Application of biomarker based on GLS2 function in tumor immunotherapy diagnosis

Abstract

The invention provides an application of GLS2K151 acetylation in a modified form of glutamine synthetase 2 (GLS2) as a biomarker in evaluating or predicting the sensitivity of a tumor patient to immunotherapy, namely a method for evaluating the immunotherapy sensitivity of the tumor patient based on GLS2K151 acetylation, which comprises the following steps: detecting the expression level of GLS2K151 acetylation in a sample from the tumor patient, and evaluating or predicting the sensitivity of the tumor patient to immunotherapy based on the detection result. The invention further provides a kit capable of quantitatively or semi-quantitatively detecting the protein, and the kit contains the acetylated antibody capable of being specifically combined with the GLS2K151. The invention also provides an application of the GLS2K151 acetylated antibody in evaluating or predicting the sensitivity of a tumor patient to immunotherapy or in preparing a tumor diagnosis kit. According to the application disclosed by the invention, by detecting the expression level of GLS2K151 acetylation, the diagnosis on the sensitivity degree of tumor immunotherapy can be realized.

Description

Technical Field

[0001] The present invention relates to the application of biomarkers based on GLS2 function in tumor immunotherapy diagnosis, and belongs to the field of tumor medicine. Background Art

[0002] Anti-PD-1 / PD-L1 therapy has shown promising clinical results in various cancer types, while 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 for many cancers to 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 patients with melanoma, gastric cancer, and non-small cell lung cancer, the PD-L1 expression pattern is spatially heterogeneous and temporally dynamic. However, the factors that regulate dynamic PD-L1 expression during tumorigenesis and progression are not fully understood, and the exact underlying mechanisms remain to be fully elucidated.

[0003] Since the mechanisms of tumor occurrence and development are very complex, there are many deficiencies in related research, which seriously hinders the development of new methods for tumor diagnosis and treatment. Metabolism is a key factor affecting the occurrence and development of tumors. During the occurrence and development of tumors, the metabolic pattern of tumor cells will change, which is called "metabolic reprogramming". As one of the important characteristics of tumors, metabolic reprogramming meets the three key needs of tumor cells: energy required for sustained growth, sufficient biomacromolecules for the synthesis of nucleic acids, proteins, and lipids, and the maintenance of redox homeostasis. Only by understanding and recognizing the mechanisms of tumor metabolism more comprehensively and systematically can we find biomarkers for tumor metabolism, break through the bottleneck of our current research on tumor diagnosis, 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 expression levels can lead to significant changes in the content of intracellular metabolites. This "classical" metabolic reprogramming plays a key role in tumor evolution. However, in recent years, more and more evidence has shown that some metabolic enzymes and metabolic small 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 the occurrence and development of tumors.

[0005] In view of the above background, in order to more accurately judge tumor development and more reasonably select treatment plans, thereby improving the effectiveness of tumor immunotherapy, it is necessary to find key markers that are related to the immune escape mechanism of tumor cells and involved in the life process of tumor cells, so as to evaluate and predict the sensitivity of tumor patients to immunotherapy. Summary of the invention

[0006] The technical problem or primary purpose to be solved by the present invention is to evaluate or predict the sensitivity of tumor patients to immunotherapy.

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

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention uses the modified form of glutamine synthetase 2 (GLS2), GLS2K151 acetylation, as a biomarker for evaluating or predicting the sensitivity of tumor patients to immunotherapy.

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

[0011] In a second aspect, the present invention provides a method for evaluating or predicting the sensitivity of tumor patients to immunotherapy, comprising detecting the expression level of GLS2K151 acetylation in a sample from the tumor patient, and evaluating or predicting the sensitivity of the tumor patient to immunotherapy based on the detection result.

[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 the sample from the tumor patient is to detect the expression level of GLS2K151 acetylation in the venous peripheral blood, paracancerous tissue and / or tumor tissue from the tumor patient.

[0014] In the scheme described in the present invention, the sensitivity of tumor patients to immunotherapy is evaluated or predicted based on the test results, specifically: when the test results show that GLS2K151 acetylation is expressed in the sample, it is judged that the tumor patient is resistant to immunotherapy; otherwise, the tumor patient is not resistant to immunotherapy.

[0015] In a third aspect, the present invention further provides an antibody, wherein the antibody is an antibody GLS2 AcK151 that can specifically bind to the acetylated GLS2K151.

[0016] In a fourth aspect, the present invention further provides a kit for quantitative or semi-quantitative detection of proteins, comprising the antibody described in the third aspect of the present invention.

[0017] In a preferred embodiment of the present invention, the kit for quantitatively or semi-quantitatively detecting proteins is a kit for detecting proteins by an enzyme-linked immunosorbent assay (Elisa) method or a Western Blot method; the 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 following group: a container, instructions for use, a positive control substance, a negative control substance, a buffer, an adjuvant or a solvent.

[0019] Among them, the instruction manual records how to use the test kit for testing, and how to use the test results to judge the development of the tumor and choose a treatment plan.

[0020] In a fifth aspect, the present invention further provides 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 application of the present invention, the tumor can be any one 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, Leigh-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteogenic sarcoma tumor, 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.

[0022] The present invention provides an application of a new biomarker, GLS2K151 acetylation, in evaluating the sensitivity of tumor patients to immunotherapy. By detecting the expression level of the biomarker in a biological sample from a patient, the sensitivity of an individual tumor patient to immunotherapy can be diagnosed.

[0023] The GLS2K151 acetylation described in the present invention, as a specific modification form of GLS2, is highly correlated with the immune escape mechanism of tumor cells and participates in the life process of tumor cells through non-metabolic pathways. Specifically, the inventors have found through research that the hypoxic microenvironment of pancreatic cancer (PDAC) can induce acetylation of the K151 site of glutamine synthetase 2 (GLS2). This acetylation can induce the transcription of PD-L1, promote the immune escape of pancreatic cancer, and thus reduce the sensitivity of an-PD-1 monoclonal antibody to pancreatic cancer treatment. In addition, in PDAC specimens, the acetylation level of GLS2 K151 is positively correlated with the expression of PD-L1 and CD4 + T, CD8 + The infiltration of T cells was negatively correlated with the poor prognosis of PDAC patients. These findings reveal a key mechanism by which the hypoxic microenvironment of tumors induces the immune escape of tumor cells by promoting the non-metabolic function of PD-L1 expression through inducing acetylation of the GLS2 K151 site, and highlight the potential of GLS2-targeted tumor immunotherapy for diagnosis and treatment.

[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 the patients' medical history. Pathological staging was assessed according to the 8th edition of the American Joint Committee on Cancer / Consortium for International Cancer Control TNM classification system.

[0030] (4) The shRNA target sequences 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: by contacting the sample with an antibody that specifically binds to the phosphorylated polypeptide and determining the amount of bound antibody, for example, by detecting or measuring the travel of the complex between the antibody and the polypeptide. The antibody may be labeled (radioactive, fluorescent, etc.) to facilitate detection of the complex.

[0033] The detection systems used for the above polypeptide levels include: radioautographic immunoassay RIA, immunofluorescence, gel electrophoresis, and Western immunoblotting.

[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 RNase-free DNaseI (Promega) for 30 min, and reverse transcribed using HiScript III-RT SuperMix for quantitative reverse transcription polymerase chain reaction (qPCR; Vazyme Biotech Co.,). qPCR was performed using ChamQ Universal SYBR qPCR Kit (Vazyme Biotech Co., Ltd.) and ABI 7500FAST Detection System (Life Technologies). The expression levels of all samples were normalized to the expression level of GAPDH.

[0037] The following primers were 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; Cell Signaling Technology). Immunoprecipitated chromatin was analyzed using a real-time qPCR detection system. The PCR primer sequences for the CD274 promoter are as follows:

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

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

[0043] (4) Reporter gene experiment: Cells were co-transfected with firefly luciferase reporter plasmid and pRL-CMV plasmid in 24-well plates using Lipofectamine 3000 (Life Technologies). Luciferase activity was assessed 48 hours after transfection using the Dual-Luciferase Reporter Gene Assay System (Promega).

[0044] (5) Mouse tumor sample acquisition and analysis: 6-week-old male immunocompetent mice were housed under specific pathogen-free conditions with a 14 h / 10 h light cycle, an ambient temperature of 21-23 °C, and a humidity of 50%, with free access to water and food. Luciferase-expressing Panc02 cells (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 6-week-old male immunocompetent mice with or without anti-PD-1 injection 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 cancer was performed using an IVIS system (AniView 100, Biolight Biotechnology, 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 hematoxylin and eosin-stained sections. Tumor volume was calculated using the formula V=1 / 2a2b (where V is volume, a is the shortest diameter, and b is the longest diameter).

[0045] (6) Immunohistochemistry: After paraffin sections were deparaffinized, rehydrated and antigen retrieval, TMA slides were incubated with primary rabbit anti-human acetylated GLS2K151 (dilution 1:200) or nonspecific IgG (as a negative control) at 4°C overnight. The slides were then incubated with anti-rabbit secondary antibody (ready-to-use solution; Cell Signaling Technology; #8114), followed by dichromatic diaminobenzidine (DAB) staining (Cell Signaling Technology) and hematoxylin staining, and fixed in xylene. Tissue slides were quantitatively scored under a microscope according to the percentage of positive cells and staining intensity. The present invention assigns the following positive cell proportion 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. The 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 (obvious color); 3, strong (very dark color). The positive cell proportion score and the staining intensity grade are added to obtain a total score (range 0-8). The reproducibility of the scoring system was independently verified by two pathologists who were unaware of the clinical information.

[0046] (7) Kaplan-Meier plots of patients' overall survival were used for data analysis using SPSS version 20.0 software (SPSS Inc., Chicago, IL, USA). The expression levels of biomarkers in tumors and normal tissues were compared using the independent sample t-test. One-way analysis of variance (ANOVA, posthoc Bonferroni test) was used to perform multiple comparisons and the least significant difference test for the correlation between the expression levels of biomarkers and the clinical pathological characteristics of patients. The Pearson correlation coefficient was used to analyze the correlation between the expression levels of biomarkers. Overall survival (OS) was defined as the duration from the date of diagnosis to the date of death or the last follow-up. K-means cluster analysis was used to classify the expression levels of relevant markers, the Kaplan-Meier method was used to draw survival curves, the log-rank test was used to compare survival rates, and the Cox regression model with a two-way Wald test was used to calculate the hazard ratio (HR) and 95% confidence intervals (CIs) for survival analysis. Censored data were used for patients who were alive at the last follow-up or were lost due to follow-up. Univariate analysis variables with a P value of less than 0.05 were included in the multivariate analysis. P < 0.05 was considered statistically significant. All statistical tests were two-sided.

[0047] 2 Results and analysis

[0048] (1)Figure 1 As shown, after Panc-1 cells were treated with hypoxia for 12 hours, immunohistochemical analysis showed that hypoxia could induce the acetylation level of GLS2.

[0049] (2) Figure 2 As shown, Co-IP combined with mass spectrometry analysis found that the GLS2K151 site in Panc-1 cells treated with hypoxia could undergo acetylation modification.

[0050] (3) Inhibition of acetylation at the GLS2 K151 site can inhibit PD-L1 expression. After expressing rGLS2 WT or rGLS2-K151R in GLS2-deficient Panc-1 and Capan-2 cells and treating with hypoxia for 12 hours, western blot results showed that the GLS2 K151R mutation could inhibit hypoxia-induced PD-L1 expression ( Figure 3 ), reporter gene experiments showed that GLS2K151R mutation could inhibit the activity of PD-L1 promoter region ( Figure 4 ), RT-PCR results showed that 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 transplanted tumors to anti-PD-1 treatment. Panc02LUC cells were injected orally into 6-week-old male immunocompetent mice and treated with anti-PD-1 at the same time. The results showed that expression of GLS2 K151 acetylation can reduce the sensitivity of transplanted tumors to anti-PD-1 treatment, and GLS2 K151R mutation can increase the sensitivity of transplanted tumors to anti-PD-1 treatment ( Figure 5 , Figure 6 ), immunohistochemistry and immunofluorescence experiments showed that acetylation of GLS2K151 site could promote PD-L1 expression and reduce CD 4 T and CD 8 T cell recruitment promotes tumor immune escape ( Figure 7 , Figure 8 ).

[0052] (5) GLS2K151 acetylation is associated with the prognosis of pancreatic cancer patients. We analyzed human primary PDAC specimens using immunohistochemical staining with validated anti-GLS2-AcK151 and found that the levels of these protein markers in PDAC tissues were significantly higher than those in adjacent normal tissues ( Fig. 9 In addition, it was observed that GLS2-AcK151 was positively correlated with PD-L1 expression, but negatively correlated with granzyme B, CD4+T, and CD8+T cells ( Fig.10). In addition, acetylation of GLS2-K151 was positively correlated with poor survival outcomes in PDAC patients ( Fig.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. By detecting the expression level of biomarkers, the sensitivity of tumors to immunotherapy can be diagnosed; 2. Samples can be collected from venous peripheral blood to complete the test, which greatly reduces the psychological burden and physical damage of the subjects. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 It reflects the acetylation level of GLS2 analyzed by immunoprecipitation and immunoblotting after cell hypoxia treatment;

[0055] Figure 2 It reflects the identification of post-translational modifications of GLS2 by mass spectrometry after hypoxia treatment of Panc-1 cells;

[0056] Figure 3 It reflects the effect of GLS2 K151R mutation on PD-L1 protein obtained by immunoprecipitation and immunoblotting analysis;

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

[0058] Figure 5 It reflects the effect of GLS2 K151R mutation on anti-PD-1 immunotherapy in orthotopic xenografts in mice;

[0059] Figure 6 for Figure 5 Photos of mouse transplanted tumors and statistical graphs of their weight;

[0060] Figure 7 Reflected Figure 5 Immunohistochemical results of transplanted tumors in mice;

[0061] Figure 8 Reflected Figure 5 Immunofluorescence results of transplanted tumors in mice;

[0062] Fig. 9 It reflects the immunohistochemical results of human pancreatic cancer and adjacent normal samples;

[0063] Fig.10 It reflects the immunohistochemical results of different pancreatic cancer patients;

[0064] Fig.11 This is the survival curve of GLS2K151 acetylation level in tumor tissue of pancreatic cancer patients and patient prognosis. DETAILED DESCRIPTION

[0065] In order to make the purpose and technical solution of the present invention more clear, the present invention is further described in detail below in conjunction with the accompanying drawings. The experimental methods described in the following experiments are all conventional methods unless otherwise specified; if no specific technology or conditions are specified in the experiment, the technology or conditions described in the literature in the field or the product instructions are used; the reagents and materials described can be obtained from commercial channels unless otherwise specified.

[0066] The present invention provides the use of a modified form of glutamine synthetase 2 (GLS2), GLS2K151 acetylation, as a biomarker in evaluating or predicting the sensitivity of tumor patients to immunotherapy.

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

[0068] The product includes a reagent for detecting the expression level of the biomarker in a sample. The sample is venous peripheral blood, paracancerous tissue and / or tumor tissue.

[0069] The reagents include a first antibody and a second antibody. The first antibody is the 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 a tumor diagnosis kit based on Elisa or Western Blot method, wherein the kit contains reagents for detecting GLS2K151 acetylation, and each reagent in the kit includes a first antibody and a second antibody, wherein the first antibody is a GLS2K151 acetylation antibody GLS2 AcK151, and the second antibody is an antibody homologous to the first antibody and labeled with horseradish peroxidase.

[0071] The kit may also include a container, instructions for use, a positive control, a negative control, a buffer, an adjuvant, and a solvent. The instructions for use describe how to use the kit for testing, and how to use the test results to judge tumor development and select a treatment plan. The components of the kit may be packaged in the form of an aqueous medium.

[0072] The present invention also provides a method for evaluating or predicting the sensitivity of tumor patients to immunotherapy, comprising:

[0073] 1) detecting the expression level of the biomarker in the sample;

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

[0075] The treated samples are used to detect the acetylation expression level of GLS2K151 by using methods such as Elisa or Western Blot;

[0076] 2) Evaluate or predict the sensitivity of tumor patients to immunotherapy based on the GLS2K151 acetylation expression level in the sample detected in 1).

[0077] The kit and the evaluation or prediction method described in the present invention can be applied to the following tumor diseases: oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory system cancer, urogenital 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, Leigh-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteogenic sarcoma tumor, 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 Blot method mentioned in the evaluation or prediction method of the present invention, i.e., protein immunoblotting experiment, is a technique commonly used to separate and identify proteins in research. It utilizes SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to separate various proteins contained in a specified sample, and then transfers the separated proteins to a nitrocellulose or PVDF membrane, and then incubates the membrane with a specific antibody to the target protein. During the membrane washing process, unbound antibodies are washed away, leaving only antibodies bound to the target protein, and finally the bound antibodies are detected by developing film or fluorescence scanning. Since the antibody is only bound to the target protein, generally only a clear band can be seen, and the thickness of the band corresponds to the amount of protein. By analyzing the position and intensity of a specific reaction, the expression information of the target protein in a given cell or tissue homogenate can be obtained. Due to the high resolution of gel electrophoresis and the strong specificity and high sensitivity of immunity, Western blot analysis can detect target proteins as low as 1ng. This method is widely used in molecular biology, biochemistry, immunogenetics and other molecular biology fields.

[0079] Based on the description of the above specific implementation methods, the present invention specifically lists the following embodiments to further illustrate the technical solution and technical effects of the present invention in detail.

[0080] Example 1.

[0081] An ELISA detection kit comprises a pre-coated microplate, a standard substance, an enzyme-labeled secondary antibody, a substrate solution, a stop solution, a washing buffer solution, a sample diluent, a control sample, an instruction manual and a sealing film; the wells of the microplate are pre-fixed with a 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 WB detection 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] Blocking solution: milk powder blocking solution;

[0088] Wash buffer: TBS / T;

[0089] Transfer buffer;

[0090] PVDF membrane;

[0091] Electroporation fluid;

[0092] Membrane staining solution;

[0093] Sample buffer;

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

[0095] Instructions for use.

[0096] Example 3.

[0097] A method for evaluating the sensitivity of a tumor patient to immunotherapy comprises the following steps:

[0098] 1. The WB detection kit described in Example 2 was used to detect the acetylation expression level of GLS2K151 in the patient's venous peripheral serum.

[0099] The specific detection steps are as follows:

[0100] 1. Sample Preparation

[0101] 1) Sample collection: Collect venous peripheral blood, place it on ice for 15 minutes, and centrifuge it at 4°C, 3500 rpm for 15 minutes to obtain serum for testing.

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

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

[0104] 4) Centrifuge at 4°C, 12,000 g for 15 min and carefully collect the supernatant into another 1.5 ml tube.

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

[0106] 6) Add 5× Loading Buffer (2.5 ml of Buffer / 10 ml of protein) to the remaining samples and cook at 95°C for 10 minutes, shaking once during the cooking.

[0107] 7) Load the sample directly onto the gel or aliquot and store at -80℃ for long term storage.

[0108] 2. SDS-PAGE polyacrylamide gel electrophoresis

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

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

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

[0112] 4) Inject the concentrated gel into the upper end of the separation gel, taking care to avoid bubbles.

[0113] 5) Insert the comb and wait for the concentrated gel to solidify (there should be a clear boundary between the gel and the comb, and the solidification time of the separation gel should be greater than 2 hours), wash the wells with double distilled water to remove the gel fragments, and then absorb the water 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 5μl of prestained marker in the marker well and add an appropriate amount of 1× loading buffer to make the total volume the same as that of the sample well. The sample loading volume is generally 15-25μl. First, use a heating block to cook at 95℃ for 5-10 minutes, shake once during the period, then quickly centrifuge and load the sample and run the gel; add an equal volume of 1× loading buffer to the well without loaded sample.

[0116] 8) Electrophoresis: Start with a constant voltage of 60-80V. After running through the concentrated 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 runs to two-thirds of the separation gel.

[0117] 3. Membrane Transfer

[0118] 1) Cut the gel according to the marker and the position of the target band (make sure to mark the gel cutting angle), and immerse the eluted gel in transfer buffer for 15 minutes.

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

[0120] 3) Prepare the "sandwich": follow the following order: fiber pad - filter paper - PVDF membrane - gel - filter paper - fiber pad; Note: each item must be aligned to ensure there are no 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 5 ml of 5% milk powder blocking solution at room temperature for 2 hours or gently shake at 4°C overnight. Since milk powder is difficult to dissolve, it should be prepared at least 1 hour in advance.

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

[0125] 4) Add 5 ml of primary antibody dilution buffer (antibodies are diluted according to the instructions), gently shake at room temperature for 2 hours or at 4°C overnight, recover the primary antibody, add sodium azide (which can inhibit bacterial growth) at 5 μl / ml primary antibody solution, and store at 4°C (uncommon antibodies can be stored at -20°C for a long time), which can be reused.

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

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

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

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

[0130] 1) Color development steps: first lay out plastic wrap, then lay out plastic wrap on absorbent paper; pour water, developer (cannot be used if the color becomes darker) and fixer into their respective trays. Mix 2.5ml of ECL-A and 2.5ml of ECL-B and protect from light. Take the ECL mixed solution, membrane, etc. into the darkroom. Close the door, lock it, and pull back the cloth. Pour the ECL mixed solution into a small box, slightly dry the membrane with absorbent paper, put it into the ECL mixed solution and shake it at room temperature for 5 minutes (so that the ECL is evenly spread on the membrane), dry it with absorbent paper and place it on the plastic wrap, wrap it face down and place it on the clip, cut the X-film (note that you can only hold the edge of the X-film with your hands) and place it on the membrane, and the cut corner side is the marker (note: when taking the film, adjust the light to the minimum, and do not face the light, but face away from the light). Adjust the exposure time according to the brightness of the strip. Generally, you can expose it for 2 minutes first, observe the depth of the strip, and then determine the best exposure time.

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

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

[0133] 2. Evaluate the patient's sensitivity to immunotherapy based on the protein expression results detected in step 1.

[0134] Diagnostic analysis of test results: If the following protein bands of corresponding sizes appear on the PVDF membrane finally obtained in step 1: GLS2K151 acetylation, it indicates that the subject may be resistant to immunotherapy.

[0135] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. Application of GLS2K151 acetylation, a modified form of glutamine synthetase 2 (GLS2), as a biomarker in evaluating or predicting the sensitivity of tumor patients to immunotherapy.

2. A method for evaluating or predicting the sensitivity of tumor patients to immunotherapy, comprising detecting the expression level of GLS2K151 acetylation in a sample from the tumor patient, and evaluating or predicting the sensitivity of the tumor patient to immunotherapy based on the detection result.

3. The evaluation or prediction method according to claim 2, characterized in that: The detection of the expression level of GLS2K151 acetylation in the sample from the tumor patient is achieved by Elisa or Western Blot method.

4. The evaluation or prediction method according to any one of claims 2 or 3, characterized in that: The detection of the expression level of GLS2K151 acetylation in the sample from the tumor patient is to detect the expression level of GLS2K151 acetylation in the venous peripheral blood, paracancerous tissue and / or tumor tissue from the tumor patient.

5. The evaluation or prediction method according to any one of claims 2 or 3, characterized in that: The evaluation or prediction of the sensitivity of tumor patients to immunotherapy based on the test results is specifically: when the test results show that GLS2K151 acetylation is expressed in the sample, it is judged that the tumor patient is resistant to immunotherapy; otherwise, the tumor patient is not resistant to immunotherapy.

6. A kit for quantitative or semi-quantitative detection of proteins, comprising an antibody that can specifically bind to acetylated GLS2K151.

7. The kit according to claim 6, characterized in that: The kit for quantitatively or semi-quantitatively detecting proteins is a kit for detecting proteins by an enzyme-linked immunosorbent assay (ELISA) method or a Western Blot method; the kit contains a first antibody and a second antibody; the first antibody is an antibody that can specifically bind to acetylated GLS2K151, and the second antibody is an enzyme-labeled antibody homologous to the first antibody.

8. Application of antibodies against GLS2K151 acetylation in evaluating or predicting the sensitivity of tumor patients to immunotherapy.

9. Application of GLS2K151 acetylated antibodies in the preparation of tumor diagnostic kits.

10. The use according to any one of claims 8 to 9, characterized in that: The tumor is any one 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, Leigh-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteogenic sarcoma tumor, 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.

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