BCMA CAR Jurkat stably transfected cell line and application thereof
By constructing a highly expressed BCMA CAR Jurkat stable transgenic cell line, the difficulty in detecting the effectiveness and safety-related characteristics of CAR-T cell products is solved, and an efficient quality evaluation tool is provided to ensure the authenticity and reliability of the test.
Patent Information
- Application Number
- CN202311498636.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-11-10
- Publication Date
- 2025-05-13
AI Technical Summary
The prior art is difficult to quickly and reliably detect the effectiveness and safety-related properties of CAR-T cell products, especially in the detection of the composition, activity and stability of protein expression of CAR-T cells.
A BCMA CAR Jurkat stable transgenic cell line was constructed. This cell line expresses a chimeric antigen receptor CAR targeting BCMA. The CAR expression density is 10 times higher than that of Jurkat cells, and was detected and verified by flow cytometry and other methods.
It provides an efficient tool for evaluating the quality and clinical efficacy of CAR-T cells, which can truly and reliably detect the formation of antibiotics or neutralizing antibodies, helping researchers to reflect the test results faster and more accurately.
Smart Images
Figure FT_1 
Figure FT_2 
Figure FT_3
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and in particular to a BCMA CAR Jurkat stable transfection cell line and uses thereof. Background Art
[0002] In recent years, with the advancement of science and technology, the field of cell gene therapy has achieved rapid development. Among them, chimeric antigen receptor T cell (CAR-T) therapy has attracted attention due to its breakthrough efficacy. At present, there are 9 CAR-T cell therapy products approved for marketing in the world, of which 3 target BCMA, and their indications are all multiple myeloma. They are Abecma (idecabtagene vicleucel), the world's first CAR-T product targeting BCMA, jointly developed by Bristol-Myers Squibb (BMS) and bluebird bio (bluebird), approved in March 2021, and Sidaquioren injection, which uses double nano antibodies as antibody binding domains and was jointly developed by Legend Biotech and Johnson & Johnson and approved in March 2022, and Ikioren injection, the first fully human BCMA CAR-T product jointly developed by Reindeer Bio and Innovent Biologics and approved in July 2023. In addition, many biopharmaceutical companies have invested in the research and development of CAR-T drugs targeting BCMA. The launch of a drug means a lot of investment in manpower, material resources, financial resources and time. How to reflect the test results faster, better and more realistically is crucial for researchers. As a "living" drug, CAR-T has different characteristics from small molecule or macromolecule drugs, that is, its composition, activity and stability of protein expression vary to a certain extent. How to test the effectiveness and safety-related characteristics of CAR-T products both realistically and reliably in the test has become very difficult for researchers.
[0003] Jurkat, CloneE6-1 (Jurkat T for short) cells are human leukemia T lymphocytes, which express CD3, CD28 and other T lymphocyte characteristic antigens on their cell membranes, and can be recognized and activated by corresponding antibodies, showing the characteristics and functions that T cells should have. When Jurkat cells express BCMACAR, they can be bound by target antigens or antibodies that can specifically bind to BCMACAR.
[0004] At present, there is no report on BCMA CAR Jurkat stable cell line. The present invention provides a BCMA CAR Jurkat stable cell line and a specific method for preparing the cell line, which is of great significance for the related research, quality evaluation, clinical efficacy evaluation, etc. of CAR-T cells. Summary of the invention
[0005] The present invention provides a BCMA CAR Jurkat stable cell line, which is a Jurkat, CloneE6-1 cell expressing a chimeric antigen receptor CAR targeting BCMA, and the CAR expression density of the BCMA CAR Jurkat stable cell line is more than 10 times that of the Jurkat, CloneE6-1 cell.
[0006] In some embodiments, the CAR positive expression rate of the BCMACAR Jurkat stably transfected cell line is not less than 90%; preferably, not less than 95%; more preferably, not less than 99%.
[0007] In other embodiments, the BCMACAR Jurkat stable cell line deposit number is CCTCCNO: C2023273.
[0008] In other embodiments, the CAR comprises an extracellular antigen binding domain, a hinge region, a transmembrane region, an intracellular co-stimulatory domain, and an intracellular signaling domain.
[0009] In certain embodiments, the antigen binding domain is a single chain antibody against BCMA.
[0010] The present invention also provides the use of the BCMACAR Jurkat stably transfected cell line in preparing a reagent for evaluating the quality or clinical efficacy of an immune cell therapy drug targeting BCMA.
[0011] In some embodiments, the BCMA-targeting immune cell therapy expresses CAR.
[0012] In certain embodiments, the amino acid sequence of the CAR is as shown in SEQ ID NO:1.
[0013] In other embodiments, the immune cell therapy drug targeting BCMA is CAR-T cells, CAR-NK cells or CAR-M cells.
[0014] The present invention also provides the use of the BCMACAR Jurkat stably transfected cell line in preparing a reagent for evaluating the formation of anti-drug antibodies or neutralizing antibodies after administration of an immune cell therapy drug targeting BCMA.
[0015] In some embodiments, the evaluation method is to contact the cells of the BCMA CAR Jurkat stably transformed cell line with the blood, plasma, serum, tissue fluid, cerebrospinal fluid, bronchoalveolar lavage fluid or urine of a subject receiving immune cell drug therapy targeting BCMA, and detect the content of anti-drug antibodies or neutralizing antibodies therein.
[0016] In other embodiments, the BCMA-targeting immune cell therapy expresses CAR.
[0017] In certain embodiments, the CAR comprises an extracellular antigen binding domain, a hinge region, a transmembrane region, an intracellular co-stimulatory domain, and an intracellular signaling domain. Preferably, the extracellular antigen binding domain is a single-chain antibody against BCMA. More preferably, the extracellular antigen binding domain is an anti-BCMA monoclonal antibody C11D5.3.
[0018] In certain embodiments, the amino acid sequence of the CAR is as shown in SEQ ID NO:1.
[0019] In other embodiments, the immune cell therapy drug is CAR-T cells, CAR-NK cells or CAR-M cells.
[0020] The present invention uses Jurkat T cells as CAR-expressing cells to construct and obtain a cell line that expresses a natural BCMA CAR structure at a high density, thereby enabling research in aspects such as antibody development, immunogenicity and functional evaluation. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 is a schematic diagram of the construction of BCMA CAR Jurkat cell line;
[0022] Figure 2 This is the flow cytometry validation diagram of the BCMA CAR Jurkat cell pool;
[0023] Figure 3 This is a flow cytometry screening image of BCMA CAR Jurkat monoclonal cells;
[0024] Figure 4 This is a schematic diagram of the BCMA CAR Jurkat cell line library construction;
[0025] Figure 5 It is a schematic diagram of the Nab detection process;
[0026] Figure 6 It is a schematic diagram of Nab detection results. DETAILED DESCRIPTION
[0027] After in-depth and extensive research, the inventors have constructed a BCMA CAR Jurkat stable cell line, which has the advantages of high CAR positive expression rate and high CAR expression density, and is of great significance for the related research, quality evaluation, clinical efficacy evaluation, etc. of CAR-T cells.
[0028] Jurkat, CloneE6-1 (abbreviated as Jurkat T) cells: human T lymphocyte leukemia cells, established by Schneider, derived from the peripheral blood of a 14-year-old boy. This clone is a clone of the Jurkat-FHCRC cell line (a derivative of Jurkat), which can produce a large amount of IL-2 after being induced by phorbol esters and lectins or anti-T3 monoclonal antibodies (requires two substances to induce together). T lymphocyte characteristic antigens such as CD3 and CD28 are expressed on its cell membrane, and can be recognized and activated by corresponding antibodies, showing the characteristics and functions that T cells should have. This cell line can be purchased through conventional channels, such as ATCC.
[0029] The "BCMA CAR Jurkat stable cell line" described herein refers to Jurkat, CloneE6-1 cells that can stably express CAR fusion protein. The CAR nucleotide sequence can be introduced into Jurkat, CloneE6-1 cells by any gene transduction method (such as viral vector-mediated gene transduction, non-viral vector-mediated gene transduction such as electroporation, etc.).
[0030] The "CAR positive expression rate" described herein refers to the proportion of cells expressing CAR in a cell population. Flow cytometry can usually be used to detect the proportion of cells expressing CAR in a BCMA CAR Jurkat stable cell population.
[0031] The "CAR expression density" described in this article is used to indicate the expression density or expression amount of the CAR fusion protein on the cell membrane of a single cell. We can detect and compare the CAR expression density of different cell lines by fluorescently labeling the CAR sequence and then detecting the mean fluorescence intensity (MFI value) by flow cytometry.
[0032] As used herein, CAR-T or chimeric antigen receptor (CAR-T) T cells refer to genetically modified T cells that can recognize specific target antigens in an MHC-unrestricted manner and continuously activate and proliferate.
[0033] CAR-modified NK cells (Chimeric Antigen Receptor-NK cells, CAR-NK) are modified by genetic engineering to express CAR, which connects antibodies (or receptors) that recognize surface antigens of target cells (such as virus-infected cells, cancer cells) with signal molecules required to activate immune cells. This can break through the limitations of inhibitory receptors and activate NK cells, thereby enhancing the specific killing of target cells by NK cells.
[0034] CAR-modified macrophages (CAR-Macrophage, or CAR-M) refer to macrophages that have been introduced with CAR. CAR M can target and engulf tumor cells, and also change the microenvironment near the tumor by secreting pro-inflammatory cytokines, present tumor antigens to T cells, activate T cells' immune response to tumors, and enhance their ability to kill tumors.
[0035] B-cell maturation antigen (BCMA), also known as CD269, consists of 184 amino acid residues, with 80 amino acid residues in the intracellular region and a very short extracellular region sequence. It has only one carbohydrate recognition domain as a B cell surface molecule. BCMA is a type I transmembrane signaling protein lacking a signal peptide and is a member of the tumor necrosis factor receptor family (TNFR). It can bind to two ligands, B cell activation factor BAFF or a proliferation induced ligand (APRIL) [Tumor necrosis factor family ligand-receptor binding. Curr Opin Struct Biol, 2004. 14 (2): p. 154-60.]. In normal tissues, BCMA is expressed on the surface of mature B cells and plasma cells. The immune system of BCMA gene knockout mice is normal, with normal spleen structure and normal development of B lymphocytes, but the number of plasma cells is significantly reduced, which proves that BCMA plays an important role in maintaining the survival of plasma cells. Its mechanism mainly includes BCMA binding to BAFF protein and upregulating anti-apoptotic genes Bcl-2, Mcl-1 and Bclw to maintain cell growth [BCMA is essential for the survival of long-lived bone marrow plasma cells. J Exp Med, 2004. 199 (1): p. 91-8.]. Similarly, this mechanism also plays a role in myeloma cells and plays an important role in promoting the malignant proliferation of myeloma cells [BAFF and APRIL protect myeloma cells from apoptosis induced by interleukin 6 deprivation and dexamethasone. Blood, 2004. 103 (8): p. 3148-57.]. Studies have shown that BCMA is commonly expressed in multiple myeloma cell lines, and detection in multiple myeloma patients has also obtained consistent results [Expression of BCMA, TACI, and BAFF-R in multiple myeloma: a mechanism for growth and survival. Blood, 2004. 103(2): p.689-94.].Based on the existing reports, Kochenderfer et al. used Q-PCR, flow cytometry and immunohistochemistry to further study the expression characteristics of BCMA, and confirmed that BCMA is not expressed in normal human tissues other than mature B cells and plasma cells, and is also not expressed in CD34+ hematopoietic cells [B-cell maturation antigen is a promising target for adoptive T-cell therapy of multiple myeloma. Clin Cancer Res, 2013. 19 (8): p. 2048-60.]. The current successful progress of anti-BCMA C-T cell therapy suggests that BCMA is one of the effective targets for cellular immunotherapy of multiple myeloma.
[0036] Typically, after applying CAR-modified immune cell therapy (e.g., anti-BCMACAR-T cell therapy), it is necessary to monitor whether anti-drug antibodies and neutralizing antibodies are produced in the subject to assess the clinical efficacy of cell therapy. Using the cell line of the present invention, samples from the subject, such as serum, plasma, lymph, blood, cerebrospinal fluid, alveolar lavage fluid or urine, can be detected to determine whether there are anti-drug antibodies or neutralizing antibodies or to determine their content.
[0037] As used herein, "subject", "patient", "object", "individual" and the like are used interchangeably herein to refer to a living organism, such as a mammal, that can elicit an immune response. Examples include, but are not limited to, humans, dogs, cats, mice, rats, and transgenic species thereof.
[0038] The present invention will be described below in the form of specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. The methods and materials used in the examples are, unless otherwise stated, materials and methods conventional in the art.
[0039] Example 1: Construction of BCMA CAR Jurkat cell line
[0040] 1) Virus transfection and cell pool construction: Coat a 24-well plate (Non-TC-treated) with Retronectin working solution at a final concentration of 15ug / mL, 250ul per well, keep away from light, and place in a refrigerator at 4°C overnight, then discard the coating solution. Add 1mL of virus solution (DCAR269B virus solution, prepared by inserting the CAR sequence shown in SEQ ID NO:1 into the retroviral vector and transfecting PG13 cells) to the above-coated 24-well culture plate, seal with sealing film, centrifuge at 32±1°C, 2000g for 2h, and discard the supernatant. Add Jurkat cells (5E5 / mL, 1mL), centrifuge at 30°C, 1000g for 10min, and place in a cell incubator. 24h after infection, aspirate the cell suspension, centrifuge at 450g for 5min, discard the supernatant, spread on a 6-well plate, 4mL / well, and culture in an incubator for 72h. See the process. Figure 1 .
[0041] 2) Detection of BCMA CAR expression in cell pool: Incubate cells with the prepared Bio-BCMA working solution for half an hour, centrifuge and discard the supernatant, wash once, incubate cells with the prepared secondary antibody working solution for half an hour, centrifuge and discard the supernatant, wash once, resuspend in PBS, and detect on flow cytometry. Observe the proportion of CAR-positive cells. Results are shown in Figure 2 .
[0042] 3) Monoclonal cell culture: After expanding the above cells, select a number of monoclonal cells under a microscope, transfer them to a 24-well or 96-well plate, and continue to culture with culture medium.
[0043] 4) Monoclonal cell screening: After each monoclonal cell is expanded and cultured, cells with poor conditions are discarded, and cells are taken for flow cytometry detection. According to the proportion of CAR-positive cells and the MFI window value, the cell line most suitable for subsequent experiments is selected for library construction. Figure 3 , it can be seen that the CAR positive expression rate and CAR density of #2 are significantly higher than those of other cell lines. The screened #2 cell line has been deposited in the China Center for Type Culture Collection (Wuhan University, China, 430072) on October 12, 2023, with the deposit number CCTCC NO: C2023273, and the culture name (classification name): Human T lymphocyte leukemia cell BCMACAR-Jurkat.
[0044] 5) Monoclonal cell bank construction: The selected monoclonal cells are expanded and cultured, and after several passages, the number of cells required for bank construction is reached, and then the expansion is stopped, the batch of cells is mixed, and the cells are cryopreserved. After cryopreservation, samples are taken for sterility, mycoplasma and cell identification experiments. See the process for details. Figure 4 .
[0045] Example 2: Human T lymphocyte leukemia cell line BCMACAR-Jurkat is used for screening of anti-CAR antibodies
[0046] 1) Take a cell from the monoclonal cell working bank for recovery, take the corresponding volume according to the number of cells required by the experimental design, centrifuge at 500g for 5 minutes, and adjust the cell density.
[0047] 2) Prepare different concentrations of the primary antibody working solution of the test antibody, Bio-BCMA antibody working solution and positive control antibody working solution for staining and incubation. After that, wash once, incubate the cells with the prepared secondary antibody working solution for half an hour, centrifuge and discard the supernatant, wash once, resuspend in PBS, and detect on a flow cytometer.
[0048] 3) Calculate the EC50 of the test antibody binding to the human T lymphocyte leukemia cell line BCMA CAR-Jurkat, and compare it with Bio-BCMA and the positive control antibody.
[0049] Example 3: Human T lymphocyte leukemia cell line BCMACAR-Jurkat is used for the detection of Nab
[0050] 1) Take the cells from the monoclonal cell working bank and culture them for two days, centrifuge and remove the supernatant, add 1 mL of culture medium to resuspend, count and adjust the cell density;
[0051] 2) Add the prepared sample to be tested to the corresponding flow cytometry tube with resuspended cells at 75uL / well and gently blow to mix. Incubate at 4 degrees for 30 minutes;
[0052] 3) After incubation, add 3 mL of washing solution to each tube and resuspend, centrifuge at 500g for 5 min, and gently discard the supernatant; then add 3 mL of washing solution to each tube and resuspend, centrifuge at 500g for 5 min, and discard the supernatant;
[0053] 4) Dilute the aliquoted bio-BCMA with cell manipulation buffer at 1:1000, add 100uL to each well to resuspend the cells, and incubate at 4 degrees for 30 minutes.
[0054] 5) After incubation, add 3 mL of washing solution to each tube and resuspend, centrifuge at 500g for 5 min, and gently discard the supernatant; then add 3 mL of washing solution to each tube and resuspend, centrifuge at 500g for 5 min, and discard the supernatant.
[0055] 6) Add 100uL staining buffer + SA-BV421 + dead and alive dye (100:0.5:0.1) to each well, resuspend, incubate at 4 degrees in the dark for 20 minutes.
[0056] 7) After incubation, add 3 mL of washing solution to each tube and resuspend, centrifuge at 500g for 5 min, and gently discard the supernatant; then add 3 mL of washing solution to each tube and resuspend, centrifuge at 500g for 5 min, and discard the supernatant.
[0057] 8) After resuspending in 80uL staining buffer, add to the 96-well plate and test in sequence.
[0058] 9) Calculate the MFI value and compare it with the screening threshold. Figure 5 The results are shown in Figure 6 .
[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A BCMA CAR Jurkat stable cell line, characterized in that: The BCMA CAR Jurkat stable cell line is a Jurkat, CloneE6-1 cell expressing a chimeric antigen receptor CAR targeting BCMA, and the CAR expression density of the BCMA CAR Jurkat stable cell line is more than 10 times that of the Jurkat, CloneE6-1 cell.
2. The BCMA CAR Jurkat stable cell line according to claim 1, characterized in that The CAR positive expression rate of the BCMA CARJurkat stably transfected cell line is not less than 90%; preferably, not less than 95%; more preferably, not less than 99%.
3. The BCMA CAR Jurkat stable cell line according to claim 1, characterized in that The deposit number of the BCMA CAR Jurkat stable cell line is CCTCC NO: C2023273.
4. The BCMA CAR Jurkat stable cell line according to claim 1, characterized in that The CAR comprises an extracellular antigen binding domain, a hinge region, a transmembrane region, an intracellular co-stimulatory domain and an intracellular signaling domain; preferably, the antigen binding domain is a single-chain antibody against BCMA.
5. Use of the BCMA CAR Jurkat stably transfected cell line according to any one of claims 1 to 4 in the preparation of a reagent for evaluating the quality or clinical efficacy of immune cell therapy drugs targeting BCMA.
6. The use according to claim 5, characterized in that: The BCMA-targeted immune cell therapy drug expresses CAR; preferably, the amino acid sequence of the CAR is as shown in SEQ ID NO:
1.
7. The use according to claim 5, characterized in that: The immune cell therapy drug targeting BCMA is CAR-T cells, CAR-NK cells or CAR-M cells.
8. Use of the BCMA CAR Jurkat stably transfected cell line according to any one of claims 1 to 4 in the preparation of a reagent for evaluating the formation of anti-drug antibodies or neutralizing antibodies after administration of an immune cell therapy drug targeting BCMA.
9. The use according to claim 8, characterized in that: The evaluation method is to contact the cells of the BCMA CARJurkat stably transfected cell line with the blood, plasma, serum, cerebrospinal fluid, bronchoalveolar lavage fluid, tissue fluid or urine of a subject receiving BCMA-targeted immune cell drug therapy, and detect the content of anti-drug antibodies or neutralizing antibodies therein.
10. The use according to claim 8, characterized in that: The BCMA-targeting immune cell therapy drug expresses CAR, and preferably, the amino acid sequence of the CAR is as shown in SEQ ID NO: 1; or the immune cell therapy drug is a CAR-T cell, a CAR-NK cell or a CAR-M cell.