WO2012057315A1 - Anticorps anti-cdh3 d'affinité élevée - Google Patents

Anticorps anti-cdh3 d'affinité élevée Download PDF

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WO2012057315A1
WO2012057315A1 PCT/JP2011/074934 JP2011074934W WO2012057315A1 WO 2012057315 A1 WO2012057315 A1 WO 2012057315A1 JP 2011074934 W JP2011074934 W JP 2011074934W WO 2012057315 A1 WO2012057315 A1 WO 2012057315A1
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antibody
nite
cadherin
cells
cdh3
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PCT/JP2011/074934
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English (en)
Japanese (ja)
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佐藤　広一
富美子 野村
石井　敬介
正 松浦
須藤　幸夫
克之 見供
克志 甲田
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株式会社ペルセウスプロテオミクス
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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
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to an anti-cadherin antibody that recognizes a specific domain of cadherin and has high affinity for cadherin.
  • 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 homophilicity 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 kinds of proteins, which play an important role in maintaining a multicellular structure.
  • Proteins belonging to the cadherin superfamily can be broadly classified into 1) classical cadherins, 2) desmosomal cadherins, 3) protocadherins, 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. Allogeneic cells adhere to each other by a mechanism in which E-cadherin recognizes and binds to E-cadherin and P-cadherin recognizes and binds to P-cadherin (FIG. 2).
  • cadherin domain 1 located at the N-terminal of the extracellular domain
  • Klingel et al. Found that when the positions 1 to 213 of the amino acid sequence of human P-cadherin (SEQ ID NO: 2) and the corresponding region of human E-cadherin were interchanged, E-cadherin did not bind and P-cadherin and (Klingel H. et al., J of Cell Science 113: 2829-36, 2000).
  • classical cadherins including E-cadherin and P-cadherin are thought to bind to each other by the same mechanism.
  • antibody drug it can be prepared by combining two elements: A: binding (accumulation) to cancer cells (responsible by antibodies) and B: drug efficacy (responsible by RI and drugs). is there.
  • Such an antibody drug can be referred to as an ARMED antibody (armed antibody) because the antibody is enhanced (armed) by B: RI, a drug, a toxin or the like.
  • ARMED antibodies, RI, drugs, toxins, and the like are responsible for the efficacy, and the antibodies only have to act to accumulate in the tumor, and may have physiological activity such as ADCC, but it is not always necessary.
  • RI, drugs, toxins, and the like are each bound to antibodies by chemical methods (modification) or molecular biological methods.
  • RI-labeled antibody is an example of ARMED antibodies, already Zevalin R (FUJIFILM RI Pharma sold) are shown practically used very strong efficacy as an antibody against malignant lymphoma, the RI-labeled antibody drug followed by Development is desired.
  • An antibody (antibody drug conjugate (ADC)) obtained by linking an antibody and a drug having a strong toxicity (toxin) is also an example of the ARMED antibody.
  • ADC antibody drug conjugate
  • toxins When administered alone to a patient, toxins can also damage normal tissues and cannot be an effective treatment.
  • by linking with an antibody that binds to a tumor cell-specific antigen it has the ability to kill only tumor cells without adversely affecting normal tissue.
  • One method for obtaining a stronger antitumor effect is to increase the amount of antibody accumulated in the tumor by using an antibody having a high affinity for the antigen, thereby increasing the drug efficacy.
  • it is necessary to obtain antibodies with high affinity but the method for obtaining such antibodies is not always clear, and has become a major issue in the development of ARMED antibody drugs. Yes.
  • An object of the present invention is to provide an anti-cadherin antibody having high affinity.
  • the present inventor has diligently studied to solve the above-mentioned problems, and when the affinity of the P-cadherin antibody to the antigen expressed on the cell surface was measured, it was found that the group tends to be divided into a high affinity group and a low affinity group. It was. Therefore, when classification is performed according to the region recognized by each antibody, it is found that an antibody with high affinity recognizes either cadherin domain 1 (EC1) or cadherin domain 2 (EC2) with a high probability. found.
  • EC1 cadherin domain 1
  • EC2 cadherin domain 2
  • the antibody according to (3), wherein the P-cadherin is soluble P-cadherin or a fragment thereof.
  • the antibody according to any one of (1) to (4) which is a monoclonal antibody.
  • the antibody according to (5), wherein the monoclonal antibody is a chimerized antibody, a humanized antibody or a human antibody.
  • a monoclonal antibody produced by a cell having NITE BP-1150 Alternatively, a monoclonal antibody produced by a cell having NITE BP-1150.
  • the cytotoxic substance is a drug, a toxin, or a radioactive substance.
  • the cytotoxic substance is a radioactive substance selected from the group consisting of yttrium (90), indium (111) and iodine (131).
  • a cytotoxic agent comprising the antibody according to any one of (1) to (15).
  • a method for damaging cadherin-expressing cells in the patient comprising administering the above-described antibody or cytotoxic agent of the present invention to a cadherin-expressing patient. Furthermore, according to the present invention, there is provided use of the above-described antibody of the present invention for the production of a cytotoxic agent.
  • the present invention provides an antibody that recognizes either cadherin domain 1 (EC1) or cadherin domain 2 (EC2) of P-cadherin and has high affinity for P-cadherin.
  • An antibody that can exhibit high affinity is useful as a material for preparing a modified antibody or a modified antibody. Since the antibody of the present invention has high affinity, a labeled antibody obtained by binding a radioactive metal having cytotoxicity to this antibody is expected to have a high anticancer activity as a cytotoxic agent when administered to a cancer patient highly expressing cadherin. it can.
  • 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 in which a human CDH3 forced expression cell line was reacted with a commercially available anti-human CDH3 antibody.
  • B CDH3 forced expression CHO cell CHO cell.
  • FIG. 4 shows typical flow cytometry results for 3 acquired antibodies and each cell line.
  • FIG. 5 shows the measurement results of the affinity KD (nM) of each antibody.
  • FIG. 6 shows the correspondence of CDH3 partial length protein fragments 1 to 6 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 CBB staining result of the CDH3 partial length protein expression (fragment 6).
  • FIG. 10 shows the results of ELISA for CDH3 partial length protein expression.
  • FIG. 10 shows the results of ELISA for CDH3 partial length protein expression.
  • FIG. 11 shows the biodistribution of 111 In-labeled PPAT-055-28c in a xenograft model.
  • FIG. 12 shows the biodistribution of 111 In-labeled PPAT-055-27c in a xenograft model.
  • FIG. 13 shows the antitumor effect of 90 Y-labeled PPAT-055-28c in a xenograft model.
  • FIG. 14 shows the antitumor effect of 90 Y-labeled PPAT-055-27c in a xenograft model.
  • the antibody of the present invention is an anti-cadherin antibody that recognizes either cadherin domain 1 (EC1) or cadherin domain 2 (EC2) and has high affinity.
  • cadherin domain 1 (EC1), cadherin domain 2 (EC2), cadherin domain 3 (EC3), cadherin domain 4 (EC4), cadherin domain 5 (EC5) of P-cadherin, E-cadherin and N-cadherin 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.
  • Antibody affinity can be measured by a known method.
  • Methods for measuring antibody affinity include FACS, BIACORE, ELISA and the like.
  • FACS Fluorescence Activated Cell Sorting
  • BIACORE BIACORE
  • ELISA ELISA
  • the method for measuring the affinity of an antibody by FACS is to obtain a point where fluorescence intensity is saturated under conditions of excess antibody, and to measure the antibody concentration that gives this half of the fluorescence intensity, this becomes the Kd value (Journal of Immunological IV Methods; 318, 147 (2007)).
  • the Kd value relating to the affinity in the specification means a case where measurement is performed under the same conditions as in Example 6. Specifically, it is as follows.
  • the affinity of the anti-CDH3 antibody was measured by a flow cytometry method (BD FACSCalibur) using a cancer cell NCI-H358 strain that is confirmed to have high expression of CDH3. That is, NCI-H358 cells were detached from the culture plate by treatment with 2 mM EDTA-PBS, and suspended in a FACS solution (1% BSA / PBS) so as to be 5 ⁇ 10 4 to 2 ⁇ 10 5 / ml. This cell suspension was seeded at 100 ⁇ 1 / well in a 96-well plate, and 100 ⁇ 1 of antibody dilution series (200 ⁇ g / ml to 0.13 ng / ml) was added, followed by reaction at 4 ° C.
  • BD FACSCalibur flow cytometry method
  • “high affinity” means that the “Kd value, which is an antibody concentration that gives 50% binding to an antigen” measured by the above method is 10 nM or less, preferably 7 nM or less. More preferably, it is 3 nM or less, More preferably, it is 1 nM or less, Especially preferably, it is 0.60 nM or less.
  • an antibody having an antibody-dependent cytotoxic activity at an antibody concentration of 1 ⁇ g / mL is less than 30%.
  • Antibody-dependent cytotoxic activity can be measured by a known method.
  • the numerical value of ADCC activity in this specification means antibody-dependent cytotoxic activity when measured under the same conditions as in Example 8. Specifically, it is as follows.
  • Bone marrow cells were collected from the femurs of C3H / HeJ Jc1 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 to a final concentration of 150 ⁇ Ci, 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
  • Cytotoxic activity (%) (AC) / (BC) ⁇ 100
  • 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 of the present invention (polyclonal antibody and monoclonal antibody) 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)]. In this specification, the “monoclonal antibody” is not limited to an antibody produced by the hybridoma method.
  • the term “monoclonal antibody” refers to an antibody that is derived from a single clone (eg, eukaryotic, prokaryotic, phage clone), regardless of the method used.
  • Monoclonal antibodies useful in the present invention can be prepared by a variety of methods known in the art. Examples of the method include a hybridoma method, a recombinant method, a phage display method, and a combination thereof.
  • cadherin domain 1 EC1
  • cadherin domain 2 EC2
  • FCA Freund's complete adjuvant
  • FIA Freund's incomplete adjuvant
  • Aluminum hydroxide adjuvant EC1
  • 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 last immunization, the antibody titer is measured by enzyme immunoassay (ELISA (Enzyme Linked Immunosorbent Assay) or EIA (enzyme immunoassay)), radioimmunoassay (RIA), etc. Blood is collected on the day of showing the antibody titer to obtain antiserum.
  • ELISA Enzyme Linked Immunosorbent Assay
  • EIA enzyme immunoassay
  • RIA radioimmunoassay
  • cadherin or a partial peptide thereof, cadherin domain 1 (EC1) or cadherin domain 2 (EC2) is used as an antigen to a mammal such as a rat, a mouse, Administer to rabbits.
  • 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. Immunization is performed mainly by injecting intravenously, subcutaneously or intraperitoneally.
  • 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 to be 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 P3 ⁇ 63-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 bound to cadherin domain 1 (EC1) or cadherin domain 2 (EC2) of cadherin by enzyme immunoassay, radioimmunoassay or the like. Hybridomas producing antibodies 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.
  • EC1 cadherin domain 1
  • EC2 cadherin domain 2
  • 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). Cultivate for ⁇ 14 days and obtain antibody 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. And synthesized by PCR. It is obtained by ligating the obtained DNA with DNA encoding a human antibody constant region, then incorporating it into an expression vector, introducing it into a host and producing it (EP239400, International Publication WO96 / 02576, etc.) .
  • CDR complementarity determining region
  • Another method for producing humanized and chimerized antibodies is to identify the framework residues that are important for antigen binding by modeling the interaction between CDRs and framework residues and compare the sequences. Thus, there is a method of identifying an unusual framework structure residue at a specific position. See, for example, Queen et al., U.S. Pat.Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,762, and 6,180,370 (each patent is incorporated herein by reference in its entirety) Do).
  • Antibodies can be humanized by a variety of methods known in the art. For example, CDR grafting (European Patent No. 239,400; PCT Publication WO 91/09967; U.S. Patent Nos.
  • the amino acid sequence of the chimerized or humanized antibody is preferably 100% identical to the amino acid sequence of the VH or VL region derived from the cDNA expressed by the deposited hybridoma, but is 90% or more identical by genetic engineering modification.
  • An antibody having a certain sequence is also preferred. Conventionally, adjustment of residue substitution for the purpose of improving antigen binding in the process of humanization and chimerization has been performed. This is because such an antibody having a partially modified sequence is basically considered to be an antibody derived from the original hybridoma.
  • the methods for producing chimerized antibodies and humanized antibodies using genetic engineering techniques are already known. That is, the VH and VL sequences of the monoclonal antibody that is the confirmed group are genetically modified and then chimerized or humanized by a conventional method.
  • a method for obtaining a human antibody is also known.
  • human lymphocytes are sensitized with a desired antigen or cells expressing the desired antigen in vitro, and the sensitized lymphocytes are fused with human myeloma cells, such as U266, to have a desired human antibody having an antigen-binding activity.
  • 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, WO94 / 25585).
  • WO96 / 34096, WO96 / 33735 ).
  • variable region of a human antibody is 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 to obtain a human antibody.
  • these antibodies recognize cadherin domain 1 (EC1) or cadherin domain 2 (EC2) and do not lose the property of being a high affinity antibody. Any of them may be a low molecular weight antibody such as an antibody fragment (fragment) or a modified antibody. Further, an antibody fragment or a low molecular weight antibody, for example, Fab, Fab ′, F (ab ′) 2, Fv, ScFv (singlechainFv), Diabody, etc. fused with the Fc portion and added with ADCC activity may be used. In order to obtain such antibodies, a gene encoding these antibodies may be constructed, introduced into an expression vector, and then expressed in an appropriate host cell.
  • a gene encoding these antibodies may be constructed, introduced into an expression vector, and then expressed in an appropriate host cell.
  • modified antibody an antibody conjugated with various molecules such as polyethylene glycol (PEG) can also be used.
  • PEG polyethylene glycol
  • drug-bound antibody sputum is useful.
  • 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 affinity, it can be used as a cytotoxic agent by binding radioactive substances, toxins, and the like.
  • the cytotoxic agent of the present invention can damage cancer cells, for example, by contacting them with cancer cells expressing cadherin.
  • ADC in which a cytotoxic substance such as a drug is bound to the antibody
  • drugs used in the present invention include, for example, duocarmycin, duocarmycin analogs and inducers, CC-1065, duocarmycin analogs based on CBI, duocarmycin analogs based on MCBI, Duocarmycin analogs based on CCBI, doxorubicin, doxorubicin conjugate, morpholino-doxorubicin, cyanomorpholino-doxorubicin, dolastatin, dressatin-10, combretastatin, calicheamicin, maytansine, maytansine analog, DM1, DM2, DM3 , DM4, DMI, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin E (MMAE), 5-benzoylvalerin Acid AE ester (AEVB), tubulin, disorazole, epothilone, paclitaxel, docetaxel, SN-38, topot
  • the binding between the drug and the antibody can be produced by a known method.
  • the antibody and the drug may be directly bonded via a linking group or the like that they have, or may be indirectly bonded via another substance such as a linker.
  • linking group when the drug is directly bonded examples include a disulfide bond using an SH group and a bond via maleimide.
  • the intramolecular disulfide bond in the Fc region of the antibody and the disulfide bond of the drug are reduced, and both are bonded by a disulfide bond.
  • Another method is to introduce cysteine into the antibody by genetic engineering.
  • the linker preferably has one or more functional groups that react with the antibody or drug or both.
  • functional groups include amino groups, carboxyl groups, mercapto groups, maleimide groups, pyridinyl groups, and the like.
  • the linker may be a peptide linker.
  • Another embodiment of the present invention includes so-called immunotoxins in which toxins and toxins are chemically or genetically bound to antibodies.
  • toxin examples include the following. Diphtheria toxin A chain, Pseudomonas endotoxin, lysine chain; sugar-free ricin A chain Gelonin (Saporin) and the like can be mentioned.
  • RI-labeled antibody obtained by binding the antibody of the present invention and a radioactive substance can be mentioned.
  • the radioactive substance is preferably a cytotoxic radioactive metal when used as a cancer therapeutic agent, and is preferably a non-cytotoxic radioactive metal when used as a cancer diagnostic agent.
  • iodine 123 (123I) and iodine 131 (131I) are also a method using iodine 123 (123I) and iodine 131 (131I).
  • cytotoxic radioactive metals examples include yttrium 90 (90Y), rhenium 186 (186Re), rhenium 188 (188Re), copper 67 (67Cu), iron 59 (59Fe), strontium 89 (89Sr), and gold 198. (198Au), mercury 203 (203Hg), lead 212 (212Pb), dysprosium 165 (165Dy), ruthenium 103 (103Ru), bismuth 212 (212Bi), bismuth 213 (213Bi), holmium 166 (166Ho), samarium 153 (153Sm) ), Lutetium 177 (177Lu) and the like.
  • radioactive metals 90Y, 153Sm, and 177Lu are preferable from the viewpoint of half-life, radiation energy, easy labeling reaction, labeling rate, complex stability, and the like.
  • non-cytotoxic radioactive metals used for diagnostic agents include technesium 99m (99mTc), indium 111 (1111n), indium 113m (113mIn), gallium 67 (67Ga), gallium 68 (68Ga), and thallium 201 (201Tl).
  • Chromium 51 (51Cr), cobalt 57 (57Co), cobalt 58 (58Co), cobalt 60 (60Co), strontium 85 (85Sr), mercury 197 (197Hg), and copper 64 (64Cu) are preferably used. It is not limited to.
  • radioactive metal elements In order to bind these radioactive metal elements to the antibody of the present invention, it is preferable to react the metal chelate reagent with the antibody and react with the radioactive metal element to form a complex. In the modified antibody thus obtained, a radioactive metal element is bound to the antibody of the present invention via a metal chelating reagent.
  • metal chelating reagents used for such complex formation include (1) 8-hydroxyquinoline, 8-acetoxyquinoline, 8-hydroxyquinaldine, oxyquinoline sulfate, 0-acetyloxin, 0-benzoyloxin, Quinoline derivatives such as 0-p-nitrobenzoyloxin, quinolone compounds having a quinoline skeleton, norfloxacin, ofloxacin, enoxacin, ciprofloxacin, lomefloxacin, tosufloxacin, fleroxacin, sparfloxacin; (2) chloranilic acid, aluminon , Thiourea, pyrogallol, cuperone, bismuthiol (II), galloyl gallic acid, thiolide, 2-mercaptobenzothiazole, tetraphenylarsonium chloride, etc .; (3) ethylenediamine Tetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (ED
  • isothiocyanobenzyl DOTA, methylisothiocyanobenzyl DTPA, and cyclohexylisothiocyanobenzyl DTPA are easy to introduce metal chelates into antibodies, labeling rate, complex stability, etc. preferable.
  • a person skilled in the art can bind a radioactive metal element to the antibody of the present invention according to a conventional method.
  • the reaction can be performed by reacting the antibody of the present invention with a metal chelate reagent, preparing a labeling precursor in advance, and then reacting with a radioactive metal element.
  • the cytotoxic agent of the present invention in addition to the antibody of the present invention (a cytotoxic substance, a radioactive substance, or a toxin may be bound if desired), the cytotoxic agent of the present invention, if necessary, a pharmaceutically acceptable carrier, Excipients, diluents and the like can be appropriately contained.
  • the cytotoxic agent of the present invention can be formulated as an injection, for example.
  • the dose of the cytotoxic agent of the present invention depends on the patient's symptom level, age and body weight, 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) 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 selected by Western blotting using an anti-c-Myc monoclonal antibody (SANTA CRUZ BIOTECHNOLOGY). As a result of selecting a cell line with a large amount of secretion into the culture supernatant and good growth, a soluble CDH3-expressing CHO cell line (EXZ1702) was obtained.
  • 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 MRL / lpr mice (Japan SLC Co., Ltd.) were injected intraperitoneally and subcutaneously to perform initial immunization. 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.
  • 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 selected 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. Shown in It was confirmed that all the selected antibodies reacted with CDH3-expressing CHO cells (EXZ1501) and NCI-H358 but not with CHO cells.
  • NITE P-987, NITE P-987 under the accession numbers NITE P-987, NITE P-987, National Institute of Technology and Evaluation of Microorganisms (Postal Code 292-0818, 2-5-8 Kazusa Kamashita, Kisarazu, Chiba, Japan) 989, NITE P-990, NITE P-991, NITE P-992, NITE P-993, NITE P-994, deposited on September 7, 2011, with accession numbers NITE BP-987, NITE BP-989, NITE BP-990, NITE BP-991, NI E BP-992, NITE BP-993, was transferred as NITE BP-994 to an international deposit under the provisions of the Budapest Treaty.
  • the hybridoma producing the antibody PPAT-055-28 was founded on September 27, 2011, by the independent microorganisms evaluation technology patent organization Microorganism Depositary Center (Zip code 292-0818, Kazusa Kamashiri, Kisarazu City, Chiba Prefecture, Japan). 5-8) is deposited internationally under the pig best treaty under the deposit number NITE BP-1146.
  • Example 4 Preparation of chimeric antibody against CDH3 Using the hybridoma prepared in Example 3, the DNA encoding the V region of the mouse monoclonal antibody against human CDH3 was cloned as follows, and the mouse variable region and the human antibody gene were cloned. A chimeric antibody was prepared by connecting to the constant region.
  • RNA from hybridoma RNA from cytoplasmic RNA from mouse hybridoma cells was analyzed using Gough, Rapid and quantitative preparation of cytoplasmic RNA from small numbers of cells, Analytical Biochemisty, 173, p93-95 (1998) (but instead of the lysis buffer described in this paper, another TNE buffer 25 mM Tris-HCl, pH 7.5; 1% NP-40; 150 mM NaCl; 1 mM EDTA, pH 8.0 was used).
  • RNA precipitate was then dissolved by adding 10-50 microliters of sterile distilled water so that the cytoplasmic RNA concentration was 0.5-2 microgram / microliter.
  • VK sense (FR1 portion) A mixture of the following 17 primers was used as a VK sense primer.
  • W represents A or T
  • R represents A or G
  • M represents A or C
  • K represents T or G
  • Y represents T or C
  • S represents G or C represents H
  • A represents A, C or T
  • B represents G
  • V represents A, G or C
  • D represents A, G or T
  • N represents A, G, C or T is shown.
  • Sequence number 19 5'-GAYATCCAGCTGAACTCAGCC-3 '(degeneracy 2)
  • J antisense (4 sets primer) J1 / J2 antisense primer (1)
  • J4 antisense primer (2) SEQ ID NO: 37: 5′-GGGACAAAGTTGGGAAATAAA-3 ′
  • J5 antisense primer (3) SEQ ID NO: 38: 5′-GGGACCAAGCTGGAGCTGAAA-3 ′ J1 / J2, J4, J5 antisense primer mixture (4)
  • VK sense signal peptide part
  • the base sequence of this primer was modified to remove the restriction enzyme site based on Novagen mouse Ig-primer set (Novagen; Merck, Cat. No. 69831-3).
  • D Set Sense Primer (Use a mixture of the following two types of primers) SEQ ID NO: 42: 5′-ATGAGRGCCCCTGCTCAGGWTYTYTGGIWTTCTT-3 ′
  • a primer having homology with the mouse heavy chain signal part (4 sets primer) at the 5 ′ end and a primer having homology with the KC part at the 3 ′ end, or a set having homology with the FR1 part at the 5 ′ end
  • a mouse immunoglobulin heavy chain variable region DNA was isolated from the cDNA by polymerase chain reaction using two types of primer sets having homology with the constant region (IGHC) of mouse heavy chain at the 3 ′ end with the primer, The sequence was analyzed.
  • the primer sequences were as follows:
  • SEQ ID NO: 56 5′-ATGGRATGSAGCTGKGTMATSCTCTT-3 ′ (degeneracy: 32)
  • SEQ ID NO: 57 5′-ATGRACTTCGGGYTGAGCTKGGTTTTT-3 ′ (degeneracy: 8)
  • SEQ ID NO: 58 5′-ATGGCTGTCTTGGGGCTGCTCTTTCT-3 ′
  • Sequence number 59 5'-ATGGRCACGRCTACWTYY-3 '(degeneracy: 32)
  • VH sense (FR1 part) This primer is based on the nucleotide sequence of the sense primer of Tan et al., “Superhumanized” Antibodies: Reduction of Immunogenic Potential by Complementarity-Determining Region Grafting with Human Germline Sequences: Application to an Anti-CD281, Journal of Immunology 169 (2002) p1119-1125. Modified and designed. SEQ ID NO: 60: 5′-SAGGTSMARKCTSAGSGATCWCGG-3 ′ (degeneracy: 256)
  • VH antisense antisense primer common to 3 and 4
  • the nucleotide sequence was designed to be degenerate so that it could be annealed with all mouse IgG isoforms.
  • the genes encoding the V region of the L chain and H chain of the cloned anti-CDH3 chimeric antibody are the gene encoding the human Ck region for the chimeric L chain expression vector and human for the chimeric H chain expression vector.
  • Genes connecting the genes encoding the Cg1 region were designed, and these L-chain and H-chain chimeric antibody genes were artificially synthesized in full length by GenScript. In that case, optimization of codon usage so that gene expression in CHO-producing cells is advantageous (Kim et al., Codon optimization for high-level expression of human erythropoietin (EPO) in MMA3, 99 301).
  • a DNA sequence essential for efficient translation (Kozak, M., J., At least six nucleotides preceding the AU initiator codon enhancement translation in Mammalian cells. 196, p947-950, 1987), mouse IGKV (k chain variable region) gene signal peptide, anti-CDH3 antibody L chain V region, human KC (k chain constant region) in this order, and both ends
  • a restriction enzyme site (NheI on the 5'-side and EcoRI on the 3'-side) was added.
  • a chimeric H chain was prepared in the same manner.
  • a pCAGGS expression vector incorporating the dhfr gene was prepared. Specifically, a dhfr gene having a CMV promoter and a poly A signal is incorporated into the transient expression vectors pCAGGS-IGH and pCAGGS-IGK.
  • a CMV promoter, a mouse dhfr gene having a Kozak sequence, and an SV40 polyA signal are each amplified by the PCR method, a mixture of these genes is connected by the PCR method, and HindIII sites are added to both ends to form a HindIII-CMV promoter-Kozak
  • a gene fragment called -dhfr-polyA-HindIII was obtained. This fragment was incorporated into the HindIII site of pCAGGS-IGH or pCAGGS-IGK to obtain pCAGGS-IGH-CMVp-dhfr-A and pCAGGS-IGK-CMVp-dhfr-A.
  • These expression vectors were able to express the chimeric antibody with the CAG promoter and the dhfr gene with the CMV promoter, and could efficiently produce the chimeric antibody using gene amplification.
  • the electroporated cells were added to Iscove's Modified Dulbecco medium (IMDM) containing 10% dialyzed FBS and not containing HT (H, hypoxanthine: T, thymidine).
  • IMDM Iscove's Modified Dulbecco medium
  • HT hypoxanthine: T, thymidine
  • neo + transformed cells were selected with 1 mg / mL G418, and a chimeric antibody-producing positive cell line clone Got.
  • gene amplification was performed using clones selected with G418.
  • Cell line PPAT-055 producing approximately 50-100 mg of chimeric CDH3 antibodies PPAT-055-27C, PPAT-055-28C per liter after amplification in 2 rounds of 0.25 mM, 1 mM methotrexate (MTX) -27C and PPAT-055-28C could be established.
  • MTX methotrexate
  • Example 5 Acquisition of Purified Antibody The antibody was purified from the culture supernatant using protein A by a conventional method to obtain a purified antibody.
  • Example 6 Measurement of Antibody Kd Value Methods for measuring antibody affinity include FACS, BIACORE, ELISA and the like. In order to measure the affinity of the antibody for the cell surface, the FACS method in which the antigen is not denatured is desirable. The method for measuring the affinity of an antibody by FACS is to obtain a Kd value by obtaining a point at which the fluorescence intensity is saturated under the condition of excess antibody, and measuring the antibody concentration that gives this 1/2 fluorescence intensity (references). : Journal of Immunological Methods; 318, 147 (2007))
  • the affinity of the anti-CDH3 antibody was measured by a flow cytometry method (BD FACSCalibur) using a cancer cell NCI-H358 strain confirmed to have high expression of CDH3. That is, NCI-H358 cells were detached from the culture plate by treatment with 2 mM EDTA-PBS, and suspended in a FACS solution (1% BSA PBS) so as to be 5 ⁇ 10 4 to 2 ⁇ 10 5 / ml. This cell suspension was seeded at 100 ⁇ l / well in a 96-well plate, and 100 ⁇ l of a mouse or chimeric antibody dilution series (200 ⁇ g / ml to 0.13 ng / ml) was added, followed by reaction at 4 ° C.
  • BD FACSCalibur flow cytometry method
  • Example 7 Epitope classification of anti-CDH3 monoclonal antibody by CDH3 partial length expression protein
  • the epitope classification of the obtained anti-CDH3 antibody is antibody PPAT-055-02, PPAT-055-09, PPAT-055-21, PPAT-055. 24, PPAT-055-25, PPAT-055-26, PPAT-055-27 were performed by Western blotting with a CDH3 partial sequence expression product, and the antibody PPAT-055-28 was confirmed to be a Western blot reaction. Since there was not, it carried out by ELISA method.
  • fragments 1 to 6 were designed so that the sequences overlap sufficiently between the fragments (FIG. 6).
  • 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.
  • the gene for fragment 6-Fc fusion protein was artificially synthesized by GenScript, a US gene synthesis contractor. At that time, NheI was added to the 5 prime side and EcoRI site was added to the 3 prime side for subcloning. In addition, the base sequence of the gene is optimized for the frequency of codon usage by GenScript.
  • the artificially synthesized gene “Fragment 6-Fc” incorporated into the pUC subcloning vector is digested with restriction enzymes NheI and EcoRI, and the mammalian expression vector pCAGGS digested with NheI and EcoRI, or the mouse DHFR gene for gene amplification Incorporated into pCAGGS-DHFR.
  • Soluble antigen expression plasmid (the plasmid was cleaved with PvuI in ampicillin resistance gene to convert the circular plasmid into a linear plasmid; the amount of the plasmid was 2 ⁇ g / sample) was 3Ax 10e3 cells (0.1 mL CHO cells) with Lmaxa Amaxa. Gene suspension).
  • Electroporated cells were added to Iscove's Modified Modified Dulbecco Medium (IMDM) containing 10% dialyzed FBS and without HT (H, Hypoxanthine ⁇ ⁇ ⁇ : T, Thymidine). 3 days after gene transfer, change medium to IMDM without 10% dialyzed FBS, 2 mM L-glutamine, HT, select neo + transformed cells with 1 mg / mL G418, positive to produce chimeric antibody Cell line clones were obtained. Next, gene amplification was performed using clones selected with G418. After two rounds of methotrexate (MTX) at 100 nM and 200 nM, cell lines were established that produced about 20-30 mg of soluble antigen per liter.
  • IMDM Iscove's Modified Modified Dulbecco Medium
  • H Hypoxanthine ⁇ ⁇ ⁇ : T, Thymidine
  • change medium to IMDM without 10% dialyzed FBS, 2 mM L
  • FIG. 9 shows a CBB staining diagram of an antigen expressed and purified by transient expression.
  • CDH3 partial length protein is suspended in PBS ( ⁇ ) at a concentration of 2.5 ⁇ g / mL and dispensed into a 96-well plate at 100 ⁇ L / well. It was left at 4 ° C. overnight. On the next day, the solution in the well was discarded, and washed with Buffer A: 50 mM Tris-HCl / 150 mM NaCl / 1 mM CaCl 2 /0.05% Tween20 (pH 7.5).
  • PPAT-055-28C diluted with Buffer A at different concentrations was prepared, dispensed at 100 ⁇ L / well, and shaken for 1 hour on a plate shaker at room temperature.
  • HRP-labeled antibody HRP-goat anti human IgG (H + L) (absorbed with mouse, rabbit, bovine IgG) (American Qualex International, cat. A-110PD)
  • HRP-goat anti human IgG H + L
  • bovine IgG American Qualex International, cat. A-110PD
  • TMB coloring solution was added at 100 ⁇ L / well, and the mixture was allowed to stand for 15 minutes in the dark to develop color.
  • Stop solution was added at 100 ⁇ L / well, and the absorbance at 450 nm was measured with a plate reader. The obtained results are shown in Table 2 and FIG. PPAT-055-28C and PPAT-055-28 were found to have a recognition epitope of 1-2.
  • 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 cytotoxic activity at an antibody concentration of 1 ug / mL was 30% or more, it was determined as S, and when it was less than 30%, it was determined as W.
  • the test results are shown in Table 3.
  • Example 9 Production of RI-labeled antibody (1) Binding of DOTA to antibody PPAT-055-28C and PPAT-055-27C antibodies were dissolved in a buffer (50 mM Bicin-NaOH, 150 mM NaCl, pH 8.5), and the antibody concentration was adjusted to 10 mg / mL. On the other hand, isothiocyanobenzyl DOTA (manufactured by Macrocyclics B-205)) was dissolved in DMSO to a concentration of 10 mg / mL. The mixture was mixed and stirred so that the molar ratio of antibody to DOTA was 1: 3, and allowed to stand at 25 ° C. for 17 hours. After completion of the reaction, the product was purified with a desalting column (PD-10, GE Healthcare 17-0435-01) using PBS.
  • a desalting column PD-10, GE Healthcare 17-0435-01
  • Marking rate (lower end count / (upper end count + lower end count)) x 100 (%)
  • the labeled antibody was purified with a desalting column (PD-10, GE Healthcare, 17-0435-01) using PBS.
  • Example 11 Medicinal effect test Lung cancer-derived cell line NCI-H1373 was cultured in RPMI1640 medium containing 10% FBS, and 5 ⁇ 10 6 cells / mouse were subcutaneously injected into the right flank of nude mice (female, 7 weeks old, CLEA Japan). It was transplanted to become. NCI-H1373 transplanted mice were prepared as 8 mice per group, and 90Y-DOTA-PPAT-055-28C or 90Y-DOTA-PPAT-055-27C antibody was administered 5.6 MBq / mouse, 3.7 MBq / mouse. . As a control, physiological saline was administered at 100 ⁇ L / mouse. Administration was performed when the average tumor diameter was 100 to 150 mm 3 in any group.

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Abstract

La présente invention résout le problème de la fourniture d'un anticorps anti-cadhérine d'affinité élevée. La présente invention concerne un anticorps anti-cadhérine qui reconnaît le domaine 1 de la cadhérine (EC1) ou le domaine 2 de la cadhérine (EC2) de la cadhérine et a une valeur de Kd, qui est la concentration d'anticorps qui confère 50% de liaison avec l'antigène, de 10 nM ou moins.
PCT/JP2011/074934 2010-10-29 2011-10-28 Anticorps anti-cdh3 d'affinité élevée WO2012057315A1 (fr)

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JP2018512860A (ja) * 2015-04-17 2018-05-24 アムゲン リサーチ （ミュンヘン） ゲーエムベーハーＡＭＧＥＮ Ｒｅｓｅａｒｃｈ（Ｍｕｎｉｃｈ）ＧｍｂＨ Ｃｄｈ３及びｃｄ３に対する二重特異性抗体構築物
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JPWO2014126198A1 (ja) * 2013-02-15 2017-02-02 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
US9644028B2 (en) 2013-02-15 2017-05-09 Perseus Proteomics Inc. Anti-CDH3 humanized antibody, drug conjugate thereof, and use thereof
US9884921B2 (en) 2014-07-01 2018-02-06 Pfizer Inc. Bispecific heterodimeric diabodies and uses thereof
US10005836B2 (en) 2014-11-14 2018-06-26 Novartis Ag Antibody drug conjugates
US10626172B2 (en) 2014-11-14 2020-04-21 Novartis Ag Antibody drug conjugates
JP2018512860A (ja) * 2015-04-17 2018-05-24 アムゲン リサーチ （ミュンヘン） ゲーエムベーハーＡＭＧＥＮ Ｒｅｓｅａｒｃｈ（Ｍｕｎｉｃｈ）ＧｍｂＨ Ｃｄｈ３及びｃｄ３に対する二重特異性抗体構築物
US11028171B2 (en) 2015-04-17 2021-06-08 Amgen Research (Munich) Gmbh Bispecific antibody constructs for CDH3 and CD3
US11926666B2 (en) 2015-04-17 2024-03-12 Amgen Research (Munich) Gmbh Bispecific antibody constructs for CDH3 and CD3
WO2018159808A1 (fr) * 2017-03-03 2018-09-07 国立研究開発法人国立がん研究センター Conjugué d'anticorps-médicament de l'anticorps anti-il-7r, et composition pharmaceutique de traitement du cancer ou de l'inflammation et contenant un conjugué d'anticorps-médicament de l'anticorps anti-il-7r et d'un médicament cytotoxique
JPWO2018159808A1 (ja) * 2017-03-03 2019-12-26 国立研究開発法人国立がん研究センター 抗ｉｌ−７ｒ抗体の抗体薬物コンジュゲートと、がんまたは炎症を処置することに用いるための、抗ｉｌ−７ｒ抗体と細胞傷害剤との抗体薬物コンジュゲートを含む医薬組成物

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