WO2023038476A1 - Procédé de production d'une cellule tueuse naturelle et son utilisation pour la prévention ou le traitement de maladies infectieuses - Google Patents

Procédé de production d'une cellule tueuse naturelle et son utilisation pour la prévention ou le traitement de maladies infectieuses Download PDF

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WO2023038476A1
WO2023038476A1 PCT/KR2022/013563 KR2022013563W WO2023038476A1 WO 2023038476 A1 WO2023038476 A1 WO 2023038476A1 KR 2022013563 W KR2022013563 W KR 2022013563W WO 2023038476 A1 WO2023038476 A1 WO 2023038476A1
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cells
virus
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spp
oskm
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Korean (ko)
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조이숙
김한섭
설빛나
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한국생명공학연구원
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4613—Natural-killer cells [NK or NK-T]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/464838—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present invention relates to a method for producing natural killer cells using direct reprogramming and a use thereof for preventing or treating infectious diseases.
  • NK (Natural killer) cells are one of the lymphoid blood cells that play an important role in innate and acquired immune responses. In particular, they recognize and immediately remove abnormal cells such as infected cells such as viruses, bacteria (bacteria), fungi, and parasites. Therefore, interest in research on the development of therapeutic agents using these functions is increasing.
  • NK cells are known to rapidly respond to pathogens and induce immune responses in the early stage of infection with various pathogens that cause the infectious diseases, that is, before the activated immune response is activated. That is, NK cells can limit viral spread and reduce inflammatory responses.
  • NK cells not only exhibit direct killing activity against bacteria and fungi, but also cytokines and interferons secreted by NK cells exhibit indirect antibacterial effects.
  • NK cells identified in viral infectious diseases such as corona virus
  • the deficiency of NK cells identified in viral infectious diseases is known to be an important cause of the patient's immunocompromised, disease severity and increase in mortality, increasing the activity and number of NK cells to treat infection Interest in developing therapies is growing rapidly.
  • NK cells both the number and high activity of NK cells are important measures of the immune effect mediated by NK cells, but technology development for securing a large amount of NK cells with high activity is still insufficient.
  • the present inventors differentiated from the conventional method of securing NK cells through an induced pluripotent stem cell reprogramming process, a new approach to secure NK cells through direct reprogramming without going through an induced pluripotent stem cell reprogramming process was developed to solve various problems in the production of NK-based therapeutics.
  • the present inventors improved the production efficiency of induced natural killer (iNK) cells from isolated cells by optimizing the direct reprogramming induction culture conditions, and the iNK cells produced through this improved the production efficiency of cells infected with viruses, bacteria, fungi, etc. It was confirmed that the present invention can be applied to the prevention or treatment of infectious and/or inflammatory diseases by exhibiting killing activity against them, thereby completing the present invention.
  • iNK induced natural killer
  • One object of the present invention is (a) introducing a reprogramming factor into the isolated cells
  • step (b) Direct reprogramming by culturing the cells in step (a) i) in a first medium containing cytokines, growth factors, and GSK3 ⁇ (Glycogen synthase kinase 3 ⁇ ) inhibitors from the next day after the introduction of the reprogramming factor and ii) promoting natural killer cell production by culturing in a second medium containing cytokines, growth factors and AHR (Aryl hydrocarbon receptor) inhibitors. It is to provide a method for preparing a composition for preventing or treating infectious diseases, including induced natural killer (iNK) cells.
  • infectious diseases including induced natural killer (iNK) cells.
  • Another object of the present invention is to provide an iNK cell prepared according to the above method.
  • Another object of the present invention is to provide a composition for preventing or treating infectious diseases, comprising iNK cells prepared according to the above method as an active ingredient.
  • Another object of the present invention is to provide a cell therapy agent for preventing or treating infectious diseases, comprising iNK cells prepared according to the above method as an active ingredient.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating infectious diseases, comprising iNK cells prepared according to the above method as an active ingredient.
  • the present invention introduces complex transcription factors into cells and constructs an induced natural killer (iNK) cell reprogramming method with improved production efficiency by using an optimized composition of cytokines, growth factors and low-molecular compounds, It was confirmed that induced natural killer (iNK) cells could be efficiently produced using this. Since the iNK cells produced through the present invention have excellent cell killing ability against cells infected with viruses, bacteria and fungi, they can be applied as cell therapy agents and compositions for preventing or treating infectious diseases.
  • iNK induced natural killer
  • Figure 1 shows the culture conditions (A) of OSKM-iNK I and OSKM-iNK II cells, which are induced natural killer (iNK) cells by direct reprogramming, cultured for different periods in the first medium and the second medium. It is a result of verifying the expression of NK-specific markers in cells cultured according to the condition (B) and FIG. 1B (C).
  • A OSKM-iNK I and OSKM-iNK II cells, which are induced natural killer (iNK) cells by direct reprogramming, cultured for different periods in the first medium and the second medium. It is a result of verifying the expression of NK-specific markers in cells cultured according to the condition (B) and FIG. 1B (C).
  • FIG. 2 is a result of confirming NK cell production using NK markers for OSKM-iNK I cells and OSKM-iNK II cells prepared according to the OSKM-iNK I culture and OSKM-iNK II culture conditions of FIG. 1A (A) and iNK cell production efficiency (B) according to OSKM-iNK I culture and OSKM-iNK II culture conditions.
  • Figure 3 shows the cell killing ability of OSKM-iNK I cells and OSKM-iNK II cells against Epstein-Barr Virus (EBV)-infected B-lymphoma Raji cells (A), CD107a + in EBV-infected Raji cells Cell frequency (B) and LMP-1 (Latent Membrane Protein 1) expression level (C) in EBV-infected Raji cells.
  • EBV Epstein-Barr Virus
  • Figure 4 shows the cell killing ability (A) and CD107a + cell frequency (B) of OSKM-iNK I cells and OSKM-iNK II cells against human immunodeficiency virus (HIV)-infected CEM T cells.
  • A cell killing ability
  • B cell frequency
  • Figure 5 shows the cell killing ability (A) and CD107a + cell frequency (B) of OSKM-iNK I cells and OSKM-iNK II cells against influenza virus-infected HEK-293T cells.
  • Figure 6 shows the cell killing ability (A) and CD107a + cell frequency (B) of HK2 proximal tubular cells infected with Papilloma virus of OSKM-iNK I cells and OSKM-iNK II cells.
  • Figure 7 shows the cell killing ability (A) and CD107a + cell frequency (B) of SNU449 liver cells infected with Hepatitis virus of OSKM-iNK I cells and OSKM-iNK II cells.
  • FIG. 9 shows the results of E. coli colony count (A) and CD107a + cell frequency (B) according to OSKM-iNK I cell and OSKM-iNK II cultures.
  • FIG. 10 is a result of analyzing antibacterial efficacy against gram-negative bacteria and gram-positive bacteria according to OSKM-iNK II cell culture.
  • FIG. 11 shows CD107a + cell frequency results according to co-culture of OSKM-iNK I cells and OSKM-iNK II cells with Candida albicans .
  • the present invention provides (a) introducing a reprogramming factor into the isolated cells; (b) Direct reprogramming by culturing the cells in step (a) i) in a first medium containing cytokines, growth factors, and GSK3 ⁇ (Glycogen synthase kinase 3 ⁇ ) inhibitors from the next day after the introduction of the reprogramming factor and ii) promoting natural killer cell production by culturing in a second medium containing cytokines, growth factors and AHR (Aryl hydrocarbon receptor) inhibitors.
  • AHR Aryl hydrocarbon receptor
  • NK cell is a key innate immune cell that immediately recognizes and removes cells infected with viruses, bacteria (bacteria), fungi and parasites, or abnormal autologous cells.
  • T cells which recognize target cells by expressing antigen-specific receptors
  • NK cells express killer immunoglobulin receptors (KIR), Balance of inhibitory and activating receptors, such as natural cytotoxicity receptors (NCR), DNAX accessory molecule-1 (DNAM-1) and NK group 2 member D (NKG2D), surface major histocompatibility complex (MHC) class ( It recognizes abnormal changes in target cells (especially infected cells), such as loss of Class I antigen, and exhibits contact-dependent cytotoxicity through various mechanisms.
  • KIR killer immunoglobulin receptors
  • NCR natural cytotoxicity receptors
  • DNAM-1 DNAX accessory molecule-1
  • NSG2D NK group 2 member D
  • MHC surface major histocompatibility complex
  • NK cells from isolated cells with a significantly improved yield of about 5 times or more was identified by optimizing the culture conditions for direct reprogramming induction (Fig. 2B), and the induced NK (iNK) cells produced thereby It was confirmed that the killing ability against the infected cells was relatively excellent (Figs. 3 to 12).
  • reprogramming refers to the production of induced pluripotent stem cells (iPSC) with pluripotency by regulating the global gene expression pattern of specific cells. Then, it means a method of transforming it into a desired cell.
  • reprogramming in the present invention refers to a method of artificially manipulating the fate of a cell to convert it into a cell having completely different characteristics. It may be performed by introducing into.
  • the reprogramming may be used interchangeably with “dedifferentiation”, “differentiation”, “conversion” or “trans-differentiation”, but is not limited thereto no.
  • direct reprogramming is differentiated from the technology of producing pluripotent induced pluripotent stem cells through a reprogramming process, and directly into a desired target cell through reprogramming culture. It is a technology that induces transformation.
  • the "direct reprogramming” may mean that "cell transformation” is performed by introducing oligonucleotides or vectors containing foreign genes or DNA into cells, and that cells change to a different state.
  • the “direct reprogramming” may mean direct dedifferentiation or direct cross-differentiation from isolated cells into NK cells.
  • direct reprogramming may be used interchangeably with “direct dedifferentiation”, “direct differentiation”, “direct conversion”, and “direct cross-differentiation”, but is not limited thereto.
  • the term “differentiation” refers to a phenomenon in which daughter cells produced by cell division acquire functions different from those of the original parent cell.
  • differentiated cells refers to a state in which cells with specialized structures or functions, that is, cells, tissues, etc. of organisms, have been changed into suitable forms and functions to perform their respective roles.
  • ectoderm, mesoderm, and endoderm cells derived from pluripotent stem cells such as embryonic stem cells are broadly differentiated cells, and narrowly, red blood cells, leukocytes, platelets, etc. derived from hematopoietic stem cells are differentiated cells. there is.
  • intermediate cell refers to cells at a stage before differentiation into NK cells, which may be lymphoid stem cells, It is not limited thereto as long as it has the possibility of differentiating into NK cells.
  • NK cells are produced through primary isolation and culture, differentiation from stem cells, or cell reprogramming for use as an immune cell therapeutic agent or the like.
  • iPSC induced pluripotent stem cell
  • 1) induced pluripotent stem cells are first prepared from isolated somatic cells, and 2) hematopoietic stem cells as a differentiation intermediate from induced pluripotent stem cells (Precursor) cells must be differentiated, and 3) additional NK cell differentiation must be induced.
  • iPSC induced pluripotent stem cell
  • the present invention directly produces NK cells from cells isolated through direct reprogramming induction without going through induced pluripotent stem cells, so that the manufacturing time is short and cost is reduced, and safety is secured. It is differentiated from technology and can provide an alternative to overcome the problems of existing NK cell sources.
  • iNK cells induced natural killer cells
  • the iNK cells of the present invention are prepared by (a) introducing a reprogramming factor into the isolated cells, and (b) culturing the cells in step (a) in the medium of the present invention from the next day after introducing the reprogramming factor. It may be prepared by directly increasing the efficiency of reprogramming and promoting NK cell production.
  • step (a) includes introducing one or more reprogramming factors into the isolated cells.
  • isolated cell is not particularly limited, but, for example, a differentiated cell whose lineage has already been specified, such as a germ cell, a somatic cell, or a progenitor cell other than NK cells. can be
  • the "somatic cell” refers to all cells that have completed differentiation constituting animals and plants, excluding reproductive cells.
  • the "progenitor cell” refers to a parental cell that does not express a differentiation trait, but has a differentiation fate (Fate) when a cell corresponding to a progeny is found to express a specific differentiation trait.
  • neuroblasts neuro stem cells
  • nerve cells nerve cells
  • myoblasts correspond to precursor cells for myotube cells.
  • the isolated cells may be cells derived from humans, but are not limited thereto, and cells derived from various organisms may also fall within the scope of the present invention.
  • the isolated cells of the present invention may include both in vivo and ex vivo cells.
  • the isolated cells may be somatic cells, and in another example, they may be somatic cells other than NK cells.
  • the isolated cells may be blood cells and fibroblasts, but are not limited thereto.
  • reprogramming factor refers to a gene (or a polynucleotide encoding the same) or a protein capable of inducing reprogramming by being introduced into a cell.
  • the reprogramming factor may vary depending on the target cell for which reprogramming is to be induced and the type of isolated cell in which reprogramming is induced.
  • the reprogramming factor may be ASC11 , KLF4 , LIN28, LMX1A, MYC, NANOG , NURR1 , OCT3 , OCT4 , PITX3 , and SOX2 , and the like, for example, OCT4 , SOX2 , KLF4 And it may include one or more factors selected from the group consisting of MYC , and in addition, may include all factors known in the art to be able to produce NK cells.
  • a person skilled in the art may select an appropriate factor depending on the type of the target cell and the cell before reprogramming, and all of these factors are included within the scope of the present invention as long as they are within the range known in the art, and are not particularly limited to the type. Since reprogramming using reprogramming genetic factors induces conversion into a target cell by regulating the entire gene expression pattern of a cell, the reprogramming genetic factor is introduced into the cell and the cell is cultured for a certain period of time to obtain the desired type. The initial cell can be reprogrammed into a target cell having the cell's gene expression pattern.
  • introduction of a reprogramming factor refers to a method of increasing the expression level of a reprogramming factor such as OCT4 , SOX2 , KLF4 and/or MYC gene present in a cell; Alternatively, it may be a method of increasing the expression level of a reprogramming factor in a cell through an expression vector, gene modification, introduction of an exogenous expression gene, treatment of a substance having an expression inducing effect, etc., but is limited as long as the expression level of the reprogramming factor is increased. It doesn't work. In particular, introducing a reprogramming factor may be a method of inducing expression of a reprogramming factor under a desired time and condition.
  • a method of introducing the reprogramming factor of step (a) into cells methods of introducing nucleic acid molecules (DNA or RNA) or proteins into cells commonly used in the art may be used without limitation.
  • a method of administering a reprogramming factor to a culture medium of a cell a method of directly injecting a reprogramming factor into a cell, or a method of transforming an expression vector containing a gene of a reprogramming factor into a cell may be used.
  • a method of directly injecting or transforming the reprogramming factor into cells may be selected and used by any method known in the art, but is not limited thereto, such as microinjection, electroporation, It can be appropriately selected and applied from among particle bombardment, direct muscle injection, insulator, and transposon methods.
  • expression vector of the present invention, as a vector capable of expressing a target protein in a suitable host cell, refers to a genetic construct comprising essential regulatory elements operably linked to express a gene insert.
  • the expression vector of the present invention contains signal sequences or leader sequences for membrane targeting or secretion in addition to expression control elements such as promoters, operators, initiation codons, stop codons, polyadenylation signals, and enhancers, and can be prepared in various ways depending on the purpose. .
  • the promoter of the expression vector may be constitutive or inducible.
  • the expression vector may include a selectable marker for selecting a host cell containing the vector, and may include an origin of replication in the case of a replicable expression vector. Expression vectors can replicate autonomously or integrate into host DNA.
  • the expression vector may include a viral vector, an episomal vector, a plasmid vector, and a cosmid vector, but is not limited thereto.
  • the viral vector is a Sendai virus, a lentivirus, a retrovirus such as HIV (Human immunodeficiency virus), MLV (Murineleukemia virus), ASLV (Avian sarcoma / Leukosis), Derived from Spleen necrosis virus (SNV), Rous sarcoma virus (RSV), Mouse mammary tumor virus (MMTV), Adenovirus, Adeno-associated virus, Herpes simplex virus, etc. It can contain one vector. In addition, it may be more specifically an RNA-based viral vector, but is not limited thereto.
  • the episomal vector of the present invention is a non-viral, non-insertable vector, and is known to have a characteristic capable of expressing a gene included in the vector without being inserted into a chromosome.
  • Cells containing an episomal vector for the purpose of the present invention may include both cases in which the episomal vector is inserted into the genome or present in the cell in a state where the episomal vector is not inserted into the genome.
  • an episomal vector may contain one or more reprogramming factors.
  • operably linked refers to functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein of interest so as to perform general functions.
  • Operational linkage with a recombinant vector can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and linkage can be performed using enzymes generally known in the art.
  • step (b) increases the efficiency of direct reprogramming by culturing the cells of step (a) in the medium of the present invention from the next day after the introduction of the reprogramming factor, and produces NK cells It may be produced by promoting.
  • the term “cultivation” refers to growing cells in appropriately controlled environmental conditions, and the culturing process of the present invention may be performed according to suitable media and culture conditions known in the art. This culture process can be easily adjusted and used by those skilled in the art according to the selected cells.
  • medium refers to a known medium used for culturing cells, and includes all known mediums for cell culture or modified mediums thereof.
  • the medium of the present invention directly reprograms cells into which reprogramming factors have been introduced into natural killer cells by increasing the efficiency of direct reprogramming or promoting NK cell production, or directly increasing the efficiency of reprogramming. It may have a composition suitable for increasing.
  • the medium of the present invention can be divided into a first medium and a second medium.
  • the first medium of the present invention may contain cytokines, growth factors, and GSK3 ⁇ inhibitors.
  • the second medium of the present invention may contain cytokines, growth factors, and AHR inhibitors.
  • the medium of the present invention may further include a third medium.
  • the third medium of the present invention may contain cytokines and growth factors.
  • cytokine is a variety of relatively small-sized proteins produced in cells and used for cell signal transduction, which can affect other cells including themselves. It is generally known to be related to the immune response to inflammation or infection.
  • the cytokine is, for example, IL (Interleukin) -2, IL-3, IL-5, IL-6, IL-7, IL-11, IL-15, IL-21, BMP4 (Bone morphogenetic protein 4) , Activin A (Acivin A), Notch ligand (Notch ligand), G-CSF (Granulocyte-colony stimulating factor) and SDF-1 (Stromal cell-derived factor-1), etc., specifically IL-2, IL -3, IL-6, IL-7 and IL-15, etc., but are not limited thereto.
  • growth factor refers to a polypeptide that promotes division, growth, and differentiation of various cells.
  • the growth factors include, for example, EGF (Epidermal growth factor), PDGF-AA (Platelet-derived growth factor-AA), IGF-1 (Insulin-like growth factor 1), TGF- ⁇ (Transforming growth factor- ⁇ ) , Fibroblast growth factor (FGF), stem cell factor (SCF), FMS-like tyrosine kinase ligand (FLT3L), and the like, and may specifically be SCF and FLT3L, but are not limited thereto.
  • EGF Epidermatitis
  • PDGF-AA Platinum-derived growth factor-AA
  • IGF-1 Insulin-like growth factor 1
  • TGF- ⁇ Transforming growth factor- ⁇
  • FGF Fibroblast growth factor
  • SCF stem cell factor
  • FLT3L FMS-like tyrosine kinase ligand
  • the cytokines and growth factors are included in a medium for directly inducing cell conversion of isolated cells into target cells, and the types of growth factors and cytokines are particularly limited as long as they can be used for direct cell conversion induction. It doesn't work.
  • GSK3 ⁇ inhibitor refers to a substance that inhibits or inhibits the activity of GSK3 ⁇ by directly/indirectly binding to a protein.
  • the GSK3 ⁇ inhibitors are, for example, 1-Azakenpaullone, 2-D08, 3F8, 5-Bromoindole, 6-Bio, A 1070722, Aloisine A, AR-A014418, Alsterpaullone, AZD-1080, AZD2858, Bikinin, BIO, BIO- acetoxime, Bisindolylmaleimide I, Bisindolylmaleimide I hydrochloride, CAS 556813-39-9, Cazpaullone, CHIR98014, CHIR98023, CHIR99021(CT99021), CP21R7, Dibromocantherelline, GSK-3 ⁇ inhibitor I, VI, VII, X, XI,XV, GSK-3 inhibitors IX
  • AHR Aryl hydrocarbon receptor inhibitor
  • TCDD Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin)
  • AHR A substance that downregulates or reduces activity.
  • the AHR antagonist may be, for example, StemRegenin I [StemRegenin I, SRI; 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenolhydrochloride, 4-(2-((2- Benzo[b]thiphen-3-yl)-9-isopropyl-9H-purin-6-yl)amino)ethyl)phenol hydrochloride], CH-223191(1-methyl-N-[2-methyl-4-[2 -(2-methylphenyl)diazenyl]phenyl-1H-pyrazole-5-carboxamide, 1-Methyl-N-[2-methyl-4-[2-(2-methylphenyl)diazenyl]phenyl-1H-pyrazole -5-carboxamide) and the like, and specifically, stemregenin I and CH-223191, etc., but are not limited thereto.
  • the inhibitor is not limited to the foregoing as long as it directly serves to increase reprogramming efficiency.
  • the first medium of the present invention may include, for example, one or more selected from the group consisting of CHIR99021, IL-3, IL-6, and combinations thereof.
  • the first medium may further include at least one selected from the group consisting of SCF, FLT3L, fetal bovine serum (FBS), antibiotics, and combinations thereof.
  • the antibiotic may be penicillin/streptomycin, but is not limited thereto.
  • the first medium may include StemSpan SFEM II containing FBS, penicillin/streptomycin, SCF, FLT3L, IL-3, IL-6, and CHIR99021 (CT99021), but is not limited thereto. More specifically, the first medium contains 8 to 12% of FBS, 0.1 to 2% of penicillin/streptomycin, 50 to 300 ng/ml of human SCF, 50 to 300 ng/ml of human FLT3L, and 5 to 50 ng/ml of human SCF.
  • the second medium of the present invention may contain, for example, at least one selected from the group consisting of stemregenin I, CH-223191, IL-2, IL-7, IL-15, and combinations thereof.
  • the second medium may further include at least one selected from the group consisting of SCF, FLT3L, FBS, antibiotics, and combinations thereof.
  • the antibiotic may be penicillin/streptomycin, but is not limited thereto.
  • the second medium may include FBS, penicillin/streptomycin, SCF, FLT3L, IL-2, IL-7, IL-15, and stemregenin I, but is not limited thereto. More specifically, the second medium contains 8 to 12% FBS, 0.1 to 2% penicillin/streptomycin, 10 to 30 ng/ml human SCF, 10 to 30 ng/ml human FLT3L, 150 to 250 IU/ml.
  • ml of human IL-2 10 to 30 ng/ml of human IL-7, 10 to 30 ng/ml of human IL-15, and 0.5 to 5 uM of Stemregenin I; Even more specifically, 9-11% FBS, 0.5-1.5% penicillin/streptomycin, 10-30 ng/ml human SCF, 10-30 ng/ml human FLT3L, 180-220 IU/ml human StemSpan SFEM II comprising IL-2, 10 to 30 ng/ml of human IL-7, 10 to 30 ng/ml of human IL-15, and 1 to 3 uM of stemregenin I, but is not limited thereto. don't
  • the third medium of the present invention may include, for example, at least one selected from the group consisting of IL-2, IL-15, and combinations thereof.
  • the third medium may further include at least one selected from the group consisting of FBS, antibiotics, and combinations thereof.
  • the antibiotic may be penicillin/streptomycin, but is not limited thereto.
  • the third medium may include FBS, penicillin/streptomycin, IL-2, and IL-15, but is not limited thereto. More specifically, the third medium comprises 8-12% FBS, 0.1-2% penicillin/streptomycin, 150-250 IU/ml human IL-2 and 10-30 ng/ml human IL-15.
  • the isolated cells introduced with the reprogramming factor may be cultured in the first medium of the present invention for 3 to 10 days and cultured in the second medium for 12 to 16 days.
  • the second medium after culturing in the second medium, it may be further cultured in the third medium for 6 days or more, but is not limited thereto.
  • NK cells prepared according to the method of the present invention may be used interchangeably with “induced natural killer cells”, “iNK” or “OSKM-iNK”.
  • Another aspect of the present invention provides an iNK cell prepared according to the method of the present invention.
  • the iNK cells produced by the present invention may express at least one selected from the group consisting of CD56 + , CD3 - and combinations thereof, but are not limited thereto.
  • the OSKM-iNK I medium condition sequentially cultured in the first medium and the second medium
  • the OSKM-iNK II medium condition sequentially cultured in the first medium, second medium, and third medium
  • the culture period in the first medium, the second medium, and the third medium and each medium of the present invention was optimized for iNK production.
  • Another aspect of the present invention provides a cell therapy agent for preventing or treating infectious and/or inflammatory diseases, including iNK cells prepared according to the method of the present invention.
  • composition of the present invention may be effective in preventing or treating infectious diseases and/or inflammatory diseases caused by the infectious diseases by including the iNK cells prepared according to the method of the present invention.
  • the infectious disease may be caused or caused by any one or more selected from the group consisting of viruses, bacteria and fungi, but is not limited thereto.
  • the virus causing the infectious disease may be an RNA virus and/or a DNA virus, but is not limited as long as it is a virus known in the art.
  • the viruses include Aviadeno virus, Alphatorque virus, Arena virus, Alphapapilloma virus, Adeno virus (Ad5), Astro virus, Aichi ( Aichi) virus, Amapari virus, Aravan virus, Aura virus, Australian bat lyssa virus, Banna virus, Barmah forest virus, Batken ( Batken) virus, Bunyamwera virus, Bunga virus, Bunya virus La crosse, BK virus, BK polyoma virus, Cercopithecine herpes virus, Cardio (Cardio) virus, Crimean-congo hemorrhagic fever virus, Chapare virus, Chandipura virus, Chandipura vesiculo virus, Chikungunya virus, Cosa virus, Cowpox virus, Coxsackie virus, Corona virus, Corona virus alpha (beta, gamma, delta), Colti virus, Cytomegalo (cytomegalo) virus, Denso virus, Dependo virus, Dependoparvo virus,
  • the virus is Epstein-Barr (Epstein-Barr) virus (EBV), hepatitis virus, human immunodeficiency (human immunodeficiency) virus (HIV), Influenza (influenza) virus, Papilloma (papilloma) virus, SARS (Severe acute respiratory tract infection) syndrome) (SARS) virus, SARS corona virus, SARS-CoV-2 virus, etc., but is not limited thereto.
  • Epstein-Barr Epstein-Barr
  • HAV human immunodeficiency virus
  • Influenza influenza virus
  • Papilloma papilloma
  • SARS severe acute respiratory tract infection
  • SARS-CoV-2 virus etc.
  • the bacteria causing the infectious disease may be gram-negative bacteria and/or gram-positive bacteria, but are not limited to bacteria known in the art.
  • the bacteria are Achromobacter species (SPP), Acinetobacter spp, Actinomyces spp, Aeromonas spp, Alternaria spp, Anthrax ( Anthrax) spp, Aspergillus spp, Bacillus spp, Bacteroides spp, Bartonella spp, Brucella spp, Borrelia, spp, Bordetella spp, Burkholderia spp, Campylobacter spp, Capnocytophaga spp, Chlamydophila spp, Chlamydia spp, Citrobacter spp, Clostridium spp, Corynebacterium spp, Coxiella spp, Diphtheria spp, Ehrlichia spp, Escherichia Enterobacter spp, Enterococcus spp, Erysipelothrix spp, Eikenella spp, Erwini
  • the bacteria are Achromobacter xylosoxidans, Acinetobacter baumannii, Acinetobacter haemolyticus, Acinetobacter junil, Acinetobacter johnsonil, Actinomyces israeli, Aeromonas hydrophilia, Aeromonas veronol, Aspergillus fumigatus, Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, Bacteroides fragilis, Bacteroides melaninogenicii, , Brucella abortus, Brucella canis, Brucella melitensis, Brucella microti, Brucella suis, Borrelia afzelii, Borrelia burgdorferi, Borrelia garinii, Borrelia recurrentis, Bordetella pertussis, Burkholderia cepacia, Burkholderia mimosarum, Burkholderia thailandensis, Campylobacter jejuni
  • the bacteria may be bacteria belonging to the genera Escherichia , Klebsiella , Burkholderia , Streptococcus , etc. More specifically, E. coli , K. pneumoniae , B. cepacia, S. pseudopneumoniae , etc., but is not limited thereto.
  • the fungus causing the infectious disease is, for example, Absidia (genus Bacteria) spp, Alternaria (genus Alternaria) spp, Aspergillus (genus Aspergillus) spp, Ascosphaera (Ascospera) spp, Ajellomyces (Ajellomyces) spp , Alternaria spp, Basidiobolus spp, Basidiomycete spp, Bipolaris spp, Blastomyces spp, Batrachochytrium spp, Beauveria Genus) spp, Bjerkandera spp, Botrytis spp, Blumeria spp, Candida spp, Coprinus spp, Chromoblastomycosis Cis) spp, Cladosporium spp, Cladophialphora spp, Chaeotomium spp, Conidiobolus spp.
  • Coccidioides spp Colletotrichum spp, Cordyceps spp, Cryptococcus spp, Cunninghamella spp, Curvularia spp, Dactylaria spp, Dacrymyces spp, Epidermophyton spp, Exophiala spp, Fusarium spp, Geotrichum spp, Geomyces (Geomyces) spp, Histoplasma spp, Lacazia spp, Lasiodiplodia spp, Leptosphaeria spp, Lomentospora spp, Malassezia Cecia spp, Madurella spp, Malassezia spp, Magnaporthe spp, Metarhizium spp, Microsporum spp, Mycosphaerella spp, Memnoniella spp, Melampsora spp, Mucor spp, Mucorales spp, Mucormycetes
  • the fungus is Absidia corymbifera, Alternaria alternate, Aspergillus alternate, Aspergillus versicolor, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Ajellomyces dermatitidis, Basidiobolus ranarum, Bipolaris spicifera, Blastomyces dermatitidis, Blastomyces gilchristii, Batrachochytrium dendrobatidis, Beauvetiscinea basseriana , Blumeria graminis, Candida albicans, Candida auris, Candida glabrata, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida krusei, Candida silvativa, Candida tropicalis, Coprinus cinereus, Cladosporium herbarum, Cladosporium immitis, Cladophialphora bantianum, Conidiobolus coronatus, Conidiobolus in
  • the fungus is Aspergillus (Aspergillus mold), Candida (Candida) , Absidia (hair fungus), Mucor (hair mold), Rhizopus (spider web mold) It may be a fungus belonging to the genus, etc., and more specifically, Candida albicans, Aspergillus fumigatus, Aspergillus fumigatus , Absidia corymbifera , Mucor circillenoides, Mucor mucedo ( Mucor mucedo ), Mucor pusillus ( Mucor pusillus ), Rhizopus oryzae ( Rhizopus oryzae ), etc., but are not limited thereto.
  • prevention of the present invention refers to any action that suppresses or delays the occurrence of an infectious disease and/or an inflammatory disease caused by the infectious disease by administration of the composition.
  • treatment refers to any activity that improves or beneficially changes symptoms caused by an infectious disease and/or an inflammatory disease caused by the infectious disease by administration of the composition.
  • cell therapy product refers to cells and tissues manufactured through isolation, culture, and special manipulation from a subject, and is a drug (US FDA regulation) used for the purpose of treatment, diagnosis, and prevention, It refers to medicines used for the purpose of treatment, diagnosis, and prevention through a series of actions, such as proliferating and selecting living autologous, allogeneic, or heterogeneous cells in vitro or changing the biological characteristics of cells in other ways to restore them.
  • the cell therapy composition may be effective in preventing or treating infectious diseases and/or inflammatory diseases caused by the infectious diseases by including the iNK cells prepared according to the method of the present invention.
  • the cell therapy composition may contain the iNK cells at 1.0x10 to 1.0x10 10 cells/ml, specifically 1.0x10 6 to 1.0x10 9 cells/ml, based on the total weight of the composition, but is not limited thereto.
  • the cell therapy composition may be formulated and administered as a unit dosage pharmaceutical preparation suitable for administration into the body of a patient according to a conventional method in the pharmaceutical field, and the preparation includes an effective dosage by one or several administrations. do.
  • Formulations suitable for this purpose may include injections such as ampoules for injection, injections such as infusion bags, and sprays such as aerosol formulations as preparations for parenteral administration.
  • the ampoule for injection may be mixed with an injection solution immediately before use, and physiological saline, glucose, mannitol, Ringer's solution, or the like may be used as the injection solution.
  • the infusion bag may be made of polyvinyl chloride or polyethylene, and may be manufactured by Baxter, Becton Dickinson, Medcep, National hospital products, or Terumo. An infusion bag of yarn can be exemplified.
  • one or more pharmaceutically acceptable conventional inert carriers for example, a preservative, pain reliever, solubilizer or stabilizer in the case of injection, and the like, in the case of formulation for topical administration
  • a base an excipient, a lubricant or a preservative may be further included.
  • the cell therapy composition of the present invention thus prepared or a pharmaceutical preparation thereof is combined with other cells used for the treatment of an infectious disease and/or an inflammatory disease caused by the infectious disease using an administration method commonly used in the art, or such It may be administered in the form of a mixture with cells, and specifically, it is possible to engraft or transplant directly to the diseased area of a patient in need of treatment, or directly transplant or inject into the abdominal cavity, but is not limited thereto.
  • the administration can be carried out by both non-surgical administration using a catheter and surgical administration methods such as injection or transplantation after incision in the diseased area.
  • parenteral for example, direct administration to a lesion according to conventional methods, transplantation by intravascular injection is also possible.
  • the cell therapy composition may be administered at a dose of 0.0001 to 1,000 mg/kg per day, specifically 0.01 to 100 mg/kg, and the administration may be administered once a day or divided into several times.
  • the actual dosage of the active ingredient should be determined in light of various related factors such as the disease to be treated, the severity of the disease, the route of administration, the weight, age and sex of the patient, and therefore, the dosage is It does not limit the scope of the present invention in any way.
  • iNK cells prepared according to the method of the present invention can exhibit antiviral effects against various viruses.
  • the antiviral effect of the OSKM-iNK I and OSKM-iNK II cells which are the iNK cells of the present invention, on virus-uninfected B-lymphoma Ramos and EBV-infected B-lymphoma Raji cells was confirmed.
  • both OSKM-iNK I cells and OSKM-iNK II cells were superior in virus-infected cell killing ability compared to PBMC-NK cells, and it was confirmed that they had relatively higher toxicity to Raji infected with EBV virus than non-infected Ramos (Fig. 3A ).
  • OSKM-iNK I cells and OSKM-iNK II cells significantly increased the frequency of CD107a + cells compared to PBMC-NK cells (Fig. 3B), and it was confirmed that the degree of decrease in LMP-1 expression was higher than that of PBMC-NK cells ( Figure 3C).
  • OSKM-iNK I and OSKM-iNK II cells are CEM T cells infected with human immunodeficiency virus (HIV), HEK-293T cells infected with Influenza virus, and infected with Papilloma virus.
  • HAV human immunodeficiency virus
  • HEK-293T cells infected with Influenza virus
  • Papilloma virus a virus that has been modified by human immunodeficiency virus
  • Papilloma virus for HK2 proximal tubular cells and SNU449 liver cells infected with Hepatitis virus, compared to PBMC-NK cells, high killing capacity even at a low E (Effector NK cell): T (Target cell) ratio (FIGS. 4A, 5A, 6A and FIGS. 7A) and high CD107a + expression frequency (FIGS. 4B, 5B, 6B and 7B).
  • iNK cells, OSKM-iNK I cells and OSKM-iNK II cells exhibited remarkable antibacterial efficacy against RNA viruses and DNA viruses compared to PBMC-NK cells and NK-92 cells.
  • iNK cells prepared according to the method of the present invention can exhibit antibacterial effects against various bacteria.
  • the killing effect of OSKM-iNK I cells and OSKM-iNK II cells on the gram-negative bacterium Escherichia coli after co-culture was measured by the number of E. coli populations (FIG. 9A) and the frequency of CD107a + expressing cells (FIG. 9A). 9B), it was confirmed that OSKM-iNK I cells and OSKM-iNK II cells had higher antibacterial efficacy than PBMC-NK cells.
  • co-culture with OSKM-iNK II cells is Gram-positive bacteria compared to positive control (PMBC-NK cells)
  • PMBC-NK cells positive control
  • iNK cells, OSKM-iNK I cells and OSKM-iNK II cells exhibited remarkable antibacterial efficacy against Gram-negative bacteria and Gram-positive bacteria compared to PBMC-NK cells and NK-92 cells.
  • iNK cells prepared according to the method of the present invention can exhibit antifungal effects against various fungi.
  • the antifungal effect of OSKM-iNK I cells and OSKM-iNK II cells against Candida Albicans , a type of Candida fungus, after co-cultivation of CD107a + expressing cell frequency (FIG. 11)
  • OSKM-iNK I cells and OSKM-iNK II cells had higher antifungal efficacy than PBMC-NK cells.
  • Another aspect of the present invention provides a pharmaceutical composition for preventing or treating infectious and/or inflammatory diseases, comprising the iNK cells prepared by the method of the present invention as an active ingredient.
  • composition of the present invention may be effective in preventing or treating infectious diseases and/or inflammatory diseases caused by the infectious diseases by including the iNK cells prepared according to the method of the present invention.
  • the pharmaceutical composition of the present invention may contain the iNK cells at 1.0x10 4 to 1.0x10 10 cells/ml, specifically 1.0x10 6 to 1.0x10 9 cells/ml, based on the total weight of the composition, but is not limited thereto. .
  • the pharmaceutical composition may further include a pharmaceutically acceptable carrier, excipient or diluent commonly used in the preparation of pharmaceutical compositions, and the carrier may include a non-naturally occurring carrier.
  • the carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • the pharmaceutical composition may be formulated according to conventional methods such as tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and transdermal formulations.
  • Absorbents, gels, lotions, ointments, creams, patches, cataplasma agents, pastes, sprays, skin emulsions, skin suspensions, transdermal delivery patches, drug-containing bandages, or suppositories may be formulated and used.
  • Solid dosage forms for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like.
  • Such a solid preparation may be prepared by mixing at least one or more excipients, for example, starch, calcium carbonate, sucrose, lactose, gelatin, and the like.
  • lubricants such as magnesium stearate and talc may also be used in addition to simple excipients.
  • Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations and suppositories.
  • Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents.
  • Witepsol, Macrogol, Tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used as a base for the suppository.
  • the pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is dependent on the type and severity of the subject, age, sex, drug activity, It may be determined according to factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field.
  • the pharmaceutical composition may be administered at a dose of 0.0001 to 1000 mg/kg per day, specifically 0.001 to 100 mg/kg, and the administration may be administered once a day or divided into several times.
  • the pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be single or multiple administrations. It is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects in consideration of all the above factors, and can be easily determined by those skilled in the art.
  • the "administration” means introducing the composition of the present invention to a subject by any suitable method, and the administration route of the composition may be administered through any general route as long as it can reach the target tissue.
  • Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration may be administered, but is not limited thereto.
  • the "individual” refers to a human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or guinea pig that has or may develop an infectious and/or inflammatory disease. all animals, including The type of subject may be included without limitation as long as the disease can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to the subject.
  • Another aspect of the present invention provides a method for treating an infectious disease and/or an inflammatory disease, comprising administering the cell therapy composition or pharmaceutical composition to a non-human subject.
  • a nucleic acid molecule DNA or RNA
  • the kit of the present invention includes a) a first container containing a nucleic acid molecule (DNA or RNA) encoding a reprogramming factor, a vector or protein containing the same, b) a second container containing the first medium of the present invention, and c) means a tool that can be used as a direct reprogramming medium for producing iNK cells, including a third container containing the second medium of the present invention.
  • the type of the kit is not particularly limited, and kits of a type commonly used in the art may be used.
  • the kit of the present invention may further include d) a fourth container containing the third medium of the present invention.
  • the kit of the present invention includes i) a nucleic acid molecule (DNA or RNA) encoding a reprogramming factor, a vector or protein containing the same; 1st medium; and a second medium or ii) a nucleic acid molecule (DNA or RNA) encoding a reprogramming factor, a vector or protein containing the same; 1st medium; second medium; And the third medium may be packaged in a form contained in an individual container or in a form contained in a container divided into one or more compartments, wherein i) a nucleic acid molecule (DNA or RNA) encoding a reprogramming factor, vector or protein containing; 1st medium; and a second medium or ii) a nucleic acid molecule (DNA or RNA) encoding a reprogramming factor, a vector or protein containing the same; 1st medium; second medium; And the third medium may be packaged in a unit dose form of a single administration dose.
  • nucleic acid molecule (DNA or RNA) encoding the reprogramming factor in the kit, a vector or protein containing the same; 1st medium; and a second medium or ii) a nucleic acid molecule (DNA or RNA) encoding a reprogramming factor, a vector or protein containing the same; 1st medium; second medium; And the third medium can be administered sequentially at an appropriate time according to the experimental plan of those skilled in the art.
  • the kit of the present invention includes i) a nucleic acid molecule (DNA or RNA) encoding a reprogramming factor, a vector or protein containing the same; 1st medium; and a second medium or ii) a nucleic acid molecule (DNA or RNA) encoding a reprogramming factor, a vector or protein containing the same; 1st medium; second medium; And it may further include an instruction manual describing each addition amount, addition method and frequency of addition of the third medium.
  • Example 1 Preparation of iNK (induced natural killer) cells through reprogramming culture and expression verification of NK (Natural killer) specific markers
  • PBMC peripheral blood mononuclear cells
  • 20 ng/ml human IL(Interleukin)-3, 20 ng/ml human IL-6, 100 ng/ml human SCF(Stem cell factor), 100 ng/ml human FLT3L(FMS-like tyrosine kinase ligand) Stempro SFEM II) was cultured for 4 days, exchanging the medium once every 2 days.
  • Sendai virus system expressing reprogramming factors [ OCT4 , SOX2 , KLF4 and MYC expressing RNA-based Sendai virus (CytoTune 2.0 Sendai reprogramming kit, Thermo Scientific); OSKM-SeV] was used.
  • the PBMC cells were cultured for 1 day in a standard culture medium (SCM medium) containing the Sendai virus (5 MOI), PBMC cells and polybrene (4 ⁇ g/ml) to sendai virus expressing reprogramming factors. After transformation, culture was performed while sequentially replacing the first medium, the second medium, or the third medium from the next day.
  • the transformed cells (2x10 5 cells/48-well plate) were placed in the 1st medium (10% FBS, 1% StemSpan SFEM II with penicillin/streptomycin, 100 ng/ml human SCF, 100 ng/ml human FLT3L, 20 ng/ml human IL-3, 20 ng/ml human IL-6, 5 uM CHIR99021 (CT99021)
  • the second medium (10% FBS, 1% penicillin / streptomycin, 20 ng / ml human SCF, 20 ng / ml human 14 in StemSpan SFEM II with FLT3L, 200 IU/ml human IL-2, 20 ng/ml human IL-7, 20 ng/ml human IL-15, 2 uM StemRegenin I (SR1) NK cells (OSKM-iNK I cells) were induced by culturing for ⁇ 16
  • the transformed cells (2x10 5 cells/48-well plate) were cultured in the first medium for 3 to 6 days and then in the second medium for 12 to 15 days. After culturing, NK cells were cultured for more than 6 days in a third medium (RPMI 1640 containing 10% FBS, 1% penicillin/streptomycin, 200 IU/ml human IL-2, and 20 ng/ml human IL-15) (OSKM-iNK II cells).
  • a third medium RPMI 1640 containing 10% FBS, 1% penicillin/streptomycin, 200 IU/ml human IL-2, and 20 ng/ml human IL-15
  • OSKM-iNK I cells or OSKM-iNK II cells of Example 1-1 were stained with CD56 antibody and CD3 antibody, and the NK cell group was analyzed using flow cytometry. (CD56 + and CD3 - ) were analyzed.
  • NK cells CD56 + CD3 -
  • NK cells were present at a ratio of 97% or more in both OSKM-iNK I and OSKM-iNK II conditions, and NK cells were highly efficient in OSKM-iNK I and OSKM-iNK II conditions. It was confirmed that it was prepared as (Fig. 2A).
  • the average NK cell yield was about 5 times higher than that of the OSKM-iNK I condition (FIG. 2B), and the NK cell yield was about 5 times higher in the OSKM-iNK II condition. It was confirmed that it can be produced with a significantly improved yield.
  • Example 2 Effect of media components on iNK cell production yield
  • Example 1B After transforming and introducing Sendai virus expressing reprogramming factors ( OCT4 , SOX2 , KLF4 and MYC ) into PBMCs in the same manner as in Example 1-1, as shown in FIG. 1B, the first medium and the second cultured in the medium for different periods of time. The third medium was used in the culture after day 18 (D18).
  • Sendai virus expressing reprogramming factors OCT4 , SOX2 , KLF4 and MYC
  • the culture conditions are as follows.
  • NK cell groups CD56 + and CD3 - .
  • NK cells CD56 + CD3 -
  • II conditions 54%
  • III conditions 65%
  • IV conditions 66%
  • V conditions 30%)
  • the first medium It was confirmed that the yield of NK cells increased when the culture time in the second medium was longer than the culture time in the culture medium (FIG. 1C).
  • the proportion of NK cells was hardly confirmed.
  • iNK cells prepared in Example 1-1 virus-infected Ramos (Human B-lymphoma) and Epstein-Barr virus (Epstein- The cell killing ability (cytotoxicity) of Raji infected with Barr virus (EBV) was measured.
  • PBMC-NK cells were used as a positive control.
  • PBMC-NK cells a positive control group, were isolated from PBMC cells using an NK isolataion kit.
  • the PBMC-NK cells were cultured for 2 days in NK medium (RPMI1640 containing 1% penicillin/streptomycin, 200 IU/ml human IL-2 and 20 ng/ml human IL-15) after isolation from PBMC cells, and then used
  • NK medium RPMI1640 containing 1% penicillin/streptomycin, 200 IU/ml human IL-2 and 20 ng/ml human IL-15
  • calcein-AM was added to a final concentration of 25 ⁇ M, and at 37° C. After culturing for 1 hour, washed with DMEM medium to prepare calcein-labeled target cells.
  • the minimum value is a measured value of a well containing only calcein-labeled target cells
  • the maximum value is a measured value of a well in which cells were completely lysed by adding 1.0% TritonX-100 to calcein-labeled target cells.
  • OSKM-iNK I cells and OSKM-iNK II cells had superior ability to kill virus-infected cells compared to PBMC-NK cells, and it was confirmed that they had relatively higher toxicity to Raji infected with EBV virus than non-infected Ramos (Fig. 3A).
  • OSKM-iNK I and OSKM-iNK II cells prepared in Example 1-1 were co-cultured with EBV-infected Raji and non-infected Ramos, and the frequency of CD107a + cells was confirmed.
  • 1x10 6 cells/ml each of EBV-infected Raji and uninfected Ramos, 1x10 6 cells/ml each of OSKM-iNK I cells, OSKM-iNK II cells, and PBMC-NK cells as a positive control were placed in a 6-well plate. After dispensing by ml, centrifuged at 400 g for 1 minute, co-cultivated in an incubator at 37°C and 5% CO 2 for 4 hours, and then the cells were washed and collected using a centrifuge, followed by FACS (Fluorescence-activated cell sorting) analysis to confirm the frequency of CD107a + cells responding to the infected cells.
  • FACS Fluorescence-activated cell sorting
  • OSKM-iNK I cells, OSKM-iNK II cells and PBMC-NK cells were each 1x10 6 cells/ml in FACS buffer containing fluorescent CD56 and antibodies against CD107a. After administration and reaction at room temperature for 20 minutes, the cells were washed and recovered using a centrifuge and subjected to FACS analysis.
  • OSKM-iNK I cells, OSKM-iNK II cells and positive control PBMC-NK cells all increased CD107a + cell frequency (%) when co-cultured with EBV-infected Raji rather than Ramos, and OSKM-iNK I cells and OSKM-iNK II cells significantly increased the frequency of CD107a + cells compared to PBMC-NK cells (FIG. 3B).
  • LMP-1 (Latent membrane protein 1)
  • OSKM-iNK I and OSKM-iNK II cells prepared in Example 1-1 were co-cultured with EBV-infected Raji and non-infected Ramos, and the expression level of LMP-1, an EBV-specific gene, was confirmed.
  • lentivirus obtained from a GFP-expressing lentiviral vector [control vector in CTIP2 (BCL11B) Human shRNA Plasmid Kit (Locus ID 64919), ORIGENE, CAT#: TL306424] was treated with 5 MOI and 8 ⁇ g/ml polybrene, , cultured in RPMI medium for 16 hours and then replaced with fresh medium, and Raji was transformed into GFP-Raji.
  • the prepared GFP-Raji 1x10 6 cells/ml and each of OSKM-iNK I cells and OSKM-iNK II cells 1x10 6 cells/ml were pipetted by dispensing 1 ml each into a 6-well plate, 37°C, 5% After co-cultivation for 24 hours in an incubator with CO 2 , the reactants were recovered by centrifugation.
  • PBMC-NK cells pNK
  • Total RNA from cell reactions was extracted using the RNeasy Mini kit (Qiagen) and reverse transcribed using the SuperScript VILOTM cDNA synthesis kit (Thermo Fisher Scientific Inc.) according to the manufacturer's instructions.
  • qRT-PCR was performed with SYBR Green and the expression level of LMP-1 was analyzed using a 7500 Fast real-time PCR system (Applied Biosystems).
  • HAV human immunodeficiency virus
  • the cell killing ability of cells, Papilloma virus-infected HK2 proximal tubular cells and Hepatitis virus-infected SNU449 liver cells and the frequency of CD107a + cells expressed through co-culture were measured in the same manner as above. and compared with PBMC-NK cells.
  • OSKM-iNK I cells and OSKM-iNK II cells showed high killing ability (FIGS. 4A, 5A, 6A and Fig. 7A) and high CD107a + expression frequency (Figs. 4B, 5B, 6B and 7B).
  • iNK cells, OSKM-iNK I cells and OSKM-iNK II cells exhibited excellent antiviral effects.
  • OSKM-iNK I and OSKM-iNK II cells which are iNK cells prepared in Example 1-1, against gram-negative bacteria, their cell killing ability and cohort against Escherichia coli (E. coli )
  • E. coli Escherichia coli
  • OSKM-iNK I cells and OSKM-iNK II cells of Example 1-1 and PBMC-NK cells as a positive control were cultured using 1X10 4 cells/100 ⁇ l, 3X10 4 cells/100 ⁇ l, and 9X10 4 cells, respectively. After dilution at a density of / 100 ⁇ l, it was dispensed into a 96-well plate. After adding 3X10 4 cells/100 ⁇ l of E.
  • OSKM-iNK I cells, OSKM-iNK II cells, and positive control PBMC-NK all showed low E. coli populations (FIG. 9A), confirming that they had high toxicity to E. coli [PBMC-iNK].
  • NK 0h (0:1); 19,825, 2h (0:1); 28,000, 2h (0.3:1); 3,670, 2h (1:1); 873, 2h (3:1); 105, OSKM-iNK I: 0h (0:1); 19,825, 2h (0:1); 28,000, 2h (0.3:1); 1,645, 2h (1:1); 173, 2h (3:1); 55, OSKM-iNK II: 0h (0:1); 19,825, 2h (0:1); 28,000, 2h (0.3:1); 1,950, 2h (1:1); 250, 2h (3:1); 60].
  • OSKM-iNK I cells and OSKM-iNK II cells had higher CD107a + cell frequencies compared to PBMC-NK cells. It was confirmed that they appeared (FIG. 9B).
  • streptococci were suspended in TSB (tryptic soy broth) and washed with RPMI 1640 medium containing 10% FBS and no antibiotics.
  • the OSKM-iNK II cells or the PBMC-NK cells as a positive control were diluted to a density of 15X10 4 cells/100 ⁇ l, respectively, using a culture medium, and then dispensed into a 96-well plate.
  • 15X10 4 cells/100 ⁇ l of streptococci were added to the above 96-well plate, incubated at 37° C. for 2 hours under 5% CO 2 conditions, and then the frequency of CD107a + cells was confirmed by flow cytometry.
  • the frequency of CD107a + cells was measured by administering OSKM-iNK II cells or PBMC-NK cells to FACS buffer containing fluorescent CD56 and CD107a antibodies, incubating them at room temperature for 20 minutes, and centrifuging the cells. Washed and harvested and subjected to FACS analysis.
  • iNK cells, OSKM-iNK I cells and OSKM-iNK II cells exhibited remarkable antibacterial efficacy against Gram-negative bacteria and Gram-positive bacteria compared to PBMC-NK cells and NK-92 cells.
  • iNK cells prepared in Example 1-1 the frequency of CD107a + cells expressed through co-culture with Candida albicans measured.
  • Candida albicans was taken from a YPD agar plate (containing 1% yeast extract, 2% peptone, 2% D-glucose, and 1% agar) and cultured in YPD medium (containing 1% yeast extract, 2% peptone, and 2% agar). D-glucose) for 2 hours at 37°C, centrifuged at 1000 xg for 5 minutes, and then washed with RPMI 1640 medium containing 10% FBS and no antibiotics.
  • the frequency of CD107a + cells was measured by injecting cultured OSKM-iNK I cells and OSKM-iNK II cells and positive control PBMC-NK cells into FACS buffer supplemented with fluorescent CD56-PE and CD107a-APC antibodies at room temperature. After reacting for 20 minutes, the cells were washed and recovered using a centrifuge and measured by FACS analysis.
  • iNK cells which are natural killer cells by direct reprogramming produced through the present invention, have excellent cell killing ability against cells infected with viruses, bacteria, and fungi, and cell therapy products and compositions for preventing or treating infectious diseases can be applied as

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Abstract

La présente invention concerne : un procédé de reprogrammation d'une cellule tueuse naturelle induite (iNK), dans lequel des facteurs de transcription complexes sont introduits dans une cellule et, en utilisant une composition optimisée de cytokines, de facteurs de croissance et de composés à petites molécules, l'efficacité de la production de cellules NK est améliorée ; et un procédé de production d'une cellule iNK en utilisant celui-ci. La présente invention concerne également un agent thérapeutique cellulaire et une composition pour la prévention ou le traitement de maladies infectieuses provoquées par des virus, des bactéries, des champignons et similaires, l'agent et la composition comprenant une cellule iNK produite par le procédé.
PCT/KR2022/013563 2021-09-10 2022-09-08 Procédé de production d'une cellule tueuse naturelle et son utilisation pour la prévention ou le traitement de maladies infectieuses WO2023038476A1 (fr)

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US20150118207A1 (en) * 2011-12-22 2015-04-30 Mogam Biotechnology Institute Method for producing natural killer cells, natural killer cells produced thereby, and composition for treating cancers and infectious diseases containing the same
KR20190033458A (ko) * 2017-09-21 2019-03-29 한국생명공학연구원 자연살해세포의 제조방법 및 그의 용도
KR20200115357A (ko) * 2019-03-28 2020-10-07 한국생명공학연구원 면역세포의 제조방법 및 그의 용도

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US20150118207A1 (en) * 2011-12-22 2015-04-30 Mogam Biotechnology Institute Method for producing natural killer cells, natural killer cells produced thereby, and composition for treating cancers and infectious diseases containing the same
KR20130084465A (ko) * 2012-01-17 2013-07-25 공주대학교 산학협력단 개 유래의 자연살해세포의 대량 증식방법
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