IL-17A antibody biological activity detection method

Abstract

According to the IL-17A antibody biological activity detection method, expression vector plasmids containing hmIL-17RA and NF-kappa B luciferase reporter genes are transfected into 293 cells, effector cells are screened, detection conditions are optimized, and a reporter gene detection method based on an IL-17A antibody action mechanism is successfully established. And determining the precision of the method through condition optimization. Establishment and prediction of the novel method will further promote screening evaluation of IL-17A antibody drugs and quality control of the IL-17A antibody drugs in the subsequent development process, so that an efficient and rapid novel method is provided for detection of the bioactivity of the IL-17A antibody, and meanwhile, a new thought and direction are provided for bioactivity evaluation of other MOA-based antibodies.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a method for detecting the biological activity of IL-17A antibody. BACKGROUND

[0002] Interleukin-17A (IL-17A) is a key pro-inflammatory cytokine mainly produced by Th17 cells, which is highly expressed in patients with various autoimmune diseases, including psoriasis, psoriatic arthritis, rheumatoid arthritis and multiple sclerosis, and is closely related to the occurrence and development of these diseases. When combined with its receptor complex, it will lead to the recruitment of ACT1 adaptor protein, which interacts with IL-17 receptor through its shared SEFIR domain. The association of ACT1 with IL-17 receptor complex leads to the recruitment of tumor necrosis factor (TNF) receptor-associated factors 6 and 3 (TRAF-6 and TRAF-3). TRAF6 activates the complex of transforming growth factor-β-activated kinase 1 (TAK1) and IκB kinase (IKK) to act on the NF-κB pathway, starting a series of transcription and expression of cytokines and growth factors. In addition, TRAF6 can also promote the activation of motigen-activated protein kinase (MAPK), further activating activator protein-1 (AP-1) to promote the transcription and expression of downstream inflammatory-related cytokines IL-6, TNF-α, G-CSF, etc., thereby triggering an inflammatory response.

[0003] At present, a variety of IL-17A monoclonal antibodies have been approved for clinical treatment of inflammatory diseases. For example, Secukinumab, Ixekizumab and Brodalumab have been approved for the treatment of inflammatory patients through clinical studies, and now they occupy the main market for the treatment of inflammatory diseases.

[0004] Current methods for detecting the biological activity of IL-17A monoclonal antibodies include surface plasmob resonance (SPR) analysis using the BIAcore T200 system and the IL-6 release inhibition assay. However, given the advantages of cell-based reporter gene analysis methods in assessing various antibody activities, such as high sensitivity, simplicity, and time savings, and their ability to closely correlate with the drug's MOA (Mean Activity Aspect), there is an urgent need to develop a novel method for detecting the biological activity of IL-17A antibodies based on the NF-κB signaling pathway. Summary of the Invention

[0005] To address the above problems, this invention provides a method for detecting the bioactivity of IL-17A antibodies.

[0006] To achieve the above technical objectives, the present invention adopts the following technical solution: a method for detecting the bioactivity of IL-17A antibody, characterized in that a reporter gene assay is used to detect the bioactivity of IL-17A antibody.

[0007] The sample processing procedure for the bioactivity detection method is as follows: a. Digest and collect effector cells, resuspend and adjust density before seeding in 96-well white plates; b. Dilute IL-17A concentration to 200-800 ng / ml, start IL-17A antibody concentration to 500-1500 μg / ml, 2-6 times 5-15 concentrations, add 20-70 μl / well to the corresponding 96-well white plate; c. Incubate the above 96-well white plates at 35-38℃ and 6-9% CO2 for 3-8 hours; d. Add 50-150 μl / well Bright-Glo reading.

[0008] The effector cells mentioned in step a are 293 / IL-17RA / NF-κB 1-D1-2-B2 cells.

[0009] In step a, the effector cell resuspension is performed by resuspending the cells in 2-5% inactivated FBS RPMI1640 / DMEM medium.

[0010] In step a, the effector cell density is adjusted to 2 × 10⁻⁶. 6 / mL~5×10 6 20-60 μL / well was inoculated into a 96-well white plate.

[0011] In step b, the IL-17A concentration is diluted to 300-700 ng / ml, the initial concentration of IL-17A antibody is increased to 800-1200 μg / ml, and 8-12 concentrations are added at 3-5 times the original concentration, with 30-60 μl / well added to the corresponding 96-well white plate.

[0012] In step c, the 96-well white plate is incubated at 36-37℃ and 7-8% CO2 for 4-7 hours.

[0013] In step d, 80-120 μl of Bright-Glo is added per well for reading.

[0014] The readings were taken using an MD SpectraMax M5 Microplate Reader instrument. Attached Figure Description

[0015] Figures 1-3 Electrocloning detection Figure 4 : Confirm report gene expression status Figure 5 Limiting dilution subcloning detection Figure 6 IL-17A antibody inhibits IL-17A-stimulated reporter cell luminescence. Figure 7 IL-17A stimulation curve Figure 8 IL-17A antibody inhibition curve Figure 9 IL-17A Antibody Reporter Gene Bioactivity Detection Method Figure 10 Method precision confirmation

[0016] Example 1: Electrocloning detection Resuscitate 293 cells, centrifuge and discard the supernatant, wash twice with serum-free medium, resuspend and count, adjust cell density to a suitable range, add hmIL-17RA and NF-κB luciferase reporter gene expression vector plasmid, mix well, place in an electroporation cuvette, electroporate, resuspend the cell suspension in complete medium, seed at 0.5 cells / well in 96-well cell culture plates, incubate in a CO2 incubator for 24 hours, and then screen: after 2-3 weeks, use G418 and hygromycin double screening to select drug-resistant clones.

[0017] The expression of the IL-17A receptor was confirmed by flow cytometry: drug-resistant clones were collected, and biotin-labeled IL-17A at 20 μg / ml was added and incubated on ice for 45 minutes. After washing, fluorescently labeled antibody was added and incubated on ice for 45 minutes. After washing, the cells were resuspended in PBS and analyzed by flow cytometry.

[0018] According to the ranking of the fluorescence intensity of the flow detection: 2-B2 > 1-D6 > 2-B4 > 2-A5 > 1-D1 > 2-B1; 2 clones were selected for limited dilution subcloning by combining with the expression of the reporter gene.

[0019] Example 2 Confirmation of the expression of the reporter gene The drug-resistant clone cells 293 / IL-17RA / NF-κB were collected and resuspended in 10% FBS RPMI1640, and the cell density was adjusted to 1×10 5 / ml and plated overnight. IL-17A was diluted to 10 μg / ml using 2% inactivated FBS RPMI1640, and 50 μl / well was added to the above plate. After 5h of culture, 50 μl / well of Bright-Glo was added for luminescence reading.

[0020] According to the ranking of the luminescence intensity of the reporter gene detection: 1-D1 > 2-A5 > 2-B2 > 1-D6 > 2-B4 > 2-B1, clones 1-D1 and 2-A5 were selected by combining with the fluorescence intensity of the flow detection, and limited dilution subcloning was performed.

[0021] Example 3 Limited dilution subclone detection The 1-D1 and 2-A5 cells were digested, centrifuged, resuspended in complete medium, counted, and the cell density was adjusted to 0.5 cells / well, 200 μl / well, plated in a 96-well cell culture plate, and placed in a carbon dioxide incubator for 2 weeks. Single clone colony cells were selected and cultured in a 24-well plate.

[0022] The drug-resistant clone cells 293 / IL-17RA / NF-κb 1-D1 and 2-A5 limited dilution subclones were collected, resuspended in 10% FBS RPMI1640, and the cell density was adjusted to 1×10 5 / ml and plated overnight. IL-17A was diluted to 10 μg / ml using 2% inactivated FBS RPMI1640, and 50 μl / well was added to the above plate. After 5h of culture, 50 μl / well of Bright-Glo was added for luminescence reading.

[0023] According to the luminescence intensity of the reporter gene detection, 1-D1-1-F12, 1-D1-2-D2, 1-D1-2-B2, 2-A5-2-D2, 2-A5-2-G7, and 2-A5-3-B4 were selected for IL-17A antibody inhibition luminescence detection.

[0024] Example 4 IL-17A antibody inhibits IL-17A stimulated reporter gene cell luminescence Collect 293 / IL-17RA / NF-κB 1-D1-2-B2 cells, resuspend in 2% inactivated FBS 16 / DM, adjust cell density to 4 x 105 / ml, 2 x 105 / ml, 1 x 105 / ml 50 μl / well plate 96 white plate.

[0025] Example 5 IL-17A stimulation curve Use cell culture solution to expand 1-D1-2-B2 cells to establish PCB, MCB, WCB respectively, and recover WCB cells for IL-17A antibody reporter gene bioactivity detection method development.

[0026] Collect 293 / IL-17RA / NF-κB 1-D1-2-B2 cells, resuspend in 2% inactivated FBS 16 / DM, adjust cell density to 4 x 105 / ml, 2 x 105 / ml, 1 x 105 / ml 50 μl / well plate 96 white plate.

[0027] Dilute IL-17A to 5 μg / ml 4-fold ratio 9 concentrations with 2% inactivated FBS 16 / DM, add 50 μl / well to the corresponding 96 white plate, incubate at 37°C 8% CO2 for 5h, add 100 μl / well Bright-Glo for reading. According to the S-curve parameter calculation, the IL-17A EC90 concentration is about 250 ng / ml, and this concentration is selected for IL-17A antibody inhibition effect.

[0028] Example 6 IL-17A antibody inhibition curve Collect 293 / IL-17RA / NF-κB 1-D1-2-B2 cells, resuspend in 2% inactivated FBS 16 / DM, adjust cell density to 4 x 105 / ml, 2 x 105 / ml, 1 x 105 / ml 50 μl / well plate 96 white plate.

[0029] IL-17A was diluted to 500 ng / ml using 2% inactivated FBS 16 / DM, and IL-17A antibody was diluted 10 times at a 4-fold ratio to 1000 μg / ml. 50 μl / well was added to the corresponding 96-well white plate and incubated at 37℃ with 8% CO2 for 5 h. 100 μl / well of Bright-Glo was then added for reading. Results showed that the IL-17A antibody inhibition curve was complete, the concentration gradient was reasonable, and different cell densities showed good results at 4×10⁻⁶ cells / well. 5 / ml, detection range better than 2×10 5 / ml and 1×10 5 / ml, select 4×10 5 Three curves were used to confirm the result at / ml.

[0030] Example 7: Validation of the IL-17A antibody reporter gene bioactivity assay method Collect 293 / IL-17RA / NF-κB 1-D1-2-B2 cells, resuspend them in 2% inactivated FBS 16 / DM, adjust the cell density to 4×10⁵ / ml, and seed 50 μl / well in 96-well white plates.

[0031] IL-17A was diluted to 500 ng / ml using 2% inactivated FBS 16 / DM at a 4-fold ratio with IL-17A antibody at 1000 μg / ml, resulting in 10 replicates. 50 μl / well was added to the corresponding 96-well white plate and incubated at 37°C with 8% CO2 for 5 h. 100 μl / well was then added to Bright-Glo for reading. The results showed that this reporter gene detection method produced good 3-replica curves and can be used for the bioactivity detection of IL-17A antibody.

[0032] Example 8: Method Precision Confirmation Collect 293 / IL-17RA / NF-κB 1-D1-2-B2 cells, resuspend them in 2% inactivated FBS 16 / DM, adjust the cell density to 4×10⁵ / ml, and seed 50 μl / well in 96-well white plates.

[0033] IL-17A was diluted to 500 ng / ml using 2% inactivated FBS 16 / DM at 4-fold ratios with IL-17A antibody at 1000 μg / ml, resulting in 10 replicates. 50 μl / well was added to the corresponding 96-well white plate and incubated at 37°C with 8% CO2 for 5 h. 100 μl / well of Bright-Glo was then added for reading. The RSD range of the detection precision was 1.98–10.53. This method can be applied to the detection of IL-17A antibody bioactivity.

Claims

1. A method for detecting the bioactivity of IL-17A antibody, characterized in that, The bioactivity of IL-17A antibody was detected using a reporter gene assay.

2. The bioactivity detection method as described in claim 1, characterized in that: a. Digest and collect effector cells, resuspend and adjust density before seeding in 96-well white plates; b. Dilute IL-17A concentration to 200-800 ng / ml, start IL-17A antibody concentration to 500-1500 μg / ml, 2-6 times 5-15 concentrations, add 20-70 μl / well to the corresponding 96-well white plate; c. Incubate the above 96-well white plates at 35-38℃ and 6-9% CO2 for 3-8 hours; d. Add 50-150 μl / well Bright-Glo reading.

3. The bioactivity detection method as described in claim 2, characterized in that, The effector cells mentioned in step a are 293 / IL-17RA / NF-κB 1-D1-2-B2 cells.

4. The bioactivity detection method as described in claim 2, characterized in that, The effector cell resuspension described in step a is resuspension in 2-5% inactivated FBS RPMI1640 / DMEM medium.

5. The bioactivity detection method as described in claim 2, characterized in that, The effector cell density is adjusted to 2 × 10⁻⁶ in step a. 6 / mL~5×10 6 20-60 μL / well was inoculated into a 96-well white plate.

6. The bioactivity detection method as described in claim 2, characterized in that, In step b, dilute the IL-17A concentration to 300-700 ng / ml, and the initial concentration of the IL-17A antibody to 800-1200 μg / ml. Add 30-60 μl / well to the corresponding 96-well white plate at 3-5 fold ratios of 8-12 concentrations.

7. The bioactivity detection method as described in claim 2, characterized in that, In step c, the above 96-well white plate is incubated at 36-37℃ and 7-8% CO2 for 4-7 hours.

8. The bioactivity detection method as described in claim 2, characterized in that, In step d, add 80-120 μl / well of Bright-Glo and take readings.

9. The reading as described in claim 8, characterized in that, Readings were taken using an MD SpectraMax M5 Microplate Reader instrument.

Images