WO2020175960A1 - 자연살해세포 활성 검출용 미세입자 및 이를 이용한 검출 방법 - Google Patents

자연살해세포 활성 검출용 미세입자 및 이를 이용한 검출 방법 Download PDF

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WO2020175960A1
WO2020175960A1 PCT/KR2020/002904 KR2020002904W WO2020175960A1 WO 2020175960 A1 WO2020175960 A1 WO 2020175960A1 KR 2020002904 W KR2020002904 W KR 2020002904W WO 2020175960 A1 WO2020175960 A1 WO 2020175960A1
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cells
complex
group
cell activity
alexa floor
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French (fr)
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김헌식
조덕
도준상
박지훈
신예린
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Asan Foundation
Samsung Life Public Welfare Foundation
University of Ulsan Foundation for Industry Cooperation
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Asan Foundation
Samsung Life Public Welfare Foundation
University of Ulsan Foundation for Industry Cooperation
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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases

Definitions

  • NK cells naturally killer cells
  • NK cells are a type of cytotoxic lymphocytes important to the innate and adaptive immune systems.
  • NK cells respond to virus-infected cells or cancer cells, typically detecting abnormalities of the major histocompatibility complex (MHC) on the cell surface.
  • MHC major histocompatibility complex
  • These NK cells secrete perforin to penetrate the cell membrane of infected cells or cancer cells. It has cytotoxicity, which kills these cells by giving it a granzyme.
  • MHC major histocompatibility complex
  • B-cell lymphoma and many cancer patients defects in the number or anticancer activity of NK cells have been found, and it is known that dysfunction of NK cells is closely related to the occurrence of these cancers (Annu. Rev. Immunol. 2013; 31:227 -258).
  • NKp46 Various activating and inhibitory receptors are involved in the activity of NK cells.
  • TAM immunoreceptor tyrosine-based activation motif
  • Receptors considered co-stimulatory are members of the signaling lymphocytic activation molecule (SLAM) family, such as 2B4 (CD244), as well as DNAM-1 (CD226), CD2, and NKp80 (KLRFl). Gene products).
  • One aspect is to provide a microparticle complex including microparticles; and peptides cleaved by cytotoxic enzymes secreted by natural killer (NK) cells.
  • NK natural killer
  • Another aspect is to provide a kit for diagnosing NK cell activity containing a complex according to one aspect.
  • Another aspect is to provide a kit for diagnosing diseases related to NK cell activity, including complexes according to one aspect.
  • Another aspect is for detecting NK cell activity, including a complex according to one aspect.
  • Another aspect involves processing a complex of one aspect into a blood sample of an individual.
  • Another aspect involves processing a complex of one aspect into an individual's blood sample.
  • Another aspect involves processing a complex of one aspect into a blood sample of an individual.
  • One aspect provides a microparticle complex containing a microparticle; and a peptide cleaved by a cytotoxic enzyme secreted by NK (Natural egg 11) cells.
  • microparticles Particles may be beads 0 ⁇ (1). Beads may be microbeads (11110'0 6&(1) or nanobeads (1 110 6&(1)).
  • beads have a diameter of 5 to 500 11111, Preferably, the diameter may have a size in the range of 200 to 500 11111.
  • the beads may be at least one selected from the group consisting of latex beads, polymer plastic beads, biocompatible polymer beads, glass beads, silica beads, and magnetic beads. have.
  • the magnetic beads can be manufactured and used through a known method, and can be purchased commercially, and for example, magnetic beads can be understood as magnetic particles that respond to magnetic force.
  • the diameter is not particularly limited, but the diameter may be small magnetic nanoparticles, and may be 11111 to 200 ⁇ , and the magnetic material of the magnetic particles may be a magnetic metal or a magnetic metal oxide.
  • the magnetic beads may be at least one selected from the group consisting of iron group metal elements, rare earth elements, currency metal elements, zinc group elements, aluminum group elements, alkaline earth metal elements, and platinum group elements. More specifically, the above-mentioned Magnetic metal is an iron group metal element, (3 ⁇ 4), rare earth element (1 Sze 06, Nd, 13 ⁇ 41, 8111,, 0(1,
  • It may be composed of one or more metals selected from aluminum group elements (new, II), alkaline earth metal elements (1 ⁇ ), and platinum group elements (, parent (1st class)) or alloys thereof.
  • aluminum group elements new, II
  • alkaline earth metal elements (1 ⁇ )
  • platinum group elements platinum group elements
  • parent (1st class) or alloys thereof.
  • the magnetic particles may contain one or more selected from the group consisting of Gd, Mo, MM'204 and MpOq.
  • the magnetic particles are passivated. It may be, for example, passivated by citrate ions.
  • NK cells or “Natural Killer cells” are
  • cytotoxic lymphocyte constituting a major component of the innate immune system
  • Lymphatic system defined as large granular lymphocyte (LGL)
  • Progenitor cells (common lymphoid progenitors, CLPs) make up a third cell differentiated from B and T lymphocytes.
  • CLPs common lymphoid progenitors
  • NK cells are additional modifications derived from any tissue source. Natural killer cells
  • Natural killer cells may contain natural killer precursor cells.
  • the natural killer cells are activated in response to interferons or macrophage-derived cytokines, and the natural killer cells are labeled with "activating receptors" and "inhibitory receptors", two types of cells that control cytotoxic activity.
  • Natural killer cells can be produced from any source, e.g. placental tissue, placental perfusate, umbilical cord blood, placental blood, peripheral blood, spleen, hematopoietic cells, e.g. hematopoietic stems or progenitors. have.
  • NK cells can mean any substance capable of increasing the activity of natural killer cells, for example, cytotoxic activity, or generating, increasing, or proliferating activated natural killer cells.
  • the NK cells are activated.
  • the substance may be a substance that stimulates factors or substances related to the activity of NK cells.
  • factor refers to all molecular factors that cause NK cell activation.
  • the factor may include intracellular and extracellular receptors, channels, and intracellular and extracellular adapters, proteins, glycoproteins, peptides, and motifs capable of binding with specific ligands.
  • stimulation acts on a factor to trigger specific signaling within and outside NK cells.
  • the method of causing the irritation is an antibody, fragment, or peptide specific for one or more of the above factors, a composition that specifically induces or inhibits, and a reversible or irreversible agonist or antagonist.
  • a composition that specifically induces or inhibits, and a reversible or irreversible agonist or antagonist. May be used, and may involve culturing with a culture containing various target cells capable of stimulating NK cells (e.g., K562, 721.221, or P815 coated with a specific receptor-stimulating antibody).
  • NK cells e.g., K562, 721.221, or P815 coated with a specific receptor-stimulating antibody.
  • Substances that activate NK cells may include immune cytokines, agonists of NK cell activating receptors, or antagonists of NK cell inhibitory receptors.
  • cytokine as used in this specification is a signaling substance that controls the body's defense system and stimulates the body, and can mean a protein that regulates physiological activity. Cytokines are autocrine. , Through paracrine action 2020/175960 1»(:1 ⁇ 1 ⁇ 2020/002904
  • the cytokines are, for example, INF (Interferon) family, IL (Interleukin) family, and TNF (Tumor necrosis factors) family. , Or a combination thereof. More specifically, the cytokine is a group consisting of IL-2, IL-5, IL-8, IL-12, IL-15, IL-18, IL-21, and combinations thereof.
  • the interferon is a type 1 interferon (e.g., interferon-aa, interferon, interferon-K, interferon- co), type 2 interferon (e.g., interferon- Y), it may be type 3 interferon or a combination thereof.
  • the TNF is a type 1 interferon (e.g., interferon-aa, interferon, interferon-K, interferon- co), type 2 interferon (e.g., interferon- Y), it may be type 3 interferon or a combination thereof.
  • the TNF is a type 1 interferon (e.g., interferon-aa, interferon, interferon-K, interferon- co), it may be type 3 interferon or a combination thereof.
  • the TNF is a type 1 interferon (e.g., interferon-aa, interferon, interferon-K, interferon- co), it may be type 3 interferon or
  • the immunocytokin may include a cytokine-antibody complex.
  • an interleukin e.g., an interleukin
  • IL-2) -Anti-interleukin (e.g., anti-IL-2) antibody complex.
  • the agonist of the NK cell activation receptor is an antibody or aptamer that binds to the activation receptor, BiKEs (Bispecific Killer Engagers) or TriKEs (Trispecific Killer
  • An example of an antibody or aptamer that binds to the activation receptor is for GITR.
  • Antibody antibody to 0X40, antibody to CD137, antibody to CD27, antibody to CD27, 0X40 functional aptamer, CD 137 functional aptamer, NKG2D ligand (e.g. MULT-1 in mice (murine UL 16 -binding protein-like-1) or H60, or MICA (MHC I-related chain A), MICB (MHC I-related chain B) or ULBPs (unique long 16-binding proteins)), or CD226 agonists in humans Can include
  • the BiKE or TriKEs contain two single-chain variable fragments (scFvs), and target
  • target cells e.g., damaged neurons, tumor cells or infected cells
  • natural killer cells to mediate apoptosis.
  • TriKES TriKES
  • BiKE specifically binds to an antigen on the surface of target cells (e.g., damaged neurons, tumor cells or infected cells), while the other scFv is a receptor on natural killer cells (e.g., CD16 and
  • target cells e.g., damaged neurons, tumor cells or infected cells
  • the other scFv is a receptor on natural killer cells (e.g., CD16 and
  • BiKE specifically binds to TAA or RAEl (Retinoic Acid Early 1) with the first scFv and an activating receptor, for example, 2020/175960 1»(:1 ⁇ 1 ⁇ 2020/002904
  • the antagonist of the NK cell inhibitory receptor may include an antibody or aptamer that binds to the inhibitory receptor.
  • NK cell inhibitory receptors may include CTLA-4, PD-1, PD-L1, Tim-3, CD96, KIR, NKG2A, or TIGIT (T-cell immunoglobulin and ITIM domain). Therefore, NK Cell activating substances may include antibodies against the receptors listed above.
  • the substance that activates the NK cells is one or more factors selected from the group consisting of NKG2D, 2B4 (CD244), DNAM-1 (CD226), CD2, CD16, NKp30, NKp44, NKp46, and NKp80. It could be something that irritates the substance.
  • various activating and inhibitory receptors can be involved in the activity of NK cells, but among the main receptors, the immune receptor tyrosine-based activity
  • Immunoreceptor tyrosine-based activation motif (IT AM)-related signaling molecule-related NKp46 and signaling molecule DAP10-related receptor NKG2D (EMBO J. 2004; 23:255.-9, Annu. Rev. Immunol. 2005; 23:225-74).
  • Receptors considered co-stimulatory are members of the signaling lymphocytic activation molecule (SLAM) family such as 2B4 (CD244), as well as DNAM-1 (CD226), CD2, and NKp80 (KLRFl gene product).
  • SLAM signaling lymphocytic activation molecule
  • ITAM-containing signaling molecules e.g., CD16, NKp46, NKp30 or NKp44, etc.
  • NKG2D 2B4 (CD244)
  • DNAM-1 CD226)
  • CD2 and NKp80 It may be to stimulate one or more substances selected from among . Bryceson Y etc. (Blood
  • NK cell activation may be possible by stimulation of various combinations of the substances listed above, as an example of a combination of the above substances. There may be 2B4 and DNAM-1, DNAM-1 and NKG2D, 2B4 and NKG2D, or 2B4, DNAM-1 and NKG2D.
  • the cytotoxic enzyme secreted by the NK cells is a group consisting of perforin, granzyme A, granzyme B, granzyme H, granzyme K, and granzyme M It may be one or more selected from. Immediate determination of the activity of NK cells may include one or more selected from the measurement of degranulation and cytotoxicity activity, and cytokines secreted by NK cell stimulation.
  • the degranulation activity and cytotoxicity activity May mean inducing target cell lysis by secretion of, for example, perforin or granzyme, which can be analyzed using flow cytometry.
  • the peptide to be cleaved is composed of 2 to 20 amino acids, and isoleucine-glycine-asparagine-arginine (IGNR, SEQ ID NO: 1)',
  • VGPDFGR a peptide comprising'valine-glycine-proline-aspartic acid-phenylalanine-glycine-arginine
  • the microparticle complex activates NK cells in the blood or sample of an individual, thereby secreting enzymes and cytokines from NK cells. Accordingly, the secreted enzymes As the peptide contained in the microparticle complex is cleaved by the activity, the activity of NK cells and detection thereof may be possible.
  • fluorescent moiety as used in this specification is a fluorescent molecule or a fluorescent molecule thereof that absorbs light of a specific frequency (eg, UV light) as it is separated from the cleavable peptide and generates a fluorescent signal. Derivative or
  • the fluorescent moiety includes coumarin and related dyes; xanthene dyes such as fluorescein, rhodole, and rhodamine; Resorupine; cyanine dye; bimanes; acridine; isoindole;
  • the fluorescent moiety may additionally include a free carboxyl group, an ester (e.g., N-hydrosuccinimide (NHS) ester) or a maleimide derivative. And may also contain streptavidin, biotin, phalloidin, amine, azide or iodoacetamide conjugates.
  • an ester e.g., N-hydrosuccinimide (NHS) ester
  • a maleimide derivative e.g., N-hydrosuccinimide (NHS) ester
  • streptavidin biotin, phalloidin, amine, azide or iodoacetamide conjugates.
  • the first fluorescent moiety is fluorescein
  • BODIPY FL BODIPY FL-Br2
  • BODIPY 530/550 BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650
  • It may be one or more selected from the group consisting of BodiP 650/665, BodiP R6G, BodiP TMR and BodiP TR.
  • the material of the fluorescent moiety may be coated on the surface of the microparticle itself. This may be coated for the purpose of more reliably discriminating the activity of NK cells through the microparticle complex according to one aspect.
  • the attached peptide is cleaved by an enzyme secreted from NK cells, the fluorescence intensity changes, and thus the activity of NK cells can be visually inspected through comparison with microparticles before treatment.
  • the substance that binds to cytokines secreted by NK cells is IFN-yMIP-la,
  • MIP-l (3TNF-a, TNF- (3RANTES, IL-8 and IL-10) may be an antibody, peptide or aptamer that binds to one or more selected from the group consisting of. From NK cells as described above, it may be an antibody, peptide, or aptamer. Analysis of secreted or expressed cytokines can be performed using FACS, intracellular cytokine staining, or ELISA.
  • linker refers to a substance for activating NK cells to microparticles, for attaching substances such as peptides and cytokines to be cleaved, or for connecting the respective substances.
  • the linker may be an immobilized compound.
  • the immobilized compound may contain a functional group that can be attached to the material.
  • the organo functional group is, for example, a thiol group or a sulfhydryl group.
  • the organofunctional group may be a thiol ester or dithiol ester, each having a RSSR' or RSR' ritual. Further, the organofunctional group may be an amino group (-NH2).
  • the linker is biotin, avidin, streptavidin, carbohydrate, poly L-lysine, thiol group, amine group, alcohol group, carboxyl group, amino group, sulfur group, aldehyde group, carbonyl group, succinimide group, maleimide group It may be one selected from the group consisting of compounds having an epoxy group, an isothiocyanate group, or a combination thereof. Examples of the compound having an amino group include 3-aminopropyltrimethoxysilane,
  • 3-Aminopropyltriethoxysilane may be included,
  • Compounds may include glutaraldehyde.
  • Examples of compounds having a thiol group may include 4-mercaptopropyltrimethoxysilane (MPTS).
  • examples of compounds having an epoxy group may include 3-glycidoxypropyl.
  • examples of compounds having trimethoxysilane and isothiocyanate groups include: 2020/175960 1»(:1 ⁇ 1 ⁇ 2020/002904
  • Examples of compounds having 4-phenylenediisothiocyanate 011, succinimide and maleimide groups include disuccinimidyl carbonate (080 or succinimidyl
  • SMPB 4-(maleimidephenyl)butyrate
  • the linker may be connected to the 5'end and/or the 3'end of each material.
  • a method of bonding through the linker is known in the art.
  • the linker may be a peptide linker.
  • the peptide linker May use various linkers known in the art, for example, may be a linker composed of a plurality of amino acids.
  • the linker is, for example, 1 to 100 or 2 to 50 arbitrary It may be a polypeptide consisting of amino acids.
  • the peptide linker may contain residues ( 3 ⁇ show 811 and 861, and may also contain neutral amino acids such as 1 ⁇ 4 and show. Amino acid sequences suitable for peptide linkers are in the art. Is known.
  • the linker may further include a polymer compound.
  • the polymer compound is polyethylene glycol £(3),
  • polyacrylic sulfonate polyhydroxyethyl methacrylate (PolyHEMA), and copolymers thereof.
  • a polymer compound at the end of the peptide to be cleaved of the complex, and an agent at the 0-end 1 The fluorescent moiety may be bound.
  • kits for diagnosing NK cell activity comprising a complex according to one aspect.
  • the kit may further include a substance for detecting cytokines secreted by NK cells.
  • the substance that detects the cytokine may be a second fluorescent moiety additionally bound.
  • the substance that detects the cytokine is a substance that tracks cytokines secreted by the NK cells, IFN-gMIP-la, TNF-pRANTES, an antibody, peptide or aptamer that binds to one or more selected from the group consisting of 1b8 and 1 10! 111 ).
  • the kit is a cytotoxicity and cytokine secretion of NK cells.
  • the microparticle complex included in the kit can activate NK cells, and accordingly, the NK cells secrete enzymes such as granzyme and cytokines such as interferon gamma (Table -?).
  • the peptide contained in the microparticle complex may be cleaved by the activity of the enzyme, resulting in a decrease in fluorescence sensitivity due to the separation of the first fluorescent moiety.
  • the cytotoxicity shown in Table 7 contained in the microparticle complex may appear.
  • Antibodies against kine can capture the table, and the captured table can increase the fluorescence intensity of the second fluorescent moiety by clustering the antibodies that detect the table ⁇ included in the kit. Simultaneous measurement of cytotoxic activity and cytokine secretion activity of NK cells 2020/175960 1»(:1 ⁇ 1 ⁇ 2020/002904
  • the second fluorescent moiety is the same as defined in the first fluorescent moiety.
  • the second fluorescent moiety is the number of moieties that can be distinguished visually from the first fluorescent moiety. have.
  • Another aspect provides a kit for diagnosing diseases related to NK cell activity, including a complex according to one aspect.
  • the disease related to the activity of NK cells is irritable immune disease
  • the irritable immune disease is one or more selected from asthma and sinusitis, or the autoimmune disease is Lupus, multiple
  • Hematopoietic lymphocytocytosis is a second type. Gunlangerhans cell histiocytosis, erythropoietic lymphocytic histiocytosis (familial, sporadic), infection-related hemagglutination syndrome, virus-related hemagglutination syndrome, histocytosis with giant lymphadenopathy, or reticulocytosis.
  • the ⁇ cancer'' may include a tumor, blood cancer or solid cancer.
  • the cancer is lung cancer, liver cancer, esophageal cancer, gastric cancer, colon cancer, small intestine cancer, pancreatic cancer, melanoma, breast cancer, oral cancer.
  • it may be selected from the group consisting of.
  • the cancer may be pancreatic cancer or B cell lymphoma.
  • the viral disease may be hepatitis B.
  • the immunodeficiency disease may be DiGeorge synderome or Chedial-Higashi syndrome.
  • Another aspect is a composition for detecting NK cell activity, including a complex of one aspect.
  • the composition may further include a substance that detects cytokines secreted by NK cells.
  • the substance that detects the cytokine is the second fluorescence.
  • the moiety may be additionally bound. Further, the composition may simultaneously detect cytotoxicity and cytokines of NK cells.
  • Another aspect involves the processing of a complex of one aspect into an individual's blood sample.
  • It provides a method for measuring NK cell activity, including.
  • the cleaved peptide may be further included.
  • the detection of the peptide is ELISA (enzyme linked immunosorbent assay),
  • Flow cytometry Fluorescence Activated Cell Sorter, FACS
  • FACS Fluorescence Activated Cell Sorter
  • protein chips may be performed by one or more selected from the group consisting of.
  • the fluorescence intensity than before the addition of the complex was performed.
  • it may include a step of determining that the activity of NK cells in the blood sample is increased.
  • the method may further include a step of treating a blood sample of an individual with a substance that detects cytokines secreted by NK cells.
  • the step of detecting cytokines secreted by the NK cells may be further included.
  • the blood sample It may include the step of judging that the activity of NK cells has increased. This is a substance that can capture cytokines by antibodies against cytokines contained in the microparticle complex, and detects cytokines by the captured cytokines. Therefore, the step of detecting the peptide cleaved from the complex and the step of detecting the cytokine secreted by the NK cells may be performed simultaneously.
  • Another aspect involves processing a complex of one aspect into an individual's blood sample.
  • It provides a method of providing information on diseases related to NK cell activity, including.
  • Another aspect involves processing a complex of one aspect into an individual's blood sample.
  • It provides a method for diagnosing diseases related to NK cell activity, including.
  • the information on the disease related to NK cell activity can be determined as abnormal NK cells when activation of NK cells in the experimental group compared to normal NK cells is not detected, or NK cells that have lost their activity against a specific receptor are In the case of abnormal activity or not, it can be determined that the target cells are associated with a specific disease.
  • a microparticle complex according to one aspect, a kit for NK cell activation including the same, a composition for detecting NK cell activity including the same, and a method for measuring NK cell activity using the same, the secretion of enzymes according to the activity of NK cells and Cytokine expression can be easily measured at the same time, and unlike other substances or methods, it is possible to effectively detect NK cell activity by showing precise measurement results at low cost.
  • 1 is a diagram showing a model of a fine particle composite.
  • FIG. 2A is a graph showing the change in fluorescence sensitivity when the granzyme B and the microparticle complex were reacted.
  • Figure 2b is a graph showing the change in fluorescence sensitivity, which appears when the IFN-Y and the microparticle complex are reacted.
  • Figure 3a is a graph measuring the secretion of Granzyme B according to the activation effect of NK cells of the microparticle complex.
  • Figure 3b shows the secretion of IFN-Y according to the NK cell activation effect of the microparticle complex.
  • FIG. 4 is an image imaged of simultaneous measurement of Granzyme B and IFN-Y secreted and activated by NK cells through a microparticle complex.
  • Granzyme b peptide sensor is Granzyme B with amino acid sequence VGPDFGR
  • flow cytometric analysis was performed as follows: First, to confirm the function of the Granzyme B sensor, the microparticle complex was placed at various concentrations consisting of 150 U/ml, 450 U/ml and 600 U/ml for 2 hours. Treated with human Granzyme B (Abcam). At the same time, PE anti-human IFN-y antibody (BioLegend, clone: 4S.B3) 2 [ xg 1, 10, to confirm the capture function for IFN-y. Recombinant human IFN-Y (Gibco) at various concentrations consisting of 100 and 1000 ng/ml was added, and microparticle complexes were mixed into each.
  • Fluorescence measurement was performed using an LSR Fortessa analyzer (BD Biosciences) to measure the fluorescence of microparticle complexes. The intensity was measured and the measurement data was analyzed with FlowJo to confirm whether each detection was made. The results are shown in Figs. 2A and 2B.
  • Test Example 2 Measurement of NK cell activity
  • P is secreted according to the activity of natural killer (NK) cells.
  • microparticles with human BD Fc block (BD Bioscience) 2020/175960 1»(:1/10 ⁇ 020/002904
  • eNK expanded NK cells
  • 50 ml of Wg/rnl PE anti-human IFN-Y antibody was incubated with the same number of microparticle complexes and 4.0xl0 6 eNK cells for 4 hours at 37 O C in IMDM medium.
  • flow cytometry was performed with an LSR Fortes sa analyzer (BD Biosciences). The detected data was analyzed with FlowJo.
  • the control group was a bead without any treatment, and Microparticles were used in which biotinylated NKG2D/2B4 antibody was replaced with biotinylated 0.5 mg/ml mouse iso antibody, IgG 20 ml. The results are shown in Figs. 3A and 3B, respectively.
  • the control group iso antibody did not stimulate NK cells.
  • the microparticle complex prevented the secretion of Granzyme B and IFN-Y of NK cells. It indicates that it has an activating effect.
  • the simultaneous measurement of Granzyme B and IFN-y according to the activity and activity of NK cells through the microparticle complex was intended to be imaged.
  • a biotin cover slip with a low surface range was added.
  • the eNK cells and IFN-Y antibody with a fluorescent moiety attached thereto were added, followed by imaging for 4 hours at 15 minutes intervals.
  • LAMBDA LS xenon lamp (175W, Sutter equipment) and filter set, FITC (Chroma S492 / 18x and 62002bs), Texas Red (Chroma S572 / 23x and 62002bs) and Cy5 (Chroma ET620 / 60x, T660LPXR and ET700 / 75m) fluorescent imaging Microscope and electric stage (MS-2000, Applied Scientific
  • the two results were overlaid, it was confirmed that the green fluorescent microparticles were changed to red microparticles. Through this, the microparticle complex was found to have the activity of NK cells and thus the activity of the NK cells. Indicates that there is.

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Abstract

미세입자 복합체, 이를 포함하는 NK 세포 활성 진단용 키트, 이를 포함하는 NK 세포 활성 검출용 조성물, 및 이를 이용한 NK 세포 활성 측정 방법을 제공한다.

Description

2020/175960 1»（：1^1{2020/002904 명세서
발명의명칭:자연살해세포활성검출용미세입자및이를이용한 검출방법
기술분야
[1] NK세포의활성검출용미세입자및이를이용한 NK세포의활성도를
검사하는방법에관한것이다.
배경기술
[2] NK세포 (natural killer cell)는선천적면역및후천적 (adaptive)면역계에중요한 세포독성림프구의한종류이다. NK세포는바이러스감염된세포나암세포에 반응하는데,전형적으로세포표면에 MHC(major histocompatibility complex)의 이상현상을감지한다.이러한 NK세포는퍼포린 (perforin)을분비해감염세포나 암세포의세포막에구멍을내고,여기에그랜자임 (granzyme)을내어이세포들을 사멸시키는세포독성을갖는다. B세포림프종및많은암환자의경우 NK 세포의수나항암활성에결함이발견되고있으며 NK세포의기능이상은이러한 암의발생과밀접한관련을가지고있음이알려져있다 (Annu. Rev. Immunol. 2013; 31:227-258).
[3] NK세포의활성에는다양한활성화및저해성수용체가관여한다.지배적이라 간주되는수용체중에는면역수용체티로신-기반활성모티프 (imnmnoreceptor tyrosine-based activation motif: ITAM)-관련신호전달분자 (예를들어 , FcR y사슬 또는 TCRC사슬)와관련된 NKp46및신호전달분자 DAP10와관련된수용체 NKG2D(EMBO J. 2004; 23:255.-9, Annu. Rev. Immunol. 2005; 23:225-74)에관련한 것들이있다.공동자극성이라간주되는수용체로는, 2B4(CD244)와같은 신호전달림프구활성분자 (signaling lymphocytic activation molecule: SLAM) 패밀리의구성원들뿐만아니라, DNAM-1(CD226), CD2,및 NKp80(KLRFl 유전자산물)과같이다양한수용체를포함한다.
[4] 상기 NK세포활성도를측정하는기존의기술은방사성동위원소,또는
형광표지탐침 (probe)을이용한등록특허 10-1926166것으로서,비용이나 시간면에서임상적으로적용하기에부적합하므로, NK세포의활성도를 간편하고신속하면서도유의적으로측정할수있는방법이다양한면역관련 질환의조기진단및예후판단에중요하게인식되고있다.
발명의상세한설명
기술적과제
[5] 일양상은미세입자;및 NK(Natural killer)세포가분비하는세포독성효소에 의해절단되는펩티드를포함하는미세입자복합체를제공하는것이다.
[6] 다른양상은일양상에따른복합체를포함하는 NK세포활성진단용키트를 제공하는것이다. 2020/175960 1»（：1^1{2020/002904
2
[7] 또다른양상은일양상에따른복합체를포함하는 NK세포활성관련질환 진단용키트를제공하는것이다.
[8] 다른양상은일양상에따른복합체를포함하는, NK세포활성검출용
조성물을제공하는것이다.
[9] 또다른양상은일양상의복합체를개체의혈액샘플에처리하는단계를
포함하는 NK세포활성도를측정하는방법을제공하는것이다.
[1이 또다른양상은일양상의복합체를개체의혈액샘플에처리하는단계를
포함하는 NK세포활성관련질환에대한정보를제공하는방법을제공하는 것이다.
[11] 또다른양상은일양상의복합체를개체의혈액샘플에처리하는단계를
포함하는 NK세포활성관련질환을진단하는방법을제공하는것이다.
과제해결수단
[12] 일양상은미세입자;및 NK（Natural난11 ）세포가분비하는세포독성효소에 의해절단되는펩티드를포함하는미세입자복합체를제공한다.
[13] 일구체예는미세입자; NK세포가분비하는세포독성효소에의해절단되는 펩티드;상기펩티드에연결된제 1형광모이어티; 세포를활성화하는물질; NK세포가분비하는사이토카인과결합하는물질;및상기펩티드, NK세포를 활성화하는물질,또는사이토카인과결합하는물질을상기미세입자와 연결하는링커를포함할수있다.일구체예에서 ,상기미세입자는비드 0^（1）인 것일수있다.비드는마이크로비드（11110'0 6&（1）또는나노비드（1 110 6&（1）일수 있다.구체적으로,비드는직경이 5내지 500 11111,바람직하게는직경이 200내지 500 11111의범위에서크기를갖을수있다.상기비드는라텍스비드,중합체 플라스틱비드,생체적합성중합체로구성된비드,유리비드,실리카비드및 자성비드로이루어진군으로부터선택된하나이상인것일수있다.
[14] 상기자성비드는공지된방법을통해서제조해서사용하는것도가능하며 , 상업적으로구입해서사용할수있으며,일예로자성비드는자력에반응하는 자성을띤입자로이해될수있다.상기자성입자의직경은특별히제한되지 않으나,직경이작은자성나노입자일수있고, 1 11111내지 200■일수있으며 , 상기자성입자의자성물질은자성금속또는자성금속산화물인것일수있다. 일구체예에서,상기자성비드는철족금속원소,희토류원소,화폐금속원소, 아연족원소,알루미늄족원소,알칼리토금속원소및백금족원소로이루어진 군으로부터선택된하나이상인것일수있다.보다구체적으로,상기자성 금속은철족금속원소 ,（¾）,희토류원소（1솨 06, Nd, 1¾1, 8111, , 0（1,
Figure imgf000004_0001
알루미늄족원소（신, II）,알칼리토금속원소（ 1切）및백금족 원소（ ,모（1등）에서하나이상선택된금속또는이들의합금으로이루어지는 것일수있다.일구체예의상기자성입자로의종류는예를들면,（¾ !,므 , 2020/175960 1»（：1^1{2020/002904
3
Gd, Mo, MM'204및 MpOq로이루어진그룹중에서선택된하나이상을포함할 수있다.일구체예에 있어서,상기자성입자가알지네이트의카르복실기와 상호작용하는것을방지하기위해,상기자성입자는부동태화된것일수 있으며,예를들면,구연산염이온에의해부동태화된것일수있다.
[15] 본명세서에서용어” NK세포”또는 "자연살해세포 (Natural Killer cells)”는
선천성면역계의주요성분을구성하는세포독성림프구로서 ,
대형과립림프구 (large granular lymphocyte, LGL)로정의되고림프계
전구세포 (common lymphoid progenitor, CLP)생성 B및 T림프구로부터분화된 제 3의세포를구성한다.상기 "자연살해세포”또는 "NK세포”는임의의조직 공급원 (source)으로부터유래된추가적인변형이없는자연살해세포를
포함하고,성숙한자연살해세포뿐아니라,자연살해전구세포를포함할수있다. 상기자연살해세포는인터페론또는대식세포-유래사이토카인에대한 반응으로활성화되고,자연살해세포는 "활성화수용체”및 "억제성수용체”로 표지되는,세포의세포독성활성을제어하는 2가지유형의표면수용체를 포함한다.자연살해세포는임의의공급원,예를들어태반조직,태반관류액, 제대혈,태반혈,말초혈,지라,간등으로부터의조혈세포,예를들어조혈줄기 또는전구체로부터생성될수있다.
[16] 본명세서에서용어 "NK세포를활성화하는물질”은생체내에투여되어
자연살해세포의활성 ,예를들면,세포독성활성을증가시키거나,활성화된 자연살해세포를생성 ,증가,또는증식시킬수있는임의의물질을의미할수 있다.일구체예에서상기 NK세포를활성화하는물질은 NK세포의활성과 관련한인자내지물질을자극하는물질일수있다.
[17] 상기용어 "인자”는 NK세포의활성화를일으키는모든분자적요소를
의미한다.구체적으로,상기인자는세포내외의수용체 ,채널 (channel),및 특이적인리간드와결합할수있는세포내외의어답터 (adaptor),단백질, 당단백질,펩티드,및모티프를포함할수있다.
[18] 상기용어 "자극”은인자에작용하여 NK세포내외의특정신호전달을
일으키는것을의미한다.상기자극을일으키는방법은상기인자의하나이상에 대해특이적인항체,그단편,또는펩티드,특이적으로유도또는억제하는 조성물,및가역적또는비가역적인작용제 (agonist)또는길항제 (antagonist)를 이용할수도있고, NK세포를자극할수있는다양한표적세포 (예를들어, K562, 721.221,또는특정수용체자극항체로코팅된 P815)를포함하는배양물과 배양하는것을포함할수있다.보다상세하게, NK의세포를활성화하는물질은 면역사이토카인, NK세포활성화수용체의작용제 ,또는 NK세포억제성 수용체의길항제를포함할수있다.
[19] 본명세서에서사용되는용어 , "사이토카인 (Cytokine)”은신체내방어체계를 제어하고,생체를자극하는신호물질로서 ,생리활성조절단백질을의미할수 있다.사이토카인은자가분비 (autocrine),파라크라인 (paracrine)작용을통하여 2020/175960 1»（：1^1{2020/002904
4 다양한세포에서세포활성화,분화,세포이동,노화,사멸유도등다양한 생물학적활성을조절한다.상기사이토카인은예를들어, INF(Interferon) 패밀리 , IL(Interleukin)패밀리 , TNF(Tumor necrosis factors)패밀리 ,또는이들의 조합일수있다.더욱상세하게는,상기사이토카인은 IL-2, IL-5, IL-8, IL-12, IL-15, IL-18, IL-21및그의조합으로이루어진군으로부터선택된어느하나인 것일수있다.더욱상세하게는,상기인터페론은 1형인터페론 (예를들면, 인터페론- aa,인터페론 ,인터페론 -K,인터페론- co), 2형인터페론 (예를들면, 인터페론 -Y), 3형인터페론또는이들의조합일수있다.또한,상기 TNF는
TNF-0C, TNF-p또는 TNF-Y등을포함할수있다.또한,상기면역사이토카인은 사이토카인-항체복합체를포함할수있다.예를들면,인터루킨 (예를들면,
IL-2) -항-인터루킨 (예를들면, anti-IL-2)항체복합체를포함할수있다.
[2이 상기 NK세포활성화수용체의작용제는,활성화수용체에결합하는항체또는 앱타머 , BiKEs(Bispecific Killer Engagers)또는 TriKEs(Trispecific Killer
Engagers)를포함할수있다.
[21] 상기활성화수용체에결합하는항체또는앱타머의예시는, GITR에대한
항체 , 0X40에대한항체 , CD137에대한항체 , CD27에대한항체 , 0X40작용성 앱타머 (angoistic aptamer), CD 137작용성앱타머 , NKG2D리간드 (예를들면, 마우스에서의 MULT - 1 (murine UL 16-binding protein-like- 1)또는 H60,또는 사람에서의 MICA(MHC I-related chain A), MICB(MHC I-related chain B)또는 ULBPs(unique long 16-binding proteins)),또는 CD226작용제를포함할수있다.
[22] 상기 BiKE또는 TriKEs는 2개의단일쇄가변단편 (scFvs)을함유하고,표적
세포사멸을중재하기위해표적세포 (예를들어,손상된신경세포,종양세포 또는감염된세포)및자연살해세포둘다에특이적으로결합하는
시약이다 (TriKES의경우,자연살해세포의두개의표면항원또는수용체에 결합한다).
[23] 이들은자연살해세포를이용하여표적세포 (예를들어,손상된신경세포,종양 세포또는감염된세포)를공동중재시키고,이에따라자연살해세포중재 항체-의존성세포성세포독성 (ADCC)을유발하는데사용된다. BiKE는당업계에 공지된,예를들어 Gleanson, M. K., et al” Mol Cancer Ther, 11: 2674-2684(2012) ; Vallera, D. A., et al., Cancer Biother Radiopharm, 28: 274-282(2013); Wiernik, A., et al., Clin Cancer Res, 19: 3844-3855(2013); Reiners, K. S et al., Mol Ther, 21:
895-903(2013); Singer, H., et al., J Immunother, 33: 599-608(2010);또는 Gleason, M. K„ et al„ Blood, 123: 3016-3026(2014)에기재된바와같은임의의방법에의해 생성될수있다. BiKE의 1종의 scFv는표적세포 (예를들어,손상된신경세포, 종양세포또는감염된세포)의표면상의항원에특이적으로결합하고,다른 scFv는자연살해세포상의수용체 (예를들어, CD16과같은 Fc수용체)에 특이적으로결합한다.일구체예에있어서, BiKE는 TAA또는 RAEl(Retinoic Acid Early 1)에특이적으로결합하는제 1 scFv와활성화수용체,예를들면, 2020/175960 1»（：1^1{2020/002904
5
CD16에특이적으로결합하는제 2 scFv를포함한다.
[24] 상기 NK세포억제성수용체의길항제는,억제성수용체에결합하는항체또는 앱타머를포함할수있다. NK세포억제성수용체의예시는, CTLA-4, PD-1, PD-L1, Tim-3, CD96, KIR, NKG2A,또는 TIGIT(T-cell immunoglobulin and ITIM domain)를포함할수있다.따라서, NK세포활성화물질은상기열거된 수용체에대한항체를포함할수있다.
[25] 일구체예에서，상기 NK세포를활성화하는물질은 NKG2D, 2B4(CD244), DNAM-1(CD226), CD2, CD16, NKp30, NKp44, NKp46및 NKp80으로이루어진 군으로부터선택된하나이상의인자내지물질을자극하는것일수있다. NK 세포의활성에는상기한바와같이 ,다양한활성화및저해성수용체가관여할 수있는데,주된수용체중에는면역수용체티로신-기반활성
모티프 (immunoreceptor tyrosine-based activation motif : IT AM)-관련신호전달 분자와관련된 NKp46및신호전달분자 DAP10와관련된수용체 NKG2D(EMBO J. 2004; 23:255.-9, Annu. Rev. Immunol. 2005; 23:225-74)에관련한것들이있다. 공동자극성이라간주되는수용체로는, 2B4(CD244)와같은신호전달림프구 활성분자 (signaling lymphocytic activation molecule: SLAM)패밀리의구성원들 뿐만아니라, DNAM-1(CD226), CD2,및 NKp80(KLRFl유전자산물)과같이 다양한수용체를포함할수있다.즉,상기 NK세포를활성화하는물질은
ITAM-함유하는신호전달분자분자 (예를들어, CD16, NKp46, NKp30또는 NKp44등)， NKG2D, 2B4(CD244), DNAM-1(CD226), CD2및 NKp80중선택된 하나이상의물질을자극하는것일수있다. Bryceson Y등 (Blood
2006;107:159-166)및 Kim HS등 (Immunity 2010;32:175-186)에의하면 NK세포 활성화는상기나열된물질의다양한조합에대한자극에의해가능할수 있으며,상기물질의조합의예로는 2B4및 DNAM-1, DNAM-1및 NKG2D, 2B4 및 NKG2D,또는 2B4, DNAM-1및 NKG2D가있을수있다.
[26] 일구체예에서 ,상기 NK세포가분비하는세포독성효소는퍼포린 (perforin), 그랜자임 A(granzyme A),그랜자임 B,그랜자임 H,그랜자임 K및그랜자임 M으로이루어진군으로부터선택된하나이상인것일수있다. NK세포의 활성도을즉정하는것은탈과립화 (degranulation)및세포독성 (cytotoxicity)활성 , 및 NK세포자극에의해분비된사이토카인의측정중선택된하나이상을 포함하는것일수있다.상기탈과립화활성및세포독성활성은예를들어, 퍼포린 (perforin)또는그랜자임 (granzyme)분비로표적세포의용해를유도하는 것을의미할수있고,이를유세포법를이용해분석할수있다.
[27] 일구체예에서 ,상기절단되는펩티드는 2내지 20개의아미노산으로이루어져 있고,，아이소류신-글라이신-아스파라진-아르기닌 (IGNR,서열번호 1)',
，아이소류신-글루탐산-프롤린-아스파르산 (IEPD,서열번호 2)’,
’프롤린-트레오닌-세린-티로신 (PTSY,서열번호 3)’,
’티로신-아르기닌-페닐알라닌-라이신 (YRFK,서열번호 4)’, 2020/175960 1»（：1^1{2020/002904
6
’라이신-발린-프롤린-류신 (KVPL,서열번호 5)’,’발린-글라이신 (VG,서열번호 6), ’페닐알라닌-글라이신-아르기닌 (FGR,서열번호 7)’,
’글라이신-프롤린-아스파르산 (GPD,서열번호 8)’,’글라이신-글루탐산 (GE, 서열번호 9)’,’프롤린-아스파르산-페닐알라닌-글라이신 (PDFG,서열번호 10)’ 또는
’발린-글라이신-프롤린-아스파르산-페닐알라닌-글라이신-아르기닌 (VGPDFGR, 서열번호 11)을포함하는펩타이드일수있다.
[28] 따라서，일실시예에 따르면,일양상에 따른미세입자복합체는개체의 혈액 또는시료중 NK세포를활성화시키고,이에 따라 NK세포로부터효소및 사이토카인들을분비시킬수있다.이에따라분비된효소들의 활성에의해 미세입자복합체내부에포함되어 있는펩티드가절단됨에 따라, NK세포의 활성 및이의 검출이가능할수있다.
[29] 본명세서에서사용되는용어 "형광성모이어티 (fluorescent moiety)”는상기 절단가능한펩티드로부터분리됨에 따라특정주파수의광 (예컨대, UV광)을 흡수하여 형광시그널을발생시키는형광분자또는이의유도체또는
컨쥬게이트를의미할수있다.예를들면,상기 형광성모이어티는쿠마린및 이와관련된염료;플루오레세인,로돌,및로다민같은크산텐 (xanthene)염료; 레소루핀;시아닌염료;바이메인염료 (bimanes);아크리딘;이소인돌;
단실 (dansyl)염료;루미놀및 이소루미놀유도체같은아미노프탈성
히드라지드 (aminophthalic hydrazides);아미노프탈이미드;아미노나프탈이미드; 아미노벤조퓨란;아미노퀴놀린;디시아노히드로퀴논; BODIPY;및유로품및 터븀복합체및 이와관련된화합물을포함할수있다.또한,상기 형광성 모이어티는자유카르복실기 ,에스테르 (예컨대, N-하이드로숙신이미드 (NHS) 에스테르)또는말레이미드유도체를추가적으로포함할수있으며,또한 스트렙타비딘,바이오틴,팔로이딘,아민,아자이드또는이오도아세트아미드 컨쥬게이트를포함할수있다.
[3이 일구체예에 있어서,상기제 1형광모이어티는플루오레신 (fluorescein),
6-FAM,로다민,텍사스레드 (Texas Red),테트라메틸로다민,카르복시로다민, 카르복시로타민 6G,카르복시로돌,카르복시로다민 110,캐스케이드
블루 (Cascade Blue),캐스케이트옐로우 (Cascade Yellow),코마린, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-크롬,피코에리트린, PerCP (페리디닌클로로필 -a단백질), PerCP-Cy5.5, JOE(6 -카르복시 - 4’,5’-디클로로- 2’,7’-디메톡시플루오레신), NED, ROX(5-(및- 6) -카르복시 - X-로다민), HEX,루시퍼 옐로우 (Lucifer Yellow),마리나 블루 (Marina Blue),오레곤그린 (Oregon Green) 488,오레곤그린 (Oregon Green) 500,오레곤그린 (Oregon Green) 514,알렉사플로어 (Alexa Fluor) 350,알렉사 플로어 430,알렉사플로어 488,알렉사플로어 532,알렉사플로어 546,알렉사 플로어 568,알렉사플로어 594,알렉사플로어 633,알렉사플로어 647,알렉사 플로어 660,알렉사플로어 680, 7 -아미노- 4 -메틸코마린- 3 -아세트산, 2020/175960 1»（：1^1{2020/002904
7 보디피 (BODIPY) FL,보디피 FL-Br2,보디피 530/550,보디피 558/568,보디피 564/570,보디피 576/589,보디피 581/591,보디피 630/650,보디피 650/665,보디피 R6G,보디피 TMR및보디피 TR로구성된군으로부터선택된하나이상의것일 수있다.일구체예에 있어서 ,상기제 1형광모이어티부분이외에도,
미세입자의표면자체에상기형광모이어티의물질이코팅될수있다.이는일 양상에따른상기미세입자복합체를통해 NK세포의활성을보다확실하게 구별하기위한목적에서코팅되는것일수있다.형광모이어티가부착된 펩티드가 NK세포로부터분비된효소에의해절단되면,형광강도가변하게 됨에따라처리전미세입자와의비교를통해 NK세포의활성도를육안으로도 검줄할수있다.
[31] 상기 NK세포가분비하는사이토카인과결합하는물질은 IFN-yMIP-la,
MIP-l(3TNF-a, TNF-(3RANTES, IL-8및 IL-10으로이루어진군으로부터선택된 하나이상의것과결합하는항체,펩티드또는앱타머 (aptamer)인것일수있다. 상기와같은 NK세포로부터분비내지발현된사이토카인에대한분석은 FACS, 세포내사이토카인염색또는 ELISA등을이용하여이루어질수있다.
[32] 본명세서에서용어 "링커”는,미세입자에 NK세포를활성화하는물질이나, 상기절단되는펩티트및사이토카인과같은물질을부착시키기위한,또는상기 각각의물질들을연결시키기위한물질을의미할수있다.상기링커는고정화 화합물일수있다.상기고정화화합물은상기물질에부착될수있는관능기를 포함할수있다.상기관능기는예를들면,티올기또는술프히드릴기를
포함하는황-함유기일수있다.상기관능기는알코올및/또는페놀의
유도체로서산소자리에황이포함된 RSH의식을가지는화합물일수있다. 또한,상기관능기는각각 RSSR'또는 RSR’의식을가지는티올에스테르 (thiol ester)또는다이티올에스테르 (di比 liol ester)일수있다.또한,상기관능기는 아미노기 (-NH2)일수있다.구체적으로,일구체예에있어서상기링커는비오틴, 아비딘,스트렙트아비딘,탄수화물,폴리 L-리신,티올기,아민기,알코올기, 카르복실기 ,아미노기,설퍼기,알데히드기,카르보닐기,숙신이미드기, 말레이미드기 ,에폭시기 ,이소티오시아네이트기를갖는화합물또는그의 조합으로이루어진군으로부터선택된하나인것일수있다.아미노기를갖는 화합물의 예에는, 3 -아미노프로필트리메톡시실란,
N-(2 -아미노에틸)- 3 -아미노프로필트리메톡시실 ¾(EDA),
트리메톡시실릴프로필디에틸렌트리아민 (DETA),
3-(2 -아미노에틸아미노프로필)트리메톡시실란,
3 -아미노프로필트리에톡시실란가포함될수있고,알데히드기를갖는
화합물에는글루타르알데히드가포함될수있다.티올기를갖는화합물의 예에는 4 -메르캅토프로필트리메톡시실란 (MPTS)가포함될수있다.또한, 에폭시기를갖는화합물의 예에는, 3 -글리시드옥시프로필트리메톡시실란, 이소티오시아네이트기를갖는화합물의 예에는, 2020/175960 1»（：1^1{2020/002904
8
4 -페닐렌디이소티오시아네이트 011 ,숙신이미드및말레이미드기를갖는 화합물의 예에는,디숙신이미딜카르보네이트 (080또는숙신이미딜
4-(말레이미드페닐)부틸레이트 (SMPB)가포함될수있다.
[33] 상기 링커는각물질의 5’말단및/또는 3’말단에 연결될수있다.상기 링커를 통한결합방법은당업계에공지되어 있다.또한,상기 링커는펩티드링커일수 있다.상기 펩티드링커는당업계에공지된다양한링커를이용할수있으며 , 예를들어,복수의아미노산으로이루어진링커일수있다.일구체예에따르면, 상기 링커는예를들어, 1내지 100개또는 2내지 50개의 임의의 아미노산으로 이루어진폴리펩티드일수있다.예를들어 ,상기 펩티드링커는 (3切쇼811및 861 잔기를포함할수있으며 , 1¾·및쇼 과같은중성 아미노산들도포함될수있다. 펩티드링커에 적합한아미노산서열은당업계에 공지되어 있다.
[34] 또한,일구체예에서상기 링커는고분자화합물을더포함하는것일수있다. 구체적으로,상기고분자화합물은폴리에틸렌글리콜 £(3),
폴리에틸렌옥사이드 묘0),폴리에틸렌이민 ),폴리프로필렌옥사이드 汉)), 폴리비닐알코올 ( ^쇼),폴리 ( 이소프로필아크릴아마이드 )(polyNIPAM), 쓸리푸마레이트,쓸리오르가노포스파젠 ,쓸리아크릴산 (]301)^4쇼(：),
폴리아크릴설포네이트,폴리하이드록시에틸메타크릴레이트 (PolyHEMA)및 이들의 공중합체로이루어진군에서선택된것일수있다.일구체예에 있어서, 상기복합체의절단되는펩티드 말단에는고분자화합물, 0-말단에는제 1 형광모이어티가결합된것일수있다.
[35] 다른양상은일양상에 따른복합체를포함하는 NK세포활성 진단용키트를 제공한다.상기 키트는 NK세포가분비하는사이토카인을검출하는물질을더 포함하는것일수있다.
[36] 일구체예에 있어서,상기사이토카인을검출하는물질은제 2형광모이어티가 추가적으로결합된것일수있다.사이토카인을검출하는물질은상기 NK 세포가분비하는사이토카인을추적하는물질로서 , IFN-gMIP-la,
Figure imgf000010_0001
TNF-pRANTES, 1ᄂ8및 1 10으로이루어진군으로부터선택된하나이상의 것과결합하는항체,펩티드또는앱타머知! 111 )인것일수있다.
[37] 일구체예에서 ,상기 키트는 NK세포의세포독성과사이토카인의분비를
동시에 검출하는것일수있다.상기 키트에포함된미세입자복합체는 NK 세포를활성시킬수있고,이에따라 NK세포는그랜자임과같은효소와 인터페론감마 (표 -?와같은사이토카인을분비하게된다.이 경우,미세입자 복합체에포함된상기 펩티드는상기 효소의활성에 의해절단되어 제 1형광 모이어티의분리에따른형광감도의감소가나타날수있다.반면,미세입자 복합체가포함하고있는표例-7과같은사이토카인에 대한항체는표別 를 포획할수있고,포획된표別 는키트내에포함된표^ 를검출하는항체를 밀집화시켜제 2형광모이어티의 형광강도가더욱강해질수있다.따라서 , 상기 키트는 NK세포의 세포독성활성과사이토카인분비 활성을동시에측정할 2020/175960 1»（：1^1{2020/002904
9 수있는것이다.
[38] 일구체예에 있어서,상기제 2형광모이어티는상기제 1형광모이어티에서 정의된것과같다.다만,제 2형광모이어티는제 1형광모이어티와육안으로 구별될수있는모이어티일수있다.
[39] 상기일양상의미세입자복합체에서언급된용어또는요소중이를포함한 NK세포활성진단용키트에서언급된용어또는요소와같은것은동일한 것으로이해된다.
[4이 또다른양상은일양상에따른복합체를포함하는 NK세포활성관련질환 진단용키트를제공한다.
[41] 일구체예에서, NK세포의활성과관련한질환은과민성면역질환,
자가면역질환,면역거부반응,면역결핍질환,조직구증,암, 2형당뇨병,기생충 감염질환및바이러스질환일수있다.상기과민성면역질환은천식및축농증 중선택된하나이상의것이거나,상기자가면역질환은루푸스,다발성
경화증 (multiple sclerosis), 1형당뇨병및류마티스관절염중선택된하나이상의 것이거나,또는상기조직구증은 HLH, XLP1,및 XLP2에서선택된어느하나 이상의것일수있다.혈구탐식성림프조직구증 (HLH)은제 2군랑게르한스세포 조직구증,적혈구탐식성림프조직구증식증 (가족성,산발성),감염연관성 혈구탐식증후군,바이러스연관성혈구탐식증후군,거대한림프절병증을 동반한조직구증,또는망상조직구증의의미를포함할수있다.상기’’암’’은 종양,혈액암또는고형암을포함할수있다.일구체예에서 ,상기암은폐암, 간암,식도암,위암,대장암,소장암,췌장암,흑색종,유방암,구강암,뇌종양, 갑상선암,부갑상선암,신장암,자궁경부암,육종,전립선암,요도암,방광암, 고환암,혈액암,림프종,피부암,건선및섬유선종으로이루어진군에서선택된 것일수있다.일구체예에서 ,상기암은췌장암또는 B세포림프종 (B cell lymphoma)일수있다.일구체예에서,상기바이러스질환은 B형간염일수있다. 일구체예에서 ,상기면역결핍질환은디조지증후군 (DiGeorge synderome)또는 체디악히가시증후군 (Chedial-Higashi syndrome)일수있다.
[42] 상기일양상의미세입자복합체에서언급된용어또는요소중이를포함한 NK세포관련질환진단용키트에서언급된용어또는요소와같은것은동일한 것으로이해된다.
[43] 다른양상은일양상의복합체를포함하는, NK세포활성검출용조성물을
제공한다.상기조성물은 NK세포가분비하는사이토카인을검출하는물질을더 포함하는것일수있다.상기사이토카인을검출하는물질은제 2형광
모이어티가추가적으로결합된것일수있다.또한,상기조성물은 NK세포의 세포독성과사이토카인를동시에검출하는것일수있다.
[44] 상기일양상의미세입자복합체에서언급된용어또는요소중이를포함한 NK세포활성검출용조성물에서언급된용어또는요소와같은것은동일한 것으로이해된다. 2020/175960 1»（：1^1{2020/002904
10
[45] 또다른양상은일양상의복합체를개체의 혈액 샘플에처리하는단계를
포함하는 NK세포활성도를측정하는방법을제공한다.또한,상기
복합체로부터 절단된펩티드를검출하는단계 ;및상기 절단된펩티드가상기 복합체를첨가하기 이전보다증가한경우,상기 혈액 샘플중 NK세포의활성이 증가한것으로판정하는단계를더포함하는것일수있다.상기 절단된 펩티드의 검줄은 ELISA(enzyme linked immunosorbent assay),
유세포분석 (Fluorescence Activated Cell Sorter, FACS)및단백질칩 (protein chip)으로이루어진군에서선택된하나이상에의해수행되는것일수있다.일 구체예에 있어서유세포법으로수행하면상기복합체를첨가하기 이전보다 형광광도가감소한경우,상기 혈액 샘플중 NK세포의활성이증가한것으로 판정하는단계를포함하는것일수있다.
[46] 상기방법은 NK세포가분비하는사이토카인을검출하는물질을개체의 혈액 샘플에 처리하는단계를더포함하는것일수있다.또한추가적으로,상기 NK 세포가분비하는사이토카인을검출하는단계 ;및상기사이토카인이상기 복합체를첨가하기 이전보다증가한경우,상기 혈액 샘플중 NK세포의활성이 증가한것으로판정하는단계를더포함하는것일수있다.상기복합체를 첨가하기 이전보다형광광도가증가한경우,상기 혈액 샘플중 NK세포의 활성이증가한것으로판정하는단계를포함하는것일수있다.이는미세입자 복합체가포함하고있는사이토카인에 대한항체는사이토카인을포획할수 있고,포획된사이토카인에의해사이토카인을검출하는물질을밀집시킬수 있기 때문이다.따라서,상기복합체로부터 절단된펩티드를검출하는단계와 상기 NK세포가분비하는사이토카인을검출하는단계는동시에수행되는것일 수있다.
[47] 상기 일양상의미세입자복합체에서 언급된용어또는요소중이를포함한 NK세포활성측정 방법에서 언급된용어또는요소와같은것은동일한것으로 이해된다.
[48] 또다른양상은일양상의복합체를개체의 혈액 샘플에처리하는단계를
포함하는 NK세포활성 관련질환에 대한정보를제공하는방법을제공한다.
[49] 또다른양상은일양상의복합체를개체의 혈액 샘플에처리하는단계를
포함하는 NK세포활성 관련질환을진단하는방법을제공한다.
[5이 상기 NK세포활성 관련질환에 대한정보는,정상 NK세포대비실험군 NK 세포의 활성화가검출되지 않는경우비정상적인 NK세포로판단할수있고, 또는특정수용체에 대해활성을상실한 NK세포가표적세포에 대해 비정상적으로활성을일으키거나일으키지 않는경우상기표적세포가특정한 질병과관련이 있음을판단할수있다.
[51] 상기 일양상의미세입자복합체에서 언급된용어또는요소중이를포함한 NK세포활성관련질환에 대한정보를제공하는방법에서 언급된용어또는 요소와같은것은동일한것으로이해된다. 2020/175960 1»（：1^1{2020/002904
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[52] 본출원에서개시된각각의설명및실시형태는각각의다른설명및실시
형태에도적용될수있다.즉,본출원에서개시된다양한요소들의모든조합이 본출원의범주에속한다.또한,하기기술된구체적인서술에의하여본출원의 범주가제한된다고볼수없다.
발명의효과
[53] 일양상에따른미세입자복합체 ,이를포함하는 NK세포활성진다용키트, 이를포함하는 NK세포활성검출용조성물,및이를이용한 NK세포활성측정 방법은 NK세포의활성에따른효소의분비와사이토카인의발현을동시에쉽게 측정할수있어다른물질또는방법과달리저비용으로정밀한측정결과를 나타냄으로써, NK세포의활성을효과적으로검출할수있다.
도면의간단한설명
[54] 도 1은미세입자복합체의모형을나타낸도면이다.
[55] 도 2a는 Granzyme B와미세입자복합체를반응시켰을때나타난형광감도의 변화를나타낸그래프이다.
[56] 도 2b는 IFN-Y와미세입자복합체를반응시켰을때나타난형광감도의변화를 나타낸그래프이다.
[57] 도 3a는미세입자복합체의 NK세포의활성효과에따른 Granzyme B의분비를 측정한그래프이다.
[58] 도 3b는미세입자복합체의 NK세포의활성효과에따른 IFN-Y의분비를
측정한그래프이다.
[59] 도 4는미세입자복합체를통하여 NK세포의활성및분비된 Granzyme B및 IFN-Y의동시측정을영상화한이미지이다.
발명의실시를위한형태
[6이 이하본발명을실시예를통하여보다상세하게설명한다.그러나,이들
실시예는본발명을예시적으로설명하기위한것으로본발명의범위가이들 실시예에한정되는것은아니다.
[61] 심시예 1.미세입자복함체의제조
[62] U .그래자임 B(Granzvme TV>펜타이드세서의제조
[63] Granzyme b펩타이드센서는아미노산서열 VGPDFGR을갖는그랜자임 B
절단가능펩티드 (Peptron, inc. A)를이용해공지된방법 (Choi, P.J. and T.J.
Mitchison, 2013. 110(16): p.6488-6493.)으로합성하였고, N-및 C-말단펩티드의 잔기각각은 NHS-PEG4-Biotin(Thermo Fisher scientific)와 Alexa Fluor™ 488 NHS Ester(InvitrogenTM)으로각각개질하였다.
[64] 1.2.미세입자복합체의제조
[65] 4.0X 108 beads/ml의다이나비드 (Dynabeads) M-450에폭시 lm모 (Thermo Fisher scientific)를탈이온화된물로세척하였고, 0.1 M인산나트륨과 0.1 M의 pH 7.4와 1 M의암모니움설페이트를포함하는완충액 1 ml에서재현탁하였다.그런다음, 2020/175960 1»（：1^1{2020/002904
12
5mg/ml의스트렙타비딘 (streptavidin, Invitrogen) 40L를상기 Dynabead현탁액에 첨가하고,비드의침강을방지하기위해부드러운회전으로실온에서 24시간 동안인큐베이션하였다.상기 Dynabeads에서미반응에폭시기 (group)를 제거하기위해, 1 ml의트리스완충액 (lOOmM의트리스, pH 7.9)을상기반응 혼합물에첨가하고,실온에서 1시간동안인큐베이션하였다.스트렙타비딘으로 코팅된 Dynabeads들을 PBS에서 3회세척하고,중탄산완충액 (100 ml중탄산 나트륨; pH 8.3)에서재현탁한후, 1 mg/ml의 Alexa Fluorä 647 NHS
Ester(Invitrogen) 0.2 ml을첨가하여 Alexa Fluorä 647로표지화시켰다.실온에서 1시간동안인큐베이션후,표지된 Dynabeads를 PBS로세척하여미반응염료를 제거하였다.그리고 streptavidin으로코팅되고, Alexa M7 -표지화된 dynabeads lml를비오틴 (biotin)화된 0.5mg/ml의인간 NKG2D/CD314항체 (R&D system; clone : 149,810) 10ml;비오틴화된 0.5mg/ml의 2B4/CD244 NTI항체 (Invi仕 ogen, clone : eBio C1.7) 10ml; 0.5mg/m公의상기 Granzyme B펩티드센서 10ml;및 비오틴화된 lmgM의항 IFN-Y항체 (Abeam, clone : MD-1) 10ml를실온에서 10분 동안서로섞어미세입자복합체를제조한후, PBS로세척하였다.상기실시예를 통해제조한미세입자복합체의모형도는도 1에나타내었다.
[66] 심험예 1.유세포분석
[67] 본실험예에서는상기실시예에서제조된미세입자복합체가정상적으로
기능하는지알아보기위해,하기와같이유세포분석을수행하였다.우선, Granzyme B센서의기능을확인하기위하여 ,미세입자복합체를 2시간동안 150U/ml, 450U/ml및 600U/ml로구성되는다양한농도의인간 Granzyme B(Abcam)로처리하였다.그리고동시에 , IFN-y에대한포획기능을확인하기 위하여 PE항-인간 IFN-y항체 (BioLegend, clone : 4S.B3) 2[xg에 1, 10, 100및 1000ng/ml로구성되는다양한농도의재조합인간 IFN-Y(Gibco)을첨가하고, 각각에미세입자복합체를혼합하였다.형광도측정은 LSR Fortessa분석기 (BD Biosciences)를사용하여미세입자복합체의형광강도를측정하고측정 데이터를 FlowJo로분석하여각검출여부를확인하였다.각각의결과는도 2a및 도 2b에나타내었다.
[68] 도 2a에나타낸바와같이 , Granzyme B를처리할경우형광도가감소하는
결과가나타났다.이는미세입자복합체내 Granzyme B효소에의해펩티드가 절단된것을의미한다.또한,도 2b에나타낸바와같이, IFN-Y처리농도가높을 경우엔형광도가상승하였는데,이는미세입자복합체내 IFN-y항체가 PE 항-인간 IFN-y항체가추적한 IFN-Y를포획하였음을나타낸다.상기결과는상기 실시예에서제조한미세입자복합체가정상적으로기능함을의미한다.
[69] 심험예 2. NK세포활성측정
P이 본실험예에서는자연살해 (natural killer : NK)세포의활성에따라분비된
Granzyme B와 IFN-y에대한미세입자복합체의 NK세포활성및이의측정 여부를실험하고자하였다.우선,인간 BD Fc블록 (BD Bioscience)으로미세입자 2020/175960 1»（：1/10公020/002904
13 복합체를처리한후,확장된 NK세포 (eNK)를사용하였다. Wg/rnl의 PE 항-인간 IFN-Y항체 50ml를 IMDM배지에서 37 OC에서 4시간동안동일한수의 미세입자복합체와 eNK세포 4.0xl06를인큐베이션하였다. PBS로 3회상기 eNK/미세입자혼합물을세척한후, LSR Fortes sa분석기 (BD Biosciences)로유동 세포계측법을수행하였다.검출된데이터를 FlowJo로분석하였다.대조군은 아무것도처리되지않은비드 (bead),비오틴화된 NKG2D/2B4항체를비오틴화된 0.5mg/ml의마우스 iso항체인 IgG 20ml로대체한미세입자를사용하였다.그 결과는도 3a및 3b에각나타내었다.
도 3a에나타낸바와같이 , iso항체로대체한미세입자의경우, NK세포에대한 활성이없어 Granzyme B의분비가일어나지않고이에따라펩티드센서의 절단이발생하지않아미세입자복합체에비해높은형광도를나타내었다.또한, 도 3b의경우엔, iso항체로대체한미세입자의 NK세포활성이없어 IFN-Y의 분비가일어나지않았고,이에따라추적에따른형광도가낮았다.이는 미세입자복합체에포함된항체인 NKG2D/2B4항체에의해 NK세포의활성이 증가는데반해,대조군인 iso항체의경우, NK세포를자극하지못하였기 때문이다.본실험예를통해 ,미세입자복합체가 NK세포의 Granzyme B및 IFN-Y의분비를활성화시키는효과가있음을나타낸다.
[72] 심험예 3.미세입자복함체의라이브셈이미징분석
본실험예에서는미세입자복합체를통하여 NK세포의활성및활성에따른 Granzyme B및 IFN-y의동시측정을영상화하고자하였다.미세입자복합체를 고정시키기위해,낮은표면범위로비오틴커버슬립에추가하였다. eNK세포및 형광모이어티가부착된 IFN-Y항체를첨가한후, 15분간격으로 4시간동안의 경과를영상화하였다.영상화과정은구체적으로, 40X(UPlanFLN, NA = 1.30) 대물렌즈와카메라 (Roper Scientific Cascade)로개조된현미경 (Olympus IX 81
73 epifluorescence)을사용하였다. LAMBDA LS크세논램프 (175W, Sutter장비)및 필터세트, FITC(Chroma S492 / 18x및 62002bs), Texas Red(Chroma S572 / 23x및 62002bs)및 Cy5(Chroma ET620 / 60x, T660LPXR및 ET700 / 75m)형광이미징에 사용되었습니다.현미경및전동스테이지 (MS-2000, Applied Scientific
Instrumentation, Inc)는 Micromanager에의해제어하였다.라이브셀이미징동안, Chamlide TC인큐베이터시스템 (Live Cell Instrument)에서세포배양조건인 37°C 및 5 % C02로유지하였다.상기의방법으로획득된이미지는 Image J.를 사용하여분석하였다.그결과는도 4에나타내었다.또한,동시에 LSR Fortessa 분석기 (BD Biosciences)로유동세포계측법을수행하였고,검출된데이터를 FlowJo로분석하여도 4에나타내었다.
[74] 도 4에나타낸바와같이, eNK가첨가된 bead에서만형광의감소가육안으로 확인되었다.또한, Granzyme B센서의경우 eNK와서로접촉되었을경우,시간이 지날수록미세입자복합체의절단펩티드에부착된형광모이어티의초록색 형광도가낮아졌고,추가로첨가된 IFN-Y항체에부착된붉은색형광모이어티에 2020/175960 1»（：1^1{2020/002904
14 의한형광도는높아지는결과가나타났다.두결과를오버레이하였을경우, 초록색의형광도를보이는미세입자가붉은색의미세입자로변화하는것을 확인할수있었다.이를통해미세입자복합체가 NK세포의활성및이에따른
Figure imgf000016_0001
있음을나타낸다.
5]
6]

Claims

2020/175960 1»（：1/10公020/002904 15 청구범위
[청구항 1] 미세입자;
NK세포가분비하는세포독성효소에의해절단되는펩티드;
상기펩티드에연결된제 1형광모이어티;
NK세포를활성화하는물질;
NK세포가분비하는사이토카인과결합하는물질;및
상기펩티드, NK세포를활성화하는물질,또는사이토카인과결합하는 물질을상기미세입자와연결하는링커를포함하는미세입자복합체.
[청구항 2] 청구항 1에있어서 ,상기미세입자는비드 (bead)인것인미세입자복합체 .
[청구항 3] 청구항 2에있어서,상기비드는라텍스비드,중합체플라스틱비드,생체 적합성중합체로구성된비드,유리비드,실리카비드및자성비드로 이루어진군으로부터선택된하나이상인것인미세입자복합체.
[청구항 4] 청구항 3에있어서,상기자성비드는철족금속원소,희토류원소,
화폐금속원소,아연족원소,알루미늄족원소,알칼리토금속원소및 백금족원소로이루어진군으로부터선택된하나이상인것인미세입자 복합체.
[청구항 5] 청구항 1에있어서,상기 NK세포가분비하는세포독성효소는
퍼포린 (perforin),그랜자임 A(granzyme A),그랜자임 B,그랜자임 H, 그랜자임 K및그랜자임 M으로이루어진군으로부터선택된하나이상인 것인미세입자복합체.
[청구항 6] 청구항 1에있어서,상기펩티드는 2내지 20개의아미노산으로이루어져 있고, , 'IGNR', 'IEPD', 'PTSY', 'YRFK', 'KVPL', 'VG', 'FGR', 'GPD', 'GE', TDFG’또는’VGPDFGR’을포함하는펩타이드인것인미세입자복합체 .
[청구항 7] 청구항 1에있어서,상기제 1형광모이어티는플루오레신 (fluorescein),
6-FAM,로다민,텍사스레드 (Texas Red),테트라메틸로다민, 카르복시로다민,카르복시로타민 6G,카르복시로돌,카르복시로다민 110,캐스케이드블루 (Cascade Blue),캐스케이트옐로우 (Cascade Yellow), 코마린, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-크롬,피코에리트린,
PerCP (페리디닌클로로필 -a단백질), PerCP-Cy5.5, JOE(6 - 카르복시 -4’,5’-디클로로- 2’,7’-디메톡시플루오레신), NED,
ROX(5-(및- 6) -카르복시 - X-로다민), HEX,루시퍼옐로우 (Lucifer Yellow), 마리나블루 (Marina Blue),오레곤그린 (Oregon Green) 488,오레곤 그린 (Oregon Green) 500,오레곤그린 (Oregon Green) 514,알렉사
플로어 (Alexa Fluor) 350,알렉사플로어 430,알렉사플로어 488,알렉사 플로어 532,알렉사플로어 546,알렉사플로어 568,알렉사플로어 594, 알렉사플로어 633,알렉사플로어 647,알렉사플로어 660,알렉사플로어 680, 7 -아미노- 4 -메틸코마린- 3 -아세트산,보디피 (BODIPY) FL,보디피 2020/175960 1»（：1^1{2020/002904
16 50,보디피 558/568,보디피 564/570,보디피 576/589, 피 630/650,보디피 650/665,보디피 1½(3,보디피
Figure imgf000018_0001
구성된군으로부터선택된하나이상의것인 미세입자복합체.
［청구항 8］ 청구항 1에있어서 ,상기 NK세포를활성화하는물질은 NKG2D,
264(00244), DNAM- 1(00226), 002, 0)16, NKp30, NKp44, NKp46,및 NKp80으로이루어진군으로부터선택된하나이상의물질을자극하는 것인미세입자복합체.
［청구항 9］ 청구항 1에있어서 ,상기 NK세포가분비하는사이토카인과결합하는 물질은 IFN-gMIP-la, MIP-l(3TNF-a, TNF-(3RANTES, 1ᄂ8및 1ᄂ10으로 이루어진군으로부터선택된하나이상의것과결합하는항체,펩티드
Figure imgf000018_0002
복합체 .
［청구항 1이 청구항 1에있어서,상기링커는비오틴,아비딘,스트렙트아비딘,
탄수화물,폴리 리신,티올기,아민기,알코올기,카르복실기,아미노기, 설퍼기,알데히드기,카르보닐기,숙신이미드기,말레이미드기,에폭시기, 이소티오시아네이트기를갖는화합물또는그의조합으로이루어진 군으로부터선택된하나인것인미세입자복합체.
［청구항 11］ 청구항 1에있어서,상기링커는고분자화합물을더포함하는것인
미세입자복합체.
［청구항 12］ 청구항 11에있어서 ,상기고분자화합물은폴리에틸렌글리콜 £(3), 폴리에틸렌옥사이드 £0),폴리에틸렌이민 ),
폴리프로필렌옥사이드 )),폴리비닐알코올 (1^쇼),
폴리 (此이소프로필아크릴아
Figure imgf000018_0003
돌리오르가노포스파젠 ,돌리아크릴산 (］301) ᅴ4(0,돌리아크릴설포네이트,
Figure imgf000018_0004
이루어진군에서선택된것인미세입자복합체 .
［청구항 13］ 청구항 11에있어서,상기절단되는펩티드의 말단에는고분자화합물,
0-말단에는제 1형광모이어티가결합된것인미세입자복합체 .
［청구항 14］ 청구항 1내지 13중어느한항의복합체를포함하는 NK세포활성
진단용키트.
［청구항 15］ 청구항 14에있어서,상기키트는 NK세포가분비하는사이토카인을
검출하는물질을더포함하는것인키트.
［청구항 16］ 청구항 14에있어서,상기사이토카인을검출하는물질은제 2형광
모이어티가추가적으로결합된것인키트.
［청구항 17］ 청구항 14에있어서,상기키트는 NK세포의세포독성과사이토카인를 동시에검출하는것인키트.
［청구항 18］ 청구항 14에있어서상기제 2형광모이어티는모이어티는
늘루오레신 (£111016 11), 6- 쇼］ ,로다민,텍사스
Figure imgf000018_0005
2020/175960 1»（：1^1{2020/002904
17 테트라메틸로다민,카르복시로다민,카르복시로타민 6G,카르복시로돌, 카르복시로다민 110,캐스케이드블루 (Cascade Blue),캐스케이트 옐로우 (Cascade Yellow),코마린, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-크롬, 피코에리트린, PerCP (페리디닌클로로필 -a단백질), PerCP-Cy5.5, JOE(6 - 카르복시 -4’,5’-디클로로- 2’,7’-디메톡시플루오레신), NED,
ROX(5-(및- 6) -카르복시 - X-로다민), HEX,루시퍼옐로우 (Lucifer Yellow), 마리나블루 (Marina Blue),오레곤그린 (Oregon Green) 488,오레곤 그린 (Oregon Green) 500,오레곤그린 (Oregon Green) 514,알렉사
플로어 (Alexa Fluor,상표명) 350,알렉사플로어 430,알렉사플로어 488, 알렉사플로어 532,알렉사플로어 546,알렉사플로어 568,알렉사플로어 594,알렉사플로어 633,알렉사플로어 647,알렉사플로어 660,알렉사 플로어 680, 7 -아미노- 4 -메틸코마린- 3 -아세트산,보디피 (BODIPY,상표명) FL,보디피 FL-Br2,보디피 530/550,보디피 558/568,보디피 564/570, 보디피 576/589,보디피 581/591,보디피 630/650,보디피 650/665,보디피 R6G,보디피 TMR및보디피 TR로구성된군으로부터선택된하나 이상의것인키트.
［청구항 19］ 청구항 15에있어서,상기 NK세포가분비하는사이토카인을검출하는 물질은 IFN-yMIP-la, MIP-l(3TNF-a, TNF-(3RANTES, IL-8및 IL-10으로 이루어진군으로부터선택된하나이상의것과결합하는항체,펩티드 또는앱타머인것인키트.
［청구항 2이 청구항 1내지 13중어느한항의복합체를포함하는 NK세포활성관련 질환진단용키트.
［청구항 21］ 청구항 20에있어서,상기 NK세포활성관련질환은과민성면역질환, 자가면역질환,면역결핍질환,조직구증,혈액암및고형암으로이루어진 군으로부터선택된것인, NK세포활성관련질환진단용키트.
［청구항 22］ 청구항 1내지 15중어느한항의복합체를포함하는, NK세포활성
검출용조성물.
［청구항 23］ 청구항 1의복합체를개체의혈액샘플에처리하는단계를포함하는 NK 세포활성도를측정하는방법 .
［청구항 24］ 청구항 23에있어서,
상기복합체로부터절단된펩티드를검출하는단계 ;및
상기절단된펩티드가상기복합체를첨가하기이전보다증가한경우, 상기혈액샘플중 NK세포의활성이증가한것으로판정하는단계를더 포함하는것인, NK세포활성도를측정하는방법 .
［청구항 25］ 청구항 24에있어서 ,
상기복합체를첨가하기이전보다형광광도가감소한경우,상기혈액 샘플중 NK세포의활성이증가한것으로판정하는단계를포함하는 것인, NK세포활성도를측정하는방법 , 2020/175960 1»（：1^1{2020/002904
18
［청구항 26］ 청구항 24에있어서 ,상기절단된펩티드의검출은 ELISA(enzyme linked immunosorbent assay),유세포분석 (Fluorescence Activated Cell Sorter, FACS)및단백질칩 (protein chip)으로이루어진군에서선택된 1개이상에 의해수행되는것인, NK세포활성도를측정하는방법 .
［청구항 27］ 청구항 23에있어서, NK세포가분비하는사이토카인을검출하는물질을 개체의혈액샘플에처리하는단계를더포함하는것인, NK세포 활성도를측정하는방법 .
［청구항 28］ 청구항 27에있어서 ,
상기 NK세포가분비하는사이토카인을검출하는단계 ;및 상기사이토카인이상기복합체를첨가하기이전보다증가한경우,상기 혈액샘플중 NK세포의활성이증가한것으로판정하는단계를더 포함하는것인, NK세포활성도를측정하는방법 .
［청구항 29］ 청구항 28에있어서,
상기복합체를첨가하기이전보다형광광도가증가한경우,상기혈액 샘플중 NK세포의활성이증가한것으로판정하는단계를포함하는 것인, NK세포활성도를측정하는방법 .
［청구항 3이 청구항 29에있어서 ,상기복합체로부터절단된펩티드를검출하는
단계와상기 NK세포가분비하는사이토카인을검출하는단계는동시에 수행되는것인, NK세포활성도를측정하는방법 .
［청구항 31］ 청구항 1의복합체를개체의혈액샘플에처리하는단계를포함하는 NK 세포활성관련질환에대한정보를제공하는방법.
［청구항 32］ 청구항 31에있어서,상기 NK세포와관련질환은과민성면역질환, 자가면역질환,면역결핍질환,조직구증,혈액암및고형암으로이루어진 군으로부터선택된것인, NK세포활성관련질환에대한정보를 제공하는방법 .
［청구항 33］ 청구항 1의복합체를개체의혈액샘플에처리하는단계를포함하는 NK 세포활성관련질환을진단하는방법.
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