Composition and method for inhibiting fibrosis

Abstract

Provided are a novel composition and method for inhibiting fibrosis. A composition for inhibiting fibrosis containing an antibody that specifically binds to PAD2 is used. Alternatively, a pharmaceutical composition for treating fibrosis containing an antibody that specifically binds to PAD2 and a pharmaceutically acceptable carrier may be used. Alternatively, a kit for inhibiting fibrosis containing an antibody that specifically binds to PAD2 may be used.

Description

Composition or method for inhibiting fibrosis

[0001] The technical field of the present invention relates to compositions and methods for the inhibition of fibrosis.

[0002] Fibrosis is known to occur when connective tissue accumulates excessively in tissues, and is said to be the cause of up to 45% of all deaths in industrialized countries (Non-Patent Document 1).

[0003] Several reports have been published in recent years regarding the causes of fibrosis. Non-Patent Document 2 (2019) describes the cause of fibrosis as increased ER stress and induction of cell death due to impaired secretion of SFTPA1 protein.

[0004] Non-patent document 3 (2020) describes NEAT1 degradation and cell death induction caused by the RBM7 protein as causes of fibrosis.

[0005] Non-patent document 4 (2021) describes fibroblast invasion mediation due to citrullinated vimentin as a cause of fibrosis.

[0006] "FIBROSIS: FROM MECHANISMS TO MEDICINES" Henderson et a., Nature. 2020 Nov; 587(7835): 555-566."A homozygous SFTPA1 mutation drives necroptosis of type II alveolar epithelial cells in patients with idiopathic pulmonary fibrosis" Takezaki et al., J Exp Med. 2019 Dec 2;216(12):2724-2735."Dysregulated Expression of the Nuclear Exosome Targeting Complex Component Rbm7 in Nonhematopoietic Cells Licenses the Development of Fibrosis" Fukushima et al., Immunity. 2020 Mar 17;52(3):542-556.e13."Citrullinated vimentin mediates development and progression of lung fibrosis" Li et al., Sci Transl Med. 2021 Mar 17;13(585):eaba2927.

[0007] Although various findings have been accumulated so far, many aspects of the causes of fibrosis remain unknown, and therefore conventional treatment strategies alone have not been sufficient.

[0008] Meanwhile, as a result of extensive research, the present inventors discovered that administration of an anti-PAD2 antibody to a fibrosis model mouse suppresses fibrosis.

[0009] This research result was unexpected from the perspective of the prior art. For example, the above-mentioned Non-Patent Documents 1-3 contain no mention of anti-PAD2 antibodies or PAD2. Non-Patent Document 4 states that "Cd / CB (cadmium / carbon black) induced the secretion of citrullinated vimentin in an Akt1- and PAD2-dependent manner" (see abstract). However, according to Non-Patent Document 4, citrullinated vimentin is produced (and subsequently secreted) by intracellular citrullination of vimentin. On the other hand, because anti-PAD2 antibodies are thought to act extracellularly, those skilled in the art would not recognize that they affect the citrullination of vimentin intracellularly. Therefore, based on literature such as Non-Patent Document 4 that describes intracellular citrullination, it is difficult to predict that anti-PAD2 antibodies would inhibit fibrosis.

[0010] According to one aspect of the present invention, there is provided a composition for inhibiting fibrosis, comprising an antibody that specifically binds to PAD2. Fibrosis can be inhibited by using this composition.

[0011] According to one aspect of the present invention, there is provided a pharmaceutical composition for treating fibrosis, comprising an antibody that specifically binds to PAD2 and a pharmaceutically acceptable carrier. This pharmaceutical composition can be used to treat fibrosis.

[0012] According to one aspect of the present invention, there is provided a method for suppressing or treating fibrosis, which comprises administering an anti-PAD2 antibody to a subject. By using this method, fibrosis can be suppressed or treated.

[0013] Figure 1 shows the results of measuring the binding affinity of anti-PAD2 antibodies to PAD2. Figure 2 shows the results of measuring the ability of anti-PAD2 antibodies to inhibit PAD2 activity. Figure 3 shows the results of measuring the rate of fibrosis after administration of anti-PAD2 antibodies to a fibrosis model. Figure 4 shows the results of measuring the ability of anti-PAD2 antibodies to inhibit PAD2 activity in a fibrosis model.

[0014] Hereinafter, embodiments of the present invention will be described in detail, with the same contents omitted as appropriate to avoid repetition.

[0015] According to one embodiment of the present invention, a novel composition for inhibiting fibrosis is provided. This composition is, for example, a composition comprising an antibody that specifically binds to PAD2. Use of such a composition can inhibit fibrosis, as demonstrated in the Examples below.

[0016] In one embodiment of the present invention, PAD2 includes a protein called peptidylarginine deiminase 2, peptidyl arginine deiminase type-2, or protein-arginine deiminase type-2. These terms can be used interchangeably. Details of PAD2, such as its amino acid sequence, can be found on websites such as NCBI or UniProt. The primary accession number for PAD2 listed in UniProt is, for example, Q9Y2J8. The amino acid sequence of human PAD2 is, for example, SEQ ID NO: 1. The biological origin of PAD2 is not limited as long as it has PAD2 activity. PAD2 activity includes, for example, citrullination activity. Citrullination activity includes, for example, the activity of catalyzing the citrullination of arginine side chains in a substrate. PAD2 activity may be evaluated by colorimetric quantification of citrulline residues after incubation of a solution containing PAD2 and a substrate. The substrate includes, for example, a compound having an arginine side chain (e.g., a protein (e.g., histone), a peptide, or a small molecule compound (e.g., BAEE)). Colorimetric assay may be performed using a mixture containing 2,3-butanedione monoxime and thiosemicarbazide. PAD2 includes, for example, PAD2 derived from humans, monkeys, mice, rats, dogs, or cats.

[0017] In one embodiment of the present invention, an anti-PAD2 antibody can suppress fibrosis. Fibrosis includes, for example, a phenomenon caused by excessive accumulation of connective tissue in tissue. Tissue hardening associated with fibrosis can occur, for example, when connective tissue composed of collagen or the like proliferates and replaces normal tissue. Fibrosis includes, for example, fibrosis occurring in tissues such as the liver, lungs, kidneys, heart, pancreas, bone marrow, or skin. In one embodiment of the present invention, fibrosis includes diseases accompanied by tissue fibrosis.

[0018] In one embodiment of the present invention, the anti-PAD2 antibody includes an antibody capable of inhibiting PAD2 activity. Inhibition of activity includes, for example, inhibition of citrullination activity. In one embodiment of the present invention, the anti-PAD2 antibody includes an antibody capable of suppressing fibrosis. In one embodiment of the present invention, the anti-PAD2 antibody includes an antibody having neutralizing activity against PAD2 (neutralizing antibody), an antibody capable of inhibiting PAD2 function, or an antibody capable of reducing the amount or proportion of fibrotic tissue in an individual or tissue. Neutralizing antibodies include antibodies capable of inhibiting PAD2 activity. In one embodiment of the present invention, the tissue includes, for example, the liver, lung, kidney, heart, pancreas, bone marrow, or skin. As used herein, antibodies that inhibit PAD2 activity include antibodies capable of inhibiting PAD2 activity or antibodies capable of inhibiting PAD2 activity. As used herein, antibodies that suppress fibrosis include antibodies capable of suppressing fibrosis or antibodies capable of suppressing fibrosis.

[0019] In one embodiment of the present invention, an anti-PAD2 antibody can be produced, for example, by immunizing a mammal or bird with PAD2 and recovering and purifying the antibody. The anti-PAD2 antibody can be produced by utilizing various techniques and modeling techniques known in the art, such as those described in "Development of therapeutic antibodies for the treatment of diseases, Lu et al., J. Biomed. Sci., volume 27, Article number: 1 (2020 Jan. 2)" and "Modeling Immunity with Rosetta: Methods for Antibody and Antigen Design, Schoeder et al., Biochemistry. 2021 Mar. 23; 60(11): 825-846." The anti-PAD2 antibody may be produced, for example, by immunizing a mammal or bird with a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 and recovering and purifying the antibody. For example, full-length PAD2 or a fragment thereof can be used as the PAD2 antigen. Anti-PAD2 antibodies may be prepared, for example, by selecting antibodies that bind to wild-type PAD2 but do not bind to mutant PAD2 (e.g., deletion mutants) at positions 341 to 357. Anti-PAD2 antibodies may also be prepared by selecting antibodies based on their binding strength to PAD. Anti-PAD2 antibodies may also be prepared by selecting antibodies with PAD2 neutralizing activity. PAD2 neutralizing activity may be assessed by (i) preparing a mixture of PAD2 and an anti-PAD2 antibody and incubating it; (ii) adding a PAD2 substrate to the mixture and incubating; or (iii) colorimetrically quantifying the citrulline residues in the substrate. In this case, PAD2 neutralizing activity is demonstrated by a decrease in the amount of citrulline residues after treatment with the anti-PAD2 antibody compared to treatment with a non-anti-PAD2 antibody or a negative control. Anti-PAD2 antibodies may also be prepared by selecting antibodies that exhibit anti-fibrosis activity.

[0020] In one embodiment of the present invention, the anti-PAD2 antibody may be, for example, PK1-16, CK1-10, or CK1-14 described below; S4, S10, S24, S47, S108, S113, S170, and S309 described in WO2019 / 244934 A1; #2, #6, and #34 described in WO2014 / 086365 A1; mSol1, mSol2, mSol3, and mSol4 described in WO2016 / 155745 A1; Ab16478 (Abcam) described in Zhou et al., Front Immunol. 2017 Sep 25;8:1200; DN6, DN18, DN31, and DN34 described in Damgaard et al., J Immunol Methods. 2014 Mar;405:15-22; or Kim et al., Cell Known anti-PAD2 antibodies such as 66386-1-Ig (Proteintech), 0G8 (Creative Diagnostics), 4D4 (Sigma), clone 9F7 (Cayman Chemical), ARG40489 (Arigo), MBS839991 (MyBioSource), AF7257 (Bio-Techne), A11711 (ABclonal, Inc.), or A2322 (BioVision Inc.), which are described in Mol Life Sci. 2022 Feb 26;79(3):155, may also be used.

[0021] In one embodiment of the present invention, the anti-PAD2 antibody includes an antibody that binds to a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or to positions 341 to 357 of PAD2. This antibody may also bind to other amino acid residues within PAD2, so long as it binds to a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or positions 341 to 357 of PAD2. For example, the anti-PAD2 antibody may be an anti-PAD2 antibody that binds to wild-type PAD2 but does not bind to mutant PAD2 (e.g., deletion mutant) consisting of positions 341 to 357. In one embodiment of the present invention, an antibody that specifically binds to a specific site includes an antibody that recognizes a specific site. In one embodiment of the present invention, "having binding ability" may refer to substantial binding ability or significant binding ability. For example, an antibody that binds to an antigen (e.g., a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2) has a KD(M) of 9.0 x 10 for that antigen. -8 The antibody may include antibodies having the following ranges (for example, including the range of KD(M) for the antigens described below). The KD(M) may be a value measured by surface plasmon resonance. In one embodiment of the present invention, "having no binding activity" may mean that the antibody has substantially no binding activity, or includes the case where the antibody has no significant binding activity. Alternatively, the EC 50 Compared to the EC for the evaluation object 50 However, if the EC is, for example, 2, 10, 100, or 10,000 times or more, it may be evaluated as not having binding activity. 50 is not determined, the antibody may be evaluated as having no binding ability. Alternatively, the antibody may be evaluated as having no binding ability when the binding ability to the target antibody is, for example, 50, 30, 10, 5, or 1% or less compared to the binding ability to the wild-type antibody.

[0022] In one embodiment of the present invention, the anti-PAD2 antibody is in the form of a monoclonal antibody, which can act on PAD2 more efficiently than a polyclonal antibody.

[0023] In one embodiment of the present invention, the anti-PAD2 antibody includes an antibody fragment (hereinafter, also referred to as an "antigen-binding fragment") that has PAD2-binding activity. The antigen-binding fragment includes an antigen-binding fragment that has any one of the CDR sets (a) to (c) described below and has PAD2-binding activity.

[0024] In one embodiment of the present invention, the anti-PAD2 antibody has a KD (M) for the antigen of, for example, 9.0 × 10 -8 , 5.0×10 -8 , 1.0×10 -8 , 9.0×10 -9 , 5.0×10 -9 , 1.0×10 -9 , 9.0×10 -10 , 5.0×10 -10 , or 1.0 × 10 -10 The KD(M) may be equal to or less than the above value, or may be within a range between any two of these values. The KD(M) may be a value measured by surface plasmon resonance.

[0025] In one embodiment of the present invention, the antibody class of the anti-PAD2 antibody is not particularly limited and may be, for example, IgM, IgD, IgG, IgA, IgE, or IgY. Furthermore, the antibody subclass is not particularly limited and may be, for example, IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.

[0026] In one embodiment of the present invention, the anti-PAD2 antibody includes an antibody that binds to PAD2 extracellularly. The extracellular may be extracellular within a living body. In one embodiment of the present invention, the anti-PAD2 antibody may be an antibody that binds to or inhibits only PAD2 of the PAD1-6 family. In one embodiment of the present invention, the anti-PAD2 antibody may be used as a monotherapy.

[0027] In one embodiment of the present invention, the anti-PAD2 antibody may be an antibody that binds to a wild-type or mutant form of PAD2. Mutant forms of PAD2 include those resulting from differences in DNA sequence between individuals, such as SNPs.

[0028] In one embodiment of the present invention, the antibody includes an antibody that binds to PAD2. Antibodies include molecules or populations thereof that can specifically bind to a specific epitope on an antigen. The antibody may be a polyclonal or monoclonal antibody. Antibodies can exist in various forms, including full-length antibodies (antibodies having Fab and Fc regions), Fv antibodies, Fab antibodies, F(ab')2 antibodies, Fab' antibodies, diabodies, single-chain antibodies (e.g., scFv), dsFv, multispecific antibodies (e.g., bispecific antibodies), antigen-binding peptides or polypeptides, chimeric antibodies, mouse antibodies, chicken antibodies, humanized antibodies, human antibodies, and their equivalents. The antibody may also be a modified or unmodified antibody. Modified antibodies may be bound to various molecules, such as polyethylene glycol. Modified antibodies can be obtained by chemically modifying antibodies using known techniques. The antibody may also be a fusion protein. A fusion protein may be an antibody having a polypeptide or oligopeptide (e.g., a His tag) attached to the N- or C-terminus thereof. The fusion protein may also be an antibody subsequence fused to a mouse, chicken, or human antibody. The antibody may also be a conjugated antibody (e.g., an antibody-drug conjugate). The drug may be, for example, a cytotoxic drug or an anticancer drug. The antibody may also be a functionally modified antibody. Such modified antibodies, fusion proteins, conjugated antibodies, and functionally modified antibodies are also included as forms of antibodies. The amino acid sequence, class, or subclass of the antibody may be derived from, for example, a human or non-human mammal (e.g., rat, mouse, rabbit, cow, monkey, etc.), or an avian species (e.g., chicken). Antibodies include, for example, isolated antibodies, purified antibodies, and recombinant antibodies. Antibodies may be used, for example, in vitro or in vivo.

[0029] In one embodiment of the present invention, polyclonal antibodies can be produced by administering an immunogen containing an antigen of interest to a mammal (e.g., rat, mouse, rabbit, cow, monkey, etc.) or bird (e.g., chicken). The immunogen may be administered via injection of one or more immunizing agents or adjuvants. Adjuvants may be used to increase the immune response and may include Freund's adjuvant (complete or incomplete), mineral gel (e.g., aluminum hydroxide), or surfactants (e.g., lysolecithin). Immunization protocols are known in the art and may be performed by any method that elicits an immune response, tailored to the host organism of choice (Protein Experiment Handbook, Yodosha (2003): 86-91).

[0030] In one embodiment of the present invention, monoclonal antibodies include antibodies in which the individual antibodies constituting the population react with substantially the same epitope. Alternatively, they may be antibodies in which the individual antibodies constituting the population are substantially identical (allowing for naturally occurring mutations). The method for producing monoclonal antibodies is not particularly limited, and they may be produced by a method similar to the hybridoma method described in "Kohler G, Milstein C., Nature. 1975 Aug. 7;256(5517):495-497." Alternatively, monoclonal antibodies may be produced by a method similar to the recombinant method described in U.S. Pat. No. 4,816,567. Alternatively, monoclonal antibodies may be isolated from a phage antibody library using a method similar to the technique described in "Clackson et al., Nature. 1991 Aug. 15;352(6336):624-628" or "Marks et al., J. Mol. Biol. 1991 Dec. 5;222(3):581-597." Alternatively, it may be prepared by the method described in "Protein Experiment Handbook, Yodosha (2003): 92-96."

[0031] In one embodiment of the present invention, a chimeric antibody is constructed by linking, for example, an antibody variable region and an antibody constant region between different species using genetic engineering techniques. Examples include chimeric antibodies derived from a non-human mammal and a human (e.g., mouse-human chimeric antibodies, chicken-human chimeric antibodies, chicken-mouse chimeric antibodies, etc.). Mouse-human chimeric antibodies can be produced, for example, by the method described in Roguska et al., Proc Natl Acad Sci USA, 1994 Feb 1;91(3):969-973. A basic method for producing a mouse-human chimeric antibody involves ligating a mouse leader sequence and variable region sequences present in a cloned cDNA to a sequence encoding a human antibody constant region already present in an expression vector for mammalian cells. Alternatively, the mouse leader sequence and variable region sequences present in a cloned cDNA may be ligated to a sequence encoding a human antibody constant region, and then ligated into a mammalian expression vector. Fragments of the human antibody constant region can be those of any human antibody H chain constant region and L chain constant region, such as Cγ1, Cγ2, Cγ3, or Cγ4 for human H chains, and Cλ or Cκ for L chains.

[0032] In one embodiment of the present invention, a humanized antibody includes, for example, an antibody that binds to a desired antigen and has one or more CDRs from a non-human species, a framework region from a human immunoglobulin, and a constant region from a human immunoglobulin. Antibody humanization can be performed by various techniques known in the art, for example, as described in "Safdari et al., Biotechnol Genet Eng Rev. 2013;29:175-86."

[0033] In one embodiment of the present invention, a human antibody is an antibody in which the regions constituting the antibody, including the heavy chain variable region and constant region and the light chain variable region and constant region, are derived from a gene encoding human immunoglobulin. Human antibodies can be produced by various techniques known in the art, such as those described in "Duvall et al., MAbs. 2011 Mar-Apr; 3(2): 203-208."

[0034] In one embodiment of the present invention, an Fv antibody is an antibody that contains an antigen-recognition site. This region comprises a non-covalently bound dimer of one heavy-chain variable region and one light-chain variable region. In this configuration, the three CDRs of each variable region can interact to form an antigen-binding site on the surface of the VH-VL dimer.

[0035] In one embodiment of the present invention, the Fab antibody is, for example, an antibody fragment obtained by treating an antibody containing a Fab region and an Fc region with the protease papain, in which approximately the N-terminal half of the H chain and the entire L chain are linked via some disulfide bonds. Fab can be obtained, for example, by treating the anti-PAD2 antibody according to the above-mentioned embodiment of the present invention, which contains a Fab region and an Fc region, with the protease papain.

[0036] In one embodiment of the present invention, the F(ab')2 antibody is an antibody that contains two regions corresponding to Fab, among fragments obtained by treating an antibody containing an Fab region and an Fc region with the protease pepsin. F(ab')2 can be obtained, for example, by treating an anti-PAD2 antibody according to an embodiment of the present invention that contains an Fab region and an Fc region with the protease pepsin. Alternatively, F(ab')2 can be prepared, for example, by forming a thioether bond or disulfide bond with the Fab' shown below.

[0037] In one embodiment of the present invention, a Fab' antibody is an antibody obtained by cleaving the disulfide bond in the hinge region of F(ab')2, for example, by treating F(ab')2 with the reducing agent dithiothreitol.

[0038] In one embodiment of the present invention, the scFv antibody includes an antibody in a form in which the VH and VL are linked via a suitable peptide linker. For example, the scFv antibody can be produced by obtaining cDNA encoding the VH and VL of the anti-PAD2 antibody according to the above-mentioned embodiment of the present invention, constructing a polynucleotide encoding the VH-peptide linker-VL, inserting the polynucleotide into a vector, and using an expression cell.

[0039] In one embodiment of the present invention, a diabody is an antibody in the form of dimerized scFv and has bivalent antigen-binding activity. The bivalent antigen-binding activities can be the same, or one of the two can have a different antigen-binding activity. For example, a diabody can be produced by constructing a polynucleotide encoding an scFv so that the amino acid sequence of the peptide linker is 8 residues or less, incorporating the resulting polynucleotide into a vector, and using an expression cell.

[0040] In one embodiment of the present invention, a dsFv is an antibody in which polypeptides having cysteine ​​residues introduced into VH and VL are linked via a disulfide bond between the cysteine ​​residues. The position at which the cysteine ​​residue is introduced can be selected based on the predicted three-dimensional structure of the antibody according to the method described by Reiter et al. (Reiter et al., Protein Eng. 1994 May;7(5):697-704.).

[0041] In one embodiment of the present invention, the antigen-binding peptide or polypeptide is an antibody comprising an antibody VH, VL, or their CDR1, 2, or 3. A peptide comprising multiple CDRs can be linked directly or via a suitable peptide linker.

[0042] The method for producing the above-mentioned Fv antibodies, Fab antibodies, F(ab')2 antibodies, Fab' antibodies, scFv antibodies, diabodies, dsFv antibodies, and antigen-binding peptides or polypeptides (hereinafter sometimes referred to as "Fv antibodies, etc.") is not particularly limited. For example, DNA encoding the Fv antibody or other domains of the anti-PAD2 antibodies according to the above-mentioned embodiments of the present invention can be inserted into an expression vector and produced using expression cells. Alternatively, they may be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBOC method (t-butyloxycarbonyl method). The antigen-binding fragment according to the above-mentioned embodiments of the present invention may be one or more of the above-mentioned Fv antibodies, etc.

[0043] In one embodiment of the present invention, the anti-PAD2 antibody comprises an antibody having a heavy chain and a light chain. The heavy chain is typically the main component of a full-length antibody. The heavy chain of a full-length antibody typically forms disulfide bonds and non-covalent interactions with the light chain. The heavy chain typically comprises a heavy chain variable region (VH) and a constant region. The light chain is typically a component of a full-length antibody that is different from the heavy chain. The light chain typically comprises a light chain variable region (VL) and a constant region.

[0044] In one embodiment of the present invention, CDRs (complementarity-determining regions) are regions of an antibody that form the antigen-binding site. Typically, CDRs are located on the Fv (variable region) of an antibody. Typically, CDRs include heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3 (sometimes abbreviated as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively). Typically, each CDR contains approximately 3 to 30 amino acid residues. It is known that the heavy chain CDRs are particularly responsible for antibody antigen binding. Among the CDRs, CDR3 is known to contribute most to antibody antigen binding. The Fv region other than the CDRs is called the framework region, which includes FR1, FR2, FR3, and FR4 and is relatively well conserved among antibodies (e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co.).

[0045] In one embodiment of the present invention, the anti-PAD2 antibody includes an antibody having HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 3, 9, or 15, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 4, 10, or 16, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 5, 11, or 17, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 6, 12, or 18, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 7, 13, or 19, or LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 8, 14, or 20. In one embodiment of the present invention, the anti-PAD2 antibody comprises (a) an HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 3, an HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 4, an HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 5, an LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 6, an LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 7, and an LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 8; (b) an HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 9, an HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, an HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 11, and an LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 12; (c) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20 (sometimes abbreviated herein as any of CDR sets (a) to (c)).

[0046] In one embodiment of the present invention, CDRs may be defined according to the Kabat definition (Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)), the IMGT definition (Lefranc et al., Dev Comp Immunol. 2003 Jan;27(1):55-77.), or the Chothia definition (Chothia et al., J. Mol. Biol., 1987;196:901-917). In one embodiment of the present invention, CDRs are preferably defined according to the Kabat definition.

[0047] In one embodiment of the present invention, the anti-PAD2 antibody includes an antibody having a heavy chain variable region or a light chain variable region, wherein the heavy chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 21, 23, or 25, and the light chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 22, 24, or 26. In one embodiment of the present invention, the anti-PAD2 antibody includes an antibody comprising: (d) a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 21 and 22, respectively; (e) a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 23 and 24, respectively; or (f) a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 25 and 26, respectively (sometimes referred to herein as any one of variable region sets (d) to (f) for short).

[0048] In one embodiment of the present invention, the anti-PAD2 antibody includes an antibody comprising a heavy chain constant region or a light chain constant region, wherein the heavy chain constant region may comprise the amino acid sequence set forth in SEQ ID NO:27 and the light chain constant region may comprise the amino acid sequence set forth in SEQ ID NO:28.

[0049] In Example 1 described below, the nucleotide and amino acid sequences of the obtained antibodies were analyzed using a DNA sequencer. The results showed that the CDR amino acid sequences of PK1-16 in Example 1 were: HCDR1: SYAMY (SEQ ID NO: 3), HCDR2: GISSSGRYTGYAPAVKG (SEQ ID NO: 4), HCDR3: DVYDSWTYANRIDA (SEQ ID NO: 5), LCDR1: SGGGRRGYYG (SEQ ID NO: 6), LCDR2: NNDERPS (SEQ ID NO: 7), and LCDR3: GSGDTTTDSGI (SEQ ID NO: 8). The amino acid sequences of the CDRs of CK1-10 in Example 1 were HCDR1: DYGMG (SEQ ID NO: 9), HCDR2: AISNRGSHTYYGAAVKG (SEQ ID NO: 10), HCDR3: DAGTCISSYGFSCVSAASIDA (SEQ ID NO: 11), LCDR1: SGGSGSYGGSYYYG (SEQ ID NO: 12), LCDR2: DNTNRPS (SEQ ID NO: 13), and LCDR3: GSIDSISDADI (SEQ ID NO: 14). The amino acid sequences of the CDRs of CK1-14 in Example 1 were HCDR1: RYAIQ (SEQ ID NO: 15), HCDR2: VINSGGRTLYAPAVKG (SEQ ID NO: 16), HCDR3: GGYAYGIET (SEQ ID NO: 17), LCDR1: SGSRYDYG (SEQ ID NO: 18), LCDR2: YNNKRPS (SEQ ID NO: 19), and LCDR3: GSTDTSNDI (SEQ ID NO: 20). The CDR sequences of PK1-16, CK1-10, and CK1-14 were all defined by the Kabat definition. The amino acid sequences of the variable regions of PK1-16 were VH: AVTLDESGGGLQTPGGGLSLVCKASGFTFRSYAMYWVRQAPGKGLEWLAGISSSGRYTGYAPAVKGRATISRDNGQSTVRLQLSNLRAEDAGTYYCAKDVYDSWTYANRIDAWGHGTEVIVSS (SEQ ID NO: 21) and VL: ALTQPSSVSANPGETVKITCSGGGRRGYYGWYQQKSPGSAPVTVIYNNDERPSNIPSRFSGFKSGSTATLTITGVQAEDEAVYYCGSGDTTTDSGIFGAGTTLTVL (SEQ ID NO: 22).The amino acid sequences of the variable regions of CK1-10 were VH: AVTLDESGGGLQTPGRALSLVCKASGFTFSDYGMGWMRQAPGKGLEWVGAISNRGSHTYYGAAVKGRATISRDNGQSTVRLQLNNLRAEDTGTYYCAKDAGTCISSYGFSCVSAASIDAWGHGTEVIVSS (SEQ ID NO: 23) and VL: ALTQPSSVSANLGGTVKITCSGGSGSYGGSYYYGWYQQKAPGSAPVTLIYDNTNRPSNIPSRFSGSKSGSTATLTITGVQADDEAVYFCGSIDSISDADIFGAGTTLTVL (SEQ ID NO: 24). The amino acid sequences of the variable regions of CK1-14 were VH: AVTLDESGGGLQTPGGALSLVCKASGFTFTRYAIQWVRQAPGKGLEWVGVINSGGRTLYAPAVKGRATISRDNGQSTVRLQLNNLRAEDTAIYYCVRGGYAYGIETWGHGTEVIVSS (SEQ ID NO: 25) and VL: ALTQPSSVSANPGETVKITCSGSRYDYGWYQQKSPGSAPVTLIYYNNKRPSDIPSRFSGSKSGSTHTLTITGVQADDEAVYFCGSTDTSNDIFGAGTTLTVL (SEQ ID NO: 26). The nucleotide sequences corresponding to the amino acid sequences shown in SEQ ID NOs: 3 to 26 were the nucleotide sequences shown in SEQ ID NOs: 29 to 52, respectively.

[0050] In Example 1 described below, expression vectors were used for antibody expression. According to these vectors, the heavy chain constant region and light chain constant region of PK1-16, CK1-10, and CK1-14 had the amino acid sequences shown in SEQ ID NOs: 27 and 28, respectively. Furthermore, the nucleotide sequences corresponding to the amino acid sequences shown in SEQ ID NOs: 27 and 28 were the nucleotide sequences shown in SEQ ID NOs: 53 and 54, respectively.

[0051] In one embodiment of the present invention, the anti-PAD2 antibody includes an antibody that competes with an anti-PAD2 antibody according to an embodiment of the present invention (e.g., an antibody having any of the CDR sets (a) to (c) above) (hereinafter, also referred to as a reference antibody) for binding to PAD2. Competition involves the inhibition of binding of another antibody to the same antigen by binding to the same antigen by an antibody. Competitive antibodies can be identified, for example, by competitive binding assays. Competitive binding assays include, for example, competitive ELISA and competitive FACS analysis (see, for example, Zhou et al., J Gen Virol. 2008 Feb;89(Pt 2):500-508). Binding may also be measured, for example, by surface plasmon resonance. A competitive binding assay may include, for example, coating an antigen on a microplate, adding a test antibody and incubating the plate to allow binding between the antigen and the test antibody, adding a labeled reference antibody to the well, incubating, and washing, or quantifying the amount of binding of the labeled reference antibody to the antigen. In this case, for example, the amount of binding of the biotinylated reference antibody may be detected by measuring absorbance at a wavelength of 450 nm using HRP-conjugated streptavidin and 3,3',5,5'-tetramethylbenzidine. The inhibition rate may be evaluated as the percentage decrease in the amount of binding of the labeled reference antibody compared to when the test antibody is not added or when a negative control is added. Inhibition of the reference antibody by a competing antibody includes 20, 30, 40, 50, 60, 70, 80, 90% or more, or 100% inhibition. This inhibition is preferably 40% or more, and particularly preferably 50% or more. The competing antibody may have binding affinity to the epitope bound by the reference antibody.

[0052] One embodiment of the present invention is a polynucleotide or vector encoding the anti-PAD2 antibody according to the above-described embodiments of the present invention (e.g., an antibody having any one of the CDR sets (a) to (c) above). A transformant can be produced by introducing this polynucleotide or vector into a cell. The transformant may be a human or non-human mammalian cell (e.g., rat, mouse, guinea pig, rabbit, cow, monkey, etc.). Examples of mammalian cells include Chinese hamster ovary cells (CHO cells), monkey COS-7 cells, and human embryonic kidney cells (e.g., HEK293 cells). Alternatively, the transformant may be Escherichia bacteria, yeast, or the like. The polynucleotide or vector may be constructed to be capable of expressing the anti-PAD2 antibody. The polynucleotide or vector may contain components necessary for protein expression, such as a promoter, an enhancer, an origin of replication, or an antibiotic resistance gene.

[0053] Examples of the vector that can be used include E. coli-derived plasmids (e.g., pET-Blue), Bacillus subtilis-derived plasmids (e.g., pUB110), yeast-derived plasmids (e.g., pSH19), animal cell expression plasmids (e.g., pA1-11, pcDNA3.1-V5 / His-TOPO), bacteriophages such as λ phage, and virus-derived vectors. The vector may be an expression vector or may be circular.

[0054] Methods for introducing the above-mentioned polynucleotides or vectors into cells include, for example, the calcium phosphate method, lipofection, electroporation, adenovirus-based methods, retrovirus-based methods, and microinjection (New Genetic Engineering Handbook, 4th revised edition, Yodosha (2003): 152-179). Antibodies can be produced using cells, for example, by the method described in Protein Experiment Handbook, Yodosha (2003): 128-142.

[0055] One embodiment of the present invention is a method for producing an anti-PAD2 antibody, comprising the step of growing cells containing the polynucleotide or vector according to any one of the above embodiments of the present invention. The growing step comprises a culturing step. This production method may also comprise the step of recovering the anti-PAD2 antibody. This production method may also comprise the step of preparing a cell culture medium. This production method may also comprise the step of purifying the anti-PAD2 antibody.

[0056] In one embodiment of the present invention, antibodies can be purified using, for example, ammonium sulfate, ethanol precipitation, protein A, protein G, gel filtration chromatography, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, or lectin chromatography (Protein Experiment Handbook, Yodosha (2003): 27-52).

[0057] One embodiment of the present invention is a composition comprising an anti-PAD2 antibody. This composition can be used to inhibit fibrosis. In one embodiment of the present invention, the composition includes, for example, a pharmaceutical composition. The composition includes, for example, a composition for treating fibrosis. The composition may include one or more pharmaceutically acceptable carriers. The form of the carrier is not particularly limited and may be, for example, solid or liquid. The carrier may be, for example, a buffer or excipient. The content of the carrier may be, for example, a pharmaceutically effective amount. The effective amount may be, for example, an amount sufficient for pharmaceutical stability or delivery of the active ingredient. For example, a buffer is effective for stabilizing the active ingredient in the vial. The composition preferably contains a therapeutically effective amount, or an amount effective to exert the desired effect (e.g., fibrosis inhibitory effect). The pharmaceutical composition may be used in any form as long as it is used for treatment. It may contain the active ingredient alone or a mixture of the active ingredient and optional ingredients. The form of the composition is not particularly limited and may be, for example, a liquid or lyophilized formulation. As described above, the anti-PAD2 antibody contained in the composition may be a PAD2 neutralizing antibody.

[0058] One embodiment of the present invention is a pharmaceutical composition for treating fibrosis, comprising an antibody that specifically binds to PAD2 and a pharmaceutically acceptable carrier. This pharmaceutical composition can be used to treat fibrosis.

[0059] One embodiment of the present invention is a method for suppressing or treating fibrosis in a subject in need thereof, comprising the step of administering to the subject an effective amount of a pharmaceutical composition comprising an antibody that specifically binds to PAD2 and a pharmaceutically acceptable carrier. This method can suppress or treat fibrosis. One embodiment of the present invention is a pharmaceutical composition for use in suppressing or treating fibrosis in a subject in need thereof, comprising an antibody that specifically binds to PAD2 and a pharmaceutically acceptable carrier. This pharmaceutical composition can suppress or treat fibrosis. One embodiment of the present invention is the use of an anti-PAD2 antibody to produce a pharmaceutical composition for suppressing or treating fibrosis. The anti-PAD2 antibody used in these methods, pharmaceutical compositions, and uses may be a PAD2 neutralizing antibody, as described above.

[0060] One embodiment of the present invention is a composition for inhibiting PAD2 activity, comprising an anti-PAD2 antibody according to the above-described embodiment of the present invention (e.g., an antibody having any of the CDR sets (a) to (c) above). One embodiment of the present invention is a method for inhibiting PAD2 activity, comprising the step of contacting PAD2 with the anti-PAD2 antibody according to the above-described embodiment of the present invention.

[0061] One embodiment of the present invention is a kit comprising an anti-PAD2 antibody. Fibrosis can be suppressed using this kit. This kit includes, for example, a kit for suppressing or treating fibrosis. This kit may include, for example, the above-described composition, and may also include an instruction manual, a buffer solution, a container, or packaging.

[0062] In one embodiment of the present invention, treatment may involve exerting an effect of improving, alleviating, suppressing, suppressing recurrence, or preventing one or more symptoms of a subject. In one embodiment of the present invention, treatment of fibrosis includes suppressing fibrosis. In one embodiment of the present invention, suppressing fibrosis may result in treatment of fibrosis, treatment of a disease accompanied by fibrosis, or treatment of a disease caused by fibrosis. In one embodiment of the present invention, a disease accompanied by fibrosis includes, for example, cirrhosis (e.g., liver cirrhosis).

[0063] The route of administration of the antibody or composition to a subject is preferably one that is effective for treatment, and may be, for example, intravenous, subcutaneous, intramuscular, or intraperitoneal. The dosage form is preferably one that is effective for treatment, and may be, for example, an injection. The aqueous solution for injection may be stored, for example, in a vial or a stainless steel container. The aqueous solution for injection may also be blended with, for example, physiological saline, sugar (e.g., trehalose), NaCl, NaOH, or the like. The composition may also be blended with, for example, an effective amount of a buffer (e.g., phosphate buffer), a pH adjuster, a stabilizer, or the like.

[0064] The dose, administration interval, and administration method of the antibody or composition can be selected appropriately depending on the age, body weight, symptoms, target organ, etc. of the patient. The dose may be, for example, 0.01 to 200 mg / kg body weight per administration. The administration interval may be, for example, once or twice every 1 to 28 days.

[0065] In one embodiment of the present invention, the subject (including the patient) is a human or non-human mammal (e.g., one or more of a mouse, guinea pig, hamster, rat, mouse, rabbit, pig, sheep, goat, cow, horse, cat, dog, marmoset, monkey, or chimpanzee, etc.). The patient may be a patient diagnosed with fibrosis or a patient in need of treatment.

[0066] In one embodiment of the present invention, the inhibitory or therapeutic effect on fibrosis may be evaluated, for example, by measuring the reduction in the amount or proportion of fibrotic tissue in an individual or tissue after administration of an anti-PAD2 antibody. The inhibitory or therapeutic effect on fibrosis may be evaluated by pathological diagnosis. In pathological diagnosis, collected biopsy tissue sections may be stained. Staining may be performed, for example, using Sirius Red staining. Furthermore, the amount or proportion of fibrotic tissue may be calculated by image analysis of the staining results. An inhibitory or therapeutic effect may be determined if the amount or proportion of fibrotic tissue after administration of an anti-PAD2 antibody is significantly reduced compared to before administration or after administration of a negative control. In one embodiment of the present invention, the inhibitory or therapeutic effect on fibrosis may be measured, for example, using the amount of a fibrosis marker (e.g., α-SMA) in a subject or a sample derived from the subject as an index. In this case, an inhibitory or therapeutic effect may be determined if the amount of the marker after administration of an anti-PAD2 antibody is significantly reduced compared to before administration of the antibody or after administration of a negative control.

[0067] In one embodiment of the present invention, the inhibitory effect on PAD2 activity may be measured, for example, using the amount of a citrullinated compound (e.g., a protein or peptide) in a subject or a sample derived from the subject as an index. In this case, the inhibitory effect may be determined to exist if the amount of the citrullinated compound after anti-PAD2 antibody treatment is significantly reduced compared to before antibody treatment or after negative control treatment. Alternatively, the inhibitory effect may be determined to exist if the amount of the citrullinated compound after anti-PAD2 antibody treatment is reduced to 90, 70, 50, 30, or 10% or less, or to 0%, compared to before antibody treatment or after negative control treatment.

[0068] One embodiment of the present invention is a composition comprising an antibody that specifically binds to PAD2 extracellularly. Use of this composition can suppress fibrosis, as demonstrated in the Examples described below. In one embodiment of the present invention, the anti-PAD2 antibody includes an antibody that inhibits PAD2 activity extracellularly, or an antibody that exists extracellularly. Anti-PAD2 antibodies include, for example, antibodies that exist extracellularly but not intracellularly after administration to a subject. Anti-PAD2 antibodies include, for example, antibodies that inhibit PAD2 activity extracellularly but do not inhibit PAD2 activity intracellularly after administration to a subject. Anti-PAD2 antibodies include, for example, antibodies that inhibit substrate citrullination extracellularly but do not inhibit substrate citrullination intracellularly after administration to a subject.

[0069] In one embodiment of the present invention, "amino acid" is a general term for organic compounds having an amino group and a carboxyl group. When an antibody according to an embodiment of the present invention comprises a "specific amino acid sequence," any amino acid in the amino acid sequence may be chemically modified. Furthermore, any amino acid in the amino acid sequence may form a salt or solvate. Furthermore, any amino acid in the amino acid sequence may be L- or D-form. Even in such cases, the antibody according to an embodiment of the present invention can be said to comprise the "specific amino acid sequence." Examples of chemical modifications that amino acids contained in proteins undergo in vivo include N-terminal modifications (e.g., acetylation, myristoylation, etc.), C-terminal modifications (e.g., amidation, glycosylphosphatidylinositol addition, etc.), and side chain modifications (e.g., phosphorylation, glycosylation, etc.).

[0070] In one embodiment of the present invention, the bond may be either a covalent bond or a non-covalent bond, for example, an ionic bond, a hydrogen bond, a hydrophobic interaction, or a hydrophilic interaction.

[0071] In one embodiment of the present invention, "significantly" may mean, for example, that a statistically significant difference is evaluated using a Student's t-test (one-tailed or two-tailed) or a Bonferroni multiple comparison test, and that the difference is p<0.001, p<0.05, or p<0.01. Alternatively, it may mean that a substantial difference is present.

[0072] All publications cited herein are incorporated by reference in their entirety. In this specification, "or" is used when "at least one or more" of the items listed in the text can be employed. The same applies to "alternative." In this specification, when it is stated that "within a range of two values," the range also includes the two values ​​themselves. In this specification, "A to B" includes A and B.

[0073] Although the embodiments of the present invention have been described above, these are merely examples of the present invention, and various other configurations may be adopted. Furthermore, the configurations described in the above embodiments may be combined and adopted.

[0074] The present invention will be further explained below with reference to examples, but is not limited to these.

[0075] Example 1: Production of anti-PAD2 antibodies Three 3-month-old Boris Brown chicks were intraperitoneally immunized with 333 μg of KLH-modified peptide antigen (SEQ ID NO: 2, positions 341 to 357 of PAD2). Complete Freund's adjuvant (014-09541, Wako) was used for the primary immunization, and incomplete Freund's adjuvant (011-09551, Wako) was used for the secondary and tertiary immunizations. The antigen diluted in phosphate buffered saline (PBS) was injected intravenously for the quaternary immunization. Blood was collected from the underwing vein every two weeks, and antibody titers were confirmed by ELISA. Immunizations were repeated up to the third immunization, with the fourth immunization being the final immunization. Three days after the final immunization, chicken spleens were harvested, lymphocytes were isolated by density gradient centrifugation using Ficoll paque PLUS (17-1440-03, Cytiva), and RNA was extracted using TRIzole Reagent (15596026, Life Technologies). cDNA was synthesized from the extracted RNA by RT-PCR using the PrimeScript II 1st Strand cDNA Synthesis Kit (6210A, TAKARA) to generate an scFv phage library. The pPDS vector was used as the expression vector. The scFv phage library was constructed according to the method described in Nakamura et al., J Vet Med Sci. 2004 Ju;66 (7): 807-814.

[0076] Panning was performed using an scFv phage antibody library on plates immobilized with full-length PAD2. Panning was performed according to the method described in Nakamura et al., J Vet Med Sci. 2004 Ju;66 (7): 807-814. After five rounds of panning, library reactivity was confirmed by ELISA using plates immobilized with synthetic peptides. Phages from libraries showing increasing reactivity were screened. For screening, phage were infected into Escherichia coli and plated on 2xYT agar plates containing ampicillin (50 μg / ml, Nacalai). The resulting colonies were cultured in ampicillin-containing 2xYT liquid medium. After infection with helper phage, phage induction was performed in 2xYT liquid medium containing ampicillin (50 μg / ml), kanamycin (25 μg / ml, Meiji Seika Kaisha), and IPTG (100 μg / ml, Nacalai). The reactivity of the scFv phage antibodies in the culture supernatant was confirmed by ELISA using an antigen-coated plate, and the obtained positive clones were sequenced to determine their sequences.

[0077] To obtain clones with different sequences, the heavy and light chain variable regions of chicken-derived antibody genes were amplified by PCR using the DNA strand encoding the scFv antibody as a template. The amplified fragments were then inserted by homologous recombination into pre-constructed expression vectors containing human heavy chain constant regions (IgG1) and human light chain constant regions using Seamless Cloning and Assembly Enzyme Mix (Thermo, A14606). The resulting heavy and light chain constructs were transfected into 293 cells, and their reactivity was confirmed by ELISA using full-length PAD2 as an immobilized antibody. Of the antibody clones obtained, PK1-16, CK1-10, and CK1-14 were used in the following experiments.

[0078] Example 2: Human PAD2 Binding Affinity of Anti-PAD2 Antibodies To evaluate the binding affinity of anti-PAD2 antibodies to human PAD2 protein, the dissociation constant (KD) was measured using a surface plasmon resonance system Biacore 8K (Cytiva). A Series S Sensor Chip Protein A (Cytiva) was used as the sensor chip. HBS-EP+ buffer was used as the running buffer. The test antibody was prepared at 0.8 μg / mL using the running buffer to serve as the ligand solution. The ligand solution was added to the flow cell at a flow rate of 10 μL / min for 60 seconds, and a flow cell without the ligand solution served as the reference cell. Human PAD2 protein was prepared at concentrations of several hundred pM to several tens of nM using the running buffer to serve as the analyte solution. The running buffer was also used as the blank solution. The blank solution or analyte solution was added for 120 seconds at a flow rate of 30 μL / min using the single-cycle method, with a dissociation time of 1200 seconds. The sensorgram of the reference cell was subtracted from the sensorgram of the flow cell to which the ligand was added when a blank solution or an analyte solution was added, and the sensorgram of the blank solution was subtracted from the sensorgram of the analyte solution. TM Binding parameters were calculated using Insight Evaluation Software (Cytiva) using a 1:1 binding model. The results are shown in Figure 1. The anti-PAD2 antibody showed good affinity.

[0079] Example 3: Inhibition of PAD2 Activity by Anti-PAD2 Antibodies The ability of anti-PAD2 antibodies to inhibit human PAD2 activity was evaluated. Human PAD2 (final concentration 10 nM) was mixed with 20 mM Tris buffer (pH 7.6) containing 1 mM DTT, 150 mM NaCl, and 0.3 mM CaCl2 to achieve final anti-PAD2 antibody concentrations of 600, 200, 66.67, 22.22, 7.41, 2.47, 0.82, 0.27, and 0.09 nM. After incubation at 37°C for 1 hour, BAEE (benzoylarginine ethyl ester) was added with stirring and mixed thoroughly (final concentration of BAEE: 10 mM). After incubation at 37°C for 3 hours, the citrullinated BAEE citrulline residues were quantified colorimetrically using a mixture containing 2,3-butanedione monoxime and thiosemicarbazide. Anti-dinitrophenyl (DNP) antibody was used as a control. The results are shown in Figure 2. The anti-PAD2 antibody inhibited human PAD2 activity in a concentration-dependent manner.

[0080] Example 4: Anti-fibrosis in a fibrosis model using anti-PAD2 antibodies 4.1 Preparation of a fibrosis model and administration of anti-PAD2 antibodies The efficacy of anti-PAD2 antibodies was evaluated using a fibrosis model. First, a mouse model of bile duct ligation-induced cholestatic liver disease (BDL) was prepared using the following procedure, and anti-PAD2 antibodies were administered. Seven-week-old male C57BL / 6J mice were anesthetized by intraperitoneal administration of a triple anesthesia mixture (medetomidine hydrochloride / mitazolam / butorphanol tartrate) at 0.75 / 4 / 5 mg / kg. After confirming anesthesia, the animal's hair was shaved around the xiphoid process of the abdomen using clippers. The shaved area was disinfected, and an approximately 1 cm laparotomy was performed. The bile duct in the duodenal region was exposed using a cotton swab or similar. The common bile duct was ligated in two locations using 7-0 silk sutures with sterilized Nesco suture needles. After ligation, the bile duct was not severed. Instead, it was returned to its original position, and the peritoneum was sutured with 3-0 silk sutures attached to a sterilized Nesco suture needle. The epidermis was sutured using an automatic suture device equipped with a suture clip. Atipamezole hydrochloride was administered intraperitoneally at 0.75 mg / kg, and the mice were then awakened and returned to their cages. They were maintained on a warming pad until fully awake from anesthesia. One hour after surgery, and on days 4, 7, and 11, anti-PAD2 antibody or control antibody (anti-DNP antibody) was administered intravenously (25 mg / kg). Sham-operated mice (thoracotomy but no bile duct ligation) served as the control group. Ten control mice, five anti-DNP antibody groups, and seven anti-PAD2 antibody groups were used for the final analysis.

[0081] 4.2 Measurement of Fibrosis Rate Liver tissues harvested on postoperative day 13 were immersed in Bouin's fixative, fixed for 24 hours at room temperature, and then embedded in paraffin. Paraffin sections were prepared and deparaffinized and hydrophilized using xylene, a 100% to 70% alcohol series, and RO water. They were then infiltrated in 0.03% Picro Sirius Red solution (Fujifilm Wako Pure Chemical Industries, Ltd.) for 60 minutes. After passing through 0.5% acetic acid solution and RO water, the stained sections were dehydrated and cleared using a 70% to 100% alcohol series and xylene, then mounted in Entelaneu (Merck Co., Ltd.) and subjected to observation. Photographs of the specimens were taken using a bright-field microscope with a CCD camera (Leica Microsystems) at 100x magnification, with five fields of view per section. Based on the captured images, the photographed area and Sirius Red-positive area in each field were measured using ImageJ software (National Institute of Health), and the fibrotic area was calculated. The results are shown in Figure 3. Administration of anti-PAD2 antibody significantly suppressed fibrosis.

[0082] 4.3 Measurement of PAD2 Activity Inhibition. Livers were harvested 13 days after surgery, and tissue proteins were extracted with RIPA buffer. Equal amounts of total protein (50 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to a PVDF membrane. The membrane was incubated with a primary antibody (Anti-peptidyl-citrulline, clone F95 Antibody; Sigma-Aldrich) followed by a horseradish peroxidase-conjugated secondary antibody (Goat anti-Mouse IgM Secondary Antibody HRP; Novus Biologicals). Finally, citrullinated proteins were detected by chemiluminescence (ECL™ Prime Western Blotting Detection Reagent; Cytiva). The results are shown in Figure 4. Administration of anti-PAD2 antibody inhibited PAD2 activity and suppressed the increase in citrullinated proteins.

[0083] As described in the above Examples, experiments have demonstrated that anti-PAD2 antibodies suppress fibrosis.

[0084] The present invention has been described above based on the embodiments. However, these embodiments are merely illustrative, and it will be understood by those skilled in the art that various modifications are possible and that such modifications are also within the scope of the present invention.

Claims

1. A composition for inhibiting fibrosis, comprising an antibody that specifically binds to PAD2 (Peptidylarginine deiminase 2).

2. The composition of claim 1, wherein the antibody is a neutralizing antibody to PAD2.

3. The composition of claim 1, wherein the antibody is an antibody that inhibits fibrosis.

4. The composition of any one of claims 1 to 3, wherein the antibody is an antibody that binds to PAD2 extracellularly.

5. The composition of claim 1 , wherein the antibody is a monoclonal antibody.

6. The antibody has a KD (M) of 9.0 × 10 for PAD2. -9 The composition of claim 1, wherein the antibody is:

7. The composition of claim 1, wherein the antibody is an antigen-binding fragment.

8. A pharmaceutical composition for treating fibrosis, comprising an antibody that specifically binds to PAD2 and a pharmaceutically acceptable carrier.

9. A kit for inhibiting fibrosis, comprising an antibody that specifically binds to PAD2.