New regulation of anti-PAD4 antibodies

Abstract

Provided is an anti-PAD4 antibody in which HCDR1 includes an amino acid sequence of SEQ ID NO: 1, HCDR2 includes an amino acid sequence of SEQ ID NO: 2, HCDR3 includes an amino acid sequence of SEQ ID NO: 3, LCDR1 includes an amino acid sequence of SEQ ID NO: 4, LCDR2 includes an amino acid sequence of SEQ ID NO: 5, and LCDR3 includes an amino acid sequence of SEQ ID NO: 6, or an antibody fragment thereof.

Description

[Technical field]

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

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

[0003] Citrullination has been reported to be related to rheumatoid arthritis (RA). For example, because vimentin, collagen, and fibrin are present as antigens in the synovium in RA, anti-cyclic citrullinated peptide antibody (anti-CCP antibody) detection kits, which are antibodies against these antigens, are commercially available as diagnostic drugs for RA.

[0004] There are also some reports on PAD4 and RA. 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. In addition, Non-Patent Document 2 reports that an anti-PAD4 antibody was used to diagnose RA. In addition, Patent Document 1 describes an attempt to suppress RA by administering a mixture of four types of anti-PAD4 antibodies to mice (see Example 2 of Patent Document 1). Furthermore, Patent Document 2 describes an anti-PAD4 antibody that has superior affinity to PAD4 and citrullination activity inhibitory ability.

[0005] Regarding PAD4 and systemic lupus erythematosus (SLE), there are reports that nephritis was suppressed in SLE models (imiquimod-induced models) using PAD4 knockout mice compared to wild-type mice, and that nephritis was suppressed in SLE model (MRL / lpr model) mice by administration of small molecule PAD inhibitors (Cl-Amidine and BB-Cl-Amidine) (Non-Patent Documents 3, 4, 5). In addition, there are reports that SLE-like symptoms developed in MRL / lpr mice in which PAD4 was knocked out, similar to wild-type mice, and that administration of a small molecule inhibitor (Cl-Amidine) to nephritis models (anti-GBM antibody-induced models and mice transferred with serum from SLE patients) did not suppress the onset of nephritis (Non-Patent Document 6).

[0006] Given the association between such diseases and PAD4, there is a demand for anti-PAD4 antibodies with superior binding affinity, stability, etc. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO / 2012 / 026309 [Patent Document 2] WO / 2016 / 143753 [Non-patent literature]

[0008] [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. [Non-Patent Document 3] "Peptidylarginine deiminases 2 and 4 modulate innate and adaptive immune responses in TLR-7-dependent lupus", Yudong et al., JCI Insight. 2018 Dec 6;3(23): e124729. [Non-Patent Document 4] "Peptidylarginine Deiminase 4 Promotes the Renal Infiltration of Neutrophils and Exacerbates the TLR7 Agonist-Induced Lupus Mice", Hanata et al., Front Immunol. 2020 Jun 23;11:1095. [Non-Patent Document 5] "Peptidylarginine deiminase inhibition disrupts NET formation and protects against kidney, skin and vascular disease in lupus-prone MRL / lpr mice", Knight et al., Ann Rheum Dis. 2015 Dec;74(12):2199-2206. [Non-Patent Document 6] "Lupus and proliferative nephritis are PAD4 independent in murine models", Gordon et al., JCI Insight. 2017 May 18;2(10):e92926. Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention has been made in consideration of the above-mentioned circumstances, and aims to provide an anti-PAD4 antibody having excellent properties, or to provide an excellent method for preventing or treating RA, arthritis, systemic lupus erythematosus, lupus nephritis, graft-versus-host disease, etc. [Means for solving the problem]

[0010] The present inventors have conducted extensive research to solve the above problems. As a result, they have found that an anti-PAD4 antibody having a specific complementarity determining region (CDR) sequence has excellent binding properties and storage stability. Furthermore, they have found that an anti-PAD4 antibody having a specific framework region (FR) sequence has excellent chemical stability, and have completed the present invention. That is, the gist of the present invention relates to the following.

[0011] [1] An anti-PAD4 antibody or an antibody fragment thereof, in which HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6. [2] An anti-PAD4 antibody or an antibody fragment thereof described in [1], wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of any of SEQ ID NOs: 7 to 10, HCDR3 comprises the amino acid sequence of SEQ ID NO: 11 or 12, LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of any of SEQ ID NOs: 13 to 15. [3] a-1) an anti-PAD4 antibody or an antibody fragment thereof, in which HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 7, HCDR3 comprises the amino acid sequence of SEQ ID NO: 11, LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 13; b-1) an anti-PAD4 antibody or an antibody fragment thereof, in which HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 8, HCDR3 comprises the amino acid sequence of SEQ ID NO: 11, LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 13; c-1) an anti-PAD4 antibody or an antibody fragment thereof, in which HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 9, HCDR3 comprises the amino acid sequence of SEQ ID NO: 12, LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 14; and d-1) an anti-PAD4 antibody or an antibody fragment thereof, in which HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 10, HCDR3 comprises the amino acid sequence of SEQ ID NO: 11, LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 15; The anti-PAD4 antibody or antibody fragment thereof according to [1] or [2], selected from the group consisting of: [4] a-2) an anti-PAD4 antibody or an antibody fragment thereof, in which HCDR1 consists of the amino acid sequence of SEQ ID NO: 1, HCDR2 consists of the amino acid sequence of SEQ ID NO: 7, HCDR3 consists of the amino acid sequence of SEQ ID NO: 11, LCDR1 consists of the amino acid sequence of SEQ ID NO: 4, LCDR2 consists of the amino acid sequence of SEQ ID NO: 5, and LCDR3 consists of the amino acid sequence of SEQ ID NO: 13; b-2) an anti-PAD4 antibody or an antibody fragment thereof, in which HCDR1 consists of the amino acid sequence of SEQ ID NO: 1, HCDR2 consists of the amino acid sequence of SEQ ID NO: 8, HCDR3 consists of the amino acid sequence of SEQ ID NO: 11, LCDR1 consists of the amino acid sequence of SEQ ID NO: 4, LCDR2 consists of the amino acid sequence of SEQ ID NO: 5, and LCDR3 consists of the amino acid sequence of SEQ ID NO: 13; c-2) an anti-PAD4 antibody or an antibody fragment thereof, in which HCDR1 consists of the amino acid sequence of SEQ ID NO: 1, HCDR2 consists of the amino acid sequence of SEQ ID NO: 9, HCDR3 consists of the amino acid sequence of SEQ ID NO: 12, LCDR1 consists of the amino acid sequence of SEQ ID NO: 4, LCDR2 consists of the amino acid sequence of SEQ ID NO: 5, and LCDR3 consists of the amino acid sequence of SEQ ID NO: 14; and d-2) an anti-PAD4 antibody or an antibody fragment thereof, in which HCDR1 consists of the amino acid sequence of SEQ ID NO: 1, HCDR2 consists of the amino acid sequence of SEQ ID NO: 10, HCDR3 consists of the amino acid sequence of SEQ ID NO: 11, LCDR1 consists of the amino acid sequence of SEQ ID NO: 4, LCDR2 consists of the amino acid sequence of SEQ ID NO: 5, and LCDR3 consists of the amino acid sequence of SEQ ID NO: 15; The anti-PAD4 antibody or antibody fragment thereof according to any one of [1] to [3], selected from the following: [5] a-3) an anti-PAD4 antibody or an antibody fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 16 and the light chain variable region comprises the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO: 17; b-3) an anti-PAD4 antibody or an antibody fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 18 and the light chain variable region comprises the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO: 19; c-3) an anti-PAD4 antibody or an antibody fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 20 and the light chain variable region comprises the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO: 21; and d-3) an anti-PAD4 antibody or an antibody fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 22 and the light chain variable region comprises the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO: 23; An anti-PAD4 antibody or an antibody fragment thereof selected from the group consisting of: [6] a-4) an anti-PAD4 antibody or an antibody fragment thereof, wherein the heavy chain variable region consists of the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 16 and the light chain variable region consists of the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO: 17; b-4) an anti-PAD4 antibody or an antibody fragment thereof, wherein the heavy chain variable region consists of the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 18 and the light chain variable region consists of the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO: 19; c-4) an anti-PAD4 antibody or an antibody fragment thereof, in which the heavy chain variable region consists of the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 20 and the light chain variable region consists of the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO: 21; and d-4) an anti-PAD4 antibody or an antibody fragment thereof, wherein the heavy chain variable region consists of the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 22 and the light chain variable region consists of the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO: 23; The anti-PAD4 antibody or antibody fragment thereof according to [5], selected from the group consisting of: [7] a-5) an anti-PAD4 antibody or an antibody fragment thereof, wherein the heavy chain consists of the amino acid sequence of SEQ ID NO: 16 and the light chain consists of the amino acid sequence of SEQ ID NO: 17; b-5) an anti-PAD4 antibody or an antibody fragment thereof, the heavy chain of which consists of the amino acid sequence of SEQ ID NO: 18 and the light chain of which consists of the amino acid sequence of SEQ ID NO: 19; c-5) an anti-PAD4 antibody or an antibody fragment thereof, the heavy chain of which consists of the amino acid sequence of SEQ ID NO: 20 and the light chain of which consists of the amino acid sequence of SEQ ID NO: 21; and d-5) an anti-PAD4 antibody or an antibody fragment thereof, the heavy chain of which consists of the amino acid sequence of SEQ ID NO: 22 and the light chain of which consists of the amino acid sequence of SEQ ID NO: 23; The anti-PAD4 antibody or antibody fragment thereof according to [5] or [6], selected from the group consisting of [8] The anti-PAD4 antibody or antibody fragment thereof according to any of [1] to [7], wherein the amino acid corresponding to the 84th amino acid in the heavy chain is serine. [9] An anti-PAD4 antibody or an antibody fragment thereof described in any of [1] to [8], which has a KD value for PAD4 of 100 pM or less.

[10] The anti-PAD4 antibody or antibody fragment thereof according to any one of [1] to [9], When the anti-PAD4 antibody or its antibody fragment is stored at 40° C. for one month, the anti-PAD4 antibody or its antibody fragment has a PAD4 binding amount that is 90% or more of the PAD4 binding amount of the anti-PAD4 antibody or its antibody fragment before storage. Anti-PAD4 antibody or antibody fragment thereof.

[0012]

[11] e-1) An anti-PAD4 antibody or an antibody fragment thereof, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 24, HCDR3 comprises the amino acid sequence of SEQ ID NO: 11, LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 25, An anti-PAD4 antibody or an antibody fragment thereof, in which the amino acid corresponding to the 84th amino acid in the heavy chain is serine.

[12] e-2) An anti-PAD4 antibody or an antibody fragment thereof, in which HCDR1 consists of the amino acid sequence of SEQ ID NO: 1, HCDR2 consists of the amino acid sequence of SEQ ID NO: 24, HCDR3 consists of the amino acid sequence of SEQ ID NO: 11, LCDR1 consists of the amino acid sequence of SEQ ID NO: 4, LCDR2 consists of the amino acid sequence of SEQ ID NO: 5, and LCDR3 consists of the amino acid sequence of SEQ ID NO: 25, The anti-PAD4 antibody or antibody fragment thereof described in

[11] , wherein the amino acid corresponding to the 84th amino acid in the heavy chain is serine.

[13] e-3) An anti-PAD4 antibody or an antibody fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 28, and the light chain variable region comprises the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO: 27.

[14] e-4) a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 28, and a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO: 27;

[13] An anti-PAD4 antibody or an antibody fragment thereof.

[15] e-5) the heavy chain consists of SEQ ID NO: 28 and the light chain consists of SEQ ID NO: 27; An anti-PAD4 antibody or an antibody fragment thereof according to

[13] or

[14] .

[16] An anti-PAD4 antibody or antibody fragment thereof according to any one of

[11] to

[15] , wherein cleavage occurring during production accounts for less than 3% of the total production amount.

[17] The anti-PAD4 antibody or antibody fragment thereof described in any of [1] to

[16] , wherein the antibody is a neutralizing antibody.

[18] An anti-PAD4 antibody or an antibody fragment thereof described in any of [1] to

[17] , wherein the antibody is a binding antibody to PAD4 represented by sequence number 29.

[19] A nucleic acid molecule comprising a base sequence encoding an anti-PAD4 antibody or an antibody fragment thereof described in any one of [1] to

[15] .

[20] A recombinant vector comprising the nucleic acid molecule described in

[19] .

[0013]

[21] A transformant comprising the recombinant vector described in

[20] .

[22] A pharmaceutical composition comprising an anti-PAD4 antibody or an antibody fragment thereof described in any one of [1] to

[18] .

[23] The pharmaceutical composition according to

[22] , which is an agent for preventing or treating rheumatoid arthritis or arthritis, systemic lupus erythematosus, lupus nephritis, or graft-versus-host disease.

[24] The pharmaceutical composition described in

[22] or

[23] , which is an inhibitor of citrullination in proteins.

[25] The pharmaceutical composition according to any one of

[22] to

[24] , which is an inhibitor of NEATosis in cells.

[26] Use of an anti-PAD4 antibody or an antibody fragment thereof described in any of [1] to

[18] , or a pharmaceutical composition described in any of

[22] to

[25] , for the prevention or treatment of rheumatoid arthritis or arthritis, systemic lupus erythematosus, lupus nephritis, or graft-versus-host disease.

[27] Use of an anti-PAD4 antibody or an antibody fragment thereof described in any of [1] to

[18] , or a pharmaceutical composition described in any of

[22] to

[25] , for the manufacture of an agent for the prevention or treatment of rheumatoid arthritis or arthritis, systemic lupus erythematosus, lupus nephritis, or graft-versus-host disease.

[28] A method for preventing or treating rheumatoid arthritis or arthritis, systemic lupus erythematosus, lupus nephritis, or graft-versus-host disease, comprising administering an effective amount of an anti-PAD4 antibody or antibody fragment thereof described in any of [1] to

[18] , or a pharmaceutical composition described in any of

[22] to

[25] . Effect of the Invention

[0014] The present invention provides an anti-PAD4 antibody having excellent properties, and an excellent method for preventing or treating RA or arthritis, systemic lupus erythematosus, lupus nephritis, or graft-versus-host disease. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 shows a method for producing a CAIA model mouse, and the effect of the anti-PAD4 antibody of the present invention on arthritis in the CAIA model. [Diagram 2] FIG. 2 shows the effect of the anti-PAD4 antibody of the present invention on arthritis in a CIA model. [Diagram 3] FIG. 3 shows the effect of the anti-PAD4 antibody of the present invention on urinary protein in a cGvHD model. [Figure 4] FIG. 4 shows the effect of the anti-PAD4 antibody of the present invention on the incidence rate in a cGvHD model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] To facilitate understanding of the present invention, the following describes terms used in the present invention.

[0017] [Neutralization] In the present invention, "neutralization" refers to the ability to bind to a target of interest and inhibit any of the functions of the target. That is, "neutralizing the activity of PAD4" refers to the inhibition of PAD4 activity by an anti-PAD4 antibody binding to PAD4. The activity of PAD4 can be evaluated by one or more of several in vitro or in vivo assays known in the art. The activity of PAD4 is, for example, the citrullination activity of arginine in a protein, and the neutralizing activity of an anti-PAD4 antibody can be evaluated by the citrullination inhibition test described herein.

[0018] [Isolated] "Isolated" as used herein for an isolated anti-PAD4 antibody means identified and separated and / or recovered from components in the natural state. Impurities in the natural state are substances that may interfere with the diagnostic or therapeutic use of the antibody, including enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In general, an anti-PAD4 antibody can be isolated by purifying it through at least one purification step, and an anti-PAD4 antibody purified through at least one purification step can be referred to as an "isolated anti-PAD4 antibody."

[0019] [Human antibody] A human antibody refers to an antibody in which both the light chain and the heavy chain are derived from human immunoglobulin. Depending on the difference in the constant region of the heavy chain, human antibodies include IgG (including IgG1, IgG2, IgG3, and IgG4) having a γ heavy chain, IgM having a μ heavy chain, IgA (including IgA1 and IgA2) having an α heavy chain, IgD having a δ heavy chain, or IgE having an ε heavy chain. In principle, the light chain includes either a κ chain or a λ chain.

[0020] [Humanized antibody] A humanized antibody refers to an antibody that is composed of a variable region consisting of a complementarity determining region of an antibody derived from a non-human animal and a framework region derived from a human antibody, and a constant region derived from a human antibody.

[0021] [Chimeric antibody] A chimeric antibody refers to an antibody in which the light chain, the heavy chain, or both, are composed of a variable region of non-human origin and a constant region of human origin.

[0022] [Anti-PAD4 antibody] In the present invention, the anti-PAD4 antibody refers to an immunoglobulin molecule that binds to PAD4 or a modified molecule thereof. The modified molecule includes a multispecific antibody, a chimeric antibody, a humanized antibody, a functionally modified antibody, and a conjugated antibody.

[0023] [Multispecific antibodies] A multispecific antibody is an asymmetric antibody that has two or more independent antigen recognition sites with two or more different antigen specificities, and examples of such antibodies include bispecific antibodies, which have two antigen specificities, and trispecific antibodies, which have three antigen specificities.

[0024] [Functionally modified antibodies] In the present invention, a functionally modified antibody refers to an antibody whose function other than the antigen-binding function, such as a cell-killing function, a complement activation function, or a function to extend the blood half-life, has been modified by modifying the antibody sequence, sugar chain, etc.

[0025] [Conjugated antibodies] In the present invention, a conjugated antibody refers to an antibody to which a functional molecule other than an antibody, such as a non-peptide polymer such as polyethylene glycol (PEG), a radioactive substance, a toxin, a low molecular weight compound, a cytokine, a growth factor, albumin, or an enzyme, is chemically or genetically bound.

[0026] [Antibody fragment] In the present invention, the antibody fragment means an antigen-binding fragment unless otherwise specified. The antibody fragment can also be called an antigen-binding molecule. The antigen-binding fragment is a protein containing a part of an antibody and capable of binding to an antigen. Examples of the antigen-binding fragment include F(ab')2, Fab', Fab, Fv (variable fragment of antibody), disulfide-linked Fv, single-chain antibody (scFv), and polymers thereof. Furthermore, the antigen-binding fragment includes conjugated antigen-binding fragments to which functional molecules other than the anti-PAD4 antibody of the present invention, such as non-peptide polymers such as polyethylene glycol (PEG), radioactive substances, toxins, low molecular weight compounds, cytokines, growth factors (TGF-β, NGF, Neurotrophin, etc.), albumin, enzymes, and other antibodies, are chemically or genetically bound.

[0027] [Complementarity determining region] Complementarity determining region (CDR) refers to a region of the variable region of an immunoglobulin molecule that forms an antigen-binding site, also called a hypervariable region, and refers to a portion in which the amino acid sequence varies greatly from one immunoglobulin molecule to another. There are three CDRs (LCDR1, LCDR2, LCDR3, and HCDR1, HCDR2, HCDR3) in each of the light chain and heavy chain. In the present invention, the CDRs of an immunoglobulin molecule are determined according to the Kabat numbering system (Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA).

[0028] [Percent (%) identity of amino acid sequence] "Percent (%) identity" with respect to an identified reference polypeptide sequence, such as a variable region, is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular reference polypeptide sequence, after aligning the sequences, introducing gaps, if necessary, to obtain the maximum percent identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of measuring percent identity can be achieved by a variety of methods within the skill of the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared. However, for purposes herein, percent identity values ​​are obtained by using the sequence comparison computer program BLAST in pairwise alignments. In the context of using BLAST for amino acid sequence comparison, the percent identity of a given amino acid sequence A to a given amino acid sequence B is calculated as follows: 100 times the fraction X / Y where X is the number of amino acid residues that score identical matches in a program alignment of A and B in the sequence alignment program BLAST, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is different from the length of amino acid sequence B, the % identity of A to B will differ from the % identity of B to A. Unless otherwise specified, all % identity values ​​herein are obtained using the BLAST computer program as set forth in the immediately preceding paragraph.

[0029] [Conflicting] In the present invention, "competing" with the anti-PAD4 antibody of the present invention means that, when measured by the surface plasmon resonance (SPR) method described in this specification, the presence of the anti-PAD4 antibody or its antigen-binding fragment significantly reduces the binding between the anti-PAD4 antibody of the present invention and PAD4.

[0030] The present invention will be described in detail below. <pad4> PAD4 is generally known as an enzyme involved in the citrullination of arginine in proteins. PAD4 is also known as an enzyme involved in cellular nematosis. Details of the amino acid sequence of PAD4 can be found on websites such as NCBI (National Center for Biotechnology Information) or HGNC (HUGO Gene Nomenclature Committee). The accession number of PAD4 listed in NCBI is, for example, NP_036519.2. The amino acid sequence of PAD4 is, for example, SEQ ID NO: 29. As long as PAD4 has PAD4 activity, its biological origin is not limited.

[0031] <Anti-PAD4 antibody> One embodiment of the present invention is a novel anti-PAD4 antibody. This antibody has better binding properties, storage stability, and chemical stability than conventional anti-PAD4 antibodies. Storage stability and chemical stability will be described later, but these are sometimes collectively referred to as stability. This antibody can be used to prevent or treat, for example, RA or arthritis, systemic lupus erythematosus, lupus nephritis, or graft-versus-host disease. This method of prevention or treatment has minimal side effects because it uses an antibody, and is therefore excellent in terms of safety. Furthermore, an anti-PAD4 antibody according to one embodiment of the present invention may be an antibody that inhibits the protein citrullination activity of PAD4. Furthermore, an anti-PAD4 antibody according to one embodiment of the present invention may be an antibody that suppresses nephrosis in cells.

[0032] The anti-PAD4 antibody according to one embodiment of the present invention has excellent binding to PAD4. The anti-PAD4 antibody of the present invention binds to PAD4, meaning that it specifically binds to PAD4. However, it is more preferable that the dissociation constant (KD) for PAD4 (preferably human PAD4) is low, and the upper limit is, for example, 100 pM or less, more preferably 90 pM or less, and even more preferably 80 pM or less, and the lower limit is not particularly limited, but may be, for example, 20 pM or more, more preferably 30 pM or more, and more preferably 40 pM or more. The dissociation constant (KD) is, for example, a value measured by the surface plasmon resonance (SPR) method described herein.

[0033] An anti-PAD4 antibody according to one embodiment of the present invention neutralizes the anti-PAD4 antibody activity of PAD4. The neutralizing activity of an anti-PAD4 antibody can be evaluated, for example, by a citrullination inhibition test shown in the Examples below. Citrullination by PAD4 is inhibited by adding an anti-PAD4 antibody. The anti-PAD4 antibody of the present invention can neutralize the citrullination activity by PAD4 by 50% or more, more preferably 80% or more, and most preferably 90% or more.

[0034] The anti-PAD4 antibody according to one embodiment of the present invention has excellent storage stability. The storage stability can be evaluated based on the decrease in binding to PAD4 after storage for a certain period of time, and the PAD4 binding amount of the anti-PAD4 antibody can be specifically measured by measuring the antigen binding activity using a surface plasmon resonance device described in Example 5. The anti-PAD4 antibody according to one embodiment of the present invention, when stored at 40°C for one month, can have a PAD4 binding amount of 90%, 93%, 94%, 95%, 96%, or 97% or more compared to the PAD4 binding amount of the anti-PAD4 antibody or its antibody fragment before storage.

[0035] The binding site of the anti-PAD4 antibody of the present invention when it binds to PAD4 is not particularly limited, but it is preferable that the antibody binds to an epitope including positions 345, 347, and 348 of PAD4 (e.g., SEQ ID NO: 29), for example.

[0036] The anti-PAD4 antibody of the present invention includes monoclonal antibodies obtained by immunizing mammals such as mice or chickens with PAD4 or a partial fragment thereof as an antigen to produce hybridomas, chimeric antibodies and humanized antibodies produced using gene recombination technology, and human antibodies produced using human antibody-producing transgenic animals. It also includes antibodies obtained by affinity maturation of the antibodies obtained above. For example, the parent antibody G8 described below can be used. When the anti-PAD4 antibody of the present invention or its antibody fragment is administered to humans as a medicine, a humanized antibody or a human antibody or an antibody fragment thereof is preferable from the viewpoint of side effects.

[0037] The antigen may be used for immunization as it is, or may be used as a complex with a carrier protein. Condensing agents such as glutaraldehyde, carbodiimide, and maleimide active ester can be used to prepare the complex of the antigen and carrier protein. Examples of carrier proteins include bovine serum albumin, thyroglobulin, hemocyanin, and KLH.

[0038] The animals to be immunized include mammals such as mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, pigs, goats, horses, and cows, and chickens, and the inoculation method includes subcutaneous, intramuscular, or intraperitoneal administration. The vaccine may be administered by mixing with complete Freund's adjuvant or incomplete Freund's adjuvant, and is usually administered once every 2 to 5 weeks. Antibody-producing cells obtained from the spleen or lymph nodes of the immunized animals are fused with myeloma cells and isolated as hybridomas. Myeloma cells derived from animals, such as mice, rats, humans, and chickens, are used.

[0039] <Monoclonal antibodies> Specifically, monoclonal antibodies can be obtained as follows. That is, the above-mentioned antigen is used as an immunogen, and the immunogen, together with Freund's adjuvant as necessary, is injected subcutaneously, intramuscularly, intravenously, into the footpad, or intraperitoneally once or several times or transplanted into the above-mentioned animal to immunize it. Usually, immunization is performed 1 to 4 times at intervals of about 1 to 14 days from the first immunization, and antibody-producing cells are obtained from the immunized animal about 1 to 5 days after the final immunization.

[0040] Monoclonal antibodies can be obtained using methods well known to those skilled in the art (e.g., Current Protocols in Molecular Biology, John Wiley & Sons (1987); Antibodies: A Laboratory Manual, Ed. Harlow and David Lane, Cold Spring Harbor Laboratory (1988)).

[0041] A "hybridoma" secreting a monoclonal antibody can be prepared according to the method of Kohler and Milstein et al. (Nature, 256, 495, 1975) or a modification method equivalent thereto. That is, the hybridoma can be prepared by cell fusion of antibody-producing cells contained in the spleen or the like obtained from an immunized animal with myeloma cells that are not capable of producing autoantibodies and are derived from an animal, preferably a mouse, rat, chicken or human.

[0042] Examples of myeloma cells that can be used in cell fusion include mouse-derived myeloma P3 / X63-AG8.653 (653), P3 / NSI / 1-Ag4-1 (NS-1), P3 / X63-Ag8.U1 (P3U1), SP2 / 0-Ag14 (Sp2 / O, Sp2), PAI, F0, or BW5147, rat-derived myeloma 210RCY3-Ag.2.3., and human-derived myeloma U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR, D1R11, or CEM-T15.

[0043] Examples of fusion promoters include polyethylene glycol, and cell fusion can usually be carried out by reacting the cells using polyethylene glycol (average molecular weight 1000 to 4000) at a concentration of about 20 to 50%, at a temperature of 20 to 40°C, preferably 30 to 37°C, with the ratio of antibody-producing cells to myeloma cells usually being about 1:1 to 10:1, for about 1 to 10 minutes.

[0044] Hybridoma clones that produce monoclonal antibodies can be screened by culturing the hybridomas, for example, in a microtiter plate and measuring the reactivity of the culture supernatant in the wells to the immunogen by an immunochemical method such as ELISA.

[0045] Cloning can be performed by limiting dilution from wells containing hybridomas that produce the desired antibody to obtain clones. Selection and breeding of hybridomas is usually performed in an animal cell medium containing 10-20% fetal bovine serum and supplemented with HAT (hypoxanthine, aminopterin, thymidine).

[0046] Monoclonal antibodies can be produced from hybridomas by culturing the hybridomas in vitro or by growing them in vivo, such as in ascites of an animal such as a mouse, rat, or chicken, and isolating the antibodies from the resulting culture supernatant or from the ascites of the animal.

[0047] When culturing in vitro, it is possible to use a nutrient medium suitable for growing, maintaining and preserving hybridomas and producing monoclonal antibodies in the culture supernatant, depending on various conditions such as the characteristics of the cell type being cultured and the culture method.

[0048] Examples of basal media include low-calcium media such as Ham'F12 medium, MCDB153 medium, and low-calcium MEM medium, and high-calcium media such as MCDB104 medium, MEM medium, D-MEM medium, RPMI1640 medium, ASF104 medium, and RD medium. The basal medium may contain, for example, serum, hormones, cytokines, and / or various inorganic or organic substances, depending on the purpose.

[0049] The isolation and purification of the monoclonal antibody can be carried out by subjecting the above-mentioned culture supernatant or ascites to saturated ammonium sulfate, the euglobulin precipitation method, the caproic acid method, the caprylic acid method, ion exchange chromatography (DEAE or DE52, etc.), affinity column chromatography such as an anti-immunoglobulin column or a protein A column, etc. Specifically, the purification of the monoclonal antibody may be carried out using a method known as a method for purifying immunoglobulin, and can be easily achieved by means of, for example, ammonium sulfate fractionation, PEG fractionation, ethanol fractionation, the use of an anion exchanger, affinity chromatography using PAD4, etc.

[0050] Monoclonal antibodies can also be obtained by phage display. In the phage display method, phages selected from any phage antibody library are screened using the target immunogen to select phages having the desired binding affinity to the immunogen. Next, the antibody-corresponding sequence contained in the phage is isolated or sequenced, and an expression vector containing a nucleic acid molecule encoding an antibody or an antigen-binding domain is constructed based on the isolated sequence or the sequence information determined. Then, a cell line transfected with such an expression vector is cultured to produce a monoclonal antibody. By using a human antibody library as the phage antibody library, a human antibody having the desired binding affinity can be generated.

[0051] A preferred embodiment of the anti-PAD4 antibody of the present invention is a chimeric antibody. An example of the "chimeric antibody" is a chimeric antibody whose variable region is derived from the immunoglobulin of a non-human animal (mouse, rat, hamster, chicken, etc.) and whose constant region is derived from human immunoglobulin. For example, the chimeric antibody can be produced by immunizing a mouse with an antigen, excising the variable region that binds to the antigen from the gene of the mouse monoclonal antibody, and combining it with an antibody constant region derived from human bone marrow. The constant region derived from human immunoglobulin has a unique amino acid sequence depending on the isotype, such as IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD, and IgE, but the constant region of the recombinant chimeric antibody of the present invention may be the constant region of human immunoglobulin belonging to any isotype. Preferably, it is the constant region of human IgG. An expression vector can be produced using the gene of the chimeric antibody produced in this way. A host cell is transformed with the expression vector to obtain a transformed cell producing a chimeric antibody, and the transformed cell is cultured to obtain the desired chimeric antibody from the culture supernatant.

[0052] Another preferred embodiment of the anti-PAD4 antibody of the present invention is a humanized antibody. The "humanized antibody" of the present invention is an antibody in which only the DNA sequence of the antigen-binding site (CDR; complementarity determining region) of a non-human animal antibody such as a mouse is transplanted (CDR grafting) into a human antibody gene. For example, it can be prepared by referring to the methods described in JP-A-4-506458 and JP-A-2912618. Specifically, it means a humanized antibody characterized in that a part or all of its CDRs are CDRs derived from a monoclonal antibody of a non-human animal (mouse, rat, hamster, chicken, etc.), the framework region of its variable region is a framework region of the variable region derived from human immunoglobulin, and its constant region is a constant region derived from human immunoglobulin.

[0053] The humanized antibody of the present invention can be produced, for example, as follows: However, it goes without saying that the production method is not limited to this.

[0054] Various techniques known in the art can be used to humanize an antibody (e.g., Almagro et al., FRont Biosci. 2008 Jan 1;13:1619-1633.), specifically, for example, CDR grafting (Ozaki et al., Blood. 1999 Jun 1;93(11):3922-3930.), Re-surfacing (roguska et al., Proc Natl Acad Sci US A. 1994 Feb 1;91(3):969-973.), or FR shuffling (Damschroder et al., Mol Immunol. 2007 Apr;44(11):3049-3060. Epub 2007 Jan 22.). To alter or improve antigen binding, amino acid residues in the human FR region may be replaced with corresponding residues from the CDR donor antibody. This FR substitution can be performed by methods well known in the art (Riechmann et al., Nature. 1988 Mar 24;332(6162):323-327.). For example, FR residues important for antigen binding may be identified by modeling the interaction between CDR and FR residues. Alternatively, unusual FR residues at specific positions may be identified by sequence comparison. Humanization may also be performed by the method described in Nishibori et al., Mol Immunol. 2006 Feb;43(6):634-42.

[0055] The isolated human heavy chain gene grafted with DNA of the mouse heavy chain CDR region is introduced into an appropriate expression vector so as to be expressible, and similarly, the human light chain gene grafted with DNA of the mouse light chain CDR region is introduced into another appropriate expression vector so as to be expressible. Alternatively, the human heavy chain and light chain genes grafted with mouse CDRs can be introduced into the same expression vector so as to be expressible. A humanized antibody-producing transformed cell is obtained by transforming a host cell with the expression vector thus prepared, and the transformed cell is cultured to obtain the desired humanized antibody from the culture supernatant.

[0056] Another preferred embodiment of the anti-PAD4 antibody of the present invention is a human antibody. A human antibody is an antibody in which all regions, including the variable region of the heavy chain and the constant region of the heavy chain, and the variable region of the light chain and the constant region of the light chain, that constitute immunoglobulin are immunoglobulins derived from genes that code for human immunoglobulins, and can be produced by introducing a human antibody gene into a mouse. Specifically, for example, a transgenic animal produced by incorporating at least a human immunoglobulin gene into the gene locus of an animal other than human, such as a mouse or chicken, can be produced in the same manner as the above-mentioned method for producing a monoclonal antibody by immunizing the animal with an antigen.

[0057] For example, transgenic mice producing human antibodies can be prepared according to the methods described in Nature Genetics, Vol.7, p.13-21, 1994; Nature Genetics, Vol.15, p.146-156, 1997; Published Japanese Translation of PCT International Publication No. 4-504365; Published Japanese Translation of PCT International Publication No. 7-509137; International Publication No. WO 94 / 25585; Nature, Vol.368, p.856-859, 1994; and Published Japanese Translation of PCT International Publication No. 6-500233. More specifically, HuMab (registered trademark) mice (Medarex, Princeton NJ), KMTM mice (Kirin Pharma Company, Japan), KM (FCγRIIb-KO) mice, etc. are included.

[0058] Specific examples of the monoclonal antibody of the present invention include: a) an anti-PAD4 antibody, in which HCDR1 consists of the amino acid sequence of SEQ ID NO: 1, HCDR2 consists of the amino acid sequence of SEQ ID NO: 7, HCDR3 consists of the amino acid sequence of SEQ ID NO: 11, LCDR1 consists of the amino acid sequence of SEQ ID NO: 4, LCDR2 consists of the amino acid sequence of SEQ ID NO: 5, and LCDR3 consists of the amino acid sequence of SEQ ID NO: 13; b) an anti-PAD4 antibody, in which HCDR1 consists of the amino acid sequence of SEQ ID NO: 1, HCDR2 consists of the amino acid sequence of SEQ ID NO: 8, HCDR3 consists of the amino acid sequence of SEQ ID NO: 11, LCDR1 consists of the amino acid sequence of SEQ ID NO: 4, LCDR2 consists of the amino acid sequence of SEQ ID NO: 5, and LCDR3 consists of the amino acid sequence of SEQ ID NO: 13; c) an anti-PAD4 antibody, in which HCDR1 consists of the amino acid sequence of SEQ ID NO: 1, HCDR2 consists of the amino acid sequence of SEQ ID NO: 9, HCDR3 consists of the amino acid sequence of SEQ ID NO: 12, LCDR1 consists of the amino acid sequence of SEQ ID NO: 4, LCDR2 consists of the amino acid sequence of SEQ ID NO: 5, and LCDR3 consists of the amino acid sequence of SEQ ID NO: 14; d) an anti-PAD4 antibody, in which HCDR1 consists of the amino acid sequence of SEQ ID NO: 1, HCDR2 consists of the amino acid sequence of SEQ ID NO: 10, HCDR3 consists of the amino acid sequence of SEQ ID NO: 11, LCDR1 consists of the amino acid sequence of SEQ ID NO: 4, LCDR2 consists of the amino acid sequence of SEQ ID NO: 5, and LCDR3 consists of the amino acid sequence of SEQ ID NO: 15; and e) an anti-PAD4 antibody, in which HCDR1 consists of the amino acid sequence of SEQ ID NO: 1, HCDR2 consists of the amino acid sequence of SEQ ID NO: 24, HCDR3 consists of the amino acid sequence of SEQ ID NO: 11, LCDR1 consists of the amino acid sequence of SEQ ID NO: 4, LCDR2 consists of the amino acid sequence of SEQ ID NO: 5, and LCDR3 consists of the amino acid sequence of SEQ ID NO: 25, in which the amino acid corresponding to the 84th amino acid of the heavy chain is serine; etc. The antibodies described in a) to d) above have significantly improved binding affinity to PAD4, and the antibodies described in a) to d) above have improved storage stability of binding activity to PAD4. The antibody described in e) above is inhibited from cleavage and has significantly improved chemical stability. The antibodies described in a) to d) above have high chemical stability equivalent to that of the antibody described in e).

[0059] The amino acid sequences described in a) to e) above correspond to the amino acid sequences of the CDRs of the antibodies 2-1-47, 3-1-39, 3-2-37, 4-2-25, and G8ss described in the Examples below. That is, the amino acid sequences of the heavy chain CDR1, 2, and 3 and the light chain CDR1, 2, and 3 of 2-1-47 are the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 13, respectively. The amino acid sequences of the heavy chain CDR1, 2, 3 and the light chain CDR1, 2, and 3 of 3-1-39 are the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 13, respectively. The amino acid sequences of the heavy chain CDR1, 2, 3 and the light chain CDR1, 2, and 3 of 3-2-37 are the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 14, respectively. The amino acid sequences of the heavy chain CDR1, 2, and 3 and the light chain CDR1, 2, and 3 of 4-2-25 are the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 15, respectively. The amino acid sequences of the heavy chain CDR1, 2, and 3 and the light chain CDR1, 2, and 3 of G8ss are the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 24, SEQ ID NO: 11, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 25, respectively.

[0060] The anti-PAD4 antibody in a) above may have the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 13 as the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3, respectively, so long as it has binding ability to PAD4 (preferably, a KD value for PAD4 of 100 pM or less, more preferably 90 pM or less, and even more preferably 80 pM or less) and maintains the property of neutralizing the citrullination activity of PAD4. The anti-PAD4 antibody in b) above may have the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 13 as the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3, respectively, so long as it has binding ability to PAD4 (preferably, a KD value for PAD4 of 100 pM or less, more preferably 90 pM or less, and even more preferably 80 pM or less) and maintains the property of neutralizing the citrullination activity of PAD4. The anti-PAD4 antibody c) above may have the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 14 as the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3, respectively, so long as it has binding ability to PAD4 (preferably, a KD value for PAD4 of 100 pM or less, more preferably 90 pM or less, and even more preferably 80 pM or less) and maintains the property of neutralizing the citrullination activity of PAD4. The anti-PAD4 antibody in d) above has binding ability to PAD4 (preferably, the KD value for PAD4 is 100 pM or less, more preferably 90 pM or less, and even more preferably 80 pM or less), and the amino acid sequences of the heavy chain CDR1, 2, and 3 and the light chain CDR1, 2, and 3 may include the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 15, respectively, so long as the property of neutralizing the citrullination activity of PAD4 is maintained. The anti-PAD4 antibody e) above may have the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 24, SEQ ID NO: 11, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 25 as the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3, respectively, so long as it has binding ability to PAD4 (preferably, a KD value for PAD4 of 100 pM or less, more preferably 90 pM or less, and even more preferably 80 pM or less) and maintains the property of neutralizing the citrullination activity of PAD4.

[0061] An anti-PAD4 antibody according to one embodiment of the present invention has the ability to bind to PAD4 (preferably, a KD value for PAD4 of 100 pM or less, more preferably 90 pM or less, and even more preferably 80 pM or less) and may have a heavy chain CDR1 that includes the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 that includes the amino acid sequence shown in SEQ ID NO: 2, a heavy chain CDR3 that includes the amino acid sequence shown in SEQ ID NO: 3, a light chain CDR1 that includes the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 that includes the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 that includes the amino acid sequence shown in SEQ ID NO: 6, so long as the antibody maintains the property of binding to PAD4 (preferably, a KD value for PAD4 of 100 pM or less, more preferably 90 pM or less, and even more preferably 80 pM or less) and neutralizing the citrullination activity of PAD4. In addition, the amino acid sequence shown in SEQ ID NO: 2 includes an amino acid sequence in which the amino acid at position 11 is Tyr, Thr or Ile, the amino acid at position 12 is Gly, Ser or Pro, the amino acid at position 13 is Thr, Val, Tyr or Pro, the amino acid at position 14 is Pro or Asn, the amino acid at position 15 is Tyr, Ala, Gln or Leu, and the amino acid at position 17 is Gly, Thr or Ser, and the amino acid at each position is any combination selected from the above amino acids. In addition, in the amino acid sequence shown in SEQ ID NO: 3, the amino acid at position 1 is Ala or Gly, and specifically includes the amino acid sequences of SEQ ID NOs: 11 and 12. In addition, in the amino acid sequence shown in SEQ ID NO:6, the amino acid at position 4 is Thr, Leu or Tyr, and specifically includes the amino acid sequences of SEQ ID NOs:13, 14 and 15.

[0062] An anti-PAD4 antibody according to one embodiment of the present invention has the ability to bind to PAD4 (preferably, a KD value for PAD4 of 100 pM or less, more preferably 90 pM or less, and even more preferably 80 pM or less) and maintains the property of neutralizing the citrullination activity of PAD4. The heavy chain CDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1, the heavy chain CDR2 may comprise the amino acid sequence shown in any of SEQ ID NOs: 7 to 10, and the heavy chain CDR3 may comprise the amino acid sequence shown in SEQ ID NO: 11 or 12, the light chain CDR1 may comprise the amino acid sequence shown in SEQ ID NO: 4, the light chain CDR2 may comprise the amino acid sequence shown in SEQ ID NO: 5, and the light chain CDR3 may comprise the amino acid sequence shown in any of SEQ ID NOs: 13 to 15.

[0063] In addition, as long as the anti-PAD4 antibody of the present invention maintains its property of having binding ability to PAD4 (preferably, KD value for PAD4 is 100 pM or less, more preferably 90 pM or less, and even more preferably 80 pM or less) and neutralizing the citrullination activity of PAD4, one to several amino acids may be substituted in one or more of these CDRs. Here, one to several means, for example, one, two, or three. In addition, the amino acid substitution is preferably a conservative substitution in order to maintain the property of the present invention. Here, "conservative substitution" means replacing an amino acid residue with another chemically similar amino acid residue so as not to substantially alter the activity of the peptide. For example, a hydrophobic residue may be replaced with another hydrophobic residue, or a polar residue may be replaced with another polar residue having the same charge. Examples of functionally similar amino acids that can be replaced in this way include non-polar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine, etc. Examples of positively charged (basic) amino acids include arginine, histidine, lysine, etc. Examples of negatively charged (acidic) amino acids include aspartic acid, glutamic acid, etc. "Maintaining the characteristics of an antibody" means that these characteristics are maintained to the same extent as compared to before modification of the amino acid sequence of the CDR, for example, 80% or more, preferably 90% or more, and more preferably 95% or more. Note that "maintaining" also includes improvement.

[0064] The regions other than the CDRs are not particularly limited as long as they maintain the structure of an antibody and can exhibit their functions, and may be any of mouse-derived sequences, human-derived sequences, sequences derived from other mammals, chicken-derived sequences, chimeric sequences thereof, and artificial sequences. When constant regions are included, the amino acid sequences of the heavy and light chain constant regions are exemplified by those described in Nucleic Acids Research vol.14, p1779, 1986, The Journal of Biological Chemistry vol.257, p1516, 1982, and Cell vol.22, p197, 1980.

[0065] As an example of a preferred embodiment of the present invention in which a region other than CDR is modified, an antibody in which the amino acid (usually asparagine) corresponding to the 84th amino acid of the antibody heavy chain (for example, SEQ ID NO: 26) is serine is mentioned. Here, the "corresponding amino acid" refers to an amino acid (usually asparagine) present at the position of the 84th amino acid, asparagine, of SEQ ID NO: 26 when the target amino acid sequence is aligned with SEQ ID NO: 26. Such an anti-PAD4 antibody of one embodiment of the present invention is excellent in the chemical stability of the antibody, specifically, in the inhibition of cleavage occurring during production. The inhibition of cleavage can be evaluated by a decrease in the amount of antibody cleaved between the 84th asparagine residue and the 85th serine residue of the heavy chain by peptide mapping, and specifically, can be evaluated by measuring the amount of cleaved peptides, for example, by the method described in Example 1. In the anti-PAD4 antibody of one embodiment of the present invention that is excellent in the inhibition of cleavage occurring during production, when stored at 4°C for 2 weeks, the amount of cleaved antibody can be 3%, 2%, 1% or less compared to the antibody before the storage.

[0066] Further examples include humanized antibodies in which the CDRs are derived from humans. Such humanized antibodies include: a) an anti-PAD4 antibody whose heavy chain variable region and light chain variable region consist of or contain the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 16 and the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO: 17, respectively; b) an anti-PAD4 antibody whose heavy chain variable region and light chain variable region consist of or contain the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 18 and the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO: 19, respectively; c) an anti-PAD4 antibody whose heavy chain variable region and light chain variable region consist of or contain the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 20 and the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO: 21, respectively; d) an anti-PAD4 antibody whose heavy chain variable region and light chain variable region consist of or contain the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 22 and the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO: 23, respectively; and e) an anti-PAD4 antibody whose heavy chain variable region and light chain variable region consist of or contain the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 26 and the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO: 27, respectively; Examples include one or more antibodies selected from the group consisting of:

[0067] Alternatively, the anti-PAD4 antibody according to one embodiment of the present invention comprises: a) an anti-PAD4 antibody, the heavy and light chains of which consist of or contain the amino acid sequences set forth in SEQ ID NOs: 16 and 17, respectively; b) an anti-PAD4 antibody whose heavy and light chains consist of or contain the amino acid sequences set forth in SEQ ID NOs: 18 and 19, respectively; c) an anti-PAD4 antibody whose heavy and light chains consist of or contain the amino acid sequences set forth in SEQ ID NOs: 20 and 21, respectively; d) an anti-PAD4 antibody, the heavy and light chains of which consist of or contain the amino acid sequences set forth in SEQ ID NOs: 22 and 23, respectively; and e) an anti-PAD4 antibody, the heavy and light chains of which consist of or contain the amino acid sequences set forth in SEQ ID NOs: 26 and 27, respectively; The antibody may be one or more antibodies selected from the group consisting of:

[0068] In addition, the heavy chain variable region and / or light chain variable region of the amino acid sequence of the humanized antibody may have a substitution, deletion, addition, or insertion of one or several amino acids (1 to 20, 1 to 10, or 1 to 5), so long as it has the ability to bind to PAD4 (preferably, the KD value for PAD4 is 100 pM or less, more preferably 90 pM or less, and even more preferably 80 pM or less) and maintains the property of neutralizing the citrullination activity of PAD4. Such substitution, deletion, or addition may be introduced into the CDR, but is preferably introduced into a region other than the CDR. Moreover, the amino acid substitution is preferably a conservative substitution in order to maintain the properties of the present invention.

[0069] The amino acid sequence of the anti-PAD4 antibody of the present invention, which includes substitutions, deletions, etc. in the heavy chain variable region and / or light chain variable region, is such that the heavy chain variable region has a similar identity to the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 16, the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 18, the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 20, the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 22, or the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 26 by 90% or more (more preferably 95%, 96%, 97%, 98%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, %, 99% or more) identity to the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO: 17, the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO: 19, the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO: 21, the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO: 23, or the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO: 27.

[0070] The anti-PAD4 antibody of the present invention may also be an anti-PAD4 antibody having a heavy chain with an amino acid sequence that is 90% or more (more preferably 95%, 96%, 97%, 98%, 99% or more) identical to SEQ ID NO: 16, 18, 20, 22, 26 in the Sequence Listing, and a light chain with an amino acid sequence that is 90% or more (more preferably 95%, 96%, 97%, 98%, 99% or more) identical to SEQ ID NO: 17, 19, 21, 23, 27 in the Sequence Listing, as long as the antibody has the ability to bind to PAD4 (preferably, a KD value for PAD4 of 100 pM or less, more preferably 90 pM or less, and even more preferably 80 pM or less) and maintains the property of neutralizing the citrullination activity of PAD4.

[0071] The anti-PAD4 antibodies of the present invention include multispecific antibodies, functionally modified antibodies, and conjugated antibodies having a CDR consisting of the above-mentioned specific amino acid sequence, or a variable region consisting of the above-mentioned specific amino acid sequence.

[0072] The anti-PAD4 antibody of the present invention can be combined with an antibody having an antigen-binding specificity other than PAD4 by genetic engineering techniques to produce a multispecific antibody such as a bispecific antibody. Such genetic engineering techniques have already been established in this field. For example, the desired bispecific antibody can be obtained by using DVD-Ig (Wu et al., Nature Biotechnology 25(11), 1290(2007)), in which variable regions are linked in series, or ART-Ig technology (Kitazawa et al., Nature Medicine 18(10), 1570(2012)), in which the heavy chains of two types of antibodies that bind to different antigens are combined by modifying the Fc region of the antibody.

[0073] The modified molecule of the anti-PAD4 antibody of the present invention includes a functionally modified antibody. The functionally modified antibody refers to an antibody whose functions, such as cell killing function, complement activation function, and blood half-life extension function, have been modified by modifying the antibody sequence, sugar chain, etc. (Shitara Kenya, Yakugaku Zasshi, 2009, Vol. 129(1), p. 3; Ishii Akiko et al., Nihon Yakurigaku Zasshi, 2010, Vol. 136(5), p. 280; Hashiguchi Shuhei et al., Seikagaku, 2010, Vol. 82(8), p. 710).

[0074] The functionally modified anti-PAD4 antibody is prepared by the following method. For example, when the anti-PAD4 antibody of the present invention is produced using CHO cells in which the α1,6-fucosyltransferase (FUT8) gene has been destroyed as a host cell, an antibody with a reduced fucose content in the sugar chain and enhanced cell killing function is obtained, and when the antibody is produced using CHO cells in which the FUT8 gene has been introduced as a host cell, an antibody with a reduced cell killing function is obtained (WO 2005 / 035586, WO 2002 / 31140, WO 00 / 61739). In addition, the complement activation function can be regulated by modifying the amino acid residues in the Fc region (U.S. Patent No. 6,737,056, U.S. Patent No. 7,297,775, U.S. Patent No. 7,317,091). Furthermore, the half-life in blood can be extended by using a mutant Fc region that enhances the binding to FcRn, one of the Fc receptors (Shuhei Hashiguchi et al., Seikagaku, 2010, Vol. 82(8), p. 710). These functionally modified antibodies can be produced by genetic engineering.

[0075] Modified molecules of the anti-PAD4 antibody of the present invention include conjugated antibodies, which are obtained by chemically or genetically binding the anti-PAD4 antibody to a functional molecule other than the anti-PAD4 antibody of the present invention, such as a non-peptide polymer such as polyethylene glycol (PEG), a radioactive substance, a toxin, a low molecular weight compound, a cytokine, a growth factor, albumin, an enzyme, or another antibody.

[0076] When PEG is used as a functional molecule, it may be of any commonly used type, and may be of either linear or branched type. PEG can be bound to an amino group of an antibody by using, for example, an NHS active group.

[0077] When a radioactive substance is used as a functional molecule, 131 I, 125 I, 90 Y, 64 Cu, 99 Tc, 77 Lu or 211 Radioactive substances can be directly bound to antibodies using the chloramine T method or the like.

[0078] When an enzyme is used as a functional molecule, luciferase (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456), malate dehydrogenase, urease, peroxidase (e.g., horseradish peroxidase (HRPO)), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidase (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidase (e.g., uricase and xanthine oxidase, etc.), lactoperoxidase, microperoxidase, etc. may be used.

[0079] Linkers used to chemically bind toxins, small molecules, or enzymes include divalent radicals (e.g., alkylene, arylene, heteroarylene), linkers represented by -(CR2)nO(CR2)n- (R is an optional substituent, n is a positive integer), repeating units of alkoxy (e.g., polyethyleneoxy, PEG, polymethyleneoxy, etc.) and alkylamino (e.g., polyethyleneamino, Jeffamine™), as well as diacid esters and amides (including succinate, succinamide, diglycolate, malonate, caproamide, etc.). Chemical modification methods for binding functional molecules have already been established in this field (DJ King., Applications and Engineering of Monoclonal antibodies., 1998 TJ International Ltd, Monoclonal Antibody-Based Therapy of Cancer., 1998 Marcel Dekker Inc; Chari et al., Cancer Res., 1992 Vol152:127; Liu et al., Proc Natl Acad Sci USA., 1996 Vol 93:8681).

[0080] An "antigen-binding fragment" of an antibody in the present invention means a partial region of an antibody having antigen-binding ability as described above, and specifically includes F(ab')2, Fab', Fab, Fv (variable fragment of antibody), disulfide-linked Fv, single-chain antibody (scFv), and polymers thereof. Furthermore, antigen-binding fragments include conjugated antigen-binding fragments to which functional molecules other than the anti-PAD4 antibody of the present invention are chemically or genetically bound, such as non-peptide polymers such as polyethylene glycol (PEG), radioactive substances, toxins, low molecular weight compounds, cytokines, growth factors (TGF-β, NGF, Neurotrophin, etc.), albumin, enzymes, and other antibodies.

[0081] Here, "F(ab')2" and "Fab" refer to antibody fragments produced by treating immunoglobulin with protease such as pepsin or papain, and digesting the antibody fragments before and after the disulfide bond between the two heavy chains in the hinge region. For example, when IgG is treated with papain, it is cleaved upstream of the disulfide bond between the two heavy chains in the hinge region to produce two homologous antibody fragments in which a light chain consisting of VL (light chain variable region) and CL (light chain constant region), and a heavy chain fragment consisting of VH (heavy chain variable region) and CHγ1 (γ1 region in the heavy chain constant region) are linked by a disulfide bond at the C-terminal region. Each of these two homologous antibody fragments is called Fab. When IgG is treated with pepsin, it is cleaved downstream of the disulfide bond between the two heavy chains in the hinge region to produce an antibody fragment that is slightly larger than the one in which the two Fabs are linked by the hinge region. This antibody fragment is called F(ab')2.

[0082] A conjugate antigen-binding fragment is an example of a modified molecule of the antigen-binding fragment of the anti-PAD4 antibody of the present invention. Examples of the conjugate antigen-binding fragment include a conjugate antigen-binding fragment in which a functional molecule other than the anti-PAD4 antibody of the present invention, such as a non-peptide polymer such as polyethylene glycol (PEG), a radioactive substance, a toxin, a low molecular weight compound, a cytokine, a growth factor, albumin, an enzyme, or another antibody, is chemically or genetically bound to a partial region of the anti-PAD4 antibody having antigen-binding activity.

[0083] When PEG is used as a functional molecule, it may be of the linear or branched type, as long as it is a commonly used functional molecule. PEG can be bound to the amino group of the anti-PAD4 antibody by using, for example, an NHS active group.

[0084] When a radioactive substance is used as a functional molecule, 131 I, 125 I, 90 Y, 64 Cu, 99 Tc, 77 Lu or 211 The radioactive substance can be directly bound to a part of the anti-PAD4 antibody that has antigen-binding activity by the chloramine T method or the like.

[0085] When an enzyme is used as a functional molecule, luciferase (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456), malate dehydrogenase, urease, peroxidase (e.g., horseradish peroxidase (HRPO)), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidase (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidase (e.g., uricase and xanthine oxidase, etc.), lactoperoxidase, microperoxidase, etc. may be used.

[0086] Linkers used to chemically bind toxins, small molecules, or enzymes include divalent radicals (e.g., alkylene, arylene, heteroarylene), linkers represented by -(CR2)nO(CR2)n- (R is an optional substituent, n is a positive integer), repeating units of alkoxy (e.g., polyethyleneoxy, PEG, polymethyleneoxy, etc.) and alkylamino (e.g., polyethyleneamino, Jeffamine™), as well as diacid esters and amides (including succinate, succinamide, diglycolate, malonate, caproamide, etc.). Chemical modification methods for binding functional molecules have already been established in this field (DJ King., Applications and Engineering of Monoclonal antibodies., 1998 TJ International Ltd, Monoclonal Antibody-Based Therapy of Cancer., 1998 Marcel Dekker Inc; Chari et al., Cancer Res., 1992 Vol152:127; Liu et al., Proc Natl Acad Sci USA., 1996 Vol 93:8681).

[0087] The anti-PAD4 antibody of the present invention comprising a CDR or variable region having a specific amino acid sequence preferably has a constant region that is a human IgG (IgG1, IgG2, IgG3, IgG4) constant region in order to maintain a long half-life in blood.

[0088] The anti-PAD4 antibody and its antigen-binding fragment of the present invention include an anti-PAD4 antibody and its antigen-binding fragment that compete with an antibody having the above-mentioned specific CDR amino acid sequence in binding to PAD4, as long as the antibody has the ability to bind to PAD4 (preferably, the KD value for PAD4 is 100 pM or less, more preferably 90 pM or less, and even more preferably 80 pM or less) and maintains the property of neutralizing the citrullination activity of PAD4. An example of an antibody that competes with an antibody having the above-mentioned specific CDR amino acid sequence in binding to PAD4 is an antibody having an epitope in a region including positions 345, 347, and 348 of PAD4. However, the G8 antibody is not included. The antibody can be obtained (screened) or evaluated by coexisting an antibody having the above-mentioned CDR sequence with PAD4 in a binding system. For example, the antibody can be obtained by screening using the surface plasmon resonance (SPR) method described in WO 2016 / 175236.

[0089] The anti-PAD4 antibody that competes with the anti-PAD4 antibody containing the amino acid sequence of the specific CDR described above for binding to PAD4 may be an antibody derived from any animal, such as a mouse antibody, human antibody, rat antibody, rabbit antibody, goat antibody, or camel antibody, or may be a chimeric antibody or humanized antibody which is a combination of these antibodies, but is preferably a chimeric antibody, humanized antibody, or human antibody.

[0090] The anti-PAD4 antibody or antibody fragment thereof of the present invention can be produced, for example, using a transformant (host) cell containing a recombinant vector comprising the nucleic acid molecule described below that encodes the anti-PAD4 antibody or antibody fragment thereof of the present invention.

[0091] <Nucleic acid molecule> One embodiment of the present invention is a nucleic acid molecule that is a polynucleotide encoding the anti-PAD4 antibody or an antibody fragment thereof of the present invention. There is no particular limitation on the nucleic acid molecule as long as it encodes a polypeptide including the above-mentioned CDR, variable region, or full-length amino acid sequence. For example, a) a heavy chain variable region and a light chain variable region, the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 16 and the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO: 17; b) a heavy chain variable region and a light chain variable region, the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 18 and the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO: 19; c) a heavy chain variable region and a light chain variable region, the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 20 and the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO: 21; d) a heavy chain variable region and a light chain variable region, the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 22 and the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO: 23, and e) Polynucleotides comprising base sequences encoding the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 28 and the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO: 27, which are the heavy chain variable region and the light chain variable region, respectively.

[0092] Other examples of nucleic acid molecules of the invention include: a) the amino acid sequences shown in SEQ ID NOs: 16 and 17, which are the heavy and light chains; b) the amino acid sequences shown in SEQ ID NOs: 18 and 19, which are the heavy and light chains; c) the amino acid sequences shown in SEQ ID NOs: 20 and 21, which are the heavy and light chains; d) the amino acid sequences set forth in SEQ ID NOs: 22 and 23, which are heavy and light chains, and e) Polynucleotides comprising base sequences encoding the amino acid sequences shown in SEQ ID NOs: 28 and 27, respectively, which are the heavy and light chains.

[0093] Other examples of nucleic acid molecules of the invention include: A polynucleotide comprising the base sequence shown in SEQ ID NO: 32, which is the full-length heavy chain of an anti-PAD4 antibody, and the base sequence shown in SEQ ID NO: 33, which is the full-length light chain of an anti-PAD4 antibody; A polynucleotide comprising the base sequence shown in SEQ ID NO: 34, which is the full-length heavy chain of an anti-PAD4 antibody, and the base sequence shown in SEQ ID NO: 35, which is the full-length light chain of an anti-PAD4 antibody; A polynucleotide comprising the base sequence shown in SEQ ID NO: 36, which is the full-length heavy chain of an anti-PAD4 antibody, and the base sequence shown in SEQ ID NO: 37, which is the full-length light chain of an anti-PAD4 antibody; A polynucleotide comprising the base sequence shown in SEQ ID NO: 38, which is the full-length heavy chain of an anti-PAD4 antibody, and the base sequence shown in SEQ ID NO: 39, which is the full-length light chain of an anti-PAD4 antibody; A polynucleotide comprising the base sequence shown in SEQ ID NO: 40, which is the full-length heavy chain of an anti-PAD4 antibody, and the base sequence shown in SEQ ID NO: 31, which is the full-length light chain of an anti-PAD4 antibody; Examples include:

[0094] The nucleic acid molecule of the present invention may contain a polynucleotide that hybridizes with a complementary DNA of the base sequence of the heavy chain variable region or the light chain variable region under stringent conditions, as long as it has a binding ability to PAD4 and encodes a monoclonal antibody that neutralizes the citrullination activity of PAD4. Here, stringent conditions include, for example, conditions in which Southern hybridization is followed by washing at 68°C with a salt concentration equivalent to 0.1×SSC and 0.1% SDS.

[0095] The nucleic acid molecule of the present invention may encode all of the heavy and light chain constant and variable regions, or may encode only the heavy and light chain variable regions. When encoding all of the constant and variable regions, the base sequences of the heavy and light chain constant regions are preferably those described in Nucleic Acids Research vol.14, p1779, 1986, The Journal of Biological Chemistry vol.257, p1516, 1982, and Cell vol.22, p197, 1980.

[0096] The nucleotide sequences encoding the full-length heavy and light chains of the humanized anti-PAD4 antibody G8 described in WO 2016 / 143753 are shown in SEQ ID NOs: 30 and 31. For example, by modifying this nucleotide sequence, it is possible to obtain a nucleic acid molecule encoding the anti-PAD4 antibody of the present invention or an antibody fragment thereof.

[0097] The nucleic acid molecule of the present invention can be obtained, for example, by the following method. First, total RNA is prepared from cells such as hybridomas using a commercially available RNA extraction kit, and cDNA is synthesized by reverse transcriptase using random primers or the like. Next, cDNA encoding an antibody is amplified by PCR using oligonucleotides of sequences conserved in the variable regions of known human antibody heavy chain genes and light chain genes as primers. The sequence encoding the constant region can be obtained by amplifying a known sequence by PCR. The base sequence of the DNA can be determined by a conventional method, such as by incorporating it into a plasmid for sequencing. Alternatively, DNA encoding the monoclonal antibody of the present invention can be obtained by chemically synthesizing the sequence of the variable region or a portion thereof and linking it to a sequence containing the constant region.

[0098] The present invention also provides a recombinant vector containing the nucleic acid molecule of the present invention and a transformant (host cell) containing the recombinant vector. The recombinant vector may be a vector (e.g., pBR322, pUC119, or a derivative thereof) that can be expressed in a prokaryotic cell such as Escherichia coli, but is preferably a vector that can be expressed in a eukaryotic cell, and more preferably a vector that can be expressed in a cell derived from a mammal. Examples of vectors that can be expressed in a cell derived from a mammal include plasmid vectors such as pcDNA3.1 (manufactured by Invitrogen), pConPlus, pcDM8, pcDNA I / Amp, pcDNA3.1, and pREP4, and viral vectors such as pDON-AI DNA (manufactured by Takara Bio). The vector may be one vector containing a heavy chain coding sequence and a light chain coding sequence, or two vectors, one containing a heavy chain coding sequence and the other containing a light chain coding sequence.

[0099] The transformant into which the recombinant vector of the present invention is introduced may be a prokaryotic cell such as Escherichia coli or Bacillus subtilis, but is preferably a eukaryotic cell, more preferably a cell derived from a mammal, such as Chinese hamster ovary cell (CHO cell), COS, myeloma, BHK, HeLa, Vero, 293, NS0, Namalwa, YB2 / 0, etc.

[0100] Anti-PAD4 antibodies or antigen-binding fragments thereof obtained by the methods described in the specification or known methods can be purified to homogeneity. Separation and purification of antibodies and the like can be performed using separation and purification methods used for normal proteins. For example, the antibody can be separated and purified by appropriately selecting and combining chromatography columns such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, etc. (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988), but is not limited thereto. Examples of columns used in affinity chromatography include protein A columns, protein G columns, anti-immunoglobulin antibody binding columns, antigen binding columns, etc. Examples of protein A columns include Hyper D, POROS, Sepharose FF (Amersham Biosciences), etc.

[0101] <Composition> One embodiment of the present invention is a composition comprising the anti-PAD4 antibody or its antibody fragment according to the embodiment of the present invention. This composition can be used to efficiently detect PAD4. It can also efficiently inhibit citrullination of proteins by PAD4. It can also efficiently inhibit nephrosis in cells. It can also prevent or treat RA or arthritis, systemic lupus erythematosus, lupus nephritis, or graft-versus-host disease. The components contained in this composition are not particularly limited as long as they contain the anti-PAD4 antibody or antibody fragment according to the embodiment of the present invention, and may contain, for example, a buffer solution. One or more of the various embodiments (e.g., carriers may be contained) of the inhibitor and pharmaceutical composition described below may be applied to this composition.

[0102] One embodiment of the present invention is a PAD4-induced protein citrullination inhibitor comprising the anti-PAD4 antibody or antibody fragment thereof according to the embodiment of the present invention. The inhibitor can be used to efficiently inhibit protein citrullination by PAD4. The reduction rate of citrullination activity by the inhibitor may be 20, 30, 40, 60, 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 PBS is used is set to 0%, for example. In one embodiment of the present invention, the "agent" includes, for example, a composition used in research or treatment. The inhibitor includes, for example, an agent for preventing or treating RA or arthritis, systemic lupus erythematosus, lupus nephritis, or graft-versus-host disease. 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 protein citrullination by PAD4, comprising a step of contacting the anti-PAD4 antibody or antibody fragment thereof according to the embodiment of the present invention with PAD4. One embodiment of the present invention is a method for inhibiting protein citrullination by PAD4, comprising a step of administering to a patient an anti-PAD4 antibody or an antibody fragment thereof according to the above-mentioned embodiment of the present invention. The above-mentioned inhibition method includes an inhibition method carried out for research or treatment. One embodiment of the present invention is use of an anti-PAD4 antibody or an antibody fragment thereof according to the above-mentioned embodiment of the present invention for producing an inhibitor of protein citrullination by PAD4.

[0103] One embodiment of the present invention is an inhibitor of cellular nephosis, comprising the anti-PAD4 antibody or antibody fragment thereof according to the embodiment of the present invention. By using this inhibitor, cellular nephosis caused by PAD4 can be efficiently inhibited. The reduction rate of cellular nephosis activity by the inhibitor may be 20, 30, 40, 60, 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 PBS is used is set to 0%, for example. 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 preventive or therapeutic agent for RA or arthritis, systemic lupus erythematosus, lupus nephritis, or graft-versus-host disease. 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 cellular nephosis, comprising a step of contacting the anti-PAD4 antibody or antibody fragment thereof according to the embodiment of the present invention with PAD4. One embodiment of the present invention is a method for inhibiting nephrosis in cells, comprising administering to a patient the anti-PAD4 antibody or antibody fragment thereof according to the above-mentioned embodiment of the present invention. The above-mentioned inhibition method includes an inhibition method carried out for research or treatment. One embodiment of the present invention is the use of the anti-PAD4 antibody or antibody fragment thereof according to the above-mentioned embodiment of the present invention for producing a nephrosis inhibitor in cells.

[0104] One embodiment of the present invention is a pharmaceutical composition comprising the anti-PAD4 antibody or antibody fragment thereof according to the above-mentioned embodiment of the present invention. This pharmaceutical composition can be used to prevent, treat, and prevent recurrence of RA or arthritis, systemic lupus erythematosus, lupus nephritis, or graft-versus-host disease. The pharmaceutical composition may contain one or more pharmacologically acceptable carriers. The pharmaceutical composition includes, for example, a pharmaceutical composition for treating RA or arthritis, systemic lupus erythematosus, lupus nephritis, or graft-versus-host disease, and a pharmaceutical composition for preventing RA or arthritis, systemic lupus erythematosus, lupus nephritis, or graft-versus-host disease. The pharmaceutical composition may contain the composition according to the above-mentioned embodiment of the present invention. One embodiment of the present invention is a method for preventing, treating, or preventing recurrence of a disease, comprising a step of administering to a patient an effective amount of an anti-PAD4 antibody or an antibody fragment thereof (or a pharmaceutical composition comprising an anti-PAD4 antibody or an antibody fragment thereof) according to the above-mentioned embodiment of the present invention. The above-mentioned disease includes, for example, RA or arthritis, systemic lupus erythematosus, lupus nephritis, or graft-versus-host disease. One embodiment of the present invention is the use of an anti-PAD4 antibody or an antibody fragment thereof (or a pharmaceutical composition comprising an anti-PAD4 antibody or an antibody fragment thereof) according to the above-mentioned embodiment of the present invention for preventing, treating, or preventing recurrence of RA or arthritis, systemic lupus erythematosus, lupus nephritis, or graft-versus-host disease. One embodiment of the present invention is the use of an anti-PAD4 antibody or an antibody fragment thereof according to the above-mentioned embodiments of the present invention (or a pharmaceutical composition containing an anti-PAD4 antibody or an antibody fragment thereof) for producing an agent for preventing, treating, or preventing recurrence of RA or arthritis, systemic lupus erythematosus, lupus nephritis, or graft-versus-host disease.

[0105] In one embodiment of the present invention, the "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, for example, by mixing the active ingredient with the above-mentioned carrier and using any method known in the technical field of pharmaceutical formulations. In addition, the pharmaceutical composition may be used in any form as long as it is used for prevention or treatment, and may be an active ingredient alone or a mixture of the active ingredient with any ingredient.

[0106] The therapeutic or / and preventive effect of the pharmaceutical composition in RA may be evaluated, for example, by arthritis score, RA score, swelling width, imaging diagnosis, modified Total Sharp score, Disease Activity Score (DAS), ACR20, ACR50, ACR70, or disease markers, which indicate the achievement rate of the rheumatoid activity evaluation criteria created by the American Rheumatism Association (ACR). When evaluating by arthritis score, for example, when the arthritis score of a patient at the time of administration of the pharmaceutical composition is significantly reduced compared to the arthritis score at the time of non-administration, it may be judged that the therapeutic or / and preventive effect is present. Alternatively, when the arthritis score of a patient at the time of administration of the pharmaceutical composition is significantly reduced compared to the arthritis score of a patient at the time of administration of a negative control substance, it may be judged that the therapeutic or / and preventive effect is present. The reduction may be, for example, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 point or less, or may be within the range of any two of these values. Alternatively, it may be determined that a therapeutic or / and preventive effect has been achieved when the arthritis score graph area of ​​a patient when the pharmaceutical composition is administered is significantly reduced compared to the arthritis score graph area when the pharmaceutical composition is not administered. Alternatively, it may be determined that a therapeutic or / and preventive effect has been achieved when the arthritis score graph area of ​​a patient when the pharmaceutical composition is administered is significantly reduced compared to the arthritis score graph area of ​​a patient when a negative control substance is administered. The reduction may be, for example, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10% or less, or may be within a range of any two of these values.

[0107] The therapeutic and / or preventive effect of the pharmaceutical composition on systemic lupus erythematosus and lupus nephritis may be evaluated, for example, by damage in each organ, urinary protein amount, urinary protein score, survival rate, pathological evaluation score, amount of autoantibody produced in blood, amount of the third and fourth complement components (C3 and C4) in blood, or disease markers. In addition, it may be evaluated by the SLE disease activity index (SLEDAI) which scores these, the British Isles Lupus Assessment Group (BILAG) index which compares disease activity with that one month before, the physician's global assessment (PGA) which evaluates the overall disease activity of the patient by a doctor, the SLE responder index (SRI) 4 which evaluates these comprehensively, etc. When evaluating by the amount of urinary protein, for example, it may be judged that the therapeutic and / or preventive effect is present when the amount of albumin or the albumin / creatinine ratio in the urine of the patient at the time of administration of the pharmaceutical composition is significantly reduced compared to that at the time of non-administration. Alternatively, it may be determined that the therapeutic or / and preventive effect is achieved when the amount of urinary albumin or the albumin / creatinine ratio of the patient when the pharmaceutical composition is administered is significantly reduced compared to the amount of urinary albumin or the albumin / creatinine ratio of the patient when the negative control substance is administered. Alternatively, it may be determined that the therapeutic or / and preventive effect is achieved when the area of ​​the graph of the amount of urinary albumin or the albumin / creatinine ratio of the patient when the pharmaceutical composition is administered is significantly reduced compared to the area of ​​the graph of the amount of urinary albumin or the albumin / creatinine ratio of the patient when the pharmaceutical composition is not administered. Alternatively, it may be determined that the therapeutic or / and preventive effect is achieved when the amount of urinary albumin or the albumin / creatinine ratio of the patient when the pharmaceutical composition is administered is significantly reduced compared to the amount of urinary albumin or the albumin / creatinine ratio of the patient when the negative control substance is administered. The reduction may be, for example, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10% or less, or may be within a range of any two of these values.

[0108] The therapeutic and / or preventive effect of the pharmaceutical composition on graft-versus-host disease can be evaluated, for example, by the effect of improving characteristic organ findings or the effect of reducing the amount of steroids. For skin lesions, the effect can be evaluated by the improvement of polymorphous cutis and scleroderma sclerosing lesions, and for liver lesions, the effect can be evaluated by the improvement of total bilirubin, ALP, AST, and ALT. For kidney lesions, the effect can be evaluated by, for example, the amount of urinary protein and the urinary albumin / creatinine ratio. In this case, the therapeutic and / or preventive effect can be evaluated in the same manner as in the case of SLE.

[0109] Here, the "patient" includes humans or non-human mammals (e.g., one or more of mice, guinea pigs, hamsters, rats, mice, rabbits, pigs, sheep, goats, cows, horses, cats, dogs, marmosets, monkeys, or chimpanzees). The patient may also be a patient diagnosed with RA or arthritis, systemic lupus erythematosus, lupus nephritis, or graft-versus-host disease. The patient may also be a patient diagnosed with a disease treatable by suppression of citrullination in cells. The patient may also be a patient diagnosed with a disease treatable by suppression of nephrosis in cells.

[0110] As used herein, "treatment" includes any treatment of a disease in a mammal, particularly a human, and includes inhibiting a symptom of the disease, i.e., arresting its progression or eliminating the disease or condition, and alleviating a symptom of the disease, i.e., causing regression of the disease or condition, or slowing the progression of the condition.

[0111] Moreover, the term "prevention" includes preventing the onset of the above-mentioned diseases in mammals, particularly humans.

[0112] Furthermore, "prevention of recurrence" includes preventing the recurrence of the above-mentioned diseases which repeat remission and relapse in mammals, particularly humans.

[0113] The administration form of the anti-PAD4 antibody or its antigen-binding fragment of the present invention, or a pharmaceutical composition containing the anti-PAD4 antibody or its antibody fragment is not particularly limited, and can be administered to mammals, including humans, via any of the following administration routes: oral administration or parenteral administration (e.g., intravenous injection, intramuscular injection, subcutaneous administration, rectal administration, transdermal administration, intracerebral administration, intraspinal administration, or other local administration).

[0114] Dosage forms for oral and parenteral administration and methods for preparing them are well known to those skilled in the art, and pharmaceutical compositions can be produced by combining the antibody of the present invention with a pharma- ceutically acceptable carrier, etc. Dosage forms for parenteral administration include injection preparations (e.g., drip injections, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, intracerebral administration preparations, intraspinal administration preparations), topical preparations (e.g., ointments, poultices, lotions), suppositories, inhalants, eye preparations, eye ointments, nasal drops, ear drops, liposomes, etc. In particular, when it is desired to directly act on the central nervous tissue, it can be continuously injected using a medical micropump of an osmotic pump, or it can be mixed with fibrin glue or the like to form a sustained release preparation and then placed in the affected tissue.

[0115] For example, an injectable formulation is usually prepared by dissolving an antibody in distilled water for injection, and if necessary, a solubilizing agent, a buffering agent, a pH adjusting agent, an isotonicity agent, a pain-reducing agent, a preservative, a stabilizer, etc. may be added. Also, a lyophilized formulation for preparation immediately before use may be prepared.

[0116] Dosage forms for oral administration include solid or liquid dosage forms, specifically, tablets, coated tablets, pills, fine granules, granules, powders, capsules, syrups, emulsions, suspensions, injections, troches, and the like.

[0117] The pharmaceutical composition of the present invention may further contain other therapeutically effective drugs, and may also contain ingredients such as bactericides, anti-inflammatory agents, vitamins, amino acids, etc., as necessary.

[0118] Examples of pharmacologically acceptable carriers include excipients, lubricants, binders and disintegrants in solid preparations, and solvents, solubilizing agents, suspending agents, isotonicity agents, buffers and soothing agents in liquid preparations. Furthermore, conventional additives such as preservatives, antioxidants, colorants, sweeteners, adsorbents and wetting agents can be used appropriately and in appropriate amounts, if necessary.

[0119] The dosage of the antibody according to the present invention is determined by a physician based on various factors such as the route of administration, type of disease, severity of symptoms, age, sex, and body weight of the patient, severity of the disease, pharmacological knowledge such as pharmacokinetic and toxicological characteristics, whether or not a drug delivery system is used, and whether the antibody is administered as part of a combination of other drugs. Generally, for an adult (body weight 60 kg), the dosage is 1 to 5,000 μg / day, preferably 10 to 2,000 μg / day, and more preferably 50 to 2,000 μg / day for oral administration, and 1 to 5,000 μg / day, preferably 5 to 2,000 μg / day, and more preferably 50 to 2,000 μg / day for injection, which can be administered once or in divided doses. In the case of systemic parenteral administration, 10 to 100,000 μg / kg of body weight, more preferably 100 to 50,000 μg / kg, and even more preferably 500 to 20,000 μg / kg can be administered once a day, once a week, once a month, or once to seven times a year. In the case of local administration using an osmotic pump or the like, the drug can be continuously infused at a rate of 10 to 100,000 μg / day, more preferably 100 to 10,000 μg / day, and even more preferably 500 to 5,000 μg / day per adult (body weight 60 kg). EXAMPLES

[0120] The present invention will be described in more detail below with reference to examples, but the scope of the present invention is not limited thereto.

[0121] <Example 1> Confirmation of chemical stability of anti-PAD4 antibody The chemical stability of the variable region containing the CDR of the humanized anti-PAD4 antibody G8 described in WO 2016 / 143753 was evaluated. A mammalian cell expression vector expressing IgG was constructed by inserting DNA encoding the amino acid sequences of the light and heavy chains of the test antibody G8 downstream of the CMV promoter. The DNA sequences of the light and heavy chains were SEQ ID NOs: 27 and 26 in the sequence table, respectively. The expression vector was introduced into ExpiCHO (LifeTechnologies) using the gene introduction reagent ExpiFectamine CHO Transfection Kit (LifeTechnologies). After culturing for 14 days after gene introduction, the culture supernatant was obtained. IgG was purified from the culture supernatant by affinity chromatography using Protein A resin (GE Healthcare, MabSelect SuRe) using an open column. The IgG bound to the Protein A resin was washed with 6 column volumes of PBS (pH 7.2). The antibody was then eluted with Arg-Antibody Elution Buffer (Nacalai) at pH 4.0, and quickly neutralized to bring the pH to near neutral. In order to increase the purity of the purified antibody, AKTA prime plus (GE Healthcare) was used, and the IgG purified using the Protein A column was purified with CHT (ceramic hydroxyapatite Type II resin) (Bio-rad). The IgG bound to CHT was eluted with a gradient of NaCI concentration, and the desired fraction was collected, and then the solution was replaced with PBS (pH 7.4) by gel filtration chromatography using PD-10 (GE Healthcare). The test antibody was concentrated to a concentration of approximately 10 mg / mL and stored at 4°C or 37°C for 2 weeks.

[0122] The chemical stability of the variable region including the CDR of the test antibody G8 was evaluated by peptide mapping analysis using liquid chromatography / mass spectrometry (LC-MS). The test antibody was reduced by reacting it with the reducing agent dithiothreitol (Wako Pure Chemicals) in the presence of guanidine hydrochloride (Wako Pure Chemicals) at 37°C for 2 hours. The alkylating agent iodoacetamide (Wako Pure Chemicals) was added and reacted for 30 minutes to alkylate the thiol groups generated by the reduction reaction. Next, the reaction solution was buffer exchanged using a desalting column Zeba Spin column (Thermo Fisher Scientific) and reacted overnight at 37°C under the conditions of 2 mol / L urea (SIGMA), 0.1 mol / L NH4HCO3 (Wako Pure Chemicals), 1 mmol / L CaCl2 (Wako Pure Chemicals), and 0.8 μg trypsin (Wako Pure Chemicals). The obtained enzyme digest was used as the LC-MS measurement sample. The peptides obtained by trypsin digestion were separated by reversed-phase chromatography, and peptide mapping analysis was performed by acquiring MS and MS / MS spectra using a mass spectrometer.

[0123] LC was performed with the following parameters: Instrument: ACQUITY UPLC (Waters) Mobile phase: A = ultrapure water containing 0.1% formic acid, B = acetonitrile containing 0.1% formic acid Separation method: Linear gradient separation (%B = 1 to 40 in 60 minutes) Flow rate: 0.2 mL / min Column: ACQUITY UPLC Peptide BEH C18 column, 300 angstroms, 1.7 μm, 2.1 x 150 mm (Waters) Column oven temperature: 40°C Sample volume: 30 μL Detection wavelength: 215 nm

[0124] MS and MS / MS were performed with the following parameters: Equipment: Synapt (Waters) TOF mode: V mode Ionization method: ESI Positive MS measurement range: 50-2000 Da MS cone voltage: 24 V Scan time: 0.4 s MS / MS method: MSE Trap CE voltage :Low energy 6V,High energy ramp 20-40V

[0125] The obtained mass data was analyzed using Biopharma Lynx Ver. 1.3.3 (Waters).

[0126] The percentage of post-translational modification was calculated using the following method. First, peptides that matched the sequence of the test antibody were extracted using Biopharma Lynx, and peptides for which MS / MS could be obtained (MS / MS b / y ion found ≧ 2) were analyzed. The percentage of post-translational modification was calculated from the ratio of the MS intensity of all detected peptides to the MS intensity of peptides containing post-translational modification.

[0127] Peptide mapping of the test antibody G8 samples stored at 4°C after purification and at 37°C for 2 weeks identified a peptide cleaved between the 84th asparagine residue and the 85th serine residue in the framework region of the heavy chain. In the sample stored at 4°C after purification, the proportion of cleaved peptides was as high as 4.6% of peptides containing the corresponding amino acid, indicating that the corresponding site may potentially be cleaved. In addition, no cleavage was identified between other asparagine residues and serine residues in the test antibody, and the cleavage was specific to the 84th asparagine residue and the 85th serine residue.

[0128] Since cleavage occurs between the asparagine and serine residues, a humanized anti-PAD4 antibody G8ss was created in which the 84th asparagine residue was replaced with a serine residue. After purification of the test antibody G8ss, peptide mapping was performed on a sample stored at 4°C and a sample stored at 40°C for two weeks. As a result, no peptide cleaved between the 84th and 85th serine residues was detected under either storage condition. From the above, it is believed that humanized anti-PAD4 antibodies with a serine residue at the 84th position have higher chemical stability. The binding affinity of the test antibodies G8 and G8ss to human PAD4 protein was determined by measuring the dissociation constant (KD) in HBS-EP+ using a surface plasmon resonance device Biacore T200 (GE Healthcare). Biotin-labeled human PAD4 protein was immobilized on a sensor chip using a Biotin Capture Kit (GE Healthcare). The PAD4 protein was prepared to 0.25 μg / mL using HBS-EP+ to serve as a ligand solution. The ligand solution was added to the flow cell at a flow rate of 10 μL / min for 30 seconds, and HBS-EP+ was added for the next 60 seconds. The flow cell to which the ligand solution was not added was used as a reference cell. The test antibody was prepared to several hundred pM to several nM using HBS-EP+ to serve as an analyte solution. The running buffer was used as a blank solution. The blank solution or analyte solution was added for 180 seconds at a flow rate of 30 μL / min using the single-cycle method, and the dissociation time was set to 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. The binding parameters were calculated using a 1:1 binding model with Biacore T200 Evaluation software (GE Healthcare). The test antibody G8 binds to human PAD4 protein with a binding rate constant k on =7.70×10 6 (1 / Ms), dissociation rate constant k off =1.65×10 -3 The antibody G8ss bound to the human PAD4 protein with a binding affinity of 1 / s and a dissociation constant KD of 214 pM. on =6.37×10 6 (1 / Ms), dissociation rate constant k off =1.76×10 -3 (1 / s) and a dissociation constant KD of 276 pM. These results demonstrate that the binding affinity of the humanized anti-PAD4 antibody to human PAD4 remains unchanged even when the 84th residue is replaced with a serine residue.

[0129] <Example 2> Determination of amino acid substitution sites for improving complementarity determining regions To improve the affinity and functionality of the humanized anti-human PAD4 antibody G8 to human PAD4, we modified the complementarity-determining region (CDR) of the antibody by Fab (meaning that the molecular form is Fab, hereafter referred to as such) phage display. The CDR was modified in two stages. In the first stage, single amino acid substitutions that could improve the affinity to human PAD4 were selected, and in the second stage, multiple combinations of these single amino acid substitutions were selected (Fujino et al., Biochem. Biophys. Res. Commun., 2012 Vol. 428(3), p395). A Fab phage display vector was constructed using the light and heavy chain variable regions of the parent clone G8. Using this as a template, a comprehensive library of single amino acid substitution mutants was constructed by substituting all amino acid residues constituting the six CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, HCDR3) of the antibody one by one with a total of 20 natural amino acids. Using the Fab phage display method (Fujino et al., Biochem. Biophys. Res. Commun., 2012 Vol. 428(3), p395), we performed several rounds of enrichment of a comprehensive library of single amino acid substitution mutants using recombinant human PAD4 protein (biotin-labeled PAD4) as bait. The base sequences of the light and heavy chain variable regions of each clone contained in the library before enrichment (immediately after construction) and after enrichment were analyzed using a next-generation sequencer (Ion GeneStudio S5 System). First, we obtained sequence data of several million reads from each library before and after enrichment, and calculated the frequency of all single amino acid substitution mutants in the complementarity determining region. Next, we calculated the fold change (enrichment ratio) in the frequency of all single amino acid substitution mutants in the library before and after enrichment, and determined single amino acid substitutions that are thought to be useful for improving affinity to human PAD4 protein using the magnitude of the enrichment ratio due to library enrichment as an index. At this time, data on single amino acid substitutions (lysine and arginine) that may affect the physical properties of the antibody were excluded.Finally, taking into consideration the total number of these single amino acid substitutions and their distribution on the amino acid sequence, the positions at which amino acid substitutions should be introduced in the custom library constructed in the second stage were determined.

[0130] In the parent clone G8, it was decided to introduce amino acid substitutions at the second asparagine of LCDR2 (Sequence Listing, SEQ ID NO: 5), the fourth aspartic acid of LCDR3 (Sequence Listing, SEQ ID NO: 25), the 11th tyrosine, the 12th glycine, the 13th alanine, the 14th alanine, the 15th valine, and the 17th glycine of HCDR2 (Sequence Listing, SEQ ID NO: 24), and the first alanine of HCDR3 (Sequence Listing, SEQ ID NO: 11).

[0131] <Example 3> Creation of humanized anti-human PAD4 antibody with improved complementarity determining region First, we designed a custom library for improving the CDRs by combining multiple useful amino acid substitutions mentioned above to improve affinity. Next, we constructed a parent clone G8 Fab phage display vector with stop codons inserted into LCDR3 and HCDR2, and performed site-directed mutagenesis by the Kunkel method (Fellouse et al., J. Mol. Biol. 2007 Vol. 373, p924) using the Fab phage display vector as a template to construct a custom library for improving the CDRs in which the CDRs were randomized based on the above design. Using human PAD4 protein (His tag PAD4, GST-biotin-PAD4) as bait, we repeated several rounds of library enrichment by Fab phage display. Since PAD4 may change its structure depending on the presence or absence of calcium ions, we performed selection in the presence and absence of calcium chloride to examine enrichment.

[0132] The recombinant protein used as the bait was prepared as follows. Human PAD4 (residues 1 to 663) was inserted into pGEX-6P-1 with a GST tag-Avi tag added to the N-terminus to construct an expression vector, and the recombinant protein was prepared. E. coli BL21(DE3)pLysS strain carrying the expression vector was pre-cultured in 5 mL of LB medium, and 1 mL of the pre-culture solution was inoculated into 50 mL of LB medium and cultured at 37°C until the logarithmic growth phase (OD600=1.0), and then expression culture was performed with 100 μM IPTG at 18°C / 200 rpm for approximately 18 hours. The collected cells were washed, then ultrasonically disrupted to lyse the cells, and the supernatant was collected. The supernatant was reacted with commercially available biotin ligase (Avidity, BirA) at 4°C for approximately 18 hours, and the supernatant was collected by centrifugation. The GST tag-Avi tag-human PAD4 contained in the supernatant was purified using GS4B resin (GE Healthcare), and the GST tag was cleaved using PreScission Protease (GE Healthcare) to recover the Avi tag-human PAD4.

[0133] Each clone contained in the Fab library enriched in the presence and absence of calcium chloride for 4 to 6 rounds was cloned, and the sequences of 384 clones randomly selected from each condition were identified using a sequencer. All Fabs of the 384 clones were converted into full-length antibodies (human IgG1 / human λ) and fragmented for expression, and the above DNA was introduced into Expi293 (LifeTechnologies) using the gene introduction reagent ExpiFectamine 293 Transfection Kits (LifeTechnologies). After culturing for 5 days after gene introduction, the culture supernatant was obtained. The test antibody (IgG) (meaning that the molecular form is IgG, hereinafter the same) was purified from the culture supernatant by affinity chromatography using Protein A resin (GE Healthcare, PreDictor MabSelect SuRe LX). The test antibody solution after purification was replaced with PBS pH 7.4 using a dialysis device (Merck, D-Tube96 Dialyzer).

[0134] The antigen binding of the test antibody was confirmed by surface plasmon resonance (SPR). In the SPR experiment, a surface plasmon resonance device Biacore T200 (GE Healthcare) was used to confirm the antigen binding of the test antibody in running buffer HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% (v / v) Surfactant P20, pH 7.4). Biotin-labeled human PAD4 protein was immobilized on a sensor chip using a Biotin Capture Kit (GE Healthcare). The PAD4 protein was prepared to 4 μg / mL using HBS-EP+ to serve as a ligand solution. The ligand solution was added to the flow cell at a flow rate of 10 μL / min for 30 seconds, and HBS-EP+ was added for the following 60 seconds. The flow cell to which the ligand solution was not added was used as a reference cell. The test antibody was prepared to 10 nM using HBS-EP+ to serve as an analyte solution. The running buffer was used as a blank solution. A blank solution or analyte solution was added at a flow rate of 30 μL / min for 120 seconds to obtain a sensorgram of the binding phase. Next, a running buffer solution was added for 180 seconds to obtain a sensorgram of the dissociation phase. The sensorgram of the reference cell was subtracted from the sensorgram of the flow cell to which the ligand was added when the blank solution or analyte solution was added, and the sensorgram of the blank solution was subtracted from the sensorgram of the analyte solution added, and the results were used for analysis. The binding signal and nonspecific adsorption were confirmed using Biacore T200 Evaluation software v3.1 (GE Healthcare).

[0135] Among the clones with improved complementarity determining regions, five clones (G8, Lib2(4R)-1-47, Lib1(6R)-1-39, Lib1(6R)-2-37, Lib2(5R)-2-25) were selected that had improved affinity for human PAD4 protein compared to G8 and had less non-specific adsorption to the sensor chip, as well as G8, to be advanced to higher level evaluation after Example 4. The CDR sequences of the five clones are shown in Table 1. The binding affinities of the five clones for human PAD4 are shown in Table 2. The human PAD4 enzyme inhibitory activity of the test antibodies (G8, Lib2(4R)-1-47, Lib1(6R)-1-39, Lib1(6R)-2-37, Lib2(5R)-2-25) was evaluated. Human PAD4 (final concentration 10 nM) was mixed with 50 mM HEPES buffer (pH 7.6) containing 1 mM DTT and 150 mM NaCl so that the final concentrations of the test antibodies were 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 (benzoyl arginine ethyl ester) was added while stirring, and CaCl2 was added and stirred well (final concentration of BAEE was 10 mM, final concentration of calcium ion was 10 mM). After incubating the solution at 37°C for 3 h, the citrulline residues in citrullinated BAEE were colorimetrically quantified using a mixture containing 2,3-butanedione monoxime and thiosemicarbazide. The test antibodies (G8, Lib2(4R)-1-47, Lib1(6R)-1-39, Lib1(6R)-2-37, Lib2(5R)-2-25) inhibited the enzymatic activity of human PAD4 with IC50 values ​​of 21.6, 9.6, 8.7, 13.4, and 8.3 nM, respectively.

[0136] [Table 1]

[0137] [Table 2]

[0138] For five clones, G8, Lib2(4R)-1-47, Lib1(6R)-1-39, Lib1(6R)-2-37, and Lib2(5R)-2-25), the 84th asparagine was replaced with serine to give G8ss, 2-1-47, 3-1-39, 3-2-37, and 4-2-25, respectively. DNAs encoding the amino acid sequences of the light and heavy chains of the test antibodies (G8ss, 2-1-47, 3-1-39, 3-2-37, and 4-2-25) were inserted downstream of the CMV promoter to construct an expression vector for mammalian cells expressing IgG. For the DNA sequences of the light chains of each clone, DNA sequences encoding SEQ ID NOs: 27, 17, 19, 21, and 23 in the Sequence Listing were used, respectively, and for the DNA sequences of the heavy chains, DNA sequences encoding SEQ ID NOs: 11 and 12 in the Sequence Listing were used, respectively. The expression vector was introduced into ExpiCHO (LifeTechnologies) using the gene introduction reagent ExpiFectamine CHO Transfection Kit (LifeTechnologies). After 14 days of culture following gene introduction, the culture supernatant was obtained. IgG was purified from the culture supernatant by affinity chromatography using Protein A resin (JSR Life Sciences, Amsphere A3 column) using AKTA pure 25M (GE Healthcare). The IgG bound to the Protein A resin was washed with 6 column volumes of 100 mM sodium carbonate buffer (pH 11.0), and then with 6 column volumes of PBS (pH 7.2) (3x). It was then eluted with Gly-HCl buffer at pH 3.5, and promptly neutralized to bring the pH to near neutral. In order to increase the purification purity, the IgG after Protein A column purification was purified with CHT (ceramic hydroxyapatite Type I resin) (Bio-Rad). The IgG bound to CHT was eluted with a gradient of NaCl concentration, and the target fraction was collected and then replaced with PBS (pH 7.4) by dialysis. Peptide mapping was performed on samples of the test antibodies (G8ss, 2-1-47, 3-1-39, 3-2-37, 4-2-25) that had been purified and stored at 4°C.As a result, no peptide cleaved between the serine residues 84 and 85 was detected.

[0139] <Example 4> Binding affinity to human PAD4 protein The binding affinity of the test antibodies (G8ss, 2-1-47, 3-1-39, 3-2-37, 4-2-25) to human PAD4 protein was determined by measuring the dissociation constant (KD) in HBS-EP+ using a surface plasmon resonance device Biacore T200 (GE Healthcare). Biotin-labeled human PAD4 protein was immobilized on a sensor chip using a Biotin Capture Kit (GE Healthcare). The PAD4 protein was prepared to 0.5 μg / mL using HBS-EP+ and used as a ligand solution. The ligand solution was added to the flow cell at a flow rate of 10 μL / min for 30 seconds, and HBS-EP+ was added for the following 60 seconds. The flow cell to which the ligand solution was not added was used as a reference cell. The test antibodies were prepared to several hundred pM to several nM using HBS-EP+ and used as an analyte solution. The running buffer was used as a blank solution. A blank solution or analyte solution was added for 180 seconds at a flow rate of 30 μL / min using the single-cycle method, and the dissociation time was 1200 seconds. The sensorgram of the reference cell was subtracted from the sensorgram of the flow cell to which the ligand was added when the blank solution or analyte solution was added, and the sensorgram of the blank solution was subtracted from the sensorgram of the analyte solution added, and the results were used for analysis. Binding parameters were calculated using a 1:1 binding model using Biacore T200 Evaluation software (GE Healthcare). The calculated binding parameters are shown in Table 3. As shown in Table 3, the test antibody G8ss binds to human PAD4 protein with a binding rate constant k on =6.15×10 6 (1 / Ms), dissociation rate constant k off =1.22×10 -3 The antibodies 2-1-47, 3-1-39, 3-2-37, and 4-2-25, which had modified complementarity-determining regions, bound to human PAD4 protein with a binding affinity of 31, 48, 73, and 20 pM, respectively.

[0140] [Table 3]

[0141] <Example 5> Evaluation of storage stability of antibody One of the indicators of the storage stability of an antibody is the retention of antigen-binding activity. It is known that storage stability varies greatly depending on the antibody. For example, in the teaching of DiCara et al., mAbs, 2018 Vol. 10(7), p. 1073, the antigen-binding ability of antibodies stored at 4°C or 40°C is compared, and the relative antigen-binding activity of the antibody stored at 40°C ranges from 30% or less to 100% of the relative antigen-binding activity of the antibody stored at 4°C. To evaluate the storage stability of the test antibodies (G8ss, 2-1-47, 3-1-39, 3-2-37, 4-2-25), the test antibodies were dissolved in citrate buffer (50 mM citric acid, 150 mM NaCl, pH 6.3) at a concentration of approximately 10 mg / mL and stored at 4°C or 40°C for 4 weeks. To examine the presence or absence of antigen binding ability after storage, antigen binding activity was measured using a surface plasmon resonance device Biacore T200 (GE Healthcare). The test antibodies stored frozen and those stored for 4 weeks at 4°C or 40°C were adjusted to 10 μg / mL using running buffer HBS-EP+. Using Series S Sensorchip Protein A (GE Healthcare), the test antibody solution was added to flow cell 2 at a flow rate of 10 μL / min for 60 seconds and immobilized on the sensor chip. Flow cell 1 was used as the reference cell. The amount of binding to flow cell 2 55 seconds after the end of the addition of the test antibody was subtracted from the amount of binding to flow cell 1 to obtain the antibody binding amount. Next, human PAD4 (Cayman Chemical Company, Cat. 10500) was adjusted to 50 nM using HBS-EP+ to use as the analyte solution. The analyte solution was added at a flow rate of 30 μL / min for 120 seconds to obtain a sensorgram of the binding phase. The amount of antigen binding was determined by subtracting the amount of binding in flow cell 2 5 seconds before the end of the addition of the analyte solution from the amount of binding in flow cell 1. The running buffer was then added for 180 seconds to obtain a sensorgram of the dissociation phase. Measurements were performed at 25°C. The amount of antibody binding and the amount of antigen binding were calculated using a data analysis program (GE Healthcare, Biacore T200 Evaluation Software v3.1), and the amount of antigen binding under each condition was divided by the amount of antibody binding to obtain the relative amount of antigen binding. The relative amount of antigen binding of the test antibody stored frozen and that stored at 4°C did not change. The relative amount of antigen binding of various test antibodies stored at 4°C for 4 weeks was compared with the relative amount of antigen binding of the test antibody stored at 40°C for 4 weeks to obtain the antigen binding activity. As shown in Table 4, the antigen binding activity of G8ss after storage at 40°C for 4 weeks was 82.2%. 2-1-47, 3-1-39, 3-2-37, and 4-2-25 retained 90% or more of their antigen binding activity after storage at 40°C for 4 weeks. For example, as taught in Pisupati et al., mAbs, 2017 Vol. 9(7), p1197, the antibody drug Remicade (registered trademark) retains 80% or more of its antigen binding activity even after storage at 40°C for 1 month. In addition, as for the stability of the biological activity of the antibody, it is desirable to retain 80% or more, preferably 90% or more of the biological activity of the antibody at the time of preparation of the antibody formulation (see, for example, International Publication WO2003 / 018056). From the above, good storage stability was observed for the test antibodies G8ss, 2-1-47, 3-1-39, 3-2-37, and 4-2-25.

[0142] [Table 4]

[0143] <Example 6> Evaluation of the enzyme inhibitory activity of human PAD4 in vitro The human PAD4 enzyme inhibitory activity of the test antibodies (G8ss, 3-2-37, 3-1-39, 4-2-25, 2-1-47) was evaluated. Human PAD4 (final concentration 10 nM) was mixed with 50 mM HEPES buffer (pH 7.4) containing 1 mM EDTA, 1 mM DTT, and 150 mM NaCl so that the final concentrations of the test antibodies were 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 (benzoyl arginine ethyl ester) was added while stirring, and CaCl2 was added and stirred well (final concentration of BAEE was 10 mM, final concentration of calcium ion was 10 mM). After incubating the solution at 37°C for 3 h, the citrulline residues in citrullinated BAEE were colorimetrically quantified using a mixture containing 2,3-butanedione monoxime and thiosemicarbazide. The test antibodies (G8ss, 3-2-37, 3-1-39, 4-2-25, and 2-1-47) inhibited the enzymatic activity of human PAD4 with IC50 values ​​of 20.3, 15.3, 8.8, 7.6, and 12.2 nM, respectively.

[0144] <Example 7> CAIA model The efficacy of the test antibodies G8ss, 2-1-47, 3-1-39, 3-2-37, and 4-2-25 was evaluated using anti-collagen antibody-induced arthritis (CAIA) model mice. CAIA model mice are model mice of rheumatoid arthritis (RA) and arthritis. The method for generating CAIA model mice followed the protocol for the mouse arthritis-inducing antibody cocktail (Chondrex Inc., Cat. 53040). The experimental conditions are outlined in Figure 1. On day 0, the anti-collagen antibody mixture (1.5 mg) was intravenously administered to the tail vein of 9-week-old female Balb / c mice (6 mice / group). On day 3, 25 μg LPS (a substance that induces inflammation) was administered intraperitoneally. On day 3, the test antibody or control antibody (human IgG1) was intravenously administered (15 mg / kg) 4 hours before LPS administration. On days 3-10, arthritis scores were assessed according to (i) to (iii) below. (i) The evaluation sites were the fingers, dorsals, and joints of the limbs. (ii) The arthritis score was assigned according to Table 5. (iii) The arthritis score was expressed as the average of the total scores of the fingers, dorsals, and joints of the limbs (maximum value: 16 / mouse). The arthritis score evaluation results are shown in Figure 1. As can be seen from these results, all test antibodies had a high therapeutic effect on RA.

[0145] [Table 5]

[0146] Example 8: CIA model The efficacy of the test antibody 3-2-37 was evaluated using collagen-induced arthritis (CIA) model mice. CIA model mice were generated by immunizing 5-7 week-old female DBA / 1 mice twice with bovine type II collagen and Freund's adjuvant on the first day and 21 days after the first immunization. 29 days after the first immunization, the test antibody was administered into the tail vein at a dose of 0.3 mg / kg, 1 mg / kg, or 3 mg / kg to the mice that had developed arthritis (9 mice per group). The same amount of 3-2-37 was administered 36 days and 43 days after the first immunization. The arthritis score was evaluated according to the following (i) to (iii). (i) The evaluation sites were the fingers, dorsals, and joints of the four limbs. (ii) The arthritis score was assigned according to Table 6. (iii) The arthritis score was expressed as the average of the total scores of the fingers, dorsals, and joints of the four limbs (maximum value was 16 / mouse). The evaluation results of the arthritis score are shown in Figure 2. As can be seen from these results, 3-2-37 at all doses established this time significantly reduced the mean arthritis score in the latter half of the study compared to the control antibody-administered group (Shirley-Williams multiple comparison test, *p<0.025, **p<0.005).

[0147] [Table 6]

[0148] Example 9: cGvHD model The efficacy of the test antibody 3-2-37 was evaluated using chronic graft-versus-host disease (cGvHD) model mice. The cGvHD model mice were generated by transferring splenocytes from 8-9 week-old female DBA / 2 mice into 8 week-old female B6D2F1 mice. These mice develop renal damage similar to lupus nephritis that occurs in SLE patients. The test antibody was administered intraperitoneally at a dose of 3 mg / kg or 30 mg / kg on the day before, 2 weeks, 4 weeks, and 6 weeks after splenocyte transfer (13 mice per group). Urine was collected once a week by pressing the lower abdomen of the mice from 2 weeks to 8 weeks after splenocyte transfer, and the protein and creatinine concentrations in the urine were measured. Urinary protein concentrations 8 weeks after splenocyte transfer were scored according to Table 7. The evaluation results of the urinary protein scores are shown in Figure 3. As can be seen from these results, a significant decrease in urinary protein score was observed in the test antibody 3-2-37 30 mg / kg administration group compared to the control antibody administration group (Shirley-Williams multiple comparison test, *p<0.025). In addition, non-symptomatic individuals were defined as those with a urinary protein / creatinine ratio of less than 3 (Pro / Cre<3), and the non-symptomatic rate in the test antibody 3-2-37 30 mg / kg administration group and the control antibody administration group was graphed over time in Figure 4. The median non-symptomatic period in the test antibody administration group was 7 weeks, while the median non-symptomatic period in the control antibody administration group was 4 weeks, indicating a significant extension of the non-symptomatic period in the test antibody administration group (LogRank test, p<0.05).

[0149] [Table 7]

[0150] <Description of sequence listing> SEQ ID NO: 1: Amino acid sequence of HCDR1 of anti-PAD4 antibody SEQ ID NO: 2: Amino acid sequence of HCDR2 of anti-PAD4 antibody (mixed sequence) SEQ ID NO: 3: Amino acid sequence of HCDR3 of anti-PAD4 antibody (mixed sequence) SEQ ID NO: 4: Amino acid sequence of LCDR1 of anti-PAD4 antibody SEQ ID NO: 5: Amino acid sequence of LCDR2 of anti-PAD4 antibody SEQ ID NO: 6: Amino acid sequence of LCDR3 of anti-PAD4 antibody (mixed sequence) SEQ ID NO: 7: Amino acid sequence of HCDR2 of anti-PAD4 antibody Lib2(4R)-1-47, 2-1-47 SEQ ID NO: 8: Amino acid sequence of HCDR2 of anti-PAD4 antibody Lib1(6R)-1-39, 3-1-39 SEQ ID NO: 9: Amino acid sequence of HCDR2 of anti-PAD4 antibody Lib1(6R)-2-37, 3-2-37 SEQ ID NO: 10: Amino acid sequence of HCDR2 of anti-PAD4 antibody Lib2(5R)-2-25, 4-2-25 SEQ ID NO: 11: Amino acid sequences of HCDR3 of anti-PAD4 antibodies G8, G8ss, Lib2(4R)-1-47, 2-1-47, Lib1(6R)-1-39, 3-1-39, Lib2(5R)-2-25, and 4-2-25 SEQ ID NO: 12: Amino acid sequence of HCDR3 of anti-PAD4 antibody Lib1(6R)-2-37, 3-2-37 SEQ ID NO: 13: Amino acid sequences of LCDR3 of anti-PAD4 antibodies Lib2(4R)-1-47, 2-1-47, Lib1(6R)-1-39, and 3-1-39 SEQ ID NO: 14: Amino acid sequence of LCDR3 of anti-PAD4 antibody Lib1(6R)-2-37, 3-2-37 SEQ ID NO: 15: Amino acid sequences of LCDR3 of anti-PAD4 antibodies Lib2(5R)-2-25 and 4-2-25 SEQ ID NO: 16: Full-length amino acid sequence of the heavy chain of anti-PAD4 antibody 2-1-47 SEQ ID NO: 17: Full-length amino acid sequence of the light chain of anti-PAD4 antibody Lib2(4R)-1-47, 2-1-47 SEQ ID NO: 18: Full-length amino acid sequence of the heavy chain of anti-PAD4 antibody 3-1-39 SEQ ID NO: 19: Full-length amino acid sequence of the light chain of anti-PAD4 antibody Lib1(6R)-1-39, 3-1-39 SEQ ID NO: 20: Amino acid sequence of the full-length heavy chain of anti-PAD4 antibody 3-2-37 SEQ ID NO: 21: Full-length amino acid sequence of the light chain of anti-PAD4 antibody Lib1(6R)-2-37, 3-2-37 SEQ ID NO: 22: Full-length amino acid sequence of the heavy chain of anti-PAD4 antibody 4-2-25 SEQ ID NO: 23: Full-length amino acid sequence of the light chain of anti-PAD4 antibody Lib2(5R)-2-25, 4-2-25 SEQ ID NO: 24: Amino acid sequence of HCDR2 of anti-PAD4 antibody G8, G8ss SEQ ID NO: 25: Amino acid sequence of LCDR3 of anti-PAD4 antibody G8, G8ss SEQ ID NO: 26: Full-length amino acid sequence of the heavy chain of anti-PAD4 antibody G8 SEQ ID NO: 27: Full-length amino acid sequence of the light chain of anti-PAD4 antibody G8, G8ss SEQ ID NO: 28: Amino acid sequence of the full-length heavy chain of anti-PAD4 antibody G8ss SEQ ID NO: 29: Amino acid sequence of PAD4 SEQ ID NO: 30: Nucleotide sequence encoding the full-length heavy chain of anti-PAD4 antibody G8 SEQ ID NO: 31: Nucleotide sequence encoding the full-length light chain of anti-PAD4 antibody G8 and G8ss SEQ ID NO: 32: Nucleotide sequence encoding the full-length heavy chain of anti-PAD4 antibody 2-1-47 SEQ ID NO: 33: Nucleotide sequence encoding the full-length light chain of anti-PAD4 antibody 2-1-47 SEQ ID NO: 34: Nucleotide sequence encoding the full-length heavy chain of anti-PAD4 antibody 3-1-39 SEQ ID NO: 35: Nucleotide sequence encoding the full-length light chain of anti-PAD4 antibody 3-1-39 SEQ ID NO: 36: Nucleotide sequence encoding the full-length heavy chain of anti-PAD4 antibody 3-2-37 SEQ ID NO: 37: Nucleotide sequence encoding the full-length light chain of anti-PAD4 antibody 3-2-37 SEQ ID NO: 38: Nucleotide sequence encoding the full-length heavy chain of anti-PAD4 antibody 4-2-25 SEQ ID NO: 39: Nucleotide sequence encoding the full-length light chain of anti-PAD4 antibody 4-2-25 SEQ ID NO: 40: Nucleotide sequence encoding the full-length heavy chain of anti-PAD4 antibody G8ss [Industrial Applicability]

[0151] INDUSTRIAL APPLICABILITY The present invention is useful for the prevention or treatment of RA or arthritis, systemic lupus erythematosus, lupus nephritis, or graft-versus-host disease, and has great utility in the pharmaceutical industry.

Claims

1. a-3) an anti-PAD4 antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 16 and the light chain variable region comprises the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO: 17; b-3) an anti-PAD4 antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 18 and the light chain variable region comprises the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO: 19; and d-3) an anti-PAD4 antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 22 and the light chain variable region comprises the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO: 23; An anti-PAD4 antibody or an antigen-binding fragment thereof selected from the group consisting of:

2. a-4) an anti-PAD4 antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region consists of the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 16 and the light chain variable region consists of the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO: 17; b-4) an anti-PAD4 antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region consists of the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 18 and the light chain variable region consists of the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO: 19; and d-4) an anti-PAD4 antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region consists of the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 22 and the light chain variable region consists of the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO: 23; The anti-PAD4 antibody or its antigen-binding fragment according to claim 1, selected from the group consisting of:

3. c-3) An anti-PAD4 antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 20 and the light chain variable region comprises the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO:

21.

4. c-4) The heavy chain variable region comprises the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO:

20. The anti-PAD4 antibody or antigen-binding fragment thereof according to claim 3, wherein the light chain variable region consists of the amino acid sequence of amino acid numbers 1 to 105 of SEQ ID NO:

21.

5. The anti-PAD4 antibody according to any one of claims 1 to 4, which has a KD value for PAD4 of 100 pM or less. or an antigen-binding fragment thereof.

6. The anti-PAD4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody is a neutralizing antibody.

7. An anti-PAD4 antibody or its antigen-binding fragment described in any one of claims 1 to 6, wherein the antibody is a binding antibody of PAD4 represented by sequence number 29.

8. A nucleic acid molecule comprising a base sequence encoding the anti-PAD4 antibody or its antigen-binding fragment described in any one of claims 1 to 7.

9. A recombinant vector comprising the nucleic acid molecule of claim 8.

10. A transformant comprising the recombinant vector according to claim 9.

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