Application of cells in drug molecule screening

Abstract

The invention relates to application of cells in drug molecule screening, a corresponding drug screening method and a drug screening system. It is found for the first time that the multiple myeloma cell line shows a remarkable concentration-dependent cell death reaction on TL1A stimulation, TL1A-targeted inhibitory drugs can remarkably inhibit TL1A-induced cell Caspase3 / 7 enzyme activity increase and / or cell death, the inhibition effect shows a clear dose-effect relationship, and the discovery shows that the multiple myeloma cell line has a significant effect on TL1A-induced cell Caspase3 / 7 enzyme activity increase and / or cell death. The multiple myeloma cell line can be used as an ideal cell model for TL1A-DR3 targeted drug screening, so that an efficient and visual tool with clinical transformation value is provided for TL1A-DR3 targeted drug screening, activity analysis and action mechanism research, and the multiple myeloma cell line has remarkable scientific significance and industrial application potential.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to use of cells in drug molecule screening. Background Art

[0002] Inflammatory bowel disease (IBD), a global chronic disease, has seen a steady increase in incidence in recent years. Characterized by chronic intestinal inflammation, ulceration, and immune dysfunction, IBD is a complex, heterogeneous group of autoimmune diseases, primarily encompassing Crohn's disease (CD) and ulcerative colitis (UC). Due to its high prevalence, low clinical cure rates, frequent complications, and high medical costs, IBD has become a significant global burden. Although its pathogenesis remains incompletely elucidated, dysregulation of the immune system is considered a core contributing factor.

[0003] Aberrant expression of tumor necrosis factor-like ligand 1A (TL1A) and its membrane-bound receptor, death receptor 3 (DR3), is closely associated with the development and progression of autoimmune diseases such as rheumatoid arthritis, psoriasis, and intraepithelial bronchitis (IBD). In recent years, TL1A inhibitors have garnered significant attention due to their demonstrated safety and efficacy in the clinical treatment of IBD. Although clinically available TL1A inhibitors have demonstrated some efficacy in the treatment of ulcerative colitis and leukopenia (CD), phase II clinical data indicate that the clinical remission rate of existing drugs remains below 50%, suggesting significant unmet clinical need in the treatment of IBD.

[0004] In the field of drug screening, evaluating biological functions based on cell models is a key step. In drug development targeting the TL1A target, apoptosis inhibition is a key screening metric. While the commonly used TF-1 cells (a human leukemia cell line that endogenously expresses the DR3 receptor) can be used for TL1A research, their in vitro culture requires interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF) for growth maintenance, and their concentration-dependent apoptosis response window to TL1A is relatively narrow, limiting the sensitivity and reliability of drug screening.

[0005] RPMI8226 cells are derived from peripheral blood B lymphocytes (IgG lambda type) from multiple myeloma patients and have been widely used in tumor pathology, drug development, and immunology research. However, there have been no reports on their application in the study of cell apoptosis mediated by the TL1A-DR3 pathway. Summary of the Invention

[0006] Purpose of the Invention In response to the needs or defects in the prior art, the present invention aims to provide a use of a multiple myeloma cell line in drug screening targeting the TL1A target. Specifically, the present invention provides the use of a multiple myeloma cell line as a cell screening model in screening drug molecules having any one or more of the following functions: (1) targeting and / or inhibiting TL1A; (2) inhibiting the TL1A-DR3 signaling pathway or inhibiting the increase in cellular Caspase3 / 7 enzyme activity and / or cell death caused by the TL1A-DR3 signaling pathway; and (3) preventing and / or treating diseases related to the TL1A-DR3 signaling pathway.

[0007] In addition, the present invention also provides a method for screening drug molecules that target and / or inhibit TL1A, inhibit the TL1A-DR3 signaling pathway, or inhibit increased cellular Caspase3 / 7 activity and / or cell death caused by the TL1A-DR3 signaling pathway and / or prevent and / or treat diseases related to the TL1A-DR3 signaling pathway, as well as a drug screening system.

[0008] Solution To achieve the above object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a use of a multiple myeloma cell line as a drug screening model in screening drug molecules having any one or more of the following effects: (1) Targeting and / or inhibiting TL1A or its receptor; (2) Inhibit the TL1A-DR3 signaling pathway or inhibit the increase in cell Caspase3 / 7 enzyme activity and / or cell death caused by the TL1A-DR3 signaling pathway; (3) Prevent and / or treat diseases related to the TL1A-DR3 signaling pathway.

[0009] For the above uses: Preferably, the multiple myeloma cell line is the RMPI8226 cell line.

[0010] Preferably, the TL1A-DR3 signaling pathway-related disease is selected from the group consisting of rheumatoid arthritis, psoriasis and inflammatory bowel disease.

[0011] Further preferably, the inflammatory bowel disease is Crohn's disease and / or ulcerative colitis.

[0012] Preferably, the multiple myeloma cell line is cultured in a medium containing cycloheximide in the presence of TL1A as a drug screening system.

[0013] Further preferably, in the drug screening system, the concentration of cycloheximide added is 1-100 μg / mL, preferably 1-50 μg / mL; in a preferred embodiment, the concentration of cycloheximide added is 10 μg / mL; Further preferably, in the drug screening system, the concentration of TL1A added is 1×10 -4 μg / mL to 1 μg / mL, preferably 1×10 -3 Between μg / mL and 1 μg / mL; Further preferably, in the drug screening system, the EC50 value of TL1A causing increased Caspase3 / 7 activity and / or cell death in the multiple myeloma cell line is lower than 1 μM.

[0014] In a second aspect, the present invention provides a method for screening drug molecules that target and / or inhibit TL1A or its receptor, inhibit the TL1A-DR3 signaling pathway, or inhibit increased cellular Caspase3 / 7 activity and / or cell death caused by the TL1A-DR3 signaling pathway and / or prevent and / or treat diseases related to the TL1A-DR3 signaling pathway, the method comprising: using the RMPI8226 cell line as a drug screening model for screening.

[0015] In a feasible embodiment, the method includes: culturing the RMPI8226 cell line in the presence of TL1A in the culture medium containing cycloheximide as a drug screening system; adding the drug molecule to be tested to the drug screening system, culturing for a predetermined time, measuring the cell death rate or Caspase3 / 7 enzyme activity, and screening the target drug molecule based on the significant reduction in the cell death rate or Caspase3 / 7 enzyme activity compared to the model control group under the same culture conditions but without the drug molecule to be tested.

[0016] Preferably, the method comprises: (1) Set up the following cell culture system, and after culturing for a predetermined period of time, detect the cell death rate or Caspase 3 / 7 enzyme activity of each group: Experimental group: Add a series of concentrations of the drug molecule to be tested into the drug screening system; Model control group: except that the drug molecules to be tested are not added, the rest are the same as the experimental group; Blank control group: except for not adding TL1A, the rest were the same as the model control group; (2) Calculate the cell death inhibition rate or Caspase 3 / 7 enzyme activity inhibition rate of the drug molecule at each concentration according to the following formula: Cell death inhibition rate (%) = (cell death rate in model control group - cell death rate in experimental group / cell death rate in model control group - cell death rate in blank control group) × 100%; Caspase 3 / 7 enzyme activity inhibition rate (%) = (Caspase 3 / 7 enzyme activity in the model control group - Caspase 3 / 7 enzyme activity in the experimental group / Caspase 3 / 7 enzyme activity in the model control group - Caspase 3 / 7 enzyme activity in the blank control group) × 100%; (3) When there is a clear positive correlation between the concentration of the drug molecule to be tested and its cell death inhibition rate or Caspase 3 / 7 enzyme activity inhibition rate, the drug molecule to be tested is determined to be the target drug molecule; Alternatively, when the EC50 value of the drug molecule to be tested for inhibiting cell death or Caspase3 / 7 enzyme activity is lower than 1 μM, the drug molecule to be tested is determined to be a target drug molecule.

[0017] Further preferably, in the drug screening system, the concentration of cycloheximide added is 1-100 μg / mL, preferably 1-50 μg / mL; in a preferred embodiment, the concentration of cycloheximide added is 10 μg / mL; Further preferably, in the drug screening system, the concentration of TL1A added is 1×10 -4 μg / mL to 1 μg / mL, preferably 1×10 -3 Between μg / mL and 1 μg / mL; Further preferably, in the drug screening system, the EC50 value of TL1A causing increased Caspase3 / 7 activity and / or cell death in the multiple myeloma cell line is lower than 1 μM.

[0018] In a feasible embodiment, the TL1A-DR3 signaling pathway-related disease is selected from the group consisting of: rheumatoid arthritis, psoriasis, and inflammatory bowel disease.

[0019] Preferably, the inflammatory bowel disease is Crohn's disease and / or ulcerative colitis.

[0020] In a third aspect, the present invention provides a drug screening system, which is a cell culture system comprising culturing the multiple myeloma cell line in a culture medium containing cycloheximide in the presence of TL1A.

[0021] Preferably, the multiple myeloma cell line is the RMPI8226 cell line; Further preferably, in the drug screening system, the concentration of cycloheximide added is 1-100 μg / mL, preferably 1-50 μg / mL; in a preferred embodiment, the concentration of cycloheximide added is 10 μg / mL; Preferably, in the drug screening system, the concentration of TL1A is 1×10 -4 μg / mL to 1 μg / mL, preferably 1×10 -3 Between μg / mL and 1μg / mL.

[0022] Beneficial effects Through systematic cell line screening, the present invention discovered that multiple myeloma cells RPMI8226 exhibit a significant concentration-dependent apoptotic response to TL1A stimulation. Furthermore, inhibitory drugs targeting TL1A (e.g., TL1A antibodies) significantly inhibit TL1A-induced apoptosis in RPMI8226 cells and Caspase 3 / 7 activity, with a clear dose-response relationship. These findings suggest that multiple myeloma cells RPMI8226 can be used as a cell model for screening drugs targeting TL1A or its receptor DR3, for example, for screening drugs that target and / or inhibit TL1A or its receptor DR3; inhibit the TL1A-DR3 signaling pathway or apoptosis induced by the TL1A-DR3 signaling pathway; and prevent and / or treat diseases associated with the TL1A-DR3 signaling pathway.

[0023] Compared with the existing TF-1 cell model, the cell model provided by the present invention has the following significant advantages: (1) Ease of operation: No need to rely on exogenous cytokines (such as IL-3, GM-CSF) to maintain culture, simplifying the experimental process; (2) Response sensitivity: The apoptosis response window of cells to TL1A stimulation is wider, the dose-effect relationship is more significant, and the sensitivity of drug screening is improved; (3) Reliability of results: Highly reproducible, and can effectively distinguish the differences in inhibitory activity of different drug molecules; (4) Wide application: Applicable to the functional screening of TL1A targeted drugs (such as monoclonal antibodies, small molecule inhibitors, etc.) with various mechanisms of action.

[0024] In summary, multiple myeloma cells RPMI8226 provide an efficient, intuitive, and clinically valuable tool for the screening, activity analysis, and mechanism of action research of TL1A-targeted drugs, which helps accelerate the development of drugs for the treatment of autoimmune diseases and has significant scientific significance and industrial application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] One or more embodiments are exemplarily illustrated by the accompanying figures, and these exemplary illustrations do not limit the embodiments. The word "exemplary" is used herein to mean "serving as an example, example, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

[0026] Figure 1 The SDS-PAGE analysis results for three TL1A antibodies are shown. Panel A shows the SDS-PAGE of antibody A219, Panel B shows the SDS-PAGE of antibody RVT-3101, and Panel C shows the SDS-PAGE of antibody TEV574. R represents the electrophoresis lane under reducing conditions, and NR represents the electrophoresis lane under non-reducing conditions.

[0027] Figure 2 The SEC-HPLC analysis results for three TL1A antibodies are shown. Figure A shows the SEC-HPLC analysis of antibody A219, Figure B shows the SEC-HPLC analysis of antibody RVT-3101, and Figure C shows the SEC-HPLC analysis of antibody TEV574.

[0028] Figure 3 The binding activity of three TL1A antibodies to the TL1A antigen protein is shown. Panel A shows the results of ELISA assay, and Panel B shows the results of flow cytometry assay.

[0029] Figure 4 Figure 1 shows the effect of TL1A on TF-1 cell death. Panel A shows the cell death rate induced by TL1A incubated with different numbers of TF-1 cells for 16 hours. Panel B shows the effect of the presence or absence of GM-CSF and different incubation times on the TL1A-induced TF-1 cell death rate. Panel C shows the effect of different concentrations of cycloheximide on TL1A-induced TF-1 cell death. Panel D shows the comparative effect of three TL1A antibodies on the inhibition of TL1A-induced TF-1 cell death.

[0030] Figure 5 Figure 1 shows the effect of TL1A on inducing cell death in different cell types. Panel A shows the ratios of cell death induced by TL1A co-cultured with cells for 24 and 48 hours, Panel B shows the ratios of cell death induced by three concentrations of TL1A co-cultured with cells for 24 hours, and Panel C shows the concentration effects of two cell types on TL1A-induced cell death.

[0031] Figure 6The figures show the concentration-dependent effect of TL1A on RPMI8826 cell death and the inhibitory effect of TL1A antibodies on RPMI8226 cell death. Panel A shows that RPMI8226 cells were co-incubated with TL1A for 16 hours, and the RLU values of viable cells were measured to determine the induced cell death of RPMI8226 cells at different concentrations. Panel B shows the cell death ratio (i.e., cell death rate (%)) induced by different concentrations of TL1A after co-incubation with RPMI8226 cells for 16 hours. Panel C shows the concentration-dependent effect of antibodies on TL1A-induced cell death of RPMI8226 cells after co-incubation with TL1A for 16 hours, and the RLU values of viable cells were measured to determine the induced cell death of RPMI8226 cells. Panel D shows the inhibition rate (i.e., cell death inhibition rate (%)) of antibodies on TL1A-induced cell death of RPMI8226 cells after co-incubation with TL1A for 16 hours.

[0032] Figure 7 Figures show the concentration-dependent effect of TL1A on the induction of RMPI8226-induced caspase 3 / 7 activity and the concentration-dependent inhibition of TL1A-induced caspase 3 / 7 activity by three TL1A antibodies. Panel A shows the concentration-dependent curves of caspase 3 / 7 activity in RPMI8226 cells after co-incubation of RPMI8226 cells with different concentrations of TL1A for 4 hours. Panel B shows the fold increase in caspase 3 / 7 activity in RPMI8226 cells compared to untreated control wells after co-incubation of RPMI8226 cells with different concentrations of TL1A for 4 hours. Panel C shows the concentration-dependent curves of the inhibition of TL1A-induced caspase 3 / 7 activity in RPMI8226 cells with antibodies after co-incubation of RPMI8226 cells with specific concentrations of TL1A for 16 hours. Panel D shows the inhibition rate (%) of TL1A-induced caspase 3 / 7 activity in RPMI8226 cells with antibodies after co-incubation of RPMI8226 cells with specific concentrations of TL1A for 16 hours. DETAILED DESCRIPTION

[0033] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0034] In addition, in order to better illustrate the present invention, numerous specific details are provided in the following detailed description. It should be understood by those skilled in the art that the present invention can be practiced without certain specific details. In some embodiments, raw materials, components, methods, means, etc. that are well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present invention.

[0035] Unless expressly stated otherwise, throughout the specification and claims, the term "comprise" or variations such as "include" or "comprising", etc., will be understood to include the stated elements or components but not to exclude other elements or other components.

[0036] The present invention is further described in detail below through examples.

[0037] In the following examples, the materials, animals, reagents, etc. used, unless otherwise specified, can be obtained through commercial channels.

[0038] In the following examples, when detecting the effects of TL1A on Caspase 3 / 7 activity and cell death, unless otherwise indicated, cycloheximide was added to the culture medium to assist in establishing the cell model.

[0039] In the following examples, the specific time point of detection, the number of cells used, and the reaction system can be selected based on routine experience, and the operation of these specific experiments does not affect the claims of the present invention.

[0040] Example 1: Culture and identification of RPMI8226 cells The RPMI8226 cell line was provided by Beijing Saifu Pharmaceutical Research Institute. The RPMI8226 cell line was cultured at 37°C, 5% CO2, in RPMI164 medium supplemented with 10% fetal bovine serum and penicillin-streptomycin. The cells were harvested and subjected to STR typing by Beijing Reader Gene Technology Co., Ltd. The results showed no cross-contamination with human cells and were consistent with the RPMI8226 cells (CCL-155) from ATCC. TM ), the data of 17 STR sites of RPMI8226 (ACC402) in DSMZ and the data of 19 STR sites of RPMI8226 (CVCL_0014) in ExPASy had a matching rate of 100%; the results showed that the cell line was indeed the multiple myeloma cell line RPMI8226.

[0041] Example 2: Expression, purification and identification of three TL1A antibodies The amino acid sequences of the light and heavy chains of three TL1A antibodies, PRA023, TEV574, and RVT3101, were extracted from public databases (see Table 1 below). The sequences of these three TL1A antibodies and their effectiveness verification have been disclosed in the prior art. The present applicant prepared these three antibodies based on the antibody sequences disclosed in the prior art and verified their antigen-binding activity using flow cytometry, ELISA, and other methods. The results demonstrated that these three antibodies are effective TL1A antibodies.

[0042] Table 1. Amino acid sequences of the light and heavy chains of TL1A antibodies PRA023, TEV574, and RVT3101 ; The specific method for preparing the antibody is as follows: The DNA coding sequences for the antibody's light and heavy chains were deduced codon-by-codon. Gene synthesis was performed, and the DNA fragments encoding the light and heavy chains were cloned into the eukaryotic expression vectors pTT5-L (containing the kappa constant region) and pTT5-H1 (containing the IgG1 heavy chain constant region), respectively, to generate recombinant expression plasmids for the antibody's light and heavy chains. The recombinant expression plasmids were extracted and co-transfected into 293F cells with the corresponding light and heavy chain plasmids. After incubation at 37°C, 5% CO2, and 120 rpm for 5 days, the culture medium was collected and purified using a Protein A column. Protein A purification procedures were as follows: the column was equilibrated with 50 mM PB, 0.15 M NaCl, pH 7.2 buffer at a flow rate of 150 cm / h for 6 column volumes. The 293F cell culture supernatant was adjusted to pH 7.2 at a flow rate of 150 cm / h. The column was then re-equilibrated with equilibration buffer at a flow rate of 150 cm / h for 6 column volumes. The column was then eluted with 50 mM citric acid-sodium citrate, pH 3.5, in a single step over 8 column volumes. The eluted peak was collected, which was the target antibody. The concentration of the target antibody protein was determined using a UV spectrophotometer (nanoDrop).

[0043] The three antibody proteins expressed and purified above were subjected to SDS-PAGE gel electrophoresis under reducing and non-reducing conditions, and the SDS-PAGE identification results of antibodies PRA023, TEV574 and RVT3101 were as follows: Figure 1 As shown in Figures A, B, and C; Figure 1 Figures A, B, and C show that for the three antibodies, the electrophoresis bands under non-reducing conditions are all single bands, and their molecular weights are consistent with the molecular weights of the corresponding antibody proteins, while the electrophoresis bands under reducing conditions are all two bands, and the molecular weights of the two bands are consistent with the molecular weights of the light and heavy chains of the corresponding antibodies, respectively. This suggests that the three target antibodies were indeed obtained through the above expression and purification procedures.

[0044] In addition, the purity of the target antibodies was tested by SEC-HPLC. The SEC-HPLC analysis results of antibodies PRA023, TEV574 and RVT3101 were as follows: Figure 2 As shown in Figures A, B, and C; Figure 2 Figures A, B, and C in the figure show that for the three antibodies, there are sharp main peaks in their HPLC spectra, and the purity of the three antibodies is above 97%.

[0045] Example 3: ELISA assay to detect the antigen binding ability of three TL1A antibodies In this example, the antigen-binding ability of three TL1A antibodies was tested by ELISA using the TL1A recombinant protein TL1A-his (Cat. No. COYOBIOAg0082, Keyuan Xinsheng) as the antigen. The specific steps are as follows: 1) Antigen coating: TL1A-his protein was diluted to 1 μg / mL with 10 mmol / L 1× PBS buffer (pH 7.4) and added to a 96-well plate at a rate of 100 μL / well (Cat. No. 9018, Corning) and incubated overnight at 4°C.

[0046] 2) Washing and sealing: The coating solution was discarded and the plate was washed four times with PBST buffer containing 0.05% Tween-20; 300 μL of 5% skim milk powder (prepared in PBS) was added to each well and blocked at 37°C for 2 hours to block nonspecific binding sites.

[0047] 3) Primary Antibody Incubation: After blocking, wash the plate 4 times with PBST and let it dry for later use; dilute the antibody to be tested 5-fold in series with PBS (a total of 10 concentration gradients, with an initial concentration of 20 μg / mL), add 100 μL of each concentration of antibody dilution to each well, and incubate at 37°C for 1 hour.

[0048] 4) Secondary antibody incubation and color development: The primary antibody solution was discarded, and the plate was washed four times with PBST. Goat anti-human IgG secondary antibody (Cat. No. 2016-05, SouthernBiotech) was diluted according to the instructions, and 100 μL of the diluted secondary antibody was added to each well, and the cells were incubated at 37°C for 1 hour. After washing the plate four times with PBST, 100 μL of TMB colorimetric solution (Cat. No. PR1200, Solebol) was added to each well. The reaction was incubated at room temperature in the dark until sufficient color was developed, and the stop solution was added to terminate the reaction. The plate was placed in an EnVision® 2105 microplate reader (Perkin Elmer), and the absorbance (OD) was read at a wavelength of 450 nm. The obtained data were statistically analyzed.

[0049] The results are as follows Figure 3As shown in Figure A, the three TL1A antibodies all exhibited a concentration-dependent binding effect on the TL1A recombinant protein. The EC50 values of A219, RVT3101, and TEV574 binding to the TL1A recombinant protein were 0.2750 μg / mL, 0.2172 μg / mL, and 0.118 μg / mL, respectively. The corresponding molar concentrations were 1.909 nM, 1.488 nM, and 0.819 nM, respectively. That is, the EC50 values of the three antibodies binding to the TL1A recombinant protein were relatively similar.

[0050] Example 4: Flow cytometry detection of antigen binding ability of three TL1A antibodies In this example, HEK293 cells overexpressing TL1A (abbreviated as HEK293-TL1A, Catalog No.: CHEK-ATP142, ACRO Biosystems) were used to detect the binding ability of three TL1A antibodies to antigens by flow cytometry. The specific steps are as follows: HEK293-TL1A cells were collected and plated at 1 × 10 cells per well. 5 The cells were seeded into a 96-well plate at a density of 10 cells / well; the 96-well plate was centrifuged at 2000 rpm for 5 minutes and the supernatant was discarded; the antibody to be tested was added to the cells and incubated at 4°C for 30 minutes to bind to the cell surface antigen; a buffer containing 1% bovine serum albumin (BSA) was added and the cells were fully suspended by gentle pipetting to wash away unbound antibodies; the 96-well plate was centrifuged again at 2000 rpm for 5 minutes and the supernatant was discarded, and this step was repeated twice; the secondary antibody (FITC-labeled goat anti-human secondary antibody, catalog number: 2045-02, SouthernBiotech) was diluted in 1% BSA buffer and added to a volume of 100 μl per well, the cells were resuspended, and the 96-well plate was incubated at 4°C in a dark environment for 30 minutes; 1% BSA buffer was added and gently mixed to remove free fluorescent antibodies. Centrifuge at 2000 rpm for 5 minutes, discard the supernatant, and repeat this step twice. Add 200 μl of 1% BSA buffer to the cell pellet, gently pipette to fully resuspend the cells, and then transfer the cell suspension to a FACS tube. Cells were analyzed using a BD Accuri™ C6Plus flow cytometer. After analysis, data were imported into FLOWJo software for preliminary analysis, and finally graphed and analyzed using GraphPad Prism software.

[0051] The experimental results are as follows Figure 3As shown in Figure B, all three antibodies exhibited concentration-dependent binding effects on TL1A-overexpressing cell lines. The half-maximal effective concentration (EC50) values of the mean fluorescence intensity of A219, RVT3101, and TEV574 antibodies binding to cells were 0.8431 μg / mL, 0.7105 μg / mL, and 0.8242 μg / mL, respectively, and the corresponding molar concentrations were 5.854 nM, 4.866 nM, and 5.723 nM, respectively. There was no significant difference in the EC50 values of the three antibodies binding to TL1A-overexpressing cell lines, and no obvious fold difference was observed.

[0052] Example 5: Detection of TL1A-induced cell death and antibody-blocked cell death In the field of cell biology research, the methods for calculating the proportion of cell apoptosis or cell death are mainly divided into two categories: one is direct quantitative analysis by measuring dead cells or apoptosis markers; the other is indirect proportion calculation by detecting the number of living cells after treatment.

[0053] 5.1 Screening of cells in which TL1A can induce death The experimental protocol for TL1A-induced cell death is as follows: First, the cells to be tested were inoculated in a suitable culture medium overnight and cultured the next day. The cells were resuspended at the desired density and seeded into a 96-well plate, and cycloheximide at the desired concentration was added. The TL1A-his recombinant protein (same as in Example 3) was serially diluted with culture medium at a starting concentration of 10 μg / mL according to the specified dilution factor and then added to the wells containing the cells. After incubation for the specified time, an equal volume of CellTiter-Glo® Reagent (CTG, G7571, Promega) was mixed with the incubated cells and shaken for 5 minutes. The microplate was placed in a PerkinElmer ENVISION microplate reader, and the fluorescence intensity (expressed as RLU value, which reflects the number of viable cells) was measured using the autofluorescence option. The cell death rate was calculated according to the following formula: Cell death rate (%) = (RLU （添加TL1A孔） -RLU （培养基空白孔） ) / (RLU （未添加TL1A孔） -RLU （培养基空白孔） )×100%.

[0054] The calculated data were input into GraphPad Prism for graphing and analysis.

[0055] Table 2. Information of cells used to induce cell death ; According to the above method, we first tested the effect of TL1A on the cell death of TF-1 cells; the results were as follows: Figure 4 As shown in the figure, although TL1A can cause a high proportion of TF-1 cell death at a high concentration (10 μg / mL), the concentration gradient effect of TL1A in causing cell death is not obvious, and the variation between different wells and different batches is large, and the cell death rate window is narrow. Although we tried using different cell numbers (see Figure 4 A in Figure ), with or without GM-CSF and different co-incubation times (see Figure 4 B in Figure 2) and different concentrations of cycloheximide (5 μg / mL, 10 μg / mL, 20 μg / mL, and 25 μg / mL, see Figure 4 C in the figure) in an attempt to optimize the mortality window of TL1A-induced TF-1 cell death. However, a wide mortality window and a significant concentration-dependent effect could not be achieved under various conditions.

[0056] We then tested the effect of TL1A on cell death in seven types of T lymphocytes, B lymphocytes, and monocytes (Table 2), including MOLT-4, OCI-Ly3, SU-DHL-6, THP-1, Raji, Jurkat, and MM1.S. Specifically, we tested 1×10 -4 The number of living cells of different cell types incubated with 1 μg / mL TL1A for 24 hours and 48 hours was calculated, and the cell death rate was calculated. Figure 5 As shown, it shows that THP1 and MM.1S have a mortality rate higher than 10% at two co-incubation times (see Figure 5 Furthermore, when we co-incubated TL1A with seven cell types at three concentrations (10 μg / mL, 0.1 μg / mL, and 0.001 μg / mL), we found that we could not obtain stable and reproducible experimental results (see Figure 5 To further verify the above results, we retested the apoptosis-inducing effects of three concentrations of TL1A (10 μg / mL, 0.2 μg / mL, and 0.004 μg / mL) on THP1 and MM.1S. The results showed that the death rate of both cells was less than 10% at the highest TL1A concentration, and when co-incubated with low concentrations of TL1A, TL1A even promoted cell proliferation (see Figure 5 These results indicate that the aforementioned cells cannot serve as an effective model for TL1A-induced cell death. This suggests that a more effective model for TL1A-induced cell death needs to be developed in academia or industry.

[0057] Finally, we examined the effect of TL1A on RPMI8226 cell death. Figure 6 As shown in Figures A and B, there is a good dose-effect relationship between TL1A and its induction of cell death in RPMI8226, as shown by the fact that when the concentration of TL1A increases, the fluorescence value obtained by measuring living cells gradually decreases (see Figure 6 Figure A shows a good S-shaped curve. The EC50 value of TL1A inducing cell death in RPMI8226 cells is 0.001324 μg / mL (approximately 59.11 pM). The concentration range of TL1A causing 0-99% cell death is 1×10 -4 μg / mL (about 5.91pM) to 1μg / mL (about 44.64nM) (see Figure 6 (B in Figure 1); whereas the irrelevant control protein CD93 did not induce cell death in RPMI8226 cells. This indicates that RPMI8226 cells have a good sensitivity and dose-response window to TL1A-induced cell death, and that the suitable concentration range of TL1A is large, with a concentration of 1×10 -4 Therefore, compared with TF-1 cells, RPMI8226 cells are more suitable for TL1A-induced cell death or proliferation inhibition, and are potentially useful for screening drugs that neutralize cell death caused by TL1A or the TL1A-DR3 pathway.

[0058] 5.2. Neutralization or Blockade of TL1A-Induced TF1 Cell Death by TL1A Antibodies Test method: In the above steps, TL1A is mixed with any of the three TL1A antibodies (A219, RVT3101, and TEV574) prepared in Example 2 and then incubated with cells to detect the number of viable cells. Briefly, the antibody is first diluted serially from a specified concentration using the corresponding cell culture medium containing cycloheximide, then mixed with a specified concentration of TL1A antigen, added to a well plate containing cells, and incubated at 37°C, 5% CO2 for 30 minutes. After treatment with CellTiter-Glo® Reagent (CTG, G7571, Promega), the microplate is placed in a PerkinElmer ENVISION microplate reader, and the fluorescence intensity (expressed as RLU value, which reflects the number of viable cells) is detected using the autofluorescence option; the cell death inhibition rate is calculated according to the following formula: Cell death inhibition rate (%) = (RLU （添加TL1A与抗体混合液细胞孔） -RLU （含有TL1A的对照细胞孔） ) / (RLU （不含TL1A的对照细胞孔） -RLU （含有TL1A的对照细胞孔） )×100%.

[0059] The obtained inhibition rate was input into Graphpad Prism for graphing and curve fitting.

[0060] Using the above method, we tested the inhibitory effects of three antibodies on TL1A-induced TF1 cell death. Specifically, the three test antibodies were serially diluted in culture medium at a starting concentration of 10 μg / mL. Each antibody dilution was mixed with 1 μg / mL TL1A and then added to a 1×10 -4 After 16 hours of co-culture, we measured the autofluorescence intensity as described above. According to the calculation formula for the inhibition rate of cell death, we obtained the cell death inhibition rate of the antibody at each concentration. The cell death inhibition rate was input into Graphpad Prism for plotting and curve fitting. The results are shown in Figure 4 Figure D in Figure 4 As shown in Figure D, all three antibodies significantly inhibited TL1A-induced cell death at higher concentrations, with the cell death inhibition rate showing a clear trend of increasing with increasing antibody concentration. This indicates that the three antibodies, RVT3101, TEV574, and A219, have a certain blocking effect on TL1A-induced TF-1 cell death under these experimental conditions, while the isotype control antibody exhibited no inhibitory effect. However, the cell death inhibition rates of the exemplified antibodies were less than 10% at concentrations ranging from 0.0005 to 10 μg / mL. This low inhibition rate requires extremely high experimental procedures and is insensitive for distinguishing candidate antibody drugs. Therefore, it is highly necessary to develop a new sensitive cell model for screening candidate molecules targeting the TL1A-DR3 signaling pathway.

[0061] 5.3. TL1A Antibody Neutralizes or Blocks TL1A-Induced RPMI8226 Cell Death Next, we tested the inhibitory effects of the three TL1A antibodies (A219, RVT3101, and TEV574) prepared in Example 2 on TL1A-induced cell death in RPMI8226 cells. The TL1A concentration used was 0.4 μg / mL (approximately 17.85 nM, i.e., the EC90 concentration that causes cell death). The highest concentration of the three TL1A antibodies and the isotype control antibody molecules was 66.66 μg / mL. Starting from this concentration, three-fold serial dilutions were made. The antibody dilutions of each concentration were mixed with TL1A and added to a 1×10 -4 After 16 hours of incubation, we measured the autofluorescence intensity as described above. We entered the autofluorescence intensity value into Graphpad Prism for plotting and curve fitting. The results are shown in Figure 6C in Figure 5.2, and the inhibition rate of the antibody on TL1A-induced cell death was calculated according to the inhibition rate formula in 5.2. The results are shown in Figure 6 Figure D in .

[0062] The results show that all three TL1A antibodies inhibited TL1A-induced cell death in RPMI8226 cells in a concentration-dependent manner, while the isotype control antibody had no inhibitory effect. Antibodies A219, RVT3101, and TEV574 exhibited IC50 values of 2.178 μg / mL, 0.7565 μg / mL, and 0.3273 μg / mL, respectively, corresponding to molar concentrations of 15.13 nM, 5.182 nM, and 3.273 nM, respectively. These results demonstrate that RPMI8226 can be effectively used to screen inhibitors of cell death induced by the TL1A-DR3 signaling pathway.

[0063] Example 6: TL1A-induced Caspase 3 / 7 activity in RPMI8226 cells and the inhibitory effect of antibodies on TL1A-induced Caspase 3 / 7 activity To further clarify the inhibitory effect of TL1A antibody on TL1A-induced Caspase3 / 7 activity in RPMI8226 cells, we used the Caspase-Glo® 3 / 7 Assay System (G8091, Promega) to measure the TL1A-induced cellular Caspase3 / 7 enzyme activity and the ability of the antibody to neutralize TL1A-induced cellular Caspase3 / 7 enzyme activity.

[0064] We first determined the effect of TL1A on the activity of Caspase 3 / 7 in RPMI8226 cells. The experimental steps were as follows: 2×10 4 TL1A was added to each well at the designated concentration, with two replicates per concentration. The cells were incubated for 4 hours. Detection reagents were added according to the instructions for the Caspase-Glo® 3 / 7 Assay System. After a 20-minute incubation, the microplate was placed in a microplate reader and autofluorescence readings were measured. Based on the fluorescence values obtained, the fold increase in TL1A-induced Caspase 3 / 7 activity was calculated according to the following formula: Caspase3 / 7 activity increase fold = (RLU （添加TL1A细胞孔） -RLU （不含TL1A的对照细胞孔） ) / (RLU （不含TL1A的对照细胞孔） -RLU （培养基对照孔） ).

[0065] The results are as follows Figure 7 shown. Figure 7The results showed that there was a good dose-effect relationship between the concentration of TL1A and the activity of Caspase3 / 7 induced by it in RPMI8226. When the concentration of TL1A increased, the fluorescence value reflecting the activity of Caspase3 / 7 gradually increased (see Figure 7 The results show that the enzyme activity of the TL1A-treated group increased by fold compared with the control wells, showing a good S-shaped curve, while the unrelated protein control group (i.e., CD93 control group) did not show a concentration-dependent effect. The enzyme activity of the TL1A-treated group increased by fold compared with the control wells, showing a good effect relationship (see Figure 7 Figure B). The EC50 value of TL1A-induced Caspase3 / 7 activity in RPMI8226 cells was 0.1391 μg / mL (approximately 6.21 nM), and the fold increase in Caspase3 / 7 activity ranged from 0 to 2.81 times.

[0066] To test the effect of the example antibodies on the activity of Caspase 3 / 7 induced by TL1A, we mixed serially diluted TL1A antibodies with 0.4 μg / ml TL1A-his protein (as described above) and incubated them for 30 min. At the same time, we collected the cultured RPMI8226 cells, resuspended them in complete medium containing cycloheximide, and added 2×10 -4 A cell suspension of 100 cells was aliquoted into a microplate. The incubated antibody and TL1A mixture was then added to the cell suspension and incubated for 4 hours. The appropriate amount of caspase 3 / 7 reagent was then added according to the Caspase-Glo® 3 / 7 Assay System kit instructions. After 3 hours at 37°C, autofluorescence was measured on a microplate reader. The resulting autofluorescence value (i.e., RLU) represents caspase 3 / 7 activity. In this assay, RPMI8226 cells treated with staurosporine (HY-15141, MedChemExpress (MCE)) served as a control group to induce changes in caspase 3 / 7 activity. The inhibition rate of TL1A antibody treatment on TL1A-induced caspase activity was calculated according to the following formula: Caspase 3 / 7 enzyme activity inhibition rate = (1-(RLU （添加TL1A与抗体混合液孔） -RLU （含有放线菌酮的对照细胞孔） ) / (RLU （添加TL1A含放线菌酮的对照细胞孔） -RLU （含放线菌酮的对照细胞孔） ))×100%.

[0067] The results are as follows Figure 7As shown in Figures C and D, using RPMI8226 as a cell model, TL1A antibody can effectively inhibit TL1A-induced Caspase3 / 7 activity, with an inhibition rate of up to 100%, which is manifested as the reduction in Caspase3 / 7 enzyme activity reaching the lowest level of control cells without TL1A addition; at the same time, the three antibodies also showed a good concentration-dependent effect on the inhibition of Caspase3 / 7 activity. The IC50 values of the three antibodies A219, REV3101 and TEV574 for inhibiting Caspase3 / 7 activity were 1.707μg / mL, 0.5777μg / mL and 0.1750μg / mL, respectively, and the corresponding molar concentrations were 11.85nM, 3.957nM and 1.215nM, respectively. The above results show that RPMI8226, as a cell model, can well distinguish and rank the inhibitory activity of the three antibodies against Caspase3 / 7. This screening method can serve as a supplement to the method of directly measuring the number of viable cells and then calculating the cell death rate inhibition assay, and the results are basically consistent with the death rate inhibition.

[0068] In summary, the present invention provides the use of the RPMI8226 cell line for screening drug molecules targeting TL1A and the corresponding drug screening method. In the above examples, we used exemplary TL1A antibodies as drug molecules in the presence of TL1A in the RPMI8226 cell line and showed that the RPMI8226 cell line as a drug screening model can accurately screen drug molecules with the following effects: (1) targeting and / or inhibiting TL1A, (2) inhibiting the TL1A-DR3 signaling pathway or inhibiting the increase in cell Caspase3 / 7 enzyme activity and / or cell death caused by the TL1A-DR3 signaling pathway, and (3) preventing and / or treating diseases related to the TL1A-DR3 signaling pathway. The cell model provided by the present invention provides an important tool that is simple, intuitive, repeatable, and discriminative for the screening, analysis, and research of drug molecules related to diseases caused by the TL1A-DR3-related pathway (not limited to autoimmune diseases).

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Use of a multiple myeloma cell line as a drug screening model for screening drug molecules having any one or more of the following effects: (1) Targeting and / or inhibiting TL1A or its receptor DR3; (2) Inhibit the TL1A-DR3 signaling pathway or inhibit the increase in cell Caspase3 / 7 enzyme activity and / or cell death caused by the TL1A-DR3 signaling pathway; (3) Prevent and / or treat diseases related to the TL1A-DR3 signaling pathway.

2. The use according to claim 1, characterized in that The multiple myeloma cell line is the RMPI8226 cell line; And / or, the TL1A-DR3 signaling pathway-related disease is selected from: rheumatoid arthritis, psoriasis and inflammatory bowel disease.

3. The use according to claim 1 or 2, characterized in that The multiple myeloma cell line was cultured in a medium containing cycloheximide in the presence of TL1A as a drug screening system.

4. The use according to claim 3, characterized in that In the drug screening system, the added concentration of cycloheximide is 1-100 μg / mL; And / or, in the drug screening system, the concentration of TL1A added is 1×10 -4 Between μg / mL and 1 μg / mL; And / or, in the drug screening system, the EC50 value of TL1A causing increased Caspase3 / 7 activity and / or cell death in the multiple myeloma cell line is lower than 1 μM.

5. A method for screening drug molecules that target and / or inhibit TL1A or its receptor DR3, inhibit the TL1A-DR3 signaling pathway, or inhibit the increase in cellular Caspase3 / 7 activity and / or cell death caused by the TL1A-DR3 signaling pathway, and / or prevent and / or treat diseases related to the TL1A-DR3 signaling pathway, characterized in that: The method comprises: using the RMPI8226 cell line as a drug screening model for screening.

6. The method according to claim 5, characterized in that The method comprises: culturing the RMPI8226 cell line in the presence of TL1A in the culture medium containing cycloheximide as a drug screening system; adding a drug molecule to be tested to the drug screening system, culturing for a predetermined time, measuring the cell death rate or Caspase3 / 7 enzyme activity, and screening the target drug molecule based on a significant decrease in the cell death rate or Caspase3 / 7 enzyme activity compared to a model control group cultured under the same conditions but without the drug molecule to be tested.

7. The method according to claim 6, characterized in that The method comprises: (1) Set up the following cell culture system, and after culturing for a predetermined period of time, detect the cell death rate or Caspase 3 / 7 enzyme activity of each group: Experimental group: Add a series of concentrations of the drug molecule to be tested into the drug screening system; Model control group: except that the drug molecules to be tested are not added, the rest are the same as the experimental group; Blank control group: except for not adding TL1A, the rest were the same as the model control group; (2) Calculate the cell death inhibition rate or Caspase 3 / 7 enzyme activity inhibition rate of the drug molecule at each concentration according to the following formula: Cell death inhibition rate (%) = (cell death rate in model control group - cell death rate in experimental group / cell death rate in model control group - cell death rate in blank control group) × 100%; Caspase 3 / 7 enzyme activity inhibition rate (%) = (Caspase 3 / 7 enzyme activity in the model control group - Caspase 3 / 7 enzyme activity in the experimental group / Caspase 3 / 7 enzyme activity in the model control group - Caspase 3 / 7 enzyme activity in the blank control group) × 100%; (3) When there is a clear positive correlation between the concentration of the drug molecule to be tested and its cell death inhibition rate or Caspase3 / 7 enzyme activity inhibition rate, the drug molecule to be tested is determined to be the target drug molecule; Alternatively, when the EC50 value of the drug molecule to be tested for inhibiting cell death or Caspase3 / 7 enzyme activity is lower than 1 μM, the drug molecule to be tested is determined to be a target drug molecule.

8. The method according to claim 6 or 7, characterized in that In the drug screening system, the added concentration of cycloheximide is 1-100 μg / mL; And / or, in the drug screening system, the concentration of TL1A added is 1×10 -4 Between μg / mL and 1 μg / mL; And / or, in the drug screening system, the EC50 value of TL1A causing increased Caspase3 / 7 activity and / or cell death in the multiple myeloma cell line is lower than 1 μM.

9. A drug screening system, characterized in that: The drug screening system is a cell culture system in which the multiple myeloma cell line is cultured in a culture medium containing cycloheximide in the presence of TL1A.

10. The drug screening system according to claim 9, characterized in that The multiple myeloma cell line was RMPI8226 cell line; And / or, in the drug screening system, the added concentration of cycloheximide is 1-100 μg / mL; And / or, in the drug screening system, the concentration of TL1A added is 1×10 -4 Between μg / mL and 1μg / mL.

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