WO2019004550A1 - Antibody specifically binding to mic-1 protein, and use thereof - Google Patents

Antibody specifically binding to mic-1 protein, and use thereof Download PDF

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WO2019004550A1
WO2019004550A1 PCT/KR2018/001171 KR2018001171W WO2019004550A1 WO 2019004550 A1 WO2019004550 A1 WO 2019004550A1 KR 2018001171 W KR2018001171 W KR 2018001171W WO 2019004550 A1 WO2019004550 A1 WO 2019004550A1
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cancer
antibody
mic
seq
light chain
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PCT/KR2018/001171
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French (fr)
Korean (ko)
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이한수
이재섭
성하영
손원준
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강원대학교 산학협력단
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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • 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
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • 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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines

Definitions

  • the present invention relates to an antibody that specifically binds to MIC-1 (macrophage inhibitory cytokine 1) protein and uses thereof, and more particularly to a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, and a heavy chain CDR3 of SEQ ID NO: A heavy chain variable region comprising a heavy chain variable region; And a light chain variable region comprising the light chain CDR1 of SEQ ID NO: 4, the light chain CDR2 of SEQ ID NO: 5, and the light chain CDR3 of SEQ ID NO: 6, a polynucleotide encoding the antibody, A polynucleotide-containing expression vector, a transformant into which the expression vector is introduced, a pharmaceutical composition for preventing or treating cancer comprising the antibody, a method for preventing or treating cancer using the antibody, and a method for diagnosing cancer including the antibody
  • a cancer diagnostic kit comprising the composition, a diagnostic method for cancer using the antibody, and a method for screening a substance for
  • Biopharmaceuticals which consist of recombinant drugs (proteins), antibodies, vaccines, cell treatments, and gene therapy, have the advantage of less side effects and excellent effects for certain diseases than chemical synthetic drugs. Accordingly, the market size is rapidly expanding, and the therapeutic monoclonal antibody market is growing at an annual average growth rate of 15.5%. In addition, the monoclonal antibody has been greatly improved in stability and efficacy of the drug due to the technological innovation, the indication is extended due to the discovery of a new target molecule, and the existing drug is resistant to a specific disease The market size is expected to increase steadily.
  • cancer cancer
  • angiogenesis monoclonal antibodies that bind to Anexelekto have been developed (Korean Patent Publication No. 10-2010-0097688), Genentech, and Novartis have developed anti-angiogenic and anti-cancer antibodies, VEGF humanized monoclonal antibodies Avastin, Lucentis, and the like.
  • avastin is very expensive and has a side effect of gastrointestinal bleeding.
  • cancer treatment targeting angiogenesis inhibition has a remarkable therapeutic and diagnostic effect on various kinds of cancer due to limited kinds of cancer that shows effects The development of antibodies is still needed.
  • MIC-1 Macrophage inhibitory cytokine-1
  • MIC-1 protein is expressed in most tissues, but its expression level is rapidly increased in pathological conditions such as tissue damage or inflammation.
  • pathological conditions such as tissue damage or inflammation.
  • MIC-1 protein is overexpressed in various cancers such as breast cancer, colon cancer, prostate cancer, pancreatic cancer, stomach cancer and the like, Cancer Res., 2003, 9: 2642-50; Koopmann J, Clin. Cancer Res. 2006, 12: 442-6).
  • the present inventors have made intensive efforts to develop an antibody that specifically binds to MIC-1 protein and exhibits excellent therapeutic and diagnostic effects against various cancers by inhibiting angiogenesis essential for cancer growth and development.
  • -MIC-1 monoclonal antibody exhibit angiogenesis inhibitory activity and have excellent anticancer effects against various cancers such as skin cancer, colon cancer, breast cancer and prostate cancer.
  • One object of the present invention is to provide a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 2, and the heavy chain CDR3 of SEQ ID NO: 3; And a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 4, a light chain CDR2 of SEQ ID NO: 5, and a light chain CDR3 of SEQ ID NO: 6, and an antibody that binds to a macrophage inhibitory cytokine 1 .
  • Another object of the present invention is to provide a diagnostic method for cancer, comprising the step of detecting the MIC-1 protein through an antigen-antibody reaction in a separate biological sample of a subject suspected of having cancer using the antibody .
  • Another object of the present invention is to provide a method for treating cancer, comprising the steps of: (a) treating a candidate cancer cell with a candidate substance for preventing or treating cancer; (b) measuring the level of MIC-1 protein using the antibody in cancer cells treated with the candidate substance; And (c) when the MIC-1 protein level of the step (b) is lower than that of the cancer cells not treated with the candidate substance, the candidate substance treated in the step (a) And a method for screening a preventive or therapeutic substance for cancer.
  • the antibody of the present invention binds to MIC-1 protein with high specificity, inhibits angiogenesis of endothelial cells, inhibits growth and development of cancer cells in vivo, and inhibits angiogenesis inside / outside of cancer tissue , And cancer in which MIC-1 protein is overexpressed.
  • anti-MIC-1 mouse antibody an anti-MIC-1 mouse IgG monoclonal antibody prepared according to an embodiment of the present invention
  • graphs where A is the tube formation assay and B is the aorta ring sprouting assay result.
  • mAb means anti-MIC-1 mouse antibody.
  • A is a skin cancer tumor present in a living body
  • B is a skin cancer tumor isolated from a mouse body
  • C is a volume change of a skin cancer tumor according to the administration of the antibody
  • D is a skin cancer tumor weight
  • E is the immunofluorescent staining result of anti-CD31 antibody against skin cancer tumor tissue sections.
  • mAb means anti-MIC-1 mouse antibody.
  • A is a colorectal cancer tumor present in a living body of a mouse
  • B is a colon cancer tumor isolated from a mouse body
  • C is a volume change of colon cancer tumor upon administration of the antibody
  • D is a colon cancer
  • E is the immunofluorescent staining result of anti-CD31 antibody against colon cancer tumor tissue sections.
  • mAb means anti-MIC-1 mouse antibody.
  • B is the volume change of the breast cancer tumor according to the administration of the antibody
  • C is the weight change of the breast cancer tumor according to the administration of the antibody
  • D is the breast cancer tumor external and internal section IB4 (Isolectin / IB4) antibody against human immunodeficiency virus.
  • mAb # 4 means anti-MIC-1 mouse antibody.
  • FIG. 5 is an image and a graph showing the growth inhibitory activity of the anti-MIC-1 mouse antibody against prostate cancer.
  • B is the volume change of the prostate cancer tumor according to the administration of the antibody
  • C is the weight change of the prostate cancer tumor according to administration of the antibody
  • D is the tumor volume of the breast cancer tumor section IB4 < / RTI > (Isolectin / IB4) antibody.
  • mAb # 4 means anti-MIC-1 mouse antibody.
  • Figure 6 shows clones from 1 to 59 expressing anti-MIC-1 humanized IgG monoclonal antibody (hereinafter referred to as "anti-MIC-1 humanized antibody "), prepared according to one embodiment of the present invention. As well as ELISA and Western blot results. ≪ Desc / Clms Page number 10 >
  • FIG. 7 is an image and a graph showing the purity of the anti-MIC-1 humanized antibody and the binding awareness to the MIC-1 protein, and relates to Coomasie blue staining, ELISA and Western blot results.
  • FIG. 8 is an image and a graph showing the angiogenesis inhibitory activity of the anti-MIC-1 humanized antibody, which relates to the results of tube formation assay using endothelial cells.
  • A shows increased angiogenesis by MIC-1 protein
  • B is treated with A549 lung cancer cells, HCT116 colorectal cancer cells, or LNCaP prostate cancer cell culture supernatant.
  • HuAb means anti-MIC-1 humanized antibody.
  • FIG. 9 is an image and a graph showing the angiogenesis inhibitory activity of various types of anti-MIC-1 antibodies, and relates to the results of tube formation assay using endothelial cells.
  • mAb means mouse whole antibody
  • HuAb means humanized whole antibody
  • scFV means fragment antibody.
  • FIG. 10 is an image and a graph showing the anti-angiogenic activity of the anti-MIC-1 humanized antibody, showing aorta ring sprouting, It is about the assay results.
  • A shows an anti-MIC-1 humanized antibody
  • B shows an angiogenesis-decreasing effect by various types of anti-MIC-1 antibody treatment.
  • mAb means mouse whole antibody
  • HuAb means humanized whole antibody
  • scFV means fragment antibody.
  • FIG. 11 is an image and a graph showing the angiogenesis inhibitory activity of the anti-MIC-1 humanized antibody, and it relates to an in vivo angiogenesis assay result using a chick chorioallantoic membrane (CAM).
  • HuAb means anti-MIC-1 humanized antibody.
  • FIG. 12 is an image and a graph showing the growth inhibitory activity of the anti-MIC-1 humanized antibody against a skin melanoma.
  • A is a skin melanoma tumor present in a mouse body
  • B is a skin melanoma tumor isolated from a mouse body
  • C is a volume change of skin melanoma tumor according to the administration of the antibody
  • D is a dose of the antibody
  • E is the result of immunofluorescent staining of anti-CD31 antibody against cutaneous melanoma tumor tissue sections
  • F is the result of immunofluorescence staining of anti-IB4 antibody against cutaneous melanoma tumor tissue sections .
  • HuAb means anti-MIC-1 humanized antibody.
  • FIG. 13 is a graph showing the anti-MIC-1 humanized antibody's growth inhibitory activity on skin melanoma, wherein A is a graph showing the concentration of plasma MIC-1 protein upon administration of the antibody, and B is a graph showing tumor size and plasma This is a graph showing the correlation of MIC-1 protein concentration.
  • A is a colorectal cancer tumor present in a living body of a mouse
  • B is a colon cancer tumor isolated from a mouse body
  • C is a volume change of colon cancer tumor upon administration of the antibody
  • D is a colon cancer
  • E is the result of immunofluorescent staining of the anti-CD31 antibody against the colon cancer tumor tissue section
  • F is the immunofluorescent staining result of the anti-IB4 antibody against the colon cancer tumor tissue section.
  • HuAb means anti-MIC-1 humanized antibody.
  • A is the level of pimonidazole detected in the colorectal tumor tissue section
  • B is the immunofluorescent staining result of the anti-Ki67 antibody against the colorectal tumor tissue section
  • C is the expression level in CoCl 2 -treated colon cancer cells
  • RTI ID 0.0 > MIC-1 < / RTI > At this time, HuAb means anti-MIC-1 humanized antibody.
  • FIG. 16 is a graph showing the anti-MIC-1 humanized antibody's growth inhibitory activity against colon cancer, wherein A is a graph showing the concentration of plasma MIC-1 protein upon administration of the antibody, and B is a graph showing tumor size and plasma MIC -1 protein concentration in the blood.
  • A is a breast cancer tumor present in the body of a mouse
  • B is a breast cancer tumor isolated from a mouse body
  • C is a volume change of a breast cancer tumor according to the administration of the antibody
  • D is weight of a breast cancer tumor
  • E is the result of immunofluorescence staining of anti-CD31 antibody against breast cancer tumor tissue sections
  • F is the result of immunofluorescence staining of anti-IB4 antibody against breast cancer tumor tissue sections.
  • HuAb means anti-MIC-1 humanized antibody.
  • FIG. 18 is an image and a graph showing anti-MIC-1 humanized antibody's cell proliferation inhibitory activity against breast cancer. Ki67 < / RTI > antibody against breast cancer tumor tissue sections. At this time, HuAb means anti-MIC-1 humanized antibody.
  • A represents a prostate cancer tumor present in a living body
  • B represents a prostate cancer tumor isolated from a mouse body
  • C represents a volume change of the prostate cancer tumor by the administration of the antibody
  • D represents a prostate cancer tumor tissue slice Results of immunofluorescent staining of anti-CD31 antibodies
  • E relates to immunofluorescence staining results of anti-IB4 (Isolectin / IB4) antibodies against prostate cancer tumor tissue sections.
  • HuAb means anti-MIC-1 humanized antibody.
  • A is the level of the sol coat is detected in prostate tumor tissue sections, wherein B is the result of immunofluorescence staining -Ki67 antibodies to prostate tumor tissue sections, and C is expressed in prostate cancer cells with CoCl 2 treatment MIC- 1 < / RTI > protein.
  • HuAb means anti-MIC-1 humanized antibody.
  • A is a pancreatic cancer tumor present in the mouse
  • B is a pancreatic cancer tumor isolated from a mouse body
  • C is a volume change of the pancreatic cancer tumor by administration of the antibody
  • D is an anti-CD31 antibody
  • E is the result of immunofluorescence staining of anti-IB4 (Isolectin / IB4) antibody against pancreatic cancer tumor tissue sections.
  • HuAb means anti-MIC-1 humanized antibody.
  • FIG. 22 is an image and a graph showing anti-MIC-1 humanized antibodies against hypoxic state and cell proliferation inhibitory activity against pancreatic cancer. Ki67 < / RTI > antibody against pancreatic cancer tumor tissue sections. At this time, HuAb means anti-MIC-1 humanized antibody.
  • HuAb means anti-MIC-1 humanized antibody.
  • FIG 24 is a graph showing the growth inhibitory activity of the anti-MIC-1 humanized antibody against gastric cancer, which relates to the volume change of gastric cancer tumor upon administration of the antibody.
  • HuAb means anti-MIC-1 humanized antibody.
  • one embodiment of the present invention is directed to a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 2, and the heavy chain CDR3 of SEQ ID NO: 3; And a light chain variable region comprising light chain CDR1 of SEQ ID NO: 4, light chain CDR2 of SEQ ID NO: 5, and light chain CDR3 of SEQ ID NO: 6.
  • MIC-1 macrophage inhibitory cytokine 1 protein
  • GDF-15 protein named GDF-15, PTGF- ⁇ , PLAB, PDF, NAG-1 or PL74
  • MIC-1 protein refers to a protein belonging to the TGF-beta superfamily that regulates inflammation and apoptosis.
  • the level of expression is markedly elevated under pathological conditions such as tissue damage or inflammation and is overexpressed in various cancers such as breast cancer, colon cancer, prostate cancer, pancreatic cancer, stomach cancer and the like, And is detected at a high level.
  • MIC-1 protein can be a target for diagnosis and treatment of cancer (Brown DA, Clin. Cancer Res., 2003, 9: 2642-50; Koopmann J, Clin. Cancer Res. 2006, 12: 442-6).
  • the MIC-1 protein may be human-derived, and its amino acid sequence and the like are known in databases such as NCBI (Accession number: NP_004855, etc.).
  • mouse antibodies and humanized antibodies that specifically bind to the MIC-1 protein have been developed. They not only inhibit angiogenesis induced by the MIC-1 protein, but also inhibit angiogenesis in a wide variety of skin cancer, colon cancer, breast cancer, It is possible to inhibit the growth and development of cancer cells.
  • antibody of the present invention means a protein molecule that acts as a receptor that specifically recognizes an antigen, including an immunoglobulin molecule that is immunologically reactive with a specific antigen.
  • the antibodies of the present invention include both polyclonal antibodies, monoclonal antibodies, whole antibodies and antibody fragments and also include chimeric antibodies and bivalent or bispecific molecules (e. G., Bispecific antibodies) , Triabodies, and tetrabodies. Also included are single chain antibodies, scabs, derivatives of antibody constant regions, and artificial antibodies based on protein scaffolds, which have a binding function for FcRn (neonatal Fc receptor).
  • the whole antibody is a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to a heavy chain by a disulfide bond.
  • the whole antibody includes IgA, IgD, IgE, IgM and IgG, and IgG is a subtype and includes IgG1, IgG2, IgG3 and IgG4.
  • the antibody fragment refers to a fragment having an antigen-binding function and includes Fc, Fd, Fab, Fab ', Fv, and F (ab') 2 .
  • the Fc refers to the tail portion of an antibody that interacts with a cell surface receptor called an Fc receptor.
  • Fd means a heavy chain portion contained in a Fab fragment
  • F (ab ') 2 means a fragment containing Fd, Fab, Fab' and Fv.
  • the Fab has one antigen-binding site in a structure having a variable region of a light chain and a heavy chain, a constant region of a light chain, and a first constant region (CH1 domain) of a heavy chain.
  • the F (ab ') 2 antibody is produced when the cysteine residue of the hinge region of the Fab' forms a disulfide bond.
  • Fv (variable fragment) refers to the smallest antibody fragment that has only a heavy chain variable region and a light chain variable region.
  • the double disulfide Fv (dsFv) is linked by a disulfide bond to a light chain variable region and a light chain variable region.
  • a short chain Fv is generally linked to a variable region of a heavy chain and a variable region of a light chain through a peptide linker by a covalent bond.
  • an antibody fragment can be obtained using a protein hydrolyzing enzyme (for example, an F (ab ') 2 fragment can be obtained by cutting a whole antibody into papain and obtaining a Fab and digesting with pepsin) Can be produced through recombinant DNA technology.
  • immunoglobulins have a heavy chain and a light chain, and each heavy and light chain includes a constant region and a variable region.
  • the variable region of the light chain and the heavy chain includes three complementarity determining regions (hereinafter referred to as " CDRs ”) and four framework regions (referred to as " FRs ”) called complementarity determining regions .
  • the CDRs of each chain primarily serve to bind to the epitope of the antigen, typically starting from the N-terminus and sequentially named CDR1, CDR2, CDR3.
  • the FR of each chain may be named FR1, FR2, FR3, FR4 sequentially starting from the N-terminus.
  • variable region of the heavy chain can be named 'VH' and the variable region of the light chain can be named 'LH'
  • CDRs of the heavy chain can be named 'VH-CDR1', 'VH-CDR2', and 'VH-CDR3' VL-CDR1 ',' VL-CDR2 ', and' VL-CDR3 ', respectively
  • the FR of the heavy chain are VH-FR1, VH-FR2, VH- -FR4 '
  • the FR of the light chain may be named' VL-FR1 ',' VL-FR2 ',' VL-FR3 ', and' VL-FR4 '.
  • antibody specifically binding to macrophage inhibitory cytokine 1 (MIC-1) protein means an antibody capable of binding to MIC-1 protein and inhibiting the activity of MIC-1 protein.
  • the antibody specifically binding to the MIC-1 protein may be a mouse antibody or a humanized antibody.
  • mouse antibody &quot means a molecule derived from a mouse immunoglobulin, wherein all of the amino acid sequences constituting the antibody are composed of an amino acid sequence derived from a mouse.
  • &quot humanized antibody " means a molecule derived from mouse and human immunoglobulin, wherein the CDR is derived from a mouse, and the region excluding it is composed of a human-derived amino acid sequence.
  • the antibody comprises a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 2, and the heavy chain CDR3 of SEQ ID NO: 3; And a light chain variable region comprising light chain CDR1 of SEQ ID NO: 4, light chain CDR2 of SEQ ID NO: 5, and light chain CDR3 of SEQ ID NO: 6.
  • the heavy chain variable region of said antibody comprises heavy chain FR2 of SEQ ID NO: 7 or 17, heavy chain FR2 of SEQ ID NO: 8 or 18, heavy chain FR3 of SEQ ID NO: 9 or 19 and heavy chain FR4 of SEQ ID NO: 10 or 20;
  • the light chain variable region of the antibody may comprise light chain FR1 of SEQ ID NO: 11 or 21, light chain FR2 of SEQ ID NO: 12 or 22, light chain FR3 of SEQ ID NO: 13 or 23 and light chain FR4 of SEQ ID NO: 14 or 24.
  • the heavy chain variable region of the antibody comprises the heavy chain FR1 of SEQ ID NO: 7, the heavy chain FR2 of SEQ ID NO: 8, the heavy chain FR3 of SEQ ID NO: 9, and the heavy chain FR4 of SEQ ID NO: 10;
  • the light chain variable region of the antibody may comprise light chain FR1 of SEQ ID NO: 11, light chain FR2 of SEQ ID NO: 12, light chain FR3 of SEQ ID NO: 13, and light chain FR4 of SEQ ID NO: 14,
  • the heavy chain variable region of the antibody comprises heavy chain FR1 of SEQ ID NO: 17, heavy chain FR2 of SEQ ID NO: 18, heavy chain FR3 of SEQ ID NO: 19 and heavy chain FR4 of SEQ ID NO: 20;
  • the light chain variable region of the antibody may include but are not limited to a light chain FR1 of SEQ ID NO: 21, a light chain FR2 of SEQ ID NO: 22, a light chain FR3 of SEQ ID NO: 23, and a light chain FR4 of SEQ ID NO:
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 15 or 25; And the light chain variable region may comprise the amino acid sequence of SEQ ID NO: 16 or 26.
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 15; and the light chain variable region may comprise the amino acid sequence of SEQ ID NO: 16,
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 25;
  • the light chain variable region may comprise the amino acid sequence of SEQ ID NO: 26, but are not limited thereto.
  • the heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 2, and the heavy chain CDR3 of SEQ ID NO: 3; And a light chain variable region comprising light chain CDR1 of SEQ ID NO: 4, light chain CDR2 of SEQ ID NO: 5, and light chain CDR3 of SEQ ID NO: 6, wherein the heavy chain variable region of the antibody comprises heavy chain FR1 of SEQ ID NO: 7, SEQ ID NO: 8 A heavy chain FR2 of SEQ ID NO: 9, and a heavy chain FR4 of SEQ ID NO: 10; And wherein the light chain variable region comprises the light chain FR1 of SEQ ID NO: 11, the light chain FR2 of SEQ ID NO: 12, the light chain FR3 of SEQ ID NO: 13 and the light chain FR4 of SEQ ID NO: 14, ≪ / RTI > And the light chain variable region may comprise the amino acid sequence of SEQ ID NO: 16,
  • the antibody comprises a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 2, and the heavy chain CDR3 of SEQ ID NO: 3; And a light chain variable region comprising light chain CDR1 of SEQ ID NO: 4, light chain CDR2 of SEQ ID NO: 5, and light chain CDR3 of SEQ ID NO: 6, wherein the heavy chain variable region comprises heavy chain FR1 of SEQ ID NO: 17, SEQ ID NO: 18 A heavy chain FR2 of SEQ ID NO: 19, and a heavy chain FR4 of SEQ ID NO: 20; And wherein the light chain variable region comprises the light chain FR1 of SEQ ID NO: 21, the light chain FR2 of SEQ ID NO: 22, the light chain FR3 of SEQ ID NO: 23, and the light chain FR4 of SEQ ID NO: 24, ≪ / RTI > And the light chain variable region may comprise the amino acid sequence of SEQ ID NO: 26, but are not
  • sequences used in the present invention are interpreted to include sequences showing substantial identity with the sequences listed in the sequence listing, considering mutations having biologically equivalent activity.
  • the term " substantial identity " means aligning the sequence of the present invention to any other sequence as much as possible, and when the aligned sequence is analyzed using an algorithm commonly used in the art, 80% homology, most specifically 90% homology.
  • the anti-MIC-1 mouse IgG monoclonal antibody that binds to the MIC-1 protein significantly reduces the angiogenic vasculature of the endothelial cells produced by the MIC-1 protein (Figure 1) Skin cancer, colon cancer, breast cancer or prostate cancer, and inhibits angiogenesis inside / outside these tissues (Figs. 2 to 5).
  • Figure 1 Skin cancer, colon cancer, breast cancer or prostate cancer, and inhibits angiogenesis inside / outside these tissues.
  • an anti-MIC-1 humanized IgG monoclonal antibody that specifically binds to the MIC-1 protein was prepared (FIGS.
  • the antibody that binds to the MIC-1 protein of the present invention can be usefully used for the diagnosis or treatment of diseases in which the MIC-1 protein is overexpressed, for example, cancer.
  • Another embodiment provides a polynucleotide encoding the antibody, an expression vector comprising the polynucleotide, and a transformant into which the expression vector is introduced.
  • the expression vector comprising the polynucleotide encoding the antibody is not particularly limited, but may be mammalian cells (e.g., human, monkey, rabbit, rat, hamster, mouse cells, etc.), plant cells, yeast cells , A vector capable of replicating and / or expressing the polynucleotide in an eukaryotic or prokaryotic cell comprising an insect cell or a bacterial cell (e. G., Escherichia coli, etc.), preferably the expression of the nucleotide in the host cell And may be a vector comprising at least one selectable marker operably linked to a suitable promoter so as to be able to be expressed.
  • the polynucleotide may be introduced into a phage, a plasmid, a cosmid, a mini-chromosome, a virus, or a retrovirus vector.
  • the expression vector comprising the polynucleotide encoding the antibody may be an expression vector comprising an expression vector containing a polynucleotide encoding the heavy or light chain of the antibody or a polynucleotide encoding the heavy or light chain, respectively.
  • the transformant into which the expression vector is introduced is not particularly limited, but bacterial cells such as Escherichia coli, Streptomyces and Salmonella typhimurium transformed with the expression vector introduced therein; Yeast cells; Fungal cells such as Pichia pastoris; Insect cells such as Drosophila and Spodoptera Sf9 cells; CHO (Chinese hamster ovary cells), SP2 / 0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), 293T, Bowmanella cells, HT-1080, BHK Animal hamster kidney cells, HEK (human embryonic kidney cells), and PERC.6 (human retinal cells); Or plant cells.
  • bacterial cells such as Escherichia coli, Streptomyces and Salmonella typhimurium transformed with the expression vector introduced therein
  • Yeast cells Fungal cells such as Pichia pastoris
  • Insect cells such as Drosophil
  • introduction means a method of delivering a vector comprising a polynucleotide encoding the antibody to a host cell. Such introduction may be accomplished by methods such as calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposomal fusion, lipofectamine and protoplast fusion Can be carried out by various methods known in the art. Transfection also means that an object is transferred into a cell using viral particles by means of infection.
  • vectors can be introduced into host cells by gene bombardment or the like. Introduction in the present invention can be used in combination with transformation or transfection.
  • Another embodiment provides a composition comprising the antibody.
  • the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the antibody.
  • the antibody shows very high specificity to the MIC-1 protein overexpressed in the cancer and significantly reduces the angiogenic vasculature of the endothelial cells produced by the MIC-1 protein, and is useful for the treatment of skin cancer, colon cancer, breast cancer, prostate cancer, pancreatic cancer, Can inhibit the growth of liver cancer or stomach cancer, inhibit angiogenesis inside or outside of these tissues, and thus can exhibit a preventive or therapeutic effect of a disease overexpressing MIC-1 protein, for example, cancer.
  • the description of the antibody is as described above.
  • cancer of the present invention refers to a mass that is abnormally grown by autonomous overgrowth of body tissue, and includes any kind of cancer that can be prevented or treated by the antibody of the present invention, Cancer of the uterus, endometrial cancer, endometrial cancer, endometrial cancer, endometrial carcinoma, endometrial carcinoma, endometrial carcinoma, endometrial carcinoma, endometrial carcinoma, endometrial carcinoma, endometrial carcinoma, endometrial cancer, Cancer of the bladder, kidney, liver, bone, connective tissue, brain, thyroid, leukemia, Hodgkin's disease, lymphoma or multiple myeloma.
  • prevention of cancer means all actions that inhibit or delay the onset of cancer by administration of the composition.
  • treatment of cancer means all the actions of improving or ameliorating symptoms of cancer by the administration of the composition.
  • Another embodiment provides a composition comprising the antibody.
  • a pharmaceutical composition for inhibiting angiogenesis comprising the antibody.
  • Diseases, diseases or conditions that can be prevented or treated by the composition of the present invention include various diseases associated with angiogenesis. Specifically, it is cancer, atherosclerosis, multiple myeloma bone disease, muscle atrophy or diabetic complication. Treatment of the disease can be achieved by the MIC-1 protein overexpression in the above-mentioned diseases, and by decreasing the expression level of the MIC-1 protein.
  • the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier and the carrier may comprise a non-naturally occuring carrier.
  • pharmaceutically acceptable carrier of the present invention means a carrier or diluent that does not irritate the organism and does not interfere with the biological activity and properties of the administered compound.
  • pharmaceutical carrier acceptable for the composition to be formulated into a liquid solution include sterilized and sterile water, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, And other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added as needed.
  • diluents, dispersants, surfactants, binders, and lubricants can be additionally added and formulated into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
  • the pharmaceutical composition may be in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories May have one formulation, and may be oral or parenterally various formulations.
  • a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.
  • Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like are also used.
  • Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like.
  • excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have.
  • Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories.
  • the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.
  • the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.
  • the pharmaceutical composition may be administered in a pharmaceutically effective amount.
  • composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.
  • the anti-MIC-1 mouse / humanized IgG monoclonal antibody that specifically binds to the MIC-1 protein significantly reduces endothelial cell neovascularization produced by the MIC-1 protein , Skin cancer, colon cancer, breast cancer, prostate cancer, pancreatic cancer, liver cancer, or stomach cancer, inhibiting angiogenesis in the inside / outside of these tissues and inhibiting proliferation of cancer cells (Figs. 1 to 5 and 8 to 24).
  • an antibody that binds to the MIC-1 protein of the present invention can be usefully used for prevention or treatment of cancer, and also for inhibiting angiogenesis.
  • Another embodiment provides a method of preventing or treating cancer, comprising administering the antibody to a subject in need thereof.
  • individual of the present invention includes mammals, birds, and the like, including, but not limited to, mice, cows, pigs, sheep, chickens, dogs, Include, but are not limited to, individuals to whom the cancer is to be treated.
  • the administration route and method of administering the composition are not particularly limited and may be appropriately selected depending on the administration route and administration mode as long as the composition can reach the desired site .
  • the composition may be administered orally or parenterally through various routes.
  • routes of administration include oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, Intramuscularly or through inhalation or the like.
  • the composition may be administered by any device capable of transferring the active agent to the target cell.
  • Another embodiment provides a cancer diagnostic composition comprising the antibody.
  • composition for cancer diagnosis of the present invention includes antibodies specifically binding to MIC-1 protein overexpressed in various kinds of cancer cells, it is useful for diagnosis of diseases related to the expression or expression level of MIC-1 protein such as cancer Can be used.
  • Another embodiment provides a cancer diagnostic kit comprising the composition.
  • composition and the description of the cancer are as described above.
  • the cancer diagnostic kit of the present invention may further comprise a composition, solution or device having one or more other components suitable for the assay method.
  • Another embodiment provides a method for diagnosing cancer, comprising the step of detecting the MIC-1 (macrophage inhibitory cytokine 1) protein in an isolated biological sample of a subject suspected of having cancer using the antibody, through an antigen- ≪ / RTI >
  • the description of the antibody, the individual, the MIC-1 protein and the cancer is as described above.
  • the antibody of the present invention which specifically binds to the MIC-1 protein, is useful for diagnosis of a disease associated with the expression or degree of expression of the MIC-1 protein, Lt; / RTI >
  • the diagnostic method of cancer can detect the MIC-1 protein by reacting the antibody specific to MIC-1 of the present invention with a separated biological sample suspected of having cancer and detecting antigen-antibody complex formation, Cancer can be diagnosed.
  • A treating the isolated antibody with a separated biological sample of a suspected cancer patient to detect MIC-1 protein through an antigen-antibody reaction; (b) comparing the level of the MIC-1 protein detected in (a) with that of the control, and determining that the level of MIC-1 protein is higher than that of the control, the cancer patient.
  • biological sample includes tissues, cells, whole blood, serum, plasma, tissue autopsy samples (brain, skin, lymph nodes, spinal cord, etc.), cell culture supernatant, ruptured eukaryotic cells and bacterial expression systems But is not limited thereto. These biological samples can be reacted with the antibody of the present invention without manipulation or manipulation to confirm the presence or absence of MIC-1 protein.
  • antigen-antibody complex refers to a combination of the MIC-1 protein antigen and the antibody of the present invention recognizing the MIC-1 protein antigen in the sample.
  • the formation of such an antigen-antibody complex can be performed by a colorimetric method, Can be detected by any method such as an electrochemical method, a fluorimetric method, a luminometry, a particle counting method, a visual assessment method, or a scintillation counting method .
  • various applications and applications are possible without being limited thereto.
  • markers can be used to detect the antigen-antibody complex.
  • enzymes, chromophores, ligands, emitters, microparticles or radioisotopes can be used, but the present invention is not limited thereto.
  • the enzyme used as a detection label include acetylcholinesterase, alkaline phosphatase,? -D-galactosidase, horseradish peroxidase,? -Lactamase, and the like.
  • the luminescent material examples include an acridinium ester, an isoluminol derivative and the like, and fine particles Colloidal gold, colored latex and the like, and the radioactive isotope includes 57 Co, 3 H, 125 I, 125 I-Bonton Hunter reagent and the like.
  • MIC-1 macrophage inhibitory cytokine 1
  • prophylactic or therapeutic candidates for cancer of the present invention is a substance expected to treat cancer, and can be used without limitation as long as it is a substance that is expected to directly or indirectly improve or improve cancer. , A gene or protein, and the like.
  • the step (a) of treating a cancer cell with a candidate substance for preventing or treating cancer can be carried out using a method known in the art.
  • the candidate substance may be treated with the cancer cell and cultured together, or the candidate substance may be administered to a living body including cancer cells, but the present invention is not limited thereto.
  • Those skilled in the art can use a method suitable for the purpose of the present invention will be.
  • the step (b) for measuring the level of MIC-1 protein can be used by any method known to a person skilled in the art.
  • it may be a step of detecting the formation of an antigen-antibody complex of an MIC-1 protein and an anti-MIC-1 antibody.
  • Specific examples include Western blot, Co-immunoprecipitation assay, ELISA, immunostaining, and FACS (Fluorescence activated cell sorter).
  • the present invention is not limited thereto, and a person skilled in the art can use a method suited to the purpose of the present invention.
  • step (c) is a step of determining whether the candidate substance can be used as a preventive or therapeutic agent for cancer, wherein the MIC-1 protein is overexpressed in cancer cells, promotes angiogenesis, A candidate substance that decreases the level of MIC-1 protein can be used as a preventive or therapeutic agent for cancer.
  • Example 1-1 Construction of anti-human MIC-1 mouse IgG monoclonal antibody
  • a human MIC-1 protein was injected into a Balb / c mouse to produce an anti-human-MIC-1 antibody-producing mouse.
  • Splenocytes were obtained from the mice, and hybridoma cells were prepared by fusing them with myeloma cells.
  • Hybrid cells were selectively cultured using HAT medium.
  • the hybridoma cell monoclon producing the anti-human MIC-1 antibody was selected by performing ELISA on the antibody produced from the hybrid cells, and the antibody present in the hybridoma cell monoclonal culture was subjected to protein-G chromatography G-agarose column chromatography to obtain an anti-human MIC-1 mouse IgG monoclonal antibody.
  • anti-MIC-1 mouse antibody anti-human MIC-1 mouse IgG monoclonal antibody
  • anticancer agent anticancer agent
  • the heavy and light chain variable regions, CDRs, and FR sequences of the anti-MIC-1 mouse antibody are shown in Table 1 below.
  • VH Anti-MIC-1 mouse antibody Heavy chain variable region
  • VH Anti-MIC-1 mouse antibody Heavy chain variable region
  • VH EVQLQQSGAELVRPGALVKLSCKASGFNIKDYYMHWVRQRPEQGLEWIGWIDPENGNTIYDPKFQGKASITTDTSSNTAYMQLSSLTCEDTAVYYCAVPDSWGQGTTLTVSSAKTTPP 15 VH-CDR1 GFNIKDYY One VH-CDR2 IDPENGNT 2 VH-CDR3 AVPDS 3 VH-FR1 EVQLQQSGAELVRPGALVKLSCKAS 7 VH-FR2 MHWVRQRPEQGLEWIGW 8 VH-FR3 IYDPKFQGKASITTDTSSNTAYMQLSSLTCEDTAVYYC 9 VH-FR4 WGQGTTLTVSSAKTTPP 10 Light chain variable region (VL) DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNT
  • human umbilical cord endothelial cells (HUVEC) were firstly dispensed into a culture vessel coated with matrigel, and the MIC-1 protein contained in the culture medium was incubated with normal IgG or anti-MIC- 1 < / RTI > monoclonal antibody. After incubation at 37 ° C and 5% CO 2 for 16 hours, the degree of tube formation was observed and its tube length was measured.
  • aortic sections obtained by cutting the aorta isolated from an SD-rat to a certain thickness were embedding in a matrigel, and then a normal IgG or anti-MIC-1 single Clone antibody and MIC-1. After incubation for 16 hours at 37 ° C and 5% CO 2 , sprouting of the endothelial cells extending from the aortic slice was measured.
  • the anti-MIC-1 mouse antibody significantly reduced the angiogenesis produced by MIC-1 protein treatment, And it is similar to the control group.
  • an HCT-116 colon cancer cell line was inoculated (1 ⁇ 10 6 cells) to each subcutaneous site of athymic nu / nu mouse, and an animal model of xenograft transplantation cancer was prepared.
  • the mice were divided into two groups, one group receiving normal IgG and the other group receiving anti-MIC-1 monoclonal antibody administered intravenously (100 ⁇ g / mouse) to the tail vein at 3-day intervals.
  • MDA-MB-231 human breast cancer cell line was mixed with matrigel (50%) and inoculated subcutaneously (1 x 10 6 cells) into a dorsal submucosa of athymic nu / nu mouse to obtain an animal model of breast cancer xenograft cancer Respectively. Then, when the tumors grow to a certain size, mice having two different tumor sizes are divided into two groups, one group is administered with normal IgG and the other group is administered with anti-MIC-1 monoclonal antibody every 3 days in the tail vein (100 [mu] g / mouse).
  • an animal model of prostate cancer xenograft cancer was prepared by inoculating PC3 human prostate cancer cell line with matrigel (50%) and inoculating subcutaneously (2 ⁇ 10 6 cells) of athymic nu / nu mouse . Thereafter, when the tumor grew to a certain size of about 100 mm 3 , mice having two different tumor sizes were divided into two groups, normal IgG in one group and anti-MIC-1 monoclonal antibody in the other group for 3 days (100 [mu] g / mouse) in the tail vein at intervals.
  • the anti-MIC-1 mouse IgG monoclonal antibody showed excellent angiogenesis inhibitory effect and inhibited the growth of various types of cancer such as skin cancer, colon cancer, breast cancer and prostate cancer , And inhibited angiogenesis in the inside or the outside of the cancer.
  • Example 2-1 Preparation of anti-MIC-1 humanized IgG monoclonal antibody
  • anti-MIC-1 mouse IgG monoclonal antibody inhibited angiogenesis and showed growth inhibitory effect on various kinds of cancer. Therefore, anti-MIC-1 mouse IgG monoclonal antibody showed anti- To prepare a humanized antibody with minimal reaction.
  • the mRNA was extracted from hydridoma cells secreting the mouse antibody and converted into cDNA.
  • the heavy and light chain variable regions (VL and VH) of mouse IgG were amplified by PCR, and the Sfi of the pComb3XSS vector I < / RTI > position, followed by analysis of the nucleotide sequence.
  • the amplified product, which was analyzed, was aligned with human antibody variable region amino acid sequence.
  • the mouse sequence was used as the CDR-1, 2 and 3, and the other region was homologous to the mouse antibody variable region
  • the amino acid sequence for the VL and VH regions of the humanized antibody was determined by substituting the amino acid of the antibody variable region of the highest human.
  • oligonucleotides were synthesized and subcloned into pdCMV-dhfrC vector, a vector expressing human IgG1 in animal cells. At this time, the light chain encoding oligonucleotide was subcloned into the HindIII & BsiWI site and the heavy chain encoding oligonucleotide was ligated to the EcoRI & ApaI site.
  • the humanized IgG1 expression vector was transfected into HEK293 cells and cultured for 24 hours.
  • the culture supernatant was reacted with recombinant MIC-1 protein on an antigen-coated plate, and then subjected to ELISA.
  • pdCMV-dhfrC co-vector there was almost no positive reaction for the culture supernatant of the control cells.
  • the culture supernatant of the cells transfected with the humanized anti-MIC-1 IgG1 expression vector Respectively.
  • anti-MIC-1 humanized antibody In order to increase the production efficiency of the produced humanized anti-MIC-1 IgG1 monoclonal antibody (hereinafter referred to as "anti-MIC-1 humanized antibody”), the expression vector was transfected into CHO cells deficient in DHFR (dihydrofolate reductase) , And cultured in a medium containing antibiotics (neomycin G418) and DHFR to obtain stable transformant clones, and each clone was separately cultured. The culture supernatants of the clones were subjected to ELISA using the MIC-1 protein as an antigen as described above.
  • DHFR dihydrofolate reductase
  • protein # G affinity chromatography was performed on the cultured supernatant obtained by treating the selected # 29 CHO cell clone with methotrexate to amplify the expression vector gene and culturing the cells in a large capacity.
  • Lt; RTI ID 0.0 > anti-MIC-1 < / RTI >
  • the purity of the anti-MIC-1 humanized IgG antibody was confirmed by coumarin blue staining and the binding affinity for the MIC-1 protein was confirmed by ELISA and Western blotting.
  • anti-MIC-1 humanized IgG antibody has high purity and binds specifically to MIC-1 protein.
  • the heavy and light chain variable regions, CDRs, and FR sequences of the anti-MIC-1 humanized antibody thus prepared are shown in Table 2 below.
  • Example 2-2-1 Identification of endothelial cell tubule formation inhibitory activity
  • Example 1 In order to confirm the activity of the anti-MIC-1 humanized antibody prepared in Example 1 as an anticancer agent, the anti-angiogenic activity was first confirmed through a tube formation assay according to Example 1-2 .
  • the anti-MIC-1 humanized antibody significantly reduced the tube formation of the endothelial cell, i.e., the angiogenesis, which was increased by the treatment with MIC-1 protein, 1 protein in the control group.
  • angiogenesis that occurs during tumor development is induced by the action of angiogenic factors secreted from tumor cells.
  • a cancer cell culture supernatant containing angiogenesis promoting factors secreted from cancer cells was treated to endothelial cells and then treated with the anti-MIC-1 humanized antibody, Formation inhibition.
  • the culture supernatant of A549 lung cancer cell line, HCT-116 colon cancer cell line or LNCaP prostate cancer cell line increased the degree of tube formation of endothelial cells, but anti-MIC- It was confirmed that the tube formation was significantly reduced by the culture supernatant of the cancer cells. In particular, it was confirmed that the degree of inhibition was similar to or better than that of the culture supernatant-untreated control.
  • mouse IgG whole antibody, humanized IgG whole antibody and scFv fragment were treated to compare the degree of tube formation.
  • anti-MIC-1 humanized antibody inhibits angiogenesis of endothelial cells and inhibits angiogenesis promoted by cancer cells to an excellent degree, and thus can be usefully used as a preventive or therapeutic agent for cancer there was.
  • Example 2-2-2 Aorta ring sprouting inhibitory activity
  • aortic ring branching assay for comparing the degree of branch formation of the rat artery slice was carried out according to the method Respectively.
  • the anti-MIC-1 humanized antibody can inhibit angiogenesis, that is, angiogenesis, of the rat artery fraction, and thus can be effectively used as a preventive or therapeutic agent for cancer.
  • Example 2-2-3 In vivo angiogenesis assay ( in vivo angiogenesis assay)
  • chicken embryos were cultured at 37 ° C for 3 days, and then the skin of the area where the air sac was formed was removed.
  • a 3M paper filter in which MIC-1 protein was mixed with normal IgG or anti-MIC-1 monoclonal antibody, was placed on the chorioallantoic membrane of the embryo removed by air sac removal. The resulting blood vessels were observed in the embryo membranes of the embryos.
  • an animal model of xenograft cancer containing the C8161 human skin melanoma cell line was prepared by the method according to Example 1-3, and then the antibody was administered.
  • the anti-MIC-1 humanized antibody can inhibit the growth and angiogenesis of skin melanoma, and thus can be usefully used as a preventive or therapeutic agent for cancer.
  • anti-MIC-1 humanized antibody significantly inhibits the growth of skin melanoma, considering that the concentration of plasma MIC-1 protein decreases as tumor size decreases (Fig. 13B) And thus it can be used as a preventive or therapeutic agent for cancer.
  • an animal model of xenograft cancer containing the HCT-116 human colon cancer cell line was prepared by the method according to Example 1-4, and then the antibody was administered.
  • the anti-MIC-1 humanized antibody inhibits the growth and angiogenesis of colon cancer, and thus can be effectively used as a preventive or therapeutic agent for cancer.
  • the HCT-116 tumor tissue was analyzed using an anti-IB4 antibody and pimonidazole.
  • the anti-MIC-1 humanized antibody was found to be a vascular endothelial cell marker IB4 levels were significantly reduced, but the level of pimonidazole, a hypoxic marker, was found to be significantly increased.
  • the HCT-116 tumor tissue was analyzed using an anti-Ki67 antibody.
  • the protein found only in the nucleus of cells proliferating / dividing by the administration of anti-MIC- And the level of Ki67 was significantly decreased.
  • the amount of MIC-1 protein in the plasma decreases when the anti-MIC-1 humanized antibody is administered, and the above-mentioned reduction effect appears to be concentration-dependent.
  • anti-MIC-1 humanized antibodies can significantly inhibit the growth of colon cancer, considering that the concentration of plasma MIC-1 protein decreases as tumor size decreases (Fig. 16B) And thus it can be used effectively as a preventive or therapeutic agent for cancer.
  • an animal model of xenograft cancer containing MDA-MB-453 human breast cancer cell line was prepared by the method according to Example 1-5, and then the antibody was administered.
  • the MDA-MB-453 tumor tissue sections were immunofluorescently stained with anti-CD31 antibody and anti-IB4 antibody, The expression of CD31 and IB4, which are cell markers, was remarkably decreased.
  • the anti-MIC-1 humanized antibody inhibits the growth and angiogenesis of breast cancer and inhibits the proliferation and division of breast cancer cells, thus being useful as a preventive or therapeutic agent for cancer.
  • an animal model of xenograft cancer containing a PC3 prostate cancer cell line was prepared by the method according to Example 1-6, and then the antibody was administered.
  • the PC3 tumor tissue sections were immunofluorescently stained with anti-CD31 antibody and anti-IB4 antibody.
  • anti-MIC-1 humanized antibody inhibits the growth and angiogenesis of prostate cancer, and thus can be effectively used as a preventive or therapeutic agent for cancer.
  • PC3 tumor tissue was analyzed using an anti-Ki67 antibody.
  • Ki67 a protein found only in the nuclei of cells proliferating / dividing by administration of anti-MIC-1 humanized antibody And the level was significantly decreased.
  • the anti-MIC-1 humanized antibody suppresses angiogenesis in the prostate cancer tissue to expand the hypoxic area, while blocking the supply of oxygen and nutrients to inhibit proliferation and division of prostate cancer cells, And inhibited the development.
  • the expression of MIC-1 which is the target protein of the antibody, is increased.
  • the antibody neutralizes the MIC-1 protein secreted from the prostate cancer cells, -1 < / RTI > expression is prevented from leading to angiogenesis in the tumor, thereby exerting an effect of inhibiting the growth of prostate cancer.
  • PANC-1 An animal model of pancreatic cancer xenograft cancer was prepared by inoculating subcutaneously (1 ⁇ 10 6 cells) of athymic nu / nu mice with a human pancreatic cancer cell line mixed with matrigel (50%). Then, when the tumors grow to a certain size, mice having two different tumor sizes are divided into two groups, one group contains normal IgG and the other group contains anti-MIC-1 monoclonal humanized antibody at 3-day intervals in the tail vein (100 [mu] g / mouse).
  • Ki67 a protein found only in the nucleus of cells proliferating / dividing by administration of anti-MIC- And the level was significantly decreased.
  • the anti-MIC-1 humanized antibody inhibits the growth and angiogenesis of pancreatic cancer and inhibits the proliferation and division of pancreatic cancer cells, thus being useful as a preventive or therapeutic agent for cancer.
  • liver cancer xenograft cancer was prepared by inoculating subcutaneously (1 ⁇ 10 6 cells) of athymic nu / nu mouse with a HepG2 human liver cancer cell line mixed with Matrigel (50%). Then, when the tumors grow to a certain size, mice having two different tumor sizes are divided into two groups, one group contains normal IgG and the other group contains anti-MIC-1 monoclonal humanized antibody at 3-day intervals in the tail vein (100 [mu] g / mouse).
  • an animal model of gastric cancer xenograft cancer was prepared by mixing AGS human gastric cancer cell line with matrigel (50%) and inoculating subcutaneously (1 ⁇ 10 6 cells) of athymic nu / nu mouse. Then, when the tumors grow to a certain size, mice having two different tumor sizes are divided into two groups, one group contains normal IgG and the other group contains anti-MIC-1 monoclonal humanized antibody at 3-day intervals in the tail vein (100 [mu] g / mouse).

Abstract

The present invention relates to an antibody specifically binding to a macrophage inhibitory cytokine 1 (MIC-1) protein, and a use thereof.

Description

MIC-1 단백질에 특이적으로 결합하는 항체 및 이의 용도Antibodies that specifically bind to MIC-1 protein and uses thereof
본 발명은 MIC-1(macrophage inhibitory cytokine 1) 단백질에 특이적으로 결합하는 항체 및 이의 용도에 관한 것으로, 구체적으로 서열번호 1의 중쇄 CDR1, 서열번호 2의 중쇄 CDR2, 및 서열번호 3의 중쇄 CDR3을 포함하는 중쇄 가변영역; 및 서열번호 4의 경쇄 CDR1, 서열번호 5의 경쇄 CDR2, 및 서열번호 6의 경쇄 CDR3을 포함하는 경쇄 가변영역을 포함하는, MIC-1 단백질에 결합하는 항체, 상기 항체를 코딩하는 폴리뉴클레오티드, 상기 폴리뉴클레오티드를 포함하는 발현 벡터, 상기 발현 벡터가 도입된 형질전환체, 상기 항체를 포함하는 암의 예방 또는 치료용 약학 조성물, 상기 항체를 이용하는 암의 예방 또는 치료 방법, 상기 항체를 포함하는 암 진단용 조성물, 상기 조성물을 포함하는 암 진단용 키트, 상기 항체를 이용하는 암의 진단 방법, 및 상기 항체를 이용하는 암의 예방 또는 치료 물질의 스크리닝 방법에 관한 것이다.The present invention relates to an antibody that specifically binds to MIC-1 (macrophage inhibitory cytokine 1) protein and uses thereof, and more particularly to a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, and a heavy chain CDR3 of SEQ ID NO: A heavy chain variable region comprising a heavy chain variable region; And a light chain variable region comprising the light chain CDR1 of SEQ ID NO: 4, the light chain CDR2 of SEQ ID NO: 5, and the light chain CDR3 of SEQ ID NO: 6, a polynucleotide encoding the antibody, A polynucleotide-containing expression vector, a transformant into which the expression vector is introduced, a pharmaceutical composition for preventing or treating cancer comprising the antibody, a method for preventing or treating cancer using the antibody, and a method for diagnosing cancer including the antibody A cancer diagnostic kit comprising the composition, a diagnostic method for cancer using the antibody, and a method for screening a substance for preventing or treating cancer using the antibody.
유전자재조합 의약품(단백질 의약품), 항체, 백신, 세포 치료제 및 유전자 치료제 등으로 구성된 바이오 의약품은 화학합성 의약품보다 부작용이 적고 특정 질환에 대한 효과가 뛰어나다는 장점을 가지고 있다. 이에, 관련 시장 규모는 급격히 팽창하고 있으며, 특히 치료용 단일클론항체 시장은 연평균 성장률이 15.5% 정도로서 빠른 성장단계에 있다. 또한, 단일클론항체는 기술혁신에 의해 약물의 안정성 및 유효성이 많이 개선되고 있고, 새로운 타겟 분자의 발굴로 적응증(indication)이 확장되고 있으며, 기존 약물이 특정 질환에 대해 저항성을 가져 치료하지 못했던 부분을 보완할 가능성이 높아 시장 규모가 지속적으로 증가할 것으로 예상된다. Biopharmaceuticals, which consist of recombinant drugs (proteins), antibodies, vaccines, cell treatments, and gene therapy, have the advantage of less side effects and excellent effects for certain diseases than chemical synthetic drugs. Accordingly, the market size is rapidly expanding, and the therapeutic monoclonal antibody market is growing at an annual average growth rate of 15.5%. In addition, the monoclonal antibody has been greatly improved in stability and efficacy of the drug due to the technological innovation, the indication is extended due to the discovery of a new target molecule, and the existing drug is resistant to a specific disease The market size is expected to increase steadily.
이 중에서도, 종양(암)을 치료 대상으로 한 시장은 2007년 기준 52.1%(65.7억 달러)를 차지할 정도로 매출규모가 가장 크며, 연평균 성장률이 약 15%에 이를 정도로 전체 치료용 항체 시장에서 가장 우위를 점하고 있다. 특히, 혈관신생을 억제하여 치료 효과를 나타내는 단일클론항체를 개발하기 위한 연구가 많이 수행되고 있다. 그 예로, Anexelekto에 결합하는 단일클론항체로서 혈관 신생 억제작용 및 항암 작용을 갖는 항체가 개발된바 있고(한국 공개특허공보 제10-2010-0097688호), Genentech 사, Novartis 사 등에서는 각각 항-VEGF 인간화 단일클론항체인 아바스틴(Avastin), 루센티스(Lucentis) 등을 개발한 바 있다. 그러나, 아바스틴은 매우 고가이고 위장관 출혈이라는 부작용을 가지고 있으며, 일반적으로 혈관신생 억제를 타켓으로 한 암 치료제는 효과를 나타내는 암의 종류가 제한적인바, 다양한 종류의 암에 대하여 현저한 치료 및 진단 효과를 갖는 항체의 개발이 여전히 필요한 실정이다.Among them, the market to treat cancer (cancer) is the largest in sales, accounting for 52.1% (US $ 6.57 billion) in 2007, and the annual average growth rate is about 15% . In particular, many studies have been conducted to develop a monoclonal antibody that exhibits therapeutic effects by inhibiting angiogenesis. As an example, monoclonal antibodies that bind to Anexelekto have been developed (Korean Patent Publication No. 10-2010-0097688), Genentech, and Novartis have developed anti-angiogenic and anti-cancer antibodies, VEGF humanized monoclonal antibodies Avastin, Lucentis, and the like. However, avastin is very expensive and has a side effect of gastrointestinal bleeding. Generally, cancer treatment targeting angiogenesis inhibition has a remarkable therapeutic and diagnostic effect on various kinds of cancer due to limited kinds of cancer that shows effects The development of antibodies is still needed.
한편, MIC-1(Macrophage inhibitory cytokine-1) 단백질은 대부분의 조직에서 발현되고 있지만, 조직 손상이나 염증과 같은 병리적인 조건에서는 발현 수준이 급격히 상승한다. 특히, MIC-1 단백질은 유방암, 대장암, 전립선암, 췌장암, 위암 등과 같은 다양한 암에서 과다 발현되고 있으며, 암조직뿐만 아니라 암환자의 혈액에서도 정상인보다 유의성 있게 높은 수준으로 검출됨이 보고되어, 암 진단의 바이오마커로서 관심이 증대되고 있다(Brown DA, Clin. Cancer Res., 2003, 9:2642-50; Koopmann J, Clin. Cancer Res. 2006, 12:442-6).On the other hand, MIC-1 (Macrophage inhibitory cytokine-1) protein is expressed in most tissues, but its expression level is rapidly increased in pathological conditions such as tissue damage or inflammation. In particular, it has been reported that MIC-1 protein is overexpressed in various cancers such as breast cancer, colon cancer, prostate cancer, pancreatic cancer, stomach cancer and the like, Cancer Res., 2003, 9: 2642-50; Koopmann J, Clin. Cancer Res. 2006, 12: 442-6).
본 발명자들은 MIC-1 단백질에 특이적으로 결합하며, 암의 성장 및 발달에 필수적인 혈관신생을 억제하여 다양한 암에 대해 우수한 치료 및 진단 효과를 나타낼 수 있는 항체를 개발하고자 예의 노력연구한 결과, 항-MIC-1 단일클론항체를 개발하였고, 이들은 혈관신생억제 활성을 나타내며, 피부암, 대장암, 유방암, 전립선암 등 다양한 암에 대해 우수한 항암 효과를 나타냄을 확인하여, 본 발명을 완성하였다.The present inventors have made intensive efforts to develop an antibody that specifically binds to MIC-1 protein and exhibits excellent therapeutic and diagnostic effects against various cancers by inhibiting angiogenesis essential for cancer growth and development. As a result, -MIC-1 monoclonal antibody. These monoclonal antibodies exhibit angiogenesis inhibitory activity and have excellent anticancer effects against various cancers such as skin cancer, colon cancer, breast cancer and prostate cancer. Thus, the present invention has been completed.
본 발명의 하나의 목적은 서열번호 1의 중쇄 CDR1, 서열번호 2의 중쇄 CDR2, 및 서열번호 3의 중쇄 CDR3을 포함하는 중쇄 가변영역; 및 서열번호 4의 경쇄 CDR1, 서열번호 5의 경쇄 CDR2, 및 서열번호 6의 경쇄 CDR3을 포함하는 경쇄 가변영역을 포함하는, MIC-1(macrophage inhibitory cytokine 1) 단백질에 결합하는 항체를 제공하는 것이다.One object of the present invention is to provide a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 2, and the heavy chain CDR3 of SEQ ID NO: 3; And a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 4, a light chain CDR2 of SEQ ID NO: 5, and a light chain CDR3 of SEQ ID NO: 6, and an antibody that binds to a macrophage inhibitory cytokine 1 .
본 발명의 다른 하나의 목적은 상기 항체를 코딩하는 폴리뉴클레오티드를 제공하는 것이다.It is another object of the present invention to provide a polynucleotide encoding said antibody.
본 발명의 또 다른 하나의 목적은 상기 폴리뉴클레오티드를 포함하는 발현 벡터를 제공하는 것이다.It is still another object of the present invention to provide an expression vector comprising the polynucleotide.
본 발명의 또 다른 하나의 목적은 상기 발현 벡터가 도입된 형질전환체를 제공하는 것이다.It is still another object of the present invention to provide a transformant into which the expression vector is introduced.
본 발명의 또 다른 하나의 목적은 상기 항체를 포함하는 조성물, 구체적으로 암의 예방 또는 치료용 약학 조성물을 제공하는 것이다.It is still another object of the present invention to provide a composition comprising the antibody, specifically, a pharmaceutical composition for preventing or treating cancer.
본 발명의 또 다른 하나의 목적은 상기 항체를 포함하는 조성물, 구체적으로 혈관신생 억제용 약학 조성물을 제공하는 것이다.It is still another object of the present invention to provide a composition comprising the antibody, specifically a pharmaceutical composition for inhibiting angiogenesis.
본 발명의 또 다른 하나의 목적은 상기 항체를 이를 필요로 하는 개체에 투여하는 단계를 포함하는, 암의 예방 또는 치료 방법을 제공하는 것이다.It is still another object of the present invention to provide a method for preventing or treating cancer, which comprises administering the antibody to a subject in need thereof.
본 발명의 또 다른 하나의 목적은 상기 항체를 포함하는, 암 진단용 조성물을 제공하는 것이다.It is another object of the present invention to provide a composition for cancer diagnosis comprising the antibody.
본 발명의 또 다른 하나의 목적은 상기 조성물을 포함하는, 암 진단용 키트를 제공하는 것이다.It is another object of the present invention to provide a cancer diagnostic kit comprising the composition.
본 발명의 또 다른 하나의 목적은 상기 항체를 이용하여, 암이 의심되는 개체의 분리된 생물학적 시료에서 MIC-1 단백질을 항원-항체 반응을 통하여 검출하는 단계를 포함하는, 암의 진단 방법을 제공하는 것이다.Another object of the present invention is to provide a diagnostic method for cancer, comprising the step of detecting the MIC-1 protein through an antigen-antibody reaction in a separate biological sample of a subject suspected of having cancer using the antibody .
본 발명의 또 다른 하나의 목적은 (a) 분리된 암세포에 암의 예방 또는 치료 후보물질을 처리하는 단계; (b) 상기 후보물질이 처리된 암세포에서 상기 항체를 이용하여, MIC-1 단백질의 수준을 측정하는 단계; 및 (c) 상기 단계 (b)의 MIC-1 단백질 수준이 후보물질을 처리하지 않은 암세포의 것보다 감소하는 경우, 상기 (a) 단계에서 처리한 후보물질을 암의 예방 또는 치료 물질로 판단하는 단계를 포함하는, 암의 예방 또는 치료 물질의 스크리닝 방법을 제공하는 것이다.Another object of the present invention is to provide a method for treating cancer, comprising the steps of: (a) treating a candidate cancer cell with a candidate substance for preventing or treating cancer; (b) measuring the level of MIC-1 protein using the antibody in cancer cells treated with the candidate substance; And (c) when the MIC-1 protein level of the step (b) is lower than that of the cancer cells not treated with the candidate substance, the candidate substance treated in the step (a) And a method for screening a preventive or therapeutic substance for cancer.
본 발명의 항체는 MIC-1 단백질에 높은 특이도를 가지고 결합하며, 내피세포의 혈관신생을 억제하고, 생체 내 암세포의 성장 및 발달을 억제할 뿐만 아니라 암 조직 내부/외부의 혈관신생을 억제하므로, MIC-1 단백질이 과발현되어 있는 암과 같은 질환의 진단 및 치료 분야에 있어 유용하게 사용될 수 있다.The antibody of the present invention binds to MIC-1 protein with high specificity, inhibits angiogenesis of endothelial cells, inhibits growth and development of cancer cells in vivo, and inhibits angiogenesis inside / outside of cancer tissue , And cancer in which MIC-1 protein is overexpressed.
도 1은 본 발명의 일 실시예에 따라 제조한, 항-MIC-1 마우스 IgG 단일클론항체(이하, "항-MIC-1 마우스 항체"로 명명)의 혈관신생(angiogenesis) 억제 활성을 보여주는 이미지 및 그래프로서, A는 튜브 형성 어세이(tube formation assay), 및 B는 대동맥 고리 가지형성 어세이(aorta ring sprouting assay) 결과에 관한 것이다. 이때, mAb는 항-MIC-1 마우스 항체를 의미한다.1 is an image showing angiogenesis inhibitory activity of an anti-MIC-1 mouse IgG monoclonal antibody (hereinafter referred to as "anti-MIC-1 mouse antibody") prepared according to an embodiment of the present invention And graphs, where A is the tube formation assay and B is the aorta ring sprouting assay result. Here, mAb means anti-MIC-1 mouse antibody.
도 2는 상기 항-MIC-1 마우스 항체의 피부암(melanoma)에 대한 성장 억제 활성을 보여주는 이미지 및 그래프이다. A는 마우스 생체 내에 존재하는 피부암 종양의 모습, B는 마우스 생체로부터 분리한 피부암 종양의 모습, C는 상기 항체의 투여에 따른 피부암 종양의 부피 변화, D는 상기 항체의 투여에 따른 피부암 종양의 무게 변화, 및 E는 피부암 종양 조직 절편에 대한 항-CD31 항체의 면역형광염색 결과에 관한 것이다. 이때, mAb는 항-MIC-1 마우스 항체를 의미한다.2 is an image and a graph showing the growth inhibitory activity of the anti-MIC-1 mouse antibody against melanoma. A is a skin cancer tumor present in a living body, B is a skin cancer tumor isolated from a mouse body, C is a volume change of a skin cancer tumor according to the administration of the antibody, D is a skin cancer tumor weight And E is the immunofluorescent staining result of anti-CD31 antibody against skin cancer tumor tissue sections. Here, mAb means anti-MIC-1 mouse antibody.
도 3은 상기 항-MIC-1 마우스 항체의 대장암에 대한 성장 억제 활성을 보여주는 이미지 및 그래프이다. A는 마우스 생체 내에 존재하는 대장암 종양의 모습, B는 마우스 생체로부터 분리한 대장암 종양의 모습, C는 상기 항체의 투여에 따른 대장암 종양의 부피 변화, D는 상기 항체의 투여에 따른 대장암 종양의 무게 변화, 및 E는 대장암 종양 조직 절편에 대한 항-CD31 항체의 면역형광염색 결과에 관한 것이다. 이때, mAb는 항-MIC-1 마우스 항체를 의미한다.3 is an image and a graph showing the growth inhibitory activity of the anti-MIC-1 mouse antibody against colon cancer. A is a colorectal cancer tumor present in a living body of a mouse, B is a colon cancer tumor isolated from a mouse body, C is a volume change of colon cancer tumor upon administration of the antibody, D is a colon cancer , And E is the immunofluorescent staining result of anti-CD31 antibody against colon cancer tumor tissue sections. Here, mAb means anti-MIC-1 mouse antibody.
도 4는 상기 항-MIC-1 마우스 항체의 유방암에 대한 성장 억제 활성을 보여주는 이미지 및 그래프이다. A는 마우스 생체로부터 분리한 유방암 종양의 모습, B는 상기 항체의 투여에 따른 유방암 종양의 부피 변화, C는 상기 항체의 투여에 따른 유방암 종양의 무게 변화, 및 D는 유방암 종양 조직 외부 및 내부 절편에 대한 항-IB4(Isolectin/IB4) 항체의 면역형광염색 결과에 관한 것이다. 이때, mAb #4는 항-MIC-1 마우스 항체를 의미한다.4 is an image and a graph showing the growth inhibitory activity of the anti-MIC-1 mouse antibody against breast cancer. B is the volume change of the breast cancer tumor according to the administration of the antibody, C is the weight change of the breast cancer tumor according to the administration of the antibody, and D is the breast cancer tumor external and internal section IB4 (Isolectin / IB4) antibody against human immunodeficiency virus. Here, mAb # 4 means anti-MIC-1 mouse antibody.
도 5는 상기 항-MIC-1 마우스 항체의 전립선암에 대한 성장 억제 활성을 보여주는 이미지 및 그래프이다. A는 마우스 생체로부터 분리한 전립선암 종양의 모습, B는 상기 항체의 투여에 따른 전립선암 종양의 부피 변화, C는 상기 항체의 투여에 따른 전립선암 종양의 무게 변화, 및 D는 유방암 종양 절편에 대한 항-IB4(Isolectin/IB4) 항체의 면역형광염색 결과에 관한 것이다. 이때, mAb #4는 항-MIC-1 마우스 항체를 의미한다.5 is an image and a graph showing the growth inhibitory activity of the anti-MIC-1 mouse antibody against prostate cancer. B is the volume change of the prostate cancer tumor according to the administration of the antibody, C is the weight change of the prostate cancer tumor according to administration of the antibody, and D is the tumor volume of the breast cancer tumor section IB4 < / RTI > (Isolectin / IB4) antibody. Here, mAb # 4 means anti-MIC-1 mouse antibody.
도 6은 본 발명의 일 실시예에 따라 제조한, 항-MIC-1 인간화 IgG 단일클론항체(이하, "항-MIC-1 인간화 항체"로 명명)를 발현하는 1 내지 59개의 클론(clone)들의 항체 발현 정도를 보여주는 그래프 및 이미지로서, ELISA 및 웨스턴블롯(Western blot) 결과에 관한 것이다. Figure 6 shows clones from 1 to 59 expressing anti-MIC-1 humanized IgG monoclonal antibody (hereinafter referred to as " anti-MIC-1 humanized antibody "), prepared according to one embodiment of the present invention. As well as ELISA and Western blot results. ≪ Desc / Clms Page number 10 >
도 7은 상기 항-MIC-1 인간화 항체의 순도 및 MIC-1 단백질에 대한 결합인지도를 보여주는 이미지 및 그래프로서, 쿠마지 블루(Coomasie blue) 염색, ELISA 및 웨스턴블롯 결과에 관한 것이다. FIG. 7 is an image and a graph showing the purity of the anti-MIC-1 humanized antibody and the binding awareness to the MIC-1 protein, and relates to Coomasie blue staining, ELISA and Western blot results.
도 8은 상기 항-MIC-1 인간화 항체의 혈관신생 억제 활성을 보여주는 이미지 및 그래프로서, 내피세포를 이용한 튜브 형성 어세이 결과에 관한 것이다. A는 MIC-1 단백질, 및 B는 A549 폐암세포, HCT116 대장암세포 또는 LNCaP 전립선암세포 배양 상층액 처리에 의해 증가한 혈관신생 감소 효과를 보여준다. 이때, HuAb는 항-MIC-1 인간화 항체를 의미한다.FIG. 8 is an image and a graph showing the angiogenesis inhibitory activity of the anti-MIC-1 humanized antibody, which relates to the results of tube formation assay using endothelial cells. A shows increased angiogenesis by MIC-1 protein, and B is treated with A549 lung cancer cells, HCT116 colorectal cancer cells, or LNCaP prostate cancer cell culture supernatant. At this time, HuAb means anti-MIC-1 humanized antibody.
도 9는 여러 유형의 항-MIC-1 항체의 혈관신생 억제 활성을 보여주는 이미지 및 그래프로서, 내피세포를 이용한 튜브 형성 어세이 결과에 관한 것이다. 이때, mAb는 마우스 전체항체, HuAb는 인간화 전체항체, 및 scFV는 단편 항체를 의미한다.FIG. 9 is an image and a graph showing the angiogenesis inhibitory activity of various types of anti-MIC-1 antibodies, and relates to the results of tube formation assay using endothelial cells. Herein, mAb means mouse whole antibody, HuAb means humanized whole antibody, and scFV means fragment antibody.
도 10은 상기 항-MIC-1 인간화 항체의 혈관신생 억제 활성을 보여주는 이미지 및 그래프로서, 대동맥 고리 가지형성(aorta ring sprouting) 어세이 결과에 관한 것이다. A는 항-MIC-1 인간화 항체, 및 B는 여러 유형의 항-MIC-1 항체 처리에 의해 증가한 혈관신생 감소 효과를 보여준다. 이때, mAb는 마우스 전체항체, HuAb는 인간화 전체항체, 및 scFV는 단편 항체를 의미한다.FIG. 10 is an image and a graph showing the anti-angiogenic activity of the anti-MIC-1 humanized antibody, showing aorta ring sprouting, It is about the assay results. A shows an anti-MIC-1 humanized antibody, and B shows an angiogenesis-decreasing effect by various types of anti-MIC-1 antibody treatment. Herein, mAb means mouse whole antibody, HuAb means humanized whole antibody, and scFV means fragment antibody.
도 11은 상기 항-MIC-1 인간화 항체의 혈관신생 억제 활성을 보여주는 이미지 및 그래프로서, CAM(chick chorioallantoic membrane)을 이용한 생체 내 혈관신생 어세이(in vivo angiogenesis assay) 결과에 관한 것이다. 이때, HuAb는 항-MIC-1 인간화 항체를 의미한다.FIG. 11 is an image and a graph showing the angiogenesis inhibitory activity of the anti-MIC-1 humanized antibody, and it relates to an in vivo angiogenesis assay result using a chick chorioallantoic membrane (CAM). At this time, HuAb means anti-MIC-1 humanized antibody.
도 12는 상기 항-MIC-1 인간화 항체의 피부흑색종에 대한 성장 억제 활성을 보여주는 이미지 및 그래프이다. A는 마우스 생체 내에 존재하는 피부흑색종 종양의 모습, B는 마우스 생체로부터 분리한 피부흑색종 종양의 모습, C는 상기 항체의 투여에 따른 피부흑색종 종양의 부피 변화, D는 상기 항체의 투여에 따른 피부흑색종 종양의 무게 변화, E는 피부흑색종 종양 조직 절편에 대한 항-CD31 항체의 면역형광염색 결과, 및 F는 피부흑색종 종양 조직 절편에 대한 항-IB4 항체의 면역형광염색 결과에 관한 것이다. 이때, HuAb는 항-MIC-1 인간화 항체를 의미한다.FIG. 12 is an image and a graph showing the growth inhibitory activity of the anti-MIC-1 humanized antibody against a skin melanoma. A is a skin melanoma tumor present in a mouse body, B is a skin melanoma tumor isolated from a mouse body, C is a volume change of skin melanoma tumor according to the administration of the antibody, D is a dose of the antibody , E is the result of immunofluorescent staining of anti-CD31 antibody against cutaneous melanoma tumor tissue sections, and F is the result of immunofluorescence staining of anti-IB4 antibody against cutaneous melanoma tumor tissue sections . At this time, HuAb means anti-MIC-1 humanized antibody.
도 13은 항-MIC-1 인간화 항체의 피부흑색종에 대한 성장 억제 활성을 보여주는 그래프로서, A는 상기 항체의 투여에 따른 혈장 MIC-1 단백질의 농도를 보여주는 그래프, 및 B는 종양 크기와 혈장 MIC-1 단백질 농도의 연관성을 보여주는 그래프이다.FIG. 13 is a graph showing the anti-MIC-1 humanized antibody's growth inhibitory activity on skin melanoma, wherein A is a graph showing the concentration of plasma MIC-1 protein upon administration of the antibody, and B is a graph showing tumor size and plasma This is a graph showing the correlation of MIC-1 protein concentration.
도 14는 상기 항-MIC-1 인간화 항체의 대장암에 대한 성장 억제 활성을 보여주는 이미지 및 그래프이다. A는 마우스 생체 내에 존재하는 대장암 종양의 모습, B는 마우스 생체로부터 분리한 대장암 종양의 모습, C는 상기 항체의 투여에 따른 대장암 종양의 부피 변화, D는 상기 항체의 투여에 따른 대장암 종양의 무게 변화, E는 대장암 종양 조직 절편에 대한 항-CD31 항체의 면역형광염색 결과, 및 F는 대장암 종양 조직 절편에 대한 항-IB4 항체의 면역형광염색 결과에 관한 것이다. 이때, HuAb는 항-MIC-1 인간화 항체를 의미한다.14 is an image and a graph showing the growth inhibitory activity of the anti-MIC-1 humanized antibody against colorectal cancer. A is a colorectal cancer tumor present in a living body of a mouse, B is a colon cancer tumor isolated from a mouse body, C is a volume change of colon cancer tumor upon administration of the antibody, D is a colon cancer E is the result of immunofluorescent staining of the anti-CD31 antibody against the colon cancer tumor tissue section, and F is the immunofluorescent staining result of the anti-IB4 antibody against the colon cancer tumor tissue section. At this time, HuAb means anti-MIC-1 humanized antibody.
도 15는 항-MIC-1 인간화 항체의 대장암에 대한 저산소 상태 유도 및 세포 증식 억제 활성을 보여주는 이미지 및 그래프이다. A는 대장암 종양 조직 절편에서 검출된 피모니다졸(Pimonidazole)의 수준, B는 대장암 종양 조직 절편에 대한 항-Ki67 항체의 면역형광염색 결과, 및 C는 CoCl2 처리된 대장암 세포에서 발현된 MIC-1 단백질의 양에 관한 것이다. 이때, HuAb는 항-MIC-1 인간화 항체를 의미한다.15 is an image and a graph showing anti-MIC-1 humanized antibody exhibiting hypoxic state and cell proliferation inhibitory activity against colon cancer. A is the level of pimonidazole detected in the colorectal tumor tissue section, B is the immunofluorescent staining result of the anti-Ki67 antibody against the colorectal tumor tissue section, and C is the expression level in CoCl 2 -treated colon cancer cells RTI ID = 0.0 > MIC-1 < / RTI > At this time, HuAb means anti-MIC-1 humanized antibody.
도 16은 항-MIC-1 인간화 항체의 대장암에 대한 성장 억제 활성을 보여주는 그래프로서, A는 상기 항체의 투여에 따른 혈장 MIC-1 단백질의 농도를 보여주는 그래프, 및 B는 종양 크기와 혈장 MIC-1 단백질 농도의 연관성을 보여주는 그래프이다.FIG. 16 is a graph showing the anti-MIC-1 humanized antibody's growth inhibitory activity against colon cancer, wherein A is a graph showing the concentration of plasma MIC-1 protein upon administration of the antibody, and B is a graph showing tumor size and plasma MIC -1 protein concentration in the blood.
도 17은 상기 항-MIC-1 인간화 항체의 유방암에 대한 성장 억제 활성을 보여주는 이미지 및 그래프이다. A는 마우스 생체 내에 존재하는 유방암 종양의 모습, B는 마우스 생체로부터 분리한 유방암 종양의 모습, C는 상기 항체의 투여에 따른 유방암 종양의 부피 변화, D는 상기 항체의 투여에 따른 유방암 종양의 무게 변화, E는 유방암 종양 조직 절편에 대한 항-CD31 항체의 면역형광염색 결과, 및 F는 유방암 종양 조직 절편에 대한 항-IB4 항체의 면역형광염색 결과에 관한 것이다. 이때, HuAb는 항-MIC-1 인간화 항체를 의미한다.17 is an image and a graph showing the growth inhibitory activity of the anti-MIC-1 humanized antibody against breast cancer. A is a breast cancer tumor present in the body of a mouse, B is a breast cancer tumor isolated from a mouse body, C is a volume change of a breast cancer tumor according to the administration of the antibody, D is weight of a breast cancer tumor , E is the result of immunofluorescence staining of anti-CD31 antibody against breast cancer tumor tissue sections, and F is the result of immunofluorescence staining of anti-IB4 antibody against breast cancer tumor tissue sections. At this time, HuAb means anti-MIC-1 humanized antibody.
도 18은 항-MIC-1 인간화 항체의 유방암에 대한 세포 증식 억제 활성을 보여주는 이미지 및 그래프이다. 유방암 종양 조직 절편에 대한 항-Ki67 항체의 면역형광염색 결과에 관한 것이다. 이때, HuAb는 항-MIC-1 인간화 항체를 의미한다.18 is an image and a graph showing anti-MIC-1 humanized antibody's cell proliferation inhibitory activity against breast cancer. Ki67 < / RTI > antibody against breast cancer tumor tissue sections. At this time, HuAb means anti-MIC-1 humanized antibody.
도 19는 상기 항-MIC-1 인간화 항체의 전립선암에 대한 성장 억제 활성을 보여주는 이미지 및 그래프이다. A는 마우스 생체 내에 존재하는 전립선암 종양의 모습, B는 마우스 생체로부터 분리한 전립선암 종양의 모습, C는 상기 항체의 투여에 따른 전립선암 종양의 부피 변화, D는 전립선암 종양 조직 절편에 대한 항-CD31 항체의 면역형광염색 결과, 및 E는 전립선암 종양 조직 절편에 대한 항-IB4(Isolectin/IB4) 항체의 면역형광염색 결과에 관한 것이다. 이때, HuAb는 항-MIC-1 인간화 항체를 의미한다.19 is an image and a graph showing the growth inhibitory activity of the anti-MIC-1 humanized antibody against prostate cancer. A represents a prostate cancer tumor present in a living body, B represents a prostate cancer tumor isolated from a mouse body, C represents a volume change of the prostate cancer tumor by the administration of the antibody, D represents a prostate cancer tumor tissue slice Results of immunofluorescent staining of anti-CD31 antibodies, and E relates to immunofluorescence staining results of anti-IB4 (Isolectin / IB4) antibodies against prostate cancer tumor tissue sections. At this time, HuAb means anti-MIC-1 humanized antibody.
도 20은 항-MIC-1 인간화 항체의 전립선암에 대한 저산소 상태 유도 및 세포 증식 억제 활성을 보여주는 이미지 및 그래프이다. A는 전립선암 종양 조직 절편에서 검출된 피모니다졸의 수준, B는 전립선암 종양 조직 절편에 대한 항-Ki67 항체의 면역형광염색 결과, 및 C는 CoCl2 처리된 전립선암 세포에서 발현된 MIC-1 단백질의 양에 관한 것이다. 이때, HuAb는 항-MIC-1 인간화 항체를 의미한다.20 is an image and a graph showing the hypoxic state induction and cell proliferation inhibitory activity of anti-MIC-1 humanized antibody against prostate cancer. A is the level of the sol coat is detected in prostate tumor tissue sections, wherein B is the result of immunofluorescence staining -Ki67 antibodies to prostate tumor tissue sections, and C is expressed in prostate cancer cells with CoCl 2 treatment MIC- 1 < / RTI > protein. At this time, HuAb means anti-MIC-1 humanized antibody.
도 21은 상기 항-MIC-1 인간화 항체의 췌장암에 대한 성장 억제 활성을 보여주는 이미지 및 그래프이다. A는 마우스 생체 내에 존재하는 췌장암 종양의 모습, B는 마우스 생체로부터 분리한 췌장암 종양의 모습, C는 상기 항체의 투여에 따른 췌장암 종양의 부피 변화, D는 췌장암 종양 조직 절편에 대한 항-CD31 항체의 면역형광염색 결과, 및 E는 췌장암 종양 조직 절편에 대한 항-IB4(Isolectin/IB4) 항체의 면역형광염색 결과에 관한 것이다. 이때, HuAb는 항-MIC-1 인간화 항체를 의미한다.21 is an image and a graph showing the growth inhibitory activity of the anti-MIC-1 humanized antibody against pancreatic cancer. A is a pancreatic cancer tumor present in the mouse, B is a pancreatic cancer tumor isolated from a mouse body, C is a volume change of the pancreatic cancer tumor by administration of the antibody, D is an anti-CD31 antibody , And E is the result of immunofluorescence staining of anti-IB4 (Isolectin / IB4) antibody against pancreatic cancer tumor tissue sections. At this time, HuAb means anti-MIC-1 humanized antibody.
도 22는 항-MIC-1 인간화 항체의 췌장암에 대한 저산소 상태 유도 및 세포 증식 억제 활성을 보여주는 이미지 및 그래프이다. 췌장암 종양 조직 절편에 대한 항-Ki67 항체의 면역형광염색 결과에 관한 것이다. 이때, HuAb는 항-MIC-1 인간화 항체를 의미한다.22 is an image and a graph showing anti-MIC-1 humanized antibodies against hypoxic state and cell proliferation inhibitory activity against pancreatic cancer. Ki67 < / RTI > antibody against pancreatic cancer tumor tissue sections. At this time, HuAb means anti-MIC-1 humanized antibody.
도 23은 상기 항-MIC-1 인간화 항체의 간암에 대한 성장 억제 활성을 보여주는 그래프로서, 상기 항체의 투여에 따른 간암 종양의 부피 변화에 관한 것이다. 이때, HuAb는 항-MIC-1 인간화 항체를 의미한다.23 is a graph showing the growth inhibitory activity of the anti-MIC-1 humanized antibody against liver cancer, which relates to the volume change of liver cancer tumors upon administration of the antibody. At this time, HuAb means anti-MIC-1 humanized antibody.
도 24는 상기 항-MIC-1 인간화 항체의 위암에 대한 성장 억제 활성을 보여주는 그래프로서, 상기 항체의 투여에 따른 위암 종양의 부피 변화에 관한 것이다. 이때, HuAb는 항-MIC-1 인간화 항체를 의미한다.24 is a graph showing the growth inhibitory activity of the anti-MIC-1 humanized antibody against gastric cancer, which relates to the volume change of gastric cancer tumor upon administration of the antibody. At this time, HuAb means anti-MIC-1 humanized antibody.
이하에서는, 본 발명을 더욱 상세히 설명한다.Hereinafter, the present invention will be described in more detail.
한편, 본원에서 개시되는 각각의 설명 및 실시형태는 각각의 다른 설명 및 실시 형태에도 적용될 수 있다. 즉, 본원에서 개시된 다양한 요소들의 모든 조합이 본 발명의 범주에 속한다. 또한, 하기 기술되는 구체적인 서술에 의하여 본 발명의 범주가 제한된다고 할 수 없다.On the other hand, each description and embodiment disclosed herein can be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein are within the scope of the present invention. In addition, it can not be said to the scope of the invention by the specific description will be described limits.
또한, 당해 기술분야의 통상의 지식을 가진 자는 통상의 실험만을 사용하여 본 출원에 기재된 본 발명의 특정 양태에 대한 다수의 등가물을 인지하거나 확인할 수 있다. 또한, 이러한 등가물은 본 발명에 포함되는 것으로 의도된다. In addition, those of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described in this application. Further, such equivalents are intended to be included in the present invention.
상기 목적을 달성하기 위하여, 본 발명의 하나의 양태는 서열번호 1의 중쇄 CDR1, 서열번호 2의 중쇄 CDR2, 및 서열번호 3의 중쇄 CDR3을 포함하는 중쇄 가변영역; 및 서열번호 4의 경쇄 CDR1, 서열번호 5의 경쇄 CDR2, 및 서열번호 6의 경쇄 CDR3을 포함하는 경쇄 가변영역을 포함하는, MIC-1(macrophage inhibitory cytokine 1) 단백질에 결합하는 항체를 제공한다.To this end, one embodiment of the present invention is directed to a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 2, and the heavy chain CDR3 of SEQ ID NO: 3; And a light chain variable region comprising light chain CDR1 of SEQ ID NO: 4, light chain CDR2 of SEQ ID NO: 5, and light chain CDR3 of SEQ ID NO: 6.
본 발명의 용어, "MIC-1(macrophage inhibitory cytokine 1) 단백질"은 GDF-15, PTGF-β, PLAB, PDF, NAG-1, PL74 등으로 명명되는 단백질로서, 조직의 손상이나 질병의 발생 중 염증 및 세포사멸 등을 조절하는 TGF-β 슈퍼패밀리(superfamily)에 속하는 단백질을 의미한다. 조직 손상이나 염증과 같은 병리적인 조건에서는 발현 수준이 급격히 상승하고, 특히 유방암, 대장암, 전립선암, 췌장암, 위암 등과 같은 다양한 암에서 과다 발현되며, 암 조직뿐만 아니라 암환자의 혈액에서도 정상인보다 유의성 있게 높은 수준으로 검출되는 것이 특징이다. 이에 따라, MIC-1 단백질은 암의 진단, 치료 등의 표적이 될 수 있음이 보고되고 있다(Brown DA, Clin. Cancer Res., 2003, 9:2642-50; Koopmann J, Clin. Cancer Res. 2006, 12:442-6). 구체적으로, 상기 MIC-1 단백질은 인간 유래인 것일 수 있으며, 이의 아미노산 서열 등은 NCBI 등의 데이터베이스에 공지되어 있다(Accession number: NP_004855 등).The term "MIC-1 (macrophage inhibitory cytokine 1) protein" of the present invention is a protein named GDF-15, PTGF-β, PLAB, PDF, NAG-1 or PL74, Refers to a protein belonging to the TGF-beta superfamily that regulates inflammation and apoptosis. The level of expression is markedly elevated under pathological conditions such as tissue damage or inflammation and is overexpressed in various cancers such as breast cancer, colon cancer, prostate cancer, pancreatic cancer, stomach cancer and the like, And is detected at a high level. Thus, it has been reported that MIC-1 protein can be a target for diagnosis and treatment of cancer (Brown DA, Clin. Cancer Res., 2003, 9: 2642-50; Koopmann J, Clin. Cancer Res. 2006, 12: 442-6). Specifically, the MIC-1 protein may be human-derived, and its amino acid sequence and the like are known in databases such as NCBI (Accession number: NP_004855, etc.).
본 발명에서는, MIC-1 단백질에 특이적으로 결합하는 마우스 항체 및 인간화 항체를 개발하였고, 이들은 MIC-1 단백질에 의해 증가한 혈관신생을 억제할 뿐만 아니라, 피부암, 대장암, 유방암, 전립선암 등 다양한 암세포의 성장 및 발달을 억제할 수 있음을 확인하였다.In the present invention, mouse antibodies and humanized antibodies that specifically bind to the MIC-1 protein have been developed. They not only inhibit angiogenesis induced by the MIC-1 protein, but also inhibit angiogenesis in a wide variety of skin cancer, colon cancer, breast cancer, It is possible to inhibit the growth and development of cancer cells.
본 발명의 용어, "항체"는 면역학적으로 특정 항원과 반응성을 갖는 면역글로불린 분자를 포함하는, 항원을 특이적으로 인식하는 수용체 역할을 하는 단백질 분자를 의미한다. 본 발명의 항체는 다클론항체, 단일클론항체, 전체(whole) 항체 및 항체 단편을 모두 포함하고, 또한 키메라 항체 및 이가(bivalent) 또는 이중특이성 분자(예를 들어, 이중특이성 항체), 디아바디, 트리아바디 및 테트라바디를 포함한다. 아울러, FcRn(neonatal Fc receptor)에 대한 결합 기능을 보유한 단쇄 항체, 스캡, 항체 불변영역의 유도체 및 단백질 스캐폴드에 기초한 인공 항체를 포함한다. The term " antibody " of the present invention means a protein molecule that acts as a receptor that specifically recognizes an antigen, including an immunoglobulin molecule that is immunologically reactive with a specific antigen. The antibodies of the present invention include both polyclonal antibodies, monoclonal antibodies, whole antibodies and antibody fragments and also include chimeric antibodies and bivalent or bispecific molecules (e. G., Bispecific antibodies) , Triabodies, and tetrabodies. Also included are single chain antibodies, scabs, derivatives of antibody constant regions, and artificial antibodies based on protein scaffolds, which have a binding function for FcRn (neonatal Fc receptor).
상기 전체 항체는 2개의 전체 길이의 경쇄 및 2개의 전체 길이의 중쇄를 갖는 구조로서, 각각의 경쇄는 중쇄와 이황화 결합(disulfide bond)으로 연결되어 있다. 상기 전체 항체는 IgA, IgD, IgE, IgM 및 IgG를 포함하며, IgG는 아형(subtype)으로, IgG1, IgG2, IgG3 및 IgG4를 포함한다. The whole antibody is a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to a heavy chain by a disulfide bond. The whole antibody includes IgA, IgD, IgE, IgM and IgG, and IgG is a subtype and includes IgG1, IgG2, IgG3 and IgG4.
상기 항체 단편은 항원결합 기능을 보유하고 있는 단편을 의미하며, Fc, Fd, Fab, Fab', Fv 및 F(ab')2 등을 포함한다. 상기 Fc는 Fc 수용체라 불리는 세포 표면 수용체와 상호작용 하는 항체의 꼬리 부분을 의미한다. 상기 Fd는 Fab 단편에 포함되어 있는 중쇄 부분을 의미하며, F(ab')2는 Fd, Fab, Fab' 및 Fv를 포함하는 단편을 의미한다. 상기 Fab는 경쇄 및 중쇄의 가변영역과 경쇄의 불변 영역 및 중쇄의 첫 번째 불변 영역(CH1 도메인)을 가지는 구조로 1개의 항원 결합 부위를 가진다. Fab'는 중쇄 CH1 도메인의 C 말단에 하나 이상의 시스테인 잔기를 포함하는 힌지 영역(hinge region)을 가진다는 점에서 Fab와 차이가 있다. F(ab')2 항체는 Fab'의 힌지 영역의 시스테인 잔기가 디설파이드 결합을 이루면서 생성된다. Fv(variable fragment)는 중쇄 가변부위 및 경쇄 가변부위만을 가지고 있는 최소의 항체조각을 의미한다. 이중디설파이드Fv(dsFv)는 디설파이드 결합으로 중쇄 가변부위와 경쇄 가변부위가 연결되어 있고 단쇄 Fv(scFv)는 일반적으로 펩타이드 링커를 통하여 중쇄의 가변 영역과 경쇄의 가변 영역이 공유 결합으로 연결되어 있다. 이러한 항체 단편은 단백질 가수분해 효소를 이용해서 얻을 수 있고(예를 들어, 전체 항체를 파파인으로 제한 절단하며 Fab를 얻을 수 있고 펩신으로 절단하면 F(ab')2 단편을 얻을 수 있다), 바람직하게는 유전자 재조합 기술을 통하여 제작할 수 있다. The antibody fragment refers to a fragment having an antigen-binding function and includes Fc, Fd, Fab, Fab ', Fv, and F (ab') 2 . The Fc refers to the tail portion of an antibody that interacts with a cell surface receptor called an Fc receptor. Fd means a heavy chain portion contained in a Fab fragment, and F (ab ') 2 means a fragment containing Fd, Fab, Fab' and Fv. The Fab has one antigen-binding site in a structure having a variable region of a light chain and a heavy chain, a constant region of a light chain, and a first constant region (CH1 domain) of a heavy chain. Fab 'differs from Fab in that it has a hinge region that contains at least one cysteine residue at the C-terminus of the heavy chain CH1 domain. The F (ab ') 2 antibody is produced when the cysteine residue of the hinge region of the Fab' forms a disulfide bond. Fv (variable fragment) refers to the smallest antibody fragment that has only a heavy chain variable region and a light chain variable region. The double disulfide Fv (dsFv) is linked by a disulfide bond to a light chain variable region and a light chain variable region. A short chain Fv (scFv) is generally linked to a variable region of a heavy chain and a variable region of a light chain through a peptide linker by a covalent bond. Such an antibody fragment can be obtained using a protein hydrolyzing enzyme (for example, an F (ab ') 2 fragment can be obtained by cutting a whole antibody into papain and obtaining a Fab and digesting with pepsin) Can be produced through recombinant DNA technology.
일반적으로, 면역글로불린은 중쇄(heavy chain) 및 경쇄(light chain)를 가지며 각각의 중쇄 및 경쇄는 불변 영역(constant region) 및 가변 영역(variable region)을 포함한다. 경쇄 및 중쇄의 가변 영역은, 상보성 결정 영역으로 불리는 3개의 다변가능한 영역(complementarity-determining region, 이하 "CDR"로 명명) 및 4개의 구조 영역(framework region, 이하 "FR"로 명명)을 포함한다. 각 사슬의 CDR은 주로 항원의 에피토프(epitope)에 결합하는 역할을 하며, 전형적으로 N-말단으로부터 시작하여 순차적으로 CDR1, CDR2, CDR3로 명명된다. 또한, 각 사슬의 FR은 N-말단으로부터 시작하여 순차적으로 FR1, FR2, FR3, FR4로 명명될 수 있다.Generally, immunoglobulins have a heavy chain and a light chain, and each heavy and light chain includes a constant region and a variable region. The variable region of the light chain and the heavy chain includes three complementarity determining regions (hereinafter referred to as " CDRs ") and four framework regions (referred to as " FRs ") called complementarity determining regions . The CDRs of each chain primarily serve to bind to the epitope of the antigen, typically starting from the N-terminus and sequentially named CDR1, CDR2, CDR3. In addition, the FR of each chain may be named FR1, FR2, FR3, FR4 sequentially starting from the N-terminus.
본 발명에서, 중쇄의 가변영역은 'VH', 경쇄의 가변영역은 'LH'로 명명될 수 있으며, 중쇄의 CDR은 각각 'VH-CDR1', 'VH-CDR2', 'VH-CDR3'으로, 경쇄의 CDR은 각각 'VL-CDR1', 'VL-CDR2', 'VL-CDR3'으로, 중쇄의 FR은 각각 'VH-FR1', 'VH-FR2', 'VH-FR3', 'VH-FR4'로, 경쇄의 FR은 'VL-FR1', 'VL-FR2', 'VL-FR3', 'VL-FR4'로 명명될 수 있다.In the present invention, the variable region of the heavy chain can be named 'VH' and the variable region of the light chain can be named 'LH', and the CDRs of the heavy chain can be named 'VH-CDR1', 'VH-CDR2', and 'VH-CDR3' VL-CDR1 ',' VL-CDR2 ', and' VL-CDR3 ', respectively, and the FR of the heavy chain are VH-FR1, VH-FR2, VH- -FR4 ', and the FR of the light chain may be named' VL-FR1 ',' VL-FR2 ',' VL-FR3 ', and' VL-FR4 '.
본 발명의 용어, "MIC-1(macrophage inhibitory cytokine 1) 단백질에 특이적으로 결합하는 항체"는 MIC-1 단백질에 결합하여 MIC-1 단백질의 활성을 저해할 수 있는 항체를 의미한다. The term " antibody specifically binding to macrophage inhibitory cytokine 1 (MIC-1) protein " of the present invention means an antibody capable of binding to MIC-1 protein and inhibiting the activity of MIC-1 protein.
구체적으로, 상기 MIC-1 단백질에 특이적으로 결합하는 항체는 마우스 항체 또는 인간화 항체일 수 있다.Specifically, the antibody specifically binding to the MIC-1 protein may be a mouse antibody or a humanized antibody.
상기 용어, '마우스 항체'는 마우스 면역글로불린으로부터 유래하는 분자로서, 항체를 구성하는 모든 아미노산 서열 전체가 마우스 유래의 아미노산 서열로 구성되어 있는 것을 의미한다. The term " mouse antibody " means a molecule derived from a mouse immunoglobulin, wherein all of the amino acid sequences constituting the antibody are composed of an amino acid sequence derived from a mouse.
상기 용어, '인간화 항체'는 마우스 및 인간 면역글로불린으로부터 유래하는 분자로서, CDR은 마우스 유래, 이를 제외한 영역은 인간 유래의 아미노산 서열로 구성되어 있는 것을 의미한다.The term " humanized antibody " means a molecule derived from mouse and human immunoglobulin, wherein the CDR is derived from a mouse, and the region excluding it is composed of a human-derived amino acid sequence.
더욱 구체적으로, 상기 항체는 서열번호 1의 중쇄 CDR1, 서열번호 2의 중쇄 CDR2, 및 서열번호 3의 중쇄 CDR3을 포함하는 중쇄 가변영역; 및 서열번호 4의 경쇄 CDR1, 서열번호 5의 경쇄 CDR2, 및 서열번호 6의 경쇄 CDR3을 포함하는 경쇄 가변영역을 포함할 수 있다.More specifically, the antibody comprises a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 2, and the heavy chain CDR3 of SEQ ID NO: 3; And a light chain variable region comprising light chain CDR1 of SEQ ID NO: 4, light chain CDR2 of SEQ ID NO: 5, and light chain CDR3 of SEQ ID NO: 6.
또한, 상기 항체의 중쇄 가변영역은 서열번호 7 또는 17의 중쇄 FR1, 서열번호 8 또는 18의 중쇄 FR2, 서열번호 9 또는 19의 중쇄 FR3 및 서열번호 10 또는 20의 중쇄 FR4를 포함하고; 및 상기 항체의 경쇄 가변영역은 서열번호 11 또는 21의 경쇄 FR1, 서열번호 12 또는 22의 경쇄 FR2, 서열번호 13 또는 23의 경쇄 FR3 및 서열번호 14 또는 24의 경쇄 FR4를 포함하는 것일 수 있다.The heavy chain variable region of said antibody comprises heavy chain FR2 of SEQ ID NO: 7 or 17, heavy chain FR2 of SEQ ID NO: 8 or 18, heavy chain FR3 of SEQ ID NO: 9 or 19 and heavy chain FR4 of SEQ ID NO: 10 or 20; And the light chain variable region of the antibody may comprise light chain FR1 of SEQ ID NO: 11 or 21, light chain FR2 of SEQ ID NO: 12 or 22, light chain FR3 of SEQ ID NO: 13 or 23 and light chain FR4 of SEQ ID NO: 14 or 24.
본 발명에서, (a) 상기 항체의 중쇄 가변영역은 서열번호 7의 중쇄 FR1, 서열번호 8의 중쇄 FR2, 서열번호 9의 중쇄 FR3 및 서열번호 10의 중쇄 FR4를 포함하고; 및 상기 항체의 경쇄 가변영역은 서열번호 11의 경쇄 FR1, 서열번호 12의 경쇄 FR2, 서열번호 13의 경쇄 FR3 및 서열번호 14의 경쇄 FR4를 포함하는 것일 수 있고, (A) the heavy chain variable region of the antibody comprises the heavy chain FR1 of SEQ ID NO: 7, the heavy chain FR2 of SEQ ID NO: 8, the heavy chain FR3 of SEQ ID NO: 9, and the heavy chain FR4 of SEQ ID NO: 10; And the light chain variable region of the antibody may comprise light chain FR1 of SEQ ID NO: 11, light chain FR2 of SEQ ID NO: 12, light chain FR3 of SEQ ID NO: 13, and light chain FR4 of SEQ ID NO: 14,
또는, (b) 상기 항체의 중쇄 가변영역은 서열번호 17의 중쇄 FR1, 서열번호 18의 중쇄 FR2, 서열번호 19의 중쇄 FR3 및 서열번호 20의 중쇄 FR4를 포함하고; 및 상기 항체의 경쇄 가변영역은 서열번호 21의 경쇄 FR1, 서열번호 22의 경쇄 FR2, 서열번호 23의 경쇄 FR3 및 서열번호 24의 경쇄 FR4를 포함하는 것일 수 있으나, 이에 제한되는 것은 아니다.Or (b) the heavy chain variable region of the antibody comprises heavy chain FR1 of SEQ ID NO: 17, heavy chain FR2 of SEQ ID NO: 18, heavy chain FR3 of SEQ ID NO: 19 and heavy chain FR4 of SEQ ID NO: 20; And the light chain variable region of the antibody may include but are not limited to a light chain FR1 of SEQ ID NO: 21, a light chain FR2 of SEQ ID NO: 22, a light chain FR3 of SEQ ID NO: 23, and a light chain FR4 of SEQ ID NO:
또한, 상기 중쇄 가변영역은 서열번호 15 또는 25의 아미노산 서열로 이루어지고; 및 상기 경쇄 가변영역은 서열번호 16 또는 26의 아미노산 서열로 이루어진 것일 수 있다.Also, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 15 or 25; And the light chain variable region may comprise the amino acid sequence of SEQ ID NO: 16 or 26.
본 발명에서, (a) 상기 중쇄 가변영역은 서열번호 15의 아미노산 서열로 이루어지고; 및 상기 경쇄 가변영역은 서열번호 16의 아미노산 서열로 이루어진 것일 수 있고,In the present invention, (a) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 15; And the light chain variable region may comprise the amino acid sequence of SEQ ID NO: 16,
또는, (b) 상기 중쇄 가변영역은 서열번호 25의 아미노산 서열로 이루어지고; 및 상기 경쇄 가변영역은 서열번호 26의 아미노산 서열로 이루어진 것일 수 있으나, 이에 제한되는 것은 아니다.Or (b) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 25; And the light chain variable region may comprise the amino acid sequence of SEQ ID NO: 26, but are not limited thereto.
가장 구체적으로, (a) 상기 항체는 서열번호 1의 중쇄 CDR1, 서열번호 2의 중쇄 CDR2, 및 서열번호 3의 중쇄 CDR3을 포함하는 중쇄 가변영역; 및 서열번호 4의 경쇄 CDR1, 서열번호 5의 경쇄 CDR2, 및 서열번호 6의 경쇄 CDR3을 포함하는 경쇄 가변영역을 포함하고, 상기 항체의 중쇄 가변영역은 서열번호 7의 중쇄 FR1, 서열번호 8의 중쇄 FR2, 서열번호 9의 중쇄 FR3 및 서열번호 10의 중쇄 FR4를 포함하고; 및 상기 항체의 경쇄 가변영역은 서열번호 11의 경쇄 FR1, 서열번호 12의 경쇄 FR2, 서열번호 13의 경쇄 FR3 및 서열번호 14의 경쇄 FR4를 포함하고, 상기 중쇄 가변영역은 서열번호 15의 아미노산 서열로 이루어지고; 및 상기 경쇄 가변영역은 서열번호 16의 아미노산 서열로 이루어진 것일 수 있으며,(A) the heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 2, and the heavy chain CDR3 of SEQ ID NO: 3; And a light chain variable region comprising light chain CDR1 of SEQ ID NO: 4, light chain CDR2 of SEQ ID NO: 5, and light chain CDR3 of SEQ ID NO: 6, wherein the heavy chain variable region of the antibody comprises heavy chain FR1 of SEQ ID NO: 7, SEQ ID NO: 8 A heavy chain FR2 of SEQ ID NO: 9, and a heavy chain FR4 of SEQ ID NO: 10; And wherein the light chain variable region comprises the light chain FR1 of SEQ ID NO: 11, the light chain FR2 of SEQ ID NO: 12, the light chain FR3 of SEQ ID NO: 13 and the light chain FR4 of SEQ ID NO: 14, ≪ / RTI > And the light chain variable region may comprise the amino acid sequence of SEQ ID NO: 16,
또는, (b) 상기 항체는 서열번호 1의 중쇄 CDR1, 서열번호 2의 중쇄 CDR2, 및 서열번호 3의 중쇄 CDR3을 포함하는 중쇄 가변영역; 및 서열번호 4의 경쇄 CDR1, 서열번호 5의 경쇄 CDR2, 및 서열번호 6의 경쇄 CDR3을 포함하는 경쇄 가변영역을 포함하고, 상기 항체의 중쇄 가변영역은 서열번호 17의 중쇄 FR1, 서열번호 18의 중쇄 FR2, 서열번호 19의 중쇄 FR3 및 서열번호 20의 중쇄 FR4를 포함하고; 및 상기 항체의 경쇄 가변영역은 서열번호 21의 경쇄 FR1, 서열번호 22의 경쇄 FR2, 서열번호 23의 경쇄 FR3 및 서열번호 24의 경쇄 FR4를 포함하고, 상기 중쇄 가변영역은 서열번호 25의 아미노산 서열로 이루어지고; 및 상기 경쇄 가변영역은 서열번호 26의 아미노산 서열로 이루어진 것일 수 있으나, 이에 제한되는 것은 아니다.Or (b) the antibody comprises a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 2, and the heavy chain CDR3 of SEQ ID NO: 3; And a light chain variable region comprising light chain CDR1 of SEQ ID NO: 4, light chain CDR2 of SEQ ID NO: 5, and light chain CDR3 of SEQ ID NO: 6, wherein the heavy chain variable region comprises heavy chain FR1 of SEQ ID NO: 17, SEQ ID NO: 18 A heavy chain FR2 of SEQ ID NO: 19, and a heavy chain FR4 of SEQ ID NO: 20; And wherein the light chain variable region comprises the light chain FR1 of SEQ ID NO: 21, the light chain FR2 of SEQ ID NO: 22, the light chain FR3 of SEQ ID NO: 23, and the light chain FR4 of SEQ ID NO: 24, ≪ / RTI > And the light chain variable region may comprise the amino acid sequence of SEQ ID NO: 26, but are not limited thereto.
본 발명에서 이용되는 서열은, 생물학적으로 균등 활성을 갖는 변이를 고려한다면, 서열목록에 기재된 서열과 실질적인 동일성(substantial identity)을 나타내는 서열도 포함하는 것으로 해석된다. 상기 용어, '실질적인 동일성'은 본 발명의 서열과 임의의 다른 서열을 최대한 대응되도록 얼라인(align)하고, 당업계에서 통상적으로 이용되는 알고리즘을 이용하여 얼라인된 서열을 분석한 경우에, 최소 80%의 상동성, 가장 구체적으로 90%의 상동성을 나타내는 서열을 의미한다.The sequences used in the present invention are interpreted to include sequences showing substantial identity with the sequences listed in the sequence listing, considering mutations having biologically equivalent activity. The term " substantial identity " means aligning the sequence of the present invention to any other sequence as much as possible, and when the aligned sequence is analyzed using an algorithm commonly used in the art, 80% homology, most specifically 90% homology.
따라서, 본 발명에서 구체적으로 제시하고 있는 항체와 생물학적으로 균등한 활성을 나타낸다면, 상기 서열번호 1 내지 26로 표시되는 서열과 높은 상동성을 갖는 아미노산 서열, 예를 들면 그 상동성이 80% 이상, 더욱 구체적으로 90% 이상의 높은 상동성을 갖는 아미노산 서열도 본 발명의 범위에 포함되는 것으로 해석되어야 한다.Therefore, it is preferable that an amino acid sequence having high homology with the sequence shown in SEQ ID NOS: 1 to 26, for example, a homologous activity of 80% or more , And more particularly, amino acid sequences having high homology of 90% or more should be interpreted as being included in the scope of the present invention.
본 발명의 구체적인 일 실시예에서는, MIC-1 단백질에 결합하는 항-MIC-1 마우스 IgG 단일클론항체는 MIC-1 단백질에 의해 생성된 내피세포의 신생 혈관을 유의하게 감소시키고(도 1), 피부암, 대장암, 유방암 또는 전립선암의 성장을 억제하며, 이들 조직 내부/외부의 혈관신생을 억제함을 확인하였다(도 2 내지 5). 또한, MIC-1 단백질에 높은 특이도로 결합하는 항-MIC-1 인간화 IgG 단일클론항체를 제조하였고(도 6 및 7), 상기 인간화 항체는 MIC-1 단백질에 의해 생성된 내피세포의 신생 혈관을 유의하게 감소시키고(도 8 내지 11), 피부암, 대장암, 유방암, 전립선암, 췌장암, 간암 또는 위암의 성장을 억제하며, 이들 조직 내부/외부의 혈관신생을 억제하고 암세포의 증식을 억제함을 확인하였다(도 12 내지 24). In one specific embodiment of the present invention, the anti-MIC-1 mouse IgG monoclonal antibody that binds to the MIC-1 protein significantly reduces the angiogenic vasculature of the endothelial cells produced by the MIC-1 protein (Figure 1) Skin cancer, colon cancer, breast cancer or prostate cancer, and inhibits angiogenesis inside / outside these tissues (Figs. 2 to 5). In addition, an anti-MIC-1 humanized IgG monoclonal antibody that specifically binds to the MIC-1 protein was prepared (FIGS. 6 and 7), and the humanized antibody was incubated with the neovasculature of endothelial cells produced by the MIC- Inhibit the growth of skin cancer, colorectal cancer, breast cancer, prostate cancer, pancreatic cancer, liver cancer or stomach cancer, inhibit angiogenesis inside / outside these tissues, and inhibit the proliferation of cancer cells (Figs. 12 to 24).
이는, 본 발명의 MIC-1 단백질에 결합하는 항체는 MIC-1 단백질이 과발현된 질환, 예를 들어 암의 진단 또는 치료 등에 유용하게 사용될 수 있음을 시사하는 것이다.This suggests that the antibody that binds to the MIC-1 protein of the present invention can be usefully used for the diagnosis or treatment of diseases in which the MIC-1 protein is overexpressed, for example, cancer.
다른 하나의 양태는 상기 항체를 코딩하는 폴리뉴클레오티드, 상기 폴리뉴클레오티드를 포함하는 발현 벡터 및 상기 발현 벡터가 도입된 형질전환체를 제공한다.Another embodiment provides a polynucleotide encoding the antibody, an expression vector comprising the polynucleotide, and a transformant into which the expression vector is introduced.
이때, 상기 항체의 설명에 대해서는 전술한 바와 같다.At this time, the description of the antibody is as described above.
본 발명에서, 상기 항체를 코딩하는 폴리뉴클레오티드를 포함하는 발현벡터는 특별히 이에 제한되지 않으나, 포유류 세포(예를 들어, 사람, 원숭이, 토끼, 래트, 햄스터, 마우스 세포 등), 식물 세포, 효모 세포, 곤충 세포 또는 박테리아 세포(예를 들어, 대장균 등)를 포함하는 진핵 또는 원핵세포에서 상기 폴리뉴클레오티드를 복제 및/또는 발현할 수 있는 벡터가 될 수 있고, 바람직하게는 숙주세포에서 상기 뉴클레오티드가 발현될 수 있도록 적절한 프로모터에 작동가능하도록 연결되며, 적어도 하나의 선별마커를 포함하는 벡터가 될 수 있다. 그 예로 파아지, 플라스미드, 코스미드, 미니-염색체, 바이러스 또는 레트로바이러스벡터 등에 상기 폴리뉴클레오티드가 도입된 형태가 될 수 있다. In the present invention, the expression vector comprising the polynucleotide encoding the antibody is not particularly limited, but may be mammalian cells (e.g., human, monkey, rabbit, rat, hamster, mouse cells, etc.), plant cells, yeast cells , A vector capable of replicating and / or expressing the polynucleotide in an eukaryotic or prokaryotic cell comprising an insect cell or a bacterial cell (e. G., Escherichia coli, etc.), preferably the expression of the nucleotide in the host cell And may be a vector comprising at least one selectable marker operably linked to a suitable promoter so as to be able to be expressed. For example, the polynucleotide may be introduced into a phage, a plasmid, a cosmid, a mini-chromosome, a virus, or a retrovirus vector.
상기 항체를 코딩하는 폴리뉴클레오티드를 포함하는 발현벡터는, 상기 항체의 중쇄 또는 경쇄를 코딩하는 폴리뉴클레오티드를 각각 포함하는 발현벡터 또는 중쇄 또는 경쇄를 코딩하는 폴리뉴클레오티드를 모두 포함하는 발현벡터일 수 있다.The expression vector comprising the polynucleotide encoding the antibody may be an expression vector comprising an expression vector containing a polynucleotide encoding the heavy or light chain of the antibody or a polynucleotide encoding the heavy or light chain, respectively.
본 발명에서, 상기 발현 벡터가 도입된 형질전환체는 특별히 이에 제한되지 않으나, 상기 발현벡터가 도입되어 형질전환된 대장균, 스트렙토미세스, 살모넬라 티피뮤리움 등의 박테리아 세포; 효모 세포; 피치아 파스토리스 등의 균류세포; 드로조필라, 스포도프테라 Sf9 세포 등의 곤충 세포; CHO(중국 햄스터 난소 세포, chinese hamster ovary cells), SP2/0(마우스 골수종), 인간 림프아구(human lymphoblastoid), COS, NSO(마우스 골수종), 293T, 보우 멜라노마 세포, HT-1080, BHK(베이비 햄스터 신장세포, baby hamster kidney cells), HEK(인간 배아신장 세포, human embryonic kidney cells), PERC.6(인간망막세포) 등의 동물 세포; 또는 식물 세포가 될 수 있다. In the present invention, the transformant into which the expression vector is introduced is not particularly limited, but bacterial cells such as Escherichia coli, Streptomyces and Salmonella typhimurium transformed with the expression vector introduced therein; Yeast cells; Fungal cells such as Pichia pastoris; Insect cells such as Drosophila and Spodoptera Sf9 cells; CHO (Chinese hamster ovary cells), SP2 / 0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), 293T, Bowmanella cells, HT-1080, BHK Animal hamster kidney cells, HEK (human embryonic kidney cells), and PERC.6 (human retinal cells); Or plant cells.
본 발명의 용어, "도입"은 상기 항체를 코딩하는 폴리뉴클레오티드를 포함하는 벡터를 숙주세포에 전달하는 방법을 의미한다. 이와 같은 도입은 칼슘 포스페이트-DNA 공침전법, DEAE-덱스트란-매개 트랜스펙션법, 폴리브렌-매개 형질감염법, 전기충격법, 미세주사법, 리포좀 융합법, 리포펙타민 및 원형질체 융합법 등의 당 분야에 공지된 여러 방법에 의해 수행될 수 있다. 또한, 형질도입은 감염(infection)을 수단으로 하여 바이러스 입자를 사용하여 목적물을 세포 내로 전달시키는 것을 의미한다. 아울러, 유전자 밤바드먼트(bombardment) 등에 의해 벡터를 숙주세포 내로 도입할 수 있다. 본 발명에서 도입은 형질전환 또는 형질주입과 혼용되어 사용될 수 있다.The term " introduction " of the present invention means a method of delivering a vector comprising a polynucleotide encoding the antibody to a host cell. Such introduction may be accomplished by methods such as calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposomal fusion, lipofectamine and protoplast fusion Can be carried out by various methods known in the art. Transfection also means that an object is transferred into a cell using viral particles by means of infection. In addition, vectors can be introduced into host cells by gene bombardment or the like. Introduction in the present invention can be used in combination with transformation or transfection.
또 다른 하나의 양태는 상기 항체를 포함하는 조성물을 제공한다. 구체적으로, 상기 항체를 포함하는 암의 예방 또는 치료용 약학 조성물을 제공한다.Another embodiment provides a composition comprising the antibody. Specifically, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the antibody.
상기 항체는 암에서 과발현되는 MIC-1 단백질에 매우 높은 특이도를 나타내며, MIC-1 단백질에 의해 생성된 내피세포의 신생 혈관을 유의하게 감소시키고, 피부암, 대장암, 유방암, 전립선암, 췌장암, 간암 또는 위암의 성장을 억제하며, 이들 조직 내부 또는 외부의 혈관신생을 억제할 수 있으므로, MIC-1 단백질이 과발현된 질환, 예를 들어 암의 예방 또는 치료 효과를 나타낼 수 있다. 이때, 상기 항체의 설명에 대해서는 전술한 바와 같다.The antibody shows very high specificity to the MIC-1 protein overexpressed in the cancer and significantly reduces the angiogenic vasculature of the endothelial cells produced by the MIC-1 protein, and is useful for the treatment of skin cancer, colon cancer, breast cancer, prostate cancer, pancreatic cancer, Can inhibit the growth of liver cancer or stomach cancer, inhibit angiogenesis inside or outside of these tissues, and thus can exhibit a preventive or therapeutic effect of a disease overexpressing MIC-1 protein, for example, cancer. At this time, the description of the antibody is as described above.
본 발명의 용어, "암"은 신체 조직의 자율적인 과잉 성장에 의해 비정상적으로 자라난 덩어리로서, 본 발명의 항체에 의해 예방 또는 치료될 수 있는 암이라면 그 종류에 제한 없이 포함하나, 구체적인 예로 피부암(구체적으로 피부흑색종), 대장암, 유방암, 전립선암, 췌장암, 위암, 담도암, 식도암, 직장암, 구강암, 인두암, 후두암, 폐암, 결장암, 난소암, 자궁경부암, 자궁내막암, 고환암, 방광암, 신장암, 간암, 골암, 결합 조직암, 뇌암, 갑상선암, 백혈병, 호지킨(Hodgkin) 질환, 림프종 또는 다발성 골수종 혈액암일 수 있다. The term " cancer " of the present invention refers to a mass that is abnormally grown by autonomous overgrowth of body tissue, and includes any kind of cancer that can be prevented or treated by the antibody of the present invention, Cancer of the uterus, endometrial cancer, endometrial cancer, endometrial cancer, endometrial carcinoma, endometrial carcinoma, endometrial carcinoma, endometrial carcinoma, endometrial carcinoma, endometrial carcinoma, endometrial cancer, Cancer of the bladder, kidney, liver, bone, connective tissue, brain, thyroid, leukemia, Hodgkin's disease, lymphoma or multiple myeloma.
본 발명의 용어, "암의 예방"은 상기 조성물의 투여에 의해 암의 발병을 억제하거나 지연시키는 모든 행위를 의미한다.The term " prevention of cancer " of the present invention means all actions that inhibit or delay the onset of cancer by administration of the composition.
본 발명의 용어, "암의 치료"는 상기 조성물의 투여에 의해 암의 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다.The term " treatment of cancer " of the present invention means all the actions of improving or ameliorating symptoms of cancer by the administration of the composition.
또 다른 하나의 양태는 상기 항체를 포함하는 조성물을 제공한다. 구체적으로, 상기 항체를 포함하는 혈관 신생 억제용 약학 조성물을 제공한다.Another embodiment provides a composition comprising the antibody. Specifically, there is provided a pharmaceutical composition for inhibiting angiogenesis comprising the antibody.
본 발명의 조성물에 의해 예방 또는 치료될 수 있는 질병, 질환 또는 상태는 혈관신생과 관련된 다양한 질환을 포함한다. 구체적으로, 암, 아테롬성 동맥경화, 다발성골수종 뼈질환(multiple myeloma bone disease), 근위축증(muscle atrophy) 또는 당뇨병 합병증이다. 상술한 질환들에서는 MIC-1 단백질이 과발현되어 있을 수 있고, 또한 상기 MIC-1 단백질의 발현수준을 감소시키면 질환의 치료가 달성될 수 있다.Diseases, diseases or conditions that can be prevented or treated by the composition of the present invention include various diseases associated with angiogenesis. Specifically, it is cancer, atherosclerosis, multiple myeloma bone disease, muscle atrophy or diabetic complication. Treatment of the disease can be achieved by the MIC-1 protein overexpression in the above-mentioned diseases, and by decreasing the expression level of the MIC-1 protein.
상기 약학 조성물은 약학적으로 허용가능한 담체를 추가로 포함할 수 있고, 상기 담체는 비자연적 담체(non-naturally occuring carrier)를 포함할 수 있다.The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier and the carrier may comprise a non-naturally occuring carrier.
본 발명의 용어, "약학적으로 허용가능한 담체"는 생물체를 자극하지 않고 투여 화합물의 생물학적 활성 및 특성을 저해하지 않는 담체 또는 희석제를 의미한다. 액상 용액으로 제제화되는 조성물에 허용되는 약학적 담체로는, 멸균 및 생체에 적합한 것으로서, 식염수, 멸균수, 링거액, 완충 식염수, 알부민 주사용액, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 하나 이상의 성분을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한, 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다. The term " pharmaceutically acceptable carrier " of the present invention means a carrier or diluent that does not irritate the organism and does not interfere with the biological activity and properties of the administered compound. Examples of the pharmaceutical carrier acceptable for the composition to be formulated into a liquid solution include sterilized and sterile water, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, And other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added and formulated into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
상기 약학적 조성물은 정제, 환제, 산제, 과립제, 캡슐제, 현탁제, 내용액제, 유제, 시럽제, 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제 및 좌제으로 이루어진 군으로부터 선택되는 어느 하나의 제형을 가질 수 있고, 경구 또는 비경구의 여러 가지 제형일 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 하나 이상의 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 탄산칼슘, 수크로오스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 스테아린산 마그네슘, 탈크 등과 같은 윤활제들도 사용된다. 경구투여를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테로 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.The pharmaceutical composition may be in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories May have one formulation, and may be oral or parenterally various formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.
또한, 상기 약학적 조성물은 약학적으로 유효한 양으로 투여될 수 있다.In addition, the pharmaceutical composition may be administered in a pharmaceutically effective amount.
본 발명에서 용어, "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 개체 종류 및 중증도, 연령, 성별, 암의 종류, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있다. 그리고 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 당업자에 의해 용이하게 결정될 수 있다.The term " pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dosage level will vary depending on the species and severity, age, sex, The type of drug, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.
본 발명의 구체적인 일 실시예에서는, MIC-1 단백질에 높은 특이도로 결합하는 항-MIC-1 마우스/인간화 IgG 단일클론항체는 MIC-1 단백질에 의해 생성된 내피세포의 신생 혈관을 유의하게 감소시키고, 피부암, 대장암, 유방암, 전립선암, 췌장암, 간암 또는 위암의 성장을 억제하며, 이들 조직 내부/외부의 혈관신생을 억제하고 암세포의 증식을 억제함을 확인하였다(도 1 내지 5, 및 도 8 내지 24).In one specific embodiment of the present invention, the anti-MIC-1 mouse / humanized IgG monoclonal antibody that specifically binds to the MIC-1 protein significantly reduces endothelial cell neovascularization produced by the MIC-1 protein , Skin cancer, colon cancer, breast cancer, prostate cancer, pancreatic cancer, liver cancer, or stomach cancer, inhibiting angiogenesis in the inside / outside of these tissues and inhibiting proliferation of cancer cells (Figs. 1 to 5 and 8 to 24).
이는, 본 발명의 MIC-1 단백질에 결합하는 항체는 암의 예방 또는 치료 용도, 또한 혈관 신생 억제 용도로서 유용하게 사용될 수 있음을 시사하는 것이다.This suggests that an antibody that binds to the MIC-1 protein of the present invention can be usefully used for prevention or treatment of cancer, and also for inhibiting angiogenesis.
또 다른 하나의 양태는 상기 항체를 이를 필요로 하는 개체에 투여하는 단계를 포함하는, 암의 예방 또는 치료 방법을 제공한다.Another embodiment provides a method of preventing or treating cancer, comprising administering the antibody to a subject in need thereof.
이때, 상기 항체 및 암의 설명에 대해서는 전술한 바와 같다.The description of the antibody and cancer is as described above.
본 발명의 용어, "개체"는 암이 발병될 가능성이 있거나 또는 발병된 쥐, 소, 돼지, 양, 닭, 개, 인간 등을 포함하는 포유동물, 조류 등을 포함하며, 본 발명의 약학 조성물의 투여에 의해 암이 치료되는 개체는 제한 없이 포함한다.The term " individual " of the present invention includes mammals, birds, and the like, including, but not limited to, mice, cows, pigs, sheep, chickens, dogs, Include, but are not limited to, individuals to whom the cancer is to be treated.
본 발명의 암의 예방 또는 치료 방법에서, 상기 조성물을 투여하는 투여 경로 및 투여 방식은 특별히 제한되지 않으며, 목적하는 해당 부위에 상기 조성물이 도달할 수 있는 한, 임의의 투여 경로 및 투여 방식에 따를 수 있다. In the method for preventing or treating cancer of the present invention, the administration route and method of administering the composition are not particularly limited and may be appropriately selected depending on the administration route and administration mode as long as the composition can reach the desired site .
구체적으로, 상기 조성물은 경구 또는 비경구의 다양한 경로를 통하여 투여될 수 있으며, 그 투여 경로의 비제한적인 예로는, 구강, 직장, 국소, 정맥내, 복강내, 근육내, 동맥내, 경피, 비측내 또는 흡입 등을 통하여 투여되는 것을 들 수 있다. 또한, 상기 조성물은 활성 물질이 표적 세포로 이동할 수 있는 임의의 장치에 의해 투여될 수 있다. Specifically, the composition may be administered orally or parenterally through various routes. Non-limiting examples of routes of administration include oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, Intramuscularly or through inhalation or the like. In addition, the composition may be administered by any device capable of transferring the active agent to the target cell.
또 다른 하나의 양태는 상기 항체를 포함하는, 암 진단용 조성물을 제공한다.Another embodiment provides a cancer diagnostic composition comprising the antibody.
이때, 상기 항체 및 암의 설명에 대해서는 전술한 바와 같다.The description of the antibody and cancer is as described above.
본 발명의 암 진단용 조성물은 다양한 종류의 암세포에서 과발현되는 MIC-1 단백질에 특이적으로 결합하는 항체들을 포함하므로, MIC-1 단백질의 발현 여부나 발현 정도와 관련된 질환, 예컨대 암의 진단에 유용하게 사용될 수 있다.Since the composition for cancer diagnosis of the present invention includes antibodies specifically binding to MIC-1 protein overexpressed in various kinds of cancer cells, it is useful for diagnosis of diseases related to the expression or expression level of MIC-1 protein such as cancer Can be used.
또 다른 하나의 양태는 상기 조성물을 포함하는, 암 진단용 키트를 제공한다.Another embodiment provides a cancer diagnostic kit comprising the composition.
이때, 상기 조성물 및 암의 설명에 대해서는 전술한 바와 같다.At this time, the composition and the description of the cancer are as described above.
본 발명의 암 진단용 키트는 분석방법에 적합한 한 종류 또는 그 이상의 다른 구성성분을 가진 조성물, 용액 또는 장치를 더 포함하여 구성될 수 있다.The cancer diagnostic kit of the present invention may further comprise a composition, solution or device having one or more other components suitable for the assay method.
또 다른 하나의 양태는 상기 항체를 이용하여, 암이 의심되는 개체의 분리된 생물학적 시료에서 MIC-1(macrophage inhibitory cytokine 1) 단백질을 항원-항체 반응을 통하여 검출하는 단계를 포함하는, 암의 진단 방법을 제공한다.Another embodiment provides a method for diagnosing cancer, comprising the step of detecting the MIC-1 (macrophage inhibitory cytokine 1) protein in an isolated biological sample of a subject suspected of having cancer using the antibody, through an antigen- ≪ / RTI >
이때, 상기 항체, 개체, MIC-1 단백질 및 암의 설명에 대해서는 전술한 바와 같다.Here, the description of the antibody, the individual, the MIC-1 protein and the cancer is as described above.
MIC-1 단백질은 다양한 종류의 암세포에서 과발현된다고 알려져 있으므로, MIC-1 단백질에 높은 특이도로 결합하는 본 발명의 항체는 MIC-1 단백질의 발현 여부나 발현 정도와 관련된 질환, 예컨대 암의 진단에 유용하게 사용될 수 있다.Since the MIC-1 protein is known to be overexpressed in various kinds of cancer cells, the antibody of the present invention, which specifically binds to the MIC-1 protein, is useful for diagnosis of a disease associated with the expression or degree of expression of the MIC-1 protein, Lt; / RTI >
상기 암의 진단 방법은, 본 발명의 MIC-1에 특이적인 항체를 암이 의심되는 개체의 분리된 생물학적 시료와 반응시키고 항원-항체 복합체 형성을 검출함으로써 MIC-1 단백질을 검출할 수 있으며, 이를 통해 암을 진단할 수 있다.The diagnostic method of cancer can detect the MIC-1 protein by reacting the antibody specific to MIC-1 of the present invention with a separated biological sample suspected of having cancer and detecting antigen-antibody complex formation, Cancer can be diagnosed.
구체적으로, (a) 분리된 상기 항체를 암이 의심되는 개체의 분리된 생물학적 시료에 처리하여 MIC-1 단백질을 항원-항체 반응을 통하여 검출하는 단계; (b) 상기 (a)에서 검출된 MIC-1 단백질의 수준을 대조군과 비교하여, 대조군에 비하여 MIC-1 단백질 수준이 높으면 암 환자로 판정하는 단계를 포함하는, 암의 진단 방법일 수 있다. (A) treating the isolated antibody with a separated biological sample of a suspected cancer patient to detect MIC-1 protein through an antigen-antibody reaction; (b) comparing the level of the MIC-1 protein detected in (a) with that of the control, and determining that the level of MIC-1 protein is higher than that of the control, the cancer patient.
본 발명의 용어, "생물학적 시료"는 조직, 세포, 전혈, 혈청, 혈장, 조직 부검 시료(뇌, 피부, 림프절, 척수 등), 세포 배양 상등액, 파열된 진핵세포 및 세균 발현계 등을 들 수 있으나, 이에 제한되는 것은 아니다. 이들 생물학적 시료를 조작하거나 조작하지 않은 상태로 본 발명의 항체와 반응시켜 MIC-1 단백질의 존재 또는 암의 유무를 확인할 수 있다.The term "biological sample" of the present invention includes tissues, cells, whole blood, serum, plasma, tissue autopsy samples (brain, skin, lymph nodes, spinal cord, etc.), cell culture supernatant, ruptured eukaryotic cells and bacterial expression systems But is not limited thereto. These biological samples can be reacted with the antibody of the present invention without manipulation or manipulation to confirm the presence or absence of MIC-1 protein.
본 발명의 용어, "항원-항체 복합체"는 시료 중의 MIC-1 단백질 항원과 이를 인지하는 본 발명의 항체의 결합물을 의미하며, 이러한 항원-항체 복합체의 형성은 비색법(colormetric method), 전기화학법(electrochemical method), 형광법(fluorimetric method), 발광법(luminometry), 입자계수법(particle counting method), 육안측정법(visual assessment) 또는 섬광계수법(scintillation counting method) 등의 임의의 방법으로 검출할 수 있다. 그러나 반드시 이들로만 제한되지 않고 다양한 응용과 적용이 가능하다.The term " antigen-antibody complex " of the present invention refers to a combination of the MIC-1 protein antigen and the antibody of the present invention recognizing the MIC-1 protein antigen in the sample. The formation of such an antigen-antibody complex can be performed by a colorimetric method, Can be detected by any method such as an electrochemical method, a fluorimetric method, a luminometry, a particle counting method, a visual assessment method, or a scintillation counting method . However, various applications and applications are possible without being limited thereto.
상기 항원-항체 복합체를 검출하기 위하여 여러 가지 표지체를 사용할 수 있다. 구체적인 예로, 효소, 형광물, 리간드, 발광물, 미소입자 또는 방사성 동위원소 등을 사용할 수 있으나, 이에 제한되는 것은 아니다. 이때, 검출 표지체로서 사용되는 효소로는 아세틸콜린에스테라제, 알칼라인 포스파타제, β-D-갈락토시다제, 호스래디쉬 퍼옥시다제, β-라타마제 등을 포함하며, 형광물로는 플루오레세인, Eu3+, Eu3+ 킬레이트 또는 크립테이트 등을 들 포함하며, 리간드로는 바이오틴 유도체 등을 포함하며, 발광물로는 아크리디늄 에스테르, 이소루미놀 유도체 등을 포함하며, 미소입자로는 콜로이드 금, 착색된 라텍스 등을 포함하며, 방사성 동위원소로는 57Co, 3H, 125I, 125I-볼톤(Bonton) 헌터(Hunter) 시약 등을 포함한다.A variety of markers can be used to detect the antigen-antibody complex. As specific examples, enzymes, chromophores, ligands, emitters, microparticles or radioisotopes can be used, but the present invention is not limited thereto. Examples of the enzyme used as a detection label include acetylcholinesterase, alkaline phosphatase,? -D-galactosidase, horseradish peroxidase,? -Lactamase, and the like. Eu 3+ , Eu 3+ chelate, or cryptate, and the ligand includes a biotin derivative and the like. Examples of the luminescent material include an acridinium ester, an isoluminol derivative and the like, and fine particles Colloidal gold, colored latex and the like, and the radioactive isotope includes 57 Co, 3 H, 125 I, 125 I-Bonton Hunter reagent and the like.
또 다른 하나의 양태는 (a) 암세포에 암의 예방 또는 치료 후보물질을 처리하는 단계; (b) 상기 후보물질이 처리된 암세포에서 상기 항체를 이용하여, MIC-1(macrophage inhibitory cytokine 1) 단백질의 수준을 측정하는 단계; 및 (c) 상기 단계 (b)의 MIC-1 단백질 수준이 후보물질을 처리하지 않은 암세포의 것보다 감소하는 경우, 상기 (a)단계에서 처리한 후보물질을 암의 예방 또는 치료 물질로 판단하는 단계를 포함하는, 암의 예방 또는 치료 물질의 스크리닝 방법을 제공한다.(A) treating the cancer cell with a candidate substance for preventing or treating cancer; (b) measuring the level of MIC-1 (macrophage inhibitory cytokine 1) protein using the antibody in cancer cells treated with the candidate substance; And (c) when the MIC-1 protein level of the step (b) is lower than that of the cancer cells not treated with the candidate substance, the candidate substance treated in the step (a) A method for screening a preventive or therapeutic agent for cancer.
이때, 상기 항체, MIC-1 단백질 및 암의 설명에 대해서는 전술한 바와 같다.The description of the antibody, MIC-1 protein and cancer is as described above.
본 발명의 용어, "암의 예방 또는 치료 후보물질"은 암을 치료할 수 있을 것으로 예상되는 물질로서, 직접 또는 간접적으로 암을 호전 또는 개선 시킬 수 있을 것으로 예상되는 물질이면 제한 없이 사용 가능하며, 화합물, 유전자 또는 단백질 등의 치료가능 예상물질을 모두 포함한다.The term " prophylactic or therapeutic candidates for cancer " of the present invention is a substance expected to treat cancer, and can be used without limitation as long as it is a substance that is expected to directly or indirectly improve or improve cancer. , A gene or protein, and the like.
본 발명에서, 암세포에 암의 예방 또는 치료 후보물질을 처리하는 상기 (a) 단계는 당업계에 공지된 방법을 사용하여 수행될 수 있다. 구체적인 예로, 암세포에 상기 후보물질을 처리하여 함께 배양하거나, 또는 암세포를 포함하는 생체 내에 상기 후보물질을 투여할 수 있으나, 이에 제한되는 것은 아니며, 당업자라면 본 발명의 목적에 맞는 방법을 사용할 수 있을 것이다. In the present invention, the step (a) of treating a cancer cell with a candidate substance for preventing or treating cancer can be carried out using a method known in the art. As a specific example, the candidate substance may be treated with the cancer cell and cultured together, or the candidate substance may be administered to a living body including cancer cells, but the present invention is not limited thereto. Those skilled in the art can use a method suitable for the purpose of the present invention will be.
또한, MIC-1 단백질의 수준을 측정하는 상기 (b) 단계는 당업자에게 알려진 어떠한 방법이든 사용 가능하다. 예컨대, MIC-1 단백질과 항-MIC-1 항체의 항원-항체 복합체(antigen-antibody complex)의 형성을 검출하는 단계일 수 있다. 또한 구체적인 예로, 웨스턴블롯(Western blot), 공동-면역침전 어세이(Co-Immunoprecipitation assay), ELISA(Enzyme Linked Immunosorbent Assay), 조직 면역염색(immunostaining) 및 FACS(Fluorescence activated cell sorter) 등이 사용될 수 있으나, 이에 제한되는 것은 아니며, 당업자라면 본 발명의 목적에 맞는 방법을 사용할 수 있을 것이다. Further, the step (b) for measuring the level of MIC-1 protein can be used by any method known to a person skilled in the art. For example, it may be a step of detecting the formation of an antigen-antibody complex of an MIC-1 protein and an anti-MIC-1 antibody. Specific examples include Western blot, Co-immunoprecipitation assay, ELISA, immunostaining, and FACS (Fluorescence activated cell sorter). However, the present invention is not limited thereto, and a person skilled in the art can use a method suited to the purpose of the present invention.
마지막으로, 상기 (c) 단계는 상기 후보물질을 암의 예방 또는 치료 물질로서 사용할 수 있는지 판단하는 단계로서, MIC-1 단백질은 암세포에서 과발현되며, 혈관신생을 촉진하고, 암세포의 성장 및 발달을 촉진하므로, MIC-1 단백질의 수준을 감소시키는 후보물질은 암의 예방 또는 치료 물질로서 사용될 수 있다.Finally, the step (c) is a step of determining whether the candidate substance can be used as a preventive or therapeutic agent for cancer, wherein the MIC-1 protein is overexpressed in cancer cells, promotes angiogenesis, A candidate substance that decreases the level of MIC-1 protein can be used as a preventive or therapeutic agent for cancer.
이하, 실시예를 통하여 본 발명을 더욱 상세하게 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and the scope of the present invention is not construed as being limited by these embodiments.
실시예 1. 항-인간 MIC-1 마우스 IgG 단일클론항체의 활성 확인Example 1. Identification of activity of anti-human MIC-1 mouse IgG monoclonal antibody
실시예 1-1. 항-인간 MIC-1 마우스 IgG 단일클론항체의 구축Example 1-1. Construction of anti-human MIC-1 mouse IgG monoclonal antibody
먼저, 인간 MIC-1 단백질을 Balb/c 마우스에 주입하여 항-인간-MIC-1 항체를 생산하는 마우스를 제조하였다. 상기 마우스로부터 지라세포(splenocyte)를 획득한 후, 이를 골수종(myeloma) 세포와 융합시켜서 혼성세포(hybridoma)를 제조하였고, HAT 배지를 이용하여 혼성세포만을 선택 배양하였다. 상기 혼성세포로부터 산출된 항체에 대해 ELISA를 수행함으로써 항-인간 MIC-1 항체를 생산하는 혼성세포 단일클론을 선별하였고, 상기 혼성세포 단일클론의 배양액에 존재하는 항체를 단백질-G 크로마토그래피(Protein G-agarose column chromatography)를 이용하여 정제함으로써 항-인간 MIC-1 마우스 IgG 단일클론항체를 수득하였다.First, a human MIC-1 protein was injected into a Balb / c mouse to produce an anti-human-MIC-1 antibody-producing mouse. Splenocytes were obtained from the mice, and hybridoma cells were prepared by fusing them with myeloma cells. Hybrid cells were selectively cultured using HAT medium. The hybridoma cell monoclon producing the anti-human MIC-1 antibody was selected by performing ELISA on the antibody produced from the hybrid cells, and the antibody present in the hybridoma cell monoclonal culture was subjected to protein-G chromatography G-agarose column chromatography to obtain an anti-human MIC-1 mouse IgG monoclonal antibody.
실시예 1-2. 혈관신생(angiogenesis) 억제 활성 확인Examples 1-2. Identification of inhibitory activity of angiogenesis
항-인간 MIC-1 마우스 IgG 단일클론항체(이하, "항-MIC-1 마우스 항체"로 명명)의 항암제로서의 활성을 확인하기 위하여, 먼저 혈관신생(angiogenesis) 억제 활성을 확인하였다. 이때, 상기 항-MIC-1 마우스 항체의 중쇄 및 경쇄 가변영역, CDR, 및 FR 서열은 하기 표 1에 기재하였다.In order to confirm the activity of anti-human MIC-1 mouse IgG monoclonal antibody (hereinafter referred to as " anti-MIC-1 mouse antibody ") as an anticancer agent, the angiogenesis inhibitory activity was first confirmed. The heavy and light chain variable regions, CDRs, and FR sequences of the anti-MIC-1 mouse antibody are shown in Table 1 below.
항-MIC-1 마우스 항체의 아미노산 서열The amino acid sequence of the anti-MIC-1 mouse antibody
항체명Antibody name 구분division 서열order 서열번호SEQ ID NO:
항-MIC-1 마우스 항체Anti-MIC-1 mouse antibody 중쇄가변영역(Heavy chain variable region; VH)Heavy chain variable region (VH) EVQLQQSGAELVRPGALVKLSCKASGFNIKDYYMHWVRQRPEQGLEWIGWIDPENGNTIYDPKFQGKASITTDTSSNTAYMQLSSLTCEDTAVYYCAVPDSWGQGTTLTVSSAKTTPPEVQLQQSGAELVRPGALVKLSCKASGFNIKDYYMHWVRQRPEQGLEWIGWIDPENGNTIYDPKFQGKASITTDTSSNTAYMQLSSLTCEDTAVYYCAVPDSWGQGTTLTVSSAKTTPP 1515
VH-CDR1VH-CDR1 GFNIKDYYGFNIKDYY 1One
VH-CDR2VH-CDR2 IDPENGNTIDPENGNT 22
VH-CDR3VH-CDR3 AVPDSAVPDS 33
VH-FR1VH-FR1 EVQLQQSGAELVRPGALVKLSCKASEVQLQQSGAELVRPGALVKLSCKAS 77
VH-FR2VH-FR2 MHWVRQRPEQGLEWIGWMHWVRQRPEQGLEWIGW 88
VH-FR3VH-FR3 IYDPKFQG KASITTDTSSNTAYMQLSSLTCEDTAVYYCIYDPKFQGKASITTDTSSNTAYMQLSSLTCEDTAVYYC 99
VH-FR4VH-FR4 WGQGTTLTVSSAKTTPPWGQGTTLTVSSAKTTPP 1010
경쇄가변영역(Light chain variable region; VL)Light chain variable region (VL) DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPDQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIKDIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPDQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIK 1616
VL-CDR1VL-CDR1 KSLLHSNGNTYKSLLHSNGNTY 44
VL-CDR2VL-CDR2 RMSRMS 55
VL-CDR3VL-CDR3 MQHLEYPFTMQHLEYPFT 66
VL-FR1VL-FR1 DIVMTQAAPSVPVTPGESVSISCRSSDIVMTQAAPSVPVTPGESVSISCRSS 1111
VL-FR2VL-FR2 LYWFLQRPDQSPQLLIYLYWFLQRPDQSPQLLIY 1212
VL-FR3VL-FR3 NLASGV PDRFSGSGSGTAFTLRISRVEAEDVGVYYCNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYC 1313
VL-FR4VL-FR4 FGSGTKLEIKFGSGTKLEIK 1414
구체적으로, 튜브 형성 어세이(tube formation assay) 및 대동맥 고리 가지형성 어세이(aorta ring sprouting assay)를 수행하였다.Specifically, a tube formation assay and an aorta ring sprouting assay were performed.
튜브 형성 어세이를 수행하기 위하여, 먼저 사람의 탯줄내피세포(HUVEC)를 마트리겔(matrigel)이 코팅되어 있는 배양용기에 분주한 후, 배양액 포함된 MIC-1 단백질을 정상 IgG 또는 항-MIC-1 단일클론항체와 혼합하여 첨가하였다. 이를 37℃, 5% CO2 조건에서 16시간 동안 배양한 후, 튜브 형성 정도를 관찰하고 이의 길이(tube length)를 측정하였다.In order to perform the tube formation assay, human umbilical cord endothelial cells (HUVEC) were firstly dispensed into a culture vessel coated with matrigel, and the MIC-1 protein contained in the culture medium was incubated with normal IgG or anti-MIC- 1 < / RTI > monoclonal antibody. After incubation at 37 ° C and 5% CO 2 for 16 hours, the degree of tube formation was observed and its tube length was measured.
대동맥 고리 가지형성 어세이를 수행하기 위하여, 먼저 SD-랫(rat)으로부터 분리한 대동맥을 일정 두께로 절단하여 얻은 대동맥 절편을 마트리겔로 둘러싼(embedding) 후, 정상 IgG 또는 항-MIC-1 단일클론항체와 혼합된 MIC-1이 첨가된 배양액에 담갔다. 이를 37℃, 5% CO2 조건에서 16시간 동안 배양한 후, 대동맥 절편으로부터 뻗어져 나온 내피세포의 가지(branch) 형성 정도(sprouting)를 측정하였다.To perform the aortic ring branching assay, aortic sections obtained by cutting the aorta isolated from an SD-rat to a certain thickness were embedding in a matrigel, and then a normal IgG or anti-MIC-1 single Clone antibody and MIC-1. After incubation for 16 hours at 37 ° C and 5% CO 2 , sprouting of the endothelial cells extending from the aortic slice was measured.
그 결과, 도 1의 A 및 B에서 볼 수 있듯이, 상기 항-MIC-1 마우스 항체는 MIC-1 단백질 처리에 의해 생성된 신생 혈관을 유의하게 감소시키며, 그 감소 정도는 MIC-1 단백질을 처리하지 않은 대조군과 유사함을 확인하였다.As a result, as shown in FIGS. 1A and 1B, the anti-MIC-1 mouse antibody significantly reduced the angiogenesis produced by MIC-1 protein treatment, And it is similar to the control group.
상기 결과를 통해, 항-MIC-1 마우스 항체는 혈관신생을 억제하므로, 암의 예방 또는 치료 물질로 유용하게 사용될 수 있음을 알 수 있었다.These results indicate that the anti-MIC-1 mouse antibody inhibits angiogenesis and thus can be useful as a preventive or therapeutic agent for cancer.
실시예 1-3. 피부암(melanoma)의 성장 억제 활성 확인Examples 1-3. Confirming the growth inhibitory activity of melanoma
상기 항-MIC-1 마우스 항체의 항암 효과를 확인하기 위하여, 피부암(melanoma)에 대한 성장 억제 활성을 확인하였다.In order to confirm the anti-cancer effect of the anti-MIC-1 mouse antibody, growth inhibitory activity against melanoma was confirmed.
구체적으로, B16F1 마우스 흑색종 피부암(melanoma) 세포주를 배양한 후, 배양된 세포를 마트리겔(50%)과 혼합하여 C57BL/6 마우스의 등쪽 피하에 접종(1 X 106 세포)함으로써 B16F1 피부암 세포주를 포함하는 이종이식(xenograft) 암 동물모델을 제작하였다. 이후, 생성된 종양의 크기가 100 ~ 150 mm3 정도일 때, 종양의 크기가 비슷한 마우스를 2개 그룹을 나누고, 한 그룹에는 정상 IgG를, 다른 그룹에는 항-MIC-1 단일클론항체를 3일 간격으로 꼬리정맥에 투여(100 ㎍/마리)하였다.Specifically, the B16F1 melanoma melanoma cell line was cultured, and the cultured cells were mixed with matrigel (50%) and inoculated subcutaneously (1 x 10 6 cells) in C57BL / 6 mice to subcutaneously inject B16F1 skin cancer cells Lt; RTI ID = 0.0 > xenograft < / RTI > Thereafter, when the size of the generated tumor was about 100 to 150 mm 3 , mice having two different tumor sizes were divided into two groups. One group received normal IgG and the other group received anti-MIC-1 monoclonal antibody (100 [mu] g / mouse) in the tail vein at intervals.
그 결과, 도 2의 A 내지 D에서 볼 수 있듯이, 정상 IgG 항체를 투여한 대조군에 비하여, 상기 항-MIC-1 마우스 항체를 투여하는 경우에는 피부암의 성장 속도가 감소하며, 종양의 크기 및 무게가 유의하게 감소하는 것을 확인하였다.As a result, as shown in FIGS. 2A to 2D, when the anti-MIC-1 mouse antibody was administered, the growth rate of skin cancer was decreased and the size and weight of the tumor were decreased compared to the control group to which the normal IgG antibody was administered Was significantly decreased.
또한, 도 2의 E에서 볼 수 있듯이, B16F1 종양 조직 절편을 항-CD31 항체로 면역형광 염색한 결과, 혈관 내피세포 마커인 CD31의 발현이 현저하게 감소하는 것을 확인하였다. As shown in FIG. 2E, immunofluorescence staining of the B16F1 tumor tissue section with the anti-CD31 antibody revealed that the expression of CD31, a vascular endothelial cell marker, was markedly decreased.
상기 결과를 통해, 항-MIC-1 마우스 항체는 피부암의 성장 및 이의 혈관신생을 억제하므로, 암의 예방 또는 치료 물질로 유용하게 사용될 수 있음을 알 수 있었다.These results indicate that the anti-MIC-1 mouse antibody suppresses the growth of skin cancer and its angiogenesis, and thus can be effectively used as a preventive or therapeutic agent for cancer.
실시예 1-4. 대장암의 성장 억제 활성 확인Examples 1-4. Identification of growth inhibitory activity of colon cancer
상기 항-MIC-1 마우스 항체의 항암 효과를 확인하기 위하여, 대장암에 대한 성장 억제 활성을 확인하였다.In order to confirm the anti-cancer effect of the anti-MIC-1 mouse antibody, growth inhibitory activity against colon cancer was confirmed.
구체적으로, HCT-116 대장암 세포주를 누드마우스(athymic nu/nu mouse)의 피하 부위에 각각 접종(1 X 106 세포)하여 대장암 이종이식 암 동물모델을 제작하였고, 종양이 일정 크기로 성장하면 종양의 크기가 비슷한 마우스를 2개 그룹을 나누고, 한 그룹에는 정상 IgG를, 다른 그룹에는 항-MIC-1 단일클론항체를 3일 간격으로 꼬리정맥에 투여(100 ㎍/마리)하였다.Specifically, an HCT-116 colon cancer cell line was inoculated (1 × 10 6 cells) to each subcutaneous site of athymic nu / nu mouse, and an animal model of xenograft transplantation cancer was prepared. The mice were divided into two groups, one group receiving normal IgG and the other group receiving anti-MIC-1 monoclonal antibody administered intravenously (100 μg / mouse) to the tail vein at 3-day intervals.
그 결과, 도 3의 A 내지 D에서 볼 수 있듯이, 상기 항-MIC-1 마우스 항체를 투여하는 경우에는 대장암의 성장 속도가 감소하며, 종양의 크기 및 무게가 유의하게 감소하는 것을 확인하였다.As a result, as shown in FIGS. 3A to 3D, it was confirmed that the growth rate of colorectal cancer was decreased when the anti-MIC-1 mouse antibody was administered, and the size and weight of the tumor were significantly decreased.
또한, 도 3의 E에서 볼 수 있듯이, HCT-116 종양 조직 절편을 항-CD31 항체로 면역형광 염색한 결과, 혈관 내피세포 마커인 CD31의 발현이 현저하게 감소하는 것을 확인하였다. As shown in FIG. 3E, immunofluorescence staining of HCT-116 tumor tissue sections with anti-CD31 antibody showed that the expression of CD31, a vascular endothelial cell marker, was significantly reduced.
상기 결과를 통해, 항-MIC-1 마우스 항체는 대장암의 성장 및 이의 혈관신생을 억제하므로, 암의 예방 또는 치료 물질로 유용하게 사용될 수 있음을 알 수 있었다.These results indicate that the anti-MIC-1 mouse antibody inhibits the growth of colon cancer and angiogenesis thereof, and thus can be effectively used as a preventive or therapeutic agent for cancer.
실시예 1-5. 유방암의 성장 억제 활성 확인Examples 1-5. Identification of growth inhibitory activity of breast cancer
상기 항-MIC-1 마우스 항체의 항암 효과를 확인하기 위하여, 유방암에 대한 성장 억제 활성을 확인하였다.In order to confirm the anti-cancer effect of the anti-MIC-1 mouse antibody, growth inhibitory activity against breast cancer was confirmed.
구체적으로, MDA-MB-231 인간 유방암 세포주를 마트리겔(50%)과 혼합하여 누드마우스(athymic nu/nu mouse)의 등쪽 피하에 접종(1 X 106 세포)함으로써 유방암 이종이식 암 동물모델을 제작하였다. 이후, 종양이 일정 크기로 성장하면, 종양의 크기가 비슷한 마우스를 2개 그룹을 나누고, 한 그룹에는 정상 IgG를, 다른 그룹에는 항-MIC-1 단일클론항체를 3일 간격으로 꼬리정맥에 투여(100 ㎍/마리)하였다.Specifically, MDA-MB-231 human breast cancer cell line was mixed with matrigel (50%) and inoculated subcutaneously (1 x 10 6 cells) into a dorsal submucosa of athymic nu / nu mouse to obtain an animal model of breast cancer xenograft cancer Respectively. Then, when the tumors grow to a certain size, mice having two different tumor sizes are divided into two groups, one group is administered with normal IgG and the other group is administered with anti-MIC-1 monoclonal antibody every 3 days in the tail vein (100 [mu] g / mouse).
그 결과, 도 4의 A 내지 C에서 볼 수 있듯이, 상기 항-MIC-1 마우스 항체를 투여하는 경우에는 유방암의 성장 속도가 감소하며, 종양의 크기 및 무게가 유의하게 감소하는 것을 확인하였다. As a result, as shown in FIGS. 4A to 4C, when the anti-MIC-1 mouse antibody was administered, the growth rate of breast cancer was decreased and the size and weight of the tumor were significantly decreased.
또한, 도 4의 D에서 볼 수 있듯이, MDA-MB-231 종양 조직 외부 및 내부 절편을 항-IB4(Isolectin/IB4) 항체로 면역형광 염색한 결과, 종양 외부에는 혈관 내피세포 마커인 IB4의 발현이 현저하게 감소하며, 종양 내부에는 IB4의 발현에 변화가 없는 것을 확인하였다.As shown in FIG. 4D, immunofluorescence staining of MDA-MB-231 tumor tissue external and internal sections with anti-IB4 (Isolectin / IB4) antibody revealed that the expression of IB4, a vascular endothelial cell marker, And there was no change in the expression of IB4 in the tumor.
상기 결과를 통해, 항-MIC-1 마우스 항체는 유방암의 성장을 억제하고, 유방암 외부의 혈관신생 억제하므로, 암의 예방 또는 치료 물질로 유용하게 사용될 수 있음을 알 수 있었다.These results indicate that the anti-MIC-1 mouse antibody inhibits the growth of breast cancer and inhibits angiogenesis outside of breast cancer, and thus can be effectively used as a preventive or therapeutic agent for cancer.
실시예 1-6. 전립선암의 성장 억제 활성 확인Examples 1-6. Identification of growth inhibitory activity of prostate cancer
상기 항-MIC-1 마우스 항체의 항암 효과를 확인하기 위하여, 전립선암에 대한 성장 억제 활성을 확인하였다.In order to confirm the anti-cancer effect of the anti-MIC-1 mouse antibody, growth inhibitory activity against prostate cancer was confirmed.
구체적으로, PC3 인간 전립선암 세포주를 마트리겔(50%)과 혼합하여 누드마우스(athymic nu/nu mouse)의 등쪽 피하에 접종(2 X 106 세포)함으로써 전립선암 이종이식 암 동물모델을 제작하였다. 이후, 종양이 약 100 mm3 정도의 일정 크기로 성장하면, 종양의 크기가 비슷한 마우스를 2개 그룹을 나누고, 한 그룹에는 정상 IgG를, 다른 그룹에는 항-MIC-1 단일클론항체를 3일 간격으로 꼬리정맥에 투여(100 ㎍/마리)하였다.Specifically, an animal model of prostate cancer xenograft cancer was prepared by inoculating PC3 human prostate cancer cell line with matrigel (50%) and inoculating subcutaneously (2 × 10 6 cells) of athymic nu / nu mouse . Thereafter, when the tumor grew to a certain size of about 100 mm 3 , mice having two different tumor sizes were divided into two groups, normal IgG in one group and anti-MIC-1 monoclonal antibody in the other group for 3 days (100 [mu] g / mouse) in the tail vein at intervals.
그 결과, 도 5의 A 내지 D에서 볼 수 있듯이, 상기 항-MIC-1 마우스 항체를 투여하는 경우에는 전립선암의 성장 속도가 감소하며, 종양의 크기 및 무게가 유의하게 감소하는 것을 확인하였다. As a result, as shown in FIGS. 5A to 5D, when the anti-MIC-1 mouse antibody was administered, the growth rate of prostate cancer was decreased, and the size and weight of the tumor were significantly decreased.
또한, 도 5의 E에서 볼 수 있듯이, PC3 종양 조직 절편을 항-IB4(Isolectin/IB4) 항체로 면역형광 염색한 결과, 혈관 내피세포 마커인 IB4의 발현이 현저하게 감소하는 것을 확인하였다.As shown in FIG. 5E, immunofluorescence staining of PC3 tumor tissue sections with anti-IB4 (Isolectin / IB4) antibody showed that the expression of IB4, a vascular endothelial cell marker, was significantly reduced.
상기 결과를 통해, 항-MIC-1 마우스 항체는 전립선암의 성장 및 이의 혈관신생을 억제하므로, 암의 예방 또는 치료 물질로 유용하게 사용될 것임을 알 수 있었다.These results indicate that the anti-MIC-1 mouse antibody inhibits the growth of prostate cancer and its angiogenesis, and thus is useful as a preventive or therapeutic agent for cancer.
상기 실시예 1의 결과들을 종합해 볼 때, 항-MIC-1 마우스 IgG 단일클론항체는 우수한 혈관신생 억제 효과를 나타내며, 피부암, 대장암, 유방암, 전립선암 등 다양한 종류의 암의 성장을 억제하고, 상기 암의 내부 또는 외부에서 혈관신생을 억제함으로써 항암 효과를 나타냄을 알 수 있었다.The anti-MIC-1 mouse IgG monoclonal antibody showed excellent angiogenesis inhibitory effect and inhibited the growth of various types of cancer such as skin cancer, colon cancer, breast cancer and prostate cancer , And inhibited angiogenesis in the inside or the outside of the cancer.
실시예 2. 항-MIC-1 인간화 IgG 단일클론항체의 활성 확인Example 2. Identification of Activity of Anti-MIC-1 Humanized IgG Monoclonal Antibodies
실시예 2-1. 항-MIC-1 인간화 IgG 단일클론항체의 제조Example 2-1. Preparation of anti-MIC-1 humanized IgG monoclonal antibody
상기 실시예 1을 통해 항-MIC-1 마우스 IgG 단일클론항체는 혈관신생을 억제하며, 다양한 종류의 암에 대하여 성장 억제 효과를 나타냄을 확인함에 따라, 상기 마우스 항체의 항암 활성은 그대로 가지면서 거부반응이 최소화된 인간화 항체를 제조하고자 하였다.In Example 1, anti-MIC-1 mouse IgG monoclonal antibody inhibited angiogenesis and showed growth inhibitory effect on various kinds of cancer. Therefore, anti-MIC-1 mouse IgG monoclonal antibody showed anti- To prepare a humanized antibody with minimal reaction.
구체적으로, 상기 마우스 항체를 분비하는 하이브리도마(hydridoma) 세포로부터 mRNA를 추출하고 cDNA로 전환한 후, 마우스 IgG의 중쇄 및 경쇄 가변영역(VL 및 VH)을 PCR로 증폭하고, pComb3XSS 벡터의 Sfi I 위치에 서브클로닝(subcloning)한 다음, 염기서열을 분석하였다. 분석이 완료된 상기 증폭산물을 사람의 항체 가변영역 아미노산 서열과 정렬하였고, CDR-1, 2, 3에 해당되는 부위는 마우스의 서열을 그대로 사용하고, 그 외 지역은 마우스 항체 가변영역과 상동성이 가장 높은 사람의 항체 가변영역의 아미노산으로 치환하는 방식으로 인간화 항체(humanized antibody)의 VL 및 VH 지역에 대한 아미노산 서열을 확정하였다. 코돈 최적화를 수행한 후, 이에 대한 올리고뉴클레오티드를 합성하여 동물세포에서 인간 IgG1를 발현하는 벡터인 pdCMV-dhfrC 벡터에 서브클로닝하였다. 이때, HindIII & BsiWI 위치에는 경쇄 암호화 올리고뉴클레오티드를, EcoRI & ApaI 위치에는 중쇄 암호화 올리고뉴클레오티드를 각각 서브클로닝하였다.Specifically, the mRNA was extracted from hydridoma cells secreting the mouse antibody and converted into cDNA. The heavy and light chain variable regions (VL and VH) of mouse IgG were amplified by PCR, and the Sfi of the pComb3XSS vector I < / RTI > position, followed by analysis of the nucleotide sequence. The amplified product, which was analyzed, was aligned with human antibody variable region amino acid sequence. The mouse sequence was used as the CDR-1, 2 and 3, and the other region was homologous to the mouse antibody variable region The amino acid sequence for the VL and VH regions of the humanized antibody was determined by substituting the amino acid of the antibody variable region of the highest human. Following codon optimization, oligonucleotides were synthesized and subcloned into pdCMV-dhfrC vector, a vector expressing human IgG1 in animal cells. At this time, the light chain encoding oligonucleotide was subcloned into the HindIII & BsiWI site and the heavy chain encoding oligonucleotide was ligated to the EcoRI & ApaI site.
이후, 상기 인간화 IgG1 발현 벡터를 HEK293 세포에 형질주입하고 24시간 동안 배양한 후, 이의 배양 상층액을 재조합 MIC-1 단백질을 항원으로 코팅한 플레이트에 넣어 반응시킨 후 ELISA를 수행한 결과, 대조군 벡터(pdCMV-dhfrC 공벡터)를 형질주입한 대조군 세포의 배양 상층액에 대해서는 양성 반응이 거의 없었으나, 상기 인간화 항-MIC-1 IgG1 발현 벡터를 형질주입한 세포의 배양 상층액에 대해서는 높은 양성 반응을 보임을 확인하였다. Then, the humanized IgG1 expression vector was transfected into HEK293 cells and cultured for 24 hours. The culture supernatant was reacted with recombinant MIC-1 protein on an antigen-coated plate, and then subjected to ELISA. As a result, (pdCMV-dhfrC co-vector), there was almost no positive reaction for the culture supernatant of the control cells. However, for the culture supernatant of the cells transfected with the humanized anti-MIC-1 IgG1 expression vector, Respectively.
상기 결과를 통해, 제조한 인간화 항체의 발현벡터는 효과적으로 인간화 항-MIC-1 IgG1 항체를 발현할 수 있음을 알 수 있었다.From the above results, it was found that the expression vector of the humanized antibody produced can effectively express the humanized anti-MIC-1 IgG1 antibody.
아울러, 제조한 인간화 항-MIC-1 IgG1 단일클론항체(이하, "항-MIC-1 인간화 항체"로 명명)의 생산 효율을 높이기 위하여, 상기 발현벡터를 DHFR(dihydrofolate reductase)이 결핍된 CHO 세포에 형질주입하고, 항생제(neomycin G418)와 DHFR이 함유된 배지에서 배양하여 안정된 형질전환체 클론들을 수득하고, 각 클론들을 분리 배양하였다. 상기 클론들의 배양 상층액에 대하여 전술한 바와 같이 MIC-1 단백질을 항원으로 하는 ELISA를 수행하였다.In order to increase the production efficiency of the produced humanized anti-MIC-1 IgG1 monoclonal antibody (hereinafter referred to as "anti-MIC-1 humanized antibody"), the expression vector was transfected into CHO cells deficient in DHFR (dihydrofolate reductase) , And cultured in a medium containing antibiotics (neomycin G418) and DHFR to obtain stable transformant clones, and each clone was separately cultured. The culture supernatants of the clones were subjected to ELISA using the MIC-1 protein as an antigen as described above.
그 결과, 도 6에서 볼 수 있듯이, 안정된 형질전환체 클론들 중 29번(#29) 클론에서 항-MIC-1 인간화 IgG1 항체가 가장 많이 발현되는 것을 확인하였다. As a result, as shown in FIG. 6, it was confirmed that anti-MIC-1 humanized IgG1 antibody was most expressed in clone # 29 (# 29) among the stable transformant clones.
이후, 선발한 #29 CHO 세포 클론에 메토트렉세이트(methotrexate)를 처리하여 발현벡터 유전자를 증폭시킨 후, 대용량으로 세포를 배양하여 얻은 배양 상층액에 대하여 단백질-G 친화 크로마토그래피(protein-G affinity chromatography)를 통해 항-MIC-1 인간화 IgG 항체를 순수 정제하였다. 상기 항-MIC-1 인간화 IgG 항체의 순도는 쿠마지 블루 염색을 통해 확인하였고, MIC-1 단백질에 대한 결합인지도는 ELISA 및 웨스턴블롯팅(Western blotting)을 통해 확인하였다.Then, protein # G affinity chromatography was performed on the cultured supernatant obtained by treating the selected # 29 CHO cell clone with methotrexate to amplify the expression vector gene and culturing the cells in a large capacity. Lt; RTI ID = 0.0 > anti-MIC-1 < / RTI > The purity of the anti-MIC-1 humanized IgG antibody was confirmed by coumarin blue staining and the binding affinity for the MIC-1 protein was confirmed by ELISA and Western blotting.
그 결과, 도 7에서 볼 수 있듯이, 항-MIC-1 인간화 IgG 항체는 높은 순도를 가지며, MIC-1 단백질에 대해서도 높은 특이도로 결합함을 확인하였다.As a result, as shown in FIG. 7, it was confirmed that anti-MIC-1 humanized IgG antibody has high purity and binds specifically to MIC-1 protein.
이와 같이 제조한 상기 항-MIC-1 인간화 항체의 중쇄 및 경쇄 가변영역, CDR, 및 FR 서열은 하기 표 2에 기재하였다.The heavy and light chain variable regions, CDRs, and FR sequences of the anti-MIC-1 humanized antibody thus prepared are shown in Table 2 below.
항-MIC-1 인간화 항체의 아미노산 서열The amino acid sequence of the anti-MIC-1 humanized antibody
항체명Antibody name 구분division 서열order 서열번호SEQ ID NO:
항-MIC-1 인간화 항체Anti-MIC-1 humanized antibody 중쇄가변영역(Heavy chain variable region; VH)Heavy chain variable region (VH) EVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYMHWVRQAPGQGLEWMGWIDPENGNTIYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAVPDSWGQGTLVTVSS≪ 2525
VH-CDR1VH-CDR1 GFNIKDYYGFNIKDYY 1One
VH-CDR2VH-CDR2 IDPENGNTIDPENGNT 22
VH-CDR3VH-CDR3 AVPDSAVPDS 33
VH-FR1VH-FR1 EVQLVQSGAEVKKPGASVKVSCKASEVQLVQSGAEVKKPGASVKVSCKAS 1717
VH-FR2VH-FR2 MHWVRQAPGQGLEWMGWMHWVRQAPGQGLEWMGW 1818
VH-FR3VH-FR3 IYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCIYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYC 1919
VH-FR4VH-FR4 WGQGTLVTVSSWGQGTLVTVSS 2020
경쇄가변영역(Light chain variable region; VL)Light chain variable region (VL) DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGNTYLYWYLQKPGQSPQLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPFTFGQGTKVEIKDIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGNTYLYWYLQKPGQSPQLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPFTFGQGTKVEIK 2626
VL-CDR1VL-CDR1 KSLLHSNGNTYKSLLHSNGNTY 44
VL-CDR2VL-CDR2 RMSRMS 55
VL-CDR3VL-CDR3 MQHLEYPFTMQHLEYPFT 66
VL-FR1VL-FR1 DIVMTQTPLSLPVTPGEPASISCRSSDIVMTQTPLSLPVTPGEPASISCRSS 2121
VL-FR2VL-FR2 LYWYLQKPGQSPQLLIYLYWYLQKPGQSPQLLIY 2222
VL-FR3VL-FR3 NLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC 2323
VL-FR4VL-FR4 FGQGTKVEIKFGQGTKVEIK 2424
실시예 2-2. 혈관신생 억제 활성 확인Example 2-2. Confirming angiogenesis inhibitory activity
실시예 2-2-1. 내피세포의 튜브 형성 억제 활성 확인Example 2-2-1. Identification of endothelial cell tubule formation inhibitory activity
상기 실시예 1에서 제조한 항-MIC-1 인간화 항체의 항암제로서의 활성을 확인하기 위하여, 먼저 혈관신생 억제 활성을 상기 실시예 1-2에 따른 튜브 형성 어세이(Tube formation assay)를 통해 확인하였다. In order to confirm the activity of the anti-MIC-1 humanized antibody prepared in Example 1 as an anticancer agent, the anti-angiogenic activity was first confirmed through a tube formation assay according to Example 1-2 .
그 결과, 도 8의 A에서 볼 수 있듯이, 상기 항-MIC-1 인간화 항체는 MIC-1 단백질 처리에 의해 증가한 내피세포의 튜브 형성, 즉 신생 혈관을 유의하게 감소시키며, 그 감소 정도는 MIC-1 단백질을 처리하지 않은 대조군과 유사함을 확인하였다.As a result, as shown in FIG. 8A, the anti-MIC-1 humanized antibody significantly reduced the tube formation of the endothelial cell, i.e., the angiogenesis, which was increased by the treatment with MIC-1 protein, 1 protein in the control group.
한편, 종양 발달 중 일어나는 혈관신생은 종양 세포로부터 분비된 혈관신생 촉진인자의 작용에 의해 유도된다. 따라서, 상기 항체의 혈관신생 억제 활성을 더욱 정확히 확인하기 위하여 암세포로부터 분비된 혈관신생 촉진인자들을 포함하는 암세포 배양 상층액을 내피세포에 처리하였고, 이후 상기 항-MIC-1 인간화 항체를 처리하여 튜브 형성 억제 정도를 확인하였다.On the other hand, angiogenesis that occurs during tumor development is induced by the action of angiogenic factors secreted from tumor cells. Thus, in order to more accurately confirm the anti-angiogenic activity of the antibody, a cancer cell culture supernatant containing angiogenesis promoting factors secreted from cancer cells was treated to endothelial cells and then treated with the anti-MIC-1 humanized antibody, Formation inhibition.
그 결과, 도 8의 B에서 볼 수 있듯이, A549 폐암 세포주, HCT-116 대장암 세포주 또는 LNCaP 전립선암 세포주의 배양 상층액은 내피세포의 튜브 형성 정도를 증가시켰지만, 항-MIC-1 인간화 항체는 상기 암세포의 배양 상층액에 의해 증가한 튜브 형성을 유의하게 감소시키는 것을 확인하였다. 특히, 그 억제 정도는 배양 상층액을 처리하지 않은 대조군과 비슷하거나 더욱 우수함을 확인하였다.As a result, as shown in Fig. 8B, the culture supernatant of A549 lung cancer cell line, HCT-116 colon cancer cell line or LNCaP prostate cancer cell line increased the degree of tube formation of endothelial cells, but anti-MIC- It was confirmed that the tube formation was significantly reduced by the culture supernatant of the cancer cells. In particular, it was confirmed that the degree of inhibition was similar to or better than that of the culture supernatant-untreated control.
아울러, 항-MIC-1 단일클론 항체 중에서도, 어느 유형의 항-MIC-1 단일클론 항체가 MIC-1에 의한 혈관신생을 효과적으로 억제하는지 확인하고자 하였다.In addition, we sought to determine which type of anti-MIC-1 monoclonal antibody effectively inhibited MIC-1-induced angiogenesis, among anti-MIC-1 monoclonal antibodies.
구체적으로, 마우스 IgG 전체 항체, 인간화 IgG 전체 항체 및 scFv 단편을 처리하여 튜브 형성 정도를 비교하였다. Specifically, mouse IgG whole antibody, humanized IgG whole antibody and scFv fragment were treated to compare the degree of tube formation.
그 결과, 도 9에서 볼 수 있듯이, 인간화 IgG 전체 항체가 가장 우수한 혈관신생 억제 활성을 나타냄을 확인하였고, 특히 그 억제 정도는 MIC-1을 처리하지 않은 대조군과 유사함을 확인하였다.As a result, as shown in FIG. 9, it was confirmed that the whole antibody of humanized IgG showed the most excellent angiogenesis inhibitory activity, and in particular, the degree of inhibition was similar to that of the control without MIC-1 treatment.
상기 결과를 통해, 항-MIC-1 인간화 항체는 내피세포의 혈관신생을 억제하며, 암세포에 의해 촉진되는 혈관신생도 우수한 정도로 억제하므로, 암의 예방 또는 치료 물질로 유용하게 사용될 수 있음을 알 수 있었다.These results indicate that the anti-MIC-1 humanized antibody inhibits angiogenesis of endothelial cells and inhibits angiogenesis promoted by cancer cells to an excellent degree, and thus can be usefully used as a preventive or therapeutic agent for cancer there was.
실시예 2-2-2. 대동맥 고리 가지형성(aorta ring sprouting) 억제 활성Example 2-2-2. Aorta ring sprouting inhibitory activity
상기 항-MIC-1 인간화 항체의 신생혈관 억제 활성을 더욱 정확히 확인하기 위하여, 랫트(rat)의 동맥절편의 가지 형성 정도를 비교하는 대동맥 고리 가지형성 어세이를 상기 실시예 1-2에 따른 방법으로 수행하였다.In order to more accurately confirm the anti-angiogenic activity of the anti-MIC-1 humanized antibody, aortic ring branching assay for comparing the degree of branch formation of the rat artery slice was carried out according to the method Respectively.
그 결과, 도 10의 A에서 볼 수 있듯이, 항-MIC-1 인간화 항체는 MIC-1 처리에 의해 증가한 가지 형성을 유의하게 감소시키는 것을 확인하였다. 특히, 그 억제 정도는 MIC-1을 처리하지 않은 대조군과 비슷함을 확인하였다.As a result, as shown in FIG. 10A, it was confirmed that the anti-MIC-1 humanized antibody significantly reduced the increased branch formation by MIC-1 treatment. In particular, it was confirmed that the degree of inhibition was similar to that of the control group not treated with MIC-1.
또한, 도 10의 B에서 볼 수 있듯이, 항-MIC-1 단일클론 항체 중에서도, 인간화 IgG 전체 항체가 가장 우수한 혈관신생 억제 활성을 나타냄을 확인하였고, 특히 그 억제 정도는 MIC-1을 처리하지 않은 대조군과 유사함을 확인하였다.In addition, as shown in FIG. 10B, it was confirmed that, among the anti-MIC-1 monoclonal antibodies, the whole antibody of humanized IgG showed the most excellent angiogenesis inhibitory activity, Similar to the control group.
상기 결과를 통해, 항-MIC-1 인간화 항체는 랫트 동맥절편의 가지 형성, 즉 혈관신생을 우수한 정도로 억제하므로, 암의 예방 또는 치료 물질로 유용하게 사용될 수 있음을 알 수 있었다.These results indicate that the anti-MIC-1 humanized antibody can inhibit angiogenesis, that is, angiogenesis, of the rat artery fraction, and thus can be effectively used as a preventive or therapeutic agent for cancer.
실시예 2-2-3. 생체 내 혈관신생 어세이(Example 2-2-3. In vivo angiogenesis assay ( in vivoin vivo angiogenesis assay) angiogenesis assay)
상기 항-MIC-1 인간화 항체의 신생혈관 억제 활성을 더욱 정확히 확인하기 위하여, CAM(chick chorioallantoic membrane)을 이용한 생체 내 혈관신생 어세이(in vivo angiogenesis assay)를 수행하였다.In order to more accurately confirm the anti-angiogenic activity of the anti-MIC-1 humanized antibody, an in vivo angiogenesis assay using a chick chorioallantoic membrane (CAM) was performed.
구체적으로, 닭의 수정란을 37℃에서 3일간 배양한 후, 공기낭(air sac)이 형성된 부위의 껍질 부분을 제거하였다. 공기낭 제거로 나출된 상기 수정란의 장요막(chorioallantoic membrane) 위에 MIC-1 단백질을 정상 IgG 또는 항-MIC-1 단일클론항체와 혼합하여 흡수시킨 3M 종이필터를 얹었고, 이를 37℃에서 4일간 배양함으로써 수정란의 장요막에 생성된 혈관을 관찰하였다.Specifically, chicken embryos were cultured at 37 ° C for 3 days, and then the skin of the area where the air sac was formed was removed. A 3M paper filter, in which MIC-1 protein was mixed with normal IgG or anti-MIC-1 monoclonal antibody, was placed on the chorioallantoic membrane of the embryo removed by air sac removal. The resulting blood vessels were observed in the embryo membranes of the embryos.
그 결과, 도 11에서 볼 수 있듯이, 항-MIC-1 인간화 항체는 MIC-1 처리에 의해 증가한 혈관 형성을 유의하게 감소시키는 것을 확인하였다. 특히, 그 억제 정도는 MIC-1을 처리하지 않은 대조군과 비슷하거나 더 감소함을 확인하였다.As a result, as shown in Fig. 11, it was confirmed that anti-MIC-1 humanized antibody significantly reduced angiogenesis by MIC-1 treatment. In particular, it was confirmed that the degree of inhibition was similar to or less than that of the control group not treated with MIC-1.
상기 결과를 통해, 항-MIC-1 인간화 항체는 생체 내 혈관신생을 우수한 정도로 억제하므로, 암의 예방 또는 치료 물질로 유용하게 사용될 수 있음을 알 수 있었다.These results indicate that the anti-MIC-1 humanized antibody inhibits angiogenesis in vivo to an excellent extent, and thus can be effectively used as a preventive or therapeutic agent for cancer.
실시예 2-3. 피부흑색종의 성장 억제 활성 확인Examples 2-3. Identify growth inhibitory activity of skin melanoma
상기 항-MIC-1 인간화 항체의 항암 효과를 확인하기 위하여, 피부흑색종에 대한 성장 억제 활성을 확인하였다. In order to confirm the anti-cancer effect of the anti-MIC-1 humanized antibody, the growth inhibitory activity against the skin melanoma was confirmed.
구체적으로, 상기 실시예 1-3에 따른 방법으로 C8161 인간 피부흑색종 세포주를 포함하는 이종이식 암 동물모델을 제작한 후, 상기 항체를 투여하였다.Specifically, an animal model of xenograft cancer containing the C8161 human skin melanoma cell line was prepared by the method according to Example 1-3, and then the antibody was administered.
그 결과, 도 12의 A 내지 D에서 볼 수 있듯이, 상기 항-MIC-1 인간화 항체를 투여하는 경우에는 피부흑색종의 성장 속도가 감소하며, 종양의 크기 및 무게가 유의하게 감소하는 것을 확인하였다. 특히, 상기 항체의 농도가 높아질수록, 항암 효과는 더욱 우수해짐을 확인하였다.As a result, as shown in FIGS. 12A to 12D, when the anti-MIC-1 humanized antibody was administered, the growth rate of skin melanoma was decreased, and the size and weight of the tumor were significantly decreased . In particular, it was confirmed that the higher the concentration of the antibody, the more excellent the anti-cancer effect.
또한, 도 12의 E 및 F에서 볼 수 있듯이, C8161 종양 조직 절편을 항-CD31 항체 및 항-IB4 항체로 면역형광 염색한 결과, 항-MIC-1 인간화 항체 투여에 의해 혈관 내피세포 마커인 CD31 및 IB4의 발현이 현저하게 감소하는 것을 확인하였다. In addition, as can be seen from FIGS. 12E and 12F, immunofluorescence staining of C8161 tumor tissue sections with anti-CD31 antibody and anti-IB4 antibody revealed that the anti-MIC-1 humanized antibody administration inhibited the vascular endothelial cell marker CD31 Lt; RTI ID = 0.0 > IB4 < / RTI >
상기 결과를 통해, 항-MIC-1 인간화 항체는 피부흑색종의 성장 및 혈관신생을 억제하므로, 암의 예방 또는 치료 물질로 유용하게 사용될 수 있음을 알 수 있었다.These results indicate that the anti-MIC-1 humanized antibody can inhibit the growth and angiogenesis of skin melanoma, and thus can be usefully used as a preventive or therapeutic agent for cancer.
나아가, 도 13의 A에서 볼 수 있듯이, 피부흑색종 동물모델에 항-MIC-1 인간화 항체를 투여하는 경우 혈장 내 MIC-1 단백질의 양이 감소하며, 상기의 감소 효과는 농도 의존적으로 나타남을 확인하였다.Furthermore, as shown in FIG. 13A, when the anti-MIC-1 humanized antibody is administered to a skin melanoma animal model, the amount of MIC-1 protein in the plasma decreases, and the above reduction effect appears to be concentration-dependent Respectively.
상기 결과를 통해, 종양의 크기가 작아질수록 혈장 MIC-1 단백질의 농도가 감소함을 고려할 때(도 13의 B), 항-MIC-1 인간화 항체는 피부흑색종의 성장을 현저하게 억제할 수 있음을 알 수 있었고, 따라서 암의 예방 또는 치료 물질로 유용하게 사용될 수 있음을 알 수 있었다.These results indicate that anti-MIC-1 humanized antibody significantly inhibits the growth of skin melanoma, considering that the concentration of plasma MIC-1 protein decreases as tumor size decreases (Fig. 13B) And thus it can be used as a preventive or therapeutic agent for cancer.
실시예 2-4. 대장암의 성장 억제 활성 확인Examples 2-4. Identification of growth inhibitory activity of colon cancer
상기 항-MIC-1 인간화 항체의 항암 효과를 확인하기 위하여, 대장암에 대한 성장 억제 활성을 확인하였다.In order to confirm the anti-cancer effect of the anti-MIC-1 humanized antibody, growth inhibitory activity against colon cancer was confirmed.
구체적으로, 상기 실시예 1-4에 따른 방법으로, HCT-116 인간 대장암 세포주를 포함하는 이종이식 암 동물모델을 제작한 후, 상기 항체를 투여하였다.Specifically, an animal model of xenograft cancer containing the HCT-116 human colon cancer cell line was prepared by the method according to Example 1-4, and then the antibody was administered.
그 결과, 도 14의 A 내지 D에서 볼 수 있듯이, 상기 항-MIC-1 인간화 항체를 투여하는 경우에는 대장암의 성장 속도가 감소하며, 종양의 크기 및 무게가 유의하게 감소하는 것을 확인하였다. 특히, 상기 항체의 농도가 높아질수록, 항암 효과는 더욱 우수해짐을 확인하였다.As a result, as shown in FIGS. 14A to 14D, when the anti-MIC-1 humanized antibody was administered, the growth rate of colorectal cancer was decreased and the size and weight of the tumor were significantly decreased. In particular, it was confirmed that the higher the concentration of the antibody, the more excellent the anti-cancer effect.
또한, 도 14의 E 및 F에서 볼 수 있듯이, HCT-116 종양 조직 절편을 항-CD31 항체 및 항-IB4 항체로 면역형광 염색한 결과, 항-MIC-1 인간화 항체 투여에 의해 혈관 내피세포 마커인 CD31 및 IB4의 발현이 현저하게 감소하는 것을 확인하였다. In addition, as can be seen in FIGS. 14E and 14F, immunohistochemical staining of HCT-116 tumor tissue sections with anti-CD31 antibody and anti-IB4 antibody revealed that the anti-MIC-1 humanized antibody Lt; RTI ID = 0.0 > CD31 < / RTI > and IB4 significantly decreased.
상기 결과를 통해, 항-MIC-1 인간화 항체는 대장암의 성장 및 혈관신생을 억제하므로, 암의 예방 또는 치료 물질로 유용하게 사용될 수 있음을 알 수 있었다.From the above results, it was found that the anti-MIC-1 humanized antibody inhibits the growth and angiogenesis of colon cancer, and thus can be effectively used as a preventive or therapeutic agent for cancer.
또한, 도 15의 A에서 볼 수 있듯이, HCT-116 종양 조직을 항-IB4 항체 및 피모니다졸(Pimonidazole)을 이용하여 분석한 결과, 항-MIC-1 인간화 항체 투여에 의해 혈관 내피세포 마커인 IB4의 수준은 현저하게 감소하였으나, 저산소증 마커인 피모니다졸의 수준은 현저하게 증가하는 것을 확인하였다.As can be seen from FIG. 15A, the HCT-116 tumor tissue was analyzed using an anti-IB4 antibody and pimonidazole. As a result, the anti-MIC-1 humanized antibody was found to be a vascular endothelial cell marker IB4 levels were significantly reduced, but the level of pimonidazole, a hypoxic marker, was found to be significantly increased.
또한, 도 15의 B에서 볼 수 있듯이, HCT-116 종양 조직을 항-Ki67 항체를 이용하여 분석한 결과, 항-MIC-1 인간화 항체 투여에 의해 증식/분열 중인 세포의 핵에서만 발견되는 단백질인 Ki67의 수준이 유의하게 감소하는 것을 확인하였다. As can be seen from FIG. 15B, the HCT-116 tumor tissue was analyzed using an anti-Ki67 antibody. As a result, the protein found only in the nucleus of cells proliferating / dividing by the administration of anti-MIC- And the level of Ki67 was significantly decreased.
한편, 도 15의 C에서 볼 수 있듯이, 저산소 상태를 야기하는 CoCl2를 HCT-116 세포에 처리한 결과, MIC-1 단백질의 발현이 증가하는 것을 확인하였다.On the other hand, as shown in FIG. 15C, when HCT-116 cells were treated with CoCl 2 causing hypoxia, the expression of MIC-1 protein was increased.
상기 결과를 통해, 항-MIC-1 인간화 항체는 대장암 조직 내에서 혈관신생을 억제하여 저산소 지역을 확대하면서 산소 및 영양분의 공급을 차단하여 대장암 세포의 증식 및 분열을 억제함으로써 대장암의 발달을 저해함을 확인하였다.The results showed that the anti-MIC-1 humanized antibody inhibited the angiogenesis in the colorectal cancer tissue, enlarged the hypoxic area, blocked the supply of oxygen and nutrients, inhibited the proliferation and division of the colon cancer cells, Respectively.
즉, 종양이 커지면서 대장암 세포가 저산소 상태에 처하게 되면 상기 항체의 타겟 단백질인 MIC-1의 발현이 증가되지만 상기 항체는 대장암 세포로부터 분비된 MIC-1 단백질을 중화시키므로, 상기 항체는 MIC-1의 발현 증가가 종양 내 혈관신생으로 이어지는 것을 차단하여 대장암의 성장을 억제하는 효과를 발휘할 수 있음을 알 수 있었다.That is, when colon cancer cells become hypoxic, the expression of MIC-1, which is a target protein of the antibody, is increased. However, since the antibody neutralizes the MIC-1 protein secreted from colon cancer cells, -1 expression is prevented from leading to angiogenesis in the tumor, thereby exerting an effect of inhibiting the growth of colon cancer.
실제로, 도 16의 A에서 볼 수 있듯이, 항-MIC-1 인간화 항체를 투여하는 경우 혈장 내 MIC-1 단백질의 양이 감소하며, 상기의 감소 효과는 농도 의존적으로 나타남을 확인하였다.In fact, as can be seen from FIG. 16A, the amount of MIC-1 protein in the plasma decreases when the anti-MIC-1 humanized antibody is administered, and the above-mentioned reduction effect appears to be concentration-dependent.
상기 결과를 통해, 종양의 크기가 작아질수록 혈장 MIC-1 단백질의 농도가 감소함을 고려할 때(도 16의 B), 항-MIC-1 인간화 항체는 대장암의 성장을 현저하게 억제할 수 있음을 알 수 있었고, 따라서 암의 예방 또는 치료 물질로 유용하게 사용될 수 있음을 알 수 있었다.The above results indicate that anti-MIC-1 humanized antibodies can significantly inhibit the growth of colon cancer, considering that the concentration of plasma MIC-1 protein decreases as tumor size decreases (Fig. 16B) And thus it can be used effectively as a preventive or therapeutic agent for cancer.
실시예 2-5. 유방암의 성장 억제 활성 확인Examples 2-5. Identification of growth inhibitory activity of breast cancer
상기 항-MIC-1 인간화 항체의 항암 효과를 확인하기 위하여, 유방암에 대한 성장 억제 활성을 확인하였다.In order to confirm the anti-cancer effect of the anti-MIC-1 humanized antibody, growth inhibitory activity against breast cancer was confirmed.
구체적으로, 상기 실시예 1-5에 따른 방법으로 MDA-MB-453 인간 유방암 세포주를 포함하는 이종이식 암 동물모델을 제작한 후, 상기 항체를 투여하였다.Specifically, an animal model of xenograft cancer containing MDA-MB-453 human breast cancer cell line was prepared by the method according to Example 1-5, and then the antibody was administered.
그 결과, 도 17의 A 내지 D에서 볼 수 있듯이, 상기 항-MIC-1 인간화 항체를 투여하는 경우에는 유방암의 성장 속도가 감소하며, 종양의 크기가 유의하게 감소하는 것을 확인하였다. 특히, 상기 항체의 농도가 높아질수록, 항암 효과는 더욱 우수해짐을 확인하였다.As a result, as shown in FIGS. 17A to 17D, when the anti-MIC-1 humanized antibody was administered, the growth rate of breast cancer was decreased and the tumor size was significantly decreased. In particular, it was confirmed that the higher the concentration of the antibody, the more excellent the anti-cancer effect.
또한, 도 17의 E 및 F에서 볼 수 있듯이, MDA-MB-453 종양 조직 절편을 항-CD31 항체 및 항-IB4 항체로 면역형광 염색한 결과, 항-MIC-1 인간화 항체 투여에 의해 혈관 내피세포 마커인 CD31 및 IB4의 발현이 현저하게 감소하는 것을 확인하였다.In addition, as can be seen from FIGS. 17E and 17F, the MDA-MB-453 tumor tissue sections were immunofluorescently stained with anti-CD31 antibody and anti-IB4 antibody, The expression of CD31 and IB4, which are cell markers, was remarkably decreased.
나아가, 도 18에서 볼 수 있듯이, MDA-MB-453 종양 조직을 항-Ki67 항체를 이용하여 분석한 결과, 항-MIC-1 인간화 항체 투여에 의해 증식/분열 중인 세포의 핵에서만 발견되는 단백질인 Ki67의 수준이 유의하게 감소하는 것을 확인하였다. Further, as shown in FIG. 18, when MDA-MB-453 tumor tissue was analyzed using an anti-Ki67 antibody, it was found that the protein found only in the nucleus of cells proliferating / dividing by the administration of anti-MIC- And the level of Ki67 was significantly decreased.
상기 결과를 통해, 항-MIC-1 인간화 항체는 유방암의 성장 및 혈관신생을 억제하고, 유방암 세포의 증식 및 분열을 억제하므로, 암의 예방 또는 치료 물질로 유용하게 사용될 수 있음을 알 수 있었다.These results indicate that the anti-MIC-1 humanized antibody inhibits the growth and angiogenesis of breast cancer and inhibits the proliferation and division of breast cancer cells, thus being useful as a preventive or therapeutic agent for cancer.
실시예 2-6. 전립선암의 성장 억제 활성 확인Examples 2-6. Identification of growth inhibitory activity of prostate cancer
상기 항-MIC-1 인간화 항체의 항암 효과를 확인하기 위하여, 전립선암에 대한 성장 억제 활성을 확인하였다.In order to confirm the anti-cancer effect of the anti-MIC-1 humanized antibody, growth inhibitory activity against prostate cancer was confirmed.
구체적으로, 상기 실시예 1-6에 따른 방법으로 PC3 전립선암 세포주를 포함하는 이종이식 암 동물모델을 제작한 후, 상기 항체를 투여하였다.Specifically, an animal model of xenograft cancer containing a PC3 prostate cancer cell line was prepared by the method according to Example 1-6, and then the antibody was administered.
그 결과, 도 19의 A 내지 C에서 볼 수 있듯이, 상기 항-MIC-1 인간화 항체를 투여하는 경우에는 전립선암의 성장 속도가 감소하며, 종양의 크기가 유의하게 감소하는 것을 확인하였다. 특히, 상기 항체의 농도가 높아질수록, 항암 효과는 더욱 우수해짐을 확인하였다.As a result, as shown in FIGS. 19A to 19C, when the anti-MIC-1 humanized antibody was administered, the growth rate of prostate cancer was decreased and the tumor size was significantly decreased. In particular, it was confirmed that the higher the concentration of the antibody, the more excellent the anti-cancer effect.
또한, 도 19의 D 및 E에서 볼 수 있듯이, PC3 종양 조직 절편을 항-CD31 항체 및 항-IB4 항체로 면역형광 염색한 결과, 항-MIC-1 인간화 항체 투여에 의해 혈관 내피세포 마커인 CD31 및 IB4의 발현이 현저하게 감소하는 것을 확인하였다. In addition, as can be seen from D and E in Fig. 19, the PC3 tumor tissue sections were immunofluorescently stained with anti-CD31 antibody and anti-IB4 antibody. As a result, anti-MIC-1 humanized antibody administration resulted in inhibition of vascular endothelial cell marker CD31 Lt; RTI ID = 0.0 > IB4 < / RTI >
상기 결과를 통해, 항-MIC-1 인간화 항체는 전립선암의 성장 및 혈관신생을 억제하므로, 암의 예방 또는 치료 물질로 유용하게 사용될 수 있음을 알 수 있었다.These results indicate that the anti-MIC-1 humanized antibody inhibits the growth and angiogenesis of prostate cancer, and thus can be effectively used as a preventive or therapeutic agent for cancer.
또한, 도 20의 A에서 볼 수 있듯이, PC3 종양 조직을 항-IB4 항체 및 피모니다졸을 이용하여 분석한 결과, 혈관 내피세포 마커인 IB4의 수준은 현저하게 감소하였으나, 저산소증 마커인 피모니다졸의 수준은 현저하게 증가하는 것을 확인하였다.As can be seen from FIG. 20A, PC3 tumor tissues were analyzed using anti-IB4 antibody and pimidazole. As a result, the level of IB4, a vascular endothelial cell marker, was remarkably decreased, but the hypoxic marker pimonidazole Of the patients were significantly increased.
또한, 도 20의 B에서 볼 수 있듯이, PC3 종양 조직을 항-Ki67 항체를 이용하여 분석한 결과, 항-MIC-1 인간화 항체 투여에 의해 증식/분열 중인 세포의 핵에서만 발견되는 단백질인 Ki67의 수준이 유의하게 감소하는 것을 확인하였다. As can be seen from FIG. 20B, PC3 tumor tissue was analyzed using an anti-Ki67 antibody. As a result, Ki67, a protein found only in the nuclei of cells proliferating / dividing by administration of anti-MIC-1 humanized antibody And the level was significantly decreased.
한편, 도 20의 C에서 볼 수 있듯이, 저산소 상태를 야기하는 CoCl2를 PC3 세포에 처리한 결과, MIC-1 단백질의 발현이 증가하는 것을 확인하였다.On the other hand, also as seen in the 20 C, the CoCl 2 to cause hypoxia was confirmed that the results of processing in PC3 cells, increased expression of MIC-1 protein.
상기 결과를 통해, 항-MIC-1 인간화 항체는 전립선암 조직 내에서 혈관신생을 억제하여 저산소 지역을 확대하면서, 산소 및 영양분의 공급을 차단하여 전립선암 세포의 증식 및 분열을 억제함으로써 전립선암의 발달을 저해함을 확인하였다. As a result, the anti-MIC-1 humanized antibody suppresses angiogenesis in the prostate cancer tissue to expand the hypoxic area, while blocking the supply of oxygen and nutrients to inhibit proliferation and division of prostate cancer cells, And inhibited the development.
즉, 종양이 커지면서 전립선암 세포가 저산소 상태에 처하게 되면 상기 항체의 타겟 단백질인 MIC-1의 발현이 증가되지만 상기 항체는 전립선암 세포로부터 분비된 MIC-1 단백질을 중화시키므로, 상기 항체는 MIC-1의 발현 증가가 종양 내 혈관신생으로 이어지는 것을 차단하여 전립선암의 성장을 억제하는 효과를 발휘할 수 있음을 알 수 있었다.That is, if the prostate cancer cells become hypoxic when the tumor is enlarged, the expression of MIC-1, which is the target protein of the antibody, is increased. However, since the antibody neutralizes the MIC-1 protein secreted from the prostate cancer cells, -1 < / RTI > expression is prevented from leading to angiogenesis in the tumor, thereby exerting an effect of inhibiting the growth of prostate cancer.
실시예 2-7. 췌장암의 성장 억제 활성 확인Examples 2-7. Identification of growth inhibitory activity of pancreatic cancer
상기 항-MIC-1 인간화 항체의 항암 효과를 확인하기 위하여, 췌장암에 대한 성장 억제 활성을 확인하였다.In order to confirm the anti-cancer effect of the anti-MIC-1 humanized antibody, growth inhibitory activity against pancreatic cancer was confirmed.
구체적으로, PANC-1 인간 췌장암 세포주를 마트리겔(50%)과 혼합하여 누드마우스(athymic nu/nu mouse)의 등쪽 피하에 접종(1 X 106 세포)함으로써 췌장암 이종이식 암 동물모델을 제작하였다. 이후, 종양이 일정 크기로 성장하면, 종양의 크기가 비슷한 마우스를 2개 그룹을 나누고, 한 그룹에는 정상 IgG를, 다른 그룹에는 항-MIC-1 단일클론 인간화 항체를 3일 간격으로 꼬리정맥에 투여(100 ㎍/마리)하였다.Specifically, PANC-1 An animal model of pancreatic cancer xenograft cancer was prepared by inoculating subcutaneously (1 × 10 6 cells) of athymic nu / nu mice with a human pancreatic cancer cell line mixed with matrigel (50%). Then, when the tumors grow to a certain size, mice having two different tumor sizes are divided into two groups, one group contains normal IgG and the other group contains anti-MIC-1 monoclonal humanized antibody at 3-day intervals in the tail vein (100 [mu] g / mouse).
그 결과, 도 21의 A 내지 D에서 볼 수 있듯이, 상기 항-MIC-1 인간화 항체를 투여하는 경우에는 췌장암의 성장 속도가 감소하며, 종양의 크기가 유의하게 감소하는 것을 확인하였다. 특히, 상기 항체의 농도가 높아질수록, 항암 효과는 더욱 우수해짐을 확인하였다.As a result, as shown in FIGS. 21A to 21D, when the anti-MIC-1 humanized antibody was administered, the growth rate of pancreatic cancer was decreased and the tumor size was significantly decreased. In particular, it was confirmed that the higher the concentration of the antibody, the more excellent the anti-cancer effect.
또한, 도 21의 E 및 F에서 볼 수 있듯이, PANC-1 종양 조직 절편을 항-CD31 항체 및 항-IB4 항체로 면역형광 염색한 결과, 항-MIC-1 인간화 항체 투여에 의해 혈관 내피세포 마커인 CD31 및 IB4의 발현이 현저하게 감소하는 것을 확인하였다. In addition, as can be seen from FIGS. 21E and 21F, immunofluorescence staining of PANC-1 tumor tissue sections with anti-CD31 antibody and anti-IB4 antibody revealed that the anti-MIC-1 humanized antibody Lt; RTI ID = 0.0 > CD31 < / RTI > and IB4 significantly decreased.
나아가, 도 22에서 볼 수 있듯이, PANC-1 종양 조직을 항-Ki67 항체를 이용하여 분석한 결과, 항-MIC-1 인간화 항체 투여에 의해 증식/분열 중인 세포의 핵에서만 발견되는 단백질인 Ki67의 수준이 유의하게 감소하는 것을 확인하였다. Further, as shown in FIG. 22, PANC-1 tumor tissue was analyzed using an anti-Ki67 antibody. As a result, Ki67, a protein found only in the nucleus of cells proliferating / dividing by administration of anti-MIC- And the level was significantly decreased.
상기 결과를 통해, 항-MIC-1 인간화 항체는 췌장암의 성장 및 혈관신생을 억제하고, 췌장암 세포의 증식 및 분열을 억제하므로, 암의 예방 또는 치료 물질로 유용하게 사용될 수 있음을 알 수 있었다.These results indicate that the anti-MIC-1 humanized antibody inhibits the growth and angiogenesis of pancreatic cancer and inhibits the proliferation and division of pancreatic cancer cells, thus being useful as a preventive or therapeutic agent for cancer.
실시예 2-8. 간암의 성장 억제 활성 확인Examples 2-8. Identify growth inhibitory activity of liver cancer
상기 항-MIC-1 인간화 항체의 항암 효과를 확인하기 위하여, 간암에 대한 성장 억제 활성을 확인하였다.In order to confirm the anti-cancer effect of the anti-MIC-1 humanized antibody, growth inhibitory activity against liver cancer was confirmed.
구체적으로, HepG2 인간 간암 세포주를 마트리겔(50%)과 혼합하여 누드마우스(athymic nu/nu mouse)의 등쪽 피하에 접종(1 X 106 세포)함으로써 간암 이종이식 암 동물모델을 제작하였다. 이후, 종양이 일정 크기로 성장하면, 종양의 크기가 비슷한 마우스를 2개 그룹을 나누고, 한 그룹에는 정상 IgG를, 다른 그룹에는 항-MIC-1 단일클론 인간화 항체를 3일 간격으로 꼬리정맥에 투여(100 ㎍/마리)하였다.Specifically, an animal model of liver cancer xenograft cancer was prepared by inoculating subcutaneously (1 × 10 6 cells) of athymic nu / nu mouse with a HepG2 human liver cancer cell line mixed with Matrigel (50%). Then, when the tumors grow to a certain size, mice having two different tumor sizes are divided into two groups, one group contains normal IgG and the other group contains anti-MIC-1 monoclonal humanized antibody at 3-day intervals in the tail vein (100 [mu] g / mouse).
그 결과, 도 23에서 볼 수 있듯이, 상기 항-MIC-1 인간화 항체를 투여하는 경우에는 간암 종양의 크기가 유의하게 감소하는 것을 확인하였다. 특히, 상기 항체의 농도가 높아질수록, 항암 효과는 더욱 우수해짐을 확인하였다.As a result, as shown in FIG. 23, when the anti-MIC-1 humanized antibody was administered, it was confirmed that the size of liver cancer tumors was significantly reduced. In particular, it was confirmed that the higher the concentration of the antibody, the more excellent the anti-cancer effect.
상기 결과를 통해, 항-MIC-1 인간화 항체는 간암의 성장을 현저히 억제하므로, 암의 예방 또는 치료 물질로 유용하게 사용될 수 있음을 알 수 있었다.These results indicate that the anti-MIC-1 humanized antibody significantly inhibits the growth of liver cancer, and thus can be effectively used as a preventive or therapeutic agent for cancer.
실시예 2-9. 위암의 성장 억제 활성 확인Examples 2-9. Identifying growth inhibitory activity of gastric cancer
상기 항-MIC-1 인간화 항체의 항암 효과를 확인하기 위하여, 위암에 대한 성장 억제 활성을 확인하였다.In order to confirm the anti-cancer effect of the anti-MIC-1 humanized antibody, growth inhibitory activity against gastric cancer was confirmed.
구체적으로, AGS 인간 위암 세포주를 마트리겔(50%)과 혼합하여 누드마우스(athymic nu/nu mouse)의 등쪽 피하에 접종(1 X 106 세포)함으로써 위암 이종이식 암 동물모델을 제작하였다. 이후, 종양이 일정 크기로 성장하면, 종양의 크기가 비슷한 마우스를 2개 그룹을 나누고, 한 그룹에는 정상 IgG를, 다른 그룹에는 항-MIC-1 단일클론 인간화 항체를 3일 간격으로 꼬리정맥에 투여(100 ㎍/마리)하였다.Specifically, an animal model of gastric cancer xenograft cancer was prepared by mixing AGS human gastric cancer cell line with matrigel (50%) and inoculating subcutaneously (1 × 10 6 cells) of athymic nu / nu mouse. Then, when the tumors grow to a certain size, mice having two different tumor sizes are divided into two groups, one group contains normal IgG and the other group contains anti-MIC-1 monoclonal humanized antibody at 3-day intervals in the tail vein (100 [mu] g / mouse).
그 결과, 도 24에서 볼 수 있듯이, 상기 항-MIC-1 인간화 항체를 투여하는 경우에는 위암 종양의 크기가 유의하게 감소하는 것을 확인하였다. 특히, 상기 항체의 농도가 높아질수록, 항암 효과는 더욱 우수해짐을 확인하였다.As a result, as shown in FIG. 24, when the anti-MIC-1 humanized antibody was administered, the size of gastric cancer tumor was significantly decreased. In particular, it was confirmed that the higher the concentration of the antibody, the more excellent the anti-cancer effect.
상기 결과를 통해, 항-MIC-1 인간화 항체는 위암의 성장을 현저히 억제하므로, 암의 예방 또는 치료 물질로 유용하게 사용될 수 있음을 알 수 있었다.These results indicate that the anti-MIC-1 humanized antibody significantly inhibits the growth of gastric cancer, and thus can be effectively used as a preventive or therapeutic agent for cancer.
이상의 설명으로부터, 본 출원이 속하는 기술분야의 당업자는 본 출원이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로 이해해야만 한다. 본 출원의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 출원의 범위에 포함되는 것으로 해석되어야 한다.From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all aspects and not restrictive. The scope of the present application is to be interpreted as being within the scope of the present application, all changes or modifications derived from the meaning and scope of the appended claims and from their equivalents rather than the detailed description.

Claims (14)

  1. 서열번호 1의 중쇄 CDR1, 서열번호 2의 중쇄 CDR2, 및 서열번호 3의 중쇄 CDR3을 포함하는 중쇄 가변영역; 및A heavy chain variable region comprising heavy chain CDR1 of SEQ ID NO: 1, heavy chain CDR2 of SEQ ID NO: 2, and heavy chain CDR3 of SEQ ID NO: 3; And
    서열번호 4의 경쇄 CDR1, 서열번호 5의 경쇄 CDR2, 및 서열번호 6의 경쇄 CDR3을 포함하는 경쇄 가변영역을 포함하는, MIC-1(macrophage inhibitory cytokine 1) 단백질에 결합하는 항체.An antibody that binds to a macrophage inhibitory cytokine 1 (MIC-1) protein comprising a light chain variable region comprising light chain CDR1 of SEQ ID NO: 4, light chain CDR2 of SEQ ID NO: 5, and light chain CDR3 of SEQ ID NO:
  2. 제1항에 있어서, 상기 항체의 중쇄 가변영역은 서열번호 7 또는 17의 중쇄 FR1, 서열번호 8 또는 18의 중쇄 FR2, 서열번호 9 또는 19의 중쇄 FR3 및 서열번호 10 또는 20의 중쇄 FR4를 포함하고; 및7. The antibody of claim 1, wherein the heavy chain variable region of the antibody comprises heavy chain FR2 of SEQ ID NO: 7 or 17, heavy chain FR2 of SEQ ID NO: 8 or 18, heavy chain FR3 of SEQ ID NO: 9 or 19 and heavy chain FR4 of SEQ ID NO: 10 or 20 and; And
    상기 항체의 경쇄 가변영역은 서열번호 11 또는 21의 경쇄 FR1, 서열번호 12 또는 22의 경쇄 FR2, 서열번호 13 또는 23의 경쇄 FR3 및 서열번호 14 또는 24의 경쇄 FR4를 포함하는 것인, 항체.The light chain variable region of said antibody comprises light chain FR1 of SEQ ID NO: 11 or 21, light chain FR2 of SEQ ID NO: 12 or 22, light chain FR3 of SEQ ID NO: 13 or 23 and light chain FR4 of SEQ ID NO: 14 or 24.
  3. 제1항에 있어서, 상기 중쇄 가변영역은 서열번호 15 또는 25의 아미노산 서열로 이루어지고; 및The method of claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 15 or 25; And
    상기 경쇄 가변영역은 서열번호 16 또는 26의 아미노산 서열로 이루어진 것인, 항체.Wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 16 or 26.
  4. 제1항 내지 제3항 중 어느 한 항의 항체를 코딩하는 폴리뉴클레오티드.A polynucleotide encoding an antibody of any one of claims 1 to 3.
  5. 제4항의 폴리뉴클레오티드를 포함하는 발현 벡터.An expression vector comprising the polynucleotide of claim 4.
  6. 제5항의 발현 벡터가 도입된 형질전환체.A transformant into which the expression vector of claim 5 is introduced.
  7. 제1항 내지 제3항 중 어느 한 항의 항체를 포함하는, 암의 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating cancer, which comprises the antibody of any one of claims 1 to 3.
  8. 제7항에 있어서, 상기 암은 피부암, 대장암, 유방암, 전립선암, 췌장암, 간암 또는 위암인 것인, 암의 예방 또는 치료용 약학 조성물.The pharmaceutical composition according to claim 7, wherein the cancer is skin cancer, colon cancer, breast cancer, prostate cancer, pancreatic cancer, liver cancer or stomach cancer.
  9. 제1항 내지 제3항 중 어느 한 항의 항체를 포함하는, 혈관신생 억제용 약학 조성물.A pharmaceutical composition for inhibiting angiogenesis, comprising the antibody of any one of claims 1 to 3.
  10. 제1항 내지 제3항 중 어느 한 항의 항체를 개체에 투여하는 단계를 포함하는, 암의 예방 또는 치료 방법.A method of preventing or treating cancer, comprising administering an antibody of any one of claims 1 to 3 to a subject.
  11. 제1항 내지 제3항 중 어느 한 항의 항체를 포함하는, 암 진단용 조성물.A cancer diagnostic composition comprising the antibody of any one of claims 1 to 3.
  12. 제11항의 조성물을 포함하는, 암 진단용 키트.A cancer diagnostic kit comprising the composition of claim 11.
  13. 제1항 내지 제3항 중 어느 한 항의 항체를 이용하여, 암이 의심되는 개체의 분리된 생물학적 시료에서 MIC-1(macrophage inhibitory cytokine 1) 단백질을 항원-항체 반응을 통하여 검출하는 단계를 포함하는, 암의 진단 방법.Comprising the step of detecting an MIC-1 (macrophage inhibitory cytokine 1) protein through an antigen-antibody reaction in a separate biological sample of a subject suspected of having cancer using the antibody of any one of claims 1 to 3 , Diagnostic methods of cancer.
  14. (a) 분리된 암세포에 암의 예방 또는 치료 후보물질을 처리하는 단계;(a) treating a candidate cancer cell for a prophylactic or therapeutic candidate for cancer;
    (b) 상기 후보물질이 처리된 암세포에서 제1항 내지 제3항 중 어느 한 항의 항체를 이용하여, MIC-1(macrophage inhibitory cytokine 1) 단백질의 수준을 측정하는 단계; 및(b) measuring the level of MIC-1 (macrophage inhibitory cytokine 1) protein using the antibody of any one of claims 1 to 3 in cancer cells treated with the candidate substance; And
    (c) 상기 단계 (b)의 MIC-1 단백질 수준이 후보물질을 처리하지 않은 암세포의 것보다 감소하는 경우, 상기 (a) 단계에서 처리한 후보물질을 암의 예방 또는 치료 물질로 판단하는 단계를 포함하는, 암의 예방 또는 치료 물질의 스크리닝 방법.(c) determining that the candidate substance treated in the step (a) is a preventive or therapeutic substance for cancer when the MIC-1 protein level of the step (b) is lower than that of cancer cells not treated with the candidate substance ≪ / RTI > wherein said method comprises the steps of:
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