New regulations for anti-PAD4 antibodies

Abstract

The objectives are to provide an anti-PAD4 antibody with superior properties, or to provide an excellent method for preventing or treating rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, or graft-versus-host disease, etc. [Solution] The present invention provides an anti-PAD4 antibody or an antibody fragment thereof, wherein HCDR1 contains the amino acid sequence of SEQ ID NO: 1, HCDR2 contains the amino acid sequence of SEQ ID NO: 2, HCDR3 contains the amino acid sequence of SEQ ID NO: 3, LCDR1 contains the amino acid sequence of SEQ ID NO: 4, LCDR2 contains the amino acid sequence of SEQ ID NO: 5, and LCDR3 contains the amino acid sequence of SEQ ID NO: 6.

Description

[Technical Field]

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

[0002] PAD4 (Peptidylarginine deiminase 4) is known as an enzyme involved in the citrullination of arginine in proteins. This citrullination is an important reaction for protein structure and reaction because it converts arginine, the most basic of the amino acids that make up proteins, into neutral citrulline.

[0003] Citrullination has been reported to be associated with rheumatoid arthritis (RA). For example, since vimentin, collagen, and fibrin are present as antigens in the synovial membrane in RA, detection kits for anti-cyclic citrullinated peptide antibodies (anti-CCP antibodies), which are antibodies against these antigens, are sold as diagnostic agents for RA.

[0004] There are also several reports on PAD4 and RA. For example, Non-Patent Document 1 reports a correlation between the onset of RA and single nucleotide polymorphisms in the PAD4 gene. Non-Patent Document 2 reports the use of anti-PAD4 antibodies to diagnose RA. Also, Patent Document 1 An attempt to suppress RA by administering a mixture of four types of anti-PAD4 antibodies to mice. It is described (see Example 2 of Patent Document 1). Furthermore, Patent Document 2 describes the affinity to PAD4. Furthermore, an anti-PAD4 antibody with superior citrullination activity inhibitory ability has been described.

[0005] Regarding reports on PAD4 and systemic lupus erythematosus (SLE), PAD4 knockout mice are one example. In an SLE model using this strain (imiquimod-induced model), nephritis was suppressed compared to the wild-type strain. There are reports that nephritis in SLE model (MRL / lpr model) mice was suppressed by administration of small molecule PAD inhibitors (Cl-Amidine and BB-Cl-Amidine) (Non-Patent Literature 3, 4, 5). In addition, there are reports that SLE-like symptoms developed in MRL / lpr mice with PAD4 knockout in the same way as in wild-type mice, and that the development of nephritis could not be suppressed even when small molecule inhibitors (Cl-Amidine) were administered to nephritis models (anti-GBM antibody-induced models and mice with serum from SLE patients) (Non-Patent Literature 6).

[0006] Given the association between these diseases and PAD4, there is a need for anti-PAD4 antibodies with superior binding affinity and stability. [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. [Overview of the project]

Problems to be Solved by the Invention

[0009] The present invention has been made in view of the above circumstances, and an object thereof is to provide an anti-PAD4 antibody having excellent characteristics, or to provide a method for preventing or treating excellent RA or arthritis, systemic lupus erythematosus, lupus nephritis, graft-versus-host disease, etc.

Means for Solving the Problems

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

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

[10] An anti-PAD4 antibody or antibody fragment thereof as described in any of [1] to [9], When the anti-PAD4 antibody or its antibody fragment is stored at 40°C for one month, the anti-PAD4 antibody before storage is... Alternatively, it may have a binding amount of 90% or more compared to the PAD4 binding amount of the antibody fragment. Anti-PAD4 antibody or a fragment thereof.

[0012]

[11] e-1) An anti-PAD4 antibody or 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 a fragment thereof, wherein the amino acid corresponding to the 84th amino acid of the heavy chain is serine.

[12] e-2) An anti-PAD4 antibody or antibody fragment thereof, wherein 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 of the heavy chain is serine.

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

[14] e-4) The heavy chain variable region consists of the amino acid sequence of amino acids 1-120 of SEQ ID NO: 28, and the light chain variable region consists of the amino acid sequence of amino acids 1-105 of SEQ ID NO: 27.

[13] The anti-PAD4 antibody or a fragment thereof described therein.

[15] e-5) The heavy chain consists of sequence number 28 and the light chain consists of sequence number 27, The anti-PAD4 antibody or antibody fragment thereof as described in

[13] or

[14] .

[16] An anti-PAD4 antibody or antibody fragment thereof as described in any of

[11] to

[15] , wherein the cleavage that occurs during production is less than 3% of the total amount produced.

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

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

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

[17] , wherein the antibody is a conjugated antibody of PAD4 represented by Sequence ID No. 29.

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

[15] . A recombinant vector containing the nucleic acid molecule described in

[20]

[19] .

[0013]

[21] A transformant comprising the recombinant vector described in

[20] . A pharmaceutical composition comprising an anti-PAD4 antibody or an antibody fragment thereof as described in any of

[22] [1] to

[18] .

[23] The pharmaceutical composition according to

[22] , which is a prophylactic or therapeutic agent for rheumatoid arthritis or arthritis, systemic lupus erythematosus, lupus nephritis, or graft-versus-host disease.

[24] The pharmaceutical composition according to

[22] or

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

[25] A pharmaceutical composition according to any one of

[22] to

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

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

[18] , or a pharmaceutical composition according to 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 according to any of [1] to

[18] , or a pharmaceutical composition according to any of

[22] to

[25] , for the manufacture of a prophylactic or therapeutic agent for 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 by administering an effective amount of an anti-PAD4 antibody or an antibody fragment thereof as described in any of [1] to

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

[22] to

[25] . [Effects of the Invention]

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

[0015] [Figure 1] Figure 1 shows the method for creating CAIA model mice and the effect of the anti-PAD4 antibody of the present invention on arthritis in the CAIA model. [Figure 2] Figure 2 shows the effect of the anti-PAD4 antibody of the present invention on arthritis in a CIA model. [Figure 3] Figure 3 shows the effect of the anti-PAD4 antibody of the present invention on urinary protein in a cGvHD model. [Figure 4] Figure 4 shows the effect of the anti-PAD4 antibody of the present invention on the incidence rate in a cGvHD model. [Modes for carrying out the invention]

[0016] To facilitate understanding of the present invention, the terms used in this invention are explained below.

[0017] [Neutralization] In this invention, "neutralization" refers to an action that binds to a target of interest and inhibits any function of that target. That is, "neutralizing PAD4 activity" means that an anti-PAD4 antibody inhibits PAD4 activity by binding to PAD4. PAD4 activity can be evaluated by one or more of several in vitro or in vivo analyses known in the art. PAD4 activity is, for example, the citrullination activity of arginine in proteins, and the neutralizing activity of an anti-PAD4 antibody can be evaluated by the citrullination inhibition test described herein.

[0018] [isolated] The term "isolated" in the context of isolated anti-PAD4 antibodies means that they have been identified, isolated, and / or recovered from components in their natural state. Natural impurities are substances that may interfere with the diagnostic or therapeutic use of the antibody and include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Generally, isolation of an anti-PAD4 antibody requires purification by at least one purification step, and an anti-PAD4 antibody purified by at least one purification step can be called an "isolated anti-PAD4 antibody."

[0019] [Human antibodies] Human antibodies refer to antibodies derived from human immunoglobulins, both in terms of light and heavy chains. Depending on the difference in the constant region of the heavy chain, IgG includes IgG1, IgG2, IgG3, and IgG4 with a γ-chain heavy chain, IgM with a μ-chain heavy chain, IgA (including IgA1 and IgA2) with an α-chain heavy chain, IgD with a δ-chain heavy chain, or IgE with an ε-chain heavy chain. In principle, the light chain contains either a κ-chain or a λ-chain.

[0020] [Humanized antibodies] A humanized antibody is an antibody that consists of a variable region comprising a complementarity-determining region of a non-human animal-derived antibody and a framework region derived from a human antibody, and a constant region derived from a human antibody.

[0021] [Chimera antibody] A chimeric antibody is an antibody in which the light chain, heavy chain, or both consist of a variable region of non-human origin and a constant region of human origin.

[0022] [Anti-PAD4 antibody] In the present invention, an anti-PAD4 antibody refers to an immunoglobulin molecule that binds to PAD4 or a modified thereof. Modified molecules include multispecific antibodies, chimeric antibodies, humanized antibodies, functionally modified antibodies, and conjugate antibodies.

[0023] [Multispecific antibody] A multispecific antibody is an asymmetric antibody that possesses two or more independent antigen recognition sites, each with two or more different antigen specificities. Examples include bispecific antibodies with two antigen specificities and trispecific antibodies with three antigen specificities.

[0024] [Modified Antibodies] In this invention, a functionally modified antibody refers to an antibody whose functions other than antigen-binding function, such as cell-killing function, complement-activating function, or blood half-life extension function, have been modified by altering the antibody sequence, sugar chain, etc.

[0025] [Conjugate antibody] In the present invention, a conjugated antibody refers to an antibody to which a functional molecule other than the antibody, such as a non-peptide polymer like polyethylene glycol (PEG), a radioactive substance, a toxin, a small molecule compound, a cytokine, a growth factor, albumin, or an enzyme, has been chemically or genetically engineered to it.

[0026] [Antibody fragment] In this invention, unless otherwise specified, "antibody fragment" refers to an antigen-binding fragment. Antibody fragments can also be called antigen-binding molecules. An antigen-binding fragment is a protein containing a part of an antibody that can bind to an antigen. Examples of antigen-binding fragments include F(ab')2, Fab', Fab, Fv (variable fragment of antibody), disulfide-bonded Fv, and single-chain antibodies (sc Examples include Fv), and polymers thereof. Furthermore, antigen-binding fragments include non-peptide polymers such as polyethylene glycol (PEG), radioactive materials, toxins, small molecule compounds, cytokines, growth factors (TGF-β, NGF, Neurotrophin, etc.), albumin, enzymes, and others. The present invention comprises a conjugate antigen-binding fragment to which a functional molecule other than the anti-PAD4 antibody of the present invention, such as an antibody, is chemically or genetically engineered to be bound.

[0027] [Complementarity Determination Area] The complementarity-determining region (CDR) is the variable region of an immunoglobulin molecule that forms the antigen-binding site. Also known as the hypervariable region, it refers to the part of the immunoglobulin molecule where the amino acid sequence changes particularly significantly. There are three CDRs in both the light chain and the heavy chain (LCDR1, LCDR2, LCDR3, and HCDR1, HCDR2, HCDR3). In this invention, the CDR of immunoglobulin molecules is assigned to the Kabat numbering system (Ka bat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health an d. Determined according to Human Services (NIH, USA).

[0028] [Percent (%) identity of amino acid sequence] "Percent (%) identity" of 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 of a specific reference polypeptide sequence, after the sequences have been aligned, gaps have been introduced if necessary to obtain the maximum % identity, and any conservative substitutions are not considered part of the sequence identity. Alignment for the purpose of measuring % identity can be performed using various methods within the scope of the skill of those skilled in the art, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR). This can be achieved by using publicly available computer software such as FUTOWER. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm necessary to achieve the greatest possible alignment with respect to the full length of the sequences being compared. However, for the purposes of this text, the % identity value is obtained by using the sequence comparison computer program BLAST in pairwise alignment. When BLAST is used for amino acid sequence comparison, the percentage identity of a given amino acid sequence A with a given amino acid sequence B is calculated as follows: 100 times the fraction X / Y Here, X is the number of amino acid residues that scored identical by the program alignment of A and B using 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 with respect to B will be different from the % identity of B with respect to A. Unless otherwise specified, all % identity values ​​here are obtained using the BLAST computer program as shown in the paragraph immediately above.

[0029] [Competing] In the present invention, "competing" with the anti-PAD4 antibody of the present invention means that, when measured by the surface plasmon resonance (SPR) method described herein, the anti-PAD4 antibody or its antigen-binding fragment is The presence of PAD4 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. It is also known as an enzyme involved in cellular netosis. For details on the amino acid sequence of PAD4, please refer to the NCBI (National Center for Biotechnology Information). It can be found on websites such as ), or HGNC (HUGO Gene Nomenclature Committee). The accession number for PAD4 listed in NCBI is, for example, NP_036519.2. The amino acid sequence of PAD4 is, for example, sequence number 29. The biological origin of PAD4 is not limited as long as it possesses PAD4 activity.

[0031] <Anti-PAD4 antibody> One embodiment of the present invention is a novel anti-PAD4 antibody. This antibody exhibits superior binding characteristics, storage stability, and chemical stability compared to conventional anti-PAD4 antibodies. Storage stability and chemical stability will be discussed later, but these are sometimes collectively referred to as stability. This antibody can be used to prevent or treat, for example, rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, or graft-versus-host disease. Because it uses antibodies, it has fewer side effects and is superior in terms of safety. Furthermore, the anti-PAD4 antibody according to one embodiment of the present invention may also be an antibody that suppresses the citrullination activity of proteins mediated by PAD4. Furthermore, the anti-PAD4 antibody according to one embodiment of the present invention may also be an antibody that suppresses netosis in cells.

[0032] An anti-PAD4 antibody according to one embodiment of the present invention exhibits excellent binding to PAD4. Binding of the anti-PAD4 antibody of the present invention to PAD4 means PAD4-specific binding, but it is even more preferable that the dissociation constant (KD) for PAD4 (preferably human PAD4) is low, with an upper limit of, for example, 100 pM or less, more preferably 90 pM or less, and even more preferably 80 pM or less. 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 a value measured, for example, 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 the anti-PAD4 antibody can be evaluated, for example, by a citrullination inhibition test as shown in the examples described below. Adding the anti-PAD4 antibody inhibits citrullination by PAD4. 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 exhibits excellent storage stability. Storage stability can be evaluated by the decrease in binding to PAD4 after storage for a certain period, and the amount of PAD4 bound by the anti-PAD4 antibody can be specifically measured by antigen binding activity measurement using a surface flame resonance spectrometer as described in Example 5. For example, when the anti-PAD4 antibody according to one embodiment of the present invention is stored at 40°C for one month, the amount of PAD4 bound by the anti-PAD4 antibody or its antibody fragment before storage decreases. In comparison, it may have PAD4 binding amounts of 90%, 93%, 94%, 95%, 96%, 97%, or more.

[0035] The binding site of the anti-PAD4 antibody of the present invention to PAD4 is not particularly limited, but for example, it may bind to epitopes 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 genetic recombination technology, and human antibodies produced using human antibody-producing transgenic animals, etc. It also includes antibodies obtained by affinity maturation of the antibodies obtained above. For example, the parent antibody can be G8 as described later. When administering the anti-PAD4 antibody or its antibody fragment to humans as a pharmaceutical, humanized antibodies, human antibodies, or their antibody fragments are preferable from the viewpoint of side effects.

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

[0038] Animals that can be immunized include mammals such as mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, pigs, goats, horses, or cattle, as well as chickens. Administration methods include subcutaneous, intramuscular, or intraperitoneal injection. The drug may be administered mixed with complete or incomplete Freund's adjuvant, and administration is usually performed once every 2-5 weeks. Antibody-producing cells obtained from the spleen or lymph nodes of immunized animals are fused with myeloma cells and isolated as hybridomas. Myeloma cells are also used. Materials derived from animals, such as mice, rats, humans, and chickens, are used.

[0039] <Monoclonal Antibody> Monoclonal antibodies can be obtained specifically as follows: using the aforementioned antigen as an immunogen, and administering the immunogen together with Freund's adjuvant as needed, subcutaneously, intramuscularly, intravenously, or intravenously in the foot pads of animals as described above. Alternatively, immunization is performed by injecting or transplanting the cells into the abdominal cavity one to several times. Typically, immunization is performed one to four times at intervals of approximately one to 14 days from the initial immunization, and antibody-producing cells are obtained from the immunized animal approximately one to five 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] The preparation of "hybridomas" that secrete monoclonal antibodies can be carried out according to the method of Kohler and Milstein et al. (Nature, 256, 495, 1975) and similar modification methods. Specifically, they are prepared by cell fusion of antibody-producing cells contained in the spleen or other organs obtained from immunosensitized animals with myeloma cells from animals, preferably mice, rats, chickens, or humans, that do not produce autoantibodies.

[0042] Examples of myeloma cells that can be used for 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. Typically, cell fusion can be performed by using polyethylene glycol at a concentration of about 20-50% (average molecular weight 1000-4000) at a temperature of 20-40°C, preferably 30-37°C, with a ratio of antibody-producing cells to myeloma cells of about 1:1-10:1, and reacting for about 1-10 minutes.

[0044] Screening for hybridoma clones that produce monoclonal antibodies can be performed by culturing the hybridomas, for example, in a microtiter plate, and measuring the reactivity of the culture supernatant from the wells to the immune antigen using an immunochemical method such as ELISA.

[0045] Clones can be obtained by further cloning using the limiting dilution method from wells containing hybridomas that produce the target antibody. Hybridoma selection and breeding are usually performed in animal cell medium containing 10-20% fetal bovine serum with the addition of HAT (hypoxanthine, aminopterin, thymidine).

[0046] Monoclonal antibodies from hybridomas can be produced by culturing hybridomas in vitro or by growing them in vivo in the ascites fluid of animals such as mice, rats, and chickens, and then isolating the resulting culture supernatant or the animal's ascites fluid.

[0047] When culturing in vitro, hybridomas are grown, maintained, and preserved according to various conditions such as the characteristics of the cell type being cultured and the culture method, and monoclonal antibodies are added to the culture supernatant. It is possible to use a nutrient medium suitable for production.

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

[0049] The isolation and purification of monoclonal antibodies can be performed by subjecting the culture supernatant or ascites fluid described above to saturated ammonium sulfate, euglobulin precipitation, caproic acid, caprylic acid, ion exchange chromatography (DEAE or DE52, etc.), or affinity column chromatography such as anti-immunoglobulin columns or protein A columns. Specifically, the purification of monoclonal antibodies can be easily achieved using known methods for purifying immunoglobulins, such as ammonium sulfate fractionation, PEG fractionation, ethanol fractionation, the use of anion exchangers, and affinity chromatography using PAD4.

[0050] Monoclonal antibodies can also be obtained by phage display. In phage display, phages selected from any phage antibody library are screened using the target immunogen to select phages with the desired binding affinity to the immunogen. Next, the antibody-corresponding sequence contained within the phage is isolated or sequenced, and an expression vector containing a nucleic acid molecule encoding the antibody or antigen-binding domain is constructed based on the isolated or sequenced information. Monoclonal antibodies can then be produced by culturing cell lines transfected with such an expression vector. By using a human antibody library as the phage antibody library, human antibodies with 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 a "chimeric antibody" is one in which the variable region is derived from an immunoglobulin of a non-human animal (such as a mouse, rat, hamster, or chicken), and the constant region is derived from a human immunoglobulin. For example, a 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 binding it to the constant region of an antibody 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 in the present invention may be the constant region of a 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 thus produced. By transforming host cells with the expression vector, chimeric antibody-producing transformed cells are obtained, and by culturing these transformed cells, the desired chimeric antibody is obtained from the culture supernatant.

[0052] Another preferred embodiment of the anti-PAD4 antibody of the present invention is a humanized antibody. In the present invention, a "humanized antibody" is an antibody obtained by transplanting only the DNA sequence of the antigen-binding site (CDR; complementarity-determining region) of a non-human animal antibody, such as a mouse, into a human antibody gene (CDR grafting). For example, it can be produced by referring to the method described in Japanese Patent Publication No. 4-506458 and Japanese Patent No. 2912618. Specifically, it refers to a humanized antibody characterized in that part or all of its CDR is derived from a monoclonal antibody of a non-human animal (mouse, rat, hamster, chicken, etc.), the framework region of its variable region is the framework region of a variable region derived from human immunoglobulin, and its constant region is the constant region derived from human immunoglobulin.

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

[0054] Various methods 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, Examples include CDR grafting (Ozaki et al., Blood. 1999 Jun 1;93(11):3922-3930.), re-surfacing (roguska et al., Proc Natl Acad Sci US A. 1994 Feb 1;91(3):969-973.), or FR shuffling (Damschroder et al., Mol Immunol. 2007 Apr;44(11):3049-3060. Epub 2007 Jan 22.). To modify or improve antigen binding, the human FR region... The amino acid residues in this region may be substituted with the corresponding residues from the CDR donor antibody. This can be carried out by methods well known in the art (Riechmann et al., Nature. 1988 Mar 24;332(6162):323-327.). For example, by modeling the interaction between CDR and FR residues. FR residues important for antigen binding may be identified. Alternatively, abnormal FR residues at specific positions may be identified by sequence comparison. (See Nishibori et al., Mol Immunol. 2006 Feb;43(6):) Humanization may be carried out using the method described in 634-42.

[0055] The DNA of the isolated mouse heavy chain CDR region is transplanted into a suitable human heavy chain gene, and this gene is then expressed into a suitable human light chain gene, which is similarly transplanted into another suitable human light chain gene, which is similarly transplanted into a mouse light chain CDR region. Alternatively, the human heavy chain and light chain genes, which have mouse CDRs transplanted into them, can be expressed into the same expression vector. By transforming host cells with the expression vector thus produced, humanized antibody-producing transformed cells are obtained, and by culturing these transformed cells, the desired humanized antibody is obtained 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 and constant region of the heavy chain and the variable region and constant region of the light chain that constitute the immunoglobulin, are derived from a gene encoding human immunoglobulin, and can be produced by introducing a human antibody gene into a mouse. Specifically, for example, a transgenic animal created by incorporating at least a human immunoglobulin gene into the gene locus of a non-human animal such as a mouse or chicken can be produced by immunizing the transgenic animal with an antigen, in the same manner as the method for producing monoclonal antibodies described above.

[0057] For example, transgenic mice that produce human antibodies are described in Nature Genetics, Vol. 7, pp. 13-21, 1994; Nature Genetics, Vol. 15, pp. 146-156, 1997; and Japanese Patent Publication No. 4-504365. Japanese Patent Publication No. 7-509137; International Publication No. 94 / 25585; Nature, Vol. 368, It can be prepared according to the methods described in pp. 856-859, 1994; and Japanese Patent Publication No. Hei 6-500233, etc. More specifically, HuMab® mouse (Medarex, Princeton NJ) KMTM mice (Kirin Pharma Company, Japan), KM(FCγRIIb-KO) mice These are some examples.

[0058] Specifically, the monoclonal antibody of the present invention is: a) 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 row of anti-PAD4 antibodies, c) 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, wherein the amino acid corresponding to the 84th amino acid of the heavy chain is serine. These are some examples. The antibodies described in a) to d) above have significantly improved binding affinity to PAD4, the antibodies described in a) to d) above have improved storage stability of binding activity to PAD4, and the antibody described in e) above has suppressed cleavage and significantly improved chemical stability. The antibodies described in a) to d) above have high chemical stability equivalent to that of the antibody described in e).

[0059] The amino acid sequences described in a) to e) above correspond to the amino acid sequences of the respective CDRs present in the 2-1-47, 3-1-39, 3-2-37, 4-2-25, and G8ss antibodies described in the examples below. In other words, 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, and 3, and the light chain CDR1, 2, and 3 of 3-1-39 are the amino acid sequences shown in SEQ ID NOs: 1, 8, 11, 4, 5, and 13, respectively. The amino acid sequences of the heavy chain CDR1, 2, and 3, and the light chain CDR1, 2, and 3 of 3-2-37 are the amino acid sequences shown in SEQ ID NOs: 1, 9, 12, 4, 5, and 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 NOs. 1, SEQ ID NOs. 10, SEQ ID NOs. 11, SEQ ID NOs. 4, SEQ ID NOs. 5, and SEQ ID NOs. 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 NOs. 1, SEQ ID NOs. 24, SEQ ID NOs. 11, SEQ ID NOs. 4, SEQ ID NOs. 5, and SEQ ID NOs. 25, respectively.

[0060] The anti-PAD4 antibody described in a) above may have the ability to bind to PAD4 (preferably with a KD value of 100 pM or less for PAD4, more preferably 90 pM or less, and even more preferably 80 pM or less) and maintain the property of neutralizing the citrullination activity of PAD4, as long as the amino acid sequences of the heavy chain CDR1, 2, 3 and the light chain CDR1, 2, and 3 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 anti-PAD4 antibody described in b) above may have the ability to bind to PAD4 (preferably with a KD value of 100 pM or less for PAD4, more preferably 90 pM or less, and even more preferably 80 pM or less) and maintain the property of neutralizing the citrullination activity of PAD4, even if the amino acid sequences of the heavy chain CDR1, 2, 3 and the light chain CDR1, 2, and 3 include the amino acid sequences shown in SEQ ID NOs: 1, 8, 11, 4, 5, and 13, respectively. stomach. The anti-PAD4 antibody described in c) above may have the ability to bind to PAD4 (preferably with a KD value of 100 pM or less for PAD4, more preferably 90 pM or less, and even more preferably 80 pM or less) and maintain the property of neutralizing the citrullination activity of PAD4, as long as the amino acid sequences of the heavy chain CDR1, 2, 3 and the light chain CDR1, 2, and 3 are the amino acid sequences shown in SEQ ID NOs: 1, SEQ ID NOs: 9, SEQ ID NOs: 12, SEQ ID NOs: 4, SEQ ID NOs: 5, and SEQ ID NOs: 14, respectively. The anti-PAD4 antibody described in d) above may have binding ability to PAD4 (preferably with a KD value of 100 pM or less for PAD4, more preferably 90 pM or less, and even more preferably 80 pM or less) and maintain the property of neutralizing the citrullination activity of PAD4, as long as the amino acid sequences of the heavy chain CDR1, 2, 3 and the light chain CDR1, 2, and 3 are included in the amino acid sequences shown in SEQ ID NOs: 1, SEQ ID NOs: 10, SEQ ID NOs: 11, SEQ ID NOs: 4, SEQ ID NOs: 5, and SEQ ID NOs: 15, respectively. The anti-PAD4 antibody described in e) above may have the ability to bind to PAD4 (preferably with a KD value of 100 pM or less for PAD4, more preferably 90 pM or less, and even more preferably 80 pM or less) and maintain the property of neutralizing the citrullination activity of PAD4, as long as the amino acid sequences of the heavy chain CDR1, 2, 3 and the light chain CDR1, 2, and 3 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.

[0061] An anti-PAD4 antibody according to one embodiment of the present invention may have the ability to bind to PAD4 (preferably with a KD value of 100 pM or less for PAD4, more preferably 90 pM or less, and even more preferably 80 pM or less), and may neutralize the citrullination activity of PAD4, as long as the properties are maintained. The heavy chain CDR1 may contain the amino acid sequence shown in SEQ ID NO: 1, the heavy chain CDR2 may contain the amino acid sequence shown in SEQ ID NO: 2, the heavy chain CDR3 may contain the amino acid sequence shown in SEQ ID NO: 3, the light chain CDR1 may contain the amino acid sequence shown in SEQ ID NO: 4, the light chain CDR2 may contain the amino acid sequence shown in SEQ ID NO: 5, and the light chain CDR3 may contain the amino acid sequence shown in SEQ ID NO: 6. Furthermore, the amino acid sequence shown in Sequence 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, with each position containing any combination of the above amino acids. Furthermore, in the amino acid sequence shown in SEQ ID NO: 3, the amino acid at position 1 is either Ala or Gly, and specifically includes the amino acid sequences of SEQ ID NOs: 11 and 12. Furthermore, 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 may have the ability to bind to PAD4 (preferably with a KD value of 100 pM or less for PAD4, more preferably 90 pM or less, and even more preferably 80 pM or less), and may neutralize the citrullination activity of PAD4, as long as the properties are maintained. The heavy chain CDR1 may contain the amino acid sequence shown in SEQ ID NO: 1, the heavy chain CDR2 may contain the amino acid sequence shown in any of SEQ ID NOs: 7 to 10, the heavy chain CDR3 may contain the amino acid sequence shown in SEQ ID NO: 11 or 12, the light chain CDR1 may contain the amino acid sequence shown in SEQ ID NO: 4, the light chain CDR2 may contain the amino acid sequence shown in SEQ ID NO: 5, and the light chain CDR3 may contain the amino acid sequence shown in any of SEQ ID NOs: 13 to 15.

[0063] Furthermore, as long as the anti-PAD4 antibody of the present invention has the ability to bind to PAD4 (preferably with a KD value of 100 pM or less for PAD4, more preferably 90 pM or less, and even more preferably 80 pM or less) and neutralizes the citrullination activity of PAD4, one of these CDRs is acceptable. 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 properties of the present invention. Here, "conservative substitution" means substituting an amino acid residue with another chemically similar amino acid residue so as not to substantially alter the activity of the peptide. Examples include substituting one hydrophobic residue with another hydrophobic residue, or substituting one polar residue with another polar residue having the same charge. Examples of functionally similar amino acids that can be substituted in this way include nonpolar (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. Furthermore, negatively charged (acidic) amino acids include aspartic acid and glutamic acid. The maintenance of antibody properties means that these properties are maintained even after the CDR amino acid sequence modification. This means maintaining the same level as before the change, for example, 80% or more, preferably 90% or more, and more preferably 95% or more. Note that "maintaining" also includes improvement.

[0064] Regions other than the CDR are not particularly restricted as long as they are sequences that maintain the antibody structure and allow it to function. The sequences may be derived from mice, humans, other mammals, chickens, chimeric sequences thereof, or artificial sequences. If a constant region is included, the amino acid sequences of the constant regions of the heavy and light chains are exemplified by those described in Nucleic Acids Research vol.14, p1779, 1986, The Journal of Biological Chemistry vol.257, p1516, 1982, and Cell vol.22, p197, 1980.

[0065] As an example of a modified region other than the CDR, a preferred embodiment of the present invention is the antibody An example of an antibody is one in which the amino acid corresponding to the 84th amino acid of the heavy chain (e.g., SEQ ID NO: 26) is serine. Here, "corresponding amino acid" refers to the amino acid (usually asparagine) that is located at the position of asparagine, the 84th amino acid 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 suppression of cleavage that occurs during production. The suppression of cleavage can be evaluated by the 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, it can be evaluated by measuring the amount of cleaved peptide by the method described in Example 1. An anti-PAD4 antibody according to one embodiment of the present invention, which is excellent in suppressing cleavage that occurs during production, may have cleaved antibody levels of 3%, 2%, or 1% or less compared to the antibody before storage when stored at 4°C for 2 weeks.

[0066] Furthermore, examples of humanized antibodies derived from human sources other than CDRs are also presented. Therefore, a) An anti-PAD4 antibody in which the heavy chain variable region and the light chain variable region consist of the amino acid sequence of amino acids 1-120 of SEQ ID NO: 16 and the amino acid sequence of amino acids 1-105 of SEQ ID NO: 17, or contain the same amino acid sequence. b) An anti-PAD4 antibody in which the heavy chain variable region and the light chain variable region consist of the amino acid sequence of amino acids 1-120 of SEQ ID NO: 18 and the amino acid sequence of amino acids 1-105 of SEQ ID NO: 19, or contain the same amino acid sequence. c) An anti-PAD4 antibody in which the heavy chain variable region and the light chain variable region consist of the amino acid sequence of amino acids 1 to 120 of SEQ ID NO: 20 and the amino acid sequence of amino acids 1 to 105 of SEQ ID NO: 21, or contains the same amino acid sequence. d) An anti-PAD4 antibody in which the heavy chain variable region and the light chain variable region consist of the amino acid sequence of amino acids 1 to 120 of SEQ ID NO: 22 and the amino acid sequence of amino acids 1 to 105 of SEQ ID NO: 23, or contain the same amino acid sequence, and e) An anti-PAD4 antibody in which the heavy chain variable region and the light chain variable region consist of the amino acid sequence of amino acids 1-120 of SEQ ID NO: 26 and the amino acid sequence of amino acids 1-105 of SEQ ID NO: 27, or contain the same amino acid sequence. One or more antibodies selected from the group consisting of the following are given as examples.

[0067] Alternatively, the anti-PAD4 antibody according to one embodiment of the present invention is a) An anti-PAD4 antibody in which the heavy chain and light chain consist of or contain the amino acid sequences shown in SEQ ID NOs. 16 and 17, respectively. b) An anti-PAD4 antibody in which the heavy chain and light chain consist of or contain the amino acid sequences shown in SEQ ID NOs. 18 and 19, respectively. c) An anti-PAD4 antibody in which the heavy chain and light chain consist of or contain the amino acid sequences shown in SEQ ID NOs. 20 and 21, respectively. d) Anti-PAD4 antibodies in which the heavy chain and light chain consist of or contain the amino acid sequences shown in SEQ ID NOs. 22 and 23, respectively, and e) Anti-PAD4 antibodies comprising or containing the amino acid sequences shown in SEQ ID NOs. 26 and 27, respectively, for the heavy and light chains. It may also be one or more antibodies selected from the group consisting of the following:

[0068] Furthermore, in the heavy chain variable region and / or light chain variable region of the amino acid sequence of the humanized antibody, one or more amino acids (1 to 20, 1 to 10, or 1 to 5) may be substituted, deleted, added, or inserted, as long as the ability to bind to PAD4 (preferably with a KD value of 100 pM or less to PAD4, more preferably 90 pM or less, and even more preferably 80 pM or less) is maintained and the property of neutralizing the citrullination activity of PAD4 is maintained. Such substitutions, deletions, or additions may be introduced into the CDR, but it is preferable that they be introduced into regions other than the CDR. In addition, it is preferable that the amino acid substitutions are conservative substitutions 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 comprises 90% or more (more preferably 95%, 96%, 97%, 98%) of the amino acid sequence of amino acids 1-120 of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 26. The amino acid sequence may have an identity of 90% or more (more preferably 95%, 96%, 97%, 98%, 99% or more), and the light chain variable region may have an amino acid sequence that has an identity of 90% or more (more preferably 95%, 96%, 97%, 98%, 99% or more) with the amino acid sequence of amino acids 1 to 105 of SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO: 27.

[0070] The anti-PAD4 antibody of the present invention may also be an anti-PAD4 antibody in which the heavy chain has an amino acid sequence having 90% or more (more preferably 95%, 96%, 97%, 98%, 99% or more) identity with sequence numbers 16, 18, 20, 22, and 26 of the sequence listing, and the light chain has an amino acid sequence having 90% or more (more preferably 95%, 96%, 97%, 98%, 99% or more) identity with sequence numbers 17, 19, 21, 23, and 27 of the sequence listing.

[0071] The anti-PAD4 antibody of the present invention includes a CDR consisting of the above-mentioned specific amino acid sequence, or a multispecific antibody, a functionally modified antibody, or a conjugate antibody having 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 produce multispecific antibodies, such as bispecific antibodies, by genetically engineering the attachment of an antibody that has antigen-binding specificity other than PAD4 to itself. This genetic engineering method is already established in this field. For example, by attaching DVD-Ig (Wu et al., Nature Biotechnology 25(11), 1290(2007)) with variable regions linked in series, or by modifying the Fc region of the antibody, it is possible to produce antibodies that bind to different antigens. By utilizing the ART-Ig technology (Kitazawa et al., Nature Medicine 18(10), 1570(2012)), in which the heavy chains of two types of antibodies are combined, a desired bispecific antibody can be obtained. .

[0073] Functionally modified antibodies are examples of modified molecules of the anti-PAD4 antibody of the present invention. Functionally modified antibodies refer to antibodies whose functions, such as cell-killing function, complement activation function, and blood half-life extension function, have been modified by altering the antibody sequence, sugar chain, etc. (Kenya Shitara, Journal of Pharmaceutical Sciences, 2009, Vol.129(1), p3; Akiko Ishii et al., Journal of Japanese Pharmacology, 2010, Vol.136(5), p280; Shuhei Hashiguchi et al., Biochemistry, 2010, Vol.82(8), p710).

[0074] Functionally modified anti-PAD4 antibodies are prepared by the following methods. 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 disrupted as the host cell, an antibody with reduced fucose content in the sugar chain and enhanced cell-killing function is obtained. When produced using CHO cells into which the FUT8 gene has been introduced as the host cell, an antibody with low cell-killing function is obtained (International Publication No. 2005 / 035586, International Publication No. 2002 / 31140, International Publication No. 00 / 61739). Furthermore, the complement activation function can be regulated by modifying the amino acid residues in the Fc region (U.S. Patent No. 6,737,056, 7,297,775, 7,317,091). Furthermore, by using mutants of the Fc region that enhance binding to FcRn, one of the Fc receptors, it is possible to extend the half-life in the blood (Shuhei Hashiguchi et al., Biochemistry, 2010, Vol.82(8), p710). Modified antibodies can be manufactured using genetic engineering.

[0075] Examples of modified molecules of the anti-PAD4 antibody of the present invention include conjugated antibodies. Conjugated antibodies include those obtained by chemically or genetically engineering a functional molecule other than the anti-PAD4 antibody of the present invention, such as a non-peptide polymer like polyethylene glycol (PEG), a radioactive substance, a toxin, a small molecule compound, a cytokine, a growth factor, albumin, an enzyme, or another antibody, to the anti-PAD4 antibody.

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

[0077] When using radioactive materials as functional molecules, 131 I, 125 I, 90 Y, 64 Cu, 99 Tc, 77 Lu or 211 At, etc., are used. Radioactive materials are directly bound to antibodies by methods such as the chloramine T method. It can be combined.

[0078] When using enzymes as functional molecules, luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Patent No. 4737456), malate dehydrogenase, urease, peroxidase (e.g., horseradish peroxidase (HRPO)), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, etc. Kaleidooxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, etc. are used.

[0079] Linkers used when chemically binding toxins, low molecular weight compounds, or enzymes include divalent radicals (e.g., alkylenes, arylenes, heteroarylenes), -(CR2)nO( Linkers and alkoxys represented as CR2)n-(where R is any substituent and n is a positive integer) Examples include polyunits (e.g., polyethyleneoxy, PEG, polymethyleneoxy, etc.) and alkylaminos (e.g., polyethyleneamino, Jeffamine™), as well as diacitates and amides (e.g., succinate, succinamide, diglycolate, malonate, and caproamide). Chemical modification methods for attaching functional molecules are already established in this field (DJKing., 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 this invention, the "antigen-binding fragment" of an antibody refers to a portion of the antibody that has antigen-binding properties, as described above, and specifically includes F(ab')2, Fab', Fab, Fv (variable fragment of antibody), disulfide bond Fv, single-chain antibody (scFv), and polymers thereof. Examples include, and furthermore, the antigen-binding fragment may include non-peptide polymers such as polyethylene glycol (PEG), radioactive materials, toxins, small molecule compounds, cytokines, growth factors (TGF-β, NGF, Neurotrophin, etc.), albumin, enzymes, and other antibodies of the present invention, as well as the anti-PAD4 antibody of the present invention. It includes conjugate antigen-binding fragments to which other functional molecules are chemically or genetically engineered.

[0081] Here, "F(ab')2" and "Fab" refer to antibody fragments produced by treating immunoglobulins with proteolytic enzymes such as pepsin or papain, and digested before and after the disulfide bond existing between two heavy chains in the hinge region. For example, when IgG is treated with papain, it is cleaved upstream of the disulfide bond existing between two heavy chains in the hinge region to produce two identical antibody fragments in which a light chain consisting of VL (variable region of the light chain) and CL (constant region of the light chain), and a heavy chain fragment consisting of VH (variable region of the heavy chain) and CHγ1 (γ1 region in the constant region of the heavy chain) are bound by a disulfide bond at the C-terminal region. These two identical antibody fragments are each called Fab. Also, when IgG is treated with pepsin, an antibody fragment that is cleaved downstream of the disulfide bond existing between two heavy chains in the hinge region and is slightly larger than that in which the two Fabs are connected in the hinge region can be produced. This antibody fragment is called F(ab')2.

[0082] Conjugated antigen-binding fragments can be mentioned as modified molecules of the antigen-binding fragment of the anti-PAD4 antibody of the present invention. Examples of the conjugated antigen-binding fragment include conjugated antigen-binding fragments in which a non-peptidic 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, etc., which are functional molecules other than the anti-PAD4 antibody of the present invention, are chemically or genetically engineered to bind to a partial region having the antigen-binding property of the anti-PAD4 antibody.

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

[0084] When using a radioactive substance as a functional molecule, 131 I, 125 I, 90 Y, 64 Cu, 99 Tc, 77 L u or 211 At, etc., is used. Radioactive materials are tested for anti-PAD4 antibodies using methods such as the chloramine T method. It can be directly bound to a portion of the region that has antigen-binding properties.

[0085] When enzymes are used as functional molecules, luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Patent No. 4737456), malate dehydrogenase, urease, peroxidase (e.g., horseradish peroxidase (HRPO)), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase, etc.), lactoperoxidase, microperoxidase, etc. are used.

[0086] Linkers used when chemically binding toxins, low molecular weight compounds, or enzymes include divalent radicals (e.g., alkylenes, arylenes, heteroarylenes), -(CR2)nO( Linkers and alkoxys represented as CR2)n-(where R is any substituent and n is a positive integer) Examples include polyunits (e.g., polyethyleneoxy, PEG, polymethyleneoxy, etc.) and alkylaminos (e.g., polyethyleneamino, Jeffamine™), as well as diacitates and amides (e.g., succinate, succinamide, diglycolate, malonate, and caproamide). Chemical modification methods for attaching functional molecules are already established in this field (DJKing., 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] In the present invention, an anti-PAD4 antibody containing a CDR or variable region having a specific amino acid sequence is preferably characterized in that the constant region is the constant region of human IgG (IgG1, IgG2, IgG3, IgG4) in order to maintain a long half-life in the blood.

[0088] The anti-PAD4 antibody and its antigen-binding fragment of the present invention have binding ability to PAD4 (preferably with a KD value of 100 pM or less for PAD4, more preferably 90 pM or less, and even more preferably 80 pM or less), and as long as the property of neutralizing the citrullination activity of PAD4 is maintained, they compete with antibodies having the above-mentioned specific CDR amino acid sequence for binding to PAD4. This also includes anti-PAD4 antibodies and their antigen-binding fragments. The amino acid sequences of specific CDRs as described above. Antibodies that compete for binding between antibodies containing PAD4 include PAD4 345, 347, and 348. Examples of antibodies that have an epitope in the region including position are given. However, G8 antibodies are not included. The antibody is coexisting with PAD4 in the binding system of the antibody having the above-mentioned CDR sequence. This allows for the acquisition (screening) and evaluation of data. For example, it can be acquired by screening using the surface plasmon resonance (SPR) method described in International Publication No. 2016 / 175236.

[0089] The anti-PAD4 antibody that competes for binding to PAD4 with the anti-PAD4 antibody containing the specific CDR amino acid sequence described above 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 it may be a chimeric antibody or humanized antibody which are combinations of these antibodies, but it is preferable that it be a chimeric antibody, humanized antibody, or human antibody.

[0090] The anti-PAD4 antibody or antibody fragment thereof in the present invention can be produced, for example, using transformant (host) cells containing a recombinant vector containing the following nucleic acid molecules encoding the anti-PAD4 antibody or antibody fragment thereof in 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 of the present invention or a fragment of that antibody. The nucleic acid molecule is not particularly limited as long as it encodes a polypeptide containing the CDR, variable region, or full-length amino acid sequence described above, but examples include: a) The heavy chain variable region and the light chain variable region, which are the amino acid sequences of amino acids 1-120 in SEQ ID NO: 16 and amino acid sequences of amino acids 1-105 in SEQ ID NO: 17. b) The heavy chain variable region and the light chain variable region, which are the amino acid sequences of amino acids 1-120 in SEQ ID NO: 18 and amino acid sequences of amino acids 1-105 in SEQ ID NO: 19. c) The heavy chain variable region and the light chain variable region, which are the amino acid sequences of amino acids 1-120 in SEQ ID NO: 20 and amino acid sequences of amino acids 1-105 in SEQ ID NO: 21. d) The heavy chain variable region and the light chain variable region, which are the amino acid sequences of amino acids 1 to 120 in SEQ ID NO: 22 and the amino acid sequences of amino acids 1 to 105 in SEQ ID NO: 23, and e) Examples include polynucleotides containing base sequences that encode the amino acid sequence of amino acid numbers 1-120 of SEQ ID NO: 28 and the amino acid sequence of amino acid numbers 1-105 of SEQ ID NO: 27, which are the heavy chain variable region and the light chain variable region, respectively.

[0092] As another example of the nucleic acid molecule of the present invention, a) The heavy chain and light chain, represented by the amino acid sequences shown in SEQ ID NOs. 16 and 17, b) The heavy chain and light chain, represented by the amino acid sequences shown in SEQ ID NOs. 18 and 19, c) The heavy chain and light chain, represented by the amino acid sequences shown in SEQ ID NOs. 20 and 21, d) The heavy chain and light chain, represented by the amino acid sequences shown in SEQ ID NOs. 22 and 23, and e) Polynucleotides comprising a heavy chain and a light chain, each containing a base sequence encoding the amino acid sequences shown in SEQ ID NOs. 28 and 27, respectively.

[0093] As another example of the nucleic acid molecule of the present invention, A polynucleotide containing the full-length heavy chain of the anti-PAD4 antibody, represented by the nucleotide sequence shown in SEQ ID NO: 32, and the full-length light chain of the anti-PAD4 antibody, represented by the nucleotide sequence shown in SEQ ID NO: 33. A polynucleotide containing the full-length heavy chain of the anti-PAD4 antibody, represented by the nucleotide sequence shown in SEQ ID NO: 34, and the full-length light chain of the anti-PAD4 antibody, represented by the nucleotide sequence shown in SEQ ID NO: 35. A polynucleotide containing the full-length heavy chain of the anti-PAD4 antibody, represented by the nucleotide sequence shown in SEQ ID NO: 36, and the full-length light chain of the anti-PAD4 antibody, represented by the nucleotide sequence shown in SEQ ID NO: 37. A polynucleotide containing the full-length heavy chain sequence of the anti-PAD4 antibody, shown in SEQ ID NO: 38, and the full-length light chain sequence of the anti-PAD4 antibody, shown in SEQ ID NO: 39. A polynucleotide containing the full-length heavy chain sequence of the anti-PAD4 antibody, shown in SEQ ID NO: 40, and the full-length light chain sequence of the anti-PAD4 antibody, shown in SEQ ID NO: 31. These are some examples.

[0094] Furthermore, the nucleic acid molecule of the present invention has the ability to bind to PAD4 and neutralizes the citrullination activity of PAD4. Insofar as it encodes a monoclonal antibody, it includes polynucleotides that hybridize under stringent conditions with the complementary DNA of the base sequence of the heavy chain variable region or light chain variable region. This may also be the case. Here, stringent conditions include, for example, washing at 68°C, with a salt concentration equivalent to 0.1% SDS, after Southern hybridization.

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

[0096] The full lengths of the heavy and light chains of the humanized anti-PAD4 antibody G8, as described in International Publication No. 2016 / 143753, The encoding base sequences are shown in SEQ ID NOs. 30 and 31. For example, by modifying these base sequences, it is possible to obtain nucleic acid molecules encoding the anti-PAD4 antibody or a fragment of that antibody according to the present invention.

[0097] Furthermore, the nucleic acid molecules 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 using reverse transcriptase with random primers. Next, the cDNA encoding the 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, respectively, 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 conventional methods, such as by incorporating it into a sequencing plasmid. Alternatively, the DNA encoding the monoclonal antibody of the present invention can also be obtained by chemically synthesizing the variable region or a part thereof and binding it to the sequence containing the constant region.

[0098] The present invention also provides a recombinant vector comprising the nucleic acid molecule of the present invention and a transformant (host cell) comprising the recombinant vector. As a recombinant vector, it is expressed in prokaryotic cells such as Echerichia coli. Any vector may be used (e.g., pBR322, pUC119, or derivatives thereof), but vectors expressible in eukaryotic cells are preferred, and vectors expressible in mammalian cells are more preferred. Examples of vectors expressible in mammalian cells include plasmid vectors such as pcDNA3.1 (Invitrogen), pConPlus, pcDM8, pcDNA I / Amp, pcDNA3.1, and pREP4, and pDON-AI DNA (Takarazuka). Examples of viral vectors include those manufactured by IO Corporation. A single vector containing both a heavy chain coding sequence and a light chain coding sequence is acceptable, or two vectors containing both a heavy chain coding sequence and a light chain coding sequence are also acceptable.

[0099] The transformants into which the recombinant vector of the present invention is introduced may be prokaryotic cells such as Escherichia coli and Bacillus subtilis, but eukaryotic cells are preferred, and mammalian-derived cells are more preferred. Examples of mammalian-derived cells include Chinese hamster ovary cells (CHO cells), COS, and Mi. Examples include Eloma, BHK, HeLa, Vero, 293, NS0, Namalwa, YB2 / 0, etc.

[0100] Antibodies and their antigen-binding fragments obtained by the methods described in this specification or by known methods can be purified until homogeneous. Separation and purification of antibodies can be performed using the same separation and purification methods 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, etc. (Antibodies: A Laboratory Manual). Ed Harlow and David Lane (Cold Spring Harbor Laboratory, 1988) are examples, but the list is not limited to these. Columns used in affinity chromatography include 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. Examples include Amersham Biosciences.

[0101] <Composition> One embodiment of the present invention is a composition comprising an anti-PAD4 antibody or an antibody fragment thereof according to the above embodiment of the present invention. This composition can be used to efficiently detect PAD4. This composition can efficiently suppress citrullination of proteins. It can also efficiently suppress netosis in cells. Furthermore, it can prevent or treat rheumatoid 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 an anti-PAD4 antibody or antibody fragment according to the embodiment of the present invention, and may otherwise contain, for example, a buffer. One or more of the various embodiments of the inhibitor and pharmaceutical composition described later (for example, being able to contain a carrier) may be applied to this composition.

[0102] One embodiment of the present invention is a PAD4-mediated protein citrullination inhibitor comprising an anti-PAD4 antibody or an antibody fragment thereof according to the above embodiment of the present invention. This inhibitor can efficiently suppress protein citrullination by PAD4. The reduction rate of citrullination activity by the inhibitor may be 20%, 30%, 40%, 60%, 80% or more, or within the range of any two of these values. For example, the reduction rate when using PBS is 0. It may also be expressed as a relative percentage. In one embodiment of the present invention, "agent" includes, for example, a composition used for research or treatment. The inhibitor includes, for example, an agent for the prevention or treatment of 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 is The composition may include the embodiments of the present invention described above. One embodiment of the present invention is a method for inhibiting protein citrullination by PAD4, comprising the step of contacting PAD4 with an anti-PAD4 antibody or an antibody fragment thereof according to the above embodiment of the present invention. Another embodiment of the present invention is a method for inhibiting protein citrullination by PAD4, comprising the step of administering an anti-PAD4 antibody or an antibody fragment thereof according to the above embodiment of the present invention to a patient. The above inhibition method includes inhibition methods performed for research or therapeutic purposes. Another embodiment of the present invention is the use of an anti-PAD4 antibody or an antibody fragment thereof according to the above embodiment of the present invention for producing an inhibitor of protein citrullination by PAD4.

[0103] One embodiment of the present invention is a cellular netosis inhibitor comprising an anti-PAD4 antibody or an antibody fragment thereof according to the above embodiment of the present invention. This inhibitor can be used to efficiently suppress PAD4-induced cellular netosis. The reduction rate of cellular netosis activity by the inhibitor may be 20%, 30%, 40%, 60%, 80% or more, and any two of these may be used. It may be within a range of one value. This reduction rate may be expressed as a relative percentage, for example, with the reduction rate when PBS is used being set to 0%. In one embodiment of the present invention, "agent" includes, for example, a composition used for research or treatment. The inhibitor includes, for example, an agent for the prevention or treatment of 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 is the same as above The composition may include an embodiment of the present invention. One embodiment of the present invention is a method for suppressing cellular netosis, comprising the step of contacting PAD4 with an anti-PAD4 antibody or an antibody fragment thereof according to the above embodiment of the present invention. Another embodiment of the present invention is a method for suppressing cellular netosis, comprising the step of administering an anti-PAD4 antibody or an antibody fragment thereof according to the above embodiment of the present invention to a patient. The above suppression method includes a suppression method performed for research or therapeutic purposes. Another embodiment of the present invention is the use of an anti-PAD4 antibody or an antibody fragment thereof according to the above embodiment of the present invention for producing a cellular netosis inhibitor.

[0104] One embodiment of the present invention is a pharmaceutical composition comprising an anti-PAD4 antibody or an antibody fragment thereof according to the above embodiment of the present invention. This pharmaceutical composition can be used to prevent, treat, or prevent recurrence of rheumatoid arthritis 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 compositions include, for example, pharmaceutical compositions for the treatment of rheumatoid arthritis or arthritis, systemic lupus erythematosus, lupus nephritis, or graft-versus-host disease, and pharmaceutical compositions for the prevention of rheumatoid arthritis or arthritis, systemic lupus erythematosus, lupus nephritis, or graft-versus-host disease. The above pharmaceutical compositions include the above The composition may include 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 the step of administering to a patient an effective amount of the anti-PAD4 antibody or antibody fragment thereof (or a pharmaceutical composition containing the anti-PAD4 antibody or antibody fragment thereof) according to the above embodiment of the present invention. The above disease includes, for example, rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, or graft-versus-host disease. One embodiment of the present invention is the use of the anti-PAD4 antibody or antibody fragment thereof (or a pharmaceutical composition containing the anti-PAD4 antibody or antibody fragment thereof) according to the above embodiment of the present invention for preventing, treating, or preventing recurrence of rheumatoid 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 containing an anti-PAD4 antibody or an antibody fragment thereof) according to the above embodiment of the present invention for the production of a prophylactic agent, therapeutic agent, or relapse prevention agent for RA or arthritis, systemic lupus erythematosus, lupus nephritis, or graft-versus-host disease.

[0105] In one embodiment of the present invention, “agent” comprises an active ingredient and one or more pharmacologically acceptable substances. The pharmaceutical composition may also include a carrier. In one embodiment of the present invention, the "pharmaceutical composition" may be manufactured, for example, by mixing the active ingredient with the carrier and using any method known in the field of pharmaceutical technology. Furthermore, the pharmaceutical composition is not limited in its form of use as long as it is used for prevention or treatment, and may consist of the active ingredient alone or a mixture of the active ingredient with any other ingredient.

[0106] The therapeutic and / or preventive effect of a pharmaceutical composition on rheumatoid arthritis (RA) may be evaluated, for example, by arthritis score, RA score, swelling width, imaging studies, modified Total Sharp score, Disease Activity Score (DAS), ACR20, ACR50, ACR70 (representing the achievement rate of the rheumatoid arthritis activity assessment criteria developed by the American College of Rheumatism (ACR)), or disease markers. When evaluating with arthritis score, for example, a therapeutic and / or preventive effect may be judged if the patient's arthritis score during administration of the pharmaceutical composition is significantly lower than the patient's arthritis score when not administered. Alternatively, a therapeutic and / or preventive effect may be judged if the patient's arthritis score during administration of the pharmaceutical composition is significantly lower than the patient's arthritis score during administration of a negative control substance. The above reduction may be, for example, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 point or less. It may be within the range of any two of those values. Or, the patient's reaction when administering the pharmaceutical composition. A therapeutic and / or preventive effect may be determined if the area of ​​the arthritis score graph is significantly reduced compared to the area of ​​the arthritis score graph when no administration was performed. Alternatively, a therapeutic and / or preventive effect may be determined if the area of ​​the arthritis score graph in patients when the pharmaceutical composition is administered is significantly reduced compared to the area of ​​the arthritis score graph in patients when the negative control substance is administered. The above reductions may be, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% or less. It may be either one of those values, or it may be within the range of any two of those values.

[0107] The therapeutic and / or preventive effects of a pharmaceutical composition in systemic lupus erythematosus and lupus nephritis may be evaluated, for example, by organ damage, urinary protein levels, urinary protein score, survival rate, pathological evaluation score, serum autoantibody production, serum complement III and IV (C3 and C4) levels, or disease markers. Alternatively, these may be evaluated using the SLE disease activity index (SLEDAI), which scores these factors; the British Isles Lupus Assessment Group (BILAG) index, which compares disease activity to that of one month prior; the physician's global assessment (PGA), which assesses the patient's overall disease activity; or the SLE responder index (SRI), which comprehensively evaluates these factors. When evaluating by urinary protein levels, for example, if the patient's urinary albumin level or albumin / creatinine ratio significantly decreases after administration of the pharmaceutical composition compared to the non-administration period, then the therapeutic effect may be evaluated. Or / and it may be judged that there was a preventive effect. Or, the amount of urinary albumin or albumin / creatinine ratio in the patient at the time of administration of the pharmaceutical composition is equal to the amount at the time of administration of the negative control substance. Compared to the urinary albumin levels or albumin / creatinine ratio of the patients, there was a significant decrease. If this occurs, it may be determined that there was a therapeutic and / or preventive effect. Alternatively, administration of the pharmaceutical composition. The area under the graph of the patient's urinary albumin level or albumin / creatinine ratio at that time was measured by the non-injection. Compared to the urinary albumin level or albumin / creatinine ratio graph area at the time of administration, the results were significantly different. If the amount decreases, it may be judged that there was a therapeutic and / or preventive effect. Alternatively, if the amount of urinary albumin or albumin / creatinine ratio in the patient at the time of administration of the pharmaceutical composition is negative, The amount of urinary albumin or albumin / creatinine ratio in patients when administering control substances. If the decrease is significant, it may be judged that there was a therapeutic and / or preventive effect. The above decrease 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 effects of pharmaceutical compositions on graft-versus-host disease can be evaluated, for example, by improvements in characteristic organ findings or steroid dose reduction. For skin lesions, it can be evaluated by improvements in polymorphic skin atrophy and scleroderma-related sclerosing lesions; for liver lesions, by improvements in total bilirubin, ALP, AST, and ALT; and for renal lesions, by improvements in urinary protein levels and urinary The lubumin / creatinine ratio may also be used for evaluation. In that case, the treatment and / or preventive effect can be assessed. This can be done in the same way as in the case of SLE described above.

[0109] Here, “patient” includes humans and one or more non-human mammals (e.g., mice, guinea pigs, hamsters, rats, rodents, rabbits, pigs, sheep, goats, cattle, horses, cats, dogs, marmosets, monkeys, or chimpanzees). Patients may also have RA or Patients may be diagnosed with arthritis, systemic lupus erythematosus, lupus nephritis, or graft-versus-host disease. Patients may also be diagnosed with a disease treatable by inhibiting citrullination in cells. Furthermore, patients may be diagnosed with a disease treatable by inhibiting netosis in cells.

[0110] Here, “treatment” includes any treatment of a disease in a mammal, particularly a human, which includes inhibiting the symptoms of the disease, i.e., stopping its progression or eliminating the disease or symptoms, and reducing the symptoms of the disease, i.e., causing a regression of the disease or symptoms or delaying the progression of the symptoms.

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

[0112] Furthermore, "prevention of relapse" includes preventing recurrence of the aforementioned diseases in mammals, particularly humans, which are characterized by repeated remissions and relapses.

[0113] The administration form of the anti-PAD4 antibody or its antigen-binding fragment, or the pharmaceutical composition containing the anti-PAD4 antibody or its antibody fragment, is not particularly limited and can be administered to mammals, including humans, by any of the following routes of administration: oral administration, 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 manufactured by combining the antibody according to the present invention with a pharmaceutically acceptable carrier or the like. Dosage forms for parenteral administration include injectable preparations (e.g., intravenous infusion, intramuscular injection, subcutaneous injection, intradermal injection, intracerebral administration preparation, intraspinal administration preparation), topical preparations (e.g., ointments, poultices, lotions), suppositories, eye preparations, eye ointments, nasal drops, ear drops, liposomal preparations, etc. In particular, if it is desired to act directly on the central nervous system tissue, it can be continuously infused using a medical micropump with osmotic pressure, or it can be mixed with fibrin glue or the like to create a sustained-release preparation which can then be placed in the affected tissue.

[0115] For example, injectable formulations are usually prepared by dissolving antibodies in distilled water for injection, but solubilizers, buffers, pH adjusters, isotonic agents, analgesics, preservatives, stabilizers, etc., can be added as needed. They can also be prepared as lyophilized formulations for immediate use.

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

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

[0118] Examples of pharmacologically acceptable carriers include excipients, lubricants, binders, and disintegrants in solid formulations, or solvents, solubilizers, suspending agents, isotonic agents, buffers, and analgesics in liquid formulations. Furthermore, if necessary, appropriate amounts of common additives such as preservatives, antioxidants, colorants, sweeteners, adsorbents, and wetting agents may be used as needed.

[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, the type of disease, the severity of symptoms, the patient's age, sex, weight, the severity of the disease, pharmacological knowledge such as pharmacokinetics and toxicological characteristics, whether or not a drug delivery system is used, and whether or not it is administered as part of a combination of other drugs. Typically, for an adult (60 kg body weight), 1 to 5000 μg / day, preferably 10 to 2000 μg / day, and more preferably 50 to 2000 μg / day can be administered orally, and 1 to 5000 μg / day, preferably 5 to 2000 μg / day, and more preferably 50 to 2000 μg / day can be administered by injection, either in one dose or in several divided doses. For systemic parenteral administration, the dose can be 10 to 100,000 μg / kg per body weight, more preferably 100 to 50,000 μg / kg, and even more preferably 500 to 20,000 μg / kg, administered once a day, once a week, once a month, or 1 to 7 times a year. For local administration using an osmotic pump or the like, it can usually 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 these examples are not intended to limit the scope of the present invention.

[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 International Publication No. 2016 / 143753, was evaluated. An expression vector for mammalian cells expressing IgG was constructed by inserting the 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 for the light and heavy chains were obtained using sequence numbers 27 and 26 from the sequence listing, respectively. The above expression vector was introduced into ExpiCHO (LifeTechnologies) using the gene transfer reagent ExpiFectamine CHO Transfection Kit (LifeTechnologies). After gene transfer, the cells were cultured for 14 days. Afterward, the culture supernatant was obtained. Using an open column, IgG was extracted from the culture supernatant by affinity chromatography using Protein A resin (GE Healthcare, MabSelect SuRe). The following steps were taken: IgG bound to Protein A resin was washed with 6 Column Volume PBS (pH 7.2). Then, it was eluted with pH 4.0 Arg-Antibody Elution Buffer (Nacalai) and quickly neutralized to bring the pH to near neutral. To improve the purity of the purification, an AKTA prime plus (GE Healthcare) was used, and the IgG after Protein A column purification was eluted with CHT (ceramic hydroxyapatite Type II resin). Purified using (Bio-rad). IgG bound to CHT was eluted with a NaCl gradient. After collecting the target fraction, the solution was replaced with PBS (pH 7.4) using gel filtration chromatography with PD-10 (GE Healthcare). The test antibody was concentrated to a concentration of approximately 10 mg / mL, and 4 It was stored at ℃ or 37℃ for two weeks.

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

[0123] LC was performed with the following parameters. Equipment: 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 over 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 using the following parameters. Device: 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 proportion of post-translational modifications was calculated using the following method. First, peptides matching the sequence of the test antibody were extracted using Biopharma Lynx, and peptides for which MS / MS was available (MS / MS b / y ion found ≥ 2) were analyzed. The MS intensity and post-translational modifications of all detected peptides were analyzed. The percentage of post-translational modifications was calculated from the percentage of MS intensities of peptides containing [the specified substance].

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

[0128] Since cleavage occurs between asparagine and serine residues, a humanized anti-PAD4 antibody, G8ss, was created by substituting the 84th asparagine residue with a serine residue. After purification of the test antibody G8ss, it was fermented at 4°C. Peptide mapping was performed on the stored samples and the samples stored at 40°C for two weeks. As a result, no peptide cleaved between the 84th and 85th serine residues was detected under either storage condition. Therefore, it is considered that in humanized anti-PAD4 antibodies, the serine residue at the 84th position is more chemically stable. The binding affinity of the test antibodies G8 and G8ss to the hitoPAD4 protein was determined by measuring the dissociation constant (KD) in HBS-EP+ using a surface plasmon resonance spectrometer, Biacore T200 (GE Healthcare). Biotin-labeled hitoPAD4 protein was then measured using the Biotin Capture Kit (GE Healthcare). The protein was immobilized onto a sensor chip. The PAD4 protein was prepared to 0.25 μg / mL using HBS-EP+ to create a ligand solution. The ligand solution was added to a flow cell at a flow rate of 10 μL / min for 30 seconds, followed by the addition of HBS-EP+ for 60 seconds. Flow cell without ligand solution This was used as the reference cell. The test antibody was prepared from several hundred pM to several nM using HBS-EP+, and The lyte solution was used. The running buffer was used as the blank solution. The blank solution or analyte solution was added at a flow rate of 30 μL / min for 180 seconds using the single-cycle method, and upon dissociation... The interval was set to 1200 seconds. For analysis, the sensorgram of the reference cell was subtracted from the sensorgram of the ligand-added flow cell when either the blank solution or analyte solution was added. Furthermore, the sensorgram of the blank solution was subtracted from the sensorgram of the analyte solution-added flow cell. Binding parameters were calculated using a 1:1 binding model with Biacore T200 Evaluation software (GE Healthcare). The test antibody G8 bound to human PAD4 protein with a binding rate constant of 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 = 214 pM. on = 6.37 × 10 6 (1 / Ms), dissociation rate constant k off = 1.76 × 10 -3 (1 / s), dissociation constant KD=276 Binding occurred due to the binding affinity of pM. Therefore, in the humanized anti-PAD4 antibody, the 84th position is serine. It was found that substitution of residues did not change the binding affinity to human PAD4.

[0129] <Example 2> Determination of amino acid substitution sites to improve the complementarity determination region To improve the affinity and functionality of the humanized anti-human PAD4 antibody G8 to human PAD4, Fab(min) (This means that the subform is Fab, and the same notation will be used below) Phase by phage display The complementarity-determining regions were improved. This improvement was carried out in two stages: in the first stage, a single amino acid substitution that could improve affinity to human PAD4 was 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-step PCR reaction using site-directed mutagenesis PCR and overlap extension PCR was performed to identify the six complementarity-determining regions of the antibody (LCDR1). A comprehensive single-amino acid substitution mutant library was constructed by substituting one amino acid residue each of the 20 native amino acids that make up (LCDR2, LCDR3, HCDR1, HCDR2, HCDR3). Recombinant phyto-PAD4 protein (biotin-labeled PAD4) was bay-displayed using the Fab phage display method (Fujino et al., Biochem. Biophys. Res. Commun., 2012 Vol. 428(3), p395). As a first step, we repeated enrichment of a comprehensive single-amino acid substitution mutant library several times. Before enrichment The nucleotide sequences of the light chain and heavy chain variable regions of each clone in the library (immediately after construction) and the enriched library were analyzed using a next-generation sequencer (Ion GeneStudio S5 System). First, several million reads of sequence data were 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 pre-enrichment library... The magnification of the change in the frequency of all single-amino acid substitution mutants in the concentrated library (concentration) We calculated the (concentration ratio) and used the magnitude of the concentration ratio obtained by library concentration as an indicator to determine a single amino acid substitution that is considered useful for improving affinity to the hitoPAD4 protein. At that time, one amino acid substitutions (lysine and arginine) that may affect the physical properties of antibodies The data was excluded. Finally, the total number of these single-amino acid substitutions and their distribution in the amino acid sequence. Taking this into consideration, we will determine the location where amino acid substitutions will be introduced in the custom library to be built in the second stage. did.

[0130] In parent clone G8, the second asparagine in LCDR2 (sequence number 5 in the sequence listing), LCDR3 ( We decided to introduce amino acid substitutions at the fourth aspartic acid position in sequence number 25 of the sequence listing, at the eleventh tyrosine, twelfth glycine, thirteenth alanine, fourteenth alanine, fifteenth valine, and seventeenth glycine position in HCDR2 (sequence number 24 of the sequence listing), and at the first alanine position in HCDR3 (sequence number 11 of the sequence listing).

[0131] <Example 3> Creation of a humanized anti-human PAD4 antibody with an improved complementarity-determining region First, a custom library for improving complementarity determination regions was designed by combining several of the above useful amino acid substitutions aimed at improving affinity. Next, a vector for displaying the parent clone G8 Fab phage was constructed by inserting stop codons into LCDR3 and HCDR2, and the Fab phage... Site-directed mutagenesis using the Kunkel method with display vectors as templates (Fellouseet By performing the procedure described in (al., J. Mol. Biol. 2007 Vol. 373, p924), the complementarity determination region is as described above. Based on the design, a custom library for improving randomized complementarity-determining regions was constructed. Using the hist-PAD4 protein (His tag PAD4, GST-biotin-PAD4) as a bait, library enrichment was repeated several times using Fab phage display. Since PAD4 may change structure depending on the presence or absence of calcium ions, selection and enrichment studies were conducted in the presence and absence of calcium chloride.

[0132] The recombinant protein used as a bait was prepared as follows: hiPAD4 (residue 1 or By inserting residue 663) with a GST tag-Avi tag attached to its N-terminus into pGEX-6P-1, An expression vector was constructed and recombinant protein was prepared. E. coli strain BL21(DE3)pLysS containing the expression vector was pre-cultured in 5 mL of LB medium, then 1 mL of the pre-culture was inoculated into 50 mL of LB medium and incubated at 37°C. After culturing until the logarithmic growth phase (OD600 = 1.0), expression culture was performed with 100 μM IPTG at 18°C / 200 rpm for approximately 18 hours. After washing the collected cells, they were lysed by sonication 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 found in GS4B resin (GE Purified using Healthcare, and GST processed using Precision Protease (GE Healthcare). The device was cut and the AviTag Human PAD4 was recovered.

[0133] The Fab library was enriched for 4-6 rounds under conditions of both the presence and absence of calcium chloride. Each included clone was cloned, and 384 clones were randomly selected from each condition. The rows were identified using a sequencer. All 384 clones of Fab were full-length antibodies (human IgG1 / H). Expression DNA was fragmented into λ) and introduced into Expi293 (LifeTechnologies) using the gene transfer reagent ExpiFectamine 293 Transfection Kits (LifeTechnologies). After culturing for 5 days following gene transfer, the culture supernatant was obtained. The test antibody (IgG) (meaning its molecular form is IgG, the same notation applies hereafter) was purified from the culture supernatant by affinity chromatography using Protein A resin (GE Healthcare, PreDictor MabSelect SuRe LX). The purified test antibody solution was replaced with PBS pH 7.4 using a dialysis device (Merck, D-Tube96 Dialyzer).

[0134] Antigen binding of the test antibody was confirmed by surface flasmon resonance (SPR). For the SPR experiment, a Biacore T200 surface flasmon resonance spectrometer (GE Healthcare) was used to confirm antigen binding of the test antibody in running buffer HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% (v / v) Surfactant P20, pH 7.4). A Biotin Capture Kit (GE Healthcare) was also used to confirm antigen binding. Otin-labeled hitoPAD4 protein was immobilized on a sensor chip. Pre- The PAD4 protein was prepared at a concentration of 4 μg / mL to form the ligand solution. The ligand solution was added to a flow cell at a flow rate of 10 μL / min for 30 seconds, followed by the addition of HBS-EP+ for 60 seconds. A flow cell without the additive was used as the reference cell. The test antibody was prepared to 10 nM using HBS-EP+. The solution was prepared and used as the analyte solution. The running buffer was used as the blank solution. The blank solution or the analyte solution was added at a flow rate of 30 μL / min for 120 seconds to obtain the bound phase sensor. Grams were obtained. Next, the dissociation phase sensor gram was obtained by adding running buffer for 180 seconds. The following results were obtained. 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. Furthermore, the sensorgram of the blank solution was subtracted from the sensorgram of the analyte solution added and used for analysis. Binding signals and non-specific adsorption were confirmed using Biacore T200 Evaluation software v3.1 (GE Healthcare).

[0135] From among the clones with improved complementarity-determining regions, a clone was selected that showed improved affinity for the hitoPAD4 protein compared to G8 and exhibited less non-specific adsorption to the sensor chip, and G8 was selected in Example 4 and later. Five clones (G8, Lib2(4R)-1-47, Lib1(6R)-1-39, Lib1(6R)-2-37, Lib2(5R)-2-25) were selected to proceed to higher-level evaluations. The CDR sequences of the five clones are shown in Table 1. The binding affinity of the five clones to human PAD4 is 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 achieve concentrations of 47, 0.82, 0.27, and 0.09 nM. The mixtures were incubated at 37°C for 1 hour. Afterward, BAEE (benzoylarginine ethyl ester) was added while stirring, and then CaCl2 was added and thoroughly mixed (final concentration of BAEE was 10 mM, final concentration of calcium ions was 10 mM). After incubating this solution at 37°C for 3 hours, the citrullinated BAEE The n residue was colorimetrically determined using a mixture containing 2,3-butanedione monooxime and thiosemicarbazide. The test antibodies (G8, Lib2(4R)-1-47, Lib1(6R)-1-39, Lib1(6R)-2-37, Lib2(5R)-2-25) inhibited the enzyme 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 For each of the following, the 84th asparagine was replaced with serine, resulting in G8ss, 2-1-47, 3-1-39, 3-2-37, and 4-2-25, respectively. Expression vectors for mammalian cells expressing IgG were constructed by inserting 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) downstream of the CMV promoter. For the light chain DNA sequences of each clone, the DNA sequences encoding sequence numbers 27, 17, 19, 21, and 23 in the sequence listing were used, respectively, and the heavy chain DNA sequences were also used. The DNA sequences used for each strand were those encoding sequence numbers 11 and 12 in the sequence listing. The expression vector was introduced into ExpiCHO (LifeTechnologies) using the ExpiFectamine CHO Transfection Kit (LifeTechnologies). The cells were cultured for 14 days after gene transfer. After incubation, the culture supernatant was obtained. AKTA pure 25M (GE Healthcare) was used to analyze Protein A Affinity chromatography using resin (JSR Life Sciences, Amsphere A3 column) IgG was purified from the culture supernatant using a filtration method. IgG bound to Protein A resin was washed with 6 Column Volume 100 mM sodium carbonate buffer (pH 11.0), then with 6 Column Volume PBS (pH 7.2) (3×). It was then eluted with pH 3.5 Gly-HCl buffer and rapidly neutralized to bring the pH to near neutral. To improve the purity, the IgG purified using the Protein A column was purified using CHT (ceramic hydroxyapatite Type I resin) (Bio-rad). IgG bound to CHT was eluted with a NaCl gradient, the target fraction was collected, and the solution was replaced with PBS (pH 7.4) using dialysis. The purification of the test antibodies (G8ss, 2-1-47, 3-1-39, 3-2-37, 4-2-25) was also performed. Peptide mapping was performed on samples stored at 4°C after preparation. As a result, the 84th peptide was identified. No peptide cleavage was detected between the serine residue at position 1 and position 85 of the serine residue.

[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 the hi-PAD4 protein was determined by measuring the dissociation constant (KD) in HBS-EP+ using a surface plasmon resonance spectrometer, Biacore T200 (GE Healthcare). A Biotin Capture Kit (GE Healthcare) was used. The biotin-labeled PAD4 protein was immobilized on a sensor chip using HBS-EP+. The PAD4 protein was prepared to a concentration of 0.5 μg / mL using the provided solution to obtain the ligand solution. The ligand solution was added to a flow cell at a flow rate of 10 μL / min for 30 seconds, followed by the addition of HBS-EP+ for 60 seconds. A flow cell without the addition of Gand solution was used as the reference cell. HBS-EP+ was used to test the antibody The ligand was prepared to a concentration of several hundred pM to several nM to create the analyte solution. The running buffer was used as the blank solution. The blank solution or analyte solution was added at a flow rate of 30 μL / min for 180 seconds using the single-cycle method, and the dissociation time was 1200 seconds. The sensorgram of the reference cell was subtracted from the sensorgram of the flow cell to which the ligand was added when the blank solution or analyte solution was added, and the sensorgram of the cell to which the analyte solution was added was then used to determine the blank solution. The sample weights obtained by subtracting the sensorgrams with the rank solution added were 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 bound to the human PAD4 protein with a binding rate constant of k on = 6.15 × 10 6 (1 / Ms), dissociation rate constant k off = 1.22 × 10 -3 The antibody bound to the PAD4 protein with a binding affinity of 1 / s and a dissociation constant KD = 199 pM. The dissociation constants for the PAD4 protein of antibodies 2-1-47, 3-1-39, 3-2-37, and 4-2-25, which had improved complementarity-determining regions, were 31, 48, 73, and 20 pM, respectively.

[0140] [Table 3]

[0141] <Example 5> Evaluation of the storage stability of antibodies One indicator of antibody storage stability is the retention of antigen-binding activity. It is well known that storage stability varies greatly depending on the antibody. For example, DiCara et al., mAbs, 2018 Vol. 10(7), p1073, compares the antigen-binding ability of antibodies stored at 4°C and 40°C, but the relative antigen-binding activity of antibodies stored at 40°C is lower than that of antibodies stored at 4°C. The relative antigen-binding activity ranges from less than 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 citrate, 150 mM NaCl, pH 6.3) at a concentration of approximately 10 mg / mL. It was dissolved and stored at 4°C or 40°C for 4 weeks. To investigate the presence or absence of antigen-binding ability after storage, antigen-binding activity was measured using a surface flame resonance spectrometer, Biacore T200 (GE Healthcare). Test antibodies stored frozen, or stored at 4°C or 40°C for 4 weeks, were prepared to 10 μg / mL using running buffer HBS-EP+. The test antibody was dissolved in flow cell 2 using Series S Sensorchip Protein A (GE Healthcare). The liquid was added at a flow rate of 10 μL / min for 60 seconds and immobilized on the sensor tip. Flow cell 1 is This was designated as a reference cell. The amount of binding 55 seconds after the completion of antibody addition in flow cell 2 was measured in the flow cell. The amount of antibody bound was calculated by subtracting it from the amount of binding of L1. Next, PAD4 (Cayman Chemical Company, Cat. 10500) was prepared 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 conjugated phase. Flowsex Subtract the amount of binding in flow cell 1 from the amount of binding in flow cell 1 five seconds before the end of adding the analyte solution in flow cell 2. This was used to determine the antigen binding amount. Subsequently, running buffer was added for 180 seconds to measure the dissociation phase sensor. Grams were obtained. Measurements were performed at 25°C. Antibody binding and antigen binding amounts were calculated using a data analysis program (GE Healthcare, Biacore T200 Evaluation Software v3.1), and the relative antigen binding amount was obtained by dividing the antigen binding amount under each condition by the antibody binding amount. There was no difference in the relative antigen binding amount of the test antibody stored frozen and the test antibody stored at 4°C. 4°C, 4 weeks The relative antigen binding amount of various test antibodies stored for a period of time and the relative antigen binding amount of test antibodies stored at 40°C for 4 weeks. The total amount was compared to determine the antigen-binding activity. As shown in Table 4, the antigen-binding activity of G8ss was 82.2% after storage at 40°C for 4 weeks. 2-1-47, 3-1-39, 3-2-37, and 4-2-25 retained over 90% antigen-binding activity after storage at 40°C for 4 weeks. For example, as taught in Pisupati et al., mAbs, 2017 Vol. 9(7), p1197, the antibody drug Remicade® retained its antigen-binding activity even after storage at 40°C for one month. The properties are retained by more than 80%. In addition, the stability of the biological activity of the antibody is maintained during the preparation of the antibody formulation. It is desirable to retain 80% or more, preferably 90% or more, of the biological activity of the antibody compared to the biological activity of the antibody (see, for example, International Publication WO2003 / 018056). Based on 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 human PAD4 enzyme inhibitory activity in vitro The human PAD4 enzyme inhibitory activity of the test antibodies (G8ss, 3-2-37, 3-1-39, 4-2-25, 2-1-47) was evaluated. 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. To achieve this, a 50 mM solution containing human PAD4 (final concentration 10 nM), 1 mM EDTA, 1 mM DTT, and 150 mM NaCl is used. HEPES buffer (pH 7.4) was mixed in. After incubation at 37°C for 1 hour, BAEE (benzoylarginine ethyl ester) was added while stirring, and then CaCl2 was added and thoroughly mixed. Mixed (final concentration of BAEE was 10 mM, final concentration of calcium ions was 10 mM). After incubation of this solution at 37°C for 3 hours, the citrulline residues of the citrullinated BAEE were removed from 2,3-buta Colorimetric quantification was performed using a mixture containing ion monooxime and thiosemicarbazide. The test antibodies (G8ss, 3-2-37, 3-1-39, 4-2-25, 2-1-47) inhibited the enzyme 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 a model of rheumatoid arthritis (RA) and arthritis. The CAIA model mice were prepared according to the protocol for the mouse arthritis induction antibody cocktail (Chondrex Inc., Cat. 53040). A summary of the experimental conditions is shown in the figure. As shown in 1, on day 0, a mixture of anti-collagen antibodies (1.5 mg) was intravenously administered into the tail vein of 9-week-old female Balb / c mice (6 mice / group). On day 3, 25 μg LPS (an inflammation-inducing substance) was administered. It was administered intraperitoneally. Four hours before LPS administration on the third day, the test antibody or control antibody (human IgG1) was administered intravenously. The drug was administered orally (15 mg / kg). From days 3 to 10, the arthritis score was evaluated according to (i) to (iii) below. (i) The evaluation sites were each finger, back of hand, and joint of the limbs. (ii) The arthritis score was assigned according to Table 5. (iii) The arthritis score was expressed as the average of the total values ​​for each finger, back of hand, and joint of the limbs (maximum value was 16 / mouse). The results of the arthritis score evaluation are shown in Figure 1. As can be seen from these results, all tested antibodies had a high therapeutic effect on rheumatoid arthritis (RA).

[0145] [Table 5]

[0146] <Example 8> CIA Model The efficacy of the test antibody 3-2-37 was evaluated using a collagen-induced arthritis (CIA) model mouse. The CIA model mice were created by feeding 5-7 week old female DBA / 1 mice with bovine type II collagen and Froy. The immunization process involves administering the adjuvant twice: once on the first day and again 21 days after the initial immunization. The test antibody was administered intratail vein at doses of 0.3 mg / kg, 1 mg / kg, or 3 mg / kg to mice that developed arthritis 29 days after the initial immunization (9 mice per group). The same dose of 3-2-37 was administered again 36 and 43 days after the initial immunization. The arthritis score was evaluated according to (i) to (iii) below: (i) The evaluation sites were each finger, back of the hand, and joint of the limb. (ii) The arthritis score was assigned according to Table 6. (iii) The arthritis score was expressed as the average of the total values ​​for each finger, back of the hand, and joint of the limb (maximum value was 16 / mouse). The evaluation results for arthritis scores are shown in Figure 2. As can be seen from these results, 3-2-37 significantly reduced the mean arthritis score compared to the control antibody group in the latter half of the study at all the doses set (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 mouse was created by transferring splenocytes from an 8-9 week old female DBA / 2 mouse into an 8-week old female B6D2F1 mouse. This mouse exhibits lupus nephritis-like renal impairment that occurs in SLE patients. Add. The test antibody was administered intraperitoneally at a dose of 3 mg / kg or 30 mg / kg on the day before splenocyte transplantation, 2 weeks later, 4 weeks later, and 6 weeks later (13 mice per group). From 2 weeks to 8 weeks after splenocyte transplantation, urine was collected by compressing the lower abdomen of the mice once a week, and the protein concentration and creatinine concentration in the urine were measured. The urinary protein concentration 8 weeks after splenocyte transplantation was scored according to Table 7. The evaluation results of the urinary protein score are shown in Figure 3. As can be seen from this result, a significant decrease in the urinary protein score was observed in the 30 mg / kg administration group of the test antibody 3-2-37 compared with the control antibody administration group (Shirley-Williams multiple comparison test, *p < 0.025). In addition, an individual with a protein / creatinine ratio in urine less than 3 (Pro / Cre < 3) was defined as an unaffected individual, and the results of graphing the incidence rates of the 30 mg / kg administration group of the test antibody 3-2-37 and the control antibody administration group over time are shown in Figure 4. The median of the unaffected period in the test antibody administration group was 7 weeks, while the median of the unaffected period in the control antibody administration group was 4 weeks, and a significant extension of the unaffected 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 in anti-PAD4 antibodies Lib1(6R)-1-39, 3-1-39 Sequence ID 9: Amino acid sequence of HCDR2 in anti-PAD4 antibodies Lib1(6R)-2-37, 3-2-37 Sequence ID 10: Amino acid sequence of HCDR2 of anti-PAD4 antibodies Lib2(5R)-2-25, 4-2-25 Sequence ID 11: Amino acid sequences of HCDR3 for anti-PAD4 antibodies G8, G8ss, Lib2(4R)-1-47, 2-1-47, Lib1(6R)-1-39, 3-1-39, Lib2(5R)-2-25, 4-2-25. SEQ ID NO: 12: Amino acid sequence of HCDR3 of anti-PAD4 antibodies Lib1(6R)-2-37, 3-2-37 Sequence ID 13: LCDR3 of anti-PAD4 antibodies Lib2(4R)-1-47, 2-1-47, Lib1(6R)-1-39, 3-1-39 amino acid sequence Sequence ID 14: Amino acid sequence of LCDR3 in anti-PAD4 antibodies Lib1(6R)-2-37, 3-2-37 Sequence ID 15: Amino acid sequence of LCDR3 in anti-PAD4 antibodies Lib2(5R)-2-25, 4-2-25 SEQ ID NO: 16: Full-length heavy chain amino acid sequence of anti-PAD4 antibody 2-1-47 SEQ ID NO: 17: Full-length light chain amino acid sequence of anti-PAD4 antibody Lib2(4R)-1-47, 2-1-47 SEQ ID NO: 18: Full-length heavy chain amino acid sequence of anti-PAD4 antibody 3-1-39 SEQ ID NO: 19: Full-length light chain amino acid sequence of anti-PAD4 antibody Lib1(6R)-1-39, 3-1-39 SEQ ID NO: 20: Full-length heavy chain amino acid sequence of anti-PAD4 antibody 3-2-37 SEQ ID NO: 21: Full-length light chain amino acid sequence of anti-PAD4 antibody Lib1(6R)-2-37, 3-2-37 SEQ ID NO: 22: Full-length heavy chain amino acid sequence of anti-PAD4 antibody 4-2-25 Sequence ID 23: Full-length light chain amino acid sequence of anti-PAD4 antibody Lib2(5R)-2-25, 4-2-25 Sequence ID No. 24: Amino acid sequence of HCDR2 in anti-PAD4 antibodies G8 and G8ss SEQ ID NO: 25: Amino acid sequence of LCDR3 in anti-PAD4 antibodies G8 and G8ss SEQ ID NO: 26: Full-length heavy chain amino acid sequence of anti-PAD4 antibody G8 SEQ ID NO: 27: Full-length light chain amino acid sequence of anti-PAD4 antibodies G8 and G8ss SEQ ID NO: 28: Full-length heavy chain amino acid sequence of the anti-PAD4 antibody G8ss SEQ ID NO: 29: Amino acid sequence of PAD4 SEQ ID NO: 30: The nucleotide sequence encoding the full-length heavy chain of the anti-PAD4 antibody G8. Sequence ID 31: The nucleotide sequence encoding the full light chain of anti-PAD4 antibodies G8 and G8ss. Sequence ID 32: The nucleotide sequence encoding the full-length heavy chain of the anti-PAD4 antibody 2-1-47. Sequence ID 33: The nucleotide sequence encoding the full light chain of the anti-PAD4 antibody 2-1-47. SEQ ID NO: 34: The nucleotide sequence encoding the full heavy chain of the anti-PAD4 antibody 3-1-39. Sequence ID 35: The nucleotide sequence encoding the full light chain of the anti-PAD4 antibody 3-1-39. SEQ ID NO: 36: The nucleotide sequence encoding the full-length heavy chain of the anti-PAD4 antibody 3-2-37. Sequence ID 37: The nucleotide sequence encoding the full light chain of the anti-PAD4 antibody 3-2-37. Sequence ID 38: The nucleotide sequence encoding the full heavy chain of the anti-PAD4 antibody 4-2-25. Sequence ID 39: The nucleotide sequence encoding the full light chain of the anti-PAD4 antibody 4-2-25. Sequence ID 40: The nucleotide sequence encoding the full-length heavy chain of the anti-PAD4 antibody G8ss. [Industrial applicability]

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

Claims

[Claim 1] The invention described in the present specification.

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