CN114262689A - Method for rapidly detecting activity of CD19/CD20-CAR-T cells - Google Patents
Method for rapidly detecting activity of CD19/CD20-CAR-T cells Download PDFInfo
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Abstract
The invention belongs to the field of biological medicine, and provides an immune cell and a method for measuring the activity of the immune cell. The invention provides an engineering cell for detecting the activity of a CD19/CD20-CAR-T cell, wherein the cell is a tumor cell strain containing an NFAT-RE-luciferase carrier and contains a coding sequence of a CD19 antibody and/or a CD20 antibody. When the engineered cells are cultured together with cells expressing target antigens, the engineered cells of the invention stimulate the expression of fluorescent genes in the cells to generate fluorescence with different intensities, so that the activation condition of the cells after CD19/20 CAR-T transfection with different proportions is judged. The method is simple and convenient to operate, efficient and rapid, can rapidly and intuitively obtain the conditions of CD19/CD20 in the CD19/CD20CAR-T cells, and evaluates the construction conditions and cell activity of the CD19/CD20CAR-T cells.
Description
Technical Field
The invention belongs to the field of biological medicine, and relates to an immune cell and a method for measuring the activity of the immune cell. In particular, the invention provides a method for rapidly detecting the activity of CAR-T (CD 19/CD20-CAR-T cells) recognizing CD19 and CD20 and application thereof.
Background
Immunotherapy, which aims to enhance the patient's own immune system against disease, can be divided into two broad categories, active and passive. In recent years, various immunotherapeutic strategies aimed at inducing, enhancing or modifying T cell responses have become promising approaches for the treatment of cancer and autoimmune diseases. Immunotherapy plays an important role in the process of generating and developing tumors, not only can prevent the generation of cancers, but also can relieve and even treat the cancers by enhancing the immunity of the organism.
Adoptive cell therapy is one of the major research directions in cellular immunotherapy. Adoptive Cell Transfer (ACT), which is a passive immunotherapy of tumors, refers to the isolation of tumor-infiltrating lymphocytes (TILs) or peripheral blood lymphocytes from tumor patients, sorting, expansion, activation in vitro, and reinfusion back into the patient. ACT currently using peripheral blood lymphocytes includes lymphokine-activated killer cells (LAK), cytokine-induced killer Cells (CIK), dendritic cell-cytokine-induced killer cells (DC-CIK), anti-CD3 antibody-induced activated killer cells (anti-CD 3 antibody-induced activated killer cells, CD3-AK cells), and the like. However, CIK and the like are non-specifically activated lymphocytes and lack tumor specific reaction capability.
In this situation, scientists have begun to introduce a T Cell Receptor (TCR) or CAR gene recognizing a tumor antigen into lymphocytes through gene modification to make them TCR gene-modified T lymphocytes (TCR-T) or CAR-T cells, thereby providing them with tumor antigen target recognition ability. The identification and quality control of the activation activity of the CAR-T cells that are prepared during the preparation of CAR-T cell therapy is a crucial element. CAR-T cell therapy combines antibody technology to recognize and target disease, is a living drug that is not only not metabolized as traditional drugs, but also replicates in the patient or forms memory cells, and can last a long time with one administration. CAR-T cell therapy has many advantages over monoclonal antibody therapy, such as strong in vitro expansion capacity, long in vivo survival, efficacy against low expression of target molecules, etc. CAR-T cell therapy has been successfully applied in clinical trials for a variety of tumor diseases, including most hematological tumors, as well as solid tumors such as kidney, liver, and neural tumors.
The traditional anti-tumor method for detecting the effect comprises blood indexes, tumor body size, cell component analysis and the like, but the methods are usually time-consuming or belong to qualitative detection results and have unclear guidance effect.
Disclosure of Invention
The technical problem to be solved by the invention is to develop a biological method based on bioluminescent reporter genes, which can rapidly and accurately evaluate the activation and activity of CAR-T cells after the preparation of CD19 and CD20CAR-T cells is completed.
The invention provides an engineered cell for detecting the activity of a CD19/CD20-CAR-T cell. The cell is a tumor cell strain containing an NFAT-RE-luciferase carrier, the tumor cell strain containing the NFAT-RE-luciferase carrier contains a CD19 antibody and/or a CD20 antibody coding sequence, and NFAT-RE in the NFAT-RE-luciferase is connected with luciferase in series.
The tumor cells can be selected from various immortalized or commercialized cells, usually from ATCC or cell banks available from the life sciences division of Chinese academy of sciences, or can be modified for specific tumor or cancer types, for example, for hematological cancers, various corresponding suspended cancer cells can be used. However, not all tumor cells are suitable for the present invention, and in a preferred embodiment of the present invention, the tumor cells are K562 cells.
The NFAT (nuclear Factor of Activated T cells) transcription Factor family plays an important role in the expression of various cytokines in immune response, and also participates in various functions such as tumor immunity, myocardial function regulation, apoptosis and the like, and various signal pathways including calmodulin and protein kinase C can play a role through NFAT. The luciferase gene expressed by NFAT control is stably integrated in the genome of the NFAT-RE-Luci stable cell strain. Activation of NFAT initiates luciferase expression, which can sensitively reflect the state of the NFAT pathway by detecting luciferase activity.
CD19 is one of the cluster differentiation antigens and is an important membrane antigen involved in B cell proliferation, differentiation, activation and antibody production. CD19 is distributed on malignant B cells such as the whole body of B cells and hairy cell leukemia cells, and follicular dendritic cells, and is therefore the best marker for diagnosis of B cell line tumors (leukemia, lymphoma) and identification of B cells.
CD20 is expressed on the surface of B cells at various stages of developmental differentiation except plasma cells (immunoglobulin-secreting B cells), and plays an important regulatory role in B cell proliferation and differentiation by acting directly on B cells through the regulation of transmembrane calcium ion flux.
The sequences of CD19 and CD20 of the present invention are obtained by screening from various sources, for example, in a preferred embodiment of the present invention, sequences of CD19 and CD20 are obtained using phage selection techniques.
In a preferred embodiment of the invention, the amino acid sequence of the CD19 antibody is:
LEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRSPTGAVEVQVPEDPVVALVGTDATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPMTFPPEA（SEQ ID NO 1）。
the amino acid sequence of the CD20 antibody is:
GGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTS（SEQ ID NO 2）。
the NFAT-RE-luciferase-K562 cells of the invention are capable of recognizing CD19 and/or CD20 signals in the system.
The invention also provides a preparation method of the cell, wherein the coding sequence of the CD19 antibody and/or the CD20 antibody and the gene of luciferase are jointly recombined and expressed in a lentiviral plasmid or the coding sequence of the CD19 antibody and/or the CD20 antibody is inserted into the NFAT-RE-luciferase vector.
Preferably, the preparation method comprises the following steps:
(1) adding a transfection reagent into K562 suspension cells, fully and uniformly mixing, and incubating;
(2) after 2-6 hours, adding culture medium to dilute the transfection reagent or replace the basal medium;
(3) continuously culturing for 3-4 days, and carrying out passage or liquid change according to the growth condition of the cells;
(4) obtaining the cell strain which stably expresses NFAT-RE-luciferase K562.
Preferably, the mixture containing the transfection reagent and the cells to be transfected is centrifuged at a speed of 120g-200 g for 3-4 hours during transfection, so that certain cells which are difficult to transfect or have low transfection efficiency can obtain good transfection effect. Preferably, the centrifugation speed is 150g-180g, such as 160g or 170g, and the specific centrifugation rotation speed can be adjusted according to the centrifuge and the actual situation. Preferably, the temperature for centrifugation is 18-26 deg.C, more preferably 20-25 deg.C. For example, room temperature, 21 ℃, 22 ℃, 23 ℃, 24 ℃, etc. are used.
In another aspect, the invention provides a method of detecting CD19/CD20-CAR-T cell activity, comprising the steps of:
s1, culturing the cells to be tested in a mixture with tumor cells containing NFAT-RE-luciferase, and
s2, co-culturing for 2-6 hours, detecting the intensity of the fluorescence signal,
s3, judging the affinity of the antigen and the cell activity of CD19/CD20CAR-T according to different fluorescence intensities generated by tumor cells containing NFAT-RE-luciferase;
wherein, in a mixed system of the cells to be detected and tumor cells containing NFAT-RE-luciferase, the alpha CD 19-CAR: carrier or α CD 20-CAR: the Carrier ratio is 0: 10-10: 0, excluding 0: 10 or 10: 0.
Bioassays to assess CD19/CD20CAR-T cell activity based on bioluminescent reporter gene based bioassays were prepared in the present invention.
In the present invention, the CD19/CD20-CAR-T cells are CAR-T cells carrying CD19 and/or CD 20. Preferably, the test cell is a CAR-T cell or Raji cell carrying a CD19 antigen or a CD20 antigen.
Preferably, the co-culture time is 3-4 hours, or the fluorescence signal intensity of NFAT-RE-luciferase-containing tumor cells is measured by using a microplate reader.
The invention also relates to a mixture of cells, said cells comprising at least: NFAT-RE-luciferase-K562 cells and CD19/CD20-CAR-T cells.
Preferably, in the cell mixture, the ratio of α CD 20-CAR: the Carrier ratio is 1:9-1: 2. The ratio range of the α CD20-CAR and Carrier vector may be in mass percent or mass percent of the substance. More preferably, the ratio of the two components is 1: 8-1: 3, even 1: 5-1: 6, the particular preferred ratio is influenced by the cells used and the other components of the cell mixture.
The invention also provides the use of a method for detecting the activity of CD19/CD20-CAR-T cells, using the NFAT-RE (CD 19/20) -luciferase combination for the design and construction of CD19/CD20-CAR-T cells, and evaluating CD19/CD20CAR-T cell activity based on a bioluminescent reporter gene bioassay.
The invention develops a bioassay method for evaluating CD19/CD20CAR-T cell activation based on a bioluminescent reporter gene, wherein a lentivirus transfected K562 cell is used for constructing an engineering cell strain for stably expressing the reporter gene, and the engineering cell strain can transmit signals when being co-cultured with a cell expressing an antigen, so that the expression of fluorescent genes in the engineered K562 cell is excited, and fluorescence with different intensities is generated, thereby judging the cell activation condition after transfection of CD19/CD20CAR-T with different proportions. The method is simple and convenient to operate, efficient and rapid, and can be used for rapidly and intuitively obtaining the conditions of CD19/CD20 in the CD19/CD20CAR-T cells and evaluating the construction conditions and the cell activity of the CD19/CD20CAR-T cells.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a schematic diagram of the construction of a vector of the engineered cell line 1.
Wherein, NFAT-RE is connected with luciferase in series. NFAT-RE-luciferase-K562 is a stable transfectant cell line, and K562 cells that use NFAT-RE to drive luciferase expression respond to CD3 but not to CD28 stimulation.
FIG. 2 is a graph showing the results of different fluorescence intensities of the NFAT-RE-luciferase-K562 cell line for CD19/CD20 CAR-T.
Wherein, the detection results are divided into two groups of Raji cell co-culture group and Raji cell-free co-culture group. The ratio from left to right is α CD 19-CAR: carrier (Carrier) = 10:0, α CD 19-CAR: carrier = 3.3: 6.6, α CD 19-CAR: carrier = 1:9, α CD 19-CAR: carrier = 0: 10; α CD 20-CAR: carrier = 10:0, α CD 20-CAR: carrier = 3.3: 6.6, α CD 20-CAR: carrier = 1:9, α CD 20-CAR: carrier = 0: 10. raji cell co-culture groups, except for two 0: in group 10, the remaining groups showed a significant increase in fluorescence intensity compared to the Raji cell-free co-cultured group, and the CAR-T cells of the present invention were able to specifically recognize Raji cells (CD 19/CD 20). Four subgroups 10:0,3.3: 6.6,1: 9,0: 10, wherein the highest ratio of alpha CD20-CAR to Carrier is 10:0, up to about 9.8 x 105. Fluorescence intensity values of 8 subsets in the Raji cell co-culture group were about 4.8 x 10 from left to right5、2.7*105、1.2*105Slightly higher than the Raji-free cell co-cultured group, 9.8 x 105、4.3*105、1.9*105Slightly higher than the Raji-free cell co-culture group. As can be seen from FIG. 2, the NFAT-RE-luciferase-K562 cell line detected CD20 with a much stronger fluorescence intensity than CD 19.
Detailed Description
The technical solutions in the embodiments of the present application will be described clearly and completely below, and it should be understood that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
Example 1 construction of NFAT-RE-luciferase-K562 Stable transgenic cell line
NFAT-RE-luciferase-K562 effector cells are K562 cells that drive luciferase expression with NFAT-RE, and respond to CD3 but not to CD28 stimulation.
1) Construction and packaging of NFAT-RE-luciferase lentiviral plasmids
The sequence of the CD19/CD20 antibody in NFAT-RE was obtained for us by phage screening technology, expressed in lentiviral plasmid pMDL by genetic recombination based on engineering and luciferase as disclosed in the UNIPROT database, and packaged and harvested using 293 cells.
Amino acid sequence of CD19 antibody:
LEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRSPTGAVEVQVPEDPVVALVGTDATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPMTFPPEA(SEQ ID NO 1)。
amino acid sequence of CD20 antibody:
GGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTS(SEQ ID NO 2)。
2) recovery and culture of K562 cells:
recovering K562 cells, adding 1640 culture solution and 10% FBS, culturing with a 50ml culture bottle, and performing subculture when the cell confluence is 70% -80%.
3) Transfection of K562 cells:
(1) at 2X 105Polybrene to 6 mug/ml and proper amount of virus are added into K562 suspension cells and mixed well. Incubation was performed at 37 ℃. Or 150g for 4 hours at room temperature.
(2) After 4 hours, an equal volume of fresh medium was added to dilute the polybrene.
(3) The culture is continued for 3-4 days. The intermediate can be passaged or changed according to the growth condition of the cells.
(4) Obtaining the cell strain which stably expresses NFAT-RE-luciferase K562.
Example 2 NFAT-RE-luciferase-K562 stable transgenic cell line for assessing the activity of the CD19/CD20CAR-T receptor
1) Raji cell culture
Raji is a tumor cell highly expressing CD19 antigen. Raji cells were recovered from a liquid nitrogen tank, and were cultured in RPMI 1640 medium containing 10% newborn calf serum, 100U/ml penicillin, 100U/ml streptomycin, 2.5 g/L glucose, 0.11 g/L sodium pyruvate, and 1.5 g/L sodium bicarbonate at pH 7.2, 5% CO2, 37 ℃ and 90% relative humidity, for 2 d passaging.
2) Activity detection of NFAT-RE-luciferase-K562 stable transgenic cell strain
Mixing and culturing Raji cells and NFAT-RE-luciferase-K562 according to different proportions, detecting the fluorescence signal intensity of the K562 cells by using a microplate reader after co-culturing for 4 hours, and judging the affinity of the antigen and the quality of CD19/CD20CAR-T design according to different fluorescence intensities generated by the K562 cells.
The detection results are divided into two groups, namely a Raji cell co-culture group and a Raji cell-free co-culture group. In each group, the ratio of alpha CD19-CAR to Carrier from left to right is 10:0,3.3: 6.6,1: 9,0: 10; the ratio of α CD20-CAR to Carrier is also 10 in order: 0,3.3: 6.6,1: 9,0: 10. raji cell co-culture groups, except for two 0: in group 10, the remaining groups showed a significant increase in fluorescence intensity compared to the Raji cell free co-cultured group. The highest ratio of alpha CD20-CAR to Carrier is 10:0, up to about 9.8 x 105Secondly, the ratio of the alpha CD19-CAR to the Carrier is 10:0, up to about 4.8 x 105. 10:0,3.3: 6.6,1: 9,0: the fluorescence intensity of the 10 four subgroups decreased in order (FIG. 2).
The above description is only for the specific embodiments of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present disclosure should be covered within the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.
SEQUENCE LISTING
<110> Shanghai nanotechnology and applied national center for engineering research Ltd
<120> a method for rapidly detecting activity of CD19/CD20-CAR-T cells
<130> 20211127
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Claims (10)
1. An engineered cell for detecting the activity of a CD19/CD20-CAR-T cell, wherein the engineered cell is a tumor cell comprising an NFAT-RE-luciferase vector,
the tumor cell containing the NFAT-RE-luciferase carrier contains a coding sequence of a CD19 antibody and/or a CD20 antibody, and NFAT-RE in the NFAT-RE-luciferase is connected with luciferase in series.
2. The engineered cell for detecting CD19/CD20-CAR-T cell activity according to claim 1, wherein said tumor cell is a K562 cell.
3. The method for producing the cell according to claim 1 or 2, wherein the coding sequence of the CD19 antibody and/or CD20 antibody is expressed in a lentiviral plasmid by co-recombination with the gene for luciferase or the coding sequence of the CD19 antibody and/or CD20 antibody is inserted into the NFAT-RE-luciferase vector; the method comprises the following steps:
(1) adding a transfection reagent into K562 suspension cells, fully and uniformly mixing, and incubating;
(2) after 2-6 hours, adding culture medium to dilute the transfection reagent or replace the basal medium;
(3) continuously culturing for 3-4 days, and carrying out passage or liquid change according to the growth condition of the cells;
(4) obtaining the K562 cell strain stably transfected with NFAT-RE-luciferase.
4. The method of claim 3, wherein the CD19 antibody has the amino acid sequence:
LEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRSPTGAVEVQVPEDPVVALVGTDATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPMTFPPEA；
alternatively, the amino acid sequence of the CD20 antibody is:
GGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTS。
5. the method according to claim 3, wherein the mixture containing the transfection reagent and the cells to be transfected is centrifuged at a speed of 120g to 200 for 3 to 4 hours at the time of transfection.
6. A method of detecting CD19/CD20-CAR-T cell activity comprising the steps of:
s1, mixing the cells to be tested with the tumor cells containing NFAT-RE-luciferase in claim 1,
s2, co-culturing for 2-6 hours, detecting the intensity of the fluorescence signal,
s3, judging the affinity of the antigen and the cell activity of CD19/CD20-CAR-T according to the difference of the fluorescence intensity generated by tumor cells containing NFAT-RE-luciferase;
wherein the content of the first and second substances,
in a mixed system of the cells to be detected and tumor cells containing NFAT-RE-luciferase, the ratio of alpha CD 19-CAR: carrier or α CD 20-CAR: the Carrier ratio is 0: 10-10: 0, excluding 0: 10 or 10: 0.
7. The method of claim 6, wherein the test cell is a CAR-T cell or Raji cell carrying CD19 antigen or CD20 antigen;
alternatively, the co-cultivation time is 3-4 hours;
alternatively, the fluorescence signal intensity of tumor cells containing NFAT-RE-luciferase is detected using a microplate reader.
8. A mixture of cells, wherein said cells comprise at least:
NFAT-RE-luciferase-K562 cells, and
CD19/CD20-CAR-T cells.
9. The cell mixture of claim 8, wherein the ratio of α CD 20-CAR: the Carrier ratio is 1:9-1: 2.
10. Use of the method of detecting the activity of CD19/CD20-CAR-T cells according to claim 6, characterized in that NFAT-RE (CD 19/20) -luciferase combination is used for the design and construction of CD19/CD20-CAR-T cells, CD19/CD20CAR-T cell activity is assessed based on a bioluminescent reporter gene bioassay.
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