Patent Application
20220267396
The present invention is drawn to a novel IL-10 variant protein and use thereof. Particularly, the present invention is drawn to a novel IL-10 variant protein whose yield of production and immunosuppressive activity is enhanced and use thereof.
Interleukin 10 (hereinafter referred to as “IL-10”) is an anti-inflammatory cytokine expressed as a non-covalently bound homodimer of about 37 kDa called cytokine synthesis inhibitory factor (CSIF). IL-10 plays an important role in the induction and maintenance of immune tolerance, and these dominant anti-inflammatory properties have been known for a long time. IL-10 inhibits the secretion of pro-inflammatory cytokines such as TNFα, IL-1, IL-6, and IL-12 as well as Th1 cytokines such as IL-2 and INFγ, and regulate the differentiation and proliferation of phagocytes, B cells and T cells (Glocker et al., Ann. NY Acad. Sci. 1246: 102-107, 2011; Moore et al., Annu. Rev. Immunol. 19: 683-765 (2001); Waal Malefyt et al., J. Exp. Med. 174: 915-924, 1991), Williams et al., Immunol. 113: 281-292, 2004). In addition, it is a potent inhibitor of antigen presentation, inhibiting MHC class II expression as well as upregulation of costimulatory factors CD80 and CD86 (Mosser & Yhang, Immunol. Rev. 226: 205-218, 2008). Because of these characteristics, various studies have been conducted to use IL-10 as a therapeutic agent for inflammatory bowel disease or immune-related diseases such as psoriasis.
However, it is known that IL-10 has a dual characteristic that also has the opposite effect of immunostimulatory activity. In this regard, specifically, IL-10 stimulates B cell activation, prolongs the survival of B cells, and may contribute to class switching of B cells. It can also stimulate NK cell proliferation and cytokine production, and may act as a growth factor promoting the proliferation of a specific subset of CD8+ T cells (Mosser & Yhang, Immunol. Rev. 226: 205-218, 2009; Cai et al., Eur. J. Immunol. 29: 2658-2665, 1999; Santin et al., J. Virol. 74: 4729-4737, 2000; Rowbottom et al., Immunol. 160: 3188-3193, 1998). Importantly, it has been reported that high doses of IL-10 (20 and 25 μg/kg, respectively) in humans can increase the production of INFγ (Lauw et al., J. Immunol., 165: 2783-2789, 2000; Tilg et al., Gut 50: 191-195, 2002).
Accordingly, it has been reported that isoleucine, the 87th amino acid of the IL-10 protein, is involved in immune activation, and when substituted with alanine, the immune activation may be suppressed (Ding et al., J. Exp. Med. 191(2): 213-223, 2000). However, in the case of the IL-10 variant protein, it has been confirmed that binding affinity with IL-10R1 is very weak compared to the wild type, and thus, it is disadvantageous in that a high dose of administration is required for equivalent immunosuppressive activity.
On the other hand, due to the structural properties of the IL-10 protein, a large amount of insoluble aggregates are generated when produced as a recombinant protein, which causes a great problem in productivity. Accordingly, a monomeric IL-10 that does not form a dimer has been developed by introducing a linker peptide of 6 a.a. between the fourth and fifth alpha helices based on the secondary structure of IL-10 (Josephson et al., J. Biol. Chem. 275(18): 13552-13557, 2000), but it has a short half-life and very low activity in the body, which is an obstacle to its use as a therapeutic agent. In order to overcome this low stability in the body, an attempt was made to express the IL-10 as a fusion protein linked to the Fc domain of IgA (Westerhof et al., PLOS ONE 7(10): e46460, 2012). But the fusion protein showed limited recovery of IL-10 activity.
The present invention is to solve various problems including the above-described problems, and the purpose of the present invention is providing a novel IL-10 variant protein which is more effective and whose in vivo stability and safety are enhanced. However, the scope of the present invention is not limited thereto.
In an aspect of the present invention, there is provided a monomeric IL-10 variant protein in which a spacer peptide with a length of 6 to 12 a.a. are inserted between asparagine, the 116th amino acid, and lysine, the 117th amino acid, based on the mature form of the human Interleukin-10 (IL-10).
In another aspect of the present invention, there is provided a fusion protein comprising a monomeric IL-10 variant protein in which a spacer peptide with a length of 6 to 12 a.a. are inserted between asparagine, the 116th amino acid, and lysine, the 117th amino acid, based on the mature form of the human Interleukin-10 (IL-10).
In another aspect of the present invention, there is provided a polynucleotide encoding the monomeric IL-10 variant protein or the fusion protein.
In another aspect of the present invention, there is provided a recombinant vector comprising the polynucleotide.
In another aspect of the present invention, there is provided a pharmaceutical composition for immunosuppression comprising the monomeric IL-10 variant protein or the fusion protein as an active ingredient.
In another aspect of the present invention, there is provided a pharmaceutical composition for treating an immune-related disease comprising the monomeric IL-10 variant protein or the fusion protein as an active ingredient.
In another aspect of the present invention, there is provided a method for treating a subject suffering from an autoimmune disease comprising administering a therapeutically effective amount of the monomeric IL-10 variant protein or the fusion protein to the subject.
The monomeric IL-10 variant protein according to an embodiment of the present invention can be used as a novel immunosuppressant and/or therapeutic agent for treating autoimmune disease, inhibiting immune activation which is one of dual actions of IL-10 protein, improving production efficiency, as well as maintaining its activity in the form of fusion protein with other physiologically active proteins.
In an aspect of the present invention, there is provided a monomeric IL-10 variant protein in which a spacer peptide with a length of 6 to 12 a.a. are inserted between asparagine, the 116th amino acid, and lysine, the 117th amino acid, based on the mature form of the human Interleukin-10 (IL-10).
In the monomeric IL-10 variant protein, the mature form of the human Interleukin-10 may be one derived from 19th to 178th amino acid sequence described in UniProtKB P22301.
In monomeric IL-10 variant protein, the spacer peptide may have 7 to 11 a.a., 8 to 10 a,a., or 9 a.a. of length, or alternatively the space peptide may have length of 6, 7, 8, 9, 10, 11 or 12 a.a. In a particular embodiment, the spacer peptide may be a peptide having amino acid sequence of GGSGGSGGS(SEQ ID NO: 4), (GGGSGG)n (unit: SEQ ID NO: 5, n is an integer of 1 or 2), (G4S)n (unit: SEQ ID NO: 12, n is an integer of 1 or 2), (GGS)n (n is an integer of 2 to 4), (GS)n (n is an integer of 3 to 6), or (GSSGGS)n (unit: SEQ ID NO: 13, n is an integer of 1 to 2). The amino acid residues consisting of the spacer peptide may be substituted with other type of amino acids as long as they do not induce any adverse immunogenic reactions and preferably may have length of 9 a.a.
The term “spacer peptide” used herein refers to a peptide that is inserted into a specific protein and plays a role in changing the structure and/or function of the protein. In this sense, it is distinguished from linker peptides connecting other fusion partners, but conventional linker peptides can be used as spacer peptides.
The monomeric IL-10 variant protein according to an embodiment of the present invention may include an amino acid sequence of SEQ ID NO: 1.
The monomeric IL-10 variant protein may be a monomeric IL-10 variant protein in which isoleucine, the 87th amino acid, is substituted with alanine based on the mature form of the human IL-10 protein, and preferably may have an amino acid sequence of SEQ ID NO: 39.
The monomeric IL-10 variant protein may have homology of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the amino acid sequence represented by SEQ ID NOs: 1 or 39 as long as it has a monomeric structure, and it may be derived from mammals.
In another aspect of the present invention, there is provided a fusion protein comprising a monomeric IL-10 variant protein in which a spacer peptide with a length of 6 to 12 a.a. are inserted between asparagine, the 116th amino acid, and lysine, the 117th amino acid, based on the mature form of the human Interleukin-10 (IL-10).
As used herein, the term “fusion protein” refers to a recombinant protein in which two or more proteins or domains responsible for specific functions in a protein are linked such that each protein or domain is responsible for an original function thereof. A linker peptide having a flexible structure can be generally inserted between the two or more proteins or domains. The linker peptide may have any one among following amino acid sequences: AAGSGGGGGSGGGGSGGGGS (SEQ ID NO: 2), GGSGG (SEQ ID NO: 3), GGSGGSGGS (SEQ ID NO: 4), GGGSGG (SEQ ID NO: 5), (G4S)n (unit: SEQ ID NO: 12, n is an integer of 1 to 10), (GGS)n (n is an integer of 1 to 10), (GS)n (n is an integer of 1 to 10), (GSSGGS)n (unit: SEQ ID NO: 13, n is an integer of 1 to 10), KESGSVSSEQLAQFRSLD (SEQ ID NO: 14), EGKSSGSGSESKST (SEQ ID NO: 15), GSAGSAAGSGEF (SEQ ID NO: 16), (EAAAK)n (unit: SEQ ID NO: 17, n is an integer of 1 to 10), CRRRRRREAEAC (SEQ ID NO: 18), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 19), GGGGGGGG (SEQ ID NO: 20), GGGGGG (SEQ ID NO: 21), AEAAAKEAAAAKA (SEQ ID NO: 22), PAPAP (SEQ ID NO: 23), (Ala-Pro)n (n is an integer of 1 to 10), VSQTSKLTRAETVFPDV (SEQ ID NO: 24), PLGLWA (SEQ ID NO: 25), TRHRQPRGWE (SEQ ID NO: 26), AGNRVRRSVG (SEQ ID NO: 27), RRRRRRRR (SEQ ID NO: 28), GFLG (SEQ ID NO: 29), GSSGGSGSSGGSGGGDEADGSRGSQKAGVDE (SEQ ID NO: 30), GGGGSGGGGSGGGGSEPKSSDKTHTCPPCP (SEQ ID NO: 31), GGGGSGGGGSGGGGS (SEQ ID NO: 34), GGGGSGGGGSGGGGSEKEKEEQEERTHTCPPCP (SEQ ID NO: 35), RNTGRGGEEKKGSKEKEEQEERETKTPECP (SEQ ID NO: 36), GGGGSGGGGSGGGGSEPKSCDKTHTCPPCP (SEQ ID NO: 37), GSGGGSGTLVTVSSESKYGPPCPPCP (SEQ ID NO: 38), EPKSSDKTHTCPPCP (SEQ ID NO: 40), EPKSCDKTHTCPPCP (SEQ ID NO: 41), THTCPPCP (SEQ ID NO: 42), GGGGSGGGGSGGGGSAKNTTAPATTRNTTRGGEEKKKEKEKEEQEERTHTCPPCP (SEQ ID NO: 43), AGSGGGGGSGGGGSGGGGS (SEQ ID NO: 44), and GGGSGGSTHTCPPCP (SEQ ID NO: 45).
The fusion protein may include one or more fusion partner proteins that perform different functions. Such fusion partner proteins may be an antibody specifically binding to a target protein, an antigen-binding fragment of the antibody, an antibody mimetic specifically binding to the target protein, an antibody Fc region, an antibody Fc region receptor, or an extracellular domain of the antibody Fc region receptor, a dimerization domain, a cytokine or an immunomodulatory peptide.
In the fusion protein, the antigen-binding fragment of the antibody may be Fab, F(ab′)2, Fab′, scFv, diabody, triabody, sdAb (single domain antibody), VNAR or VHH, and the antibody mimetic may be affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, DARPin, Fynomer, Kunitz domain peptide, monobody, repebody, VLR, or nanoCLAMP. The Fc region may an Fc region of IgG, IgA, IgD, IgE, IgM or the Fc region may be a hybrid Fc (hyFc) in which two or more domains (hinge, CH2, and CH3 domain) of Fc regions of the above-described Ig subclasses are mixed, and the IgG may be IgG1, IgG2, IgG3, or IgG4. More specifically, the Fc region may be a variant Fc region whose functional parts (effectors) responsible for antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) are mutated in order to lower affinity for Fc gamma receptor (FcγRc) and a complement (C1q) and/or a variant Fc region engineered to improve selective affinity to neonatal Fc receptor (FcRn) having elevated blood half-life thereby. Among these, the hybrid Fc may be those described in Korean Patent Nos. 897938, 1380732, and 1380729, etc., or the variant Fc region may be a modified immunoglobulin Fc protein (NTIG) described in International Patent Application PCT/KR2020/006346. More specifically, the Fc region may contain an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 to 9. The term ‘NTIG’ used herein refers to a modified Fc domain protein in which 18th and 196th amino acids of the hybrid Fc protein including an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 7 are mutated to other amino acids, that lacks effector functions such as ADCC and CDC, but has improved blood half-life by increasing selective affinity for FcRn. The NTIG may contain an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9.
The dimerization domain may be a hinge domain of antibody, LIM/double zinc-finger motif, RAG1 domain, HAT dimerization domain, TRFH dimerization domain, Stat3 dimerization domain, or LFB1/HNF1 dimerization domain, but not limited thereto. The cytokine may be IL-4, IL-6, IL-1α or TGF-β. The immunomodulatory peptide may be PD-1L or CTLA-4 (CD152). In particular, if an Fc domain is a fusion partner protein, a dimer may be formed by intermolecular disulfide bonds generated between cysteine groups present in the hinge region. The fusion partner protein may be linked to either the N-terminus or the C-terminus of the monomeric IL-10 variant protein according to an embodiment of the present invention. In addition, in this case, the fusion partner protein may be linked to the monomeric IL-10 variant protein through various linker peptides described above.
As used herein, the term “antibody” refers to an immunoglobulin molecule which is a hetero-tetrameric protein produced by binding two identical heavy chains and two identical light chains, and performs antigen-specific binding through an antigen-binding site composed of a variable region (VL) of the light chain and a variable region (VH) of the heavy chain, thereby causing an antigen-specific humoral immune response.
As used herein, the term “antigen-binding fragment of an antibody” refers to a fragment which has antigen-binding ability derived from an antibody and includes both a fragment produced by cleaving an antibody with a protein cleaving enzyme as well as a single-chain fragment produced in a recombinant manner, and examples thereof include Fab, F(ab′)2, scFv, diabody, triabody, sdAb, and VHH.
As used herein, the term “Fab” refers to an antigen-binding antibody fragment (fragment antigen-binding) which is produced by cleaving an antibody molecule with a proteolytic enzyme, papain, is a heterodimer of two peptides of VH-CH1 and VL-CL, and the other fragment produced by the papain is referred to as Fc (fragment crystallizable).
As used herein, the term “F(ab′)2” refers to a fragment that includes an antigen-binding site among fragments produced by cleaving an antibody with pepsin, which is a proteinase, and is in a form of a tetramer in which the two Fab's are linked by a disulfide bond. The other fragment produced by the pepsin is referred to as pFc′.
As used herein, the term “Fab” refers to a molecule having a similar structure to that of Fab produced by separating the abovementioned F(ab′)2 under weak reducing conditions.
As used herein, the term “scFv” is an abbreviation for a “single chain variable fragment”, and refers to a fragment which is not a fragment of an actual antibody, but is a kind of fusion protein prepared by linking the heavy-chain variable region (VH) to the light-chain variable region (VL) of the antibody through a linker peptide having a size of about 25 a.a., and is known to have antigen-binding ability even though the fragment is not a unique antibody fragment (Glockshuber et al., Biochem. 29(6): 1362-1367, 1990).
As used herein, the terms “diabody” and “triabody” refer to antibody fragments in a form of two and three scFv's linked by a linker, respectively.
As used herein, the term “single domain antibody (sdAb)” refers to an antibody fragment which is also referred to as a nanobody and consists of a single variable region fragment of an antibody. The sdAb derived from the heavy chain is mainly used, but a single variable region fragment derived from the light chain is also reported to specifically bind to an antigen. VNAR composed of variable region fragments of a shark antibody and VHH composed of variable region fragments of a camelid antibody, which consist only of dimers of single chains unlike conventional antibodies composed of a heavy chain and a light chain, are also included in sdAb.
As used herein, the term “antibody mimetic” or alternatively “antibody analog” is a concept including a protein having similar functions to those of antibodies prepared from non-antibody-derived protein scaffolds such as a monobody and a variable lymphocyte receptor (VLR), that is, having antigen-binding ability, unlike a normal full-length antibody in which two heavy chains and two light chains form a quaternary structure of a hetero-tetramer to exhibit functions. Examples of such an antibody mimetic include Affibody derived from a Z domain of protein A (Nygren, P. A., FEBS J. 275(11): 2668 to 2676, 2008), Affilin derived from Gamma-B crystallin or Ubiquitin (Ebersbach et al., J. Mol. Biol. 372(1): 172-185, 2007), Affimer derived from Cystatin (Johnson et al., Anal. Chem. 84(15): 6553-6560, 2012), Affitin derived from Sac7d (Krehenbrink et al., J. Mol. Biol. 383 (5): 1058-1068, 2008), Alphabody derived from a triple helix coiled coil protein (Desmet et al., Nat. Commun. 5: 5237, 2014), Anticalin derived from lipocalin (Skerra et al., FEBS J. 275(11): 2677-2683, 2008), Avimer derived from domains of various membrane receptors (Silverman et al., Nat. Biotechnol. 23(12): 1556-1561, 2005), DARPin derived from Ankyrin repeat motif (Stumpp et al., Drug Discov. Today. 13(15-16): 695-701, 2008), Fynomer derived from a SH3 domain of a Fyn protein (Grabulovski et al., J. Biol. Chem. 282(5): 3196-3204, 2007), Kunitz domain peptides derived from Kunitz domains of various protein inhibitors (Nixon and Wood, Curr. Opin. Drug Discov. Dev. 9(2): 261-268, 2006), a monobody derived from the 10th type 3 domain of fibronectin (Koide and Koide, Methods Mol. Biol. 352: 95-109, 2007), nanoCLAMP derived from carbohydrate-binding module 32-2 (Suderman et al., Protein Exp. Purif. 134: 114-124, 2017), a variable lymphocyte receptor (VLR) derived from a hagfish (Boehm et al., Ann. Rev. Immunol. 30: 203-220, 2012), and a repebody engineered to enhance antigen affinity based on the VLR (Lee et al., Proc. Natl. Acad. Sci. USA, 109: 3299-3304, 2012).
In the fusion protein, the antibody Fc region receptor may be an extracellular domain of alpha subunit of IgE Fc receptor.
In another aspect of the present invention, there is provided a polynucleotide encoding the monomeric IL-10 variant protein or the fusion protein.
In another aspect of the present invention, there is provided a recombinant vector comprising the polynucleotide.
In the recombinant vector, the polynucleotide may be contained in a form of a gene construct operably linked to a regulatory sequence.
As used herein, the term “operably linked to” means that a target nucleic acid sequence (for example, in vitro transcription/translation system or in a host cell) is linked to the regulatory sequence in such a way that the target nucleic acid sequence can be expressed.
As used herein, the term “regulatory sequence” is meant to include a promoter, an enhancer, and other regulatory elements (for example, polyadenylation signal). Examples of the regulatory sequence include a sequence which directs such that a target nucleic acid is constantly expressed in many host cells, a sequence (for example, a tissue-specific regulatory sequence) which directs such that a target nucleic acid is expressed only in a specific tissue cell, and a sequence (for example, an inducible regulatory sequence) which directs such that expression is induced by a specific signal. Those skilled in the art could understand that the design of an expression vector may vary depending on factors such as the selection of a host cell to be transformed and the desired level of protein expression. The expression vector of the present invention can be introduced into a host cell to express the fusion protein. Regulatory sequences which enable expression in the eukaryotic cell and the prokaryotic cell are well known to those skilled in the art. As described above, these regulatory sequences generally include regulatory sequences responsible for transcription initiation, and optionally, a poly-A signal responsible for transcription termination and stabilization of a transcript. Additional regulatory sequences may include a translation enhancing factor and/or a naturally-combined or heterologous promoter region, in addition to the transcription regulatory factor. For example, possible regulatory sequences which enable expression in a mammalian host cell include a CMV-HSV thymidine kinase promoter, SV40, an RSV (Rous sarcoma virus)-promoter, a human kidney urea 1α-promoter, a glucocorticoid-inducing MMTV (Moloney mouse tumor virus)-promoter, a metallothionein- or tetracycline-inducible promoter, or an amplifying agent such as a CMV amplifying agent and an SV40 amplifying agent. It is considered that for expression in a nerve cell, a neurofilament-promoter, a PGDF-promoter, an NSE-promoter, a PrP-promoter, or a thy-1-promoter can be used. The abovementioned promoters are known in the art, and are described in the literature (Charron, J. Biol. Chem. 270: 25739 to 25745, 1995). For the expression in the prokaryotic cell, a number of promoters, including a lac-promoter, a tac-promoter, or a trp promoter, have been disclosed. In addition to the factors capable of initiating transcription, the regulatory sequences may include a transcription termination signal, such as an SV40-poly-A site and a TK-poly-A site, on the downstream of the polynucleotide according to one exemplary embodiment of the present invention. In the present specification, suitable expression vectors are known in the art, and examples thereof include Okayama-Berg cDNA expression vector pcDV1 (Parmacia), pRc/CMV, pcDNA1, pcDNA3 (Invitrogen), pSPORT1 (GIBCO BRL), pGX-27 (Korean Patent No. 1442254), pX (Pagano et al., Science 255: 1144-1147, 1992), a yeast two-hybrid vector such as pEG202 and dpJG4-5 (Gyuris et al., Cell 75: 791-803, 1995), and a prokaryotic expression vector such as lambda ga 1 and pGEX (Amersham Pharmacia). The vector may further include a polynucleotide encoding a secretion signal, in addition to the nucleic acid molecules of the present invention. The secretion signals are well known to those skilled in the art. Moreover, depending on the used expression system, a leader sequence which can lead the fusion protein according to one exemplary embodiment of the present invention to a cellular compartment is combined with a coding sequence of the polynucleotide according to one exemplary embodiment of the present invention, and is preferably a leader sequence capable of directly secreting a decoded protein or the protein thereof into a pericytoplasmic or extracellular medium.
In addition, the vector of the present invention can be prepared, for example, by a standard recombinant DNA technique, and examples of the standard recombinant DNA technique include ligation of a smooth terminus and an adhesion terminus, a restriction enzyme treatment to provide a proper terminus, removal of a phosphate group by an alkaline phosphatase treatment to prevent inappropriate binding, and enzymatic linkage by T4 DNA ligase. The vector of the present invention can be prepared by recombining DNA encoding a signal peptide obtained by chemical synthesis or a genetic recombination technique, the immunoglobulin Fc domain variant protein according to one exemplary embodiment of the present invention, or DNA encoding a fusion protein containing the same with a vector containing an appropriate regulatory sequence. The vector containing a regulatory sequence can be commercially purchased or prepared, and in one exemplary embodiment of the present invention, a pBispecific backbone vector (Genexine, Inc., Korea), a pAD15 vector, pGP30 (Genexine, Inc. Korea), or a pN293F vector (Y-Biologics, Inc., Korea) was used as a backbone vector.
The expression vector may further include a polynucleotide encoding a secretion signal sequence, and the secretion signal sequence induces the extracellular secretion of the recombinant protein expressed in the cell, and may be a tissue plasminogen activator (tPA) signal sequence, a herpes simplex virus glycoprotein Ds (HSV gDs) signal sequence, or a growth hormone signal sequence.
The expression vector according to one exemplary embodiment of the present invention may be an expression vector capable of expressing the protein in a host cell, and the expression vector may be in any form such as a plasmid vector, a viral vector, a cosmid vector, a phagemid vector, or an artificial human chromosome.
In another aspect of the present invention, there is provided a pharmaceutical composition for immunosuppression comprising the monomeric IL-10 variant protein or the fusion protein as an active ingredient.
The pharmaceutical composition may further contain a known immunosuppressant component (a cytokine with immunosuppressive effects, a Decoy receptor, a ligand involved in the activation and differentiation of immune cells and an antibody against the ligand, and an antibody capable of inhibiting immune cell activity, etc.). These known immunosuppressants include a glucocorticoid, a cytostatic agent, an anti-CD20 antibody, an anti-CD3 antibody, an anti-IL-2 antibody, an immunophilin inhibitor, interferon (3, opioid, TNFα binding protein, mycophenolate, fingolimod or myriocin. The glucocorticoid may be prednisone, dexamethasone, or hydrocortisone. The cytostatic agent may be nitrogen mustard, nitrosourea, platinum coordination complex, folic acid analogue, azathioprine, mercaptopurine, fluorouracil, methotrexate, dactinomycin, anthracycline, mitomycin C, bleomycin or mithramycin. The immunophilin inhibitor may be cyclosporin, tacrolimus, sirolimus, or everolimus.
According to another aspect of the present invention, there is provided a pharmaceutical composition for the treatment of an immune-related disease comprising the monomeric IL-10 variant protein or fusion protein as an active ingredient.
In the pharmaceutical composition, the immune-related disease may be type 1 diabetes, alopecia areata, anti-phospholipid antibody syndrome, rheumatoid arthritis, psoriasis or psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease, Addison's disease, Graves' disease, Sjögren's syndrome, Guillain-Barre syndrome, Hashimoto's thyroiditis, Myasthenia gravis, inflammatory myopathy, autoimmune vasculitis, autoimmune hepatitis, hemorrhagic anemia, idiopathic thrombocytopenic purpura, primary biliary cirrhosis, scleroderma, vitiligo, pernicious anemia, allergic disease or chronic celiac disease.
The composition may contain a pharmaceutically acceptable carrier, and may further include a pharmaceutically acceptable adjuvant, excipient, or diluent in addition to the carrier.
As used herein, the term “pharmaceutically acceptable” refers to a composition which is physiologically acceptable and generally does not cause an allergic reaction, such as a gastrointestinal disorder and dizziness, or a similar reaction when administered to a human Examples of the carrier, the excipient, and the diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Moreover, a filler, an anti-aggregation agent, a lubricant, a wetting agent, a fragrance, an emulsifier, and an antiseptic agent may be further contained.
Furthermore, when the pharmaceutical composition according to one exemplary embodiment of the present invention is administered to a mammal, the pharmaceutical composition can be formulated using methods known in the art to allow rapid, sustained, or delayed release of the active ingredient. Examples of the formulation include powder, a granule, a tablet, an emulsion, a syrup, an aerosol, a soft or hard gelatin capsule, a sterile injectable solution, and a sterile powder form.
The composition according to one exemplary embodiment of the present invention may be administered by various routes such as oral administration and parenteral administration, for example, suppository, transdermal, intravenous, intraperitoneal, intramuscular, intralesional, nasal, or intravertebral administration, and may be administered using an implantation device for sustained release or continuous or repeated release. The administration may be performed once or several times a day within a desired range, and can be performed at an interval such as once a week, twice a week, and once a month, and the duration of administration is also not particularly limited.
The composition according to one exemplary embodiment of the present invention may be formulated in a suitable formulation with a conventional pharmaceutically acceptable carrier. Examples of the pharmaceutically acceptable carrier include carriers for parenteral administration such as water, an appropriate oil, a saline solution, aqueous glucose, and glycol, and a stabilizer and a preservative may be included additionally. Examples of the stabilizer include antioxidants such as sodium hydrogen sulfite, sodium sulfite, or ascorbic acid. Examples of the suitable preservative include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Moreover, the composition according to an embodiment of the present invention may contain a suspending agent, a solubilizer, a stabilizer, an integerotonic agent, a preservative, an adsorption inhibitor, a surfactant, a diluent, an excipient, a pH adjuster, a painless agent, a buffer agent, an antioxidant, or the like if necessary, depending on the administration method or the formulation. The pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in the literature [Remington's Pharmaceutical Sciences, latest edition].
The dosage of the composition to a patient depends on many factors including a height of the patient, a body surface area, an age, a specific compound administered, a gender, a time and a route of administration, general health, and other drugs administered simultaneously. A pharmaceutically active protein can be administered in an amount of 100 ng/body weight (kg) to 10 mg/body weight (kg), more preferably 1 to 500 μg/kg (body weight), and most preferably 5 to 50 μg/kg (body weight), but the dosage can be adjusted in consideration of the abovementioned factors.
In another aspect of the present invention, there is provided a method of suppressing immune response in a subject in need of immunosuppression comprising administering a therapeutically effective amount of the monomeric IL-10 variant protein or the fusion protein to the subject.
The subject may be human or mammal except human, and maybe a patient who has received an organ transplant or a patient with immune-related disease that requires immunosuppression.
In addition, the monomeric IL-10 variant protein or fusion protein of the present invention can be administered in a therapeutically effective amount.
As used herein, the term “therapeutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type and severity of the subject, Age, sex, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including concurrent drugs and other factors well known in the medical field. The therapeutically effective amount of the composition of the present invention may be 0.1 mg/kg to 1 g/kg, more preferably 1 mg/kg to 500 mg/kg, but the effective dosage may be adjusted appropriately according to the patient's age, sex and condition.
Hereinafter, the present invention will be described in more detail through Examples and Experimental Examples. However, the present invention is not limited to Examples and Experimental Examples described below, and may be implemented in various other forms, and Examples and Experimental Examples described below are provided to enable the disclosure of the present invention to be complete and to fully convey the scope of the invention to those skilled in the art to which the present invention belongs.
The present inventors have designed various monomeric human IL-10 variants. Specifically, the structure of the IL-10 protein was analyzed, and the minimum linker length was devised to allow the N-terminus and C-terminus of the IL-10 of a single molecule to form a pair, considering that the N-terminus of an IL-10 molecule were paired to the C-terminus of another IL-10 molecule and thus they form a dimer through the pairing. It was expected that a linear distance of 17.3 Å was required to bind the N-terminus and C-terminus of a single IL-10 molecule, requiring a linker length of at least 7 a.a. between the N-terminus and C-terminus. In this case, since the N-terminus part and the C-terminus part within a single molecule should be combined, it is expected that the linker that is too long can act as an obstacle to the binding between the both terminus so that 9 a.a. is configured as an optimal linker length and may be designed to include a spacer peptide of up to length of 12 a.a.
In particular, the present inventors determined a human IL-10 variant protein sequence having a configuration shown in Table 1 below and prepared vector constructs comprising polynucleotides encoding various IL-10 variant proteins, respectively, by reacting an NTIG Fc sub-vector, IL-10Vm sub-vector, and a backbone vector (pBispecific Backbone; Genexine, Inc.) in a single tube using Type II restriction enzyme, BsaI and T4 ligase.
As described in the above Table 1, in one embodiment of this invention, an Fc region was linked to the N-terminus of the IL-10 protein, a dimer was formed thereby and the IL-10 variant protein was devised in a form that could facilitate the separation and purification step of the fusion protein. The Fc protein used herein is a modified Fc region (SEQ ID NOs: 8 and 9), consisting of the hinge of IgG1, and CH2 and CH3 which is a hybrid proteins of IgD and IgG4, and the same one described in international patent application PCT/KR2020/006346 was used. For convenience the modified Fc region was referred to as “NTIG”. The PCT application is incorporated herein by reference. It was designed to insert a linker peptide consisting of an amino acid sequence of SEQ ID NOs: 2 or 3 between the modified Fc region and the IL-10 variant protein according to an embodiment of the present invention. The IL-10 protein used in the Comparative Example 1 is a variant protein (SEQ ID NO: 10) that suppresses immune activation by substituting isoleucine, the 87th amino acid of the wild-type human IL-10 protein, with alanine, and the Examples 2 and 3 also include the same substitution of the 87th amino acid. Meanwhile, the IL-10 proteins (SEQ ID NOs: 1 and 11) used in the Examples 1 and 2 and Comparative Example 2 have spacer peptides inserted between asparagine (N), which is the 116th amino acid, and lysine (K), which is the 117th amino acid, and in particular, spacer peptides consisting of amino acid represented by SEQ ID NOs: 4 or 5. In particular, the IL-10 protein (SEQ ID NO: 11) comprising a linker peptide having amino acid sequence represented by SEQ ID NO: 5 according to the Comparative Example 2 contains the same monomeric IL-10 protein that Joshepson et al. devised (Josephson et al., J. Biol. Chem. 275(18): 13552-13557, 2000). Polynucleotides encoding the components of each fusion protein devised as described above were prepared by PCR amplification and oligonucleotide synthesis, and then prepared a final vector construct by reacting reaction mixture in a single tube after subcloning them into NTIG sub-vector and IL-10Vm sub-vector and a backbone vector (pBispecific vector, Genexine, Inc., Korea) using BsaI restriction enzyme and T4 ligase. The vector constructs prepared as described above were temporarily expressed using an ExpiCHO kit manufactured by Thermo Fisher. Specifically, after mixing the above-prepared vector constructs and the ExpiFectamine reagent contained in the kit with the ExpiCHO-S cell, incubation was performed for 1 day in an incubator with conditions of 8% CO2 and 37° C., and the temperature was lowered to 32° C. to 7 days.
And then, protein A capture purification was performed, and it was confirmed whether the candidate materials were purified through SDS-PAGE analysis under non-reduction and reduction conditions, and formulation was performed with a formulation buffer considering the pI value of the candidate material. The candidate materials whose formulation was completed were quantified using Nano Drop, and the final purity was determined using SEC-HPLC.
As a result, as shown in Table 2 and
3-1: Transient Expression
The present inventors devised a fusion protein in which FcεRIα, a receptor specifically binding to IgE, and the monomeric IL-10 variant proteins (IL-10Vm) of the Example 1 and Comparative Example 2 are linked to Fc proteins.
Specifically, the present inventors prepared polynucleotides encoding fusion proteins having configurations shown in the below Table 3 using oligonucleotide synthesis and PCR amplification, and prepared a final vector construct by reacting reaction mixture in a single tube after subcloning them into FcεRIα sub-vector, NTIG Fc sub-vector, IL-10Vm sub-vector and a backbone vector in the same manner as in the Example 1.
The present inventors designated the fusion protein, FcεRIα-Fc-IL-10Vm of the Example 3 as “PG075-8”, and FcεRIα-Fc-IL-10Vm of the Comparative Example 3 as “PG075-9”. The vector constructs prepared as described above were transiently expressed using ExpiCHO kit manufactured by Thermo Fisher. Specifically, after mixing the above-prepared vector construct and the ExpiFectamine reagent contained in the kit with ExpiCHO-S cells in the kit, the mixture was incubated at 8% CO2 and 37° C. for 1 day. Thereafter, the temperature was lowered to 32° C. and cultured on a 250 mL scale until the 7th day.
And then, protein A capture purification was performed, and it was confirmed whether the candidate materials were purified through SDS-PAGE analysis under non-reduction and reduction conditions. And then, formulation was performed with a formulation buffer considering the pI value of the candidate materials. The formulated candidate materials were quantified using Nano Drop, and the final purity was confirmed using SEC-HPLC.
As shown in
3-2: Establishment and Production of Stable Cell Lines
Accordingly, the present inventors inserted the gene construction encoding the fusion protein constructed in the Example 3-1 into a pAD15 expression vector (WO2015/009052A) and then transfected it using a Neon-transfection system (CHODG44) cell. As the first screening process, HT screening was performed using 10% dFBS (Gibco, USA, 30067-334), MEMα (Gibco, 12561, USA, Cat No. 12561-049), and HT+ (Gibco, USA, 11067-030) medium without HT (5-hydroxypypamine) Subsequently, methotrexate (MTX) amplification was performed using HT-selected clones in order to amplify the expression gene using a DHFR (dihydrofolate reductase) system. MTX amplification was performed in the form of a mini-pool on the plate to isolate high productivity clones, and after screening for one species whose amplification was confirmed, limiting dilution cloning (LDC) (96 wells, 30 plates) was performed to isolate the final cell line.
The final isolated cell line was incubated 700 ml in the hyCellCHO medium, and the purified proteins were identified by performing Protein A purification on the culture medium obtained on the 5th day of cultivation, and yield and purity of the purified proteins were analyzed through SDS-PAGE and SEC-HPLC.
Consequently, as confirmed in
The present inventors analyzed the mixed lymphocyte reaction using whole blood provided from a plurality of donors in order to confirm the immunosuppressive activity of the fusion protein prepared in the examples.
Specifically, the present inventors separated peripheral blood mononuclear cells (PBMC) by centrifuging the whole blood provided from 10 donors by Ficoll gradient centrifugation. After counting the number of separated monocytes, the cells were mixed, and cell stocks were prepared with 5×106 cells per vial (1 mL).
Then, the cells received from the donors and cells of the recipients were thawed, the number of cells was counted, and then resuspended at a concentration of 1×106 cells/ml. After separating the cells of recipients into reactive cells and stimulating cells, the recipient's stimulating cells and donor cells were stimulated by irradiating 3,000 rad gamma rays. Then, the recipient's reactive cells were stained with cell trace violet (V450) and the recipient's stimulating cells and donor cells were stained with cell trace red (APC). 1×105 of reactive cells from the donors and 1×105 of reactive cells from the recipients were added to 200 μl of RPMI medium supplemented with 10% FBS and mixed. The mixed cells were cultured for 6 days at 37° C. and 5% CO2. The fusion proteins of the Comparative Examples 1 and 2 and Examples 1 and 2 were treated at a concentration of 0.5 μM, respectively. After completion of the culture, FACS analysis was performed. Antibodies used in FACS analysis are BV650-conjugated anti-CD3 antibodies, PE (phycoerythrin)-conjugated anti-PD1-antibodies, PE-TR-conjugated anti-CD-14 antibodies, PE-TR-conjugated anti-CD19 antibodies, PerCp-conjugated anti-CD4 antibodies, Cy5.5-conjugated CD4 antibodies, APC (allophycocyanine)-conjugated anti-CD8 antibodies, H7-conjugated anti-CD8 antibodies.
As a result of the FACS analysis, when reactive cells from the recipients and stimulating cells from the recipients were reacted (autologous, abbreviated as ‘Auto’ hereinafter), no proliferation of CD4+ T cells and CD8+ T cells in the recipients was found, whereas when the reactive cells from the recipients and stimulating cells from the donors were reacted (allogenic, abbreviated as “Allo”) proliferation of CD4+ T cells and CD8+ T cells in the recipients was confirmed. As shown in
The present inventors have attempted to investigate the immune-stimulatory activity of the monomeric IL-10 fusion protein of the present invention.
In particular, bone marrow-derived mast cells (BMMC) of 5×103 cells/100 μl/well were seeded in a 96-well plate containing an RPMI medium containing 10% FBS, 1% antibiotics, rmSCF 20 ng/ml and rmIL-3 10 ng/ml, and treated the cells with the recombinant human IL-10 and IL-10 fusion protein according to the Comparative Examples 1 and 2 and Examples 1 and 2 after diluting them at an appropriate concentration. Subsequently, 20 μl of MTS reagents were dispensed into 96 well plates to measure the degree of proliferation of mast cells, and then the 96 well plates were put into a CO2 incubator at a temperature of 37° C. and reacted for 2 hours, and then absorbance was measured at 595 nm using a microplate reader.
As a result, as shown in
As a result, as shown in Table 5 and
Following the analysis of mast cell proliferation, the effect of IL-10 fusion protein on the reduction of TNF-α-secretion activity in mast cells, i.e., immunosuppressive activity, was investigated.
3-1: Analysis of TNF-α-Secretion Inhibitory Activity in Mast Cells
To this end, specifically, bone marrow-derived mast cells (BMMC) of 1×104 cells/50 μl/well were seeded in a 96-well plate containing an RPMI medium containing 10% FBS, 1% antibiotics, rmSCF 20 ng/ml and rmIL-3 10 ng/ml, and treated the cells with the recombinant human IL-10 and IL-10 fusion protein according to the Comparative Examples 1 and 2 and Examples 1 and 2 were diluted to appropriate concentration. And then, anti-DNP IgE diluted to 3 μg/mL and 50 μl was added to a 96-well plate and incubated for 24 hours at 37° C. and 5% CO2 conditions. Subsequently, 50 μl of DNP-BSA (Antigen) diluted to 400 ng/mL was added to 96 well plates, and then reacted overnight at 37° C. and 5% CO2 conditions. Then, centrifugation was performed at 4° C. at a speed of 1,500 rpm for 5 minutes, the supernatant 150 μl recovered was dispensed in a new 96-well plate, and the concentration of TNF-α was measured using a TNF-α ELISA kit (Biolegend, USA).
As a result, in Comparative Example 1 (IL-10M), concentration-dependent TNF-α inhibitory activity was shown, as shown in Tables 6 and 7 and
In particular, it was confirmed that the IL-10 variant protein according to an embodiment of the present invention exhibited concentration-dependent TNF-α secretion inhibitory activity compared to the IL-10 variant protein of the Comparative Example 2, which is similarly expressed in a monomeric form, and it had a significant inhibitory activity of allergy reaction when it is used as a fusion protein in which a FcεR1α is linked thereto (Example 3).
3-2: Analysis of LPS-Induced TNF-α Secretion Inhibitory Activity in Macrophages
The inventors investigated whether TNF-α secretion inhibitory activity of PG075-8 protein according to an embodiment of the present invention in macrophages which are immune cells closely related to TNF-α-mediated inflammatory reaction, confirming that TNF-α secretion inhibitory activity of PG075-8 protein according to an embodiment of the present invention in mast cells.
To this end, 100 μL of RAW264.7 cells prepared at a concentration of 5×105 cells/mL were dispensed into 96 well plates, and then cultured for 12 hours in a 5% CO2 and 37° C. incubator. RAW264.7 cells in culture were treated with the PG075-8 protein according to an embodiment of the present invention after diluting sequentially the protein at an appropriate concentration. In this case, TNF-α expression was induced by treating 100 μL of LPS (400 ng/mL) on 96 well plates. Then, 150 μL of supernatant was separated after 12 hours of incubation at 37° C. and 5% CO2 incubator, and the amount of TNF-α expression level contained in the supernatant was measured in the same manner as in the Experimental Example 3-1 (Table 8 and
Consequently, as shown in Table 8 and
Based on the experimental results of the Experimental Examples 1 to 3, the present inventors analyzed binding affinity of the fusion proteins of the Comparative Example 1 and Example 1 of the present invention to IL-10 receptor 1 (IL-10R1) through bio-layer interferometry (BLI) analysis. At this time, as controls, a fusion protein in which FcεRIα is not connected and a dimeric IL-10 variant protein (IL-10V) having amino acid sequence represented by SEQ ID NO: 10 whose immuno-stimulating activity is inhibited by substituting isoleucine, 87th amino acid of wild type IL-10 protein, with alanine is linked to a hybrid Fc region, and a fusion protein (NTIG-IL-10Vm) in which a monomeric IL-10 variant protein having an inserted spacer peptide (GGSGGSGGS) between the 116th amino acid, asparagine (N) and the 117th amino acid, lysine (K) were used. The NTIG-IL-10Vm is the same as PG075-8 according to an embodiment of the present invention, except that FcεRIα is not connected.
To this end, first of all, these inventors attached IL-10R His-tag protein to a 96-well plate using the Dip and Read™ Amine Reactive 2nd Generation (AR2G) Reagent Kit (forteBio, Cat No. 18-5092). Specifically, after dispensing 200 μl of D.W. in a 96-well plate, an amine biosensor included in the kit was inserted and hydrated for 10 minutes. Subsequently, after dispensing 200 μl of D.W. additionally to the plate, EDC:NHS was mixed in a 1:1 ratio in a volume corresponding to 1/20 of the required sample, then diluted in D.W. and 200 μl was dispensed into the 96-well plate. Subsequently, IL-10R His-tag protein was diluted to 10 μg/ml in 10 mM acetic acid solution (pH 5.0), and 200 μl was dispensed into the 96-well plate. Then, 200 μl of 1 M ethanolamine was added to the 96-well plate, and the biosensor plate and the sample plate were inserted into the Octet® K2 BLI analyzer and signals were measured. After the measurement was completed, 200 μl of the 1× Kinetics buffer was added to the sample plate, and then a baseline was determined. Subsequently, the NTIG-IL-10 fusion protein prepared in the above Example was diluted in a 1× Kinetic buffer solution at various concentrations (0, 62.5, 125, 250, 500, and 1,000 nM), then dispensed to the sample plate at 200 μl, and then BLI analysis was performed to measure binding affinity using Octet® K2 BLI analyzer.
As a result, as shown in
The present inventors analyzed binding affinity of the fusion protein (FcεRIα-Fc-IL-10Vm) prepared in Example 3 to mouse IgE and human IgE through biolayer interferometry (BLI) analysis.
To this end, first of all, the present inventors attached FcεRIα-Fc fusion protein not containing IL-10Vm (control), and the fusion protein prepared in the Example 3 to 96-well plates, respectively using Dip and Read™ Amine Reactive 2nd Generation (AR2G) Reagent Kit (forteBio, Cat No. 18-5092). Specifically, after dispensing 200 μl of D.W. to the 96-well plate, the amine biosensor included in the kit was inserted and hydrated for 10 minutes. Then, after dispensing additionally 200 μl of D.W. to the 96-well plate, EDC:NHS was mixed in a 1:1 ratio in a volume corresponding to 1/20 of the required sample then diluted in D.W. and 200 μl was dispensed into the 96-well plate. Subsequently, the FcεRIα-Fc fusion protein and the FcεRIα-Fc-IL-10Vm fusion protein was diluted to 10 μg/ml in 10 mM acetic acid solution (pH 5.0), and 200 μl was dispensed into the 96-well plate. Then, 200 μl of 1 M ethanolamine was added to the 96-well plate, and the biosensor plate and the sample plate were inserted into the Octet® K2 BLI analyzer and signals were measured. After the measurement was completed, 200 μl of the 1× Kinetics buffer was added to the sample plate, and then a baseline was determined. Subsequently, anti-DNP mouse IgE antibodies (Sigma, USA) were diluted in a 1× Kinetic buffer solution at various concentrations (50 pM to 3.125 nM), then dispensed to the sample plate at 200 μl, and then BLI analysis was performed to measure binding affinity using Octet® K2 BLI analyzer.
As a result, as shown in
As a result, as shown in
The present inventors performed pharmacokinetic analysis to confirm how stable the fusion protein of the present invention is when administered in vivo.
Specifically, the fusion protein (PG075-8) according to an embodiment of the present invention was administered to 3 SD rats in each group by intravenous injection, intraperitoneal injection, intramuscular injection, and subcutaneous injection at a dose of 1 mg/Kg body weight, respectively. After administration in volumes of 1.0 mL/kg, 1.0 mL/kg, 0.4 mL/kg and 1.0 mL/kg, respectively, at 0 min, 5 min, 1 hours, 5 hours, 10 hours, 24 hours, 48 hours, 72 hours, 120 hours, 168 hours, 240 hours and 336 hours, in case of intravenous injection, and in the case of other administration methods (intraperitoneal injection, intramuscular injection, and subcutaneous injection) at the same time as the intravenous injection, except for measurement after 5 minutes, serum was collected and the fusion protein was quantified using human IgG4 Fc ELISA.
As a result, as shown in
Conventional recombinant IL-10 has a side effect of anemia symptoms due to decreasing concentration of hemoglobin in blood and thrombocytopenia due to the decrease of concentration of platelets in blood when the administration dose is increased or repeated administration is applied (Tilg et al., J. Immunol. 164(4): 2204-2209, 2002; Fedorak et al., Gastroendocrinol. 119(6): 1473-1482, 2000). Accordingly, the present inventors investigated whether the fusion protein according to an embodiment of the present invention would exhibit such side effects.
To this end, specifically, the present inventors prepared 3 female ICR mice in each group, and administered PG075-8 at doses of 0, 50, 150 and 300 mg/kg to each group, and collected blood on the 11th day after drug administration. Then, the present inventors counted the white blood cell differential count, total red blood cells (RBCs), and total platelets using an automatic hematology analyzer (XN-V, SYSMEX, JAPAN). As a result, as shown in
The present inventors investigated whether symptom of diarrhea which is a representative symptom allergy that occur when OVA is administrated orally to experimental animals sensitized with albumin (OVA) was alleviated by the administration of the fusion protein PG075-8 according to an embodiment of the present invention to the experimental animals in order to confirm of the therapeutic effect of the fusion protein on allergy diseases.
Specifically, Balb/c mice (Koatech, Korea) were sensitized by intraperitoneally administering a solution of 50 μg of OVA (ovalbumin) and 1 mg of Alum twice at an interval of 14 days. Then, on days 28, 30, 32, 34, and 36, 50 mg of OVA was orally administered at 2-day intervals over a total of 5 times to induce food allergy in the intestine. In the process, the drugs constituting each group were administered on the 31st day. Phosphate buffer solution (PBS) in the first group (control group), IgETRAP (5 mg/kg) in the second group, and PG075-8 (7.3 mg/kg) according to an embodiment of the present invention in the third group, respectively, was administered after calculating the number of moles, respectively. Diarrhea occurrence was scored while oral administration of OVA was administered 5 times in total. On day 37, the mice were sacrificed and autopsied. Then, the number of mast cells in the small intestine, blood IgE concentration, and concentration of degranulation enzyme concentration (MCPT-1, Mast cell protease-1) in blood mast cells in the mice of each group were analyzed. As a result, as shown in
Accordingly, the fusion protein which IL-10Vm and FcεRIα as another fusion partner are applied according to an embodiment of the present invention inhibits the activation of the immune cells and their functions, particularly the secretion of anti-inflammatory cytokines, antigen presentation activity, etc. Thus, it can be used for the treatment of various immune-related diseases caused by overactivated immune function, for example, allergic diseases such as atopic disease, food allergy, chronic spontaneous urticaria, asthma, and the like.
The present invention has been described with reference to the above-described embodiments and experimental examples, but these are merely exemplary, and those of ordinary skill in the art will appreciate that various modifications and equivalent other embodiments are possible therefrom. Therefore, the true technical protection scope of the present invention should be determined by the technical spirit of the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2019-0082149 | Jul 2019 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2020/008871 | 7/7/2020 | WO |
