WO2019004550A1 - Anticorps se liant spécifiquement à une protéine mic-1, et utilisation associée - Google Patents

Anticorps se liant spécifiquement à une protéine mic-1, et utilisation associée Download PDF

Info

Publication number
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
Authority
WO
WIPO (PCT)
Prior art keywords
cancer
antibody
mic
seq
light chain
Prior art date
Application number
PCT/KR2018/001171
Other languages
English (en)
Korean (ko)
Inventor
이한수
이재섭
성하영
손원준
Original Assignee
강원대학교 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 강원대학교 산학협력단 filed Critical 강원대학교 산학협력단
Publication of WO2019004550A1 publication Critical patent/WO2019004550A1/fr

Links

Images

Classifications

    • 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).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Toxicology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un anticorps se liant spécifiquement à une protéine de cytokine 1 inhibitrice de macrophages (MIC-1) ; et une utilisation associée.
PCT/KR2018/001171 2017-06-26 2018-01-26 Anticorps se liant spécifiquement à une protéine mic-1, et utilisation associée WO2019004550A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR20170080653 2017-06-26
KR10-2017-0080653 2017-06-26

Publications (1)

Publication Number Publication Date
WO2019004550A1 true WO2019004550A1 (fr) 2019-01-03

Family

ID=64741654

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2018/001171 WO2019004550A1 (fr) 2017-06-26 2018-01-26 Anticorps se liant spécifiquement à une protéine mic-1, et utilisation associée

Country Status (2)

Country Link
KR (1) KR101998029B1 (fr)
WO (1) WO2019004550A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101346181B1 (ko) * 2013-02-18 2014-01-03 강원대학교산학협력단 Mic-1의 혈관신생유도 기능을 억제하는 항 mic-1 단일클론항체 mbm-12
EP2441466B1 (fr) * 2004-04-13 2014-07-23 St Vincent's Hospital Sydney Limited Agent inhibiteur de MIC-1

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100679666B1 (ko) 2004-01-20 2007-02-07 한국생명공학연구원 인간 대식세포 억제 사이토카인-1 특이적 단일클론항체,이를 분비하는 하이브리도마 및 이를 포함하는 위암 진단키트
KR101346132B1 (ko) 2013-02-18 2013-12-31 강원대학교산학협력단 Mic-1의 혈관신생유도 기능을 억제하는 항 mic-1 단일클론항체 mbm-14

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2441466B1 (fr) * 2004-04-13 2014-07-23 St Vincent's Hospital Sydney Limited Agent inhibiteur de MIC-1
KR101346181B1 (ko) * 2013-02-18 2014-01-03 강원대학교산학협력단 Mic-1의 혈관신생유도 기능을 억제하는 항 mic-1 단일클론항체 mbm-12

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BOYLE, GLEN M.: "Macrophage inhibitory cytokine-1 is overexpressed in malignant melanoma and is associated with tumorigenicity", JOURNAL OF INVESTIGATIVE DERMATOLOGY, 2009, pages 383 - 391, XP055330204, DOI: 10.1038/jid.2008.270 *
JIN, Y-J.: "Regulation of endothelial cell angiogenic activity by extracellular matrix and macrophage inhibitory cytokine-l", DOCTORAL THESIS, KANGWON NATIONAL UNIVERSITY, vol. 114, September 2012 (2012-09-01), pages 1 - 150 *
M. HUANG, ET AL: "A Complex Of Extracellular Domain Of Tissue Factor With An Inhibitory Fab (5g9)", FULL WWPDB X-RAY STRUCTURE VALIDATION REPORT, 1 January 1997 (1997-01-01), pages 1 - 37, XP055679972 *
SUNG, H.Y.: "Development of Humanized Monoclonal Antibody IgG blocking Pro-angiogenic Activity of MIC-1", MASTER'S THESIS, KANGWON NATIONAL UNIVERSITY, February 2017 (2017-02-01), pages 1 - 36 *

Also Published As

Publication number Publication date
KR101998029B1 (ko) 2019-07-08
KR20190001538A (ko) 2019-01-04

Similar Documents

Publication Publication Date Title
WO2016137108A1 (fr) Nouvel anticorps se liant à la tfpi et composition le comprenant
WO2015005632A1 (fr) Nouvelles protéines à double cible se liant spécifiquement à dll4 et vegf et leur utilisation
WO2015058573A1 (fr) Anticorps monoclonal pour l'antagonisme et l'inhibition de la liaison de mort programmée (pd-1) à son ligand et sa séquence codante et son utilisation
WO2015133817A1 (fr) Anticorps monoclonal reconnaissant spécifiquement des cellules de lymphome à cellules b, et son utilisation
WO2014077648A1 (fr) Anticorps se liant spécifiquement à la protéine l1cam humaine et murine, et son utilisation
WO2014090053A1 (fr) Anticorps monoclonal pour l'antagonisation et l'inhibition de la liaison du facteur de croissance cellulaire endothélial vasculaire et de son récepteur, et sa séquence codante et son utilisation
WO2022039490A1 (fr) Anticorps bispécifiques anti-b7-h4/anti-4-1bb et leurs utilisations
WO2021020846A1 (fr) Anticorps bispécifique anti-her2/anti-4-1bb et son utilisation
WO2018026248A1 (fr) Nouvel anticorps dirigé contre la protéine programmée de mort cellulaire (pd-1) et son utilisation
WO2020101365A1 (fr) Anticorps anti-c-met présentant une stabilité améliorée ou des fragments de liaison à l'antigène de celui-ci
WO2022177394A1 (fr) Anticorps bispécifique à domaine unique dirigé contre pd-l1 et cd47 et son utilisation
WO2021101346A1 (fr) Anticorps bispécifiques anti-ror1/anti-4-1bb et leurs utilisations
WO2021020845A1 (fr) Anticorps bispécifique anti-egfr/anti-4-1bb et son utilisation
WO2016199964A1 (fr) Anticorps se liant spécifiquement à un peptide isolé dérivé de la vimentine, ou à un fragment de liaison dudit peptide
WO2020251316A1 (fr) ANTICORPS BISPÉCIFIQUE DIRIGÉ CONTRE α-SYN/IGF1R ET UTILISATION ASSOCIÉE
WO2022085905A1 (fr) Anticorps se liant spécifiquement à la protéine de spicule du sars-cov-2 et utilisation associée
WO2020250204A1 (fr) Nouveaux anticorps spécifiques de cthrc1 et leur utilisation
WO2019045477A1 (fr) Composition pour prévenir et traiter une maladie de la peau comprenant une substance se liant spécifiquement à un peptide dérivé de la vimentine
WO2018026249A1 (fr) Anticorps dirigé contre le ligand 1 de mort programmée (pd-l1) et son utilisation
WO2019004550A1 (fr) Anticorps se liant spécifiquement à une protéine mic-1, et utilisation associée
WO2022035201A1 (fr) Protéine de fusion comprenant il-12 et anticorps anti-fap et utilisation associée
WO2019022274A1 (fr) Anticorps se liant spécifiquement à une protéine pauf, et utilisation associée
WO2020204305A1 (fr) Protéine de fusion comprenant un anticorps anti-mésothéline, un anticorps anti-cd3 ou un anticorps anti-egfr, anticorps bispécifique ou trispécifique les comprenant, et utilisations associées
WO2016200220A1 (fr) Anticorps se liant spécifiquement à des peptides dérivés de la vimentine isolés ou fragment de liaison du peptide
WO2019022281A1 (fr) Anticorps se liant spécifiquement à une protéine pauf et utilisation correspondante

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18824556

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18824556

Country of ref document: EP

Kind code of ref document: A1