WO2016122250A1 - Anti-eprs monoclonal antibody and use thereof - Google Patents

Anti-eprs monoclonal antibody and use thereof Download PDF

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Publication number
WO2016122250A1
WO2016122250A1 PCT/KR2016/001004 KR2016001004W WO2016122250A1 WO 2016122250 A1 WO2016122250 A1 WO 2016122250A1 KR 2016001004 W KR2016001004 W KR 2016001004W WO 2016122250 A1 WO2016122250 A1 WO 2016122250A1
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antibody
seq
amino acid
acid sequence
fragment
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PCT/KR2016/001004
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French (fr)
Korean (ko)
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김성훈
최연식
심현보
이남주
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재단법인 의약바이오컨버젼스연구단
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Publication of WO2016122250A1 publication Critical patent/WO2016122250A1/en

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    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • 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
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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
    • C07K2317/90—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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y601/00—Ligases forming carbon-oxygen bonds (6.1)
    • C12Y601/01—Ligases forming aminoacyl-tRNA and related compounds (6.1.1)
    • C12Y601/01014—Glycine-tRNA ligase (6.1.1.14)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/9015—Ligases (6)

Definitions

  • the present invention relates to an anti-EPRS antibody that selectively binds to EPRS glutamyl-prolyl tRNA synthetase), and more specifically to an antibody or fragment thereof that binds to human EPRS, and a pharmaceutical composition for treating cancer comprising the same. It's about
  • Aminoacyl-tRNA synthetase is an enzyme that attaches a specific amino acid to its corresponding tRNA.
  • Higher organisms consist of 23 enzymes, including 3 species involved in the formation of mult i synthetase complexes, including ⁇ 1 ( ⁇ 43), (AIMP2) p38, and (AIMP3) pl8.
  • ⁇ 1 ⁇ 43
  • AIMP2 p38
  • AIMP3 p38
  • EPRS glutamyl-prolyl tRNA synthetase
  • EPRS is a form in which two enzyme active domains are linked by a linker, which is a form in which the WHEP domain is repeated three times.
  • This WHEP domain is similar in structure to some ARSs enzymes such as WRS, HRS, GRS, MRS, etc., but unlike EPRS, it does not have a repeated structure.
  • Interferon-gamma IFN- ⁇
  • GIT gamma- int eron-activated inhibitor of translation complex
  • VEGFA Vascular endothelial growth factor A
  • EPRS Ray, PS et al. A stress-responsive RNA swi tch regulates VEGFA expression. Nature 457, 915919 (2009)).
  • EPRS may be an important regulator of cancer, immune and inflammatory disease gene expression and may be used as an important diagnostic biomarker.
  • ARSs have similarities in protein structure, antibodies obtained from an immune response from animals show cross reactions that bind to other ARSs, and in many cases no high-sensitivity antibodies are produced. Antibodies of the present invention are expected to be of high diagnostic and industrial availability as well as for research in view of their excellent sensitivity and no cross-reaction between ARS.
  • Another object of the present invention is to provide a polynucleotide encoding the antibody or fragment thereof.
  • Another object of the present invention is to provide a vector comprising the polynucleotide.
  • Another object of the present invention is to provide a cell comprising the vector.
  • Another object of the present invention is to provide a method for producing an antibody or fragment thereof that binds to human EPRS.
  • Another object of the present invention is to provide an EPRS specific detection method comprising contacting an antibody or fragment thereof with a sample and detecting the antibody or fragment thereof. It is.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising the antibody or fragment thereof as an active ingredient.
  • Another object of the present invention is to provide a cancer diagnostic composition comprising the antibody or fragment thereof as an active ingredient.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating immune diseases, comprising the antibody or fragment thereof as an active ingredient.
  • Another object of the present invention to provide an immunological disease diagnostic composition comprising the antibody or fragment thereof as an active ingredient.
  • Another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising the antibody or fragment thereof as an active ingredient.
  • Another object of the present invention is to provide a composition for diagnosing inflammatory diseases comprising the antibody or fragment thereof as an active ingredient.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating islet emulsification comprising the antibody or fragment thereof as an active ingredient.
  • Another object of the present invention is to provide a composition for diagnosing ischemic disease comprising the antibody or fragment thereof as an active ingredient.
  • Another object of the present invention is to provide a method for treating or diagnosing cancer, immune disease, inflammatory disease and fibrosis, comprising administering an effective amount of the antibody or fragment thereof to a subject in need thereof.
  • the present invention provides a method for determining the complementarity determining region (CDR) comprising an amino acid sequence represented by SEQ ID NO: 2).
  • L2 the complementarity determining region (CDR) comprising the amino acid sequence represented by SEQ ID NO: 3
  • the antibody light chain variable region (VL) comprising the L3
  • the complementarity determining region (CDR) Hl comprising the amino acid sequence represented by SEQ ID NO: 4
  • An antibody heavy chain variable region (VH) comprising a complementarity determining region (CDR) H2 comprising an amino acid sequence represented by SEQ ID NO: 5, a complementarity determining region (CDR) H3 comprising an amino acid sequence represented by SEQ ID NO: 6 Containing antibody;
  • CDR Ll an antibody light chain variable region comprising a complementarity determining region (CDR) L2 comprising an amino acid sequence represented by SEQ ID NO: 16, a complementarity determining region (CDR) L3 comprising an amino acid sequence represented by SEQ ID NO: 17
  • Complementarity Determining Site (CDR) H1 comprising an amino acid sequence represented by V and SEQ ID NO: 18, Complementarity Determining Site (CDR) H2 comprising an amino acid sequence represented by SEQ ID NO: 19, and an amino acid sequence represented by SEQ ID NO: 20
  • An antibody comprising an antibody heavy chain variable region (VH) comprising a complementarity determining region (CDR) H3; and
  • CDR Ll an antibody light chain variable region comprising a complementarity determining region (CDR) L2 comprising an amino acid sequence represented by SEQ ID NO: 30, a complementarity determining region (CDR) L3 comprising an amino acid sequence represented by SEQ ID NO: 31 VL) and the complementarity determining region (CDR) Hl comprising the amino acid sequence represented by SEQ ID NO: 32, the complementarity determining region (CDR) H2 comprising the amino acid sequence represented by SEQ ID NO: 33, amino acid sequence represented by SEQ ID NO: 34
  • An antibody comprising an antibody recombination region (VH) comprising a complementarity determining region (CDR) H3 comprising a;
  • antibodies or fragments thereof that bind to human EPRS selected from the group consisting of:
  • the present invention provides a polynucleotide encoding the antibody or fragment thereof.
  • the present invention provides a vector comprising the polynucleotide.
  • the present invention provides a cell comprising the vector.
  • the present invention provides a method for producing a polypeptide comprising a light chain and a heavy chain variable region by culturing the cells under a condition in which the polynucleotide is expressed, and the cells or the same. It provides a method for producing an antibody or fragment thereof that binds to human EPRS, comprising recovering the polypeptide from the culture medium.
  • the present invention provides an EPRS specific detection method comprising contacting an antibody or fragment thereof with a sample and detecting the antibody or fragment thereof.
  • the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the antibody or fragment thereof as an active ingredient.
  • the present invention provides a composition for diagnosing cancer comprising the antibody or fragment thereof as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating immune diseases, comprising the antibody or fragment thereof as an active ingredient.
  • the present invention provides a composition for diagnosing immunological diseases comprising the antibody or fragment thereof as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases, comprising the antibody or fragment thereof as an active ingredient.
  • the present invention is directed to the antibody or fragment thereof. It provides a composition for diagnosing inflammatory diseases comprising as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating fibrosis comprising the antibody or fragment thereof as an active ingredient.
  • the present invention provides a composition for diagnosing fibrosis comprising the antibody or fragment thereof as an active ingredient.
  • the present invention provides a method for treating cancer, immune disease, inflammatory disease and fibrosis, or a method for diagnosing cancer, comprising administering the antibody or fragment thereof in an effective amount to a subject in need thereof. to provide.
  • the present invention provides the antibody or fragment thereof for use in the manufacture of a medicament or diagnostic agent for the treatment of cancer, immune diseases, inflammatory diseases and fibrosis.
  • Complementarity Determining Site (CDR) Ll comprising an amino acid sequence represented by SEQ ID NO: 1
  • Complementarity Determining Site (CDR) L2 comprising an amino acid sequence represented by SEQ ID NO: 2
  • SEQ ID NO: 3 An antibody light chain variable region (VL) comprising a complementary determining region (CDR) L3 comprising an indicated amino acid sequence and a complementarity determining region (CDR) Hl comprising an amino acid sequence represented by SEQ ID NO: 4, represented by SEQ ID NO: 5
  • Complementarity determining site comprising the amino acid sequence represented by SEQ ID NO: 15
  • CDR Ll an antibody light chain variable region comprising a complementarity determining region (CDR) L2 comprising an amino acid sequence represented by SEQ ID NO: 16, a complementarity determining region (CDR) L3 comprising an amino acid sequence represented by SEQ ID NO: 17 VL) and the complementarity determining region (CDR) Hl comprising the amino acid sequence represented by SEQ ID NO: 18, the complementarity determining region (CDR) H2 comprising the amino acid sequence represented by SEQ ID NO: 19, the amino acid sequence represented by SEQ ID NO: 20 Comprising an antibody heavy chain variable region (VH) comprising a complementarity determining region (CDR) H3 comprising Antibodies; And
  • CDR Ll an antibody light chain variable region comprising a complementarity determining region (CDR) L2 comprising an amino acid sequence represented by SEQ ID NO: 30, a complementarity determining region (CDR) L3 comprising an amino acid sequence represented by SEQ ID NO: 31 VL) and the complementarity determining region (CDR) Hl comprising the amino acid sequence represented by SEQ ID NO: 32, the complementarity determining region (CDR) H2 comprising the amino acid sequence represented by SEQ ID NO: 33, amino acid sequence represented by SEQ ID NO: 34
  • An antibody comprising a antibody heavy chain variable region (VH) comprising a complementarity determining region (CDR) H3 comprising an antibody or fragment thereof that binds to human EPRS selected from the group consisting of:
  • EPRS Glutamyl-prolyl—tRNA synthetase
  • ARS amino acid-tRNA synthetase
  • antibody anti-EPRS antibody
  • humanized anti-EPRS antibody humanized anti-EPRS antibody
  • modified humanized anti-EPRS antibody ant i -EPRS ant ibody
  • monoclonal antibodies monoclonal antibodies, including full length monoclonal antibodies
  • polyclonal antibodies polyclonal antibodies
  • multispecific antibodies eg bispecific antibodies
  • antibody fragments eg variable Region and other portions of the antibody that exhibit the desired biological activity (eg, binding to EPRS).
  • Antibodies of the present invention are antibodies in which specific amino acid sequences are included in the light and heavy chain CDRs so as to selectively bind to EPRS, and include both monoclonal and polyclonal antibodies, preferably monoclonal. It may be an antibody.
  • the antibody of the present invention includes all chimeric antibodies, humanized antibodies, human antibodies, and preferably human antibodies.
  • Monoclonal antibodies of the invention refer to antibodies obtained from a substantially homogeneous population of antibodies, ie, the individual antibodies that make up the population, except for naturally occurring mutations that may be present in small amounts. Is the same. Monoclonal antibodies only Binds very specifically to one antigen epitope.
  • the term "monoclonal" refers to the characteristics of an antibody as it is obtained from a substantially homologous population and does not necessarily produce the antibody by a particular method.
  • Antibodies of the invention specifically include chimeric antibodies, wherein some of the heavy and / or light chains originate from a particular species or show the same or homologous as the corresponding sequence of a particular antibody, but the remainder As long as the antibodies of the invention exhibit desirable biological activity (eg, selective binding to EPRS), they may be from other species or exhibit the same or homologous as the corresponding sequences of other antibodies.
  • Humanized antibodies are antibodies that contain the sequences of both human and non-human (eg rat, rat) antibodies. Generally, the rest of the human antibody, except for the region that binds to the epitope (CDR), belongs to a human antibody. The site that binds the epitope (CDR) may comprise a non-human derived sequence.
  • Complete human antibodies refer to antibodies comprising only human immunoglobulin protein sequences, and can be produced in mice, mouse cells, or hybridomas derived from mouse cells, or produced by phage display.
  • Natural antibodies produced in vivo are typically hetero-tetrameric glycoproteins of about 150,000 Daltons, composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by one covalent disulfide bond, but the disulfide chain number varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains.
  • VH variable domain
  • Each light chain has a variable dome at one end Phosphorus (VL) and a constant domain at its other end;
  • the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
  • Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
  • variable region or “variable domain” of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody.
  • the variable region of the heavy chain is described as “VH” or “V H "
  • the variable region of the light chain is described as “VL” or “V L.”
  • These domains are generally the most variable portions of an antibody, Antigen binding site.
  • variable refers to the fact that several sequences within the variable region differ widely in sequence between antibodies and directly affect the binding and specificity of each particular antibody to its specific antigenic determinants. It refers to the fact that it contains the residues involved.
  • CDR complementarity determining regions
  • HVL hypervariable loops
  • CDR-L1 is located approximately residues 24-34 in the light chain variable region
  • CDR-L2 is approximately residues 50-56 and CDRL3 is approximately residues 89-97
  • CDR-H1 is located approximately residues 31-35 in the heavy chain variable region
  • CDR-H2 is approximately residues 50-65
  • CDR-H3 is approximately residues 95-102.
  • the three CDRs in each of the heavy and light chains are separated by site (FR), which contains sequences that tend to be less variable.
  • site FR
  • the large ⁇ sheet arrangement of the FRs brings the CDRs within each chain close to each other as well as to CDRs from other chains.
  • the resulting form contributes to the antigen binding site (see Kabat et al., 1991, NIH Publ. No. 91-3242, Vol. I, pages 647-669), but not all CDR residues need to be directly involved in antigen binding.
  • each CDR belonging to the light and heavy chain variable regions has a specific sequence. Characterized in that it specifically binds to EPRS, specifically, the antibody of the present invention i) Complementarity determining region (CDR) Ll comprising the amino acid sequence represented by SEQ ID NO: 1, represented by SEQ ID NO: 2 Complementarity determining region (CDR) L2 comprising an amino acid sequence, antibody light chain variable region (VL) comprising a complementarity determining region (CDR) L3 comprising an amino acid sequence represented by SEQ ID NO: 3, and an amino acid sequence represented by SEQ ID NO: 4 Complementarity determining region (CDR) H1 comprising a column, complementarity determining region (CDR) H2 comprising an amino acid sequence represented by SEQ ID NO: 5, complementarity determining region (CDR) H3 comprising an amino acid sequence represented by SEQ ID NO: An antibody comprising an antibody heavy chain variable region (VH) comprising; ⁇ i03> i
  • CDR Ll an antibody light chain variable region comprising a complementarity determining region (CDR) L2 comprising an amino acid sequence represented by SEQ ID NO: 16, a complementarity determining region (CDR) L3 comprising an amino acid sequence represented by SEQ ID NO: 17 VL) and the complementarity determining region (CDR) Hl comprising the amino acid sequence represented by SEQ ID NO: 18, the complementarity determining region (CDR) H2 comprising the amino acid sequence represented by SEQ ID NO: 19, the amino acid sequence represented by SEQ ID NO: 20
  • An antibody comprising an antibody heavy chain variable region (VH) comprising a complementarity determining region (CDR) H3 comprising;
  • CDR Ll an antibody light chain variable region comprising a complementarity determining region (CDR) L2 comprising an amino acid sequence represented by SEQ ID NO: 30, a complementarity determining region (CDR) L3 comprising an amino acid sequence represented by SEQ ID NO: 31 VL) and the complementarity determining region (CDR) Hl comprising the amino acid sequence represented by SEQ ID NO: 32, the complementarity determining region (CDR) H2 comprising the amino acid sequence represented by SEQ ID NO: 33, amino acid sequence represented by SEQ ID NO: 34
  • It may be an antibody selected from the group consisting of an antibody comprising an antibody heavy chain variable region (VH) comprising a complementarity determining region (CDR) H3 comprising a.
  • the antibody of the present invention may preferably comprise a specific light chain variable region and a heavy chain variable region, specifically
  • An antibody comprising amino acid sequences 132 to 241 of SEQ ID NO: 13 as a light chain variable region and comprising amino acid sequences 1 to 116 of SEQ ID NO: 13 as a heavy chain variable region;
  • It may be an antibody selected from the group consisting of ⁇ >.
  • the antibody of the present invention is most preferably SEQ ID NO: 13, SEQ ID NO: 27 and SEQ ID NO:
  • It may be an antibody comprising an amino acid sequence represented by a sequence number selected from the group consisting of 41.
  • Antibody fragments, fragments or “fragments” of the invention typically retain at least a portion of the binding specificity of the parent antibody, typically at least a portion or variable region of the antigen binding of the parent antibody (eg, one or more CDRs). Fragments or derivatives of the antibody comprising a. Examples of antibody fragments include Fab, Fab ', F (ab') 2 and Fv fragments; Diabodies; Linear antibodies; S ingle-chain antibody molecules such as sc-Fv; And multispecific antibodies formed from antibody fragments. Typically, an antibody fragment or derivative retains at least 10% of its EPRS binding activity when its activity is expressed on a molar basis.
  • the antibody fragment or derivative has at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the EPRS binding affinity as the parent antibody.
  • EPRS antibody fragments may also include conservative amino acid substitutions (called conservative variants of the antibody) that do not substantially alter their biological activity.
  • conservative variants of the antibody refer to both antibodies and fragments thereof.
  • Fab consists of one light chain and one heavy chain of CH1 (first constant domain) and variable region.
  • the heavy chain of the Fab molecule cannot form disulfide bonds with other heavy chain molecules.
  • the ⁇ i i6> Fc region contains two heavy chain fragments comprising the CH1 and CH2 domains of an antibody. Two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interaction of the CH3 domain.
  • F (ab ') 2 is two light chains, and the intra-chain disulfide bond is between the two heavy chains It contains two heavy chains that contain part of the fixed region between the CHI and CH2 domains to form.
  • the F (ab ') 2 fragment consists of two Fab ' fragments held together by disulfide bonds between the two heavy chains.
  • Fv is an antibody fragment which includes both heavy and light chain variable regions but lacks a fixed region.
  • Single-chain Fv or scFv refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen binding.
  • scFv see Pluckthim (1994) THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Moore eds. Springer-Ver lag, New York, pp. 269-315. See also WO0 88/01649 and US Pat. Nos. 4, 946, 778 and 5,26, 203.
  • Diabodies refer to small antibody fragments having two antigen-binding sites, wherein the fragments comprise a heavy chain variable domain (VH) (VH-VL) linked to the light chain variable domain (VL) in the same polypeptide chain. Include. Using a short linker that does not allow pairing between two domains on the same chain, the domains are forcibly paired with the complementary domains of the other chain to create two antigen-binding sites. Diabodies are described, for example, in European Patent No. 404,097; WO 93/11161 and Hol l inger et al. , Proc. Nat l. Acad. Sci. USA, 90: 6444-6448 (1993).
  • a linear antibody consists of a pair of tandem Fd fragments (VH- which form a pair of antigen binding sites).
  • Linear antibodies are described, for example, in Zapata et al. 1995, Protein Eng. 8 (10): 1057-1062, which may be bispecific or monospecific.
  • Domain antibodies are immunologically functional immunoglobulin fragments containing only the variable region of the heavy chain or the variable region of the light chain.
  • two or more VH regions are covalently linked to a peptide linker to produce a bivalent domain antibody.
  • Two VH regions of a bivalent domain antibody may target the same or different antigens.
  • Bivalent antibodies comprise two antigen binding regions. In some instances, the two binding regions have the same antigen specificity. However, bivalent antibodies may be bispecific (bispeci f ic). Can be. Antibodies or fragments thereof of the invention can be produced using methods known in the art, such as phage display methods or yeast cell surface expression systems. As a method for preparing scFv, the method described in US Pat. Nos. 4, 946, 778 and 5, 258, 498 may be used, and recombinantly generating Fab, Fab 'and F (ab') 2 fragments. As the above method, the method described in WO 92/22324 may be used.
  • Antibodies of the invention can be derived from any animal, including mammals, birds, and the like, including humans.
  • the antibody may be an antibody of human, mouse, donkey, sheep, rabbit, goat, guinea pig, camel, horse or chicken.
  • Human antibodies are antibodies having the amino acid sequence of human immunoglobulins, which include antibodies isolated from human immunoglobulin libraries or antibodies isolated from animals transfected against one or more human immunoglobulins and not expressing endogenous immunoglobulins ( US Patent No. 5, 939, 598).
  • the antibody of the present invention may be conjugated to an enzyme, a fluorescent substance, a radioactive substance, a protein, or the like, but is not limited thereto.
  • methods of conjugating the material to antibodies are well known in the art.
  • the invention also provides a polynucleotide encoding an antibody or fragmentol thereof according to the invention as described above.
  • Polynucleotides may also be described as ligonucleotides or nucleic acids, and are generated using DNA molecules (eg cDNA or genomi c DNA), RNA molecules (eg mRNA), nucleotide analogues Analogues of DNA or RNA (eg, peptide nucleic acids and non-naturally occurring nucleotide analogues) and hybrids thereof.
  • DNA molecules eg cDNA or genomi c DNA
  • RNA molecules eg mRNA
  • nucleotide analogues Analogues of DNA or RNA (eg, peptide nucleic acids and non-naturally occurring nucleotide analogues) and hybrids thereof.
  • the polynucleotide may be single-stranded or double-stranded ( If the polynucleotide of the present invention is to encode the antibody or fragment thereof of the present invention, the sequence is not particularly limited, but preferably
  • ii) a polynucleotide represented by SEQ ID NOs: 21 to 26; And iii) a polynucleotide represented by SEQ ID NOs: 35 to 40; It may be to include a polynucleotide selected from the group consisting of.
  • Polynucleotides encoding the antibodies of the present invention or fragments thereof can be obtained by methods well known in the art. For example, oligonucleotide synthesis techniques well known in the art, such as polymerase chain reaction (PCR), etc., are based on DNA sequences or corresponding amino acid sequences encoding portions or all of the heavy and light chains of the antibody. Can be synthesized using PCR, etc.
  • the present invention also provides a vector comprising the polynucleotide.
  • the vector of the present invention is used for the purpose of replicating or expressing a polynucleotide of the present invention for recombinant production of an antibody or fragment thereof of the present invention, and generally includes a signal sequence, a copy origin, one or more marker genes, an enhancer. At least one of an urea, a promoter and a transcription termination sequence.
  • the vector of the present invention may preferably be an expression vector, more preferably a vector comprising a polynucleotide of the present invention operably linked to a regulatory sequence, for example, a promoter.
  • Plasmid refers to a linear or circular double helix DNA molecule into which external polynucleotide fragments can be joined.
  • Other forms of vectors are viral vectors (e.g., repl i cat ion defect ive retrovises, adenoviruses and adeno-associated viruses). Wherein additional DNA fragments can be introduced into the viral genome.
  • viral vectors e.g., repl i cat ion defect ive retrovises, adenoviruses and adeno-associated viruses.
  • additional DNA fragments can be introduced into the viral genome.
  • Certain vectors include bacterial vectors, including host cells into which they are introduced (eg, bacterial or igin and epi somal mammalian vectors). Autonomous repl icat ion can be achieved in Other vectors (eg, non-epi soma 1 mammal i an vectors) are integrated into the genome of the host cell by introduction into host cells and By the host genome.
  • Expression vectors are a form of expressible vector of selected polynucleotides.
  • One polynucleotide sequence is operatively linked to the regulatory sequence when the regulatory sequence affects the expression (eg, level, timing or location of expression) of the polynucleotide sequence.
  • the regulatory sequence is a sequence that affects the expression (eg, level, timing or location of expression) of the nucleic acid to which it is operably linked.
  • the regulatory sequence is influenced by, for example, its effect directly or through the action of one or more other molecules (eg, the regulatory sequence and / or polypeptides that bind the nucleic acid) to the regulated nucleic acid. Can be crazy.
  • the regulatory sequence includes promoters, enhancers and other expression control elements.
  • the vector of the present invention may preferably be a pCom3x (phagmid) vector containing a scF Insert at the Sfil site.
  • the present invention provides a cell comprising the vector of the present invention.
  • the cell of the present invention is not particularly limited as long as the cell can be used to express the polynucleotide of the present invention.
  • the cell of the present invention may be a prokaryote (e.g., E. coli), eukaryote (e.g., yeast or other fungus), plant cell (e.g. tobacco or tomato plant cell), animal cell (e.g., Human cells, monkey cells, hamster cells, rat cells, mouse cells or tortilla cells) or hybridomas.
  • a prokaryote e.g., E. coli
  • eukaryote e.g., yeast or other fungus
  • plant cell e.g. tobacco or tomato plant cell
  • animal cell e.g., Human cells, monkey cells, hamster cells, rat cells, mouse cells or tortilla cells
  • hybridomas e.g., human cells, monkey cells, hamster cells, rat cells, mouse cells or tortilla cells
  • Prokaryotes suitable for this purpose are Gram negative or Gram positive organisms, for example Enterobacteriaceae, for example Escherichia, for example E. coli. E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example Salmonella typhimurium, Serratia, For example, Serratia marcescans and Shigella, and Bacilli, for example B. Subtilis (B. subtil is) and b. B. licheniformis, Pseudomonas, for example blood. P. aeruginosa and Streptomyces.
  • the cells of the present invention are not particularly limited as long as they can express the vector of the present invention, but preferably.
  • Coli for example but not limited to. E. coli ER2537, this. Coli B, this. E. coli X1776 (ATCC 31,537). E. coli W3110 (ATCC 27,325) or LacZ expressing. May be coli, more preferably E. coli. E. coli ER2537 ⁇
  • eukaryotes are present in Saccharomyces cerevisiae
  • K. K.lactis K. K. fragilis
  • K. K. wickeramii ATCC 24,178
  • K. K. waltii ATCC 56,500
  • K. Drosophilarum ATCC 36,906
  • K. Termorerans ((K. thermotolerans) and K.
  • the cells of the present invention may be animal cells, particularly vertebrate cells.
  • Proliferation of vertebrate cells in culture has become a routine method and techniques are widely available.
  • useful mammalian host cells include, but are not limited to, monkey kidney CV1 line (COS-7, ATCC CRL 1651) transformed by SV40, human embryonic kidney lys (293 or 293 subcloned from suspension culture). Cells [Graham et al., 1977, J Gen Virol. 36: 59]), baby hamster kidney cells (BHK, ATCC CCL10), Chinese hamster ovary cells / -DHFR "(CHO, Urlaub et al., 1980, Proc. Natl. Acad Sci.
  • MRC 5 cells MRC 5 cells, FS4 cells, human liver cancer cell line (Hep G2), HEK 293 cells (human embryonic kidney cells) and Expi293F cells, preferably CHO cells , HEK 293 cells (human embryonic kidney cells) or Expi293 cells.
  • the cells of the present invention are cultured cells that can be transformed or transfected with the polynucleotide of the present invention or a vector comprising the same, and can be expressed continuously in the host cell.
  • Recombinant cell refers to a cell transformed or transfected with a polynucleotide to be expressed.
  • the cells of the invention may also be cells which contain a polynucleotide of the invention but which do not express it to the desired level unless a regulatory sequence is introduced into the cell so as to be operably linked to the polynucleotide. .
  • Cells of the invention can be cultured in a variety of media.
  • Commercially available media such as Ham 's F10 (Si ma-Aldrich Co., St. Louis, MO), minimum essential medium (MEM, Sigma-Aldrich Co.), RPMI-1640 (Sigma-Aldrich Co.), and Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich Co.) are suitable for culturing cells.
  • the medium may be added with hormones and / or other growth factors, salts, buffers, nucleotides, antibiotics, trace elements and glucose or equivalent energy sources if necessary.
  • the medium of the present invention is preferably SB (Bactotrytone 30g, yeast extract 20g,
  • the present invention is to culture the cells under the condition that the polynucleotide is expressed, to produce a polypeptide comprising a light chain and heavy chain variable region and to recover the polypeptide from the cell or culture medium cultured thereof It provides an antibody or fragment thereof and a production method that binds to human EPRS comprising the step.
  • the cells of the production method of the present invention are as described above, and contain a polynucleotide encoding the antibody of the present invention.
  • the polypeptide of the production method of the present invention may be an antibody of the present invention or a fragment thereof, and an amino acid sequence other than the antibody of the present invention or a fragment thereof may be further bound. In this case, it can be removed from the antibody or fragment thereof of the present invention using methods well known to those skilled in the art.
  • the medium composition and culture conditions may vary depending on the type of the cells. Which can be properly selected and adjusted by those skilled in the art.
  • the antibody molecule may accumulate in the cytoplasm of the cell, be secreted from the cell, or targeted to periplasm or extracellular medium by appropriate signal sequences, and the periplasm or extracellular medium. It is also desirable to refold the produced antibody molecule and to have a conformal ion using methods well known to those skilled in the art.
  • the recovery of the polypeptide may vary depending on the nature of the produced polypeptide and the properties of the cells, which can be appropriately selected and controlled by one of ordinary skill in the art.
  • the polypeptide may be produced intracellularly, in the surrounding cytoplasmic space, or directly secreted into the medium. If the polypeptide is produced in a cell, the cell can be destroyed to release the protein as a step 1 step. Particulate debris, host cells or lysed fragments are removed, for example, by centrifugation or ultrafiltration. When the antibody is secreted into the medium, the supernatant from this expression system is generally first concentrated using a commercially available protein enrichment filter, for example Amicon or Mi l ipore Pel l icon ultrafiltration unit. Protease inhibitors, such as PMSF, may be included in any preceding step to inhibit proteolysis, and antibiotics may be included to prevent accidental growth of contaminants.
  • PMSF Protease inhibitors, such as PMSF, may be included in any preceding step to inhibit proteolysis, and antibiotics may be included to prevent accidental growth of contaminants.
  • Antibodies prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis and affinity chromatography, and the antibodies of the invention are preferably via affinity chromatography. It can be purified.
  • the antibody or fragment thereof of the present invention specifically binds to EPRS and is therefore useful in diagnostic assays for detecting and quantifying EPRS proteins, for example for detecting EPRS expression in specific cells, tissues, or serum. .
  • the present invention provides an EPRS specific detection method comprising contacting the antibody or fragment thereof with a sample and detecting the antibody or fragment thereof.
  • the antibody or fragment thereof may generally be labeled with a detectable moiety.
  • Radioactivity can be measured, for example, by scintillation counting, and fluorescence can be quantified using a fluorometer.
  • luciferases include luciferases, luciferins such as choparic luciferase and bacterial luciferase (US Pat. No. 4,737,456).
  • luciferins such as choparic luciferase and bacterial luciferase (US Pat. No. 4,737,456).
  • 2,3 dihydrophthalazine diones peroxides such as malate dehydrogenase, urase, horseradish peroxidase (HRP0), alkaline phosphatase, ⁇ -gal Lactosidase, glucoamylase, lysozyme, saccharide oxidase (e.g.
  • Labels can be indirectly conjugated with antibodies using a variety of known techniques.
  • the antibody may be conjugated to biotin and any labels belonging to the three broad categolic rings mentioned above may be conjugated with avidin and vice versa. Biotin binds selectively to avidin and thus the label can be conjugated to the antibody in this indirect manner.
  • the antibody may be conjugated with a small hapten (eg digoxin) and one of the different types of labels mentioned above may be anti- Conjugated to hapten antibodies (eg, anti-dioxine antibodies).
  • hapten antibodies eg, anti-dioxine antibodies
  • Antibodies or fragments thereof of the present invention may be used in any known assay method such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays.
  • Antibodies or fragments thereof of the present invention may be used in a diagnostic kit, i.e., a diagnostic kit for performing a diagnostic assay, i.e., a packaged combination of reagents in a predetermined amount with instructions for use.
  • a diagnostic kit for performing a diagnostic assay, i.e., a packaged combination of reagents in a predetermined amount with instructions for use.
  • the kit will contain the cofactors required by the enzyme as substrate precursors to provide the substrate and chromophores or fluorophores. Can be.
  • other additives may be included, such as stabilizers, complete fluids (eg, blocked complete fluids or dissolved complete fluids).
  • the relative amounts of the various reagents can be varied widely to provide concentrations in solution of the reagents that further optimize the sensitivity of the assay.
  • the reagent may be provided as a generally lyophilized, dry powder, comprising an excipient that will provide a reagent solution with the appropriate concentration upon
  • EPRS is involved in the transcription process of regulators involved in various inflammatory and immune responses by forming complexes by binding to the 3-UTR portion of mRNA of various regulatory proteins (Sampath, P. et al. Noncanonical funct ion of glut amy 1 ro lyltRNA synthetase: gene-speci fic si lencing of translat ion. Cell 119, 195208 (2004)).
  • protein synthesis of Vascular Endothelial Growth Factor A (YEGFA) an important factor in angiogenesis, has been shown to be regulated by EPRS (Ray, P. S. et al. A stress-responsive).
  • RNA swi tch regulates VEGFA expression.Nature 457, 915919 (2009)).
  • EPRS can be used as a diagnostic marker for the diagnosis of certain cancers, inflammatory diseases, immune diseases and fibrosis, the progression of disease and the prognosis before and after treatment through detection.
  • Diagnosis and prognosis of cancer, inflammatory diseases, immune diseases, and fibrosis according to the present invention can be performed by detecting EPRS proteins in biological samples.
  • the present invention provides a composition for diagnosing cancer, inflammatory disease, immune disease, and fibrosis, comprising the antibody or fragment thereof of the present invention as an active ingredient.
  • the biological sample includes solid tissue samples such as blood and other liquid samples of biological origin, biopsy samples, tissue cultures or cells derived therefrom. More specifically, it may be, for example, but not limited to, tissues, extracts, cell lysates, whole blood, plasma, serum, saliva, ocular fluid, cerebrospinal fluid, sweat, urine, milk, ascites fluid, synovial fluid, peritoneal fluid, and the like. All.
  • the sample can be obtained from an animal, preferably a mammal, most preferably a human.
  • the sample may be pretreated before use for detection. For example, it may include filtration, distillation, extraction, concentration, inactivation of interference components, addition of reagents, and the like.
  • nucleic acids and proteins can be separated from the sample and used for detection.
  • the detection is as described above.
  • the type of the cancer is not particularly limited, and for example, breast cancer, colon cancer, Lung cancer, small cell lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular myeloma, uterine cancer, ovarian cancer, rectal cancer, anal muscle cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma Cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, Bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma and pituitary adenoma, preferably metastatic
  • fibrosis is not particularly limited, for example, liver fibrosis, renal fibrosis, pulmonary fibrosis, interstitial fibrosis, systemic scleroderma, macular degeneration, pancreatic fibrosis, spleen Fibrosis, cardiac fibrosis, mediastinal fibrosis, myelofibrosis, vascular fibrosis, skin fibrosis, eye fibrosis, joint fibrosis, myofiber, thyroid fibrosis, endocardial myocardial fibrosis, peritoneal fibrosis, Post-peritoneal fibrosis, progressive mass necrotic fibrosis, systemic systemic fibrosis, fibrotic complications of surgery and infectious fibrosis.
  • the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the antibody or fragment thereof as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating immune diseases, comprising the antibody or fragment thereof as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases, comprising an antibody or fragment thereof as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating fibrosis comprising an antibody or fragment thereof as an active ingredient.
  • composition of the present invention is characterized by comprising the antibody of the present invention or a fragment thereof as an active ingredient.
  • the pharmaceutical composition according to the present invention may include the antibody of the present invention or a fragment thereof alone or further include one or more pharmaceutically acceptable carriers.
  • “Pharmaceutically effective amount” refers to an amount that exhibits a higher response than a negative control, preferably an amount sufficient to treat cancer.
  • pharmaceutically acceptable means a physiologically acceptable and, when administered to humans, does not inhibit the action of the active ingredient and usually does not cause an allergic reaction, such as gastrointestinal disorders, dizziness or similar reactions. Refers to non-toxic compositions.
  • the antibody or fragment thereof may be administered in a variety of oral and parenteral formulations during clinical administration, and when formulated, it is a commonly used layering agent, extender, and binder. It may be prepared using a diluent or excipient such as a wetting agent, a disintegrant, a surfactant.
  • Solid preparations for oral administration include tablets, patients, powders, granules, capsules, troches, and the like, and the solid preparations may be at least one aryl derivative of Formula 1 of the present invention, or a pharmaceutical thereof. May be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose or gelatin with an acceptable salt. In addition to the simple excipients, lubricants such as magnesium stearate, talc and the like may also be used.
  • Liquid preparations for oral administration include suspensions, solvents, emulsions, or syrups.
  • various excipients such as wetting agents, sweeteners, fragrances, and preservatives can be used. Can be.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories.
  • the therapeutic composition of the present invention may be any physiologically acceptable carrier, excipient or stabilizer (Remington: The Science and Pract ice of Pharmacy, 19th Edit ion, Al fonso, R., ed, Mack Publ i shing). Co. (East on, PA: 1995)) and antibodies with the desired purity can be prepared in the form of lyophilized cakes or aqueous solutions for storage. Acceptable carriers, excipients or stabilizers may be used at the dosages and concentrations employed.
  • Non-toxic to recipients, complete solutions such as phosphoric acid, citric acid and other organic acids; Antioxidants including ascorbic acid; Low molecular weight (less than about 10 residues) polypeptides; Proteins such as serum albumin, gelatin or immunoglobulins; Hydrophilic polymers such as polyvinylpyridone; Amino acids such as glycine, glutamine, asparagine, arginine or lysine; Monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; Chelating agents such as EDTA; Sugar alcohols such as manny or sorbbi; Salt-forming counterions such as sodium; And / or nonionic surfactants such as tween, pluronics or polyethylene glycol (PEG).
  • Antioxidants including ascorbic acid
  • Low molecular weight (less than about 10 residues) polypeptides Proteins such as serum albumin, gelatin or immunoglobulins
  • Hydrophilic polymers
  • the dosage of the antibody or fragment thereof of the present invention to the human body may vary depending on the age, weight, sex, dosage form, health condition and degree of disease of the patient, and generally 0.01-100 mg / kg / week, preferably from 0.1 to 20 mg / kg / week, more preferably from 5 to 10 mg / kg / week. It may also be administered in divided doses at regular intervals, as determined by the physician or pharmacist.
  • the route of administration of the composition of the present invention may be administered or injected by known methods, for example intravenous, intraperitoneal, intracranial, subcutaneous, intramuscular, intraocular, intraarterial, cerebrospinal, or intralesional routes, Injection or injection by the sustained release system described below.
  • the antibody may be administered systemically.
  • compositions of the present invention may be used alone or in combination with methods using surgery, hormonal therapy, chemotherapy and biological response modifiers for the prevention or treatment of cancer.
  • a human VH3-23 / VLlg gene is used as a skeleton and a library for inserting random sequences into CDRs, and phages that selectively bind to EPRS are isolated and purified. The sequence was analyzed.
  • the affinity between the anti-EPRS scFv antibody and EPRS of the present invention was measured by surface resonance analysis. As a result, it was confirmed that the antibody of the present invention had a relatively high affinity with an equilibrium dissociation constant of about 10 nM.
  • the cross reaction properties of the anti-EPRS scFv antibodies of the present invention were measured.
  • the antibody of the present invention was ARS except for EPRS. It is confirmed that it does not bind to WRS and HRS which have WHEP domain.
  • the usefulness of the anti-EPRS scFv antibody of the present invention as a diagnostic antibody was tested. After preparing the coated with the present invention and antibody, the reaction was diluted by the concentration of EPRS WHEP domains standards, and the binding was measured by ELISA. As a result, it was confirmed that the antibody binding is measured in a concentration-dependent manner with respect to the EPRS WHEP domains standard, it was confirmed that the antibody of the present invention can be used for the diagnosis of cancer, inflammatory diseases, immune diseases and ischemia.
  • the present invention provides a method or diagnostic method for treating cancer, immune disease, inflammatory disease and fibrosis, comprising administering to a subject in need thereof an effective amount of the antibody or fragment thereof.
  • the present invention also provides the antibody or fragment thereof for use in the manufacture of a medicament or diagnostic agent for the treatment of cancer, immune disease, inflammatory disease and fibrosis.
  • the term 'effective amount' refers to an amount that exhibits the therapeutic and / or prophylactic effect of cancer, immune disease, inflammatory disease and fibrosis
  • the term 'subject' refers to an animal, preferably May be an animal including a mammal, especially a human, and may be a cell, tissue, organ or the like derived from an animal. The subject may be a patient in need of treatment.
  • the antibody or fragment thereof of the present invention specifically binds to human EPRS and does not have cross-reaction with other proteins including the same ARS fami ly, so that EPRS can be detected and suppressed. It is effective in the diagnosis and treatment of human cancer, inflammatory disease, immune disease and ischemia.
  • Figure 2 is a graph of the result of measuring the binding affinity of the anti-EPRS scFv Biocon-EPl antibody of the present invention by surface plasmon resonance (SPR) (Kd: equilibrium dissociation constant, horizontal axis: time (seconds), Vertical axis: response unit (RU)).
  • SPR surface plasmon resonance
  • Figure 3 is a graph of the result of measuring the binding affinity of the antibody of the present invention anti-EPRS scFv Biocon_EP2 by surface plasmon resonance (SPR) (Kd: equilibrium dissociation constant, horizontal axis: time (seconds), vertical axis: response unit (RU)).
  • SPR surface plasmon resonance
  • FIG. 4 is a graph showing the result of measuring the binding affinity of the antibody of the present invention, anti-EPRS scFv Biocon_EP3, by surface plasmon resonance (SPR) (Kd: Equilibrium dissociation constant, horizontal axis: time (sec), vertical axis: response unit (RU)).
  • SPR surface plasmon resonance
  • FIG. 5 is a graph of the cross-activity of the anti-EPRS scFv Biocon-EPl antibody, which is an antibody of the present invention, measured by Luminex Multiplex Assay method (vertical axis-(FI): fluorescence intensity, CRS: Cysteinyl-tRNA synthetase, DRS: Aspartyl-tRNA synthetase, EPRS: Glutamyl-prolyl tRNA synthetase, HRS : Histidyl-tRNA synthetase, WRS : Tryptophanyl-tRNA synthetase, AIMP1 : AS-binding multifunctional protein 1 (Aminoacyl-tRNA-synthetase-interacting multifunctional protein 1)), AIMP3: ARS-binding multifunctional protein 3 (Aminoacyl-tRNA-synthetase—interacting multifunctional protein 3)).
  • Luminex Multiplex Assay method vertical axis-(FI)
  • FIG. 6 is a graph illustrating the cross-activity of the anti-EPRS scFv Biocon-EP2 antibody, which is an antibody of the present invention, measured by Luminex Multiplex Assay method (vertical axis (FI): fluorescence intensity, CRS) : Cysteinyl-tRNA synthetase, DRS: Aspartyl—tRNA synthetase, EPRS: Glutamyl-prolyl tRNA synthetase, HRS : Histidyl-tRNA synthetase, WRS: Tryptophanyl-tRNA synthetase, AIMP1: ARS binding multifunctional protein 1 (Aminoacyl-tRNA-synthetase-interacting multifunctional protein) D), AIMP3: ARS-binding multifunctional protein 3 (Aminoacyl-tRNA-synthetase-interacting multifunctional protein 3)).
  • FI fluorescence intensity
  • CRS Cysteinyl-t
  • Figure 7 is a graph of the cross activity of the anti-EPRS scFv Biocon_EP3 antibody of the present invention measured by Luminex Multiplex Assay method (vertical axis (FI): fluorescence intensity, CRS: cysteinyl- CyRNAl-tRNA synthetase, DRS: Aspartyl-tRNA synthetase, EPRS: Glutamyl-prolyl tRNA synthetase, HRS : histidyl-tRNA Histyl-tRNA synthetase, WRS: Tryptophanyl-tRNA synthetase, AIMP1: Ami noacyl-tRNA-synthetase-interacting multifunctional protein 1), AIMP3 : ARS binding multifunctional protein 3 (Aminoacyl-tRNA-synthetase— interacting multifunctional protein 3).
  • FI vertical axis
  • Fig. 8 shows the results of ELISA experiments to determine whether the antibody of the present invention can detect EPRS (vertical axis: 450 nra absorbance, horizontal axis: EPRS WHEP domains standard concentration (ng / ml)).
  • Libraries are designed by placing human VH3-23 / VLlg genes in the backbone and inserting random sequences into complementarity determining regions (CDRs), and the beta lactase genes as selection markers.
  • CDRs complementarity determining regions
  • beta lactase genes as selection markers.
  • CDRs complementarity determining regions
  • plasmid vector immediately after the beta-lactamase gene leader sequence, restriction enzyme fragment sequences were introduced to prepare a pFDV plasmid vector, an scFv gene library was inserted, transfected into E. coli and cultured. Through this process, it is possible to improve the quality of the library by removing most scFv gene sequences having abnormal stop codons or frameshi ft in the library.
  • Sequences from which the error was removed from the cultured library were amplified by polymerase chain reaction, from which the scFv gene library was recombined and inserted into the pComb3X phagemid vector, and E. coli ER2537 strain E. coli was transfected to obtain a final library.
  • the library was incubated in 400 mL of SB (Super broth) medium containing carbenicillin, and the absorbance at 600 nanometers was 0.5, adding 10 13 CFU of VCSM13 auxiliary phage and stirring at 80 rpm for 1 hour. Infected at 37 degrees.
  • the final 70 yg / mL kanamycin antibiotic was added and stirred overnight at 30 degrees Celsius, 200 rpm to incubate overnight to produce scFv surface-treated phage.
  • the precipitated phage was dissolved in 50 mL of complete PBS solution, precipitated again in the same manner as above, and finally dissolved in 2 mL of PBS buffer.
  • the phage scFv library was obtained by centrifugation to remove foreign substances.
  • the final phage library contains more than 10 13 CFU / mL phage particles. .
  • ⁇ 26i> The eluted phage was neutralized with 1.0M Tr i s-HCl buffer (pH 7.8) and then infected with ER2537 Escherichia coli at 37 ° C for 1 hour, and the infected E. coli containing carbenicillin LB (Lur ia-Bertani) was applied to agar medium and incubated at 37 ° C. The next day, E. coli cultured was suspended in 3 mL of super broth (SB) -carbenicillin culture and 15% glycerol was added to keep some at -80 ° C, and 50 microliters of 20 mL of SB- Carbenicillin -2% glucose solution was inoculated and incubated at 37 ° C.
  • SB super broth
  • the antigen-specific clone was concentrated by repeating the panning process using 1 mL of the supernatant containing phage particles as a library.
  • E. coli was suspended in a solution of 40 IX TES (50 mM Tr is, 1 mM EDTA, 20% Sucrose, pH 8.0), and then mixed with 60 uL of 0.2X TES solution, mixed for 30 minutes at 4 degrees Celsius, and centrifuged. The periplasm was extracted with supernatant.
  • IX TES 50 mM Tr is, 1 mM EDTA, 20% Sucrose, pH 8.0
  • the scFv antibody extracted from periplasm was used to determine whether the scFv antibody binds to EPRS originally expressed in human cells using immunoblot technique. After 50 ug of Hela cel l lysate was electrophoresed through SDS PAGE, it was transferred to Ni trocel lulos membrane by Wet transfer method, blocked with 3% skim milk, and then extracted by adding scFv antibody. For detection, the reacted scFv reacted with an Ant i-HA secondary antibody linked with HRPChorseradi sh peroxidase) and was then photosensitive in the dark room using ECL reagent as a substrate. A band corresponding to the size of the EPRS was identified in comparison with the prominent standard molecular markers.
  • the identified antigen-specific antibody clones were cultured overnight in 10 ml of SB medium containing carbenicillin, plasmid DNA was extracted using plasmid miniprep kit, and the base was obtained through Capillary sequencing service (Macrogen Co). The sequences were analyzed and CDR sequences were analyzed based on Kabat protein seqeunce database and IMGKthe internat ional ImMunoGeneTics informat ion system.
  • the number of scFv bound to the EPRS antigen was 3 in total, and their nucleotide sequences were found to be SEQ ID NOs: 14, 28 and 42.
  • the binding affinity of the antibody of the present invention to the EPRS WEHP domain antigen was measured using a Prote0nTMXPR36 surface plasmon resonance (SPR) biosensor (Bio—Rad). Specifically, about 10 ug / ml of EPRS antigen was added to the GLC chip (Bio-Rad 6 X 6 sensor chip, Compact capacity amine coupling for protein-protein interact ions) according to the manufacturer's instructions. After immobilization, 30 ⁇ l of purified scFv antibody (250-30 nM) of the present invention diluted to various concentrations using PBS was injected into the chip at a rate of 50 ul / min at 25 ° C. to interact with antigen. Dragon was quantified.
  • SPR surface plasmon resonance
  • the surface of the chip was regenerated with 0.85% phosphoric acid, the association rate and dissociation rate were calculated using ProteOn Manager software, and the equilibrium dissociation constant (KD) was calculated as the dissociation rate / association rate ratio. As shown in [4], it was confirmed that the antibody of the present invention had a maximum KD 10 nM value and had a very high EPRS affinity.
  • Anti-EPRS scFv antibody cross activity measurement In order to determine whether the scFv antibody is reactive with other antigens, cross reaction between the antibody of the present invention and EPRS and other ARS family proteins was measured using Luminex bead.
  • the beads connected to each ARS were washed twice with PBS and then added with a blocking buffer to react at room temperature for 30 minutes to block unbound residues. After washing twice with PBS, the beads were suspended in LOOul PBS and placed in a light-blocked tube. Stored. The number of bound beads was recorded by measuring with a hemocytometer.
  • ⁇ 3 ⁇ > antibody of the present invention extracted from the periplasm was subjected to sample dilution (PBST + 2% BSA)
  • the solution was first diluted at 1: 400 concentration, then diluted twice with sample diluent in 96 well filter plates, and 50ul were dispensed.
  • Each ARS-coupled bead was placed in a bead mix tube so that 2000 beads per well were added in a volume capable of dispensing 50 ul of sample diluent per well.
  • each we ll was dispensed and reacted in the dark for 1 hour.
  • the vacuum manifold was washed three times with PBS (200 ul / well), and then 50 ul Anti- (HA) -biotin secondary antibody was added and reacted in the dark for 1 hour.
  • an EPRS sandwich EL ISA pairing test was performed.
  • the capture antibody was used as the antibody of the present invention and the detection antibody was used as a rabbit polyclonal product. With antibody Pairing tests were performed to determine whether a quantitative curve for EPRS standards was produced.
  • the purified antibody of the present invention was coated with a coat ing buffer (0.1 M sodium carbonate H).
  • the plate was washed three times with PBST, and then HRP-linked ant i-rabbit IgG was added and reacted at room temperature for 1 hour for binding.
  • HRP HRP-linked ant i-rabbit IgG was added and reacted at room temperature for 1 hour for binding.
  • wash the plate 3 times with PBST add 50 ul of TRP solut ion (HRP substrate) for 10 minutes, and stop the color reaction by adding 50 ul of 2N sulfuric acid.
  • Absorbance was measured at nm.
  • the antibody of the present invention can be used for the diagnosis of cancer and immune diseases.
  • the antibody or fragment thereof of the present invention specifically binds to human EPRS, and does not have cross-reaction with other proteins including the same ARS fami ly, thus enabling EPRS detection and inhibition.

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  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The present invention relates to an anti-EPRS (glycyl-tRNA synthetase) antibody which selectively binds to EPRS and, more specifically, to an antibody which binds to human EPRS, or a fragment thereof, a production method thereof, and a pharmaceutical composition for diagnosing and treating cancers, inflammatory diseases, immune disorders and fibrosis comprising the same. The antibody or a fragment thereof of the present invention can be used for the purpose of detecting EPRS and diagnosing and treating cancers, inflammatory diseases, immune disorders and fibrosis, which are diseases related to EPRS, since antibody or a fragment thereof specifically binds to human EPRS and has no cross-reactivity with other proteins including the same ARS family, thereby enabling detection and inhibition of EPRS.

Description

【명세서】  【Specification】
【발명의 명칭】  [Name of invention]
항 EPRS모노클로날 항체 및 이의 용도 【기술분야】  Anti-EPRS Monoclonal Antibodies and Uses thereof
<ι> 본 출원은 2015년 1월 29일에 출원된 대한민국 특허출원 계 10-2015-0014616 호를 우선권으로 주장하고， 상기 명세서 전체는 본 출원의 참고문헌이다.  <ι> This application claims the priority of Korean Patent Application No. 10-2015-0014616, filed on January 29, 2015, the entirety of which is a reference of the present application.
<2>  <2>
<3> 본 발명은 EPRS glutamyl-prolyl tRNA synthetase)에 선택적으로 결합하는 항 EPRS 항체에 관한 것으로 보다 구체적으로는 인간 EPRS에 결합하는 항체 또는 그 단편 이의 생산방법 및 이를 포함하는 암 치료용 약학적 조성물에 관한 것이 다.  The present invention relates to an anti-EPRS antibody that selectively binds to EPRS glutamyl-prolyl tRNA synthetase), and more specifically to an antibody or fragment thereof that binds to human EPRS, and a pharmaceutical composition for treating cancer comprising the same. It's about
<4>  <4>
【배경기술】  Background Art
<5> Aminoacyl-tRNA synthetase(ARS)는 특정 아미노산을 그 해당하는 tRNA에 붙 여주는 역할을 하는 효소이며. 고등생물의 경우 아미노산 종류에 따른 20 개의 효 소외에 ΑΙΜΡ1(ρ43), (AIMP2)p38, (AIMP3)pl8 등 mult i synthetase complex 형성에 관여하는 3종류를 포함하여 23종의 효소로 구성되어 있으며 mul ti synthetase complex에 참여하는 효소외에 몇몇은 free form 형태로도 존재한다. 그러나 최근 들어 기본적인 기능외에 특정 환경에서 다양한 다른 활성기능을 가지고 있음이 보 고 되었는데 EPRS(glutamyl-prolyl tRNA synthetase)가 그중의 하나이다. EPRS는 두가지 효소 활성 도메인 (domain)이 링커 (linker)로 연결되어 있는 형태이며 이 링 커는 WHEP이라는 도메인이 3개가 반복된 형태로 되어있다. 이 WHEP 도메인 이라는 구조는 WRS, HRS, GRS, MRS 등과 같은 몇몇 ARSs 효소에도 유사한 구조가 있으나 EPRS와는 달리 반복된 구조를 가지고 있지는 않다. EPRS와 질환과의 관련성으로는 인터페론 감마 (IFN-γ)에 의해 EPRS는 여러 조절 단백질의 mRNA의 3-UTR 부분에 결 합하여 복합체 (GAIT, gamma- int erf eron-act ivated inhibitor of translation complex)를 형성함으로 인해 여러 염증반응 및 면역반응에 관여하는 조절인자의 전 사 과정에 관여하는 것으로 알려져 있다 ( Sampath, P. et al . Noncanonical function of glut amylprolyl tRNA synthetase: gene-specific silencing of translation. Cell 119， 195208 (2004)). 또한 혈관신생 (angiogenesis)에 중요한 인자로 알려진 VEGFA (Vascular endothelial growth factor A)의 단백질 합성과정 도 EPRS에 의해 조절되는 것으로 밝혀졌다 (Ray, P. S. et al. A stress- responsive RNA swi tch regulates VEGFA expression. Nature 457, 915919 (2009) ) . 따라서 EPRS는 암과 면역질환 및 염증질환 관련 유전자 발현에 있어 중요한 조절자 로 관여할 수 있으며 중요한 진단 바이오마커로 사용될 가능성이 있다. <5> Aminoacyl-tRNA synthetase (ARS) is an enzyme that attaches a specific amino acid to its corresponding tRNA. Higher organisms consist of 23 enzymes, including 3 species involved in the formation of mult i synthetase complexes, including ΑΙΜΡ1 (ρ43), (AIMP2) p38, and (AIMP3) pl8. In addition to the enzymes involved in the ti synthetase complex, some also exist in free form. Recently, however, it has been reported to have various other active functions in a specific environment in addition to the basic function, and one of them is EPRS (glutamyl-prolyl tRNA synthetase). EPRS is a form in which two enzyme active domains are linked by a linker, which is a form in which the WHEP domain is repeated three times. This WHEP domain is similar in structure to some ARSs enzymes such as WRS, HRS, GRS, MRS, etc., but unlike EPRS, it does not have a repeated structure. Interferon-gamma (IFN-γ) is a link between EPRS and disease, and EPRS binds to the 3-UTR portion of mRNAs of several regulatory proteins to form a complex (GAIT, gamma- int eron-activated inhibitor of translation complex) Formation is known to be involved in the transcriptional processes of regulators involved in various inflammatory and immune responses (Sampath, P. et al. Noncanonical function of glut amylprolyl tRNA synthetase: gene-specific silencing of translation. Cell 119, 195208 (2004)). Protein synthesis of Vascular endothelial growth factor A (VEGFA), also known as an important factor in angiogenesis It has also been shown to be regulated by EPRS (Ray, PS et al. A stress-responsive RNA swi tch regulates VEGFA expression. Nature 457, 915919 (2009)). Thus, EPRS may be an important regulator of cancer, immune and inflammatory disease gene expression and may be used as an important diagnostic biomarker.
<6> 하지만 EPRS를 비롯한 ARS들에 대한 바이오마커로서의 중요성에도 불구하고 <6> However, despite its importance as a biomarker for ARSs including EPRS
ARS들은 단백질 구조상 유사한 점이 많아 동물로부터 면역반응으로 얻어지는 항체 는 다른 ARS에도 결합하는 교차 반웅을 보이고 아예 고 감도의 항체가 생성되지 않 은 경우가 많다. 본 발명의 항체는 우수한 감도와 ARS간 교차반응이 없는 점에서 연구용 뿐만 아니라 진단용 및 산업상 이용가능성이 높은 항체라 예상된다. Since ARSs have similarities in protein structure, antibodies obtained from an immune response from animals show cross reactions that bind to other ARSs, and in many cases no high-sensitivity antibodies are produced. Antibodies of the present invention are expected to be of high diagnostic and industrial availability as well as for research in view of their excellent sensitivity and no cross-reaction between ARS.
<7>  <7>
【발명의 상세한 설명】  [Detailed Description of the Invention]
【기술적 과제】  [Technical problem]
<8> 본 발명자들은 EPRS에 특이적으로 결합하는 항체를 연구하던 중， 파지디스플 레이 방법으로 만들어진 라이브러리에서 EPRS에 특이적으로 결합하는 단편들을 찾 아내고, 이의 서열 및 결합특이성을 밝혀내어 본 발명을 완성하였다.  While studying the antibody specifically binding to EPRS, the present inventors searched for fragments that specifically bind to EPRS in a library made by phage display method, and found the sequence and binding specificity thereof. The invention has been completed.
<9>  <9>
<ιο> 따라서 본 발명의 목적은 인간 EPRS에 결합하는 항체 또는 그 단편을 제공하 는 것이다.  It is therefore an object of the present invention to provide antibodies or fragments thereof that bind to human EPRS.
<1 1>  <1 1>
<12> 본 발명의 다른 목적은 상기 항체 또는 그 단편을 암호화 하는 폴리뉴클레오 티드를 제공하는 것이다.  Another object of the present invention is to provide a polynucleotide encoding the antibody or fragment thereof.
<13>  <13>
<14> 본 발명의 다른 목적은 상기 폴리뉴클레오티드를 포함하는 백터를 제공하는 것이다.  Another object of the present invention is to provide a vector comprising the polynucleotide.
<15>  <15>
<16> 본 발명의 다른 목적은 상기 백터를 포함하는 세포를 제공하는 것이다.  Another object of the present invention is to provide a cell comprising the vector.
<17>  <17>
<18> 본 발명의 다른 목적은 인간 EPRS에 결합하는 항체 또는 그 단편의 생산방법 을 제공하는 것이다.  Another object of the present invention is to provide a method for producing an antibody or fragment thereof that binds to human EPRS.
<19>  <19>
<20> 본 발명의 다른 목적은 항체 또는 그 단편을 시료와 접촉시키는 단계 및 상 기 항체 또는 그 단편을 검출하는 단계를 포함하는 EPRS 특이적 검출 방법을 제공 하는 것이다. Another object of the present invention is to provide an EPRS specific detection method comprising contacting an antibody or fragment thereof with a sample and detecting the antibody or fragment thereof. It is.
<21>  <21>
<22> 본 발명의 다른 목적은 상기 항체 또는 그 단편을 유효성분으로 포함하는 암 예방 또는 치료용 약학적 조성물을 제공하는 것이다.  Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising the antibody or fragment thereof as an active ingredient.
<23>  <23>
<24> 본 발명의 다른 목적은 상기 항체 또는 그 단편을 유효성분으로 포함하는 암 진단용 조성물을 제공하는 것이다.  Another object of the present invention is to provide a cancer diagnostic composition comprising the antibody or fragment thereof as an active ingredient.
<25>  <25>
<26> 본 발명의 다른 목적은 상기 항체 또는 그 단편을 유효성분으로 포함하는 면 역질환 예방 또는 치료용 약학적 조성물을 제공하는 것이다.  Another object of the present invention is to provide a pharmaceutical composition for preventing or treating immune diseases, comprising the antibody or fragment thereof as an active ingredient.
<27>  <27>
<28> 본 발명의 다른 목적은 상기 항체 또는 그 단편을 유효성분으로 포함하는 면 역질환 진단용 조성물을 제공하는 것이다.  Another object of the present invention to provide an immunological disease diagnostic composition comprising the antibody or fragment thereof as an active ingredient.
<29>  <29>
<30> 본 발명의 다른 목적은 상기 항체 또는 그 단편을 유효성분으로 포함하는 염 증질환 예방 또는 치료용 약학적 조성물을 제공하는 것이다.  Another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising the antibody or fragment thereof as an active ingredient.
<31>  <31>
<32> 본 발명의 다른 목적은 상기 항체 또는 그 단편을 유효성분으로 포함하는 염 증질환 진단용 조성물을 제공하는 것이다.  Another object of the present invention is to provide a composition for diagnosing inflammatory diseases comprising the antibody or fragment thereof as an active ingredient.
<33>  <33>
<34> 본 발명의 다른 목적은 상기 항체 또는 그 단편을 유효성분으로 포함하는 섬 유화증 예방 또는 치료용 약학적 조성물을 제공하는 것이다.  Another object of the present invention is to provide a pharmaceutical composition for preventing or treating islet emulsification comprising the antibody or fragment thereof as an active ingredient.
<35>  <35>
<36> 본 발명의 다른 목적은 상기 항체 또는 그 단편을 유효성분으로 포함하는 섬 유화증 진단용 조성물을 제공하는 것이다.  Another object of the present invention is to provide a composition for diagnosing ischemic disease comprising the antibody or fragment thereof as an active ingredient.
<37>  <37>
<38> 본 발명의 다른 목적은 상기 항체 또는 그 단편을 필요로 하는 개체에 유효 량으로 투여하는 단계를 포함하는 암， 면역질환, 염증질환 및 섬유화증 치료 방법 또는 진단 방법을 제공하는 것이다.  Another object of the present invention is to provide a method for treating or diagnosing cancer, immune disease, inflammatory disease and fibrosis, comprising administering an effective amount of the antibody or fragment thereof to a subject in need thereof.
<39>  <39>
<40> 본 발명의 다른 목적은 암， 면역질환， 염증질환 및 섬유화증 치료용 약제 또 는 진단 제제 제조의 용도를 위한 상기 항체 또는 그 단편을 제공하는 것이다. <41> It is another object of the present invention to provide such an antibody or fragment thereof for use in the manufacture of a medicament or diagnostic agent for the treatment of cancer, immune diseases, inflammatory diseases and fibrosis. <41>
【기술적 해결방법】  Technical Solution
<42> 상기의 목적을 달성하기 위해 본 발명은 i ) 서열번호 1로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)Llᅳ 서열번호 2로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)L2, 서열번호 3으로 표시되는 아미노산 서열을 포함 하는 상보성 결정부위 (CDR)L3를 포함하는 항체 경쇄가변영역 (VL) 및 서열번호 4로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)Hl, 서열번호 5로 표시되 는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)H2 , 서열번호 6으로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)H3를 포함하는 항체 중쇄가변영역 (VH)을 포함하는 항체；  In order to achieve the above object, the present invention provides a method for determining the complementarity determining region (CDR) comprising an amino acid sequence represented by SEQ ID NO: 2). ) L2, the complementarity determining region (CDR) comprising the amino acid sequence represented by SEQ ID NO: 3, the antibody light chain variable region (VL) comprising the L3, and the complementarity determining region (CDR) Hl comprising the amino acid sequence represented by SEQ ID NO: 4 An antibody heavy chain variable region (VH) comprising a complementarity determining region (CDR) H2 comprising an amino acid sequence represented by SEQ ID NO: 5, a complementarity determining region (CDR) H3 comprising an amino acid sequence represented by SEQ ID NO: 6 Containing antibody;
<43> i i ) 서열번호 15로 표시되는 아미노산 서열을 포함하는 상보성 결정부위  I i) Complementarity determining site comprising the amino acid sequence represented by SEQ ID NO: 15
(CDR)Ll, 서열번호 16으로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)L2 , 서열번호 17로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)L3를 포함하는 항체 경쇄가변영역 (V 및 서열번호 18로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)Hl , 서열번호 19로 표시되는 아미노산 서열 을 포함하는 상보성 결정부위 (CDR)H2 , 서열번호 20으로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)H3를 포함하는 항체 중쇄가변영역 (VH)을 포함하는 항체 ; 및  (CDR) Ll, an antibody light chain variable region comprising a complementarity determining region (CDR) L2 comprising an amino acid sequence represented by SEQ ID NO: 16, a complementarity determining region (CDR) L3 comprising an amino acid sequence represented by SEQ ID NO: 17 Complementarity Determining Site (CDR) H1 comprising an amino acid sequence represented by V and SEQ ID NO: 18, Complementarity Determining Site (CDR) H2 comprising an amino acid sequence represented by SEQ ID NO: 19, and an amino acid sequence represented by SEQ ID NO: 20 An antibody comprising an antibody heavy chain variable region (VH) comprising a complementarity determining region (CDR) H3; and
<44> i i i ) 서열번호 29로 표시되는 아미노산 서열을 포함하는 상보성 결정부위  I i) complementarity determining site comprising the amino acid sequence represented by SEQ ID NO: 29
(CDR)Ll, 서열번호 30으로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)L2, 서열번호 31로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)L3를 포함하는 항체 경쇄가변영역 (VL) 및 서열번호 32로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)Hl , 서열번호 33으로 표시되는 아미노산 서 열을 포함하는 상보성 결정부위 (CDR)H2, 서열번호 34로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)H3를 포함하는 항체 증쇄가변영역 (VH)을 포함하는 항체；  (CDR) Ll, an antibody light chain variable region comprising a complementarity determining region (CDR) L2 comprising an amino acid sequence represented by SEQ ID NO: 30, a complementarity determining region (CDR) L3 comprising an amino acid sequence represented by SEQ ID NO: 31 VL) and the complementarity determining region (CDR) Hl comprising the amino acid sequence represented by SEQ ID NO: 32, the complementarity determining region (CDR) H2 comprising the amino acid sequence represented by SEQ ID NO: 33, amino acid sequence represented by SEQ ID NO: 34 An antibody comprising an antibody recombination region (VH) comprising a complementarity determining region (CDR) H3 comprising a;
<45> 로 이루어지는 군에서 선택된 인간 EPRS에 결합하는 항체 또는 그 단편을 제 공한다.  Provided are antibodies or fragments thereof that bind to human EPRS selected from the group consisting of:
<46>  <46>
<47> 본 발명의 다른 목적을 달성하기 위해 본 발명은 상기 항체 또는 그 단편을 암호화 하는 폴리뉴클레오티드를 제공한다.  In order to achieve another object of the present invention, the present invention provides a polynucleotide encoding the antibody or fragment thereof.
<48> <49> 본 발명의 다른 목적을 달성하기 위해 본 발명은 상기 폴리뉴클레오티드를 포함하는 백터를 제공한다. <48> In order to achieve another object of the present invention, the present invention provides a vector comprising the polynucleotide.
<50>  <50>
<51> 본 발명의 다른 목적을 달성하기 위해 본 발명은 상기 백터를 포함하는 세포 를 제공한다.  In order to achieve another object of the present invention, the present invention provides a cell comprising the vector.
<52>  <52>
<53> 본 발명의 다른 목적을 달성하기 위해 본 발명은 상기 세포를 폴리뉴클레오 티드가 발현되는 조건하에서 배양하여, 경쇄 및 중쇄가변영역을 포함하는 폴리펩타 이드를 생산하는 단계 및 상기 세포 또는 이를 배양한 배양 배지로부터 상기 폴리 펩타이드를 회수하는 단계를 포함하는 인간 EPRS에 결합하는 항체 또는 그 단편의 생산방법을 제공한다.  In order to achieve another object of the present invention, the present invention provides a method for producing a polypeptide comprising a light chain and a heavy chain variable region by culturing the cells under a condition in which the polynucleotide is expressed, and the cells or the same. It provides a method for producing an antibody or fragment thereof that binds to human EPRS, comprising recovering the polypeptide from the culture medium.
<54>  <54>
<55> 본 발명의 다른 목적을 달성하기 위해 본 발명은 항체 또는 그 단편을 시료 와 접촉시키는 단계 및 상기 항체 또는 그 단편을 검출하는 단계를 포함하는 EPRS 특이적 검출 방법을 제공한다.  In order to achieve another object of the present invention, the present invention provides an EPRS specific detection method comprising contacting an antibody or fragment thereof with a sample and detecting the antibody or fragment thereof.
<56>  <56>
<57> 본 발명의 다른 목적을 달성하기 위해 본 발명은 상기 항체 또는 그 단편을 유효성분으로 포함하는 암 예방 또는 치료용 약학적 조성물을 제공한다 .  In order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the antibody or fragment thereof as an active ingredient.
<58>  <58>
<59> 본 발명의 다른 목적을 달성하기 위해 본 발명은 상기 항체 또는 그 단편을 유효성분으로 포함하는 암 진단용 조성물을 제공한다.  In order to achieve another object of the present invention, the present invention provides a composition for diagnosing cancer comprising the antibody or fragment thereof as an active ingredient.
<60>  <60>
<61> 본 발명의 다른 목적을 달성하기 위해 본 발명은 상기 항체 또는 그 단편을 유효성분으로 포함하는 면역질환 예방 또는 치료용 약학적 조성물을 제공한다. In order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating immune diseases, comprising the antibody or fragment thereof as an active ingredient.
<62> <62>
<63> 본 발명의 다른 목적을 달성하기 위해 본 발명은 상기 항체 또는 그 단편을 유효성분으로 포함하는 면역질환 진단용 조성물을 제공한다.  In order to achieve another object of the present invention, the present invention provides a composition for diagnosing immunological diseases comprising the antibody or fragment thereof as an active ingredient.
<64>  <64>
<65> 본 발명의 다른 목적을 달성하기 위해 본 발명은 상기 항체 또는 그 단편을 유효성분으로 포함하는 염증질환 예방 또는 치료용 약학적 조성물을 제공한다. In order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases, comprising the antibody or fragment thereof as an active ingredient.
<66> <66>
<67> 본 발명의 다른 목적을 달성하기 위해 본 발명은 상기 항체 또는 그 단편을 유효성분으로 포함하는 염증질환 진단용 조성물을 제공한다. In order to achieve another object of the present invention, the present invention is directed to the antibody or fragment thereof. It provides a composition for diagnosing inflammatory diseases comprising as an active ingredient.
<68>  <68>
<69> 본 발명의 다른 목적을 달성하기 위해 본 발명은 상기 항체 또는 그 단편을 유효성분으로 포함하는 섬유화증 예방 또는 치료용 약학적 조성물을 제공한다 . In order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating fibrosis comprising the antibody or fragment thereof as an active ingredient.
<70> <70>
<71> 본 발명의 다른 목적을 달성하기 위해 본 발명은 상기 항체 또는 그 단편을 유효성분으로 포함하는 섬유화증 진단용 조성물을 제공한다.  In order to achieve the another object of the present invention, the present invention provides a composition for diagnosing fibrosis comprising the antibody or fragment thereof as an active ingredient.
<72>  <72>
<73> 본 발명의 다른 목적올 달성하기 위해 본 발명은 상기 항체 또는 그 단편을 필요로 하는 개체에 유효량으로 투여하는 단계를 포함하는 암， 면역질환， 염증질환 및 섬유화증 치료 방법 또는 진단 방법을 제공한다.  In order to achieve another object of the present invention, the present invention provides a method for treating cancer, immune disease, inflammatory disease and fibrosis, or a method for diagnosing cancer, comprising administering the antibody or fragment thereof in an effective amount to a subject in need thereof. to provide.
<74>  <74>
<75> 본 발명의 다른 목적을 달성하기 위해 본 발명은 암， 면역질환， 염증질환 및 섬유화증 치료용 약제 또는 진단 제제 제조의 용도를 위한 상기 항체 또는 그 단편 을 제공한다.  In order to achieve another object of the present invention, the present invention provides the antibody or fragment thereof for use in the manufacture of a medicament or diagnostic agent for the treatment of cancer, immune diseases, inflammatory diseases and fibrosis.
<76>  <76>
<77> 이하 본 발명을 상세히 설명한다 .  Hereinafter, the present invention will be described in detail.
<78>  <78>
<79> 본 발명은 i ) 서열번호 1로 표시되는 아미노산 서열을 포함하는 상보성 결정 부위 (CDR)Ll , 서열번호 2로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)L2, 서열번호 3으로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)L3를 포함하는 항체 경쇄가변영역 (VL) 및 서열번호 4로 표시되는 아미노산 서 열을 포함하는 상보성 결정부위 (CDR)Hl , 서열번호 5로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDF H2 , 서열번호 6으로 표시되는 아미노산 서열을 포함 하는 상보성 결정부위 (CDR)H3를 포함하는 항체 중쇄가변영역 (VH)을 포함하는 항체 ; <80> i i ) 서열번호 15로 표시되는 아미노산 서열을 포함하는 상보성 결정부위  I) Complementarity Determining Site (CDR) Ll comprising an amino acid sequence represented by SEQ ID NO: 1, Complementarity Determining Site (CDR) L2 comprising an amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 3 An antibody light chain variable region (VL) comprising a complementary determining region (CDR) L3 comprising an indicated amino acid sequence and a complementarity determining region (CDR) Hl comprising an amino acid sequence represented by SEQ ID NO: 4, represented by SEQ ID NO: 5 An antibody comprising an antibody heavy chain variable region (VH) comprising a complementarity determining region (CDF H2) comprising an amino acid sequence as defined herein (CDF H2, complementary determining region (CDR) H3 comprising an amino acid sequence represented by SEQ ID NO: 6); ) Complementarity determining site comprising the amino acid sequence represented by SEQ ID NO: 15
(CDR)Ll , 서열번호 16으로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)L2, 서열번호 17로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)L3를 포함하는 항체 경쇄가변영역 (VL) 및 서열번호 18로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)Hl , 서열번호 19로 표시되는 아미노산 서열 을 포함하는 상보성 결정부위 (CDR)H2, 서열번호 20으로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)H3를 포함하는 항체 중쇄가변영역 (VH)을 포함하는 항체; 및 (CDR) Ll, an antibody light chain variable region comprising a complementarity determining region (CDR) L2 comprising an amino acid sequence represented by SEQ ID NO: 16, a complementarity determining region (CDR) L3 comprising an amino acid sequence represented by SEQ ID NO: 17 VL) and the complementarity determining region (CDR) Hl comprising the amino acid sequence represented by SEQ ID NO: 18, the complementarity determining region (CDR) H2 comprising the amino acid sequence represented by SEQ ID NO: 19, the amino acid sequence represented by SEQ ID NO: 20 Comprising an antibody heavy chain variable region (VH) comprising a complementarity determining region (CDR) H3 comprising Antibodies; And
<8i> i i i ) 서열번호 29로 표시되는 아미노산 서열을 포함하는 상보성 결정부위  <8i> i i) complementarity determining site comprising the amino acid sequence represented by SEQ ID NO: 29
(CDR)Ll , 서열번호 30으로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)L2, 서열번호 31로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)L3를 포함하는 항체 경쇄가변영역 (VL) 및 서열번호 32로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)Hl , 서열번호 33으로 표시되는 아미노산 서 열을 포함하는 상보성 결정부위 (CDR)H2, 서열번호 34로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)H3를 포함하는 항체 중쇄가변영역 (VH)을 포함하는 항체;로 이루어지는 군에서 선택된 인간 EPRS에 결합하는 항체 또는 그 단편을 제 공한다.  (CDR) Ll, an antibody light chain variable region comprising a complementarity determining region (CDR) L2 comprising an amino acid sequence represented by SEQ ID NO: 30, a complementarity determining region (CDR) L3 comprising an amino acid sequence represented by SEQ ID NO: 31 VL) and the complementarity determining region (CDR) Hl comprising the amino acid sequence represented by SEQ ID NO: 32, the complementarity determining region (CDR) H2 comprising the amino acid sequence represented by SEQ ID NO: 33, amino acid sequence represented by SEQ ID NO: 34 An antibody comprising a antibody heavy chain variable region (VH) comprising a complementarity determining region (CDR) H3 comprising an antibody or fragment thereof that binds to human EPRS selected from the group consisting of:
<82>  <82>
<83> 글루타밀-프로릴— tRNA 합성효소 (EPRS, Glutamyl-prolyl tRNA synthetase)는 글리타밀 (Glutamyl ) 및 프로릴 (glycyl )의 tRNA의 결합을 촉진시키는 효소로 ARS(Aminoacyl-tRNA synthetase)의 일종이다. 본 발명의 EPRS는 일반적으로 천연형 또는 재조합 인간 EPRS, 및 인간 EPRS의 비 -인간 동족체를 나타낸다.  Glutamyl-prolyl—tRNA synthetase (EPRS) is an enzyme that promotes the binding of glutamyl and prolyl (glycyl) tRNAs to the amino acid-tRNA synthetase (ARS). It is a kind. EPRS of the present invention generally refers to native or recombinant human EPRS, and non-human homologs of human EPRS.
<84>  <84>
<85> "항체"， "항 EPRS 항체" , "인간화 항 EPRS 항체" 및 "변형 인간화 항 EPRS 항체"， " ant i -EPRS ant ibody"라는 용어는 본 발명에서 가장 광의의 의미로 사용되 며， 구체적으로 단일클론 항체 (모노클로날 항체， 완전 길이 단일클론 항체 포함)， 다클론 항체 (폴리클로날 항체)， 다중특이 항체 (예를 들어 이중특이 항체)， 및 항체 단편 (예를 들어 가변 영역 및 목적하는 생물 활성 (예를 들어 EPRS와의 결합)을 나 타내는 항체의 다른 부분)을 포함한다.  The terms "antibody", "anti-EPRS antibody", "humanized anti-EPRS antibody" and "modified humanized anti-EPRS antibody", "ant i -EPRS ant ibody" are used in the broadest sense of the present invention. Specifically, monoclonal antibodies (monoclonal antibodies, including full length monoclonal antibodies), polyclonal antibodies (polyclonal antibodies), multispecific antibodies (eg bispecific antibodies), and antibody fragments (eg variable Region and other portions of the antibody that exhibit the desired biological activity (eg, binding to EPRS).
<86>  <86>
<87> 본 발명의 항체는 EPRS와 선택적으로 결합할 수 있도록 특정 아미노산 서열 이 경쇄 및 중쇄 CDR에 포함되어 있는 항체로 모노클로날 항체 및 폴리클로날 항체 를 모두 포함하며， 바람직하게는 모노클로날 항체일 수 있다. 또한 본 발명의 항체 는 키메라 항체， 인간화된 항체， 인간항체를 모두 포함하며 바람직하게는 인간항체 일 수 있다.  Antibodies of the present invention are antibodies in which specific amino acid sequences are included in the light and heavy chain CDRs so as to selectively bind to EPRS, and include both monoclonal and polyclonal antibodies, preferably monoclonal. It may be an antibody. In addition, the antibody of the present invention includes all chimeric antibodies, humanized antibodies, human antibodies, and preferably human antibodies.
<88>  <88>
<89> 본 발명의 모노클로날 항체는 실질적으로 동질 항체의 집단으로부터 수득된 항체를 나타내며, 즉, 집단을 구성하는 개개의 항체는 소량으로 존재할 수 있는 가 능한 천연적으로 존재하는 돌연변이를 제외하고는 동일하다. 모노클로날 항체는 단 일 항원 에피토프에 매우 특이적으로 결합한다. Monoclonal antibodies of the invention refer to antibodies obtained from a substantially homogeneous population of antibodies, ie, the individual antibodies that make up the population, except for naturally occurring mutations that may be present in small amounts. Is the same. Monoclonal antibodies only Binds very specifically to one antigen epitope.
<90> "모노클로날"이라는 말은 항체가 실질적인 상동성 집단으로부터 수득되는 것 과 같이 항체의 특성을 나타내는 말이며， 반드시 항체를 특정 방법에 의해 생산해 야 한다는 것은 아니다.  The term "monoclonal" refers to the characteristics of an antibody as it is obtained from a substantially homologous population and does not necessarily produce the antibody by a particular method.
<9i> 예를 들어， 본 발명의 모노클로날 항체는 문헌 [참조: Kohler et al . ( 1975)  <9i> For example, monoclonal antibodies of the invention are described in Kohler et al. (1975)
Nature 256： 495]에 처음 기재된 하이브리도마 방법에 의해 제조할 수 있거나， 또 는 재조합 DNA 방법 (참조: 미국 특허 제 4, 816,567호)에 의해 제조할 수 있다. 또 한， 예를 들어， 문헌 [참조: Clackson et al . (1991) Nature 352： 624-628 및 Marks et al . (1991) J . Mol . Biol . 222： 581-597 및 Presta (2005) J . Al lergy Cl in. Immunol . 116:731]에 기술된 기술을 사용하여 파아지 항체 라이브러리로부터 분리 할 수 있다.  Nature 256: 495, which may be prepared by the hybridoma method described first, or by recombinant DNA methods (see US Pat. No. 4, 816,567). See also, for example, Clackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol. 222: 581-597 and Presta (2005) J. Al lergy Cl in. Immunol. 116: 731 can be used to isolate from phage antibody libraries.
<92> 본 발명의 항체는 구체적으로 키메라 항체를 포함하며， 이 경우 중쇄 및 /또 는 경쇄의 일부는 특정 종으로부터 기원하거나 또는 특정 항체의 상응하는 서열과 동일하거나 상동성을 보이지만， 나머지 부분은 본 발명의 항체가 바람직한 생물학 적 활성 (예를 들어 EPRS와의 선택적 결합)을 나타내는 한, 다른 종으로부터 기원하 거나 또는 다른 항체의 상응하는 서열과 동일하거나 상동성을 나타내는 것이어도 무방하다 [참조: 미국 특허 제 4, 816, 567호; 및 Morri son et al . , (1984) Proc. Nat l . Acad. Sci . USA 81： 6851-6855] .  Antibodies of the invention specifically include chimeric antibodies, wherein some of the heavy and / or light chains originate from a particular species or show the same or homologous as the corresponding sequence of a particular antibody, but the remainder As long as the antibodies of the invention exhibit desirable biological activity (eg, selective binding to EPRS), they may be from other species or exhibit the same or homologous as the corresponding sequences of other antibodies. Patents 4, 816, 567; And Morri son et al. , (1984) Proc. Nat l. Acad. Sci. USA 81 ： 6851-6855].
<93> 인간화된 항체는 인간 및 비 -인간 (예: 쥐， 랫트) 항체의 서열을 모두 포함하 는 항체로 일반적으로， 에피토프와 결합하는 부위 (CDR)을 제외한 나머지 부분은 인 간 항체의 것이며, 에피토프와 결합하는 부위 (CDR)는 비 -인간 유래의 서열을 포함 할 수 있다.  Humanized antibodies are antibodies that contain the sequences of both human and non-human (eg rat, rat) antibodies. Generally, the rest of the human antibody, except for the region that binds to the epitope (CDR), belongs to a human antibody. The site that binds the epitope (CDR) may comprise a non-human derived sequence.
<94> 완전한 인간항체는 사람 면역글로불린 단백질 서열만을 포함하는 항체를 말 하며， 마우스, 마우스 세포， 또는 마우스 세포로부터 기원한 하이브리도마에서 생 산하거나， 파지 디스플레이 방법으로 생산할 수 있다.  Complete human antibodies refer to antibodies comprising only human immunoglobulin protein sequences, and can be produced in mice, mouse cells, or hybridomas derived from mouse cells, or produced by phage display.
<95>  <95>
<%> 생체에서 생산되는 천연 항체는 통상적으로, 2개의 동일한 경쇄 (L)와 2개의 동일한 중쇄 (H)로 구성된， 약 150,000 달톤의 이종-사량체성 당단백질이다. 각 경 쇄는 1개의 공유 디설파이드 결합에 의해 중쇄와 연결되지만, 디설파이드 연쇄수는 상이한 면역글로불린 이소형의 중쇄들 간에 다양하다. 각 중쇄 및 경쇄는 규칙적으 로 이격된 쇄내 디설파이드 브릿지를 또한 갖고 있다. 각 중쇄는 한 말단에 가변 도메인 (VH)에 이어 수 많은 불변 도메인을 갖는다. 각 경쇄는 한 말단에 가변 도메 인 (VL)을 갖고， 그의 다른 말단에 불변 도메인을 갖는데; 경쇄의 불변 도메인은 중 쇄의 제 1 불변 도메인과 정렬되고, 경쇄 가변 도메인은 중쇄의 가변 도메인과 정렬 된다. 특별한 아미노산 잔기가 경쇄 가변 도메인과 중쇄 가변 도메인 간에 계면을 형성하는 것으로 여겨진다. <%> Natural antibodies produced in vivo are typically hetero-tetrameric glycoproteins of about 150,000 Daltons, composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by one covalent disulfide bond, but the disulfide chain number varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable dome at one end Phosphorus (VL) and a constant domain at its other end; The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
<97> 항체의 "가변 영역" 또는 "가변 도메인 "은 항체의 중쇄 또는 경쇄의 아미노- 말단 도메인을 지칭한다. 중쇄의 가변 영역은 "VH" 또는' 'VH"로 기재하며, 경쇄의 가 변 영역은 "VL" 또는 "VL"로 기재한다. 이들 도메인은 일반적으로， 항체의 가장 가 변 부분이고， 항원 결합 부위를 포함한다. The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable region of the heavy chain is described as "VH" or "V H ", and the variable region of the light chain is described as "VL" or "V L. " These domains are generally the most variable portions of an antibody, Antigen binding site.
<98>  <98>
<99> "초가변성 (hypervariable) "이란 용어는 상기 가변 영역 내의 몇몇 서열들이 항체들간 서열에 있어서 광범위하게 상이하며 그의 특이적인 항원 결정인자들에 대 한 각각의 특정 항체의 결합 및 특이성에 직접적으로 관련되는 잔기들을 포함한다 는 사실을 지칭한다.  The term "hypervariable" refers to the fact that several sequences within the variable region differ widely in sequence between antibodies and directly affect the binding and specificity of each particular antibody to its specific antigenic determinants. It refers to the fact that it contains the residues involved.
<ιοο> 경쇄 및 중쇄 가변 영역 모두에 있어서 초가변성은 상보성 결정부위 (CDR) 또 는 초가변성 루프 (HVL)로서 공지된 3 개의 분절들에 집중된다. CDR은 문헌 [Kabat 등，■ 1991， In: Sequences of Proteins of Immunological Interest , 5th Ed. Publ ic Health Service, Nat ional Inst i tutes of Heal th, Bethesda, MD. ]에서의 서열 비교 에 의해 한정되는 반면， HVL은 문헌 [Chothia and Le나 1987， J . Mol . Biol . 196:901— 91기에 개시된 바와 같이, 상기 가변 영역의 3 차원 구조에 따라 구조적으 로 한정된다. 카바트 (Kabat )에 의해 한정된 바와 같이， CDR-L1은 경쇄 가변 영역 에서 대략 잔기 24-34에 , CDR-L2는 대략 잔기 50-56에， CDRL3은 대략 잔기 89-97에 위치하며; CDR-H1은 중쇄 가변 영역에서 대략 잔기 31-35에， CDR-H2는 대략 잔기 50-65에， CDR-H3은 대략 잔기 95-102에 위치한다. In both light and heavy chain variable regions, hypervariability is concentrated in three segments known as complementarity determining regions (CDRs) or hypervariable loops (HVLs). CDR was the literature [Kabat, etc., ■ 1991, In: Sequences of Proteins of Immunological Interest, 5th Ed. Publ ic Health Service, Nat ional Inst i tutes of Heal th, Bethesda, MD. ], While HVL is described by Chothia and Le or 1987, J. Mol. Biol. 196: 901—As described at 91, structurally defined by the three-dimensional structure of the variable region. As defined by Kabat, CDR-L1 is located approximately residues 24-34 in the light chain variable region, CDR-L2 is approximately residues 50-56 and CDRL3 is approximately residues 89-97; CDR-H1 is located approximately residues 31-35 in the heavy chain variable region, CDR-H2 is approximately residues 50-65 and CDR-H3 is approximately residues 95-102.
<ιοι> 상기 중쇄 및 경쇄 각각 내의 3 개의 CDR들은 를 부위 (FR)에 의해 분리되 며， 상기 부위는 덜 가변적인 경향이 있는 서열들을 포함한다. 상기 중쇄 및 경쇄 가변 영역의 아미노 말단에서부터 카복시 말단까지， 상기 FR 및 CDR은 하기의 순서 로 배열된다: FRl , CDR1 , FR2 , CDR2, FR3 , CDR3 및 FR4. 상기 FR의 큰 β 시트 배 치는 상기 각각의 쇄 내부의 CDR올 서로뿐만 아니라 다른 쇄로부터의 CDR에 가깝게 한다. 생성되는 형태는 항원 결합 부위에 기여하지만 (Kabat 등， 1991 , NIH Publ . No. 91-3242, Vol . I , pages 647-669 참조) , 모든 CDR 잔기들이 항원 결합에 직접 관여할 필요는 없다.  The three CDRs in each of the heavy and light chains are separated by site (FR), which contains sequences that tend to be less variable. From the amino terminus to the carboxy terminus of the heavy and light chain variable regions, the FRs and CDRs are arranged in the following order: FRl, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The large β sheet arrangement of the FRs brings the CDRs within each chain close to each other as well as to CDRs from other chains. The resulting form contributes to the antigen binding site (see Kabat et al., 1991, NIH Publ. No. 91-3242, Vol. I, pages 647-669), but not all CDR residues need to be directly involved in antigen binding.
<102> 본 발명의 항체는 경쇄 및 중쇄 가변영역에 속하는 각 CDR이 각각 특정 서열 을 포함하여， EPRS와 특이적으로 결합하는 것을 특징으로 하며, 구체적으로 본 발 명의 항체는 i ) 서열번호 1로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)Ll , 서열번호 2로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)L2 , 서열번호 3으로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)L3를 포함하는 항체 경쇄가변영역 (VL) 및 서열번호 4로 표시되는 아미노산 서 열을 포함하는 상보성 결정부위 (CDR)Hl , 서열번호 5로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)H2 , 서열번호 6으로 표시되는 아미노산 서열을 포함 하는 상보성 결정부위 (CDR)H3를 포함하는 항체 중쇄가변영역 (VH)을 포함하는 항체; <i03> i i ) 서열번호 15로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 <102> In the antibody of the present invention, each CDR belonging to the light and heavy chain variable regions has a specific sequence. Characterized in that it specifically binds to EPRS, specifically, the antibody of the present invention i) Complementarity determining region (CDR) Ll comprising the amino acid sequence represented by SEQ ID NO: 1, represented by SEQ ID NO: 2 Complementarity determining region (CDR) L2 comprising an amino acid sequence, antibody light chain variable region (VL) comprising a complementarity determining region (CDR) L3 comprising an amino acid sequence represented by SEQ ID NO: 3, and an amino acid sequence represented by SEQ ID NO: 4 Complementarity determining region (CDR) H1 comprising a column, complementarity determining region (CDR) H2 comprising an amino acid sequence represented by SEQ ID NO: 5, complementarity determining region (CDR) H3 comprising an amino acid sequence represented by SEQ ID NO: An antibody comprising an antibody heavy chain variable region (VH) comprising; <i03> ii) complementarity determining region comprising the amino acid sequence represented by SEQ ID NO: 15
(CDR)Ll , 서열번호 16으로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)L2, 서열번호 17로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)L3를 포함하는 항체 경쇄가변영역 (VL) 및 서열번호 18로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)Hl , 서열번호 19로 표시되는 아미노산 서열 을 포함하는 상보성 결정부위 (CDR)H2, 서열번호 20으로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)H3를 포함하는 항체 중쇄가변영역 (VH)을 포함하는 항체 ; 및  (CDR) Ll, an antibody light chain variable region comprising a complementarity determining region (CDR) L2 comprising an amino acid sequence represented by SEQ ID NO: 16, a complementarity determining region (CDR) L3 comprising an amino acid sequence represented by SEQ ID NO: 17 VL) and the complementarity determining region (CDR) Hl comprising the amino acid sequence represented by SEQ ID NO: 18, the complementarity determining region (CDR) H2 comprising the amino acid sequence represented by SEQ ID NO: 19, the amino acid sequence represented by SEQ ID NO: 20 An antibody comprising an antibody heavy chain variable region (VH) comprising a complementarity determining region (CDR) H3 comprising; And
<i04> i i i ) 서열번호 29로 표시되는 아미노산 서열을 포함하는 상보성 결정부위  <i04> i i i) complementarity determining site comprising the amino acid sequence represented by SEQ ID NO: 29
(CDR)Ll , 서열번호 30으로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)L2, 서열번호 31로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)L3를 포함하는 항체 경쇄가변영역 (VL) 및 서열번호 32로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)Hl , 서열번호 33으로 표시되는 아미노산 서 열을 포함하는 상보성 결정부위 (CDR)H2, 서열번호 34로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)H3를 포함하는 항체 중쇄가변영역 (VH)을 포함하는 항체로 이루어진 군에서 선택된 항체일 수 있다.  (CDR) Ll, an antibody light chain variable region comprising a complementarity determining region (CDR) L2 comprising an amino acid sequence represented by SEQ ID NO: 30, a complementarity determining region (CDR) L3 comprising an amino acid sequence represented by SEQ ID NO: 31 VL) and the complementarity determining region (CDR) Hl comprising the amino acid sequence represented by SEQ ID NO: 32, the complementarity determining region (CDR) H2 comprising the amino acid sequence represented by SEQ ID NO: 33, amino acid sequence represented by SEQ ID NO: 34 It may be an antibody selected from the group consisting of an antibody comprising an antibody heavy chain variable region (VH) comprising a complementarity determining region (CDR) H3 comprising a.
<105>  <105>
<106> 본 발명의 항체는 바람직하게는 특정 경쇄가변영역 및 중쇄가변영역을 포함 할 수 있으며， 구체적으로  The antibody of the present invention may preferably comprise a specific light chain variable region and a heavy chain variable region, specifically
<107> 0 경쇄가변영역으로 서열번호 13의 132 번째 내지 241 번째 아미노산 서열 을 포함하며， 중쇄가변영역으로 서열번호 13의 1 번째 내지 116 번째 아미노산 서 열을 포함하는 항체；  An antibody comprising amino acid sequences 132 to 241 of SEQ ID NO: 13 as a light chain variable region and comprising amino acid sequences 1 to 116 of SEQ ID NO: 13 as a heavy chain variable region;
<108> i i ) 경쇄가변영역으로 서열번호 27의 132 번째 내지 241 번째 아미노산 서열 을 포함하며， 중쇄가변영역으로 서열번호 27의 1 번째 내지 116 번째 아미노산 서 열을 포함하는 항체； 및 Ii) the light chain variable region comprising the 132th to 241th amino acid sequence of SEQ ID NO: 27, and the heavy chain variable region to the 1st to 116th amino acid sequence of SEQ ID NO: 27; An antibody comprising a fever; and
<109> i i i ) 경쇄가변영역으로 서열번호 41의 132 번째 내지 241 번째 아미노산 서 열을 포함하며, 중쇄가변영역으로 서열번호 41의 1 번째 내지 116 번째 아미노산 서열을 포함하는 항체 ;  I i i) an light chain variable region comprising the 132 th to 241 th amino acid sequences of SEQ ID NO: 41, and a heavy chain variable region comprising the 1 th to 116 th amino acid sequences of SEQ ID NO: 41;
<ιιο> 로 이루어진 군에서 선택된 항체일 수 있다.  It may be an antibody selected from the group consisting of <ιιο>.
<111>  <111>
<112> 본 발명의 항체는 가장 바람직하게는 서열번호 13， 서열번호 27 및 서열번호  The antibody of the present invention is most preferably SEQ ID NO: 13, SEQ ID NO: 27 and SEQ ID NO:
41로 이루어진 군에서 선택된 서열번호로 표시되는 아미노산 서열을 포함하는 항체 일 수 있다.  It may be an antibody comprising an amino acid sequence represented by a sequence number selected from the group consisting of 41.
<113>  <113>
<114> 본 발명의 항체 단편， 단편 또는 "그 단편" 은 모 항체의 결합 특이성의 적 어도 일부를 보유하는， 전형적으로 적어도 모 항체의 항원 결합의 일부 또는 가변 영역 (예를 들어， 하나 이상의 CDR)를 포함하는 항체의 단편 또는 유도체를 포함한 다. 항체 단편의 예는 Fab , Fab ' , F(ab ' )2 및 Fv 단편; 디아바디; 선형 항체; 일본 쇄 (s ingle-chain) 항체 분자， 예를 들어， sc-Fv; 및 항체 단편으로부터 형성된 다 특이적 항체를 포함하지만， 이에 제한되지 않는다. 전형적으로,.항체 단편 또는 유 도체는 해당 활성이 몰 기준으로 표현되는 경우 이의 EPRS 결합 활성의 10% 이상을 보유한다. 바람직하게는， 항체 단편 또는 유도체는 모 항체로서의 EPRS 결합 친화 도의 적어도 20% , 50% , 70%, 80%, 90% , 95% 또는 100% 또는 그 이상을 보유한다. 또한， EPRS 항체 단편은 이의 생물학적 활성을 실질적으로 변경하지 않는 보존적 아미노산 치환 (항체의 보존적 변이체라고 함)을 포함할 수 있다. 본 발명의 "결합 화합물 "은 항체 및 그 단편 둘 다를 나타낸다.  Antibody fragments, fragments or “fragments” of the invention typically retain at least a portion of the binding specificity of the parent antibody, typically at least a portion or variable region of the antigen binding of the parent antibody (eg, one or more CDRs). Fragments or derivatives of the antibody comprising a. Examples of antibody fragments include Fab, Fab ', F (ab') 2 and Fv fragments; Diabodies; Linear antibodies; S ingle-chain antibody molecules such as sc-Fv; And multispecific antibodies formed from antibody fragments. Typically, an antibody fragment or derivative retains at least 10% of its EPRS binding activity when its activity is expressed on a molar basis. Preferably, the antibody fragment or derivative has at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the EPRS binding affinity as the parent antibody. EPRS antibody fragments may also include conservative amino acid substitutions (called conservative variants of the antibody) that do not substantially alter their biological activity. "Binding compounds" of the invention refer to both antibodies and fragments thereof.
<i i5> Fab는 하나의 경쇄， 및 하나의 중쇄의 CH1 (제 1 불변 도메인) 및 가변 영역으 로 이루어진다. Fab 분자의 중쇄는 다른 중쇄 분자와 디설파이드 결합을 형성할 수 없다. <i i5> Fab consists of one light chain and one heavy chain of CH1 (first constant domain) and variable region. The heavy chain of the Fab molecule cannot form disulfide bonds with other heavy chain molecules.
<i i6> Fc 영역은 항체의 CH1 및 CH2 도메인을 포함하는 두 개의 중쇄 단편을 함유 한다. 두 개의 중쇄 단편은 두 개 이상의 디설파이드 결합에 의해 및 CH3 도메인의 소수성 상호작용에 의해 함께 유지된다.  The <i i6> Fc region contains two heavy chain fragments comprising the CH1 and CH2 domains of an antibody. Two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interaction of the CH3 domain.
<i i7> Fab '은 하나의 경쇄， 및 VH 도메인과 CH1 도메인 및 CH1과 CH2 도메인 사이 의 영역을 함유하여 쇄내디설파이드 결합이 두 개의 Fab ' 단편의 두 개의 중쇄 사 이에 형성되어 F(ab ' )2 분자를 형성하도록 하는 하나의 중쇄의 일부를 함유한다. <i i7> Fab 'contains one light chain and a region between the VH domain and the CH1 domain and between the CH1 and CH2 domains such that an intrachain disulfide bond is formed between two heavy chains of two Fab' fragments, resulting in F (ab ') It contains part of one heavy chain that allows it to form two molecules.
<i i8> F(ab ' )2는 두 개의 경쇄， 및 쇄내 디설파이드 결합이 두 개의 중쇄 사이에 형성되도록 CHI 및 CH2 도메인 사이의 고정 영역의 일부를 함유하는 두 개의 중쇄 를 함유한다. 따라서， F(ab' )2 단편은 두 개의 중쇄 사이의 디설파이드 결합에 의해 함께 유지되는 두 개의 Fab ' 단편으로 이루어진다. <i i8> F (ab ') 2 is two light chains, and the intra-chain disulfide bond is between the two heavy chains It contains two heavy chains that contain part of the fixed region between the CHI and CH2 domains to form. Thus, the F (ab ') 2 fragment consists of two Fab ' fragments held together by disulfide bonds between the two heavy chains.
<119> Fv는 중쇄와 경쇄 가변 영역을 모두 포함하지만， 고정 영역이 결여되어 있는 항체 단편이다. Fv is an antibody fragment which includes both heavy and light chain variable regions but lacks a fixed region.
<120> 일본쇄 (single-chain) Fv 또는 scFv는 항체의 VH 및 VL 도메인을 포함하는 항체 단편을 나타내며, 여기서， 이들 도메인은 단일 폴리펩타이드 쇄내에 존재한 다. 일반적으로， Fv 폴리펩타이드는 scFv가 항원 결합을 위해 바람직한 구조를 형 성하도록 하는 , VH 및 VL 도메인 사이의 폴리펩타이드 링커를 추가로 포함한다. scFv의 개요에 대해서는 문헌 [참조: Pluckthim (1994) THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol . 113， Rosenburg and Moore eds . Springer-Ver lag, New York, pp. 269-315]을 참조한다. 또한, 국제 특허 공개공보 제 W0 88/01649호 및 미국 특허 제 4, 946, 778호 및 제 5 ,26으 203호를 참조한다.  Single-chain Fv or scFv refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In general, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen binding. For an overview of scFv, see Pluckthim (1994) THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Moore eds. Springer-Ver lag, New York, pp. 269-315. See also WO0 88/01649 and US Pat. Nos. 4, 946, 778 and 5,26, 203.
<121> 디아바디는 2개의 항원 -결합 부위를 갖는 작은 항체 단편을 의미하며， 여기 서 단편은 동일한 폴리펩티드쇄에서 경쇄 가변 도메인 (VL)에 연결된 중쇄 가변 도 메인 (VH) (VH-VL)을 포함한다. 동일 쇄 상의 2개 도메인 사이에 페어링을 허용하 지 않는 짧은 링커를 사용하여, 도메인을 다른 쇄의 상보성 도메인과 강제로 페어 링시켜 2개의 항원 -결합 부위를 생성시킨다. 디아바디는, 예를 들어 유럽 특허 제 404,097호; W0 93/11161； 및 문헌 [Hol l inger et al . , Proc . Nat l . Acad. Sci . USA, 90: 6444-6448 (1993) ]에 보다 상세히 기재되어 있다.  Diabodies refer to small antibody fragments having two antigen-binding sites, wherein the fragments comprise a heavy chain variable domain (VH) (VH-VL) linked to the light chain variable domain (VL) in the same polypeptide chain. Include. Using a short linker that does not allow pairing between two domains on the same chain, the domains are forcibly paired with the complementary domains of the other chain to create two antigen-binding sites. Diabodies are described, for example, in European Patent No. 404,097; WO 93/11161 and Hol l inger et al. , Proc. Nat l. Acad. Sci. USA, 90: 6444-6448 (1993).
<122> 선형 항체는 한 쌍의 항원 결합 부위를 형성하는 한 쌍의 직렬 Fd 단편 (VH- A linear antibody consists of a pair of tandem Fd fragments (VH- which form a pair of antigen binding sites).
CH1-VH-CH1)을 포함하는 항체를 지칭한다. 선형 항체는 예를 들어 문헌 [Zapata 등 1995, Protein Eng. 8 (10)： 1057-1062]에 개시된 바와 같이 이중특이적 또는 단일 특이적일 수 있다. Refers to an antibody comprising CH1-VH-CH1). Linear antibodies are described, for example, in Zapata et al. 1995, Protein Eng. 8 (10): 1057-1062, which may be bispecific or monospecific.
<123>  <123>
<124> "도메인 항체' '는 중쇄의 가변 영역 또는 경쇄의 가변 영역만을 함유하는 면 역 기능성 면역글로불린 단편이다.  "Domain antibodies" are immunologically functional immunoglobulin fragments containing only the variable region of the heavy chain or the variable region of the light chain.
<125> 몇몇 예에서， 두 개 이상의 VH 영역은 펩타이드 링커와 공유결합하여 2가 도 메인 항체를 생성한다. 2가 도메인 항체의 두 개의 VH 영역은 동일하거나 상이한 항원을 표적화할 수 있다. In some instances, two or more VH regions are covalently linked to a peptide linker to produce a bivalent domain antibody. Two VH regions of a bivalent domain antibody may target the same or different antigens.
<126> 2가 항체는 두 개의 항원 결합 영역을 포함한다. 몇몇 예에서， 두 개의 결합 영역은 동일한 항원 특이성을 갖는다. 그러나， 2가 항체는 이특이적 (bispeci f ic)일 수 있다. 본 발명의 항체 또는 그 단편은 당업계에 알려져 있는 방법， 예를 들어, 파 지 디스플레이 방법 또는 효모 세포 표면 발현 시스템을 사용하여 생성될 수 있다 . scFv를 제조하는 방법으로는 미국특허 제 4, 946 , 778호 및 제 5 , 258 , 498호에 기재된 방법이 사용될 수 있으며， Fab , Fab ' 및 F(ab ' )2 단편을 재조합적으로 생성하기 위 한 방법으로는 W0 92/22324 등에 기재된 방법이 사용될 수 있다. 본 발명의 항체는 인간을 포함하는 포유동물， 조류 등을 포함한 임의의 동물 로부터 유래한 것일 수 있다. 바람직하게는, 상기 항체는 인간， 생쥐， 당나귀， 양, 토끼， 염소， 기니피그, 낙타, 말 또는 닭의 항체일 수 있다. Bivalent antibodies comprise two antigen binding regions. In some instances, the two binding regions have the same antigen specificity. However, bivalent antibodies may be bispecific (bispeci f ic). Can be. Antibodies or fragments thereof of the invention can be produced using methods known in the art, such as phage display methods or yeast cell surface expression systems. As a method for preparing scFv, the method described in US Pat. Nos. 4, 946, 778 and 5, 258, 498 may be used, and recombinantly generating Fab, Fab 'and F (ab') 2 fragments. As the above method, the method described in WO 92/22324 may be used. Antibodies of the invention can be derived from any animal, including mammals, birds, and the like, including humans. Preferably, the antibody may be an antibody of human, mouse, donkey, sheep, rabbit, goat, guinea pig, camel, horse or chicken.
인간 항체는 인간 면역글로불린의 아미노산 서열을 가진 항체로서， 인간 면 역글로불린 라이브러리로부터 분리된 항체 또는 하나 이상의 인간 면역글로불린에 대하여 형질 이식되고 내재적 면역글로불린은 발현하지 않는 동물로부터 분리된 항 체가 포함된다 (미국특허 제 5 , 939 , 598호 참조) .  Human antibodies are antibodies having the amino acid sequence of human immunoglobulins, which include antibodies isolated from human immunoglobulin libraries or antibodies isolated from animals transfected against one or more human immunoglobulins and not expressing endogenous immunoglobulins ( US Patent No. 5, 939, 598).
본 발명의 항체는 효소， 형광 물질， 방사선 물질 및 단백질 등과 접합된 것 일 수 있으나， 이에 한정되지 않는다. 또한， 항체에 상기 물질을 접합하는 방법은 당업계에 잘 알려져 있다. 본 발명은 또한 상기한 바와 같은 본 발명에 따른 항체 또는 그 단편올 암호 화 하는 폴리뉴클레오티드를 제공한다.  The antibody of the present invention may be conjugated to an enzyme, a fluorescent substance, a radioactive substance, a protein, or the like, but is not limited thereto. In addition, methods of conjugating the material to antibodies are well known in the art. The invention also provides a polynucleotide encoding an antibody or fragmentol thereof according to the invention as described above.
폴리뉴클레오티드는 을리고뉴클레오티드 또는 핵산으로 기재될 수도 있으며， DNA분자들 (예를 들어， cDNA 또는 유전체 (genomi c DNA) , RNA 분자들 (예를 들어， mRNA) , 뉴클레오티드 유사체들을 사용하여 생성된 상기 DNA 또는 RNA의 유사체들( 예를 들어， 펩티드 핵산들 및 비 -자연적으로 발생하는 뉴클레오티드 유사체들) 및 이들의 하이브리드들이 포함된다. 상기 폴리뉴클레오티드는 단일 -가닥 (single- stranded) 또는 이중 -가닥 (double-stranded)이 될 수 있다. 본 발명의 폴리뉴클레오티드는 본 발명의 항체 또는 그 단편을 암호화하는 것이면, 그 서열이 특별히 제한되지 아니하나 바람직하게는  Polynucleotides may also be described as ligonucleotides or nucleic acids, and are generated using DNA molecules (eg cDNA or genomi c DNA), RNA molecules (eg mRNA), nucleotide analogues Analogues of DNA or RNA (eg, peptide nucleic acids and non-naturally occurring nucleotide analogues) and hybrids thereof.The polynucleotide may be single-stranded or double-stranded ( If the polynucleotide of the present invention is to encode the antibody or fragment thereof of the present invention, the sequence is not particularly limited, but preferably
i ) 서열번호 7 내지 12로 표시되는 폴리뉴클레오티드;  i) a polynucleotide represented by SEQ ID NOs: 7-12;
i i ) 서열번호 21 내지 26으로 표시되는 폴리뉴클레오티드; 및 <i40> i i i ) 서열번호 35 내지 40으로 표시되는 폴리뉴클레오티드; 로 이루어진 군 에서 선택된 폴리뉴클레오티드를 포함하는 것일 수 있다. ii) a polynucleotide represented by SEQ ID NOs: 21 to 26; And iii) a polynucleotide represented by SEQ ID NOs: 35 to 40; It may be to include a polynucleotide selected from the group consisting of.
<141> 본 발명의 항체 또는 그 단편을 암호화하는 폴리뉴클레오티드는 당업계에 잘 알려진 방법에 의하여 얻어질 수 있다. 예를 들어, 상기 항체의 중쇄 및 경쇄의 일 부분 또는 전부를 코딩하는 DNA 서열 또는 해당 아미노산 서열에 근거하여 , 당분야 에 잘 알려진 올리고뉴클레오타이드 합성기법, 예를 들아 중합효소 연쇄 반웅 (PCR)법 등을 사용하여 합성할 수 있다. Polynucleotides encoding the antibodies of the present invention or fragments thereof can be obtained by methods well known in the art. For example, oligonucleotide synthesis techniques well known in the art, such as polymerase chain reaction (PCR), etc., are based on DNA sequences or corresponding amino acid sequences encoding portions or all of the heavy and light chains of the antibody. Can be synthesized using
<142>  <142>
<143> 또한 본 발명은 상기 폴리뉴클레오티드를 포함하는 백터를 제공한다.  The present invention also provides a vector comprising the polynucleotide.
<144> 본 발명의 백터는 본 발명의 항체 또는 그 단편의 재조합 생산을 위하여 본 발명의 폴리뉴클레오티드의 복제 또는 발현의 목적으로 이용되며， 일반적으로 시그 날 서열， 복제 기원， 하나 이상의 마커 유전자， 인핸서 요소， 프로모터 및 전사 종 결 서열 중 하나 이상을 포함한다.  The vector of the present invention is used for the purpose of replicating or expressing a polynucleotide of the present invention for recombinant production of an antibody or fragment thereof of the present invention, and generally includes a signal sequence, a copy origin, one or more marker genes, an enhancer. At least one of an urea, a promoter and a transcription termination sequence.
<145> 본 발명의 백터는 바람직하게는 발현백터일 수 있으며, 더욱 바람직하게는 조절시퀀스， 예를 들어 프로모터에 작동 가능하게 연결된 본 발명의 폴리뉴클레오 티드를 포함하는 백터일 수 있다. The vector of the present invention may preferably be an expression vector, more preferably a vector comprising a polynucleotide of the present invention operably linked to a regulatory sequence, for example, a promoter.
<146> 백터의 일종인 플라스미드 (plasmid)는 외부의 폴리뉴클레오티드 단편들이 결 합될 수 있는 선형 또는 원형의 이중 나선의 DNA 분자를 의미한다. 백터의 다른 형 태는 바이러스성 백터 (vi ral vector ； 예를 들어, 복제 -결핍 레트로바이러스 (repl i cat ion defect ive retrovi ruses) , 아데노바이러스들 및 아데노 -연관 바이러 스들 (adenoassociated viruses) )이며， 여기에서 부가의 DNA 단편들은 상기 바이러 스성 게놈 (vi ral genome) 내로 도입될 수 있다. 특정의 백터들은 그 안으로 이들이 도입되는 숙주세포 (예를 들어， 박테리아 유래 (bacter ial or igin) 및 에피좀의 포유 류 백터 (epi somal mammal ian vectors)를 포함하는 박테리아성 백터들 (bacter i al vectors) ) 내에서의 자가복제 (autonomous repl icat ion)를 할 수 있다. 다른 백터들 (예를 들어, 비-에피좀의 포유동물 백터들 (non-epi soma 1 mammal i an vectors) )이 숙 주세포 내로의 도입에 의한 숙주세포의 게놈 내로 통합 ( integrated)되고 그리고 그 에 의하여 상기 숙주 게놈과 함께 복제된다.  Plasmid, a type of vector, refers to a linear or circular double helix DNA molecule into which external polynucleotide fragments can be joined. Other forms of vectors are viral vectors (e.g., repl i cat ion defect ive retrovises, adenoviruses and adeno-associated viruses). Wherein additional DNA fragments can be introduced into the viral genome. Certain vectors include bacterial vectors, including host cells into which they are introduced (eg, bacterial or igin and epi somal mammalian vectors). Autonomous repl icat ion can be achieved in Other vectors (eg, non-epi soma 1 mammal i an vectors) are integrated into the genome of the host cell by introduction into host cells and By the host genome.
<147> 발현백터 (expression vector)는 선택된 폴리뉴클레오타드의 발현할 수 있는 백터의 한 형태이다. 하나의 폴리뉴클레오티드 시뭔스는， 조절 시퀀스가 상기 폴리 뉴클레오티드 시뭔스의 발현 (예를 들어, 수준， 타이밍 또는 발현의 위치)에 영향을 주는 경우, 상기 조절 시뭔스 (regulatory sequence)에 "작동가능하게 연결"된다. 상기 조절 시퀀스는 그것이 작동가능하게 연결되는 핵산의 발현 (예를 들어， 수준， 타이밍 또는 발현의 위치)에 영향을 주는 서열이다. 상기 조절 시뭔스는， 예를 들 어， 조절된 핵산에 직접적으로 또는 하나 또는 그 이상의 다른 분자들 (예를 들어， 상기 조절 시퀀스 및 /또는 상기 핵산에 결합하는 폴리펩티드들)의 작용을 통하여 그의 영향이 미치도록 할 수 있다. 상기 조절 시퀀스에는 프로모터 (promoters), 인 핸서 (enhancers) 및 다른 발현 조절 요소들이 포함된다. 본 발명의 백터는 바람직 하게는 Sfil site에 scF Insert가 포함된 pCom3x (phagmid) vector일 수 있다.Expression vectors are a form of expressible vector of selected polynucleotides. One polynucleotide sequence is operatively linked to the regulatory sequence when the regulatory sequence affects the expression (eg, level, timing or location of expression) of the polynucleotide sequence. "do. The regulatory sequence is a sequence that affects the expression (eg, level, timing or location of expression) of the nucleic acid to which it is operably linked. The regulatory sequence is influenced by, for example, its effect directly or through the action of one or more other molecules (eg, the regulatory sequence and / or polypeptides that bind the nucleic acid) to the regulated nucleic acid. Can be crazy. The regulatory sequence includes promoters, enhancers and other expression control elements. The vector of the present invention may preferably be a pCom3x (phagmid) vector containing a scF Insert at the Sfil site.
<148> <148>
<149> 한편 본 발명은 본 발명의 백터를 포함하는 세포를 제공한다.  On the other hand, the present invention provides a cell comprising the vector of the present invention.
<150> 본 발명의 세포는 본 발명의 폴리뉴클레오티드를 발현하는데 사용될 수 있는 세포면 그 종류는 특별히 제한되지 아니한다.  The cell of the present invention is not particularly limited as long as the cell can be used to express the polynucleotide of the present invention.
<151> 본 발명의 세포는 숙주세포는 원핵생물 (예를 들어 대장균)， 진핵생물 (예를 들어 효모 또는 다른 균류)， 식물 세포 (예를 들어 담배 또는 토마토 식물 세포)， 동물 세포 (예를 들어 인간 세포， 원숭이 세포， 햄스터 (hamster) 세포， 집쥐 세포 (rat cell), 생쥐 세포 (mouse cell) 또는 곤층 세포) 또는 하이브리도마가 될 수 있다. The cell of the present invention may be a prokaryote (e.g., E. coli), eukaryote (e.g., yeast or other fungus), plant cell (e.g. tobacco or tomato plant cell), animal cell (e.g., Human cells, monkey cells, hamster cells, rat cells, mouse cells or tortilla cells) or hybridomas.
<152>  <152>
<153> 본 목적에 적합한 원핵생물은 그람 음성 또는 그람 양성 유기체， 예를 들어 엔테로박테리아새 (Enterobacteriaceae), 예를 들어 에스케리치아 (Escherichia), 예를 들어 이. 콜라이， 엔테로박터 (Enterobacter), 에르위니아 (Erwinia), 클렙시 엘라 (Klebsiella), 프로테우스 (Proteus), 살모넬라 (Salmonella), 예를 들어， 살 모넬라 티피무륨 (Salmonella typhimurium)， 세라티아 (Serratia), 예를 들어， 세 라티아 마르세스칸스 (Serratia marcescans) 및 시겔라 (Shigella), 및 바실리 (Bacilli), 예를 들어， 비. 섭틸리스 (B. subtil is) 및 비. 리케니포르미스 (B. licheniformis), 슈도모나스 (Pseudomonas)， 예를 들어 피. 애루기노사 (P. aeruginosa) 및 스트렙토마이세스 (Streptomyces)를 포함한다. 본 발명의 세포는 본 발명의 백터를 발현가능 한 것이면， 특별히 제한되지 아니하나， 바람직하게는 이 . 콜라이 , 이에 한정되지 아니하나 예를 들어， 이 . 콜라이 ER2537, 이 . 콜라이 B, 이 . 콜라이 X1776 (ATCC 31,537), 이 . 콜라이 W3110 (ATCC 27,325) 또는 LacZ가 발현 가능한 이. 콜라이일 수 있으며， 더욱 바람직하게는 이. 콜라이 ER2537^ 수 있다.  Prokaryotes suitable for this purpose are Gram negative or Gram positive organisms, for example Enterobacteriaceae, for example Escherichia, for example E. coli. E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example Salmonella typhimurium, Serratia, For example, Serratia marcescans and Shigella, and Bacilli, for example B. Subtilis (B. subtil is) and b. B. licheniformis, Pseudomonas, for example blood. P. aeruginosa and Streptomyces. The cells of the present invention are not particularly limited as long as they can express the vector of the present invention, but preferably. Coli, for example but not limited to. E. coli ER2537, this. Coli B, this. E. coli X1776 (ATCC 31,537). E. coli W3110 (ATCC 27,325) or LacZ expressing. May be coli, more preferably E. coli. E. coli ER2537 ^
<154> <155> 본 발명의 세포로서 진핵생물은 사카로마이세스 세레비지아에<154> <155> As a cell of the present invention, eukaryotes are present in Saccharomyces cerevisiae
(Saccharomyces cerevisiae)가 가장 흔히 사용된다. 그러나， 많은 다른 속， 종 및 균주, 이에 한정되지 아니하나， 예를 들어 쉬조사카로마이세스폼베 (Schizosaccharomyces pombe) , 클루이베로마이세스 숙주, 예를 들어 케이 . 락티스 (K.lactis), 케이. 프라길리스 (K. fragilis) (ATCC 12,424), 케이. 불가리쿠스 (K. bulgaricus) (ATCC 16,045)， 케이. 위커라미 (K. wickeramii) (ATCC 24,178)， 케이 . 왈티 (K. waltii) (ATCC 56,500), 케이 . 드로소필라룸 (Κ· drosophi larum) (ATCC 36,906), 케이. 테르모를레란스 ((K. thermotolerans) 및 케이. 마르시아누 스 (K. mar xi anus); 야로위아 (yarrowia) (EP 402,226)； 피키아 파스토리스 (Pichia pastoris) (EP 183,070)； 칸디다 (Candida); 트리코데르마 레에시아 (Trichoderma reesia (EP 244,234))； 뉴로스포라 크라사 (Neurospora crassa); 쉬바 니오마이세스 ( Schwann iomyces), 예를 들어 쉬바니오마이세스 옥시덴탈리스 (occidental is); 및 필라멘트성 진균, 예를 들어 뉴로스포라， 페니실리움 (Penici Ilium), 를리포클라디움 (Tolypocladium) 및 아스퍼질러스 (Aspergillus) 숙 주， 예를 들어 에이. 니둘란스 (nidulans) 및 에이. 니거 (niger)가 사용가능 하 다 · (Saccharomyces cerevisiae) is the most commonly used. However, many other genera, species and strains include, but are not limited to, for example, Schizosaccharomyces pombe, Kluyveromyces hosts, such as K. K.lactis, K. K. fragilis (ATCC 12,424), k. B. bulgaricus (ATCC 16,045), K. K. wickeramii (ATCC 24,178), K. K. waltii (ATCC 56,500), K. Drosophilarum (ATCC 36,906), K. Termorerans ((K. thermotolerans) and K. mar xi anus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida Trichoderma reesia (EP 244,234); Neurospora crassa; Schwann iomyces, e.g. Schwaniomyses occidental is And filamentous fungi such as neurospora, Penici Ilium, tolypocladium and Aspergillus hosts, for example A. nidulans and A. Niger is available
<156> 한편 본 발명의 세포는 동물세포 특히 척추동물 세포일 수 있다. 배양 (조직 배양)에서 척추동물 세포의 증식은 일상적인 방법이 되었고 기술들이 폭넓게 이용 가능하다. 이에 제한되지 아니하나， 유용한 포유동물 숙주 세포의 예는 SV40에 의 해 형질전환된 원숭이 신장 CV1 라인 (COS-7, ATCC CRL 1651)， 인간 배아 신장 라 이 (현탁 배양으로부터 서브클로닝된 293 또는 293 세포 [Graham 등, 1977, J Gen Virol. 36： 59])， 새끼 햄스터 신장 세포 (BHK, ATCC CCL10) , 차이니즈 햄스터 난 소 세포 /-DHFR" (CHO, Urlaub등， 1980， Proc. Natl. Acad. Sci. USA 77： 4216； 예 를 들어， DG44), 마우스 세르를리 세포 (TM4, Mather , 1980， Biol. Reprod. 23:243-251), 원숭이 신장 세포 (CV1 ATCC CCL 70)， 아프리카 녹색 원숭이 신장 세 포 (VERO-76, ATCC CRL- 1587) , 인간 경부 암세포 (HELA, ATCC CCL 2), 개 신장 세 포 (MDCK, ATCC CCL 34)， 버팔로 랫트 간세포 (BRL 3A, ATCC CRL 1442)， 인간 폐세 포 ( 38,ATCC CCL 75), 인간 간세포 (H印 G2, HB 8065)， 마우스 유방 종양 (醒 T 060562, ATCC CCL51), TRI 세포 (Mather 등， 1982， Annals NY. Acad. Sci. 383： 44- 68 )， MRC 5 세포， FS4 세포， 인간 간암 세포주 (Hep G2), HEK 293 cell (human embryonic kidney cell) 및 Expi293F cell일 수 있으며, 바람직하게는 CHO cell, HEK 293 cell (human embryonic kidney cell) 또는 Expi293 cell일 수 있다. <157> Meanwhile, the cells of the present invention may be animal cells, particularly vertebrate cells. Proliferation of vertebrate cells in culture (tissue culture) has become a routine method and techniques are widely available. Examples of useful mammalian host cells include, but are not limited to, monkey kidney CV1 line (COS-7, ATCC CRL 1651) transformed by SV40, human embryonic kidney lys (293 or 293 subcloned from suspension culture). Cells [Graham et al., 1977, J Gen Virol. 36: 59]), baby hamster kidney cells (BHK, ATCC CCL10), Chinese hamster ovary cells / -DHFR "(CHO, Urlaub et al., 1980, Proc. Natl. Acad Sci. USA 77: 4216; for example DG44), mouse serli cells (TM4, Mather, 1980, Biol. Reprod. 23: 243-251), monkey kidney cells (CV1 ATCC CCL 70), African green Monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL 2), dog kidney cells (MDCK, ATCC CCL 34), buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442), Human lung cells (38, ATCC CCL 75), human hepatocytes (H 印 G2, HB 8065), mouse breast tumors (醒 T 060562, ATCC CCL51), TRI cells (Mather et al., 1982 Annals NY.Acad. Sci. 383: 44-68), MRC 5 cells, FS4 cells, human liver cancer cell line (Hep G2), HEK 293 cells (human embryonic kidney cells) and Expi293F cells, preferably CHO cells , HEK 293 cells (human embryonic kidney cells) or Expi293 cells. <157>
<158> 본 발명의 세포는 본 발명의 폴리뉴클레오티드 또는 이를 포함하는 백터로 형질전환되거나 또는 형질감염 (transfected)될 수 있는 배양된 세포이고， 이는 계 속해서 상기 숙주세포 내에서 발현될 수 있다. 재조합 세포는 발현되어야 할 폴리 뉴클레오티드로 형질전환되거나 또는 형질감염된 세포를 말한다. 본 발명의 세포는 또한 본 발명의 폴리뉴클레오티드를 포함하나， 조절 시퀀스가 상기 폴리뉴클레오티 드에 작동 가능하게 연결되도록 상기 세포 내로 도입되지 않는 한 이를 원하는 수 준으로 발현하지 않는 세포가 될 수 있다.  The cells of the present invention are cultured cells that can be transformed or transfected with the polynucleotide of the present invention or a vector comprising the same, and can be expressed continuously in the host cell. Recombinant cell refers to a cell transformed or transfected with a polynucleotide to be expressed. The cells of the invention may also be cells which contain a polynucleotide of the invention but which do not express it to the desired level unless a regulatory sequence is introduced into the cell so as to be operably linked to the polynucleotide. .
<159>  <159>
<160> 본 발명의 세포는 다양한 배지에서 배양될 수 있다. 상업적으로 이용가능한 배지， 예컨대 햄 (Ham' s) F10 (Si ma-Aldrich Co. , St . Louis , MO) , 최소 필수 배 지 (MEM, Sigma-Aldrich Co . ) , RPMI-1640 (Sigma-Aldrich Co. ) , 및 둘베코 (Dulbecco ' s) 개질 이글 (Eagle ' s) 배지 (DMEM, Sigma-Aldrich Co. )가 세포를 배양 하기에 적합하다. 상기 배지는 필요하다면 호르몬 및 /또는 다른 성장 인자， 염， 완 충액， 뉴클레오티드， 항생제， 미량 원소 및 글루코스 또는 동등 에너지원이 추가될 수 있다.  Cells of the invention can be cultured in a variety of media. Commercially available media such as Ham 's F10 (Si ma-Aldrich Co., St. Louis, MO), minimum essential medium (MEM, Sigma-Aldrich Co.), RPMI-1640 (Sigma-Aldrich Co.), and Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich Co.) are suitable for culturing cells. The medium may be added with hormones and / or other growth factors, salts, buffers, nucleotides, antibiotics, trace elements and glucose or equivalent energy sources if necessary.
<161> 본 발명의 배지는 바람직하게는 SB (Bactotrytone 30g, yeast extract 20g,  The medium of the present invention is preferably SB (Bactotrytone 30g, yeast extract 20g,
MOPS buf fer lOg/L) Medium, FreeStyle™ 293 Medium또는 Expi293™ Medium일 수 있 다.  MOPS buf fer lOg / L) Medium, FreeStyle ™ 293 Medium, or Expi293 ™ Medium.
<162>  <162>
<163> 한편 본 발명은 상기 세포를 폴리뉴클레오티드가 발현되는 조건하에서 배양 하여， 경쇄 및 중쇄가변영역을 포함하는 폴리펩타이드를 생산하는 단계 및 상기 세 포 또는 이를 배양한 배양 배지로부터 상기 폴리펩타이드를 회수하는 단계를 포함 하는 인간 EPRS에 결합하는 항체 또는 그 단편와 생산방법을 제공한다.  On the other hand, the present invention is to culture the cells under the condition that the polynucleotide is expressed, to produce a polypeptide comprising a light chain and heavy chain variable region and to recover the polypeptide from the cell or culture medium cultured thereof It provides an antibody or fragment thereof and a production method that binds to human EPRS comprising the step.
<164>  <164>
<165> 본 발명 생산방법의 세포에 대하여는 상기 기술한 바와 같으며 , 본 발명의 항체를 암호화하는 폴리뉴클레오티드를 포함하고 있다.  The cells of the production method of the present invention are as described above, and contain a polynucleotide encoding the antibody of the present invention.
<166> 본 발명 생산방법의 폴리펩타이드는 본 발명의 항체 또는 그 단편 그 자체일 수 있으며 , 본 발명의 항체 또는 그 단편 외 다른 아미노산서열이 추가로 결합된 것일 수 있다. 이 경우 본 기술분야의 통상의 기술자에게 잘 알려져 있는 방법을 이용하여 본 발명의 항체 또는 그 단편으로부터 제거할 수 있다 .  The polypeptide of the production method of the present invention may be an antibody of the present invention or a fragment thereof, and an amino acid sequence other than the antibody of the present invention or a fragment thereof may be further bound. In this case, it can be removed from the antibody or fragment thereof of the present invention using methods well known to those skilled in the art.
<167> 상기 배양은 상기 세포의 종류에 따라 배지조성 및 배양 조건이 달라질 수 있으며， 이는 본 기술분야의 통상의 기술자가 적절히 선택 및 조절할 수 있다. In the culture, the medium composition and culture conditions may vary depending on the type of the cells. Which can be properly selected and adjusted by those skilled in the art.
<168> 상기 항체 분자는 세포의 세포질 내에 축적되거나, 세포로부터 분비되거나， 적절한 신호 서열에 의하여 페리플라즘 또는 세포외 배지 (supernatant )로 표적화 (targeted)될 수 있으며， 페리플라즘 또는 세포외 배지로 표적화되는 것이 바람직 하다ᅳ 또한， 생산된 항체 분자를 본 기술분야의 통상의 기술자에게 잘 알려져 있는 방법을 이용하여 리폴딩 (refolding)시키고 기능적 형태 (conformat ion)를 갖도록 하는 것이 바람직하다. The antibody molecule may accumulate in the cytoplasm of the cell, be secreted from the cell, or targeted to periplasm or extracellular medium by appropriate signal sequences, and the periplasm or extracellular medium. It is also desirable to refold the produced antibody molecule and to have a conformal ion using methods well known to those skilled in the art.
<169> 상기 폴리펩타이드의 회수는 생산된 폴리펩타이드의 특성 및 세포의 특성에 따라 달라질 수 있으며， 이는 본 기술분야의 통상의 지식을 가진자가 적절히 선택 및 조절할 수 있다.  The recovery of the polypeptide may vary depending on the nature of the produced polypeptide and the properties of the cells, which can be appropriately selected and controlled by one of ordinary skill in the art.
<170> 상기 폴리펩타이드는 세포내， 주변 세포질 공간에 생산되거나 배지 내로 직 접 분비될 수 있다. 만약 폴리펩타이드가 세포 내에서 생산되면， 이 세포는 계 1 단 계로서 단백질을 방출하기 위하여 파괴될 수 있다. 입자형 파편， 숙주 세포 또는 용해된 단편은 예를 들어 원심분리 또는 한외여과에 의해 제거된다. 항체가 배지 내로 분비되는 경우， 이러한 발현 시스템으로부터의 상등액을 일반적으로 먼저 상 업적으로 이용가능한 단백질 농축 필터， 예를 들어 Amicon 또는 Mi l l ipore Pel l icon 한외여과 유닛을 사용하여 농축시킨다. 단백분해를 억제하기 위하여 프로 테아제 억제제, 예를 들어 PMSF가 임의의 선행 단계에 포함될 수 있고, 우발적인 오염물의 성장을 방지하기 위하여 항생제가 포함될 수 있다.  The polypeptide may be produced intracellularly, in the surrounding cytoplasmic space, or directly secreted into the medium. If the polypeptide is produced in a cell, the cell can be destroyed to release the protein as a step 1 step. Particulate debris, host cells or lysed fragments are removed, for example, by centrifugation or ultrafiltration. When the antibody is secreted into the medium, the supernatant from this expression system is generally first concentrated using a commercially available protein enrichment filter, for example Amicon or Mi l ipore Pel l icon ultrafiltration unit. Protease inhibitors, such as PMSF, may be included in any preceding step to inhibit proteolysis, and antibiotics may be included to prevent accidental growth of contaminants.
<171> 세포로부터 제조된 항체는 예를 들어 하이드톡시아파타이트 크로마토그래피, 겔 전기영동， 투석 및 친화도 크로마토그래피를 사용하여 정제될 수 있고, 본 발명 의 항체는 바람직하게는 친화도 크로마토그래피를 통하여 정제할 수 있다.Antibodies prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis and affinity chromatography, and the antibodies of the invention are preferably via affinity chromatography. It can be purified.
<172> <172>
<173> 본 발명의 항체 또는 그 단편은 EPRS와 특이적으로 결합하므로 예를 들어， 특정 세포， 조직, 또는 혈청 내 EPRS 발현을 검출하는， EPRS 단백질을 검출하고 정 량하기 위한 진단 분석에 유용하다.  The antibody or fragment thereof of the present invention specifically binds to EPRS and is therefore useful in diagnostic assays for detecting and quantifying EPRS proteins, for example for detecting EPRS expression in specific cells, tissues, or serum. .
<174> 따라서 본 발명은 상기 항체 또는 그 단편을 시료와 접촉시키는 단계 및 상 기 항체 또는 그 단편을 검출하는 단계를 포함하는 EPRS 특이적 검출 방법을 제공 한다.  Accordingly, the present invention provides an EPRS specific detection method comprising contacting the antibody or fragment thereof with a sample and detecting the antibody or fragment thereof.
<175> 상기 항체 또는 그 단편을 '검출 '하기 위하여， 항체 또는 그 단편은 일반적 으로 검출가능 모이어티로 표지될 수 있다.  In order to 'detect' the antibody or fragment thereof, the antibody or fragment thereof may generally be labeled with a detectable moiety.
<176> 예를 들어， 문헌 [Current Protocols in Immunology, Volumes 1 and 2， 1991 , Col igen 등， Ed. Wi ley-Interscience , New York, N. Y.， Pubs]에 기술된 기 술을 이용하여， 방사성 동위원소 또는 형광표지로 표지될 수 있다. 방사능은， 예를 들어， 신틸레이션 계수 (scint i l lat ion count ing)에 의해 측정될 수 있으며， 형광은 형광계를 이용하여 정량될 수 있다. <176> See, eg, Current Protocols in Immunology, Volumes 1 and 2, 1991, Col igen et al., Ed. Wi ley-Interscience, New York, NY, Pubs] can be labeled with radioisotopes or fluorescent labels using the techniques described. Radioactivity can be measured, for example, by scintillation counting, and fluorescence can be quantified using a fluorometer.
<177> 또는 다양한 효소 -기질 표지가 이용가능하며， 상기 효소적 표지의 예는 초파 리 루시러파제 및 세균 루시퍼라제 (미국 특허 제 4，737，456호)와 같은 루시퍼라제, 루시페린 ( luci ferin) , 2，3_다이하이드로프탈라진디오네스， 말레이트 디하이드로게 나제， 유라제 (urase) , 호스래디쉬 퍼옥시다제 (HRP0)와 같은 퍼옥시다제， 알칼라 인 포스파타제， β -갈락토시다제, 글루코아밀라제， 라이소자임， 사카라이드 옥시다 제 (예를 들어 글루코스옥시다제， 갈락토스 옥시다제, 및 글루코스 -6-포스페이트 디하이드로게나제) , 헤테로사이클릭 옥시다제 (예를 들어 유리카제 및 잔틴 옥시다 제)， 락토퍼옥시다제， 마이크로퍼옥시다제 등을 포함한다. 항체에 효소를 접합시키 는 기술은 예를 들어, 문헌 [0' Sul l ivan 등， 1981， Methods for the Preparat ion of Enzyme-항체 Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (J . Langone & H. Van Vunakis , eds . ) , Academic press , N. Y. , 73： 14그66]에 기 술되어 있다. Alternatively, various enzyme-substrate labels are available, examples of which include luciferases, luciferins such as choparic luciferase and bacterial luciferase (US Pat. No. 4,737,456). ), 2,3 dihydrophthalazine diones , peroxides such as malate dehydrogenase, urase, horseradish peroxidase (HRP0), alkaline phosphatase, β-gal Lactosidase, glucoamylase, lysozyme, saccharide oxidase (e.g. glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidase (e.g. uricase and Xanthine oxidase), lactoperoxidase, microperoxidase and the like. Techniques for conjugating enzymes to antibodies are described, for example, in 0 'Sul l ivan et al., 1981, Methods for the Preparat ion of Enzyme-antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (J. Langone & H. Van Vunakis, eds.), Academic press, NY, 73:14.
<178>  <178>
<179> 표지는 다양한 공지된 기술을 이용하여 항체에 간접적으로 접합될 수 있다 .  Labels can be indirectly conjugated with antibodies using a variety of known techniques.
예를 들어， 항체는 바이오틴에 접합될 수 있고 상기에 언급된 3종의 광범위한 카테 고리에 속하는 임의의 표지들이 아비딘과， 또는 그 반대로 접합될 수 있다. 바이오 틴은 아비딘에 선택적으로 결합하고， 따라서 이 표지는 이러한 간접적 방식으로 항 체에 접합될 수 있다. 또는， 항체에 표지의 간접적 접합을 달성하기 위하여， 항체 는 작은 합텐 (hapten) (예를 들어， 딕옥신 [digoxin] )과 접합될 수 있고 상기에 언급된 서로 다른 유형의 표지들의 하나가 항 -합텐 항체에 접합될 수 있다 (예컨 대, 항-딕옥신 항체) . 따라서, 항체에 대한 표지의 간접적 접합이 달성될 수 있다. <180> 본 발명의 항체 또는 그 단편은 경쟁적 결합 분석， 직접 및 간접 샌드위치 분석， 및 면역침전 분석과 같은 임의의 공지된 분석 방법에 사용될 수 있다. For example, the antibody may be conjugated to biotin and any labels belonging to the three broad categolic rings mentioned above may be conjugated with avidin and vice versa. Biotin binds selectively to avidin and thus the label can be conjugated to the antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the label to the antibody, the antibody may be conjugated with a small hapten (eg digoxin) and one of the different types of labels mentioned above may be anti- Conjugated to hapten antibodies (eg, anti-dioxine antibodies). Thus, indirect conjugation of the label to the antibody can be achieved. Antibodies or fragments thereof of the present invention may be used in any known assay method such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays.
<181> <181>
<182> 본 발명의 항체 또는 그 단편은 진단 킷트 즉， 진단 분석을 수행하기 위한 진단 킷트， 즉 사용설명서와 함께 미리 지정된 양으로 시약들의 포장된 조합에 사 용될 수 있다. 항체가 효소로 표지된 경우에， 킷트는 기질 및 발색단 또는 형광단 을 제공하는 기질 전구체로서 효소에 의해 요구되는 보조인자 (cofactor)를 포함할 수 있다. 또한， 안정화제, 완층액 (예를 들어 , 차단 완층액 또는 용해 완층액) 등 과 같은 다른 첨가제들이 포함될 수 있다. 다양한 시약들의 상대적인 양은 분석의 민감도를 층분히 최적화시키는 시약의 용액 내 농도를 제공하기 위해 폭넓게 변화 될 수 있다. 시약은 용해 시 적절한 농도를 갖는 시약 용액을 제공하게 될 부형제 를 포함하는, 일반적으로 동결건조된， 건조 분말로서 제공될 수 있다. Antibodies or fragments thereof of the present invention may be used in a diagnostic kit, i.e., a diagnostic kit for performing a diagnostic assay, i.e., a packaged combination of reagents in a predetermined amount with instructions for use. If the antibody is labeled with an enzyme, the kit will contain the cofactors required by the enzyme as substrate precursors to provide the substrate and chromophores or fluorophores. Can be. In addition, other additives may be included, such as stabilizers, complete fluids (eg, blocked complete fluids or dissolved complete fluids). The relative amounts of the various reagents can be varied widely to provide concentrations in solution of the reagents that further optimize the sensitivity of the assay. The reagent may be provided as a generally lyophilized, dry powder, comprising an excipient that will provide a reagent solution with the appropriate concentration upon dissolution.
<183>  <183>
<184> 진단용 바이오마커로서의 EPRS의 가능성을 살펴보면 인터페론 감마 ( IFN- Y )  <184> Looking at the possibility of EPRS as a diagnostic biomarker, interferon gamma (IFN- Y)
에 의해 EPRS가 여러 조절 단백질의 mRNA의 3-UTR 부분에 결합하여 복합체를 형성 함으로 인해 여러 염증반응 및 면역반응에 관여하는 조절인자의 전사 과정에 관여 하는 것으로 알려져 있다 ( Sampath, P . et al . Noncanonical funct ion of glut amy 1 ro lyltRNA synthetase : gene-speci f ic si lencing of trans lat ion. Cell 119， 195208 (2004) ) . 또한 혈관신생 (angiogenesi s)에 중요한 인자로 알려진 YEGFA (Vascular endothel i al growth factor A)의 단백질 합성과정도 EPRS에 의해 조절되 는 것으로 밝혀졌다 (Ray, P . S . et al. A stress-responsive RNA swi tch regulates VEGFA expression. Nature 457, 915919 (2009) ) .  It is known that EPRS is involved in the transcription process of regulators involved in various inflammatory and immune responses by forming complexes by binding to the 3-UTR portion of mRNA of various regulatory proteins (Sampath, P. et al. Noncanonical funct ion of glut amy 1 ro lyltRNA synthetase: gene-speci fic si lencing of translat ion. Cell 119, 195208 (2004)). In addition, protein synthesis of Vascular Endothelial Growth Factor A (YEGFA), an important factor in angiogenesis, has been shown to be regulated by EPRS (Ray, P. S. et al. A stress-responsive). RNA swi tch regulates VEGFA expression.Nature 457, 915919 (2009)).
<i85> 따라서， EPRS는 검출을 통해 특정 암， 염증성 질환， 면역질환 및 섬유화증의 진단， 병의 진행 상태 및 치료전후의 예후의 평가를 위한 진단 마커로서 사용될 수 있다. 본 발명에 따른 암, 염증성 질환， 면역질환， 및 섬유화증의 진단 및 예후 평 가는 생물학적 시료 중에서 EPRS 단백질을 검출함으로써 수행될 수 있다. 따라서 본 발명은 본 발명의 항체 또는 그 단편을 유효성분으로 포함하는 암, 염증성 질 환， 면역질환 및 섬유화증 진단용 조성물을 제공한다.  Thus, EPRS can be used as a diagnostic marker for the diagnosis of certain cancers, inflammatory diseases, immune diseases and fibrosis, the progression of disease and the prognosis before and after treatment through detection. Diagnosis and prognosis of cancer, inflammatory diseases, immune diseases, and fibrosis according to the present invention can be performed by detecting EPRS proteins in biological samples. Accordingly, the present invention provides a composition for diagnosing cancer, inflammatory disease, immune disease, and fibrosis, comprising the antibody or fragment thereof of the present invention as an active ingredient.
<186>  <186>
<187> 상기 생물학적 시료는 혈액 및 생물학적 기원의 기타 액상 시료, 생검 표본， 조직 배양과 같은 고형 조직 시료 또는 이로부터 유래된 세포가 포함된다. 보다 구 체적으로 예를 들면 이에 한정되지는 않으나 조직, 추출물， 세포 용해물， 전혈， 혈 장, 혈청， 침, 안구액， 뇌척수액, 땀， 뇨， 젖, 복수액， 활액， 복막액 등일 수 있 다. 상기 시료는 동물, 바람직하게는 포유동물， 가장 바람직하게는 인간으로부터 수득될 수 있다. 상기 시료는 검출에 사용하기 전에 전처리할 수 있다. 예를 들어， 여과, 증류， 추출, 농축， 방해 성분의 불활성화， 시약의 첨가 등올 포함할 수 있 다. 또한， 상기 시료로부터 핵산 및 단백질을 분리하여 검출에 사용할 수 있다. The biological sample includes solid tissue samples such as blood and other liquid samples of biological origin, biopsy samples, tissue cultures or cells derived therefrom. More specifically, it may be, for example, but not limited to, tissues, extracts, cell lysates, whole blood, plasma, serum, saliva, ocular fluid, cerebrospinal fluid, sweat, urine, milk, ascites fluid, synovial fluid, peritoneal fluid, and the like. All. The sample can be obtained from an animal, preferably a mammal, most preferably a human. The sample may be pretreated before use for detection. For example, it may include filtration, distillation, extraction, concentration, inactivation of interference components, addition of reagents, and the like. In addition, nucleic acids and proteins can be separated from the sample and used for detection.
<188> 상기 검출에 관하여는 앞서 설명한 바와 같다. The detection is as described above.
<189> 상기 암은 그 종류가 특별히 제한되지 아니하며， 예를 들어 유방암， 대장암， 폐암， 소세포폐암, 위암, 간암， 혈액암， 골암， 췌장암， 피부암， 두부 또는 경부암， 피부 또는 안구내 혹색종, 자궁암， 난소암， 직장암， 항문부근암， 결장암, 유방암， 나팔관암종, 자궁내막암종， 자궁경부암， 질암, 음문암종， 호지킨병， 식도암， 소장 암， 내분비선암， 갑상선암, 부갑상선암， 부신암， 연조직 육종， 요도암， 음경암， 전 립선암, 만성 또는 급성 백혈병， 림프구 림프종， 방광암， 신장 또는 수뇨관 암， 신 장세포 암종， 신장골반 암종， CNS 종양， 1차 CNS 림프종， 척수 종양， 뇌간신경교종 및 뇌하수체 선종 일 수 있으며， 바람직하게는 전이가 일어나는 암종일 수 있다. <190> 또한, 상기 섬유화증은 그 종류가 특별히 제한되지 아니하며, 예를 들어 간 섬유화증， 신장 섬유화증， 폐 섬유화증， 간질성 섬유화증， 전신성 강피증, 황반 변 성, 췌장 섬유화증， 비장 섬유화증， 심장 섬유화증 종격막 섬유화증， 골수 섬유화 증, 혈관 섬유화증， 피부 섬유화증， 눈 섬유화증， 관절 섬유화증, 근 섬유화증, 갑 상선 섬유화증, 심내막심근 섬유화증， 복막 섬유화증， 복막후 섬유화증， 진행성 종 괴성 섬유화증， 신원성 전신섬유화증， 외과수술의 섬유성 합병증 및 감염 섬유화증 일 수 있다. The type of the cancer is not particularly limited, and for example, breast cancer, colon cancer, Lung cancer, small cell lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular myeloma, uterine cancer, ovarian cancer, rectal cancer, anal muscle cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma Cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, Bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma and pituitary adenoma, preferably metastatic carcinoma. In addition, the type of fibrosis is not particularly limited, for example, liver fibrosis, renal fibrosis, pulmonary fibrosis, interstitial fibrosis, systemic scleroderma, macular degeneration, pancreatic fibrosis, spleen Fibrosis, cardiac fibrosis, mediastinal fibrosis, myelofibrosis, vascular fibrosis, skin fibrosis, eye fibrosis, joint fibrosis, myofiber, thyroid fibrosis, endocardial myocardial fibrosis, peritoneal fibrosis, Post-peritoneal fibrosis, progressive mass necrotic fibrosis, systemic systemic fibrosis, fibrotic complications of surgery and infectious fibrosis.
<191>  <191>
<192> 따라서 본 발명은 항체 또는 그 단편을 유효성분으로 포함하는 암 예방 또는 치료용 약학적 조성물을 제공한다.  Accordingly, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the antibody or fragment thereof as an active ingredient.
<193>  <193>
<194> 또한， 본 발명은 항체 또는 그 단편을 유효성분으로 포함하는 면역질환 예방 또는 치료용 약학적 조성물을 제공한다.  In addition, the present invention provides a pharmaceutical composition for preventing or treating immune diseases, comprising the antibody or fragment thereof as an active ingredient.
<195>  <195>
<196> 또한， 본 발명은 항체 또는 그 단편을 유효성분으로 포함하는 염증질환 예방 또는 치료용 약학적 조성물을 제공한다.  In addition, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases, comprising an antibody or fragment thereof as an active ingredient.
<197>  <197>
<198> 또한， 본 발명은 항체 또는 그 단편을 유효성분으로 포함하는 섬유화증 예방 또는 치료용 약학적 조성물을 제공한다.  In addition, the present invention provides a pharmaceutical composition for preventing or treating fibrosis comprising an antibody or fragment thereof as an active ingredient.
<199>  <199>
<200> 본 발명의 조성물은 본 발명의 항체 또는 그 단편을 유효성분으로 포함하는 것을 특징으로 한다.  The composition of the present invention is characterized by comprising the antibody of the present invention or a fragment thereof as an active ingredient.
<201>  <201>
<202> 본 발명에 따른 약학적 조성물은 본 발명의 항체 또는 그 단편을 단독으로 포함하거나 하나 이상의 약학적으로 허용되는 담체를 추가로 포함할 수 있다. 상기 에서 "약학적으로 유효한 양" 이란 음성 대조군에 비해 그 이상의 반응을 나타내 는 양을 말하며 바람직하게는 암을 치료하기에 층분한 양을 말한다. The pharmaceutical composition according to the present invention may include the antibody of the present invention or a fragment thereof alone or further include one or more pharmaceutically acceptable carriers. remind "Pharmaceutically effective amount" refers to an amount that exhibits a higher response than a negative control, preferably an amount sufficient to treat cancer.
<203> 상기에서 "약학적으로 허용되는" 이란 생리학적으로 허용되고 인간에게 투 여될 때， 활성성분의 작용을 저해하지 않으며 통상적으로 위장 장애， 현기증과 같 은 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 비독성의 조성물을 말한 다. As used herein, "pharmaceutically acceptable" means a physiologically acceptable and, when administered to humans, does not inhibit the action of the active ingredient and usually does not cause an allergic reaction, such as gastrointestinal disorders, dizziness or similar reactions. Refers to non-toxic compositions.
<204> 본 발명에 따른 약학적 조성물에 있어서, 상기 항체 또는 그 단편은 임상 투 여시에 경구 및 비경구의 여러 가지 제형으로 투여될 수 있는데, 제제화할 경우에 는 보통 사용하는 층전제， 증량제， 결합제， 습윤제， 붕해제， 계면활성제 등의 회석 제 또는 부형제를 사용하여 제조될 수 있다.  In the pharmaceutical composition according to the present invention, the antibody or fragment thereof may be administered in a variety of oral and parenteral formulations during clinical administration, and when formulated, it is a commonly used layering agent, extender, and binder. It may be prepared using a diluent or excipient such as a wetting agent, a disintegrant, a surfactant.
<205> 경구투여를 위한 고형 제제에는 정제, 환자， 산제, 과립제, 캡슐제, 트로키 제 등이 포함되며， 이러한 고형 제제는 하나 이상의 본 발명의 상기 화학식 1의 아 릴 유도체， 또는 이의 약학적으로 허용가능한 염에 적어도 하나 이상의 부형제 예 를 들어， 전분， 탄산칼슘， 수크로오스 (sucrose) 또는 락토오스 ( lactose) 또는 젤라 틴 등을 섞어 조제될 수 있다. 또한， 단순한 부형제 외에 스테아린산 마그네슘， 탈 크 등과 같은 윤활제들도 사용될 수 있다. 경구 투여를 위한 액상 제제로는 현탁 제， 내용액제， 유제 또는 시럽제 등이 해당되는데， 흔히 사용되는 단순 회석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제 , 예를 들어 습윤제， 감미제， 방향제 , 보존제 등이 포함될 수 있다.  Solid preparations for oral administration include tablets, patients, powders, granules, capsules, troches, and the like, and the solid preparations may be at least one aryl derivative of Formula 1 of the present invention, or a pharmaceutical thereof. May be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose or gelatin with an acceptable salt. In addition to the simple excipients, lubricants such as magnesium stearate, talc and the like may also be used. Liquid preparations for oral administration include suspensions, solvents, emulsions, or syrups.In addition to the commonly used simple diluents, water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives can be used. Can be.
<206> 비경구 투여를 위한 제제에는 멸균된 수용액， 비수성용제， 현탁용제, 유제， 동결건조제제, 좌제가포함된다.  Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories.
<207> 본 발명의 치료용 조성물을 임의의 생리학적으로 허용가능한 담체, 부형제 또는 안정화제 (Remington: The Science and Pract ice of Pharmacy , 19th Edit ion, Al fonso , R. , ed, Mack Publ i shing Co . (East on, PA: 1995) )와 바람직한 순도를 갖 는 항체를 흔합하여 저장하기 위해 동결건조된 케이크 또는 수용액의 형태로 제조 할 수 있다. 허용가능한 담체， 부형제 또는 안정화제는 사용된 투여량 및 농도에서 . 수용자에게 비독성이고, 완층용액, 예를 들어 인산, 시트르산 및 다른 유기산; 아 스코르브산을 비롯한 항산화제 ; 저분자량 (약 10개 미만의 잔기) 폴리펩티드; 단백 질， 예를 들어 혈청 알부민, 젤라틴 또는 면역글로블린; 친수성 중합체， 예를 들어 폴리비닐피를리돈; 아미노산, 예를 들어 글리신, 글루타민， 아스파라긴， 아르기닌 또는 리신; 단당류， 이당류， 및 글루코스， 만노스 또는 덱스트린을 비롯한 다른 탄 수화물; 킬레이트제， 예를 들어 EDTA; 당 알콜， 예를 들어 만니를 또는 소르비를; 염 -형성 반대이온, 예를 들어 나트륨; 및 (또는) 비이온성 계면활성제, 예를 들어 트윈， 플루로닉스 또는 폴리에틸렌 글리콜 (PEG)이 포함된다. <207> The therapeutic composition of the present invention may be any physiologically acceptable carrier, excipient or stabilizer (Remington: The Science and Pract ice of Pharmacy, 19th Edit ion, Al fonso, R., ed, Mack Publ i shing). Co. (East on, PA: 1995)) and antibodies with the desired purity can be prepared in the form of lyophilized cakes or aqueous solutions for storage. Acceptable carriers, excipients or stabilizers may be used at the dosages and concentrations employed. Non-toxic to recipients, complete solutions such as phosphoric acid, citric acid and other organic acids; Antioxidants including ascorbic acid; Low molecular weight (less than about 10 residues) polypeptides; Proteins such as serum albumin, gelatin or immunoglobulins; Hydrophilic polymers such as polyvinylpyridone; Amino acids such as glycine, glutamine, asparagine, arginine or lysine; Monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; Chelating agents such as EDTA; Sugar alcohols such as manny or sorbbi; Salt-forming counterions such as sodium; And / or nonionic surfactants such as tween, pluronics or polyethylene glycol (PEG).
<208>  <208>
<209> 또한, 본 발명의 항체 또는 그 단편의 인체에 대한 투여량은 환자의 나이， 몸무게， 성별， 투여형태, 건강상태 및 질환 정도에 따라 달라질 수 있으며, 일반적 으로 0.01 - 100 mg/kg/week이며, 바람직하게는 0. 1 - 20 mg/kg/week이며， 더욱 바 람직하게는 5 - 10 mg/kg/week일 수 있다. 또한 의사 또는 약사의 판단에 따라 일 정 간격으로 분할 투여할 수도 있다.  In addition, the dosage of the antibody or fragment thereof of the present invention to the human body may vary depending on the age, weight, sex, dosage form, health condition and degree of disease of the patient, and generally 0.01-100 mg / kg / week, preferably from 0.1 to 20 mg / kg / week, more preferably from 5 to 10 mg / kg / week. It may also be administered in divided doses at regular intervals, as determined by the physician or pharmacist.
<210>  <210>
<211> 본 발명의 조성물의 투여 경로는 공지된 방법， 예를 들어 정맥내， 복강내, 뇌내, 피하, 근육내， 안내， 동맥내， 뇌척수내， 또는 병변내 경로에 의한 주사 또는 주입, 또는 하기 기재된 서방성 (sustained release) 시스템에 의한 주사 또는 주 입이다. 바람직하게는 항체는 전신으로 투여될 수 있다.  The route of administration of the composition of the present invention may be administered or injected by known methods, for example intravenous, intraperitoneal, intracranial, subcutaneous, intramuscular, intraocular, intraarterial, cerebrospinal, or intralesional routes, Injection or injection by the sustained release system described below. Preferably the antibody may be administered systemically.
<212> 본 발명의 약학적 조성물은 암 예방 또는 치료를 위하여 단독으로， 또는 수 술， 호르몬 치료， 화학 치료 및 생물학적 반웅 조절제를 사용하는 방법들과 병용하 여 사용할 수 있다. The pharmaceutical compositions of the present invention may be used alone or in combination with methods using surgery, hormonal therapy, chemotherapy and biological response modifiers for the prevention or treatment of cancer.
<213>  <213>
<214> 본 발명의 일실시예에서는 인간의 VH3-23/VLlg 유전자를 골격으로 하고 CDR 에 무작위 서열을 삽입하는 라이브러리를 구축하고， EPRS와 선택적으로 결합하는 파지를 선별하여 항체를 분리， 정제 하고 염기서열을 분석하였다.  In an embodiment of the present invention, a human VH3-23 / VLlg gene is used as a skeleton and a library for inserting random sequences into CDRs, and phages that selectively bind to EPRS are isolated and purified. The sequence was analyzed.
<215>  <215>
<216> 본 발명의 다른 일실시예에서는 정제한 항체가 EPRS와 결합하는지 western blot 방법으로 확인하였다. 그 결과 본 발명의 항체는 EPRS와 결합하는 것을 확인 하였다.  In another embodiment of the present invention it was confirmed by the western blot method whether the purified antibody binds to EPRS. As a result, it was confirmed that the antibody of the present invention binds to EPRS.
<217>  <217>
<218> 본 발명의 다른 일실시예에서는 본 발명의 항 EPRS scFv항체와 EPRS의 친화 도를 표면공명분석방법으로 측정하였다. 그 결과 본 발명의 항체는 평형해리상수 약 10으 300nM 값을 가져 비교적 높은 친화도를 나타내는 것을 확인하였다. In another embodiment of the present invention, the affinity between the anti-EPRS scFv antibody and EPRS of the present invention was measured by surface resonance analysis. As a result, it was confirmed that the antibody of the present invention had a relatively high affinity with an equilibrium dissociation constant of about 10 nM.
<219> <219>
<220> 본 발명의 다른 일실시예에서는 본 발명의 항 EPRS scFv 항체의 교차반웅성 을 측정하였다. Luminex bead를 이용하여 본 발명의 항체와 EPRS를 포함한 7종의 유사 단백질과의 반응성올 측정한 결과 본 발명의 항체는 EPRS를 제외한 다른 ARS 들히 WHEP domain을 가지고 있는 WRS, HRS에도 결합하지 않는 것으로 보아 매우 특 이적임을 확인하였다. In another embodiment of the present invention, the cross reaction properties of the anti-EPRS scFv antibodies of the present invention were measured. As a result of measuring the reactivity of the antibody of the present invention with seven similar proteins including EPRS using Luminex bead, the antibody of the present invention was ARS except for EPRS. It is confirmed that it does not bind to WRS and HRS which have WHEP domain.
<221>  <221>
<222> 본 발명의 다른 일실시예에서는 본 발명의 항 EPRS scFv항체의 진단용 항체 로의 유용성 여부를 실험하였다. 본 발명와 항체로 코팅된 pl ate를 제작한 후， EPRS WHEP domains 표준물질올 농도별로 희석하여 반웅시키고, 결합여부를 ELISA로 측정하였다. 그 결과 EPRS WHEP domains 표준물질에 대하여 농도 의존적으로 항체 결합이 측정되는 것을 확인하여, 본 발명의 항체가 암， 염증질환, 면역질환 및 섬 유화증 진단용으로 사용될 수 있음을 확인하였다.  In another embodiment of the present invention, the usefulness of the anti-EPRS scFv antibody of the present invention as a diagnostic antibody was tested. After preparing the coated with the present invention and antibody, the reaction was diluted by the concentration of EPRS WHEP domains standards, and the binding was measured by ELISA. As a result, it was confirmed that the antibody binding is measured in a concentration-dependent manner with respect to the EPRS WHEP domains standard, it was confirmed that the antibody of the present invention can be used for the diagnosis of cancer, inflammatory diseases, immune diseases and ischemia.
<223>  <223>
<224> 본 발명은 상기 항체 또는 그 단편을 필요로 하는 개체에 유효량으로 투여하 는 단계를 포함하는 암, 면역질환， 염증질환 및 섬유화증 치료 방법 또는 진단 방 법을 제공한다.  The present invention provides a method or diagnostic method for treating cancer, immune disease, inflammatory disease and fibrosis, comprising administering to a subject in need thereof an effective amount of the antibody or fragment thereof.
<225>  <225>
<226> 또한， 본 발명은 암， 면역질환， 염증질환 및 섬유화증 치료용 약제 또는 진 단 제제 제조의 용도를 위한 상기 항체 또는 그 단편을 제공한다.  The present invention also provides the antibody or fragment thereof for use in the manufacture of a medicament or diagnostic agent for the treatment of cancer, immune disease, inflammatory disease and fibrosis.
<227>  <227>
<228> 상기에서 '유효량' 이란 개체에게 투여하였을 때， 암, 면역질환， 염증질환 및 섬유화증의 치료 및 /또는 예방 효과를 나타내는 양을 말하며, 상기 '개체 (subject )' 란 동물， 바람직하게는 포유동물, 특히 인간올 포함하는 동물일 수 있 으며, 동물에서 유래한 세포， 조직, 기관 등일 수도 있다. 상기 개체는 치료가 필 요한 환자 (pat ient )일 수 있다.  As used herein, the term 'effective amount' refers to an amount that exhibits the therapeutic and / or prophylactic effect of cancer, immune disease, inflammatory disease and fibrosis, and the term 'subject' refers to an animal, preferably May be an animal including a mammal, especially a human, and may be a cell, tissue, organ or the like derived from an animal. The subject may be a patient in need of treatment.
<229>  <229>
【유리한 효과]  Advantageous Effects
<230> 따라서， 본 발명의 항체 또는 그 단편은 인간 EPRS에 특이적으로 결합하며， 동일한 ARS fami ly를 포함한 다른 단백질과 교차반웅성이 없어 EPRS 검출 및 억제 가 가능하므로 EPRS 검출 및 EPRS가 관련된 질환인 암， 염증질환， 면역질환 및 섬 유화증 진단 및 치료에 효과적이다.  Therefore, the antibody or fragment thereof of the present invention specifically binds to human EPRS and does not have cross-reaction with other proteins including the same ARS fami ly, so that EPRS can be detected and suppressed. It is effective in the diagnosis and treatment of human cancer, inflammatory disease, immune disease and ischemia.
<231>  <231>
【도면의 간단한 설명】  [Brief Description of Drawings]
<232> 도 1은 본 발명의 항체가 목적단백질인 EPRS에 결합하는지를 확인한 western blot 실험 결과이다. <233> 1 is a western blot experiment confirming whether the antibody of the present invention binds to EPRS, the protein of interest. <233>
<234> 도 2는 본 발명의 항체인 anti-EPRS scFv Biocon-EPl의 결합친화도를 표면 플라즈몬 공명 (SPR)으로 측정한 결과 그래프이다 (Kd ： 평형해리상수, 가로축 ： 시 간 (초)， 세로축 ： response unit(RU)).  Figure 2 is a graph of the result of measuring the binding affinity of the anti-EPRS scFv Biocon-EPl antibody of the present invention by surface plasmon resonance (SPR) (Kd: equilibrium dissociation constant, horizontal axis: time (seconds), Vertical axis: response unit (RU)).
<235>  <235>
<236> 도 3은 본 발명의 항체인 anti-EPRS scFv Biocon_EP2의 결합친화도를 표면 플라즈몬 공명 (SPR)으로 측정한 결과 그래프이다 (Kd ： 평형해리상수, 가로축 ： 시 간 (초)， 세로축 ： response unit(RU)).  Figure 3 is a graph of the result of measuring the binding affinity of the antibody of the present invention anti-EPRS scFv Biocon_EP2 by surface plasmon resonance (SPR) (Kd: equilibrium dissociation constant, horizontal axis: time (seconds), vertical axis: response unit (RU)).
<237>  <237>
<238> 도 4는 본 발명의 항체인 anti-EPRS scFv Biocon_EP3의 결합친화도를 표면 플라즈몬 공명 (SPR)으로 측정한 결과 그래프이다 (Kd ： 평형해리상수, 가로축 ： 시 간 (초)， 세로축 ： response unit(RU)).  4 is a graph showing the result of measuring the binding affinity of the antibody of the present invention, anti-EPRS scFv Biocon_EP3, by surface plasmon resonance (SPR) (Kd: Equilibrium dissociation constant, horizontal axis: time (sec), vertical axis: response unit (RU)).
<239>  <239>
<240> 도 5는 본 발명의 항체인 anti-EPRS scFv Biocon-EPl의 교차반응성 (cross activity)을 Luminex Multiplex Assay 방법으로 측정한 결과 그래프이다 (세로축 - (FI) ： 형광강도 (fluorescent intensity), CRS: 시스테닐 -tRNA 합성효소 (Cysteinyl-tRNA synthetase), DRS: 아스파틸 -tRNA 합성효소 (Aspartyl—tRNA synthetase), EPRS: 글루타밀-프로릴 -tRNA 합성효소 (Glutamyl— prolyl tRNA synthetase), HRS ： 히스티딜— tRNA 합성효소 (Histidyl-tRNA synthetase), WRS ： 트 립토파닐 -tRNA 합성효소 (Tryptophanyl-tRNA synthetase), AIMP1 ： A S 결합 다기 능 단백질 1 ( Aminoacyl-tRNA-synthetase-interact ing multifunctional protein 1)), AIMP3: ARS 결합 다기능 단백질 3 ( Aminoacyl-tRNA-synthetase— interacting multifunctional protein 3)).  FIG. 5 is a graph of the cross-activity of the anti-EPRS scFv Biocon-EPl antibody, which is an antibody of the present invention, measured by Luminex Multiplex Assay method (vertical axis-(FI): fluorescence intensity, CRS: Cysteinyl-tRNA synthetase, DRS: Aspartyl-tRNA synthetase, EPRS: Glutamyl-prolyl tRNA synthetase, HRS ： Histidyl-tRNA synthetase, WRS ： Tryptophanyl-tRNA synthetase, AIMP1 ： AS-binding multifunctional protein 1 (Aminoacyl-tRNA-synthetase-interacting multifunctional protein 1)), AIMP3: ARS-binding multifunctional protein 3 (Aminoacyl-tRNA-synthetase—interacting multifunctional protein 3)).
<241>  <241>
<242> 도 6은 본 발명의 항체인 anti-EPRS scFv Biocon-EP2의 교차반응성 (cross activity)을 Luminex Multiplex Assay 방법으로 측정한 결과 그래프이다 (세로축 (FI) ： 형광강도 (fluorescent intensity), CRS: 시스테닐 -tRNA 합성효소 (Cysteinyl-tRNA synthetase), DRS: 아스파틸— tRNA 합성효소 (Aspartyl—tRNA synthetase), EPRS: 글루타밀-프로릴 -tRNA 합성효소 (Glutamyl-prolyl tRNA synthetase), HRS ： 히스티딜 -tRNA 합성효소 (Hist idyl -tRNA synthetase), WRS ： 트 립토파닐 -tRNA 합성효소 (Tryptophanyl-tRNA synthetase), AIMP1 ： ARS 결합 다기 능 단백질 1 ( Aminoacyl-tRNA-synthetase-interact ing multifunctional protein D), AIMP3: ARS 결합 다기능 단백질 3 ( Aminoacyl-tRNA-synthetase-interacting multifunctional protein 3)). 도 7은 본 발명의 항체인 anti-EPRS scFv Biocon_EP3의 교차반웅성 (cross activity)을 Luminex Multiplex Assay 방법으로 측정한 결과 그래프이다 (세로축 (FI) ： 형광강도 (fluorescent intensity), CRS: 시스테닐 -tRNA 합성효소 (Cysteinyl-tRNA synthetase), DRS: 아스파틸 -tRNA 합성효소 (Aspartyl-tRNA synthetase), EPRS: 글루타밀-프로릴 -tRNA 합성효소 (Glutamyl-prolyl tRNA synthetase), HRS ： 히스티딜 -tRNA 합성효소 (Histidyl-tRNA synthetase), WRS ： 트 립토파닐 -tRNA 합성효소 (Tryptophanyl-tRNA synthetase), AIMP1 ： ARS 결합 다기 능 단백질 1 ( Ami noacyl-tRNA-synthetase- interacting multifunctional protein 1))， AIMP3: ARS 결합 다기능 단백질 3 ( Aminoacyl-tRNA-synthetase— interact ing multifunctional protein 3)) . 도 8은 본 발명의 항체가 EPRS를 detection 할 수 있는지를 확인하기 위한 ELISA 실험 결과이다 (세로축 ： 450nra 흡광도， 가로축 ： EPRS WHEP domains 표준물 질의 농도 (ng/ml)). FIG. 6 is a graph illustrating the cross-activity of the anti-EPRS scFv Biocon-EP2 antibody, which is an antibody of the present invention, measured by Luminex Multiplex Assay method (vertical axis (FI): fluorescence intensity, CRS) : Cysteinyl-tRNA synthetase, DRS: Aspartyl—tRNA synthetase, EPRS: Glutamyl-prolyl tRNA synthetase, HRS ： Histidyl-tRNA synthetase, WRS: Tryptophanyl-tRNA synthetase, AIMP1: ARS binding multifunctional protein 1 (Aminoacyl-tRNA-synthetase-interacting multifunctional protein) D), AIMP3: ARS-binding multifunctional protein 3 (Aminoacyl-tRNA-synthetase-interacting multifunctional protein 3)). Figure 7 is a graph of the cross activity of the anti-EPRS scFv Biocon_EP3 antibody of the present invention measured by Luminex Multiplex Assay method (vertical axis (FI): fluorescence intensity, CRS: cysteinyl- CyRNAl-tRNA synthetase, DRS: Aspartyl-tRNA synthetase, EPRS: Glutamyl-prolyl tRNA synthetase, HRS ： histidyl-tRNA Histyl-tRNA synthetase, WRS: Tryptophanyl-tRNA synthetase, AIMP1: Ami noacyl-tRNA-synthetase-interacting multifunctional protein 1), AIMP3 : ARS binding multifunctional protein 3 (Aminoacyl-tRNA-synthetase— interacting multifunctional protein 3). Fig. 8 shows the results of ELISA experiments to determine whether the antibody of the present invention can detect EPRS (vertical axis: 450 nra absorbance, horizontal axis: EPRS WHEP domains standard concentration (ng / ml)).
【발명의 실시를 위한 형태】 [Form for implementation of invention]
이하， 본 발명을 실시예에 의하여 상세히 설명한다.  Hereinafter, the present invention will be described in detail by way of examples.
단， 하기 실시예는 본 발명을 예시하는 것일 뿐， 본 발명의 내용이 하기 실 시예에 한정되는 것은 아니다.  However, the following examples are merely to illustrate the present invention, the content of the present invention is not limited to the following embodiments.
<실시예 1> <Example 1>
scFv라이브러리 구축 및 항 EPRS scFv선1 j <!-!> scFv라이브러리 구축 scFv library building and EPRS scFv line building 1 j <!-!> scFv library building
scFv 라이브러리는 대한민국특허 10-0961392호에 나온 방법에 따라 구축하였 다.  scFv library was constructed according to the method described in Korean Patent No. 10-0961392.
인간의 VH3-23/VLlg 유전자를 골격으로 하고 상보성 결정부위 (complementarity determining regions, CDR)에 무작위 서열을 삽입하는 것으로 라 이브러리를 설계하고， 베타락타메이즈 유전자를 선택마커 (selection marker)로 가 지는 플라스미드 백터에서 베타락타메이즈 유전자의 리더서열 직후에 제한효소 절 단서열들을 도입하여 pFDV 플라스미드 백터를 제조하고 scFv 유전자 라이브러리를 삽입한 후， 대장균에 형질감염 시킨 후 이를 배양하여 라이브러리를 구축하였다. 이 과정을 통하여 라이브러리 내에서 비정상적인 정지코돈 혹은 프레임시프트 ( frameshi ft )를 가지는 scFv 유전자 서열을 대부분 제거하여 라이브러리의 품질을 향상시키는 것이 가능하다. 배양된 라이브러리로부터 오류가 제거된 서열들을 중합 효소 연쇄반웅으로 증폭한 후 이로부터 scFv 유전자 라이브러리를 재조합하여 pComb3X 파아지미드 백터에 삽입하고, 이.콜라이 ER2537 스트레인의 대장균을 형질 감염하여 최종 라이브러리를 획득하였다. 라이브러리를 카르베니실린을 함유하는 SB (Super broth) 배지 400 mL에 배양하고， 600 나노미터에서의 흡광도가 0.5가 되 었을 때 1013 CFU의 VCSM13 보조파아지를 가하여 80 rpm에서 교반하며 1시간동안 섭 씨 37도에서 감염시켰다. 여기에 최종 70 y g/mL의 카나마이신 항생제를 넣고 섭씨 30도， 200 rpm에서 교반하며 밤새 배양하여 scFv가 표면제시된 파지를 생산하였다. 다음날 아침에 배양액을 원심분리하고， 배양액 속의 파지를 4%의 폴리에틸렌글리콜 -8000과 3¾의 염화소듐을 가하여 침전시켰다. 침전된 파지를 50 mL의 PBS 완층용액 에 녹이고， 위와 같은 방식으로 재차 침전하여 최종적으로 2 mL의 PBS 완충용액에 녹였다. 이를 원심분리하여 이물질을 제거함으로써 파지 scFv 라이브러리를 얻었으 며 일반적으로 최종 파지 라이브러리에는 1013 CFU/mL 이상의 파지 입자가 포함된 다. . Libraries are designed by placing human VH3-23 / VLlg genes in the backbone and inserting random sequences into complementarity determining regions (CDRs), and the beta lactase genes as selection markers. In the plasmid vector, immediately after the beta-lactamase gene leader sequence, restriction enzyme fragment sequences were introduced to prepare a pFDV plasmid vector, an scFv gene library was inserted, transfected into E. coli and cultured. Through this process, it is possible to improve the quality of the library by removing most scFv gene sequences having abnormal stop codons or frameshi ft in the library. Sequences from which the error was removed from the cultured library were amplified by polymerase chain reaction, from which the scFv gene library was recombined and inserted into the pComb3X phagemid vector, and E. coli ER2537 strain E. coli was transfected to obtain a final library. . The library was incubated in 400 mL of SB (Super broth) medium containing carbenicillin, and the absorbance at 600 nanometers was 0.5, adding 10 13 CFU of VCSM13 auxiliary phage and stirring at 80 rpm for 1 hour. Infected at 37 degrees. The final 70 yg / mL kanamycin antibiotic was added and stirred overnight at 30 degrees Celsius, 200 rpm to incubate overnight to produce scFv surface-treated phage. The next morning the culture was centrifuged, and the phage in the culture was precipitated by adding 4% polyethylene glycol -8000 and 3¾ sodium chloride. The precipitated phage was dissolved in 50 mL of complete PBS solution, precipitated again in the same manner as above, and finally dissolved in 2 mL of PBS buffer. The phage scFv library was obtained by centrifugation to remove foreign substances. Generally, the final phage library contains more than 10 13 CFU / mL phage particles. .
<257>  <257>
<258> <1-2> 항 EPRS scFv 선별  <1-2> Anti-EPRS scFv Screening
<259> 면역시험관 ( immunotube)에 10 ug/ml 농도의 EPRS WHEP domain 항원올 첨가하 여 1시간동안 시험관 표면에 단백질을 흡착시킨 후 분말우유 3¾ 용액을 시험관에 첨가하여 EPRS가 흡착되지 않은 표면을 보호하였다. 시험관을 비운 후 여기에 분말 우유 3% 용액에 분산된 1012 CFU의 항체파지 라이브러리를 넣어 항원과 결합시켰다. 비특이적으로 결합한 파지를 TBST (tri s buf fered sal ine - tween20) 용액으로 3회 씻어낸 후, 남아있는 항원특이적 파지 항체를 100 mM 트리에틸아민 용액을 이용하 여 용리하였다. <259> After adsorbing the protein on the surface of the test tube for 1 hour by adding 10 ug / ml EPRS WHEP domain antigen to the immunotube, the powdered milk 3¾ solution was added to the test tube to remove the surface where the EPRS was not adsorbed. Protected. After emptying the test tube, the antibody phage library of 10 12 CFU dispersed in a 3% solution of powdered milk was added thereto to bind the antigen. Nonspecifically bound phage was washed three times with TBST (tri s buf fered sal ine-tween20) solution, and the remaining antigen-specific phage antibody was eluted with 100 mM triethylamine solution.
<260>  <260>
<26i> 용리된 파지를 1.0M 농도의 Tr i s-HCl 버퍼 (pH 7.8)로 중화시킨 후 ER2537 대장균에 37°C에서 1시간 감염시키고， 감염된 대장균을 카르베니실린을 함유하는 LB(Lur ia-Bertani ) 한천배지에 도포하여 37°C에서 배양하였다. 다음날 배양된 대장 균을 3 mL의 SB (super broth) - 카르베니실린 배양액에 현탁하고 15% 글리세를을 첨가하여 일부는 -80°C에 보관하고， 나머지 중 50마이크로리터를 20 mL의 SB-카르 베니실린 -2% 포도당 용액에 접종하여 37°C에서 배양하였다. 배양액의 600 나노미터 광선의 흡광도가 0.5가 되면 원심분리하여 박테리아만을 분리하고， 이를 다시 20 mL의 SB-카르베니실린 배양액에 현탁한 후 1012PFU (플라크 형성단위)의 VCSM13 보 조파지를 넣고 서서히 교반하며 37°C에서 배양하였다. <26i> The eluted phage was neutralized with 1.0M Tr i s-HCl buffer (pH 7.8) and then infected with ER2537 Escherichia coli at 37 ° C for 1 hour, and the infected E. coli containing carbenicillin LB (Lur ia-Bertani) was applied to agar medium and incubated at 37 ° C. The next day, E. coli cultured was suspended in 3 mL of super broth (SB) -carbenicillin culture and 15% glycerol was added to keep some at -80 ° C, and 50 microliters of 20 mL of SB- Carbenicillin -2% glucose solution was inoculated and incubated at 37 ° C. When the absorbance of the 600 nanometer light of the culture medium is 0.5, only the bacteria are separated by centrifugation, and suspended again in 20 mL of SB-carbenicillin culture medium, and then added 10 12 PFU (plaque forming unit) VCSM13 auxiliary phage. Incubated at 37 ° C with stirring.
<262> 1시간 후 카나마이신 70ug/ml을 첨가하고 30°C에서 빠르게 교반하며 (250 rpm) 밤새 배양하였다. 다음날 배양액을 원심분리한 후, 파지 입자를 포함하는 상 층액 1 mL을 라이브러리로 사용하여 위의 패닝 과정을 반복함으로써 항원특이적 클 론을 농축시켰다. After 1 hour, 70 ug / ml of kanamycin was added and incubated overnight with rapid stirring at 250 ° C. (250 rpm). The next day, after centrifuging the culture, the antigen-specific clone was concentrated by repeating the panning process using 1 mL of the supernatant containing phage particles as a library.
<263>  <263>
<264> <실시예 2>  <264> <Example 2>
<265> 항 EPRS scFv항체 발현 및 정제  <265> Expression and Purification of Anti-EPRS scFv Antibodies
<266>  <266>
<267> 3-4회 정도의 반복적인 패닝 후 항체유전자를 포함하는 대장균올 카르베니실 린을 함유하는 LB 한천배지에 도포， 배양하여 단일 콜로니들을 얻고， 이를 200 uL SB-카르베니실린 용액에 접종， 배양한 후 IPTG로 유도하여 scFv 단백질을 대장균의 페리플라즘 (per iplasm)에서 발현하였다. 대장균을 40 의 IX TES (50 mM Tr i s , 1 mM EDTA, 20% Sucrose , pH 8.0) 용액에 현탁한 후 여기에 0.2X TES 용액 60 u L 를 가하고 섞어서 섭씨 4도에서 30분 이상 처리하고 원심분리하여 상층액으로 페리 플라즘을 추출하였다.  After repeated panning about 3-4 times, LB agar medium containing Escherichia coli carbenicillin containing antibody genes was applied and cultured to obtain single colonies, which were obtained in 200 uL SB-carbenicillin solution. After inoculation and incubation, IPTG was induced to express the scFv protein in E. coli periplasm. E. coli was suspended in a solution of 40 IX TES (50 mM Tr is, 1 mM EDTA, 20% Sucrose, pH 8.0), and then mixed with 60 uL of 0.2X TES solution, mixed for 30 minutes at 4 degrees Celsius, and centrifuged. The periplasm was extracted with supernatant.
<268>  <268>
<269> <2-1> EPRS에 대해 선별된 scFv 항체 발현  <2-1> scFv Antibody Expression Selected for EPRS
<270> 선별된 EPRS에 대한 scFv 양성 단일 콜로니 클론을 카르베니실린을 함유하 는 SB 배지 (Bactotrytone 30g, yeast extract 20g,M0PS buf fer lOg/L)에 5 ml에 배 양하여 seed cul ture를 시작하여 overnight 배양후 500ml 카네니실린함유 SB배지에 옮겨 0D 600 = 0.5 정도 되었을때 IPTG를 ImM되게 넣어 30도에서 overnight 배양하 여 scFv 단백질을 대장균의 페리플라즘 (per iplasm)에서 발현하였다. 다음날 원심분 리하여 수득한 대장균을 IX TES buf fer에 (50 mM Tr i s , 1 mM EDTA, 20% Sucrose , pH 8.0) 현탁한 후 0.2 X TES를 1.5배 첨가하여 섞어주고 원심분리하여 페리플라즘 을 추출하였다. <271> ScFv positive single colony clones for selected EPRS were incubated in 5 ml of SB medium containing carbenicillin (30 g Bactotrytone, 20 g yeast extract, M0PS buf fer lOg / L) to initiate seed culture. After overnight incubation, the cells were transferred to 500ml cannicillin-containing SB medium, when IPD was imM at 0D 600 = 0.5, and cultured overnight at 30 ° C to express scFv protein in E. coli periplasm. The E. coli obtained by centrifugation the next day was suspended in IX TES buf fer (50 mM Tr is, 1 mM EDTA, 20% Sucrose, pH 8.0), and mixed with 1.5 times 0.2 X TES, followed by centrifugation. Extracted. <271>
<272> <2-2> 선별된 scFv항체 정제  <2-2> Purification of Selected scFv Antibodies
<273> 페리플라즘에서 추출한 scFv 항체에 최종 5 mM MgS04를 가하고 이를 미리 The final 5 mM MgS0 4 was added to the scFv antibody extracted from Periplasm and preliminarily
PBS에 평형시킨 Ni-NTA bead 와 섞어 1시간 동안 넁장에서 교반하여 Ni- bead에 결 합시킨후 친화도 크로마토그래피를 수행하여 결합하지 않은 단백질을 PBS로 충분히 씻어내었다. 5 mM Imidazole이 함유된 buf fer로 더 층분히 씻어 준 다음 결합한 scFv 항체는 200 mM Imidazole buf fer를 이용하여 용출하였다. 용출된 항체는 투석 하여 전기이동을 통해 순도를 확인하고 BCA 방법으로 단백질 정량을 하여 정제된 항체양을 기록후 일정양을 분주하여 냉동 보관하였다 After mixing with Ni-NTA bead equilibrated in PBS, the mixture was stirred in the intestine for 1 hour to bind to Ni-bead, and affinity chromatography was performed to wash out the unbound protein sufficiently with PBS. After further washing with buf fer containing 5 mM Imidazole, the bound scFv antibody was eluted using 200 mM Imidazole buf fer. The eluted antibody was dialyzed to confirm purity through electrophoresis, the protein was quantified by BCA method, and the amount of purified antibody was recorded.
<274>  <274>
<275> <2-3> 면역블롯 및 sequencing  <275> <2-3> Immunoblotting and Sequencing
<276> 페리플라즘에서 추출한 scFv 항체는 면역블롯 (western blot ) 기법을 사용하 여 human 세포에서 원래 발현된 EPRS에 scFv 항체가 결합하는지 여부를 확인하는 데 사용하였다. 50ug의 Hela cel l lysate를 SDS PAGE를 통해 전기이동한 후 Wet transfer 방법으로 Ni trocel lulos membrane에 옮겨 3% skim mi lk로 blocking 한후 추출한 scFv 항체를 첨가하여 결합시켰다. 검출을 위해 결합한 scFv에 HRPChorseradi sh peroxidase)가 연결된 Ant i-HA 이차항체를 반응시킨후 기질로 ECL reagent를 이용하여 암실에서 필름 감광하였다. 감광된 띄는 표준 분자 마커와 비 교하여 EPRS의 사이즈에 해당하는 띠를 확인하였다.  The scFv antibody extracted from periplasm was used to determine whether the scFv antibody binds to EPRS originally expressed in human cells using immunoblot technique. After 50 ug of Hela cel l lysate was electrophoresed through SDS PAGE, it was transferred to Ni trocel lulos membrane by Wet transfer method, blocked with 3% skim milk, and then extracted by adding scFv antibody. For detection, the reacted scFv reacted with an Ant i-HA secondary antibody linked with HRPChorseradi sh peroxidase) and was then photosensitive in the dark room using ECL reagent as a substrate. A band corresponding to the size of the EPRS was identified in comparison with the prominent standard molecular markers.
<277>  <277>
<278> 이로부터 확인된 항원특이적 항체 클론은 카베니실린이 함유된 SB 배지 10ml 에 overnight 배양하여 plasmid miniprep ki t를 사용하여 plasmid DNA를 추출하여 Capi l lary sequencing service (Macrogen Co) 를 통해 염기서열을 분석하고 Kabat protein seqeunce database와 IMGKthe internat ional ImMunoGeneTics informat ion system) 분석방법에 근거하여 CDR서열을 분석하였다.  The identified antigen-specific antibody clones were cultured overnight in 10 ml of SB medium containing carbenicillin, plasmid DNA was extracted using plasmid miniprep kit, and the base was obtained through Capillary sequencing service (Macrogen Co). The sequences were analyzed and CDR sequences were analyzed based on Kabat protein seqeunce database and IMGKthe internat ional ImMunoGeneTics informat ion system.
<279>  <279>
<280> 그 결과 EPRS 항원과 결합한 scFv의 개수는 총 3 건이며， 이들의 염기서열은 서열번호 14， 28 및 42 인 것으로 확인되었다.  As a result, the number of scFv bound to the EPRS antigen was 3 in total, and their nucleotide sequences were found to be SEQ ID NOs: 14, 28 and 42.
<281>  <281>
<282> <실시예 3>  <282> <Example 3>
<283> 항 EPRS scFv항체의 친화도 측정  Affinity Measurement of Anti-EPRS scFv Antibodies
<284> EPRS WEHP domain 항원에 대한 본 발명 항체의 결합 친화도를 Prote0nTMXPR36 SPR (surface plasmon resonance) 바이오센서 (Bio—Rad 사)를 이용 하여 측정하였다. 구체적으로， 약 10 ug/ml의 EPRS 항원을 제조사의 설명서에 따라 GLC 칩 (Bio-Rad 6 X 6 sensor chip, Compact capacity amine coupling for protein- protein interact ions)에 약 2,000 내지 4,000 반웅 단위 (response unit)로 고정 화시킨 후， PBS를 이용하여 다양한 농도로 희석한 본 발명의 정제된 scFv 항체 (250-30nM) 30ul씩을 25°C에서 50 ul/min의 속도로 칩에 주입하여 항원과의 상호작 용을 정량하였다. 칩의 표면은 0.85% phosphoric acid로 재생하였으며， ProteOn Manager 소프트웨어를 이용하여 연합 속도와 해리속도를 계산하였고， 평형 해리 상 수 (KD)는 해리속도 /연합속도 비로서 계산하였다 그 결과 [도 2-4]에서 보는 바와 같이， 본 발명의 항체는 최대 KD 10nM 값을 가져 매우 높은 EPRS 친화도를 가지는 것을 확인하였다. <284> The binding affinity of the antibody of the present invention to the EPRS WEHP domain antigen was measured using a Prote0nTMXPR36 surface plasmon resonance (SPR) biosensor (Bio—Rad). Specifically, about 10 ug / ml of EPRS antigen was added to the GLC chip (Bio-Rad 6 X 6 sensor chip, Compact capacity amine coupling for protein-protein interact ions) according to the manufacturer's instructions. After immobilization, 30 μl of purified scFv antibody (250-30 nM) of the present invention diluted to various concentrations using PBS was injected into the chip at a rate of 50 ul / min at 25 ° C. to interact with antigen. Dragon was quantified. The surface of the chip was regenerated with 0.85% phosphoric acid, the association rate and dissociation rate were calculated using ProteOn Manager software, and the equilibrium dissociation constant (KD) was calculated as the dissociation rate / association rate ratio. As shown in [4], it was confirmed that the antibody of the present invention had a maximum KD 10 nM value and had a very high EPRS affinity.
<실시예 4> <Example 4>
항 EPRS scFv항체 교차 반응성 (cross activity) 측정 scFv 항체가 다른 항원과 반응성이 있는지 판단하기 위하여 Luminex bead를 이용하여 본 발명의 항체와 EPRS 및 다른 ARS family protein과의 교차 반웅성을 측정하였다.  Anti-EPRS scFv antibody cross activity measurement In order to determine whether the scFv antibody is reactive with other antigens, cross reaction between the antibody of the present invention and EPRS and other ARS family proteins was measured using Luminex bead.
<4-1>각각의 protein이 결합된 Luminex bead제조 <4-1> Manufacture of Luminex bead with each protein
Code No가 다른 각각의 bead에 protein의 amine 잔기를 결합시키기 위해 Bio-rad사의 Amine coupling kit의 실험과정에 따라 coupling을 수행하였다. 먼저  Coupling was performed according to the experimental procedure of Bio-rad's Amine coupling kit to bind amine residues of protein to each bead with different Code No. first
1 xlO6 에 해당하는 각각의 bead를 96well filter pi ate로 옮겨 activation buffer 로 vacuum manifold를 이용하여 세척 후 50 mg/ml S~NHS(N- hydroxysulfosuccinimide)와 50 mg/ml EDAC ( 1-e t hy-3- [ 3- dimethylaminopropyl]carbodiimide)를 첨가하여 상온에서 20 분 활성화시켰다. 활 성화된 각각의 bead는 PBS로 세척 후 순도 90¾이상의 정제된 각각의 재조합 ARS 항 원 즉 CRS,DRS, EPRS WHEP domain, HRS, WRS, AIMPl(p43), AIMP3(pl8) 10 ug을 첨 가하여 상온에서 2시간 반응시켰다. 각각의 ARS에 연결된 bead는 PBS로 2번 세척 후 Blocking buffer를 첨가하여 30분간 상온에서 반응시켜 결합하지 않은 잔기를 blocking 시킨후 PBS로 2번 세척 후 lOOul PBS에 다시 현탁하고 빛이 차단된 tube 에 보관하였다. 결합된 bead의 수는 hemocytometer로 측정하여 기록하였다.Transfer each bead corresponding to 1 xlO 6 to a 96well filter piate and wash it using a vacuum manifold as an activation buffer, and then wash it from 50 mg / ml S to NHS (N-hydroxysulfosuccinimide) and 50 mg / ml EDAC (1-et hy- 3- [3-dimethylaminopropyl] carbodiimide) was added and activated at room temperature for 20 minutes. Each activated bead was washed with PBS and purified with each purified ARS antigen of purified purity of 90¾ or higher, namely CRS, DRS, EPRS WHEP domain, HRS, WRS, AIMPl (p43), AIMP3 (pl8). It was added and reacted at room temperature for 2 hours. The beads connected to each ARS were washed twice with PBS and then added with a blocking buffer to react at room temperature for 30 minutes to block unbound residues. After washing twice with PBS, the beads were suspended in LOOul PBS and placed in a light-blocked tube. Stored. The number of bound beads was recorded by measuring with a hemocytometer.
<298> <298>
<299> <4-2> Multiplex Assay를 이용한 Cross activity측정  <299> <4-2> Cross activity measurement using multiplex assay
<3θο> 페리플라즘에서 추출된 본 발명의 항체를 검체 희석액 (PBST + 2% BSA)에  <3θο> antibody of the present invention extracted from the periplasm was subjected to sample dilution (PBST + 2% BSA)
1:400농도로 우선 회석한 다음 96 well filter 플레이트에 검체 회석액으로 순차적 으로 2배씩 희석하여 50ul 씩 분주하였고 항체가 없는 Blank well에는 검체 회석액 만올 첨가하였다. ARS가 결합된 각각의 bead는 well 당 2000 개 bead가 되도록 bead mix tube에 각각 넣고 검체 회석액을 각 well당 50 ul 씩 분주 할 수 있는 부 피만큼 넣어주었다. bead mix용액을 잘 섞어 준뒤 각 well에 분주하여 암실에서 1 시간 동안 반응 시켰다. 반응이 끝난 후 vacuum manifold를 이용하여 PBS (200 ul/well)로 3번 세척 후 50 ul Anti-(HA)-biotin 이차 항체를 첨가하여 암실에서 1 시간동안 추가로 반응시켰다. 이차 항체와 결합한 bead는 PBS로 3번 세척후 형광 표지를 위해 SA-PE (Streptavidin- Phycoerythrin)을 2 ug/ml 되게 ¾가하여 암실 에서 30분간 반응시킨 후 PBS 3번 세척과정을 거쳐 PBS lOOul에 다시 현탁하였다. 형광 표지된 bead는 Bio—Plex (Luminex) 200 장비와 Bio-Plex manager 프로그램을 이용하여 형광 세기를 측정한 결과 값을 분석하여 각각의 ARS에 대한 항체의 결합 여부를 확인하였다. The solution was first diluted at 1: 400 concentration, then diluted twice with sample diluent in 96 well filter plates, and 50ul were dispensed. Each ARS-coupled bead was placed in a bead mix tube so that 2000 beads per well were added in a volume capable of dispensing 50 ul of sample diluent per well. After mixing the bead mix solution well, each we ll was dispensed and reacted in the dark for 1 hour. After the reaction, the vacuum manifold was washed three times with PBS (200 ul / well), and then 50 ul Anti- (HA) -biotin secondary antibody was added and reacted in the dark for 1 hour. Secondary antibody-bound bead was washed three times with PBS and SA-PE (Streptavidin-Phycoerythrin) was added to ¾ 2 ug / ml for fluorescent labeling, reacted in the dark for 30 minutes, and washed three times with PBS. Suspended. Fluorescently labeled bead was analyzed by fluorescence intensity measurement using Bio-Plex (Luminex) 200 instrument and Bio-Plex manager program to confirm the binding of antibody to each ARS.
<301>  <301>
<302> 그 결과 [도 5] 내지 [도 기에서 보는 바와 같이， 본 발명의 항체는 모든 농 도에서 EPRS를 제외한 다른 ARS family protein과 반응하지 않는 것을 확인하였다. <303> 따라서 본 발명의 항체는 다른 단백질과 교차 반웅성이 없는 것을 확인하였 다.  As a result, as shown in FIG. 5 to FIG. 5, it was confirmed that the antibody of the present invention did not react with other ARS family proteins except EPRS at all concentrations. Therefore, the antibody of the present invention was confirmed that there is no cross reaction with other proteins.
<304>  <304>
<305> <실시예 5>  <305> <Example 5>
<306> 정제된 항 -EPRS scFv항체를 이용한 sandwich EL ISA pairing test  Sandwich EL ISA pairing test using purified anti-EPRS scFv antibody
<307>  <307>
<308> 본 발명의 항체가 진단용 항체로의 유용성 여부를 알아보기 위해 EPRS sandwich EL ISA pairing test를 수행하였다 ELISA 구성중 capture 항체를 본 발명 의 항체로 사용하고 detection 항체를 기존 제품의 rabbit 폴리클로날 항체로 pairing test를 해보아 EPRS 표준물질에 대한 정량 곡선이 만들어 지는 지를 확인 하였다. To determine whether the antibody of the present invention is useful as a diagnostic antibody, an EPRS sandwich EL ISA pairing test was performed. The capture antibody was used as the antibody of the present invention and the detection antibody was used as a rabbit polyclonal product. With antibody Pairing tests were performed to determine whether a quantitative curve for EPRS standards was produced.
<309> 먼저 본 발명의 정제된 항체를 coat ing buffer (0.1M sodium carbonate H  First, the purified antibody of the present invention was coated with a coat ing buffer (0.1 M sodium carbonate H).
9.0)에 회석하여 wel l 당 100 - 400ng 되게 ELISA plate에 lOOul 씩 분주한 후 상 온에서 3시간 방치하였다. PBST로 3번 세척후 3% skim mi lk를 함유한 PBST 350 ul 를 첨가하여 상온에서 1시간 blocking한후 PBST로 3번 세척하였다. 항체를 coat ing 한 plate wel l에 정제된 재조합 EPRS 표준물질을 농도별로 검체 희석액에 2배씩 희 석하여 100 ul씩 넣고 상온에서 1시간 반응시켰다. 반응후 plate는 PBST로 3번 세 척후 회석된 detect ion 항체 (4 ug/ral ) 100 ul 첨가하여 추가로 상온에서 1시간 반 응하였다. 검출을 위해 plate를 PBST 3번 세척후 HRP가 연결된 ant i-rabbit IgG를 넣어 상온에서 1시간 반응시켜 결합시켰다. HRP에 의한 발색반응을 보고자 plate를 PBST로 3번 세척후 HRP 기질인 TMB solut ion 50 ul를 첨가하여 10분동안 발색반응 을 보고 2N 황산 50 ul 첨가하여 발색반응을 멈춘후 ELISA reader를 이용하여 450 nm에서 흡광도를 측정하였다.  9.0) was dispensed 100 liters per 100 l per wel l on the ELISA plate and left for 3 hours at room temperature. After washing three times with PBST, 350 ul PBST containing 3% skim mi lk was added, and then blocked at room temperature for 1 hour, and then washed three times with PBST. The recombinant EPRS standard purified on the plate wel l coated with the antibody was diluted twice in the sample dilution for each concentration, and 100 ul was added and reacted at room temperature for 1 hour. After the reaction, the plate was washed three times with PBST and added 100 ul of the detectable ion antibody (4 ug / ral), which was further reacted at room temperature for 1 hour. For detection, the plate was washed three times with PBST, and then HRP-linked ant i-rabbit IgG was added and reacted at room temperature for 1 hour for binding. To see the color reaction by HRP, wash the plate 3 times with PBST, add 50 ul of TRP solut ion (HRP substrate) for 10 minutes, and stop the color reaction by adding 50 ul of 2N sulfuric acid. Absorbance was measured at nm.
<3io> 그 결과 도 8에서 보는 바와 같이 재조합 EPRS WHEP domain표준물질에 대해  <3io> As a result, as shown in Figure 8 for the recombinant EPRS WHEP domain standard
50 ng/ml 농도이하에서부터 해당하는 표준 직선이 그려짐을 확인 할 수 있었다. 이 로서 본 발명의 항체가 암 및 면역질환의 진단용으로 사용 될 수 있는 것을 확인하 였다.  From the concentration below 50 ng / ml, the corresponding standard straight line was drawn. It was confirmed that the antibody of the present invention can be used for the diagnosis of cancer and immune diseases.
<31 1>  <31 1>
【산업상 이용가능성】  Industrial Applicability
<312> 이상 살펴본 바와 같이， 본 발명의 항체 또는 그 단편은 인간 EPRS에 특이적 으로 결합하며， 동일한 ARS fami ly를 포함한 다른 단백질과 교차반웅성이 없어 EPRS 검출 및 억제가 가능하므로 EPRS 검출 및 EPRS가 관련된 질환인 암， 염증질 환， 면역 질환 및 섬유화증 진단 및 치료 목적으로 사용될 수 있어 산업상 이용가 능성이 높다.  As described above, the antibody or fragment thereof of the present invention specifically binds to human EPRS, and does not have cross-reaction with other proteins including the same ARS fami ly, thus enabling EPRS detection and inhibition. Can be used for the diagnosis and treatment of related diseases such as cancer, inflammatory diseases, immune diseases and fibrosis, and thus have high industrial utility.
<314>  <314>

Claims

【청구의 범위】  [Range of request]
【청구항 11  [Claim 11
i ) 서열번호 1로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)Ll , 서열번호 2로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)L2, 서열번호 3으로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)L3를 포함하는 항체 경쇄가변영역 (VL) 및 서열번호 4로 표시되는 아미노산 서 열을 포함하는 상보성 결정부위 (CDR)Hl , 서열번호 5로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)H2, 서열번호 6으로 표시되는 아미노산 서열을 포함 하는 상보성 '결정부위 (CDR)H3를 포함하는 항체 중쇄가변영역 (VH)을 포함하는 항체; i i ) 서열번호 15로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)Ll, 서열번호 16으로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)L2 , 서열번호 17로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)L3를 포함하는 항체 경쇄가변영역 (VL) 및 서열번호 18로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)Hl , 서열번호 19로 표시되는 아미노산 서열 을 포함하는 상보성 결정부위 (CDR)H2 , 서열번호 20으로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDFOH3를 포함하는 항체 중쇄가변영역 (VH)을 포함하는 항체 ; 및 i) Complementarity Determining Site (CDR) Ll comprising an amino acid sequence represented by SEQ ID NO: 1, Complementarity Determining Site (CDR) L2 comprising an amino acid sequence represented by SEQ ID NO: 2, and comprising an amino acid sequence represented by SEQ ID NO: 3 An antibody light chain variable region (VL) comprising a complementarity determining region (CDR) L3 and a complementarity determining region (CDR) Hl comprising an amino acid sequence represented by SEQ ID NO: 4, comprising an amino acid sequence represented by SEQ ID NO: 5 antibody comprising the complementarity determining region (CDR) H2, SEQ ID NO: complementarity comprising the amino acid sequence shown as 6 'determining regions (CDR) an antibody heavy chain variable region comprising a H3 (VH); ii) Complementarity Determining Site (CDR) Ll comprising an amino acid sequence represented by SEQ ID NO: 15, Complementarity Determining Site (CDR) L2 comprising an amino acid sequence represented by SEQ ID NO: 16, and an amino acid sequence represented by SEQ ID NO: 17 Complementarity comprising the antibody light chain variable region (VL) comprising the complementarity determining region (CDR) L3 and the complementarity determining region (CDR) Hl comprising the amino acid sequence represented by SEQ ID NO: 18, the amino acid sequence represented by SEQ ID NO: 19 Determination region (CDR) H2, Complementarity determination region comprising the amino acid sequence represented by SEQ ID NO: 20 (An antibody comprising an antibody heavy chain variable region (VH) comprising CDFOH3; And
i i i ) 서열번호 29로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)Ll , 서열번호 30으로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)L2, 서열번호 31로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)L3를 포함하는 항체 경쇄가변영역 (VL) 및 서열번호 32로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)Hl , 서열번호 33으로 표시되는 아미노산 서 열을 포함하는 상보성 결정부위 (CDR)H2 , 서열번호 34로 표시되는 아미노산 서열을 포함하는 상보성 결정부위 (CDR)H3를 포함하는 항체 중쇄가변영역 (VH)을 포함하는 항체;  iii) Complementarity Determining Site (CDR) Ll comprising the amino acid sequence represented by SEQ ID NO: 29, Complementarity Determining Site (CDR) L2 comprising the amino acid sequence represented by SEQ ID NO: 30, and the amino acid sequence represented by SEQ ID NO: 31 An antibody light chain variable region (VL) comprising a complementarity determining region (CDR) L3 and a complementarity determining region (CDR) Hl comprising an amino acid sequence represented by SEQ ID NO: 32, and an amino acid sequence represented by SEQ ID NO: 33 An antibody comprising an antibody heavy chain variable region (VH) comprising a complementarity determining region (CDR) H2, a complementarity determining region (CDR) H3 comprising the amino acid sequence represented by SEQ ID NO: 34;
로 이루어지는 군에서 선택된 인간 EPRS에 결합하는 항체 또는 그 단편.  An antibody or fragment thereof that binds to human EPRS selected from the group consisting of:
【청구항 2】 [Claim 2]
제 1항에 있어서， 상기 항체는  The method of claim 1, wherein the antibody is
i ) 경쇄가변영역으로 서열번호 13의 132 번째 내지 241 번째 아미노산 서열 을 포함하며， 중쇄가변영역으로 서열번호 13의 1 번째 내지 116 번째 아미노산 서 열을 포함하는 항체； i i ) 경쇄가변영역으로 서열번호 27의 132 번째 내지 241 번째 아미노산 서열 을 포함하며， 중쇄가변영역으로 서열번호 27의 1 번째 내지 116 번째 아미노산 서 열을 포함하는 항체 ; 및 i) an antibody comprising the 132th to 241th amino acid sequence of SEQ ID NO: 13 as a light chain variable region and comprising the 1st to 116th amino acid sequence of SEQ ID NO: 13 as a heavy chain variable region; ii) an antibody comprising the 132 th to 241 th amino acid sequences of SEQ ID NO: 27 as a light chain variable region and the 1 th to 116 th amino acid sequences of SEQ ID NO: 27 as a heavy chain variable region; And
i i i ) 경쇄가변영역으로 서열번호 41의 132 번째 내지 241 번째 아미노산 서 열을 포함하며， 중쇄가변영역으로 서열번호 41의 1 번째 내지 116 번째 아미노산 서열을 포함하는 항체;  i i i) an antibody comprising the 132 th to 241 th amino acid sequences of SEQ ID NO: 41 as the light chain variable region, and comprising the 1 th to 116 th amino acid sequences of SEQ ID NO: 41 as the heavy chain variable region;
로 이루어진 군에서 선택된 항체인 것을 특징으로 하는 인간 EPRS에 결합하 는 항체 또는 그 단편 .  An antibody or fragment thereof that binds to human EPRS, which is an antibody selected from the group consisting of:
【청구항 3】 [Claim 3]
제 1항에 있어서, 상기 항체는 서열번호 13， 서열번호 27, 서열번호 41로 이 루어진 군에서 선택된 서열번호로 표시되는 아미노산서열을 포함하는 것을 특징으 로 하는 항체 또는 그 단편 .  The antibody or fragment thereof of claim 1, wherein the antibody comprises an amino acid sequence represented by SEQ ID NO: 13 selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 27, SEQ ID NO: 41.
【청구항 4】 [Claim 4]
제 1항에 있어서 , 상기 단편은 디아바디， Fab, Fab' , F(ab)2 > F(ab' )2, Fv 및 scFv로 이루어진 군에서 선택되는 단편인 것을 특징으로 하는 항체 또는 그 단편. The antibody or fragment thereof according to claim 1, wherein the fragment is a fragment selected from the group consisting of diabodies, Fab, Fab ', F (ab) 2> F (ab') 2 , Fv, and scFv.
【청구항 5] [Claim 5]
제 1항의 항체 또는 그 단편을 암호화 하는 폴리뉴클레오티드.  A polynucleotide encoding the antibody of claim 1 or a fragment thereof.
【청구항 6】 [Claim 6]
제 5항에 있어서, 상기 폴리뉴클레오티드는  The method of claim 5, wherein the polynucleotide
i ) 서열번호 7 내지 12로 표시되는 폴리뉴클레오티드;  i) a polynucleotide represented by SEQ ID NOs: 7-12;
i i ) 서열번호 21 내지 26으로 표시되는 폴리뉴클레오티드; 및  i i) a polynucleotide represented by SEQ ID NOs: 21 to 26; And
i i i ) 서열번호 35 내지 40으로 표시되는 폴리뉴클레오티드;  i i i) a polynucleotide represented by SEQ ID NOs: 35 to 40;
로 이루어진 군에서 선택된 폴리뉴클레오티드를 포함하는 것을 특징으로 하 는 폴리뉴클레오티드.  Polynucleotide comprising a polynucleotide selected from the group consisting of.
【청구항 7] [Claim 7]
제 5항의 폴리뉴클레오티드를 포함하는 백터. A vector comprising the polynucleotide of claim 5.
【청구항 8] [Claim 8]
제 7항의 백터를 포함하는 세포.  A cell comprising the vector of claim 7.
【청구항 9] [Claim 9]
제 8항의 세포를 폴리뉴클레오티드가 발현되는 조건하에서 배양하여， 경쇄 및 중쇄가변영역을 포함하는 폴리펩타이드를 생산하는 단계 및 상기 세포 또는 이를 배양한 배양 배지로부터 상기 폴리펩타이드를 회수하는 단계를 포함하는 인간 EPRS 에 결합하는 항체 또는 그 단편의 생산방법 .  A human comprising culturing the cell of claim 8 under conditions in which the polynucleotide is expressed, producing a polypeptide comprising a light chain and a heavy chain variable region, and recovering the polypeptide from the cell or the culture medium in which the cell is cultured. Method for producing antibody or fragment thereof that binds EPRS.
【청구항 10] [Claim 10]
제 1항의 항체 또는 그 단편을 시료와 접촉시키는 단계 및 상기 항체 또는 그 단편을 검출하는 단계를 포함하는 EPRS특이적 검출 방법.  The method of claim 1 comprising contacting the antibody or fragment thereof with a sample and detecting the antibody or fragment thereof.
【청구항 11] [Claim 11]
제 1항의 항체 또는 그 단편을 유효성분으로 포함하는 암 예방 또는 치료용 약학적 조성물.  A pharmaceutical composition for preventing or treating cancer, comprising the antibody of claim 1 or a fragment thereof as an active ingredient.
【청구항 12] [Claim 12]
제 1항의 항체 또는 그 단편을 유효성분으로 포함하는 암 진단용 조성물.  Cancer diagnostic composition comprising the antibody of claim 1 or a fragment thereof as an active ingredient.
【청구항 13] [Claim 13]
제 1항의 항체 또는 그 단편을 유효성분으로 포함하는 면역질환 예방 또는 치 료용 약학적 조성물.  A pharmaceutical composition for preventing or treating immune diseases, comprising the antibody of claim 1 or a fragment thereof as an active ingredient.
【청구항 14] [Claim 14]
제 1항의 항체 또는 그 단편을 유효성분으로 포함하는 면역질환 진단용 조성 물.  A composition for diagnosing autoimmune diseases comprising the antibody of claim 1 or a fragment thereof as an active ingredient.
【청구항 15] [Claim 15]
제 1항의 항체 또는 그 단편을 유효성분으로 포함하는 염증질환 예방 또는 치 료용 약학적 조성물. Inflammatory disease preventing or treating pharmaceutical composition comprising the antibody of claim 1 or a fragment thereof as an active ingredient.
【청구항 16] [Claim 16]
제 1항의 항체 또는 그 단편을 유효성분으로 포함하는 염증질환 진단용 조성 물  Inflammatory disease diagnostic composition comprising the antibody or fragment thereof of claim 1 as an active ingredient
【청구항 17】 [Claim 17]
제 1항의 항체 또는 그 단편을 유효성분으로 포함하는 섬유화증 예방 또는 치 료용 약학적 조성물.  A pharmaceutical composition for preventing or treating fibrosis, comprising the antibody of claim 1 or a fragment thereof as an active ingredient.
【청구항 18] [Claim 18]
거 U항의 항체 또는 그 단편을 유효성분으로 포함하는 섬유화증 진단용 조성 물.  A composition for diagnosing fibrosis comprising a U-antibody or a fragment thereof as an active ingredient.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110891977A (en) * 2017-05-11 2020-03-17 温口特 Monoclonal antibody specifically binding to MRS

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100961392B1 (en) * 2008-04-15 2010-06-07 이화여자대학교 산학협력단 Production method of antibody phage display library, the antibody phage display library produced thereby and the phagemid vector comprising said antibody phage display library
KR20110046521A (en) * 2008-08-18 2011-05-04 재단법인서울대학교산학협력재단 How to regulate cancer metastasis or cancer cell migration by regulating intracellular levels of lysyl TNNA synthase
KR20130032286A (en) * 2011-09-22 2013-04-01 재단법인 의약바이오컨버젼스연구단 Novel use of leucyl trna synthetase
CN104297486A (en) * 2013-07-17 2015-01-21 张曼 Application of urine glutamoyl-prolyl tRNA synthetic enzyme

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100961392B1 (en) * 2008-04-15 2010-06-07 이화여자대학교 산학협력단 Production method of antibody phage display library, the antibody phage display library produced thereby and the phagemid vector comprising said antibody phage display library
KR20110046521A (en) * 2008-08-18 2011-05-04 재단법인서울대학교산학협력재단 How to regulate cancer metastasis or cancer cell migration by regulating intracellular levels of lysyl TNNA synthase
KR20130032286A (en) * 2011-09-22 2013-04-01 재단법인 의약바이오컨버젼스연구단 Novel use of leucyl trna synthetase
CN104297486A (en) * 2013-07-17 2015-01-21 张曼 Application of urine glutamoyl-prolyl tRNA synthetic enzyme

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MIRANDO, ADAM C. ET AL.: "Regulation of Angiogenesis by Aminoacyl-tRNA Synthetases", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 15, 2014, pages 23725 - 23748 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110891977A (en) * 2017-05-11 2020-03-17 温口特 Monoclonal antibody specifically binding to MRS
CN110891977B (en) * 2017-05-11 2023-10-27 温口特 Monoclonal antibodies that specifically bind MRS

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