Cell lines for FSH receptor expression

Abstract

The present invention, as described herein, relates to a novel cell line for expressing the human FSH receptor together with a luciferase reporter gene. Methods for preparing such cell lines are also described. The cell lines of the present invention expressing the FSH receptor and luciferase reporter gene can be used in methods for determining the presence and biological activity of r-FSH samples.

Description

Technical Field

[0001] This specification describes novel cell lines useful for the development of bioassays related to the production of recombinant human follicle-stimulating hormone (r-hFSH). This cell line expresses both the human FSH receptor and a luciferase reporter gene (HEK293T FSHR luciferase) under the control of the cAMP response element (CRE).

Background Art

[0002] Bioassays are used to evaluate the efficacy of pharmaceutical products before they are released onto the market. In order to estimate the potency of a drug, the assay needs to reflect or mimic the known mechanism of action (or a part thereof) of the product as it occurs in the human body, in order to reliably evaluate the biological activity of the product.

[0003] An in vivo efficacy assay is a method of treating animals with a reference substance and a test substance, evaluating the relationship between the product concentration and the resulting response, and estimating the efficacy. Instead, in a cell-based (or in vitro) efficacy assay, the use of cell lines that respond to specific ligands or infectious agents is envisaged. The nature of the cell lines varies, and includes those derived from tumors, factor-dependently immortalized ones, or engineered cell lines transfected with the appropriate target of interest. One of the main requirements is that these cell lines must be stable in order to guarantee reproducible and reliable results for the assay over time. The cellular response to the protein of interest is related to the mechanism of action of the drug and the exposure period. Some drug responses include cell proliferation, cell death, cell differentiation, secretion of cytokines or other molecules, activation of intracellular responses, and activation of enzymes.

[0004] Currently, bioassays for determining the presence and potency of recombinant human FSH involve in vivo assays using animals and newly developed in vitro assays. This cell-based assay anticipates the use of cell lines used to determine the presence and potency of r-hFSH.

Summary of the Invention

[0005] This invention provides a solution to the dependence on commercially available kits currently used in cell-based assays for conducting tests. This solution provides a novel mammalian cell line that highly expresses the human FSH receptor and conjugates it to a reporter. Thus, by binding r-FSH to the human FSH receptor on the novel cells of this invention, the biological activity of r-FSH can be quantified.

[0006] In one embodiment, a mammalian cell line is provided that already expresses a luciferase reporter gene under the control of a cAMP response element (CRE) for expressing a human FSH receptor, which includes a nucleic acid molecule encoding a human FSH receptor under the control of a promoter for protein expression.

[0007] In another embodiment, a method for generating human FSH receptors in mammalian cells is provided, the method comprising: a) transfecting mammalian cells with a lentivirus containing a gene encoding an FSH receptor under the control of a promoter for protein expression; b) adding a first selective agent to the transfected cells of step a) to induce stably expressed human FSH receptor cells and a luciferase reporter; and d) collecting cells that stably express the human FSH receptor and luciferase reporter (HEK293-T FSHR luciferase).

[0008] In yet another embodiment, a method is provided for quantifying the biological activity of an r-hFSH sample, the method comprising: a) stimulating mammalian cells expressing a human FSH receptor and a luciferase reporter with the sample; b) adding a luciferase enzyme luminescence substrate to a cell culture; c) detecting luminescence from the cells; d) quantifying the amount of luminescence; and e) determining the potency of r-hFSH. [Brief explanation of the drawing]

[0009] [Figure 1]Figures 1A and 1B: FRET analysis for FSH receptor activity quantification to evaluate transfection efficiency. Response of HEK293-T FSHR luciferase (1A) versus HEK293-T WT (1B) to r-hFSH stimulation. [Figure 2] Figure 2: Representative RGA dose-response curve for quantifying the efficacy of r-hFSH samples. [Figure 3] Figure 3: FSHR staining of HEK293-T FSHR luciferase. Data were acquired using BD FACS Aria Fusion and analyzed using Flowjo software. FACS staining of HEK293-T WT (left panel) and HEK293-T FSHR luciferase (right panel) with anti-human FSH R Alexa Fluor® 488 labeled antibody. [Figure 4] Figure 4: Monitoring of cell transfection stability within a defined passage range. FACS staining of HEK293-T LHCGR luciferase (right panel) over time using anti-human FSH® Alexa Fluor® 488 labeled antibody. [Figure 5] Figure 5: Cell behavior within a defined passage range. A) Analysis of cell viability, B) Analysis of cell doubling. Figure 5A. Cell line viability expressed as a percentage, measured using a Nucleocounter instrument. Figure 5B. Doubling time results for HEK293-T FSHR luciferase cells range from 16 to 20 hours. [Modes for carrying out the invention]

[0010] Currently, efficacy testing for each batch release of new FSH formulations is performed using either in vivo or in vitro methods. In vivo methods use rats for quality control purposes and should be carried out in accordance with the requirements and descriptions of regulatory authorities and guidelines. Cell-based assays are performed for batch releases of the active pharmaceutical ingredient in the list of countries where the switch to the new method has been approved.

[0011] Currently, the only commercially available cell line is suitable for the development of in vitro cell-based analytical methods, intended for the quantification of cAMP produced by cells after stimulation with r-hFSH. Quantification involves measuring cAMP released from cells using a commercially available kit. This technique restricts QC staff to using expensive and difficult-to-replace kits. On the other hand, it is preferable to use commercially available alternatives for the quantification reagents.

[0012] The present invention provides such cell lines, methods for producing such cell lines, and methods for in vitro testing of r-hFSH from production batches using these new cell lines.

[0013] The cell line of the present invention is based on the human embryonic kidney (HEK) cell line, more specifically HEK293-T.

[0014] The development of the cell line and the in vitro method were carried out according to the mechanism of action of r-hFSH in accordance with the guidelines and requirements of health authorities.

[0015] Cell line development began with HEK293-T luciferase cells. The HEK293-T luciferase cell line was stably transfected with the human FSH receptor.

[0016] The newly created cells faithfully reproduce part of the in vivo mechanism of action of r-hFSH, namely the first step in the metabolic cascade that occurs when the receptor binds to the hormone.

[0017] The transfection process resulted in the creation of a novel cell line of HEK293T cells expressing the human FSH receptor and luciferase reporter (HEK293-T FSHR luciferase). The transfected cells were selected and expanded while freezing in various cell banks. After the cell banks were created, two different functional assays were developed to obtain a method for preliminarily evaluating the efficiency of transfection from a functional perspective.

[0018] We developed the first functional assay to quantify cAMP after cell stimulation using a commercially available FRET kit. This method allowed us to evaluate FSH receptor activation in HEK293-T FSHR luciferase cells. This assay enabled us to evaluate the differences between transfected and untransfected cells, confirming that the FSH receptor was expressed only in transfected cells.

[0019] A second functional assay was developed to evaluate reporter gene transcription after intracellular cAMP accumulation. This assay involved stimulating cells with r-hFSH, resulting in adenylyl cyclase activation. This stimulation increased intracellular cAMP, necessitating the evaluation of reporter gene transcription in transfected cells. Again, differences in luminescence generation after cell stimulation were observed between transfected and untransfected cells. This assay allowed for the confirmation of the luciferase reporter gene vector's presence and functionality in the HEK293-T FSHR luciferase cell line.

[0020] Preliminary characterization of HEK293-T FSHR luciferase was performed over a defined passage range from passage 20 to passage 37. During this study, cell behavior was monitored while evaluating specific parameters such as FSH receptor expression on the cell membrane, cell viability, and cell proliferation (cell doubling time). Previously developed functional assays were also included in the characterization, allowing for monitoring of transfection stability over time.

[0021] FSH receptor expression in the FSHR luciferase membrane of HEK293-T cells was evaluated compared to untransfected cells. These experiments confirmed the presence of FSH receptors in transfected cells, with the percentage of FSH-positive cells being approximately 95-99%.

[0022] Phenotypic and functional characterization of the HEK293-T FSHR luciferase cell line showed that the cells exhibited stable viability and stable growth during the selected passage range.

[0023] The stability of receptor expression in the cell membrane was evaluated by stimulating the cells with FACS analysis and r-hFSH, and the stability and functionality of transfection over time were confirmed.

[0024] In addition to all activities related to the development and characterization of the cell line, an in vitro assay was developed to quantify the potency of r-hFSH samples. The procedure of the developed method was very rapid and simple. These features ensure the optimal application of the measurement method in quality control routines.

Example

[0025] Culture of HEK293T luciferase cells The recommended cell culture medium is Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 0.5 μg / mL puromycin. The doubling time of the cells is approximately 16 - 20 hours. The cells were cultured in a humidified atmosphere at 37 ± 1°C and 5 ± 1% CO2. All of these cell characteristics are described in the cell data sheet provided by the supplier, and the cell passage conditions have been specified in our laboratory.

[0026] Transfection of HEK293T luciferase cells The HEK293T luciferase cell line was transfected with the FSH receptor using an appropriate lentivirus (provided by Vector Builder) consisting of a nucleic acid molecule encoding the human FSH receptor. The vector was a mammalian gene expression vector and was 8085 bp in length. For stable transfection, hygromycin B was used as a selection marker. Prior to transfection, the minimum hygromycin B concentration that could block the growth of HEK293-T in 10 days was identified, and a kill curve assay was performed. A concentration of 200 μg / mL was optimal.

[0027] A lentivirus containing the FSH receptor gene was introduced into HEK293-T luciferase cells using cell transduction. The commercially available reagents used for the transduction were included in the VectorBuilder kit sold together with the lentivirus.

[0028] The culture medium used for transduction was OptiMEM (Life Technologies, 31985). Different infection MOIs were tested using different amounts of vector DNA. The transduction process and various experimental conditions followed the protocol suggested by the supplier. 24 hours after transduction, the medium was replaced with DMEM and 10% FBS to remove the lentivirus. 24 hours after the medium change, antibiotic selection was added to the cells to select only the population resistant to the antibiotic concentrations identified during the cell death curve experiment.

[0029] Resistant cells were expanded, and different cell banks belonging to each transfection condition were cryopreserved. The usual approach to creating cell banks is to freeze a master cell bank (MCB). If necessary, a working cell bank (WCB) is created from the MCB as a starting point. The purpose of bank creation is to ensure a sufficient number of cells during assay development and implementation. Bank creation depends on the proliferation characteristics of the cells, and the size of the bank depends on the number of cells required for each assay and the frequency of assay execution.

[0030] Initial Functional Assay Evaluation To evaluate transfection efficiency, transfected cells were stimulated with r-hFSH, and cAMP production was quantified using a commercially available kit based on fluorescence resonance energy transfer (FRET). The FRET kit used for cAMP quantification was provided by Cisbio. This kit contains cAMP conjugated to d2 (acceptor) and an anti-cAMP antibody conjugated to a cryptotate (donor, europium). FRET is detected using a plate reader equipped with HTRF (Homogeneous Time Resolve Fluorescence) detector technology. All experiments were performed according to the datasheet provided by the kit supplier.

[0031] For data interpretation, the ratio of acceptor and donor emission signals for each well needs to be calculated using the following formula: Ratio = (Signal at 665nm) / (Signal at 620nm) × 10000

[0032] All raw data (proportions) obtained from the FRET kit were analyzed and graphed using the statistical software GraphPad Prism.

[0033] Cells were plated in 96-well plates at the same concentration and stimulated with different amounts of r-hFSH to create dose-response curves. Untransfected HEK293-T cells were also stimulated as a negative control. After 30 minutes of incubation, FRET reagent was added to each well of the plate. The plates were again cultured with shaking for 60 minutes, and finally, results were obtained using a plate reader. Panels A and B in Figure 1 show how the responses of the two cell lines differed. Transfected cells (Figure 1A) responded to r-hFSH stimulation, while untransfected HEK293-T cells (Figure 1B) failed to produce a dose-related response after stimulation, confirming the absence of receptors on the cell membrane.

[0034] Second functional assay evaluation. To evaluate transfection efficiency, a receptor gene assay (RGA) was performed. This assay is suggested in the Promega vector datasheet. HEK293-T FSHR luciferase cells were stimulated with r-hFSH. The luminescence released after reporter gene transcription was quantified using commercially available reagents.

[0035] HEK293-T FSHR luciferase cells were seeded in 96-well plates and stimulated with different concentrations of r-hFSH. After incubation, the ONE-Glo EX luciferase assay system (Promega, E8120) was added to each well, and after shaking for 10 minutes, results were obtained using a plate reader.

[0036] Figure 2 shows the luminescence response (expressed as relative luminous units, RLU) obtained in each well after plate reading. The HEK293-T FSHR luciferase response was shown to be dose-dependent. These data confirmed the success of the transfection.

[0037] Characterization of cell lines. The objective of this study was to monitor and evaluate various parameters to investigate the behavior of cell lines over time. Evaluating cell line behavior is important for monitoring cell viability, proliferation rate, target receptor expression, and functional performance over a predetermined number of cell passages. The parameters monitored for HEK293-T FSH luciferase cells are as follows: ● Presence of FSH receptor expression in the cell membrane and its stability over time ● Cell viability ●Cell proliferation (doubling time)

[0038] The cells were monitored over a long period, from passage 20 to passage 37.

[0039] Each parameter was evaluated within a specified passage range. Data were analyzed using linear regression analysis with a regression significance of 95% (p-value < 0.05). All statistical analyses were performed using GraphPad Prism.

[0040] Evaluation of FSH receptor expression in the cell membrane and its stability over time. The proportion of transfected cells expressing the human FSH receptor on the cell membrane was evaluated. For this range, cells were stained and analyzed using a FACS Aria Fusion instrument (BD Biosciences). Data analysis of the staining experiments was performed using FlowJo software. The cell lines used for staining were HEK293-T FSHR luciferase and untransfected HEK293-T WT. Primary FSHR-conjugating antibody (Human FSH R Alexa Fluor® 488-conjugating antibody, R&D Systems, FAB65591G) was used for staining.

[0041] The antibody was diluted with PBS and 2% FBS and incubated at 4°C for 30 minutes. After incubation, the cells were washed with PBS and 2% FBS and resuspended in PBS and 2% FBS. The results were obtained using a FACS instrument.

[0042] As shown in Figure 3, differences were observed between untransfected cells (HEK293-T WT) and transfected cells (HEK293-T LHCGR luciferase). In fact, HEK293-T FSHR luciferase showed 98.6% of FSH receptor-positive cells compared to 0% positively stained cells in the HEK293-T WT sample. This staining experiment confirmed the success of transfection. Analysis of the temporal stability of FSH receptor expression revealed a stable working window, as shown in Figure 4.

[0043] Survival rate and doubling time of HEK293-T LHCGR luciferase Cell line viability was measured using a Nucleocounter instrument (Chemometec). The results showed a viability of over 95%, indicating stability within the analyzed passage range (Figure 5A).

[0044] The doubling time (Figure 5B) was calculated using the following formula:

[0045]

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Claims

1. a. A human FSH receptor comprising a nucleic acid molecule encoding a human FSH receptor under the control of a promoter for protein expression, and b. A reporter gene containing a nucleic acid molecule encoding a luciferase reporter gene under the control of the cAMP response element (CRE). Mammalian cells for expressing the human FSH receptor and luciferase reporter genes, including [specific genes / data].

2. The mammalian cell according to claim 1, wherein the mammalian cell is a mammalian cell culture.

3. The mammalian cell according to claim 2, wherein the mammalian cell culture is an adherent cell culture.

4. A mammalian cell according to any one of claims 1 to 3, wherein the promoter for protein expression is a potent promoter for high-level protein expression such as CAG.

5. a. The nucleic acid molecule encoding the human FSH receptor is a vector containing the FSH receptor gene and the hygromycin resistance gene. b. The nucleic acid molecule encoding the luciferase reporter gene is a vector containing the luciferase reporter gene under the control of the cAMP response element (CRE), c. A mammalian cell according to any one of claims 1 to 4, wherein the gene is a puromycin resistance gene.

6. The mammalian cell according to any one of claims 1 to 5, wherein the mammalian cell is a human embryonic kidney (HEK) 293 cell.

7. The mammalian cell line according to claim 6, wherein the HEK293 cells are HEK293T cells.

8. A method for generating the human FSH receptor and luciferase reporter gene in mammalian cells, a. A step of transfecting a mammalian cell with an isolated nucleic acid molecule encoding an FSH receptor under the control of a promoter for protein expression, wherein the mammalian cell line is a mammalian cell line stably transfected with a luciferase reporter gene under the control of a cAMP response element (CRE), b. A step of adding a selective agent to the transfected cells from step a) to induce a human FSH receptor cell line that stably expresses the receptor, e. The process of collecting cells that stably express the human FSH receptor and luciferase reporter. The above method, including.

9. The method according to claim 8, wherein the selective agent is hygromycin.

10. The method according to claim 8 or 9, wherein the promoter for protein expression is a potent promoter for high-level protein expression, such as CAG.

11. A method for determining the biological activity of an r-hFSH sample, a. A step of bringing the sample into contact with a cell culture of mammalian cells according to claims 1 to 7, b. The step of adding a luciferase enzyme luminescent substrate to the cell culture, c. A step to detect luminescence from cells, d. A process for quantifying the amount of light emitted, e. A step to determine the biological activity of the r-hFSH sample. The above method, including.

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