WO2020175960A1

WO2020175960A1 - Microparticles for detecting natural killer cell activity and detection method using same

Info

Publication number: WO2020175960A1
Application number: PCT/KR2020/002904
Authority: WO
Inventors: 김헌식; 조덕; 도준상; 박지훈; 신예린
Original assignee: 재단법인 아산사회복지재단; 사회복지법인 삼성생명공익재단; 울산대학교 산학협력단
Priority date: 2019-02-28
Filing date: 2020-02-28
Publication date: 2020-09-03
Also published as: KR102210455B1; KR20200105339A

Abstract

Provided are a microparticle complex, a kit comprising same for diagnosing NK cell activity, a composition comprising same for detecting NK cell activity, and a method for measuring NK cell activity by using same.

Description

2020/175960 1»（：1^1{2020/002904 specification

Name of the invention: Fine particles for detecting natural killer cell activity and detection method using the same

Technical field

[1] NK cell activity detection microparticles and NK cell activity using the same

It's about how to test.

Background

[2] NK cells (natural killer 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. 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. In the case of 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).

[3] Various activating and inhibitory receptors are involved in the activity of NK cells. Among the receptors considered dominant, the immunoreceptor tyrosine-based activation motif (ITAM)-related signaling molecules (for example, , FcR y chain or TCRC chain) related to NKp46 and the receptor NKG2D related to the signaling molecule DAP10 (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 products).

[4] Existing techniques for measuring the NK cell activity are radioisotopes, or

As a registered patent 10-1926166 using a fluorescent probe, it is unsuitable for clinical application in terms of cost and time, so it is a method that can measure the activity of NK cells easily, quickly, and meaningfully, in the early stages of various immune-related diseases. It is recognized as important for diagnosis and prognosis.

Detailed description of the invention

Technical task

[5] One aspect is to provide a microparticle complex including microparticles; and peptides cleaved by cytotoxic enzymes secreted by natural killer (NK) cells.

[6] Another aspect is to provide a kit for diagnosing NK cell activity containing a complex according to one aspect. 2020/175960 1»（：1^1{2020/002904

2

[7] Another aspect is to provide a kit for diagnosing diseases related to NK cell activity, including complexes according to one aspect.

[8] Another aspect is for detecting NK cell activity, including a complex according to one aspect.

It is to provide the composition.

[9] Another aspect involves processing a complex of one aspect into a blood sample of an individual.

It is to provide a method of measuring the NK cell activity, including.

[1] Another aspect involves processing a complex of one aspect into an individual's blood sample.

It is to provide a method of providing information on diseases related to NK cell activity, including.

[11] Another aspect involves processing a complex of one aspect into a blood sample of an individual.

It is to provide a method for diagnosing diseases related to NK cell activity, including.

Problem solving means

[12] One aspect provides a microparticle complex containing a microparticle; and a peptide cleaved by a cytotoxic enzyme secreted by NK (Natural egg 11) cells.

[13] One specific example is fine particles; A peptide cleaved by a cytotoxic enzyme secreted by NK cells; a first fluorescent moiety linked to the peptide; Substances that activate cells; A substance that binds to cytokines secreted by NK cells; And a linker that connects the peptide, a substance that activates NK cells, or a substance that binds to cytokines with the microparticles. In one embodiment, the microparticles Particles may be beads 0^(1). Beads may be microbeads (11110'0 6&(1) or nanobeads (1 110 6&(1)). Specifically, 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.

[14] 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. In one embodiment, 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, (¾), rare earth element (1 Sze 06, Nd, 1¾1, 8111,, 0（1,

It may be composed of one or more metals selected from aluminum group elements (new, II), alkaline earth metal elements (1 切), and platinum group elements (, parent (1st class)) or alloys thereof. For example, (¾!,M, 2020/175960 1»（：1^1{2020/002904

3

It may contain one or more selected from the group consisting of Gd, Mo, MM'204 and MpOq. In one embodiment, in order to prevent the magnetic particles from interacting with the carboxyl groups of alginate, the magnetic particles are passivated. It may be, for example, passivated by citrate ions.

[15] In this specification, the terms "NK cells" or "Natural Killer cells" are

As a 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. The "natural killer cells" or "NK cells" are additional modifications derived from any tissue source. Natural killer cells

And, as well as mature 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.

[16] In this specification, the term "a substance that activates NK cells" is administered to the body

It 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. In one embodiment, the NK cells are activated. The substance may be a substance that stimulates factors or substances related to the activity of NK cells.

[17] The term "factor" refers to all molecular factors that cause NK cell activation.

Specifically, 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.

[18] The term "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. ) 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). Substances that activate NK cells may include immune cytokines, agonists of NK cell activating receptors, or antagonists of NK cell inhibitory receptors.

[19] The term "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

4 It regulates various biological activities such as cell activation, differentiation, cell migration, aging, and induction of apoptosis in various cells. 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. More specifically, 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. In addition, the TNF is

TNF-0 _C , TNF-p or TNF-Y, etc. Also, the immunocytokin may include a cytokine-antibody complex. For example, an interleukin (e.g., an interleukin)

IL-2) -Anti-interleukin (e.g., anti-IL-2) antibody complex.

[2] 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

Engagers).

[21] 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

[22] The BiKE or TriKEs contain two single-chain variable fragments (scFvs), and target

Specific binding to both target cells (e.g., damaged neurons, tumor cells or infected cells) and natural killer cells to mediate apoptosis.

It is a reagent (for TriKES, it binds to two surface antigens or receptors of natural killer cells).

[23] They use natural killer cells to co-mediate target cells (eg damaged neurons, tumor cells, or infected cells), thereby inducing natural killer cell-mediated antibody-dependent cellular cytotoxicity (ADCC). do. BiKE is known in the art, for example 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); or by a random method as described in Gleason, M. K„ et al„ Blood, 123: 3016-3026 (2014). Can be created. One scFv of 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 In one embodiment, 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

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And a second scFv that specifically binds to CD16.

[24] The antagonist of the NK cell inhibitory receptor may include an antibody or aptamer that binds to the inhibitory receptor. Examples of 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.

[25] In one embodiment, 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. As described above, 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). ) May contain various receptors, that is, the substance that activates the NK cells

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

2006;107:159-166) and Kim HS et al. (Immunity 2010;32:175-186), 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.

[26] In one embodiment, 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.

[27] In one embodiment, the peptide to be cleaved is composed of 2 to 20 amino acids, and isoleucine-glycine-asparagine-arginine (IGNR, SEQ ID NO: 1)',

，Isoleucine-glutamic acid-proline-aspartic acid (IEPD, SEQ ID NO: 2)’,

'Proline-threonine-serine-tyrosine (PTSY, SEQ ID NO: 3)',

'Tyrosine-arginine-phenylalanine-lysine (YRFK, SEQ ID NO: 4)', 2020/175960 1»（：1^1{2020/002904

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'Lysine-valine-proline-leucine (KVPL, SEQ ID NO: 5)','valine-glycine (VG, SEQ ID NO: 6),'phenylalanine-glycine-arginine (FGR, SEQ ID NO: 7)',

'Glycine-proline-aspartic acid (GPD, SEQ ID NO: 8)','Glycine-glutamic acid (GE, SEQ ID NO: 9)','Proline-aspartic acid-phenylalanine-glycine (PDFG, SEQ ID NO: 10)' or

It may be a peptide comprising'valine-glycine-proline-aspartic acid-phenylalanine-glycine-arginine (VGPDFGR, SEQ ID NO: 11).

Thus, according to one embodiment, the microparticle complex according to one aspect 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.

[29] The term "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

Conjugates. For example, the fluorescent moiety includes coumarin and related dyes; xanthene dyes such as fluorescein, rhodole, and rhodamine; Resorupine; cyanine dye; bimanes; acridine; isoindole;

Dansyl dyes; aminophthalic properties such as luminol and isoluminol derivatives

Hydrazides (aminophthalic hydrazides); aminophthalimide; aminonaphthalimide; Aminobenzofuran; aminoquinoline; dicyanohydroquinone; BODIPY; and euros and terbium complexes and compounds related thereto. In addition, 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.

[3 In one embodiment, the first fluorescent moiety is fluorescein,

6-FAM, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, carboxyrotamine 6G, carboxyrodol, carboxyrhodamine 110, cascade

Blue (Cascade Blue), Cascade Yellow, Comarin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-chrome, phycoerythrin, PerCP (peridinine chlorophyll-a protein), PerCP -Cy5.5, JOE (6-carboxy- 4',5'-dichloro- 2',7'-dimethoxyfluorescein), NED, ROX (5- (and-6) -carboxy-X-rhodamine ), HEX, Lucifer Yellow, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor 350, Alexa Floor 430, Alexa Floor 488, Alexa Floor 532, Alexa Floor 546, Alexa Floor 568, Alexa Floor 594, Alexa Floor 633, Alexa Floor 647, Alexa Floor 660, Alexa Floor 680, 7 -Amino-4 -Methylcommarin- 3-acetic acid, 2020/175960 1»（：1^1{2020/002904

7 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. In one embodiment, in addition to the first fluorescent moiety part,

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. When 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.

[31] 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.

[32] In the present specification, the term "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.

It may be a sulfur-containing group containing. The functional groups of alcohols and/or phenols

As a derivative, it may be a compound having the formula of RSH containing sulfur in the oxygen site. In addition, 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). Specifically, one In a specific embodiment, 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,

N-(2 -aminoethyl)-3 -aminopropyltrimethoxysyl ¾(EDA),

Trimethoxysilylpropyldiethylenetriamine (DETA),

3-(2-aminoethylaminopropyl)trimethoxysilane,

3-Aminopropyltriethoxysilane may be included,

Compounds may include glutaraldehyde. Examples of compounds having a thiol group may include 4-mercaptopropyltrimethoxysilane (MPTS). In addition, 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

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Examples of compounds having 4-phenylenediisothiocyanate 011, succinimide and maleimide groups include disuccinimidyl carbonate (080 or succinimidyl

4-(maleimidephenyl)butyrate (SMPB) may be included.

[33] 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. In addition, 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. According to one embodiment, the linker is, for example, 1 to 100 or 2 to 50 arbitrary It may be a polypeptide consisting of amino acids. For example, the peptide linker may contain residues ⁽ 3切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.

[34] In addition, in one embodiment, the linker may further include a polymer compound. Specifically, the polymer compound is polyethylene glycol £(3),

Polyethylene oxide seed 0), polyethyleneimine), polypropylene oxide)), polyvinyl alcohol (^ show), poly (isopropylacrylamide) (polyNIPAM), salifumarate, saliorganophosphazene, saliacrylic acid () 301)^4 show(：),

It may be selected from the group consisting of polyacrylic sulfonate, polyhydroxyethyl methacrylate (PolyHEMA), and copolymers thereof. In one embodiment, 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.

[35] Another aspect provides a kit 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.

[36] In one embodiment, 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 ).

[37] In one embodiment, 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. On the other hand, 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

9 can be.

[38] In one embodiment, the second fluorescent moiety is the same as defined in the first fluorescent moiety. However, the second fluorescent moiety is the number of moieties that can be distinguished visually from the first fluorescent moiety. have.

[39] Among the terms or elements mentioned in the above one aspect of the microparticle complex, the terms or elements mentioned in the NK cell activity diagnostic kit including ear are understood to be the same.

[4] Another aspect provides a kit for diagnosing diseases related to NK cell activity, including a complex according to one aspect.

[41] In one embodiment, the disease related to the activity of NK cells is irritable immune disease,

It may be an autoimmune disease, an immune rejection reaction, an immunodeficiency disease, histocytosis, cancer, type 2 diabetes, a parasitic infection, or a viral disease. The irritable immune disease is one or more selected from asthma and sinusitis, or the autoimmune disease is Lupus, multiple

Either one or more selected from multiple sclerosis, type 1 diabetes and rheumatoid arthritis, or the histocytosis may be any one or more selected from HLH, XLP1, and XLP2. Hematopoietic lymphocytocytosis (HLH) 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. In one embodiment, the cancer is lung cancer, liver cancer, esophageal cancer, gastric cancer, colon cancer, small intestine cancer, pancreatic cancer, melanoma, breast cancer, oral cancer. , Brain tumor, thyroid cancer, parathyroid cancer, kidney cancer, cervical cancer, sarcoma, prostate cancer, urethral cancer, bladder cancer, testicular cancer, hematologic cancer, lymphoma, skin cancer, psoriasis, and fibroadenoma. In one embodiment, it may be selected from the group consisting of. , The cancer may be pancreatic cancer or B cell lymphoma. In one embodiment, the viral disease may be hepatitis B. In one embodiment, the immunodeficiency disease may be DiGeorge synderome or Chedial-Higashi syndrome.

[42] Among the terms or elements mentioned in the one-part microparticle complex, it is understood that the terms or elements mentioned in the NK cell-related disease diagnosis kit including ear are the same.

[43] 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.

[44] It is understood that the terms or elements mentioned in the composition for detecting NK cell activity including ear are the same among the terms or elements mentioned in the one-part microparticle complex. 2020/175960 1»（：1^1{2020/002904

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[45] 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.

Detecting the cleaved peptide from the complex; And when the cleaved peptide is increased than before the addition of the complex, determining that the activity of NK cells in the blood sample is increased. 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) and protein chips may be performed by one or more selected from the group consisting of. In one embodiment, when performed by flow cytometry, the fluorescence intensity than before the addition of the complex was performed. In the case where the is decreased, it may include a step of determining that the activity of NK cells in the blood sample is increased.

[46] 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. In addition, the step of detecting cytokines secreted by the NK cells; And When the cytokine is increased than before the addition of the complex, the step of determining that the activity of NK cells in the blood sample is increased may be further included. When the fluorescence intensity is increased than before the addition of the complex, 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.

[47] Among the terms or elements mentioned in the one-part microparticle complex, the terms or elements mentioned in the method for measuring NK cell activity including ear are understood to be the same.

[48] 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.

[49] 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.

[5] 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.

[51] Among the terms or elements mentioned in the one-part microparticle complex, the terms or elements mentioned in the method for providing information on NK cell activity-related diseases including ear are understood to be the same. 2020/175960 1»（：1^1{2020/002904

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[52] Each description and embodiment disclosed in this application is a different description and implementation.

Forms may also be applied, i.e., all combinations of the various elements disclosed in this application fall within the scope of this application, and the specific descriptions described below do not limit the scope of this application.

Effects of the Invention

[53] 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.

Brief description of the drawing

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.

[58] Figure 3b shows the secretion of IFN-Y according to the NK cell activation effect of the microparticle complex.

It is a measured graph.

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.

Modes for the implementation of the invention

[6] Hereinafter, the present invention will be described in more detail through examples. However, these

The examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

[61] Simulated example 1. Manufacture of fine particle complex

[62] U. Grazyme B (Granzvme TV>Pentide production

[63] Granzyme b peptide sensor is Granzyme B with amino acid sequence VGPDFGR

A known method using a cleavable peptide (Peptron, inc. A) (Choi, P.J. and T.J.

Mitchison, 2013. 110(16): p.6488-6493.), and the residues of N- and C-terminal peptides, respectively, were NHS-PEG4-Biotin (Thermo Fisher scientific) and Alexa Fluor™ 488 NHS Ester (Invitrogen ^TM ) respectively.

[64] 1.2.Manufacturing of fine particle complex

[65] 4.0X 10 ⁸ beads/ml of Dynabeads M-450 epoxy lm (Thermo Fisher scientific) was washed with deionized water, 0.1 M sodium phosphate and 0.1 M of pH 7.4 and 1 M of ammonium Resuspended in 1 ml of buffer solution containing um sulfate. Then, 2020/175960 1»（：1^1{2020/002904

12

40 L of 5 mg/ml streptavidin (Invitrogen) was added to the Dynabead suspension, and incubated for 24 hours at room temperature with gentle rotation to prevent sedimentation of the beads. Unreacted epoxy groups were removed from the Dynabeads. To do this, 1 ml of Tris buffer (100 mM Tris, pH 7.9) was added to the reaction mixture, and incubated for 1 hour at room temperature. Dynabeads coated with streptavidin were washed three times in PBS, and bicarbonate buffer ( 100 ml sodium bicarbonate; pH 8.3) after resuspension, 1 mg/ml of Alexa Fluorä 647 NHS

0.2 ml of Ester (Invitrogen) was added and labeled with Alexa Fluorä 647. After incubation for 1 hour at room temperature, the labeled Dynabeads were washed with PBS to remove unreacted dyes. And coated with streptavidin, Alexa M7-labeled. dynabeads lml biotinylated 0.5mg/ml human NKG2D/CD314 antibody (R&D system; clone: 149,810) 10ml; biotinylated 0.5mg/ml 2B4/CD244 NTI antibody (Invi仕 ogen, clone: eBio C1) .7) 10 ml; 10 ml of the Granzyme B peptide sensor of 0.5 mg/m; and 10 ml of biotinylated lmgM anti-IFN-Y antibody (Abeam, clone: MD-1) were mixed with each other for 10 minutes at room temperature to prepare a microparticle complex, and then PBS A schematic diagram of the microparticle composite manufactured through the above example is shown in FIG. 1.

[66] Experimental Example 1. Flow cytometric analysis

[67] In this experimental example, the fine particle composite prepared in the above example is normally

To see if it was functioning, 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.

[68] As shown in Fig. 2a, when granzyme B is treated, the fluorescence intensity is reduced.

The results were shown. This means that the peptide was cleaved by the enzyme Granzyme B in the microparticle complex. In addition, as shown in Fig. 2b, when the concentration of IFN-Y treatment was high, the fluorescence increased, which means that in the microparticle complex It indicates that the IFN-y antibody captured the IFN-Y tracked by the PE anti-human IFN-y antibody. The above results imply that the microparticle complex produced in the above example functions normally.

[69] Test Example 2. Measurement of NK cell activity

In this experimental example, P is secreted according to the activity of natural killer (NK) cells.

We tried to test the NK cell activity of the microparticle complex against Granzyme B and IFN-y and whether it was measured. First, microparticles with human BD Fc block (BD Bioscience) 2020/175960 1»（：1/10公020/002904

After treating the 13 complex, expanded NK cells (eNK) were used. 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. After washing the eNK/fine particle mixture three times with PBS, 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.

As shown in Figure 3a, in the case of the microparticles replaced with the iso antibody, the secretion of Granzyme B did not occur because there was no activity on NK cells, and accordingly, cleavage of the peptide sensor did not occur, indicating a higher fluorescence intensity compared to the microparticle complex. In addition, in the case of Fig. 3b, the secretion of IFN-Y did not occur due to the lack of NK cell activity of the microparticles replaced with the iso antibody, and accordingly, the fluorescence according to the trace was low. This is the NKG2D/2B4 antibody, which is an antibody contained in the microparticle complex. This is because, while the activity of NK cells increases due to the increase in the activity of NK cells, the control group iso antibody did not stimulate NK cells. In this experimental example, the microparticle complex prevented the secretion of Granzyme B and IFN-Y of NK cells. It indicates that it has an activating effect.

[72] Experimental Example 3. Analysis of Live Imaging of Microparticle Complex

In this experimental example, 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. To fix the microparticle complex, 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. The imaging process is specifically, 40X (UPlanFLN, NA = 1.30) objective lens and camera ( Roper Scientific Cascade) modified microscope (Olympus IX 81)

⁷³ epifluorescence) was used. 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

Instrumentation, Inc.) was controlled by Micromanager. During live cell imaging, the cell culture conditions were maintained at 37°C and 5% CO ₂ in a Chamlide TC incubator system (Live Cell Instrument). The images acquired by the above method were maintained in the above manner. The analysis was performed using Image J. The results are shown in Fig. 4. In addition, flow cytometry was performed simultaneously with an LSR Fortessa analyzer (BD Biosciences), and the detected data were analyzed with FlowJo and shown in Fig. 4.

[74] As shown in Fig. 4, the reduction of fluorescence was visually confirmed only in the bead to which eNK was added. In addition, in the case of the Granzyme B sensor, when the eNK and the eNK were in contact with each other, it adhered to the cutting peptide of the microparticle complex as time passed. The green fluorescence of the fluorescent moiety was lowered, and the red fluorescent moiety attached to the IFN-Y antibody was added. 2020/175960 1»（：1^1{2020/002904

When 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.

5]

6]

Claims

2020/175960 1»（：1/10公020/002904 15 Claims

[Claim 1] Fine particles;

Peptides cleaved by cytotoxic enzymes secreted by NK cells;

A first fluorescent moiety linked to the peptide;

Substances that activate NK cells;

A substance that binds to cytokines secreted by NK cells; And

A microparticle complex comprising a linker connecting the peptide, a substance that activates NK cells, or a substance that binds to cytokines with the microparticles.

[Claim 2] In claim 1, the fine particles are bead (bead) of the fine particle complex.

[Claim 3] The microparticle composite according to claim 2, wherein the bead is at least one selected from the group consisting of latex beads, polymer plastic beads, biocompatible polymer beads, glass beads, silica beads and magnetic beads.

[Claim 4] In claim 3, the magnetic bead is an iron group metal element, a rare earth element,

A complex of fine particles that is one or more selected from the group consisting of a currency metal element, a zinc group element, an aluminum group element, an alkaline earth metal element, and a platinum group element.

[Claim 5] In claim 1, the cytotoxic enzyme secreted by the NK cells is

Perforin (perforin), granzyme A (granzyme A), granzyme B, granzyme H, granzyme K, and granzyme M is one or more selected from the group consisting of a microparticle complex.

[Claim 6] In claim 1, the peptide is composed of 2 to 20 amino acids,'IGNR','IEPD','PTSY','YRFK','KVPL','VG','FGR', A microparticle complex which is a peptide containing'GPD','GE', TDFG' or'VGPDFGR'.

[Claim 7] In claim 1, wherein the first fluorescent moiety is fluorescein,

6-FAM, Rhodamine, Texas Red, Tetramethylrhodamine, Carboxyrhodamine, Carboxyrotamine 6G, Carboxyrhodol, Carboxyrhodamine 110, Cascade Blue, Cascade Yellow ), Komarin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-chrome, phycoerythrin,

PerCP (peridinine chlorophyll-a protein), PerCP-Cy5.5, JOE (6-carboxy -4', 5'-dichloro-2', 7'-dimethoxyfluorescein), NED,

ROX (5- (and-6) -carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa

Floor (Alexa Fluor) 350, Alexa Floor 430, Alexa Floor 488, Alexa Floor 532, Alexa Floor 546, Alexa Floor 568, Alexa Floor 594, Alexa Floor 633, Alexa Floor 647, Alexa Floor 660, Alexa Floor 680, 7-Amino -4 -Methylcomarin-3 -Acetic acid, BODIPY FL, Body Pe 2020/175960 1»（：1^1{2020/002904

16 50, Body P 558/568, Body P 564/570, Body P 576/589, Body P 630/650, Body P 650/665, Body P 1½ ⁽ 3, Body P

A microparticle complex that is one or more selected from the group consisting of.

[Claim 8] In claim 1, the substance that activates NK cells is NKG2D,

264(00244), DNAM-1(00226), 002, 0)16, NKp30, NKp44, NKp46, and NKp80 to stimulate one or more substances selected from the group consisting of a microparticle complex.

[Claim 9] In claim 1, the substance that binds to cytokines secreted by NK cells is IFN-gMIP-la, MIP-l (3TNF-a, TNF-(3RANTES, 1b8 and 1b10) Antibodies and peptides that bind to one or more selected from the group

Complex.

[In claim 1, claim 1, wherein the linker is biotin, avidin, streptavidin,

From the group consisting of compounds having carbohydrates, polylysine, thiol groups, amine groups, alcohol groups, carboxyl groups, amino groups, sulfur groups, aldehyde groups, carbonyl groups, succinimide groups, maleimide groups, epoxy groups, isothiocyanate groups, or combinations thereof The fine particle complex that is the chosen one.

[Claim 11] In claim 1, the linker further contains a polymer compound.

Microparticle complex.

[Claim 12] In claim 11, the polymer compound is polyethylene glycol £(3), polyethylene oxide £0), polyethyleneimine),

Polypropylene oxide)), polyvinyl alcohol (1^show),

Poly(此isopropylacrylic acid)

Dolyorganophosphazene _, dolyacrylic acid ()301 ₎ ᅴ4(0, dolyacrylic sulfonate,

Fine particle complex, selected from the group consisting of.

[Claim 13] In claim 11, at the end of the peptide to be cleaved, a polymer compound,

A microparticle complex in which the first fluorescent moiety is attached to the 0-terminus.

[Claim 14] NK cell activity containing the complex of any one of claims 1 to 13

Diagnostic kit.

[Claim 15] In claim 14, the kit contains cytokines secreted by NK cells.

A kit containing more substances to be detected.

[Claim 16] In claim 14, the substance detecting the cytokine is the second fluorescence

A kit in which the moiety is additionally incorporated.

[Claim 17] The kit according to claim 14, wherein the kit detects cytotoxicity of NK cells and cytokines at the same time.

[Claim 18] In claim 14, the second fluorescent moiety is

Nuluoresin (11 £111016), 6-show], Rhodamine, Texas

2020/175960 1»（：1^1{2020/002904

17 Tetramethylrhodamine, Carboxyrhodamine, Carboxyrotamine 6G, Carboxyrodol, Carboxyrhodamine 110, Cascade Blue, Cascade Yellow, Komarin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-chrome, phycoerythrin, PerCP (peridine chlorophyll-a protein), PerCP-Cy5.5, JOE (6-carboxy -4',5'-dichloro-2',7'- Dimethoxyfluorescein), NED,

Floor (Alexa Fluor, trademark) 350, Alexa Floor 430, Alexa Floor 488, Alexa Floor 532, Alexa Floor 546, Alexa Floor 568, Alexa Floor 594, Alexa Floor 633, Alexa Floor 647, Alexa Floor 660, Alexa Floor 680, 7 -Amino-4 -Methylcomarin-3 -Acetic acid, BODIPY (trade name) FL, BODIPY FL-Br2, BODIPY 530/550, BODYPE 558/568, BODIPY 564/570, BODIPY 576/ 589, Body P 581/591, Body P 630/650, Body P 650/665, Body P R6G, Body P TMR and Body P TR A kit that is one or more selected from the group consisting of.

[Claim 19] In claim 15, the substance for detecting cytokines secreted by NK cells is IFN-yMIP-la, MIP-l (3TNF-a, TNF-(3RANTES, IL-8 and IL-10) A kit which is an antibody, peptide, or aptamer that binds to one or more selected from the group.

[Claim 2 is a kit for diagnosing diseases related to NK cell activity, comprising the complex of any one of claims 1 to 13.

[Claim 21] In claim 20, the NK cell activity-related disease is selected from the group consisting of irritable immune disease, autoimmune disease, immunodeficiency disease, histocytosis, blood cancer, and solid cancer. .

[Claim 22] NK cell activity comprising the complex of any one of claims 1 to 15

Composition for detection.

[Claim 23] A method for measuring NK cell activity comprising the step of treating the complex of claim 1 in a blood sample of an individual.

[Claim 24] In claim 23,

Detecting the peptide cleaved from the complex; And

A method for measuring NK cell activity further comprising the step of determining that the activity of NK cells in the blood sample is increased when the cleaved peptide is increased than before adding the complex.

[Claim 25] In claim 24,

A method for measuring NK cell activity, comprising the step of determining that the activity of NK cells in the blood sample is increased when the fluorescence intensity is reduced than before the addition of the complex, 2020/175960 1»（：1^1{2020/002904

18

[Claim 26] In claim 24, the detection of the cleaved peptide is performed on at least one selected from the group consisting of ELISA (enzyme linked immunosorbent assay), flow cytometry (Fluorescence Activated Cell Sorter, FACS), and protein chip. A method for measuring NK cell activity, which is carried out by.

[Claim 27] The method for measuring NK cell activity according to claim 23, further comprising the step of processing a substance that detects cytokines secreted by NK cells into a blood sample of an individual.

[Claim 28] In claim ²⁷ ,

The step of detecting the cytokine secreted by the NK cells; And when the cytokine is increased than before the addition of the complex, the step of determining that the activity of the NK cells in the blood sample is increased, NK cell activity How to measure.

[Claim 29] In claim 28,

A method for measuring NK cell activity, comprising the step of determining that the activity of NK cells in the blood sample is increased when the fluorescence intensity is increased than before the addition of the complex.

[Claim 3 according to claim 29, to detect the peptide cleaved from the complex

The step and the step of detecting the cytokine secreted by the NK cells are performed at the same time, a method of measuring NK cell activity.

[Claim 31] A method of providing information on diseases related to NK cell activity comprising the step of treating the complex of claim 1 in a blood sample of an individual.

[Claim 32] In claim 31, the NK cell-related disease is selected from the group consisting of irritable immune disease, autoimmune disease, immunodeficiency disease, histocytosis, blood cancer, and solid cancer. How to provide information.

[Claim 33] A method for diagnosing diseases related to NK cell activity, comprising the step of treating the complex of claim 1 in a blood sample of an individual.