WO2020189942A1

WO2020189942A1 - Immune cell with improved cancer killing ability

Info

Publication number: WO2020189942A1
Application number: PCT/KR2020/003321
Authority: WO
Inventors: 장미희; 전은성; 주안나
Original assignee: 한국과학기술연구원; 재단법인 아산사회복지재단; 울산대학교 산학협력단
Priority date: 2019-03-15
Filing date: 2020-03-10
Publication date: 2020-09-24

Abstract

The present invention relates to a genetically modified immune cell expressing a chimeric antigen receptor (CAR) including an antigen binding domain binding specifically to cancer cells and/or expressing TRAIL, a composition comprising the immune cell for prevention or treatment of cancer, a cell therapy product, a method for providing information on cancer diagnosis, and a method for preparing a genetically modified immune cell.

Description

Immune cells with improved cancer killing ability

Genetically modified immune cells expressing chimeric antigen receptor (CAR) containing an antigen-binding domain that specifically binds to cancer cells or producing TRAIL protein, a composition for preventing or treating cancer comprising the immune cells, It relates to cell therapy, a method of providing information for diagnosis of cancer, and a method of producing genetically modified immune cells.

Cancer immunotherapy, which has recently emerged, is a treatment that uses the human body's own immune system to minimize damage to normal cells and removes cancer cells more specifically.It is in various fields (antibody therapy, immune cell therapy, Viral immunotherapy, nanotechnology immunotherapy, etc.) are being actively researched. Among these, immune cell therapy includes lymphocytes obtained from the patient's blood, natural killer cells, natural killer T cells, T cells, B cells, and dendritic cells. It is a method of treating cancer by increasing the number of cells in the body, enhancing its function in vitro , and returning it back to the patient's body. Therapies using these immune cells show good effects in immune response control therapy, and are evaluated to be excellent in terms of toxicity and safety.

In particular, recently, there is increasing interest in cell therapy methods in which immune cells in the body are taken out, strengthened, or genetically engineered to be re-inserted through immune cell therapy. Representative examples thereof include Tumor Infiltrating Lymphocytes (TIL), Chimeric Antigen Receptor (CAR), and T-Cell Receptor (TCR) technology. Research using the receptor CAR is being actively conducted.

The chimeric antigen receptor (CAR) is an artificial receptor designed to transmit antigen specificity to T cells. They include antigen specific components, transmembrane components, and intracellular components selected to activate T cells and provide specific immunity. Chimeric antigen receptor expressing T cells can be used in a variety of therapies, including cancer therapy.

However, while treatments such as CAR-T are effective against tumors, in some cases these treatments have caused side effects due to partially non-specific attacks on healthy tissues. In order to overcome this, research on the 3rd generation CAR-T is currently underway, which has two signal domains that serve as auxiliary stimulus signals and an additional engineered receptor to increase the'cancer cell antigen recognition ability', thereby preventing normal cells. It is characterized by trying to minimize the side effects of attacking.

Nevertheless, the current CAR-T technology is manufactured to recognize only one protein expressed in cancer cells, so it is too expensive to develop individual therapeutic agents, and once CAR-T is injected, toxic T cells are produced. Even after cancer cells are removed, their function continues to lead to toxicity, and if there are normal cells representing the target protein, they also cause non-specific attacks and cause fatal side effects, which cannot be reversed. It is hindering the development of cell therapy products.

Therefore, immune cell therapy using NK cells has the advantage of being the only cell therapy that can use other people's immune cells without side effects, and interest in it is increasing. Therefore, for the past 10 years, tumor immunity therapy using the patient's immune system has been steadily developed, and'cell therapy products' using this have also been commercialized. Therefore, in order to promote patient-tailored treatment, interest in cell therapy products that separate and cultivate immune cells (e.g., CAR-NK cells) introduced with chimeric antigen receptors from healthy healthy blood and inject them into cancer patients. The situation is rising.

One aspect provides a genetically modified immune cell expressing a chimeric antigen receptor (CAR) and/or TRAIL comprising an antigen binding domain that specifically binds to cancer cells.

One aspect provides genetically modified immune cells that produce TRAIL proteins.

One aspect provides a pharmaceutical composition for preventing or treating cancer, comprising the genetically modified immune cells as an active ingredient.

One aspect provides a cell therapy comprising the genetically modified immune cells as an active ingredient.

Another aspect provides a method of providing information for diagnosis of cancer, comprising the step of contacting the genetically modified immune cells with a sample isolated from an individual.

Another aspect comprises introducing a recombinant vector comprising i) an antigen-binding domain that specifically binds to cancer, ii) comprising a TRAIL gene, or iii) a combination thereof, into immune cells isolated from the human body It provides a method for producing genetically modified immune cells.

Another aspect provides a method of preventing or treating cancer comprising administering the genetically modified immune cells to an individual in need thereof.

In order to achieve the above object, a genetically modified immune cell expressing a chimeric antigen receptor (CAR) comprising an antigen-binding domain that specifically binds to cancer cells is provided.

Genetically modified immune cells that produce TRAIL proteins are provided.

It provides a pharmaceutical composition for preventing or treating cancer, comprising the genetically modified immune cells as an active ingredient.

It provides a cell therapy comprising the genetically modified immune cells as an active ingredient.

It provides a method of providing information for diagnosis of cancer, comprising the step of contacting the genetically modified immune cells with a specimen isolated from an individual.

Genetically modified comprising the step of introducing a recombinant vector containing i) an antigen binding domain that specifically binds to FOLR1, ii) containing a TRAIL gene, or iii) a combination thereof into immune cells isolated from the human body It provides a method of manufacturing the immune cells.

When the genetically modified immune cells according to one aspect are used, excellent cytotoxicity to cancer cells including FOLR1, DR4, DR5, or a combination thereof is recognized, and can be effectively used as an anticancer agent. Therefore, the genetically modified immune cells produced according to one aspect exhibits highly efficient anticancer effects, and thus can be usefully used in gene therapy.

FIG. 1 shows the results of a Western blotting experiment confirming the expression of FOLR1 identified in pancreatic cancer cell lines PNAC-1, Aspc1 and Miapaca2 and patient-derived cells (PDC) SNU213, 410, 2491 and 110621. It is a diagram shown.

2 is a diagram showing the expression of FOLR1, DR4 and DR5 identified in cancer cell lines PNAC-1, Aspc1, Miapaca2, SNU213, 410, 2491 and 110621 through FACS analysis.

3 is a diagram schematically showing a transformed NK cell and a vector introduced into the NK cell.

Figure 4a is a diagram showing the result of confirming the FACS analysis in the prepared CAR-NK cells.

Figure 4b is a diagram showing the results of confirming the Western blotting (Figure 4B) confirmed through the primary antibody against α-CD3zeta.

4C is a diagram showing the results of confirming the expression of CAR through a multifocal fluorescence microscope.

Figure 5a is a diagram showing the cell death effect by treating the prepared CAR-NK cells to the cancer cell line SNU2491.

5B is a graph showing the cell-specific killing effect by treating the prepared CAR-NK cells with SNU2491 and SNU2469.

6A is a diagram showing the results of confirming the ability to inhibit proliferation through the effect of reducing the volume of cancer cells appearing on day 18 after administration of the prepared CAR-NK cells to a cancer model mouse.

Figure 6b is a diagram showing the results showing the tumor volume measured 3, 6, 9, 12, 15, and 18 days after administration of CAR-NK cells.

Figure 6c is a diagram showing the results of apoptosis through Tunel staining of a group administered with GFP, Fra CAR, and TRAIL, respectively, and a group administered with Fra CAR and TRAIL in combination with SNU2491 cancer cells.

Figure 6d is a diagram showing the results of apoptosis through H&E staining of a group administered with GFP, Fra CAR, and TRAIL, respectively, and a group administered with Fra CAR and TRAIL in combination.

The immune cells may be any one selected from the group consisting of macrophages, B lymphocytes, T lymphocytes, mast cells, monocytes, dendritic cells, eosinophils, natural killer cells, basophils, and neutrophils.

The "natural killer cell (NK cell)" is a type of white blood cell in the blood responsible for innate immunity, and is also called a "natural killer cell." The main function of the NK cells is to directly attack and destroy virus-infected cells or cancer cells. In particular, it is known that NK cells attack cancer cells to prevent the occurrence, proliferation, and metastasis of cancer cells, and it is also known that it can effectively control cancer stem cells, which play the most important role in recurrence of cancer. Treatment continues to be studied, and the need for research on CAR-NK incorporating chimeric antigen receptors is emerging.

The types of cancer cell antigens, that is, cancer-related antigens, include folate receptor alpha (FOLR1), HER2, HER2/neu, NKG2D, PSMA, CEA, IL13Roc2, EphA2, BCMA, CSPG4, CD138, survivin, CD19, CD20, CD22, k light chain, CD30, CD33, CD123, CD38, ROR1, ErbB2,ErbB3/4, ErbB dimers, EGFr vIII, carcinoembryonic antigen, EGP2, EGP40, mesothelin, TAG72, PSMA, NKG2D ligands, B7- H6, IL-13 receptor a2, MUC 1, MUC 16, CA9, GD2, GD3, HMW-MAA, CD171, Lewis Y, G250/CAIX, HLA-AI MAGE Al, HLA-A2 NY- ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7/8, avb6 integrin, 8H9, NCAM, VEGF receptors, 5T4, Baff, GPC2, CD56, Foetal AchR, NKG2D ligands, or CD44v6.

The chimeric antigen receptor may comprise an antigen binding domain that specifically binds to the above-mentioned antigen.

In one embodiment, the chimeric antigen receptor may be an antigen binding domain that specifically binds to folate receptor alpha (FOLR1).

The FOLR1 protein is encoded by the FOLR1 gene and is known as a member of the folate receptor (FOLR) family, and the protein has high affinity for folic acid and various folic acid derivatives, and 5-methyltetrahydrofolate (5 -methyltetrahydrofolate) is known to mediate delivery.

The genetically modified immune cells, for example natural killer cells, include those introduced by a recombinant vector containing a sequence encoding a chimeric antigen receptor. The term “vector” refers to a means for expressing a gene of interest in a host cell. For example, a viral vector selected from the group consisting of a plasmid vector, a cosmid vector, a bacteriophage vector, a herpes simplex virus, and a basinia virus, an adenovirus vector, a retroviral vector, a lentiviral expression vector and an adeno-associated virus vector Include. Vectors that can be used as the recombinant vector are, for example, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series and pUC19 Plasmids often used in the art, such as λgt4λB, λ-Charon, λΔz1, and phages such as M13, or viruses such as SV40 may be fabricated.

In the recombinant vector, the polynucleotide sequence encoding the fusion protein is operably linked to a promoter. The term “operatively linked” refers to a functional linkage between a nucleotide expression control sequence, such as a promoter sequence, and another nucleotide sequence, whereby the control sequence facilitates transcription and/or translation of the other nucleotide sequence. Will be adjusted.

The recombinant vector may be an expression vector capable of stably expressing the fusion protein in a host cell. The expression vector may be a conventional one used in the art to express foreign proteins in plants, animals or microorganisms. The recombinant vector can be constructed through various methods known in the art.

The recombinant vector can be constructed using prokaryotic or eukaryotic cells as a host. For example, when the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (e.g., pLλ promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc. ), a ribosome binding site for initiation of translation and a transcription/translation termination sequence are generally included. In the case of eukaryotic cells as a host, the origin of replication operating in eukaryotic cells included in the vector includes the f1 origin of replication, SV40 origin of replication, pMB1 origin of replication, adeno origin of replication, AAV origin of replication, BBV origin of replication, etc. It is not limited. In addition, a promoter derived from the genome of a mammalian cell (e.g., metallotionine promoter) or a promoter derived from mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, The cytomegalovirus promoter and the tk promoter of HSV) can be used and generally have a polyadenylation sequence as a transcription termination sequence.

The cells, e.g., eukaryotic cells, are yeast, fungi, protozoa, plants, higher plants and insects, or cells of amphibians, or cells of mammals such as CHO, HeLa, HEK293, and COS-1. Can be, for example, cultured cells (in vitro), transplanted cells (graft cells) and primary cell cultures (in vitro and ex vivo), and in vivo ( in vivo) cells, and also mammalian cells, including humans. In addition, the organism may be yeast, fungi, protozoa, plants, higher plants and insects, amphibians, or mammals.

Genetically modified immune cells expressing the chimeric antigen receptor, for example, genetically modified natural killer cells (hereinafter "CAR-NK or CAR-natural killer cells") are cancer immunity using conventional CAR-T therapeutic agents. Reaction initiation (on) / stop (off) switch for problems due to persistent toxicity of treatment, risk of autoimmune disease, problems of graft versus host disease (GVHD) and non-target toxicity problems for xenogeneic cell transplantation. Not only can it be solved through this, but it can be used as a general-purpose therapeutic agent by allowing it to target various cancer cells. Since the CAR-NK cells can be allografted, high-efficiency cells can be premade compared to CAR-T, which uses the patient's own immune cells, thus shortening the time of administration of therapeutic agents to increase therapeutic efficacy, as well as development and treatment. It can be usefully used in the development of therapeutic agents for various diseases according to cost reduction.

In the present invention, "antibody" refers to a substance produced by stimulation of an antigen in the immune system, and the kind is not particularly limited. In addition, in the present specification, the antibody includes, but is not limited to, fragments of an antibody having antigen-binding ability, such as Fab, Fab', F(ab')2 and Fv.

"Chimeric antibody" refers to an antibody originating in an animal whose variable region or complementarity determining region (CDR) thereof is different from the rest of the antibody.

In such an antibody, for example, the antibody variable region may be derived from an animal other than human (eg, mouse, rabbit, poultry, etc.), and the antibody constant region may be an antibody derived from human. Such chimeric antibodies can be prepared by methods such as gene recombination known in the art.

The "heavy chain" is a variable region domain VH containing an amino acid sequence of a variable region sufficient to confer antigen specificity, and a full-length heavy chain including three constant region domains CH1, CH2 and CH3, and fragments thereof. It is called.

As used herein, "light chain" refers to both a full-length light chain including a variable region domain VL and a constant region domain CL including an amino acid sequence of a variable region sufficient to confer antigen specificity and a fragment thereof.

The antigen-binding domain included in the chimeric antigen receptor according to one aspect is a site through which the main signal is transmitted and is outside the cell membrane and refers to a site that recognizes the cell membrane ligand (a substance that binds to and activates a receptor) of a target cell with a specific antigen. In a specific aspect, an antigen-binding domain that specifically binds to FOLR1 was used, and an antibody or antibody fragment that specifically binds to FOLR1 was used as the antigen-binding domain. In addition, the fragment of the antibody may be scFv, and the fragment of the antibody may be, for example, a nucleotide sequence represented by SEQ ID NO: 1, but is not limited thereto.

The chimeric antigen receptor according to one aspect may include an intracellular signal transduction domain.

The intracellular signal transduction domain, which is a component of the chimeric antigen receptor, can be used without limitation in the intracellular signal transduction domain known in the art. As an embodiment of the present invention, the intracellular signal transduction domain may be a costimulatory domain, CD3z, or a combination thereof, but is not limited thereto. The co-stimulatory domain may be at least one selected from the group consisting of ICOS, CD27, CD28, 4-1BB, and OX40.

The chimeric antigen receptor according to an aspect may exhibit a killing effect on cancer cells with high activity of NK cells by using a costimulatory domain, CD3z, or a combination thereof as an intracellular signaling domain. The CD3z (zeta) may function as an NK cell activation domain. Of the costimulatory domains, CD27 may be, for example, a nucleotide sequence represented by SEQ ID NO: 2, and CD3z may be, for example, a nucleotide sequence represented by SEQ ID NO: 3, but is not limited thereto. In addition, in a specific aspect, the chimeric antigen receptor may be, for example, a nucleotide sequence represented by SEQ ID NO: 6.

The recombinant vector according to an aspect may further include a TNF-related apoptosis-inducing ligand (TRAIL) gene to produce a TRAIL protein in cells into which the recombinant vector has been introduced. In addition, the genetically modified immune cells that produce the TRAIL protein, for example, genetically modified natural killer cells, may be introduced by the introduction of a recombinant vector containing the TRAIL gene. The TRAIL gene may be, for example, a nucleotide sequence represented by SEQ ID NO: 4, but is not limited thereto.

Genetically modified immune cells according to one aspect are i) cytotoxic against cancer cells expressing a cancer cell-specific antigen (eg, FOLR), ii) expressing DR4 or DR5, or iii) a combination thereof. It may be to represent.

In one embodiment, immune cells genetically modified by the introduction of a recombinant vector comprising a single chain variable fragment of a FOLR1 antibody and TRAIL, for example, a genetically modified natural killer cell, is a single chain variable fragment of the FOLR1 antibody or TRAIL. It was confirmed that the recombinant vector including a showed an anticancer effect superior to that of various cancer cells (Example 3). In addition, the NK cells expressing CAR according to one aspect have excellent cytotoxic effects on cancer cells expressing FOLR.

In one aspect, genetically modified immune cells, such as genetically modified natural killer cells, that express chimeric antigen receptor (CAR) containing an antigen-binding domain that specifically binds to cancer cells or produce TRAIL protein. It provides a pharmaceutical composition comprising. The pharmaceutical composition can be used for the prevention and/or treatment of cancer.

The term "prevention" refers to any action that prevents the disease by eliminating the etiology or early detection of cancer.

The term “treatment” refers to any action in which symptoms caused by cancer are improved or beneficially altered.

The term'cancer' refers to a group of diseases characterized by excessive cell proliferation and invasion into surrounding tissues when the normal apoptosis balance is broken. The cancer is, for example, lung cancer, laryngeal cancer, gastric cancer, colon / rectal cancer, liver cancer, gallbladder cancer, pancreatic cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, prostate cancer, kidney cancer, carcinoma derived from epithelial cells such as skin cancer, Group consisting of sarcoma derived from connective tissue cells such as bone cancer, muscle cancer, fat cancer, fibroblast cancer, hematopoietic cells such as leukemia, lymphoma, and multiple myeloma, and tumors occurring in nerve tissue It may be one or more selected from, but is not limited thereto.

Genetically modified immune cells that express chimeric antigen receptor (CAR) containing an antigen-binding domain that specifically binds to cancer cells according to an aspect or produce TRAIL protein, for example, genetically modified natural killer cells The pharmaceutical composition comprising i) exhibits excellent cancer cell killing effect against cancer cells expressing i) a cancer cell-specific antigen (eg, FOLR), ii) expressing DR4 or DR5, or iii) a combination thereof. .

In addition to the genetically modified immune cells that express chimeric antigen receptor (CAR) containing an antigen-binding domain that specifically binds to cancer cells, or produce TRAIL protein, the pharmaceutical composition for preventing or treating cancer Acceptable carriers such as saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and one or more of these components can be mixed and used, and antioxidants as needed , May further include other conventional additives such as a buffer solution. In addition, diluents, dispersants, surfactants, binders, and/or lubricants may be additionally added to prepare injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets. Further, it may be preferably formulated according to each component by an appropriate method in the art or by using a method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA. The pharmaceutical composition of the present invention is not particularly limited in its formulation, but is preferably formulated as an injection or inhalant.

The method of administering the pharmaceutical composition according to an aspect 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 to treat 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 one 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 1,000-10,000 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/cycle, may be dividedly administered 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 an object in need of treatment for cancer, vascular disease, or inflammatory disease, and more specifically, human or non-human primates, mice, rats, dogs, cats, horses, and It means mammals such as cattle.

The pharmaceutical composition according to an aspect comprises a pharmaceutical composition comprising a genetically modified immune cell that expresses a chimeric antigen receptor (CAR) containing an antigen-binding domain that specifically binds to cancer cells or produces a TRAIL protein. to provide. In addition, if the immune cells are included as an active ingredient, it can be used as a cell therapy for the treatment and prevention of cancer. The pharmaceutical composition or cell therapy may be used for the prevention and/or treatment of cancer.

The term “cell therapy” refers to a therapeutic agent using autologous, allogenic, and xenogenic cells to restore tissue functions, and refers to a therapeutic agent used to suppress cancer. Including the immune cells, for example, genetically modified natural killer cells as an active ingredient, can be used as a cell therapy for the treatment and prevention of cancer.

The cell therapy agent may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be used by mixing, for example, saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, human serum albumin (HSA), and one or more of these components. In addition, other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as needed.

In addition, the cell therapy agent, if necessary, depending on the formulation, suspending agent, solubilizing aid, stabilizer, isotonic agent, preservative, adsorption inhibitor, surfactant, diluent, excipient, pH adjuster, painless agent, buffer, sulfur-containing reducing agent, antioxidant Etc. can be added appropriately. Examples of the suspending agent include methyl cellulose, polysorbate 80, hydroxyethyl cellulose, gum arabic, tragant mal, sodium carboxymethyl cellulose, polyoxyethylene sorbitan monolaurate, and the like.

Examples of the solution adjuvant include polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinic acid amide, polyoxyethylene sorbitan monolaurate, macrogol, castor oil fatty acid ethyl ester and the like. As a stabilizer, dextran 40, methylcellulose, gelatin, sodium sulfite, sodium metasulfate, etc. are mentioned.

Examples of the tonicity agent include D-mannitol and sorbitol.

Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, sorbic acid, phenol, cresol, and chlorocresol.

Examples of the adsorption inhibitor include human serum albumin, lecithin, dextran, ethylene oxide propylene oxide copolymer, hydroxypropyl cellulose, methyl cellulose, polyoxyethylene hydrogenated castor oil, polyethylene glycol, and the like.

Examples of the sulfur-containing reducing agent include N-acetylcysteine, N-acetylhomocysteine, thioctoic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodium thiosulfate, glutathione, carbon atom number. And those having a sulfhydryl group such as 1 to 7 thioalkanoic acid.

Examples of antioxidants include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbic acid palmitate, L-ascorbic acid. And chelating agents such as stearate, sodium hydrogen sulfite, sodium sulfite, triamyl gallic acid, propyl gallic acid or sodium ethylenediamine tetraacetate (EDTA), sodium pyrophosphate, and sodium metaphosphate.

In addition, when the cell therapy product is 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 ~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 injection product according to the present invention may be prepared in the form of a filled injection by taking an amount commonly known in the art according to the constitution of the patient and the type of defect.

The term “therapeutic agent” or “pharmaceutical composition” refers to a molecule or compound that imparts several beneficial effects upon administration to a subject. The beneficial effect is to enable diagnostic decisions; Improvement of a disease, symptom, disorder or condition; Reducing or preventing the onset of a disease, symptom, disorder or condition; And the response of a disease, symptom, disorder or condition in general.

As used herein, “treatment” or “treating” or “relaxing” or “improving” are used interchangeably. These terms refer to methods of obtaining beneficial or desired results, including but not limited to therapeutic benefits and/or prophylactic benefits. A therapeutic benefit refers to any therapeutically significant improvement or effect thereon of one or more diseases, disorders or symptoms under treatment. In a prophylactic benefit, the composition may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of the disease, even if the disease, condition, or symptom is not yet present.

The term “effective amount” or “therapeutically effective amount” refers to an amount of an agent sufficient to produce an advantageous or desired result. The therapeutically effective amount may vary according to one or more of the subject and condition to be treated, the weight and age of the subject, the severity of the condition, the mode of administration, and the like, which can be easily determined by those skilled in the art. Further, the term applies to the capacity to provide an image for detection by any of the imaging methods described herein. The specific dosage may vary depending on one or more of the particular agent selected, the dosage regimen that follows, whether it is administered in combination with other compounds, the timing of administration, the tissue being imaged, and the body delivery system carrying it.

In one embodiment, the genetically modified immune cells expressing CAR-NK have a synergistic effect when administered in combination with TRAIL.

In addition, in another aspect, a chimeric antigen receptor (CAR) containing an antigen-binding domain that specifically binds to folate receptor alpha (FOLR1) according to an aspect is expressed or genetically produced by a TRAIL protein. A composition for diagnosis of cancer and a kit for diagnosis of cancer, including modified immune cells, immune cells, for example, genetically modified natural killer cells. Another aspect of the present invention is a gene that expresses a chimeric antigen receptor (CAR) or produces a TRAIL protein comprising an antigen-binding domain that specifically binds to one aspect of folate receptor alpha (FOLR1). A method of providing information for diagnosis of cancer, comprising contacting a composition comprising entirely modified immune cells with a sample isolated from an individual.

The term “diagnosis” refers to determining an object's susceptibility to a particular disease or condition, determining whether an object currently has a particular disease or condition, or the prognosis of an object suffering from a particular disease or condition. determining prognosis, or therametrics (eg, monitoring the condition of an object to provide information about treatment efficacy).

Another aspect is the step of introducing a recombinant vector comprising i) an antigen-binding domain that specifically binds to cancer cells, ii) including a TRAIL gene, or iii) a combination thereof, into immune cells isolated from the human body. It provides a method for producing a genetically modified immune cell comprising, for example, a genetically modified natural killer cell.

The method may further include the step of administering TRAIL in combination, and the TRAIL may be administered simultaneously, separately or sequentially in combination with the immune cells. When the genetically modified immune cells and TRAIL are administered in combination with two active ingredients, the therapeutic effect on cancer may be additional or synergistic. The genetically modified immune cells can be administered to individuals in need thereof by various routes. All modes of administration can be expected and can be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injection.

Redundant content is omitted in consideration of the complexity of the present specification, and terms not otherwise defined herein have meanings commonly used in the technical field to which the present invention belongs.

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.

실시예 1. 암세포 특이적 표적항원의 확인Example 1. Identification of target antigen specific to cancer cells

Expression in pancreatic cancer cell lines PNAC-1, Aspc1 and Miapaca2 and patient-derived cancer cell lines (Patient-derived cells: PDC) SNU213, 410, 2491 and 110621 to select target antigens commonly expressed in various types of cancer cells Experiments to identify these markers were performed through western blotting and fluorescence activated cell sorter (FACS) analysis.

After pretreatment for analysis of pancreatic cancer cell lines PNAC-1, Aspc1 and Miapaca2 and patient-derived cells (PDC), SNU213, 410, 2491 and 110621, RIPA supplemented with protease inhibitor (Thermo Scientific) Lysis was performed by adding lysis buffer (Sigma). A total of 50 μg of protein was analyzed for immunoblotting, incubated overnight at 4° C. and detected using a primary antibody specific for FOLR1 (ABcam), and then at room temperature with a horseradish peroxidase-conjugated secondary antibody. Incubated for 2 hours. Protein was visualized by chemiluminescence using a Western ECL substrate (Bio-Rad), and the obtained luminescence image was analyzed with LAS-3000 (Fujifilm). The band intensity was quantified using ImageJ software to confirm the result, and the result is shown in FIG. 1.

Thereafter, the stained cells were collected using a Guava easyCyte flow cytometer (Merck Millipore) and analyzed with FlowJo version 10.2 (TreeStar). FOLR1, DR4 and DR5 were detected by APC-conjugated anti-human FOLR1, APC-conjugated anti-human DR4, and PE-conjugated anti-human DR5 antibodies, respectively, and FACS analysis was performed and the results are shown in FIG. Indicated.

As shown in FIG. 1, it was confirmed that the pancreatic cancer cell line PNAC-1, Aspc1, and PDC SNU410 and 110621 hardly showed FOLR1. In addition, in Miapaca2, α-FOLR1 was shown to be weak, but in cancer cell lines SNU2491 and SNU213 derived from patients, α-FOLR1 was found to be at a strong level.

In addition, as confirmed in Fig. 2, as a result of confirming in cancer cell lines PNAC-1, Aspc1, Miapaca2, SNU213, 410, 2491 and 110621, unlike FOLR1 expression in some cancer cells, TRAIL receptors DR4 and DR5 are the seven cancer cell lines. It was confirmed that all were expressed in large amounts.

Taken together, it was confirmed that FOLR1 was expressed only in some pancreatic cancer cell lines, but it was confirmed that TRAIL receptors DR4 and DR5 appeared in various types of cancer cell lines. Accordingly, it was confirmed that FOLR1 can be used for specific targeting of pancreatic cancer cell lines, and it was confirmed that DR4 and DR5 can be used as cancer cell-specific target antigen markers for targeting more various types of cancer cells.

실시예 2. 키메릭 항원 수용체(Chimeric Antigen Receptor : CAR)-NK 세포의 생산Example 2. Production of chimeric antigen receptor (CAR)-NK cells

In the case of producing CAR-NK cells expressing the chimeric antigen receptor specific to FOLR1 identified in Example 1, an experiment was designed to confirm whether a cancer cell-specific killing effect could be exhibited. In addition, DR4 and DR5, which are identified as markers in various cancer cells, are known as TRAIL receptors. When TRAIL is expressed in NK cells, which can exhibit anticancer effects by exhibiting cytotoxicity against cancer cells, expression of FOLR1-specific chimeric antigen receptor In order to confirm that NK cells expressing TRAIL together with the CAR-NK described above can be effectively killed various cancer cells, an experiment for producing CAR-NK was designed.

A second-generation CAR vector expressing a fusion protein composed of a single chain variable fragment (scFv) of the FOLR1 antibody and a signal transduction region composed of CD27 and CD3z was constructed. FOLR1-CAR DNA, including scFv, CD27 and CD3z of FOLR1, was purchased from Creative biolabs (USA). Lentiviral expression vectors containing pLVXPuro (Cat. #632164) were obtained from Addgene. In order to generate the GFP-expressing CAR-NK direct folate receptor, a CAR vector sequence was prepared by inserting a DNA fragment containing GFP below the P2A sequence. Thereafter, a vector was prepared in which the TRAIL gene was further introduced into the vector into which the single-chain variable fragment for FOLR1 was introduced, and a vector to express only the TRAIL gene was prepared and inserted into GFP-expressing CAR-NK or NK cells. The transformed NK cells and the vectors introduced into the NK cells are shown in Fig. 3, respectively.

The experimental method is specifically as follows: Invitrogen Lipofectamin 3000 reagent (Invitrogen, L3000-015) 293T cells through a lentivirus-expression vector and viral power lentiviral packaging MIX (Invitrogen, 44)-mediated transformation method -2050) was used to transform. After 48 hours, the culture medium was harvested and centrifuged for 5 minutes at 1300 rpm. A solution in which the centrifuged supernatant was passed through a 0.45 μm filter, and then a solution supplemented with IL-2 200 IU/ml and 8 μg/ml polybrene (santa cruz, sc-134220) in a volume ratio of 1:1 In addition to, 2 x 10 ⁵ NK-92 cells were incubated with lentiviral vector. Cells were added with 2 μg/ml concentration of puromycin (Sigma-Aldrich, USA), cultured for 2 weeks, and then selected. Subsequently, fluorescence activated cell sorter (FACS) analysis, western blotting, and multifocal fluorescence confirmed through a primary antibody against α-CD3zeta in CAR-NK cells prepared through the above selection process. The expression of CAR was confirmed through a microscope, and the specific experimental method is as follows.

Expression of NK cell lines transfected with CAR was evaluated using a Guava easyCyte Flow cytometer (Merck Millipore), and analyzed with FlowJo version 10.2 (TreeStar). 2 x 10 ⁵ cells were analyzed and GFP expressing cells were counted into the FL1 channel. In addition, 3 x 10 ⁴ NK cells transduced with CAR were inoculated into an IVID dish, and expression was confirmed with a multifocal fluorescence microscope (Carl-zeiss, Germany). The CAR-transduced 3×10 ⁴ NK cells were lysed using RIPA lysis buffer (Sigma) supplemented with a protease inhibitor (Thermo Scientific). A total of 40 μg of protein was analyzed for immunoblotting, incubated overnight at 4° C., and detected using CD3ZETA (Santa cruz, 1: 1000), and then horseradish peroxidase-binding secondary antibody (Bethy Laboratories, 1: 5000) at room temperature for 2 hours. Then, the reacted protein was visualized by chemiluminescence using a Western ECL substrate (Bio-Rad), the visualized luminescence image was analyzed with LAS-3000 (Fujifilm), and the measured band intensity was quantified using ImageJ software. And the results are shown in Figures 4A, 4B and 4C.

4A As shown in FIGS. 4B and 4C, it was confirmed that NK cells into which the chimeric antigen receptor was introduced express the chimeric antigen receptor of the present invention, and NK cells into which TRAIL was introduced also secrete the substance. , It was confirmed that the vector prepared according to this example can effectively genetically modify CAR-NK cells.

실시예 3. 제조된 CAR-NK세포를 통한 암세포주의 세포독성 효과의 확인Example 3. Confirmation of the cytotoxic effect of cancer cell lines through the prepared CAR-NK cells

A cytotoxicity test was performed to confirm whether the CAR-NK cells prepared in Example 2 can exhibit actual anticancer effects even in various cell lines. CAR-transduced NK-92 cells were added to target cancer cells (2 x 10 ⁵ cells), which are cancer cell lines SNU2491 and SNU2469 derived from patients, at a ratio of 2.5:1 and 5:1, and co-cultured in culture medium for 4 hours. I did. SNU2491 cells are cancer cell lines with high expression of FOLR1. Then, in order to visualize the NK cells, FITC was labeled with conjugated CD56 (Biolegend). In order to evaluate the killing effect of cancer cells, target cells were isolated and stained with 7-aminoactinomycin D (7-AAD), a red fluorescent probe that labels dead cells and necrotic target cells in cytotoxicity analysis, and Analysis was performed using FACS. FITC-CD556-labeled cells were measured in the FL1 channel and 7-AAD stained cells were measured in the FL3 channel, and the results are shown in FIG. 5A.

In addition, specific cell lysis was confirmed for a cancer cell line with low expression of FOLR1 and DR4/5 (SNU2469) and a cancer cell line with high expression of FOLR1 and DR4/5 (SNU2491), which is shown in FIG. 5B. Indicated.

As shown in Figure 5a, the SNU2491 cancer cell line with high expression of FOLR1 and DR4/5 induces a higher degree of cytotoxicity than when co-cultured with NK cells expressing Fra GFP CAR and TRAIL compared to a control expressing only GFP. Confirmed. In addition, it was confirmed that the group containing the single-chain variable fragment of the FOLR1 antibody (NK-Fra CAR) and the group expressing TRAIL (NK-TRAIL) exhibited better apoptosis effect. In particular, it was confirmed that NK cells (NK-Combi) expressing a combination of Fra and TRAIL exhibit an improved synergistic effect, which is more than twice as excellent as the apoptosis effect of the FRa GFP group.

In addition, as shown in FIG. 5B, it was confirmed that there was little difference between the control group (GFP) and the group expressing the Fra GFP CAR in SNU2469, which showed low expression of FOLR1 and DR4/5. In contrast, in the SNU2491 cancer cell line with high expression of FOLR1 and DR4/5, NK cells expressing a combination of Fra and TRAIL (NK-Combi) are about twice as high as those expressing each of the Fra and TRAIL cells. It was confirmed that a synergistic cell killing effect was exhibited.

Overall, in CAR-NK cells, CAR-NK cells into which the Fra GFP vector was introduced, CAR-NK cells into which the TRAIL-GFP vector was introduced, and NK cells expressing a combination of Fra and TRAIL (FRa-TRAIL-GFP) in order. As a result, it was confirmed that the cytotoxic effect on cancer cells was excellent. In addition, in the cancer cell line to be treated, it was confirmed that the CAR-NK cells prepared according to this example can exhibit better apoptosis effect in the cancer cell line with high expression of FOLR1 and DR4 and 5.

실시예 4. 제조된 CAR-NK세포를 통한 동물 세포에서 암세포주 증식 억제 효과의 확인Example 4. Confirmation of the effect of inhibiting cancer cell line proliferation in animal cells through the prepared CAR-NK cells

4.1 동물 세포에서 암세포 크기 감소 효과의 확인4.1 Confirmation of the effect of reducing cancer cell size in animal cells

In order to confirm whether the CAR-NK cells transfected with the CAR prepared in Example 2 can effectively inhibit the proliferation of cancer cells, the CAR-NK cells were injected into a cancer disease model mouse and the volume of cancer cells of the disease model mouse was determined. An experiment to measure was performed.

In order to prepare cancer disease model mice, after culturing SNU2491 cancer cells 1.5 to 10 ⁷ in a medium mixed with 50% Matrigel solution (BD Biosciences), then female NSG (NOD / SCID / IL-2Rgcnull) mice It was injected subcutaneously in the left flank. Thereafter, when the tumor size of the mouse reached 0.1 cm ³ , the cultured NK cells prepared in Example 2 were administered intravenously three times at 3 days intervals at a density of 5 Х 10 ⁶ cells. Each individual tumor volume was measured every 3 days, the volume of the tumor was monitored for a total of 18 days, and the volume of the tumor was determined by the formula V = (A Х B ² )/2. In the above formula, A is the largest diameter in the tumor and B is the shortest diameter in the tumor. On the 28th day after administration, the mice were sacrificed, and the mice were dissected to further investigate the tumor, and the results of confirming the volume of cancer cells in the group to which the CAR-NK cells were administered are shown in FIG. 6A, wherein the CAR-NK cells were administered. The results showing the tumor volume measured after 3, 6, 9, 12, 15 and 18 days are shown in Fig. 6B. Mock relates to a group in which NK cells were not treated as a negative control group.

As shown in FIG. 6A, it was confirmed that the volume of cancer cells was reduced in the group to which NK cells into which GFP and TRAIL were introduced compared to the control Mock. It was confirmed that the volume of cancer cells in the group administered with the Fra GFP CAR and the group expressing Fra GFP + TRAIL was significantly smaller than that of the control Mock. In addition, as shown in FIG. 6B, when the NK cell group into which GFP, TRAIL, and Fra GFP CAR was introduced was introduced, it was confirmed that the volume of the first cancer cell decreased by about 20% when 18 days elapsed after the administration of NK cells. . Particularly, in the group into which Fra GFP + TRAIL was introduced, the volume of cancer cells significantly decreased from the beginning of transduction, and when 18 days had elapsed after administration, the volume of cancer cells decreased by about 60%.

4.2 동물 세포에서 암세포 살상 효과의 확인4.2 Confirmation of cancer cell killing effect in animal cells

In order to confirm whether the CAR-NK cells transfected with the CAR prepared in Example 2 can effectively inhibit the proliferation of cancer cells, the extent of cancer cell tissue cell death or necrosis through the administration of CAR-NK cells in an animal model TUNEL staining and hematoxylin-eosin (H&E) staining were performed to confirm.

In order to evaluate the cell death of cancer tissues in vivo, in the animal model prepared in Example 4.1, when the tumor size of the mouse reaches 0.1 cm ³ , the cultured NK cells prepared in Example 2 were converted to 5 × 10 ⁶ cells. It was administered intravenously 3 times at 3 days intervals at a density of. On the 28th day after NK cell administration, the mice were sacrificed, and the mice were dissected to further investigate the tumor, and TUNEL staining was performed using an in situ cell death detection kit (Roche) according to the manufacturer's instructions. Tumor tissue sections were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 and 0.1% sodium citrate. Subsequently, tumor sections were incubated in a dark room at 37° C. for 1 hour with a TUNEL reaction mixture in a humid atmosphere. After washing three times with PBS, the slides were mounted with DAPI mounting medium (Vector Laboratories) and confirmed with a Zeiss LSM 700 confocal microscope, and the results of the confirmation are shown in FIG. 6C. Mock relates to a group in which NK cells were not treated as a negative control group.

In addition, H&E staining was also performed on the 28th day after administration of NK cells. The tumor was fixed in 4% paraformaldehyde overnight, embedded in paraffin, and then sliced so that the tumor section had a 6 μm cross section using a microtome (Leica). The prepared sections were de-paraffinized with xylene and the sections were rehydrated using a graded ethanol wash. Tumor sections were stained with hematoxylin and eosin (H&E) and observed under an optical microscope (Olympus), and the results are shown in FIG. 6D. Mock relates to a group in which NK cells were not treated as a negative control group.

As shown in Figure 6C, in Tunel staining, it was confirmed that apoptosis appeared in the group administered with each of GFP, Fra CAR, and TRAIL in mice bearing SNU2491 cancer cells, but the combined treatment group administered with Fra GFP + TRAIL (Combi) It was confirmed that the degree of apoptosis of cancer cells was significantly detected. In addition, as shown in FIG. 6D, even when H&E staining was performed, the necrosis of cancer cells was significantly increased in the group (Combi) administered with FRa and TRAIL together compared to the group administered with each of GFP, Fra CAR, and TRAIL. I could confirm that.

Therefore, when the CAR and TRAIL genes of the present invention are simultaneously introduced and expressed, a greater synergistic effect than the cancer cell proliferation inhibitory effect that occurs in the NK cell group introduced with the Fra GFP CAR can occur, resulting in a remarkable anti-cancer effect. It was confirmed through.

Claims

Genetically modified immune cells expressing chimeric antigen receptor (CAR) and TRAIL (TNF-related apoptosis-inducing ligand) containing an antigen-binding domain that specifically binds to cancer cells.
The genetically modified immune cell according to claim 1, wherein the genetically modified immune cell is by introduction of a chimeric antigen receptor recombinant vector comprising an antigen binding domain that specifically binds to a cancer antigen.
The group consisting of a plasmid vector, a cosmid vector, a bacteriophage vector, a herpes simplex virus, and a basinia virus, an adenovirus vector, a retroviral vector, a lentivirus expression vector, and an adeno-associated virus vector. Is selected from, genetically modified immune cells.
The genetically modified immune cell according to claim 1, wherein the chimeric antigen receptor comprises an antigen binding domain that specifically binds to folate receptor alpha (FOLR1).
The genetically modified immune cell of claim 1, wherein the antigen-binding domain is an antibody or fragment of an antibody that specifically binds to a cancer antigen.
The genetically modified immune cell of claim 5, wherein the fragment of the antibody is an scFv.
The genetically modified immune cell of claim 1, wherein the chimeric antigen receptor comprises an intracellular signal transduction domain.
The genetically modified immune cell of claim 7, wherein the intracellular signal transduction domain comprises a costimulatory domain, CD3z, or a combination thereof.
Genetically modified immune cells that produce TRAIL protein.
The genetically modified immune cell according to claim 9, wherein the genetically modified immune cell is by introduction of a recombinant vector containing a TRAIL gene.
The genetically modified immune cells of claim 1 or 9 exhibit cytotoxicity to cancer cells expressing a cancer cell-specific antigen, a genetically modified immune cell.
A pharmaceutical composition for preventing or treating cancer, comprising the genetically modified immune cells of claim 1 or 9 as an active ingredient.
The method of claim 12, wherein the cancer is lung cancer, laryngeal cancer, gastric cancer, colon/rectal cancer, liver cancer, gallbladder cancer, pancreatic cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, prostate cancer, kidney cancer, carcinoma derived from epithelial cells, One or more pharmaceuticals selected from the group consisting of bone cancer, muscle cancer, fat cancer, sarcoma derived from connective tissue cells, leukemia, lymphoma, blood cancer derived from hematopoietic cells such as multiple myeloma, and tumors occurring in nerve tissue Composition.
Cell therapy comprising the genetically modified immune cells of claim 1 or 9 as an active ingredient.
A method of providing information for diagnosis of cancer, comprising the step of contacting the genetically modified immune cells of claim 1 or 9 with a specimen isolated from an individual.
Genetically comprising the step of introducing a recombinant vector containing i) an antigen-binding domain that specifically binds to cancer cells, ii) containing a TRAIL gene, or iii) a combination thereof, into immune cells isolated from the human body Method for producing modified immune cells.
A method for preventing or treating cancer, comprising administering the genetically modified immune cells of claim 1 or 9 to an individual in need thereof.