Composition for alleviating SFTS virus infection using il27 inhibitor

Abstract

The present invention discloses a composition for alleviating SFTS virus infection using an IL27 inhibitor on the basis of experimental results such as an improved survival rate of mice and a decrease in the number of virus replications when IL27 action is inhibited in mice infected with SFTS virus.

Description

Composition for improving SFTS virus infection using IL27 inhibitor

[0001] The present invention relates to a composition for improving SFTS virus infection using an IL27 inhibitor.

[0002] Severe fever with thrombocytopenia syndrome (SFTS) is an infectious disease caused by the SFTS virus (SFTSV). First reported in China in 2009, outbreaks have since been reported in Asian countries, including China, South Korea, and Japan. SFTSV is divided into six genotypes (genotypes A to F), with genotype B prevalent in South Korea and Japan, while genotypes A, B, D, and F are predominant in China, depending on the region (Scientific Reports 6(1):19563, 2016).

[0003] SFTSV has three segmented RNA genomes, L (large), M (medium), and S (small). The L genome encodes RNA-dependent RNA polymerase (RdRp), the M genome encodes two envelope glycoproteins (Gn, Gc), and the S genome encodes the nucleocapsid (Np) and nonstructural protein (Ns) (Rev Med Virol. 2014;24(2):90-102). SFTSV is a single-stranded negative-sense RNA virus, but the S genome, which encodes the nucleocapsid (Np) and nonstructural proteins (Ns), is a duplex RNA genome containing negative-sense RNA for the Np protein and positive-sense RNA for the Ns protein (Front Microbiol. 2021 Aug 16;12:709517; Experimental & Molecular Medicine volume 53, pages713-722, 2021). The Np protein forms a complex with RNA (RNP complex) to participate in virus assembly, and the Ns protein inhibits host interferon production (Virologica Sin. 32, 51-62, 2017; J. Acute Dis. 4, 277-279, 2015).

[0004] Infection with this virus can cause high fever, gastrointestinal complications, leukopenia, and thrombocytopenia, with a mortality rate of 5–20% (Lancet Infect Dis 2018, 18, 1127-1137, doi:10.1016 / S1473-3099(18)30293-7). There is currently no effective treatment, but vaccine development is ongoing.

[0005] Meanwhile, interleukin 27 (IL27), first identified in 1996 in a B cell line transformed with Epstein-Barr virus (EBV), is composed of the IL27p28 and Ebi3 (EBV-induced gene 3) subunits (Proc Natl Acad Sci USA 1997;94:12041-6; Immunity 2002;16:779-90). The IL27p28 subunit was identified using a bioinformatics approach based on structural homology with α-helical cytokines of the IL-6 family, and was later found to be coexpressed with its partner Ebi3 (Immunity 2002;16:779-90). IL27p28 and Ebi3 are not secreted as disulfide-bonded dimers, and therefore the nature of their binding in vivo remains unclear. Co-expression of Ebi3 and IL27p28 may not occur in the same cell but in different cell types for the production of biologically active IL27 cytokines.

[0006] The IL27 receptor (IL27R) consists of the gp130 protein and IL-27Rα (WSX-1 / TCCR), and the biological effects of IL27 are mediated through the activation of JAK1, JAK2, TYK2, STAT1, and STAT3 (J Immunol 2004;173:715-20). As shown in studies of Il27ra knockout mice encoding WSX-1 / TCCR, one of the major functions of IL27 is to limit the intensity and duration of T cell responses and suppress Th1, Th2, and Th17 responses by inhibiting CD28-mediated IL-2 production through SOCS3 (suppressor of cytokine signaling 3) (J Immunol 2006;176:2773-80; J Immunol 2006;176:237-47). IL27 is also known to limit autoimmune encephalomyelitis by inhibiting the development of Th17 cells and inducing the expansion of the IL-10-secreting T cell population (Nat Immunol 2007;8:1372-9; Nat Immunol 2007;8:1380-9; Nat Immunol 2006;7:929-36).

[0007] The present invention discloses that inhibition of IL27 can improve SFTSV infection.

[0008] The purpose of the present invention is to provide a composition for improving SFTS virus infection using an IL27 inhibitor.

[0009] Other or specific purposes of the present invention will be presented below.

[0010] The present invention was completed based on experimental results showing that, as confirmed in the examples below, when an IL27 inhibitory antibody is administered to an animal infected with SFTS virus, the survival rate is significantly improved and the number of viral replications is reduced.

[0011] Considering the above, the present invention can be understood as a composition for improving SFTS virus infection, which comprises an IL27 inhibitor as an active ingredient.

[0012] In the present invention, the IL27 inhibitor may be a molecule that inhibits the biological function of IL27, specifically a molecule that specifically binds to IL27 and prevents IL27 from binding to its receptor, IL27R, thereby inhibiting the function of IL27, or a molecule that specifically binds to IL27R, the receptor for IL27, thereby preventing IL27 from binding to the receptor, IL27R, thereby inhibiting the function of IL27. The molecule that specifically binds to IL27R, the receptor for IL27, may be a molecule that specifically binds to gp130 protein and inhibits the binding of IL27 to the receptor, given that IL27R is composed of gp130 protein and IL-27Rα subunit, or a molecule that specifically binds to IL-27Rα and inhibits the binding of IL27 to the receptor.

[0013] In the present invention, the active ingredient means a component that exhibits the desired activity alone or can exhibit the activity together with a pharmaceutically acceptable carrier or the like that is inactive on its own.

[0014] In addition, in the present invention, specifically binding to inhibit the function of IL27 means that the specific binding molecule forms a complex with the target protein, i.e., IL27 or its receptor, IL27R, and substantially does not form such a complex with other proteins, and also refers to a case where the function of IL27 can be inhibited by binding to a site of IL27 that binds to the receptor, IL27R (an epitope on IL27) or a site of IL27R that binds to IL27 (an epitope on IL27R). Such epitopes can be identified by epitope determination methods known and commonly used in the art, such as peptide arrays, phage display, cryo-EM (cryo-electron microscopy), X-ray crystallography, etc.

[0015] In the present invention, the IL27 inhibitor may be an antibody or an aptamer, whether it specifically binds to IL27 and inhibits its biological function, or specifically binds to IL27R, a receptor of IL27, and inhibits the biological function of IL27.

[0016] In the present invention, the antibody may be any antibody that can specifically bind to the target protein IL27 or IL27R and inhibit the function of IL27. Accordingly, in addition to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (i.e., antibodies having binding specificities for two or more antigens or two or more epitopes, such as bispecific antibodies), humanized antibodies, and human antibodies, it also includes antibody fragments, recombinant antibodies, and chemically modified antibodies that have the ability to specifically bind to the target protein IL27 or IL27R.

[0017] Humanized antibodies are antibodies in which the complementarity determining region (CDR) of human immunoglobulin is replaced with the CDR of a non-human species such as a mouse, rabbit, rat, or primate, thereby minimizing immune rejection. Methods for producing such antibodies are known in the art, and for details, see references to literature [Riechmann L. et al., Nature, 332; 323-327, 1988], literature [Nakatani T. et al., Protein Engineering, 7; 435-443, 1994], literature [Jones et al., Nature, 321:522-525 (1986)], literature [Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].

[0018] Human antibodies are antibodies obtained by introducing human immunoglobulin genes into an animal, such as a mouse, whose genes capable of producing endogenous immunoglobulins have been destroyed, and then immunizing the animal with a specific antigen. For details, see [Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993)], [Jakobovits et al., Nature, 362: 255-258 (1993)], [Bruggermann et al., Year in Immuno., 7: 33 (1993)], [Hoogenboom et al., J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Bio., 222: 581-597 (1991)].

[0019] Examples of antibody fragments include Fab, F(ab'), F(ab')2, scFv (an antibody in which Fv of a heavy or light chain is connected with an appropriate linker), Fv fragment, Fab / c (an antibody having one Fab and a complete Fc), linear antibodies (Zapata et al., Protein Eng. 8(10):1057-1062(1995)), antibody fragments obtained by treating antibodies with protein-cleaving enzymes such as papain or pepsin, and recombinant antibody fragments obtained by introducing and expressing a gene for a fragment into a host cell by genetic recombination. The globulin type of the antibody is not particularly limited as long as it specifically binds to the target protein IL27 or IL27R, and the globulin type may be any of IgG, IgM, IgA, IgE, IgY, and IgD.

[0020] Polyclonal antibodies can be produced by immunizing birds (e.g., chickens, etc.) and mammals (e.g., rabbits, goats, horses, sheep, mice, non-human primates (monkeys, chimpanzees, gorillas)) with the target protein IL27 or IL27R. Typically, 6 to 60 days after the final immunization, antibody titers are measured using enzyme-linked immunosorbent assays (EIA and ELISA), radioimmunoassay (RIA), etc., and blood is drawn when the highest antibody titer is observed. Antibodies can be purified from the blood of the immunized animal using methods known and commonly used in the art, such as ion exchange chromatography and affinity chromatography.

[0021] Monoclonal antibodies can be obtained by hybridoma cell lines that produce monoclonal antibodies specific for the target protein IL27 or IL27R. Methods for producing such hybridoma cell lines include, for example, immunizing an animal (e.g., a mouse) with the target protein IL27 or IL27R, collecting spleen cells from the immunized animal, fusing the spleen cells to a myeloma cell line to produce hybridoma cells, and isolating and culturing the hybridoma cell line that produces the desired monoclonal antibody. Monoclonal antibodies can be recovered using techniques known and commonly used in the art. Examples of such techniques known and commonly used in the art include ammonium sulfate salting-out, ion exchange chromatography, affinity chromatography, gel filtration chromatography, and the like, or combinations thereof. Specific examples of the production of monoclonal antibodies are described in the literature [Lu et al. Journal of Biomedical Science, 2020; 27:1], [Journal of Immunological Methods, 35 (1980) 1-21], [ILAR J. 2005;46(3):269-79], etc.

[0022] The production of the monoclonal antibody of the present invention can also utilize genetic recombination technology, which involves cloning the antibody gene from a hybridoma, inserting it into an appropriate vector, and introducing and expressing it in an appropriate host cell (Vandamme, AM et al., Eur. J. Biochem., 192, 767-775, 1990). Specifically, mRNA encoding the variable region (V region) of the antibody of the present invention is obtained from a hybridoma producing the antibody of the present invention. The mRNA is obtained by obtaining total RNA using a method known and commonly used in the art, such as the guanidine ultracentrifugation method (Chirgwin, JM et al., Biochemistry., Vol. 18, 5294-5299, 1979), the AGPC method (Chomczynski, P. et al., Anal. Biochem., 162, 156-159), etc., and then obtaining the desired mRNA from the total mRNA using an mRNA Purification Kit (Pharmacia). Alternatively, the desired mRNA can be obtained directly using the QuickPrep mRNA Purification Kit (Pharmacia). The cDNA of the antibody variable region is synthesized from the obtained mRNA using reverse transcriptase. If necessary, a RACE PCR method, etc. can be applied to the cDNA synthesis and amplification. The cDNA encoding the variable region thus obtained is inserted into an expression vector containing the DNA encoding the constant region (C region) of the antibody. These expression vectors may contain regulatory sequences such as a promoter, enhancer, replication origin, polyadenylation signal, and ribosome binding site. When this expression vector is transformed into a host cell, antibody production becomes possible.Expression of antibody genes may be achieved by simultaneously transforming host cells by recombining DNA encoding antibody heavy chain (H chain) or light chain (L chain) into expression vectors, or by transforming host cells by inserting DNA encoding heavy chain and light chain into a single expression vector (WO 94 / 11523).

[0023] Meanwhile, the target protein of the present invention, IL27 or IL27R, which is an immunogen used to obtain the antibody of the present invention, can be obtained by a DNA recombination technique known and commonly used in the art. Typically, the cDNA of the target protein of the present invention, IL27 or IL27R, is prepared, the cDNA is inserted into an expression vector, the expression vector is transformed into a prokaryotic or eukaryotic host cell, the transformed host cell is cultured in a suitable medium, and the cDNA is obtained from the culture medium or cells. The cDNA can be prepared within the ordinary ability of a person skilled in the art based on the gene sequence provided by a gene / protein database such as GenBank or the sequence provided in the present specification.

[0024] For more specific information regarding the production of recombinant antibodies, see Front Bioeng Biotechnol. 2022 Mar 4; 10:856049, Front Immunol. 2013 Jul 29; 4:217, Appl Microbiol Biotechnol (2016) 100:3451-3461, Curr Opin Biotechnol. 2019 December; 60: 153-158.

[0025] In the present invention, the IL27 inhibitor may be an aptamer that specifically binds to IL27 or IL27R and inhibits the function of IL27.

[0026] An aptamer, like an antibody, refers to a nucleic acid ligand that can specifically bind to a target molecule such as a target antigen. If it can specifically bind to a target molecule, the aptamer can be a single-stranded DNA aptamer or a single-stranded RNA aptamer. The production and selection methods for aptamers that can specifically bind to such target molecules are all known in the art, and in particular, SELEX technology or improved technologies such as Counter-SELEX technology for increasing specificity for target molecules (Science 263(5152):1425-1429, 1994), Toggle SELEX technology that takes nonclinical animal testing into account in the development of aptamer therapeutics (Mol Ther 4(6):567-573, 2001), and Spiegelmer technology that utilizes mirror images of the target molecule and aptamer (Chem Biol 9(3):351-359, 2002) can be used. The above SELEX technology is an abbreviation for "Systematic Evolution of Ligands by EXponential enrichment", and for the technology, reference can be made to literature Science 249 (4968):505-510, 1990, U.S. Patent No. 5,475,096, U.S. Patent No. 5,270,163, International Patent Publication No. WO 91 / 19813, etc. For specific methods for selecting aptamers or the use of appropriate reagents and materials, reference can be made to literature Methods Enzymol 267:275-301, 1996, and literature Methods Enzymol 318:193-214, 2000, etc.

[0027] Aptamers may be modified with sugars, phosphates, and / or bases to enhance their in vivo half-life. Nucleotides modified with such sugars, phosphates, and / or bases are specifically known in the art, including methods for their preparation. For example, nucleotides modified in sugars include those in which the hydroxyl group (2'-OH group) of the sugar is modified with a halogen group (especially fluorine (F)), an aliphatic group, an ether group, an amine group, or especially modified with OMe, O-alkyl, O-allyl, S-alkyl, S-allyl, or a halogen, or those in which the sugar ribose or deoxyribose itself is replaced with a sugar analogue such as α-anomeric sugars, epimeric sugars such as arabinose, xyloses, or lyxoses, pyranose sugars, or furanose sugars. Also, for example, modifications in phosphate include modifications of phosphate to P(O)S(thioate), P(S)S(dithioate), P(O)NR2(amidate), P(O)R, P(O)OR', CO or CH2(formacetal). Here, R or R' is H or substituted or unsubstituted alkyl, etc., and when modified in phosphate, the linking group becomes -O-, -N-, -S- or -C-, and adjacent nucleotides are bonded to each other through this linking group. It is well known in the art that the binding ability of an aptamer to a target molecule remains unchanged or is maintained even if the binding ability is reduced, even if one or more nucleotides are added, substituted, or deleted at the site where it binds to the target molecule or at a site other than the binding site, or even if the aptamer is chemically modified or another sequence is added to both ends (Molecules. 2020 Jan; 25(1):3; Int J Mol Sci. 2017 Aug; 18(8):1683).For specific information regarding the use of therapeutic aptamers, see [Nature Reviews Drug Discovery, 9:537-550, 2010], [ACS Appl Mater Interfaces. 2021 Mar 3;13(8):9500-9519], etc.

[0028] In the present invention, with respect to the synthesis of aptamers, techniques known in the art, such as solid-phase oligonucleotide synthesis techniques and solution-phase synthesis techniques such as triester synthesis methods, can be used. For specific details, reference can be made to literature [Nucl Acid Res 14:5399-5467, 1986], literature [Tet Lett 27:5575-5578, 1986], literature [Nucl Acid Res 4:2557, 1977], literature [Lett, 28:2449, 1978], etc.

[0029] The composition of the present invention can be manufactured into a pharmaceutical composition for preventive and therapeutic use against SFTS virus infection.

[0030] In the pharmaceutical composition of the present invention, the IL27 inhibitor of the present invention, which is the active ingredient, may be included in any appropriate amount (effective amount) depending on the disease to which it is applied, formulation, administration route, etc., as long as it can exhibit preventive and therapeutic efficacy against SFTS virus infection, etc., and a typical effective amount will be determined within the range of 0.001 wt % to 20.0 wt % based on the total weight of the composition. Here, the "effective amount" refers to the amount of the active ingredient included in the composition of the present invention, which can exhibit the intended medical and pharmacological effect, such as the effect of improving SFTS virus infection, when the composition of the present invention is administered to a mammal, preferably a human, which is the subject of application, for an administration period as recommended by a medical professional. This effective amount can be experimentally determined within the normal ability of a person skilled in the art.

[0031] The pharmaceutical composition of the present invention may be prepared as an oral or parenteral formulation according to the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, the route of administration may be any appropriate route, including the topical route, the oral route, the intravenous route, the intramuscular route, and direct absorption through mucosal tissues, and a combination of two or more routes may be used. An example of a combination of two or more routes is a case where two or more drug formulations according to the route of administration are combined, for example, one drug is administered intravenously first, and the other drug is administered topically second.

[0032] Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or formulation, and specific examples can be found in the pharmacopoeias of each country, including the “Korean Pharmacopoeia.”

[0033] When the pharmaceutical composition of the present invention is prepared as an oral dosage form, it can be prepared in the form of powder, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions, wafers, etc., using a suitable carrier and a method known in the art. Examples of suitable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol; starches such as corn starch, potato starch, and wheat starch; cellulosics such as cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyols, vegetable oils, ethanol, and glycerol. When formulating, appropriate binders, lubricants, disintegrants, colorants, diluents, etc. may be included as needed. Suitable binders include starch, magnesium aluminum silicate, starch peristalsis, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweetener, sodium alginate, polyethylene glycol, wax, etc., and lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, magnesium salts and calcium salts thereof, polyethylene glycol, etc., and disintegrants include starch, methyl cellulose, agar, bentonite, xanthan gum, starch, alginic acid, or sodium salts thereof. Other diluents include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine.

[0034] When the pharmaceutical composition of the present invention is prepared as a parenteral dosage form, it can be formulated in the form of injections, transdermal administration, nasal inhalants, and suppositories using a suitable carrier according to a method known in the art. When formulated as an injection, an aqueous isotonic solution or suspension can be used as a suitable carrier, and specifically, an isotonic solution such as PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, or 5% dextrose can be used. When formulated as a transdermal dosage form, it can be formulated in the form of an ointment, cream, lotion, gel, external solution, paste, liniment, aerosol, etc. In the case of nasal inhalation, it can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, or carbon dioxide, and in the case of formulating it as a suppository, the carrier can be witepsol, tween 61, polyethylene glycol, cacao butter, laurin butter, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene stearate, sorbitan fatty acid ester, or the like.

[0035] The pharmaceutical composition of the present invention can also be administered in the form of liposomal drug delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be prepared from various phospholipids containing cholesterol, stearylamine, or phosphatidylcholine. Liposomal drug delivery systems are well known in the art; see, for example, U.S. Patent No. 5,262,564.

[0036] With regard to the formulation and manufacture of other pharmaceutical compositions, reference may also be made to literature known in the art, such as Remington's Pharmaceutical Sciences (19th ed., 1995).

[0037] The preferred dosage of the pharmaceutical composition of the present invention may range from 0.001 mg / kg to 1,000 mg / kg per day, and preferably from 0.001 mg / kg to 1 g / kg, depending on the patient's condition, weight, sex, age, severity of the condition, and route of administration. For example, when administered orally, the dosage may range from about 0.05 mg / kg to 7,500 mg / kg per day. Administration may be administered once a day or divided into several doses. These dosages are described solely for illustrative purposes and should not be construed as limiting the scope of the present invention in any way.

[0038] As described above, according to the present invention, a composition for improving SFTS virus infection using an IL27 inhibitor can be provided.

[0039] The composition of the present invention can be used as a pharmaceutical composition for preventive and therapeutic purposes for SFTS virus infection.

[0040] Figure 1 shows the results of evaluating the effect of IL27 blockade on the severity of SFTSV infection in SFTSV-infected mice. (A) Experimental schematic; (B) Mouse survival rate (left panel) and body weight change (right panel).

[0041] Figure 2 shows the results evaluating the effects of IL27 (p28) blockade on bone marrow cell density and immune cell profiles in SFTSV-infected mice. Each circle in the graph represents an individual mouse.

[0042] (a) Quantification of bone marrow cell density in mice from each experimental group 4 days after infection (n = 8).

[0043] (b) Lymphocytes (CD45) within viable cells + CD11b - Ly6G - cell) ratio (n = 8).

[0044] (c) Flow cytometry analysis of lower lymphocyte lineage cells in the bone marrow 4 days after infection (graph shows CD45+ (representing the frequency of each cell type within a cell).

[0045] (d) Flow cytometry analysis of submyeloid cells in the bone marrow 4 days after infection (graph shows CD45 + (representing the frequency of each cell type within a cell).

[0046] Figure 3 shows the results evaluating the effect of IL27 (p28) blockade on the recovery of B lymphocyte production in SFTSV-infected mice. Each circle in the graph represents an individual mouse.

[0047] (a) Lin of bone marrow HSPCs 4 days after infection - Results quantified as cell ratio.

[0048] (b) Proportion of myeloid lineage progenitor cells in mouse bone marrow 4 days after infection.

[0049] (c) Proportion of common lymphocyte progenitors (CLPs) in mouse bone marrow 4 days after infection.

[0050] Statistical analysis was performed using the Mann-Whitney test. Each circle in the graph represents an individual mouse. HSPC; hematopoietic stem and progenitor cell, CMP; common myeloid progenitor cell, GMP; granulocyte-monocyte progenitor cell, MDP; monocyte-dendritic cell progenitor cell, MEP; megakaryocyte-erythroid progenitor cell, CLP; common lymphocyte progenitor cell

[0051] Figure 4 shows the results evaluating the effect of IL27 (p28) blockade on peripheral immune cell profiles in SFTSV-infected mice. Each circle in the graph represents an individual mouse.

[0052] (a) Quantification of spleen cell density in each experimental group 4 days after infection (n = 8).

[0053] (b) Percentage of lymphocytes within viable cells (CD45+CD11b-Ly6G- cells counted as lymphocytes).

[0054] (cd) Complete blood count (CBC) analysis results from blood samples collected from each mouse. PLT; platelets.

[0055] (e) Flow cytometry analysis of lower lymphocyte lineage cells in the spleen 4 days after infection (graph shows the frequency of each cell type within CD45+ cells).

[0056] (f) Flow cytometry analysis of lower hematopoietic lineage cells in the spleen 4 days after infection (graph shows the frequency of each cell type within CD45+ cells).

[0057] Figure 5 shows the results of evaluating the effect of IL27 (p28) blockade on B cell-mediated adaptive immune responses in SFTSV-infected mice. Each circle in the graph represents an individual mouse.

[0058] (a) Quantification of viral load in plasma 4 days after infection.

[0059] (b) Anti-NP IgG response measured by ELISA 4 days after infection.

[0060] (c) Spleen cells were collected from mice 4 days after infection. IFN-γ and TNF-α production secreted by CD4+ and CD8+ T cells were analyzed by flow cytometry after stimulation with the indicated antigens. Data are expressed as mean ± SD from duplicate experiments using four mice per group. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. pep: SFTSV-NP peptide; NP: SFTSV-NP protein; (+): stimulation with PMA + ionomycin.

[0061] The present invention will be described below with reference to examples. However, the scope of the present invention is not limited to these examples.

[0062]

[0063] <Example> Experimental study on the improvement activity of IL27-targeting antibodies against SFTS virus infection

[0064] 1. Experimental method

[0065] 1.1 Animal testing

[0066] Female C57BL / 6 mice, 8–12 weeks old, were used for infection experiments and maintained in a hospital-free environment at Seoul National University College of Medicine. They were then transferred to a level 3 animal biosafety facility at Seoul National University Hospital for infection studies. Because normal mice are not susceptible to SFTSV infection, each mouse received 500 μg of anti-mouse IFNAR1 antibody (Leinco, MO, USA) or isotype IgG1 (BioXCell, NH, USA) as a control by intraperitoneal injection 24 hours before virus inoculation. Isotype IgG1 has the same subtype as the anti-mouse IFNAR1 antibody described above and is nonreactive to IFNAR1. SFTSV infection was administered subcutaneously, and health indicators, including body weight and survival rate, were monitored daily. Blood, spleen, and bones from the femur and tibia were collected for further analysis. In a related experiment to evaluate the effect of IL27(p28) neutralization, mice were intraperitoneally injected with 400 μg of anti-mouse IL27(p28) antibody (BioXCell, NH, USA) or an isotype control IgG2a anti-mouse IL27(p28) antibody (BioXCell, NH, USA) 24 hours after virus infection.

[0067] 1.2 Isolation of mouse bone marrow cells and spleen cells

[0068] After euthanasia, tissues, including the spleen and bone marrow from the femur and tibia, were collected for analysis. To isolate splenocytes, the spleens were minced, and the cells were dissociated in RPMI 1640 medium (Thermo Fisher, DE, USA) and filtered using a 70 μm cell strainer (BD Biosciences, CA, USA). Bone marrow cells were collected by excising the femur and tibia at the joint area and flushing the bone marrow with PBS through a 25-gauge needle. The resulting cell suspension was filtered through a 70 μm filter. Cells isolated from the spleen and bone marrow were centrifuged and washed several times with PBS, and red blood cells were lysed using RBC lysis buffer (Sigma, MO, USA). To increase lysis efficiency, the cells were resuspended in 2 ml of lysis buffer and incubated at room temperature for 4 minutes. After lysis of red blood cells, cells resuspended in the medium were counted using trypan blue staining.

[0069] 1.3 Flow cytometry

[0070] Flow cytometry was performed using a CytoFLEX S system (Beckman Coulter, CA, USA), and data were analyzed using Flowjo software version 10.6 (Tree Star, OR, USA). Before antibody staining, cells were blocked with ultra-block solution containing 10% rat serum, 10% hamster serum, and 10% mouse serum (Sigma, MA, USA). For hematopoietic stem and progenitor cell analysis, cells were stained with the following antibodies: anti-mouse Lineage Cocktail Pacific Blue (containing anti-CD3, anti-Ly6G, anti-Ly6C, anti-CD11b, anti-CD45R, anti-Ter119), anti-mouse CD117 (c-Kit) BV605, anti-mouse Ly-6A / E (Sca-1) APC / Fire 750, anti-mouse CD127 (IL-7Rα) FITC, anti-mouse CD34 PE, anti-mouse CD16 / 32 APC (Biolegend, CA, USA), anti-mouse CD135 (FLT3) PE-Cy7 (BD Pharmingen, CA, USA), anti-mouse CD115 (CSF-1R) BV711 (BD Biosciences, NJ, USA). For mature immune cell analysis, cells were stained with the following antibodies: anti-mouse CD45 APC / Fire750, anti-mouse CD11b FITC, anti-mouse Ly6G BV605, anti-mouse Ly6C PerCP, anti-mouse F4 / 80 PE-Cy7, anti-mouse CD3 BV711, anti-mouse CD4 APC, anti-mouse CD8 Alexa Fluor 594 (Biolegend, CA, USA), anti-mouse CD19 PE (eBioscience, CA, USA).For B-cell lineage analysis, cells were stained with the following antibodies: anti-mouse CD45 APC / Fire750, anti-mouse CD3 BV711, anti-mouse IgM PE / Dazzle594, anti-mouse IgD PE, anti-mouse B220 APC (Biolegend, CA, USA), and anti-mouse CD138 (BD Pharmingen, CA, USA). Live and dead cells were distinguished using Zombie Aqua viability dye (Biolegend, CA, USA).

[0071] 1.4 Virus quantification

[0072] Total RNA was extracted using Trizol Reagent (Life Technologies, CA, USA) according to the manufacturer's instructions. RNA was reverse transcribed into cDNA using the HiSenScript RH (-) RT Premix kit (Intron, Seongnam, Republic of Korea). cDNA quantification was performed using the SensiFAST Probe Lo-ROX kit (Bioline) with primers targeting the SFTSV NP gene (forward: 5'-CCTTCAGGTCATGACAGCTGG-3', reverse: 5'-ACCAGGCTCTCAATCACTCCTGT-3') and a detection probe (5'-6FAM-AGCACATGTCCAAGTGGGAAGGCTCTG-BHQ1-3'). qRT-PCR was performed on a Bio-Rad CFX Connect Real-Time PCR System (Bio-Rad, CA, USA).

[0073] 1.5 Statistical Analysis

[0074] Data were analyzed using GraphPad Prism 5.01 software (GraphPad Software, CA, USA) or R (ver. 4.1.0). Statistical analysis was performed using the Mann-Whitney test or the Kruskal-Wallis test followed by the Bonferroni correction for between-group comparisons. Data are expressed as mean ± standard deviation (SD). Statistical analysis of survival was performed using the Mantel-Cox Log Rank test. A p value < 0.05 was considered statistically significant (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

[0075] 2. Experimental Results

[0076] 2.1 Neutralization of IL27(p28) Increases Survival in SFTSV-Infected Mice

[0077] To evaluate whether IL27 (p28) antibody administration improves the survival rate in SFTSV-infected mice, C57BL / 6 mice were infected with SFTSV after being administered anti-IFNAR1 antibody, and 24 hours after infection, anti-IL27 (p28) blocking antibody or isotype IgG as a control was administered, and then their body weight changes and survival rates were observed (Fig. 1a).

[0078] After infection, both IL27-blocking antibodies and isotype control antibodies gradually decreased body weight (Fig. 1c). However, while 9 out of 10 mice administered isotype control antibodies died, resulting in a survival rate of only 10%, IL27 (p28)-blocking antibodies significantly increased survival to 60% in mice administered a single dose (Fig. 1b). These results suggest that IL27 (p28) antibody administration may be useful in the treatment of SFTSV infection.

[0079] 2.2 Neutralization of IL27 (p28) contributes to the improvement of immune cell production and myeloid / lymphoid cell ratio in bone marrow in SFTSV-infected mice.

[0080] Because a significant difference in body weight loss was observed between the IL27 (p28) antibody-administered group and the isotype antibody-administered group on day 4 after infection (Fig. 1c), immune cells were analyzed at this time point.

[0081] Four days after infection, the number of bone marrow cells in IL27 (p28) antibody-treated mice tended to increase compared to the isotype antibody-treated group (p = 0.065; Fig. 2a). Considering that a decrease in bone marrow cell count is a typical feature of myeloid cell overproduction and bone marrow dysfunction (Pang WW et al., Human bone marrow hematopoietic stem cells are increased in frequency and myeloid-biased with age., PNAS., 2011), these results suggest that IL27 antibody administration may be useful for SFTSV infection by improving bone marrow function.

[0082] In addition, when comparing mature immune cells in the bone marrow, viable lymphocytes significantly increased in the IL27(p28) antibody-treated group (Fig. 2b), and CD19+ B cells also significantly increased (Fig. 2c). In contrast, T cells showed no difference between the two groups (Fig. 2c). Among bone marrow cells, monocytes and Ly6Chigh monocytes significantly decreased in mice treated with IL27(p28) antibody, whereas neutrophils maintained a level similar to that of mice treated with isotype IgG (Fig. 2d).

[0083] Among hematopoietic cells, granulocyte / monocyte progenitor cells (GMPs) significantly decreased, while common lymphoid progenitor cells (CLPs) significantly increased in the IL27 (p28) antibody treatment group (Fig. 3 b and c). These results indicate that IL27 (p28) blockade restores B lymphocyte production. There were no significant changes in hematopoietic stem cells and progenitor cells (HSPCs), common myeloid progenitor cells (CMPs), monocyte-dendritic progenitor cells (MDPs), and megakaryocyte-erythrocyte progenitor cells (MEPs) (Fig. 3 b and c).

[0084] In addition, to determine whether peripheral immune cell profiles were affected, splenic immune cells were analyzed, and similar to the results in bone marrow, CD19+ B cells in the spleen of the IL27(p28) antibody-administered group significantly increased compared to the isotype antibody-treated group (Fig. 4e), and the monocyte ratio in the IL27(p28) antibody-administered group also tended to decrease compared to the isotype antibody-administered group (P = 0.060) (Fig. 4f). The remaining cell counts, viable lymphocytes, platelets (PLT), T cells, and Ly6C high Monocytes and neutrophils remained at similar levels or showed no significant difference compared to mice treated with isotype IgG.

[0085] Taken together, these results suggest that in vivo blockade of IL27 (p28) restores B lymphocyte production and improves abnormal myelopoiesis in a SFTS mouse model.

[0086] 2.3 In vivo IL27 (p28) neutralization restores B cell-mediated adaptive immune responses against SFTSV.

[0087] To determine whether anti-IL27 (p28) antibody administration restores the adaptive immune response to SFTSV, viremia, anti-SFTSV antibody levels, and SFTSV-specific T cell responses were evaluated in the anti-IL27 (p28) and isotype antibody groups.

[0088] As expected, the number of viral copies in the plasma of the anti-IL27 (p28)-treated group was significantly reduced 4 days after infection compared to the isotype antibody-treated group (Fig. 5a), which was consistent with the improved survival rate (Fig. 1b). In addition, the anti-SFTSV NP IgG level was significantly increased in the blood of the anti-IL27 (p28) antibody-treated group (Fig. 5b), indicating a correlation with the increase in splenic CD19+ B cells in the anti-IL27 (p28) antibody-treated group. However, the T cell response, as measured by cytokine-secreting T cells in the spleen, did not show any difference between the two groups (Fig. 5c).

[0089] These results suggest that in vivo IL27 (p28) neutralization restores B cell-mediated adaptive immune responses against SFTSV.

[0090] The present invention can be used industrially as a pharmaceutical product.

Claims

1. A composition for improving SFTS virus infection, comprising an IL27 inhibitor as an active ingredient.

2. In paragraph 1, A composition characterized in that the improvement of the above SFTS virus infection is due to inhibition of SFTS virus replication.

3. In paragraph 1, The IL27 inhibitor is a molecule that inhibits the biological function of IL27, and is a composition characterized in that it is a molecule that specifically binds to IL27 and inhibits the function of IL27 by preventing IL27 from binding to its receptor, IL27R.

4. In paragraph 1, A composition characterized in that the IL27 inhibitor is a molecule that inhibits the function of IL27 by specifically binding to IL27R, a receptor of IL27, thereby preventing IL27 from binding to the receptor IL27R.

5. In paragraph 4, A composition characterized in that the molecule is a molecule that specifically binds to the gp130 protein, a subunit of IL27R, and inhibits the binding of IL27 to the receptor, or a molecule that specifically binds to IL-27Rα and inhibits the binding of IL27 to the receptor.

6. In any one of paragraphs 3 to 5, A composition characterized in that the above molecule is an antibody or an aptamer.

7. In paragraph 6, A composition characterized in that the antibody is a monoclonal antibody, a polyclonal antibody, a multispecific antibody, a humanized antibody, a human antibody, an antibody fragment, a recombinant antibody, or a chemically modified antibody.

8. In paragraph 6, A composition characterized in that the above aptamer is an RNA aptamer.

9. In paragraph 6, A composition characterized in that the above aptamer is an RNA aptamer, the aptamer is a modified aptamer, and the modified aptamer is an aptamer chemically modified at any one or more of the sugar position, phosphate position, base position, 5' terminal position, and 3' terminal position of a ribonucleotide.

10. In paragraph 9, An aptamer-based targeted combination anticancer agent, characterized in that the modified RNA aptamer is modified with one of OMe, O-alkyl, O-allyl, S-alkyl, S-allyl, and halogen at the 2'-OH group of the sugar.

11. In paragraph 1, A composition characterized in that the above composition is a pharmaceutical composition.

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