WO2010126137A1 - 抗カドヘリン抗体 - Google Patents
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- WO2010126137A1 WO2010126137A1 PCT/JP2010/057694 JP2010057694W WO2010126137A1 WO 2010126137 A1 WO2010126137 A1 WO 2010126137A1 JP 2010057694 W JP2010057694 W JP 2010057694W WO 2010126137 A1 WO2010126137 A1 WO 2010126137A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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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
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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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
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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- 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
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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
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
Definitions
- the present invention relates to an anti-cadherin antibody that recognizes a specific domain of cadherin and has high antibody-dependent cytotoxic activity.
- Cancer is an important disease that occupies the top causes of death, but its therapeutic needs have not yet been met.
- cancer treatment with molecular targeted drugs that design and treat drugs targeting specific molecules specifically expressed in cancer cells Has been actively studied.
- Cadherin is an example of a molecule that can be a target for molecular therapeutics in cancer.
- Cadherin is a membrane protein discovered as a molecule that is involved in cell adhesion with allophilic affinity in a calcium-dependent manner (Yoshida and Takeichi, Cell 28: 217-224, 1982).
- Proteins having cadherin repeats (ECs) consisting of about 110 amino acid residues having high homology to each other are called cadherin superfamily, and there are over 120 types of proteins, which play an important role in maintaining a multicellular structure.
- ADCC Antibody-dependent cellular cytotoxicity
- Proteins belonging to the cadherin superfamily can be broadly classified into 1) classical cadherin, 2) desmosomal cadherin, 3) protocadherin, and 4) others according to their structural characteristics.
- Classical cadherins, major members of the cadherin superfamily are highly homologous to each other ( Figure 1). That is, it is a single transmembrane protein that is supposed to form a dimer, and has five cadherin domains of EC1-EC5 and an intracellular domain in the extracellular region.
- Classical cadherin-mediated cell adhesion is characterized by adhesion between allogeneic cells, and is performed by cells recognizing the same type of cadherin molecules that are specifically expressed differently depending on the cell type.
- EC1 cadherin domain 1
- ADCC antibody-dependent cellular cytotoxicity
- An object of the present invention is to provide an anti-cadherin antibody having high antibody-dependent cytotoxic activity.
- the present inventor has intensively studied to solve the above-mentioned problems and measured the antibody-dependent cellular cytotoxicity (ADCC) activity of the P-cadherin antibody, and found that it tends to be divided into two groups depending on the strength of the ADCC activity. .
- ADCC antibody-dependent cellular cytotoxicity
- an antibody with strong ADCC activity recognizes either the upstream region of EC1, cadherin domain 4 (EC4) or cadherin domain 5 (EC5) with high probability.
- Elements that define the ADCC activity of the antibody include the affinity of the Fc portion of the antibody for the Fc receptor of the effector cell, the affinity of the antibody for the antigen, and the epitope recognized by the antibody.
- the antibody In order to exert ADCC activity, it is essential that the antibody binds to the antigen and the Fc receptor of the effector cell binds to the Fc site of the antibody. However, due to the difference in the region of CDH3 to which the antibody binds, the effector cell It is presumed that there is a spatial restriction in the binding of the antibody to the Fc site, resulting in a difference in ADCC activity.
- the present invention has been completed based on these findings.
- the following inventions are provided.
- the upstream region of EC1 indicates a region of 24 amino acid residues upstream of EC1 of E-cadherin, P-cadherin, N-cadherin, and a corresponding region of other cadherins.
- the anti-cadherin antibody of the present invention recognizes a cadherin domain of cadherin upstream region of EC1, cadherin domain 4 (EC4) or cadherin domain 5 (EC5), and has high antibody-dependent cytotoxic activity. And An antibody that can exert high cytotoxic activity is useful as a material for preparing a modified antibody or a modified antibody.
- an anticancer action based on antibody-dependent cytotoxic activity can be exerted. That is, the anti-cadherin antibody of the present invention is useful as an anticancer agent.
- FIG. 1 shows the mature protein sequence excluding E-cadherin (CDH1), N-cadherin (CDH2), P-cadherin (CDH3) signals and propeptide sequences.
- FIG. 2 shows the adhesion mechanism of molecules belonging to the classical cadherin family.
- FIG. 3 shows the results of flow cytometry obtained by reacting a human CDH3 forced expression cell line with a commercially available anti-human CDH3 antibody.
- B CHO cell.
- b 0.1 ⁇ g / ml of anti-CDH3 antibody
- c 1 ⁇ g / ml of anti-CDH3 antibody FIG.
- FIG. 4 shows typical flow cytometry results for 3 acquired antibodies and each cell line.
- A CDH3 forced expression CHO cell
- B CHO cell
- C Lung cancer-derived cell line NCI-H358.
- a anti-CDH3 antibody 0.01 ⁇ g / ml
- b anti-CDH3 antibody 0.1 ⁇ g / ml
- c anti-CDH3 antibody 1 ⁇ g / ml.
- FIG. 5 shows the ADCC activity of each antibody.
- FIG. 6 shows the correspondence of CDH3 partial length protein fragments 1 to 5 with the CDH3 extracellular region.
- FIG. 7 shows the expression result of CDH3 partial length protein.
- FIG. 8 shows the reaction of the CDH3 partial length protein and each antibody by Western blotting.
- FIG. 9 shows the results of epitope analysis of PPMX13 using a peptide array. The numerical value on the X axis indicates the number of the peptide array.
- FIG. 10 shows the expression results of CDH3 mRNA in various tumor tissues.
- FIG. 11 shows the expression results of CDH3 in various tumor tissues.
- FIG. 12 shows the antitumor effect of PPMX12-producing antibody in a xenograft transplanted with human lung cancer-derived cell line NCI-H351.
- FIG. 13 shows the antitumor effect of PPMX12-producing antibody in a xenograft transplanted with human pancreatic cancer-derived cell line PK-45P.
- FIG. 14 shows the antitumor effect of PPMX12-producing antibody in a xenograft transplanted with human skin cancer-derived cell line A431.
- the antibody of the present invention recognizes the cadherin domain of either the upstream region of EC1, cadherin domain 4 (EC4) or cadherin domain 5 (EC5), and has antibody-dependent cytotoxic activity when the antibody concentration is 1 ⁇ g / mL.
- an anti-cadherin antibody 30% or more of an anti-cadherin antibody, an upstream region of EC1, a cadherin domain of either cadherin domain 4 (EC4) or cadherin domain 5 (EC5), and an antibody concentration of 0.1 ⁇ g / mL
- an anti-cadherin antibody characterized by having an antibody-dependent cytotoxic activity of 25% or more (for example, stronger than the activity of PPMX5), or an upstream region of EC1, cadherin domain 4 (EC4) or cadherin domain 5 ( EC5) recognizes any cadherin domain, and ADCC has a maximum activity of 35 % Of an anti-cadherin antibody that is at least% (eg stronger than the activity of PPMX6).
- the maximum activity of ADCC refers to the ADCC activity when the antibody concentration is increased and the increase in ADCC activity reaches a plateau.
- the upstream region of EC1 of P-cadherin, P-cadherin and N-cadherin, cadherin domain 1 (EC1), cadherin domain 2 (EC2), cadherin domain 3 (EC3), cadherin domain 4 (EC4), cadherin Domain 5 (EC5) indicates the following areas, respectively.
- the corresponding region of other cadherins can be determined by comparing the sequences of known cadherin proteins obtained from Genbank et al.
- ADCC activity Antibody-dependent cytotoxic activity
- the numerical value of ADCC activity in the present specification means antibody-dependent cytotoxic activity when measured under the same conditions as in Example 4. Specifically, it is as follows. (1) Preparation of effector cells Bone marrow cells were collected from the femurs of C3H / HeJ Jcl mice (8 weeks old, male, Claire Japan) and prepared to 2 ⁇ 10 6 cells / mL in RPMI1640 medium containing 10% FBS. The cells were cultured for 6 days in the presence of human IL-2 (PEPROTECH) 50 ng / mL and mouse GM-CSF (PEPROTECH) 10 ng / mL.
- human IL-2 PEPROTECH
- mouse GM-CSF PEPROTECH
- target cells full-length CDH3-expressing CHO cells (EXZ1501) were used. After the cells are detached from the plate, suspended in HAM medium containing 10% FBS to 1 ⁇ 10 7 cells / mL, 51 Cr is added to a final concentration of 150 uCi, and 1.5% at 37 ° C. in a 5% carbon dioxide incubator. Incubate for hours.
- the cells were washed twice with a medium, 10% FBS-containing HAM medium was added, and the cells were seeded on a 96-well U-bottom plate (NUNC) at 1 ⁇ 10 4 cells / well to serve as target cells.
- NUNC 96-well U-bottom plate
- the culture supernatant is collected, and the radioactivity in 100 ⁇ L of the culture supernatant is measured with a scintillation counter.
- cadherin recognized by the antibody of the present invention is preferably classic cadherin. Examples include, but are not limited to, E-cadherin, N-cadherin, and P-cadherin.
- cadherin or a partial peptide thereof can be used as an antigen for preparing the antibody of the present invention.
- soluble CDH3 protein or the like can be used, but is not limited thereto.
- the antibody of the present invention may be a polyclonal antibody or a monoclonal antibody.
- the antibody (polyclonal antibody and monoclonal antibody) of the present invention can be produced by any of various methods. Methods for producing such antibodies are well known in the art [see, for example, Sambrook, J et al., Molecular Cloning, Cold Spring Harbor Press (1989)].
- cadherin or the cadherin domain of either cadherin domain 4 (EC4) or cadherin domain 5 (EC5) is preferred.
- the dose of the antigen per animal is 0.1 to 100 mg when no adjuvant is used, and 1 to 100 ⁇ g when an adjuvant is used.
- adjuvants include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant.
- FCA Freund's complete adjuvant
- FIA Freund's incomplete adjuvant
- Immunization is performed mainly by injecting intravenously, subcutaneously, intraperitoneally, or the like.
- the immunization interval is not particularly limited, and immunization is performed 1 to 10 times, preferably 2 to 5 times at intervals of several days to several weeks, preferably 2 to 5 weeks. Then, 6 to 60 days after the final immunization, the antibody titer is measured by enzyme immunoassay (ELISA (enzyme-linked immunosorbent assay) or EIA (enzyme immunoassay)), radioimmunoassay (RIA; radioimmunoassay), etc. On the day when the maximum antibody titer is shown, blood is collected to obtain antiserum.
- ELISA enzyme-linked immunosorbent assay
- EIA enzyme immunoassay
- RIA radioimmunoassay
- EC1 cadherin or its partial peptide, cadherin domain 4 (EC4) or cadherin domain 5 (EC5)
- FCA Freund's complete adjuvant
- FIA Freund's incomplete adjuvant
- Aluminum hydroxide adjuvant aluminum hydroxide adjuvant
- the immunization interval is not particularly limited, and immunization is performed 1 to 10 times, preferably 2 to 5 times at intervals of several days to several weeks, preferably 2 to 5 weeks. Then, antibody-producing cells are collected 1 to 60 days, preferably 1 to 14 days after the final immunization day.
- antibody-producing cells include spleen cells, lymph node cells, peripheral blood cells, etc., but spleen cells or local lymph node cells are preferred.
- cell fusion between antibody-producing cells and myeloma cells is performed.
- myeloma cells to be fused with antibody-producing cells generally available cell lines of animals such as mice can be used.
- the cell line used has drug selectivity and cannot survive in a HAT selection medium (including hypoxanthine, aminopterin, and thymidine) in an unfused state, but can survive only in a state fused with antibody-producing cells.
- HAT selection medium including hypoxanthine, aminopterin, and thymidine
- myeloma cells include P3X63-Ag. 8).
- Examples include mouse myeloma cell lines such as U1 (P3U1) and NS-1.
- the myeloma cell and the antibody-producing cell are fused.
- Cell fusion is performed by using 1 ⁇ 10 6 to 1 ⁇ 10 7 antibody-producing cells and 2 ⁇ 10 5 to 2 ⁇ 10 6 in animal cell culture media such as serum-free DMEM and RPMI-1640 medium.
- 1 / ml myeloma cells are mixed (a cell ratio of antibody-producing cells to myeloma cells is preferably 5: 1), and a fusion reaction is performed in the presence of a cell fusion promoter.
- the cell fusion promoter polyethylene glycol having an average molecular weight of 1000 to 6000 daltons can be used.
- antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device utilizing electrical stimulation (for example, electroporation).
- the target hybridoma is selected from the cells after cell fusion treatment.
- the cell suspension is appropriately diluted with, for example, a fetal bovine serum-containing RPMI-1640 medium, and then plated on a microtiter plate at about 3 ⁇ 10 5 cells / well, and a selective medium is added to each well.
- the culture is carried out with the selective medium changed. As a result, cells that grow from about 14 days after the start of culture in the selective medium can be obtained as hybridomas.
- Hybridoma screening is not particularly limited, and may be performed according to ordinary methods. For example, a part of the culture supernatant contained in a well grown as a hybridoma is collected, and an antibody that binds to the upstream region of EC1 of cadherin, EC4 or EC5 domain is produced by enzyme immunoassay, radioimmunoassay or the like. Hybridomas can be screened. Cloning of the fused cells is performed by limiting dilution or the like, and finally, a hybridoma that is a monoclonal antibody-producing cell can be established.
- a normal cell culture method or ascites collection method can be employed as a method for collecting a monoclonal antibody from the established hybridoma.
- the hybridoma is cultured in an animal cell culture medium such as RPMI-1640 medium containing 10% fetal bovine serum, MEM medium, or serum-free medium under normal culture conditions (for example, 37 ° C., 5% CO 2 concentration). Culturing for 7 to 14 days, and antibodies are obtained from the culture supernatant.
- hybridomas In the case of the ascites collection method, about 1 ⁇ 10 7 hybridomas are administered into the abdominal cavity of a myeloma cell-derived mammal and a homologous animal, and the hybridomas are proliferated in large quantities. Ascites are collected after 1-2 weeks.
- known methods such as ammonium sulfate salting-out method, ion exchange chromatography, gel filtration, affinity chromatography are appropriately selected, or a combination thereof is used. Can be purified.
- the type of the antibody of the present invention is not particularly limited, and it is artificial for the purpose of reducing the foreign antigenicity against humans such as mouse antibody, human antibody, rat antibody, rabbit antibody, sheep antibody, camel antibody, avian antibody, etc. Any of a recombinant antibody modified into, for example, a chimeric antibody or a humanized antibody may be used.
- the recombinant antibody can be produced using a known method.
- a chimeric antibody is an antibody comprising a non-human mammal, for example, a mouse antibody heavy chain and light chain variable region and a human antibody heavy chain and light chain constant region, and a DNA encoding the murine antibody variable region.
- a humanized antibody is obtained by transplanting the complementarity determining region (CDR) of a mammal other than a human, for example, a mouse antibody, to the complementarity determining region of a human antibody, and its general gene recombination technique is also known. . Specifically, several oligonucleotides were prepared so that the DNA sequence designed to link the CDR of the mouse antibody and the framework region (FR) of the human antibody had an overlapping portion at the end. It is synthesized from nucleotides by PCR. Obtained DNA can be obtained by ligating with human antibody DNA encoding constant region, and then incorporating it into an expression vector, introducing it into a host and producing it (EP239400, International Publication WO96 / 02576, etc.) .
- a method for obtaining a human antibody is also known.
- human lymphocytes are sensitized in vitro with a desired antigen or cells expressing the desired antigen, and the sensitized lymphocytes are fused with human myeloma cells, such as U266, to form a desired human antibody having binding activity to the antigen.
- a desired human antibody can be obtained by immunizing a transgenic animal having all repertoires of human antibody genes with a desired antigen (WO93 / 12227, WO92 / 03918, WO94 / 02602, 25WO94 / 25585).
- WO 96/34096, WO 96/33735 ).
- variable region of a human antibody can be expressed as a single chain antibody (scFv) on the surface of the phage by the phage display method, and a phage that binds to the antigen can be selected.
- scFv single chain antibody
- the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. If the DNA sequence of scFv that binds to the antigen is clarified, an appropriate expression vector can be prepared from the sequence and a human antibody can be obtained.
- These antibodies recognize the cadherin domain of either the upstream region of EC1, cadherin domain 4 (EC4) or cadherin domain 5 (EC5), and antibody-dependent cytotoxic activity when the antibody concentration is 1 ⁇ g / mL. Is 30% or more, recognizes the cadherin domain of either the upstream region of EC1, cadherin domain 4 (EC4) or cadherin domain 5 (EC5), and antibody-dependent cytotoxic activity at an antibody concentration of 0.1 ⁇ g / mL Recognizes an cadherin domain of an antibody or an upstream region of EC1, cadherin domain 4 (EC4) or cadherin domain 5 (EC5), and ADCC has maximum activity Loss of the characteristic of being an antibody of 35% or more (for example, stronger than PPMX6 activity) Unless, monovalent antibodies, bivalent antibodies may be either a multivalent antibody may be an antibody fragment (fragment) minibodies or antibody modifications of such.
- an antibody fragment or a low molecular weight antibody for example, Fab, Fab ′, F (ab ′) 2 , Fv, ScFv (single chain Fv), diabody, etc., fused with Fc portion and added with ADCC activity may be used.
- a gene encoding these antibodies may be constructed, introduced into an expression vector, and then expressed in an appropriate host cell.
- a modified antibody an antibody conjugated with various molecules such as polyethylene glycol (PEG) can be used. It is also possible to bind a radioisotope, a chemotherapeutic agent or the like to the antibody rod, and particularly a radiolabeled antibody rod is useful. Such a modified antibody can be obtained by chemically modifying the obtained antibody bag. Antibody modification methods are known to those skilled in the art.
- the antibody of the present invention exhibits high antibody-dependent cytotoxic activity, it can be used as a cytotoxic agent.
- the cytotoxic agent of the present invention can damage cancer cells, for example, by contacting them with cancer cells expressing cadherin.
- the cytotoxic agent of the present invention can appropriately contain a pharmaceutically acceptable carrier, excipient, diluent and the like as necessary.
- the cytotoxic agent of the present invention can be formulated as an injection, for example.
- the dosage of the cytotoxic agent of the present invention depends on the symptom level, age and body weight of the patient, administration method, etc., and the weight of the antibody as the active ingredient is usually in the range of about 10 ng to about 100 mg / kg body weight. It is.
- Example 1 Establishment of a CDH3-expressing CHO cell line
- a CHO cell expressing full-length CDH3 was established.
- (1) Preparation of CDH3 gene expression vector In order to insert the full-length human CDH3 DNA shown in SEQ ID NO: 1 into the mammalian expression vector pEF4 / myc-HisB (Invitrogen), two types of restriction enzymes KpnI (Takara Bio) and XbaI (Takara Bio) Biotechnology) at 37 ° C.
- CDH3 full-length expression CHO clones were selected by Western blotting using an anti-c-Myc monoclonal antibody (SANTA CRUZ BIOTECHNOLOGY). As a result, the CDH3 full-length expression CHO cell line with high expression and good growth ( EXZ1501) was obtained. The results of measurement of this cell line and a commercially available anti-CDH3 antibody (R & D SYSTEMS) using a flow cytometer are shown in FIG.
- Soluble CDH3 (sCDH3) protein lacking the C-terminal transmembrane region was prepared for use as an immunogen for producing anti-CDH3 antibody.
- soluble CDH3 antigen expression vector Forward designed to amplify a portion corresponding to the CDH3 extracellular region (corresponding to 1-654 of SEQ ID NO: 2, hereinafter referred to as sCDH3 cDNA) using CDH3 full-length cDNA as a template PCR reaction was performed using a primer (SEQ ID NO: 7: CGCGGGTACCATGGGGCTCCCTCGT, (hCDH3FullFW)) and a reverse primer (SEQ ID NO: 8: CCGTCTAGATAACCCTCCCTTCAGGGTCC, (hCDH3SolbRV)). The reaction was performed using KOD-Plus (Toyobo Co., Ltd.) under the reaction conditions of 94 ° C. for 15 seconds, 55 ° C. for
- this sCDH3 cDNA was treated with two types of restriction enzymes KpnI and XbaI, and then treated with p4 / myc-HisB treated with KpnI and XbaI using T4 DNA ligase.
- the insertion was performed according to the method to obtain the expression vector pEF4-sCDH3-myc-His.
- soluble CDH3-expressing CHO cells were performed by Western blotting using an anti-c-Myc monoclonal antibody (SANTA CRUZ BIOTECHNOLOGY).
- SANTA CRUZ BIOTECHNOLOGY an anti-c-Myc monoclonal antibody
- EXZ1702 a soluble CDH3-expressing CHO cell line with a large amount of secretion into the culture supernatant and good growth.
- the selected soluble CDH3-expressing CHO cell line (EXZ1702) uses 3 roller bottles with a culture area of 1,500 cm 2 , and 333 mL of serum-free medium CHO-S-SFM-II (Invitrogen) per roller bottle. The culture supernatant was collected for 72 hours.
- the obtained culture supernatant is soluble by affinity chromatography using a HisTrap (registered trademark) HP column (GE Healthcare Bioscience) and gel filtration chromatography using a Superdex (registered trademark) 200 pg column (GE Healthcare Bioscience).
- Type CDH3 protein was obtained.
- Example 3 Preparation of anti-CDH3 monoclonal antibody (1) Preparation of monoclonal antibody using soluble CDH3 protein as immunogen 50 ⁇ g of soluble CDH3 protein dissolved in physiological saline and Titer-MAX Gold (registered trademark) (Titer Max) was mixed in an equal amount, and initial immunization was performed by intraperitoneal and subcutaneous injection of MRL / lpr mice (Japan SLC Co., Ltd.). The second and subsequent immunizations were carried out by mixing the similarly prepared soluble CDH3 protein equivalent to the amount of 25 ⁇ g protein and Titer-MAX Gold and injecting them intraperitoneally and subcutaneously.
- Titer-MAX Gold registered trademark
- mice Three days after the final immunization, spleen cells were aseptically prepared from mice, and cell fusion with mouse myeloma cells SP2 / O-Ag14 or P3-X63-Ag8.653 was carried out by the polyethylene glycol method according to a conventional method.
- Anti-CDH3 antibody was selected by flow cytometry using a CHO cell line (EXZ1501) that expresses full-length CDH3.
- a CHO cell line (EXZ1501) expressing full-length CDH3 was treated with 2 mM EDTA-PBS, detached from the culture plate, and then suspended in a FACS solution so as to be 1 ⁇ 10 6 cells / mL.
- This cell suspension was seeded in a 96-well plate at 50 ⁇ L / well, the hybridoma culture supernatant was added, reacted at 4 ° C. for 60 minutes, washed twice with FACS solution (200 ⁇ L / well), and then AlexaFluor 488. Labeled anti-mouse IgG • goat F (ab ′) 2 (Invitrogen) was added and reacted at 4 ° C. for 30 minutes. Then, after washing twice with a FACS solution, flow cytometry was performed to select hybridomas that showed a strong reaction in CDH3-expressing CHO cells.
- FIG. 4 shows typical reaction results of an antibody obtained from the hybridoma and a CDH3-expressing CHO cell (EXZ1501), a parental CHO cell, and a cancer cell NCI-H358 in which CDH3 is confirmed to be highly expressed. Show. It was confirmed that all the selected hybridomas reacted with CDH3-expressing CHO cells (EXZ1501) and NCI-H358, but not with CHO cells.
- ADCC activity is measured by a method in which an antibody is allowed to act on a radiolabeled target cell in the presence of an effector cell and its free radioactivity is measured. It was.
- (1) Preparation of effector cells Bone marrow cells were collected from the femurs of C3H / HeJ Jcl mice (8 weeks old, male, Claire Japan) and prepared to 2 ⁇ 10 6 cells / mL in RPMI1640 medium containing 10% FBS. The cells were cultured for 6 days in the presence of human IL-2 (PEPROTECH) 50 ng / mL and mouse GM-CSF (PEPROTECH) 10 ng / mL. On the day of measurement, the cells were collected, washed with HAM medium containing 10% FBS, and used as an effector cell solution.
- human IL-2 PEPROTECH
- mouse GM-CSF PEPROTECH
- target cells full-length CDH3-expressing CHO cells (EXZ1501) were used. After detaching the cells from the plate, the cells are suspended in HAM medium containing 10% FBS to 1 ⁇ 10 7 cells / mL, 51 Cr is added so that the final concentration is 150 uCi, and then 1. is added at 37 ° C. in a 5% carbon dioxide incubator. Cultured for 5 hours. The cells were washed twice with a medium, 10% FBS-containing HAM medium was added, and the cells were seeded on a 96-well U-bottom plate (NUNC) at 1 ⁇ 10 4 cells / well to serve as target cells.
- NUNC 96-well U-bottom plate
- Cytotoxic activity (%) (AC) / (BC) ⁇ 100
- the test was performed in triplicate, and the cytotoxic activity (%) was calculated from the average value.
- the test results are shown in Table 1 and FIG.
- An antibody having an ADCC activity of 30% or more at an antibody concentration of 1 ⁇ g / mL was defined as a high ADCC activity group, and an antibody having a concentration of less than 30% was defined as a low ADCC activity group.
- R & D-104805 indicates a commercially available anti-CDH3 antibody (R & D SYSTEMS).
- BD-610227 indicates a commercially available anti-CDH3 antibody (BD BIOSCIENCE).
- Negative Abs 1 and 2 represent antibodies that recognize antigens unrelated to CDH3. * S: High ADCC activity (more than 30% at antibody concentration of 1 ⁇ g / mL) W: Low ADCC activity (less than 30% when antibody concentration is 1 ⁇ g / mL)
- the hybridoma PPMX12 that produces the antibody PPMX12 was founded on January 20, 2010 in the independent administrative corporation Product Evaluation Technology Foundation Patent Microorganism Depositary Center (Postal Code 292-0818, Kazusa Kamashika 2-5-8, Kisarazu City, Chiba Prefecture, Japan). Deposited under the Budapest Treaty under the deposit number NITE BP-865.
- Example 5 Epitope classification of anti-CDH3 monoclonal antibody by CDH3 partial length expression protein Epitope classification of the obtained anti-CDH3 antibody was performed by Western blotting with a CDH3 partial sequence expression product.
- CDH3 partial sequence expression fragments 1 to 5 were designed so that the sequences sufficiently overlapped between the fragments (FIG. 6).
- CDH3 partial length protein expression vector PCR reaction was performed using the full-length CDH3 cDNA of Example 1 as a template and a primer set described below.
- iProof High Fidelity DNA polymerase Bio-Rad
- a gel containing a band close to the target size was cut out by agarose gel electrophoresis, and the target CDH3 cDNA fragment was obtained using a QIA (registered trademark) quick gel extraction kit.
- Fragment 1 (positions 108-236 of SEQ ID NO: 2)
- Forward primer TATGGAGCTCCGTACCCGATGGGGTGGTTGCTCCCAAATTCG (SEQ ID NO: 9)
- Reverse primer AGATTACCTATCTAGACTACTGCCATCACAGAAGTACCTGGTAGG (SEQ ID NO: 10)
- Fragment 2 (positions 132-348 of SEQ ID NO: 2)
- Forward primer TATGGAGCTCCGTACCAAGTCTAATAAAGATAGAGACACCAAG (SEQ ID NO: 11)
- Reverse primer AGATACTACCTTAGTAGACTACTCTCTCACCCATCATGGCCCACTGCATTCTCA (SEQ ID NO: 12)
- Fragment 3 (positions 237 to 461 of SEQ ID NO: 2)
- Forward primer TATGGAGCTCCGTACCGTGACAGCCACGGGATGAGGATGATG (SEQ ID NO: 13)
- Reverse primer AGATTACCTATCTAGACTAGACACACACAGGCGCCCCCAGTG (SEQ ID NO: 14)
- Fragment 4 (positions 349 to 550 of SEQ ID NO: 2)
- Forward primer TATGGAGCTCCGTACCCCTGACGGTTCACTGATCTGGACG (SEQ ID NO: 15)
- Reverse primer AGATTACCTATTAGTAGTAGGGCTCAGGGGACTGGGCCATGGTCATTTG (SEQ ID NO: 16)
- Fragment 5 (positions 462 to 654 of SEQ ID NO: 2)
- Forward primer TATGGAGCTCCGTACTACTACACTGCAGAAGACCCTGACAAGG (SEQ ID NO: 17)
- Reverse primer AGATTACCTATCTAGACTAACCCTCCCTTCCAGGGTCCAGGGCAGGTTTC (SEQ ID NO: 18)
- the expression of the CDH3 partial length protein was confirmed by the presence of a band at the expected position by carrying out Western blotting with an anti-Penta-His antibody (Qiagen) after electrophoresis of an E. coli culture solution. That is, an electrophoresis buffer corresponding to 1/10 volume of the above-mentioned E. coli culture solution was added, charged to a 5-20% gradient gel (Bio-Rad) under reducing conditions, and after electrophoresis, Immobilon (registered trademark) P (Millipore).
- the transfer film was lightly washed with TBS-T (0.05% Tween (registered trademark) 20, TBS), shaken with TBS containing 40% BSA for 1 hour, and then 10% Block Ace (registered trademark) (Snow Brand Milk Products).
- TBS-T 0.05% Tween (registered trademark) 20, TBS
- Block Ace registered trademark
- Each anti-CDH3 antibody diluted with TBS-T was added and shaken for 1 hour.
- HRP-anti-mouse IgG antibody GE Healthcare Biosciences
- X-ray film RX-u Fluji Film
- ECL registered trademark
- -Plus GE Healthcare Bioscience
- the region recognized by each antibody was determined by the reactivity with each CDH3 partial length protein (Table 2).
- the correspondence relationship with the region recognized by each antibody on the CDH3 sequence shown in SEQ ID NO: 2 is shown below.
- R & D-104805 indicates a commercially available anti-CDH3 antibody (R & D SYSTEMS).
- BD-610227 indicates a commercially available anti-CDH3 antibody (BD BIOSCIENCE).
- Negative Abs 1 and 2 represent antibodies that recognize an antigen unrelated to CDH3. * S: High ADCC activity (more than 30% at antibody concentration of 1 ⁇ g / mL) W: Low ADCC activity (less than 30% when antibody concentration is 1 ⁇ g / mL)
- Example 6 Anti-CDH3 Monoclonal Antibody Epitope Determination Using Peptide Array
- a peptide array (Reptopepe manufactured by JPT Peptide Technologies) was used for antibody PPMX13 considered to correspond to the border region. ) To determine the epitope in more detail.
- a peptide (108, which was synthesized by sliding a region corresponding to the extracellular region of CDH3 (corresponding to positions 108 to 563 of SEQ ID NO: 2) by 13 amino acids from the N-terminal by 2 amino acids. -120, 110-122 ... 551-563) are immobilized on a glass slide and blocked with SuperBlock® (PIERCE). And reacted. After washing 3 times with TBS-T, detection was performed with DyLight649 fluorescently labeled anti-mouse antibody (PIERCE). Those that did not react with the epitope search target antibody were measured as negative controls. The measurement results are shown in FIG. A strong signal was observed in the region corresponding to the amino acid sequence at positions 446-472, 490-504 of CDH3 shown in SEQ ID NO: 2, and it was presumed to be an epitope of the antibody.
- ADCC highly active antibodies were concentrated in the upstream region of EC1, EC4 and EC5. Indicated.
- RNA was prepared from normal human tissue and various cancer tissues from a sample collected by laser microdissection method using ISOGEN (Nippon Gene) according to a standard method. did. Gene expression of each 10 ng of RNA was analyzed using GeneChip U-133B (Affymetrix) according to Expression Analysis Technical Manual (Affymetrix). When an average expression score of all genes was set to 100 and a gene whose expression was enhanced in cancer cells was searched, CDH3 was highly expressed in lung cancer, colon cancer and pancreatic cancer (FIG. 10B). Further, when the expression of CDH3 mRNA in pancreatic cancer tissues having different degrees of differentiation was examined, tissues with high expression were recognized regardless of the degree of differentiation (FIG. 10C).
- Example 8 Expression of CDH3 protein in cancer tissue by immunohistochemical staining
- immunostaining was performed on a cancer specimen tissue array.
- the cancer cell tissue array is manufactured by Shanghai Outdo Biotech Co., Ltd., pancreatic cancer (adenocarcinoma), lung cancer (adenocarcinoma), lung cancer (squamous cell carcinoma) and colon cancer (glandular gland). Cancer).
- Each tissue array slide was deparaffinized and activated with 10 mM Tris 1 mM EDTA (pH 9.0) at 95 ° C. for 40 minutes.
- Example 9 Anti-Tumor Effect in Xenograft Model
- the anti-tumor effect of anti-CDH3 antibody was determined using a xenograft grafted with human lung cancer-derived cell line NCI-H358, human skin cancer-derived cell line A431, and human pancreatic cancer-derived cell line PK-45P. Confirmed with. NCI-H358 and PK-45P were cultured in 10% FBS-containing RPMI1640 medium, A431 was cultured in 10% FBS-containing DMEM medium, and 5 ⁇ subcutaneously in the right ventral region of SCID mice (female, 7 weeks old, Japan Marie). The cells were transplanted to 106 cells / mouse.
- RCB1205-producing antibody anti-pertussis toxin mouse IgG antibody
- 7.5 mg / kg was administered from the tail vein. The administration was started when the average tumor diameter reached 90 mm3, and was administered twice a week (every 3 days or 4 days).
- Administered intravenously The administration was started when the average tumor diameter reached 120 mm3, and was administered twice a week (every 3 days or 4 days).
- Administration was started when the average tumor diameter reached 110 mm 3 and was performed twice a week (every 3 or 4 days) 6 times.
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Abstract
Description
カドヘリン相互の同種/異種の認識は細胞外ドメインのN端に位置するカドヘリンドメイン1(EC1)によるとされる(Nose A. et al., Cell 61:147-155, 1990)。Klingelらは、ヒトP-カドヘリンのアミノ酸配列の第1位~第213位(配列番号2)とヒトE-カドヘリンの対応する領域を入れ替えると、E-カドヘリンとは結合せず、P-カドヘリンと結合することを示している(Klingel H. et al., J of Cell Science 113:2829ー36, 2000)。このように、E-カドヘリン、P-カドヘリンをはじめとする古典的カドヘリンは同一の機序で相互に結合すると考えられる。
抗体のADCC活性を規定する要素としては、抗体のFc部分のエフェクター細胞のFcレセプターへの親和性、抗体の抗原に対する親和性、抗体が認識するエピトープが挙げられる。ADCC活性が発揮されるためには、抗原に抗体が結合し、抗体のFc部位にエフェクター細胞のFcレセプターが結合することが必須であるが、抗体が結合するCDH3の領域の違いにより、エフェクター細胞の抗体のFc部位への結合に空間的な制限がありADCC活性の差を生じていることが推定される。本発明はこれらの知見に基づいて完成したものである。
(1)EC1の上流領域、カドヘリンドメイン4(EC4)またはカドヘリンドメイン5(EC5)の何れかのカドヘリンドメインを認識し、かつ抗体濃度1μg/mLのときの抗体依存性細胞傷害活性が30%以上である、抗カドヘリン抗体。
(2)カドヘリンがP-カドヘリンである、(1)に記載の抗体。
(3)可溶型P-カドヘリンを免疫原として投与した免疫動物から取得した抗体産生細胞が産生する抗体である、(1)又は(2)に記載の抗体。
(4)モノクローナル抗体である、(1)から(3)の何れかに記載の抗体。
(5)(4)に記載の抗体を産生するハイブリドーマ。
(6)(1)から(4)の何れかに記載の抗体を含む、細胞傷害剤。
(7)癌細胞に投与される、(6)に記載の細胞障害剤。
なお、本明細書においてEC1の上流領域とは、E-カドヘリン、P-カドヘリン、N-カドヘリンのEC1の上流側24アミノ酸残基の領域、および、他のカドヘリンの対応する領域を示す。
本発明の抗体は、EC1の上流領域、カドヘリンドメイン4(EC4)またはカドヘリンドメイン5(EC5)の何れかのカドヘリンドメインを認識し、かつ抗体濃度1μg/mLのときの抗体依存性細胞傷害活性が30%以上であることを特徴とする抗カドヘリン抗体、EC1の上流領域、カドヘリンドメイン4(EC4)またはカドヘリンドメイン5(EC5)の何れかのカドヘリンドメインを認識し、かつ抗体濃度0.1μg/mLのときの抗体依存性細胞傷害活性が25%以上(例えばPPMX5の活性より強い)であることを特徴とする抗カドヘリン抗体、または、EC1の上流領域、カドヘリンドメイン4(EC4)またはカドヘリンドメイン5(EC5)の何れかのカドヘリンドメインを認識し、かつADCCの最大活性が35%以上(例えばPPMX6の活性より強い)である抗カドヘリン抗体である。ここで、ADCCの最大活性とは、抗体濃度を上げてADCC活性の上昇がプラトーに達したときのADCC活性をいう。
本明細書においてP-カドヘリン、P-カドヘリンおよびN-カドヘリンのEC1の上流領域、カドヘリンドメイン1(EC1)、カドヘリンドメイン2(EC2)、カドヘリンドメイン3(EC3)、カドヘリンドメイン4(EC4)、カドヘリンドメイン5(EC5)は、それぞれ次の領域を示す。また、他のカドヘリンの対応する領域は、Genbank等より得られる公知のカドヘリンタンパク質の配列を比較することによって決定することができる。配列の比較は公知のプログラム、例えばClustalW2(Thompson JD et al., Nucleic Acids Research 22 (22): 3673ー3680、1994)またはClustalX2(Thompson JD et al., Nucleic Acids Research 25 (24): 4876ー4882, 1997)などを用いて行うことが可能である。
・P-カドヘリン(CDH3)
EC1の上流領域:配列番号2のアミノ酸配列の第108位~第131位
カドヘリンドメイン1(EC1):配列番号2のアミノ酸配列の第132位~第236位
カドヘリンドメイン2(EC2):配列番号2のアミノ酸配列の第237位~第348位
カドヘリンドメイン3(EC3):配列番号2のアミノ酸配列の第349位~第461位
カドヘリンドメイン4(EC4):配列番号2のアミノ酸配列の第462位~第550位
カドヘリンドメイン5(EC5):配列番号2のアミノ酸配列の第551位~第654位
・E-カドヘリン(CDH1)
EC1の上流領域:配列番号4のアミノ酸配列の第155位~第178位
カドヘリンドメイン1(EC1):配列番号4のアミノ酸配列の第179位~第283位
カドヘリンドメイン2(EC2):配列番号4のアミノ酸配列の第284位~第395位
カドヘリンドメイン3(EC3):配列番号4のアミノ酸配列の第396位~第507位
カドヘリンドメイン4(EC4):配列番号4のアミノ酸配列の第508位~第597位
カドヘリンドメイン5(EC5):配列番号4のアミノ酸配列の第598位~第704位
・N-カドヘリン(CDH2)
EC1の上流領域:配列番号6のアミノ酸配列の第160位~第183位
カドヘリンドメイン1(EC1):配列番号6のアミノ酸配列の第184位~第288位
カドヘリンドメイン2(EC2):配列番号6のアミノ酸配列の第289位~第402位
カドヘリンドメイン3(EC3):配列番号6のアミノ酸配列の第403位~第518位
カドヘリンドメイン4(EC4):配列番号6のアミノ酸配列の第519位~第607位
カドヘリンドメイン5(EC5):配列番号6のアミノ酸配列の第608位~第719位
(1)エフェクター細胞の調製
C3H/HeJ Jclマウス(8週齢、オス、日本クレア社)の大腿骨から骨髄細胞を採取し、10%FBS含有RPMI1640培地中で2x106個/mLに調製し、ヒトIL-2(PEPROTECH社)50ng/mL、マウスGM-CSF(PEPROTECH社)10ng/mL存在下で6日間培養した。測定当日に細胞を回収し、10%FBS含有HAM培地で洗浄後、エフェクター細胞溶液とした。
(2)標的細胞の調製
標的細胞として、全長CDH3発現CHO細胞(EXZ1501)を用いた。細胞をプレートから剥離後、1x107個/mLとなるように10%FBS含有HAM培地に懸濁し、終濃度150uCiとなるように51Crを加え、5%炭酸ガスインキュベーター中37℃で1.5時間培養した。この細胞を培地で2回洗浄後、10%FBS含有HAM培地を加え、1x104個/ウェルとなるように96ウェルU底プレート(NUNC社)に播き、標的細胞とした。
(3)ADCC活性の測定
標的細胞にそれぞれ0.001、0.01、0.1、1μg/mL濃度に調製した抗体溶液を50μL/ウェル分注した後、室温で15分間インキュベーションし、次いでエフェクター細胞100μL(1x105細胞/ウェル)を分注し、炭酸ガスインキュベーター中で4時間培養する。培養上清を回収し、培養上清100μL中の放射活性をシンチレーションカウンターで測定する。細胞傷害活性は以下の式で求めることができる。
細胞傷害活性(%)=(A-C)/(B-C)×100
A:各抗体添加ウェルの放射活性値(cpm)
B:標的細胞に2%NP40溶液100μL、 10%FBS含有RPMI培地50μLを添加したウェルの放射活性値(cpm)
C:標的細胞にエフェクター細胞を含む10%FBS含有培地150μLを加えたウェルの放射活性値(cpm)
ポリクローナル抗体を作製するためには、カドヘリン又はその部分ペプチドであるEC1の上流領域、カドヘリンドメイン4(EC4)またはカドヘリンドメイン5(EC5)の何れかのカドヘリンドメインが好ましい)を抗原として、これを哺乳動物、例えばラット、マウス、ウサギなどに投与する。抗原の動物1匹当たりの投与量は、アジュバントを用いないときは0.1~100mgであり、アジュバントを用いるときは1~100μgである。アジュバントとしては、フロイント完全アジュバント(FCA)、フロイント不完全アジュバント(FIA)、水酸化アルミニウムアジュバント等が挙げられる。免疫は、主として静脈内、皮下、腹腔内等に注入することにより行われる。また、免疫の間隔は特に限定されず、数日から数週間間隔、好ましくは2~5週間間隔で、1~10回、好ましくは2~5回免疫を行う。そして、最終の免疫日から6~60日後に、酵素免疫測定法(ELISA(enzumeーlinked immunosorbent assy)又は EIA(enzyme immunoassay))、放射性免疫測定法(RIA;radioimmuno assay)等で抗体価を測定し、最大の抗体価を示した日に採血し、抗血清を得る。抗血清から抗体の精製が必要とされる場合は、硫安塩析法、イオン交換クロマトグラフィー、ゲル濾過、アフィニティークロマトグラフィーなどの公知の方法を適宜選択して、又はこれらを組み合わせることにより精製することができる。
モノクローナル抗体を作製するためには、先ず、カドヘリン又はその部分ペプチドであるEC1の上流領域、カドヘリンドメイン4(EC4)またはカドヘリンドメイン5(EC5)の何れかのカドヘリンドメインが好ましい)を抗原として、哺乳動物、例えばラット、マウス、ウサギなどに投与する。抗原の動物1匹当たりの投与量は、アジュバントを用いないときは0.1~100mgであり、アジュバントを用いるときは1~100μgである。アジュバントとしては、フロイント完全アジュバント(FCA)、フロイント不完全アジュバント(FIA)、水酸化アルミニウムアジュバント等が挙げられる。免疫は、主として静脈内、皮下、腹腔内に注入することにより行われる。また、免疫の間隔は特に限定されず、数日から数週間間隔、好ましくは2~5週間間隔で、1~10回、好ましくは2~5回免疫を行う。そして、最終の免疫日から1~60日後、好ましくは1~14日後に抗体産生細胞を採集する。抗体産生細胞としては、脾臓細胞、リンパ節細胞、末梢血細胞等が挙げられるが、脾臓細胞又は局所リンパ節細胞が好ましい。
抗CDH3抗体スクリーニング用細胞株を得るため、全長CDH3を発現するCHO細胞の樹立を行った。
(1)CDH3遺伝子発現ベクターの作製
配列番号1に示す全長ヒトCDH3DNAを哺乳類発現ベクターpEF4/myc-HisB(インビトロジェン社)へ挿入するため、2種類の制限酵素KpnI(タカラバイオ社)及びXbaI(タカラバイオ社)で37℃、1時間処理した後、同じくKpnI及びXbaIで処理したpEF4/myc-HisBへT4 DNAリガーゼ(プロメガ社)により常法に従って挿入し、発現ベクターpEF4―CDH3―myc-Hisを得た。
FuGENE(登録商標)6トランスフェクション試薬(ロシュ・ダイアグノスティックス社)のプロトコールに準じ、トランスフェクション前日に径10cmディッシュに8×105細胞のCHO細胞を播種し一晩培養後、8μgの発現ベクターpEF4―CDH3―myc-Hisと16μLのFuGENE6試薬を400μLの無血清RPMI1640培地(SIGMA-ALDRICH社)に混合し15分間室温放置後、細胞培養液に加えトランスフェクションを行った。トランスフェクション翌々日に選択試薬(Zeocin(登録商標))を用いて限界希釈法にてクローニングを行った。
抗CDH3抗体作製の免疫原とするため、C末端膜貫通領域以降を欠損させた可溶型CDH3(sCDH3)タンパク質を作製した。
(1)可溶型CDH3抗原発現ベクターの作製
CDH3全長cDNAをテンプレートとして、CDH3細胞外領域に相当する部分(配列番号2の1-654に相当、以下sCDH3cDNA)を増幅するように設計されたフォワードプライマー (配列番号7:CGCGGTACCATGGGGCTCCCTCGT、(hCDH3FullFW))とリバースプライマー (配列番号8:CCGTCTAGATAACCTCCCTTCCAGGGTCC、(hCDH3SolbRV))を用いてPCR反応を行った。反応にはKOD-Plus(東洋紡社)を用い、94℃-15秒、55℃-30秒、68℃-90秒、30サイクルの反応条件で行った。
FuGENE6トランスフェクション試薬のプロトコールに準じ、トランスフェクション前日に径10cmディッシュに8×105 個のCHO細胞を播種し一晩培養後、8μgの発現ベクターpEF4-sCDH3-myc-Hisと16μLのFuGENE6試薬を400μLの無血清RPMI1640培地に混合、15分間室温放置後、細胞培養液に加えトランスフェクションを行った。トランスフェクション翌々日に選択試薬(Zeocin)を用いて限界希釈法にてクローニングを行った。
(1)可溶型CDH3タンパク質を免疫原としたモノクローナル抗体の作製
生理食塩水に溶解した50μgの可溶型CDH3タンパク質とTiter-MAX Gold(登録商標)(タイターマックス社)を等量混合し、MRL/lprマウス(日本エスエルシー株式会社)の腹腔内および皮下に注射する事により初回免疫を行った。2回目以降の免疫は同様に調製した25μgタンパク質量相当の可溶型CDH3タンパク質とTiter-MAX Goldを混合して腹腔内および皮下に注射することにより実施した。最終免疫から3日後にマウスから脾臓細胞を無菌的に調製し、常法に従って、ポリエチレングリコール法によりマウスミエローマ細胞SP2/O-Ag14あるいはP3-X63-Ag8.653との細胞融合を行った。
抗CDH3抗体の選抜は、全長CDH3を発現するCHO細胞株(EXZ1501)を用いたフローサイトメトリで行った。
ADCC活性の測定は、放射性ラベルした標的細胞にエフェクター細胞存在下、抗体を作用させ、その遊離放射活性を測定する方法によった。
(1)エフェクター細胞の調製
C3H/HeJ Jclマウス(8週齢、オス、日本クレア社)の大腿骨から骨髄細胞を採取し、10%FBS含有RPMI1640培地中で2x106個/mLに調製し、ヒトIL-2(PEPROTECH社)50ng/mL、マウスGM-CSF(PEPROTECH社)10ng/mL存在下で6日間培養した。測定当日に細胞を回収し、10%FBS含有HAM培地で洗浄後、エフェクター細胞溶液とした。
標的細胞として、全長CDH3発現CHO細胞(EXZ1501)を用いた。細胞をプレートから剥離後、1x107個/mLとなるように10%FBS含有HAM培地に懸濁し、終濃度)150uCiとなるように51Crを加え、5%炭酸ガスインキュベーター中37℃で1.5時間培養した。この細胞を培地で2回洗浄後、10%FBS含有HAM培地を加え、1x104個/ウェルとなるように96ウェルU底プレート(NUNC社)に播き、標的細胞とした。
標的細胞にそれぞれ0.001、0.01、0.1、1μg/mL濃度になるよう調製した抗体溶液を50μL/ウェル分注した後、室温で15分間インキュベートした。次いで、エフェクター細胞100μL(1x105細胞/ウェル)を分注し、炭酸ガスインキュベーター中で4時間培養した。培養上清を回収し、培養上清100μL中の放射活性をシンチレーションカウンターで測定した。
細胞傷害活性(%)=(A-C)/(B-C)×100
A:各抗体添加ウェルの放射活性値(cpm)
B:標的細胞に2%NP40溶液100μL、 10%FBS含有RPMI培地50μLを添加したウェルの放射活性値(cpm)
C:標的細胞にエフェクター細胞を含む10%FBS含有培地150μLを加えたウェルの放射活性値(cpm)
試験結果を表1および図5に示す。ADCC活性を有する抗体の中でも特に強い活性を持つ抗体群が認められた。抗体濃度1μg/mLのときのADCC活性が30%以上であった抗体を高ADCC活性群、30%未満であった抗体を低ADCC活性群とした。
BDー610227は、市販抗CDH3抗体(BD BIOSCIENCE社)を示す。
Negative Ab1、2は、CDH3とは無関係な抗原を認識する抗体を示す。
*S:高ADCC活性(抗体濃度1μg/mLのときに30%以上)
W:低ADCC活性(抗体濃度1μg/mLのときに30%未満)
得られた抗CDH3抗体のエピトープ分類は、CDH3部分配列発現物とのウェスタンブロット法により行った。CDH3部分配列発現物は各断片間で十分に配列が重複するように断片1から5を設計した(図6)。
実施例1の全長CDH3cDNAをテンプレートとし、後述のプライマーセットを用いてPCR反応を行った。この反応にはiProof High Fidelity DNAポリメラーゼ(バイオラッド社)を用い、条件は98℃-10秒、60℃-10秒、72℃-30秒、35サイクルで行なった。アガロースゲル電気泳動により目的のサイズに近いバンドを含むゲルを切り出し、QIA(登録商標)クイックゲル抽出キットを用いて、目的のCDH3cDNA断片を得た。
以下のプライマーセットを用いてPCR反応を行い各断片を得た。
フォワードプライマー:TATGGAGCTCGGTACCGATTGGGTGGTTGCTCCAATATCTG(配列番号:9)
リバースプライマー:AGATTACCTATCTAGACTACTGCATCACAGAAGTACCTGGTAGG(配列番号:10)
フォワードプライマー:TATGGAGCTCGGTACCAAGTCTAATAAAGATAGAGACACCAAG(配列番号:11)
リバースプライマー:AGATTACCTATCTAGACTACCTCTGCACCTCATGGCCCACTGCATTCTCA(配列番号:12)
フォワードプライマー:TATGGAGCTCGGTACCGTGACAGCCACGGATGAGGATGATG(配列番号:13)
リバースプライマー:AGATTACCTATCTAGACTAGACACACACAGGCTCCCCAGTG(配列番号:14)
フォワードプライマー:TATGGAGCTCGGTACCCTGACGGTCACTGATCTGGACG(配列番号:15)
リバースプライマー:AGATTACCTATCTAGACTAGGGCTCAGGGACTGGGCCATGGTCATTG(配列番号:16)
フォワードプライマー:TATGGAGCTCGGTACCTACACTGCAGAAGACCCTGACAAGG(配列番号:17)
リバースプライマー:AGATTACCTATCTAGACTAACCTCCCTTCCAGGGTCCAGGGCAGGTTTCG(配列番号:18)
(1)のCDH3断片の発現ベクターを使用して、常法により大腸菌Rossetta(登録商標)2(メルク社)を形質転換し、LB培地中で培養。600nmの吸光値が0.4となった段階で、30分間氷冷し、イソプロピルーβーチオガラクトピラノシド(IPTG)濃度を0.5mMとし、20℃で18時間培養後、回収した。
即ち、上述した大腸菌培養液量の1/10量に相当する泳動緩衝液を加え、還元条件下5-20%グラジエントゲル(バイオラッド社)にチャージして電気泳動後、イモビロン(登録商標)P(ミリポア社)に転写した。転写膜は、TBS-T(0.05%Tween(登録商標)20、TBS)で軽く洗った後、40%BSA入りTBSで1時間振とう後、10%ブロックエース(登録商標)(雪印乳業社)入りTBS-Tで希釈した各抗CDH3抗体を加え1時間振とうした。TBS-Tで洗った後10%ブロックエース入りTBS-Tで希釈したHRP-抗マウスIgG抗体(GEヘルスケアバイオサイエンス社)で1時間振とう後、TBS-Tで洗った。発色はメーカーの解説書に従い、ECL(登録商標)-Plus(GEヘルスケアバイオサイエンス社)を用いて、X線フィルムRX-u(富士フイルム社)にて検出した。得られた結果を図7に示す。
上述の各CDH3部分長タンパク質を発現させた大腸菌溶解物を還元条件下、5-20%グラジエントゲル(バイオラッド社)にチャージして電気泳動後、ブロッティング装置(バイオラッド社)を用いて、イモビロンP(ミリポア社)に転写した。転写膜は、TBS-T(0.05%Tween20、TBS)で軽く洗った後、40%BSA入りTBSで1時間振とう後、短冊状に等間隔で切断し、10%ブロックエース入りTBS-Tで希釈した各抗CDH3抗体を加え1時間振とうした。TBS-Tで洗った後10%ブロックエース入りTBS-Tで希釈したHRP-抗マウスIgG抗体(GEヘルスケアバイオサイエンス社)で1時間振とう後、TBS-Tで洗った。発色はメーカーの解説書に従い、ECL-Plus(GEヘルスケアバイオサイエンス社)を用いて、X線Film RX-u(富士フイルム社)にて検出した。得られた結果を図8に示す。
配列番号2で示されるCDH3配列上の各抗体が認識する領域との対応関係を以下に示す。
EC1:132位―236位
EC2:237位―348位
EC3:349位―461位
EC4:462位―550位
EC5:551位―654位
BDー610227は、市販抗CDH3抗体(BD BIOSCIENCE社)を示す。
Negative Ab1、2は、CDH3とは無関係の抗原を認識する抗体を示す。
*S:高ADCC活性(抗体濃度1μg/mLのときに30%以上)
W:低ADCC活性(抗体濃度1μg/mLのときに30%未満)
前述したCDH3部分長発現タンパク質によるエピトープ決定において、その境界領域に相当したと考えられる抗体PPMX13を対象に、ペプチドアレイ(JPT Peptide Technologies社製Replitope)を用いてより詳細にエピトープ決定を行った。
正常ヒト組織および各種癌組織より、レーザーマイクロダイセクション法(Laser Capture Microdissection)で回収したサンプルよりISOGEN(ニッポンジーン社)を用い定法に従って全RNAを調製した。RNA各10ngをGeneChipU-133B(Affimetrix社)を用い、Expression Analysis Technical Manual(Affimetrix社)に準じて遺伝子発現を解析した。全遺伝子の発現スコアの平均値を100とし、癌細胞において発現が亢進する遺伝子を探索したところ、CDH3は肺癌、大腸癌、膵臓癌で発現が高かった(図10B)。また、分化度の異なる膵臓癌組織におけるCDH3mRNAの発現を検討したところ、分化度に関わらず発現が高い組織が認められた(図10C)。
癌臨床検体でのCDH3タンパク質の発現を確認するため、癌検体組織アレイで免疫染色を行った。
癌細胞組織アレイは、上海芯超生物科技有限公司社(Shanghai Outdo Biotech Co.,Ltd.)製の、膵癌(腺癌)、肺癌(腺癌)、肺癌(扁平上皮癌)および大腸癌(腺癌)を使用した。
各組織アレイスライドを脱パラフィン処理し、10mMTris 1mM EDTA(pH9.0)で95℃40分賦活化を行った。ENVISION+Kit(Dako社)付属のブロッキング試薬にて内在性ペルオキシダーゼの不活性化を行った後、抗CDH3抗体610227(BD BIOSCIENCE社)、およびネガティブコントロールとして抗HBs抗体Hyb-3423と5μg/mLの濃度で4℃一晩反応させた。抗体溶液を洗い流した後に、ENVISION+Kit付属のポリマー二次抗体試薬と室温30分間反応させた。ENVISION+Kit付属の発色試薬にて発色を行い、ヘマトキシリンエオジン溶液にて核染色を行った。
図11に結果を示す。癌細胞は抗CDH3抗体で染色され正常細胞は染色されなかった。
抗CDH3抗体の抗腫瘍効果を、ヒト肺癌由来細胞株NCI-H358、ヒト皮膚癌由来細胞株A431、およびヒト膵臓癌由来細胞株PK-45Pを移植したゼノグラフトで確認した。
NCI-H358およびPK-45Pは10%FBS含有RPMI1640培地、A431は10%FBS含有DMEM培地を用いて培養し、SCIDマウス(メス、7週齢、日本クレア)の右腹側部皮下に5×106個/マウスとなるように移植した。
NCI-H358移植マウスを6群に分け(n=8)、各群にPPMX12産生抗体を0.01mg/kg、0.06mg/kg、0.3mg/kg、1.5mg/kg、対照抗体としてRCB1205産生抗体(抗百日咳毒素マウスIgG抗体)7.5mg/kgを尾静脈より投与した。投与は、平均腫瘍径が90mm3となった時点で開始し、週2回(3日または4日毎)に8回行った。
A431移植マウスを2群に分け(n=8)、各群にPPMX12産生抗体を7.5mg/kg、対照抗体としてRCB1205産生抗体(抗百日咳毒素マウスIgG抗体)7.5mg/kgを尾静脈より投与した。投与は、平均腫瘍径が110mm3となった時点で開始し、週2回(3日または4日毎)に6回行った。
Claims (7)
- EC1の上流領域、カドヘリンドメイン4(EC4)またはカドヘリンドメイン5(EC5)の何れかのカドヘリンドメインを認識し、かつ抗体濃度1μg/mLのときの抗体依存性細胞傷害活性が30%以上である、抗カドヘリン抗体。
- カドヘリンがP-カドヘリンである、請求項1に記載の抗体。
- 可溶型P-カドヘリンを免疫原として投与した免疫動物から取得した抗体産生細胞が産生する抗体である、請求項1又は2に記載の抗体。
- モノクローナル抗体である、請求項1から3の何れかに記載の抗体。
- 請求項4に記載の抗体を産生するハイブリドーマ。
- 請求項1から4の何れかに記載の抗体を含む、細胞傷害剤。
- 癌細胞に投与される、請求項6に記載の細胞障害剤。
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US13/886,936 US20130245232A1 (en) | 2009-05-01 | 2013-05-03 | Highly effective anti-cadherin antibody for induction of antibody-dependent cellular cytotoxicity in vivo |
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EP2634194A4 (en) * | 2010-10-29 | 2014-05-28 | Perseus Proteomics Inc | ANTI-CDH3 ANTIBODIES WITH HIGH INTERNALIZATION ABILITY |
WO2014126198A1 (ja) * | 2013-02-15 | 2014-08-21 | 株式会社ペルセウスプロテオミクス | 抗cdh3ヒト化抗体、その薬剤コンジュゲート、及びそれらの使用 |
WO2016166360A1 (en) | 2015-04-17 | 2016-10-20 | Bayer Pharma Aktiengesellschaft | Bispecific antibody constructs for cdh3 and cd3 |
JP2018524373A (ja) * | 2015-07-15 | 2018-08-30 | ザイムワークス,インコーポレイテッド | 薬物がコンジュゲートされた二重特異性抗原結合コンストラクト |
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2010
- 2010-04-30 AU AU2010242338A patent/AU2010242338B2/en not_active Ceased
- 2010-04-30 JP JP2011511471A patent/JP5723270B2/ja not_active Expired - Fee Related
- 2010-04-30 KR KR1020117028845A patent/KR20120051603A/ko not_active Application Discontinuation
- 2010-04-30 WO PCT/JP2010/057694 patent/WO2010126137A1/ja active Application Filing
- 2010-04-30 CN CN2010800281876A patent/CN102753579A/zh active Pending
- 2010-04-30 US US13/318,422 patent/US8455249B2/en not_active Expired - Fee Related
- 2010-04-30 EP EP10769835A patent/EP2426149A4/en not_active Withdrawn
- 2010-04-30 CA CA2760642A patent/CA2760642A1/en not_active Abandoned
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2013
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Also Published As
Publication number | Publication date |
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AU2010242338B2 (en) | 2013-12-19 |
KR20120051603A (ko) | 2012-05-22 |
CN102753579A (zh) | 2012-10-24 |
US20130245232A1 (en) | 2013-09-19 |
JP5723270B2 (ja) | 2015-05-27 |
US20130243771A1 (en) | 2013-09-19 |
JPWO2010126137A1 (ja) | 2012-11-01 |
EP2426149A1 (en) | 2012-03-07 |
AU2010242338A1 (en) | 2011-12-01 |
US8455249B2 (en) | 2013-06-04 |
EP2426149A4 (en) | 2013-01-23 |
US20120136140A1 (en) | 2012-05-31 |
US9017683B2 (en) | 2015-04-28 |
CA2760642A1 (en) | 2010-11-04 |
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