WO2019182425A1 - Lignée de cellules nk génétiquement modifiée ayant un nouveau gène codant pour le récepteur chimérique de l'antigène et son utilisation - Google Patents

Lignée de cellules nk génétiquement modifiée ayant un nouveau gène codant pour le récepteur chimérique de l'antigène et son utilisation Download PDF

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WO2019182425A1
WO2019182425A1 PCT/KR2019/003432 KR2019003432W WO2019182425A1 WO 2019182425 A1 WO2019182425 A1 WO 2019182425A1 KR 2019003432 W KR2019003432 W KR 2019003432W WO 2019182425 A1 WO2019182425 A1 WO 2019182425A1
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cancer
cell
cell line
cells
gene
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성영철
김세원
강문철
황인정
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주식회사 에스엘바이젠
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • 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
    • A61K39/464466—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to a genetically modified immune cell line transformed with a chimeric antigen receptor coding gene and its use, and more particularly to a genetically modified NK cell line transduced with a novel chimeric antigen receptor coding gene having improved anticancer activity and its use. will be.
  • Cancer immune cell therapy is a therapeutic technology that inhibits and eliminates the proliferation of cancer cells by extracting and propagating their own immune cells and then administering them back to the patient.
  • dendritic cells DCs
  • NKs natural killer cells
  • CTLs cytotoxic T lymphocytes
  • CTLs have an advantage of having an immunological memory function, antigen specificity and excellent in vivo proliferation ability, and thus, much research is being performed in comparison with dendritic cells and natural killer cells.
  • T cell therapies have been developed to the 3rd generation until now.
  • the first generation of T cell therapies has been applied to patients by proliferating all the T cells present in the blood or cancerous tissues.
  • the second-generation T cell therapy showed enhanced treatment effect by separating / bulk-cultured tumor antigen-specific T cells only to cancer patients.
  • the complexity of the process has arisen, and the third generation of T cell therapeutics has the potential to: 1) directly introduce TCR genes that recognize specific cancer antigens into T cells, or 2) antigen recognition sites (scFv) of monoclonal antibodies that recognize specific antigens.
  • scFv antigen recognition sites
  • the third generation T cell therapeutics described above are genetically engineered to express so-called chimeric antigen receptors (hereinafter, abbreviated as 'CAR'), which are still approved through clinical trials. Does not exist.
  • 'CAR' chimeric antigen receptors
  • ALL leukemia
  • NHL non-Hodgkin's in lymphoma
  • the CD19 target CAR-introduced T cells have only the indications of ALL and NHL, which are types of hematological cancers, and thus have a disadvantage in that they are not applicable to solid cancers because of their poor versatility and target B cell-specific antigens.
  • CD1a, CD3, CD4, CD8, CD14, CD20, CD23, CD34, TCR ⁇ and TCR ⁇ are negative.
  • a cell therapy agent for treating cancer containing a therapeutically effective amount of the genetically modified NK cell line as an active ingredient.
  • 3D is a CD107a, perforin, granzyme, FasL and TRAIL involved in NK cell dependent cytotoxicity in NK101 cells It is a histogram showing the flow cytometry results for expression, Figure 3e is a histogram showing the flow cytometry results for cytokine receptor expression in NK101 cells, Figure 3f is for the expression of various CC chemokine receptors and CXC chemokine receptors in NK101 cells Histogram showing flow cytometry results.
  • Figure 4a is a histogram showing the results of flow cytometry confirming the CD56 expression of NK101, NK-92 and peripheral culture CD56 + peripheral blood NK cells
  • Figure 4b is a two-dimensional contour line showing the flow cytometry results confirming the CD56 and CD62L expression of the cells It is a graph.
  • FIG. 5A is a graph confirming the proliferation rate of NK101 cells after treatment with each indicated cytokine in a culture solution for 3 days (left) and the production of NK activated cytokine IFN- ⁇ by ELISA (right),
  • FIG. 5b is a graph showing the results obtained by multiplex analysis of cytokines secreted after NK101 cells were co-cultured with K562 or THP-1 cancer cell lines for 24 hours.
  • Figure 6a is a graph showing the results of confirming the killing of cancer cells by co-culture of various cancer cell lines and NK101 cells at various cell ratios for 24 hours
  • Figure 6b is acute myeloid treatment of the neutralizing antibodies to the surface antibodies respectively marked on NK101 cells The cell death was measured after 24 hours co-culture with leukemia cell line THP-1 and 4: 1 ratio
  • Figure 6c is treated with neutralizing antibodies to the surface antibody labeled on NK101 cells and chronic myeloid leukemia cell line K562 and 4
  • FIG. 6D shows a neutral lymphocytic leukemia cell line Jurkat and 4: 1 ratio treated with neutralizing antibodies to surface antibodies labeled on NK101 cells, respectively. After 24 hours co-culture with a graph showing the results of measuring apoptosis, Figure 6e is treated with DNAM-1, CD54 or DNAM-1 and CD54 neutralizing antibodies to NK101, respectively, It is a graph showing the result of confirming the synergistic inhibition by the simultaneous neutralization of markers.
  • Figure 7a is a histogram showing the results of confirming the expression patterns of CD7, CD28 costimulatory factors in NK101 and the established cell lines KHYG-1, and NK-92 through flow cytometry
  • Figure 7b is a cancer cell killing effect of NK101 cells
  • Figure 7c is a schematic diagram of the gene construct introduced for enhancement
  • Figure 7b is a flow cytometric analysis of CD7, CD28 expression in the NK101 cells (named SL-K01) introduced with the gene construct shown in FIG. It is a histogram showing a result
  • FIG. 7D is a photograph showing the result confirmed by reverse transcription PCR of CD :: UPRT expression in SL-K01 cells.
  • Figure 8a is a graph showing the results of measuring cancer cell death frequency through flow cytometry after co-culture of HDLM-2, IM-9, JEKO-1 and K562 cancer cells with NK101 or SL-K01 cells at 4: 1 ratio for 24 hours
  • 8B is a graph showing the results obtained after 48 hours of treatment with various concentrations of 5-FC in NK101 or SL-K01 cells through MTS analysis
  • FIG. 8C is an IM-9 cell line and NK101 or SL. It is a graph showing the result of measuring the frequency of IM-9 cancer cell death by flow cytometry when 5-K01 cells are co-cultured in a 2: 1, 1: 1 or 0.5: 1 ratio with or without 5-FC.
  • FIG. 10A is a graph showing the population doubling level according to the presence or absence of IL-2 in the culture of SL-K01 and NK111 cells
  • FIG. 10B is a flow chart of NKG2D expression in NK101, SL-K01 and NK111 cells. The histogram showing the results confirmed through the analysis
  • FIG. 10C shows the IM according to the presence or absence of 5-FC when co-culture of the IM-9 cell line and SL-K01 or NK111 cells in a 2: 1, 1: 1, or 0.5: 1 ratio.
  • FIG. -9 is a graph showing the result of measuring the death frequency of cancer cells through flow cytometry
  • Figure 10d is co-culture of OVCAR-3 or THP-1 cell line with SL-K01 and NK111 cells, after treatment with various concentrations of TGF ⁇ 1
  • FIG. 12A shows the anti-EpCAM CAR construct in the parental cell line NK111 (left) and in the transgenic NK cell line transfected with the anti-EpCAM CAR construct according to one embodiment of the invention (SL-K10, right). It is a histogram showing the results of the expression pattern measured by flow cytometry, Figure 12b is a NK111 and the gene introduced by the anti-EpCAM CAR construct in the transgenic NK cell line (SL-K10) according to an embodiment of the present invention Expression is a photograph showing the results confirmed by Western blot analysis after electrophoresis of reducing and non-reducing conditions.
  • Figure 13a is a graph showing the cell growth during long-term passage of the genetically modified NK cell line (SL-K10) according to an embodiment of the present invention
  • Figure 13b is a genetically modified NK cell line (SL- according to an embodiment of the present invention) K10) is a series of histograms showing the results of analyzing the expression pattern of the gene introduced into the long-term passage through flow cytometry
  • Figure 13c is a long-term passage of the genetically modified NK cell line (SL-K10) according to an embodiment of the present invention This is a series of histograms showing the results of flow cytometry analysis of major NK cell markers.
  • Figure 14a is a histogram showing the results of analysis of the EpCAM expression patterns of RMG-1 (left) and KOC-2S (right), the EpCAM high expression ovarian cancer cell lines used in the present invention by flow cytometry
  • Figure 145b is Genetic modified NK cell line (SL-K10) according to an embodiment, NK101 and NK111 control group treated with RMG-1 (left) and KOC-2S (right) at various E: T ratio and then cancer cell specific apoptosis
  • Figure 14c is a graph showing the results of measurement
  • Figure 14c is a genetically modified NK cell line (SL-K10), and the control group NK111 according to an embodiment of the present invention as a culture medium when co-culture with cancer cells RMG-1 and KOC-2S
  • Figure 14d is a gamma of a genetically modified NK cell line (SL-K10) according to an embodiment of the
  • FIG. 14F shows a variety of irradiation genetically modified NK cell lines in RMG-1 (left), which is an EpCAM high-expressing cancer cell, and KOC-2S (right), which is an EpCAM non-expressing cancer cell, according to an embodiment of the present invention. It is a graph which shows the result of measuring cancer cell specific cell death after processing in E: T ratio.
  • the NK cell coactivator may be any one or more selected from the group consisting of Ly49, natural cytotoxicity receptor (NCR), CD7, CD16, and CD28.
  • apoptotic genes may be further transduced.
  • the apoptosis gene is a uracil phosphoribosyltransferase (UPRT) gene, herpes simplex virus thymidine phosphorylation gene (HSV TK), varicella zoster virus thymidine kinase (VZV TK) Gene, cytosine deminase gene, carboxyl esterase gene, nitroreductase gene, carboxypeptide G2 gene, or inducible caspase 9 (iCas9) gene.
  • UPRT uracil phosphoribosyltransferase
  • HSV TK herpes simplex virus thymidine phosphorylation gene
  • VZV TK varicella zoster virus thymidine kinase
  • polynucleotides encoding cytoplasmic domain deleted TGF ⁇ receptors may be further transduced.
  • the cytoplasmic domain deleted TGF ⁇ receptor may be cytoplasmic domain deleted TGF ⁇ receptor II.
  • the cancer antigen is CD19, CD22, prostate specific antigen (PSA), carcinoembryonic antigen (CEA), CA-125, mucin 1, alphafetoprotein (AFP), epipithelial tumor antigen (ETA), tyrosine Naese, CD52, PD-L1 (programmed death-ligand 1), CTLA4 (cytotoxic T-lymphocyte-associated protein 4), CD20, MAGE (melanoma-associated antigen), FAP (fibroblast activation protein), FLT3 (fms like tyrosine kinase 3), IL13R ⁇ 2 or an epithelial cell adhesion molecule (EpCAM).
  • PSA prostate specific antigen
  • CEA carcinoembryonic antigen
  • CA-125 CA-125
  • mucin 1 mucin 1
  • AFP alphafetoprotein
  • ETA epipithelial tumor antigen
  • tyrosine Naese CD52
  • PD-L1 programmed death-ligand
  • the chimeric antigen receptor may be a fusion protein comprising a ligand or antibody analog-transmembrane domain-co-stimulatory factor-cellular signaling domain that specifically binds to cancer antigen.
  • the antibody analog may be scFv, sdAb, nanobody, V H H, V NAR , VLR, or monobody.
  • the costimulatory factors are CD28, inducible costimulator (ICOS), cytotoxic T lymphocyte associated protein 4 (CTLA4), programmed cell death protein 1 (PD1), B and T lymphocyte associated protein (BTLA), Death receptor 3 (DR3), 4-1BB, CD2, CD7, CD40, CD30, CD27, signaling lymphocyte activation molecule (SLAM), 2B4 (CD244), NKp30, NKp44, NKp46, NKp80, NKG2D (natural-killer group 2, member D) / DAP12 (DNAX-activating protein 12), DAP10, DNAM-1, NTB-A, TIM1 (T-Cell immunoglobulin and mucin domain containing protein 1), TIM2, TIM3, TIGIT, CD226, CD160, LAG3 (lymphocyte activation gene 3), B7-1, B7-H1, glucocorticoid-induced TNFR family related protein (GITR), herpesvirus
  • the intracellular signaling domain may be a CD3 ⁇ domain, a CD16, NKp30, NKp44, NKp46, NKp80, DAP10, or DAP12 of a T cell receptor.
  • one or more genes may be removed as necessary.
  • the gene may be determined according to the genotype of the patient, and may be a gene capable of causing side effects or inhibiting the activity of the administered cells by inducing an excessive immune response upon administration to the patient. Genome editing tools such as TALEN or CRISPR can be used to remove these genes.
  • a pharmaceutical composition for preventing and treating cancer comprising any one of the above genetically modified NK cell line as an active ingredient.
  • the suicide inducing agent is gancyclovir or 6-methoxypurine arabinonucleoside, when the suicide gene is HSV TK or VZV TK, respectively.
  • 5-fluorocytosine 5-FC
  • UPRT uracil phosphoribosyl transferase
  • CPT-11 cytosine deminase
  • CB1954 5 (aziridin-1-yl) -2,4-dinitrobenzamide
  • 4- when the apoptosis gene is carboxypeptide G2.
  • a method for treating cancer in a subject comprising administering to a subject with cancer a therapeutically effective amount of any one of the above genetically modified NK cell lines and optionally suicide inducing agent. Is provided.
  • a transgenic NK cell line transduced to express NK cell coactivator, NK cell proliferation factor, polynucleotides encoding cytoplasmic domain-deleted TGF ⁇ receptor and apoptotic genes, respectively, in an isolated NK cell line Is provided.
  • the NK cell coactivator may be selected from the group consisting of Ly49, natural cytotoxicity receptor (NCR), CD7, CD16, and CD28.
  • NCR natural cytotoxicity receptor
  • the NK cell coactivator may be CD7 and / or CD28.
  • the NK cell proliferation factor is at least one cytokine or cytokine selected from the group consisting of IL-2, IL-12, IL-15, IL-18 and IL-21. It may be a variant.
  • the apoptosis gene is a uracilphosphoribosyltransferase (UPRT) gene, herpes simplex virus thymidine phosphorylation gene (HSV TK), varicella zoster virus thymidine kinase (VZV TK) Gene, cytosine deminase gene, carboxyl esterase gene, nitroreductase gene, carboxypeptide G2 gene, or inducible caspase 9 (iCas9) gene.
  • UPRT uracilphosphoribosyltransferase
  • HSV TK herpes simplex virus thymidine phosphorylation gene
  • VZV TK varicella zoster virus thymidine kinase
  • one or more genes may be removed as necessary.
  • the gene may be determined according to the genotype of the patient, and may be a gene capable of causing side effects or inhibiting the activity of the administered cells by inducing an excessive immune response upon administration to the patient. Genome editing tools such as TALEN or CRISPR can be used to remove these genes.
  • the genetically modified NK cell line may be further transduced with a polynucleotide encoding a chimeric antigen receptor that specifically recognizes a cancer antigen.
  • the cancer antigen can be used any known cancer antigen
  • the cancer antigen is CD19, CD22, PSA (prostate specific antigen), CEA (carcinoembryonic antigen), CA-125, mucin 1, AFP (alphafetoprotein), Epithelial tumor antigen (ETA), tyrosinase, CD52, programmed death-ligand 1 (PD-L1), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), CD20, melanoma-associated antigen (MAGE), fibroblast activation protein), FLT3 (fms like tyrosine kinase 3), IL13R ⁇ 2, or epithelial cell adhesion molecule (EpCAM).
  • the chimeric antigen receptor may be a fusion protein comprising a ligand or an antibody analog-transmembrane domain-co-stimulatory factor-cellular signaling domain that specifically binds to cancer antigens, wherein the antibody analog is scFv, sdAb, It may be a nanobody, V H H, V NAR , VLR, or monobody, the co-stimulatory factors are CD28, inducible costimulator (ICOS), cytotoxic T lymphocyte associated protein 4 (CTLA4), programmed cell death protein 1 (PD1) , B and T lymphocyte associated protein (BTLA), death receptor 3 (DR3), 4-1BB, CD2, CD7, CD40, CD30, CD27, signaling lymphocyte activation molecule (SLAM), 2B4 (CD244), NKp30, NKp44, NKp46 , NKp80, NKG2D (natural-killer group 2, member D) / DAP12 (DNAX-activating protein 12), DAP10,
  • the NK cell coactivator, NK cell proliferation factor, cytoplasmic domain deletion TGF ⁇ receptor and apoptosis genes are expressed individually, simultaneously expressed in one gene construct, or two or more genes. Divided into constructs can be expressed. For example, some of the introduced genes are expressed in the form of a fusion protein linked by a linker or linked to a protease recognition site and automatically cleaved by a protease expressed in a cell to be expressed as a mature protein, or IRES. It is also possible to be expressed as a single mRNA linked to the back, and then expressed as individual proteins during translation.
  • genes constructs are operably linked to one or more promoters and can be inserted into expression vectors optimized for expression in mammalian cells, particularly NK cells, and transduced with a variety of eukaryotic transduction methods.
  • transduction can be performed using a variety of methods, including lipopexen, calcium phosphate transfection, gene gun, electroporation, and genome editing tools such as TALEN or CRISPR for more sophisticated intranuclear transduction. Can be used.
  • 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 to be transcribed into mRNA and translated into the protein by the action of the promoter.
  • An operably linked polynucleotide encoding another protein may allow the particular protein to be expressed in the form of a fusion protein with the other protein.
  • 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.
  • neurofiber-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.
  • the vector may further comprise a polynucleotide encoding a secretory signal.
  • the secretion signals are well known to those skilled in the art.
  • a leader sequence capable of directing the peptide of the present invention to the cell compartment is combined with the coding sequence of the polynucleotide according to an embodiment of the present invention, and preferably the translated protein.
  • 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.
  • a pharmaceutical composition for cancer treatment and cancer prevention comprising the genetically modified NK cell line as an active ingredient.
  • the cancer may be hematological cancer or solid cancer
  • the solid cancer is liver cancer, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, gallbladder cancer, gastric cancer, biliary cancer, colon cancer, head Cervical cancer, esophageal cancer, thyroid cancer, brain tumor, malignant melanoma, prostate cancer, testicular cancer, tongue cancer, lymphoma or leukemia
  • the solid cancer may be metastatic cancer.
  • the pharmaceutical composition of the present invention may contain at least one known active ingredient having an anticancer effect together with the genetically modified NK cell line.
  • the composition for treating cancer of the present invention means a pharmaceutical composition formulated by mixing the genetically modified NK cell line and a known active ingredient together with a pharmaceutically acceptable carrier, and if not formulated together, separately packaged. And may be administered simultaneously or sequentially. In the latter case, it may be referred to as a kit rather than a composition.
  • composition may further include a pharmaceutically acceptable excipient or diluent in addition to the pharmaceutically acceptable carrier.
  • the cell therapy agent may further include a pharmaceutically acceptable carrier, diluent, or excipient in addition to the carrier.
  • compositions according to one embodiment of the invention can be administered by a variety of routes, for example, oral, parenteral, e.g. suppositories, transdermal, intravenous, abdominal, intramuscular, intracranial, intralesional, nasal It can be administered intrathecal, and can also be administered using a sustained release or implantable device for continuous or repeated release.
  • the frequency of administration can be administered once a day or divided into several times within the desired range, the administration period is not particularly limited.
  • kits for treating cancer comprising the genetically modified NK cell line and suicide inducing agent.
  • the cancer treatment kit includes the genetically modified NK cell line and suicide inducer, but the two components are not provided in a mixed form, but are provided separately packaged, and these two components may be administered at the same time or through different routes. It can be, but is distinguished from the general pharmaceutical composition in that it is administered at regular intervals according to the doctor's prescription.
  • the kit of the present invention first administers the genetically modified NK cell line, and then, at an appropriate time point, such as, for example, administration of the genetically modified NK cell line, 1 day, 2 days, 3 days, 4 days, or 5 days after administration.
  • CAR construct is an abbreviation for “chimeric antigen receptor construct” and typically passes through a single chain-based antibody analogue-cell membrane, such as scFv, sdAb, to the antigen recognition site. It is a synthetic protein composed of domain-co-stimulatory factor-intracellular signaling domain, which is transduced into immune cells such as T cells to recognize cancer cell-specific antigens, thereby improving the anticancer activity of these cancer cells of immune cells expressing CAR constructs. It is well known to make.
  • cell suicide gene refers to a gene that induces cytotoxicity or triggers apoptosis mechanisms to induce the death of cells in which the gene is expressed.
  • gene expression itself does not trigger apoptosis, but the treatment of a particular prodrug (prodrug) can lead to cell death by the metabolites of the prodrug caused by the apoptosis gene triggers a cytotoxic or apoptosis mechanism.
  • prodrug prodrug
  • These apoptosis genes include the herpes herpes simplex virus thymidine phosphorylation gene (HSV TK) and 6-methoxypurine arabinonucleoside, which use gancyclovir as a suicide induction signal.
  • Cytosine deminase and uracil phosphoribosyltransferase UPRT
  • irinotecan Varicela zoster virus thymidine kinase (VZV TK)
  • 5-fluorocytosine 5-FC
  • CB1954 aziridin-1-yl) -2,4-dinitrobenzamide
  • CB1954 CPT-11
  • Suicide induction signal using 4-[(2-chloroethyl) (2-mesyloxyethyl) amino] benzoyl-l-glutamic acid (CMDA) as a suicide induction signal, carboxypeptides G2, an intermolecular dimerization dimerizer Inductive casing Izu is a 9 (iCas9) are
  • CD1a, CD3, CD4, CD8, CD14, CD20, CD23, CD34, TCR ⁇ and TCR ⁇ are negative.
  • the isolated NK cell line may be an NK101 cell line deposited with accession number KCTC 13305BP.
  • the scFv may be composed of an amino acid sequence set forth in SEQ ID NO: 3, and the modified Ig Fc domain may consist of an amino acid sequence set forth in SEQ ID NO: 5, wherein the CD28 transmembrane domain May be composed of the amino acid sequence set forth in SEQ ID NO: 7, wherein the DAP10 may consist of the amino acid sequence set forth in SEQ ID NO: 9, the DAP12 may consist of the amino acid sequence set forth in SEQ ID NO: 11, and
  • the CD3 ⁇ domain of the T cell receptor may consist of an amino acid sequence consisting of SEQ ID NO: 13.
  • the EpCAM specific chimeric antigen receptor may be entirely composed of an amino acid sequence as set forth in SEQ ID NO: 1, and the polynucleotide encoding the EpCAM specific chimeric antigen receptor may be composed of a nucleic acid sequence as set forth in SEQ ID NO: 2.
  • DAP10 refers to a transmembrane signal adapter that forms an immune receptor complex, which refers to a hematopoietic cell signal transmitter encoded by a hematopoietic cell signal transducer (HCST) gene, and is used for activation of NK cells and T It is known to play an important role in the survival and proliferation of cells by cellular responses.
  • HCST hematopoietic cell signal transducer
  • DAP12 is a 12 kDa transmembrane protein with high homology with DAP10 and is recognized as an important signaling receptor in NK cells.
  • the genetically modified NK cell line may additionally be transduced with a polynucleotide encoding a NK cell coactivator, thereby expressing the NK cell activator.
  • polynucleotides encoding at least one or more NK cell proliferation factors may be further transduced.
  • the IL-15 may be membrane-bound IL-15.
  • apoptotic genes may be further transduced.
  • the apoptotic gene is a uracil phosphoribosyltransferase (UPRT) gene, herpes simplex virus thymidine phosphorylation gene (HSV TK), varicella zoster virus thymidine kinase (VZV TK) Gene, cytosine deminase gene, carboxyl esterase gene, nitroreductase gene, carboxypeptide G2 gene, or inducible caspase 9 (iCas9) gene.
  • UPRT uracil phosphoribosyltransferase
  • HSV TK herpes simplex virus thymidine phosphorylation gene
  • VZV TK varicella zoster virus thymidine kinase
  • polynucleotides encoding cytoplasmic domain deleted TGF ⁇ receptors may be further transduced.
  • the cytoplasmic domain deleted TGF ⁇ receptor may be cytoplasmic domain deleted TGF ⁇ receptor II.
  • chimeric antigen receptor refers to a kind of fusion protein prepared by fusing a binding portion (variable region) to an antigen of a monoclonal antibody, an intracellular signaling site derived from a lymphocyte activating receptor. .
  • CAR chimeric antigen receptor
  • MHC major histocompatibility complex
  • the protein encoded by the cancer antigen specific chimeric antigen receptor and the apoptosis gene is expressed in the form of a fusion protein, or cloned into a single gene construct, and then transfected with host immune cells. It can be expressed or cloned into separate gene constructs, respectively, and then coexpressed by cotransfecting host immune cells.
  • the polynucleotides encoding the cancer antigen specific chimeric antigen receptor and the apoptotic gene polynucleotides are cloned into the single gene construct, they are operably linked and expressed in separate promoters, or both polynucleotides operate on a single promoter.
  • polynucleotide encoding the cancer antigen specific chimeric antigen receptor and the apoptotic gene polynucleotide are linked to an internal ribosome entry site (IRES) to thereby be expressed polycistronic. can do.
  • IRS internal ribosome entry site
  • 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 to be transcribed into mRNA and translated into the protein by the action of the promoter.
  • An operably linked polynucleotide encoding another protein may allow the particular protein to be expressed in the form of a fusion protein with the other protein.
  • the gene construct may be cloned into an expression vector to transfect host immune cells, which may include various regulators that allow expression of the cloned gene inside, including a promoter.
  • regulators are well known to those skilled in the art. As mentioned above, these usually include regulators responsible for transcription initiation and, optionally, poly-A signals responsible for transcription termination and stabilization of the transcript. Additional regulators may include, in addition to transcriptional regulators, translation enhancers and / or naturally-combined or heterologous promoter regions.
  • regulators that allow expression in mammalian host cells include the CMV-HSV thymidine kinase promoter, SV40, the RSV-promoter (Louse sarcoma virus), human kidney element 1 ⁇ -promoter, glucocorticoid-induced MMTV- Promoters (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, Cell 75: 791-803, 2005).
  • a cell therapy agent for treating cancer containing a therapeutically effective amount of the genetically modified NK cell line as an active ingredient.
  • the cell therapy agent is a kind of pharmaceutical composition, and the pharmaceutically acceptable carrier, additive or excipient required for the formulation of the pharmaceutical composition is as described above.
  • the dosage of the cell therapeutic agent according to an embodiment of the present invention may be 10 7 to 10 11 cells, but may be adjusted according to the sex, age, disease progression, treatment purpose of the patient. In general, this amount will be sufficient to obtain localization in the target cell, eg, cancer antigen overexpressing cancer cell, and to kill the cancer cell, eg, by phagocytosis or lysis.
  • the cell therapy agent may further include a pharmaceutically acceptable carrier, diluent, or excipient in addition to the carrier.
  • compositions according to one embodiment of the 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. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid.
  • Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.
  • the cell therapy according to the present invention if necessary according to the administration method or dosage form, suspensions, dissolution aids, stabilizers, isotonic agents, preservatives, adsorption agents, surfactants, diluents, excipients, pH adjusters, analgesics, buffers And antioxidants may be included as appropriate.
  • Pharmaceutically acceptable carriers and formulations suitable for the present invention including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, latest edition.
  • cell therapeutic agents according to one embodiment of the present invention can be formulated using methods known in the art to allow for rapid release, or sustained or delayed release of the active ingredient when administered to a mammal.
  • Formulations include powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powder forms.
  • Dosage to the patient of the pharmaceutical composition depends on many factors including the patient's height, body surface area, age, specific compound administered, sex, time and route of administration, general health, and other drugs administered simultaneously.
  • Pharmaceutically active protein material may be present in an amount of 1 ng-10 mg / kg body weight per administration; Administration below or above this exemplary range is also contemplated, especially with regard to the above factors. If the dosing regimen is a continuous infusion, it should be in the range of 1 ⁇ g-10 mg units per kilogram of body weight per minute.
  • kits for treating cancer comprising the genetically modified NK cell line and suicide inducing agent.
  • the cancer treatment kit of the present invention includes the genetically modified immune cells and suicide inducing agents, but these two components are not provided in a mixed form and are provided separately packaged, and these two components are provided at the same time or in different routes. It can be administered, but is distinguished from the general composition in that it is administered at regular intervals according to the doctor's prescription.
  • the kit of the present invention first administers the genetically modified immune cells, and then, at an appropriate time point, such as at the time of administration of the genetically modified immune cells, 1 day, 2 days, 3 days, 4 days, 5 days after administration , After 6 days, or after 1 week, after 10 days, after 2 weeks, after 15 days, after 20 days, after 3 weeks, after 25 days, after 4 weeks, or after 30 days, the first dose It may then be administered in multiple doses two or more times at intervals of two days, three days, four days, five days, six days, or one week.
  • the suicide inducing agent depends on the type of apoptotic genes, for example, when the apoptotic gene is HSV TK or VZV TK, respectively, gancyclovir or 6-methoxypurine arabinonucleoside (6-mehoxypurine arabinonucleoside). ), 5-fluorocytosine (5-FC) for cytosine diaminase, and irinotecan (CPT-11) for carboxyl esterase.
  • nitroreductase may be 5 (aziridin-1-yl) -2,4-dinitrobenzamide (CB1954), and in the case of carboxypeptides G2, 4-[(2-chloroethyl) (2-mesyloxyethyl) amino] benzoyl-L-glutamic acid (CMDA), iCas9 may be an iCas9 dimer, and iCas9 dimer may be AP20187 or AP1903.
  • CB1954 carboxypeptides G2
  • CMDA 4-[(2-chloroethyl) (2-mesyloxyethyl) amino] benzoyl-L-glutamic acid
  • iCas9 may be an iCas9 dimer
  • iCas9 dimer may be AP20187 or AP1903.
  • Cancer that can be treated through the use of the cancer treatment kit of the present invention may be blood cancer or solid cancer, the solid cancer is liver cancer, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, gallbladder cancer, gastric cancer, biliary tract cancer And colorectal cancer, head and neck cancer, esophageal cancer, thyroid cancer, brain tumor, malignant melanoma, prostate cancer, testicular cancer, tongue cancer, or bone marrow cancer.
  • the solid cancer is liver cancer, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, gallbladder cancer, gastric cancer, biliary tract cancer And colorectal cancer, head and neck cancer, esophageal cancer, thyroid cancer, brain tumor, malignant melanoma, prostate cancer, testicular cancer, tongue cancer, or bone marrow cancer.
  • the present inventors have isolated a new NK cell line having the characteristics shown in Table 1 from cancer tissues of patients with NK lymphoma, and as a result of investigating various characteristics thereof, as shown in FIGS. It was confirmed that it is a multifunctional NK cell line having both cancer cell killing ability and immunomodulatory ability. In particular, the proliferative capacity is significantly higher than that of NK-92, the only NK cell line currently undergoing clinical trials, and it is identified as an economically viable cell. These cells are named 'NK101' and are located at 181, Sinpsin-gil, Jeongeup-si, Jeollabuk-do, Korea.
  • KCTC Korean Collection for Type Culture
  • Figure 7b is a schematic diagram showing the structure of the gene construct CD7-CD28-CD :: UPRT used in the production of the genetically modified NK cell line SL-K01 according to an embodiment of the present invention to achieve the above object;
  • 9A is a schematic diagram schematically showing the structure of the gene construct mbIL-15-mTGF ⁇ II ⁇ cyto used for the preparation of the genetically modified NK cell line NK111 according to one embodiment of the present invention.
  • NK101 cells accession number KCTC 13305BP
  • the TGF ⁇ receptor was introduced to construct an NK111 cell line capable of enhancing anticancer effects of the NK cells and proliferating NK cells independently of cytokine supplements (see FIGS. 9B and 9C).
  • Figure 7 shows the cell surface markers of the genetically modified NK cell line (SL-K01) and the parent cell line NK101 of the SL-K01, and the conventionally constructed NK cell line KHYG-1 and NK-92 according to an embodiment of the present invention
  • Figure 7a shows the definition of the term CD7 in NK101, KHYG-1 and NK-92 :
  • the term "scFv” is an abbreviation for "single chain variable fragment” and is not a fragment of an actual antibody.
  • the heavy chain variable region (VH) and light chain variable region (VL) of the antibody are referred to as a linker peptide having a size of about 25 aa. It is a kind of fusion protein produced by ligation and is known to have antigen-binding ability despite not being an inherent antibody fragment (Glockshuber et al ., Biochem . 29 (6): 1362-1367, 1990).
  • CD1a, CD3, CD4, CD8, CD14, CD20, CD23, CD34, TCR ⁇ and TCR ⁇ are negative.
  • the EpCAM specific chimeric antigen receptor may be composed of scFv, modified Ig Fc domain, CD28 transmembrane domain, CD3 ⁇ domain of DAP10, DAP12 and T cell receptor that specifically binds to EpCAM. .
  • the scFv may be composed of an amino acid sequence set forth in SEQ ID NO: 3, and the modified Ig Fc domain may consist of an amino acid sequence set forth in SEQ ID NO: 5, wherein the CD28 transmembrane domain May be composed of the amino acid sequence set forth in SEQ ID NO: 7, wherein the DAP10 may consist of the amino acid sequence set forth in SEQ ID NO: 9, the DAP12 may consist of the amino acid sequence set forth in SEQ ID NO: 11, and
  • the CD3 ⁇ domain of the T cell receptor may consist of an amino acid sequence consisting of SEQ ID NO: 13.
  • the genetically modified NK cell line may additionally be transduced with a polynucleotide encoding a NK cell coactivator, thereby expressing the NK cell activator.
  • the NK cell coactivator may be any one or more selected from the group consisting of Ly49, natural cytotoxicity receptor (NCR), CD7, CD16, and CD28.
  • the NK cell coactivator may be CD7 and / or CD28.
  • polynucleotides encoding at least one or more NK cell proliferation factors may be further transduced.
  • the NK cell proliferation factor is at least one cytokine or cytokine selected from the group consisting of IL-2, IL-12, IL-15, IL-18 and IL-21. It may be a variant.
  • the IL-15 may be membrane-bound IL-15.
  • apoptotic genes may be further transduced.
  • the apoptotic gene is a uracil phosphoribosyltransferase (UPRT) gene, herpes simplex virus thymidine phosphorylation gene (HSV TK), varicella zoster virus thymidine kinase (VZV TK) Gene, cytosine deminase gene, carboxyl esterase gene, nitroreductase gene, carboxypeptide G2 gene, or inducible caspase 9 (iCas9) gene.
  • UPRT uracil phosphoribosyltransferase
  • HSV TK herpes simplex virus thymidine phosphorylation gene
  • VZV TK varicella zoster virus thymidine kinase
  • polynucleotides encoding cytoplasmic domain deleted TGF ⁇ receptors may be further transduced.
  • the cytoplasmic domain deleted TGF ⁇ receptor may be cytoplasmic domain deleted TGF ⁇ receptor II.
  • chimeric antigen receptor refers to a kind of fusion protein prepared by fusing a binding portion (variable region) to an antigen of a monoclonal antibody, an intracellular signaling site derived from a lymphocyte activating receptor. .
  • CAR chimeric antigen receptor
  • MHC major histocompatibility complex
  • the protein encoded by the cancer antigen specific chimeric antigen receptor and the apoptosis gene is expressed in the form of a fusion protein, or cloned into a single gene construct, and then transfected with host immune cells. It can be expressed or cloned into separate gene constructs, respectively, and then coexpressed by cotransfecting host immune cells.
  • the polynucleotides encoding the cancer antigen specific chimeric antigen receptor and the apoptotic gene polynucleotides are cloned into the single gene construct, they are operably linked and expressed in separate promoters, or both polynucleotides operate on a single promoter.
  • polynucleotide encoding the cancer antigen specific chimeric antigen receptor and the apoptotic gene polynucleotide are linked to an internal ribosome entry site (IRES) to thereby be expressed polycistronic. can do.
  • IRS internal ribosome entry site
  • regulators that allow expression in mammalian host cells include the CMV-HSV thymidine kinase promoter, SV40, the RSV-promoter (Louse sarcoma virus), human kidney element 1 ⁇ -promoter, glucocorticoid-induced MMTV- Promoters (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.
  • the cell therapy agent is a kind of pharmaceutical composition, and the pharmaceutically acceptable carrier, additive or excipient required for the formulation of the pharmaceutical composition is as described above.
  • the cell therapy agent may further include a pharmaceutically acceptable carrier, diluent, or excipient in addition to the carrier.
  • kits for treating cancer comprising the genetically modified NK cell line and suicide inducing agent.
  • the suicide inducing agent is dependent on the type of apoptotic genes, for example, when the suicide gene is HSV TK or VZV TK, respectively, gancyclovir or 6-methoxypurine arabinonucleoside (6-methoxypurine arabinonucleoside). ), 5-fluorocytosine (5-FC) for cytosine diaminase, and irinotecan (CPT-11) for carboxyl esterase.
  • nitroreductase may be 5 (aziridin-1-yl) -2,4-dinitrobenzamide (CB1954), and in the case of carboxypeptides G2, 4-[(2-chloroethyl) (2-mesyloxyethyl) amino] benzoyl-L-glutamic acid (CMDA), iCas9 may be an iCas9 dimer, and iCas9 dimer may be AP20187 or AP1903.
  • CB1954 carboxypeptides G2
  • CMDA 4-[(2-chloroethyl) (2-mesyloxyethyl) amino] benzoyl-L-glutamic acid
  • iCas9 may be an iCas9 dimer
  • iCas9 dimer may be AP20187 or AP1903.
  • the genetically modified NK cell line and the suicide inducing agent may be administered at the same time, but as described above, may be divided into appropriate administration intervals for optimal effect, the administration interval may be adjusted to maximize the therapeutic activity. .
  • Cancer that can be treated through the use of the cancer treatment kit of the present invention may be blood cancer or solid cancer, the solid cancer is liver cancer, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, gallbladder cancer, gastric cancer, biliary tract cancer And colorectal cancer, head and neck cancer, esophageal cancer, thyroid cancer, brain tumor, malignant melanoma, prostate cancer, testicular cancer, tongue cancer, or bone marrow cancer.
  • the solid cancer is liver cancer, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, gallbladder cancer, gastric cancer, biliary tract cancer And colorectal cancer, head and neck cancer, esophageal cancer, thyroid cancer, brain tumor, malignant melanoma, prostate cancer, testicular cancer, tongue cancer, or bone marrow cancer.
  • the present inventors have isolated a new NK cell line having the characteristics shown in Table 1 from cancer tissues of patients with NK lymphoma, and as a result of investigating various characteristics thereof, as shown in FIGS. It was confirmed that it is a multifunctional NK cell line having both cancer cell killing ability and immunomodulatory ability. In particular, the proliferative capacity is significantly higher than that of NK-92, the only NK cell line currently undergoing clinical trials, and it is identified as an economically viable cell. These cells are named 'NK101' and are located at 181, Sinpsin-gil, Jeongeup-si, Jeollabuk-do, Korea.
  • Figure 7b is a schematic diagram showing the structure of the gene construct CD7-CD28-CD :: UPRT used in the production of the genetically modified NK cell line SL-K01 according to an embodiment of the present invention to achieve the above object;
  • 9A is a schematic diagram schematically showing the structure of the gene construct mbIL-15-mTGF ⁇ 1I ⁇ cyto used for the preparation of the genetically modified NK cell line NK111 according to an embodiment of the present invention.
  • NK101 cells accession number KCTC 13305BP
  • the TGF ⁇ receptor was introduced to construct an NK111 cell line capable of enhancing anticancer effects of the NK cells and proliferating NK cells independently of cytokine supplements (see FIGS. 9B and 9C).
  • Figure 7 shows the cell surface markers of the genetically modified NK cell line (SL-K01) and the parent cell line NK101 of the SL-K01, and the conventionally constructed NK cell line KHYG-1 and NK-92 according to an embodiment of the present invention
  • Figure 7a is a histogram showing the results of flow cytometry analysis of the expression of CD7 and CD28 in NK101, KHYG-1 and NK-92
  • Figure 7c is a gene construct shown in Figure 7b It is a histogram showing the result of confirming the expression of CD7 and CD28 in the transgenic cell line SL-K01 cells according to an embodiment of the present invention by flow cytometry.
  • NK-92 which are known to have high cancer cell killing ability among conventionally constructed NK cell lines, have a characteristic of expressing CD7, and NK-92 cells have CD7 as well as CD28. While NK101 has the characteristic of expressing at the same time, it was confirmed that the expression of the two co-stimulatory factor is not at all. Therefore, the present inventors attempted to evaluate whether cancer cell killing ability is enhanced by introducing CD7 and CD28 into NK101 cells.
  • FIG. 8 is a result of analyzing apoptosis of various cancer cells of the genetically modified NK cell line SL-K01 and the parent cell line NK101 of the SL-K01 according to an embodiment of the present invention
  • FIG. 8A illustrates HDLM-2 and IM-. 9
  • JEKO-1 and K562 cancer cells were cocultured with NK101 or SL-K01 cells at 4: 1 ratio for 24 hours, and then the cancer cell death frequency was measured by flow cytometry.
  • FIG. 8 is a result of analyzing apoptosis of various cancer cells of the genetically modified NK cell line SL-K01 and the parent cell line NK101 of the SL-K01 according to an embodiment of the present invention
  • FIG. 8A illustrates HDLM-2 and IM-. 9
  • JEKO-1 and K562 cancer cells were cocultured with NK101 or SL-K01 cells at 4: 1 ratio for 24 hours, and then the cancer cell death frequency was measured by flow cytometry.
  • FIG. 8B is a graph showing various concentrations of 5 -FC treated with NK101 or SL-K01 cells for 48 hours, the cell growth rate is a graph showing the results confirmed by MTS analysis
  • Figure 8c is an IM-9 cell line and NK101 or SL-K01 cells 2: 1, 1 When co-cultured at a ratio of 1: 1 or 0.5: 1, the graph shows the result of measurement of the frequency of IM-9 cancer cell death by flow cytometry with or without 5-FC.
  • the genetically modified NK cell line SL-K01 shows excellent killing ability in various cancer cell lines compared to NK101, the killing ability of NK101 cells was enhanced through the introduction of CD7, CD28 I could confirm it.
  • Figure 9 shows the construction of a genetically modified NK cell line NK111 cell line according to another embodiment of the present invention
  • Figure 9a is an additional cancer cell killing effect of SL-K01 cells and resistance to the immunosuppressive factor
  • TGF- ⁇ 9B is a schematic diagram of the gene construct introduced for induction
  • FIG. 9B shows IL-15 expression on the surface of SL-K01 and SL-K01 cells (named NK111) into which the gene construct shown in FIG. 9A was introduced.
  • It is a histogram showing the results confirmed by flow cytometry
  • Figure 9c is a histogram showing the results confirmed by flow cytometry TGF ⁇ RII ⁇ cyto expression in SL-K01 and NK111 cells.
  • FIG. 9a is an additional cancer cell killing effect of SL-K01 cells and resistance to the immunosuppressive factor
  • TGF- ⁇ 9B is a schematic diagram of the gene construct introduced for induction
  • FIG. 9B shows IL-15 expression on the surface of SL-K01 and
  • CD7 / CD28 introduction showed a moderate effect on the killing of NK101 cells.
  • the present inventors tried to transduce the membrane-bound IL-15, which is a representative NK cell activating factor, into the SL-K01 cells and to the immunosuppressive factor TGF- ⁇ .
  • TGF- ⁇ immunosuppressive factor
  • FIG. 10 is a result of comparing and analyzing the anticancer activity and safety of NK111, a genetically modified NK cell line according to an embodiment of the present invention
  • Figure 10a shows the number of cells according to the presence or absence of IL-2 in the culture of SL-K01 and NK111 cells
  • 10B is a histogram showing the results of confirming NKG2D expression in NK101, SL-K01 and NK111 cells through flow cytometry
  • FIG. 10C is an IM-9 cell line and SL-K01. Or when NK111 cells are co-cultured at a ratio of 2: 1, 1: 1, or 0.54: 1, the killing frequency of IM-9 cancer cells with or without 5-FC is measured by flow cytometry, and FIG. 10D.
  • NK111 according to an embodiment of the present invention is capable of cell growth independently of IL-2, which increases the convenience in production.
  • NKG2D expression a representative activating receptor of NK cells, was upregulated through the introduction of IL-15, thereby enhancing killing ability against cancer cells.
  • the inventors of the present invention when using a genetically modified NK cell line according to an embodiment of the present invention when transducing a chimeric antigen receptor capable of antigen-specific cell therapy more effective anti-cancer treatment for cancers that express high cancer antigens
  • a genetically modified NK cell line according to an embodiment of the present invention when transducing a chimeric antigen receptor capable of antigen-specific cell therapy more effective anti-cancer treatment for cancers that express high cancer antigens
  • a chimeric antigen receptor construct targeting EpCAM known to be overexpressed in various solid cancers, such as ovarian cancer FIG. 11
  • NK cell-derived cell line To prepare an NK cell-derived cell line, the following process was carried out. A patient-derived extranudal NK lymphoma is placed on a 40 ⁇ m strainer and Cellgro ® stem cell growth medium containing 20% fetal bovine serum (GE Healthcare, USA) and 1% antibiotic (Gibco, USA) SCGM (CellGenix, Germany, hereinafter referred to as 'NK media') was added to 10 mL and then separated into single cells using the shear force of the piston of a 5 mL syringe and suspended.
  • 'NK media' 20% fetal bovine serum
  • SCGM CellGenix, Germany
  • NK cells in single cell suspensions were isolated using NK isolation kit (Milltenyi Biotec, Germany) and then 1000 U / mL of recombinant human IL-2 (rhIL-2; Prometheus Laboratories Inc., USA) Incubated for 3 weeks in the added NK media.
  • NK media containing IL-2 was added twice a week, and it was confirmed that stable cell lines were formed by continuously culturing dividing cell lines up to 30 passages (FIG. 1A). Since the cell line expresses CD56 without expressing CD3, CD20, and CD16, it was confirmed that the origin of the cells is NK cells (FIG. 1B). The cell line was confirmed under a microscope to form a spheroid in culture (Fig.
  • CFDA Carboxyfluorescein Diacetate
  • NK101 is an immortalized cell that can be passaged continuously, forms a colony in culture, and has a characteristic that the phenotype and function are consistent with previously known NK cells.
  • the cell line was named 'NK101' by identifying the cell line and the characteristics of the NK cell.
  • KCTC was deposited on August 7, 2017, and received the accession number of KCTC 13305BP on August 24, 2017. The depositary body is an international depositary body under the Budapest Treaty.
  • the NK101 cell line of the present invention exhibits the phenotype of activated NK cells, and is distinguished from other NK cell lines in that CD25 is highly expressed.
  • CD25 is an indicator of activated NK cells and is a marker of NK cells with high division ability.
  • CD56 dim and CD56 bright can be classified according to the expression of CD56, which is thought to be composed of two populations, each of which is more dominant in cytotoxicity or cytokine production.
  • CD56 bright NK cells show high cell proliferation ability, secrete IFN- ⁇ upon activation by cytokines, and show low cancer cell killing ability.
  • CD56 dim NK cells have low cell proliferation ability, as opposed to CD56 bright NK cells, Secrete IFN- ⁇ and have high cytotoxicity.
  • Recently validated CD56 dim CD62L + NK cells Juelke, K et al ,, Blood , 116 (8): 1299-1307, 2010; Luetke-Eversolh, M et al ., Front.
  • the NK101 cell-administered group showed cell killing ability against various human cancer cell lines.
  • THP-1 (FIG. 6B) after treatment with neutralizing antibodies to CD25, CD62L, DNAM-1, and CD54 (ICAM-1), which are highly expressed in NK cells, to identify the major markers of cell killing ability of NK101 cells, Apoptosis was analyzed after co-culture with K562 (FIG. 6C), Jurkat (FIG. 6D) and an effector cell-to-target cell ratio of 4: 1.
  • K562 FIG. 6C
  • Jurkat FIG. 6D
  • an effector cell-to-target cell ratio of 4: 1 As a result, the cell killing ability of NK101 was reduced by treatment of neutralizing antibodies such as DNAM-1 and CD54 in KP, CD562 in K562 and CD25, CD62L and CD54 in Jurkat.
  • KHYG-1 and NK-92 are known to have high cancer cell killing ability among the established NK cell lines, and they have the characteristic of expressing CD7 and CD28 in common, whereas NK101 does not express both co-stimulatory factors at all.
  • the present inventors attempted to evaluate whether cancer cell killing ability is enhanced when the NK101 is transduced with genes encoding CD7 and CD28.
  • the lentiviral-concentrator mixture was centrifuged at 4000 rpm for 60 minutes, the supernatant was removed, and the recovered lentivirus in pellet form was diluted with 1-2 mL of culture and stored at -80 ° C until use. .
  • Total RNA was isolated using RNA extraction kit (iNtRON, South Korea).
  • Reverse transcription polymerase chain reaction was carried out using a QuantiTect Reverse Transcription Kit (QIAGEN, Germany) to synthesize cDNA, and then mixed the synthesized cDNA with Taq polymerase and primers, and then subjected to heavy enzyme chain reaction (PCR).
  • the amplified PCR product was electrophoresed on a 1% agarose gel to confirm the presence of the CD :: UPRT gene. As a result, as shown in Figure 7d, it was confirmed that the transduced CD :: UPRT gene is normally expressed.
  • NK101 and SL-K01 cells were suspended in the culture medium at a desired ratio, and then 1 mL was dispensed into 24-well plates containing the target tumor cell line and co-cultured at 37 ° C. for 24 hours. As a result, it was confirmed that the cell killing ability increased in all target cell lines (Fig. 8a).
  • apoptosis was further increased in the experimental group treated with 5-FC.
  • the therapeutic effect can be maximized by the bystander effect of killing not only NK cells into which apoptotic genes have been introduced, but also target cancer cells.
  • PWPT-mbIL-15-TGF ⁇ RII ⁇ cyto was prepared by cloning into the transfer vector pWPT (FIG. 9A).
  • MbIL-15-TGF ⁇ RII lentiviral was prepared in the same manner as described in Example 6, and then infected with SL-K01 cells, and only SL-K01 cells expressing the transgene were selectively isolated using a fluorescent activated cell separator. Expression of membrane surface proteins IL-15 and TGF ⁇ RII in the isolated SL-K01-mbIL-15-TGF ⁇ RII cell line was confirmed by flow cytometry (FIG. 9B).
  • the SL-K01 cell line expressing mbIL-15-TGF ⁇ RII was named 'NK111'.
  • NK111-based cell line therapeutics may have high productivity with only a deficiency condition of IL-2, a culture supplement.
  • expression of CD314 (NKG2D), one of the natural killer cell activation receptors, was confirmed by flow cytometry in NK101, SL-K01, and NK111 cell lines (FIG. 10B).
  • IM9 human lymphoblasts
  • C9-labeled IM9 cells were treated with 5-FC together with SL-K01 cells and NK111 cells, and cultured for 48 hours, and cell death was confirmed by flow cytometry.
  • NK111 cells into which mbIL-15 was introduced increased cancer cell killing ability against target cells, and apoptosis was significantly increased in the experimental group treated with 5-FC (FIG. 10C).
  • SL-K01 cells significantly increased the cancer cell killing ability compared to the parental cell line NK101 while maintaining the main characteristics of the parental cell line NK101, and further enhanced the performance of the SL-K01
  • the NK111 cells have increased cancer cell killing ability significantly compared to SL-K01 cells, and can avoid the inhibition of TGF ⁇ -induced cell killing ability, and thus, it is expected that the NK111 cells can be used as a very effective cancer treatment agent when administered in vivo.
  • a gene construct encoding a polynucleotide encoding SEQ ID NO: 13 (SEQ ID NO: 14) is sequentially encoded to the anti-EpCAM single chain variable fragment (scFv). It was to connect to the polynucleotide, and named it as' anti-EpCAM scFv-CAR gene construct (SEQ ID NO: 2) (Fig. 11). Subsequently, the prepared anti-EpCAM scFv-CAR gene construct was inserted into a transfer vector pWPT for lentiviral production to prepare a pWPT / anti-EpCAM scFv-CAR vector.
  • the lentivirals collected in the medium were collected, centrifuged for 5 minutes (4 ° C., 4000 rpm), the supernatant was collected, and the cell debris was removed using a 0.45 ⁇ m filter (Millipore, USA). It was. To concentrate the lentiviral, Lenti-X concentrator (Clontech, USA) reagent was added to the filtered lentiviral supernatant and stored at 4 ° C. overnight.
  • the lentiviral-concentrator mixture was centrifuged at 4000 rpm for 60 minutes, the supernatant was removed, and the recovered lentivirus in pellet form was diluted with 1-2 mL of culture and stored at -80 ° C until use. .
  • the anti-EpCAM scFv-CAR prepared according to the embodiment of the present invention is overexpressed on the surface of the NK111 cell.
  • the anti-EpCAM chimeric antigen of the present invention prepared in the above process Western blot analysis was performed to verify intracellular expression of the receptor. 50 ⁇ g of protein quantified from lysate of anti-EpCAM scFv-CAR expressing NK cell line was isolated by 8% polyacrylamide gel electrophoresis under reducing or non-reducing conditions.
  • the anti-EpCAM scFv-CAR prepared according to the embodiment of the present invention is expressed in the NK111 cell line.
  • the present inventors named the genetically modified NK cell line expressing the anti-EpCAM scFv-CAR as "SL-K10".
  • SL-K10 cell line was passaged for 50 days in NK media. Passage was performed every 3 days except for 2 days immediately after the initial thawing. After 15 passages, flow cytometry was performed for markers related to transgene and NK characteristics using flow cytometry.
  • SL-K10 cell line shows a significantly higher cell killing ability compared to the control NK101 and NK111 for RMG-1, EpCAM overexpressing cells, EpCAM not expressed It was confirmed that KOC-2S cells, which are cells, showed cell killing ability equivalent to NK101 and NK111.
  • KOC-2S cells which are cells, showed cell killing ability equivalent to NK101 and NK111.
  • the amount of granzyme B and IFN- ⁇ secretion involved in cytotoxicity and immune activation of NK cells was measured by ELISA, as shown in FIG. 14C.
  • SL-K10 cell line shows significantly higher levels of Granzyme B and IFN- ⁇ secretion of EpCAM overexpressing cells, RMG-1, compared to control NK111 cells, whereas KOC-2S cells, which are EpCAM non-expressing cells, As for, it was confirmed that the granzyme B and IFN- ⁇ secretion similar to NK111 cells.

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Abstract

La présente invention concerne une cellule immunitaire génétiquement modifiée et son utilisation, la cellule immunitaire génétiquement modifiée possédant dans l'ordre afin de permettre une immunothérapie plus efficace contre des tumeurs solides, une cellule immunitaire hôte transformée de manière à exprimer une protéine du récepteur antigénique chimérique spécifique d'un antigène du cancer (CARD) comprenant un anticorps monoclonal spécifique d'un antigène du cancer ou un fragment fonctionnel de celui-ci, un domaine transmembranaire et un domaine CD3ζ d'un récepteur des lymphocytes T.
PCT/KR2019/003432 2018-03-23 2019-03-25 Lignée de cellules nk génétiquement modifiée ayant un nouveau gène codant pour le récepteur chimérique de l'antigène et son utilisation WO2019182425A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021058563A1 (fr) 2019-09-23 2021-04-01 King's College London Polypeptides de fusion dap10/dap12
WO2022095902A1 (fr) * 2020-11-03 2022-05-12 Hangzhou Qihan Biotechnology Co., Ltd. Systèmes et procédés pour des immunothérapies améliorées
WO2023078287A1 (fr) * 2021-11-03 2023-05-11 Hangzhou Qihan Biotechnology Co., Ltd. Systèmes et procédés pour des immunothérapies améliorées
WO2023180511A1 (fr) * 2022-03-25 2023-09-28 F. Hoffmann-La Roche Ag Récepteurs chimériques améliorés

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102517943B1 (ko) 2019-09-10 2023-04-04 에이치엘만도 주식회사 자동차 조향컬럼
US20210338723A1 (en) * 2020-01-21 2021-11-04 Washington University Compositions and methods for inhibiting or screening for cd8 and methods and assays for detecting cd8 in cells
KR102559355B1 (ko) * 2020-01-31 2023-07-25 주식회사 제넥신 항-taa 항체, 항-pd-l1 항체 및 il-2를 포함하는 융합단백질 및 이의 용도
EP4259285A1 (fr) * 2020-12-09 2023-10-18 Affyimmune Therapeutics, Inc. Double récepteur antigénique chimérique ciblant epcam et icam-1
CA3216524A1 (fr) * 2021-04-12 2022-10-20 Medgene Therapeutics, Inc. Methode d'activation et de proliferation de lymphocytes t cd8+ epuises, de lymphocytes t cd8+ a activite accrue prepares par cette methode, et leur utilisation

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020068044A1 (en) * 1997-04-30 2002-06-06 Hans Klingemann Natural killer cell lines and methods of use
KR20170075785A (ko) * 2014-11-05 2017-07-03 보드 오브 리전츠, 더 유니버시티 오브 텍사스 시스템 유전자 변형된 면역 효과기 세포 및 면역 효과기 세포의 증식용 가공된 세포
KR20180008862A (ko) * 2015-06-10 2018-01-24 난트케이웨스트, 인크. 암을 치료하기 위한 변형된 nk-92 세포

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020068044A1 (en) * 1997-04-30 2002-06-06 Hans Klingemann Natural killer cell lines and methods of use
KR20170075785A (ko) * 2014-11-05 2017-07-03 보드 오브 리전츠, 더 유니버시티 오브 텍사스 시스템 유전자 변형된 면역 효과기 세포 및 면역 효과기 세포의 증식용 가공된 세포
KR20180008862A (ko) * 2015-06-10 2018-01-24 난트케이웨스트, 인크. 암을 치료하기 위한 변형된 nk-92 세포

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BURGA, RACHEL A. ET AL.: "Improving efficacy of cancer immunotherapy by genetic modification of natural killer cells", CYTOTHERAPY, vol. 18, no. 11, 2016, pages 141 0 - 1421, XP055637768 *
POLI, AURELIE ET AL.: "CD 56bright natural killer (NK) cells: an important NK cell subset", IMMUNOLOGY, vol. 126, 2009, pages 458 - 465, XP002536329 *
SAHM, CHRISTIANE ET AL.: "Expression of IL -15 in NK cells results in rapid enrichment and selective cytotoxicity of gene -modified effectors that carry a tumor-specific antigen receptor", CANCER IMMUNOLOGY IMMUNOTHERAPY, vol. 61, no. 9, 2012, pages 1451 - 1461, XP035103279, DOI: 10.1007/s00262-012-1212-x *
TSUCHIYAMA, JUNJIRO ET AL.: "Characterization of a novel human natural killer- cell line (NK-YS) established from natural killer cell lymphoma/leukemia associated with Epstein-Barr virus infection", BLOOD, vol. 92, no. 4, 1998, pages 1374 - 1383, XP055499557 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021058563A1 (fr) 2019-09-23 2021-04-01 King's College London Polypeptides de fusion dap10/dap12
WO2022095902A1 (fr) * 2020-11-03 2022-05-12 Hangzhou Qihan Biotechnology Co., Ltd. Systèmes et procédés pour des immunothérapies améliorées
WO2023078287A1 (fr) * 2021-11-03 2023-05-11 Hangzhou Qihan Biotechnology Co., Ltd. Systèmes et procédés pour des immunothérapies améliorées
WO2023180511A1 (fr) * 2022-03-25 2023-09-28 F. Hoffmann-La Roche Ag Récepteurs chimériques améliorés

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