Method for detecting GP73 content in T cell and application thereof

Abstract

The invention relates to a method for detecting GP73 protein level in T lymphocytes through flow cytometry and application of the method. The method disclosed by the invention can be used for conveniently and accurately evaluating the GP73 level in the T lymphocyte of the subject, so that the method can be used for diagnosis or auxiliary diagnosis of cancers, prognosis evaluation of cancers or evaluation of applicability of cancer patients to immunotherapy, and has a very good clinical application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a method for detecting the content of GP73 in T cells and application thereof. BACKGROUND

[0002] In recent years, the incidence of tumors has been rising, leading to a total cure rate of less than 20%, which has caused a serious threat to human health. At present, the clinical treatment methods for tumors mainly include surgical resection, radiotherapy and chemotherapy, however, they each have limitations. Tumor immunotherapy has significantly prolonged the survival time of tumor patients such as melanoma, prostate cancer and lung cancer in clinical application, and has gradually become the fourth tumor treatment method after surgery, radiotherapy and chemotherapy. This method is widely recognized as the only method that can completely eliminate cancer cells at present, and is the most active and promising treatment in the comprehensive treatment mode of tumors, and is widely recognized by scholars around the world. The core of tumor immunotherapy is to eliminate tumor cells by stimulating T lymphocytes, therefore, studying the immune checkpoint regulatory molecule mechanism in T cells becomes the core of stimulating T cells against tumor immunity.

[0003] Studies have shown that abnormal high expression of GP73 in cells promotes the growth and metastasis of tumors by activating multiple signaling pathways, and GP73 in serum mediates the transmission of endoplasmic reticulum stress between hepatocytes and immune cells, which induces the recruitment of tumor macrophages and causes immune tolerance in the tumor microenvironment. At the same time, we found that GP73 antibody can prolong the survival of tumor-bearing mice, and T cells play an important role in it; secondly, the mortality of mice after blocking T cell clearance induced by GP73 antibody is much lower than that of wild-type mice, which indicates that GP73 is likely to affect the anti-tumor function of T cells. The expression level of GP73 in the process of T cell activation is increasing.

[0004] Current research mainly focuses on the serum detection of GP73 and its expression in hepatocytes, and there is no specific method for detecting GP73 in T cells. In order to further understand the function and mechanism of GP73 in T cells, a method for detecting the level of GP73 in T cells needs to be developed, which can provide important theoretical basis for studying immune checkpoint-sensitive clinical markers, improving the efficacy of immune checkpoint drugs, reducing side effects, and deepening the understanding of the mechanism of combination methods. SUMMARY

[0005] Invention objectives

[0006] In order to meet the needs of the above-mentioned prior art, the purpose of the present application is to provide a method for detecting the content of GP73 in T cells and application thereof.

[0007] Solution

[0008] To achieve the object of the present application, the technical scheme adopted by the present application is as follows:

[0009] In a first aspect, the present application provides a method for detecting the level of GP73 protein in T lymphocytes by flow cytometry, the method comprising:

[0010] (1) lysing red blood cells

[0011] Take a whole blood sample for anticoagulant treatment, and add a red blood cell lysing solution to lyse the red blood cells; after lysis is complete, centrifuge to obtain a cell pellet, wash the cell pellet and resuspend it to obtain a cell suspension I;

[0012] (2) cell surface staining

[0013] Add anti-CD45 antibody and anti-CD3 antibody with first and second fluorescent labels, respectively, to the cell suspension I obtained in step (1), and incubate in the dark to perform cell surface staining; after staining is complete, wash the cells and resuspend them to obtain a cell suspension II;

[0014] Since CD45 and CD3 are characteristic surface antigens of T lymphocytes, T lymphocytes can be screened and determined by screening these two surface antigens;

[0015] (3) cell fixation and membrane rupture

[0016] Add a membrane rupture agent Foxp3 / Transcription Factor Fix / Perm Concentrate 1x to the cell suspension II obtained in step (2), and incubate in the dark; after incubation is complete, wash the cells using Flow Cytometry Perm Buffer 1x and resuspend them to obtain a cell suspension III;

[0017] (4) intracellular staining

[0018] Add anti-GP73 antibody with a third fluorescent label to the cell suspension III obtained in step (3), and incubate in the dark to perform intracellular staining; after staining is complete, wash the cells and resuspend them to obtain a cell suspension IV;

[0019] (5) flow cytometry detection

[0020] Detect the cell suspension IV obtained in step (4) on a flow cytometer, and obtain the average fluorescence intensity of GP73 in T lymphocytes by gate strategy, which reflects the level of GP73 protein in T lymphocytes;

[0021] The first, second, and third fluorescent labels are each different.

[0022] For the above method:

[0023] Optionally, in step (1), the red blood cell lysis solution is red blood cell lysis solution (Solarbio, R1010); preferably, the lysis is to the cell solution clear and transparent, indicating that the lysis is complete.

[0024] And / or, the solution for washing the cell precipitate and / or for resuspending the cells is a PBS solution or physiological saline.

[0025] Optionally, in step (2), the anti-CD45 antibody with a first fluorescent label is an anti-CD45 antibody with an APC / Cyanine7 fluorescent label, preferably APC / Cyanine7 anti-human CD45;

[0026] And / or, the anti-CD3 antibody with a second fluorescent label is an anti-CD3 antibody with a PerCP-Cyanine5.5 fluorescent label, preferably Anti-Hu CD3, eBioscience™ PerCP-Cyanine5.5;

[0027] And / or, the light-protected incubation is carried out at 2-8°C for 20-40 minutes, preferably 30 minutes;

[0028] And / or, the solution for washing the cells and / or for resuspending the cells is a PBS solution or physiological saline.

[0029] Optionally, in step (3), the light-protected incubation is carried out at 2-8°C for 30-60 minutes, preferably 40 minutes.

[0030] Optionally, in step (4), the anti-GP73 antibody with a third fluorescent label is an anti-GP73 antibody with a FITC fluorescent label, preferably GOLPH2 Recombinant Rabbit Monoclonal Antibody (024), FITC (antibody);

[0031] And / or, the light-protected incubation is carried out at 2-8°C for 30-40 minutes;

[0032] And / or, the solution for washing the cells and / or for resuspending the cells is a PBS solution or physiological saline.

[0033] Optionally, in step (5), the gating strategy comprises:

[0034] Gating a cell population with the first fluorescence and the second fluorescence, which is a T lymphocyte; calculating the average intensity of the third fluorescence in the gated T lymphocyte, which reflects the GP73 protein level in the T lymphocyte.

[0035] In a second aspect, the present application provides uses of reagents for the method of the first aspect above in any one or more of the following aspects:

[0036] (1) preparing a product for detecting the level of GP73 protein in T lymphocytes;

[0037] (2) preparing a product for diagnosing or aiding in the diagnosis of cancer;

[0038] (3) preparing a product for evaluating the prognosis of a cancer patient;

[0039] (4) preparing a product for evaluating the degree of benefit of a cancer patient to immunotherapy.

[0040] The uses of the above (2) and (3) are based on the inventors' unexpected finding that the level of GP73 in T lymphocytes of a cancer patient can serve as a marker for cancer, a marker for predicting the prognosis or the degree of benefit to immunotherapy, and a cancer diagnosis model, a cancer prognosis evaluation model or a degree of benefit to immunotherapy evaluation model constructed based on the marker can diagnose or aid in the diagnosis of cancer, accurately predict the prognosis of a cancer patient and the degree of benefit to immunotherapy; in view of this, the method for detecting the content of GP73 in T cells and the reagents involved in the method can be used for the diagnosis or aid in the diagnosis of cancer, the prognosis evaluation of a cancer patient and / or the suitability evaluation of a cancer patient for immunotherapy.

[0041] For the above uses:

[0042] In a feasible embodiment, the cancer is lung cancer;

[0043] In a feasible embodiment, the immunotherapy is an immunotherapy using an immune checkpoint inhibitor, preferably an immunotherapy using an anti-PD-1 or anti-PD-L1 antibody.

[0044] Beneficial effects

[0045] The inventors of the present application found in their research that the level of GP73 in T lymphocytes of a cancer patient can serve as a marker for predicting the prognosis or the degree of benefit to immunotherapy. In order to use this marker to predict the prognosis of a cancer patient or to evaluate the degree of benefit to immunotherapy of a cancer patient, the inventors developed a method capable of detecting the level of GP73 in T lymphocytes, which can conveniently and accurately evaluate the level of GP73 in T lymphocytes of a patient, and thus can be used for the diagnosis or aid in the diagnosis of cancer, the prognosis evaluation of cancer or the suitability evaluation of a cancer patient for immunotherapy, and has good clinical application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 Figure 9 shows the average fluorescence intensity of GP73 in T lymphocytes detected by EasyCell flow cytometer in healthy and lung cancer groups; the results were analyzed by T test, and *** indicates P<0.001 compared with the healthy group;

[0047] Figure 2 Figure 10 shows the average fluorescence intensity of GP73 in T lymphocytes detected by BD FACSCanto flow cytometer in healthy and lung cancer groups; the results were analyzed by T test, and * indicates P<0.05 compared with the healthy group;

[0048] Figure 3 Figure 11 shows the receiver operating characteristic curve (i.e., ROC curve) based on the average fluorescence intensity of GP73 in T lymphocytes detected by EasyCell flow cytometer in healthy and lung cancer groups; AUC=0.775;

[0049] Figure 4 Figure 12 shows the receiver operating characteristic curve (i.e., ROC curve) based on the average fluorescence intensity of GP73 in T lymphocytes detected by BD FACSCanto flow cytometer in healthy and lung cancer groups; AUC=0.750;

[0050] Figure 5 Figure 13 shows that the expression level of GP73 in T lymphocytes of lung cancer patients is positively correlated with the efficacy of anti-PD-1 therapy; wherein, panel A shows the fold change of the average fluorescence intensity of GP73 in T lymphocytes of patients in the partial remission group (i.e., “PR” group) and the stable disease group (i.e., “SD” group) compared with the healthy control group (i.e., “H” group); the results were analyzed by T test, ***p<0.001, *p<0.05; panel B shows the receiver operating characteristic curve (i.e., ROC curve) based on the average fluorescence intensity of GP73 in T lymphocytes of patients in the partial remission group and the stable disease group, AUC=0.812. DETAILED DESCRIPTION

[0051] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0052] In addition, for a better understanding of the present application, a number of specific details are given in the following detailed description. It will be readily apparent to those skilled in the art that the present application can be practiced without some of these specific details. In some instances, well-known components, materials and methods have not been described in detail in order to avoid obscuring the subject matter of the present application.

[0053] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure, and will not be interpreted in an overly literal sense unless expressly so defined herein.

[0054] Example 1: Flow cytometry detection of GP73 levels in T cells of lung cancer patients and normal healthy patients

[0055] In this example, two flow cytometers (EasyCell flow cytometer and BD FACSCanto flow cytometer) were used to detect the mean fluorescence intensity of GP73 in T cells of lung cancer patients and normal healthy patients by the method of the present application. Specifically, blood samples from lung cancer patients and normal healthy patients who had been pathologically diagnosed with lung cancer were used, which were from the Third Medical Center of General Hospital of People's Liberation Army. The specific detection steps are as follows:

[0056] I. Lysis of red blood cells

[0057] 1. Each blood sample was subjected to anticoagulation treatment (purple tube EDTA anticoagulant), and 120ul of the obtained anticoagulated blood was placed in a 5mL polystyrene round-bottom tube / flow tube. The blood was placed at room temperature and detected within 8 hours, otherwise discarded.

[0058] 2. Add 6-10 times the volume of the cell volume of red blood cell lysate (Solarbio, R1010) 1mL to the flow tube, mix gently, lyse for 10 minutes until the tube is clear and transparent. This step can be performed at room temperature or 4 degrees.

[0059] 3. Place the 5mL polystyrene round-bottom tube / flow tube in a horizontal centrifuge and centrifuge at 500g for 5 minutes, discard the red supernatant. 4°C centrifugation is better.

[0060] 4*. If it is found that the red blood cell lysis is not complete, the above steps 2 and 3 can be repeated once. Generally, a very small amount of red blood cells will not affect the subsequent detection.

[0061] 5. Wash 1-2 times; add appropriate amount of 1 ml 1 x PBS solution / saline to each 5 mL polystyrene round-bottom / flow tube, resuspend pellet, place 5 mL polystyrene round-bottom / flow tube in a horizontal centrifuge at 500 g for 5 minutes, discard supernatant. May repeat 1 more time for a total of 1-2 washes. The volume of wash solution should typically be at least 5 times the volume of the cell pellet, and centrifugation at 4°C is preferred.

[0062] 6. Collect cell pellet.

[0063] II. Cell surface staining

[0064] 1. Resuspend cells from Step 1; specifically, add 80-100 ul of PBS buffer containing 0.3% BSA filtered through a 0.22 um filter to each 5 mL polystyrene round-bottom / flow tube to resuspend the cells.

[0065] 2. Collect T lymphocytes; add APC / Cyanine 7 anti-human CD45 antibody (3 ul) and Anti-Hu CD3, eBioscience™ PerCP-Cyanine 5.5 antibody (3 ul) to each 5 mL polystyrene round-bottom / flow tube. The antibodies can be mixed together prior to addition to the 5 mL polystyrene round-bottom / flow tube. After addition of the antibodies, incubate at 2-8°C for 30 minutes in the dark.

[0066] 3. Add 1 ml 1 x PBS solution / saline to each 5 mL polystyrene round-bottom / flow tube, resuspend pellet, place 5 mL polystyrene round-bottom / flow tube in a horizontal centrifuge at 500 g for 5 minutes, discard supernatant.

[0067] 4. Repeat Step 3, add 1 ml 1 x PBS solution / saline to each 5 mL polystyrene round-bottom / flow tube, resuspend pellet, place 5 mL polystyrene round-bottom / flow tube in a horizontal centrifuge at 500 g for 5 minutes, discard supernatant.

[0068] III. Cell fixation and membrane rupture

[0069] 1. Add 1 mL of working concentration of Fixation and Permeabilization Solution (Foxp3 / Transcription Factor Fix / Perm Concentrate 1 x) to each 5 mL polystyrene round-bottom / flow tube.

[0070] 2. Incubate 5 mL polystyrene round-bottom / flow tube at 2-8°C for 30-60 minutes in the dark.

[0071] 3. After the end of the incubation, wash twice with Flow Cytometry Perm Buffer 1x. Resuspend the pellet, place the 5 mL polystyrene round-bottom / flow tube tube in a horizontal centrifuge at 500g for 5 minutes, and discard the supernatant.

[0072] IV. Intracellular staining

[0073] 1. Resuspend the cells; specifically, add 80-100 ul of PBS buffer containing 0.3% BSA filtered through a 0.22um filter to each 5 mL polystyrene round-bottom / flow tube to resuspend the cells.

[0074] 2. Add GOLPH2 Recombinant Rabbit Monoclonal Antibody (024), FITC (antibody) antibody (2.5ul) to the 5 mL polystyrene round-bottom / flow tube, and incubate at 2-8°C in the dark for 30-40 minutes.

[0075] 3. Add 1 mL of 1x PBS solution / normal saline to each 5 mL polystyrene round-bottom / flow tube, resuspend the pellet, place the 5 mL polystyrene round-bottom / flow tube in a horizontal centrifuge at 500g for 5 minutes, and discard the supernatant.

[0076] 4. Repeat step 3, add 1 mL of 1x PBS solution / normal saline to each 5 mL polystyrene round-bottom / flow tube, resuspend the pellet, place the 5 mL polystyrene round-bottom / flow tube in a horizontal centrifuge at 500g for 5 minutes, and discard the supernatant.

[0077] 5. Add 500uL of 1x PBS solution / normal saline to each tube, and proceed to detection.

[0078] V. Flow cytometry detection

[0079] 1. Perform the detection procedure according to the flow cytometer operation manual.

[0080] 2. Draw a detection scheme template according to the gate strategy. Detect the average fluorescence intensity (MFI) of GP73 in T lymphocytes.

[0081] Specifically, circle the cell population with the first fluorescence and the second fluorescence, which is T lymphocytes; calculate the average fluorescence intensity of the third fluorescence in the circled T lymphocytes, which reflects the level of GP73 protein in T lymphocytes.

[0082] VI. Data saving and analysis

[0083] 1. Save all experimental results in all batches in FCS format.

[0084] 2. Analyze the experimental results by FlowJo_v10.8.1.

[0085] The results of the average fluorescence intensity of GP73 in T lymphocytes of the healthy group and the lung cancer group detected by EasyCell flow cytometry are shown in Figure 1 The ROC curve based on the average fluorescence intensity of GP73 in T lymphocytes of the healthy group and the lung cancer group detected by EasyCell flow cytometry is shown in Figure 3 .

[0086] The results of the average fluorescence intensity of GP73 in T lymphocytes of the healthy group and the lung cancer group detected by BD FACSCanto flow cytometry are shown in Figure 2 The ROC curve based on the average fluorescence intensity of GP73 in T lymphocytes of the healthy group and the lung cancer group detected by BD FACSCanto flow cytometry is shown in Figure 4 .

[0087] Figure 1 and Figure 2 show that the average fluorescence intensity of GP73 in T lymphocytes of the lung cancer group is significantly enhanced compared with the healthy group; Figure 3 and Figure 4 show that the average fluorescence intensity of GP73 in T lymphocytes can effectively distinguish the lung cancer group from the healthy group, and thus can be used as a diagnostic marker for lung cancer, which is consistent with the findings of the inventors' contemporaneous research.

[0088] Example 2: Detecting the level of GP73 in T cells by the method of the present application and determining its relationship with the efficacy of anti-PD-1 treatment

[0089] In this example, the level of GP73 in T cells of the subjects is detected by the method of the present application, and the relationship between the level of GP73 in T cells thus detected and the efficacy of anti-PD-1 antibody immunotherapy is explored.

[0090] Specifically, lung cancer patient cases treated with the immunotherapeutic drug anti-PD-1 antibody are collected, and according to the CT imaging report, the patients are divided into the partial remission group (referred to as "PR") and the stable disease group (referred to as "PD") according to the solid tumor clinical efficacy evaluation index (RECIST1.1), and at the same time, healthy people without underlying diseases in the physical examination population are selected as healthy controls (referred to as "H"); blood samples of lung cancer patients at the last treatment time are taken, and blood samples of healthy controls are also collected, and on the day of sample collection, the fluorescence intensity of GP73 in T cells is detected by flow cytometry, and the experimental results are analyzed by FlowJo_v10.8.1.

[0091] The specific detection process is the same as in Example 1.

[0092] After the detection was completed, the fold change of the average fluorescence intensity of GP73 in T lymphocytes of the patients in the partial remission group (i.e., the "PR" group) and the stable disease group (i.e., the "SD" group) relative to the healthy control group was made into a column chart, and T test was used for statistical analysis, and the results are shown in Figure 5 A of FIG. 1, which shows that, compared with the patients with stable disease, we observed a significant increase in the level of GP73 in T lymphocytes in the patients with partial remission to anti-PD-1 therapy.

[0093] The receiver operating characteristic curve (ROC curve) made by the average fluorescence intensity of GP73 in T lymphocytes of the patients in the partial remission group and the stable disease group is shown in Figure 5 B of FIG. 1, which shows that the area under the ROC curve AUC is 0.812, which indicates that: based on the difference in the average fluorescence intensity of GP73 in T lymphocytes, the partial remission group and the stable disease group can be well distinguished.

[0094] The above results show that the expression level of GP73 in T lymphocytes is positively correlated with the efficacy of anti-PD-1 immunotherapy, and also prove that the method for detecting the content of GP73 in T lymphocytes of the present application is convenient and feasible, and the results can accurately indicate the level of the content of GP73 in T lymphocytes.

[0095] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for detecting GP73 protein levels in T lymphocytes by flow cytometry, characterized in that, The method includes: (1) Lysis of red blood cells Whole blood samples were anticoagulated, and red blood cell lysis buffer was added to lyse the red blood cells. After lysis, the samples were centrifuged to obtain a cell pellet. The cell pellet was washed and resuspended to obtain cell suspension I. (2) Cell surface staining Add anti-CD45 antibody and anti-CD3 antibody with first and second fluorescent labels respectively to cell suspension I obtained in step (1), and incubate in the dark to stain the cell surface; after staining, wash the cells and resuspend the cells to obtain cell suspension II; (3) Cell fixation and membrane rupture Add Foxp3 / Transcription Factor Fix / Perm Concentrate 1× to the cell suspension II obtained in step (2), and incubate in the dark; after incubation, wash the cells with Flow Cytometry Perm Buffer 1× and resuspend the cells to obtain cell suspension III; (4) Intracellular staining Add the anti-GP73 antibody with a third fluorescent label to the cell suspension III obtained in step (3), and incubate in the dark to perform intracellular staining; after staining, wash the cells and resuspend the cells to obtain cell suspension IV; (5) Flow cytometry detection The cell suspension IV obtained in step (4) was detected on a flow cytometer. The average fluorescence intensity of GP73 in T lymphocytes was obtained by using a gating strategy, which reflects the level of GP73 protein in T lymphocytes. The first, second, and third fluorescent labels are all different.

2. The method according to claim 1, characterized in that, In step (1), the red blood cell lysis buffer is red blood cell lysis buffer with catalog number R1010 from Solarbio; preferably, the lysis is performed until the cell fluid is clear and transparent; And / or, the solution used for washing cell pellets and / or for cell resuspending is PBS or physiological saline.

3. The method according to claim 1 or 2, characterized in that, In step (2), the anti-CD45 antibody with the first fluorescent label is an anti-CD45 antibody with the APC / Cyanine7 fluorescent label, preferably APC / Cyanine7 anti-humanCD45; And / or, the anti-CD3 antibody with a second fluorescent label is an anti-CD3 antibody with a PerCP-Cyanine5.5 fluorescent label, preferably Anti-Hu CD3, eBioscience™ PerCP-Cyanine5.5; And / or, the light-protected incubation is carried out at 2-8°C for 20-40 minutes, preferably 30 minutes; And / or, the solution used for washing cells and / or for resuspending cells is PBS or physiological saline.

4. The method according to any one of claims 1-3, characterized in that, In step (3), the light-protected incubation is carried out at 2-8°C for 30-60 minutes, preferably 40 minutes.

5. The method according to any one of claims 1-4, characterized in that, In step (4), the anti-GP73 antibody with the third fluorescent label is an anti-GP73 antibody with the FITC fluorescent label, preferably GOLPH2 Recombinant Rabbit Monoclonal Antibody (024) or FITC (antibody); And / or, the light-protected incubation is carried out at 2-8°C for 30-40 minutes; And / or, the solution used for washing cells and / or for resuspending cells is PBS or physiological saline.

6. The method according to any one of claims 1-5, characterized in that, In step (5), the gate strategy includes: Circle the cell populations exhibiting first and second fluorescence, which are T lymphocytes; calculate the average fluorescence intensity of the third fluorescence in the circled T lymphocytes, which reflects the GP73 protein level within the T lymphocytes.

7. Use of the reagents used in the method according to any one of claims 1-6 in one or more of the following aspects: (1) Prepare a product for detecting the level of GP73 protein in T lymphocytes; (2) To prepare products for the diagnosis or auxiliary diagnosis of cancer; (3) Prepare products for assessing the prognosis of cancer patients; (4) Prepare products for evaluating the benefits of immunotherapy to cancer patients.

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