Anti-αvβ3 integrin chimeric antigen receptor (CAR) and use thereof

Abstract

The present invention relates to: an αvβ3 integrin-targeting chimeric antigen receptor (CAR); CAR-NK cells comprising same; and a pharmaceutical composition for preventing or treating cancer. The inventors found that the present invention can accurately target and thereby efficiently recognize and eliminate cancer cells overexpressing αvβ3 integrin. Therefore, the present invention provides improved efficacy compared to conventional therapies, and can be effectively utilized in cancer treatments using αvβ3 integrin-targeting CAR-NK cells.

Description

Anti-αvβ3 integrin chimeric antigen receptor (CAR) and its uses

[0001] One example of the present invention is α v The invention relates to a β3-integrin (Integrin) target chimeric antigen receptor; a nucleic acid encoding the same; a vector; a virus; a CAR-NK cell; a method for manufacturing a CAR-NK cell; and a pharmaceutical composition for the prevention or treatment of cancer.

[0002] Integrins are receptors present on the surface of cells that play an important role in regulating major physiological functions such as cell adhesion, migration, differentiation, and proliferation. These proteins have a heterodimer structure in which two subunits, α and β, are connected by non-covalent bonds, and 22 types of integrin families are formed through various combinations of these two subunits.

[0003] Integrins primarily interact with extracellular matrix (ECM) proteins, with fibronectin, collagen, laminin, vWF, and fibrinogen being representative ligands. Each integrin possesses binding specificity to a particular ligand, and some integrins exhibit the ability to bind to multiple ligands simultaneously.

[0004] Especially α v β3-integrin is known to be overexpressed in various types of cancer. This integrin shows high expression in diverse cancer types, including skin cancer, prostate cancer, breast cancer, cervical cancer, colorectal cancer, lung cancer, gallbladder cancer, pancreatic cancer, and gastric cancer. α v β3-integrin plays a role in increasing the malignancy of cancer by regulating the growth, survival, and invasion of tumor cells.

[0005] α v β3-integrin promotes tumor growth and metastasis through intracellular signaling and is also involved in the survival of adhesion-independent tumor cells. In addition, α vβ3 integrin is primarily expressed in neovascularization and is closely associated with angiogenesis. Given that neovascularization is one of the essential factors for early solid tumor growth, it serves as an important target in the tumor microenvironment for most solid tumors.

[0006] Based on these characteristics, α v When a single-domain antibody targeting β3-integrin is applied to CAR-NK cells, α v It is expected to exhibit high selectivity and anticancer efficacy in tumors with high expression of β3-integrin.

[0007] CAR (Chimeric Antigen Receptor) immunotherapy is an innovative immunotherapy method in which a patient's immune cells are genetically engineered to specifically recognize and attack cancer cells. This treatment is primarily being developed using T cells and NK cells, and is referred to as CAR-T and CAR-NK, respectively.

[0008] The basic structure of a CAR consists of an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain is primarily a single-chain fragment (scFv) of an antibody that recognizes specific cancer antigens. The transmembrane domain stably anchors the CAR to the cell membrane, while the intracellular domain transmits signals necessary for immune cell activation.

[0009] CAR-T cells have shown excellent efficacy primarily in the treatment of blood cancers. Currently, they are FDA-approved for the treatment of acute lymphoblastic leukemia and diffuse large B-cell lymphoma. However, CAR-T cells can cause serious side effects such as cytokine release syndrome and neurotoxicity, and show limited efficacy in the treatment of solid tumors.

[0010] To overcome these limitations of CAR-T, CAR-NK cell therapy is garnering attention. NK cells are part of the innate immune system and possess natural anticancer activity, allowing them to recognize and destroy cancer cells even without a CAR. Compared to CAR-T, CAR-NK cells have fewer side effects and can utilize allogeneic cells, giving them the potential to be developed as an "immediately available" treatment.

[0011] In the future, CAR immunotherapy is expected to be applied to more types of cancer, and improving efficacy, particularly in the treatment of solid tumors, remains a major research challenge.

[0012] Accordingly, the present invention comprises (a) an extracellular domain (Ecto domain); and (b) an intracellular domain (Endo domain), α v The purpose is to provide a β3-integrin target chimeric antigen receptor (CAR).

[0013] In addition, the present invention aims to provide a nucleic acid encoding the chimeric antigen receptor (CAR).

[0014] In addition, the present invention aims to provide an expression vector comprising the above nucleic acid.

[0015] In addition, the present invention aims to provide a virus comprising the above-mentioned expression vector.

[0016] In addition, the present invention aims to provide a CAR-NK cell that expresses the chimeric antigen receptor (CAR) on its surface.

[0017] In addition, the present invention aims to provide a method for manufacturing CAR-NK cells, comprising the step of introducing a nucleic acid encoding the chimeric antigen receptor (CAR) into NK cells.

[0018] In addition, the present invention aims to provide a pharmaceutical composition for cancer prevention or treatment comprising the above-mentioned CAR-NK cells.

[0019] In addition, the present invention aims to provide a method for preventing or treating cancer using the above-mentioned CAR-NK cells.

[0020] In addition, the present invention aims to provide a use for cancer prevention or treatment using the above-mentioned CAR-NK cells.

[0021] In order to achieve the above objective,

[0022] The present invention comprises (a) an extracellular domain (Ecto domain); and (b) an intracellular domain (Endo domain), α v Provides a β3-integrin-targeted chimeric antigen receptor (CAR).

[0023] In one embodiment of the present invention, the extracellular domain may be composed of an amino acid sequence of any one of SEQ ID NOs 1 to 3.

[0024] In another embodiment of the present invention, the intracellular domain may be CD28-CD3ζ or 41BB-CD3ζ.

[0025] In another embodiment of the present invention, the extracellular domain may be composed of the amino acid sequence of SEQ ID NO. 1, and the intracellular domain may be CD28-CD3ζ.

[0026] In addition, the present invention provides a nucleic acid encoding the chimeric antigen receptor (CAR).

[0027] In addition, the present invention provides an expression vector comprising the nucleic acid.

[0028] In addition, the present invention provides a virus comprising the above-mentioned expression vector.

[0029] In one embodiment of the present invention, the virus may be selected from the group consisting of retrovirus, adenovirus, adeno-associated virus (AAV), simian virus, vaccinia virus, Sendai virus, Epstein-Barr virus (EBV), and herpes simplex virus (HSV).

[0030] In addition, the present invention provides CAR-NK cells that express the chimeric antigen receptor (CAR) on their surface.

[0031] In one embodiment of the present invention, the NK cell may be an NK-92 or a stem cell-derived NK cell.

[0032] In addition, the present invention provides a method for manufacturing CAR-NK cells, comprising the step of introducing a nucleic acid encoding the chimeric antigen receptor (CAR) into NK cells.

[0033] In one embodiment of the present invention, the NK cell may be an NK-92 or a stem cell-derived NK cell.

[0034] In another embodiment of the present invention, the nucleic acid may be introduced into NK cells by a viral vector.

[0035] In another embodiment of the present invention, the virus vector may be selected from the group consisting of retrovirus vectors, adenovirus vectors, adeno-associated virus (AAV) vectors, simian virus vectors, vaccinia virus vectors, Sendai virus vectors, Epstein-Barr virus (EBV) vectors, and herpes simplex virus (HSV) vectors.

[0036] In addition, the present invention provides a pharmaceutical composition for cancer prevention or treatment comprising the above-mentioned CAR-NK cells.

[0037] In one embodiment of the present invention, the NK cell may be an NK-92 or a stem cell-derived NK cell.

[0038] In another embodiment of the present invention, the arm is αv It may be a cancer that overexpresses β3-integrin.

[0039] In another embodiment of the present invention, the cancer is α which is highly expressed not only in the cancer cells themselves but also in the neovascularization constituting the cancer tissue. v It may be a cancer that reflects β3-integrin.

[0040] In another embodiment of the present invention, the cancer may be one or more selected from the group consisting of breast cancer, uterine cancer, esophageal cancer, stomach cancer, brain cancer, rectal cancer, colorectal cancer, lung cancer, skin cancer, ovarian cancer, cervical cancer, blood cancer, pancreatic cancer, prostate cancer, testicular cancer, laryngeal cancer, oral cancer, head and neck cancer, thyroid cancer, liver cancer, bladder cancer, osteosarcoma, lymphoma, and leukemia.

[0041] In another embodiment of the present invention, the CAR-NK cells may have increased production of IFN-γ or TNF-α.

[0042] In addition, the present invention provides a method for preventing or treating cancer using the CAR-NK cells.

[0043] In addition, the present invention provides a use for cancer prevention or treatment using the above-mentioned CAR-NK cells.

[0044] The inventors α v β3-integrin (Integrin) targeted chimeric antigen receptor; and CAR-NK cells containing the same α v It was confirmed that cancer cells overexpressing β3-integrin can be accurately targeted, efficiently recognized, and eliminated. The CAR-NK cells of the present invention exhibit anticancer activity against various cancer cells, such as solid tumors and hematological cancers, and activate an immune response and induce cytotoxic effects through increased production of IFNγ and TNF-α. In particular, α v It acts efficiently on cancer cells that overexpress β3-integrin. Accordingly, the present invention can be expected to provide improved efficacy compared to existing treatments.

[0045] The effects of the present invention are not limited to those mentioned above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description or claims.

[0046] Figure 1 shows a schematic diagram of an experiment conducted to implement the present invention.

[0047] Figure 2 shows a schematic diagram of lentivirus production performed to implement the present invention.

[0048] Figure 3 is an image of Hek293FT cells expressing a lentivirus vector.

[0049] Figure 4 is α v This shows the cell sorting results of an infected NK-92 cell line containing a β3integrin-targeted nanobody (or single-domain antibody) CAR.

[0050] Figure 5 shows the α of the NK-92 cell line. v This shows the results of confirming the expression efficiency of β3integrin nanobody CAR.

[0051] Figure 6 shows the α of the cancer cell line. v This shows the results of confirming β3integrin protein cell surface expression.

[0052] Figure 7 shows the results of confirming whether the ITGB3 gene is expressed in cancer cell lines.

[0053] Figure 8 is α v This shows the results of the H1975 cancer cell killing ability of β3integrin nanobody CAR NK cells.

[0054] Fig. 9 is α v This shows the bioluminescence (BLI) results according to the E / T (Effector:Target) ratio for β3integrin nanobody CAR NK cells against H1975 cancer cells.

[0055] Figure 10 shows α vThis shows the results of evaluating the H1975 cancer cell killing ability of β3integrin nanobody CAR NK cells through an LDH assay.

[0056] Fig. 11 shows α v This shows the results of the cancer cell killing ability of U87MG in β3integrin nanobody CAR NK cells.

[0057] Fig. 12 is α v This shows the results of the SKOV cancer cell killing ability of β3integrin nanobody CAR NK cells.

[0058] Fig. 13 shows α v This shows the results of the killing ability of β3integrin nanobody CAR NK cells on K562 cancer cells.

[0059] Figure 14 shows the results of verifying the target specificity of CAR-NK cells.

[0060] Figure 15 shows the results of measuring the amount of IFNγ produced by CAR-NK cells.

[0061] Figure 16 shows the results of measuring the amount of TNF-α produced by CAR-NK cells.

[0062] Figure 17 shows the antigen binding ability results of pSLCAR-Nb13-28z CAR-NK cells.

[0063] Fig. 18 is α v This shows the results of the MCF7 cancer cell killing ability of β3integrin nanobody CAR NK cells.

[0064] Fig. 19 is α v This shows the results of evaluating the cancer cell killing ability of β3integrin nanobody CAR-NK cells of various cancer types using an LDH assay.

[0065] Figure 20 shows cord blood-derived CD34 +The process of manufacturing CAR-NK cells by using hematopoietic stem cells as initial cells, followed by CAR lentivirus infection and differentiation into the NK lineage, is shown in chronological order.

[0066] Figure 21 shows cord blood-derived CD34 + This shows the results confirming that differentiation into NK cells occurred in cells differentiated from hematopoietic stem cells using CD56 expression as an indicator.

[0067] Figure 22 shows cord blood-derived CD34 + Hematopoietic stem cell-based α v This shows the results of evaluating the antitumor activity of β3integrin nanobody CAR-NK cells against SKOV3 cancer cells.

[0068] The present invention will be described in detail below.

[0069] The present invention comprises (a) an extracellular domain (Ecto domain); and (b) an intracellular domain (Endo domain); α v Provides a β3-integrin-targeted chimeric antigen receptor (CAR).

[0070] As used herein, the term "Chimeric Antigen Receptor (CAR)" refers to a synthetic protein expressed in immune cells through genetic engineering, comprising an extracellular domain capable of recognizing a specific antigen and an intracellular domain that induces intracellular signal transduction. The CAR of the present invention is α v It is designed to target β3-integrin and α-integrin overexpressed in cancer cells v It can be used to eliminate cancer cells by specifically recognizing β3-integrin and activating immune cells.

[0071] Terms used in this specification, "α vβ3-integrin is a receptor molecule present on the cell surface that acts when cells adhere to the extracellular matrix, such as fibronectin and collagen. It is a transmembrane glycoprotein composed of a heterodimer of two subunits, α and β, and the existence of 21 types of integrins has been identified to date. Among them, α v β3-integrin has been reported to play a very important role in maintaining the structure of the cardiovascular system and bone tissue.

[0072] As used herein, the term "extracellular domain" refers to a protein region located on the cell surface that interacts with the external environment and mediates intercellular signaling or the recognition of specific molecules. In the chimeric antigen receptor (CAR) of the present invention, the extracellular domain is α v It is designed to specifically recognize β3-integrin.

[0073] In one embodiment of the present invention, the extracellular domain may be composed of an amino acid sequence of any one of SEQ ID NOs 1 to 3.

[0074] As used in this specification, the term "endo domain" refers to a protein region located within the cell membrane that regulates intracellular signal transduction processes and activates or inhibits cell functions and responses. In the chimeric antigen receptor (CAR) of the present invention, the endo domain enhances the signal transduction ability of the CAR to induce the activation of immune cells.

[0075] In another embodiment of the present invention, the intracellular domain may be CD28-CD3ζ or 41BB-CD3ζ.

[0076] In another embodiment of the present invention, the extracellular domain may be composed of the amino acid sequence of SEQ ID NO. 1, and the intracellular domain may be CD28-CD3ζ.

[0077] The chimeric antigen receptor (CAR) of the present invention is α vVariants of the amino acid sequences listed in the sequence list may be included within a range capable of specifically recognizing β3-integrin. For example, the amino acid sequences of the present invention may exhibit 80% or more homology with the amino acid sequences of each corresponding sequence number, preferably 90%, and more preferably 95% or more homology. Additionally, changes may be made to the amino acid sequences of the antibody to improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletion, insertion, and / or substitution of amino acid sequence residues of the antibody.

[0078]

[0079] In addition, the present invention provides a nucleic acid encoding the chimeric antigen receptor (CAR).

[0080] As used herein, the term "nucleic acid" refers to a molecule that stores and transmits genetic information within an organism, including DNA (deoxyribonucleic acid) or RNA (ribonucleic acid). Nucleic acids are classified into two main types, each playing an important role in holding genetic information and providing instructions for protein synthesis. The present invention provides a nucleic acid molecule that can be used as a gene encoding a chimeric antigen receptor (CAR).

[0081] The concept of a nucleic acid molecule that can be used as a gene encoding the chimeric antigen receptor (CAR) of the present invention includes functional equivalents of the nucleic acid molecule constituting it, for example, variants in which some base sequences of the nucleic acid molecule have been modified by deletion, substitution, or insertion, but which can perform the same function as the nucleic acid molecule. Specifically, the said gene may include a base sequence having sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% with respect to the base sequence of the present invention. The "% of sequence homology" for a polynucleotide is determined by comparing two optimally arranged sequences with a comparison region, and a portion of the polynucleotide sequence in the comparison region may include additions or deletions (i.e., gaps) compared to the reference sequence (which does not include additions or deletions) for the optimal arrangement of the two sequences.

[0082]

[0083] In addition, the present invention provides an expression vector comprising the nucleic acid.

[0084] As used herein, the term "vector" refers to a composition of material comprising isolated nucleic acids that can be used to deliver the isolated nucleic acids into a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.

[0085] Accordingly, the term "vector" includes self-replicating plasmids or viruses. The term should also be understood to include non-plasmid and non-viral compounds that facilitate the delivery of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, etc.

[0086] According to a preferred embodiment of the present invention, a nucleic acid molecule encoding a chimeric antigen receptor (CAR) in the vector of the present invention is operatively linked to a promoter.

[0087] As used herein, the term "operationally coupled" means a functional coupling between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) and another nucleic acid sequence, thereby allowing the regulatory sequence to regulate the transcription and / or translation of the other nucleic acid sequence.

[0088]

[0089] In addition, the present invention provides a virus comprising the above-mentioned expression vector.

[0090] As used herein, the term "Virus" refers to a biological particle used to deliver or express genetic material into a host cell, and can be utilized in various biotechnological applications, such as gene therapy or vaccine development. The virus of the present invention comprises an expression vector and delivers a specific gene into a host cell to α v It was designed to express a β3-integrin specific chimeric antigen receptor (CAR).

[0091] In one embodiment of the present invention, the virus may include a retrovirus, and the retrovirus may include a lentivirus capable of gene transfer even in non-dividing cells. In addition, the virus may be selected from the group consisting of adenovirus, adeno-associated virus (AAV), simian virus, vaccinia virus, Sendai virus, Epstein-Barr virus (EBV), and herpes simplex virus (HSV), although not limited thereto.

[0092]

[0093] In addition, the present invention provides a CAR-NK cell that expresses the chimeric antigen receptor (CAR) on its surface.

[0094] The above terms, such as "chimeric antigen receptor," may be within the aforementioned range.

[0095] In one embodiment of the present invention, the NK cell may be an NK-92 or a stem cell-derived NK cell.

[0096] The above-mentioned NK-92 cells are a homogeneous cell line that is easy to culture and genetically manipulate, and α v It is useful as a model cell for verifying the expression, target specificity, and anticancer efficacy of β3-integrin-targeted CARs. In the embodiments of the present invention, α v In cancer cell lines with high expression of β3-integrin (H1975, U87MG, SKOV3, etc.), increased cancer cell killing ability and increased production of IFNγ and TNF-α by CAR-NK cells were observed, whereas in cell lines with low expression, no anticancer activity was observed, confirming that the CAR-NK cells of the present invention have selective anticancer activity dependent on target antigen expression.

[0097] In addition, the above stem cell-derived NK cells are cord blood-derived CD34 +It can be manufactured by introducing a CAR at the early progenitor cell stage, such as hematopoietic stem cells, and differentiating them into NK cells; in this case as well, CAR expression was stably maintained and reproducible cytotoxic activity against tumor cells was confirmed. Accordingly, the present invention applies α not only to NK-92 cell lines but also to cases where stem cell-derived NK cells are applied. v It includes the maintenance of functional expression and antitumor effects of the β3-integrin-targeted CAR.

[0098] As used herein, the term "CAR-immune cell" refers to an immune cell that expresses a chimeric antigen receptor (CAR) on its cell surface. By expressing the CAR, this immune cell recognizes a specific antigen and performs the function of inducing an immune response or targeting and eliminating cancer cells. CAR-immune cells play an important role in immunotherapy, and in the present invention, α v Provides immune cells expressing a CAR capable of specifically recognizing β3-integrin.

[0099] The term "CAR-NK" as used in this specification refers to an immune cell that recognizes a specific antigen and directly attacks cancer cells by expressing a chimeric antigen receptor (CAR) on natural killer cells (NK cells). Although CAR-NK cells recognize cancer cells similarly to CAR-T cells, NK cells generally have the characteristic of directly attacking cancer cells, so CAR-NK cells are attracting attention as a new approach in cancer treatment.

[0100]

[0101] In addition, the present invention provides a method for manufacturing CAR-NK cells, comprising the step of introducing a nucleic acid encoding the chimeric antigen receptor (CAR) into NK cells.

[0102] The above terms "chimeric antigen receptor," "CAR-NK," etc. may be within the aforementioned range.

[0103] As used herein, the term "introduction" refers to the process of delivering nucleic acids or genetic material into specific cells or tissues so that they can be expressed within the cells. This process is primarily required for gene therapy or genetic modification of immune cells. "Introduction" can be performed through various methods, and the present invention provides a method for introducing nucleic acids encoding a chimeric antigen receptor (CAR) into immune cells using a viral vector. This process causes the immune cells to express the CAR, thereby enabling the targeting of specific antigens and the elimination of cancer cells.

[0104] In one embodiment of the present invention, the NK cell may be an NK-92 or a stem cell-derived NK cell.

[0105] In another embodiment of the present invention, the nucleic acid may be introduced into NK cells by a viral vector.

[0106] In another embodiment of the present invention, the virus may include a retrovirus, and the retrovirus may include a lentivirus capable of gene transfer even in non-dividing cells. In addition, the virus may be selected from the group consisting of adenovirus, adeno-associated virus (AAV), simian virus, vaccinia virus, Sendai virus, Epstein-Barr virus (EBV), and herpes simplex virus (HSV), although not limited thereto.

[0107]

[0108] In addition, the present invention provides a pharmaceutical composition for cancer prevention or treatment comprising the above-mentioned CAR-NK cells.

[0109] The above terms "CAR-NK," etc., may be within the aforementioned scope.

[0110] As used herein, the term "cancer" refers to a class of diseases characterized by the occurrence of abnormal cells that proliferate uncontrollably and have the ability to infiltrate and destroy normal body tissues.

[0111] As used in this specification, the term "prevention" may mean any act of suppressing cancer in an individual or delaying its onset by administering a pharmaceutical composition according to one aspect.

[0112] As used herein, the term "treatment" may mean any act in which the symptoms of cancer in an individual are improved or beneficially altered by the administration of a pharmaceutical composition according to one aspect.

[0113] In one embodiment of the present invention, the NK cell may be an NK-92 or a stem cell-derived NK cell.

[0114] In another embodiment of the present invention, the arm is α v It may be a cancer that overexpresses β3-integrin.

[0115] In another embodiment of the present invention, the cancer is α from the cancer cell itself. v Not only in cases where β3-integrin is overexpressed, but also in the neovascularization constituting cancer tissue, α v It may be a cancer that reflects the characteristic of relatively high expression of β3-integrin. In such a case, the above α v β3-integrin can contribute to target recognition by CAR-NK cells within the tumor microenvironment.

[0116] As used herein, the term “overexpression” refers to a state in which a specific gene or protein is increased relative to normal tissue or reference cells. That is, overexpression may manifest as an increase in the transcription level of a specific gene, a consequent increase in protein expression, or an increase in the expression density of cell surface proteins. Overexpression often occurs when cells respond to external stimuli or in disease states, and in particular, overexpression of specific proteins in cancer cells plays a significant role in the development, progression, and metastasis of cancer.

[0117] In an embodiment of the present invention, α on the surface of cancer cells v As a result of analyzing β3-integrin expression, in lung cancer cell line H460, α v While β3-integrin expression is relatively low, in H1975, U87MG, and SKOV3 cell lines, α v It was confirmed that β3-integrin was expressed at a relatively high level (Fig. 6). In addition, α v Analysis of ITGB3 gene expression, which is involved in the expression of the β3 subunit of β3-integrin, revealed that ITGB3 gene expression was higher in various cancer cells, including H1975, U87MG, and SKOV3 cells, compared to H460 cells (Fig. 7). From these results, α v It has been confirmed that whether β3-integrin is overexpressed can be used as a criterion for distinguishing cancers to which the CAR-NK cells of the present invention are applied.

[0118] In the present invention, α v It proposes a treatment method targeting cancers overexpressing β3-integrin, and α v Overexpression of β3-integrin serves as an important target for the targeting of corresponding cancer cells and the enhancement of immune responses using CAR-NK cells. Overexpressed α v β3-integrin is recognized by CAR-NK cells and can induce a specific immune response against cancer cells.

[0119] In another embodiment of the present invention, the cancer may be a solid tumor or a blood cancer, and more specifically, may be one or more selected from the group consisting of breast cancer, uterine cancer, esophageal cancer, stomach cancer, brain cancer, rectal cancer, colorectal cancer, lung cancer, skin cancer, ovarian cancer, cervical cancer, blood cancer, pancreatic cancer, prostate cancer, testicular cancer, laryngeal cancer, oral cancer, head and neck cancer, thyroid cancer, liver cancer, bladder cancer, osteosarcoma, lymphoma, and leukemia.

[0120] In another embodiment of the present invention, the CAR-NK cells may have increased production of IFN-γ or TNF-α.

[0121] As used herein, the term "IFN-γ (interferon gamma)" refers to a type of cytokine that plays an important role in the immune response and is primarily produced by T cells and NK cells. IFN-γ is known to activate signal transduction between immune cells and to exert antiviral and antitumor effects. In particular, when the production of IFN-γ increases when immune cells are activated, the immune response is more strongly promoted, thereby enhancing the function of immune cells targeting cancer cells.

[0122] As used herein, the term "TNF-α (tumor necrosis factor-alpha)" refers to a representative cytokine involved in immune and inflammatory responses, which is primarily produced by activated T cells, NK cells, and macrophages. TNF-α performs functions such as inducing apoptosis in target cells or regulating immune responses in the tumor microenvironment, and is known to play an important role in the induction and amplification of anti-tumor immune responses. In particular, when the production of TNF-α increases with the activation of immune cells, not only is the direct cytotoxic effect on tumor cells enhanced, but the activation and influx of other immune cells are also promoted, which can improve the overall anti-cancer immune response.

[0123] The above pharmaceutical composition may be provided as a pharmaceutical composition comprising an active ingredient alone or comprising one or more pharmaceutically acceptable excipients or diluents.

[0124] When the above pharmaceutical composition is formulated, it may be prepared using diluents or excipients such as commonly used lubricants, sweeteners, flavorings, emulsifiers, suspending agents, preservatives, fillers, volume expanders, binders, wetting agents, disintegrants, and surfactants. Solid dosage forms for oral administration may include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the above composition. In addition, lubricants such as magnesium stearate and talc may also be used in addition to simple excipients. Liquid formulations for oral administration include suspensions, oral liquids, emulsions, syrups, etc., and may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents like water and liquid paraffin. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. For non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. For suppository bases, witepsol, macrogol, tween 61, cacao oil, laurin oil, glycerogelatin, etc. may be used, and known diluents or excipients may be used when manufactured in the form of ophthalmic preparations.

[0125] In another embodiment of the present invention, the composition may be administered parenterally by one or more routes selected from the group consisting of oral administration or external application to the skin or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intra-arterial injection, intramedullary injection, intracardiac injection, intrathecal injection, transdermal injection, intranasal injection, intra-intestinal injection, local injection, sublingual injection, or intrathoracic injection.

[0126] The above pharmaceutical composition is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0127] The dosage varies depending on the patient's condition and weight, the severity of the disease, the form of the drug, the route of administration, and the time, but can be appropriately selected by a person skilled in the art.

[0128] The composition of the present invention may be administered alone, or may be administered in combination with other anticancer agents to enhance the efficacy of anticancer treatment or to alleviate side effects. Combined administration may be simultaneous, sequential, or alternating. Other therapeutic agents that can be used in combination may be selected according to the purpose of the present invention and the type of cancer to which it is applied, and may include, but are not limited to, (i) immune checkpoint inhibitors (anti-PD-1, anti-PD-L1, anti-CTLA-4, etc.), (ii) anticancer chemotherapy agents, (iii) targeted anticancer agents (e.g., EGFR, ALK, VEGF / VEGFR, HER2, BRAF / MEK, PARP, PI3K / AKT / mTOR pathway inhibitors, etc.), (iv) radiation therapy, (v) antibody therapeutic agents or antibody-drug conjugates (ADCs), (vi) anti-angiogenic agents, (vii) adjuvants or cytokines (e.g., IL-2, IL-15, IL-21, IFN-α, IFN-β, etc.) and / or cytokine modulators, (viii) tumor microenvironment modulators (e.g., TGF-β pathway inhibitors, etc.), (ix) oncolytic virus therapeutic agents, (x) cancer vaccines, etc.

[0129] As used herein, the terms “drug” or “drug formulation” are used broadly to refer to any prophylactic, therapeutic, or diagnostic agent, or any other substance suitable for introduction into biological tissue, including pharmaceutical excipients and substances for tattooing, cosmetics, and other uses. A drug may be a substance having biological activity. A drug formulation may include various forms, such as liquid solutions, gels, solid particles (e.g., microparticles, nanoparticles), or combinations thereof. A drug may include small molecules, large (i.e., macro)molecules, or combinations thereof. A drug may be selected from suitable proteins, peptides, and fragments thereof, which may be produced naturally, synthetically, or recombinantly.

[0130]

[0131] In addition, the present invention provides a method for preventing or treating cancer using the CAR-NK cells.

[0132] In addition, the present invention provides a use for cancer prevention or treatment using the above-mentioned CAR-NK cells.

[0133]

[0134] To aid in understanding the present invention, it will be explained in more detail below through examples. However, the following examples are merely illustrative for explaining the content of the present invention, and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the technical field to which the invention belongs.

[0135]

[0136] Examples

[0137] 1. Experimental Method

[0138] (1) α v CAR expression lentiviral vector for β3integrin and production of lentivirus

[0139] The process of producing the chimeric antigen receptor (CAR) of the present invention and the lentivirus expressing the CAR is shown in Figures 1 and 2.

[0140] The eight plasmid vectors used for lentivirus production are classified into two groups. The first is the negative control group, the Deletion Ecto Domain CAR NK group, characterized by the removal of the extracellular domain, while the intracellular domain consists of CD28-CD3ζ and 41BB-CD3ζ. The plasmids in this group were named pSLCAR-dEcto-28z and pSLCAR-dEcto-BBz. The second is Integrin α vThe β3CAR NK group contains clones 13, 22, and 25 (represented by the nucleotide sequences of SEQ ID NOs 4, 5, and 6, respectively) in the extracellular domain, and the intracellular domain consists of CD28-CD3ζ and 41BB-CD3ζ. The plasmids of this group were named pSLCAR-Nb13-28z, pSLCAR-Nb13-BBz, pSLCAR-Nb22-28z, pSLCAR-Nb22-BBz, pSLCAR-Nb25-28z, and pSLCAR-Nb25-BBz.

[0141] Additionally, to analyze CAR target specificity, pSLCAR-CD19-28z and pSLCAR-CD19-BBz (Addgene #135992, 135991) were purchased as controls and used in the experiment.

[0142] The inventors α v HEK293FT cell lines were used to construct CAR lentiviruses for each β3integrin clone. HEK293FT cells were cultured in Hyclone high glucose DMEM medium supplemented with 10% FBS, 1% P / S, 500 μg / ml G418, and 2 mM L-glutamine. Specific methods for the culture and maintenance of HEK293FT cell lines are presented in Table 1.

[0143] [Table 1] Culture and Maintenance of HEK293FT Cell Line

[0144]

[0145] To produce lentiviruses, four types of plasmids, including eight types of pSLCAR lentivirus plasmid vectors and packaging vectors (pLP / VSVG, pLP1, pLP2), were transformed into HEK293FT cell lines using Turbofect transformation reagent. Since the pSLCAR plasmid vector contains green fluorescent protein (GFP), gene expression after transformation was confirmed by fluorescence microscopy (Fig. 3).

[0146]

[0147] (2) Lentivirus generation

[0148] The lentivirus generation process is as follows. First, serum-free media was dispensed into ep tubes in amounts of 100-200 μl, and the packaging vector and target DNA were mixed in the ratio of PLP / VSVG (2.7 μg): PLP1 (4.5 μg): PLP2 (1.8 μg): target DNA (3 μg). Turbofect reagent was added at twice the amount of DNA (24 μl), and the reaction was carried out at room temperature for 15 minutes. The transformation mixture was drop-treated on an HEK293FT culture dish, and 10% FBS was added after 4 hours. The mixture was then incubated for 48 hours to confirm fluorescent protein expression under a microscope, and the supernatant was harvested.

[0149] 8-9 ml of the harvested virus supernatant was placed into 100 kD filter tubes (Amicon Ultra Ultracel 100K) and concentrated by centrifugation at 4000-4500 rpm for 20 minutes. The filtered medium in the lower layer of the filter tube was discarded, and the above process was repeated to concentrate the supernatant to approximately 50 times its original volume. The concentrated virus was collected in ep tubes and stored at -80°C.

[0150]

[0151] (3) Lentivirus titer measurement

[0152] The titer of the synthesized lentivirus was measured using the abm qPCR Lentivirus Titer Kit. 2 μl of the viral sample was mixed with 18 μl of Virus Lysis Buffer and reacted for 5 minutes, followed by centrifugation. A standard curve was generated by diluting the Standard Control DNA 10-fold four times. The qPCR reaction mixture consisted of 2X qPCR MasterMix (10 μl), Primer Mix (2 μl), Sample / Negative Control / Standard DNA (2 μl each), and Nuclease-Free H₂O (6 μl). The qPCR conditions were set to reverse transcription at 42°C for 20 minutes, enzyme activation at 95°C for 10 minutes, denaturation at 95°C for 15 seconds (34 cycles), and binding and extension at 62°C for 1 minute. Viral titers were calculated by generating a standard curve regression equation based on the Ct values.

[0153]

[0154] (4) Lentivirus infection of NK-92 cells

[0155] NK-92 cell lines were infected with the generated CAR lentivirus using TransDux reagent. Virus soup was infected with the MOI (Multiplicity of Infection) set to 10.

[0156] MOI was calculated as follows:

[0157] MOI = (Virus titer × Infection volume) / Number of target cells

[0158] Here, Virus titer is given in units of IU / ml (Infectious Units per milliliter), Infection volume refers to the volume (ml) used to infect with the virus, and Number of target cells refers to the number of cells to be infected.

[0159] In other words, the cells to be infected are 1×10 6 If the MOI is set to 10, 1×10 virus particles 7 The infection was carried out using IU.

[0160] GFP-positive cells were identified using a flow cytometry system (Fig. 4). Infected GFP-positive cells were separated into 96-well plates using a cell sorter, subcultured for 3 weeks to proliferate the cells, and cryopreserved for use in subsequent experiments.

[0161] After releasing and culturing the cryopreserved CAR-NK cells, GFP-expressing cells were identified through flow cytometry, and a CAR expression rate of approximately 80% was confirmed (Fig. 5).

[0162]

[0163] 2. Experimental Results

[0164] (1) α in cancer cells v Confirmation of β3Integrin expression

[0165] α of cancer cells v β3integrin expression was analyzed using flow cytometry (FACS). The antibodies used for the analysis were as follows: RB780 Mouse IgG1, κ Isotype Control (concentration: 0.2 mg / ml) was used as the control antibody, and RB780 Mouse Anti-Human CD51 / CD61 (concentration: 0.2 mg / ml) was used as the target antibody. CD51 / CD61 is α v As a marker representing β3integrin, α of cancer cells through this antibody v β3integrin expression was directly measured. An isotype control was used to confirm non-specific binding and control background signals, and both antibodies were used at the same concentration to enable accurate comparison of results.

[0166] As a result, the lung cancer cell line H460 is α v While β3integrin expression was low, high expression was confirmed in H1975, U87MG, and SKOV3 cell lines (Fig. 6). These expression patterns varied depending on the characteristics of each cell, and α v Higher anticancer effects can be expected in cell lines with high expression of β3integrin.

[0167] (2) ITGB3 gene expression analysis

[0168] α v The expression of the ITGB3 gene, which is involved in β3 integrin protein expression among β3 integrin heterodimers, was analyzed (Fig. 7). As a result, ITGB3 gene expression was higher in cells such as H1975, U87MG, and SKOV3 compared to H460 cells. In addition, ITGB3 gene expression was higher in T98G, K562, U138MG, MDA-MB-231, A549, N87, MCF7, PLC / PRF5, and HepG2 cells compared to H460 cells. This is α v This suggests that it can be a good target for NK cells expressing β3integrin CAR. Subsequently, the inventors selected cells with high ITGB3 gene expression that did not overlap with cancer types and analyzed the anticancer effect.

[0169] (3) Evaluation of the anticancer activity of CAR-NK cells in H1975 non-small cell lung cancer cells

[0170] α v To investigate the cancer cell killing ability of β3integrin CAR NK cells, bioluminescence imaging was performed after co-culturing with H1975-Efluc cells, which express a bioluminescent protein, on H1975 cancer cells.

[0171] As a result of co-culturing H1975 cancer cells and CAR-NK cells with an Effector:Target ratio (E / T ratio) set to 5:1 (Fig. 8), the highest anticancer activity was confirmed in pSLCAR-Nb13-28z CAR-NK cells. Furthermore, as the E / T ratio increased, the bioluminescence (BLI) levels analyzed in living cancer cells decreased, confirming the cancer cell killing effect (Fig. 9). In the Lactate Dehydrogenase (LDH) assay as well, pSLCAR-Nb13-28z CAR-NK cells demonstrated superior cancer cell killing ability compared to other CAR-NK cell groups under the same conditions (Fig. 10).

[0172] (4) Anticancer activity of CAR-NK cells in U87MG glioblastoma cells

[0173] In addition to the H1975 cell line, α v To investigate the cancer-killing ability of β3integrin CAR-NK cells, U87MG glioblastoma (GBM) cancer cells were co-cultured with CAR-NK cells using U87MG-Efluc cells, which express a bioluminescent protein, and bioluminescence imaging was performed. As a result (Fig. 11), it was confirmed that CAR-NK cells effectively killed U87MG cells. This indicates that CAR-NK cells exhibit effective anticancer activity against other solid tumor cells.

[0174] (5) Anticancer activity of CAR-NK cells in SKOV3 ovarian adenocarcinoma cells

[0175] Additionally, it was confirmed that ITGB3 gene expression was relatively high in SKVO3 cells. Accordingly, to confirm the anticancer activity of CAR-NK cells against SKVO3 cells, when they were co-cultured with SKVO3 cells, the bioluminescence (BLI) levels analyzed in living cancer cells decreased as the E / T ratio increased, confirming the cancer cell killing effect (Fig. 12). This suggests that CAR-NK cells exhibit effective anticancer efficacy even in ovarian adenocarcinoma cells.

[0176] (6) Anticancer activity of CAR-NK cells in K562 chronic myeloid leukemia cells

[0177] In addition to solid tumor cell lines, it was confirmed that ITGB3 gene expression was relatively high in K562 cells, a hematological cancer cell line. Accordingly, to confirm the anticancer activity of CAR-NK cells against K562 cells, when they were co-cultured with K562-Efluc-GFP cells, the bioluminescence (BLI) levels analyzed in living cancer cells decreased as the E / T ratio increased, confirming a cancer cell killing effect (Fig. 13). This suggests that CAR-NK cells exhibit effective anticancer efficacy in hematological cancer cells as well.

[0178] (7) Verification of target specificity and anticancer efficacy of CAR-NK cells

[0179] Targeting pSLCAR-Nb13-28z CAR-NK cells, which have the highest anticancer efficacy, α v The anticancer effect was investigated in cancer cells with different levels of β3integrin expression. In the H460 cell line, no anticancer activity was observed in any of the CAR-NK cell populations when co-cultured at an Effector:Target ratio of 5:1, so this result played an important role in confirming the target specificity of CAR-NK cells (Fig. 14).

[0180] (8) Measurement of IFNγ production in CAR-NK cells

[0181] To confirm the mechanism of anticancer efficacy of CAR-NK cells, the amount of IFNγ produced during co-culture was measured, and the results showed that α v Higher IFNγ production was confirmed in β3integrin clones 13 and 22 compared to the control NK-92 cell line when co-cultured with H1975 cancer cells (Fig. 15). Therefore, α v It was confirmed that CAR-NK cells expressing β3integrin nanobody clones 13 and 22 have a mechanism of exerting anticancer activity through cytotoxic action and immune activation by increasing the production of IFNγ in co-culture with cancer cells.

[0182] (9) Measurement of TNF-α production in CAR-NK cells

[0183] To confirm the mechanism of anticancer efficacy of CAR-NK cells, the amount of TNF-α produced during co-culture was measured, and the results showed that α v Higher TNF-α production was confirmed in β3integrin pSLCAR-Nb13-28z compared to the control NK-92 cell line and other experimental groups when co-cultured with H1975 or U87MG cancer cells (Fig. 16). Therefore, the α of pSLCAR-Nb13-28z v It was confirmed that CAR-NK cells expressing β3integrin nanobody have a mechanism of exerting anticancer activity through cytotoxic action and immune activation by increasing the production of TNF-α in co-culture with cancer cells.

[0184] (10) Analysis of antigen binding specificity of CAR-NK cells

[0185] To analyze antigen binding specificity, his tagged α v Analysis using β3integrin protein and anti-His-PE antibody (Fig. 17) confirmed that pSLCAR-Nb13-28z CAR-NK cells exhibited the highest binding ability to the antigen. This indicates that the corresponding CAR-NK cells possess high specificity α vIt demonstrates that it expresses β3integrin CAR.

[0186] (11) Anticancer activity of CAR-NK cells in MCF7 breast cancer cells

[0187] α v To determine whether the anticancer activity of CAR-NK cells expressing β3integrin nanobodies is effectively demonstrated in breast cancer cells, the cancer-killing ability of CAR-NK cells was evaluated using the MCF7-Luc breast cancer cell line expressing a luminescent protein. MCF7-Luc cells were used as target cells, and α was used as the effector cell. v CAR-NK cells containing β3integrin nanobody were used, and the ratio of NK cells to cancer cells (E:T ratio) was set to 5:1. The degree of cancer cell death was analyzed through a decrease in luminescence signal (Fig. 18).

[0188] As a result, α v Significant death of MCF7-Luc breast cancer cells was observed in CAR-NK cells containing Nb13, a nanobody specific to β3integrin. In particular, in Nb13 CAR-NK cells containing a CD28 co-stimulation domain, the luminescence signal was significantly reduced, confirming that a very high level of cancer cell killing effect was induced against MCF7 breast cancer cells.

[0189] (12) Evaluation of the cancer cell killing ability of CAR-NK cells targeting additional cancer types

[0190] α vTo determine whether the anticancer efficacy of CAR-NK cells expressing β3integrin nanobody is reproducibly observed in various solid tumors rather than being limited to specific cancer types, the cancer cell killing ability was evaluated on cell lines of liver cancer (HepG2, PLC / PRF / 5), breast cancer (MDA-MB-231, BT474), glioblastoma (U87MG), and gastric cancer (AGS). Cell lines that do not express luminescent proteins were used as cancer cells, and after setting the ratio of NK cells to cancer cells (E:T ratio) to 5:1, the degree of cancer cell lysis was quantitatively analyzed using an LDH assay (Fig. 19).

[0191] As a result, in the HepG2 cell line, the highest LDH release was observed in Nb13-containing CAR-NK cells, particularly those containing the CD28ζ co-stimulation domain, confirming a potent cancer cell killing effect. In U87MG glioblastoma as well, relatively high cytotoxicity was induced in Nb13-based CAR-NK cells, and α v It was confirmed that it exhibits selective killing ability in cancers with high expression of β3integrin. Meanwhile, although the overall killing efficiency was low in the MDA-MB-231 cell line, relatively high cancer cell lysis was observed in Nb13 CAR-NK cells, indicating that differences in efficacy were reflected in the level of target expression or target recognition specificity.

[0192] In addition, high cancer cell killing ability was confirmed in Nb13 CAR-28ζ and Nb22 CAR-BBζ cells in AGS gastric cancer cell lines, and in PLC / PRF / 5 liver cancer cell lines, Nb13 CAR-28ζ and Nb22 CAR-BBζ cells showed significantly higher cytotoxicity compared to the control group, confirming that the effect was CAR-dependent targeted killing rather than an effect caused by non-specific NK activity. In BT474 breast cancer cell lines, overall intermediate to high levels of cancer cell killing ability were observed in various CAR-NK cells, suggesting that the CAR-NK cells according to the present invention can be applied to cancer types with different target expression environments.

[0193] From the above results, it was confirmed that the CAR-NK cells according to the present invention exhibit cancer cell killing ability against various cancer cell lines, not limited to specific cancer cell lines, and that different efficacy characteristics can be shown depending on the type of cancer depending on the combination of the target recognition domain and the co-stimulation domain.

[0194] (13) Cord blood-derived CD34 + Preparation of αvβ3 integrin CAR-NK cells using cells as progenitor cells and evaluation of antitumor activity

[0195] The inventors additionally α v In the production of NK cells expressing a chimeric antigen receptor (CAR) targeting β3integrin, cord blood-derived CD34 + Hematopoietic stem cells were used as initiation cells. That is, CD34 cells were used as the starting cells for CAR introduction and NK cell differentiation. + We verified whether functionally effective CAR-NK cells could be produced even when extended to the hematopoietic stem cell stage.

[0196] 1) CD34 + Derived CAR-NK cell differentiation method

[0197] The inventors of the present invention used STEMCELL’s Human Cord Blood CD34+, Mixed Donors (cat#70008, Lot#2402529009) cord blood-derived CD34 + Hematopoietic stem and progenitor cells (HSPCs) were used as initiation cells. The above CD34 + The cells are hematopoietic cells in a very early stage collected immediately after birth, and they possess excellent differentiation and proliferation capabilities and are immunologically immature, giving them advantageous characteristics for the development of allogeneic cell therapies. Subsequently, a chimeric antigen receptor (CAR) was introduced using a lentiviral gene delivery method, and differentiation into CAR-expressing NK cells (CAR-NK) was induced.

[0198] CD34 + To induce CAR-NK cells from cells, CD34+ cells were induced into lymphoid progenitors using the StemSpan™ NK Cell Generation Kit (Catalog #09960) and then differentiated stepwise into NK cells (Fig. 21). Specifically, on Day 0, a non-tissue culture-treated 6-cell culture vessel was coated using the StemSpan™ Lymphoid Differentiation Coating Material, and then thawed umbilical cord blood-derived CD34 + Cells were seeded. At this time, cell viability was confirmed to be over 85% via Trypan Blue staining, and CD34 through flow cytometry +The cell ratio was confirmed to be 97.17%. PE Mouse IgG1, κ Isotype Control antibody (BD Bioscience, cat#555749) and Anti-Human CD34-PE antibody (Clone 8G12, BD Bioscience, cat#348057) were used for flow cytometry analysis. 2.5 × 10⁴ cells were placed in each well of a coated 6-cell cell culture dish. 4 34 CDs + Cells were seeded and cultured for 3 days at 37°C and 5% CO2.

[0199] On the 3rd day of culture (Day 3), the CD34 stabilized as described above + CAR gene introduction was performed on cells. Specifically, lentiviruses expressing nanobody-based CAR genes designed to recognize cancer cell target antigens were infected using System Biosciences' TransDux™ MAX Lentivirus Transduction Reagent (Cat# LV860A-1). The experimental groups consisted of a non-transfected control group, a non-targeted control group (dEcto-CD28-CAR), and a nanobody-targeted group (Nb#13-CD28-CAR). 2.5 × 10⁶ cells per well 4 10 CD34+ cells were resuspended in 1.6 mL of medium, 400 μL of MAX Enhancer, and 8 μL of TransDux™, followed by the addition of 16 μL of 1 M HEPES buffer and the lentivirus concentrate containing the CAR gene. Subsequently, centrifugation was performed at 1,500 × g for 99 minutes, after which medium was added, and the cells were incubated for 3 days at 37°C under 5% CO2 conditions. During this process, the CAR gene was CD34 + It was stably inserted into the cell genome so that it could be continuously expressed throughout the entire NK differentiation process. Figure 20 shows umbilical cord blood CD34 +This shows the series of processes in chronological order, from the cell acquisition stage to CAR lentivirus infection and differentiation into NK lineages.

[0200] From day 7 to day 13, the differentiation and proliferation of lymphoid progenitor cells were promoted by replacing half of the StemSpan™ Lymphoid Progenitor Expansion medium every three days. Subsequently, on day 14, lymphoid progenitor cells were harvested, and cell viability was confirmed via Trypan Blue staining, followed by 2.5 × 10 5 NK cell differentiation was initiated by seeding cells into non-adherent 6-cell culture dishes with StemSpan™ NK Cell Differentiation medium. From day 17 to day 28, maturation into NK cells was induced by replacing half of the NK differentiation medium at 3-day intervals.

[0201] On day 28, the finally differentiated cells were recovered, and the differentiation into NK cells was confirmed using CD56 expression as an indicator; as a result, umbilical cord blood-derived CD34 + It was confirmed that NK cells expressing CAR were successfully induced from cells (Fig. 21).

[0202] 2) CD34 + Confirmation of anticancer activity of derived CAR-NK cells

[0203] The above cord blood-derived CD34 + We confirmed whether hematopoietic stem cell-based CAR-NK cells exhibit anti-tumor activity against tumor cells (Fig. 22). Specifically, umbilical cord blood-derived CD34 + The above CD34 obtained by introducing CAR at the cell stage and differentiating into NK cells + Cytotoxic activity against tumor cells (SKOV3) was evaluated using derived CAR-NK cells. NK-92 cell lines were used as a control for comparing anticancer efficacy.

[0204] As a result, while the survival rate of tumor cells remained high in the group cultured with tumor cells alone and in the group treated with NK cells differentiated from non-transduced CD34+ cells, the CD34 expressing the CAR of the present invention + In the group treated with derived CAR-NK cells, tumor cell survival was significantly reduced. In addition, throughout the repeated experiments, the aforementioned CD34 + Consistently low tumor cell survival rates were observed in the CAR-NK cell-derived treatment group.

[0205] The above results are cord blood-derived CD34 + This demonstrates that CAR-NK cells produced by the method of the present invention, in which a CAR is introduced at the cellular level and then differentiated into NK cells, reproducibly exhibit cytotoxic activity. These results are related to the CD34 of the present invention. + This experimentally demonstrates that the cell-based CAR-NK manufacturing method is a useful manufacturing platform for the development of clinically applicable CAR-NK cell therapies.

[0206]

[0207] 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. (a) Extracellular domain (Ecto domain); and (b) Endo domain; α including v β3-Integrin Target Chimeric Antigen Receptor (CAR) 2. A chimeric antigen receptor according to claim 1, wherein the extracellular domain is composed of an amino acid sequence of any one of SEQ ID NOs 1 to 3.

3. A chimeric antigen receptor according to claim 1, characterized in that the intracellular domain is CD28-CD3ζ or 41BB-CD3ζ.

4. A chimeric antigen receptor according to claim 1, characterized in that the extracellular domain is composed of the amino acid sequence of SEQ ID NO. 1 and the intracellular domain is CD28-CD3ζ.

5. A nucleic acid encoding the chimeric antigen receptor (CAR) of any one of claims 1 to 4.

6. An expression vector comprising the nucleic acid of claim 5.

7. A virus comprising the expression vector of claim 6.

8. The virus of claim 7, wherein the virus is selected from the group consisting of retrovirus, adenovirus, adeno-associated virus (AAV), simian virus, vaccinia virus, Sendai virus, Epstein-Barr virus (EBV), and herpes simplex virus (HSV).

9. A CAR-NK cell expressing a chimeric antigen receptor (CAR) of any one of claims 1 to 4 on its surface.

10. A CAR-NK cell according to claim 9, characterized in that the NK cell is an NK-92 or stem cell-derived NK cell.

11. A method for producing CAR-NK cells comprising the step of introducing a nucleic acid encoding a chimeric antigen receptor (CAR) according to any one of claims 1 to 4 into NK cells.

12. A method of manufacturing according to claim 11, characterized in that the NK cells are NK-92 or stem cell-derived NK cells.

13. A method of manufacturing according to claim 11, wherein the nucleic acid is introduced into an NK cell by a viral vector.

14. A method of manufacturing according to claim 13, wherein the virus vector is selected from the group consisting of a retrovirus vector, an adenovirus vector, an adeno-associated virus (AAV) vector, a Simian virus vector, a vaccinia virus vector, a Sendai virus vector, an Epstein-Barr virus (EBV) vector, and a herpes simplex virus (HSV) vector.

15. A pharmaceutical composition for the prevention or treatment of cancer, comprising the CAR-NK cells of claim 9.

16. A pharmaceutical composition according to claim 15, characterized in that the NK cells are NK-92 or stem cell-derived NK cells.

17. In claim 15, the arm is α v A pharmaceutical composition characterized by being a cancer that overexpresses β3-integrin.

18. A pharmaceutical composition according to claim 15, wherein the cancer is one or more selected from the group consisting of breast cancer, uterine cancer, esophageal cancer, stomach cancer, brain cancer, rectal cancer, colorectal cancer, lung cancer, skin cancer, ovarian cancer, cervical cancer, blood cancer, pancreatic cancer, prostate cancer, testicular cancer, laryngeal cancer, oral cancer, head and neck cancer, thyroid cancer, liver cancer, bladder cancer, osteosarcoma, lymphoma, and leukemia.

19. A pharmaceutical composition according to claim 15, wherein the CAR-NK cells are characterized by an increased production of IFN-γ or TNF-α.

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