WO2019182422A1 - Novel genetically modified natural killer cell line and use thereof - Google Patents

Abstract

The present invention relates to a novel genetically modified NK cell line and a use thereof and, more specifically, provides: a genetically modified NK cell line into which polynucleotides encoding CD7, CD28, cell membrane-bound IL-15 and a TGFβ receptor, respectively, and an apoptotic gene are transduced into an isolated NK cell line; and a use thereof, such as in a pharmaceutical composition for treating cancer, containing the cell line.

Description

New genetically modified natural killer cell lines and uses thereof

This application claims priority to Korean Patent Application No. 2018-0034079, filed March 23, 2018. The matters described in this patent application are incorporated herein by reference.

The present invention relates to novel cell lines and their uses, and more particularly to novel genetically modified natural killer cell lines and their use.

Natural killer cells (hereinafter referred to as 'NK cells') have the ability to kill abnormal autologous cells, or cancer cells, present in the body. The difference between NK cells and cytotoxic T cells (hereinafter CTL) is the presence or absence of antigen specificity. Cytotoxic T cells (hereinafter CTLs) that specifically recognize MHC and antigen peptides of target cells using antigen specific antigen receptors (hereinafter CTL) have a responsiveness to a single target, whereas for NK cells, NK activation on the surface of cancer cells Relatively broad range because it can kill cells by recognizing various abnormal changes that occur in cells, such as the expression of Killer cell activation receptors (KAR) and KIR (Killer cell inhibitory receptors) and the absence of surface MHC I antigens Of cancer cell death (Min Cheng, 20131, Cellular & Molecular immunology). Since NK cells remove leukemia cells or cancer cells without causing graft-versus-host disease, they have been regarded as suitable cells for adoptive cell therapy in that their side effects are very low (Glienke et. al ., Front. Pharmacol ., 6: 21, 2015). In addition, NK cells, unlike T cells, do not have immune memory, and thus are killed by themselves after being activated by cancer cells. Therefore, anticancer therapy using NK cells has very little possibility of side effects compared to the therapy based on T cells that have formed immune memory in the body for a long time.

However, in the case of immune cell treatment using NK cells, although therapeutic agents are established by isolating, activating, and amplifying primary NK cells of patient-derived cells or allogeneic cells, the cellular resources can be obtained. Over-amplification is limited, the process is complicated by using a feeder cell and a production process using various cytokines, and the limitation due to the possibility of batch-to-batch variation of the therapeutic agent There is. In addition, the cells can be proliferated for a limited time in vitro, thus enhancing the effect through gene insertion / modification may be somewhat limited. In order to overcome these limitations, the development of a cell therapy based on a single NK cell line may be an effective method. Through this, it is possible to overcome the limitation of cellular resources, the simplicity of the process, the possibility of mass production, and the homogeneity of the therapeutic agent. have. In addition, several factors may be necessary for NK cells to be used as an effective therapeutic agent, which enhances cell killing ability of NK cell lines, secures additional safety when administering patients, facilitates the cultivation process during production, and the immunosuppressive environment in patients. It can be prevented by the loss of function, etc., cell line-based therapeutic agent is very easy to construct a single cell-derived cell therapy with an additional therapeutic gene compared to the primary cultured cells.

However, even in established NK cell lines, since expression of a factor necessary for showing optimal cell killing ability is often lacking, it is difficult to use the established NK cells as an effective substance for anticancer drugs.

NK cells require NK cell activation receptors (KARs), cell adhesion molecules, NK costimulatory molecules, etc. in order to exhibit assassination effects. In addition, in the case of cell line-based therapeutics, the origin of the cell line may be cancerous or immortalized, and thus, a problem such as tumor formation may occur when introduced into a patient, and an immune cell line having high immune activity may cause unexpected hyperactivity in the patient. This may need to be prepared.

In addition, in the production of cell lines, the use of minimal supplemental cells and cytokine supplements can simplify the process and increase production efficiency.

Lastly, in the case of immune cells, various immunosuppressive environments (regulatory T cells, myeloid derived suppressor cells, immunosuppressive cytokines) exist in the patient's body. Active function may be inhibited. Therefore, there is a need to establish a genetically modified NK cell line that can maximize the anticancer cell killing ability of the isolated and constructed NK cell line, improve its proliferation, and minimize side effects.

Such genetically modified NK cell lines include NK-92 cells transformed to express the Fc receptor (CD16) (US20060292156A1).

However, the genetically modified NK cell line (NK-92-CD16) of the prior document only enhanced the cancer cell killing ability (ADCC) of NK cells by the antibody, did not increase its own cancer cell killing ability, and the cell proliferation and side effects of It has nothing to do with minimization.

Accordingly, an object of the present invention is to provide a genetically modified NK cell line that can be easily proliferated and minimize side effects due to immune and activation while further improving cell killing ability based on NK cells.

Still another object of the present invention is to provide a pharmaceutical composition for treating cancer comprising the genetically modified NK cell line as an active ingredient.

According to one aspect of the present invention, there is provided a genetically modified NK cell line in which a polynucleotide encoding an NK cell coactivator is transduced into an isolated NK cell line having the following characteristics such that the NK cell coactivator is expressed:

CD2, CD11a, CD25, CD45, CD54, DNAM-1, CD62L and CD56 are positive; And

CD1a, CD3, CD4, CD8, CD14, CD20, CD23, CD34, TCRαβ and TCRγδ are negative.

According to another aspect of the invention, there is provided a pharmaceutical composition for the treatment and prevention of cancer comprising the genetically modified NK cell line as an active ingredient.

According to one aspect of the present invention, a transgenic NK cell line transduced with a polynucleotide encoding a NK cell coactivator, an NK cell proliferation factor, a cytoplasmic domain-deleted TGFβ receptor, and / or an apoptosis gene in an isolated NK cell line Is provided.

According to another aspect of the invention, there is provided a pharmaceutical composition for the treatment and prevention of cancer comprising the genetically modified NK cell line as an active ingredient.

In addition, according to another aspect of the present invention, there is provided a kit for treating cancer comprising the genetically modified NK cell line and suicide inducing agent.

According to another aspect of the invention, the treatment of a subject with cancer comprising the step of administering a therapeutically effective amount of any one of the above genetically modified NK cell lines and optionally suicide inducing agent to the subject with cancer A method is provided.

According to another aspect of the invention, the isolated NK cell line is transduced with polynucleotides and apoptosis genes encoding the NK cell coactivator, cell membrane-bound IL-15, and TGFβ receptor, respectively, cancer antigen specific chimeric antigen A polynucleotide encoding a receptor is transduced to provide a genetically modified NK cell line expressing the cancer antigen specific chimeric antigen receptor.

Genetically modified NK cell line according to an embodiment of the present invention is superior to the conventionally reported NK cell line, the economic efficiency is very high, the cancer cell killing ability is significantly improved compared to the parent cell line, cancer antigen specific using chimeric antigen receptor It has been proved experimentally that it can be used as a platform for chemotherapy, and thus it can be used very efficiently as a cell therapy for cancer treatment.

Figure 1a is a graph showing the degree of cell proliferation according to the passage of the NK101 cell line of the present invention, Figure 1b is a dot graph confirming that NK101 cells are CD3, CD20, and CD16 negative and CD56 positive NK cells. Figure 1c is a photograph taken by using a microscope of the morphology of the NK101 cells when cultured, Figure 1d is a photograph of the shape of the NK101 cells using Wright-Giemsa staining method, Figure 1e is the NK101 cells It is confirmed by the fluorescent staining technique that expresses the major cell death factors of NK cells, Perforin (green) and Granzyme (red), Figure 1f is a cancer cell of NK101 through co-culture of NK101 and K562, MHC class I negative cells This graph shows the result of confirming the killing ability.

Figure 2a is a graph of comparative analysis of the IL-2 concentration-dependent cell growth rate and sensitivity of NK101 and NK-92 using MTS analysis, Figure 2b is a flow cytometric analysis of the difference in the expression of IL-2 receptor subunits of NK101 and NK-92 Figure 2c is a graph showing the results of comparing the cell proliferation and survival rate from the thawing time of NK101 and NK-92 in the same culture conditions, Figure 2d is a comparison of the growth and doubling time after culture adaptation It is a graph.

Figure 3a is a histogram showing the results of flow cytometry analysis of the main lineage or progenitor marker expression in NK101 cells, Figure 3b shows the flow cytometry results for the expression of activated and inactivated receptors in NK101 cells Histogram, FIG. 3C is a histogram showing the results of flow cytometry analysis of cell adhesion factor expression in NK101 cells, and FIG. 3D is a CD107a, perforin, granzyme, FasL and TRAIL involved in NK cell dependent cytotoxicity in NK101 cells It is a histogram showing the flow cytometry results for expression, Figure 3e is a histogram showing the flow cytometry results for cytokine receptor expression in NK101 cells, Figure 3f is for the expression of various CC chemokine receptors and CXC chemokine receptors in NK101 cells Histogram showing flow cytometry results.

Figure 4a is a histogram showing the results of flow cytometry confirming the CD56 expression of NK101, NK-92 and peripheral culture CD56 ⁺ peripheral blood NK cells, Figure 4b is a two-dimensional contour line showing the flow cytometry results confirming the CD56 and CD62L expression of the cells It is a graph.

5A is a graph confirming the proliferation rate of NK101 cells after treatment with each indicated cytokine in a culture solution for 3 days (left) and the production of NK activated cytokine IFN-γ by ELISA (right), FIG. 5b is a graph showing the results obtained by multiplex analysis of cytokines secreted after NK101 cells were co-cultured with K562 or THP-1 cancer cell lines for 24 hours.

Figure 6a is a graph showing the results of confirming the killing of cancer cells by co-culture of various cancer cell lines and NK101 cells at various cell ratios for 24 hours, Figure 6b is acute myeloid treatment of the neutralizing antibodies to the surface antibodies respectively marked on NK101 cells The cell death was measured after 24 hours co-culture with leukemia cell line THP-1 and 4: 1 ratio, Figure 6c is treated with neutralizing antibodies to the surface antibody labeled on NK101 cells and chronic myeloid leukemia cell line K562 and 4 A graph showing the result of measuring apoptosis after co-culture at 1: 1 ratio for 24 hours, and FIG. 6D shows a neutral lymphocytic leukemia cell line Jurkat and 4: 1 ratio treated with neutralizing antibodies to surface antibodies labeled on NK101 cells, respectively. After 24 hours co-culture with a graph showing the results of measuring apoptosis, Figure 6e is treated with DNAM-1, CD54 or DNAM-1 and CD54 neutralizing antibodies to NK101, respectively, It is a graph showing the result of confirming the synergistic inhibition by the simultaneous neutralization of markers.

Figure 7a is a histogram showing the results of confirming the expression patterns of CD7, CD28 costimulatory factors in NK101 and the established cell lines KHYG-1, and NK-92 through flow cytometry, Figure 7b is a cancer cell killing effect of NK101 cells Figure 7c is a schematic diagram of the gene construct introduced for enhancement, Figure 7b is a flow cytometric analysis of CD7, CD28 expression in the NK101 cells (named SL-K01) introduced with the gene construct shown in FIG. It is a histogram showing a result, and FIG. 7D is a photograph showing the result confirmed by reverse transcription PCR of CD :: UPRT expression in SL-K01 cells.

Figure 8a is a graph showing the results of measuring cancer cell death frequency through flow cytometry after co-culture of HDLM-2, IM-9, JEKO-1 and K562 cancer cells with NK101 or SL-K01 cells at 4: 1 ratio for 24 hours 8B is a graph showing the results obtained after 48 hours of treatment with various concentrations of 5-FC in NK101 or SL-K01 cells through MTS analysis, and FIG. 8C is an IM-9 cell line and NK101 or SL. It is a graph showing the result of measuring the frequency of IM-9 cancer cell death by flow cytometry when 5-K01 cells are co-cultured in a 2: 1, 1: 1 or 0.5: 1 ratio with or without 5-FC.

Figure 9a is a schematic diagram of the gene constructs introduced for the enhancement of additional cancer cell killing effect of SL-K01 cells and the induction of resistance to the immunosuppressive factor TGF-β, Figure 9b is a gene shown in SL-K01 and Figure 9a A histogram showing the results of confirming IL-15 expression on the surface of the construct-induced SL-K01 cells (named NK111) through flow cytometry, and FIG. 9c shows the flow cytometry of TGFβRIIΔcyto expression in SL-K01 and NK111 cells. This is a histogram showing the result of checking through.

FIG. 10A is a graph showing the population doubling level according to the presence or absence of IL-2 in the culture of SL-K01 and NK111 cells, and FIG. 10B is a flow chart of NKG2D expression in NK101, SL-K01 and NK111 cells. The histogram showing the results confirmed through the analysis, FIG. 10C shows the IM according to the presence or absence of 5-FC when co-culture of the IM-9 cell line and SL-K01 or NK111 cells in a 2: 1, 1: 1, or 0.5: 1 ratio. -9 is a graph showing the result of measuring the death frequency of cancer cells through flow cytometry, Figure 10d is co-culture of OVCAR-3 or THP-1 cell line with SL-K01 and NK111 cells, after treatment with various concentrations of TGFβ1 , Is a graph showing the effect on cancer cell killing ability.

FIG. 11A is a schematic diagram of a chimeric antigen receptor gene construct introduced to enhance EpCAM specific cancer cell killing ability of NK111 cells according to one embodiment of the present invention, and FIG. 11B is a gene construct shown in FIG. 11A in NK111. It is a histogram showing the result of the flow cytometry for confirming the expression of the chimeric antigen receptor on the surface of the NK111-EpCAM-CAR cell introduced by the truck, Figure 11c is a human ovarian cancer cell line RMG-1 (left) and KOC-2S (right) ) Is a graph showing the histogram showing the results of analyzing the expression patterns of EpCAM antigen (top) and the degree of cancer cell death during co-culture of the cancer cells with NK101, NK111, or NK111-EpCAM-CAR, and FIG. The culture supernatant of the co-culture sample was collected and the graph showing the result of ELISA analyzing the level of IFN-γ and granzyme B.

Definition of Terms:

As used herein, the term “CAR construct” is an abbreviation for “chimeric antigen receptor construct” and is typically a single chain-based antibody analogue such as a ligand or scFv, sdAb as an antigen recognition site. Anti-cancer activity against immune cells of immune cells expressing CAR constructs by transducing immune cells such as T cells and expressing cancer cell-specific antigens as a synthetic protein composed of a cell membrane transmembrane domain-co-stimulatory factor-intracellular signaling domain It is well known to improve.

As used herein, the term "scFv" is an abbreviation for "single chain variable fragment" and is not a fragment of an actual antibody. The linker is about 25 aa in size for the heavy chain variable region (V _H ) and the light chain variable region (V _L ). It is a kind of fusion protein prepared by linking with peptides and is known to have antigen binding ability despite not being an inherent antibody fragment (Glockshuber et al ., Biochem . 29 (6): 1362-1367, 1990).

The term "sdAb (single domain antibody)" as used herein is referred to as a nanobody and is an antibody fragment consisting of a single variable region fragment of an antibody. Although sdAbs mainly derived from heavy chains are used, single variable region fragments derived from light chains have also been reported to have specific binding to antigens.

As used herein, "antibody mimetic" is the smallest unit that maintains antigen binding ability, unlike conventional full-length antibodies in which two heavy and two light chains function by forming a quaternary structure of heterotetramers. Fragments (eg, Fab, F (ab ') ₂ , Fab' or single-chain variable fragment (scFv), which is an artificial fragment linking the variable regions of the heavy and light chains with a linker, without the light chain Concepts include antibody-like proteins made from camelaceae cartilage-derived antibody fragments (V _H H, V _NAR, etc.) consisting of heavy chains only, or protein scaffolds derived from non-antibodies such as nanobody, monobody, variable lymphocyte receptor (VLR) to be.

As used herein, the term "costimulatory domain" refers to the transmembrane domain and cytoplasmic domain of costimulatory factor, an immune-related protein that aids T / NK activation. These costimulatory domains are CD28, inducible costimulator (ICOS), cytotoxic T lymphocyte associated protein 4 (CTLA4), programmed cell death protein 1 (PD1), B and T lymphocyte associated protein (BTLA), death receptor 3 (DR3), 4 -1BB, CD2, CD7, CD40, CD30, CD27, signaling lymphocyte activation molecule (SLAM), 2B4 (CD244), NKp30, NKp44, NKp46, NKp80, natural-killer group 2, member D), DAP12 (DNAX- activating protein 12), DAP10, DNAM-1, NTB-A, T-Cell immunoglobulin and mucin domain containing protein 1), TIM2, TIM3, TIGIT, CD226, CD160, LAG3 (lymphocyte activation gene 3), B7-1 , B7-H1, glucocorticoid-induced TNFR family related protein (GITR), herpesvirus entry mediator (HVEM) or cytoplasmic domain of OX40L [ligand for CD134 (OX40), CD252], or two or more of these.

As used herein, the term "intracellular signaling domain" refers to a domain that generates signals that induce activation of immune cells when antigen recognition sites specifically bind to cancer antigens in CAR constructs. TCR) CD3ξ domain and the like are known. In addition to the CD3ξ domain, it is also possible to use CD16, NKp30, NKp44, NKp46, NKp80, DAP10, DAP12 and the like.

The term "genetically modified" as used herein refers to a host cell or host cell or polynucleotide or vector according to one embodiment of the present invention introduced into one of the predecessors / parents. Means to include in addition to their genome. In addition, the polynucleotide or vector according to an embodiment of the present invention may exist in a genetically modified host cell as an independent molecule, preferably a replicable molecule outside the genome, or stably into the genome of the host immune cell. Can be inserted.

As used herein, the term "cell suicide gene" refers to a gene that induces cytotoxicity or triggers apoptosis mechanisms to induce the death of cells in which the gene is expressed. In particular, gene expression itself does not trigger apoptosis, but metabolites produced by metabolizing prodrugs by apoptotic genes when treating certain prodrugs (prodrugs) trigger cell cytotoxicity or apoptosis mechanisms. It can lead to death. These apoptosis genes include the herpes herpes simplex virus thymidine phosphorylation gene (HSV TK) and 6-methoxypurine arabinonucleoside, which use gancyclovir as a suicide induction signal. Cytosine deminase and uracil phosphoribosyltransferase (UPRT), irinotecan (Varicela zoster virus thymidine kinase (VZV TK), 5-fluorocytosine (5-FC) used as a suicide induction signal Nitrodeductase using carboxyl esterase, 5 (aziridin-1-yl) -2,4-dinitrobenzamide (CB1954), using CPT-11) as a suicide induction signal Suicide induction signal using 4-[(2-chloroethyl) (2-mesyloxyethyl) amino] benzoyl-l-glutamic acid (CMDA) as a suicide induction signal, carboxypeptides G2, an intermolecular dimerization dimerizer Inductive casing Izu is a 9 (iCas9) are reported.

Detailed description of the invention:

According to one aspect of the present invention, there is provided a genetically modified NK cell line in which a polynucleotide encoding an NK cell coactivator is transduced into an isolated NK cell line having the following characteristics such that the NK cell coactivator is expressed:

CD2, CD11a, CD25, CD45, CD54, DNAM-1, CD62L and CD56 are positive; And

CD1a, CD3, CD4, CD8, CD14, CD20, CD23, CD34, TCRαβ and TCRγδ are negative.

In the genetically modified NK cell line, the isolated NK cell line may be an NK101 cell line deposited with accession number KCTC 13305BP.

In the genetically modified NK cell line, the NK cell coactivator may be any one or more selected from the group consisting of Ly49, natural cytotoxicity receptor (NCR), CD7, CD16, and CD28.

In the genetically modified NK cell line, the NK cell coactivator may be CD7 and / or CD28.

In the genetically modified NK cell line, polynucleotides encoding at least one or more NK cell proliferation factors may be further transduced.

In the genetically modified NK cell line, the NK cell proliferation factor is at least one cytokine or cytokine selected from the group consisting of IL-2, IL-12, IL-15, IL-18 and IL-21. It may be a variant.

In the genetically modified NK cell line, the IL-15 may be membrane-bound IL-15.

In the genetically modified NK cell line, apoptotic genes may be further transduced.

In the genetically modified NK cell line, the apoptosis gene is a uracil phosphoribosyltransferase (UPRT) gene, herpes simplex virus thymidine phosphorylation gene (HSV TK), varicella zoster virus thymidine kinase (VZV TK) Gene, cytosine deminase gene, carboxyl esterase gene, nitroreductase gene, carboxypeptide G2 gene, or inducible caspase 9 (iCas9) gene.

In such genetically modified NK cell lines, polynucleotides encoding cytoplasmic domain deleted TGFβ receptors may be further transduced.

In the genetically modified NK cell line, the cytoplasmic domain deleted TGFβ receptor may be cytoplasmic domain deleted TGFβ receptor II.

In the genetically modified NK cell line, a polynucleotide encoding a chimeric antigen receptor that specifically recognizes a cancer antigen may be further transduced.

In the genetically modified NK cell line, the cancer antigen is CD19, CD22, prostate specific antigen (PSA), carcinoembryonic antigen (CEA), CA-125, mucin 1, alphafetoprotein (AFP), epipithelial tumor antigen (ETA), tyrosine Naese, CD52, PD-L1 (programmed death-ligand 1), CTLA4 (cytotoxic T-lymphocyte-associated protein 4), CD20, MAGE (melanoma-associated antigen), FAP (fibroblast activation protein), FLT3 (fms like tyrosine kinase 3), IL13Rα2 or an epithelial cell adhesion molecule (EpCAM).

In the genetically modified NK cell line, the chimeric antigen receptor may be a fusion protein comprising a ligand or antibody analog-transmembrane domain-co-stimulatory factor-cellular signaling domain that specifically binds to cancer antigen.

In the genetically modified NK cell line, the antibody analog may be scFv, sdAb, nanobody, V _H H, V _NAR , VLR, or monobody.

In the genetically modified NK cell line, the costimulatory factors are CD28, inducible costimulator (ICOS), cytotoxic T lymphocyte associated protein 4 (CTLA4), programmed cell death protein 1 (PD1), B and T lymphocyte associated protein (BTLA), Death receptor 3 (DR3), 4-1BB, CD2, CD7, CD40, CD30, CD27, signaling lymphocyte activation molecule (SLAM), 2B4 (CD244), NKp30, NKp44, NKp46, NKp80, NKG2D (natural-killer group 2, member D) / DAP12 (DNAX-activating protein 12), DAP10, DNAM-1, NTB-A, TIM1 (T-Cell immunoglobulin and mucin domain containing protein 1), TIM2, TIM3, TIGIT, CD226, CD160, LAG3 (lymphocyte activation gene 3), B7-1, B7-H1, glucocorticoid-induced TNFR family related protein (GITR), herpesvirus entry mediator (HVEM) or cytoplasmic domain of ligand for CD134 (OX40), CD252] or two or more of these It may be a linking body.

In the genetically modified NK cell line, the intracellular signaling domain may be a CD3ξ domain, a CD16, NKp30, NKp44, NKp46, NKp80, DAP10, or DAP12 of a T cell receptor.

In the genetically modified NK cell line, one or more genes may be removed as necessary. The gene may be determined according to the genotype of the patient, and may be a gene capable of causing side effects or inhibiting the activity of the administered cells by inducing an excessive immune response upon administration to the patient. Genome editing tools such as TALEN or CRISPR can be used to remove these genes.

According to another aspect of the invention, there is provided a pharmaceutical composition for preventing and treating cancer comprising any one of the above genetically modified NK cell line as an active ingredient.

According to another aspect of the present invention, there is provided a cancer treatment kit comprising any one of the above genetically modified NK cell line and suicide inducing agent.

In the cancer treatment kit, the suicide inducing agent is gancyclovir or 6-methoxypurine arabinonucleoside, if the apoptosis gene is HSV TK or VZV TK, respectively. 5-fluorocytosine (5-FC) when the apoptosis gene is uracil phosphoribosyl transferase (UPRT) or cytosine deminase, irinotecan (CPT-11) when the apoptosis gene is carboxyl esterase, 5 (aziridin-1-yl) -2,4-dinitrobenzamide (CB1954) when the apoptosis gene is nitroreductase, and 4- [when the apoptosis gene is carboxypeptide G2. (2-chloroethyl) (2-mesyloxyethyl) amino] benzoyl-l-glutamic acid (CMDA), iCas9 dimerizer when the apoptosis gene is iCas9.

According to another aspect of the invention, there is provided a method for treating cancer in a subject comprising administering to a subject with cancer a therapeutically effective amount of any one of the above genetically modified NK cell lines and optionally suicide inducing agent. Is provided.

According to another aspect of the present invention, there is provided a genetically modified NK cell line transduced with a polynucleotide and an apoptotic gene encoding the NK cell coactivator, NK cell proliferation factor, cytoplasmic domain deletion TGFβ receptor, respectively, to an isolated NK cell line do.

In the genetically modified NK cell line, the isolated NK cell line may have the following characteristics:

CD2, CD11a, CD25, CD45, CD54, DNAM-1, CD62L, and CD56 are positive; And

CD1a, CD3, CD4, CD8, CD14, CD20, CD23, CD34, TCRαβ and TCRγδ are negative.

In the genetically modified NK cell line, the isolated NK cell line may be an NK101 cell line deposited with accession number KCTC 13305BP.

In the genetically modified NK cell line, the NK cell coactivator may be selected from the group consisting of Ly49, natural cytotoxicity receptor (NCR), CD7, CD16, and CD28. Preferably, the NK cell coactivator may be CD7 and / or CD28.

In the genetically modified NK cell line, the NK cell proliferation factor is at least one cytokine or cytokine selected from the group consisting of IL-2, IL-12, IL-15, IL-18 and IL-21. It may be a variant.

In the genetically modified NK cell line, the cytoplasmic domain deleted TGFβ receptor may be cytoplasmic domain deleted TGFβ receptor II.

In the genetically modified NK cell line, the apoptosis gene is a uracilphosphoribosyltransferase (UPRT) gene, herpes simplex virus thymidine phosphorylation gene (HSV TK), varicella zoster virus thymidine kinase (VZV TK) Gene, cytosine deminase gene, carboxyl esterase gene, nitroreductase gene, carboxypeptide G2 gene, or inducible caspase 9 (iCas9) gene.

In the genetically modified NK cell line, one or more genes may be removed as necessary. The gene may be determined according to the genotype of the patient, and may be a gene capable of causing side effects or inhibiting the activity of the administered cells by inducing an excessive immune response upon administration to the patient. Genome editing tools such as TALEN or CRISPR can be used to remove these genes.

The genetically modified NK cell line may be further transduced with a polynucleotide encoding a chimeric antigen receptor that specifically recognizes a cancer antigen.

At this time, the cancer antigen can be used any known cancer antigen, the cancer antigen is CD19, CD22, PSA (prostate specific antigen), CEA (carcinoembryonic antigen), CA-125, mucin 1, AFP (alphafetoprotein), Epithelial tumor antigen (ETA), tyrosinase, CD52, programmed death-ligand 1 (PD-L1), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), CD20, melanoma-associated antigen (MAGE), fibroblast activation protein), FLT3 (fms like tyrosine kinase 3), IL13Rα2, or epithelial cell adhesion molecule (EpCAM).

In addition, the chimeric antigen receptor may be a fusion protein comprising a ligand or an antibody analog-transmembrane domain-co-stimulatory factor-cellular signaling domain that specifically binds to cancer antigens, wherein the antibody analog is scFv, sdAb, It may be a nanobody, V _H H, V _NAR , VLR, or monobody, the co-stimulatory factors are CD28, inducible costimulator (ICOS), cytotoxic T lymphocyte associated protein 4 (CTLA4), programmed cell death protein 1 (PD1) , B and T lymphocyte associated protein (BTLA), death receptor 3 (DR3), 4-1BB, CD2, CD7, CD40, CD30, CD27, signaling lymphocyte activation molecule (SLAM), 2B4 (CD244), NKp30, NKp44, NKp46 , NKp80, NKG2D (natural-killer group 2, member D) / DAP12 (DNAX-activating protein 12), DAP10, DNAM-1, NTB-A, TIM1 (T-Cell immunoglobulin and mucin domain containing protein 1), TIM2, TIM3, TIGIT, CD226, CD160, lymphocyte activation gene 3 (LAG3), B7-1, B7-H1, glucocorticoid-induced TNFR family related protein (GITR) , A herpesvirus entry mediator (HVEM) or a cytoplasmic domain of OX40L [ligand for CD134 (OX40), CD252] or two or more of these, wherein the intracellular signaling domain is the CD3ξ domain of the T cell receptor, CD16, NKp30, It may be NKp44, NKp46, NKp80, DAP10 or DAP12.

In the genetically modified NK cell line, the NK cell coactivator, NK cell proliferation factor, cytoplasmic domain deletion TGFβ receptor and apoptosis genes are expressed individually, simultaneously expressed in one gene construct, or two or more genes. Divided into constructs can be expressed. For example, some of the introduced genes are expressed in the form of a fusion protein linked by a linker or linked to a protease recognition site and automatically cleaved by a protease expressed in a cell to be expressed as a mature protein, or IRES. It is also possible to be expressed as a single mRNA linked to the back, and then expressed as individual proteins during translation. These gene constructs are operably linked to one or more promoters and can be inserted into expression vectors optimized for expression in mammalian cells, particularly NK cells, and transduced with a variety of eukaryotic transduction methods. Such transduction can be performed using a variety of methods, including lipopexen, calcium phosphate transfection, gene gun, electroporation, and genome editing tools such as TALEN or CRISPR for more sophisticated intranuclear transduction. Can be used.

As used herein, "operably linked to" means that a particular polynucleotide is linked to another polynucleotide so that it can function. In other words, the operably linked polynucleotide encoding a particular protein means that the polynucleotide encoding the specific protein is linked to be transcribed into mRNA and translated into the protein by the action of the promoter. An operably linked polynucleotide encoding another protein may allow the particular protein to be expressed in the form of a fusion protein with the other protein.

Regulatory factors that allow expression in the eukaryotic and prokaryotic cells are well known to those skilled in the art. As mentioned above, these usually include regulators responsible for transcription initiation and, optionally, poly-A signals responsible for transcription termination and stabilization of the transcript. Additional regulators may include, in addition to transcriptional regulators, translation enhancers and / or naturally-combined or heterologous promoter regions. For example, possible regulators that allow expression in mammalian host cells include the CMV-HSV thymidine kinase promoter, SV40, RSV (Loose Sarcoma Virus) promoter, human kidney element 1α-promoter, glucocorticoid-induced MMTV-promoter (Molony mouse tumor virus), metallothionein-induced or tetracycline-induced promoters or amplification agents such as CMV amplifiers or SV40-amplifiers. For expression in neural cells, it is contemplated that neurofiber-promoter, PGDF-promoter, NSE-promoter, PrP-promoter or thy-1-promoter may be used. Such promoters are known in the art and described in Charron, J. Biol. Chem. 1995, 270: 25739-25745. For prokaryotic expression, a number of promoters have been disclosed, including lac-promoters, tac-promoters or trp promoters. In addition to factors capable of initiating transcription, these regulators include transcription termination signals, such as the SV40-poly-A site or the TK-poly-A site, downstream of the polynucleotide according to one embodiment of the invention. You may. In this document, suitable expression vectors are known in the art, for example, the Okayama-Berg cDNA expression vector pcDV1 (Parmacia), pRc / CMV, pcDNA1, pcDNA3 (In-vitrogene), pSPORT1 (GIBCO BRL). ), pX (Pagano (1992) Science 255, 1144-1147), yeast two-hybrid vectors such as pEG202 and dpJG4-5 (Gyuris et al ., Cell 75, 791-803, 1995) or prokaryotic Expression vectors such as lambda gt11 or pGEX (Amersham-Pharmacia). In addition to the nucleic acid molecules of the invention, the vector may further comprise a polynucleotide encoding a secretory signal. The secretion signals are well known to those skilled in the art. And, depending on the expression system used, a leader sequence capable of directing the peptide of the present invention to the cell compartment is combined with the coding sequence of the polynucleotide according to an embodiment of the present invention, and preferably the translated protein. Or a leader sequence capable of secreting its protein directly around the cytoplasm or into an extracellular medium. Optionally, the heterologous sequence can encode a fusion protein comprising a C-terminal or N-terminal tag peptide that confers desired properties such as stabilization or simple purification of the expressed recombinant product. Such tags include, but are not limited to, FLAG, GST (glutathione S transferase), HisX6, and the like. When the vector according to one embodiment of the present invention is transduced into a suitable host cell or non-human host object, the host cell or host object is maintained under conditions suitable for high level expression of the nucleotide sequence.

According to another aspect of the invention, there is provided a pharmaceutical composition for cancer treatment and cancer prevention comprising the genetically modified NK cell line as an active ingredient.

In the pharmaceutical composition, the cancer may be hematological cancer or solid cancer, the solid cancer is liver cancer, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, gallbladder cancer, gastric cancer, biliary cancer, colon cancer, head Cervical cancer, esophageal cancer, thyroid cancer, brain tumor, malignant melanoma, prostate cancer, testicular cancer, tongue cancer, lymphoma or leukemia, the solid cancer may be metastatic cancer.

The pharmaceutical composition of the present invention may contain at least one known active ingredient having an anticancer effect together with the genetically modified NK cell line. In this case, the composition for treating cancer of the present invention means a pharmaceutical composition formulated by mixing the genetically modified NK cell line and a known active ingredient together with a pharmaceutically acceptable carrier, and if not formulated together, separately packaged. And may be administered simultaneously or sequentially. In the latter case, it may be referred to as a kit rather than a composition.

In addition, the composition may further include a pharmaceutically acceptable excipient or diluent in addition to the pharmaceutically acceptable carrier.

Such pharmaceutically acceptable carriers include, for example, carriers for parenteral administration such as water, suitable oils, saline, aqueous glucose and glycols, and the like, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. In addition, the cell therapy according to the present invention, if necessary according to the administration method or dosage form, suspensions, dissolution aids, stabilizers, isotonic agents, preservatives, adsorption agents, surfactants, diluents, excipients, pH adjusters, analgesics, buffers And antioxidants may be included as appropriate. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, latest edition.

On the other hand, the dosage of the pharmaceutical composition according to an embodiment of the present invention may be 10 ⁷ to 10 ¹¹ cells on the basis of the cell number, the dosage is depending on the sex, age, disease progression, treatment purpose of the patient Can be adjusted. In general, this amount will be sufficient to obtain localization in the target cell, eg, cancer antigen overexpressing cancer cell, and to kill the cancer cell, eg, by phagocytosis or lysis.

The cell therapy agent may further include a pharmaceutically acceptable carrier, diluent, or excipient in addition to the carrier.

In addition, as used herein, the term "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction, such as gastrointestinal disorders, dizziness, or the like when administered to a human.

In addition, cell therapeutic agents according to one embodiment of the present invention can be formulated using methods known in the art to allow for rapid release, or sustained or delayed release of the active ingredient when administered to a mammal. Formulations include powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powder forms.

Pharmaceutical compositions according to one embodiment of the invention can be administered by a variety of routes, for example, oral, parenteral, e.g. suppositories, transdermal, intravenous, abdominal, intramuscular, intracranial, intralesional, nasal It can be administered intrathecal, and can also be administered using a sustained release or implantable device for continuous or repeated release. The frequency of administration can be administered once a day or divided into several times within the desired range, the administration period is not particularly limited.

In addition, according to another aspect of the present invention, there is provided a kit for treating cancer comprising the genetically modified NK cell line and suicide inducing agent.

The cancer treatment kit includes the genetically modified NK cell line and suicide inducer, but the two components are not provided in a mixed form, but are provided separately packaged, and these two components may be administered at the same time or through different routes. It can be, but is distinguished from the general pharmaceutical composition in that it is administered at regular intervals according to the doctor's prescription. The kit of the present invention first administers the genetically modified NK cell line, and then, at an appropriate time point, such as, for example, administration of the genetically modified NK cell line, 1 day, 2 days, 3 days, 4 days, or 5 days after administration. , After 6 days, or after 1 week, after 10 days, after 2 weeks, after 15 days, after 20 days, after 3 weeks, after 25 days, after 4 weeks, or after 30 days, the first dose It may then be administered in multiple doses two or more times at intervals of two days, three days, four days, five days, six days, or one week. In addition, the kit may be the same or different route of administration of each component. For example, after administering the genetically modified NK cell line by intravenous injection, the suicide-inducing agent may be administered orally, or intraperitoneally or intravenously, at about the same time interval or at predetermined intervals. The route of administration of the suicide inducing agent may be appropriately determined according to the characteristics of the suicide inducing agent. For example, in the case of 5-FC and iriokecan, either intraperitoneal injection, intravenous injection or oral administration is possible. Pharmaceutically acceptable carriers included in the pharmaceutical composition for cancer treatment comprising the genetically modified NK cell line and suicide inducer as active ingredients are as described above.

The suicide inducing agent depends on the type of suicide gene. For example, when the suicide gene is HSV TK or VZV TK, gancyclovir or 6-methoxypurine arabinonucleoside, respectively. ), 5-fluorocytosine (5-FC) for uracil phosphoribosyl transferase (UPRT) or cytosine deminase, and irinotecan (CPT-11) for carboxyl esterase. have. In addition, nitroreductase may be 5 (aziridin-1-yl) -2,4-dinitrobenzamide (CB1954), and in the case of carboxypeptides G2, 4-[(2-chloroethyl) (2-mesyloxyethyl) amino] benzoyl-L-glutamic acid (CMDA), iCas9 may be an iCas9 dimer, and iCas9 dimer may be AP20187 or AP1903.

According to another aspect of the present invention, there is provided a method for treating a subject with cancer comprising administering a therapeutically effective amount of said genetically modified NK cell line or optionally suicide inducing agent to the subject with cancer. .

As used herein, "therapeutically effective amount" means an amount that significantly inhibits the death of cancer cells or at least the growth of cancer tissue.

Cancer that can be treated through the use of a pharmaceutical composition for treating cancer or a kit for cancer treatment according to an embodiment of the present invention is liver cancer, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, gallbladder cancer, gastric cancer, biliary tract Cancer, colorectal cancer, head and neck cancer, esophageal cancer, thyroid cancer, brain tumor, malignant melanoma, prostate cancer, testicular cancer, tongue cancer, lymphoma or leukemia.

According to another aspect of the present invention, the isolated NK cell line is transduced with polynucleotides and apoptosis genes encoding NK cell coactivator, cell membrane-bound IL-15, and TGFβ receptor, respectively, and is a cancer antigen specific chimeric antigen receptor. A polynucleotide encoding a gene is transduced to provide a genetically modified NK cell line expressing the cancer antigen specific chimeric antigen receptor.

The cancer antigen is CD19, CD22, prostate specific antigen (PSA), carcinoembryonic antigen (CEA), CA-125, mucin 1, alphafetoprotein (AFP), epipithelial tumor antigen (ETA), tyrosinase, CD52, PD-L1 ( programmed death-ligand 1), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), CD20, melanoma-associated antigen (MAGE), fibroblast activation protein (FAP), fms like tyrosine kinase 3 (FLT3), IL13Rα2 or epipithelial (EpCAM) cell adhesion molecule).

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings.

The present inventors have isolated a new NK cell line having the characteristics shown in Table 1 from cancer tissues of patients with NK lymphoma, and as a result of investigating various characteristics thereof, as shown in FIGS. It was confirmed that it is a multifunctional NK cell line having both cancer cell killing ability and immunomodulatory ability. In particular, the proliferative capacity is significantly higher than that of NK-92, the only NK cell line currently undergoing clinical trials, and it is identified as an economically viable cell.These cells are named 'NK101' and are located at 181, Sinpsin-gil, Jeongeup-si, Jeollabuk-do, Korea. It was deposited on August 7, 2017 by the Korean Collection for Type Culture (KCTC) in the Korea Research Institute of Bioscience and Biotechnology, and received an accession number of KCTC 13305BP on August 24, 2017. However, the gene expression analysis of the NK101 cell line revealed that the IL-2 receptor CD25 is highly expressed and has a phenotype of CD56 ^dim CD62L ⁺ , but is a NK cell coactivator that directly affects the anticancer activity of NK cells. By confirming that CD7 and CD28 do not express, anticancer activity itself was expected not to be higher than conventionally constructed NK cell lines.

Thus, the present inventors intended to prepare a genetically modified NK cell line based on NK101 cells in order to enhance the anticancer activity of NK101 cells according to an embodiment of the present invention. Figure 7b is a schematic diagram showing the structure of the gene construct CD7-CD28-CD :: UPRT used in the production of the genetically modified NK cell line SL-K01 according to an embodiment of the present invention to achieve the above object; 9A is a schematic diagram schematically showing the structure of the gene construct mbIL-15-mTGFβIIΔcyto used for the preparation of the genetically modified NK cell line NK111 according to one embodiment of the present invention.

In the present invention, in order to complement the aforementioned elements, the NK101 cells (accession number KCTC 13305BP) previously established by the present inventors, coactivating receptors of NK cells, apoptosis genes, membrane bound cytokines, mutations The TGFβ receptor was introduced to construct an NK111 cell line capable of enhancing anticancer effects of the NK cells and proliferating independently of cytokine supplements (see FIGS. 9B and 9C).

FIG. 7 shows cell surface markers of a genetically modified NK cell line (SL-K01) and a parent cell line NK101 of the SL-K01, and conventionally constructed NK cell lines KHYG-1 and NK-92 according to an embodiment of the present invention. As a result of analyzing the expression pattern by flow cytometry, Figure 7a is a histogram showing the results of flow cytometry analysis of the expression of CD7 and CD28 in NK101, KHYG-1 and NK-92, Figure 7c is shown in Figure 7b It is a histogram showing the result of confirming the expression of CD7 and CD28 in the genetically modified cell line SL-K01 cells according to an embodiment of the present invention transduced by flow cytometry. As shown in FIG. 7, KHYG-1 and NK-92, which are known to have high cancer cell killing ability among conventionally constructed NK cell lines, have a characteristic of expressing CD7, and NK-92 cells have CD7 as well as CD28. While NK101 has the characteristic of expressing at the same time, it was confirmed that both co-stimulatory factors are not expressed at all. Therefore, the present inventors attempted to evaluate whether cancer cell killing ability is enhanced by introducing CD7 and CD28 into NK101 cells.

FIG. 8 is a result of analyzing apoptosis of various cancer cells of the genetically modified NK cell line SL-K01 and the parent cell line NK101 of the SL-K01 according to an embodiment of the present invention, and FIG. 8A illustrates HDLM-2 and IM-. 9, JEKO-1 and K562 cancer cells were cocultured with NK101 or SL-K01 cells at 4: 1 ratio for 24 hours, and then the cancer cell death frequency was measured by flow cytometry. FIG. 8B is a graph showing various concentrations of 5 -FC treated with NK101 or SL-K01 cells for 48 hours, the cell growth rate is a graph showing the results confirmed by the MTS analysis, Figure 8c is an IM-9 cell line and NK101 or SL-K01 cells 1: 1, 2 When co-cultured in the ratio of 1: 1 or 4: 1, the graph shows the result of measuring the flow rate of IM-9 cancer cells by flow cytometry with or without 5-FC. As shown in Figures 8a to 8c, the genetically modified NK cell line SL-K01 according to an embodiment of the present invention shows excellent killing ability in various cancer cell lines compared to NK101, the killing ability of NK101 cells was enhanced through the introduction of CD7, CD28 I could confirm it. In addition, it was confirmed that not only can secure safety by inducing the removal of SL-K01 cells through 5-FC treatment, but also induces bystander killing effect to remove surrounding cancer cells.

Figure 9 shows the construction of a genetically modified NK cell line NK111 cell line according to another embodiment of the present invention, Figure 9a is an additional cancer cell killing effect of SL-K01 cells and resistance to the immunosuppressive factor TGF-β 9A is a schematic diagram of a gene construct introduced for induction, and FIG. 9B shows IL-15 expression on the surface of SL-K01 and SL-K01 cells (named 'NK111') into which the gene construct shown in FIG. 9A is introduced. Is a histogram showing the results confirmed by flow cytometry, Figure 9c is a histogram showing the results confirmed by flow cytometry TGFβRIIΔcyto expression in SL-K01 and NK111 cells. As confirmed in FIG. 8, CD7 / CD28 introduction showed a moderate effect on the killing of NK101 cells. In order to maximize the cancer cell killing ability of SL-K01 cells, the present inventors tried to transduce SL-K01 cells with a membrane bound IL-15 (mbIL-15), a representative NK cell coactivator, To induce resistance to the inhibitor TGF-β overexpressed TGFβRIIΔcyto deleted from the cytoplasmic domain to act as a decoy receptor. As a result, as shown in Figures 9b and 9c, IL-15 and TGFβRII that was not expressed in the parent cell line SL-K01 was confirmed that the expression in the NK111 cell line according to an embodiment of the present invention.

10 is a result of comparing and analyzing the anticancer activity and safety of NK111, a genetically modified NK cell line according to an embodiment of the present invention, Figure 10a shows the number of cells according to the presence or absence of IL-2 in the culture of SL-K01 and NK111 cells 10B is a histogram showing the results of confirming NKG2D expression in NK101, SL-K01 and NK111 cells through flow cytometry, and FIG. 10C is an IM-9 cell line and SL-K01. Or when the NK111 cells are co-cultured at a ratio of 1: 1, 2: 1, or 4: 1, the death frequency of IM-9 cancer cells with or without 5-FC is measured by flow cytometry, and FIG. 10D. Is a graph showing the effect on cancer cell killing ability after co-culture of OVCAR-3 or THP-1 cell line with SL-K01 and NK111 cells after treatment with various concentrations of TGFβ1. As shown in Figure 10a, through the introduction of membrane-bound IL-15, NK111 according to an embodiment of the present invention is capable of cell growth independently of IL-2, which increases the convenience in production. In addition, as confirmed in FIG. 10B, it was confirmed that NKG2D expression, a representative activating receptor of NK cells, was upregulated through the introduction of IL-15, thereby enhancing killing ability against cancer cells. In addition, as can be seen in Figure 10c, bystander killing effect (bystander killing effect) by the 5-FC treatment, killing ability was increased enough to induce cancer cell death to about 90% at 1: 1 ratio. In addition, as shown in Figure 10d, through the introduction of TGFβRIIΔcyto, a bait receptor for TGFβ1, an over-secreted immunosuppressive factor in the tumor microenvironment, it was not affected by the killing ability of TGFβ1, which would later decrease in vivo activity. It is a characteristic that expects to overcome to some extent.

In addition, the inventors of the present invention to determine whether the NK111 cells according to an embodiment of the present invention can act as a platform for delivery of cancer antigen-specific chimeric antigen receptor (CAR) that can achieve antigen-specific anticancer activity By constructing a CAR construct targeting EpCAM, a cancer antigen (FIG. 11A), a SL-K10 cell line transduced with NK111 cells was prepared, and the SL-K10 cell line normally expressed an EpCAM specific CAR ( 11b), KOC, which is a control cancer cell that does not express EpCAM, was analyzed as a result of analyzing cancer cell killing ability when co-cultured at RGF-1 cells overexpressing EpCAM under in vitro conditions at various effector: target (E: T) ratios. Unlike -2S, not only the anticancer activity was significantly increased (FIG. 11C), but the superior anticancer activity was confirmed to be due to the increased expression of the anticancer inflammatory cytokines INF-γ and granzyme B. It could be done (FIG. 11D). These results suggest that NK111 cells according to an embodiment of the present invention can be used very efficiently as a platform for the next generation CAR-expressing cell therapy which can be used for the treatment of solid cancer as well as blood cancer.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, the present invention is not limited to the Examples and Experimental Examples disclosed below, but can be implemented in various forms, and the following Examples and Experimental Examples to complete the disclosure of the present invention, the present invention It is provided to fully inform the person skilled in the art the scope of the invention.

Example 1 Preparation of NK Cell Lines of the Invention

To prepare an NK cell-derived cell line, the following process was carried out. A patient-derived extranudal NK lymphoma is placed on a 40 μm strainer and Cellgro ^® stem cell growth medium containing 20% fetal bovine serum (GE Healthcare, USA) and 1% antibiotic (Gibco, USA) SCGM (CellGenix, Germany, hereinafter referred to as 'NK media') was added to 10 mL and then separated into single cells using the shear force of the piston of a 5 mL syringe and suspended. NK cells in single cell suspensions were isolated using NK isolation kit (Milltenyi Biotec, Germany) and then 1000 U / mL of recombinant human IL-2 (rhIL-2; Prometheus Laboratories Inc., USA) Incubated for 3 weeks in the added NK media. NK media containing IL-2 was added twice a week, and it was confirmed that stable cell lines were formed by continuously culturing dividing cell lines up to 30 passages (FIG. 1A). Since the cell line expresses CD56 without expressing CD3, CD20, and CD16, it was confirmed that the origin of the cells is NK cells (FIG. 1B). The cell line was confirmed under a microscope to form a spheroid in culture (Fig. 1c), and the characteristics of large granular lymphocytes with NK101 cells in morphological analysis using Wright-Giemsa staining method. It was confirmed that it has (Fig. 1d). Fluorescent staining of cell death factors, Perforin (green) and Granzyme B (Granzyme B, red), NK cell characteristics, confirmed that NK101 expressed Perforin and Granzyme B (FIG. 1E). Coculture was performed with K562, a MHCI negative cell that responds sensitively to NK cells. K562 cells labeled with Carboxyfluorescein Diacetate (CFDA; Invitrogen, USA) were seeded in 24-well plates (Corning, USA) at a concentration of 3x10 ⁵ cells / mL and NK101 cells were treated with various effector cells. It was suspended in 1 mL medium at a target ratio (E: T ratio = 1: 1, 2: 1, 4: 1, 10: 1), and then incubated with the cancer cells for 24 hours. After incubation all cells were recovered from each well and centrifuged, and the cell pellet was suspended in FACS buffer. By suspending the cell pellet formed by centrifuging again in ^{1 μL LIVE / DEAD ® Fixable Near} -IR Dead Stain Kit FACS buffer, 100 μL insert the (Invitrogen, USA), was reacted at 4 ℃ 20 minutes. After washing twice with FACS buffer, the cell pellet was suspended in a solution diluted with 5 μL of Annexin V APC (Biolegend, USA) in 100 μL of 1 × Annexin V binding buffer, and reacted at room temperature for 20 minutes. Cell death was determined by flow cytometry using viable cells (annexin V-negative / LIVE / DEAD-negative), early apoptotic cells (annexin V-positive / LIVE / DEAD-negative), late apoptotic cells (annexin V-positive / LIVE / DEAD-positive) and necrotic cells (annexin V-negative / LIVE / DEAD-positive). 1 shows that NK101 is an immortalized cell that can be passaged continuously, forms a colony in culture, and has a characteristic that the phenotype and function are consistent with previously known NK cells. The cell line was named 'NK101' by identifying the cell line and the characteristics of the NK cell. KCTC) was deposited on August 7, 2017, and received the accession number of KCTC 13305BP on August 24, 2017. The depositary body is an international depositary body under the Budapest Treaty.

Example 2 Analysis of Cell Division Capacity of NK101

In order to establish culture conditions and compare the sequencing ability of the NK101 cell line prepared in Example 1, IL-2 at various concentrations in SCGM culture medium containing 20% fetal bovine serum in NK101 cells and NK-92 cells as control cells After treatment, the cell growth of the two cell lines was compared by MTS analysis. As shown in FIG. 2A, NK101 started to grow at an IL-2 concentration of about 8 pM, and stagnated at 500 pM (EC ₅₀ = 23.3 pM), and NK-92 confirmed cell growth in the presence of 30 pM IL-2. The growth was stagnant at the concentration of 2000 pM (EC ₅₀ = 128.3 pM), indicating that NK101 requires a lower concentration of IL-2 than NK-92. As a result of confirming the expression of the IL-2 receptor subunit confirmed in FIG. 2B by flow cytometry, it was confirmed that NK101 expresses CD25, which is a high affinity IL-2 receptor, higher than that of NK-92. In addition, NK101 and NK-92 after freezing cell growth rate and survival rate were analyzed under the same culture conditions, NK101 recovered cell growth after 2 days (1 passage) after thawing, but NK-92 10 days after thawing (5 passages) Since it was confirmed that reached a certain cell growth (Fig. 2c). After cell growth of the two cells was stabilized, when comparing the cell proliferation of the two cells, it was confirmed that the total cell line of NK101 cells obtained after 16 days of passage was about 100 times the expected number of cells obtained from NK-92 cells (FIG. 2d). This suggests that the productivity of the NK101 cells of the present invention is superior to that of the conventional NK-92 cells, and that the NK101 cells of the present invention are very advantageous in terms of economy.

The comprehensive characteristics of NK101 cells of the present invention are summarized in the following [Table 1].

Main features of NK101 cells of the invention

Item	NK101 cells
Clinical data
Age / gender	56 year old male
race	Asian
Diagnosis	Extraranodal NK / T lymphoma
Cell culture
Growth pattern	Multicellular Aggregates in Suspension
Doubling Time	18-32 hours
Cell concentration	1.2x10 6 cells / ml
Minimum cell concentration	0.5x10 5 cells / ml
Cytokine dependence	IL-2 dependency (500 IU / ml)
Optimal cleavage	Every 2-3 days
Immunological properties
T / NK marker	CD2 +, CD3 -, CD4 - , CD7 -, CD8 -, CD16 -, CD56 +
B cell marker	CD10 -, CD19 -, CD20 -
Bone Supranuclear Markers	CD13 -, CD14 -, CD33 +
NK cell activating receptor	NKp46 + , NKp30 + , NKG2D +
NK cell inhibitory receptor	KIR2DL1 -, KIR2DL2 -, ILT2 -
Offspring / activation markers	CD34 -, FAS +
Attachment marker	CD11a + , CD18 + , CD54 +
Functional characteristics
NK activity	Pro-inflammatory cytokine secretion (immune activity) and proliferation
Cytokine production	Secretion of anti-inflammatory cytokines such as IFN-γ, TNF-α, GM-CSF, IL-2, and secretion of anti-inflammatory cytokines such as IL-1 receptor antagonists and IL-10
Cytokine receptors	CD25 +, CD122 +, CD132 + , CD127 -
Chemokine receptor	CCR4 + , CCR6 + , CCR7 + , CCR8 + , CXCR3 + , CXCR4 +

Example 3 Surface Marker Analysis The NK101 cell line of the present invention prepared in Example 1 had the characteristics of floating cells, and the expression level of the surface antigen of the cells was confirmed by flow cytometry. In the case of the T / NK cell marker, the NK101 cell line of the present invention expresses CD2 and CD56, which are surface antigens of NK cells, but does not express CD16, but CD3, CD4, CD8, TCRαβ and TCRγδ and B cells, which are surface antigens of T cells. It had a phenotype of NK cells not expressing the surface antigen CD20 and monocyte expressing antigen CD14 (FIG. 3A). In addition, the NK101 cell line of the present invention expressed NKG2D, NKp30, NKp46, DNAM-1, 2B4, which are activating receptors of NK cells, and expressed inactive receptors, CD94, NKG2A, but KIR2DL1 / S1 / S3 / S5, KIR2DL2 / DL3. , And CD85j and the like were not expressed (FIG. 3B). In addition, they express the cell adhesion molecules CD2, CD11a, CD19, ICAM-1, CD7 was not expressed (Fig. 3c). In addition, the NK101 cell line of the present invention is highly expressed CD107a, Perforin, Granzyme B, which is involved in the cytotoxicity and immune activation of NK cells, and TRAIL and FASL are expressed at a low level (Fig. 3d), cytokines of NK cells Among the caine receptors, it was confirmed that they express CD25, IL-2 receptors (CD122, CD132) and IL-15Ra, which are IL-2 high affinity receptors (FIG. 3E). Among chemokine receptors involved in the mobility of NK cell lines, CCR4, CCR6, CCR7, CXCR3, and CXCR4 were expressed, but other CCRs and CXCRs were not expressed (FIG. 3F). In conclusion, the NK101 cell line of the present invention exhibits the phenotype of activated NK cells, and is distinguished from other NK cell lines in that CD25 is highly expressed. CD25 is an indicator of activated NK cells and is a marker of NK cells with high division ability. (Clausen, J. et al ., Immunobiology , 207 (2): 85-93, 2003), it can be seen that the NK101 cell line of the present invention is a very suitable cell line for mass production of cell therapeutics. Expression of various cell markers of cells is summarized in Table 2 below.

Marker Properties of NK101 Cells of the Invention

antigen	Expression	antigen	Expression
Strain marker		Functional marker
CD1a	-	CD95 (FAS)	+++
CD2	+++	CD178 (FAS-L)	+
CD3	-	CD107a	+++
CD4	-	TRAIL (CD253)	+
CD5	-	Perforin	+++
CD8	-	Granzyme B	+++
CD10	-	IFNγ	+++
CD11a	+++	Chemokine receptor
CD11c	-	CCR1	+
CD13	+	CCR2	-
CD14	+	CCR3	-
CD16	-	CCR4	+++
CD18	+++	CCR5	+
CD19	-	CCR6	+++
CD23	-	CCR7	+++
CD33	+++	CCR8	++
CD45	+++	CCR9	+
CD56	+++	CXCR1	-
CD57	-	CXCR2	-
CD161	++	CXCR3	+++
Activating receptor		CXCR4	+++
2B4	+++	CXCR5	-
NKp30	++	CXCR6	-
NKp46	+++	CXCR7	-
NKG2D	++	Cytokine receptors
Inhibitory receptor		CD25 (IL-2Ra)	+++
CD85j (ILT2)	-	CD122 (IL-2Rb)	++
CD94	+++	CD132 (common γ chain)	+++
CD158 (KIR2DL1 / S1 / S3 / S5)	-	CD127 (IL-7Ra)	-
CD158b (KIR2DL2 / DL3)	-	Guitar markers
CD159a	+++	TCRαβ	-
Adhesion molecule		TCRγδ	-
DNAM-1 (CD226)	+++
ICAM-1 (CD54)	+++
CD62L	++
-, voice; +, <10% positive; ++, 10-69% positive; +++, 70-100% positive (% represents percentage of positive cells in cell population)

Example 4 Identification of CD56 and D62L Expression of NK101 In order to analyze the characteristics of NK101 cells, expression of CD56 and CD62L, markers of NK cells, was identified by flow cytometry in comparison with NK-92 and primary cultured NK cells. It was. As shown in FIG. 4A, NK101 cells were identified as CD56 ^dim NK cells having a lower CD56 expression level than NK-92 cells. In addition, as shown in the contour graph of FIG. 4B, NK101 cells express high CD62L markers, which are unexpressed in NK-92 cells and are expressed in limited numbers in some cultured NK cells. In general cultured NK cells, CD56 ^dim and CD56 ^bright can be classified according to the expression of CD56, which is thought to be composed of two populations, each of which is more dominant in cytotoxicity or cytokine production. CD56 ^bright NK cells show high cell proliferation ability, secrete IFN-γ upon activation by cytokines, and show low cancer cell killing ability. CD56 ^dim NK cells have low cell proliferation ability, as opposed to CD56 ^bright NK cells, Secrete IFN-γ and have high cytotoxicity. Recently validated CD56 ^dim CD62L ⁺ NK cells (Juelke, K et al ,, Blood , 116 (8): 1299-1307, 2010; Luetke-Eversolh, M et al ., Front. Immunol ., 4: 499, 2013 ) CD56 ^bright , CD56 ^dim NK cells are reported as a multi-functional NK cells that have both characteristics. NK101 can secrete proliferation and IFN- [gamma] by cytokine stimulation (FIG. 5a), and it has been confirmed that it secretes various types of cytokines even upon recognition of target cells (FIG. 5b). In conclusion, NK101 has the phenotype and characteristics of CD56 ^dim CD62L ⁺ NK cells, and NK101 has been reported for either primary cultured NK cells or ex vivo expanded primary cultured NK cells expressing CD56 ^dim CD62L ⁺ characteristic markers. Is an immortalized NK cell line, which is distinguished from them, which can be said to be a unique characteristic not found among known NK cell lines. Example 5: In vitro cancer cell killing ability and cytotoxicity mechanism of NK101

In order to confirm the cancer cell killing ability of the NK101 cell line prepared in Example 1 and confirmed phenotype, the following experiment was performed. Specifically, CFDA-labeled human-derived cancer cell lines THP-1, KG-1, HL-60 (acute myeloid leukemia), HCT116 (colon cancer), U373 (brain cancer), A2780 (ovarian cancer), A549 (lung adenocarcinoma) ), And SK-BR3 (breast cancer) cells were seeded in 1-well at 3x10 ⁵ cells / mL concentration in 24-well plates, respectively. NK101 cells and controls were then suspended in 1 mL medium at various effector cell-to-target wash ratios (E: T ratio = 1: 1, 2: 1, and 4: 1) and then incubated with the cancer cells for 24 hours. After incubation all cells were collected and cells were harvested from each well, centrifuged and the cell pellet suspended in FACS buffer. By suspending the cell pellet formed by centrifuging again in 1 μL LIVE / DEAD ^® Fixable FACS buffer, Near-IR Dead Stain 100 μL diluted Kit, was reacted at 4 ℃ 20 minutes. After washing twice with FACS buffer, the cell pellet was suspended in a solution diluted with 5 μL of Annexin V APC (Biolegend, USA) in 100 μL of 1 × Annexin V binding buffer, and reacted at room temperature for 20 minutes. Cell death was determined by flow cytometry using viable cells (annexin V-negative / LIVE / DEAD-negative), early apoptotic cells (annexin V-positive / LIVE / DEAD-negative), late apoptotic cells (annexin V-positive / LIVE / DEAD-positive) and necrotic cells (annexin V-negative / LIVE / DEAD-positive). As shown in FIG. 6A, the NK101 cell-administered group showed cell killing ability against various human cancer cell lines. THP-1 (FIG. 6B) after treatment with neutralizing antibodies to CD25, CD62L, DNAM-1, and CD54 (ICAM-1), which are highly expressed in NK cells, to identify the major markers of cell killing ability of NK101 cells, Apoptosis was analyzed after co-culture with K562 (FIG. 6C), Jurkat (FIG. 6D) and an effector cell-to-target cell ratio of 4: 1. As a result, the cell killing ability of NK101 was reduced by treatment of neutralizing antibodies such as DNAM-1 and CD54 in KP, CD562 in K562 and CD25, CD62L and CD54 in Jurkat. In addition, in co-culture of THP-1 and NK101 in FIG. 6E, synergistically, apoptosis was reduced when the neutralizing antibodies of DNAM-1 and CD54 were simultaneously treated. Based on the results, it can be seen that markers such as DNAM-1, CD25, CD62L, and CD54 are mainly involved in apoptosis by NK101.

Example 6: Preparation of enhanced NK cell line

KHYG-1 and NK-92 are known to have high cancer cell killing ability among the established NK cell lines, and they have the characteristic of expressing CD7 and CD28 in common, whereas NK101 does not express both co-stimulatory factors at all. With this in mind, the present inventors attempted to evaluate whether cancer cell killing ability is enhanced when the NK101 is transduced with genes encoding CD7 and CD28.

6-1: Preparation of NK Cell Activator and Apoptosis Gene Construct

The present inventors prepared a nucleic acid molecule (SEQ ID NO: 16) and a CD28 immune cell co-stimulatory factor (SEQ ID NO: 17) encoding CD7 (SEQ ID NO: 15) to the NK101 cells prepared in Example 1 to produce a functionally enhanced NK cell line. The nucleic acid molecule encoding (SEQ ID NO: 18) is linked to the nucleic acid molecule encoding the 2A peptide (SEQ ID NO: 20), and the nucleic acid molecule encoding the CD :: UPRT (SEQ ID NO: 21) is added. Gene constructs linked with IRES (SEQ ID NO: 23) were prepared and cloned into the transfer vector pWPT for lentiviral production to prepare pWPT-CD7-CD28-CD :: UPRT (FIG. 7B).

6-2: Lentivirus Production

In the Lenti-X cells cultured for 48 hours or 72 hours for lentiviral production, 12 μg of the transfer plasmid prepared in Example 6-1, 12 μg of the packaging plasmid psPAX2 and 2.4 μg of the envelope plasmid pMD2.G were lipofectamine. Invitrogen, USA) and transduced Lenti-X 293T cells (Clontech, USA). After 6 hours of incubation at 37 ° C, the medium was removed and fresh growth medium was added. After 48 hours of incubation, the lentivirals collected in the medium were collected, centrifuged for 5 minutes (4 ° C., 4000 rpm), the supernatant was collected, and the cell debris was removed using a 0.45 μm filter (Millipore, USA). It was. To concentrate the lentiviral, Lenti-X concentrator (Clontech, USA) reagent was added to the filtered lentiviral supernatant and stored at 4 ° C. overnight. As a final step, the lentiviral-concentrator mixture was centrifuged at 4000 rpm for 60 minutes, the supernatant was removed, and the recovered lentivirus in pellet form was diluted with 1-2 mL of culture and stored at -80 ° C until use. .

6-3: Production of NK Cell Activating Factor and Apoptotic Gene Line

The CD7-CD28-CD :: UPRT lentivirus prepared in Example 6-2 was transfected into NK101 cells using protamine sulfate (Sigma, USA) and then incubated at 37 ° C for 4 hours. After two times of infection, 72 hours after infection, the expression of CD7 and CD28, the immune cell co-stimulatory factors, was confirmed by flow cytometry, and 1 week later, the fluorescence activated cell sorter (BD FACSMelody, BD, USA) was used to selectively isolate only NK101 cells expressing the transgene. Expression of the membrane surface proteins CD7 and CD28 in the isolated and propagated NK101-CD7-CD28-CD :: UPRT cell lines was confirmed by flow cytometry (FIG. 7C). We named the genetically modified NK101 cell line expressing CD7-CD28-CD :: UPRT as 'SL-K01'.

6-4: Reverse Transcription Polymerase Chain Reaction (RT-PCR)

CD :: UPRT, an intracellular expression protein, isolated RNA and confirmed its expression using reverse transcriptase polymerase chain reaction (RT-PCR) (FIG. 7D). Total RNA was isolated using RNA extraction kit (iNtRON, South Korea). Reverse transcription polymerase chain reaction was carried out using a QuantiTect Reverse Transcription Kit (QIAGEN, Germany) to synthesize cDNA, and then mixed the synthesized cDNA with Taq polymerase and primers, and then subjected to heavy enzyme chain reaction (PCR). The amplified PCR product was electrophoresed on a 1% agarose gel to confirm the presence of the CD :: UPRT gene. As a result, as shown in Figure 7d, it was confirmed that the transduced CD :: UPRT gene is normally expressed.

Example 7: Confirmation of Increased NK Cell Activation by NK Cell Coactivator and Apoptosis Gene

7-1: Verification of NK cell killing ability by introducing CD7 and CD28

In order to analyze the cell killing ability of the SL-K01 cells prepared in Example 6, HDLM-2 (human Hodgkin's lymphoma), IM9 (human lymphoblastoma), Jeko-1 (human mantle cell lymphoma) and K562 (Chronic myeloid leukemia) and co-culture were performed. Target tumor cell lines labeled with Celltracker violet dye (CTV; Invitrogen, USA) were prepared at a concentration of 3 × 10 5 / mL, and then dispensed in 1 mL portions in 24-well plates. In contrast to the target tumor cell line, NK101 and SL-K01 cells were suspended in the culture medium at a desired ratio, and then 1 mL was dispensed into 24-well plates containing the target tumor cell line and co-cultured at 37 ° C. for 24 hours. As a result, it was confirmed that the cell killing ability increased in all target cell lines (Fig. 8a).

7-2: Confirmation of NK cell death following apoptotic gene precursor administration

In order to analyze the effect of CD :: UPRT, which is an apoptosis gene introduced into SL-K01 cells prepared in Example 6, the cells were prepared in 20% FBS composition in SCGM medium with 2x10 ⁴ cells / 90 μL / well Aliquots in 96-well plates (Corning, USA) and diluted to concentrations (0, 0.1, 1, 10, 100, 1000 μg / mL, respectively), 5-Fluorocytosine (5-FC; Sigma, USA). ) Or positive control group (1% Triton-X) was inoculated in 10 μL of the wells, and incubated for 48 hours at 5% CO ₂ , 37 ° C. Subsequently, MTS solution (Promega, USA) was placed in a 96-well plate at 20 μL per well and reacted for 5 hours in a 5% CO ₂ , 37 ° C. incubator, followed by 490 using a SpectraMax 190 Microplate Reader (Molecular Device, USA). Absorbance was measured at nm. As a result, it was confirmed that 5-FC concentration-dependent cell death in cells expressing CD :: UPRT (FIG. 8B). C9-labeled IM9 cells were treated with SL-K01 cells and 100 μg / mL 5-FC together for 48 hours, and then cell death was confirmed by flow cytometry. As confirmed by the results shown in FIG. 8C, apoptosis was further increased in the experimental group treated with 5-FC. In conclusion, when the precursor is administered, the therapeutic effect can be maximized by the bystander effect of killing not only NK cells into which apoptotic genes have been introduced, but also target cancer cells.

Example 8: Introduction of mbIL-15 and TGFβRIIΔcyto

The present inventors of the nucleic acid molecule (SEQ ID NO: 25) and TGFβRII encoding the membrane-bound IL-15 (mbIL-15, SEQ ID NO: 24) to further enhance the cell killing capacity of the SL-K01 cells prepared in Example 6 Nucleic acid molecule (SEQ ID NO: 27) encoding TGFβRIIΔcyto (SEQ ID NO: 26) from which the cytoplasmic domain was removed was prepared by constructing a gene construct linked to a nucleic acid molecule (SEQ ID NO: 20) encoding a 2A peptide (SEQ ID NO: 19) for the production of lentiviral. PWPT-mbIL-15-TGFβRIIΔcyto was prepared by cloning into the transfer vector pWPT (FIG. 9A). MbIL-15-TGFβRII lentiviral was prepared in the same manner as described in Example 6, and then infected with SL-K01 cells, and only SL-K01 cells expressing the transgene were selectively isolated using a fluorescent activated cell separator. Expression of membrane surface proteins IL-15 and TGFβRII in the isolated SL-K01-mbIL-15-TGFβRII cell line was confirmed by flow cytometry (FIG. 9B). The SL-K01 cell line expressing mbIL-15-TGFβRII was named 'NK111'.

Example 9 Confirmation of Cell Division Capability by Introducing mbIL-15 and Confirmation of Inhibition of Immune Resistance by Endogenous TGFβ by Introduction of TGFβRIIΔcyto

9-1: IL-2 Dependent Proliferation Assay

In order to confirm the IL-2 independent NK cell division ability of the NK111 cells prepared in Example 8, the SL-K01 cells and NK111 cells were 48 × under 250 U / mL IL-2 at a concentration of 2 × 10 ⁵ cells / mL. After culturing in a time cycle, the cells were collected and the number of viable cells was measured by trypan blue staining (Trypan Blue). As can be seen in Figure 10a, SL-K01 cells were not cell proliferation under IL-2 deficiency conditions, cell growth was confirmed in the IL-2 treatment conditions. It was observed that NK111 cells introduced with mbIL-15 proliferated similarly to SL-K01 under IL-2 treatment conditions. Proliferation was observed while maintaining a constant PDL even in an IL-2 deficient condition (FIG. 10A). The results suggest that NK111-based cell line therapeutics may have high productivity with only a deficiency condition of IL-2, a culture supplement. In addition, expression of CD314 (NKG2D), one of the natural killer cell activation receptors, was confirmed by flow cytometry in NK101, SL-K01, and NK111 cell lines (FIG. 10B).

9-2: Cancer Cell Killing Capacity Analysis

Coculture with IM9 (human lymphoblasts) was performed to confirm cell killing ability of NK111 cells following the introduction of mbIL-15. C9-labeled IM9 cells were treated with 5-FC together with SL-K01 cells and NK111 cells, and cultured for 48 hours, and cell death was confirmed by flow cytometry. NK111 cells into which mbIL-15 was introduced increased cancer cell killing ability against target cells, and apoptosis was significantly increased in the experimental group treated with 5-FC (FIG. 10C).

9-3: Analysis of effect of inhibition of apoptosis by TGFβ1

In addition, the present inventors performed the following experiments to determine whether the TGFβ1 immunosuppressive effect, which is well known as an immunosuppressive factor present in a large amount in the patient's body, is inhibited by TGFβRIIΔcyto introduced into NK111 cells. CTV-labeled OVCAR-3 (human ovarian cancer) cells and THP-1 (acute myeloid leukemia) cells and effector cells SL-K01 and NK111 cells were prepared at a concentration of 3x10 ⁵ cells / mL and 1 mL in a 24-well plate. After each aliquot (E: T = 1: 1), 0, 0.3, 1, 3, and 10 μg / mL of TGFβ1 were treated and incubated at 5% CO ₂ , 37 ° C. for 24 hours. As a result, as can be seen in Figure 10d, SL-K01 cells decreased the cancer cell killing ability as the concentration of TGFβ1 increased, but the NK111 cells were analyzed to have no or insignificant activity inhibitory effect by TGFβ1.

As described above, SL-K01 cells according to an embodiment of the present invention significantly increased the cancer cell killing ability compared to the parental cell line NK101 while maintaining the main characteristics of the parental cell line NK101, and further enhanced the performance of the SL-K01 In addition, the NK111 cells have increased cancer cell killing ability significantly compared to SL-K01 cells, and can avoid the inhibition of TGFβ-induced cell killing ability, and thus, it is expected that the NK111 cells can be used as a very effective cancer treatment agent when administered in vivo.

Example 10 Preparation of EpCAM Targeted Chimeric Antigen Receptor NK Cell Lines

The present inventors investigated whether antigen-specific cancer cell killing ability is enhanced when NK111 prepared in Example 8 is transduced with a polynucleotide encoding a chimeric antigen receptor. To this end, the present inventors specifically prepared an EpCAM targeting chimeric antigen receptor gene construct and transduced it into NK111 to prepare NK111-EpCAM-CAR cells and evaluated their function.

10-1: Preparation of anti-EpCAM scFv-CAR Gene Construct

The present inventors used scFv and modified human immunoglobulin (Fc) domain, CD28 transmembrane domain, DAP10 intracellular activation domain, scFv targeting EpCAM to NK111 cells prepared in Example 8 to produce tumor target NK cell lines. Gene constructs comprising a DAP12 intracellular activation domain and a polynucleotide encoding the anti-EpCAM scFv-CAR protein (SEQ ID NO: 14) consisting of CD3z (SEQ ID NO: 15) were cloned into the transfer vector pWPT for lentiviral preparation. PWPT-EpCAM scFv-CAR was prepared (FIG. 11A).

10-2: Lentivirus Preparation

Then, the inventors of the present invention, Lenti-X cells cultured for 48 hours or 72 hours for lentiviral production, 12 μg of the transfer vector pWPT-EpCAM scFv-CAR prepared in Example 10-1 and 12 μg of the packaging plasmid psPAX2 and the outer skin plasmid. 2.4 μg of pMD2.G was mixed with Lipofectamine Invitrogen, USA to transduce Lenti-X 293T cells (Clontech, USA). After 6 hours of incubation at 37 ° C., the medium was removed and fresh growth medium was added. After 48 hours of incubation, the lentiviral produced in the medium was collected, centrifuged for 5 minutes (4 ° C., 4000 rpm), the supernatant was collected, and the cell debris was removed using a 0.45 μm filter (Millipore, USA). It was. To concentrate the lentiviral, Lenti-X concentrator (Clontech, USA) reagent was added to the filtered lentiviral supernatant and stored at 4 ° C. overnight. As a final step, the lentiviral-concentrator mixture was centrifuged at 4000 rpm for 60 minutes, the supernatant was removed, and the recovered lentivirus in pellet form was diluted with 1-2 mL of culture and stored at -80 ° C until use. .

10-3: NK111-EpCAM-CAR Cell Line Construction

The inventors of the anti-EpCAM scFv-CAR lentivirus prepared in Example 10-2 was infected with NK111 cells using protamine sulfate (Sigma, USA) and incubated for 4 hours at 37 ℃. After performing a total of two infection processes, the expression of the chimeric antigen receptor was confirmed by flow cytometry 72 hours after infection, and one week later, the transgene was introduced using a fluorescence activated cell sorter (BD FACSMelody, BD, USA). Only NK111 cells expressing T cells were selectively isolated. Expression of EpCAM-CAR in the isolated and propagated NK111-EpCAM-CAR cell line was confirmed by flow cytometry (FIG. 11B).

10-4: Analysis of cancer cell co-culture and cancer cell death active substance

The present inventors performed the following experiment using NK111 and NK111-EpCAM-CAR cell lines to verify target specific cancer cell killing ability of the NK111-EpCAM-CAR cell line prepared in Example 10-3. Specifically, EpCAM-positive RMG-1 cells and EpCAM-negative KOC-2S in human-derived ovarian cancer cell lines labeled with CTV (celltracker violet dye) were seeded 1 ml at a concentration of 3x10 ⁵ cells / ml in a 24-well culture dish. . The NK111 and NK111-EpCAM-CAR cell lines were then suspended in 1 ml medium at various effector cell-to-target wash ratios (E: T ratio = 1: 1, 2: 1, 4: 1) and then with the cancer cells for 24 hours. Incubated. After incubation all cells were collected and cells were harvested from each well, centrifuged and the cell pellet suspended in FACS buffer. By suspending the cell pellet formed by centrifuging again in FACS buffer of 100 ㎕ diluted ^{1 ㎕ LIVE / DEAD ® Fixable Near} -IR Dead Stain Kit (Life Technologies), was reacted at 4 ℃ 20 minutes. After washing twice with FACS buffer, the cell pellet was suspended in a solution diluted with 5 μl Annexin V APC (Biolegend, USA) in 100 μl of 1 × Annexin V binding buffer, and reacted at room temperature for 20 minutes. Cell death was determined by flow cytometry using viable cells (annexin V-negative / LIVE / DEAD-negative), early apoptotic cells (annexin V-positive / LIVE / DEAD-negative), late apoptotic cells (annexin V-positive / LIVE / DEAD-positive) and necrotic cells (annexin V-negative / LIVE / DEAD-positive). As a result, as shown in FIG. 11C, NK111-EpCAM-CAR cells showed significantly higher cell killing ability than ENK-expressing cells RMG-1, compared to the control group NK111, whereas ENKC cell lines showed equivalent cell killing ability. It was confirmed. In addition, ELISA experiments were carried out using the coculture supernatant obtained in the above experiments. As a result, co-culture with EpCAM-expressing RMG-1 cells and NK111 expressing EpCAM-CAR when co-cultured with cancer cell killing such as interferon gamma and granzyme B It was confirmed that the secretion of the active substance was high. Through the above experiments, it can be confirmed that NK111 according to one embodiment of the present invention is a cell platform suitable for enhancing antigen-specific cancer cell killing ability through the chimeric antigen receptor.

Examples of preparations for the compositions of the present invention are illustrated below.

Formulation Example 1 Preparation of Injection

Recombinant NK111 cells 1x10 ⁸ to 5x10 ¹² cells

pH adjuster

Stabilizer

Up to 100% sterile distilled water for injection

According to the conventional method for preparing an injection, the amount of the above ingredient was prepared per ampoule (2 ml).

Although the present invention has been described with reference to the above-described examples and experimental examples, these are merely exemplary, and those skilled in the art will understand that various modifications and equivalent other embodiments are possible. Therefore, the true technical protection scope of the present invention will be defined by the technical spirit of the appended claims.

Genetically modified NK cell line according to an embodiment of the present invention can be used in the production of a medicament for the treatment of cancer.

Claims (40)

1. A genetically modified NK cell line wherein the isolated NK cell line is transduced with a polynucleotide encoding a NK cell coactivator so that the NK cell coactivator is expressed:

   CD2, CD11a, CD25, CD45, CD54, DNAM-1, CD62L and CD56 are positive; And

   CD1a, CD3, CD4, CD8, CD14, CD20, CD23, CD34, TCRαβ and TCRγδ are negative.
2. The method of claim 1,

   The NK cell co-activating factor is any one or more selected from the group consisting of Ly49, natural cytotoxicity receptor (NCR), CD7, CD16 and CD28, genetically modified NK cell line.
3. The method of claim 2,

   Wherein said NK cell coactivator is CD7 and / or CD28.
4. The method of claim 1,

   A genetically modified NK cell line, wherein the polynucleotide encoding at least one or more NK cell proliferation factors is further transduced to express the NK cell proliferation factor.
5. The method of claim 4, wherein

   Wherein said NK cell proliferation factor is at least one cytokine selected from the group consisting of IL-2, IL-12, IL-15, IL-18 and IL-21 or a variant of said cytokine, genetically modified NK cell line.
6. The method of claim 5,

   Wherein said IL-15 is membrane-bound IL-15.
7. The method of claim 1,

   Genetically modified NK cell line, further transduced with apoptotic genes.
8. The method of claim 7, wherein

   The apoptosis genes include uracil phosphoribosyltransferase (UPRT) gene, herpes herpes simplex virus thymidine phosphorylation gene (HSV TK), varicella zoster virus thymidine kinase (VZV TK) gene, cytosine deminase gene, The transgenic NK cell line, which is a carboxyl esterase gene, nitroreductase gene, carboxypeptides G2 gene, or inducible caspase 9 (iCas9) gene.
9. The method of claim 1,

   A genetically modified NK cell line, wherein the polynucleotide encoding the cytoplasmic domain deleted TGFβ receptor is further transduced to express the cytoplasmic domain deleted TGFβ receptor.
10. The method of claim 9,

    Wherein said cytoplasmic domain deleted TGFβ receptor is cytoplasmic domain deleted TGFβ receptor II.
11. The method of claim 1,

    A genetically modified NK cell line in which a polynucleotide encoding a chimeric antigen receptor that specifically recognizes a cancer antigen is further transduced to express the chimeric antigen receptor.
12. The method of claim 11,

    The cancer antigen is CD19, CD22, prostate specific antigen (PSA), carcinoembryonic antigen (CEA), CA-125, mucin 1, alphafetoprotein (AFP), epipithelial tumor antigen (ETA), tyrosinase, CD52, PD-L1 ( programmed death-ligand 1), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), CD20, melanoma-associated antigen (MAGE), fibroblast activation protein (FAP), fms like tyrosine kinase 3 (FLT3), IL13Rα2 or epipithelial (EpCAM) cell adhesion molecule).
13. The method of claim 11,

    The chimeric antigen receptor is a genetically modified NK cell line, which is a fusion protein comprising a ligand or antibody analog-transmembrane domain-co-stimulatory factor-cellular signaling domain that specifically binds to a cancer antigen.
14. The method of claim 13,

    The antibody analog is scFv, sdAb, nanobody, V _H H, V _NAR , VLR, or monobody, genetically modified NK cell line.
15. The method of claim 13,

    The co-stimulatory factors are CD28, inducible costimulator (ICOS), cytotoxic T lymphocyte associated protein 4 (CTLA4), programmed cell death protein 1 (PD1), B and T lymphocyte associated protein (BTLA), death receptor 3 (DR3), 4 -1BB, CD2, CD7, CD40, CD30, CD27, signaling lymphocyte activation molecule (SLAM), 2B4 (CD244), NKp30, NKp44, NKp46, NKp80, natural-killer group 2, member D), DAP12 (DNAX- activating protein 12), DAP10, DNAM-1, NTB-A, T-Cell immunoglobulin and mucin domain containing protein 1), TIM2, TIM3, TIGIT, CD226, CD160, LAG3 (lymphocyte activation gene 3), B7-1 , B7-H1, glucocorticoid-induced TNFR family related protein (GITR), herpesvirus entry mediator (HVEM) or cytoplasmic domain of ligand for CD134 (OX40), CD252] or a combination of two or more thereof.
16. The method of claim 11,

    Wherein said intracellular signaling domain is a CD3ξ domain of a T cell receptor, CD16, NKp30, NKp44, NKp46, NKp80, DAP10, or DAP12.
17. A pharmaceutical composition for treating cancer and preventing cancer comprising the genetically modified NK cell line of any one of claims 1 to 16 as an active ingredient.
18. A cancer treatment kit comprising the genetically modified NK cell line of claim 7 or 8 and a suicide inducing agent.
19. The method of claim 18,

    The suicide inducing agent is gancyclovir or 6-methoxypurine arabinonucleoside when the suicide gene is HSV TK or VZV TK, respectively, and the suicide gene is uracil phosphorus. 5-fluorocytosine (5-FC) in case of transferase (UPRT) or cytosine deminase, irinotecan (CPT-11) in case the apoptosis gene is carboxyl esterase, and the apoptosis gene is nitroriducte 5 (aziridin-1-yl) -2,4-dinitrobenzamide (CB1954) for izudose and 4-[(2-chloroethyl) (2 if the apoptotic gene is carboxypeptides G2. -Mesyloxyethyl) amino] benzoyl-L-glutamic acid (CMDA), iCas9 dimerizer when the apoptosis gene is iCas9, cancer treatment kit.
20. A method of treating cancer in a subject, comprising administering a therapeutically effective amount of the genetically modified NK cell line of any one of claims 1 to 16 and optionally a suicide inducing agent to the subject having cancer.
21. A genetically modified NK cell line transduced with polynucleotides and apoptosis genes encoding NK cell coactivator, NK cell proliferation factor and TGFβ receptor, respectively, in an isolated NK cell line.
22. The method of claim 21,

    The isolated NK cell line has the following characteristics, genetically modified NK cell line:

    CD2, CD11a, CD25, CD45, CD54, DNAM-1, CD62L, and CD56 are positive; And

    CD1a, CD3, CD4, CD8, CD14, CD20, CD23, CD34, TCRαβ and TCRγδ are negative.
23. The method of claim 21,

    The NK cell coactivator is Ly49, natural cytotoxicity receptor (NCR), CD7, CD16 and CD28 selected from the group consisting of any one, genetically modified NK cell line.
24. The method of claim 23, wherein

    Wherein said NK cell coactivator is CD7 and / or CD28.
25. The method of claim 21,

    Wherein said NK cell proliferation factor is at least one cytokine selected from the group consisting of IL-2, IL-12, IL-15, IL-18 and IL-21 or a variant of said cytokine, genetically modified NK cell line.
26. The method of claim 21,

    The NK cell coactivator, NK cell proliferation factor, TGFβ receptor and apoptosis genes are expressed individually, simultaneously expressed in one gene construct, or divided into two or more gene constructs. .
27. The method of claim 21,

    Wherein said TGFβ receptor is cytoplasmic domain deleted TGFβ receptor II.
28. The method of claim 21,

    The apoptosis genes include uracil phosphoribosyltransferase (UPRT) gene, herpes herpes simplex virus thymidine phosphorylation gene (HSV TK), varicella zoster virus thymidine kinase (VZV TK) gene, cytosine deminase gene, The transgenic NK cell line, which is a carboxyl esterase gene, nitroreductase gene, carboxypeptides G2 gene, or inducible caspase 9 (iCas9) gene.
29. The method of claim 21,

    A genetically modified NK cell line, further transduced with a polynucleotide encoding a chimeric antigen receptor that specifically recognizes a cancer antigen.
30. The method of claim 29,

    The cancer antigen is CD19, CD22, prostate specific antigen (PSA), carcinoembryonic antigen (CEA), CA-125, mucin 1, alphafetoprotein (AFP), epipithelial tumor antigen (ETA), tyrosinase, CD52, PD-L1 ( programmed death-ligand 1), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), CD20, melanoma-associated antigen (MAGE), fibroblast activation protein (FAP), fms like tyrosine kinase 3 (FLT3), IL13Rα2 or epipithelial (EpCAM) cell adhesion molecule).
31. The method of claim 29,

    The chimeric antigen receptor is a genetically modified NK cell line, which is a fusion protein comprising a ligand or antibody analog-transmembrane domain-co-stimulatory factor-cellular signaling domain that specifically binds to a cancer antigen.
32. The method of claim 31, wherein

    The antibody analog is scFv, sdAb, nanobody, VHH, VNAR, VLR, or monobody, genetically modified NK cell line.
33. The method of claim 31, wherein

    The co-stimulatory factors are CD28, inducible costimulator (ICOS), cytotoxic T lymphocyte associated protein 4 (CTLA4), programmed cell death protein 1 (PD1), B and T lymphocyte associated protein (BTLA), death receptor 3 (DR3), 4 -1BB, CD2, CD7, CD40, CD30, CD27, signaling lymphocyte activation molecule (SLAM), 2B4 (CD244), NKp30, NKp44, NKp46, NKp80, natural-killer group 2, member D), DAP12 (DNAX- activating protein 12), DAP10, DNAM-1, NTB-A, T-Cell immunoglobulin and mucin domain containing protein 1), TIM2, TIM3, TIGIT, CD226, CD160, LAG3 (lymphocyte activation gene 3), B7-1 , B7-H1, glucocorticoid-induced TNFR family related protein (GITR), herpesvirus entry mediator (HVEM) or cytoplasmic domain of ligand for CD134 (OX40), CD252] or a combination of two or more thereof.
34. The method of claim 31, wherein

    Wherein said intracellular signaling domain is a CD3ξ domain of a T cell receptor, CD16, NKp30, NKp44, NKp46, NKp80, DAP10, or DAP12.
35. 35. A pharmaceutical composition for the treatment and prevention of cancer comprising the genetically modified NK cell line of any one of claims 21 to 34 as an active ingredient.
36. 35. A kit for treating cancer, comprising the genetically modified NK cell line of claim 21 and a suicide inducing agent.
37. The method of claim 36,

    The suicide inducing agent is gancyclovir or 6-methoxypurine arabinonucleoside when the suicide gene is HSV TK or VZV TK, respectively, and the suicide gene is uracil phosphorus. 5-fluorocytosine (5-FC) in case of transferase (UPRT) or cytosine deminase, irinotecan (CPT-11) in case the apoptosis gene is carboxyl esterase, and the apoptosis gene is nitroriducte 5 (aziridin-1-yl) -2,4-dinitrobenzamide (CB1954) for izudose and 4-[(2-chloroethyl) (2 if the apoptotic gene is carboxypeptides G2. -Mesyloxyethyl) amino] benzoyl-L-glutamic acid (CMDA), iCas9 dimerizer when the apoptosis gene is iCas9, cancer treatment kit.
38. 34. A method of treating cancer in a subject, comprising administering to the subject suffering from cancer a therapeutically effective amount of the genetically modified NK cell line of any one of claims 21 to 34 and optionally suicide.
39. The isolated NK cell line is transduced with polynucleotides and apoptosis genes encoding NK cell coactivator, membrane-bound IL-15, and TGFβ receptor, respectively, and transduced with polynucleotides encoding cancer antigen specific chimeric antigen receptor. Transgenic NK cell line expressing said cancer antigen specific chimeric antigen receptor.
40. The method of claim 39,

    The cancer antigen is CD19, CD22, prostate specific antigen (PSA), carcinoembryonic antigen (CEA), CA-125, mucin 1, alphafetoprotein (AFP), epipithelial tumor antigen (ETA), tyrosinase, CD52, PD-L1 ( programmed death-ligand 1), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), CD20, melanoma-associated antigen (MAGE), fibroblast activation protein (FAP), fms like tyrosine kinase 3 (FLT3), IL13Rα2 or epipithelial (EpCAM) cell adhesion molecule).

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