Novel Antibody for Preventing or Treating Fibrotic Disease

Abstract

The present invention relates to a method for predicting the occurrence of fibrotic disease or preventing or treating fibrotic disease by using an antibody that specifically binds to an asporin protein involved in fibrosis, or by inhibiting the activity of the said protein. The present invention provides an effective antibody that can be used for the diagnosis, prevention, and treatment of idiopathic pulmonary fibrosis, a disease for which there has been a lack of effective treatment methods due to the unknown pathogenesis mechanism. Furthermore, the present invention can be usefully utilized to efficiently block fibrosis in various tissues by ultimately providing a method for fundamentally inhibiting the pathological mechanism of fibrosis.

Description

Technology Field

[0001] The present invention relates to a method for predicting the occurrence of fibrotic disease or preventing or treating fibrotic disease by using an antibody that specifically binds to an asporin (ASPN) protein involved in fibrosis, or by inhibiting the activity of the said protein. Background Technology

[0003] Most organs undergo an inflammatory response and healing process following tissue damage; however, in cases of continuous damage or irreversible irritation, tissue fibrosis occurs during the healing process, leading to impaired organ function. Meanwhile, idiopathic pulmonary fibrosis (IPF) is a representative disease that causes fibrosis in the lungs. This disease typically develops between the ages of 50 and 70, and is known to have a poor prognosis, with a five-year survival rate of only 20 to 30%. To date, the exact cause of the disease is unknown, and aside from treatment through lung transplantation, only a few treatments with very limited efficacy exist, making the development of new therapeutic drugs urgently needed.

[0004] Fibrosis is a regenerative or reactive process characterized by the formation and deposition of excessive fibrous connective tissue, acting as a major etiologist for various fibrotic and chronic diseases. In particular, while the precise mechanism of pulmonary fibrosis is not fully understood, it is known to result from the excessive deposition of the extracellular matrix (ECM) and damage to abnormal alveolar epithelial cells. Under normal conditions, fibroblasts play a crucial role in wound healing and connective tissue formation; however, uncontrolled and excessive fibroblast expression leads to the accumulation of excessive immune cells and ECM proteins in foci, resulting in the development of a pathological condition. Beyond this general mechanism, the predictors and pathogenesis of pulmonary fibrosis remain ambiguous; therefore, elucidating the precise molecular and biological mechanisms is a critical factor in the diagnosis and treatment of the disease.

[0005] TGF-β is upregulated and activated in fibrosis and fibrotic diseases, serving as an important signaling mechanism for fibrosis. TGF-β leads to an increase in the Extracellular Matrix Gene Set, and matricellular proteins are non-structural proteins found in the ECM. Rather than functioning as stable structural elements within the ECM, matricellular proteins act as pro-fibrotic elements that activate fibroblasts as regulators within the tissue. Pamrevlumab, currently undergoing clinical trials as a treatment for pulmonary fibrosis, is also a targeted therapy that targets CTGF, a member of the matricellular protein group.

[0006] However, research on treatment methods for fibrosis, particularly pulmonary fibrosis, targeting asporin proteins, which belong to the SLRP (Small Leucine-rich Repeat Proteoglycan) protein family of ECM proteins, is currently insufficient.

[0007] Accordingly, the inventors focused on the association between the asporin protein and fibrosis, developed a monoclonal antibody specific to asporin using the said protein as a therapeutic target for fibrosis, and intended to demonstrate the anti-fibrotic effect of said antibody.

[0009] Throughout this specification, numerous papers and patent documents are referenced and cited. The disclosures of the cited papers and patent documents are incorporated by reference into this specification in their entirety to more clearly explain the state of the art to which the present invention pertains and the content of the present invention. Prior art literature

[0011] Patent Document 1. U.S. Patent Publication No. 2009-0048163 The problem to be solved

[0012] The inventors have made diligent research efforts to discover an efficient treatment method for idiopathic pulmonary fibrosis (IPF), an intractable disease with a high global prevalence and no effective treatment methods other than lung transplantation. As a result, the role and function of asporin in the pathogenesis of pulmonary fibrosis were identified, and the present invention was completed by developing a novel monoclonal neutralizing antibody that specifically recognizes and inhibits asporin and confirming the preventive or therapeutic effect on pulmonary fibrosis.

[0013] Therefore, the object of the present invention is to provide an antibody against an asporin protein or its antigen-binding fragment and a nucleic acid molecule encoding the same.

[0014] Another objective of the present invention is to provide a composition for the prevention or treatment of fibrotic disease.

[0015] Another objective of the present invention is to provide a diagnostic composition comprising the antibody or its antigen-binding fragment of the present invention as an active ingredient.

[0016] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims, and drawings. means of solving the problem

[0018] According to one aspect of the present invention, the present invention provides an antibody against an asporin protein or an antigen-binding fragment thereof comprising a heavy chain variable region having a heavy chain CDR (complementarity determining region) amino acid sequence of HCDR1 consisting of the sequence of SEQ ID NO. 1, HCDR2 consisting of the sequence of SEQ ID NO. 2, and HCDR3 consisting of the sequence of SEQ ID NO. 3.

[0019] The inventors have made diligent research efforts to discover an efficient treatment method for idiopathic pulmonary fibrosis (IPF), an intractable disease with a high global prevalence and no effective treatment methods other than lung transplantation. As a result, they identified that the asporin protein causes fibroblast-mediated fibrosis (fibrosis) in the pathogenesis of pulmonary fibrosis and confirmed that the asporin protein can be a therapeutic target. Furthermore, by developing a novel monoclonal neutralizing antibody that recognizes the asporin protein with high affinity and inhibits it, they have completed the present invention.

[0020] In this specification, the term “antibody” refers to an antibody against an asporin protein that specifically recognizes and binds to a specific epitope of the asporin protein, and includes not only the complete antibody form but also antigen-binding fragments (antibody fragments) of the antibody molecule.

[0021] A complete antibody has a structure consisting of two full-length light chains and two full-length heavy chains, with each light chain connected to the heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and has subclasses gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The light chain constant region has kappa (κ) and lambda (λ) types.

[0022] In this specification, the term “antigen-binding fragment of an antibody” refers to a fragment within the entire antibody molecule that possesses antigen-antibody binding function, and includes Fab, F(ab'), F(ab')2, and Fv, etc. Among the antibody fragments, Fab comprises a variable region of the light chain and heavy chain, a constant region of the light chain, and a first constant region of the heavy chain (C H1 It has a structure containing ) and possesses one antigen-binding site. Fab' is heavy chain C H1It differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the domain. F(ab')2 antibodies are produced when the cysteine ​​residues in the hinge region of Fab' form disulfide bonds. A recombinant technology for generating Fv fragments from minimal antibody fragments having only a heavy chain variable region and a light chain variable region is disclosed in PCT international published patent applications WO 88 / 10649, WO 88 / 106630, WO 88 / 07085, WO 88 / 07086, and WO 88 / 09344. Two-chain Fvs have a heavy variable region and a light variable region connected by non-covalent bonds, while single-chain Fvs generally have a heavy variable region and a single variable region connected by covalent bonds via a peptide linker or directly connected at the C-terminus, allowing them to form a dimer-like structure similar to two-chain Fvs. These antibody fragments can be obtained using proteolytic enzymes (for example, restriction cleavage of a whole antibody with papain yields Fab, and cleavage with pepsin yields the F(ab')2 fragment), or they can be produced using recombinant DNA technology.

[0023] In this specification, the term “heavy chain” refers to a variable region domain V comprising an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen. H and 3 invariant domains C H1 , C H2 and C H3 It refers to both the full-length heavy chain containing and its fragments.

[0024] In this specification, the term “CDR (complementarity determining region)” refers to the amino acid sequence of the hypervariable region of the immunoglobulin heavy chain and light chain (Kabat et al . Sequences of Proteins of Immunological Interest, 4th Ed., US Department of Health and Human Services, National Institutes of Health (1987)). The heavy chains (HCDR1, HCDR2, and HCDR3) and light chains (LCDR1, LCDR2, and LCDR3) each contain three CDRs, and these CDRs provide major contact residues for the antibody to bind to an antigen or epitope.

[0025] The range of the antibody or antibody fragment of the present invention includes variants having conservative amino acid substitutions in the CDR region. Additionally, the antibody or antibody fragment of the present invention may include variants of the amino acid sequences described in the attached sequence list within a range capable of specifically recognizing phosphorylated PLCγ2. For example, additional changes may be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletion, insertion, and / or substitution of amino acid sequence residues of the antibody. Such amino acid modifications are made based on the relative similarity of amino acid side chain substituents, e.g., hydrophobicity, hydrophilicity, charge, size, etc. By analysis of the size, shape, and type of amino acid side chain substituents, it can be seen that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be considered biologically functional equivalents.

[0026] In introducing mutations, the hydropathic index of the amino acids may be considered. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0027] The hydrophobic amino acid index is very important in conferring interactive biological functions of proteins. It is a known fact that similar biological activity can be achieved by substituting with amino acids having similar hydrophobic indices. When introducing a mutation based on the hydrophobic index, substitutions are made between amino acids that exhibit a difference in hydrophobic index within ± 2 in one specific example, within ± 1 in another specific example, and within ± 0.5 in yet another specific example.

[0028] Meanwhile, it is also well known that substitution between amino acids having similar hydrophilicity values ​​results in proteins having uniform biological activity. As disclosed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​are assigned to each amino acid residue: arginine (+3.0); lysine (+3.0); aspalate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); Phenylalanine (-2.5); tryptophan (-3.4). When introducing a variation based on hydrophilicity values, substitutions are made between amino acids that show a difference in hydrophilicity values ​​within ± 2 in one specific example, within ± 1 in another specific example, and within ± 0.5 in yet another specific example.

[0029] Amino acid exchanges in proteins that do not change the overall activity of the molecule are known in the art (H. Neurath, RLHill, The Proteins, Academic Press, New York, 1979). The most common exchanges are exchanges between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0030] Considering the variant having the aforementioned biologically equivalent activity, the antibody of the present invention or the nucleic acid molecule encoding it is interpreted to include a sequence that exhibits substantial identity with the sequence listed in the sequence list. The above substantial identity means a sequence that, when any other sequence is aligned to correspond as much as possible with the sequence of the present invention and the aligned sequence is analyzed using an algorithm commonly used in the art, exhibits at least 61% homology, 70% homology according to one specific example, 80% homology according to another specific example, and 90% homology according to yet another specific example. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are described in Smith and Waterman, Adv. Appl. Math . (1981) 2:482 Needleman and Wunsch, J. Mol. Bio . (1970) 48:443; Pearson and Lipman, Methods in Mol. Biol . (1988) 24: 307-31; Higgins and Sharp, Gene (1988) 73:237-44; Higgins and Sharp, CABIOS (1989) 5:151-3; Corpet et al . Nuc. Acids Res . (1988) 16:10881-90; Huang et al . Comp. Appl. BioSci . (1992) 8:155-65 and Pearson et al . Meth. Mol. Biol It is disclosed in . (1994) 24:307-31. NCBI Basic Local Alignment Search Tool(BLAST)(Altschul et al . J. Mol. Biol. (1990) 215:403-10) is available from NBCI and other sources and can be used online in conjunction with sequencing analysis programs such as blastp, blasm, blastx, tblastn, and tblastx. BLSAT is accessible at www.ncbi.nlm.nih.gov / BLAST / . Methods for comparing sequence homology using this program can be found at www.ncbi.nlm.nih.gov / BLAST / blast_help.html.

[0031] In the present invention, the term “asporin protein” refers to, in humans ASPN It refers to a protein encoded by a gene that belongs to the leucine-rich repeat (LRR) protein family associated with the cartilage matrix. The asporin protein may also be referred to as OS3, PLAP-1, PLAP1, or SLRR1C.

[0033] According to a specific embodiment of the present invention, the heavy chain variable region of the antibody or its antigen-binding fragment of the present invention has the amino acid sequence of SEQ ID NO. 11.

[0035] According to a specific embodiment of the present invention, the antibody or its antigen-binding fragment further comprises a light chain variable region having a light chain CDR amino acid sequence of LCDR1 consisting of the sequence of SEQ ID NO. 4, LCDR2 consisting of the sequence of SEQ ID NO. 5, and LCDR3 consisting of the sequence of SEQ ID NO. 6.

[0036] The term “light chain” in this specification refers to a variable region domain V comprising an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen. L and invariant domain C L It refers to the entire length light chain and all fragments thereof that include it.

[0037] According to a specific embodiment of the present invention, the light chain variable region of the antibody or its antigen-binding fragment of the present invention has the amino acid sequence of SEQ ID NO. 16.

[0038] According to a specific embodiment of the present invention, the antigen-binding fragment of the present invention is a Fab fragment, an F(ab') fragment, an F(ab')2 fragment, or an Fv fragment.

[0039] The antibodies of the present invention include, but are not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, short-chain Fvs(scFV), short-chain antibodies, Fab fragments, F(ab') fragments, disulfide-bound Fvs(sdFV) and anti-idiotype (anti-Id) antibodies, and epitope-bound fragments of said antibodies.

[0041] According to a specific embodiment of the present invention, the antibody of the present invention is a monoclonal antibody.

[0042] In this specification, the term “monoclonal antibody” means an antibody molecule of a single molecular composition obtained from substantially the same group of antibodies, and the monoclonal antibody exhibits single binding specificity and affinity for a specific epitope.

[0044] According to another aspect of the present invention, the present invention provides a composition for the prevention or treatment of fibrotic disease comprising an antibody or an antigen-binding fragment thereof as an active ingredient.

[0045] In this specification, the term “prevention” means suppressing the occurrence of a disease or illness in subjects who have not been diagnosed with having such a disease or illness but are at risk of developing such a disease or illness.

[0046] In this specification, the term “treatment” means (a) inhibition of the progression of a disease, illness, or symptom; (b) alleviation of a disease, illness, or symptom; or (c) elimination of a disease, illness, or symptom. When the composition of the present invention is administered to a subject, it inhibits the activity of asporin proteins that cause fibrosis, thereby preventing excessive fibrosis and serving to inhibit, eliminate, or alleviate the progression of symptoms caused by fibrotic disease. Accordingly, the composition of the present invention may serve as a treatment for these diseases on its own, or it may be applied as an adjuvant for treatment of said diseases when administered together with other pharmacological components. Accordingly, in this specification, the terms “treatment” or “therapeutic agent” include the meaning of “therapeutic aid” or “therapeutic adjuvant.”

[0047] In this invention, the term “fibrotic disease” collectively refers to all pathological conditions involving the progression of fibrosis in pathological tissues and having such fibrosis as a direct or indirect cause. Furthermore, the term “fibrosis” refers to a disease in which fibrous connective tissue (components of the extracellular matrix, such as collagen and fibronectin) accumulates excessively within and around inflamed or damaged tissues, which can lead to permanent scarring, organ dysfunction, and ultimately death.

[0049] According to a specific embodiment of the present invention, the fibrotic disease is selected from the group consisting of liver fibrosis; renal fibrosis and pulmonary fibrosis.

[0050] In the present invention, the term “liver fibrosis” refers to a condition in which fibrous tissue proliferates due to chronic damage to the liver, and may occur due to various causes such as biliary fibrosis, scarring after necrosis, and diffuse liver fibrosis.

[0051] In the present invention, the term “renal fibrosis” refers to a disease characterized by tubulointerstitial fibrosis and glomerular sclerosis, and as a final sign of chronic kidney disease, renal fibrosis is characterized by the excessive accumulation and deposition of extracellular matrix components.

[0052] In the present invention, the term “pulmonary fibrosis” refers to a disease in which the lungs become fibrotic due to repeated damage and injury to the lungs or lung tissues, and is accompanied by symptoms of shortness of breath; dry cough; fatigue; weight loss; and nail clubbing.

[0054] According to a specific embodiment of the present invention, the pulmonary fibrosis of the present invention is idiopathic pulmonary fibrosis (IPF).

[0055] In the present invention, the term “idiopathic pulmonary fibrosis” refers to a progressive respiratory disease characterized by the thickening and stiffening of lung tissue and the formation of scar tissue, and is a type of chronic scarring lung disease characterized by a gradual and irreversible decline in lung function, and is referred to as “idiopathic” because the clear cause is unknown.

[0057] According to another aspect of the present invention, the present invention provides a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof.

[0058] In this specification, the term “nucleic acid molecule” has a meaning that comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic building blocks of nucleic acid molecules, include not only natural nucleotides but also analogues in which sugar or base sites are modified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, (1990) 90:543-584). The sequence of a nucleic acid molecule encoding the heavy and light chain variable regions of the present invention may be modified. Such modification includes the addition, deletion, or non-conservative or conservative substitution of nucleotides.

[0059] The nucleic acid molecule of the present invention is interpreted to include a nucleotide sequence that exhibits substantial identity with respect to the nucleotide sequence described above. The substantial identity described above refers to a nucleotide sequence that exhibits at least 80% homology, at least 90% homology in one specific example, and at least 95% homology in another specific example when any other sequence is aligned to correspond as much as possible with the nucleotide sequence of the present invention described above and the aligned sequence is analyzed using an algorithm commonly used in the art.

[0061] According to another aspect of the present invention, the present invention provides a composition for diagnosing a fibrotic disease comprising an antibody or an antigen-binding fragment thereof as an active ingredient.

[0062] According to the present invention, the asporin protein of the present invention can be detected by an immunoassay method using an antigen-antibody reaction and used to analyze whether fibrotic disease has occurred. Such immunoassay can be performed according to various immunoassay or immunostaining protocols developed in the past.

[0063] For example, when the method of the present invention is carried out according to a radioimmunoassay method, a radioactive isotope (e.g., C 14 , I 125 , P 32 and S 35 An antibody labeled with ) may be used. In the present invention, the antibody that specifically recognizes the asporin protein is a polyclonal or monoclonal antibody, preferably a monoclonal antibody.

[0064] The antibodies of the present invention are produced by methods commonly practiced in the art, for example, fusion methods (Kohler and Milstein, European Journal of Immunology , 6:511-519 (1976)), recombinant DNA method (U.S. Patent No. 4,816,567) or phage antibody library method (Clackson et al, Nature , 352:624-628 (1991) and Marks et al. J. Mol. Biol. It can be manufactured according to , 222:58, 1-597(1991)). The general process for antibody production is Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual It is described in detail in Cold Spring Harbor Press, New York (1999).

[0065] By analyzing the intensity of the final signal obtained through the aforementioned immunoassay process, it is possible to predict whether or not a tumor has metastasized or the likelihood of metastasis. That is, if the signal for asporin protein in an individual's sample appears stronger than in a normal sample, the individual's Metastasis has occurred or is highly likely to occur in the future It is judged that...

[0066] In this specification, the term “diagnosis” includes the determination of an individual’s susceptibility to a specific disease, the determination of whether an individual currently possesses a specific disease, and the determination of the prognosis of an individual afflicted with a specific disease.

[0067] In this specification, the term “diagnostic composition” refers to a conventional mixture or device comprising a means for measuring the expression level of an asporin protein or a gene encoding it, for determining whether a fibrotic disease has occurred in a subject or predicting the likelihood of its occurrence, and may also be expressed as a “diagnostic kit.”

[0069] According to another aspect of the present invention, the present invention provides an antibody against an asporin protein or an antigen-binding fragment thereof comprising a light chain variable region having a light chain CDR (complementarity determining region) amino acid sequence of LCDR1 consisting of the sequence of SEQ ID NO. 4, LCDR2 consisting of the sequence of SEQ ID NO. 5, and LCDR3 consisting of the sequence of SEQ ID NO. 6.

[0070] Since the light chain CDR (LCDR); light chain variable region; and the antibody or antigen-binding fragment containing the same used in the present invention have already been described above, their description is omitted to avoid excessive duplication. Effects of the invention

[0072] The features and advantages of the present invention are summarized as follows:

[0073] (a) The present invention provides a method for predicting whether a fibrotic disease will occur by using an antibody that specifically binds to an asporin protein involved in fibrosis, or for preventing or treating a fibrotic disease by inhibiting the activity of the said protein.

[0074] (b) The present invention provides an effective antibody that can be used for the diagnosis, prevention, and treatment of idiopathic pulmonary fibrosis, for which there is no effective treatment method due to the unknown clear pathogenesis.

[0075] (c) In addition, the present invention can be usefully utilized to efficiently block fibrosis in various tissues by ultimately providing a method for fundamentally inhibiting the pathological fibrosis mechanism. Brief explanation of the drawing

[0077] Figure 1a is a figure showing the results of immunohistochemical staining of human lung tissue, the left side showing the staining results in a control group and a patient with immediate pulmonary fibrosis (IPF), and the right side is a graph quantitatively comparing the expression of asporin in a control group and an IPF patient with asporin expression on the y-axis. Figure 2a shows the immunohistochemical staining results for asporin (left) and a graph (right) showing the expression level of asporin in a mouse lung fibrosis model in which fibrosis was advanced by injecting bleomycin. Figure 2b shows the immunohistochemical staining results for asporin (left) and a graph (right) showing the expression level of asporin in a mouse lung fibrosis model in which fibrosis was advanced through TGF-β transgenic transplantation. Figure 3a shows the results of immunohistochemical staining for asporin (top left), observation of collagen via Sirius Red staining (bottom left), a graph showing asporin expression levels (top right), and a graph showing the results of measuring the area of ​​fibrosis via Sirius Red staining (bottom right) in a unilateral ureter obstruction (UUO) mouse model of renal fibrosis. Figure 3b shows the results of immunohistochemical staining for asporin (top left), observation of collagen via Sirius Red staining (bottom left), a graph showing asporin expression levels (top right), and a graph showing the results of measuring the area of ​​fibrosis via Sirius Red staining (bottom right) in a choline-deficient high-fat diet (CD-HFD) mouse model of liver fibrosis. Figure 4 is a table showing monoclonal antibodies #1 to #11 produced for the development of asporin-targeted therapeutic agents. Figure 5a shows the results of dot blotting performed on two clones with good affinity. Figure 5b shows the results of peptide blocking performed on MRC5 lung fibroblasts. Figure 6a is a graph showing the results confirming that TGF-βdp-induced cell proliferation in human fibroblasts is reduced upon treatment with asporin antibodies. Figure 6b shows the target genes of TGF-βdp-induced fibrosis in human fibroblasts ( Acta 2 , Col1a1 and Col3a1 This is a graph showing the results confirming that ) is reduced due to asporin antibody treatment. Figure 7a is a graph showing the results of comparing the fibroblast proliferation inhibitory ability of the anti-asporin antibodies (#9 and #10) of the present invention with that of the CTGF antibody (CTGF Ab.). Figure 7b is a figure showing the results of verifying the specificity of the antibody by measuring the degree of peptide blocking through the fibroblast proliferation inhibitory ability of the present invention. Figure 7c shows the fibrosis target gene of the anti-asporin antibodies (#9 and #10) of the present invention using qRT-PCR ( Acta2 , Col1a1 , Col3a1 This is a figure illustrating the results of verifying ). Figure 8a is a graph showing the pattern of tissue fibrosis following the administration of the antibody (#10) of the present invention in a pulmonary fibrosis mouse model and the quantitative results of fibrosis according to the MTS alc α-SMA method. Figure 8b is a figure showing the results of counting cells contained in bronchoalveolar lavage fluid collected from a pulmonary fibrosis mouse model. Figure 8c is a figure showing the results of measuring and quantifying water-soluble collagen in a pulmonary fibrosis mouse model. Specific details for implementing the invention

[0078] The present invention will be described in more detail below through examples. These examples are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these examples according to the gist of the invention.

[0080] Examples

[0081] Experimental methods and analysis methods

[0082] animal models

[0083] Mice were housed in a specific pathogen-free animal facility with controlled temperature and humidity under a 12-hour light / 12-hour dark cycle. Prior to BLM administration, 8-week-old mice were injected with 10 mg / kg tamoxifen (Merck, Darmstadt, Germany) three times a week for one week. Mice were anesthetized by intraperitoneal injection of Zoletil 50 (30 mg / kg) and Rompun (10 mg / kg). Mice were injected intratracheally with PBS (vehicle control) or 4 mg / kg BLM (Santa Cruz Biotechnology, Dallas, TX, USA). An average of 8 mice were used in each group.

[0085] Immunostaining

[0086] To prepare for immunohistochemistry (IHC), lung section slides were rehydrated through a graded alcohol series and then antigens were retrieved in 10 mM sodium citrate. The IHC staining protocol was performed by sequential application of a peroxidase blocking reagent, a primary antibody (O / N), a secondary antibody (1 hr) molecule, and horseradish peroxidase linked to a polymer backbone; and visualization using a chromogenic reagent (Dako, Santa Clara, CA, USA) containing a hydrogen peroxide substrate along with 3,3'-diaminobenzidine tetrahydrochloride (DAB). The intensity of asporin proteins was calculated using ImageJ. Additionally, the asporin antibody was detected using Thermo PA5-28124.

[0088] Surgery in a mouse fibrosis model of unilateral ureter obstruction (UUO).

[0089] Mechanical stress was applied to mice to induce renal fibrosis. Mice were anesthetized by intraperitoneal injection of Zolletil (50 mg / ml) and Lumpun (23.32 mg / ml), and the skin and peritoneum were incised using forceps and medical ophthalmic scissors (Iris Scissors). One kidney was located, the ureter was severed with a blunt hook, and the ureter was tied twice with black silk thread. Subsequently, the peritoneum and skin were sutured with black silk thread and sterilized with povidone iodine. The surgical mice were sacrificed on the 8th day after unilateral ureteral obstruction surgery.

[0091] Preparation of kidney tissue sections for histological observation

[0092] 4% formaldehyde was injected into no-capsule kidneys, and the kidneys were fixed by applying pressure at 4°C for 24 hours. After tissue treatment, the tissues were embedded in paraffin blocks and cut into 4 μm pieces.

[0094] Sirius Red staining for collagen observation

[0095] FFPE renal tissue was used, paraffin was removed with xylene, and the tissue was hydrated with 100%, 90%, and 80% EtoH. After hydration, picro Sirius red (ab150681, Abcam, Cambridge, UK) was instilled into the renal tissue for 1 hour, and the tissue was washed twice with 0.5% acidic water. The tissue was mounted with a hydrophobic mounting solution.

[0097] Choline-deficient High Fat Diet (CD-HFD mouse model)

[0098] In order to manufacture a CD-HFD model, Int J Exp PatholSee Apr;94(2):93-103. (2013). Additionally, an HFD containing CDAA, 0.1% methionine (CDAHFD; #A06071302) was obtained from Research Diets (New Brunswick, NJ, USA). This HFD provides 5.2 kcal per gram and contains 320 g / kg of lard-based fat for 8 weeks.

[0100] Dot blotting

[0101] Dot blotting was performed through the following steps.

[0102] (1) Quality control of delivery antibodies: Dot blot detection of the corresponding peptide antigen

[0103] (2) BSA-conjugated polypeptides were attached to a nitrocellulose membrane (each point was 2 μL). The point groups contained 25 ng, 5 ng, 1 ng, 0.25 ng, 0.05 ng, and 0.01 ng of conjugated polypeptides from right to left (Fig. 5a). Blocked using 5% skim milk / TBST.

[0104] (3) After incubating with 1:1000 diluted antibody for 1 hour, wash with TBST 3 times and incubate with secondary antibody for 45 minutes.

[0105] (4) After adding ECL reagent and reacting for 1 minute, a preservative film was added and the X-ray film was developed by exposing it in a dark room (exposure was performed several times).

[0107] Peptide blocking and Western blotting

[0108] Cells were lysed in lysis buffer (20 mM Tris-Cl, 150 mM NaCl, 1% Triton X-100, 1.5% MgCl2, 1 mM EDTA, 1 mM Na2VO4, 1 mM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitor cocktail, pH 7.5). The lysate was briefly vortexed and sonicated, and impurities were removed by centrifugation at 12,000 xg for 20 minutes at 4°C. The supernatant was collected and transferred to a new tube. Protein concentration was determined using a 660 nm protein assay reagent (Thermo Scientific, Waltham, MA, USA). Equal amounts of protein extracts were electrophoresed on an SDS-polyacrylamide gel and then transferred to a nitrocellulose transfer membrane (Whatman, Dassel, Germany). The membrane is 0.1% (v / v) Tween 20 (Sigma-Aldrich, St. Louis, MO, USA) and 5% (w / v) fat-free Difco TM The membrane was blocked with PBS containing skim milk (BD Biosciences, Sparks, MD, USA) and 3% BSA (Affymetrix, Santa Clara OH, USA), and irradiated using anti-asporin (Abmart, China) as the primary antibody. Then, the membrane was washed with 1x PBST and incubated for 1 hour with an appropriate secondary anti-mouse horseradish peroxidase-conjugated antibody (Thermo Scientific, Rockford IL, USA). Visualization was performed using a LAS-3000 system (Fujifilm, Stamford, CT, USA) with an enhanced chemiluminescence detection reagent (Thermo Scientific).

[0109] For peptide blocking, the diluted primary antibody was incubated with the ASPN sequence peptide for 4 hours. The incubated antibody was used for blotted membrane incubation. Additionally, the sequence of the asporine peptide is as follows: CPFGCQCYSRVVHCSDLGLT (Sequence No. 23).

[0111] Media concentration

[0112] Medium was harvested from 60-mm cell culture dishes in which MRC5 cells were cultured, passed through a 0.45 μm filter (Sartorius, Stonehouse, UK), and used as conditioned medium. Additionally, the culture medium was applied to a VIVASPIN6 column (Sartorius) and centrifuged at 10,000 xg for 2 hours; the concentrated protein was counted using a protein analyzer and then immunoblotted using antibodies.

[0114] cell culture

[0115] Human fibroblast cell line MRC5 was purchased from the Korean Cell Line Bank (Seoul, Korea). Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v / v) fetal bovine serum and 1% antibiotic / antifungal solution (Corning, Manassas, VA, USA) at 37°C and 5% CO2.

[0117] MRC5 human fibroblast proliferation

[0118] Cells were serum-depleted for 2 hours prior to 24 hours of treatment with TGF-β1 (20 ng / ml) and monoclonal antibodies. Cells were trypsinized and counted with a cell counter.

[0120] RNA isolation and quantitative reverse transcription polymerase chain reaction (qRT-PCR)

[0121] Total RNA was extracted using the Trizol reagent according to the manufacturer's protocol (Takara, Kyoto, Japan). 500 µl of Trizol was added to a cell culture dish (6-well), and cells were collected in tubes. 100 µl of chloroform was added to the sample, and the sample was vortexed. The sample was incubated at room temperature for 3 minutes and centrifuged at 12,000 xg for 15 minutes at 4°C. 250 µl of the supernatant was collected and transferred to a new tube. 250 µl of isopropanol was added to the sample, and the sample was centrifuged at 12,000 xg for 10 minutes at 4°C. The supernatant was removed, and 500 µl of 75% ethanol was added to the sample. The sample was centrifuged at 13,000 xg for 5 minutes at 4°C. The supernatant was removed, and the pellet was dried at room temperature. 30 µl of DEPC was added to the dried pellet. RNA concentration was measured using Nanodrop1000 (Thermo Scientific, Waltham, MA, USA). After RNA isolation, 1 μg of total RNA was used, and cDNA was synthesized using CellScript (CellSafe, Seoul, Korea) according to the manufacturer's protocol.

[0122] qRT-PCR analysis was performed using the ABI PRISM 7000 Sequence Detection System instrument and software (Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer's protocol with some modifications. Briefly, an appropriate amount of reverse transcription mixture was amplified with specific primers using SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA, USA). The PCR primer sequences for qRT-PCR are listed in Table 1.

[0123] Target Sensedak antisense strand human COL1a1 CCTCAAGGGCTCCAAC (Sequence No. 33) GGTTTTGTATTCAATCACTGTCTTGC(Sequence No. 34) human COL3a1 TGGTCTGCAAGGAATGCCTGGA(Sequence No. 35) TCTTTCCCTGGGACACCATCAG(Sequence No. 36) human α-SMA CTGGCATCGTGCTGGACTCT(Sequence No. 37) GATCTCGGCCAGCCAGATC (Sequence No. 38)

[0124] Statistical analysis

[0125] The results were visualized and analyzed using Prism software version 5 (GraphPad Software, San Diego, CA, USA). One-way ANOVA was performed to determine statistical significance when comparing two or more sample groups. Data values ​​were expressed as mean ± standard deviation (SD). Student's t-test was used to determine the statistical significance of two groups. A value of P < 0.05 was considered statistically significant.

[0128] Experimental results

[0129] It was confirmed that asporin protein expression is increased in IPF patients.

[0130] Observation of lung section slides from IPF patients via the aforementioned immunohistochemical staining revealed that the expression of asporin protein was increased in IPF patients. The left image of Figure 1 shows the results of immunohistochemical staining in the control group and IPF patients, with the areas stained by asporin expression in IPF patients indicated by arrows. The right image of Figure 1 is a graph showing the expression levels of ASPN (asporin, y-axis) in the control group and IPF patients. According to the experimental results, it was confirmed that the expression level of asporin was significantly increased in IPF patients compared to the control group.

[0132] We confirmed that asporin expression significantly increased in mouse pulmonary fibrosis models.

[0133] Since bleomycin causes side effects such as lung injury and pulmonary fibrosis, a mouse model with advanced pulmonary fibrosis was obtained using it. Additionally, a mouse model with induced pulmonary fibrosis was prepared through the transgenic transfer of the TGF-β gene. Immunohistochemical staining of mouse lung tissues obtained from these pulmonary fibrosis-induced mouse models confirmed that asporin expression was significantly increased in all of them.

[0135] We confirmed that asporin expression significantly increased in renal fibrosis mouse models and liver fibrosis mouse models.

[0136] In the Unilateral Ureter Obstruction (UUO) mouse model, a renal fibrosis model, and the choline-deficient high-fat diet (CD-HFD) mouse model, a liver fibrosis model, it was confirmed that asporin expression significantly increased with the induction of fibrosis. Figure 3a illustrates the experimental results for UUO, and Figure 3b for CD-HFD; for each model, the results of immunohistochemical staining and graphs showing asporin expression levels and the area of ​​fibrosis are depicted. From these results, it was clearly evident that asporin increases due to fibrosis in the kidneys and livers.

[0138] Antibodies with high specificity were selected by investigating the affinity of monoclonal antibody candidates for asporin.

[0139] As shown in Figure 4, clones of anti-asporin antibodies #1 to #11 were obtained. Among these, two clones with high specificity were selected by investigation through dot blotting and peptide blocking, and the fibrosis inhibitory effect of one of them (#10) was confirmed.

[0141] The fibrosis-inhibiting effect of selected monoclonal antibodies was confirmed.

[0142] It was experimentally confirmed that the selected monoclonal antibody (#10) has inhibitory effects on fibroblast proliferation and fibrosis target gene expression. Through this, the inhibitory effects of the selected monoclonal antibody on fibrosis and its preventive or therapeutic effects against fibrotic diseases could be confirmed.

[0144] The antifibrotic efficacy of selected antibodies in cell models was confirmed.

[0145] It was confirmed that TGF-β-induced cell proliferation in human fibroblasts was reduced upon treatment with asporin antibodies. In addition, it was confirmed that TGF-β-induced fibrosis target genes in human fibroblasts were reduced after treatment with asporin antibodies. Through this, the anti-fibrotic efficacy of the anti-asporin antibodies of the present invention was finally confirmed.

[0147] The CDR sequences of the selected antibodies are as follows.

[0148] HCDR1 : GFTFSDYW (Sequence No. 1)

[0149] HCDR2 : IRNKPYNYETY (Sequence No. 2)

[0150] HCDR3 : TAYYRYDVLFDY (Sequence No. 3)

[0151] LCDR1 : QDISNY (Sequence No. 4)

[0152] LCDR2 : YTS (Sequence No. 5)

[0153] LCDR3 : QQSKTLPLT (Sequence No. 6)

[0155] HFR1 : EVKLVETGGGLVQPGRPMKLSCVAS (Sequence No. 7)

[0156] HFR2 : MNWVRQSPEKGLEWVVQ (Sequence No. 8)

[0157] HFR3 : YYSDSVKGRFTISRDDSKSCVYLQMNNLRAEDMGIYYC (Sequence No. 9)

[0158] HFR4 : WGQGTSLTVSS (Sequence No. 10)

[0159] Heavy chain variable region: EVKLVETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVVQIRNKPYNYETYYSDSVKGRFTISRDDSKSCVYLQMNNLRAEDMGIYYCTAYYRYDVLFDYWGQGTSLTVSS (Sequence No. 11)

[0160] LFR1 : DIQMTQTTSSLSASLGDRVTISCRAS (Sequence No. 12)

[0161] LFR2 : LNWYQQKPEGTVKLLIY (Sequence No. 13)

[0162] LFR3 : RLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFC (Sequence No. 14)

[0163] LFR4 : FGAGTKLELK (Sequence No. 15)

[0164] Light chain variable region: DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPEGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQSKTLPLTFGAGTKLELK (Sequence No. 16)

[0166] nucleic acid sequence

[0167] HCDR1 : ggattcacttttagtgactactgg (Sequence No. 17)

[0168] HCDR2 : attagaaacaaaccttataattatgaaaca (sequence number 18)

[0169] HCDR3 : acagcctactataggtacgacgtcctcttcgactac (Sequence No. 19)

[0170] LCDR1 : caggacattagcaattat (sequence number 20)

[0171] LCDR2 : tacacatca (sequence number 21)

[0172] LCDR3 : caacagagtaaaacgcttcctctcacg (sequence number 22)

[0174] HFR1: gaggtgaagctggttgagactggaggaggcttggtgcaacctgggaggcccatgaaactctcctgtgttgcctct (SEQ ID NO: 23)

[0175] HFR2 : atgaactgggtccgccagtctccagagaaaggactggagtgggtagtacaa (sequence number 24)

[0176] HFR3: tattattcagattctgtgaaaggcagattcaccatctcaagagatgattccaaaagttgtgtctacctgcaaatgaacaacttaagagctgaagacatgggtatctattactgt (SEQ ID NO: 25)

[0177] HFR4 : tggggccaaggcacctctctcacagtctcctcag (Sequence No. 26)

[0178] Heavy chain variable region: gaggtgaagctggttgagactggaggaggcttggtgcaacctgggaggcccatgaaactctcctgtgtttgcctctggattcacttttagtgactactggatgaactgggtccgccagtctccagagaaaggactggagtgggtagtacaaattagaaacaaaccttataattatgaaacata ttatcagattctgtgaaaggcagattcaccatctcaagagatgattccaaaagttgtgtctacctgcaaatgaacaacttaagagctgaagacatgggtatctattactgtacagcctactataggtacgacgtcctcttcgactactggggccaaggcacctctctcacagtctcctcag (SEQ ID NO: 27)

[0180] LFR1: gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagt (SEQ ID NO: 28)

[0181] LFR2: ttaaactggtatcagcagaaaccagaaggaactgttaaactcctgatctac (SEQ ID NO: 29)

[0182] LFR3 : agattacactcaggagtcccatcaagattcagtggcagtgggtggaacagattattctctcaccattagcaacctggaacaagaagatattgccacttacttttgt (sequence number 30)

[0183] LFR4 : ttcggagctgggaccaagttggagctgaaac (sequence number 31)

[0184] Light chain variable region: gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagcaattatttaaactggtatcagcagaaaccagaaggaactgttaaactcctgatctactacacatcaagatt acactcaggagtcccatcaagattcagtggcagtgggtctggaacagattattctctcaccattagcaacctggaacaagaagatattgccacttacttttgtcaacagagtaaaacgcttcctctcacgttcggagctgggaccaagttggagctgaaac (SEQ ID NO: 32)

[0186] The efficacy of anti-asporin antibodies was evaluated in a mouse model of pulmonary fibrosis.

[0187] To evaluate the efficacy of the aforementioned #10 anti-asporin antibody, the inhibitory effect on fibrosis in an animal model was further confirmed. A pulmonary fibrosis mouse model was used.

[0188] Pulmonary fibrosis was induced in 8-10 week old C57BL / 6 mice by intrabronchial injection of bleomycin at a dose of 2 mg / kg. Starting the next day, an asporin antibody at 10 mg / kg was administered intravenously every other day for 5 doses, and the mice were sacrificed on the 12th day for use in the experiment. Intratissue fibrosis was observed in paraffin sections of mouse lungs using Masson's trichrome staining (MTS) and α-SMA immunohistochemical staining, and quantified using ImageJ software. It was observed that pulmonary fibrosis induced by bleomycin decreased upon injection of the anti-asporin antibody of the present invention (Fig. 8a).

[0189] Bronchoalveolar lavage fluid was taken from the lungs of mice and cells were counted. It was observed that the number of cells increased by bleomycin was significantly reduced in the group treated with the anti-asporin antibody of the present invention (Fig. 8b).

[0190] In addition, water-soluble collagen was measured in the lungs of mice. When measured using the Sircol collagen assay kit (Biocolor, UK), it was observed that the amount of collagen increased by bleomycin was significantly reduced upon injection of the anti-asporin antibody of the present invention (Fig. 8c).

[0191] In conclusion, the excellent fibrosis inhibitory effect of the anti-asporin antibody of the present invention could be verified using an animal model.

[0193] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

Claim 1 An antibody against an asporin protein or its antigen-binding fragment, comprising: a heavy chain variable region having a heavy chain CDR (complementarity determining region) amino acid sequence of HCDR1 consisting of the sequence of SEQ ID NO. 1, HCDR2 consisting of the sequence of SEQ ID NO. 2, and HCDR3 consisting of the sequence of SEQ ID NO. 3; and a light chain variable region having a light chain CDR amino acid sequence of LCDR1 consisting of the sequence of SEQ ID NO. 4, LCDR2 consisting of the sequence of SEQ ID NO. 5, and LCDR3 consisting of the sequence of SEQ ID NO.

6. Claim 2 An antibody or its antigen-binding fragment according to claim 1, characterized in that the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.

11. Claim 3 delete Claim 4 An antibody or its antigen-binding fragment according to claim 1, characterized in that the light chain variable region comprises the amino acid sequence of SEQ ID NO.

16. Claim 5 An antibody or its antigen-binding fragment according to claim 1, characterized in that the antigen-binding fragment is a Fab fragment, an F(ab') fragment, an F(ab')2 fragment, or an Fv fragment. Claim 6 An antibody or its antigen-binding fragment characterized in that, in claim 1, the antibody is a monoclonal antibody. Claim 7 A composition for the prevention or treatment of a fibrotic disease comprising, as an active ingredient, an antibody or an antigen-binding fragment thereof according to any one of claims 1, 2, and 4 to 6, wherein the fibrotic disease is a disease selected from the group consisting of liver fibrosis; renal fibrosis; and pulmonary fibrosis. Claim 8 delete Claim 9 A composition according to claim 7, characterized in that the pulmonary fibrosis is idiopathic pulmonary fibrosis (IPF). Claim 10 A nucleic acid molecule encoding the antibody or its antigen-binding fragment of any one of claims 1, 2, and 4 to 6. Claim 11 A composition for diagnosing a fibrotic disease comprising, as an active ingredient, an antibody or an antigen-binding fragment thereof according to any one of claims 1, 2, and 4 to 6, wherein the fibrotic disease is a disease selected from the group consisting of liver fibrosis; renal fibrosis; and pulmonary fibrosis. Claim 12 delete

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