CN113913458A

CN113913458A - Preparation of NK cells with stable and high expression of chimeric receptors by non-viral method

Info

Publication number: CN113913458A
Application number: CN202010657827.5A
Authority: CN
Inventors: 肖�琳; 裘新红; 彭群武; 张亮; 朱苏闽; 徐芳芳
Original assignee: Hangzhou Youkairui Pharmaceutical Technology Co ltd
Current assignee: Hangzhou Shuoxi Biopharmaceutical Co ltd
Priority date: 2020-07-09
Filing date: 2020-07-09
Publication date: 2022-01-11
Also published as: WO2022007665A1

Abstract

The present invention provides a method for preparing natural killer (NK) cells that stably and highly expresses chimeric receptors by using a non-viral method and its application. Transferring into natural killer cells to obtain original chimeric receptor-modified natural killer cells; (2) using artificial antigen-presenting cells to expand the original chimeric receptor-modified natural killer cells. Compared with the prior art, the method of the present invention is simple, fast, efficient, accurate, repeatable, cost-effective, and easy to operate. It has good safety, and the chimeric receptor-modified NK cells prepared from this have strong tumor-killing ability, and show a good anti-tumor effect in vitro, which is used in various fields of biomedicine, especially cell therapy of diseases The field has broad application prospects.

Description

Non-viral method for preparing NK (natural killer) cells of stable high-expression chimeric receptor

Technical Field

The invention belongs to the technical field of medical bioengineering, and relates to a method for preparing stable high-expression chimeric receptor modified NK cells by using a non-viral method and application thereof.

Background

Natural Killer (NK) cells are an important component of the innate immune system, with a proportion of NK cells in peripheral blood lymphocytes of about 10%, the third largest lymphocyte population following B-cells and T-cells. NK cells have multiple biological functions and are the first line of defense against infection and tumors in humans. NK cell function is regulated by a delicate and complex network of activating and inhibitory receptors. Most circulating NK cells are in the resting phase and can be activated by cytokines and infiltrate into pathogen infected or malignant cell containing tissues. NK cells can also secrete certain cytokines, such as Interferon (IFN) - γ, to exert immunomodulatory effects when their receptors bind to corresponding ligands. One of the main functions of NK cells is to carry out immune surveillance on the body, and researchers have found that NK cells are involved in controlling the development of various diseases. Several in vitro studies on mammalian cells (including human cells) and in vivo studies in mice and rats have shown that NK cells can recognize tumor cells as targets and control the growth, metastasis and spread of tumor cells in vivo. One 11-year follow-up epidemiological survey showed that a decrease in peripheral blood NK cell activity increases the risk of cancer in adults. Moreover, in vitro experiments and animal experiments also find that the NK cells have good inhibition and elimination capability on SARS, H5N1, Ebola, Zika, dengue fever, HIV and other outbreak virus infection diseases.

Unlike MHC-restricted T cells, NK cells do not require HLA matching, and thus can be used as a heterologous stock-cell drug for immunotherapy of patients, and have important clinical application prospects. Adoptive therapies based on autologous or allogeneic NK cells have been clinically applied to anti-tumor, anti-viral infection, and particularly allogeneic NK cells show good therapeutic effects for the treatment of hematologic cancers.

Although NK cells have a powerful immune effector function, many cancer cells and viruses suppress NK cell function through mechanisms such as antigen escape and immunosuppression. For these reasons, scientists have been exploring, over the past decade, ways to enhance the functional activity of NK cells and avoid immunosuppression by genetic engineering methods, such as enhancing the proliferation capacity of NK cells following reinfusion by expressing endogenous cytokines, suppressing immunosuppressive signals or enhancing the killing function of NK cells, etc. The latter approach currently redirects NK cells to tumor cells primarily through modification of Chimeric Antigen Receptors (CARs). The chimeric antigen receptor is formed by fusing an antibody ScFv with an intracellular signal transduction structure, wherein the ScFv is used for mediating the recognition and the binding of tumor cell surface antigens.

However, primary NK cells are more resistant to gene transfection, which results in low uptake of NK cells into various vector systems, low transgene expression of NK cells, and higher gene transfer efficiency in primary NK cells only with viral vectors. Currently, the modification of chimeric receptors for primary NK cells is generally achieved by either electroporating the mRNA of the gene encoding the chimeric receptor or by infection with lentiviruses/retroviruses. The mRNA electrotransformation only can ensure that the chimeric receptor coding gene can be transiently expressed, the cost for synthesizing the mRNA in vitro is high, multiple injections are required clinically, and the treatment cost is high. Lentivirus/retrovirus transfection can prepare the NK cell with stably expressed chimeric receptor gene, however, compared with NK cell strains such as NK92, the infection difficulty of primary NK cells, especially peripheral blood NK cells is very high, so far, only few scientific research teams successfully apply lentivirus/retrovirus to prepare the chimeric antigen receptor modified primary NK cells, after repeated infection for many times, the expression proportion of transgene in the final product is between 20% and 80%, and the transfection efficiency of retrovirus is higher than that of lentivirus. However, since retroviruses and/or lentiviruses are themselves pathogenic, with the potential for insertional mutagenesis, they constitute a significant regulatory hurdle to the implementation of human clinical trials. Moreover, the large scale production of viral vectors in cell lines has the problems of high cost and low efficiency, and poses a barrier to the clinical transformation of chimeric receptor-modified NK cells, significantly increasing production costs.

To overcome the above limitations of viral vector systems, researchers have further explored the feasibility of non-viral vectors. Generally, the non-viral vector system is an artificial synthetic system, which does not depend on any viral components or mammalian cells for production and manufacture, and is low in cost, and simultaneously, is beneficial to avoiding the immune problems caused by the use of viral vectors. The DNA transposon is a typical non-viral vector system and has the advantages of gene sustainable expression, low immunogenicity, low cost, high efficiency and the like.

In nature, DNA transposons are discrete DNA fragments that contain a transposase gene, flanked by Inverted Terminal Repeats (ITRs) that contain transposon binding sites. The process of transposition is that the transposase binds to the TIRs, "splicing" the transposition from one location and "sticking" to another new location. Transposon-based vector systems, such as the Sleeping Beauty (SB) system, piggybac (pb) system, Tol transposon system, are methods that utilize transposition to introduce transgenes into the host genome. Similar to retroviral vectors, stable genomic integration into the host can achieve long-term efficient transgene expression. Both SB and PB transposons have been successfully used to modify T cells to express CD19 specific CARs. However, there is no report of using a transposon system to prepare a primary NK cell that is genetically modified.

Disclosure of Invention

In order to overcome the problems of low efficiency of performing chimeric receptor modification on primary NK cells by the current technical means, supervision obstacle and high production cost caused by using retrovirus, the invention provides a non-viral method for preparing chimeric receptor modified NK cells, which introduces a stable and high-expression chimeric receptor into NK cells by using a transposon system to realize high-efficiency genetic modification on the NK cells.

(1) Transferring the coding gene of the chimeric receptor into NK cells by using a transposon system;

(2) amplifying the chimeric receptor-modified NK cells using artificial antigen presenting cells.

The NK cell is a human primary NK cell, and can be derived from human peripheral blood, umbilical cord blood, Induced Pluripotent Stem Cells (iPSCs) or the like.

The chimeric receptors include chimeric antigen receptors, chimeric switch receptors, or other chimeric receptors synthesized using recombinant DNA techniques.

The transposon system in the step (1) comprises a piggyBac transposon system, a Sleeping Beauty transposon system or a Tol2 transposon system and the like.

Preferably, the transposon system of step (1) comprises a transposon element and a transposase element; the transposon element comprises a transposon 5 'inverted terminal repeat (5' ITR) and a 3 'inverted terminal repeat (3' ITR), two insulators are arranged between the 5 'ITR and the 3' ITR, a promoter and a coding gene of a chimeric receptor are arranged between the two insulators, and the transposon can be a plasmid or a linearized nucleic acid segment; the transposase element can be encoded by a plasmid or a linearized nucleic acid segment, can be encoded by an mRNA, and can be a protein.

Preferably, the nucleic acid sequence of the transposase and the nucleic acid sequence of the transposon are on one vector or on two different vectors.

Preferably, the method for transferring a gene encoding a chimeric receptor into an NK cell using a transposon system comprises: electroporation, chemical transfection, and other non-viral transfection methods.

More preferably, the vector of interest of the transposon system is transferred into primary NK cells by electroporation.

Preferably, the NK cells may or may not be pre-activated before transferring the transgene into the NK cells using the transposon system as described in step (1).

Preferably, if NK cells are activated in advance, artificial antigen-presenting cells, feeder cells, cytokines, antibodies, compounds, or the like may be used for activation.

Preferably, if the NK cell is previously activated, the gene encoding the chimeric receptor may be transferred into the NK cell using the transposon system 0 to 14 days, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days after NK cell activation.

More preferably, the coding gene of the chimeric receptor is transferred into the NK cell by applying a transposon system 1-7 days after the NK cell is activated.

More preferably, the gene encoding the chimeric receptor is transferred into the NK cell using the transposon system 2 days after NK cell activation.

Preferably, if the NK cells are not activated, CD3+ cells can be removed by magnetic sorting before transferring the gene encoding the chimeric receptor into NK cells using the transposon system; or removing CD3+ cells by magnetic sorting 0-14 days after the encoding gene of the chimeric receptor is transferred into the NK cells by using the transposon system.

More preferably, CD3+ cells can be removed by magnetic sorting 1 to 8 days, for example, 1, 2, 3, 4, 5, 6, 7 or 8 days after the gene encoding the chimeric receptor is transferred into NK cells using the transposon system.

The artificial antigen presenting cell of step (2) comprises a cell-based, synthetic or exosome-based artificial antigen presenting cell.

The artificial antigen presenting cell based on cells is selected from human myeloid leukemia K562 cell, human Burkitt lymphoma Daudi cell, EBV transformed B lymphoblastoid cell (EBV-LCL) cell or mouse embryo fibroblast cell line NIH/3T3 cell.

Preferably, the cell-based artificial antigen presenting cells are selected from K562 cells.

Preferably, the cell-based artificial antigen presenting cell is an engineered cell that expresses an antigen recognized by a chimeric receptor.

Preferably, the engineered cells express ligand molecules, cytokines, etc., required for NK cell activation, wherein the cytokines may be secreted or membrane-fixed.

More preferably, the ligand molecules required for NK cell activation include CD137L and/or OX40L, and the cytokines required for NK activation include any one or a combination of at least two of human IL-12, human IL-15, human IL-18 or human IL-21.

Preferably, the cell-based artificial antigen-presenting cells and feeder cells are treated with gamma radiation (100Gy) or mitomycin C (20. mu.g/mL).

More preferably, both NK cell activation and CAR-NK cell expansion can be performed using artificial antigen presenting cells.

Preferably, the NK cell modified by the chimeric receptor is stimulated to proliferate 0-14 days after the coding gene of the chimeric receptor is transferred into the NK cell by using the transposon system in the step (1).

Preferably, the chimeric receptor-modified NK cells in the step (2) are amplified for multiple times, and each amplification cycle is 3-14 days.

Preferably, in the preparation of functional chimeric receptor-modified NK cells using the transposon system, cytokine compositions may be added: hrIL-12, hrIL-15, and hrIL-18.

Preferably, the cytokine composition may be added at any day during the preparation of the chimeric receptor-modified NK cells.

More preferably, the hrIL-12 final concentration is 1-100 ng/mL, rIL-15 final concentration is 1-100 ng/mL, rIL-18 final concentration is 1-100 ng/mL, such as 1ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL or 100 ng/mL.

In the present invention, the culture of the chimeric receptor-modified NK cell is carried out using a liquid medium, which may be a cell culture medium commonly used in the art, for example, AIM V^TM(Gibco)、X-VIVO^TM 15(Lonza)、SCGM^TM(CellGenix)、RPMI、NK MACS^TM(Miltenyi)、OpTmizer^TM(Gibco) or StemSpan^TM(STEMCELL)。

As a preferred technical scheme, the present invention provides a method for preparing a chimeric receptor-modified NK cell using a piggyBac transposon system, the method comprising:

(1) co-culturing isolated Peripheral Blood Mononuclear Cells (PBMCs) with gamma-ray treated engineered K562 cells;

(2) after 2 days, the helper plasmid encoding the piggybac (pb) transposon system and the donor plasmid encoding the chimeric acceptor were electroporated into NK cells using a Lonza 4D-Nucleofector device;

(3) after 5-9 days of electrotransfer, co-culturing the NK cells modified by the chimeric receptor and the engineered K562 cells treated by gamma rays, and adding the hrIL-12, the hrIL-15 and the hrIL-18 when the culture is started;

wherein the cell culture solution is AIM V culture solution added with 1-5% of human AB serum/human autologous plasma, and the hrIL-2 exists in the culture process.

Compared with the prior art, the invention has the following beneficial effects:

(1) at present, the modification of a primary NK cell for a chimeric receptor mainly depends on lentivirus or retrovirus mediated gene integration or mRNA electroporation mediated gene transient expression, because a virus vector preparation process is complex, potential safety hazards exist in the preparation and use processes, the quality of viruses is uneven, the mRNA transient expression and the preparation cost are high, and the clinical exploration of the chimeric receptor modified NK cell in solid tumor treatment is obviously hindered, the invention provides the preparation of the chimeric receptor modified NK primary cell by applying a transposon system and an artificial antigen presenting cell, wherein the combination of the transposon system and the artificial antigen presenting cell is necessary, and the invention also finds and provides that the time point of transferring transgenes into the NK cell by using the transposon system is very important for the amplification efficiency, the purity and the positive rate of the chimeric receptor, according to the invention, through theoretical and experimental researches, the sequence, time point, concentration of used reagents or co-culture proportion and the like of each step are optimized, and the positive rate of the chimeric receptor in NK cells, the amplification multiple of NK, the purity, the tumor killing capacity and the like are obviously improved;

(2) at present, in the process of preparing the chimeric receptor modified NK cell, a target gene is mainly delivered by depending on a lentiviral vector or a retroviral vector, but the production process of lentivirus or retrovirus is complex and high in cost, so that the production cost of the genetically modified NK cell is greatly increased, for example, Kymria (anti-CD19 CAR-T) of Nowa is priced to be $ 47.5 ten thousand, and the expensive price can not be borne by many tumor patients, so that the requirement on the production cost of the chimeric receptor modified immune cell is extremely urgent;

(3) the use of lentiviral vectors or retroviral vectors also brings certain potential safety hazards to operators, and moreover, when the lentiviral vectors or the retroviral vectors are used for preparing chimeric receptor modified NK cells, the potential risk of pollution of NK cell products by replication-competent retroviruses (RCR) or replication-competent lentiviruses (RCL) also exists;

(4) by applying the method, the integration efficiency of the coding gene of the chimeric receptor in the NK cell is between 40 and 80 percent, and the same level of the prior art that retrovirus is adopted to modify primary NK cells is achieved.

Drawings

FIG. 1A is a map of plasmid 3, and FIG. 1B is a map of plasmid 4;

FIG. 2A is the expression of membrane-fixed human interleukin 15 on the cell surface of K562-NK1, FIG. 2B is the expression of membrane-fixed interleukin 21 on the cell surface of K562-NK1, with the abscissa being the expression intensity of membrane-fixed human interleukin IL-15 or IL-21 and the ordinate being the cell count;

FIG. 3 is a flow diagram of CAR-NK preparation using piggyBac transposon systems;

FIG. 4A is the proportion of NK cells in the cell population at

days

10, 17, and 24 of culture for different CAR-NK preparation methods, and FIG. 4B is the proportion of CAR in NK cells at

days

10, 17, and 24 of culture for different CAR-NK preparation methods;

FIG. 5 is the fold expansion of total cell number at

days

10, 17, 24 of culture for different CAR-NK preparation methods;

FIG. 6A is the proportion of NK cells in the cell population at

days

10, 17, 24, 31 of culture for different CAR-NK preparation methods, and FIG. 6B is the proportion of CAR in NK cells at

days

10, 17, 24, 31 of culture for different CAR-NK preparation methods;

FIG. 7 is the fold expansion of total cell number at

days

10, 17, 24, 31 of culture for different CAR-NK preparation methods;

fig. 8A is a real-time killing map of NK, CAR-NK and cCAR-NK cells on day 17 against human ovarian cancer cell line SKOV3, with the abscissa being the time after tumor cell plating (i.e. after the start of the experiment) in hours and the ordinate being the cell index, and fig. 8B being the data analysis performed 24 hours after effector cells were added, and the tumor growth inhibition ratios were calculated, with the abscissa being different experimental groups and the ordinate being the tumor growth inhibition ratio;

FIG. 9A shows the expression of GFP in NK cells after 1 day of electroporation of pmax-GFP plasmid, using NK cells amplified on day 17 as NK cells, the abscissa shows the expression intensity of GFP, and the ordinate shows the cell count, FIG. 9B shows the numbers of NK cells before electroporation of pmax-GFP plasmid and after 1 day of electroporation, using NK cells amplified on day 17 as NK cells, and the abscissas show the numbers of cells before electroporation and after 1 day of electroporation, respectively.

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

Unless otherwise stated, cell cultures were maintained at 37 ℃ with 5% CO₂And (c) humidification (relative humidity of 95%).

The experimental materials used in the examples are as follows:

PBMCs are extracted from peripheral blood of solid tumor patients or healthy donors;

human ovarian cancer cell line SKOV3-luc, wild type K562 cells from ATCC;

the culture medium of SKOV3-luc is McCoy 5A (Gibco) + 10% FBS (Gibco), and the culture medium of K562 is IMDM (Gibco) + 10% FBS (Gibco);

the K562-NK1 cells used in the examples were genetically engineered K562 cells encoding membrane-fixed human IL-15(mbiL15), membrane-fixed human IL-21(mbiL21), eGFP and Puromycin that were gamma-treated (cells were irradiated with 100Gy of gamma radiation for half an hour). Since K562 cells naturally express NKG2D ligand, the NKG2D CAR-recognized antigen is not additionally loaded into K562-NK1 cells here. The membrane-immobilized human IL-15 is formed by fusing GM-CSF signal peptide (amino acids 1-22 of UniprotKB P15509), human IL-15 (amino acids 30-162 of UniprotKB P40933) and a hinge transmembrane region (amino acids 128-213 of UniprotKB P01732) of human CD 8; the membrane-immobilized human IL-21 is formed by fusing GM-CSF signal peptide (amino acids 1-22 of UniprotKB P15509), human IL-21 (amino acids 25-162 of UniprotKB Q9HBE 4), a hinge constant region (amino acids 99-327 of UniprotKB P01861) of human IGHG4 and a transmembrane region (amino acids 397-418 of UniprotKB P01730) of human CD4, wherein the amino acid sequence of eGFP is shown as SEQ ID No. 3, and the amino acid sequence of Puromycin is shown as SEQ ID No. 4;

SEQ ID NO:3：

MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTY GVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGN ILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQ SALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK；

SEQ ID NO:4：

MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERVTELQELFLTRVGLDIGKVWVA DDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQ GKGLGSAVVLPGVEAAERAGVPAFLETSAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA；

human recombinant IL-2(hrIL2) was purchased from Beijing Erlu, human recombinant IL-12(hrIL12), human recombinant IL-15(hrIL15), human recombinant IL-18 (hrIL18) from nearshore protein;

NK cell culture solution: AIM medium (Gibco) + 5% human AB serum (Gemini);

p3 Primary Cell 4D-Nucleofector X Kit was purchased from Lonza;

PE-conjugated anti-human CD3 antibody, APC-conjugated anti-human NKG2D antibody were purchased from BD, FITC-conjugated anti-Strep-tag II antibody was purchased from Kinshire, Biotin conjugated anti-human IL-15 antibody and APC conjugated Streptavidin were purchased from Biolegend, Biotin conjugated anti-human IL-21 antibody was purchased from eBioscience;

flow cytometry was purchased from BD, model C6 Sampler;

the real-time killing detector is purchased from ACEA Bio company and has the model of xCELLIGENCE RTCA DP;

the amino acid sequence of PiggyBac transposase is from GenBank: AAA 87375.2;

NKG2D CAR is: NKG2D-CD28-4-1BB-CD3 ζ, an antigen binding domain from amino acids 83 to 216 of NKG2D (UniProtKB-P26718), a hinge region from amino acids 99 to 110 of IgG4(UniProtKB-P01861), a transmembrane region from amino acids 153 to 179 of CD28 (UniProtKB-P10747), an intracellular domain from amino acids 209 to 255 of 4-1BB (UniProtKB-Q07011), and amino acids 52 to 164 of CD3 ζ (UniProtKB-P20963);

pmax-GFP was purchased from Lonza.

Example 1 vector construction

(1) Construction of vector for preparing engineered K562-NK1 cell

DNA fragment 1: the nucleotide sequence of the CMV promoter, the nucleotide sequence encoding puromycin and the nucleotide sequence encoding EGFP are synthesized by an outsourcing service company;

DNA fragment 2: the CMV promoter nucleotide sequence, the nucleotide sequence encoding bmIL-15, the nucleotide sequence encoding P2A, and the nucleotide sequence encoding mbiL-21 were synthesized by outsourced service companies;

the DNA fragment 1 and the DNA fragment 2 were cloned into pFastBac1 plasmid (purchased from Thermofeisher) and designated as plasmid 1.

(2) Construction of CAR vectors

To construct NKG2D CAR vectors, the extracellular antigen binding domain (ED) of NKG2D was fused to the IgG4 hinge region, CD28 transmembrane region, 4-1BB, and CD3 zeta signaling domain to generate a second generation NKG2D CAR vector; to facilitate detection of CAR expression by flow cytometry, 3 repeats of Strept-tag II (ST2) were added to each CAR sequence;

the CAR fragment was synthesized by an outsourcing service company, and the 5 'and 3' ends of the sequence included restriction sites EcoRI and SalI;

the synthesized DNA fragment was digested with EcoRI and SalI, and cloned into pFastBac1 plasmid (purchased from Thermofeisher) and designated plasmid 2.

(3) Construction of piggyBac transposon vector (Donor vector)

The 5 'inverted terminal repeat (SEQ ID NO:1) of piggyBac transposon, chicken beta-globin chromatin insulator cHS4 (GenBank: AY040835.1), EF1 alpha promoter, EcoRI and SalI restriction site sequences, SV40 polyA sequence, reverse complementary cHS4 sequence and 3' inverted terminal repeat (SEQ ID NO:2) were fused, synthesized by an outsourcing service company, cloned into pmaxCloning vector (from Lonza) by BsaI, named pZTS4, and the expression of the fusion gene was controlled by EF1 alpha promoter;

SEQ ID NO:1：

5’-ccctagaaagataatcatattgtgacgtacgttaaagataatcatgtgtaaaattgacgcatg-3’；

SEQ ID NO:2：

5’-catgcgtcaattttacgcagactatctttctaggg-3’；

to construct a transposon donor vector containing the NKG2D CAR gene, the CAR sequence in plasmid 2 was cut with EcoRI and SalI and cloned into pZTS4, designated plasmid 3, as shown in fig. 1A, and the expression of the CAR gene was controlled by the EF 1A promoter.

(4) Construction of piggyBac transposase vector (auxiliary vector)

In order to construct a piggyBac transposase encoding plasmid, AgeI and SacI enzyme cutting sites are respectively added at two ends of a piggyBac transposase gene (GenBank: EF587698.1), sequences are synthesized by an outsourcing service company, and are cloned into a pmaxCloning vector through the two enzyme cutting sites, namely a plasmid 4, as shown in FIG. 1B, and a CMV promoter in the pmaxCloning vector controls the expression of the piggyBac transposase.

EXAMPLE 2 preparation of engineered K562-NK1 cells

Resuspending wild type K562 cells in 10mL Opti-MEM, centrifuging at 300 Xg for 10min, resuspending the cell pellet in 100. mu. L P3 buffer, adding 10. mu.g plasmid 1, mixing well, and transferring to a Lonza electric shock cup;

placing the electric shock cup in a Lonza 4D-nucleofector X Unit (in a single electric shock cup module), carrying out electric conversion, slowly transferring K562 cell suspension in a cuvette to one hole of a 6-hole plate after the electric conversion is finished, and adding a K562 culture medium (IMDM + 10% FBS) into the hole in advance;

on day 5 after electroporation, puromycin screening was performed at a concentration of 200 μ g/mL for 1 month with medium changed every 2 days;

after 1 month, single cells were sorted into a 96-well plate using a cell sorter BD FACSAria (BD Biosciences), and the amplified single cell clones were analyzed by flow cytometry to detect the expression of the fusion gene, and the detection antibodies were APC-bound Streptavidin, biotin-bound anti-human IL-15 antibody, and biotin-bound anti-human IL-21 antibody, respectively, and the screened single cell clones were named as K562-NK 1.

As shown in FIG. 2A and FIG. 2B, K562-NK1 cell surface expressed high levels of human IL-15 and human-IL 21, with positive rates of 100% and 79%, respectively.

Example 3 preparation of CAR-NK Using piggyBac transposon System

The preparation flow chart is shown in figure 3:

on day 0, 5X 10⁶PBMC and 5X 10⁶K562-NK1(100Gy gamma ray irradiation) is resuspended in 10mL NK cell culture medium, inoculated in a T25 cell culture flask, and hrIL-2 is added to make the final concentration 100 IU/mL;

on day 2, the suspended cells were counted and centrifuged at 300 Xg for 10 min; resuspend the cell pellet with 10mL Opti-MEM, centrifuge at 300 Xg for 10 min; resuspending the cell pellet in 100. mu. L P3 buffer, adding 5. mu.g plasmid 4 and 10. mu.g plasmid 3, mixing well, and transferring to electric Lonza electric shock cup; placing an electric shock cup in a Lonza 4D-nucleofector X Unit (in a single electric shock cup module), carrying out electric conversion, slowly transferring NK cell suspension in a cuvette into a new T25 cell culture bottle after the electric conversion is finished, adding 10mL of NK cell culture solution containing hrIL-2 with the final concentration of 100IU/mL, gently mixing uniformly, and placing a T25 cell culture bottle in a 37 ℃ cell culture box for culture;

adding a proper amount of fresh NK cell culture solution containing hrIL-2 according to the growth condition of the cells from the 3 rd to the 10 th days;

on day 10, all cells were collected and counted, 2X 10⁶Flow cytophenotyping of individual cells (anti-human CD3 antibody, anti-human CD56 antibody, anti-Strep-tag II antibody); 2 x 10 to⁶Each cell and 2X 10⁶The K562-NK1 cells (100Gy gamma ray irradiation) were resuspended in 10mL of medium and hrIL-2 was added to a final concentration of 100 IU/mL;

adding a proper amount of fresh NK cell culture solution containing hrIL-2 according to the growth condition of the cells from 10 days to 17 days;

on day 17, all cells were collected and counted, 2X 10⁶Flow cytophenotyping of individual cells (anti-human CD3 antibody, anti-human CD56 antibody, anti-Strep-tag II antibody); 2 x 10 to⁶Each cell and 2X 10⁶The K562-NK1 cells (100Gy gamma ray irradiation) were resuspended in 10mL of medium and hrIL-2 was added to a final concentration of 100 IU/mL;

adding a proper amount of fresh NK cell culture solution containing hrIL-2 according to the growth condition of the cells from the 17 th to the 24 th days;

on day 24, all cells were collected and counted, 2X 10⁶Flow cytophenotypic analysis of individual cells (anti-human CD3 antibody, anti-human CD56 antibody, anti-human CD3 antibody, anti-human CD56 antibody, anti-human CD-binding antibody, anti-human-binding antibody, anti-human-binding antibody, anti-human-CD-binding antibody, anti-CD-human-CD-beta-glucosidase, anti-beta-gamma-beta-gamma-beta-gamma,anti-Strep-tag II antibodies); 2 x 10 to⁶Each cell and 2X 10⁶The K562-NK1 cells (100Gy gamma ray irradiation) were resuspended in 10mL of medium and hrIL-2 was added to a final concentration of 100 IU/mL;

adding a proper amount of fresh NK cell culture solution containing hrIL-2 according to the growth condition of the cells from the 24 th day to the 31 th day;

on day 31, all cells were collected and counted, 2X 10⁶Flow cytophenotypic analysis was performed on individual cells (anti-human CD3 antibody, anti-human CD56 antibody, anti-Strep-tag II antibody).

Example 4 preparation of cCAR-NK Using piggyBac transposon System

On day 0, 5X 10⁶PBMC and 5X 10⁶K562-NK1(100Gy gamma ray irradiation) was resuspended in 10mL of NK cell medium, inoculated into a T25 cell culture flask, and hrIL-2 was added to give a final concentration of 100 IU/mL;

on day 10, all cells were collected and counted, 2X 10⁶Flow cytophenotyping of individual cells (anti-human CD3 antibody, anti-human CD56 antibody, anti-Strep-tag II antibody); 2 x 10 to⁶Each cell and 2X 10⁶Resuspending K562-NK1(100Gy gamma ray irradiation) in 10mL of culture medium, adding hrIL-2, hrIL-15, and hrIL-18 to make their final concentrations 10ng/mL, 50ng/mL, and 50ng/mL, respectively;

on day 11, all cells were collected, collected at 300 Xg, centrifuged for 10min, resuspended in 20mL of fresh NK cell medium, and hrIL-2 was added to a final concentration of 100 IU/mL;

adding a proper amount of fresh NK cell culture solution containing hrIL-2 according to the growth condition of the cells from day 12 to day 17;

on day 17, all cells were collected and counted, 2X 10⁶Flow cytophenotyping of individual cells (anti-human CD3 antibody, anti-human CD56 antibody, anti-Strep-tag II antibody); 2 x 10 to⁶Each cell and 2X 10⁶Resuspending K562-NK1(100Gy gamma ray irradiation) in 10mL of culture medium, and adding hriL-2 to make the final concentration 100 IU/mL;

on day 24, all cells were collected and counted, 2X 10⁶Flow cytophenotyping of individual cells (anti-human CD3 antibody, anti-human CD56 antibody, anti-Strep-tag II antibody); 2 x 10 to⁶Each cell and 2X 10⁶Resuspending K562-NK1(100Gy gamma ray irradiation) in 10mL of culture medium, and adding hriL-2 to make the final concentration 100 IU/mL;

Example 5 Effect of different electrotransfer conditions on the expansion fold and cell phenotype of CAR-NK cells

This example compares the proportion of NK cells in CAR-NK, the proportion of CAR expressed in NK cells and the fold expansion of total cells obtained by electroporation before NK cell activation or by electroporation at day 2 or 4 after NK cell activation.

The method of example 3 was performed by electroporation on day 2 after NK cell activation; the method of electroporation prior to NK cell activation differs from example 3 in that 5X 10 cells were used⁶PBMC were electroporated according to the method of example 3After completing the electric conversion, the electric conversion is immediately combined with 5X 10⁶Co-culturing K562-NK1(100Gy gamma ray irradiation); the difference between the electroporation carried out on the 4 th day after NK cell activation and example 3 is that 5X 10 cells were used⁶PBMC and 5X 10⁶After 4 days of K562-NK1(100Gy gamma irradiation), the suspension cells were again removed and subjected to electroporation.

When CAR-NK cells were prepared from 1 normal donor sample by the above three methods and the obtained NK cell purities (CD3-CD56+) were compared, as shown in fig. 4A, the proportion of NK cells in CAR-NK cells prepared in example 3 was 68%, 77%, and 85% on

days

10, 17, and 24, respectively, while the proportion of NK cells in CAR-NK cells prepared by electroporation before NK cell activation was 11% and 13% on

days

10 and 17, respectively, and the proportion of NK cells in CAR-NK cells prepared by electroporation 4 days after NK cell activation was 46%, 81%, and 88% on

days

10, 17, and 24, respectively. Shows that the proportion of NK cells is higher in CAR-NK cells prepared by electrotransformation after NK cell activation than that before NK activation.

Further, comparing the proportion of CAR in the obtained NK cells, the results are shown in fig. 4B, and the proportion of CAR in NK cells in CAR-NK cells prepared in example 3 was 14%, 30%, 45% at

days

10, 17, and 24 of culture, respectively, while the proportion of CAR in NK cells in CAR-NK cells prepared by electroporation before NK cell activation was 14% and 27% at

days

10 and 17 of culture, respectively, and the proportion of CAR in NK cells prepared by electroporation 4 days after NK cell activation was 2%, 15%, and 19% at

days

10, 17, and 24 of culture, respectively. Indicating that the earlier the electrotransfer was performed, the higher the proportion of CAR in NK cells.

The results of comparing the total cell number amplification factors obtained in this example are shown in FIG. 5, and the total cell number amplification factor obtained in 24 days of amplification by the method of example 3 is 3022 times; performing electrotransformation and total amplification for 17 days before NK cell activation, wherein the total cell amplification multiple is 133 times; after 4 days of NK cell activation, the total cell number expansion multiple is 736 times after the cells are electrically transferred and expanded for 24 days. Indicating that the cells obtained by the electroporation performed on day 2 after NK cell activation had the highest expansion fold.

Combining these three parameters, the CAR-NK cells prepared by the method of example 3, i.e. performing electroporation 2 days after NK cell activation, had the relatively best NK cell ratio, CAR ratio in NK cells and expansion factor of total cell number.

Example 6 Effect of different cytokine compositions on the expansion fold and cell phenotype of CAR-NK cells

This example compares the effect of whether the addition of cytokine compositions (IL-12, IL-15, IL-18) on the growth of CAR-NK cells on CAR-NK production day 10, when co-cultured with K562-NK1 for the second time.

CAR-NK panel, i.e. CAR-NK preparation on day 10 in second coculture with K562-NK1, only hrIL-2 was added (example 3);

the cCAR-NK panel, CAR-NK preparation day 10, was co-cultured with K562-NK1 for a second time with cytokine combinations IL-12, IL-15, IL-18 (example 4).

CAR-NK cells were prepared from 1 normal donor sample by the above two methods, and the obtained NK cell purities (CD3-CD56+) were compared, and as a result, as shown in fig. 6A, the proportion of NK cells in CAR-NK cells prepared in example 3 was 79%, 81%, 90%, 93% at

days

10, 17, 24, 31 of culture, respectively, while the proportion of NK cells in CAR-NK cells prepared in example 4 was 79%, 87%, 93% at

days

10, 17, 24, 31 of culture, respectively. It is demonstrated that the addition of cytokine combinations IL-12, IL-15, IL-18 slightly increased the proportion of NK cells when co-cultured with K562-NK1 for the second time on the 10 th day of CAR-NK preparation.

Further, comparing the proportion of CAR in the obtained NK cells, the results are shown in fig. 6B, and the proportion of CAR in NK cells in CAR-NK cells prepared in example 3 was 34%, 47%, 49%, 74% at

days

10, 17, 24, 31 of culture, respectively, while the proportion of CAR in NK cells in cCAR-NK cells prepared in example 4 was 34%, 57%, 58%, 85% at

days

10, 17, 24, 31 of culture, respectively. The results show that when the CAR-NK is prepared on the 10 th day and is co-cultured with K562-NK1 for the second time, the proportion of CAR in NK cells can be remarkably improved by adding cytokine compositions IL-12, IL-15 and IL-18.

This example also compares the total expansion fold of the cells obtained, and the results are shown in FIG. 7, and the CAR-NK cells prepared in example 3 had total expansion fold of 27, 21, 9, 8 fold at days 0-10, 10-17, 17-24 and 24-31, respectively; and the cCAR-NK cells prepared in example 4 had total expansion fold of 27, 9, 7 fold on days 0-10, 10-17, 17-24 and 24-31, respectively. It is demonstrated that the addition of cytokine compositions IL-12, IL-15, IL-18 slightly increases the expansion fold of NK cells when co-cultured with K562-NK1 for the second time on the 10 th day of CAR-NK preparation.

Combining these three parameter comparisons, the addition of cytokine compositions IL-12, IL-15, IL-18, facilitated CAR + CAR-NK cell expansion by the method of example 4, i.e. at CAR-NK production day 10 when co-cultured with K562-NK1 for the second time.

Example 7CAR-NK preparation day 10 Effect of cytokine supplementation on the growth of human ovarian cancer cell line SKOV3 (RTCA)

This example compares the effect of the addition of cytokine compositions (IL-12, IL-15, IL-18) on CAR-NK killing capacity in vitro on day 10 of NKG2D CAR-NK preparation, when co-cultured with K562-NK1 for the second time.

RTCA experiment started at 0 hour, SKOV3 was plated in 16-well electrode plates (ACEA Bio) at 5000 cells/well; after about 24 hours, NKG2D CAR-NK cells, NKG2D cCAR-NK cells and NK cells (effector cells) on day 17 were seeded in electrode plates at a ratio of effector to target cells of 1:1 or 2:1, respectively, in a total volume of 200 μ L, with each set of experiments repeated 2 times; wherein the NK cells were prepared by the method of example 2, but without electroporation; the effect of NKG2D CAR-NK cells on the growth of SKOV3 was examined with xCELLigence RTCA.

As shown in fig. 8A and 8B, tumor cells continued to grow for up to 70 hours without the addition of the effector cell group (shown as "SKOV 3"); when the effector cell is tumor cell 1:1, the growth of the tumor cell can be obviously inhibited after adding NK cell (shown as SKOV3: NK 1:1) or CAR-NK cell (shown as SKOV3: CAR-NK 1:1) which has obviously better tumor inhibition effect than the NK cell, thus indicating the effect of CAR in NKG2D CAR-NK cell; whereas, upon addition of cCAR-NK cells (shown as "SKOV 3: cCAR-NK 1: 1") (E: T1: 1) or CAR-NK cells (shown as "SKOV 3: CAR-NK 1: 2") (E: T2: 1), tumor cells were almost completely killed.

Analysis of data at 24h after addition of effector cells (shown in FIG. 8A by vertical dashed lines) tumor growth inhibition was calculated according to the following formula:

100% × (SKOV3 cell index-Experimental group cell index)/SKOV3 cell index

It can be found that the tumor inhibition rate of the added cCAR-NK cell group (shown as 'SKOV 3: cCAR-NK 1: 1') and the added CAR-NK cell group (shown as 'SKOV 3: CAR-NK 1: 2') is about 80%, which indicates that the growth inhibition effect of CAR-NK cells on tumor cells can be remarkably improved by adding cytokine compositions (IL-12, IL-15 and IL-18) when NKG2D CAR-NK is prepared for 10 days and is co-cultured with K562-NK1 for the second time, and the reasons can be that the CAR-NK cell proportion is improved and the self-killing effect of NK cells is enhanced by the cytokine compositions (IL-12, IL-15 and IL-18).

Example 8 direct electroporation of NK cells, expression efficiency of transgene and NK cell survival

In this example, a green fluorescent protein GFP gene was used as a reporter gene, and the NK cells amplified for 17 days were directly electroporated with pmax-GFP plasmid, and the expression ratio of GFP in NK cells and the survival rate of NK cells were measured 1 day after electroporation. The NK cell expansion protocol was as follows: on day 0, 2X 10⁶PBMC and 2X 10⁶K562-NK1(100Gy gamma ray irradiation) was resuspended in 10mL of NK cell culture medium, inoculated into a T75 cell culture flask (vertical culture), and hrIL-2 was added to give a final concentration of 100 IU/mL; adding a proper amount of fresh NK cell culture solution containing hrIL-2 according to the growth condition of the cells from the 2 nd to the 10 th days; on day 10, all cells were collected and counted, 2X 10⁶Flow cytophenotyping of individual cells (anti-human CD3 antibody, anti-human CD56 antibody); 2 x 10 to⁶Each cell and 2X 10⁶Resuspending K562-NK1(100Gy gamma irradiation) in 10mL of culture medium, and adding hriL-2 to make the final concentration 100 IU/mL; day 10 to day 10Adding appropriate amount of fresh NK cell culture solution containing hrIL-2 according to cell growth condition for 17 days; on day 17, all cells were collected and counted, 2X 10⁶Flow cytophenotypic analysis was performed on individual cells (anti-human CD3 antibody, anti-human CD56 antibody). The NK cells on day 17 of amplification were collected and electroporated by the method of example 3, and the number of electroporated NK cells was 2X 10⁶And the plasmid was replaced with the pmax-GFP plasmid. The following day after the electrotransfer, the proportion of GFP in the NK cells was examined by cell counting and flow cytometry. As shown in FIGS. 9A and 9B, the survival rate of NK cells was only 3.2% although the expression ratio of GFP was 17.99% by day after the electrotransfer.

The results show that if only the NK cells are subjected to electric transformation and artificial antigen presenting cells are not used for enrichment and amplification, the activity rate of the NK cells and the expression rate of transgenes are low. Therefore, the combination of the transposon system and the artificial antigen presenting cell is necessary, the survival and the expansion multiple of the NK cell can be obviously improved, and the proportion of the NK cell modified by the chimeric receptor can be obviously improved.

In conclusion, the invention adopts a non-viral method, utilizes the transposon system to introduce stable and high-expression transgenes into the NK cells, realizes high-efficiency genetic modification of the NK cells, has short production period and low production cost, and the prepared transgenic modified NK cells have high purity and good safety and have obvious killing and/or inhibiting effects on tumor cells.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

SEQUENCE LISTING

<110> Hangzhou Youkayi pharmaceutical science and technology Co., Ltd

<120> non-viral method for preparing NK cells stably and highly expressing chimeric receptor

<130> 20200707

<160> 4

<170> PatentIn version 3.3

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Claims

1. a method utilizing non-viral transposon system and artificial antigen presenting cell to prepare the natural killer cell of chimeric receptor modification, it is characterised in that the method comprises:

(1) Using the transposon system to transfer the encoding gene of the chimeric receptor into natural killer cells to obtain the original natural killer cells modified by the chimeric receptor;

(2) Using artificial antigen-presenting cells to expand the original chimeric receptor-modified natural killer cells.

2. The method of claim 1, wherein the natural killer cells comprise human primary natural killer cells;

The human primary natural killer cells are derived from peripheral blood, umbilical cord blood or induced pluripotent stem cells.

3. The method of claim 1, wherein the chimeric receptor comprises a chimeric antigen receptor and/or a chimeric switch receptor.

4 . The method according to claim 1 , wherein the transposon system of step (1) comprises piggyBac transposon system, Sleeping Beauty transposon system or Tol2 transposon system. 5 .

5. The method according to claim 1, wherein the transposon system in step (1) comprises a transposon element and a transposase element.

6. The method of claim 5, wherein the transposon element comprises a transposon 5' inverted terminal repeat and a 3' inverted terminal repeat;

There are two insulators between the 5' inverted terminal repeat and the 3' inverted terminal repeat;

A gene encoding a promoter and a chimeric receptor is contained between the two insulators;

The transposon element is a plasmid or a linearized nucleic acid fragment.

7. The method of claim 5, wherein the transposase element comprises a transposase protein or a nucleic acid molecule encoding a transposase;

The nucleic acid molecules encoding transposases include plasmids, linearized nucleic acid fragments, or mRNA.

8. The method of claim 5, wherein the transposon element and the transposase element are linked on the same expression vector.

9. The method of claim 5, wherein the transposon element and the transposase element are on different expression vectors.

10 . The method according to claim 1 , wherein the transposon system in step (1) is transferred into natural killer cells by electroporation and/or chemical reagents. 11 .

11. The method of claim 1, further comprising a step of activating natural killer cells before step (1).

12. The method of claim 11, wherein the activation of natural killer cells is performed using artificial antigen presenting cells, feeder cells, cytokines, antibodies or compounds.

13 . The method according to claim 11 , wherein the transposon system is transferred into natural killer cells after 0-14 days of activation of the natural killer cells. 14 .

14. The method according to claim 1, characterized in that, before step (1), it further comprises the step of removing CD3+ cells by magnetic sorting.

15. The method according to claim 1, characterized in that, after step (1), it further comprises the step of removing CD3+ cells by magnetic sorting after culturing the initial chimeric receptor-modified natural killer cells for 0-14 days .

16. The method according to claim 1 or 12, wherein the artificial antigen-presenting cells comprise cell-based artificial antigen-presenting cells, artificially synthesized artificial antigen-presenting cells or exosome-based artificial antigens presenting cells.

17. The method of claim 16, wherein the cell-based artificial antigen presenting cells comprise human myeloid leukemia K562 cells, human Burkitt lymphoma Daudi cells, and EBV transformed B lymphoblastoid cells Like cells or mouse embryonic fibroblast cell line NIH/3T3 cells.

18. The method of claim 16, wherein the cell-based artificial antigen presenting cells are engineered cells;

the engineered cells express an antigen recognized by the chimeric receptor;

The engineered cells express ligand molecules and/or cytokines for activating natural killer cells;

The ligand molecule includes CD137L and/or OX40L;

The cytokine includes any one or a combination of at least two of human IL-21, human IL-15 or human IL-18;

The cytokines include secreted cytokines and/or membrane-fixed cytokines;

The engineered cells were treated with gamma rays or mitomycin C.

19 . The method according to claim 1 , wherein the initial chimeric receptor-modified natural killer cells are expanded 0 to 14 days after the gene encoding the chimeric receptor is transferred into the natural killer cells. 20 .

20 . The method according to claim 1 , wherein the amplification is performed multiple times, and each round of amplification cycle is 3-14 days. 21 .

21. method according to claim 1 is characterized in that, in the preparation process of the natural killer cell of described chimeric receptor modification, is added with cytokine composition hrIL-12, hrIL-15 and hrIL- 18.

22. Chimeric receptor-modified natural killer cells, characterized by being prepared by the method of any one of claims 1-21.

23. Use of the chimeric receptor-modified natural killer cell of claim 22 in the preparation of a tumor therapeutic drug and/or a tumor preventive drug.