DKK-1 antibody biological activity detection method

Abstract

According to the DKK-1 antibody biological activity detection method provided by the invention, a luciferase expression plasmid containing a TCF-LEF reaction element is transfected into an HEK 293 cell with an active Wnt / beta-catenin signal channel, and a reporter gene detection method based on DKK-1 antibody MOA is successfully established by screening effector cells and optimizing detection conditions. And then carrying out practicability confirmation of the RGA method through condition optimization. The establishment and prediction of the new method can further promote the screening evaluation of DKK-1 antibody drugs and the quality control of the DKK-1 antibody drugs in the subsequent development process, so that an efficient and rapid new method is provided for the biological activity detection of the DKK-1 antibody, and a new thought and direction are provided for the biological activity evaluation of other MOA-based antibodies.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a method for detecting the bioactivity of DKK-1 antibody. Background Technology

[0002] With the increasing aging of society, osteoporosis has become a growing public health challenge. According to the World Health Organization (WHO) definition, approximately 11.7% of men and 23.1% of women worldwide suffer from osteoporosis. Antibody drugs are gaining increasing attention due to their high targeting specificity, fewer side effects, and significant therapeutic effects. Targeted therapy for osteoporosis has gradually matured, with denosumab, a marketed antibody drug, being widely used clinically. However, while there are many drugs available for treating osteoporosis, their efficacy varies, and many suffer from slow onset of action and significant side effects. This has prompted the academic community to continuously seek more precise therapeutic targets. The classical Wnt signaling pathway plays a crucial role in bone biology and bone-related diseases; therefore, regulating the classical Wnt signaling pathway to treat osteoporosis is of great significance. As a Wnt / β-catenin signaling pathway antagonist, DKK-1 is considered a promising new target.

[0003] The bioactivity of antibody drugs is highly correlated with their efficacy. Globally, bioactivity is considered a critical quality attribute (CQA) for antibody drugs, a point emphasized in the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH Q6B) guidelines: "Quality Standards: Methods of Examination and Acceptance Criteria for Biotechnology Products and Biological Products" and pharmacopoeias. According to ICH Q6B guidelines, the higher-order structure of complex molecules cannot be fully determined solely by physicochemical information, but it can be indirectly reflected by assessing their bioactivity, highlighting the central role of bioactivity in drug quality control. Furthermore, bioactivity testing methods can accurately reflect the mechanism of action (MOA) of a drug.

[0004] As an emerging class of target drugs, DKK-1 antibodies have shown great potential in the treatment of osteoporosis and cancer. Studies have confirmed the bone balance correlation of DKK-1. Therefore, developing bioactive DKK-1 antibodies provides a new avenue for osteoporosis treatment. Accurate assessment of the bioactivity of DKK-1 antibodies is crucial for the screening, evaluation, and quality control of such drugs. However, current evaluations of DKK-1 antibody bioactivity mainly rely on alkaline phosphatase activity assays, a method that is not only cumbersome and prone to variability but also time-consuming. Given the advantages of cell-based reporter gene analysis (RGA) in assessing various antibody activities, including high sensitivity, simplicity, and time efficiency, and its ability to closely correlate with the drug's MOA (Mean Activity Aspect), there is an urgent need to develop a novel reporter gene method for DKK-1 antibody bioactivity based on the Wnt / β-catenin signaling pathway. Currently, research on reporter gene methods for detecting DKK-1 antibody bioactivity based on the Wnt / β-catenin signaling pathway is relatively limited, which to some extent hinders the research and quality control of DKK-1 antibodies. This study aims to fill this gap and provide strong support for the development and application of DKK-1 antibodies. Summary of the Invention

[0005] To address the above problems, this invention provides a method for detecting the bioactivity of DKK-1 antibodies.

[0006] To achieve the above technical objectives, the technical solution adopted by the present invention is as follows: A method for detecting the bioactivity of DKK-1 antibody, characterized in that: a. Collect RGA effector cells by digestion, resuspend and adjust density before seeding in 96-well plates; b. Dilute Wnt3a supernatant 1:2 to 1:5, dilute DKK-1 to 90-120 μg / mL, and dilute DKK-1 antibody to 0.1-0.5 μg / mL or 90-135 μg / mL, and add to 96-well plates; c. After incubation at 35-38℃ and 6.5-9% CO2, the sample is tested.

[0007] In step a, the RGA effector cells are HEK 293 / TCF-LEF / Luc2p cells.

[0008] In step a, the RGA effector cell resuspension is performed in 10% FBS RPMI1640 / DMEM medium.

[0009] In step a, the RGA effector cell density is adjusted to 1.1 × 10⁻⁶. 6 / mL~1.5×10 620-60 μL / well were seeded into a 96-well plate.

[0010] In step b, the Wnt3a supernatant is diluted 1:3 to 1:4.5 times, and DKK-1 is diluted to 95-110 μg / mL, with 20-60 μL / well added to each well of a 96-well plate.

[0011] The DKK-1 antibody mentioned in step b includes mouse DKK-1 antibody and human DKK-1 antibody.

[0012] More preferably, the concentration of mouse DKK-1 antibody is 0.25~0.35 μg / mL, and 20-40 μL / well is added to a 96-well plate. The concentration of human DKK-1 antibody is 95~125 μg / mL as the starting point, 3-fold ratio, 6 concentrations, and 20-40 μL / well is added to a 96-well plate.

[0013] The incubation time in step c is 12-17 hours.

[0014] In step c, the detection involves adding 50-120 μL of Bright-Glo™ Luciferase Assay System after incubation and using a Promega GloMax bioluminescence detector to detect the relative light units. Attached Figure Description

[0015] Figure 1 RGA effector cell screening Figure 2 Optimization of Wnt3a and DKK-1 protein concentrations Figure 3 RGA method for evaluating the biological activity of DKK-1 antibody. Implementation

[0016] Collect 1×10⁶ HEK 293 cells in logarithmic growth phase (ATCC, USA) 7 700 μL of pGL4.49 [Luc2p / TCF-LEF RE / Hygro] plasmid (Promega, USA) was added to the cells. The total amount was 40 μg, centrifuged at 10000 rpm for 5 min, diluted with purified water to 100 μL, and then added to the cells. The mixed suspension was then added to a 0.4 cm... 2Cells were placed in electroporation cuvettes and then on an electroporator (BIO-RAD, USA) under the following conditions: Voltage (V), 300; Pulse length (ms), 20; Number of Pulse, 1; Pulse interval (sec), 0; Cuvette, 4; Drop, 30. The electroporated cell-plasmid mixture was then transferred to 10% FBS RPMI 1640 / DMEM medium. 500 μL / well was added to a 24-well plate and incubated at 37°C with 5% CO2. After 24 h, cells were subjected to pressurization with 10% FBS RPMI 1640 / DMEM selection medium containing 0.5 μg / mL hygromycin. Cells survived under hygromycin pressurization throughout the entire culture and assay period.

[0017] Cell lines requiring subcloning were diluted with culture medium to 375 cells / 15 mL and seeded at 200 μL / well in a 96-well cell culture plate. Then, an equal volume of selection medium was added to the remaining cell suspension and seeded at 200 μL / well in the second column of 96-well plates. This process was repeated 2-fold at 12 different concentrations in 12 separate 96-well cell culture plates. Cells were incubated at 37°C and 5% CO2 for approximately 7–10 days. After observing colony formation, single colonies were marked with a marker, and the culture was expanded until selection.

[0018] HEK 293 / TCF-LEF / Luc2p cells (Shanghai Maitaijunao Biotechnology Co., Ltd., China) were collected and resuspended in culture medium, with the density adjusted to 7 × 10⁵ / mL. Wnt3a Expi supernatant was diluted 1:8, and 50 μL each of cells and detection proteins were added to 96-well assay plates. After incubation at 37℃ and 8% CO₂ for 16 h, 100 μL of Bright-Glo™ Luciferase Assay System (Vazyme, Nanjing) was added, and relative light units were measured using a Promega GloMax bioluminescence analyzer (Promega, USA). Once effector cells were identified, cell libraries were constructed for long-term experimental use.

[0019] The schematic diagram of the reaction elements of plasmid pGL4.49[Luc2p / TCF-LEF RE / Hygro] is shown below. Figure 1a. After plasmid amplification and preparation, the cells were used for cell transfection. The pGL4.49[Luc2p / TCF-LEF RE / Hygro] vector was introduced into HEK293 cells (ATCC, USA) using an electroporator to obtain effector cells expressing the luciferase gene under the regulation of the TCF-LEF response element. Subsequently, cell lines in the hygromycin B resistance pool were screened using Wnt3a (Shanghai Maitaijunao Biotechnology Co., Ltd., China). Among the eight maternal clones we identified, 1-B5 showed a high luciferase response signal (…). Figure 1 b). To ensure the monoclonal nature of the detected cells and achieve better methodological precision, we used a limiting dilution method to screen subclones of the 1-B5 mother clone. The screening results showed that subclone 1-B5-3-H6 not only exhibited a high response value but also had the optimal signal-to-noise ratio (SNR). Figure 1 c), therefore, 1-B5-3-H6 was selected as the cell line for subsequent reporter gene analysis (RGA) experiments to ensure the stability and consistency of the experimental results.

[0020] Example 2: RGA Method Optimization To determine the optimal reaction conditions for reporter gene assay of DKK-1 antibody, we optimized the working concentrations of Wnt3a and DKK-1 (Shanghai Maitaijunao Biotechnology Co., Ltd., China). HEK 293 / TCF-LEF / Luc2p cells were cultured in complete medium at 7 × 10⁻⁶ ppm. 5 At a density of / mL, 50 μL / well was seeded into 96-well plates. Wnt3a Expi supernatant was initially diluted 1:2, 50 μL / well, at a 2-fold ratio, for 9 concentrations, with a total volume of 100 μL. For simplified data analysis, the initial 1:2 concentration was set to 500. The dose-response curve was fitted (…). Figure 2 a) We found that the Wnt3a stimulation produced the strongest luminescence signal at a dilution ratio of 1:16. Based on this result, we selected a dilution ratio of 1:16 as the optimal working concentration of Wnt3a.

[0021] At a fixed concentration of Wnt3a 1:16, DKK-1 antigen was started at 100 μg / mL, 25 μL / well, in 2-fold ratios, with 8 concentrations, for a total system volume of 100 μL. The dose-response curve was fitted (…). Figure 2 (b) To ensure that the antibody has a sufficient anti-inhibition signal range, the optimal working concentration of DKK-1 was finally determined to be 25 μg / mL, which ensured the sensitivity and reliability of the experiment.

[0022] Example 3: RGA method for evaluating the biological activity of DKK-1 antibody To verify the practicality and accuracy of this method, the activity of mouse-derived and humanized DKK-1 antibodies (Shanghai Maitaijunao Biotechnology Co., Ltd., China) in the laboratory was evaluated. RGA effector cells were collected after digestion with 0.02% EDTA and 0.05% Trypsin, and then resuspended in 10% FBS RPMI 1640 / DMEM medium, adjusting the density to 1.4 × 10⁻⁶. 6 Wnt3a supernatant was diluted 1:4, and DKK-1 was diluted to 100 μg / mL, with 25 μL / well added to each well of a 96-well plate. Mouse DKK-1 antibody 0.37 μg / mL was added to each well of a 96-well plate; humanized DKK-1 antibody was added to each well starting at 100 μg / mL, in 3-fold increments (6 concentrations), with 25 μL / well added to each well. After incubation at 37°C and 8% CO2 for 16 h, 100 μL of Bright-Glo™ Luciferase Assay System was added, and relative light units were measured using a Promega GloMax bioluminescence detector.

[0023] The results showed that, in the RGA method, the two murine antibodies increased the response value by reducing the DKK-1 inhibitory ability. Figure 3 a) effectively reflects their biological activity. For humanized antibodies, we obtained the EC50 values ​​of each antibody through complete dose-response curves (a), which effectively reflects their biological activity. Figure 3 (b) This not only effectively assessed antibody activity but also provided reliable data support for screening humanized antibodies with optimal biological activity.

Claims

1. A method for detecting the bioactivity of DKK-1 antibody, characterized in that: a. Collect RGA effector cells by digestion, resuspend and adjust density before seeding in 96-well plates; b. Dilute Wnt3a supernatant 1:2 to 1:5, DKK-1 to 90 to 120 μg / mL, and DKK-1 antibody to 0.1 to 0.5 μg / mL or 90 to 135 μg / mL, and add to a 96-well plate; c. After incubation at 35-38℃ and 6.5-9% CO2, the sample is tested.

2. The bioactivity detection method as described in claim 1, characterized in that, The RGA effector cells mentioned in step a are HEK 293 / TCF-LEF / Luc2p cells.

3. The bioactivity detection method as described in claim 1, characterized in that, The RGA effector cell resuspension described in step a is performed in 10% FBS RPMI 1640 / DMEM medium.

4. The bioactivity detection method as described in claim 1, characterized in that, The RGA effector cell density was adjusted to 1.1 × 10⁻⁶ cells in step a. 6 / mL~1.5×10 6 20-60 μL / well were seeded into a 96-well plate.

5. The bioactivity detection method as described in claim 1, characterized in that, The Wnt3a supernatant described in step b was diluted 1:3 to 1:4.5 times, and DKK-1 was diluted to 95 to 110 μg / mL. 20 to 60 μL of each solution was added to a 96-well plate.

6. The bioactivity detection method as described in claim 1, characterized in that, The DKK-1 antibody mentioned in step b includes mouse DKK-1 antibody and human DKK-1 antibody.

7. The bioactivity detection method as described in claim 1, characterized in that, The incubation time described in step c is 12-17 hours.

8. The bioactivity detection method as described in claim 1, characterized in that, The detection described in step c involves adding 50-120 μL of Bright-Glo™ Luciferase Assay System after incubation and using a Promega GloMax bioluminescence detector to measure the relative light units.

9. The DKK-1 antibody as described in claim 6, characterized in that, The concentration of mouse DKK-1 antibody was 0.25~0.35 μg / mL, and 20-40 μL / well was added to a 96-well plate. The concentration of human DKK-1 antibody was 95~125 μg / mL as the starting concentration, in 3-fold increments, with 6 concentrations, and 20-40 μL / well was added to a 96-well plate.

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