Application of GLS2-based biomarkers in the diagnosis of tumor immunotherapy

Abstract

This invention provides the application of GLS2K151 acetylation, a modified form of glutamine synthase 2 (GLS2), as a biomarker in assessing or predicting the sensitivity of cancer patients to immunotherapy. Specifically, it provides a method for assessing the sensitivity of cancer patients to immunotherapy based on GLS2K151 acetylation, including detecting the expression level of GLS2K151 acetylation in samples from said cancer patients and assessing or predicting the sensitivity of cancer patients to immunotherapy based on the detection results. This invention also provides a kit for quantitative or semi-quantitative detection of proteins containing antibodies that specifically bind to GLS2K151 acetylation. Furthermore, this invention provides the application of antibodies against GLS2K151 acetylation in assessing or predicting the sensitivity of cancer patients to immunotherapy or in the preparation of cancer diagnostic kits. The applications described in this invention, by detecting the expression level of GLS2K151 acetylation, enable the diagnosis of cancer immunotherapy sensitivity.

Description

Technical Field

[0001] This invention relates to the application of GLS2-based biomarkers in the diagnosis of tumor immunotherapy, and belongs to the field of oncology medicine. Background Technology

[0002] Anti-PD-1 / PD-L1 therapy has shown promising clinical outcomes across various cancer types, but resistance to this therapy, including primary, adaptive, and acquired resistance, is prevalent in solid tumors. Aberrant expression of PD-L1 on the surface of cancer cells is a central mechanism by which many cancers evade antitumor immune responses, and PD-L1 expression serves as a predictive biomarker for a favorable response to immune checkpoint blockade. Although previous studies have reported increased PD-L1 expression in 13% to 72% of tumors in melanoma, gastric cancer, and non-small cell lung cancer patients, PD-L1 expression patterns are spatially heterogeneous and temporally dynamic. However, the factors regulating dynamic PD-L1 expression during tumorigenesis and development are not fully understood, and the exact underlying mechanisms remain to be elucidated.

[0003] The mechanisms of tumor development and progression are highly complex, and related research has many shortcomings, severely hindering the development of new diagnostic and therapeutic methods for tumors. Metabolism is a key factor influencing tumor development and progression. During tumor development, tumor cells undergo changes in their metabolic patterns, a phenomenon known as "metabolic reprogramming." As a crucial characteristic of tumors, metabolic reprogramming fulfills three key needs of tumor cells: the energy required for sustained growth, sufficient biomolecules for the synthesis of nucleic acids, proteins, and lipids, and the maintenance of redox homeostasis. Only through a more comprehensive and systematic understanding of the mechanisms of tumor metabolism can we identify biomarkers targeting tumor metabolism, overcome current bottlenecks in tumor diagnosis research, and truly achieve early diagnosis of tumors from the perspective of metabolic abnormalities.

[0004] In tumor cells, mutations in metabolic enzyme genes or changes in their expression levels lead to significant alterations in the levels of intracellular metabolites. This "classical" metabolic reprogramming plays a crucial role in tumor progression. However, increasing evidence in recent years suggests that some metabolic enzymes and small metabolic molecules can also participate in various important life processes of tumor cells through "non-classical" functions that are not dependent on metabolic pathways, thereby promoting tumor development and progression.

[0005] Given the above background, in order to more accurately determine tumor development, more rationally select treatment options, and thus improve the effectiveness of tumor immunotherapy, it is necessary to identify key biomarkers related to the immune escape mechanism of tumor cells and involved in the life process of tumor cells, so as to assess and predict the sensitivity of cancer patients to immunotherapy. Summary of the Invention

[0006] The technical problem or primary objective of this invention is to assess or predict the sensitivity of cancer patients to immunotherapy.

[0007] Another object of the present invention is to provide a product for assessing or predicting the sensitivity of cancer patients to immunotherapy.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In one aspect, the present invention uses the acetylation of the modified form of glutamine synthase 2 (GLS2) GLS2K151 as a biomarker to assess or predict the sensitivity of cancer patients to immunotherapy.

[0010] In the present invention, GLS2K151 acetylation refers to the acetylation of amino acid position 151 of GLS2, and the NCBI sequence number of GLS2 is NP_001267727.1.

[0011] Secondly, the present invention provides a method for assessing or predicting the sensitivity of cancer patients to immunotherapy, comprising detecting the expression level of GLS2K151 acetylation in a sample from the cancer patient, and assessing or predicting the sensitivity of the cancer patient to immunotherapy based on the detection results.

[0012] In a preferred embodiment of the present invention, the detection of the expression level of GLS2K151 acetylation in the sample from the tumor patient is achieved by ELISA or Western Blot method.

[0013] In a preferred embodiment of the present invention, the detection of the expression level of GLS2K151 acetylation in samples from the tumor patient is to detect the expression level of GLS2K151 acetylation in peripheral venous blood, adjacent normal tissue, and / or tumor tissue from the tumor patient.

[0014] In the scheme described in this invention, the assessment or prediction of the sensitivity of cancer patients to immunotherapy based on the detection results specifically means that: when the detection results show that GLS2K151 acetylation is expressed in the sample, the cancer patient is determined to be resistant to immunotherapy; otherwise, the cancer patient is not resistant to immunotherapy.

[0015] Thirdly, the present invention also provides an antibody, wherein the antibody is an antibody GLS2 AcK151 that can specifically bind to the acetylated GLS2K151.

[0016] Fourthly, the present invention also provides a kit for quantitative or semi-quantitative detection of proteins, containing the antibody described in the third aspect of the present invention.

[0017] In a preferred embodiment of the present invention, the reagent kit for quantitative or semi-quantitative detection of proteins is a reagent kit for detecting proteins by enzyme-linked immunosorbent assay (ELISA) or Western blotting; the reagent kit contains a first antibody and a second antibody; the first antibody is the antibody GLS2AcK151 described in the third aspect of the present invention, and the second antibody is an enzyme-labeled antibody homologous to the first antibody.

[0018] In a further preferred embodiment of the present invention, the kit further comprises one or more substances selected from the group consisting of: a container, instructions for use, a positive control, a negative control, a buffer, an adjuvant, or a solvent.

[0019] The instruction manual describes how to use the kit for testing and how to use the test results to assess tumor development and select treatment options.

[0020] Fifthly, the present invention also provides the use of the antibody described in the third aspect of the present invention in the preparation of a kit for tumor diagnosis.

[0021] In the applications described in this invention, the tumor can be any of the following tumor diseases: oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory system cancer, genitourinary system cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine system cancer, hematopoietic system cancer, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, kidney cancer, biliary system cancer, pheochromocytoma, islet cell carcinoma, Levi-Flaumeni tumor, thyroid cancer, parathyroid cancer, pituitary adenoma, adrenal adenoma, bone-derived sarcoma, neuroendocrine system tumor, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer.

[0022] This invention provides a novel application of the biomarker GLS2K151 acetylation in assessing the sensitivity of cancer patients to immunotherapy. By detecting the expression level of this biomarker in biological samples from patients, the sensitivity of individual cancer patients to immunotherapy can be diagnosed.

[0023] The GLS2K151 acetylation described in this invention, as a specific modification of GLS2, is highly correlated with the immune escape mechanism of tumor cells and participates in tumor cell life processes through non-metabolic pathways. Specifically, the inventors discovered that the hypoxic microenvironment of pancreatic cancer (PDAC) can induce acetylation at the K151 site of glutamine synthase 2 (GLS2). This acetylation can induce PD-L1 transcription, promoting immune escape from pancreatic cancer and thus reducing the sensitivity of pancreatic cancer to an-PD-1 monoclonal antibody therapy. Furthermore, in PDAC specimens, the acetylation level of GLS2 K151 is positively correlated with PD-L1 expression and CD4+ expression. + T, CD8 + T infiltration was negatively correlated with poor prognosis in PDAC patients. These findings reveal a key mechanism by which the hypoxic tumor microenvironment induces immune escape from tumor cells by promoting the non-metabolic function of PD-L1 expression through acetylation at the GLS2 K151 site, and highlight the potential of GLS2-targeted tumor immunotherapy for both diagnostic and therapeutic purposes.

[0024] The above findings were obtained through the following research:

[0025] 1. Materials and Methods

[0026] 1.1 Materials Used

[0027] (1) Cell types: Panc-1 cells and Capan-2 cells, from ATCC;

[0028] (2) Mice: 6-week-old C57BL / 6 mice;

[0029] (3) Patient Samples: Human PDAC and adjacent matched non-tumor tissue samples were obtained from the Second Affiliated Hospital of Zhejiang University School of Medicine. Clinical data were obtained by reviewing patients' medical histories. Pathological staging was assessed according to the 8th edition of the American Joint Committee on Cancer / International Federation for Cancer Control TNM Classification System.

[0030] (4) The target sequences of the shRNA used for gene knockdown are as follows: GLS2: 5′-CGCATCTTTGAGGATGTCAAA-3′ and 5′-CGGAATTATGCCATCGGCTAT-3′.

[0031] 1.2 Test Methods

[0032] (1) Detection of phosphorylated protein levels: This involves contacting a sample with an antibody that specifically binds to a phosphorylated peptide and determining the amount of antibody bound, for example, by detecting or measuring the path of the complex between the antibody and the peptide. The antibody may be labeled (radioactive, fluorescent, etc.) to facilitate the detection of the complex.

[0033] Systems used for detecting the above-mentioned peptide levels include: autoradiographic immunoassay (RIA), immunofluorescence, gel electrophoresis, and Western blotting.

[0034] Antibodies used (added): FLAG-Tag (14793S), HA-Tag (3724), Myc-Tag (2278), YAP1 (14074), pYAP1 (S127)(4911), 14-3-3 (8312), Pan-TEAD (13295), CTGF (86641), CYR61 (14479), GLS2 (85934s), GLS1 (88964S), LATS1 (3477), LATS2 (5888), H3 (4499)

[0035] Acetylated-Lysine (9441), Hif1-α (36169), GST-Tag (2622), CD4 (25229), CD8 (85336), Granzyme B (46890s), PD-L1 (human, 13684), PD-L1 (mouse, 64988), and β-Actin (4967) were purchased from CST. GLS2 AcK151 and YAP1 GluE100 specific antibodies were synthesized by Abclonal.

[0036] (2) RT-PCR: Total RNA was extracted using TRIzol reagent (Life Technologies), treated with RNased DNase I (Promega) for 30 minutes, and reverse transcribed using HiScript III-RT SuperMix for quantitative reverse transcription polymerase chain reaction (qPCR; Vazyme Biotech Co.). qPCR was performed using the ChamQ Universal SYBR qPCR kit (Vazyme Biotech Co., Ltd.) and an ABI 7500FAST detection system (Life Technologies). The expression levels of all samples were normalized to GAPDH expression levels.

[0037] The following primers are used for qRT-PCR:

[0038] CD274, 5′-CTGCACTTTTAGGAGATTAGATC-3′ and 5′-CTACACCAAGGCATAATAAGAT G-3′;

[0039] GAPDH, 5′-AGGGCATCCTGGGCTACAC-3′ and 5′-GCCAAATTCGTTGTCATACCAG-3′.

[0040] (3) ChIP Analysis: ChIP experiments were performed using the SimpleChIP Enzymatic Chromatin IP Kit (9003; CellSignaling Technology). Immunoprecipitated chromatin was analyzed using a real-time qPCR detection system. The PCR primer sequences for the CD274 promoter are as follows:

[0041] Positive: 5′-CATCGGATTACCACGCTGA-3′;

[0042] Reverse: 5′-TTCGTCCATTAGGCG-3′.

[0043] (4) Reporter gene assay: Cells were co-transfected in 24-well plates with firefly luciferase reporter plasmid and pRL-CMV plasmid using a Lipofectamine 3000 (Life Technologies). Luciferase activity was assessed 48 hours post-transfection using a dual-luciferase reporter gene assay system (Promega).

[0044] (5) Acquisition and analysis of mouse tumor samples: Six-week-old male immunocompetent mice were housed under specific pathogen-free conditions with a 14-hour / 10-hour light cycle, an ambient temperature of 21-23°C, and 50% humidity, and had free access to water and food. Panc02 cells expressing luciferase (3 × 10⁵) were suspended in 20 μl PBS and injected directly into the pancreas, or suspended in 50 μl PBS and injected subcutaneously into the liver of six-week-old male immunocompetent mice, with or without anti-PD-1 injection starting from day 2. For anti-PD-1 blockade, each mouse received 200 μg of anti-PD-1 antibody or control IgG2a intraperitoneally every 3 days. Bioluminescence imaging of in situ carcinomas was performed using an IVIS system (AniView 100, BiolightBiotechnology, Guangzhou, China). Subsequently, the animals were euthanized, and the tumors of each mouse were dissected, fixed in 4% formaldehyde, and embedded in paraffin. Tumor formation and phenotype were assessed by histological analysis of sections stained with hematoxylin and eosin. Tumor volume was calculated using the formula V = 1 / 2a²b (where V is the volume, a is the shortest diameter, and b is the longest diameter).

[0045] (6) Immunohistochemistry: After dewaxing paraffin sections, rehydration, and antigen retrieval, TMA slides were incubated overnight at 4°C with primary antibody rabbit anti-human acetylated GLS2K151 (dilution 1:200) or nonspecific IgG (as a negative control). The slides were then incubated with anti-rabbit secondary antibody (ready-to-use solution; CellSignaling Technology; #8114), followed by dichromatographic diaminobenzidine (DAB) staining (CellSignaling Technology) and hematoxylin staining, and fixed on xylene. The tissue slides were quantitatively scored under a microscope based on the percentage of positive cells and staining intensity. This invention assigns the following positive cell percentage scores: 0 points, 0% of cells are positive; 1 point, 0% to 1% of cells are positive; 2 points, 2% to 10% of cells are positive; 3 points, 11% to 30% of cells are positive; 4 points, 31% to 70% of cells are positive; and 5 points, 71% to 100% of cells are positive. Staining intensity is also rated on a scale of 0 to 3: 0, negative (i.e., no visible staining); 1, weak (very light color); 2, moderate (distinct color); 3, strong (very dark color). The positive cell percentage score and staining intensity grade are summed to obtain a total score (range 0-8). The reproducibility of the scoring system was independently verified by two pathologists without clinical knowledge.

[0046] (7) Overall survival Kaplan-Meier plotting was performed using SPSS version 20.0 (SPSS Inc., Chicago, Illinois, USA). Independent samples t-tests were used to compare biomarker expression levels in tumor and normal tissues. One-way ANOVA (posthoc-Bonferroni test) was used for multiple comparisons and minimum significance tests to assess the correlation between biomarker expression levels and patient clinicopathological features. Pearson correlation coefficients were used to analyze the correlation between biomarker expression levels. Overall survival (OS) was defined as the duration from diagnosis to death or the last follow-up. K-means clustering analysis was used to classify the expression levels of relevant biomarkers, Kaplan-Meier survival curves were plotted, log-rank tests were used to compare survival rates, and Cox regression models with two-way Wald tests were used to calculate hazard ratios (HR) and 95% confidence intervals (CIs) for survival analysis. Data reviewed were for patients who were alive at the last follow-up or who were lost at follow-up. Variables with p-values ​​less than 0.05 were included in multivariate analyses. A p-value < 0.05 is considered statistically significant. All statistical tests are two-sided.

[0047] 2 Results and Analysis

[0048] (1) AsFigure 1 As shown, after 12 hours of hypoxia treatment, immunoassay results of Panc-1 cells showed that hypoxia could induce the acetylation level of GLS2.

[0049] (2) Figure 2 As shown, Co-IP combined with mass spectrometry analysis revealed that the GLS2K151 site in Panc-1 cells underwent acetylation modification after hypoxia treatment.

[0050] (3) Inhibition of GLS2 K151 acetylation can suppress PD-L1 expression. In GLS2-deficient Panc-1 and Capan-2 cells, expression of rGLS2 WT or rGLS2-K151R, after 12 hours of hypoxia treatment, Western blot results showed that the GLS2 K151R mutation could inhibit hypoxia-induced PD-L1 expression. Figure 3 ), reporter gene experiments showed that the GLS2K151R mutation can inhibit the activity of the PD-L1 promoter region ( ), Figure 4 RT-PCR results showed that the GLS2 K151R mutation could inhibit hypoxia-induced PD-L1 mRNA transcription. Figure 5 ).

[0051] (4) Inhibition of GLS2 K151 acetylation can increase the sensitivity of xenografts to anti-PD-1 therapy. Panc02LUC cells were orally injected into 6-week-old male immunocompetent mice, and simultaneously treated with anti-PD-1. The results showed that GLS2 K151 acetylation decreased the sensitivity of xenografts to anti-PD-1 therapy, while GLS2 K151R mutation increased the sensitivity of xenografts to anti-PD-1 therapy. Figure 5 , Figure 6 Immunohistochemical and immunofluorescence experiments showed that acetylation at the GLS2K151 site could promote PD-L1 expression and reduce CD4+ expression. 4 T and CD 8 T cell recruitment promotes tumor immune escape. Figure 7 , Figure 8 ).

[0052] (5) GLS2K151 acetylation is associated with prognosis in pancreatic cancer patients. We analyzed human primary PDAC specimens using validated anti-GLS2-AcK151 immunohistochemical staining and found that the levels of these protein markers in PDAC tissues were significantly higher than in adjacent normal tissues. Figure 9 Furthermore, GLS2-AcK151 was observed to be positively correlated with PD-L1 expression, but negatively correlated with granzyme B, CD4+ T cells, and CD8+ T cells. Figure 10Furthermore, GLS2-K151 acetylation was positively correlated with poor survival outcomes in PDAC patients. Figure 11 ).

[0053] Compared with the prior art, the beneficial effects of the present invention are: 1. Based on the mechanism of tumor metabolism, the present invention has found new biomarkers for tumor metabolism, and the sensitivity of tumors to immunotherapy can be diagnosed by detecting the expression level of biomarkers; 2. The test can be completed by collecting peripheral venous blood samples, which greatly reduces the psychological burden and physical damage to the subjects. Attached Figure Description

[0054] Figure 1 This reflects the acetylation level of GLS2 after immunoprecipitation and immunoblotting analysis following cellular hypoxia treatment;

[0055] Figure 2 This demonstrates the post-translational modifications of GLS2 in Panc-1 cells after hypoxia treatment, as identified by mass spectrometry.

[0056] Figure 3 This demonstrates the effect of the GLS2 K151R mutation on PD-L1 protein, as obtained by immunoprecipitation and Western blot analysis.

[0057] Figure 4 This demonstrates the effect of the GLS2 K151R mutation detected by RT-PCR on PD-L1 mRNA expression.

[0058] Figure 5 This demonstrates the impact of GLS2 K151R mutations in mouse orthotopic xenografts on anti-PD-1 immunotherapy;

[0059] Figure 6 for Figure 5 Photographs and weight statistics of xenografts in mice;

[0060] Figure 7 It reflects Figure 5 Immunohistochemical results of xenografts in medullomyeloid mice;

[0061] Figure 8 It reflects Figure 5 Immunofluorescence results of xenografts in medullomyeloid mice;

[0062] Figure 9 This reflects the immunohistochemical results of human pancreatic cancer and adjacent normal samples;

[0063] Figure 10 This reflects the immunohistochemical results of different pancreatic cancer patients;

[0064] Figure 11 The relationship between GLS2K151 acetylation levels in tumor tissue of pancreatic cancer patients and patient prognosis is shown in the survival curve. Detailed Implementation

[0065] To make the objectives and technical solutions of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings. Unless otherwise specified, the experimental methods described in the following tests are conventional methods; for tests where specific techniques or conditions are not specified, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions; unless otherwise specified, the reagents and materials described are commercially available.

[0066] This invention provides the application of the modified form of glutamine synthase 2 (GLS2), GLS2K151 acetylation, as a biomarker in assessing or predicting the sensitivity of cancer patients to immunotherapy.

[0067] This invention also provides the application of GLS2K151 acetylation, a biomarker based on GLS2 function, in the preparation of diagnostic products for tumor immunotherapy.

[0068] The product includes reagents for detecting the expression levels of the biomarkers in a sample. The sample is peripheral venous blood, adjacent normal tissue, and / or tumor tissue.

[0069] The reagents include a first antibody and a second antibody. The first antibody is a GLS2K151 acetylated antibody, GLS2AcK151. The second antibody is an antibody homologous to the first antibody and labeled with horseradish peroxidase.

[0070] The present invention also provides tumor diagnostic kits based on ELISA or Western Blot methods, the kits containing reagents for detecting GLS2K151 acetylation, each kit containing reagents including a first antibody and a second antibody, the first antibody being a GLS2K151 acetylation antibody GLS2 AcK151, and the second antibody being an antibody homologous to the first antibody and labeled with horseradish peroxidase.

[0071] The kit may also include a container, instructions for use, positive control, negative control, buffer, auxiliaries, and solvent. The instructions for use describe how to use the kit for detection and how to use the results to assess tumor development and select treatment options. The kit components may be packaged in an aqueous medium.

[0072] This invention also provides a method for assessing or predicting the sensitivity of cancer patients to immunotherapy, including:

[0073] 1) Detect the expression levels of the biomarkers mentioned in the samples;

[0074] When using peripheral venous blood as a sample, the sample processing method includes: after collecting peripheral venous blood, it should be placed on ice for 15 minutes, and then centrifuged at 3500 rpm for 15 minutes to obtain serum or plasma, which can then be tested or stored at -80℃. Samples that cannot be tested immediately should be stored at -80℃ and thawed on ice before testing.

[0075] The GLS2K151 acetylation expression level in the processed samples was detected using methods such as ELISA or Western Blot.

[0076] 2) Assess or predict the sensitivity of cancer patients to immunotherapy based on the GLS2K151 acetylation expression level in the samples obtained from 1).

[0077] The kits and assessment or prediction methods described in this invention can be applied to the following tumor diseases: oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory system cancer, genitourinary system cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine system cancer or hematopoietic system cancer, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, kidney cancer, biliary system cancer, pheochromocytoma, islet cell carcinoma, Levi-Flaumeni tumor, thyroid cancer, parathyroid cancer, pituitary adenoma, adrenal adenoma, bone-derived sarcoma, neuroendocrine system tumor, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer.

[0078] The Western blotting method mentioned in the evaluation or prediction method described in this invention, also known as protein immunoblotting, is a technique commonly used in research for the isolation and identification of proteins. It utilizes SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to separate various proteins contained in a specified sample. The separated proteins are then transferred to a nitrocellulose or PVDF membrane, which is subsequently incubated with a specific antibody against the target protein. During membrane washing, unbound antibodies are washed away, leaving only antibodies bound to the target protein. Finally, the bound antibodies are detected by developing film or fluorescence scanning. Because the antibody binds only to the target protein, typically only a single, clear band is observed, with the band thickness corresponding to the protein content. By analyzing the location and intensity of specific reactions, information about the expression of the target protein in a given cell or tissue homogenate can be obtained. Due to the high resolution of gel electrophoresis and the high specificity and sensitivity of immunoblotting, Western blotting analysis can detect target proteins as low as 1 ng. This method is widely used in molecular biology fields such as molecular biology, biochemistry, and immunogenetics.

[0079] Based on the above description of specific embodiments, the present invention provides the following examples to further illustrate the technical solution and its technical effects.

[0080] Example 1.

[0081] An ELISA detection kit contains a pre-coated microplate, standards, enzyme-labeled secondary antibody, substrate solution, stop solution, washing buffer, sample diluent, control sample, instructions, and sealing film; the wells of the microplate are pre-immobilized with the capture antibody GLS2 AcK151; the enzyme-labeled secondary antibody is an antibody homologous to the capture antibody GLS2 AcK151 and labeled with horseradish peroxidase.

[0082] Example 2.

[0083] A Western blot (WB) test kit containing the following reagents and items:

[0084] Primary antibody GLS2 AcK151;

[0085] Secondary antibody (antibody homologous to GLS2 AcK151 and labeled with horseradish peroxidase);

[0086] Substrate solution;

[0087] Sealing solution: Milk powder sealing solution;

[0088] Washing buffer: TBS / T;

[0089] Transfer buffer;

[0090] PVDF membrane;

[0091] Electro-hydraulic transfer;

[0092] Membrane staining solution;

[0093] Sample buffer solution;

[0094] Standard protein or marker protein: used to estimate the molecular weight of the target protein;

[0095] Instruction manual.

[0096] Example 3.

[0097] A method for assessing the sensitivity of cancer patients to immunotherapy includes the following steps:

[0098] I. The expression level of GLS2K151 acetylation in the peripheral serum of patients was detected using the WB detection kit described in Example 2.

[0099] The specific testing steps are as follows:

[0100] 1. Prepare samples

[0101] 1) Sample collection: Collect peripheral blood from the vein, let it stand on ice for 15 minutes, centrifuge at 3500 rpm for 15 minutes at 4℃ to obtain serum, and then perform the test.

[0102] 2) Prepare ice packs; prepare cell lysis buffer: determine the required lysis buffer based on the number of cells: 50 μl / well of a six-well plate; lysis buffer formula: 100 μl cell lysis buffer + 0.1 μl phosphatase inhibitor mixture + 1 μl protease inhibitor mixture.

[0103] 3) Prepare the cells according to the experimental requirements: Remove the culture medium, wash 3 times with 1×PBS (to remove the serum from the culture medium), and add 50 μl of lysis buffer containing protease / phosphatase inhibitors to each well (6-well plate). Quickly scrape the cells off with a cell scraper and transfer them to a 1.5 ml centrifuge tube. Incubate on ice for 20 min to lyse, vortex to mix, and then incubate on ice for another 10 min.

[0104] 4) Centrifuge at 4℃, 12000g for 15 minutes, and carefully collect the supernatant into another 1.5ml tube.

[0105] 5) Take 2.5 μl of sample and dilute it with 22.5 μl of triple-distilled water for use in the BCA method to determine protein concentration.

[0106] 6) Add 5× Loading Buffer (2.5ml Buffer / 10ml protein) to the remaining samples and boil at 95℃ for 10 minutes, shaking once during the process.

[0107] 7) Directly load the sample for gel running or dispense it for long-term storage at -80℃.

[0108] 2. SDS-PAGE polyacrylamide gel electrophoresis

[0109] 1) Prepare the separating gel (6 ml / gel).

[0110] 2) Carefully inject the separating gel, leaving about 2cm of space for the concentrating gel, cover the top with deionized water, and let stand for about 30 minutes.

[0111] 3) Prepare the concentrated gel (2 ml / gel).

[0112] 4) Pour the stacking gel into the top of the separating gel, being careful to avoid air bubbles.

[0113] 5) Insert the comb and wait for the concentrated gel to solidify (there is a clear boundary between the gel and the comb, and the solidification time of the separating gel should be greater than 2 hours). Clean the hole with double-distilled water to remove gel fragments, and then blot dry with filter paper.

[0114] 6) Place the gel into the electrophoresis tank, and add 1× electrophoresis buffer to both the upper and lower tanks (do not reuse more than 3 times).

[0115] 7) Sample loading: Take 5 μl of prestained marker into the marker well and add an appropriate amount of 1× loading buffer to make the total volume the same as the sample well. The sample loading volume is generally 15-25 μl. First, boil it with heating block at 95℃ for 5-10 minutes, shaking once during the process, then quickly centrifuge and load the sample for gel running; add an equal volume of 1× loading buffer to the wells without sample loading.

[0116] 8) Electrophoresis: Start with a constant voltage of 60-80V. After running through the stacking gel, increase the current to 100-120V. The electrophoresis time is determined according to the size of the target protein and the position of the marker. Generally, the target protein is ready when it reaches two-thirds of the separating gel.

[0117] 3. Membrane transfer

[0118] 1) Cut the gel according to the marker indication and the position of the target band (note to mark the cut corner of the gel). Immerse the eluted gel in the transfer buffer for 15 minutes.

[0119] 2) After marking the PVDF membrane, immerse it in methanol for 1 minute, then immerse it along with 4 sheets of 3mm filter paper and a sponge in transfer buffer for 15 minutes.

[0120] 3) Prepare the "sandwich": Follow the order below: fiber mat -- filter paper -- PVDF membrane -- gel -- filter paper -- fiber mat; Note: Align each item as it is added to ensure there are no air bubbles.

[0121] 4) Transfer: The transfer time is determined according to 1). One side of the PVDF membrane is connected to the positive electrode (red), and the other side of the gel is connected to the negative electrode (black).

[0122] 4. Membrane blocking and antibody incubation 1) After transfer, wash the membrane with 10 ml of 1×TBS at room temperature for 10 minutes.

[0123] 2) Incubate 5ml of 5% milk powder sealing solution at room temperature for 2 hours or at 4°C with gentle shaking overnight. Because milk powder is relatively difficult to dissolve, it should be prepared at least 1 hour in advance.

[0124] 3) Wash the membrane with 10ml of TBS / T solution 3 times, 5 minutes each time.

[0125] 4) Add 5 ml of primary antibody dilution buffer (divide the antibody according to the instructions), incubate at room temperature for 2 h or at 4 °C with gentle shaking overnight, recover the primary antibody, add sodium azide (which can inhibit bacterial growth) at 5 μl / ml of primary antibody solution, and store at 4 °C (less frequently used antibodies can be stored at -20 °C for a long time). It can be reused.

[0126] 5) Wash the membrane three times with 10ml of TBS / T solution, each time for 5 minutes.

[0127] 6) Add the secondary antibody (usually diluted 1:2000) and shake gently at room temperature for 1 hour.

[0128] 7) Wash the membrane three times with 10ml of TBS / T solution, each time for 5 minutes.

[0129] 5. Development and fixing (or direct fluorescence scanning after incubation with fluorescently labeled secondary antibody)

[0130] 1) Developing steps: First, lay down plastic wrap, then place another layer of plastic wrap on top of absorbent paper. Pour water, developer (if the color darkens, do not use), and fixer into their respective trays. Mix 2.5ml of ECL-A and 2.5ml of ECL-B, and keep away from light. Take the ECL mixture, film, etc., into the darkroom. Close and lock the door, and pull back the curtain. Pour the ECL mixture into a small box. Slightly pat the film dry with absorbent paper and place it in the ECL mixture, shaking at room temperature for 5 minutes (to ensure the ECL is evenly spread over the film). After patting dry with absorbent paper, place it on plastic wrap, film side down, and place it on a clip. Cut an X-film (note that you can only hold the edge of the X-film) and place it on the film, cutting off one corner as a marker (Note: When handling the film, turn the light to minimum and do not face the light; keep it away from the light). Adjust the exposure time according to the brightness of the strips. Generally, you can expose for 2 minutes first, observe the depth of the strips, and then determine the optimal exposure time.

[0131] 2) Development and fixing: After taking out the X-film and placing it in the developer for a certain period of time (depending on the background and the intensity of the target band), wash it once with water and then place it in the fixer for at least 5 minutes.

[0132] Finally, the target protein band on the PVDF membrane is obtained. By comparing the size with the protein marker, the size and location of the target protein can be determined (GLS2 protein size is 60KD), and the expression result of the target protein can be obtained.

[0133] II. Evaluate the sensitivity of patients to immunotherapy based on the protein expression results detected in step one.

[0134] Diagnostic analysis of test results: If the following protein bands of the size corresponding to GLS2K151 acetylation appear on the PVDF membrane obtained in step one, it suggests that the subject may be resistant to immunotherapy.

[0135] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. The use of a reagent for detecting the modified form of the biomarker glutamine synthase 2 (GLS2) acetylated protein GLS2K151 in the preparation of a drug for assessing or predicting the sensitivity of pancreatic cancer patients to immunotherapy; wherein the immunotherapy is PD-1 or PD-L1 inhibitor therapy.

2. The application as described in claim 1, characterized in that: The assessment or prediction of pancreatic cancer patients' sensitivity to immunotherapy includes detecting the expression level of GLS2K151 acetylated protein in samples from said pancreatic cancer patients, and assessing or predicting the sensitivity of pancreatic cancer patients to immunotherapy based on said detection results.

3. The application as described in claim 2, characterized in that: The detection of the expression level of GLS2K151 acetylated protein in samples from the pancreatic cancer patient was performed using ELISA or Western Blot methods.

4. The application as described in any one of claims 2 or 3, characterized in that: The detection of the expression level of GLS2K151 acetylated protein in samples from the pancreatic cancer patient involves detecting the expression level of GLS2K151 acetylated protein in peripheral venous blood, adjacent normal tissue, and / or tumor tissue from the pancreatic cancer patient.

5. The application as described in any one of claims 2 or 3, characterized in that: The method of assessing or predicting the sensitivity of pancreatic cancer patients to immunotherapy based on the test results is as follows: when the test results show that GLS2K151 acetylated protein is expressed in the sample, the pancreatic cancer patient is judged to be resistant to immunotherapy; otherwise, the pancreatic cancer patient is not resistant to immunotherapy.

6. A kit for quantitative or semi-quantitative detection of proteins, containing an antibody that specifically binds to GLS2K151 acetylated protein.

7. The kit as described in claim 6, characterized in that: The aforementioned kit for quantitative or semi-quantitative protein detection is a kit that can detect proteins using enzyme-linked immunosorbent assay (ELISA) or Western blotting. The kit contains a first antibody and a second antibody. The first antibody is an antibody that specifically binds to GLS2K151 acetylated protein, and the second antibody is an enzyme-labeled antibody homologous to the first antibody.

8. The use of antibodies against GLS2K151 acetylated protein in the preparation of medicaments for assessing or predicting the sensitivity of pancreatic cancer patients to immunotherapy; wherein the immunotherapy is PD-1 or PD-L1 inhibitor therapy.

9. Application of GLS2K151 acetylated protein antibody in the preparation of pancreatic cancer diagnostic kit.

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