Method for determining biological activity of human IL-36/IL36R/IL1RAcP pathway inhibitor

Abstract

The invention provides a method for determining the biological activity of a human IL-36 / IL36R / IL1RAcP pathway inhibitor. The method comprises the following steps of: with a cell for stably expressingIL36R, IL1RacP and NF-kappa B reporter genes as an effector cell, mixing the reporter gene with IL-36 and a human IL-36 / IL36R / IL1RAcP pathway inhibitor, incubating, and determining the biological activity of the human IL-36 / IL36R / IL1RAcP pathway inhibitor according to the measured signal value of the reporter gene. The determination method provided by the invention does not need any human primarytissue-derived cells or other components, has the advantages of stable color development result, short experimental period and simple operation, and avoids cell pollution and errors caused by long-time incubation and multi-step operation; in addition, the measured biological activity is related to the clinical curative effect, and the technical requirements related to NMPA are met.

Description

technical field

[0001] The invention relates to the field of biological activity detection of biological medicines, in particular, the invention relates to a method for rapidly, accurately and quantitatively measuring the biological activity of IL-36 / IL36R / IL1RAcP pathway inhibitors. Background technique

[0002] Monoclonal antibody is a highly uniform antibody produced by a single B cell clone that only targets a specific epitope, and has become an important part of the global pharmaceutical market. The determination of the biological activity of monoclonal antibodies at the cellular level plays an important role in the discovery and development of monoclonal antibody drugs. Currently, most biological activity detection methods use cell-based biological activity assays, including cell proliferation inhibition methods. , cytotoxicity, complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, cell ELISA and reporter gene method.

[0003] The Interleuk...

Examples

Embodiment 1

[0084] Example 1 Exploration of electric shock transfection conditions for Jurkat cells

[0085] Jurkat cells are suspended human peripheral blood leukemia T cells, and the transfection is very difficult. The chemical transfection method is difficult to transfect the plasmid into the cells, so the electroporation transfection method is chosen.

[0086] (1) Electric shock transfection condition 1: take 2×10 6 Jurkat cells (Cell Bank, Chinese Academy of Sciences), 10 μg pDSRed plasmid (Clontech, 632406) (red fluorescent plasmid is used to identify transfection efficiency), electric shock voltage 1100v, electric shock time 50ms, electric shock frequency 1 time;

[0087] (2) Electric shock transfection condition 2: take 2×10 6 1 Jurkat cell, 10 μg pDSRed plasmid, electric shock voltage 1300v, electric shock time 20ms, electric shock times 2 times;

[0088] (3) Electric shock transfection condition 3: take 2×10 6 1 Jurkat cell, 10 μg pDSRed plasmid, electric shock voltage 1400v,...

Embodiment 2

[0090] Example 2 Preparation of effector cells expressing Jurkat / IL36R / IL1RAcP / NF-κB

[0091] Using the electroporation transfection condition 3 screened in Example 1, 3.33 μg pGL4.32[luc2P / NF-κB-RE / Hygro] (Promega (Beijing) Biotechnology Co., Ltd.) plasmid, 3.33 μg pcDNA3.1 ( +) / IL36R plasmid and 3.33 μg pcDNA3.1(+) / IL1RAcP were transfected into Jurkat cells (Cell Bank, Chinese Academy of Sciences), and then treated with 0.1 mg / mL hygromycin B, 0.1 mg / mL G418, 0.05 mg / mL Jurkat / IL36R / IL1RAcP / NF-κB effector cells were obtained by culturing in Zeocine medium.

[0092] Dilute recombinant human IL-36β (ACROBiosystems, Cat. No. IL8-H5115) to 200ng / mL, 66.66ng / mL, 22.22ng / mL, 7.40ng / mL, 2.46ng / mL, 0.82ng in 1640+10% FBS medium / mL, 0.26ng / mL and other concentrations were added to a 96-well plate (Corning, cat. 3610) at 50 μL / well, and then 100,000 per well and 50 μL / well were used for different Jurkat / IL36R / IL1RAcP / NF-κB effects Cell clones were added to the above 96-well plate, 37...

Embodiment 3

[0097] Example 3 Optimization and establishment of IL36R monoclonal antibody biological activity assay method

[0098] The Jurkat / IL36R / IL1RAcP / NF-κB-25# screened out in Example 2 was selected as the effector cell strain used in optimizing and establishing the IL36R monoclonal antibody biological activity assay method.

[0099] (1) Optimize cell density:

[0100] IL36β preparation: use 1640+10% FBS medium to dilute 0.2mg / mL IL36β to 100ng / mL;

[0101] Drug preparation: use 1640+10% FBS medium to dilute IL-36R monoclonal antibody BI-655130 to 50000ng / mL, 12500ng / mL, 3125ng / mL, 781.25ng / mL, 195.31ng / mL, 48.82ng / mL, 12. 20ng / mL, 3.02ng / mL and other concentrations, the above-mentioned IL36β and different concentrations of BI-655130 were mixed 1:1 according to 100μL and 100μL, and added to a 96-well plate according to 50μL / well;

[0102] Preparation of effector cell suspension: Dilute Jurkat / IL36R / IL1RAcP / NF-κB-25# effector cells to 4×10 5 pcs / mL, 1×10 6 pcs / mL, 2×10 6 ...

technical field

[0001] The invention relates to the field of biological activity detection of biological medicines, in particular, the invention relates to a method for rapidly, accurately and quantitatively measuring the biological activity of IL-36 / IL36R / IL1RAcP pathway inhibitors. Background technique

[0002] Monoclonal antibody is a highly uniform antibody produced by a single B cell clone that only targets a specific epitope, and has become an important part of the global pharmaceutical market. The determination of the biological activity of monoclonal antibodies at the cellular level plays an important role in the discovery and development of monoclonal antibody drugs. Currently, most biological activity detection methods use cell-based biological activity assays, including cell proliferation inhibition methods. , cytotoxicity, complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, cell ELISA and reporter gene method.

[0003] The Interleuk...

Examples

Embodiment 1

[0084] Example 1 Exploration of electric shock transfection conditions for Jurkat cells

[0085] Jurkat cells are suspended human peripheral blood leukemia T cells, and the transfection is very difficult. The chemical transfection method is difficult to transfect the plasmid into the cells, so the electroporation transfection method is chosen.

[0086] (1) Electric shock transfection condition 1: take 2×10 6 Jurkat cells (Cell Bank, Chinese Academy of Sciences), 10 μg pDSRed plasmid (Clontech, 632406) (red fluorescent plasmid is used to identify transfection efficiency), electric shock voltage 1100v, electric shock time 50ms, electric shock frequency 1 time;

[0087] (2) Electric shock transfection condition 2: take 2×10 6 1 Jurkat cell, 10 μg pDSRed plasmid, electric shock voltage 1300v, electric shock time 20ms, electric shock times 2 times;

[0088] (3) Electric shock transfection condition 3: take 2×10 6 1 Jurkat cell, 10 μg pDSRed plasmid, electric shock voltage 1400v,...

Embodiment 2

[0090] Example 2 Preparation of effector cells expressing Jurkat / IL36R / IL1RAcP / NF-κB

[0091] Using the electroporation transfection condition 3 screened in Example 1, 3.33 μg pGL4.32[luc2P / NF-κB-RE / Hygro] (Promega (Beijing) Biotechnology Co., Ltd.) plasmid, 3.33 μg pcDNA3.1 ( +) / IL36R plasmid and 3.33 μg pcDNA3.1(+) / IL1RAcP were transfected into Jurkat cells (Cell Bank, Chinese Academy of Sciences), and then treated with 0.1 mg / mL hygromycin B, 0.1 mg / mL G418, 0.05 mg / mL Jurkat / IL36R / IL1RAcP / NF-κB effector cells were obtained by culturing in Zeocine medium.

[0092] Dilute recombinant human IL-36β (ACROBiosystems, Cat. No. IL8-H5115) to 200ng / mL, 66.66ng / mL, 22.22ng / mL, 7.40ng / mL, 2.46ng / mL, 0.82ng in 1640+10% FBS medium / mL, 0.26ng / mL and other concentrations were added to a 96-well plate (Corning, cat. 3610) at 50 μL / well, and then 100,000 per well and 50 μL / well were used for different Jurkat / IL36R / IL1RAcP / NF-κB effects Cell clones were added to the above 96-well plate, 37...

Embodiment 3

[0097] Example 3 Optimization and establishment of IL36R monoclonal antibody biological activity assay method

[0098] The Jurkat / IL36R / IL1RAcP / NF-κB-25# screened out in Example 2 was selected as the effector cell strain used in optimizing and establishing the IL36R monoclonal antibody biological activity assay method.

[0099] (1) Optimize cell density:

[0100] IL36β preparation: use 1640+10% FBS medium to dilute 0.2mg / mL IL36β to 100ng / mL;

[0101] Drug preparation: use 1640+10% FBS medium to dilute IL-36R monoclonal antibody BI-655130 to 50000ng / mL, 12500ng / mL, 3125ng / mL, 781.25ng / mL, 195.31ng / mL, 48.82ng / mL, 12. 20ng / mL, 3.02ng / mL and other concentrations, the above-mentioned IL36β and different concentrations of BI-655130 were mixed 1:1 according to 100μL and 100μL, and added to a 96-well plate according to 50μL / well;

[0102] Preparation of effector cell suspension: Dilute Jurkat / IL36R / IL1RAcP / NF-κB-25# effector cells to 4×10 5 pcs / mL, 1×10 6 pcs / mL, 2×10 6 ...

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