WO2019182425A1 - Genetically modified nk cell line having novel chimeric antigen receptor-encoding gene transduced therein, and use thereof - Google Patents
Genetically modified nk cell line having novel chimeric antigen receptor-encoding gene transduced therein, and use thereof Download PDFInfo
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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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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- A61K39/4631—Chimeric Antigen Receptors [CAR]
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- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
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- 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
The present invention provides a genetically modified immune cell and a use thereof, the genetically modified immune cell having, in order to enable more efficient immunotherapy for solid tumors, a host immune cell transformed so as to express a cancer antigen-specific chimeric antigen receptor (CAR) protein comprising a cancer antigen-specific monoclonal antibody or a functional fragment thereof, a transmembrane domain, and a CD3ζ domain of a T cell receptor.
Description
본 출원은 2018년 3월 23일자로 출원된 대한민국 특허출원 제2018-0034079 호에 대한 우선권을 주장한다. 상기 특허출원서에 기재된 사항은 본 문서에 참조로 삽입된다.This application claims priority to Korean Patent Application No. 2018-0034079, filed March 23, 2018. The matters described in this patent application are incorporated herein by reference.
본 발명은 키메라 항원 수용체 암호화 유전자로 형질전환된 유전자 변형 면역세포주 및 그의 용도에 관한 것으로서, 보다 상세하게는 항암활성이 향상된 신규 키메라 항원 수용체 암호화 유전자가 형질도입된 유전자 변형 NK 세포주 및 그의 용도에 관한 것이다. 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.
암 면역세포치료는 암환자 자신의 면역세포를 추출하여 증식시킨 뒤, 환자에게 다시 투여하여 암세포의 증식을 억제·제거하는 치료기술이다. 암 면역세포치료에는 다양한 종류의 면역세포 중 수지상세포(dendritic cells, 이하 DCs), 자연살해세포(natural killer cells, 이하 NKs) 및 세포 독성 T 림프구(cytotoxic T lymphocytes, 이하 CTLs)가 주로 사용되고 있다. 특히, CTLs의 경우, 면역기억(immunological memory) 기능, 항원 특이성 및 뛰어난 생체 내 증식 능을 가지고 있다는 장점이 있어, 수지상 세포와 자연살해 세포에 비하여 상대적으로 많은 연구가 이루어지고 있는 실정이다.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. In cancer immune cell therapy, dendritic cells (DCs), natural killer cells (NKs), and cytotoxic T lymphocytes (CTLs) among various kinds of immune cells are mainly used. In particular, 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 세포치료제는 현재까지 3세대까지 개발되고 있는데 제1세대 T 세포치료제는 혈액 또는 암 조직 내 존재하는 모든 T 세포(bulk T cells)를 증식시켜 환자에게 투여하였기 때문에 암세포에 대한 특이성이 낮아 효력을 기대할 수 없었고, 제2세대 T 세포치료제는 종양 항원 특이적 T 세포만(Ag-specific T cells)을 분리/대량 배양하여 암 환자에게 투여하는 방법으로 증진된 치료 효과를 보였으나, 배양기간이 길고 공정이 복잡하다는 문제가 대두되었으며, 제3세대 T 세포치료제는 1) 특정 암항원을 인식하는 TCR 유전자를 T 세포에 직접 도입하거나, 2) 특정 항원을 인식하는 단클론항체의 항원인식부위(scFv)에 T세포 활성화 도메인(T cell activation domain)을 결합시켜 T 세포에 도입함으로써, 항원 특이성을 높이고 제조기간을 단축하였을 뿐 아니라, 그 치료 효능 또한 매우 뛰어나 일부 백혈병 및 림프종에서 100%에 가까운 치료 효과를 유도하였다.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. Unexpectedly, 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. By incorporating a T cell activation domain into the T cells, not only increased antigen specificity and production time, but also the therapeutic efficacy was very high. Words or in some leukemia and lymphoma therapeutic effects induced near 100 percent.
상기와 같은 제3세대 T 세포치료제는 이른바 키메라 항원 수용체(chimeric antigen receptor, 이하, 'CAR'라고 약칭함)를 발현하도록 유전자조작된 것을 특징으로 하고 있는데, 아직까지 임상시험을 통해 승인을 받은 치료제는 존재하지 않는다.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.
상기 CAR 도입 T 세포 기술을 기반으로 가장 앞서가고 있는 기업은 미국의 Novartis, Juno Therapeutics사 그리고 Kite Pharma사로 모두 B 세포 특이 항원인 CD19를 표적화하는 CAR 도입 T 세포를 개발하였으며, 저항성/재발성 급성 림프구성 백혈병(ALL) 및 비호지킨 림프종(NHL)에서 80 내지 90%에 육박하는 높은 치료율을 보여 표적지향성 면역세포치료제 분야의 선두주자로 자리매김하고 있다(Hartmann et al., EMBO Mol. Med. 9(9): 1183Δ1197, 2017).The leading companies based on the CAR-introduced T-cell technology have developed CAR-introduced T-cells targeting CD19, a B-cell-specific antigen, to Novartis, Juno Therapeutics, and Kite Pharma of the United States. leukemia (ALL) and non-Hodgkin's in lymphoma (NHL) demonstrate a high cure rate of nearly 80% to 90% has established itself as a leader in immune targeting of cell therapy field (Hartmann et al., EMBO Mol . Med. 9 (9): 1183Δ1197, 2017).
그러나 상기 CD19 표적 CAR 도입 T 세포의 경우 혈액암의 일종인 ALL 및 NHL만을 적응증으로 하고 있어, 범용성이 떨어지고 B 세포 특이 항원을 표적으로 하고 있다는 점에서 고형암에는 적용될 수 없다는 단점을 가지고 있다.However, 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.
본 발명은 상기 문제점을 포함한 다양한 문제점을 해결하기 위한 것으로서, 고형암의 치료에 효과적인 신규 키메라 항원 수용체 및 세포자살 유전자를 동시에 발현하도록 형질전환된 유전자 변형 NK 세포주, 상기 유전자 변형 NK 세포주를 이용한 암 치료 방법을 제공하는 것을 목적으로 한다. The present invention is to solve a variety of problems, including the above problems, transgenic NK cell line transformed to simultaneously express a novel chimeric antigen receptor and apoptosis gene effective in the treatment of solid cancer, cancer treatment method using the genetically modified NK cell line The purpose is to provide.
본 발명의 일 관점에 따르면, 하기의 특성을 갖는 분리된 NK 세포주에 EpCAM 특이적 키메라 항원 수용체를 암호화하는 폴리뉴클레오타이드가 형질도입되어 상기 EpCAM 특이적 키메라 항원 수용체를 발현하는 유전자 변형 NK 세포주가 제공된다:According to an aspect of the present invention, a polynucleotide encoding an EpCAM specific chimeric antigen receptor is transduced into an isolated NK cell line having the following characteristics to provide a genetically modified NK cell line expressing the EpCAM specific chimeric antigen receptor. :
CD2, CD11a, CD25, CD45, CD54, DNAM-1, CD62L 및 CD56은 양성; 및CD2, CD11a, CD25, CD45, CD54, DNAM-1, CD62L and CD56 are positive; And
CD1a, CD3, CD4, CD8, CD14, CD20, CD23, CD34, TCRαβ 및 TCRγδ는 음성.CD1a, CD3, CD4, CD8, CD14, CD20, CD23, CD34, TCRαβ and TCRγδ are negative.
본 발명의 다른 일 관점에 따르면, 치료적으로 유효한 양의 상기 유전자 변형 NK 세포주를 유효성분으로 함유하는 암 치료용 세포치료제가 제공된다.According to another aspect of the present invention, there is provided a cell therapy agent for treating cancer containing a therapeutically effective amount of the genetically modified NK cell line as an active ingredient.
아울러 본 발명의 또 다른 일 관점에 따르면, 상기 유전자 변형 NK 세포주 및 자살유도제를 포함하는 암 치료용 키트가 제공된다.In addition, according to another aspect of the present invention, there is provided a kit for treating cancer comprising the genetically modified NK cell line and suicide inducing agent.
본 발명의 다른 일 관점에 따르면, 치료적으로 유효한 양의 상기 유전자 변형 NK 세포주 및 선택적으로 자살유도제를 암의 치료를 필요로 하는 개체에 투여하는 단계를 포함하는 상기 개체의 암 치료 방법이 제공된다.According to another aspect of the invention, there is provided a method of treating cancer in a subject comprising administering to the subject in need thereof a therapeutically effective amount of the genetically modified NK cell line and optionally suicide. .
본 발명의 일 실시예에 따른 암항원 특이적인 키메라 항원 수용체 단백질 및 세포자살 유전자를 동시에 발현하는 유전자 변형 면역세포 및 상기 유전자 변형 면역세포 및 자살유도제를 포함하는 암치료용 키트는 암 세포의 사멸에 매우 효율적이기 때문에, 새로운 면역세포치료제로 암의 치료에 유용하게 사용될 수 있다. A cancer therapy kit comprising a genetically modified immune cell expressing a cancer antigen-specific chimeric antigen receptor protein and an apoptotic gene simultaneously and a genetically modified immune cell and a suicide inducing agent according to an embodiment of the present invention are used for killing cancer cells. Because of its high efficiency, new immune cell therapies may be useful for the treatment of cancer.
도 1a는 본 발명의 NK101 세포주의 계대에 따른 세포증식 정도를 나타내는 그래프이고, 도 1b는 NK101 세포가 CD3, CD20, 및 CD16 음성이며 CD56 양성인 NK세포임을 확인한 도트 그래프이다. 도 1c는 상기 NK101 세포의 배양시 확인되는 세포형태를 현미경을 이용하여 촬영한 사진이며, 도 1d는 Wright-Giemsa 염색법을 이용하여 NK101 세포의 형태를 촬영한 사진이고, 도 1e는 상기 NK101 세포가 NK세포의 주요 세포 사멸인자인 Perforin(녹색) 및 Granzyme(적색)을 발현함을 형광염색 기법을 통해 확인한 사진이며, 도 1f는 NK101와 MHC class I 음성 세포인 K562의 공배양을 통해 NK101의 암세포 사멸능을 확인한 결과를 나타내는 그래프이다. 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 dot graph confirming that NK101 cells are CD3, CD20, and CD16 negative and CD56 positive NK cells. Figure 1c is a photograph taken by using a microscope of the morphology of the NK101 cells when cultured, Figure 1d is a photograph of the shape of the NK101 cells using Wright-Giemsa staining method, Figure 1e is the NK101 cells It is confirmed by the fluorescent staining technique that expresses the major cell death factors of NK cells, Perforin (green) and Granzyme (red), Figure 1f is a cancer cell of NK101 through co-culture of NK101 and K562, MHC class I negative cells This graph shows the result of confirming the killing ability.
도 2a NK101와 NK-92의 IL-2 농도 의존적 세포 성장률 및 민감도를 MTS 분석을 이용하여 비교 분석한 그래프이고, 도 2b는 NK101 및 NK-92의 IL-2 수용체 소단위의 발현도의 차이를 유세포 분석을 이용해 확인한 히스토그램이며, 도 2c는 NK101 및 NK-92의 해동 시점부터 세포증식 및 생존율을 동일 배양조건에서 비교한 결과를 나타내는 그래프이고, 도 2d는 배양적응 후 증식정도 및 배가시간을 비교하여 나타낸 그래프이다. Figure 2a is a graph of comparative analysis of the IL-2 concentration-dependent cell growth rate and sensitivity of NK101 and NK-92 using MTS analysis, Figure 2b is a flow cytometric analysis of the difference in the expression of IL-2 receptor subunits of NK101 and NK-92 Figure 2c is a graph showing the results of comparing the cell proliferation and survival rate from the thawing time of NK101 and NK-92 in the same culture conditions, Figure 2d is a comparison of the growth and doubling time after culture adaptation It is a graph.
도 3a는 NK101 세포에서의 주요 계통 (lineage) 또는 조상 표지자 (progenitor marker) 발현에 대한 유세포 분석 결과를 나타내는 히스토그램이고, 도 3b는 NK101 세포에서의 활성화 수용체 및 비활성화 수용체 발현에 대한 유세포 분석 결과를 나타내는 히스토그램이며, 도 3c는 NK101 세포에서의 세포부착 인자 발현에 대한 유세포 분석 결과를 나타내는 히스토그램이고, 도 3d는 NK101 세포에서의 NK 세포 의존적 세포독성에 관여하는 CD107a, 퍼포린, 그랜자임, FasL 및 TRAIL 발현에 대한 유세포 분석 결과를 나타내는 히스토그램이며, 도 3e는 NK101 세포에서의 사이토카인 수용체 발현에 대한 유세포 분석 결과를 나타내는 히스토그램이고, 도 3f는 NK101 세포에서의 다양한 C-C 케모카인 수용체 및 C-X-C 케모카인 수용체 발현에 대한 유세포 분석 결과를 나타내는 히스토그램이다.Figure 3a is a histogram showing the results of flow cytometry analysis of the main lineage or progenitor marker expression in NK101 cells, Figure 3b shows the flow cytometry results for the expression of activated and inactivated receptors in NK101 cells Histogram, FIG. 3C is a histogram showing the results of flow cytometry analysis of cell adhesion factor expression in NK101 cells, and FIG. 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.
도 4a는 NK101, NK-92 및 초도배양 CD56+ 말초혈액 NK 세포의 CD56 발현을 확인한 유세포 분석 결과를 나타내는 히스토그램이고, 도 4b는 상기 세포들의 CD56 및 CD62L 발현을 확인한 유세포 분석 결과를 나타내는 2차원 등고선 그래프이다. 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.
도 5a는 각각 표기된 사이토카인을 3일간 배양액에 처리한 후 NK101 세포의 증식률을 MTS 분석법으로 확인한 그래프(좌측) 및 NK 활성화 사이토카인인 IFN-γ의 생성을 ELISA로 확인한 그래프(우측)이고, 도 5b는 NK101 세포를 K562 또는 THP-1 암세포주와 24시간 공배양한 후 분비되는 사이토카인을 Multiplex 분석법으로 확인한 결과를 나타내는 그래프이다.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-γ 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.
도 6a는 다양한 암세포주와 NK101 세포를 다양한 세포비율로 24시간 공배양하여 암세포의 사멸도를 확인한 결과를 나타내는 그래프이고, 도 6b는 NK101 세포에 각각 표기된 표면항체에 대한 중화항체를 처리하고 급성골수성백혈병 세포주 THP-1과 4:1 비율로 24시간 공배양한 후 세포사멸도를 측정한 그래프이며, 도 6c는 NK101 세포에 각각 표기된 표면항체에 대한 중화항체를 처리하고 만성골수성백혈병 세포주 K562와 4:1 비율로 24시간 공배양한 후 세포사멸도를 측정한 결과를 나타내는 그래프이고, 도 6d는 NK101 세포에 각각 표기된 표면항체에 대한 중화항체를 처리하고 급성림프구성백혈병 세포주 Jurkat과 4:1 비율로 24시간 공배양한 후 세포사멸도를 측정한 결과를 나타내는 그래프이며, 도 6e는 NK101에 각각 DNAM-1, CD54 혹은 DNAM-1 및 CD54 중화항체를 처리한 후, 각 표지자의 동시 중화에 의한 상승적 저해작용(synergistic inhibition)을 확인한 결과를 나타내는 그래프이다.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 A graph showing the result of measuring apoptosis after co-culture at 1: 1 ratio for 24 hours, and 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.
도 7a는 NK101 및 기존에 구축된 세포주 KHYG-1, 및 NK-92에서의 CD7, CD28 보조자극인자의 발현 양상을 유세포 분석을 통해 확인한 결과를 나타내는 히스토그램이고, 도 7b는 NK101 세포의 암세포 사멸 효과 증진을 위해 도입된 유전자 컨스트럭트의 개요도이며, 도 7c는 상기 도 7b에 도시된 유전자 컨스트럭트가 도입된 NK101 세포(SL-K01로 명명)에서의 CD7, CD28 발현을 유세포 분석을 통해 확인한 결과를 나타내는 히스토그램이고, 도 7d는 SL-K01 세포에서의 CD::UPRT 발현을 역전사 PCR을 통해 확인한 결과를 나타내는 사진이다.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, and FIG. 7D is a photograph showing the result confirmed by reverse transcription PCR of CD :: UPRT expression in SL-K01 cells.
도 8a는 HDLM-2, IM-9, JEKO-1 및 K562 암세포를 NK101 또는 SL-K01 세포와 4:1 비율로 24시간 공배양한 후 암세포 사멸빈도를 유세포 분석을 통해 측정한 결과를 나타내는 그래프이고, 도 8b는 다양한 농도의 5-FC를 NK101 또는 SL-K01 세포에 48시간 처리한 뒤, 세포 성장률을 MTS 분석을 통하여 확인한 결과를 나타내는 그래프이며, 도 8c는 IM-9 세포주와 NK101 또는 SL-K01 세포를 2:1, 1:1 또는 0.5:1 비율로 공배양 시, 5-FC 유무에 따른 IM-9 암세포 사멸 빈도를 유세포 분석을 통해 측정한 결과를 나타내는 그래프이다. 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, and 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.
도 9a는 SL-K01 세포의 추가적 암세포 사멸 효과 증진 및 면역억제인자 TGF-β에 대한 저항성 유도를 위해 도입된 유전자 컨스트럭트의 개요도이고, 도 9b는 SL-K01 및 상기 도 9a에 도시된 유전자 컨스트럭트가 도입된 SL-K01 세포(NK111로 명명) 표면에서의 IL-15 발현을 유세포 분석을 통해 확인한 결과를 나타내는 히스토그램이며, 도 9c는 SL-K01 및 NK111 세포에서의 TGFβRIIΔcyto 발현을 유세포 분석을 통해 확인한 결과를 나타내는 히스토그램이다. Figure 9a is a schematic diagram of the gene constructs introduced for the enhancement of additional cancer cell killing effect of SL-K01 cells and the induction of resistance to the immunosuppressive factor TGF-β, Figure 9b is a gene shown in SL-K01 and Figure 9a A histogram showing the results of confirming IL-15 expression on the surface of the construct-induced SL-K01 cells (named NK111) through flow cytometry, and FIG. 9c shows the flow cytometry of TGFβRIIΔcyto expression in SL-K01 and NK111 cells. This is a histogram showing the result of checking through.
도 10a는 SL-K01 및 NK111 세포의 배양 시, IL-2 유무에 따른 세포수 증식수준(population doubling level)을 나타낸 그래프이고, 도 10b는 NK101, SL-K01 및 NK111 세포에서의 NKG2D 발현을 유세포 분석을 통해 확인한 결과를 나타내는 히스토그램이며, 도 10c는 IM-9 세포주와 SL-K01 또는 NK111 세포를 2:1, 1:1, 또는 0.5:1 비율로 공배양 시, 5-FC 유무에 따른 IM-9 암세포의 사멸빈도를 유세포 분석을 통해 측정한 결과를 나타내는 그래프이고, 도 10d는 OVCAR-3 또는 THP-1 세포주를 SL-K01 및 NK111 세포와 공배양시, 다양한 농도의 TGFβ1을 처리한 후, 암세포 사멸능에 미치는 영향을 나타낸 그래프이다.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, and 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. -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 TGFβ1 , Is a graph showing the effect on cancer cell killing ability.
도 11은 본 발명의 일 실시예에 따른 항-EpCAM CAR 컨스트럭트인 anti-EpCAM scFv-CAR-NK 컨스트럭트의 구조를 개략적으로 도시한 개요도이다.11 is a schematic diagram schematically showing the structure of an anti-EpCAM scFv-CAR-NK construct, an anti-EpCAM CAR construct, according to an embodiment of the present invention.
도 12a는 모세포주인 NK111(좌측) 및 본 발명의 일 실시예에 따른 anti-EpCAM CAR 컨스트럭트가 형질도입된 유전자 변형 NK 세포주(SL-K10, 우측)에서의 anti-EpCAM CAR 컨스트럭트의 발현 양상을 유세포 분석으로 측정한 결과를 나타내는 히스토그램이고, 도 12b는 NK111 및 본 발명의 일 실시예에 따른 유전자 변형 NK 세포주(SL-K10)에서 anti-EpCAM CAR 컨스트럭트에 의해 도입된 유전자의 발현을 환원조건 및 비환원조건의 전기영동 후 웨스턴블랏 분석을 통해 확인한 결과를 나타내는 사진이다.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.
도 13a은 본 발명의 일 실시예에 따른 유전자 변형 NK 세포주(SL-K10)의 장기간 계대배양시 세포성장을 나타내는 그래프이고, 도 13b는 본 발명의 일 실시예에 따른 유전자 변형 NK 세포주(SL-K10)의 장기계대시 도입 유전자의 발현양상을 유세포 분석을 통해 분석한 결과를 나타내는 일련의 히스토그램이며, 도 13c는 본 발명의 일 실시예에 따른 유전자 변형 NK 세포주(SL-K10)의 장기계대시 주요 NK 세포 표지자의 발현양상을 유세포 분석을 통해 분석한 결과를 나타내는 일련의 히스토그램이다.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.
도 14a는 본 발명에서 사용된 EpCAM 고발현 난소암 세포주인 RMG-1(좌측) 및 KOC-2S(우측)의 EpCAM 발현양상을 유세포 분석으로 분석한 결과를 나타내는 히스토그램이고, 도 145b는 본 발명의 일 실시예에 따른 유전자 변형 NK 세포주(SL-K10), 대조군인 NK101 및 NK111을 RMG-1(좌측) 및 KOC-2S(우측)에 다양한 E:T 비율로 처리한 후 암세포 특이적 세포사멸능을 측정한 결과를 나타내는 그래프이며, 도 14c는 본 발명의 일 실시예에 따른 유전자 변형 NK 세포주(SL-K10), 및 대조군인 NK111를 암세포인 RMG-1 및 KOC-2S와 공배양시 배양액으로 분비되는 INF-γ(좌측) 및 그랜자임 B(우측)의 농도를 ELISA로 분석한 결과를 나타내는 그래프이고, 도 14d는 본 발명의 일 실시예에 따른 유전자 변형 NK 세포주(SL-K10)의 감마 방사선 조사시 시간의 경과에 따른 세포수(좌측) 및 세포 생존도(우측)를 측정한 결과를 나타내는 그래프이며, 도 14e는 본 발명의 일 실시예에 따른 유전자 변형 NK 세포주(SL-K10)의 방사선 조사 전후의 형질도입 유전자 및 주요 NK 세포 표지자의 발현양상을 비교분석한 유세포 분석 결과를 나타내는 일련의 히스토그램이고, 도 14f는 본 발명의 일 실시예에 따른 방사선 조사 유전자 변형 NK 세포주를 EpCAM 고발현 암세포인 RMG-1(좌측) 및 EpCAM 미발현 암세포인 KOC-2S(우측)에 다양한 E:T 비율로 처리한 후, 암세포 특이적인 세포사 정도를 측정한 결과를 나타내는 그래프이다. 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 It is a graph showing the results of analysis of the concentration of secreted INF-γ (left) and Granzyme B (right) by ELISA, Figure 14d is a gamma of a genetically modified NK cell line (SL-K10) according to an embodiment of the present invention The number of cells (left) and cell viability (right) over time when irradiated 14E is a graph showing a result, and flow cytometry results of comparing and analyzing expression patterns of transgenic genes and major NK cell markers before and after irradiation of the transgenic NK cell line (SL-K10) according to an embodiment of the present invention. 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.
도 15a는 본 발명의 일 실시예에 따른 유전자 변형 NK 세포주(SL-K10)의 생체내 항암활성을 확인하기 위한 동물실험 설계를 나타내는 투여스케쥴이고, 도 15b는 상기 도 15a의 투여 스케쥴대로 본 발명의 일 실시예에 따른 유전자 변형 NK 세포주(SL-K10)를 정맥내 또는 복강내 투여시 시간의 경과에 따른 종양 성장정도를 나타낸 그래프이며, 도 15c는 본 발명의 일 실시예에 따른 유전자 변형 NK 세포주(SL-K10)를 단독으로 또는 세포자살 유도제인 5-FC와의 병용투여(i.p.)에 의한 효과를 확인하기 위한 동물실험 설계를 나타내는 투여스케쥴이고, 도 15d는 상기 도 15c의 투여 스케쥴 대로 유전자 변형 NK 세포주(SL-K10) 단독 또는 5-FC와의 병용 투여시(i.p.) 시간의 경과에 따른 종양 성장정도를 나타내는 그래프이이며, 도 15e는 상기 도 15d의 실험동물로부터 수득된 생체내 생물발광 이미지이다.Figure 15a is a dosing schedule showing the animal experiment design for confirming the anticancer activity of the genetically modified NK cell line (SL-K10) according to an embodiment of the present invention, Figure 15b is the invention according to the dosing schedule of Figure 15a Genetically modified NK cell line (SL-K10) according to an embodiment of the intravenous or intraperitoneal administration is a graph showing the degree of tumor growth over time, Figure 15c genetically modified NK according to an embodiment of the present invention Dosage schedule showing the animal experiment design for confirming the effect of the cell line (SL-K10) alone or in combination with the apoptosis inducer 5-FC (ip), Figure 15d is a gene according to the administration schedule of Figure 15c This is a graph showing the degree of tumor growth over time when modified NK cell line (SL-K10) alone or in combination with 5-FC (ip), FIG. 15E shows in vivo obtained from the experimental animals of FIG. 15D. A water emission image.
본 발명의 일 관점에 따르면, 하기 특성을 갖는 분리된 NK 세포주에 NK 세포 보조활성화 인자를 암호화하는 폴리뉴클레오타이드가 형질도입되어 상기 NK 세포 보조활성화 인자가 발현되는, 유전자 변형 NK 세포주가 제공된다: According to one aspect of the present invention, there is provided a genetically modified NK cell line in which a polynucleotide encoding an NK cell coactivator is transduced into an isolated NK cell line having the following characteristics such that the NK cell coactivator is expressed:
CD2, CD11a, CD25, CD45, CD54, DNAM-1, CD62L 및 CD56은 양성; 및CD2, CD11a, CD25, CD45, CD54, DNAM-1, CD62L and CD56 are positive; And
CD1a, CD3, CD4, CD8, CD14, CD20, CD23, CD34, TCRαβ 및 TCRγδ는 음성.CD1a, CD3, CD4, CD8, CD14, CD20, CD23, CD34, TCRαβ and TCRγδ are negative.
상기 유전자 변형 NK 세포주에 있어서, 상기 분리된 NK 세포주는 수탁번호 KCTC 13305BP로 기탁된 NK101 세포주일 수 있다.In the genetically modified NK cell line, the isolated NK cell line may be an NK101 cell line deposited with accession number KCTC 13305BP.
상기 유전자 변형 NK 세포주에 있어서, 상기 NK 세포 보조활성화 인자는 Ly49, NCR(natural cytotoxicity receptor), CD7, CD16 및 CD28로 구성되는 군으로부터 선택되는 어느 하나 이상일 수 있다.In the genetically modified NK cell line, 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.
상기 유전자 변형 NK 세포주에 있어서, 상기 NK 세포 보조활성화 인자는 CD7 및/또는 CD28일 수 있다.In the genetically modified NK cell line, the NK cell coactivator may be CD7 and / or CD28.
상기 유전자 변형 NK 세포주에 있어서, 적어도 하나 이상의 NK 세포 증식 인자를 암호화하는 폴리뉴클레오타이드가 추가로 형질도입될 수 있다.In the genetically modified NK cell line, polynucleotides encoding at least one or more NK cell proliferation factors may be further transduced.
상기 유전자 변형 NK 세포주에 있어서, 상기 NK 세포 증식 인자는 IL-2, IL-12, IL-15, IL-18 및 IL-21로 구성되는 군으로부터 선택되는 적어도 하나 이상의 사이토카인 또는 상기 사이토카인의 변이체일 수 있다.In the genetically modified NK cell line, 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.
상기 유전자 변형 NK 세포주에 있어서, 상기 IL-15는 막결합 IL-15일 수 있다.In the genetically modified NK cell line, the IL-15 may be membrane-bound IL-15.
상기 유전자 변형 NK 세포주에 있어서, 세포자살 유전자가 추가로 형질도입될 수 있다.In the genetically modified NK cell line, apoptotic genes may be further transduced.
상기 유전자 변형 NK 세포주에 있어서, 상기 세포자살 유전자는 우라실 포스포리보실전이효소(UPRT) 유전자, 헤르페스 단순포진 바이러스 티미딘 인산화 유전자(HSV TK), 바리셀라 조스터 바이러스 티미딘 인산화효소(VZV TK) 유전자, 시토신 디아미네이즈 유전자, 카르복실 에스터레이즈 유전자, 니트로리덕테이즈 유전자, 카르복시펩티데이즈 G2 유전자, 또는 유도성 카스페이즈 9(iCas9)유전자일 수 있다.In the genetically modified NK cell line, 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.
상기 유전자 변형 NK 세포주에 있어서, 세포질 도메인 결실 TGFβ 수용체를 암호화하는 폴리뉴클레오타이드가 추가로 형질도입될 수 있다.In such genetically modified NK cell lines, polynucleotides encoding cytoplasmic domain deleted TGFβ receptors may be further transduced.
상기 유전자 변형 NK 세포주에 있어서, 상기 세포질 도메인 결실 TGFβ 수용체는 세포질 도메인 결실 TGFβ 수용체II일 수 있다.In the genetically modified NK cell line, the cytoplasmic domain deleted TGFβ receptor may be cytoplasmic domain deleted TGFβ receptor II.
상기 유전자 변형 NK 세포주에 있어서, 암항원을 특이적으로 인식하는 키메라 항원 수용체를 암호화하는 폴리뉴클레오타이드가 추가로 형질도입될 수 있다.In the genetically modified NK cell line, a polynucleotide encoding a chimeric antigen receptor that specifically recognizes a cancer antigen may be further transduced.
상기 유전자 변형 NK 세포주에 있어서, 상기 암항원은 CD19, CD22, PSA(prostate specific antigen), CEA(carcinoembryonic antigen), CA-125, mucin 1, AFP(alphafetoprotein), ETA(epithelial tumor antigen), 티로시네이즈, 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), IL13Rα2 또는 EpCAM(epithelial cell adhesion molecule)일 수 있다.In the genetically modified NK cell line, 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), IL13Rα2 or an epithelial cell adhesion molecule (EpCAM).
상기 유전자 변형 NK 세포주에 있어서, 상기 키메라 항원 수용체는 암항원에 특이적으로 결합하는 리간드 또는 항체유사체-막통과 도메인-보조자극인자-세포내 신호전달 도메인을 포함하는 융합단백질일 수 있다.In the genetically modified NK cell line, 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.
상기 유전자 변형 NK 세포주에 있어서, 상기 항체유사체는 scFv, sdAb, 나노바디, VHH, VNAR, VLR, 또는 모노바디일 수 있다.In the genetically modified NK cell line, the antibody analog may be scFv, sdAb, nanobody, V H H, V NAR , VLR, or monobody.
상기 유전자 변형 NK 세포주에 있어서, 상기 보조자극인자는 CD28, ICOS(inducible costimulator), CTLA4(cytotoxic T lymphocyte associated protein 4), PD1(programmed cell death protein 1), BTLA(B and T lymphocyte associated protein), DR3(death receptor 3), 4-1BB, CD2, CD7, CD40, CD30, CD27, SLAM(signaling lymphocyte activation molecule), 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, GITR(glucocorticoid-induced TNFR family related protein), HVEM(herpesvirus entry mediator) 또는 OX40L[ligand for CD134(OX40), CD252]의 세포질 도메인 또는 이들 중 둘 이상의 연결체일 수 있다.In the genetically modified NK cell line, 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 entry mediator (HVEM) or cytoplasmic domain of ligand for CD134 (OX40), CD252] or two or more of these It may be a linking body.
상기 유전자 변형 NK 세포주에 있어서, 상기 세포내 신호전달 도메인은 T 세포 수용체의 CD3ξ 도메인, CD16, NKp30, NKp44, NKp46, NKp80, DAP10, 또는 DAP12일 수 있다.In the genetically modified NK cell line, the intracellular signaling domain may be a CD3ξ domain, a CD16, NKp30, NKp44, NKp46, NKp80, DAP10, or DAP12 of a T cell receptor.
상기 유전자 변형 NK 세포주에 있어서, 필요에 따라 하나 또는 둘 이상의 유전자가 제거될 수 있다. 상기 유전자는 환자의 유전자형에 따라서 결정될 수 있는데, 환자에게 투여시 과도한 면역반응을 유도함으로써 부작용을 일으키거나 투여된 세포의 활성을 저해할 수 있는 유전자일 수 있다. 이러한 유전자의 제거에는 TALEN, 또는 CRISPR와 같은 게놈 편집 도구가 사용될 수 있다.In the genetically modified NK cell line, 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.
본 발명의 다른 일 관점에 따르면, 상기 중 어느 하나의 유전자 변형 NK 세포주를 유효성분으로 포함하는 암 예방 및 치료용 약학적 조성물이 제공된다.According to another aspect of the invention, there is provided a pharmaceutical composition for preventing and treating cancer comprising any one of the above genetically modified NK cell line as an active ingredient.
본 발명의 다른 일 관점에 따르면, 상기 중 어느 하나의 유전자 변형 NK 세포주 및 자살유도제를 포함하는 암 치료용 키트가 제공된다.According to another aspect of the present invention, there is provided a cancer treatment kit comprising any one of the above genetically modified NK cell line and suicide inducing agent.
상기 암 치료용 키트에 있어서, 상기 자살유도제는 상기 세포자살 유전자가 HSV TK 또는 VZV TK인 경우에는 각각 간시클로비르(gancyclovir) 또는 6-메톡시퓨린 아라비노뉴클레오사이드(6-mehoxypurine arabinonucleoside), 상기 세포자살 유전자가 우라실 포스포리보실 전이효소(UPRT) 또는 시토신 디아미네이즈인 경우 5-플루오로시토신(5-FC), 상기 세포자살 유전자가 카르복실 에스터라제인 경우 이리노테칸(CPT-11), 상기 세포자살 유전자가 니트로리덕테이즈인 경우에는 5(아지리딘-1-일)-2,4-디니트로벤자마이드(CB1954), 상기 세포자살 유전자가 카르복시펩티데이즈 G2인 경우에는 4-[(2-클로로에틸)(2-메실록시에틸)아미노]벤조일-엘-글루탐산(CMDA), 상기 세포자살 유전자가 iCas9일 경우 iCas9 이량화제(dimerizer)일 수 있다.In the cancer treatment kit, 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) when the apoptosis gene is uracil phosphoribosyl transferase (UPRT) or cytosine deminase, irinotecan (CPT-11) when the apoptosis gene is carboxyl esterase, 5 (aziridin-1-yl) -2,4-dinitrobenzamide (CB1954) when the apoptosis gene is nitroreductase, and 4- [when the apoptosis gene is carboxypeptide G2. (2-chloroethyl) (2-mesyloxyethyl) amino] benzoyl-l-glutamic acid (CMDA), iCas9 dimerizer when the apoptosis gene is iCas9.
본 발명의 다른 일 관점에 따르면, 치료적으로 유효한 양의 상기 중 어느 하나의 유전자 변형 NK 세포주 및 선택적으로 자살유도제를 추가로 암에 걸린 개체에 투여하는 단계를 포함하는 상기 개체의 암 치료방법이 제공된다.According to another aspect of the invention, there is provided 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.
본 발명의 다른 일 관점에 따르면, 분리된 NK 세포주에 NK 세포 보조활성화 인자, NK 세포 증식 인자, 세포질 도메인 결실 TGFβ 수용체를 각각 암호화하는 폴리뉴클레오타이드 및 세포자살 유전자가 발현되도록 형질도입된 유전자 변형 NK 세포주가 제공된다. According to another aspect of the present invention, a transgenic NK cell line transduced to express NK cell coactivator, NK cell proliferation factor, polynucleotides encoding cytoplasmic domain-deleted TGFβ receptor and apoptotic genes, respectively, in an isolated NK cell line Is provided.
상기 유전자 변형 NK 세포주에 있어서, 상기 분리된 NK 세포주는 하기의 특성을 가질 수 있다:In the genetically modified NK cell line, the isolated NK cell line may have the following characteristics:
CD2, CD11a, CD25, CD45, CD54, DNAM-1, CD62L, 및 CD56은 양성; 및CD2, CD11a, CD25, CD45, CD54, DNAM-1, CD62L, and CD56 are positive; And
CD1a, CD3, CD4, CD8, CD14, CD20, CD23, CD34, TCRαβ 및 TCRγδ는 음성.CD1a, CD3, CD4, CD8, CD14, CD20, CD23, CD34, TCRαβ and TCRγδ are negative.
상기 유전자 변형 NK 세포주에 있어서, 상기 분리된 NK 세포주는 수탁번호 KCTC 13305BP로 기탁된 NK101 세포주일 수 있다.In the genetically modified NK cell line, the isolated NK cell line may be an NK101 cell line deposited with accession number KCTC 13305BP.
상기 유전자 변형 NK 세포주에 있어서, 상기 NK 세포 보조활성화 인자는 Ly49, NCR(natural cytotoxicity receptor), CD7, CD16 및 CD28로 구성되는 군으로부터 선택되는 일 수 있다. 바람직하게는, 상기 NK 세포 보조활성화 인자는 CD7 및/또는 CD28일 수 있다.In the genetically modified NK cell line, the NK cell coactivator may be selected from the group consisting of Ly49, natural cytotoxicity receptor (NCR), CD7, CD16, and CD28. Preferably, the NK cell coactivator may be CD7 and / or CD28.
상기 유전자 변형 NK 세포주에 있어서, 상기 NK 세포 증식 인자는 IL-2, IL-12, IL-15, IL-18 및 IL-21로 구성되는 군으로부터 선택되는 적어도 하나 이상의 사이토카인 또는 상기 사이토카인의 변이체일 수 있다.In the genetically modified NK cell line, 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.
상기 유전자 변형 NK 세포주에 있어서, 상기 세포질 도메인 결실 TGFβ 수용체는 세포질 도메인 결실 TGFβ 수용체II일 수 있다.In the genetically modified NK cell line, the cytoplasmic domain deleted TGFβ receptor may be cytoplasmic domain deleted TGFβ receptor II.
상기 유전자 변형 NK 세포주에 있어서, 상기 세포자살 유전자는 우라실포스포리보실전이효소(UPRT) 유전자, 헤르페스 단순포진 바이러스 티미딘 인산화 유전자(HSV TK), 바리셀라 조스터 바이러스 티미딘 인산화효소(VZV TK) 유전자, 시토신 디아미네이즈 유전자, 카르복실 에스터레이즈 유전자, 니트로리덕테이즈 유전자, 카르복시펩티데이즈 G2 유전자, 또는 유도성 카스페이즈 9(iCas9)유전자일 수 있다.In the genetically modified NK cell line, 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.
상기 유전자 변형 NK 세포주에 있어서, 필요에 따라 하나 또는 둘 이상의 유전자가 제거될 수 있다. 상기 유전자는 환자의 유전자형에 따라서 결정될 수 있는데, 환자에게 투여시 과도한 면역반응을 유도함으로써 부작용을 일으키거나 투여된 세포의 활성을 저해할 수 있는 유전자일 수 있다. 이러한 유전자의 제거에는 TALEN, 또는 CRISPR와 같은 게놈 편집 도구가 사용될 수 있다.In the genetically modified NK cell line, 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.
상기 유전자 변형 NK 세포주는 암항원을 특이적으로 인식하는 키메라 항원 수용체를 암호화하는 폴리뉴클레오타이드가 추가로 형질도입된 것일 수 있다.The genetically modified NK cell line may be further transduced with a polynucleotide encoding a chimeric antigen receptor that specifically recognizes a cancer antigen.
이 때, 상기 암항원은 공지된 어떠한 암항원도 사용이 가능한데, 상기 암항원은 CD19, CD22, PSA(prostate specific antigen), CEA(carcinoembryonic antigen), CA-125, mucin 1, AFP(alphafetoprotein), ETA(epithelial tumor antigen), 티로시네이즈, 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), IL13Rα2 또는 EpCAM(epithelial cell adhesion molecule)일 수 있다.At this time, 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), IL13Rα2, or epithelial cell adhesion molecule (EpCAM).
아울러, 상기 키메라 항원 수용체는 암항원에 특이적으로 결합하는 리간드 또는 항체유사체-막통과 도메인-보조자극인자-세포내 신호전달 도메인을 포함하는 융합단백질을 수 있고, 상기 항체유사체는 scFv, sdAb, 나노바디, VHH, VNAR, VLR, 또는 모노바디일 수 있으며, 상기 보조자극인자는 CD28, ICOS(inducible costimulator), CTLA4(cytotoxic T lymphocyte associated protein 4), PD1(programmed cell death protein 1), BTLA(B and T lymphocyte associated protein), DR3(death receptor 3), 4-1BB, CD2, CD7, CD40, CD30, CD27, SLAM(signaling lymphocyte activation molecule), 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, GITR(glucocorticoid-induced TNFR family related protein), HVEM(herpesvirus entry mediator) 또는 OX40L[ligand for CD134(OX40), CD252]의 세포질 도메인 또는 이들 중 둘 이상의 연결체일 수 있고, 상기 세포내 신호전달 도메인은 T 세포 수용체의 CD3ξ 도메인, CD16, NKp30, NKp44, NKp46, NKp80, DAP10 또는 DAP12일 수 있다.In addition, 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, DNAM-1, NTB-A, 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-induced TNFR family related protein (GITR) , A herpesvirus entry mediator (HVEM) or a cytoplasmic domain of OX40L [ligand for CD134 (OX40), CD252] or two or more of these, wherein the intracellular signaling domain is the CD3ξ domain of the T cell receptor, CD16, NKp30, It may be NKp44, NKp46, NKp80, DAP10 or DAP12.
상기 유전자 변형 NK 세포주에 있어서, 상기 NK 세포 보조활성화 인자, NK 세포 증식 인자, 세포질 도메인 결실 TGFβ 수용체 및 세포자살 유전자는 개별적으로 발현되거나, 하나의 유전자 컨스트럭트 내에서 동시에 발현되거나, 둘 이상의 유전자 컨스트럭트로 나뉘어 발현될 수 있다. 예컨대, 상기 도입 유전자들 중 일부는 링커로 연결된 융합단백질의 형태로 발현되거나 단백질 분해효소 인식부위로 연결되어 세포내에서 발현되는 단백질 분해효소에 의해 자동적으로 절단되어 성숙된 단백질로 발현이 되거나, IRES 등으로 연결되어 하나의 mRNA로 발현된 후, 번역시 개별적인 단백질로 발현되는 것도 가능하다. 이들 유전자 컨스트럭트들은 하나 또는 그 이상의 프로모터에 작동 가능하게 연결되어 포유동물 세포 특히 NK 세포에서의 발현에 최적화된 발현벡터에 삽입되어 다양한 진핵세포 형질도입법으로 형질도입될 수 있다. 이러한 형질도입법에는 리포펙센, 인산칼슘 형질감염, 유전자총(gene gun), 전기천공법(electroporation) 등 다양한 방법이 사용될 수 있으며, 더욱 정교한 핵내 형질도입을 위해 TALEN, 또는 CRISPR와 같은 게놈 편집 도구가 사용될 수 있다.In the genetically modified NK cell line, the NK cell coactivator, NK cell proliferation factor, cytoplasmic domain deletion TGFβ 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. These gene 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. Such 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.
본 문서에서 사용되는 "작동가능하게 연결된(operably linked to)"은 특정 폴리뉴클레오타이드가 그 기능을 발휘할 수 있게 다른 폴리뉴클레오타이드에 연결된 것을 의미한다. 즉, 특정 단백질을 암호화하는 폴리뉴클레오타이드가 프로모터에 작동가능하게 연결되었다는 것은 당해 프로모터의 작용에 의해 mRNA로 전사되고 당해 단백질로 번역까지 될 수 있게 연결되었다는 것을 의미하고, 특정 단백질을 암호화하는 폴리뉴클레오타이드가 다른 단백질을 암호화하는 폴리뉴클레오타이드에 작동 가능하게 연결되었다는 것은 당해 특정 단백질이 다른 단백질과 융합단백질의 형태로 발현될 수 있다.As used herein, "operably linked to" means that a particular polynucleotide is linked to another polynucleotide so that it can function. In other words, 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.
상기 진핵세포 및 원핵세포에서 발현을 가능하게 하는 조절 인자들은 당업자에게 잘 알려져 있다. 상술한 바와 같이, 이들은 보통 전사개시를 담당하는 조절인자들 및, 선택적으로 전사물의 전사종결 및 안정화를 담당하는 폴리-A 신호를 포함한다. 추가적인 조절인자들은 전사조절인자 외에도 번역 증진인자 및/또는 천연-조합 또는 이종성 프로모터 영역을 포함할 수 있다. 예를 들어 포유류 숙주 세포에서 발현을 가능하게 하는 가능한 조절인자들은 CMV-HSV 티미딘 키나아제 프로모터, SV40, RSV(로우스 육종 바이러스) 프로모터, 인간 신장 요소 1α-프로모터, 글루코코르티코이드-유도성 MMTV-프로모터(몰로니 마우스 종양 바이러스), 메탈로티오네인-유도성 또는 테트라사이클린-유도성 프로모터 또는, CMV 증폭제 또는 SV40-증폭제와 같은 증폭제를 포함한다. 신-경 세포 내 발현을 위해, 신경미세섬유-프로모터(neurofilament-promoter), PGDF-프로모터, NSE-프로모터, PrP-프로모터 또는 thy-1-프로모터들이 사용될 수 있다는 것이 고려되고 있다. 상기 프로모터들은 당 분야에 알려져 있으며, 문헌(Charron, J. Biol. Chem. 1995, 270: 25739-25745)에 기술되어 있다. 원핵세포내 발현을 위해, lac-프로모터, tac-프로모터 또는 trp 프로모터를 포함하는 다수의 프로모터들이 개시되어 있다. 전사를 개시할 수 있는 인자들 외에, 상기 조절인자들은 본 발명의 일 실시예에 따른 폴리뉴클레오타이드의 하류(downstream)에 SV40-폴리-A 부위 또는 TK-폴리-A 부위와 같은 전사 종결 신호를 포함할 수도 있다. 본 문서에서, 적당한 발현 벡터들은 당 분야에 알려져 있으며, 그 예로는 오카야마-베르그(Okayama-Berg) cDNA 발현 벡터 pcDV1(Parmacia), pRc/CMV, pcDNA1, pcDNA3(In-vitrogene), pSPORT1(GIBCO BRL), pX(Pagano (1992) Science 255, 1144-1147), 효모 2-혼성(two-hybrid) 벡터, 가령 pEG202 및 dpJG4-5(Gyuris et al., Cell 75, 791-803, 1995) 또는 원핵 발현 벡터, 가령 람다 gt11 또는 pGEX(Amersham-Pharmacia)가 있다. 본 발명의 핵산 분자들 외에, 벡터는 분비 신호를 암호화하는 폴리뉴클레오타이드를 추가로 포함할 수 있다. 상기 분비신호들은 당업자에게 잘 알려져 있다. 그리고, 사용된 발현 시스템에 따라, 본 발명의 펩타이드를 세포 구획으로 이끌 수 있는 리더서열(leader sequence)이 본 발명의 일 실시예에 따른 폴리뉴클레오타이드의 코딩 서열에 조합되며, 바람직하게는 해독된 단백질 또는 이의 단백질을 세포질 주변 또는 세포외 매질로 직접 분비할 수 있는 리더 서열이다. 선택적으로, 이종 서열은 발현된 재조합 생성물의 안정화 또는 간단한 정제와 같은 목적하는 특성들을 부여하는 C-말단 또는 N-말단 태그(tag) 펩타이드를 포함하는 융합단백질(fusion protein)을 코딩할 수 있다. 이러한 태그로는 FLAG, GST(glutathione S transferase), HisX6 등이 존재하나, 이로 제한되는 것은 아니다. 본 발명의 일 실시예에 따른 벡터가 적당한 숙주세포 또는 비인간 숙주개체에 형질도입되면, 상기 숙주세포 또는 숙주개체는 뉴클레오티드 서열의 고수준 발현에 적당한 조건하에서 유지된다. Regulatory factors that allow expression in the eukaryotic and prokaryotic cells 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. For example, 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 1α-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. For expression in neural cells, it is contemplated that neurofiber-promoter, PGDF-promoter, NSE-promoter, PrP-promoter or thy-1-promoter may be used. Such promoters are known in the art and described in Charron, J. Biol. Chem. 1995, 270: 25739-25745. For prokaryotic expression, a number of promoters have been disclosed, including lac-promoters, tac-promoters or trp promoters. In addition to factors capable of initiating transcription, 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. In this document, 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). In addition to the nucleic acid molecules of the invention, the vector may further comprise a polynucleotide encoding a secretory signal. The secretion signals are well known to those skilled in the art. And, depending on the expression system used, 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. Or a leader sequence capable of secreting its protein directly around the cytoplasm or into an extracellular medium. Optionally, 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. When the vector according to one embodiment of the present invention is transduced into a suitable host cell or non-human host object, the host cell or host object is maintained under conditions suitable for high level expression of the nucleotide sequence.
본 발명의 다른 일 관점에 따르면, 상기 유전자 변형 NK 세포주를 유효성분으로 포함하는 암치료용 및 암 예방용 약학적 조성물이 제공된다.According to another aspect of the invention, there is provided a pharmaceutical composition for cancer treatment and cancer prevention comprising the genetically modified NK cell line as an active ingredient.
상기 약학적 조성물에 있어서, 상기 암은 혈액암 또는 고형암일 수 있고, 상기 고형암은 간암, 폐암, 췌장암, 유방암, 난소암, 자궁내막암, 자궁경부암, 담낭암, 위암, 담도암, 대장암, 두경부암, 식도암, 갑상선암, 뇌종양, 악성 흑색종, 전립선암, 고환암, 설암, 림프종 또는 백혈병일 수 있으며, 상기 고형암은 전이성 암일 수 있다.In the pharmaceutical composition, 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.
본 발명의 약학적 조성물은 상기 유전자 변형 NK 세포주와 함께 항암효과를 갖는 공지의 유효성분을 1종 이상 함유할 수 있다. 이 경우, 본 발명의 암 치료용 조성물은 상기 유전자 변형 NK 세포주와 공지의 유효성분이 약학적으로 허용가능한 담체와 함께 혼합되어 제형화된 약학적 조성물을 의미하며, 함께 제형화되지 않을 경우에는 별도로 포장되어 동시에 또는 시간차를 두고 순차적으로 투여될 수도 있다. 후자의 경우에는 조성물이라기 보다는 키트라고 할 수 있을 것이다.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. In this case, 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.
아울러, 상기 조성물은 약학적으로 허용 가능한 담체 외에 약학적으로 허용 가능한 부형제 또는 희석제를 추가적으로 포함할 수 있다.In addition, the composition may further include a pharmaceutically acceptable excipient or diluent in addition to the pharmaceutically acceptable carrier.
상기 약학적으로 허용 가능한 담체로는 예를 들면, 물, 적합한 오일, 식염수, 수성 글루코스 및 글리콜 등과 같은 비경구 투여용 담체 등이 있으며 안정화제 및 보존제를 추가로 포함할 수 있다. 적합한 안정화제로는 아황산수소나트륨, 아황산나트륨 또는 아스코르브산과 같은 항산화제가 있다. 적합한 보존제로는 벤즈알코늄 클로라이드, 메틸- 또는 프로필-파라벤 및 클로로부탄올이 있다. 또한 본 발명에 따른 세포치료제는 그 투여방법이나 제형에 따라 필요한 경우, 현탁제, 용해보조제, 안정화제, 등장화제, 보존제, 흡착방지제, 계면활성화제, 희석제, 부형제, pH 조정제, 무통화제, 완충제, 산화방지제 등을 적절히 포함할 수 있다. 상기에 예시된 것들을 비롯하여 본 발명에 적합한 약학적으로 허용되는 담체 및 제제는 문헌[Remington's Pharmaceutical Sciences, 최신판]에 상세히 기재되어 있다. Such pharmaceutically acceptable carriers include, for example, carriers for parenteral administration such as water, suitable oils, saline, 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. In addition, 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.
한편, 본 발명의 일 실시예에 따른 약학적 조성물의 투여량은 세포수 기준으로 107 내지 1011 cells일 수 있지만, 상기 투여량은 환자의 성별, 나이, 질병의 진행정도, 치료 목적에 따라 조절될 수 있다. 일반적으로, 이러한 양은 표적 세포, 예를 들어, 암항원 과발현 암 세포에서의 국소화를 수득하고, 상기 암세포를, 예를 들어, 포식작용 또는 용해에 의해 죽이는데 충분할 것이다.On the other hand, the dosage of the pharmaceutical composition according to an embodiment of the present invention may be 10 7 to 10 11 cells on the basis of the cell number, the dosage is depending on the sex, age, disease progression, treatment purpose of the patient Can be adjusted. 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.
상기 세포치료제는 상기 담체 외에 약학적으로 허용가능한 담체, 희석제, 또는 부형제를 추가적으로 포함할 수 있다.The cell therapy agent may further include a pharmaceutically acceptable carrier, diluent, or excipient in addition to the carrier.
아울러 본 문서에서 사용되는 용어 "약학적으로 허용 가능한"이란 생리학적으로 허용되고 인간에게 투여될 때, 통상적으로 위장 장애, 현기증과 같은 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 조성물을 말한다. In addition, as used herein, the term "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction, such as gastrointestinal disorders, dizziness, or the like when administered to a human.
또한, 본 발명의 일 실시예에 따른 세포 치료제는 포유동물에 투여 시, 활성 성분의 신속한 방출, 또는 지속 또는 지연된 방출이 가능하도록 당업계에 공지된 방법을 사용하여 제형화될 수 있다. 제형은 분말, 과립, 정제, 에멀젼, 시럽, 에어로졸, 연질 또는 경질 젤라틴 캅셀, 멸균 주사용액, 멸균 분말 형태를 포함한다. In addition, 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.
본 발명의 일 실시예에 따른 약학적 조성물은 다양한 경로로 투여될 수 있으며, 예를 들면, 경구, 비경구, 예를 들면 좌제, 경피, 정맥, 복강, 근육내, 두개강내, 병변내, 비강, 척추관내로 투여될 수 있으며, 또한 서방형 또는 연속적 또는 반복적 방출을 위한 이식장치를 사용하여 투여될 수 있다. 투여횟수는 원하는 범위 내에서 하루에 1회, 또는 수회로 나누어 투여할 수 있으며, 투여 기간도 특별히 한정되지 않는다.Pharmaceutical 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.
아울러 본 발명의 또 다른 일 관점에 따르면, 상기 유전자 변형 NK 세포주 및 자살유도제를 포함하는 암 치료용 키트가 제공된다.In addition, according to another aspect of the present invention, there is provided a kit for treating cancer comprising the genetically modified NK cell line and suicide inducing agent.
상기 암 치료용 키트는 상기 유전자 변형 NK 세포주와 자살유도제를 포함하되 이들 두 구성성분이 혼합된 형태로 제공되지 않고 별도로 포장되어 제공되며, 이들 두 구성성분은 같은 시점에 같거나 다른 경로로 투여될 수 있으나, 의사의 처방에 따라 일정한 간격을 두고 투여된다는 점에서 일반적인 약학적 조성물과 구분된다. 본 발명의 키트는 먼저 상기 유전자 변형 NK 세포주를 투여한 후, 적절한 시점, 예컨대 유전자 변형 NK 세포주 투여와 같은 시점, 투여로부터 1일 후, 2일 후, 3일 후, 4일 후, 5일 후, 6일 후, 또는 1주일 후, 10일 후, 2주 후, 15일 후, 20일 후, 3주 후, 25일 후, 4주 후, 또는 30일 후에 투여될 수 있고, 첫 번째 투여 이후 이틀, 사흘, 나흘, 닷새, 엿새, 일주일간의 간격으로 두 차례 이상 복수로 투여될 수도 있다. 아울러, 상기 키트는 각각의 구성성분의 투여 경로가 같을 수도 있고 다를 수도 있다. 예컨대, 상기 유전자 변형 NK 세포주를 정맥주사로 투여한 후, 거의 같은 시간대 또는 소정의 간격을 투고 상기 자살유 용어의 정의:
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. , 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. In addition, the kit may be the same or different route of administration of each component. For example, after the administration of the transgenic NK cells intravenously, contribute about the same time or at predetermined intervals defined in the suicidal oil terms:
본 문서에서 사용되는 용어 "CAR 컨스트럭트"는 "키메라 항원 수용체(chimeric antigen receptor) 컨스트럭트"의 약어로, 통상적으로 항원 인식부위로 scFv, sdAb와 같은 단일쇄 기반의 항체 유사체-세포막 통과 도메인-보조자극인자-세포내 신호전달 도메인으로 구성된 합성 단백질로서 T 세포 등 면역세포에 형질도입되어 암세포 특이적인 항원을 인식하여 CAR 컨스트럭트를 발현하는 면역세포의 이들 암세포에 대한 항암 활성을 향상시키는 것으로 잘 알려져 있다.As used herein, the term “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.
본 문서에서 사용되는 용어 "scFv"는 "single chain variable fragment"의 약어로서 실제 항체의 단편은 아니며, 항체의 중쇄 가변영역(VH)과 경쇄 가변영역(VL)을 약 25 a.a. 크기의 링커 펩타이드로 연결하여 제조한 일종의 융합단백질로서 고유의 항체 단편이 아님에도 불구하고 항원 결합능을 지닌 것으로 알려지고 있다(Glockshuber et al., Biochem. 29(6): 1362-1367, 1990).As used herein, 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).
본 문서에서 사용되는 용어 "유전자 변형(genetically modified)"이라는 용어는 숙주세포, 또는 선행종(predecessors)/모종(parents) 중 하나로 도입된 본 발명의 일 실시예에 따른 폴리뉴클레오타이드 또는 벡터를 숙주세포가 자신의 게놈 외에 포함하는 것을 의미한다. 아울러, 본 발명의 일 실시예에 따른 폴리뉴클레오타이드 또는 벡터는 게놈 외부의 독립적 분자, 바람직하게는 복제할 수 있는 분자로서 유전적으로 변형된 숙주세포 내에 존재할 수 있거나, 또는 숙주 면역세포의 게놈으로 안정적으로 삽입될 수 있다.The term "genetically modified" as used herein refers to a host cell or host cell or polynucleotide or vector according to one embodiment of the present invention introduced into one of the predecessors / parents. Means to include in addition to their genome. In addition, the polynucleotide or vector according to an embodiment of the present invention may exist in a genetically modified host cell as an independent molecule, preferably a replicable molecule outside the genome, or stably into the genome of the host immune cell. Can be inserted.
본 문서에서 사용되는 용어 "세포자살 유전자(cell suicide gene)"는 세포독성을 유발하거나 또는 세포사멸 기전을 촉발시킴으로써 해당 유전자가 발현되는 세포가 사멸하도록 유도하는 유전자를 의미한다. 특히 유전자 발현 자체로는 세포사멸이 촉발되지 않으나 특정 프로드러그(prodrug)를 처리시 세포자살 유전자에 의한 프로드러그의 대사산물이 세포독성 또는 세포사멸 기전을 촉발함으로써 세포를 사멸에 이르게 할 수 있다. 이러한 세포자살 유전자들에는 간시클로비르를 자살 유도신호로 사용하는 헤르페스 단순포진 바이러스 티미딘 인산화 유전자(HSV TK), 6-메톡시퓨린 아라비노뉴클레오사이드(6-mehoxypurine arabinonucleoside)를 자살 유도 신호로 사용하는 바리셀라 조스터 바이러스 티미딘 인산화효소(VZV TK), 5-플루오로시토신(5-FC)를 자살 유도신호로 사용하는 시토신 디아미네이즈 및 우라실 포스포리보실전이효소(UPRT), 이리노테칸(CPT-11)을 자살 유도신호로 사용하는 카르복실 에스터라제, 5(아지리딘-1-일)-2,4-디니트로벤자마이드(CB1954)를 자살 유도신호로 사용하는 니트로리덕테이즈, 4-[(2-클로로에틸)(2-메실록시에틸)아미노]벤조일-엘-글루탐산(CMDA)을 자살 유도신호로 사용하는 카르복시펩티데이즈 G2, 분자간 이량화 유도제(dimerizer)를 자살 유도신호로 사용하는 유도성 카스페이즈 9(iCas9)이 보고된 바 있다. As used herein, the term "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. In particular, 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. 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) used as a suicide induction signal Nitrodeductase using carboxyl esterase, 5 (aziridin-1-yl) -2,4-dinitrobenzamide (CB1954), using CPT-11) as a suicide induction signal 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 reported.
발명의 상세한 설명:Detailed description of the invention:
본 발명의 일 관점에 따르면, 하기 특성을 갖는 분리된 NK 세포주에 EpCAM 특이적 키메라 항원 수용체를 암호화하는 폴리뉴클레오타이드가 형질도입되어 상기 EpCAM 특이적 키메라 항원 수용체를 발현하는 유전자 변형 NK 세포주가 제공된다:According to one aspect of the present invention, a transgenic NK cell line expressing the EpCAM specific chimeric antigen receptor is transfected with a polynucleotide encoding an EpCAM specific chimeric antigen receptor to an isolated NK cell line having the following characteristics:
CD2, CD11a, CD25, CD45, CD54, DNAM-1, CD62L 및 CD56은 양성; 및CD2, CD11a, CD25, CD45, CD54, DNAM-1, CD62L and CD56 are positive; And
CD1a, CD3, CD4, CD8, CD14, CD20, CD23, CD34, TCRαβ 및 TCRγδ는 음성.CD1a, CD3, CD4, CD8, CD14, CD20, CD23, CD34, TCRαβ and TCRγδ are negative.
상기 유전자 변형 NK 세포주에 있어서, 상기 분리된 NK 세포주는 수탁번호 KCTC 13305BP로 기탁된 NK101 세포주일 수 있다.In the genetically modified NK cell line, the isolated NK cell line may be an NK101 cell line deposited with accession number KCTC 13305BP.
상기 유전자 변형 NK 세포주에 있어서, 상기 EpCAM 특이적 키메라 항원 수용체는 EpCAM에 특이적으로 결합하는 scFv, 변형 Ig Fc 도메인, CD28 막통과 도메인, DAP10, DAP12 및 T 세포 수용체의 CD3ζ 도메인으로 구성될 수 있다. In the genetically modified NK cell line, the EpCAM specific chimeric antigen receptor may be composed of scFv, modified Ig Fc domain, CD28 transmembrane domain, CD3ζ domain of DAP10, DAP12 and T cell receptor that specifically binds to EpCAM. .
상기 유전자 변형 NK 세포주에 있어서, 상기 scFv는 서열번호 3으로 기재되는아미노산 서열로 구성될 수 있고, 상기 변형 Ig Fc 도메인은 서열번호 5로 기재되는 아미노산 서열로 구성될 수 있으며, 상기 CD28 막통과 도메인은 서열번호 7로 기재되는 아미노산 서열로 구성될 수 있고, 상기 DAP10은 서열번호 9로 기재되는 아미노산 서열로 구성될 수 있으며, 상기 DAP12는 서열번호 11로 기재되는 아미노산 서열로 구성될 수 있고, 상기 T 세포 수용체의 CD3ζ 도메인은 서열번호 13으로 구성되는 아미노산 서열로 구성될 수 있다. 상기 EpCAM 특이적 키메라 항원 수용체는 전체적으로 서열번호 1로 기재되는 아미노산 서열로 구성될 수 있고, 상기 EpCAM 특이적 키메라 항원 수용체를 암호화하는 폴리뉴클레오타이드는 서열번호 2로 기재되는 핵산서열로 구성될 수 있다.In the genetically modified NK cell line, 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 CD3ζ 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.
본 문서에서 사용되는 용어 "DAP10"은 면역 수용체 복합체를 형성하는 막통과 신호 어댑터로서, HCST(hematopoietic cell signal transducer) 유전자에 의해 암호화되는 조혈성 세포 신호 전달자를 의미하며, NK 세포의 활성화에 및 T 세포 반응에 의한 세포의 생존 및 증식에 중요한 역할을 하는 것으로 알려지고 있다. As used herein, the term “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.
본 문서에서 사용되는 용어 "DAP12"는 12 kDa 크기의 막통과 단백질로서 상기 DAP10와 높은 상동성을 가지고 있으며, NK 세포에서 중요한 신호전달 수용체로 인식되고 있다.As used herein, the term "DAP12" is a 12 kDa transmembrane protein with high homology with DAP10 and is recognized as an important signaling receptor in NK cells.
상기 유전자 변형 NK 세포주는 추가적으로 NK 세포 보조활성화 인자를 암호화하는 폴리뉴클레오타이드가 형질도입되어, 상기 NK 세포 활성화 인자를 발현할 수 있다. The genetically modified NK cell line may additionally be transduced with a polynucleotide encoding a NK cell coactivator, thereby expressing the NK cell activator.
상기 유전자 변형 NK 세포주에 있어서, 상기 NK 세포 보조활성화 인자는 Ly49, NCR(natural cytotoxicity receptor), CD7, CD16 및 CD28로 구성되는 군으로부터 선택되는 어느 하나 이상일 수 있다.In the genetically modified NK cell line, 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.
상기 유전자 변형 NK 세포주에 있어서, 상기 NK 세포 보조활성화 인자는 CD7 및/또는 CD28일 수 있다.In the genetically modified NK cell line, the NK cell coactivator may be CD7 and / or CD28.
상기 유전자 변형 NK 세포주에 있어서, 적어도 하나 이상의 NK 세포 증식 인자를 암호화하는 폴리뉴클레오타이드가 추가로 형질도입될 수 있다.In the genetically modified NK cell line, polynucleotides encoding at least one or more NK cell proliferation factors may be further transduced.
상기 유전자 변형 NK 세포주에 있어서, 상기 NK 세포 증식 인자는 IL-2, IL-12, IL-15, IL-18 및 IL-21로 구성되는 군으로부터 선택되는 적어도 하나 이상의 사이토카인 또는 상기 사이토카인의 변이체일 수 있다.In the genetically modified NK cell line, 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.
상기 유전자 변형 NK 세포주에 있어서, 상기 IL-15는 막결합 IL-15일 수 있다.In the genetically modified NK cell line, the IL-15 may be membrane-bound IL-15.
상기 유전자 변형 NK 세포주에 있어서, 세포자살유전자가 추가로 형질도입될 수 있다.In the genetically modified NK cell line, apoptotic genes may be further transduced.
상기 유전자 변형 NK 세포주에 있어서, 상기 세포자살유전자는 우라실 포스포리보실전이효소(UPRT) 유전자, 헤르페스 단순포진 바이러스 티미딘 인산화 유전자(HSV TK), 바리셀라 조스터 바이러스 티미딘 인산화효소(VZV TK) 유전자, 시토신 디아미네이즈 유전자, 카르복실 에스터레이즈 유전자, 니트로리덕테이즈 유전자, 카르복시펩티데이즈 G2 유전자, 또는 유도성 카스페이즈 9(iCas9)유전자일 수 있다.In the genetically modified NK cell line, 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.
상기 유전자 변형 NK 세포주에 있어서, 세포질 도메인 결실 TGFβ 수용체를 암호화하는 폴리뉴클레오타이드가 추가로 형질도입될 수 있다.In such genetically modified NK cell lines, polynucleotides encoding cytoplasmic domain deleted TGFβ receptors may be further transduced.
상기 유전자 변형 NK 세포주에 있어서, 상기 세포질 도메인 결실 TGFβ 수용체는 세포질 도메인 결실 TGFβ 수용체II일 수 있다.In the genetically modified NK cell line, the cytoplasmic domain deleted TGFβ receptor may be cytoplasmic domain deleted TGFβ receptor II.
본 문서에서 사용되는 용어 "키메라 항원 수용체(CAR)"는 단일클론항체의 항원에 결합 부분(가변 영역)을 림프구 활성화 수용체로부터 유래된 세포 내 신호전달 부위를 융합하여 제조된 일종의 융합단백질을 지칭한다. 이들 키메라 항원 수용체가 인간 T 세포들에서 발현되었을 때 MHC(major histocompatibility complex) 제한의 한계성 없이 강력한 세포 작용 기작들을 유도할 수 있다. 이러한 접근법의 잠재성은 CAR를 발현하는 T 세포들이 췌장암 환자들에게 주사된 임상적 연구에서 보고되었다. 지속적인 효능과 현저한 객관적인 반응(objective responses)이 저항성을 보이는 환자들에서 관찰되었고, CAR 기술의 활용이 좀 더 광범위하게 암 치료에서 사용될 것으로 전망되고 있으며, 현재 1세대, 2세대를 거쳐 3세대 CAR 분자가 보고되고 있다.As used herein, the term “chimeric antigen receptor (CAR)” 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. . When these chimeric antigen receptors are expressed in human T cells, they can induce potent cellular mechanisms without the limitation of major histocompatibility complex (MHC) limitations. The potential of this approach has been reported in clinical studies in which CAR-expressing T cells are injected into pancreatic cancer patients. Sustained efficacy and significant objective responses have been observed in patients who are resistant, and the use of CAR technology is expected to be used in cancer treatment more widely, and is now undergoing first generation, second generation, and third generation CAR molecules. Is being reported.
본 문서에서 사용되는 용어 "유전자 변형된(genetically modified)"이라는 용어는 숙주세포, 또는 선행종(predecessors)/모종(parents) 중 하나로 도입된 본 발명의 일 실시예에 따른 폴리뉴클레오타이드 또는 벡터를 숙주세포가 자신의 게놈 외에 포함하는 것을 의미한다. 아울러, 본 발명의 일 실시예에 따른 폴리뉴클레오타이드 또는 벡터는 게놈 외부의 독립적 분자, 바람직하게는 복제할 수 있는 분자로서 유전적으로 변형된 숙주세포 내에 존재할 수 있거나, 또는 숙주 면역세포의 게놈으로 안정적으로 삽입될 수 있다.As used herein, the term "genetically modified" refers to a host or polynucleotide or vector according to one embodiment of the invention introduced into one of the predecessors / parents. It means that cells contain outside their genome. In addition, the polynucleotide or vector according to an embodiment of the present invention may exist in a genetically modified host cell as an independent molecule, preferably a replicable molecule outside the genome, or stably into the genome of the host immune cell. Can be inserted.
보다 구체적으로, 상기 암항원 특이적 키메라 항원 수용체와 상기 세포자살 유전자에 의해 암호화되는 단백질은 융합단백질의 형태로 발현이 되거나, 단일 유전자 컨스트럭트 내에 클로닝된 후, 숙주 면역세포를 형질감염하여 공발현되거나, 별도의 유전자 컨스트럭트 내에 각각 클로닝된 후, 숙주 면역세포를 공형질감염함으로써 공발현될 수 있다. 상기 단일 유전자 컨스트럭트 내로 암항원 특이적 키메라 항원 수용체를 암호화하는 폴리뉴클레오타이드 및 세포자살 유전자 폴리뉴클레오타이드가 클로닝되는 경우 별도의 프로모터에 각각 작동 가능하게 연결되어 발현되거나, 두 폴리뉴클레오타이드 모두 단일 프로모터에 작동가능하게 연결되되, 상기 암항원 특이적 키메라 항원 수용체를 암호화하는 폴리뉴클레오타이드와 상기 세포자살 유전자 폴리뉴클레오타이드가 내부 리보솜 진입 부위(internal ribosome entry site, IRES)로 연결됨으로써 폴리시스트로닉하게(polycistronic) 발현되도록 할 수 있다.More specifically, 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. When 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. Possibly linked, wherein the 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.
본 문서에서 사용되는 "작동가능하게 연결된(operably linked to)"은 특정 폴리뉴클레오타이드가 그 기능을 발휘할 수 있게 다른 폴리뉴클레오타이드에 연결된 것을 의미한다. 즉, 특정 단백질을 암호화하는 폴리뉴클레오타이드가 프로모터에 작동가능하게 연결되었다는 것은 당해 프로모터의 작용에 의해 mRNA로 전사되고 당해 단백질로 번역까지 될 수 있게 연결되었다는 것을 의미하고, 특정 단백질을 암호화하는 폴리뉴클레오타이드가 다른 단백질을 암호화하는 폴리뉴클레오타이드에 작동 가능하게 연결되었다는 것은 당해 특정 단백질이 다른 단백질과 융합단백질의 형태로 발현될 수 있다.As used herein, "operably linked to" means that a particular polynucleotide is linked to another polynucleotide so that it can function. In other words, 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.
상기 유전자 컨스트럭트는 발현벡터에 클로닝되어 숙주 면역세포를 형질감염시킬 수 있는데, 이러한 벡터에는 프로모터를 포함한 내부에 클로닝된 유전자의 발현을 가능케하는 다양한 조절인자들을 포함할 수 있다. 상기 조절인자들은 당업자에게 잘 알려져 있다. 상술한 바와 같이, 이들은 보통 전사개시를 담당하는 조절인자들 및, 선택적으로 전사물의 전사종결 및 안정화를 담당하는 폴리-A 신호를 포함한다. 추가적인 조절인자들은 전사조절인자 외에도 번역 증진인자 및/또는 천연-조합 또는 이종성 프로모터 영역을 포함할 수 있다. 예를 들어 포유류 숙주 세포에서 발현을 가능하게 하는 가능한 조절인자들은 CMV-HSV 티미딘 키나아제 프로모터, SV40, RSV-프로모터(로우스 육종 바이러스), 인간 신장 요소 1α-프로모터, 글루코코르티코이드-유도성 MMTV-프로모터(몰로니 마우스 종양 바이러스), 메탈로티오네인-유도성 또는 테트라사이클린-유도성 프로모터 또는, CMV 증폭제 또는 SV40-증폭제와 같은 증폭제를 포함한다. 신경 세포 내 발현을 위해, 신경미세섬유-프로모터(neurofilament-promoter), PGDF-프로모터, NSE-프로모터, PrP-프로모터 또는 thy-1-프로모터들이 사용될 수 있다는 것이 고려되고 있다. 상기 프로모터들은 당 분야에 알려져 있으며, 문헌(Charron, J. Biol. Chem. 1995, 270: 25739-25745)에 기술되어 있다. 전사를 개시할 수 있는 인자들 외에, 상기 조절인자들은 본 발명의 일 실시예에 따른 폴리뉴클레오타이드의 하류(downstream)에 SV40-폴리-A 부위 또는 TK-폴리-A 부위와 같은 전사 종결 신호를 포함할 수도 있다. 본 문서에서, 적당한 발현 벡터들은 당 분야에 알려져 있으며, 그 예로는 오카야마-베르그(Okayama-Berg) cDNA 발현 벡터 pcDV1(Parmacia), pRc/CMV, pcDNA1, pcDNA3(In-vitrogene), pSPORT1(GIBCO BRL), pX(Pagano (1992) Science 255, 1144-1147), 효모 2-혼성(two-hybrid) 벡터, 가령 pEG202 및 dpJG4-5(Gyuris, Cell 75: 791-803, 2005)가 있다. 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. Such 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. For example, possible 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 1α-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. For expression in neurons, it is contemplated that nerve microfiber-promoter, PGDF-promoter, NSE-promoter, PrP-promoter or thy-1-promoter may be used. Such promoters are known in the art and described in Charron, J. Biol. Chem. 1995, 270: 25739-25745. In addition to factors capable of initiating transcription, 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. In this document, 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).
본 발명의 다른 일 관점에 따르면, 치료적으로 유효한 양의 상기 유전자 변형 NK 세포주를 유효성분으로 함유하는 암치료용 세포치료제가 제공된다.According to another aspect of the present invention, there is provided a cell therapy agent for treating cancer containing a therapeutically effective amount of the genetically modified NK cell line as an active ingredient.
상기 세포치료제는 일종의 약학적 조성물로서, 약학적 조성물의 제형에 필요한 약학적으로 허용 가능한 담체, 첨가제 또는 부형제는 상술한 바와 같다. 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.
한편, 본 발명의 일 실시예에 따른 세포치료제의 투여량은 세포의 수는 107 내지 1011개일 수 있지만, 환자의 성별, 나이, 질병의 진행정도, 치료 목적에 따라 조절될 수 있다. 일반적으로, 이러한 양은 표적 세포, 예를 들어, 암항원 과발현 암 세포에서의 국소화를 수득하고, 상기 암세포를, 예를 들어, 포식작용 또는 용해에 의해 죽이는데 충분할 것이다.On the other hand, 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.
상기 세포치료제는 상기 담체 외에 약학적으로 허용가능한 담체, 희석제, 또는 부형제를 추가적으로 포함할 수 있다.The cell therapy agent may further include a pharmaceutically acceptable carrier, diluent, or excipient in addition to the carrier.
아울러 상기 "약학적으로 허용 가능한"이란 생리학적으로 허용되고 인간에게 투여될 때, 통상적으로 위장 장애, 현기증과 같은 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 조성물을 말한다. 본 발명에 일 실시예에 따른 조성물은 일반적으로 사용되는 약학적으로 허용가능한 담체와 함께 적합한 형태로 제형화될 수 있다. 약학적으로 허용되는 담체로는 예를 들면, 물, 적합한 오일, 식염수, 수성 글루코스 및 글리콜 등과 같은 비경구 투여용 담체 등이 있으며 안정화제 및 보존제를 추가로 포함할 수 있다. 적합한 안정화제로는 아황산수소나트륨, 아황산나트륨 또는 아스코르브산과 같은 항산화제가 있다. 적합한 보존제로는 벤즈알코늄 클로라이드, 메틸- 또는 프로필-파라벤 및 클로로부탄올이 있다. 또한 본 발명에 따른 세포치료제는 그 투여방법이나 제형에 따라 필요한 경우, 현탁제, 용해보조제, 안정화제, 등장화제, 보존제, 흡착방지제, 계면활성화제, 희석제, 부형제, pH 조정제, 무통화제, 완충제, 산화방지제 등을 적절히 포함할 수 있다. 상기에 예시된 것들을 비롯하여 본 발명에 적합한 약학적으로 허용되는 담체 및 제제는 문헌[Remington's Pharmaceutical Sciences, 최신판]에 상세히 기재되어 있다. In addition, the "pharmaceutically acceptable" refers to a composition which, when administered physiologically and humanly, does not usually cause an allergic reaction such as gastrointestinal disorders, dizziness or the like. 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. In addition, 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.
또한, 본 발명의 일 실시예에 따른 세포 치료제는 포유동물에 투여 시, 활성 성분의 신속한 방출, 또는 지속 또는 지연된 방출이 가능하도록 당업계에 공지된 방법을 사용하여 제형화될 수 있다. 제형은 분말, 과립, 정제, 에멀젼, 시럽, 에어로졸, 연질 또는 경질 젤라틴 캅셀, 멸균 주사용액, 멸균 분말 형태를 포함한다. In addition, 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.
본 발명의 일 실시예에 따른 조성물은 다양한 경로로 투여될 수 있으며, 예를 들면, 경구, 비경구, 예를 들면 좌제, 경피, 정맥, 복강, 근육내, 병변내, 두 개강내, 비강, 척추관내로 투여될 수 있으며, 또한 서방형 또는 연속적 또는 반복적 방출을 위한 이식장치를 사용하여 투여될 수 있다. 투여횟수는 원하는 범위 내에서 하루에 1회, 또는 수회로 나누어 투여할 수 있으며, 투여 기간도 특별히 한정되지 않는다.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, intracranial, nasal, It may be administered intrathecalally, and may also be administered using an extended 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.
상기 약학적 조성물의 환자에 대한 투여량은 환자의 신장, 체표면적, 연령, 투여되는 특정 화합물, 성별, 투여 시간 및 경로, 일반적인 건강, 및 동시에 투여되는 다른 약물들을 포함하는 많은 요소들에 따라 다르다. 약학적으로 활성인 단백질 물질은 투여마다 1 ng - 10 ㎎/체중(㎏)의 양으로 존재할 수 있지만; 상기 예시 범위 이하 또는 이상의 투여도 특히 상기 요소들을 고려하여 고려된다. 투여법이 연속 주입이면, 1분당 체중 1 ㎏ 당 1 ㎍ - 10 ㎎ 단위의 범위 내에 있어야 한다.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 μg-10 mg units per kilogram of body weight per minute.
아울러 본 발명의 또 다른 일 관점에 따르면, 상기 유전자 변형 NK 세포주 및 자살유도제를 포함하는 암 치료용 키트가 제공된다.In addition, according to another aspect of the present invention, there is provided a kit for treating cancer comprising the genetically modified NK cell line and suicide inducing agent.
본 발명의 암 치료용 키트는 상기 유전자 변형 면역세포와 자살유도제를 포함하되 이들 두 구성성분이 혼합된 형태로 제공되지 않고 별도로 포장되어 제공되며, 이들 두 구성성분은 같은 시점에 같거나 다른 경로로 투여될 수 있으나, 의사의 처방에 따라 일정한 간격을 두고 투여된다는 점에서 일반적인 조성물과 구분된다. 본 발명의 키트는 먼저 상기 유전자 변형 면역세포를 투여한 후, 적절한 시점, 예컨대 유전자 변형 면역세포 투여와 같은 시점, 투여로부터 1일 후, 2일 후, 3일 후, 4일 후, 5일 후, 6일 후, 또는 1주일 후, 10일 후, 2주 후, 15일 후, 20일 후, 3주 후, 25일 후, 4주 후, 또는 30일 후에 투여될 수 있고, 첫 번째 투여 이후 이틀, 사흘, 나흘, 닷새, 엿새, 일주일간의 간격으로 두 차례 이상 복수로 투여될 수도 있다.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.
상기 자살유도제는 세포자살 유전자의 종류에 따라 달라지는데, 예컨대, 세포자살 유전자가 HSV TK 또는 VZV TK일 경우에는 각각 간시클로비르(gancyclovir) 또는 6-메톡시퓨린 아라비노뉴클레오사이드(6-mehoxypurine arabinonucleoside)일 수 있고, 시토신 디아미네이즈의 경우 5-플루오로시토신(5-FC)일 수 있으며, 카르복실 에스터라제의 경우 이리노테칸(CPT-11)일 수 있다. 아울러 니트로리덕테이즈의 경우에는 5(아지리딘-1-일)-2,4-디니트로벤자마이드(CB1954)일 수 있고, 카르복시펩티데이즈 G2의 경우에는 4-[(2-클로로에틸)(2-메실록시에틸)아미노]벤조일-엘-글루탐산(CMDA)일 수 있으며, iCas9일 경우 iCas9 이량화제(dimerizer)일 수 있고, 상기 iCas9 이량화제는 AP20187 또는 AP1903일 수 있다. 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. In addition, 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.
본 발명의 다른 일 관점에 따르면, 치료적으로 유효한 양의 상기 유전자 변형 NK 세포주 및 선택적으로 자살유도제를 암의 치료를 필요로 하는 개체에 투여하는 단계를 포함하는 상기 개체의 암 치료 방법이 제공된다.According to another aspect of the invention, there is provided a method of treating cancer in a subject comprising administering to the subject in need thereof a therapeutically effective amount of the genetically modified NK cell line and optionally suicide. .
상기 유전자 변형 NK 세포주와 상기 자살유도제는 동시에 투여될 수 있으나, 상술한 바와 같이, 최적의 효과를 위하여 적절한 투여간격으로 나누어 투여될 수 있으며, 상기 투여간격은 치료활성의 극대화를 위해 조절될 수 있다.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. .
본 발명의 암 치료용 키트의 사용을 통해 치료 가능한 암은 혈액암 또는 고형암일 수 있는데, 상기 고형암은 간암, 폐암, 췌장암, 유방암, 난소암, 자궁내막암, 자궁경부암, 담낭암, 위암, 담도암, 대장암, 두경부암, 식도암, 갑상선암, 뇌종양, 악성 흑색종, 전립선암, 고환암, 설암, 또는 골수암일 수 있다.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.
이하, 본 발명을 첨부되는 도면을 이용하여 보다 상세히 설명한다.Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings.
본 발명자들은 NK 림프암종 환자의 암조직으로부터 표 1에 기재된 특성을 갖는 새로운 NK 세포주를 분리하였으며, 이에 대한 다양한 특성을 조사한 결과, 도 1 내지 6에 나타난 바와 같이, IL-2 의존적 증식능을 나타내며, 암세포 사멸능과 면역조절능을 모두 갖는 다기능성 NK 세포주임을 확인할 수 있었다. 특히, 현재 임상시험이 진행중인 유일한 NK 세포주인 NK-92에 비해 증식능이 현저하게 높아서 경제적으로 생산가능한 세포임을 확인하여, 이를 'NK101' 세포주로 명명하고 이를 대한민국 전라북도 정읍시 입신길 181번지에 소재하고 있는 한국생명공학연구원 내 한국유전자은행(Korean Collection for Type Culture, KCTC)에 2017년 8월 7일자로 기탁하여, 2017년 8월 24일자로 KCTC 13305BP의 수탁번호를 부여받았다. 그러나, 상기 NK101 세포주는 유전자 발현 분석 결과, IL-2 수용체인 CD25는 고발현이고, CD56dimCD62L+의 표현형을 갖는 것으로 확인되었으나, NK 세포의 항암 활성에 직접적인 영향을 주는 NK 세포 활성화 인자인 CD7 및 CD28은 발현하지 않음을 확인함으로써, 항암 활성 자체는 종래에 구축된 NK 세포주 보다 높지 않을 것으로 예상하게 되었다. 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. It was deposited on August 7, 2017 by the Korean Collection for Type Culture (KCTC) in the Korea Research Institute of Bioscience and Biotechnology, and received an accession number of KCTC 13305BP on August 24, 2017. However, the gene expression analysis of the NK101 cell line revealed that the IL-2 receptor CD25 was highly expressed and had a phenotype of CD56 dim CD62L + , but CD7, an NK cell activating factor that directly affects the anticancer activity of NK cells. And by confirming that CD28 does not express, anticancer activity itself was expected to not be higher than conventionally constructed NK cell line.
이에, 본 발명자들은 본 발명의 일 실시예에 따른 NK101 세포의 항암활성을 강화하기 위해, NK101 세포에 기반한 유전자 변형 NK 세포주를 제조하고자 하였다. 도 7b는 상기 목적을 달성하기 위해, 본 발명의 일 실시예에 따른 유전자 변형 NK 세포주 SL-K01의 제조에 사용된 유전자 컨스트럭트 CD7-CD28-CD::UPRT의 구조를 개략적으로 나타낸 개요도이고 및 도 9a는 본 발명의 일 실시예에 따른 유전자 변형 NK 세포주 NK111의 제조를 위해 사용된 유전자 컨스트럭트 mbIL-15-mTGFβIIΔcyto의 구조를 개략적으로 나타낸 개요도이다.Thus, the present inventors intended to prepare a genetically modified NK cell line based on NK101 cells in order to enhance the anticancer activity of NK101 cells according to an embodiment of the present invention. 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-mTGFβIIΔcyto used for the preparation of the genetically modified NK cell line NK111 according to one embodiment of the present invention.
본 발명에서는 앞서 언급한 요소를 보완하기 위하여, 본 발명자들에 의해 기확립된 NK101 세포(수탁번호 KCTC 13305BP)에 NK 세포의 보조활성화 수용체, 세포자살유전자, 세포막 고정(membrane bound) 사이토카인, 돌연변이 TGFβ 수용체를 도입하여 해당 NK 세포의 항암효과 증진, 및 사이토카인 보충제(cytokine supplement) 비의존적으로 NK 세포의 증식이 가능한 NK111 세포주를 구축하였다(도 9b 및 9c 참조). In the present invention, in order to complement the aforementioned elements, co-activating receptors, apoptotic genes, membrane bound cytokines, mutations of NK cells in NK101 cells (accession number KCTC 13305BP) previously established by the present inventors The TGFβ 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).
도 7은 본 발명의 일 실시예에 따른 유전자 변형 NK 세포주(SL-K01) 및 상기 SL-K01의 모세포주인 NK101, 그리고 종래구축된 NK 세포주인 KHYG-1 및 NK-92에서의 세포 표면 표지자의 발현 여부를 분석한 결과로서, 도 7a는 NK101, KHYG-1 및 NK-92에서 CD7 용어의 정의:
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 As a result of analyzing the expression, Figure 7a shows the definition of the term CD7 in NK101, KHYG-1 and NK-92 :
본 문서에서 사용되는 용어 "CAR 컨스트럭트"는 "키메라 항원 수용체(chimeric antigen receptor) 컨스트럭트"의 약어로, 통상적으로 항원 인식부위로 scFv, sdAb와 같은 단일쇄 기반의 항체 유사체-세포막 통과 도메인-보조자극인자-세포내 신호전달 도메인으로 구성된 합성 단백질로서 T 세포 등 면역세포에 형질도입되어 암세포 특이적인 항원을 인식하여 CAR 컨스트럭트를 발현하는 면역세포의 이들 암세포에 대한 항암 활성을 향상시키는 것으로 잘 알려져 있다.As used herein, the term “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.
본 문서에서 사용되는 용어 "scFv"는 "single chain variable fragment"의 약어로서 실제 항체의 단편은 아니며, 항체의 중쇄 가변영역(VH)과 경쇄 가변영역(VL)을 약 25 a.a. 크기의 링커 펩타이드로 연결하여 제조한 일종의 융합단백질로서 고유의 항체 단편이 아님에도 불구하고 항원 결합능을 지닌 것으로 알려지고 있다(Glockshuber et al., Biochem. 29(6): 1362-1367, 1990).As used herein, 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).
본 문서에서 사용되는 용어 "유전자 변형(genetically modified)"이라는 용어는 숙주세포, 또는 선행종(predecessors)/모종(parents) 중 하나로 도입된 본 발명의 일 실시예에 따른 폴리뉴클레오타이드 또는 벡터를 숙주세포가 자신의 게놈 외에 포함하는 것을 의미한다. 아울러, 본 발명의 일 실시예에 따른 폴리뉴클레오타이드 또는 벡터는 게놈 외부의 독립적 분자, 바람직하게는 복제할 수 있는 분자로서 유전적으로 변형된 숙주세포 내에 존재할 수 있거나, 또는 숙주 면역세포의 게놈으로 안정적으로 삽입될 수 있다.The term "genetically modified" as used herein refers to a host cell or host cell or polynucleotide or vector according to one embodiment of the present invention introduced into one of the predecessors / parents. Means to include in addition to their genome. In addition, the polynucleotide or vector according to an embodiment of the present invention may exist in a genetically modified host cell as an independent molecule, preferably a replicable molecule outside the genome, or stably into the genome of the host immune cell. Can be inserted.
본 문서에서 사용되는 용어 "세포자살 유전자(cell suicide gene)"는 세포독성을 유발하거나 또는 세포사멸 기전을 촉발시킴으로써 해당 유전자가 발현되는 세포가 사멸하도록 유도하는 유전자를 의미한다. 특히 유전자 발현 자체로는 세포사멸이 촉발되지 않으나 특정 프로드러그(prodrug)를 처리시 세포자살 유전자에 의한 프로드러그의 대사산물이 세포독성 또는 세포사멸 기전을 촉발함으로써 세포를 사멸에 이르게 할 수 있다. 이러한 세포자살 유전자들에는 간시클로비르를 자살 유도신호로 사용하는 헤르페스 단순포진 바이러스 티미딘 인산화 유전자(HSV TK), 6-메톡시퓨린 아라비노뉴클레오사이드(6-methoxypurine arabinonucleoside)를 자살 유도 신호로 사용하는 바리셀라 조스터 바이러스 티미딘 인산화효소(VZV TK), 5-플루오로시토신(5-FC)를 자살 유도신호로 사용하는 시토신 디아미네이즈 및 우라실 포스포리보실전이효소(UPRT), 이리노테칸(CPT-11)을 자살 유도신호로 사용하는 카르복실 에스터라제, 5(아지리딘-1-일)-2,4-디니트로벤자마이드(CB1954)를 자살 유도신호로 사용하는 니트로리덕테이즈, 4-[(2-클로로에틸)(2-메실록시에틸)아미노]벤조일-엘-글루탐산(CMDA)을 자살 유도신호로 사용하는 카르복시펩티데이즈 G2, 분자간 이량화 유도제(dimerizer)를 자살 유도신호로 사용하는 유도성 카스페이즈 9(iCas9)이 보고된 바 있다. As used herein, the term "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. In particular, 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. These apoptosis genes include the herpes herpes simplex virus thymidine phosphorylation gene (HSV TK) and 6-methoxypurine arabinonucleoside, which use gancyclovir as a suicide signal. Cytosine deminase and uracil phosphoribosyltransferase (UPRT), irinotecan (Varicela zoster virus thymidine kinase (VZV TK), 5-fluorocytosine (5-FC) used as a suicide induction signal Nitrodeductase using carboxyl esterase, 5 (aziridin-1-yl) -2,4-dinitrobenzamide (CB1954), using CPT-11) as a suicide induction signal 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 Phase 9 (iCas9) has been reported.
발명의 상세한 설명:Detailed description of the invention:
본 발명의 일 관점에 따르면, 하기 특성을 갖는 분리된 NK 세포주에 EpCAM 특이적 키메라 항원 수용체를 암호화하는 폴리뉴클레오타이드가 형질도입되어 상기 EpCAM 특이적 키메라 항원 수용체를 발현하는 유전자 변형 NK 세포주가 제공된다:According to one aspect of the present invention, a transgenic NK cell line expressing the EpCAM specific chimeric antigen receptor is transfected with a polynucleotide encoding an EpCAM specific chimeric antigen receptor to an isolated NK cell line having the following characteristics:
CD2, CD11a, CD25, CD45, CD54, DNAM-1, CD62L 및 CD56은 양성; 및CD2, CD11a, CD25, CD45, CD54, DNAM-1, CD62L and CD56 are positive; And
CD1a, CD3, CD4, CD8, CD14, CD20, CD23, CD34, TCRαβ 및 TCRγδ는 음성.CD1a, CD3, CD4, CD8, CD14, CD20, CD23, CD34, TCRαβ and TCRγδ are negative.
상기 유전자 변형 NK 세포주에 있어서, 상기 분리된 NK 세포주는 수탁번호 KCTC 13305BP로 기탁된 NK101 세포주일 수 있다.In the genetically modified NK cell line, the isolated NK cell line may be an NK101 cell line deposited with accession number KCTC 13305BP.
상기 유전자 변형 NK 세포주에 있어서, 상기 EpCAM 특이적 키메라 항원 수용체는 EpCAM에 특이적으로 결합하는 scFv, 변형 Ig Fc 도메인, CD28 막통과 도메인, DAP10, DAP12 및 T 세포 수용체의 CD3ζ 도메인으로 구성될 수 있다. In the genetically modified NK cell line, the EpCAM specific chimeric antigen receptor may be composed of scFv, modified Ig Fc domain, CD28 transmembrane domain, CD3ζ domain of DAP10, DAP12 and T cell receptor that specifically binds to EpCAM. .
상기 유전자 변형 NK 세포주에 있어서, 상기 scFv는 서열번호 3으로 기재되는아미노산 서열로 구성될 수 있고, 상기 변형 Ig Fc 도메인은 서열번호 5로 기재되는 아미노산 서열로 구성될 수 있으며, 상기 CD28 막통과 도메인은 서열번호 7로 기재되는 아미노산 서열로 구성될 수 있고, 상기 DAP10은 서열번호 9로 기재되는 아미노산 서열로 구성될 수 있으며, 상기 DAP12는 서열번호 11로 기재되는 아미노산 서열로 구성될 수 있고, 상기 T 세포 수용체의 CD3ζ 도메인은 서열번호 13으로 구성되는 아미노산 서열로 구성될 수 있다. 상기 EpCAM 특이적 키메라 항원 수용체는 전체적으로 서열번호 1로 기재되는 아미노산 서열로 구성될 수 있고, 상기 EpCAM 특이적 키메라 항원 수용체를 암호화하는 폴리뉴클레오타이드는 서열번호 2로 기재되는 핵산서열로 구성될 수 있다.In the genetically modified NK cell line, 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 CD3ζ 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.
본 문서에서 사용되는 용어 "DAP10"은 면역 수용체 복합체를 형성하는 막통과 신호 어댑터로서, HCST(hematopoietic cell signal transducer) 유전자에 의해 암호화되는 조혈성 세포 신호 전달자를 의미하며, NK 세포의 활성화에 및 T 세포 반응에 의한 세포의 생존 및 증식에 중요한 역할을 하는 것으로 알려지고 있다. As used herein, the term “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.
본 문서에서 사용되는 용어 "DAP12"는 12 kDa 크기의 막통과 단백질로서 상기 DAP10와 높은 상동성을 가지고 있으며, NK 세포에서 중요한 신호전달 수용체로 인식되고 있다.As used herein, the term "DAP12" is a 12 kDa transmembrane protein with high homology with DAP10 and is recognized as an important signaling receptor in NK cells.
상기 유전자 변형 NK 세포주는 추가적으로 NK 세포 보조활성화 인자를 암호화하는 폴리뉴클레오타이드가 형질도입되어, 상기 NK 세포 활성화 인자를 발현할 수 있다. The genetically modified NK cell line may additionally be transduced with a polynucleotide encoding a NK cell coactivator, thereby expressing the NK cell activator.
상기 유전자 변형 NK 세포주에 있어서, 상기 NK 세포 보조활성화 인자는 Ly49, NCR(natural cytotoxicity receptor), CD7, CD16 및 CD28로 구성되는 군으로부터 선택되는 어느 하나 이상일 수 있다.In the genetically modified NK cell line, 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.
상기 유전자 변형 NK 세포주에 있어서, 상기 NK 세포 보조활성화 인자는 CD7 및/또는 CD28일 수 있다.In the genetically modified NK cell line, the NK cell coactivator may be CD7 and / or CD28.
상기 유전자 변형 NK 세포주에 있어서, 적어도 하나 이상의 NK 세포 증식 인자를 암호화하는 폴리뉴클레오타이드가 추가로 형질도입될 수 있다.In the genetically modified NK cell line, polynucleotides encoding at least one or more NK cell proliferation factors may be further transduced.
상기 유전자 변형 NK 세포주에 있어서, 상기 NK 세포 증식 인자는 IL-2, IL-12, IL-15, IL-18 및 IL-21로 구성되는 군으로부터 선택되는 적어도 하나 이상의 사이토카인 또는 상기 사이토카인의 변이체일 수 있다.In the genetically modified NK cell line, 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.
상기 유전자 변형 NK 세포주에 있어서, 상기 IL-15는 막결합 IL-15일 수 있다.In the genetically modified NK cell line, the IL-15 may be membrane-bound IL-15.
상기 유전자 변형 NK 세포주에 있어서, 세포자살유전자가 추가로 형질도입될 수 있다.In the genetically modified NK cell line, apoptotic genes may be further transduced.
상기 유전자 변형 NK 세포주에 있어서, 상기 세포자살유전자는 우라실 포스포리보실전이효소(UPRT) 유전자, 헤르페스 단순포진 바이러스 티미딘 인산화 유전자(HSV TK), 바리셀라 조스터 바이러스 티미딘 인산화효소(VZV TK) 유전자, 시토신 디아미네이즈 유전자, 카르복실 에스터레이즈 유전자, 니트로리덕테이즈 유전자, 카르복시펩티데이즈 G2 유전자, 또는 유도성 카스페이즈 9(iCas9)유전자일 수 있다.In the genetically modified NK cell line, 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.
상기 유전자 변형 NK 세포주에 있어서, 세포질 도메인 결실 TGFβ 수용체를 암호화하는 폴리뉴클레오타이드가 추가로 형질도입될 수 있다.In such genetically modified NK cell lines, polynucleotides encoding cytoplasmic domain deleted TGFβ receptors may be further transduced.
상기 유전자 변형 NK 세포주에 있어서, 상기 세포질 도메인 결실 TGFβ 수용체는 세포질 도메인 결실 TGFβ 수용체II일 수 있다.In the genetically modified NK cell line, the cytoplasmic domain deleted TGFβ receptor may be cytoplasmic domain deleted TGFβ receptor II.
본 문서에서 사용되는 용어 "키메라 항원 수용체(CAR)"는 단일클론항체의 항원에 결합 부분(가변 영역)을 림프구 활성화 수용체로부터 유래된 세포 내 신호전달 부위를 융합하여 제조된 일종의 융합단백질을 지칭한다. 이들 키메라 항원 수용체가 인간 T 세포들에서 발현되었을 때 MHC(major histocompatibility complex) 제한의 한계성 없이 강력한 세포 작용 기작들을 유도할 수 있다. 이러한 접근법의 잠재성은 CAR를 발현하는 T 세포들이 췌장암 환자들에게 주사된 임상적 연구에서 보고되었다. 지속적인 효능과 현저한 객관적인 반응(objective responses)이 저항성을 보이는 환자들에서 관찰되었고, CAR 기술의 활용이 좀 더 광범위하게 암 치료에서 사용될 것으로 전망되고 있으며, 현재 1세대, 2세대를 거쳐 3세대 CAR 분자가 보고되고 있다.As used herein, the term “chimeric antigen receptor (CAR)” 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. . When these chimeric antigen receptors are expressed in human T cells, they can induce potent cellular mechanisms without the limitation of major histocompatibility complex (MHC) limitations. The potential of this approach has been reported in clinical studies in which CAR-expressing T cells are injected into pancreatic cancer patients. Sustained efficacy and significant objective responses have been observed in patients who are resistant, and the use of CAR technology is expected to be used in cancer treatment more widely, and is now undergoing first generation, second generation, and third generation CAR molecules. Is being reported.
본 문서에서 사용되는 용어 "유전자 변형된(genetically modified)"이라는 용어는 숙주세포, 또는 선행종(predecessors)/모종(parents) 중 하나로 도입된 본 발명의 일 실시예에 따른 폴리뉴클레오타이드 또는 벡터를 숙주세포가 자신의 게놈 외에 포함하는 것을 의미한다. 아울러, 본 발명의 일 실시예에 따른 폴리뉴클레오타이드 또는 벡터는 게놈 외부의 독립적 분자, 바람직하게는 복제할 수 있는 분자로서 유전적으로 변형된 숙주세포 내에 존재할 수 있거나, 또는 숙주 면역세포의 게놈으로 안정적으로 삽입될 수 있다.As used herein, the term "genetically modified" refers to a host or polynucleotide or vector according to one embodiment of the invention introduced into one of the predecessors / parents. It means that cells contain outside their genome. In addition, the polynucleotide or vector according to an embodiment of the present invention may exist in a genetically modified host cell as an independent molecule, preferably a replicable molecule outside the genome, or stably into the genome of the host immune cell. Can be inserted.
보다 구체적으로, 상기 암항원 특이적 키메라 항원 수용체와 상기 세포자살 유전자에 의해 암호화되는 단백질은 융합단백질의 형태로 발현이 되거나, 단일 유전자 컨스트럭트 내에 클로닝된 후, 숙주 면역세포를 형질감염하여 공발현되거나, 별도의 유전자 컨스트럭트 내에 각각 클로닝된 후, 숙주 면역세포를 공형질감염함으로써 공발현될 수 있다. 상기 단일 유전자 컨스트럭트 내로 암항원 특이적 키메라 항원 수용체를 암호화하는 폴리뉴클레오타이드 및 세포자살 유전자 폴리뉴클레오타이드가 클로닝되는 경우 별도의 프로모터에 각각 작동 가능하게 연결되어 발현되거나, 두 폴리뉴클레오타이드 모두 단일 프로모터에 작동가능하게 연결되되, 상기 암항원 특이적 키메라 항원 수용체를 암호화하는 폴리뉴클레오타이드와 상기 세포자살 유전자 폴리뉴클레오타이드가 내부 리보솜 진입 부위(internal ribosome entry site, IRES)로 연결됨으로써 폴리시스트로닉하게(polycistronic) 발현되도록 할 수 있다.More specifically, 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. When 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. Possibly linked, wherein the 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.
본 문서에서 사용되는 "작동가능하게 연결된(operably linked to)"은 특정 폴리뉴클레오타이드가 그 기능을 발휘할 수 있게 다른 폴리뉴클레오타이드에 연결된 것을 의미한다. 즉, 특정 단백질을 암호화하는 폴리뉴클레오타이드가 프로모터에 작동가능하게 연결되었다는 것은 당해 프로모터의 작용에 의해 mRNA로 전사되고 당해 단백질로 번역까지 될 수 있게 연결되었다는 것을 의미하고, 특정 단백질을 암호화하는 폴리뉴클레오타이드가 다른 단백질을 암호화하는 폴리뉴클레오타이드에 작동 가능하게 연결되었다는 것은 당해 특정 단백질이 다른 단백질과 융합단백질의 형태로 발현될 수 있다.As used herein, "operably linked to" means that a particular polynucleotide is linked to another polynucleotide so that it can function. In other words, 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.
상기 유전자 컨스트럭트는 발현벡터에 클로닝되어 숙주 면역세포를 형질감염시킬 수 있는데, 이러한 벡터에는 프로모터를 포함한 내부에 클로닝된 유전자의 발현을 가능케하는 다양한 조절인자들을 포함할 수 있다. 상기 조절인자들은 당업자에게 잘 알려져 있다. 상술한 바와 같이, 이들은 보통 전사개시를 담당하는 조절인자들 및, 선택적으로 전사물의 전사종결 및 안정화를 담당하는 폴리-A 신호를 포함한다. 추가적인 조절인자들은 전사조절인자 외에도 번역 증진인자 및/또는 천연-조합 또는 이종성 프로모터 영역을 포함할 수 있다. 예를 들어 포유류 숙주 세포에서 발현을 가능하게 하는 가능한 조절인자들은 CMV-HSV 티미딘 키나아제 프로모터, SV40, RSV-프로모터(로우스 육종 바이러스), 인간 신장 요소 1α-프로모터, 글루코코르티코이드-유도성 MMTV-프로모터(몰로니 마우스 종양 바이러스), 메탈로티오네인-유도성 또는 테트라사이클린-유도성 프로모터 또는, CMV 증폭제 또는 SV40-증폭제와 같은 증폭제를 포함한다. 신경 세포 내 발현을 위해, 신경미세섬유-프로모터(neurofilament-promoter), PGDF-프로모터, NSE-프로모터, PrP-프로모터 또는 thy-1-프로모터들이 사용될 수 있다는 것이 고려되고 있다. 상기 프로모터들은 당 분야에 알려져 있으며, 문헌(Charron, J. Biol. Chem. 1995, 270: 25739-25745)에 기술되어 있다. 전사를 개시할 수 있는 인자들 외에, 상기 조절인자들은 본 발명의 일 실시예에 따른 폴리뉴클레오타이드의 하류(downstream)에 SV40-폴리-A 부위 또는 TK-폴리-A 부위와 같은 전사 종결 신호를 포함할 수도 있다. 본 문서에서, 적당한 발현 벡터들은 당 분야에 알려져 있으며, 그 예로는 오카야마-베르그(Okayama-Berg) cDNA 발현 벡터 pcDV1(Parmacia), pRc/CMV, pcDNA1, pcDNA3(In-vitrogene), pSPORT1(GIBCO BRL), pX(Pagano (1992) Science 255, 1144-1147), 효모 2-혼성(two-hybrid) 벡터, 가령 pEG202 및 dpJG4-5(Gyuris, Cell 75: 791-803, 2005)가 있다. 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. Such 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. For example, possible 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 1α-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. For expression in neurons, it is contemplated that nerve microfiber-promoter, PGDF-promoter, NSE-promoter, PrP-promoter or thy-1-promoter may be used. Such promoters are known in the art and described in Charron, J. Biol. Chem. 1995, 270: 25739-25745. In addition to factors capable of initiating transcription, 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. In this document, 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).
본 발명의 다른 일 관점에 따르면, 치료적으로 유효한 양의 상기 유전자 변형 NK 세포주를 유효성분으로 함유하는 암치료용 세포치료제가 제공된다.According to another aspect of the present invention, there is provided a cell therapy agent for treating cancer containing a therapeutically effective amount of the genetically modified NK cell line as an active ingredient.
상기 세포치료제는 일종의 약학적 조성물로서, 약학적 조성물의 제형에 필요한 약학적으로 허용 가능한 담체, 첨가제 또는 부형제는 상술한 바와 같다. 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.
한편, 본 발명의 일 실시예에 따른 세포치료제의 투여량은 세포의 수는 107 내지 1011개일 수 있지만, 환자의 성별, 나이, 질병의 진행정도, 치료 목적에 따라 조절될 수 있다. 일반적으로, 이러한 양은 표적 세포, 예를 들어, 암항원 과발현 암 세포에서의 국소화를 수득하고, 상기 암세포를, 예를 들어, 포식작용 또는 용해에 의해 죽이는데 충분할 것이다.On the other hand, 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.
상기 세포치료제는 상기 담체 외에 약학적으로 허용가능한 담체, 희석제, 또는 부형제를 추가적으로 포함할 수 있다.The cell therapy agent may further include a pharmaceutically acceptable carrier, diluent, or excipient in addition to the carrier.
아울러 상기 "약학적으로 허용 가능한"이란 생리학적으로 허용되고 인간에게 투여될 때, 통상적으로 위장 장애, 현기증과 같은 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 조성물을 말한다. 본 발명에 일 실시예에 따른 조성물은 일반적으로 사용되는 약학적으로 허용가능한 담체와 함께 적합한 형태로 제형화될 수 있다. 약학적으로 허용되는 담체로는 예를 들면, 물, 적합한 오일, 식염수, 수성 글루코스 및 글리콜 등과 같은 비경구 투여용 담체 등이 있으며 안정화제 및 보존제를 추가로 포함할 수 있다. 적합한 안정화제로는 아황산수소나트륨, 아황산나트륨 또는 아스코르브산과 같은 항산화제가 있다. 적합한 보존제로는 벤즈알코늄 클로라이드, 메틸- 또는 프로필-파라벤 및 클로로부탄올이 있다. 또한 본 발명에 따른 세포치료제는 그 투여방법이나 제형에 따라 필요한 경우, 현탁제, 용해보조제, 안정화제, 등장화제, 보존제, 흡착방지제, 계면활성화제, 희석제, 부형제, pH 조정제, 무통화제, 완충제, 산화방지제 등을 적절히 포함할 수 있다. 상기에 예시된 것들을 비롯하여 본 발명에 적합한 약학적으로 허용되는 담체 및 제제는 문헌[Remington's Pharmaceutical Sciences, 최신판]에 상세히 기재되어 있다. In addition, the "pharmaceutically acceptable" refers to a composition which, when administered physiologically and humanly, does not usually cause an allergic reaction such as gastrointestinal disorders, dizziness or the like. 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. In addition, 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.
또한, 본 발명의 일 실시예에 따른 세포 치료제는 포유동물에 투여 시, 활성 성분의 신속한 방출, 또는 지속 또는 지연된 방출이 가능하도록 당업계에 공지된 방법을 사용하여 제형화될 수 있다. 제형은 분말, 과립, 정제, 에멀젼, 시럽, 에어로졸, 연질 또는 경질 젤라틴 캅셀, 멸균 주사용액, 멸균 분말 형태를 포함한다. In addition, 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.
본 발명의 일 실시예에 따른 조성물은 다양한 경로로 투여될 수 있으며, 예를 들면, 경구, 비경구, 예를 들면 좌제, 경피, 정맥, 복강, 근육내, 병변내, 두개강내, 비강, 척추관내로 투여될 수 있으며, 또한 서방형 또는 연속적 또는 반복적 방출을 위한 이식장치를 사용하여 투여될 수 있다. 투여횟수는 원하는 범위 내에서 하루에 1회, 또는 수회로 나누어 투여할 수 있으며, 투여 기간도 특별히 한정되지 않는다.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, intracranial, nasal, spinal It may be administered intravenously, 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.
상기 약학적 조성물의 환자에 대한 투여량은 환자의 신장, 체표면적, 연령, 투여되는 특정 화합물, 성별, 투여 시간 및 경로, 일반적인 건강, 및 동시에 투여되는 다른 약물들을 포함하는 많은 요소들에 따라 다르다. 약학적으로 활성인 단백질 물질은 투여마다 1 ng - 10 ㎎/체중(㎏)의 양으로 존재할 수 있지만; 상기 예시 범위 이하 또는 이상의 투여도 특히 상기 요소들을 고려하여 고려된다. 투여법이 연속 주입이면, 1분당 체중 1 ㎏ 당 1 ㎍ - 10 ㎎ 단위의 범위 내에 있어야 한다.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 μg-10 mg units per kilogram of body weight per minute.
아울러 본 발명의 또 다른 일 관점에 따르면, 상기 유전자 변형 NK 세포주 및 자살유도제를 포함하는 암 치료용 키트가 제공된다.In addition, according to another aspect of the present invention, there is provided a kit for treating cancer comprising the genetically modified NK cell line and suicide inducing agent.
본 발명의 암 치료용 키트는 상기 유전자 변형 면역세포와 자살유도제를 포함하되 이들 두 구성성분이 혼합된 형태로 제공되지 않고 별도로 포장되어 제공되며, 이들 두 구성성분은 같은 시점에 같거나 다른 경로로 투여될 수 있으나, 의사의 처방에 따라 일정한 간격을 두고 투여된다는 점에서 일반적인 조성물과 구분된다. 본 발명의 키트는 먼저 상기 유전자 변형 면역세포를 투여한 후, 적절한 시점, 예컨대 유전자 변형 면역세포 투여와 같은 시점, 투여로부터 1일 후, 2일 후, 3일 후, 4일 후, 5일 후, 6일 후, 또는 1주일 후, 10일 후, 2주 후, 15일 후, 20일 후, 3주 후, 25일 후, 4주 후, 또는 30일 후에 투여될 수 있고, 첫 번째 투여 이후 이틀, 사흘, 나흘, 닷새, 엿새, 일주일간의 간격으로 두 차례 이상 복수로 투여될 수도 있다.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.
상기 자살유도제는 세포자살 유전자의 종류에 따라 달라지는데, 예컨대, 세포자살 유전자가 HSV TK 또는 VZV TK일 경우에는 각각 간시클로비르(gancyclovir) 또는 6-메톡시퓨린 아라비노뉴클레오사이드(6-methoxypurine arabinonucleoside)일 수 있고, 시토신 디아미네이즈의 경우 5-플루오로시토신(5-FC)일 수 있으며, 카르복실 에스터라제의 경우 이리노테칸(CPT-11)일 수 있다. 아울러 니트로리덕테이즈의 경우에는 5(아지리딘-1-일)-2,4-디니트로벤자마이드(CB1954)일 수 있고, 카르복시펩티데이즈 G2의 경우에는 4-[(2-클로로에틸)(2-메실록시에틸)아미노]벤조일-엘-글루탐산(CMDA)일 수 있으며, iCas9일 경우 iCas9 이량화제(dimerizer)일 수 있고, 상기 iCas9 이량화제는 AP20187 또는 AP1903일 수 있다. 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. In addition, 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.
본 발명의 다른 일 관점에 따르면, 치료적으로 유효한 양의 상기 유전자 변형 NK 세포주 및 선택적으로 자살유도제를 암의 치료를 필요로 하는 개체에 투여하는 단계를 포함하는 상기 개체의 암 치료 방법이 제공된다.According to another aspect of the invention, there is provided a method of treating cancer in a subject comprising administering to the subject in need thereof a therapeutically effective amount of the genetically modified NK cell line and optionally suicide. .
상기 유전자 변형 NK 세포주와 상기 자살유도제는 동시에 투여될 수 있으나, 상술한 바와 같이, 최적의 효과를 위하여 적절한 투여간격으로 나누어 투여될 수 있으며, 상기 투여간격은 치료활성의 극대화를 위해 조절될 수 있다.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. .
본 발명의 암 치료용 키트의 사용을 통해 치료 가능한 암은 혈액암 또는 고형암일 수 있는데, 상기 고형암은 간암, 폐암, 췌장암, 유방암, 난소암, 자궁내막암, 자궁경부암, 담낭암, 위암, 담도암, 대장암, 두경부암, 식도암, 갑상선암, 뇌종양, 악성 흑색종, 전립선암, 고환암, 설암, 또는 골수암일 수 있다.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.
이하, 본 발명을 첨부되는 도면을 이용하여 보다 상세히 설명한다.Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings.
본 발명자들은 NK 림프암종 환자의 암조직으로부터 표 1에 기재된 특성을 갖는 새로운 NK 세포주를 분리하였으며, 이에 대한 다양한 특성을 조사한 결과, 도 1 내지 6에 나타난 바와 같이, IL-2 의존적 증식능을 나타내며, 암세포 사멸능과 면역조절능을 모두 갖는 다기능성 NK 세포주임을 확인할 수 있었다. 특히, 현재 임상시험이 진행중인 유일한 NK 세포주인 NK-92에 비해 증식능이 현저하게 높아서 경제적으로 생산가능한 세포임을 확인하여, 이를 'NK101' 세포주로 명명하고 이를 대한민국 전라북도 정읍시 입신길 181번지에 소재하고 있는 한국생명공학연구원 내 한국유전자은행(Korean Collection for Type Culture, KCTC)에 2017년 8월 7일자로 기탁하여, 2017년 8월 24일자로 KCTC 13305BP의 수탁번호를 부여받았다. 그러나, 상기 NK101 세포주는 유전자 발현 분석 결과, IL-2 수용체인 CD25는 고발현이고, CD56dimCD62L+의 표현형을 갖는 것으로 확인되었으나, NK 세포의 항암 활성에 직접적인 영향을 주는 NK 세포 활성화 인자인 CD7 및 CD28은 발현하지 않음을 확인함으로써, 항암 활성 자체는 종래에 구축된 NK 세포주 보다 높지 않을 것으로 예상하게 되었다. 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. It was deposited on August 7, 2017 by the Korean Collection for Type Culture (KCTC) in the Korea Research Institute of Bioscience and Biotechnology, and received an accession number of KCTC 13305BP on August 24, 2017. However, the gene expression analysis of the NK101 cell line revealed that the IL-2 receptor CD25 was highly expressed and had a phenotype of CD56 dim CD62L + , but CD7, an NK cell activating factor that directly affects the anticancer activity of NK cells. And by confirming that CD28 does not express, anticancer activity itself was expected to not be higher than conventionally constructed NK cell line.
이에, 본 발명자들은 본 발명의 일 실시예에 따른 NK101 세포의 항암활성을 강화하기 위해, NK101 세포에 기반한 유전자 변형 NK 세포주를 제조하고자 하였다. 도 7b는 상기 목적을 달성하기 위해, 본 발명의 일 실시예에 따른 유전자 변형 NK 세포주 SL-K01의 제조에 사용된 유전자 컨스트럭트 CD7-CD28-CD::UPRT의 구조를 개략적으로 나타낸 개요도이고 및 도 9a는 본 발명의 일 실시예에 따른 유전자 변형 NK 세포주 NK111의 제조를 위해 사용된 유전자 컨스트럭트 mbIL-15-mTGFβ1IΔcyto의 구조를 개략적으로 나타낸 개요도이다.Thus, the present inventors intended to prepare a genetically modified NK cell line based on NK101 cells in order to enhance the anticancer activity of NK101 cells according to an embodiment of the present invention. 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-mTGFβ1IΔcyto used for the preparation of the genetically modified NK cell line NK111 according to an embodiment of the present invention.
본 발명에서는 앞서 언급한 요소를 보완하기 위하여, 본 발명자들에 의해 기확립된 NK101 세포(수탁번호 KCTC 13305BP)에 NK 세포의 보조활성화 수용체, 세포자살유전자, 세포막 고정(membrane bound) 사이토카인, 돌연변이 TGFβ 수용체를 도입하여 해당 NK 세포의 항암효과 증진, 및 사이토카인 보충제(cytokine supplement) 비의존적으로 NK 세포의 증식이 가능한 NK111 세포주를 구축하였다(도 9b 및 9c 참조). In the present invention, in order to complement the aforementioned elements, co-activating receptors, apoptotic genes, membrane bound cytokines, mutations of NK cells in NK101 cells (accession number KCTC 13305BP) previously established by the present inventors The TGFβ 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).
도 7은 본 발명의 일 실시예에 따른 유전자 변형 NK 세포주(SL-K01) 및 상기 SL-K01의 모세포주인 NK101, 그리고 종래구축된 NK 세포주인 KHYG-1 및 NK-92에서의 세포 표면 표지자의 발현 여부를 분석한 결과로서, 도 7a는 NK101, KHYG-1 및 NK-92에서 CD7 및 CD28의 발현여부를 분석한 유세포 분석 결과를 나타내는 히스토그램이고, 도 7c는 상기 도 7b에 도시된 유전자 컨스트럭트를 형질도입한 본 발명의 일 실시예에 따른 유전자 변형 세포주 SL-K01 세포에서의 CD7 및 CD28 발현을 유세포 분석을 통해 확인한 결과를 나타내는 히스토그램이다. 상기 도 7에서 확인되는 바와 같이, 기존에 구축된 NK세포주 들 중 암세포 사멸능이 높다고 알려져 있는 KHYG-1 및 NK-92는 공통적으로 CD7을 발현하는 특징을 갖고, NK-92 세포는 CD7은 물론 CD28까지도 동시에 발현하는 특성을 가지고 있는 반면, NK101은 두 보조자극 인자의 발현이 전혀 되지 않음을 확인할 수 있었다. 이에, 본 발명자들은 NK101 세포에 CD7 및 CD28 도입을 통해 암세포 사멸능이 증진되는지의 여부를 평가하고자 하였다. 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 As a result of analyzing the expression, 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. As shown in FIG. 7, KHYG-1 and 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.
도 8은 본 발명의 일 실시예에 따른 유전자 변형 NK 세포주 SL-K01 및 상기 SL-K01의 모세포주인 NK101의 다양한 암세포에 대한 세포사멸능을 분석한 결과로서, 도 8a는 HDLM-2, IM-9, JEKO-1 및 K562 암세포를 NK101 또는 SL-K01 세포와 4:1 비율로 24시간 공배양한 후 암세포 사멸빈도를 유세포 분석을 통해 측정한 결과를 나타내는 그래프이고, 도 8b는 다양한 농도의 5-FC를 NK101 또는 SL-K01 세포에 48시간 처리한 뒤, 세포 성장률을 MTS 분석을 통하여 확인한 결과를 나타내는 그래프이며, 도 8c는 IM-9 세포주와 NK101 또는 SL-K01 세포를 2:1, 1:1, 또는 0.5:1 비율로 공배양 시, 5-FC 유무에 따른 IM-9 암세포 사멸 빈도를 유세포 분석을 통해 측정한 결과를 나타내는 그래프이다. 도 8a 내지 8c에서 확인되듯이, 본 발명의 일 실시예에 따른 유전자 변형 NK 세포주 SL-K01은 NK101 대비 다양한 암세포주에서 우수한 살상능을 보여, CD7, CD28 도입을 통해 NK101 세포의 살상능이 증진되었음을 확인할 수 있었다. 또한, 5-FC 처리를 통해 SL-K01 세포의 제거를 유도함으로써 안전성을 확보할 수 있을 뿐 아니라, 방관자 살상 효과까지 유도하여 주변의 암세포를 제거할 수 있음을 확인하였다. 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, and 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. As shown in Figures 8a to 8c, the genetically modified NK cell line SL-K01 according to an embodiment of the present invention 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. In addition, it was confirmed that not only can secure safety by inducing the removal of SL-K01 cells through 5-FC treatment, but also induces bystander killing effect to remove surrounding cancer cells.
도 9는 본 발명의 또 다른 일 실시예에 따른 유전자 변형 NK 세포주인 NK111 세포주의 구축과정을 나타내는 것으로서, 도 9a는 SL-K01 세포의 추가적 암세포 사멸 효과 증진 및 면역억제인자 TGF-β에 대한 저항성 유도를 위해 도입된 유전자 컨스트럭트의 개요도이고, 도 9b는 SL-K01 및 상기 도 9a에 도시된 유전자 컨스트럭트가 도입된 SL-K01 세포(NK111로 명명) 표면에서의 IL-15 발현을 유세포 분석을 통해 확인한 결과를 나타내는 히스토그램이며, 도 9c는 SL-K01 및 NK111 세포에서의 TGFβRIIΔcyto 발현을 유세포 분석을 통해 확인한 결과를 나타내는 히스토그램이다. 도 8에서 확인된 바와 같이, CD7/CD28 도입은 NK101 세포의 살상능 증진에 중간 정도의 효과를 나타냈다. 이에 본 발명자들은 SL-K01 세포의 암세포 사멸능을 극대화하기 위해, 대표적 NK 세포 활성화 인자인 막결합(membrane bound) IL-15을 SL-K01 세포에 형질도입하고자 하였고, 면역억제인자 TGF-β에 대한 저항성 유도를 위해 세포질 도메인(cytoplasmic domain)이 결실된 TGFβRIIΔcyto을 과발현시켜 미끼 수용체(decoy receptor)로 작용하도록 하였다. 그 결과, 도 9b 및 9c에서 확인되는 바와 같이, 모세포주인 SL-K01에서는 발현이 되지 않던 IL-15 및 TGFβRII가 본 발명의 일 실시예에 따른 NK111 세포주에서는 발현이 됨이 확인되었다.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-β 9B is a schematic diagram of the gene construct introduced for induction, and 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 TGFβRIIΔcyto expression in SL-K01 and NK111 cells. As confirmed in FIG. 8, CD7 / CD28 introduction showed a moderate effect on the killing of NK101 cells. In order to maximize the cancer cell killing ability of the SL-K01 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-β. To induce resistance to TGFβRIIΔcyto-deleted cytoplasmic domain overexpressed to act as a decoy receptor (decoy receptor). As a result, as shown in Figures 9b and 9c, IL-15 and TGFβRII that was not expressed in the parent cell line SL-K01 was confirmed that the expression in the NK111 cell line according to an embodiment of the present invention.
도 10은 본 발명의 일 실시예에 따른 유전자 변형 NK 세포주인 NK111의 항암활성 및 안전성을 비교분석한 결과로서, 도 10a는 SL-K01 및 NK111 세포의 배양 시, IL-2 유무에 따른 세포수 증식수준(population doubling level)을 나타낸 그래프이고, 도 10b는 NK101, SL-K01 및 NK111 세포에서의 NKG2D 발현을 유세포 분석을 통해 확인한 결과를 나타내는 히스토그램이며, 도 10c는 IM-9 세포주와 SL-K01 또는 NK111 세포를 2:1, 1:1, 또는 0.54:1 비율로 공배양 시, 5-FC 유무에 따른 IM-9 암세포의 사멸빈도를 유세포 분석을 통해 측정한 결과를 나타내는 그래프이고, 도 10d는 OVCAR-3 또는 THP-1 세포주를 SL-K01 및 NK111 세포와 공배양시, 다양한 농도의 TGFβ1을 처리한 후, 암세포 사멸능에 미치는 영향을 나타낸 그래프이다. 도 10a에서 확인되듯이, 막결합 IL-15의 도입을 통해, 본 발명의 일 실시예에 따른 NK111는 IL-2 독립적으로 세포성장이 가능하게 되었으며, 이는 생산시 편의성을 높여준다. 또한, 도 10b에서 확인되는 바와 같이, IL-15 도입을 통해 NK 세포의 대표적 활성화 수용체인 NKG2D 발현이 상향조절됨으로써 암세포에 대한 살상능을 증진시킴을 확인할 수 있었다. 아울러, 도 10c에서 확인되는 바와 같이, 5-FC 처리에 의한 방관자 살상 효과(bystander killing effect)가 더해지면, 1:1 비율에서 암세포 사멸을 90% 정도로 유도할 수 있을 만큼 살상능이 증진되었다. 또한, 도 10d에서 확인되는 바와 같이, 종양미세환경에서 과다분비되는 면역억제인자인 TGFβ1에 대한 미끼 수용체인 TGFβRIIΔcyto 도입을 통해 TGFβ1에 대한 살상능 저하에도 영향을 받지 않게 되었는데, 이는 추후 생체내 활성 감소를 어느 정도 극복할 수 있음을 기대하게 하는 특성이다.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, and 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. Is a graph showing the effect on cancer cell killing ability after co-culture of OVCAR-3 or THP-1 cell line with SL-K01 and NK111 cells after treatment with various concentrations of TGFβ1. As shown in Figure 10a, through the introduction of membrane-bound IL-15, NK111 according to an embodiment of the present invention is capable of cell growth independently of IL-2, which increases the convenience in production. In addition, as confirmed in FIG. 10B, it was confirmed that NKG2D expression, a representative activating receptor of NK cells, was upregulated through the introduction of IL-15, thereby enhancing killing ability against cancer cells. In addition, as can be seen in Figure 10c, bystander killing effect (bystander killing effect) by the 5-FC treatment, killing ability was increased enough to induce cancer cell death to about 90% at 1: 1 ratio. In addition, as shown in Figure 10d, through the introduction of TGFβRIIΔcyto, a bait receptor for TGFβ1, an over-secreted immunosuppressive factor in the tumor microenvironment, it was not affected by the killing ability of TGFβ1, which would later decrease in vivo activity. It is a characteristic that expects to overcome to some extent.
더 나아가, 본 발명자들은 본 발명의 일 실시예에 따른 유전자 변형 NK 세포주를 이용하여 항원 특이적 세포치료가 가능한 키메라 항원 수용체를 형질도입할 경우 해당 암항원을 고발현하는 암에 대하여 보다 효과적인 항암치료가 가능할 수 있을 것이라는 가정하에 난소암 등 다양한 고형암에서 과발현되는 것으로 알려진 EpCAM을 표적으로 한 키메라 항원 수용체 컨스트럭트를 고안한 후(도 11), 이를 제작하여 상기에서 제조한 NK111 세포주에 추가로 형질도입시킨 결과, 장기계대시에도 모세포(NK101)의 특성은 그대로 유지하면서도 형질도입된 유전자들을 정상적으로 발현시킬 뿐만 아니라(도 12a 내지 도 14b), EpCAM을 과발현하는 암세포에 대하여 시험관내 조건은 물론 생체내 조건에서 매우 효과적인 항암활성을 나타냄을 실험적으로 입증함으로써(도 15a 내지 15e) 본 발명을 완성하였다. Furthermore, 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 Under the assumption that it may be possible, after devising a chimeric antigen receptor construct targeting EpCAM known to be overexpressed in various solid cancers, such as ovarian cancer (FIG. 11), it was further transformed into the NK111 cell line prepared above. As a result of introduction, not only the expression of the transduced genes normally while maintaining the characteristics of the parent cell (NK101) even during long-term passage, but also in vitro conditions for cancer cells overexpressing EpCAM, as well as in vivo By experimentally demonstrating a very effective anticancer activity under the conditions (FIG. 15A If 15e), thereby completing the present invention.
이하, 실시예 및 실험예를 통하여 본 발명을 더 상세히 설명한다. 그러나 본 발명은 이하에서 개시되는 실시예 및 실험예에 한정되는 것이 아니라 서로 다른 다양한 형태로 구현될 수 있는 것으로, 이하의 실시예 및 실험예는 본 발명의 개시가 완전하도록 하며, 통상의 지식을 가진 자에게 발명의 범주를 완전하게 알려주기 위해 제공되는 것이다. Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, the present invention is not limited to the examples and experimental examples disclosed below, but may be embodied in various different forms. The following examples and experimental examples are provided to make the disclosure of the present invention complete and the general knowledge. It is provided to fully inform those who have the scope of the invention.
실시예 1: 본 발명의 NK 세포주의 제조 과정Example 1 Preparation of NK Cell Lines of the Invention
NK 세포 유래 세포주를 제작하기 위하여 다음과 같은 과정을 거쳤다. 환자에서 유래한 림프절외(extranodal) NK 림프암종을 40 μm 스트레이너에 올려두고, 20% 우태아혈청(GE Healthcare, USA)와 1% 항생제(Gibco, USA)가 포함된 Cellgro® 줄기세포 성장배지(SCGM; CellGenix, Germany, 이하 'NK media'라 함) 10 mL을 첨가한 후 5 mL 주사기의 피스톤의 전단력을 이용하여 단일세포로 떼어낸 후 현탁하였다. 단일세포 현탁액 중 NK 세포를 NK 분리키트(Milltenyi Biotec, Germany)를 이용하여 분리한 후 1000 U/mL의 인간 재조합 IL-2(recombinant human IL-2; rhIL-2; Prometheus Laboratories Inc., USA)가 첨가된 NK media에서 3주간 배양하였다. IL-2가 포함된 NK media를 주 2회 첨가하였으며, 분열 세포주를 30계대까지 지속 배양하여 안정적인 세포주가 형성되었음을 확인하였다(도 1a). 상기 세포주는 CD3, CD20, CD16는 발현하지 않으면서 CD56을 발현하므로, 해당 세포의 기원이 NK 세포임을 확인하였다(도 1b). 해당 세포주는 배양 시 군집(spheroid)을 형성하는 특징이 있다는 것을 현미경 상에서 확인할 수 있었으며(도 1c), Wright-Giemsa 염색법을 이용한 형태학적 분석에서 NK101 세포가 큰 과립성 림프구(large granular lymphocyte)의 특성을 가짐을 확인하였다(도 1d). NK 세포 특성인 세포 사멸인자 퍼포린(Perforin, green) 및 그랜자임 B(Granzyme B, red)의 형광염색을 통해 NK101이 퍼포린과 그랜자임 B를 발현함을 확인하였다(도 1e). NK 세포에 민감하게 반응하는 MHCⅠ 음성 세포인 K562와 공배양을 수행하였다. 카르복시플루오레세인 디아세테이트(CFDA; Invitrogen, USA)로 표지한 K562 세포를 3x105 cells/mL 농도로 24-웰 플레이트(well-plate; Corning, USA)에 파종한 뒤 NK101 세포를 다양한 효과기 세포 대 표적 비율(E:T 비율 = 1:1, 2:1, 4:1, 10:1)로 1 mL 배지에 부유한 후 상기 암세포와 24시간 동안 함께 배양하였다. 배양 이후 모든 세포를 각 웰로부터 회수하여 원심 분리한 후, 세포 펠렛을 FACS 완충액으로 현탁시켰다. 다시 원심분리하여 형성된 세포 펠렛을 1 μL LIVE/DEAD® Fixable Near-IR Dead Stain Kit(Invitrogen, USA)를 넣은 FACS 완충액 100 μL에 현탁시켜, 4℃에서 20분간 반응하였다. FACS 완충액으로 2번 세척한 뒤, 1X Annexin V binding buffer 100 μL에 Annexin V APC 5 μL(Biolegend, USA)를 희석한 용액에 세포 펠렛을 현탁시켜, 실온에서 20분간 반응하였다. 세포의 사멸 여부는 유세포 분석법을 이용하여 생존 세포(annexin V-음성/LIVE/DEAD-음성), 초기 아폽토시스 세포(annexin V-양성/LIVE/DEAD-음성), 후기 아폽토시스 세포(annexin V-양성/LIVE/DEAD-양성), 괴사(necrotic) 세포(annexin V-음성/LIVE/DEAD-양성)으로 구분하였다. 도 1의 결과를 통하여 NK101은 지속적인 계대가 가능한 불멸화 세포이고, 배양 시 군집을 형성하며, 표현형 및 기능이 기존에 알려진 NK 세포와 일치하는 특성을 가짐을 알 수 있었다. 해당세포의 세포주화 및 NK 세포의 특성을 확인함으로써 해당 세포주를 'NK101'으로 명명하였으며, 이를 대한민국 전라북도 정읍시 입신길 181번지에 소재하고 있는 한국생명공학연구원 내 한국유전자은행(Korean Collection for Type Culture, KCTC)에 2017년 8월 7일자로 기탁하여, 2017년 8월 24일자로 KCTC 13305BP의 수탁번호를 부여받았다. 상기 기탁기관은 부다페스트 조약상 국제기탁기관이다.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 μm 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 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. 1c), and the characteristics of large granular lymphocytes with NK101 cells in morphological analysis using Wright-Giemsa staining method. It was confirmed that it has (Fig. 1d). Fluorescent staining of cell death factors, Perforin (green) and Granzyme B (Granzyme B, red), NK cell characteristics, confirmed that NK101 expressed Perforin and Granzyme B (FIG. 1E). Coculture was performed with K562, a MHCI negative cell that responds sensitively to NK cells. K562 cells labeled with Carboxyfluorescein Diacetate (CFDA; Invitrogen, USA) were seeded in 24-well plates (Corning, USA) at a concentration of 3x10 5 cells / mL and NK101 cells were treated with various effector cells. It was suspended in 1 mL medium at a target ratio (E: T ratio = 1: 1, 2: 1, 4: 1, 10: 1), and then incubated with the cancer cells for 24 hours. After incubation all cells were recovered from each well and centrifuged, and the cell pellet was suspended in FACS buffer. By suspending the cell pellet formed by centrifuging again in 1 μL LIVE / DEAD ® Fixable Near -IR Dead Stain Kit FACS buffer, 100 μL insert the (Invitrogen, USA), was reacted at 4 ℃ 20 minutes. After washing twice with FACS buffer, the cell pellet was suspended in a solution diluted with 5 μL of Annexin V APC (Biolegend, USA) in 100 μL 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). 1 shows that 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.
실시예 2 : NK101의 세포 분열능 분석Example 2 Analysis of Cell Division Capacity of NK101
상기 실시예 1에서 제조된 NK101 세포주에 대한 배양 조건 확립 및 분열능 비교를 위하여, NK101 세포와 대조군 세포인 NK-92 세포에 20% 우태아 혈청이 포함된 SCGM 배양배지에 다양한 농도의 IL-2를 처리한 후 MTS 분석법을 통해 두 세포주의 세포생장을 비교하였다. 도 2a와 같이 NK101은 약 8pM의 IL-2 농도에서 생장이 시작되어 500 pM에서 생장이 정체되었으며(EC50=23.3 pM), NK-92는 30 pM 농도의 IL-2 존재 시 세포 생장이 확인되며 2000 pM 농도에서 생장이 정체되어(EC50=128.3 pM) 세포생장에 있어 NK101이 NK-92 대비 낮은 농도의 IL-2를 필요로 함을 알 수 있었다. 도 2b에서 확인한 IL-2 수용체 소단위체 발현을 유세포 분석으로 확인한 결과, NK101이 고친화 IL-2 수용체인 CD25를 NK-92 대비 높게 발현함을 확인하였다. 또한 NK101 및 NK-92의 동결 후 세포 생장률 및 생존율을 동일 배양조건에서 분석한 결과 NK101은 해동 후 2일(1 계대) 이후 세포생장이 회복되나, NK-92는 해동 후 10일(5 계대) 이후 일정한 세포생장에 도달함을 확인할 수 있었다(도 2c). 두 세포의 세포생장이 안정화된 후, 두 세포의 세포증식을 비교하였을 때 16일 계대 후 수득되는 NK101 세포의 총 세포주가 NK-92 세포의 수득 예상 세포수의 약 100배임을 확인할 수 있었다(도 2d). 이는, 본 발명의 NK101 세포의 생산성이 종래의 NK-92 세포에 비해 월등하여, 경제성의 측면에서 본 발명의 NK101 세포가 매우 유리함을 시사하는 것이다.In order to establish culture conditions and compare the sequencing ability of the NK101 cell line prepared in Example 1, IL-2 at various concentrations in SCGM culture medium containing 20% fetal bovine serum in NK101 cells and NK-92 cells as control cells After treatment, the cell growth of the two cell lines was compared by MTS analysis. As shown in FIG. 2A, NK101 started to grow at an IL-2 concentration of about 8 pM, and stagnated at 500 pM (EC 50 = 23.3 pM), and NK-92 confirmed cell growth in the presence of 30 pM IL-2. The growth was stagnant at the concentration of 2000 pM (EC 50 = 128.3 pM), indicating that NK101 requires a lower concentration of IL-2 than NK-92. As a result of confirming the expression of the IL-2 receptor subunit confirmed in FIG. 2B by flow cytometry, it was confirmed that NK101 expresses CD25, which is a high affinity IL-2 receptor, higher than that of NK-92. In addition, NK101 and NK-92 after freezing cell growth rate and survival rate were analyzed under the same culture conditions, NK101 recovered cell growth after 2 days (1 passage) after thawing, but NK-92 10 days after thawing (5 passages) Since it was confirmed that reached a certain cell growth (Fig. 2c). After cell growth of the two cells was stabilized, when comparing the cell proliferation of the two cells, it was confirmed that the total cell line of NK101 cells obtained after 16 days of passage was about 100 times the expected number of cells obtained from NK-92 cells (FIG. 2d). This suggests that the productivity of the NK101 cells of the present invention is superior to that of the conventional NK-92 cells, and that the NK101 cells of the present invention are very advantageous in terms of economy.
본 발명의 NK101 세포의 종합적인 특성은 하기 [표 1]로 정리하였다.The comprehensive characteristics of NK101 cells of the present invention are summarized in the following [Table 1].
항목Item | NK101 세포NK101 cells |
임상 데이터Clinical data | |
연령/성별Age / gender | 56세 남성56 year old male |
인종race | 아시안Asian |
진단Diagnosis | 절외 NK/T 림프종(extranodal NK/T lymphoma)Extraranodal NK / T lymphoma |
세포배양Cell culture | |
성장 양상Growth pattern | 현탁상태에서 다중세포 응집체Multicellular Aggregates in Suspension |
배가시간Doubling Time | 18-32시간 18-32 hours |
최대 세포농도Cell concentration | 1.2x106 cells/㎖1.2x10 6 cells / ml |
최소 세포농도Minimum cell concentration | 0.5x105 cells/㎖0.5x10 5 cells / ml |
사이토카인 의존성Cytokine dependence | IL-2 의존성(500 IU/㎖)IL-2 dependency (500 IU / ml) |
최적 분열Optimal cleavage | 매 2-3일Every 2-3 days |
면역학적 특성Immunological properties | |
T/NK 마커T / NK marker | CD2+, CD3-, CD4-, CD7-, CD8-, CD16-, CD56+ CD2 +, CD3 -, CD4 - , CD7 -, CD8 -, CD16 -, CD56 + |
B 세포 마커B cell marker | CD10-, CD19-, CD20- CD10 -, CD19 -, CD20 - |
골수단핵구성 마커Bone Supranuclear Markers | CD13-, CD14-, CD33+ CD13 -, CD14 -, CD33 + |
NK 세포 활성화 수용체NK cell activating receptor | NKp46+, NKp30+, NKG2D+ NKp46 + , NKp30 + , NKG2D + |
NK 세포 저해성 수용체NK cell inhibitory receptor | KIR2DL1-, KIR2DL2-, ILT2- KIR2DL1 -, KIR2DL2 -, ILT2 - |
자손/활성화 마커Offspring / activation markers | CD34-, FAS+ CD34 -, FAS + |
부착 마커Attachment marker | CD11a+, CD18+, CD54+ CD11a + , CD18 + , CD54 + |
기능적 특성Functional characteristics | |
NK 활성NK activity | 호-염증성(pro-inflammatory) 사이토카인 분비능(면역활성능) 및 증식능Pro-inflammatory cytokine secretion (immune activity) and proliferation |
사이토카인 생산Cytokine production | IFN-γ, TNF-α, GM-CSF, IL-2 등 호-염증성 사이토카인 분비 및 IL-1 수용체 길항제 및 IL-10 등 항-염증성 사이토카인 미분비Secretion of anti-inflammatory cytokines such as IFN-γ, TNF-α, GM-CSF, IL-2, and secretion of anti-inflammatory cytokines such as IL-1 receptor antagonists and IL-10 |
사이토카인 수용체Cytokine receptors | CD25+, CD122+, CD132+, CD127- CD25 +, CD122 +, CD132 + , CD127 - |
케모카인 수용체Chemokine receptor | CCR4+, CCR6+, CCR7+, CCR8+, CXCR3+, CXCR4+ CCR4 + , CCR6 + , CCR7 + , CCR8 + , CXCR3 + , CXCR4 + |
실시예 3 : 표면 마커 분석 상기 실시예 1에서 제조된 본 발명의 NK101 세포주는 부유세포의 특성을 가지며, 해당 세포의 표면항원의 발현도를 유세포 분석으로 확인하였다. T/NK 세포 마커의 경우, 본 발명의 NK101 세포주는 NK 세포의 표면항원인 CD2, CD56을 발현하나 CD16은 발현하지 않으며, T 세포의 표면항원인 CD3, CD4, CD8, TCRαβ 및 TCRγδ과 B세포 표면항원인 CD20, 단핵구 표현항원 CD14를 발현하지 않는 NK 세포의 표현형을 가지고 있었다(도 3a). 또한, 본 발명의 NK101 세포주는 NK 세포의 활성화 수용체인 NKG2D, NKp30, NKp46, DNAM-1, 2B4를 발현하였고, 비활성화 수용체인 CD94, NKG2A는 발현하였으나, KIR2DL1/S1/S3/S5, KIR2DL2/DL3, 및 CD85j 등은 발현하지 않았다(도 3b). 또한 세포부착 분자인 CD2, CD11a, CD19, ICAM-1을 발현하며, CD7은 미발현하였다(도 3c). 추가적으로 본 발명의 NK101 세포주는 NK 세포의 세포독성 및 면역활성화에 관여하는 CD107a, 퍼포린, 그랜자임 B은 고발현되고, TRAIL 및 FASL은 낮은 수준이긴 하나 발현되며(도 3d), NK 세포의 사이토카인 수용체 중에서는 IL-2 고친화 수용체인 CD25, IL-2 수용체(CD122, CD132) 및 IL-15 수용체인 IL-15Ra를 발현함을 확인하였다(도 3e). NK 세포주의 이동성에 관련된 케모카인 수용체 중에서는 CCR4, CCR6, CCR7, CXCR3, 및 CXCR4를 발현하였으나, 그 외의 CCR 및 CXCR은 발현되지 않았다(도 3f). 결론적으로, 본 발명의 NK101 세포주는 활성화된 NK 세포의 표현형을 보이며, 특히 CD25가 고발현된다는 점에서 다른 NK 세포주와 구분되는데, CD25는 활성화된 NK 세포의 지표로 특히 분열능이 높은 NK 세포의 표지자로 알려 있어(Clausen, J. et al., Immunobiology, 207(2): 85-93, 2003), 본 발명의 NK101 세포주는 세포치료제의 대량생산에 매우 적합한 세포주임을 알 수 있다. Example 3 Surface Marker Analysis The NK101 cell line of the present invention prepared in Example 1 had the characteristics of floating cells, and the expression level of the surface antigen of the cells was confirmed by flow cytometry. In the case of the T / NK cell marker, the NK101 cell line of the present invention expresses CD2 and CD56, which are surface antigens of NK cells, but does not express CD16, but CD3, CD4, CD8, TCRαβ and TCRγδ and B cells, which are surface antigens of T cells. It had a phenotype of NK cells not expressing the surface antigen CD20 and monocyte expressing antigen CD14 (FIG. 3A). In addition, the NK101 cell line of the present invention expressed NKG2D, NKp30, NKp46, DNAM-1, 2B4, which are activating receptors of NK cells, and expressed inactive receptors, CD94, NKG2A, but KIR2DL1 / S1 / S3 / S5, KIR2DL2 / DL3. , And CD85j and the like were not expressed (FIG. 3B). In addition, they express the cell adhesion molecules CD2, CD11a, CD19, ICAM-1, CD7 was not expressed (Fig. 3c). In addition, the NK101 cell line of the present invention is highly expressed CD107a, Perforin, Granzyme B, which is involved in the cytotoxicity and immune activation of NK cells, and TRAIL and FASL are expressed at a low level (Fig. 3d), cytokines of NK cells Among the caine receptors, it was confirmed that they express CD25, IL-2 receptors (CD122, CD132) and IL-15Ra, which are IL-2 high affinity receptors (FIG. 3E). Among chemokine receptors involved in the mobility of NK cell lines, CCR4, CCR6, CCR7, CXCR3, and CXCR4 were expressed, but other CCRs and CXCRs were not expressed (FIG. 3F). In conclusion, 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. (Clausen, J. et al ., Immunobiology , 207 (2): 85-93, 2003), it can be seen that the NK101 cell line of the present invention is a very suitable cell line for mass production of cell therapeutics.
본 발명의 NK101 세포의 다양한 세포표지자의 발현여부는 하기 [표 2]로 정리하였다.Expression of various cell markers of NK101 cells of the present invention is summarized in the following [Table 2].
항원antigen | 발현여부Expression | 항원antigen | 발현여부Expression |
계통 마커Strain marker | 기능성 마커Functional marker | ||
CD1aCD1a | -- | CD95 (FAS)CD95 (FAS) | ++++++ |
CD2CD2 | ++++++ | CD178 (FAS-L)CD178 (FAS-L) | ++ |
CD3CD3 | -- | CD107aCD107a | ++++++ |
CD4CD4 | -- | TRAIL (CD253)TRAIL (CD253) | ++ |
CD5CD5 | -- | 퍼포린Perforin | ++++++ |
CD8CD8 | -- | 그랜자임 BGranzyme B | ++++++ |
CD10CD10 | -- | IFNγIFNγ | ++++++ |
CD11aCD11a | ++++++ | 케모카인 수용체Chemokine receptor | |
CD11cCD11c | -- | CCR1CCR1 | ++ |
CD13CD13 | ++ | CCR2CCR2 | -- |
CD14CD14 | ++ | CCR3CCR3 | -- |
CD16CD16 | -- | CCR4CCR4 | ++++++ |
CD18CD18 | ++++++ | CCR5CCR5 | ++ |
CD19CD19 | -- | CCR6CCR6 | ++++++ |
CD23CD23 | -- | CCR7CCR7 | ++++++ |
CD33CD33 | ++++++ | CCR8CCR8 | ++++ |
CD45CD45 | ++++++ | CCR9CCR9 | ++ |
CD56CD56 | ++++++ | CXCR1CXCR1 | -- |
CD57CD57 | -- | CXCR2CXCR2 | -- |
CD161CD161 | ++++ | CXCR3CXCR3 | ++++++ |
활성화 수용체Activating receptor | CXCR4CXCR4 | ++++++ | |
2B42B4 | ++++++ | CXCR5CXCR5 | -- |
NKp30NKp30 | ++++ | CXCR6CXCR6 | -- |
NKp46NKp46 | ++++++ | CXCR7CXCR7 | -- |
NKG2DNKG2D | ++++ | 사이토카인 수용체Cytokine receptors | |
저해 수용체Inhibitory receptor | CD25(IL-2Ra)CD25 (IL-2Ra) | ++++++ | |
CD85j(ILT2)CD85j (ILT2) | -- | CD122(IL-2Rb)CD122 (IL-2Rb) | ++++ |
CD94CD94 | ++++++ | CD132(공통 γ 사슬)CD132 (common γ chain) | ++++++ |
CD158(KIR2DL1/S1/S3/S5)CD158 (KIR2DL1 / S1 / S3 / S5) | -- | CD127(IL-7Ra)CD127 (IL-7Ra) | -- |
CD158b(KIR2DL2/DL3)CD158b (KIR2DL2 / DL3) | -- | 기타 마커Guitar markers | |
CD159aCD159a | ++++++ | TCRαβTCRαβ | -- |
부착분자Adhesion molecule | TCRγδTCRγδ | -- | |
DNAM-1(CD226)DNAM-1 (CD226) | ++++++ | ||
ICAM-1(CD54)ICAM-1 (CD54) | ++++++ | ||
CD62LCD62L | ++++ | ||
-, 음성; +, < 10% 양성; ++, 10-69% 양성; +++, 70-100% 양성(%는 세포 집단 내 양성 세포의 비율을 나타냄)-, voice; +, <10% positive; ++, 10-69% positive; +++, 70-100% positive (% represents percentage of positive cells in cell population) |
실시예 4 : NK101의 CD56 및 D62L 발현양상 확인 NK101 세포의 특성을 분석하기 위하여 NK 세포의 표지마커인 CD56 및 CD62L의 발현양상을 NK-92와 초도배양 NK 세포와 비교하여 유세포 분석을 이용하여 확인하였다. 도 4a에서와 같이 NK101 세포는 NK-92 세포 대비 CD56 발현 정도가 낮은 CD56dim NK 세포로 확인되었다. 또한 도 4b의 등고선 그래프와 같이 NK101 세포는 CD62L 마커를 고발현하는데, 이는 NK-92 세포에서는 미발현되고, 일부 초도배양 NK 세포에서 한정적으로 발현되는 마커이다. 일반적인 초도배양 NK 세포에서는 CD56의 발현에 따라 CD56dim, CD56bright으로 구분할 수 있고 이는 각각 세포독성 또는 사이토카인 생산이 더 우세한 특성을 가진 2개의 군집으로 구성된다고 여겨진다. CD56bright NK 세포는 높은 세포 증식능, 사이토카인에 의한 활성화 시 IFN-γ를 분비, 낮은 암세포 사멸능을 보이며, CD56dim NK 세포는 CD56bright NK 세포와는 반대로 세포 증식능은 낮고, 표적세포의 인지에 의해서 IFN-γ를 분비하며, 높은 세포독성을 가지는 특성이 있다. 최근 검증된 CD56dimCD62L+ NK 세포는(Juelke, K et al,, Blood, 116(8): 1299-1307, 2010; Luetke-Eversolh, M et al., Front. Immunol., 4: 499, 2013) CD56bright, CD56dim NK 세포의 특성을 모두 가지고 있는 다기능성의 NK 세포로 보고된다. NK101은 사이토카인 자극에 의해 증식 및 IFN-γ를 분비할 수 있고(도 5a), 표적세포 인지 시에도 다양한 종류의 사이토카인을 분비함이 확인되었다(도 5b). 결론적으로 NK101은 CD56dimCD62L+ NK 세포의 표현형과 특성을 가지고 있으며, CD56dimCD62L+ 특성 마커를 발현하는 초도배양 NK 세포 혹은 체외 증식(ex vivo expanded) 초도배양 NK 세포가 보고된 바가 있다 하더라도 NK101은 불멸화된 NK 세포주로서 이들과는 차별되며, 이는 기존 알려진 NK 세포주 중에서도 찾아볼 수 없는 고유 특성이라고 할 수 있다. Example 4 Identification of CD56 and D62L Expression of NK101 In order to analyze the characteristics of NK101 cells, expression of CD56 and CD62L, markers of NK cells, was identified by flow cytometry in comparison with NK-92 and primary cultured NK cells. It was. As shown in FIG. 4A, NK101 cells were identified as CD56 dim NK cells having a lower CD56 expression level than NK-92 cells. In addition, as shown in the contour graph of FIG. 4B, NK101 cells express high CD62L markers, which are unexpressed in NK-92 cells and are expressed in limited numbers in some cultured NK cells. In general cultured NK cells, 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-γ 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-γ 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. Immunol ., 4: 499, 2013 ) CD56 bright , CD56 dim NK cells are reported as a multi-functional NK cells that have both characteristics. NK101 can secrete proliferation and IFN- [gamma] by cytokine stimulation (FIG. 5a), and it has been confirmed that it secretes various types of cytokines even upon recognition of target cells (FIG. 5b). In conclusion, NK101 has the phenotype and characteristics of CD56 dim CD62L + NK cells, and NK101 has been reported for either primary cultured NK cells or ex vivo expanded primary cultured NK cells expressing CD56 dim CD62L + characteristic markers. Is an immortalized NK cell line, which is distinguished from them, which can be said to be a unique characteristic not found among known NK cell lines.
실시예 5 : NK101의 시험관내 암세포 사멸능 및 세포독성 메커니즘 규명Example 5: In vitro cancer cell killing ability and cytotoxicity mechanism of NK101
상기 실시예 1에서 제조되어 표현형이 확인된 NK101 세포주의 암세포 사멸능을 확인하기 위하여 하기와 같은 실험을 수행하였다. 구체적으로, CFDA로 표지한 인간-유래 암세포주인 THP-1, KG-1, HL-60(급성골수성백혈병), HCT116(대장암), U373(뇌암), A2780(난소암), A549(폐 선암종), 및 SK-BR3(유방암) 세포를 각각 24-웰 플레이트에 3x105 cells/mL 농도로 1 mL 씩 파종하였다. 이후 NK101 세포 및 대조군을 다양한 효과기 세포 대 표적 세표 비율(E:T 비율=1:1, 2:1, 및 4:1)로 1 mL 배지에 부유한 후 상기 암세포와 24시간 동안 함께 배양하였다. 배양 이후 모든 세포를 수집한 뒤 각 웰로부터 세포를 회수하여 원심분리한 후, 세포 펠렛을 FACS 완충액으로 현탁시켰다. 다시 원심분리하여 형성된 세포 펠렛을 1 μL LIVE/DEAD® Fixable Near-IR Dead Stain Kit를 희석한 100 μL의 FACS 완충액에 현탁시켜, 4℃에서 20분간 반응하였다. FACS 완충액으로 2번 세척한 뒤, 1X Annexin V binding buffer 100 μL에 Annexin V APC 5 μL (Biolegend, USA)를 희석한 용액에 세포 펠렛을 현탁시켜, 실온에서 20분간 반응하였다. 세포의 사멸 여부는 유세포 분석법을 이용하여 생존 세포(annexin V-음성/LIVE/DEAD-음성), 초기 아폽토시스 세포(annexin V-양성/LIVE/DEAD-음성), 후기 아폽토시스 세포(annexin V-양성/LIVE/DEAD-양성), 괴사(necrotic) 세포(annexin V-음성/LIVE/DEAD-양성)으로 구분하였다. 도 6a에서 확인되는 바와 같이 NK101 세포 투여군의 경우 다양한 인간 암세포주에 대하여 세포 살상능을 보임을 확인할 수 있었다. NK101 세포의 세포 살상능의 주요 표지 인자를 확인하기 위하여 NK 세포에 고발현되는 CD25, CD62L, DNAM-1, CD54(ICAM-1)에 대한 중화항체를 처리한 후 THP-1(도 6b), K562(도 6c), Jurkat(도 6d)와 4:1의 효과기 세포 대 표적세포 비율로 공배양한 뒤 세포사멸을 분석하였다. 그 결과 THP-1에서는 DNAM-1 및 CD54, K562에서는 CD54, Jurkat에서는 CD25, CD62L, CD54 등의 중화항체 처리에 의해 NK101의 세포 사멸능이 감소함을 확인할 수 있었다. 또한 도 6e에서 THP-1과 NK101의 공배양 시 DNAM-1 및 CD54의 중화항체를 동시 처리할 경우 상승성으로(synergestic) 세포사멸능이 감소함을 확인할 수 있었다. 해당 결과를 토대로 NK101에 의한 세포살상능에 DNAM-1, CD25, CD62L 및 CD54 등의 표지인자가 주로 관여함을 알 수 있다.In order to confirm the cancer cell killing ability of the NK101 cell line prepared in Example 1 and confirmed phenotype, the following experiment was performed. Specifically, CFDA-labeled human-derived cancer cell lines THP-1, KG-1, HL-60 (acute myeloid leukemia), HCT116 (colon cancer), U373 (brain cancer), A2780 (ovarian cancer), A549 (lung adenocarcinoma) ), And SK-BR3 (breast cancer) cells were seeded in 1-well at 3x10 5 cells / mL concentration in 24-well plates, respectively. NK101 cells and controls were then suspended in 1 mL medium at various effector cell-to-target wash ratios (E: T ratio = 1: 1, 2: 1, and 4: 1) and then incubated with the cancer cells for 24 hours. After incubation all cells were collected and cells were harvested from each well, centrifuged and the cell pellet suspended in FACS buffer. By suspending the cell pellet formed by centrifuging again in 1 μL LIVE / DEAD ® Fixable FACS buffer, Near-IR Dead Stain 100 μL diluted Kit, was reacted at 4 ℃ 20 minutes. After washing twice with FACS buffer, the cell pellet was suspended in a solution diluted with 5 μL of Annexin V APC (Biolegend, USA) in 100 μL 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). As shown in FIG. 6A, 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. 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. In addition, in co-culture of THP-1 and NK101 in FIG. 6E, synergistically, apoptosis was reduced when the neutralizing antibodies of DNAM-1 and CD54 were simultaneously treated. Based on the results, it can be seen that markers such as DNAM-1, CD25, CD62L, and CD54 are mainly involved in apoptosis by NK101.
실시예 6: 기능강화 NK 세포주 제조Example 6: Preparation of enhanced NK cell line
KHYG-1 및 NK-92는 기존에 구축된 NK세포주 들 중, 암세포 사멸능이 높다고 알려져있고 이들은 공통적으로 CD7, CD28을 발현하는 특징을 갖는 반면, NK101은 두 보조자극인자의 발현이 전혀 되지 않음에 착안하여, 본 발명자들은 상기 NK101에 CD7, 및 CD28를 암호화하는 유전자를 형질도입할 경우 암세포 사멸능이 증진되는지의 여부를 평가하고자 하였다. 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. With this in mind, the present inventors attempted to evaluate whether cancer cell killing ability is enhanced when the NK101 is transduced with genes encoding CD7 and CD28.
6-1: NK 세포 활성화 인자 및 세포자살 유전자 도입 컨스트럭트의 제조6-1: Preparation of NK Cell Activator and Apoptosis Gene Construct
본 발명자들은 기능강화 NK 세포주를 제작하기 위하여 상기 실시예 1에서 제조된 NK101 세포에 CD7(서열번호 15)를 암호화하는 핵산분자(서열번호 16) 및 CD28 면역세포 보조자극인자(서열번호 17)를 암호화하는 핵산분자(서열번호 18)가 2A 펩타이드(19)를 암호화하는 핵산분자(서열번호 20)로 연결되고, 추가적으로 CD::UPRT(서열번호 21)을 암호화하는 핵산분자(서열번호 22)가 IRES(서열번호 23)로 연결된 유전자 컨스트럭트를 제조하여 렌티바이러스 제조용 트랜스퍼 벡터 pWPT에 클로닝하여 pWPT-CD7-CD28-CD::UPRT를 제조하였다(도 7b).The present inventors prepared a nucleic acid molecule (SEQ ID NO: 16) and a CD28 immune cell co-stimulatory factor (SEQ ID NO: 17) encoding CD7 (SEQ ID NO: 15) to the NK101 cells prepared in Example 1 to produce a functionally enhanced NK cell line. The nucleic acid molecule encoding (SEQ ID NO: 18) is linked to the nucleic acid molecule encoding the 2A peptide (SEQ ID NO: 20), and the nucleic acid molecule encoding the CD :: UPRT (SEQ ID NO: 21) is added. Gene constructs linked with IRES (SEQ ID NO: 23) were prepared and cloned into the transfer vector pWPT for lentiviral production to prepare pWPT-CD7-CD28-CD :: UPRT (FIG. 7B).
6-2: 렌티바이러스 제조6-2: Lentivirus Production
렌티바이러스 생산을 위하여 48시간 또는 72시간 배양한 Lenti-X 세포에 상기 실시예 6-1에서 제조된 트랜스퍼 플라스미드 12 μg와 패키징 플라스미드 psPAX2 12 μg 및 외피 플라스미드 pMD2.G 2.4 μg을 리포펙타민(Lipofectamine; Invitrogen, USA)과 혼합하여 Lenti-X 293T 세포(Clontech, USA)에 형질도입하였다. 37℃에서 6시간 배양 후, 배지를 제거하고 신선한 성장배지를 첨가하였다. 세포를 추가로 48시간 배양 후, 배지 내에 생선된 렌티바이러스를 수거하여 5분간 원심분리(4℃, 4000 rpm)하고 상층액을 취한 후 0.45 μm 필터(Millipore, USA)를 이용하여 세포 찌꺼기들을 제거하였다. 렌티바이러스를 농축하기 위하여 여과된 렌티바이러스 상층액에 Lenti-X concentrator (Clontech, USA) 시약을 넣은 후 4℃에서 밤새 보관하였다. 최종단계로, 렌티바이러스-concentrator 혼합액을 4000 rpm에서 60분간 원심분리한 후 상층액을 제거하고 펠렛 형태로 회수된 렌티바이러스를 1~2 mL의 배양액으로 희석하고 사용 시까지 -80℃에 보관하였다.In the Lenti-X cells cultured for 48 hours or 72 hours for lentiviral production, 12 μg of the transfer plasmid prepared in Example 6-1, 12 μg of the packaging plasmid psPAX2 and 2.4 μg of the envelope plasmid pMD2.G were lipofectamine. Invitrogen, USA) and transduced Lenti-X 293T cells (Clontech, USA). After 6 hours of incubation at 37 ° C, the medium was removed and fresh growth medium was added. After 48 hours of incubation, 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 μm 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. As a final step, 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. .
6-3: NK 세포 활성화 인자 및 세포자살 유전자 도입 세포주 제작6-3: Production of NK Cell Activating Factor and Apoptotic Gene Line
상기 실시예 6-2에서 제조된 CD7-CD28-CD::UPRT 렌티바이러스를 프로타민 설페이트(Sigma, USA)를 이용하여 NK101 세포에 형질감염한 후 37℃에서 4시간 배양한다. 총 2번의 감염과정을 수행한 후, 감염 72시간 후 면역세포 공동자극인자인 CD7과 CD28의 발현을 유세포 분석으로 확인하고, 1주일 뒤 형광활성 세포분리기(Fluorescence activated cell sorter, BD FACSMelody, BD, USA)를 이용하여 도입유전자를 발현하는 NK101 세포만을 선택적으로 분리하였다. 분리 증식한 NK101-CD7-CD28-CD::UPRT 세포주에서 막표면 단백질인 CD7과 CD28의 발현은 유세포 분석으로 확인하였다(도 7c). 본 발명자들은 CD7-CD28-CD::UPRT를 발현하는 유전자 변형 NK101 세포주를 'SL-K01'으로 명명하였다.The CD7-CD28-CD :: UPRT lentivirus prepared in Example 6-2 was transfected into NK101 cells using protamine sulfate (Sigma, USA) and then incubated at 37 ° C for 4 hours. After two times of infection, 72 hours after infection, the expression of CD7 and CD28, the immune cell co-stimulatory factors, was confirmed by flow cytometry, and 1 week later, the fluorescence activated cell sorter (BD FACSMelody, BD, USA) was used to selectively isolate only NK101 cells expressing the transgene. Expression of the membrane surface proteins CD7 and CD28 in the isolated and propagated NK101-CD7-CD28-CD :: UPRT cell lines was confirmed by flow cytometry (FIG. 7C). We named the genetically modified NK101 cell line expressing CD7-CD28-CD :: UPRT as 'SL-K01'.
6-4: 역전사 중합효소 연쇄반응(RT-PCR)6-4: Reverse Transcription Polymerase Chain Reaction (RT-PCR)
세포 내 발현 단백질인 CD::UPRT는 RNA를 분리하여 역전사 중합효소 연쇄반응(RT-PCR)을 이용하여 발현을 확인하였다(도 7d). 전체 RNA는 RNA 추출 키트(iNtRON, South Korea)을 이용하여 분리하였다. 역전사 중합효소 연쇄반응은 QuantiTect Reverse Transcription Kit(QIAGEN, Germany)을 이용하여 cDNA를 합성한 후 합성된 cDNA를 Taq 폴리머라제와 프라이머 등과 혼합한 후 중횹효소연쇄반응(PCR)을 수행하였다. 증폭된 PCR 산물은 1% 아가로스 겔에서 전기영동하여 CD::UPRT 유전자 존재여부를 확인하였다. 그 결과 도 7d에서 확인되는 바와 같이, 형질도입된 CD::UPRT 유전자는 정상적으로 발현이 됨을 확인할 수 있었다.CD :: UPRT, an intracellular expression protein, isolated RNA and confirmed its expression using reverse transcriptase polymerase chain reaction (RT-PCR) (FIG. 7D). 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.
실시예 7: NK 세포 공동활성 인자 및 세포자살 유전자에 의한 NK 세포 활성화 증가 확인Example 7: Confirmation of Increased NK Cell Activation by NK Cell Coactivator and Apoptosis Gene
7-1: CD7 및 CD28 도입에 의한 NK 세포 살상능 검증7-1: Verification of NK cell killing ability by introducing CD7 and CD28
상기 실시예 6에서 제조한 SL-K01 세포의 세포 살상능을 분석하기 위하여 HDLM-2(인간 호지킨 림프종, Hodgkin's lymphoma), IM9(인간 림프아세포종), Jeko-1(인간 외투세포 림프종) 및 K562(만성골수성백혈병)와 공배양을 수행하였다. Celltracker violet dye(CTV; Invitrogen, USA)로 표지한 타깃 종양 세포주를 3x105/mL의 농도로 준비한 후, 24-웰 플레이트에 1 mL씩 분주하였다. 타깃 종양 세포주에 대비하여 NK101 및 SL-K01 세포를 원하는 비율로 배양액에 현탁한 후 상기 타깃 종양 세포주가 들어있는 24-웰 플레이트에 1 mL씩 분주하여 37℃에서 24시간동안 공배양하였다. 이 결과, 모든 타깃 세포주에서 세포 살상능이 증가함을 확인할 수 있었다(도 8a).In order to analyze the cell killing ability of the SL-K01 cells prepared in Example 6, HDLM-2 (human Hodgkin's lymphoma), IM9 (human lymphoblastoma), Jeko-1 (human mantle cell lymphoma) and K562 (Chronic myeloid leukemia) and co-culture were performed. Target tumor cell lines labeled with Celltracker violet dye (CTV; Invitrogen, USA) were prepared at a concentration of 3 × 10 5 / mL, and then dispensed in 1 mL portions in 24-well plates. In contrast to the target tumor cell line, 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).
7-2: 세포자살 유전자 전구물질 투여에 따른 NK 세포 사멸 확인7-2: Confirmation of NK cell death following apoptotic gene precursor administration
상기 실시예 6에서 제조한 SL-K01 세포에 도입된 세포자살 유전자인 CD::UPRT의 효과를 분석하기 위하여, 20% FBS 조성의 SCGM 배지에 세포수가 2x104 cells/90 μL/well이 되도록 준비하여 96-웰 플레이트(Corning, USA)에 각각 분주하고, 농도별(각각 0, 0.1, 1, 10, 100, 1000 μg/mL)로 희석한 전구물질 5-Fluorocytosine(5-FC; Sigma, USA) 또는 양성대조군(1% Triton-X)을 웰에 10 μL씩 접종하여, 5% CO2, 37℃ 조건에서 48시간 동안 배양하였다. 이어서 MTS 용액(Promega, USA)을 96-웰 플레이트에 웰당 20 μL씩 넣어주고 5% CO2, 37℃ 배양기에서 4시간 동안 반응시킨 다음, SpectraMax 190 Microplate Reader(Molecular Device, USA)를 이용하여 490 nm에서 흡광도를 측정하였다. 그 결과 CD::UPRT를 발현하는 세포에서 5-FC 농도 의존적 세포 사멸을 확인할 수 있었다(도 8b). CTV로 표지한 IM9 세포에 SL-K01 세포와 100 μg/mL 5-FC를 함께 처리하여 48시간 배양 후 유세포 분석법으로 세포 사멸 효과를 확인하였다. 도 8c에 나타난 결과에서 확인된 바와 같이, 5-FC를 처리한 실험군에서 세포사멸이 더욱 증가하였다. 결론적으로, 전구물질을 투여하였을 때, 세포자살 유전자가 도입된 NK 세포뿐 아니라, 표적 암세포까지 사멸하는 방관자 효과(bystander effect)에 의해 치료 효과를 극대화시킬 수 있다.In order to analyze the effect of CD :: UPRT, which is an apoptosis gene introduced into SL-K01 cells prepared in Example 6, the cells were prepared in 20% FBS composition in SCGM medium with 2x10 4 cells / 90 μL / well Aliquots in 96-well plates (Corning, USA) and diluted to concentrations (0, 0.1, 1, 10, 100, 1000 μg / mL, respectively), 5-Fluorocytosine (5-FC; Sigma, USA). ) Or positive control group (1% Triton-X) was inoculated in 10 μL of the wells, and incubated for 48 hours at 5% CO 2 , 37 ° C. Subsequently, MTS solution (Promega, USA) was placed in a 96-well plate at 20 μL per well and reacted for 5 hours in a 5% CO 2 , 37 ° C. incubator, followed by 490 using a SpectraMax 190 Microplate Reader (Molecular Device, USA). Absorbance was measured at nm. As a result, it was confirmed that 5-FC concentration-dependent cell death in cells expressing CD :: UPRT (FIG. 8B). C9-labeled IM9 cells were treated with SL-K01 cells and 100 μg / mL 5-FC together for 48 hours, and then cell death was confirmed by flow cytometry. As confirmed by the results shown in FIG. 8C, apoptosis was further increased in the experimental group treated with 5-FC. In conclusion, when the precursor is administered, 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.
실시예 8: mbIL-15과 TGFβRIIΔcyto 도입Example 8: Introduction of mbIL-15 and TGFβRIIΔcyto
본 발명자들은 상기 실시예 6에서 제조된 SL-K01 세포의 세포 살상능을 더욱 증진시키기 위하여 막 결합 IL-15(mbIL-15, 서열번호 24)를 암호화하는 핵산분자(서열번호 25)와 TGFβRII의 세포질 도메인을 제거한 TGFβRIIΔcyto(서열번호 26)를 암호화하는 핵산분자(서열번호 27)가 2A 펩타이드(서열번호 19)를 암호화하는 핵산분자(서열번호 20)로 연결된 유전자 컨스트럭트를 제조하여 렌티바이러스 제조용 트랜스퍼 벡터 pWPT에 클로닝하여 pWPT-mbIL-15-TGFβRIIΔcyto를 제조하였다(도 9a). 실시예 6에서 명시한 것과 동일한 방법으로 mbIL-15-TGFβRII 렌티바이러스를 제조한 후 SL-K01 세포에 감염하였으며, 형광활성 세포분리기를 이용하여 도입유전자를 발현하는 SL-K01 세포만을 선택적으로 분리하였다. 분리증식한 SL-K01-mbIL-15-TGFβRII 세포주에서 막표면 단백질인 IL-15과 TGFβRII의 발현은 유세포 분석법으로 확인하였다(도 9b). mbIL-15-TGFβRII를 발현하는 SL-K01 세포주를 'NK111'으로 명명하였다.The present inventors of the nucleic acid molecule (SEQ ID NO: 25) and TGFβRII encoding the membrane-bound IL-15 (mbIL-15, SEQ ID NO: 24) to further enhance the cell killing capacity of the SL-K01 cells prepared in Example 6 Nucleic acid molecule (SEQ ID NO: 27) encoding TGFβRIIΔcyto (SEQ ID NO: 26) from which the cytoplasmic domain was removed was prepared by constructing a gene construct linked to a nucleic acid molecule (SEQ ID NO: 20) encoding a 2A peptide (SEQ ID NO: 19) for the production of lentiviral. PWPT-mbIL-15-TGFβRIIΔcyto was prepared by cloning into the transfer vector pWPT (FIG. 9A). MbIL-15-TGFβRII 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 TGFβRII in the isolated SL-K01-mbIL-15-TGFβRII cell line was confirmed by flow cytometry (FIG. 9B). The SL-K01 cell line expressing mbIL-15-TGFβRII was named 'NK111'.
실시예 9: mbIL-15 도입에 의한 세포 분열능 확인 및 TGFβRIIΔcyto 도입에 따른 내인성 TGFβ에 의한 면역저항성 억제 확인Example 9 Confirmation of Cell Division Capability by Introducing mbIL-15 and Confirmation of Inhibition of Immune Resistance by Endogenous TGFβ by Introduction of TGFβRIIΔcyto
9-1: IL-2 의존성 증식 분석9-1: IL-2 Dependent Proliferation Assay
상기 실시예 8에서 제조된 NK111 세포의 IL-2 비의존적 NK 세포 분열능을 확인하기 위하여, SL-K01 세포 및 NK111 세포를 2x105 cells/mL 농도로 250 U/mL IL-2 유무 조건에서 48시간 주기로 배양한 후 세포를 회수하여 트립판 블루 염색법(Trypan Blue)으로 생존 세포 수를 측정하였다. 도 10a에서 확인할 수 있듯이, SL-K01 세포는 IL-2 결핍조건에서는 세포증식이 이루어지지 않으나, IL-2 처리 조건에서는 세포 성장을 확인할 수 있었다. mbIL-15을 도입한 NK111 세포는 IL-2 처리 조건에서 SL-K01과 유사하게 증식함을 관찰하였다. IL-2 결핍조건에서도 일정한 PDL을 유지하며 증식하는 것을 관찰하였다(도 10a). 해당 결과는 NK111 기반 세포주 치료제의 경우 배양보조제인 IL-2의 결핍 조건만으로도 높은 생산성을 가질 수 있음을 시사한다. 아울러, NK101, SL-K01, NK111 세포주에서 자연 살상 세포 활성화 수용체 중 하나인 CD314(NKG2D)의 발현이 유세포 분석에 의해 확인되었다(도 10b). In order to confirm the IL-2 independent NK cell division ability of the NK111 cells prepared in Example 8, the SL-K01 cells and NK111 cells were 48 × under 250 U / mL IL-2 at a concentration of 2 × 10 5 cells / mL. After culturing in a time cycle, the cells were collected and the number of viable cells was measured by trypan blue staining (Trypan Blue). As can be seen in Figure 10a, SL-K01 cells were not cell proliferation under IL-2 deficiency conditions, cell growth was confirmed in the IL-2 treatment conditions. It was observed that NK111 cells introduced with mbIL-15 proliferated similarly to SL-K01 under IL-2 treatment conditions. Proliferation was observed while maintaining a constant PDL even in an IL-2 deficient condition (FIG. 10A). The results suggest that NK111-based cell line therapeutics may have high productivity with only a deficiency condition of IL-2, a culture supplement. In addition, 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).
9-2: 암세포 사멸능 분석9-2: Cancer Cell Killing Capacity Analysis
mbIL-15 도입에 따른 NK111 세포의 세포 사멸능 확인을 위하여 IM9(인간 림프아세포)와 공배양을 수행하였다. CTV로 표지한 IM9 세포에 SL-K01 세포 및 NK111 세포와 함께 5-FC를 처리하여 48시간 배양 후 유세포 분석법으로 세포 사멸 효과를 확인하였다. mbIL-15이 도입된 NK111 세포는 표적세포에 대한 암세포 사멸능이 증가하였으며, 5-FC를 처리한 실험군에서는 세포사멸이 현저히 증가하였다(도 10c).Coculture with IM9 (human lymphoblasts) was performed to confirm cell killing ability of NK111 cells following the introduction of mbIL-15. 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).
9-3: TGFβ1에 의한 세포사멸 저해 영향 분석9-3: Analysis of effect of inhibition of apoptosis by TGFβ1
아울러, 본 발명자들은 환자의 체내에 다량 존재하는 면역억제 인자로 잘 알려진 TGFβ1 면역억제 효과가 NK111 세포에 도입된 TGFβRIIΔcyto에 의하여 저해되는지의 여부를 확인하기 위하여 하기와 같은 실험을 수행하였다. CTV로 표지한 OVCAR-3(인간 난소암), THP-1(급성골수성백혈병) 세포와 효과기 세포인 SL-K01 및 NK111 세포를 3x105 cells/mL의 농도로 준비하여 24-웰 플레이트에 1 mL씩 분주한 뒤(E:T=1:1), 각각 0, 0.3, 1, 3, 및 10 μg/mL의 TGFβ1를 처리하여, 5% CO2, 37℃ 조건에서 24시간 동안 배양하였다. 그 결과, 도 10d에서 확인할 수 있듯이, SL-K01 세포는 TGFβ1의 농도가 높아짐에 따라 암세포 사멸능이 감소하였으나, NK111 세포의 경우 TGFβ1에 의한 활성 억제 효과가 없거나 미미한 수준으로 분석되었다.In addition, the present inventors performed the following experiments to determine whether the TGFβ1 immunosuppressive effect, which is well known as an immunosuppressive factor present in a large amount in the patient's body, is inhibited by TGFβRIIΔcyto introduced into NK111 cells. CTV-labeled OVCAR-3 (human ovarian cancer) cells and THP-1 (acute myeloid leukemia) cells and effector cells SL-K01 and NK111 cells were prepared at a concentration of 3x10 5 cells / mL and 1 mL in a 24-well plate. After each aliquot (E: T = 1: 1), 0, 0.3, 1, 3, and 10 μg / mL of TGFβ1 were treated and incubated at 5% CO 2 , 37 ° C. for 24 hours. As a result, as can be seen in Figure 10d, SL-K01 cells decreased the cancer cell killing ability as the concentration of TGFβ1 increased, but the NK111 cells were analyzed to have no or insignificant activity inhibitory effect by TGFβ1.
상술한 바와 같이, 본 발명의 일 실시예에 따른 SL-K01 세포는 모세포주인 NK101의 주된 특징은 그대로 유지하면서도 모세포주인 NK101과 비교하여 암세포사멸능이 현저히 증가하였고, 상기 SL-K01의 성능을 더 강화시킨 NK111 세포는 암세포 살상능이 SL-K01 세포에 비해 현저하게 증가하였을 뿐만 아니라, TGFβ로 인한 세포살상능의 저해를 회피할 수 있어서 체내 투여시 매우 효율적인 암 치료제로 사용이 가능할 것으로 기대가 된다.As described above, SL-K01 cells according to an embodiment of the present invention 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 In addition, the NK111 cells have increased cancer cell killing ability significantly compared to SL-K01 cells, and can avoid the inhibition of TGFβ-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.
실시예 10: anti-EpCAM scFV-CAR 유전자 컨스트럭트의 제조Example 10 Preparation of anti-EpCAM scFV-CAR Gene Construct
본 발명자들은 키메라 항원 수용체(이하, 'CAR'라 약칭함)를 발현하는 NK111 세포주를 제조하기에 앞서, 항-EpCAM 단일클론항체의 가변영역과 NK 세포 활성화 인자를 연결시킨 키메라 항원 수용체 발현 렌티바이러스 벡터를 제조하였다. Prior to preparing an NK111 cell line expressing a chimeric antigen receptor (hereinafter, abbreviated as 'CAR'), the present inventors have directed a chimeric antigen receptor expressing lentiviral that connects a variable region of an anti-EpCAM monoclonal antibody to an NK cell activating factor. Vectors were prepared.
구체적으로 항-EpCAM 단일클론항체의 중쇄 가변영역 및 경쇄가변영역을 링커로 연결시킨 단일쇄 가변단편(scFv, 서열번호 3)을 암호화하는 폴리뉴클레오타이드(서열번호 4)를 클로닝한 후, 상기 폴리뉴클레오타이드와 인간 IgD의 변형 힌지(hinge), IgG4의 CH2 및 CH3 부위로 구성되는 변형 FC 단백질(서열번호 5)을 암호화하는 폴리뉴클레오타이드(서열번호 6), 인간 CD28 막통과도메인(서열번호 7)를 암호화하는 폴리뉴클레오타이드(서열번호 8), DAP10 단백질(서열번호 9)를 암호화하는 폴리뉴클레오타이드(서열번호 10), DAP12 단백질(서열번호 11)을 암호화하는 폴리뉴클레오타이드(서열번호 12) 및 인간 CD3ζ 세포질 도메인(서열번호 13)을 암호화하는 폴리뉴클레오타이드(서열번호 14)가 순차적으로 연결된 유전자 컨스트럭트를 상기 항-EpCAM 단일쇄 가변단편(scFv)를 암호화하는 폴리뉴클레오타이드에 연결하였으며, 이를 'anti-EpCAM scFv-CAR 유전자 컨스트럭트(서열번호 2)'로 명명하였다(도 11). 이어, 상기 제작된 anti-EpCAM scFv-CAR 유전자 컨스트럭트를 렌티바이러스 제조용 트랜스퍼 벡터 pWPT에 삽입시켜 pWPT/anti-EpCAM scFv-CAR 벡터를 제조하였다. Specifically, after cloning a polynucleotide (SEQ ID NO: 4) encoding a single chain variable fragment (scFv, SEQ ID NO: 3) linking the heavy chain variable region and the light chain variable region of the anti-EpCAM monoclonal antibody with a linker, the polynucleotide And a modified hinge of human IgD, a polynucleotide encoding the modified FC protein (SEQ ID NO: 5) consisting of the CH2 and CH3 sites of IgG4 (SEQ ID NO: 6), a human CD28 transmembrane domain (SEQ ID NO: 7) A polynucleotide (SEQ ID NO: 8), a polynucleotide encoding the DAP10 protein (SEQ ID NO: 9), a polynucleotide encoding the DAP12 protein (SEQ ID NO: 11), and a human CD3ζ cytoplasmic domain (SEQ ID NO: 12). 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.
실시예 11: anti-EpCAM-CAR 렌티바이러스의 제조Example 11: Preparation of anti-EpCAM-CAR Lentivirus
렌티바이러스 생산을 위하여, 상기 실시예 10에서 제조된 12 ㎍의 pWPT/anti-EpCAM scFv-CAR와 패키징 플라스미드 psPAX2 12 μg 및 외피 플라스미드 pMD2.G 2.4 μg을 리포펙타민(Lipofectamine; Invitrogen, USA)과 혼합하여 Lenti-X 293T 세포(Clontech, USA)에 형질도입하였다. 37℃에서 6시간 배양 후, 배지를 제거하고 신선한 성장배지를 첨가하였다. 세포를 추가로 48시간 배양 후, 배지 내에 생선된 렌티바이러스를 수거하여 5분간 원심분리(4℃, 4000 rpm)하고 상층액을 취한 후 0.45 μm 필터(Millipore, USA)를 이용하여 세포 찌꺼기들을 제거하였다. 렌티바이러스를 농축하기 위하여 여과된 렌티바이러스 상층액에 Lenti-X concentrator(Clontech, USA) 시약을 넣은 후 4℃에서 밤새 보관하였다. 최종단계로, 렌티바이러스-concentrator 혼합액을 4000 rpm에서 60분간 원심분리한 후 상층액을 제거하고 펠렛 형태로 회수된 렌티바이러스를 1~2 mL의 배양액으로 희석하고 사용 시까지 -80℃에 보관하였다.For lentiviral production, 12 μg of pWPT / anti-EpCAM scFv-CAR prepared in Example 10 and 12 μg of packaging plasmid psPAX2 and 2.4 μg of envelope plasmid pMD2.G were combined with Lipofectamine (Invitrogen, USA). Mixed and transduced Lenti-X 293T cells (Clontech, USA). After 6 hours of incubation at 37 ° C, the medium was removed and fresh growth medium was added. After 48 hours of incubation, 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 μm 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. As a final step, 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. .
실시예 12: anti-EpCAM scFV-CAR 발현 NK111 세포주의 제조Example 12 Preparation of anti-EpCAM scFV-CAR Expressing NK111 Cell Line
상기 EpCAM 표적 키메라 항원 수용체를 발현하는 NK111 세포주를 제작하기 위하여, 상기 실시예 11에서 제조된 anti-EpCAM scFv-CAR 렌티바이러스 및 프로타민 설페이트(protamine sulfate)를 이용하여 37℃에서 4시간동안 반응시켜 상기 실시예 8에서 제조된 NK111 세포에 감염시켰다. 총 2번의 감염과정을 수행한 후, 감염 72시간 후 키메라 항원 수용체의 발현을 유세포 분석법으로 확인하고, 1주일 뒤 형광활성세포분류기(fluorescence activated cell sorter)를 이용하여 항-EpCAM 키메라 항원 수용체를 발현하는 NK111 세포만을 선택적으로 분리하였다. 분리 후 증식한 세포주에서 키메라 항원 수용체의 발현을 유세포 분석법을 통해 세포 표면에서의 키메라 항원 수용체 발현을 측정하였다(도 12a).In order to prepare the NK111 cell line expressing the EpCAM target chimeric antigen receptor, the anti-EpCAM scFv-CAR lentivirus prepared in Example 11 and protamine sulfate (protamine sulfate) were reacted for 4 hours at 37 ° C. Infected with NK111 cells prepared in Example 8. After performing a total of two infections, 72 hours after infection, the expression of the chimeric antigen receptor was confirmed by flow cytometry, and one week later, the anti-EpCAM chimeric antigen receptor was expressed using a fluorescence activated cell sorter. Only NK111 cells were selectively isolated. Expression of the chimeric antigen receptor in the cell line proliferated after isolation was measured by flow cytometry to measure the expression of the chimeric antigen receptor on the cell surface (FIG. 12A).
그 결과 도 12a에 나타난 바와 같이, 본 발명의 실시예에 따라 제조된 anti-EpCAM scFv-CAR가 NK111 세포 표면에서 과발현되고 있음을 확인할 수 있다.상기 과정에서 제조된 본 발명의 항-EpCAM 키메라 항원 수용체의 세포 내 발현을 검증하기 위하여 웨스턴 블랏 분석을 수행하였다. anti-EpCAM scFv-CAR 발현 NK 세포주의 용해물로부터 정량한 단백질 50 ㎍을 환원조건 또는 비환원조건으로 8% 폴리아크릴아마이드 겔 전기영동을 통해 분리하였다. 전기영동한 겔을 미리 적셔둔 다공성 패드(porous pad)와 3MM 페이퍼(Whatman laboratory Products, Maidstone, UK) 위에 올려놓고 그 위에 PVDF 막(PVDF Western Blottng Membrane, Roche)과 3MM 페이퍼(Whatman), 다공성 패드를 공기 방울이 생기지 않게 잘 덮고 전이 카세트(transfer cassette)로 압착하였다. 이것을 전극 전이 키트(electrode transfer kit)에 넣고 전이완충액(2.5mM Tris, 6.9mM 글리신, 20% 메탄올)에 완전히 잠기게 한 후 전기영동하였다. 단백질이 완전하게 전기영동된 막을 차단용액(blocking solution)[TBS-T(1% Tween 20) 내 5% 탈지분유]에 넣고 1시간 동안 진탕 배양하였다. TBS-T(0.1% Tween 20)로 차단용액을 30분 동안 세척한 후, 일차 항체인 항 CD3ξ 단일클론항체를 TBS-T(0.1% Tween 20)에 1/1,000로 희석하여 넣고 4℃에서 16시간 동안 진탕 배양하였다. TBS-T(0.1% Tween 20)로 막을 10분 동안 3회에 걸쳐 헹구어준 후, 이차 항체(Anti-mouse Ig(H+L), horseradish peroxidase linked conjugate; cat# 170-6516, Biorad, USA)를 1/3,000으로 희석하여 넣고 실온에서 2시간 동안 진탕 배양하였다. TBS-T(0.1% Tween 20)로 막을 20분 동안 3회 헹구어 준 후, 막에 웨스턴 검출용액(western detection solution)을 도포한 후 암실에서 필름에 감광시켜 현상하였다(도 12b). As a result, as shown in Figure 12a, it can be seen that 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 μg 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. Place electrophoretic gel on pre-soaked porous pads and 3MM paper (Whatman laboratory Products, Maidstone, UK) and on top of them PVDF membrane (PVDF Western Blottng Membrane, Roche) and 3MM paper (Whatman), porous pads Was covered well with no air bubbles and pressed into a transfer cassette. This was placed in an electrode transfer kit (electrode transfer kit) and completely immersed in a transition buffer (2.5mM Tris, 6.9mM glycine, 20% methanol) and electrophoresed. Membrane with complete protein electrophoresis was placed in a blocking solution (5% skim milk powder in TBS-T (1% Tween 20)) and shaken for 1 hour. After washing the blocking solution with TBS-T (0.1% Tween 20) for 30 minutes, the primary antibody, anti-CD3ξ monoclonal antibody, was diluted to 1 / 1,000 in TBS-T (0.1% Tween 20) and 16 at 4 ° C. Shake incubation for hours. After rinsing the membrane with TBS-T (0.1% Tween 20) three times for 10 minutes, the secondary antibody (Anti-mouse Ig (H + L), horseradish peroxidase linked conjugate; cat # 170-6516, Biorad, USA) Was diluted to 1 / 3,000 and shaken for 2 hours at room temperature. After rinsing the membrane with TBS-T (0.1% Tween 20) three times for 20 minutes, a Western detection solution was applied to the membrane, and the photosensitive film was developed in a dark room (FIG. 12B).
결과 도 12b에 나타난 바와 같이, 본 발명의 실시예에 따라 제조된 anti-EpCAM scFv-CAR가 NK111 세포주에서 발현 되고 있음을 확인할 수 있다. 이에, 본 발명자들은 상기 anti-EpCAM scFv-CAR를 발현하는 유전자 변형 NK 세포주를 "SL-K10"으로 명명하였다. As shown in Figure 12b, it can be seen that the anti-EpCAM scFv-CAR prepared according to the embodiment of the present invention is expressed in the NK111 cell line. Thus, the present inventors named the genetically modified NK cell line expressing the anti-EpCAM scFv-CAR as "SL-K10".
실시예 13: SL-K10 세포의 다양한 특성 분석Example 13: Various Characterization of SL-K10 Cells
13-1: SL-K10의 증식능 분석13-1: Analysis of proliferative capacity of SL-K10
상기 실시예 12에서 제조된 SL-K10 세포의 IL-2 비의존적인 NK 세포 분열능을 확인하기 위하여, SL-K10 세포를 IL-2가 결핍된 NK media에서 48~72 시간 주기로 배양한 후 세포를 회수하여 트립판 블루 염색법(Trypan Blue)으로 생존 세포 수를 측정하였다. 도 13에서 확인할 수 있듯이, SL-K10 세포는 IL-2 결핍조건에서도 일정한 군집배가율(PDL)을 유지하며 증식하는 것을 관찰하였다. In order to confirm IL-2 independent NK cell division ability of the SL-K10 cells prepared in Example 12, SL-K10 cells were cultured in IL-2 deficient NK media for 48 to 72 hours and then The number of viable cells was collected and collected by trypan blue staining. As can be seen in Figure 13, SL-K10 cells were observed to proliferate while maintaining a constant cluster doubling rate (PDL) even under IL-2 deficient conditions.
13-2: 장기계대 안정성 시험13-2: Long-term Passage Stability Test
본 발명자들은 상기에서 구축된 SL-K10 연구용 세포은행(RCB)으로부터 임상시험용 세포은행(MCB) 및 최종 제품생산용 세포은행(WBC) 생산 시점까지 고려하여 장기계대배양시 안정성 분석을 수행하였다.The inventors performed the stability analysis during long-term passages in consideration of the above-described production time of the SLB-K10 research cell bank (RCB) and the clinical test cell bank (MCB) and the final product cell bank (WBC) production.
이를 위해 SL-K10 세포주를 NK media에서 50일 가량 장기 계대 배양하였다. 계대는 초기 해동 직후 2일을 제외하고는 3일 주기로 하였고, 15계대 뒤, 유세포 분석을 이용하여 도입유전자 및 NK 특성과 관련된 표지인자에 대한 유세포 분석을 수행하였다.To this end, 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.
그 결과, 도 13b에서 확인되는 바와 같이, 도입 유전자인 CD7, CD28, TGFβRII, mbIL-15, 및 EpCAM-CAR가 SL-K10 세포 표면에서 발현되며, 도입유전자의 발현이 장기계대 시에도 유지됨을 확인할 수 있었다.As a result, as shown in FIG. 13B, the transgenes CD7, CD28, TGFβRII, mbIL-15, and EpCAM-CAR were expressed on the surface of the SL-K10 cell, and it was confirmed that expression of the transgene was maintained even during long passage. Could.
뿐만 아니라, 도 13c에서 확인되는 바와 같이, 모든 NK 활성 관련 표지자 모두 장기 계대 시에도 안정적으로 발현됨을 확인할 수 있었다. 따라서, 본 발명의 일 실시예에 따른 SL-K10 세포주는 세포치료제의 생산에 매우 적합한 세포주임을 알 수 있다.In addition, as confirmed in Figure 13c, it was confirmed that all the NK activity-related markers are stably expressed even at long-term passage. Therefore, the SL-K10 cell line according to an embodiment of the present invention can be seen that the cell line is very suitable for the production of cell therapy.
실시예 14: SL-K10 세포의 항암 활성 분석Example 14 Analysis of Anticancer Activity of SL-K10 Cells
14-1: 시험관내 항암활성 분석14-1: In Vitro Anticancer Activity Analysis
본 발명자들은 본 발명의 일 실시예에 따른 SL-K10 세포주가 암세포 사멸에 있어서 효과가 있는 지를 in vitro 상에서 확인하기 위하여, 암세포와 SL-K10 세포를 함께 배양하여 세포사멸이 일어난 세포와 죽은 세포를 분석하였다. 암세포로는 EpCAM이 고발현되는 RMG-1 및 EpCAM이 발현되지 않는 KOC-2S 난소암 세포를 선정하였다. RMG-1 및 KOC-2S 세포의 EpCAM 발현 양상은 유세포 분석을 이용하여 검증하였다(도 14a).In order to confirm in vitro whether the SL-K10 cell line according to an embodiment of the present invention is effective in killing cancer cells, the present inventors have cultured cancer cells and SL-K10 cells together to detect dead cells and dead cells. Analyzed. As cancer cells, RMG-1 expressing high EpCAM and KOC-2S ovarian cancer cells not expressing EpCAM were selected. EpCAM expression patterns of RMG-1 and KOC-2S cells were verified using flow cytometry (FIG. 14A).
구체적으로, 암세포 특이적인 세포사멸을 확인하기 위하여 CFDA (caroxyfluorescein diacetate)를 표시한 암세포 주를 24 well plate에 300,000 cells/ ml 농도로 파종하였다. 이후 SL-K10 세포 및 대조군 NK101 및 NK111 세포를 다양한 효과기 세포 대 표적 세표 비율(E:T 비율 : 1:1, 2:1, 4:1)로 1 ml 배지에 부유한 후 상기 암세포와 24시간 동안 함께 배양하였다. 배양 이후 모든 세포를 모은 뒤 각 웰로부터 세포를 회수하여 원심분리한 후, 세포 펠렛을 FACS 완충액으로 현탁시켰다. 다시 원심분리하여 형성된 세포 펠렛을 1 ㎕ LIVE/DEAD® Fixable Near-IR Dead Stain Kit(Life Technologies)를 희석한 100 ㎕의 FACS 완충액에 현탁시켜, 4℃에서 20분간 반응하였다. FACS 완충액으로 2번 세척한 뒤, 1X Annexin V binding buffer 100 ㎕에 Annexin V APC 5 ㎕(Biolegend, USA)를 희석한 용액에 세포 펠렛을 현탁시켜, 실온에서 20분간 반응하였다. 세포의 사멸 여부는 유세포 분석법을 이용하여 생존 세포(annexin V-음성/LIVE/DEAD-음성), 초기 아폽토시스 세포(annexin V-양성/LIVE/DEAD-음성), 후기 아폽토시스 세포(annexin V-양성/LIVE/DEAD-양성), 괴사(necrotic) 세포(annexin V-음성/LIVE/DEAD-양성)으로 구분하였다(도 14b). Specifically, in order to confirm cancer cell-specific apoptosis, cancer cell lines expressing CFDA (caroxyfluorescein diacetate) were seeded at 300,000 cells / ml in 24 well plates. The SL-K10 cells and the control NK101 and NK111 cells were then suspended in 1 ml medium at various effector cell-to-target cell ratios (E: T ratios: 1: 1, 2: 1, 4: 1) and after 24 hours with the cancer cells. Were incubated together. After incubation all cells were collected and cells were harvested from each well, centrifuged and the cell pellet suspended in FACS buffer. By suspending the cell pellet formed by centrifuging again in FACS buffer of 100 ㎕ diluted 1 ㎕ LIVE / DEAD ® Fixable Near -IR Dead Stain Kit (Life Technologies), was reacted at 4 ℃ 20 minutes. After washing twice with FACS buffer, the cell pellet was suspended in a solution diluted with 5 μl Annexin V APC (Biolegend, USA) in 100 μl 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).
그 결과, 도 14b에서 확인되는 바와 같이 본 발명의 일 실시예에 따른 SL-K10 세포주는 EpCAM 과발현 세포인 RMG-1에 대해서 대조군 NK101 및 NK111에 비해 현저히 높은 세포 살상능을 보이는 반면, EpCAM 미발현 세포인 KOC-2S 세포에 대해서는 NK101 및 NK111와 동등한 세포 살상능을 보임을 확인하였다. 뿐만 아니라, 상기 실험에서 수득한 공배양 상등액을 이용하여 ELISA 법으로 NK 세포의 세포독성 및 면역활성화에 관여하는 그랜자임 B 및 IFN-γ 분비량을 측정한 결과, 도 14c에서 확인되는 바와 같이, 본 발명의 일 실시예에 따른 SL-K10 세포주는 EpCAM 과발현 세포인 RMG-1에 대해서 대조군인 NK111 세포에 비해 현저히 높은 그랜자임 B 및 IFN-γ 분비량을 보이는 반면, EpCAM 미발현 세포인 KOC-2S 세포에 대해서는 NK111 세포와 유사한 그랜자임 B 및 IFN-γ 분비량을 보임을 확인하였다. As a result, as shown in Figure 14b SL-K10 cell line according to an embodiment of the present invention 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. In addition, by using the co-culture supernatant obtained in the above experiments, the amount of granzyme B and IFN-γ secretion involved in cytotoxicity and immune activation of NK cells was measured by ELISA, as shown in FIG. 14C. SL-K10 cell line according to an embodiment of the invention shows significantly higher levels of Granzyme B and IFN-γ 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-γ secretion similar to NK111 cells.
이는 SL-K10 세포의 암세포 EpCAM 발현 특이적 살상 효과 및 사이토카인 분비능을 증명하는 결과이다. This demonstrates the cancer cell EpCAM expression specific killing effects and cytokine secretion of SL-K10 cells.
14-2: 방사선 조사에 따른 세포 특성 변화 여부 분석14-2: Analysis of Changes in Cell Characteristics Following Irradiation
본 발명의 일 실시예에 따른 SL-K10 세포주는 NK 림프종 유래 세포주인 NK101에 렌티바이러스를 이용한 유전자 도입을 수행한 세포이므로, 치료제로 이용되기 위해서는 방사선 조사를 통해 세포 성장을 억제하여 종양원성을 제거함과 동시에 동등한 정도의 항암 활성을 유지하여야 한다. 이를 검증하기 위하여, 본 발명자들은 SL-K10 세포주에 5 Gy 또는 10 Gy의 감마 방사선을 조사한 뒤, 세포의 성장 억제, 도입유전자 및 NK 특성과 관련된 표지인자의 발현양상 및 암세포 살상능을 평가하였다. SL-K10 cell line according to an embodiment of the present invention is a cell subjected to gene introduction using a lentivirus into NK lymphoma-derived cell line NK101, in order to be used as a therapeutic agent by inhibiting cell growth through irradiation to remove tumorigenicity At the same time, anticancer activity should be maintained at an equal level. In order to verify this, the inventors investigated the SL-K10 cell line with 5 Gy or 10 Gy gamma radiation, and then evaluated the expression patterns and cancer cell killing ability related to growth inhibition, transgene and NK characteristics of the cells.
그 결과, 도 14d에서 확인되는 바와 같이, 방사선을 조사하지 않은 SL-K10 세포는 지속적으로 증식하는 반면, 5 Gy 또는 10 Gy 방사선을 조사한 SL-K10 세포는 세포성장이 저해되어 약 96 시간 이후 대부분의 NK 세포가 사멸됨을 확인하였다. 단, 10 Gy의 방사선이 조사되었을 때, 세포 생존률이 7일째 0%에 도달함을 확인하였다. As a result, as shown in FIG. 14D, SL-K10 cells which did not irradiate continuously proliferated, while SL-K10 cells which were irradiated with 5 Gy or 10 Gy radiation inhibited cell growth, mostly after about 96 hours. It was confirmed that NK cells were killed. However, when 10 Gy of radiation was irradiated, it was confirmed that the cell survival rate reached 0% on day 7.
또한, 도 14e에서 확인되는 바와 같이, 도입유전자인 EpCAM-CAR 및 NK 활성 관련 표지자인 CD56, NKG2D, NKp30, 및 2B4 모두 10 Gy 방사선 조사 후에도 안정적으로 유지됨을 확인할 수 있었다.In addition, as confirmed in FIG. 14e, it was confirmed that transgenes EpCAM-CAR and NK activity related markers CD56, NKG2D, NKp30, and 2B4 were all stably maintained after 10 Gy irradiation.
뿐만 아니라, 도 14f에서 확인되는 바와 같이, 10 Gy의 방사선을 조사한 SL-K10 세포주는 EpCAM 과발현 세포인 RMG-1에 대해서 대조군 NK101 및 NK111에 비해 현저히 높은 세포 살상능을 보이는 반면, EpCAM 미발현 세포인 KOC-2S 세포에 대해서는 NK101 및 NK111와 동등한 세포 살상능을 보여, 방사선 조사 후에도 방사전 조사 전과 동등한 살상능을 가짐을 확인할 수 있었다. In addition, as confirmed in FIG. 14F, the 10-Gy-irradiated SL-K10 cell line showed significantly higher cell killing ability than the control NK101 and NK111 cells for RMG-1, an EpCAM overexpressing cell, whereas the EpCAM unexpressed cells About KOC-2S cells, the cell killing ability was the same as that of NK101 and NK111, and it was confirmed that the cell killing ability was the same as before the radiation before irradiation.
따라서, 본 발명의 일 실시예에 따른 SL-K10 세포주는 방사선 조사를 통해 체내 증식에 대한 우려 없이, 체내의 암세포를 제거하는 용도로 사용될 수 있음을 알 수 있다.Therefore, it can be seen that the SL-K10 cell line according to an embodiment of the present invention can be used for removing cancer cells in the body without concern for proliferation in the body through irradiation.
14-3: 생체내 항암활성 분석14-3: In vivo anticancer activity assay
본 발명자들은 본 발명의 일 실시예에 따른 SL-K10 세포주가 실제 생체내 조건에서도 유의한 항암활성을 나타내는지 확인하기 위해, 이종이식(xenograft) 종양 모델 마우스를 이용하여 SL-K10 세포주의 항암 활성을 분석하였다.In order to confirm whether the SL-K10 cell line according to an embodiment of the present invention exhibits significant anticancer activity even under actual in vivo conditions, the present inventors have anti-cancer activity of a SL-K10 cell line using a xenograft tumor model mouse. Was analyzed.
구체적으로, EpCAM 및 루시퍼레이즈를 발현하는 인간 난소암세포주인 RMG-1-luc-EGFP 5x106 cell을 7주령의 면역력 제거 실험쥐(NSG)에 복강접종하고 7일 경과 후, 대조군(성장 배지), 및 10 Gy의 방사선을 조사한 SL-K10 세포 5x106 cell을 정맥내 또는 복강내 2일 간격으로 네 차례 투여한 후 암세포에서 방사되는 루시퍼레이즈에 의한 생물발광 정도를 투여시점부터 1주일 간격으로 네 차례 측정함으로써 본 발명의 일 실시예에 따른 SL-K10 세포주의 in vivo 항암 활성을 조사하였다(도 15a). Specifically, RMG-1-luc-EGFP 5x10 6 cells, which are human ovarian cancer cell lines expressing EpCAM and luciferase, were intraperitoneally inoculated into 7-week-old immunized mice (NSG), and after 7 days, the control group (growth medium), And 5x10 6 cells of 10 Gy irradiated SL-K10 cells 4 times at 2 day intervals intravenously or intraperitoneally, and then bioluminescence by luciferase radiated from cancer cells 4 times at 1 week intervals from the time of administration. By measuring the in vivo anticancer activity of the SL-K10 cell line according to an embodiment of the present invention (Fig. 15a).
그 결과, 도 15b에서 확인되는 바와 같이, 정맥투여 및 복강투여 모두에서도 매우 우수한 생체내 항암활성이 확인되었다. 특히, 복강투여시 암세포가 거의 자라지 않았음을 알 수 있었다.As a result, as shown in FIG. 15B, very good in vivo anticancer activity was confirmed in both intravenous administration and intraperitoneal administration. In particular, it was found that cancer cells hardly grew during intraperitoneal administration.
아울러, 본 발명자들은 다중투여에 따른 효과 및5-FC와의 병용투여에 따른 항암 활성능 증진 여부를 확인하기 위해, 상기 도 15b에서 더 좋은 효과가 확인된 복강내 투여 경로를 채택하여 투여량을 증가하여 반복 투여 및 5-FC 병용 처리 시의 항암활성을 비교분석하였다.In addition, the present inventors adopted the intraperitoneal route of administration, in which the better effect is confirmed in FIG. 15B, to increase the dosage in order to confirm the effects of the multi-dose and the improvement of anti-cancer activity according to the co-administration with 5-FC. The anticancer activity of the repeated administration and 5-FC combination treatment was compared.
구체적으로 EpCAM 및 루시퍼레이즈를 발현하는 인간 난소암세포주인 RMG-1-luc-EGFP 5x106 cell을 7주령의 면역력 제거 실험쥐(NSG)에 복강접종하고 7일 경과 후, 10 Gy의 방사선을 조사한 SL-K10 및 대조군 NK111 세포주를 3x107 cells의 투여량으로 3일 또는 4일 간격으로 총 8회 투여한 후 4주간 SL-K10 세포 투여시점부터 7일, 14일, 21일 및 28일째에 암세포에서 방사되는 생체내 생물발광 정도를 측정함으로써, 암세포의 성장 정도를 분석하였다. 또한, 5-FC 병용 처리군의 경우, 500 mg/kg의 5-FC를 SL-K10 세포 투여시점부터 28일째까지 매일 복강투여 하였다(도 15c). 상기 생체내 생물발광 이미지는 마취된 실험동물을 냉각된 전하쌍 장치 카메라를 장착한 IVIS 이미지시스템(Caliper Life Sciences, USA)의 광차단 챔버에 둔 후, 암세포로부터 방출된 광자를 포집하여 1분동안 통합하였고, 광자수를 가리키는 가상색 이미지를 Living Image software v. 2.50(Caliper Life Science, USA)를 이용하여 수득한 마우스의 이미지 위에 중첩시킴으로써 수득하였다. Specifically, 7 mg of RMG-1-luc-EGFP 5x10 6 cells, a human ovarian cancer cell line expressing EpCAM and luciferase, were intraperitoneally inoculated into 7-week-old immunization mice (NSG), and after 7 days, SL was irradiated with 10 Gy of radiation. -K10 and control NK111 cell lines were administered eight times at three or four-day intervals at a dose of 3x10 7 cells, followed by cancer cells at 7, 14, 21 and 28 days from SL-K10 cell administration for 4 weeks. By measuring the degree of bioluminescence emitted in vivo, the growth of cancer cells was analyzed. In addition, in the 5-FC combination treatment group, 500 mg / kg of 5-FC was intraperitoneally administered daily from the time of SL-K10 cell administration to the 28th day (FIG. 15C). The in vivo bioluminescence image was placed in a light-blocking chamber of an IVIS Imaging System (Caliper Life Sciences, USA) equipped with a cooled charge pair device camera, and captured photons emitted from cancer cells for 1 minute. And integrated virtual color images indicating photon counts. Obtained by superimposing on images of mice obtained using 2.50 (Caliper Life Science, USA).
그 결과, 도 15d 및 도 15e에서 확인되는 바와 같이 본 발명의 일 실시예에 따른 SL-K10 세포는 3x107 cells의 투여량으로 반복 투여시 동일량을 투여한 NK111 대조군 대비 암세포의 생장이 현저하게 억제됨을 알 수 있었다. 또한, 5-FC 병용 투여시 SL-K10 단독투여군 대비 월등히 뛰어난 암세포 성장 억제능을 보여, SL-K10 투여 시점 대비 암세포가 전혀 자라지 않았음을 확인하였다. As a result, as shown in Figure 15d and 15e SL-K10 cells according to an embodiment of the present invention is significantly growth of cancer cells compared to the NK111 control group administered the same amount when repeated doses of 3x10 7 cells It can be seen that it is suppressed. In addition, 5-FC co-administration showed a significantly superior cancer cell growth inhibitory effect compared to the SL-K10 alone administration group, confirming that the cancer cells did not grow at all compared to the time of SL-K10 administration.
이는 세포자살 유전자 발현, 키메라 항원 수용체 도입 면역세포와 전구물질 병용투여를 통해 암세포 사멸 유도의 시너지를 실험적으로 입증한 최초의 사례이다. This is the first case to demonstrate experimentally the synergy of apoptosis gene expression, chimeric antigen receptor-introduced immune cell and precursor co-administration.
상술한 바와 같이, 본 발명의 일 실시예에 따른 anti-EpCAM CAR 발현 유전자 변형 NK111 세포주는 유전자 조작에도 불구하고 장기간 계대시 모세포주의 특성이 그대로 유지되면서도 시험관내 조건은 물론 생체내 조건에서 EpCAM 고발현 종양에 대한 매우 효과적인 항암활성을 나타냈다. 따라서, 본 발명의 일 실시예에 따른 유전자 변형 NK 세포주는 종래 CAR 컨스트럭트를 이용한 항암치료의 한계를 돌파하여 고형암에 대한 면역치료제로 매우 효과적으로 사용될 수 있을 것으로 예상된다.As described above, the anti-EpCAM CAR expression genetically modified NK111 cell line according to an embodiment of the present invention high expression of EpCAM under in vitro conditions as well as in vitro conditions while maintaining the characteristics of the parental cell line during long-term passage despite genetic manipulation. It showed very effective anticancer activity against tumors. Therefore, the genetically modified NK cell line according to one embodiment of the present invention is expected to be used very effectively as an immunotherapeutic agent for solid cancer by breaking the limitations of chemotherapy using conventional CAR constructs.
하기에 본 발명의 조성물을 위한 제제예를 예시한다.Examples of preparations for the compositions of the present invention are illustrated below.
제제예 1: 주사제의 제조Formulation Example 1 Preparation of Injection
유전자 재조합 SL-K10 세포 1x108 내지 5x1012 cellsRecombinant SL-K10 cells 1x10 8 to 5x10 12 cells
pH 조절제 적량pH adjuster
안정화제 적량Stabilizer
주사용 멸균 증류수 100% 까지Up to 100% sterile distilled water for injection
통상의 주사제의 제조방법에 따라 1 앰플 당(2 ㎖) 상기의 성분 함량으로 제조하였다.According to the conventional method for preparing an injection, the amount of the above ingredient was prepared per ampoule (2 ml).
본 발명은 상술한 실시예 및 실험예를 참고로 설명되었으나 이는 예시적인 것에 불과하며, 당해 기술분야에서 통상의 지식을 가진 자라면 이로부터 다양한 변형 및 균등한 다른 실시예가 가능하다는 점을 이해할 것이다. 따라서 본 발명의 진정한 기술적 보호 범위는 첨부된 특허청구범위의 기술적 사상에 의하여 정해져야 할 것이다.Although the present invention has been described with reference to the above-described examples and experimental examples, these are merely exemplary, and those skilled in the art will understand that various modifications and equivalent other embodiments are possible. Therefore, the true technical protection scope of the present invention will be defined by the technical spirit of the appended claims.
본 발명의 일 실시예에 따른 신규 키메라 항원 수용체 암호화 유전자가 형질도입된 유전자 변형 NK 세포주는 암치료를 위한 의약의 생산에 사용될 수 있다.Genetically modified NK cell line transduced with a novel chimeric antigen receptor coding gene according to an embodiment of the present invention can be used in the production of a medicament for the treatment of cancer.
Claims (26)
- 하기 특성을 갖는 분리된 NK 세포주에 EpCAM 특이적 키메라 항원 수용체를 암호화하는 폴리뉴클레오타이드가 형질도입되어 상기 EpCAM 특이적 키메라 항원 수용체를 발현하는 유전자 변형 NK 세포주:A genetically modified NK cell line expressing the EpCAM specific chimeric antigen receptor by transducing a polynucleotide encoding an EpCAM specific chimeric antigen receptor into an isolated NK cell line having the following characteristics:CD2, CD11a, CD25, CD45, CD54, DNAM-1, CD62L 및 CD56은 양성; 및CD2, CD11a, CD25, CD45, CD54, DNAM-1, CD62L and CD56 are positive; AndCD1a, CD3, CD4, CD8, CD14, CD20, CD23, CD34, TCRαβ 및 TCRγδ는 음성.CD1a, CD3, CD4, CD8, CD14, CD20, CD23, CD34, TCRαβ and TCRγδ are negative.
- 제1항에 있어서,The method of claim 1,상기 EpCAM 특이적 키메라 항원 수용체는 EpCAM에 특이적으로 결합하는 scFv, 변형 Ig Fc 도메인, CD28 막통과 도메인, DAP10, DAP12 및 T 세포 수용체의 CD3ζ 도메인으로 구성되는, 유전자 변형 NK 세포주.Wherein said EpCAM specific chimeric antigen receptor consists of a scFv, a modified Ig Fc domain, a CD28 transmembrane domain, a CD3ζ domain of DAP10, DAP12 and a T cell receptor that specifically binds to EpCAM.
- 제2항에 있어서, The method of claim 2,상기 scFv는 서열번호 3으로 기재되는 아미노산 서열로 구성되는, 유전자 변형 NK 세포주.Wherein said scFv consists of the amino acid sequence set forth in SEQ ID NO: 3. Genetically modified NK cell line.
- 제2항에 있어서,The method of claim 2,상기 변형 Ig Fc 도메인은 서열번호 5로 기재되는 아미노산 서열로 구성되는, 유전자 변형 NK 세포주.Wherein said modified Ig Fc domain consists of the amino acid sequence set forth in SEQ ID NO: 5. Genetically modified NK cell line.
- 제2항에 있어서,The method of claim 2,상기 CD28 막통과 도메인은 서열번호 7로 기재되는 아미노산 서열로 구성되는, 유전자 변형 NK 세포주. Wherein said CD28 transmembrane domain consists of the amino acid sequence set forth in SEQ ID NO: 7. Genetically modified NK cell line.
- 제2항에 있어서,The method of claim 2,상기 DAP10은 서열번호 9로 기재되는 아미노산 서열로 구성되는, 유전자 변형 NK 세포주.Said DAP10 is composed of the amino acid sequence set forth in SEQ ID NO: 9, Genetically modified NK cell line.
- 제2항에 있어서,The method of claim 2,상기 DAP12는 서열번호 11로 기재되는 아미노산 서열로 구성되는, 유전자 변형 NK 세포주.The DAP12 is a genetically modified NK cell line, consisting of the amino acid sequence set forth in SEQ ID NO: 11.
- 제2항에 있어서,The method of claim 2,상기 T 세포 수용체의 CD3ζ 도메인은 서열번호 13으로 구성되는 아미노산 서열로 구성되는, 유전자 변형 NK 세포주.The CD3ζ domain of the T cell receptor is composed of an amino acid sequence consisting of SEQ ID NO: 13, genetically modified NK cell line.
- 제1항에 있어서,The method of claim 1,상기 EpCAM 특이적 키메라 항원 수용체는 서열번호 1로 기재되는 아미노산 서열로 구성되는, 유전자 변형 NK 세포주.Wherein said EpCAM specific chimeric antigen receptor consists of the amino acid sequence set forth in SEQ ID NO: 1.
- 제1항에 있어서,The method of claim 1,NK 세포 보조활성화 인자를 암호화하는 폴리뉴클레오타이드가 추가적으로 형질도입되어 상기 NK 세포 보조활성화 인자가 발현되는, 유전자 변형 NK 세포주.A genetically modified NK cell line wherein a polynucleotide encoding a NK cell coactivator is additionally transduced to express the NK cell coactivator.
- 제10항에 있어서,The method of claim 10,상기 NK 세포 보조활성화 인자는 Ly49, NCR(natural cytotoxicity receptor), CD7, CD16 및 CD28로 구성되는 군으로부터 선택되는 어느 하나 이상인, 유전자 변형 NK 세포주.The NK cell co-activating factor is any one or more selected from the group consisting of Ly49, natural cytotoxicity receptor (NCR), CD7, CD16 and CD28, genetically modified NK cell line.
- 제11항에 있어서,The method of claim 11,상기 NK 세포 보조활성화 인자는 CD7 및/또는 CD28인, 유전자 변형 NK 세포주.Wherein said NK cell coactivator is CD7 and / or CD28.
- 제10항에 있어서, The method of claim 10,적어도 하나 이상의 NK 세포 증식 인자를 암호화하는 폴리뉴클레오타이드가 추가로 형질도입된, 유전자 변형 NK 세포주.A genetically modified NK cell line further transduced with a polynucleotide encoding at least one or more NK cell proliferation factors.
- 제13항에 있어서,The method of claim 13,상기 NK 세포 증식 인자는 IL-2, IL-12, IL-15, IL-18 및 IL-21로 구성되는 군으로부터 선택되는 적어도 하나 이상의 사이토카인 또는 상기 사이토카인의 변이체인, 유전자 변형 NK 세포주.Wherein said NK cell proliferation factor is at least one cytokine selected from the group consisting of IL-2, IL-12, IL-15, IL-18 and IL-21 or a variant of said cytokine, genetically modified NK cell line.
- 제14항에 있어서,The method of claim 14,상기 IL-15는 막결합 IL-15인, 유전자 변형 NK 세포주.Wherein said IL-15 is membrane-bound IL-15.
- 제10항에 있어서,The method of claim 10,세포자살 유전자가 추가로 형질도입되는, 유전자 변형 NK 세포주.The transgenic NK cell line, wherein the apoptotic gene is further transduced.
- 제16항에 있어서,The method of claim 16,상기 세포자살 유전자는 우라실 포스포리보실전이효소(UPRT) 유전자, 헤르페스 단순포진 바이러스 티미딘 인산화 유전자(HSV TK), 바리셀라 조스터 바이러스 티미딘 인산화효소(VZV TK) 유전자, 시토신 디아미네이즈 유전자, 카르복실 에스터레이즈 유전자, 니트로리덕테이즈 유전자, 카르복시펩티데이즈 G2 유전자, 또는 유도성 카스페이즈 9(iCas9)유전자인, 유전자 변형 NK 세포주.The apoptosis genes include uracil phosphoribosyltransferase (UPRT) gene, herpes herpes simplex virus thymidine phosphorylation gene (HSV TK), varicella zoster virus thymidine kinase (VZV TK) gene, cytosine deminase gene, The transgenic NK cell line, which is a carboxyl esterase gene, nitroreductase gene, carboxypeptides G2 gene, or inducible caspase 9 (iCas9) gene.
- 제10항에 있어서, The method of claim 10,세포질 도메인 결실 TGFβ 수용체를 암호화하는 폴리뉴클레오타이드가 추가로 형질도입되는, 유전자 변형 NK 세포주.The genetically modified NK cell line, which is further transduced with a polynucleotide encoding a cytoplasmic domain deleted TGFβ receptor.
- 제18항에 있어서,The method of claim 18,상기 세포질 도메인 결실 TGFβ 수용체는 세포질 도메인 결실 TGFβ 수용체II인, 유전자 변형 NK 세포주.Wherein said cytoplasmic domain deleted TGFβ receptor is cytoplasmic domain deleted TGFβ receptor II.
- 제1항 내지 제19항 중 어느 한 항의 치료적으로 유효한 양의 상기 유전자 변형 NK 세포주를 유효성분으로 함유하는 암치료용 세포치료제.A cell therapy agent for cancer treatment, comprising the therapeutically effective amount of the genetically modified NK cell line of any one of claims 1 to 19 as an active ingredient.
- 제1항 내지 제19항 중 어느 한 항의 유전자 변형 NK 세포주 및 자살유도제를 포함하는 암 치료용 키트.20. A kit for treating cancer comprising the genetically modified NK cell line of claim 1 and a suicide inducing agent.
- 제21항에 있어서,The method of claim 21,상기 자살유도제는 상기 세포자살 유전자가 HSV TK 또는 VZV TK일 경우에는 각각 간시클로비르(gancyclovir) 또는 6-메톡시퓨린 아라비노뉴클레오사이드(6-mehoxypurine arabinonucleoside)이고, 상기 세포자살 유전자가 시토신 디아미네이즈인 경우 5-플루오로시토신(5-FC)이며, 상기 세포자살 유전자가 카르복실 에스터라제인 경우 이리노테칸(CPT-11)이고, 상기 세포자살 유전자가 니트로리덕테이즈인 경우 5(아지리딘-1-일)-2,4-디니트로벤자마이드(CB1954)이며, 상기 세포자살 유전자가 카르복시펩티데이즈 G2인 경우 4-[(2-클로로에틸)(2-메실록시에틸)아미노]벤조일-엘-글루탐산(CMDA)이고, 상기 세포자살 유전자가 iCas9인 경우 iCas9 이량화제(dimerizer)인, 암 치료용 키트.The suicide inducing agent is gancyclovir or 6-methoxypurine arabinonucleoside when the suicide gene is HSV TK or VZV TK, respectively, and the suicide gene is cytosine dia 5-Fluorocytosine (5-FC) for minase, irinotecan (CPT-11) if the apoptosis gene is carboxyl esterase, 5 (aziridine) for the apoptosis gene is nitroreductase -1-yl) -2,4-dinitrobenzamide (CB1954) and 4-[(2-chloroethyl) (2-mesyloxyethyl) amino] benzoyl when the apoptosis gene is carboxypeptides G2 -L-glutamic acid (CMDA), iCas9 dimerizer if the apoptosis gene is iCas9, cancer kit.
- 치료적으로 유효한 양의 제1항 내지 제19항 중 어느 한 항의 유전자 변형 NK 세포주 및 선택적으로 자살유도제를 암의 치료를 필요로 하는 개체에 투여하는 단계를 포함하는 상기 개체의 암 치료 방법.A method of treating cancer in a subject comprising administering to the subject in need thereof a therapeutically effective amount of the genetically modified NK cell line of any one of claims 1-19 and optionally suicide.
- 제23항에 있어서,The method of claim 23, wherein상기 자살유도제는 상기 세포자살 유전자가 HSV TK 또는 VZV TK일 경우에는 각각 간시클로비르(gancyclovir) 또는 6-메톡시퓨린 아라비노뉴클레오사이드(6-mehoxypurine arabinonucleoside)이고, 상기 세포자살 유전자가 시토신 디아미네이즈인 경우 5-플루오로시토신(5-FC)이며, 상기 세포자살 유전자가 카르복실 에스터라제인 경우 이리노테칸(CPT-11)이고, 상기 세포자살 유전자가 니트로리덕테이즈인 경우 5(아지리딘-1-일)-2,4-디니트로벤자마이드(CB1954)이며, 상기 세포자살 유전자가 카르복시펩티데이즈 G2인 경우 4-[(2-클로로에틸)(2-메실록시에틸)아미노]벤조일-엘-글루탐산(CMDA)이고, 상기 세포자살 유전자가 iCas9인 경우 iCas9 이량화제(dimerizer)인, 암 치료 방법.The suicide inducing agent is gancyclovir or 6-methoxypurine arabinonucleoside when the suicide gene is HSV TK or VZV TK, respectively, and the suicide gene is cytosine dia 5-Fluorocytosine (5-FC) for minase, irinotecan (CPT-11) if the apoptosis gene is carboxyl esterase, 5 (aziridine) for the apoptosis gene is nitroreductase -1-yl) -2,4-dinitrobenzamide (CB1954) and 4-[(2-chloroethyl) (2-mesyloxyethyl) amino] benzoyl when the apoptosis gene is carboxypeptides G2 -L-glutamic acid (CMDA) and iCas9 dimerizer when the apoptosis gene is iCas9.
- 제23항에 있어서,The method of claim 23, wherein상기 암은 혈액암 또는 고형암인, 암 치료 방법.The cancer is hematological cancer or solid cancer.
- 제25항에 있어서,The method of claim 25,상기 고형암은 간암, 폐암, 췌장암, 유방암, 난소암, 자궁내막암, 자궁경부암, 담낭암, 위암, 담도암, 대장암, 두경부암, 식도암, 갑상선암, 뇌종양, 악성 흑색종, 전립선암, 고환암, 또는 설암인, 암 치료 방법.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, colon cancer, head and neck cancer, esophageal cancer, thyroid cancer, brain tumor, malignant melanoma, prostate cancer, testicular cancer, or Sulfur cancer, cancer treatment method.
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WO2023180511A1 (en) * | 2022-03-25 | 2023-09-28 | F. Hoffmann-La Roche Ag | Improved chimeric receptors |
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KR20190111844A (en) | 2019-10-02 |
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