A cell and method for detecting the activity of GLP-1R / GIPR dual-target agonist drugs

Abstract

This invention discloses a cell line and method for detecting the activity of GLP-1R / GIPR dual-target agonist drugs. The invention relates to a cell line co-expressing GLP-1R, GIPR, and CRE-miniP-luciferase reporter genes, and provides a method for detecting the biological activity of GLP-1R / GIPR dual-target agonist drugs based on this cell line. This method is simple to operate, has a short experimental cycle, is highly sensitive, exhibits low variability, strong specificity, and high accuracy. It can be used for product release testing and is of great significance for the quality control and clinical application of GLP-1R / GIPR dual-target agonist drugs.

Description

Technical Field

[0001] This invention belongs to the field of biological activity detection of biopharmaceuticals. Specifically, this invention relates to a cell and method for detecting the activity of a GLP-1R / GIPR dual-target agonist drug. Background Technology

[0002] GLP-1R / GIPR dual-target agonists are novel drugs that simultaneously activate glucagon-like peptide-1 receptor (GLP-1R) and gastric inhibitory peptide receptor (GIPR) to achieve multiple biological effects, including lowering blood sugar, weight loss, and improving metabolism. This drug design aims to integrate the beneficial effects of multiple gastrointestinal hormones to improve metabolic disorders, particularly metabolic diseases such as obesity and diabetes.

[0003] Biological activity reflects the correctness of the higher-order structure of therapeutic proteins, the integrity of their biological functions, and batch-to-batch consistency, making it a key quality attribute of GLP-1R / GIPR dual-target agonist drugs. Traditional methods for determining the biological activity of GLP-1R / GIPR dual-target agonist drugs include using luminescent beads to detect cAMP and Promega kits to detect residual ATP. However, these methods are highly susceptible to environmental influences, have short half-lives, are costly, and exhibit poor stability. Therefore, exploring new methods for detecting the activity of GLP-1R / GIPR dual-target agonist drugs is of great significance. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the purpose of this invention is to provide a cell and method for detecting the activity of GLP-1R / GIPR dual-target agonist drugs.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention provides a cell line.

[0006] Furthermore, the cell line was named GLP1R / GIPR-luc-293, deposited at the China Culture Collection Committee, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46309, classified as Human HEK293 cells, and deposited on December 25, 2024.

[0007] Furthermore, the cell line co-expresses GLP-1R, GIPR, and CRE-miniP-luciferase reporter genes; CRE-miniP is a fusion gene formed by linking the CRE gene and the miniP gene.

[0008] Furthermore, the sequence of the CRE gene is shown in SEQ ID NO.1, the sequence of the miniP gene is shown in SEQ ID NO.2, and the sequence of the CRE-miniP gene is shown in SEQ ID NO.3.

[0009] In this invention, the applicant transfected HEK293 cells with the CRE-miniP-luciferase reporter gene, GLP-1 receptor (GLP-1R) expression gene, and GIP receptor (GIPR) expression gene. Five stable monoclonal cell lines expressing these genes were obtained using pressure selection, monoclonal isolation culture, and flow cytometry. Functional validation revealed that the five cell lines responded to the GLP-1R / GIPR dual-target agonist telpolide to varying degrees. Among them, monoclonal cell C9 exhibited the strongest response to telpolide, GLP-1, and GIP, with upper and lower asymptote ratios of 162, 173, and 166, respectively. Therefore, this cell line was selected for preservation and named GLP1R / GIPR-luc-293.

[0010] A second aspect of the present invention provides a cell preparation.

[0011] Furthermore, the cell preparation includes the cell line described in the first aspect of the present invention.

[0012] Preferably, the cell preparation further includes a luciferase reaction substrate.

[0013] Preferably, the cell preparation includes suspension-type cell preparations, solution-type cell preparations, and solid dispersion-type cell preparations.

[0014] The third aspect of this invention provides an evaluation system.

[0015] Furthermore, the evaluation system includes the following components: the cell line described in the first aspect of the present invention, a GLP-1R / GIPR dual-target agonist drug sample, and a reference product.

[0016] In this invention, the term "system" refers to a pharmaceutical quality control system, which refers to the control of various factors in the pharmaceutical production process through scientific management methods to ensure that the quality of pharmaceuticals meets predetermined quality standards and requirements.

[0017] In this invention, the term "sample" refers to a test sample to be identified and measured. In this invention, a GLP-1R / GIPR dual-target agonist drug sample includes a drug sample produced in industrial production for which its biological activity is to be tested.

[0018] The fourth aspect of the present invention provides a product.

[0019] Furthermore, the product includes the cell line described in the first aspect of the present invention or the cell preparation described in the second aspect of the present invention.

[0020] Preferably, the kit further includes a luciferase reaction substrate and a diluent.

[0021] Preferably, the product is a reagent kit.

[0022] In this invention, the components of the kit can be packaged in an aqueous medium or in a lyophilized form. Suitable containers in the kit typically include at least one vial, test tube, long-necked flask, PET bottle, syringe, or other container, in which one component can be placed, and preferably, appropriately aliquoted. When more than one component is present in the kit, the kit will also typically include a second, third, or other additional container in which the additional components are placed separately. However, different combinations of components may be contained in a single vial. The kit of this invention will also typically include a container for containing the reactants, sealed for commercial sale. Such a container may include injection-molded or blow-molded plastic containers in which the desired vials can be held.

[0023] The fifth aspect of the present invention provides a method for constructing the cell line described in the first aspect of the present invention.

[0024] Furthermore, the method includes: 1) Transfect effector cells with vectors expressing the GLP-1R gene, GIPR gene, and / or CRE-miniP gene; 2) Screening yielded monoclonal cell lines co-expressing GLP-1R, GIPR, and CRE-miniP.

[0025] Furthermore, the CRE-miniP is a fusion gene formed by linking the CRE gene and the miniP gene.

[0026] Furthermore, the sequence of the CRE gene is shown in SEQ ID NO.1, the sequence of the miniP gene is shown in SEQ ID NO.2, and the sequence of the CRE-miniP gene is shown in SEQ ID NO.3.

[0027] Those skilled in the art can choose different methods to synthesize the CRE-miniP gene based on the CRE gene and miniP gene sequences. In a specific embodiment of the present invention, the CRE gene and miniP gene were sent to Suzhou Dongling Biotechnology Co., Ltd. for ligation using total gene synthesis technology.

[0028] Furthermore, the effector cells are HEK293 cells.

[0029] Furthermore, the expression vector includes plasmid vectors, phage vectors, and viral vectors.

[0030] Preferably, the expression vector is a plasmid vector.

[0031] Furthermore, the transfection methods include physical-mediated, chemical-mediated, and biological-mediated transfection.

[0032] Preferably, the transfection method is biologically mediated.

[0033] More preferably, the transfection method is lentiviral infection.

[0034] In some implementations, the cell types typically used for transfection are diverse, including but not limited to the following: 1) Primary cells and their derivatives: These cells are directly isolated from biological tissues, without long-term in vitro culture, and maintain high physiological activity and differentiation potential; 2) Tumor cell lines: such as human breast cancer cells (MCF-7), human lung cancer cells (A549), and human cervical cancer cells (HeLa). These cell lines have high division rates and stability, and are often used for gene function research and drug screening; 3) Cell lines derived from specific tissues: Human embryonic kidney cells (HEK-293): Possessing high transfection efficiency and stability, they are commonly used for gene cloning and vaccine production. Human embryonic lung cells (HEL): Used as recipient cells in some experiments; Human umbilical vein endothelial cells (HUVECs): Used for research on vascular biology and cardiovascular diseases; 4) Animal cell lines: Young hamster kidney cells (BHK-21): Widely used in virology research and other biomedical research; Mouse embryonic fibroblasts (NIH3T3): Possessing low immunogenicity and high division rate, they are commonly used for gene knockout and drug screening. Other special cell types: Neurons: Used in neurobiological research; Rat hepatocytes: used for drug metabolism and toxicity studies; Rat glioma cells (C6): These cells exhibit high differentiation and stability, and are commonly used in drug screening and tumor research. Those skilled in the art can select different effector cells based on existing technology. In a specific embodiment of the present invention, the effector cell is HEK293.

[0035] In this invention, the term "reporter gene" refers to a gene that encodes a protein or enzyme that is easily detectable against an endogenous protein background. The reporter gene is linked to a target gene or regulatory sequence, and by indirectly or directly detecting the signal of the reporter gene's encoded product, such as a reporter protein, mRNA, or enzyme, it directly reflects the transcriptional activity or expression level of the gene within the cell. Generally, reporter genes are characterized by being non-toxic, non-immunogenic, easily detectable, and possessing a certain degree of specificity.

[0036] In a specific embodiment of the present invention, the reporter gene used is the LUC gene, namely the luciferase reporter gene. Luciferase is a class of enzymes derived from bioluminescent organisms in nature, capable of catalyzing the oxidation of luciferin or fatty aldehydes to produce light. Depending on the source organism, luciferases can be divided into firefly luciferase (FL) and bacterial luciferase (BL). Currently, the luciferase gene derived from North American fireflies is the most widely used; this gene encodes a luciferase protein of 550 amino acids. The FL gene originates from fireflies and is found in Mg... 2+ With the participation of ATP and O2, luciferase catalyzes the oxidative decarboxylation of D-luciferin, producing activated oxidized luciferin, which emits photons and generates fluorescence at 550-580 nm. Luciferase reporter gene detection is an important tool in modern molecular biology research for analyzing the interactions between potential cis-elements (such as promoters, enhancers, and silencers) and trans-acting factors in flanking regions of structural genes. The luciferase reporter gene system is a reporter system that uses luciferin as a substrate to detect the activity of firefly luciferase. Luciferase catalyzes the oxidation of luciferin to oxidized luciferin, during which bioluminescence is emitted, which is then measured using a chemiluminescence analyzer or liquid scintillation analyzer.

[0037] In this invention, the term "plasmid" refers to a naked, double-stranded circular DNA molecule independent of the DNA in the cell nucleus. It possesses the ability to replicate autonomously and can be stably inherited within the cell. Plasmids are primarily found in bacteria, but can also be found in eukaryotes. Introducing specific DNA sequences encoding one or more specific proteins into a plasmid confers new traits or functions on the host cell. For example, a plasmid can carry a target gene for gene therapy, a coding sequence for protein production, or impart new functions to cells through transfection. In this invention, those skilled in the art can select different plasmids to introduce the GLP-1R gene, the GIPR gene, and the CRE-miniP-luciferase reporter gene into cells. In a specific embodiment of this invention, the vector for introducing the GLP-1R gene and the GIPR gene into cells is the pLU.cmv.neo plasmid, and the vector for introducing the CRE-miniP-luciferase reporter gene into cells is the pLU.CMV.Luc.PGK.PURO plasmid.

[0038] In this invention, the term "transfection" refers to the process of introducing foreign DNA (such as plasmids) into eukaryotic cells, enabling the expression of foreign genes within the cells. Plasmid transfection is an important technique in genetic engineering and is widely used in fields such as gene function research and protein production.

[0039] Cell transfection methods include physical, chemical, and biological mediation. Physical mediation includes electroporation, microinjection, and gene gun methods; chemical mediation includes calcium phosphate coprecipitation, cationic polymer methods, and cationic liposome methods; and biological mediation is mainly virus-mediated, such as lentiviruses and adenoviruses. In a specific embodiment of this invention, the transfection method is lentiviral transfection. Furthermore, the screening includes the following steps: pressure screening, single-clone isolation culture, and flow cytometry analysis.

[0040] Preferably, the pressure screening is performed by adding antibiotics.

[0041] Furthermore, the antibiotics are bleomycin, puromycin, and G418.

[0042] More preferably, the concentrations of bleomycin, puromycin, and G418 are each 200 μg·mL. -1 3 μg·mL -1 and 1000 μg·mL -1 .

[0043] In this invention, the term "pressure screening" refers to the selection and retention of successfully transfected cells through specific selection pressure. Typically, pressure screening methods include antibiotic screening and gene expression product screening. In a specific embodiment of this invention, the pressure screening is performed by adding antibiotics.

[0044] In cell selection, antibiotics are commonly used as a tool for resistance screening to distinguish successfully transfected cells from untransfected cells. These antibiotics allow successfully transfected cells that express antibiotic resistance genes to survive by inhibiting the growth of untransfected cells or killing them.

[0045] Furthermore, the antibiotics include, but are not limited to: penicillin-streptomycin mixture, G-418 (Geneticin), blastcinin, hygromycin B, puromycin, 6-thioguanine, ampicillin, and zeocin. In a specific embodiment of the present invention, the antibiotics are bleomycin, puromycin, and G418.

[0046] The sixth aspect of this invention provides a method for detecting the biological activity of a GLP-1R / GIPR dual-target agonist drug.

[0047] Furthermore, the method includes: adding a GLP-1R / GIPR dual-target agonist drug to the cell line described in the first aspect of the present invention for incubation, adding an enzyme reaction substrate after incubation, and determining the biological activity of the GLP-1R / GIPR dual-target agonist drug based on the measured reporter gene signal value.

[0048] Furthermore, when the GLP-1R / GIPR dual-target agonist drug is incubated with the cell line described in the first aspect of this invention, its initial concentration is 25 nmol·mL⁻¹. -1 ~100 nmol·mL -1 .

[0049] Preferably, the initial concentration of the GLP-1R / GIPR dual-target agonist drug is 25 nmol·mL. -1 .

[0050] Furthermore, the dilution factor of the GLP-1R / GIPR dual-target agonist drug is 4 to 6 times.

[0051] More preferably, the dilution factor of the GLP-1R / GIPR dual-target agonist drug is 4 times.

[0052] Furthermore, the GLP-1R / GIPR dual-target agonist drugs include telpoitide, BGM0504, and HS-20094.

[0053] Preferably, the GLP-1R / GIPR dual-target agonist drug is telpolide.

[0054] In this invention, the applicant optimized the initial concentration and dilution factor of the GLP-1R / GIPR dual-target agonist drug telpotetide, and found that the optimal initial concentration was 25 nmol·mL. -1 The optimal dilution factor is 4-fold.

[0055] In this invention, the biological activity of a GLP-1R / GIPR dual-target agonist drug is determined by fitting a four-parameter curve after measuring the reporter gene signal value. The term "fitting a four-parameter curve" is a mathematical method used to describe a set of data by fitting a curve and obtaining a functional equation that describes the relationship between the data, in order to predict future data. A four-parameter curve is typically defined by four parameters that have practical significance in a biological context. For example, in detecting drug biological activity, these four parameters might represent: the lower limit of absorbance (D), the upper limit of absorbance (A), i.e., the minimum and maximum absorbance values; the half-maximum response concentration (C), i.e., the value at which the drug concentration reaches half of the maximum response; and the slope of the curve (B). These parameters allow for a more accurate understanding and analysis of experimental data, thus providing deeper insights for biological research. The seventh aspect of the present invention provides for application in any of the following aspects.

[0056] Furthermore, the applications include: 1) The use of the cell line described in the first aspect of the present invention or the cell preparation described in the second aspect of the present invention in the preparation of products for detecting the biological activity of GLP-1R / GIPR dual-target agonist drugs; 2) Application of the cell line described in the first aspect of the present invention, the cell preparation described in the second aspect of the present invention, or the evaluation system described in the third aspect of the present invention in the quality control of GLP-1R / GIPR dual-target agonist drugs; 3) The application of the evaluation system described in the third aspect of this invention in evaluating the biological activity of GLP-1R / GIPR dual-target agonist drugs.

[0057] Advantages and beneficial effects of the present invention: This invention provides a cell and method for detecting the activity of a GLP-1R / GIPR dual-target agonist. The principle of this method is that after the GLP-1 receptor and GIP receptor of the CRE-miniP-luc-GLP-1R-GIPR HEK293 monoclonal cell line bind to telpolide, a series of signal transduction pathways activate the expression of the reporter gene luciferase. The activity of telpolide is quantified by detecting changes in luciferase expression after telpolide stimulation. This method has the advantages of simple operation, short experimental cycle, high sensitivity, low variability, high specificity, and high accuracy. It can be used for product release testing and is of great significance for the quality control and clinical application of telpolide. Attached Figure Description

[0058] Figure 1 The results show the confirmation that the CRE-miniP-luc-GLP-1R-GIPR HEK293 monoclonal cell line expresses GLP1-R; Figure 2 The results show the confirmation that the HEK293 monoclonal cell line expresses GIPR; Figure 3 The response of the CRE-miniP-luc-GLP-1R-GIPR HEK293 monoclonal cell line to telposide, GLP-1, and GIP was shown. Figure 4 The graph shows the results of optimizing the initial concentration of telpoeptide; Figure 5 The graph shows the results of optimizing the dilution factor of telpolide. Detailed Implementation

[0059] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents.

[0060] Unless otherwise specified, the experimental methods described in the following examples are generally performed under standard conditions or as recommended by the manufacturer.

[0061] Example 1: Cell Construction and Positive Cell Screening I. Experimental Methods The GLP-1R gene was constructed into the pLU.cmv.neo vector using the BamHI and SalI cloning sites, the GIPR gene into the pLU.cmv.zeo vector using the BamHI and SalI cloning sites, and the CRE-miniP gene into the pLU.CMV.Luc.PGK.PURO vector using the ClaI and XbaI cloning sites. The plasmids were then amplified and purified, packaged into lentiviruses using a viral packaging kit, and used to infect HEK293T cells. Finally, 200 μg / mL... -1 zeocin, 3 μg·mL -1 puromycin, 1000 μg·mL -1 Stable transfected cells were obtained by screening G418 cells. Finally, the expression of GLP-1R and GIPR on the cell surface was analyzed by flow cytometry to identify positive monoclonal cells.

[0062]

[0063] II. Experimental Results Five monoclonal cell lines were obtained using the limiting dilution method. The GLP-1R positivity rates in the five monoclonal cell lines (B6, B9, C9, E10, and G8) were 99.2%, 99.3%, 96.0%, 91.7%, and 96.9%, respectively; the GIPR positivity rates were 96.3%, 98.8%, 98.4%, 98.1%, and 97.4%, respectively. Figure 1 and Figure 2 Five cell lines were respectively fed with 4.0 × 10⁻⁶ cells. 4 Cells were seeded at a density of cells / well in slabs, and the biological activities of the same telpoeptide, GLP-1, and GIP were measured. Figure 3 As shown, all five cell lines responded to telposide stimulation with a dose-response relationship, and the level of luciferase expression increased with increasing drug concentration. The ratio of the upper and lower asymptotes of the S-curves fitted by the four-parameter model for all five cell lines was greater than 40. Among them, the C9 monoclonal cell line showed the strongest response to telposide, GLP-1, and GIP, with upper and lower asymptote ratios of 162, 173, and 166, respectively. Therefore, C9 monoclonal cells were used in subsequent experiments.

[0064] Example 2: Method Parameter Optimization I. Experimental Methods Dilute telpolide to an initial concentration of 100 nmol·mL⁻¹ -1 50 nmol·mL -1 25 nmol·mL -1 Subsequently, 6-fold serial dilutions were performed, resulting in 10 concentration points, to verify the response of the C9 cell line to telpolide. The optimal starting concentration was then selected to further optimize the dilution factor.

[0065] II. Experimental Results like Figure 4 As shown, all three initial concentrations reached the plateau on the curve, with the initial concentration being 25 nmol·mL⁻¹. -1 At that point, the curve had formed a clear upper plateau, but there were only two points in the linear portion, so 25 nmol·mL⁻¹ was chosen. -1 To further optimize the dilution factor for the initial concentration, 25 nmol·mL⁻¹ telpoide was diluted 4-, 5-, and 6-fold, resulting in 10 concentration points for each dilution. At a 4-fold dilution, the curve showed 3 points on both the upper and lower plateaus, with the linear portion having the most points. Figure 5 Therefore, the optimal starting concentration for the method was determined to be 25 nmol·mL⁻¹. -1 The optimal dilution factor is 4-fold.

[0066] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A cell line, characterized in that, The cell line was named GLP1R / GIPR-luc-293 and deposited with the China Culture Collection Committee, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo.46309. It was classified as Human HEK293 cells and the deposit date was December 25, 2024.

2. The cell line according to claim 1, characterized in that, The cell line co-expresses GLP-1R, GIPR, and CRE-miniP-luciferase reporter genes; CRE-miniP is a fusion gene formed by linking the CRE gene and the miniP gene.

3. The cell line according to claim 2, characterized in that, The sequence of the CRE gene is shown in SEQ ID NO.1, the sequence of the miniP gene is shown in SEQ ID NO.2, and the sequence of the CRE-miniP gene is shown in SEQ ID NO.

3.

4. A cell preparation, characterized in that, The cell preparation includes the cell line described in claim 1.

5. The cell preparation according to claim 4, characterized in that, The cell preparation also includes a luciferase reaction substrate.

6. The cell preparation according to claim 4, characterized in that, The cell preparations include suspension-type cell preparations, solution-type cell preparations, and solid dispersion-type cell preparations.

7. An evaluation system, characterized in that, The evaluation system comprises the following components: the cell line described in any one of claims 1-3, a GLP-1R / GIPR dual-target agonist drug sample, and a reference product.

8. A product characterized in that, The product includes the cell line according to any one of claims 1-3 or the cell preparation according to any one of claims 4-6.

9. The product according to claim 8, characterized in that, The product is a reagent kit.

10. The product according to claim 9, characterized in that, The kit also includes a luciferase reaction substrate and a diluent.

11. A method for detecting the biological activity of a GLP-1R / GIPR dual-target agonist drug, characterized in that, The method includes: adding a GLP-1R / GIPR dual-target agonist drug to the cell line described in any one of claims 1-3 for incubation, adding an enzyme reaction substrate after incubation, and determining the biological activity of the GLP-1R / GIPR dual-target agonist drug based on the measured reporter gene signal value.

12. The method according to claim 11, characterized in that, When the GLP-1R / GIPR dual-target agonist drug is incubated with the cell line according to any one of claims 1-3, its initial concentration is 25 nmol·mL⁻¹ to 100 nmol·mL⁻¹.

13. The method according to claim 12, characterized in that, The initial concentration of the GLP-1R / GIPR dual-target agonist drug was 25 nmol·mL⁻¹.

14. The method according to claim 11, characterized in that, The dilution factor of the GLP-1R / GIPR dual-target agonist drug is 4 to 6 times.

15. The method according to claim 14, characterized in that, The GLP-1R / GIPR dual-target agonist drug was diluted 4 times.

16. The method according to claim 11, characterized in that, The GLP-1R / GIPR dual-target agonist drugs include telpoitide, BGM0504, and HS-20094.

17. The method according to claim 16, characterized in that, The GLP-1R / GIPR dual-target agonist drug is telpolide.

18. The use of the cell line according to any one of claims 1-3 or the cell preparation according to any one of claims 4-6 in the preparation of products for detecting the biological activity of GLP-1R / GIPR dual-target agonist drugs.

19. The application of the cell line according to any one of claims 1-3, the cell preparation according to any one of claims 4-6, or the evaluation system according to claim 7 in the quality control of GLP-1R / GIPR dual-target agonist drugs.

20. The application of the evaluation system described in claim 7 in evaluating the biological activity of GLP-1R / GIPR dual-target agonist drugs.

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