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• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/40—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against enzymes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
  • C12Y305/03—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amidines (3.5.3)
  • C12Y305/03015—Protein-arginine deiminase (3.5.3.15)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

• a pharmaceutical composition for treating fibrosis comprising an antibody that specifically binds to PAD2 and a pharmaceutically acceptable carrier.
• This pharmaceutical composition can be used to treat fibrosis.
• anti-PAD2 antibodies can suppress fibrosis.
• Fibrosis includes, for example, a phenomenon caused by excessive accumulation of connective tissue in a tissue. Tissue hardening associated with fibrosis may occur, for example, when connective tissue containing collagen and the like grows and replaces normal tissue. Fibrosis includes, for example, fibrosis that occurs in tissues such as the liver, lungs, kidneys, heart, pancreas, bone marrow, or skin. In one embodiment of the invention, fibrosis includes diseases that involve tissue fibrosis.
• "having no binding property” includes cases where the binding property does not have to be substantially or it does not have the binding property significantly.
• the EC 50 for the evaluation target is, for example, 2, 10, 100, or 10,000 times or more higher than the EC 50 for the wild type, it may be evaluated that the binding property is not present. At this time, if the EC 50 is Not determined, it may be evaluated that there is no binding property.
• the binding property to the evaluation target is 50, 30, 10, 5, or 1% or less compared to the binding property to the wild type, it may be evaluated that the protein has no binding property.
• the anti-PAD2 antibody includes the form of a monoclonal antibody.
• Monoclonal antibodies can act against PAD2 more efficiently than polyclonal antibodies.
• the anti-PAD2 antibody may be an antibody that binds to wild type or mutant type of PAD2.
• Variants of PAD2 include those caused by differences in DNA sequence between individuals, such as SNPs.
• the antibody includes an antibody that has binding ability to PAD2.
• Antibodies include molecules or populations thereof that are capable of specifically binding a particular epitope on an antigen.
• Antibodies may be polyclonal or monoclonal antibodies.
• Antibodies can exist in various forms, such as full-length antibodies (antibodies with Fab and Fc regions), Fv antibodies, Fab antibodies, F(ab') 2 antibodies, Fab' antibodies, diabodies, single antibodies, etc.
• a chimeric antibody is one in which the variable region of an antibody between different species and the constant region of an antibody are linked, and can be constructed by genetic recombination technology.
• examples include chimeric antibodies derived from non-human mammals and humans (eg, mouse-human chimeric antibodies, chicken-human chimeric antibodies, chicken-mouse chimeric antibodies, etc.).
• Mouse-human chimeric antibodies can be produced, for example, by the method described in "Roguska et al., Proc Natl Acad Sci USA.
• the Fv antibody is an antibody that includes an antigen recognition site.
• This region comprises a dimer of one heavy chain variable region and one light chain variable region in non-covalent association.
• the three CDRs of each variable region can interact to form an antigen binding site on the surface of the VH-VL dimer.
• a Fab antibody is a fragment obtained by treating an antibody containing a Fab region and an Fc region with the proteolytic enzyme papain, in which approximately half of the N-terminal side of the H chain and the entire L chain are combined. This is an antibody that is linked via a disulfide bond between two parts.
• Fab can be obtained, for example, by treating the anti-PAD2 antibody according to the embodiment of the present invention, which includes a Fab region and an Fc region, with the proteolytic enzyme papain.
• the F(ab') 2 antibody is, for example, a fragment obtained by treating an antibody containing a Fab region and an Fc region with the protease pepsin, which contains two sites corresponding to Fab. It is an antibody.
• F(ab') 2 can be obtained, for example, by treating the anti-PAD2 antibody according to the embodiment of the present invention, which includes a Fab region and an Fc region, with the protease pepsin. Further, for example, it can be produced by linking Fab' shown below with a thioether bond or a disulfide bond.
• the anti-PAD2 antibody includes an antibody having a heavy chain and a light chain.
• Heavy chains are typically the major component of full-length antibodies.
• the heavy chain of a full-length antibody typically forms disulfide bonds and non-covalent interactions with the light chain.
• Heavy chains typically include a heavy chain variable region (VH) and a constant region.
• Light chains are typically components of full-length antibodies that are distinct from heavy chains.
• Light chains typically include a light chain variable region (VL) and a constant region.
• CDR complementarity determining region
• CDRs are located on the Fv (variable region) of an antibody.
• the CDRs include heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3 (sometimes abbreviated as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively).
• CDRs each have about 3 to 30 amino acid residues. It is known that heavy chain CDRs particularly contribute to the binding of antibodies to antigens.
• CDR3 is known to have the highest contribution to antibody binding to antigens.
• the CDR is defined by Kabat's definition (Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)), IMGT's definition (Lefranc et al. ., Dev Comp Immunol . 2003 Jan;27(1):55-77.) or Chothia's definition (Chothia et al., J. Mol. Biol., 1987;196:901-917). good.
• CDRs are preferably defined by Kabat's definition.
• the amino acid sequences of the CDRs of CK1-10 in Example 1 are HCDR1:DYGMG (SEQ ID NO: 9), HCDR2: AISNRGSHTYYGAAVKG (SEQ ID NO: 10), HCDR3: DAGTCISSYGFSCVSAASIDA (SEQ ID NO: 11), LCDR1: SGGSGSYGGSYYYG (SEQ ID NO: 12). ), LCDR2: DNTNRPS (SEQ ID NO: 13), and LCDR3: GSIDSISDADI (SEQ ID NO: 14).
• amino acid sequences of the variable region of PK1-16 are VH: AVTLDESGGGLQTPGGGLSLVCKASGFTFRSYAMYWVRQAPGKGLEWLAGISSSGRYTGYAPAVKGRATISRDNGQSTVRLQLSNLRAEDAGTYYCAKDVYDSWTYANRIDAWGHGTEVIVSS (SEQ ID NO: 21) and VL: ALTQPSSVSANPGETVKITCSGGGRRGYYGWYQQKSPGSAPVTVIY NNDERPSNIPSRFSGFKSGSTATLTITGVQAEDEAVYYCGSGDTTTDSGIFGAGTTLTVL (SEQ ID NO: 22).
• amino acid sequences of the variable region of CK1-14 are VH: AVTLDESGGGLQTPGGALSLVCKASGFTFTRYAIQWVRQAPGKGLEWVGVINSGGRTLYAPAVKGRATISRDNGQSTVRLQLNNLRAEDTAIYYCVRGGYAYGIETWGHGTEVIVSS (SEQ ID NO: 25) and VL: ALTQPSSVSANPGETVKITCSGSRYDYGWYQQKSPGSAPVTLIYYNNKRP SDIPSRFSGSKSGSTHTLTITGVQADDEAVYFCGSTDTSNDIFGAGTTLTVL (SEQ ID NO: 26). Furthermore, the nucleotide sequences corresponding to the amino acid sequences shown by SEQ ID NOs: 3 to 26 were the nucleotide sequences shown by SEQ ID NOs: 29 to 52, respectively.
• Example 1 an expression vector was used to express the antibody.
• the heavy chain constant region and light chain constant region of PK1-16, CK1-10 and CK1-14 had the amino acid sequences shown by SEQ ID NOs: 27 and 28, respectively.
• the base sequences corresponding to the amino acid sequences shown by SEQ ID NOs: 27 and 28 were the base sequences shown by SEQ ID NOs: 53 and 54, respectively.
• the transformant may be Escherichia bacteria, yeast, or the like.
• the above polynucleotide or vector may be constructed to be able to express an anti-PAD2 antibody.
• the polynucleotide or vector may contain components necessary for protein expression, such as a promoter, enhancer, origin of replication, or antibiotic resistance gene.
• "significantly” means, for example, statistically significant differences are evaluated using Student's t-test (one-sided or two-tailed) or Bonferroni's multiple comparison test, p ⁇ 0.001, p ⁇ 0.001, p ⁇ The condition may be 0.05 or p ⁇ 0.01. Alternatively, the state may be such that a substantial difference has occurred.
• panning was performed using a plate immobilized with full-length PAD2. Panning was performed according to the method described in the reference document: "Nakamura et al., J Vet Med Sci. 2004 Ju;66 (7): 807-814". After performing panning five times, the reactivity of the library was confirmed by ELISA using a plate immobilized with synthetic peptides, and phages were screened from the library whose reactivity began to increase. For screening, E.
• the chicken-derived antibody gene H chain variable region and L chain variable region are amplified by PCR using the DNA chain encoding the scFv antibody as a template, and the amplified fragments are used as human H chain constant region (IgG1). and inserted into a preconstructed expression vector into which the human L chain constant region had been incorporated, by homologous recombination using Seamless Cloning and Assembly Enzyme Mix (Thermo, A14606). After transfecting the prepared H chain and L chain constructs into 293 cells, reactivity was confirmed by ELISA using immobilized full-length PAD2.
• PK1-16, CK1-10, and CK1-14 were used in the following experiments.
• Example 4 Anti-fibrosis in fibrosis model using anti-PAD2 antibody 4.1 Preparation of fibrosis model and administration of anti-PAD2 antibody The efficacy of anti-PAD2 antibody was evaluated using a fibrosis model. First, a bile duct ligation-induced cholestatic liver disease (BDL) model mouse was created using the following procedure, and an anti-PAD2 antibody was administered. Seven-week-old male C57BL/6J mice were anesthetized by intraperitoneally administering triple-mixed anesthesia (medetomidine hydrochloride/mitazolam/butorphanol tartrate) at 0.75/4/5 mg/kg.
• triple-mixed anesthesia medetomidine hydrochloride/mitazolam/butorphanol tartrate
• Atipamezole hydrochloride was administered intraperitoneally at 0.75 mg/kg to awaken the mice and return them to their cages. The animals were kept on a heating pad until they fully awoke from anesthesia.
• An anti-PAD2 antibody or a control antibody (anti-DNP antibody) was administered intravenously (25 mg/kg) 1 hour after surgery, and on the 4th, 7th, and 11th days.
• the sham-operated (mice with thoracotomy but no bile duct ligation) group served as the control group.
• Ten model mice were used in the control group, five in the anti-DNP antibody group, and seven in each anti-PAD2 antibody group for the final analysis.

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