CN110819678A - Method for evaluating effectiveness of CART cells - Google Patents
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Abstract
The invention provides a method for evaluating the preclinical pharmacodynamics of CART cells. The system systematically expounds pharmacodynamics detection indexes of CART cells before entering clinic, and the evaluation is divided into in-vivo experiment evaluation and in-vitro experiment evaluation.
Description
Technical Field
The invention relates to the field of medical biology, and in particular relates to a preparation method of CART cells.
Background
Chimeric Antigen Receptor-T cell (CAR-T) T cell refers to a T cell that is genetically modified to recognize a specific Antigen of interest in an MHC non-limiting manner and to continuously activate expanded T cells. The international cell therapy association (interna) in 2012 indicates that biological immune cell therapy has become a fourth means for treating tumors besides surgery, radiotherapy and chemotherapy, and will become a necessary means for treating tumors in the future. CAR-T cell back-infusion therapy is the most clearly effective form of immunotherapy in current tumor therapy. A large number of studies show that the CAR-T cells can effectively recognize tumor antigens, cause specific anti-tumor immune response and remarkably improve the survival condition of patients.
Chimeric Antigen Receptors (CARs) are a core component of CAR-T, conferring on T cells the ability to recognize tumor antigens in an HLA-independent manner, which enables CAR-engineered T cells to recognize a broader range of targets than native T cell surface receptor TCRs. The basic design of a CAR includes a tumor-associated antigen (TAA) binding region (usually the scFV fragment from the antigen binding region of a monoclonal antibody), an extracellular hinge region, a transmembrane region, and an intracellular signaling region. The choice of antigen of interest is a key determinant for the specificity, efficacy of the CAR and safety of the genetically engineered T cells themselves.
Before clinical experiments, the CART cells need systematic evaluation of drug effect, pharmacokinetics, pharmacology and toxicology, and the safety and effectiveness of the CART cells are comprehensively researched. The present invention systematically describes a CART preclinical efficacy assessment method.
Disclosure of Invention
One aspect of the invention provides a method for assessing the effectiveness of CD19-CART cells, including in vitro assay assessment methods and in vivo assay assessment methods.
A second aspect of the invention provides an in vitro assay evaluation method comprising:
the method comprises the following steps of (I) detecting cell phenotype;
(II) CAR targeting assay;
(III) intracellular staining analysis of cytokines;
(IV) cell killing experiment analysis.
Preferably, the phenotypic detection of the cell comprises a CAR successfully infected with the virus+Cellular ratio, CD8/CD4 ratio, CD45RO/CD62L ratio.
Preferably, the CART cell targeting assay comprises an affinity assay of the CAR protein for tumor cells.
Preferably, the cytokine intracellular staining assay comprises INF γ and CD107a detection assays.
Preferably, the cell killing assay is a killing assay of tumor cells by CART cells.
A third aspect of the invention provides an in vivo experimental evaluation method comprising:
(I) an immunodeficient mouse lymphoma model;
(II) a normal immune mouse lymphoma model.
Preferably, the immunodeficient mouse is a NOG mouse.
Preferably, the normally immunized mouse is a Balb/c mouse.
Preferably, the immunodeficient mouse lymphoma model comprises a single dose injection of the CART cell model and a multiple dose injection of the CART cell model to assess the efficacy of CART cells on tumor cells in vivo.
The invention has the following beneficial effects:
the invention relates to a method for comprehensively evaluating the effectiveness of CART cells.
Drawings
FIG. 1 CART cell capable of remarkably prolonging survival time of tumor-bearing mice in vivo
Detailed Description
Examples
The present invention is described in further detail by referring to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Accordingly, the present invention should in no way be construed as limited to the following examples, but rather should be construed to include any and all variations which become apparent in light of the teachings provided herein.
Example 1: cell phenotype detection
1. Infected and uninfected cells were taken as 1X 10^6 suspensions, the cell suspensions were centrifuged to remove the medium, the cells were washed twice with PBS, 300g, and centrifuged at 4 ℃ for 5 minutes. The supernatant was discarded, the cells resuspended in PBS, placed in a flow-up tube, and labeled, with approximately 100. mu.l of cell suspension per sample (10)6A cell).
2. Appropriate amounts of CD3, CD4, CD8, CD45RO, CD62L, FITC-F (ab)' antibody (all antibodies from Biolegged) were added to each tube and incubated on ice protected from light for 15-20 minutes.
3. Cells were washed 1 time with 1ml PBS per tube, centrifuged at 300g for 5 minutes, and the supernatant carefully aspirated.
4. The test tube is tapped to mix the cells evenly, then a proper amount of 7-AAD dye is added, and the cells are incubated for 15 to 20 minutes on ice in a dark place.
5. Cells were washed 1 time with 1ml PBS per tube, centrifuged at 300g for 5 minutes, and the supernatant carefully aspirated.
6. Gently tap the tube and mix the cells, and resuspend the cells in 100-.
7. The data was detected and analyzed by an up-flow cytometer.
Example 2: CAR-targeted assays
1. The use of scFv antibodies with high affinity for the tumor target is critical in determining CAR-T cell function. CAR targeting assay, i.e. assay of the affinity of CART cells for tumor antigens.
2. The invention constructs PG13 cells (PG13-CD19) carrying CD19 overexpression genes. Affinity was determined with PG13-CD19 and Raji as CD19 positive cells.
Example 3: intracellular staining assay for cytokines
1. Taking prepared CAR-T cells, resuspending the CAR-T cells in Lonza culture medium, and adjusting the cell concentration to be 1 × 106/mL。
2. Experiment group each hole contains 2X 10 target cells or negative control cells52X 10 CAR-T/NT cells5200. mu.l of Lonza medium without IL-2. Mix well and add to 96-well plate. BD GolgiStop (containing monesin, 1. mu.l BD GolgiStop per 1ml of medium) was added thereto, and after mixing well, the mixture was incubated at 37 ℃ for 5 hours. Cells were collected as experimental groups.
3. Cells were washed 1 time with 1mL of PBS per tube and centrifuged at 300g for 5 minutes. The supernatant was discarded, and an appropriate amount of specific surface antibody CAR, CD3 was added to each tube, the volume of resuspension was 100ul, and incubation was carried out for 30 minutes on ice in the absence of light.
After washing the cells with PBS, 250. mu.l/EP tube Fixation/Permeabilization solution was added and incubated at 4 ℃ for 20 minutes to fix the cells and rupture the membranes. Using 1 XBD Perm/WashTMbuffer washes cells 2 times, 1 mL/time.
5. Staining with intracellular factor, taking appropriate amount of IFN-gamma cytokine fluorescent antibody or negative control, and performing BD Perm/WashTMbuffer diluted to 50. mu.l. Resuspending the fixed and disrupted cells thoroughly with the antibody dilution, incubating at 4 ℃ in the dark for 30min, 1 XBD Perm/WashTMbuffer 1 mL/wash cells 2 times, then use PBS heavy suspension.
6. Flow cytometry detected CAR, CD3, IFN- γ.
Example 4 cell killing assay
The K562 cells (negative control cells) were resuspended in serum-free medium (1640) at a cell concentration of 1X 106Perml, the fluorescent dye BMQC (2,3,6,7-tetrahydro-9-bromomethyl-1H,5Hquinolizino (9,1-gh) coumarins) was added to a final concentration of 5. mu.M.
2. Mixing, and incubating at 37 deg.C for 30 min.
3. Centrifugation was carried out at 1500rpm for 5min at room temperature, the supernatant was discarded and the cells resuspended in cytotoxic medium (phenol red-free 1640+ 5% AB serum) and incubated for 60min at 37 ℃.
4. Fresh cytotoxic Medium cells were washed twice and resuspended in fresh cytotoxic Medium at a density of 1X 106/ml。
Raji cells (containing CD19, as target cells) were suspended in PBS containing 0.1% BSA at a concentration of 1X 106/ml。
6. The fluorescent dye CFSE (fluorescent dye) (CFSE) was added to a final concentration of 1. mu.M.
7. Mixing, and incubating at 37 deg.C for 10 min.
8. After the incubation was completed, FBS in an equal volume to the cell suspension was added and incubated at room temperature for 2min to terminate the labeling reaction.
9. Cells were washed and resuspended in fresh cytotoxic medium at a density of 1X 106/ml。
10. Effector T cells were washed and suspended in cytotoxic medium at a concentration of 5X 106/ml。
11. In all experiments, cytotoxicity of CAR-infected effector T cells (CAR-T cells) was compared to that of uninfected negative control effector T cells (NT cells), and these effector T cells were from the same patient.
CAR-T and negative control effector T cells, according to T cell: the target cells were cultured in 5ml sterile test tubes (BD Biosciences) at a ratio of 5:1, 1: 1. In each co-culture group, the target cells were Raji cells (100,000 cells) (50. mu.l), and the negative control cells were K562 cells (100,000 cells) (50. mu.l). A panel was set up containing only Raj i target cells and K562 negative control cells.
13. The co-cultured cells were incubated at 37 ℃ for 5 h.
14. After incubation was complete, cells were washed with PBS and immediately followed by rapid addition of 7-AAD (7-aminoactomycin D) at the concentrations recommended by the instructions and incubation on ice for 30 min.
15. The Flow-type detection is directly carried out without cleaning, and the data is analyzed by Flow Jo.
16. Assay 7AAD negative viable cells were gated and the ratio of viable target cells to viable negative control cells was determined after co-culture of T cells and target cells.
The% cytotoxic killer cells was (1- (number of live target cells containing effector cells/number of live K562 cells containing effector cells)/(number of live target cells without effector cells/number of live K562 cells without effector cells)). 100%.
Example 5 in vivo model detection of killing effect of CART cells on tumor cells
NOG mice, female, 6 weeks old. The kit is divided into a solvent control group, a NT cell control group, a CAR-T low dose group, a CAR-T medium dose group and a CAR-T high dose group.
Nalm6 tumor cells, 3X 105And (4) injecting each cell/cell and tail vein, and establishing a blood system tumor model.
3. Three days later, the tail vein was injected with the corresponding solvent or CAR-T injection, and each mouse was administered with a volume of 200. mu.l. CAR-T Low dose group administration 1X 1065X 10 in the middle-dose group of CAR-T61X 10 cells/cell, CAR-T high dose group7One cell/one. The day of dosing was marked 0D.
4. The survival time of each group of mice was observed and a survival curve was plotted.
The results of this example are shown in the figure, the mean survival time of the vehicle control group is 24 days, the mean survival time of the NT control group is 27.5 days, and no significant difference exists between the two groups. The mean survival time of the CAR-T low dose group is 64 days, compared with that of the NT group, the mean survival time of the CAR-T group is prolonged by 133%, and the survival time of tumor model mice is remarkably prolonged by CAR-T cells (P < 0.0001). By 140 days after T cell reinfusion, 3 survived CAR-T medium dose groups and 1 survived CAR-T high dose groups. Still under continued observation.
Claims (10)
1. One method for assessing the effectiveness of CD19-CART cells includes in vitro assay assessment methods and in vivo assay assessment methods.
2. The in vitro assay of claim 1 wherein said assay comprises a cell phenotype assay, a CART cell targeting assay, a cytokine intracellular staining assay, a cell killing assay.
3. The in vivo experimental evaluation as claimed in claim 1, wherein said in vivo experimental evaluation comprises an immunodeficiency mouse lymphoma model and a normal immune mouse lymphoma model.
4. The cellular phenotype assay of claim 2 comprising a CAR successfully infected with a virus+Cellular ratio, CD8/CD4 ratio, CD45RO/CD62L ratio.
5. The CART cell targeting assay of claim 2, comprising an affinity assay of the CAR protein for tumor cells.
6. The cytokine intracellular staining assay of claim 2 comprising INF γ and CD107a detection assays.
7. The cell killing assay of claim 2, which is a killing assay of tumor cells by CART cells.
8. The immunodeficient mouse of claim 3 is a NOG mouse.
9. The normally immunized mouse of claim 3 is a Balb/c mouse.
10. The immunodeficient mouse lymphoma model according to claim 3 comprising a single dose injection of the CART cell model and a multiple dose injection of the CART cell model to assess the efficacy of the CART cell on tumor cells in vivo.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113109572A (en) * | 2021-04-01 | 2021-07-13 | 苏州才博医学科技有限公司 | Method for detecting somatic immunogenicity of autologous CART cells |
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Cited By (1)
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| CN113109572A (en) * | 2021-04-01 | 2021-07-13 | 苏州才博医学科技有限公司 | Method for detecting somatic immunogenicity of autologous CART cells |
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