WO2018066585A1 - Fzd10に対するモノクローナル抗体およびその使用 - Google Patents
Fzd10に対するモノクローナル抗体およびその使用 Download PDFInfo
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- WO2018066585A1 WO2018066585A1 PCT/JP2017/036081 JP2017036081W WO2018066585A1 WO 2018066585 A1 WO2018066585 A1 WO 2018066585A1 JP 2017036081 W JP2017036081 W JP 2017036081W WO 2018066585 A1 WO2018066585 A1 WO 2018066585A1
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
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- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Definitions
- the present invention relates to a monoclonal antibody against FZD10, a method for diagnosing FZD10-related diseases using the antibody, a method for detecting FZD10 protein, a method for determining the efficacy after treatment with an FZD10 inhibitor, and a high therapeutic effect with an FZD10 inhibitor
- the present invention relates to a method for screening a subject and a diagnostic reagent containing the antibody.
- Frizzled proteins are a family of G protein-coupled receptors that have binding sites for Wnt protein ligands. Genetic analysis has so far identified 18 Wnt genes and 10 Frizzled genes (FZD1 to FZD10), all of which are highly structurally similar. Frizzled protein is a seven-transmembrane protein with an extracellular cysteine-rich domain at the N-terminus. This cysteine-rich domain is the Wnt ligand binding site. The binding between the Wnt ligand and the Frizzled receptor is not necessarily one-to-one, and it has been confirmed that one type of Wnt ligand binds to multiple types of Frizzled receptors and one type of Frizzled receptor binds to multiple types of Wnt ligands.
- Wnt signaling pathway is activated by the binding of Wnt ligand and Frizzled receptor.
- Wnt signaling pathways There are several Wnt signaling pathways that activate the ⁇ -catenin pathway and several pathways that do not mediate ⁇ -catenin, and it is thought that different pathways are activated by combinations of Wnt ligands and Frizzled receptors. ing.
- the Wnt / ⁇ -catenin signaling pathway activated by receptor binding is mediated by Dishevelled (Dsh), a cytoplasmic protein that interacts directly with the Frizzled receptor, to stabilize and accumulate ⁇ -catenin in the cytoplasm Bring.
- Dsh Dishevelled
- ⁇ -catenin localizes to a cytoplasmic degradation complex that includes the tumor suppressor proteins colon adenomatous polyposis (APC) and auxin. These proteins serve as critical scaffolds for glycogen synthase kinase (GSK) -3 ⁇ to bind to and phosphorylate ⁇ -catenin, which is designated for degradation via the ubiquitin / proteasome pathway.
- APC tumor suppressor proteins colon adenomatous polyposis
- auxin serve as critical scaffolds for glycogen synthase kinase (GSK) -3 ⁇ to bind to and phosphorylate ⁇ -catenin, which is designated for degradation via the ubiquitin / proteasome pathway.
- Patent Document 1 ⁇ -catenin-independent pathways have been shown to be involved in a number of processes, but are involved in intracellular planar polarity (PCP), cell motility and adhesion involved in the control of the cytoskeletal system
- PCP planar polarity
- Wnt / Ca 2+ pathways involved in the control of myogenesis via protein kinase A, and the like.
- the Frizzled receptor can be dimerized, and it has been reported that this dimerization is involved in the activation of the Wnt signal pathway (Non-patent Document 1).
- FZD10 (reference sequence: Genbank accession number NM_007197.3 (SEQ ID NO: 21)) mRNA is found in many cancer cell lines, including cervical, gastrointestinal and glioblastoma cell lines, and approximately 40% There are reports that it is up-regulated in primary gastric cancer, primary colon cancer and most synovial sarcoma tissues (Patent Documents 1 and 2, Non-Patent Documents 2 and 3).
- Diseases associated with overexpression of FZD10 protein include synovial sarcoma, colorectal cancer (colon cancer), gastric cancer, chronic myelogenous leukemia (CML), and acute myeloid leukemia (AML) (Patent Literature) 3-5).
- FZD10 is considered to be a suitable target for anticancer drugs, and the growth of synovial sarcoma cells is suppressed by siRNA specific for FZD10, and the antibody against FZD10 is a mouse graft model of synovial sarcoma. Have antitumor activity (Patent Documents 3 and 4).
- monoclonal antibodies against FZD10 can be expected to serve as diagnostic agents in therapy.
- monoclonal antibodies such as trastuzumab diagnostics, rituximab diagnostics, and bevacizumab diagnostics for breast cancer, malignant lymphoma, and colon cancer
- monoclonal antibodies against other molecular targets are under development and their diagnostic effects are under evaluation. These diagnostic agents are expected to lead to more effective treatments from the viewpoint of effective patient selection in therapeutic agents.
- Diagnostic agents in the treatment of tumors with molecularly targeted therapeutics are important to detect cellular proteins that are overexpressed in the majority of the target tumor and are not expressed in normal tissue or minimally expressed .
- it is difficult to specifically detect a protein expressed in a tumor with high sensitivity and it is also difficult to obtain an antibody against such a protein.
- several antibodies are commercially available for FZD10, which is considered a target for anticancer drugs.
- FZD10-expressing cells with a commercially available antibody obtained by the present inventors, a positive signal (false positive) was sometimes generated even in cells with a low expression level of FZD10.
- an object of the present invention is to provide an antibody that binds to FZD10 specifically and with high sensitivity.
- the present inventors searched for an antibody that specifically binds to FZD10 from monoclonal antibodies obtained by immunizing mice with FZD10 antigen, and the specific antibody specifically binds to recombinant human FZD10 protein.
- the present invention relates to: [1] CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1; CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2; and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3
- a heavy chain variable region comprising, and CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4; CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5; and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6
- the heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 7 and the light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 8, or both, An antibody or antigen-binding fragment thereof.
- [3] The antibody or antigen-binding fragment thereof according to [1] or [2], which specifically recognizes a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 9.
- [5] The antibody or antigen-binding fragment thereof according to any one of [1] to [4], which is bound to an affinity label, an enzyme label, a radioisotope label, or a fluorescent label.
- [7] comprising the antibody or antigen-binding fragment thereof according to any one of [1] to [5], diagnosing an FZD10-related disease, determining efficacy after treatment with an FZD10 inhibitor, or therapeutic effect with an FZD10 inhibitor Reagents for screening high subjects.
- a method for diagnosing an FZD10-related disease or a predisposition for developing the disease in a subject comprising the following steps: (a) contacting the sample isolated from the subject with the antibody or antigen-binding fragment thereof according to any one of [1] to [5]; (b) detecting FZD10 protein in the sample by detecting binding of the antibody or antigen-binding fragment thereof to the sample: and (c) comparing the FZD10 protein level in the sample to a control, wherein the subject is suffering from or at risk of developing the disease when the FZD10 protein level is high compared to the control. Stage.
- the cancer is selected from the group consisting of synovial sarcoma, lung cancer, esophageal cancer, colorectal cancer (colon cancer), gastric cancer, chronic myeloid leukemia (CML) and acute myeloid leukemia (AML)
- CML chronic myeloid leukemia
- AML acute myeloid leukemia
- a method for detecting FZD10 protein in a sample comprising the following steps: (a) contacting a sample isolated from a subject with the antibody or antigen-binding fragment thereof according to any one of [1] to [5]; and (b) detecting FZD10 protein in the sample by detecting binding of the antibody or antigen-binding fragment thereof to the sample.
- a method for determining efficacy after treatment with an FZD10 inhibitor in a subject comprising the following steps: (a) contacting the sample isolated from the subject with the antibody or antigen-binding fragment thereof according to any one of [1] to [5]; (b) detecting FZD10 protein in the sample by detecting binding of the antibody or antigen-binding fragment thereof to the sample; (c) comparing the FZD10 protein level in the sample with the expression level before drug administration, indicating that the subject had a medicinal effect when the FZD10 protein level was low compared to before drug administration , Stage.
- a method for screening a subject having a high therapeutic effect by an FZD10 inhibitor comprising the following steps: (a) contacting the sample isolated from the subject with the antibody or antigen-binding fragment thereof according to any one of [1] to [5]; (b) detecting FZD10 protein in the sample by detecting binding of the antibody or antigen-binding fragment to the sample; (c) comparing the FZD10 protein level in the sample with a control, wherein the FZD10 inhibitor has a higher therapeutic effect in the subject when the FZD10 protein level is comparable or higher than the control It is shown that the stage.
- a method for producing an antibody capable of binding to FZD10 protein or a partial peptide thereof comprising the following steps: (a) culturing a cell containing a vector into which the polynucleotide according to [6] is introduced; and (b) recovering the antibody from the cell culture or culture medium.
- the invention further relates to: [16] A method for detecting a diagnostic marker for an FZD10-related disease or a predisposition to develop the disease, comprising the following steps: (a) contacting a sample isolated from the subject with the antibody or antigen-binding fragment thereof according to any one of [1] to [5]; and (b) detecting FZD10 protein in the sample as the marker by detecting binding of the antibody or antigen-binding fragment to the sample. [17] The antibody or antigen-binding fragment thereof according to any one of [1] to [5] for use in diagnosis of an FZD10-related disease or a predisposition to developing the disease.
- [18] Use of the antibody or antigen-binding fragment thereof according to any one of [1] to [5] in the manufacture of a reagent for diagnosing a FZD10-related disease or a predisposition to the disease.
- a method for detecting a drug efficacy marker of an FZD10 inhibitor comprising the following steps: (a) contacting a sample isolated from the subject with the antibody or antigen-binding fragment thereof according to any one of [1] to [5]; and (b) detecting FZD10 protein in the sample as the marker by detecting binding of the antibody or antigen-binding fragment to the sample.
- a method for detecting an FZD10 inhibitor treatment responsive marker comprising the following steps: (a) contacting a sample isolated from the subject with the antibody or antigen-binding fragment thereof according to any one of [1] to [5]; and (b) detecting FZD10 protein in the sample as the marker by detecting binding of the antibody or antigen-binding fragment to the sample.
- FIG. 1 is a diagram showing the expression of FZD10 mRNA in each cancer cell line in Cancer Line Encyclopedia (CCLE), which is an mRNA expression analysis database using cell lines for each cancer type.
- FIG. 2 is a photomicrograph showing the specificity of the anti-FZD10 antibody (10A8H4G4) in immunohistochemical staining using the FZD10 forced expression system cell line.
- an anti-FZD10 antibody (10A8H4G4) specific staining was observed in a paraffin section prepared from the FZD10 forced expression cell line (FZD10 / DLD1), but an Empty vector was introduced as a negative control. It was not stained with the cell line (Mock / DLD1).
- FZD10 (L164) Ab, FZD10PolyclonalAb
- staining was observed in both FZD10 / DLD1 and Mock / DLD1 as a result of immunohistochemical staining.
- the specificity of the anti-FZD10 antibody (10A8H4G4) was revealed compared with the commercially available antibody.
- the graph below the photo shows the ratio (%) of FZD10 positive cells in the number of forced expression cells based on the results of immunohistochemical staining.
- FIG. 3 shows the correlation between expression in flow cytometry and immunohistochemical staining using various cell lines with different expression levels of FZD10.
- A A histogram showing the results of flow cytometry using 10A8H4G4 in which the expression of FZD10 on the cell membrane of each cell line was detected.
- SYO-1, T.T, and H727 were FZD10 expressing cell lines, and LoVo was a FZD10 non-expressing cell line.
- B Photomicrograph showing the results of immunohistochemical staining using paraffin sections prepared from each cell line. In addition, the graph below the photograph shows the ratio (%) of FZD10-positive cells in each cell line calculated from the results of immunohistochemical staining. Staining was observed in FZD10-expressing cell lines such as SYO-1, T.T, and H727, but not in LoVo (non-expressing cell line).
- FIG. 4 shows the correlation between immunohistochemical staining and expression in RealTime-PCR using paraffin sections prepared from mouse Xenograft tumors of SYO-1 and COLO201 cell lines and lung cancer clinical specimens 1-6.
- the ratio of the number of FZD10-positive cells in the tumor cells was calculated from the results of immunohistochemical staining of B: A and shown in the graph.
- C The results correlated with immunohistochemical staining were obtained by the expression analysis of the same case by RealTime-PCR. Analysis using clinical specimens revealed high specificity of the anti-FZD10 antibody (10A8H4G4) of the present invention in the immunohistochemical staining method.
- the present invention provides an anti-FZD10 monoclonal antibody capable of specifically binding to FZD10 protein or a partial peptide thereof.
- the present invention provides evidence that the anti-FZD10 monoclonal antibody of the present invention has high specificity in the detection of FZD10 protein in immunohistochemical staining methods.
- the anti-FZD10 antibody (10A8H4G4) of the present invention has the following amino acid sequence in at least the variable region.
- 10A8H4G4G4, light chain variable region amino acid sequence (excluding signal sequence): DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRKSGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPVTFGAGTKLELKRAD SEQ ID NO: 8
- the antibody of the present invention can also be produced by a recombinant technique using DNA
- the antibody of the present invention was obtained by screening and selecting an antibody having binding ability to FZD10 by ELISA from a plurality of antibody-producing hybridomas obtained by immunization of mice.
- the antibodies selected by ELISA were further selected by immunostaining.
- Antibodies that show negative results were selected.
- an antibody having a weak binding force is attached as a background.
- the antibody of the present invention specifically binds to FZD10. Therefore, the antibody of the present invention is useful as a tool for detecting FZD10 or a cell or tissue expressing FZD10. Further, the antibody of the present invention can be used as a label bound to a label capable of detecting the antibody, and the label is, for example, a cancer cell or cancer expressing FZD10 such as colorectal cancer. It is more preferable in detecting the tissue.
- the label that binds to the antibody of the present invention may be any label that can detect an antibody bound to FZD10, such as an affinity label (eg, biotin, avidin, etc.), an enzyme label (eg, horseradish peroxidase, alkaline phosphatase, etc.) And fluorescent labels (for example, FITC, rhodamine and the like).
- an affinity label eg, biotin, avidin, etc.
- an enzyme label eg, horseradish peroxidase, alkaline phosphatase, etc.
- fluorescent labels for example, FITC, rhodamine and the like.
- the antibody of the present invention when used as a diagnostic agent for selection of cancer treatment patients, the antibody of the present invention can be used as it is, but it can be made into a composition suitable for various usage forms.
- an isolated or purified antibody refers to an antibody that is substantially free of other cellular material, such as carbohydrates, lipids, and other contaminating proteins, from the cell or tissue source from which the antibody is derived.
- the antibodies of the invention are isolated or purified.
- polypeptide and “protein” are used interchangeably herein and refer to a polymer of amino acid residues. This term applies to non-natural amino acid polymers containing one or more non-natural amino acid residues as well as natural amino acid polymers. Non-natural amino acids include amino acid analogs and amino acid mimetics.
- polynucleotide oligonucleotide
- nucleic acid refers to a polymer of nucleotides.
- FZD10-related disease is a cancer that expresses FZD10.
- cancer refers to cancers that express FZD10, preferably those that overexpress the FZD10 gene, such as synovial sarcoma, lung cancer, esophageal cancer, colon Examples include, but are not limited to, rectal cancer (colon cancer), gastric cancer, chronic myeloid leukemia (CML), and acute myeloid leukemia (AML).
- rectal cancer colon cancer
- CML chronic myeloid leukemia
- AML acute myeloid leukemia
- cancer cells expressing FZD10 from mRNA expression analysis database “Cancer Cell Cell Line Encyclopedia” (CCLE) using a cell line for each cancer type. Also in this application, remarkable up-regulation was confirmed in lung cancer, esophageal cancer, and colorectal cancer (colorectal cancer) (FIG. 1).
- antibody is intended to include immunoglobulins and fragments thereof that have specific reactivity to the designated protein or peptide thereof.
- Antibodies can include antibodies fused with other proteins or labels, and antibody fragments.
- antibodies are used herein in the broadest sense, specifically formed from intact monoclonal antibodies, polyclonal antibodies, at least two intact antibodies as long as they exhibit the desired biological activity. Multispecific antibodies (eg, bispecific antibodies), and antibody fragments are covered.
- Antibody refers to all classes (eg, IgA, IgD, IgE, IgG, and IgM).
- antibody fragment is a portion of an intact antibody and generally includes one or more antigen binding or variable regions of the intact antibody.
- antibody fragments can include one or more antigen binding portions of an intact antibody.
- the term “antigen-binding portion” or “antigen-binding fragment” of an antibody refers to one or more immunology of an antibody that retains the ability to specifically bind to an antigen (eg, FZD10). Active fragment. It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antibody fragments include Fab, Fab ′, F (ab ′) 2 , and Fv fragments, linear antibodies, and single chain antibody molecules.
- an antibody fragment binds with the same antigen that is recognized by the intact antibody.
- antibody fragment also refers to a synthetic or genetically engineered polypeptide that binds to a specific antigen, eg, a polypeptide comprising a light chain variable region, an “Fv” fragment comprising heavy and light chain variable regions, a light chain It also includes a recombinant single-chain polypeptide molecule ("scFv protein") in which the variable regions of the chain and heavy chain are linked by a peptide linker, as well as a minimal recognition unit consisting of amino acid residues that mimic the hypervariable region.
- scFv protein single-chain polypeptide molecule
- an FZD10 protein and an antibody can be evaluated by, for example, competition between antibodies.
- an antibody of the present invention for example, an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and an antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8 as the reference antibody.
- the specificity of the candidate antibody can be evaluated.
- a representative reference antibody is 10A8H4G4.
- the amount of antibody that binds to FZD10 protein in the absence of the candidate antibody is 10%, 20%, 30%, or 40% when the reference antibody and FZD10 protein are reacted in the presence of the candidate antibody.
- the binding of the reference antibody is inhibited by 50% or more, it can be determined that competition between the antibodies has occurred.
- a partial peptide thereof can be used as long as the reference antibody binds.
- a preferred partial peptide is the N-terminal extracellular domain peptide of FZD10 protein, for example, a partial peptide comprising the amino acid sequence of SEQ ID NO: 9.
- an anti-FZD10 monoclonal antibody is used. Such antibodies are provided by methods well known in the art. Exemplary antibody production techniques used by the present invention are described below.
- Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies that make up the population are identical except for natural mutations that may be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture with separate antibodies. For example, monoclonal antibodies can be produced using the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or can also be produced by recombinant DNA methods (US 4,816,567). sell.
- mice or other suitable host animals can be immunized with FZD10 polypeptide (FZD10 protein or a partial polypeptide thereof) to produce or produce antibodies that specifically bind to FZD10 polypeptide.
- FZD10 polypeptide FZD10 protein or a partial polypeptide thereof
- lymphocytes may be immunized with FZD10 polypeptide in vitro.
- the lymphocytes are then fused with myeloma cells using a suitable fusing agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)) .
- the prepared hybridoma cells are seeded in an appropriate culture medium that preferably contains one or more substances that inhibit the growth or survival of unfused parent myeloma cells, and grown in that medium.
- an appropriate culture medium that preferably contains one or more substances that inhibit the growth or survival of unfused parent myeloma cells, and grown in that medium.
- the culture medium for hybridomas can typically include hypoxanthine, aminopterin, and thymidine. (HAT medium), these substances interfere with the growth of HGPRT-deficient cells.
- Preferred myeloma cells are those that fuse efficiently, assist stable high level antibody production by selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
- Preferred myeloma cell lines include mouse myeloma lines such as those derived from MOPC-21 and MPC-11 mouse tumors available from Salk Institute Cell Distribution Center, San Diego, California USA, and American Type Culture Collection, Manassas , SP-2 cells or X63-Ag8-653 cells available from Virginia, USA.
- Human myeloma cell lines and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 300 1 (1984); Brodeur et al., OcMonoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
- Culture medium in which hybridoma cells are growing is analyzed for production of monoclonal antibodies directed against the antigen.
- the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
- RIA radioimmunoassay
- ELISA enzyme-linked immunosorbent assay
- the binding affinity of a monoclonal antibody can be determined, for example, by the 30 Scatchard analysis of Munson et al., Anal. Biochem., 107: 220 (1980).
- hybridoma cells After identifying hybridoma cells producing antibodies of the desired specificity, affinity, and / or activity, the clones can be subcloned by limiting dilution and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Examples of culture media suitable for this purpose include D-MEM medium or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo, such as ascites tumors in animals.
- the monoclonal antibody secreted by the subclone may be purified to homogeneity.
- separation and purification of antibodies can be performed according to separation and purification methods used for general proteins. For example, but not limited to, appropriately selecting and combining the use of column chromatography such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, and isoelectric focusing
- column chromatography such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, and isoelectric focusing
- proteins can be appropriately separated and isolated from culture medium, ascites fluid, or serum (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)).
- Protein A and protein G columns can be used as affinity columns.
- Exemplary protein A columns to be used include, for example, Hyper®D, POROS, and Sepharose® F.F. (Pharmacia).
- Exemplary chromatography includes, in addition to affinity chromatography, for example, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography, etc. (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press (1996)). Chromatographic procedures can be performed by liquid phase chromatography such as HPLC and FPLC.
- the DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (eg, by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the murine antibody). It is determined. Hybridoma cells serve as a preferred source of such DNA. Once the DNA is isolated, it is placed in an expression vector which is then E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells that do not otherwise produce immunoglobulin proteins, or Transfection into host cells such as myeloma cells can achieve the synthesis of monoclonal antibodies in recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5: 256-262 (1993) and Pluckthun, Immunol. Revs., 130: 151- 188 (1992) is included.
- Another method for producing a specific antibody or antibody fragment that is reactive to FZD10 is an expression library encoding an immunoglobulin gene or a portion thereof expressed in bacteria, FZD10 protein or its Screening with partial peptides.
- complete Fab fragments, VH regions, and Fv regions can be expressed in bacteria using phage expression libraries. See, for example, Ward et al., Nature 341: 544-546 (1989); Huse et al., Science 246: 1275-1281 (1989); and McCafferty et al., Nature 348: 552-554 (1990).
- I want For example, by screening such a library using the FZD10 peptide, immunoglobulin fragments reactive to FZD10 can be identified.
- SCID-hu mice available from Genpharm) may be used to produce antibodies or fragments thereof.
- the antibody or antibody fragment can be isolated from an antibody phage library produced using the techniques described in McCafferty et al., Nature, 348: 552-554 (1990). Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J MoL BioL, 222: 581-597 (1991), on isolation of mouse and human antibodies using phage libraries Each is listed.
- the present invention provides antibodies suitable for diagnosis of FZD10-related diseases, determination of drug efficacy after treatment with FZD10 inhibitors, and screening of subjects with high therapeutic effects with FZD10 inhibitors.
- the present invention has succeeded in establishing a mouse monoclonal antibody clone (10A8H4G4) capable of detecting FZD10 protein with high specificity in immunohistochemical staining and flow cytometry.
- this antibody clone is used for immunohistochemical staining of lung cancer clinical specimens, positive findings are observed in lung cancer specimens that have been confirmed to have high FZD10 expression levels by RealTime-PCR, but low expression levels of FZD10 have been confirmed. Lung cancer specimens proved to be hardly stained.
- the amino acid sequences of the heavy chain variable region (H chain V region) and light chain variable region (L chain V region) of the anti-FZD10 mouse monoclonal antibody clone (10A8H4G4) of the present invention are shown in SEQ ID NO: 7 and 8, respectively. ing.
- the CDR (complementarity determining region) contained in the heavy chain variable region and the light chain variable region can be determined according to a method well known in the art. For example, by Kabat et al. (Kabat E. A. et al. (1991) Sequence of Proteins of Immunological Interest. 5th Edition) or Chothia et al. (Chothia et al. J. Mol. The described method is commonly used for CDR determination.
- the CDR1, 2, and 3 of the heavy chain variable region of the anti-FZD10 mouse monoclonal antibody clone (10A8H4G4) of the present invention determined by Kabat definition are shown in SEQ ID NO: 1, 2, and 3, respectively.
- the CDRs 1, 2, and 3 of the light chain variable region are shown in SEQ ID NO: 4, 5, and 6, respectively.
- the present invention provides an antibody or antigen-binding fragment thereof capable of binding to FZD10 protein or a partial peptide thereof, comprising either or both of a heavy chain variable region and a light chain variable region, wherein the heavy chain variable
- the area is CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1; CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2; and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3
- the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4; CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5; and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6
- An antibody or antigen-binding fragment thereof is provided.
- the partial peptide of FZD10 protein to which the antibody of the present invention binds is preferably the N-terminal extracellular domain peptide of FZD10 protein, for example, corresponding to positions 21-161 of FZD10 protein (SEQ ID NO: 22) It comprises an amino acid sequence (SEQ ID NO: 9) and consists of an amino acid sequence selected from SEQ ID NO: 22. More preferably, the partial peptide of FZD10 protein in the present invention can consist of the amino acid sequence of SEQ ID NO: 9.
- One example of a variable region is a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7.
- One example of a variable region is a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8.
- the invention includes either or both of a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8
- a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7
- a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8
- the antibody of the present invention can be prepared by a conventional method.
- an antibody can be prepared by incorporating a polynucleotide encoding an antibody polypeptide into an appropriate vector, introducing the vector into a host, and producing the antibody from the host.
- Vandamme, A. M. et al., Eur. J. Biochem. (1990) 192, 767-75 See, for example, Vandamme, A. M. et al., Eur. J. Biochem. (1990) 192, 767-75).
- the nucleic acid sequence of the polynucleotide encoding the variable region (V region) of the antibody of the present invention can be deduced from the amino acid sequence of the V region of the antibody of the present invention.
- the nucleic acid sequences encoding the heavy chain variable region (VH) and light chain variable region (VL) of the antibody clone of the present invention for example, the nucleic acid sequences shown in SEQ ID NO: 10 and 11 can be used, respectively. .
- Polynucleotide encoding the V region of the antibody of the present invention is a solid phase technology (Beaucage SL & Iyer RP, Tetrahedron (1992) 48, 2223-311; Matthes et al., EMBO J (1984) 3, 801-5) And can be synthesized based on the sequence information by a conventional method such as oligonucleotide synthesis technology (Jones et al. Nature (1986) 321, 522-5).
- a polynucleotide encoding the antibody V region is incorporated into an expression vector comprising a polynucleotide encoding the antibody constant (C) region.
- a polynucleotide encoding the antibody is incorporated into an expression vector so that the antibody gene can be expressed under the control of an expression control element (eg, enhancer, promoter).
- An expression vector is used to transform host cells to express the antibody.
- the polynucleotide encoding the antibody H chain and the polynucleotide encoding the L chain may be incorporated into separate expression vectors, and then the resulting recombinant expression vector is cotransformed into a host cell.
- both the polynucleotide encoding the antibody H chain and the polynucleotide encoding the L chain may be incorporated together into a single expression vector, and then the resulting recombinant expression vector is transformed into a host cell ( For example, International Publication No. 94/11523).
- the antibody gene can be expressed by a known method.
- a conventional useful promoter for expression in mammalian cells, a conventional useful promoter, the expressed antibody gene, and a poly (A) signal (located downstream from the 3 ′ end of the antibody gene) can be operably linked.
- a human cytomegalovirus immediate early promoter / enhancer system can be used as a useful promoter / enhancer system.
- promoter / enhancer systems such as those derived from viruses (eg, retroviruses, polyomaviruses, adenoviruses and simian virus 40 (SV40)), and those derived from mammalian cells (eg, human elongation factor 1 ⁇ ( HEF1 ⁇ )) can also be used for the expression of antibodies in the present invention.
- viruses eg, retroviruses, polyomaviruses, adenoviruses and simian virus 40 (SV40)
- HEF1 ⁇ human elongation factor 1 ⁇
- gene expression can be easily performed by the method of Mulligan et al. (Nature (1979) 277, 108-14.).
- HEF1 ⁇ promoter / enhancer system gene expression can be easily performed by the method of Mizushima et al. (Nucleic Acids Res. (1990) 18, 5322.).
- a conventional useful promoter a signal sequence for secreting the antibody of interest, and an antibody gene can be operably linked.
- the promoter lacZ promoter or araB promoter can be used.
- gene expression can be performed by the method of Ward et al. (Nature (1098) 341, 544-6 .; FASBE J. (1992) 6, 2422-7.), while the araB promoter. Is used, gene expression can be performed by the method of Better et al. (Science (1988) 240, 1041-3.).
- the pelB signal sequence (Lei, SP et al, J. Bacteriol. (1987) 169, 4379-83.) Can be used.
- the antibody secreted into the periplasmic space is isolated and then refolded so that the antibody assumes the proper conformation.
- a replication origin derived from a virus eg, SV40, polyoma virus, adenovirus, bovine papilloma virus (BPV)
- a virus eg, SV40, polyoma virus, adenovirus, bovine papilloma virus (BPV)
- expression vectors include aminoglycoside phosphotransferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene and dihydrofolate reductase (dhfr )
- a selection marker gene such as a gene may be further included.
- any expression system can be used, including eukaryotic and prokaryotic cell systems.
- Eukaryotic cells include established cell lines of animals (eg, mammals, insects, molds and fungi, yeast).
- Prokaryotic cells include bacterial cells such as E. coli cells.
- the antibody used in the present invention is preferably expressed in mammalian cells such as CHO cells, COS cells, myeloma cells, BHK cells, Vero cells, and HeLa cells.
- the transformed host cell is cultured in vitro or in vivo to produce the antibody of interest.
- the host cell can be cultured by any known method.
- the culture medium that can be used herein may be DMEM, MEM, RPMI-1640 or IMDM medium.
- the culture medium may contain a serum supplement such as fetal calf serum (FCS).
- transgenic animals can also be used as hosts for the production of recombinant antibodies.
- a fusion gene is prepared by inserting an antibody gene into a predetermined site of a gene encoding a protein (eg, ⁇ -casein) originally produced in animal breast milk.
- a DNA fragment containing the fusion gene into which the antibody gene has been introduced is injected into a non-human animal embryo, which is then introduced into a female animal.
- the female animal having the embryo in its body produces a transgenic non-human animal.
- the antibody of interest is secreted into the breast milk from the transgenic non-human animal or its progeny.
- an appropriate hormone can be administered to the transgenic animal (Ebert, K.M. et al, Bio / Technology (1994) 12, 699-702).
- the antibody expressed and produced as described above can be isolated from the body of the cell or host animal and purified. Isolation and purification of the antibody used in the present invention can be performed on an affinity column. Other methods conventionally used for antibody isolation and purification can also be used; therefore, the method is not particularly limited. For example, a variety of chromatography, filtration, ultrafiltration, salting out and dialysis can be used alone or in combination to isolate and purify the antibody of interest (Antibodies A Laboratory Manual. Ed. Harlow, David Lane, Cold Spring Harbor Laboratory, atory1988).
- Antibody fragments Various techniques have been developed for the production of antibody fragments. Previously, these fragments were obtained through proteolytic digestion of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) and Brennan et al., Science , 229: 81 (1985)). However, these fragments can now be produced directly by recombinant host cells. For example, antibody fragments can be isolated from antibody phage libraries. Alternatively, Fab′-SH fragments may be recovered directly from E. coli and chemically coupled to form F (ab ′) 2 fragments (Carter et al., Bio / Technology 10: 163-167 ( 1992)).
- F (ab ′) 2 fragments can be isolated directly from recombinant host cell culture.
- the optimal antibody is a single chain Fv fragment (scFv). See WO 93/16185; US 5,571,894; and US 5,587,458.
- the antibody fragment may also be a “linear antibody” as described in, for example, US Pat. No. 5,641,870. Such linear antibody fragments can have monospecificity or bispecificity.
- the antibody of the present invention is optionally conjugated with an affinity label, enzyme label, radioisotope label, fluorescent label, or chemiluminescent label.
- an affinity label for example, the presence of a detectable label that is present in a cancer tissue that expresses FZD10 makes it possible to determine the presence or absence of a cancer or tumor in the subject being diagnosed. Further, the spread of the disease can be determined by the localization of the label in the cancer tissue.
- Suitable labels for use include, for example, fluorescent labels such as fluorescein and rhodamine; and enzyme labels such as luciferase.
- the detectable / detectable label used is selected according to the imaging modality used.
- conjugates of label and antibody can be made using protocols and techniques known in the art.
- the antibody of the present invention may be bound to a desired label immediately before use, or may be provided as an antibody bound to the label.
- Conjugates of antibody and label are N-succinimidyl-3- (2-pyridylditriol) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate, iminothiolane (IT), imide Bifunctional derivatives of esters (eg dimethyl adipate HCL), active esters (eg disuccinimidyl suberate), aldehydes (eg glutaraldehyde), bis-azide compounds (eg bis (p-azidobenzoyl) hexanediamine), Bis-diazonium derivatives (eg bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (eg toluene 2,6-diisocyanate), and bis-active fluorine compounds (eg 1,5-difluoro-2,4-dinitrobenzene)
- SPDP N-succ
- a fusion protein comprising the antibody and a label can be made, eg, by recombinant techniques or peptide synthesis.
- a fusion protein include a fusion protein of a labeled protein such as ECFP, EYFP, or EGFP and an antibody.
- FZD10 is useful as a diagnostic marker for FZD10-related diseases and as a marker for evaluating the responsiveness to FZD10 inhibitors and the efficacy of the inhibitors in subjects suffering from the diseases. Therefore, the antibody of the present invention is a marker for diagnosing FZD10-related diseases such as cancer, for screening subjects with a high therapeutic effect by FZD10 inhibitors, or for determining the efficacy after treatment with FZD10 inhibitors. It can be used as a detection reagent.
- the present invention provides a method for diagnosing a FZD10-related disease or a predisposition for developing the disease in a subject by detecting FZD10 protein using the antibody of the present invention in a sample isolated from the subject, and a therapeutic effect of an FZD10 inhibitor A method for screening a subject having a high incidence and a method for determining the efficacy after treatment with an FZD10 inhibitor are provided.
- These methods include the following steps: (a) contacting a sample isolated from a subject with an antibody of the present invention or an antigen-binding fragment thereof; (b) detecting FZD10 protein in the sample by detecting binding of the antibody or antigen-binding fragment thereof to the sample: and (c) comparing the FZD10 protein level in the sample with a control.
- the sample is a cell or tissue isolated from the subject, preferably a tissue isolated from the subject. Therefore, typically, each method of the invention is performed in vitro on a sample isolated from a subject.
- Techniques for isolating tissues and cells from an object by techniques such as biopsy (biopsy) and blood collection are known.
- a biological sample that has been removed from a subject through a therapeutic medical procedure such as a surgical operation
- Cells and tissues isolated from the subject can be appropriately treated before contact with the antibody.
- a tissue sample generally obtained from a subject can be sectioned after freezing and further fixed with alcohol, formalin, or the like to obtain a sample for immunohistological analysis.
- a tissue sample, cultured cells or the like can be fixed with formalin and then embedded in paraffin to obtain a section for immunohistological analysis.
- the binding of the antibody of the present invention or the antigen-binding fragment thereof to a sample can be detected by methods known to those skilled in the art. More specifically, after contacting the antibody of the present invention with the sample, those that did not bind to FZD10 in the sample are removed by washing, and then the antibody remaining in the sample is detected, thereby detecting the antibody of the present invention. And binding of FZD10 protein in the sample can be detected. At this time, when the antibody is directly labeled, the presence of the antibody of the present invention bound to the FZD10 protein can be detected by detecting the label.
- the label is detectable such as an enzyme, a fluorescent substance, a luminescent substance, or particles
- these labels can be detected immediately.
- an affinity substance binding substance
- biotin affinity label
- the presence of the antibody can be captured by a binding partner such as labeled avidin.
- the antibody of the present invention can be detected with a binding reagent for the antibody.
- an antibody binding reagent an antibody against protein A or an antibody can be labeled and used to detect the antibody.
- step (c) compared to the control level (normal control level, preferably the expression level of FZD10 protein in a sample isolated from a healthy subject not suffering from FZD10-related disease)
- control level normal control level, preferably the expression level of FZD10 protein in a sample isolated from a healthy subject not suffering from FZD10-related disease
- a high FZD10 protein level indicates that the subject is suffering from or at risk of developing a FZD10-related disease.
- a control level preferably, an expression level of FZD10 protein in a tissue of a subject diagnosed with an FZD10-related disease.
- the FZD10 protein level is comparable or higher, it indicates that the therapeutic effect of the FZD10 inhibitor in the subject is high.
- a control level preferably, the expression level of FZD10 protein in a sample isolated from a subject before drug administration.
- a patient who has been shown to have an FZD10-related disease by the diagnostic method of the present invention is likely to be treated with an FZD10 inhibitor. Therefore, following the diagnostic method of the present invention, an FZD10 inhibitor can be administered to a patient who has been shown to have an FZD10-related disease. Alternatively, the FZD10 inhibitor can be administered to a patient who has been shown to have a high therapeutic effect of the FZD10 inhibitor via screening. Furthermore, even if a patient who has been administered an FZD10 inhibitor is shown to have a therapeutic effect of the inhibitor, the FZD10 inhibitor can be subsequently administered to the same patient.
- the present invention relates to a method for treating an FZD10-related disease, comprising the step of identifying any patient selected from the following group by the method of the present invention and administering an FZD10 inhibitor to the patient; A patient shown by the diagnostic method of the invention to have an FZD10-related disease; Patients who have been shown to be likely to benefit from FZD10 inhibitors; and Patients who have been treated with FZD10 inhibitors and have been shown to have therapeutic effects of the inhibitors.
- a known compound can be used as the FZD10 inhibitor administered to a patient.
- antibodies showing cytotoxic activity against FZD10-expressing cells and double-stranded RNA molecules that suppress FZD10 expression are also included in the FZD10 inhibitor.
- examples of such antibodies and double-stranded RNA molecules include antibodies against FZD10 disclosed in WO2005 / 004912 or WO2007 / 148417, and siRNA specific for FZD10 disclosed in WO2006 / 013733. it can.
- a control level measured from a biological sample known not to suffer from an FZD10-related disease is referred to as a “normal control level”. If the FZD10 protein level in the sample isolated from the subject is high compared to the normal control level, the subject can be diagnosed as having an FZD10-related disease to be treated.
- a control level measured from a biological sample known to suffer from an FZD10-related disease eg, cancerous
- a “disease control level eg, cancerous control level
- the subject can be diagnosed as having a high therapeutic effect with the FZD10 inhibitor. Also, if the FZD10 protein level in a sample isolated from a subject after treatment with an FZD10 inhibitor is lower than the subject's disease control level prior to drug administration, the treatment was effective, i.e., the subject was treated with an FZD10 inhibitor. It can be diagnosed that the therapeutic effect is high.
- normal cells (or tissues) obtained from an unaffected area (eg, non-cancerous area) of an organ having an FZD10-related disease (eg, cancer) to be treated are used as normal controls.
- the control level is a result obtained by analyzing pre-measured FZD10 protein levels in a sample from a subject with known disease state (eg, cancerous or non-cancerous). Based on this, it may be determined by a statistical method.
- the control level can be derived from a database of expression patterns from previously tested samples (cells or tissues). When the sample to be evaluated is a tissue sample, a sample derived from the same tissue is preferably used as the control sample.
- the FZD10 protein level in a biological sample may be compared to a plurality of control levels measured from a plurality of reference samples. It is preferred to use a control level measured from a reference sample derived from a tissue type similar to the tissue type of the subject-derived biological sample. Furthermore, it is preferred to use a reference value for FZD10 protein level in a population with known disease state.
- the reference value can be obtained by any method known in the art. For example, the range of the average value +/ ⁇ 2 S.D. or the average value +/ ⁇ 3 S.D. can be used as the reference value.
- the FZD10 protein level in the sample is 10%, 25%, or 50% higher than the control level, or more than 1.1, 1.5, 2.0, 5.0, 10.0, or more If it is higher, it can be considered high.
- the FZD10 protein level in the sample is, for example, 10%, 25%, or 50% lower than the control level, or greater than 1.1, 1.5, 2.0, 5.0, 10.0, or If it is lower, it can be regarded as low.
- the FZD10-related disease is a cancer that expresses FZD10.
- Cancers that express FZD10 are, for example, synovial sarcoma, lung cancer, esophageal cancer, colorectal cancer (colon cancer), gastric cancer, chronic myeloid leukemia (CML), and acute myeloid leukemia (AML) Although there is, it is not limited to these.
- the present invention is a method for detecting a diagnostic marker for an FZD10-related disease or a predisposition to developing the disease, wherein the FZD10 protein in a sample is detected using the antibody of the present invention or an antigen-binding fragment thereof
- a method comprising the step of detecting as FZD10 has been shown to have increased expression in certain cancer cells compared to normal tissues. Therefore, if the expression level of FZD10 can be specifically detected, it is useful as a diagnostic marker for the disease to which it is related.
- a diagnostic marker for an FZD10-related disease or predisposition to the disease is FZD10 in a sample isolated from a subject detected by binding to an antibody of the invention or an antigen-binding fragment thereof.
- the control level is a normal control level, preferably the expression level of FZD10 protein in a sample isolated from a healthy subject not suffering from an FZD10-related disease.
- the control level is preferably the expression level in the same tissue as the tissue from which the cancer cells to be detected for the diagnostic marker are derived.
- the invention also provides an antibody of the invention or an antigen-binding fragment thereof for use in diagnosing an FZD10-related disease or a predisposition to the disease.
- the present invention provides the use of an antibody of the present invention or an antigen-binding fragment thereof in the manufacture of a reagent for diagnosing an FZD10-related disease or a predisposition for developing the disease.
- the present invention provides a method for detecting an FZD10 inhibitor treatment responsive marker, comprising the step of detecting FZD10 protein in a sample as the responsive marker using the antibody of the present invention or an antigen-binding fragment thereof.
- a method of including is provided.
- the expression of FZD10 is specifically enhanced in certain types of cancer cells, and it has been clarified that the proliferation of such cancer cells is suppressed by FZD10 inhibitors (WO2005 / 004912, WO2006 / 013733, WO2007 / 148417). That is, responsiveness to FZD10 inhibitors can be predicted using FZD10 expression as an index.
- an FZD10 inhibitor treatment responsive marker is an FZD10 protein in a sample isolated from a subject that is detected by binding to an antibody of the present invention or an antigen-binding fragment thereof, When the expression level is comparable or higher than the control level, the subject is shown to have a high therapeutic effect by the FZD10 inhibitor.
- the control level is preferably a disease control level, i.e.
- the present invention also provides an antibody of the present invention or an antigen-binding fragment thereof for use in screening a subject having a high therapeutic effect with an FZD10 inhibitor.
- the present invention provides the use of an antibody of the present invention or an antigen-binding fragment thereof in the manufacture of a reagent for screening a subject with a high therapeutic effect by an FZD10 inhibitor.
- the present invention further provides a method for detecting a drug efficacy marker of an FZD10 inhibitor, the method comprising the step of detecting FZD10 protein in a sample as the drug efficacy marker using the antibody of the present invention or an antigen-binding fragment thereof.
- the expression of FZD10 is specifically enhanced in certain types of cancer cells, and it has been clarified that the proliferation of such cancer cells is suppressed by FZD10 inhibitors (WO2005 / 004912, WO2006 / 013733, WO2007 / 148417).
- FZD10 inhibitors WO2005 / 004912, WO2006 / 013733, WO2007 / 148417.
- cancerous tissue with such cancer cells can be reduced or killed by FZD10 inhibitors.
- the efficacy of the FZD10 inhibitor in a subject having such cancer cells can be evaluated using the expression level of FZD10 as an index. If the expression level of FZD10 in a sample isolated from tissue that had FZD10 positive cancer cells is reduced compared to the sample isolated before treatment with the FZD10 inhibitor, the FZD10 inhibitor This is because FZD10-positive cancer cells can be considered to have decreased. Therefore, if the expression level of FZD10 can be specifically detected, it is useful as a drug efficacy marker for FZD10 inhibitors.
- a FZD10 inhibitor drug efficacy marker is a sample in a sample isolated from a subject that has been administered an FZD10 inhibitor that is detected by binding to an antibody of the present invention or an antigen-binding fragment thereof.
- FZD10 protein characterized in that when the expression level is low compared to the control level, the subject was shown to have a medicinal effect of the FZD10 inhibitor.
- the control level is preferably the expression level of FZD10 protein in a sample isolated from the disease site of interest prior to drug administration.
- the present invention also provides an antibody of the present invention or an antigen-binding fragment thereof for use in determining the efficacy after treatment with an FZD10 inhibitor.
- the present invention provides the use of an antibody of the present invention or an antigen-binding fragment thereof in the manufacture of a reagent for determining drug efficacy after treatment with an FZD10 inhibitor.
- kits for diagnosis of FZD10-related diseases for diagnosis of FZD10-related diseases, screening of subjects with high therapeutic effects with FZD10 inhibitors, or determination of drug efficacy after treatment with FZD10 inhibitors
- the present invention relates to diagnosis of FZD10-related diseases, FZD10 inhibitors
- the present invention provides a reagent or kit for screening a subject having a high therapeutic effect by, or for determining a drug effect after treatment with an FZD10 inhibitor.
- these kits contain the antibody of the present invention or an antigen-binding fragment thereof as a detection reagent for FZD10 protein.
- an antibody for a diagnostic reagent or kit of the present invention can be labeled with a fluorescent material, a luminescent material, or a radioisotope.
- a fluorescent material e.g., a fluorescent material
- a luminescent material e.g., a fluorescent material
- a radioisotope e.g., a radioisotope
- the kit can contain a combination of the antibody of the present invention or an antigen-binding fragment thereof and another marker detection reagent.
- the kit can further include positive and negative control reagents for FZD10 and a secondary antibody for detecting the antibody of the present invention.
- a culture section or tissue sample of a cell line known to be highly expressing FZD10 can serve as a useful positive control reagent.
- tissue samples obtained from healthy subjects or non-cancerous tissues can serve as useful negative control reagents.
- the secondary antibody for detecting the antibody of the present invention is preferably labeled with a fluorescent substance, a luminescent substance, a radioisotope, or an enzyme.
- the kit of the present invention further includes buffers, diluents, filters, needles, syringes, and package inserts with instructions for use (eg, written, tape, CD-ROM, etc.) for commercial or user standpoints. May contain other materials desired. These reagents and the like can be held in a labeled container. Suitable containers include bottles, vials, and test tubes. The container can be formed from a variety of materials such as glass or plastic.
- FZD10 forced expression cell line (FZD10 / DLD1) in which the FZD10 expression vector was introduced into DLD1, a human colon cancer cell line purchased from ATCC, and an empty vector introduced as a negative control (Mock / DLD1) was produced.
- DLD1 a human colon cancer cell line purchased from ATCC
- Mock / DLD1 an empty vector introduced as a negative control
- SYO-1 a human synovial sarcoma cell line distributed by the National Cancer Center Rare Cancer Center, is 10% fetal bovine serum (FBS) and 1% at 37 ° C in a humidified atmosphere of 5% CO 2 Maintained in DMEM supplemented with penicillin / streptomycin.
- TT a human esophageal cancer cell line purchased from JCRB, is DMEM / F12 (1: 5) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37 ° C. in a humidified atmosphere of 5% CO 2. 1) maintained in.
- H727 a human lung cancer cell line purchased from ATCC, was maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37 ° C. in a humidified atmosphere of 5% CO 2 .
- COLO201 a human colon cancer cell line purchased from ATCC, is 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, 10 mM HEPES, 1 mM sodium pyruvate and 4.5 at 37 ° C. in a humidified atmosphere of 5% CO 2.
- LoVo a human colon cancer cell line purchased from ATCC, was maintained in F12 supplemented with 20% fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37 ° C. in a humidified atmosphere of CO 2 .
- FBS fetal bovine serum
- penicillin / streptomycin at 37 ° C. in a humidified atmosphere of CO 2 .
- Immunohistochemical staining Formalin-fixed paraffin-sectioned FZD10 / DLD1, Mock / DLD1, SYO1, TT, H727, LoVo, COLO201 and clinical specimens were immersed in xylene three times for 3 minutes and then deparaffinized, and then twice in 100% ethanol for 1 minute , 90%, 70%, and 50% ethanol for 1 minute each to rehydrate.
- Sections immersed in antigen activation solution pH9 (Nichirei Bioscience) for antigen activation treatment are FZD10 / DLD1, Mock / DLD1, 125 ° C for 30 seconds, SYO1, TT, H727, LoVo, COLO201, and 95 clinical specimens.
- FZD10Ab (10A8H4G4), FZD10 (L164) pAb, FZD10 PolyclonalAb were appropriately reduced in a wet box, allowed to stand for 60 minutes, and then washed 3 times with Wash Buffer for 5 minutes.
- An appropriate amount of Histofine Simple Stain MAX-PO was used as a secondary antibody in a wet box and allowed to stand for 30 minutes. Wash 3 times for 5 minutes with Wash Buffer. Color development reaction was performed with DAB substrate solution (Nichirei Bioscience). The slide section was immersed in hematoxylin (Dako) for 20 seconds and washed with running water.
- Dehydration was performed by immersion in 50%, 70%, and 90% ethanol for 1 minute each and twice in 100% ethanol for 1 minute each. Finally, the sections were immersed in xylene twice for 3 minutes to be clarified, and mounted by Mount-Quick (DAIDO SANGYO).
- RNeasy Mini kit QIAGEN
- the frozen tissue piece was soaked in TRIzol Reagent (Invitrogen) and ground and extracted with chloroform. An approximately equal amount of 70% ethanol was added to the obtained extract, and total RNA was extracted using RNeasy Mini kit (QIAGEN).
- CDNA was synthesized from total RNA using reverse transcriptase of SuperScript III Reverse Transcriptase (Invitrogen). PCR reaction was performed using KAPA SYBR FAST ABI Prism qPCR kit (KAPA Biosystems) using cDNA as a template.
- the target gene for expression analysis was FZD10, and the housekeeping gene was GAPDH (glyceraldehyde-3-phosphate dehydrogenase).
- FZD10 uses FZD10-11F: 5'-GTGTGCAGCCGTAGGTTAAAG-3 '(SEQ ID NO: 12), FZD10 R5: 5'-GACTGGGCAGGGATCTCATA-3' (SEQ ID NO: 13) primer set, GAPDH is GAPDH RT-PCR Fw: 5'-ACAACAGCCTCAAGATCATCAG-3 '(SEQ ID NO: 14), GAPDH RT-PCR Re: 5'-GGTCCACCACTGACACGTTG-3' (SEQ ID NO: 15) I did it.
- the amount of PCR amplification product was monitored over time, and a Ct value was calculated by setting a threshold in a region where the PCR amplification product was exponentially amplified.
- the concentration of the unknown sample was calculated by applying the Ct value to the calibration curve.
- FCM Flow cytometry
- Example 1 Production of anti-FZD10 monoclonal antibody (1) Acquisition of anti-FZD10 antibody-producing hybridoma using soluble FZD10 protein as an immunogen Since FZD10 is a membrane protein, it can be used to produce antibodies that react with immobilized membrane proteins. The extracellular region can be an immunogen. Since the extracellular region other than the N-terminus of FZD10 was very short and thought to be difficult to use as an antigen, the N-terminal extracellular region (1-161 amino acids) was selected as the immunogen, and the signal peptide Monoclonal antibodies were prepared using soluble FZD10 protein (21-161 amino acid region) (SEQ ID NO: 9) except for.
- mice After adding 50 ⁇ g of the antigenic peptide to Freund Adjuvant, it was emulsified and injected for the first time by subcutaneous injection into Balb / c mice (Japan SLC Co., Ltd.). The second and subsequent immunizations were carried out by injecting subcutaneously the equivalent amount of 25 ⁇ g antigen peptide prepared in the same manner. Three days after the final immunization, spleen cells were aseptically prepared from mice, and cell fusion with mouse myeloma cells SP2 / 0 was performed by the polyethylene glycol method according to a conventional method.
- Anti-FZD10 antibody was selected by first screening for an antibody having binding ability to FZD10 by ELISA using soluble FZD10 protein. The antibodies selected by ELISA were further selected by immunostaining. Selection by immunostaining was performed by immunohistochemical staining using FZD10 forced expression cell line FZD10 / DLD1 expressing full-length FZD10 protein (SEQ ID NO: 22). That is, FZD10 forced expression cell line FZD10 / DLD1 in which full-length FZD10 protein was forcibly expressed was sectioned with formalin-fixed paraffin and then immunostained to select hybridomas in which a strong reaction was observed.
- FIG. 2 shows the results of immunohistochemical staining for the hybridoma clone 10A8H4G4 that was confirmed to produce a high level of FZD10-specific antibody among the tested hybridomas.
- FIG. 2 shows the results of immunohistochemical staining for the hybridoma clone 10A8H4G4 that was confirmed to produce a high level of FZD10-specific antibody among the tested hybridomas.
- FIG. 2 shows the results of immunohistochemical staining for the hybridoma clone 10A8H4G4 that was confirmed to produce a high level of FZD10-specific antibody among the tested hybridomas.
- FIG. 2 shows the results of immunohistochemical staining for the hybridoma clone 10A8H4G4 that was confirmed to produce a high level of FZD10-specific antibody among the tested hybridomas.
- the hybridoma clone 10A8H4G4 is useful as a hybridoma producing an antibody that specifically detects FZD10 in immunohistochemical staining.
- staining was performed using commercially available antibodies (FZD10 (L164) Ab, FZD10 Polyclonal Ab), staining was observed in both FZD10 / DLD1 and Mock / DLD1 (a signal was detected in a high proportion of cells). Therefore, it was suspected that the detection results with these commercially available antibodies contained a false positive signal.
- the anti-FZD10 antibody (10A8H4G4) of the present invention has an advantageous property that FZD10 can be detected with high specificity in the immunohistochemical staining method as compared with a commercially available anti-FZD10 antibody. Became clear.
- This hybridoma clone 10A8H4G4 was selected to produce antibodies for further experiments.
- the hybridoma clone 10A8H4G4 was mass-cultured, and the culture solution was collected after 2 to 3 weeks.
- the antibody was purified from the culture using a protein A column (GE Healthcare, NJ).
- the antibody of the present invention is also referred to as clone 10A8H4G4.
- Example 2 Evaluation of specificity of anti-FZD10 monoclonal antibody
- 10A8H4G4 was evaluated using various cell lines with different expression levels of FZD10. That is, the results of immunohistochemical staining and flow cytometry using 10A8H4G4 were compared, and it was examined whether staining corresponding to the expression level of FZD10 protein was observed.
- CCLE cancer cell line encyclopedia
- an mRNA expression analysis database using cell lines for each cancer type marked up-regulation is shown in lung cancer, esophageal cancer, and colorectal cancer (colon cancer). ( Figure 1). Therefore, cell lines derived from these cancer types were used for the following analysis.
- endogenous FZD10 protein on the cell membrane of various human cancer tissue-derived cell lines was confirmed by flow cytometry using 10A8H4G4.
- 10A8H4G4 in addition to human synovial sarcoma-derived SYO-1, which is known to express endogenous FZD10 protein, in human esophageal cancer-derived cell line TT and human lung cancer-derived cell line H727 A positive signal was detected by flow cytometry using 10A8H4G4.
- no positive signal was detected by flow cytometry using 10A8H4G4 in the human colon cancer-derived cell line LoVo, which is known to be an FZD10 non-expressing cell line.
- paraffin sections prepared from these cell lines were subjected to immunohistochemical staining using 10A8H4G4.
- staining with 10A8H4G4 revealed staining in paraffin sections prepared from cell lines SYO-1, TT, and H727 that were confirmed to express FZD10. .
- a positive signal was detected in a high proportion of cells in correlation with the flow cytometry results.
- paraffin sections prepared from LoVo which is a non-cell line of FZD10, were hardly stained, and positive signals were hardly detected.
- the anti-FZD10 antibody (10A8H4G4) of the present invention is a useful tool capable of detecting FZD10 protein in a sample with high specificity in both flow cytometry and immunohistochemical staining. .
- Example 3 Detection of FZD10 protein in tumor transplantation models and clinical specimens Specificity in immunohistochemical staining of the anti-FZD10 antibody (10A8H4G4) of the present invention was determined using a paraffin section prepared from a mouse Xenograft tumor of a cancer cell line, and Evaluation was performed using clinical specimens of lung cancer. Paraffin sections prepared from mouse Xenograft tumors of SYO-1 and COLO201 cell lines and expression of FZD10 protein and RNA in tumor and non-tumor areas of lung cancer clinical specimens 1-6 by immunohistochemical staining and RealTime-PCR, respectively The detection results are shown in FIG. As shown in FIG.
- lung cancer clinical specimens 5 in which the expression of tumor region-specific FZD10 RNA was confirmed cells stained with 10A8H4G4, that is, FZD10-positive cells were detected, but tumor region-specific FZD10 RNA expression was detected.
- the other lung cancer specimens that were not confirmed were hardly stained and few FZD10 positive cells were detected. This result indicates that 10A8H4G4 can specifically detect FZD10 protein even in clinical specimens.
- Example 4 Analysis of amino acid sequence of variable region of anti-FZD10 monoclonal antibody
- the amino acid sequence of the variable region of the anti-FZD10 antibody (10A8H4G4) of the present invention was analyzed.
- Total RNA was extracted from hybridoma 10A8H4G4 using RNeasy mini kit (QIAGEN).
- CDNA was synthesized from total RNA using Super Script II Reverse Transcriptase (Invitrogen). Primers for cDNA synthesis are as follows.
- MIGCUniRv for heavy chain 3 'primer 5'-CTGGGAAGGTGTGCACAC-3 '(SEQ ID NO: 16)
- MIGKRv2 light chain 3 'primer 5'-GTTGTTCAAGAAGCACACGAC-3 '(SEQ ID NO: 17)
- a 5 'RACE System for Rapid Amplification of cDNA Ends (Invitrogen) is used to add a dC polymer to the cDNA end, and Platinum Taq DNA Polymerase High Fidelity (Invitrogen) is used to encode a polynucleotide encoding the variable region of a monoclonal antibody. Amplified. Primers for amplification are as follows.
- Base “I” in the primer sequence indicates inosine.
- 5 'RACE Abridged Anchor Primer for heavy chain 5' and light chain 5 'primers 5'-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3 '(SEQ ID NO: 18), MIGCUniRv2 for heavy chain 3 'primer: 5′-TGGACAGGGATCCAGAGTTCC-3 ′ (SEQ ID NO: 19), as well as light chain 3 ′ primer mIGKNesRv2: 5′-CAGATGTTAACTGCTCACTGGATGG-3 ′ (SEQ ID NO: 20).
- the PCR product was cloned into pGEM-T Easy Vector (Promega). The insert fragment region was sequenced, and the nucleic acid sequence of the variable region of 10A8H4G4 (excluding the signal sequence) was determined.
- amino acid sequence and nucleic acid sequence of the heavy chain variable region and light chain variable region of the mouse monoclonal antibody were determined as follows. 10A8H4G4, heavy chain variable region amino acid sequence (excluding signal sequence): QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGLGVSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITSVDTADTATYYCARRAYYGNYYALDYWGQGTSVTVSS (SEQ ID NO: 7 encoded by the nucleic acid sequence shown in SEQ ID NO: 10) 10A8H4G4, heavy chain variable region nucleic acid sequence: 5'-CAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCTGGTCTGGGTGTGAGCTGGATTCGTCAGCCTTCAGGAAAGGGTCTGGAGTGGCTGGCACACATTTACTGGGATGATGACAAGCG
- the CDR sequence of the antibody of the present invention (10A8H4G4) determined by Kabat's definition is as follows.
- Heavy chain CDR1 (CDR-H1) TSGLGVS (SEQ ID NO: 1);
- Heavy chain CDR2 (CDR-H2) HIYWDDDKRYNPSLKS (SEQ ID NO: 2);
- Light chain CDR2 (CDR-L2) WASTRKS (SEQ ID NO: 5); and
- Light chain CDR3 (CDR-L3) QNDYSYPVT (SEQ ID NO: 6)
- the present invention has succeeded in producing an anti-FZD10 antibody capable of detecting FZD10 protein in a sample isolated from a subject such as a clinical specimen with high specificity.
- FZD10 antibody of the present invention FZD10 protein in a sample can be detected with high sensitivity and low background. Therefore, the antibody of the present invention is useful for diagnosis of FZD10-related diseases such as cancers that express FZD10.
- the antibody of the present invention can detect FZD10 according to the expression level of FZD10 in a sample, screening of subjects with high therapeutic effects by FZD10 inhibitors (pre-treatment diagnosis) and FZD10 inhibition in subjects It is also useful in the determination of drug efficacy after treatment with agents (post-treatment diagnosis).
Abstract
Description
Frizzledタンパクは7回膜貫通型タンパクであり、N末端に細胞外システインリッチドメインを有する。このシステインリッチドメインがWntリガンドの結合部位である。WntリガンドとFrizzled受容体との結合は必ずしも一対一ではなく、一種のWntリガンドが複数種のFrizzled受容体と、一種のFrizzled受容体に複数種のWntリガンドが結合することが確認されている。
受容体結合により活性化されたWnt/β-カテニンシグナル伝達経路は、Frizzled受容体と直接相互作用する細胞質タンパク質であるDishevelled(Dsh)によって仲介されて、β-カテニンの細胞質での安定化および蓄積をもたらす。Wntシグナルが存在しない場合、β-カテニンは腫瘍抑制タンパク質である大腸腺腫様ポリポーシス(APC)およびオーキシンを含む細胞質の分解複合体に局在する。これらのタンパク質はグリコーゲン合成酵素キナーゼ(GSK)-3βがβ-カテニンに結合してリン酸化し、これをユビキチン/プロテアソーム経路を介した分解用に指定するための重大な足場として機能する。(特許文献1)β-カテニン非依存経路は多数のプロセスに関係していることが示されているが、細胞骨格系の制御に関与する細胞内平面極性(PCP)、細胞の運動および接着に関与しているWnt/Ca2+経路、プロテインキナーゼAを介して筋新生の制御に関与する経路などが存在する。Frizzled受容体は2量体化することができ、この2量体化はWntシグナル経路の活性化に関与しているとの報告がある(非特許文献1)。
〔1〕SEQ ID NO:1に示されるアミノ酸配列を含むCDR1;
SEQ ID NO:2に示されるアミノ酸配列を含むCDR2;および
SEQ ID NO:3に示されるアミノ酸配列を含むCDR3
を含む重鎖可変領域、ならびに
SEQ ID NO:4に示されるアミノ酸配列を含むCDR1;
SEQ ID NO:5に示されるアミノ酸配列を含むCDR2;および
SEQ ID NO:6に示されるアミノ酸配列を含むCDR3
を含む軽鎖可変領域
のいずれかまたは両方を含む、FZD10タンパク質またはその部分ペプチドに結合することができる、抗体またはその抗原結合性断片。
〔2〕SEQ ID NO:7に示されるアミノ酸配列を含む重鎖可変領域およびSEQ ID NO:8に示されるアミノ酸配列を含む軽鎖可変領域のいずれかまたは両方を含む、〔1〕に記載の抗体またはその抗原結合性断片。
〔3〕SEQ ID NO:9に示されるアミノ酸配列からなるポリペプチドを特異的に認識する、〔1〕または〔2〕に記載の抗体またはその抗原結合性断片。
〔4〕FZD10への特異的な結合について、〔1〕~〔3〕のいずれかに記載の抗体と競合する、抗体またはその抗原結合性断片。
〔5〕アフィニティー標識、酵素標識、放射性同位体標識、または蛍光標識と結合している、〔1〕~〔4〕のいずれかに記載の抗体またはその抗原結合性断片。
〔6〕〔1〕~〔5〕のいずれかに記載の抗体またはその抗原結合性断片をコードするポリヌクレオチド。
〔7〕〔1〕~〔5〕のいずれかに記載の抗体またはその抗原結合性断片を含む、FZD10関連疾患を診断、FZD10阻害剤による治療後の薬効を判定、またはFZD10阻害剤による治療効果の高い対象をスクリーニングするための試薬。
〔8〕以下の段階を含む、対象におけるFZD10関連疾患または該疾患の発症素因を診断する方法:
(a) 該対象から単離した試料を〔1〕~〔5〕のいずれかに記載の抗体またはその抗原結合性断片と接触させる段階;
(b) 該抗体またはその抗原結合性断片と該試料との結合を検出することによって、該試料中のFZD10タンパク質を検出する段階:および
(c) 該試料中のFZD10タンパク質レベルを対照と比較する段階であって、対照と比較してFZD10タンパク質レベルが高いときに該対象が該疾患を患っているかまたはその発症リスクを有することが示される、段階。
〔9〕前記FZD10関連疾患が、FZD10を発現しているがんである、〔7〕に記載の試薬または〔8〕に記載の方法。
〔10〕前記がんが、滑膜肉腫、肺がん、食道がん、結腸直腸がん(大腸がん)、胃がん、慢性骨髄性白血病(CML)および急性骨髄性白血病(AML)からなる群より選択される、〔9〕に記載の試薬または方法。
〔11〕以下の段階を含む、試料中のFZD10タンパク質を検出する方法:
(a) 対象から単離した試料を〔1〕~〔5〕のいずれかに記載の抗体またはその抗原結合性断片と接触させる段階;および
(b) 該抗体またはその抗原結合性断片と該試料との結合を検出することによって、該試料中のFZD10タンパク質を検出する段階。
〔12〕以下の段階を含む、対象におけるFZD10阻害剤による治療後の薬効を判定する方法:
(a) 該対象から単離した試料を〔1〕~〔5〕のいずれかに記載の抗体またはその抗原結合性断片と接触させる段階;
(b) 該抗体またはその抗原結合性断片と該試料との結合を検出することによって、該試料中のFZD10タンパク質を検出する段階;
(c) 該試料中のFZD10タンパク質レベルを薬剤投与前の発現レベルと比較する段階であって、薬剤投与前と比較してFZD10タンパク質レベルが低いときに該対象における薬効があったことが示される、段階。
〔13〕以下の段階を含む、FZD10阻害剤による治療効果の高い対象をスクリーニングする方法:
(a) 該対象から単離した試料を〔1〕~〔5〕のいずれかに記載の抗体またはその抗原結合性断片と接触させる段階;
(b) 該抗体または抗原結合性断片と該試料との結合を検出することによって、該試料中のFZD10タンパク質を検出する段階;
(c) 該試料中のFZD10タンパク質レベルを対照と比較する段階であって、対照と比較してFZD10タンパク質レベルが同程度かそれよりも高いときに、該対象におけるFZD10阻害剤による治療効果が高いことが示される、段階。
〔14〕前記試料が、前記対象から単離した細胞または組織である、〔8〕~〔13〕のいずれかに記載の方法。
〔15〕以下の工程を含む、FZD10タンパク質またはその部分ペプチドに結合することができる抗体を製造する方法:
(a) 〔6〕に記載のポリヌクレオチドが導入されたベクターを含む細胞を培養する段階;および
(b) 該細胞の培養物または培養培地から前記抗体を回収する段階。
〔16〕以下の段階を含む、FZD10関連疾患または該疾患の発症素因の診断マーカーを検出する方法:
(a) 該対象から単離した試料を〔1〕~〔5〕のいずれかに記載の抗体またはその抗原結合性断片と接触させる段階;および
(b) 該抗体または抗原結合性断片と該試料との結合を検出することによって、該試料中のFZD10タンパク質を該マーカーとして検出する段階。
〔17〕FZD10関連疾患または該疾患の発症素因の診断における使用のための、〔1〕~〔5〕のいずれかに記載の抗体またはその抗原結合性断片。
〔18〕FZD10関連疾患または該疾患の発症素因を診断するための試薬の製造における、〔1〕~〔5〕のいずれかに記載の抗体またはその抗原結合性断片の使用。
〔19〕以下の段階を含む、FZD10阻害剤の薬効マーカーを検出する方法:
(a) 該対象から単離した試料を〔1〕~〔5〕のいずれかに記載の抗体またはその抗原結合性断片と接触させる段階;および
(b) 該抗体または抗原結合性断片と該試料との結合を検出することによって、該試料中のFZD10タンパク質を該マーカーとして検出する段階。
〔20〕FZD10阻害剤による治療後の薬効の判定における使用のための、〔1〕~〔5〕のいずれかに記載の抗体またはその抗原結合性断片。
〔21〕FZD10阻害剤による治療後の薬効を判定するための試薬の製造における、〔1〕~〔5〕のいずれかに記載の抗体またはその抗原結合性断片の使用。
〔22〕以下の段階を含む、FZD10阻害剤治療応答性マーカーを検出する方法:
(a) 該対象から単離した試料を〔1〕~〔5〕のいずれかに記載の抗体またはその抗原結合性断片と接触させる段階;および
(b) 該抗体または抗原結合性断片と該試料との結合を検出することによって、該試料中のFZD10タンパク質を該マーカーとして検出する段階。
〔23〕FZD10阻害剤による治療効果の高い対象のスクリーニングにおける使用のための、〔1〕~〔5〕のいずれかに記載の抗体またはその抗原結合性断片。
〔24〕FZD10阻害剤による治療効果の高い対象をスクリーニングするための試薬の製造における、〔1〕~〔5〕のいずれかに記載の抗体またはその抗原結合性断片の使用。
本発明の態様を実施または試験するにあたって、本明細書に記載の方法および材料と類似のまたは同等の任意の方法および材料を用いることができるが、好ましい方法、装置、および材料をここに記載する。しかしながら、本発明の材料および方法について記載する前に、本明細書に記載の特定の大きさ、形状、寸法、材料、方法論、プロトコル等は慣例的な実験法および最適化に応じて変更可能であるため、本発明がこれらに限定されないことが理解されるべきである。本記載に使用する専門用語は特定の型または態様のみを説明する目的のためのものであり、添付の特許請求の範囲によってのみ限定される本発明の範囲を限定することは意図されないことも、また理解されるべきである。
10A8H4G4、重鎖可変領域アミノ酸配列(シグナル配列を除く):
QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGLGVSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITSVDTADTATYYCARRAYYGNYYALDYWGQGTSVTVSS(SEQ ID NO:7)
10A8H4G4、軽鎖可変領域アミノ酸配列(シグナル配列を除く):
DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRKSGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPVTFGAGTKLELKRAD(SEQ ID NO:8)
本発明の抗体は、前記アミノ酸配列をコードするDNAを用いる組み換え技術によって製造することもできる。
本明細書で用いる「1つの(a)」、「1つの(an)」および「その(the)」という単語は、他に特記されない限り「少なくとも1つの」を意味する。
物質(例えば、ペプチド、抗体、ポリヌクレオチド等)に関して用いる「単離された」および「精製された」という用語は、該物質がそうでなければ天然源中に含まれ得る少なくとも1種の物質を実質的に含まないことを示す。したがって、単離または精製された抗体は、その抗体が由来する細胞もしくは組織源からの他の細胞材料、例えば糖質、脂質および他の混入タンパク質を実質的に含まない抗体を指す。好ましい態様では、本発明の抗体は単離または精製されている。
本発明では、抗FZD10モノクローナル抗体を用いる。当該抗体は当技術分野において周知の方法によって提供される。
本発明によって用いられる例示的な抗体産生技術について以下に記載する。
モノクローナル抗体は実質的に均一な抗体の集団から得られる。すなわち、集団を構成する個々の抗体は、わずかな量で存在しうる可能性のある天然の変異を除き同一である。このように、「モノクローナル」という修飾語は、別個の抗体との混合物ではないという抗体の特徴を示す。
例えばモノクローナル抗体は、Kohler et al., Nature, 256: 495 (1975)によって最初に記載されたハイブリドーマ法を用いて産生することができ、または組換えDNA法(US4,816,567号)によっても産生されうる。
モノクローナル抗体の結合親和性は、例えば、Munson et al., Anal. Biochem., 107: 220 (1980)の30スキャッチャード分析によって判定することができる。
所望の特異性、親和性、および/または活性の抗体を産生するハイブリドーマ細胞を同定した後に、このクローンを限界希釈法によってサブクローニングし、標準的な方法によって増殖させることができる(Goding, Monoclonal Antibodies : Principles and Practice, pp. 59-103 (Academic Press, 1986))。この目的に適した培養培地の例として、D-MEM培地またはRPMI-1640培地が含まれる。さらに、ハイブリドーマ細胞は動物における腹水腫瘍のようなインビボで増殖させることもできる。
SEQ ID NO:1に示されるアミノ酸配列を含むCDR1;
SEQ ID NO:2に示されるアミノ酸配列を含むCDR2;および
SEQ ID NO:3に示されるアミノ酸配列を含むCDR3
を含み、該軽鎖可変領域は、
SEQ ID NO:4に示されるアミノ酸配列を含むCDR1;
SEQ ID NO:5に示されるアミノ酸配列を含むCDR2;および
SEQ ID NO:6に示されるアミノ酸配列を含むCDR3
を含む、抗体またはその抗原結合性断片を提供する。
本発明において用いられる抗体の産生のため、該抗体(抗体遺伝子)をコードするポリヌクレオチドを、発現制御要素(例えば、エンハンサー、プロモーター)の制御下で抗体遺伝子が発現できるように、発現ベクターに組み込む。発現ベクターを用いて宿主細胞を形質転換し、抗体を発現させる。
他のプロモーター/エンハンサー系、例えば、ウイルス(例えば、レトロウイルス、ポリオーマウイルス、アデノウイルスおよびサルウイルス40(SV40))に由来するもの、ならびに哺乳類細胞に由来するもの(例えば、ヒト伸長因子1α(HEF1α))を本発明における抗体の発現に用いることもできる。
SV40プロモーター/エンハンサー系が用いられる場合、遺伝子発現はMulliganら(Nature (1979) 277, 108-14.)の方法によって容易に行うことができる。HEF1αプロモーター/エンハンサー系が用いられる場合、遺伝子発現はMizushimaら(Nucleic Acids Res. (1990) 18, 5322.)の方法によって容易に行うことができる。
さまざまな技術が抗体断片の産生のために開発されている。従来は、インタクトな抗体のタンパク質分解消化を介してこれらの断片が得られていた(例えば、Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992)およびBrennan et al., Science, 229: 81 (1985)を参照されたい)。しかしながら、これらの断片は現在、組換え宿主細胞によって直接産生することができる。例えば、抗体断片を抗体ファージライブラリーから単離することができる。あるいは、Fab'-SH断片を大腸菌から直接回収し、化学的にカップリングして、F(ab')2断片を形成させてもよい(Carter et al., Bio/Technology 10: 163-167 (1992))。別のアプローチによれば、F(ab')2断片を組換え宿主細胞培養物から直接単離することができる。抗体断片の産生のための他の技術は、当業者には明らかであろう。他の態様において、最適な抗体とは一本鎖Fv断片(scFv)である。WO93/16185号;US5,571,894号;およびUS5,587,458号を参照されたい。また抗体断片は、例えばUS5,641,870号に例えば記載されているように、「直鎖状抗体」であってもよい。そのような直鎖状抗体断片は単一特異性または二重特異性を有しうる。
本発明の抗体を、任意で、アフィニティー標識、酵素標識、放射性同位体標識、蛍光標識、または化学発光標識と結合させる。例えば、FZD10を発現しているがん組織に存在しかつ検出可能な標識の存在によって、診断される対象におけるがんまたは腫瘍の有無の判定が可能になる。また、がん組織における標識の局在性によって、疾患の拡大を判定することもできる。
使用に適した標識には、例えば、フルオレセインおよびローダミン等の蛍光標識;ならびにルシフェラーゼ等の酵素標識が含まれる。使用される検出可能な/検出用の標識は、使用するイメージング様式にしたがって選択される。そのような標識と抗体との結合体は、当技術分野で公知のプロトコルおよび技術を用いて作製することができる。本発明において、本発明の抗体は使用の直前に所望の標識と結合されてもよいし、標識と結合した抗体として提供されてもよい。
FZD10は、FZD10関連疾患の診断マーカーとして、ならびに当該疾患に罹患している対象におけるFZD10阻害剤に対する応答性および当該阻害剤の薬効を評価するためのマーカーとして有用である。したがって、本発明の抗体は、がんなどのFZD10関連疾患を診断するため、FZD10阻害剤による治療効果の高い対象をスクリーニングするため、またはFZD10阻害剤による治療後の薬効を判定するためのマーカーの検出試薬として用いることができる。
より具体的には、本発明の抗体によって対象から単離した試料中のFZD10タンパク質を検出し、FZD10関連疾患の診断、FZD10阻害剤による治療効果の高い対象のスクリーニング、またはFZD10阻害剤による治療後の薬効の判定を実施することができる。したがって、本発明は、対象から単離した試料において本発明の抗体を用いてFZD10タンパク質を検出することによる、対象においてFZD10関連疾患または該疾患の発症素因を診断する方法、FZD10阻害剤による治療効果の高い対象をスクリーニングする方法、および、FZD10阻害剤による治療後の薬効を判定する方法を提供する。これらの方法は以下の段階を含む:
(a) 対象から単離した試料を本発明の抗体またはその抗原結合性断片と接触させる段階;
(b) 該抗体またはその抗原結合性断片と該試料との結合を検出することによって、該試料中のFZD10タンパク質を検出する段階:および
(c) 該試料中のFZD10タンパク質レベルを対照と比較する段階。
また、FZD10阻害剤による治療効果の高い対象のスクリーニングの場合、前記段階(c)において、対照レベル(好ましくは、FZD10関連疾患と診断された対象の組織におけるFZD10タンパク質の発現レベル)と比較してFZD10タンパク質レベルが同程度かそれよりも高いときに、対象におけるFZD10阻害剤による治療効果が高いことが示される。
一方、FZD10阻害剤による治療後の薬効の判定の場合、前記段階(c)において、対照レベル(好ましくは、薬剤投与前の対象から単離した試料中のFZD10タンパク質の発現レベル)と比較してFZD10タンパク質レベルが低いときに、対象における薬効があったことが示される。
すなわち本発明は、以下の群から選択されるいずれかの患者を本発明の方法によって特定し、当該患者にFZD10阻害剤を投与する工程を含む、FZD10関連疾患の治療方法に関する;
本発明の診断方法によって、FZD10関連疾患を有することが示された患者;
FZD10阻害剤の治療効果が高い可能性が示された患者;そして
FZD10阻害剤の投与を受けた患者において、当該阻害剤の治療効果が有ったことが示された患者。
一方、FZD10関連疾患を患っている(例えば、がん性である)とわかっている生体試料から測定された対照レベルは「疾患対照レベル(例えば、がん性対照レベル)」と称される。FZD10阻害剤による治療前の対象から単離された試料におけるFZD10タンパク質レベルが疾患対照レベルと同程度かそれよりも高い場合、対象はFZD10阻害剤による治療効果が高い、と診断されうる。
また、FZD10阻害剤による治療後の対象から単離された試料におけるFZD10タンパク質レベルが薬剤投与前の同対象の疾患対照レベルより低い場合、治療による薬効があった、すなわち、対象はFZD10阻害剤による治療効果が高い、と診断されうる。
本発明はまた、FZD10関連疾患または該疾患の発症素因の診断における使用のための、本発明の抗体またはその抗原結合性断片を提供する。あるいは、本発明は、FZD10関連疾患または該疾患の発症素因を診断するための試薬の製造における、本発明の抗体またはその抗原結合性断片の使用を提供する。
本発明はまた、FZD10阻害剤による治療効果の高い対象のスクリーニングにおける使用のための、本発明の抗体またはその抗原結合性断片を提供する。あるいは、本発明は、FZD10阻害剤による治療効果の高い対象をスクリーニングするための試薬の製造における、本発明の抗体またはその抗原結合性断片の使用を提供する。
本発明はまた、FZD10阻害剤による治療後の薬効の判定における使用のための、本発明の抗体またはその抗原結合性断片を提供する。あるいは、本発明は、FZD10阻害剤による治療後の薬効を判定するための試薬の製造における、本発明の抗体またはその抗原結合性断片の使用を提供する。
本発明は、FZD10関連疾患の診断、FZD10阻害剤による治療効果の高い対象のスクリーニング、またはFZD10阻害剤による治療後の薬効の判定のための試薬またはキットを提供する。具体的には、これらのキットは、FZD10タンパク質の検出試薬として、本発明の抗体またはその抗原結合性断片を含む。一態様において、本発明の診断試薬またはキットのための抗体を蛍光物質、発光物質、または放射性同位体で標識することができる。抗体を標識するための方法および標識抗体を検出するための方法は、当技術分野において周知であり、任意の標識および方法を本発明のために利用することができる。
細胞培養
ATCCから購入したヒト大腸がん細胞株であるDLD1にFZD10発現ベクターを導入させたFZD10強制発現細胞株(FZD10/DLD1)を作製し、陰性対照としてEmpty Vectorを導入させた細胞株(Mock/DLD1)を作製した。5%CO2の加湿雰囲気中37℃で、10%ウシ胎仔血清(FBS)、1%ペニシリン/ストレプトマイシンおよび0.8mg/mL GENETICINを補充したRPMI-1640中で維持した。国立がん研究センター希少がんセンターから分与されたヒト滑膜肉腫細胞株であるSYO-1は、5%CO2の加湿雰囲気中37℃で、10%ウシ胎仔血清(FBS)および1%ペニシリン/ストレプトマイシンを補充したDMEM中で維持した。JCRBから購入したヒト食道がん細胞株であるT.Tは、5%CO2の加湿雰囲気中37℃で、10%ウシ胎仔血清(FBS)および1%ペニシリン/ストレプトマイシンを補充したDMEM/F12(1:1)中で維持した。ATCCから購入したヒト肺がん細胞株であるH727は、5%CO2の加湿雰囲気中37℃で、10%ウシ胎仔血清(FBS)および1%ペニシリン/ストレプトマイシンを補充したRPMI-1640中で維持した。ATCCから購入したヒト大腸がん細胞株であるCOLO201は、5%CO2の加湿雰囲気中37℃で、10%ウシ胎仔血清(FBS)、1%ペニシリン/ストレプトマイシン、10mM HEPES、1mM Sodium pyruvateおよび4.5g/L グルコースを補充したRPMI-1640中で維持した。ATCCから購入したヒト大腸がん細胞株であるLoVoは、CO2の加湿雰囲気中37℃で、20%ウシ胎仔血清(FBS)および1%ペニシリン/ストレプトマイシンを補充したF12中で維持した。
外科的に切除された肺がんからの組織試料(ホルマリン固定パラフィン切片、凍結組織)、ならびにそれらの対応する臨床的情報は、書面によるインフォームドコンセントをとった上で、神奈川がんセンターから入手した。
ホルマリン固定パラフィン切片化したFZD10/DLD1、Mock/DLD1、SYO1、T.T、H727、LoVo、COLO201および臨床検体を、キシレンに3分間3回浸漬して脱パラフィンした後、100%エタノールに1分間2回、90%、70%、および50%エタノールにてそれぞれ1分間浸漬して再水和させた。抗原賦活化処理のために抗原賦活化液pH9(ニチレイバイオサイエンス)に浸漬させた切片をFZD10/DLD1、Mock/DLD1は125℃30秒間、SYO1、T.T、H727、LoVo、COLO201および臨床検体は95℃40分間インキュベートした。インキュベート後、20分間室温放置し、流水にて5分間水洗した。3.0%過酸化水素水にて10分間浸漬して内因性ペルオキシダーゼのブロッキングを行ない、Wash Buffer(TBS-T(Takara Bio))にて、5分間3回洗浄した。さらに、非特異的な反応をブロックする目的で切片上にProtein Block液(Dako)を適量滴下し、10分間静置した。湿潤箱にて1次抗体(FZD10Ab(10A8H4G4)、FZD10(L164)pAb、FZD10 PolyclonalAb)をそれぞれ適量適下し、60分間静置後、Wash Bufferにて、5分間3回洗浄した。湿潤箱にて2次抗体としてヒストファイン シンプルステイン MAX-PO(ニチレイバイオサイエンス)を適量適下し、30分間静置した。Wash Bufferにて、5分間3回洗浄した。DAB基質溶液(ニチレイバイオサイエンス)により発色反応を行なった。スライド切片をヘマトキシリン(Dako)に20秒間浸漬し、流水で洗浄した。50%、70%、および90%エタノールにてそれぞれ1分間、および100%エタノールに1分間2回浸漬して脱水を行なった。最後に、切片をキシレンで3分間2回浸漬して透徹し、Mount-Quick(DAIDO SANGYO)によって封入した。
RNeasy Mini kit(QIAGEN)を用いてがん細胞株(SYO-1、COLO201)より総RNAを抽出した。臨床凍結組織(肺扁平上皮がん)については、TRIzol Reagent(Invitrogen)に凍結組織片を浸けてすり潰しクロロホルム抽出を行なった。得られた抽出液におおよそ等量の70%エタノールを加えて、RNeasy Mini kit(QIAGEN)を用いて総RNAを抽出した。SuperScript III Reverse Transcriptase(Invitrogen)の逆転写酵素を用いて総RNAよりcDNAを合成した。cDNAを鋳型としてKAPA SYBR FAST ABI Prism qPCR kit(KAPA Biosystems)を用いてPCR反応を行なった。発現解析目的遺伝子はFZD10、ハウスキーピング遺伝子はGAPDH(glyceraldehyde-3-phosphate dehydrogenase)とした。FZD10は、FZD10-11F:5'-GTGTGCAGCCGTAGGTTAAAG-3'(SEQ ID NO:12)、FZD10 R5:5'-GACTGGGCAGGGATCTCATA-3'(SEQ ID NO:13)のプライマーセットを用いて、GAPDHは、GAPDH RT-PCR Fw:5'-ACAACAGCCTCAAGATCATCAG-3'(SEQ ID NO:14)、GAPDH RT-PCR Re:5'-GGTCCACCACTGACACGTTG-3'(SEQ ID NO:15)のプライマーセットを用いてリアルタイムPCR反応を行なった。PCR増幅産物量を経時的にモニタリングし、PCR増幅産物が指数関数的に増幅している領域に閾値を設定してCt値を算出した。検量線にCt値をあてはめて未知サンプルの濃度を算出した。FZD10遺伝子の定量値をGAPDH遺伝子の定量値で割ることにより、サンプル間の鋳型量の標準化を行ない、FZD10遺伝子の発現量を比較した。
SYO1、T.T、H727、およびLoVoを、初期コンフルエント(early-confluent)までそれぞれ推奨される培養培地中で維持した。細胞表面抗原の変性を回避するため、次に細胞を、細胞剥離用溶液(Cell Dissociation Solution; SIGMA, MO)で分離させ、0.5% BSA/PBSで懸濁した。セルストレイナーに懸濁した細胞を通して塊をのぞき細胞数をカウントした。細胞を96Wellアッセイプレートに分注した(2×105個/50μL)。希釈済みの試験抗体もしくは培養上清50μLを添加し、4℃で1時間静置した。プレートを1500rpm、4℃で5分間遠心し、上清を除去した後、0.5% BSA/PBS 150μLで2度洗浄した。Alexa Fluor488ヤギ抗マウスIgG(H+L)(Invitrogen, CA)20 mcg/mLと混合し、暗所中4℃で1時間静置した。プレートを1500rpm、4℃で5分間遠心し、上清を除去した後、0.5% BSA/PBS 150μLで2度洗浄し、120μLの0.5% BSA/PBSで細胞を懸濁した。試験抗体の結合様式および特異性をFACS Calibur(Becton Dickinson, NJ)により製造元の使用説明書にしたがって評価した。
(1)可溶型FZD10タンパク質を免疫原とした抗FZD10抗体産生ハイブリドーマの取得
FZD10は膜タンパク質であるため、固定された膜タンパク質に反応する抗体作製には細胞外領域が免疫原となりうる。FZD10のN末端以外の細胞外領域は非常に短く、抗原とするには困難であると考えられたことから、N末端の細胞外領域(1-161アミノ酸)を免疫原として選択し、シグナルペプチドを除く可溶型FZD10タンパク質(21-161アミノ酸領域)(SEQ ID NO:9)を免疫原としてモノクローナル抗体を作製した。Freund Adjuvantに抗原ペプチド50μgを加えた後、エマルジョン化して、Balb/cマウス(日本エスエルシー株式会社)の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抗原ペプチド量相当を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SP2/0との細胞融合を行った。
抗FZD10抗体の選抜は、まず可溶性FZD10タンパク質をもちいたELISA法によりFZD10と結合性を有する抗体をスクリーニングした。ELISA法で選択された抗体について更に免疫染色法による選抜を行った。免疫染色法による選抜は、全長FZD10タンパク質(SEQ ID NO:22)を発現するFZD10強制発現細胞株FZD10/DLD1を用いた免疫組織化学染色により行った。すなわち、全長FZD10タンパク質を強制発現させたFZD10強制発現細胞株FZD10/DLD1をホルマリン固定パラフィン切片化した後、免疫染色を実施し、強い反応が認められたハイブリドーマを選抜した。
一方、市販抗体(FZD10(L164)Ab、FZD10PolyclonalAb)を用いて染色した場合、FZD10/DLD1とMock/DLD1いずれにおいても染色が観察された(高い割合の細胞にシグナルが検出された)。そのため、これらの市販の抗体による検出結果には偽陽性シグナルが含まれている可能性が疑われた。
以上の結果より、本発明の抗FZD10抗体(10A8H4G4)は、免疫組織化学染色法において、市販の抗FZD10抗体と比較して高い特異性でFZD10を検出できるという有利な性質を有していることが明らかとなった。
次に、FZD10の発現量が異なる種々の細胞株を用いて、10A8H4G4の特異性を評価した。すなわち、10A8H4G4を用いた免疫組織化学染色およびフローサイトメトリーの結果を比較し、FZD10タンパク質の発現量に応じた染色が認められるか検討した。ここで、各がん種毎の細胞株を用いたmRNAの発現解析データベース Cancer Cell Line Encyclopedia(CCLE)において、肺がん、食道がん、結腸直腸がん(大腸がん)で顕著な上方制御が示されている(図1)。そこで、これらのがん種に由来する細胞株を以下の解析に用いた。
まず、10A8H4G4を用いたフローサイトメトリーにより、種々のヒトがん組織由来細胞株の細胞膜上における内因性のFZD10タンパク質の発現を確認した。図3に示されているように、内因性のFZD10タンパク質の発現が知られているヒト滑膜肉腫由来SYO-1の他、ヒト食道がん由来細胞株T.Tおよびヒト肺がん由来細胞株H727においても、10A8H4G4を用いたフローサイトメトリーによって陽性シグナルが検出された。一方、FZD10非発現細胞株であることが知られているヒト大腸がん由来細胞株LoVoにおいては、10A8H4G4を用いたフローサイトメトリーによって陽性シグナルが検出されなかった。
続いて、これらの細胞株から作製されたパラフィン切片について、10A8H4G4を用いて免疫組織化学染色を行った。図3Bに示されているように、10A8H4G4を用いて染色した結果、FZD10を発現していることが確認された細胞株SYO-1、T.T、H727から作製されたパラフィン切片において染色が認められた。これらのパラフィン切片には、フローサイトメトリーの結果と相関して高い割合の細胞に陽性のシグナルが検出された。一方、FZD10非細胞株であるLoVoから作製されたパラフィン切片はほとんど染色されず、陽性のシグナルがほとんど検出されなかった。
以上の結果より、本発明の抗FZD10抗体(10A8H4G4)は、フローサイトメトリーにおいても免疫組織化学染色法においても高い特異性で試料中のFZD10タンパク質を検出できる有用なツールであることが示された。
本発明の抗FZD10抗体(10A8H4G4)の免疫組織化学染色における特異性を、がん細胞株のマウスXenograft腫瘍から作製されたパラフィン切片ならびに肺がん臨床検体を用いて評価した。SYO-1、COLO201細胞株のマウスXenograft腫瘍から作製されたパラフィン切片ならびに肺がん臨床検体1~6の腫瘍領域および非腫瘍領域におけるFZD10のタンパク質およびRNAの発現をそれぞれ免疫組織化学染色およびRealTime-PCRによって検出した結果を図4に示す。
図4に示されているように、FZD10のRNAの高発現が確認された細胞株SYO-1のマウスXenograft腫瘍から作製されたパラフィン切片においては、10A8H4G4により染色された細胞、すなわちFZD10陽性細胞が検出されたが、FZD10のRNAの発現量が少なかった細胞株COLO201のマウスXenograft腫瘍から作製されたパラフィン切片においては、10A8H4G4により染色された細胞、すなわちFZD10陽性細胞も少なかった。この結果は、本発明の抗FZD10抗体(10A8H4G4)は、免疫組織化学染色法において試料中のFZD10タンパク質の発現量と相関した検出結果を得るために有用なツールであることを示している。
また、腫瘍領域特異的なFZD10のRNAの発現が確認された肺がん臨床検体5においては10A8H4G4により染色された細胞、すなわちFZD10陽性細胞が検出されたが、腫瘍領域特異的なFZD10のRNAの発現が確認されなかった他の肺がん臨床検体はほとんど染色されず、FZD10陽性細胞がほとんど検出されなかった。この結果は、10A8H4G4は臨床検体においても特異的にFZD10タンパク質を検出できることを示している。
本発明の抗FZD10抗体(10A8H4G4)の可変領域のアミノ酸配列を分析した。
RNeasy mini kit(QIAGEN)を用いて、総RNAをハイブリドーマ10A8H4G4から抽出した。Super Script II Reverse Transcriptase(Invitrogen)を用いて、総RNAからcDNAを合成した。cDNA合成用のプライマーは以下の通りである。
重鎖3'プライマーのmIGCUniRv:
5'-CTGGGAAGGTGTGCACAC-3'(SEQ ID NO:16)
軽鎖3'プライマーのmIGKRv2:
5'-GTTGTTCAAGAAGCACACGAC-3'(SEQ ID NO:17)
5' RACE System for Rapid Amplification of cDNA Ends(Invitrogen)を用いてcDNA末端にdCのポリマーを付加し、Platinum Taq DNA Polymerase High Fidelity(Invitrogen)を用いて、モノクローナル抗体の可変領域をコードするポリヌクレオチドを増幅した。増幅用のプライマーは以下の通りである。プライマー配列中の塩基「I」は、イノシンを示す。
重鎖5'および軽鎖5'プライマーの5' RACE Abridged Anchor Primer:
5'-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3'(SEQ ID NO:18)、
重鎖3'プライマーのmIGCUniRv2:
5'-TGGACAGGGATCCAGAGTTCC-3'(SEQ ID NO:19)、ならびに
軽鎖3'プライマーのmIGKNesRv2:
5'-CAGATGTTAACTGCTCACTGGATGG-3'(SEQ ID NO:20)。
PCR産物をpGEM-T Easy Vector(Promega)にクローニングした。挿入断片領域を配列決定し、10A8H4G4の可変領域(シグナル配列を除く)の核酸配列を決定した。
10A8H4G4、重鎖可変領域アミノ酸配列(シグナル配列を除く):
QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGLGVSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITSVDTADTATYYCARRAYYGNYYALDYWGQGTSVTVSS(SEQ ID NO:7)(SEQ ID NO:10に示される核酸配列によってコードされる)
10A8H4G4、重鎖可変領域核酸配列:
5'-CAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCTGGTCTGGGTGTGAGCTGGATTCGTCAGCCTTCAGGAAAGGGTCTGGAGTGGCTGGCACACATTTACTGGGATGATGACAAGCGCTATAACCCATCCCTGAAGAGCCGGCTCACAATCTCCAAGGATACCTCCAGCAACCAGGTATTCCTCAAGATCACCAGTGTGGACACTGCAGATACTGCCACATACTACTGTGCTCGAAGAGCCTACTATGGTAATTACTATGCTTTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA-3'(SEQ ID NO:10)
10A8H4G4、軽鎖可変領域アミノ酸配列(シグナル配列を除く):
DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRKSGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPVTFGAGTKLELKRAD(SEQ ID NO:8)(SEQ ID NO:11に示される核酸配列によってコードされる)
10A8H4G4、軽鎖可変領域核酸配列:
5'-GACATTGTGATGACACAGTCTCCATCCTCCCTGACTGTGACAGCAGGAGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAACAGTGGAAATCAAAAGAACTACTTGACCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAACTGTTGATCTACTGGGCATCCACTAGGAAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGAATGATTATAGTTATCCGGTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGGCTGAT-3'(SEQ ID NO:11)
重鎖CDR1(CDR-H1):TSGLGVS(SEQ ID NO:1);
重鎖CDR2(CDR-H2):HIYWDDDKRYNPSLKS(SEQ ID NO:2);
重鎖CDR3(CDR-H3):RAYYGNYYALDY(SEQ ID NO:3);
軽鎖CDR1(CDR-L1):KSSQSLLNSGNQKNYLT(SEQ ID NO:4);
軽鎖CDR2(CDR-L2):WASTRKS(SEQ ID NO:5);および
軽鎖CDR3(CDR-L3):QNDYSYPVT(SEQ ID NO:6)
Claims (15)
- SEQ ID NO:1に示されるアミノ酸配列を含むCDR1;
SEQ ID NO:2に示されるアミノ酸配列を含むCDR2;および
SEQ ID NO:3に示されるアミノ酸配列を含むCDR3
を含む重鎖可変領域、ならびに
SEQ ID NO:4に示されるアミノ酸配列を含むCDR1;
SEQ ID NO:5に示されるアミノ酸配列を含むCDR2;および
SEQ ID NO:6に示されるアミノ酸配列を含むCDR3
を含む軽鎖可変領域
のいずれかまたは両方を含む、FZD10タンパク質またはその部分ペプチドに結合することができる、抗体またはその抗原結合性断片。 - SEQ ID NO:7に示されるアミノ酸配列を含む重鎖可変領域およびSEQ ID NO:8に示されるアミノ酸配列を含む軽鎖可変領域のいずれかまたは両方を含む、請求項1に記載の抗体またはその抗原結合性断片。
- SEQ ID NO:9に示されるアミノ酸配列からなるポリペプチドを特異的に認識する、請求項1または2に記載の抗体またはその抗原結合性断片。
- FZD10への特異的な結合について、請求項1~3のいずれか一項に記載の抗体と競合する、抗体またはその抗原結合性断片。
- アフィニティー標識、酵素標識、放射性同位体標識、または蛍光標識と結合している、請求項1~4のいずれか一項に記載の抗体またはその抗原結合性断片。
- 請求項1~5のいずれか一項に記載の抗体またはその抗原結合性断片をコードするポリヌクレオチド。
- 請求項1~5のいずれか一項に記載の抗体またはその抗原結合性断片を含む、FZD10関連疾患を診断、FZD10阻害剤による治療後の薬効を判定、またはFZD10阻害剤による治療効果の高い対象をスクリーニングするための試薬。
- 以下の段階を含む、対象におけるFZD10関連疾患または該疾患の発症素因を診断する方法:
(a) 該対象から単離した試料を請求項1~5のいずれか一項に記載の抗体またはその抗原結合性断片と接触させる段階;
(b) 該抗体またはその抗原結合性断片と該試料との結合を検出することによって、該試料中のFZD10タンパク質を検出する段階:および
(c) 該試料中のFZD10タンパク質レベルを対照と比較する段階であって、対照と比較してFZD10タンパク質レベルが高いときに該対象が該疾患を患っているかまたはその発症リスクを有することが示される、段階。 - 前記FZD10関連疾患が、FZD10を発現しているがんである、請求項7に記載の試薬または請求項8に記載の方法。
- 前記がんが、滑膜肉腫、肺がん、食道がん、結腸直腸がん(大腸がん)、胃がん、慢性骨髄性白血病(CML)および急性骨髄性白血病(AML)からなる群より選択される、請求項9に記載の試薬または方法。
- 以下の段階を含む、試料中のFZD10タンパク質を検出する方法:
(a) 対象から単離した試料を請求項1~5のいずれか一項に記載の抗体またはその抗原結合性断片と接触させる段階;および
(b) 該抗体またはその抗原結合性断片と該試料との結合を検出することによって、該試料中のFZD10タンパク質を検出する段階。 - 以下の段階を含む、対象におけるFZD10阻害剤による治療後の薬効を判定する方法:
(a) 該対象から単離した試料を請求項1~5のいずれか一項に記載の抗体またはその抗原結合性断片と接触させる段階;
(b) 該抗体またはその抗原結合性断片と該試料との結合を検出することによって、該試料中のFZD10タンパク質を検出する段階;
(c) 該試料中のFZD10タンパク質レベルを薬剤投与前の発現レベルと比較する段階であって、薬剤投与前と比較してFZD10タンパク質レベルが低いときに該対象における薬効があったことが示される、段階。 - 以下の段階を含む、FZD10阻害剤による治療効果の高い対象をスクリーニングする方法:
(a) 該対象から単離した試料を請求項1~5のいずれか一項に記載の抗体またはその抗原結合性断片と接触させる段階;
(b) 該抗体またはその抗原結合性断片と該試料との結合を検出することによって、該試料中のFZD10タンパク質を検出する段階;
(c) 該試料中のFZD10タンパク質レベルを対照と比較する段階であって、対照と比較してFZD10タンパク質レベルが同程度かそれよりも高いときに、該対象におけるFZD10阻害剤による治療効果が高いことが示される、段階。 - 前記試料が、前記対象から単離した細胞または組織である、請求項8~13のいずれか一項に記載の方法。
- 以下の工程を含む、FZD10タンパク質またはその部分ペプチドに結合することができる抗体またはその抗原結合性断片を製造する方法:
(a) 請求項6に記載のポリヌクレオチドが導入されたベクターを含む細胞を培養する段階;および
(b) 該細胞の培養物または培養培地から前記抗体を回収する段階。
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CN110835372B (zh) * | 2019-11-06 | 2020-12-01 | 上海健康医学院 | 一种靶向Frizzled7单克隆抗体及其制备方法与应用 |
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CA3038789A1 (en) | 2018-04-12 |
EP3524677B1 (en) | 2023-05-31 |
RU2765431C2 (ru) | 2022-01-31 |
TWI762516B (zh) | 2022-05-01 |
EP3524677A4 (en) | 2020-03-11 |
KR20190059318A (ko) | 2019-05-30 |
EP3524677A1 (en) | 2019-08-14 |
CN110036110B (zh) | 2023-02-03 |
JPWO2018066585A1 (ja) | 2019-07-25 |
BR112019006422A2 (pt) | 2019-06-25 |
RU2019112825A (ru) | 2020-11-09 |
TW201827465A (zh) | 2018-08-01 |
JP2022068145A (ja) | 2022-05-09 |
JP7039039B2 (ja) | 2022-03-22 |
US20200174002A1 (en) | 2020-06-04 |
CN110036110A (zh) | 2019-07-19 |
US11079386B2 (en) | 2021-08-03 |
KR102531006B1 (ko) | 2023-05-09 |
RU2019112825A3 (ja) | 2021-02-26 |
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