Method for evaluating specific killing ability of engineered natural killer cells

Abstract

The invention relates to a method for evaluating the specific killing ability of engineered natural killer cells. Specifically, the invention provides a method for dynamically detecting the killing ability of the immune effector cell in real time, a target cell capable of stably expressing fluorescent protein is constructed, and the real-time killing of the immune effector cell on the target cell is accurately reflected through the disappearance condition of a fluorescence signal of the target cell. In the system provided by the invention, target cells can be added again for continuous challenge, tumor invasion is simulated, and the in-vitro killing function of the natural killer cells of the chimeric antigen receptor is comprehensively evaluated. The method provided by the invention provides effective and accurate evaluation for process development of immune effector cells, especially engineered and modified NK cell products, and provides effective analysis support for researching immune curative effects of NK cells and the like.

Description

Technical Field

[0001] The present invention relates to the field of immune cell therapy, and in particular to a method for evaluating the specific killing ability of engineered natural killer cells. Background Art

[0002] Natural Killer Cell (NK cell) is a type of innate lymphocyte that was originally discovered to have the ability to actively and spontaneously kill target cells, which is essential for controlling viral infections and eliminating malignant cells. NK cells can recognize tumor cells through a unique mechanism that relies on NK cell activation and inhibitory receptors that can recognize whether tumor cells express relevant ligands. If tumor cells express relevant ligands, NK cells will be activated, giving them the ability to quickly kill tumor cells.

[0003] Activated NK cells release perforins and granzymes, cytotoxic granules that directly lyse tumor cells. Not only that, NK cells can also mediate apoptosis of tumor cells through death receptors, including tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL). The overall safety of NK cells has been demonstrated in early clinical trials, even in an allogeneic setting. NK cells do not require HLA matching. Their ability to act in an antigen-independent manner makes them a viable option for "off-the-shelf" therapies that can be mass-produced and easily distributed to cancer patients.

[0004] The detection of NK cell cytotoxicity reflects the activity of NK cells in killing tumors. A suitable method for evaluating the killing ability of NK cell drugs in vitro will help the development of NK cell drugs. Therefore, the method of evaluating NK cell cytotoxicity is a very important task in basic medicine and clinical research.

[0005] Currently, common methods for evaluating immune cell cytotoxicity include luciferase reporter gene method, flow cytometry-based detection method, real-time label-free cell analyzer (RTCA), ethidium uptake and trypan blue dye exclusion test (EuTDA), enzyme-linked immunosorbent assay (ELISA), MTT method and Cr51 radioisotope method, etc. However, the above methods are applicable under limited conditions, and most of them are endpoint methods, which cannot achieve real-time dynamic detection.

[0006] Therefore, there is an urgent need in this field to develop a more universal method that can dynamically evaluate the multi-round cell killing ability in real time. Summary of the invention

[0007] The purpose of the present invention is to provide a more universal method that can realize real-time dynamic evaluation of multiple rounds of cell killing ability.

[0008] In a first aspect of the present invention, a method for real-time dynamic detection of effector cell killing ability is provided, comprising the steps of:

[0009] A. Construction of target cells stably expressing fluorescent proteins;

[0010] B. providing effector cells to be detected; dividing the target cells in step A into an experimental group and a control group, wherein the effector cells are added to the experimental group for co-culture, and the control group contains only target cells, and other experimental conditions of the experimental group and the control group are kept consistent;

[0011] C. At one or more time points during the co-culture process, the fluorescence intensity of the fluorescent protein in the experimental group and the control group is detected; for each time point, the fluorescence intensity of the experimental group is recorded as R1, and the fluorescence intensity of the control group is recorded as R0, and the cell killing ability C at that time point is calculated using the following formula I:

[0012] C(%)=(1-R1 / R0)×100% (Formula I).

[0013] In another preferred embodiment, step C further comprises: calculating the cell killing ability at multiple time points, and drawing a curve chart showing the change of the cell killing ability over time.

[0014] In another preferred embodiment, step A further comprises:

[0015] A1. Construct a recombinant vector with a fluorescent protein encoding gene;

[0016] A2. transfecting the recombinant vector into target cells;

[0017] A3. Screen stably transfected cell lines to obtain target cells that stably express the fluorescent protein.

[0018] In another preferred embodiment, in step B, the ratio E:T of the added effector cells to the target cells is (0.1:1) to (10:1).

[0019] In another preferred embodiment, the ratio E:T of the added effector cells to the target cells is (0.3:1) to (9:1).

[0020] In another preferred embodiment, the ratio E:T of the added effector cells to the target cells is (1:1) to (9:1).

[0021] In another preferred embodiment, the ratio E:T of the added effector cells to the target cells is (3:1) to (9:1).

[0022] In another preferred embodiment, the fluorescent protein is green fluorescent protein (GFP).

[0023] In another preferred embodiment, the recombinant vector is a plasmid or a viral vector.

[0024] In another preferred embodiment, the plasmid carries a resistance gene for screening, such as the Puro resistance gene.

[0025] In another preferred embodiment, the recombinant vector is a lentiviral vector.

[0026] In another preferred embodiment, the transfection is a stable transfection.

[0027] In another preferred embodiment, the transfection method includes: viral transfection, electroporation, liposome transfection, or a combination thereof.

[0028] In another preferred embodiment, the viral transfection includes lentiviral transfection.

[0029] In another preferred embodiment, the target cells are tumor cells.

[0030] In another preferred embodiment, the target cells are K562 cells or NALM6 cells.

[0031] In another preferred embodiment, the target cells are tumor cells cultured in an adherent or suspended manner.

[0032] In another preferred embodiment, in step B, the effector cells are genetically engineered or unengineered cells selected from the group consisting of NK cells, T cells, or a combination thereof.

[0033] In another preferred embodiment, the effector cells are genetically engineered or unengineered NK cells.

[0034] In another preferred embodiment, the NK cells include: NK cells derived from pluripotent stem cells, NK cells derived from peripheral blood, and NK cells derived from umbilical cord blood.

[0035] In another preferred embodiment, the NK cells are NK cells derived from pluripotent stem cells, preferably NK cells derived from induced pluripotent stem cells (iPSCs).

[0036] In another preferred embodiment, the genetic engineering modification includes: introducing exogenous genes into the cell genome, delivering exogenous vectors into cells, expressing exogenous proteins, knocking out predetermined endogenous genes in the cell genome, etc.

[0037] In another preferred embodiment, the genetic engineering modification is to express a chimeric antigen receptor (CAR).

[0038] In another preferred embodiment, the effector cells are chimeric antigen receptor-NK (CAR-NK) cells.

[0039] In another preferred embodiment, in step B, the number of starting target cells in each group is 10,000-40,000, preferably 15,000-30,000, and more preferably 15,000-25,000.

[0040] In another preferred embodiment, in step B, different numbers of effector cells are added for co-culture with target cells to obtain the killing ability of effector cells under different effector-target ratios.

[0041] In another preferred embodiment, the detection in step C is started after 6 hours of co-cultivation in step B.

[0042] In another preferred embodiment, the detection of step C is performed at one or more time points during the co-cultivation of 6h to 72h in step B; preferably, the detection of step C is performed at one or more time points during the co-cultivation of 12h to 60h; more preferably, the detection of step C is performed at one or more time points during the co-cultivation of 24h to 48h.

[0043] In another preferred embodiment, the detection of step C is performed at intervals of time t in step B; preferably, t is selected from 0.5h to 4h, for example, t is 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, etc.

[0044] In another preferred embodiment, in step C, the fluorescence intensity is detected using an automatic cell imaging system.

[0045] In another preferred embodiment, in step C, the process of detecting the fluorescence intensity does not affect the co-culture process of the cells.

[0046] In another preferred embodiment, in step C, the fluorescence intensity is detected multiple times during the co-culture process.

[0047] In another preferred embodiment, the method further comprises the steps of:

[0048] D. Add target cells to the experimental group again, repeat step C after co-culture, and calculate the killing ability of effector cells on target cells in the current round;

[0049] E. Repeat step D to calculate the killing ability of effector cells on multiple rounds of target cells.

[0050] In another preferred embodiment, in step D, the detection of step C is performed at one or more time points during the co-culture of 6h to 72h; preferably, the detection of step C is performed at one or more time points during the co-culture of 12h to 60h; more preferably, the detection of step C is performed at one or more time points during the co-culture of 24h to 48h.

[0051] In another preferred embodiment, the method is a quality control method used for quality control of immune product production.

[0052] In another preferred embodiment, the immune product is a NK cell product.

[0053] In a second aspect of the present invention, a system for real-time dynamic detection of effector cell killing ability is provided, comprising the following modules:

[0054] (M1) an experimental module, which is configured to include a cell culture container, and effector cells to be detected and target cells located in the container, wherein the target cells stably express fluorescent proteins; the experimental module is used to co-culture the effector cells to be detected and the target cells;

[0055] (M2) a control module, which is configured to include a cell culture container, wherein the container contains only target cells; the control module is used to culture the target cells as a control;

[0056] (M3) a fluorescence detection module, used for real-time dynamic detection of the fluorescence intensity in the experimental module and the control module;

[0057] (M4) An output module is used to output the fluorescence intensity value detected by the fluorescence detection module, wherein the fluorescence intensity of the experimental module is recorded as R1, and the fluorescence intensity of the control module is recorded as R0, and the cell killing ability C is calculated and output using the formula of the following formula I:

[0058] C(%)=(1-R1 / R0)×100% (Formula I).

[0059] In another preferred embodiment, the experimental module and the control module are under the same experimental conditions.

[0060] In another preferred example, the fluorescence detection module includes an automatic cell imaging system.

[0061] In another preferred example, the system further includes: (M5) a control module, which is used to control the (M3) fluorescence detection module to perform fluorescence intensity detection at one or more time points during the operation of the (M1) experimental module and the (M2) control module for 6h to 72h, preferably 12 to 60h, and more preferably 24 to 48h.

[0062] In another preferred example, the (M5) control module is used to control the (M3) fluorescence detection module to perform fluorescence intensity detection at time intervals t; preferably, the t is selected from 0.5h to 4h, for example, t is 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, etc.

[0063] In the third aspect of the present invention, there is provided the use of the method according to the first aspect of the present invention in detecting effector cell immune activity, quality control of immune products or evaluation of individual immune function.

[0064] In another preferred embodiment, the effector cells are NK cells.

[0065] In another preferred embodiment, the effector cells are CAR-NK cells.

[0066] In another preferred embodiment, the immune product is a NK cell product.

[0067] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0068] The beneficial effects of the present invention include:

[0069] 1. The method of the present invention can monitor the death of tumor cells in real time. Compared with the endpoint method, it can continuously detect the changes in cell killing ability over a long period of time.

[0070] 2. The method of the present invention can be used to simulate tumor invasion in vitro, detect the sustained killing ability of cells in multiple rounds, and comprehensively evaluate the survival of cell products in vitro and their toxicity to tumor cells.

[0071] 3. The method of the present invention can be applied to both adherent type target cells and suspended type target cells, and is more versatile.

[0072] 4. The method of the present invention utilizes tumor cells with GFP labels to replace other reagents to detect the death of target cells, which greatly reduces the cost and has high sensitivity, good repeatability and simple operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 A schematic diagram of the construction of an exemplary recombinant vector of the present invention is shown.

[0074] Figure 2 The expression abundance of green fluorescent protein in K562-GFP and NALM6-GFP cells is shown.

[0075] Figure 3 The results show the consistency between the apoptosis indicators (Annexin V, 7AAD) of target cells K562-GFP and NALM6-GFP under co-culture conditions detected by flow cytometry and the disappearance of green fluorescent protein signals.

[0076] Figure 4A The figure shows the proliferation curve of K562 cells seeded at different densities in a 96-well plate detected by a live cell real-time dynamic imaging system.

[0077] Figure 4B The correlation between different seeded cell numbers and total fluorescence intensity is shown.

[0078] Figure 5A The live cell real-time dynamic imaging system is used to monitor the cytotoxicity of NK cells against K562 and NALM6.

[0079] Figure 5B The 4PL model curves of the real-time cytotoxicity of NK cells to K562 and NALM6 at different time points (unit: hours) are shown.

[0080] Figure 5C Real-time EC50 toxicity assay results of NK cells against K562 and NALM6 are shown.

[0081] Figure 5D The image shows the killing process of K562 and NALM6 by NK cells monitored by the live cell real-time dynamic imaging system.

[0082] Fig. 6A The real-time dynamic imaging system is used to monitor multiple rounds of NK cell killing in vitro.

[0083] Figure 6B It shows the evaluation of the sustained killing effect of natural killer cells in vitro by adding multiple rounds of target cells to evaluate different types, different differentiation conditions, different expansion conditions, different freezing conditions and other processes. DETAILED DESCRIPTION

[0084] After extensive and in-depth research, the inventors have developed for the first time a method for dynamically detecting the killing ability of immune effector cells (such as NK cells) in real time, constructed target cells that stably express fluorescent proteins, and accurately reflected the real-time killing of target cells by immune effector cells by the disappearance of target cell fluorescent signals. Furthermore, target cells can be added again to the experimental system for continuous challenges to simulate tumor invasion. By adding multiple rounds of target cells, the killing function of natural killer cells of chimeric antigen receptors in vitro is comprehensively evaluated. The method of the present invention provides an effective and accurate evaluation for the process development of immune effector cells, especially engineered NK cell products, and provides effective analytical support for studying the immunotherapeutic effects of NK cells, etc.

[0085] On this basis, the present invention has been completed.

[0086] Killing effect of immune effector cells

[0087] As used herein, the terms "immune effector cells" and "effector cells" are used interchangeably and refer to cells in the immune system that can effectively fight tumor cells, including NK cells and T cells (such as cytotoxic T lymphocytes, CTLs), etc.

[0088] Among them, NK cells are a type of innate lymphocytes that have the ability to actively and spontaneously kill target cells and play an important role in the elimination of malignant cells by the immune system.

[0089] As used herein, the terms "killing ability", "cytotoxicity" and "toxic function" are used interchangeably, all referring to the ability of immune effector cells (such as NK cells) to directly mediate the death of target cells (such as tumor cells). NK cells are activated after recognizing tumor cells, releasing perforins and granzymes, and these cytotoxic granules directly lyse tumor cells. In addition, NK cells can also mediate apoptosis of tumor cells through death receptors, including tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL). The detection of NK cell toxicity reflects the activity of NK cells in killing tumors, and a suitable method for evaluating the killing ability of NK cell drugs in vitro will help the development of NK cell drugs. Therefore, the method of evaluating the cytotoxicity of NK cells is a very important task in basic medicine and clinical research.

[0090] In the present invention, immune effector cells include genetically engineered immune effector cells, including: introducing exogenous genes into the cell genome, delivering exogenous vectors into cells, expressing exogenous proteins, knocking out predetermined endogenous genes in the cell genome, etc. Typically, the immune effector cells of the present invention are cells expressing exogenous chimeric antigen receptors (CARs), such as CAR-NK cells or CAR-T cells. Convenient, accurate and universal methods for detecting engineered NK cell cytotoxicity are beneficial to the process development and quality assessment of NK cell products.

[0091] Methods for evaluating immune cytotoxicity

[0092] Currently, the common methods for evaluating the cytotoxicity of immune effector cells are mainly divided into two categories: endpoint method and real-time dynamic method. Most common methods belong to endpoint method, that is, toxicity is detected after the effector cells kill the target cells, such as luciferase reporter gene method, flow cytometry-based detection method, enzyme-linked immunosorbent assay (ELISA) method and Cr51 radioisotope, etc. These methods cannot continuously detect the killing ability and have limitations.

[0093] For the real-time dynamic method, the typical one is the real-time label-free cell analyzer (RTCA) method, which uses the change of resistance value to feedback the death of target cells. The detection consumables are expensive, and due to technical limitations, only adherent target cells can be detected. In the present invention, a real-time dynamic imaging detection system is provided, which can be used to continuously and multi-roundly detect the killing ability of immune effector cells and target cells during co-culture.

[0094] Method and system of the present invention

[0095] The present invention provides a method and system for real-time dynamic detection of effector cell killing ability. The living cell real-time dynamic imaging system of the present invention is used to monitor the process of effector cells killing target cells and obtain real-time effector cell killing ability data.

[0096] The method of the present invention constructs a target cell to be killed with a fluorescent marker; uses a live cell real-time dynamic imaging system to detect the whole process of multiple rounds of specific killing of target cells by effector cells, and obtains real-time imaging data; by analyzing the data of total fluorescence intensity, a 4PL mathematical model is constructed to calculate EC50, and the killing ability of effector cells in vitro is comprehensively evaluated by the value of EC50. The specific steps are as follows:

[0097] First, a target cell stably expressing a fluorescent protein is constructed. Preferably, a gene encoding green fluorescent protein is integrated into the targeted genome using a stable transfection method. The stable transfection method includes, but is not limited to, methods well known to those skilled in the art such as viral transfection, electroporation, and liposome transfection.

[0098] In a preferred embodiment, K562 cells and NALM6 cells are infected with a lentiviral vector to obtain K562-GFP cells and NALM6-GFP cells that stably express green fluorescent protein. Cells that are observed to have green fluorescence under a fluorescence microscope are K562-GFP and NALM6-GFP cells, and K562-GFP and NALM6-GFP stable transfected cells are obtained by puro resistance screening. The constructed stable transfected cells are determined to have uniform green fluorescence by flow cytometry.

[0099] Secondly, effector cells with a certain effector-target ratio are added to co-culture with fluorescent target cells, and a group containing only target cells without effector cells is used as a control group. In a preferred embodiment, cells with different effector-target ratios are added to detect changes in the killing ability of effector cells under different effector-target ratios.

[0100] During the co-culture process, the fluorescence intensity of the experimental group and the control group was detected, and the cell killing ability was calculated using the following formula I:

[0101] Cell killing ability = 1-fluorescence intensity of the experimental group / fluorescence intensity of the control group (Formula I).

[0102] The fluorescence signal can be detected by conventional methods in the art, such as detection using a Cellcyte instrument.

[0103] The experiment of the present invention verifies that for target cells stably transfected with green fluorescent protein, the disappearance of the fluorescent signal indicates the death of the target cells, especially after a period of co-culture, the detection result is more accurate. In a preferred embodiment, the fluorescence intensity is detected after 6 hours of co-culture, preferably after 12 hours of co-culture, and more preferably after 24 hours of co-culture.

[0104] In a preferred embodiment, during the co-culture process, the cell killing ability is detected multiple times at multiple time points to obtain the change of the cell killing ability over time. For example, during the co-culture for 24h-48h, the fluorescence signal is detected every 0.5h or 1h to calculate the cell killing ability.

[0105] The method of the present invention can also be used to detect the multi-round killing ability of effector cells. During the co-culture process, target cells are added again at a certain interval to detect the changes in the killing ability of effector cells to target cells in multiple rounds, so as to obtain more information on the persistent killing ability of effector cells.

[0106] The system for real-time dynamic detection of effector cell killing ability of the present invention is also called a living cell real-time dynamic imaging system, which comprises:

[0107] (1) A co-culture module: comprising a cell culture container, and effector cells and target cells to be detected located in the container, wherein the target cells stably express fluorescent proteins;

[0108] (2) a control module: comprising a control cell culture container, wherein the container contains only the target cells;

[0109] (3) Fluorescence detection module: real-time dynamic detection of the fluorescence intensity in the co-culture module and the control module;

[0110] (4) Output module: The fluorescence intensity of the co-culture module is recorded as R1, and the fluorescence intensity of the control module is recorded as R0. The cell killing capacity C is calculated and output using the following formula I:

[0111] C(%)=(1-R1 / R0)×100% (Formula I).

[0112] application

[0113] The live cell real-time dynamic imaging system of the present invention can provide a platform for full-process visualization in the co-culture stage of effector cells and target cells, can detect adherent and suspended target cells, and is simple to operate, low in consumables, highly sensitive, and reproducible, and can monitor the killing of target cells in real time. In addition, the method of the present invention can repeat multiple rounds of in vitro killing ability evaluation methods of NK cells simulating tumor cell invasion, and comprehensively evaluate the potency of NK cell killing ability in vitro.

[0114] In a preferred embodiment, the differences in the multi-round killing ability of NK cell products produced by different processes are compared. As can be seen from the results, in the first few rounds of killing, the killing ability of NK cell products of each process is not much different, but after continuing multiple rounds of killing, it can be seen that the NK cell products of each process show differences in sustained killing ability. It shows that the method of the present invention can be used to more accurately and comprehensively evaluate the effect of immune cell products (such as NK cells or engineered NK cell products).

[0115] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually performed under normal conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and fractions are weight percentages and weight fractions.

[0116] Materials and equipment

[0117] The names and sources of the reagents, consumables and instruments used in the examples of the present invention are shown in Table 1:

[0118] Table 1 Names and sources of reagents, consumables and instruments

[0119]

[0120]

[0121] Example 1: Construction of green fluorescent labeled target cells

[0122] Tumor cells K562 cells and NALM6 cells were used as target cells and infected with lentivirus to obtain fluorescently labeled K562-GFP cells and NALM6-GFP cells. The specific steps are as follows:

[0123] 1. Lentivirus packaging: Construct a recombinant plasmid with GFP gene, as shown in the schematic diagram Figure 1 The total length of the plasmid vector is 8118 base pairs (bp), including the following key functional elements:

[0124] (1) CMV promoter, located near the 5'LTR. A strong promoter from cytomegalovirus that can efficiently initiate the expression of downstream genes in various types of mammalian cells and is used to drive the expression of target genes or reporter genes (such as GFP);

[0125] (2) 5'LTR (Long Terminal Repeat), located at the 5' end of the plasmid. The long terminal repeat sequence is one of the essential components of retroviruses. The 5'LTR initiates the transcription of viral RNA and contains promoter and enhancer elements.

[0126] (3) HIV-1ψ (Psi), located downstream of the 5'LTR. The packaging signal sequence of HIV-1 virus marks the RNA to be packaged into the virus particle, ensuring that it is recognized and packaged into the virus particle during virus packaging.

[0127] (4) CopGFP, located near the right side of the plasmid, next to the T2A sequence. It is a green fluorescent protein used as a reporter gene to facilitate observation and screening of transformed cells by fluorescence microscopy or flow cytometry.

[0128] (5) T2A sequence, which is located between CopGFP and Puro genes. The 2A peptide sequence allows two separate but functionally independent proteins to be translated from one open reading frame (ORF). This system allows the protein generated by the translation of the same mRNA to be split into two independent proteins at the T2A sequence.

[0129] (6) Puro (puromycin resistance gene), located immediately to the right of the T2A sequence, provides resistance to puromycin, allowing mammalian cells carrying the plasmid to survive and grow under puromycin selection pressure, and is used to screen successfully transformed cells.

[0130] (7) WPRE (Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element), located downstream of the Puro gene. Transcriptional regulatory elements increase protein expression levels by enhancing the expression of downstream genes and mRNA stability.

[0131] (8) SV40 polyA-R (SV40 polyadenylation signal-Reverse), near the left side of the plasmid. The polyadenylation signal from the SV40 virus is arranged in reverse order and is used to terminate transcription and add a polyA tail, thereby increasing the stability of the mRNA.

[0132] (9) ori (Origin of Replication), located near the left side of the plasmid. The origin of replication is the sequence that initiates the autonomous replication of the plasmid in the bacterial cell, ensuring the replication and maintenance of the plasmid in the host cell.

[0133] (10) AmpR (Ampicillin Resistance), near the lower left side of the plasmid. It encodes the ampicillin resistance protein, which enables bacteria carrying the plasmid to grow in medium containing ampicillin and is used for screening plasmids in bacteria.

[0134] (11) 3'LTR (Long Terminal Repeat), located near the lower left of the plasmid. Long terminal repeat sequence is one of the essential components of retroviruses. 3'LTR is involved in the integration of retroviral DNA into the host genome and contains a signal to terminate transcription.

[0135] The recombinant plasmid constructed by the present invention contains multiple elements for gene expression, screening and expression rate enhancement, and is a multifunctional vector suitable for complex gene manipulation experiments in mammalian cells.

[0136] The above recombinant plasmid (Transfer Vector) was co-transfected with auxiliary packaging plasmids (Envelope Vector and Packaging Vector) into 293T cells, and the cell supernatant rich in lentiviral particles was collected to obtain lentiviral supernatant, and the titer was determined.

[0137] 2. Infect target cells: Add lentivirus to K562 and NALM6 respectively, replace the culture medium 5 hours after infection, and cells that can be observed to have green fluorescence under a fluorescence microscope after 48 hours of culture are target cells infected with GFP.

[0138] 3. Screening of stably transfected target cells: Screen target cells infected with lentivirus through Puro resistance, and select target cells with uniform fluorescence through monoclonal screening.

[0139] 4. Detect the abundance of K562-GFP and NALM6-GFP green fluorescent proteins by flow cytometry. If the expression of GFP in the cells is clearly observed to be at a uniform level in the flow cytometry graph, the target cells are successfully constructed.

[0140] The flow cytometry results are as follows Figure 2 As shown, red is the control cell that is not transfected with GFP, and blue is the stable cell that stably expresses GFP. The results show that GFP is expressed uniformly in the cells, indicating that the target cells K562-GFP and NALM6-GFP that stably express GFP have been successfully obtained.

[0141] Example 2: Relationship between fluorescence signal and target cell apoptosis

[0142] In this implementation, in order to verify that the disappearance of GFP fluorescence signal can represent cell apoptosis, flow cytometry was used to detect the disappearance of GFP fluorescence signal during apoptosis of K562-GFP and NALM6-GFP cells, and the early apoptosis index (Annexin V) and late apoptosis index (7AAD) of cells were detected to determine the correlation between cell apoptosis and the disappearance of GFP fluorescence signal. The specific steps are as follows:

[0143] 1. Target cell preparation: Calculate the number of live cells and perform Celltrace-Farred staining on the target cells according to the cell counting results. 1E6 cells require 1μl of dye in 1mL of serum-free medium. Place the cells being stained in a cell culture incubator for 20 minutes. After staining, add 4ml of complete medium and incubate at room temperature for 5 minutes. After incubation at room temperature, centrifuge at 300g for 5 minutes. After centrifugation, remove the supernatant and add complete medium to adjust the target cell density to 0.3E6 cells / mL.

[0144] 2. Effector cell preparation: The effector cells are CAR-NK cells targeting CD19. Calculate the number of viable cells and prepare the required number of effector cells according to the desired effector-target ratio.

[0145] 3. Co-culture of effector cells and target cells: Add the diluted effector cells to the 96-well U-shaped plate according to the layout, 100 μL per well. Add the stained target cells to the 96-well U-shaped plate according to the layout, 100 μL per well. After adding the cells, place the 96-well U-shaped plate in a carbon dioxide incubator and co-culture for 24 hours.

[0146] 4. Annexin V / 7AAD staining: After co-culture, place the U-shaped 96-well plate in a centrifuge and centrifuge at 300g for 5 minutes. Prepare the staining solution. The reagents required for one sample are 100μL binding buffer + 2.5μL Annexin V + 2.5μL 7AAD. After centrifugation, add 100μL staining solution to each well. Incubate at room temperature for 10 minutes. After incubation, add 100μL binding buffer. Detect on flow cytometer.

[0147] 5. Calculate cytotoxicity by GFP, Annexin V, and 7AAD respectively:

[0148] (1) Cytotoxicity was calculated by analyzing the early apoptosis index of tumor cells (Annexin V+) during co-culture. The formula is as follows:

[0149] Cytotoxicity%=1-((1-Co-culture Group Annexin V+ ) / (1-Target Only Group Annexin V+ )),

[0150] Among them, Cytotoxicity means cytotoxicity, Co-culture Group Annexin V+ Indicates the proportion of Annexin V+ in the co-culture group, Target Only Group Annexin V+ It indicates the proportion of Annexin V+ in the target cell group only.

[0151] (2) Cytotoxicity was calculated by analyzing the tumor cell death index (7AAD+) during co-culture, using the following formula:

[0152] Cytotoxicity%=1-((1-Co-culture Group 7AAD+ ) / (1-Target OnlyGroup 7AAD+ )),

[0153] Among them, Cytotoxicity means cytotoxicity, Co-culture Group 7AAD+ Represents the proportion of 7AAD+ in the co-culture group, Target Only Group 7AAD+ It indicates the proportion of 7AAD+ in the target cell group only.

[0154] (3) Cytotoxicity was calculated by analyzing green fluorescent protein (GFP+) during co-culture using the following formula:

[0155] Cytotoxicity%=1-(Co-culture Group GFP+ / Target Only Group GFP+ ),

[0156] Among them, Cytotoxicity means cytotoxicity, Co-culture Group GFP+ Indicates the intensity of GFP+ in the co-culture group, Target Only Group GFP+ It indicates the intensity of GFP+ in the target cell group only.

[0157] The results are as follows Figure 3 As shown. The results calculated by different indicators show that in the early stage of target cell apoptosis, the AnnexinV indicator can indicate the occurrence of apoptosis faster, while the GFP signal indicator is slightly faster than the 7AAD indicator. The death of target cells requires a certain process, so as the co-culture time increases, it can be seen that the difference between the three analysis methods is gradually narrowing. There is no significant difference after 6 hours of co-culture, and the data are almost consistent after 24 hours of co-culture.

[0158] The above results show that the disappearance of the target cell GFP signal can reflect the survival of the tumor cells, that is, when the tumor cells are GFP+, the tumor cells are considered to be living cells, and when the tumor cells are GFP-, the tumor cells are considered to be dead cells.

[0159] Example 3: Linear relationship of the number of target cells detected by the live cell real-time dynamic imaging system

[0160] Based on the conclusion of Example 2, the GFP fluorescence signal of the target cells can be used to characterize the apoptosis of the target cells, thereby calculating the killing ability of the effector cells, thereby constructing a live cell real-time dynamic imaging detection system. Since the system detects the apoptosis of the target cells in the co-culture system in real time and dynamically, the number of target cells will affect the accuracy of the detection results. Therefore, in order to obtain more effective and accurate results, it is necessary to determine the most suitable number of target cells in the co-culture system. The specific method is as follows:

[0161] 1. Pre-treat a 96-well flat-bottom plate by adding 50 μl of poly-L-ornithine solution to each well to enhance cell attachment to the plate. Leave the plate at room temperature for 1 hour. After 1 hour, remove the poly-L-ornithine solution and dry the plate at room temperature for 1 hour.

[0162] 2. Count the target cells and obtain a target cell suspension with a density of 1.2E6 Cells / ml. Then, perform two-fold gradient dilution to obtain 9 cell suspensions with different densities.

[0163] 3. Inoculate the cell suspension in a 96-well flat-bottom plate, place it in the instrument, monitor it in real time for 2 days, and analyze and obtain the fluorescence intensity data. If the number of added target cells shows a good linear relationship, the analysis method is considered to have high sensitivity.

[0164] Figure 4A The results show that when the number of tumor cells plated is 80,000 or 40,000 cells, the maximum fluorescence detection value will be reached too early, which is not suitable for multiple rounds of cell addition. When the number of tumor cells plated is less than 10,000, the detection platform will be reached later, and the Bar value changes too high. When the number of tumor cells plated is 20,000 cells, the detection platform can be reached in about 48 hours, and the feedback fluorescence value is stable, and the Bar value is low. Therefore, the co-culture system of this detection method selects 20,000 target cells as the plating amount as the optimal system, which can obtain more stable response data and accurately feedback the killing of target cells.

[0165] This detection method obtains the intensity of fluorescent protein through the real-time dynamic imaging system of living cells to provide real-time feedback on the activity of target cells. Figure 4B The results show that this method can well reflect the relationship between the intensity of fluorescent protein and the number of living cells (R 2≥0.9972), so in subsequent experiments the survival of target cells can be reflected by the change of real-time fluorescence intensity.

[0166] Example 4: Detection of NK cell killing efficiency against target cells based on live cell real-time dynamic imaging system

[0167] In this example, the live cell real-time dynamic imaging system constructed by the present invention was used to detect the killing efficiency of CD19-targeted CAR-iNK cells (19CAR-iNK) on target cells K562-GFP and NALM6-GFP. The specific steps are as follows:

[0168] 1. Pre-treat a 96-well flat-bottom plate, add 50 μl of poly-L-ornithine solution to each well, and leave at room temperature for 1 hour. After 1 hour, remove the poly-L-ornithine solution and dry at room temperature for 1 hour.

[0169] 2. Count K562-GFP cells. Resuspend K562-GFP cells in F12K medium and adjust the density to 2E5 cells / mL. Inoculate cells in a 96-well flat-bottom plate at 100 μl / well, i.e., 20,000 cells per well.

[0170] 3. Place the 96-well flat-bottom plate into the automated cell imaging system Cellcyte, set the instrument parameters, turn on the white light channel (enhance); green fluorescence channel (Green), and set the photography parameters to 200ms, 4Gain. The photography duration is 48h, and the photography interval is 1h.

[0171] 4. Set the fluorescence analysis parameters and use the fluorescence intensity and size as a reference for analysis.

[0172] 5. Dilute the natural killer cells in advance according to the effector-target ratio of 9:1, 3:1, 1:1, 0.3:1, and 0.1:1. The corresponding cell densities are 9E6 cells / mL, 3E6 cells / mL, 1E6 cells / mL, 0.3E6 cells / mL, and 0.1E6 cells / mL, respectively.

[0173] 6. After 2 hours, wait until the next shooting interval, take out the 96-well flat-bottom plate, add the proportionally diluted natural killer cells to the corresponding wells, 20 μl per well, and put it back into the Cellcyte. Add 20 μl of culture medium without effector cells to the blank control (target cell group only). Wait until the fixed shooting time to continue acquiring data.

[0174] 7. After 24 hours of co-culture, the killing efficiency corresponding to each group's effector-target ratio was calculated using the total fluorescence intensity obtained.

[0175] 8. Data processing: In order to reduce the variation between experiments and the influence of background, and make the data under different experimental conditions comparable, the data of each group were normalized to minimize the error between groups. The normalization method is: each group of cells uses the fluorescence intensity of the group of cells at 2h (the time point of adding effector cells) as the baseline, and the formula is: Normalized fluorescence intensity (tx) = fluorescence intensity (tx) / fluorescence intensity (t2); where fluorescence intensity (tx) represents the fluorescence intensity at different time points; fluorescence intensity (t2) represents the fluorescence intensity at 2h.

[0176] The EC50 value of the killing plateau phase is calculated by calculating the killing efficiency obtained at each moment, which is used to evaluate the efficiency of natural single killing and obtain corresponding imaging data.

[0177] like Figure 5A As shown, 2 hours after the target cells were added, iNK cells of different proportions were added, and the fluorescence intensity of tumor cells obtained in real time (data collected every 0.5 hours) was able to feedback the changes in fluorescence intensity of different co-culture ratios. The results showed that when the effector-target ratio was larger, the fluorescence degradation signal disappeared faster, that is, more tumor cells died. Figure 5A The tabular data are shown in Table A and Table B below.

[0178] Table A

[0179]

[0180] Table B

[0181]

[0182] pass Figure 5A The fluorescence intensity results can be used to calculate the cytotoxicity results of each effector-target ratio group at each time (every 0.5h), such as Figure 5B The formula is as follows:

[0183] Cytotoxicity%=1-(Co-culture Group Normalized Total Intension / Target OnlyGroup Normalized Total Intension ),

[0184] Among them, Cytotoxicity means cytotoxicity, Co-culture Group Normalized Total Intension Represents the normalized fluorescence intensity of the co-culture group, Target Only Group Normalized Total Intension The normalized fluorescence intensity of the target cell group only is shown.

[0185] By obtaining the fluorescence intensity at each moment (every 0.5h), the toxicity results of different effector-target ratios (0.1:1, 0.3:1, 1:1, 3:1, 9:1) can be calculated. The cytotoxicity 4PL model curve at each moment can be drawn to obtain the EC50 value at each moment, that is, the effector-target ratio required to achieve 50% target cell killing. The results are shown in Figure 5C shown.

[0186] By plotting the EC50 values ​​at each time, we can find that before 6 hours of co-culture, it takes a certain amount of time for apoptosis to cell death, so the EC50 value jumps a lot, but after 6 hours of co-culture, the value tends to be stable. Therefore, the EC50 value after 6 hours of co-culture can characterize the killing ability of NK cells on target cells.

[0187] Since NALM6 is a CD19-positive cell, 19CAR-iNK is a specific killer of NALM6, but a non-specific killer of K562. Figure 5C The results show that the killing effect of 19CAR-iNK on NALM6 (red curve) is significantly better than that on K562 (black curve), which can reflect the good specificity of this method. Figure 5D shown.

[0188] Example 5: Detection of NK cell killing efficiency against multiple rounds of target cells based on a live cell real-time dynamic imaging system

[0189] In this example, the live cell real-time dynamic imaging system constructed by the present invention is used to detect the killing efficiency of 19CAR-iNK cells on multiple rounds of target cells. The specific steps are as follows:

[0190] 1. Pre-treat a 96-well flat-bottom plate, add 50 μl of poly-L-ornithine solution to each well, and leave at room temperature for 1 hour. After 1 hour, remove the poly-L-ornithine solution and dry at room temperature for 1 hour.

[0191] 2. Count K562-GFP cells. Resuspend K562-GFP cells in F12K medium and adjust the density to 2E5 cells / mL. Inoculate cells in a 96-well flat-bottom plate at 100 μl / well, i.e., 20,000 cells per well.

[0192] 3. Place the 96-well flat-bottom plate in the Cellcyte, set the instrument parameters, turn on the white light channel (enhance); green fluorescence channel (Green), and set the camera parameters to 200ms, 4Gain. The camera duration is 240h, and the camera interval is 1h.

[0193] 4. Set the fluorescence analysis parameters and use the fluorescence intensity and size as a reference for analysis.

[0194] 5. Dilute the natural killer cells according to the effector-target ratio of 9:1, and the corresponding cell density is 9E6 cells / mL.

[0195] 6. After 2 hours, wait until the next shooting interval, take out the 96-well flat-bottom plate, add the proportionally diluted natural killer cells to the corresponding wells, and add 20μl to each well. After adding the effector cells, put it back into the Cellcyte. Add 20μl of culture medium without effector cells to the blank control (target cell group only). Wait until the fixed shooting time to continue to acquire data.

[0196] 7. After 24 hours of co-culture, if the killing of target cells by NK cells continues, target cells can be added again through real-time data of target cell fluorescence.

[0197] 8. Count K562-GFP cells. Resuspend K562-GFP cells in F12K medium and adjust the density to 1E6 cells / mL. Add cells to a 96-well flat-bottom plate at 20 μl / well, i.e., 20,000 cells per well.

[0198] 9. Subsequent operations are still performed according to step 7 until NK cells no longer inhibit the proliferation of target cells, at which point the experiment is stopped.

[0199] This multiple rounds of target cell invasion can be used to evaluate the killing efficiency and duration of natural killer cells in vitro, which will be more representative than the evaluation of a single killing.

[0200] The results of real-time dynamic imaging system monitoring the multiple rounds of NK cell killing of K562 cells in vitro are shown in Fig. 6A As shown, it is shown that the system of the present invention is able to detect the killing of tumor cells added in multiple rounds, which is used to model tumor invasion and provide multi-dimensional feedback on the killing of tumor cells by NK cell products.

[0201] The NK products prepared by different processes were named G1 to G8, and the continuous killing of 19CAR-iNK cells in each group was detected by real-time dynamic imaging system. The results are as follows Figure 6B shown.

[0202] The results showed that when there was only one round of killing results, the toxicity of the cell products of each process could not be distinguished. However, after adding K562 cells again, the NK cells produced under G5 conditions could no longer kill K562; after adding the second round of K562 cells, it can be seen that the NK cells produced under G3 conditions could no longer kill K562; after the fourth round of killing process, it can be clearly judged that the NK cells produced under G4 process conditions have the strongest cytotoxicity to K562 cells.

[0203] This indicates that the real-time dynamic imaging system of the present invention can sensitively evaluate the cytotoxicity of NK products under different process conditions.

[0204] Example 6: Comparison of the evaluation method of the present invention with the existing killing detection method

[0205] It can be seen from the results of the above examples that the real-time dynamic imaging system of the present invention can accurately, in real time, and in multiple rounds detect the killing ability of NK cells. In this example, the conventional cytotoxicity or cell killing ability detection method in the art is compared with the method of the present invention to illustrate the advantages of the method of the present invention. The conventional methods are as follows:

[0206] 1. Luciferase reporter gene: The instrument required is widely used, cheap, and supports high-throughput detection. However, since the luciferin substrate needs to be released only after the target cells are completely ruptured, this may cause unstable data.

[0207] 2. Flow cytometry-based detection method: Target cells are usually labeled with cell tracking reagents, and then stained with live and dead dyes. The high sensitivity of flow cytometry can obtain more accurate and stable killing results, but due to the limitations of flow cytometry, only suspended cell type target cells can be detected.

[0208] 3. Real-time label-free cell analyzer (RTCA): The change in resistance value is used to feedback the death of target cells. The advantage is that the survival curve of tumor cells can be monitored in real time, but the consumables are expensive and only adherent target cells can be detected.

[0209] 4. Ethidium uptake and trypan blue dye exclusion assay (EuTDA): The target cells need to be labeled, and the release of EuTDA after cell lysis reflects the death of the target cells. The disadvantage is that EuTDA will only be released after the target cells are completely ruptured, which may cause unstable data.

[0210] 5. Enzyme-linked immunosorbent assay (ELISA): It has high detection sensitivity and good repeatability. It usually detects the release levels of IFN-γ, TNF-α, Granzyme B, etc. to feedback the cell killing situation. However, it is an indirect method for detecting killing, and the detection experiment process is long and complicated.

[0211] 6.MTT: The principle is that succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble formazan. It has high accuracy, but is not suitable for cell detection in co-culture groups.

[0212] 7. Cr51 radioactive isotope: Target cells need to be labeled with radioactive elements. It has high sensitivity, requires specific instruments, and has radioactive hazards.

[0213] The comparison between the method of the present invention and the above method in terms of application scope, type, etc. is shown in Table 2 below:

[0214] Table 2 Comparison of the evaluation method of the present invention with the existing killing detection method

[0215]

[0216] The above comparison shows that the current conventional cytotoxicity detection is usually an endpoint method, which cannot perform continuous multiple rounds of detection and has certain limitations; and the label-free cell analyzer (RTCA) method that can realize real-time dynamic detection is costly and can only detect target cells of the adherent type. The cytotoxicity detection method based on the real-time dynamic imaging system of living cells of the present invention can detect the sustained killing ability of cells by adding K562 tumor cells in multiple rounds, and can better distinguish the cytotoxicity of NK cell products. In addition, the cell types and supported culture systems of the method of the present invention are more extensive, low cost, and easy to operate, and it is a more general cytotoxicity detection method.

[0217] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for real-time dynamic detection of effector cell killing ability, characterized in that: Includes steps: A. Construction of target cells stably expressing fluorescent proteins; B. providing effector cells to be detected; dividing the target cells in step A into an experimental group and a control group, wherein the effector cells are added to the experimental group for co-culture, and the control group contains only target cells, and other experimental conditions of the experimental group and the control group are kept consistent; C. At one or more time points during the co-culture process, the fluorescence intensity of the fluorescent protein in the experimental group and the control group is detected; for each time point, the fluorescence intensity of the experimental group is recorded as R1, and the fluorescence intensity of the control group is recorded as R0, and the cell killing ability C at that time point is calculated using the following formula I: C(%)=(1-R1 / R0)×100% (Formula I).

2. The method according to claim 1, characterized in that Step A further comprises: A1. Construct a recombinant vector with a fluorescent protein encoding gene; A2. transfecting the recombinant vector into target cells; A3. Screen stably transfected cell lines to obtain target cells that stably express the fluorescent protein.

3. The method according to claim 1, characterized in that The fluorescent protein is green fluorescent protein (GFP).

4. The method according to claim 1, characterized in that The target cells are tumor cells cultured in adherent or suspended form.

5. The method according to claim 1, characterized in that In step B, the effector cells are genetically engineered or unengineered cells selected from the group consisting of NK cells, T cells, or a combination thereof.

6. The method according to claim 5, characterized in that The effector cells are chimeric antigen receptor-NK (CAR-NK) cells.

7. The method according to claim 1, characterized in that After 6 h of co-culture in step B, the detection in step C was performed.

8. The method according to claim 1, characterized in that The method further comprises the steps of: D. Add target cells to the experimental group again, repeat step C after co-culture, and calculate the killing ability of effector cells on target cells in the current round; E. Repeat step D to calculate the killing ability of effector cells on multiple rounds of target cells.

9. The method according to claim 1, characterized in that The method is a quality control method, which is used for quality control of the production of immune products.

10. A system for real-time dynamic detection of effector cell killing ability, characterized in that: Includes the following modules: (M1) an experimental module, which is configured to include a cell culture container, and effector cells to be detected and target cells located in the container, wherein the target cells stably express fluorescent proteins; the experimental module is used to co-culture the effector cells to be detected and the target cells; (M2) a control module, which is configured to include a cell culture container, wherein the container contains only target cells; the control module is used to culture the target cells as a control; (M3) a fluorescence detection module, used for real-time dynamic detection of the fluorescence intensity in the experimental module and the control module; (M4) An output module is used to output the fluorescence intensity value detected by the fluorescence detection module, wherein the fluorescence intensity of the experimental module is recorded as R1, and the fluorescence intensity of the control module is recorded as R0, and the cell killing ability C is calculated and output using the formula of the following formula I: C(%)=(1-R1 / R0)×100% (Formula I).

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