Biomarker for predicting stomach cancer PD-1 monoclonal antibody immunotherapy effect and application thereof

Abstract

The invention provides an application of a biomarker in predicting a gastric cancer immunotherapy effect and a related product. The biomarker is clustered protein. The gastric cancer treatment effect of the anti-PD-1 antibody can be predicted by detecting the expression level of clusterin or mRNA thereof in a gastric cancer tumor cell or tumor tissue sample. Furthermore, by inhibiting the expression level of clusterin, the gastric cancer treatment effect of the anti-PD-1 antibody can be improved. A new biomarker cluster protein is provided for predicting the gastric cancer treatment effect, and the cluster protein can be used as a novel target spot of the gastric cancer, so that a new direction is provided for screening drugs for treating the gastric cancer and treating the gastric cancer.

Description

Technical Field

[0001] This application belongs to the field of biomedicine and involves the application of biomarkers in predicting the effect of immunotherapy for gastric cancer and related products. Background Art

[0002] In recent years, tumor immunotherapy has made significant progress in the treatment of advanced malignancies and has become a hot topic in medical research. Among these, checkpoint inhibitor-based immunotherapy, particularly immune checkpoint blockade targeting the programmed death receptor-1 (PD-1) on the surface of T cells and the programmed death receptor ligand 1 (PD-L1) on tumor cells, has become a reliable and highly effective cancer treatment. This treatment approach blocks the PD-1 / PD-L1 interaction, reactivating the immune killing effect of T cells against tumor cells, thereby inhibiting tumor growth and metastasis.

[0003] However, while PD-1 / PD-L1 immune checkpoint blockade has achieved significant efficacy in some patients, responses to immune checkpoint inhibitors vary significantly among patients. This variability may stem from individual differences in patient genetics, tumor type, tumor microenvironment, and other factors. Therefore, optimizing checkpoint immunotherapy to achieve precise treatment for each patient remains a pressing challenge in the field of tumor immunotherapy. Summary of the Invention

[0004] In order to overcome the deficiencies of the prior art, one of the objectives of the present application is to provide a reagent for detecting biomarkers for use in the preparation of a product for predicting the effect of immunotherapy for gastric cancer.

[0005] The second purpose of this application is to provide a product for predicting the effect of immunotherapy for gastric cancer.

[0006] The third object of the present application is to provide a reagent for inhibiting clusterin expression for use in the preparation of a drug for enhancing the effect of anti-PD-1 antibodies in treating gastric cancer.

[0007] The first aspect of the present application provides: use of a reagent for detecting a biomarker in preparing a product for predicting the effect of immunotherapy for gastric cancer, wherein the biomarker is clusterin.

[0008] In some embodiments, the reagent is used to detect the expression level of clusterin or its mRNA in a sample.

[0009] In some embodiments, the gastric cancer immunotherapy is anti-PD-1 antibody therapy for gastric cancer.

[0010] In some embodiments, the sample is a gastric cancer tumor cell or tumor tissue sample.

[0011] In some embodiments, the expression level of the clusterin protein or its mRNA in gastric cancer tumor cells or tumor tissue samples is negatively correlated with the efficacy of anti-PD-1 antibody immunotherapy in gastric cancer patients.

[0012] The second aspect of the present application provides: a product for predicting the effect of immunotherapy for gastric cancer, wherein the product comprises a reagent for detecting biomarkers as described in the first aspect of the present application.

[0013] In some embodiments, the product is used to detect the expression level of the biomarker.

[0014] In some embodiments, the product is a kit.

[0015] The third aspect of the present application provides the use of an agent for inhibiting clusterin expression in the preparation of a drug for enhancing the effect of anti-PD-1 antibodies in treating gastric cancer.

[0016] In some embodiments, the anti-PD-1 antibody is camrelizumab.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] The present application provides an application of a biomarker in predicting the effect of immunotherapy for gastric cancer and related products. The biomarker is clusterin. By detecting the expression level of clusterin or its mRNA in gastric cancer tumor cells or tumor tissue samples, the effect of anti-PD-1 antibodies in treating gastric cancer can be predicted. Furthermore, inhibiting the expression level of clusterin can enhance the effect of anti-PD-1 antibodies in treating gastric cancer. The present application provides a new biomarker clusterin for predicting the effect of gastric cancer treatment, and clusterin can be used as a new target for gastric cancer, providing a new direction for screening drugs for treating gastric cancer and treating gastric cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The expression levels of CLU in AGS, BGC-823, HGC-27, MGC-803, MKN-1, NCI-N87, and MKN-45 cells are shown;

[0020] Figure 2 A shows the expression levels of CLU in MKN-1, AGS, and NCI-N87 cells and the changes in their expression levels after siRNA knockdown; Figure 2 B shows that before and after CLU knockdown, camrelizumab enhances the growth inhibition rate of T cells against MKN-1, AGS, and NCI-N87 cells in vitro;

[0021] Figure 3 A shows the Western Blot method to verify the CLU knockout situation, Figure 3 B. Figure 3 C shows the tumor inhibition of carrelizumab in humanized tumor-bearing mice transplanted with MKN-1 cells in vivo before and after CLU knockout. DETAILED DESCRIPTION

[0022] Below, in conjunction with the accompanying drawings and specific embodiments, the present application is further described. It should be noted that, under the premise of no conflict, the various embodiments described below or the various technical features can be arbitrarily combined to form new embodiments. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0023] In order to optimize checkpoint immunotherapy and achieve precise treatment for patients, the development of predictive biomarkers is particularly important. Predictive biomarkers refer to specific biological indicators that can predict a patient's response to immunotherapy. These markers can help doctors screen for the groups that will benefit from immunotherapy, thereby more accurately selecting treatment options. Currently, PD-L1 protein expression is one of the most commonly used biomarkers for PD-1 / PD-L1 monoclonal antibody immunotherapy, but its sensitivity and specificity are limited, and the reproducibility of the test results is a challenge. Therefore, there is an urgent need to discover more effective predictive markers for immunotherapy effects that are applicable to a variety of cancers.

[0024] The inventors have discovered that CLU (Clusterin), as an important cancer regulatory factor, has demonstrated its important role in tumor progression. CLU can control a variety of cancer-related cellular events, including cancer cell proliferation, stemness, survival, metastasis, epithelial-mesenchymal transition (EMT), treatment resistance, and inhibition of programmed cell death, thereby supporting cancer growth and recurrence. The inventors also found that CLU can further promote tumor growth, metastasis, EMT, inflammation, and resistance to chemotherapy by activating survival pathways in cancer cells and cancer stem cells (CSCs). Therefore, exploring the role of CLU in tumor immunotherapy and its potential as a predictive biomarker for immunotherapy has important scientific significance and clinical application value.

[0025] Therefore, developing effective biomarkers to predict the effectiveness of immunotherapy, especially biomarkers based on cancer regulatory factors such as CLU, is of great significance for optimizing tumor immunotherapy and achieving precision treatment for patients. At the same time, this also provides new ideas and methods for future tumor immunotherapy research.

[0026] The application of the cluster protein of the present application in predicting the effect of gastric cancer immunotherapy and the product for detecting the expression level of cluster protein are further described below with reference to the accompanying drawings.

[0027] In this application, the term "high level of CLU expression in gastric cancer tissue" means gastric cancer with a CLU expression score of 3+ in immunohistochemistry (IHC), and gastric cancer with a CLU expression score of 2+ in IHC and CLU expression determined to be high in in situ hybridization (ISH). The in situ hybridization method includes fluorescence in situ hybridization (FISH) and dual-color in situ hybridization (DISH).

[0028] In the present application, the term "low-level CLU expression in gastric cancer tissue" is not particularly limited, as long as it is recognized by those skilled in the art as gastric cancer with low CLU expression. Preferred examples of gastric cancer with low CLU expression include gastric cancer with a CLU expression score of 2+ in IHC and low CLU expression determined by in situ hybridization, and gastric cancer with a CLU expression score of 1+ in IHC.

[0029] In this application, the term "anti-PD-1 antibody" refers to an antibody that specifically binds to PD-1 (programmed death receptor 1). Specifically, the anti-PD-1 antibody refers to camrelizumab. Furthermore, high levels of CLU expression in gastric cancer tissue indicate poor immunotherapy efficacy for gastric cancer, while low levels of CLU expression in gastric cancer tissue indicate good immunotherapy efficacy for gastric cancer.

[0030] In the present application, the term "reagent for detecting CLU expression" is not particularly limited, as long as it can detect the expression level of CLU protein or mRNA. For example, reagents for detecting CLU expression can include reagents used in the following methods: Western blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay, immunohistochemical staining, mass spectrometry, immunoprecipitation analysis, complement fixation assay, flow cytometry fluorescence separation technology, and protein chip method.

[0031] In the present application, the reagents for inhibiting CLU expression include one or more nucleic acid molecules, carbohydrates, lipids, small molecule compounds, antibodies, polypeptides, proteins, gene editing vectors, lentiviruses or adeno-associated viruses that inhibit CLU expression. The inhibition of CLU expression can be achieved by gene mutation, gene silencing, gene knockout, gene editing or gene knockdown techniques well known to those skilled in the art. For example, RNA interference (RNAi) technology can be used to specifically eliminate or shut down the expression of a specific gene; the tools using gene editing technology can be CRISPR / Cas9 technology, zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs) technology, etc., but are not limited thereto. The gene knockdown technology includes RNA interference, Morpholino interference, antisense nucleic acids, ribozymes or dominant negative inhibitory mutations, but are not limited thereto. Utilizing shRNA or siRNA expressed by viruses (such as lentiviruses, adeno-associated viruses) to inhibit gene expression, gene silencing is well known to those skilled in the art. In some embodiments, the aforementioned nucleic acid molecules may include sgRNA, shRNA, microRNA, siRNA and / or antisense oligonucleotides.

[0032] Example 1

[0033] Identification of CLU expression in gastric cancer cell lines

[0034] AGS, BGC-823, HGC-27, MGC-803, MKN-1, NCI-N87, and MKN-45 cells were cultured in RPMI-1640 or DMEM supplemented with 10% fetal bovine serum at 37°C and 5% CO2. Cells were grown as monolayers and harvested when they reached approximately 80% confluence. Total protein was extracted, and Western blot analysis was used to verify CLU expression in all gastric cancer cell lines.

[0035] The expression results of CLU in the above gastric cancer cells are as follows Figure 1 As shown by Figure 1 It can be seen that the expression level of CLU is highest in MKN-1 cells, moderate in AGS cells, and lowest in NCI-N87 cells. Therefore, this application subsequently selected these three gastric cancer cell lines as research subjects.

[0036] Example 2

[0037] Effects of low CLU expression

[0038] siRNA was used to knock down the expression level of CLU in AGS, MKN-1, and NCI-N87 cells. The CLU siRNA interference sequence was 5'-CCAGACGGUCUCAGACAAUTT-3' (sense strand); 5'-AUUGUCUGAGACCGUCUGGTT-3' (antisense strand). The knockdown process was as follows: (1) Cell plating: After treating AGS, MKN-1, and NCI-N87 cells in good condition, an appropriate amount of cells were evenly plated in a culture dish. During transfection, the transfection effect was best when the cell density was maintained at 40% to 50%.

[0039] (2) Cell transfection: After ensuring that the cells are completely attached to the culture dish, discard the culture medium and replace it with 2 mL of serum-free culture medium. During the operation, please use a sterile and RNase-free pipette tip. First, prepare the siRNA system: 50 μL blank culture medium + 4 μL siRNA, and use a pipette to thoroughly mix the system. Next, prepare the transfection reagent system: 50 μL blank culture medium + 6 μL H4000 transfection reagent, mix well and let the two systems stand for 5 minutes. Finally, add the transfection reagent system to the siRNA system, mix well, incubate for 15 minutes, and then add it to the culture dish.

[0040] (3) Change the medium: Decide whether to change the medium based on the condition of the cells after transfection. Generally speaking, the medium can be changed 4 to 6 hours after transfection. If the cells are in good condition, overnight transfection can be selected.

[0041] (4) Verification: 48 hours after transfection, the cells can be collected, part of which can be used for upstream and downstream experiments, and part of which can be used for WB verification of the transfection results.

[0042] like Figure 2 As described in A, this example successfully knocked down the expression of CLU protein in AGS and MKN-1. However, due to the low expression of CLU in NCI-N87, the knockdown effect was not obvious.

[0043] CLU knockdown AGS, MKN-1, and NCI-N87 cells were digested and collected using a mixture of 0.25% trypsin and 0.02% EDTA. Cell counts were performed and each cell was diluted to 3 × 104 cells / mL with fresh RPMI-1640 medium. 100 μL / well was seeded into 96-well plates. Six replicate wells were plated and cultured in a 37°C, 5% CO2, saturated humidity incubator. After 24 hours of culture, the medium was replaced, and T cells were added to each well at a 5:1 effector-target ratio. Camrelizumab treatment was then added. Each cell type was assigned to a blank control group (no cells), a negative control group (tumor cells + cell culture medium), a non-treated group (tumor cells + T cells), a Camrelizumab (100 μg / mL) treatment group (tumor cells + T cells + Camrelizumab).

[0044] After 72 hours of incubation, discard the culture medium from each well, gently wash away any remaining T cells and dead cells with PBS, add 10 μL of CCK8 solution and 90 μL of culture medium to each well, and continue incubation for another 2 hours. Measure the absorbance (A450) of each well at 450 nm using a microplate reader. Calculate the cell growth inhibition rate according to the following formula:

[0045]

[0046] The results are as follows Figure 2 As shown in B, after knocking down CLU, carrelizumab can further enhance the killing ability of T cells against MKN-1 and AGS, but has no such effect on NCI-N87 cells that themselves lowly express CLU.

[0047] According to the results of Example 2, the expression level of CLU is correlated with the therapeutic effect of anti-PD-1 antibodies. After knocking down CLU, PD-1 antibodies further enhance the killing ability of T cells against tumor cells.

[0048] Example 3

[0049] Knockout of CLU enhances the therapeutic efficacy of anti-PD-1 antibodies in an in vivo humanized mouse model

[0050] In this example, a sgRNA sequence specific for the CLU gene was designed and constructed. The CLU sgRNA sequence is GCACAGCAGGAGAATCTTCA. This sgRNA sequence was then cloned into a stable expression plasmid, and the plasmid containing the sgRNA was packaged into viral particles using a viral packaging system. The virus was transfected into MKN-1 cells, followed by puromycin selection and single-clone screening to generate CLU-knockout MKN-1 cells.

[0051] The knockout process of CLU is as follows: (1) MKN-1 cell plating: cells in good condition are evenly seeded in a 96-well plate so that the confluence is 40%-50% during virus transfection.

[0052] (2) Virus infection: Based on the cell MOI = 100, prepare the virus infection system: 20 μL virus + 40 μL complete culture medium + 2 μL infection enhancement solution A / P, and add them to the corresponding 96-well plate.

[0053] (3) Fluid exchange: The decision to change the fluid depends on the state of the cells after transfection. Usually, within 12 to 24 hours after viral infection, the viral infection fluid that infected the cells should be discarded and 0.5 mL of the corresponding complete culture medium should be added to continue the culture.

[0054] (4) Culture: Within 48 to 72 hours after virus infection, observe the expression of green fluorescent protein under an inverted fluorescence microscope. If green fluorescent protein expression can be observed, it means that the infection is successful.

[0055] (5) Puromycin screening: Taking advantage of the puromycin resistance gene carried by viral vectors, we set up a gradient of different puromycin concentrations to screen target cells. For example, for MKN-1 cells, the puromycin concentration gradient was set to 2 μg / mL. The target cells were cultured in a culture system containing puromycin until they showed stable growth and no cell death. Then, the cells were switched to complete medium for further culture.

[0056] (6) Result verification: After collecting some of the screened cells and extracting their total protein, the CLU knockout was verified by Western Blot. The results are as follows: Figure 3 As shown in A.

[0057] Fifty female 4-5-week-old NSG mice were anesthetized and injected subcutaneously into the left forelimb axilla with 200 μL of gastric cancer cell line MKN-1 cells at a cell concentration of 2 × 10 7 / mL, and observe the tumor growth and general condition of the mice every two days after inoculation to understand the formation time and growth of the tumor, and observe the general activity and nutritional status of the mice. Use a vernier caliper to measure the long diameter of the tumor every two days. When the diameter of the tumor grows to 5-7mm, it indicates that the model is successful. 40 mice with successful modeling were screened for humanization, and human peripheral blood mononuclear cells (PBMCs) were injected into each mouse through the tail vein. Each mouse was inoculated with 1×10 7 One week later, the humanized mice were randomly divided into 4 groups, with 10 mice in each group, namely MKN-1+PBS group, MKN-1+carrellis beads group, MKN-1-sgRNA+PBS group, and MKN-1-sgRNA+carrellis beads group.

[0058] The dosages for each group were as follows: MKN-1+PBS group: intravenous injection of an equal volume of normal saline; MKN-1+carrelizumab group: intravenous injection of an equal volume of carrelizumab (5 mg / kg); MKN-1-sgRNA+PBS group: intravenous injection of an equal volume of normal saline; MKN-1-sgRNA+carrelizumab group: intravenous injection of an equal volume of carrelizumab (5 mg / kg).

[0059] Each group was dosed twice a week for 2 consecutive weeks. The mice's condition, such as weight, appetite, and mental state, was observed daily. Tumor volume was measured every 2 days. Tumors were removed and weighed after 2 weeks. The tumor volume was calculated using the formula:

[0060]

[0061] The formula for calculating the tumor inhibition rate is:

[0062]

[0063] The results are as follows Figure 3 B. Figure 3 As shown in C, after CLU knockout, the tumor growth rate slowed down and the therapeutic effect of carrelizumab was further improved, further proving that inhibiting the expression of CLU can significantly enhance the therapeutic effect of anti-PD-1 antibodies on gastric cancer.

[0064] The above-mentioned embodiments are only preferred embodiments of the present application and cannot be used to limit the scope of protection of the present application. Any non-substantial changes and replacements made by technicians in this field based on the present application shall fall within the scope of protection required by the present application.

Claims

1. Use of a reagent for detecting a biomarker in the preparation of a product for predicting the effect of immunotherapy for gastric cancer, characterized in that: The biomarker is clusterin.

2. The use according to claim 1, characterized in that The reagent is used to detect the expression level of clusterin or its mRNA in a sample.

3. The use according to claim 1, characterized in that The gastric cancer immunotherapy is gastric cancer anti-PD-1 antibody treatment.

4. The use according to claim 2, characterized in that The sample is a gastric cancer tumor cell or tumor tissue sample.

5. The use according to claim 4, characterized in that The expression level of the cluster protein or its mRNA in gastric cancer tumor cells or tumor tissue samples is negatively correlated with the efficacy of anti-PD-1 antibody immunotherapy in gastric cancer patients.

6. A product for predicting the effect of gastric cancer immunotherapy, characterized in that: The product comprises the reagent for detecting a biomarker according to any one of claims 1 to 5.

7. The product according to claim 6, characterized in that The product is used to detect the expression level of the biomarker.

8. The product according to claim 7, characterized in that The product is a test kit.

9. Use of an agent that inhibits clusterin expression in the preparation of a drug for enhancing the effect of anti-PD-1 antibodies in treating gastric cancer.

10. The use according to claim 9, characterized in that The anti-PD-1 antibody is carrelizumab.

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