Use of novel surface protein lrig-1 specifically present in regulatory t cells

Abstract

The present invention relates to a cell therapeutic agent comprising immune cells expressing Lrig-1, as an active ingredient. The cell therapeutic agent comprising immune cells expressing Lrig-1 as an active ingredient, provided in the present invention, maintains or promotes the differentiation of regulatory T cells, or maintains or activates functions thereof, and maintains or reduces the activity of effector T cells, and thus can be very efficiently used for the prevention, amelioration or treatment of various diseases.

Description

Uses of LRIG-1, a novel surface protein specifically present on regulator T cells

[0001] The present invention relates to the use of a novel surface protein Lrig-1 specifically present in regulator T cells, i.e., a cell therapy agent.

[0002]

[0003] Cell therapy involves directly injecting living cells into patients. Cell therapy technology, which cultivates living cells to treat and prevent diseases, is gaining attention as a treatment for various intractable diseases, including cancer, by overcoming the limitations of existing pharmaceuticals. Because cell therapy products rely on living cells as their raw material, they require not only precise cell culture technology but also rigorous quality control. Technical capabilities across the entire process are emerging as a key strategy for commercialization success.

[0004] In addition, cell therapeutics refers to medicines used for treatment, diagnosis, and prevention purposes by proliferating and selecting living autologous, allogenic, and xenogenic cells in vitro or by changing the biological characteristics of cells through other methods to restore the functions of cells and tissues.

[0005] Cell therapies can be categorized into somatic cell therapies and stem cell therapies, depending on whether they are differentiated. They can also be categorized by origin (autologous, allogeneic, or xenogeneic cell therapies) and the type of final cell (immune cells, cardiomyocytes, etc.). However, distinguishing between differentiated and undifferentiated stem cells is difficult in industry. Current legal classifications (such as the Pharmaceutical Affairs Act) do not distinguish between somatic cell therapies and stem cell therapies, instead referring to them collectively as cell therapies.

[0006] Cell therapy has mechanisms such as cell replacement or regeneration, paracrine signaling, immunomodulation, and scaffold-based approaches. In particular, the immunomodulatory mechanism of cell therapy is that specific types of cells used in cell therapy, such as regulatory T cells (Tregs) and dendritic cells (DCs), exert therapeutic effects through immunomodulation. For example, regulatory T cells help regulate the immune response by suppressing excessive inflammation and preventing autoimmunity, and dendritic cells are involved in antigen presentation and immune system activation. These cells can be used to treat autoimmune diseases, inflammatory disorders, and transplant rejection by regulating the immune response.

[0007]

[0008] One object of the present invention is to provide a cell therapeutic agent for preventing or treating a neurological disease or an immune-related disease, which comprises an immune cell expressing Lrig-1 as an active ingredient.

[0009] Another object of the present invention is to provide a cell therapeutic agent in which the immune cells expressing Lrig-1 are expressed on the cell surface of regulatory T cells (Treg) or induced regulatory T cells (iTreg).

[0010] Another object of the present invention is to provide a method for manufacturing the cell therapeutic agent.

[0011]

[0012] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0013]

[0014] Various embodiments of the present invention are described with reference to the drawings. In the following description, various specific details, such as specific configurations, compositions, and processes, are set forth to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and configurations. In other instances, well-known processes and manufacturing techniques are not described in specific detail so as not to unnecessarily obscure the present invention. Reference throughout this specification to an embodiment means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the appearance of an embodiment in various places throughout this specification does not necessarily indicate the same embodiment of the present invention. Additionally, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0015]

[0016] In one embodiment of the present invention, a cell therapeutic agent for preventing or treating a neurological disease is provided, which comprises Lrig-1+ immune cells as an active ingredient.

[0017] In the present invention, "cell therapy" refers to a medicine (US FDA regulation) used for the purposes of treatment, diagnosis, and prevention by isolating, culturing, and manufacturing cells and tissues from humans through special processing, and is a medicine used for the purposes of treatment, diagnosis, and prevention through a series of actions such as proliferating and selecting living autologous, allogeneic, or xenogeneic cells in vitro or changing the biological characteristics of cells through other methods to restore the function of cells or tissues. Cell therapy products are broadly classified into somatic cell therapy products and stem cell therapy products depending on the degree of cell differentiation.

[0018] The cell therapeutic agent of the present invention can preferably be used for the treatment of neurological diseases and immune-related diseases.

[0019] The cell therapy composition of the present invention may further comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" refers to a composition that is non-toxic to cells or humans exposed to the composition. Any carrier known in the art, such as a buffer, preservative, analgesic, solubilizer, isotonic agent, stabilizer, carrier, excipient, lubricant, or preservative, may be used without limitation. The cell therapy composition of the present invention may be manufactured in various formulations using commonly used techniques.

[0020] In addition, the cell therapeutic agent of the present invention can be administered through any route as long as it can induce movement to the diseased site. In some cases, a method of loading the cell therapeutic agent into a vehicle having a means for directing the cell therapeutic agent to the lesion may be considered. Therefore, the cell therapeutic agent of the present invention can be administered through various routes including topical (including buccal, sublingual, cutaneous and intraocular administration), parenteral (including subcutaneous, intradermal, intramuscular, drip, intravenous, intraarterial, intraarticular and intracerebrospinal fluid) or transdermal administration, preferably parenterally, and most preferably directly to the affected site. In one embodiment, the cell therapeutic agent is diluted in a suitable diluent at a concentration of about 1X10 3 Inside 1X108 It can be administered to an individual by suspension at a concentration of cells / ml, and this diluent is used for the purpose of protecting and maintaining the cells and facilitating use when injected into the target tissue. The diluent may include a buffer solution such as physiological saline, phosphate buffer solution, HBSS, plasma, cerebrospinal fluid, or blood components.

[0021] In addition, the cell therapeutic agent of the present invention can be administered by any device so that the active substance can move to the target cell. The preferred method of administration and formulation is an injection. The injection can be prepared using an aqueous solvent such as saline solution, Ringer's solution, Hank's solution or a sterilized aqueous solution, a vegetable oil such as olive oil, a higher fatty acid ester such as ethyl oleic acid, and a non-aqueous solvent such as ethanol, benzyl alcohol, propylene glycol, polyethylene glycol or glycerin, and for mucosal penetration, a non-penetrating agent known in the art suitable for the barrier to pass through can be used, and as a stabilizer to prevent deterioration, ascorbic acid, sodium bisulfite, BHA, tocopherol, EDTA, etc., and an emulsifier, a buffer for pH adjustment, a preservative to inhibit microbial growth such as phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc., can additionally be included.

[0022] In the present invention, the Lrig-1 protein is a transmembrane protein consisting of 1091 amino acids present on the surface of regulatory T cells, and is composed of a leucine-rich repeat (LRR) sequence on the extracellular or lumen side, three immunoglobulin-like domains, a transmembrane sequence, and a cytoplasmic tail. The LRIG gene family consists of LRIG1, LRIG2, and LRIG3, and the amino acids between them are highly conserved. The LRIG1 gene is highly expressed in normal skin, and can regulate the proliferation of epithelial stem cells by being expressed in basal and hair follicle cells. Therefore, it plays an important role in maintaining epidermal homeostasis, and its absence can lead to psoriasis or skin cancer. It has been reported that when the chromosome 3p14.3 where LRIG1 is located is truncated, there is a possibility of developing into cancer cells, and in fact, it has been confirmed that the expression of LRIG1 is greatly reduced in renal cell carcinoma and cutaneous squamous cell carcinoma. However, it has recently been revealed that only about 20-30% of cancers express Lrig-1. Meanwhile, for the purpose of the present invention, the Lrig-1 protein may be a protein existing in humans or mice, but is not limited thereto.

[0023] In another example of the present invention, the Lrig-1 protein may be, but is not limited to, the extracellular domain of the Lrig-1 protein.

[0024] The Lrig-1 extracellular domain of the present invention may be an extracellular domain of an Lrig-1 protein derived from a mammal, for example, a primate such as a human or monkey, or a rodent such as a mouse or rat. For the purposes of the present invention, the extracellular protein of the Lrig-1 protein may be an extracellular domain of an Lrig-1 protein derived from a human or mouse, but is not limited thereto.

[0025] In the present invention, the Lrig-1 protein may be a polypeptide represented by SEQ ID NO: 1 of human origin or a polypeptide represented by SEQ ID NO: 3 of mouse origin, but is not limited thereto.

[0026] In addition, in the present invention, the Lrig-1 protein represented by the sequence number 1 may be encoded by a polynucleotide represented by the sequence number 2, but is not limited thereto.

[0027] Additionally, in the present invention, the Lrig-1 protein represented by the sequence number 3 may be encoded by a polynucleotide represented by the sequence number 4, but is not limited thereto.

[0028] The most important characteristic of all normal organisms is the ability to recognize and eliminate non-self antigens while not reacting harmfully to antigenic substances that constitute the self. This lack of response of the body to self-antigens is called immunologic unresponsiveness or tolerance. Self-tolerance occurs by eliminating lymphocytes that may have specific receptors for self-antigens, or by inactivating the body's ability to respond to self-antigens after exposure to them. If there is a problem in inducing or maintaining self-tolerance, an immune response to self-antigens occurs, and the resulting disease is called an autoimmune disease.

[0029] Since the concept of suppressor T cells, which are T cells capable of controlling and suppressing the effector function of conventional T cells, was first proposed by Gershon in the early 1970s for the treatment of the above autoimmune diseases, research has been conducted in many fields of immunology to elucidate the biological characteristics and functions of regulatory T cells.

[0030] Regulatory T cells (Tregs) play a crucial role in naturally preventing excessive inflammation and immune responses. However, it has been reported that the function and number of regulatory T cells are significantly reduced in autoimmune and chronic inflammatory diseases. Therefore, for patients with immune and inflammatory diseases, it is crucial to ensure normal levels of regulatory T cells, which may be a potential treatment option.

[0031] To date, research has been conducted on genes and proteins that are specific to regulatory T cells, and it has been suggested that substances such as CD25, CTLA4, CD62L, CD38, CD103, GITR, and CD45RB may be markers. However, genes and proteins that can target only regulatory T cells do not yet exist.

[0032] The “neurological disease” that the cell therapy provided in the present invention is intended to prevent, improve, or treat may be a neurodegenerative disease or a neuroinflammatory disease.

[0033] The “neurodegenerative disease” and “neuroinflammatory disease” of the present invention may be selected from the group consisting of stroke, dementia, Alzheimer's disease, multiple sclerosis, Parkinson's disease, Huntington's disease, Niemann-Pick disease, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy dementia, amyotrophic lateral sclerosis, paraneoplastic syndrome, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, nervous system autoimmune disease, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury, and tauopathy. However, it is not limited to this.

[0034] In the present invention, the “multiple sclerosis” may be selected from the group consisting of relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple sclerosis, myelitis, neuromyelitis optica, transverse myelitis, and encephalomyelitis, but is not limited thereto.

[0035] Multiple sclerosis (MS) is a chronic inflammatory disease that affects the central nervous system, including the brain, spinal cord, and optic nerves. It is also known as a type of neurological disease. Because MS is known to be caused by immune cells damaging nerves, controlling the immune response could potentially treat various neurological diseases caused by immune system disorders (particularly immune overactivation). Clinically, more than 80% of MS cases have relapsing-remitting MS, characterized by recurrent flare-ups and improvements. An estimated 2.3 million people worldwide (as of 2018) are estimated to have MS. The incidence is rapidly increasing among young adults aged 20-40, and with a lifespan similar to that of the general population, MS leads to lifelong treatment regimens, making it the largest market for central nervous system diseases. Initial symptoms at onset include sensory symptoms or sudden vision loss due to optic neuritis, followed by motor disorders such as hemiplegia, paraplegia, and quadriplegia, as well as speech disorders and cognitive dysfunction.

[0036] The cause of multiple sclerosis is known to be activated T lymphocytes that pass through the blood-brain barrier (BBB) ​​and enter the central nervous system, damaging the nerves. Since multiple sclerosis lesions can occur anywhere in the central nervous system, various symptoms appear depending on the location of the lesion. If the lesion is in the optic nerve, symptoms such as decreased vision and central blind spots appear. If the lesion is in the cerebrum, symptoms such as sensory and motor disorders and cognitive dysfunction appear. If the lesion is in the brainstem, double vision and dizziness may appear. If it is in the cerebellum, dizziness and tremors may appear. If it is in the spinal cord, symptoms such as sensory and motor disorders and urinary problems may appear.

[0037] Neuromyelitis optica (NMO) is a serious inflammatory disease of the central nervous system, characterized by recurrent, severe, and disabling relapses. Previously, NMO was considered a clinical variant of multiple sclerosis.

[0038]

[0039] In one embodiment of the present invention, a cell therapeutic agent for preventing or treating an immune-related disease is provided, which comprises Lrig-1+ immune cells as an active ingredient.

[0040] In particular, the cell therapeutic agent comprising Lrig-1+ immune cells provided in the present invention as an active ingredient can treat various immune-related diseases, including autoimmune diseases caused by excessive activity of effector T cells, by suppressing the function of effector T cells.

[0041] The “immune-related disease” of the present invention may be at least one selected from the group consisting of autoimmune disease, graft-versus-host disease, organ transplant rejection, asthma, atopy, and acute or chronic inflammatory disease, but is not limited thereto.

[0042] The autoimmune disease of the present invention may be at least one selected from the group consisting of rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosus, atopic dermatitis, psoriasis, alopecia areata, asthma, Crohn's disease, Behcet's disease, Sjogren's syndrome, Guillain-Barré syndrome, chronic thyroiditis, multiple sclerosis, polymyositis, ankylosing spondylitis, fibromyalgia, and polyarteritis nodosa, but is not limited thereto.

[0043] Inflammatory bowel disease (IBD) is a chronic, abnormal inflammation of the intestines characterized by recurrent episodes of inflammation. Ulcerative colitis and Crohn's disease are representative examples. These immune-related diseases typically develop in people in their 20s. Ulcerative colitis is characterized by inflammation of the colonic mucosa extending from the rectum to the proximal colon, while Crohn's disease can occur anywhere along the gastrointestinal tract, from the mouth to the anus. IBD typically develops in people in their 20s and is thought to be caused by a complex interplay of genetic factors, immunological abnormalities, changes in gut microbiota, and environmental factors such as a Westernized diet. Symptoms often improve and worsen, and if left uncontrolled, treatment such as hospitalization or surgery can be necessary, significantly reducing quality of life. While distributed worldwide, IBD is most prevalent in North America and Northern Europe. Recently, the prevalence and incidence have been rapidly increasing in Asian countries, including Korea, and other developing countries. Furthermore, IBD is believed to be caused by normal gut bacteria inappropriately triggering an immune response.

[0044]

[0045] Meanwhile, in the present invention, “prevention” may include, without limitation, any act of blocking, suppressing or delaying the symptoms of a disease by using the pharmaceutical composition of the present invention.

[0046] Additionally, in the present invention, “treatment” may include, without limitation, any act that improves or benefits the symptoms of a disease by using the pharmaceutical composition of the present invention.

[0047] In the present invention, the pharmaceutical composition may be characterized as being in the form of a capsule, tablet, granule, injection, ointment, powder, or beverage, and the pharmaceutical composition may be characterized as being intended for humans.

[0048] In the present invention, the pharmaceutical composition is not limited to these, but may be formulated and used in the form of oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, etc., external preparations, suppositories, and sterile injection solutions, respectively, according to conventional methods. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, fragrances, etc. for oral administration, and buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. for injections, and bases, excipients, lubricants, preservatives, etc. for topical administration. The formulation of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with the pharmaceutically acceptable carriers described above. For example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injections, it can be manufactured in the form of unit dose ampoules or multiple doses. In addition, it can be formulated in the form of solutions, suspensions, tablets, capsules, sustained-release preparations, etc.

[0049] Meanwhile, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, or mineral oil. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, and the like may be additionally included.

[0050] The routes of administration of the pharmaceutical composition of the present invention include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Oral or parenteral administration is preferred.

[0051] The term "parenteral" in the present invention includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.

[0052] The pharmaceutical composition of the present invention may vary depending on various factors including the activity of the specific compound used, age, body weight, general health, sex, dosage form, administration time, administration route, excretion rate, drug combination, and severity of the specific disease to be prevented or treated, and the dosage of the pharmaceutical composition may vary depending on the patient's condition, body weight, degree of disease, form of medicine, administration route, and period, but may be appropriately selected by those skilled in the art, and may be administered at 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. Administration may be administered once a day or divided into several times. The dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as a pill, a dragee, a capsule, a liquid, a gel, a syrup, a slurry, or a suspension.

[0053]

[0054] In one embodiment of the present invention, a cell therapeutic agent for preventing or treating a neurological disease is provided, which comprises an immune cell expressing Lrig-1 as an active ingredient.

[0055] In another embodiment of the present invention, a cell therapeutic agent is provided, wherein the immune cells expressing Lrig-1 are regulatory T cells (Treg) or induced regulatory T cells (iTreg).

[0056] In another embodiment of the present invention, a cell therapeutic agent is provided, wherein the immune cells expressing Lrig-1 are expressed on the cell surface of regulatory T cells (Treg) or induced regulatory T cells (iTreg).

[0057] In another embodiment of the present invention, a cell therapeutic agent is provided, wherein the neurological disease is a neurodegenerative disease or a neuroinflammatory disease.

[0058] In another embodiment of the present invention, the neurodegenerative disease or neuroinflammatory disease is at least one selected from the group consisting of stroke, dementia, Alzheimer's disease, multiple sclerosis, Parkinson's disease, Huntington's disease, Niemann-Pick disease, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy dementia, amyotrophic lateral sclerosis, paraneoplastic syndrome, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, nervous system autoimmune disease, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury, and tauopathy. It provides cell therapy, which is one of them.

[0059] In another embodiment of the present invention, a cell therapeutic agent is provided, wherein the multiple sclerosis is selected from the group consisting of relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple sclerosis, myelitis, neuromyelitis optica, transverse myelitis, and encephalomyelitis.

[0060] In another embodiment of the present invention, the immune cells are 1x10 4 1x10 10 / cells, 1x10 4 1x10 8 / cells, 1x10 5 1x10 8 / cells, 1x10 5 1x10 7 / cells, 1x10 6 1x10 7 / cells, 1x10 6 9x10 6 / cells, 1x10 6 8x10 6 / Provides cell therapy by administering cells.

[0061] In one embodiment of the present invention, a cell therapeutic agent for preventing or treating an immune-related disease is provided, which comprises an immune cell expressing Lrig-1 as an active ingredient.

[0062] In another embodiment of the present invention, a cell therapeutic agent is provided, wherein the immune cells expressing Lrig-1 are regulatory T cells (Treg) or induced regulatory T cells (iTreg).

[0063] In another embodiment of the present invention, a cell therapeutic agent is provided, wherein the immune cells expressing Lrig-1 are expressed on the cell surface of regulatory T cells (Treg) or induced regulatory T cells (iTreg).

[0064] In another embodiment of the present invention, a cell therapy for an immune-related disease is provided, wherein the immune-related disease is at least one selected from the group consisting of autoimmune disease, graft-versus-host disease, organ transplant rejection, asthma, atopy, and acute or chronic inflammatory disease.

[0065] In another embodiment of the present invention, a cell therapy agent for preventing or treating an immune-related disease is provided, wherein the autoimmune disease is at least one selected from the group consisting of rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosus, atopic dermatitis, psoriasis, alopecia areata, asthma, Crohn's disease, Behcet's disease, Sjogren's syndrome, Guillain-Barré syndrome, chronic thyroiditis, multiple sclerosis, polymyositis, ankylosing spondylitis, fibromyalgia, and polyarteritis nodosa.

[0066] In one embodiment of the present invention, a method for producing a cell therapeutic agent is provided, comprising the steps of: a) obtaining immune cells from a biological sample isolated from a patient; and b) isolating immune cells expressing Lrig-1 protein from the immune cells.

[0067] In another embodiment of the present invention, a method for manufacturing a cell therapeutic agent is provided, wherein the technology for separating cells in step b) is at least one of fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and affinity chromatography.

[0068] In another embodiment of the present invention, a method for producing a cell therapeutic agent is provided, wherein the immune cells expressing Lrig-1 are regulatory T cells (Treg) or induced regulatory T cells (iTreg).

[0069] In another embodiment of the present invention, a method for producing a cell therapeutic agent is provided, wherein the immune cells expressing Lrig-1 are expressed on the cell surface of regulatory T cells (Treg) or induced regulatory T cells (iTreg).

[0070]

[0071] The cell therapeutic agent comprising an immune cell expressing Lrig-1 provided in the present invention as an active ingredient can be used very effectively for the prevention, improvement or treatment of various diseases by maintaining or promoting the differentiation of regulatory T cells or maintaining or activating their function and maintaining or reducing the activity of effector T cells.

[0072]

[0073] Figure 1 shows the expression level of Lrig-1 mRNA according to one embodiment of the present invention.

[0074] Figure 2 shows the expression level of Lrig-1 mRNA according to one embodiment of the present invention.

[0075] Figure 3 shows the expression level of Lrig-1 mRNA according to one embodiment of the present invention.

[0076] Figure 4 shows the expression levels of Lrig-1, Lrig-2, and Lrig-3 mRNA according to one embodiment of the present invention.

[0077] Figure 5 shows the results of comparing the expression levels of Lrig-1 protein in regulatory T cells and non-regulatory T cells according to one embodiment of the present invention.

[0078] Figure 6 shows the expression of Lrig-1 protein on the surface of regulatory T cells according to one embodiment of the present invention.

[0079] Figure 7 shows the mouse iTreg culture period.

[0080] Figure 8 is FACS data showing that induction, differentiation, and proliferation into iTreg were well demonstrated on day D10.

[0081] Figure 9 is data showing the treatment effect of an immunocytochemical treatment agent according to the present invention on multiple sclerosis.

[0082] Figure 10 is data showing the treatment effect of an immunocytochemical treatment agent according to the present invention on inflammatory bowel disease.

[0083] Figure 11 is data comparing the distribution of T cells in the spinal cord when an immunocytochemical treatment agent according to the present invention is administered.

[0084]

[0085] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0086]

[0087] Example

[0088]

[0089] [Preparation Example] T cell subset cell culture

[0090] To confirm that Lrig-1 protein is expressed only in regulatory T cells (Treg), T cell subsets such as Th0, Th1, Th2, Th17, and iTreg were prepared. Unlike naturally isolated nTreg, iTreg refers to cells whose differentiation was artificially induced in a medium containing the following composition.

[0091] T cell subtypes were first isolated from naive T cells obtained from the spleen of mice, and then differentiated into each cell type by adding each of the components in Table 1 to RPMI1640 (Invitrogen Gibco, Grand Island, NY) nutrient medium containing 10% fetal bovine serum (FBS; hyclone, Logan, UT) and culturing for 72 hours in a 5% CO2 incubator at 37°C.

[0092] Differentiated cell composition Th0 anti-CD3, anti-CD28 Th1 IL-12, anti-IL-4 antibody Th2 IL-4, anti-IFNβ Th17 IL-6, TGFβ, anti-IFNβ, anti-IL-4 i Treg IL-2, TGFβ

[0093]

[0094] [Example 1] Confirmation of specific expression of Lrig-1 protein in regulatory T cells

[0095] [Example 1-1] Confirmation of specific expression of Lrig-1 mRNA in regulatory T cells

[0096] We verified whether Lrig-1 protein can act as a biomarker specific for regulatory T cells.

[0097] For the above verification, CD4 beads were sorted from the spleen of mice using magnet-activated cell sorting (MACS). + T cells were isolated. Afterwards, regulatory T (CD4) cells were sorted using a fluorescence-activated cell sorter (FACS) using CD25 antibody. + CD25 + T) cells and non-regulatory T (CD4 + CD25 -T) cells were isolated. Each cell and the cells differentiated in Preparation Example 1 were subjected to mRNA extraction using Trizol, and then gDNA was removed from genomic RNA using a gDNA extraction kit (Qiagen) according to the protocol provided by the company. The mRNA from which gDNA was removed was synthesized into cDNA using a BDsprint cDNA synthesis kit (Clonetech).

[0098] To quantitatively confirm the expression level of Lrig-1 mRNA in the above cDNA, real-time polymerase chain reaction (real-time PCR) was performed.

[0099] The above real-time polymerase chain reaction was performed using SYBR Green (Molecular Probes) according to the protocol provided by the company, under the conditions of 40 cycles of 95°C for 3 minutes, 61°C for 15 seconds, and 72°C for 30 seconds each, using the primers in Table 2 below. The relative gene expression level was calculated using the △CT method and normalized using HPRT, and the results are shown in Figures 3 to 6.

[0100] Primer direction Sequence number Mouse Lrig-1 forward 5' - GAC GGA ATT CAG TGA GGA GAA CCT - 3' SEQ ID NO: 7 reverse 5' - CAA CTG GTA GTG GCA GCT TGT AGG - 3' SEQ ID NO: 8 Mouse Lrig-2 forward 5' - TCA CAA GGA ACA TTG TCT GAA CCA- 3' SEQ ID NO: 9 reverse 5' - GCC TGA TCT AAC ACA TCC TCC TCA- 3' SEQ ID NO: 10 Mouse Lrig-3 forward 5' - CAG CAC CTT GAG CTG AAC AGA AAC - 3' SEQ ID NO: 11 reverse 5' - CCA GCC TTT GGT AAT CTC GGT TAG - 3' SEQ ID NO: 12 Mouse FOXP3 forward 5' - CTT TCA CCT ATC CCA CCC TTA TCC - 3'SEQ ID NO: 13reverse 5' - ATT CAT CTA CGG TCC ACA CTG CTC - 3'SEQ ID NO: 14ACTG1forward 5' - GGC GTC ATG GTG GGC ATG GG - 3'SEQ ID NO: 15reverse 5' - ATG GCG TGG GGA AGG GCG TA - 3'SEQ ID NO: 16

[0101]

[0102] As shown in Figure 1, non-regulatory T (CD4 + CD25 - Regulatory T (CD4) cells compared to T cells + CD25 + It was found that the expression of Lrig-1 was 18.1 times higher in T cells. This was approximately 10 times higher than that of Lag3 and Ikzf4, which are previously known markers of regulatory T cells.

[0103] Additionally, as shown in Figures 2 and 3, the expression of Lrig-1 mRNA was significantly higher in naturally isolated regulatory T cells (nTreg) compared to other types of immune cells, especially in induced regulatory T cells (iTreg).

[0104] Additionally, as shown in Figure 4, among Lrig-1, Lrig-2, and Lrig-3, which belong to the Lrig family, the expression of Lrig-1 was the highest.

[0105] The above results show that the Lrig-1 protein according to the present invention is specifically expressed in regulatory T cells, particularly in naturally existing regulatory T cells.

[0106]

[0107] [Example 1-2] Confirmation of specific expression of Lrig-1 protein in regulatory T cells

[0108] We confirmed that Lrig-1 protein expressed from Lrig-1 mRNA is specifically expressed only in regulatory T cells.

[0109] Using mice injected with FOXP3-RFP, a regulatory T cell-specific transcription factor, in which RFP (Red fluorescence protein) is linked to the FOXP3 promoter, CD4 cells were sorted from the spleen of the mice using magnet-activated cell sorting (MACS) with CD4 beads. + T cells were isolated. Afterwards, regulatory T (CD4) cells were sorted using a fluorescence-activated cell sorter (FACS) using RFP protein. + RFP + T cells and non-regulatory T (CD4) + RFP -T) cells were isolated and obtained. Each of the above cells was stained with the purchased Lrig-1 antibody and the negative control isotype, and the expression level of Lrig-1 was measured using a fluorescence-activated cell sorter, and the results are shown in Fig. 5.

[0110] As shown in Figure 5, for non-regulatory T cells indicated by a dotted line, the expression level of Lrig-1 was almost the same as that of the negative control group, but for regulatory T cells, there were many cells with high expression levels of Lrig-1.

[0111] The above results show that the Lrig-1 protein according to the present invention is specifically expressed in regulatory T cells.

[0112]

[0113] [Example 1-3] Confirmation of specific expression of Lrig-1 protein on the surface of regulatory T cells

[0114] Since Lrig-1 protein must be expressed on the surface of regulatory T cells to be a target for cell therapy, and thus target therapy can be more effective, we confirmed whether Lrig-1 protein was expressed on the surface.

[0115] Each differentiated T cell subtype of the above Preparation Example 1 was stained with anti-CD4-APC and anti-Lrig-1-PE antibodies, and the expression level of Lrig-1 on the surface of each cell was measured using a fluorescence-activated cell sorter (FACS), and the results are shown in Fig. 8.

[0116] As shown in Fig. 6, Lrig-1 expression was expressed in amounts ranging from 0.77 to 15.3 in activated T cells, Th1 cells, Th2 cells, Th17 cells, and naive T cells, whereas it was highly expressed at 83.9 in differentiated T cells (iTreg).

[0117] The above results show that the Lrig-1 protein according to the present invention is not only specifically expressed in regulatory T cells (Treg) cells, but is also expressed at a higher level, particularly on the surface of regulatory T cells.

[0118]

[0119] [Example 2] Therapeutic effect of Lrig-1+ immune cell therapy

[0120] [Example 2-1] Lrig-1 + immune cell culture

[0121] Eight-week-old female C57BlL / 6 mice were prepared, and mouse iTreg were cultured. As shown in the experimental plan in Fig. 7, specifically, from D-3 to D0, cells and Dynabeads were cultured at a 1:1 ratio under conditions of mIL-2 (20 ng / mL) and mTGF-β (5 ng / mL), to induce and differentiate iTreg. Subsequently, from D0 to D3, cells and Dynabeads were cultured at a 1:4 ratio under conditions of hIL-2 (1000 U / mL) and mTGF-β (5 ng / mL), to stimulate and expand iTreg. From D3 to D7, iTreg resting was performed under hIL-2 (1000 U / mL) conditions, and from D7 to D10, iTreg restimulation was performed by culturing cells and Dynabeads at a 1:1 ratio under hIL-2 (1000 U / mL) and mTGF-β (5 ng / mL) conditions.

[0122] As a result of FACS analysis on day 10, it was confirmed that induction, differentiation, and proliferation into mouse iTreg had occurred, as shown in Figure 8.

[0123]

[0124] [Example 2-2] Verification of the therapeutic effect of Lrig-1 + immune cells on neurological diseases.

[0125] First, we aimed to verify the therapeutic effect of multiple sclerosis in an Experimental Autoimmune Encephalomyelitis (EAE) mouse model. To create a multiple sclerosis mouse model, 7-week-old C57BL / 6 mice were injected subcutaneously with MOG peptide on day 0, and Mycobacterium tuberculosis toxin was injected intraperitoneally. On day 2, the Mycobacterium tuberculosis toxin was injected once more to enhance the immune system of the mice. On day 9, the mice were injected with 1x10 mouse iTreg induced, differentiated, and expanded in Example 2-1. 6 / cells and 5x10 6 / cells concentration, endo-free PBS was administered as a control group, and anti-IL-17a antibody was administered as a positive control group.

[0126] Then, from the 10th day, the clinical score was calculated according to the evaluation criteria below to evaluate the treatment effect on multiple sclerosis in mice, and the results are shown in Fig. 9.

[0127] <Evaluation Criteria>

[0128] 0 points: No symptoms and moves normally

[0129] 1 point: Part of the tip of the tail is paralyzed and hangs down.

[0130] 2 points: The entire tail is paralyzed and the entire tail is drooping.

[0131] 3 points: Partial paralysis of one hind leg, but able to respond to stimulation

[0132] 4 points: One hind leg is completely paralyzed, does not respond to stimuli, and drags the leg.

[0133] 5 points: All hind legs are paralyzed, so the body is dragged around with the front legs.

[0134] 6 points: The animal has difficulty dragging itself with its front legs, but the front legs still respond to stimulation.

[0135] 7 points: The mouse cannot move, but one forelimb responds to stimulation.

[0136] 8 points: The mouse is immobile and neither forelimb responds to stimulation.

[0137] 9 points: Mouse cannot move and breathing is irregular.

[0138] 10 points: Mouse dies

[0139] As shown in Figure 9, when the immune cell therapeutic agent according to the present invention was administered to a multiple sclerosis-induced model, it was confirmed that multiple sclerosis was cured, and in particular, from day D21, mouse iTreg was increased to 5x10 6 / cells concentration as well as 1x10 6 / It was confirmed that even when cells were administered, multiple sclerosis and other neurological diseases, especially degenerative brain diseases, were cured.

[0140]

[0141] [Example 2-2] Verification of the therapeutic effect of Lrig-1 + immune cells on autoimmune diseases.

[0142] In order to confirm the therapeutic effect of iTreg induced, differentiated and proliferated in Example 2-1 on autoimmune diseases, CD45RB (high) cells were adoptively transferred into RAG-1- / - mice to induce inflammatory bowel disease (IBD), an autoimmune disease, and then 1x10 mouse iTreg induced, differentiated and proliferated in Example 2-1 were injected. 6 / cells and 5x10 6 / cells concentration, endo-free PBS was administered as a control group, and anti-IL-17a antibody was administered as a positive control group.

[0143] As shown in Fig. 10, it was confirmed that the immune cell therapeutic agent according to the present invention significantly inhibited the body weight reduction effect in mice induced with inflammatory bowel disease. Through this, it can be seen that the immune cell therapeutic agent according to the present invention can effectively prevent, improve, or treat immune-related diseases such as autoimmune diseases, graft-versus-host diseases, organ transplant rejection, asthma, atopy, or acute or chronic inflammatory diseases induced by excessive activation and expression of various immune cells and inflammatory cells.

[0144]

[0145] [Example 3] Confirmation of T cell distribution in the spinal cord after administration of immunocytochemical therapy

[0146] In order to determine the effect of the immune cells induced, differentiated, and proliferated in Example 2-1 above on the distribution of T cells within the spinal cord, the distribution of CD4+ Fox3+ Tregs and Foxp3+ Lrig-1+ was confirmed in total T cells and CD4 T cells.

[0147] As shown in Fig. 11, it was confirmed that the immune cells provided by the present invention can suppress the function of effector T cells by increasing the distribution of Tregs in the spinal cord, thereby ultimately treating neurological diseases including multiple sclerosis and autoimmune diseases including inflammatory bowel disease.

[0148]

[0149] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A cell therapy agent for the prevention or treatment of neurological diseases, comprising immune cells expressing Lrig-1 as an active ingredient.

2. In paragraph 1, A cell therapeutic agent, wherein the immune cells expressing the above Lrig-1 are regulatory T cells (Treg) or induced regulatory T cells (iTreg).

3. In paragraph 2, A cell therapeutic agent wherein the immune cells expressing the above Lrig-1 are expressed on the cell surface of regulatory T cells (Treg) or induced regulatory T cells (iTreg).

4. In paragraph 3, The above neurological disease is a neurodegenerative disease or neuroinflammatory disease, cell therapy.

5. In paragraph 4, A cell therapeutic agent, wherein the neurodegenerative disease or neuroinflammatory disease is at least one selected from the group consisting of stroke, dementia, Alzheimer's disease, multiple sclerosis, Parkinson's disease, Huntington's disease, Niemann-Pick disease, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy dementia, amyotrophic lateral sclerosis, paraneoplastic syndrome, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, nervous system autoimmune disease, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury, and tauopathy.

6. In paragraph 5, A cell therapeutic agent, wherein the above multiple sclerosis is selected from the group consisting of relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple sclerosis, myelitis, neuromyelitis optica, transverse myelitis, and encephalomyelitis.

7. A cell therapy agent for preventing or treating immune-related diseases, comprising immune cells expressing Lrig-1 as an active ingredient.

8. In paragraph 7, A cell therapeutic agent, wherein the immune cells expressing the above Lrig-1 are regulatory T cells (Treg) or induced regulatory T cells (iTreg).

9. In paragraph 8, A cell therapeutic agent wherein the immune cells expressing the above Lrig-1 are expressed on the cell surface of regulatory T cells (Treg) or induced regulatory T cells (iTreg).

10. In paragraph 9, A cell therapy agent for preventing or treating an immune-related disease, wherein the immune-related disease is at least one selected from the group consisting of autoimmune disease, graft-versus-host disease, organ transplant rejection, asthma, atopy, and acute or chronic inflammatory disease.

11. In paragraph 10, A cell therapy agent for preventing or treating an immune-related disease, wherein the autoimmune disease is at least one selected from the group consisting of rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosus, atopic dermatitis, psoriasis, alopecia areata, asthma, Crohn's disease, Behcet's disease, Sjogren's syndrome, Guillain-Barré syndrome, chronic thyroiditis, multiple sclerosis, polymyositis, ankylosing spondylitis, fibromyalgia, and polyarteritis nodosa. 12.a) A step of obtaining immune cells from a biological sample isolated from a patient; and b) A method for producing a cell therapeutic agent, comprising the step of isolating immune cells expressing Lrig-1 protein from the above immune cells.

13. In paragraph 12, A method for manufacturing a cell therapeutic agent, wherein the technology for separating cells in step b) is at least one of FACS (fluorescence-activated cell sorting), MACS (magnetic-activated cell sorting), and affinity chromatography.

14. In paragraph 13, A method for manufacturing a cell therapeutic agent, wherein the immune cells expressing the above Lrig-1 are regulatory T cells (Treg) or induced regulatory T cells (iTreg).

15. In paragraph 14, A method for manufacturing a cell therapeutic agent, wherein the immune cells expressing the above Lrig-1 are expressed on the cell surface of regulatory T cells (Treg) or induced regulatory T cells (iTreg).

16. A method for preventing or treating a neurological disease comprising administering to a subject immune cells expressing Lrig-1.

17. In paragraph 16, A method wherein the immune cells expressing the above Lrig-1 are regulatory T cells (Treg) or induced regulatory T cells (iTreg).

18. In paragraph 17, A method wherein the immune cells expressing the above Lrig-1 are expressed on the cell surface of regulatory T cells (Treg) or induced regulatory T cells (iTreg).

19. In paragraph 18, The above-mentioned brain and nervous system disease is a neurodegenerative disease or a neuroinflammatory disease.

20. In paragraph 19, A method according to claim 1, wherein the neurodegenerative disease or neuroinflammatory disease is at least one selected from the group consisting of stroke, dementia, Alzheimer's disease, multiple sclerosis, Parkinson's disease, Huntington's disease, Niemann-Pick disease, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy dementia, amyotrophic lateral sclerosis, paraneoplastic syndrome, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, nervous system autoimmune disease, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury, and tauopathy.

21. In paragraph 20, A method wherein the above multiple sclerosis is selected from the group consisting of relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple sclerosis, myelitis, neuromyelitis optica, transverse myelitis, and encephalomyelitis.

22. A method for preventing or treating an immune-related disease comprising administering to a subject immune cells expressing Lrig-1.

23. In paragraph 22, A method wherein the immune cells expressing the above Lrig-1 are regulatory T cells (Treg) or induced regulatory T cells (iTreg).

24. In paragraph 23, A method wherein the immune cells expressing the above Lrig-1 are expressed on the cell surface of regulatory T cells (Treg) or induced regulatory T cells (iTreg).

25. In paragraph 24, A method for preventing or treating an immune-related disease, wherein the immune-related disease is at least one selected from the group consisting of autoimmune disease, graft-versus-host disease, organ transplant rejection, asthma, atopy, and acute or chronic inflammatory disease.

26. In paragraph 25, A method for preventing or treating an immune-related disease, wherein the autoimmune disease is at least one selected from the group consisting of rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosus, atopic dermatitis, psoriasis, alopecia areata, asthma, Crohn's disease, Behcet's disease, Sjogren's syndrome, Guillain-Barré syndrome, chronic thyroiditis, multiple sclerosis, polymyositis, ankylosing spondylitis, fibromyalgia, and polyarteritis nodosa.

27. In either of paragraphs 21 or 26, The above method is a method for preventing or treating an immune-related disease, which comprises injecting or infusing the immune cells orally, intravenously, intramuscularly, intraarterially, intraspinally, intramedullaryly, intrathecally, intracardiacly, transdermally, subcutaneously, intraperitoneally, intranasally, intestinally, topically, sublingually, rectally, intradermally, intraarticularly, intrasynovially, intrasternally, intrathecally, intralesionally, and intracranially into a patient suffering from a neurological disease.

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