Novel Anti-pad4 antibody

Abstract

To provide an anti-PAD4 antibody with excellent properties, or to provide an excellent preventive or therapeutic method for RA or arthritis, systemic lupus erythematosus, lupus nephritis, graft-versus-host disease, and the like.SOLUTION: Provided is 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.SELECTED DRAWING: Figure 2

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 attempted to suppress RA by administering a mixture of four anti-PAD4 antibodies to mice. (See Example 2 of Patent Document 1). Furthermore, Patent Document 2 also describes the affinity to PAD4. and anti-PAD4 antibodies with superior citrullination activity inhibitory ability have been described.

[0005] Regarding PAD4 and systemic lupus erythematosus (SLE), the PAD4 knockout mouse In an SLE model (imiquimod-induced model) using There are reports that administration of small molecule PAD inhibitors (Cl-Amidine and BB-Cl-Amidine) suppressed nephritis in SLE model (MRL / lpr model) mice (Non-Patent Documents 3, 4, 5). There are also 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 model 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, the present inventors discovered that an anti-PAD4 antibody having a specific framework region (FR) sequence has excellent chemical stability, thereby completing 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) HCDR1 has the amino acid sequence of SEQ ID NO: 1 and HCDR2 has the amino acid sequence of SEQ ID NO: 7 an anti-PAD4 antibody or an antibody fragment thereof, comprising an HCDR3 having the amino acid sequence of SEQ ID NO: 11, an LCDR1 having the amino acid sequence of SEQ ID NO: 4, an LCDR2 having the amino acid sequence of SEQ ID NO: 5, and an LCDR3 having 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 antibody fragment thereof was stored at 40° C. for one month, the anti-PAD4 antibody before storage or an antibody fragment thereof having a PAD4 binding amount of 90% or more compared to the PAD4 binding amount of the antibody fragment. 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 is an antibody in which both the light chain and the heavy chain are derived from human immunoglobulin. Depending on the difference in the heavy chain constant region, they include IgG (including IgG1, IgG2, IgG3, and IgG4) with γ heavy chains, IgM with μ heavy chains, IgA (including IgA1 and IgA2) with α heavy chains, IgD with δ heavy chains, and IgE with ε heavy chains. In principle, the light chains include either κ or λ chains.

[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, unless otherwise specified, the term "antibody fragment" refers to an antigen-binding fragment. An antibody fragment may also be called an antigen-binding molecule. An antigen-binding fragment is a protein that contains a portion of an antibody and is capable of binding to an antigen. Examples of antigen-binding fragments include F(ab') 2, Fab', Fab, Fv (variable fragment of antibody), disulfide-linked Fv, single-chain antibody (sc Furthermore, antigen-binding fragments include 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, etc. The present invention also includes conjugated antigen-binding fragments to which functional molecules other than the anti-PAD4 antibody of the present invention, such as the antibody, are chemically or genetically linked.

[0027] [Complementarity determining region] Complementarity determining region (CDR) refers to the region that forms the antigen binding site in the variable region of an immunoglobulin molecule, also called hypervariable region, and refers to the part where the amino acid sequence varies greatly from one immunoglobulin molecule to another. There are three CDRs (LCDR1, LCDR2, LCDR3, and HCDR1, HCDR2, HCDR3) in the light chain and the heavy chain. In the present invention, the CDRs of an immunoglobulin molecule are numbered according to the Kabat numbering system (Ka bat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health an d 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 the particular reference polypeptide sequence after aligning the sequences, introducing gaps, if necessary, to achieve the maximum percent identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent identity can be performed using a variety of methods within the skill of the art, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. This can be achieved by using publicly available computer software such as BLAST 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. For purposes herein, however, 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 the anti-PAD4 antibody or antigen-binding fragment thereof has a higher affinity for the antibody than the anti-PAD4 antibody or antigen-binding fragment thereof when measured by the surface plasmon resonance (SPR) method described herein. This means that the presence of such a substance 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 at NCBI (National Center for Biotechnology Information). ), or the website of 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. The organism from which PAD4 is derived is not limited, so long as it has PAD4 activity.

[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. Because it uses antibodies, it has minimal side effects and is therefore excellent from a safety standpoint. 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. Storage stability can be evaluated based on a 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. When the anti-PAD4 antibody according to one embodiment of the present invention is stored, for example, at 40° C. for one month, it has a PAD4 binding amount that is higher than that of the anti-PAD4 antibody or its antibody fragment before storage. In comparison, the PAD4 binding amount may be 90%, 93%, 94%, 95%, 96%, 97% or more.

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

[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] Examples of animals to be immunized include mammals such as mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, pigs, goats, horses, and cows, as well as chickens. Inoculation methods include subcutaneous, intramuscular, or intraperitoneal administration. The antibody 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 an immunized animal are fused with myeloma cells and isolated as hybridomas. Myeloma cells can be used in the manufacture of animal models. For example, those derived from mice, rats, humans, chickens, etc. are used.

[0039] <Monoclonal antibodies> Specifically, monoclonal antibodies can be obtained as follows: The above-mentioned antigen is used as an immunogen, and the immunogen is injected subcutaneously, intramuscularly, intravenously, or intrafootpad into the above-mentioned animals together with Freund's adjuvant, if necessary. Alternatively, immunization is performed by intraperitoneal injection or transplantation once or several times. 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, hybridomas are grown, maintained and preserved according to various conditions such as the characteristics of the cell type to be cultured and the culture method, and monoclonal antibodies are produced in the culture supernatant. Any suitable nutrient medium can be used for production.

[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 antibodies. For example, Almagro et al., FRont Biosci. 2008 Jan 1;13:1619-1633.), specifically, for example 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.) can be used. The amino acid residues in the FR region may be replaced with the corresponding residues from the CDR donor antibody. This can be done by methods well known in the art (Riechmann et al., Nature. 1988 Mar. 24;332(6162):323-327.), for example, by modeling the interactions of CDR and FR residues. Alternatively, sequence comparison may be used to identify unusual FR residues at specific positions. It may also be humanized by the methods described in US Pat. No. 6,334-42.

[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 are 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; JP-T-7-509137A; WO 94 / 25585; Nature, Vol. 368, p.856-859, 1994; and JP-A-6-500233. More specifically, HuMab (registered trademark) mouse (Medarex, Princeton NJ) , KMTM mice (Kirin Pharma Company, Japan), KM (FCγRIIb-KO) mice etc.

[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) 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. a column of an anti-PAD4 antibody; 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, and the antibody described in e) above is inhibited from being cleaved 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) above.

[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 a 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. stomach. 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 properties 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 or more of these CDRs may be selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 29, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, In the above, one to several amino acids may be substituted. Here, one to several means, for example, one, two, or three. It is preferable that the amino acid substitution is a conservative substitution in order to maintain the characteristics 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 substituted 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, and cysteine. Examples of positively charged (basic) amino acids include arginine, histidine, and lysine. Examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid. The antibody characteristics are maintained when these characteristics are maintained by modifying the amino acid sequence of the CDR. It means that the condition is maintained to the same extent as before the change, for example, 80% or more, preferably 90% or more, and more preferably 95% or more. Note that "maintained" also includes "improvement."

[0064] There are no particular restrictions on the sequence of the regions other than the CDRs, as long as they maintain the structure of the antibody and can exert their functions. The constant region may be any of a mouse-derived sequence, a human-derived sequence, a sequence derived from another mammal, a chicken-derived sequence, a chimeric sequence thereof, and an artificial sequence. In the case where the constant region is 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 a preferred embodiment of the present invention in which a region other than CDR is modified, An example of such an antibody is an antibody in which the amino acid (usually asparagine) corresponding to the 84th amino acid of the heavy chain (for example, SEQ ID NO: 26) is serine. 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 according to 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. An anti-PAD4 antibody according to one embodiment of the present invention that is excellent in the inhibition of cleavage occurring during production can be, for example, 3%, 2%, or 1% or less of cleaved antibody when stored at 4°C for 2 weeks compared to the antibody before the storage.

[0066] Further, humanized antibodies in which the CDRs are not derived from humans are also exemplified. So, 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 used to prepare multispecific antibodies such as bispecific antibodies by linking to an antibody having an antigen-binding specificity other than PAD4 by genetic engineering techniques. Such genetic engineering techniques have already been established in this field. For example, DVD-Ig (Wu et al., Nature Biotechnology 25(11), 1290(2007)), in which the variable regions are linked in series, and antibodies that bind to different antigens by modifying the Fc region of the antibody can be used. The ART-Ig technology (Kitazawa et al., Nature Medicine 18(10), 1570(2012)), which combines the heavy chains of two different antibodies, can be used to obtain the desired bispecific antibodies. .

[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 binding to FcRn, one of the Fc receptors (Shuhei Hashiguchi et al., Seikagaku, 2010, Vol. 82(8), p. 710). The functionally modified antibodies can be produced by genetic engineering techniques.

[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 are directly bound to antibodies using the chloramine T method. It can be combined.

[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, saccharomyces cerevisiae, and the like can be used. Examples of oxidases that can be used include kalide oxidase (eg, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (eg, uricase and xanthine oxidase), lactoperoxidase, and microperoxidase.

[0079] Linkers used to chemically link toxins, small molecules, or enzymes include divalent radicals (e.g., alkylene, arylene, heteroarylene), -(CR 2 )nO( CR 2 )n-(R is an arbitrary substituent, n is a positive integer) These include repeat units (e.g., polyethyleneoxy, PEG, polymethyleneoxy, etc.) and alkylamino (e.g., polyethyleneamino, Jeffamine™), as well as diacid esters and amides (succinate, succinamide, diglycolate, malonate, caproamide, etc.). Chemical modification methods for binding functional molecules have already been established in the 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] In the present invention, the "antigen-binding fragment" of an antibody refers to a partial region of the antibody having antigen-binding ability, specifically, F(ab') 2 , Fab', Fab, Fv (variable fragment of antibody), disulfide-linked Fv, single-chain antibody (scFv), and polymers thereof. and the like. Furthermore, the antigen-binding fragment includes 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 (TGF-β, NGF, Neurotrophin, etc.), albumin, an enzyme, or another antibody. Conjugated antigen-binding fragments include those to which functional molecules other than the above are chemically or genetically linked.

[0081] Here, "F(ab') 2 " and "Fab" refer to an antibody fragment produced by treating immunoglobulin with a proteolytic enzyme such as pepsin or papain, and digesting it before and after the disulfide bond present 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 present between the two heavy chains in the hinge region, producing 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 the IgG is treated with pepsin, the disulfide bond between the two heavy chains in the hinge region is cleaved downstream to produce an antibody that is slightly larger than the one in which the two Fabs are connected at the hinge region. The antibody fragment can be produced as F(ab') 2 He said.

[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 L u or 211 Radioactive substances are used for the detection of anti-PAD4 antibodies by the Chloramine T method, etc. It can be directly linked to a region of the antigen-binding portion.

[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 link toxins, small molecules, or enzymes include divalent radicals (e.g., alkylene, arylene, heteroarylene), -(CR 2 )nO( CR 2 )n-(R is an arbitrary substituent, n is a positive integer) These include repeat units (e.g., polyethyleneoxy, PEG, polymethyleneoxy, etc.) and alkylamino (e.g., polyethyleneamino, Jeffamine™), as well as diacid esters and amides (succinate, succinamide, diglycolate, malonate, caproamide, etc.). Chemical modification methods for binding functional molecules have already been established in the 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 antigen-binding fragment of the present invention have 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 competes in binding to PAD4 with an antibody having the amino acid sequence of the specific CDR as described above, so long as the antibody maintains the property of neutralizing the citrullinating activity of PAD4. Anti-PAD4 antibodies and antigen-binding fragments thereof are also included. Antibodies that compete with the antibody having the formula (I) for binding to PAD4 include 345, 347, and 348 of PAD4. Examples of such antibodies include antibodies having an epitope in a region including the site, but do not include G8 antibody. The antibody is allowed to coexist in a binding system between an antibody having the above-mentioned CDR sequence and PAD4. 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 has a binding ability to PAD4 and neutralizes the citrullinating activity of PAD4. As long as the polynucleotide encodes a monoclonal antibody having the same sequence as the heavy chain variable region or the light chain variable region, the polynucleotide may hybridize under stringent conditions with a complementary DNA sequence of the heavy chain variable region or the light chain variable region. Here, examples of stringent conditions include conditions in which, after Southern hybridization, washing is performed 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 constant and variable regions of the heavy and light chains, or may encode only the variable regions of the heavy and light chains. When encoding all of the constant and variable regions, the base sequences of the heavy and light chain constant regions are as described in Nucleic Acids Research vol.14, p1779, 1986, The Journal of Biological Chemistry vol.257, p1516, 1982 and Cell vol. 22, p197, 1980 are preferred.

[0096] The full length heavy and light chains of the humanized anti-PAD4 antibody G8 described in WO 2016 / 143753 The encoding nucleotide sequences 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 or an antibody fragment thereof of the present invention.

[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. As a recombinant vector, expression in prokaryotic cells such as E. coli is possible. The vector may be a vector capable of expression in eukaryotic cells (e.g., pBR322, pUC119, or a derivative thereof), but a vector capable of expression in eukaryotic cells is preferred, and a vector capable of expression in cells derived from mammals is more preferred. Examples of vectors capable of expression in cells derived from mammals include plasmid vectors such as pcDNA3.1 (manufactured by Invitrogen), pConPlus, pcDM8, pcDNA I / Amp, pcDNA3.1, and pREP4, pDON-AI DNA (Takarabata), and the like. Examples of the vector include viral vectors such as those manufactured by Io Pharmaceuticals, Inc. The vector may be one 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. Examples of cells derived from a mammal include Chinese hamster ovary cells (CHO cells), COS, mycobacterium tuberculosis, and the like. Examples include Eloma, BHK, HeLa, Vero, 293, NS0, Namalwa, and YB2 / 0.

[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 that are commonly used for proteins. For example, antibodies 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, and the like (Antibodies: A Laboratory Manual. Examples of columns used in affinity chromatography include, but are not limited to, Protein A columns, Protein G columns, anti-immunoglobulin antibody binding columns, and antigen binding columns. For example, Protein A columns include Hyper D, POROS, and Sepharose FF. (Amersham Biosciences), etc.

[0101] <Composition> One embodiment of the present invention is a composition comprising the anti-PAD4 antibody or antibody fragment thereof according to the above-mentioned embodiment of the present invention. By using this composition, PAD4 can be efficiently detected. It can efficiently inhibit citrullination of proteins caused by erythropoietin. It can also efficiently inhibit nephosis 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., the inclusion of a carrier, etc.) related to the inhibitor and pharmaceutical composition described below may be applied to this composition.

[0102] One embodiment of the present invention is an inhibitor of protein citrullination by PAD4, comprising the anti-PAD4 antibody or antibody fragment thereof according to the above embodiment of the present invention. By using this inhibitor, protein citrullination by PAD4 can be efficiently inhibited. The reduction rate of citrullination activity by the inhibitor may be 20, 30, 40, 60, 80% or more, or may be within a range of any two of these values. This reduction rate is, for example, 0% lower than the reduction rate when PBS is used. %. 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 can be used in any of the above-mentioned ways. The composition according to the embodiment of the present invention may include the composition according to the embodiment of the present invention. One embodiment of the present invention is a method for suppressing protein citrullination by PAD4, comprising a step of contacting PAD4 with an anti-PAD4 antibody or an antibody fragment thereof according to the embodiment of the present invention. One embodiment of the present invention is a method for suppressing protein citrullination by PAD4, comprising a step of administering to a patient an anti-PAD4 antibody or an antibody fragment thereof according to the embodiment of the present invention. The suppression method includes a suppression method carried out for research or treatment. One embodiment of the present invention is the use of an anti-PAD4 antibody or an antibody fragment thereof according to the 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 nematosis, comprising the anti-PAD4 antibody or its antibody fragment according to the embodiment of the present invention. By using this inhibitor, cellular nematosis caused by PAD4 can be efficiently suppressed. The rate of reduction of cellular nematosis activity by the inhibitor may be 20, 30, 40, 60, 80% or more, or any two of them. The reduction rate may be within a range of one value. This reduction rate may be expressed as a relative percentage, for example, when the reduction rate when PBS is used is set to 0%. In one embodiment of the present invention, the "agent" includes, for example, a composition used in research or treatment. The above-mentioned inhibitor includes, for example, an agent for preventing or treating RA or arthritis, systemic lupus erythematosus, lupus nephritis, or graft-versus-host disease. The above-mentioned inhibitor can be used, for example, in vitro or in vivo. The above-mentioned inhibitor can be used, for example, in the above-mentioned The present invention may include a composition according to an embodiment of the present invention. One embodiment of the present invention is a method for suppressing nephosis in a cell, comprising a step of contacting an anti-PAD4 antibody or an antibody fragment thereof according to an embodiment of the present invention with PAD4. One embodiment of the present invention is a method for suppressing nephosis in a cell, comprising a step of administering an anti-PAD4 antibody or an antibody fragment thereof according to an embodiment of the present invention to a patient. The suppression method includes a suppression method performed for research or treatment. One embodiment of the present invention is the use of an anti-PAD4 antibody or an antibody fragment thereof according to an embodiment of the present invention for producing a nephosis inhibitor in a cell.

[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, or 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. It may include a composition according to an 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 term "agent" refers to a compound containing an active ingredient and one or more pharmacologically acceptable The pharmaceutical composition may be a pharmaceutical composition containing a carrier. In one embodiment of the present invention, the "pharmaceutical composition" may be prepared by, for example, mixing an active ingredient with the carrier and using any method known in the technical field of pharmaceutical formulations. 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 an active ingredient and any other ingredient.

[0106] The therapeutic or / and preventive effect of the pharmaceutical composition on 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 assessment 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 determined 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 determined 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, The range may be any two of these values. 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 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 the pharmaceutical composition 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 between 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, 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 urinary protein amount, for example, when the amount of albumin or albumin / creatinine ratio in the urine of the patient at the time of administration of the pharmaceutical composition is significantly reduced compared to when not administered, it is considered that the treatment is effective. Or, it may be determined that 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 greater than or equal to the amount of albumin or creatinine in the urine of the patient at the time of administration of the negative control substance. The urinary albumin level or albumin / creatinine ratio in patients with If the therapeutic and / or prophylactic effect is observed, it may be determined that the pharmaceutical composition has a therapeutic or / and prophylactic effect. The area of ​​the graph of the amount of urinary albumin or albumin / creatinine ratio of the patient at the time of administration is Significantly higher than the amount of urinary albumin or the albumin / creatinine ratio graph area at the time of administration. If the amount of albumin in the urine of the patient or the albumin / creatinine ratio at the time of administration of the pharmaceutical composition is decreased, it may be judged that the therapeutic and / or prophylactic effect has been achieved. The amount of albumin in the urine or the albumin / creatinine ratio of the patient when the control substance was administered When the amount of the antibody or antibody that is administered is significantly reduced, the antibody or antibody that is administered may be determined to have a therapeutic or / and preventive effect. The reduction may be, for example, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10% or less. It may be within the range of any two of those 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 and 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 the improvement of urinary protein amount, urinary alfa, and the like. The albumin / creatinine ratio may be used to evaluate the efficacy of treatment and / or prevention. can be done as for SLE above.

[0109] Here, the term "patient" includes humans and 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, chimpanzees, etc.). The patient may be a patient diagnosed with arthritis, systemic lupus erythematosus, lupus nephritis, or graft-versus-host disease. The patient may be a patient diagnosed with a disease treatable by inhibition of citrullination in cells. The patient may be a patient diagnosed with a disease treatable by inhibition 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 including 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 as shown in SEQ ID NOs: 27 and 26 in the sequence listing, respectively. The above expression vector was introduced into ExpiCHO (Life Technologies) using the gene introduction reagent ExpiFectamine CHO Transfection Kit (Life Technologies). After gene introduction, the cells were cultured for 14 days. The culture supernatant was obtained after the incubation. IgG was purified from the culture supernatant by affinity chromatography using an open column and Protein A resin (GE Healthcare, MabSelect SuRe). The IgG bound to the Protein A resin was washed with 6 column volumes of PBS (pH 7.2). It was then eluted with Arg-Antibody Elution Buffer (Nacalai) at pH 4.0 and promptly neutralized to a near-neutral pH. To increase the purity of the purified IgG, the IgG after Protein A column purification was eluted on a CHT (ceramic hydroxyapatite Type II resin) using AKTA prime plus (GE Healthcare). The IgG bound to CHT was eluted with a gradient of NaCl concentration. After collecting the target fraction, 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 The mixture was stored at 37°C or 40°C for 2 weeks.

[0122] The chemical stability of the variable region including the CDR of the test antibody G8 was evaluated by liquid chromatography / mass spectrometry. The antibody was evaluated by peptide mapping analysis using LC-MS. The mixture was reduced by reacting with the reducing agent dithiothreitol (Wako Pure Chemicals) in the presence of guanidine (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 then desalted with 2 mol / L urea (SIGMA), 0.1 mol / L NH 4 HCO 3 (Wako Pure Chemical Industries), 1 mmol / L CaCl 2 (Wako Pure Chemical Industries, Ltd.) and 0.8 μg trypsin (Wako Pure Chemical Industries, Ltd.) at 37° C. overnight. The resulting enzyme digest was used as the LC-MS measurement sample. The peptides obtained by trypsin digestion were separated by reverse 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 modifications 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 MS intensity and post-translational modifications of all detected peptides were The proportion of post-translational modifications was calculated from the proportion of MS intensities of peptides containing the above.

[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 samples, the proportion of cleaved peptides was as high as 4.6% of peptides containing the amino acid in question, indicating that the site in question may be potentially cleaved. Furthermore, no cleavage was identified between other asparagine and serine residues in the tested antibody, and the cleavage was specific to the region between asparagine residue 84 and serine residue 85.

[0128] Since the 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 the preserved samples and the samples stored at 40°C for 2 weeks. As a result, no peptide cleaved between serine residues 84 and 85 was detected under either storage condition. From the above, it is considered that humanized anti-PAD4 antibodies with a serine residue at position 84 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). The protein was immobilized on a sensor chip. The PAD4 protein was adjusted to 0.25 μg / mL using HBS-EP+ to prepare a ligand solution. The ligand solution was added to the flow cell at a flow rate of 10 μL / min for 30 seconds, and then HBS-EP+ was added for the next 60 seconds. Flow cell without addition of ligand solution The test antibody was adjusted to several hundred pM to several nM using HBS-EP+. The blank solution or analyte solution was added at a flow rate of 30 μL / min for 180 seconds using the single-cycle method. The time interval 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 a blank solution or analyte solution was added, and the sensorgram of the blank solution was subtracted from the sensorgram of the analyte solution. 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), dissociation constant KD=276 The humanized anti-PAD4 antibody bound to the 84th amino acid sequence with a binding affinity of 100 pM. It was found that the substitution of these residues did not affect the binding affinity to human PAD4.

[0129] <Example 2> Determination of amino acid substitution sites for improving complementarity determining regions In order to improve the affinity and functionality of the humanized anti-human PAD4 antibody G8 to human PAD4, (This means that the molecular structure is Fab, hereafter the same) The complementarity determining regions were improved. The improvement of the complementarity determining regions was carried out in two stages. In the first stage, single amino acid substitutions that could improve affinity to human PAD4 were determined, and in the second stage, multiple combinations of these single amino acid substitutions were determined (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 multi-stage PCR reaction using site-directed mutagenesis PCR and overlap extension PCR was carried out to identify the six complementarity determining regions (LCDR1, LCDR2, and LCDR3) of the antibody. We constructed a comprehensive library of single amino acid substitution mutants in which all amino acid residues constituting the PAD4 gene (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) were replaced one by one with all 20 natural amino acids. Recombinant human PAD4 protein (biotin-labeled PAD4) was displayed on phages by Fab phage display (Fujino et al., Biochem. Biophys. Res. Commun., 2012 Vol. 428(3), p395) to generate a phages-display library of single amino acid substitution mutants. As a result, the comprehensive single amino acid substitution mutant library was enriched for several rounds. The base sequences of the light and heavy chain variable regions of each clone contained in the library (immediately after construction) and the library after enrichment were analyzed using a next-generation sequencer (Ion GeneStudio S5 System). First, sequence data of several million reads was obtained from each library before and after enrichment, and the frequency of all single amino acid substitution mutants in the complementarity determining region was calculated. Next, the library before enrichment The fold change in the frequency of all single amino acid substitution variants in the library after enrichment and The enrichment ratio was calculated, and the magnitude of the enrichment ratio due to library enrichment was used as an index to determine a single amino acid substitution that is considered to be useful for improving affinity for human PAD4 protein. When using antibodies with single amino acid substitutions (lysine and arginine) that may affect the physical properties of the antibody, The data was excluded. Finally, the total number of these single amino acid substitutions and their distribution on the amino acid sequence were Considering this, the positions to introduce amino acid substitutions in the custom library to be constructed in the second stage are determined. did.

[0130] In the parent clone G8, the second asparagine of LCDR2 (SEQ ID NO: 5 in the Sequence Listing) and the second asparagine of LCDR3 (SEQ ID NO: 5 in the Sequence Listing) were It was decided to introduce amino acid substitutions into the 4th aspartic acid of HCDR1 (SEQ ID NO: 25 in the Sequence Listing), the 11th tyrosine, the 12th glycine, the 13th alanine, the 14th alanine, the 15th valine, and the 17th glycine of HCDR2 (SEQ ID NO: 24 in the Sequence Listing), and the 1st alanine of HCDR3 (SEQ ID NO: 11 in the Sequence Listing).

[0131] <Example 3> Creation of humanized anti-human PAD4 antibody with improved complementarity determining region First, we designed a custom library for full-scale improvement of the complementarity-determining regions by combining multiple useful amino acid substitutions mentioned above for the purpose of improving affinity. Next, we constructed a parent clone G8 Fab phage display vector with stop codons inserted into LCDR3 and HCDR2, and displayed the Fab phage. Site-directed mutagenesis using the Kunkel method with a display vector as a template (Fellouseet al., J. Mol. Biol. 2007 Vol. 373, p924) to obtain the above-mentioned complementarity determining regions. Based on the design of the above, a randomized custom library for improving the complementarity determining regions was constructed. Using human PAD4 protein (His tag PAD4, GST-biotin-PAD4) as bait, several rounds of library enrichment by Fab phage display were repeated. Since PAD4 may change its structure depending on the presence or absence of calcium ions, selection was performed 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 (residue 1 to The residues 663 to 665) were inserted into pGEX-6P-1 with a GST tag and Avi tag attached to the N-terminus. An expression vector was constructed and a recombinant protein was prepared. E. coli BL21(DE3)pLysS strain carrying the expression vector was precultured in 5 mL of LB medium, and 1 mL of the preculture was inoculated into 50 mL of LB medium and incubated at 37°C. After culturing to logarithmic growth phase (OD600 = 1.0), 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 ligated with GS4B resin (GE The GST protein was purified using PreScission Protease (GE Healthcare) and purified using GST protein synthesis. The tag was cleaved to recover Avi-tag-human PAD4.

[0133] Fab libraries enriched for 4 to 6 rounds in the presence and absence of calcium chloride Each included clone was cloned and the arrangement of 384 randomly selected clones from each condition was analyzed. The Fabs of the 384 clones were all full-length antibodies (human IgG1 / human IgG2). The DNA for expression was fragmented and transformed into Expi293 (LifeTechnologies) using the gene transfer reagent ExpiFectamine 293 Transfection Kits (LifeTechnologies). The above DNA was transferred into Expi293 (LifeTechnologies). After 5 days of culture following gene transfer, the culture supernatant was obtained. The test antibody (IgG) (meaning that the molecular form is IgG, hereafter the same) was purified from the culture supernatant by affinity chromatography using Protein A resin (GE Healthcare, PreDictor MabSelect SuRe LX). After purification, the test antibody solution was replaced with PBS pH 7.4 using a dialysis device (Merck, D-Tube96 Dialyzer).

[0134] Antigen binding of the test antibodies was confirmed by surface plasmon resonance (SPR). For the SPR experiment, a surface plasmon resonance device Biacore T200 (GE Healthcare) was used to confirm the antigen binding of the test antibodies in the running buffer HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% (v / v) Surfactant P20, pH 7.4). Biotin Capture Kit (GE Healthcare) was used to capture the antigen binding of the test antibodies. Human PAD4 protein labeled with thiocyanin was immobilized on a sensor chip. The PAD4 protein was adjusted to 4 μg / mL to prepare a ligand solution. The ligand solution was added to the flow cell at a flow rate of 10 μL / min for 30 seconds, and then HBS-EP+ was added for the next 60 seconds. Ligand solution The flow cell without addition of IgG was used as the reference cell. The blank solution or analyte solution was added at a flow rate of 30 μL / min for 120 seconds to measure the binding phase of the sensor. Next, the running buffer was added for 180 seconds to obtain the sensorgram of the dissociation phase. The results were used for analysis by subtracting the sensorgram of the reference cell from the sensorgram of the flow cell to which the ligand was added when a blank solution or analyte solution was added, and further subtracting the sensorgram of the blank solution from the sensorgram of the analyte solution. 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, clones with improved affinity for human PAD4 protein and less non-specific adsorption to the sensor chip compared to G8, and clones with improved affinity for human PAD4 protein compared to G8 compared to Example 4 and above, Five clones (G8, Lib2(4R)-1-47, Lib1(6R)-1-39, Lib1(6R)-2-37, and Lib2(5R)-2-25) were selected for further evaluation. The CDR sequences of the five clones are shown in Table 1. The binding affinities of the five clones to 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. The final concentrations of the test antibodies were 600, 200, 66.67, 22.22, 7.41, and 2. Human PAD4 (final concentration 10 nM) was mixed with 50 mM HEPES buffer (pH 7.6) containing 1 mM DTT and 150 mM NaCl to give concentrations of 47, 0.82, 0.27, and 0.09 nM. Incubation was performed at 37°C for 1 hour. Then, while stirring, add BAEE (benzoyl arginine ethyl ester) and then CaCl 2 and mixed thoroughly (final concentration of BAEE: 10 mM, final concentration of calcium ions: 10 mM). After incubating the solution at 37°C for 3 hours, the citrullinated BAEE was The amine residues were determined colorimetrically 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] 5 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. DNA 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, 4-2-25) was inserted downstream of the CMV promoter to construct an expression vector for mammalian cells expressing IgG. The DNA sequences encoding SEQ ID NOs: 27, 17, 19, 21, and 23 in the sequence listing were used for the light chain DNA sequences of each clone, and the heavy chain DNA sequences were The DNA sequences of the strands were those encoding SEQ ID NOs: 11 and 12 in the sequence listing. The above expression vector was introduced into ExpiCHO (LifeTechnologies) using a gene introduction reagent, ExpiFectamine CHO Transfection Kit (LifeTechnologies). After gene introduction, the cells were cultured for 14 days. After culturing, the culture supernatant was obtained. Protein A was detected using AKTA pure 25M (GE Healthcare). Affinity matrix using resin (JSR Life Sciences, Amsphere A3 column) IgG was purified from the culture supernatant by filtration. The IgG bound to the Protein A resin was washed with 6 column volumes of 100 mM sodium carbonate buffer (pH 11.0), then with 6 column volumes of PBS (pH 7.2) (3x). It was then eluted with Gly-HCl buffer (pH 3.5) and promptly neutralized to a near-neutral pH. In order to increase the purity of the purified IgG, 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. Purification of the test antibodies (G8ss, 2-1-47, 3-1-39, 3-2-37, 4-2-25) Peptide mapping was performed on the samples stored at 4°C after preparation. No peptide cleaved between the serine residue at position 1 and the serine residue at position 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 HBS-EP+. The PAD4 protein was adjusted to 0.5 μg / mL using 100 μL / min to prepare a ligand solution. The ligand solution was added to the flow cell at a flow rate of 10 μL / min for 30 seconds, and then HBS-EP+ was added for the next 60 seconds. The flow cell without the addition of the guard solution was used as the reference cell. The ligand was prepared at a concentration of several hundred pM to several nM and used as the analyte solution. The running buffer solution was used as the 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 the blank solution or analyte solution was added, and the blank solution was calculated from the sensorgram of the flow cell to which the analyte solution was added. The sensorgrams with the rank solution added were subtracted and used for analysis. Binding parameters were calculated using a 1:1 binding model with 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 study taught by DiCara et al., mAbs, 2018 Vol. 10(7), p. 1073, the antigen-binding ability of antibodies stored at 4°C or 40°C was compared, but the relative antigen-binding activity of the antibody stored at 40°C was compared with that of the antibody stored at 4°C. Relative antigen binding activity ranges from below 30% to 100%. 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. The solution was dissolved in 100 ml of ...% ethanol 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 at 4°C or 40°C for 4 weeks were adjusted to 10 μg / mL using running buffer HBS-EP+. A Series S Sensorchip Protein A (GE Healthcare) was used to measure the amount of the test antibody dissolved in flow cell 2. The solution was added at a flow rate of 10 μL / min for 60 seconds and immobilized on the sensor chip. The amount of binding in flow cell 2 55 seconds after the addition of the test antibody was determined as the flow cell reference cell. The amount of antibody binding was subtracted from the amount of binding of antibody 1 to obtain the amount of antibody binding. Next, human PAD4 (Cayman Chemical Company, Cat. 10500) was adjusted to 50 nM using HBS-EP+ to obtain 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 binding in flow cell 2 5 seconds before the end of the addition of the analyte solution was subtracted from the amount of binding in flow cell 1. The amount of antigen binding was determined. The running buffer was then added for 180 seconds to measure the amount of antigen binding in the dissociation phase of the sensor. 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. There was no change in the relative amount of antigen binding of the test antibody stored frozen and at 4°C. After 4°C for 4 weeks, The relative antigen binding amount of various test antibodies stored at 40℃ for 4 weeks was compared with that of the test antibodies stored at 40℃ for 4 weeks. The combined amounts were compared to determine 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 more than 90% of their antigen binding activity after storage at 40°C for 4 weeks. For example, as taught by Pisupati et al., mAbs, 2017 Vol. 9(7), p1197, the antibody drug Remicade (registered trademark) retained its antigen binding activity even after storage at 40°C for one month. The antibody's biological activity is maintained at 80% or more during preparation of the antibody formulation. It is desirable that the biological activity of the antibody retains 80% or more, preferably 90% or more, of the biological activity of the antibody in the culture medium (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, and 2-1-47) was evaluated. 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. Human PAD4 (final concentration 10 nM) and 50 mM EDTA, 1 mM DTT, and 150 mM NaCl were added to the 50 mM HEPES buffer (pH 7.4) was mixed. After incubation at 37°C for 1 hour, BAEE (benzoyl arginine ethyl ester) was added with stirring, and CaCl 2 Add and stir well. The final concentration of BAEE was 10 mM, and the final concentration of calcium ions was 10 mM. This solution was incubated at 37°C for 3 hours, after which the citrulline residue of citrullinated BAEE was converted to 2,3-butanol. The determination was carried out colorimetrically using a mixture containing benzene 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 CAIA model mice were generated according to the protocol for the mouse arthritis-inducing antibody cocktail (Chondrex Inc., Cat. 53040). The experimental conditions are summarized in Figure 1. On day 0, 9-week-old female Balb / c mice (6 mice / group) were intravenously administered an anti-collagen antibody mixture (1.5 mg) into the tail vein. On day 3, 25 μg LPS (an inflammation-inducing substance) was administered. On the third day, 4 hours before LPS administration, the test antibody or control antibody (human IgG1) was intravenously administered. The rats were administered 15 mg / kg intravenously. On days 3-10, the arthritis score was evaluated according to the following (i) to (iii). (i) The evaluation sites were the fingers, dorsals, and joints of each limb. (ii) The arthritis score was calculated according to Table 5. (iii) The arthritis score was expressed as the average of the total scores of the fingers, dorsals, and joints of each limb (maximum score was 16 / mouse). The arthritis score evaluation results are shown in Figure 1. As can be seen from these results, all of the 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. The CIA model mice were generated by injecting bovine type II collagen and fructose into 5- to 7-week-old female DBA / 1 mice. The vaccine was created by immunizing the mice twice with the anti-adjuvant on the first day and 21 days after the first immunization. Twenty-nine 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 (i) to (iii) below. (i) The evaluation sites were the fingers, dorsals, and joints of each limb. (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 each limb (maximum value was 16 / mouse). The arthritis score evaluation results are shown in Figure 2. As can be seen from these results, 3-2-37 significantly reduced the mean arthritis score in the latter half of the study at all doses established this time compared to the control antibody administration 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 a mouse model of chronic graft-versus-host disease (cGvHD). The cGvHD model mice were generated by transferring splenocytes from 8- to 9-week-old female DBA / 2 mice to 8-week-old female B6D2F1 mice. These mice developed lupus nephritis-like kidney damage that occurs in SLE patients. The day before, 2 weeks, 4 weeks, and 6 weeks after splenocyte transfer, the test antibody was intraperitoneally administered at a dose of 3 mg / kg or 30 mg / kg (13 mice per group). From 2 weeks after splenocyte transfer until 8 weeks after, urine was collected once a week by pressing the lower abdomen of the mice, and the protein concentration and creatinine concentration 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 score are shown in Figure 3. As can be seen from these results, a significant decrease in the 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, a non-symptomatic individual was defined as one 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, and a significant extension of the non-symptomatic period was observed 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: LCDR3 of anti-PAD4 antibodies Lib2(4R)-1-47, 2-1-47, Lib1(6R)-1-39, and 3-1-39 Amino acid sequence 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

[Claim 1] The invention described herein.

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