KR102453266B1 - Gene delivery syetem comprising specially-coated nanoparticles and EGFR-CAR gene with a second generation domain - Google Patents

Abstract

It relates to nanoparticles and NK cells for immunotherapy and pharmaceutical compositions comprising them, and according to the nanoparticles according to an aspect, it is possible to express genes by delivering genes to immune cells, and to NK cells into which the nanoparticles are introduced. According to the report, it effectively treats cancer without the side effects of existing CAR T cells and at the same time has the effect of tracking cells.

Description

Gene delivery system comprising specially-coated nanoparticles and EGFR-CAR gene with a second generation domain {Gene delivery syetem comprising specially-coated nanoparticles and EGFR-CAR gene with a second generation domain}

It relates to specially coated nanoparticles and their use.

Cancer is a disease that ranks first in the nation's mortality rate, and the need to develop anticancer drugs is constantly emerging.

Looking at the anticancer drug development process, there were chemical anticancer drugs that attack dividing cells using the characteristics of rapidly proliferating tumor cells and targeted anticancer drugs that attack specific molecules or signal transduction systems of tumor cells. Immune anticancer drugs that can minimize side effects by using the innate immunity of

Immunotherapy is a cancer treatment that activates the body's immune system to fight cancer cells. Immune chemotherapy uses the immune system to attack only cancer cells and has fewer side effects than conventional chemotherapy, and has the advantage of being able to obtain long-term anticancer effects because it utilizes the memory and adaptability of the immune system. As described above, immunotherapy, 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 the research of the year in 2013.

Immune anticancer drugs can be divided into antibody therapeutics that target tumor antigens (Rituximab, etc.), immune checkpoint inhibitors that reactivate immune cells (Immune checkpoint inhibitors, etc.), and immune cell therapies that directly administer immune cells (Immune cell therapy). (Oiseth et al, 2017).

Among the methods of immunotherapy, immune cell therapy refers to a treatment that is genetically modified using immune cells in the body and then put back into the body. Immunotherapy is aimed at strengthening cellular immunity. Early immunotherapeutic agents attempted to treat patients with malignant melanoma using autologous T cells activated in vitro. However, T cells that are not given antigen specificity had a limitation in that they did not obtain sufficient effects on malignant tumors. became However, chimeric antigen receptor T-cells (CAR-T cells) have disadvantages such as complicated genetic manipulation and high treatment costs, and there are also problems that cytokine release syndrome and various side effects are caused due to the characteristics of T cells. . Therefore, in the art, in order to overcome the above problems, the development of CAR-T cells that do not induce immunogenicity or inject a suicide gene into CAR-T cells is continuously attempted, but the effect is insufficient.

In addition, until now, a gene transfer technology using a virus has been used as a method for expressing a 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 gene transfer using viruses is difficult due to their characteristics. Existing methods using nanoparticles also have limitations that are unsuitable for immune cells.

Therefore, the present inventors used nanoparticles rather than the conventional method using viruses, and tried to overcome the existing limitations by effectively delivering genes to immune cells through specially coated nanoparticles.

One aspect includes a first layer comprising a coated caffeic acid (CA); It provides nanoparticles comprising a second layer comprising polydopamine (PDA) coated on the first layer.

Another aspect provides a gene delivery system comprising the nanoparticles.

Another aspect provides immune cells comprising nanoparticles and genes.

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

Another aspect is to provide a pharmaceutical composition for treating or preventing cancer comprising immune cells as an active ingredient.

One aspect includes a first layer comprising a coated caffeic acid (CA); It provides nanoparticles (Nanoparticles) including a second layer including polydopamine (PDA) coated on the first layer.

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

The term “nanoparticles” may refer to particles having a size of 1 to 100 nm having a surface. “Multifunctional Nanoparticles (MF-NPs)” may refer to nanoparticles used in the field of anticancer or drug delivery. Multifunctional nanoparticles and nanoparticles may be used interchangeably. The structure of the nanoparticles may be such that a central core and a surface exist.

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 cation layer, It may be at least one 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 Polyethylenimine (PEI).

The term “coating” means changing the properties of the surface by covering the surface of the underlying material with a film of a different component. Surface modification and surface coating may be used interchangeably.

The term “caffeic acid” is a kind of hydroxycinnamic acid, and may be yellow and have a phenol group and an acryl group. The caffeic acid may have the structure of Formula 1 below.

[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 polyethylamine (PEI) layer in order. it may have been For example, the PEI layer top may be cationic.

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

In one embodiment, the nanoparticles may have a fluorescent label (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 combined with a fluorescent material. The bond may refer to a covalent bond or an ionic bond. The term “fluorescent material” may refer to a material emitting fluorescence by irradiation of an ultraviolet irradiation device.

In one embodiment, the fluorescent material is rhodamine DAPI, ethidium bromide, acridine orange, propidium iodine (Propidium Iodide) or cyanine 7 (Cyaine 7) amine: Cy7). 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 magnetism 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 a magnetic or fluorescent material, tracking or imaging may be possible using a device. For example, the tracking or imaging may be performed using magnetic resonance imaging (MRI) or fluorescence optical imaging to enable imaging of the location and movement of nanoparticles after administration in vivo or into cells.

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

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

The drug may be delivered into the cell by nanoparticles. The types of drugs include anticancer drugs, immunotherapy drugs, signal transduction inhibitors, cell cycle inhibitors, epigenetic regulators, angiogenesis inhibitors, blood vessel destruction drugs, immunomodulators, immunostimulants, 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.

In one embodiment, the drug may be an anticancer drug. In one embodiment, the drug may be an immuno-cancer drug. In addition, the anticancer agent, immunotherapy agent, signal transduction inhibitory agent, 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 refer to a material that transports and helps a gene for treatment to be properly expressed in a gene therapy process. It may be replicable and may include all genetic elements capable of transferring a gene sequence between cells, and is also called a vector. The gene carrier may be a viral or non-viral carrier. 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 carrier may be non-viral. In one embodiment, it may be nanoparticles.

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

In one embodiment, the coating layer is an oleylamine (OA) layer that is a first layer, a caffeic acid (CA) layer that is a second layer, a polydopamine (PDA) layer that is a third layer, and a fourth layer The polyethylamine (Polyethylenimine: PEI) layer may be coated in order.

In one embodiment, the gene delivery system may include a CAR gene, and may be a gene that causes CAR expression in cells. 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 second-generation.

The term "CAR (Chimeric antigen receptor)" may refer to an artificial membrane-bound protein that induces immune cells (eg, T lymphocytes) against an antigen and stimulates the immune cells to kill antigen-presenting cells . The CAR may have a structural feature that has both the structure of a T cell and an antigen-binding site. More specifically, the structure of a CAR may include an extracellular domain that binds 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 a second generation or a third generation. For example, it may be the second generation. The second generation may mean adding a signaling domain of a costimulatory receptor such as CD28 or 41BB (CD137) to CD3-zeta as a signaling domain.

The gene comprising the sequence of SEQ ID NO: 1 may include a sequence having a similarity thereto, for example, about 70% or more, about 75% or more, about 80% or more, about the sequence of SEQ ID NO: 1 It may encode a sequence having 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 additionally on the surface.

In one embodiment, the gene delivery system may be one that can be tracked or imaged using a device.

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

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

In another embodiment, the manufacturing method may further include the step of forming a fluorescent label (Fluorescently-label) on the surface of the cation layer using a fluorescent material.

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

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

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

Another aspect provides the use of said immune cell, cell population thereof, or culture medium thereof for use in the manufacture of a cell therapy product, pharmaceutical composition or formulation.

Another aspect provides a method for treating a disease, comprising administering the immune cell, a cell population thereof, or a culture solution thereof to a subject.

The term nanoparticle, CAR gene is as described above.

The term "prevention" refers to any act of inhibiting or delaying the occurrence of cancer by administration of the composition according to the present invention.

The term "treatment" refers to or includes the alleviation, inhibition of progression or prevention of a disease, disorder or condition, or one or more symptoms thereof, and "active ingredient" or the term "pharmaceutically effective amount" refers to the 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 the progression or prevent one or more symptoms thereof.

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

The term “T cell” refers to lymphocytes derived from the thymus or bone marrow, and is a type of immune lymphocytes. It may be mainly 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-oncology. More specifically, the CAR receptor may be expressed. The CAR T cells may be used for cancer treatment.

The term “NK cells” refers to Natural Killer cells. The cells are cytotoxic lymphocytes that constitute a major component of the innate immune system, and are defined as large granular lymphocytes (LGL) and are a third type of lymphocyte differentiated from common lymphoid progenitor (CLP)-producing B and T lymphocytes. It may constitute a cell. In addition, the cells may include natural killer cells, mature natural killer cells and natural killer progenitor cells derived from any tissue source without further modification. The NK cells are activated in response to interferon or macrophage-derived cytokines, and NK cells are of two types that control the cytotoxic activity of cells, labeled with “activating receptors” and “inhibiting receptors”. surface receptors. The natural killer cells may also be those 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 one produced from hematopoietic stems or precursors.

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 intrinsic immunomodulatory ability of natural killer cells is activated compared to parental cells, for example, hematopoietic cells, or natural killer progenitor cells. More specifically, it may be that natural killer cells first mature in the liver or bone marrow, and then undergo an activation step to change into cells that apoptosis of abnormal cells.

In addition, the natural killer cells may have a lower risk of cytokine storm due to weak memory function and clonal express function compared to T cells.

The term "genetic modification" or "genetic 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 have improved target specificity and/or antigen specificity. More specifically, it may refer to genetically modified CAR NK cells.

The term “CAR-NK cell” may refer to a cell in which a CAR has been introduced into the NK cell. The CAR NK cells may be generated by introducing CAR into NK cells in order to overcome the disadvantages of the existing CAR T cells. Cytokine storm (Cytokine Storm) due to the fact that T cells have to go through a complex genetic manipulation process using the patient's own T cells, are expensive customized treatments worth hundreds of millions of won for each patient, and divide rapidly when they meet target cells. 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 the disadvantage that the same side effects may occur unexpectedly at any time in case of recurrence, so NK cells with relatively low memory function and division function were selected. In the case of using NK cells, there may also be time and economic advantages compared to CAR T cells, which need to 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 costimulatory domains. More specifically, since there are cases in which the function of NK cells is lowered due to suppression of the expression of costimulatory molecules of immune cells in a general tumor environment, the intracellular signaling domain of a factor capable of overcoming the immune suppression response is selected as an antigen-binding domain. CAR can be prepared by fusion with When all other conditions are met, the CAR is expressed on the surface of, eg, a T lymphocyte, eg, a primary T lymphocyte, and the extracellular domain of the CAR binds an antigen, the extracellular signaling domain is a T lymphocyte It activates and/or proliferates by transmitting a signal to, and also kills cells expressing the antigen when the antigen is present on the cell surface. Some immune cells, such as T lymphocytes and natural killer cells, require two signals for maximal activation, a primary activation signal and a costimulatory signal, and CARs also indicate that binding of antigen to the extracellular domain is the primary activation It may include a costimulatory domain to cause transmission of both the signal and the costimulatory signal.

In a specific embodiment, the NK cell 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 the composition according to one embodiment to a desired site. It may mean a batch of a composition according to an embodiment. Administration may be by any suitable route that delivers at least a portion of a cell or cellular component of a composition according to one embodiment to a desired location in a surviving individual. The survival period of the cells after administration to a subject may be as short as several hours, for example, from 24 hours to several days or as long as several years.

The term "isolated cell", such as "isolated immune cell" and the like, may refer to a cell substantially isolated from the tissue from which the cell originates.

The composition of the present invention may be used in a method for treating or recognizing a tumor or cancer derived from a neoplasm. The neoplasm may be malignant or benign, and the cancer may be primary or metastatic; The neoplasm or cancer may be early or late. Non-limiting examples of neoplasms or cancers that can be treated include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendicitis cancer, astrocytoma (pediatric cerebellar or cerebrum). ), basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brainstem glioma, brain tumor (cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymocytoma, medulloblastoma, supratenoral primitive neuroectoderm tumor, visual pathway and hypothalamic glioma), Breast cancer, bronchial adenoma/carcinoid, Burkitt's lymphoma, carcinoid tumor (pediatric, gastrointestinal tract), unknown primary carcinoma, central nervous system lymphoma (primary), cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, cervical cancer, Childhood cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, colon cancer, cutaneous T-cell lymphoma, fibrous tissue-forming small round cell tumor, endometrial cancer, ependymocytoma, esophageal cancer, Ewing's sarcoma with Ewing's tumor, extracranial reproduction Cell tumor (pediatric), extratesticular germ cell tumor, extrahepatic cholangiocarcinoma, eye cancer (intraocular melanoma, retinoblastoma), gallbladder cancer, gastric (gastric) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germ cell tumor (pediatric) extracranial, extratesticular, ovarian), gestational villous tumor, glioma (adult, juvenile brainstem, juvenile cerebral astrocytoma, 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 glioma (pediatric), intraocular melanoma, islet cell carcinoma, Kaposi's sarcoma, kidney cancer (renal cell cancer), laryngeal cancer, leukemia (acute lymphoblastic, acute myeloid, chronic lymphocyte) , chronic myeloid, hair 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), melanoma, intraocular melanoma, Merkel cell Carcinoma, mesothelioma (adult malignant, pediatric), latent primary metastatic head and neck squamous cell carcinoma, oral cancer, multiple endocrine tumor syndrome (pediatric), multiple myeloma/plasma cell neoplasia, mycosis fungoides, myelodysplastic syndrome, myelodysplasia/ Myeloproliferative disease, myeloid leukemia (chronic), myeloid 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 bone, ovarian cancer, ovarian epithelial cancer (superficial epithelial-stromal tumor), ovarian germ cell tumor, ovarian low malignant latent tumor, pancreatic cancer , pancreatic cancer (islet cell), sinus and sinus cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal seed cell tumor, pineal blastoma and supratenoral primitive neuroectodermal tumor (pediatric), pituitary adenoma, Plasma cell neoplasia, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvic and ureteral transitional cell cancer, retinoblastoma, rhabdomyosarcoma (pediatric), salivary gland cancer, sarcoma (Ewing family) tumors of, Kaposi, soft tissue, fallopian tubes), Sezary syndrome, skin cancer (non-melanoma, melanoma), skin carcinoma (Merkel cell), small cell lung cancer, small-cell cancer, soft tissue sarcoma, squamous cell carcinoma, potentially primary (metastatic) head and neck Squamous Cell Cancer, Gastric Cancer, Supratentorial Primitive Neuroectodermal Tumor (Child), T-Cell Lymphoma (Skin), Testicular Cancer, Throat Cancer, Thymoma (Child), Thymoma and Thymic Carcinoma, Thyroid Cancer, Thyroid Cancer (Child), Renal Pelvic and Ureter Transition cell carcinoma, villous tumor (pregnancy), unknown primary site (adult, pediatric), ureter and renal pelvic transition cell carcinoma, urethral cancer, fallopian tube cancer (endometrium), tubal sarcoma, vaginal cancer, visual pathway and hypothalamic glioma (pediatric), vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor (pediatric).

The term “hematologic cancer” is a cancer affecting the blood, bone marrow and lymphatic system. There are three main groups of blood cancers: leukemia, lymphoma and myeloma. There are four broad classifications of leukemia: acute lymphocytic leukemia (ALL), acute myeloid 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, either rapidly growing (high-grade) or slowly growing (low-grade). There are 14 types of B-cell non-Hodgkin's lymphoma. The others are T-cell lymphomas, named after different cancerous white blood cells, or 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, stomach cancer, colorectal cancer, lung cancer, liver cancer, prostate cancer, pancreatic cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin's lymphoma, oral cancer, labial cancer, testicular cancer, acute myeloid leukemia, basal cell cancer ; , it may be at least one 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 intravenously, subcutaneously, intraperitoneally, inhalation or topical application, depending on the desired method. The dosage varies according to the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of disease. A daily dose refers to an amount of a therapeutic agent according to one aspect sufficient to treat a disease state ameliorated by administration to a subject in need thereof. The effective amount of a therapeutic agent will depend on the particular compound, the disease state and its severity, and the individual in need of treatment, which can be routinely determined by one of ordinary skill in the art. As a non-limiting example, the dosage for the human body of the composition according to one aspect may vary depending on the patient's age, weight, sex, dosage form, health status, and disease degree. Based on an adult patient weighing 70 kg, for example, about 1,000-10,000 cells/time, 1,000-100,000 cells/time, 1,000-1000,000 cells/time, 1,000-10,000,000, 1,000-100,000,000 cells/time, 1,000 to 1,000,000,000 cells/time, 1,000 to 10,000,000,000 cells/time, may be administered in divided doses once or several times a day at regular time intervals, or may be administered several times at regular time intervals.

The term 'individual' refers to a subject in need of treatment for 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 one 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 materials such as biopolymers, inorganic materials such as hydroxyapatite, specifically collagen matrix, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers and chemical derivatives thereof, etc. may also be combined for topical administration. can When the pharmaceutical composition according to one embodiment is formulated in a dosage form suitable for injection, immune cells, immune cells, or substances that increase their activity are dissolved in a pharmaceutically acceptable carrier or in a dissolved solution state. It may be frozen.

The pharmaceutical composition according to one embodiment, if necessary according to its administration method or formulation, a suspending agent, solubilizing agent, stabilizer, isotonic agent, preservative, anti-adsorption agent, surfactant, diluent, excipient, pH adjuster, analgesic agent, Buffers, reducing agents, antioxidants and the like may be included as appropriate. Pharmaceutically acceptable carriers and agents 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 one 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 of ordinary skill in the art to which the present invention pertains. It can be prepared as or by putting it in a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oily or aqueous medium, or in the form of a powder, granules, tablets or capsules.

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

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

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

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

The drug is the same as described above.

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

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

Another aspect provides the use of said immune cell, cell population thereof, or culture medium thereof for use in the manufacture of a cell therapy product, pharmaceutical composition or formulation.

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

The terms NK cell, gene, CAR, cancer, therapeutic, prophylactic and immune cells 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 the 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 the step of preparing the gene delivery system. Specifically, it may be to go through the step of attaching the target gene to the surface of the nanoparticles. Attachment may be using a known genetic engineering method. The gene may be DNA, RNA, siRNA, miRNA, dsRNA or iRNA, specifically plasmid RNA. The attached nanoparticles are a gene carrier, and the gene carrier may be introduced into the prepared NK cells. The introduction may be by using a known genetic engineering method, and in one embodiment, NK cells and the nanoparticles may be cultured together. It may be that the intracellular insertion process is made due to the cationicity of the NK cell surface during the culture process. The introduced nanoparticles can be degraded by intracellular digestion processes that are innately present in the cells. The introduced nanoparticles may include a step of 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. The core particles remaining in the cell include a magnetic or fluorescent material, and may be imaged or traceable by a device. For example, the device may be by MRI or fluorescence visual imaging.

In one embodiment, the gene delivery system may include a CAR (Chimeric Antigen Receptor) expression gene. More specifically, it may be an EGFR:CAR gene. In addition, when the CAR gene is introduced into the cell, the CAR receptor is expressed on the NK cell surface to generate CAR NK cells.

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

1 is a schematic diagram of an experiment showing the step of introducing nanoparticles into NK cells in this experiment and tracking the step when injected into an individual.
2A is a schematic diagram showing a method for preparing multifunctional nanoparticles.
Figure 2b is an image showing the chemical structure of the surface of the nanoparticles in the manufacturing method.
3 is a graph of the ex vivo cell compatibility of multifunctional nanoparticles in NK-92MI cells; a is a graph analyzing the cytotoxicity caused by multifunctional nanoparticles in NK-92MI cells through MTT analysis, b is a graph showing apoptosis of a single cell or a cell population group for 48 hours at the nanoparticle concentration (x-axis) , c is a graph showing the phenotypic change over 48 hours of nanoparticles-treated NK cells and the control group, d is a graph showing flow cytometry analysis for 48 hours in NK cells treated with 16ug/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 through 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 Graph of EGFR-CAR expression and cancer cell killing ability of NK-92MI analyzed by staining, e is a graph showing quantitative analysis of c and d data, f is cancer cell MDA-MB-231 xenograft mouse model A graph showing the anti-tumor effect of EGFR-CAR-expressing NK-92MI cells against; On the left is a biofluorescence imaging image, on the right is a measurement of tumor size in mice, and black arrows indicate injection of NK cells via tail vein on days 7. 10. and 13.
5a is an image of observing magnetic nanoparticles, CA-coated magnetic nanoparticles, CA, PDA-coated magnetic nanoparticles, PEI, CA, PDA-coated magnetic nanoparticles from the left under a TEM microscope.
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.
Figure 5c, d shows the thermogravimetric 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, and PDA-coated magnetic nanoparticles.
6 is a graph showing the in vitro gene transfer ability of nanoparticles in 293T cells and NK-92MI cells; 6a is a graph of electrophoresis of nanoparticles-plasmid DNA at each concentration in 1% agarose gel, 6b is a fluorescence image of 293T cells transfected with the nanoparticle-qGFP complex for 24 hours, 6c is a nanoparticle-qGFP complex 24 Fluorescence images 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 nanoparticles-treated NK-92MI cells, 7b is an optical imaging image of a mouse transplanted with fluorescently labeled nanoparticles-treated NK-92MI cells, and 7c is an in vitro fluorescence image of NK-92MI cells treated with nanoparticles. , 7d is a magnetic resonance image of nanoparticle-treated NK-92MI cells injected into a mouse in vivo; The yellow dotted line and the white arrow indicate the implantation site of the nanoparticle-treated cells.
8 is a graph evaluating the toxicity of nanoparticles to the degree of glucose uptake in NK-92MI cells; Glucose concentration was determined by culturing 5 x 105 cells in 1 mL of fresh medium for 24 h, then harvesting the supernatant and analyzing the glucose concentration.
9 is an image observed with a bio-TEM microscope of native NK cells and NK cells treated with the pDNA / MF-NP complex.
10 is a graph analyzing CAR expression by quantitative RT-PCR after treating NK-92MI single cells or a population of cells with MF-NP/pEGFR-CAR for 48 hours.
FIG. 11 is a graph in which the CAR gene-attached nanoparticles were measured by FACS after 24 hours of incubation with NK NK cells; From the left, untreated control, 0, 2, 4, 8ul / ml concentration of nanoparticles are treated conditions.
12A is a T2-mapping image at various iron concentrations.
12B is a graph showing 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 of the present invention, and the scope of the present invention is not limited to these examples.

1 is an image showing a schematic diagram of the experiment. In Example 1.2, specially coated nanoparticles were prepared, and in Example 2, using the genetic engineering method, the NK cells were transduced and the effect thereof was 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 purchase it

Iron (III) acetylacetonate, oleylamine, caffeic acid, and Tris base were purchased from Tokyo Chemical Industry, 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 (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 (0.5 g) in DMSO for 12 hours at room temperature, and purified by dialysis with distilled water at room temperature for 48 hours. After freeze-drying, it was stored in a freezer (-20 °C).

1.2 Preparation of NK cells

As NK cells, NK-92MI cells were used, and NK-92MI cells were purchased from ATCC (American Type Culture Collection). Next, the purchased cells were added to 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 0.02 mM folic acid were added and incubated. Cells were maintained at a density of 2.5 X 105 cells/ml and passaged every 3-4 days.

1.3 Method of manufacturing multifunctional nanoparticles

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

As a first step, magnetic nanoparticles coated with oleylamine (OA) are synthesized by thermal decomposition of Zn/Fe-based nanoparticles. 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 in 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 6 mL of 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 and mixed, and 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 was increased by about 35 nm compared to the nanoparticles coated with OA.

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

Finally, polydopamine-coated nanoparticles were dispersed in 2 mL distilled water. 40 mg of polyethyleneimine or fluorescently-labeled polyethyleneimine 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 make cationic, or fluorescent Labeled PEI was immobilized on the PDA layer. Finally, the polyethyleneimine-coated nanoparticles were purified using a centrifuge 3 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 component 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 from -31 to -21, whereas PEI/PDA/CA/MNPs had a cationicity of +35 mV. This means that the cationic PEI was successfully immobilized on the surface.

1.1 Preparation of animal models

As the 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 6-week-old female mice. After 7, 10, and 13 days, NK-92MI or EGFR-CAR- NK-92MI cells were injected by tail vein injection. Tumor size was measured using a Pearl Impurse bioluminescence system. The formula used here is as follows: 0.5236 X A X B2 (A : major axis, B : perpendicular to major axis).

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 binding to EGFR, and the CD8 hinge domain, CD28 transmembrane/endodomain, and CD3zeta domain were combined after the heavy and light chain of scFv. This DNA sequence was cloned into pcDNA3.1 vector and the structure is shown in FIG. 4 .

Example 2. Introduction of NK cells of magnetic multilayer structure multifunctional nanoparticles and confirmation of effect thereof

2.1 Investigating the effect of nanoparticles on NK cell activity to confirm their potential for use as a transfection tool

It is known in the art that genetic manipulation of NK cells is difficult, and it has been confirmed that conventional transfection tools such as viral vectors and electroporation are not suitable for NK. Therefore, in order to confirm that the nanoparticles produced in Example 1.2 are an effective transfection tool for NK cells, cytotoxicity was first evaluated by MTT assay (data not shown). As NK cells, NK-92MI cells were used. When the cells were treated with nanoparticles at various concentrations, the cell viability exceeded 90% at the concentration of 32 μg/mL, but the cell viability was less than 60% at the 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 not 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 Identification of the effect of nanoparticle delivery of genetic material and inducing 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 gel retardation assay. The experimenters attached various concentrations of nanoparticles to the plasmid and then applied them to agarose gel electroporation. Next, the nanoparticles were labeled with RITC, a fluorescent substance, and bound to qGFP, and then these particles were administered to 293T cells to confirm the ability of the nanoparticles to deliver qDNA and induce exogenous proteins.

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, flow cytometry analysis revealed that the nanoparticles produced 293T cells at higher levels than conventional non-viral transfection (data not shown).

As a result of introducing into NK-92MI cells to confirm the effect of gene delivery and protein expression in NK cells as well as T cells, it was confirmed that the genes were successfully introduced and proteins were also expressed as shown in FIG. 6c . More specifically, red is the nanoparticle and green is the GFP fluorescence intensity, and in conclusion, it indicates that the gene was successfully delivered to NK-92MI cells by the nanoparticles. Accordingly, it was confirmed that the nanoparticles can effectively express the target protein by transferring the gene to the 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 second-generation CAR EGFR

It is known that CAR-based immunotherapy requires the successful expression of CAR gene as an essential factor, and CAR-NK is attracting attention as an alternative to solve the problem of CAR-T cells despite difficulties in several studies. Accordingly, the present inventors constructed a second-generation EGFR targeting 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. 4 a, and SEQ ID NO: 1 is the same as SEQ ID NO: 1.

2.3.2 Transformation method

As a transformation method, 1 X 106 cells of 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 with the cells. After 48 hours, the cells were harvested and analyzed for CAR expression. Each cell was analyzed for CAR expression level 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 confirmed that pEGFR-CAR was successfully transferred to 293T cells by nanoparticles. In addition, in order to confirm that nanoparticles can manipulate NK cells to express EGFR-CAR, NK-92MI cells or a population thereof were incubated with the nanoparticles-EGFR-CAR complex for 24 hours. As a result, as shown in FIG. 10 , it was found that the gene transfer efficiency of nanoparticles was more effective in a cell population than in a single cell configuration.

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

The above results can control the level of CAR expression according to the concentration of nanoparticles, and the higher the level of CAR expression, the higher the cancer cell killing ability. .

2.3.4 Confirmation of Cytokine Activity and Antitumor Effect of Transformed Cells

Next, the present inventors investigated whether the expression of EGFR-CAR can enhance the cytokine activity of NK cells. As a result, as shown in FIG. 4d , the cytokine activity according to CAR expression was investigated. It was confirmed that the 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 EGFR-CAR-expressing NK cells. A tumor xenograft model was generated using NK cell-free NOG mice (NOD/Shi-scid/IL2Rγnull), and MDA-MB-231-GFP-Luc cells were ectopic injected into the mammary gland fat pad of each mouse. Seven days after cancer cell inoculation, NK cells were injected via tail vein 3 times every 3 days. As a result, as shown in FIG. 4f , it was confirmed that the tumor size was the smallest when EGFR-CAR-expressing NK cells were injected. This suggests that the nanoparticles of the present invention successfully transformed NK cells in order to improve the cell viability of MDA-MB-231 cells against xenografted tumors in vivo. In addition, through immunohistochemical staining, it was confirmed that the tumor invasion ability of NK-92MI cells was not affected by CAR expression (data not shown). Consequently, 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 imaging results of NK-92MI cells transformed with nanoparticles in vivo

3.1 Imaging method

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

More specifically, 1 × 107 nanoparticle-treated NK cells 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 Figure 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 in vivo imaging

Next, in order to find out whether fluorescently-labeled NK cells can be traced by fluorescence imaging in vivo, the present inventors injected nanoparticles-treated NK cells into mice. As a result, as shown in FIG. 7B, a clear fluorescence signal could be observed by scanning. As shown in Figure 7c, nanoparticle-treated NK cells were also confirmed that nanoparticles were photographed by imaging compared to NK cells that were not treated with nanoparticles. 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 radiation exposure. In T2-weighted MR imaging, the relaxation coefficient (r2) is known to have a significant effect on the enhancement of the 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 the nanoparticles. It was estimated that the excellent T2 weighting effect of nanoparticles as described above is 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 significantly reduced. This suggests that the colloidal stability of the nanoparticles was reduced during complex formation. It was confirmed that nanoparticle-labeled NK cells exhibited greater T2 weight than simple nanoparticles.

Due to this, it was possible to confirm the feasibility of in vivo tracking using nanoparticles. As a result, through the above cell tracking results, when the nanoparticles-treated NK cells of the present invention are injected into an individual, it is confirmed that the nanoparticles can be stably tracked for a period of time, suggesting the possibility of application for in vivo tracking. will be.

<110> SUNGKWANG MEDICAL FOUNDATION CHA University Industry-Academic Cooperation Foundation <120> Gene delivery syetem comprising specially-coated nanoparticles and EGFR-CAR gene with a second generation domain <130> PN142475 <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 (1)

nanoparticles; And in the gene delivery system comprising the EGFR-CAR gene comprising the sequence of SEQ ID NO: 1,
The nanoparticles may include a magnetic core;
Caffeic acid (Caffeic acid: CA) comprising a first coating layer;
a second coating layer comprising polydopamine (PDA) on an upper portion of the first coating layer; and
A third coating layer comprising a cationic material on top of the second coating layer,
The EGFR-CAR gene includes a signaling domain in which a signaling site of a costimulatory receptor is added to CD3-zeta,
The gene delivery system is for genetic manipulation of NK-92MI cells.

Images