WO2018056736A1 - Nouvelle lignée de cellules tueuses naturelles et utilisation correspondante - Google Patents

Nouvelle lignée de cellules tueuses naturelles et utilisation correspondante Download PDF

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WO2018056736A1
WO2018056736A1 PCT/KR2017/010438 KR2017010438W WO2018056736A1 WO 2018056736 A1 WO2018056736 A1 WO 2018056736A1 KR 2017010438 W KR2017010438 W KR 2017010438W WO 2018056736 A1 WO2018056736 A1 WO 2018056736A1
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cells
cell
cancer
isolated
cell line
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성영철
김세원
강문철
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주식회사 에스엘바이젠
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • 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
    • 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
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • 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
    • 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
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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
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
    • C—CHEMISTRY; METALLURGY
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to novel cell lines and their use, and more particularly to novel natural killer (NK) cell lines and their use.
  • NK natural killer
  • the basic direction of cancer immune cell therapy currently being performed is to adopt autoimmune immune cells of cancer patients to proliferate and activate cancer-specific immune cells in vitro, and then administer them to the patient to remove cancer cells.
  • ACT cell therapy
  • the main immune cells used to treat cancer cells are largely cytotoxic T lymphocytes (CTLs), dendritic cells (DCs), and natural killer cells.
  • CTLs cytotoxic T lymphocytes
  • DCs dendritic cells
  • NK which is the most widely used method since cell therapy using T cells from patients was first introduced in 1985 by Dr. Steven Rosenberg of NIH, USA.
  • T-cell therapies have been developed to the third generation so far, and the first-generation T-cell therapies proliferate all T cells present in the blood or cancerous tissues and administer them to patients. Can not expect effect.
  • the tumor-specific T cells were isolated / bulk-cultured and administered to cancer patients. The problem is that the process is long and complex.
  • the third generation of T cell therapeutics includes 1) a TCR gene that recognizes a specific cancer antigen directly into T cells, or 2) a T cell activation domain (T cell) in the antigen recognition site (scFv) of a monoclonal antibody that recognizes a specific cancer antigen.
  • CAR chimeric antigen receptor
  • CART therapy with T cells still has some limitations (Challice L Bonifant, 2016, Molecular Therapy).
  • CART chimeric antigen receptor T cell
  • ACT chimeric antigen receptor T cell
  • the cycle of separating, purifying and amplifying patient's blood cells is long, so it is difficult to perform immediate treatment and cell purification process and amplification system tailored to each patient.
  • treatment costs are significantly higher than conventional therapy.
  • immune cells present in patients after chemotherapy are significantly less functional than immune cells in healthy patients, and the number of immune cells available due to the rapid decrease in the number of blood cells after chemotherapy. Also less.
  • CART cells can be activated by cytokine release syndrome such as IL-6, bystander activation of existing TCR, neurotoxicity, or by allogeneic transplantation. It is known to cause allograft graft versus host disease, which can cause serious side effects. Therefore, in order to overcome this disadvantage, there exists an alternative to cell lineage of allogenic immune cells, the most likely of which is NK cells.
  • NK cells have the ability to kill abnormal autologous cells, ie cancer cells, similar to cytotoxic T cells (CTL). Unlike T cells expressing antigen-specific antigen receptors, NK cells have no antigen specificity and are characterized by killer cell activation receptors (KAR) and killer cell inhibitory receptors (KIR) on the surface of cancer cells. Cell death by recognizing abnormal changes in cells, such as the expression and absence of surface MHC I antigens (Cheng et al ., Cell Mol . Immunol ., 10: 230-252, 2013). In particular, NK cells are attracting attention as cells suitable for ACT because they remove leukemia cells or cancer cells without causing graft-versus-host disease (Glienke et al ., Front.
  • NK cells unlike T cells, do not have immune memory and are killed by themselves after being activated by cancer cells. Therefore, anticancer therapy using NK cells has very little possibility of side effects compared to the therapy based on T cells that have formed immune memory in the body for a long time.
  • the method of producing a therapeutic agent by simply separating and amplifying a patient's autologous cells or allogeneic NK cells is limited due to limited cell resources and due to the limitation of production, complexity and variability of the production process. There may be batch to batch variation due to the heterogeneity of the therapeutic agent itself since it is a delay in time point, not a therapeutic agent of single cell origin.
  • cell therapy based on a single NK cell line may be a more effective method, which simplifies the cell production process, reduces the time difference between the production and administration of the drug, and has homogeneity.
  • Cell therapies can be prepared.
  • NK-92 is the only cell line used in clinical trials (see Table 1).
  • attempts to enhance cancer-specific killing effects by introducing CAR into the NK-92 cell line exist in experimental and non-clinical trial stages and have been confirmed in actual clinical safety (Cheng et al ., Cell Mol . Immunol . , 10: 230-252, 2013).
  • treatment with NK-92 cells alone does not show sufficient anti-cancer efficacy and that the persistence in the body is low, such as being removed within 48 hours in vivo in a single administration (Tonn et al ., Cytotherapy , 15 (12): 1563-1570, 2013).
  • NK cell lines and properties name Establishment year Nationality disease origin Phenotype EBV infection Other YT 1985 Japan Acute Lymphoblastic Leukemia and Thymoma Pericardial fluid unidentified + First established NK cell line NK92 1994 Canada Non-Hodgkin's Lymphoma Pericardial fluid CD56 + CD2 + CD7 + CD3 - - Only cell line to enter clinical trial NKL 1996 United States of America Giant granulocyte leukemia Pericardial fluid CD56 dim CD16 - CD2 + CD3 - - NK92 and other anticancer spectrum shown HANK-1 1998 Japan NK / T lymphoma Lymph node ⁇ SCID mouse CD56 + CD2 + CD3e + HLA-DR + + Type 2 EBV latent (LMP-1 + EBNA2 -) NK-YS 1998 Japan NK cell lymphoma Skin lesions CD56 + CD2 + CD5 + CD3 - CD16 - + Coculture with Mouse Stromal Cell Line
  • Another object of the present invention is to provide a recombinant NK cell in which a polynucleotide encoding one or more foreign proteins is introduced into the isolated NK cell.
  • Another object of the present invention to provide a pharmaceutical composition for treating cancer comprising the NK cell line or recombinant NK cells as an active ingredient.
  • isolated NK cells from humans having the following characteristics:
  • CD2 CD11a, CD25, CD45, CD54, CD56 and HLA-DR are positive;
  • CD1a, CD3, CD4, CD8, CD14, CD16, CD20, CD23, CD34, TCR ⁇ and TCR ⁇ are negative.
  • the isolated NK cells are transduced with a polynucleotide encoding one or more foreign proteins operably linked to a promoter to express the foreign protein Genetically modified NK cells are provided.
  • a pharmaceutical composition for the treatment and prevention of cancer containing the isolated NK cells, and / or the recombinant NK cells as an active ingredient.
  • a method of treating a subject with cancer comprising administering a therapeutically effective amount of the isolated NK cells and / or genetically modified NK cells to the subject with cancer.
  • NK101 cells according to an embodiment of the present invention is not only excellent in proliferative capacity compared to conventionally established NK cell lines, but also has a feature of high expression of INF ⁇ upon IL-2 treatment, and thus is very useful as a cell therapeutic agent for anticancer immunotherapy. It can be usefully used.
  • Figure 1a is a graph showing the degree of cell proliferation according to the passage of the NK101 cell line of the present invention
  • Figure 1b is a photograph taken by using a microscope of the cell morphology confirmed during the culture of the NK101 cells
  • Figure 1c of the NK101 cells A photomicrograph showing the IL-2 dependent cell division.
  • FIG. 1D is a dot graph showing the flow cytometry results of CD3 and CD16, which are major cell markers of the NK101 cells
  • FIG. 1E is IL- of the NK101 cells. 2 Graph showing dependent cleavage ability.
  • Figure 2a is a graph showing the cell division capacity (CPDL, cumulative population doubling level, left) and cell viability according to the culture medium of the NK101 cell line of the present invention (right)
  • Figure 2b is the NK101 cell of the present invention
  • a graph showing the cumulative population doubling level (left) and cell viability in the same medium of the NK-92 cell line which is a conventionally established NK cell line (right).
  • Figure 3 is a histogram showing the flow cytometry results for T / NK markers in NK101 cells of the present invention.
  • Figure 4 is a histogram showing the results of flow cytometry analysis of the major lineage, progenitor markers in NK101 cells of the present invention.
  • Figure 5a is a histogram showing the results of performing a flow cytometry analysis of the NK cells activating receptors in the NK101 cells of the present invention
  • Figure 5b shows a flow cytometry results of the inactivating receptors of NK cells in the NK101 cells of the present invention
  • 5C is a histogram showing the results of flow cytometry on the co-receptors of T cell adhesion and NK cell activation in NK101 cells of the present invention.
  • Figure 6 is a histogram showing the results of flow cytometry for the expression of perforin (IF), IFN- ⁇ and TRAIL involved in the cytotoxicity and immune activation of NK cells in NK101 cells of the present invention.
  • Figure 7 is a histogram showing the results of flow cytometry for cytokine receptors in NK101 cells of the present invention.
  • Figure 8a is a histogram showing the results of flow cytometry analysis of the expression of various CC chemokines (CCR) in the NK101 cell line of the present invention
  • Figure 8b is the expression of various CXC chemokines (CXCR) in the NK101 cell line of the present invention Histogram showing flow cytometry results.
  • Figure 9a is a graph showing the anticancer effect on various human cancer cell lines according to the cell mixing ratio of the NK101 cell line of the present invention
  • Figure 9b is a graph comparing the anticancer effect of NK101 and NK-92 in THP-1 cancer cells
  • Figure 9c is a graph comparing the concentration of the effector molecule (INF- ⁇ ) observed in the culture when co-culture in NK101 cells and NK-92 cells
  • Figure 9d is a leukemia transplanted THP-1 cancer cell line in mice It is a graph showing the results confirmed by comparing the anticancer effect of NK101 in the model with NK-92 cells.
  • Figure 10a is a schematic diagram showing the structure of the TRAIL expression construct prepared for transforming the NK101 cell line of the present invention
  • Figure 10b is an anticancer effect of the recombinant NK101 cells transduced with the TRAIL expression construct It is a histogram showing the result of the confirmed flow cytometry.
  • Figure 11a is a schematic diagram showing the structure of the CD7-CD28 expression construct prepared for transforming the NK101 cell line of the present invention
  • Figure 11a is a recombinant NK101 cell transduced with the CD7-CD28 expression construct
  • Figure 11c is a graph comparing the cancer cell imagination of various cancer cells of the control NK101 cells and the recombinant NK101 cells transduced with the CD7-CD28 expression construct .
  • Figure 12a is a graph measuring the survival rate of cells over time when irradiated with different radiation dose to the NK101 cell line of the present invention
  • Figure 12b is a graph showing the cytotoxicity when co-culture with HCT116 cells according to the radiation dose to be.
  • isolated NK cells from humans having the following characteristics:
  • CD2 CD11a, CD25, CD45, CD54, CD56 and HLA-DR are positive;
  • CD1a, CD3, CD4, CD8, CD14, CD16, CD20, CD23, CD34, TCR ⁇ and TCR ⁇ are negative.
  • the isolated NK cells may further comprise the following features in addition to the phenotype:
  • CD18, CD33, CD122, CD132, CD159 and FAS are positive;
  • CD7, CD10, CD11c, CD13, CD19, CD127, CD158a (KIR2DL1), CD158b (KIR2DL2), NKG2C, and ILT2 are negative.
  • CCR4, CCR6, CCR7, CCR8, CXCR3, and CXCR4 express
  • CCR1, CCR2, CCR3, CCR5, CCR9, CXCR1, CXCR2, CXCR5, CXCR6 and CXCR7 do not express.
  • the isolated NK cells are transduced with a polynucleotide encoding one or more foreign proteins operably linked to a promoter to express the foreign protein Genetically modified NK cells are provided.
  • the foreign protein may be any one for immunostimulation of NK cells, and the foreign protein includes a protein, an auxiliary that specifically binds to a cancer cell specific receptor or ligand for cancer cell targeting.
  • Immunomodulatory polypeptides, chemokine receptors or apoptosis-inducing ligands, including costimulation domains can be included.
  • cancer cell specific receptor or ligand refers to a cell surface receptor or ligand that is specifically expressed in cancer cells, and such cancer cell specific receptor or ligand includes epidermal growth factor receptor (EGFR), somatostatin receptor (SSTR), ⁇ v ⁇ 5 integrin, vascular endothelial growth factor receptor (VEFGR), human epidermal growth factor receptor 2 (HER2), androgen receptor (AR), estrogen receptor (ER), progesterone receptor (PR), sigma-2 Receptor, bombesin receptor, prostate-specific G-protein coupled receptor, PD-1 ligand (PD-1L), MUC1, MUC2, MUC3, folate receptor, ErbB2, transferrin receptor, TAG- 72, G M3 , Le x , CD10, CD20, or CEA.
  • EGFR epidermal growth factor receptor
  • SSTR somatostatin receptor
  • HER2 human epidermal growth factor receptor 2
  • AR HER2
  • AR estrogen receptor
  • PR progesterone receptor
  • a protein that specifically binds to the cancer cell specific receptor or ligand may include an antibody, a functional fragment thereof, or an antibody analog thereof that specifically binds to the cancer cell specific receptor or ligand, and includes the cancer cell specific receptor or ligand. It may be a protein that specifically binds, such as a protein having an RGD domain that specifically binds to integrins that are specifically expressed in cancer cells.
  • antibody is also called immunoglobulin and refers to the Y-shaped protein produced from plasma cells used by the immune system to identify or neutralize foreign substances such as bacteria or viruses.
  • Such antibodies as used herein include various "functional fragments” derived from antibodies, such as Fab, F (ab ') 2, Fab', ScFv, and sdAb.
  • the term "functional fragment of an antibody” as used in a separate document is a fragment having an antigen-binding ability derived from an antibody, and includes both a fragment generated by cleaving the antibody with a protein cleavage enzyme and a single chain fragment generated in a recombinant manner.
  • Fab refers to a fragment produced by cleaving an antibody molecule with papain, a protease, as a fragment of a dimer of two peptides, VH-CH1 and VL-CL.
  • Another fragment produced by papain is referred to as fragment crystallizable (FC).
  • F (ab ') 2 refers to antigens in fragments produced by cleaving antibodies with pepsin, a protease.
  • the Fab is in the form of a tetramer connected by two disulfide bonds.
  • the other fragment produced is called pFc '.
  • Fab ' is a molecule similar in structure to the Fab produced by separating F (ab') 2 under mild reducing conditions.
  • the term "ScFv” is an abbreviation for "single chain variable fragment” and is not a fragment of an actual antibody, and the heavy chain variable region (VH) and light chain variable region (VL) of the antibody are approximately 25 a.a. It is a kind of fusion protein prepared by linking with a linker peptide having a size and is known to have antigen binding ability even though it is not an original antibody fragment (Glockshuber et al., Biochem. 29 (6): 1362-1367, 1990).
  • sdAb single domain antibody
  • nanobody an antibody fragment consisting of a single variable region fragment of an antibody.
  • sdAbs mainly derived from heavy chains are used, single variable region fragments derived from light chains have also been reported to have specific binding to antigens.
  • the "antibody mimetic" is the smallest unit that maintains antigen binding ability, unlike conventional full-length antibodies in which two heavy and two light chains form the quaternary structure of the heterodimer. Fragments (eg, Fab, F (ab ′) 2, Fab ′ or single-chain variable fragments (scFv), which are artificial fragments linking the variable regions of heavy and light chains with linkers, without light chains) It is a concept including an antibody-like protein prepared from an antibody fragment (VHH, VNAR, etc.) derived from camelaceae or cartilaginous fish consisting of heavy chains, or a protein scaffold derived from non-antibodies such as nanobody, monobody, and variable lymphocyte receptor (VLR).
  • VHH antibody fragment
  • VNAR variable lymphocyte receptor
  • costimulatory domain refers to the cytoplasmic domain responsible for the T cell costimulatory function of costimulatory factor, an immune-related protein that aids T / NK activation. it means.
  • costimulatory domains are CD28, inducible costimulator (ICOS), cytotoxic T lymphocyte associated protein 4 (CTLA4), programmed cell death protein 1 (PD1), B and T lymphocyte associated protein (BTLA), death receptor 3 (DR3), 4 -1BB, CD2, CD40, CD30, CD27, signaling lymphocyte activation molecule (SLAM), 2B4 (CD244), natural-killer group 2, member D (NKG2D) / DNAX-activating protein 12 (DAP12), TIM1 (T-Cell) immunoglobulin and mucin domain containing protein 1), TIM2, TIM3, TIGIT, CD226, CD160, lymphocyte activation gene 3 (LAG3), B7-1, B7-H1, glucocorticoid-
  • the immunoregulatory polypeptide is CD28, ICOS, CTLA4, PD1, BTLA, DR3, 4-1BB, CD2, CD40, CD30, CD27, SLAM, 2B4, NKG2D) / DAP12, TIM1, TIM2 , TIM3, TIGIT, CD226, CD160, LAG3, B7-1, B7-H1, GITR, HVEM or OX40L or fragments comprising costimulatory domains thereof, but are not limited thereto (Chen, L. and Flies , DB, Nat. Rev. Immunol . 13 (4): 227-242, 2013).
  • chemokine' refers to a chemotactic cytokine that regulates the movement and location of cells by activating a G protein-linked chemokine receptor (GPCR) comprising a 7-membrane moiety.
  • GPCR G protein-linked chemokine receptor
  • Chemokines are divided into four subfamilies: CC, CXC, CX3C and XC, depending on the location of the first two N-terminal cysteine residues.
  • 'TME' tumor microenvironment
  • chemokines are also involved in other tumor-related processes, including tumor cell growth, neovascularization and metastasis.
  • polynucleotides encoding chemokine receptors that are not expressed in NK101 of the present invention can be transduced and expressed so as to increase specific mobility of cancer cells of NK101 cells of the present invention (Yang et al . J. Immunother) . Cancer , 3 (Suppl 2): P24, 2015).
  • the chemokine receptor may be CCR or CXCR
  • the CCR may be CCR1, CCR2, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9 or CCR10
  • the CXCR is It may be CXCR1, CXCR2, CXCR3, CXCR3B, CXCR4, CXCR5, CXCR6, or CXCR7.
  • apoptosis inducing ligand refers to a protein that binds to a receptor on the cell surface and induces apoptosis of a target cell.
  • Representative apoptosis inducing ligands include TRAIL (TNF-related apoptosis-inducing ligand) and FasL (Fas ligand).
  • the apoptosis inducing ligand may be TRAIL or FasL.
  • operably linked to means that a particular polynucleotide is linked to another polynucleotide so that it can function.
  • the operably linked polynucleotide encoding a particular protein means that the polynucleotide encoding the specific protein is linked so that it can be transcribed into mRNA and translated into the protein by the action of the promoter.
  • Linkage to a polynucleotide encoding another protein means that the particular protein can be expressed in the form of a fusion protein with the other protein.
  • regulators responsible for transcription initiation and, optionally, poly-A signals responsible for transcription termination and stabilization of the transcript usually include, in addition to transcriptional regulators, translation enhancers and / or naturally-combined or heterologous promoter regions.
  • possible regulators that allow expression in mammalian host cells include the CMV-HSV thymidine kinase promoter, SV40, RSV (Loose Sarcoma Virus) promoter, human kidney element 1 ⁇ -promoter, glucocorticoid-induced MMTV-promoter (Molony mouse tumor virus), metallothionein-induced or tetracycline-induced promoters or amplification agents such as CMV amplifiers or SV40-amplifiers.
  • nerve microfiber-promoter, PGDF-promoter, NSE-promoter, PrP-promoter or thy-1-promoter may be used.
  • promoters are known in the art and described in Charron, J. Biol. Chem. 1995, 270: 25739-25745.
  • these regulators include transcription termination signals, such as the SV40-poly-A site or the TK-poly-A site, downstream of the polynucleotide according to one embodiment of the invention. You may.
  • suitable expression vectors are known in the art, for example, the Okayama-Berg cDNA expression vector pcDV1 (Parmacia), pRc / CMV, pcDNA1, pcDNA3 (In-vitrogene), pSPORT1 (GIBCO BRL). ), pX (Pagano (1992) Science 255, 1144-1147), yeast two-hybrid vectors such as pEG202 and dpJG4-5 (Gyuris et al ., Cell 75, 791-803, 1995) or prokaryotic Expression vectors such as lambda gt11 or pGEX (Amersham-Pharmacia).
  • the vector may further comprise a polynucleotide encoding a secretion signal.
  • the secretion signals are well known to those skilled in the art.
  • a leader sequence capable of leading the peptide of the present invention to the cell compartment is combined with the coding sequence of the polynucleotide according to an embodiment of the present invention, and preferably the translated protein.
  • the heterologous sequence can encode a fusion protein comprising a C-terminal or N-terminal tag peptide that confers desired properties such as stabilization or simple purification of the expressed recombinant product.
  • Such tags include, but are not limited to, FLAG, GST (glutathione S transferase), HisX6, and the like.
  • the term "genetic recombinant or genetic engineering” refers to a poly according to one embodiment of the invention introduced into one of a host cell or host entity, or of predecessors / parents.
  • a nucleotide or vector is meant to include a host cell or host entity outside its genome.
  • the polynucleotide or vector according to an embodiment of the present invention may exist in a genetically modified host cell or host entity as an independent molecule, preferably a replicable molecule, outside the genome, or a host cell or host entity Can be stably inserted into the genome.
  • a pharmaceutical composition for the treatment and prevention of cancer containing the isolated NK cells and / or the recombinant NK cell line as an active ingredient.
  • the cancer is lung cancer, stomach cancer, liver cancer, bone cancer, pancreatic cancer, gallbladder cancer, cholangiocarcinoma, skin cancer, head and neck cancer, skin melanoma, uterine cancer, ovarian cancer, rectal cancer, colon cancer, colon cancer, breast cancer, uterine sarcoma, Fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, esophageal cancer, laryngeal cancer, small intestine cancer, thyroid cancer, parathyroid cancer, sarcoma of soft tissue, urethral cancer, penis cancer, prostate cancer, solid tumor of childhood, bladder cancer, kidney Cancer, renal cell carcinoma, renal pelvic carcinoma, spinal contraction tumor, glioma or pituitary adenoma, and the like.
  • the cancer may be metastatic cancer.
  • composition of the present invention may contain one or more known active ingredients having an anticancer effect together with the isolated NK cells and / or the recombinant NK cells.
  • composition may further include a pharmaceutically acceptable excipient or diluent in addition to the carrier.
  • the "pharmaceutically acceptable” refers to a composition which, when administered physiologically and is human, does not normally cause an allergic reaction such as gastrointestinal disorders, dizziness or the like.
  • carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.
  • compositions according to one embodiment of the present invention may be formulated using methods known in the art to allow for rapid release, or sustained or delayed release of the active ingredient when administered to a mammal.
  • Formulations include powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powder forms.
  • compositions according to one embodiment of the invention can be administered by a variety of routes, for example, oral, parenteral, such as suppositories, transdermal, intravenous, intraperitoneal, intramuscular, intralesional, nasal, intrathecal administration It may also be administered, or may be administered using an implant for sustained release or continuous or repeated release.
  • routes for example, oral, parenteral, such as suppositories, transdermal, intravenous, intraperitoneal, intramuscular, intralesional, nasal, intrathecal administration It may also be administered, or may be administered using an implant for sustained release or continuous or repeated release.
  • the frequency of administration can be administered once a day or divided into several times within the desired range, the administration period is not particularly limited.
  • the route of administration of the composition according to one embodiment of the invention can be administered via any general route as long as it can reach the desired tissue.
  • Such route of administration may be, but is not limited to, parenteral administration, eg, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intravitreal administration.
  • compositions according to one embodiment of the present invention may be formulated in a suitable form with a pharmaceutically acceptable carrier generally used.
  • pharmaceutically acceptable carriers include, for example, water, suitable oils, saline, carriers for parenteral administration such as aqueous glucose and glycols, and the like, and may further include stabilizers and preservatives.
  • stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid.
  • Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.
  • compositions according to the present invention if necessary according to the administration method or dosage form, suspensions, dissolution aids, stabilizers, isotonic agents, preservatives, adsorption agents, surfactants, diluents, excipients, pH adjusters, analgesics, buffers, Antioxidant etc. can be contained suitably.
  • Pharmaceutically acceptable carriers and formulations suitable for the present invention including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, latest edition.
  • NK cells for cell therapy are usually administered at around 10 9 to 10 10 cells per m 2 of body surface area per dose. Therefore, 6 ⁇ 10 10 cells are appropriately administered on the basis of a general adult (about 60 kg), but the dosage may vary according to various conditions of the patient and the type and amount of the drug to be co-administered as described above. .
  • isolated NK cells or recombinant NK cells of the present invention that are pharmaceutically active can be administered in an amount of 10 6 to 10 10 cells / kg (body weight) and administration below or above the above-described ranges may in particular Considered. If the dosing is continuous infusion, it should be in the range of 10 3 to 10 9 cell units per kilogram of body weight per minute.
  • a method of treating a subject with cancer comprising administering a therapeutically effective amount of the isolated NK cells and / or genetically modified NK cells to the subject with cancer.
  • terapéuticaally effective amount means an amount that significantly inhibits the death of cancer cells or at least the growth of cancer tissue.
  • NK cell-derived cell line To prepare an NK cell-derived cell line, the following process was carried out. Cellgro ® containing 20% fetal bovine serum (GE healthcare, Pittsburgh, USA) and 1% antibiotics (Life technologies, Maryland, USA), with patient-derived lymphadenopathic (NK) lymphoma on a 40 ⁇ m strainer 10 ml of stem cell growth medium (SCGM; CellGenix, Freiburg, Germany, hereinafter referred to as 'NK media') was added and separated into single cells using the shear force of the piston of a 5 ml syringe and then suspended.
  • SCGM stem cell growth medium
  • NK cells in a single cell suspension were isolated using an NK separation kit (Milltenyi Biotec, Bergisch Gladbach, Germany), and then 1000 U / ml of human recombinant IL-2 (rhIL-2; Prometheus, San Diego, USA) was added. Incubated for 3 weeks in NK media. NK media containing rhIL-2 was added twice a week during the culturing, and it was confirmed that stable cell lines were formed by continuously culturing dividing cell lines up to 30 passages (FIG. 1A). The cell line can be confirmed under a microscope to form a colony (spheroid) in the culture (Fig. 1b), the cell proliferation ability was dependent on IL-2 (Fig. 1c).
  • rhIL-2 human recombinant IL-2
  • the cell line expresses CD56 without expressing CD3, CD20, CD16, confirming that the origin of the cell is NK cells (FIG. 1D), and the cell division ability is dependent on IL-2 (FIG. 1E).
  • the cell line was named 'NK101 cell line' by confirming cell culture and characteristics of NK cell, and it was named Korea Collection for Type Culture (KCTC) in Korea Research Institute of Bioscience and Biotechnology located at 181, Yeopsin-gil, Jeongeup-si, Jeollabuk-do, Korea. Was deposited on August 7, 2017, and received the accession number of KCTC 13305BP on August 24, 2017. The Depositary is an International Depositary under the Butafest Treaty.
  • KCTC Korea Collection for Type Culture
  • Example 2 NK cell line culture conditions and cell division analysis of the present invention
  • NK101 cells of the present invention The comprehensive characteristics of NK101 cells of the present invention are summarized in Table 2 below.
  • NK101 cells of the invention Item NK101 cells Clinical data Age / gender 56 year old male race Asian Diagnosis Extraranodal NK / T lymphoma Treatment status At diagnosis Established year 2016 Cell culture Growth pattern Multicellular Aggregates in Suspension Doubling Time 18-32 hours Cell concentration 1.2 ⁇ 10 6 cells / ml Minimum cell concentration 1 ⁇ 10 5 cells / ml Cytokine dependence IL-2 dependency (500 IU / ml) Optimal cleavage Every 2-3 days Immunological properties T / NK marker CD2 +, CD3 -, CD4 - , CD7 -, CD8 -, CD16 -, CD56 + B cell marker CD10 -, CD19 -, CD20 - Bone Supranuclear Markers CD13 -, CD14 -, CD33 + NK cell activating receptor NKp46 +, NKp44 -, NKp30 + , NKG2D +, NKG2C - NK cell inhibitor
  • Example 3 of the present invention NK Cell Line Phenotyping
  • NK101 cell line of the present invention prepared in Example 1 has the characteristics of floating cells, the expression level of the surface antigen of the cell was confirmed by flow cytometry.
  • the NK101 cell line of the present invention expresses CD2, CD56, which are surface antigens of NK cells, but does not express CD16, and does not express CD3, CD4, CD8, TCR ⁇ , and TCR ⁇ , which are surface antigens of T cells. Did not have the phenotype of NK cells (FIG. 3).
  • B cell surface antigens CD10, CD19, CD20
  • granulocyte expressing antigens CD13, CD23
  • dendritic and monocyte surface antigens CD1a, CD11c, CD14
  • progenitor surface antigens CD34
  • NK101 cell line of the present invention expressed NKp30, NKp46, and NKG2D, which are activating receptors of NK cells (FIG. 5A), but did not express inactive receptors ILT2, KIR2DL1, and KIR2DL2 / DL3, but CD94 and CD159a.
  • Fig. 5b It was confirmed that they express CD11a, CD18, CD54, DNAM-1, 2B4, and the like, which act as co-receptors for T cell adhesion and NK cell activation (FIG. 5C).
  • the NK101 cell line of the present invention has high expression of perforin, IFN- ⁇ , which is involved in cytotoxicity and immune activation of NK cells, while low expression of TRAIL (FIG. 6), among cytokine receptors of NK cells.
  • TRAIL FIG. 6
  • CD25 and IL-2 receptors CD122 and CD132
  • CD127 which is an IL-7 receptor
  • the role of IFN- ⁇ is typically NK cell activation, macrophage activation, IgG antibody isomorphism induction, Th2 inhibition, increased MHC expression, etc., and high INF- ⁇ activity has the advantage of inducing strong anticancer activity.
  • the NK101 cell line of the present invention is distinguished from other NK cell lines in that CD25 is highly expressed, which is an indicator of activated NK cells and is particularly known as a marker of NK cells with high dividing capacity (Clausen, J. et al. , Immunobiology , 207 (2): 85-93, 2003) It can be seen that the cell line is very suitable for mass production of cell therapy products.
  • NK101 cell lines of the present invention express CD2, CD11a, CD26, CD45, CD54, CD56 and HLA-DR, and do not express CD1a, CD3, CD4, CD8, CD14, CD16, CD20, CD23, and CD34. It had a phenotype of activated NK cells ( Figures 3, 4 and 5c).
  • the NK101 cell line of the present invention is distinguished from NK-92 cells in that it is HLA-DR positive unlike HLA-DR negative NK-92.
  • the NK101 cell line of the present invention expresses NKp30, NKp46, low levels of NKG2D, CD94 and KRLB-1, which are known as NK cell activation receptors (FIG. 5A), and express inactive receptors ILT2, KIR2DL1, and KIR2DL2 / DL3. It was confirmed that perforin, IFN- ⁇ , and TRAIL, which are involved in cytotoxicity and immune activation of NK cells, were expressed (FIG. 6B). Among the cytokine receptors of NK cells, CD25, an IL-2 high affinity receptor, was expressed and CD127, an IL-7 receptor, was not expressed (FIG. 7).
  • NK101 cell line of the present invention is distinguished from other NK cell lines in that CD25 is highly expressed.
  • CD25 is an indicator of activated NK cells, and is known as a marker of NK cells with high dividing ability (Clausen, J. et al ., Immunobiology , 207 (2): 85-93, 2013), NK101 cell line of the present invention can be seen that the cell line is very suitable for mass production of cell therapy.
  • human-derived cancer cell lines NCI-H460 lung cancer
  • U373 brain cancer
  • A2780 ovarian cancer
  • HCT116 colon cancer
  • SK-BR3 breast cancer
  • THP-1 which are labeled with caroxyfluorescein diacetate (CFDA) (Acute myeloid leukemia) and K562 (chronic myeloid leukemia) were seeded at a concentration of 3 ⁇ 10 4 cells / ml in a 24-well culture dish.
  • CFDA caroxyfluorescein diacetate
  • K562 chronic myeloid leukemia
  • the cell pellet was suspended in a solution of 5 ⁇ l Annexin V APC (Biolegend, USA) in 100 ⁇ l of 1 ⁇ Annexin V binding buffer, and reacted at room temperature for 20 minutes.
  • Cell death was determined by flow cytometry using viable cells (annexin V-negative / LIVE / DEAD-negative), early apoptotic cells (annexin V-positive / LIVE / DEAD-negative), late apoptotic cells (annexin V-positive / LIVE / DEAD-positive) and necrotic cells (annexin V-negative / LIVE / DEAD-positive).
  • the NK101-administered group shows cell killing ability against various human cancer cell lines.
  • NK101 and NK-92 showed similar cancer cell killing (FIG. 9B).
  • IFN ⁇ was confirmed by ELISA in the co-culture of THP-1, NK101 and NK-92, it was confirmed that the amount of INF ⁇ expressed in the NK101 cell line was significantly higher than that expressed in NK-92 (FIG. 9C).
  • NK101 cells have cancer cell killing ability similar to NK-92 cells, they have the advantage of immunocytokinetic secretion ability such as IFN ⁇ , which can cause additional anticancer immune response. Can be.
  • NK101 cells In order to confirm the anticancer effect of NK101 cells in vivo , the present inventors have intravenously administered 10 6 THP-1 cell lines expressing luciferase to human immunodeficiency mice (NSG mice). After constructing acute myeloid leukemia xenograft model, 5x10 6 NK101 cells or NK-92 cells were administered 4 times at 2 days intervals from 3 days after cancer administration, followed by intraperitoneal administration of 15 mg / kg of luciferin in IVIS spectrum in The therapeutic effect of these NK cells was confirmed by contrast imaging through an in vivo imaging system (Perkin Elmer, USA) (FIG. 9D). As a result, both NK101 cells and NK-92 cells showed similar anticancer effects.
  • Example 5 Induction of Apoptosis Preparation of Transgenic NK101 Cells Expressing Ligand TRAIL and Evaluation of Anticancer Activity
  • TRAIL GeneBank No. AAH32722.1
  • FIG. 10a a transfer vector for lentiviral production, developed in-house and pBD2.5.
  • / TRAIL vector was constructed.
  • the cell saturation of the prepared 12 ⁇ g pBD2.5 / TRAIL plasmid, 12 ⁇ g packaging psPAX2 plasmid, and 2.4 ⁇ g pMD.G plasmid with Lipofectamine 200 (Lipofectamine 2000, Invitrogen, USA) 90% of 293T cells (Invitrogen, Carlsbad, CA, USA) were transduced. After incubation for about 48-72 hours, the virus was obtained and mixed with the lentiviral enrichment kit (Lenti X Concentrator, Clontech Laboratories, USA). After overnight incubation at 4 ° C., the viruses were harvested by 4,000 RPM centrifugation and released again into the culture.
  • NK101 cell line expressing TRAIL To prepare an NK cell line expressing TRAIL, the lentiviral and protamine sulfate prepared above were reacted at 37 ° C. for 4 hours to infect the NK101 cell line. After a total of 2 infections, 72 hours after infection, TRAIL expression was confirmed by flow cytometry, and after 1 week, only NK101 cells expressing TRAIL were selectively isolated using a fluorescence activated cell sorter. .
  • NK101 cell line expressing TRAIL enhances the effect on cancer cell death
  • incubation of cancer cells and NK101-TRAIL cells together to identify apoptosis-treated and dead cells The method was used according to the analysis.
  • Target cancer cells are HCT116 human colorectal cancer cell line.
  • NK101 cell line which does not express the transgene was used as a control.
  • NK101 cells expressing TRAIL kill about 60% of cancer cells at E: T ratio 1: 1, 75% at 3: 1.
  • T ratio 1 1, 75% at 3: 1.
  • NK101 cells significantly increase anticancer effects due to the introduction of apoptosis inducing ligands such as the TNF family.
  • Example 6 Preparation of transformed NK101 cells expressing NK cell costimulatory factors CD7, CD28 and evaluation of anticancer activity
  • Nucleic acid molecule encoding the P2A sequence such that CD7 (GenBank No. AAH13297.1) and CD28 (GenBank No. AAH93698.1) which are not expressed in the NK101 cell line of the present invention are coexpressed (SEQ ID NO: 1)
  • the DNA cassette (FIG. 11a) was connected to the pBD2.5, which is a transfer vector for producing a lentiviral, which was developed in-house, to construct a pBD2.5 / CD7-CD28 vector, which was prepared as a lentiviral as in Example 5. Then, the NK101 / CD7-CD28 transformed cell line was isolated by introducing into the NK101 cell line.
  • the cells were co-cultured at 1: 1 ratio with NK101 / CD7-CD28 cell line and cancer cells in vitro .
  • the target cancer cells used were Jeko-1, which expresses SECTM-1, a ligand of CD7, and U937, KG-1, and THP-1, which expresses CD80, CD86, which are ligands of CD28, and NK101 as a control cell. It was.
  • the cancer cell specific killing ability of NK101 / CD7-CD28 was 30% (Jeko-1), 56.6% (U937), 41.3% (KG-1), 47.4 (THP), respectively. -1)% at about 1.6-fold compared to 15% (Jeko-1), 35.9% (U937), 24.9% (KG-1), and 23% (THP-1), the cancer cell killing capacity of NK101 used as a control. It could be confirmed that the increase up to 2 times.
  • NK101 cells of the present invention are cell lines derived from human cancer, and may be proliferated in the body when used as a therapeutic agent, which may cause safety problems.
  • the NK101 cells of the present invention were irradiated with 1, 5, 10, and 20 Gy of radiation, respectively, to observe the proliferation of the cells (FIG. 12A).
  • the proliferation of NK101 cell lines was inhibited when the radiation was irradiated over 5 Gy.
  • the cytotoxicity of the NK101 cell line after irradiation was 4: 1 co-cultured with HCT116 cells, it was confirmed that the cytotoxicity of NK101 did not decrease significantly when the radiation was about 5 Gy or less (FIG. 12B).
  • NK101 cells can maintain cancer cell killing ability without proliferation in human body through human irradiation. Therefore, NK101 cells of the present invention is expected to be used as a cell therapy for more efficient and economical cancer treatment.
  • Isolated NK101 Cells or Recombinant NK101 Cells 1 ⁇ 10 6 to 1 ⁇ 10 9 cells
  • the amount of the above ingredient was prepared per ampoule (2 ml).
  • Isolated NK cells and recombinant NK cells according to an embodiment of the present invention can be usefully used as a cell therapy for anticancer therapy in the medical field.

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Abstract

La présente invention concerne une nouvelle lignée de cellules tueuses naturelles et une utilisation correspondante et, plus particulièrement, une nouvelle lignée de cellules tueuses naturelles qui est positive à CD2, CD11a, CD25, CD45, CD54, CD56 et HLA-DR ; et négative à CD1a, CD3, CD4, CD8, CD14, CD16, CD20, CD23, CD34, TCRαβ et TCRγδ et l'utilisation correspondante comme composition pharmaceutique la comprenant pour le traitement du cancer.
PCT/KR2017/010438 2016-09-23 2017-09-22 Nouvelle lignée de cellules tueuses naturelles et utilisation correspondante WO2018056736A1 (fr)

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WO2021025626A1 (fr) * 2019-08-08 2021-02-11 National University Of Singapore Cellules nk modifiées et utilisations associées
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US20220333079A1 (en) * 2019-09-18 2022-10-20 Slbigen Inc. Genetically modified nk cell line transduced with gene encoding novel chimeric antigen receptor and use thereof
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