CN115466726A

CN115466726A - High-efficiency gene transduction scheme of NK (natural killer) cells

Info

Publication number: CN115466726A
Application number: CN202211080597.6A
Authority: CN
Inventors: 贾兴龙; 韩瑞胜; 张晓艳; 黄园园; 杨月峰; 郭雷鸣; 王立燕
Original assignee: Beijing Jingda Biotechnology Co ltd
Current assignee: Beijing Jingda Biotechnology Co ltd
Priority date: 2022-09-05
Filing date: 2022-09-05
Publication date: 2022-12-13
Anticipated expiration: 2042-09-05
Also published as: CN115466726B

Abstract

The invention belongs to the technical field of biomedicine and biology, and particularly relates to a high-efficiency gene transduction scheme of NK cells. Specifically, a cell processing method for improving transduction efficiency in virus transduction is provided, the method comprises the steps of activating and/or promoting transduction, wherein a culture medium used for activating at least cytokines IL-2, IL-15 and IL-1 beta is contained, and the medium used for promoting transduction contains Polybrene and/or BX795.

Description

High-efficiency gene transduction scheme of NK (Natural killer) cells

Technical Field

The invention belongs to the technical field of biomedicine and biology, and particularly relates to a high-efficiency gene transduction scheme of NK cells.

Background

Natural Killer (NK) cells derived from CD34 ⁺ Lymphoid progenitor cells, which have the ability to secrete Interferon gamma (IFN- γ), are the first line of defense in humans against infected or abnormal cells. NK cells are not only the primary effector cells of the innate immune system, but also play an important role in the process of adaptive immune activation. Studies have shown that NK cell activation induces "CD4 ⁺ The key link of T lymphocyte independent Cytotoxic T Lymphocytes (CTLs) is the following mechanism: by activating NK cells to generate IFN-gamma, DC is further activated to generate IL-12, CTLs are finally induced, and the effects of killing tumors, monitoring relapse and the like are further exerted. With the continuous deepening of the basic research of NK cells and the continuous improvement of treatment technologies, NK cells from different sources, such as peripheral blood, umbilical cord blood, embryonic stem cells, induced pluripotent stem cells, tumor cells and other sources, are widely applied to basic and clinical research. Compared with T lymphocytes, NK cells have the characteristics of wider antigen spectrum, no MHC restriction, no initiation of cytokine storm and the like, and are increasingly concerned by researchers.

The great success of Chimeric antigen receptor T lymphocytes (CAR-T) in hematological tumors has greatly promoted the development of immune cell therapy. To date, 7 CRA-T products have been approved for global use in hematological tumor therapy. However, limitations of CAR-T also develop in clinical treatment, such as poor efficacy against solid tumors; individual preparation is needed, and the preparation period is long; is easy to generate neurotoxicity and potential safety hazard caused by cytokine storm, etc. In recent years, the adoption of a strategy similar to CAR-T to prepare CAR-NK cells has made a major breakthrough in both basic and clinical research. Its main advantages include: (1) The product has no MHC limitation, the foreign body application basically does not generate Graft-versus-host disease (GVHD), and the product can be prepared into a 'shelf' product; (2) The killing process mainly secretes IFN-gamma, interleukin (IL) -3, granulocyte-macrophage colony stimulating factor (GM-CSF), and the like, tumor necrosis factor alpha (TNF-alpha), IL-6, and the like, and has low expression level, low risk of cytokine storm and neurotoxicity; (3) In addition to CAR-mediated cell killing, multiple mechanisms can exert anti-tumor effects through CAR-independent pathways; (4) The NK cells are low in PD1 expression, and the solid tumor immunosuppressive microenvironment is expected to be broken through.

Compared with NK cells from other sources, the NK cells from peripheral blood have the characteristics of high CD16 expression level, strong cell killing activity and the like, and are ideal effector cells of Antibody-dependent cell-mediated cytotoxicity (ADCC). However, there are many difficulties in gene transduction using recombinant lentiviruses based on the antiviral properties of terminally differentiated NK cells. The recombinant lentivirus can enter cells by recognizing Low-Density Lipoprotein (LDL) receptors on the surfaces of target cells, but because the LDL receptors of NK cells are Low in expression, the gene transduction efficiency of the unmodified recombinant lentivirus on the NK cells is Low.

Disclosure of Invention

In order to solve the problem of low transduction efficiency of the recombinant lentivirus to the NK cell, the invention provides a technical scheme capable of remarkably improving the gene transduction efficiency of the NK cell, and solves the technical problem of low transduction efficiency of the recombinant lentivirus based on VSV-G to the NK cell.

The following technical scheme is specifically proposed:

product(s)

In one aspect, the invention provides a transduction promoting medium for NK cells, which contains a transduction promoting agent, wherein the transduction promoting agent comprises 1 or 2 of Vectofusin-1, polybrene, BX795, dextran and Rosuvastatin.

Preferably, the transduction promoting medium is X-VIVO15 medium containing 2000U/mL IL-2 and 100ng/mL IL-15, and the transduction promoting reagent is added.

Preferably, the concentration of the Vectofusin-1 is 2-20 mug/mL; more preferably, 10. Mu.g/mL.

Preferably, the concentration of Polybrene is 1.0 μ g/mL to 10.0 μ g/mL; more preferably, 8. Mu.g/mL.

Preferably, the concentration of BX795 is 0.05 μ M to 1.0 μ M; more preferably, 0.1. Mu.M.

Preferably, the concentration of said Dextran is 1.0 μ g/mL to 5.0 μ g/mL; more preferably, 2.0. Mu.g/mL.

Preferably, the concentration of the Rosuvastatin is 100.0 μ M to 1.0 μ M; more preferably, 2.0. Mu.M.

Most preferably, the transduction promoting medium provided by the invention is X-VIVO15 medium containing 2000U/mL IL-2 and 100ng/mL IL-15, and any one of the following transduction promoting reagents is added into the medium:

(1) Polybrene 8. Mu.g/mL and BX795 0.1. Mu.M;

(2) 0.1. Mu.M BX795;

(3) 8. Mu.g/mL Polybrene.

As demonstrated in the specific examples of the present invention, the improvement of transduction efficiency by the transduction promoting reagents of the (1) th and (2) th groups is better than that of the (3) th groups, while the cells treated with the transduction promoting reagent of the (2) th group in the (1) th and (2) th groups have higher amplification ability, so that the most preferred transduction promoting reagent used in the transduction promoting medium is the above transduction promoting reagent of the (2) th group.

In another aspect, the invention provides a cytokine combination comprising IL-2, IL-15, and further comprising one or more of IL-1 β, IL-18 and IL-21.

Preferably, the cytokine combination is composed of IL-2, IL-15 and IL-1 β.

In another aspect, the invention provides an activation medium comprising the aforementioned combination of cytokines.

Preferably, the basal medium of the activation medium is X-VIVO15 medium.

Preferably, the IL-2 concentration is 500-2000U/mL; more preferably, 2000U/mL.

Preferably, the IL-15 concentration is 20-200ng/mL; more preferably, 100ng/mL.

Preferably, the IL-1 beta concentration is 20-200U/mL; more preferably, 100U/mL.

Preferably, the IL-18 concentration is 10-200ng/mL; more preferably, 20ng/mL.

Preferably, the IL-21 concentration is 10-200ng/mL; more preferably, 30ng/mL.

Preferably, the cytokine combination is composed of IL-2, IL-15 and IL-1 β. Most preferably, the activation medium provided by the present invention is X-VIVO15 medium containing 2000U/mL IL-2, 100ng/mL IL-15 and 100U/mL IL-1 β.

The cells cultured in the above activation medium for 2 days as demonstrated in example 2 of the present invention significantly increased the expression level of CD 16.

Method

In another aspect, the invention provides a method of treating a cell to increase transduction efficiency when conducting viral transduction, the method comprising the step of activating and/or promoting transduction.

Preferably, the step of activating comprises culturing the cells using an activation medium.

More specifically, the step of activating is collecting the cells and culturing the cells using an activation medium.

Preferably, the activated culture environment is 37 ℃, 5% CO ₂ And saturated humidity.

Specifically, the step of activating is collecting the cells, suspending the cells with an activating medium, and culturing for at least 2 days; preferably, 2, 3 or 4 days; more preferably, 2 days (about 48 hours).

Preferably, the step of promoting transduction comprises culturing the cell using a transduction promoting medium.

Specifically, the step of promoting transduction is collecting the cells, resuspending the cells with a transduction promoting medium, and culturing for at least 1 hour.

Preferably, the culturing is continued for 1, 2, 3 or 4 hours; most preferably, 4 hours;

preferably, the transduction-promoting culture environment is 37 ℃, 5% CO ₂ And saturated humidity.

More specifically, the step of promoting transduction further comprises the step of supplementing a double volume of complete medium for 1 day (about 24 hours) after culturing the cells using the activation medium as above, and culturing the cells using an expansion medium (CAR-NK expansion medium) after collecting the cells. This amplification medium was used until viral transduction was performed.

Preferably, the complete medium of the invention is VIVO15 medium containing 5% inactivated plasma, 2000U/mL IL-2, 100ng/mL IL-15, 100U/mL IL-12.

Preferably, the amplification medium (CAR-NK amplification medium) is VIVO15 medium containing 2% inactivated plasma 2000U/mL IL-2, 100ng/mL IL-15, 20ng/mL IL-18.

Preferably, the purity of the activated NK cells is greater than 85%; more preferably, greater than 90%.

Preferably, CD16 expression in activated cells is greater than 60%; more preferably, greater than 70%.

Preferably, the transduction density of NK cells is 1.0 to 5.0X 10 ⁶ Cells/ml; more preferably, 2.0X 10 ⁶ Cells/ml.

The method of "harvesting cells" according to the invention is conventional, as is well known to the person skilled in the art, and in particular the method used in the examples is centrifugation at 1800rpm for 10min

The "cell processing method for improving transduction efficiency in viral transduction" according to the present invention may also be referred to as "a method for preparing a cell that is easily transduced by a virus", and when a cell prepared by the method is virally transduced, the expression level of CD16 is significantly improved and the transduction efficiency of a virus is significantly improved as compared to a cell that has not been treated by the method, and thus the "cell processing method for improving transduction efficiency in viral transduction" according to the present invention may also be referred to as "a method for improving the expression level of CD16 in a cell".

More preferably, the sample is peripheral blood.

Preferably, the cells targeted by the method comprise primary NK cells isolated from a sample of healthy volunteers, NK cells obtained by stem cell induced differentiation, NK tumor cell lines.

Preferably, the healthy volunteer samples comprise peripheral blood, cord blood.

Preferably, the stem cells include embryonic stem cells, hematopoietic stem cells, iPSCs.

Preferably, the cells are obtained by treating the following steps:

(1) Obtaining a mononuclear cell; preferably, the obtained are Peripheral Blood Mononuclear Cells (PBMCs),

(2) Inducing NK cells: culturing the mononuclear cells obtained in step 1) in a medium containing 2000IU/mL of IL-2, 100ng/mL of IL-15, 100IU/mL of IL-12, 5. Mu.g/mL of CD137 and 10% inactivated plasma,

(3) Liquid supplementing for the first time: on day 3, the medium of (2) was supplemented with a medium containing 5% inactivated plasma, IL-2 (2000 IU/mL), IL-15 (100 ng/mL), and IL-12 (100 IU/mL).

(4) And (3) second liquid supplement: on day 5, the medium of (3) was supplemented with a medium containing 5% inactivated plasma, 2000IU/mL IL-2, 100ng/mL IL-15, 100IU/mL IL-12.

Preferably, the initial cell concentration at which the step (2) is performed on the cells is 2.0X 10 ⁶ One per ml.

Preferably, the volume of the cell culture medium after the solution supplementing in step (3) is changed from one volume to two volumes, that is, the volume of the culture medium added in the solution supplementing step is the same as the volume of the culture medium in step (2), and as used in the specific embodiment, 20ml of cells are inoculated in step (2), and the solution supplementing in step (3) is supplemented with 20ml of the culture medium.

Preferably, the volume of the cell culture medium after the solution supplementing in the step (4) is changed from two times to six times, that is, the volume of the culture medium added in the solution supplementing is four times of the volume of the culture medium in the step (2), as used in the specific embodiment, the step (2) is used for inoculating 20ml of cells, and the solution supplementing in the step (2) is used for supplementing 80ml of culture medium.

Preferably, the step of treating the cells further comprises the step (5) of performing a third fluid replacement: on day 7, the medium of (4) was supplemented with a medium containing 5% inactivated plasma, 2000IU/mL IL-2, 100ng/mL IL-15, 100IU/mL IL-12.

Preferably, the volume of the cell culture medium after the solution supplementing in the step (5) is changed from six times to twelve times, that is, the volume of the culture medium added in the solution supplementing step is six times of the volume of the culture medium in the step (2), as used in the specific embodiment, 20ml of cells are inoculated in the step (2), and 120ml of culture medium is supplemented in the solution supplementing step (3).

Preferably, the culture environment of the above steps (1) - (5) are all 37 ℃, 5% ₂ And saturated humidity.

Preferably, the basal medium of the medium used in the above steps (1) - (5) is X-VIVO15 medium.

The most predominant cell type among the cells subjected to the treatment of said steps (1) to (4) or steps (1) to (5) is NK cells. As will be understood by those skilled in the art, blood is a complex composition, and the cells obtained after the steps (1) - (4) or the steps (1) - (5) are still the composition, but invariably, NK cells in the cells obtained after the steps (1) - (4) or the steps (1) - (5) are the largest cell type in proportion, so the cell population with NK cells as the most main cells is also called NK cells in the embodiment of the invention.

The method of "obtaining mononuclear cells" or "obtaining peripheral blood mononuclear cells" according to the present invention is a conventional method, for example, the mononuclear cell is collected by the apheresis machine mentioned in the embodiment 1 of the present invention, or the mononuclear cell is separated by the Ficoll density gradient centrifugation method.

Preferably, the patient is a cancer patient. As shown in the specific embodiment 3 of the invention, CAR123-NK cells obtained after the recombinant lentivirus JD-LV-123-1 is transferred into the cells prepared by the method can be greatly amplified, keep high survival rate, have strong killing effect on AML (acute myeloid leukemia) tumor cells and have high clinical use value.

Preferably, the virus is a recombinant lentivirus.

More preferably, the recombinant lentivirus comprises a VSV-G, baboon-based recombinant lentivirus; more preferably, the VSV-G based recombinant lentivirus.

Preferably, the recombinant lentivirus is purified by column chromatography and ultracentrifugation; more preferably, column chromatography purification.

Preferably, the infection intensity of the recombinant lentivirus is 1-10MOI (Multiplicity of infection), specifically including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; more preferably, 4MOI.

Other products and uses thereof

In another aspect, the invention provides a combination of media comprising the aforementioned activation media and a transduction promoting media.

In another aspect, the invention provides the use of a combination of the aforementioned media to confer high transduction efficiency to a cell when transduced by a virus (or to confer a trait on a cell that is readily transduced).

In another aspect, the invention provides cells (cell populations) treated by the above methods of preparation and uses in the preparation of genetically modified cells.

The genetically modified cell may also be referred to as a genetically modified NK cell, and may also be referred to as a CAR-NK cell after transduction of the CAR molecule by a virus. The transduction efficiency of the cells reaches more than 30 percent, and the CD3 of the cells ^- CD56 ⁺ The proportion of cells is greater than 98%.

Preferably, the virus is a recombinant lentivirus.

Preferably, the obtained genetically modified NK cell or CAR-NK cell has upregulated NK cell activating receptor expression; the activating receptors include CD69, NKp30, NKp44, NKp46.

In another aspect, the invention provides the use of a genetically modified cell in the manufacture of a medicament for the treatment of a disease.

Preferably, said use includes, but is not limited to, any genetically modified NK cell or CAR-NK cell use, in particular, in the manufacture of a medicament for cell therapy, in the manufacture of a medicament for combating viral infections, in the manufacture of a medicament for the treatment of cancer or autoimmune diseases; use in the combination therapy of genetically modified NK cells or CAR-NK cells with antibody drugs, nucleic acid drugs, small molecule drugs, oncolytic virus drugs and cellular drugs; the application of the gene modified NK cell or CAR-NK cell in combination with radiotherapy, chemotherapy drugs, stem cell transplantation operation, interventional therapy, ablation therapy and other treatment means.

The term "cancer" as used herein encompasses any type of cancer, including solid and non-solid cancers. In particular, the cancer includes cervical cancer, seminoma, testicular lymphoma, prostate cancer, ovarian cancer, lung cancer, rectal cancer, breast cancer, cutaneous squamous cell carcinoma, colon cancer, liver cancer, pancreatic cancer, gastric cancer, esophageal cancer, thyroid cancer, transitional epithelial carcinoma of the bladder, leukemia, brain tumor, gastric cancer, peritoneal cancer, head and neck cancer, endometrial cancer, kidney cancer, cancer of the female reproductive tract, carcinoma in situ, neurofibroma, bone cancer, skin cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma.

Drawings

FIG. 1 is a graph showing the results of flow cytometry of cellular components after PBMCs have been isolated.

FIG. 2 shows the results of the detection of transduction efficiency, amplification factor and activity rate of NK cells in D5 after transduction of different transduction promoting agents mediated by recombinant lentivirus JD-LV-19-1, A: transduction efficiency, B, cell viability, fold C amplification.

FIG. 3 is a graph showing the results of flow measurement of NK cell purity and CD16 expression on day 7 of PBMCs induction culture, wherein A is measurement of CD56 and CD3 in KA050 cells, B is measurement of CD16, C is measurement of CD56 and CD3 in KA054 cells, and D is measurement of CD 16.

FIG. 4 is a graph showing the results of flow assay of NK cell purity and CD16 expression 2 days after NK cell activation, wherein A is assay of CD56 and CD3 of KA050 cells, B is assay of CD16, C is assay of CD56 and CD3 of KA054 cells, and D is assay of CD 16.

FIG. 5 is a graph showing the results of flow assay of cells before activation.

FIG. 6 is a graph showing the results of flow assay of cells after activation.

FIG. 7 is a graph showing the results of flow assays performed on cells prior to activation and pro-transduction.

FIG. 8 is a graph showing the results of flow assays on cells after activation and pro-transduction.

FIG. 9 shows the killing effect of CAR123-NK cells on CD123+ AML tumor cell lines MOLM-13, KG-1a, THP1 and CD123-AML tumor cell line HL60 as measured by Calcein-AM method until day 12 after CAR123-NK cells were cultured, wherein A is THP1, B is MOLM-13, C is KG-1a, and D is HL60.

FIG. 10 shows CAR123-NK cells cultured to day 12, and the Calcein-AM method examined the killing effect of CAR123-NK cells on CD123+ primary AML tumor cells, where A is AML02 and B is AML03.

Figure 11 is flow cytometric detection of cytokine IFN- γ expression following CAR123-NK cell co-incubation with tumor cells.

FIG. 12 is the amplification curve of three cells after JD-LV-123-1 transduction.

FIG. 13 is the expansion curve of JD-LV-123-1 after transduction of KA052 cells cultured until 22 days after transduction.

FIG. 14 is a graph of the results of measurements of killing of MOLM-13 by CAR123-NK cells 22 days after JD-LV-123-1 transduction.

Detailed Description

The invention is described in detail below with reference to the drawings and examples, so that those skilled in the art can understand and implement the invention and further recognize the innovative points of the present invention. Unless defined otherwise in the present specification, all technical terms used herein are used in accordance with their customary definitions commonly used and understood by those of ordinary skill in the art. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.

The following description is only exemplary of the present invention, and is not intended to limit the present invention in any way, and those skilled in the art may modify the present invention by the following claims and their equivalents. Any simple modifications or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.

Example 1 Effect of different Protransduction Agents on the transduction efficiency of recombinant lentiviruses

Step 1, preparation of peripheral blood-derived NK cells:

the Peripheral blood sample is a Peripheral blood whole blood sample or Peripheral Blood Mononuclear Cells (PBMCs) collected by a single-sampling machine. The following is illustrated by taking a whole blood sample of peripheral blood as an example:

1. pretreating a culture flask: 12.5mL of a physiological saline solution containing 8. Mu.g/mL of CD137, 8. Mu.g/mL of CD28 and 8. Mu.g/mL of CD3 was added to a T175 cell culture flask (Corning), and the solution was sufficiently dispersed at the bottom of the flask and kept at 4 ℃ overnight.

Isolation of PBMCs: centrifuging the collected heparin sodium anticoagulated peripheral blood sample at room temperature and 1800rpm for 10min to collect plasma, and inactivating the plasma at 56 ℃ for 30min for later use; the blood cell pellet was diluted with physiological saline, and PBMCs were separated by Ficoll density gradient centrifugation. Specifically, the mixture was carefully added to a 50ml centrifuge tube containing a Ficoll layer and centrifuged at 1800rmp for 30min (up to 9 and down to 0) at room temperature; the buffy coat was aspirated and washed twice with normal saline.

Quality detection of PBMCs: the PBMCs obtained by separation are used for detecting the expression of CD3, CD56, CD19, CD14 and CD16 by flow cytometry, and the result shows that the CD3 ^- CD56 ⁺ The proportion of NK cells was 10% or more, and induction culture of NK cells was performed in accordance with the standard (as shown in FIG. 1).

Induced culture of NK cells: PBMCs are prepared according to the specification of 2.0 x 10 ⁶ Cell concentration per mL cells were cultured in X-VIVO15 medium containing 2000IU/mL IL-2, 100ng/mL IL-15, 100IU/mL IL-12, 5. Mu.g/mL CD137 and 10% inactivated plasma, and 20mL cells were inoculated into pre-treated flasks. In an incubator (37 ℃, 5% CO) ₂ Saturated humidity).

And 5, infusion of NK cells:

on day 3, the flasks were supplemented with 20mL of X-Vivo15 medium containing 5% inactivated plasma, IL-2 (2000 IU/mL), IL-15 (100 ng/mL), IL-12 (100 IU/mL);

on day 5, the flasks were continuously supplemented with 80mL of 5% inactivated plasma, IL-2 (2000 IU/mL), IL-15 (100 ng/mL), IL-12 (100 IU/mL) in X-Vivo15 medium;

on day 7, the flasks were continuously supplemented with 120mL of X-Vivo15 medium with 5% inactivated plasma, IL-2 (2000 IU/mL), IL-15 (100 ng/mL), IL-12 (100 IU/mL).

Step 2, transduction promoting treatment and virus transduction:

to compare the efficiency of the different transduction-promoting agents in promoting transduction of recombinant lentiviruses, PBMCs-derived NK cells were cultured to day 9 according to the above procedure for virus transduction (taking recombinant lentivirus JD-LV-19-1 as an example). The recombinant lentivirus JD-LV-19-1 is a recombinant lentivirus vector formed by expressing CAR molecules targeting CD 19. The CAR molecule is formed by sequentially connecting a CD8 signal peptide, a scFv region of a CD19 antibody, a CD8 hinge and transmembrane region, a 4-1BB costimulatory region and a CD3 zeta region. After the CAR molecule is constructed into a lentivirus main plasmid vector, a four-plasmid packaging system is adoptedAfter 293T cells were transduced, recombinant lentivirus JD-LV-19-1 was obtained. JD-LV-19-1 was further purified by column chromatography, virus was diluted in gradient, infected with K562 cells, flow-cytometrically detected CAR19 expression, and the titer of infection was calculated to be 1.15X 10 ⁸ TU/mL。

A transduction promoting step: PBMCs-derived NK cells were cultured by the previous procedure until after day 9: cells were harvested by centrifugation at 1800rpm for 10min at room temperature, resuspended in X-VIVO15 medium containing 2000U/mL IL-2 and 100ng/mL IL-15, and adjusted to a density of 2.0X 10 ⁶ Cells/ml. 6-well plates were inoculated at 2.0 ml/well, divided into three groups A, B, C:

polybrene with 8. Mu.g/mL transduction promoting reagent and 0.1. Mu.M BX795 were added to group A;

group B was supplemented with 0.1. Mu.M of transduction-promoting agent BX795;

polybrene was added to group C at 8. Mu.g/mL of transduction-promoting reagent.

And, the three groups of ABC are divided into a control group and an experimental group: control group, only transduction-promoting reagent was added, and virus (A1, B1, C1) was not added; in the experimental group, 4MOI of recombinant lentivirus JD-LV-19-1 and transduction-promoting reagents (A2, B2 and C2) are added in sequence.

Mixing them, and adding CO at 37 deg.C and 5% ₂ And culturing for 4 hours in an incubator with saturated humidity. Supplementing 2mL of fresh complete medium (VIVO 15 medium containing 5% inactivated plasma, 2000U/mL IL-2, 100ng/mL IL-15, and 100U/mL IL-12) and continuing to culture for 24h; taking out, collecting cells, centrifuging at room temperature of 1800rpm for 10min, and harvesting precipitates; CAR-NK amplification Medium (VIVO 15 Medium containing 2% inactivated plasma 2000U/mL IL-2, 100ng/mL IL-15, 20ng/mL IL-18) was used at 1X 10 ⁶ Expanding cells/ml, supplementing 1-2 times of original culture medium every two days, and maintaining cell density at 0.5-2 × 10 ⁶ Cells/ml.

The experimental results are as follows: and culturing until 5 days after transduction, and detecting the transduction efficiency, the cell survival rate and the amplification multiple of the NK cells. The results show that the transduction efficiency of groups a and B is significantly higher than that of group C; the amplification capacity of group B was higher than that of groups A and C (as shown in FIG. 2).

Therefore, a combination of different transduction-promoting agents on NK cellsEffect of cell transfer efficiency on the resulting transferred Influence of the expansion Capacity of NK cells (CAR-NK cells), 0.1. Mu.M BX795 is preferred as a transducibility-promoting agent.

Example 2: activation of peripheral blood-derived NK cells before transduction

In order to further increase the transduction efficiency of NK cells, when the peripheral blood mononuclear cells were cultured up to 5 to 7 days and the proportion of NK cells was more than 60%, i.e., prior to the transduction described in step 2 of example 1, according to the method for preparing peripheral blood-derived NK cells described in step 1 of example 1, the NK cells were activated (primary cells KA050, KA054, and KA060 were used as examples).

PBMCs were induced to culture until day 7 and flow cytometry was used to detect a NK cell proportion of greater than 60% (shown in FIG. 3).

However, compared with cells separated from PBMCs, the expression of CD16 is remarkably reduced, namely the expression of CD16 is influenced by inducing and culturing NK cells, and CD16 is an important mark for the transformation of NK cells to cytotoxic NK cell subtypes and can only be the killing activity of the NK cells. For this purpose, the NK cells need to be activated, and the experimental steps for activation are as follows:

cells were harvested by centrifugation at 1800rpm for 10min at room temperature, resuspended in X-VIVO medium containing 2000U/mL IL-2, 100ng/mL IL-15 and 100U/mL IL-1 β, and cell density adjusted to 1.0X 10 ⁶ cells/mL, inoculated in a T175 flask, at 37 ℃ and 5% ₂ And continuously culturing in an incubator with saturated humidity.

The experimental results are as follows: after 2 days of activation, flow cytometry results show that after activation, the purity of NK cells is further improved, and the expression level of CD16 is remarkably up-regulated (figure 4). The statistics of the expression change of CD16 in the culture processes of KA050 and KA054 are shown in the following table, and the experimental results show that the activation treatment provided by the invention can obviously improve the expression of CD 16. Furthermore, upon activation, activating receptors for NK cells, such as NKG2D (fig. 5C, 6C), NKP30 (fig. 5D, 6D), CD69 (fig. 5F, 6F), etc., were significantly upregulated, while NKP46 was not significantly altered (fig. 5E, 6E). The above results indicate that NK cells are significantly activated in the culture system.

TABLE 1 statistics of CD16 changes during culture

	PBMCs	Before activation	After activation
				KA050	94.6％	32.32% (FIG. 3B)	70.61% (FIG. 4B)
KA054	93.34％	9.57% (FIG. 3D)	42.65% (FIG. 4D)
				KA060	98.46％	38.63% (FIG. 5B)	64.15% (FIG. 6B)

Example 3: use of optimized recombinant lentivirus transduction system index CAR123-NK cells

Activated NK cells of example 2 were transduced (recombinant lentivirus JD-LV-123-1 as an example) and the activity of the obtained CAR123-NK cells was verified.

The recombinant lentivirus JD-LV-123-1 is a recombinant lentivirus vector expressing a CAR molecule targeting CD 123. Wherein the CAR molecule is formed by sequentially connecting a CD8 signal peptide, a scFv region of a CD123 antibody, a CD8 hinge and transmembrane region, a 4-1BB costimulatory region and a CD3 zeta region. After the CAR molecule is constructed into a lentivirus main plasmid vector, a four-plasmid packaging system is adopted to transduce 293T cells to obtain the recombinant lentivirus JD-LV-123-1.JD-LV-123-1 was further purified by column chromatography, virus was diluted in gradient, infected with K562 cells, flow-cytometrically detected the expression of CAR123, and the titer of infection was calculated to be 5.93X 10 ⁷ TU/ml。

Collecting activated or non-activated KA059 and KA060 cells, centrifuging at 1800rpm for 10min at room temperature to collect the precipitate, resuspending the cells in X-VIVO15 medium containing 2000U/mL IL-2 and 100ng/mL IL-15, adjusting the density to 2.0X 10 ⁶ Cells/ml; inoculating 6-well plate at 2.0 ml/well, sequentially adding 4MOI recombinant lentivirus JD-LV-123-1 and 0.1. Mu.M BX795, mixing, and removing CO at 37 deg.C and 5% ₂ Culturing for 4 hours in an incubator with saturated humidity; supplementing 2ml of fresh complete culture medium and continuing to culture for 24 hours; after removal, the cells were collected and centrifuged at 1800rpm for 10min at room temperature to harvest the pellet. CAR-NK expansion medium (VIVO 15 medium containing 2% inactivated plasma 2000U/mL IL-2, 100ng/mL IL-15, 20ng/mL IL-18) was used at 1X 10 ⁶ Expanding the cell/ml density, supplementing 1-2 times of original culture medium every two days, and maintaining the cell density at 0.5-2 × 10 ⁶ Cells/ml.

12 days after the recombinant lentivirus JD-LV-123-1 transduces NK cells, cells are collected by centrifugation at 1800rpm for 10min at room temperature, and CAR123-NK cells are obtained.

The experimental results are as follows:

and (3) respectively conducting transduction on the activated and non-activated NK cells, wherein the cell survival rate is over 90% and the cell proliferation times are more than 5 times after 5 days of transduction. The transduction efficiency of NK cells of the figures after activation and pro-transduction treatment according to the invention was significantly higher than that of non-activated NK cells (as shown in figures 7-8).

CAR123-NK cell killing efficacyInjured CD123+ tumor cells

Contacting CAR-123-NK cells with CD123 ⁺ Acute Myeloid Leukemia (AML) tumor cells, such as MOLM-13, KG-1a, THP1 and CD123 ^- The AML tumor cell HL60 is incubated for 4 hours according to different effect-target ratios, and the killing effect is detected. CAR123 compared to unmodified NK cells ^- NK cell pair CD123 ⁺ The killing effect of the cells is obviously improved; to CD123 ^- AML cells, no significant enhancement (as shown in figure 9); in addition, it is to CD123 ⁺ The primary AML cells of (a) also had a significant killing effect (as shown in figure 10). At the same time, with CD123 ⁺ The co-incubation of tumor cells (such as MOLM-13 and THP 1) can obviously enhance the expression of IFN-gamma; and CD123 ^- Tumor cells (e.g., HL 60) were unable to increase IFN-. Gamma.expression levels upon co-incubation (see FIG. 11).

CAR-123-NK cell long-term culture:

after transduction of NK cells by recombinant lentiviruses, CAR123-NK cells were obtained. After the expansion to the 5 th day, the cell density is maintained at 0.5-2 × 10 by supplementing 1-2 times of original culture medium every other day ⁶ The expansion culture of CAR-NK cells was performed at a cell/ml. 3 primary cells (KA 050, KA052, KA 054) from different patients were prepared into CAR-NK cells by the above steps, and the amplification times of the CAR-NK cells on day 12 were all above 100-fold (as shown in FIG. 12). Especially, the amplification multiple of the KA052 sample reaches more than 900 times (as shown in FIG. 13) after the sample is amplified to 22 days after transduction; meanwhile, the obtained cells still maintain the survival rate of more than 90 percent and have the survival rate on CD123 ⁺ AML tumor cells had a strong killing effect (as shown in figure 14).

Claims

1. A transduction promoting medium for NK cells comprising a transduction promoting agent comprising 1 or 2 of Vectofusin-1, polybrene, BX795, dextran, and Rosuvastatin;

preferably, the transduction promoting medium is X-VIV015 medium containing 2000U/mL IL-2 and 100ng/mL IL-15 with the addition of transduction promoting reagent;

preferably, the concentration of the Vectofusin-1 is 2-20 mug/mL; more preferably, 10 μ g/mL;

preferably, the concentration of Polybrene is 1.0 μ g/mL to 10.0 μ g/mL; more preferably, 8 μ g/mL;

preferably, the concentration of BX795 is 0.05 μ M to 1.0 μ M; more preferably, 0.1 μ M;

preferably, the concentration of said Dextran is between 1.0 μ g/mL and 5.0 μ g/mL; more preferably, 2.0. Mu.g/mL;

preferably, the concentration of the Rosuvastatin is 100.0 μ M to 1.0 μ M; more preferably, 2.0 μ M;

most preferably, the transduction promoting medium is X-VIV015 medium containing 2000U/mL IL-2 and 100ng/mL IL-15, supplemented with any one of the following transduction promoting agents:

(1) 8. Mu.g/mL Polybrene and 0.1. Mu.M BX795;

(2) 0.1. Mu.M BX795;

(3) Polybrene 8. Mu.g/mL.

2. A cytokine combination comprising IL2, IL15 and one or more of IL-1 β, IL18 and IL 21;

preferably, the cytokine combination is composed of IL-2, IL-15 and IL-1 β.

3. An activation medium comprising the combination of cytokines of claim 2;

preferably, the basal medium of the activation medium is X-VIV015 medium;

preferably, the IL-2 concentration is 500-2000U/mL; more preferably, 2000U/mL;

preferably, the IL-15 concentration is 20-200ng/mL; more preferably, 100ng/mL;

preferably, the IL-1 beta concentration is 20-200U/mL; more preferably, 100U/mL;

preferably, the IL-18 concentration is 10-200ng/mL; more preferably, 20ng/mL;

preferably, the IL-21 concentration is 10-200ng/mL; more preferably, 30ng/mL;

preferably, the cytokine combination is composed of IL-2, IL-15 and IL-1 beta;

most preferably, the activation medium is X-VIV015 medium containing 2000U/mL IL-2, 100ng/mL IL-15 and 100U/mL IL-1 β.

4. A method of cell processing to increase transduction efficiency when performing viral transduction, the method comprising the steps of activating and/or promoting transduction;

preferably, the step of activating comprises culturing the cells with the activation medium of claim 3;

preferably, the culturing lasts for 2-4 days; more preferably, the culturing lasts for 2 days;

preferably, the step of promoting transduction comprises culturing the cell with the transduction promoting medium of claim 1;

preferably, the culturing lasts for 1-4 hours; most preferably, 4 hours.

5. The method of claim 4, wherein the cells targeted by the method comprise cells isolated from a sample from a subject or NK tumor cell lines;

preferably, the subject comprises a healthy or cancer patient;

preferably, the sample comprises peripheral blood, cord blood, iPSC;

more preferably, the sample is peripheral blood;

preferably, the cells are obtained by treating the following steps:

(1) Obtaining a mononuclear cell; preferably, the obtaining is peripheral blood mononuclear cells;

(2) Inducing NK cells: culturing the mononuclear cells obtained in step 1) with a medium containing 2000IU/mL of IL-2, 100ng/mL of IL-15, 100IU/mL of IL-12, 5. Mu.g/mL of CD137 and 10% inactivated plasma;

(3) First liquid supplementing: on day 3, the medium of (2) was supplemented with a medium containing 5% inactivated plasma, 2000IU/mL IL-2, 100ng/mL IL-15, 100IU/mL IL-12;

(4) And (3) second liquid supplement: on day 5, the medium of (3) was supplemented with a medium containing 5% inactivated plasma, 2000IU/mL IL-2, 100ng/mL IL-15, 100IU/mL IL-12;

preferably, the step of treating the cells further comprises the step (5) of performing a third fluid replacement: on day 7, the medium of (4) was supplemented with a medium containing 5% inactivated plasma, 2000IU/mL IL-2, 100ng/mL IL-15, 100IU/mL IL-12;

preferably, the basal medium of the medium used in the above steps (1) to (5) is X-VIVO15 medium;

preferably, the patient is a cancer patient, said cancer comprising cervical cancer, seminoma, testicular lymphoma, prostate cancer, ovarian cancer, lung cancer, rectal cancer, breast cancer, cutaneous squamous cell carcinoma, colon cancer, liver cancer, pancreatic cancer, gastric cancer, esophageal cancer, thyroid cancer, transitional epithelial carcinoma of the bladder, leukemia, brain tumor, gastric cancer, peritoneal cancer, head and neck cancer, endometrial cancer, kidney cancer, cancer of the female reproductive tract, carcinoma in situ, neurofibroma, bone cancer, skin cancer, gastrointestinal stromal tumors, mast cell tumors, multiple myeloma, melanoma, glioma.

6. The method of claim 4, wherein the virus is a recombinant lentivirus;

more preferably, the recombinant lentivirus comprises a VSV-G, baboon-based recombinant lentivirus; more preferably, a VSV-G based recombinant lentivirus;

preferably, the recombinant lentivirus is purified by column chromatography and ultracentrifugation; more preferably, column chromatography purification;

preferably, the infection intensity of the recombinant lentivirus is 1-10MOI; more preferably, 4MOI.

7. A media combination comprising the activation medium of claim 3 and the transduction promoting medium of claim 1.

8. Use of one or more media from the combination of media according to claim 7 for high transduction efficiency of cells transduced with a virus.

9. The cells produced by the method of claim 4 or their use in the production of genetically modified NK cells;

preferably, the NK cell is upregulated in expression of an activating receptor; the activating receptor comprises CD69, NKp30, NKp44, NKp46, or the CD3-CD56 ⁺ The proportion of cells is greater than 98%.

10. Use of genetically modified NK cells produced from cells produced by the method of claim 4 for the preparation of a medicament, a pharmaceutical composition;

preferably, the medicament comprises a cell therapy medicament, a medicament for preparing antiviral infection, a medicament for preparing cancer or autoimmune disease;

preferably, the cell composition further comprises an antibody drug, a nucleic acid drug, a small molecule drug, an oncolytic virus drug and a cell drug.