WO2018174544A2 - Muc1에 특이적으로 결합하는 항체 및 그의 용도 - Google Patents
Muc1에 특이적으로 결합하는 항체 및 그의 용도 Download PDFInfo
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- WO2018174544A2 WO2018174544A2 PCT/KR2018/003267 KR2018003267W WO2018174544A2 WO 2018174544 A2 WO2018174544 A2 WO 2018174544A2 KR 2018003267 W KR2018003267 W KR 2018003267W WO 2018174544 A2 WO2018174544 A2 WO 2018174544A2
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- antibody
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Classifications
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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
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- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
- C12N5/12—Fused cells, e.g. hybridomas
- C12N5/16—Animal cells
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- 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
Definitions
- the present invention relates to an anti-MUC1 antibody that specifically binds to MUC1 (Mucin 1) and its use, and more particularly, to an anti-MUC1 antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof -Drug conjugates or bispecific antibodies, pharmaceutical compositions for the prevention or treatment of cancer comprising the same, nucleic acids encoding the antibodies or antigen-binding fragments thereof, vectors and host cells comprising the nucleic acids, anti-MUC1 antibodies or the same It relates to a method for preparing an antigen binding fragment.
- Mucin 1 (MUC1, Mucin 1) is a transmembrane glycoprotein comprising a number of glycated extracellular domains. MUC1 length is 200-500 nm at the cell surface and MUC1 is located in the apical membrane of normal epithelial cells. MUC1 is expressed in glandular or luminal epithelial cells of many organs such as the breast, stomach, esophagus, pancreas, urethra, lungs, kidneys and gallbladder. The negatively charged carbohydrates of MUC1 in normal tissues form physical barriers that protect the basal epithelium from dehydration, pH changes, pollen and microorganisms.
- the MUC1 gene encodes a single transcript. After translation, MUC1 is autocleaved in the GSVVV motif located in the sea urchin sperm protein enterokinase and agrin (SEA) domains. These consist of two peptide fragments, the N-terminal subunit (MUC1-N) and the C-terminal subunit (MUC1-C).
- GSVVV motif located in the sea urchin sperm protein enterokinase and agrin (SEA) domains.
- SEA sea urchin sperm protein enterokinase and agrin
- the MUC1-N terminal subunit has a variable number of Tandem Repeats and consists of a Proline / Threonine / Serine-rich (PTS) domain and a SEA domain.
- the MUC1-C terminal subunit consists of 58 amino acid extracellular domains (ECDs), 28 transmembrane domains (TMDs) and 72 amino acid cytoplasmic domains (CDs).
- ECDs extracellular domains
- TMDs transmembrane domains
- CDs 72 amino acid cytoplasmic domains
- MUC1 extensively O-linked glycosylation in the extracellular domain. Depending on the extent of the N-glycosylation pattern, the MUC1-C size ranges from 23 to 25 kDa, or 17 kDa in the absence of N-glycosylation.
- MUC1 is bound to a cell by a noncovalent interaction between a part that can be released from the cell and cleavage occurs by an enzyme called Sheddase.
- MUC1 complexes are isolated by stimulation of cytokines such as IFN- ⁇ and TNF- ⁇ .
- MUC1-N release is caused by enzymes including TNF- ⁇ Converting Enzyme (TACE) and Matrix Metallo-protease (MMP). These enzymes cleave the ECD of MUC1-C into two fragments, and as cancer progresses, these extracellular fragments leave the cancer cells and float in the body's blood, causing the fragments that are bound to the cells to continue with the cancer cells. Will remain in a combined state.
- TACE TNF- ⁇ Converting Enzyme
- MMP Matrix Metallo-protease
- MUC1 is important for cancer cell growth because it binds to cell membrane proteins related to cancer cell proliferation present in other cancer cells, and thus plays a crucial role in cancer cell proliferation by sending continuous cell proliferation signals.
- this part is always the same fate until cancer cell growth and extinction, which is a good target of cancer detection and also a definitive biomarker capable of removing cancer.
- this part is known to be the only part that does not glycosylation, and was thought to show a distinct difference between MUC1 in cancer and normal cells. Therefore, the present inventors have developed the antibody of the present invention by binding to MUC1 cells and sharing the fate with cancer, and looking at this fragment which can distinguish between MUC1 of normal cells and MUC1 of cancer cells as the optimal antigen of the antibody.
- the antibody-drug conjugate is a cytotoxic drug bound to the antibody through a linker. Since monoclonal antibodies exhibit target specific properties, drugs in antibody-drug conjugates can be delivered to tumors expressing antigens / targets recognized by monoclonal antibodies with selective target capacity. Ideally, the antibody-drug conjugate in the prodrug state in the blood after administration should be non-toxic, and when the antibody binds to the target tumor antigen and then internalizes into the cancer cell, the drug is released in active form to kill the tumor cell.
- targets / antigens to which antibodies bind have been cell surface proteins that predominantly, and ideally, cancer cell specific expression (overexpression) in tumor cells.
- the present inventors have developed an antibody that specifically binds to cancer cells-MUC1 that recognizes a part different from MUC1 expressed in normal cells, and maximizes ADC efficacy by binding to cancer cells due to ADC characteristics.
- Anti-MUC1 antibodies and antibody-drug conjugates that bind to "parts that bind to cells" that share fate with cancer cells can specifically bind to MUC1 expressing cells to treat diseases caused by MUC1 expression It was confirmed that the present invention was completed.
- An object of the present invention is an antibody or antigen-binding fragment thereof that specifically binds to a “part bound to a cell” of MUC1, an antibody-drug conjugate conjugated with a drug to the antibody or antigen-binding fragment thereof, and the antibody or antigen thereof It is to provide a bispecific antibody comprising a binding fragment.
- Another object of the present invention to provide a hybridoma (KCLRFBP 00395) for the production of the anti-MUC1 antibody.
- Still another object of the present invention is to provide a composition and a method for treating or preventing cancer comprising the anti-MUC1 antibody or antigen-binding fragment thereof, the antibody-drug conjugate or bispecific antibody.
- Still another object of the present invention is to provide a composition for diagnosing and diagnosing cancer comprising the anti-MUC1 antibody or antigen-binding fragment thereof.
- It is another object of the present invention to provide an immunogenic composition comprising a complex encapsulating a MUC1-C terminal region, a SEA domain of MUC1 or a C-terminal extracellular domain of MUC1 and CpG-DNA in liposomes.
- Still another object of the present invention is to provide a method of preparing an anti-MUC1 monoclonal antibody comprising inoculating a mouse with the immunogenic composition.
- Still another object of the present invention is to provide a nucleic acid encoding the anti-MUC1 antibody or antigen-binding fragment thereof, a vector and a host cell comprising the nucleic acid, a method for producing an anti-MUC1 antibody or antigen-binding fragment thereof using the same. There is.
- the present invention provides an anti-MUC1 antibody or antigen-binding fragment thereof that recognizes a polypeptide comprising five or more contiguous amino acids in the C-terminal extracellular domain of MUC1.
- the anti-MUC1 antibody or antigen binding fragment thereof comprises six complementarity determining regions (CDRs), wherein the antibody or antigen binding fragment thereof is a heavy chain CDR1 of SEQ ID NO: 1 (GYTFTSYWMH); A heavy chain CDR2 of SEQ ID NO: 2 (YINPGTGYIEYNQKFKD); A heavy chain CDR3 of SEQ ID NO: 3 (STAPFDY); Light chain CDR1 of SEQ ID NO: 4 (KASQDIKSYLS); Light chain CDR2 of SEQ ID NO: 5 (YATRLAD); And light chain CDR3 of SEQ ID NO: 6 (LQYDESPYT) may be characterized in that it comprises one or more sequences selected from the group consisting of.
- CDRs complementarity determining regions
- the present invention also provides hybridomas (KCLRFBP 00395) for the production of the anti-MUC1 antibody.
- the invention also provides antibody-drug conjugates and bispecific antibodies comprising anti-MUC1 antibodies or antigen binding fragments thereof.
- the present invention also provides a pharmaceutical composition and a method for treating or preventing cancer comprising the anti-MUC1 antibody or antigen-binding fragment thereof, antibody-drug conjugate or bispecific antibody.
- the invention also provides the use of said anti-MUC1 antibody or antigen binding fragment thereof for the prevention or treatment of cancer.
- the invention also provides the use of said anti-MUC1 antibody or antigen binding fragment thereof for the manufacture of a medicament for the prevention or treatment of cancer.
- the present invention also provides a diagnostic composition and diagnostic method for cancer comprising the anti-MUC1 antibody or antigen-binding fragment thereof.
- the present invention also relates to an immunogenic composition
- an immunogenic composition comprising a complex encapsulating (1) the C-terminal region of MUC1, the SEA domain of MUC1 or the C-terminal extracellular domain of MUC1, and (2) CpG-DNA in a liposome and said immunity
- an anti-MUC1 monoclonal antibody manufacturing method comprising the step of inoculating a mouse with the original composition.
- the present invention also provides a nucleic acid encoding the anti-MUC1 antibody or antigen-binding fragment thereof, a vector and host cell comprising the nucleic acid, a method for producing an anti-MUC1 antibody or antigen-binding fragment thereof using the same.
- 1A is a graph showing the total amount of IgG in mice immunized with rhMUC1-C protein.
- 1b and 1c show the results of screening hybridoma cells isolated from immunized mice
- 1b is a screening result using HAT medium
- Figure 1c is a screening result using HT medium.
- 2A is a graph showing the results of ELISA on ascites of mice immunized with hybridoma cells (hMUC1-1H7 clone), showing the presence of rhMUC1-C specific antibodies.
- Figure 2b shows the results of analysis of the purified anti-hMUC1 monoclonal antibody by SDS-PAGE and Commassie staining.
- Figure 2c is a graph showing the results of confirming the iso type of the anti-MUC1 antibody through ELISA.
- Figure 3a shows the results of Western blotting using breast cancer cell lysate using anti-hMUC1 monoclonal antibody and anti-MUC1-CT antibody purified from hMUC1-1H7 clone.
- FIG. 3B immunoprecipitates cell lysates of MCF-7, MDA-MB-231, T47D and ZR75-1 cells with anti-hMUC1 monoclonal antibody purified from mouse normal IgG or hMUC1-1H7 clone, and anti-MUC1- The results of immunoblotting using CT antibodies are shown.
- Figure 3C shows the results of Western blotting using T47D cell lysate with PNGase F and anti-hMUC1 monoclonal antibody purified from hMUC1-1H7 clone compared to the case with other antibodies.
- FIG. 3D shows immunoblot of T47D cell lysate treated with PNGase F with anti-hMUC1 monoclonal antibody purified from hMUC1-1H7 clone, followed by immunoblotting with anti-MUC1-CT or anti-MUC1-CT2 antibody. Shows.
- Figure 4a shows the results of Western blotting on pancreatic cancer cell lysates using anti-MUC1-CT antibodies, anti-hMUC1 antibodies purified from hMUC1-1H7 clones, or anti-beta-actin antibodies.
- FIG. 4B shows immunoprecipitated with anti-hMUC1 monoclonal antibody purified from mouse normal IgG or hMUC1-1H7 clone against pancreatic cancer cell lysate and immunoblocked using anti-MUC1-CT antibody and anti-hMUC1 monoclonal antibody Show results.
- FIG. 5A shows fluorescence images obtained by treating anti-hMUC1 antibody purified from hMUC1-1H7 clone at 4 ° C. (Surface) or lysing cells with 0.1% Triton X-100 to intracellular breast cancer cells. Shows.
- 5B shows fluorescence images obtained after treating anti-hMUC1 antibodies purified from fluorescently probed hMUC1-1H7 clones and incubating at 37 ° C. for 6 hours.
- FIG. 6 shows fluorescence images obtained by treating anti-hMUC1 antibodies purified from hMUC1-1H7 clones at 4 ° C. (Surface) or lysing cells with 0.1% Triton X-100 on intracellular pancreatic cancer cells (intracellular). Shows.
- FIG. 7 shows fluorescence images obtained after treatment of pan-cancer cancer cells with anti-hMUC1 antibodies purified from fluorescently probed hMUC1-1H7 clones and incubated at 37 ° C. for 24 hours.
- 8A is a cleavage map schematically showing the recombinant expression plasmid pFabE-hMUC1-1H7.
- 8B schematically shows the recombinant protein expressed by the recombinant expression plasmid pFabE-hMUC1-1H7.
- 8C is a fluorescence image obtained by treating Fab fragments of anti hMUC1 antibodies derived from hMUC1-1H7 clones in MCF-7, MDA-MB-231, T47D and ZR75-1 cells.
- FIG. 9 is a graph showing changes in cell proliferation when MDA-MB-231, T47D and ZR75-1 cells were treated with anti-hMUC1 antibody derived from hMUC1-1H7 clone.
- 10A to 10H are fluorescence images obtained by intravenous administration of anti-hMUC1 monoclonal antibodies derived from fluorescently labeled hMUC1-1H7 clones to mice with breast cancer.
- 11A to 11C are fluorescence images obtained by intravenous administration of anti-hMUC1 monoclonal antibodies derived from fluorescently labeled hMUC1-1H7 clones to mice with pancreatic cancer.
- 12A shows tumor tissue extracted from xenograft mouse models.
- 12B is a graph showing the size ((width 2 * length) / 2) of tumors extracted from xenograft mouse models administered with anti-hMUC1 monoclonal antibodies derived from hMUC1-1H7 clones.
- 12C is a graph showing tumor weights extracted from xenograft mouse models administered with anti-hMUC1 monoclonal antibody.
- 12D is a graph showing the body weight of a xenograft mouse model administered with anti-hMUC1 monoclonal antibody.
- 16 is a result showing the cancer cell proliferation inhibitory effect of hMUC1-1H7 antibody-drug conjugate in breast cancer tissue.
- 17 shows the results of confirming the binding affinity between the humanized antibody (hMUC1-G3) and hMUC1-C through ELISA.
- FIG. 18 shows the results of ELISA confirming the epitope homogeneity of hMUC1-C recognized by hMUC1-G3 and hMUC1-1H7 antibodies.
- hMUC1-G3 antibody-drug conjugate selectively expresses breast cancer cells in MUC1-expressing cell lines (ZR75-1, T47D) and MUC1-free cell lines (MDA-MB-231). The result shows killing.
- Figure 23 is a result showing the effect of selectively inhibiting the cancer cells of the hMUC1-G3 antibody-drug conjugate in xenograft mouse model produced through TNBC tissue transplantation derived from breast cancer patients.
- MUC1 Moc 1, Mucin 1
- MUC1 is generally expressed on one side of normal epithelial cells (apical membrane) and protects the basal epithelium from drying, pH changes, contamination, and microorganisms.
- MUC1 is abnormally expressed at high levels in various human carcinomas, has a reduced degree of glycation, is expressed evenly on the entire surface of the cell, and is involved in promoting cancer cell proliferation, invasion, metastasis and angiogenesis.
- MUC1 is a target for treating cancer specificity.
- MUC1 comprises an N-terminal subunit (MUC1-N) and a C-terminal subunit (MUC1-C), wherein MUC1-N and MUC1-C are cleavage sites in the sea urchin sperm protein enterokinase and agrin domain. It is formed by cutting itself from above.
- SEA domains contribute to the formation of stable heterodimeric complexes.
- extracellular MUC1-N (the N-terminal subunit of MUC1) can be released to isolate many anti-MUC1-SEA antibodies.
- Most anti-MUC1-SEA antibodies known to date are known to target the MUC1-N repeat sequence domain (Prinssen et al. 1998; Gillespie et al. 2000).
- MUC1-N is not found directly at the cell surface but is observed in the peripheral circulation, there is a limitation that only limited circulating anti-MUC1-N antibodies can be used in MUC1-positive tumor cells.
- the present invention provides an antibody that specifically binds to the novel MUC1 targeting the MUC1-C terminal region (extracellular domain) remaining on the cell surface after MUC1 cleavage.
- the present invention in one aspect, relates to an anti-MUC1 antibody or antigen binding fragment thereof that specifically binds to MUC1. More specifically, it relates to an anti-MUC1 antibody or antigen binding fragment thereof that recognizes a polypeptide comprising five or more contiguous amino acids in the C-terminal extracellular domain of MUC1.
- MUC1 is heavily glycosylated, but this part occurs mainly in the cut-out part, and is relatively glycosylated in the part where the cell is bound. In the present invention, this fact was recognized and expressed in E. coli without glycosylation using 190 amino acids bound to cells after cleavage.
- the antibody according to the present invention is made of an antigen expressed in Escherichia coli, it recognizes an antigen that is not glycosylated.
- an antibody was developed using mouse hybridoma technology, and the developed antibody specifically recognized that the tertiary structure was recognized without recognizing the primary structure of the amino acid sequence.
- the FACS using the breast cancer cell lines T47D and ZR75-1 was confirmed that the MUC-1 specific, but in the lysate obtained by crushing the cells recognized in the Western recognized primary structure It wasn't.
- the antibody was detected in the antibody according to the present invention, since the antigen was detected when Western cells were washed by Immunoprecipition method using the cell lysate and the antibody according to the present invention. Proved to be an antibody.
- the C-terminal extracellular domain of MUC1 may be characterized by consisting of the amino acid sequence of SEQ ID NO: 10 (SVV VQLTLAFREG TINVHDVETQ FNQYKTEAAS RYNLTISDVS VSDVPFPFSA QS).
- the term "antibody” is a generic term for a substance produced by stimulation of an antigen in the immune system, and the kind thereof is not particularly limited.
- the antibody is an immunoglobulin molecule that is immunologically reactive with a specific antigen, and refers to a protein molecule that serves as a receptor that specifically recognizes an antigen, and includes a polyclonal antibody and a monoclonal antibody (monoclonal antibody). ) And all antibodies and antibody fragments.
- the antibody may be one produced unnaturally, such as one produced recombinantly or synthetically.
- the antibody may be an animal antibody (eg, a mouse antibody, etc.), chimeric antibody, humanized antibody or human antibody.
- the antibody may be a monoclonal antibody.
- Antibodies can also be understood to include antigen binding fragments of antibodies that possess antigen binding capacity, unless otherwise noted.
- CDRs complementarity-determining regions
- the anti-MUC1 antibody may be produced from hybridoma hMUC1-1H7 (KCLRF-BP-00395).
- the anti-MUC1 antibody or antigen-binding fragment thereof is complementary determining regions (CDR-H1, CDR-H2, CDR-H3, CDR-) of the antibody produced from the hybridoma hMUC1-1H7 (KCLRF-BP-00395) L1, CDR-L2 and CDR-L3) or heavy chain variable region and light chain variable region may be characterized.
- the anti-MUC1 antibody or antigen-binding fragment thereof comprises six complementarity determining regions (CDRs).
- the antibody or antigen-binding fragment thereof may comprise a heavy chain CDR1 of SEQ ID NO: 1 (GYTFTSYWMH); A heavy chain CDR2 of SEQ ID NO: 2 (YINPGTGYIEYNQKFKD); A heavy chain CDR3 of SEQ ID NO: 3 (STAPFDY); Light chain CDR1 of SEQ ID NO: 4 (KASQDIKSYLS); Light chain CDR2 of SEQ ID NO: 5 (YATRLAD); And light chain CDR3 of SEQ ID NO: 6 (LQYDESPYT) may be characterized in that it comprises one or more sequences selected from the group consisting of.
- the antibody or antigen-binding fragment thereof is a heavy chain CDR1 of SEQ ID NO: 1; A heavy chain CDR2 of SEQ ID NO: 2; A heavy chain CDR3 of SEQ ID NO: 3; 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 antibody or antigen-binding fragment thereof may be characterized by comprising a heavy chain variable region of SEQ ID NO: 22 or 24 and a light chain variable region of SEQ ID NO: 23 or 25. More specifically.
- the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region of SEQ ID NO: 22 and a light chain variable region of SEQ ID NO: 23; Or a heavy chain variable region of SEQ ID NO: 24 and a light chain variable region of SEQ ID NO: 25.
- the anti-MUC1 antibody or antigen-binding fragment thereof has an inhibitory effect on MUC1.
- the MUC1 gene encodes one transcript, and after translation the MUC1 protein is autocleaved at "G" in the GSVVV motif located in the SEA domain.
- the MUC1 protein may be a human MUC1 protein, for example GenBank Accession No. Sequence identity with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the amino acid sequences of P15941 (SEQ ID NO: 7) It may be to have.
- the extracellular domain of the MUC1 protein may be the extracellular domain of human MUC1 protein, for example GenBank Accession No.
- the extracellular domain of the MUC1 protein comprises an SEA domain, and in one embodiment, the SEA domain is GenBank Accession No. It may be a protein fragment (SEQ ID NO: 9) comprising 119 amino acids from 1034 to 1152 of the amino acid sequence (SEQ ID NO: 7) of P15941.
- G is a cleavage site, and when cleavage occurs, the site after "G” (in the C-terminal direction) is the C-term extracellular domain (or ' MUC1-C terminal (site) extracellular domain 'or' MUC1-C subunit '(SEQ ID NO: 10).
- MUC1 protein site Explanation order Full amino acid sequence of the MUC1 protein Full amino acid sequence of P15941 MTPGTQSPFF LLLLLTVLTV VTG SGHASST PGGEKETSAT QRSSVPSSTE KNAVSMTSSV LSSHSPGSGS STTQGQDVTL APATEPASGS AATWGQDVTS VPVTRPALGS TTPPAHDVTS APDNKPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS AP
- the anti-MUC1 antibody or antigen-binding fragment thereof is MUC1 protein (eg SEQ ID NO: 7), specifically 5, 7 in the C-terminal extracellular domain (eg SEQ ID NO: 10) of the MUC1 protein. It may be characterized in that it recognizes or specifically binds a polypeptide (epitope) comprising dog, 10, 12 or more or preferably 15 or more amino acids.
- the antibody or antigen-binding fragment thereof may comprise an extracellular domain of MUC1 protein (eg SEQ ID NO: 8), an SEA domain of MUC1 protein (eg SEQ ID NO: 9) or a C-terminal extracellular domain of MUC1 protein (eg SEQ ID NO: 10). ), And / or specifically binds thereto.
- MUC1 specific antibody or “an antibody that specifically binds to MUC1” refers to an antibody that binds to MUC1 and results in inhibition of the biological activity of MUC1, and is used interchangeably with “anti-MUC1 antibody”. Used.
- Anti-MUC1 antibody in the present invention may be an animal antibody (eg a mouse antibody), a chimeric antibody (eg a mouse-human chimeric antibody) or a humanized antibody, may be a monoclonal antibody or a polyclonal antibody
- monoclonal antibodies may be an animal antibody (eg a mouse antibody), a chimeric antibody (eg a mouse-human chimeric antibody) or a humanized antibody
- monoclonal antibodies may be a monoclonal antibody or a polyclonal antibody
- the concept includes both a polyclonal antibody and a monoclonal antibody (monoclonal antibody, monoclonal antibody), preferably a monoclonal antibody, it may have a whole antibody (whole antibody) form.
- the whole antibody is a structure having two full-length light chains and two full-length heavy chains, and includes a constant region, and each light chain is connected by heavy and disulfide bonds.
- the total antibody of the anti-MUC1 antibody according to the present invention includes the IgA, IgD, IgE, IgM and IgG forms, wherein IgG is a subtype and includes IgG1, IgG2, IgG3 and IgG4.
- a full IgG antibody has two full length light chains and two full length heavy chains, each of which is linked by a heavy chain and disulfide bond.
- the constant region of the antibody is divided into a heavy chain constant region and a light chain constant region, and the heavy chain constant region has a gamma ( ⁇ ), mu ( ⁇ ), alpha ( ⁇ ), delta ( ⁇ ) and epsilon ( ⁇ ) type, subclass Gamma 1 ( ⁇ 1), gamma 2 ( ⁇ 2), gamma 3 ( ⁇ 3), gamma 4 ( ⁇ 4), alpha 1 ( ⁇ 1) and alpha 2 ( ⁇ 2).
- the constant regions of the light chains have kappa ( ⁇ ) and lambda ( ⁇ ) types.
- the term “heavy chain” refers to the variable region domain VH and the three constant region domains C H1 , C H2 and C H3 comprising an amino acid sequence having a variable region sequence sufficient to confer specificity to the antigen. It is interpreted to include both full-length heavy chains and fragments thereof including and hinges.
- the term “light chain” refers to both the full-length light chain and fragment thereof comprising the variable region domain V L and the constant region domain C L comprising an amino acid sequence having sufficient variable region sequence to confer specificity to the antigen. It is interpreted to include.
- complementarity determining region refers to the amino acid sequence of the hypervariable region of the heavy and light chains of immunoglobulin.
- the heavy and light chains may each comprise three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3).
- the CDRs can provide key contact residues for the antibody to bind antigen or epitope.
- Antigen binding fragment of the anti-MUC1 antibody means a fragment having the function of binding to the antigen of the anti-MUC1 antibody, namely MUC1, Fab, Fab ', F (ab') 2 , scFv (scFv) 2 , scFv-Fc, Fv, and the like, and the like.
- antibody fragment is used interchangeably.
- the antigen binding fragment may be, for example, but not limited to scFv, (scFv) 2 , Fab, Fab 'or F (ab') 2 .
- Fab of the antigen-binding fragment has one antigen binding site in a structure having a variable region of the light and heavy chains, a constant region of the light chain and a first constant region of the heavy chain (C H1 ).
- F (ab ') 2 antibodies are produced when the cysteine residues of the hinge region of Fab' form disulfide bonds. Recombinant techniques for generating Fv fragments with minimal antibody fragments in which Fv has only heavy chain variable regions and light chain variable regions are well known in the art.
- Double-chain Fv is a non-covalent bond in which the heavy chain variable region and the light chain variable region are linked, and the single-chain Fv is generally shared by the variable region of the heavy chain and the short chain variable region through a peptide linker. It may be linked by bond or directly at the C-terminus to form a dimer-like structure such as a double chain Fv.
- the antigen binding fragments can be obtained using proteolytic enzymes (e.g., restriction digestion of the entire antibody with papain can yield Fab and cleavage with pepsin can yield F (ab ') 2 fragment). It can be produced through genetic recombination technology.
- the term "hinge region” is a region included in the heavy chain of the antibody, and exists between the C H1 and C H2 regions, and provides a function of providing flexibility of the antigen binding site in the antibody. It means the area to do.
- the anti-MUC1 antibody may be a monoclonal antibody.
- Monoclonal antibodies can be prepared according to methods well known in the art. For example, it may be manufactured using a phage display technique. Alternatively, anti-MUC1 antibodies can be used to prepare monoclonal antibodies derived from mice by conventional methods.
- individual monoclonal antibodies can be screened based on their ability to bind to MUC1 using a typical Enzyme-Linked ImmunoSorbent Assay (ELISA) format.
- Assays can be assayed for inhibitory activity through functional assays such as competitive ELISA or cell-based assays to assay molecular interactions for the conjugates.
- Each affinity (Kd values) for MUC1 is then assayed for selected monoclonal antibody members based on strong inhibitory activity.
- anti-MUC1 antibody or antigen-binding fragment thereof while having any one or more of CDR1 to CDR3 of light and heavy chains contained in the anti-MUC1 antibody or antigen-binding fragment thereof, as such, peptides and aptamers having the binding capacity and specificity for the same MUC1 antigen are also included.
- the present invention relates to a hybridoma that produces the anti-MUC1 antibody.
- the hybridoma is Accession No. KCLRF-BP-00395.
- the present invention provides an anti-MUC1 antibody or antigen-binding fragment thereof produced by the hybridoma.
- Another example is the heavy chain complementarity determining region (CDR-H1, CDR-H2, CDR-H3, or a combination thereof) of the anti-MUC1 antibody produced by the hybridoma, light chain complementarity determining region (CDR-L1, CDR-L2) , CDR-L3, or a combination thereof, or a combination thereof;
- an anti-MUC1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region, a light chain variable region, or a combination thereof of the anti-MUC1 antibodies produced by the hybridoma.
- the complementarity determining portion may be determined by any conventional method, for example, IMGT definition (http://www.imgt.org/IMGT_vquest/share/textes/) or Kabat definition (http: // www. bioinf.org.uk/abs/), but is not limited thereto.
- IMGT definition http://www.imgt.org/IMGT_vquest/share/textes/
- Kabat definition http: // www. bioinf.org.uk/abs/
- the anti-MUC1 antibody or antigen-binding fragment thereof specifically recognizes the MUC1-C terminal extracellular domain, whereby the MUC1-C terminal extracellular domain is expressed at a higher level and less glycosylation than normal cells. It can act specifically in cancer or tumor cells and can also recognize / bind MUC1 protein expressed on one surface of the cell as well as on the entire surface.
- the anti-MUC1 antibody or antigen-binding fragment thereof not only binds MUC1 protein, in particular the MUC1-C terminal extracellular domain, but also internalizes into cells (see Examples 9-8 and 18) to mediate MUC1-mediated pathways. It can be effectively inhibited to maximize the pharmacological effect.
- the internalization properties of the anti-MUC1 antibody or antigen-binding fragment thereof have the advantage of effectively delivering the conjugated drug into the cell when applied as an antibody-drug conjugate (ADC).
- the present invention provides a chimeric antigen receptor (CAR) -T cell therapeutic agent and / or chimeric antigen receptor (CAR) -NK (natural killer cell) comprising an anti-MUC1 antibody or an antigen-binding fragment thereof according to the present invention. It is about.
- the form of the anti-MUC1 antibody or antigen-binding fragment thereof contained in the CAR-T or CAR-NK is preferably scFv, but is not limited thereto.
- the present invention relates to an antibody-drug conjugate (ADC) in which a drug is conjugated to the anti-MUC1 antibody or antigen-binding fragment thereof.
- ADC antibody-drug conjugate
- the drug of the antibody-drug conjugate is monomethyl auristatin E (MMAE).
- MMAE monomethyl auristatin E
- the antibody-drug conjugate binds to MUC1 expressing cells, then internalizes to MUC1 expressing tumor cells and selectively releases MMAE into target MUC1 cells by proteolytic cleavage.
- the released MMAE binds to tubulin, it disrupts the intracellular microtubule network, induces cell cycle arrest, and apoptosis progresses while microtubule division occurs.
- ADCs Antibody-drug conjugates require the anticancer drug to be stably bound to the antibody until the anticancer drug is delivered to the target cancer cell. Drug delivered to the target must be released from the antibody to induce killing of the target cell. This requires that the drug binds to the antibody stably and at the same time has sufficient cytotoxicity to induce the death of the target cell when released from the target cell.
- the cytotoxic substances including drugs such as the anti-MUC1 antibody or antigen-binding fragment thereof and an anticancer agent are bound to each other (eg, by covalent bonds, peptide bonds, etc.) to conjugate or fusion proteins (cytotoxicity).
- cytotoxicity In the form of a substance and / or a label).
- the cytotoxic substance may be any substance that is toxic to cancer cells, particularly solid cancer cells, and may be one or more selected from the group consisting of radioisotopes, cytotoxic compounds, cytotoxic proteins, anticancer agents, and the like. It is not limited to this.
- the cytotoxin protein is selected from the group consisting of lysine (ricin), saporin (saporin), gelonin (gelonin), momordin (momordin), deboganin (debouganin), diphtheria toxin, pseudomonas toxin, etc. It may be one or more, but is not limited thereto.
- the radioisotope may be at least one selected from the group consisting of 131I, 188Rh, 90Y, and the like, but is not limited thereto.
- the cytotoxin compound is duocarmycin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), N2'-diacetyl-N2 '-( 3-mercapto-1-oxopropyl) maytansine (N2'-deacetyl-N2 '-(3-mercapto-1-oxopropyl) maytansine; DM1), PBD (Pyrrolobenzodiazepine) dimer, etc.
- MMAE monomethyl auristatin E
- MMAF monomethyl auristatin F
- N2'-diacetyl-N2 '-( 3-mercapto-1-oxopropyl) maytansine N2'-deacetyl-N2 '-(3-mercapto-1-oxopropyl) maytansine
- DM1 PBD (Pyrrolobenzodiazepine) dimer, etc.
- PBD
- the antibody-drug conjugate may be according to techniques well known in the art.
- the antibody-drug conjugate may be characterized in that the antibody or antigen-binding fragment thereof is bound to the drug through a linker.
- the linker may be a cleavable linker or a non-cleavable linker.
- the linker is the linking site between the anti-MUC1 antibody and the drug, for example the linker is in a form that is cleavable under intracellular conditions, i.e., the drug can be released from the antibody through cleavage of the linker in the intracellular environment.
- the linker may be cleaved by a cleavage agent present in an intracellular environment such as a lysosomal or endosome, and may be a peptide linker that may be cleaved by an intracellular peptidase or protease enzyme such as a lysosomal or endosomal protease.
- Peptide linkers generally have at least two amino acids in length.
- the cleavage agent may include cathepsin B and cathepsin D, plasmin, and may hydrolyze the peptide to release the drug into target cells.
- the peptide linker may be cleaved by thiol dependent protease cathepsin-B, which is highly expressed in cancer tissue, for example Phe-Leu or Gly-Phe-Leu-Gly linkers can be used.
- the peptide linker may be cleaved by, for example, an intracellular protease, and may be a Val-Cit linker or a Phe-Lys linker.
- the cleavable linker is pH sensitive, and may be sensitive to hydrolysis at a specific pH value.
- pH sensitive linkers can be hydrolyzed under acidic conditions.
- acid labile linkers that can be hydrolyzed in lysosomes such as hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, Ketal and the like.
- the linker may be cleaved under reducing conditions, for example disulfide linkers.
- SATA N-succinimidyl-S-acetylthioacetate
- SPDP N-succinimidyl-3- (2-pyridyldithio) propionate
- SPDB N-succinimidyl-3- (2-pyridyldithio) butyrate
- N-succinimidyl-oxycarbonyl SMPT Various alpha disulfide bonds can be formed using -alpha-methyl-alpha- (2
- the drug and / or drug-linker may be conjugated randomly through lysine of the antibody or through cysteine which is exposed when the disulfide bond chain is reduced.
- the linker-drug may be bound via a genetically engineered tag, such as cysteine present in a peptide or protein.
- the genetically engineered tag eg, peptide or protein, may comprise an amino acid motif that can be recognized by, for example, an isoprenoid transferase.
- the peptide or protein has a deletion at the carboxy terminus of the peptide or protein, or has an addition via covalent attachment of a spacer unit to the carboxy (C) terminus of the peptide or protein.
- the peptide or protein may be directly covalently linked to an amino acid motif or covalently linked to a spacer unit to be linked to an amino acid motif.
- the amino acid spacer unit is composed of 1 to 20 amino acids, of which a glycine unit is preferable.
- the linker may comprise a beta-glucuronide linker which is present in a large number in lysosomes or is hydrolyzed by beta-glucuronidase which is overexpressed in some tumor cells.
- the hydrophilicity is high, and when combined with drugs having high hydrophobic properties, the solubility of the antibody-drug complex can be increased.
- the present invention relates to a beta-glucuronide linker disclosed in Korean Patent Publication No. 2015-0137015, for example, a beta-glucuronide linker comprising a self-immolative group. Can be used.
- linker may be, for example, a non-cleavable linker, and the drug is released through only one step of antibody hydrolysis to produce, for example, an amino acid-linker-drug complex.
- This type of linker may be a thioether group or maleimidocaproyl, and may maintain stability in blood.
- the drug may be characterized as a chemotherapeutic agent, toxin, micro RNA (miRNA), siRNA, shRNA or radioisotope.
- the drug may be bound to the antibody with an agent that exhibits a pharmacological effect.
- the chemotherapeutic agent may be a cytotoxic agent or an immunosuppressant. Specifically, it may include a microtubulin inhibitor, a mitosis inhibitor, a topoisomerase inhibitor, or a chemotherapeutic agent that can function as a DNA intercalator. It may also include immunomodulatory compounds, anticancer agents, antiviral agents, antibacterial agents, antifungal agents, antiparasitic agents or combinations thereof.
- Such drugs include, for example, maytansinoids, orstatin, aminopterin, actinomycin, bleomycin, thalisomycin, camptothecin, N8-acetyl spermidine, 1- (2 chloroethyl) -1, 2-dimethyl sulfonyl hydrazide, esperamycin, etoposide, 6-mercaptopurine, dolastatin, tricortesene, calicheamicin, taxol, taxanes, paclitaxel, docetaxel, Methotrexate, vincristine, vinblastine, doxorubicin, melphalan, mitomycin A, mitomycin C, chlorambucil, duocarmycin, L-asparaginase, mercaptopurine, thioguanine (thioguanine), hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea,
- the drug is an amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate which can react to form covalent bonds with electrophilic groups on linkers and linker reagents. And one or more nucleophilic groups selected from the group consisting of arylhydrazide groups.
- the present invention relates to a bispecific antibody comprising the anti-MUC1 antibody or antigen-binding fragment thereof.
- Said bispecific antibody comprises of two arms of the antibody, one arm comprises an anti-MUC1 antibody or antigen binding fragment thereof according to the invention, and the other arm is other than MUC1. It refers to a form comprising an antibody or an antigen-binding fragment thereof that specifically binds to an antigen, preferably an antibody related to a cancer-associated antigen or an immune gateway protein antigen, or an antigen-cell-associated antigen.
- the antigen to which the antibody other than the anti-MUC1 antibody included in the double antibody according to the present invention binds is preferably a cancer-related antigen or an immune gateway protein antigen, such as Her2, EGFR, VEGF, VEGF-R, CD-20, MUC16, CD30. , CD33, CD52, PD-1, PD-L1, CTLA4, BTLA4, EphB2, E-selectin, EpCam, CEA, PSMA, PSA, ERB3, c-MET, etc.
- TCR / CD3, CD16 (Fc ⁇ RIIIa) CD44, CD56, CD69, CD64 (Fc ⁇ RI), CD89, and CD11b / CD18 (CR3) may be selected, but are not limited thereto.
- the present invention relates to a pharmaceutical composition for preventing and / or treating MUC1-related diseases comprising the anti-MUC1 antibody or antigen-binding fragment thereof, the antibody-drug conjugate or the bispecific antibody.
- the MUC1-related disease may be a disease associated with expression or overexpression of MUC1, expression on all surfaces of cells of MUC1, and / or reduced glycation of MUC1 protein relative to normal cells, for example cancer.
- the normal cell may be a non-tumor cell. Accordingly, the MUC1-related disease is preferably cancer or tumor, but is not limited thereto.
- cancer refers to or means the physiological state of a mammal, which is typically characterized by unregulated cell growth / proliferation.
- the cancer or carcinoma that can be treated with the composition of the present invention is not particularly limited and includes both solid and hematological cancers.
- Examples of such cancers include skin cancers such as melanoma, liver cancer, hepatocellular carcinoma, hepatocellular cancer, gastric cancer, breast cancer, lung cancer, ovarian cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer, colon cancer, and Cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, parathyroid cancer, kidney cancer, esophageal cancer, biliary tract cancer, testicular cancer, rectal cancer, head and neck cancer, cervical cancer, ureter cancer, osteosarcoma, neuroblastoma, fibrosarcoma, rhabdomyosarcoma , But may be selected from the group consisting of astrocytoma, neuroblastoma and glioma, but is not limited thereto.
- the cancer is characterized by the expression of MUC1 protein, breast cancer, pancreatic cancer, prostate cancer, lung cancer, thyroid cancer, gastric cancer, ovarian cancer, colon cancer, liver cancer, gallbladder cancer, kidney cancer, cervical cancer, or bladder cancer
- the cancer may be primary or metastatic cancer.
- the MUC1-related disease may be, but is not limited to, Non-Alcoholic SteatoHepatitis (NASH) or TGF- ⁇ -mediated immune disease.
- NASH Non-Alcoholic SteatoHepatitis
- TGF- ⁇ -mediated immune disease TGF- ⁇ -mediated immune disease.
- the anti-MUC1 antibody or antigen-binding fragment thereof in the pharmaceutical composition, method and use for preventing and / or treating the cancer, is provided as a single active ingredient, or a cytotoxic substance such as an anticancer agent. It may be administered in combination or in the form of an antibody-drug conjugate (ADC) conjugated with a cytotoxic substance such as an anticancer agent.
- ADC antibody-drug conjugate
- the anti-MUC1 antibody or antigen-binding fragment thereof, and a pharmaceutical composition comprising the same according to the present invention can be used for use in combination with a conventional therapeutic agent.
- the anti-MUC1 antibody or antigen-binding fragment thereof according to the present invention, and a pharmaceutical composition comprising the same may be used in the same or administered sequentially with a conventional therapeutic agent such as an anticancer agent.
- the present invention comprises administering a therapeutically effective amount of the anti-MUC1 antibody or antigen-binding fragment thereof or the antibody-drug conjugate to a patient in need of preventing and / or treating MUC1-associated diseases.
- the prevention and / or treatment method may further comprise identifying a patient in need of prevention and / or treatment of the disease prior to the administering step.
- antibody conjugates for topical delivery of the drug in the composition allows the drug to be delivered to cells exhibiting an antigen targeted with an anti-MUC1 antibody.
- the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier.
- the pharmaceutically acceptable carrier is conventionally used in the formulation of drugs, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose , Polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate, mineral oil, etc. may be one or more selected from the group, but is not limited thereto. It doesn't happen.
- the pharmaceutical composition may also further include one or more selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like conventionally used in the manufacture of pharmaceutical compositions.
- the pharmaceutical composition may be administered orally or parenterally.
- parenteral administration it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, intranasal administration, pulmonary administration, rectal administration, or topical administration of lesion site.
- the protein or peptide is digested so that the oral composition can be formulated to coat the active agent or protect it from degradation in the stomach.
- the composition may be administered by any device in which the active substance may migrate to the target cell.
- the content or dosage of the anti-MUC1 antibody or antigen-binding fragment thereof in the pharmaceutical composition may vary depending on the method of formulation, mode of administration, age, weight, sex, morbidity, food, time of administration, interval of administration, route of administration, rate of excretion Various prescriptions can be made by factors such as response sensitivity.
- the daily dose of the anti-MUC1 antibody or antigen-binding fragment thereof is 0.001 to 1000 mg / kg, specifically 0.01 to 100 mg / kg, more specifically 0.1 to 50 mg / kg, more specifically 0.1 to 20 It may be in the mg / kg range, but is not limited thereto.
- the daily dosage may be formulated into one formulation in unit dosage form, may be formulated in appropriate quantities or may be prepared within a multi-dose container.
- compositions may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media or may be formulated in the form of extracts, powders, powders, granules, tablets or capsules, and for the purpose of formulation, dispersants or stabilizers It may further include.
- the patient to be administered the pharmaceutical composition may be a mammal including a primate including a human, a monkey, and the like, a rodent including a mouse, a rat, and the like.
- the treatment of cancer may mean any anti-cancer action that prevents, alleviates or improves the symptoms of cancer, such as inhibiting the proliferation of cancer cells, killing cancer cells, inhibiting metastasis, or partially or completely eradicate cancer.
- the invention relates to the use of said anti-MUC1 antibody or antigen binding fragment thereof for the prevention or treatment of cancer.
- the present invention relates to the use of said anti-MUC1 antibody or antigen-binding fragment thereof for the manufacture of a medicament for the prevention or treatment of cancer.
- the present invention provides a composition for detecting a MUC1 protein, such as an MUC1-C-terminal extracellular domain, comprising the anti-MUC1 antibody or antigen-binding fragment thereof, and a biological sample, wherein the anti-MUC1 antibody or It relates to a method for detecting MUC1 comprising processing an antigen binding fragment.
- the detection method may further comprise the step of determining whether the antigen-antibody reaction after the step of the treatment.
- the detection method when an antigen-antibody reaction is detected, it may be determined (determined) that MUC1, such as MUC1-C-terminal extracellular domain, is present in the biological sample. Accordingly, the detection method may further include determining that MUC1 is present in the biological sample when the antigen-antibody reaction is detected after the identifying step.
- the biological sample may be selected from the group consisting of (isolated) cells, tissues, body fluids, cultures thereof, etc. obtained from mammals such as humans (eg cancer patients).
- the present invention is in another aspect Treating (administering) the anti-MUC1 antibody or antigen-binding fragment thereof, comprising a composition for detecting or diagnosing a disease, such as cancer, including an anti-MUC1 antibody or antigen-binding fragment thereof, and a biological sample isolated from the subject It relates to a method for detecting or diagnosing cancer or providing information for detecting or diagnosing a cancer.
- the detecting or diagnosing method may further include determining whether an antigen-antibody reaction is performed after the treating.
- an antigen-antibody response when an antigen-antibody response is detected, it may be determined (determined) that a MUC1-related disease, such as cancer, is present in the biological sample or in a patient from which the biological sample is derived.
- the method may further comprise, after the identifying step, determining that the biological sample or the patient is a MUC1-related disease patient, such as a cancer patient, when an antigen-antibody response is detected.
- the biological sample may be selected from the group consisting of (isolated) cells, tissues, body fluids, cultures thereof, etc. obtained from mammals such as humans (eg cancer patients).
- Determining whether the antigen-antibody reaction can be carried out through various methods known in the art. For example, it can be measured through conventional enzymatic reaction, fluorescence, luminescence and / or radiation detection, and specifically, immunochromatography, immunohistochemistry, enzyme linked immunosorbent assay assay: ELISA, radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), Western blotting ), Microarrays, immunoprecipitation assays, etc. may be measured by a method selected from the group consisting of, but is not limited thereto.
- the anti-MUC1 antibody or antigen-binding fragment thereof may further include a label.
- the labeling substance may be at least one selected from the group consisting of radioisotopes, fluorescent substances, chromogens, dyeing substances, and the like.
- the labeling substance may be linked (linked) to the antibody or antigen-binding fragment by conventional methods (eg, chemical bonds such as covalent bonds, coordination bonds, and ionic bonds).
- the binding of the antibody (or antigen-binding fragment) and the labeling material may be according to techniques well known in the art.
- the C-terminus of MUC1 was cloned from breast cancer cell lines and expressed in Rosetta competent cells to prepare monoclonal antibodies against MUC1.
- the C-terminal portion of MUC1 was formulated with CpG-DNA (eg MB-ODN 4531 (O)) and encapsulated in the DOPE: CHEMS complex and used for mouse immunization.
- CpG-DNA eg MB-ODN 4531 (O)
- DOPE CHEMS complex
- anti-hMUC1-SEA monoclonal antibodies are obtained by fusing splenocytes obtained from mice immunized with [hMUC1-SEA]-[MB-ODN 4531 (O)]-[DOPE: CHEMS] complex with SP2 / 0 cells. Can be.
- the present invention relates to an immunogenic composition
- an immunogenic composition comprising the C-terminal region of MUC1, the SEA domain of MUC1, or the C-terminal extracellular domain of MUC1.
- Another example provides an immunogenic composition comprising a complex encapsulating (1) the C-terminal region of MUC1, the SEA domain of MUC1, or the C-terminal extracellular domain of MUC1 and (2) CpG-DNA in liposomes.
- the immunogenic composition refers to a composition having the ability to generate an antibody by inducing an immune response upon injection (inoculation) into a living organism in vivo.
- the liposome is a cationic liposome, such as dioleoylphosphatidylethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS) is 1: 0.5 to 1: 2, more specifically 1: 0.67 to 1: 1.5 (DOPE: CHEMS) molar ratio
- DOPE dioleoylphosphatidylethanolamine
- CHEMS cholesteryl hemisuccinate
- molar ratio such as, but not limited to, a mixture mixed at a molar ratio of about 1: 1, or one obtained therefrom (eg, a solvent-free lipid film, etc.).
- the CpG-DNA means an oligodeoxynucleotide (ODN: Oligodeoxynucleotide) including a total of 10 to 20 nucleotides including one or more CpG motifs, for example, 1 to 3.
- ODN Oligodeoxynucleotide
- the CpG-DNA may include a nucleic acid sequence of 'AGCAG CG TT CG TGT CG GCCT' (SEQ ID NO: 11), but is not limited thereto.
- the liposomes and oligodeoxynucleotides can function as an adjuvant.
- Adjuvant along with the immune system, help to recognize antigens more easily and enhance the immune response.
- Bacterial DNA containing synthetic oligodeoxynucleotides (ODNs) and unmethylated CpG dinucleotides flanked by specific nucleotide sequences have important immunomodulatory effects in B lymphocytes, natural killer cells, macrophages, and dendritic cells .
- Cationic liposomes eg lipofectamine, phosphatidyl-beta-oleoyl-gamma-palmitoyl ethanolamine (DOPE): cholesterol hemisuccinate (CHEMS) increase antibody production, antibody transport and CTL reactions To increase. Therefore, CpG-DNA (Lipoplex (O)) encapsulated in the DOPE: CHEMS (1: 1 ratio) complex can promote an effective immune response in human and mouse cells.
- DOPE phosphatidyl-beta-oleoyl-gamma-palmitoyl ethanolamine
- CHEMS cholesterol hemisuccinate
- the present invention provides a C-terminal region of MUC1, an SEA domain of MUC1, or a C-terminal extracellular domain or liposome of MUC1, including (1) a C-terminal region of MUC1, an SEA domain of MUC1, or a MUC1.
- a method for producing an anti-MUC1-SEA monoclonal antibody comprising inoculating a C-terminal extracellular domain and (2) a complex encapsulating CpG-DNA into a mammal, such as a mouse.
- the method for preparing the antibody may further include, after the inoculating step, separating and / or purifying the antibody from the serum of the mouse in a conventional manner.
- nucleic acids may be present in cells, cell lysates, or in partially purified or substantially pure form. Nucleic acids are prepared by other cellular components or other contaminants, for example, by standard techniques, including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. When purified from nucleic acids or proteins of other cells, they are "isolated” or “substantially pure.” Nucleic acids of the invention may be, for example, DNA or RNA, and may or may not include intron sequences.
- the nucleic acid encoding the anti-MUC1 antibody may be characterized in that it comprises any one or more sequences selected from the group consisting of SEQ ID NO: 34 to SEQ ID NO: 45.
- the polynucleotide sequence encoding the heavy chain of the antibody according to the present invention is SEQ ID NO: 34 to 39 and / or the polynucleotide sequence encoding the light chain of the antibody according to the present invention is SEQ ID NO: 40 to 45.
- the present invention relates to a recombinant expression vector comprising the nucleic acid.
- DNA encoding partial or full-length light and heavy chains can be selected from standard molecular biology techniques (e.g., PCR amplification or hybridomas expressing the antibody of interest). CDNA cloning used), and the DNA can be "bind to work" to transcription and translation control sequences and inserted into the expression vector.
- binding to work may mean that the gene encoding the antibody is ligated into the vector such that the transcriptional and translational control sequences in the vector serve the intended function of regulating the transcription and translation of the antibody gene.
- the expression vector and expression control sequences are chosen to be compatible with the expression host cell used.
- the light chain gene of the antibody and the heavy chain gene of the antibody are inserted into separate vectors, or both genes are inserted into the same expression vector.
- Antibodies are inserted into expression vectors by standard methods (eg ligation of complementary restriction enzyme sites on antibody gene fragments and vectors, or blunt terminal ligation if no restriction enzyme sites are present).
- the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from the host cell.
- the antibody chain gene can be cloned into the vector such that the signal peptide binds to the amino terminus of the antibody chain gene in frame.
- the signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide derived from an immunoglobulin non-protein).
- the recombinant expression vector has a regulatory sequence that controls the expression of the antibody chain gene in the host cell.
- a “regulatory sequence” can include promoters, enhancers and other expression control elements (eg, polyadenylation signals) that control the transcription or translation of antibody chain genes.
- promoters e.g., promoters, enhancers and other expression control elements (eg, polyadenylation signals) that control the transcription or translation of antibody chain genes.
- expression control elements e.g, polyadenylation signals
- the design of the expression vector can vary by differently selecting regulatory sequences depending on factors such as the selection of host cells to be transformed, the expression level of the protein, and the like.
- the present invention relates to a host cell comprising the nucleic acid or the vector.
- the host cell according to the present invention is preferably selected from the group consisting of animal cells, plant cells, yeast, E. coli and insect cells, but is not limited thereto.
- the host cell according to the present invention is E. coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis or Staphyllo Prokaryotic cells, such as the Staphylococcus sp.
- fungi such as Aspergillus sp., Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp.
- Neuro Eukaryotic cells such as yeast, such as Neurospora crassa, other lower eukaryotic cells, and cells of higher eukaryotes such as cells from insects.
- COS7 cells monkey kidney cells (COS7) cells, NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells and HEK293 cells and the like are available, but are not limited to these.
- COS7 cells monkey kidney cells
- NSO cells NSO cells
- SP2 / 0 Chinese hamster ovary (CHO) cells W138
- BHK baby hamster kidney
- MDCK myeloma cell lines
- HuT 78 cells and HEK293 cells and the like are available, but are not limited to these.
- CHO cells can be used.
- the nucleic acid or the vector is transfected or transfected into a host cell.
- Various expression host / vector combinations can be used to express anti-glycancan 3 antibodies according to the invention
- Expression vectors suitable for eukaryotic hosts include, but are not limited to, expression control sequences derived from SV40, bovine papillomavirus, anenovirus, adeno-associated virus, cytomegalovirus and retroviruses.
- Expression vectors include Escherichia coli such as pET, pRSET, pBluescript, pGEX2T, pUC vector, col E1, pCR1, pBR322, pMB9 and derivatives thereof. Resulting bacterial plasmids, plasmids with a broader host range such as RP4, phage DNA exemplified by a wide variety of phage lambda derivatives such as ⁇ gt10 and ⁇ gt11, NM989, and DNA phages of M13 and filamentary single strands; Other DNA phages such as, etc. Expression vectors useful for yeast cells are 2 ° C. plasmids and derivatives thereof.
- the present invention provides a method for producing an anti-MUC1 antibody or antigen-binding fragment thereof according to the present invention comprising culturing a host cell to express an anti-MUC1 antibody or antigen-binding fragment thereof according to the present invention. It is about.
- the antibody When a recombinant expression vector capable of expressing the anti-MUC1 antibody or antigen-binding fragment thereof is introduced into a mammalian host cell, the antibody is cultivated for a period sufficient to allow the antibody to be expressed in the host cell, or more preferably the host cell is cultured. It can be prepared by culturing the host cell for a period sufficient to allow the antibody to be secreted into the culture medium.
- the expressed antibody may be purified from the host cell to be homogeneous. Separation or purification of the antibody can be carried out by separation, purification methods, such as chromatography, which are used in conventional proteins.
- the chromatography can include, for example, affinity chromatography comprising a Protein A column, Protein G column, ion exchange chromatography or hydrophobic chromatography.
- the antibody can be separated and purified by further combining filtration, ultrafiltration, salting out, dialysis and the like.
- the MUC1-C terminus (hereinafter referred to as the "hMUC1-C protein"; a protein comprising 192 amino acids from 961 to 1152 of P15941, wherein 1034-1152 aa is the MUC-SEA domain); Encoding human cDNA was obtained and amplified by RT-PCR using the following primer sets.
- Sense primer hMUC1 C-Nco I-S3 5'-CC ATG GCC TCA GGC TCT GCA TC-3 '(SEQ ID NO: 12);
- Antisense Primer hMUC1 C-Xho I-AS4 5'-CTC GAG AGA CTG GGC AGA GAA AGG AAA T-3 '(SEQ ID NO: 13).
- the obtained hMUC1-C terminal protein coding sequence was confirmed by DNA sequencing, GenBank Accession No. It was confirmed that it had the same sequence as the nucleic acid sequence containing 576 nucleotides of 2954 th-3529 of J05582.1 (SEQ ID NO: 26).
- the amplified cDNA fragment was cloned into expression vector pET-22b (Novagen, Darmstadt, Germany) containing C-terminal His-tag.
- the obtained plasmids were transformed into Escherichia coli Rosetta TM (Invitrogen, Carlsbad, CA) receptor cells, and using 1 mM isopropyl-D-1-thioglactopyranoside (IPTG, Sigma-Aldrich, Saint Louis, MO). Induction at 37 ° C. for 8 hours.
- the obtained cells were lysed in ice lysis buffer (50 mM Tris-HCl, 100 mM NaCl, 5 mM EDTA, 0.5% Triton X100, 1 g / ml lysozyme, proteinase inhibitor cocktail). After centrifugation, the inclusion body fraction was mixed with buffer B (100 mM NaH 2 PO 4 , 10 mM Tris-HCl, 8 M urea, pH 8.0), using a Ni-NTA agarose (Qiagen, Valencia, CA) system. Purified.
- the resulting mixture was loaded onto a Ni-NTA column and washed with wash buffer C (100 mM NaH 2 PO 4 , 10 mM Tris-HCl, 8 M urea, pH 6.3). Bound proteins were eluted with elution buffer (100 mM NaH 2 PO 4 , 10 mM Tris-HCl, 8 M urea, pH 4.5) and analyzed by SDS-PAGE and Western blotting. Western blots were performed using anti-His-tag antibody (Santa Cruz).
- MB-ODN 4531 consists of 20 bases containing three CpG motifs (underlined) (AGCAG CG TT CG TGT CG GCCT: SEQ ID NO: 11).
- the CpG oligodeoxynucleotide (ODN) (hereinafter referred to as "CpG-DNA 4531 (O)") was purchased from Samchully Pharm (Seoul, Korea) and GenoTech (Daejeon, Korea).
- DOPE Phosphatidyl-beta-oleoyl-gamma-palmitoylethanolamine
- CHEMS cholesterol hemisuccinate
- Solvent-free lipid film was resuspended in a mixture containing 50 ⁇ g of the same volume of water-soluble CpG-DNA 4531 (O) and 50 ⁇ g of rhMUC1-C protein (Example 2), and vigorously stirred at room temperature for 30 minutes to give DOPE and CHEMS CpG-DNA 4531 (O) and rhMUC1-C proteins coencapsulated within the complex were prepared.
- “Lipoplex (O) Complex” refers to CpG-DNA 4531 (O) encapsulated in DOPE and CHEMS complexes.
- the rhMUC1-C protein and Lipoplex (O) complexes were sonicated for 30 seconds by sonicator, the solution was filtered using a 0.22 ⁇ m filter and freeze-thawed three times with liquid nitrogen. Used for.
- mice Four-week-old female BALB / c mice (OrientBio, Inc. (Seoul, Korea)) and BALB / cAnNCri-nu / nu mice (four-week-old) were purchased from Nara Biotech, Inc. (Seoul, Korea). The mice were maintained at 20-25 ° C. and 32-37% humidity in sterile conditions without specific pathogens. All animal testing procedures were conducted in accordance with the "Guide for the Care and Use of Laboratory Animals of the National Veterinary Research & Quarantine Service of Korea" with the approval of the Institutional Animal Care and Use Committee. Every effort was made to sacrifice the mice through isoflurane inhalation and to minimize pain.
- 50 ⁇ g of rhMUC1-C protein and 50 ⁇ g of CpG-DNA 4531 comprise a complex co-encapsulated in phosphatidyl-beta-oleoyl-gamma-palmitoylethanolamine: cholesterol hemisuccinate (DOPE: CHEMS complex)
- DOPE cholesterol hemisuccinate
- rhMUC1-C specific IgG was measured by ELISA.
- 96-well immunoplates were coated with 10 ⁇ g / ml rhMUC1-C protein (Example 2) and 1% (w / v) in PBS-T (PBS with 0.1% (v / v) Tween-20) Blocked with bovine serum albumin (BSA).
- BSA bovine serum albumin
- Immunized mouse serum, hybridoma cell culture supernatants, or purified antibodies in Example 5 were diluted with PBS-T and incubated at room temperature for 2 hours. Plates were washed three times with PBS-T and incubated with goat anti-mouse IgG HRP-conjugated secondary antibody for 1 hour.
- TMB substrate solutions A and B (1: 1 ratio) (Kirkegaard and Perry Laboratories, Gaithersburg, MD) were developed and 450 nm using a Spectra Max 250 micro plate reader (Molecular Devices, Sunnyvale, USA) Absorbance at was measured and colorimetric assay was performed.
- Splenocytes of mice immunized with rhMUC1-C protein in Example 5 were fused with SP2 / 0 myeloma cells (ATCC) using polyethylene glycol (PEG, Sigma-Aldrich).
- the fused cells were cultured and selected as hypoxanthine-aminopterin-thymidine (HAT, Sigma-Aldrich) medium.
- the culture supernatants of the selected hybridoma cells were tested for binding to rhMUC1-C protein via ELISA and screened for hybridoma cells showing binding positive, ie, producing rhMUC1-C protein specific antibodies.
- Hybridoma clones were selected according to standard hybridoma technology (Yokoyama et al. 2006).
- the obtained hybridoma clone (KCLRF-BP-00395; hereinafter referred to as “hMUC1-1H7” for hybridoma cells and antibodies produced therefrom) was cultured in hypoxanthine-thymidine (HT) medium.
- HT hypoxanthine-thymidine
- ELISA-positive hybridoma cell populations were subcloned.
- mice were hybridized with i.p. hybridoma cells (hMUC1-1H7 clone). Inoculation. After 10 days, the peritoneal fluid was collected and centrifuged at 3,000 rpm for 30 minutes. The supernatant was purified using Protein-A chromatography (Repligen, Waltham, MA) to anti-hMUC1 monoclonal antibody that binds to rhMUC1-C protein.
- 96-well immunoplate (Nalgen Nunc International, Penfield, USA) was coated with 1 ⁇ g / ml of hMUC1-C protein, 1 Blocking with 0.05% Tween-20 (PBST) in PBS containing% BSA.
- PBST Tween-20
- Anti-hMUC1-C monoclonal antibodies were added to the top row of each plate and a series of 1: 4 dilutions in PBST were placed in the next row. Plates were incubated for 2 hours at room temperature and washed with PBST.
- Anti-mouse total IgG, IgG1, IgG2a, IgG3b, IgG3 antibodies combined with Horseradish Peroxidase (HRP) (BD Pharmingen) were then added to each well (1: 500 dilution) and 1 at room temperature. Incubated for hours.
- Whole volume analysis using TMB substrate solutions A and B (1: 1 ratio) (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA), using a Spectra Max 250 microplate reader (Molecular Devices, Sunnydale, CA, USA) was measured at 450 nm absorbance.
- MCF-7 Human breast cancer cell lines
- Capan-2 pancreatic cancer cell lines
- ATCC American Type Culture Collection
- T47D human breast cancer cell lines
- ZR75- 1 pancreatic cancer cell lines
- PANC-1 Korean Cell Line Bank
- MCF-7 cells were cultured in Eagle's Minimum Essential Medium supplemented with 0.01 mg / ml human recombinant insulin; MDA-MB-231 cells were cultured in Leibovitz's L-15 medium (Thermo Fisher Scientific); T47D, ZR75-1, and Capan-1 cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific).
- Capan-2 cells in McMcoy's 5A medium (Thermo Fisher Scientific), CFPAC-1 cells in Iscove's modified Dulbecco's medium (IMDM, Thermo Fisher Scientific), PANC-1 cells in Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher Scientific) Each was incubated.
- MCF-7, T47D, ZR75-1 Capan-1, Capan-2, CFPAC-1, and PANC-1 cells were cultured at 37 ° C. and 5% CO 2 conditions, and MDA-MB-231 cells were CO 2 at 37 ° C. Incubated in the absence of conditions.
- anti-MUC1-CT antibodies and anti-MUC1-CT2 antibodies were obtained from Abcam (Cambridge, UK).
- Anti-MUC1-CT antibodies and anti-MUC1-CT2 antibodies recognize the cytoplasmic tail region of MUC1.
- Anti-beta-actin antibodies were purchased from Sigma-Aldrich.
- the cells were lysed in lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 100 mM NaF, 2 mM Na 3 Vo 4 , Protease Inhibitor Cocktail, 1% (w / v) NP-40 ) And centrifuged at 14,000 rpm for 20 minutes at 4 ° C. The same amount of protein was separated on a 4-12% Bis-Tris gradient gel (Thermo Fisher Scientific) and transferred to a nitrocellulose membrane blocked with 3% (w / v) BSA in PBS-T for 1 hour at room temperature. The nitrocellulose membrane was left overnight at 4 ° C.
- anti-hMUC1 antibody hMUC1-1H7; Example 7
- anti-MUC1-CT antibody Abacam, EPR1023
- anti-MUC1-CT2 antibody Abcam, ab80952
- anti Incubated with beta-actin antibody Sigma-Aldrich.
- Immune reactive proteins were measured using horseradish peroxidase bound secondary antibodies and enhanced chemiluminescent reagents (Thermo Fisher Scientific).
- Cell lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 100 mM NaF, 2 mM Na 3 Vo 4 , Protease Inhibitor Cocktail, 1% (w / v) NP-40) Incubated overnight at 4 ° C. with anti-hMUC1 antibody. Protein A beads were added to the mixture and incubated at 4 ° C. for 1 hour. The immune complexes collected by centrifugation were washed and analyzed by western blotting.
- Membranes are combined with anti-hMUC1 antibody (hMUC1-1H7), anti-MUC1-CT antibody (Abacam, EPR1023), anti-MUC1-CT2 antibody (Abcam, ab80952), or anti-beta-actin antibody (Sigma-Aldrich) antibody. Incubated.
- Cell lysates of T47D cells were extracted with lysis buffer (0.5% (w / v) SDS, 1% (w / v) beta-mercapto ethanol) and boiled at 100 ° C. for 10 minutes. Thereafter, the sample was incubated with PNGase F (Elpis-Biotech, Daejeon, Korea) for 2 hours at 37 °C, boiled for 10 minutes at 100 °C. The resulting sample was diluted with lysis buffer and immunoprecipitated with anti-hMUC1 monoclonal antibody. The resulting immune complexes were analyzed by western blotting with anti-MUC-CT antibody or anti-MUC1-CT2 antibody.
- Cells were cultured on glasscover slips coated with poly-L-lysine in 12-well culture plates.
- For cell surface staining cells were fixed with 4% (w / v) paraformaldehyde, blocked with 3% (w / v) BSA and stained with anti-hMUC1 antibody for 2 hours on ice.
- For intracellular staining cells were fixed with 4% (w / v) paraformaldehyde, permeabilized with 0.1% (w / v) Triton X-100, blocked with 3% (w / v) BSA, Stained with anti-hMUC1 antibody for 2 hours at room temperature.
- Anti-hMUC1 monoclonal antibodies were labeled with DyLight 488 according to the manufacturer's instructions (Thermo Fisher Scientific). Breast cancer cells and pancreatic cancer cells were treated with DyLight 488 labeled anti-hMUC1 antibody and incubated at 37 ° C. for the indicated time. Fluorescent signals generated from cells in which the antibody was internalized were detected by CLSM (LSM 710, Carl Zeiss).
- Anti-hMUC1 monoclonal antibody (hMUC1-1H7; Example 7) was incubated using a mouse monoclonal antibody isotype typing kit (Dipstick format, Bibco BRL or Roche, Mannheim, Germany). Total RNA was extracted from the hybridoma cells using RNeasy Mini Kit (Qiagen) to generate cDNA. To clone the heavy and light chain variable regions (VH and VL) of the anti-hMUC1 monoclonal antibody generated from hMUC1-1H7, the generated cDNA was subjected to Vent polymerase (NEB) having the following primer sets. Amplified using.
- Heavy chain primers IGG1 (5'-GGA AGA TCT ATA GAC AGA TGG GGG TGT CGT TTT GGC-3 '; SEQ ID NO: 14) and 5'MH2 (5'-CTT CCG GAA TTC SAR GTN MAG CTG SAG SAG TCW GG- 3 '; SEQ ID NO: 15)
- -hMUC1 monoclonal antibody (hMUC1-1H7; Example 7) of the light chain variable region and heavy chain variable regions (VH and VL) for amplifying the coding sequence, and each with a Sfi I and BstX I serves as a bacterial expression vector FabE Cloning (Jeon et al., Mol. Immunol. 44: 827-836 (2007), Kwon et al. Oncol. Rep. 18: 513-517 (2007)) and the resulting recombinant expression plasmid with pFabE-hMUC1-1H7 (FIG. 8A). Named it.
- the primer used at this time is as follows.
- Reverse primer 5'-GGC CGT GCT GGC CCC GAC AGA TGG GGG TGT CGT TTT GGC-3 '(SEQ ID NO: 19),
- Reverse primer 5'-CCA CCG TAC TGG CGG ATA CAG TTG GTG CAG CAT C-3 '(SEQ ID NO: 21).
- pFabE-hMUC1-1H7 Recombinant expression plasmid pFabE-hMUC1-1H7 was confirmed by restriction analysis and DNA sequencing. pFabE-hMUC1-1H7 was transformed into TG1 Escherichia coli cells, optimized for expression of recombinant protein, and confirmed by western blotting with anti-His antibody.
- FabE-hMUC1-1H7 For large scale production and purification of recombinant Fab, 500 ml of hybridoma hMUC1-1H7 clone was treated with 0.5 mM IPTG and incubated at 18 ° C. for 16 hours. The culture was centrifuged to harvest the culture supernatant and placed in a Ni-NTA affinity column (Clontech) to separate Fab proteins that were properly folded and assembled using His tags in the VH-CH fusion protein.
- Recombinant Fab bound to the column was eluted with 10 mM imidazole (pH 8.0), the protein solution was centrifuged (3,500 xg) at 4 ° C, dialyzed and concentrated using centricon.
- Capan-2 cells in 50% (w / v) Matrigel were subcutaneously inoculated into the dorsal right flank of 4 week old male BALB / cAnCrj-nu / nu mice.
- 17 beta-estradiol pellets US, FL, Sarasota, Innovative Research of America, pellets, 60 day release
- 5 ⁇ 10 6 cells (T47D, ZR75-1) in 50% (w / v) Matrigel were subcutaneously inoculated into the dorsal right flank of the mouse.
- mice When the tumor volume reached an average of about 100 mm 3 , the mice were injected intravenously with normal mouse IgG-DyLight 755 (5 mg / kg) or anti-hMUC1 antibody-DyLight 755 (5 mg / kg).
- the antibody target images were then monitored using an in vivo imaging system (IVIS 200, Xenogen Corporation, Mass.) At 0, 24, 48 hours.
- IVIS 200 In vivo imaging system
- DyLight 488-labeled anti-hMUC1 antibody 5 mg / kg was injected intravenously. After 2 days, tumor tissue was harvested and frozen sections were performed. Internalization of the antibody was detected in tumor sections with CLSM (LSM 710, Carl Zeiss).
- Paraffin-depressed human breast cancer tissue sections were purchased from ISU ABXIS (Seoul, Korea). The tissue sections were treated with xylene for 30 minutes to remove paraffin, rehydrated with ethanol, and incubated with 3% hydrogen peroxide solution for 10 minutes. Antigen retrieval was performed in citric acid solution (pH 6.0). The sections were blocked for 30 minutes using normal horse serum and incubated with anti-hMUC1 antibody (1 ⁇ g / slide) for 2 hours at room temperature. The sections were washed and incubated with biotinylated anti-mouse IgG antibody Vector Laboratories, Burlingame, CA for 1 hour. It was then washed and incubated with HRP-streptavidin for 30 minutes.
- FIG. 1A the control is a PBS inoculation group, and rhMUC1-C-1, -2, -3, and -4 denote each mouse. As shown in Figure 1a, it can be seen that rhMUC1-C specific IgG induced significant.
- FIGS. 1B and 1C hybridoma cells isolated from the immunized mice were screened according to Example 7 and shown in FIGS. 1B and 1C.
- Figure 1b is a screening result using HAT medium
- Figure 1c is a screening result using HT medium. 1B and 1C
- # is the number of 96-well plates
- a to H designate horizontal sections of the plate. From the results in FIGS. 1B and 1C, hMUC1-1H7 clones were selected as hybridomas producing rhMUC1-C specific monoclonal antibodies.
- the selected hybridoma hMUC1-1H7 cells were deposited on March 8, 2017 with the Korea Cell Line Research Foundation (KCLRF) located in Yeongun-dong, Jongno-gu, Seoul, Korea, and received accession number KCLRF-BP-00395.
- KCLRF Korea Cell Line Research Foundation
- FIG. 2a is a result of injecting the hybridoma hMUC1-1H7 clone into the abdominal cavity of the mouse, ascites was recovered, the analysis of the presence of rhMUC1-C specific antibody by ELISA, control serum means the result of the PBS inoculation group do. As a result, it was confirmed that rhMUC1-C specific antibody was present in ascites of mice injected with hMUC1-1H7 clone, and isolated and purified.
- HC represents a heavy chain
- LC represents a light chain
- MUC1 protein in breast cancer cells MUC1 protein in normal cells is composed of dimers of MUC1-N subunit domain and MUC1-C subunit domain and is hyperglycosylated in cancer cells
- MCF-7, MDA-MB-231, T47D and ZR75-1 cell lysates of breast cancer cells
- the anti-MUC1-CT antibody detected MUC1 protein in MCF-7, T47D and ZR75-1, but not MDA-MB-231.
- anti-hMUC1 monoclonal antibodies did not recognize MUC1 protein in all cell samples. Since no MUC-1 protein was detected in MDA-MB-231 cells, this cell line was used as a negative control in all tests.
- results of FIG. 3A show that the anti-hMUC1 monoclonal antibody does not recognize the denatured MUC1 protein used in Western blotting
- results of FIG. 3B show that the anti-hMUC1 monoclonal antibody is unique in cancer through immunoprecipitation. It can be seen that the recognition of the MUC1 protein of the anti-hMUC1 monoclonal antibody recognizes the unique tertiary structure of the MUC1 protein.
- the extracellular domain of MUC1 is densely glycated from 200 to 500 nm from the cell surface. Therefore, it was investigated whether the anti-hMUC1 monoclonal antibody could recognize the deglycosylated protein core (see Example 9.6).
- T47D cell lysates were treated with PNGase F and Western blotting using anti-MUC1-CT antibody, anti-MUC1-CT2 antibody, anti-hMUC1 monoclonal antibody (Example 7), or anti-beta-actin antibody
- FIG. 3C it can be seen that the anti-hMUC1 monoclonal antibody does not detect the MUC1 protein.
- T47D cell lysates treated with PNGase F were immunoprecipitated with anti-hMUC1 monoclonal antibody, and then immunoblotted with anti-MUC1-CT or anti-MUC1-CT2 antibody.
- anti-hMUC1 monoclonal antibody could immunoprecipitate deglycosylated MUC1 protein in T47D cells.
- pancreatic cancer cells Capan-1, Capan-2, CFPAC-1, and PANC-1
- pancreatic cancer cells Capan-1, Capan-2, CFPAC-1, and PANC-1
- anti-hMUC1 monoclonal antibody recognizes an intact MUC1 protein in pancreatic cancer cells
- the lysates were separated by SDS-PAGE and western blotting was performed using anti-MUC1-CT antibodies, anti-hMUC1 antibodies, or anti-beta-actin antibodies, and the results are shown in FIG. 4A.
- the anti-hMUC1 monoclonal antibody was not able to recognize MUC1 protein in all cell samples tested.
- MCF-7 breast cancer cells
- MDA-MB-231 MDA-MB-231
- T47D and ZR75-1 pancreatic cancer cells
- Capan- 1, Capan-2, CFPAC-1, PANC-1 immunofluorescence staining and the results were analyzed by confocal microscopy (see Example 9-7).
- MCF-7, MDA-MB-231, T47D and ZR75-1 cells were treated with anti-hMUC1 monoclonal antibody or mouse normal IgG at 4 ° C. (for cell surface MUC1 protein) or at room temperature (intracellular MUC1 protein). After incubation in), and incubated with Alexa 488 (green) -conjugated secondary antibody for 1 hour, the cell nuclei were stained with Hoechst 33258 (blue). The obtained fluorescence image was observed with a confocal microscope (CLSM, LSM 710, Carl Zeiss, Jena, Germany), which is shown in A of FIG. 5 (scale bar: 10 ⁇ m).
- MCF-7, MDA-MB-231, T47D and ZR75-1 cells were treated with DyLight 488 (green) -labeled anti-hMUC1 monoclonal antibody. And incubated at 37 ° C. for 6 hours, the nuclei were stained with Hoechst 33258, and the resulting fluorescence images were observed by confocal microscopy (CLSM, LSM 710, Carl Zeiss, Jena, Germany). (Scale bar: 10 ⁇ m).
- pancreatic cancer cell lines Capan-1, Capan-2, CFPAC-1 and PANC-1 cells were incubated with anti-hMUC1 monoclonal antibody or mouse normal IgG at 4 ° C. (surface) or at room temperature (in cells), then Alexa 488 Incubated with conjugated secondary antibody for 1 hour and nuclei stained with Hoechst 33258.
- the obtained fluorescence image was observed with a confocal microscope (CLSM, LSM 710, Carl Zeiss, Jena, Germany) and shown in FIG. 6 (scale bar: 10 mu m).
- the anti-hMUC1 antibody stains MUC1 distinctly in breast cancer cells (MCF-7, T47D and ZR75-1) and pancreatic cancer cells (Capan-2 and CFPAC-1). It can be seen that they are located in the cell surface and in the cell.
- the antibody recognizes the extracellular region of the MUC1 C-terminal subunit.
- the efficacy of the antibody as a therapeutic agent can be said to depend more on cell internalization. Therefore, the anti-hMUC1 antibody was combined with DyLight 488, and treated with breast cancer cells and pancreatic cancer cells for 6 hours, and then the cell internalization of the antibody was confirmed through fluorescence images.
- Pancreatic cancer cells (Capan-1, Capan-2, CFPAC-1 and PANC-1) were treated with DyLight 488-labeled anti-hMUC1 monoclonal antibody, 0, 30 minutes, 1 hour, 3 hours, 6 hours, 12 Incubated at 37 ° C. for 24 h and 24 h. Nuclei were stained with Hoechst 33258.
- the results confirm that anti-hMUC1 monoclonal antibodies can target and internalize MUC1 protein in living cells.
- variable regions of the heavy and light chains of the anti-hMUC1 monoclonal antibody were cloned from the hybridoma cells producing the anti-hMUC1 monoclonal antibody (hMUC1-1H7) and DNA sequenced (Example 10).
- the sequence identified by the DNA sequencing was analyzed by the BLAST program (http://www.ncbi.nlm.nih.gov) to analyze homology with known sequences.
- the cDNA (heavy chain variable region coding cDNA: 393bp; light chain variable region coding cDNA: 396bp) encoding the variable regions of the heavy and light chains of the hMUC1-1H7 antibody was encoded by the heavy and light chain variable regions of the known mouse immunoglobulin (IgG1). Sequence homology of about 80-95% and 93-98% with the sequence, respectively.
- CDRs of the heavy and light chains were identified by a known method (kabat CDR definition) (Table 2).
- the variable region sequences of the heavy and light chains are shown in Table 3 below.
- Recombinant Fab was expressed in E. coli using the expression vector pFabE (Example 11).
- Vector pFabE is a light chain constant region (CL), of the heavy chain constant region (CH1), the light chain variable region (VL), and a heavy chain variable region including two cloning sites BstX I and Sfi I in (VH) may be inserted respectively, do.
- Recombinant expression plasmid pFabE-hMUC1-1H7 was constructed by sequentially subcloning the VL and VH sequences of hMUC1-1H7 (FIG. 8A). The recombinant plasmid bi-cistronic expression of the VL-CL fusion protein and the VH-CH1 fusion protein under control of the LacZ promoter in E. coli.
- VL-CL fusion proteins include N-terminal OmpA tags and C-terminal Pre-S1 tags, while VH-CH1 fusion proteins include N-terminal pelB tags and C-terminal His tags (FIG. 8B). Since the recombinant VH-CH1 fusion protein contained His tag, large scale culture of E. coli and recombinant Fab purification of hMUC1-1H7 were performed using Ni-NTA affinity column chromatography.
- recombinant Fab-hMUC1-1H7 breast cancer cells
- An immunofluorescence staining test was performed for 1).
- Breast cancer cells MCF-7, MDA-MB-231, T47D and ZR75-1 cells were incubated with recombinant Fab-hMUC1-1H7, respectively, followed by incubation with Alexa 488-conjugated secondary antibody, and nuclei stained with Hoechst 33258. It was.
- the obtained fluorescence image was observed with a confocal microscope (CLSM, LSM 710, Carl Zeiss, Jena, Germany), and is shown in FIG. 8C (scale bar: 10 mu m).
- the confocal image clearly stained MUC1 in recombinant Fab-hMUC1-1H7 breast cancer cells (MCF-7, T47D and ZR75-1), indicating that the recombinant Fab-hMUC1-1H7 stained breast cancer cells. It means to recognize MUC1.
- MUC1 is overexpressed in various types of cancerous tissues to promote cell proliferation.
- anti-hMUC1 monoclonal antibodies As cancer therapeutics, the effects of anti-hMUC1 monoclonal antibodies on breast cancer cell proliferation were tested.
- MDA-MB-231, T47D and ZR75-1 cells were treated with anti-hMUC1 antibody (10 ⁇ g / ml; Example 7) or normal mouse IgG1 (10 ⁇ g / ml) and MTT assay at the time intervals shown in FIG. 9. The effect on cell proliferation was tested by (Example 12).
- control is a group not treated with the antibody.
- FIG. 9 it was confirmed that the treatment of anti-hMUC1 antibody significantly delayed the proliferation of T47D and ZR75-1 cells compared to the control IgG treatment.
- anti-hMUC1 antibodies did not alter proliferation of MDA-MB-231 cells.
- FIGS. 10A to 10H breast cancer
- 11A to 11C pancreatic cancer
- 10A to 10D show the results of tumors induced by subcutaneous injection of T47D cells, which are breast cancer cells
- FIGS. 10E to 10H show results of tumors induced by tumors induced by tumor injection of ZR75-1 cells, which are breast cancer cells.
- 10B and 10F imaging of dissected mice was performed using a real time IVIS imaging system 200.
- 10C and 10G show the results of antibody distribution by separating several organs and tumors.
- 10D and 10H show tumor sections stained with DAPI for nuclei and evaluated by confocal microscopy (scale bar: 10 ⁇ m).
- anti-hMUC1 monoclonal antibody labeled with DyLight shows a clear staining result in tumor sections, whereas normal IgG labeled with DyLight does not show this effect.
- tumor-specific intracellular localization of anti-hMUC1 monoclonal antibodies was further confirmed by the confocal images of FIGS. 10D, 10H and 11C.
- anti-hMUC1 monoclonal antibodies can be used to specifically target breast and pancreatic cancer in animal models.
- a xenograft mouse model was used to test the effect of MUC1 targeted monoclonal antibodies on the growth of pancreatic cancer cells in vivo.
- FIG. 12A The tumor tissue extracted from the xenograft mouse model is shown in FIG. 12A and the tumor size ((width 2 * length) / 2), weight, and mouse weight are shown in FIGS. 12B, 12C, and 12D, respectively. It was. As shown in Figure 12a to 12d, administration of anti-hMUC1 monoclonal antibody is attenuated the progression of pancreatic tumors, it can be seen that the antibody treatment does not adversely affect the body weight of the test body.
- MUC1 protein in breast and pancreatic cancer tissues was examined by immunostaining. Breast cancer tissue and pancreatic cancer tissue were tested using normal tissue as a control. The results are shown in FIGS. 13 (breast cancer) and 14 (pancreatic cancer). 13 and 14, it was confirmed that MUC1 is expressed in most breast cancer tissues and pancreatic cancer tissues, whereas MUC1 is not expressed in normal breast tissues.
- ADC was prepared by binding MMAE to 1H7 antibody in Levena Biopharma (USA). The ratio of MMAE (DAR) to antibody of the prepared ADC was 5.80.
- DAR MMAE
- three cell lines of MDA-MB-231, T47D, and ZR75-1 were used. Each cell line was cultured in a 96-well plate, and 24 hours after the start of the culture, 1H7-ADC at the concentration set as shown in FIG. 15 was treated. 72 hours after 1H7-ADC treatment, cell viability was compared using CCK-8 kit (Dojindo, USA).
- the treatment method of CCK-8 was performed according to the manual provided, and the absorbance value was measured using an I3X microplate reader (Molecular Devices, USA). It was confirmed that the survival rate of T47D and ZR75-1 cells treated with 1H7-ADC was significantly lower than the control group not treated with 1H7-ADC. In contrast, survival of MDA-MB-231 cells treated with 1H7-ADC showed no significant difference from the control. These results indicate that 1H7-ADC selectively shows anticancer effects on cancer cells expressing MUC1.
- Humanized antibodies were prepared based on the 1H7 antibody which confirmed the cytotoxicity and anticancer effects in animal models through ADC preparation. Humanized antibodies were produced by the New Drug Development Support Center of Osong Advanced Medical Industry Promotion Foundation (Osong, Korea) and Fusion Antibodies (UK). The antigen recognition sites of the 1H7 antibody were identified and the variable region sequences except for the antigen recognition sites were changed to prepare 6 heavy chain sequences and 6 light chain sequences. Each amino acid sequence is shown in Table 6.
- the affinity measurement for binding of the anti-hMUC1-C humanized antibody and hMUC1-C protein prepared in Example 27 was measured by ELISA method. Affinity analysis via ELISA was performed to coat the prepared anti-hMUC1-C humanized antibody in 96-well immune plate at the same concentration and blocked with super block solution. Thereafter, the MBP-hMUC1-C protein was diluted and placed in an immune plate coated with an anti-hMUC1-C humanized antibody, and left standing in a 37 ° C. incubator to bind. Plates were washed three times with wash solution and reacted with anti-MBP antibody conjugated with HRP. The reaction was developed using TMB substrate solution and the reaction was terminated with 2N HCl.
- MBP-hMUC1-C protein was diluted and coated on 96-well immune plates at various concentrations. Subsequently, the biotin-labeled 50 nM anti-hMUC1-C humanized antibody and anti-hMUC1-C mouse antibody were serially diluted from 5 uM and simultaneously placed in a plate coated with MBP-hMUC1-C protein and reacted in a stationary state.
- HRP-labeled streptavidin was treated at a constant concentration to detect biotin labeled on anti-hMUC1-C humanized antibody.
- TMB substrate solution used for the confirmation of affinity was used.
- Absorption value by the reaction was analyzed by measuring through an I3X microplate reader (Molecular Devices, USA). The results are shown in Figure 18, the decrease in absorbance with increasing concentration of anti-hMUC1-C mouse antibody unlabeled biotin was the same epitope of the anti-hMUC1-C humanized antibody and anti-hMUC1-C mouse antibody Recognize.
- FACS analysis was performed to analyze cell binding patterns of h-MUCI-humanized antibodies against hMUCI-C in cell lines where the expression of hMUCI was known.
- Each cell line was prepared by dividing a predetermined number of cells into each tube after culturing in a culture medium. Then 4% paraformaldehyde was fixed and washed once with FACS assay solution after centrifugation.
- the prepared cell line was treated with anti-hMUC1 humanized antibody and treated to bind the corresponding humanized antibody to the cells through culture at 4 ° C. Thereafter, FITC-labeled anti-human IgG antibodies were treated, and FACS analysis was performed using BD FACS Canto (BD, USA). The analysis results are shown in FIG.
- Anti-hMUC1-C humanized antibody (G3) and anti-hMUC1-C mouse antibody (1H7) were treated at various concentrations in the ZR75-1 cell line where the expression of hMUC1 was confirmed. Confirmation through analysis. The analysis results are shown in FIG.
- ADCs were prepared by binding MMAE to anti-hMUC1 humanized antibodies in Alteogen (Daejeon, Korea). The ratio of MMAE (DAR) to the antibody of the prepared ADC was 4.8.
- DAR MMAE
- three cell lines of MDA-MB-231 MUC1-, HER2-
- T47D MUC1 +, HER2 +
- ZR75-1 MUC1 +, HER2 +
- Each cell line was incubated in a 96-well plate, 24 hours after the start of the culture, MUC1-ADC (G3-ADC) and Kadcyla (HER2-ADC) used as a control was treated at the corresponding concentration.
- Cytotoxicity in myeloid leukemia cell lines was confirmed using the same anti-hMUC1 humanized antibody ADC used in Example 31-1.
- the myeloid leukemia cell lines used were K562 (MUC1 +) and KG-1 (MUC1-). Each cell line was incubated in a 96-well plate, and 24 hours after the start of the culture, anti-hMUC1 humanized antibody ADCs were treated at the corresponding concentrations. 72 hours after anti-hMUC1 humanized antibody ADC treatment, cell viability was compared by MTT assay. It was confirmed that the survival rate of the cells treated with the anti-hMUC1 humanized antibody ADC was significantly lower than that of the untreated control. This is shown in Figure 22, the cytotoxicity was confirmed only in the K562 cell line confirmed MUC1 expression of the two cell lines.
- the anti-hMUC1-C humanized antibody-drug conjugates mentioned in Example 31 above have been shown to inhibit cancer growth in animal models.
- the size of the cancer tissues of the mice administered with the anti-hMUC1-C humanized antibody-drug conjugate was reduced, but the size of the cancer tissues of control mice not administered the anti-hMUC1-C humanized antibody-drug conjugate. Showed no statistically significant difference.
- the results indicate that the anti-hMUC1-C humanized antibody-drug conjugate has an effect of inhibiting the growth of TNBC breast cancer tissues against cancer tissues expressing MUC1. No statistically significant weight change was observed in all tested animal subjects, and it was confirmed that it effectively inhibited cancer cell growth in a concentration-dependent manner. Statistical analysis was done with Two-way ANOVA.
- an antibody or antigen-binding fragment thereof that specifically binds to MUC1 exhibits good affinity and avidity for MUC1
- the antibody-drug conjugate to which the drug is conjugated to the antibody or antigen-binding fragment thereof is a MUC1 expressing cell.
- the drug can be delivered efficiently, specificly or selectively.
- the anti-MUC1 antibodies and antibody-drug conjugates according to the invention can be usefully applied in the treatment of MUC1-related diseases such as cancer.
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Abstract
Description
MUC1 단백질 부위 | 설명 | 서열 |
MUC1 단백질의 전체 아미노산 서열 | P15941의 전체 아미노산 서열 | MTPGTQSPFF LLLLLTVLTV VTGSGHASST PGGEKETSAT QRSSVPSSTE KNAVSMTSSV LSSHSPGSGS STTQGQDVTL APATEPASGS AATWGQDVTS VPVTRPALGS TTPPAHDVTS APDNKPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDNRPALGS TAPPVHNVTS ASGSASGSAS TLVHNGTSAR ATTTPASKST PFSIPSHHSD TPTTLASHST KTDASSTHHS SVPPLTSSNH STSPQLSTGV SFFFLSFHIS NLQFNSSLED PSTDYYQELQ RDISEMFLQI YKQGGFLGLS NIKFRPGSVV VQLTLAFREG TINVHDVETQ FNQYKTEAAS RYNLTISDVS VSDVPFPFSA QSGAGVPGWG IALLVLVCVL VALAIVYLIA LAVCQCRRKN YGQLDIFPAR DTYHPMSEYP TYHTHGRYVP PSSTDRSPYE KVSAGNGGSS LSYTNPAVAA TSANL(서열번호 7) |
MUC1 단백질의 세포외 도메인(C 말단 방향) | P15941 중 961-1152의 192개 아미노산 포함 | ASGSASGSAS TLVHNGTSAR ATTTPASKST PFSIPSHHSD TPTTLASHST KTDASSTHHS SVPPLTSSNH STSPQLSTGV SFFFLSFHIS NLQFNSSLED PSTDYYQELQ RDISEMFLQI YKQGGFLGLS NIKFRPGSVV VQLTLAFREG TINVHDVETQ FNQYKTEAAS RYNLTISDVS VSDVPFPFSA QS(서열번호 8; 밑줄: SEA 도메인; "G": 절단부위) |
MUC1 단백질의 SEA 도메인 | P15941 중 1034-1152의 119개 아미노산 포함 | PQLSTGV SFFFLSFHIS NLQFNSSLED PSTDYYQELQ RDISEMFLQI YKQGGFLGLS NIKFRPG SVV VQLTLAFREG TINVHDVETQ FNQYKTEAAS RYNLTISDVS VSDVPFPFSA QS(서열번호 9; 밑줄: C-말단 세포외 도메인) |
MUC1-C 말단부위 세포외 도메인 | MUC1 단백질의 SEA 도메인에서 절단되어 생성된 C-말단 서브유닛 | SVV VQLTLAFREG TINVHDVETQ FNQYKTEAAS RYNLTISDVS VSDVPFPFSA QS(서열번호 10) |
Claims (27)
- MUC1의 C-말단 세포외 도메인 내의 연속하는 5개 이상의 아미노산을 포함하는 폴리펩타이드를 인식하는 항-MUC1 항체 또는 이의 항원 결합 단편.
- 제1항에 있어서, 상기 MUC1의 C-말단 세포외 도메인은 서열번호 10의 아미노산 서열로 이루어진 것을 특징으로 하는 항-MUC1 항체 또는 이의 항원 결합 단편.
- 제1항에 있어서,서열번호 1의 중쇄 CDR1; 서열번호 2의 중쇄 CDR2; 서열번호 3의 중쇄 CDR3; 및서열번호 4의 경쇄 CDR1; 서열번호 5의 경쇄 CDR2; 서열번호 6의 경쇄 CDR3를 포함하는 것을 특징으로 하는 항-MUC1 항체 또는 이의 항원 결합 단편.
- 제3항에 있어서,서열번호 22 또는 24의 중쇄 가변영역 및 서열번호 23 또는 25의 경쇄 가변영역을 포함하는 항-MUC1 항체 또는 이의 항원 결합 단편.
- 제4항에 있어서,서열번호 22의 중쇄 가변영역 및 서열번호 23의 경쇄 가변영역; 또는서열번호 24의 중쇄 가변영역 및 서열번호 25의 경쇄 가변영역을 포함하는 항-MUC1 항체 또는 이의 항원 결합 단편.
- 기탁번호 KCLRF-BP-00395인 항-MUC1 항체 생산을 위한 하이브리도마.
- 제1항의 항-MUC1 항체 또는 이의 항원 결합 단편에 약물이 접합된 항체-약물 접합체.
- 제7항에 있어서, 상기 항체 또는 이의 항원 결합 단편은 링커를 통하여 약물과 결합되는 것을 특징으로 하는 항체-약물 접합체.
- 제8항에 있어서, 상기 링커는 절단성 링커 또는 비절단성 링커인 것을 특징으로 하는 항체-약물 접합체.
- 제7항에 있어서, 상기 약물은 화학요법제, 독소, 마이크로 RNA(miRNA), siRNA, shRNA 또는 방사성 동위원소인 것을 특징으로 하는 항체-약물 접합체.
- 제1항의 항-MUC1 항체 또는 이의 항원 결합 단편을 포함하는 이중특이 항체(bispecific antibody).
- 제11항에 있어서, 상기 이중특이 항체는 항-MUC1 항체 또는 이의 항원 결합 단편과, 암 관련 항원 또는 면역관문 단백질 항원 또는 면역효능세포 특이적 표적분자에 대한 결합능을 가지는 항체로 이루어진 것을 특징으로 하는 이중특이 항체.
- 제1항 내지 제5항 중 어느 한 항의 항-MUC1 항체 또는 이의 항원 결합 단편, 제7항 내지 제10항 중 어느 한 항의 항체-약물 접합체 또는 제11항 내지 제12항 중 어느 한 항의 이중특이 항체를 포함하는 암의 예방 또는 치료용 약학 조성물.
- 제13항에 있어서, 상기 암은 MUC1 단백질이 발현된 것을 특징으로 하는 암의 예방 또는 치료용 약학 조성물.
- 제14항에 있어서, 상기 암은 유방암, 췌장암, 전립선암, 폐암, 갑상선암, 위암, 난소암, 대장암, 간암, 담낭암, 신장암, 자궁경부암, 또는 방광암인 암의 예방 또는 치료용 약학 조성물.
- 제1항 내지 제5항 중 어느 한 항의 항-MUC1 항체 또는 이의 항원 결합 단편을 포함하는 암의 진단용 조성물.
- 제16항에 있어서, 상기 암은 MUC1 단백질이 발현된 것을 특징으로 하는 암의 진단용 조성물.
- 리포좀 내에 (1) MUC1의 C-말단 부위, MUC1의 SEA 도메인, 또는 MUC1의 C-말단 세포외 도메인 및 (2) CpG-DNA를 캡슐화한 복합체를 포함하는 면역원성 조성물.
- 제18항에 있어서, 상기 리포좀은 dioleoylphosphatidylethanolamine(DOPE) 및 cholesteryl hemisuccinate(CHEMS)가 1:0.5 내지 1:2의 몰비로 혼합된 혼합물인 것을 특징으로 하는 면역원성 조성물.
- 제18항에 있어서, 상기 CpG-DNA는 CpG 모티프를 1 내지 3개 포함하는 총 10 내지 20개의 뉴클레오티드를 포함하는 올리고데옥시뉴클레오티드인 것을 특징으로 하는 면역원성 조성물.
- 제18항 내지 제20항 중 어느 한 항의 면역원성 조성물을 마우스에 접종하는 단계를 포함하는 항 MUC1-SEA 단일클론 항체의 제조 방법.
- 제1항 내지 제5항 중 어느 한 항에 따른 항-MUC1 항체 또는 이의 항원 결합 단편을 코딩하는 핵산.
- 제22항에 따른 핵산을 포함하는 재조합 발현 벡터.
- 제23항에 따른 재조합 발현 벡터로 형질전환된 숙주세포.
- 제24항에 있어서, 동물세포, 식물세포, 효모, 대장균 및 곤충세포로 구성된 군에서 선택된 것을 특징으로 하는 숙주세포.
- 제25항에 있어서, 원숭이 신장 세포7(COS7: monkey kidney cells) 세포, NSO 세포, SP2/0 세포, 차이니즈 햄스터 난소(CHO: Chinese hamster ovary) 세포, W138, 어린 햄스터 신장(BHK: baby hamster kidney) 세포, MDCK, 골수종 세포주, HuT 78 세포 및 HEK293 세포, 대장균, 바실러스 서브틸리스(Bacillus subtilis), 스트렙토마이세스 속(Streptomyces sp.), 슈도모나스 속(Pseudomonas sp.), 프로테우스 미라빌리스(Proteus mirabilis)또는 스타필로코쿠스 속(Staphylococcus sp.), 아스페르길러스 속(Aspergillus sp.), 피치아 파스토리스(Pichiapastoris),사카로마이세스 세레비지애(Saccharomyces cerevisiae), 쉬조사카로마세스 속(Schizosaccharomyces sp.) 및 뉴로스포라 크라사(Neurosporacrassa)으로 구성된 군에서 선택된 것을 특징으로 하는 숙주세포.
- 제24항 내지 제26항 중 어느 한 항에 따른 숙주세포를 배양하는 단계를 포함하는 항-MUC1 항체 또는 이의 항원 결합 단편의 제조 방법.
Priority Applications (19)
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CA3056582A CA3056582C (en) | 2017-03-21 | 2018-03-21 | Antibody binding specifically to muc1 and use thereof |
EP20212863.3A EP3825328A1 (en) | 2017-03-21 | 2018-03-21 | Antibody binding specifically to muc1 and use thereof |
RU2019132900A RU2746413C1 (ru) | 2017-03-21 | 2018-03-21 | Антитело, специфически связывающееся с muc1, и его применение |
BR112019019650-1A BR112019019650A2 (pt) | 2017-03-21 | 2018-03-21 | ligação de anticorpos especificamente à muc1 e utilização dos mesmos |
EP18771928.1A EP3604334A4 (en) | 2017-03-21 | 2018-03-21 | ANTIBODIES BINDING SPECIFICALLY TO MUC1 AND ITS USE |
US16/495,373 US11472887B2 (en) | 2017-03-21 | 2018-03-21 | Antibody binding specifically to MUC1 and use thereof |
MX2019011101A MX2019011101A (es) | 2017-03-21 | 2018-03-21 | Anticuerpo que se une especificamente a muc1 y uso del mismo. |
JP2019552556A JP7032425B2 (ja) | 2017-03-21 | 2018-03-21 | Muc1に特異的に結合する抗体及びその用途 |
IL269488A IL269488B2 (en) | 2017-03-21 | 2018-03-21 | Conjugation of an antibody specifically for muc1 and its use |
CN201880026127.7A CN111201240B (zh) | 2017-03-21 | 2018-03-21 | 特异性地结合muc1的抗体及其用途 |
AU2018238165A AU2018238165C1 (en) | 2017-03-21 | 2018-03-21 | Antibody binding specifically to MUC1 and use thereof |
ZA2019/06813A ZA201906813B (en) | 2017-03-21 | 2019-10-16 | Antibody binding specifically to muc1 and use thereof |
AU2020281132A AU2020281132B2 (en) | 2017-03-21 | 2020-12-04 | Antibody binding specifically to MUC1 and use thereof |
AU2020281130A AU2020281130B2 (en) | 2017-03-21 | 2020-12-04 | Antibody binding specifically to MUC1 and use thereof |
US17/116,265 US11718684B2 (en) | 2017-03-21 | 2020-12-09 | Antibody binding specifically to MUC1 and use thereof |
JP2021158852A JP7158547B2 (ja) | 2017-03-21 | 2021-09-29 | Muc1に特異的に結合する抗体及びその用途 |
JP2021158853A JP7158548B2 (ja) | 2017-03-21 | 2021-09-29 | Muc1に特異的に結合する抗体及びその用途 |
US17/811,845 US11739158B2 (en) | 2017-03-21 | 2022-07-11 | Antibody binding specifically to MUC1 and use thereof |
US17/811,904 US11739159B2 (en) | 2017-03-21 | 2022-07-12 | Antibody binding specifically to MUC1 and use thereof |
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KR1020180032592A KR102127421B1 (ko) | 2017-03-21 | 2018-03-21 | Muc1에 특이적으로 결합하는 항체 및 그의 용도 |
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US17/116,265 Division US11718684B2 (en) | 2017-03-21 | 2020-12-09 | Antibody binding specifically to MUC1 and use thereof |
US17/811,845 Division US11739158B2 (en) | 2017-03-21 | 2022-07-11 | Antibody binding specifically to MUC1 and use thereof |
US17/811,904 Division US11739159B2 (en) | 2017-03-21 | 2022-07-12 | Antibody binding specifically to MUC1 and use thereof |
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CN110174515A (zh) * | 2019-05-09 | 2019-08-27 | 青岛海兰深生物科技有限公司 | 一种检测抗肺癌天然抗体的组合物、试剂盒和方法 |
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WO2022139537A2 (ko) | 2020-12-24 | 2022-06-30 | 주식회사 엘지화학 | 뮤신 1에 특이적인 폴리펩티드 및 이의 이용 |
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CN110174515A (zh) * | 2019-05-09 | 2019-08-27 | 青岛海兰深生物科技有限公司 | 一种检测抗肺癌天然抗体的组合物、试剂盒和方法 |
CN110174515B (zh) * | 2019-05-09 | 2022-07-01 | 青岛海兰深生物科技有限公司 | 一种检测抗肺癌天然抗体的组合物、试剂盒和方法 |
WO2020252472A3 (en) * | 2019-06-14 | 2021-01-21 | Dana-Farber Cancer Institute, Inc. | Antibodies against muc1 and methods of use thereof |
WO2022139537A2 (ko) | 2020-12-24 | 2022-06-30 | 주식회사 엘지화학 | 뮤신 1에 특이적인 폴리펩티드 및 이의 이용 |
WO2022148736A1 (en) * | 2021-01-05 | 2022-07-14 | Transgene | Vectorization of muc1 t cell engager |
CN113444181A (zh) * | 2021-09-01 | 2021-09-28 | 北京华睿博生物科技有限公司 | 抗kl-6双特异性抗体及基因、重组载体、药物、试剂盒 |
CN117547600A (zh) * | 2023-11-15 | 2024-02-13 | 华中科技大学同济医学院附属协和医院 | 一种靶向hdac的脂质体疫苗的制备及应用 |
CN117547600B (zh) * | 2023-11-15 | 2024-04-30 | 华中科技大学同济医学院附属协和医院 | 一种靶向hdac的脂质体疫苗的制备及应用 |
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