratio 1:500). Place in a wet box and incubate in a 4°C refrigerator overnight for 16 hours;

[0049] (8) Take out from 4°C, warm to room temperature for 15 min, and then wash with 0.01M PBS for 5 min × 4;

[0050] (9) Add the secondary antibody (horseradish peroxidase-labeled goat anti-mouse IgG, purchased from Jiangsu KeyGen Biotech Co., Ltd., dilution ratio 1:200) and incubate for 45 min at 37°C;

[0051] (10) Wash with 0.01M PBS for 5 min × 4, develop with DAB for 2 - 10 min, and observe under the microscope;

[0052] (11) Stop the color development with double-distilled water and counterstain with hematoxylin for 10 seconds;

[0053] (12) Differentiate and then return to blue with tap water, soak in distilled water;

[0054] (13) Dehydrate and clarify, cover with a coverslip;

[0055] (14) Observe positive staining under the microscope, randomly select 3 fields of view in renal clear cell carcinoma tissue and take pictures;

[0056] (15) Semi - quantitative determination was performed by comprehensively considering the staining intensity and the proportion of positive cells under a high - power microscope. The specific scoring is as follows:

[0057] 1. Scoring criteria for the staining intensity of tumor cells: negative is 0 points, weakly positive is 1 point, moderately positive is 2 points, and strongly positive is 3 points.

[0058] 2. Scoring criteria for the proportion of tumor positive cells: 0 points: <1%, 1 point: 1% - 25%, 2 points: 26% - 50%, 3 points: 51% - 75%, 4 points: >76%.

[0059] 3. The staining intensity score is multiplied by the score of the proportion of positive cells to obtain the IRS score, with a range of 0 - 12 points.

[0060] The expression of DIAPH1 protein in tumor tissues was detected by the above - mentioned experimental steps of immunohistochemistry. When the score is less than or equal to 2 points, it indicates low expression of DIAPH1 protein, the patient is not sensitive to immunotherapy, and the clinical benefit is uncertain. When the immunohistochemistry score is higher than 2 points, it indicates high expression of DIAPH1 protein, and the patient is sensitive to immunotherapy with significant clinical benefit.

[0061] The test results are as Figures 1-4 shown. From the above test results, it can be seen that by using immunohistochemistry to detect the expression level of DIAPH1 protein molecule, the results show that there is a significant correlation between the expression level of DIAPH1 protein and the efficacy of immunotherapy in patients. Therefore, using DIAPH1 protein as a molecular marker and detecting its expression level can be used to predict the efficacy of immunotherapy in patients. Correspondingly, antibodies specific to DIAPH1 protein, including monoclonal antibodies and polyclonal antibodies, can be used to prepare a kit for predicting the efficacy of tumor immunotherapy.

[0062] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art can make various equivalent variations or substitutions without departing from the spirit of the present invention, and these equivalent variations or substitutions are all included within the scope defined by the claims of this application.

Claims

1. Use of a kit in the preparation of a reagent for predicting the efficacy of tumor immunotherapy, characterized in that, The kit includes DIAPH1 monoclonal antibody or polyclonal antibody and immunohistochemistry reagents; the efficacy of the tumor immunotherapy is the efficacy of anti-PD-1 immunotherapy for clear cell renal carcinoma.

2. The use according to claim 1, characterized in that, The immunohistochemistry reagents include the following: 2-4% H2O2 solution, 0.5-1.5% BSA blocking solution, DAB chromogenic reagent, hematoxylin, horseradish peroxidase-labeled goat anti-mouse IgG, xylene, ethanol.

3. The use according to claim 1, characterized in that, The application includes the following steps: Using DIAPH1 protein as a molecular marker, and using the kit to detect the expression level of DIAPH1 protein in tumor tissues, and predicting the efficacy of immunotherapy for patients according to the expression level of DIAPH1 protein in tumor tissues.

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