New regulation of anti-PAD4 antibodies

Abstract

To provide an anti-PAD4 antibody that binds to active PAD4 or an anti-PAD4 antibody that inhibits the functions of active PAD4.SOLUTION: The present invention provides an anti-PAD4 antibody binding specifically to a peptide consisting of a specific amino acid sequence, or an anti-PAD4 antibody binding specifically to positions 633-645 of human PAD4, which binds to active PAD4 or inhibits its functions. This anti-PAD4 antibody optionally may inhibit the citrullination activity of PAD4.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an anti-PAD4 antibody. [Background technology]

[0002] PAD4 (peptidylarginine deiminase 4) is known to be an enzyme involved in the citrullination of arginine in proteins. This citrullination is important for the structure and reaction of proteins because it converts arginine, the most basic amino acid in proteins, into neutral citrulline.

[0003] There are several reports showing that PAD4 is involved in the pathology of rheumatoid arthritis (RA) and arthritis. For example, Non-Patent Document 1 reports that there is a correlation between the onset of RA and a single nucleotide polymorphism in the PAD4 gene. Non-Patent Document 2 reports that an anti-PAD4 antibody was used to diagnose RA. Patent Document 1 describes an attempt to suppress RA by administering a mixture of four types of anti-PAD4 antibodies to mice. Patent Document 2 describes that RA or arthritis was suppressed by administering an anti-PAD4 antibody that binds to a specific epitope to mice.

[0004] There are two types of PAD4: active PAD4 and inactive PAD4. The active type is Ca 2+ It binds Ca 2+ It is also called bound PAD4. The inactive form is Ca 2+ It does not bind Ca 2+ It is also called unbound PAD4.

[0005] PAD4 and Ca 2+For example, Patent Document 3 is a report on this. Patent Document 3 describes an attempt to measure the amount of PAD4 in the serum of healthy subjects and patients with rheumatoid arthritis by ELISA using an anti-PAD4 antibody. In the examples of Patent Document 3, it is described that when performing ELISA, appropriate measurement values ​​could not be obtained when untreated serum was used, but appropriate measurement values ​​were obtained when EDTA-treated serum was used. At this time, EDTA was added with Ca 2+ It is thought that as a result of trapping, inactive PAD4 reacted with the anti-PAD4 antibody. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO / 2012 / 026309 [Patent Document 2] WO / 2016 / 143753 [Patent Document 3] JP5252339 (B2) [Non-patent literature]

[0007] [Non-Patent Document 1] "Functional haplotypes of PADI4, encoding citrullinating enzyme peptidylarginine deiminase 4, are associated with rheumatoid arthritis.", Suzuki et al., Nat Genet. 2003 Aug;34(4):395-402. [Non-Patent Document 2] "Two novel sandwich ELISAs identify PAD4 levels and PAD4 autoantibodies in patients with rheumatoid arthritis.", Ishigami et al., Mod Rheumatol. 2013 Jul;23(4):794-803. Summary of the Invention [Problem to be solved by the invention]

[0008] Although antibodies that bind to inactive PAD4 have been obtained, no antibodies that bind to active PAD4 have been obtained, and no antibodies that inhibit the function of active PAD4 have been obtained.

[0009] The present invention has been made in consideration of the above circumstances, and aims to provide an anti-PAD4 antibody that binds to active PAD4 or an anti-PAD4 antibody that inhibits the function of active PAD4. [Means for solving the problem]

[0010] As described in the Examples below, the present inventors have surprisingly found that an anti-PAD4 antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 (corresponding to positions 633 to 645 of PAD4) binds to active PAD4 and inhibits its function. Based on these results, the present invention has been completed.

[0011] That is, according to one aspect of the present invention, an anti-PAD4 antibody that binds to active PAD4 is provided. This antibody can be used to detect active PAD4. This antibody may be an antibody that inhibits the function of active PAD4.

[0012] According to another aspect of the present invention, there is provided an anti-PAD4 antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1, or an anti-PAD4 antibody that specifically binds to positions 633 to 645 of human PAD4. The antibody can be used to detect or inhibit the function of active PAD4.

[0013] Furthermore, according to one aspect of the present invention, there is provided a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1. This peptide can be used to produce a binding antibody or a function-inhibiting antibody against activated PAD4. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 shows the amino acid sequences of the variable regions of the antibodies according to the examples. [Diagram 2] FIG. 2 shows the base sequences of the variable regions of the antibodies according to the examples. [Diagram 3] FIG. 3 shows the base sequences of the variable regions of the antibodies according to the examples. [Figure 4] FIG. 4 is a graph showing the results of investigating the citrullination rate when an antibody according to an example was used. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Hereinafter, the embodiments of the present invention will be described in detail. Note that, in order to avoid repetition and complexity, the description of similar contents will be omitted as appropriate.

[0016] One embodiment of the present invention is a novel anti-PAD4 antibody. This antibody may be, for example, an anti-PAD4 antibody that binds to active PAD4. This antibody can be used to detect, for example, active PAD4. This antibody may be, for example, an antibody that inhibits the function of active PAD4. This antibody can be used to inhibit, for example, the citrullination activity of active PAD4. This antibody can also be used to treat, for example, rheumatoid arthritis (RA) or arthritis. This treatment method uses an antibody, so it has few side effects and is excellent in terms of safety.

[0017] The anti-PAD4 antibody according to one embodiment of the present invention may be, for example, an anti-PAD4 antibody that specifically binds to the peptide shown in SEQ ID NO: 1, or an anti-PAD4 antibody that specifically binds to positions 633 to 645 of human PAD4. This antibody can be used to detect, for example, active PAD4. This antibody can also be used to inhibit, for example, the citrullination activity of active PAD4. This antibody can also be used to treat, for example, RA or arthritis. This treatment method is excellent in terms of safety, with fewer side effects due to the use of an antibody.

[0018] PAD4 is generally known as an enzyme involved in the citrullination of arginine in proteins. Details of the amino acid sequence of PAD4, etc., can be found on the websites of NCBI (National Center for Biotechnology Information) or HGNC (HUGO Gene Nomenclature Committee), etc. The accession number of PAD4 listed in NCBI is, for example, NP_036519.2. The amino acid sequence of human PAD4 is, for example, SEQ ID NO: 2. The biological origin of PAD4 is not limited as long as it has PAD4 activity. In the present specification, PAD4 may be active PAD4 or inactive PAD4. In the present specification, active PAD4 is Ca 2+ The inactive PAD4 may be a Ca-bound PAD4. 2+ The active PAD4 may be a Ca 2+ It contains PAD4 that is activated by binding to it.

[0019] In one embodiment of the present invention, the "anti-PAD4 antibody" includes an antibody having binding activity to PAD4. The method for producing this anti-PAD4 antibody is not particularly limited, and for example, the antibody may be produced by immunizing a mammal or bird with a peptide consisting of the amino acid sequence shown in SEQ ID NO:1.

[0020] The anti-PAD4 antibody according to one embodiment of the present invention may be an antibody that inhibits the function of PAD4. The function includes, for example, citrullination activity. In one embodiment of the present invention, the anti-PAD4 antibody that binds to active PAD4 includes an antibody that has binding affinity to inactive PAD4.

[0021] The anti-PAD4 antibody according to one embodiment of the present invention may be a monoclonal antibody. A monoclonal antibody can act on PAD4 more efficiently than a polyclonal antibody. From the viewpoint of efficiently producing an anti-PAD4 monoclonal antibody having a desired effect, it is preferable to immunize chickens with PAD4. Unless otherwise specified, the PAD4 used as an antigen includes full-length PAD4 or a PAD4 peptide fragment.

[0022] The anti-PAD4 antibody according to one embodiment of the present invention is not limited to a full-length antibody, but includes an antibody fragment having PAD4 binding activity (hereinafter, also referred to as an "antigen-binding antibody fragment"). The antigen-binding antibody fragment has the effect of increasing the stability or production efficiency of the antibody.

[0023] The anti-PAD4 antibody according to one embodiment of the present invention may be a fusion protein. This fusion protein may be one in which a polypeptide or oligopeptide is bound to the N- or C-terminus of PAD4. Here, the oligopeptide may be a His tag. The fusion protein may also be one in which a mouse, human, or chicken antibody partial sequence is fused. Such fusion proteins are also included in one embodiment of the anti-PAD4 antibody according to this embodiment.

[0024] The anti-PAD4 antibody according to one embodiment of the present invention may be an antibody obtained by immunizing chickens with PAD4. It may also be an antibody having a CDR set of an antibody obtained by immunizing chickens with PAD4. The CDR set is a set of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3.

[0025] The anti-PAD4 antibody according to one embodiment of the present invention has a KD(M) of, for example, 9.9×10 -8 , 9.0×10 -8 , 8.0×10 -8 , 7.0×10 -8 , 6.0×10 -8 , 5.0×10 -8 , 4.0×10 -8 , 3.0×10 -8, 2.0×10 -8 , or 1.0×10 -8 From the viewpoint of enhancing the therapeutic effect of the antibody, the KD(M) is preferably 9.0×10 or less, or may be within the range of any two of these values. -8 The following is preferred:

[0026] The anti-PAD4 antibody according to one embodiment of the present invention may be an antibody that binds to wild-type or mutant PAD4. Mutations include those resulting from differences in DNA sequences between individuals, such as SNPs. The amino acid sequence of wild-type or mutant PAD4 may conserve positions 633 to 645 with respect to the amino acid sequence shown in SEQ ID NO: 2, and may have a homology of preferably 95% or more, particularly preferably 98% or more. The anti-PAD4 antibody according to one embodiment of the present invention may be an antibody that has or does not have a binding ability to mouse PAD4.

[0027] An anti-PAD4 antibody according to one embodiment of the present invention may be an antibody that has binding affinity to wild-type PAD4 and has no binding affinity to mutant PAD4 having a mutation in human PAD4 at positions 633 to 645. "Having no binding affinity" may mean that the antibody has substantially no binding affinity.

[0028] An anti-PAD4 antibody according to one embodiment of the present invention may be an antibody obtained by a production method including a step of selecting an antibody that shows significant reactivity to wild-type PAD4, or a step of selecting an antibody that does not bind to mutant PAD4 having a mutation at positions 633 to 645 of human PAD4.

[0029] In one embodiment of the present invention, an antibody that specifically binds to positions 633 to 645 of human PAD4 includes an antibody that has or does not have binding affinity to other amino acid residues, so long as it has binding affinity to positions 633 to 645 of human PAD4. An antibody that specifically binds to a specific site may be an antibody that recognizes a specific site.

[0030] An anti-PAD4 antibody according to one embodiment of the present invention may be an antibody that specifically binds to an epitope including positions 2, 6, 7, and 8 of a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1. An anti-PAD4 antibody according to one embodiment of the present invention may be an antibody that specifically binds to an epitope including positions 634, 638, 639, and 640 of human PAD4.

[0031] In one embodiment of the present invention, the term "antibody" includes a molecule or a group thereof that can specifically bind to a specific epitope on an antigen. The antibody may be a polyclonal antibody or a monoclonal antibody. The form of the antibody is not particularly limited, and includes, for example, one or more forms selected from the group consisting of a full-length antibody (an antibody having a Fab region and an Fc region), an Fv antibody, a Fab antibody, an F(ab')2 antibody, an Fab' antibody, a single-chain antibody (e.g., scFv), a dsFv, an antigen-binding peptide, an antibody-like molecule, a chimeric antibody, a mouse antibody, a chicken antibody, a humanized antibody, a human antibody, or an equivalent thereof (or an equivalent thereof). The antibody also includes modified or unmodified antibodies. The modified antibody may be bound to an antibody with various molecules such as polyethylene glycol. The modified antibody can be obtained by chemically modifying the antibody using a known method. The amino acid sequence, class, or subclass of the antibody may be derived from, for example, human, mammals other than human (e.g., rat, mouse, rabbit, cow, monkey, etc.), or birds (e.g., chicken). The antibody class is not particularly limited, and may be, for example, IgM, IgD, IgG, IgA, IgE, or IgY. The antibody subclass is not particularly limited, and may be, for example, IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. Furthermore, the antibody includes, for example, an isolated antibody, a purified antibody, or a recombinant antibody. Furthermore, the antibody may be used, for example, in vitro or in vivo.

[0032] In one embodiment of the present invention, a "polyclonal antibody" can be produced by administering an immunogen containing an antigen of interest to, for example, a mammal (e.g., rat, mouse, rabbit, cow, monkey, etc.) or bird (e.g., chicken). The administration of the immunogen may be by 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 surfactant (e.g., lysolecithin), etc. Immunization protocols are known in the art and may be performed by any method that induces an immune response depending on the host organism selected (Protein Experiment Handbook, Yodosha (2003):86-91.).

[0033] In one embodiment of the present invention, the term "monoclonal antibody" includes antibodies in which each antibody constituting the population reacts with substantially the same epitope. Alternatively, the term may refer to antibodies in which each antibody constituting the population is substantially identical (allowing for naturally occurring mutations). Monoclonal antibodies are highly specific and differ from conventional polyclonal antibodies, which typically contain different antibodies corresponding to different epitopes. There is no particular limitation on the method for producing monoclonal antibodies, 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 phage antibody libraries using techniques similar to those 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, they may be produced by the methods described in "Protein Experiment Handbook, Yodosha (2003):92-96."

[0034] In one embodiment of the present invention, an "Fv antibody" is an antibody that includes an antigen recognition site. This region includes a dimer of one heavy chain variable domain and one light chain variable domain that are non-covalently bound. In this configuration, the three CDRs of each variable domain can interact with each other to form an antigen binding site on the surface of the VH-VL dimer. In one embodiment of the present invention, a "Fab antibody" is, for example, an antibody in which about the N-terminal half of the H chain and the entire L chain are bound via some disulfide bonds, among fragments obtained by treating an antibody including a Fab region and an Fc region with the protease papain. Fab can be obtained, for example, by treating an anti-PAD4 antibody according to the above embodiment of the present invention, including a Fab region and an Fc region, with the protease papain.

[0035] In one embodiment of the present invention, the "F(ab')2 antibody" is, for example, an antibody that contains two sites corresponding to Fab among fragments obtained by treating an antibody containing a Fab region and an Fc region with a protease pepsin. F(ab')2 can be obtained, for example, by treating an anti-PAD4 antibody according to the embodiment of the present invention that contains a Fab region and an Fc region with a protease pepsin. In addition, for example, it can be prepared by forming a thioether bond or disulfide bond with the following Fab'. In one embodiment of the present invention, the "Fab' antibody" is, for example, an antibody obtained by cleaving a disulfide bond in the hinge region of F(ab')2. For example, it can be obtained by treating F(ab')2 with a reducing agent dithiothreitol.

[0036] In one embodiment of the present invention, the "scFv antibody" is an antibody in which VH and VL are linked via a suitable peptide linker. The scFv antibody can be produced, for example, by obtaining cDNA encoding the VH and VL of the anti-PAD4 antibody according to the embodiment of the present invention, constructing a polynucleotide encoding VH-peptide linker-VL, incorporating the polynucleotide into a vector, and using an expression cell. In one embodiment of the present invention, the "dsFv" is an antibody in which polypeptides in which cysteine ​​residues have been introduced into VH and VL are linked via disulfide bonds between the cysteine ​​residues. The position at which the cysteine ​​residue is introduced can be selected based on the three-dimensional structure prediction of the antibody according to the method shown by Reiter et al. (Reiter et al., Protein Eng. 1994 May;7(5):697-704.).

[0037] In one embodiment of the present invention, the "antigen-binding peptide" includes, for example, a peptide that is composed of VH and VL of an antibody, or CDR1, 2, and 3 thereof, and has binding ability to PAD4. A peptide containing multiple CDRs can be linked directly or via a suitable peptide linker. In one embodiment of the present invention, the "antibody-like molecule" is a molecule that has binding ability to PAD4, and includes, for example, affibodies, DARPins, anticalins, monobodies, adnectins, surrobodies, avimers, or affilins. Details of the characteristics and preparation methods of antibody-like molecules are described in "Helma et al., J Cell Biol. 2015 Jun 8;209(5):633-44." and "Angeline et al., Future Med Chem. 2017 Aug;9(12):1301-1304." and references cited in those documents.

[0038] The method for producing the above-mentioned Fv antibody, Fab antibody, F(ab')2 antibody, Fab' antibody, scFv antibody, dsFv antibody, and antigen-binding peptide (hereinafter sometimes referred to as "Fv antibody, etc.") is not particularly limited. For example, DNA encoding a region of the Fv antibody, etc. in the anti-PAD4 antibody according to the embodiment of the present invention is inserted into an expression vector, and the antibody can be produced using an expression cell. Alternatively, the antibody may be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tBOC method (t-butyloxycarbonyl method). The antigen-binding antibody fragment according to the embodiment of the present invention may contain one or more of the above-mentioned Fv antibodies, etc.

[0039] In one embodiment of the present invention, the "chimeric antibody" is, for example, an antibody in which the variable region of an antibody and the constant region of an antibody between different organisms are linked, and can be constructed by gene recombination technology. Examples of the chimeric antibody include mouse-human chimeric antibody, chicken-human chimeric antibody, and chicken-mouse chimeric antibody. The mouse-human chimeric antibody can be prepared, for example, by the method described in "Roguska et al., Proc Natl Acad Sci US A. 1994 Feb 1;91(3):969-973.". The basic method for preparing a mouse-human chimeric antibody is, for example, to link a mouse leader sequence and a variable region sequence 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 the variable region sequence present in a cloned cDNA may be linked to a sequence encoding a human antibody constant region, and then linked to a mammalian cell expression vector. The fragment of the human antibody constant region can be any human antibody H chain constant region or L chain constant region, for example, Cγ1, Cγ2, Cγ3, or Cγ4 for the human H chain, and Cλ or Cκ for the L chain.

[0040] In one embodiment of the present invention, a "humanized antibody" is, for example, an antibody that has one or more CDRs derived from a non-human species, a framework region derived from a human immunoglobulin, and a constant region derived from a human immunoglobulin, and binds to a desired antigen. Various techniques known in the art may be used to humanize an antibody. In one embodiment of the present invention, a "human antibody" is, for example, an antibody in which the regions comprising the heavy chain variable region and constant region and the light chain variable region and constant region are derived from a gene encoding a human immunoglobulin. Various techniques known in the art may be used to prepare a human antibody.

[0041] In one embodiment of the present invention, the "heavy chain" is typically a major component of a full-length antibody. The heavy chain is usually bound to the light chain by disulfide bonds and non-covalent bonds. The N-terminal domain of the heavy chain contains a region called a variable region (VH), whose amino acid sequence is not constant even in antibodies of the same species and class, and it is generally known that the VH contributes greatly to the specificity and affinity for an antigen. For example, "Reiter et al., J Mol Biol. 1999 Jul 16;290(3):685-98." describes that when a molecule consisting of only VH was prepared, it bound specifically to the antigen with high affinity. Furthermore, "Wolfson W, Chem Biol. 2006 Dec;13(12):1243-1244." describes that there are antibodies of camels that are composed only of heavy chains and have no light chains.

[0042] In one embodiment of the present invention, the "CDR (complementarity determining region)" is a region of an antibody that actually contacts an antigen to form a binding site. Generally, the CDR is located on the Fv (variable region: including the heavy chain variable region (VH) and the light chain variable region (VL)) of an antibody. Generally, the CDR includes CDR1, CDR2, and CDR3, each of which is composed of about 5 to 30 amino acid residues. It is known that the CDR of the heavy chain particularly contributes to the binding of the antibody to the antigen. It is also known that, among the CDRs, CDR3 contributes most to the binding of the antibody to the antigen. For example, "Willy et al., Biochemical and Biophysical Research Communications Volume 356, Issue 1, 27 April 2007, Pages 124-128" describes that the binding ability of an antibody is increased by modifying the heavy chain CDR3. The Fv region other than the CDRs is called the framework region (FR), which consists of FR1, FR2, FR3 and FR4, and is relatively well conserved among antibodies (Kabat et al., "Sequence of Proteins of Immunological Interest", US Dept. Health and Human Services, 1983).

[0043] There are several reported methods for defining CDRs and determining their positions. For example, the Kabat definition (Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) or Chothia's definition (Chothia et al., J. Mol. Biol., 1987; 196: 901-917) may be adopted. In one embodiment of the present invention, the Kabat definition is adopted as a preferred example, but is not necessarily limited thereto. In some cases, the definition may be determined taking into consideration both the Kabat definition and the Chothia definition. For example, the overlapping portion of CDRs according to each definition, or a portion including both CDRs according to each definition, may be regarded as a CDR. A specific example of such a method is the method of Martin et al. (Proc. Natl. Acad. Sci. USA, 1989; 86: 9268-9272) using Oxford Molecular's AbM antibody modeling software, which is a compromise between the Kabat and Chothia definitions.

[0044] One embodiment of the present invention is one or more anti-PAD4 antibodies selected from the group consisting of: (a) an antibody in which heavy chain CDR1-3 and light chain CDR1-3 comprise the amino acid sequences shown in SEQ ID NOs: 19-24, respectively; (b) an antibody in which heavy chain CDR1-3 and light chain CDR1-3 comprise the amino acid sequences shown in SEQ ID NOs: 25-30, respectively; (c) an antibody in which heavy chain CDR1-3 and light chain CDR1-3 comprise the amino acid sequences shown in SEQ ID NOs: 31-36, respectively; and (d) an antibody in which heavy chain CDR1-3 and light chain CDR1-3 comprise the amino acid sequences shown in SEQ ID NOs: 37-42, respectively. The use of this antibody can inhibit the citrullination activity of active PAD4. The CDR sequences of the above antibodies (a) to (d) correspond to the CDR sequences of the antibodies P1 to P4 described in the Examples below, respectively. Another embodiment of the present invention is an anti-PAD4 antibody comprising at least one of the above-listed sets of amino acid sequences of heavy chain CDR1 to 3 and light chain CDR1 to 3.

[0045] The antibody (a) may have heavy chain FR1-4 and light chain FR1-4 each having the amino acid sequence shown in SEQ ID NO: 43-50. The antibody (b) may have heavy chain FR1-4 and light chain FR1-4 each having the amino acid sequence shown in SEQ ID NO: 51-58. The antibody (c) may have heavy chain FR1-4 and light chain FR1-4 each having the amino acid sequence shown in SEQ ID NO: 59-66. The antibody (d) may have heavy chain FR1-4 and light chain FR1-4 each having the amino acid sequence shown in SEQ ID NO: 67-74. These FR sequences correspond to the FR sequences of the antibodies P1-P4 described in the Examples below. Another embodiment of the present invention is an anti-PAD4 antibody that contains at least one of the sets of amino acid sequences of heavy chain FR1-4 and light chain FR1-4 listed above.

[0046] An antibody according to one embodiment of the present invention may have a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 19, 25, 31 or 37, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 20, 26, 32 or 38, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 21, 27, 33 or 39, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 22, 28, 34 or 40, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 23, 29, 35 or 41, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 24, 30, 36 or 42.

[0047] In one embodiment of the antibody, heavy chain FR1 may comprise the amino acid sequence shown in SEQ ID NO: 43, 51, 59, or 67; heavy chain FR2 may comprise the amino acid sequence shown in SEQ ID NO: 44, 52, 60, or 68; heavy chain FR3 may comprise the amino acid sequence shown in SEQ ID NO: 45, 53, 61, or 69; heavy chain FR4 may comprise the amino acid sequence shown in SEQ ID NO: 46, 54, 62, or 70; light chain FR1 may comprise the amino acid sequence shown in SEQ ID NO: 47, 55, 63, or 71; light chain FR2 may comprise the amino acid sequence shown in SEQ ID NO: 48, 56, 64, or 72; light chain FR3 may comprise the amino acid sequence shown in SEQ ID NO: 49, 57, 65, or 73; and light chain FR4 may comprise the amino acid sequence shown in SEQ ID NO: 50, 58, 66, or 74.

[0048] The anti-PAD4 antibody according to one embodiment of the present invention may be in the form of scFv, in which case it may have a linker between the heavy chain and the light chain. A representative example of the linker is, but is not limited to, a sequence of 0 to 5 amino acids consisting of G and P. The linker may have, for example, the amino acid sequence shown in SEQ ID NO: 75. The linker is not essential and may not be present.

[0049] An anti-PAD4 antibody according to one embodiment of the present invention may have a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 3, 4, 5, or 6, and a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 11, 12, 13, or 14.

[0050] In one embodiment of the present invention, "amino acid" is a general term for an organic compound having an amino group and a carboxyl group. When the antibody according to the embodiment of the present invention contains a "specific amino acid sequence", any of the amino acids in the amino acid sequence may be chemically modified. Any of the amino acids in the amino acid sequence may form a salt or a solvate. Any of the amino acids in the amino acid sequence may be L-type or D-type. Even in such cases, the antibody according to the embodiment of the present invention can be said to contain the above-mentioned "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, sugar chain addition, etc.).

[0051] One embodiment of the present invention is a polynucleotide or vector encoding the anti-PAD4 antibody according to the embodiment of the present invention. A transformant can be produced by introducing this polynucleotide or vector into a cell. The transformant may be a cell of a human or a mammal other than a human (e.g., rat, mouse, guinea pig, rabbit, cow, monkey, etc.). Examples of mammalian cells include Chinese hamster ovary cells (CHO cells), monkey cells COS-7, and human embryonic kidney cells (e.g., HEK293 cells). Alternatively, the transformant may be Escherichia genus bacteria, yeast, etc. The polynucleotide or vector may be constructed so as to be capable of expressing an anti-PAD4 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. The polynucleotide or vector may have a base sequence derived from a heterologous species. The heterologous base sequence may contain base sequences derived from two or more organisms selected from the group consisting of humans and non-human organisms (e.g., bacteria, archaea, yeast, insects, birds, viruses, or non-human mammals, etc.).

[0052] Examples of the vector that can be used include plasmids derived from Escherichia coli (e.g., pET-Blue), plasmids derived from Bacillus subtilis (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, vectors derived from viruses, etc. The vector may be an expression vector or may be circular.

[0053] 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, microinjection, etc. (Revised 4th Edition, New Genetic Engineering Handbook, Yodosha Publishing (2003): 152-179.). Methods for producing antibodies using cells include, for example, the methods described in "Protein Experiment Handbook, Yodosha Publishing (2003): 128-142."

[0054] One embodiment of the present invention is a cell containing the polynucleotide or vector according to the above-mentioned embodiment of the present invention. One embodiment of the present invention is a method for producing an anti-PAD4 antibody, comprising the step of growing the above-mentioned cell. The growing step comprises a culturing step. This production method may also comprise the step of recovering the anti-PAD4 antibody. This production method may also comprise the step of preparing a cell culture solution. This production method may also comprise the step of purifying the anti-PAD4 antibody.

[0055] 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 Publishing (2003): 27-52).

[0056] One embodiment of the present invention is a composition comprising the anti-PAD4 antibody according to the embodiment of the present invention. This composition can be used to efficiently detect active PAD4. In addition, the citrullination of active PAD4 can be efficiently inhibited. In addition, RA or arthritis can be treated. The components contained in this composition are not particularly limited, and may contain, for example, a buffer solution. This composition may be applied to one or more of the various embodiments (e.g., the inhibitor and pharmaceutical composition described below may contain a carrier, etc.) described later.

[0057] One embodiment of the present invention is a PAD4 function inhibitor comprising the anti-PAD4 antibody according to the embodiment of the present invention. By using this inhibitor, the citrullination activity of active PAD4 can be efficiently inhibited. The reduction rate of the citrullination activity of active PAD4 by the inhibitor may be 15, 20, 30, 40, 50, 60, 70, 80% or more, or may be within the range of any two of these values. This reduction rate may be expressed as a relative percentage when the reduction rate when TBS Buffer is used is set to 0%. This reduction rate is preferably 20% or more, more preferably 30% or more. In one embodiment of the present invention, the "agent" includes, for example, a composition used in research or treatment. The inhibitor includes, for example, a therapeutic agent for RA or arthritis. The inhibitor can be used, for example, in vitro or in vivo. The inhibitor may include a composition according to the embodiment of the present invention. One embodiment of the present invention is a method for inhibiting the function of PAD4, comprising a step of contacting the anti-PAD4 antibody according to the embodiment of the present invention with PAD4. One embodiment of the present invention is a method for inhibiting PAD4 function, comprising administering the anti-PAD4 antibody according to the embodiment of the present invention to a patient. The inhibition method includes an inhibition method performed for research or treatment. One embodiment of the present invention is the use of the anti-PAD4 antibody according to the embodiment of the present invention for producing a PAD4 function inhibitor. It should be noted that, again, PAD4 may be active PAD4.

[0058] One embodiment of the present invention is a pharmaceutical composition comprising the anti-PAD4 antibody according to the embodiment of the present invention. This pharmaceutical composition can be used to treat RA or arthritis. The pharmaceutical composition may comprise one or more pharmacologically acceptable carriers. The pharmaceutical composition includes, for example, a pharmaceutical composition for treating RA or arthritis. The pharmaceutical composition may comprise a composition according to the embodiment of the present invention. One embodiment of the present invention is a method for treating a disease, comprising administering to a patient an anti-PAD4 antibody according to the embodiment of the present invention (or a pharmaceutical composition comprising an anti-PAD4 antibody). The disease includes, for example, RA or arthritis. One embodiment of the present invention is the use of the anti-PAD4 antibody according to the embodiment of the present invention for producing a pharmaceutical composition.

[0059] It has been reported that the number of RA patients worldwide is as high as 70 million. Currently, several therapeutic drugs are commercially available, but there is a certain percentage of patients for whom existing drugs are ineffective, and it is said that 60 to 80% of patients do not receive satisfactory treatment. In addition, problems with side effects of existing drugs have been pointed out. By using the anti-PAD4 antibody according to the embodiment of the present invention described above, RA can be treated with a new mechanism.

[0060] One embodiment of the present invention is a diagnostic agent for RA or arthritis, comprising the anti-PAD4 antibody according to the embodiment of the present invention. Using this diagnostic agent, RA or arthritis can be efficiently diagnosed. One embodiment of the present invention is a diagnostic method for RA or arthritis, comprising a step of contacting a patient sample with the anti-PAD4 antibody according to the embodiment of the present invention. One embodiment of the present invention is a detection reagent for PAD4, comprising the anti-PAD4 antibody according to the embodiment of the present invention. Using this reagent, PAD4 can be efficiently detected. One embodiment of the present invention is a detection method for PAD4, comprising a step of contacting a test sample with the anti-PAD4 antibody according to the embodiment of the present invention. This method can appropriately detect PAD4 even without EDTA treatment as described in Patent Document 3. One embodiment of the present invention is a kit, comprising the anti-PAD4 antibody according to the embodiment of the present invention. Using this kit, for example, disease treatment, diagnosis, or PAD4 detection can be performed. This kit may, for example, comprise a composition, inhibitor, pharmaceutical composition, diagnostic agent, or detection reagent according to the embodiment of the present invention, and may comprise an instruction manual, a buffer solution, a container (e.g., a vial or syringe), or packaging. As used herein, a patient sample or test sample may be blood, serum, or plasma.

[0061] In one embodiment of the present invention, "treatment" includes exerting a symptom-improving effect, a suppressive effect, or a preventive effect on a patient's disease or one or more symptoms associated with the disease. In one embodiment of the present invention, the "therapeutic agent" may be a pharmaceutical composition containing an active ingredient and one or more pharmacologically acceptable carriers. In one embodiment of the present invention, the "pharmaceutical composition" may be produced by, for example, mixing the active ingredient with the carrier and any method known in the technical field of pharmaceuticals. In addition, the pharmaceutical composition is not limited in its form of use as long as it is used for treatment, and may be an active ingredient alone or a mixture of the active ingredient and any ingredient. In addition, the shape of the carrier is not particularly limited, and may be, for example, a solid or liquid (e.g., a buffer solution). The content of the carrier may be, for example, a pharmacy-effective amount. This effective amount may be, for example, an amount sufficient for pharmacy-based stability or delivery of the active ingredient. For example, a buffer solution is effective for stabilizing the active ingredient in a vial.

[0062] The route of administration of the pharmaceutical composition is preferably one that is effective for treatment, and may be, for example, intravenous, subcutaneous, intramuscular, intraperitoneal, or oral administration. The dosage form may be, for example, an injection, capsule, tablet, or granule. When administering an antibody, it is effective to use it as an injection. The aqueous solution for injection may be stored, for example, in a vial or stainless steel container. The aqueous solution for injection may also be blended with, for example, physiological saline, sugar (for example, trehalose), NaCl, or NaOH. The pharmaceutical composition may also be blended with, for example, an effective amount of a buffer (for example, a phosphate buffer), a pH adjuster, a stabilizer, or the like.

[0063] The dosage, administration interval, and administration method are not particularly limited and may be appropriately selected depending on the age and weight of the patient, symptoms, target organ, etc. In addition, the pharmaceutical composition preferably contains a therapeutically effective amount, or an effective amount for exerting a desired effect, of an active ingredient.

[0064] The therapeutic effect of the pharmaceutical composition may be evaluated, for example, by arthritis score, RA score, swelling width, imaging diagnosis, modified Total Sharp score, or disease marker. When evaluating by swelling width, for example, the therapeutic effect may be determined to be present when the swelling width of the affected area when the pharmaceutical composition is administered is significantly reduced compared to the swelling width when not administered. Alternatively, the therapeutic effect may be determined to be present when the swelling width of the affected area when the pharmaceutical composition is administered is significantly reduced compared to the swelling width of the affected area when a negative control substance is administered. The amount of reduction may be, for example, 40, 50, 60, 70, 80, 90, or 100%, or may be within the range of any two of these values.

[0065] In one embodiment of the present invention, a "patient" includes a human or a non-human mammal (e.g., one or more of a mouse, a guinea pig, a hamster, a rat, a mouse, a rabbit, a pig, a sheep, a goat, a cow, a horse, a cat, a dog, a marmoset, a monkey, or a chimpanzee). The patient may also be a patient diagnosed with RA or arthritis. The patient may also be a patient diagnosed with a disease treatable by inhibition of citrullination.

[0066] One embodiment of the present invention is a method for enhancing the citrullination activity inhibitory ability or therapeutic effect of a composition, comprising a step of increasing the proportion of anti-PAD4 antibodies that specifically bind to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or anti-PAD4 antibodies that specifically bind to positions 633 to 645 of human PAD4 in the composition. One embodiment of the present invention is a composition containing an anti-PAD4 antibody, in which 90% or more of the anti-PAD4 antibody molecules in the composition are anti-PAD4 antibodies that specifically bind to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or anti-PAD4 antibodies that specifically bind to positions 633 to 645 of human PAD4. One embodiment of the present invention is an antibody population containing an anti-PAD4 antibody, in which 90% or more of the anti-PAD4 antibody molecules in the antibody population are anti-PAD4 antibodies that specifically bind to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or anti-PAD4 antibodies that specifically bind to positions 633 to 645 of human PAD4. The above-mentioned 90% or more may be, for example, 90, 95, 96, 97, 98, 99% or more, or 100%, or may be within a range between any two of these values.

[0067] One embodiment of the present invention is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1. By using this peptide, an antibody that binds to activated PAD4 can be produced. In addition, an antibody that binds to activated PAD4 can be detected. The peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 may be chemically modified (e.g., KLH modified), and such chemically modified peptides are also included in one form of the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1. The peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 may be an isolated, purified, or concentrated peptide.

[0068] One embodiment of the present invention is an antigen composition comprising a peptide having an amino acid sequence as shown in SEQ ID NO: 1. This antigen composition may comprise, for example, a buffer solution. One embodiment of the present invention is a method for producing an anti-PAD4 antibody, comprising a step of immunizing a mammal or bird with a peptide having an amino acid sequence as shown in SEQ ID NO: 1. One embodiment of the present invention is a method for producing an anti-PAD4 antibody, comprising a step of contacting a peptide having an amino acid sequence as shown in SEQ ID NO: 1 with an antibody or an antibody library. One embodiment of the present invention is a method for detecting an antibody that binds to active PAD4, comprising a step of contacting a peptide having an amino acid sequence as shown in SEQ ID NO: 1 with a test sample containing an anti-PAD4 antibody. One embodiment of the present invention is a composition for detecting an antibody that binds to active PAD4, comprising a peptide having an amino acid sequence as shown in SEQ ID NO: 1.

[0069] In one embodiment of the present invention, a "bond" may be a covalent bond or a non-covalent bond, for example, an ionic bond, a hydrogen bond, a hydrophobic interaction, or a hydrophilic interaction.

[0070] In one embodiment of the present invention, "significantly" may mean, for example, a state where a statistically significant difference is evaluated using a Student's t-test (one-tailed or two-tailed) and p<0.05 or p<0.01, or a state where a substantial difference is generated.

[0071] All publications, publications (patents or patent applications) cited herein are incorporated by reference in their entirety.

[0072] In this specification, "or" is used when "at least one or more" of the items listed in the text can be adopted. The same applies to "alternative." When it is specified in this specification that "within a range of two values," the range includes the two values ​​themselves. In this specification, "A to B" means A or more and B or less. In this specification, "respectively" is synonymous with "respectively, in order."

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

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

[0075] <Example 1> Preparation of anti-PAD4 antibody Three 3-month-old Boris Brown chickens were intraperitoneally immunized with 333 μg of KLH-modified FFTYHIRHGEVHC (SEQ ID NO: 1) peptide per chicken. The peptide used was a peptide antigen corresponding to positions 633-645 of PAD4 (SEQ ID NO: 2). Complete Freund's adjuvant (Wako, 014-09541) was used for the first immunization, and incomplete Freund's adjuvant (Wako, 011-09551) was used for the second and third immunizations. The antigen diluted in PBS (phosphate buffered saline) was intravenously injected for the fourth immunization. Blood was collected from the underwing vein every two weeks, and the antibody titer was confirmed by ELISA. Three chickens were immunized up to the third immunization, and the fourth immunization was performed on the individual that showed the highest increase in antibody titer, with the fourth immunization being the final immunization. Three days after the final immunization, chicken spleens were collected, lymphocytes were isolated by density gradient centrifugation using Ficoll paque PLUS (GE Healthcare, 17-1440-03), and RNA was extracted using TRIzole Reagent (Life Technologies, 15596026). cDNA was synthesized from the extracted RNA by RT-PCR using PrimeScript II 1st Strand cDNA Synthesis Kit (TAKARA, 6210A), and an scFv phage library was prepared. The expression vector used was a type in which chicken λ chain was inserted instead of mouse κ chain of pPDS. The scFv phage library was prepared according to the method described in the reference: "Nakamura et al., J Vet Med Sci. 2004 Ju;66 (7): 807-814".

[0076] Using the scFv phage antibody library, panning was performed on plates immobilized with the above peptide antigens. Panning was performed according to the method described in the reference "Nakamura et al., J Vet Med Sci. 2004 Ju;66 (7): 807-814". After five rounds of panning, the reactivity of the library was confirmed by ELISA using plates immobilized with BSA-modified peptide antigens, and phages were screened from libraries in which reactivity began to increase. In the screening, the phages were infected into E. coli and plated on 2xYT Agar plates containing 50 μg / ml ampicillin (nacalai, 02739-32), and the obtained colonies were cultured in ampicillin-containing 2xYT liquid medium. After infection with helper phage, phage induction was performed in 2xYT liquid medium containing 50 μg / ml ampicillin, 25 μg / ml kanamycin (Meiji Seika Kaisha, GS1-RSS), and 100 μg / ml IPTG (Nacalai, 19742-94). The reactivity of the scFv phage antibody in the culture supernatant was confirmed by ELISA using an antigen-immobilized plate. The obtained positive clones were sequenced using a DNA sequencer (Applied Biosystems, ABI PRISM 3100-Genetic Analyzer) to determine the sequence.

[0077] For clones with different sequences, the DNA strand encoding the scFv antibody was used as a template to amplify the chicken-derived antibody gene H-chain variable region and L-chain variable region by PCR, and the PCR product was then treated with restriction enzymes SacII (BioLabs, Cat#R0157S) and NheI (BioLabs, Cat#R0131S). Next, the H-chain variable region and L-chain variable region were each recombined into a mouse chimeric antibody (IgG1) expression vector (H-chain expression vector: pcDNA4 / myc-His, L-chain expression vector: pcDNA3 / myc-His, Invitrogen) that had been similarly treated with restriction enzymes. The constructed H-chain and L-chain constructs were transfected into CHO cells, and the reactivity of the culture supernatant was confirmed by ELISA using BSA-modified peptide antigen and full-length recombinant human PAD4 protein as immobilized antigen. The mouse chimeric expression vector used was the vector described in Tateishi et al., J Vet Med Sci. 2008 Apr;70(4): 397-400. As described above, antibody clones P1, P2, P3, and P4 were obtained. The amino acid sequences of the heavy chain variable regions of P1, P2, P3, and P4 are SEQ ID NOs:3 to 6, DNA sequences are SEQ ID NOs:7 to 10, amino acid sequences of the light chain variable regions are SEQ ID NOs:11 to 14, DNA sequences are SEQ ID NOs:15 to 18 (FIGS. 1 to 3). Hereinafter, these antibodies may be collectively referred to as "P1, etc."

[0078] To mass-produce antibody clones such as P1, the H-chain and L-chain expression vectors were transfected into cultured mammalian cells using the Expi293 Expression system (Invitrogen, A14635), and the expressed antibodies were purified using Protein G Sepharose 4 Fast Flow (GE healthcare, 17-018-02).

[0079] <Example 2> Evaluation of affinity of anti-PAD4 antibody Biacore (GE Healthcare, Biacore T200) was performed to evaluate the affinity of P1 and other molecules to human PAD4. A Mouse Antibody Capture Kit (GE Healthcare, BR-1008-38) was used to measure affinity. Specifically, rabbit anti-mouse polyclonal antibodies were immobilized on the surface of a CM5 chip by the amine coupling method using the free carboxyl groups on the CM5 chip surface using NHS / EDC according to the standard protocol provided by the manufacturer. Next, P1 and other molecules were captured by the rabbit anti-mouse polyclonal antibodies. Various concentrations of human PAD4 were applied to the Biacore T200, and kinetic sensorgrams were created.

[0080] The affinity measurement showed that Kd (M) was 7.3x10 for P1. -8 , P2 is 3.1x10 -8 , P3 is 2.4x10 -8 , P4 is 1.5x10 -8 As can be seen from these results, P1 and the like all showed high affinity for human PAD4.

[0081] Example 3: Evaluation of binding and inhibitory activity to activated PAD4 (1) Protocol 1 (adding calcium after antibody-enzyme reaction) Anti-PAD4 antibody (P1, etc.) and mouse IgG (negative control) were prepared at 450 μg / mL each in TBS as a solvent. 5 μL of antibody solution (final concentration in 50 μL of reaction system is 45 μg / mL (300 nM)) and 5 μL of 7.5 μg / mL (100 nM) human PAD4 were mixed with 20 mM Tris-HCl buffer (pH.7.6) containing 1 mM EDTA and 1 mM DTT to a total volume of 40 μL. After incubation at 37°C for 60 minutes, 5 μL of 100 mM BAEE (benzoyl arginine ethyl ester) was added, and 5 μL of 100 mM CaCl2 was added (total volume 50 μL, final concentration of BAEE is 10 mM, final concentration of calcium ion is 10 mM) and incubated. As controls, samples were similarly prepared by adding a solvent (TBS), an anti-DNP antibody (negative control) solution (final concentration 300 nM), or an L207 (anti-PAD4 antibody L207-11 described in the Examples of WO / 2012 / 026309) solution (final concentration 300 nM) instead of 5 μL of the antibody solution. This solution was reacted at 37° C. for 3 hours.

[0082] (2) Protocol 2 (adding calcium before the reaction between antibody and enzyme) In protocol 2, PAD4 was made active by reacting it with calcium in advance. Specifically, the procedure was as follows. 5 μL of 7.5 μg / mL (100 nM) human PAD4 was mixed with 5 μL of 100 mM CaCl2, and then mixed with 20 mM Tris-HCl buffer (pH 7.6) containing 1 mM EDTA and 1 mM DTT to a total volume of 40 μL. After mixing, preincubation was performed at 37°C for 60 minutes. Anti-PAD4 antibody (P1, etc.) and mouse IgG (negative control) were each prepared at 450 μg / mL in TBS as a solvent. 5 μL of the prepared antibody solution (final concentration in 50 μL of reaction system is 45 μg / mL (300 nM)) and 5 μL of 100 mM BAEE were added to the pre-incubated reaction solution to make a total volume of 50 μL (final concentration of BAEE is 10 mM, final concentration of calcium ion is 10 mM). As a control, samples were similarly prepared in which the solvent (TBS), anti-DNP antibody solution (final concentration is 300 nM), or L207 solution (final concentration is 300 nM) was added instead of 5 μL of the antibody solution. These solutions were reacted at 37°C for 3 hours.

[0083] (3) Common Protocol After 3 hours of reaction at 37°C, 12.5μL of 5M perchloric acid was added to stop the reaction. 40μL of this stopped solution was mixed with 150μL of reaction solution (reaction solution 1 (98% H2SO4:85% H3PO4:H2O=25:20:55, 0.0416% FeCl3·6H2O): reaction solution 2 (1% 2,3-butanedione oxime:0.02% thiosemicarbazide=1:1)=2:1 mixture) and reacted at 98°C for 6 minutes. After leaving the mixture on ice for 1 minute, the amount of citrullinated BAEE in the supernatant was colorimetrically quantified. The measured value of the control sample in which the solvent was used instead of the antibody solution was set to 100, and the citrullination rate when each antibody was used was calculated.

[0084] The results of the PAD4 inhibitory activity measurement are shown in Figure 4. As can be seen from these results, P1 and the like bound to active PAD4 and strongly inhibited its citrullination activity (citrullination rates were 63% for P1, 62% for P2, 80% for P3, and 82% for P4). On the other hand, anti-DNP antibody and L207 did not inhibit the citrullination activity of active PAD4.

[0085] The above examples have demonstrated that an anti-PAD4 antibody that specifically binds to positions 633 to 645 of PAD4 has the property of binding to active PAD4 and inhibiting citrullination activity.

[0086] 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 the function of PAD4, comprising a peptide consisting of the amino acid sequence shown in SEQ ID NO:1, or an anti-PAD4 antibody that has specific binding affinity to positions 633 to 645 of human PAD4 (peptidylarginine deiminase 4) and inhibits the citrullination activity of active PAD4.

2. The composition described in claim 1, wherein the anti-PAD4 antibody is a monoclonal antibody.

3. The composition described in claim 1 or 2, wherein the anti-PAD4 antibody is an antigen-binding antibody fragment.

4. A composition according to any one of claims 1 to 3 for inhibiting the function of activated PAD4.

5. A composition described in any of claims 1 to 4, wherein the anti-PAD4 antibody has specific binding affinity to a peptide consisting of the amino acid sequence shown in sequence number 1 and inhibits the citrullination activity of active PAD4.

6. A composition described in any one of claims 1 to 5, comprising one or more pharmacologically acceptable carriers.

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