KR20210034280A - Nanoparticles including improved coatings and their uses - Google Patents

Abstract

The present invention relates to nanoparticles and NK cells for immunotherapy, and a pharmaceutical composition containing the same. By using nanoparticles according to an aspect, it is possible to express a gene by delivering the gene to immune cells, and by using NK cells into which the nanoparticles are introduced, an effect of effectively treating cancer and tracking cells without side effects of existing chimeric antigen receptor (CAR) T cells can be provided.

Description

Specially coated nanoparticles and their uses {Nanoparticles including improved coatings and their uses}

It relates to specially coated nanoparticles and their use.

Cancer is a disease that records the number 1 mortality rate among Koreans, and the need for anticancer drug development is steadily emerging.

Looking at the development process of anticancer drugs, there were chemical anticancer drugs that attack dividing cells using the characteristics of rapidly proliferating tumor cells, and target anticancer drugs that attack specific molecules or signaling systems of tumor cells, but there were several side effects. Immuno-cancer drugs that can minimize side effects have emerged using the innate immunity of.

Immuno-chemotherapy refers to cancer therapy that activates the body's immune system to fight cancer cells. Immune chemotherapy uses the immune system to attack only cancer cells, so it has fewer side effects than conventional chemotherapy, and has the advantage of obtaining a long-term anticancer effect because it uses the memory and adaptability of the immune system. As described above, immune chemotherapy, which overcomes the shortcomings of existing anticancer drugs, is in the spotlight as a new paradigm for cancer treatment, and Science magazine selected immunotherapy as a study of the year in 2013.

Anticancer drugs can be divided into antibody treatments targeting tumor antigens (Rituximab, etc.), immune checkpoint inhibitors (Immune checkpoint inhibitors, etc.) that reactivate immune cells, and immune cell therapies (Immune cell theraphy) that directly administer immune cells. (Oiseth et al, 2017).

Among the methods of immune chemotherapy, immune cell therapy refers to a therapeutic agent that is genetically engineered using immune cells in the body and then injected into the body again. Immuno-chemotherapy targets to strengthen cellular immunity. Early immunotherapy drugs were attempted to treat malignant melanoma patients using autologous T cells activated by in vitro culture. However, there was a limitation in that T cells without antigen specificity were not able to obtain a sufficient effect on malignant tumors, and to overcome this, a chimeric antigen receptor (CAR) was developed that combined an antibody recognition site with an activation domain in T cells. Became. However, chimeric antigen receptor T-cells (CAR-T cells) have disadvantages of complicated genetic manipulation and high treatment cost, and also have problems that cause cytokine release syndrome and various side effects due to the characteristics of T cells. . Therefore, in the present technical field, in order to overcome the above-described problems continuously, attempts have been made to inject a suicide gene into CAR-T cells or develop CAR-T cells that do not cause immunogenicity, but the effect is insufficient.

In addition, until now, gene transfer technology using viruses has been used as a method for expressing chimeric antigen receptor (CAR) in immune cells, but the above method has a problem of potential danger due to viruses. . Also, among immune cells, NK cells have a limitation in that it is difficult to transfer genes using viruses due to their characteristics. Conventional methods using nanoparticles also have limitations that are unsuitable for immune cells.

Accordingly, the present inventors used nanoparticles, not conventional virus-based methods, and aimed to overcome existing limitations by effectively delivering genes to immune cells through specially coated nanoparticles.

One aspect is a first layer comprising a coated caffeic acid (CA); Nanoparticles including a second layer including polydopamine (PDA) coated on the first layer are provided.

Another aspect provides a gene delivery system comprising the nanoparticles.

Another aspect provides an immune cell comprising nanoparticles and genes.

Another aspect provides an immune cell comprising the EGFR-CAR gene.

Another aspect is to provide a pharmaceutical composition for the treatment or prevention of cancer comprising immune cells as an active ingredient.

One aspect is a first layer comprising a coated caffeic acid (CA); Nanoparticles including a second layer including polydopamine (PDA) coated on the first layer are provided.

In one embodiment, the nanoparticles may include a third layer including a cationic layer on top of the second layer.

The term “nanoparticles” may mean particles having a size of 1 to 100 nm having a surface. “Multifunctional Nanoparticles (MF-NPs)” may refer to nanoparticles used in anticancer fields or delivering drugs. Multifunctional nanoparticles and nanoparticles may be used interchangeably. The structure of the nanoparticles may have a core and a surface as a center.

In one embodiment, the coating layer of the nanoparticles is an amine layer, a hydroxycinnamic acid layer, a polydopamine layer and a polyethylenimine (PEI) layer, a layer containing a fluorescent material, a cationic layer, It may be one or more selected from the group consisting of an anion layer.

In one embodiment, the coating layer may be an Oleylamine (OA) layer, a Caffeic acid (CA) layer, a Polydopamine (PDA) layer, or a Polyethylenimine (PEI) layer.

The term “coating” refers to changing the properties of the surface by covering the surface of the underlying material with a film of other components. Surface modification and surface coating can be mixed.

The term “caffeic acid” is a kind of hydroxynamic acid, which has a yellow color and may have a phenol group and an acrylic group. The caffeic acid may have a structure represented by the following formula (1).

[Formula 1]

The term “polydopamine” is a kind of dopamine polymer and may include the structure of Formula 2 below.

[Formula 2]

In one embodiment, the coated caffeic acid or polydopamine may include a derivative or salt of caffeic acid or polydopamine.

In a specific embodiment, the coating layer is an oleylamine (OA) layer, a caffeic acid (CA) layer, a polydopamine (PDA) layer, and a polyethylenimine (PEI) layer in that order. It may have been. For example, the upper portion of the PEI layer may be cationic.

In one embodiment, the surface of the nanoparticles may be additionally coated or modified, or may be combined with other genes, proteins, polypeptides, or drugs.

In one embodiment, the nanoparticles may have a fluorescently-label formed on the third layer.

The fluorescent label may be made of a fluorescent material. More specifically, the cation of the fourth layer may be a combination of a fluorescent material. The bond may mean a covalent bond or an ionic bond. The term “fluorescent material” may mean a material that emits fluorescence by irradiation of an ultraviolet irradiation device.

In one embodiment, the fluorescent material is Rhodamine DAPI, Ethidium bromide, Acridine orange, Propidium Iodide, or Cyaine 7 amine: Cy7) may be included. In certain embodiments, it may be Rhodamine or Cy7.

In one embodiment, the nanoparticles may be magnetic nanoparticles.

In one embodiment, the nanoparticles may be magnetic nanoparticles. More specifically, the core of the nanoparticles may include Zn or Fe. In one embodiment of the present invention, the nanoparticles may have magnetic properties due to the core layer. The magnetic nanoparticles may be prepared by thermal synthesis.

In one embodiment, the nanoparticles may be traceable or imageable using a device. More specifically, since the nanoparticles contain magnetic or fluorescent materials, tracking or imaging may be possible using a device. For example, the tracking or imaging may be performed using a magnetic resonance apparatus (MRI) or fluorescence optical imaging to enable imaging of the position and movement of nanoparticles after administration in vivo or in cells.

In one embodiment, the nanoparticles may include a gene delivery system or a drug on the surface.

In one embodiment, the gene delivery system may include a drug in addition to the surface.

The drug may be transported into the cell by nanoparticles. Types of the above drugs include anticancer agents, immune anticancer agents, signal transduction inhibitors, cell cycle inhibitors, epigenetic modulators, angiogenesis inhibitors, blood vessel destruction drugs, immunomodulators, immune promoters, immunosuppressants, antihistamines, nutrients, proliferation inhibitors, antibiotics, Anti-inflammatory agents, gene therapy agents, recombinant DNA products, recombinant RNA products, collagen, collagen derivatives, protein analogs, saccharides, saccharide derivatives, cell regeneration promoters, or combinations thereof may be included, but are not limited thereto.

In one embodiment, the drug may be an anticancer agent. In one embodiment, the drug may be an anticancer agent. In addition, the anticancer agent, immune anticancer agent, signal transduction inhibitor, epigenetic modulator, or angiogenesis inhibitor may be one that destroys cancer cells or inhibits proliferation.

Another aspect provides a gene delivery system comprising the nanoparticles.

The nanoparticles are as described above.

The term “gene carrier (Vector)” refers to a material that transfers a gene, and specifically, may mean a material that transports and helps to properly express a therapeutic gene in the process of gene therapy. It may be replicable and may include all genetic elements capable of transferring gene sequences between cells, and is also referred to as a vector. The gene delivery system may be a viral or non-viral delivery system. For example, it may be a nanoparticle, a plasmid, a phage, a transposon, a cosmid, a chromosome, a virus, or a virion.

In one embodiment, the gene delivery system may be non-viral. In one embodiment, it may be a nanoparticle.

In one embodiment, the nanoparticles may be the nanoparticles of claim 1. More specifically, the nanoparticles include a first layer containing caffeic acid (CA); It may include a second layer including polydopamine (PDA) coated on the first layer. For example, the nanoparticles may include a second layer including a cationic layer on the second layer. It may include three layers.

In one embodiment, the coating layer is an oleylamine (OA) layer as a first layer, a caffeic acid (CA) layer as a second layer, a polydopamine (PDA) layer as a third layer, and a fourth layer. It may be coated in the order of polyethylenimine (PEI) layers.

In one embodiment, the gene delivery system may include a CAR gene, and may be a gene that allows cells to express CAR. In one embodiment, the CAR gene may be an EGRF-CAR gene. In addition, the EGRF-CAR gene may include the sequence of SEQ ID NO: 1, and may be the second generation.

The term “Chimeric antigen receptor (CAR)” may refer to an artificial membrane-binding protein that induces immune cells (eg, T lymphocytes) against an antigen and kills cells representing the antigen by stimulating the immune cells. . The CAR may be structurally characterized by having both a structure of a T cell and an antigen binding site. More specifically, the structure of the CAR may include an extracellular domain that binds to an antigen on a cell, a transmembrane domain, and an intracellular signaling domain that transmits a primary activation signal to an immune cell, and/or a co-stimulatory domain. . The target domain is a structure that recognizes an antigen such as a cancer cell, and may be located in an extracellular domain. In addition, the signaling domain may include an Immunoreceptor Tyrosine-based Activation Motif (ITAM) sequence. In one embodiment, the extracellular domain of the CAR may be an antigen binding domain.

In addition, the CAR may be the second generation or the third generation. For example, it may be the second generation. The second generation may mean that a signaling site of a costimulatory receptor such as CD28 or 41BB (CD137) is added to CD3-zeta as a signaling domain.

The gene comprising the sequence of SEQ ID NO: 1 may include a sequence having similar chemistry, for example, about 70% or more, about 75% or more, about 80% or more, about the sequence of SEQ ID NO: 1 At least 85%, at least about 90%, at least about 92%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% homology.

In one embodiment, the gene delivery system may include a drug in addition to the surface.

In one embodiment, the gene delivery system may be one capable of being tracked or imaged using a device.

Another aspect is the step of coating the nanoparticles with caffeic acid; And it provides a method for producing a multifunctional nanoparticles comprising the step of coating with polydopamine on the top of the caffeic acid layer.

In one embodiment, the manufacturing method may further include coating the polydopamine layer with a cationic layer using an agent having a charge on the top of the polydopamine layer. In one embodiment, the agent having the charge may be one containing polyethylenimine (PEI).

In another embodiment, the manufacturing method may further include forming a fluorescently-label on the surface of the cation layer by using a fluorescent material.

The terms magnetic, nanoparticle, fluorescent material, coating and coating layer are the same as described above.

Another aspect provides an immune cell comprising the nanoparticles and genes.

Another aspect provides a pharmaceutical composition, cell therapy agent or preparation for the treatment or prevention of cancer comprising the immune cells, population of immune cells, or a culture medium thereof as an active ingredient.

Another aspect provides the use of the immune cells, cell populations thereof, or cultures thereof for use in the manufacture of cell therapy agents, pharmaceutical compositions or preparations.

Another aspect provides a method of treating a disease comprising administering the immune cell, a population of cells, or a culture thereof to an individual.

The terms nanoparticle and CAR gene are as described above.

The term "prevention" refers to any action that suppresses or delays the occurrence of cancer by administration of the composition according to the present invention.

The term “treatment” refers to, or includes the alleviation, inhibition or prevention of progression of a disease, disorder or condition, or one or more symptoms thereof, and the “active ingredient” or the term “pharmaceutically effective amount” refers to a disease, disorder or condition, or It may mean any amount of a composition used in the practice of the invention provided herein sufficient to alleviate, inhibit or prevent progression of one or more symptoms thereof.

In one embodiment, the immune cells may be NK cells or T cells.

The term “T cell” is a lymphocyte derived from the thymus or bone marrow, and is a type of immune lymphocyte. It may be primarily involved in cellular immunity. In one embodiment, the T cell may be a CAR T cell expressing a CAR gene.

The term “CAR T (Chimeric antigen receptor T cells) cells” may refer to cells that have been genetically engineered to produce artificial T cell receptors for immuno-cancer. More specifically, the CAR receptor may be expressed. The CAR T cells may be used for cancer treatment.

The term “NK cells” means natural killer cells. The cells are cytotoxic lymphocytes constituting the main component of the innate immune system, defined as large granular lymphocytes (LGL), and a third differentiated from B and T lymphocytes producing common lymphoid progenitors (CLPs). It may be what constitutes a cell. In addition, the cells may include natural killer cells, mature natural killer cells, and natural killer progenitor cells without additional modifications derived from any tissue source. The natural killer cells are activated in response to interferon or macrophage-derived cytokines, and the natural killer cells are labeled with "activating receptors" and "inhibitory receptors". Surface receptors. In addition, natural killer cells may be produced from hematopoietic cells from any source, for example, placental tissue, placental perfusate, umbilical cord blood, placental blood, peripheral blood, spleen, liver, and the like. For example, it may be produced from a hematopoietic stem or precursor.

In one embodiment, the natural killer cells may be activated natural killer cells. The activated natural killer cells may refer to cells in which cytotoxicity or natural immunomodulatory ability of natural killer cells is activated compared to a parent cell, for example, a hematopoietic cell or a natural killer progenitor cell. More specifically, it may be that the natural killer cells first mature in the liver or bone marrow and then undergo an activation step to change into cells that kill abnormal cells.

In addition, the natural killer cells may have a low risk of cytokine storms due to poor memory function and clonal express function compared to T cells.

The term “genetical modification” or “genetical engineering” includes artificially altering the composition or structure of a cell's genetic material.

In one embodiment, immune cells (eg, natural killer cells) may be genetically modified to improve target specificity and/or antigen specificity. More specifically, it may mean a genetically modified CAR NK cell.

The term “CAR-NK cell” may refer to a cell into which CAR is introduced into an NK cell. The CAR NK cells may be generated by introducing CARs into NK cells in order to overcome the disadvantages of existing CAR T cells. T cells have to go through a complex genetic manipulation process using the patient's own T cells, and it is an expensive personalized treatment worth hundreds of millions of dollars for one patient, and due to the characteristics of rapidly dividing when a target cell is encountered, Cytokine Storm (Cytokine Storm) storm, Cytokine release syndrome, CRS) and central nervous system edema have been reported as disadvantages. In addition, the characteristics of T cells that remain in the body for a long time as memory also have a disadvantage that the same side effects may occur at any time if they recur, so NK cells with relatively low memory and division functions were selected. When using NK cells, there may also be temporal and economic advantages compared to CAR T cells, which must use only the patient's own immune cells. According to an embodiment of the present invention, CAR NK cells may react with various antigens. The antigen may be a cancer cell.

In one embodiment, the CAR NK cells may have one or more co-stimulatory domains. More specifically, in a general tumor environment, the expression of co-stimulatory molecules of immune cells is suppressed and the function of NK cells is lowered, so the intracellular signaling domain of the factor capable of overcoming the immune suppression reaction is used as an antigen-binding domain. CAR can be prepared by fusion with. When all other conditions are met and the CAR is expressed on the surface of, for example, a T lymphocyte, e.g., a primary T lymphocyte, and the extracellular domain of the CAR binds to the antigen, the extracellular signaling domain is a T lymphocyte. It transmits a signal to and activates and/or proliferates, and when the antigen is present on the cell surface, it kills the cell expressing the antigen. Some immune cells, such as T lymphocytes and natural killer cells, require two signals for maximum activation: a primary activation signal and a costimulatory signal, and CARs also require the binding of an antigen to an extracellular domain to be the primary activation. A costimulatory domain may be included to cause transmission of both a signal and a costimulatory signal.

In a specific embodiment, the NK cells may be NK92MI.

As used herein, the terms "administering," "introducing" and "implanting" are used interchangeably and into a subject by a method or route that results in at least partial localization of a composition according to one embodiment to a desired site. It may mean the arrangement of the composition according to one embodiment. It can be administered by any suitable route that delivers at least a portion of the cells or cellular components of the composition according to one embodiment to a desired location within a surviving individual. The survival period of the cells after administration to a subject may be several hours if short, for example from 24 hours to several days to several years if long.

The term "isolated cell", such as "isolated immune cell" or the like, may mean a cell that is substantially separated from a tissue from which the cell originates.

The composition of the present invention can be used in a method of treating or recognizing a tumor or cancer derived from a neoplasm. Neoplasms can be malignant or benign, cancers can be primary or metastatic; Neoplasms or cancers can be early or late. Non-limiting examples of neoplasms or cancers that can be treated include acute lymphoblastic leukemia, acute myelogenous leukemia, adrenal cortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma (pediatric cerebellar or cerebral ), basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brainstem gliomas, brain tumors (cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ventricular cell tumor, medulloblastoma, suppository primitive neuroectodermal tumor, visual pathway and hypothalamic glioma), Breast cancer, bronchial adenoma/carsinoid, Burkitt lymphoma, carcinoid tumor (pediatric, gastrointestinal), unknown primary carcinoma, central nervous system lymphoma (primary), cerebellar astrocyte tumor, cerebral astrocyte/malignant glioma, cervical cancer, Pediatric cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, colon cancer, cutaneous T-cell lymphoma, fibroblastic small round cell tumor, endometrial cancer, ventricular cell tumor, esophageal cancer, Ewing sarcoma with Ewing tumor, extracranial reproduction Cell tumors (infants), extratesticular germ cell tumors, extrahepatic bile duct cancer, eye cancer (intraocular blackoma, retinoblastoma), gallbladder cancer, gastric (gastric) cancer, gastrointestinal carcinoid tumor, gastrointestinal interstitial tumor, germ cell tumor (child Extracranial, extra-testicular, ovary), pregnant chorionic tumor, gliomas (adult, pediatric brainstem, pediatric cerebral astrocyte tumor, pediatric visual pathway and hypothalamus), gastric carcinoid, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer , Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway gliomas (infants), intraocular blackoma, islet cell carcinoma, Kaposi's sarcoma, kidney cancer (renal cell carcinoma), laryngeal cancer, leukemia (acute lymphoblasts, acute myeloid, chronic lymphocytes) , Chronic myeloid, hairy cell), lip and oral cancer, liver cancer (primary), lung cancer (non-small cell, small cell), lymphoma (AIDS-related, Burkitt, skin T-cell, Hodgkin, non-Hodgkin, Primary central nervous system), macroglobulinemia (Waldenstrom), malignant fibrous histiocytoma/osteosarcoma of bone, medulloblastoma (pediatric), black tumor, intraocular black tumor, Merkel cell Carcinoma, mesothelioma (adult malignancies, children), potential primary metastatic squamous cell carcinoma of the head and neck, oral cancer, multiple endocrine tumor syndrome (child), multiple myeloma/plasma cell neoplasm, mycosis myeloma, myelodysplastic syndrome, myelodysplasia/ Myeloproliferative disease, myelogenous leukemia (chronic), myelogenous leukemia (adult acute, childhood acute), multiple myeloma, myeloproliferative disorder (chronic), nasal and sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, non-small cell Lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma/malignant fibrous histiocytoma of the bone, ovarian cancer, ovarian epithelial cancer (superficial epithelial-stromal tumor), ovarian germ cell tumor, ovarian hypomalignant latent tumor, pancreatic cancer , Pancreatic cancer (islet cell), sinus and nasal cancer, parathyroid gland cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal seed cell tumor, pineal stem cell tumor, and primitive neuroectodermal tumor (pediatric), pituitary adenoma, Plasma cell neoplasm, pleural pulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureter transition cell carcinoma, retinoblastoma, rhabdomyosarcoma (pediatric), salivary gland cancer, sarcoma (Ewing and Tumor, Kaposi, soft tissue, fallopian tube), Sezary syndrome, skin cancer (non-black and black tumor), skin carcinoma (Merkel cell), small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, potential primary (metastatic) head and neck Squamous cell carcinoma, gastric cancer, primitive neuroectodermal tumor (child), T-cell lymphoma (skin), testicular cancer, throat cancer, thymoma (child), thymoma and thymic carcinoma, goiter carcinoma, thyroid carcinoma (child), renal pelvis and ureter transition Cell carcinoma, villous tumor (pregnant), unknown primary site (adult, child), ureter and renal pelvic transition cell carcinoma, urethral cancer, fallopian tube cancer (endometrial), fallopian tube sarcoma, vaginal cancer, visual pathway and hypothalamus glioma (child), Vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor (infant).

The term “blood cancer” is a cancer that affects the blood, bone marrow and lymphatic system. There are three main groups of hematologic cancers: leukemia, lymphoma and myeloma. The four broad categories of leukemia are: acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myelogenous leukemia (CML). Lymphomas fall into two categories: Hodgkin's lymphoma and non-Hodgkin's lymphoma. Most non-Hodgkin's lymphomas are B-cell lymphomas and grow rapidly (high-grade) or slowly (low-grade). There are 14 types of B-cell non-Hodgkin lymphomas. The rest are different cancer leukocytes, or T-cell lymphomas named after lymphocytes. Because myeloma occurs frequently in many areas of the bone marrow, it is often referred to as multiple myeloma.

In one embodiment, the cancer is breast cancer, thyroid cancer, gastric cancer, colon cancer, lung cancer, liver cancer, prostate cancer, pancreatic cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin's lymphoma, oral cancer, cleft lip cancer, testicular cancer, acute myeloid leukemia, basal cell cancer. , Ovarian epithelial cancer, brain tumor, multiple myeloma, blood cancer, chronic myelogenous leukemia, chronic lymphocytic leukemia, bladder cancer, peritoneal cancer, tongue cancer, non-small cell lung cancer, small cell lung cancer, small intestine cancer, esophageal cancer, kidney cancer, heart cancer, malignant lymphoma, urethral cancer , It may be one or more selected from the group consisting of cervical cancer, rectal cancer, tonsil cancer and laryngeal cancer.

In one embodiment, the method of administering the pharmaceutical composition is not particularly limited, but may be administered parenterally or orally, such as intravenous, subcutaneous, intraperitoneal, inhalation or topical application, depending on the intended method. The dosage range varies depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of disease. The daily dosage refers to an amount of a therapeutic substance according to an aspect sufficient for treatment of a disease state alleviated by being administered to an individual in need of treatment. The effective amount of a therapeutic substance depends on the particular compound, the disease state and its severity, the individual in need of treatment, which can be determined routinely by a person skilled in the art. As a non-limiting example, the dosage of the composition according to an aspect to the human body may vary depending on the age, weight, sex, dosage form, health condition, and degree of disease of the patient. Based on an adult patient weighing 70 kg, for example, about 1,000 to 10,000 cells/time, 1,000 to 100,000 cells/time, 1,000 to 100,000 cells/time, 1,000 to 10,000,000, 1,000 to 100,000,000 cells/time, 1,000~1,000,000,000 cells/time, 1,000~10,000,000,000 cells/time, may be dividedly administered once or several times a day at regular time intervals, or several times at regular time intervals.

The term'individual' refers to a subject in need of treatment of a disease, and more specifically refers to mammals such as human or non-human primates, mice, rats, dogs, cats, horses and cattle. do.

The pharmaceutical composition according to an embodiment may include a pharmaceutically acceptable carrier and/or additive. For example, sterile water, physiological saline, conventional buffers (phosphoric acid, citric acid, other organic acids, etc.), stabilizers, salts, antioxidants (ascorbic acid, etc.), surfactants, suspending agents, isotonic agents, or preservatives, etc. can do. For topical administration, organic substances such as biopolymers, inorganic substances such as hydroxyapatite, specifically collagen matrix, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers, and chemical derivatives thereof, etc. may be included. I can. When the pharmaceutical composition according to an embodiment is prepared in a dosage form suitable for injection, the immune cells, immune cells, or substances that increase the activity thereof are dissolved in or dissolved in a pharmaceutically acceptable carrier. It may be frozen.

The pharmaceutical composition according to an embodiment, if necessary depending on the administration method or formulation, is a suspension agent, a solubilizer, a stabilizer, an isotonic agent, a preservative, an adsorption inhibitor, a surfactant, a diluent, an excipient, a pH adjuster, a painless agent, Buffering agents, reducing agents, antioxidants, and the like may be appropriately included. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, 19th ed., 1995. The pharmaceutical composition according to an embodiment is formulated in a unit dosage form by using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the technical field to which the present invention pertains. It may be made of, or may be prepared by placing it in a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of powder, granules, tablets, or capsules.

In one embodiment, the gene may be a CAR expression gene or a cancer therapy gene. More specifically, it may be an EFGR-CAR expression gene. In addition, the gene may be the second generation, and may include the sequence number of SEQ ID NO: 1.

In one embodiment, the immune cells may be traceable using a device because the core of the nanoparticles remain therein.

In addition, the core of the nanoparticles may be traceable using a device. More specifically, the tracking may be possible by magnetic or fluorescent materials of the nanoparticle core in the cell. In addition, the device may be an MRI or a fluorescence imaging device in an embodiment of the present invention.

In one embodiment, the cells may additionally contain a drug.

The drug is the same as described above.

Another aspect provides an immune cell comprising the EGFR-CAR gene.

Another aspect is to provide a pharmaceutical composition, cell therapy agent, or preparation for cancer treatment or prevention comprising immune cells, a cell population thereof, or a culture medium thereof as an active ingredient.

Another aspect provides the use of the immune cells, cell populations thereof, or cultures thereof for use in the manufacture of cell therapy agents, pharmaceutical compositions or preparations.

Another aspect provides a method of treating a disease comprising administering the NK cells, a cell population thereof, or a culture medium thereof to an individual.

The terms NK cell, gene, CAR, cancer, treatment, prophylaxis and immune cell are the same as described above.

In one embodiment, the immune cells may include NK cells or T cells.

In one embodiment, the gene may include the sequence of SEQ ID NO: 1.

Another aspect is to provide a method for producing an immune cell comprising the step of introducing the gene delivery system into an immune cell.

In one embodiment, the immune cells may be NK cells or T cells.

The terms nanoparticle, gene carrier, NK cell, and CAR are the same as described above.

The method may include preparing the gene delivery system. Specifically, it may be to go through the step of attaching a gene to be transferred to the surface of the nanoparticles. Attachment may be by using a known genetic engineering method. The gene may be DNA, RNA, siRNA, miRNA, dsRNA, or iRNA, and specifically, may be plasmid RNA. The attached nanoparticles are a gene delivery system, and the gene delivery system may be introduced into a prepared NK cell. The introduction may be performed using a known genetic engineering method, and in one embodiment, NK cells and the nanoparticles may be cultured together. During the culture process, the intracellular insertion process may be performed due to the cationic nature of the surface of NK cells. The introduced nanoparticles can be degraded by intracellular digestion processes that are inherently present in the cell. The introduced nanoparticles may include introducing a gene into a cell, and expressing the introduced gene. In one embodiment, the intracellular digestion process may not be able to remove the core of the nanoparticles. Core particles remaining in the cell contain magnetic or fluorescent materials, and may be imaged or traceable by a device. For example, the device may be based on MRI or fluorescence visual images.

In one embodiment, the gene delivery system may include a chimeric antigen receptor (CAR) expression gene. More specifically, it may be an EGFR:CAR gene. In addition, when introduced into the CAR gene cells, CAR receptors may be expressed on the surface of NK cells, thereby generating CAR NK cells.

According to the nanoparticles and gene delivery system according to one aspect, it is possible to express genes in immune cells with high efficiency, and according to the immune cells according to one aspect and a pharmaceutical composition containing the same, when administered to an individual, tracking is possible, and various There is an effect that can be used to effectively treat cancer.

1 is a schematic diagram of an experiment showing the steps of introducing nanoparticles into NK cells in this experiment and the steps of tracking when injected into an individual.
Figure 2a is a schematic showing a method for producing a multifunctional nanoparticles.
2B is an image showing the chemical structure of the surface of nanoparticles in the manufacturing method.
3 is a graph of the ex vivo cellular compatibility of multifunctional nanoparticles in NK-92MI cells; a is a graph showing cytotoxicity by multifunctional nanoparticles in NK-92MI cells through MTT analysis, and b is a graph showing apoptosis of a single cell or a group of cells for 48 hours at the concentration of nanoparticles (x-axis) , c is a graph showing the phenotypic change for 48 hours between the nanoparticle-treated NK cells and the control group, and d is a graph showing the flow cytometric analysis for 48 hours in the NK cells treated with 16 ug/ml nanoparticles (*p < 0.05, and ****p <0.0001).
Figure 4a is an image showing the structure of the second generation CAR: EGFR. b is a graph showing the expression of EGFR-CAR in 293T cells measured by quantitative RT-PCR, c is a graph showing CAR expression on the surface of NK-92MI cells measured by flow cytometry, d is CFSE/7AAD A graph of EGFR-CAR expression analyzed by staining and the ability of NK-92MI to kill cancer cells, e is a graph showing quantitative analysis of c and d data, and f is a cancer cell MDA-MB-231 xenograft mouse model It is a graph showing the anti-tumor effect of EGFR-CAR-expressing NK-92MI cells against; The left is the biofluorescence imaging image, the right is the measurement of the tumor size in mice, and the black arrows indicate the injection of NK cells through the tail vein at 7. 10. and 13 days.
5A is an image of magnetic nanoparticles, CA-coated magnetic nanoparticles, CA, PDA-coated magnetic nanoparticles, PEI, CA, PDA-coated magnetic nanoparticles observed with a TEM microscope from the left.
5B is a graph analyzing the sizes of magnetic nanoparticles, CA-coated magnetic nanoparticles, CA, PDA-coated magnetic nanoparticles, PEI, CA, and PDA-coated magnetic nanoparticles from the left.
Figures 5c and d show thermal gravimetric analysis (TGA) and Fourier transform infrared spectroscopy (FT-IR) of CA-coated magnetic nanoparticles, CA, PDA-coated magnetic nanoparticles, PEI, CA, PDA-coated magnetic nanoparticles. It is a graph.
5E is a graph analyzing the zeta potential of CA-coated magnetic nanoparticles, CA, PDA-coated magnetic nanoparticles, PEI, CA, PDA-coated magnetic nanoparticles.
6 is a graph of the in vitro gene transfer ability of nanoparticles observed in 293T cells and NK-92MI cells; 6a is a graph of electrophoresis of each concentration of nanoparticle-plysmid DNA on a 1% agarose gel, 6b is a fluorescence image of 293T cells transfected with a nanoparticle-qGFP complex for 24 hours, and 6c is a nanoparticle-qGFP complex. This is a fluorescence image of time-transfected NK-92MI cells.
7 is a graph showing in vivo imaging of nanoparticle-treated immune cells; 7a is an in vitro fluorescence image of nanoparticle-treated NK-92MI cells, 7b is an optical imaging image of a mouse transplanted with fluorescently labeled nanoparticle-treated NK-92MI cells, and 7c is an in vitro nanoparticle-treated NK-92MI cell. The magnetic resonance image of, 7d is a magnetic resonance image of NK-92MI cells treated with nanoparticles injected into the living body of a mouse; The yellow dotted line and white arrow indicate the implantation site of the nanoparticle-treated cells.
8 is a graph evaluating the degree of glucose uptake in NK-92MI cells for toxicity of nanoparticles; The glucose concentration was obtained by incubating 5 x 10 5 cells in 1 mL of fresh medium for 24 hours, and then harvesting the supernatant and analyzing the glucose concentration.
9 is an image observed with a bio-TEM microscope of natural NK cells and NK cells treated with a pDNA/MF-NP complex.
FIG. 10 is a graph showing NK-92MI single cells or a population of cells treated with MF-NP/pEGFR-CAR for 48 hours, and then CAR expression was analyzed by quantitative RT-PCR.
Figure 11 is a graph of the nanoparticles to which the CAR gene is attached to NK NK cells and cultured with NK NK cells 24 hours after the cells are measured by FACS; From the left, it is the treatment conditions of the untreated control group, 0, 2, 4, 8ul/ml of nanoparticles.
12A is an image of T2-mapping at various iron concentrations.
12B is a graph showing the R2 values in CA/MNP, MF-NP, and MF-NP/pEGFR-CAR at various iron concentrations.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

FIG. 1 is an image showing a schematic diagram of an experiment. In Example 1.2, specially coated nanoparticles were prepared, and in Example 2, NK cells were transduced using a genetic engineering method, and the effects thereof were examined. Next, NK cells treated with nanoparticles in Example 3 were injected into mice to confirm MR and fluorescence image imaging.

Example 1. Preparation of materials and animal models

1.1 Preparation of the compound or where to buy it

Iron (III) acetylacetonate, oleylamine, caffeic acid, and Tris base were purchased from Tokyo Chemical Industry, and zinc chloride, oleic acid, trioctylamine, tetrahydrofuran (THF), dopamine hydrochloride, rhodamine B isothiocyanate (RITC) and polyethyleneimine (PEI; Mw 25 kDa) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Toluene, ethanol, DMSO, and NaOH were purchased from Daejung Chemical & Metals Co., Ltd (Gyeonggi-do, Korea). The nanoparticles of the present invention were synthesized by a known thermal decomposition method (a specific synthesis method is described), and PEI labeled with a fluorescent molecule (RITC or Cy7-NHS) was also synthesized according to a known method. Specifically, RITC or Cy7-NHS (1.4 mg) was reacted with PEI (0.5g) in DMSO for 12 hours at room temperature, and purified by dialysis for 48 hours with distilled water at room temperature. Thereafter, it was freeze-dried and stored in a freezer (-20 °C).

1.2 Preparation of NK cells

NK cells were NK-92MI cells, and NK-92MI cells were purchased from ATCC (American Type Culture Collection). Next, the purchased cells were placed in MEM-a (Minimum Essential Medium-alpha) with fetal bovine serum 12.5%, penicillin/streptomycin 1%, 2mM L-glutamine, 1.5g/L sodium bicarbonate, 0.2mM inositol, 0.1mM 2-mercaptoethanol. And 0.02mM folic acid was added and cultured. Cells were maintained at a density of 2.5 X 10 5 cells/ml and passaged every 3-4 days.

1.3 Manufacturing method of multifunctional nanoparticles

MF-NPs were synthesized by a three-step process, as shown in FIG. 2A. Figure 2b shows the chemical formula of the nanoparticle surface in each step.

As a first step, magnetic nanoparticles coated with oleylamine (OA) are synthesized by thermal decomposition of nanoparticles containing Zn/Fe as a core. After synthesis, the structure was analyzed by HR-TEM. As a result, it was confirmed that Zn and Fe were synthesized in large quantities, and the ratios were 87% and 13%, respectively.

Next, the magnetic nanoparticles capped with oleylamine were dispersed into water in an organic solvent through a ligand exchange reaction with caffeic acid. More specifically, 50 mg of caffeic acid and 5 mg of magnetic nanoparticles were mixed and dissolved in a 6 mL tetrahydrofuran solution. Next, after reacting at 40° C. for 4 hours, 500 µl of 0.5 N NaOH was added to the solution in which the nanoparticles were dispersed, mixed, and then the nanoparticles dispersed in NaOH were purified using a magnet. After purification, the nanoparticles capped with caffeic acid were dispersed in 2 mL distilled water. It was confirmed that the diameter of the nanoparticles coated with CA additionally increased by about 35 nm than the nanoparticles coated with OA.

Next, in order to synthesize the polydopamine-coated nanoparticles, 0.25 mg of the nanoparticles capped with caffeic acid were dispersed in a Tris-HCl buffer. Next, 5 mg of dopamine hydrochloride was added, followed by stirring at room temperature for 3 hours. Nanoparticles coated with polydopamine (PDA) were purified using a centrifuge three times at 12,000 rpm for 10 minutes.

Finally, polydopamine-coated nanoparticles were dispersed in 2 mL distilled water. Polyethyleneimine or fluorescently labeled polyethyleneimine 40 mg is dissolved in 8 mL of distilled water, and polydopamine-coated nanoparticles are added to the polyethyleneimine solution and stirred at room temperature for 12 hours to cationic or fluorescent. The labeled PEI was immobilized on the PDA layer. Finally, the polyethyleneimine-coated nanoparticles were purified using a centrifuge three times at 12,000 rpm for 10 minutes.

As a result of TGA analysis of the nanoparticles prepared by the above method, as shown in by 1, the composition ratio of each composition was 34%, 70%, and 83% for MNP and PEI/PDA/CA/MNP, respectively.

In addition, Fourier transform infrared spectroscopy was performed to confirm the chemical change of the surface after surface modification. As a result, specific characteristic peaks of CA, PDA, and PEI were detected in each nanoparticle sample. In addition, it was confirmed that CA/MNPs and PDA/CA/MNPs were negatively charged to -31 to -21, whereas PEI/PDA/CA/MNPs had a cationic property of +35mV. This means that the cationic PEI was successfully immobilized on the surface.

1.1 Preparation of the animal model

As an animal model, female mice were used, and NOD/Shi-scid/IL-2Rgnull purchased from Coretech was used. Tumors were generated by injecting 1 X 106 MDA-MB-231 GFP-Luc cells into a 6-week-old female mouse, and NK-92MI or EGFR-CAR- after 7, 10, and 13 days in tumor-generated mice. NK-92MI cells were injected by tail vein injection. Tumor size was measured using the Pearl Impurse bioluminescence system. The formula used is as follows: 0.5236 X A X B2 (A: major axis, B: major axis repair).

1.2 Preparation of EGFR CAR plasmid

The single chain fragment variable (scFv) DNA sequence of the EGFR CAR plasmid was obtained from a monoclonal antibody that binds to EGFR, and the CD8 hinge domain, CD28 transmembrane/endodomain, and CD3zeta domain were combined after the heavy chain and light chain of scFv. This DNA sequence was cloned into pcDNA3.1 vector and this structure is shown in FIG . 4.

Example 2. NK cell introduction of magnetic multilayer structure multifunctional nanoparticles and confirmation of the effect thereof

2.1 Confirmation of the possibility of use as a transfection tool by investigating the effect of nanoparticles on NK cell activity

It is known in the art that genetic manipulation of NK cells is difficult, and it has been confirmed that conventional viral vectors and transfection tools such as electroporation are not suitable for NK. Therefore, in order to confirm whether the nanoparticles produced in Example 1.2 is an effective transfection tool for NK cells, cytotoxicity was first evaluated by MTT analysis (data not shown). NK cells were used as NK-92MI cells. When the cells were treated with various concentrations of nanoparticles, the cell viability exceeded 90% at a concentration of 32 μg/mL, but the cell viability was less than 60% at a concentration of 64 μg/mL. In addition, typical markers related to NK activity were not changed by nanoparticle treatment (FIG. 3D ).

In addition, as shown in FIG. 8, glucose uptake was disturbed at a concentration of 64 μg/mL, but glucose uptake was not disturbed in the case of NK-92MI cells at a concentration of 32 μg/mL. These results suggest that the appropriate concentration of nanoparticles does not affect NK cell activity.

2.2 Confirmation of the effect of nanoparticles inducing genetic material delivery and protein expression in vitro

As a first step for delivering plasmid DNA, the present inventors conducted an experiment to find the optimal ratio of nanoparticles and qDNA in a gel retardation assay. The present experimenters attached nanoparticles of various concentrations to the plasmid and then applied to agarose gel electroporation. Next, the nanoparticles were labeled with a fluorescent substance, RITC, and bound to qGFP, and then these particles were administered to 293T cells to confirm the qDNA delivery ability and the ability to induce exogenous proteins of the nanoparticles.

According to Example 2.1, nanoparticles were used at a concentration of 16 μg/mL. As a result, as shown in FIG. 6B, both RITC and GFP signals were detected in the nanoparticle-treated group. This suggests that nanoparticles can be used as transfection reagents. In addition, as a result of flow cytometry, the nanoparticles were found to produce 293T cells at higher levels than conventional non-viral transfection (data not shown).

As a result of introducing the gene into NK-92MI cells to confirm the effect of gene transfer and protein expression in not only T cells but also NK cells, it was confirmed that the gene was successfully introduced and the protein was also expressed as shown in FIG. 6C. More specifically, red represents nanoparticles, and green represents GFP fluorescence intensity, and consequently, genes were successfully transferred to NK-92MI cells by nanoparticles. Accordingly, it was confirmed that the nanoparticles can effectively express the target protein by transferring the gene to NK cells.

In addition, in order to confirm the distribution of the qDNA-nanoparticle complex in NK-92MI cells, as a result of observing the cells through a bio-TEM microscope, as shown in FIG. 9, it was confirmed that the complex was observed in both the cell membrane and endosomes. Could. These results suggest that nanoparticles are introduced into NK cells through endocytosis.

2.3 Confirmation of NK-92MI transfection of pEGFR-CAR using nanoparticles in vitro

2.3.1 Structure of 2nd generation CAR EGFR

It is known that CAR-based immunotherapy requires the successful expression of the CAR gene as an essential element, and despite the difficulties in various studies, CAR-NK is drawing attention as an alternative to solve the problem of CAR-T cells. Accordingly, the present inventors constructed a second generation EGFR targeting the CAE expression plasmid (pEGFR-CAR) to investigate whether nanoparticles can be efficiently delivered to NK-92MI cells to express CAR. The structure of the second generation EGFR is as shown in Fig. 4a, and the sequence number is as shown in SEQ ID NO: 1 below.

2.3.2 Transformation method

As a transformation method, 1×10 6 NK-92MI cells were counted and seeded in a 6-well plate, and each concentration of MF-NP and plasmid were combined at room temperature for 15 minutes and treated on the cells. After 48 hours, cells were harvested and analyzed for CAR expression. Each cell was analyzed for CAR expression by flow cytometry using anti-Fab-biotin antibody and streptavidin-FITC.

2.3.3 Confirmation of Transformation in T Cells and NK Cells

First, the transfection efficiency of pEGFR-CAR through nanoparticles in 293T cells was investigated. As a result, as shown in FIG. 4B, as a result of quantitative RT-PCR, it was found that pEGFR-CAR was successfully transferred to 293T cells by nanoparticles. In addition, in order to confirm that the nanoparticles can manipulate NK cells to express EGFR-CAR, NK-92MI cells or a population thereof were cultured with the nanoparticle-EGFR-CAR complex for 24 hours. As a result, as shown in FIG. 10, it was found that the gene transfer efficiency of the nanoparticles is more effective in a cell population than in a single cell composition.

In addition, as shown in Figure 4c, as a result of flow cytometry, it was confirmed that the pEGFR-CAR NK cell population increased in proportion to the concentration of pEGFR-CAR and nanoparticles. When CAR plasmid was delivered using MF-NPs, MF- It was confirmed that the level of CAR expression increased up to 60% depending on the NPs concentration. In addition, as shown in Figure 11, it was confirmed that the target cell killing ability increased according to the CAR expression ratio among NK cells. More specifically, it showed 85% killing ability at the maximum concentration of 8NP.

The above results suggest that the level of CAR expression can be adjusted according to the concentration of the nanoparticles, and the higher the level of CAR expression is, the higher the ability to kill cancer cells, thus suggesting that CAR NK cells using nanocells have an excellent cancer cell killing effect. .

2.3.4 Confirmation of cytokine activity and anti-tumor effect of transformed cells

Next, the present inventors investigated whether the expression of EGFR-CAR can improve the cytokine activity of NK cells. As a result, as shown in Fig. 4d, next, cytokine activity according to CAR expression was investigated. It was confirmed that cytokine activity increased in proportion to the concentration of nanoparticles and pEGFR-CAR. This suggests that there is a positive correlation between the percentage of EGFR-CAR positive NK cells and cytokine activity as shown in FIG. 4E.

Finally, the present inventors analyzed the anti-tumor activity of NK cells expressing EGFR-CAR. A tumor xenograft model was generated using NK cell-free NOG mice (NOD/Shi-scid/IL2Rγnull), and MDA-MB-231-GFP-Luc cells were ectopically injected into the mammary fat pad of each mouse. Seven days after inoculation of cancer cells, NK cells were injected through the tail vein three times every three days. As a result, as shown in FIG. 4F, it was confirmed that the tumor size was the smallest when NK cells expressing EGFR-CAR were injected. This suggests that the nanoparticles of the present invention successfully transformed NK cells in order to improve cell footing of MDA-MB-231 cells to xenografted tumors in vivo. In addition, through the immunohistochemical staining results, it was confirmed that the tumor invasion ability of NK-92MI cells was not affected by CAR expression (data not shown). As a result, the above results suggest that the nanoparticles of the present invention can be used as a tool for expressing CAR in NK cells.

Example 3. Confirmation of the imaging results of NK-92MI cells transformed with nanoparticles in vivo

3.1 Imaging method

Since the nanoparticles of the present invention have magnetic properties, the use of a near-infrared (NIR) fluorescent dye (eg, Cy7) allows simultaneous imaging with MR and fluorescence imaging contrast. To use this, the present inventors imaged in vivo with fluorescent imaging devices using cells labeled with nanoparticles.

More specifically, 1×107 NK cells treated with nanoparticles were injected into mice, and imaging was performed 12 hours after cell injection. Fluorescence imaging was performed using a fluorescence-labeled bioimaging instrument. Magnetic resonance imaging was performed using a 9.4 T/160mm animal magnetic resonance imaging system.

As a result, as shown in FIG. 7A, normal NK cells did not have a fluorescent signal, but cells labeled with nanoparticles could observe a strong signal.

3.2 Confirmation of possible use of imaging in vivo

Next, in order to find out whether fluorescence-labeled NK cells can be traced in vivo by fluorescence imaging, the present inventors injected nanoparticle-treated NK cells into mice. As a result, as shown in Fig. 7B, a clear fluorescent signal could be observed by scanning. As shown in Figure 7c, it was confirmed that the nanoparticles were imaged inside the nanoparticle-treated NK cells as well as the NK cells that were not, and when injected directly into the mouse, as shown in FIG. Tracking was possible.

Such imaging is advantageous for tracking cells and has the advantage of obtaining high-resolution images from soft tissues such as muscles, ligaments, brain, nervous system, and tumors without exposure to radiation. In T2 weighted MR imaging, the relaxation factor (r2) is known to have an important effect on the improvement of contrast medium. Interestingly, as shown in FIG. 12, it was confirmed that the nanoparticles had an r2 value that was about 4 times higher than that of FIG. The excellent T2 weighting effect of the nanoparticles as described above was estimated to be due to the dopant effect of Zn+. However, as the surface of the nanoparticles of the present invention was modified by surface modification, the r2 value in MR was remarkably decreased. This suggests that the colloidal stability of the nanoparticles was lowered during complex formation. It was confirmed that nanoparticle-labeled NK cells exhibited greater T2 weighting than simple nanoparticles.

For this reason, it was possible to confirm the validity of in vivo tracking using nanoparticles. As a result, through the above-described cell tracking results, it was confirmed that when the NK cells treated with the nanoparticles of the present invention are injected into an individual, tracking is possible during the time that the nanoparticles stay stable, suggesting the possibility of utilizing in vivo tracking. will be.

<110> SUNGKWANG MEDICAL FOUNDATION CHA University Industry-Academic Cooperation Foundation <120> Nanoparticles including improved coatings and their uses <130> PN128395 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1578 <212> DNA <213> Artificial Sequence <220> <223> the DNA sequences of pEGFR-CAR. single chain fragment variable (scFv) DNA of EGFR CAR plasmid. <400> 1 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccgaagcttg acattctaat gacccaatct ccactctccc tgcctgtcag tcttggagat 120 caagcctcct acctgcaaag gccaggccag tctccaaagc tcctgatcta caaagtttcc 180 gaccgatttt acctgcaaag gccaggccag tctccaaagc tcctgatcta caaagtttcc 240 gaccgatttt ctggggtccc agacaggttc agtggcagtg gatcagggac agatttcaca 300 ctcaagatca gcagagtaga ggctgaggat ctgggaattt attactgctt tcaaggttca 360 catattcctc ccacgttcgg aggggggacc aagctggaaa tcaaacgtgc ggccaagctt 420 ggtggaggcg gttcaggcgg aggtggcagc ggcggtggcg ggtcgggtac ccaggtccag 480 ctgcagcagt ctgggtctga gatggcgagg cctggagctt cagtgaagct gccctgcaag 540 gcttctggcg acacattcac cagttactgg atgcactggg tgaagcagag gcatggacat 600 ggccctgagt ggatcggaaa tatttatcca ggtagtggtg gtactaacta cgctgagaag 660 ttcaagaaca aggtcactct gactgtagac aggtcctccc gcacagtcta catgcacctc 720 agcaggctga catctgagga ctctgcggtc tattattgta caagatcggg gggtccctac 780 ttctttgact actggggcca aggcaccact ctcacagtct cctccggatc cgccctgagc 840 aactccatca tgtacttcag ccacttcgtg ccggtcttcc tgccagcgaa gcccaccacg 900 acgccagcgc cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc cctgtccctg 960 cgcccagagg cgtgccggcc agcggcgggg ggcgcagtgc acacgagggg gctggacgat 1020 atcttttggg tgctggtggt ggttggtgga gtcctggctt gctatagctt gctagtaaca 1080 gtggccttta ttattttctg ggtgaggagt aagaggagca ggctcctgca cagtgactac 1140 atgaacatga ctccccgccg ccccgggccc acccgcaagc attaccagcc ctatgcccca 1200 ccacgcgact tcgcagccta tcgctccgcg gccgcaagag tgaagttcag caggagcgca 1260 gacgcccccg cgtaccagca gggccagaac cagctctata acgagctcaa tctaggacga 1320 agagaggagt acgatgtttt ggacaagaga cgtggccggg accctgagat ggggggaaag 1380 ccgcagagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1440 gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1500 ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1560 gccctgcccc ctcgctaa 1578

Claims (19)

A first layer comprising coated caffeic acid (CA); And
Nanoparticles comprising a second layer containing polydopamine (PDA) coated on the first layer. The nanoparticle of claim 1, comprising a third layer including a cationic layer on top of the second layer. The nanoparticle of claim 1, wherein a fluorescently-label is formed on the third layer. The nanoparticle of claim 1, wherein the nanoparticle is a magnetic nanoparticle. The nanoparticle of claim 1, wherein the nanoparticle is capable of being tracked or imaged using a device. The nanoparticle of claim 1, wherein the nanoparticle contains a gene delivery system or a drug on the surface. The gene delivery system according to claim 1, comprising the nanoparticles. The delivery system of claim 7, wherein the gene delivery system comprises a chimeric antigen receptor (CAR) gene. The delivery system of claim 7, wherein the gene delivery system further comprises a drug on the surface. The delivery system of claim 7, wherein the gene delivery system is capable of being tracked or imaged using a device. Immune cells comprising the nanoparticles and genes of claim 1. The cell of claim 11, wherein the gene is a CAR expression gene. The cell of claim 11, wherein the immune cell is an NK cell. The cell of claim 11, wherein the immune cell has a core of nanoparticles remaining therein and can be tracked using a device. EGFR: immune cells containing the CAR gene. The cell of claim 15, wherein the immune cell is an NK cell. The cell of claim 15, wherein the gene comprises the sequence of SEQ ID NO: 1. A pharmaceutical composition for the treatment or prevention of cancer comprising the immune cells of claim 15 or a group of cells thereof as an active ingredient. The method of claim 16, wherein the cancer is breast cancer, thyroid cancer, blood cancer, renal cell cancer, gastric cancer, colon cancer, lung cancer, liver cancer, prostate cancer, pancreatic cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin's lymphoma, oral cancer, cleft lip cancer, testicular cancer, acute myeloid cancer. Leukemia, basal cell cancer, ovarian epithelial cancer, brain tumor, multiple myeloma, chronic myelogenous leukemia, chronic lymphocytic leukemia, bladder cancer, peritoneal cancer, tongue cancer, non-small cell lung cancer, small cell lung cancer, small intestine cancer, esophageal cancer, kidney cancer, heart cancer, malignant lymphoma, A composition that is one or more selected from the group consisting of urethral cancer, cervical cancer, rectal cancer, tonsil cancer and laryngeal cancer.

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