CN113122579B

CN113122579B - Method for transfecting immune cells by lentivirus

Info

Publication number: CN113122579B
Application number: CN202110403031.1A
Authority: CN
Inventors: 李建强; 牛星; 邢思捷
Original assignee: Hebei Senlang Biotechnology Co ltd
Current assignee: Hebei Senlang Biotechnology Co ltd
Priority date: 2021-04-14
Filing date: 2021-04-14
Publication date: 2022-03-29
Anticipated expiration: 2041-04-14
Also published as: CN113122579A

Abstract

The invention discloses a method for transfecting immune cells by lentivirus. The method comprises transfecting an immune cell with a lentivirus comprising RNA transcribed from a chimeric antigen receptor gene in a transfection system comprising prostaglandin E2. The method can be applied to the transfection of alpha beta T cells, gamma delta T cells and NK cells, has high transfection efficiency and can reduce the influence of the transfection on immune cells.

Description

Method for transfecting immune cells by lentivirus

Technical Field

The invention relates to the technical field of biology, in particular to a method for transfecting immune cells by lentiviruses.

Background

CAR-T, collectively known as Chimeric Antigen Receptor T-Cell Immunotherapy, refers to the direct binding of T lymphocytes to specific antigens on the surface of tumor cells to activate them by transferring genetic material (CAR) carrying specific Antigen recognition domains and T Cell activation signals into the T lymphocytes by genetic modification techniques. After the T lymphocyte is activated, on one hand, the tumor cell is directly killed and killed by releasing perforin, granzyme B and the like; on the other hand, the cell factor is released to recruit the endogenous immune cells of the human body to kill the tumor cells so as to achieve the aim of treating the tumor. Meanwhile, immunological memory T cells can be formed, so that a specific anti-tumor long-acting mechanism is obtained. CAR-T therapy has significant efficacy in the treatment of acute leukemias and non-hodgkin's lymphomas, and is considered to be one of the most promising modes of tumor treatment.

Human T lymphocytes are classified into two major groups, α β T cells and γ δ T cells, according to their surface T cell antigen receptors. The alpha beta T cells account for about 95 percent, namely the T cells are commonly called. The gamma delta T cell accounts for 1-10% of the T cell, but the function of the gamma delta T cell is independent of MHC recognition, so that the escape of tumors can be resisted. The key of CAR-T therapy is to equip T lymphocytes with CAR molecules that recognize specific cancer cells, so that they can specifically recognize and kill cancer cells, thereby achieving the effect of treating cancer. However, the gene sequence of the Chimeric Antigen Receptor (CAR) to be transferred into the immune cell is large, so that the transfection efficiency is low, and the function of the CAR-T cell is influenced. Therefore, the research of the transfection assisting reagent which can improve the transfection efficiency of immune cells, particularly gamma delta T cells and the like which are difficult to transfect has important significance for clinical treatment.

CAR-NK therapy is an adoptive cell therapy based on natural killer cells (NK), using genetic engineering techniques to modify NK cells with CAR (chimeric antigen receptor) molecules that navigate T cell localization to form CAR-NK hybrid cells. NK cells are important immune cells in the body and are involved in anti-tumor, anti-viral infection and immune regulation. Since the killing activity of NK cells is not MHC-restricted, is independent of antibodies, and is therefore called natural killing activity, similar to the action of γ δ T cells, and is also difficult to transfect,

however, conventional transfection assisting reagents such as Polybrene, PS (Protamine Sulfate) and the like cannot achieve high transfection efficiency on immune cells, and thus a more efficient method for transfection needs to be found.

Disclosure of Invention

The present invention provides a method for transfecting an immune cell with a lentivirus comprising RNA transcribed from a chimeric antigen receptor gene, comprising transfecting an immune cell with a lentivirus in a transfection system comprising prostaglandin E2.

The transfection system may also include serum-free media. The serum-free medium consists of KBM581 and IL-2. The content of the IL-2 in the serum-free culture medium is 200 IU/ml.

The concentration of prostaglandin E2 in the transfection system is, for example, 0.5-2. mu.M.

Optionally, the ratio of the number of immune cells to the transfection system is 3 × 10⁵Is one 5X10⁵The method comprises the following steps: 500 μ L.

The lentivirus expresses the chimeric antigen receptor gene in the immune cell. The chimeric antigen receptor gene is the CAR gene. The CAR gene expresses a chimeric antigen receptor. The chimeric antigen receptor targeted antigens include, but are not limited to, CD19, CD20, CD22, CD30, HER2, GD2, EGFR, EGFRvIII, EphA2, IL13Ra2, CD133, ROR1, IGF1R, and/or L1 CAM.

For example, the CAR gene encodes a protein that includes a single chain antibody, an extracellular hinge region (e.g., a CD8a hinge region), a transmembrane region (e.g., a CD8a transmembrane region), and an intracellular region (e.g., 4-1BB, CD3 ζ). If desired, the protein encoded by the CAR gene may further include a T2A peptide, a tfegfr protein. For example, the CAR gene encodes a protein comprising a single chain antibody, an extracellular hinge region, a transmembrane region and an intracellular region, a T2A peptide, and a tfegfr protein linked together. Further, the CAR gene encodes a protein that can be formed by linking a single chain antibody, an extracellular hinge region, a transmembrane region, an intracellular region, a T2A peptide, and a tfegfr protein. In one embodiment of the invention, the sequence of the CAR gene is shown in SEQ ID No. 1.

Optionally, the immune cell is an NK cell or a T lymphocyte according to the method described above; the T lymphocyte is an alpha beta T cell or a gamma delta T cell.

The immune cell is an NK cell or a γ δ T cell, and the method may comprise the steps of:

(1) activation culture: the immune cells were cultured on a culture plate coated with an activating antibody using an activating medium comprising KBM581, OK432, IFN- γ, IL-2, IL-15, IL-21, and 10% autologous plasma. The time for activation culture may be 2-8 days, for example 2, 3, 4, 5, 6, 7, 8 days. The immune cells are NK cells, and the activation culture time can be 8 days. The immune cells are gamma delta T cells, and the activation culture time can be 6 days. The immune cell is an NK cell, and the antibody can be Anti-CD16 monoclonal antibody.

The immune cells are γ δ T cells and the antibody may be an anti- γ δ TCR antibody, such as the Purifield danti-human g/d TCR from Biolegend, cat # 331204.

(2) Transfection: transfecting the activated cultured immune cells with the lentivirus. The transfection time is, for example, 1 day. The transfection method may be 32-35 ℃ and the transfection system is centrifuged. The lentivirus is transfected with the dose of MOI 2-50. The immune cells are NK cells or gamma delta T cells, and the dosage of lentivirus transfection can be MOI 50. The immune cells are alpha beta T cells, and the dosage of lentivirus transfection can be MOI-2.

(3) Amplification culture: transfected immune cells were cultured in expansion medium including KBM581, IL-2 and 5% autologous plasma.

The activation medium may consist of KBM581, OK432, IFN-. gamma.IL-2, IL-15, IL-21, 10% autologous plasma (inactivated). OK432 may be present in the activation medium in an amount of 80-120ng/mL (e.g., 100ng/mL), IFN-. gamma.may be present in the activation medium in an amount of 800-1200IU/mL (e.g., 1000IU/mL), IL-2 may be present in the activation medium in an amount of 500-1500IU/mL (e.g., 1000IU/mL), IL-15 may be present in the activation medium in an amount of 80-120ng/mL (e.g., 100ng/mL), IL-21 may be present in the activation medium in an amount of 80-120ng/mL (e.g., 100ng/mL), and autologous plasma (inactivated) may be present in the activation medium in an amount of 10% by volume.

The amplification medium may consist of KBM581, IL-2, 5% autologous plasma. IL-2 may be present in the amplification medium in an amount of 1000IU/mL and autologous plasma (inactivated) may be present in the amplification medium in an amount of 5% by volume.

The use of prostaglandin E2 is also within the scope of the present invention. The application specifically refers to the application in at least one of A1) -A6),

A1) the application of the lentivirus in improving the transfection rate of the lentivirus to immune cells;

A2) the application in preparing products for improving the transfection efficiency of lentivirus to immune cells;

A3) use in the manufacture of a respective recombinant immune cell which is a CAR- α β T cell, a CAR- γ δ T cell and/or a CAR-NK cell;

A4) the application in the preparation of the recombinant immune cell product;

A5) the application in promoting the expansion of immune cells;

A6) application in preparing products for promoting immune cell expansion.

Alternatively, in the above application, the recombinant immune cell may comprise a chimeric antigen receptor gene. The recombinant immune cell expresses a chimeric antigen receptor encoded by the chimeric antigen receptor gene.

Optionally, in the above use, the immune cell is an NK cell or a T lymphocyte; the T lymphocyte is an alpha beta T cell or a gamma delta T cell.

The present invention employs the use of 4 compounds to enhance the transduction of immune cells, and it is expected that sufficient CAR positive cells can be prepared in a certain amount of peripheral blood or umbilical cord blood for clinical treatment. Experimental research shows that PGE2 can reduce the influence of transfection on cell state and ensure the positive rate of transfected cells.

In conclusion, the invention provides a method for transfecting immune cells by lentiviruses, which can be applied to the transfection of alpha beta T cells, gamma delta T cells and NK cells, has high transfection efficiency and can reduce the influence of the transfection on the immune cells.

Drawings

FIG. 1 is a representation of the results of flow assays on peripheral blood-derived α β T cells using four transfection-assisting reagents of example 2.

FIG. 2 is a statistical chart of transfection efficiency using four transfection assisting agents in example 2.

FIG. 3 is a statistical plot of cell viability using four cotransfection-assisting agents in example 2.

FIG. 4 is a cell expansion curve of the PS group and the PGE2 group of example 2.

FIG. 5 is a representative flow assay of example 3 using four cotransfection-assisting reagents on peripheral blood-derived γ δ T cells.

FIG. 6 is a statistical chart of transfection efficiency using four transfection assisting reagents in example 3.

FIG. 7 is a statistical plot of cell viability using four cotransfection-assisting agents in example 3.

FIG. 8 is a cell expansion curve of the PS group and the PGE2 group in example 3.

FIG. 9 is a representative flow assay of example 4 using four transfection-assisting reagents on cord blood-derived γ δ T cells.

FIG. 10 is a statistical chart of transfection efficiency using four transfection assisting reagents in example 4.

FIG. 11 is a statistical plot of cell viability using the four transfection-assisting reagents of example 4.

FIG. 12 is a graph showing cell expansion curves of the PS group and the PGE2 group in example 4.

FIG. 13A is a representative flow chart of purity change of NK cells of example 5.

FIG. 13B is the statistical result of the percentage change of NK cells of example 5.

FIG. 14 is a representative flow diagram of example 5 on cord blood-derived NK cells using four transfection assisting reagents.

FIG. 15 is a statistical chart of transfection efficiency using four transfection assisting reagents in example 5.

FIG. 16 is a statistical plot of cell viability using four cotransfection-assisting agents in example 5.

FIG. 17 is a cell expansion curve of the PS group and the PGE2 group of example 5.

FIG. 18 is a graph showing the results of the experiment for optimizing transfection time of cord blood-derived γ δ T cells in example 6.

FIG. 19 is a graph showing the results of the experiment for optimizing the transfection time of cord blood-derived NK cells in example 6.

Wherein the content of the first and second substances,^***is that p is less than 0.001,^**is that p is less than 0.01,^*p < 0.05, ns is no statistical difference.

Detailed Description

Animal virus: the biological material is available to the applicant for the public in accordance with the relevant national biosafety regulations, and is only used for repeating the relevant experiments of the present invention, and is not used for other purposes.

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The data were processed using GraphPad Prism8 statistical software and the results were expressed as mean ± standard deviation using the ttest test.

The reagents and kits used in the following examples are specifically shown in Table 1.

TABLE 1 reagents and kits

The transfection assisting reagents used in the following examples are specifically as follows:

(1) PS manufacturers: yuekang pharmaceutical industry group Limited, product number H11020246, full name Protamine SuLfate injection, Protamine SuLfate injection;

(2) PGE2 manufacturer: MedChemexpress, cat No. 363-24-6, full name Prostaglandin E2, Prostaglandin E2;

(3) the P407 manufacturer: dalian Melam Biotechnology Ltd, Cat No. HY-D1005, known as Poloxamer 407, Poloxamer 407;

(4) stau manufacturer: MedChemexpress, cat # 41248, known as Staurosporine, Staurosporine.

Example 1

Materials and methods

1. Acquisition of PBMC (peripheral blood mononuclear cells) and CBMC (cord blood mononuclear cells):

taking peripheral blood or umbilical cord blood of a healthy person, adding physiological saline with the same volume at room temperature for dilution, slowly adding the diluted blood and the lymphocyte separation liquid into the lymphocyte separation liquid along the centrifugal tube wall, and enabling the proportion of the diluted blood to the lymphocyte separation liquid to be 2: 1. 2000rpm (equivalent to 872g), centrifuging for 20min, and separating into four layers from the bottom of the tube to the liquid surface, namely a red blood cell and granulocyte layer, a liquid separation layer, a mononuclear cell layer and a plasma layer. Sucking the mononuclear cell layer, putting into a new centrifuge tube, adding normal saline not less than 3 times of the volume of the mononuclear cell layer to wash the cells, performing cell counting twice at 2000rpm for 5 min. Obtaining PBMC or CBMC.

2. Sorting and culturing of α β T cells:

2.1 sorting of α β T cells Using the CD3 sorting kit for MACS

(1) Preparing 0.5% HSA buffer solution with the formula of 1.25mLHSA +48.75 mLDPBS;

(2) centrifuging the CBMC or PBMC obtained above at 2000r/min for 5min, discarding supernatant, and adding 80uL/1 × 10⁷Resuspending CBMC or PBMC in buffer;

(3) adding 20 uL/1X 10⁷CD3 magnetic beads, mixing, incubating at 4 deg.C for 15min, and shaking once every 5 min;

(4) adding 1-2 mL/1X 10⁷Washing cells with buffer solution at 2000r/min, centrifuging for 5min, and discarding supernatant;

(5) the number of cells is less than 1 × 10⁸Adding 500uL buffer solution in an amount of more than 1 × 10⁸The buffer was added proportionally and the cells were resuspended.

(6) Preparing a magnetic sorting column, rinsing the column with 3mL of buffer solution for 1 time, adding 500uL of cell suspension for sorting cells, and then adding 3mL of buffer solution for washing the column for 2 times, wherein the sorted positive cells are the alpha beta T cells.

(7) Cell counting:

the cells were mixed well and sampled for trypan blue staining counting.

2.2 α β T cell culture:

inoculating the selected alpha beta T cells on a 24-well plate, wherein the inoculation concentration is 3x10⁵One/well/500 ul. 5% C0 at 37 ℃₂Culturing in an incubator.

The culture medium consists of KBM581 and IL-2. The content of IL-2 in the culture medium is 200 IU/mL.

Sorting and culturing of 3 gamma delta T cells

3.1 sorting of Gamma Delta T cells Using the Human TCR Gamma Delta sorting kit of MACS

(3) adding 20 uL/1X 10⁷Mixing the Biotin-Antibody Cocktail, incubating at 4 ℃ for 10min, and shaking up every 5 min;

(5) adding 80 uL/1X 10⁷Buffer resuspend cells and 20 uL/1X 10⁷Anti-Biotin MicroBeads, mixing, incubating at 4 deg.C for 15min, and shaking once every 5 min;

(6) adding 1-2 mL/1X 10⁷Washing cells with buffer solution at 2000r/min, centrifuging for 5min, and discarding supernatant;

(7) the number of cells is less than 1 × 10⁸Adding 500uL buffer solution in an amount of more than 1 × 10⁸The buffer was added proportionally and the cells were resuspended.

(8) Preparing a magnetic sorting column, rinsing the column with 3mL of buffer solution for 1 time, adding 500uL of cell suspension for sorting cells, adding 3mL of buffer solution for washing the column for 2 times, and obtaining sorting negative cells, namely gamma delta T cells.

(9) Cell counting:

mixing the cells, sampling, counting by trypan blue staining, and collecting 1x10⁵One cell was subjected to flow assay and the remaining cells were inoculated.

3.2 culture of Gamma Delta T cells

Coating a 96-well plate by using an antibody, wherein the specific coating method is secondary antibody coating; anti-mouse 1gG working concentration is 10ug/mL, each well of a 96-well plate is 100uL of antibody diluent, incubation is carried out for 1h at 37 ℃, secondary antibody is discarded, and DPBS is added to wash the 96-well plate; primary anti-coating: the working concentration of the Purified anti-human g/d TCR (BI) is 100ng/mL, 100uL of antibody diluent is added into each hole of a 96-hole plate, the incubation is carried out for 1h at room temperature, primary antibody is discarded, and 200uL of DPBS (double stranded block copolymer) is added into the 96-hole plate to be soaked for later use.

The gamma delta T cells selected above were seeded on antibody-coated 96-well plates at a concentration of 1.5X10⁵One/well/200 ul. 5% CO at 37 ℃₂Culturing in an incubator, wherein the activated culture medium is used 6 days before culturing, and the amplification culture medium is used completely after 7 days.

The activation medium consists of KBM581, OK432, IFN-gamma, IL-2, IL-15, IL-21, 10% autologous plasma (inactivated). OK432 was 100ng/mL in the activation medium, IFN- γ was 1000IU/mL in the activation medium, IL-2 was 1000IU/mL in the activation medium, IL-15 was 100ng/mL in the activation medium, IL-21 was 100ng/mL in the activation medium, and autologous plasma (inactivated) was 10 vol% in the activation medium. The amplification medium consisted of KBM581, IL-2, 5% autologous plasma. IL-2 was present in the amplification medium at 1000IU/mL and autologous plasma (inactivated) was present in the amplification medium at 5 vol%.

Sorting and culturing of 4CD4-CD 8-cells

4.1 sorting of CD4-CD 8-cells Using the CD4, CD8 sorting kit from MACS

(2) centrifuging CBMC or PBMC at 2000r/min for 5min, discarding supernatant, adding 80uL/1 × 10⁷Resuspending CBMC in buffer solution;

(3) adding 20 uL/1X 10⁷CD4 and CD8 magnetic beads are mixed evenly and incubated for 15min at 4 ℃, and the mixture is shaken up once every 5 min;

(6) Preparing a magnetic sorting column, rinsing the column with 3mL of buffer solution for 1 time, adding 500uL of cell suspension for sorting cells, adding 3mL of buffer solution for washing the column for 2 times, and collecting negative cells, namely CD4-CD 8-cells.

(7) Cell counting:

the cells were mixed well and sampled for trypan blue staining counting. Inoculation concentration of 2X10⁶One/well/1 mL.

4.2 culture of CD4-CD 8-cells

Coating a 24-pore plate by using an antibody, wherein the specific coating method is as follows; Anti-CD16 with the working concentration of 1ug/mL, incubating the 24-well plate with 300uL of antibody diluent in each well at 37 ℃ for 1h, discarding the antibody, and adding 300 uL/well DPBS to soak the 24-well plate for later use.

The above-selected cells were seeded on antibody-coated 24-well plates at a concentration of 1 × 10⁶One/well/500 ul. 5% CO at 37 ℃₂Culturing in incubator, activating 6 days before culturingMedium, all amplification medium used after day 7.

The activation medium consists of KBM581, OK432, IFN-gamma, IL-2, IL-15, IL-21, 10% autologous plasma (inactivated). OK432 was 100ng/mL in the activation medium, IFN- γ was 1000IU/mL in the activation medium, IL-2 was 1000IU/mL in the activation medium, IL-15 was 100ng/mL in the activation medium, IL-21 was 100ng/mL in the activation medium, and autologous plasma (inactivated) was 10 vol% in the activation medium. Amplification medium consisted of KBM581, IL-2, 5% autologous plasma (inactivated). IL-2 was present in the amplification medium at 1000IU/mL and autologous plasma (inactivated) was present in the amplification medium at 5 vol%.

Transforming the sorted CD4-CD 8-cells into NK cells under the action of CD16 antibody and cytokine, and detecting the purity ratio of the NK cells by adopting a flow method before transfection, wherein the antibody is as follows: 7AAD, CD56-FITC, CD 3-APC.

5. And (3) slow virus packaging:

(1) cell plating:

A. DMEM, PBS and TrypLETM EXPRESS containing 10% fetal bovine serum are placed at 37 ℃ in 5% CO₂The cell culture box is preheated for 30 min.

B. 293FT grown to 80% -90% was washed once with PBS, 2mL of TrypLETM EXPRESS was added to each T175 cell culture flask and allowed to lay down well on the cell surface, the flask was covered, placed under a microscope for observation until the cells became round, 4mL of DMEM medium containing 10% fetal bovine serum was added to stop digestion, the cells on the flask wall were blown down with a pipette, the cell broth was transferred to a 15mL sterile centrifuge tube, centrifuged at 1500rpm for 5min, the supernatant was discarded, the cells were resuspended and counted.

C.293FT cells at 9X 10⁶Inoculating each 145mm plate, adding 20mL DMEM complete medium to each 145mm plate, shaking uniformly, and standing at 37 deg.C with 5% CO₂The cells in the cell culture box are continuously cultured.

(2) And (3) packaging the plasmid: plating the next day for plasmid packaging.

A. Will be provided with

buffer、

The four plasmids pMD2.G, pMDLg/pRRE, pRSV/REV, U6-SR8-MND1904 were returned to room temperature. Wherein, three auxiliary plasmids of pMD2.G, pMDLg/pRRE and pRSV/REV are purchased from Wuhan vast Ling company. The U6-SR8-MND1904 plasmid is constructed according to the following method:

the CAR gene shown in SEQ ID No.1 is cloned between recognition sites of restriction enzymes Cla I and Spe I of a multiple cloning site of a vector (Addgene) of pLenti, and a U6-SR8-MND1904 plasmid is obtained. In SEQ ID No.1, the nucleotide sequence of the U6 promoter is at positions 1-249, the nucleotide sequence of the SRb2m-8 encoding gene is at positions 252-310, the nucleotide sequence of the MND promoter is at positions 311-710, the recognition site sequence of restriction enzyme cleavage Pac I is at positions 711-718, the Kozak sequence is at position 719-724, the colony stimulating factor signal peptide sequence is at position 725-790, the nucleotide sequence of a single-stranded antibody (anti-CD 19-19 VH-linker-anti-CD19VL) against CD19 is at positions 791-1525, the nucleotide sequence of CD 8-8 a hinge region and transmembrane region is at positions 1526-1731861, the nucleotide sequence of 4-1BB is at positions 1736-1862-186191, the nucleotide sequence of CD3 ζ, the nucleotide sequence of EGFR 2262207-2260 is T2A, and the nucleotide sequence of EGFR 22634 is at positions 3334-T-22634.

B. 1.25mL per 145mm dish

The above solutions were mixed well in amounts of buffer, 15. mu. g U6-SR8-MND1904 plasmid, 7.5. mu.g psPAX2, 3.75. mu.g pMD2. G. Adding into the mixed solution

62.5. mu.L/145 mm dish, mixed well again, and left to stand at room temperature for 10 min.

C.293FT cells for packaging virus 5% CO from 37 ℃₂The cell culture box is taken out, firstly 75 percent alcohol is sprayed on the surface of a cell culture dish, and then the cell culture dish is placedPlacing in a safety cabinet, distributing the above mixed solution into culture medium of 145mm plate, shaking, and adding 5% CO at 37 deg.C₂An incubator.

3. Liquid changing device

After 4h of packaging, the old medium was discarded, 5mL of pre-warmed PBS was added to wash the cells, 20mL of fresh pre-warmed DMEM medium containing 10% fetal calf serum was added, and the cells were placed in a 37 ℃ 5% CO2 incubator.

4. Collection of viruses

A. And collecting the virus stock solution 48-72 h after the plasmid is packaged. The stock solution was collected in 50ml centrifuge tubes and centrifuged at 1500rpm for 5 min.

B. The supernatant was left 1mL for virus-related detection. The remainder was filtered through a 0.45 μm filter into a new centrifuge tube and centrifuged at 18300g at 4 ℃ for 2 h.

C. The supernatant was discarded as clean as possible and serum-free medium was added to resuspend the virus particles. Adding culture medium and virus stock solution at ratio of 1: 500 to obtain virus concentrated solution. Packaging the concentrated solution and storing in a refrigerator at-80 deg.C. The virus concentrated solution is lentivirus U6-SR8-MND 1904. Lentiviral U6-SR8-MND1904 includes the CAR gene (chimeric antigen receptor gene). Lentivirus U6-SR8-MND1904 expresses the CAR gene shown in SEQ ID No.1 in immune cells.

Example 2 Lentiviral transfection of peripheral blood-derived α β T cells

The peripheral blood-derived α β T cells cultured for 2 days in example 1 were used and classified into PS group, PGE2 group, P407 group and Stau group. According to 3X10⁵Each well was inoculated with a 24-well plate, serum-free medium (KBM581+200IU/mL IL-2) was added to each well, and the virus concentrate prepared in example 1, U6-SR8-MND1904, PS group, PGE2 group, P407 group and Stau group were added at concentrations of PS 8. mu.g/mL, PGE 21. mu.M, P407100. mu.g/mL and Stau 200nM, respectively, to give a final volume of 500. mu.l/well. Centrifuging at 35 deg.C and 2000rpm for 2h, taking out, adding 5% CO at 37 deg.C₂The culture medium is KBM581+200IU/ml IL-2, and the test is carried out after 96 h.

The transfection efficiency is detected by a flow cytometer, and the specifically used antibodies are EGFR-APC, CD3-APC-cy7, CD4-FITC, CD8-PB, 7AAD and SA-PE. Cell counts were performed using trypan blue staining.

The experimental results are shown in FIGS. 1 to 4. FIG. 1 is a representative flow chart of four transfection assisting reagents on peripheral blood-derived α β T cells, in which the transfection efficiencies of PS, PGE2, P407, and Stau transfection assisting reagents are: 66.00%, 85.10%, 34.29%, 60.00%. FIG. 2 shows the statistics of the transfection efficiency of peripheral blood-derived α β T cells, and the average transfection efficiency of PS, PGE2, P407, and Stau co-transfection reagents are: 50.33%, 77.33%, 33.67%, 60.00%. FIG. 3 shows statistics of cell viability, and the average viability of cells transfected with PS, PGE2, P407 and Stau is: 97.13%, 98.03%, 81.33%, 69.67%. FIG. 4 is a graph showing the amplification curves of cells transfected by the transfection assisting reagents PS and PGE2, wherein the amplification times of the cells on the day after PS transfection (D0), the fourth day (D4), the seventh day (D7), the tenth day (D10), the twelfth day (D12) and the fourteenth day (D14) are 1, 3.01, 29, 72.67, 101 and 94, respectively; the amplification fold of D0, D4, D7, D10, D12 and D14 after PGE2 transfection is 1, 4.9, 54, 105.3, 133 and 131 respectively.

This example illustrates that the transfection-assisting reagents PGE2 and Stau can significantly improve the transfection efficiency of α β T cells derived from peripheral blood, but the cell viability is significantly reduced after the Stau transfection-assisting reagent is added, suggesting that the transfection-assisting reagent has a greater toxicity to cells; PGE2 assisted transfection reagent not only did not reduce cell viability, but also was more favorable to cell growth.

Example 3 Lentiviral transfection of peripheral blood derived γ δ T cells

The peripheral blood-derived γ δ T cells cultured for 6 days in example 1 were used and classified into PS group, PGE2 group, P407 group and Stau group. According to 1 × 10⁶Cells were inoculated at a concentration of one/mL in a serum-free medium (KBM581+1000IU/mL IL-2), and the virus concentrates U6-SR8-MND1904, PS, PGE2, P407, and Stau prepared in example 1 were added at an MOI of 50, respectively, in a concentration of PS 8. mu.g/mL, PGE 21. mu.M, P407100. mu.g/mL, and Stau 200nM, respectively, with different transfection assisting reagents, to give a final volume of 500. mu.l/well. After centrifugation at 35 ℃ for 2 hours, the culture was continued. The next day, the medium was changed to the amplification medium described in example 1. At 96h post-transfection, samples were taken for detection.

The transfection efficiency is detected by a flow cytometer, and the specifically used antibodies are CD3-APC-Cy7, TCR alpha beta PE-Cy7, EGFR-APC and 7 AAD. Cell counts were performed using trypan blue staining.

The experimental results are shown in fig. 5-8. FIG. 5 shows the flow-based assay results of the primary transfection efficiency, wherein the transfection efficiencies of PS, PGE2, P407 and Stau transfection assisting reagents are: 16.6%, 23.8%, 15.8%, 23.3%. Fig. 6 shows statistics of transfection efficiencies of γ δ T cells derived from peripheral blood, and the average transfection efficiencies of PS, PGE2, P407, and Stau transfection assisting agents are: 17.2%, 23.7%, 13.05%, 26.60%. FIG. 7 is the statistics of the cell viability of each group after 96h of transfection, and the average viability of the cells after PS, PGE2, P407 and Stau transfection is: 94.78%, 95.53%, 88.50%, 32.95%. FIG. 8 shows the amplification curves of cells transfected by the transfection assisting reagents PS and PGE2, wherein the amplification times of the cells on the day after PS transfection (D0), the fourth day (D4), the eighth day (D8), the twelfth day (D12) and the eighteenth day (D18) are 1, 9.23, 25.79, 25.59 and 21.63 respectively; after transfection with PGE2, the amplification factors of D0, D4, D8, D12 and D18 were 1, 13.80, 39.80, 48.63 and 38.33, respectively.

The example proves that the transfection assisting reagents PGE2 and Stau can remarkably improve the transfection efficiency of the gamma delta T cells from peripheral blood, but the cell survival rate is obviously reduced after the Stau transfection assisting reagent is added, which indicates that the transfection assisting reagent has higher toxicity to the cells; PGE2 assisted transfection reagent not only did not reduce cell viability, but also was more favorable to cell growth.

Example 4 Lentiviral transfection of cord blood-derived γ δ T cells

Cord blood-derived γ δ T cells cultured for 6 days in example 1 were used and classified into PS group, PGE2 group, P407 group and Stau group. According to 1 × 10⁶Cells were inoculated at a concentration of one/mL in serum-free medium (KBM581+1000IU/mL IL-2), and virus concentrate U6-SR8-MND1904, PS group, PGE2 group, P407 group and Stau group prepared in example 1 were added at an MOI of 50 to the cells at concentrations of PS 8 μ g/mL, PGE21 μ M, P407100 μ g/mL and Stau 200nM, respectively, with different transfection assisting reagents, to give a final volume of 500 μ l/well. After centrifugation at 35 ℃ for 2 hours, the culture was continued. The next day, the medium was changed to practiceAmplification medium as described in example 1. At 96h post-transfection, samples were taken for detection.

The experimental results are shown in fig. 9-12. FIG. 9 shows representative flow-based measurements of transfection efficiencies of γ δ T cells derived from cord blood, wherein the transfection efficiencies of PS, PGE2, P407, and Stau transfection assisting reagents are: 46.0%, 66.5%, 48.8%, 68.4%. FIG. 10 is a statistical result of transfection efficiency of cord blood-derived γ δ T cells, and the average transfection efficiency of PS, PGE2, P407, and Stau co-transfection reagent is: 45.47%, 68.1%, 48.27%, 69.87%. FIG. 11 shows the statistics of cell viability, and the average viability of cells after transfection with PS, PGE2, P407 and Stau is: 98.75%, 97.72%, 79.33%, 34.00%. FIG. 12 is a graph showing the amplification curves of cells transfected by the transfection assisting reagents PS and PGE2, wherein the amplification factors on the day after PS transfection (D0), the fourth day (D4), the eighth day (D8), the twelfth day (D12), the eighteenth day (D18) and the twenty-second day (D22) are 1, 10.20, 24.50, 50.73, 92.00 and 120.33 respectively; after PGE2 transfection, the amplification fold of D0, D4, D8, D12, D18 and D22 were 1, 14.17, 49.93, 111.77, 227.72 and 178.43 respectively.

The transfection efficiency of the cord blood-derived γ δ T was overall higher than that of the peripheral blood-derived γ δ T (example 3), but the transfection efficiencies of the four cotransfection reagents were the same as the peripheral blood trend, again showing that PGE2 is a more suitable transfection reagent.

Example 5 Lentiviral transfection of cord blood-derived NK cells

The purity of NK cells was examined before transfection by sorting negative cells using cord blood-derived CD4 and CD8 cultured for 8 days in example 1, and the cells were classified into PS group, PGE2 group, P407 group and Stau group at the time of transfection. According to 1 × 10⁶Cells were inoculated at a concentration of one/mL in serum-free medium (KBM581+1000IU/mL IL-2), and the virus concentrate prepared in example 1, U6-SR8-MND1904, PS, PGE2, P407 and Stau, at an MOI of 50, were added to different transfection-assisting tests at concentrations of PS 8. mu.g/mL, PGE 21. mu.M, P407100. mu.g/mL and Stau 200nM, respectivelyThe final volume of the agent and the system is 500 ul/hole. After centrifugation at 35 ℃ for 2 hours, the culture was continued. The next day, the medium was changed to the amplification medium described in example 1. At 96h post-transfection, samples were taken for detection.

The transfection efficiency was measured by flow cytometry, and the specific antibodies used were CD3-APC-Cy7, CD56-PE, EGFR-APC, 7 AAD. Cell counts were performed using trypan blue staining.

The experimental results are shown in FIGS. 13A-17. FIG. 13A is a flow chart showing the change in the ratio of NK cells among cord blood-derived CD4-CD 8-cells, in which NK cell detection was carried out for 28.2%, 60.9%, 87.6%, and 96.6% on the day of culture (D0), 7 days of culture (D7), 14 days of culture (D14), and 21 days of culture (D21), respectively. FIG. 13B shows the statistical results of the percentage change of NK cells in cord blood-derived CD4-CD 8-cells, and the average percentage of NK cells in D0, D7, D14 and D21 detected cells is 25.23%, 61.83%, 83.77% and 96.47%. FIG. 14 is a representative flow chart of the measurement of transfection efficiency of cord blood-derived NK cells, in which the transfection efficiencies of PS, PGE2, P407, and Stau co-transfection reagents are: 20.2%, 45.0%, 23.7%, 45.7%. FIG. 15 is a statistic result of transfection efficiency of cord blood-derived NK cells, and the average transfection efficiencies of PS, PGE2, P407, and Stau co-transfection reagents were: 21.20%, 44.50%, 23.07%, 45.00%. FIG. 16 shows the statistics of cell viability, and the average viability of cells after transfection with PS, PGE2, P407 and Stau is: 96.77%, 96.40%, 79.10%, 32.43%. FIG. 17 is a graph showing the amplification curves of cells transfected with the transfection assisting reagents PS and PGE2, wherein the amplification factors on the day after PS transfection (D0), the seventh day (D7), the fourteenth day (D14), the twenty-first day (D21) and the twenty-eighth day (D28) are 1, 8.50, 36.00, 53.17 and 45.13, respectively; after transfection with PGE2, amplification factors of D0, D7, D14, D21 and D28 were 1, 12.83, 55.50, 69.67 and 69.97, respectively.

This example illustrates that PGE2 also has a promoting effect on NK cell transfection.

Example 6 transfection time optimization

Optimization of transfection time of umbilical cord blood-derived gamma delta T cells

The γ δ samples were collected at 5 days (D5), 6 days (D6), 7 days (D7), 8 days (D8) and 9 days (D9) of the culture in example 1, respectivelyT cells, replaced with transfection medium (KBM581+200IU/ml IL-2); according to 1 × 10⁶Cells were seeded at a concentration of one/mL, the transfection assisting reagent PGE2 was added, the lentivirus PLV _ M1904 prepared in example 6 was added at a MOI of 50 using a concentration of 1 μ M, and centrifuged at 2000rpm for 2h at 32 ℃; the next day, the medium was changed to the amplification medium described in example 1; and performing sampling flow detection on the transfection rate at the fourth day after transfection, wherein the antibodies used for detection are CD3-APC-Cy7, TCR alpha beta PE-Cy7, EGFR-APC and 7 AAD.

The number of test replicates was 3 and the data were processed as mean ± standard deviation.

As shown in fig. 18, since the γ δ T cells were transfected by the cultured D5, D6, D7, D8 and D9, the average transfection efficiencies (average values) were 5.1%, 34.2%, 27.87%, 22.03% and 9.4%, respectively, D6 was the optimal time for γ δ T cell transfection.

Second, optimization of transfection time of cord blood-derived NK cells

Cord blood-derived CD4 and CD8 sorting negative cells cultured for 5 days (D5), 8 days (D8), and 11 days (D11) in example 1 were collected, respectively, and purity of NK cells was examined before transfection. Are respectively in accordance with 1 × 10⁶Cells were seeded at a concentration of one/mL in serum-free medium (KBM581+1000IU/mL IL-2), virus concentrate U6-SR8-MND1904 prepared in example 1 was added at an MOI of 50, and transfection aid reagent was added at a concentration of PGE21 μ M to a final volume of 500 ul/well. After centrifugation at 35 ℃ for 2 hours, the culture was continued. The next day, the medium was changed to the amplification medium described in example 1. At 96h post-transfection, samples were taken for detection.

The transfection efficiency was measured by flow cytometry, and the specific antibodies used were CD3-APC-Cy7, CD56-PE, EGFR-APC, 7 AAD.

As shown in FIG. 19, the NK cells were transfected into cultured D5, D8 and D11, and the average transfection efficiencies were 18.83%, 23.8% and 16.10%, respectively, so that D8 was the most suitable time for NK cell transfection.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> Hebei Senlang Biotech Co., Ltd

<120> method for transfecting immune cells by lentivirus

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<170> SIPOSequenceListing 1.0

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gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60

ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120

aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180

atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240

cgaaacaccg gcagcagaga atggaaagtc aactcgagtt gactttccat tctctgctgt 300

ttttgaattc ttttatttag tctccagaaa aaggggggaa tgaaagaccc cacctgtagg 360

tttggcaagc taggatcaag gttaggaaca gagagacagc agaatatggg ccaaacagga 420

tatctgtggt aagcagttcc tgccccggct cagggccaag aacagttgga acagcagaat 480

atgggccaaa caggatatct gtggtaagca gttcctgccc cggctcaggg ccaagaacag 540

atggtcccca gatgcggtcc cgccctcagc agtttctaga gaaccatcag atgtttccag 600

ggtgccccaa ggacctgaaa tgaccctgtg ccttatttga actaaccaat cagttcgctt 660

ctcgcttctg ttcgcgcgct tctgctcccc gagctcaata aaagagccca ttaattaagc 720

caccatgctg ctgctggtga ccagcctgct gctgtgcgag ctgccccacc ccgcctttct 780

gctgatcccc gacatccaga tgacccagac cacctccagc ctgagcgcca gcctgggcga 840

ccgggtgacc atcagctgcc gggccagcca ggacatcagc aagtacctga actggtatca 900

gcagaagccc gacggcaccg tcaagctgct gatctaccac accagccggc tgcacagcgg 960

cgtgcccagc cggtttagcg gcagcggctc cggcaccgac tacagcctga ccatctccaa 1020

cctggaacag gaagatatcg ccacctactt ttgccagcag ggcaacacac tgccctacac 1080

ctttggcggc ggaacaaagc tggaaatcac cggcagcacc tccggcagcg gcaagcctgg 1140

cagcggcgag ggcagcacca agggcgaggt gaagctgcag gaaagcggcc ctggcctggt 1200

ggcccccagc cagagcctga gcgtgacctg caccgtgagc ggcgtgagcc tgcccgacta 1260

cggcgtgagc tggatccggc agccccccag gaagggcctg gaatggctgg gcgtgatctg 1320

gggcagcgag accacctact acaacagcgc cctgaagagc cggctgacca tcatcaagga 1380

caacagcaag agccaggtgt tcctgaagat gaacagcctg cagaccgacg acaccgccat 1440

ctactactgc gccaagcact actactacgg cggcagctac gccatggact actggggcca 1500

gggcaccagc gtgaccgtga gcagcactac cccagcaccg cggccaccca ccccggctcc 1560

taccatcgcc tcccagcctc tgtccctgcg tccggaggca tgtagacccg cagctggtgg 1620

ggccgtgcat acccggggtc ttgacttcgc ctgcgatatc tacatttggg cccctctggc 1680

tggtacttgc ggggtcctgc tgctttcact cgtgatcact ctttactgta agcgcggtcg 1740

gaagaagctg ctgtacatct ttaagcaacc cttcatgagg cctgtgcaga ctactcaaga 1800

ggaggacggc tgttcatgcc ggttcccaga ggaggaggaa ggcggctgcg aactgcgcgt 1860

gaaattcagc cgcagcgcag atgctccagc ctacaagcag gggcagaacc agctctacaa 1920

cgaactcaat cttggtcgga gagaggagta cgacgtgctg gacaagcgga gaggacggga 1980

cccagaaatg ggcgggaagc cgcgcagaaa gaatccccaa gagggcctgt acaacgagct 2040

ccaaaaggat aagatggcag aagcctatag cgagattggt atgaaagggg aacgcagaag 2100

aggcaaaggc cacgacggac tgtaccaggg actcagcacc gccaccaagg acacctatga 2160

cgctcttcac atgcaggccc tgccgcctcg gctcgagggc ggcggagagg gcagaggaag 2220

tcttctaaca tgcggtgacg tggaggagaa tcccggccct aggatgcttc tcctggtgac 2280

aagccttctg ctctgtgagt taccacaccc agcattcctc ctgatcccac gcaaagtgtg 2340

taacggaata ggtattggtg aatttaaaga ctcactctcc ataaatgcta cgaatattaa 2400

acacttcaaa aactgcacct ccatcagtgg cgatctccac atcctgccgg tggcatttag 2460

gggtgactcc ttcacacata ctcctcctct ggatccacag gaactggata ttctgaaaac 2520

cgtaaaggaa atcacagggt ttttgctgat tcaggcttgg cctgaaaaca ggacggacct 2580

ccatgccttt gagaacctag aaatcatacg cggcaggacc aagcaacatg gtcagttttc 2640

tcttgcagtc gtcagcctga acataacatc cttgggatta cgctccctca aggagataag 2700

tgatggagat gtgataattt ccggaaacaa aaatttgtgc tatgcaaata caataaactg 2760

gaaaaaactg tttgggacct ccggtcagaa aaccaaaatt ataagcaaca gaggtgaaaa 2820

cagctgcaag gccacaggcc aggtctgcca tgccttgtgc tcccccgagg gctgctgggg 2880

cccggagccc agggactgcg tctcttgccg gaatgtcagc cgaggcaggg aatgcgtgga 2940

caagtgcaac cttctggagg gtgagccaag ggagtttgtg gagaactctg agtgcataca 3000

gtgccaccca gagtgcctgc ctcaggccat gaacatcacc tgcacaggac ggggaccaga 3060

caactgtatc cagtgtgccc actacattga cggcccccac tgcgtcaaga cctgcccggc 3120

aggagtcatg ggagaaaaca acaccctggt ctggaagtac gcagacgccg gccatgtgtg 3180

ccacctgtgc catccaaact gcacctacgg atgcactggg ccaggtcttg aaggctgtcc 3240

aacgaatggg cctaagatcc cgtccatcgc cactgggatg gtgggggccc tcctcttgct 3300

gctggtggtg gccctgggga tcggcctctt catgtga 3337

Claims

1. A method for transfecting an immune cell with a lentivirus in vitro, comprising: the method comprises transfecting an immune cell with a lentivirus comprising RNA transcribed from a chimeric antigen receptor gene in a transfection system comprising prostaglandin E2.

2. The method of claim 1, wherein: the immune cell is an NK cell or a T lymphocyte; the T lymphocyte is an alpha beta T cell or a gamma delta T cell.

3. The method of claim 2, wherein: the immune cell is an NK cell or a gamma delta T cell, and the method comprises the following steps:

(1) activation culture: culturing said immune cells in an activation medium comprising KBM581, OK432, IFN- γ, IL-2, IL-15, IL-21, and 10% autologous plasma on a culture plate coated with an activating antibody;

(2) transfection: transfecting the activated cultured immune cells with the lentivirus;

4. The method of claim 3, wherein: the time of the activation culture in the step (1) is 2-8 days.

5. The method according to any one of claims 1-4, wherein: the concentration of prostaglandin E2 in the transfection system was 0.5-2. mu.M.

6. The method of claim 1, wherein: the lentivirus was transfected at an MOI = 2-50.

7. The method of claim 1, wherein: the transfection system also included serum-free medium consisting of KBM581 and IL-2.

8. The prostaglandin E2 is applied to the following A1) or A2),

A1) the use of lentiviruses in vitro to increase the transfection efficiency of immune cells;

A2) the application of the lentivirus in preparing products for improving the transfection rate of lentivirus to immune cells.

9. Use according to claim 8, characterized in that: the immune cell is an NK cell or a T lymphocyte; the T lymphocyte is an alpha beta T cell or a gamma delta T cell.