WO2019131769A1 - Novel anti-pad4 antibody - Google Patents

Novel anti-pad4 antibody Download PDF

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WO2019131769A1
WO2019131769A1 PCT/JP2018/047871 JP2018047871W WO2019131769A1 WO 2019131769 A1 WO2019131769 A1 WO 2019131769A1 JP 2018047871 W JP2018047871 W JP 2018047871W WO 2019131769 A1 WO2019131769 A1 WO 2019131769A1
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antibody
pad4
present
amino acid
seq
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PCT/JP2018/047871
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French (fr)
Japanese (ja)
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智 金澤
佐藤　衛
道之 山田
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公立大学法人名古屋市立大学
公立大学法人横浜市立大学
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Priority to JP2019562109A priority Critical patent/JPWO2019131769A1/en
Publication of WO2019131769A1 publication Critical patent/WO2019131769A1/en
Priority to JP2023221077A priority patent/JP2024038166A/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to anti-PAD4 antibodies.
  • PAD4 Peptidylarginine deiminase 4
  • PAD4 is known as an enzyme involved in citrullination of arginine in proteins. This citrullination is important for protein structure and reaction because it is a reaction in which the most basic arginine among the amino acids constituting the protein is converted to neutral citrulline.
  • Non-Patent Document 1 reports that there is a correlation between the onset of RA and the single nucleotide polymorphism of PAD4 gene.
  • Non-Patent Document 2 reports that an anti-PAD4 antibody was used to diagnose RA.
  • Patent Document 1 describes an attempt to suppress RA by administering a mixture of four anti-PAD4 antibodies to mice.
  • Patent Document 2 describes that RA or arthritis was suppressed by administering an anti-PAD4 antibody that binds to a specific epitope to mice.
  • active PAD4 and "inactive PAD4" are present.
  • the active form binds Ca 2+ and is also called Ca 2+ -linked PAD4.
  • Non active form not bound to Ca 2+ also referred to as Ca 2+ unconjugated PAD4.
  • Patent Document 3 exists as a report on PAD4 and Ca 2+ .
  • Patent Document 3 describes an attempt to measure the amount of PAD4 in the serum of healthy subjects and rheumatoid arthritis patients by ELISA using an anti-PAD4 antibody.
  • an appropriate measurement value was not obtained using untreated serum in performing ELISA, it was described that an appropriate measurement value was obtained using EDTA-treated serum. It is done. At this time, as a result of Ca 2+ being trapped in EDTA, it is considered that non-activated PAD 4 reacted with the anti-PAD 4 antibody.
  • the present invention has been made in view of the above circumstances, and an object thereof is to provide an anti-PAD4 antibody that binds to active PAD4, or an anti-PAD4 antibody that inhibits the function of active PAD4.
  • the present inventors have surprisingly found that an anti-PAD4 antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 (corresponding to positions 633-644 of PAD4), as described in the examples below. Were found to bind to activated PAD4 and to inhibit the function. And based on the result, the present invention was completed.
  • an anti-PAD4 antibody that binds to active PAD4.
  • active PAD4 can be detected.
  • This antibody may be a functional inhibition antibody of active PAD4.
  • an anti-PAD4 antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1, or an anti-PAD4 antibody that specifically binds to positions 634-644 of human PAD4. Be done. This antibody can be used to detect or inhibit the function of active PAD4.
  • a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1.
  • FIG. 1 is a diagram showing the amino acid sequence of the variable region of the antibody according to the example.
  • FIG. 2 is a diagram showing the nucleotide sequence of the variable region of the antibody according to the example.
  • FIG. 3 is a diagram showing the nucleotide sequence of the variable region of the antibody according to the example.
  • FIG. 4 is a graph showing the results of examining the percentage inhibition of active PAD 4 by the antibodies according to the examples.
  • One embodiment of the present invention is a novel anti-PAD4 antibody.
  • This antibody may be, for example, an anti-PAD4 antibody that binds to active PAD4. With this antibody, for example, active PAD4 can be detected.
  • This antibody may be, for example, a function-inhibiting antibody of active PAD4.
  • This antibody can be used, for example, to inhibit the citrullinating activity of active PAD4.
  • this antibody can be used, for example, to treat rheumatoid arthritis (RA) or arthritis.
  • RA rheumatoid arthritis
  • the anti-PAD4 antibody is, for example, an anti-PAD4 antibody that specifically binds to the peptide shown by SEQ ID NO: 1, or an anti-PAD4 antibody that specifically binds to positions 634-644 of human PAD4. It may be. With this antibody, for example, active PAD4 can be detected. Moreover, if this antibody is used, for example, the citrullinating activity of active PAD 4 can be inhibited. Also, this antibody can be used, for example, to treat RA or arthritis. This therapeutic method has small side effects due to the use of antibodies and is excellent in terms of safety.
  • PAD4 is generally known as an enzyme involved in the citrullination of arginine in proteins. Details such as the amino acid sequence of PAD4 can be viewed from the website such as NCBI (National Center for Biotechnology Information) or HGNC (HUGO Gene Nomenclature Committee). The accession number of PAD 4 described in NCBI is, for example, NP_036519.2. The amino acid sequence of human PAD4 is, for example, SEQ ID NO: 2. The origin of PAD4 is not limited as long as it has PAD4 activity. In the present specification, PAD4 may be active PAD4 or inactive PAD4. In the present specification, active PAD4 may be Ca 2+ -bound PAD4, and non-active PAD 4 may be non-Ca 2+ -bound PAD4. Active PAD4 contains PAD4 activated by binding of Ca 2+ .
  • the "anti-PAD4 antibody” comprises an antibody having binding to PAD4.
  • the method for producing this anti-PAD4 antibody is not particularly limited, and for example, it may be produced by immunizing a mammal or birds with a peptide consisting of the amino acid sequence shown by SEQ ID NO: 1.
  • the anti-PAD4 antibody may be an antibody that inhibits the function of PAD4. Functions include, for example, citrullinating activity.
  • an anti-PAD4 antibody that binds to active PAD4 comprises an antibody that has binding to non-active PAD4.
  • the anti-PAD4 antibody according to one embodiment of the present invention may be a monoclonal antibody. If it is a monoclonal antibody, it can be made to act on PAD4 efficiently compared with a polyclonal antibody. From the viewpoint of efficiently producing an anti-PAD4 monoclonal antibody having a desired effect, it is preferable to immunize chickens with PAD4.
  • PAD4 used as an antigen includes PAD4 full-length or PAD4 peptide fragment, unless otherwise specified.
  • the anti-PAD4 antibody according to one embodiment of the present invention is not limited to a full-length antibody, and includes an antibody fragment having PAD4 binding activity (hereinafter sometimes referred to as "antigen-binding antibody fragment").
  • Antigen-binding antibody fragments have effects such as increased stability or antibody production efficiency.
  • the anti-PAD4 antibody according to one embodiment of the present invention may be a fusion protein.
  • This fusion protein may be one in which a polypeptide or an oligopeptide is linked to the N or C terminus of PAD4.
  • the oligopeptide may be a His tag.
  • the fusion protein may also be a fusion of mouse, human or chicken antibody partial sequences. Such fusion proteins are also included in one form of the anti-PAD4 antibody according to the present embodiment.
  • the anti-PAD4 antibody may be an antibody obtained through the step of immunizing PAD4 to a chicken.
  • it may be an antibody having a CDR set of an antibody obtained through the step of immunizing chicken with PAD4.
  • the CDR set is a set of heavy chain CDRs 1, 2, 3 and light chain CDRs 1, 2, and 3.
  • the anti-PAD 4 antibody has a KD (M) of, for example, 9.9 ⁇ 10 ⁇ 8 , 9.0 ⁇ 10 ⁇ 8 , 8.0 ⁇ 10 ⁇ 8 , 7.0 ⁇ 10 ⁇ 8 , 6.0 ⁇ 10 ⁇ 8. , 5.0 ⁇ 10 ⁇ 8 , 4.0 ⁇ 10 ⁇ 8 , 3.0 ⁇ 10 ⁇ 8 , 2.0 ⁇ 10 ⁇ 8 , or 1.0 ⁇ 10 ⁇ 8 or less, any of which is within the range of two values It is also good.
  • the KD (M) is preferably 9.0 ⁇ 10 -8 or less.
  • the anti-PAD4 antibody according to one embodiment of the present invention may be an antibody that binds to wild type or mutant form of PAD4. Variants include those that result from differences in DNA sequences between individuals, such as SNPs.
  • the amino acid sequence of wild-type or mutant PAD 4 is homologous to the amino acid sequence shown in SEQ ID NO: 2, with positions 633-644 conserved and preferably 95% or more, particularly preferably 98% or more homologous May be
  • the anti-PAD4 antibody according to one embodiment of the present invention may be an antibody having binding ability to or not having mouse PAD4.
  • An anti-PAD4 antibody according to one embodiment of the present invention is an antibody having binding to wild-type PAD4 and having no binding to mutant PAD4 having a mutation at positions 633-644 of human PAD4. It may be The "non-binding" may be substantially non-binding.
  • the anti-PAD4 antibody according to one embodiment of the present invention is a step of selecting an antibody showing significant reactivity to wild-type PAD4, or binding to mutant PAD4 having a mutation at position 633-644 of human PAD4. And b) selecting an antibody not showing.
  • an antibody that specifically binds to positions 633-644 of human PAD4 has an antibody that binds to other amino acid residues, or an antibody that binds to other amino acid residues as long as it has binding to positions 633-644 of human PAD4. It contains the antibody which does not have.
  • the antibody that specifically binds to a specific site may be an antibody that recognizes a specific site.
  • the anti-PAD4 antibody according to one embodiment of the present invention may be an antibody that specifically binds to an epitope including positions 2, 6, 7 and 8 of the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1.
  • the anti-PAD4 antibody according to one embodiment of the present invention may be an antibody that specifically binds to an epitope including positions 634, 638, 639, and 640 of human PAD4.
  • an "antibody” comprises a molecule or population thereof capable of specifically binding to a particular epitope on an antigen.
  • the antibody may also be a polyclonal antibody or a monoclonal antibody.
  • the form of the antibody is not particularly limited. For example, a full-length antibody (an antibody having a Fab region and an Fc region), an Fv antibody, a Fab antibody, an F (ab ') 2 antibody, an Fab' antibody, a single chain antibody (for example, scFv) ), DsFv, antigen binding peptide, antibody-like molecule, chimeric antibody, mouse antibody, chicken antibody, humanized antibody, human antibody, or an equivalent (or equivalent) thereof including.
  • Antibodies also include modified or unmodified antibodies.
  • the modified antibody may be bound to an antibody and various molecules such as polyethylene glycol.
  • Modified antibodies can be obtained by chemically modifying antibodies using known techniques.
  • the amino acid sequence, class, or subclass of the antibody may be derived from, for example, humans, mammals other than humans (eg, rats, mice, rabbits, cattle, monkeys, etc.), or birds (eg, chickens).
  • the antibody class is not particularly limited, and may be, for example, IgM, IgD, IgG, IgA, IgE, or IgY.
  • the antibody subclass is not particularly limited, and may be, for example, IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.
  • Antibodies also include, for example, isolated antibodies, purified antibodies, or recombinant antibodies. The antibodies can also be used, for example, in vitro or in vivo.
  • the "polyclonal antibody” administers an immunogen containing an antigen of interest to, for example, mammals (eg, rats, mice, rabbits, cattle, monkeys etc.) or birds (eg, chickens) etc. It is possible to generate by Administration of immunogen may be infused with one or more immunizing agents, or an adjuvant. Adjuvants may also be used to increase the immune response, and may include Freund's adjuvant (complete or incomplete), mineral gel (such as aluminum hydroxide), or surfactant (such as lysolecithin), etc. . Immunization protocols are known in the art and may be performed by any method that elicits an immune response, depending on the host organism chosen (Protein Experimental Handbook, Yodosha (2003): 86-91 .).
  • monoclonal antibodies include antibodies wherein individual antibodies making up a population respond to substantially the same epitope.
  • the individual antibodies that make up the population may be antibodies that are substantially identical (naturally occurring mutations are acceptable).
  • Monoclonal antibodies are highly specific and differ from normal polyclonal antibodies, which typically include different antibodies corresponding to different epitopes.
  • the method for producing a monoclonal antibody is not particularly limited, but, for example, a method similar to the hybridoma method as disclosed in "Kohler G, Milstein C., Nature. 1975 Aug 7; 256 (5517): 495-497.” It may be produced by Alternatively, monoclonal antibodies may be made by methods similar to recombinant methods as described in US Pat. No.
  • the monoclonal antibody may be “Clackson et al., Nature. 1991 Aug 15; 352 (6336): 624-628.” Or “Marks et al., J MoI Biol. 1991 Dec 5; 222 (3): 581. It may be isolated from phage antibody libraries using methods similar to the techniques as described in “-597.” Alternatively, it may be prepared by the method described in "Protein Experiment Handbook, Yodosha (2003): 92-96.”
  • an "Fv antibody” is an antibody that comprises an antigen recognition site. This region contains a dimer of one heavy chain variable domain and one light chain variable domain by noncovalent binding. In this configuration, the three CDRs of each variable domain can interact to form an antigen binding site on the surface of the VH-VL dimer.
  • the “Fab antibody” is, for example, about N-terminal half of the H chain and the entire L chain among fragments obtained by treating an antibody containing the Fab region and the Fc region with the proteolytic enzyme papain Is an antibody linked through some disulfide bonds.
  • Fab can be obtained, for example, by treating the anti-PAD4 antibody according to the above-mentioned embodiment of the present invention containing a Fab region and an Fc region with the proteolytic enzyme papain.
  • F (ab ′) 2 antibody is, for example, a fragment corresponding to Fab in a fragment obtained by treating an antibody containing Fab region and Fc region with proteolytic enzyme pepsin
  • Antibodies that contain F (ab ′) 2 can be obtained, for example, by treating the anti-PAD 4 antibody according to the above-described embodiment of the present invention containing a Fab region and an Fc region with the proteolytic enzyme pepsin.
  • it can be produced by thioether bond or disulfide bond of the following Fab ′.
  • the “Fab ′ antibody” is, for example, an antibody obtained by cleaving a disulfide bond in the hinge region of F (ab ′) 2 .
  • F (ab ') 2 can be obtained by treatment with a reducing agent dithiothreitol.
  • the "scFv antibody” is an antibody in which VH and VL are linked via a suitable peptide linker.
  • the scFv antibody obtains cDNAs encoding VH and VL of the anti-PAD4 antibody according to the above-mentioned embodiment of the present invention, constructs a polynucleotide encoding VH-peptide linker-VL, and makes the polynucleotide a vector And can be produced using cells for expression.
  • dsFv is an antibody in which a polypeptide having a cysteine residue introduced into VH and VL is linked via a disulfide bond between the above-mentioned cysteine residues.
  • the position to be introduced into the cysteine residue should be selected based on the prediction of the three-dimensional structure of the antibody according to the method shown by Reiter et al. (Reiter et al., Protein Eng. 1994 May; 7 (5): 697-704.). Can.
  • the “antigen-binding peptide” includes, for example, a VH and VL of an antibody, or CDRs 1, 2 and 3 thereof, and a peptide having PAD4 binding activity. Peptides containing multiple CDRs can be linked directly or via appropriate peptide linkers.
  • the “antibody-like molecule” is a molecule having a binding property to PAD4 and includes, for example, affibody, DARPins, anticalin, monobody, adnectin, salobody, avimer, or affilin. Details of the properties and preparation methods of antibody-like molecules are described in "Helma et al., J Cell Biol. 2015 Jun 8; 209 (5): 633-44.” And “Angeline et al., Future Med Chem. 2017 Aug. 9 (12): 1301-1304. "And references cited therein.
  • Fv antibody etc. The method for producing the above Fv antibody, Fab antibody, F (ab ') 2 antibody, Fab' antibody, scFv antibody, dsFv antibody, antigen-binding peptide (hereinafter sometimes referred to as "Fv antibody etc.") is particularly limited do not do.
  • a DNA encoding a region such as an Fv antibody in the anti-PAD 4 antibody according to the embodiment of the present invention described above can be incorporated into an expression vector and produced using expression cells.
  • they may be produced by a chemical synthesis method such as Fmoc method (fluorenylmethyloxycarbonyl method), tBOC method (t-butyloxycarbonyl method) or the like.
  • the antigen-binding antibody fragment according to the embodiment of the present invention may contain one or more of the Fv antibody and the like.
  • a "chimeric antibody” is, for example, one obtained by linking the variable region of an antibody between heterologous organisms and the constant region of the antibody, and can be constructed by genetic recombination technology.
  • mouse-human chimeric antibodies, chicken-human chimeric antibodies, chicken-mouse chimeric antibodies and the like can be mentioned.
  • a mouse-human chimeric antibody can be produced, for example, by the method described in "Roguska et al., Proc Natl Acad Sci US A. 1994 Feb 1; 91 (3) 969-973.”.
  • a basic method for producing mouse-human chimeric antibodies is, for example, encoding the mouse leader and variable region sequences present in the cloned cDNA, and human antibody constant regions already present in mammalian cell expression vectors.
  • the mouse leader sequence and variable region sequence present in the cloned cDNA may be ligated to a sequence encoding a human antibody constant region and then ligated to a mammalian cell expression vector.
  • the fragments of the human antibody constant region can be those of the heavy chain constant region of any human antibody and the light chain constant region of the human antibody, eg, for human heavy chain, C ⁇ 1, C ⁇ 2, C ⁇ 3 or C ⁇ 4 As for the L chain one, C ⁇ or C ⁇ ⁇ ⁇ can be mentioned respectively.
  • a "humanized antibody” has, for example, one or more CDRs from a non-human species, and a framework region derived from human immunoglobulin, and further a constant region derived from human immunoglobulin. Is an antibody that binds to the antigen of For humanization of antibodies, various techniques known in the art may be used.
  • the “human antibody” is derived from, for example, a region including the variable region and constant region of heavy chain constituting the antibody, the variable region and constant region of light chain, from a gene encoding human immunoglobulin Antibody.
  • Various techniques known in the art may be used to generate human antibodies.
  • variable region In one embodiment of the invention "heavy chain” is typically the main component of full-length antibodies.
  • the heavy chain is usually linked to the light chain by disulfide bonds and noncovalent bonds.
  • VH variable region
  • the amino acid sequence is not constant even in the same kind of antibody of the same class.
  • VH has a large affinity to the antigen. It is known to contribute.
  • a molecule consisting only of VH was produced in "Reiter et al., J Mol Biol. 1999 Jul 16; 290 (3): 685-98.”, It specifically bound to the antigen with high affinity. Is described.
  • Wolfson W Chem Biol. 2006 Dec; 13 (12): 1243-1244.
  • CDR complementarity determining region
  • Fv variable region: including heavy chain variable region (VH) and light chain variable region (VL)
  • CDRs include CDR1, CDR2 and CDR3 consisting of about 5 to 30 amino acid residues.
  • heavy chain CDRs particularly contribute to antibody binding to antigen.
  • CDR3 is known to have the highest contribution in antibody binding to antigen.
  • CDRs CDRs and methods of determining their location.
  • Kabat Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)
  • Chothia Chothia et al., J. Mol. Biol. , 1987; 196: 901-917
  • the definition of Kabat is taken as a preferred example but not necessarily limited thereto.
  • a portion including overlapping portions of CDRs according to each definition or portions including both CDRs according to each definition It can also be a CDR.
  • Martin et al.'S method Proc. Natl. Acad. Sci. USA, 1989; 86; 86
  • Oxford Molecular's AbM antibody modeling software which is a compromise between the Kabat definition and the Chothia definition. : 9268-9272).
  • One embodiment of the present invention is an antibody comprising (a) an amino acid sequence as shown in SEQ ID NOs: 19 to 24, and (b) heavy chain CDRs 1 to 3 and light chain, respectively.
  • An antibody comprising an amino acid sequence as shown in SEQ ID NO: 25-30
  • An antibody comprising an amino acid sequence as shown in SEQ ID NO: 31-36
  • (c) heavy chain CDR1-3 and light chain CDR1-3 are one or more anti-PAD4 antibodies selected from the group consisting of This antibody can be used to inhibit the citrullinating activity of active PAD4.
  • the CDR sequences of the above-mentioned antibodies (a) to (d) correspond to the CDR sequences possessed by the P1 to P4 antibodies described in the examples below, respectively.
  • another embodiment of the present invention is an anti-PAD4 antibody comprising at least one set of the heavy chain CDR1-3 and light chain CDR1-3 amino acid sequences listed above.
  • the heavy chain FR1 to 4 and the light chain FR1 to 4 may contain the amino acid sequences shown by SEQ ID NOs: 43 to 50, respectively.
  • heavy chain FR1 to 4 and light chain FR1 to 4 may contain the amino acid sequences shown by SEQ ID NOs: 51 to 58, respectively.
  • the heavy chain FR1 to 4 and the light chain FR1 to 4 may contain the amino acid sequences shown by SEQ ID NOs: 59 to 66, respectively.
  • the heavy chain FR1 to 4 and the light chain FR1 to 4 may contain the amino acid sequences shown by SEQ ID NOs: 67 to 74, respectively.
  • FR sequences correspond to the FR sequences possessed by the P1 to P4 antibodies described in the examples below, respectively.
  • another embodiment of the present invention is an anti-PAD4 antibody comprising at least one set of the heavy chain FR1-4 and light chain FR1-4 amino acid sequence sets listed above.
  • the antibody according to one embodiment of the present invention may comprise the amino acid sequence shown in SEQ ID NO: 19, 25, 31 or 37 in heavy chain CDR1 and SEQ ID NO: 20, 26, 32 or in heavy chain CDR2.
  • the heavy chain CDR3 may contain the amino acid sequence shown in SEQ ID NO: 21, 27, 33 or 39
  • the light chain CDR1 may contain the amino acid sequence shown in SEQ ID NO: 22, 28, 34 Or 40
  • the light chain CDR2 may contain the amino acid sequence shown in SEQ ID NO: 23, 29, 35, or 41
  • the light chain CDR3 may contain SEQ ID NO: 24, 30
  • the amino acid sequence shown by 36, or 42 may be included.
  • the antibody according to one embodiment of the present invention may comprise the amino acid sequence shown in SEQ ID NO: 43, 51, 59 or 67 in heavy chain FR1, and SEQ ID NO: 44, 52, 60 or in heavy chain FR2.
  • the heavy chain FR3 may contain the amino acid sequence shown in SEQ ID NO: 68
  • the heavy chain FR3 may contain the amino acid sequence shown in SEQ ID NO: 45, 53, 61 or 69
  • the heavy chain FR4 may contain the amino acid sequence shown in SEQ ID NO: 46, 54, 62
  • the amino acid sequence represented by SEQ ID NO: 47, 55 may contain the amino acid sequence represented by SEQ ID NO: 47, 55, 63, or 71 in the light chain FR1; , 64, or 72.
  • the light chain FR3 may include the amino acid sequence represented by SEQ ID NO: 49, 57, 65, or 73, and the light chain FR4 may include the amino acid sequence of SEQ ID NO: 50.
  • the amino acid sequence shown by 58, 66, or 74 may be included.
  • the anti-PAD4 antibody according to one embodiment of the present invention may be in the form of scFv, in which case it may have a linker between the heavy chain and the light chain.
  • the linker is, for example, typically, but not limited to, a sequence of 0 to 5 amino acids consisting of G and P.
  • the linker may have, for example, the amino acid sequence set forth in SEQ ID NO: 75. The linker is not required and may not be present.
  • the heavy chain variable region may contain the amino acid sequence shown by SEQ ID NO: 3, 4, 5, or 6, and the light chain variable region comprises SEQ ID NO: 11, 12 , 13 or 14 may be included.
  • amino acid is a generic term for organic compounds having an amino group and a carboxyl group.
  • any amino acid in the amino acid sequence may be chemically modified.
  • any amino acid in the amino acid sequence may form a salt or a solvate.
  • any amino acid in the amino acid sequence may be L-form or D-form. Even in those cases, it can be said that the antibody according to the embodiment of the present invention comprises the above-mentioned "specific amino acid sequence".
  • Examples of chemical modifications that amino acids contained in proteins undergo in vivo include, for example, N-terminal modification (eg, acetylation, myristoylation etc.), C-terminal modification (eg amidification, glycosylphosphatidylinositol addition etc.), or side chain Modifications (eg, phosphorylation, glycosylation, etc.) and the like are known.
  • N-terminal modification eg, acetylation, myristoylation etc.
  • C-terminal modification eg amidification, glycosylphosphatidylinositol addition etc.
  • side chain Modifications eg, phosphorylation, glycosylation, etc.
  • One embodiment of the present invention is a polynucleotide or a vector encoding the anti-PAD4 antibody according to the above-mentioned embodiment of the present invention.
  • Transformants can be produced by introducing this polynucleotide or vector into cells.
  • the transformant may be a cell of human or a mammal other than human (eg, rat, mouse, guinea pig, rabbit, bovine, monkey, etc.).
  • Mammalian cells include, for example, Chinese hamster ovary cells (CHO cells), monkey cells COS-7, human fetal kidney cells (eg, HEK 293 cells) and the like.
  • the transformant may be Escherichia genus, yeast or the like.
  • the polynucleotide or vector may be constructed so as to express an anti-PAD4 antibody.
  • the polynucleotide or vector may contain, for example, components necessary for protein expression, such as a promoter, an enhancer, an origin of replication, or an antibiotic resistance gene.
  • the polynucleotide or vector may have a base sequence of heterologous origin.
  • the base sequence derived from different species is, for example, two or more organisms selected from the group consisting of humans and non-human organisms (eg, bacteria, archaea, yeast, insects, birds, viruses, mammals other than human, etc.) You may contain the base sequence derived from.
  • the vector examples include a plasmid derived from E. coli (eg, pET-Blue), a plasmid derived from Bacillus subtilis (eg, pUB110), a yeast-derived plasmid (eg, pSH19), an animal cell expression plasmid (eg, pA1-11, pcDNA3.1- Bacteriophages such as V5 / His-TOPO), ⁇ phage, and vectors derived from viruses can be used.
  • the vector may be an expression vector or may be circular.
  • a method for introducing the above polynucleotide or vector into cells for example, calcium phosphate method, lipofection method, electroporation method, method by adenovirus, method by retrovirus, microinjection, etc. can be used (revised 4 th ed. New Genetic Engineering Handbook, Yodosha (2003): 152-179.).
  • a method for producing an antibody using cells for example, the method described in "Protein Experimental Handbook, Yodosha (2003): 128-142.” Can be used.
  • One embodiment of the present invention is a cell containing the polynucleotide or vector according to the above-mentioned embodiment of the present invention.
  • One embodiment of the present invention is a method of producing an anti-PAD4 antibody, which comprises the step of growing the cells described above. The growing step includes a culturing step. The production method may also include the step of recovering the anti-PAD4 antibody. The production method may also include the step of preparing a cell culture solution. The production method may also include the step of purifying the anti-PAD4 antibody.
  • purification of the antibody is carried out, for example, with ammonium sulfate, ethanol precipitation, protein A, protein G, gel filtration chromatography, anion, cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography Chromatography, affinity chromatography, hydroxylapatite chromatography, or lectin chromatography can be used (Protein Experimental Handbook, Yodosha (2003): 27-52.).
  • One embodiment of the present invention is a composition comprising the anti-PAD4 antibody according to the above-mentioned embodiment of the present invention.
  • active PAD 4 can be detected efficiently.
  • citrullination of active PAD 4 can be efficiently inhibited.
  • RA or arthritis can be treated.
  • the components contained in this composition are not particularly limited, and may contain, for example, a buffer.
  • one or more of the various embodiments (for example, the possibility of containing a carrier, etc.) of the inhibitors and pharmaceutical compositions described below may be applied.
  • One embodiment of the present invention is a functional inhibitor of PAD4 comprising the anti-PAD4 antibody according to the above-mentioned embodiment of the present invention.
  • This inhibitor can be used to effectively inhibit the citrullinating activity of active PAD4.
  • the reduction rate of the citrullinating activity of active PAD 4 by the inhibitor may be 15, 20, 30, 40, 50, 60, 70, 80% or more, and any of these is within the range of two values. May be This reduction rate may be expressed, for example, as a relative ratio when the reduction rate when TBS Buffer is used is 0%.
  • the reduction rate is preferably 20% or more, and more preferably 30% or more.
  • an "agent" comprises, for example, a composition used for research or treatment.
  • the inhibitor includes, for example, a therapeutic agent for RA or arthritis.
  • the above inhibitors can be used, for example, in vitro or in vivo.
  • the inhibitor may comprise the composition according to the embodiment of the invention described above.
  • One embodiment of the present invention is a method for inhibiting the function of PAD4, comprising the step of bringing an anti-PAD4 antibody according to the above-mentioned embodiment of the present invention into contact with PAD4.
  • One embodiment of the present invention is a method for inhibiting PAD4 function comprising administering an anti-PAD4 antibody according to the above-mentioned embodiment of the present invention to a patient.
  • the above inhibition methods include inhibition methods performed for research or treatment.
  • One embodiment of the present invention is the use of the anti-PAD4 antibody according to the above-mentioned embodiment of the present invention for producing a functional inhibitor of PAD4. Note that, again, PAD 4 may be active PAD 4.
  • One embodiment of the present invention is a pharmaceutical composition comprising the anti-PAD4 antibody according to the above-mentioned embodiment of the present invention.
  • This pharmaceutical composition can be used to treat RA or arthritis.
  • the pharmaceutical composition may comprise one or more pharmacologically acceptable carriers.
  • the above pharmaceutical composition includes, for example, a pharmaceutical composition for treating RA or arthritis.
  • the above-mentioned pharmaceutical composition may contain the composition concerning the above-mentioned embodiment of the present invention.
  • One embodiment of the present invention is a method for treating a disease, which comprises the step of administering to a patient an anti-PAD4 antibody (or a pharmaceutical composition containing the anti-PAD4 antibody) according to the above-mentioned embodiment of the present invention.
  • the diseases include, for example, RA or arthritis.
  • One embodiment of the present invention is the use of the anti-PAD4 antibody according to the above-mentioned embodiment of the present invention for producing a pharmaceutical composition.
  • RA can be treated by a novel mechanism.
  • One embodiment of the present invention is a diagnostic agent for RA or arthritis, which comprises the anti-PAD4 antibody according to the above-mentioned embodiment of the present invention.
  • This diagnostic agent can be used to diagnose RA or arthritis efficiently.
  • One embodiment of the present invention is a method for diagnosing RA or arthritis, which comprises the step of contacting a patient sample with the anti-PAD4 antibody according to the above-mentioned embodiment of the present invention.
  • One embodiment of the present invention is a detection reagent for PAD4 comprising the anti-PAD4 antibody according to the above-mentioned embodiment of the present invention. By using this reagent, PAD 4 can be detected efficiently.
  • One embodiment of the present invention is a method of detecting PAD4 comprising the step of bringing a test sample into contact with the anti-PAD4 antibody according to the above-described embodiment of the present invention. This method can properly detect PAD 4 even when it is not treated with EDTA as described in Patent Document 3 above.
  • One embodiment of the present invention is a kit comprising the anti-PAD4 antibody according to the above-mentioned embodiment of the present invention. With this kit, for example, treatment of diseases, diagnosis or detection of PAD4 can be performed.
  • This kit may contain, for example, the composition, the inhibitor, the pharmaceutical composition, the diagnostic agent, or the detection reagent according to the above-mentioned embodiment of the present invention, and the instruction manual, the buffer, the container (for example, a vial) Or a syringe) or a package.
  • a patient sample or test sample may be blood, serum or plasma.
  • treatment includes the ability to exert a symptom ameliorating effect, a suppressing effect or a preventing effect on a disease of a patient or one or more symptoms associated with the disease.
  • the "therapeutic agent” may be a pharmaceutical composition comprising an active ingredient and one or more pharmacologically acceptable carriers.
  • the “pharmaceutical composition” may be produced, for example, by mixing the active ingredient and the above-mentioned carrier, by any method known in the pharmaceutical arts. The use of the pharmaceutical composition is not limited as long as it is used for treatment, and the active ingredient may be used alone, or may be a mixture of the active ingredient and an optional ingredient.
  • the shape of the carrier is not particularly limited, and may be, for example, a solid or a liquid (for example, buffer).
  • the content of the carrier may be, for example, a pharmaceutically effective amount.
  • the effective amount may be, for example, an amount sufficient for pharmaceutical stability or delivery of the active ingredient.
  • buffers are effective in stabilizing the active ingredient in the vial.
  • the route of administration of the pharmaceutical composition is preferably that which is effective in treatment, and may be, for example, intravenous, subcutaneous, intramuscular, intraperitoneal or oral administration.
  • the administration mode may be, for example, an injection, a capsule, a tablet, a granule and the like.
  • the aqueous solution for injection may be stored, for example, in a vial or a stainless steel container.
  • the aqueous solution for injection may be formulated with, for example, physiological saline, sugar (eg, trehalose), NaCl, or NaOH.
  • the pharmaceutical composition may also contain, for example, an effective amount of a buffer (eg, phosphate buffer), a pH adjuster, a stabilizer, and the like.
  • the dosage, administration interval, and administration method are not particularly limited, and may be appropriately selected depending on the age and weight of the patient, symptoms, target organs and the like.
  • the pharmaceutical composition also preferably contains a therapeutically effective amount, or an effective amount of an active ingredient that exerts a desired effect.
  • the therapeutic effect of the pharmaceutical composition may be evaluated by, for example, arthritis score, RA score, swelling width, diagnostic imaging, modified Total Sharp score, or disease marker.
  • evaluating the swelling width for example, when the swelling width of the affected area at the time of administration of the pharmaceutical composition is significantly reduced as compared to the swelling width at the time of non-administration, it may be judged that the therapeutic effect was obtained.
  • the amount of reduction may be, for example, 40, 50, 60, 70, 80, 90, or 100%, and may be in the range of any two values.
  • the “patient” is a human or a mammal other than a human (eg, mouse, guinea pig, hamster, rat, rat, rat, rabbit, pig, sheep, goat, cow, horse, cat, dog, marmoset) , Monkey or chimpanzee etc.).
  • the patient may also be a patient diagnosed as having RA or arthritis.
  • the patient may also be a patient diagnosed as having a disease treatable by suppression of citrullination.
  • One embodiment of the present invention is an anti-PAD4 antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 in the composition, or an anti-PAD4 that specifically binds to positions 634-644 of human PAD4.
  • a method of promoting the ability to inhibit the citrullinating activity or the therapeutic effect of a composition comprising the step of increasing the proportion of antibodies.
  • One embodiment of the present invention is a composition containing an anti-PAD4 antibody, wherein 90% or more of the anti-PAD4 antibody molecules in the composition specifically bind to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 Or PAD4 antibody that specifically binds to positions 633-644 of human PAD4.
  • One embodiment of the present invention is an antibody population comprising an anti-PAD4 antibody, wherein 90% or more of the anti-PAD4 antibody molecules in the antibody population specifically bind to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 Antibody population, or an anti-PAD4 antibody that specifically binds to positions 633-644 of human PAD4.
  • the 90% or more may be, for example, 90, 95, 96, 97, 98, 99% or more, or 100%, and may be in the range of any two values.
  • One embodiment of the present invention is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1.
  • This peptide can be used to produce an antibody that binds to active PAD4.
  • antibodies that bind to active PAD4 can be detected.
  • the peptide consisting of the amino acid sequence shown by SEQ ID NO: 1 may be chemically modified (for example, KLH modified), and such chemically modified peptide is also one form of the peptide consisting of the amino acid sequence shown by SEQ ID NO: 1 included.
  • the peptide consisting of the amino acid sequence shown by SEQ ID NO: 1 may be an isolated, purified or enriched peptide.
  • One embodiment of the present invention is an antigen composition comprising a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1.
  • the antigenic composition may, for example, comprise a buffer.
  • One embodiment of the present invention is a method for producing an anti-PAD4 antibody, which comprises the step of immunizing a mammal or a bird with a peptide consisting of the amino acid sequence shown by SEQ ID NO: 1.
  • One embodiment of the present invention is a method for producing an anti-PAD4 antibody, which comprises the step of contacting an antibody or antibody library with a peptide consisting of the amino acid sequence shown by SEQ ID NO: 1.
  • One embodiment of the present invention is a method of detecting an antibody that binds to active PAD4, comprising the step of contacting a peptide consisting of the amino acid sequence shown by SEQ ID NO: 1 with a test sample containing an anti-PAD4 antibody.
  • One embodiment of the present invention is a composition for detecting an antibody that binds to active PAD4, comprising a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1.
  • binding may be either covalent or non-covalent, for example, ionic, hydrogen bonding, hydrophobic interaction, or hydrophilic interaction.
  • “Significantly” in one embodiment of the present invention is, for example, a condition where statistical significance is evaluated using Student's t-test (one side or both sides) and p ⁇ 0.05 or p ⁇ 0.01. It is also good. Or, it may be in a state where a difference is substantially generated.
  • Example 1 Preparation of anti-PAD4 antibody
  • the peptide of KLH-modified FFTYHIRHGEVHC (SEQ ID NO: 1) was immunized in the abdominal cavity at 333 ⁇ g per 3 rabbits of 3 months old Boris Brown.
  • the peptide used is the peptide antigen corresponding to positions 633-644 of PAD4 (SEQ ID NO: 2).
  • Antigens were immunized with complete Freund's adjuvant (Wako, 014-09541) for primary immunization and incomplete Freund's adjuvant (Wako, 011-09551) for secondary and tertiary immunization.
  • the fourth immunization was intravenously injected with antigen diluted in PBS (phosphate buffered saline). Blood was collected from the sub wing vein every other week, and the antibody titer was confirmed by ELISA. A third immunization was performed on three birds, and a fourth immunization was performed on one individual with the highest antibody titer, and the fourth immunization was used as a final immunization. Three days after the final immunization, spleens of chickens are collected, and lymphocytes are isolated by density gradient centrifugation using Ficoll paque PLUS (GE Healthcare, 17-1440-03), using TRIzole Reagent (Life Technologies, 1559 026) RNA was extracted.
  • PBS phosphate buffered saline
  • CDNA was synthesized from the extracted RNA by RT-PCR using a PrimeScript II 1st Strand cDNA Synthesis Kit (TAKARA, 6210A) to prepare a scFv phage library.
  • the expression vector used was a type of expression vector in which a chicken ⁇ chain was inserted instead of the mouse ⁇ chain of pPDS.
  • the preparation of the scFv phage library was performed according to the method described in the reference: "Nakamura et al., J Vet Med Sci. 2004 Ju; 66 (7): 807-814".
  • the scFv phage antibody library was used to perform panning on a plate on which the above-mentioned peptide antigen was immobilized. Panning was performed according to the method described in the reference "Nakamura et al., J Vet Med Sci. 2004 Ju; 66 (7): 807-814". After five rounds of panning, the reactivity of the library was confirmed by ELISA using a plate on which BSA-modified peptide antigen was immobilized, and phages were screened from the library whose reactivity began to increase. For screening, phage was infected into E.
  • Induction of The reactivity of the scFv phage antibody in the obtained culture supernatant was confirmed by ELISA using an antigen-immobilized plate.
  • the obtained positive clones were sequenced and sequenced using a DNA sequencer (Applied Biosystems, ABI PRISM 3100-Genetic Analyzer).
  • amplification of chicken antibody gene H chain variable region and L chain variable region is performed by PCR using a DNA chain encoding scFv antibody as a template, and then the PCR product is SacII (BioLabs, Cat # R0157S) and NheI (BioLabs, Cat # R0131S) restriction enzymes were treated.
  • SacII BioLabs, Cat # R0157S
  • NheI BioLabs, Cat # R0131S
  • a mouse chimeric antibody (IgG1) expression vector H chain expression vector: pcDNA4 / myc-His, L chain expression vector
  • restriction enzymes for each of the H chain variable region and the L chain variable region It recombined into pcDNA3 / myc-His, Invitrogen).
  • the mouse chimeric expression vector used was the vector described in Tateishi et al., J Vet Med Sci. 2008 Apr; 70 (4): 397-400. Thus, antibody clones of P1, P2, P3 and P4 were obtained.
  • amino acid sequences of the heavy chain variable regions of P1, P2, P3 and P4 are sequentially SEQ ID NO: 3 to 6, the DNA sequence is sequentially SEQ ID NO: 7 to 10, the amino acid sequence of the light chain variable region is sequentially SEQ ID NO 11 to 14, DNA
  • sequences are, in order, SEQ ID NO: 15-18 (FIGS. 1-3).
  • these antibodies may be collectively referred to as "P1 etc.”
  • the purified antibody is purified by Protein G Sepharose 4 Fast Flow (GE healthcare, 17-018-02) was performed.
  • Mouse Antibody Capture Kit GE Healthcare, BR-1008-38
  • a rabbit anti-mouse polyclonal antibody was immobilized on the surface of the CM5 chip by amine coupling using free carboxyl groups on the surface of the CM5 chip using NHS / EDC according to a standard protocol provided by the manufacturer.
  • P1 etc. were captured on a rabbit anti-mouse polyclonal antibody.
  • Various concentrations of human PAD4 were applied to Biacore T200 to generate kinetic sensorgrams.
  • Protocol 1 (Calcium added after reaction of antibody and enzyme)
  • the anti-PAD4 antibody (P1 etc.) and mouse IgG (negative control) were each adjusted to 450 ⁇ g / mL using TBS as a solvent.
  • 5 ⁇ L of antibody solution final concentration in 50 ⁇ L of reaction system is 45 ⁇ g / mL (300 nM)
  • the mixture was mixed with 20 mM Tris-HCl buffer (pH. 7.6).
  • BAEE benzoylarginine ethyl ester
  • CaCl 2 total volume 50 ⁇ L, final concentration of BAEE is 10 mM, final concentration of calcium ion is 10 mM
  • a solvent TBS
  • a solution of anti-DNP antibody negative control
  • final concentration 300 nM
  • L207 the anti-PAD4 antibody L207- described in the example of WO / 2012/026309
  • Protocol 2 add calcium before reaction of antibody and enzyme
  • PAD4 was activated by reacting calcium and PAD4 in advance. Specifically, the procedure was as follows. Mix 5 ⁇ L of 7.5 ⁇ g / mL (100 nM) human PAD 4 with 5 ⁇ L of 100 mM CaCl 2 to make a total volume of 40 ⁇ L in 20 mM Tris-HCl buffer (pH. 7.6) containing 1 mM EDTA, 1 mM DTT. And mixed. After mixing, pre-incubation was performed at 37 ° C. for 60 minutes.
  • the anti-PAD4 antibody (P1 etc.) and mouse IgG (negative control) were each adjusted to 450 ⁇ g / mL using TBS as a solvent.
  • the final concentration of ions was 10 mM).
  • samples were also prepared in which solvent (TBS), a solution of anti-DNP antibody (final concentration 300 nM) or a solution of L207 (final concentration 300 nM) was added instead of 5 ⁇ L of antibody solution.
  • TBS solvent
  • a solution of anti-DNP antibody final concentration 300 nM
  • a solution of L207 final concentration 300 nM
  • citrullinated BAEE contained in the supernatant was subjected to colorimetric determination.
  • the inhibition value of each antibody was calculated by setting the measured value of the control sample using a solvent instead of the antibody solution as 100.
  • the anti-PAD4 antibody that specifically binds to positions 633-644 of PAD4 has the property of binding to active PAD4 and inhibiting the citrullinating activity.

Abstract

An anti-PAD4 antibody that binds to active PAD4 or an anti-PAD4 antibody that inhibits the function of active PAD4 is achieved. An anti-PAD4 antibody is used which binds to active PAD4. Or, an anti-PAD4 antibody which binds specifically to a peptide formed from the amino acid sequence represented by sequence number 1, or an anti-PAD4 antibody which binds specifically to positions 633-644 of human PAD4 is used. This anti-PAD4 antibody optionally inhibits the citrullination activity of PAD4.

Description

新規抗ＰＡＤ４抗体Novel anti-PAD4 antibody
　本発明は、抗PAD4抗体に関する。 The present invention relates to anti-PAD4 antibodies.
　PAD4（Peptidylarginine deiminase 4）は、蛋白質中のアルギニンのシトルリン化に関与する酵素として知られている。このシトルリン化は、蛋白質を構成するアミノ酸の中で最も塩基性の強いアルギニンが中性のシトルリンに変換される反応であるため、蛋白質の構造と反応にとって重要である。 PAD4 (Peptidylarginine deiminase 4) is known as an enzyme involved in citrullination of arginine in proteins. This citrullination is important for protein structure and reaction because it is a reaction in which the most basic arginine among the amino acids constituting the protein is converted to neutral citrulline.
　PAD4が関節リウマチ（RA）や関節炎の病態に関与することを示す報告がいくつか存在する。例えば、非特許文献1には、RAの発症とPAD4遺伝子の一塩基多型との間に相関があることが報告されている。非特許文献2には、RAを診断するために抗PAD4抗体を使用したことが報告されている。特許文献1には、4種類の抗PAD4抗体の混合物をマウスに投与することによって、RAを抑制する試みが記載されている。特許文献2には、特定のエピトープに結合する抗PAD4抗体をマウスに投与することによって、RA又は関節炎を抑制したことが記載されている。 There are several reports showing that PAD4 is involved in the pathogenesis of rheumatoid arthritis (RA) and arthritis. For example, Non-Patent Document 1 reports that there is a correlation between the onset of RA and the single nucleotide polymorphism of PAD4 gene. Non-Patent Document 2 reports that an anti-PAD4 antibody was used to diagnose RA. Patent Document 1 describes an attempt to suppress RA by administering a mixture of four anti-PAD4 antibodies to mice. Patent Document 2 describes that RA or arthritis was suppressed by administering an anti-PAD4 antibody that binds to a specific epitope to mice.
　PAD4には、「活性型PAD4」と「非活性型PAD4」が存在する。活性型はCa2+を結合しており、Ca2+結合型PAD4とも呼ばれる。非活性型はCa2+を結合しておらず、Ca2+非結合型PAD4とも呼ばれる。 In PAD4, "active PAD4" and "inactive PAD4" are present. The active form binds Ca 2+ and is also called Ca 2+ -linked PAD4. Non active form not bound to Ca 2+, also referred to as Ca 2+ unconjugated PAD4.
　PAD4とCa2+に関する報告としては、例えば、特許文献3が存在する。この特許文献3には、健常者及び関節リウマチ患者の血清中のPAD4量を、抗PAD4抗体を用いたELISAで測定する試みが記載されている。特許文献3の実施例には、ELISAを行うに当たり、未処理の血清を用いると適切な測定値が得られなかったが、EDTA処理した血清を用いると適切な測定値が得られたことが記載されている。このとき、EDTAにCa2+がトラップされた結果、非活性型PAD4が抗PAD4抗体と反応したと考えられる。 For example, Patent Document 3 exists as a report on PAD4 and Ca 2+ . Patent Document 3 describes an attempt to measure the amount of PAD4 in the serum of healthy subjects and rheumatoid arthritis patients by ELISA using an anti-PAD4 antibody. In the example of Patent Document 3, although an appropriate measurement value was not obtained using untreated serum in performing ELISA, it was described that an appropriate measurement value was obtained using EDTA-treated serum. It is done. At this time, as a result of Ca 2+ being trapped in EDTA, it is considered that non-activated PAD 4 reacted with the anti-PAD 4 antibody.
WO/2012/026309WO / 2012/026309 WO/2016/143753WO / 2016/143753 JP5252339 (B2)JP5252339 (B2)
　従来、非活性型PAD4に結合する抗体は得られていたが、活性型PAD4に結合する抗体は得られていなかった。また、活性型PAD4の機能を阻害する抗体は得られていなかった。 Conventionally, although an antibody that binds to non-activated PAD4 has been obtained, an antibody that binds to active PAD4 has not been obtained. In addition, no antibody that inhibits the function of active PAD4 has been obtained.
　本発明は上記事情に鑑みてなされたものであり、活性型PAD4に結合する抗PAD4抗体、又は活性型PAD4の機能を阻害する抗PAD4抗体を提供すること等を目的とする。 The present invention has been made in view of the above circumstances, and an object thereof is to provide an anti-PAD4 antibody that binds to active PAD4, or an anti-PAD4 antibody that inhibits the function of active PAD4.
　本願発明者らは、後述する実施例に記載の通り、配列番号1で示されるアミノ酸配列からなるペプチド（PAD4の633～644位に相当）に特異的に結合する抗PAD4抗体が、驚くべきことに、活性型PAD4に結合し、機能を阻害することを見いだした。そして、その結果に基づき本発明を完成させた。 The present inventors have surprisingly found that an anti-PAD4 antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 (corresponding to positions 633-644 of PAD4), as described in the examples below. Were found to bind to activated PAD4 and to inhibit the function. And based on the result, the present invention was completed.
　即ち本発明の一態様によれば、活性型PAD4に結合する、抗PAD4抗体が提供される。この抗体を用いれば、活性型PAD4を検出することができる。この抗体は、活性型PAD4の機能阻害抗体であってもよい。 That is, according to one aspect of the present invention, there is provided an anti-PAD4 antibody that binds to active PAD4. By using this antibody, active PAD4 can be detected. This antibody may be a functional inhibition antibody of active PAD4.
　また本発明の一態様によれば、配列番号1で示されるアミノ酸配列からなるペプチドに特異的に結合する抗PAD4抗体、又はヒトPAD4の633～644位に特異的に結合する抗PAD4抗体が提供される。この抗体を用いれば、活性型PAD4の検出又は機能阻害をすることができる。 Moreover, according to one aspect of the present invention, there is provided an anti-PAD4 antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1, or an anti-PAD4 antibody that specifically binds to positions 634-644 of human PAD4. Be done. This antibody can be used to detect or inhibit the function of active PAD4.
　また本発明の一態様によれば、配列番号1で示されるアミノ酸配列からなるペプチドが提供される。このペプチドを用いれば、活性型PAD4に対する結合性抗体又は機能阻害抗体を生産することができる。 Further, according to one aspect of the present invention, there is provided a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1. By using this peptide, it is possible to produce a binding antibody or a function inhibiting antibody against active PAD4.
図1は、実施例に係る抗体の可変領域のアミノ酸配列を表した図である。FIG. 1 is a diagram showing the amino acid sequence of the variable region of the antibody according to the example. 図2は、実施例に係る抗体の可変領域の塩基配列を表した図である。FIG. 2 is a diagram showing the nucleotide sequence of the variable region of the antibody according to the example. 図3は、実施例に係る抗体の可変領域の塩基配列を表した図である。FIG. 3 is a diagram showing the nucleotide sequence of the variable region of the antibody according to the example. 図4は、実施例に係る抗体による活性型PAD4阻害率を調べた結果を表したグラフである。FIG. 4 is a graph showing the results of examining the percentage inhibition of active PAD 4 by the antibodies according to the examples.
　以下、本発明の実施の形態について詳細に説明する。なお、同様な内容については繰り返しの煩雑を避けるために、適宜説明を省略する。 Hereinafter, embodiments of the present invention will be described in detail. In addition, in order to avoid the repetition complexity about the same content, description is abbreviate | omitted suitably.
　本発明の一実施形態は、新規の抗PAD4抗体である。この抗体は、例えば、活性型PAD4に結合する抗PAD4抗体であってもよい。この抗体を用いれば、例えば、活性型PAD4を検出することができる。この抗体は、例えば、活性型PAD4の機能阻害抗体であってもよい。この抗体を用いれば、例えば、活性型PAD4のシトルリン化活性を阻害することができる。またこの抗体を用いれば、例えば、関節リウマチ（RA）又は関節炎の治療を行うことができる。この治療方法は、抗体を使用するため副作用が小さく、安全性の観点から優れている。 One embodiment of the present invention is a novel anti-PAD4 antibody. This antibody may be, for example, an anti-PAD4 antibody that binds to active PAD4. With this antibody, for example, active PAD4 can be detected. This antibody may be, for example, a function-inhibiting antibody of active PAD4. This antibody can be used, for example, to inhibit the citrullinating activity of active PAD4. In addition, this antibody can be used, for example, to treat rheumatoid arthritis (RA) or arthritis. This therapeutic method has small side effects due to the use of antibodies and is excellent in terms of safety.
　本発明の一実施形態に係る抗PAD4抗体は、例えば、配列番号1で示されるペプチドに特異的に結合する抗PAD4抗体、又はヒトPAD4の633～644位に特異的に結合する抗PAD4抗体であってもよい。この抗体を用いれば、例えば、活性型PAD4を検出することができる。またこの抗体を用いれば、例えば、活性型PAD4のシトルリン化活性を阻害することができる。またこの抗体を用いれば、例えば、RA又は関節炎の治療を行うことができる。この治療方法は、抗体を使用するため副作用が小さく、安全性の観点から優れている。 The anti-PAD4 antibody according to one embodiment of the present invention is, for example, an anti-PAD4 antibody that specifically binds to the peptide shown by SEQ ID NO: 1, or an anti-PAD4 antibody that specifically binds to positions 634-644 of human PAD4. It may be. With this antibody, for example, active PAD4 can be detected. Moreover, if this antibody is used, for example, the citrullinating activity of active PAD 4 can be inhibited. Also, this antibody can be used, for example, to treat RA or arthritis. This therapeutic method has small side effects due to the use of antibodies and is excellent in terms of safety.
　PAD4は、一般的に、蛋白質中のアルギニンのシトルリン化に関与する酵素として知られている。PAD4のアミノ酸配列等の詳細は、NCBI（National Center for Biotechnology Information）、又はHGNC（HUGO Gene Nomenclature Committee)等のWEBサイトから見ることができる。NCBIに記載されているPAD4のアクセッションナンバーは、例えば、NP_036519.2である。ヒトPAD4のアミノ酸配列は、例えば、配列番号2である。PAD4は、PAD4活性を有していれば、その生物由来は限定されない。本願明細書において、PAD4は、活性型PAD4又は非活性型PAD4であってもよい。本願明細書において、活性型PAD4はCa2+結合型PAD4であってもよく、非活性型PAD4はCa2+非結合型PAD4であってもよい。活性型PAD4はCa2+が結合したことにより活性化したPAD4を含む。 PAD4 is generally known as an enzyme involved in the citrullination of arginine in proteins. Details such as the amino acid sequence of PAD4 can be viewed from the website such as NCBI (National Center for Biotechnology Information) or HGNC (HUGO Gene Nomenclature Committee). The accession number of PAD 4 described in NCBI is, for example, NP_036519.2. The amino acid sequence of human PAD4 is, for example, SEQ ID NO: 2. The origin of PAD4 is not limited as long as it has PAD4 activity. In the present specification, PAD4 may be active PAD4 or inactive PAD4. In the present specification, active PAD4 may be Ca 2+ -bound PAD4, and non-active PAD 4 may be non-Ca 2+ -bound PAD4. Active PAD4 contains PAD4 activated by binding of Ca 2+ .
　本発明の一実施形態において「抗PAD4抗体」は、PAD4に結合性を有する抗体を含む。この抗PAD4抗体の生産方法は特に限定されないが、例えば、配列番号1で示されるアミノ酸配列からなるペプチドを哺乳類又は鳥類に免疫することによって生産してもよい。 In one embodiment of the present invention, the "anti-PAD4 antibody" comprises an antibody having binding to PAD4. The method for producing this anti-PAD4 antibody is not particularly limited, and for example, it may be produced by immunizing a mammal or birds with a peptide consisting of the amino acid sequence shown by SEQ ID NO: 1.
　本発明の一実施形態に係る抗PAD4抗体は、PAD4の機能を阻害する抗体であってもよい。機能は、例えば、シトルリン化活性を含む。本発明の一実施形態において、活性型PAD4に結合する抗PAD4抗体は、非活性型PAD4に結合性を有する抗体を含む。 The anti-PAD4 antibody according to one embodiment of the present invention may be an antibody that inhibits the function of PAD4. Functions include, for example, citrullinating activity. In one embodiment of the present invention, an anti-PAD4 antibody that binds to active PAD4 comprises an antibody that has binding to non-active PAD4.
　本発明の一実施形態に係る抗PAD4抗体は、モノクローナル抗体であってもよい。モノクローナル抗体であれば、ポリクローナル抗体に比べて、効率的にPAD4に対して作用させることができる。所望の効果を有する抗PAD4モノクローナル抗体を効率的に生産する観点からは、PAD4をニワトリに免疫することが好ましい。抗原として使用するPAD4は、特に指定しない限り、PAD4全長又はPAD4ペプチド断片を含む。 The anti-PAD4 antibody according to one embodiment of the present invention may be a monoclonal antibody. If it is a monoclonal antibody, it can be made to act on PAD4 efficiently compared with a polyclonal antibody. From the viewpoint of efficiently producing an anti-PAD4 monoclonal antibody having a desired effect, it is preferable to immunize chickens with PAD4. PAD4 used as an antigen includes PAD4 full-length or PAD4 peptide fragment, unless otherwise specified.
　本発明の一実施形態に係る抗PAD4抗体は、全長抗体に限定されず、PAD4結合活性を有する抗体断片（以下、「抗原結合性抗体断片」と称することもある）を含む。抗原結合性抗体断片は、安定性又は抗体の生産効率が上昇する等の効果がある。 The anti-PAD4 antibody according to one embodiment of the present invention is not limited to a full-length antibody, and includes an antibody fragment having PAD4 binding activity (hereinafter sometimes referred to as "antigen-binding antibody fragment"). Antigen-binding antibody fragments have effects such as increased stability or antibody production efficiency.
　本発明の一実施形態に係る抗PAD4抗体は、融合蛋白質であってもよい。この融合蛋白質は、PAD4のN又はC末端に、ポリペプチド又はオリゴペプチドが結合したものであってもよい。ここで、オリゴペプチドは、Hisタグであってもよい。また融合蛋白質は、マウス、ヒト、又はニワトリの抗体部分配列を融合したものであってもよい。それらのような融合蛋白質も、本実施形態に係る抗PAD4抗体の一形態に含まれる。 The anti-PAD4 antibody according to one embodiment of the present invention may be a fusion protein. This fusion protein may be one in which a polypeptide or an oligopeptide is linked to the N or C terminus of PAD4. Here, the oligopeptide may be a His tag. The fusion protein may also be a fusion of mouse, human or chicken antibody partial sequences. Such fusion proteins are also included in one form of the anti-PAD4 antibody according to the present embodiment.
　本発明の一実施形態に係る抗PAD4抗体は、PAD4をニワトリに免疫する工程を経て得られる抗体であってもよい。また、PAD4をニワトリに免疫する工程を経て得られる抗体のCDRセットを有する抗体であってもよい。CDRセットとは、重鎖CDR1、2、3、軽鎖CDR1、2、及び3のセットである。 The anti-PAD4 antibody according to one embodiment of the present invention may be an antibody obtained through the step of immunizing PAD4 to a chicken. Alternatively, it may be an antibody having a CDR set of an antibody obtained through the step of immunizing chicken with PAD4. The CDR set is a set of heavy chain CDRs 1, 2, 3 and light chain CDRs 1, 2, and 3.
　本発明の一実施形態に係る抗PAD4抗体は、KD（M）が、例えば、9.9×10-8、9.0×10-8、8.0×10-8、7.0×10-8、6.0×10-8、5.0×10-8、4.0×10-8、3.0×10-8、2.0×10-8、又は1.0×10-8以下であってもよく、それらいずれか2つの値の範囲内であってもよい。抗体による治療効果を高める観点からは、KD（M）は9.0×10-8以下が好ましい。 The anti-PAD 4 antibody according to one embodiment of the present invention has a KD (M) of, for example, 9.9 × 10 −8 , 9.0 × 10 −8 , 8.0 × 10 −8 , 7.0 × 10 −8 , 6.0 × 10 −8. , 5.0 × 10 −8 , 4.0 × 10 −8 , 3.0 × 10 −8 , 2.0 × 10 −8 , or 1.0 × 10 −8 or less, any of which is within the range of two values It is also good. From the viewpoint of enhancing the therapeutic effect of the antibody, the KD (M) is preferably 9.0 × 10 -8 or less.
　本発明の一実施形態に係る抗PAD4抗体は、PAD4の野生型又は変異型に結合する抗体であってもよい。変異型は、SNPsのように、個体間のDNA配列の差異に起因するものを含む。野生型又は変異型のPAD4のアミノ酸配列は、配列番号2に示すアミノ酸配列に対し、633～644位は保存され、且つ好ましくは95％以上、特に好ましくは98％以上の相同性を有していてもよい。本発明の一実施形態に係る抗PAD4抗体は、マウスPAD4に結合性を有する抗体又は有さない抗体であってもよい。 The anti-PAD4 antibody according to one embodiment of the present invention may be an antibody that binds to wild type or mutant form of PAD4. Variants include those that result from differences in DNA sequences between individuals, such as SNPs. The amino acid sequence of wild-type or mutant PAD 4 is homologous to the amino acid sequence shown in SEQ ID NO: 2, with positions 633-644 conserved and preferably 95% or more, particularly preferably 98% or more homologous May be The anti-PAD4 antibody according to one embodiment of the present invention may be an antibody having binding ability to or not having mouse PAD4.
　本発明の一実施形態に係る抗PAD4抗体は、野生型PAD4に対して結合性を有し、且つヒトPAD4の633～644位に変異を有する変異型PAD4に対して結合性を有さない抗体であってもよい。「結合性を有さない」は、実質的に結合性を有さなければよい。 An anti-PAD4 antibody according to one embodiment of the present invention is an antibody having binding to wild-type PAD4 and having no binding to mutant PAD4 having a mutation at positions 633-644 of human PAD4. It may be The "non-binding" may be substantially non-binding.
　本発明の一実施形態に係る抗PAD4抗体は、野生型のPAD4に有意な反応性を示す抗体を選抜する工程、又はヒトPAD4の633～644位に変異を有する変異型PAD4に対して結合性を示さない抗体を選抜する工程、を含む生産方法によって得られる抗体であってもよい。 The anti-PAD4 antibody according to one embodiment of the present invention is a step of selecting an antibody showing significant reactivity to wild-type PAD4, or binding to mutant PAD4 having a mutation at position 633-644 of human PAD4. And b) selecting an antibody not showing.
　本発明の一実施形態において、ヒトPAD4の633～644位に特異的に結合する抗体は、ヒトPAD4の633～644位に対する結合性がある限り、他のアミノ酸残基に結合性を有する抗体又は有さない抗体を含む。特定の部位に特異的に結合する抗体は、特定の部位を認識する抗体であってもよい。 In one embodiment of the present invention, an antibody that specifically binds to positions 633-644 of human PAD4 has an antibody that binds to other amino acid residues, or an antibody that binds to other amino acid residues as long as it has binding to positions 633-644 of human PAD4. It contains the antibody which does not have. The antibody that specifically binds to a specific site may be an antibody that recognizes a specific site.
　本発明の一実施形態に係る抗PAD4抗体は、配列番号1で示されるアミノ酸配列からなるペプチドの2、6、7、及び8位を含むエピトープに特異的に結合する抗体であってもよい。本発明の一実施形態に係る抗PAD4抗体は、ヒトPAD4の634、638、639、及び640位を含むエピトープに特異的に結合する抗体であってもよい。 The anti-PAD4 antibody according to one embodiment of the present invention may be an antibody that specifically binds to an epitope including positions 2, 6, 7 and 8 of the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1. The anti-PAD4 antibody according to one embodiment of the present invention may be an antibody that specifically binds to an epitope including positions 634, 638, 639, and 640 of human PAD4.
　本発明の一実施形態において「抗体」は、抗原上の特定のエピトープに特異的に結合することができる分子又はその集団を含む。また抗体は、ポリクローナル抗体又はモノクローナル抗体であってもよい。抗体の形態は特に限定されず、例えば、全長抗体（Fab領域とFc領域を有する抗体）、Fv抗体、Fab抗体、F(ab')2抗体、Fab'抗体、一本鎖抗体（例えば、scFv）、dsFv、抗原結合性ペプチド、抗体様分子、キメラ抗体、マウス抗体、ニワトリ抗体、ヒト化抗体、ヒト抗体、又はそれらの同等物（又は等価物）からなる群から選ばれる1種以上の形態を含む。また抗体は、抗体修飾物又は抗体非修飾物を含む。抗体修飾物は、抗体と、例えばポリエチレングリコール等の各種分子が結合していてもよい。抗体修飾物は、抗体に公知の手法を用いて化学的な修飾を施すことによって得ることができる。抗体のアミノ酸配列、クラス、又はサブクラスは、例えば、ヒト、ヒトを除く哺乳類（例えば、ラット、マウス、ウサギ、ウシ、サル等）、又は鳥類（例えば、ニワトリ）等由来であってもよい。抗体クラスは特に限定されないが、例えばIgM、IgD、IgG、IgA、IgE、又はIgYであってもよい。抗体サブクラスは特に限定されないが、例えばIgG1、IgG2、IgG3、IgG4、IgA1、又はIgA2であってもよい。また抗体は、例えば、単離抗体、精製抗体、又は組換抗体を含む。また抗体は、例えば、in vitro又はin vivoで使用できる。 In one embodiment of the invention an "antibody" comprises a molecule or population thereof capable of specifically binding to a particular epitope on an antigen. The antibody may also be a polyclonal antibody or a monoclonal antibody. The form of the antibody is not particularly limited. For example, a full-length antibody (an antibody having a Fab region and an Fc region), an Fv antibody, a Fab antibody, an F (ab ') 2 antibody, an Fab' antibody, a single chain antibody (for example, scFv) ), DsFv, antigen binding peptide, antibody-like molecule, chimeric antibody, mouse antibody, chicken antibody, humanized antibody, human antibody, or an equivalent (or equivalent) thereof including. Antibodies also include modified or unmodified antibodies. The modified antibody may be bound to an antibody and various molecules such as polyethylene glycol. Modified antibodies can be obtained by chemically modifying antibodies using known techniques. The amino acid sequence, class, or subclass of the antibody may be derived from, for example, humans, mammals other than humans (eg, rats, mice, rabbits, cattle, monkeys, etc.), or birds (eg, chickens). The antibody class is not particularly limited, and may be, for example, IgM, IgD, IgG, IgA, IgE, or IgY. The antibody subclass is not particularly limited, and may be, for example, IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. Antibodies also include, for example, isolated antibodies, purified antibodies, or recombinant antibodies. The antibodies can also be used, for example, in vitro or in vivo.
　本発明の一実施形態において「ポリクローナル抗体」は、例えば、哺乳類（例えば、ラット、マウス、ウサギ、ウシ、サル等）又は鳥類（例えば、ニワトリ）等に、目的の抗原を含む免疫原を投与することによって生成することが可能である。免疫原の投与は、1つ以上の免疫剤、又はアジュバントを注入してもよい。アジュバントは、免疫応答を増加させるために使用されることもあり、フロイントアジュバント（完全又は不完全）、ミネラルゲル（水酸化アルミニウム等）、又は界面活性物質（リゾレシチン等）等を含んでいてもよい。免疫プロトコルは、当該技術分野で公知であり、選択する宿主生物に合わせて、免疫応答を誘発する任意の方法によって実施される場合がある（タンパク質実験ハンドブック, 羊土社(2003):86-91.）。 In one embodiment of the present invention, the "polyclonal antibody" administers an immunogen containing an antigen of interest to, for example, mammals (eg, rats, mice, rabbits, cattle, monkeys etc.) or birds (eg, chickens) etc. It is possible to generate by Administration of immunogen may be infused with one or more immunizing agents, or an adjuvant. Adjuvants may also be used to increase the immune response, and may include Freund's adjuvant (complete or incomplete), mineral gel (such as aluminum hydroxide), or surfactant (such as lysolecithin), etc. . Immunization protocols are known in the art and may be performed by any method that elicits an immune response, depending on the host organism chosen (Protein Experimental Handbook, Yodosha (2003): 86-91 .).
　本発明の一実施形態において「モノクローナル抗体」は、集団を構成する個々の抗体が、実質的に同じエピトープに反応する場合の抗体を含む。又は、集団を構成する個々の抗体が、実質的に同一（自然発生可能な突然変異は許容される）である場合の抗体であってもよい。モノクローナル抗体は高度に特異的であり、異なるエピトープに対応する異なる抗体を典型的に含むような、通常のポリクローナル抗体とは異なる。モノクローナル抗体の作製方法は特に限定されないが、例えば、"Kohler G, Milstein C., Nature. 1975 Aug 7;256(5517):495-497."に掲載されているようなハイブリドーマ法と同様の方法によって作製してもよい。あるいは、モノクローナル抗体は、米国特許第4816567号に記載されているような組換え法と同様の方法によって作製してもよい。又は、モノクローナル抗体は、"Clackson et al., Nature. 1991 Aug 15;352(6336):624-628."、又は"Marks et al., J Mol Biol. 1991 Dec 5;222(3):581-597."に記載されているような技術と同様の方法を用いてファージ抗体ライブラリーから単離してもよい。又は、"タンパク質実験ハンドブック, 羊土社(2003):92-96."に掲載されている方法によって作製してもよい。 In one embodiment of the invention "monoclonal antibodies" include antibodies wherein individual antibodies making up a population respond to substantially the same epitope. Alternatively, the individual antibodies that make up the population may be antibodies that are substantially identical (naturally occurring mutations are acceptable). Monoclonal antibodies are highly specific and differ from normal polyclonal antibodies, which typically include different antibodies corresponding to different epitopes. The method for producing a monoclonal antibody is not particularly limited, but, for example, a method similar to the hybridoma method as disclosed in "Kohler G, Milstein C., Nature. 1975 Aug 7; 256 (5517): 495-497." It may be produced by Alternatively, monoclonal antibodies may be made by methods similar to recombinant methods as described in US Pat. No. 4,816,567. Alternatively, the monoclonal antibody may be “Clackson et al., Nature. 1991 Aug 15; 352 (6336): 624-628.” Or “Marks et al., J MoI Biol. 1991 Dec 5; 222 (3): 581. It may be isolated from phage antibody libraries using methods similar to the techniques as described in "-597." Alternatively, it may be prepared by the method described in "Protein Experiment Handbook, Yodosha (2003): 92-96."
　本発明の一実施形態において「Fv抗体」は、抗原認識部位を含む抗体である。この領域は、非共有結合による1つの重鎖可変ドメイン及び1つの軽鎖可変ドメインの二量体を含む。この構成において、各可変ドメインの3つのCDRは相互に作用してVH-VL二量体の表面に抗原結合部位を形成することができる。本発明の一実施形態において「Fab抗体」は、例えば、Fab領域及びFc領域を含む抗体を蛋白質分解酵素パパインで処理して得られる断片のうち、H鎖のN末端側約半分とL鎖全体が一部のジスルフィド結合を介して結合した抗体である。Fabは、例えば、Fab領域及びFc領域を含む上記の本発明の実施形態に係る抗PAD4抗体を、蛋白質分解酵素パパインで処理して得ることができる。 In one embodiment of the invention an "Fv antibody" is an antibody that comprises an antigen recognition site. This region contains a dimer of one heavy chain variable domain and one light chain variable domain by noncovalent binding. In this configuration, the three CDRs of each variable domain can interact to form an antigen binding site on the surface of the VH-VL dimer. In one embodiment of the present invention, the “Fab antibody” is, for example, about N-terminal half of the H chain and the entire L chain among fragments obtained by treating an antibody containing the Fab region and the Fc region with the proteolytic enzyme papain Is an antibody linked through some disulfide bonds. Fab can be obtained, for example, by treating the anti-PAD4 antibody according to the above-mentioned embodiment of the present invention containing a Fab region and an Fc region with the proteolytic enzyme papain.
　本発明の一実施形態において「F(ab')2抗体」は、例えば、Fab領域及びFc領域を含む抗体を蛋白質分解酵素ペプシンで処理して得られる断片のうち、Fabに相当する部位を2つ含む抗体である。F(ab')2は、例えば、Fab領域及びFc領域を含む上記の本発明の実施形態に係る抗PAD4抗体を、蛋白質分解酵素ペプシンで処理して得ることができる。また、例えば、下記のFab'をチオエーテル結合あるいはジスルフィド結合させることで、作製することができる。本発明の一実施形態において「Fab'抗体」は、例えば、F(ab')2のヒンジ領域のジスルフィド結合を切断して得られる抗体である。例えば、F(ab')2を還元剤ジチオスレイトール処理して得ることができる。 In one embodiment of the present invention, “F (ab ′) 2 antibody” is, for example, a fragment corresponding to Fab in a fragment obtained by treating an antibody containing Fab region and Fc region with proteolytic enzyme pepsin, Antibodies that contain F (ab ′) 2 can be obtained, for example, by treating the anti-PAD 4 antibody according to the above-described embodiment of the present invention containing a Fab region and an Fc region with the proteolytic enzyme pepsin. Also, for example, it can be produced by thioether bond or disulfide bond of the following Fab ′. In one embodiment of the present invention, the “Fab ′ antibody” is, for example, an antibody obtained by cleaving a disulfide bond in the hinge region of F (ab ′) 2 . For example, F (ab ') 2 can be obtained by treatment with a reducing agent dithiothreitol.
　本発明の一実施形態において「scFv抗体」は、VHとVLとが適当なペプチドリンカーを介して連結した抗体である。scFv抗体は、例えば、上記の本発明の実施形態に係る抗PAD4抗体のVHおよびVLをコードするcDNAを取得し、VH-ペプチドリンカー-VLをコードするポリヌクレオチドを構築し、そのポリヌクレオチドをベクターに組み込み、発現用の細胞を用いて生産できる。本発明の一実施形態において「dsFv」は、VH及びVL中にシステイン残基を導入したポリペプチドを、上記システイン残基間のジスルフィド結合を介して結合させた抗体である。システイン残基に導入する位置はReiterらにより示された方法（Reiter et al., Protein Eng. 1994 May;7(5):697-704.）に従って、抗体の立体構造予測に基づいて選択することができる。 In one embodiment of the present invention, the "scFv antibody" is an antibody in which VH and VL are linked via a suitable peptide linker. For example, the scFv antibody obtains cDNAs encoding VH and VL of the anti-PAD4 antibody according to the above-mentioned embodiment of the present invention, constructs a polynucleotide encoding VH-peptide linker-VL, and makes the polynucleotide a vector And can be produced using cells for expression. In one embodiment of the present invention, "dsFv" is an antibody in which a polypeptide having a cysteine residue introduced into VH and VL is linked via a disulfide bond between the above-mentioned cysteine residues. The position to be introduced into the cysteine residue should be selected based on the prediction of the three-dimensional structure of the antibody according to the method shown by Reiter et al. (Reiter et al., Protein Eng. 1994 May; 7 (5): 697-704.). Can.
　本発明の一実施形態において「抗原結合性ペプチド」は、例えば、抗体のVH及びVL、又はそれらのCDR1、2、及び3を含んで構成され、且つPAD4に対する結合性を有するペプチドを含む。複数のCDRを含むペプチドは、直接又は適当なペプチドリンカーを介して結合させることができる。本発明の一実施形態において「抗体様分子」は、PAD4に対する結合性を有する分子であって、例えば、アフィボディ、DARPins、アンチカリン、モノボディ、アドネクチン、サロボディ、アビマー、又はアフィリンを含む。抗体様分子の特性及び作製法等の詳細は、"Helma et al., J Cell Biol. 2015 Jun 8;209(5):633-44."や"Angeline et al., Future Med Chem. 2017 Aug;9(12):1301-1304."及びそれらの文献に記載された引用文献等に記載されている。 In one embodiment of the present invention, the “antigen-binding peptide” includes, for example, a VH and VL of an antibody, or CDRs 1, 2 and 3 thereof, and a peptide having PAD4 binding activity. Peptides containing multiple CDRs can be linked directly or via appropriate peptide linkers. In one embodiment of the present invention, the “antibody-like molecule” is a molecule having a binding property to PAD4 and includes, for example, affibody, DARPins, anticalin, monobody, adnectin, salobody, avimer, or affilin. Details of the properties and preparation methods of antibody-like molecules are described in "Helma et al., J Cell Biol. 2015 Jun 8; 209 (5): 633-44." And "Angeline et al., Future Med Chem. 2017 Aug. 9 (12): 1301-1304. "And references cited therein.
　上記のFv抗体、Fab抗体、F(ab')2抗体、Fab' 抗体、scFv抗体、dsFv抗体、抗原結合性ペプチド（以下、「Fv抗体等」と称することもある）の生産方法は特に限定しない。例えば、上記の本発明の実施形態に係る抗PAD4抗体におけるFv抗体等の領域をコードするDNAを発現用ベクターに組み込み、発現用細胞を用いて生産できる。又は、Fmoc法（フルオレニルメチルオキシカルボニル法）、tBOC法（t-ブチルオキシカルボニル法）などの化学合成法によって生産してもよい。なお上記の本発明の実施形態に係る抗原結合性抗体断片は、上記Fv抗体等の1種以上を含んでいてもよい。 The method for producing the above Fv antibody, Fab antibody, F (ab ') 2 antibody, Fab' antibody, scFv antibody, dsFv antibody, antigen-binding peptide (hereinafter sometimes referred to as "Fv antibody etc.") is particularly limited do not do. For example, a DNA encoding a region such as an Fv antibody in the anti-PAD 4 antibody according to the embodiment of the present invention described above can be incorporated into an expression vector and produced using expression cells. Alternatively, they may be produced by a chemical synthesis method such as Fmoc method (fluorenylmethyloxycarbonyl method), tBOC method (t-butyloxycarbonyl method) or the like. The antigen-binding antibody fragment according to the embodiment of the present invention may contain one or more of the Fv antibody and the like.
　本発明の一実施形態において「キメラ抗体」は、例えば、異種生物間における抗体の可変領域と、抗体の定常領域とを連結したもので、遺伝子組換え技術によって構築できる。例えば、マウス-ヒトキメラ抗体、ニワトリ-ヒトキメラ抗体、ニワトリ-マウスキメラ抗体等が挙げられる。マウス-ヒトキメラ抗体は、例えば、"Roguska et al., Proc Natl Acad Sci U S A. 1994 Feb 1;91(3):969-973."に記載の方法で作製できる。マウス-ヒトキメラ抗体を作製するための基本的な方法は、例えば、クローン化されたcDNAに存在するマウスリーダー配列及び可変領域配列を、哺乳類細胞の発現ベクター中にすでに存在するヒト抗体定常領域をコードする配列に連結する。又は、クローン化されたcDNAに存在するマウスリーダー配列及び可変領域配列をヒト抗体定常領域をコードする配列に連結した後、哺乳類細胞発現ベクターに連結してもよい。ヒト抗体定常領域の断片は、任意のヒト抗体のH鎖定常領域及びヒト抗体のL鎖定常領域のものとすることができ、例えばヒトH鎖のものについてはCγ1、Cγ2、Cγ3又はCγ4を、L鎖のものについてはCλ又はCκを各々挙げることができる。 In one embodiment of the present invention, a "chimeric antibody" is, for example, one obtained by linking the variable region of an antibody between heterologous organisms and the constant region of the antibody, and can be constructed by genetic recombination technology. For example, mouse-human chimeric antibodies, chicken-human chimeric antibodies, chicken-mouse chimeric antibodies and the like can be mentioned. A mouse-human chimeric antibody can be produced, for example, by the method described in "Roguska et al., Proc Natl Acad Sci US A. 1994 Feb 1; 91 (3) 969-973.". A basic method for producing mouse-human chimeric antibodies is, for example, encoding the mouse leader and variable region sequences present in the cloned cDNA, and human antibody constant regions already present in mammalian cell expression vectors. Linked to the sequence Alternatively, the mouse leader sequence and variable region sequence present in the cloned cDNA may be ligated to a sequence encoding a human antibody constant region and then ligated to a mammalian cell expression vector. The fragments of the human antibody constant region can be those of the heavy chain constant region of any human antibody and the light chain constant region of the human antibody, eg, for human heavy chain, Cγ1, Cγ2, Cγ3 or Cγ4 As for the L chain one, Cλ or C の も の can be mentioned respectively.
　本発明の一実施形態において「ヒト化抗体」は、例えば、非ヒト種由来の1つ以上のCDR、及びヒト免疫グロブリン由来のフレームワーク領域、さらにヒト免疫グロブリン由来の定常領域を有し、所望の抗原に結合する抗体である。抗体のヒト化には、当該技術分野で既知の種々の手法を使用してもよい。本発明の一実施形態において「ヒト抗体」は、例えば、抗体を構成する重鎖の可変領域及び定常領域、軽鎖の可変領域及び定常領域を含む領域が、ヒトイムノグロブリンをコードする遺伝子に由来する抗体である。ヒト抗体の作製には、当該技術分野で既知の種々の手法を使用してもよい。 In one embodiment of the present invention, a "humanized antibody" has, for example, one or more CDRs from a non-human species, and a framework region derived from human immunoglobulin, and further a constant region derived from human immunoglobulin. Is an antibody that binds to the antigen of For humanization of antibodies, various techniques known in the art may be used. In one embodiment of the present invention, the “human antibody” is derived from, for example, a region including the variable region and constant region of heavy chain constituting the antibody, the variable region and constant region of light chain, from a gene encoding human immunoglobulin Antibody. Various techniques known in the art may be used to generate human antibodies.
　本発明の一実施形態において「重鎖」は、典型的には、全長抗体の主な構成要素である。重鎖は、通常、軽鎖とジスルフィド結合及び非共有結合によって結合している。重鎖のN末端側のドメインには、同種の同一クラスの抗体でもアミノ酸配列が一定しない可変領域（VH）と呼ばれる領域が存在し、一般的に、VHが抗原に対する特異性、親和性に大きく寄与していることが知られている。例えば、"Reiter et al., J Mol Biol. 1999 Jul 16;290(3):685-98."にはVHのみの分子を作製したところ、抗原と特異的に、高い親和性で結合したことが記載されている。さらに、"Wolfson W, Chem Biol. 2006 Dec;13(12):1243-1244."には、ラクダの抗体の中には、軽鎖を持たない重鎖のみの抗体が存在していることが記載されている。 In one embodiment of the invention "heavy chain" is typically the main component of full-length antibodies. The heavy chain is usually linked to the light chain by disulfide bonds and noncovalent bonds. In the domain at the N-terminal side of the heavy chain, there is a region called variable region (VH) where the amino acid sequence is not constant even in the same kind of antibody of the same class. Generally, VH has a large affinity to the antigen. It is known to contribute. For example, when a molecule consisting only of VH was produced in "Reiter et al., J Mol Biol. 1999 Jul 16; 290 (3): 685-98.", It specifically bound to the antigen with high affinity. Is described. Furthermore, in "Wolfson W, Chem Biol. 2006 Dec; 13 (12): 1243-1244.", There is a heavy chain-only antibody which does not have a light chain among camel antibodies. Have been described.
　本発明の一実施形態において「CDR（相補性決定領域）」は、抗体において、実際に抗原に接触して結合部位を形成している領域である。一般的にCDRは、抗体のFv（可変領域：重鎖可変領域（VH）及び軽鎖可変領域（VL）を含む）上に位置している。また一般的にCDRは、5～30アミノ酸残基程度からなるCDR1、CDR2、CDR3が存在する。そして、重鎖のCDRが抗体の抗原への結合に特に寄与していることが知られている。またCDRの中でも、CDR3が抗体の抗原への結合における寄与が最も高いことが知られている。例えば、"Willy et al., Biochemical and Biophysical Research Communications Volume 356, Issue 1, 27 April 2007, Pages 124-128"には、重鎖CDR3を改変させることで抗体の結合能を上昇させたことが記載されている。CDR以外のFv領域はフレームワーク領域（FR）と呼ばれ、FR1、FR2、FR3およびFR4からなり、抗体間で比較的よく保存されている（Kabat et al.,「Sequence of Proteins of Immunological Interest」US Dept. Health and Human Services, 1983.）。 In one embodiment of the present invention, "CDR (complementarity determining region)" is a region in an antibody that actually contacts an antigen to form a binding site. Generally, the CDRs are located on the Fv (variable region: including heavy chain variable region (VH) and light chain variable region (VL)) of the antibody. Generally, CDRs include CDR1, CDR2 and CDR3 consisting of about 5 to 30 amino acid residues. And, it is known that heavy chain CDRs particularly contribute to antibody binding to antigen. Among CDRs, CDR3 is known to have the highest contribution in antibody binding to antigen. For example, "Willy et al., Biochemical and Biophysical Research Communications Volume 356, Issue 1, 27 April 2007, Pages 124-128" describes that the binding ability of the antibody was increased by modifying the heavy chain CDR3. It is done. Fv regions other than CDRs are called framework regions (FR) and consist of FR1, FR2, FR3 and FR4 and are relatively well conserved among antibodies (Kabat et al., "Sequence of Proteins of Immunological Interest") US Dept. Health and Human Services, 1983.).
　CDRの定義およびその位置を決定する方法は複数報告されている。例えば、Kabatの定義 （Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)）、又はChothiaの定義（Chothia et al., J. Mol. Biol.,1987;196:901-917）を採用してもよい。本発明の一実施形態においては、Kabatの定義を好適な例として採用するが、必ずしもこれに限定されない。また、場合によっては、Kabatの定義とChothiaの定義の両方を考慮して決定しても良く、例えば、各々の定義によるCDRの重複部分を、又は各々の定義によるCDRの両方を含んだ部分をCDRとすることもできる。そのような方法の具体例としては、Kabatの定義とChothiaの定義の折衷案である、Oxford Molecular's AbM antibody modeling softwareを用いたMartinらの方法（Proc.Natl.Acad.Sci.USA,1989;86:9268-9272）がある。 Several definitions of CDRs and methods of determining their location have been reported. For example, the definition of Kabat (Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)), or Chothia (Chothia et al., J. Mol. Biol. , 1987; 196: 901-917) may be adopted. In one embodiment of the present invention, the definition of Kabat is taken as a preferred example but not necessarily limited thereto. Also, in some cases, it may be determined in consideration of both the definition of Kabat and the definition of Chothia, for example, a portion including overlapping portions of CDRs according to each definition or portions including both CDRs according to each definition It can also be a CDR. As a specific example of such a method, Martin et al.'S method (Proc. Natl. Acad. Sci. USA, 1989; 86; 86) using Oxford Molecular's AbM antibody modeling software, which is a compromise between the Kabat definition and the Chothia definition. : 9268-9272).
　本発明の一実施形態は、(a)重鎖CDR1～3及び軽鎖CDR1～3が、それぞれ配列番号19～24で示されるアミノ酸配列を含む抗体、(b)重鎖CDR1～3及び軽鎖CDR1～3が、それぞれ配列番号25～30で示されるアミノ酸配列を含む抗体、(c)重鎖CDR1～3及び軽鎖CDR1～3が、それぞれ配列番号31～36で示されるアミノ酸配列を含む抗体、及び(d)重鎖CDR1～3及び軽鎖CDR1～3が、それぞれ配列番号37～42で示されるアミノ酸配列を含む抗体、からなる群から選ばれる1種以上の抗PAD4抗体である。この抗体を用いれば、活性型PAD4のシトルリン化活性を阻害することができる。上記(a)～(d)の抗体のCDR配列は、後述の実施例に記載のP1～P4の抗体が有するCDR配列にそれぞれ対応している。また本発明の別の実施形態は、上に列挙した重鎖CDR1～3及び軽鎖CDR1～3のアミノ酸配列のセットのうち、少なくとも1つのセットを含む抗PAD4抗体である。 One embodiment of the present invention is an antibody comprising (a) an amino acid sequence as shown in SEQ ID NOs: 19 to 24, and (b) heavy chain CDRs 1 to 3 and light chain, respectively. An antibody comprising an amino acid sequence as shown in SEQ ID NO: 25-30, (c) An antibody comprising an amino acid sequence as shown in SEQ ID NO: 31-36, and (c) heavy chain CDR1-3 and light chain CDR1-3. And (d) an antibody comprising an amino acid sequence represented by SEQ ID NOs: 37 to 42, respectively, heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3 are one or more anti-PAD4 antibodies selected from the group consisting of This antibody can be used to inhibit the citrullinating activity of active PAD4. The CDR sequences of the above-mentioned antibodies (a) to (d) correspond to the CDR sequences possessed by the P1 to P4 antibodies described in the examples below, respectively. In addition, another embodiment of the present invention is an anti-PAD4 antibody comprising at least one set of the heavy chain CDR1-3 and light chain CDR1-3 amino acid sequences listed above.
　上記(a)の抗体は、重鎖FR1～4及び軽鎖FR1～4が、それぞれ配列番号43～50で示されるアミノ酸配列を含んでいてもよい。上記(b)の抗体は、重鎖FR1～4及び軽鎖FR1～4が、それぞれ配列番号51～58で示されるアミノ酸配列を含んでいてもよい。上記(c)の抗体は、重鎖FR1～4及び軽鎖FR1～4が、それぞれ配列番号59～66で示されるアミノ酸配列を含んでいてもよい。上記(d)の抗体は、重鎖FR1～4及び軽鎖FR1～4が、それぞれ配列番号67～74で示されるアミノ酸配列を含んでいてもよい。これらのFR配列は、後述の実施例に記載のP1～P4の抗体が有するFR配列にそれぞれ対応している。また本発明の別の実施形態は、上に列挙した重鎖FR1～4及び軽鎖FR1～4のアミノ酸配列のセットのうち、少なくとも1つのセットを含む抗PAD4抗体である。 In the antibody of (a) above, the heavy chain FR1 to 4 and the light chain FR1 to 4 may contain the amino acid sequences shown by SEQ ID NOs: 43 to 50, respectively. In the antibody (b), heavy chain FR1 to 4 and light chain FR1 to 4 may contain the amino acid sequences shown by SEQ ID NOs: 51 to 58, respectively. In the antibody of (c) above, the heavy chain FR1 to 4 and the light chain FR1 to 4 may contain the amino acid sequences shown by SEQ ID NOs: 59 to 66, respectively. In the antibody of (d) above, the heavy chain FR1 to 4 and the light chain FR1 to 4 may contain the amino acid sequences shown by SEQ ID NOs: 67 to 74, respectively. These FR sequences correspond to the FR sequences possessed by the P1 to P4 antibodies described in the examples below, respectively. Also, another embodiment of the present invention is an anti-PAD4 antibody comprising at least one set of the heavy chain FR1-4 and light chain FR1-4 amino acid sequence sets listed above.
　本発明の一実施形態に係る抗体は、重鎖CDR1に配列番号19、25、31、又は37で示されるアミノ酸配列を含んでいてもよく、重鎖CDR2に配列番号20、26、32、又は38で示されるアミノ酸配列を含んでいてもよく、重鎖CDR3に配列番号21、27、33、又は39で示されるアミノ酸配列を含んでいてもよく、軽鎖CDR1に配列番号22、28、34、又は40で示されるアミノ酸配列を含んでいてもよく、軽鎖CDR2に配列番号23、29、35、又は41で示されるアミノ酸配列を含んでいてもよく、軽鎖CDR3に配列番号24、30、36、又は42で示されるアミノ酸配列を含んでいてもよい。 The antibody according to one embodiment of the present invention may comprise the amino acid sequence shown in SEQ ID NO: 19, 25, 31 or 37 in heavy chain CDR1 and SEQ ID NO: 20, 26, 32 or in heavy chain CDR2. The heavy chain CDR3 may contain the amino acid sequence shown in SEQ ID NO: 21, 27, 33 or 39, and the light chain CDR1 may contain the amino acid sequence shown in SEQ ID NO: 22, 28, 34 Or 40, and the light chain CDR2 may contain the amino acid sequence shown in SEQ ID NO: 23, 29, 35, or 41, and the light chain CDR3 may contain SEQ ID NO: 24, 30 The amino acid sequence shown by 36, or 42 may be included.
　本発明の一実施形態に係る抗体は、重鎖FR1に配列番号43、51、59、又は67で示されるアミノ酸配列を含んでいてもよく、重鎖FR2に配列番号44、52、60、又は68で示されるアミノ酸配列を含んでいてもよく、重鎖FR3に配列番号45、53、61、又は69で示されるアミノ酸配列を含んでいてもよく、重鎖FR4に配列番号46、54、62、又は70で示されるアミノ酸配列を含んでいてもよく、軽鎖FR1に配列番号47、55、63、又は71で示されるアミノ酸配列を含んでいてもよく、軽鎖FR2に配列番号48、56、64、又は72で示されるアミノ酸配列を含んでいてもよく、軽鎖FR3に配列番号49、57、65、又は73で示されるアミノ酸配列を含んでいてもよく、軽鎖FR4に配列番号50、58、66、又は74で示されるアミノ酸配列を含んでいてもよい。 The antibody according to one embodiment of the present invention may comprise the amino acid sequence shown in SEQ ID NO: 43, 51, 59 or 67 in heavy chain FR1, and SEQ ID NO: 44, 52, 60 or in heavy chain FR2. The heavy chain FR3 may contain the amino acid sequence shown in SEQ ID NO: 68, and the heavy chain FR3 may contain the amino acid sequence shown in SEQ ID NO: 45, 53, 61 or 69, and the heavy chain FR4 may contain the amino acid sequence shown in SEQ ID NO: 46, 54, 62 Or the amino acid sequence represented by SEQ ID NO: 47, 55 may contain the amino acid sequence represented by SEQ ID NO: 47, 55, 63, or 71 in the light chain FR1; , 64, or 72. The light chain FR3 may include the amino acid sequence represented by SEQ ID NO: 49, 57, 65, or 73, and the light chain FR4 may include the amino acid sequence of SEQ ID NO: 50. The amino acid sequence shown by 58, 66, or 74 may be included.
　本発明の一実施形態に係る抗PAD4抗体は、scFvの形態であってもよく、その場合、重鎖と軽鎖間にリンカーを有していてもよい。リンカーは、例えば、代表的には、GとPからなる0～5アミノ酸の配列が挙げられるがこれに限定されない。リンカーは、例えば、配列番号75で示されるアミノ酸配列を有していてもよい。リンカーは必須ではなく、存在しなくてもよい。 The anti-PAD4 antibody according to one embodiment of the present invention may be in the form of scFv, in which case it may have a linker between the heavy chain and the light chain. The linker is, for example, typically, but not limited to, a sequence of 0 to 5 amino acids consisting of G and P. The linker may have, for example, the amino acid sequence set forth in SEQ ID NO: 75. The linker is not required and may not be present.
　本発明の一実施形態に係る抗PAD4抗体は、重鎖可変領域が配列番号3、4、5、又は6で示されるアミノ酸配列を含んでいてもよく、軽鎖可変領域が配列番号11、12、13、又は14で示されるアミノ酸配列を含んでいてもよい。 In the anti-PAD 4 antibody according to one embodiment of the present invention, the heavy chain variable region may contain the amino acid sequence shown by SEQ ID NO: 3, 4, 5, or 6, and the light chain variable region comprises SEQ ID NO: 11, 12 , 13 or 14 may be included.
　本発明の一実施形態において「アミノ酸」は、アミノ基とカルボキシル基を持つ有機化合物の総称である。本発明の実施形態に係る抗体が「特定のアミノ酸配列」を含むとき、そのアミノ酸配列中のいずれかのアミノ酸が化学修飾を受けていてもよい。また、そのアミノ酸配列中のいずれかのアミノ酸が塩、又は溶媒和物を形成していてもよい。また、そのアミノ酸配列中のいずれかのアミノ酸がL型、又はD型であってもよい。それらのような場合でも、本発明の実施形態に係る抗体は、上記「特定のアミノ酸配列」を含むといえる。蛋白質に含まれるアミノ酸が生体内で受ける化学修飾としては、例えば、N末端修飾（例えば、アセチル化、ミリストイル化等）、C末端修飾（例えば、アミド化、グリコシルホスファチジルイノシトール付加等）、又は側鎖修飾（例えば、リン酸化、糖鎖付加等）等が知られている。 In one embodiment of the present invention, "amino acid" is a generic term for organic compounds having an amino group and a carboxyl group. When an antibody according to an embodiment of the present invention contains a "specific amino acid sequence", any amino acid in the amino acid sequence may be chemically modified. In addition, any amino acid in the amino acid sequence may form a salt or a solvate. In addition, any amino acid in the amino acid sequence may be L-form or D-form. Even in those cases, it can be said that the antibody according to the embodiment of the present invention comprises the above-mentioned "specific amino acid sequence". Examples of chemical modifications that amino acids contained in proteins undergo in vivo include, for example, N-terminal modification (eg, acetylation, myristoylation etc.), C-terminal modification (eg amidification, glycosylphosphatidylinositol addition etc.), or side chain Modifications (eg, phosphorylation, glycosylation, etc.) and the like are known.
　本発明の一実施形態は、上記の本発明の実施形態に係る抗PAD4抗体をコードするポリヌクレオチド又はベクターである。このポリヌクレオチド又はベクターを細胞に導入することによって、形質転換体を作製できる。形質転換体は、ヒト又はヒトを除く哺乳動物（例えば、ラット、マウス、モルモット、ウサギ、ウシ、サル等）の細胞であってもよい。哺乳動物細胞としては、例えば、チャイニーズハムスター卵巣細胞(CHO細胞)、サル細胞COS-7、ヒト胎児由来腎臓細胞（例えば、HEK293細胞）などが挙げられる。又は、形質転換体はEscherichia属菌、酵母等であってもよい。上記ポリヌクレオチド又はベクターは、抗PAD4抗体を発現可能に構築されていてもよい。上記ポリヌクレオチド又はベクターは、例えば、プロモーター、エンハンサー、複製開始点、又は抗生物質耐性遺伝子など、蛋白質発現に必要な構成要素を含んでいてもよい。上記ポリヌクレオチド又はベクターは、異種由来の塩基配列を有していてもよい。異種由来の塩基配列は、例えば、ヒト及びヒトを除く生物（例えば、細菌、古細菌、酵母、昆虫、鳥類、ウイルス、又はヒトを除く哺乳動物等）からなる群から選ばれる2種以上の生物由来の塩基配列を含んでいてもよい。 One embodiment of the present invention is a polynucleotide or a vector encoding the anti-PAD4 antibody according to the above-mentioned embodiment of the present invention. Transformants can be produced by introducing this polynucleotide or vector into cells. The transformant may be a cell of human or a mammal other than human (eg, rat, mouse, guinea pig, rabbit, bovine, monkey, etc.). Mammalian cells include, for example, Chinese hamster ovary cells (CHO cells), monkey cells COS-7, human fetal kidney cells (eg, HEK 293 cells) and the like. Alternatively, the transformant may be Escherichia genus, yeast or the like. The polynucleotide or vector may be constructed so as to express an anti-PAD4 antibody. The polynucleotide or vector may contain, for example, components necessary for protein expression, such as a promoter, an enhancer, an origin of replication, or an antibiotic resistance gene. The polynucleotide or vector may have a base sequence of heterologous origin. The base sequence derived from different species is, for example, two or more organisms selected from the group consisting of humans and non-human organisms (eg, bacteria, archaea, yeast, insects, birds, viruses, mammals other than human, etc.) You may contain the base sequence derived from.
　上記のベクターとしては、例えば大腸菌由来のプラスミド（例えばpET-Blue）、枯草菌由来のプラスミド（例えばpUB110）、酵母由来プラスミド（例えばpSH19）、動物細胞発現プラスミド（例えばpA1-11、pcDNA3.1-V5/His-TOPO）、λファージなどのバクテリオファージ、ウイルス由来のベクターなどを用いることができる。ベクターは発現ベクターであってもよく、環状であってもよい。 Examples of the vector include a plasmid derived from E. coli (eg, pET-Blue), a plasmid derived from Bacillus subtilis (eg, pUB110), a yeast-derived plasmid (eg, pSH19), an animal cell expression plasmid (eg, pA1-11, pcDNA3.1- Bacteriophages such as V5 / His-TOPO), λ phage, and vectors derived from viruses can be used. The vector may be an expression vector or may be circular.
　上記のポリヌクレオチド又はベクターの細胞への導入方法としては、例えば、リン酸カルシウム法、リポフェクション法、エレクトロポレーション法、アデノウイルスによる方法、レトロウイルスによる方法、又はマイクロインジェクションなどを使用できる（改訂第4版 新 遺伝子工学ハンドブック, 羊土社(2003):152-179.）。細胞を用いた抗体の生産方法としては、例えば、"タンパク質実験ハンドブック,羊土社(2003):128-142."に記載の方法を使用できる。 As a method for introducing the above polynucleotide or vector into cells, for example, calcium phosphate method, lipofection method, electroporation method, method by adenovirus, method by retrovirus, microinjection, etc. can be used (revised 4 th ed. New Genetic Engineering Handbook, Yodosha (2003): 152-179.). As a method for producing an antibody using cells, for example, the method described in "Protein Experimental Handbook, Yodosha (2003): 128-142." Can be used.
　本発明の一実施形態は、上記の本発明の実施形態に係るポリヌクレオチド又はベクターを含有する細胞である。本発明の一実施形態は、上記の細胞を増殖させる工程を含む、抗PAD4抗体の生産方法である。上記増殖させる工程は、培養する工程を含む。またこの生産方法は、抗PAD4抗体を回収する工程を含んでいてもよい。またこの生産方法は、細胞培養液を調製する工程を含んでいてもよい。またこの生産方法は、抗PAD4抗体を精製する工程を含んでいてもよい。 One embodiment of the present invention is a cell containing the polynucleotide or vector according to the above-mentioned embodiment of the present invention. One embodiment of the present invention is a method of producing an anti-PAD4 antibody, which comprises the step of growing the cells described above. The growing step includes a culturing step. The production method may also include the step of recovering the anti-PAD4 antibody. The production method may also include the step of preparing a cell culture solution. The production method may also include the step of purifying the anti-PAD4 antibody.
　本発明の一実施形態において、抗体の精製は、例えば、硫酸アンモニウム、エタノール沈殿、プロテインA、プロテインG、ゲルろ過クロマトグラフィー、陰イオン、陽イオン交換クロマトグラフィー、ホスホセルロースクロマトグラフィー、疎水性相互作用クロマトグラフィー、アフィニティークロマトグラフィー、ヒドロキシルアパタイトクロマトグラフィー、又はレクチンクロマトグラフィーなどを用いることができる（タンパク質実験ハンドブック, 羊土社(2003):27-52.）。 In one embodiment of the present invention, purification of the antibody is carried out, for example, with ammonium sulfate, ethanol precipitation, protein A, protein G, gel filtration chromatography, anion, cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography Chromatography, affinity chromatography, hydroxylapatite chromatography, or lectin chromatography can be used (Protein Experimental Handbook, Yodosha (2003): 27-52.).
　本発明の一実施形態は、上記の本発明の実施形態に係る抗PAD4抗体を含む、組成物である。この組成物を用いれば、活性型PAD4を効率的に検出できる。また、活性型PAD4のシトルリン化を効率的に阻害することができる。また、RA又は関節炎を治療することができる。この組成物が含有する成分は特に限定されず、例えば、緩衝液を含んでいてもよい。この組成物に対して、後述の阻害剤及び医薬組成物に係る種々の実施形態（例えば、担体を含有可能なこと等）の1つ以上を適用してもよい。 One embodiment of the present invention is a composition comprising the anti-PAD4 antibody according to the above-mentioned embodiment of the present invention. Using this composition, active PAD 4 can be detected efficiently. In addition, citrullination of active PAD 4 can be efficiently inhibited. Also, RA or arthritis can be treated. The components contained in this composition are not particularly limited, and may contain, for example, a buffer. To this composition, one or more of the various embodiments (for example, the possibility of containing a carrier, etc.) of the inhibitors and pharmaceutical compositions described below may be applied.
　本発明の一実施形態は、上記の本発明の実施形態に係る抗PAD4抗体を含む、PAD4の機能阻害剤である。この阻害剤を用いれば、活性型PAD4のシトルリン化活性を効率的に阻害することができる。上記阻害剤による活性型PAD4のシトルリン化活性の低下率は、15、20、30、40、50、60、70、80％以上であってもよく、それらいずれか2つの値の範囲内であってもよい。この低下率は、例えば、TBS Bufferを用いたときの低下率を0％としたときの相対割合で表してもよい。この低下率は、20％以上が好ましく、30％以上がより好ましい。本発明の一実施形態において「剤」は、例えば、研究又は治療に用いられる組成物を含む。上記阻害剤は、例えば、RA又は関節炎の治療剤を含む。上記阻害剤は、例えば、in vitro又はin vivoで用いることができる。上記阻害剤は、上記の本発明の実施形態に係る組成物を含んでいてもよい。本発明の一実施形態は、上記の本発明の実施形態に係る抗PAD4抗体と、PAD4とを接触させる工程を含む、PAD4の機能阻害方法である。本発明の一実施形態は、上記の本発明の実施形態に係る抗PAD4抗体を患者に投与する工程を含む、PAD4の機能阻害方法である。上記阻害方法は、研究又は治療のために行われる阻害方法を含む。本発明の一実施形態は、PAD4の機能阻害剤を生産するための、上記の本発明の実施形態に係る抗PAD4抗体の使用である。なお、繰り返しになるが、PAD4は活性型PAD4であってもよい。 One embodiment of the present invention is a functional inhibitor of PAD4 comprising the anti-PAD4 antibody according to the above-mentioned embodiment of the present invention. This inhibitor can be used to effectively inhibit the citrullinating activity of active PAD4. The reduction rate of the citrullinating activity of active PAD 4 by the inhibitor may be 15, 20, 30, 40, 50, 60, 70, 80% or more, and any of these is within the range of two values. May be This reduction rate may be expressed, for example, as a relative ratio when the reduction rate when TBS Buffer is used is 0%. The reduction rate is preferably 20% or more, and more preferably 30% or more. In one embodiment of the invention an "agent" comprises, for example, a composition used for research or treatment. The inhibitor includes, for example, a therapeutic agent for RA or arthritis. The above inhibitors can be used, for example, in vitro or in vivo. The inhibitor may comprise the composition according to the embodiment of the invention described above. One embodiment of the present invention is a method for inhibiting the function of PAD4, comprising the step of bringing an anti-PAD4 antibody according to the above-mentioned embodiment of the present invention into contact with PAD4. One embodiment of the present invention is a method for inhibiting PAD4 function comprising administering an anti-PAD4 antibody according to the above-mentioned embodiment of the present invention to a patient. The above inhibition methods include inhibition methods performed for research or treatment. One embodiment of the present invention is the use of the anti-PAD4 antibody according to the above-mentioned embodiment of the present invention for producing a functional inhibitor of PAD4. Note that, again, PAD 4 may be active PAD 4.
　本発明の一実施形態は、上記の本発明の実施形態に係る抗PAD4抗体を含む、医薬組成物である。この医薬組成物を用いれば、RA又は関節炎を治療することができる。上記医薬組成物は、薬理学的に許容される1つ以上の担体を含んでいてもよい。上記医薬組成物は、例えば、RA又は関節炎の治療用医薬組成物を含む。上記医薬組成物は、上記の本発明の実施形態に係る組成物を含んでいてもよい。本発明の一実施形態は、上記の本発明の実施形態に係る抗PAD4抗体（又は抗PAD4抗体を含む医薬組成物）を患者に投与する工程を含む、疾患の治療方法である。上記疾患は、例えば、RA又は関節炎を含む。本発明の一実施形態は、医薬組成物を生産するための、上記の本発明の実施形態に係る抗PAD4抗体の使用である。 One embodiment of the present invention is a pharmaceutical composition comprising the anti-PAD4 antibody according to the above-mentioned embodiment of the present invention. This pharmaceutical composition can be used to treat RA or arthritis. The pharmaceutical composition may comprise one or more pharmacologically acceptable carriers. The above pharmaceutical composition includes, for example, a pharmaceutical composition for treating RA or arthritis. The above-mentioned pharmaceutical composition may contain the composition concerning the above-mentioned embodiment of the present invention. One embodiment of the present invention is a method for treating a disease, which comprises the step of administering to a patient an anti-PAD4 antibody (or a pharmaceutical composition containing the anti-PAD4 antibody) according to the above-mentioned embodiment of the present invention. The diseases include, for example, RA or arthritis. One embodiment of the present invention is the use of the anti-PAD4 antibody according to the above-mentioned embodiment of the present invention for producing a pharmaceutical composition.
　RA発症患者数は全世界で7000万人にも上ると報告されている。現在いくつかの治療薬が市販されているが、既存薬の効かない患者が一定割合存在しており、患者の60～80％は満足のいく治療を受けられていないともいわれている。また、既存薬には、副作用の問題も指摘されている。上記の本発明の実施形態に係る抗PAD4抗体を用いれば、新規のメカニズムでRAを治療することができる。 The number of RA cases has been reported to be as high as 70 million worldwide. Several therapeutic agents are currently marketed, but a certain percentage of patients do not respond to the existing agents, and it is said that 60 to 80% of patients do not receive satisfactory treatment. In addition, there are also problems with side effects in existing drugs. Using the anti-PAD4 antibody according to the embodiment of the present invention described above, RA can be treated by a novel mechanism.
　本発明の一実施形態は、上記の本発明の実施形態に係る抗PAD4抗体を含む、RA又は関節炎の診断薬である。この診断薬を用いれば、効率的にRA又は関節炎を診断できる。本発明の一実施形態は、上記の本発明の実施形態に係る抗PAD4抗体と、患者サンプルを接触させる工程を含む、RA又は関節炎の診断方法である。本発明の一実施形態は、上記の本発明の実施形態に係る抗PAD4抗体を含む、PAD4の検出試薬である。この試薬を用いれば、効率的にPAD4を検出できる。本発明の一実施形態は、上記の本発明の実施形態に係る抗PAD4抗体と、試験サンプルを接触させる工程を含む、PAD4の検出方法である。この方法は、上記特許文献3に記載されているようなEDTA処理をしない場合でも、PAD4を適切に検出できる。本発明の一実施形態は、上記の本発明の実施形態に係る抗PAD4抗体を含む、キットである。このキットを用いれば、例えば、疾患の治療、診断、又はPAD4の検出ができる。このキットは、例えば、上記の本発明の実施形態に係る組成物、阻害剤、医薬組成物、診断薬、又は検出試薬を含んでいてもよく、取扱説明書、緩衝液、容器（例えば、バイアル、又はシリンジ）、又は包装を含んでいてもよい。本明細書において、患者サンプル又は試験サンプルは、血液、血清、又は血漿であってもよい。 One embodiment of the present invention is a diagnostic agent for RA or arthritis, which comprises the anti-PAD4 antibody according to the above-mentioned embodiment of the present invention. This diagnostic agent can be used to diagnose RA or arthritis efficiently. One embodiment of the present invention is a method for diagnosing RA or arthritis, which comprises the step of contacting a patient sample with the anti-PAD4 antibody according to the above-mentioned embodiment of the present invention. One embodiment of the present invention is a detection reagent for PAD4 comprising the anti-PAD4 antibody according to the above-mentioned embodiment of the present invention. By using this reagent, PAD 4 can be detected efficiently. One embodiment of the present invention is a method of detecting PAD4 comprising the step of bringing a test sample into contact with the anti-PAD4 antibody according to the above-described embodiment of the present invention. This method can properly detect PAD 4 even when it is not treated with EDTA as described in Patent Document 3 above. One embodiment of the present invention is a kit comprising the anti-PAD4 antibody according to the above-mentioned embodiment of the present invention. With this kit, for example, treatment of diseases, diagnosis or detection of PAD4 can be performed. This kit may contain, for example, the composition, the inhibitor, the pharmaceutical composition, the diagnostic agent, or the detection reagent according to the above-mentioned embodiment of the present invention, and the instruction manual, the buffer, the container (for example, a vial) Or a syringe) or a package. As used herein, a patient sample or test sample may be blood, serum or plasma.
　本発明の一実施形態において「治療」は、患者の疾患、もしくは疾患に伴う1つ以上の症状の、症状改善効果、抑制効果、又は予防効果を発揮しうることを含む。本発明の一実施形態において「治療薬」は、有効成分と、薬理学的に許容される1つ以上の担体とを含む医薬組成物であってもよい。本発明の一実施形態において「医薬組成物」は、例えば有効成分と上記担体とを混合し、製剤学の技術分野において知られる任意の方法により製造してもよい。また医薬組成物は、治療のために用いられる物であれば使用形態は限定されず、有効成分単独であってもよいし、有効成分と任意の成分との混合物であってもよい。また上記担体の形状は特に限定されず、例えば、固体又は液体（例えば、緩衝液）であってもよい。上記担体の含有量は、例えば、製剤学上有効量であってもよい。この有効量は、例えば、有効成分の製剤学的な安定性又は送達のために十分量であってもよい。例えば、緩衝液は、バイアル中における有効成分の安定化に有効である。 In one embodiment of the present invention "treatment" includes the ability to exert a symptom ameliorating effect, a suppressing effect or a preventing effect on a disease of a patient or one or more symptoms associated with the disease. In one embodiment of the present invention, the "therapeutic agent" may be a pharmaceutical composition comprising an active ingredient and one or more pharmacologically acceptable carriers. In one embodiment of the present invention, the “pharmaceutical composition” may be produced, for example, by mixing the active ingredient and the above-mentioned carrier, by any method known in the pharmaceutical arts. The use of the pharmaceutical composition is not limited as long as it is used for treatment, and the active ingredient may be used alone, or may be a mixture of the active ingredient and an optional ingredient. Further, the shape of the carrier is not particularly limited, and may be, for example, a solid or a liquid (for example, buffer). The content of the carrier may be, for example, a pharmaceutically effective amount. The effective amount may be, for example, an amount sufficient for pharmaceutical stability or delivery of the active ingredient. For example, buffers are effective in stabilizing the active ingredient in the vial.
　医薬組成物の投与経路は、治療に際して効果的なものを使用するのが好ましく、例えば、静脈内、皮下、筋肉内、腹腔内、又は経口投与等であってもよい。投与形態としては、例えば、注射剤、カプセル剤、錠剤、顆粒剤等であってもよい。抗体を投与する場合には、注射剤として用いることが効果的である。注射用の水溶液は、例えば、バイアル、又はステンレス容器で保存してもよい。また注射用の水溶液は、例えば生理食塩水、糖(例えばトレハロース)、NaCl、又はNaOH等を配合してもよい。また医薬組成物は、例えば、有効量の緩衝剤(例えばリン酸塩緩衝液)、pH調整剤、安定剤等を配合してもよい。 The route of administration of the pharmaceutical composition is preferably that which is effective in treatment, and may be, for example, intravenous, subcutaneous, intramuscular, intraperitoneal or oral administration. The administration mode may be, for example, an injection, a capsule, a tablet, a granule and the like. When the antibody is administered, it is effective to use it as an injection. The aqueous solution for injection may be stored, for example, in a vial or a stainless steel container. The aqueous solution for injection may be formulated with, for example, physiological saline, sugar (eg, trehalose), NaCl, or NaOH. The pharmaceutical composition may also contain, for example, an effective amount of a buffer (eg, phosphate buffer), a pH adjuster, a stabilizer, and the like.
　投与量、投与間隔、投与方法は、特に限定されず、患者の年齢や体重、症状、対象臓器等により、適宜選択してもよい。また医薬組成物は、治療有効量、又は所望の作用を発揮する有効量の有効成分を含むことが好ましい。 The dosage, administration interval, and administration method are not particularly limited, and may be appropriately selected depending on the age and weight of the patient, symptoms, target organs and the like. The pharmaceutical composition also preferably contains a therapeutically effective amount, or an effective amount of an active ingredient that exerts a desired effect.
　医薬組成物の治療効果は、例えば、関節炎スコア、RAスコア、腫脹幅、画像診断、modified Total Sharpスコア、又は疾患マーカーにより評価してもよい。腫脹幅で評価する場合、例えば、医薬組成物投与時の患部の腫脹幅が、非投与時の腫脹幅に比べて有意に減少した場合に、治療効果があったと判断してもよい。又は、医薬組成物投与時の患部の腫脹幅が、ネガティブコントロール物質投与時の患部の腫脹幅に比べて、有意に減少した場合に、治療効果があったと判断してもよい。上記減少の量は、例えば、40、50、60、70、80、90、又は100％であってもよく、それらいずれか2つの値の範囲内であってもよい。 The therapeutic effect of the pharmaceutical composition may be evaluated by, for example, arthritis score, RA score, swelling width, diagnostic imaging, modified Total Sharp score, or disease marker. In the case of evaluating the swelling width, for example, when the swelling width of the affected area at the time of administration of the pharmaceutical composition is significantly reduced as compared to the swelling width at the time of non-administration, it may be judged that the therapeutic effect was obtained. Alternatively, it may be determined that the therapeutic effect has been achieved if the swelling width of the affected area upon administration of the pharmaceutical composition is significantly reduced compared to the swelling width of the affected area upon administration of the negative control substance. The amount of reduction may be, for example, 40, 50, 60, 70, 80, 90, or 100%, and may be in the range of any two values.
　本発明の一実施形態において「患者」は、ヒト、又はヒトを除く哺乳動物（例えば、マウス、モルモット、ハムスター、ラット、ネズミ、ウサギ、ブタ、ヒツジ、ヤギ、ウシ、ウマ、ネコ、イヌ、マーモセット、サル、又はチンパンジー等の1種以上）を含む。また患者は、RA又は関節炎を発症していると診断された患者であってもよい。また患者は、シトルリン化の抑制によって治療可能な疾患を発症していると診断された患者であってもよい。 In one embodiment of the present invention, the “patient” is a human or a mammal other than a human (eg, mouse, guinea pig, hamster, rat, rat, rat, rabbit, pig, sheep, goat, cow, horse, cat, dog, marmoset) , Monkey or chimpanzee etc.). The patient may also be a patient diagnosed as having RA or arthritis. The patient may also be a patient diagnosed as having a disease treatable by suppression of citrullination.
　本発明の一実施形態は、組成物中の、配列番号1で示されるアミノ酸配列からなるペプチドに特異的に結合する抗PAD4抗体、又はヒトPAD4の633～644位に特異的に結合する抗PAD4抗体の割合を増加させる工程を含む、組成物のシトルリン化活性阻害能又は治療効果を促進させる方法である。本発明の一実施形態は、抗PAD4抗体を含有する組成物であって、組成物中の抗PAD4抗体分子の90％以上が、配列番号1で示されるアミノ酸配列からなるペプチドに特異的に結合する抗PAD4抗体、又はヒトPAD4の633～644位に特異的に結合する抗PAD4抗体である、組成物である。本発明の一実施形態は、抗PAD4抗体を含有する抗体集団であって、抗体集団中の抗PAD4抗体分子の90％以上が、配列番号1で示されるアミノ酸配列からなるペプチドに特異的に結合する抗PAD4抗体、又はヒトPAD4の633～644位に特異的に結合する抗PAD4抗体である、抗体集団である。上記90％以上は、例えば、90、95、96、97、98、99％以上、又は100％であってもよく、それらいずれか2つの値の範囲内であってもよい。 One embodiment of the present invention is an anti-PAD4 antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 in the composition, or an anti-PAD4 that specifically binds to positions 634-644 of human PAD4. A method of promoting the ability to inhibit the citrullinating activity or the therapeutic effect of a composition, comprising the step of increasing the proportion of antibodies. One embodiment of the present invention is a composition containing an anti-PAD4 antibody, wherein 90% or more of the anti-PAD4 antibody molecules in the composition specifically bind to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 Or PAD4 antibody that specifically binds to positions 633-644 of human PAD4. One embodiment of the present invention is an antibody population comprising an anti-PAD4 antibody, wherein 90% or more of the anti-PAD4 antibody molecules in the antibody population specifically bind to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 Antibody population, or an anti-PAD4 antibody that specifically binds to positions 633-644 of human PAD4. The 90% or more may be, for example, 90, 95, 96, 97, 98, 99% or more, or 100%, and may be in the range of any two values.
　本発明の一実施形態は、配列番号1で示されるアミノ酸配列からなるペプチドである。このペプチドを用いれば、活性型PAD4に結合する抗体を生産することができる。また、活性型PAD4に結合する抗体を検出することができる。配列番号1で示されるアミノ酸配列からなるペプチドは、化学修飾（例えば、KLH修飾）を受けていてもよく、そのような化学修飾ペプチドも配列番号1で示されるアミノ酸配列からなるペプチドの一形態に含まれる。配列番号1で示されるアミノ酸配列からなるペプチドは、単離、精製、又は濃縮されたペプチドであってもよい。 One embodiment of the present invention is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1. This peptide can be used to produce an antibody that binds to active PAD4. In addition, antibodies that bind to active PAD4 can be detected. The peptide consisting of the amino acid sequence shown by SEQ ID NO: 1 may be chemically modified (for example, KLH modified), and such chemically modified peptide is also one form of the peptide consisting of the amino acid sequence shown by SEQ ID NO: 1 included. The peptide consisting of the amino acid sequence shown by SEQ ID NO: 1 may be an isolated, purified or enriched peptide.
　本発明の一実施形態は、配列番号1で示されるアミノ酸配列からなるペプチドを含む抗原組成物である。この抗原組成物は、例えば、緩衝液を含んでいてもよい。本発明の一実施形態は、配列番号1で示されるアミノ酸配列からなるペプチドを哺乳類又は鳥類に免疫する工程を含む、抗PAD4抗体の生産方法である。本発明の一実施形態は、配列番号1で示されるアミノ酸配列からなるペプチドと、抗体又は抗体ライブラリーを接触させる工程を含む、抗PAD4抗体の生産方法である。本発明の一実施形態は、配列番号1で示されるアミノ酸配列からなるペプチドと、抗PAD4抗体を含む試験サンプルと、を接触させる工程を含む、活性型PAD4に結合する抗体の検出方法である。本発明の一実施形態は、配列番号1で示されるアミノ酸配列からなるペプチドを含む、活性型PAD4に結合する抗体の検出用組成物である。 One embodiment of the present invention is an antigen composition comprising a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1. The antigenic composition may, for example, comprise a buffer. One embodiment of the present invention is a method for producing an anti-PAD4 antibody, which comprises the step of immunizing a mammal or a bird with a peptide consisting of the amino acid sequence shown by SEQ ID NO: 1. One embodiment of the present invention is a method for producing an anti-PAD4 antibody, which comprises the step of contacting an antibody or antibody library with a peptide consisting of the amino acid sequence shown by SEQ ID NO: 1. One embodiment of the present invention is a method of detecting an antibody that binds to active PAD4, comprising the step of contacting a peptide consisting of the amino acid sequence shown by SEQ ID NO: 1 with a test sample containing an anti-PAD4 antibody. One embodiment of the present invention is a composition for detecting an antibody that binds to active PAD4, comprising a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1.
　本発明の一実施形態において「結合」は、共有結合又は非共有結合のいずれであってもよく、例えば、イオン結合、水素結合、疎水性相互作用、又は親水性相互作用であってもよい。 In one embodiment of the present invention, "binding" may be either covalent or non-covalent, for example, ionic, hydrogen bonding, hydrophobic interaction, or hydrophilic interaction.
　本発明の一実施形態において「有意に」は、例えば、統計学的有意差をスチューデントのt検定（片側又は両側）を使用して評価し、p＜0.05又はp＜0.01である状態であってもよい。又は、実質的に差異が生じている状態であってもよい。 "Significantly" in one embodiment of the present invention is, for example, a condition where statistical significance is evaluated using Student's t-test (one side or both sides) and p <0.05 or p <0.01. It is also good. Or, it may be in a state where a difference is substantially generated.
　本明細書において引用しているあらゆる刊行物、公報類（特許、又は特許出願）は、その全体を参照により援用する。 All publications, publications (patents or patent applications) cited herein are incorporated by reference in their entirety.
　本明細書において「又は」は、文章中に列挙されている事項の「少なくとも1つ以上」を採用できるときに使用される。「もしくは」も同様である。本明細書において「2つの値の範囲内」と明記した場合、その範囲には2つの値自体も含む。本明細書において「A～B」は、A以上B以下を意味するものとする。本明細書において「それぞれに」は、「それぞれ順に」と同義である。 In the present specification, “or” is used when “at least one or more” of the items listed in the text can be adopted. The same applies to "or". In the present specification, when “within the range of two values” is specified, the range also includes the two values themselves. In the present specification, “A to B” mean A or more and B or less. In the present specification, “in each” is synonymous with “in each order”.
　以上、本発明の実施形態について述べたが、これらは本発明の例示であり、上記以外の様々な構成を採用することもできる。また、上記実施形態に記載の構成を組み合わせて採用することもできる。
As mentioned above, although embodiment of this invention was described, these are the illustrations of this invention, and various structures other than the above can also be employ | adopted. Further, the configurations described in the above embodiments can be combined and adopted.
　以下、実施例によりさらに説明するが、本発明はこれらに限定されるものではない。 The present invention will be further described by way of examples, but the present invention is not limited thereto.
　＜実施例1＞抗PAD4抗体の作製
　KLH修飾されたFFTYHIRHGEVHC（配列番号1）のペプチドを、3ヵ月齢のボリスブラウン3羽に対して、1羽当たり333μgずつ腹腔に免疫した。使用したペプチドはPAD4（配列番号2）の633～644位に対応するペプチド抗原である。1次免疫には完全フロイントアジュバンド（Wako、014-09541）、2次及び3次免疫には不完全フロイントアジュバンド（Wako、011-09551）を用いて抗原を免疫した。四次免疫はPBS（phosphate buffered saline）に希釈した抗原を静脈注射した。隔週で翼下静脈より採血を行い、ELISAによって抗体価の確認を行った。3羽に対して3次免疫まで実施し、最も抗体価の上昇が見られた個体1羽に対して、4次免疫を実施し、4次免疫を最終免疫とした。最終免疫から3日後、ニワトリの脾臓を回収し、Ficoll paque PLUS（GE Healthcare、17-1440-03）を用いた密度勾配遠心によりリンパ球を単離し、TRIzole Reagent（Life Technologies、15596026）を用いてRNAを抽出した。抽出したRNAからPrimeScript II 1st Strand cDNA Synthesis Kit（TAKARA、6210A）を用いたRT-PCRによりcDNAの合成を行い、scFvファージライブラリーを作製した。発現ベクターはpPDSのマウスκ鎖の代わりにニワトリλ鎖を挿入したタイプの発現ベクターを使用した。scFvファージライブラリーの作製は、参考文献："Nakamura et al., J Vet Med Sci. 2004 Ju;66 (7): 807-814"に記載の方法に従って行った。 <Example 1> Preparation of anti-PAD4 antibody The peptide of KLH-modified FFTYHIRHGEVHC (SEQ ID NO: 1) was immunized in the abdominal cavity at 333 μg per 3 rabbits of 3 months old Boris Brown. The peptide used is the peptide antigen corresponding to positions 633-644 of PAD4 (SEQ ID NO: 2). Antigens were immunized with complete Freund's adjuvant (Wako, 014-09541) for primary immunization and incomplete Freund's adjuvant (Wako, 011-09551) for secondary and tertiary immunization. The fourth immunization was intravenously injected with antigen diluted in PBS (phosphate buffered saline). Blood was collected from the sub wing vein every other week, and the antibody titer was confirmed by ELISA. A third immunization was performed on three birds, and a fourth immunization was performed on one individual with the highest antibody titer, and the fourth immunization was used as a final immunization. Three days after the final immunization, spleens of chickens are collected, and lymphocytes are isolated by density gradient centrifugation using Ficoll paque PLUS (GE Healthcare, 17-1440-03), using TRIzole Reagent (Life Technologies, 1559 026) RNA was extracted. CDNA was synthesized from the extracted RNA by RT-PCR using a PrimeScript II 1st Strand cDNA Synthesis Kit (TAKARA, 6210A) to prepare a scFv phage library. The expression vector used was a type of expression vector in which a chicken λ chain was inserted instead of the mouse 鎖 chain of pPDS. The preparation of the scFv phage library was performed according to the method described in the reference: "Nakamura et al., J Vet Med Sci. 2004 Ju; 66 (7): 807-814".
　scFvファージ抗体ライブラリーを用いて、上記のペプチド抗原を固相化したプレートによるパニングを行った。パニングは参考文献"Nakamura et al., J Vet Med Sci. 2004 Ju;66 (7): 807-814"に記載の方法に従って行った。5回パニングを行った後、ライブラリーの反応性を、BSA修飾ペプチド抗原を固相化したプレートを用いたELISAによって確認し、反応性が上昇し始めたライブラリーからファージのスクリーニングを行った。スクリーニングでは、ファージを大腸菌に感染させ、アンピシリン50 μg/ml（nacalai、02739-32）を含む2×YT Agar plateにプレーティングし、得られたコロニーをアンピシリン含有2×YT液体培地中で培養した。ヘルパーファージを感染させた後、アンピシリン50 μg/ml、カナマイシン25 μg/ml（明治製菓株式会社、GS1-RSS）、IPTG100 μg/ml（nacalai、19742-94）含有2×YT液体培地中でファージの誘導を行った。得られた培養上清中のscFvファージ抗体の反応性を、抗原固相化プレートを用いたELISAによって確認した。得られた陽性クローンはDNAシークエンサー（Applied Biosystems、ABI PRISM 3100-Genetic Analyzer）を用いてシークエンスを行い、配列を決定した。 The scFv phage antibody library was used to perform panning on a plate on which the above-mentioned peptide antigen was immobilized. Panning was performed according to the method described in the reference "Nakamura et al., J Vet Med Sci. 2004 Ju; 66 (7): 807-814". After five rounds of panning, the reactivity of the library was confirmed by ELISA using a plate on which BSA-modified peptide antigen was immobilized, and phages were screened from the library whose reactivity began to increase. For screening, phage was infected into E. coli, plated on 2 × YT Agar plate containing 50 μg / ml of ampicillin (nacalai, 02749-32), and the obtained colonies were cultured in 2 × YT liquid medium containing ampicillin. . After infection with helper phage, phages in 2 × YT liquid medium containing ampicillin 50 μg / ml, kanamycin 25 μg / ml (Meiji Seika Co., Ltd., GS1-RSS), IPTG 100 μg / ml (nacalai, 19742-94) Induction of The reactivity of the scFv phage antibody in the obtained culture supernatant was confirmed by ELISA using an antigen-immobilized plate. The obtained positive clones were sequenced and sequenced using a DNA sequencer (Applied Biosystems, ABI PRISM 3100-Genetic Analyzer).
　配列の異なるクローンについて、scFv抗体をコードするDNA鎖を鋳型にして、ニワトリ由来抗体遺伝子H鎖可変領域及びL鎖可変領域の増幅をPCRで行った後、PCR産物をSacII（BioLabs社, Cat#R0157S）及びNheI（BioLabs社, Cat#R0131S）制限酵素処理した。次に、H鎖可変領域及びL鎖可変領域のそれぞれについて、同じように制限酵素処理したマウスキメラ抗体（IgG1）発現ベクター（H鎖用発現ベクター：pcDNA4/myc-His、L鎖用発現ベクター：pcDNA3/myc-His、Invitrogen）に組換えた。作製したH鎖及びL鎖のコンストラクトをCHO細胞にトランスフェクトした後、培養上清をBSA修飾ペプチド抗原、及び完全長の組換えヒトPAD4タンパク質を固相したELISAで反応性の確認を行った。使用したマウスキメラ発現ベクターはTateishi et al., J Vet Med Sci. 2008 Apr;70(4): 397-400に記載されているベクターを使用した。以上により、P1、P2、P3、及びP4の抗体クローンを得た。P1、P2、P3、及びP4の重鎖可変領域のアミノ酸配列は順に配列番号3～6、DNA配列は順に配列番号7～10、軽鎖可変領域のアミノ酸配列は順に配列番号11～14、DNA配列は順に配列番号15～18である（図1～3）。以下ではこれらの抗体を纏めて「P1等」と称することもある。 For clones with different sequences, amplification of chicken antibody gene H chain variable region and L chain variable region is performed by PCR using a DNA chain encoding scFv antibody as a template, and then the PCR product is SacII (BioLabs, Cat # R0157S) and NheI (BioLabs, Cat # R0131S) restriction enzymes were treated. Next, a mouse chimeric antibody (IgG1) expression vector (H chain expression vector: pcDNA4 / myc-His, L chain expression vector) similarly treated with restriction enzymes for each of the H chain variable region and the L chain variable region: It recombined into pcDNA3 / myc-His, Invitrogen). After transfecting the prepared H-chain and L-chain constructs into CHO cells, the culture supernatant was subjected to ELISA in which BSA-modified peptide antigen and full-length recombinant human PAD4 protein were solid-phased to confirm the reactivity. The mouse chimeric expression vector used was the vector described in Tateishi et al., J Vet Med Sci. 2008 Apr; 70 (4): 397-400. Thus, antibody clones of P1, P2, P3 and P4 were obtained. The amino acid sequences of the heavy chain variable regions of P1, P2, P3 and P4 are sequentially SEQ ID NO: 3 to 6, the DNA sequence is sequentially SEQ ID NO: 7 to 10, the amino acid sequence of the light chain variable region is sequentially SEQ ID NO 11 to 14, DNA The sequences are, in order, SEQ ID NO: 15-18 (FIGS. 1-3). Hereinafter, these antibodies may be collectively referred to as "P1 etc."
　P1等の抗体クローンを大量に製造するため、作製したH鎖およびL鎖の発現ベクターをほ乳類培養細胞にExpi293Expression system(Invitrogen、A14635)を利用しトランスフェクトした後、発現した抗体の精製をProteinG Sepharose 4 Fast Flow（GE healthcare、17-018-02）を用いて行った。 In order to produce a large amount of antibody clones such as P1, etc., after transfecting the prepared H-chain and L-chain expression vectors into mammalian cultured cells using the Expi293 Expression system (Invitrogen, A14635), the purified antibody is purified by Protein G Sepharose 4 Fast Flow (GE healthcare, 17-018-02) was performed.
　＜実施例2＞抗PAD4抗体の親和性評価
　Biacore（GE Healthcare、Biacore T200）を行い、P1等のヒトPAD4に対する親和性を評価した。親和性の測定には、Mouse Antibody Capture Kit（GE Healthcare、BR-1008-38）を用いた。具体的には、メーカー提供の標準プロトコルに従い、NHS/EDCを使用し、CM5チップ表面のフリーカルボキシル基を利用したアミンカップリング法によって、ウサギ抗マウスポリクローナル抗体をCM5チップ表面に固定化した。次に、P1等をウサギ抗マウスポリクローナル抗体にキャプチャーした。Biacore T200に種々の濃度のヒトPAD4を供し、カイネティクスセンサーグラムを作成した。 <Example 2> Affinity evaluation of anti-PAD4 antibody Biacore (GE Healthcare, Biacore T200) was performed to evaluate the affinity of P1 or the like to human PAD4. For measurement of affinity, Mouse Antibody Capture Kit (GE Healthcare, BR-1008-38) was used. Specifically, a rabbit anti-mouse polyclonal antibody was immobilized on the surface of the CM5 chip by amine coupling using free carboxyl groups on the surface of the CM5 chip using NHS / EDC according to a standard protocol provided by the manufacturer. Next, P1 etc. were captured on a rabbit anti-mouse polyclonal antibody. Various concentrations of human PAD4 were applied to Biacore T200 to generate kinetic sensorgrams.
　親和性測定の結果、Kd (M)は、P1が7.3x10-8、P2が3.1x10-8、P3が2.4x10-8、P4が1.5x10-8であった。この結果からわかるように、P1等はいずれもヒトPAD4に対する高い親和性を示した。 Results Affinity measurement, Kd (M) is, P1 is 7.3x10 -8, P2 is 3.1 × 10 -8, P3 is 2.4 × 10 -8, P4 was 1.5 × 10 -8. As can be seen from this result, P1 and the like all showed high affinity for human PAD4.
　＜実施例3＞活性型PAD4への結合及び阻害活性の評価
　(1)プロトコル1（抗体と酵素の反応後にカルシウムを添加）
　抗PAD4抗体（P1等）、マウスIgG（ネガティブコントロール）を、TBSを溶媒として、それぞれ450μg/mLに調製した。5μLの抗体溶液（反応系50μL中における終濃度は45μg/mL（300 nM））と5μLの7.5μg/mL（100 nM）ヒトPAD4を全量が40μLになるように1 mM EDTA、1 mM DTTを含む20 mM Tris-HCl緩衝液（pH.7.6）に混合した。37℃で60分間インキュベーションしたのち、5μLの100mM BAEE（ベンゾイルアルギニンエチルエステル）を加え、さらに5μLの100 mM CaCl2を加えて（全量50μL、BAEEの終濃度は10 mM、カルシウムイオンの終濃度は10 mM）インキュベーションした。コントロールとして5μLの抗体溶液の代わりに、溶媒（TBS）、抗DNP抗体（ネガティブコントロール）の溶液（終濃度は300nM）、又はL207（WO/2012/026309の実施例に記載の抗PAD4抗体L207-11）の溶液（終濃度は300nM）を添加したサンプルも同様に調製した。この溶液を37℃で3時間反応させた。 <Example 3> Evaluation of binding to inhibitory PAD 4 and inhibition activity (1) Protocol 1 (Calcium added after reaction of antibody and enzyme)
The anti-PAD4 antibody (P1 etc.) and mouse IgG (negative control) were each adjusted to 450 μg / mL using TBS as a solvent. 5 μL of antibody solution (final concentration in 50 μL of reaction system is 45 μg / mL (300 nM)) and 5 μL of 7.5 μg / mL (100 nM) human PAD4 in a total volume of 40 μL with 1 mM EDTA, 1 mM DTT The mixture was mixed with 20 mM Tris-HCl buffer (pH. 7.6). After incubation at 37 ° C. for 60 minutes, 5 μL of 100 mM BAEE (benzoylarginine ethyl ester) is added, and 5 μL of 100 mM CaCl 2 is further added (total volume 50 μL, final concentration of BAEE is 10 mM, final concentration of calcium ion is 10 mM) incubation. As a control, a solvent (TBS), a solution of anti-DNP antibody (negative control) (final concentration is 300 nM) instead of 5 μL of the antibody solution, or L207 (the anti-PAD4 antibody L207- described in the example of WO / 2012/026309) A sample to which the solution of 11) (final concentration 300 nM) was added was similarly prepared. The solution was allowed to react at 37 ° C. for 3 hours.
　(2)プロトコル2（抗体と酵素の反応前にカルシウムを添加）
　プロトコル2では、事前にカルシウムとPAD4を反応させることでPAD4を活性型にした。具体的には以下の手順で行った。5μLの7.5μg/mL（100 nM）ヒトPAD4と5μLの100 mM CaCl2を混合し、全量が40μLになるように1 mM EDTA、1mM DTTを含む20 mM Tris-HCl緩衝液（pH.7.6）とを混合した。混合後、37℃で60分間プレインキュベーションを行った。抗PAD4抗体（P1等）、マウスIgG（ネガティブコントロール）を、TBSを溶媒として、それぞれ450μg/mLに調製した。調製した5μLの抗体溶液（反応系50μL中における終濃度は45μg/mL（300 nM））と5μLの100 mM BAEEをプレインキュベーションした反応液に加え、全量50μL（BAEEの終濃度は10 mM, カルシウムイオンの終濃度は10 mM）とした。コントロールとして5μLの抗体溶液の代わりに、溶媒（TBS）、抗DNP抗体の溶液（終濃度は300nM）、又はL207の溶液（終濃度は300nM）を添加したサンプルも同様に調製した。これらの溶液を37℃で3時間反応させた。 (2) Protocol 2 (add calcium before reaction of antibody and enzyme)
In Protocol 2, PAD4 was activated by reacting calcium and PAD4 in advance. Specifically, the procedure was as follows. Mix 5 μL of 7.5 μg / mL (100 nM) human PAD 4 with 5 μL of 100 mM CaCl 2 to make a total volume of 40 μL in 20 mM Tris-HCl buffer (pH. 7.6) containing 1 mM EDTA, 1 mM DTT. And mixed. After mixing, pre-incubation was performed at 37 ° C. for 60 minutes. The anti-PAD4 antibody (P1 etc.) and mouse IgG (negative control) were each adjusted to 450 μg / mL using TBS as a solvent. Add 5 μL of prepared antibody solution (final concentration in 50 μL of reaction system is 45 μg / mL (300 nM)) and 5 μL of 100 mM BAEE to the reaction solution preincubated, and make a total volume of 50 μL (final concentration of BAEE is 10 mM, calcium) The final concentration of ions was 10 mM). As a control, samples were also prepared in which solvent (TBS), a solution of anti-DNP antibody (final concentration 300 nM) or a solution of L207 (final concentration 300 nM) was added instead of 5 μL of antibody solution. The solutions were allowed to react at 37 ° C. for 3 hours.
　(3)共通プロトコル
　37℃で3時間反応させた後、5Mの過塩素酸を12.5μLを加えて反応を停止させた。この反応停止させた溶液40μLと反応溶液（反応液1（98% H2SO4：85% H3PO4：H2O＝25：20：55、0.0416% FeCl3・6H2O）：反応液2（1% 2,3-butanedione oxime：0.02% thiosemicarbazide=1：1）＝2：1混合液）150μLを混合したのち、98℃で6分間反応させた。1分間氷冷下で静置後、上清に含まれるシトルリン化されたBAEEを比色定量した。抗体溶液の代わりに溶媒を用いたコントロールサンプルの測定値を100とし、各抗体の阻害率を算出した。 (3) Common protocol After reacting at 37 ° C. for 3 hours, the reaction was stopped by adding 12.5 μL of 5 M perchloric acid. 40 μL of this quenched solution and the reaction solution (Reaction solution 1 (98% H 2 SO 4 : 85% H 3 PO 4 : H 2 O = 25: 20: 55, 0.0416% FeCl 3 · 6H 2 O): reaction After 150 μL of solution 2 (1% 2,3-butanedione oxime: 0.02% thiosemicarbamide = 1: 1 mixed solution) was mixed, the mixture was reacted at 98 ° C. for 6 minutes. After standing for 1 minute under ice-cooling, citrullinated BAEE contained in the supernatant was subjected to colorimetric determination. The inhibition value of each antibody was calculated by setting the measured value of the control sample using a solvent instead of the antibody solution as 100.
　PAD4阻害活性測定の結果を図4に示す。この結果からわかるように、P1等は活性型PAD4に結合し、シトルリン化活性を強く阻害した（阻害率はP1が63％、P2が62％、P3が80％、P4が82％）。一方で、抗DNP抗体及びL207は活性型PAD4のシトルリン化活性を阻害しなかった。 The results of PAD4 inhibitory activity measurement are shown in FIG. As can be seen from this result, P1 and the like bound to active PAD4 and strongly inhibited the citrullinating activity (the inhibition rate is 63% for P1, 62% for P2, 80% for P3, and 82% for P4). On the other hand, anti-DNP antibody and L207 did not inhibit the citrullinating activity of active PAD4.
　以上の実施例により、PAD4の633～644位に特異的に結合する抗PAD4抗体が、活性型PAD4に結合し、シトルリン化活性を阻害する特性を有していることが明らかとなった。 From the above examples, it became clear that the anti-PAD4 antibody that specifically binds to positions 633-644 of PAD4 has the property of binding to active PAD4 and inhibiting the citrullinating activity.
　以上、本発明を実施例に基づいて説明した。この実施例はあくまで例示であり、種々の変形例が可能なこと、またそうした変形例も本発明の範囲にあることは当業者に理解されるところである。 The present invention has been described above based on the embodiments. It is understood by those skilled in the art that this embodiment is merely an example, and that various modifications are possible, and such modifications are also within the scope of the present invention.

Claims (13)

  1. 　活性型PAD4（Peptidylarginine deiminase 4）に結合する、抗PAD4抗体。 An anti-PAD4 antibody that binds to activated PAD4 (Peptidylarginine deiminase 4).
  2. 　活性型PAD4のシトルリン化活性を阻害する、請求項1に記載の抗PAD4抗体。 The anti-PAD4 antibody according to claim 1, which inhibits the citrullinating activity of active PAD4.
  3. 　配列番号1で示されるアミノ酸配列からなるペプチド、又はヒトPAD4の633～644位に特異的に結合する、請求項1又は2に記載の抗PAD4抗体。 The anti-PAD4 antibody according to claim 1 or 2, which specifically binds to a peptide consisting of the amino acid sequence shown by SEQ ID NO: 1 or positions 633-644 of human PAD4.
  4. 　モノクローナル抗体である、請求項1～3いずれかに記載の抗PAD4抗体。 The anti-PAD4 antibody according to any one of claims 1 to 3, which is a monoclonal antibody.
  5. 　抗原結合性抗体断片である、請求項1～4いずれかに記載の抗PAD4抗体。 The anti-PAD4 antibody according to any one of claims 1 to 4, which is an antigen-binding antibody fragment.
  6. 　請求項1～5いずれかに記載の抗PAD4抗体をコードする、ポリヌクレオチド又はベクター。 A polynucleotide or a vector encoding the anti-PAD4 antibody according to any one of claims 1 to 5.
  7. 　請求項1～5いずれかに記載の抗PAD4抗体を含む、PAD4の機能阻害剤。 A functional inhibitor of PAD4 comprising the anti-PAD4 antibody according to any one of claims 1 to 5.
  8. 　請求項6に記載のポリヌクレオチド又はベクターを含有する細胞を増殖させる工程を含む、抗PAD4抗体の生産方法。 A method for producing an anti-PAD4 antibody, comprising the step of growing cells containing the polynucleotide or vector according to claim 6.
  9. 　配列番号1で示されるアミノ酸配列からなるペプチド。 A peptide consisting of the amino acid sequence shown in SEQ ID NO: 1.
  10. 　化学修飾ペプチドである、請求項9に記載のペプチド。 The peptide according to claim 9, which is a chemically modified peptide.
  11. 　請求項9又は10に記載のペプチドを含む抗原組成物。 An antigen composition comprising the peptide according to claim 9 or 10.
  12. 　請求項9又は10に記載のペプチドで哺乳類又は鳥類を免疫する工程を含む、抗PAD4抗体の生産方法。 A method for producing an anti-PAD4 antibody, comprising the step of immunizing a mammal or avian with the peptide according to claim 9 or 10.
  13. 　請求項9又は10に記載のペプチドと、抗PAD4抗体を含む試験サンプルと、を接触させる工程を含む、活性型PAD4に結合する抗体の検出方法。 A method for detecting an antibody that binds to active PAD4, comprising the step of contacting the peptide according to claim 9 with a test sample containing an anti-PAD4 antibody.
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EP3992287A4 (en) * 2020-07-13 2022-11-02 Shandong Xinchuang Biological Technology Co., Ltd Antigen prepared on basis of taking padi4 as tumor marker, and antibody and use thereof
WO2024020579A1 (en) * 2022-07-22 2024-01-25 Bristol-Myers Squibb Company Antibodies binding to human pad4 and uses thereof

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