CN108872207A - A kind of vitro detection tool and method for evaluating RANKL target spot compound biological activity - Google Patents

Abstract

The invention provides an in vitro detection tool and method for evaluating the biological activity of a RANKL target compound. In the present invention, two plasmids containing the target gene are transferred into HEK293 cells (human embryonic kidney cells), and detection tool cells are obtained through drug screening, and then the tool cells are used to evaluate the in vitro biological activity of monoclonal antibody drugs, compared with traditional detection methods , the method has the advantages of short experimental period, simple operation, high accuracy and precision, and ensures the stability and reliability of the detection results. The detection tool cell of the present invention can be used for biological activity evaluation of therapeutic monoclonal antibodies targeting RANKL (nuclear factor kappa B receptor activator ligand), anti-drug neutralizing antibodies (Neutralizing Antibodies) in animal experiments and clinical trials detection etc.

Description

An in vitro detection tool for evaluating the biological activity of RANKL target compounds and method

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to a tool and method for evaluating RANKL target compounds in vitro.

Background technique

Bone metastasis of malignant tumors or metastatic bone disease (Metastatic Bone Diseases, MBD) is a common disease of advanced tumors. Bone metastases of malignant tumors often lead to severe bone lesions, which can greatly reduce the quality of life of tumor patients. In severe cases, the condition will deteriorate rapidly or even die, which greatly affects the prolongation of the patient's survival period. Therefore, while controlling the primary disease, active prevention and treatment of SREs (skeletal metastases and bone-related events) also have important clinical significance in the treatment of malignant tumor bone metastases.

Preclinical studies have found that the occurrence of bone metastasis is a complex system involving multiple protein interactions and multiple pathways. The OPG/RANKL/RANK system is a key molecule involved in the formation, function and survival of osteoclasts. RANK and its ligand RANKL are important regulators of bone metabolism. The combination of RANK expressed by osteoclasts and their precursor cells and RANKL can stimulate the formation, activation, adhesion and survival of osteoclasts, which ultimately leads to increased bone resorption.

In the development process of biotherapeutic drugs, it is necessary to detect biological activity based on the cell level, and this detection method also needs to meet the following conditions: First, the experimental mechanism of the method and the mechanism of the drug's in vivo action should be as close as possible. In this way, the efficacy of the drug in the body can be truly reflected; secondly, the method must be able to pass methodological verification, so as to ensure the authenticity, accuracy and reliability of the test results; finally, the method should be as simple as possible.

The precursor cells of osteoclasts will express RANK on the cell surface, and RANK will be activated by its ligand RANKL, and then recruit TRAF6 in the cytoplasm, activate the NF-κB signaling pathway, and accelerate the maturation of osteoclast precursor cells. Differentiate into osteoclasts. Cell biology experiments designed according to this principle can be used to detect the biological activity of RANKL protein, and then detect the biological activity of monoclonal antibody drugs targeting RANKL. The traditional cell used for viability measurement is RAW264.7 cell (monocyte-macrophage cell line). The monocytes can be differentiated into multinucleated osteoclast-like cells in vitro in the presence of RANKL, and secrete and express tartrate-resistant acid phosphatase (Tartrate-Resistant Acid Phosphatase, TRAP). TRAP enzyme activity is an important marker of osteoclasts, and its activity is positively correlated with osteoclast activity. However, the traditional method has a long experimental cycle, cumbersome operation, poor method accuracy and precision, and it is difficult to guarantee the stability and reliability of the test results.

Contents of the invention

The present invention aims at the above existing problems and deficiencies, and provides a reporter gene detection method for evaluating the in vitro biological activity of drugs at the cellular level during the development of therapeutic RANKL target compounds. This method can meet the requirements of specificity, accuracy, precision and robustness during method validation. Compared with the traditional biological activity evaluation method, the reporter gene activity assay has the advantages of simple operation, short test period, good method accuracy and precision, and stable and reliable detection results.

To this end, the present invention discloses a detection tool and method for evaluating the in vitro biological activity of RANKL target compounds, including the preparation of detection tool cells and the development of detection methods.

The invention relates to a tool cell for detecting the biological activity of a RANKL target compound, and the cell is a HEK293 cell stably expressing RANKL and NF-κB fusion luciferase. The cells were passed through multiple passages or conditions changed, but the expression level remained stable. The preparation process of the tool cell is to transfer a plasmid expressing RANK and NF-κB fusion luciferase in HEK293 cells, the two genes transferred can be on the same plasmid or located on two different plasmids, and the plasmid is also Contains genes encoding anti-resistant drugs, and the transfected cells are screened by adding corresponding resistant drugs to obtain monoclonal cells.

The RANKL target compound of the present invention is selected from one or a combination of monoclonal antibodies against RANKL targets or neutralizing antibodies, Fc fusion proteins, small molecule compounds, preferably anti-RANKL monoclonal antibodies.

The RANK gene is a wild-type or mutant gene, and its sequence is preferably SEQ ID No.1.

The NF-κB fusion luciferase is full-length or part of the NF-κB gene fusion luciferase, the luciferase is a wild type or a mutant type with enhanced enzyme activity, and the mutant luciferase gene with enhanced enzyme activity corresponds to The signal amplification effect is enhanced, and the sequence is preferably SEQ ID No.2.

The transfection method is selected from electroporation transfection method, liposome transfection method or calcium ion transfection method.

The resistant drug is selected from one of G418, Hygromycin B, Puromycin or a combination thereof.

The monoclonal cell screening method is a limiting dilution method.

The present invention also relates to a method for detecting the biological activity of a RANKL target compound, said method comprising the steps of:

(1) Prepare RANKL target compound reference substance solution and need testing solution respectively, set dilution concentration gradient;

(2) After cultivating HEK293 cells stably expressing RANKL and NF-κB fusion luciferase, add RANKL solution, then add the reference substance solution and the test solution prepared in step (1) to treat the cells;

(3) Use a luminescent luciferase detection kit to detect the change in the luciferase level in the cells after treatment with the RANKL target compound, and read the corresponding chemiluminescent units with a microplate reader;

(4) Take the dilution factor or drug concentration as the abscissa, and take the relative chemiluminescence unit as the ordinate to obtain the dose-effect curve of the RANKL target compound and calculate the biological activity of the test product.

The gradient dilution is 2-5 times dilution.

The luciferase detection kit is Luciferase Assay System.

The invention can quickly, accurately and efficiently detect the biological activity of the RANKL target therapeutic drug. The method has good specificity, strong specificity, high accuracy, simple and fast operation, and the activity measurement range reaches 50%-150%; the accuracy is in the range of 80.0%-120.0%, and the repeatability and day-to-day precision RSD are all less than 20 %. If this method is used to detect anti-drug neutralizing antibodies, it also has the characteristics of high serum tolerance. In addition, the present invention does not require expensive instruments and complicated operations, and is fully capable of detecting the biological activity of monoclonal antibody drugs targeting RANKL and the detection of neutralizing antibodies.

The biological material preservation information in the present invention is as follows,

Name of depository unit: China Center for Type Culture Collection (CCTCC)

Address of Preservation Unit: Wuhan University, Wuhan, China

Storage date: 2018.7.1

Deposit number: CCTCC NO: C2018157

Classification Name: Human Embryonic Kidney 293 Transfected Cell HEK293-RANK&NF-κB-RE-luc

Description of drawings

Figure 1 Specificity evaluation of biological activity detection of monoclonal antibody targeting RANKL

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the following examples are usually according to conventional conditions or according to the conditions suggested by the manufacturer. Unless otherwise defined, all professional and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the method of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

Resistance Drug Concentration Screening in Example 1 Tool Cell Construction

HEK293 cells (purchased from ATCC) were plated on 24-well plates, and when the cells grew to a confluence of 90%, the cells were digested, and 50% of the cells were mixed with different concentrations of Hygromycin B (20 μg/ml, 25 μg/ml, 40 μg/ml ml, 75 μg/ml) and G418 (200 μg/ml, 400 μg/ml, 600 μg/ml, 800 μg/ml) were mixed and spread on a 24-well plate. Culture for 1 to 2 weeks, so that the lowest concentration of Hygromycin B and G418 used to make all the cells die is the resistance screening concentration. During this period, when the confluence of the cells in the plate reached about 90%, they were passaged at a ratio of 1:4. The results showed that the concentrations of resistance screening drugs G418 and Hygromycin B were 800 μg/ml and 25 μg/ml, respectively.

The construction of embodiment 2 tool cell construction

Transfer the blank HEK293 cells to a T75 flask and culture them until the confluence reaches over 90%, collect the cells, add plasmid A (self-constructed) and plasmid B (purchased from Promega) at a certain ratio at the same time, electric shock at 250V for 30ms, and culture after 24 hours The base was replaced with fresh DMEM medium (containing 10% FBS). The cell density reached 90% after 72 hours of transfection, inoculated in a new T25 flask at a ratio of 1:1, and replaced with a medium containing Hygromycin B and G418 for screening, and screened for a period of time to obtain a stable transfected cell line Afterwards, monoclonal cells were picked and expanded using the limiting dilution method. Add RANKL stimulation to the candidate monoclonal cell line, detect the response value, and select the monoclonal cell line that meets the requirements, that is, the clone strain with high background value and S/N, as the tool cell.

Plasmid A construction process: The human RANK open reading frame DNA sequence obtained by synthesis or PCR amplification was ligated into the eukaryotic expression plasmid by double restriction digestion.

Plasmid B construction process: The human NF-κB response element tandem luciferase reporter gene DNA sequence obtained by synthesis or PCR amplification was ligated into the eukaryotic expression plasmid by double digestion.

Embodiment 3 specificity evaluation experiment

Using the monoclonal antibody drug against the RANKL target (TK006, refer to CN 103232539B for the specific preparation method), the monoclonal antibody drug against the VEGF target (TK001, refer to CN101935349 B for the specific preparation method), and the drug excipient solution as materials, the above detection The method steps are tested, and the results are shown in Figure 1. When this method is used for anti-VEGF monoclonal antibody and drug adjuvant buffered saline solution, the results cannot be fitted with four parameters. The detection results of anti-RANKL monoclonal antibody can be drawn in the figure. A complete dose-effect curve, while satisfying the fitting coefficient R ² >0.98, and the CV% of multiple wells <20%. The experimental results show that the method of the present invention has good specificity.

Embodiment 4 accuracy evaluation experiment

TK006 working reference product (the active standard product used in the determination of TK006 biological activity within the enterprise) is produced by Jiangsu Taikang Biomedical Co., Ltd. and has undergone strict testing (see Example 3 for the specific preparation method). Samples with different polymer contents were prepared. Using these samples as materials, the above detection method was used to detect the activity of samples with different polymer contents. Subsequently, using the reference product as a material, the samples to be tested with relative activities of 50%, 70%, 100%, 130%, and 150% were prepared, and the activities of these samples were detected by the above detection method.

As shown in Table 1 and Table 2, the data acquisition and analysis were completed by the SoftMaxPro 7.0.2 software developed by Molecular Deveices, and the collected signal and drug concentration were fitted with four parameters. The four parameter equation is shown in Figure 1, and the curve fitting constant R ² are all satisfied greater than 0.98, and the CV% of the RLU value of multiple wells in all concentration points is satisfied to be less than 20%. Through calculation, the method can detect that the activity of the working reference product decreases with the increase of the polymer content. The experimental results show that the method can sensitively detect the change of the quality of the sample, so as to better indicate the quality of the sample.

Four-parameter fitting equation: y=(A-D)/(1+(x/C)^B)+D Table 1 Table 2

Table 1 Summary table of evaluation results of biological activity detection accuracy of RANKL target monoclonal antibody (different polymers)

Aggregate content% relative activity% control 100.0% 2.2 92.8% 4.3 90.8% 6.7 87.9% 9.3 86.4%

Table 2 Summary table of evaluation results of biological activity detection accuracy of RANKL target monoclonal antibody (different activities)

Embodiment 5 precision evaluation experiment

Using the working reference product and the sample to be tested as materials, the precision described in the present invention is evaluated. First, an experimenter uses the above-mentioned detection method steps to select a sample to be tested for biological activity detection, and performs parallel experiments 6 times to investigate the repeatability of the method, and then two experimenters use the above-mentioned detection method steps within 2 days Three parallel experiments were completed to investigate the intermediate precision of the method.

As shown in Table 3, using the above-mentioned detection method, one experimenter carried out 6 consecutive parallel experiments. As a result, the fitted four-parameter equation and the curve fitting constant R2 ^were all greater than 0.98, and the RLU value of the duplicate wells in all concentration points was greater than 0.98. The CV% is less than 20%, and the deviation of all results is less than 10%, indicating that the method of the present invention has high repeatability.

As shown in Table 4, using the above-mentioned detection method, two experimenters conducted three parallel experiments in at least two days, and the four-parameter equation fitted by the results, the curve fitting constant R2 satisfies greater ^than 0.98, and all concentration points The CV% of the RLU value of the multiple wells satisfies less than 20%, and the deviation of the results obtained by different experimenters is less than 10%, indicating that the method of the present invention has high intermediate precision.

Table 3 Summary of the evaluation results of the repeatability evaluation of the biological activity of the RANKL target monoclonal antibody

Table 4 Summary of intermediate precision evaluation results for the biological activity detection of RANKL target monoclonal antibodies

Example 6 Cell Subculture Stability Evaluation Experiment

Using the working reference product as the material, select the tool cells of different cell generations (3, 4, 7, 11, 21), and use the above detection method steps to detect the biological activity, and collect the RANKL stimulation produced by the cells of different generations. The EC ₅₀ of the dose-response curve is used as the basis for evaluating whether the cells are usable. The results are shown in Table 5, which shows that the cells have no obvious influence on the detection results after 21 passages.

Table 5 Test stability for cell biological activity of RANKL target monoclonal antibody drug

(Cell Generation) Evaluation Results Summary Table

Embodiment 7 durability evaluation experiment

Using the working reference product and the sample to be tested as materials, using different incubation times, color development times and different cell numbers, the same sample to be tested is detected by the above detection method.

Table 6 Durability of Cell Biological Activity Detection of RANKL Target Monoclonal Antibody Drugs

(Cell number) evaluation result summary table

Table 7 Durability of Cell Biological Activity Detection of RANKL Target Monoclonal Antibody Drugs

(Incubation and color development time) evaluation results summary table

In summary, the present invention constructs a tool cell, and establishes a biological activity detection method for RANKL target therapeutic drugs. The tool cell and method can be used to quickly, efficiently, and mass-produce drugs targeting RANKL target The biological activity detection operation of biotherapeutic drugs, through the verification of method specificity, accuracy, precision and durability, etc., proves that the method has strong stability and good applicability, and can meet the biological requirements of RANKL target therapeutic drugs. Activity detection. In principle, this method can also be used for the detection of anti-drug neutralizing antibodies in serum/plasma samples collected in preclinical and clinical trials, because the test cycle is short and should have a high serum/plasma matrix tolerance.

The scope of the invention is not to be limited by the specific embodiments described, which are presented as single examples illustrating various aspects of the invention, and functionally equivalent methods and components are also intended to be within the scope of the invention. In fact, in addition to the content described herein, those skilled in the art can easily grasp various modifications to the present invention by referring to the above description and accompanying drawings. Such modifications also fall within the scope of the appended claims. Each of the above-mentioned references is hereby incorporated by reference in its entirety.

sequence listing

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Claims (9)

1. a kind of for detecting the vehicles cells of RANKL target spot compound biological activity, the cell is to stablize expression RANK With the HEK293 cell of NF- κ B fusion luciferase.

2. vehicles cells described in claim 1 detection selected from for RANKL target spot monoclonal antibody or in which with antibody, Fc Application in one or a combination set of fusion protein, small molecule compound.

3. a kind of detection method of RANKL target spot compound biological activity, described method includes following steps：

（1）RANKL target spot compound control product solution and test solution are prepared respectively, and diluted concentration gradient is set；

（2）After the HEK293 cell of expression RANK and NF- κ B fusion luciferase is stablized in culture, RANKL solution is added, adds Step（1）The reference substance solution and test solution of middle preparation handle cell；

（3）The intracellular luciferase level after anti-RANKL antibody is handled is detected with luminescence method luciferase assay reagents box Variation reads corresponding chemiluminescence unit with microplate reader；

（4）Using extension rate or drug concentration as abscissa, using relatively chemical flat light emission as ordinate, RANKL target spot is obtained The dose-effect curve of compound and the biological activity for calculating test sample.

4. detection method according to claim 3, it is characterised in that the gradient dilution is 2 ~ 5 times of dilutions.

5. detection method according to claim 3, it is characterized in that the luciferase assay reagents box is Luciferase Assay System。

6. detection method according to claim 3, it is characterized in that calculating the biological activity using quadruplex parameters.

7. detection method according to claim 3, it is characterized in that the RANK sequence is SEQ ID No.1.

8. detection method according to claim 3, it is characterized in that NF- κ B fusion luciferase sequence is SEQ ID No.2。

9. detection method according to claim 3 detection for RANKL target spot monoclonal antibody or in which with antibody, Application in the drug neutralizing antibody of one or a combination set of Fc fusion protein, small molecule compound.