Reporter gene cell strain and application thereof

Abstract

The invention belongs to the technical field of biology, and provides a reporter gene cell strain and application thereof. Specifically, the invention provides a cell strain for detecting the biological activity of an antibody or an antigen binding fragment thereof, and also provides a kit containing the cell strain and a method for using the cell strain.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology and relates to a reporter gene cell line and a construction method and application thereof. Background Art

[0002] In recent years, antibody drugs have been widely used in the clinical treatment of various diseases due to their high specificity, safety and effectiveness. Interleukin 33 (IL-33) is a member of the IL-1 cytokine family and is mainly expressed by tissue endothelial cells or epithelial cells. When cells receive stimuli such as stress, infection and injury, IL-33 is expressed in the cells and transmits external antigen stimulation signals to the Th2 pathway. IL-33 binds to the IL-1 receptor-related protein (IL-1RL1, also known as ST2) on the cell surface, recruits IL-1 receptor auxiliary protein (IL-1RAP), causes intracellular TIR aggregation and then recruits MyD88, activates the NF-κB signaling pathway, and induces mast cells, Th2 cells, Treg cells and ILC2 cells to secrete pro-inflammatory factors such as IL-5, IL-6, IL-13, TNF, INF-γ and chemokines such as CCL17, CCL22, CXCL8, and induces inflammatory response. The expression of IL-33 is abnormally elevated in diseases of mucosal tissue inflammation, joint inflammation and chronic skin inflammation, such as allergic rhinitis (Kamekura Ret al., (2012) Clin Exp Allergy. 42: 218-28), rheumatoid arthritis, ankylosing spondylitis, atopic dermatitis (Savinko T et al., (2012) J Invest Dermatol. 132: 1392-1400), and psoriasis; and IL-33 / ST2 signaling pathway dysregulation is closely related to immune-mediated diseases such as asthma, chronic obstructive pulmonary disease (Hacker, Lambers et al., (2009) J Clin Lab Anal. 23: 372-9), bronchitis, inflammatory bowel disease (Beltran CJ et al., (2010) Inflamm Bowel Dis. 16: 1097-107), systemic lupus erythematosus, liver fibrosis, and systemic sclerosis. Anti-ST2 / IL-33 axis antibodies block the binding of IL-33 to ST2, inhibit the IL-33 / ST2 signaling pathway and the inflammatory response it induces, and become an important means of treating inflammatory and immune-related diseases.

[0003] Biological activity is a key quality attribute of antibody drugs, which directly affects the quality of antibody drugs and needs to be strictly controlled throughout the entire process. Therefore, a cell and method for accurately and rapidly evaluating the biological activity of anti-ST2 / IL-33 axis antibodies are provided, which is suitable for large-scale antibody drug testing, provides a favorable tool for the research and screening of anti-IL-33 / ST2 axis antibody drugs, and has broad application prospects. Summary of the invention

[0004] The present application provides a cell strain, wherein the cell strain is a mammalian cell, and a first expression cassette and a second expression cassette are integrated into the genome of the cell strain, wherein the first expression cassette expresses an exogenous ST2 gene, and the second expression cassette expresses an exogenous reporter gene regulated by NF-κB.

[0005] In some embodiments, the exogenous ST2 gene is a human ST2 gene or a monkey ST2 gene. In some embodiments, the ST2 gene includes a cDNA gene. In some embodiments, the ST2 gene includes a wild-type and a mutant gene.

[0006] In some embodiments, the transcriptional expression of the reporter gene depends on the activation of the NF-κB response element (NF-κB / RE). In some embodiments, the second expression cassette includes a NF-κB response element and a reporter gene coding sequence. In some embodiments, the reporter gene is a luciferase reporter gene (Luciferase gene). In some embodiments, the reporter gene is a luc2P gene (Photinus pyralis).

[0007] In some embodiments, the cell line is a human kidney epithelial cell line. In some embodiments, the cell line is a human embryonic kidney 293 (HEK293) cell or a cell derived therefrom. In some embodiments, the cell line is a human embryonic kidney 293T (HEK293T) cell.

[0008] In some specific embodiments, the deposit number of the cell line is CCTCC NO: C202397.

[0009] In some specific embodiments, the cell line is HEK293T cell, which was deposited in China Center for Type Culture Collection (CCTCC) on May 9, 2023, and classified and named as: Human embryonic kidney 293T-ST2-NFκB reporter gene cell HEK293T-ST2-NFκB Luciferase, with a collection number of CCTCC NO: C202397, and a collection address of: Wuhan University, Wuhan, China, 430072.

[0010] In some embodiments, the cell line has one or more of the following characteristics:

[0011] (a) can be used to detect the biological activity of anti-IL-33 / ST2 axis antibodies or antigen-binding fragments thereof in vitro;

[0012] (b) can be used to determine or evaluate the inhibitory or blocking effect of anti-IL-33 / ST2 axis antibodies or their antigen-binding fragments on IL-33 / ST2 interaction in vitro;

[0013] (c) good specificity. Specifically, the fitting curve of the binding of the anti-IL-33 / ST2 axis antibody or its antigen-binding fragment to the cell shows a good "S"-shaped trend;

[0014] (d) good stability over generations. Specifically, the cell line can be stably passaged for 5 generations or more, 10 generations or more, 15 generations or more, 20 generations or more, 25 generations or more, 30 generations or more, 35 generations or more, 40 generations or more, 45 generations or more, 50 generations or more, or more generations.

[0015] On the other hand, the present application also provides a cell line for detecting the biological activity of an anti-IL-33 / ST2 axis antibody or its antigen-binding fragment, wherein the cell line is a mammalian cell, and a first expression cassette and a second expression cassette are integrated into the genome of the cell line, wherein the first expression cassette expresses an exogenous ST2 gene, and the second expression cassette expresses an exogenous NF-κB-regulated reporter gene.

[0016] In some embodiments, the exogenous ST2 gene is a human ST2 gene or a monkey ST2 gene. In some embodiments, the ST2 gene includes a cDNA gene. In some embodiments, the ST2 gene includes a wild-type and a mutant gene.

[0017] In some embodiments, the transcriptional expression of the reporter gene depends on the activation of the NF-κB response element (NF-κB / RE). In some embodiments, the second expression cassette includes a NF-κB response element and a reporter gene coding sequence. In some embodiments, the reporter gene is a luciferase reporter gene (Luciferase gene). In some embodiments, the reporter gene is a luc2P gene (Photinus pyralis).

[0018] In some embodiments, the cell line is a human kidney epithelial cell line. In some embodiments, the cell line is a HEK293 cell or a cell derived therefrom. In some embodiments, the cell line is a HEK293T cell.

[0019] In some specific embodiments, the deposit number of the cell line is CCTCC NO: C202397.

[0020] In some specific embodiments, the cell line is HEK293T cell, which was deposited in China Center for Type Culture Collection (CCTCC) on May 9, 2023, and classified and named as: Human embryonic kidney 293T-ST2-NFκB reporter gene cell HEK293T-ST2-NFκB Luciferase, with a collection number of CCTCC NO: C202397, and a collection address of: Wuhan University, Wuhan, China, 430072.

[0021] The anti-IL-33 / ST2 axis antibody or antigen binding fragment thereof described in the present application refers to an antibody or antigen binding fragment thereof that can inhibit or block the IL-33 / ST2 interaction, including but not limited to anti-ST2 antibodies or antigen binding fragments thereof and anti-IL-33 antibodies or antigen binding fragments thereof. In some embodiments, the anti-IL-33 / ST2 axis antibody or antigen binding fragment thereof is an anti-ST2 antibody or antigen binding fragment thereof. In some embodiments, the anti-IL-33 / ST2 axis antibody or antigen binding fragment thereof is an anti-IL-33 antibody or antigen binding fragment thereof.

[0022] On the other hand, the present application also provides a kit comprising the cell strain described in the present application. In some embodiments, the kit also includes instructions for use of the cell strain described in the present application. In some embodiments, the kit can be used to detect the biological activity of anti-IL-33 / ST2 axis antibodies or antigen-binding fragments thereof. In some embodiments, the kit also includes instructions for detecting the biological activity of anti-IL-33 / ST2 axis antibodies or antigen-binding fragments thereof using the cell strain described in the present application.

[0023] On the other hand, the present application also provides the use of the cell line described in the present application for detecting the biological activity of an antibody or an antigen-binding fragment thereof.

[0024] On the other hand, the present application also provides the use of the cell line described in the present application in preparing a reagent for detecting the biological activity of an antibody or an antigen-binding fragment thereof.

[0025] In some embodiments, the antibody or antigen binding fragment thereof is an anti-IL-33 / ST2 axis antibody or antigen binding fragment thereof. In some embodiments, the anti-IL-33 / ST2 axis antibody or antigen binding fragment thereof is an anti-IL-33 antibody or antigen binding fragment thereof or an anti-ST2 antibody or antigen binding fragment thereof.

[0026] In some embodiments, the present application also provides use of the cell line described herein for detecting the biological activity of anti-IL-33 / ST2 axis antibodies or antigen-binding fragments thereof.

[0027] In some embodiments, the present application provides the use of the cell line described in the present application in the preparation of an anti-IL-33 / ST2 axis antibody or an antigen-binding fragment thereof biological activity detection reagent.

[0028] In some embodiments, the biological activity includes the binding activity of the antibody or its antigen-binding fragment (e.g., the binding activity of the anti-ST2 antibody or its antigen-binding fragment to ST2) and the blocking activity (e.g., the blocking activity of the anti-ST2 antibody or its antigen-binding fragment on the IL-33 / ST2 interaction).

[0029] On the other hand, the present application also provides a method for detecting the biological activity of an anti-IL-33 / ST2 axis antibody or an antigen-binding fragment thereof, comprising the steps of:

[0030] (1) Providing the cell line described in the present application;

[0031] (2) contacting the antibody or its antigen-binding fragment with IL-33, or contacting the antibody or its antigen-binding fragment with a cell line;

[0032] (3) placing the antibody or its antigen-binding fragment, IL-33 and the cell line in the same reaction system for incubation;

[0033] (4) Detecting the expression level of the reporter gene in the cell line to obtain the biological activity of the antibody or its antigen-binding fragment.

[0034] In some embodiments, the detection method further comprises adding a luciferase detection reagent after step (3). In some embodiments, the reporter gene is a luciferase reporter gene. In some embodiments, the reporter gene is a luc2P gene.

[0035] In some specific embodiments, in the detection method, detecting the expression level of the reporter gene in the cell line in step (4) is achieved by detecting fluorescence intensity.

[0036] In some specific embodiments, in the detection method, step (2) is: gradiently diluting the antibody or its antigen-binding fragment into different concentrations of the antibody or its antigen-binding fragment, and allowing each concentration of the antibody or its antigen-binding fragment to contact with IL-33, or allowing each concentration of the antibody or its antigen-binding fragment to contact with the cell line, respectively.

[0037] In some specific embodiments, in the detection method, step (3) is: placing IL-33, cell lines and each concentration of antibody or antigen-binding fragment thereof in the same reaction system for incubation.

[0038] In some specific embodiments, in the detection method, step (4) is: detecting the fluorescence intensity in each reaction system to detect the expression level of the luciferase reporter gene in the cell line, obtaining an inhibition curve based on the fluorescence intensity and the concentration of the antibody or its antigen-binding fragment, and obtaining the IC value of the antibody or its antigen-binding fragment to block the IL-33 / ST2 interaction. 50 value, thereby obtaining the biological activity of the antibody or its antigen-binding fragment.

[0039] On the other hand, the present application also provides a method for detecting the biological activity of an anti-IL-33 / ST2 axis antibody or an antigen-binding fragment thereof, comprising the steps of:

[0040] (a) providing the cell line described in the present application;

[0041] (b) contacting the antibody or antigen-binding fragment thereof with IL-33;

[0042] (c) placing the antibody or antigen-binding fragment thereof, IL-33 and the cell line in the same reaction system for incubation;

[0043] (d) adding luciferase detection reagent;

[0044] (e) The expression level of the luciferase reporter gene in the cell line is detected by detecting the fluorescence intensity, thereby obtaining the biological activity of the antibody or its antigen-binding fragment.

[0045] In some embodiments, in the detection method, step (b) is: gradiently diluting the antibody or its antigen-binding fragment into different concentrations of the antibody or its antigen-binding fragment, so that each concentration of the antibody or its antigen-binding fragment is contacted with IL-33 respectively.

[0046] In some specific embodiments, in the detection method, step (c) is: placing IL-33, cell lines and each concentration of the antibody or its antigen-binding fragment in the same reaction system for incubation.

[0047] In some specific embodiments, in the detection method, the step (e) is: detecting the fluorescence intensity in each reaction system to detect the expression level of the luciferase reporter gene in the cell line, obtaining an inhibition curve based on the fluorescence intensity and the concentration of the antibody or its antigen-binding fragment, and obtaining the IC value of the antibody or its antigen-binding fragment blocking the IL-33 / ST2 interaction. 50 value, thereby obtaining the biological activity of the antibody or its antigen-binding fragment.

[0048] On the other hand, the present application also provides a method for detecting the biological activity of an anti-IL-33 / ST2 axis antibody or an antigen-binding fragment thereof, comprising the steps of:

[0049] (i) providing the cell line described in the present application;

[0050] (ii) contacting the antibody or antigen-binding fragment thereof with the cell line;

[0051] (iii) placing the antibody or antigen-binding fragment thereof, IL-33 and the cell line in the same reaction system for incubation;

[0052] (iv) adding luciferase detection reagent;

[0053] (v) detecting the expression level of the luciferase reporter gene in the cell line by detecting the fluorescence intensity, thereby obtaining the biological activity of the antibody or its antigen-binding fragment.

[0054] In some specific embodiments, in the detection method, step (ii) is: gradiently diluting the antibody or its antigen-binding fragment into different concentrations of the antibody or its antigen-binding fragment, and allowing each concentration of the antibody or its antigen-binding fragment to contact the cell line respectively.

[0055] In some specific embodiments, in the detection method, step (iii) is: placing IL-33, cell lines and each concentration of the antibody or its antigen-binding fragment in the same reaction system for incubation.

[0056] In some specific embodiments, in the detection method, step (v) is: detecting the fluorescence intensity in each reaction system to detect the expression level of the luciferase reporter gene in the cell line, obtaining an inhibition curve based on the fluorescence intensity and the concentration of the antibody or its antigen-binding fragment, and obtaining the IC value of the antibody or its antigen-binding fragment to block the IL-33 / ST2 interaction. 50 value, thereby obtaining the biological activity of the antibody or its antigen-binding fragment.

[0057] In some embodiments, the detection method is an in vitro detection method.

[0058] In some embodiments, the time of incubation is 1 hour to 48 hours. In some embodiments, the time of incubation is 2 hours to 24 hours. In some embodiments, the time of incubation is 4 hours to 8 hours. In some embodiments, the time of incubation is 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, or the range formed by any of the above values. In some embodiments, the time of incubation is 4 hours, 5 hours, 6 hours, 8 hours, or the range formed by any of the above values. In some embodiments, the time of incubation is 5 hours.

[0059] In some embodiments, the concentration of IL-33 in the reaction system is 0.01 ng / mL to 10 ng / mL. In some embodiments, the concentration of IL-33 in the reaction system is 0.03 ng / mL to 8.5 ng / mL. In some embodiments, the concentration of IL-33 in the reaction system is 0.03 ng / mL to 0.48 ng / mL. In some embodiments, the concentration of IL-33 in the reaction system is 0.06 ng / mL to 0.3 ng / mL. In some embodiments, the concentration of IL-33 in the reaction system is 0.03ng / mL, 0.06ng / mL, 0.09ng / mL, 0.12ng / mL, 0.15ng / mL, 0.18ng / mL, 0.2ng / mL, 0.24ng / mL, 0.3ng / mL, 0.36ng / mL, 0.48ng / mL, 0.72ng / mL, 0.96ng / mL, 1ng / mL, 2ng / mL, 4ng / mL, 6ng / mL, 8ng / mL, 8.5ng / mL, or a range formed by any of the above values. In some embodiments, the concentration of IL-33 in the reaction system is 0.03ng / mL, 0.06ng / mL, 0.09ng / mL, 0.18ng / mL, 0.48ng / mL, or a range formed by any of the above values. In some embodiments, the concentration of IL-33 in the reaction system is 0.18ng / mL.

[0060] In some embodiments, the concentration of the cell line in the reaction system is 1×10 4 / mL to 1×10 6 In some embodiments, the concentration of the cell line in the reaction system is 1×10 5 / mL to 1×10 6 In some embodiments, the concentration of the cell line in the reaction system is 2×10 5 / mL to 6×10 5In some embodiments, the concentration of the cell line in the reaction system is 1×10 5 / mL, 1.5×10 5 / mL, 2×10 5 / mL, 2.5×10 5 / mL, 3×10 5 / mL, 3.5×10 5 / mL, 4×10 5 / mL, 4.5×10 5 / mL, 5×10 5 / mL, 5.5×10 5 / mL, 6×10 5 / mL, 6.5×10 5 / mL, 7×10 5 / mL, 7.5×10 5 / mL, 8×10 5 / mL, 9×10 5 / mL, 1×10 6 In some embodiments, the concentration of the cell line in the reaction system is 2×10 5 / mL, 2.5×10 5 / mL, 3×10 5 / mL, 3.5×10 5 / mL, 4×10 5 / mL, 4.5×10 5 / mL, 5×10 5 / mL, 5.5×10 5 / mL, 6×10 5 In some embodiments, the concentration of the cell line in the reaction system is 4×10 5 Pieces / mL.

[0061] In some embodiments, the maximum concentration of the antibody or its antigen-binding fragment in the reaction system is 10 μg / mL to 1000 μg / mL. In some embodiments, the maximum concentration of the antibody or its antigen-binding fragment in the reaction system is 100 μg / mL to 800 μg / mL. In some embodiments, the maximum concentration of the antibody or its antigen-binding fragment in the reaction system is 100 μg / mL to 500 μg / mL. In some embodiments, the maximum concentration of the antibody or its antigen-binding fragment in the reaction system is 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 350 μg / mL, 400 μg / mL, 450 μg / mL, 500 μg / mL, 600 μg / mL, 700 μg / mL, 800 μg / mL, or a range formed by any of the above values. In some embodiments, the maximum concentration of the antibody or antigen-binding fragment thereof in the reaction system is 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, or a range formed by any of the above values. In some embodiments, the maximum concentration of the antibody or antigen-binding fragment thereof in the reaction system is 200 μg / mL.

[0062] In some embodiments, the dilution multiple of the gradient dilution of the antibody or its antigen-binding fragment in the reaction system is 1-10 times. In some embodiments, the dilution multiple of the gradient dilution of the antibody or its antigen-binding fragment in the reaction system is 2-6 times. In some embodiments, the dilution multiple of the gradient dilution of the antibody or its antigen-binding fragment in the reaction system is 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, or the range formed by any of the above values. In some embodiments, the dilution multiple of the gradient dilution of the antibody or its antigen-binding fragment in the reaction system is 2 times, 3 times, 4 times, 5 times, 6 times, or the range formed by any of the above values. In some embodiments, the dilution multiple of the gradient dilution of the antibody or its antigen-binding fragment in the reaction system is 4 times.

[0063] In some embodiments, the contact time is 1 minute to 180 minutes. In some embodiments, the contact time is 5 minutes to 120 minutes. In some embodiments, the contact time is 10 minutes to 60 minutes. In some embodiments, the contact time is 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes or the range formed by any of the above values. In some embodiments, the contact time is 30 minutes.

[0064] In some specific embodiments, the method for detecting the biological activity of the anti-IL-33 / ST2 axis antibody or its antigen-binding fragment comprises the steps of:

[0065] The anti-ST2 antibody or its antigen-binding fragment is gradiently diluted to different concentrations of the anti-ST2 antibody or its antigen-binding fragment, and each concentration of the anti-ST2 antibody or its antigen-binding fragment is contacted with IL-33, wherein the dilution multiple of the gradient dilution of the anti-ST2 antibody or its antigen-binding fragment is 4 times, and the contact time is 30 minutes;

[0066] Then, IL-33, cell lines and each concentration of anti-ST2 antibody or its antigen-binding fragment were placed in the same reaction system for incubation. The incubation time was 5 hours. The deposit number of the cell line was CCTCC NO: C202397. The concentration of IL-33 in the reaction system was 0.18 ng / mL. The maximum concentration of the anti-ST2 antibody or its antigen-binding fragment in the reaction system was 200 μg / mL. The concentration of the cell line in the reaction system was 4×10 5 Pieces / mL;

[0067] Then, a luciferase detection reagent is added to the reaction system, wherein the luciferase detection reagent comprises a cell lysate and a luciferase substrate;

[0068] Then, the fluorescence intensity in each reaction system was detected, and the inhibition curve was obtained based on the fluorescence intensity and the concentration of the anti-ST2 antibody or its antigen-binding fragment, and the IC value of the anti-ST2 antibody or its antigen-binding fragment in blocking the IL-33 / ST2 interaction was obtained. 50 value, thereby obtaining the biological activity of the anti-ST2 antibody or its antigen-binding fragment.

[0069] In some specific embodiments, the method for detecting the biological activity of the anti-IL-33 / ST2 axis antibody or its antigen-binding fragment comprises the steps of:

[0070] The anti-ST2 antibody or its antigen-binding fragment is gradiently diluted to different concentrations of the anti-ST2 antibody or its antigen-binding fragment, and each concentration of the anti-ST2 antibody or its antigen-binding fragment is contacted with IL-33, wherein the dilution multiple of the gradient dilution of the anti-ST2 antibody or its antigen-binding fragment is 4 times, the contact environment is 37° C., containing 5% CO2, and the contact time is 30 minutes;

[0071] Then, IL-33, cell lines and each concentration of anti-ST2 antibody or its antigen-binding fragment were placed in the same reaction system for incubation, wherein the incubation environment was 37°C and contained 5% CO2, the incubation time was 5 hours, the preservation number of the cell line was CCTCC NO: C202397, the concentration of IL-33 in the reaction system was 0.18 ng / mL, the maximum concentration of anti-ST2 antibody or its antigen-binding fragment in the reaction system was 200 μg / mL, and the concentration of the cell line in the reaction system was 4×10 5 Pieces / mL;

[0072] Then, a luciferase detection reagent is added to the reaction system, wherein the luciferase detection reagent comprises a cell lysate and a luciferase substrate;

[0073] Then, the fluorescence intensity in each reaction system was detected, and the inhibition curve was obtained based on the fluorescence intensity and the concentration of the anti-ST2 antibody or its antigen-binding fragment, and the IC value of the anti-ST2 antibody or its antigen-binding fragment in blocking the IL-33 / ST2 interaction was obtained. 50 value, thereby obtaining the biological activity of the anti-ST2 antibody or its antigen-binding fragment.

[0074] In some specific embodiments, the method for detecting the biological activity of the anti-IL-33 / ST2 axis antibody or its antigen-binding fragment comprises the steps of:

[0075] The anti-ST2 antibody or its antigen-binding fragment is gradiently diluted to different concentrations of the anti-ST2 antibody or its antigen-binding fragment, wherein the dilution multiple of the gradient dilution of the anti-ST2 antibody or its antigen-binding fragment is 4 times, each concentration of the anti-ST2 antibody or its antigen-binding fragment is added to the cell culture plate with IL-33, and the cell culture plate is placed in an environment of 37° C. and 5% CO2 for 30 minutes;

[0076] Then, the cell line with the deposit number of CCTCC NO: C202397 was added to the cell culture plate. At this time, the density of the cell line in the cell culture plate was 4×10 5 / mL, the concentration of IL-33 in the cell culture plate is 0.18ng / mL, the maximum concentration of the anti-ST2 antibody or its antigen-binding fragment in the cell culture plate is 200μg / mL, and the cell culture plate is placed in an environment of 37°C and 5% CO2 for 5 hours;

[0077] Then, a luciferase detection reagent is added into each well of the cell culture plate, wherein the luciferase detection reagent comprises a cell lysate and a luciferase substrate;

[0078] Then, the fluorescence intensity in each well of the cell culture plate is detected, and an inhibition curve is obtained based on the fluorescence intensity and the concentration of the anti-ST2 antibody or its antigen-binding fragment, and the IC value of the anti-ST2 antibody or its antigen-binding fragment in blocking the IL-33 / ST2 interaction is obtained. 50 value, thereby obtaining the biological activity of the anti-ST2 antibody or its antigen-binding fragment.

[0079] In some specific embodiments, the method for detecting the biological activity of the anti-IL-33 / ST2 axis antibody or its antigen-binding fragment comprises the steps of:

[0080] IL-33 was diluted to 0.72 ng / mL and added to the cell culture plate at a volume of V1 / well;

[0081] Then, the anti-ST2 antibody or its antigen-binding fragment is diluted in a gradient manner, wherein the starting concentration of the anti-ST2 antibody or its antigen-binding fragment is 800 μg / mL and the dilution multiple is 4 times, and added to the cell culture plate at a volume of V2 / well, and the cell culture plate is placed in an environment of 37° C. and 5% CO2 for 30 minutes;

[0082] Then, the cell line with the deposit number of CCTCC NO: C202397 was taken and the cell density was adjusted to 8×10 5 / mL, and added into the cell culture plate at a volume of V3 / well, and placed the cell culture plate in an environment of 37°C and 5% CO2 for 5 hours;

[0083] Then, a luciferase detection reagent is added into each well of the cell culture plate at a volume of V4 / well, wherein the luciferase detection reagent comprises a cell lysate and a luciferase substrate;

[0084] Then, the fluorescence intensity in each well of the cell culture plate is detected, and an inhibition curve is obtained based on the fluorescence intensity and the concentration of the anti-ST2 antibody or its antigen-binding fragment, and the IC value of the anti-ST2 antibody or its antigen-binding fragment in blocking the IL-33 / ST2 interaction is obtained. 50 value, thereby obtaining the biological activity of the anti-ST2 antibody or its antigen-binding fragment;

[0085] Among them, V1:V2:V3:V4 is 1:1:2:4.

[0086] In some specific embodiments, the method for detecting the biological activity of the anti-IL-33 / ST2 axis antibody or its antigen-binding fragment comprises the steps of:

[0087] IL-33 was diluted to 0.72 ng / mL and added to the cell culture plate at a volume of 25 / well;

[0088] Then, the anti-ST2 antibody or its antigen-binding fragment is diluted in a gradient manner, wherein the starting concentration of the anti-ST2 antibody or its antigen-binding fragment is 800 μg / mL and the dilution multiple is 4 times, and the anti-ST2 antibody or its antigen-binding fragment is added to the cell culture plate at a volume of 25 / well, and the cell culture plate is placed in an environment of 37° C. and 5% CO2 for 30 minutes;

[0089] Then, the cell line with the deposit number of CCTCC NO: C202397 was taken and the cell density was adjusted to 8×10 5 pcs / mL, and added into the cell culture plate at a volume of 50 / well, and placed the cell culture plate in an environment of 37°C and 5% CO2 for 5 hours;

[0090] Then, a luciferase detection reagent with a volume of 100 μL / well was added into each well of the cell culture plate, wherein the luciferase detection reagent comprises a cell lysate and a luciferase substrate;

[0091] Then, the fluorescence intensity in each well of the cell culture plate is detected, and an inhibition curve is obtained based on the fluorescence intensity and the concentration of the anti-ST2 antibody or its antigen-binding fragment, and the IC value of the anti-ST2 antibody or its antigen-binding fragment in blocking the IL-33 / ST2 interaction is obtained. 50 value, thereby obtaining the biological activity of the anti-ST2 antibody or its antigen-binding fragment.

[0092] The detection method of the present application has good accuracy, and the recovery rate is 80% to 120%, and the recovery rate is the ratio of the measured value to the theoretical value of the biological activity of the antibody or its antigen-binding fragment. In some embodiments, the recovery rate is 85% to 95%, 90% to 115%, 90% to 110%, or 95% to 105%. In some embodiments, the recovery rate is 89.58% to 112.11%, 95.77% to 110.18%, 95.93% to 112.11%, 98.76% to 111.00%, 89.58% to 110.53%, or 90.99% to 105.31%. In some embodiments, the recovery is between 86.32% and 119.12%, between 86.32% and 117.73%, between 86.67% and 116.38%, between 86.89% and 111.00%, between 87.71% and 119.12%, or between 88.29% and 118.24%. In some embodiments, the recovery is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, or 120%.

[0093] The detection method of the present application has good linearity, and the coefficient of determination of the linear regression equation fitting of the measured value and the theoretical value of the biological activity of the antibody or its antigen-binding fragment is greater than 0.9. In some embodiments, the coefficient of determination is greater than 0.91, greater than 0.92, greater than 0.93, greater than 0.94, greater than 0.95, greater than 0.96, greater than 0.97, greater than 0.98 or greater than 0.99. In some embodiments, the coefficient of determination is greater than 0.95. In some embodiments, the coefficient of determination is greater than 0.99.

[0094] The detection method of the present application has good precision, and the relative standard deviation (RSD) of multiple measurements of the biological activity of the antibody or its antigen-binding fragment is less than 12%, preferably less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1%.

[0095] In some embodiments, the detection method has good specificity. The specificity is also called specificity. In some embodiments, the detection method of the present application can specifically detect the biological activity of anti-IL-33 / ST2 axis antibodies or antigen-binding fragments thereof. In some embodiments, the detection method can specifically detect the biological activity of anti-ST2 antibodies or antigen-binding fragments thereof or anti-IL-33 antibodies or antigen-binding fragments thereof. In some embodiments, the detection method can be used for in vitro determination or evaluation of the inhibitory or blocking effect of anti-IL-33 / ST2 axis antibodies or antigen-binding fragments on IL-33 / ST2 interactions. In some embodiments, the detection method can be used for in vitro determination or evaluation of the inhibitory or blocking effect of anti-ST2 antibodies or antigen-binding fragments thereof or anti-IL-33 antibodies or antigen-binding fragments on IL-33 / ST2 interactions. In some embodiments, the detection method is not suitable for the detection of biological activity of non-anti-IL-33 / ST2 axis antibodies or antigen-binding fragments thereof.

[0096] In some embodiments, the detection method has a wide measurement range and can accurately measure within the biological activity range of 20% to 200%; preferably, the detection method can accurately measure within the biological activity range of 60% to 160%.

[0097] On the other hand, the present application provides a method for constructing the cell line described in the present application, comprising the steps of:

[0098] (a) providing a first vector and a second vector, wherein the first vector contains a first expression cassette, and the second vector contains a second expression cassette, wherein the first expression cassette expresses an exogenous ST2 gene, and the second expression cassette expresses an exogenous reporter gene regulated by NF-κB;

[0099] (b) introducing the first vector and the second vector into cells, and screening to obtain the cell line described in the present application.

[0100] In some embodiments, the first vector and the second vector are the same vector or different vectors.

[0101] In some embodiments, the first vector and the second vector are introduced into the cell sequentially, or are introduced into the cell simultaneously.

[0102] In some embodiments, the cell line is a human kidney epithelial cell line. In some embodiments, the cell line is a HEK293 cell or a cell derived therefrom. In some embodiments, the cell line is a HEK293T cell.

[0103] In some embodiments, the screening includes resistance screening. In some embodiments, the screening includes first stage screening and second stage screening. In some embodiments, the first stage screening and second stage screening both use resistance screening. In some embodiments, the first stage screening and second stage screening use blasticidin, zeocin, geneticin and / or kanamycin resistance screening. In some embodiments, the first stage screening uses blasticidin resistance screening. In some embodiments, the first stage screening uses zeocin resistance screening. In some embodiments, the second stage screening uses zeocin and blasticidin resistance screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0104] Figure 1 The figure shows the dose-effect curve of monoclonal cells obtained after the first round of screening under TNF-α stimulation during the construction of reporter gene cell lines;

[0105] Figure 2 The binding curve of anti-ST2 antibody to V-01 cells is shown, and the binding of isotype control antibody IgG2 is shown as a negative control;

[0106] Figures 3A-3F The V-01 cells were expressed as 1×10 5 / mL, 2×10 5 / mL, 4×10 5 / mL and 8×10 5 After plating at a cell density of 100 cells / mL, the cells were incubated with IL-33 for 2 h ( Figure 3A )、4h( Figure 3B )、5h( Figure 3C )、6h( Figure 3D )、8h( Figure 3E ) or 24h( Figure 3F ) after the dose-effect curve;

[0107] Figure 4 The dose-effect curve of the inhibitory effect of anti-ST2 antibody on the expression of luciferase in V-01 cells stimulated by different concentrations of IL-33 is shown, where EC 50 ,EC 60 ,EC 70 ,EC 80 ,EC 90 and EC 100 corresponding to 0.03 ng / mL, 0.06 ng / mL, 0.09 ng / mL, 0.18 ng / mL, 0.48 ng / mL, and 8.5 ng / mL of IL-33;

[0108] Figures 5A-5C The dose-effect curve of the inhibitory effect of anti-ST2 antibody on IL-33-stimulated V-01 cell luciferase expression is shown. Figure 5A The starting point of the dilution of anti-ST2 antibody was 800 μg / mL (final concentration in the plate), and the dilution was 4-fold. Figure 5B The starting point of the dilution of anti-ST2 antibody was 500 μg / mL (final concentration in the plate), 4-fold serial dilution or 5-fold serial dilution. Figure 5C The starting point of dilution of anti-ST2 antibody was 200 μg / mL (final concentration in the plate), and the dilution was 4-fold;

[0109] Figure 6 The dose-effect curves showing the inhibitory effects of different antibodies on IL-33-stimulated V-01 cell luciferase expression;

[0110] Fig. 7A The dose-effect curves show the inhibitory effect of anti-ST2 antibody on the expression of luciferase in V-01 cells of different generations stimulated by IL-33; Figure 7B The signal-to-noise ratio of the inhibitory effect of anti-ST2 antibody on luciferase expression in V-01 cells of different passages stimulated by IL-33 is shown;

[0111] Fig. 8A The dose-effect curves showing the inhibitory effect of anti-ST2 antibody on luciferase expression in V-01 cells stimulated by IL-33 in the presence of different amounts of luciferase detection reagent; Figure 8B The signal-to-noise ratio of the inhibitory effect of anti-ST2 antibody on luciferase expression in V-01 cells stimulated by IL-33 in the presence of different amounts of luciferase detection reagent is shown;

[0112] Fig. 9A The dose-effect curve of the inhibitory effect of anti-ST2 antibody on luciferase expression in V-01 cells stimulated by IL-33 after adding luciferase detection reagent and incubating for different time periods is shown; Fig. 9B The signal-to-noise ratio of the inhibitory effect of anti-ST2 antibody on luciferase expression in V-01 cells stimulated by IL-33 is shown after adding luciferase detection reagent and incubating for different time periods;

[0113] Fig. 10A and 10B The dose-effect curves of different antibodies on the inhibition of IL-33-stimulated V-01 cells expressing luciferase are shown. Fig. 10A Anti-IL-33 antibody, Fig. 10B is anti-ST2 antibody; Fig. 10C The dose-effect curves show the inhibitory effect of anti-ST2 antibody on luciferase expression in V-01 cells stimulated by IL-33 in the presence of different luciferase detection reagents.

[0114] Definition and Explanation

[0115] Unless otherwise specified, the following terms used in this application have the following meanings. A particular term should not be considered to be uncertain or unclear in the absence of a special definition, but should be understood according to the common meaning in the art. When a trade name appears in this article, it is intended to refer to the corresponding commodity or its active ingredient.

[0116] The term "antibody" is used in the broadest sense to cover natural antibodies and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies and multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies), and various antibody structures of single-chain antibodies, as long as they show the desired antigen-binding activity.

[0117] The term "multi-specific" means that the antibody can specifically bind to a variety of different antigenic determinants, for example, it can specifically bind to two (i.e., "bispecific") or more different antigenic determinants. Antigenic determinants in this article are synonymous with antigenic epitopes. Generally, bispecific antibodies contain two antigen binding sites, each of which is specific for a different antigenic determinant. Different antigenic determinants can be expressed on the same or different cells. Different antigenic determinants can be different due to different antigen types, or they can exist on the same antigen. Antigenic determinants are special chemical groups with a certain composition and structure on the surface of antigenic molecules or other parts, which can specifically bind to their corresponding antibodies or sensitized lymphocytes.

[0118] The "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the function of specifically binding to an antigen (e.g., ST2 protein). It has been confirmed that the antigen-binding function of an antibody can be implemented by a fragment of a full-length antibody. Examples encompassed in the term "antigen-binding fragment" of an antibody include: (i) Fab fragment: a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragment, a bivalent fragment of two Fab fragments connected by a disulfide bridge in the hinge region; (iii) Fd fragment consisting of VH and CH1 domains; (iv) Fv fragment consisting of VL and VH domains of an antibody single arm; (v) dAb fragment consisting of VH domains (see Ward et al., Nature. 341: 544-546 (1989)); and (vi) nanobodies, an antibody comprising a single variable domain and two constant domains. In addition, although the two domains VL and VH of the Fv fragment are encoded by different genes, VH and VL can be connected into a single protein chain by a recombinant method through a linker, wherein VL and VH are paired to form a monovalent molecule called single-chain Fv (scFv) (see Bird et al., Science. 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. 85: 5879-5883 (1988)), and these single-chain antibodies are also included in the term antigen-binding fragment. These antibody fragments can be obtained by conventional techniques known to those skilled in the art, and the fragments can be functionally screened by the same method as full-length antibodies.

[0119] As used herein, the term “EC 50 ", i.e., half maximal effective concentration, refers to the concentration of an antibody that induces a response that is 50% of the maximal response, i.e., halfway between the maximal response and the baseline. As used herein, "EC 60 ”, “EC 70 ”, “EC 80 ” or “EC 90 ” refers to the concentration of antibody that induces a response that is 60%, 70%, 80% or 90% of the maximum response, respectively. “EC 100 ” refers to the concentration of antibody that induces the maximum response. 50 ”, “EC 60 ”, “EC 70 ”, “EC 80 ”, “EC 90 ” and “EC 100 " can be measured by ELISA or FACS analysis or any other method known in the art.

[0120] As used herein, "about" means within the acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean within 1 or more than 1 standard deviation according to the practice in the art. Alternatively, "about" may mean a range of up to ±5%, such as fluctuations within ±2%, within ±1%, or within ±0.5% of a given specific numerical range. When a specific value is given in the scope of the present application, unless otherwise stated, the meaning of "about" should be considered to be within the acceptable error range for that specific value. In this article, unless otherwise stated, the values ​​of recovery, determination coefficient, relative standard deviation, step parameters or conditions (such as concentration, time, temperature, etc.) are all modified by "about" by default.

[0121] The terms "comprise," "comprises," or "comprising" and their equivalents (for example, contain, contains, containing, include, includes, and including) should be understood as "including but not limited to," meaning that in addition to the listed elements, components, and steps, other unspecified elements, components, and steps may also be included.

[0122] Herein, singular terms include plural referents and vice versa unless the context clearly dictates otherwise. DETAILED DESCRIPTION

[0123] The present application also provides the following specific implementation schemes, but the protection scope of the present application is not limited thereto:

[0124] Embodiment 1. A cell line, wherein the cell line is a mammalian cell, and a first expression cassette and a second expression cassette are integrated into the genome of the cell line, wherein the first expression cassette expresses an exogenous ST2 gene, and the second expression cassette expresses an exogenous reporter gene regulated by NF-κB.

[0125] Embodiment 2. The cell line according to Embodiment 1, wherein the exogenous ST2 gene is a human ST2 gene or a monkey ST2 gene.

[0126] Embodiment 3. The cell line according to Embodiment 1 or 2, wherein the transcriptional expression of the reporter gene is dependent on the activation of an NF-κB response element.

[0127] Embodiment 4. A cell line according to any one of Embodiments 1-3, wherein the reporter gene is a luciferase reporter gene.

[0128] Embodiment 5. The cell line according to any one of Embodiments 1-4, wherein the cell line is a HEK293 cell or a cell derived therefrom; preferably, the cell line is a HEK293T cell.

[0129] Embodiment 6. A cell line according to any one of Embodiments 1-5, wherein the preservation number of the cell line is CCTCC NO: C202397.

[0130] Embodiment 7. A cell line, wherein the deposit number of the cell line is CCTCC NO: C202397.

[0131] Embodiment 8. A kit comprising the cell line described in any one of Embodiments 1-7.

[0132] Embodiment 9. Use of the cell line according to any one of Embodiments 1 to 7 for detecting the biological activity of anti-IL-33 / ST2 axis antibodies or antigen-binding fragments thereof.

[0133] Embodiment 10. The use according to Embodiment 9, wherein the anti-IL-33 / ST2 axis antibody or its antigen-binding fragment comprises an anti-ST2 antibody or its antigen-binding fragment and an anti-IL-33 antibody or its antigen-binding fragment.

[0134] Embodiment 11. The use according to embodiment 9 or 10, wherein the biological activity includes binding activity and blocking activity.

[0135] Embodiment 12. A method for detecting the biological activity of an anti-IL-33 / ST2 axis antibody or an antigen-binding fragment thereof, comprising the steps of:

[0136] (1) Providing a cell line according to any one of embodiments 1-7;

[0137] (2) contacting the antibody or its antigen-binding fragment with IL-33, or contacting the antibody or its antigen-binding fragment with a cell line;

[0138] (3) placing the antibody or its antigen-binding fragment, IL-33 and the cell line in the same reaction system for incubation;

[0139] (4) Detecting the expression of the reporter gene in the cell line to obtain the biological activity of the antibody or its antigen-binding fragment.

[0140] Embodiment 13. The method according to embodiment 12, wherein the incubation time is 1 hour to 48 hours, 2 hours to 24 hours, or 4 hours to 8 hours, or the incubation time is 4 hours, 5 hours, 6 hours or 8 hours.

[0141] Embodiment 14. The method according to embodiment 12 or 13, wherein the concentration of IL-33 in the reaction system is 0.01 ng / mL to 10 ng / mL, 0.03 ng / mL to 8.5 ng / mL, 0.03 ng / mL to 0.48 ng / mL, or 0.06 ng / mL to 0.3 ng / mL, or the concentration of IL-33 in the reaction system is 0.03 ng / mL, 0.06 ng / mL, 0.09 ng / mL, 0.18 ng / mL or 0.48 ng / mL.

[0142] Embodiment 15. The method according to any one of embodiments 12-14, wherein the concentration of the cell line in the reaction system is 1×10 4 / mL to 1×10 6 / mL, 1×10 5 / mL to 1×10 6 / mL, or 2×10 5 / mL to 6×10 5 / mL, or the concentration of the cell line in the reaction system is 2×10 5 / mL, 2.5×10 5 / mL, 3×10 5 / mL, 3.5×10 5 / mL, 4×10 5 / mL, 4.5×10 5 / mL, 5×10 5 / mL, 5.5×10 5 / mL or 6×10 5 Pieces / mL.

[0143] Embodiment 16. The method according to any one of embodiments 12-15, wherein:

[0144] The step (2) comprises: gradient diluting the antibody or its antigen-binding fragment into different concentrations of the antibody or its antigen-binding fragment, so that each concentration of the antibody or its antigen-binding fragment is contacted with IL-33, or so that each concentration of the antibody or its antigen-binding fragment is contacted with the cell line; the dilution multiple of the gradient dilution is 1-10 times, or 2-6 times, or the dilution multiple of the gradient dilution is 2 times, 3 times, 4 times, 5 times or 6 times; and,

[0145] The step (3) is: placing IL-33, the cell line and each concentration of the antibody or its antigen-binding fragment in the same reaction system for incubation.

[0146] Embodiment 17. A method according to any one of Embodiments 12-16, wherein the maximum concentration of the antibody or antigen-binding fragment thereof in the reactant is 10 μg / mL to 1000 μg / mL, 100 μg / mL to 800 μg / mL, or 100 μg / mL to 500 μg / mL, or the maximum concentration of the antibody or antigen-binding fragment thereof in the reactant is 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL or 500 μg / mL.

[0147] Embodiment 18. A method according to any one of Embodiments 12-17, wherein the contact time is 1 minute to 180 minutes, 5 minutes to 120 minutes, or 10 minutes to 60 minutes, or the contact time is 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes or 60 minutes.

[0148] Embodiment 19. A method according to any one of Embodiments 12-18, wherein the anti-IL-33 / ST2 axis antibody or its antigen-binding fragment is an anti-ST2 antibody or its antigen-binding fragment or an anti-IL-33 antibody or its antigen-binding fragment.

[0149] Embodiment 20. The method according to embodiment 19, wherein the method comprises the steps of:

[0150] (1) diluting the anti-ST2 antibody or its antigen-binding fragment in a gradient manner to different concentrations of the anti-ST2 antibody or its antigen-binding fragment, and contacting each concentration of the anti-ST2 antibody or its antigen-binding fragment with IL-33, wherein the dilution multiple of the gradient dilution of the anti-ST2 antibody or its antigen-binding fragment is 4 times, and the contact time is 30 minutes;

[0151] (2) IL-33, cell lines, and each concentration of anti-ST2 antibody or its antigen-binding fragment were placed in the same reaction system for incubation. The incubation time was 5 hours. The concentration of IL-33 in the reaction system was 0.18 ng / mL. The highest concentration of anti-ST2 antibody or its antigen-binding fragment in the reaction system was 200 μg / mL. The concentration of cell lines in the reaction system was 4×10 5 Pieces / mL;

[0152] (3) adding a luciferase detection reagent into the reaction system, wherein the luciferase detection reagent comprises a cell lysate and a luciferase substrate;

[0153] (4) Detecting the fluorescence intensity in each reaction system, obtaining an inhibition curve based on the fluorescence intensity and the concentration of the anti-ST2 antibody or its antigen-binding fragment, and obtaining the IC value of the anti-ST2 antibody or its antigen-binding fragment in blocking the IL-33 / ST2 interaction. 50 value, thereby obtaining the biological activity of the anti-ST2 antibody or its antigen-binding fragment.

[0154] Embodiment 21. A method for detecting the biological activity of an anti-ST2 antibody or an antigen-binding fragment thereof, comprising the steps of:

[0155] (1) diluting the anti-ST2 antibody or its antigen-binding fragment in a gradient manner to different concentrations of the anti-ST2 antibody or its antigen-binding fragment, and contacting each concentration of the anti-ST2 antibody or its antigen-binding fragment with IL-33, wherein the dilution multiple of the gradient dilution of the anti-ST2 antibody or its antigen-binding fragment is 4 times, and the contact time is 30 minutes;

[0156] (2) IL-33, the cell line described in any one of Embodiments 1 to 7, and each concentration of the anti-ST2 antibody or its antigen-binding fragment are placed in the same reaction system for incubation. The incubation time is 5 hours. The concentration of IL-33 in the reaction system is 0.18 ng / mL. The maximum concentration of the anti-ST2 antibody or its antigen-binding fragment in the reaction system is 200 μg / mL. The concentration of the cell line in the reaction system is 4×10 5 Pieces / mL;

[0157] (3) adding a luciferase detection reagent into the reaction system, wherein the luciferase detection reagent comprises a cell lysate and a luciferase substrate;

[0158] (4) Detecting the fluorescence intensity in each reaction system, obtaining an inhibition curve based on the fluorescence intensity and the concentration of the anti-ST2 antibody or its antigen-binding fragment, and obtaining the IC value of the anti-ST2 antibody or its antigen-binding fragment in blocking the IL-33 / ST2 interaction. 50 value, thereby obtaining the biological activity of the anti-ST2 antibody or its antigen-binding fragment.

[0159] Embodiment 22. A method for constructing the cell line of any one of embodiments 1-7, comprising the steps of:

[0160] (a) providing a first vector and a second vector, wherein the first vector contains a first expression cassette, and the second vector contains a second expression cassette, wherein the first expression cassette expresses an exogenous ST2 gene, and the second expression cassette expresses an exogenous reporter gene regulated by NF-κB; (b) introducing the first vector and the second vector into cells, and obtaining the cell line through screening.

[0161] For the sake of clarity, the present application is further described with examples, but the examples do not limit the scope of the present application. The heavy chain amino acid sequence of the anti-ST2 antibody used in the examples is shown in SEQ ID NO: 1, and the light chain amino acid sequence is shown in SEQ ID NO: 2.

[0162] Example 1: Construction of reporter gene cell line

[0163] The NF-κB response element-luciferase reporter gene sequence (NF-κB / RE-Luc2P gene sequence) was obtained and cloned into the pLenti6.3 vector containing the blasticidin resistance gene to construct a recombinant vector A; the gene sequence encoding ST2 was synthesized and cloned into the pLenti6.3 vector containing the zeocin resistance gene to construct a recombinant vector B.

[0164] The recombinant vector A was transfected into HEK293T cells (source: Chinese Academy of Sciences Cell Bank), and cultured with DMEM medium (Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 5 μg / mL blasticidin (InvivoGen, catalog number: ant-bl-1), and the first round of mixed clone cell suspension was collected. The first round of mixed clone cell suspension was diluted and monoclonal culture was performed, and dozens of monoclonal cells were collected for expansion culture, with a concentration of 2.5×10 5 1000 cells / well were plated in a 96-well white cell culture plate, and gradient dilutions (the plate concentration was 20 ng / mL, 4-fold gradient dilution) of TNF-α (R&D, catalog number: 10291-TA-020) were added to the plate, and incubated in a 37°C, 5% CO2 incubator for 5-6 hours. After incubation, ONE-Glo TM The luciferase detection reagent was used to detect the chemiluminescent signal value on an ELISA instrument (PE company). A dose-effect curve was drawn for each clone. The clone with the largest ratio of the maximum to the minimum luminescent signal value on the dose-effect curve was selected as the monoclonal cell after the first round of screening. The dose-effect curve is shown in Figure 1 As shown, EC 50 It is 2.3ng / mL.

[0165] The recombinant vector B was transfected into the monoclonal cells obtained after the first round of screening, and cultured with DMEM medium containing 10% FBS, 400 μg / mL bleomycin (Zeocin, Gibco, catalog number: 10131-027) and 5 μg / mL blasticidin, and the second round of mixed clone cell suspension was collected. The second round of mixed clone cell suspension was diluted and monoclonal culture was performed, and dozens of monoclonal cells were collected for expansion culture, and PE-labeled anti-hST2 antibody (R&D, catalog number: FAB5231P) was added for incubation, and detected by flow cytometry. After 4 weeks of screening, the reporter gene cell line V-01 with stable high expression of ST2 was obtained.

[0166] The obtained reporter gene cell line V-01 was deposited in the China Center for Type Culture Collection (CCTCC) on May 9, 2023, and was classified and named: Human embryonic kidney 293T-ST2-NFκB reporter gene cell HEK293T-ST2-NFκB Luciferase, with a deposit number of CCTCC NO: C202397, and a deposit address of: Wuhan University, Wuhan, China, 430072.

[0167] Anti-ST2 antibody was diluted to 133 nM with PBS, and then diluted to 0.008 nM with a 1:3 dilution gradient, for a total of 8 concentration gradients. V-01 cells in the logarithmic growth phase were taken and the cell density was adjusted to 2×10 6 / mL, inoculated into 96-well plates at 100μL / well. Then, the diluted anti-ST2 antibody was added to the 96-well plates, 100μL / well, and incubated at 4℃ in the dark for 1 hour. After washing the cells with PBS, PE Donkey-anti-human working solution (Jackson Immuno Reseach) was added, 100μL / well, and incubated at 4℃ in the dark for 30 minutes. After washing the cells with PBS, PBS was added to resuspend the cells. Detected by flow cytometry (BD Company), the results are as follows Figure 2 As shown, the binding of anti-ST2 antibody to EC 50 The maximum binding MFI was 0.4676 nM and 204065, indicating that the anti-ST2 antibody can bind specifically to V-01 cells.

[0168] Example 2: Antibody biological activity detection

[0169] V-01 cells in the logarithmic growth phase were taken and Opti-MEM medium (Gibco) was added to adjust the density to 1×10 5 / mL, 2×10 5 / mL, 4×10 5 / mL, 8×10 5 / mL. Cells of different densities were inoculated into 96-well cell culture plates, 50μL / well. IL-33 protein (Sino Biological Technology Co., Ltd.) was diluted with Opti-MEM medium to an initial dilution concentration of 50ng / mL (final concentration in the plate), and then 4-fold gradient dilution was performed, for a total of 12 concentration gradients. The prepared IL-33 solution was added to the 96-well cell culture plates with cells at a volume of 50μL / well, and incubated in a 37°C, 5% CO2 cell culture incubator for 2h, 4h, 5h, 6h, 8h, and 24h, respectively. After incubation, take out the 96-well cell culture plate and let it stand to room temperature. Then, Bright-Glo luciferase detection reagent (Promega) was added to each well at a volume of 100 μL, and incubated at room temperature in the dark for 2-3 minutes to completely lyse the cells. The chemiluminescence value of the 96-well cell culture plate was then read using a Spark multi-function microplate reader (Tecan) to accurately read the relative light unit (RLU) value.

[0170] The results are as follows Figures 3A-3F As shown in the figure, when the cell plating density is different, the higher the cell density, the higher the signal-to-noise ratio (sample RLU / blank control RLU). Therefore, the cell plating density of 8×10 5 After 2 hours of incubation, there was no upper platform. After 24 hours of incubation, the curve had no upper platform. The curves of 4 hours, 5 hours, 6 hours and 8 hours of incubation had good morphology and high signal-to-noise ratio. The incubation time of 5 hours was short and the signal value and signal-to-noise ratio were both at a high level. Therefore, the incubation time of 5 hours was selected.

[0171] Take V-01 cells in the logarithmic growth phase and adjust the cell density to 8×10 5 / mL, inoculated into a 96-well cell culture plate at a volume of 50μL / well. Dilute the IL-33 protein with Opti-MEM medium to an initial dilution concentration of 50ng / mL (final concentration in the plate), and then dilute it 4 times, for a total of 12 concentration gradients. Add the prepared IL-33 solution to the 96-well cell culture plate with cells at a volume of 50μL / well, incubate for 5h in a 37°C, 5% CO2 cell culture incubator, add luciferase detection reagent (Promega), and use a microplate reader to detect the expression of luciferase. The four-parameter regression calculation method is used, with the logarithm of the IL-33 concentration as the horizontal axis and the average chemiluminescence value as the vertical axis to calculate the EC of IL-33. 50 ,EC 60 ,EC 70 ,EC 80 ,EC 90 and EC 100The values ​​were 0.03ng / mL, 0.06ng / mL, 0.09ng / mL, 0.18ng / mL, 0.48ng / mL and 8.5ng / mL respectively.

[0172] Anti-ST2 antibody was diluted with Opti-MEM medium. The anti-ST2 antibody used 500 μg / mL (final concentration in the plate) as the starting point of dilution, and 5-fold gradient dilution was performed, with a total of 12 concentration gradients. The prepared anti-ST2 antibody solution was added to the 96-well cell all-white plate at a volume of 25 μL / well. IL-33 was diluted according to the above 6 different EC values ​​(i.e., IL-33 was diluted to a final concentration of 0.03 ng / mL, 0.06 ng / mL, 0.09 ng / mL, 0.18 ng / mL, 0.48 ng / mL and 8.5 ng / mL in the plate), and added to the 96-well cell all-white plate at a volume of 25 μL / well, and mixed and incubated in a cell culture incubator at 37°C and 5% CO2 for 30 minutes. Take out the 96-well cell all-white plate, add 50 μL of V-01 cell suspension (density 8×10 5 / mL), and cultured at 37°C, 5% CO2 for 5 hours. Then, luciferase detection reagent (Promega) was added, and the expression of luciferase was detected by microplate reader. The results are shown in Figure 4 As shown, except EC 100 Except for 8.5 ng / mL, other concentrations can produce good dose-effect curves. Among them, 0.18 ng / mL of IL-33 produced the best inhibition curve and better signal-to-noise ratio. Therefore, 0.18 ng / mL was selected as the stimulation concentration of IL-33.

[0173] The anti-ST2 antibody was diluted to 800 μg / mL (final concentration in the plate) as the starting point, and the dilution was performed in 4-fold gradients, with a total of 12 concentration gradients. The prepared anti-ST2 antibody solution was added to a 96-well plate at a volume of 25 μL / well, and then 25 μL / well of IL-33 (final concentration in the plate was 0.18 ng / mL) was added, and mixed and incubated in a carbon dioxide incubator at 37°C for half an hour. The V-01 cells in the logarithmic growth phase were removed and the cell density was adjusted to 8×10 5 pcs / mL, and added to the 96-well plate at a volume of 50 μL per well after the incubation. Incubate for 5 h at 37°C and 5% CO2, then add luciferase detection reagent (Promega) for detection. The optimal starting concentration of anti-ST2 antibody was selected based on the upper and lower platforms and linear range of the dose-effect curve obtained. Figure 5A As shown in the figure, a complete dose-effect curve was obtained. In order to obtain a more optimized four-parameter curve, we selected 500 μg / mL as the starting dilution concentration of the anti-ST2 antibody.

[0174] Select 4-fold and 5-fold dilution multiples for gradient dilution, and use 500 μg / mL (final concentration in the plate) as the starting concentration of anti-ST2 antibody. Repeat the above experiment. After incubation, add luciferase detection reagent (Promega) for detection. Select the optimal dilution multiple of anti-ST2 antibody based on whether the upper and lower platforms of the dose-effect curve and the points in the linear range are evenly distributed. The results are shown in Figure 5B As shown in the figure, the curves obtained by the two dilution multiples are very complete, but the 5-fold dilution multiple is higher and prone to errors, so 4-fold is selected as the dilution multiple of the anti-ST2 antibody. In order to reduce the number of upper and lower platform points of the dose-effect curve, 200 μg / mL (final concentration in the plate) is selected as the starting dilution concentration of the anti-ST2 antibody, and 4-fold gradient dilution is performed. The dose-effect curve is shown in the figure below. Figure 5C shown.

[0175] Example 3: Methodological Validation

[0176] 1. Methodological Validation Experimental Steps

[0177] IL-33 protein (Sino Biological Technology Co., Ltd.) was diluted to 0.72 ng / mL with Opti-MEM medium (Gibco) and added to a 96-well culture plate at a volume of 25 μL / well. Anti-ST2 antibody was diluted 4 times with Opti-MEM medium at 200 μg / mL (final concentration in the plate) as the starting point of dilution. The prepared anti-ST2 antibody solution was added to the 96-well culture plate with IL-33 at a volume of 25 μL / well, and mixed and incubated in a cell culture incubator at 37°C and 5% CO2 for 30 minutes.

[0178] Take V-01 cells in the logarithmic growth phase and adjust the cell density to 8×10 5 After the incubation, 50 μL / well of the 96-well culture plate was added, and the plate was incubated at 37°C and 5% CO2 for 5 hours. Then, luciferase detection reagent was added at 100 μL / well.

[0179] 2. Accuracy

[0180] Accuracy refers to the degree of closeness or consistency between the true value or publicly recognized reference value and the actual measured value obtained by measuring the sample according to this method. It is sometimes also called authenticity. The accuracy of the method is usually expressed in terms of recovery rate. The closer the actual measured value is to the reference value, the better the accuracy of the method.

[0181] The anti-ST2 antibody with an initial dilution concentration (final concentration in the plate) of 200 μg / mL was used as the 100% reference titer level for accuracy verification, and was diluted 4 times to obtain a gradient dilution anti-ST2 antibody reference product; the anti-ST2 antibody with an initial dilution concentration (final concentration in the plate) of 128 μg / mL, 160 μg / mL, 200 μg / mL, 250 μg / mL and 312 μg / mL was used as 64%, 80%, 100%, 125% and 156% titer levels, and was diluted 4 times to obtain 5 groups of gradient dilution anti-ST2 antibody samples with different titer levels. The three experimental operators referred to the above "methodological verification experimental steps" and conducted parallel experiments for 3 days (respectively recorded as D1, D2 and D3) for each titer level, with 3 effective parallel experiments per day, and 2 replicates were set for each experiment. The IC was calculated by fitting the curve using the four-parameter regression method, with the logarithm of the ratio of the anti-ST2 antibody concentration at each titer level to the corresponding titer level as the abscissa and the average chemiluminescence value of two replicate wells as the ordinate. 50 Value, through IC 50 The relative biological activity was calculated based on the relative biological activity, and the recovery rate was calculated based on the relative biological activity.

[0182] The calculation formula is as follows:

[0183] Recovery rate (%) = measured value of relative biological activity / theoretical value of relative biological activity × 100%;

[0184] Relative biological activity (%) = anti-ST2 antibody reference IC 50 / Anti-ST2 Antibody Sample IC 50 ×100%.

[0185] The recovery rate and its range results of a single experiment by each experimental operator are shown in Table 1. The recovery rate range is 86.32%-119.12%, which is within the required range of 80%-120%, indicating that the accuracy of this method is good.

[0186] Table 1. Recovery rates of samples at different titer levels

[0187]

[0188]

[0189] 3. Linear

[0190] The measured values ​​of the relative biological activities of anti-ST2 antibodies of five different titer concentrations obtained by three experimental operators in the “accuracy” experiment were linearly fitted with the theoretical values, where the theoretical value is X and the measured value is Y. The results are shown in Table 2. R of the three linear equations 2 All of them are greater than 0.99, indicating that the linearity of this method is good.

[0191] Table 2. Linear fitting of measured values ​​and theoretical values ​​of relative biological activity

[0192] Linear equations <![CDATA[R 2 ]]> Operator 1 Y=0.9312*X+9.287 0.9969 Operator 2 Y=1.01*X-0.6177 0.9982 Operator 3 Y=0.9204*X+9.717 0.9969

[0193] 4. Precision

[0194] Precision refers to the closeness between the results obtained by comparing and analyzing the final measured results by using this method to perform multiple sampling and measurement on the same uniform sample under specific conditions. Precision includes three aspects: repeatability, intermediate precision and reproducibility. Repeatability refers to the coefficient of variation between multiple measurement results obtained by the same experimental operator under the same operating conditions and within a short time interval, which can also be called intra-day precision; intermediate precision refers to the coefficient of variation between experimental results measured when the internal conditions of the same laboratory change, such as those measured at different times are called inter-day precision, and those measured by different analytical measurement experimental operators are called inter-personnel intermediate precision; reproducibility refers to the coefficient of variation between experimental results measured by different experimental operators in different laboratories, which is usually used for collaborative research on statutory standards. The precision verification experiment of this method is mainly completed by measuring repeatability and intermediate precision.

[0195] The anti-ST2 antibody with an initial dilution concentration (final concentration in the plate) of 200 μg / mL was used as the 100% reference titer level for accuracy verification, and was diluted 4 times to obtain a gradient diluted anti-ST2 antibody reference product; the anti-ST2 antibody with an initial dilution concentration (final concentration in the plate) of 128 μg / mL, 160 μg / mL, 200 μg / mL, 250 μg / mL and 312 μg / mL was used as 64%, 80%, 100%, 125% and 156% titer levels, respectively, and was diluted 4 times to obtain 5 groups of gradient diluted anti-ST2 antibody samples with different titer levels.

[0196] Repeatability: The same experimental operator performed parallel experiments for 3 days according to the "Methodology Validation Experimental Procedures", and conducted 3 effective parallel experiments every day, with 2 replicate wells in each experiment. The four-parameter regression calculation method was used, with the logarithm of the ratio of the anti-ST2 antibody concentration at each titer level to the corresponding titer level as the horizontal axis and the average chemiluminescence value of the 2 replicate wells as the vertical axis, and the curve was fitted to calculate the IC 50 value.

[0197] Intermediate precision: Under the same experimental conditions, three different experimental operators performed parallel experiments for three days according to the "Methodology Validation Experimental Procedures", with three effective parallel experiments per day and two replicate wells per experiment. The four-parameter regression calculation method was used, with the logarithm of the ratio of the anti-ST2 antibody concentration at each titer level to the corresponding titer level as the abscissa and the average chemiluminescence value of the two replicate wells as the ordinate, to fit the curve and calculate the IC 50 value.

[0198] The repeatability result of this method was obtained by the coefficient of variation of the relative biological activity of antibodies at each titer level obtained by parallel experiments of each experimental operator within one day. The intermediate precision result of this method was obtained by the coefficient of variation of the relative biological activity of antibodies at each titer level obtained by parallel experiments of 3 experimental operators within 3 days.

[0199] The calculation formula of relative biological activity refers to the formula in "Accuracy".

[0200] The repeatability results are shown in Table 3-1, and the intermediate precision results are shown in Table 3-2. The results show that the precision of this method is good.

[0201] Table 3-1. Repeatability of relative biological activity of anti-ST2 antibodies at different titer levels

[0202]

[0203]

[0204] Table 3-2 Intermediate precision of relative biological activity of anti-ST2 antibodies at different titer levels

[0205]

[0206] 5. Exclusivity

[0207] Specificity, also known as specificity, refers to the ability to correctly determine the components of the sample to be tested without interference from other components, such as impurities, degradation products, matrix, etc. The specificity verification experiment of this method is mainly completed by analyzing the specificity of V-01 cells to antibodies with different targets and the specificity to antibodies with different degrees of denaturation.

[0208] (1) Specificity of antibodies for different targets

[0209] Five non-anti-IL-33 / ST2 axis antibodies were selected: Trastuzumab (anti-HER2 antibody), Denosumab (anti-RANKL antibody), Pembrolizumab (anti-PD-1 antibody), Ramucirumab (anti-VEGF antibody) and Belimumab (anti-BLyS antibody). Anti-ST2 antibody and the above five non-anti-IL-33 / ST2 axis antibodies were parallelly tested according to the "Methodology Validation Experimental Procedures" to detect the expression of cellular luciferase.

[0210] The results are as follows Figure 6 As shown, the five non-anti-IL-33 / ST2 axis antibodies could not inhibit the expression of luciferase in cells, while the anti-ST2 antibody inhibited the expression of luciferase in cells in a dose-dependent manner. The results showed that this method can specifically test the biological activity of anti-ST2 antibodies.

[0211] (2) Specificity for denatured antibodies

[0212] After adding the oxidant AAPH (CAS No. 2997-92-4), the anti-ST2 antibody was subjected to a forced degradation test under different temperature conditions, and the conditions are shown in Table 4. Then, the anti-ST2 antibody treated by the forced degradation test was used to perform the experimental operation according to the "Methodology Validation Experimental Procedures", and the expression of cell luciferase was detected to perform a titer analysis. As shown in Table 4, the relative biological activity of the antibody after the forced degradation test was significantly reduced, indicating that this method can make a judgment on the denaturation of the antibody and has good specificity.

[0213] Relative biological activity (%) = IC of anti-ST2 antibody after forced degradation 50 / Untreated anti-ST2 antibody IC 50 ×100%.

[0214] Table 4. Conditions of forced degradation test and relative biological activity of antibodies after forced degradation treatment

[0215] Test conditions Relative biological activity After adding 2% AAPH, place at 25℃ for 2 days 87％ After adding 2% AAPH, place at 25℃ for 7 days 67％ After adding 2% AAPH, place at 40℃ for 2 days 63％ After adding 2% AAPH, place at 40℃ for 7 days 28％

[0216] 6. Stability

[0217] V-01 cells were cultured continuously, and cells of different generations were frozen when the cells were in good growth condition. In this experiment, cells of the 5th, 15th, and 30th generations were thawed and revived, and cultured for 1-2 generations. Before the experimental determination, the cell state was ensured, and the survival rate was maintained above 95% before subsequent experiments. Specific experimental operations were carried out according to the "Methodology Validation Experimental Steps" to detect the expression of luciferase in cells of different generations.

[0218] The results are as follows Fig. 7A and 7B As shown, there was no significant difference between the dose-effect curves, and there was no significant difference in the signal-to-noise ratio between cells of different generations, P>0.05, with no statistical difference, indicating that the cell line constructed by this method had good generation stability.

[0219] 7. Durability

[0220] Robustness refers to the ability of the method to remain stable and provide reliability within the normal range after slight changes to specific parameters in the analytical method. This methodological experiment verifies whether the experimental results are affected by changing the amount of luciferase detection reagent added and the incubation time.

[0221] (1) Amount of luciferase detection reagent added

[0222] The experimental operation was carried out according to the "Methodology Validation Experimental Procedures", and the luciferase detection reagent was added at a volume of 80 μL / well, 100 μL / well, and 120 μL / well, and the expression of luciferase was detected respectively. The dose-effect curve is shown in Fig. 8A As shown, after adding 80 μL / well, 100 μL / well, or 120 μL / well luciferase assay reagent, the IC of anti-ST2 antibody 50 The values ​​were 0.0909 μg / mL, 0.102 μg / mL, and 0.0937 μg / mL, respectively; Figure 8B As shown, there was no significant difference in the signal-to-noise ratio after adding different amounts of luciferase detection reagent, P>0.05, and there was no statistical difference.

[0223] (2) Incubation time after adding luciferase detection reagent

[0224] The experimental operation was carried out according to the "Methodology Validation Experimental Procedures", and the luciferase expression was detected after incubation for 2 minutes, 10 minutes, and 30 minutes. Fig. 9A As shown, after adding the same luciferase detection reagent and incubating for 2 minutes, 10 minutes, or 30 minutes, the IC of anti-ST2 antibody 50 The values ​​were 0.0902μg / mL, 0.0947μg / mL and 0.103μg / mL respectively; Fig. 9B As shown, there was no significant difference in the signal-to-noise ratio detected at different incubation times, P>0.05, and there was no statistical difference.

[0225] The above results show that the method has good durability.

[0226] 8. Applicability

[0227] The applicability of this method was verified by using different antibodies and different luciferase detection reagents to determine the final experimental results.

[0228] (1) Different antibodies

[0229] Select anti-ST2 antibody and anti-IL-33 antibody, refer to the "Methodology Validation Experimental Procedures" to detect the expression of luciferase in the two antibodies. By analyzing the experimental results, a four-parameter curve is fitted, such as Fig. 10A and 10B As shown in Figure 2, under the stimulation of IL-33 antigen, the selected anti-ST2 antibody and anti-IL-33 antibody (Itepekimab) can inhibit the expression of luciferase in V-01 cells. 50 There are different differences, but both can fit a good dose-effect curve. It is proved that this method can be applied to the determination of biological activity of anti-ST2 antibodies and anti-IL-33 antibodies. In other words, this method has good applicability for different anti-IL-33 / ST2 axis antibodies.

[0230] (2) Different luciferase detection reagents

[0231] Follow the "Methodology Validation Experimental Procedures" and add four different luciferase detection reagents Bright-Glo, Bio-Glo, Steady-Glo or One-Glo (all from Promega) to measure the chemiluminescence value. Fig. 10C As shown in the figure, although the response values ​​measured by different luciferase detection reagents are different, the four-parameter curve fitting is good. The IC values ​​of anti-ST2 antibodies detected after adding Bright-Glo, Bio-Glo, Steady-Glo or One-Glo are 50 The values ​​were 0.2625 μg / mL, 0.3214 μg / mL, 0.3026 μg / mL and 0.2605 μg / mL, respectively, with no significant difference, which met the requirements of the detection experiment, indicating that this method has good applicability to different types of luciferase detection reagents.

[0232] All patents, patent applications and other identified publications are expressly incorporated herein by reference for the purposes of description and disclosure. These publications are provided solely because their disclosure predates the filing date of the present application. All statements regarding the dates of these documents or representations of the contents of these documents are based on the information available to the applicant and do not constitute any admission as to the correctness of the dates of these documents or the contents of these documents. Furthermore, any reference to these publications herein does not constitute an admission that the publications become part of the common general knowledge in the art in any country.

[0233] Although the present application has been described in detail above with general descriptions and specific implementation schemes, it is obvious to those skilled in the art that some modifications or improvements may be made to the present application. Therefore, these modifications or improvements made without departing from the spirit of the present application are within the scope of protection claimed in the present application.

Claims

1. A cell line, wherein: The cell line is a mammalian cell, and a first expression cassette and a second expression cassette are integrated into the genome of the cell line, wherein the first expression cassette expresses an exogenous ST2 gene, and the second expression cassette expresses an exogenous reporter gene regulated by NF-κB.

2. The cell line according to claim 1, wherein The exogenous ST2 gene is a human ST2 gene or a monkey ST2 gene.

3. The cell line according to claim 1 or 2, wherein The reporter gene is a luciferase reporter gene. Optionally, the transcriptional expression of the reporter gene depends on the activation of an NF-κB response element.

4. The cell line according to any one of claims 1 to 3, wherein The cell line is a HEK293 cell or a cell derived therefrom; preferably, the cell line is a HEK293T cell.

5. The cell line according to any one of claims 1 to 4, wherein The deposit number of the cell line is CCTCCNO: C202397.

6. A cell line, wherein: The deposit number of the cell line is CCTCC NO: C202397.

7. A kit comprising the cell line according to any one of claims 1 to 6.

8. Use of the cell line according to any one of claims 1 to 6 for detecting the biological activity of an anti-IL-33 / ST2 axis antibody or an antigen-binding fragment thereof.

9. The use according to claim 8, wherein The anti-IL-33 / ST2 axis antibodies or antigen-binding fragments thereof include anti-ST2 antibodies or antigen-binding fragments thereof and anti-IL-33 antibodies or antigen-binding fragments thereof.

10. The use according to claim 8 or 9, wherein The biological activity includes binding activity and blocking activity.

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