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WO2025220989A1 - Cellules nourricières pour amélioration de l'amplification des cellules tueuses naturelles et de l'efficacité de transduction - Google Patents

Cellules nourricières pour amélioration de l'amplification des cellules tueuses naturelles et de l'efficacité de transduction

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Publication number
WO2025220989A1
WO2025220989A1 PCT/KR2025/005052 KR2025005052W WO2025220989A1 WO 2025220989 A1 WO2025220989 A1 WO 2025220989A1 KR 2025005052 W KR2025005052 W KR 2025005052W WO 2025220989 A1 WO2025220989 A1 WO 2025220989A1
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Prior art keywords
cells
natural killer
killer cells
feeder
present
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English (en)
Korean (ko)
Inventor
정수영
윤상원
서민구
정희중
홍병석
박진호
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Uci Therapeutics Inc
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Uci Therapeutics Inc
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Publication of WO2025220989A1 publication Critical patent/WO2025220989A1/fr
Pending legal-status Critical Current
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    • 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
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C07K14/525—Tumour necrosis factor [TNF]
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    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5443—IL-15
    • 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
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • 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
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C12N5/0694—Cells of blood, e.g. leukemia cells, myeloma cells
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/30—Coculture with; Conditioned medium produced by tumour cells
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20211—Vesiculovirus, e.g. vesicular stomatitis Indiana virus
    • C12N2760/20241—Use of virus, viral particle or viral elements as a vector
    • C12N2760/20243—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to genetically engineered feeder cells that can promote the proliferation and expansion of natural killer cells and increase the efficiency of introducing foreign genes into natural killer cells.
  • Transformation is the technique of introducing foreign DNA, such as plasmids, into microorganisms and is a fundamental process of genetic engineering.
  • Commonly used bacterial transformation methods can be categorized into chemical and physical transformation methods, depending on how the bacterial cell wall is treated to allow DNA penetration.
  • NK cells natural killer cells
  • NK cells cytotoxic lymphocytes that constitute a major component of the innate immune system.
  • target cells including virus-infected cells and many malignant cells, non-specifically and without prior immune sensitization to antigens.
  • NK cells have been used as anticancer treatments in the past, but recently, research and development of CAR-NK cell therapies that genetically engineer NK cells to enhance their immune efficacy and enable them to specifically bind to cancer cells has been actively conducted.
  • NK cells when introducing foreign genes, such as CAR constructs, into NK cells, the transduction efficiency into NK cells is significantly lower than that of other immune cells, such as T cells. Furthermore, using NK cells as immunotherapeutic agents requires a process for activating them, which further reduces vector transduction efficiency, hindering the development of genetically engineered NK cell therapies, such as CAR-NK cell therapies.
  • One object of the present invention is to provide genetically engineered feeder cells that can not only promote the proliferation and amplification of natural killer cells but also increase the efficiency of introducing foreign genes into natural killer cells.
  • Another object of the present invention is to provide a method for promoting the proliferation or amplification of natural killer cells or increasing the efficiency of introducing a desired foreign gene into natural killer cells using the genetically engineered feeder cells described above.
  • the present invention relates to a cell line genetically engineered to express lunasin protein or a fragment thereof as feeder cells of natural killer cells.
  • feeder cells genetically engineered to express the lunasin protein or a fragment thereof as described above can promote the proliferation and expansion of natural killer cells when co-cultured with natural killer cells, and can also increase the efficiency of introducing a foreign gene, particularly a vector containing a desired foreign gene, into natural killer cells.
  • the "lunasin” is a peptide derived from soybeans (glycine max), and has been suggested to have anticancer activity that inhibits mitosis of cancer cells and kills them due to the characteristic of binding to chromatin within cells (Galvez and de Lumen, 1999).
  • the lunasin may be derived from nature, for example, isolated and purified from soybeans, barley, wheat, soybeans, etc., or synthesized by various existing methods, or artificially recombined using E. coli or yeast, etc., and its origin is not particularly limited.
  • the lunasin may be a protein consisting of a 43 amino acid sequence represented by any one of SEQ ID NOs: 1 to 4, a protein encoded by a base sequence represented by SEQ ID NO: 5, or a fragment thereof, a derivative thereof, etc., without limitation.
  • the term “genetic engineering” or “genetically engineered” refers to the act of introducing one or more genetic modifications into a cell or a cell produced thereby.
  • the term “transgenic,” “recombinant,” or “genetically modified” as used herein refers to a host cell that replicates a foreign gene or expresses a peptide or protein encoded by a foreign gene. Such a cell is described as being transformed by one or more foreign genes or as being transformed by the introduction of one or more foreign genes.
  • a recombinant or transformed cell may contain genes that are not found in the original (non-recombinant) form of the cell.
  • a recombinant cell may also contain genes that are found in the original form of the cell, but which are altered by artificial means and reintroduced into the cell.
  • the term also includes cells comprising a nucleic acid endogenous to said cell, which has been modified or whose expression has been modified without removing the nucleic acid from the cell; such modifications include modifications obtained by gene replacement, promoter replacement, site-specific mutation, and related techniques.
  • a "recombinant polypeptide” or “recombinant protein” is a polypeptide produced by a recombinant cell.
  • NK cells natural killer cells
  • LGL large granular lymphocytes
  • NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus and enter the circulation.
  • the NK cells may include any type of NK cell without limitation, and may include, but are not limited to, cultured NK cells, for example, primary NK cells, NK cells from a cultured NK cell line, or NK cells obtained from a mammal.
  • the NK cells When the NK cells are obtained from a mammal, the NK cells may be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or body fluids.
  • the NK cells may be concentrated or purified.
  • the NK cells may preferably be human NK cells (e.g., isolated from a human).
  • NK cell lines are available from, for example, the American Type Culture Collection (ATCC), and include, for example, NK-92 cells (ATCC CRL-2407), NK92MI cells (ATCC CRL-2408), or derivatives thereof.
  • the "feeder cells” are also called support cells or culture auxiliary cells, and are cells that do not have the ability to divide and proliferate by irradiation or other treatments, but have metabolic activity and thus produce various metabolites to help the proliferation of target natural killer cells.
  • the genetically engineered feeder cells provided in the present invention not only enhance the simple proliferation of natural killer cells, but also enable the production of an optimal number of natural killer cells as a cell therapy by amplifying natural killer cells from collected blood cells, and play a role in helping the efficiency of introducing foreign genes into natural killer cells.
  • the type of genetically engineered cell may be an animal cell line, such as a human chronic myelogenous leukemia cell line (e.g., K562 cell), RPMI8866, EBV_LCL, 721.221, HFWT, etc., and preferably a K562 cell, but is not limited thereto.
  • a human chronic myelogenous leukemia cell line e.g., K562 cell
  • RPMI8866 e.g., RPMI8866, EBV_LCL, 721.221, HFWT, etc.
  • the above “vector” refers to a recombinant vector that can be introduced into a suitable host cell to express a target protein, and a genetic construct that includes essential regulatory elements operably linked to allow the gene insert to be expressed.
  • operably linked means that a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein are functionally linked to perform a general function.
  • the operably linked vector can be produced using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be easily performed using enzymes generally known in the art.
  • various types of vectors such as nanoparticles, plasmids, viruses, and cosmids can be used as the expression vector.
  • the type of recombinant vector is not particularly limited as long as it has the function of expressing a desired gene and producing a desired protein in various host cells of prokaryotic and eukaryotic cells.
  • a vector that has a promoter that exhibits strong activity and a strong expression ability while being able to mass-produce a foreign protein in a form similar to that in the natural state can be used.
  • Expression vectors suitable for eukaryotic hosts include, but are not limited to, expression control sequences derived from SV40, bovine papillomavirus, adenovirus, adeno-associated virus, cytomegalovirus, and retrovirus.
  • Expression vectors that can be used in bacterial hosts include, but are not limited to, bacterial plasmids obtained from Escherichia coli, including pET, pRSET, pBluescript, pGEX2T, pUC vectors, col E1, pCR1, pBR322, pMB9 or derivatives thereof, plasmids having a wider host range such as RP4, phage DNA such as phage lambda derivatives such as ⁇ gt10, ⁇ gt11 or NM989, and other DNA phages such as M13 and filamentous single-stranded DNA phages.
  • Yeast cells may be used with the 2°C plasmid or derivatives thereof, and insect cells may be used with the pVL941 or the like.
  • Suitable expression vectors of the present invention may include signal sequences for membrane targeting or secretion, in addition to expression control elements such as a promoter, initiation codon, termination codon, polyadenylation signal, and enhancer.
  • the initiation and termination codons are generally considered to be part of the nucleotide sequence encoding the immunogenic target protein, and must be functional in a subject when the genetic construct is administered, and must be in frame with the coding sequence.
  • Common promoters can be constitutive or inducible and include, but are not limited to, the lac, tac, T3 and T7 promoters in prokaryotes, and the simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoters, human immunodeficiency virus (HIV), e.g., the long terminal repeat (LTR) promoter of HIV, Moloney virus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), Rous sarcoma virus (RSV) promoters, as well as the ⁇ -actin promoter, human hemoglobin, human muscle creatine and human metallothionein promoters in eukaryotes.
  • SV40 simian virus 40
  • MMTV mouse mammary tumor virus
  • HAV human immunodeficiency virus
  • LTR long terminal repeat
  • CMV cytomegalovirus
  • EBV Epstein-Barr virus
  • RSV Rous sarcoma virus
  • the expression vector is a nanoparticle, a plasmid, a cosmid, a virus (bacteriophage, an animal virus and a plant virus) and an artificial chromosome (e.g., YAC), for example, a retroviral vector (derived from Moloney murine leukemia virus vector (MoMLV), MSCV, SFFV, MPSV, SNV, etc.), a lentiviral vector (derived from HIV-1, HIV-2, SIV, BIV, FIV, VSV, etc.), an adenovirus (Ad) vector including its replication competent, replication deficient and anaplastic forms, an adeno-associated virus (AAV) vector, a simian virus 40 (SV-40) vector, a bovine papillomavirus vector, an Epstein-Barr virus vector, a herpes virus vector, a varicella virus vector, a Harvey murine sarcoma virus vector, a murine mammary tumor virus vector
  • the vector may include a foreign genetic insert intended to be expressed in natural killer cells.
  • exogenous or heterologous used in relation to the gene, nucleic acid, polynucleotide, etc. in the present invention encompass all cases where the gene, nucleic acid, polynucleotide, etc. originates or is derived from a source other than the host organism species, or where the polynucleotide, gene, or nucleic acid originating or derived from the host organism species is a modified form, or where the polynucleotide, gene, or nucleic acid originating or derived from the host organism species is inserted into a location other than the original location in the genome.
  • the foreign gene in the present invention may include without limitation one or more genetic inserts capable of altering the structure and/or function of the host cell, that is, the natural killer cell.
  • the foreign gene in the present invention may include an appropriate marker gene necessary for selecting the transformed host cell.
  • the foreign gene may be a foreign gene that can have a desirable effect on activating or amplifying natural killer cells, or may be a foreign gene that can contribute to increasing the efficiency of preventing, improving, or treating various diseases using natural killer cells, but is not limited thereto.
  • the foreign gene may be, but is not limited to, a nucleic acid encoding a chimeric antigen receptor that can contribute to increasing the effectiveness of preventing, improving, or treating tumors using natural killer cells.
  • the "chimeric antigen receptor (CAR)" is a modular, synthetic, single-chain protein comprising three functional domains: (i) a binding receptor (extracellular domain); (ii) a spacer and transmembrane region; and (iii) a cytoplasmic signaling domain (intracellular) (Zhang, C., et al., Biomark Res, 2017. 5: p. 22).
  • a cleavable leader or signal peptide is positioned at the N-terminus of the protein to direct passage through the endoplasmic reticulum and promote display on the plasma membrane.
  • Each “module” can be derived from a protein to achieve specific target binding and desired cellular responses induced via a cytoplasmic signaling domain, e.g., the CD3 ⁇ domain (Daniyan, A.F. and R.J. Brentjens, J Leukoc Biol, 2016. 100(6): p. 1255-1264; Oluwole, O.O. and M.L. Davila, J Leukoc Biol, 2016. 100(6): p. 1265-1272).
  • a cytoplasmic signaling domain e.g., the CD3 ⁇ domain
  • binding of cell surface-expressed chimeric receptors to appropriate targets results in clustering and activation of CAR-presenting cells.
  • the foreign gene may be or include a marker gene.
  • the marker gene may be an antibiotic resistance gene or a fluorescent protein gene, and the antibiotic resistance gene may be selected from the group consisting of, but not limited to, a hygromycin resistance gene, a kanamycin resistance gene, a chloramphenicol resistance gene, and a tetracycline resistance gene.
  • the fluorescent protein gene may be selected from the group consisting of, but not limited to, a yeast-enhanced green fluorescent protein (yEGFP) gene, a green fluorescent protein (GFP) gene, a blue fluorescent protein (BFP) gene, and a red fluorescent protein (RFP) gene.
  • the method for introducing the vector into the natural killer cells may use electroporation, lipofection, calcium-phosphate precipitation, DEAE-dextran transfection, nucleofection, squeeze-poration, sonoporation, optical transfection, magnetofection, impelfection, etc., or may include the use of viruses.
  • various techniques are known to those skilled in the art, and are disclosed, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
  • the feeder cells may be genetically engineered to additionally express interleukin and/or 4-1BB ligand in addition to the lunasin protein or a fragment thereof.
  • the interleukin belongs to a group of cytokines expressed and secreted in leukocytes and some other somatic cells.
  • the interleukin genetically engineered to be expressed in the feeder cells may be at least one 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.
  • interleukin includes membrane-bound interleukin, and also includes variants that have the same function as interleukin.
  • membrane-bound interleukin refers to interleukin bound to a cell membrane, and may have a distinct meaning from interleukin secreted extracellularly.
  • the interleukin may preferably include IL-15, IL-21, or a combination thereof.
  • the IL-15 includes the full-length sequence of IL-15, a fragment thereof, or a mutant variant that performs a function equivalent to IL-15.
  • the IL-15 also includes one containing a signal peptide at the N-terminus.
  • the IL-15 may include the amino acid sequence of SEQ ID NO: 6, or may be a protein encoded by the base sequence of SEQ ID NO: 7 or 8, but is not limited thereto.
  • the IL-21 also includes the full-length sequence of IL-21, a fragment thereof, or a mutant variant that performs a function equivalent to IL-21. Furthermore, the IL-21 also includes one containing a signal peptide at the N-terminus.
  • the IL-21 may include the amino acid sequence of SEQ ID NO: 9 or 10, or may be a protein encoded by the base sequence of SEQ ID NO: 11 or 12, but is not limited thereto.
  • the IL-21 may include the amino acid sequence of SEQ ID NO: 9, or may be composed of the sequence, but is not limited thereto.
  • the feeder cells of the present invention are genetically engineered to express IL-15 and/or IL-21
  • the feeder cells may be genetically engineered to express a polypeptide comprising IL-15 and/or IL-21.
  • the polypeptide may further comprise a transmembrane domain in addition to the amino acid sequence of IL-15 or IL-21. Furthermore, within the polypeptide, IL-15 and the transmembrane domain may be optionally linked by a peptide linker and/or a hinge. Furthermore, the polypeptide in the present invention may further comprise a signal peptide.
  • the "transmembrane domain” may contain one or more additional amino acids adjacent to the transmembrane domain, for example, one or more amino acids associated with the extracellular region of the protein from which the transmembrane domain is derived (e.g., amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to a maximum of 15 of the extracellular region) and/or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to a maximum of 15 of the intracellular region).
  • transmembrane domains include transmembrane regions of the alpha, beta or zeta chains of T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8 alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, etc.
  • the transmembrane domain in the present invention may comprise an amino acid sequence of any one of SEQ ID NOs: 15 to 17, or may be encoded by a base sequence of any one of SEQ ID NOs: 18 to 20, but is not limited thereto.
  • the "peptide linker” may mean an oligopeptide comprising 1 to 100 amino acids, particularly 2 to 50 amino acids, each of which may be any kind of amino acid without limitation.
  • a non-limiting example of the peptide linker may be expressed as (G m S l ) n (m, l, and n are the number of "G", “S”, and "(G m S l )", and each may be independently selected from an integer of about 1 to about 10, particularly, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).
  • the peptide linker in the present invention may include, but is not limited to, an amino acid sequence of SEQ ID NO: 31 or 32, or encoded by a base sequence of SEQ ID NO: 33 or 34.
  • the "hinge” refers to a flexible polypeptide connector region (also referred to herein as a "hinge region”) that provides structural flexibility and spacing to the flanking polypeptide region, and may be composed of a natural or synthetic polypeptide.
  • the hinge include a hinge region derived from IgG, CD8, CD28, 4-1BB, OX40, all or part of the CD3 zeta ( ⁇ ) chain, T cell receptor ⁇ or ⁇ chain, CD28, CD3 ⁇ , CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, etc.
  • the hinge in the present invention may include, but is not limited to, an amino acid sequence of SEQ ID NO: 35 or encoded by a base sequence of SEQ ID NO: 36.
  • the "signal peptide”, also called a “leader sequence,” typically directs protein localization.
  • This signal peptide/leader sequence is linked to the N-terminus of the protein of interest.
  • the signal peptide may be included in the amino-terminus (N-ter) of IL-15 or IL-21. However, this signal peptide may be selectively cleaved from IL-15 or IL-21 during IL-15 or IL-21 processing in the cell and localization to the cell membrane.
  • Non-limiting examples of the signal peptide may include IL-2 signal peptide, CD8 signal peptide (21 amino acids), CD33 signal peptide (17 amino acids), CD4 signal peptide (25 amino acids), IL-2R (CD25) signal peptide (21 amino acids), trypsinogen-2 signal peptide (15 amino acids), VEGFR1 signal peptide (26 amino acids), EGFR signal peptide (24 amino acids), GMCSFR signal peptide (22 amino acids), IgVL signal peptide, IgVK signal peptide, or Ig VH signal peptide.
  • the signal peptide may include an amino acid sequence of any one of SEQ ID NOs: 21 to 25, or may be encoded by a base sequence of any one of SEQ ID NOs: 26 to 30, but is not limited thereto.
  • the polypeptide comprising IL-15 may comprise IL-15; a peptide linker and/or hinge; and a transmembrane domain in the direction from the N-terminus to the C-terminus.
  • the polypeptide comprising IL-15 in the present invention may comprise an amino acid sequence of any one of SEQ ID NOs: 37 to 39, or may be encoded by a base sequence of any one of SEQ ID NOs: 40 to 43, but is not limited thereto.
  • polypeptide comprising IL-15 in the present invention may further comprise a signal peptide at the N-terminus, and specifically, may comprise a signal peptide; IL-15; a peptide linker and/or hinge; and a transmembrane domain in the direction from the N-terminus to the C-terminus.
  • the polypeptide comprising IL-15 in the present invention may comprise an amino acid sequence of any one of SEQ ID NOs: 44 to 47, or may be encoded by a base sequence of any one of SEQ ID NOs: 48 to 52, but is not limited thereto.
  • the polypeptide comprising IL-21 may comprise IL-21; a peptide linker and/or hinge; and a transmembrane domain in the direction from the N-terminus to the C-terminus.
  • the polypeptide comprising IL-21 in the present invention may comprise the amino acid sequence of SEQ ID NO: 53 or may be encoded by the base sequence of SEQ ID NO: 54, but is not limited thereto.
  • polypeptide comprising IL-21 in the present invention may further comprise a signal peptide at the N-terminus, and specifically, may comprise a signal peptide; IL-21; a peptide linker and/or hinge; and a transmembrane domain in the direction from the N-terminus to the C-terminus.
  • the polypeptide comprising IL-21 in the present invention may comprise an amino acid sequence of SEQ ID NO: 55 or may be encoded by a base sequence of SEQ ID NO: 56, but is not limited thereto.
  • the 4-1BB ligand (4-1BBL) may include an amino acid sequence of SEQ ID NO: 13, or may be a protein encoded by a base sequence of SEQ ID NO: 14, but is not limited thereto.
  • the feeder cells may be genetically engineered to express IL-15, IL-21 or a combination thereof together with the lunasin protein or a fragment thereof.
  • the feeder cells may be genetically engineered to express IL-21 together with the lunasin protein or a fragment thereof.
  • the feeder cells may be genetically engineered to express IL-15 together with the lunasin protein or a fragment thereof.
  • the feeder cells may be genetically engineered to express IL-15 and IL-21 together with the lunasin protein or a fragment thereof.
  • the feeder cells may be genetically engineered to express 4-1BBL together with the lunasin protein or a fragment thereof.
  • the feeder cells may be genetically engineered to express IL-15 and 4-1BBL together with the lunasin protein or a fragment thereof.
  • the feeder cells may be genetically engineered to express IL-21 and 4-1BBL together with the lunasin protein or a fragment thereof.
  • the feeder cells may be genetically engineered to express IL-15, IL-21 and 4-1BBL together with the lunasin protein or a fragment thereof.
  • the feeder cell includes not only a case where a gene encoding a lunasin protein or a fragment thereof, a gene encoding an interleukin, and/or a gene encoding 4-1BBL are all transduced into the cell line via a single vector, but also a case where all of the above genes are transduced into the cell line via separate vectors, or a case where any two of the above genes are transduced into the cell line via one vector and the remaining one is transduced into the cell line via another vector, all of which are included within the scope of the present invention.
  • the feeder cells may be inactivated to be replication incapable.
  • the inactivation method is not particularly limited, and any method known in the art may be used without limitation, but for example, gamma ray or X-ray irradiation, mitomycin-C treatment, electric pulse, mild chemical fixation (e.g., using formaldehyde or glutaraldehyde), or suicide genes may be used.
  • composition comprising feeder cells provided by the present invention.
  • composition of the present invention can be used as a composition for culturing natural killer cells for the purpose of promoting the proliferation of natural killer cells.
  • composition of the present invention can be used as a composition for amplifying natural killer cells for the purpose of amplifying natural killer cells from a blood sample or blood cells.
  • composition of the present invention can be used as a composition for enhancing the transduction efficiency of natural killer cells, for the purpose of enhancing the introduction efficiency of foreign genes (vectors) into natural killer cells.
  • composition of the present invention can be added to a culture medium for natural killer cells.
  • co-culture of natural killer cells with feeder cells within the composition can promote the proliferation of natural killer cells.
  • composition of the present invention can be added to a blood sample containing natural killer cells or a culture medium containing blood cells, in which case the blood cells and the feeder cells in the composition are co-cultured, thereby inducing the promotion or expansion of the proliferation of natural killer cells.
  • composition of the present invention can be added to a culture medium before, at the time of, or during introduction of a vector into natural killer cells, in which case the efficiency of introduction of a vector, particularly a viral vector, into natural killer cells is increased, so that expression of a desired foreign gene insert contained in the vector is increased, expression of a desired protein encoded by the foreign gene is increased, or the proportion of natural killer cells expressing the desired foreign gene or protein is increased.
  • a method for proliferation or amplification of natural killer cells comprising the step of treating a feeder cell provided in the present invention or a composition containing the feeder cell to a blood sample containing the natural killer cell or the blood sample containing the feeder cell.
  • the method of the present invention may include a step of contacting natural killer cells or a blood sample containing the same with the feeder cells.
  • the contacting step may be performed by co-culturing the natural killer cells or blood cells with the feeder cells.
  • the blood sample is isolated from a donor and may be blood, whole blood, serum, plasma, lymph, urine, feces, tissue, cells, organs, bone marrow, saliva, sputum, cerebrospinal fluid, or a combination thereof containing natural killer cells.
  • the blood sample may include PBMCs, purified NK cells, or primary resting cells (i.e., cells isolated directly from blood).
  • the co-culture may be performed for 1 to 30 days, preferably 7 to 21 days, or 7 to 14 days, or 14 to 21 days, but is not limited thereto.
  • the type of medium used in the co-culture is not particularly limited, and any medium used in the art for culturing or amplifying natural killer cells can be used without limitation.
  • Non-limiting examples thereof include KBM501 medium (Kojin Bio Co., Ltd.), CellGro SCGM medium (Cellgenex, Iwai Chemical Industry Co., Ltd.), X-VIVO15 medium (Lonza, Takara Bio Co., Ltd.), IMDM, MEM, DMEM, RPMI-1640, Xpander (Gibco), etc.
  • the co-culture may be performed in a medium to which cytokines are added.
  • the 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 (Interferon) family, interleukin (IL) family, TNF (Tumor necrosis factors) 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 interleukin may be at least one 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, IL-36 and variants thereof, and preferably may include IL-2, IL-15 or a combination thereof, but is not limited thereto.
  • IL-2 may be added to the medium in an amount of 10 to 1,000 IU/ml, 100 to 500 IU/ml, or 100 to 300 IU/ml, but is not limited thereto.
  • IL-15 may be added to the medium in an amount of 1 to 100 ng/ml, 5 to 50 ng/ml, or 10 to 30 ng/ml, but is not limited thereto.
  • the co-culture may be performed under conditions of 3% to 7% by volume of CO2 at a temperature of about 35°C to 38°C, but is not limited thereto.
  • the medium or other culture conditions used in the co-culture can be appropriately selected and changed from among methods used in the art, and are not limited to the conditions described above.
  • a method for improving the efficiency of introducing a foreign gene into natural killer cells comprising the step of treating natural killer cells or a blood sample containing the same with a feeder cell provided in the present invention or the composition containing the same.
  • the method of the present invention may include a step of contacting natural killer cells or a blood sample containing the same with the feeder cells.
  • the contacting step may be performed by co-culturing the natural killer cells or blood cells with the feeder cells.
  • the blood sample is isolated from a donor and may be blood, whole blood, serum, plasma, lymph, urine, feces, tissue, cells, organs, bone marrow, saliva, sputum, cerebrospinal fluid, or a combination thereof containing natural killer cells.
  • the blood sample may include PBMCs, purified NK cells, or primary resting cells (i.e., cells isolated directly from blood).
  • the co-culture may be performed for 1 to 30 days, preferably 7 to 21 days, or 7 to 14 days, or 14 to 21 days, but is not limited thereto.
  • the type of medium used in the co-culture is not particularly limited, and any medium used in the art for the expansion or proliferation of NK cells can be used without limitation.
  • Non-limiting examples thereof include KBM501 medium (Kojin Bio Co., Ltd.), CellGro SCGM medium (Cellgenex, Iwai Chemical Industry Co., Ltd.), X-VIVO15 medium (Lonza, Takara Bio Co., Ltd.), IMDM, MEM, DMEM, RPMI-1640, Xpander (Gibco), etc.
  • the co-culture may be performed in a medium to which cytokines are added.
  • the 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 (Interferon) family, interleukin (IL) family, TNF (Tumor necrosis factors) 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 interleukin may be at least one 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, IL-36 and variants thereof, and preferably may include IL-2, IL-15 or a combination thereof, but is not limited thereto.
  • IL-2 may be added to the medium in an amount of 10 to 1,000 IU/ml, 100 to 500 IU/ml, or 100 to 300 IU/ml, but is not limited thereto.
  • IL-15 may be added to the medium in an amount of 1 to 100 ng/ml, 5 to 50 ng/ml, or 10 to 30 ng/ml, but is not limited thereto.
  • the co-culture may be performed under conditions of 3% to 7% by volume of CO2 at a temperature of about 35°C to 38°C, but is not limited thereto.
  • co-culture with the feeder cells may be performed before, at the time of, or during introduction of the vector into the natural killer cells, and is not limited thereto, but preferably may be performed before introducing the vector into the natural killer cells.
  • the natural killer cells may be treated with a vector containing a desired foreign gene after 6 hours to 7 days, 12 hours to 7 days, 1 day to 7 days, 1 day to 5 days, or 1 day to 3 days after the co-culture.
  • the method of the present invention may further include a step of culturing or amplifying natural killer cells before, during, or after the co-culturing.
  • the method of culturing or amplifying natural killer cells may be performed by culturing natural killer cells with feeder cells that are different from the feeder cells provided in the present invention and may or may not be irradiated, or by adding one or more agents that promote expansion, such as one or more cytokines, to a medium containing natural killer cells, but is not limited thereto.
  • the cytokines may include IL-2, IL-15, IL-18, and/or IL-21.
  • the above medium may be KBM501 medium (Kojin Bio Co., Ltd.), CellGro SCGM medium (Cellgenex, Iwai Chemical Industry Co., Ltd.), X-VIVO15 medium (Lonza, Takara Bio Co., Ltd.), IMDM, MEM, DMEM, RPMI-1640, Xpander (Gibco), etc., but any medium used in the art for the expansion or proliferation of NK cells may be used without limitation.
  • the expansion or amplification of the NK cells may be performed by culturing at a temperature of about 35°C to 38°C under conditions of 3% to 7% by volume of CO2 , but is not limited thereto.
  • the method of the present invention may include a step of introducing a foreign gene into natural killer cells.
  • the foreign gene includes all cases where it originates or is derived from a source other than the host organism species, or where a polynucleotide, gene or nucleic acid originating or derived from the host organism species is a modified form, or where a polynucleotide, gene or nucleic acid originating or derived from the host organism species is inserted into another location in the genome, etc.
  • the foreign gene in the present invention is one that can change the structure and/or function of the host cell, and any vector containing one or more genes may be included without limitation.
  • the vector may be a viral vector, and preferably a lentiviral vector, but is not limited thereto.
  • the method of the present invention may further include, if necessary, a step of culturing or amplifying natural killer cells after introducing a vector into the natural killer cells.
  • the secondary culturing or amplifying method for the natural killer cells may be the same as the primary culturing or amplifying method for the natural killer cells described above, but may be performed under different conditions from the primary amplification or expansion method, such as using different media, adding different exogenous compounds, or having different culturing/expansion periods.
  • the present invention relates to natural killer cells induced by the method of the present invention.
  • Natural killer cells derived from the present invention may exhibit increased expression of the low-density lipoprotein receptor (LDLR) compared to natural killer cells isolated from a donor, particularly a subject. Natural killer cells with such increased LDLR expression exhibit a high efficiency in introducing foreign genes. Therefore, this may be advantageous for developing genetically engineered natural killer cell therapeutics.
  • LDLR low-density lipoprotein receptor
  • the feeder cells provided by the present invention allows for the amplification of natural killer cells with high purity and multiplication rate. Furthermore, using the feeder cells provided by the present invention can enhance the efficiency of introducing foreign genes into natural killer cells. Therefore, the feeder cells of the present invention can be useful in the development of natural killer cell-based cell therapeutics.
  • Figures 1a to 1d show the results of evaluating the expression levels of lunasin, IL-15, IL-21, and 4-1BBL in feeder cells manufactured according to the present invention in Experimental Example 1 of the present invention by flow cytometry.
  • Figure 2 is a graph showing the change in the total number of cells per day of culture while co-culturing peripheral blood mononuclear cells (PBMC) and feeder cells according to the present invention for 14 days in Experimental Example 2 of the present invention.
  • PBMC peripheral blood mononuclear cells
  • Figure 3 is a graph showing the results of measuring the ratio of CD3-CD56+ natural killer cells through flow cytometry after co-culturing peripheral blood mononuclear cells (PBMCs) and feeder cells according to the present invention for 14 days in Experimental Example 2 of the present invention.
  • PBMCs peripheral blood mononuclear cells
  • Figure 4 is a graph showing the results of measuring the ratio of LDLR+ natural killer cells through flow cytometry after co-culturing peripheral blood mononuclear cells (PBMCs) and feeder cells according to the present invention for 7 days in Experimental Example 3 of the present invention.
  • PBMCs peripheral blood mononuclear cells
  • feeder cells of natural killer cells are genetically engineered to express lunasin protein or a fragment thereof.
  • the above feeder cells may be selected from the group consisting of K562, RPMI8866, EBV_LCL, and HFWT cells.
  • the feeder cells may be genetically engineered to additionally express membrane bound interleukin.
  • the feeder cells may be genetically engineered to additionally express a membrane bound interleukin 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.
  • a membrane bound interleukin 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
  • the feeder cells may be genetically engineered to express more 4-1BBL.
  • the feeder cells may be genetically engineered to additionally express 4-1BBL and membrane bound interleukin.
  • the present invention relates to a composition for promoting proliferation or amplification of natural killer cells including the feeder cells.
  • the present invention relates to a composition for enhancing the efficiency of transduction of a foreign gene into natural killer cells including the feeder cells.
  • a method for proliferation or amplification of natural killer cells comprising the step of contacting feeder cells genetically engineered to express lunasin protein or a fragment thereof with natural killer cells or a blood sample containing the same.
  • the above contacting step can be performed by co-culturing the natural killer cells and feeder cells for 1 to 30 days.
  • the above feeder cells may be selected from the group consisting of K562, RPMI8866, EBV_LCL, and HFWT cells.
  • the feeder cells may be genetically engineered to additionally express membrane bound interleukin.
  • the feeder cells may be genetically engineered to additionally express a membrane bound interleukin 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.
  • a membrane bound interleukin 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
  • the feeder cells may be genetically engineered to express more 4-1BBL.
  • the feeder cells may be genetically engineered to additionally express 4-1BBL and membrane bound interleukin.
  • a method for improving the efficiency of introducing a foreign gene into natural killer cells comprising the step of contacting feeder cells genetically engineered to express lunasin protein or a fragment thereof with natural killer cells or a blood sample containing the same.
  • the above contacting step can be performed by co-culturing the natural killer cells and feeder cells for 1 to 30 days.
  • the above feeder cells may be selected from the group consisting of K562, RPMI8866, EBV_LCL, and HFWT cells.
  • the feeder cells may be genetically engineered to additionally express membrane bound interleukin.
  • the feeder cells may be genetically engineered to additionally express a membrane bound interleukin 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.
  • a membrane bound interleukin 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
  • the feeder cells may be genetically engineered to express more 4-1BBL.
  • the feeder cells may be genetically engineered to additionally express 4-1BBL and membrane bound interleukin.
  • the above foreign gene may be a viral vector containing the desired foreign gene.
  • K562 cells purchased from ATCC were transduced with lunasin-myc, IL-15, 4-1BBL, and IL-21 as shown in Table 1 below.
  • the transduction order was to first introduce lunasin-myc and IL-15, and then simultaneously introduce 4-1BBL and IL-21.
  • the lunasin-myc gene (SEQ ID NO: 5), the gene encoding a polypeptide (IgKsp-IL15-IL15RaTM) containing membrane-bound IL-15 (SEQ ID NO: 6) (SEQ ID NO: 48), the 4-1BBL gene (SEQ ID NO: 14), and the gene encoding a polypeptide (CD8sp-IL21-CD8 hinge-CD8TM) containing membrane-bound IL-21 (SEQ ID NO: 9) (SEQ ID NO: 56) were individually cloned into a lentiviral vector, vesicular stomatitis virus G-pseudotype lentivirus (VSVG-LV), to produce a vector for producing a recombinant lentivirus.
  • VSVG-LV vesicular stomatitis virus G-pseudotype lentivirus
  • RPMI Fetal Bovine Serum
  • VSVG-LV vesicular stomatitis virus G-pseudotyped lentivirus
  • RPMI (10% FBS) medium was added to achieve a final cell concentration of 2 X 10 5 cells/mL and cultured for 2 days at 37°C, 5% CO 2.
  • the culture medium used thereafter was RPMI [10% FBS, 1X penicillin-streptomycin (PS)], and in the case of antibiotic selection, antibiotics were added to the culture medium and used.
  • PS penicillin-streptomycin
  • Example 1 Lunacin-myc expression
  • Example 2 Lunacin-myc/IL-15 expression
  • Example 3 Lunacin-myc/4-1BBL expression
  • Example 4 Lunacin-myc/IL-21 expression
  • Example 5 Lunacin-myc/4-1BBL/IL-15 expression
  • Example 6 Lunacin-myc/4-1BBL/IL-21 expression
  • Example 7 Lunacin-myc/4-1BBL/IL-15/IL-21 expression
  • K562 feeder cells To inactivate K562 feeder cells to be used for culturing natural killer cells, they were treated with mitomycin C (MMC). Specifically, K562 cells under each condition during culture were washed once with DPBS, prepared at a concentration of 1 X 10 6 cells/mL using RPMI (10% FBS) medium, and then mitomycin C was added to a final concentration of 10 ⁇ g/mL. Afterwards, they were cultured for 2 hours in a shaking incubator under conditions of 37°C and 5% CO 2 , washed three times with DPBS, and then suspended in Xpander medium and prepared.
  • MMC mitomycin C
  • Xpander is a final culture medium containing 5% Immune Cell Serum Replacement (ICSR), 1X Supplement, 1X Penicillin-Streptomycin (PS), 200 IU/mL rhIL-2, and 20 ng/mL rhIL-15, respectively.
  • ICSR Immune Cell Serum Replacement
  • PS Penicillin-Streptomycin
  • K562 (ATCC) cells genetically engineered to express 4-1BBL and/or IL-21 were prepared using the same method as in the above examples, as shown in Table 2 below, and then inactivated.
  • inactivated wild-type K562 (ATCC) cells were prepared.
  • FIGS. 1A to 1D it was confirmed that lunasin, IL-15, IL-21, and 4-1BBL were stably expressed in the feeder cells genetically engineered according to the present invention.
  • the ratio of lunasin-expressing cells increased to 99.58%
  • the ratio of IL-15-expressing cells increased to 99.98%
  • the ratio of IL-21-expressing cells increased to 99.25%
  • the ratio of 4-1BBL-expressing cells increased to 99.84%.
  • PBMCs Human peripheral blood mononuclear cells
  • the amplification rate of natural killer cells from PBMC cells was significantly increased by more than 5 times compared to when using K562 feeder cells that were not genetically engineered, and it was confirmed that the amplification rate of natural killer cells was higher than when using K562 feeder cells genetically engineered to express only 4-1BBL and IL-21 without lunasin.
  • the receptor of lentiviruses corresponds to the low-density lipoprotein receptor (LDLR).
  • LDLR low-density lipoprotein receptor
  • the expression level of LDLR is very low in T cells, B cells, and natural killer cells, and the transduction efficiency of lentiviruses, etc. is also very low.
  • the change in the expression level of LDLR in natural killer cells after co-culture with each feeder cell was confirmed.
  • the thawed PBMC stock was suspended in the Xpander final culture medium, and then PBMC (1.5 X 10 7 cells) or genetically engineered K562 (2.5 X 10 6 cells) of Example 7 or Comparative Example 4 was added to an Erlenmeyer flask (125 mL) so that the final culture volume was 10 mL.
  • Immune cells such as natural killer cells generally have very low expression levels of LDLR, but when its expression increases due to external or internal factors, the transduction efficiency of lentiviruses into the cells increases, as has already been experimentally confirmed in several papers (Bari R. et al., Front Immunol (2019) 10:102975; Ying G. et al., ORIGINAL ARTICLE
  • the present invention relates to genetically engineered feeder cells that can promote the proliferation and expansion of natural killer cells and increase the efficiency of introducing foreign genes into natural killer cells.

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Abstract

La présente invention concerne : des cellules nourricières de cellules tueuses naturelles, les cellules nourricières étant génétiquement modifiées pour exprimer une protéine lunasine ; et un procédé pour favoriser la prolifération ou l'amplification de cellules tueuses naturelles ou améliorer l'efficacité de transduction d'un gène étranger dans des cellules tueuses naturelles, en l'utilisant.
PCT/KR2025/005052 2024-04-16 2025-04-14 Cellules nourricières pour amélioration de l'amplification des cellules tueuses naturelles et de l'efficacité de transduction Pending WO2025220989A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030229038A1 (en) * 1997-09-25 2003-12-11 The Regents Of The University Of California Lunasin peptides
KR20190135912A (ko) * 2018-05-29 2019-12-09 사회복지법인 삼성생명공익재단 Ox40l을 발현하는 배양보조세포 및 이를 이용한 자연살해세포 배양 방법
WO2020104676A1 (fr) * 2018-11-23 2020-05-28 Technische Universität Dresden Lignées de cellules nourricières hla positives artificielles pour cellules nk et utilisations correspondantes
KR20220163286A (ko) * 2021-06-01 2022-12-09 주식회사 박셀바이오 배양보조세포를 포함하는 자연살해세포 증식용 조성물
KR20230058167A (ko) * 2020-09-02 2023-05-02 엔카르타, 인크. 조작된 자연 살해 세포의 향상된 증폭 및 세포 독성 및 그의 용도

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030229038A1 (en) * 1997-09-25 2003-12-11 The Regents Of The University Of California Lunasin peptides
KR20190135912A (ko) * 2018-05-29 2019-12-09 사회복지법인 삼성생명공익재단 Ox40l을 발현하는 배양보조세포 및 이를 이용한 자연살해세포 배양 방법
WO2020104676A1 (fr) * 2018-11-23 2020-05-28 Technische Universität Dresden Lignées de cellules nourricières hla positives artificielles pour cellules nk et utilisations correspondantes
KR20230058167A (ko) * 2020-09-02 2023-05-02 엔카르타, 인크. 조작된 자연 살해 세포의 향상된 증폭 및 세포 독성 및 그의 용도
KR20220163286A (ko) * 2021-06-01 2022-12-09 주식회사 박셀바이오 배양보조세포를 포함하는 자연살해세포 증식용 조성물

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LIU, J. ET AL.: "Lunasin as a promising health-beneficial peptide", EUROPEAN REVIEW FOR MEDICAL AND PHARMACOLOGICAL SCIENCE, vol. 18, 2014, pages 2070 - 2075, XP093162498 *

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