WO2016122250A1 - Anticorps monoclonal anti-eprs et son utilisation - Google Patents

Anticorps monoclonal anti-eprs et son utilisation 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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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/fr

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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.

Abstract

La présente invention concerne un anticorps anti-EPRS (glycyl-ARNt synthétase) qui se lie sélectivement à EPRS et, plus particulièrement, un anticorps qui se lie à l'EPRS humaine ou un fragment correspondant, un procédé de production correspondant et une composition pharmaceutique pour le diagnostic et le traitement de cancers, de maladies inflammatoires, de troubles immuns et de fibrose le comprenant. L'anticorps ou un fragment correspondant de la présente invention peut être utilisé dans le but de détecter l'EPRS et de diagnostiquer et de traiter des cancers, des maladies inflammatoires, des troubles immuns et la fibrose, qui sont des maladies liées à l'EPRS, étant donné que l'anticorps ou un fragment correspondant se lie spécifiquement à l'EPRS humaine et ne présente pas de réactivité croisée avec d'autres protéines, comprenant la même famille des ARS, ce qui permet ainsi la détection et l'inhibition de l'EPRS.
PCT/KR2016/001004 2015-01-29 2016-01-29 Anticorps monoclonal anti-eprs et son utilisation WO2016122250A1 (fr)

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KR1020150014616A KR20160093502A (ko) 2015-01-29 2015-01-29 항 eprs 모노클로날 항체 및 이의 용도
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