CN115466726B

CN115466726B - NK cell efficient gene transduction scheme

Info

Publication number: CN115466726B
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: 2023-09-19
Anticipated expiration: 2042-09-05
Also published as: CN115466726A

Abstract

The invention belongs to the field of biological medicine and biotechnology, and particularly relates to a high-efficiency gene transduction scheme of NK cells. In particular, a method for treating cells is provided for improving transduction efficiency in viral transduction, the method comprises the steps of activating and/or transduction, the activating uses a culture medium containing at least cytokines IL-2, IL-15 and IL-1 beta, and the transduction medium contains Polybrene and/or BX795.

Description

NK cell efficient gene transduction scheme

Technical Field

The invention belongs to the field of biological medicine and biotechnology, and particularly relates to a high-efficiency gene transduction scheme of NK cells.

Background

Natural Killer (NK) cells are derived from CD34 ⁺ Lymphoid progenitor cells, which have the ability to secrete Interferon gamma (IFN-gamma), are the first line of defense of the human body against infection or abnormal cells. NK cell failureIs only the main effector cell of the natural immune system and also plays an important role in the adaptive immune activation process. Studies have shown that NK cell activation is an induction of "CD4 ⁺ The key link of T lymphocyte independent Cytotoxic T Lymphocytes (CTLs) is that the main mechanism is: through activating NK cells to generate IFN-gamma, DC is further activated to generate IL-12, and CTLs are finally induced, so that the effects of killing tumors, monitoring recurrence and the like are exerted. Along with the continuous deep research of NK cell foundation and the continuous improvement of treatment technology, NK cells from different sources, such as peripheral blood, umbilical cord blood, embryonic stem cells, induced pluripotent stem cells, tumor cells and the like, are widely applied to foundation and clinical research. Compared with T lymphocytes, NK cells have the characteristics of wider antigen spectrum, no MHC restriction, no cytokine storm initiation and the like, and are increasingly attracting attention of researchers.

The great success of chimeric antigen receptor T lymphocytes (Chimeric antigen receptors-modified T cells, CAR-T) in hematological tumors has greatly driven the development of immune cell therapies. To date, 7 CRA-T products have been marketed in bulk worldwide for the treatment of hematological neoplasms. However, in clinical treatment, limitations of CAR-T are also emerging, such as poor efficacy against solid tumors; the 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 preparation of CAR-NK cells by using a strategy similar to CAR-T has made a major breakthrough in both basic and clinical studies. Its main advantages include: (1) Without MHC restriction, allograft use does not substantially produce graft versus host disease (Graft versus host disease, GVHD), and "shelf" products can be made; (2) The killing process mainly secretes IFN-gamma, interleukin (IL) -3, granulocyte-macrophage colony stimulating factor (Granulocyte-macrophage colony-stimulating factor, GM-CSF) and the like, and the expression level of tumor necrosis factor alpha (Tumor necrosis factor, TNF-alpha), IL-6 and the like is low, and the risk of cytokine storm and neurotoxicity is low; (3) Besides the CAR-mediated cell killing, the anti-tumor effect can be exerted by multiple mechanisms through a CAR independent path; (4) NK cells low-express PD1, and are expected to break through the solid tumor immunosuppression microenvironment.

Compared with other NK cells, the peripheral blood NK cells have the characteristics of high CD16 expression level, strong cell killing activity and the like, and are ideal effector cells for Antibody-dependent cell-mediated cytotoxicity (ADCC). However, gene transduction using recombinant lentiviruses has a number of difficulties based on the antiviral properties of terminally differentiated NK cells. Recombinant lentiviruses can enter cells by recognizing Low-density lipoprotein (Low-Density Lipoprotein, LDL) receptors on the surface of target cells, but since NK cell LDL receptors are expressed Low, the gene transduction efficiency of NK cells by unmodified recombinant lentiviruses is Low.

Disclosure of Invention

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

The technical scheme is specifically as follows:

product(s)

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

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

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

Preferably, the Polybrene concentration is 1.0 μg/mL-10.0 μg/mL; more preferably 8. Mu.g/mL.

Preferably, the concentration of BX795 is 0.05 μM to 1.0 μM; more preferably 0.1 μm.

Preferably, the concentration of the Dextran is 1.0 μg/mL-5.0 μg/mL; more preferably 2.0. Mu.g/mL.

Preferably, the concentration of Rosuvastatin is 100.0 μm to 1.0 μm; more preferably 2.0 μm.

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

(1) Polybrene at 8 μg/mL and BX795 at 0.1 μM;

(2) 0.1 μm BX795;

(3) Polybrene 8 μg/mL.

As demonstrated by the specific examples of the present invention, the improvement of the transduction efficiency of the two groups (1) and (2) of transduction reagent is superior to that of the group (3), and the cells treated with the transduction reagent of the group (2) in the two groups (1) and (2) have higher expansion capacity, so that the transduction reagent used in the most preferred transduction medium is the transduction reagent of the group (2) above.

In another aspect, the invention provides a cytokine combination comprising IL2, IL15, and further comprising one or more of IL-1β, IL18, and IL 21.

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

In another aspect, the invention provides an activation medium comprising a combination of the aforementioned 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-1β 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 consists of IL-2, IL-15 and IL-1β. Most preferably, the activation medium provided by the present invention is an X-VIVO15 medium containing 2000U/mL IL-2, 100ng/mL IL-15, and 100U/mL IL-1β.

Cells cultured for 2 days in the above activation medium 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 in performing viral transduction, the method comprising the step of activating and/or transducing.

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

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

Preferably, the activated culture environment is 37℃and 5% CO ₂ Saturated humidity.

Specifically, the step of activating is collecting cells, resuspending the cells with an activation 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 transducing comprises culturing the cells using a transduction promoting medium.

Specifically, the step of transduction is collecting 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℃and 5% CO ₂ Saturated humidity.

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

Preferably, the complete medium of the present invention is a 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 a 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, the CD16 expression in the activated cells is greater than 60%; more preferably greater than 70%.

Preferably, NK cells have a transduction density of 1.0-5.0X10 ⁶ Cells/ml; more preferably 2.0X10 ⁶ Cells/ml.

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

The "method for treating cells to improve transduction efficiency when performing viral transduction" according to the present invention may also be referred to as "a method for preparing cells that are easily transduced by viruses", in which the expression level of CD16 is significantly improved and the transduction efficiency of viruses is significantly improved when performing viral transduction on cells prepared by the method, as compared with cells that have not been treated by the method, and therefore, the "method for treating cells to improve transduction efficiency when performing viral transduction" according to the present invention may also be referred to as "a method for improving the expression level of CD16 in cells".

More preferably, the sample is peripheral blood.

Preferably, the cells to which the method is directed include primary NK cells isolated from healthy volunteer samples, NK cells obtained by stem cell induced differentiation, NK tumor cell lines.

Preferably, the healthy volunteer sample comprises peripheral blood, cord blood.

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

Preferably, the cells are obtained by treatment with:

(1) Obtaining a mononuclear cell; preferably, the peripheral blood mononuclear cells (Peripheral blood mononuclear cells, PBMCs) are obtained,

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

(3) First fluid infusion: on day 3, the medium of (2) was supplemented with medium containing 5% of inactivated plasma, IL-2 (2000 IU/mL), IL-15 (100 ng/mL), and IL-12 (100 IU/mL).

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

Preferably, the initial cell concentration when cells are performed in step (2) is 2.0X10 ⁶ And each ml.

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

Preferably, the volume of the cell culture medium after the fluid infusion in step (4) is changed from twice to six times, i.e. the volume of the culture medium added in the fluid infusion is four times the volume of the culture medium in step (2), and as used in the specific example, step (2) is inoculated with 20ml of cells and step (2) is supplemented with 80ml of the culture medium.

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

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

Preferably, the culture environments of the steps (1) - (5) are 37 ℃ and 5% CO ₂ Saturated humidity.

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

The most predominant cell type in the cells treated by the steps (1) - (4) or steps (1) - (5) is NK cells. It will be appreciated by those skilled in the art that blood is a complex composition and that cells obtained after the treatment of steps (1) - (4) or steps (1) - (5) remain a composition, but that NK cells are the cell type having the greatest percentage of NK cells among cells obtained after the treatment of steps (1) - (4) or steps (1) - (5), so that this NK cell-based cell population is also referred to as NK cells in the embodiments of the present invention.

The method of "obtaining mononuclear cells" or "obtaining peripheral blood mononuclear cells" according to the present invention is conventional, such as the single harvester mentioned in the specific example 1 of the present invention for collecting mononuclear cells, or Ficoll density gradient centrifugation method for separating mononuclear cells.

Preferably, the patient is a cancer patient. As proved by the specific example 3 of the invention, the CAR123-NK cells obtained after the recombinant lentivirus JD-LV-123-1 is transduced into the cells prepared by the method of the invention can be greatly amplified while maintaining higher activity, and has strong killing effect on AML (acute myelogenous leukemia) tumor cells and higher 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, VSV-G based recombinant lentiviruses.

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

Preferably, the infection strength 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 transduction promoting media.

In another aspect, the invention provides the use of the aforementioned combination of media for providing cells with high transduction efficiency (or in providing cells with properties that are easily transduced) when the cells are transduced by a virus.

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

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

Preferably, the virus is a recombinant lentivirus.

Preferably, the obtained genetically modified NK cells or CAR-NK cells, the NK cell activating receptor expression of which is up-regulated; 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, the use includes, but is not limited to, the use of any genetically modified NK cells or CAR-NK cells, in particular, the use in the manufacture of a cell therapy medicament, the manufacture of a medicament for the treatment of an antiviral infection, the manufacture of a medicament for the treatment of cancer or an autoimmune disease; the application in the combined treatment of the gene modified NK cells or the CAR-NK cells, antibody drugs, nucleic acid drugs, small molecule drugs, oncolytic virus drugs and cell drugs; the application of the modified NK cells or the CAR-NK cells in combination with radiotherapy, chemotherapeutics, stem cell transplantation, interventional therapy, ablation therapy and other therapeutic means.

The term "cancer" as used herein encompasses any type of cancer, including both solid and non-solid cancers. In particular, the cancer comprises 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, stomach cancer, esophageal cancer, thyroid cancer, transitional bladder epithelial cancer, leukemia, brain tumor, stomach cancer, peritoneal cancer, head and neck cancer, endometrial cancer, renal cancer, female genital tract cancer, 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-type detection of cellular components after separation of PBMCs.

FIG. 2 shows the results of detection of D5, NK cell transduction efficiency, fold expansion and viability following transduction by recombinant lentivirus JD-LV-19-1 mediated by different transduction promoting agents, A: transduction efficiency, B, cell viability, C expansion fold.

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

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

FIG. 5 is a graph showing the results of flow-through detection of cells prior to activation.

FIG. 6 is a graph showing the results of flow assays performed on cells after activation.

FIG. 7 is a graph of the results of a flow assay on cells prior to activation and transduction.

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

FIG. 9 is a graph showing 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, A is THP1, B is MOLM-13, C is KG-1a and D is HL60, by CAR123-NK cell culture until day 12, calcein-AM assay.

FIG. 10 is a graph showing the killing effect of CAR123-NK cells on CD123+ primary AML tumor cells by the Calcein-AM method, A is AML02 and B is AML03, cultured until day 12.

FIG. 11 is a flow cell detection of cytokine IFN- γ expression after co-incubation of CAR123-NK cells with tumor cells.

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

FIG. 13 is an amplification curve of cells cultured to 22 days after KA052 transduction by JD-LV-123-1.

FIG. 14 is the result of detection of killing of MOLM-13 by CAR123-NK cells 22 days after JD-LV-123-1 transduction.

Detailed Description

The present invention will be described in detail below with reference to the drawings and examples to facilitate understanding and practice of the invention and to further realize the innovative features of the present invention by those skilled in the art. Unless otherwise defined in the specification of the present invention, all technical terms herein are used according to conventional 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, unless otherwise specified, are commercially available.

The following description is of preferred embodiments of the invention and is not intended to limit the invention in any way, but rather to enable any person skilled in the art to make and use the invention as disclosed below with equivalent embodiments. Any simple modification or equivalent variation of the following embodiments according to the technical substance of the present invention falls within the scope of the present invention.

Example 1 Effect of different transduction reagents on transduction efficiency of recombinant lentiviruses

Step 1, preparation of NK cells from peripheral blood:

the peripheral blood sample is a peripheral blood whole blood sample or peripheral blood mononuclear cells (Peripheral blood mononuclear cells, PBMCs) collected by a single harvester. The following description will be made taking a peripheral blood whole blood sample as an example:

1. pretreatment of culture flask: 12.5mL of 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 liquid was allowed to spread well at the bottom of the flask and left to stand overnight at 4 ℃.

Isolation of PBMCs: centrifuging the collected peripheral blood sample anticoagulated by heparin sodium 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 the PBMCs were isolated by Ficoll density gradient centrifugation. Specifically, the above mixture was carefully added to a 50ml centrifuge tube containing a Ficoll layer, and centrifuged at 1800rmp for 30min (rise 9 and fall 0) at room temperature; the white membrane layer was sucked and washed twice with physiological saline.

Quality detection of PBMCs: isolated PBMCs were examined for expression of CD3, CD56, CD19, CD14 and CD16 by flow cytometry, and the results showed that CD3 ^- CD56 ⁺ The NK cells were 10% or more, which meets the standard, and induction culture of NK cells was possible (as shown in FIG. 1).

Induction culture of nk cells: PBMCs were prepared according to 2.0X10 ⁶ Cell concentration per mL cells were cultured using 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 the pretreated flask. In an incubator (37 ℃, 5% CO) ₂ Saturated humidity).

NK cell replacement fluid:

on day 3, 20mL of X-Vivo15 medium containing 5% of inactivated plasma, IL-2 (2000 IU/mL), IL-15 (100 ng/mL) and IL-12 (100 IU/mL) was added to the flask;

on day 5, 80mL of X-Vivo15 medium, 5% of inactivated plasma, IL-2 (2000 IU/mL), IL-15 (100 ng/mL), IL-12 (100 IU/mL) was continuously fed into the flask;

on day 7, the flask was continued to be supplemented with 120mL of X-Vivo15 medium containing 5% of inactivated plasma, IL-2 (2000 IU/mL), IL-15 (100 ng/mL) and IL-12 (100 IU/mL).

Step 2, transduction promotion treatment and virus transduction:

to compare the efficiency of transduction of recombinant lentivirus viruses against different transduction-promoting agent pairs, PBMCs-derived NK cells were cultured to day 9 as described above for viral transduction (exemplified by recombinant lentivirus JD-LV-19-1). The recombinant lentivirus JD-LV-19-1 is a recombinant lentivirus vector formed by expressing a CD 19-targeted CAR molecule. The CAR molecule is formed by sequentially connecting a CD8 signal peptide, a scFv region of a CD19 antibody, a CD8 hinge, a transmembrane region, a 4-1BB costimulatory region and a CD3 zeta region. After the CAR molecule is constructed into a lentiviral main plasmid vector, a four-plasmid packaging system is adopted to transduce 293T cells to obtain the recombinant lentiviral JD-LV-19-1.JD-LV-19-1 was further purified by column chromatography, and after dilution of the virus gradient, K562 cells were infected, and the flow cell was examined for expression of CAR19, and the infection titer was calculated to be 1.15X10 ⁸ TU/mL。

Step of transduction promotion: after the 9 th day of culture of PBMCs-derived NK cells according to the above-mentioned procedure: cells were collected 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 the density was adjusted to 2.0X10 ⁶ Cells/ml. 6-well plates were seeded at 2.0 ml/well and divided into A, B, C three groups:

group A was added Polybrene at 8 μg/mL and BX795 at 0.1 μM of transduction enhancing agent;

group B was added with 0.1 μm BX795 as transduction enhancing agent;

group C was added Polybrene at 8 μg/mL of transduction enhancing agent.

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

Mixing the above materials, and heating to 37deg.C and 5% CO ₂ Culturing in an incubator with saturated humidity for 4 hours. 2mL of fresh complete medium (VIVO 15 medium containing 5% inactivated plasma, 2000U/mL IL-2, 100ng/mL IL-15, 100U/mL IL-12) was supplemented for further culture for 24h; after taking out, collecting cells, and centrifuging at 1800rpm at room temperature for 10min to obtain a precipitate; the 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 according to 1X 10 ⁶ The cell/ml density is amplified, 1-2 times of the original culture medium is supplemented every two days, and the cell density is maintained at 0.5-2×10 ⁶ Cells/ml.

Experimental results: on day 5 after culturing to transduction, NK cells were examined for transduction efficiency, cell viability and fold expansion. The results show that the transduction efficiency of groups A and B is significantly higher than that of group C; group B had higher amplification capacity than groups a and C (as shown in figure 2).

Thus, the effect of different transduction-promoting agents on NK cell transduction efficiency, on the resulting transduction were taken into account comprehensively Influence of the expansion capacity of NK cells (CAR-NK cells), BX795 of 0.1. Mu.M is preferred as a transduction promoting agent.

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

In order to further improve the transduction efficiency of NK cells, peripheral blood mononuclear cells were cultured for 5 to 7 days according to the preparation method of peripheral blood-derived NK cells described in step 1 of example 1, and the NK cells were activated when the NK cell proportion was more than 60% (for example, primary cells KA050, KA054, KA 060) before transduction described in step 2 of example 1 was performed.

PBMCs were induced to culture to day 7 and NK cell fractions greater than 60% were detected by flow cytometry (shown in figure 3).

However, in contrast to cells isolated from PBMCs, CD16 expression is significantly down-regulated, i.e., the expression level of CD16 is affected by NK cell induction and culture, and CD16 is an important marker for NK cell conversion to cytotoxic NK cell subtype, which may be just the killing activity of NK cells. For this reason, NK cells need to be activated, and the experimental steps of activation are as follows:

cells were collected by centrifugation at 1800rpm for 10min at room temperature, resuspended in X-VIVO medium containing 2000U/mL of IL-2, 100ng/mL of IL-15 and 100U/mL of IL-1β, and the cell density was adjusted to 1.0X10 ⁶ cell/mL, inoculated in T175 flask at 37℃in 5% CO ₂ Culturing is continued in an incubator with saturated humidity.

Experimental results: after 2d of activation, flow cytometry detection showed a further increase in NK cell purity following activation, with significant up-regulation of CD16 expression levels (fig. 4). The statistics of the variation of the expression quantity of CD16 in the culture process of KA050 and KA054 are shown in the following table, and experimental results show that the activation treatment provided by the invention can remarkably improve the expression quantity of CD 16. In addition, after activation, NK cell activating receptors such as NKG2D (fig. 5C, 6C), NKP30 (fig. 5D, 6D), CD69 (fig. 5F, 6F), etc. were significantly upregulated, whereas NKP46 was not significantly altered (fig. 5E, 6E). The above results indicate that NK cells are significantly activated in the in-situ culture system.

TABLE 1 statistical variation of CD16 during cultivation

	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 lentiviral transduction system index CAR123-NK cells

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

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

Activated or non-activated KA059 and KA060 cells were collected, the pellet was collected by centrifugation at 1800rpm for 10min at room temperature, the cells were resuspended in X-VIVO15 medium containing 2000U/mL IL-2 and 100ng/mL IL-15, and the density was adjusted to 2.0X10 ⁶ Cells/ml; inoculating 6-well plate according to 2.0 ml/well, sequentially adding 1MOI or 3MOI recombinant lentivirus JD-LV-123-1, 0.1 μBX795 of M was mixed uniformly and then heated to 37℃with 5% CO ₂ Culturing in a saturated humidity incubator for 4 hours; 2ml of fresh complete culture medium is added for continuous culture for 24 hours; after removal, the cells were collected and centrifuged at 1800rpm at room temperature for 10min to harvest the pellet. The 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 according to 1X 10 ⁶ The cell/ml density is amplified, 1-2 times of the original culture medium is supplemented every two days, and the cell density is maintained at 0.5-2×10 ⁶ Cells/ml.

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

Experimental results:

the NK cells after activation and without activation are transduced respectively, the cell activation rate is above 90% after transduction for 5 days, and the cell proliferation times are more than 5 times. NK cells of the figures after the activation and transduction promoting treatments of the present invention were significantly more transduction efficient than non-activated NK cells (as shown in FIGS. 7-8).

CAR123-NK cells effectively kill CD123+ tumor cells

CAR-123-NK cells and CD123 ⁺ Acute myeloid leukemia (Acute myeloid leukemia, AML) tumor cells, such as MOLM-13, KG-1a, THP1 and CD123 ^- The killing effect of AML tumor cells HL60 is detected by incubating for 4 hours according to different target effect ratios. CAR123 compared to unmodified NK cells ^- NK cell pair CD123 ⁺ The killing effect of cells is obviously improved; and to CD123 ^- AML cells were not significantly enhanced (as shown in figure 9); in addition, it is against CD123 ⁺ Also the primary AML cells of (a) have a significant killing effect (as shown in figure 10). At the same time with CD123 ⁺ Co-incubation of tumor cells (e.g., MOLM-13 and THP 1) can significantly enhance IFN- γ expression; and with CD123 ^- Co-incubation of tumor cells (e.g., HL 60) failed to elevate IFN- γ expression levels (as shown in fig. 11).

Long-term culture of car-123-NK cells:

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

Claims

1. A cell treatment method for improving viral transduction efficiency, the method comprising NK cell activation treatment comprising the steps of: the cell density was adjusted to 1X 10 using X-VIVO15 medium containing only 2000U/mL of IL-2, 100ng/mL of IL-15 and 100U/mL of IL-1β ⁶ Individual cells/ml, activated for 2 days;

the method further comprises resuspending the activated NK cells with an X-VIVO15 medium containing only 2000U/mL IL-2 and 100ng/mL IL-15, and adjusting the density to 2.0X10 ⁶ Inoculating 6-hole plates with cells/ml according to 2.0 ml/hole, sequentially adding recombinant lentivirus and 0.1 mu M BX795, and culturing for 4 hours; 2mL of X-VIVO15 medium containing only 5% of inactivated plasma, 2000IU/mL of IL-2, 100ng/mL of IL-15 and 100IU/mL of IL-12 was supplemented for culture for 24 hours; after cell collection, the cells were cultured with X-VIVO15 medium containing only 2% of inactivated plasma, 2000U/mL of IL-2, 100ng/mL of IL-15 and 20ng/mL of IL-18;

the NK cells are obtained by the following treatment:

(1) Pretreatment of culture flask: physiological saline solution containing only CD137 at 8 μg/mL, CD28 at 8 μg/mL and CD3 at 8 μg/mL was added to the cell culture flask and the solution was allowed to disperse well at the bottom of the flask overnight at 4 ℃;

(2) Obtaining peripheral blood mononuclear cells;

(3) Induction of NK cells: will step by stepPeripheral blood mononuclear cells obtained in step (2) were obtained according to a ratio of 2.0X10 ⁶ Cell concentration per mL, using X-VIVO15 medium containing only 2000IU/mL IL-2, 100ng/mL IL-15, 100IU/mL IL-12, 5 μg/mL CD137 and 10% inactivated plasma, inoculating 20mL cells into the culture flask pretreated in step (1);

(4) First fluid infusion: on day 3, 20mL of X-VIVO15 medium containing only 5% of inactivated plasma, 2000IU/mL of IL-2, 100ng/mL of IL-15 and 100IU/mL of IL-12 was fed into the flask;

(5) And (3) supplementing liquid for the second time: 80mL of X-VIVO15 medium containing only 5% of inactivated plasma, 2000IU/mL of IL-2, 100ng/mL of IL-15 and 100IU/mL of IL-12 was continuously fed into the flask on day 5;

the infection intensity of the recombinant lentivirus is 1MOI or 3MOI; the recombinant lentivirus is a VSV-G based recombinant lentivirus.

2. The method of claim 1, wherein the NK cell treatment further comprises step (6) of third fluid replacement: on day 7, 120mL of X-VIVO15 medium containing only 5% of inactivated plasma, 2000IU/mL of IL-2, 100ng/mL of IL-15, and 100IU/mL of IL-12 was continuously fed into the flask.

3. The method of claim 1, wherein the recombinant lentivirus is purified by column chromatography.