Method for detecting biological activity of lentivirus infected T cells

Abstract

The invention relates to the technical field of biology, in particular to a method for detecting the biological activity of lentivirus infected T cells. The invention provides a method for detecting the biological activity of lentivirus infected T cells, which comprises the following steps: activating T cells, controlling the density of the activated T cells to be 1.0 * 10 < 6 >-2.0 * 10 < 6 > cells / mL, mixing the activated T cells with lentivirus containing a CAR gene, and culturing for 70-74 hours after mixing to obtain CAR-T cells; the method does not comprise liquid supplementing; mixing the obtained CAR-T cells with a CD3 antibody and a CAR antibody, and incubating for the first time; dyeing, incubating for the second time, and washing; and detecting the percentage of CD3 + CAR + cells. The method provided by the invention can accurately evaluate the biological activity of the lentivirus, has the advantages of strong specificity, high accuracy, good repeatability and stable and reliable detection result, and can be used for quality research, release detection, stability research, comparability research and the like of the lentivirus.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a method for detecting the biological activity of lentivirus-infected T cells. Background Art

[0002] Lentivirus is a viral vector modified from human immunodeficiency virus (HIV-1), which can efficiently introduce foreign genes into the host cell genome and achieve stable expression. Because it can infect both dividing and non-dividing cells, it has been widely used in cell and gene therapy. As a key vector, the biological activity of lentivirus directly determines whether the target gene can be effectively integrated into the host cell and stably expressed.

[0003] At present, the commonly used methods for detecting the biological activity of lentivirus are: 1) co-culture of lentivirus with 293T or Jurkat cell lines, and detection of CAR molecule expression level after lentivirus infection of target cells by flow cytometry and calculation of functional titer. This method can evaluate the content of virus with infection efficacy, but only represents the efficiency of lentivirus transduction of 293T or Jurkat cell lines; 2) p24 protein detection, using ELISA method to detect HIV-1p24 antigen content in lentivirus samples, this method can quickly detect the p24 content in lentivirus supernatant, but the lentivirus content includes empty shell viruses without infection activity, which cannot directly reflect the infection ability of lentivirus; 3) Real-time fluorescence quantitative PCR (RT-qPCR), lentivirus and 293T or Jurkat cell lines are co-cultured, and RT-qPCR is used to detect the lentivirus copy number in target cells. This method is simple to operate, but when measuring the target cell DNA titer, defects or other non-infectious particles and free-floating vector genomes in the supernatant are calculated at the same time, thereby overestimating the functional titer. Therefore, it is urgent to develop new methods for detecting the biological activity of lentivirus. Summary of the invention

[0004] In order to solve the defects of the above-mentioned prior art, the present application has developed a method for detecting the biological activity of lentivirus-infected T cells. By optimizing the preparation process of CAR-T cells in the detection method, the preparation time of CAR-T cells in the detection method is greatly shortened, and there is no need to calculate the titer of the lentivirus, thereby reducing the detection time of the biological activity of lentivirus-infected T cells, thereby being able to screen out lentiviruses with high infection activity. The method for detecting the biological activity of lentivirus-infected T cells of the present application can accurately evaluate the biological activity of lentivirus, has strong specificity, high accuracy, good repeatability, and stable and reliable detection results.

[0005] The first aspect of the present application provides a method for detecting the biological activity of lentivirus-infected T cells, comprising the following steps:

[0006] 1) Preparation of CAR-T cells;

[0007] 2) mixing the CAR-T cells obtained in 1) with CD3 antibody and CAR antibody and incubating them for the first time;

[0008] 3) staining and second incubation, washing;

[0009] 4) Detection of CD3 + CAR + The percentage of cells was used to evaluate the biological activity of lentivirus-infected T cells.

[0010] The preparation of CAR-T cells comprises the following steps: activating T cells, wherein the density of activated T cells is 1.0×10 6 ~2.0×10 6 cells / mL, the activated T cells were mixed with the lentivirus containing the CAR gene, and cultured for 70-74 hours to obtain CAR-T cells.

[0011] The culture medium used in preparing the CAR-T cells is a complete T cell culture medium, which contains the following components: 96% to 98% by volume of X-VIVO 15 culture medium, 2% to 4% by volume of serum replacement, and a final concentration of 200 to 250 IU / mL of IL-2.

[0012] The second aspect of the present application provides the use of the above-mentioned method for detecting the biological activity of lentivirus-infected T cells in lentivirus quality control and detection, or lentivirus vector production, or preparation of products for preventing and / or treating cancer, or preparation of T cell therapeutic drugs.

[0013] The beneficial effects of this application are:

[0014] In the prior art, the use of target cells such as 293T cells or Jurkat cells to detect the infection titer of lentivirus to detect the infection efficiency of lentivirus to cell lines cannot represent the infection efficiency of lentivirus to T cells. The present application provides a method for detecting the biological activity of lentivirus-infected T cells, which does not require the calculation of lentivirus titer and can directly reflect the infection ability and biological activity of lentivirus-infected T cells. By optimizing the preparation process of CAR-T cells during the detection process, the detection process is simplified as a whole, the detection time of detecting the biological activity of lentivirus to T cells is shortened, and there is no need to detect the titer. Only 5 days and 3 steps are required to complete the detection: D0 recovery and activation of T cells, D1 lentivirus transduction of T cells, D4 cell harvesting and detection, to achieve accurate detection of the biological activity of lentivirus-infected T cells in a shorter time, and on the premise of simplifying and optimizing the detection process, lentivirus can still achieve efficient infection of T cells, thereby accurately reflecting the biological activity of lentivirus-infected T cells. Through the method of detecting the biological activity of lentivirus-infected T cells in the present application, lentiviruses with the best infection activity and infection conditions can be screened. The method of this application has completed methodological validation, has strong specificity, high accuracy, good repeatability, and stable and reliable test results. It can be used for quality research, release testing, stability research, comparability research, etc. of lentiviruses, and has broad market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a schematic diagram of the optimization process in Example 1.

[0016] Figure 2 This is the “Live cells” gating logic used in Example 1.

[0017] Figure 3 The lentivirus transduced T cells CD3 at different dilutions in Example 8 + CAR + %.

[0018] Figure 4 It is the linear curve fitted by different dilution multiples in Example 8.

[0019] Figure 5 CD3 detected at D4 in Example 1 + CAR + Detection data.

[0020] Figure 6 CD3 detected by groups 1 and 2 at D7 in Example 1 + CAR + Detection data.

[0021] Figure 7 CD3 detected by groups 3 and 4 at D7 in Example 1 +CAR + Detection data.

[0022] Figure 8 This is the specificity detection result in Example 4.

[0023] Fig. 9 This is the accuracy test result in Example 5.

[0024] Fig.10 This is the repeatability test result in Example 6.

[0025] Fig.11 This is the intermediate precision test result in Example 7.

[0026] Fig.12 This is the linear detection result in Example 8. DETAILED DESCRIPTION

[0027] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0028] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0029] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0030] In the present application, the biological activity of lentivirus-infected T cells refers to the ability of lentiviral vectors to infect T cells, which describes the ability of lentiviral vectors to infect target T cells and integrate exogenous genes into the host cell genome.

[0031] The present application first provides a method for detecting the biological activity of a lentivirus, comprising the following steps:

[0032] 1) Preparation of CAR-T cells;

[0033] 2) mixing the CAR-T cells obtained in 1) with CD3 antibody and CAR antibody and incubating them for the first time;

[0034] 3) staining and second incubation, washing;

[0035] 4) Detection of CD3 + CAR + The percentage of cells was used to evaluate the biological activity of lentivirus-infected T cells.

[0036] In a specific embodiment of the present application, in step 1), the preparation of CAR-T cells comprises the following steps: activating T cells, adjusting the density of activated T cells to 1.0×10 6 ~2.0×10 6 cells / mL, the activated T cells were mixed with the lentivirus containing the CAR gene, and cultured for 70-74 hours to obtain CAR-T cells.

[0037] The T cells, namely T lymphocytes, are a key white blood cell in the immune system, mainly responsible for cellular immune responses, including autologous T cells, allogeneic (allogeneic) T cells, memory T cells, γδT cells, regulatory T cells, etc.

[0038] The activated T cells refer to T lymphocytes that have been activated by specific antigen stimulation. T cells can be activated by the following methods, such as antigen recognition: T cells recognize antigen peptide fragments presented by major histocompatibility complex (MHC) molecules on the surface of antigen presenting cells (APCs) through their T cell receptors (TCR); co-stimulatory signals: T cell activation requires co-stimulatory signals, such as the interaction between CD28 and B7 molecules (CD80 / CD86); CD3 / CD28 antibody activation: using anti-CD3 and anti-CD28 antibodies to jointly stimulate T cells, simulating the dual signal effect of T cell activation in vivo; magnetic bead method: using CD3 / CD28 antibody-coupled magnetic beads to physically bind T cells to antibodies and activate T cells; multiple Polymer method: Streptamer polymers coupled to CD3 / CD28 antibodies are used to transmit activation signals through multivalent binding; Addition of cytokines: Cytokines such as IL-2 can be used in combination with CD3 / CD28 antibodies to promote the activation and proliferation of T cells; Dynabeads technology: Use magnetic beads with antibodies (such as Dynabeads) to activate T cells and provide physiological activation; Chemical induction: Use chemicals such as phorbol ester 12-myristate 13-acetate (PMA) and ionomycin to induce T cell activation; Artificial antigen presenting cells: Under laboratory conditions, artificial antigen presenting cells can be used to activate T cells.

[0039] In a specific embodiment of the present application, in step 1), the method for activating T cells is: suspending T cells with T cell complete culture medium to prepare a T cell suspension, mixing the T cell suspension with a T cell activator at a volume ratio of 90 to 110:1, and activating at a temperature of 36 to 38° C. and a carbon dioxide concentration of 4.5 to 5.5% for 22 to 26 hours.

[0040] The T cell activator can be phytohemagglutinin, anti-CD3 monoclonal antibody, anti-CD28 monoclonal antibody, phorbol ester (PMA) and ionomycin (Ionomycin), Dynabeads, cytokines, etc. In a specific embodiment of the present application, the T cell activator can be the T cell Transact product of Miltenyi Biotec, which is a new type of T cell activation reagent that couples humanized CD3 and CD28 antibodies on a nanoscale polymer matrix.

[0041] In a specific embodiment of the present application, in step 1), the amount of T cell activator added can be a volume ratio of T cell suspension to T cell activator of 90 to 110:1, for example, it can be 90:1, 92:1, 94:1, 96:1, 98:1, 100:1, 102:1, 104:1, 106:1, 108:1, 110:1.

[0042] In a specific embodiment of the present application, in step 1), the activation temperature can be 36-38°C, for example, 36.0°C, 36.5°C, 37.0°C, 37.5°C, 38.0°C.

[0043] In a specific embodiment of the present application, in step 1), the concentration of carbon dioxide during activation may be 4.5-5.5%, for example, 4.5%, 5.0%, or 5.5%.

[0044] In a specific embodiment of the present application, in step 1), the activation time can be 22 to 26 hours, for example, 22 hours, 23 hours, 24 hours, 25 hours, or 26 hours.

[0045] In a specific embodiment of the present application, in step 1), the activated T cells are suspended in a complete T cell culture medium, and the density of the activated T cells is adjusted.

[0046] In a specific embodiment of the present application, in step 1), the density of activated T cells can be adjusted to 1.0×10 6 ~2.0×10 6 cells / mL, for example, 1.0×10 6 cells / mL,1.2×10 6 cells / mL,1.4×10 6 cells / mL,1.6×10 6 cells / mL,1.8×10 6 cells / mL,2.0×10 6 cells / mL.

[0047] In a preferred embodiment of the present application, in step 1), the density of the activated T cells can be adjusted to 1.0×10 6 cells / mL, after optimization in this application, only 1.0×10 6 Cells / mL can complete efficient lentiviral infection of T cells.

[0048] In the present application, the lentivirus is a lentivirus containing a CAR gene, which is transduced into T cells via the lentivirus and expressed on the surface of T cells. In a certain embodiment of the present application, the CAR gene is a CD19 CAR gene.

[0049] The CAR gene refers to a gene encoding a chimeric antigen receptor (CAR), which is a special protein that usually includes an antigen binding domain, a hinge region, a transmembrane domain, and a signal transduction domain. It is designed to redirect the immune response of T cells (a type of immune cell) to specific antigens. This genetically engineered T cell can recognize and attack cells that express specific antigens, such as cancer cells.

[0050] The preparation method of the lentivirus containing the CAR gene can be conventional and adjusted, and it is only necessary to make the lentivirus carry the required CAR gene. For example, the following steps may be included: vector design: design a lentiviral vector containing the CAR gene, and the lentiviral vector usually contains long terminal repeats (LTRs), a packaging signal (Ψ), a reverse transcriptase coding sequence, and necessary promoter and enhancer elements; plasmid preparation: prepare three main plasmids: a transfer plasmid (containing the CAR gene and LTRs), a packaging plasmid (encoding viral proteins such as gag / pol), and an envelope plasmid (usually encoding VSV-G protein); cell culture, using HEK293T cells for virus packaging; transient transfection, co-transfecting the above three plasmids (transfection reagents such as calcium phosphate, polyethyleneimine or liposomes can be used) into HEK293T cells to produce lentiviral particles, virus collection and concentration, and after transfection, collecting the culture supernatant containing the lentiviral particles, and clarifying, concentrating and purifying.

[0051] In certain embodiments of the present application, the lentivirus may be a lentivirus stock solution.

[0052] In other embodiments of the present application, the lentivirus may be a lentivirus suspension diluted with a complete T cell culture medium, and the dilution may be a gradient dilution.

[0053] In a specific embodiment of the present application, the dilution multiple is 80 to 1280 times, by setting a series of lentiviral gradient dilutions and passing through CD3 + CAR + The percentage results showed that the appropriate dilution factor of the lentivirus was selected. When the lentivirus suspensions at all dilution factors infected T cells, higher CD3 + CAR + If the percentage of cells infected with the lentivirus is higher than that of infected T cells, the lentivirus has a higher biological activity of infecting T cells.

[0054] In a specific embodiment of the present application, in step 1), the ratio of the volume of the lentiviral stock solution or lentiviral suspension to the volume of the activated T cell suspension can be 0.8-1.2:1, for example, it can be 0.8:1, 0.9:1, 1.0:1, 1.1:1, 1.2:1; under this condition, it is beneficial to improve the efficiency of lentiviral transduction of T cells.

[0055] In a specific embodiment of the present application, in step 1), the activated T cell suspension is mixed with a lentivirus or lentivirus suspension containing a CAR gene, and the mixture is cultured for 70 to 74 hours to obtain CAR-T cells. The culture time can be, for example, 70 hours, 71 hours, 72 hours, 73 hours, or 74 hours. After optimization of the present application, it is found that a higher CAR positivity rate can be obtained with a culture time of 72 hours ± 2 hours (D4 sampling) compared with a conventional culture time of 144 hours ± 2 hours (D7 sampling). This indicates that a culture time of 72 hours ± 2 hours is more conducive to lentivirus infection of T cells, can achieve high lentivirus infection of T cells, optimizes and simplifies the detection process, and shortens the detection time for detecting the biological activity of lentivirus-infected T cells.

[0056] In a specific embodiment of the present application, in step 1), the culture temperature can be 36-38°C, for example, 36°C, 36.5°C, 37°C, 37.5°C, 38°C.

[0057] In a specific embodiment of the present application, in step 1), the carbon dioxide concentration during the culture may be 4.5-5.5%, for example, 4.5%, 5.0%, or 5.5%.

[0058] In the specific embodiment of the present application, in step 1), the method for preparing CAR-T cells does not include fluid replenishment, which means that fluid replenishment is not included in the entire process of preparing CAR-T cells. Fluid replenishment means that during the cell culture process, due to the consumption of nutrients and the accumulation of metabolic waste, fresh culture medium or specific nutrients need to be added to the culture medium regularly or as needed to maintain the growth environment and activity of the cells. The present application found that fluid replenishment or no fluid replenishment has a significant effect on CD3 + CAR + The positive rate was not significantly affected; direct rehydration or rehydration after centrifugation had no significant effect on CD3 + CAR + The positive rate was not significantly affected; the present application simplifies the culture process, and can still obtain a high CAR positive rate without the need for fluid replacement, thereby achieving efficient lentivirus infection of T cells, and optimizes and simplifies the detection process, shortening the detection time for detecting the biological activity of lentivirus-infected T cells.

[0059] Through the above-mentioned optimization and simplification of the steps for preparing CAR-T cells, the detection time for detecting the biological activity of T cells infected with lentivirus is shortened. Under the premise of simplifying the steps and shortening the detection time, the lentivirus can still efficiently infect T cells, thereby accurately reflecting the biological activity of T cells infected with lentivirus, and making the detection method for detecting the biological activity of T cells infected with lentivirus have high accuracy, repeatability and precision.

[0060] In this application, the above-mentioned T cell complete medium refers to a medium specifically used to support the growth, expansion and maintenance of T cells, including basal culture medium, serum, cytokines, antibiotics and other necessary supplements, such as RPMI-1640 complete culture medium, X-VIVO series complete culture medium, GIBCO OpTmizer TM CTS TM T cell expansion SFM culture medium and serum-free culture medium. In a specific embodiment of the present application, the T cell complete culture medium X-VIVO 15 complete culture medium comprises the following components: 96-98% by volume of X-VIVO 15 culture medium, 2-4% by volume of serum replacement, and IL-2 with a final concentration of 200-250 IU / mL, all of which can be obtained through conventional channels. In the present application, the culture medium is optimized, the components of the culture medium are simplified, and costs are saved.

[0061] In a specific embodiment of the present application, in step 2), the CD3 antibody refers to a monoclonal antibody against the CD3 molecule on the surface of T cells. The CD3 antibody can be prepared and synthesized based on a known amino acid sequence, or it can be an existing commercial product.

[0062] In a specific embodiment of the present application, in step 2), the CAR antibody can specifically recognize and bind to CARs for different targets, and the different targets include but are not limited to CD19, CD20, CD22, CD33, CD123, BCMA, HER2, EGFR, MUC1, GD2, PLL3, GPC3, B7H3, IL-13Rα2, GPRC5D, CD7, MSLN, Claudin 18.2, CEA, PSMA, CLL1, and the CAR that can specifically recognize and bind to different targets refers to the ability to recognize and bind to a specific part of the CAR molecule (e.g., scFv in CAR), and the CAR antibody includes but is not limited to anti-CD19 CAR antibody, anti-CD20 CAR antibody, anti-CD22 CAR antibody, anti-CD33 CAR antibody, anti-CD123 CAR antibody, anti-BCMA CAR antibody, anti-HER2 CAR antibody, anti-EGFR CAR antibody, anti-MUC1 CAR antibody, anti-GD2 CAR antibody, anti-DLL3 CAR antibody, anti-GPC3 CAR antibody, anti-B7H3 CAR antibody, anti-IL-13Rα2CAR antibody, anti-GPRC5D CAR antibody, anti-CD7 CAR antibody, anti-MSLN CAR antibody, anti-Claudin18.2CAR antibody, anti-CEA CAR antibody, anti-PSMA CAR antibody, anti-CLL1 CAR antibody, for example, the anti-CD19 CAR antibody can be FMC63 antibody, which specifically targets the FMC63 scFv antigen recognition epitope, recognizes and binds to the anti-CD19 CAR constructed based on FMC63 scFv.

[0063] In one embodiment of the present application, the CAR antibody is an anti-CD19 CAR antibody, specifically an anti-FMC63 antibody.

[0064] Based on the fact that the lentiviral vector exemplified in the present application contains the CD19 CAR gene, and the corresponding anti-CD19CAR antibody (FMC63) is used for detection to accurately detect the activity of the lentivirus, it can be expected that when the lentivirus contains other CAR genes, the anti-CAR antibody corresponding to the CAR gene in the lentivirus is used to detect the activity of the lentivirus, which can also achieve the effect of the present application, that is, as long as the anti-CAR antibody has a corresponding relationship with the CAR expressed by the CAR gene and can specifically recognize and bind to the CAR expressed by the corresponding CAR gene, other anti-CAR antibodies are also included in the protection scope of the present application.

[0065] In a specific embodiment of the present application, in step 2), the CD3 antibody and CAR antibody carry a marker, and the marker can be fluorescein, enzyme, or biotin. The CD3 antibody and CAR antibody with a marker can be obtained by covalently linking the marker to the antibody; for example, a ready-made direct-labeled antibody can be directly used, and the direct-labeled antibody refers to a primary antibody that has been directly coupled to fluorescein or other visual markers (such as enzymes, biotin, etc.). For CD3 antibodies, direct-labeled antibodies can be antibodies that are directly bound to fluorescein or other markers such as FITC, PE, CY3, CY5, PE, etc.; for CAR antibodies, it refers to antibodies that have been directly coupled to fluorescein or other visual markers, which can recognize different parts of CAR, including antigen binding sites, light chains, or hinge regions, etc., without the need for additional secondary antibodies; intermediate-labeled antibodies can also be used.

[0066] In a specific embodiment of the present application, in step 2), after adding CD3 antibody and CAR antibody, the incubation temperature for the first incubation can be 2-8°C, for example, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C. The incubation time for the first incubation can be 15-20 minutes, for example, 15min, 16min, 17min, 18min, 19min, 20min.

[0067] In the present application, CD3 antibodies are used to identify markers on the surface of T cells, CAR antibodies are used to identify CAR expressed on the surface of T cells after being transduced into T cells by lentivirus, and the fluorescent signals of markers on CD3 and CAR antibodies are analyzed by flow cytometry to obtain the expression level of CAR on the surface of CAR-T cells to evaluate the biological activity of the lentivirus.

[0068] In a specific embodiment of the present application, in step 3), after the first incubation is completed, a dye is added to stain the T cells, and the dye is a viability dye for distinguishing live cells from dead cells. The viability dye can be propidium iodide, 7-AAD, SYTOX series dyes, LIVE / DEAD Fixable series dyes, Fixable Viability Stain 780. In certain embodiments of the present application, the dye is Fixable Viability Stain 780.

[0069] In a specific embodiment of the present application, in step 3), a second incubation is performed after the dye is added, and the temperature of the second incubation can be 2 to 8° C., for example, 2° C., 3° ​​C., 4° C., 5° C., 6° C., 7° C., 8° C. The incubation time of the second incubation can be 15 to 20 minutes, for example, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min.

[0070] In a specific embodiment of the present application, the washing includes adding PBS to mix, and discarding the supernatant after centrifugation, which is used for unbound CD3 antibodies and CAR antibodies and excess dyes, etc.

[0071] In a specific embodiment of the present application, the washing can be performed more than twice to ensure that unbound CD3 antibodies and CAR antibodies and excess dyes are completely washed away.

[0072] In a specific embodiment of the present application, the centrifugal speed can be 300-400g, for example, 300g, 320g, 340g, 360g, 380g, 400g; the centrifugal time can be 5-10 minutes, for example, 5min, 6min, 7min, 8min, 9min, 10min.

[0073] In a specific embodiment of the present application, the CD3 + CAR + It means that both are positive at the same time, that is, cells expressing CD3 and CAR are detected at the same time, which are T cells expressing CAR after being infected with lentivirus.

[0074] The present application also provides the use of the above method in lentivirus quality control and detection, lentiviral vector production, and preparation of T cell therapeutic drugs.

[0075] The lentiviral quality control and testing refers to a series of tests and verifications to evaluate and monitor the quality, safety, purity, activity, potency and stability of the viral vector during the production process of the lentiviral vector, which may include release testing, a series of tests performed on the final product during the lentiviral production process to ensure that the product meets the predetermined quality standards and safety requirements.

[0076] The lentiviral vector production refers to the process of preparing lentiviral vectors using bioengineering technology, and quality testing is performed at each stage of the production process to ensure the quality of the viral vector.

[0077] The preparation of T cell therapy products refers to the process of modifying T cells using genetic engineering technology, such as using gene delivery systems such as lentiviral vectors to introduce specific chimeric antigen receptors (CARs) or other genes into T cells, enabling them to specifically recognize and attack specific targets (such as tumor cells or virus-infected cells). During the preparation process, the transduction efficiency of the lentiviral vector is tested.

[0078] The present application is further described below by way of examples, but the scope of the present application is not limited thereby. Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields conventional in the art. These techniques have been fully described in the existing literature. Unless otherwise stated, the materials, instruments, reagents, etc. used in the examples can be obtained by conventional means.

[0079] Reagent preparation:

[0080] Preparation of IL-2 stock solution: Add IL-2 powder to 1 mL of 0.9% sodium chloride injection to prepare an IL-2 stock solution with an initial concentration of 200,000 IU / mL. After fully dissolved, divide the solution into 120 μL / tube and store it at -20°C.

[0081] X-VIVO 15 complete medium preparation: 98mL X-VIVO 15 medium + 2mL SR (CTS TM Immune cell serum replacement) + 0.1mL IL-2 (final concentration 200IU / mL).

[0082] 10-fold dilution of the dead and viable dye: Take out the Fixable Viability Stain 780 dye stock solution from the -20℃ refrigerator, wait for it to melt, dilute the 10× Fixable Viability Stain 780 dye stock solution to 1× with flow cytometry solution (PBS), mix well by pipetting, and place in a 2-8℃ refrigerator for later use.

[0083] The lentivirus used in the embodiment is: a lentivirus carrying the CAR (comprising a single-chain antibody variable region targeting the antigen CD19, a CD8 transmembrane region, a 4-1BB co-stimulatory domain, and a CD3ζ signal transduction domain) gene, which is obtained by conventional lentiviral vector construction (inserting the CAR gene into the transfer plasmid of the lentiviral vector) and lentiviral packaging (in cell culture, using packaging cells (such as HEK293T cells) to co-transfect the transfer plasmid containing the CAR gene, the packaging plasmid and the envelope plasmid to produce lentiviral particles carrying the CAR gene).

[0084] Example 1 Optimization of lentivirus biological activity detection conditions

[0085] This example optimizes the process of lentiviral infection of T cells, including: 1) the cell plating amount of T cells activated by D1 lentiviral transduction (1×10 5 cells / well, 2×10 5 cells / well, 4×10 5cells / well); 2) the time point of rehydration (rehydration on D2 or no rehydration) and the method of rehydration (direct rehydration on D4 or rehydration after centrifugation); 3) the total infection time (72±2 hours (sample collection on D4) or 144±2 hours (sample collection on D7)).

[0086] Experimental methods:

[0087] 1.D0: Recovery and activation of cryopreserved T cells

[0088] (1) Turn on the electric constant temperature water bath in advance, take out the frozen T cells from the liquid nitrogen tank, transfer them to the water bath, wait until the temperature of the water bath stabilizes at 37°C, take out the cells and start to thaw (shake the cryopreserved tube while thaw), and take them out when the cells in the cryopreserved tube are about to melt completely (only a small amount of ice crystals remain). Wipe the surface of the cryopreserved tube with a dust-free cloth and wipe and disinfect it, then transfer it to the biological safety cabinet. Use a pipette to carefully transfer the thawed cells to complete culture medium, centrifuge at 300g for 5 minutes, discard the supernatant, add X-VIVO 15 complete culture medium to resuspend evenly, take 0.3-0.5mL of the cell suspension into a 1.5mL centrifuge tube, and count the cells according to the "QC1062 Cell Counter (NC-200) Use, Maintenance and Operation Procedures".

[0089] (2) According to the counting results, the T cell density was adjusted to 1.0×10 6 cells / mL, transfer the cell suspension to a culture flask, add T cell Transact at a volume ratio of 100:1 for activation, gently mix after addition, place the culture flask in a 37.0±1℃, 5.0±0.5% carbon dioxide incubator, and perform virus infection 24±2 hours after activation.

[0090] 2.D1: Lentiviral transduction

[0091] (1) The activated T cells were centrifuged at 300 g for 5 minutes, the cell pellet was resuspended in complete medium and the cells were counted. The cell density was adjusted to 1×10 based on the counting results. 6 cells / mL, 2×10 6 cells / mL、4×10 6 cells / mL.

[0092] (2) Prepare four 96-well flat-bottom cell culture plates (Group 1, Group 2, Group 3, and Group 4). Add 100 μL of 1×10 6 cells / mL, 2×10 6 cells / mL and 4×10 6 cells / mL cell suspension (i.e., the number of cells in each well was 1×10 5 cells, 2×10 5cells, 4×10 5 cells), Group 2, Group 3, and Group 4 were the same as Group 1, and each of Groups 1 to 4 contained the above three cell densities).

[0093] (3) Take out the lentivirus from the -80℃ freezer and place it in a 4℃ ice box to thaw. After the lentivirus is thawed, dilute the lentivirus in a gradient manner using X-VIVO 15 complete medium.

[0094] (4) After dilution, add 100 μL of lentiviral stock solution or diluent to each well of the experimental group, and add 100 μL of X-VIVO 15 complete medium to each well of the control group. Set up 2 parallel wells for each dilution.

[0095] 3.D2: Fluid rehydration

[0096] (1) Group 1 did not receive any fluid replacement on the second day.

[0097] (2) The 96-well flat-bottom cell culture plates of Group 2, Group 3, and Group 4 were removed from the incubator, centrifuged at 300 g for 5 minutes, 100 μL of supernatant was discarded, and 100 μL of complete culture medium was added to resuspend the cells. After completion, the plates were placed in an incubator at 37.0±1°C and 5.0±0.5% carbon dioxide for further culture.

[0098] 4.D4: Fluid refill and sampling test

[0099] (1) Take out the 96-well flat-bottom cell culture plate of "Group 1" from the CO2 incubator, mix it by pipetting, and take 100 μL of the cell suspension to detect CD3 + CAR + (%), add 150 μL complete culture medium to the remaining cell suspension, mix well and place it back in a 37.0±1°C, 5.0±0.5% carbon dioxide incubator for further culture.

[0100] (2) Take out the 96-well flat-bottom cell culture plate of "Group 2" from the CO2 incubator, mix it by pipetting, and take 100 μL of the cell suspension to detect CD3 + CAR + (%), add 150 μL complete culture medium to the remaining cell suspension, mix well and place it back in a 37.0±1°C, 5.0±0.5% carbon dioxide incubator for further culture.

[0101] (3) Take out the 96-well flat-bottom cell culture plate of "Group 3" from the CO2 incubator, mix it by pipetting, and take 50 μL of the cell suspension for cell counting. According to the counting results, adjust the cell density of the remaining cell suspension to 1.0×10 6 cells / mL, mix well and place back in a 37.0±1℃, 5.0±0.5% carbon dioxide incubator for further culture.

[0102] (4) Centrifuge the 96-well flat-bottom cell culture plate of "Group 4" at 300 g for 5 minutes, discard 100 μL of supernatant, add 150 μL of complete culture medium to resuspend the cells, mix well by pipetting, and then place in a 37.0±1°C, 5.0±0.5% carbon dioxide incubator for continued cultivation.

[0103] (5)CD3 + CAR + (%) Detection

[0104] Turn on the flow cytometer: Follow the startup process and complete the quality control procedure in accordance with the "AD1004 MACS Quant 10 Flow Cytometer Use, Maintenance and Maintenance Standard Operating Procedures". After the quality control is completed, the machine will display "PASS" and can be used normally.

[0105] The cells were collected into flow cytometry tubes, 1 mL of PBS was added to each tube, vortexed and mixed, and then centrifuged at 300 g for 5 minutes, and the washing was repeated once.

[0106] Discard the supernatant and add 100 μL PBS to each tube.

[0107] Antibody incubation was performed using CD3 direct-labeled antibody and CAR direct-labeled antibody FMC63, and both antibodies were added to each experimental tube and control tube.

[0108] Incubation: After adding the antibody, shake to mix and incubate in a dark place at 2-8℃ for 15 minutes.

[0109] After incubation, add 1 μL 1× Fixable Viability Stain 780 to each tube for staining, mix well, and place in a 2-8°C refrigerator incubated in the dark for 15 minutes.

[0110] After incubation, add 1 mL of PBS to each tube, shake to mix, and centrifuge at 300 g for 5 min.

[0111] Discard the supernatant, add 1 mL of PBS to each tube, shake to mix, and centrifuge at 300 g for 5 minutes.

[0112] Discard the supernatant, add 200 μL PBS to each tube, and vortex to mix.

[0113] Set under the "Live cells" circle to collect about 10,000 events.

[0114] The logic of the circle is as follows Figure 2 shown.

[0115] 5. Data Analysis

[0116] CD3 + CAR+ (%) = (Experimental tube 1: CD3 + CAR + (%) + Experimental tube 2: CD3 + CAR + (%)) / 2

[0117] When the positive rate of the control group is less than 2%, the experimental results are valid.

[0118] 4.D7: Sampling and testing

[0119] (1) Take out four sets of 96-well flat-bottom cell culture plates from the CO2 incubator, mix them by pipetting, and transfer the cell suspension to a flow cytometer for CD3 + CAR + (%) detection.

[0120] (2) CD3 + CAR + (%) detection, compared with the above D4 "CD3 + CAR + (%) detection" is the same.

[0121] Experimental results:

[0122] Optimize the experimental process such as Figure 1 shown.

[0123] At D4, CD3 + CAR + Test data such as Figure 5 As shown. At D7, CD3 + CAR + Test data such as Figure 6 and Figure 7 shown.

[0124] Figure 5 , Figure 6 , Figure 7 The test results showed that: 1) When the number of T cells increased after lentiviral transduction and activation, the lentivirus had an effect on the CD3 + CAR + The positive rate did not increase; 2) Whether or not to replenish fluid on D2 had any effect on CD3 + CAR + The positive rate was not significantly affected; the rehydration method of direct rehydration or rehydration after centrifugation at D4 had no significant effect on CD3 + CAR + The positive rate was not significantly affected; 3) From the analysis of the total infection duration, the CD3 + CAR + The positive rate was lower than that of CD3 collected on D4 + CAR+ .

[0125] In summary, the experimental conditions of the present application are as follows: 1) The number of T cells activated by D1 lentiviral transduction is 1×10 5 cells / well; 2) no "replenishment" operation was performed on D2 during transduction; 3) the total infection time was (72±2 hours (sample collection on D4)).

[0126] Example 2 Detection of lentiviral biological activity

[0127] Based on the condition optimization of Example 1, this example provides a method for detecting the biological activity of T cells infected by a lentivirus.

[0128] 1.D0: Recovery and activation of cryopreserved T cells

[0129] (1) Turn on the electric constant temperature water bath in advance, take out the frozen T cells from the liquid nitrogen tank, transfer them to the water bath, wait until the temperature of the water bath stabilizes at 37°C, take out the cells and start to thaw (shake the cryopreserved tube while thaw), and take them out when the cells in the cryopreserved tube are about to melt completely (only a small amount of ice crystals remain). Wipe the surface of the cryopreserved tube with a dust-free cloth and wipe and disinfect it, then transfer it to the biological safety cabinet. Use a pipette to carefully transfer the thawed cells to complete culture medium, centrifuge at 300g for 5 minutes, discard the supernatant, add X-VIVO 15 complete culture medium to resuspend evenly, take 0.3-0.5mL of the cell suspension into a 1.5mL centrifuge tube, and count the cells according to the "QC1062 Cell Counter (NC-200) Use, Maintenance and Operation Procedures".

[0130] (2) According to the counting results, the T cell density was adjusted to 1.0×10 6 cells / mL, transfer the cell suspension to a culture flask, add T cell Transact at a volume ratio of 100:1 for activation, gently mix after addition, place the culture flask in a 37.0±1℃, 5.0±0.5% carbon dioxide incubator, and perform virus infection 24±2 hours after activation.

[0131] 2.D1: Lentiviral transduction

[0132] (1) The activated T cells were centrifuged at 300 g for 5 minutes, the cell pellet was resuspended in X-VIVO 15 complete medium, and the cells were counted. The cell density was adjusted to 1.0×10 based on the counting results. 6 cells / mL.

[0133] (2) Prepare a 96-well flat-bottom cell culture plate and add 100 μL of the mixed cell suspension to each well (i.e., the number of cells per well is 1.0×10 5 cells).

[0134] (3) Take out the lentivirus from the -80℃ freezer and place it in a 4℃ ice box to thaw. After the lentivirus is thawed, dilute the lentivirus in a gradient manner using X-VIVO 15 complete medium.

[0135] (4) After dilution, add 100 μL of lentiviral stock solution or diluent to each well of the experimental group, and add 100 μL of X-VIVO 15 complete medium to each well of the control group. Set up 2 parallel wells for each dilution.

[0136] (5) Gently shake the culture plate to mix the lentivirus and cells, and culture in a 37.0±1°C, 5.0±0.5% carbon dioxide incubator for 72±2 hours.

[0137] 3.D4: Sample collection and testing

[0138] (1) Take out the 96-well flat-bottom cell culture plate from the CO2 incubator, mix it by pipetting, and transfer the cell suspension to a flow cytometry tube to detect CD3 + CAR + (%).

[0139] (2) CD3 + CAR + (%) Detection

[0140] Turn on the flow cytometer: Follow the startup process and complete the quality control procedure in accordance with the "AD1004 MACS Quant 10 Flow Cytometer Use, Maintenance and Maintenance Standard Operating Procedures". After the quality control is completed, the machine will display "PASS" and can be used normally.

[0141] The cells were collected into flow cytometry tubes, 1 mL of PBS was added to each tube, vortexed and mixed, and then centrifuged at 300 g for 5 minutes, and the washing was repeated once.

[0142] Discard the supernatant and add 100 μL PBS to each tube.

[0143] Antibody incubation was performed using CD3 direct-labeled antibody and CAR direct-labeled antibody FMC63, and both antibodies were added to each experimental tube and control tube.

[0144] Incubation: After adding the antibody, shake to mix and incubate in a dark place at 2-8℃ for 15 minutes.

[0145] After incubation, add 1 μL 1× Fixable Viability Stain 780 to each tube for staining, mix well, and place in a 2-8°C refrigerator incubated in the dark for 15 minutes.

[0146] After incubation, add 1 mL of PBS to each tube, shake to mix, and centrifuge at 300 g for 5 min.

[0147] Discard the supernatant, add 1 mL of PBS to each tube, shake to mix, and centrifuge at 300 g for 5 minutes.

[0148] Discard the supernatant, add 200 μL PBS to each tube, and vortex to mix.

[0149] Set the "Live cells" gate and collect about 10,000 events. (When using an intermediate antibody or multiple antibodies to detect CAR, use the antibodies according to the instructions or other methods.)

[0150] The logic of the circle is as follows Figure 1 shown.

[0151] 4. Data Analysis

[0152] CD3 + CAR + (%) = (Experimental tube 1: CD3 + CAR + (%) + Experimental tube 2: CD3 + CAR + (%)) / 2

[0153] When the positive rate of the control group is less than 2%, the experimental results are valid.

[0154] Example 3 Detection of lentiviral biological activity

[0155] Based on the optimization of the method conditions in Example 1, this example provides a method for detecting the biological activity of lentivirus-infected T cells.

[0156] 1.D0: Recovery and activation of cryopreserved T cells

[0157] (1) Turn on the electric constant temperature water bath in advance, take out the frozen T cells from the liquid nitrogen tank, transfer them to the water bath, wait until the temperature of the water bath stabilizes at 37°C, take out the cells and start to thaw (shake the cryopreserved tube while thaw), and take them out when the cells in the cryopreserved tube are about to melt completely (only a small amount of ice crystals remain). Wipe the surface of the cryopreserved tube with a dust-free cloth and wipe and disinfect it, then transfer it to the biological safety cabinet. Use a pipette to carefully transfer the thawed cells to complete culture medium, centrifuge at 300g for 5 minutes, discard the supernatant, add X-VIVO 15 complete culture medium to resuspend evenly, take 0.3-0.5mL of the cell suspension into a 1.5mL centrifuge tube, and count the cells according to the "QC1062 Cell Counter (NC-200) Use, Maintenance and Operation Procedures".

[0158] (2) According to the counting results, the T cell density was adjusted to 2.0×10 6cells / mL, transfer the cell suspension to a culture flask, add T cell Transact at a volume ratio of 110:1 for activation, gently mix after addition, place the culture flask in a 37.0±1℃, 5.0±0.5% carbon dioxide incubator, and perform virus infection 24±2 hours after activation.

[0159] 2.D1: Lentiviral transduction

[0160] (1) The activated T cells were centrifuged at 300 g for 5 minutes, the cell pellet was resuspended in X-VIVO 15 complete medium, and the cells were counted. The cell density was adjusted to 2.0×10 based on the counting results. 6 cells / mL.

[0161] (2) Prepare a 96-well flat-bottom cell culture plate and add 100 μL of the mixed cell suspension to each well (i.e., the number of cells per well is 2.0×10 5 cells).

[0162] (3) Take out the lentivirus from the -80℃ freezer and place it in a 4℃ ice box to thaw. After the lentivirus is thawed, dilute the lentivirus in a gradient manner using X-VIVO 15 complete medium.

[0163] (4) After dilution, 120 μL of lentiviral stock solution or dilution was added to each well of the experimental group, and 120 μL of X-VIVO 15 complete medium was added to each well of the control group. Two parallel wells were set up for each dilution.

[0164] (5) Gently shake the culture plate to mix the lentivirus and cells, and culture in a 37.0±1°C, 5.0±0.5% carbon dioxide incubator for 72±2 hours.

[0165] 3.D4: Sample collection and testing

[0166] (1) Take out the 96-well flat-bottom cell culture plate from the CO2 incubator, mix it by pipetting, and transfer the cell suspension to a flow cytometry tube to detect CD3 + CAR + (%).

[0167] (2) CD3 + CAR + (%) Detection

[0168] Turn on the flow cytometer: Follow the startup process and complete the quality control procedure in accordance with the "AD1004 MACS Quant 10 Flow Cytometer Use, Maintenance and Maintenance Standard Operating Procedures". After the quality control is completed, the machine will display "PASS" and can be used normally.

[0169] The cells were collected into flow cytometry tubes, 1 mL of PBS was added to each tube, vortexed and mixed, and then centrifuged at 300 g for 5 minutes, and the washing was repeated once.

[0170] Discard the supernatant and add 100 μL PBS to each tube.

[0171] Antibody incubation was performed using CD3 direct-labeled antibody and CAR direct-labeled antibody FMC63, and both antibodies were added to each experimental tube and control tube.

[0172] Incubation: After adding the antibody, shake to mix and incubate in a dark place at 2-8℃ refrigerator for 20 minutes.

[0173] After incubation, add 1 μL 1× Fixable Viability Stain 780 to each tube for staining, mix well, and place in a 2-8°C refrigerator incubated for 20 minutes away from light.

[0174] After incubation, add 1 mL of PBS to each tube, shake to mix, and centrifuge at 400 g for 10 min.

[0175] Discard the supernatant, add 1 mL of PBS to each tube, shake to mix, and centrifuge at 300 g for 5 minutes.

[0176] Discard the supernatant, add 200 μL PBS to each tube, and vortex to mix.

[0177] Set the "Live cells" gate and collect about 10,000 events. (When using an intermediate antibody or multiple antibodies to detect CAR, use the antibodies according to the instructions or other methods.)

[0178] The logic of the circle is as follows Figure 2 shown.

[0179] 4. Data Analysis

[0180] CD3 + CAR + (%) = (Experimental tube 1: CD3 + CAR + (%) + Experimental tube 2: CD3 + CAR + (%)) / 2

[0181] When the positive rate of the control group is less than 2%, the experimental results are valid.

[0182] Example 4 Specificity of the detection method

[0183] According to the detection method of Example 2, the CD3 + CAR+ (%) are all 0.0%, lentivirus test sample 800-fold dilution group CD3 + CAR + (%) is 27.8%. The specificity test results are shown in Figure 8 , with good specificity.

[0184] Example 5 Accuracy of the detection method

[0185] According to the detection method of Example 2, sample A (400-fold dilution) and sample B (1600-fold dilution) were mixed in a ratio of 3:1, 1:1, and 1:3, and the sample recovery rate was 99% to 107%. The accuracy test results are shown in Fig. 9 , with good accuracy.

[0186] Example 6 Repeatability of the detection method

[0187] Take the lentivirus test sample, dilute it into 800-fold dilution using X-VIVO15 complete medium, and make 6 parallels. According to the detection method of Example 2, the CD3 + CAR + (%) The coefficient of variation (CV) was 3%. The repeatability test results are shown in Fig.10 , with good repeatability.

[0188] Example 7 Intermediate Precision of the Detection Method

[0189] Two analysts tested the same batch of lentivirus test samples according to the detection method of Example 2. The CV of the test results of 6 parallel samples by the same analyst was <20%, and the CV of the test results of 12 parallel samples by two analysts was <25%. The intermediate precision test results are shown in Fig.11 .

[0190] Example 8 Linearity of the Detection Method

[0191] The lentivirus test sample was diluted at different times, and the positive rate was detected according to the method of Example 2. The relative concentration of the dilution multiple and the positive rate were used as the horizontal and vertical coordinates, respectively, and a linear curve was fitted. The R2 was 0.9956, and the CV of the parallel wells was <20%. The linear detection results are shown in Fig.12 , flow chart see Figure 3 , standard curve see Figure 4 , which has a good linear relationship.

[0192] The above embodiments are merely illustrative of the principles and effects of the present application and are not intended to limit the present application. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by a person of ordinary skill in the art without departing from the spirit and technical ideas disclosed in the present application shall still be covered by the claims of the present application.

Claims

1. A method for detecting the biological activity of lentivirus-infected T cells, comprising the following steps: 1) Preparation of CAR-T cells; 2) mixing the CAR-T cells obtained in 1) with CD3 antibody and CAR antibody and incubating them for the first time; 3) staining and second incubation, washing; 4) Detection of CD3 + CAR + The percentage of cells was used to evaluate the biological activity of lentivirus-infected T cells; The preparation of CAR-T cells comprises the following steps: activating T cells, wherein the density of activated T cells is 1.0×10 6 ~2.0×10 6 cells / mL, the activated T cells were mixed with the lentivirus containing the CAR gene, and cultured for 70-74 hours to obtain CAR-T cells; The culture medium used in preparing the CAR-T cells is a complete T cell culture medium, which contains the following components: 96% to 98% by volume of X-VIVO 15 culture medium, 2% to 4% by volume of serum replacement, and a final concentration of 200 to 250 IU / mL of IL-2.

2. The method according to claim 1, characterized in that The preparation of CAR-T cells does not include rehydration.

3. The method according to claim 1, characterized in that The method for activating T cells is as follows: suspending T cells in a complete T cell culture medium to prepare a T cell suspension, mixing the T cell suspension with a T cell activator at a volume ratio of 90 to 110:1, and activating the T cell suspension at a temperature of 36 to 38° C. and a carbon dioxide concentration of 4.5 to 5.5% for 22 to 26 hours.

4. The method according to claim 1, characterized in that The activated T cells are mixed with the lentivirus containing the CAR gene by mixing the activated T cell suspension with the lentivirus stock solution or the lentivirus suspension diluted with the T cell complete culture medium; the ratio of the volume of the lentivirus stock solution or the lentivirus suspension to the volume of the activated T cell suspension is 0.8-1.2:

1.

5. The method according to claim 4, characterized in that The dilution multiple of the dilution is 80 to 1280 times.

6. The method according to claim 1, characterized in that The culture temperature is 36-38°C; And / or, the carbon dioxide concentration during the culturing is 4.5-5.5%.

7. The method according to claim 1, characterized in that The CD3 antibody and CAR antibody carry markers.

8. The method according to claim 1, characterized in that The label is selected from fluorescein, enzyme, and biotin.

9. The method according to claim 1, characterized in that: The temperature of the first incubation is 2 to 8°C, and the time of the first incubation is 15 to 20 minutes; And / or, the temperature of the second incubation is 2 to 8° C., and the time of the second incubation is 15 to 20 minutes; And / or, the washing includes adding PBS, mixing, and discarding the supernatant after centrifugation.

10. Use of the method for detecting the biological activity of lentivirus-infected T cells according to any one of claims 1 to 9 in lentivirus quality control and detection, or lentivirus vector production, or preparation of products for preventing and / or treating cancer, or preparation of T cell therapy products.

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