Preparation method and application of monoclonal cell with positive CAR (chimeric antigen receptor)

Abstract

The invention discloses a preparation method and application of a monoclonal cell with positive CAR (chimeric antigen receptor). The cell preparation method comprises the following steps: acquiring tumor cells and a lentiviral vector containing a target CAR gene, and transferring the tumor cells and the lentiviral vector to a 24-hole culture plate for mixed transfection culture; after culture is finished, centrifuging the culture solution, removing supernatant, and resuspending cell precipitate by using a culture medium; sorting the cell suspension, transplanting the sorted cells into a 96-well plate, and culturing for 7-14 days; and after the culture is finished, centrifuging the culture solution, removing the supernatant, and washing the cell precipitate with PBD to obtain the CAR-positive monoclonal cell. A series of cell strain lines capable of stably expressing the CAR gene are constructed through lentivirus transfection, a stable positive quality control product is provided for CAR-T cell detection, standardized and systematized operation of CAR-T detection is improved, it is ensured that a CAR-T detection result is accurate, credible and reliable, and it is ensured that CAR-T cells are safely and effectively used clinically.

Description

Technical Field

[0001] The present invention relates to the field of cell culture technology, and in particular to a method for preparing CAR-positive monoclonal cells and an application thereof in cell-positive quality control detection. Background Art

[0002] CAR-T therapy, or Chimeric Antigen Receptor T-Cell Immunotherapy, is a form of adoptive cell therapy and a new type of precision targeted therapy. Its effectiveness in clinical cancer treatment has been demonstrated in recent years. It is a highly promising new tumor immunotherapy approach that is precise, rapid, and highly effective, potentially curing cancer.

[0003] At present, domestic standard products and positive control products for immune cell therapy mainly rely on imports or are provided by a few institutions, and there are problems such as large batch differences and insufficient stability. There are no standardized control products approved by the National Medical Products Administration for immune cell therapy for solid tumors, and companies need to build their own working control products. In the process of developing internal standard products or working control products, companies often face technical research and development problems, such as the preparation of working control products, including long preparation time, difficulty in obtaining high-purity products, and long-term stability after preparation. These problems bring various adverse factors to the subsequent use of working control products, affecting their normal use, and even bringing unstable factors to the quality control of CAR-T cells, ultimately affecting the product quality of CAR-T preparations.

[0004] Based on the above problems, the present invention designs a technology that is easy to prepare and more suitable for CAR-T quality testing needs in use, and provides new ideas for peers. Summary of the Invention

[0005] Based on the above problems, the problem to be solved by the present invention is to provide a method for preparing monoclonal cells with CAR positivity. This method can construct a diversified positive quality control sample library by screening different positive monoclonal cells, and prepare quality control samples of different complexities by mixing different monoclonal cell lines in proportion, which can be used in different detection scenarios to meet more immune cell detection quality control needs.

[0006] The technical solutions of the present invention are as follows:

[0007] A method for preparing CAR-positive monoclonal cells comprises the following steps:

[0008] Prepare tumor cells and lentiviral vectors containing the target CAR gene;

[0009] The tumor cells were resuspended in complete culture medium and transferred to a 24-well plate. Complete culture medium and lentiviral vector were added to each well, and the cells were cultured for 3 days after transfection.

[0010] After 3 days of transfection culture, the transfection culture medium was collected and centrifuged to remove the supernatant. The cell pellet was resuspended in complete culture medium and the resulting cell suspension was transferred to a 12-well plate and incubated for another 5 days.

[0011] After the 12-well plate incubation is completed, the incubation medium is collected and centrifuged to remove the supernatant, and the cell pellet is resuspended in complete culture medium to obtain a cell suspension.

[0012] The cell suspension is added with a sorting buffer, and the cells are sorted using a flow cytometer; the sorted cells are then transferred to a 96-well culture plate and cultured for 7 to 14 days;

[0013] After 4 days of culture, the culture medium was collected and the supernatant was removed by centrifugation. The cell pellet was washed 2 to 3 times with PBS to obtain CAR-positive monoclonal cells.

[0014] In one embodiment, in the preparation method, the tumor cells are obtained by suspension growth, and the cell viability is ≥95%.

[0015] In one embodiment, in the preparation method, the tumor cells are Jurkat cells, K562 cells or Raji cells.

[0016] In one embodiment, in the preparation method, when the lentiviral vector and tumor cells are transfected and cultured in a 24-well plate, the amount of the complete culture medium added is based on a cell density of 2.0E+05 cells / 0.5 mL per well; the amount of the lentiviral vector added is based on an MOI of 20.

[0017] In one embodiment, in the preparation method, after the lentiviral vector and tumor cells are transfected and cultured in a 24-well plate for 6 hours, complete culture medium is added to each well to a total volume of 1 mL.

[0018] In one embodiment, in the preparation method, when the cell suspension is incubated and cultured in a 12-well plate, the cell density in the cell suspension is 8.0E+05 cells / mL.

[0019] In one embodiment, in the preparation method, when the cell suspension is cultured in a 96-well plate, the cell density in the cell suspension is controlled to be 5.0E+05 to 1.0E+07 cells / mL.

[0020] In one embodiment, in the preparation method, the separation buffer is DPBS containing 2% FBD; 1 mL of separation buffer is added to every 50 μL of cell suspension.

[0021] In one embodiment, in the preparation method, the centrifugation is performed at 200g for 5 minutes at room temperature.

[0022] The monoclonal cells prepared by the above preparation method can be used in cell culture and cell positive quality control detection.

[0023] In the present invention, the complete culture medium used is RPMI-1640 culture medium containing 10% FBS.

[0024] The present invention provides a preparation method for improving the CAR positivity of CAR-T cells. It constructs a series of cell lines that can stably express the CAR gene through lentiviral transfection, provides stable positive quality control products for CAR-T cell detection, improves the standardization and systematization of CAR-T detection, ensures that the CAR-T detection results are accurate, credible and reliable, and guarantees the safe and effective use of CAR-T cells in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The following are images of Jurkat-M1 cells observed under a microscope after culture in a 12-well plate in Example 1, using a 10x eyepiece x 10x objective lens. Image a represents a brightfield image of Jurkat-M1 cells; image b represents a darkfield image of Jurkat-M1 cells.

[0026] Figure 2 In Example 1, Jurkat-M1 cells were cultured in 96-well plates for 4 days and monoclonal images were observed under a microscope using a 10x eyepiece x 10x objective lens;

[0027] Figure 3 This is a graph showing the changing trend of GFP positive rates of Jurkat-M1 monoclonal cells at generations P0, P15, and P30 in Example 1;

[0028] Figure 4 This is a titration trend diagram of 6 groups of CAR antibodies after dilution in Example 2. DETAILED DESCRIPTION

[0029] The preferred embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0030] In the clinical application of CAR-T cells, the detection of CAR expression rate runs through the entire process of efficacy evaluation from cell preparation to clinical application in humans. The accuracy of its detection has a huge impact on the safe and effective use of CAR-T in clinical practice, especially for the accurate control of the infusion dose of CAR-T prepared in vitro and the judgment of clinical prognosis based on changes in CAR-T in the patient's body.

[0031] Flow cytometry (FCM) is one of the most commonly used detection methods in laboratories. Whether it is a scientific research unit, enterprise or hospital, flow cytometers are essential detection instruments. Therefore, using FCM to monitor the CAR expression rate of CAR-T cells is the simplest and fastest method, and it can also further detect immunophenotyping and T cell function. In flow cytometry, although the performance of the flow cytometer can be tested using flow quality control microspheres, it cannot accurately reflect the complexity of the cell sample. In addition, samples from different individual sources and different test items vary greatly. Relying solely on isotype control antibody quality control cannot provide reliable judgment for complex result analysis.

[0032] ddPCR, qPCR and other technologies are used to detect the vector copy number (VCN) in CAR-T cells and the pharmacokinetics (PK) after CAR-T infusion. Although the two technologies can complement each other to a certain extent, there is the problem of false positives caused by high background noise. At the same time, factors such as primers, probes, and external contaminants have a great impact on the result analysis, and various quality control methods need to be used to eliminate interfering factors.

[0033] Therefore, the preparation method for improving the CAR positivity of CAR-T cells provided by the present invention is to specifically analyze the CAR gene by lentiviral transduction into tumor cells, such as Jurkat cells, K562 cells or Raji cells, to construct an immortalized cell line that stably expresses the CAR gene, and to confirm the transfer efficiency by fluorescent reporter gene, and to enrich positive monoclonal cells by flow cytometry or limiting dilution. After screening the positive monoclonal cells of different backgrounds, a multivariate positive quality control sample cell library is constructed. Monoclonal cell lines of different backgrounds are mixed in proportion to prepare quality control samples of different complexities for use in different detection scenarios to meet more quality control requirements.

[0034] The preparation method for improving CAR-T cell CAR positivity provided by the present invention comprises the following steps:

[0035] S1. Prepare tumor cells and a lentiviral vector containing the target CAR gene and GFP gene. Tumor cells are preferably suspension-cultured tumor cell lines with a cell viability >95%, such as Jurkat, K562, and Raji cells. The GFP gene in the lentiviral vector is a marker gene that serves only as a fluorescent marker for microscopic observation.

[0036] S2. Resuspend the tumor cells from step S1 in complete culture medium, such as RPMI-1640 medium containing 10% FBS, and control the cell density to 2.0E+05 cells / 0.5 mL. Plate the resulting cell suspension into a 24-well plate. Add the lentiviral vector to each well at an MOI of 20. After transfection and culture for 6 hours, add complete culture medium to a total volume of 1 mL / well. Continue transfection and culture for 3 days.

[0037] S3. After the 3-day transfection culture in step S2 is completed, a small amount of tumor cells transfected with the lentiviral vector in the 24-well cell culture plate is taken for cell viability detection. The fluorescence intensity of the GFP gene in the cells is observed under a fluorescence microscope to confirm whether the CAR gene has been transferred into the tumor cells, and the tumor cell viability is calculated by sampling;

[0038] S4. Remove the supernatant of the transfection culture in step S3, collect the lower layer of the transfection culture and centrifuge at 200g for 5 min, remove the supernatant, resuspend the cell pellet in complete culture medium, and adjust the cell density to 8.0E+05 cells / mL. Transfer the resulting cell suspension to a 12-well plate and continue incubation for 5 days;

[0039] After the incubation in step S4 is completed, the culture medium is collected and centrifuged at 200 g for 5 min. The supernatant is removed and the cell pellet is resuspended in complete culture medium to control the cell density to 5.0E+05 to 1.0E+07 cells / mL.

[0040] S6. Add 1 mL of sorting buffer per 50 μL of cell suspension, take the cell suspension in step S5, add sorting buffer, such as 2% FBD buffer in DPBS, and perform cell sorting using a flow cytometer;

[0041] S7. The sorted cells are placed in a 96-well culture plate and cultured for 7 to 14 days. During this period, the GFP fluorescence signal of the cells is regularly observed, and the culture wells where the fluorescence signal is observed are marked.

[0042] S8. Transfer the tumor cells in the culture wells labeled in step S7 to a TC75 culture flask, continue culturing for 1 month, and again detect cell autofluorescence using a flow cytometer to preliminarily confirm that the cells are monoclonal cells;

[0043] S9. Collect the culture medium in step S8 and centrifuge at 200 g for 5 minutes. Remove the supernatant and wash the cell pellet 2 to 3 times with PBS to obtain CAR-positive monoclonal cells, which are frozen for future use.

[0044] The technical solution of the present invention is further described below through specific embodiments.

[0045] Example 1

[0046] 1.1. Prepare lentivirus M1, which contains CAR and GFP genes. Jurkat cells were selected as the target tumor cells. The M1 activity titer was determined to be 2.18E+08TU / mL. Jurkat cells were resuspended in 10% FBS + RPMI-1640 medium at a titer of 2.0E+05 cells / 0.5mL / well and added to a 24-well cell culture plate. Based on an MOI of 20 for Jurkat cells, lentivirus M1 was added. After gentle shaking, the 24-well plate was transferred to a 37°C, 5% CO2 incubator for transfection. After 6 hours, 10% FBS + RPMI-1640 medium was added to the culture wells again to a total volume of 1mL / well. Transfection culture was continued for 3 days.

[0047] 1.2. After 3 days of transfection culture, a small amount of CAR-transfected Jurkat cells in the 24-well plate was taken for cell viability detection; the GFP fluorescence intensity of the CAR-transfected cells was observed at the same time to confirm the presence of successfully transfected Jurkat-M1 cells; the upper layer of the transfection culture was then removed, and the lower layer of the transfection culture was collected and centrifuged at 200g for 5 minutes at room temperature. The supernatant was discarded, and the cells were resuspended in new 10% FBS + RPMI-1640 medium and the cell density was controlled to 8.0E+05 cells / mL. The resuspended cell solution was transferred to a 12-well plate and incubated in an incubator at 37°C and 5% carbon dioxide for another 5 days;

[0048] 1.3. After the incubation, take a small amount of Jurkat-M1 cells from the 12-well plate and observe them under a fluorescence microscope. Figure 1 As shown; confirm that some of the Jurkat cells transfected with lentivirus after MI are Jurkat-M1 cells; collect the incubation culture medium and centrifuge at 200g for 5 minutes, remove the supernatant, and collect the cell pellet; then take 2.0E+06 to 4.0E+06 cells, resuspend and dilute them in 10% FBS+RPMI-1640 medium, and control the cell density to 5.0E+05 to 1.0E+07 cells / mL; then take 100 μL of cell suspension (i.e., 1.0E+06 cells), add 2 mL of 2% FBD+DPBS sorting buffer, and sort them using a flow cytometer;

[0049] 1.4. Transfer 1.0E+06 cells after sorting to a 96-well plate for culture. Observe the cells under a microscope every 3 days. Figure 2 As shown; confirm the growth of positive monoclonal cells and mark the culture wells where fluorescent signals are observed;

[0050] 1.5. Transfer the positive monoclonal cells in the labeled culture wells to TC75 culture flasks and continue culturing for 1 month. Detect cell autofluorescence again using flow cytometry to further confirm that the cells are monoclonal cells.

[0051] 1.6. The culture medium in step S8 was collected and centrifuged at 200 g for 5 minutes. The supernatant was removed and the cell pellet was washed 2-3 times with PBS to obtain CAR-positive monoclonal cells, which were frozen for future use. At the same time, the total number of Jurkat-M1 positive monoclonal cells obtained was expanded and cultured to more than 3.0E+08 cells, and the cell generation was defined as P0. They were frozen and a master cell bank was established. The master cell bank was then expanded and cultured in sequence to establish a working cell bank. The cell generations were defined as P15, P30, etc.; Jurkat-M1 cells of P0, P15, and P30 generations were subjected to flow cytometry and CAR copy number detection, respectively. The results were as follows: Figure 3 and as shown in Table 1.

[0052] Table 1 CAR copy number results of Jurkat-M1 monoclonal cells at different culture passages

[0053]

[0054] Depend on Figure 3 As shown in Table 1, the sorted Jurkat-M1 cells are monoclonal cells, indicating that monoclonal cells can be obtained by sorting.

[0055] Example 2

[0056] Use of positive monoclonal cells: Resuspend the cells according to the live cell ratio in flow cytometry and mix them in different proportions according to the test needs to form test groups such as negative control quality control samples, 100% positive control quality control samples, and mixed control quality control samples. The prepared quality control samples can be used for multiple test projects, such as CAR-T cell positive ratio detection, VCN detection, CAR-T cell PD-1 ratio detection, CAR-T cell related flow cytometry antibody titer calibration, etc.

[0057] This example uses the titration of CAR detection antibodies as an example: the number of fixed cells in this detection system is 1.0E+06 / 100 μL / tube, and the flow cytometry antibody system is diluted 2-fold;

[0058] A small amount of Jurkat-M1 monoclonal cells prepared in Example 1 were taken, centrifuged, and resuspended in 2% FBS + DPBS flow cytometry buffer to a cell density of 1.0E+06 cells / 50 μL / tube. The cells were aliquoted into six 1.5 mL centrifuge tubes and diluted with the antibody to be titrated to an intermediate concentration, as shown in Table 2.

[0059] Table 2 Antibody dilution gradient

[0060]

[0061] Take the intermediate concentration solutions of the antibodies in each gradient ratio in Table 2 and add them to the aliquoted Jurkat-M1 monoclonal cell suspension. Incubate at room temperature in the dark for 30 minutes, then analyze on a flow cytometer. Count the median fluorescence intensity (MFI value) corresponding to the negative and positive populations, and calculate the staining index (SI) of each group. The calculation formula is as follows:

[0062]

[0063] Flow cytometry was used to detect each dilution group of the antibody (groups 1 to 6). The median fluorescence intensity values ​​corresponding to the negative and positive groups, as well as the rSD (relative standard deviation) values ​​of the negative group, were collected. The corresponding SI values ​​were calculated according to the above calculation formula. The results are shown in Table 3.

[0064] Table 3 CAR antibody titer staining index

[0065]

[0066] According to the data in Table 3, the SI value trend graph of each dilution group was drawn to determine the optimal amount of antibody that can be used for detection. The results are as follows: Figure 4 shown. Figure 4 The point in the box at the top of the curve represents the antibody dosage that produces the highest specific staining with minimal background. To achieve optimal detection, the CAR antibody dilution ratio with the highest staining index (SI = 14.58) was used in sample testing, i.e., the optimal concentration was 1.25 μg / 100 μL.

[0067] The above tests show that the CAR positivity rate of CAR-T cells is of great significance in clinical treatment. It is related to the accurate calculation of the CAR-T cell reinfusion dose and the reduction of safety issues caused by excessive reinfusion doses, such as severe CRS reactions. Since the stability of antibodies still varies under the daily optimal storage conditions, it is necessary to use CAR-positive monoclonal cell controls again to confirm the optimal usage of CAR antibodies during the CAR-T cell positivity rate test.

[0068] It should be understood that the above description of the preferred embodiments of the present invention is relatively detailed and cannot be regarded as limiting the scope of patent protection of the present invention. The scope of patent protection of the present invention shall be based on the appended claims.

Claims

1. A method for preparing CAR-positive monoclonal cells, characterized in that: The steps include: Prepare tumor cells and lentiviral vectors containing the target CAR gene; The tumor cells were resuspended in complete culture medium and transferred to a 24-well plate. Complete culture medium and lentiviral vector were added to each well, and the cells were cultured for 3 days after transfection. After 3 days of transfection culture, the transfection culture medium was collected and centrifuged, and the supernatant was removed. The cell pellet was resuspended in complete culture medium, and the resulting cell suspension was transferred to a 12-well plate and incubated for another 5 days. After 5 days of incubation, the culture medium was collected and centrifuged, the supernatant was removed, and the cell pellet was resuspended in complete culture medium to obtain a cell suspension; The cell suspension is added with a sorting buffer and then cell sorting is performed, and the sorted cells are transferred to a 96-well culture plate and cultured for 7 to 14 days; After the 96-well culture was completed, the culture medium was collected and centrifuged, and the supernatant was removed. The cell pellet was washed 2 to 3 times with PBS to obtain CAR-positive monoclonal cells.

2. The preparation method according to claim 1, characterized in that The tumor cells are obtained by suspension growth, and the cell viability is ≥95%.

3. The preparation method according to claim 1 or 2, characterized in that The tumor cells are Jurkat cells, K562 cells or Raji cells.

4. The preparation method according to claim 1, characterized in that When the lentiviral vector and tumor cells were transfected and cultured in a 24-well plate, the volume of the complete medium was added according to a cell density of 2.0E+05 cells / 0.5 mL per well, and the volume of the lentiviral vector was added according to an MOI of 20.

5. The preparation method according to claim 1, characterized in that After the lentiviral vector and tumor cells were transfected and cultured in a 24-well plate for 6 hours, complete culture medium was added to each well to a total volume of 1 mL.

6. The preparation method according to claim 1, characterized in that When the cell suspension is incubated and cultured in a 12-well plate, the cell density in the cell suspension is 8.0E+05 cells / mL.

7. The preparation method according to claim 1, characterized in that When the cell suspension is cultured in a 96-well plate, the cell density in the cell suspension is controlled to be 5.0E+05 to 1.0E+07 cells / mL.

8. The preparation method according to claim 1, characterized in that When the cell suspension was cultured in a 96-well plate, 1 mL of the sorting buffer was added to every 50 μL of the cell suspension, and the sorting buffer was a DPBS buffer containing 2% FBS.

9. The preparation method according to any one of claims 1 to 8, characterized in that The complete culture medium is RPMI-1640 culture medium containing 10% FBS.

10. Use of the monoclonal cells prepared by the preparation method according to any one of claims 1 to 9 in cell culture and cell positive quality control detection.

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