Immune cell comprising tumor-targeting chimeric antigen receptor and uses thereof

Abstract

The present invention relates to immune cells comprising a novel tumor-targeting chimeric antigen receptor and to an anticancer immunotherapy composition including same. The immune cells and the anticancer immunotherapy composition according to one aspect can function as therapeutic agents for a variety of tumors by introducing of a pan-tumor chimeric antigen receptor, and exhibit improved stability of immune cells, thus being advantageously applicable to immune cell therapy for cancer treatment.

Description

Immune cells containing tumor-targeted chimeric antigen receptors and their uses

[0001] The present invention relates to an immune cell comprising a novel chimeric antigen receptor and the use thereof.

[0002] The field of immuno-oncology is a method of treating cancer by utilizing the power of adaptive immunity, such as by increasing the number of lymphocytes obtained from a patient's blood—including natural killer (NK) cells, natural killer T cells, T cells, B cells, and dendritic cells—and enhancing their in vitro functions to return them to the patient's body. Therapeutic methods using immune cells are evaluated as having good efficacy in modulating immune responses and being excellent in terms of toxicity and safety.

[0003] Immune cells are sometimes redesigned using chimeric antigen receptors (CARs). A CAR refers to a recombinant receptor for an antigen that allows the specificity and function of an immune cell to be redesignated within a single molecule. These include antigen-specific components, transmembrane components, and intracellular components selected to activate immune cells and provide specific immunity, and can be used in various therapies, including cancer therapy.

[0004] However, immune cells can exhibit a decline in overall function and a decrease in surface activating receptors due to TGF β, a representative immunosuppressant secreted by cancer cells. To address these issues, clinical practice involves administering inhibitors to patients alongside immunosuppressants or developing immune cells with the receptor genes for immunosuppressants removed. Furthermore, since the clinical benefits of T cell therapy are not extended to all patients or tumor indications, utilizing NK cells as powerful agents of innate immune responses is emerging as a promising approach; however, existing chimeric antigen receptors are built on T cells and have the disadvantage of lacking a signaling pathway optimized for NK cells.

[0005] Accordingly, the researchers also made research efforts to develop a structure capable of enhancing immune cell efficiency, and completed the present invention by confirming that immune cells equipped with a chimeric antigen receptor having the novel structure of the present invention exhibit excellent anti-tumor effects, superior adaptability in immunosuppressive environments, and outstanding cell activity and persistence in clinical settings.

[0006] One aspect is to provide a chimeric antigen receptor (CAR) comprising the extracellular domain of NKG2D; the transmembrane domain; the intracellular signaling domain of CD244; and the immunoreceptor tyrosine-based activation motif (ITAM) of DAP12.

[0007] Another aspect is to provide a composition comprising a gene encoding the above-mentioned chimeric antigen receptor.

[0008] Another aspect is to provide a vector containing the above-mentioned work.

[0009] Another aspect is to provide an immune cell comprising the chimeric antigen receptor, the construct, and / or the vector.

[0010] Another aspect provides an immune cell comprising a gene encoding the chimeric antigen receptor; shRNA targeting a gene encoding a cytokine receptor; and / or a gene encoding IL-15 or a fragment thereof.

[0011] Another aspect is to provide a composition comprising the chimeric antigen receptor, the construct, the vector, and / or the immune cell.

[0012] Another aspect is to provide a pharmaceutical composition for the prevention or treatment of cancer comprising the chimeric antigen receptor, the construct, the vector, and / or the immune cell.

[0013] Another aspect is to provide a use for the prevention or treatment of a disease (e.g., cancer) of a composition comprising the chimeric antigen receptor, the construct, the vector, and / or the immune cell.

[0014] Another aspect is to provide a use for the manufacture of a medicine for the prevention or treatment of a disease (e.g., cancer) of the above chimeric antigen receptor, the above construct, the above vector, the above immune cell, and / or the above composition.

[0015] Another aspect provides a method for preventing or treating a disease (e.g., cancer), comprising the step of administering the chimeric antigen receptor, the construct, the vector, the immune cell, and / or the composition to an individual in need thereof.

[0016] Another aspect provides a method for producing immune cells comprising the step of introducing the above-mentioned composition and / or the above-mentioned vector into immune cells.

[0017] One aspect provides a chimeric antigen receptor (CAR) comprising the extracellular domain of NKG2D; the transmembrane domain; the intracellular signaling domain of CD244; and the immunoreceptor tyrosine-based activation motif (ITAM) of DAP12.

[0018] In this specification, the term "Chimeric antigen receptor (CAR)" refers to a synthetic complex designed to induce an immune response against a target antigen and a cell expressing said antigen when the complex recognizes and binds to the target antigen. The chimeric antigen receptor may include an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain. The chimeric antigen receptor is expressed on the surface of an immune cell and, through an antigen binding site included in the extracellular domain, recognizes and binds to a specific antigen, such as an antigen specifically expressed on the surface of a cancer cell, thereby inducing signal transduction within the immune cell and altering the activity of the immune cell, thus enabling an immune response to be induced by targeting a specific antigen.

[0019] In this specification, the term "extracellular domain" refers to a domain that protrudes outside the cell and binds to ligands, etc.

[0020] The chimeric antigen receptor of the present invention may include the extracellular domain of NKG2D. "NKG2D" is a major activating receptor belonging to the NKG2 family of C-type lectin-like receptors and recognizes eight distinct ligands that are upregulated in tumor cells: MICA (MHC class I chain-related protein A), MICB (MHC class I chain-related protein B), and ULBPs (UL-16-binding proteins) designated as ULBP1 to ULBP6. NKG2D ligands are not expressed or are expressed at low levels in normal cells, but are overexpressed on the cell surface when cells are exposed to various stresses such as infection, deformation, or aging; when NKG2D binds to one or more of these ligands, it triggers downstream signaling in effector cells. NKG2D is expressed not only in NK cells but also in specific T cell populations such as CD8+, NKT, and γ / δ T cells. That is, the CAR of the present invention is a pan-tumor CAR capable of recognizing various cancer-specific ligands present on the surface of cancer cells by including the extracellular domain of NKG2D, and in particular, can recognize MICA / B and / or ULBPs.

[0021] In one embodiment, the extracellular domain may comprise the amino acid sequence of SEQ ID NO. 5, a part thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with these.

[0022] In this specification, the terms “transmembrane domain” or “transmembrane domain” refer to a domain located on the cell membrane that connects an extracellular domain and an intracellular domain, and refers to a region that connects and fuses the extracellular domain and the intracellular signaling domain to each other and serves to anchor a chimeric antigen receptor to the plasma membrane of an immune cell. The transmembrane domain may be derived from natural, synthetic, semi-synthetic, or recombinant sources. The transmembrane domain may be any one of the transmembrane domains selected from the group consisting of the alpha (α), beta (β), or zeta (ζ) chain of a T-cell receptor (TCR), CD28, CD3 epsilon (ε), CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, but is not limited thereto.

[0023] In one embodiment, the transmembrane domain may comprise the amino acid sequence of SEQ ID NO. 7, a part thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with these.

[0024] In this specification, the terms "intracellular domain" or "intracellular signaling domain" refer to a region located inside the cell membrane, i.e., in the cytoplasm, which transmits a signal into the cell through the binding of a ligand or the like to an extracellular domain.

[0025] The chimeric antigen receptor of the present invention may include an intracellular signaling domain of CD244. "CD244" is a type I transmembrane protein belonging to the SLAMF (signaling lymphocytic activation molecule family of receptors) family, expressed in various types of hematopoietic cells, and involved in the regulation of the immune system. CD244 is expressed in all types of NK cells and also in a subset of effector and effector memory CD8+ T cells, and plays a role in activating the proliferation, cytotoxicity, and IFNγ production of these cells. CD244 is also known as "2B4" or "SLAMF4". By utilizing the intracellular signaling domain of NK cell-based CD244, the chimeric antigen receptor of the present invention may have a signaling system suitable for NK cells compared to T cell-based signaling domains such as CD28, 4-1BB, CD3ζ, etc.

[0026] In one embodiment, the intracellular domain may comprise the amino acid sequence of SEQ ID NO. 8, a part thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with these.

[0027] The chimeric antigen receptor of the present invention may include the ITAM (immunoreceptor tyrosine-based activation motif) of DAP12. The ITAM of DAP12 plays a major role in signal transduction within immune cells and can prevent excessive activation of NK cells and prevent them from becoming exhausted within a short period of time.

[0028] In one embodiment, the ITAM of the DAP12 may be a tandem repeat domain of two or more ITAMs. Specifically, it may be a tandem structure in which the active domains of the DAP12 are linked by a linker.

[0029] In one embodiment, the linker connecting the active domain of the DAP12 may be a rigid linker. When a plurality of ITAMs include the linker, they may be arranged in a straight line without bending toward each other, and through this structure, they can interact efficiently with adapter proteins. As a result, the signal transduction efficiency of the CAR structure according to one embodiment may be improved. Specifically, the rigid linker may be (EAAAK)n, (A(EAAAK)mA)n, (EAAAKEAAAKEAAAK)n, etc., but is not limited thereto, and n may be a natural number greater than or equal to 1.

[0030] More specifically, the DAP12 ITAM may comprise the amino acid sequence of SEQ ID NO. 9 or 14, a part thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with these.

[0031] In one embodiment, the chimeric antigen receptor may additionally include a hinge domain. The extracellular domain of the NKG2D may be connected to a transmembrane domain through the hinge domain. The hinge domain is a portion that allows the antigen binding site to be physically separated from the surface of the immune cell on which the chimeric antigen receptor is expressed, so as to enable appropriate cell / cell contact, appropriate binding of the antigen / antigen binding site, and appropriate activation of the chimeric antigen receptor, and may play an important role in localizing the extracellular domain.

[0032] The hinge domain may be a hinge region derived from the extracellular domain of a type 1 membrane protein such as CD8, CD4, CD28, or CD7, but any capable of connecting the antigen binding site, the transmembrane domain, and the intracellular signaling domain across the cell membrane may be used without limitation. Additionally, it may be a wild-type hinge region from these molecules or may be modified.

[0033] The chimeric antigen receptor may include one or more hinge domains between the extracellular domain and the transmembrane domain.

[0034] In one embodiment, the hinge domain may comprise the amino acid sequence of SEQ ID NO. 6, a part thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with these.

[0035] In one embodiment, the chimeric antigen receptor may further comprise a signal peptide (sp). A signal peptide generally refers to a peptide chain for guiding protein delivery and may be a short peptide having a length of 5 to 30 amino acids. The signal peptide may be used interchangeably with "sp," "leader sequence," etc.

[0036] The above signal peptide may be a signal peptide of a molecule selected from the group consisting of CD8, GM-CSF receptor α, Ig-kappa, and IgG1 heavy chain, but any of them may be used without limitation.

[0037] The above signal peptide may be located in front of the above extracellular domain.

[0038] In one embodiment, the signal peptide may comprise the amino acid sequence of SEQ ID NO. 4, a part thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with these.

[0039] In one embodiment, the chimeric antigen receptor may comprise one or more amino acid sequences of SEQ ID NOs 1 to 3, a part thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with these. The amino acid sequences of SEQ ID NOs 1 to 3 are as shown in Table 1 below.

[0040] Sequence Number Target Amino Acid Sequence 1NKG2D Extracellular domain + CD8 hinge domain + CD28 transmembrane domain + 2B4+ tandem DAP12MALPVTALLLPLALLLHAARPFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSL LVTVAFIIFWVWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYSRVKFSRSAEAATRKQRITETESPYQELQGQRSDVYSDLNTQEAAAKAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK2NKG2D Extracellular domain + CD8 hinge domain + CD28 transmembrane domain + 2B4+ tandem DAP12+ P2A + solubleIL-15MALPVTALLLPLALLLHAARPFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYSRVKFSRSAEAATRKQRITETESPYQELQGQRSDVYSDLNTQEAAAKAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKGSAAAGSGATNFSLLKQAGDVEENPGPMRRMQLLLLIALSLALVTNSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS3NKG2D 세포외 도메인 + CD8 힌지 도메인 + CD28 막 횡단 도메인 + 2B4+ tandem DAP12+ P2A + GFP + T2A solubleIL-15MALPVTALLLPLALLLHAARPFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYSRVKFSRSAEAATRKQRITETESPYQELQGQRSDVYSDLNTQEAAAKAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKGSAAAGSGATNFSLLKQAGDVEENPGPPAMEIECRITGTLNGVEFELVGGGEGTPKQGRMTNKMKSTKGALTFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYRYEAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDGGYYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFARSRAQSSNSAVDGTAGPGSTGSRTSGSGEGRGSLLTCGDVEENPGPMRRMQLLLLIALSLALVTNSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0041] In one embodiment, the chimeric antigen receptor may further comprise IL-15 or a fragment thereof. IL-15 may increase or enhance the persistence or activity of immune cells in immune cells comprising or expressing the chimeric antigen receptor. As specified herein, a fragment of IL-15 means a fragment having a function equivalent to or similar to IL-15.

[0042] The above IL-15 may be in a state not bound to the cell surface and may be in a form that can circulate freely. That is, the above IL-15 may be soluble IL-15.

[0043] In one embodiment, the IL-15 may comprise the amino acid sequence of SEQ ID NO. 11, a part thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with these.

[0044] In one embodiment, the chimeric antigen receptor may further comprise a signal peptide. The IL-15 or a fragment thereof may be linked to the ITAM domain through a signal peptide, for example, a 2A peptide.

[0045] The above 2A peptide may be T2A, P2A, E2A, and / or F2A. That is, the IL-15 or a fragment thereof may be linked to the ITAM domain through a P2A or T2A peptide.

[0046] Specifically, the 2A peptide may comprise the amino acid sequence of SEQ ID NO. 10 or 13, a part thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with these.

[0047] In one embodiment, the chimeric antigen receptor may have the structure of a first-generation CAR to a fifth-generation CAR.

[0048] Each domain of the chimeric antigen receptors according to the present invention may be directly or optionally connected by a short oligopeptide or polypeptide linker. The linker is not particularly limited in its length or type, and any linker known in the art may be applied without limitation.

[0049] In one embodiment, the linker may be a polypeptide composed of any amino acid. The linker may be a flexible linker. For example, it may be a peptide linker composed of glycine, serine, alanine, or proline, and more specifically, it may be (GS)n, (GGGGS)n, (GGGGA)n, (GGGGP)n, (GGGA)n, (GGS)n, (GSGGS)n, or (GGGS)n, etc. The copy number "n" is any natural number and can be adjusted considering the optimization of the linker.

[0050] Each domain of the chimeric antigen receptor according to the present invention may include the aforementioned domains as well as modified forms of each of the domains. In this case, the modification may be performed by substituting, deleting, or adding one or more amino acids in the amino acid sequence of the wild-type antibody and domain without altering the function of the antibody and domains. Typically, the substitution may be performed by a conservative amino acid substitution that does not affect the charge, polarity, or hydrophobicity of the entire protein.

[0051]

[0052] Another aspect provides a composition comprising a gene encoding the above-mentioned chimeric antigen receptor.

[0053] In this specification, the term "construct" refers to a macromolecule or molecular complex comprising a polynucleotide that is delivered to a host cell in vitro, in vivo, or ex vivo.

[0054] The description of each domain of the above chimeric antigen receptor applies equally to the above-mentioned work.

[0055] In one embodiment, the chimeric antigen receptor and / or construct may further comprise a gene encoding a short hairpin RNA (shRNA) that targets a gene encoding a cytokine receptor. The cytokine receptor may bind to a cytokine secreted by a cancer cell.

[0056] The above shRNA can inhibit or reduce the protein expression of the cytokine receptor. As the protein expression of the above cytokine receptor is inhibited, the binding between the immunosuppressive substance or immunoinhibitor secreted by the cancer cell and the active receptor of the immune cell is blocked, thereby increasing or enhancing the immune stimulation response of the immune cell containing the chimeric antigen receptor or the construct containing the gene encoding it.

[0057] The above cytokines are IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-21, IFN-γ, IL-1α, IL-1β, IL1RA, IL-18, IL-33, IL-36α, IL-36β, IL-36γ, IL-36RA, IL-37, IL-38, IL-3, IL-5, IL-6, IL-11, IL-13, IL-23, granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF), leukemia inhibitory factor (LIF), stem cell factor (SCF), thrombopoietin (TPO), and macrophage-colony stimulation It may be a factor (M-CSF: macrophage-colony stimulating factor), erythropoietin (EPO: erythropoieticn), Flt-3, IFN-α, IFN-β, IFN-γ, IL-19, IL-20, IL-22, IL-24, TNF-α, TNF-β, BAFF, APRIL, lymphotoxin beta (TNF-γ: lymphotoxin beta), IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-25, TSLP, IL-35, IL-27, or TGF-β, but is not limited thereto.

[0058] In one embodiment, the shRNA may comprise the gene sequence of SEQ ID NO. 17 or 18, a part thereof, or a gene sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with these.

[0059] In one embodiment, the composition may further comprise a gene encoding IL-15 or a fragment thereof.

[0060] In one embodiment, the composition may comprise a gene encoding the chimeric antigen receptor; a gene encoding the shRNA; and / or a gene encoding IL-15 or a fragment thereof.

[0061] In one embodiment, the composition may further include a promoter operably connected thereto for the expression of the said genes.

[0062] Specifically, the above-mentioned composition may further comprise a promoter sequence operably linked to a gene encoding the chimeric antigen receptor. The promoter may be an SFFV promoter (spleen focus-forming virus promoter). The SFFV promoter may comprise the gene sequence of SEQ ID NO. 20, a part thereof, or a gene sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with these.

[0063] Specifically, the above-mentioned composition may include a promoter operably linked to shRNA for the expression of shRNA, specifically a U6 promoter, etc. The U6 promoter may include the gene sequence of SEQ ID NO. 19, a part thereof, or a gene sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with these.

[0064] In one embodiment, the above-mentioned composition may comprise the gene sequence of SEQ ID NO. 15 or 16, a part thereof, or a gene sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with these.

[0065] That is, the chimeric antigen receptor may include an amino acid sequence encoded by the gene sequence of SEQ ID NO. 15 or 16.

[0066]

[0067] Another aspect is to provide a vector containing the above-mentioned work.

[0068] The description of each domain of the above chimeric antigen receptor and the above construct applies equally to the above vector.

[0069] In this specification, the term “vector” refers to any nucleic acid construct capable of delivering or directing the transfer of foreign genetic material to a target cell where said polynucleotide may be replicated and / or expressed. As used herein, the term “vector” includes the construct to be delivered. The vector may be a linear molecule or a circular molecule. The vector may be integrating or non-integrating.

[0070] Major types of vectors include, but are not limited to, plasmids, episomal vectors, viral vectors, nonviral vectors, cosmids, and artificial chromosomes. The viral or nonviral vectors may be used without limitation as long as they can stably express the chimeric antigen receptor within host cells by transduction or transfection of animal cells, particularly NK cells, by infection.

[0071] In one embodiment, the virus vector may be any one selected from the group consisting of retrovirus, lentivirus, adenovirus, adeno-associated virus, and vaccinia virus, but is not limited thereto.

[0072] In one embodiment, the nonviral vector is preferably a transposon system (Hackett et al., US 6,489,458 B), but is not limited thereto, and it is obvious to a person skilled in the art that any nonviral vector that is suitable for the purpose of the present invention can be used.

[0073] In one embodiment, the virus or non-viral vector may comprise the genes of the bispecific chimeric protein and the chimeric antigen receptor.

[0074] Even if the present application is described as "comprising a gene sequence / amino acid sequence of a specific sequence number" or "having a gene sequence / amino acid sequence of a specific sequence number," it is obvious that a gene sequence / amino acid sequence having some sequences deleted, modified, substituted, or added may also be used in the present application if it has the same or equivalent function as that composed of the gene sequence / amino acid sequence of the said sequence number. Furthermore, gene sequences and base sequences may be used interchangeably in the present application.

[0075] For example, if the chimeric protein, the chimeric antigen receptor, or the composition has the same or corresponding function, it is obvious that the addition of a meaningless sequence within or at the end of the sequence of the said sequence number, or the deletion of a part of the sequence within or at the end of the said sequence number, falls within the scope of the present invention.

[0076] Homology and identity refer to the degree of association between two given base sequences and can be expressed as a percentage. The terms homology and identity are often used interchangeably. Whether any two sequences have homology or identity can be determined using known computer algorithms, such as the "FASTA" program, with default parameters as in, for example, Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) (GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] (Including Academic Press, San Diego, 1994, and [CARILLO ETA / .](1988) SIAM J Applied Math 48: 1073). For example, homology or identity of sequences can be determined using BLAST from the National Biotechnology Information Database Center or ClustalW.

[0077]

[0078] Another aspect is to provide an immune cell comprising the above-mentioned chimeric antigen receptor, the above-mentioned construct, or a vector comprising the above-mentioned construct.

[0079] The descriptions regarding each domain of the chimeric antigen receptor, the composition, and the vector also apply equally to the immune cells.

[0080] Another aspect provides an immune cell comprising a gene encoding the chimeric antigen receptor; shRNA targeting a gene encoding a cytokine receptor; and / or a gene encoding IL-15 or a fragment thereof.

[0081] In one embodiment, the immune cell may be a macrophage, B lymphocyte, T lymphocyte, mast cell, monocyte, dendritic cell, eosinophil, natural killer cell, basophil, and neutrophil, or a combination thereof, but is not limited thereto.

[0082] In this specification, the term "natural killer cells (NK cells)" refers to a type of cytotoxic lymphocyte derived from bone marrow. Natural killer cells account for 5 to 20 percent of total lymphocytes and are responsible for innate immunity; they are characterized by providing an immediate immune response against virus-infected cells, cancer cells, and other modified cells, even though they lack a major histocompatibility complex or antibodies on their surface. Natural killer cells include natural killer cells isolated from an individual as well as natural killer cells cultured from or modified therefrom, and commercially available natural killer cell lines may also be used. That is, the natural killer cells of the present invention are not limited to a specific type, and it is sufficient for them to possess molecular characteristics and biological activities identical or similar to those of natural killer cells. In one embodiment, the natural killer cells may be selected from, for example, HANK1, NKL, NK92, NK-YS, YT, NOI-90, and NK101, but this is merely an example and is not limited thereto. The natural killer cell expressing a chimeric antigen receptor according to the present invention may be a CAR-NK cell (Chimeric antigen receptor natural killer cell).

[0083] Natural killer cells expressing a chimeric antigen receptor according to the present invention can be activated more rapidly or to a higher level compared to natural killer cells that do not express the chimeric antigen receptor when they recognize a target through the receptor, and can kill target cells more rapidly or effectively.

[0084]

[0085] Another aspect provides a composition comprising the chimeric antigen receptor, the gene, the construct, the vector, or the immune cell.

[0086] The descriptions regarding each domain of the chimeric antigen receptor, the composition, the gene, the vector, and the immune cell also apply to the composition.

[0087] Another aspect provides the use of the chimeric antigen receptor, the gene, the construct, the vector, the immune cell, or the composition for the prevention or treatment of a disease (e.g., cancer).

[0088] In this specification, the term "prevention" refers to any act of suppressing or delaying a disease by administering the composition of the present invention to an individual. For preventive benefits, the composition may be administered to a subject at risk of developing a specific disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of a disease, even if the disease, condition, or symptom has not yet appeared.

[0089] In this specification, the term "treat" refers to any act of administering the composition of the present invention to an individual to improve or benefit from the symptoms of a disease. As used herein, "treat," "alleviate," or "improvement" may be used interchangeably. A therapeutic benefit means any therapeutically significant improvement of one or more diseases, conditions, or symptoms under treatment, or an effect thereon.

[0090] In this specification, the terms “administering,” “introducing,” and “implanting” are used interchangeably and may mean the placement of a composition according to one embodiment into an individual by a method or route that results in at least partial localization of the composition according to one embodiment to a desired site. The composition according to one embodiment may be administered by any suitable route that delivers at least a portion of the cells or cellular components to a desired location within a living individual. The survival period of the cells after administration to the individual may be as short as a few hours, for example, from 24 hours to several days, or as long as several years.

[0091] The cancer of the present invention is a cancer that expresses a target antigen recognized by the chimeric antigen receptor of the present invention. That is, the cancer is a cancer that expresses a tumor antigen that can be recognized by the chimeric antigen receptor of the present invention.

[0092] In one embodiment, the cancer or tumor may express MICA (MHC class I chain-related protein A), MICB (MHC class I chain-related protein B), ULBP1 (UL-16-binding protein 1), ULBP2, ULBP3, ULBP4, ULBP5, and / or ULBP6. That is, the composition may be a composition for the prevention or treatment of cancer, solid tumors, and / or hematological malignancies in which MICA / B and / or ULBPs are expressed.

[0093] The above cancer, tumor, or carcinoma is not particularly limited and includes solid tumors and blood cancers. Specifically, it may include gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colorectal cancer, colon cancer, pancreatic cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, parathyroid cancer, kidney cancer, esophageal cancer, bile duct cancer, testicular cancer, rectal cancer, head and neck cancer, cervical cancer, ureteral cancer, osteosarcoma, neuroblastoma, melanoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, or glioma, etc.

[0094] The content of immune cells expressing the CAR, expression vectors, and / or cells containing the expression vectors in the composition of the present invention can be appropriately adjusted according to the symptoms of the disease, the degree of progression of symptoms, the condition of the patient, etc. For example, it may be 0.0001 to 99.9% by weight or 0.001 to 50% by weight based on the total weight of the composition, but is not limited thereto. The above content ratio is a value based on the dry weight after removing the solvent.

[0095] The composition according to the present invention may further include a pharmaceutically acceptable carrier. For oral administration, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavors, etc. may be used; for injectables, buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. may be mixed and used; and for topical administration, bases, excipients, lubricants, preservatives, etc. may be used. The formulations of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with the pharmaceutically acceptable carriers described above. For example, for oral administration, it may be prepared in the form of tablets, troches, capsules, ellipsis, suspensions, syrups, wafers, etc., and for injectables, it may be prepared in the form of unit dosing ampoules or multi-dose formulations. In addition, the anticancer composition may typically include a surfactant that facilitates movement across a membrane. These surfactants include those derived from steroids, cationic lipids such as N-[1-(2,3-dioleoyl)propyl-N,N,N-trimethylammonium chloride (DOTMA), or various compounds such as cholesterol hemisuccinate and phosphatidyl glycerol.

[0096] The above composition may be administered in combination with additional anticancer agents. Examples of additional anticancer agents may include alkylating agents, antimetabolites, spindle inhibitors, plant alkaloids, cytotoxic / antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Examples of the above anticancer agents may include compounds used in targeted therapy and conventional chemotherapy. In addition, examples of the above antibodies include alemtuzumab, apolizumab, aselizumab, atlizumab, bapineuzumab, bevacizumab, vivatuzumab mertansine, cantuzumab mertansine, cedelizumab, sertolizumab pegol, sidfucituzumab, sidtuzumab, daclizumab, eculizumab, epalizumab, efratuzumab, erlizumab, felbizumab, pontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, rabetuzumab, lintuzumab, matuzumab, mepolizumab, motabizumab, motobizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, Peckfucituzumab, pectuzumab, pertuzumab, pexelizumab, ralibizumab, ranibizumab, reslibizumab, reslibizumab, resaibizumab, lovelizumab, luplizumab, cibrotuzumab, ciplizumab, sontuzumab, tacatuzumab, tetraxetane, tadocizumab, talizumab, tepivazumab, tocilizumab, toralizumab, trastuzumab, tucotuzumab, selmoleukin, tucucituzumab, umavizumab, urtoxazumab, and bicilizumab may be included.

[0097] Another aspect provides a use of a composition comprising the chimeric antigen receptor, the construct, the vector, and / or the immune cell for the prevention or treatment of a disease (e.g., cancer).

[0098] Another aspect provides a use for the manufacture of a medicine for the prevention or treatment of a disease (e.g., cancer) of the above chimeric antigen receptor, the above construct, the above vector, the above immune cell, and / or the above composition.

[0099] Another aspect provides a method for preventing or treating a disease (e.g., cancer), comprising the step of administering the chimeric antigen receptor, the construct, the vector, the immune cell, and / or the composition to an individual in need thereof.

[0100]

[0101] Another aspect provides a method for producing immune cells comprising the step of introducing the above gene, the above construct, and the above vector into immune cells.

[0102] The descriptions regarding each domain of the chimeric antigen receptor, the gene, the construct, the vector, and the immune cell also apply to the manufacturing method.

[0103] In one embodiment, the manufacturing method may include the step of introducing the gene, the construct, or a vector containing the construct into an immune cell.

[0104] In one embodiment, the manufacturing method may additionally include the step of introducing into an immune cell a gene encoding shRNA targeting a gene encoding a cytokine receptor and / or a gene encoding IL-15 or a fragment thereof.

[0105] In one embodiment, each gene may be introduced into immune cells independently, simultaneously, sequentially, or in reverse order. In this case, the independent introduction of the genes may be performed according to methods known in the art. For example, a vector containing the first gene and a vector containing the second gene may be used independently, but are not limited thereto.

[0106]

[0107] The present invention is capable of various modifications and may have various embodiments. Specific embodiments are illustrated in the drawings and described in detail in the detailed description below. However, this is not intended to limit the present invention to specific embodiments, and it should be understood that it includes all modifications, equivalents, and substitutions that fall within the spirit and scope of the present invention. In describing the present invention, detailed descriptions of related prior art are omitted if it is determined that such detailed descriptions may obscure the essence of the present invention.

[0108] An immune cell containing a chimeric antigen receptor according to one aspect and a composition thereof for anticancer immunotherapy can provide an immune cell with enhanced stability, such as preventing excessive activation of the immune cell and improving cell persistence, thereby exhibiting improved tumor treatment efficiency and being useful for immune cell therapy for cancer treatment.

[0109] Figure 1 is a schematic diagram showing the structure of a CAR vector according to a working pattern.

[0110] Figure 2 is a schematic diagram modeling an engineered tandem DAP12 structure within a CAR vector according to a working pattern.

[0111] Figure 3 shows the results of CAR expression rate flow cytometry analysis to measure CAR lentivirus titers according to daily patterns.

[0112] Figure 4 is the result of flow cytometry analysis confirming the CAR expression rate of NK cells transduced with CAR vectors according to one pattern.

[0113] Figure 5 is a graph showing the cell proliferation rate of NK cells transduced with a CAR vector according to the pattern.

[0114] Figure 6 shows the results of analyzing Interleukin-15 secretion from NK cells transduced with a CAR vector according to a specific pattern using the ELISA method.

[0115] Figure 7 is the result of flow cytometry analysis confirming changes in surface marker expression of NK cells transfected with a CAR vector according to a specific pattern.

[0116] Figure 8 shows the results of flow cytometry analysis confirming the expression of the CAR target ligand MICA / B in various solid tumor cell lines according to the pattern.

[0117] Figure 9 is a graph analyzing the cancer cell death of NK cells transfected with a CAR vector according to the pattern.

[0118] Preferred embodiments are presented below to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the invention and do not limit the scope of the invention. Since the embodiments are subject to various modifications, they are not limited to the embodiments disclosed below but can be implemented in various forms.

[0119]

[0120] Example 1. Preparation and Production of CAR Lentivirus

[0121] 1.1. Construction of the CAR Lentivirus Vector

[0122] To construct a lentivirus encoding CAR, the genes presented in the CAR blueprint were inserted into a pCDH bicistronic lentivector (System Biosciences, SBI lentivector system). Specifically, each gene was synthesized using GenScript (GenScript Biotech Corp.) and the IDT gBlock system (Integrated DNA Technologies, Inc.), and then linked to the lentivector using the Gibson gene assembly method (NEB, NEBuilder HiFi DNA assembly). The constructed CAR-lentivector was transformed into NEB Stbl3 competent cells (NEB) and amplified. The CAR construct consists of genes encoding the proteins presented in Table 1 below; the corresponding amino acid sequences are shown in Table 3, and the schematic diagram is shown in Figure 1.

[0123]

[0124] Composition #1 shRNA for TGFβ receptor 2 + SFFV promoter + CD8 leader + NKG2D extracellular domain + CD8 hinge domain + CD28 transmembrane domain + 2B4 + tandem DAP12 + P2A + soluble IL-15 #2 shRNA for TGFβ receptor 2 + SFFV promoter + CD8 leader + NKG2D extracellular domain + CD8 hinge domain + CD28 transmembrane domain + 2B4 + tandem DAP12 + P2A + GFP + T2A + soluble IL-15

[0125]

[0126] Domain sequence number RNA or amino acid sequence TGFβ2 receptor shRNA17GGCCUGUAUAUAAAUAUGAAUAGCUUCAAGAGAGCUAUUCAUAUUUAUAUACAGGCUUUUUUUGGAUCUACUAAUGAAAAAUUGUUCUUUCAAGAAGAACAAUUUUUUCAUUAGUAGAUUUUUUUGCD8 sp4MALPVTALLLPLALLLHAARPNKG2D ED5FNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVCD8a hinge6TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDCD28a TM7FWVLVVVGGVLACYSLLVTVAFIIFWV2B48WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQN PARLSRKELENFDVYSDAP129RVKFSRSAEAATRKQRITETESPYQELQGQRSDVYSDLNTQEAAAKAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKP2A10ATNFSLLKQAGDVEENPGPSolubleIL1511MRRMQLLLLIALSLALVTNSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSN GNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGFP12PAMEIECRITGTLNGVEFELVGGGEGTPKQGRMTNKMKSTKGALTFSPYLLSHVMGYGFYH FGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYRYEAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDGGY YSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFARSRAQSSNSAVDGTAGPGSTGSRT2A13EGRGSLLTCGDVEENPGP

[0127]

[0128] 1.2 Intracellular Signaling Domains

[0129] DAP12, a signaling domain responsible for intracellular signaling of the chimeric antigen receptor (CAR), was modified from a single ITAM motif into a tandem DAP12 in which the ITAM motif sequence is repeated twice to enhance signaling. DAP12 has the disadvantage of weak signaling due to having only one ITAM motif. Tandem DAP12 is a tandem sequence in which the ITAM motif is repeated twice to overcome this disadvantage. In particular, a structure in which the ITAM motif is simply repeated twice exists in a bent shape as shown in [Fig. 2, left], which has the disadvantage that it is difficult for adaptor proteins to bind; however, the CAR structure according to one embodiment overcomes this structural disadvantage by connecting the ITAM motif of DAP12 with a linker. The above structure facilitates the binding of adaptor proteins and is effective for intracellular signaling.

[0130]

[0131] - tandem DAP12 (sequence number 14): RVKFSRSAEAATRKQRITETESPYQELQGQRSDVYSDLNTQAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK

[0132]

[0133] - engineered tandem DAP12 (SEQ ID NO: 9): RVKFSRSAEAATRKQRITETESPYQELQGQRSDVYSDLNTQEAAAKAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK

[0134]

[0135] 1.3 Production of CAR Lentivirus

[0136] Lentiviruses were produced using a third-generation lentivirus system. Specifically, using a CAR-expressing transfer plasmid 14 µg, pCgpV packaging plasmid 7 µg, pRSV-Rev packaging plasmid 3.5 µg, and BaEV (baboon envelope) envelope plasmid 3.5 µg, 5 x 10 6HEK293T cells were transduced with Lipofectamine 3000 (ThermoFisher, USA). After incubation at 37°C, the lentiviruses produced at 24, 48, and 72 hours were collected, and the HEK293T cells were replaced with fresh medium. The harvested lentivirus supernatant was passed through a 0.45 μm filter to remove cellular debris, and to concentrate the lentivirus, the filtered supernatant was mixed with Lenti-X concentrator (Clontech, USA) reagent in a 3:1 ratio and stored at 4°C for 24 hours. As a final step, the lentivirus-concentrator mixture was centrifuged at 1500 xg for 60 minutes, the supernatant was removed, and the recovered lentivirus pellet was diluted with 0.5 mL of 1x PBS and stored at -80°C until use.

[0137]

[0138] 1.3 Titration of CAR Lentivirus

[0139] Additionally, the titer of the functional lentivirus was determined using HEK cells. First, the lentivirus stock was serially diluted tenfold a total of six times (10 2 -10 6 Dilution factor up to). After treating HEK cells with the diluted solution, fluorescence expression was checked to see if the expressed GFP fluorescence decreased in proportion to the dilution factor, and then the results of measuring the fluorescence expressed in the cells using flow cytometry were presented. For lentivirus titers, only HEK cell results within the 10–40% range, where a linear relationship is formed between the proportion of expressed cells and the number of virus particles, were used. Viral titers were calculated using the following formula, and the results calculated using the Baboon envelope vector are shown in Figure 3.

[0140] [Mathematical Formula 2]

[0141] Titer (TU / mL) = (Number of HEK cells × GFP expression percentage × Dilution factor) / 100

[0142]

[0143] The lentivirus with the inserted structure was diluted to a factor of 10 in experiments 4 It showed 28.07% expression, and according to the formula, 8.42x10 7 It was confirmed that it exhibited a titer of TU / mL. The produced CAR lentivirus was transduced into NK cells at various MOIs.

[0144]

[0145] 1.4 Transduction of CAR Lentivirus into NK Cells

[0146] Based on the quantitative value of the lentivirus obtained in Example 1.3, the lentivirus was transduced into NK cells.

[0147] Specifically, the lentivirus particles containing the CAR of Table 1 were mixed with polybrene 8 μg / ml (Sigma Aldrich), and then 1 x 10 5 After treating 48-well plates seeded with NK-92 cells, the cells were centrifuged at 1,800 xg for 90 minutes, and then cultured in a 37°C incubator for 5 to 7 days. The CAR expression level was then checked using a flow cytometer. In the case of CAR expression, the antibody against NKG2D and GFP were inserted, the GFP expression level was checked using a flow cytometer.

[0148]

[0149] Example 2. Evaluation of CAR expression in CAR-NK cells

[0150] CAR expression in CAR-NK cells according to one embodiment was evaluated.

[0151] Specifically, CAR expression in NK92 cells was confirmed by flow cytometry 5 days after lentivirus infection. NK cells are cells that intrinsically express NKG2D, and to distinguish them from the expression of externally introduced CAR (NKG2D), NK92 cells stained with the same antibody were used as a control, and the results of the flow cytometry analysis are shown in Figure 4.

[0152] As shown in Figure 4, when infected with lentivirus MOI 5 or MOI 10 and then stained with an antibody against CAR-NKG2D, it was confirmed that the NKG2D transduction efficiency was over 90% and GFP expression was 70-80%.

[0153]

[0154] Example 3. Determination of the cell proliferation ability of CAR-NK cells.

[0155] To confirm the cell proliferation ability of CAR-NK cells according to one embodiment, the following experiment was performed.

[0156] 1x10 5 Dog NK92 cells were treated with CAR lentivirus at an MOI of 5 and cultured for 12 days. Cells were stained using trypan blue staining, and the cell count was determined using an automated cell counter (LUNA-II, Logos Biosystem). The results are shown in Figure 5.

[0157] As shown in Fig. 5, NK92 cells not treated with lentivirus for 12 days 1 x 10 5 9.67x10 from dogs 5 It increased 9.6-fold, and NK92 cells treated with the CAR lentivirus according to one embodiment were 1.2 x 10 6 It was confirmed that the number increased 12.2 times.

[0158]

[0159] Example 4. Elucidation of IL-15 production and secretion capabilities of CAR-NK cells

[0160] The present structure is linked to a signal peptide capable of producing and secreting the cytokine IL-15 via P2A and IL-15. To confirm the IL-15 production and secretion ability of CAR-NK cells according to one embodiment, the following experiment was performed.

[0161]

[0162] First, NK92 cells were treated with CAR lentivirus and cultured for 12 days, and the culture medium was collected daily. The concentration of IL-15 secreted in the cell culture medium was analyzed using a human IL-15 ELISA kit (R&D system), and the results are shown in Figure 6.

[0163] As shown in Figure 6, it was confirmed through ELISA that CAR-NK92 cells containing a soluble IL-15 sequence continuously secreted IL-15 into the culture medium at concentrations of 50 ng / mL to 250 ng / mL, and that almost no secretion occurred in NK92 cells without the CAR structure inserted.

[0164]

[0165] Example 5. Confirmation of changes in surface marker expression in CAR-NK cells

[0166] To determine whether the introduction and expression of CAR using a lentivirus alters the characteristics of NK cells, representative surface markers CD56, CD16, NKG2D, Nkp46, and NKG2A were tested using a flow cytometer. After introducing the CAR lentivirus into NK92 cells, the test group (CAR-NK92) and the control group (NK cells) were collected on day 5 and analyzed using a flow cytometer.

[0167] As shown in Figure 7, no significant changes were observed in the expression of each cell surface marker following the introduction of CAR-lentivirus, and in the case of NKG2D, the increase in expression caused by CAR was reconfirmed.

[0168]

[0169] Example 6. Identification of the apoptotic ability of CAR-NK cells.

[0170] To confirm the cancer cell killing ability of CAR-NK cells according to one embodiment, the following experiment was performed.

[0171] First, the expression of the CAR target antigen MICA / B was confirmed in solid tumor cell lines SK-OV3, A2780, OVCAR3, HepG2, and Huh7 using a flow cytometer.

[0172] Specifically, ovarian cancer cell lines SK-OV-3 and A2780, which express the target antigen, and the hepatocellular carcinoma cell line HepG2 were stained with the cell trace dye CFSE (CellTrace™ CFSE Cell Proliferation Kit), and then 5 x 10⁴ were placed in a 96-well plate. 5100 μL of cells were seeded at a concentration of cells / mL. Subsequently, Pan-NKG2D-GFP-sIL15 cells according to one embodiment and control NK-92 cells were cultured with the cancer cells for 4, 24, or 48 hours after setting various effector cell (E) to target cell (T) ratios (E:T = 5:1, 2:1, 1:1, 0.5:1). After culture, all cells were collected and centrifuged. The cells were then suspended in FACS buffer, 1 μL of 1X 7-AAD was added to the cells, and the mixture was reacted at room temperature for 5 minutes. Cell death was determined using flow cytometry by classifying viable cells (7-AAD negative / CFSE-positive) and necrotic or apoptotic cells (7-AAD positive / CFSE-positive), and the degree of cell death was analyzed. The results are shown in Figures 8 and 9.

[0173] As shown in Figures 8 and 9, a cell death effect more than twice as strong was observed in NK cells to which the CAR according to one embodiment was introduced, compared to control NK cells. This cell death ability showed a similar pattern in two types of ovarian cancer cell lines, and it was confirmed that the cell death ability increased over time. In addition, in the hepatocellular carcinoma cell line HepG2, it was confirmed that more than 95% cell death ability was observed through 4 hours of co-culture compared to control NK cells.

[0174]

[0175] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. Extracellular domain of NKG2D; transmembrane domain; The intracellular signaling domain of CD244; and ITAM (immunoreceptor tyrosine-based activation motif) of DAP12 A chimeric antigen receptor (CAR) including 2. The chimeric antigen receptor of claim 1, wherein the transmembrane domain comprises any one transmembrane domain selected from the group consisting of the alpha (α), beta (β), or zeta (ζ) chain of a T-cell receptor (TCR), CD28, CD3 epsilon (ε), CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

3. The chimeric antigen receptor of claim 1, further comprising any one hinge domain selected from the group consisting of CD8, CD4, CD28 and CD7.

4. A construct comprising a gene encoding the chimeric antigen receptor of Claim 1.

5. The composition of claim 4, further comprising an SFFV promoter (spleen focus-forming virus promoter) operably linked to the gene.

6. A vector comprising the composition of claim 4 or 5.

7. The vector of claim 6, wherein the vector is any one selected from the group consisting of retrovirus, lentivirus, adenovirus, adeno-associated virus, and vaccinia virus.

8. An immune cell comprising a chimeric antigen receptor of any one of claims 1 to 3; a construct of claim 4 or 5; or a vector comprising said construct.

9. In claim 8, the immune cell is one or more selected from the group consisting of T cells, NK cells, NKT cells, and macrophages.

10. A pharmaceutical composition for treating cancer, comprising: a chimeric antigen receptor of any one of claims 1 to 3; a construct of claim 4 or 5; a vector comprising said construct; and an immune cell comprising said chimeric antigen receptor, said construct, or said vector.

11. A method for producing CAR-expressing immune cells comprising the step of introducing a gene encoding the chimeric antigen receptor of Claim 1 into an immune cell.

12. A method of manufacturing according to claim 11, wherein the step comprises introducing the construct of claim 4 or 5; or a vector containing said construct into an immune cell.

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