Cell composition, preparation method and application thereof

Abstract

The invention relates to the field of biological detection, in particular to a cell composition as well as a preparation method and application thereof. The invention provides a cell composition. The cell composition comprises effector cells and target cells, the effector cells are obtained by performing primary activation, secondary activation, culture and cell collection on PBMC cells; the first activation adopts a CD3 monoclonal antibody; in the second activation, rhIL-2, rhIL-4, rhG-CSF, rhTNF-alpha and rhIFN-gamma are adopted; the target cells are obtained after HLA high-resolution typing screening. The invention provides a method for detecting the killing activity of a human in-vitro immune cell activation preparation on tumor cells. The effectiveness of the prepared preparation for treating tumor diseases is quickly, effectively and intuitively evaluated.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a cell composition, a preparation method and application thereof. Background Art

[0002] Human in vitro activated immune cell preparation is an autologous immune cell prepared by in vitro activation process using antigen presenting cells combined with CD3 monoclonal antibody and specific cytokines, which has both the non-specific killing activity of CIK and the specific killing activity of DC. At present, human in vitro activated immune cell preparation has obvious effects on autologous breast cancer, colorectal cancer, melanoma and non-small cell lung cancer.

[0003] Safety, effectiveness, and quality control are the basic attributes of drugs. In vitro activated immune cell preparations are therapeutic biological products, and effectiveness is an important evaluation indicator. In the prior art, the methods for evaluating the effectiveness of immune cell preparations mainly include model animal method, isotope labeled target cell method, enzyme reaction colorimetry, flow cytometry, etc. The model animal method has high accuracy, but the cycle is too long and the success rate is low, so it cannot be used for routine release testing of cell preparations. The isotope release test in the isotope labeling method has high protection requirements, threats to human health and the environment, and other factors, and can only detect the mortality rate of target cells. The enzyme reaction colorimetry method has low sensitivity and repeatability, and the time required is long, and the batch-to-batch difference is large. Flow cytometry is not suitable for detecting cells in an early apoptotic state. Therefore, it is particularly important to develop a method that can be visualized, dynamic, fast, accurate, and stable to evaluate and release the effectiveness of in vitro activated immune cells. Summary of the invention

[0004] In view of this, the present invention provides a cell composition, a preparation method and its application. The present invention provides a method for detecting the killing activity of human in vitro activated immune cell preparations on tumor cells, which quickly, effectively and intuitively evaluates the effectiveness of the prepared preparations in treating tumor diseases. The present invention screens out tumor cell lines whose HLA-A, B, and C loci are homozygous and whose allele distribution frequency in Chinese people is greater than 2.500% through HLA high-resolution typing. It can match with human in vitro activated immune cell preparations with a high probability, reduce the interference caused by irrelevant HLA, and more accurately evaluate the killing activity of human in vitro activated immune cell preparations; then use lentivirus to transfect target cells that can stably express GFP and Luc dual fluorescence, and after co-culturing with effector cells for a certain period of time, the fluorescence intensity of GFP can be observed by fluorescence microscopy, which can intuitively, visually and dynamically reflect the degree of killing, and provide an intuitive basis for the detection of the killing activity of human in vitro activated immune cells on tumor cells. On the other hand, the present invention can also quantitatively and simply obtain Luc RLU through an ELISA instrument to calculate the killing activity of in vitro activated immune cells against tumor cells, providing data support for the detection of the killing activity of in vitro activated immune cells against tumor cells, and the two complement each other.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a cell composition, comprising: effector cells and target cells;

[0007] The effector cells are obtained by first activating, second activating, culturing and collecting PBMC cells;

[0008] The first activation uses CD3 monoclonal antibody; the second activation uses: rhIL-2, rhIL-4, rhG-CSF, rhTNF-α and rhIFN-γ;

[0009] The target cells are obtained after HLA high-resolution typing screening;

[0010] The HLA high-resolution typing includes: HLA-A locus typing, HLA-B locus typing and HLA-C locus typing;

[0011] The screening criteria include: the HLA-A site typing, the HLA-B site typing and the HLA-C site typing alleles are all homozygous, and the distribution frequency of the HLA-A site typing, the HLA-B site typing and the HLA-C site typing alleles is not less than 2.500%.

[0012] In some embodiments of the present invention, in the above-mentioned cell composition, the concentration of the CD3 monoclonal antibody is 2 μg / mL; the concentrations of the cytokines are: 600 IU / mL of rhIL-2, 60 IU / mL of rhIL-4, 6000 IU / mL of rhG-CSF, 300 IU / mL of rhTNF-α and 6000 IU / mL of rhIFN-γ.

[0013] In some embodiments of the present invention, in the above-mentioned cell composition, the first activation time is 3 hours; the second activation time is 5 hours.

[0014] In some embodiments of the present invention, in the above cell composition, the concentration of the PBMC cells is 1×10 6 / mL.

[0015] In some embodiments of the present invention, in the above-mentioned cell composition, the target cells are marked with a detection signal, and the detection signal includes a fluorescent protein and / or a luciferase; the fluorescent protein includes: green fluorescent protein; the luciferase includes: firefly luciferase.

[0016] In some embodiments of the present invention, in the above cell composition, the target cells include: cell lines of non-small cell lung cancer.

[0017] In some embodiments of the present invention, in the above-mentioned cell composition, the target cells include: one or more of A549 cells, H1975 cells, H1299 cells, PC-9 cells, NCI-H1975 cells and HCC827 cells.

[0018] In some embodiments of the present invention, in the above cell composition, the target cell is: HCC827 cell.

[0019] In some embodiments of the present invention, in the above-mentioned cell composition, the effector-target ratio of the effector cells to the target cells is: (10-20):1.

[0020] In some embodiments of the present invention, in the above-mentioned cell composition, the effector-target ratio is 10:1, 15:1 or 20:1.

[0021] In some embodiments of the present invention, in the above-mentioned cell composition, the step of freezing after collecting cells is further included; the freezing step uses a freezing solution; the freezing solution includes: 15%~35% (v / v) human serum albumin, 4%~6% (v / v) DMSO, 15%~50% (v / v) hydroxyethyl starch solution, 100~200μg / mL recombinant human interleukin-2 and 10~50μg / mL human interferon γ.

[0022] The present invention also provides a method for preparing the above cell composition, wherein the effector cells are inoculated into the target cells and cultured to obtain the cell composition; the culture time is 50 to 76 hours.

[0023] In some embodiments of the present invention, in the above preparation method, the culturing time is 50 h, 54 h, 70 h or 76 h.

[0024] In some embodiments of the present invention, in the above preparation method, the culturing time is 50 hours.

[0025] The present invention also provides a freezing solution, comprising: 15% to 35% (v / v) human serum albumin, 4% to 6% (v / v) DMSO, 15% to 50% (v / v) hydroxyethyl starch solution, 100 to 200 μg / mL of recombinant human interleukin-2 and 10 to 50 μg / mL of human interferon γ.

[0026] In some embodiments of the present invention, the freezing solution comprises: 25% (v / v) human serum albumin, 5% (v / v) DMSO, 35% (v / v) hydroxyethyl starch solution, 150 μg / mL recombinant human interleukin-2 and 25 μg / mL human interferon γ.

[0027] The present invention also provides the use of the above cell composition and / or the cell composition obtained by the above preparation method in preparing a product for detecting the killing activity of effector cells on target cells.

[0028] In some embodiments of the present invention, in the above application, the detection comprises the following steps:

[0029] S1: The first activation of the effector cells: the PBMC cells are inoculated into a culture carrier pre-coated with the CD3 monoclonal antibody;

[0030] S2: the second activation of the effector cells: mixing the cells obtained by the first activation with the cytokines;

[0031] S3: Obtaining effector cells: culturing, centrifuging, collecting, and freezing the cells obtained by the second activation to obtain the effector cells;

[0032] S4: Screening of target cells: The target cells are tumor cell lines that are homozygous for HLA-A, HLA-B, and HLA-C alleles by HLA high-resolution typing, and whose distribution frequencies of the alleles of the HLA-A locus typing, the HLA-B locus typing, and the HLA-C locus typing are not less than 2.500%;

[0033] S5: Construction of the target cell labeled with the detection signal: transfecting the target cell with a lentiviral vector containing the detection signal to obtain the target cell labeled with the detection signal;

[0034] S6: co-culture: the effector cells obtained in S3 are inoculated into the target cells obtained in S5 to obtain a test group, serum-free culture medium is inoculated into the target cells obtained in S5 to obtain a control group, and the test group and the control group are co-cultured;

[0035] S7: Obtain the test results: After taking a picture of the cell composition after S6 co-culture using 488nm excitation light, discard the supernatant, mix the precipitate with the substrate solution, and incubate, obtain the test results based on the fluorescence intensity value, and judge the killing activity of the effector cells based on the picture and the value; the substrate solution includes: D-fluorescein sodium and a cell disruptor.

[0036] In some embodiments of the present invention, in the above application, the freezing is carried out using a freezing solution, which includes: 15%~35% (v / v) human serum albumin, 4%~6% (v / v) DMSO, 15%~50% (v / v) hydroxyethyl starch solution, 100~200μg / mL recombinant human interleukin-2 and 10~50μg / mL human interferon γ.

[0037] In some embodiments of the present invention, in the above application, the detection comprises the following steps:

[0038] S1: The first activation of the effector cells: the PBMC cells are inoculated into a culture carrier pre-coated with the CD3 monoclonal antibody, and the concentration of the CD3 monoclonal antibody is 2 μg / mL;

[0039] S2: The second activation of the effector cells: the cells obtained by the first activation are mixed with the cytokines, the types and concentrations of the cytokines are: 600 IU / mL of rhIL-2, 60 IU / mL of rhIL-4, 6000 IU / mL of rhG-CSF, 300 IU / mL of rhTNF-α and 6000 IU / mL of rhIFN-γ;

[0040] S3: Obtaining effector cells: culturing, centrifuging, collecting and freezing the cells obtained by the second activation to obtain the effector cells, wherein the freezing solution used for the freezing includes: 15%~35% (v / v) human serum albumin, 4%~6% (v / v) DMSO, 15%~50% (v / v) hydroxyethyl starch solution, 100~200μg / mL recombinant human interleukin-2 and 10~50μg / mL human interferon γ;

[0041] S4: Screening of target cells: The target cells are tumor cell lines that are homozygous for HLA-A, HLA-B, and HLA-C alleles by HLA high-resolution typing, and whose distribution frequencies of the alleles of the HLA-A locus typing, the HLA-B locus typing, and the HLA-C locus typing are not less than 2.500%;

[0042] S5: Construction of the target cell marked with a detection signal: transfecting the target cell with a lentiviral vector containing a detection signal to obtain the target cell marked with a detection signal; the detection signal includes a fluorescent protein and / or a luciferase; the fluorescent protein includes: green fluorescent protein; the luciferase includes: firefly luciferase;

[0043] S6: Co-culture: The effector cells obtained in S3 are inoculated into the target cells obtained in S5 to obtain a test group, and serum-free culture medium is inoculated into the target cells obtained in S5 to obtain a control group. The test group and the control group are co-cultured for 50-76 hours, and the effector-target ratio of the effector cells to the target cells is: (10-20):1;

[0044] S7: Obtain the test results: After taking a picture of the cell composition after S6 co-culture using 488nm excitation light, discard the supernatant, mix the precipitate with the substrate solution, and incubate, obtain the test results based on the fluorescence intensity value, and judge the killing activity of the effector cells based on the picture and the value; the substrate solution includes: D-fluorescein sodium and a cell disruptor.

[0045] In some embodiments of the present invention, in the above application, the green fluorescent protein includes: GFP; the luciferase includes: Luciferase.

[0046] In some embodiments of the present invention, in the above application, the method for constructing the lentiviral vector containing the detection signal comprises the following steps:

[0047] S1: obtaining a recombinant vector containing the detection signal, transforming and / or transfecting competent cells, incubating, and obtaining a host;

[0048] S2: extracting the host plasmid and the auxiliary plasmid to transform and / or transfect 293T cells, culturing, centrifuging, collecting the supernatant, and obtaining the lentiviral vector containing the fluorescent protein and / or the luciferase.

[0049] In some embodiments of the present invention, in the above application, the recombinant vector is pCDH-MSCV-MCS-luc-EF1α-GFP-T2A-Puro, including: MSCV promoter, multiple cloning site, EF1α promoter, T2A and Puro sites.

[0050] In some embodiments of the present invention, in the above application, the auxiliary plasmid includes: psPAX2 and pMD2G.

[0051] In some embodiments of the present invention, in the above application, the concentration of the target cells is 0.6SYMBOL 180 \f "Symbol" \s 14´SYMBOL 180 \f "Symbol" \s 14SYMBOL 180 \f "Symbol" \s 14´´SYMBOL 180 \f "Symbol" \s 14´10 6 / mL.

[0052] In some embodiments of the present invention, in the above application, the substrate solution comprises: 600 μM D-fluorescein sodium and 2.0% (v / v) Triton-X.

[0053] In some embodiments of the present invention, the target cells further include the steps of recovery and plating; the cell concentration during recovery is 1.7-2×10 6 The cell concentration during plating was 1×10 5 Pieces / mL.

[0054] In some embodiments of the present invention, in the above application, the calculation formula of the test result is: (1-fluorescence value RLU of the test group / fluorescence value RLU of the control group)×100%.

[0055] The beneficial effects of the present invention include:

[0056] (1) The detection method of the present invention is simple and has a wide range of applications. It can visually and dynamically observe the killing situation and provide detection data values, thereby providing intuitive and reliable image evaluation basis and data basis for the effectiveness of the cell preparation of the present invention.

[0057] (2) The effector cells of the present invention have no spontaneous fluorescence and do not interfere with the detection of GFP and Luc during detection, and only reflect the killing activity on target cells. The detection method of the present invention has high precision and repeatability.

[0058] (3) The present invention provides the optimal parameters for evaluating the killing activity of human in vitro activated immune cell preparations on tumor cells by exploring the culture system, which greatly improves the accuracy of the test.

[0059] (4) The present invention optimizes the freezing and resuscitation conditions of effector cells, and the effector cells still maintain a high activity after being frozen for 6 months, which provides a reference basis for release testing in quality research and can achieve rapid release of products. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0061] Figure 1 Luc PCR amplification product is shown;

[0062] Figure 2 Schematic diagram of virus titer determination in GFP Jurka cell FITC channel; A shows negative control; B shows lentivirus solution dilution of 10 -5 ; C shows that the dilution of the lentivirus solution is 10 -4 ; D shows that the dilution of the lentivirus solution is 10 -3 ; E shows that the dilution of the lentivirus solution is 10 -2 ; F shows that the dilution of the lentivirus solution is 10 -1 ;

[0063] Figure 3 The figure shows the construction of HCC827-Luc-GFP target cells and the detection of GFP expression (magnification is 10×); A shows HCC827 cells growing on the wall; B shows the GFP gene expression in HCC827-Luc-GFP cells observed under an inverted fluorescence microscope;

[0064] Figure 4 The figure shows the killing of HCC827-Luc-GFP cells by in vitro activated immune cell preparations at different effector-target ratios (co-culture time is 50 h) (magnification is 10×); among them: A is the target cell control; B is E / T of 5:1; C is E / T of 10:1; D is E / T of 15:1; E is E / T of 20:1;

[0065] Figure 5 The figure shows the killing of HCC827-Luc-GFP cells by in vitro activated immune cell preparations at different co-culture times (effect-target ratio is 5:1, 10:1); wherein: A is the target cell control; B is E / T 5:1, 50h; C is E / T 5:1, 54h; D is: E / T 5:1, 72h; E is E / T 5:1, 76h; F is the target cell control; G is E / T 10:1, 50h; H is E / T 10:1, 54h; I is E / T 10:1, 72h; J is E / T 10:1, 76h;

[0066] Figure 6 The killing effect of in vitro activated immune cells on HCC827-Luc-GFP cells at different effector-target ratios (co-culture time is 50h);

[0067] Figure 7The figure shows the killing effect of in vitro activated immune cells on HCC827-Luc-GFP cells at different co-culture times (effect-target ratios are 5:1 and 10:1). DETAILED DESCRIPTION

[0068] The invention discloses a cell composition, a preparation method and application thereof.

[0069] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0070] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0071] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0072] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0073] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0074] The relevant terms involved in the present invention are:

[0075] Luc: Luciferase (Luc for short) is a class of enzymes that can catalyze substrates to produce biological fluorescence.

[0076] GFP: Green Fluorescent Protein, a protein of great value in biological research, is a protein composed of 238 amino acids. It has the characteristics of self-luminescence and high stability.

[0077] RLU: Relative light unit, is a relative value used to measure the luminous intensity of a sample.

[0078] Killing activity: In the present invention, human in vitro activated immune cell preparations are used as effector cells, and tumor cells stably expressing green fluorescent protein and luciferase are used as target cells. They are co-cultured in a certain ratio for a certain period of time and the death rate of target cells is detected by the detection method of the present invention. That is, the death rate of target cells can be used to express the killing activity of effector cells.

[0079] The human in vitro activated immune cell preparation described in the present invention is the effector cell.

[0080] The raw materials and reagents used in Examples 1 to 6 of the present invention can all be purchased from the market.

[0081] The present invention will be further described below in conjunction with embodiments:

[0082] Example 1 Preparation of human in vitro activated immune cell preparation

[0083] 1.1 Methods

[0084] 1.1.1 Isolation of mononuclear cells (PBMC): After single blood or peripheral blood is diluted with compound electrolyte injection, it is separated by density gradient centrifugation using lymphocyte separation solution; after the cells are mixed with serum-free culture medium, the viability of the obtained cells is analyzed by automatic image analysis.

[0085] 1.1.2 Mononuclear cells were cultured in serum-free medium at a concentration of 1×10 6 / mL was inoculated into a T25 bottle pre-coated with CD3 monoclonal antibody for the first step of activation for 3 hours, and the CD3 monoclonal antibody concentration was 2μg / mL.

[0086] 1.1.3 Take out the pre-cultured PBMCs and add a combination of cytokines including rhIL-2, rhIL-4, rhGM-CSF, rhTNF-α, and rhIFN-γ for the second step of in vitro activation culture. Continue culturing for 5 hours. The concentrations of rhIL-2, rhIL-4, rhGM-CSF, rhTNF-α, and rhIFN-γ are 6000 IU / mL, 300 IU / mL, and 6000 IU / mL, respectively.

[0087] 1.1.4 Collect the cell suspension and centrifuge it. Mix the cells with serum-free medium and add 3.0×10 5 The cells were inoculated into culture flasks or culture bags at a concentration of 10 viable cells / mL and cultured for 120 h.

[0088] 1.1.5 After collecting the cell suspension and centrifuging it, mix the cells with compound electrolyte injection and analyze the viability of the obtained cells using automatic image analysis.

[0089] 1.2 Results

[0090] The results in Table 1 show that the viability of immune cells obtained by the method in 1.1 is as high as 93.7%.

[0091] Table 1 Viability of immune cells activated in vitro

[0092]

[0093] Example 2 Optimization of Effector Cell Cryopreservation Solution Composition

[0094] 2.1 Methods

[0095] Cryopreservation solutions with different component contents were prepared as experimental groups and control groups were set up. The experimental groups are shown in Table 2. The components and contents of the control group were: 75% (v / v) serum-free medium, 15% (v / v) FBS, and 10% (v / v) DMSO. The in vitro activated immune cells obtained according to Example 1 were placed in the cryopreservation solution and frozen at 38.5°C. After 2 days of freezing, the cells were revived and the cell viability after resuscitation was measured.

[0096] The method for determining cell viability is as follows: take 50 μL of the resuscitated cell suspension into a 96-well cell culture plate, add 50 μL of AO / PI dye solution to the cell suspension, and mix by pipetting with a 100 μL pipette. Take 20 μL from the middle of the suspension, add it to one side of a disposable cell counting plate, and insert the end of the sample into the sample slot of the Cellometer K2 dual fluorescence cell analyzer. Use the program set by the analyzer software to count live cells and evaluate viability.

[0097] Table 2 Cryopreservation solution composition and dosage

[0098]

[0099] 2.2 Results

[0100] The results in Table 3 show that the viability of the test group was significantly improved compared with the control group. The viability of immune cells after being cryopreserved in cryopreservation solution for 2 days can be maintained at more than 90%, and can reach up to 95%.

[0101] Table 3 Effects of cryopreservation solution on the viability of human immune cells activated in vitro

[0102]

[0103] Example 3 Screening of tumor cell lines for detection

[0104] 3.1 Methods

[0105] A549 cells, H1975 cells, H1299 cells, PC-9 cells, NCI-H1975 cells, and HCC827 cells were obtained from the working library for HLA typing detection with an accuracy of 4 quartiles.

[0106] 3.2 Results

[0107] HCC827 HLA-A*11:01:01, HLA-B*52:01:01, HLA-C*12:02:02, are all genotypes with a high probability of distribution in Chinese people, with distribution frequencies of 20.893%, 2.991%, and 3.139%, respectively. One is selected from A549 cells, H1975 cells, H1299 cells, PC-9 cells, NCI-H1975 cells, and HCC827 cells; preferably HCC827.

[0108] Example 4 Method for constructing a Luc-GFP dual fluorescently labeled target cell line

[0109] 4.1 Construction of the pCDH-MSCV-MCS-Luc-EF1α-GFP-T2A-Puro lentiviral vector containing green fluorescent protein gene and luciferase gene

[0110] (Note: pCDH-MSCV-MCS-Luc-EF1α-GFP-T2A-Puro lentiviral vector backbone: MSCV promoter: Murine Stem Cell Virus (MSCV) promoter, which can drive gene expression in specific cell types.

[0111] Multiple cloning site (MCS): provides multiple restriction endonuclease recognition sites to facilitate the insertion of target genes.

[0112] EF1α promoter: Elongation factor 1α promoter, which has high transcriptional activity and can drive downstream gene expression.

[0113] GFP: Green fluorescent protein gene, which can make cells expressing this plasmid emit green fluorescence and is used for cell marking and tracking.

[0114] T2A: A self-cleaving peptide sequence that promotes the independent expression of two proteins during translation.

[0115] Puro: Puromycin resistance gene, used to select stable transfected cells.

[0116] 4.1.1 PCR amplification of luciferase gene

[0117] Using psiCHECK-2 as a template, according to the restriction sites at both ends of the pCDH-MSCV-MCS-EF1α-GFP (Cat. No.: CD713B-1) vector backbone, Primer Premier 6 software was used to design the primer Luc-F: 5'-TG CTAGA GAAGACGCCAAAAACATAAAGAAAGGC-3' (as shown in SEQ ID NO: 1), Luc-R: 5'-TCA CCAAG CAATTTGGACTTTCCGCCCTTC-3' (as shown in SEQ ID NO:2) (Note: the underlined CTAGA is the XbaⅠ restriction site; the underlined CCAAG is the BamHI restriction site) was PCR amplified (95℃ 60s1cycle, 95℃ 50s, 68℃ 90s 35 cycles, 68℃ 180s) Luc target gene. The PCR product was electrophoresed by 1.0% agarose gel and analyzed by UVITEC gel imaging system, and a 1653bp fragment was selected. The target gene fragment was recovered using the Invitrogen DNA purification and recovery kit. The target gene and pCDH-MSCV-MCS-EF1α-GFP vector were double-digested with XbaⅠ and BamHI sites, and then the pCDH-MSCV-MCS-Luc-EF1α-GFP vector recombinant plasmid was constructed using T4 DNA ligase.

[0118] 4.1.2 Transformation of competent cells into DH5α cells with pCDH-MSCV-MCS-Luc-EF1α-GFP vector

[0119] 1): Take 3 tubes of Subcloning Efficiency™ DH5α™ competent cells, 50μL each, add 1~5μL of pCDH-MSCV-MCS-Luc-EF1α-GFP vector, numbered as tube A, 2.5μL of pUC 19 control as positive control, numbered as B, and 2.5μL of sterilized ddH2O as negative control, numbered as C. Ice-bathe the ABC 3-tube transformation system for 30 min, heat-shock at 42℃ for 20 seconds, and then ice-bathe for 2 min. Add 50μL of antibiotic-free LB liquid medium to each tube and incubate in a 37℃ shaker at 225 rpm / min for 1h.

[0120] 2): Take 50μL, 100μL, 150μL of tube A, 100μL of tube B, and 100μL of tube C from step 1) and spread them on LB plates containing 60 μg / mL ampicillin, and culture at 37°C for 16-18h.

[0121] 3): Use an inoculation loop to pick up a single clone, select a small, transparent, full, isolated single clone and add it to a 250mL LB conical flask, and place it in a 37℃ shaker at 225 rpm / min for 18~24h.

[0122] 4): Collect the bacterial solution and use the PureLink™ Expi Endotoxin-Free Large Plasmid Purification Kit to extract the plasmid. Place the obtained plasmid in an Eppendorf tube and store it at -20°C for later use.

[0123] 5): The concentration and purity of the recombinant plasmid were detected by the multifunctional microplate reader A260 / A280, which should be between 1.8 and 2.0.

[0124] 4.2 Preparation of recombinant lentivirus solution

[0125] 4.2.1 Take out the working bank 293T cells, resuscitate at 37℃, and adjust the concentration to 3.5~7.0×10 6 Inoculate 1 mL of the culture medium into a 15 cm cell culture dish, add the culture medium to 20-25 mL, place in a 5% CO2 incubator and culture at 37°C until the cell confluence reaches 80%-90%, then take out for later use.

[0126] 4.2.2 Dissolve the three plasmids containing 11μg pCDH-MSCV-MCS-Luc-EF1α-GFP, 8.5μg psPAX2, and 2.8μg pMD2G in 1mL OPTI-MEM respectively, and mix them to make transfection solution 1; take 48μL Invitrogen Lipofectamine™ 2000 transfection reagent and dilute it in 1mL OPTI-MEM to make transfection solution 2; then mix 1 and 2 and let it stand at room temperature for 10-15min to form DNA-Lipofectamine complex.

[0127] 4.2.3 Slowly add the transfection solution in step 4.2.2 to the cell culture dish in step 4.2.1, place the dish in a 5% CO2 incubator for 8-12 hours, discard the culture medium containing the transfection solution, and add 20mL-25mL of DMEM containing 1mmol / L sodium pyruvate and 10% FBS at 37°C. Collect the virus-containing supernatant twice 48h-72h after transfection, centrifuge at 2000rpm for 10min at 4°C, take the supernatant, and store it at -80°C for later use.

[0128] 4.2.4 Virus titer determination

[0129] 1): Take out the Jurka cells from the working library, resuscitate at 37°C, and adjust the concentration to 4.0×10 5 / mL, take 0.5mL and inoculate into 18 wells of a 24-well cell culture plate, put it in a 5% CO2 incubator at 37℃ for overnight, and then take it out for use.

[0130] 2): Add 10 -1 Up to 10 -5 0.5mL of virus supernatant of different dilutions, and no virus supernatant was added to the other 3 wells as negative control, with 3 replicates for each gradient. Samples were taken after 48h, and 150μL of the above 6 groups of different cell suspensions were added to 150μL of luciferase substrate respectively, and immediately placed in a multifunctional microplate reader to detect Luc RLU. The remaining cells were washed twice with DPBS, centrifuged at 350g for 5 min, and the supernatant was discarded. The cell pellet was resuspended with 200~500μL Cell staining buffer, and then loaded and tested with a Beckman CytoFLEX flow cytometer. The sample data was obtained using its supporting CytExpert2.5 software, and the gate was set according to the cell light scattering characteristics, and the data was analyzed using NovoExpress software. The FITC channel histogram was created based on the gate, and the negative control gate was used to determine the positive proportion of the FITC channel, and then the virus titer was calculated according to the formula: cell positive rate × total number of cells at transduction × virus dilution multiple / virus volume.

[0131] 4.3 Construction of Luc-GFP dual fluorescently labeled non-small cell lung cancer cell line

[0132] 4.3.1 Take HCC827 cells with good growth status and adjust the cell density to 0.6SYMBOL 180 \f "Symbol"\s 14´SYMBOL 180 \f "Symbol" \s 14SYMBOL 180 \f "Symbol" \s 14´´SYMBOL 180 \f"Symbol" \s 14´10 6 / mL, 1 mL per well was inoculated in a 6-well culture plate, and the culture medium was added to each well to a total culture volume of 2 mL, for a total of 4 wells. 5% CO2, saturated humidity, 37°C static culture until the confluence reached 80%.

[0133] 4.3.2 1 mL, 0.75 mL, 0.50 mL, and 0.25 mL of the GFP fluorescent protein and Luc lentiviral vector prepared in Example 3 were added to construct target cells, which were numbered A, B, C, D, and E, respectively, and placed in 5% CO2, saturated humidity, and cultured at 37°C.

[0134] 4.3.3 When the cell confluence reaches 90%, transfer to T25 for expansion culture, use puromycin to select HCC827-Luc-GFP resistant cells, and take photos under a fluorescence microscope to detect fluorescence when the cell confluence reaches about 90% again. Use ImagPro Plus 6.0 to analyze the photos, select cells with a positive rate greater than 95% and the best cell growth status, expand culture again, and establish a target cell master cell bank.

[0135] 4.4 Results

[0136] 4.4.1 Lentivirus titer determination results

[0137] Figure 1 The results showed that the Luc target gene had been successfully inserted into the recombinant plasmid. The results in Table 4 showed that as the dilution of the virus solution increased, the fluorescence value of Jurkat Luc gradually decreased, indicating that the Luc gene was successfully transfected. Figure 2 A- Figure 2 The results of F showed that the harvested lentiviral supernatant was used to infect Jurka cells at different dilutions. After 48 hours, the FITC channel positive percentage measured by flow cytometry gradually decreased with the increase of virus liquid dilution, with the highest being 90.45%. According to the formula: Luc-GFP lentiviral titer = cell positive rate × total number of cells at transduction × virus dilution multiple / virus volume, it was calculated to be 1.83×10 7 GFPTU / mL.

[0138] Table 4 Jurka cell luciferase activity assay data of this example

[0139]

[0140] 4.4.2 Results of Luc-GFP dual fluorescent labeling of non-small cell lung cancer cell lines

[0141] The results of Imag Pro Plus 6.0 analysis showed that the HCC827-Luc-GFP positive rate was 96% in group A, 82% in group B, 61% in group C, 48% in group D, and 35% in group E, and the cells were growing well. Figure 3 The results of group A are shown. The cell morphology of HCC827-Luc-GFP is shown in Figure 3 A, Fluorescence image of GFP expression Figure 3 B.

[0142] Example 5 Establishment of a method for detecting the killing activity of human in vitro activated immune cell preparations on tumor cells

[0143] 5.1 Basic steps

[0144] 5.1.1 Target cell resurrection culture

[0145] After the HCC827-Luc-GFP prepared according to Example 4 was revived, it was directly plated or cultured for 48 to 72 hours after resuscitation, the viability was counted, the concentration was adjusted, and then plated, and then co-cultured with effector cells for a period of time to examine its killing activity.

[0146] 5.1.2 Target cell plating

[0147] Adjust the cell concentration in step 5.1 to 1×10 5 100 μL / well, plated on a 96-well plate, set up a target cell control group and an experimental group. Each well has 6 replicates. Place in 5% CO2, incubate at 37℃ in the dark for 20-24h, and culture the target cells in a monolayer on the bottom of the well.

[0148] 5.1.3 Resuscitation of human in vitro activated immune cell preparations

[0149] The human in vitro activated immune cell preparation obtained in Example 1 was taken out and placed in a 38.5°C resuscitation apparatus for resuscitation. The cell concentration was adjusted to 1×10 6 Pieces / mL.

[0150] 5.1.4 Effector cell plating

[0151] Take the cell culture plate from step 5.1.2 to a sterile area, discard the culture medium, and add the immune cell preparation from step 5.1.3 according to a certain effector-target ratio, 100 μL per well.

[0152] 5.1.5 Incubate at 5% CO2 and 37℃ for a period of time.

[0153] 5.1.6 Observe the killing situation under a fluorescence microscope. Use 488nm excitation light to take GFP images. Use the fluorescence microscope software provided by Nikon to collect images.

[0154] 5.1.7 Adding substrate solution

[0155] Aspirate the culture medium in the culture plate, add substrate solution at 100 μL / well, and mix on a horizontal shaker at room temperature for 10 min to 15 min.

[0156] 5.1.8 Luminescence Detection

[0157] The average fluorescence value data of the luminescence assay was measured using a multifunctional microplate reader with a chemiluminescence detection module 528 / 20 nm and the killing activity was calculated according to the formula.

[0158] 5.2 Optimization of effector-target ratio and co-culture time

[0159] 5.2.1 Initial optimization of effector-target ratio and co-culture time

[0160] According to the basic steps of 5.1, test groups were set up for the same batch of human in vitro activated immune cell preparations, with the effector-target ratios set to 5:1, 10:1, 15:1, and 20:1, and the co-culture time was set to 24h, 27h, 30h, 33h, 36h, 43h, and 46h, and the killing activity was calculated.

[0161] The results in Table 5 show that the killing activity of different effector-target ratios becomes negative before 33 hours of culture, and the killing activity is low between 33 and 46 hours, so the killing activity of effector cells cannot be evaluated. The optimal co-culture time should be more than 46 hours.

[0162] Table 5 Results of initial optimization of effector-target ratio and co-culture time

[0163]

[0164] 5.2.2 Second optimization of effector-target ratio and co-culture time

[0165] According to the basic steps in 5.1, for the same batch of human in vitro activated immune cell preparations, the co-culture time was set to 50h, 54h, 70h, and 76h, and the effector-target ratio was 1:1, 5:1, 10:1, and 40:1, respectively, and the killing activity was calculated.

[0166] The results in Table 6 show that when the effector-target ratio is 1:1, the killing activity is low and the killing activity of effector cells cannot be evaluated. When the effector-target ratio is 5:1-40:1, the killing activity is 59% or more at 50h-76h, and the highest can reach 99.52%. Therefore, the preferred effector-target ratio is between 5:1 and 40:1, and the preferred co-culture time is 50-76h, more preferably 50-70h. Since the effector cells required for 40:1 are too high, the preferred effector-target ratio is below 40:1.

[0167] Table 6 Results of the second optimization of effector-target ratio and co-culture time

[0168]

[0169] 5.2.3 Third optimization of effector-target ratio and co-culture time

[0170] According to the basic steps in 5.1, the effector-target ratio was set to 5:1, 10:1, 15:1, and 20:1, the co-culture time was selected to be 50 h, and the killing activity was calculated.

[0171] The results in Table 7 show that when the effector-target ratio is 10:1-20:1, the immune cells have a higher killing activity, especially when the effector-target ratio is 15:1 and above, the killing activity can reach more than 92%. Therefore, the preferred effector-target ratio is 10:1-20:1, and the more preferred effector-target ratio is 15:1-20:1.

[0172] Table 7 Results of the third optimization of effector-target ratio and co-culture time

[0173]

[0174] 5.3 Optimization of cryopreservation and thawing conditions of effector cells

[0175] According to the basic steps in 5.1, the effector cells were frozen for 0 month, 1 month, 3 months and 6 months and then revived. The revivification time conditions were 5±3℃ for 0h and 4h; 23±2℃ for 0h and 4h, and the survival rate of effector cells and the killing activity against target cells were tested.

[0176] The results in Table 8 show that the killing activity of effector cells against target cells after being revived after being frozen for 0, 1, 3, and 6 months is similar, and the cell viability fluctuates slightly, and the state is relatively stable. After being frozen for 6 months, the cell viability and killing activity can still be maintained at more than 90%. The results in Table 8 show that the killing activity of effector cells against target cells after being revived and stored at 5±3℃ and 23±2℃ for 0h and 4h is similar, and the cell viability fluctuates slightly, and the state is relatively stable.

[0177] Table 8 Killing activity of two batches of test products after thawing at different freezing times

[0178]

[0179] Table 9 Killing activity of two batches of test products under different storage conditions after resuscitation

[0180]

[0181] Example 6 Verification of the applicability of a method for detecting the killing activity of human in vitro activated immune cell preparations on tumor cells

[0182] 6.1 Methods

[0183] 6.1.1 Exclusivity

[0184] A target cell control group (HCC827 target cells), an effector cell control group (human in vitro activated immune cell preparation), a negative control group (lethal target cells), and a quality control group QC (target cell live cells + target cell dead cells) were set up. The detection was performed according to Example 5, and the fluorescence value RLU was determined. The fluorescence value CV of each sample was counted, compared with the target cell control group, and the recovery rate was calculated.

[0185] 6.1.2 Precision

[0186] In this example, three batches of test products were used, and six replicate samples were prepared from each batch of samples for parallel testing. The test was co-cultured for 72 hours at an effector-target ratio of 10:1, and the example data were obtained by two different testers.

[0187] 6.2 Results

[0188] 6.2.1 Specificity results

[0189] The average RLU of the negative control group and the effector cell control group were 10 and 3 respectively; the average RLU of the target cell control group was 1944 respectively; the RLU CV value of the target cell control group was less than 15%; the RLU CV value of the quality control group 1 and the quality control group 2 was less than 15%. Compared with the target cell control group, the recovery rate was between 94.39 and 97.07%. It shows that the method of the present invention has strong specificity, and the co-culture system of the method is not interfered by target cells and effector cells. Specific values ​​are shown in Table 10.

[0190] Table 10 Test data of the experimental group confirmation test

[0191]

[0192] 6.2.2 Precision results

[0193] The results in Table 11 show that two testers conducted three batches of independent repeated tests, and the repeatability (CV%) of each tester was 0.80-1.86%, and the precision (CV%) of the two testers was 1.00-1.58%, and their CV% was less than 5%. And no significant difference was found through variance analysis (P>0.05). It shows that the present invention has good repeatability. And it is not affected by the operation of different testers.

[0194] Table 11 Data of the precision (repeatability, intermediate precision) test example of the present invention

[0195]

[0196] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A cell composition, characterized in that include: effector cells and target cells; The effector cells are obtained by first activating, second activating, culturing and collecting PBMC cells; The first activation uses CD3 monoclonal antibody; the second activation uses: rhIL-2, rhIL-4, rhG-CSF, rhTNF-α and rhIFN-γ; The target cells are obtained after HLA high-resolution typing screening; The HLA high-resolution typing includes: HLA-A locus typing, HLA-B locus typing and HLA-C locus typing; The screening criteria include: the HLA-A site typing, the HLA-B site typing and the HLA-C site typing alleles are all homozygous, and the distribution frequency of the HLA-A site typing, the HLA-B site typing and the HLA-C site typing alleles is not less than 2.500%.

2. The cell composition according to claim 1, characterized in that The concentration of the CD3 monoclonal antibody is 2 μg / mL; the concentrations of the cytokines are: 600 IU / mL of rhIL-2, 60 IU / mL of rhIL-4, 6000 IU / mL of rhG-CSF, 300 IU / mL of rhTNF-α and 6000 IU / mL of rhIFN-γ.

3. The cell composition according to claim 1, characterized in that The first activation time is 3 hours; the second activation time is 5 hours.

4. The cell composition according to claim 1, characterized in that The target cells are marked with a detection signal, and the detection signal includes fluorescent protein and / or luciferase; The fluorescent protein includes green fluorescent protein; the luciferase includes firefly luciferase.

5. The cell composition according to claim 1, characterized in that The effector-target ratio of the effector cells to the target cells is (10-20):

1.

6. The cell composition according to claim 1, characterized in that The method further includes a freezing step after collecting cells; the freezing step uses a freezing solution; the freezing solution includes: 15%~35% (v / v) human serum albumin, 4%~6% (v / v) DMSO, 15%~50% (v / v) hydroxyethyl starch solution, 100~200μg / mL recombinant human interleukin-2 and 10~50μg / mL human interferon γ.

7. The method for preparing the cell composition according to any one of claims 1 to 6, characterized in that: The effector cells are inoculated into the target cells and cultured to obtain the cell composition; the culture time is 50 to 76 hours.

8. Use of the cell composition according to any one of claims 1 to 6 and / or the cell composition obtained by the preparation method according to claim 7 in preparing a product for detecting the killing activity of effector cells on target cells.

9. The use according to claim 8, characterized in that The detection comprises the following steps: S1: The first activation of the effector cells: the PBMC cells are inoculated into a culture carrier pre-coated with the CD3 monoclonal antibody; S2: the second activation of the effector cells: mixing the cells obtained by the first activation with the cytokines; S3: Obtaining effector cells: culturing, centrifuging, collecting, and freezing the cells obtained by the second activation to obtain the effector cells; S4: Screening of target cells: The target cells are tumor cell lines that are homozygous for HLA-A, HLA-B, and HLA-C alleles by HLA high-resolution typing, and whose distribution frequencies of the alleles of the HLA-A locus typing, the HLA-B locus typing, and the HLA-C locus typing are not less than 2.500%; S5: Construction of the target cell labeled with the detection signal: transfecting the target cell with a lentiviral vector containing the detection signal to obtain the target cell labeled with the detection signal; S6: co-culture: the effector cells obtained in S3 are inoculated into the target cells obtained in S5 to obtain a test group, the target cells obtained in S5 are inoculated into a serum-free medium to obtain a control group, and the test group and the control group are co-cultured; S7: Obtaining the test result: After taking a picture of the cell composition after co-culture in S6 using 488 nm excitation light, discarding the supernatant, mixing the precipitate with the substrate solution, and incubating, obtaining the test result according to the value of the fluorescence intensity, and judging the killing activity of the effector cells in combination with the picture and the value; The substrate solution comprises: D-fluorescein sodium and a cell disruptor.

Images