WO2025150751A1 - Cellule nourricière pour l'activation et l'amplification des cellules nk et son utilisation - Google Patents

Cellule nourricière pour l'activation et l'amplification des cellules nk et son utilisation

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Publication number
WO2025150751A1
WO2025150751A1 PCT/KR2024/021499 KR2024021499W WO2025150751A1 WO 2025150751 A1 WO2025150751 A1 WO 2025150751A1 KR 2024021499 W KR2024021499 W KR 2024021499W WO 2025150751 A1 WO2025150751 A1 WO 2025150751A1
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WIPO (PCT)
Prior art keywords
cells
cell
culture
membrane
hla
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Pending
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PCT/KR2024/021499
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English (en)
Korean (ko)
Inventor
조덕
김현영
김진호
조유정
이미정
한병우
강진모
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Sungkyunkwan University
Samsung Life Public Welfare Foundation
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Sungkyunkwan University
Samsung Life Public Welfare Foundation
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Application filed by Sungkyunkwan University, Samsung Life Public Welfare Foundation filed Critical Sungkyunkwan University
Priority claimed from KR1020240200768A external-priority patent/KR20250110716A/ko
Publication of WO2025150751A1 publication Critical patent/WO2025150751A1/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • 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 culture auxiliary cells for activation and expansion of NK cells and their uses.
  • Natural killer cells are one of the cytotoxic lymphocytes that are important in innate immunity, and they mainly react to virus-infected cells or cancer cells. This response is influenced by signals generated by the interaction of various activating receptors and inhibitory receptors of NK cells with ligands of target cells.
  • Normal cells have ligands (e.g., MHC) for the inhibitory receptors of NK cells, which transmit inhibitory signals, thereby activating the defense mechanism against NK cells.
  • infected cells or abnormal cells have ligands for the activating receptors of NK cells, which transmit activation signals, and when the activation signals are strengthened by stimuli such as cytokines, the balance with the inhibitory signals is broken, and the NK cell attack begins.
  • NK cells secrete perforin to create holes in the cell membranes of infected cells or cancer cells, and release granzymes to activate apoptosis signals in cells, thereby inducing cell death.
  • NK cells which have the ability to directly recognize and destroy cancer cells, are currently undergoing various studies and clinical trials as one of the recent anticancer treatment strategies.
  • adaptive NK cells that respond to stimuli such as tumors or infections and remember the characteristics of conventional NK cells have recently begun to receive attention.
  • Adaptive NK cells show similar characteristics to memory cells, such as increased expression of NKG2C activating receptors after HCMV infection and long-term survival. Research is being conducted to apply these adaptive NK cells to anticancer treatment to complement the limitations of conventional NK cells.
  • the purpose of the present invention is to provide culture auxiliary cells for activating and amplifying NK cells.
  • the present invention aims to provide a composition for activating and amplifying NK cells including the above-mentioned culture auxiliary cells.
  • the antibody includes not only a complete form having two full-length light chains and two full-length heavy chains, but also a functional fragment of an antibody molecule.
  • the functional fragment of the antibody molecule means a fragment having at least an antigen-binding function, and may be Fab, F(ab'), F(ab') 2 , Fv, etc.
  • the culture auxiliary cells may express human leukocyte antigen E (HLA-E) and membrane-bound anti-CD16 antibody (mbCD16).
  • HLA-E human leukocyte antigen E
  • mbCD16 membrane-bound anti-CD16 antibody
  • the culture auxiliary cells may express human leukocyte antigen E (HLA-E), membrane-bound interleukin-12 (mbIL-12), and membrane-bound interleukin-18 (mbIL-18).
  • HLA-E human leukocyte antigen E
  • mbIL-12 membrane-bound interleukin-12
  • mbIL-18 membrane-bound interleukin-18
  • the culture auxiliary cells may express human leukocyte antigen E (HLA-E), membrane-bound anti-CD16 antibody (mbCD16), membrane-bound interleukin-12 (mbIL-12), and membrane-bound interleukin-18 (mbIL-18).
  • HLA-E human leukocyte antigen E
  • mbCD16 membrane-bound anti-CD16 antibody
  • mbIL-12 membrane-bound interleukin-12
  • mbIL-18 membrane-bound interleukin-18
  • One aspect of the present invention provides a composition for activating and amplifying NK cells, including the culture auxiliary cells.
  • the NK cells in the present invention include NK cells without additional modification derived from any tissue source, and may include mature NK cells as well as NK progenitor cells.
  • the NK cells are activated in response to interferon or macrophage-derived cytokines, and contain two types of surface receptors that control the cytotoxic activity of the cells, which are labeled as activating receptors and inhibitory receptors.
  • the NK cells can be generated from hematopoietic cells, for example, hematopoietic stem or progenitor cells, from any source, for example, placental tissue, placental perfusate, umbilical cord blood, placental blood, peripheral blood, spleen, liver, and the like.
  • the cytokine of the present disclosure may be a naturally occurring cytokine or may be a mutated version of a naturally occurring cytokine.
  • Naturally occurring herein may also be referred to as wild type, and includes allelic variants.
  • the "mutated version of a naturally occurring cytokine” or “mutation” refers to specific mutations made to a naturally occurring sequence so as to alter the function, activity, and/or specificity of the cytokine. In one embodiment, the mutations may enhance or decrease the function, activity, and/or specificity of the cytokine.
  • the mutations may include deletions or additions of one or more amino acid residues of the cytokine.
  • the above cytokine may be at least one selected from the group consisting of BMP (Bone morphogenetic protein) family, CCL (Cheomkine ligands) family, CMTM (CKLF-like MARVEL transmembrane domain containing member) family, CXCL (C-X-C motif ligand ligand) family, GDF (Growth/differentiation factor) family, growth hormone, IFN family, IL family, TNF family, GPI (glycophosphatidylinositol), SLUPR-1 (Secreted Ly-6/uPAR-Related Protein 1), SLUPR-2 (Secreted Ly-6/uPAR-Related Protein 2), and combinations thereof.
  • the composition may further comprise interleukin or a mutant thereof as a cytokine.
  • helper CD4 T lymphocytes as well as monocytes, macrophages, and endothelial cells, and can promote the development and differentiation of T and B lymphocytes and hematopoietic cells.
  • the interleukin may further include, but is not limited to, one or more selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35 and IL-36.
  • One aspect of the present invention provides a method for activating and amplifying NK cells, comprising the step of contacting NK cells with a composition for activating and amplifying NK cells comprising the cultured auxiliary cells or the cultured auxiliary cells.
  • the above sample may be a biological sample derived from an individual, for example, a mammal including a human.
  • the biological sample may be isolated from the individual, and may be, for example, but not limited to, blood, whole blood, serum, plasma, lymph, urine, feces, tissue, cells, organs, bone marrow, saliva, sputum, cerebrospinal fluid, or a combination thereof.
  • the NK cells may be derived from PBMCs, and the cultured feeder cells may be separately treated to inactivate proliferation.
  • the NK cells that have been co-cultured according to the present invention may be activated.
  • the activated NK cells may refer to cells in which cytotoxicity or the inherent immune-regulatory ability of NK cells is activated compared to parent cells, for example, hematopoietic cells, or NK progenitor cells.
  • the activated NK cells or the population enriched in activated NK cells can be assessed by detecting at least one or more functionally relevant markers, for example, CD16, CD57, CD69, CD94, CD161, CD158a, CD158b, NKp30, NKp44, NKp46, DNAM-1,2B4, KIR (e.g., KIR2DL1, KIR2DL2/3, KIR3DL1) and the NKG2 family of activating receptors (e.g., NKG2A, NKG2C, NKG2D).
  • KIR e.g., KIR2DL1, KIR2DL2/3, KIR3DL1
  • NKG2 family of activating receptors e.g., NKG2A, NKG2C, NKG2D
  • the method for activating and amplifying NK cells according to the present invention may further include a step of recovering activated NK cells after the step of contacting the NK cells with cultured auxiliary cells or a composition for activating and amplifying NK cells containing the cultured auxiliary cells.
  • the recovery of the above NK cells may be performed using any cell recovery method known in the art without limitation.
  • One aspect of the present invention provides a method for examining the activity of NK cells, comprising the steps of: i) contacting NK cells with the culture auxiliary cells or the composition to activate NK cells; and ii) analyzing the degree of activation of the activated NK cells.
  • one aspect of the present invention provides a method for providing information for diagnosing an NK cell-related disease, comprising the steps of: i) activating NK cells by contacting the culture auxiliary cells or the composition to the NK cells; and ii) analyzing the degree of activation of the activated NK cells.
  • step i) above overlaps with the method for activating and amplifying NK cells described above, and the specific details of the culture auxiliary cells and composition are as described above.
  • the NK cells of step i) may exist together with other blood cells or lymphocytes, and may be stimulated after being separated from a blood sample, if necessary.
  • the separation step may be performed after stimulation, and in order to use only NK cells as lymphocytes, the stimulation may be performed after the separation step.
  • the degree of purification of the separated NK cells and the composition of the sample may vary to the extent necessary for the experiment, and are not particularly limited as known in the art.
  • the NK cells may be used as they are purified from the sample, if necessary, or may be used after being proliferated to secure conditions or cell amounts suitable for the experiment.
  • the step of isolating the NK cells may not be essential when using a combination of factors specific to NK cells in the method of the present invention.
  • the above step ii) is a process of measuring the degree of NK cell activation according to a method known in the art and comparing it with a normal control group.
  • the above degranulation activity may mean, for example, inducing lysis of target cells by secretion of perforin or granzyme, and this can be analyzed using FACS.
  • a method can be used in which CD107a expression, which is proportional to degranulation, is measured using a fluorochrome-conjugated antibody after stimulating PBMCs isolated from whole blood samples or pure isolated NK cells.
  • the cytotoxic activity can be measured, for example, by incubating a culture containing target cells capable of activating NK cells labeled with a europium fluorescent dye, and then measuring the amount of fluorescent dye released through target cell lysis using a microplate reader.
  • the cytokine secreted by the above NK cell stimulation may be at least one selected from the group consisting of IFN- ⁇ , TNF- ⁇ , TNF- ⁇ , MIP1- ⁇ , MIP-1 ⁇ , PANTES, IL-8, and IL-10.
  • the analysis of the expression of the immune activator of the NK cell as described above can be performed using FACS, intracellular cytokine staining, ELISA, etc. Specifically, a method can be used in which the surface of the NK cell is stained using a specific antibody conjugated to fluorochrome, the cells are permeabilized, and the cytokines, etc. are stained with another specific immune activator (e.g., IFN- ⁇ ) antibody conjugated to fluorochrome to measure the expression of the immune activator in the NK cell.
  • another specific immune activator e.g., IFN- ⁇
  • step ii) the degree of activation of the activated NK cells may be compared with that of normal NK cells.
  • normal NK cells when normal NK cells are stimulated with the above-mentioned cultured feeder cells under conditions equivalent to those of the experimental group as a control group, if the activation phenomenon shown in the normal NK cells is significantly higher in the experimental group, or does not appear or the degree is significantly lower, it is judged as abnormal.
  • pathological signs of diseases related to abnormal NK cells, viral infection, the presence of cancer cells, and specific cancers can be judged, and the prognosis for the above diseases can be predicted.
  • the "normal NK cell” refers to an NK cell possessed by or derived from an individual who does not have a disease, and the individual who does not have the disease does not have at least a physical, genetic, or external condition known to affect NK cell activity.
  • the NK cell-related disease is one showing abnormal NK cell activity, and may be, for example, a hypersensitive immune disease, an autoimmune disease, an immune rejection reaction, an immunodeficiency disease, a histiocytosis, cancer, type 2 diabetes, a parasitic infection disease, and a viral disease.
  • the hypersensitive immune disease may be at least one selected from asthma and sinusitis, or the autoimmune disease may be at least one selected from lupus, multiple sclerosis, type 1 diabetes, and rheumatoid arthritis, or the histiocytosis may be at least one selected from HLH, XLP1, and XLP2.
  • Hemophagocytic lymphohistiocytosis may include group II Langerhans cell histiocytosis, erythrophagocytic lymphohistiocytosis (familial, sporadic), infection-associated hemophagocytic syndrome, virus-associated hemophagocytic syndrome, histiocytosis with massive lymphadenopathy, or reticulohistiocytosis.
  • the "cancer” may include a tumor, a hematological cancer or a solid cancer, which impairs the synergistic activity of NK cells of the subject or does not cause the synergistic activity of NK cells as target cells under certain conditions.
  • the cancer may be selected from the group consisting of lung cancer, liver cancer, esophageal cancer, stomach cancer, colon cancer, small intestine cancer, pancreatic cancer, melanoma, breast cancer, oral cancer, brain cancer, thyroid cancer, parathyroid cancer, kidney cancer, cervical cancer, sarcoma, prostate cancer, urethral cancer, bladder cancer, testicular cancer, hematological cancer, lymphoma, skin cancer, psoriasis and fibroadenoma.
  • the cancer can be pancreatic cancer or B cell lymphoma.
  • the viral disease can be hepatitis B.
  • the immunodeficiency disease can be DiGeorge syndrome or Chediak-Higashi syndrome.
  • Information on the disease related to the above NK cell activity can be determined as an abnormal NK cell if no activation of the NK cells of the experimental group is detected compared to normal NK cells, or can be determined as the target cell being related to a specific disease if the NK cells that have lost activity for a specific receptor abnormally or do not cause activity for a target cell.
  • NK cells NK cells, cell therapy agents, pharmaceutical compositions, uses of NK cells, and methods for treating diseases
  • One aspect of the present invention provides NK cells manufactured by a method for activating and amplifying the NK cells.
  • one aspect of the present invention provides a cell therapy agent comprising the NK cell as an effective ingredient.
  • one aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer or infectious disease, comprising the NK cell as an effective ingredient.
  • one aspect of the present invention provides a use of said NK cells for the manufacture of a medicament.
  • one aspect of the present invention provides a disease treatment method for preventing or treating cancer or an infectious disease, comprising a step of administering the NK cells to a subject.
  • the NK cells may comprise NK cells or a population thereof.
  • Disease as used herein may mean a pathological condition, particularly cancer, an infectious disease, an inflammatory disease, a metabolic disease, an autoimmune disorder, a degenerative disease, a disease involving apoptosis, and graft rejection.
  • treatment refers to or includes the alleviation, inhibition of progression, or prevention of a disease, disorder or condition, or one or more symptoms thereof
  • active ingredient or “pharmaceutically effective amount” may mean any amount of the composition used in the course of practicing the invention provided herein that is sufficient to alleviate, inhibit progression, or prevent a disease, disorder or condition, or one or more symptoms thereof.
  • administering may refer to placement of a composition according to one embodiment into a subject by a method or route that results in at least partial localization of the composition to a desired site.
  • Administration may be by any suitable route that delivers at least a portion of the cells or cellular components of the composition according to one embodiment to a desired location in a viable subject.
  • the survival period of the cells after administration to a subject may be as short as several hours, for example 24 hours, to several days, or as long as several years.
  • isolated cell as used in the present invention, such as “isolated immune cell”, means a cell that is substantially separated from the tissue from which the cell originates, such as a hematopoietic cell.
  • the method of administering the pharmaceutical composition is not particularly limited, but may be administered parenterally, such as intravenously, subcutaneously, intraperitoneally, by inhalation, or topical application, or may be administered orally, depending on the intended method.
  • the dosage may vary depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity.
  • the daily dosage refers to the amount of a therapeutic substance according to one aspect that is sufficient to treat the alleviated disease state by being administered to a subject requiring treatment.
  • the effective amount of a therapeutic substance varies depending on a specific compound, a disease state and its severity, and a subject requiring treatment, and this can be routinely determined by a person skilled in the art.
  • the dosage of the composition according to one aspect to a human body may vary depending on the patient's age, body weight, sex, administration form, health condition, and disease severity.
  • the dosage of the composition according to one aspect to a human body may vary depending on the patient's age, body weight, sex, administration form, health condition, and disease severity.
  • an adult patient weighing 70 kg for example, about 1,000 to 10,000 cells/time, 1,000 to 100,000 cells/time, 1,000 to 1,000,000 cells/time, 1,000 to 10,000,000, 1,000 to 100,000,000 cells/time, 1,000 to 1,000,000,000 cells/time, 1,000 to 10,000,000,000 cells/time, can be divided into administration once or several times a day at regular intervals, or can be administered multiple times at regular intervals.
  • subject means a subject requiring treatment of a disease, and more specifically, means a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, or cow.
  • the pharmaceutical composition may include a pharmaceutically acceptable carrier and/or additive.
  • a pharmaceutically acceptable carrier and/or additive may include sterile water, saline solution, a conventional buffer (such as phosphoric acid, citric acid, or other organic acids), a stabilizer, a salt, an antioxidant (such as ascorbic acid), a surfactant, a suspending agent, an isotonic agent, or a preservative.
  • a pharmaceutically acceptable carrier may include sterile water, saline solution, a conventional buffer (such as phosphoric acid, citric acid, or other organic acids), a stabilizer, a salt, an antioxidant (such as ascorbic acid), a surfactant, a suspending agent, an isotonic agent, or a preservative.
  • an organic substance such as a biopolymer, an inorganic substance such as hydroxyapatite, specifically, a collagen matrix, a polylactic acid polymer or copolymer, a polyethylene glycol polymer or copoly
  • the pharmaceutical composition may appropriately contain, depending on the administration method or formulation, a suspending agent, a solubilizing agent, a stabilizer, an isotonic agent, a preservative, an anti-adsorption agent, a surfactant, a diluent, an excipient, a pH adjuster, a soothing agent, a buffer, a reducing agent, an antioxidant, etc., if necessary.
  • a suspending agent e.g., a solubilizing agent, a stabilizer, an isotonic agent, a preservative, an anti-adsorption agent, a surfactant, a diluent, an excipient, a pH adjuster, a soothing agent, a buffer, a reducing agent, an antioxidant, etc.
  • the pharmaceutical composition according to one specific example may be manufactured in a unit dose form or may be manufactured by introducing it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person skilled in the art to which the present invention pertains.
  • the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of a powder, granules, tablets, or capsules.
  • the NK cell culture auxiliary cell stimulates NK cells by expressing at least one of mbCD16, mbIL-12 and mbIL-18 together with HLA-E, thereby increasing the activation and expansion of NK cells, and such NK cells can be used as a cell therapeutic agent and a pharmaceutical composition for preventing or treating cancer or infectious diseases.
  • Figure 1 shows the HLA-E expression levels of K562 (K562-HLA-E) expressing HLA-E, K562 (K562-HLA-E UL40F) expressing HLA-E and UL40F, and K562 (K562-HLA-E UL40I) expressing HLA-E and UL40I as culture auxiliary cells according to one embodiment of the present invention.
  • FIG. 2 shows the expression levels of mbCD16-1 or mbCD16-2 of K562 (K562-HLA-E-mbCD16-1) expressing HLA-E and mbCD16-1, and K562 (K562-HLA-E-mbCD16-2) expressing HLA-E and mbCD16-2, as culture auxiliary cells according to one embodiment of the present invention.
  • FIG. 3 shows the expression levels of mbCD16-1 or mbCD16-2 of K562 (K562-HLA-E UL40F-mbCD16-1) expressing HLA-E and mbCD16-1, and K562 (K562-HLA-E UL40F-mbCD16-2) expressing HLA-E and mbCD16-2, as culture auxiliary cells according to one embodiment of the present invention.
  • FIG. 4 shows the expression levels of mbCD16-1 or mbCD16-2 of K562 (K562-HLA-E UL40I-mbCD16-1) expressing HLA-E and mbCD16-1, and K562 (K562-HLA-E UL40I-mbCD16-2) expressing HLA-E and mbCD16-2, as culture auxiliary cells according to one embodiment of the present invention.
  • FIG. 5 shows the expression levels of mbIL-12 and mbIL-18 of K562 (K562-HLA-E UL40F-mbCD16-1-mbIL12/18) expressing HLA-E, mbCD16-1, mbIL-12 and mbIL-18 as a culture auxiliary cell according to one embodiment of the present invention.
  • Figure 6 shows the level of gNK expression co-cultured with cultured auxiliary cells according to one embodiment of the present invention, with A and B using PBMCs from different donors.
  • Figure 7 shows the cell killing ability of gNK co-cultured with culture auxiliary cells according to one embodiment of the present invention.
  • K562 human myelogenous leukemia cell line
  • ATCC American Type Culture Collection
  • the cells were cultured in RPMI 1640 medium supplemented with 10% FBS (heat-inactivated) (Gibco), 100 U/mL penicillin, and 100 ⁇ g/mL streptomycin (Invitrogen) at 37°C in a humidified 5% CO 2 cell incubator.
  • K562 was then used to produce feeder cells.
  • K562 expressing HLA-E was produced, and the CMV peptide UL40F or UL40I was additionally expressed to promote the amplification of NK cells.
  • human HLA-E gene (SEQ ID NO: 1), UL40F (SEQ ID NO: 2), or UL40I (SEQ ID NO: 3) was cloned into the lentiviral vector pCDH-CMV-EF1-GFP to produce recombinant lentivirus vectors Vector_HLA-E (SEQ ID NO: 4), Vector_HLA-E-UL40F (SEQ ID NO: 5), and Vector_HLA-E-UL40I (SEQ ID NO: 6). Then, each of the constructed vectors was transfected into 293FT cells together with the packaging vector using lipofectamin3000 (Invitrogen) for virus production.
  • lipofectamin3000 Invitrogen
  • the medium was replaced with fresh medium, and the cells were cultured for 48 hours, and then the medium containing the virus was collected.
  • the recovered medium was centrifuged at 500 ⁇ g for 10 minutes, and only the pure medium containing the virus was separated using a 0.45 ⁇ m filter to produce HLA-E, HLA-E + UL40F, or HLA-E + UL40I expressing lentivirus.
  • 1 mL of each lentivirus was dissolved in 9 mL of medium containing K562, and the mixture was added together with polybrene (8 ⁇ g/ml), and cell culture was performed for 48 hours. Thereafter, infected cells were selected using flow cytometry (fluorescence-activated cell sorting, FACS).
  • NK cells and gNK were stained.
  • the purity of NK cells and gNK was confirmed by the expression of CD3-CD56+ and CD56+FcR ⁇ -/CD3-CD56+, respectively.
  • Fixation/Permeabilization Kit (BD, 554714) was used. After treating the cells with 100 ul of 1X Perm/Wash buffer containing 1 ul of Fc ⁇ RI ( ⁇ subunit-FITC) (Merck, FCABS400F), staining was performed in the dark at 4°C for 30 minutes. After washing with 1X Perm/Wash buffer, the supernatant was removed by centrifugation. After adding 200 ul of 1X Perm/Wash buffer to the cells, flow cytometry analysis was performed.
  • K562 expressing HLA-E was named 'K562-HLA-E'
  • K562 expressing UL40F or UL440I together with HLA-E was named 'K562-HLA-E UL40F' and 'K562-HLA-E UL40I', respectively.
  • lipofectamin3000 Invitrogen
  • the medium was replaced with fresh medium, and the cells were cultured for 48 hours, and then the medium containing the virus was collected.
  • the recovered medium was centrifuged at 500 xg for 10 minutes, and only the pure medium containing the virus was separated using a 0.45 ⁇ m filter to produce mbCD16-1 or mbCD16-2 expressing lentivirus.
  • 1 mL of each lentivirus was dissolved in 9 mL of the medium containing K562-HLA-E, K562-HLA-E UL40F, or K562-HLA-E UL40I produced in Example 1-1, and the mixture was added together with polybrene (8 ⁇ g/ml), and cell culture was performed for 48 hours.
  • K562 expressing HLA-E and mbCD16-1 were named 'K562-HLA-E-mbCD16-1', 'K562-HLA-E UL40F-mbCD16-1', and 'K562-HLA-E UL40I-mbCD16-1'
  • K562 expressing HLA-E and mbCD16-2 were named 'K562-HLA-E-mbCD16-2', 'K562-HLA-E UL40F-mbCD16-2', and 'K562-HLA-E UL40I-mbCD16-2'.
  • gNK cells have reduced expression of receptors for cytokines IL-12 or IL-18, which may weaken their responsiveness to IL-12 or IL-18 during NK cell amplification. Therefore, in order to provide cytokine stimulation to supplement memory function during the amplification step, cultured feeder cells expressing membrane-bound forms of IL-12 and IL-18 (mbIL-12/18) together with HLA-E and mbCD16 were produced.
  • mbIL-12 (SEQ ID NO: 11) and mbIL-18 (SEQ ID NO: 12) were cloned into the lentiviral vector pCDH-CMV-EF1-GFP to produce a recombinant lentivirus vector, Vector_mbIL12/18 (SEQ ID NO: 13). Then, for virus production, the constructed vectors were transfected into 293FT cells together with the packaging vector using lipofectamin3000 (Invitrogen). After a period of time, the medium was replaced with fresh medium, and the cells were cultured for 48 hours. Then, the medium containing the viruses was collected.
  • the collected medium was centrifuged at 500 ⁇ g for 10 minutes, and only the pure medium containing the viruses was separated using a 0.45 ⁇ m filter to produce mbIL-12 and mbIL-18 expressing lentiviruses. Thereafter, 1 mL of the lentivirus was dissolved in 9 mL of the medium containing K562-HLA-E UL40F-mbCD16-1 produced in Example 1-2, and the solution was added together with polybrene (8 ⁇ g/ml), and cell culture was performed for 48 hours. Thereafter, infected cells were selected using flow cytometry, and the expression of mbIL-12 and mbIL-18 in the selected cells was confirmed using RT-qPCR (Quantitative reverse transcription PCR) (Fig.
  • the cultured feeder cells were lysed to extract mRNA.
  • 1 mg of the extracted mRNA 1 mg was synthesized, and mbIL-12 and mbIL-18 were amplified using this as a template.
  • PCR was performed at 60°C for 30 cycles using the primers in Table 1 below.
  • Primer name Primer sequence (5'-3') Sequence number mbIL18_F GAA TGA CCA AGT TCT CTT CAT TGA CCA AG 14 mbIL18_R GTT CTC ACA GGA GAG AGT TGA AAT TTT CTC 15 mbIL12_F GAT GCC GTT CAC AAG CTC AAG 16 mbIL12_R GAA CGC AGA ATG TCA GGG AGA AGT AG 17
  • PBMCs Human peripheral blood mononuclear cells

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Abstract

La présente invention concerne une cellule nourricière pour l'activation et l'amplification des cellules NK et son utilisation. La cellule nourricière des cellules NK exprime au moins l'un des éléments suivants : mbCD16, mbIL-12 et mbIL-18, ainsi que HLA-E, stimulant ainsi les cellules NK pour augmenter l'activation et l'amplification des cellules NK, et ces cellules NK peuvent être utilisées comme agent thérapeutique cellulaire et composition pharmaceutique pour la prévention ou le traitement du cancer ou des maladies infectieuses.
PCT/KR2024/021499 2024-01-11 2024-12-30 Cellule nourricière pour l'activation et l'amplification des cellules nk et son utilisation Pending WO2025150751A1 (fr)

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KR10-2024-0004642 2024-01-11
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KR10-2024-0200768 2024-12-30
KR1020240200768A KR20250110716A (ko) 2024-01-11 2024-12-30 Nk 세포의 활성화 및 증폭용 배양보조세포 및 이의 용도

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20190111804A (ko) * 2018-03-23 2019-10-02 주식회사 녹십자랩셀 자연살해세포의 제조방법
KR20210152934A (ko) * 2020-06-09 2021-12-16 사회복지법인 삼성생명공익재단 Nk 세포의 활성화 및 증폭을 위해 유전적으로 조작된 세포주, 및 그의 용도
KR20220038439A (ko) * 2019-07-31 2022-03-28 엔카르타, 인크. 자연 살해 세포의 향상된 증폭 및 세포 독성을 위한 방법 및 조성물
KR20220064163A (ko) * 2020-11-11 2022-05-18 주식회사 티제이씨라이프 세포 배양 면적 조절을 이용한 자연살해세포의 증식 촉진 방법
WO2023278811A1 (fr) * 2021-07-01 2023-01-05 Indapta Therapeutics, Inc. Cellules tueuses naturelles (nk) modifiées et méthodes associées
KR20230078501A (ko) * 2021-11-25 2023-06-02 사회복지법인 삼성생명공익재단 Hla-e를 발현하도록 유전적으로 조작된 배양보조세포주, 및 그의 용도

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20190111804A (ko) * 2018-03-23 2019-10-02 주식회사 녹십자랩셀 자연살해세포의 제조방법
KR20220038439A (ko) * 2019-07-31 2022-03-28 엔카르타, 인크. 자연 살해 세포의 향상된 증폭 및 세포 독성을 위한 방법 및 조성물
KR20210152934A (ko) * 2020-06-09 2021-12-16 사회복지법인 삼성생명공익재단 Nk 세포의 활성화 및 증폭을 위해 유전적으로 조작된 세포주, 및 그의 용도
KR20220064163A (ko) * 2020-11-11 2022-05-18 주식회사 티제이씨라이프 세포 배양 면적 조절을 이용한 자연살해세포의 증식 촉진 방법
WO2023278811A1 (fr) * 2021-07-01 2023-01-05 Indapta Therapeutics, Inc. Cellules tueuses naturelles (nk) modifiées et méthodes associées
KR20230078501A (ko) * 2021-11-25 2023-06-02 사회복지법인 삼성생명공익재단 Hla-e를 발현하도록 유전적으로 조작된 배양보조세포주, 및 그의 용도

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