CN114591907A - Preparation and amplification method and application of gamma delta T cells - Google Patents

Abstract

The invention belongs to the technical field of cell biology, and relates to a method for separating and expanding peripheral blood-derived γδT cells. The present invention provides an in vitro method for isolating, expanding and modifying in vitro expanded γδT cells, comprising: obtaining or isolating γδT cells; and/or in the presence of zoledronic acid, human recombinant interleukin 2 (IL-2 ) and human recombinant interleukin 7 (IL-7) in the culture medium to expand the isolated γδ T cells. The present invention also provides the modification of γδT cells by using the chimeric antigen receptor, and the application thereof in the preparation of anti-tumor and anti-AIDS products.

Description

A kind of preparation and expansion method of γδT cell and application

technical field

The invention belongs to the technical field of cell biology, and relates to the separation, preparation, expansion and application of γδT cells. The invention also relates to a method for separating and expanding γδT cells derived from peripheral blood, a method for in vitro gene modification of γδT cells, and the application of the modified γδT cells in the field of anti-tumor.

Background technique

At present, T-cell immunotherapy for cancer continues to develop and achieve good clinical efficacy. However, barriers to allogeneic therapy remain. γδT cells do not need specific antigen stimulation when they exert their killing activity, are not restricted by major histocompatibility complex (MHC), have good anti-tumor activity and a variety of biological functions, and are the immune system of tumor cells. A powerful tool for healing.

According to the different T cell receptors (TCRs), human T lymphocytes are divided into αβ T cells and γδ T cells. αβT cells are the main immune T cells in the body and have the cytotoxic function of specific antigen recognition mediated by the major histocompatibility complex (MHC). In recent years, γδT cells with non-MHC restriction have gradually attracted the attention of researchers. Under normal circumstances, γδT cells account for less than 5% of the total number of peripheral blood T cells, mainly distributed in the skin, small intestine, esophagus, lung, reproductive organs, etc., and are non-specific immune cells. As important effector T cells, γδT cells can secrete important cytokines that kill tumor cells, such as interferon γ (IFN-γ) and interleukin (IL)-17A, so they have strong killing activity.

Salt baked shows that when the γδT cells are engineered to express chimeric co-stimulatory receptors, they can specifically recognize tumor cell targets and thereby specifically kill target cells. Moreover, compared with single-antibody-based CARs that cannot distinguish between tumors and healthy cells expressing the target antigen, genetically modified γδ T cells do not have the risk of tumor off-target (on-target/off-tumor) toxicity. Compared with CAR-T cells with antigen-independent (tonic) signaling, genetically modified γδ T cells have a stronger ability to proliferate and are less prone to exhaustion.

To date, commonly used expansion methods such as IL-2, IL-15, IL-18 and other culture compound components expand γδT cells, which can increase total γδT cells 100-1000-fold within 14 days; after that, the expansion rate decreases , which is consistent with an increase in cell death. Therefore, traditional γδT expansion protocols cannot generate sufficient numbers of cells to qualify for a commercially viable allogeneic product.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method for expanding γδT cells derived from human peripheral blood in a large amount and rapidly based on the current state of the art.

Another object of the present invention is to prepare γδT cells with the effect of targeting and killing tumor cells.

Another object of the present invention is to provide the application of the above-mentioned γδT cells.

In one aspect, the present invention provides methods of isolating and expanding γδT cells comprising isolating γδT cells from a blood sample of a human subject, expanding the isolated γδT cells in the presence of zoledronic acid and a cytokine composition Cells can be expanded up to 1500-fold.

The present invention provides a method for expanding γδT cells in vitro, the method comprising:

obtaining or isolating γδ T cells; and/or

The γδ T cells were expanded in culture medium containing zoledronic acid, human recombinant interleukin 2 and human recombinant interleukin 7.

Preferably, the γδ T cells are isolated using magnetic beads.

Preferably, zoledronic acid is present in the culture medium (medium) of the live expansion at a concentration of 0.5-2.0 μM.

Preferably, the culture medium (medium) also contains interleukin, wherein IL-2 is present at a concentration of 5 ng/ml to 10 ng/ml. Preferably, IL-7 can also be included, and the concentration of IL-7 is 0.5-5ng/ml.

Preferably, when the cell density reaches 0.5-2.0× ^{10 6} cells/mL, passage 1:1-1:4. The research of the present invention shows that although the control of the cell density usually takes 2-3 days to change the medium, or to judge whether to change the medium according to the color of the indicator in the medium, the test results show that the usual method can maintain the cell growth, but fully reach the It is less than the expansion fold of the method for expanding (cultivating) γδ T cells of the present invention.

In another aspect, the present application relates to a method for enhancing the efficiency of viral transduction in γδT cells, comprising transducing the expanded γδT cells with a recombinant viral vector. Preferably, the viral vector is a lentiviral vector. The method also includes:

(1) Using the expression vector of chimeric antigen receptor to transfect 293T cells to obtain a lentiviral vector;

(2) using the lentiviral vector obtained in step (1) to transduce γδ T lymphocytes;

The chimeric antigen receptors include: single-chain antibody ScFv targeting specific indication antigens, IgG4 hinge region, CD8 transmembrane region, 4-1BB;

The amino acid sequences of the ScFv, IgG4 hinge region, CD8 transmembrane region, and 4-1BB are shown in the sequence table, respectively.

Preferably, the step (2) includes:

The lentivirus recombined with the above-mentioned chimeric antigen receptor expression vector is added to the culture environment of γδT cells;

Lentiviral infection was performed overnight (24 hours later) with medium changes.

The present invention also provides a γδT cell, the γδT cell is prepared by the above method, and the method includes:

obtaining or isolating γδ T cells; and/or expanding said γδ T cells in a culture medium containing zoledronic acid, human recombinant interleukin 2, and human recombinant interleukin 7.

The method may also include:

(1) Using the expression vector of chimeric antigen receptor to transfect 293T cells to obtain a lentiviral vector;

(2) using the lentiviral vector obtained in step (1) to transduce γδ T lymphocytes;

The chimeric antigen receptors include: single-chain antibody ScFv targeting specific indication antigens, IgG4 hinge region, CD8 transmembrane region, 4-1BB;

The amino acid sequence and nucleic acid coding sequence of the ScFv, IgG4 hinge region, CD8 transmembrane region, 4-1BB are shown in SEQ ID NO. 1, 3, 5, and 7, respectively.

In the γδT cells of the present invention, the γδT cells are genetically modified using the above-mentioned chimeric antigen receptor.

In another aspect, the present invention provides a chimeric antigen receptor comprising:

The single chain antibody ScFv, IgG4 hinge region, CD8 transmembrane region, 4-1BB targeting HIV-1 gp120, the sequence of which is shown in SEQ ID NO. 1-8.

Among them, the single-chain antibody ScFv targeting HIV-1 gp120 is a ScFv that can recognize and bind to HIV-1 gp120 protein of HIV. The single-chain antibody ScFv can recognize gp120 on the surface of HIV virus-infected cells, and is obtained by concatenating variable regions of antibody light chain and heavy chain against gp120 on the surface of HIV virus-infected cells. The single-chain antibody ScFv serves as the extracellular binding domain of the entire CAR molecule, and its amino acid sequence is derived from the 3BNC117-pTRPE plasmid.

The chimeric antigen receptor can be used to prepare genetically modified γδ lymphocytes to target HIV-1 gp120.

The IgG4 hinge region, ie IgG4 hinge, is a hinge molecule linking the 3BNC117 ScFv and the CD8 transmembrane region, and its sequence can be found in SEQ ID NO.3-4.

The CD8 transmembrane domain is a transmembrane molecule linking the extracellular domain structure and the intracellular domain structure of the chimeric antigen receptor, and its sequence can be found in SEQ ID NO.5-6.

4-1BB is an intracellular signaling co-stimulatory domain, the sequence of which can be found in SEQ ID NO. 7-8.

3BNC117-IgG4 Hinge-CD8跨膜区-4-1BB-CD3ζ氨基酸序列为：MLLLVTSLLLCELPHPAFLLIPQVQLLQSGAAVTKPGASVRVSCEASGYNIRDYFIHWWRQAPGQGLQWVGWINPKTGQPNNPRQFQGRVSLTRHASWDFDTFSFYMDLKALRSDDTAVYFCARQRSDYWDFDVWGSGTQVTVSSASTKGPGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDTVTITCQANGYLNWYQQRRGKAPKLLIYDGSKLERGVPSRFSGRRWGQEYNLTINNLQPEDIATYFCQVYEFVVPGTRLDLKRTVAAPESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*(SEQ ID NO.1)

The corresponding nucleotide sequence is:

ATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCCCACCCTGCCTTTCTGCTGATCCCCCAGGTGCAGCTGCTGCAGAGCGGAGCCGCCGTGACAAAGCCTGGCGCTTCTGTGCGGGTGTCCTGCGAGGCCAGCGGCTACAACATCCGGGACTACTTCATCCACTGGTGGCGGCAGGCCCCAGGCCAGGGACTGCAGTGGGTGGGATGGATCAACCCCAAGACCGGCCAGCCCAACAACCCCCGGCAGTTCCAGGGCCGGGTGTCCCTGACAAGACACGCCAGCTGGGACTTCGACACCTTCAGCTTCTACATGGACCTGAAGGCCCTGCGGAGCGACGATACCGCCGTGTACTTCTGCGCCAGACAGCGGAGCGACTACTGGGATTTCGACGTGTGGGGCAGCGGCACCCAGGTCACAGTGTCCAGCGCCAGCACAAAGGGACCTGGCGGCGGAGGATCTGGCGGAGGCGGAAGTGGCGGAGGGGGCAGCGATATTCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACACCGTGACCATCACCTGTCAGGCCAACGGATACCTGAACTGGTATCAGCAGCGGAGAGGCAAGGCCCCCAAGCTGCTGATCTACGACGGCAGCAAGCTGGAACGGGGCGTGCCCAGCCGGTTCAGCGGCAGAAGATGGGGCCAAGAGTACAACCTGACCATCAACAACCTGCAGCCCGAGGATATTGCCACATACTTTTGCCAGGTGTACGAGTTCGTGGTGCCCGGGACCCGGCTGGATCTGAAGAGAACCGTGGCCGCTCCCGAGAGCAAATACGGGCCCCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACG GCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACAC CTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA (SEQ ID NO. 2).

IgG4 Hinge氨基酸序列为：ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKD(SEQ ID NO.3)。

相应的核苷酸序列为：GAGAGCAAATACGGGCCCCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGGAT(SEQ ID NO.4)。

The amino acid sequence of CD8 transmembrane region is:

IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO. 5).

The corresponding nucleotide sequence is: ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC (SEQ ID NO. 6).

The amino acid sequence of 4-1BB is:

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO. 7).

The corresponding nucleotide sequence is: AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO. 8).

On the other hand, the present invention provides the application of the γδT lymphocytes, including the application of the gene-modified γδT lymphocytes in the preparation of anti-tumor or anti-AIDS living cell drugs; wherein, the indications are selected from lung cancer, breast cancer Cancer, pancreatic cancer, liver cancer, stomach cancer, colorectal cancer, leukemia or ovarian cancer, or the indication is AIDS.

The present invention provides a gene-modified γδT lymphocyte, and the surface of the γδT lymphocyte expresses the above-mentioned chimeric antigen receptor.

In a preferred embodiment of the present invention, the genetically modified γδ T lymphocytes are prepared by the following method:

Mononuclear cells PBMCs were isolated from peripheral blood, and then γδT cells were obtained by magnetic bead positive selection. After expansion and culture, the lentivirus with recombinant 3B-CAR molecule was added to infect for 24 hours and then the medium was changed (MOI=10). From the fourth day after virus infection, the cells ^were counted and the medium was supplemented according to the cell state and proliferation. Cells were further expanded until the number of cells for reinfusion was met.

The present invention has carried out in vitro proliferation and activity experiments, and the results show that the γδT cells separated and expanded by the method have higher purity and stronger proliferation ability.

In the constructed HIV-1 in vitro cell model, the present invention finds that 3B-γδT cells show better proliferation ability, cell killing ability and cytokine release ability.

The tumor cells are derived from lung cancer, breast cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, leukemia or ovarian cancer.

The present invention provides a new type of allogeneic anti-HIV γδ T cells, which enables HIV-1 infected cells to be efficiently, lastingly and specifically cleared, and lays the foundation for the allogeneic treatment for long-term control of HIV-1 viral load in patients.

The present invention also provides the application of the above-mentioned gene-modified γδ T lymphocytes in the preparation of allogeneic anti-HIV-infected live cell drugs.

The present invention combines HIV CAR-T therapy with γδT, provides an optional method for HIV CAR-γδT therapy, and provides a feasible idea for the exploration of HIV-1 functional cure.

After screening, comparison and optimization, the present invention obtains a method that can rapidly expand and prepare γδT cells in large quantities, and the amplification multiple reaches 1500 times or more within 14 days. In vitro proliferation and activity experiments show that the γδT cells isolated and expanded by the method of the present invention have higher purity and stronger proliferation ability. On this basis, in the constructed HIV-1 in vitro cell model, it was found that 3B-γδT cells showed good proliferation ability, tumor cell killing ability and cytokine release ability; the genetically modified γδT cells constructed in the present invention had better proliferation ability Strong anti-tumor cell-killing effect is helpful for functional cure of HIV-1.

Description of drawings

Figure 1. Purity map of γδT cells after isolation,

Among them, the abscissa represents the expression of CD3, and the ordinate represents the expression of γδTCR.

Figure 2. The results of the detection of γδT cell proliferation,

The abscissa represents the expansion time after separation, and the ordinate represents the number of γδT cells. It can be seen that γδT cells can be significantly expanded by the method of the present invention.

Figure 3. Schematic diagram of 3B CAR recombinant vector.

Figure 4. 3B CAR-γδ T cell phenotype detection results,

Among them, UTD is γδT cells without any treatment as control,

The vertical axis FS-A is the anterior scattered light, and the abscissa CAR represents the proportion of CAR positive rate detected by FITC-goat anti-human IgG flow antibody staining;

The results showed that the CAR molecule expression efficiency of the prepared 3B CAR-γδ T cells was about 90%.

Figure 5. Detection results of 3B CAR-γδT cells targeting and killing HIV-1 cells, wherein,

The ordinate is the killing percentage Specific lysis, the unit is %, which refers to the proportion of target cells that are killed. The calculation formula is

The abscissa ratio refers to the ratio of the number of effector cells to the number of target cells;

The results showed that 3B CAR-γδT cells had a killing effect on LEL6 cells under different co-incubation ratios.

Figure 6. 3B CAR-γδT cell cytokine release ability test results, in which,

The ordinate is the concentration of the three cytokines. The results showed that after co-incubation with LEL6 cells, the release of TNF-α, IL-2 and IFN-γ cytokines in the 3B CAR-γδT group increased significantly. ***p<0.001.

Detailed ways

Example 1 Isolation of γδ T cells

Take the anticoagulated peripheral blood sample, after balancing, centrifuge at 20°C, 800×g for 20 minutes; transfer the upper plasma to a new sterile centrifuge tube. Use sterile phosphate buffered saline (PBS) 100ml to resuspend and dilute the cell pellet, and slowly attach the cell suspension to the human lymphocyte separation solution in stages (the volume ratio of cell suspension to human lymphocyte separation solution is 1:1: 1); Centrifuge at 20°C, 800×g for 20 minutes (1 increase, 0 decrease). Use a 10ml pipette to gently insert 0.5cm above the middle layer of thin cloud-like mononuclear cells, aspirate the layer of cells along the tube wall, transfer the middle layer of cells to a new 50ml sterile centrifuge tube, use 30ml sterile PBS was centrifuged at 20°C, 800 xg, for 20 minutes (1 9, 9 lower), and washed twice. Discard the supernatant, resuspend the cell pellet in 4ml x-vivo 15 medium, harvest PBMC, store at 4°C for later use.

Configuration of sorting buffer: Add 0.5% calf serum and 2 mM EDTA in sterile PBS pH 7.2 and place in an ice bath to pre-cool.

Magnetic Bead Labeling: After counting the cells to determine the number of cells, centrifuge the cell suspension at 300 x g for 10 minutes. Resuspend the cell pellet in 80 ul of buffer per 10 ⁷ cells. 20ul of biotin-labeled antibody mixture was added to each 107 cells, mixed well, and refrigerated for 10 minutes (4-8°C). Add 1-2 mL of buffer per 107 cells to wash the cells and centrifuge at 300 × g for 10 min. Then add ^80ul buffer solution and 20ul anti-biotin magnetic beads to 107 cells. Mix well and refrigerate for 15 minutes (4-8°C). Add 1-2 mL of buffer per 10 ⁷ cells to wash the cells and centrifuge at 300×g for 10 minutes. Magnetic separation was performed by resuspending cells in 500 ul buffer.

Magnetic separation: According to the total number of cells and the number of TCRγ/δ+ cells, select an appropriate sorting column and corresponding sorter, place the sorting column in the magnetic field of the corresponding MACS sorter, and rinse the column with an appropriate amount of buffer to prepare .

The resuspended cell suspension was added to the sorting column, and after the liquid was exhausted, an appropriate amount of buffer was added to elute the sorting column 3 times. Remove the sorting column, remove the magnetic field, and quickly collect the magnetic bead-labeled γδT cells on the sorting column into a new centrifuge tube for use.

Example 2 γδT amplification

Use 25ml of the prepared γδT cell culture medium to resuspend the γδT cells obtained by magnetic bead sorting, transfer the cell suspension to a 75cm2 culture flask, and place the culture flask in a saturated humidity, 37°C, 5.0% CO2 incubator to cultivate. The growth of cells in the flasks was observed by microscope every other day. According to the cell growth state, observe the cell density in time and control it at 0.5-2×10 ⁶ cells/ml. When the density exceeds this range, change the medium with γδT cell culture medium, and adjust the cell density to within this range. After 14-16 days of continuous culture, the γδT cells were harvested, and the entire cell culture process should be performed aseptically. Cell culture supernatants were taken at 0, 3, 7, 14, and 16 days of cell culture, and counted under a microscope to determine cell viability according to the above method; The total number and expansion fold of γδT cells, and the growth curve was drawn.

γδT cell culture medium composition: x-vivo 15 serum-free medium, 10% serum, 5ng/ml IL-2, 2ng/mL IL-7, 1.0 μM zoledronic acid.

Example 3 In vitro construction of a chimeric antigen receptor expression vector targeting HIV-1 gp120

Using the pCDH-CMV-MCS-EF1α-Puro plasmid as the backbone, the MCS-EF1α-Puro fragment was removed by double digestion with EcoRI and SalI endonucleases. Then, using the pTRPE-3BNC117-G4H-BBz plasmid as a template, using onestep-3BNC117-F, onestep-3BNC117-R primers to PCR amplify ScFv, IgG4 hinge, CD8 molecules containing the variable region of HIV broadly neutralizing antibody Transmembrane region, 3BNC117 CAR fragment of 4-1BB. Finally, the double-enzyme digested product and the PCR product were gel recovered and then connected by Onestep homologous recombination. The ligated product was transformed in DH5α competent and then coated on Amp+ plate to screen positive clones. After the positive clones were expanded and cultured, the plasmids were extracted and sequenced for verification. The positive plasmid pCDH-CMV-3BNC117 ScFv-IgG4-CD8Tm-4-1BB was obtained and named 3BNC117 CAR (abbreviated as 3B CAR).

Example 4 Detection of purity and proliferation ability of γδT cells

Collect the cells cultured for 6, 8, 11, and 14 days with flow cytometry and analyze the results, while using the PBMC control tube before culture. Collect 1 × 106 cells/tube in a centrifuge tube, place them in a low-temperature high-speed centrifuge after balancing, centrifuge at 800 rpm for 5 min at 4°C, discard the supernatant, resuspend with 500ul-1ml PBS, centrifuge at 800 rpm for 5 min, discard Remove the supernatant, then resuspend the cells with 100ul PBS, add 1ul anti-γδTCR-FITC and CD3-Pe to each tube, carry out double immunofluorescence staining, incubate at 4°C for 30 minutes in the dark, add 1ml PBS, After mixing by gentle pipetting, centrifuge at 4°C and 3000 rpm for 5 minutes and wash 3 times. The supernatant was discarded, and 500 μl of PBS was added to resuspend the cell pellet, and then the proportion of γδ T cells was detected by a flow cytometer (fluorescence activating cell sorter, FACs).

According to the cell growth state, the medium was changed with γδT cell culture medium every 1-3 days, and the cells were counted. After 14-16 days of continuous culture, the γδT cells were harvested, and the aseptic operation was observed during the entire cell culture process. On days 0, 3, 7, 14, and 16 of cell culture, the cell culture supernatants of each group were collected, stained with trypan blue according to the above method, counted under a microscope, and determined the cell viability; and detected by flow cytometry The proportion of γδT cells after culture was calculated, the total number and expansion fold of γδT cells were calculated, and the growth curve was drawn. Amplification fold=(total number of cells after expansion×proportion of γδT cells after expansion)/(total number of cells before expansion×proportion of γδT cells before expansion).

The results show that, within 14 days using the method in this example, the expansion fold of γδT cells reaches 1500 times, while using the currently commonly used better methods, such as the presence of human recombinant interleukin 2 (IL-2) and human recombinant leukocytes. Expansion of γδ T cells in the presence of interleukin 15 (IL-15) or selected from the group consisting of IL-21, stromal cell-derived factor (SDF), IL-1β, IL-12, IL-18 and IL-33 In the case of a factor, the amplification fold is usually more than 100 times and less than 1000 times.

Example 5 Preparation of 3B CAR-γδ T cells

In order to obtain lentiviral particles expressing 3B CAR, the present invention co-transfected the lentiviral backbone plasmid, △8.91 and VSVG into 293T cells, collected the virus supernatant after 48 hours, filtered, concentrated by ultracentrifugation and placed it at -80°C Save for backup.

Effector cells were prepared by infecting healthy human γδ T lymphocytes with lentiviruses carrying 3B CAR elements. Anti-HIV CAR-γδT effector cells were prepared by infecting γδT cells with MOI=10 static infection, and uninfected γδT cells were used as control.

After 6 days, it was labeled with Fluorescein(FITC)-conjugated AffiniPure F(ab')2Fragment GoatAnti-Human IgG(H+L). The results showed that the CAR positive rate in the 3B CAR group was about 90% compared with the UTD group.

Example 6 Detection of immune function of 3B CAR-γδ T cells

杀伤百分比越高，证实越多的靶细胞被杀灭。The present invention preliminarily verified its killing effect on LEL6 target cells of HIV-1 env ⁺ cell model in vitro. We co-incubated unmodified γδT cells, 3B CAR-γδT cells with LEL6 cells, and used Jurkat cells as a negative control to detect the cell killing effect by lactate dehydrogenase (LDH) method. The results showed that compared with the UTD group, under the three different co-incubation ratios of 1:1, 5:1, and 10:1, the 3B CAR group could effectively kill the target cells, and the killing effect could reach about 90%. In contrast to HIV-1 env ^- Jurkat cells, the effector cells did not produce non-specific killing. Specific Lysis is the kill percentage, and the formula is:

The higher the killing percentage, the more target cells were confirmed to be killed.

To further test the function of Anti-HIV CAR-γδT effector cells, Anti-HIV CAR-T effector cells were co-incubated with LEL6 target cells at a ratio of 10:1, and IL-2, TNF- The release of α, IFN-γ 3 cytokines, the results showed that after co-incubation with LEL6 target cells, the secretion of 3 cytokines in the 3B CAR group was significantly higher than that in the UTD group.

The above are only specific embodiments of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed in the present application, All should be covered within the scope of protection of this application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

sequence listing

<110> Fudan University

<120> A kind of preparation and expansion method and application of γδT cells

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Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Gln Val Gln Leu Leu Gln Ser Gly Ala Ala

20 25 30

Val Thr Lys Pro Gly Ala Ser Val Arg Val Ser Cys Glu Ala Ser Gly

35 40 45

Tyr Asn Ile Arg Asp Tyr Phe Ile His Trp Trp Arg Gln Ala Pro Gly

50 55 60

Gln Gly Leu Gln Trp Val Gly Trp Ile Asn Pro Lys Thr Gly Gln Pro

65 70 75 80

Asn Asn Pro Arg Gln Phe Gln Gly Arg Val Ser Leu Thr Arg His Ala

85 90 95

Ser Trp Asp Phe Asp Thr Phe Ser Phe Tyr Met Asp Leu Lys Ala Leu

100 105 110

Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys Ala Arg Gln Arg Ser Asp

115 120 125

Tyr Trp Asp Phe Asp Val Trp Gly Ser Gly Thr Gln Val Thr Val Ser

130 135 140

Ser Ala Ser Thr Lys Gly Pro Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

165 170 175

Leu Ser Ala Ser Val Gly Asp Thr Val Thr Ile Thr Cys Gln Ala Asn

180 185 190

Gly Tyr Leu Asn Trp Tyr Gln Gln Arg Arg Gly Lys Ala Pro Lys Leu

195 200 205

Leu Ile Tyr Asp Gly Ser Lys Leu Glu Arg Gly Val Pro Ser Arg Phe

210 215 220

Ser Gly Arg Arg Trp Gly Gln Glu Tyr Asn Leu Thr Ile Asn Asn Leu

225 230 235 240

Gln Pro Glu Asp Ile Ala Thr Tyr Phe Cys Gln Val Tyr Glu Phe Val

245 250 255

Val Pro Gly Thr Arg Leu Asp Leu Lys Arg Thr Val Ala Ala Pro Glu

260 265 270

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu

275 280 285

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

290 295 300

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

305 310 315 320

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

325 330 335

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

340 345 350

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

355 360 365

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

370 375 380

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

385 390 395 400

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

405 410 415

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

420 425 430

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

435 440 445

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr

450 455 460

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

465 470 475 480

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

485 490 495

Ser Leu Gly Lys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys

500 505 510

Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly

515 520 525

Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val

530 535 540

Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu

545 550 555 560

Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp

565 570 575

Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn

580 585 590

Leu Gly Arg Arg Glu Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg

595 600 605

Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly

610 615 620

Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu

625 630 635 640

Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu

645 650 655

Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His

660 665 670

Met Gln Ala Leu Pro Pro Arg

675

<210> 2

<211> 2040

<212> DNA

<213> Homo sapiens

<400> 2

atgctgctgc tggtgacaag cctgctgctg tgcgagctgc cccaccctgc ctttctgctg 60

atcccccagg tgcagctgct gcagagcgga gccgccgtga caaagcctgg cgcttctgtg 120

cgggtgtcct gcgaggccag cggctacaac atccgggact acttcatcca ctggtggcgg 180

caggccccag gccagggact gcagtgggtg ggatggatca accccaagac cggccagccc 240

aacaaccccc ggcagttcca gggccgggtg tccctgacaa gacacgccag ctgggacttc 300

gacaccttca gcttctacat ggacctgaag gccctgcgga gcgacgatac cgccgtgtac 360

ttctgcgcca gacagcggag cgactactgg gatttcgacg tgtggggcag cggcacccag 420

gtcacagtgt ccagcgccag cacaaaggga cctggcggcg gaggatctgg cggaggcgga 480

agtggcggag ggggcagcga tattcagatg acccagagcc ccagcagcct gagcgccagc 540

gtgggcgaca ccgtgaccat cacctgtcag gccaacggat acctgaactg gtatcagcag 600

cggagaggca aggcccccaa gctgctgatc tacgacggca gcaagctgga acggggcgtg 660

cccagccggt tcagcggcag aagatggggc caagagtaca acctgaccat caacaacctg 720

cagcccgagg atattgccac atacttttgc caggtgtacg agttcgtggt gcccgggacc 780

cggctggatc tgaagagaac cgtggccgct cccgagagca aatacgggcc cccctgcccc 840

ccttgccctg cccccgagtt cctgggcgga cccagcgtgt tcctgttccc ccccaagccc 900

aaggacaccc tgatgatcag ccggaccccc gaggtgacct gtgtggtggt ggacgtgtcc 960

caggaggacc ccgaggtcca gttcaactgg tacgtggacg gcgtggaggt gcacaacgcc 1020

aagaccaagc cccgggagga gcagttcaat agcacctacc gggtggtgtc cgtgctgacc 1080

gtgctgcacc aggactggct gaacggcaag gaatacaagt gtaaggtgtc caacaagggc 1140

ctgcccagca gcatcgagaa aaccatcagc aaggccaagg gccagcctcg ggagccccag 1200

gtgtacaccc tgccccctag ccaagaggag atgaccaaga accaggtgtc cctgacctgc 1260

ctggtgaagg gcttctaccc cagcgacatc gccgtggagt gggagagcaa cggccagccc 1320

gagaacaact acaagaccac cccccctgtg ctggacagcg acggcagctt cttcctgtac 1380

agccggctga ccgtggacaa gagccggtgg caggagggca acgtctttag ctgctccgtg 1440

atgcacgagg ccctgcacaa ccactacacc cagaagagcc tgagcctgtc cctgggcaag 1500

gatatctaca tctgggcgcc cttggccggg acttgtgggg tccttctcct gtcactggtt 1560

atcacccttt actgcaaacg gggcagaaag aaactcctgt atatattcaa acaaccattt 1620

atgagaccag tacaaactac tcaagaggaa gatggctgta gctgccgatt tccagaagaa 1680

gaagaaggag gatgtgaact gagagtgaag ttcagcagga gcgcagacgc ccccgcgtac 1740

aagcagggcc agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat 1800

gttttggaca agagacgtgg ccgggaccct gagatgggggg gaaagccgag aaggaagaac 1860

cctcaggaag gcctgtacaa tgaactgcag aaagataaga tggcggaggc ctacagtgag 1920

attgggatga aaggcgagcg ccggaggggc aaggggcacg atggccttta ccagggtctc 1980

agtacagcca ccaaggacac ctacgacgcc cttcacatgc aggccctgcc ccctcgctaa 2040

<210> 3

<211> 230

<212> PRT

<213> Homo sapiens

<400> 3

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 10 15

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys Asp

225 230

<210> 4

<211> 690

<212> DNA

<213> Homo sapiens

<400> 4

gagagcaaat acgggccccc ctgcccccct tgccctgccc ccgagttcct gggcggaccc 60

agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagccg gacccccgag 120

gtgacctgtg tggtggtgga cgtgtcccag gaggaccccg aggtccagtt caactggtac 180

gtggacggcg tggaggtgca caacgccaag accaagcccc gggaggagca gttcaatagc 240

acctaccggg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggaa 300

tacaagtgta aggtgtccaa caagggcctg cccagcagca tcgagaaaac catcagcaag 360

gccaagggcc agcctcggga gccccaggtg tacaccctgc cccctagcca agaggagatg 420

accaagaacc aggtgtccct gacctgcctg gtgaagggct tctaccccag cgacatcgcc 480

gtggagtggg agagcaacgg ccagcccgag aacaactaca agaccacccc ccctgtgctg 540

gacagcgacg gcagcttctt cctgtacagc cggctgaccg tggacaagag ccggtggcag 600

gagggcaacg tctttagctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 660

aagagcctga gcctgtccct gggcaaggat 690

<210> 5

<211> 24

<212> PRT

<213> Homo sapiens

<400> 5

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210> 6

<211> 72

<212> DNA

<213> Homo sapiens

<400> 6

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gc 72

<210> 7

<211> 42

<212> PRT

<213> Homo sapiens

<400> 7

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 8

<211> 126

<212> DNA

<213> Homo sapiens

<400> 8

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

Claims (10)

1. A method for expanding γ δ T cells in vitro, comprising:

obtaining or isolating γ δ T cells, the γ δ T cells isolated using magnetic beads; and/or

Amplifying the gamma delta T cells in a culture medium containing zoledronic acid, human recombinant interleukin 2 and human recombinant interleukin 7, wherein zoledronic acid is present in live amplification at a concentration of 0.5-2.0 muM.

2. The method of claim 1, wherein the concentration of IL-2 is from 5ng/ml to 10 ng/ml.

3. The method of claim 1, wherein the concentration of IL-7 is from 0.5ng/ml to 5 ng/ml.

4. The method of claim 1, wherein the cell density reaches 0.5-2.0x10⁶1/mL, 1: 1-1: 4 passages.

5. The method of any one of claims 1-4, further comprising:

(1) transfecting 293T cells by using an expression vector of a chimeric antigen receptor to obtain a lentiviral vector;

(2) transducing γ δ T lymphocytes using the lentiviral vector obtained in step (1);

the chimeric antigen receptor comprises: single chain antibodies ScFv, IgG4 hinge region, CD8 transmembrane region, 4-1BB targeting specific indication antigens;

the amino acid sequences of the ScFv, the IgG4 hinge region, the CD8 transmembrane region and the 4-1BB are respectively shown as SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO. 7; or

The nucleic acid sequences of the ScFv, the IgG4 hinge region, the CD8 transmembrane region and the 4-1BB are respectively shown as SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO. 8.

6. The method of claim 5, wherein said step (2) comprises:

adding lentivirus recombined with the chimeric antigen receptor expression vector into a culture environment of the gamma delta T cells;

fluid changes were made 24 hours after lentivirus infection.

7. A γ δ T cell prepared using the method of claim 1.

8. The γ δ T-cell according to claim 7, wherein the γ δ T-cell is genetically modified with the chimeric antigen receptor of claim 5.

9. Use of the γ δ T-cell according to claim 7 or 8, wherein the genetically modified γ δ T-lymphocyte is used for preparing a live cell medicament for anti-tumor or anti-aids.

10. The use according to claim 9, wherein the tumor is selected from the group consisting of lung cancer, breast cancer, pancreatic cancer, liver cancer, stomach cancer, colorectal cancer, leukemia and ovarian cancer.

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