WO2022055093A1 - Immunotherapy composition with ability for escaping tumor microenvironment - Google Patents

Immunotherapy composition with ability for escaping tumor microenvironment Download PDF

Info

Publication number
WO2022055093A1
WO2022055093A1 PCT/KR2021/008232 KR2021008232W WO2022055093A1 WO 2022055093 A1 WO2022055093 A1 WO 2022055093A1 KR 2021008232 W KR2021008232 W KR 2021008232W WO 2022055093 A1 WO2022055093 A1 WO 2022055093A1
Authority
WO
WIPO (PCT)
Prior art keywords
cancer
present
immune
cells
immune cell
Prior art date
Application number
PCT/KR2021/008232
Other languages
French (fr)
Korean (ko)
Inventor
정수영
Original Assignee
주식회사 유씨아이테라퓨틱스
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 주식회사 유씨아이테라퓨틱스 filed Critical 주식회사 유씨아이테라퓨틱스
Publication of WO2022055093A1 publication Critical patent/WO2022055093A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4613—Natural-killer cells [NK or NK-T]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • the present invention relates to an immune cell therapy composition.
  • Immune anticancer drugs can be divided into antibody therapeutics that target tumor antigens (Rituximab, etc.), immune checkpoint inhibitors that reactivate immune cells (Immune checkpoint inhibitors, etc.), and immune cell therapies that directly administer immune cells (Immune cell therapy). There is (Oiseth et al, 2017).
  • NK cells natural killer cells
  • the natural killer cells correspond to cells having a function of immediately recognizing and removing abnormal cells such as tumor cells or virus-infected cells in a human immune response. Since these natural killer cells have relatively weak memory function and self-replication function compared to T cells, there is an advantage that side effects such as cytokine storm may be relatively small.
  • One object of the present invention is to provide an immune cell therapy composition.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing, improving or treating cancer.
  • Another object of the present invention is to provide a method for preparing an immune cell therapy composition.
  • Another object of the present invention is to provide a method for screening for immune cell therapy reactivity.
  • Another object of the present invention is to provide a method for preventing or treating cancer.
  • One embodiment of the present invention provides an immune cell therapy composition.
  • the immunotherapy composition may include an antigen binding domain that specifically binds to a cancer cell surface protein
  • a chimeric antigen receptor comprising an intracellular signaling domain includes
  • the immune cells may be natural killer cells (NK cells).
  • NK cells natural killer cells
  • the expression vector may include an operably linked inducible promoter.
  • the antigen-binding domain may be an antibody or antibody fragment that specifically binds to the cancer cell surface protein.
  • the fragment of the antibody may be scFv.
  • Another embodiment of the present invention provides a pharmaceutical composition for preventing or treating cancer.
  • the pharmaceutical composition of the present invention comprises an antigen-binding domain that specifically binds to a cancer cell surface protein
  • It contains a chimeric antigen receptor comprising an intracellular signaling domain; as an active ingredient.
  • the cancer is esophageal cancer, stomach cancer, colorectal cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer , connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's disease, soft tissue sarcoma, lymphoma, and multiple myeloma may be selected from the group consisting of blood cancer.
  • Another embodiment of the present invention provides a food composition for preventing or improving cancer.
  • an antigen binding domain that specifically binds to a cancer cell surface protein
  • It contains a chimeric antigen receptor comprising an intracellular signaling domain; as an active ingredient.
  • the cancer is esophageal cancer, stomach cancer, colorectal cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer , connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's disease, soft tissue sarcoma, lymphoma, and multiple myeloma may be selected from the group consisting of blood cancer.
  • Another embodiment of the present invention provides a method for manufacturing an immune cell therapy product.
  • the manufacturing method of the present invention comprises the steps of culturing immune cells.
  • the immune cells may be natural killer cells.
  • the transfection may be performed using a viral or non-viral transfection method.
  • the chimeric antigen receptor is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoe-2-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • an antigen binding domain that specifically binds to a cancer cell surface protein
  • Another embodiment of the present invention provides a method for screening an immune cell therapy agent.
  • the screening method of the present invention includes an antigen-binding domain that specifically binds to a cancer cell surface protein in a biological sample isolated from a subject of interest; transmembrane domain; and a chimeric antigen receptor comprising an intracellular signaling domain; and treating an immune cell comprising an expression vector containing a gene encoding the same.
  • the method further includes measuring the size of cancer organoids, apoptosis, or the degree of invasion into cancer tissues in the biological sample after the treatment of the immune cells.
  • the measured size of the cancer organoids or the degree of invasion into cancer tissues is reduced compared to before the treatment of the immune cells; or when the degree of apoptosis is increased, determining the immune cell as an immune cell therapy agent.
  • the present invention relates to an immune cell therapy composition, and the immune cell therapy composition of the present invention can very effectively kill not only blood cancer cell lines but also solid cancer cell lines by recognizing cancer cells very effectively.
  • FIG. 1 shows a schematic diagram of an immune cell therapy agent according to an embodiment of the present invention.
  • an antigen binding domain that specifically binds to a cancer cell surface protein; transmembrane domain; and a chimeric antigen receptor comprising an intracellular signaling domain; provides an immune cell therapy composition comprising an immune cell comprising as an active ingredient.
  • an antigen binding domain that specifically binds to a cancer cell surface protein; transmembrane domain; and a chimeric antigen receptor comprising an intracellular signaling domain; provides a pharmaceutical composition for the prevention or treatment of cancer comprising immune cells as an active ingredient.
  • culturing immune cells and transfecting the cultured immune cells with a vector containing a cassette encoding a chimeric antigen receptor.
  • an antigen-binding domain that specifically binds to a cancer cell surface protein; transmembrane domain; and a chimeric antigen receptor comprising an intracellular signaling domain; It provides a method of screening an immune cell therapy, comprising the step of treating immune cells comprising a.
  • an antigen binding domain that specifically binds to a cancer cell surface protein; transmembrane domain; and a chimeric antigen receptor comprising an intracellular signaling domain; It provides a method for preventing or treating cancer, comprising administering a pharmaceutically effective amount of an immune cell therapy composition comprising immune cells as an active ingredient to a subject.
  • One embodiment of the present invention provides an immune cell therapy composition.
  • the immune cell therapy composition of the present invention is an immune cell therapy agent comprising immune cells as an active ingredient, and may be used as a pharmaceutical composition or a food composition.
  • the immune cell therapy composition comprises an antigen binding domain that specifically binds to a cancer cell surface protein; and a transmembrane domain; A chimeric antigen receptor comprising an intracellular signaling domain; includes an immune cell containing as an active ingredient.
  • the “immune cell therapy agent” of the present invention generally uses immune cells such as dendritic cells, natural killer cells, and T cells to activate an immune response in the body and is used for the purpose of treating diseases. means medicine.
  • the "chimeric antigen receptor" of the present invention refers to a receptor engineered to confer a desired specificity for an antigen to immune cells, for example, natural killer cells and T cells.
  • the chimeric antigen receptor comprises an antigen binding domain; transmembrane domain; and an intracellular signaling domain.
  • the "immune cell” of the present invention may include any cell capable of inducing an immune response by being localized around a target cell, tissue or tumor microenvironment by the chimeric antigen receptor, for example, natural It may be a natural killer cell (NK cell), but is not limited thereto.
  • NK cell natural killer cell
  • the "natural killer cells" of the present invention are cells having the function of immediately recognizing and removing abnormal cells such as tumor cells or virus-infected cells in a human immune response, compared with immune cells such as T cells. Therefore, since the memory function and self-replication function are relatively weak, side effects such as cytokine storm may be relatively few, and there is an advantage that allogeneic cell therapy is possible.
  • the antigen-binding domain of the present invention may be derived from an antibody that is localized on the surface of an immune cell and can specifically bind to a desired antigen, that is, a cancer cell surface protein.
  • the "cancer cell surface protein" of the present invention may be, for example, a protein that is specifically present only in cancer cells compared to normal cells.
  • the “antibody” of the present invention refers to a substance produced by stimulation of an antigen in the immune system, and the type thereof is not particularly limited.
  • the term “antibody” includes, but is not limited to, fragments of an antibody having antigen-binding ability, such as Fab, Fab', F(ab')2, Fv and scFv.
  • the "scFv" of the present invention corresponds to the smallest unit that has excellent tissue permeability and maintains antigen reactivity because its size is only 1/6 of an antibody molecule.
  • the antigen-binding domain may be, for example, an scFv, but is not limited thereto.
  • the antibody of the present invention is a chimeric antibody, humanized antibody, bivalent, bispecific molecule, minibody, domain antibody, bispecific antibody, antibody mimic, unibody (unibody), diabody (diabody), triabody (triabody), tetrabody (tetrabody) or a fragment thereof, but is not limited thereto.
  • the "chimeric antibody” of the present invention refers to an antibody derived from an animal in which the antibody variable region or its complementarity determining region (CDR) is different from the rest of the antibody.
  • the antibody variable region may be derived from a non-human animal (eg, mouse, rabbit, poultry, etc.), and the antibody constant region may be an antibody derived from a human.
  • Such chimeric antibodies can be prepared by methods such as genetic recombination known in the art.
  • the "humanized antibody” of the present invention refers to an antibody in which the protein sequence of an antibody derived from a non-human species is modified to be similar to an antibody variant naturally produced in humans.
  • the humanized antibody can be prepared by recombination of a mouse-derived CDR with a human antibody-derived FR to prepare a humanized variable region, and recombination with the constant region of a desired human antibody to produce a humanized antibody.
  • the "Unibody” of the present invention is manufactured by a technology for producing a stable small antibody format so that the therapeutic effect can be exerted for a longer period of time compared to a general antibody. , it may be designed so that only one region capable of binding to the target exists. Compared to the full-size IgG4 antibody, the unibody is very superior in terms of stability. Such a unibody can be manufactured with reference to WO2007/059782 and (Kolfschoten et al. (2007) Science 317: 1554-1557).
  • the "heavy chain” of the present invention is a full-length heavy chain comprising a variable region domain VH and three constant region domains CH1, CH2 and CH3 comprising an amino acid sequence of a variable region sufficient to confer specificity for an antigen, and a full-length heavy chain thereof refers to all fragments.
  • the "light chain” of the present invention refers to both a full-length light chain comprising a variable region domain VL and a constant region CL comprising an amino acid sequence of a variable region sufficient to confer specificity to an antigen, and a fragment thereof.
  • the transmembrane domain refers to a site connecting the antigen-binding domain and the costimulatory and essential signaling domains between the cell membrane.
  • the transmembrane domain comprises a hydrophobic polypeptide spanning the cell membrane and may span from one side of the cell membrane (extracellular) through the other side of the cell membrane (intracellular or cytoplasmic).
  • the transmembrane domain of the present invention may be in the form of an alpha helix or beta barrel, or a combination thereof.
  • the transmembrane domain may include a polyprotein having a plurality of transmembrane fragments, each alpha-helical, beta sheet, or a combination thereof.
  • the transmembrane domain of the present invention includes, for example, all or part of the CD3 zeta ( ⁇ ) chain, CD28, CD3 ⁇ , CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof, and combinations thereof.
  • artificially designed as the transmembrane domain of the present invention may be a polypeptide mainly comprising hydrophobic residues such as leucine and valine.
  • a triplet of phenylalanine, tryptophan and valine can be found at each terminus of the synthetic membrane variable domain.
  • the "intracellular signal transduction domain" of the present invention refers to a portion of a chimeric antigen receptor that is found or engineered to be found inside an immune cell.
  • the intracellular signaling domain of the present invention may comprise a polypeptide that provides for activation of an immune cell to stimulate or activate at least some aspect of an immune cell signaling pathway.
  • the intracellular signaling domain of the present invention includes all or part of CD3 zeta ( ⁇ ), consensus FcR gamma (FcER1G), FcgammaR2a, FcRbeta (Fc epsilon rib), CD3gamma, CD3delta, CD3 epsilon, CD79a, CD79b, DNAX-activating protein 10 (DAP10), DNAX-activating protein 12 (DAP12), active fragments thereof, functional derivatives thereof, and combinations thereof.
  • CD3 zeta ⁇
  • FcER1G consensus FcR gamma
  • FcR2a FcgammaR2a
  • FcRbeta Fc epsilon rib
  • CD3gamma CD3delta
  • CD3 epsilon CD79a
  • CD79b CD79b
  • DAP10 DNAX-activating protein 10
  • DAP12 DNAX-activating protein 12
  • active fragments thereof functional derivative
  • Another embodiment of the present invention provides a pharmaceutical composition for preventing or treating cancer.
  • the pharmaceutical composition of the present invention comprises an antigen-binding domain that specifically binds to a cancer cell surface protein; and a transmembrane domain; A chimeric antigen receptor comprising an intracellular signaling domain; contains the containing immune cell as an active ingredient.
  • composition of the present invention the contents related to cancer cell surface protein, immune cell, antigen binding domain, transmembrane domain, intracellular signaling domain, chimeric antigen receptor, immune cell, etc. are described above in 1. Immune cell therapy composition Same as one bar and omitted.
  • the cancer of the present invention may be a solid cancer or a blood cancer, for example, esophageal cancer, stomach cancer, colorectal cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer Cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's disease, soft tissue sarcoma, lymphoma and multiple myeloma blood cancer It may be selected from the group consisting of, but is not limited thereto.
  • prevention of the present invention means a reduction in the degree of occurrence or damage to or loss of pathological cells in an animal. Prevention may be complete or partial. In this case, it may refer to a phenomenon in which the generation of pathological cells or abnormal immune action in an individual is decreased compared to the case where the composition for preventing and treating cancer is not used.
  • the "treatment" of the present invention means any action that clinically intervenes in order to change the natural process of a subject or cell to be treated, and may be performed while a clinical pathology is in progress or to prevent it.
  • the desired therapeutic effect is to prevent the occurrence or recurrence of a disease, alleviate symptoms, reduce any direct or indirect pathological consequences of the disease, prevent metastasis, reduce the rate of disease progression, alleviating or temporarily ameliorating the condition, or improving the prognosis. That is, the treatment may be interpreted to encompass all actions in which symptoms of cancer are improved or cured by the composition.
  • the pharmaceutical composition of the present invention may be characterized in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and the pharmaceutical composition may be characterized in that it is targeted to humans.
  • the pharmaceutical composition of the present invention is not limited thereto, but each is formulated in the form of oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories, and sterile injection solutions according to conventional methods to be used.
  • 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, dyes, fragrances, etc., for oral administration, and in the case of injections, buffers, preservatives, pain-freezing agents Agents, solubilizers, isotonic agents, stabilizers, etc.
  • the dosage form of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above.
  • a pharmaceutically acceptable carrier as described above.
  • it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. for oral administration, and in the case of injections, it can be prepared in the form of unit dosage ampoules or multiple dosage forms. there is.
  • it can be formulated as a solution, suspension, tablet, capsule, sustained release formulation, and the like.
  • suitable carriers, excipients and diluents 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, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil may be used.
  • fillers, anti-agglomeration agents, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives and the like may be further included.
  • the route of administration of the pharmaceutical composition of the present invention is, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal. Oral or parenteral administration is preferred.
  • the "parenteral" of 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.
  • the pharmaceutical composition of the present invention depends on several factors including the activity of the specific compound used, age, weight, general health, sex, formula, administration time, administration route, excretion rate, drug formulation, and the severity of the specific disease to be prevented or treated. It can be variously changed, and the dosage of the pharmaceutical composition varies depending on the patient's condition, body weight, degree of disease, drug form, administration route and period, but may be appropriately selected by those skilled in the art, and 0.0001 to 50 mg/day per day kg or 0.001 to 50 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. The above 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 pills, dragees, capsules, solutions, gels, syrups, slurries, and suspensions.
  • Another embodiment of the present invention provides a food composition for preventing or improving cancer.
  • the food composition of the present invention comprises an antigen binding domain that specifically binds to a cancer cell surface protein; transmembrane domain; And a chimeric antigen receptor comprising an intracellular signaling domain; includes immune cells containing the as an active ingredient.
  • the cancer of the present invention may be a solid cancer or a blood cancer, for example, esophageal cancer, stomach cancer, colorectal cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer Cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's disease, soft tissue sarcoma, lymphoma and multiple myeloma blood cancer It may be selected from the group consisting of, but is not limited thereto.
  • prevention of the present invention means a reduction in the degree of occurrence or damage to or loss of pathological cells in an animal. Prevention may be complete or partial. In this case, it may refer to a phenomenon in which the generation of pathological cells or abnormal immune action in an individual is decreased compared to the case where the composition for preventing and treating cancer is not used.
  • the "improvement" of the present invention means any action that clinically intervenes in order to change the natural process of a target or cell to be improved, and can be performed while a clinical pathology is in progress or to prevent it.
  • the desired effect of improvement is preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, alleviating or temporarily ameliorating the disease state, or improving the prognosis. That is, the improvement may be interpreted as encompassing all actions in which symptoms are improved or cured by the composition.
  • the food composition of the present invention may be prepared in the form of various foods, for example, beverages, gums, tea, vitamin complexes, powders, granules, tablets, capsules, confectionery, rice cakes, bread, and the like.
  • the amount may be added in a proportion of 0.1 to 50% of the total weight, but is not limited thereto.
  • the food composition of the present invention is prepared in the form of a beverage, there is no particular limitation except for including the food composition in the indicated ratio, and it may contain various flavoring agents or natural carbohydrates as additional ingredients, like a conventional beverage.
  • natural carbohydrates monosaccharides such as glucose, disaccharides such as fructose, and polysaccharides such as sucrose, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol are used as natural carbohydrates.
  • the flavoring agent may be a natural flavoring agent (taumartin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agent (saccharin, aspartame, etc.).
  • stevia extract eg, rebaudioside A, glycyrrhizin, etc.
  • synthetic flavoring agent sacharin, aspartame, etc.
  • the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, It may further include a pH adjuster, a stabilizer, a preservative, glycerin, alcohol, a carbonation agent used in carbonated beverages, and the like.
  • the above components of the present invention may be used independently or in combination.
  • the proportion of the additive is not a key element of the present invention, but may be selected from 0.1 to about 50 parts by weight per 100 parts by weight of the food composition of the present invention, but is not limited thereto.
  • Another embodiment of the present invention provides a method for manufacturing an immune cell therapy product.
  • the manufacturing method of the present invention comprises the steps of culturing immune cells; and transfecting the cultured immune cells with a vector containing a cassette encoding a chimeric antigen receptor.
  • the "cassette” of the present invention includes a polynucleotide encoding a chimeric antigen receptor according to the present invention, a promoter capable of regulating its expression, an enhancer, a polyadenylation signal, a transcription terminator or an internal ribosome entry site (IRES), etc. It may include an expression control sequence.
  • the promoter may include, for example, an SFFV promoter, a human elongation factor 11 ⁇ (EF) promoter, or a CAG (chicken beta-actin promoter with a CMV enhancer) promoter, but is not limited thereto.
  • the "vector" of the present invention may be, for example, a plasmid, a transposon, or a cosmid, but is not limited thereto.
  • the transfection step of the present invention includes microinjection, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene -Mediated transfection (polybrene-mediated transfection), electroporation (electroporation), gene gun (gene gun) or other known methods for introducing nucleic acids into the cell can be introduced into the cell, but is limited thereto No (Wu et al., J. Bio. Chem., 267:963-967, 1992; Wu and Wu, J. Bio. Chem., 263:14621-14624, 1988).
  • the transfection step of the present invention may be using a viral transfection method or a non-viral transfection method.
  • a viral transfection method for the purpose of the present invention, since immune cells recombined in vitro are directly injected into the subject as an active ingredient as an active ingredient, compared with the case of using a virus, It is very useful in that it can reduce side effects.
  • Another embodiment of the present invention provides a method for screening an immune cell therapy agent.
  • the screening method of the present invention includes an antigen-binding domain that specifically binds to a cancer cell surface protein in a biological sample isolated from a subject of interest; transmembrane domain; and a chimeric antigen receptor comprising an intracellular signaling domain;
  • the "target individual” refers to an individual whose treatment responsiveness to an immune cell therapy agent is uncertain, and an individual who has developed a disease or is highly likely to develop the disease.
  • the "biological sample” of the present invention refers to any material, biological fluid, tissue or cell obtained from or derived from an individual, for example, whole blood, leukocytes, peripheral blood mononuclear blood, sputum, tears, mucus, nasal washes, including peripheral blood mononuclear cells, buffy coat, plasma and serum ), nasal aspirate, breath, urine, semen, saliva, peritoneal washings, pelvic fluids, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspiration It may include bronchial aspirate, synovial fluid, joint aspirate, organ secretions, cell, cell extract or cerebrospinal fluid, However, the present invention is not limited thereto.
  • the screening method of the present invention may include measuring the size of cancer organoids, apoptosis, or the degree of invasion into cancer tissues in the biological sample after the treatment of the immune cells.
  • organ analogue refers to a three-dimensional cell aggregate formed through a process of self-renewal or self-organization using stem cells or cells derived from a patient. Since such organoids are made by aggregating and recombination of cells through a three-dimensional culture method, they contain specific cells of a model organ.
  • the size of the cancer organoid is reduced or maintained. Occation; Alternatively, when the degree of infiltration into cancer organoids is suppressed, the immune cells may be determined as an immune cell therapy agent.
  • the "apoptosis" of the present invention means a step leading to death due to genetic properties while maintaining a functional role of the cell.
  • the expression is expressed in cancer cells or cancer organoids derived from the subject of interest.
  • the expression level of a gene or protein related to apoptosis is measured, and when apoptosis is increased, the immune cells can be determined as an immune cell therapy agent.
  • the method of measuring the expression level of the protein of the present invention is a protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser) Desorption/Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunodiffusion method, Oukteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, 2D electrophoresis analysis, liquid chromatography-mass Analysis (liquid chromatography-Mass Spectrometry, LC-MS), LC-MS/MS (liquid chromatography-Mass Spectrometry/ Mass Spectrometry), Western blotting and ELISA (enzyme linked immunosorbent assay) may be at least one selected from the group consisting of there is.
  • MALDI-TOF Microx Assisted Laser De
  • the expression level of the protein of the present invention can be measured using an agent capable of measuring the expression level of the protein.
  • the agent capable of measuring the expression level of the protein may be at least one selected from the group consisting of an antibody, oligopeptide, ligand, PNA (peptide nucleic acid) and aptamer that specifically binds to the protein. there is.
  • the "antibody” of the present invention refers to a substance that specifically binds to an antigen and causes an antigen-antibody reaction.
  • an antibody refers to an antibody that specifically binds to said protein.
  • Antibodies of the present invention include polyclonal antibodies, monoclonal antibodies and recombinant antibodies.
  • the antibody can be readily prepared using techniques well known in the art.
  • the polyclonal antibody can be produced by a method well known in the art, which includes the process of injecting an antigen of the protein into an animal and collecting blood from the animal to obtain a serum containing the antibody.
  • Such polyclonal antibodies can be prepared from any animal such as goat, rabbit, sheep, monkey, horse, pig, cow, dog, and the like.
  • monoclonal antibodies can be prepared using the hybridoma method well known in the art (see Kohler and Milstein (1976) European Journal of Immunology 6:511-519), or the phage antibody library technology (Clackson et al, Nature, 352:624-628, 1991; Marks et al, J. Mol. Biol., 222:58, 1-597, 1991).
  • the antibody prepared by the above method may be separated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography.
  • the antibody of the present invention includes a complete form having two full-length light chains and two full-length heavy chains, as well as functional fragments of the antibody.
  • the functional fragment of the antibody of the present invention means a fragment having at least an antigen-binding function, and includes Fab, F(ab'), F(ab')2 and Fv.
  • PNA Peptide Nucleic Acid
  • DNA has a phosphate-ribose sugar backbone
  • PNA has a repeated N-(2-aminoethyl)-glycine backbone linked by peptide bonds, which greatly increases binding strength and stability to DNA or RNA, resulting in molecular biology , diagnostic assays and antisense therapy.
  • PNA is described in Nielsen PE, Egholm M, Berg RH, Buchardt O (December 1991). "Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide". Science 254 (5037): 1497-1500.
  • the "aptamer” of the present invention is an oligonucleic acid or peptide molecule, and the general description of the aptamer is described in Bock LC et al., Nature 355(6360):5646(1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine”. J Mol Med. 78(8):42630(2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library”. Proc Natl Acad Sci USA. 95(24): 142727 (1998).
  • the agent capable of measuring the expression level of the protein of the present invention is based on the amino acid sequence constituting the proteins of the present invention, which corresponds to an antibody, PNA, and aptamer that specifically binds to the protein by those skilled in the art.
  • the formulation can be easily manufactured.
  • the method of measuring the expression level of the gene of the present invention is reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase reaction (Real-time RT-PCR), RNase It may be at least one selected from the group consisting of a protection assay (RPA; RNase protection assay), Northern blotting, and a DNA chip.
  • RT-PCR reverse transcription polymerase reaction
  • Competitive RT-PCR competitive reverse transcription polymerase reaction
  • Real-time RT-PCR real-time reverse transcription polymerase reaction
  • RNase It may be at least one selected from the group consisting of a protection assay (RPA; RNase protection assay), Northern blotting, and a DNA chip.
  • the expression level of the gene of the present invention can be measured using an agent capable of measuring the expression level of the gene.
  • the agent capable of measuring the expression level of the gene may be at least one selected from the group consisting of primers, probes and antisense nucleotides that complementarily bind to the gene.
  • the "primer” of the present invention is a fragment recognizing a target gene sequence, and includes a pair of forward and reverse primers, but preferably, a primer pair that provides analysis results having specificity and sensitivity.
  • the nucleic acid sequence of the primer is a sequence that is inconsistent with a non-target sequence present in the sample, and thus amplifies only the target gene sequence containing the complementary primer binding site and does not cause non-specific amplification, high specificity can be conferred. .
  • the "probe” of the present invention means a substance capable of complementary binding to a target substance to be detected in a sample, and means a substance capable of specifically confirming the presence of a target substance in the sample through the binding.
  • the type of probe is not limited as a material commonly used in the art, but preferably PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA or DNA, and most preferably It is PNA.
  • the probe is a biomaterial derived from or similar thereto, or manufactured in vitro, and includes, for example, enzymes, proteins, antibodies, microorganisms, animal and plant cells and organs, neurons, DNA, and It may be RNA, and DNA includes cDNA, genomic DNA, and oligonucleotides, and RNA includes genomic RNA, mRNA, and oligonucleotides.
  • LNA Locked nucleic acids
  • LNA nucleosides include common nucleic acid bases in DNA and RNA, and can form base pairs according to Watson-Crick base pairing rules. However, due to the 'locking' of the molecule due to the methylene bridge, the LNA does not form an ideal shape at the Watson-Crick bond.
  • LNA When LNA is incorporated into DNA or RNA oligonucleotides, LNA can pair with complementary nucleotide chains more rapidly, increasing the stability of the double helix.
  • the "antisense nucleotide” of the present invention is a nucleotide sequence in which an antisense oligomer is hybridized with a target sequence in RNA by Watson-Crick base pairing, typically mRNA and RNA: oligomeric heteroduplex formation in the target sequence. and oligomers having an inter-subunit backbone.
  • An oligomer may have exact sequence complementarity or approximate complementarity to a target sequence.
  • the agent capable of measuring the expression level of the gene of the present invention is based on the nucleotide sequence of the gene of the present invention, and the agent corresponding to the primer, probe, etc. that complementarily binds to the gene can be easily prepared by a person skilled in the art. can be
  • Another embodiment of the present invention provides a method for preventing or treating cancer.
  • the prophylactic or therapeutic method of the present invention includes an antigen-binding domain that specifically binds to a cancer cell surface protein; transmembrane domain; And a chimeric antigen receptor comprising an intracellular signaling domain; it comprises the step of administering to the subject a pharmaceutically effective amount of the immune cell therapy composition comprising immune cells comprising the as an active ingredient.
  • the "individual” of the present invention refers to an individual in need of prevention or treatment of cancer, and includes all mammals such as cattle, horses, sheep, pigs, goats, camels, antelopes, dogs, and cats as well as primates such as humans. It may include, but is not limited to.
  • the "administration" of the present invention means the process of introducing the active ingredient of the present invention to an individual by any suitable method, and the administration method in the treatment method of the present invention is through various routes such as oral or parenteral. may be administered.
  • the present invention relates to an immune cell therapy composition having an ability to avoid tumor microenvironment, and the immune cell therapy composition of the present invention can very effectively kill not only blood cancer cell lines but also solid cancer cell lines by recognizing cancer cells very effectively.

Abstract

The present invention relates to an immunotherapy composition with an ability for escaping tumor microenvironments, the immunotherapy composition of the present invention recognizing cancerous cells highly effectively to kill not only hematological malignancy strains but also solid cancer strains highly effectively.

Description

종양미세환경 회피능을 갖는 면역세포치료제 조성물Immune cell therapy composition having ability to avoid tumor microenvironment
본 발명은 면역세포치료제 조성물에 관한 것이다.The present invention relates to an immune cell therapy composition.
빠르게 증식하는 종양세포의 특성을 이용하여 분열하는 세포를 공격하는 화학항암제와 종양 세포의 특정 분자나 세포신호전달체계를 표적하는 표적항암제에 대한 개발이 과거에 다수 수행되었으나, 최근에는 면역학적 방법을 이용하여 종양을 치료하는 면역항암제로 그 항암제 개발 과정이 변화되고 있다. 면역항암제는 종양 항원을 표적하는 항체치료제(Rituximab 등), 면역세포를 다시 활성화시키는 면역관문억제제(Immune checkpoint inhibitor 등) 및 면역세포를 직접 투여하는 면역세포치료제(Immune cell theraphy) 등으로 구분될 수 있다(Oiseth et al, 2017).Using the characteristics of rapidly proliferating tumor cells, the development of chemical anticancer agents that attack dividing cells and targeted anticancer agents that target specific molecules or cell signaling systems of tumor cells have been conducted in the past, but recently, immunological methods have been developed. The process of developing anti-cancer drugs is changing as an immuno-oncology drug that treats tumors using chemotherapy. Immune anticancer drugs can be divided into antibody therapeutics that target tumor antigens (Rituximab, etc.), immune checkpoint inhibitors that reactivate immune cells (Immune checkpoint inhibitors, etc.), and immune cell therapies that directly administer immune cells (Immune cell therapy). There is (Oiseth et al, 2017).
상기 면역세포치료제의 경우, 1980년대 중반 NIH(National Institutes of Health)를 중심으로 체외 배양되어 활성화된 자가 T 세포를 이용한 악성 흑색종 환자의 치료와 관련된 임상연구가 시도되면서 많은 연구가 수행되어 왔다. 그러나, 항원 특이성이 부여되지 않은 T 세포는 악성 종양을 제거하는데 충분한 효과가 발휘되지 못하였다. 이와 관련하여, 2010년 덴드레온(Dendreon)사에서 FDA로부터 허가받은 수지상세포치료제인 프로벤지(Provenge)가 최초 의약품에 해당한다. 그러나, 이와 같은 치료제는 가격 대비 낮은 유효성 등의 이유로 시장에서 큰 호응을 받지 못하였다.In the case of the immune cell therapy, many studies have been conducted in the mid-1980s as clinical studies related to the treatment of malignant melanoma patients using in vitro cultured and activated autologous T cells centered on the National Institutes of Health (NIH) were attempted. However, T cells to which antigen specificity is not conferred did not exhibit sufficient effect to remove malignant tumors. In this regard, Provenge, a dendritic cell therapy approved by the FDA by Dendreon in 2010, is the first drug. However, such therapeutic agents have not been well-received in the market due to low efficacy compared to the price.
이와 같은 한계점에 따라, T 세포에 항원 특이성을 부여하기 위한 다양한 공정이 시도되어 왔다. 특히, 항체인식부위(scFv)와 T 세포의 세포내 활성화 도메인을 결합시킨 키메릭항원수용체(Chimeric antigen receptor; CAR)를 이용한 방법이 매우 효과적으로 T 세포에 항원 특이성을 부여할 수 있는 것으로 각광받고 있다. 이와 관련하여, 노바티스(Norvatis)사에 의해 개발된 CD19-CAR-T인 Kymriah(제품명)는 2017년 8월 미국 FDA에 의해 허가되었으며, 이러한 흐름에 맞추어 면역세포치료제의 본격적인 상업화가 시작되었다.According to these limitations, various processes for conferring antigen specificity to T cells have been tried. In particular, a method using a chimeric antigen receptor (CAR) that combines an antibody recognition site (scFv) with an intracellular activation domain of T cells is in the spotlight as being able to confer antigen specificity to T cells very effectively. . In this regard, Kymriah (product name), a CD19-CAR-T developed by Novartis, was approved by the US FDA in August 2017, and in line with this trend, full-scale commercialization of immune cell therapy began.
그러나, 상기 CAR-T 세포의 경우 우선적으로 환자 자신에게서 T 세포를 채취한 뒤, 채취된 T 세포를 이용하여 복잡한 유전자 조작 공정을 거쳐야 하기 때문에 비용이 과도하다는 단점이 존재한다. 뿐만 아니라, 표적 세포와 접촉하는 경우 T 세포는 빠르게 분열하므로 사이토카인폭풍(Cytokine release syndrome; CRS) 및 중추 신경계의 부종과 같은 심각한 부작용이 보고되고 있으며, T 세포는 메모리 T 세포에 의해 치료받은 환자에게 장기간 존재하여 언제든 동일한 부작용이 발생될 수 있다. 나아가, 이와 같은 CAR-T 세포는 면역억제적인 종양미세환경(Tumor microenvironment)과 암세포의 이질성(Heterogeneity) 뿐만 아니라, 혈액암에 비해 상대적으로 CAR-T 세포의 침투가 어렵기 때문에 고형암의 치료에 적용하기 위해서는 한계점이 있다.However, in the case of the CAR-T cells, there is a disadvantage in that the cost is excessive because T cells are first collected from the patient themselves, and then a complex genetic manipulation process is performed using the collected T cells. In addition, since T cells divide rapidly when in contact with target cells, serious side effects such as cytokine release syndrome (CRS) and central nervous system edema have been reported, and T cells are treated by memory T cells in patients The same side effects can occur at any time due to its long-term presence in Furthermore, such CAR-T cells are applied to the treatment of solid cancers because of their immunosuppressive tumor microenvironment and heterogeneity of cancer cells, as well as relatively difficult penetration of CAR-T cells compared to blood cancers. There are limits to this.
이러한 문제점을 해결하기 위하여 최근 자연살해세포(Natural killer cell; NK cell)를 이용한 항암면역세포치료제의 개발이 활발하게 진행되고 있다. 상기 자연살해세포는 인간의 면역반응에서 종양 세포나 바이러스가 감염된 세포와 같은 비 정상적인 세포를 즉각적으로 인식하여 제거할 수 있는 기능을 갖는 세포에 해당한다. 이러한 자연살해세포는 T 세포와 비교하여, 메모리 기능과 자가복제 기능이 상대적으로 약하기 때문에, 사이토카인폭풍과 같은 부작용이 상대적으로 적을 수 있다는 장점이 존재한다.In order to solve this problem, the development of anticancer immune cell therapy using natural killer cells (NK cells) is being actively conducted. The natural killer cells correspond to cells having a function of immediately recognizing and removing abnormal cells such as tumor cells or virus-infected cells in a human immune response. Since these natural killer cells have relatively weak memory function and self-replication function compared to T cells, there is an advantage that side effects such as cytokine storm may be relatively small.
본 발명의 일 목적은 면역세포치료제 조성물을 제공하는 것이다.One object of the present invention is to provide an immune cell therapy composition.
본 발명의 다른 목적은 암의 예방, 개선 또는 치료용 약학 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for preventing, improving or treating cancer.
본 발명의 또 다른 목적은 면역세포치료제 조성물의 제조 방법을 제공하는 것이다.Another object of the present invention is to provide a method for preparing an immune cell therapy composition.
본 발명의 또 다른 목적은 면역세포치료제 반응성을 스크리닝하는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for screening for immune cell therapy reactivity.
본 발명의 또 다른 목적은 암의 예방 또는 치료 방법을 제공하는 것이다.Another object of the present invention is to provide a method for preventing or treating cancer.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당 업계에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those of ordinary skill in the art from the following description.
본 발명의 일 구현 예에서는 면역세포치료제 조성물을 제공한다.One embodiment of the present invention provides an immune cell therapy composition.
상기 면역세포치료 조성물은 암 세포 표면 단백질에 특이적으로 결합하는 항원 결합 도메인;The immunotherapy composition may include an antigen binding domain that specifically binds to a cancer cell surface protein;
막관통 도메인; 및transmembrane domain; and
세포내 신호전달 도메인을 포함하는 키메릭 항원 수용체; 를 포함한다.a chimeric antigen receptor comprising an intracellular signaling domain; includes
상기 면역세포는 자연살해세포(Natural killer cell; NK cell)인 것일 수 있다.The immune cells may be natural killer cells (NK cells).
상기 발현 벡터는 작동가능하게 연결된 유도성 프로모터를 포함하는 것일 수 있다.The expression vector may include an operably linked inducible promoter.
상기 항원 결합 도메인은 상기 암 세포 표면 단백질에 특이적으로 결합하는 항체 또는 항체의 단편인 것인 것일 수 있다.The antigen-binding domain may be an antibody or antibody fragment that specifically binds to the cancer cell surface protein.
상기 항체의 단편은 scFv인 것일 수 있다.The fragment of the antibody may be scFv.
본 발명의 다른 구현 예에서는 암의 예방 또는 치료용 약학 조성물을 제공한다.Another embodiment of the present invention provides a pharmaceutical composition for preventing or treating cancer.
본 발명의 상기 약학 조성물은 암 세포 표면 단백질에 특이적으로 결합하는 항원 결합 도메인;The pharmaceutical composition of the present invention comprises an antigen-binding domain that specifically binds to a cancer cell surface protein;
막관통 도메인; 및transmembrane domain; and
세포내 신호전달 도메인을 포함하는 키메릭 항원 수용체;를 유효성분으로 포함한다.It contains a chimeric antigen receptor comprising an intracellular signaling domain; as an active ingredient.
상기 암은 식도암, 위암, 대장암, 직장암, 구강암, 인두암, 후두암, 폐암, 결장암, 유방암, 자궁경부암, 자궁내막암, 난소암, 전립선암, 고환암, 방광암, 신장암, 간암, 췌장암, 골암, 결합 조직암, 피부암, 뇌암, 갑상선암, 백혈병, 호지킨(Hodgkin) 질환, 연부조직육종(soft tissue sarcoma), 림프종 및 다발성 골수종 혈액암으로 구성된 군으로부터 선택되는 것일 수 있다.The cancer is esophageal cancer, stomach cancer, colorectal cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer , connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's disease, soft tissue sarcoma, lymphoma, and multiple myeloma may be selected from the group consisting of blood cancer.
본 발명의 또 다른 구현 예에서는 암의 예방 또는 개선용 식품 조성물을 제공한다.Another embodiment of the present invention provides a food composition for preventing or improving cancer.
암 세포 표면 단백질에 특이적으로 결합하는 항원 결합 도메인;an antigen binding domain that specifically binds to a cancer cell surface protein;
막관통 도메인; 및transmembrane domain; and
세포내 신호전달 도메인을 포함하는 키메릭 항원 수용체;를 유효성분으로 포함한다.It contains a chimeric antigen receptor comprising an intracellular signaling domain; as an active ingredient.
상기 암은 식도암, 위암, 대장암, 직장암, 구강암, 인두암, 후두암, 폐암, 결장암, 유방암, 자궁경부암, 자궁내막암, 난소암, 전립선암, 고환암, 방광암, 신장암, 간암, 췌장암, 골암, 결합 조직암, 피부암, 뇌암, 갑상선암, 백혈병, 호지킨(Hodgkin) 질환, 연부조직육종(soft tissue sarcoma), 림프종 및 다발성 골수종 혈액암으로 구성된 군으로부터 선택되는 것일 수 있다.The cancer is esophageal cancer, stomach cancer, colorectal cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer , connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's disease, soft tissue sarcoma, lymphoma, and multiple myeloma may be selected from the group consisting of blood cancer.
본 발명의 또 다른 구현 예에서는 면역세포치료제의 제조 방법을 제공한다.Another embodiment of the present invention provides a method for manufacturing an immune cell therapy product.
본 발명의 상기 제조 방법은 면역세포를 배양하는 단계; 및The manufacturing method of the present invention comprises the steps of culturing immune cells; and
상기 배양된 면역세포에 키메릭 항원 수용체를 암호화하는 카세트가 포함된 벡터를 형질 주입하는 단계;를 포함한다.and transfecting the cultured immune cells with a vector containing a cassette encoding a chimeric antigen receptor.
상기 면역세포는 자연살해세포인 것일 수 있다.The immune cells may be natural killer cells.
상기 형질 주입하는 단계는 바이러스성 또는 비바이러스성 형질 주입 방법을 이용하는 것 일 수 있다.The transfection may be performed using a viral or non-viral transfection method.
상기 키메릭 항원 수용체는The chimeric antigen receptor is
암 세포 표면 단백질에 특이적으로 결합하는 항원 결합 도메인;an antigen binding domain that specifically binds to a cancer cell surface protein;
막관통 도메인; 및transmembrane domain; and
세포내 신호전달 도메인을 포함하는 것일 수 있다.It may include an intracellular signaling domain.
본 발명의 또 다른 구현 예에서는 면역세포치료제를 스크리닝하는 방법을 제공한다.Another embodiment of the present invention provides a method for screening an immune cell therapy agent.
본 발명의 상기 스크리닝하는 방법은 목적하는 개체로부터 분리된 생물학적 시료에서, 암 세포 표면 단백질에 특이적으로 결합하는 항원 결합 도메인; 막관통 도메인; 및 세포내 신호전달 도메인을 포함하는 키메릭 항원 수용체;를 암호화하는 유전자가 포함된 발현 벡터를 포함하는 면역세포를 처리하는 단계를 포함한다.The screening method of the present invention includes an antigen-binding domain that specifically binds to a cancer cell surface protein in a biological sample isolated from a subject of interest; transmembrane domain; and a chimeric antigen receptor comprising an intracellular signaling domain; and treating an immune cell comprising an expression vector containing a gene encoding the same.
상기 면역세포의 처리 후 상기 생물학적 시료에서 암 오가노이드의 크기, 세포사멸 또는 암 조직으로 침윤 정도를 측정하는 단계를 더 포함한다.The method further includes measuring the size of cancer organoids, apoptosis, or the degree of invasion into cancer tissues in the biological sample after the treatment of the immune cells.
상기 측정된 암 오가노이드의 크기 또는 암 조직으로의 침윤 정도가 상기 면역세포의 처리 전에 비교하여 감소되거나; 또는 세포사멸 정도가 증가된 경우, 상기 면역세포를 면역세포치료제로 판단하는 단계를 포함한다.the measured size of the cancer organoids or the degree of invasion into cancer tissues is reduced compared to before the treatment of the immune cells; or when the degree of apoptosis is increased, determining the immune cell as an immune cell therapy agent.
본 발명은 면역세포치료제 조성물에 관한 것으로서, 본 발명의 상기 면역세포치료제 조성물은 암 세포를 매우 효과적으로 인지함으로써 혈액암 세포주 뿐만 아니라, 고형암 세포주 역시 매우 효과적으로 사멸시킬 수 있다.The present invention relates to an immune cell therapy composition, and the immune cell therapy composition of the present invention can very effectively kill not only blood cancer cell lines but also solid cancer cell lines by recognizing cancer cells very effectively.
도 1은 본 발명의 일 실시예 따른 면역세포치료제의 모식도를 나타낸 것이다.1 shows a schematic diagram of an immune cell therapy agent according to an embodiment of the present invention.
본 발명의 일 실시예에서는 암 세포 표면 단백질에 특이적으로 결합하는 항원 결합 도메인; 막관통 도메인; 및 세포내 신호전달 도메인을 포함하는 키메릭 항원 수용체;를 포함하는 면역세포를 유효성분으로 포함하는 면역세포치료제 조성물을 제공한다.In one embodiment of the present invention, an antigen binding domain that specifically binds to a cancer cell surface protein; transmembrane domain; and a chimeric antigen receptor comprising an intracellular signaling domain; provides an immune cell therapy composition comprising an immune cell comprising as an active ingredient.
본 발명의 다른 실시예에서는 암 세포 표면 단백질에 특이적으로 결합하는 항원 결합 도메인; 막관통 도메인; 및 세포내 신호전달 도메인을 포함하는 키메릭 항원 수용체;를 포함하는 면역세포를 유효성분으로 포함하는 암의 예방 또는 치료용 약학 조성물을 제공한다.In another embodiment of the present invention, an antigen binding domain that specifically binds to a cancer cell surface protein; transmembrane domain; and a chimeric antigen receptor comprising an intracellular signaling domain; provides a pharmaceutical composition for the prevention or treatment of cancer comprising immune cells as an active ingredient.
본 발명의 또 다른 실시예에서는 면역세포를 배양하는 단계; 및 상기 배양된 면역세포에 키메릭 항원 수용체를 암호화하는 카세트가 포함된 벡터를 형질 주입하는 단계;를 포함하는 면역세포치료제의 제조 방법을 제공한다.In another embodiment of the present invention, culturing immune cells; and transfecting the cultured immune cells with a vector containing a cassette encoding a chimeric antigen receptor.
본 발명의 또 다른 실시예에서는 목적하는 개체로부터 분리된 생물학적 시료에서, 암 세포 표면 단백질에 특이적으로 결합하는 항원 결합 도메인; 막관통 도메인; 및 세포내 신호전달 도메인을 포함하는 키메릭 항원 수용체; 를 포함하는 면역세포를 처리하는 단계를 포함하는, 면역세포치료제를 스크리닝하는 방법을 제공한다.In another embodiment of the present invention, in a biological sample isolated from a subject of interest, an antigen-binding domain that specifically binds to a cancer cell surface protein; transmembrane domain; and a chimeric antigen receptor comprising an intracellular signaling domain; It provides a method of screening an immune cell therapy, comprising the step of treating immune cells comprising a.
본 발명의 또 다른 실시예에서는 암 세포 표면 단백질에 특이적으로 결합하는 항원 결합 도메인; 막관통 도메인; 및 세포내 신호전달 도메인을 포함하는 키메릭 항원 수용체; 를 포함하는 면역세포를 유효성분으로 포함하는 면역세포치료제 조성물을 약학적으로 유효량 개체에 투여하는 단계를 포함하는, 암의 예방 또는 치료 방법을 제공한다.In another embodiment of the present invention, an antigen binding domain that specifically binds to a cancer cell surface protein; transmembrane domain; and a chimeric antigen receptor comprising an intracellular signaling domain; It provides a method for preventing or treating cancer, comprising administering a pharmaceutically effective amount of an immune cell therapy composition comprising immune cells as an active ingredient to a subject.
이하, 본 발명을 하기의 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are only illustrative of the present invention, and the content of the present invention is not limited by the following examples.
1. One. 면역세포치료제 조성물Immune cell therapy composition
본 발명의 일 구현 예에서는 면역세포치료제 조성물을 제공한다.One embodiment of the present invention provides an immune cell therapy composition.
본 발명의 상기 면역세포치료제 조성물은, 면역세포를 유효성분으로 포함하는 면역세포치료제로서, 약학 조성물 또는 식품 조성물로 사용될 수 있다.The immune cell therapy composition of the present invention is an immune cell therapy agent comprising immune cells as an active ingredient, and may be used as a pharmaceutical composition or a food composition.
상기 면역세포치료제 조성물은 암 세포 표면 단백질에 특이적으로 결합하는 항원 결합 도메인; 및 막관통 도메인; 세포내 신호전달 도메인을 포함하는 키메릭 항원 수용체;를 포함하는 면역세포를 유효성분으로 포함한다.The immune cell therapy composition comprises an antigen binding domain that specifically binds to a cancer cell surface protein; and a transmembrane domain; A chimeric antigen receptor comprising an intracellular signaling domain; includes an immune cell containing as an active ingredient.
본 발명의 상기 "면역세포치료제"는 일반적으로 수지상세포(dendritic cell), 자연살해세포(natural killer cell) 및 T 세포와 같은 면역세포를 이용하여 체내 면역반응을 활성화시켜 질병을 치료할 목적으로 사용되는 의약품을 의미한다.The "immune cell therapy agent" of the present invention generally uses immune cells such as dendritic cells, natural killer cells, and T cells to activate an immune response in the body and is used for the purpose of treating diseases. means medicine.
본 발명의 상기 "키메릭 항원 수용체"는 항원에 대한 원하는 특이성을 면역세포, 예를 들면 자연살해세포 및 T 세포에 부여할 수 있도록 조작된 수용체를 의미한다. 상기 키메릭 항원 수용체는 항원 결합 도메인; 막관통 도메인; 및 세포 내 신호전달 도메인을 포함한다.The "chimeric antigen receptor" of the present invention refers to a receptor engineered to confer a desired specificity for an antigen to immune cells, for example, natural killer cells and T cells. The chimeric antigen receptor comprises an antigen binding domain; transmembrane domain; and an intracellular signaling domain.
본 발명의 상기 "면역세포"는 상기 키메릭 항원 수용체에 의해 목적하는 세포, 조직 또는 종양미세환경 주변으로 위치화됨으로써, 면역반응을 유도할 수 있는 세포라면 모두 포함될 수 있고, 예를 들면, 자연살해세포(Natural killer cell; NK cell)일 수 있으나, 이에 제한되는 것은 아니다.The "immune cell" of the present invention may include any cell capable of inducing an immune response by being localized around a target cell, tissue or tumor microenvironment by the chimeric antigen receptor, for example, natural It may be a natural killer cell (NK cell), but is not limited thereto.
본 발명의 상기 "자연살해세포"는 인간의 면역반응에서 종양 세포나 바이러스가 감염된 세포와 같은 비 정상적인 세포를 즉각적으로 인식하여 제거할 수 있는 기능을 갖는 세포로서, T 세포와 같은 면역세포와 비교하여 메모리 기능과 자가복제 기능이 상대적으로 약하기 때문에, 사이토카인폭풍과 같은 부작용이 상대적으로 적을 수 있고 동종 세포 치료가 가능하다는 이점이 존재한다.The "natural killer cells" of the present invention are cells having the function of immediately recognizing and removing abnormal cells such as tumor cells or virus-infected cells in a human immune response, compared with immune cells such as T cells. Therefore, since the memory function and self-replication function are relatively weak, side effects such as cytokine storm may be relatively few, and there is an advantage that allogeneic cell therapy is possible.
본 발명의 상기 키메릭 항원 수용체의 각 구성에 대해 이하 자세히 설명한다.Each configuration of the chimeric antigen receptor of the present invention will be described in detail below.
1) 암 세포 표면 단백질에 특이적으로 결합하는 항원 결합 도메인1) an antigen binding domain that specifically binds to cancer cell surface proteins
본 발명의 상기 항원 결합 도메인은 면역 세포의 표면에 위치화되어 목적하는 항원, 즉 암 세포 표면 단백질에 특이적으로 결합할 수 있는 항체로부터 유래된 것일 수 있다.The antigen-binding domain of the present invention may be derived from an antibody that is localized on the surface of an immune cell and can specifically bind to a desired antigen, that is, a cancer cell surface protein.
본 발명의 상기 "암 세포 표면 단백질"은 예를 들면, 정상 세포와 비교하여 암 세포에만 특이적으로 존재하는 단백질일 수 있다.The "cancer cell surface protein" of the present invention may be, for example, a protein that is specifically present only in cancer cells compared to normal cells.
본 발명의 상기 "항체"는, 면역계 내에서 항원의 자극에 의하여 만들어지는 물질을 의미하는 것으로서 그 종류는 특별히 제한되지 않는다. 또한 본 명세서에서 항체란 항원 결합능을 보유한 항체의 단편, 예컨대, Fab, Fab', F(ab')2, Fv 및 scFv등을 포함하며, 이에 제한되지 않는다.The "antibody" of the present invention refers to a substance produced by stimulation of an antigen in the immune system, and the type thereof is not particularly limited. In the present specification, the term "antibody" includes, but is not limited to, fragments of an antibody having antigen-binding ability, such as Fab, Fab', F(ab')2, Fv and scFv.
본 발명의 상기 "scFv"는 그 크기가 항체 분자의 1/6에 불과하여 조직 투과성이 뛰어남과 동시에, 항원 반응성을 유지하는 가장 작은 단위에 해당한다. 본 발명의 목적상 상기 항원 결합 도메인은 예를 들면, scFv일 수 있으나, 이에 제한되는 것은 아니다.The "scFv" of the present invention corresponds to the smallest unit that has excellent tissue permeability and maintains antigen reactivity because its size is only 1/6 of an antibody molecule. For the purposes of the present invention, the antigen-binding domain may be, for example, an scFv, but is not limited thereto.
본 발명의 상기 항체는 키메릭 항체, 인간화 항체(humanized antibody), 이가(bivalent), 양특이성 분자, 미니바디(minibody), 도메인 항체, 이중특이적 항체(bispecific antibody), 항체 모방체, 유니바디(unibody), 디아바디(diabody), 트리아바디(triabody), 테트라바디(tetrabody) 또는 이의 단편일 수 있으나, 이에 제한되는 것은 아니다.The antibody of the present invention is a chimeric antibody, humanized antibody, bivalent, bispecific molecule, minibody, domain antibody, bispecific antibody, antibody mimic, unibody (unibody), diabody (diabody), triabody (triabody), tetrabody (tetrabody) or a fragment thereof, but is not limited thereto.
본 발명의 상기 "키메릭 항체"는, 항체 가변영역(variable region) 또는 이의 상보성 결정 영역(complementarity determining region, CDR)이 항체의 나머지 부분과 상이한 동물에서 기원된 항체를 말한다. 이러한 항체는, 예를 들어, 항체 가변 영역은 인간 이외의 동물 (예를 들면, 마우스, 토끼, 가금류 등)에서 유 래하고, 항체 불변영역 (constant region)은 인간에서 유래한 항체일 수 있다. 이러한 키메릭 항체는 당업계에 공지된 유전자 재조합 등의 방법으로 제조될 수 있다.The "chimeric antibody" of the present invention refers to an antibody derived from an animal in which the antibody variable region or its complementarity determining region (CDR) is different from the rest of the antibody. In such an antibody, for example, the antibody variable region may be derived from a non-human animal (eg, mouse, rabbit, poultry, etc.), and the antibody constant region may be an antibody derived from a human. Such chimeric antibodies can be prepared by methods such as genetic recombination known in the art.
본 발명의 상기 "인간화 항체"는 인간이 아닌 종에서 유래한 항체의 단백질 서열을 인간에서 자연적으로 생산된 항체 변이체와 유사하도록 변형시킨 항체를 의미한다. 그 예로 상기 인간화 항체는 생쥐 유래의 CDR을 인간 항체 유래의 FR과 재조합시켜 인간화 가변 영역을 제조하고, 이를 바람직한 인간 항체의 불변 영역과 재조합시켜 인간화 항체를 제조할 수 있다.The "humanized antibody" of the present invention refers to an antibody in which the protein sequence of an antibody derived from a non-human species is modified to be similar to an antibody variant naturally produced in humans. For example, the humanized antibody can be prepared by recombination of a mouse-derived CDR with a human antibody-derived FR to prepare a humanized variable region, and recombination with the constant region of a desired human antibody to produce a humanized antibody.
본 발명의 상기 "유니바디"는 일반적인 항체에 비하여 더 오랜 시간 동안 치료 효과 등이 발휘될 수 있도록 안정적인 작은 항체 형식을 생산하는 기술에 의해 제작된 것으로서, 유니바디는 IgG4 항체의 힌지 영역을 제거하여, 표적에 결합할 수 있는 영역이 하나만 존재하도록 제작된 것일 수 있다. 전체 크기의 IgG4 항체와 비교하여 상기 유니바디는 안정성 측면에서 매우 뛰어나다. 이와 같은 유니바디는 제 WO2007/059782 호 및 (Kolfschoten et al. (2007) Science 317: 1554-1557)를 참고하여 제작할 수 있다.The "Unibody" of the present invention is manufactured by a technology for producing a stable small antibody format so that the therapeutic effect can be exerted for a longer period of time compared to a general antibody. , it may be designed so that only one region capable of binding to the target exists. Compared to the full-size IgG4 antibody, the unibody is very superior in terms of stability. Such a unibody can be manufactured with reference to WO2007/059782 and (Kolfschoten et al. (2007) Science 317: 1554-1557).
본 발명의 "중쇄"는, 항원에 대한 특이성을 부여하기 위해 충분한 가변영역의 아미노산 서열을 포함하는 가변영 역 도메인 VH 및 3 개의 불변영역 도메인인 CH1, CH2 및 CH3을 포함하는 전체 길이 중쇄 및 이의 단편을 모두 일컫는다.The "heavy chain" of the present invention is a full-length heavy chain comprising a variable region domain VH and three constant region domains CH1, CH2 and CH3 comprising an amino acid sequence of a variable region sufficient to confer specificity for an antigen, and a full-length heavy chain thereof refers to all fragments.
본 발명의 "경쇄"는, 항원에 특이성을 부여하기 위해 충분한 가변영역의 아미노산 서열을 포함하는 가변영역 도 메인 VL 및 불변영역 도메인 CL을 포함하는 전체 길이 경쇄 및 이의 단편을 모두 일컫는다.The "light chain" of the present invention refers to both a full-length light chain comprising a variable region domain VL and a constant region CL comprising an amino acid sequence of a variable region sufficient to confer specificity to an antigen, and a fragment thereof.
2) 막관통 도메인2) transmembrane domain
본 발명의 상기 "막통과 도메인"은 항원 결합 도메인과 보조자극, 필수 신호전달 도메인을 세포막 사이로 연결하는 부위를 의미한다. 상기 막통과 도메인은 세포막에 걸친 소수성 폴리펩타이드를 포함하며, 세포막의 한 면(세포 외)에서 세포막의 다른 면 (세포 내 또는 세포질)을 통하여 걸쳐 있을 수 있다.The "transmembrane domain" of the present invention refers to a site connecting the antigen-binding domain and the costimulatory and essential signaling domains between the cell membrane. The transmembrane domain comprises a hydrophobic polypeptide spanning the cell membrane and may span from one side of the cell membrane (extracellular) through the other side of the cell membrane (intracellular or cytoplasmic).
본 발명의 상기 막통과 도메인은 알파 나선 또는 베타 배럴, 또는 이들의 조합의 형태일 수 있다. 또한, 본 발명에서 상기 막통과 도메인은 다수의 막통과 조각, 각 알파-나선형, 베타 시트, 또는 이들의 조합을 갖는 다원 단백질을 포함할 수 있다.The transmembrane domain of the present invention may be in the form of an alpha helix or beta barrel, or a combination thereof. In addition, in the present invention, the transmembrane domain may include a polyprotein having a plurality of transmembrane fragments, each alpha-helical, beta sheet, or a combination thereof.
본 발명의 상기 막통과 도메인으로는, 예를 들면 CD3 제타(ζ) 사슬의 전부 또는 일부, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, 이들의 기능적 유도체 및 이들의 조합을 포함할 수 있으나, 이에 제한되는 것은 아니다. The transmembrane domain of the present invention includes, for example, all or part of the CD3 zeta (ζ) chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof, and combinations thereof.
본 발명의 상기 막통과 도메인으로 예를 들어 인공적으로 설계된 것은 주로 류신 및 발린과 같은 소수성 잔기를 포함하는 폴리펩타이드일 수 있다. 본 발명의 일 실시양태에서, 페닐알라닌, 트립토판 및 발린의 삼중체는 합성 막 변이 도메인의 각 말단에서 발견될 수 있다.For example, artificially designed as the transmembrane domain of the present invention may be a polypeptide mainly comprising hydrophobic residues such as leucine and valine. In one embodiment of the invention, a triplet of phenylalanine, tryptophan and valine can be found at each terminus of the synthetic membrane variable domain.
3) 세포내 신호전달 도메인3) intracellular signaling domains
본 발명의 상기 "세포내 신호 전달 도메인"은 면역세포 내부에서 발견되거나 발견되도록 조작된 키메릭 항원 수용체의 부분을 의미한다.The "intracellular signal transduction domain" of the present invention refers to a portion of a chimeric antigen receptor that is found or engineered to be found inside an immune cell.
본 발명의 상기 세포내 신호전달 도메인은 면역세포 신호전달 경로의 적어도 일부 면을 자극 또는 활성화시키기 위해 면역세포의 활성화를 제공하는 폴리펩타이드를 포함할 수 있다.The intracellular signaling domain of the present invention may comprise a polypeptide that provides for activation of an immune cell to stimulate or activate at least some aspect of an immune cell signaling pathway.
본 발명의 상기 세포내 신호전달 도메인은 CD3 제타(ζ)의 전부 또는 일부, 공통 FcR 감마 (FcER1G), Fc감마R²a, FcR베타 (Fc 엡실론 립), CD3감마, CD3델타, CD3 엡실론, CD79a, CD79b, DNAX- 활성화 단백질 10 (DAP10), DNAX- 활성화 단백질 12 (DAP12), 이의 활성 단편, 이의 기능적 유도체 및 이들의 조합을 포함하는 폴리펩타이드로부터 유래한 기능 신호전달 도메인을 포함할 수 있으나, 이에 제한되는 것은 아니며, 이러한 신호전달 도메인은 당업계에 공지되어 있다.The intracellular signaling domain of the present invention includes all or part of CD3 zeta (ζ), consensus FcR gamma (FcER1G), FcgammaR²a, FcRbeta (Fc epsilon rib), CD3gamma, CD3delta, CD3 epsilon, CD79a, CD79b, DNAX-activating protein 10 (DAP10), DNAX-activating protein 12 (DAP12), active fragments thereof, functional derivatives thereof, and combinations thereof. Without limitation, such signaling domains are known in the art.
2. 2. 암의 예방 또는 치료용 약학 조성물Pharmaceutical composition for preventing or treating cancer
본 발명의 또 다른 구현 예에서는 암의 예방 또는 치료용 약학 조성물을 제공한다.Another embodiment of the present invention provides a pharmaceutical composition for preventing or treating cancer.
본 발명의 상기 약학 조성물은 암 세포 표면 단백질에 특이적으로 결합하는 항원 결합 도메인; 및 막관통 도메인; 세포내 신호전달 도메인을 포함하는 키메릭 항원 수용체;가 포함된 면역세포를 유효성분으로 포함한다.The pharmaceutical composition of the present invention comprises an antigen-binding domain that specifically binds to a cancer cell surface protein; and a transmembrane domain; A chimeric antigen receptor comprising an intracellular signaling domain; contains the containing immune cell as an active ingredient.
본 발명의 상기 약학 조성물에서, 암 세포 표면 단백질, 면역세포, 항원 결합도메인, 막관통 도메인, 세포내 신호전달 도메인, 키메릭 항원 수용체, 면역세포 등과 관련된 내용은 앞서 1. 면역세포치료제 조성물에서 기재한 바와 동일하여 생락한다.In the pharmaceutical composition of the present invention, the contents related to cancer cell surface protein, immune cell, antigen binding domain, transmembrane domain, intracellular signaling domain, chimeric antigen receptor, immune cell, etc. are described above in 1. Immune cell therapy composition Same as one bar and omitted.
본 발명의 상기 암은 고형암 또는 혈액암일 수 있고, 예를 들면, 식도암, 위암, 대장암, 직장암, 구강암, 인두암, 후두암, 폐암, 결장암, 유방암, 자궁경부암, 자궁내막암, 난소암, 전립선암, 고환암, 방광암, 신장암, 간암, 췌장암, 골암, 결합 조직암, 피부암, 뇌암, 갑상선암, 백혈병, 호지킨(Hodgkin) 질환, 연부조직육종(soft tissue sarcoma), 림프종 및 다발성 골수종 혈액암으로 구성된 군으로부터 선택되는 것일 수 있으나, 이에 제한되는 것은 아니다.The cancer of the present invention may be a solid cancer or a blood cancer, for example, esophageal cancer, stomach cancer, colorectal cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer Cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's disease, soft tissue sarcoma, lymphoma and multiple myeloma blood cancer It may be selected from the group consisting of, but is not limited thereto.
본 발명의 상기 "예방" 은 동물의 병리학적 세포의 발생 또는 세포의 손상, 소실의 정도의 감소를 의미한다. 예방은 완전할 수 있으며 또는 부분적일 수도 있다. 이 경우에는 개체 내의 병리학적 세포의 발생 또는 비정상적인 면역 작용 등이 상기 암의 예방 및 치료용 조성물을 사용하지 않은 경우와 비교하여 감소하는 현상을 의미할 수 있다.The "prevention" of the present invention means a reduction in the degree of occurrence or damage to or loss of pathological cells in an animal. Prevention may be complete or partial. In this case, it may refer to a phenomenon in which the generation of pathological cells or abnormal immune action in an individual is decreased compared to the case where the composition for preventing and treating cancer is not used.
본 발명의 상기 "치료"는 치료하고자 하는 대상 또는 세포의 천연 과정을 변경시키기 위하여 임상적으로 개입하는 모든 행위를 의미하며, 임상 병리 상태가 진행되는 동안 또는 이를 예방하기 위하여 수행할 수 있다. 목적하는 치료 효과는 질병의 발생 또는 재발을 예방하거나, 증상을 완화시키거나, 질병에 따른 모든 직접 또는 간접적인 병리학적 결과를 저하시키거나, 전이를 예방하거나, 질병 진행 속도를 감소시키거나, 질병 상태를 경감 또는 일시적 완화시키거나, 예후를 개선시키는 것을 포함할 수 있다. 즉, 상기 치료는 상기 조성물에 의해 암의 증세가 호전되거나 완치되는 모든 행위를 포괄하는 것으로 해석될 수 있다.The "treatment" of the present invention means any action that clinically intervenes in order to change the natural process of a subject or cell to be treated, and may be performed while a clinical pathology is in progress or to prevent it. The desired therapeutic effect is to prevent the occurrence or recurrence of a disease, alleviate symptoms, reduce any direct or indirect pathological consequences of the disease, prevent metastasis, reduce the rate of disease progression, alleviating or temporarily ameliorating the condition, or improving the prognosis. That is, the treatment may be interpreted to encompass all actions in which symptoms of cancer are improved or cured by the composition.
본 발명의 상기 면역세포는 1Х107 내지 1Х108, 1Х108 내지 2Х108, 2Х108 내지 4Х108, 4Х108 내지 6Х108, 6Х108 내지 8Х108, 8Х108 내지 1Х109, 1Х109 내지 2Х109, 2Х109 내지 4Х109, 4Х109 내지 1Х1010, 2Х108 내지 6Х108, 6Х108 내지 1Х109, 1Х108 내지 2Х108, 2Х108 내지 2Х109, 1Х107 내지 1Х108, 1Х108 내지 1Х109, 1Х109 내지 1Х1010 또는 1Х107 내지 1Х109개 중 어느 하나의 세포/㎏의 투여량으로 투여될 수 있으나, 이에 제한되는 것은 아니다.The immune cells of the present invention are 1Х10 7 to 1Х10 8 , 1Х10 8 to 2Х10 8 , 2Х10 8 to 4Х10 8 , 4Х10 8 to 6Х10 8 , 6Х10 8 to 8Х10 8 , 8Х10 8 to 1Х10 9 , 1Х10 9 to 2Х10 9 , 9 to 4Х10 9 , 4Х10 9 to 1Х10 10 , 2Х10 8 to 6Х10 8 , 6Х10 8 to 1Х10 9 , 1Х10 8 to 2Х10 8 , 2Х10 8 to 2Х10 9 , 1Х10 7 to 1Х10 8 , 1Х10 9 , 1Х10 8 , 1Х10 8 to 1Х10 9 , 1Х10 10 or 1Х10 7 to 1Х10 9 may be administered at a dose of any one cell/kg, but is not limited thereto.
본 발명의 상기 약학 조성물은 캡슐, 정제, 과립, 주사제, 연고제, 분말 또는 음료 형태임을 특징으로 할 수 있으며, 상기 약학 조성물은 인간을 대상으로 하는 것을 특징으로 할 수 있다. The pharmaceutical composition of the present invention may be characterized in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and the pharmaceutical composition may be characterized in that it is targeted to humans.
본 발명의 상기 약학 조성물은 이들로 한정되는 것은 아니지만, 각각 통상의 방법에 따라 산제, 과립제, 캡슐, 정제, 수성 현탁액 등의 경구형 제형, 외용제, 좌제 및 멸균 주사 용액의 형태로 제형화되어 사용될 수 있다. 본 발명의 약학 조성물은 약학적으로 허용 가능한 담체를 포함할 수 있다. 약학적으로 허용되는 담체는 경구 투여 시에는 결합제, 활탁제, 붕해제, 부형제, 가용화제, 분산제, 안정화제, 현탁화제, 색소, 향료 등이 사용될 수 있으며, 주사제의 경우에는 완충제, 보존제, 무통화제, 가용화제, 등장제, 안정화제 등이 혼합되어 사용될 수 있으며, 국소투여용의 경우에는 기제, 부형제, 윤활제, 보존제 등이 사용될 수 있다. 본 발명의 약학 조성물의 제형은 상술한 바와 같은 약학적으로 허용되는 담체와 혼합하여 다양하게 제조될 수 있다. 예를 들어, 경구 투여시에는 정제, 트로키, 캡슐, 엘릭서(elixir), 서스펜션, 시럽, 웨이퍼 등의 형태로 제조할 수 있으며, 주사제의 경우에는 단위 투약 앰플 또는 다수 회 투약 형태로 제조할 수 있다. 기타, 용액, 현탁액, 정제, 캡슐, 서방형 제제 등으로 제형화할 수 있다.The pharmaceutical composition of the present invention is not limited thereto, but each is formulated in the form of oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories, and sterile injection solutions according to conventional methods to be used. can 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, dyes, fragrances, etc., for oral administration, and in the case of injections, buffers, preservatives, pain-freezing agents Agents, solubilizers, isotonic agents, stabilizers, etc. may be mixed and used, and in the case of topical administration, bases, excipients, lubricants, preservatives, etc. may be used. The dosage form of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above. For example, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. for oral administration, and in the case of injections, it can be prepared in the form of unit dosage ampoules or multiple dosage forms. there is. In addition, it can be formulated as a solution, suspension, tablet, capsule, sustained release formulation, and the like.
한편, 제제화에 적합한 담체, 부형제 및 희석제의 예로는, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 또는 광물유 등이 사용될 수 있다. 또한, 충진제, 항 응집제, 윤활제, 습윤제, 향료, 유화제, 방부제 등을 추가로 포함할 수 있다.Meanwhile, examples of suitable carriers, excipients and diluents 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, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil may be used. In addition, fillers, anti-agglomeration agents, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives and the like may be further included.
본 발명의 상기 약학 조성물의 투여 경로는 이들로 한정되는 것은 아니지만 구강, 정맥내, 근육내, 동맥내, 골수내, 경막내, 심장내, 경피, 피하, 복강내, 비강내, 장관, 국소, 설하 또는 직장이 포함된다. 경구 또는 비경구 투하가 바람직하다. The route of administration of the pharmaceutical composition of the present invention is, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal. Oral or parenteral administration is preferred.
본 발명의 상기 "비경구"란, 피하, 피내, 정맥내, 근육내, 관절내, 활액낭내, 흉골내, 경막내, 병소내 및 두개골내 주사 또는 주입기술을 포함한다. 본 발명의 약학 조성물은 또한 직장 투여를 위한 좌제의 형태로 투여될 수 있다.The "parenteral" of 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.
본 발명의 상기 약학 조성물은 사용된 특정 화합물의 활성, 연령, 체중, 일반적인 건강, 성별, 정식, 투여 시간, 투여 경로, 배출율, 약물 배합 및 예방 또는 치료될 특정 질환의 중증을 포함한 여러 요인에 따라 다양하게 변할 수 있고, 상기 약학 조성물의 투여량은 환자의 상태, 체중, 질병의 정도, 약무 형태, 투여 경로 및 기간에 따라 다르지만 당업자에 의해 적절하게 선택될 수 있고, 1일 0.0001 내지 50 mg/kg 또는 0.001 내지 50 mg/kg으로 투여할 수 있다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다. 본 발명에 따른 의약 조성물은 환제, 당의정, 캡슐, 액제, 겔, 시럽, 슬러리, 현탁제로 제형화될 수 있다.The pharmaceutical composition of the present invention depends on several factors including the activity of the specific compound used, age, weight, general health, sex, formula, administration time, administration route, excretion rate, drug formulation, and the severity of the specific disease to be prevented or treated. It can be variously changed, and the dosage of the pharmaceutical composition varies depending on the patient's condition, body weight, degree of disease, drug form, administration route and period, but may be appropriately selected by those skilled in the art, and 0.0001 to 50 mg/day per day kg or 0.001 to 50 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. The above 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 pills, dragees, capsules, solutions, gels, syrups, slurries, and suspensions.
3. 3. 암의 예방 또는 개선용 식품 조성물Food composition for preventing or improving cancer
본 발명의 또 다른 구현 예에서는 암의 예방, 또는 개선용 식품 조성물을 제공한다.Another embodiment of the present invention provides a food composition for preventing or improving cancer.
본 발명의 상기 식품 조성물은 암 세포 표면 단백질에 특이적으로 결합하는 항원 결합 도메인; 막관통 도메인; 및 세포내 신호전달 도메인을 포함하는 키메릭 항원 수용체;가 포함된 면역세포를 유효성분으로 포함한다.The food composition of the present invention comprises an antigen binding domain that specifically binds to a cancer cell surface protein; transmembrane domain; And a chimeric antigen receptor comprising an intracellular signaling domain; includes immune cells containing the as an active ingredient.
본 발명의 상기 식품 조성물에서, 암 세포 표면 단백질, 면역세포, 항원 결합도메인, 막관통 도메인, 세포내 신호전달 도메인, 키메릭 항원 수용체, 면역세포 등과 관련된 내용은 앞서 1. 면역세포치료제 조성물에서 기재한 바와 동일하여 생략한다.In the food composition of the present invention, the contents related to cancer cell surface protein, immune cell, antigen binding domain, transmembrane domain, intracellular signaling domain, chimeric antigen receptor, immune cell, etc. are described above in 1. Immune cell therapy composition It is omitted as it is the same as the previous one.
본 발명의 상기 암은 고형암 또는 혈액암일 수 있고, 예를 들면, 식도암, 위암, 대장암, 직장암, 구강암, 인두암, 후두암, 폐암, 결장암, 유방암, 자궁경부암, 자궁내막암, 난소암, 전립선암, 고환암, 방광암, 신장암, 간암, 췌장암, 골암, 결합 조직암, 피부암, 뇌암, 갑상선암, 백혈병, 호지킨(Hodgkin) 질환, 연부조직육종(soft tissue sarcoma), 림프종 및 다발성 골수종 혈액암으로 구성된 군으로부터 선택되는 것일 수 있으나, 이에 제한되는 것은 아니다.The cancer of the present invention may be a solid cancer or a blood cancer, for example, esophageal cancer, stomach cancer, colorectal cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer Cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's disease, soft tissue sarcoma, lymphoma and multiple myeloma blood cancer It may be selected from the group consisting of, but is not limited thereto.
본 발명의 상기 "예방" 은 동물의 병리학적 세포의 발생 또는 세포의 손상, 소실의 정도의 감소를 의미한다. 예방은 완전할 수 있으며 또는 부분적일 수도 있다. 이 경우에는 개체 내의 병리학적 세포의 발생 또는 비정상적인 면역 작용 등이 상기 암의 예방 및 치료용 조성물을 사용하지 않은 경우와 비교하여 감소하는 현상을 의미할 수 있다.The "prevention" of the present invention means a reduction in the degree of occurrence or damage to or loss of pathological cells in an animal. Prevention may be complete or partial. In this case, it may refer to a phenomenon in which the generation of pathological cells or abnormal immune action in an individual is decreased compared to the case where the composition for preventing and treating cancer is not used.
본 발명의 상기 "개선"은 개선시키고자 하는 대상 또는 세포의 천연 과정을 변경시키기 위하여 임상적으로 개입하는 모든 행위를 의미하며, 임상 병리 상태가 진행되는 동안 또는 이를 예방하기 위하여 수행할 수 있다. 목적하는 개선의 효과는 질병의 발생 또는 재발을 예방하거나, 증상을 완화시키거나, 질병에 따른 모든 직접 또는 간접적인 병리학적 결과를 저하시키거나, 전이를 예방하거나, 질병 진행 속도를 감소시키거나, 질병 상태를 경감 또는 일시적 완화시키거나, 예후를 개선시키는 것을 포함할 수 있다. 즉, 상기 개선은 상기 조성물에 의해 증세가 호전되거나 완치되는 모든 행위를 포괄하는 것으로 해석될 수 있다.The "improvement" of the present invention means any action that clinically intervenes in order to change the natural process of a target or cell to be improved, and can be performed while a clinical pathology is in progress or to prevent it. The desired effect of improvement is preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, alleviating or temporarily ameliorating the disease state, or improving the prognosis. That is, the improvement may be interpreted as encompassing all actions in which symptoms are improved or cured by the composition.
본 발명의 상기 면역세포는 1Х107 내지 1Х108, 1Х108 내지 2Х108, 2Х108 내지 4Х108, 4Х108 내지 6Х108, 6Х108 내지 8Х108, 8Х108 내지 1Х109, 1Х109 내지 2Х109, 2Х109 내지 4Х109, 4Х109 내지 1Х1010, 2Х108 내지 6Х108, 6Х108 내지 1Х109, 1Х108 내지 2Х108, 2Х108 내지 2Х109, 1Х107 내지 1Х108, 1Х108 내지 1Х109, 1Х109 내지 1Х1010 또는 1Х107 내지 1Х109개 중 어느 하나의 세포/㎏의 투여량으로 투여될 수 있으나, 이에 제한되는 것은 아니다.The immune cells of the present invention are 1Х10 7 to 1Х10 8 , 1Х10 8 to 2Х10 8 , 2Х10 8 to 4Х10 8 , 4Х10 8 to 6Х10 8 , 6Х10 8 to 8Х10 8 , 8Х10 8 to 1Х10 9 , 1Х10 9 to 2Х10 9 , 9 to 4Х10 9 , 4Х10 9 to 1Х10 10 , 2Х10 8 to 6Х10 8 , 6Х10 8 to 1Х10 9 , 1Х10 8 to 2Х10 8 , 2Х10 8 to 2Х10 9 , 1Х10 7 to 1Х10 8 , 1Х10 9 , 1Х10 8 , 1Х10 8 to 1Х10 9 , 1Х10 10 or 1Х10 7 to 1Х10 9 may be administered at a dose of any one cell/kg, but is not limited thereto.
본 발명의 상기 식품 조성물은 각종 식품류, 예를 들어, 음료, 껌, 차, 비타민 복합제, 분말, 과립, 정제, 캡슐, 과자, 떡, 빵 등의 형태로 제조될 수 있다. The food composition of the present invention may be prepared in the form of various foods, for example, beverages, gums, tea, vitamin complexes, powders, granules, tablets, capsules, confectionery, rice cakes, bread, and the like.
본 발명의 상기 유효성분이 식품 조성물에 포함될 때 그 양은 전체 중량의 0.1 내지 50%의 비율로 첨가할 수 있으나, 이에 제한되는 것은 아니다.When the active ingredient of the present invention is included in the food composition, the amount may be added in a proportion of 0.1 to 50% of the total weight, but is not limited thereto.
본 발명의 상기 식품 조성물이 음료 형태로 제조되는 경우 지시된 비율로 상기 식품 조성물을 포함하는 것 외에 특별한 제한점은 없으며, 통상의 음료와 같이 다양한 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 구체적으로, 천연 탄수화물로서 포도당 등의 모노사카라이드, 과당 등의 디사카라이드, 수크로스 등의 및 폴리사카라이드, 덱스트린, 시클로덱스트린 등과 같은 통상적인 당 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜 등을 포함할 수 있다. 상기 향미제로서는 천연 향미제(타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진등) 및 합성 향미제(사카린, 아스파르탐 등) 등일 수 있다. When the food composition of the present invention is prepared in the form of a beverage, there is no particular limitation except for including the food composition in the indicated ratio, and it may contain various flavoring agents or natural carbohydrates as additional ingredients, like a conventional beverage. . Specifically, as natural carbohydrates, monosaccharides such as glucose, disaccharides such as fructose, and polysaccharides such as sucrose, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol are used as natural carbohydrates. may include The flavoring agent may be a natural flavoring agent (taumartin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agent (saccharin, aspartame, etc.).
본 발명의 상기 식품 조성물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 더 포함할 수 있다.The food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, It may further include a pH adjuster, a stabilizer, a preservative, glycerin, alcohol, a carbonation agent used in carbonated beverages, and the like.
본 발명의 상기 성분은 독립적 또는 조합하여 사용할 수 있다. 상기 첨가제의 비율은 본 발명의 핵심적인 요소에 해당하지 아니하지만, 본 발명의 식품 조성물 100 중량부 당 0.1 내지 약 50 중량부의 범위에서 선택될 수 있으나, 이에 제한되는 것은 아니다.The above components of the present invention may be used independently or in combination. The proportion of the additive is not a key element of the present invention, but may be selected from 0.1 to about 50 parts by weight per 100 parts by weight of the food composition of the present invention, but is not limited thereto.
4. 4. 면역세포치료제의 제조 방법Manufacturing method for immune cell therapy
본 발명의 또 다른 구현 예에서는 면역세포치료제의 제조 방법을 제공한다.Another embodiment of the present invention provides a method for manufacturing an immune cell therapy product.
본 발명의 상기 제조 방법은 면역세포를 배양하는 단계; 및 상기 배양된 면역세포에 키메릭 항원 수용체를 암호화하는 카세트가 포함된 벡터를 형질 주입하는 단계;를 포함한다.The manufacturing method of the present invention comprises the steps of culturing immune cells; and transfecting the cultured immune cells with a vector containing a cassette encoding a chimeric antigen receptor.
본 발명의 상기 "카세트"는 본 발명에 따른 키메릭 항원 수용체를 암호화하는 폴리뉴클레오타이드, 이의 발현을 조절할 수 있는 프로모터, 인핸서, 폴리아데닐화 신호, 전사 종결요소 또는 내부 리보솜 진입부위(IRES) 등과 같은 발현 제어 서열을 포함하는 것일 수 있다.The "cassette" of the present invention includes a polynucleotide encoding a chimeric antigen receptor according to the present invention, a promoter capable of regulating its expression, an enhancer, a polyadenylation signal, a transcription terminator or an internal ribosome entry site (IRES), etc. It may include an expression control sequence.
본 발명에서 상기 프로모터로는 예를 들어, SFFV 프로모터, 인간연장 인자 11α (EF) 프로모터 또는 CAG (CMV 증강인자를 갖는 닭 베타-액틴 프로모터) 프로모터를 포함할 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the promoter may include, for example, an SFFV promoter, a human elongation factor 11α (EF) promoter, or a CAG (chicken beta-actin promoter with a CMV enhancer) promoter, but is not limited thereto.
본 발명의 상기 "벡터"는 예를 들면, 플라스미드, 트랜스포손 또는 코스미드일 수 있으나, 이에 제한되는 것은 아니다.The "vector" of the present invention may be, for example, a plasmid, a transposon, or a cosmid, but is not limited thereto.
본 발명의 상기 형질 주입하는 단계는 미세주사, 세포융합, 칼슘 포스페이트 침전법, 리포좀 매개된 형질 주입(liposome-mediated transfection), DEAE 덱스트란-매개된 형질 주입(DEAE Dextran- mediated transfection), 폴리브렌-매개된 형질 주입(polybrene-mediated transfection), 전기침공법(electroporation), 유전자 총(gene gun) 또는 세포 내로 핵산을 유입시키기 위한 다른 공지의 방법에 의해 세포 내로 도입할 수 있으며, 이에 제한되는 것은 아니다(Wu et al., J. Bio. Chem., 267:963-967, 1992; Wu and Wu, J. Bio. Chem.,263:14621-14624, 1988).The transfection step of the present invention includes microinjection, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene -Mediated transfection (polybrene-mediated transfection), electroporation (electroporation), gene gun (gene gun) or other known methods for introducing nucleic acids into the cell can be introduced into the cell, but is limited thereto No (Wu et al., J. Bio. Chem., 267:963-967, 1992; Wu and Wu, J. Bio. Chem., 263:14621-14624, 1988).
본 발명의 상기 형질 주입하는 단계는 바이러스성 형질 주입 방법 또는 비바이러스성 형질 주입 방법을 이용하는 것일 수 있다. 본 발명의 목적상 비 바이러스성 형질 주입 방법을 이용하는 경우에는 시험관 내에서 재조합된 면역세포 자체가 유효성분으로서, 대상이 되는 개체에 직접 투입되는 것이므로 바이러스를 사용한 경우와 비교하여 개체에 발생될 수 있는 부작용을 줄일 수 있다는 점에서 매우 유용하다.The transfection step of the present invention may be using a viral transfection method or a non-viral transfection method. In the case of using the non-viral transfection method for the purpose of the present invention, since immune cells recombined in vitro are directly injected into the subject as an active ingredient as an active ingredient, compared with the case of using a virus, It is very useful in that it can reduce side effects.
5. 5. 면역세포치료제를 스크리닝하는 방법How to screen for immune cell therapy
본 발명의 또 다른 구현 예에서는 면역세포치료제를 스크리닝하는 방법을 제공한다.Another embodiment of the present invention provides a method for screening an immune cell therapy agent.
본 발명의 상기 스크리닝하는 방법은 목적하는 개체로부터 분리된 생물학적 시료에서, 암 세포 표면 단백질에 특이적으로 결합하는 항원 결합 도메인; 막관통 도메인; 및 세포내 신호전달 도메인을 포함하는 키메릭 항원 수용체;를 포함하는 면역세포를 처리하는 단계를 포함한다. The screening method of the present invention includes an antigen-binding domain that specifically binds to a cancer cell surface protein in a biological sample isolated from a subject of interest; transmembrane domain; and a chimeric antigen receptor comprising an intracellular signaling domain;
본 발명에서 상기 "목적하는 개체"란 면역세포치료제에 의한 치료 반응성이 불확실한 개체로, 질환이 발병되었거나, 또는 발병 가능성이 높은 개체를 의미한다.In the present invention, the "target individual" refers to an individual whose treatment responsiveness to an immune cell therapy agent is uncertain, and an individual who has developed a disease or is highly likely to develop the disease.
본 발명의 상기 "생물학적 시료"는 개체로부터 얻어지거나 개체로부터 유래된 임의의 물질, 생물학적 체액, 조직 또는 세포를 의미하는 것으로, 예를 들면, 전혈(whole blood), 백혈구(leukocytes), 말초혈액 단핵 세포(peripheral blood mononuclear cells), 백혈구 연층(buffy coat), 혈장(plasma) 및 혈청(serum)을 포함하는) 혈액, 객담(sputum), 눈물(tears), 점액(mucus), 세비액(nasal washes), 비강 흡인물(nasal aspirate), 호흡(breath), 소변(urine), 정액(semen), 침(saliva), 복강 세척액(peritoneal washings), 골반 내 유체액(pelvic fluids), 낭종액(cystic fluid), 뇌척수막 액(meningeal fluid), 양수(amniotic fluid), 선액(glandular fluid), 췌장액(pancreatic fluid), 림프액(lymph fluid), 흉수(pleural fluid), 유두 흡인물(nipple aspirate), 기관지 흡인물(bronchial aspirate), 활액(synovial fluid), 관절 흡인물(joint aspirate), 기관 분비물(organ secretions), 세포(cell), 세포 추출물(cell extract) 또는 뇌척수액(cerebrospinal fluid)을 포함할 수 있으나, 이에 제한되는 것은 아니다.The "biological sample" of the present invention refers to any material, biological fluid, tissue or cell obtained from or derived from an individual, for example, whole blood, leukocytes, peripheral blood mononuclear blood, sputum, tears, mucus, nasal washes, including peripheral blood mononuclear cells, buffy coat, plasma and serum ), nasal aspirate, breath, urine, semen, saliva, peritoneal washings, pelvic fluids, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspiration It may include bronchial aspirate, synovial fluid, joint aspirate, organ secretions, cell, cell extract or cerebrospinal fluid, However, the present invention is not limited thereto.
본 발명의 상기 스크리닝 방법에서, 암 세포 표면 단백질, 항원 결합도메인, 막관통 도메인, 세포내 신호전달 도메인, 키메릭 항원 수용체, 면역세포 및 발현 벡터 등과 관련된 내용은 앞서 1. 면역세포치료제 조성물에서 기재한 바와 동일하여 생략한다.In the screening method of the present invention, the contents related to cancer cell surface protein, antigen binding domain, transmembrane domain, intracellular signaling domain, chimeric antigen receptor, immune cell and expression vector, etc. are described above in 1. Immune cell therapy composition It is omitted as it is the same as the previous one.
본 발명의 상기 스크리닝하는 방법에서, 상기 면역세포의 처리 후 상기 생물학적 시료에서 암 오가노이드의 크기, 세포사멸 또는 암 조직으로 침윤 정도를 측정하는 단계를 포함할 수 있다.In the screening method of the present invention, it may include measuring the size of cancer organoids, apoptosis, or the degree of invasion into cancer tissues in the biological sample after the treatment of the immune cells.
본 발명의 상기 "오가노이드"는 장기유사체라고도 불리우며, 줄기세포 또는 환자로부터 유래된 세포를 이용하여 자가재생 또는 자가 조직화 과정을 통해 형성된 3차원 세포 집합체를 의미한다. 이와 같은 오가노이드는 세포를 3차원 배양법을 통해 응집 재조합하여 만들기 때문에, 모델 장기의 특이적인 세포를 포함하고 있다.The "organoid" of the present invention is also called an organ analogue, and refers to a three-dimensional cell aggregate formed through a process of self-renewal or self-organization using stem cells or cells derived from a patient. Since such organoids are made by aggregating and recombination of cells through a three-dimensional culture method, they contain specific cells of a model organ.
본 발명의 상기 스크리닝하는 방법에서, 상기 목적하는 개체로부터 유래된 암 세포를 이용하여 제조된 암 오가노이드와 본 발명에 따른 상기 면역세포를 공배양한 뒤, 암 오가노이드의 크기가 감소 또는 유지되는 경우; 또는 암 오가노이드로의 침윤 정도가 억제되는 경우에는 상기 면역세포를 면역세포치료제로 판단할 수 있다.In the screening method of the present invention, after co-culturing a cancer organoid prepared using cancer cells derived from the target individual with the immune cell according to the present invention, the size of the cancer organoid is reduced or maintained. Occation; Alternatively, when the degree of infiltration into cancer organoids is suppressed, the immune cells may be determined as an immune cell therapy agent.
본 발명의 상기 "세포사멸"은 세포가 기능적인 역할은 유지하면서 유전적 성질에 의해 죽음으로 이르는 단계를 의미한다.The "apoptosis" of the present invention means a step leading to death due to genetic properties while maintaining a functional role of the cell.
본 발명의 상기 스크리닝하는 방법에서, 상기 목적하는 개체로부터 유래된 암 세포 또는 암 오가노이드와 본 발명에 따른 상기 면역세포를 공배양한 뒤에 목적하는 개체로부터 유래된 암 세포 또는 암 오가노이드에서 발현되는 세포사멸과 관련된 유전자 또는 단백질의 발현 수준을 측정하고, 세포사멸이 증가된 경우 상기 면역세포를 면역세포치료제로 판단할 수 있다.In the screening method of the present invention, after co-culturing the cancer cells or cancer organoids derived from the subject of interest with the immune cells according to the present invention, the expression is expressed in cancer cells or cancer organoids derived from the subject of interest. The expression level of a gene or protein related to apoptosis is measured, and when apoptosis is increased, the immune cells can be determined as an immune cell therapy agent.
본 발명의 상기 단백질의 발현 수준을 측정하는 방법은 단백질 칩 분석, 면역측정법, 리간드 바인딩 어세이, MALDI-TOF(Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) 분석, SELDI-TOF(Sulface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry) 분석, 방사선 면역 분석, 방사 면역 확산법, 오우크테로니 면역 확산법, 로케트 면역전기영동, 조직면역 염색, 보체 고정 분석법, 2차원 전기영동 분석, 액상 크로마토그래피-질량분석(liquid chromatography-Mass Spectrometry, LC-MS), LC-MS/MS(liquid chromatography-Mass Spectrometry/ Mass Spectrometry), 웨스턴 블랏팅 및 ELISA(enzyme linked immunosorbentassay)로 이루어진 군으로부터 선택되는 적어도 하나인 것일 수 있다.The method of measuring the expression level of the protein of the present invention is a protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser) Desorption/Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunodiffusion method, Oukteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, 2D electrophoresis analysis, liquid chromatography-mass Analysis (liquid chromatography-Mass Spectrometry, LC-MS), LC-MS/MS (liquid chromatography-Mass Spectrometry/ Mass Spectrometry), Western blotting and ELISA (enzyme linked immunosorbent assay) may be at least one selected from the group consisting of there is.
본 발명의 상기 단백질의 발현 수준은 단백질의 발현 수준을 측정할 수 있는 제제를 이용하여 측정될 수 있다. 상기 단백질의 발현 수준을 측정할 수 있는 제제는 상기 단백질에 특이적으로 결합하는 항체, 올리고펩타이드, 리간드, PNA(Peptide nucleic acid) 및 앱타머(aptamer)로 이루어진 군으로부터 선택되는 적어도 하나인 것일 수 있다.The expression level of the protein of the present invention can be measured using an agent capable of measuring the expression level of the protein. The agent capable of measuring the expression level of the protein may be at least one selected from the group consisting of an antibody, oligopeptide, ligand, PNA (peptide nucleic acid) and aptamer that specifically binds to the protein. there is.
본 발명의 상기 "항체"는 항원과 특이적으로 결합하여 항원-항체 반응을 일으키는 물질을 가리킨다. 본 발명의 목적상, 항체는 상기 단백질에 대해 특이적으로 결합하는 항체를 의미한다. 본 발명의 항체는 다클론 항체, 단일클론 항체 및 재조합 항체를 모두 포함한다. 상기 항체는 당업계에 널리 공지된 기술을 이용하여 용이하게 제조될 수 있다. 예를 들어, 다클론 항체는 상기 단백질의 항원을 동물에 주사하고 동물로부터 채혈하여 항체를 포함하는 혈청을 수득하는 과정을 포함하는 당업계에 널리 공지된 방법에 의해 생산될 수 있다. 이러한 다클론 항체는 염소, 토끼, 양, 원숭이, 말, 돼지, 소, 개 등의 임의의 동물로부터 제조될 수 있다. 또한, 단일클론 항체는 당업계에 널리 공지된 하이브리도마 방법(hybridoma method; Kohler 및 Milstein (1976) European Journal of Immunology 6:511-519 참조), 또는 파지 항체 라이브러리 기술(Clackson et al, Nature, 352:624-628, 1991; Marks et al, J. Mol. Biol., 222:58, 1-597, 1991 참조)을 이용하여 제조될 수 있다. 상기 방법으로 제조된 항체는 겔 전기영동, 투석, 염 침전, 이온교환 크로마토그래피, 친화성 크로마토그래피 등의 방법을 이용하여 분리, 정제될 수 있다. 또한, 본 발명의 항체는 2개의 전장의 경쇄 및 2개의 전장의 중쇄를 갖는 완전한 형태뿐만 아니라, 항체의 기능적인 단편을 포함한다.The "antibody" of the present invention refers to a substance that specifically binds to an antigen and causes an antigen-antibody reaction. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to said protein. Antibodies of the present invention include polyclonal antibodies, monoclonal antibodies and recombinant antibodies. The antibody can be readily prepared using techniques well known in the art. For example, the polyclonal antibody can be produced by a method well known in the art, which includes the process of injecting an antigen of the protein into an animal and collecting blood from the animal to obtain a serum containing the antibody. Such polyclonal antibodies can be prepared from any animal such as goat, rabbit, sheep, monkey, horse, pig, cow, dog, and the like. In addition, monoclonal antibodies can be prepared using the hybridoma method well known in the art (see Kohler and Milstein (1976) European Journal of Immunology 6:511-519), or the phage antibody library technology (Clackson et al, Nature, 352:624-628, 1991; Marks et al, J. Mol. Biol., 222:58, 1-597, 1991). The antibody prepared by the above method may be separated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography. In addition, the antibody of the present invention includes a complete form having two full-length light chains and two full-length heavy chains, as well as functional fragments of the antibody.
본 발명의 상기 항체의 기능적인 단편이란, 적어도 항원 결합 기능을 보유하고 있는 단편을 의미하며, Fab, F(ab'), F(ab')2 및 Fv 등이 있다.The functional fragment of the antibody of the present invention means a fragment having at least an antigen-binding function, and includes Fab, F(ab'), F(ab')2 and Fv.
본 발명에 상기 "PNA(Peptide Nucleic Acid)"는 인공적으로 합성된, DNA 또는 RNA와 비슷한 중합체를 가리키며, 1991년 덴마크 코펜하겐 대학교의 Nielsen, Egholm, Berg와 Buchardt 교수에 의해 처음으로 소개되었다. DNA는 인산-리보스당 골격을 갖는데 반해, PNA는 펩타이드 결합에 의해 연결된 반복된 N-(2-아미노에틸)-글리신 골격을 가지며, 이로 인해 DNA 또는 RNA에 대한 결합력과 안정성이 크게 증가되어 분자 생물학, 진단 분석 및 안티센스 치료법에 사용되고 있다. PNA는 문헌[Nielsen PE, Egholm M, Berg RH, Buchardt O (December 1991). "Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide". Science 254 (5037): 1497-1500]에 상세하게 개시되어 있다.In the present invention, the "PNA (Peptide Nucleic Acid)" refers to an artificially synthesized, DNA or RNA-like polymer, and was first introduced by Professors Nielsen, Egholm, Berg and Buchardt of the University of Copenhagen, Denmark in 1991. Whereas DNA has a phosphate-ribose sugar backbone, PNA has a repeated N-(2-aminoethyl)-glycine backbone linked by peptide bonds, which greatly increases binding strength and stability to DNA or RNA, resulting in molecular biology , diagnostic assays and antisense therapy. PNA is described in Nielsen PE, Egholm M, Berg RH, Buchardt O (December 1991). "Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide". Science 254 (5037): 1497-1500.
본 발명의 상기 "앱타머"는 올리고핵산 또는 펩타이드 분자이며, 앱타머의 일반적인 내용은 문헌[Bock LC et al., Nature 355(6360):5646(1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78(8):42630(2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95(24): 142727(1998)]에 상세하게 개시되어 있다.The "aptamer" of the present invention is an oligonucleic acid or peptide molecule, and the general description of the aptamer is described in Bock LC et al., Nature 355(6360):5646(1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78(8):42630(2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95(24): 142727 (1998).
본 발명의 상기 단백질의 발현 수준을 측정할 수 있는 제제는 본 발명의 상기 단백질들을 이루는 아미노산 서열을 토대로, 통상의 기술자에 의해 상기 단백질에 특이적으로 결합하는 항체, PNA 및 앱타머 등에 해당하는 상기 제제가 쉽게 제작될 수 있다.The agent capable of measuring the expression level of the protein of the present invention is based on the amino acid sequence constituting the proteins of the present invention, which corresponds to an antibody, PNA, and aptamer that specifically binds to the protein by those skilled in the art. The formulation can be easily manufactured.
본 발명의 상기 유전자의 발현 수준을 측정하는 방법은 역전사 중합효소반응(RT-PCR), 경쟁적 역전사 중합효소반응(Competitive RT-PCR), 실시간 역전사 중합효소반응(Real-time RT-PCR), RNase 보호 분석법(RPA; RNase protection assay), 노던 블랏팅(Northern blotting) 및 DNA 칩으로 이루어진 군으로부터 선택되는 적어도 하나인 것일 수 있다.The method of measuring the expression level of the gene of the present invention is reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase reaction (Real-time RT-PCR), RNase It may be at least one selected from the group consisting of a protection assay (RPA; RNase protection assay), Northern blotting, and a DNA chip.
본 발명의 상기 유전자의 발현 수준은 유전자의 발현 수준을 측정할 수 있는 제제를 이용하여 측정될 수 있다. 상기 유전자의 발현 수준을 측정할 수 있는 제제는 상기 유전자에 상보적으로 결합하는 프라이머, 프로브 및 안티센스 뉴클레오티드로 이루어진 군으로부터 선택되는 적어도 하나인 것일 수 있다.The expression level of the gene of the present invention can be measured using an agent capable of measuring the expression level of the gene. The agent capable of measuring the expression level of the gene may be at least one selected from the group consisting of primers, probes and antisense nucleotides that complementarily bind to the gene.
본 발명의 상기 "프라이머"는 표적 유전자 서열을 인지하는 단편으로서, 정방향 및 역방향의 프라이머 쌍을 포함하나, 바람직하게는, 특이성 및 민감성을 가지는 분석 결과를 제공하는 프라이머 쌍이다. 프라이머의 핵산 서열이 시료 내 존재하는 비-표적 서열과 불일치하는 서열이어서, 상보적인 프라이머 결합 부위를 함유하는 표적 유전자 서열만 증폭하고 비특이적 증폭을 유발하지 않는 프라이머일 때, 높은 특이성이 부여될 수 있다.The "primer" of the present invention is a fragment recognizing a target gene sequence, and includes a pair of forward and reverse primers, but preferably, a primer pair that provides analysis results having specificity and sensitivity. When the nucleic acid sequence of the primer is a sequence that is inconsistent with a non-target sequence present in the sample, and thus amplifies only the target gene sequence containing the complementary primer binding site and does not cause non-specific amplification, high specificity can be conferred. .
본 발명의 상기 "프로브"란 시료 내의 검출하고자 하는 표적 물질과 상보적으로 결합할 수 있는 물질을 의미하며, 상기 결합을 통하여 특이적으로 시료 내의 표적 물질의 존재를 확인할 수 있는 물질을 의미한다. 프로브의 종류는 당업계에서 통상적으로 사용되는 물질로서 제한은 없으나, 바람직하게는 PNA(peptide nucleic acid), LNA(locked nucleic acid), 펩타이드, 폴리펩타이드, 단백질, RNA 또는 DNA일 수 있으며, 가장 바람직하게는 PNA이다. 보다 구체적으로, 상기 프로브는 바이오 물질로서 생물에서 유래되거나 이와 유사한 것 또는 생체 외에서 제조된 것을 포함하는 것으로, 예를 들어, 효소, 단백질, 항체, 미생물, 동식물 세포 및 기관, 신경세포, DNA, 및 RNA일 수 있으며, DNA는 cDNA, 게놈 DNA, 올리고뉴클레오타이드를 포함하며, RNA는 게놈 RNA, mRNA, 올리고뉴클레오타이드를 포함한다.The "probe" of the present invention means a substance capable of complementary binding to a target substance to be detected in a sample, and means a substance capable of specifically confirming the presence of a target substance in the sample through the binding. The type of probe is not limited as a material commonly used in the art, but preferably PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA or DNA, and most preferably It is PNA. More specifically, the probe is a biomaterial derived from or similar thereto, or manufactured in vitro, and includes, for example, enzymes, proteins, antibodies, microorganisms, animal and plant cells and organs, neurons, DNA, and It may be RNA, and DNA includes cDNA, genomic DNA, and oligonucleotides, and RNA includes genomic RNA, mRNA, and oligonucleotides.
본 발명의 상기 "LNA(Locked nucleic acids)"란, 2'-O, 4'-C 메틸렌 브릿지를 포함하는 핵산 아날로그를 의미한다 [J Weiler, J Hunziker and J Hall Gene Therapy (2006) 13, 496.502]. LNA 뉴클레오사이드는 DNA와 RNA의 일반적 핵산 염기를 포함하며, Watson-Crick 염기 쌍 규칙에 따라 염기 쌍을 형성할 수 있다. 하지만, 메틸렌 브릿지로 인한 분자의 'locking'으로 인해, LNA는 왓슨-크릭 결합에서 이상적 형상을 형성하지 못하게 된다. LNA가 DNA 또는 RNA 올리고뉴클레오티드에 포함되면, LNA는 보다 빠르게 상보적 뉴클레오티드 사슬과 쌍을 이루어 이중 나선의 안정성을 높일 수 있다.The "LNA (Locked nucleic acids)" of the present invention means a nucleic acid analog comprising a 2'-O, 4'-C methylene bridge [J Weiler, J Hunziker and J Hall Gene Therapy (2006) 13, 496.502) ]. LNA nucleosides include common nucleic acid bases in DNA and RNA, and can form base pairs according to Watson-Crick base pairing rules. However, due to the 'locking' of the molecule due to the methylene bridge, the LNA does not form an ideal shape at the Watson-Crick bond. When LNA is incorporated into DNA or RNA oligonucleotides, LNA can pair with complementary nucleotide chains more rapidly, increasing the stability of the double helix.
본 발명의 상기 "안티센스 뉴클레오티드"는 안티센스 올리고머가 왓슨-크릭 염기쌍 형성에 의해 RNA 내의 표적 서열과 혼성화되어, 표적서열 내에서 전형적으로 mRNA와 RNA:올리고머 헤테로 이중체의 형성을 허용하는, 뉴클레오티드 염기서열 및 서브유닛간 백본을 갖는 올리고머를 의미한다. 올리고머는 표적 서열에 대한 정확한 서열 상보성 또는 근사 상보성을 가질 수 있다.The "antisense nucleotide" of the present invention is a nucleotide sequence in which an antisense oligomer is hybridized with a target sequence in RNA by Watson-Crick base pairing, typically mRNA and RNA: oligomeric heteroduplex formation in the target sequence. and oligomers having an inter-subunit backbone. An oligomer may have exact sequence complementarity or approximate complementarity to a target sequence.
본 발명의 상기 유전자의 발현 수준을 측정할 수 있는 제제는 본 발명의 상기 유전자들의 염기서열을 토대로, 통상의 기술자에 의해 상기 유전자에 상보적으로 결합하는 프라이머, 프로브 등에 해당하는 상기 제제가 쉽게 제작될 수 있다.The agent capable of measuring the expression level of the gene of the present invention is based on the nucleotide sequence of the gene of the present invention, and the agent corresponding to the primer, probe, etc. that complementarily binds to the gene can be easily prepared by a person skilled in the art. can be
6. 6. 암의 예방 또는 치료 방법How to prevent or treat cancer
본 발명의 또 다른 구현 예에서는 암의 예방 또는 치료 방법을 제공한다.Another embodiment of the present invention provides a method for preventing or treating cancer.
본 발명의 상기 예방 또는 치료 방법은 암 세포 표면 단백질에 특이적으로 결합하는 항원 결합 도메인; 막관통 도메인; 및 세포내 신호전달 도메인을 포함하는 키메릭 항원 수용체;를 포함하는 면역세포를 유효성분으로 포함하는 면역세포치료제조성물을 약학적으로 유효량 개체에 투여하는 단계를 포함한다.The prophylactic or therapeutic method of the present invention includes an antigen-binding domain that specifically binds to a cancer cell surface protein; transmembrane domain; And a chimeric antigen receptor comprising an intracellular signaling domain; it comprises the step of administering to the subject a pharmaceutically effective amount of the immune cell therapy composition comprising immune cells comprising the as an active ingredient.
본 발명의 상기 "개체"란, 암의 예방 또는 치료가 필요한 개체로서, 영장류 예를 들면 인간뿐만 아니라, 소, 말, 양, 돼지, 염소, 낙타, 영양, 개, 고양이 등의 포유동물을 모두 포함할 수 있으나, 이에 제한되는 것은 아니다.The "individual" of the present invention refers to an individual in need of prevention or treatment of cancer, and includes all mammals such as cattle, horses, sheep, pigs, goats, camels, antelopes, dogs, and cats as well as primates such as humans. It may include, but is not limited to.
본 발명의 상기 "투여"란, 임의의 적절한 방법으로 개체에게 본 발명의 유효성분을 도입하는 과정을 의미하는 것으로서, 본 발명의 상기 치료 방법에서 투여 방법은 경구 또는 비경구 등의 다양한 경로를 통해 투여될 수 있다.The "administration" of the present invention means the process of introducing the active ingredient of the present invention to an individual by any suitable method, and the administration method in the treatment method of the present invention is through various routes such as oral or parenteral. may be administered.
본 발명의 상기 예방 또는 치료 방법에서, 암 세포 표면 단백질, 항원 결합도메인, 막관통 도메인, 세포내 신호전달 도메인, 키메릭 항원 수용체, 면역세포 및 발현 벡터 등과 관련된 내용은 앞서 1. 면역세포치료제 조성물에서 기재한 바와 동일하여 생략한다.In the prevention or treatment method of the present invention, information related to cancer cell surface proteins, antigen binding domains, transmembrane domains, intracellular signaling domains, chimeric antigen receptors, immune cells and expression vectors, etc. are described above in 1. Immune cell therapy composition As described above, it is omitted.
본 발명은 종양미세환경 회피능을 갖는 면역세포치료제 조성물에 관한 것으로서, 본 발명의 상기 면역세포치료제 조성물은 암 세포를 매우 효과적으로 인지함으로써 혈액암 세포주 뿐만 아니라, 고형암 세포주 역시 매우 효과적으로 사멸시킬 수 있다.The present invention relates to an immune cell therapy composition having an ability to avoid tumor microenvironment, and the immune cell therapy composition of the present invention can very effectively kill not only blood cancer cell lines but also solid cancer cell lines by recognizing cancer cells very effectively.

Claims (12)

  1. 암 세포 표면 단백질에 특이적으로 결합하는 항원 결합 도메인;an antigen binding domain that specifically binds to a cancer cell surface protein;
    막관통 도메인; 및transmembrane domain; and
    세포내 신호전달 도메인을 포함하는 키메릭 항원 수용체; 를 포함하는 면역세포를 유효성분으로 포함하는 면역세포치료제 조성물.a chimeric antigen receptor comprising an intracellular signaling domain; Immune cell therapy composition comprising immune cells as an active ingredient comprising a.
  2. 제1항에 있어서,The method of claim 1,
    상기 면역세포는 자연살해세포(Natural killer cell; NK cell)인 것인, 면역세포치료제 조성물.The immune cell is a natural killer cell (Natural killer cell; NK cell) will, immune cell therapy composition.
  3. 제1항에 있어서,According to claim 1,
    상기 항원 결합 도메인은 상기 암 세포 표면 단백질에 특이적으로 결합하는 항체 또는 항체의 단편인 것인, 면역세포치료제 조성물.The antigen-binding domain is an antibody or antibody fragment that specifically binds to the cancer cell surface protein, immune cell therapy composition.
  4. 제3항에 있어서,4. The method of claim 3,
    상기 항체의 단편은 scFv인 것인, 면역세포치료제 조성물.The antibody fragment is scFv, immune cell therapy composition.
  5. 암 세포 표면 단백질에 특이적으로 결합하는 항원 결합 도메인;an antigen binding domain that specifically binds to a cancer cell surface protein;
    막관통 도메인; 및transmembrane domain; and
    세포내 신호전달 도메인을 포함하는 키메릭 항원 수용체; 를 포함하는 면역세포를 유효성분으로 포함하는 암의 예방 또는 치료용 약학 조성물.a chimeric antigen receptor comprising an intracellular signaling domain; A pharmaceutical composition for preventing or treating cancer comprising immune cells as an active ingredient, comprising:
  6. 면역세포를 배양하는 단계; 및culturing immune cells; and
    상기 배양된 면역세포에 키메릭 항원 수용체를 암호화하는 카세트가 포함된 벡터를 형질 주입하는 단계;를 포함하는 면역세포치료제의 제조 방법.A method for producing an immune cell therapy comprising; transfecting the cultured immune cells with a vector containing a cassette encoding a chimeric antigen receptor.
  7. 제6항에 있어서,7. The method of claim 6,
    상기 면역세포는 자연살해세포인 것인, 제조 방법.The immune cells are natural killer cells, the production method.
  8. 제6항에 있어서,7. The method of claim 6,
    상기 형질 주입하는 단계는 바이러스성 또는 비바이러스성 형질 주입 방법을 이용하는 것인, 제조 방법.The transfection step is to use a viral or non-viral transfection method, the manufacturing method.
  9. 목적하는 개체로부터 분리된 생물학적 시료에서, 암 세포 표면 단백질에 특이적으로 결합하는 항원 결합 도메인; 막관통 도메인; 및 세포내 신호전달 도메인을 포함하는 키메릭 항원 수용체; 를 포함하는 면역세포를 처리하는 단계를 포함하는, 면역세포치료제를 스크리닝하는 방법.In a biological sample isolated from a subject of interest, an antigen-binding domain that specifically binds to a cancer cell surface protein; transmembrane domain; and a chimeric antigen receptor comprising an intracellular signaling domain; A method of screening an immune cell therapy, comprising the step of treating immune cells comprising a.
  10. 제9항에 있어서,10. The method of claim 9,
    상기 면역세포의 처리 후 상기 생물학적 시료에서 암 오가노이드의 크기, 세포사멸 또는 암 조직으로 침윤 정도를 측정하는 단계를 더 포함하는 것인, 스크리닝하는 방법.The screening method, further comprising the step of measuring the size of cancer organoids, apoptosis, or the degree of invasion into cancer tissues in the biological sample after the treatment of the immune cells.
  11. 제9항에 있어서,10. The method of claim 9,
    상기 측정된 암 오가노이드의 크기 또는 암 조직으로의 침윤 정도가 상기 면역세포의 처리 전에 비교하여 감소되거나; 또는 세포사멸 정도가 증가된 경우, 상기 면역세포를 면역세포치료제로 판단하는 단계를 포함하는 것인, 스크리닝하는 방법.the measured size of the cancer organoids or the degree of invasion into cancer tissues is reduced compared to before the treatment of the immune cells; Or, when the degree of apoptosis is increased, the screening method comprising the step of determining the immune cell as an immune cell therapy.
  12. 암 세포 표면 단백질에 특이적으로 결합하는 항원 결합 도메인; 막관통 도메인; 및 세포내 신호전달 도메인을 포함하는 키메릭 항원 수용체; 를 포함하는 면역세포를 유효성분으로 포함하는 면역세포치료제 조성물을 약학적으로 유효량 개체에 투여하는 단계를 포함하는, 암의 예방 또는 치료 방법.an antigen binding domain that specifically binds to a cancer cell surface protein; transmembrane domain; and a chimeric antigen receptor comprising an intracellular signaling domain; A method for preventing or treating cancer, comprising administering to an individual a pharmaceutically effective amount of an immune cell therapy composition comprising immune cells as an active ingredient, comprising:
PCT/KR2021/008232 2020-09-11 2021-06-30 Immunotherapy composition with ability for escaping tumor microenvironment WO2022055093A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR20200116899 2020-09-11
KR10-2020-0116899 2020-09-11

Publications (1)

Publication Number Publication Date
WO2022055093A1 true WO2022055093A1 (en) 2022-03-17

Family

ID=80631915

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2021/008232 WO2022055093A1 (en) 2020-09-11 2021-06-30 Immunotherapy composition with ability for escaping tumor microenvironment

Country Status (2)

Country Link
KR (1) KR20220034648A (en)
WO (1) WO2022055093A1 (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019204827A2 (en) * 2018-04-20 2019-10-24 The Regents Of The University Of California Treatment of prostate cancer using chimeric antigen receptors

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019204827A2 (en) * 2018-04-20 2019-10-24 The Regents Of The University Of California Treatment of prostate cancer using chimeric antigen receptors

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
E LIU, Y TONG, G DOTTI, H SHAIM, B SAVOLDO, M MUKHERJEE, J ORANGE, X WAN, X LU, A REYNOLDS, M GAGEA, P BANERJEE, R CAI, M H BDAIWI: "Cord blood NK cells engineered to express IL-15 and a CD19-targeted CAR show long-term persistence and potent antitumor activity", LEUKEMIA, vol. 32, no. 2, 1 February 2018 (2018-02-01), London, pages 520 - 531, XP055664776, ISSN: 0887-6924, DOI: 10.1038/leu.2017.226 *
KRUSCHINSKI ANNA, MOOSMANN ANDREAS, POSCHKE ISABEL, NORELL HÅKAN, CHMIELEWSKI MARKUS, SELIGER BARBARA, KIESSLING ROLF, BLANKENSTEI: "Engineering antigen-specific primary human NK cells against HER-2 positive carcinomas", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 105, no. 45, 11 November 2008 (2008-11-11), pages 17481 - 17486, XP055911709, ISSN: 0027-8424, DOI: 10.1073/pnas.0804788105 *
NADJA MüLLER, SUSANNE MICHEN, STEFANIE TIETZE, KATRIN TöPFER, ALEXANDER SCHULTE, KATRIN LAMSZUS, MARC SCHMITZ, GABRIELE : "Engineering NK Cells Modified With an EGFRvIII-specific Chimeric Antigen Receptor to Overexpress CXCR4 Improves Immunotherapy of CXCL12/SDF-1α-secreting Glioblastoma :", HHS PUBLIC ACCESS AUTHOR MANUSCRIPT, vol. 38, no. 5, 1 June 2015 (2015-06-01), pages 1 - 28, XP055511758, DOI: 10.1097/CJI.0000000000000082 *
SCHNALZGER THERESA E, GROOT MARNIX HP, ZHANG CONGCONG, MOSA MOHAMMED H, MICHELS BIRGITTA E, RÖDER JASMIN, DARVISHI TAHMINEH, WELS : "3D model for CAR-mediated cytotoxicity using patient-derived colorectal cancer organoids", THE EMBO JOURNAL / EUROPEAN MOLECULAR BIOLOGY ORGANIZATION, IRL PRESS, OXFORD, vol. 38, no. 12, 17 June 2019 (2019-06-17), Oxford , pages 1 - 15, XP055911690, ISSN: 0261-4189, DOI: 10.15252/embj.2018100928 *

Also Published As

Publication number Publication date
KR20220034648A (en) 2022-03-18

Similar Documents

Publication Publication Date Title
WO2020076105A1 (en) Novel anti-c-kit antibody
CN112442132A (en) Recombinant difunctional fusion protein targeting tumor and application thereof
KR20210143096A (en) Antibody specific for CD22 and uses thereof
Rosenbaum et al. Expression of neurophysin-related precursor in cell membranes of a small-cell lung carcinoma.
WO2022055093A1 (en) Immunotherapy composition with ability for escaping tumor microenvironment
WO2022131889A1 (en) Use of taci protein
WO2019216675A1 (en) Epitope of regulatory t cell surface antigen and antibody specifically binding thereto
KR20210032412A (en) Peripheral nerve disorder, peripheral nerve disorder, or a method of preventing or treating pain due to a disease recognized as a stellate cell disorder
WO2022015113A1 (en) Antibody specifically binding to ptk7 and use thereof
WO2022231032A1 (en) Anti-cntn4-specific antibodies and use thereof
WO2007083759A1 (en) Bone destruction inhibitor comprising anti-ccl20 antibody
WO1991012272A1 (en) Protein factor
KR20120113580A (en) Monoclonal antibody specific to vibrio vulnificus rtxa1, hybridoma producing the monoclonal antibody and diagnostic kit comprising the monoclonal antibody
CA3160181A1 (en) Novel ddr1 antibodies and uses thereof
EP4234575A1 (en) Cell surface antigen of activated immune cell and various uses thereof
WO2020251297A1 (en) Composition for preventing or treating cancer
WO2018128429A1 (en) Pharmaceutical composition for preventing or treating inflammatory diseases, containing inositol polyphosphate multikinase inhibitor as active ingredient
WO2018079997A1 (en) Aglycosylated antibody fc region for treating cancer
WO2023191117A1 (en) Affinity matured cd47-specific humanized antibody
WO2022124764A1 (en) Antibody specific for cd47 and use thereof
WO2023043124A1 (en) GLYCATED FC VARIANTS WITH IMPROVED BINDING AFFINITY FOR FCγRIIIA
WO2019231188A1 (en) Pharmaceutical composition for preventing or treating cancer, containing cd300c expression inhibitor or activity inhibitor
EP4286412A1 (en) Novel binding molecule and use thereof
WO2021225378A1 (en) Pd-1 variants having increased pd-l1 affinity
JP6233932B2 (en) BBF2H7 (BBF2 human homologon on chromosome 7) A composition for regulating cell growth comprising a peptide having a partial amino acid sequence or an antibody binding thereto

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21866963

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21866963

Country of ref document: EP

Kind code of ref document: A1