WO2022075482A1 - がん治療用医薬 - Google Patents
がん治療用医薬 Download PDFInfo
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- WO2022075482A1 WO2022075482A1 PCT/JP2021/037494 JP2021037494W WO2022075482A1 WO 2022075482 A1 WO2022075482 A1 WO 2022075482A1 JP 2021037494 W JP2021037494 W JP 2021037494W WO 2022075482 A1 WO2022075482 A1 WO 2022075482A1
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Definitions
- the present invention relates to a medicine and a method for treating cancer.
- liver cancer is the second leading cause of cancer-related death in the world, and about 750,000 people die from liver cancer every year in the world. About 80% of the annual new cases of about 780,000 are concentrated in the Asian region including Japan and China. Hepatocellular carcinoma accounts for 85 to 90% of all liver cancers. In Japan, the number of patients with hepatocellular carcinoma is about 42,000 and the annual number of deaths is about 26,000. It is reported that. Unresectable hepatocellular carcinoma is a disease with limited treatment, extremely poor prognosis, and high unmet medical needs. Multikinase inhibitors for multiple receptor tyrosine kinases have been used as therapeutic agents for unresectable advanced hepatocellular carcinoma. Specifically, sorafenib and lenvatinib are used as first-line drugs, and regorafenib and cabozantinib are used as second-line drugs, but their effects are limited.
- fibroblast growth factor (FGF) -19 has been reported in about 30% of patients (Non-Patent Document 1 and Non-Patent Document 2), and overexpression from liver cancer cells.
- the secreted FGF-19 acts in an autoclinic manner on its receptor, fibroblast growth factor receptor 4 (FGFR4), and excessively activates FGFR4 in liver cancer cells, thereby causing liver cancer. It has been reported that it promotes cell proliferation and exacerbates liver cancer.
- Fibroblast growth factor receptor 4 (FGFR4) a tyrosine kinase receptor for FGF, is involved in a variety of cellular processes including regulation of cell proliferation, differentiation, migration, metabolism, and bile acid biosynthesis, and is involved in FGFR4.
- FGF401, H3B-6527, BLU-554 are small molecule TKIs (tyrosine kinase inhibitors) that specifically bind to Cys552 in the FGFR4-specific ATP binding region. It is a mechanism of action that inhibits kinase activity.
- TKIs tyrosine kinase inhibitors
- FGFR4 selective small molecule tyrosine kinase inhibitors are undergoing clinical trials in patients with advanced hepatocellular carcinoma expressing FGF-19 overexpression or FGFR4. Information on the efficacy of each FGFR4 inhibitor is limited because it is in the development stage of Phase I or Phase I / II, but the therapeutic effect of each of the above inhibitors by monotherapy is limited. be.
- FGFR4 selective tyrosine kinases are used for various cancers, especially cancers in which FGFR4 is expressed and / or FGFR4 tyrosine kinase activity is observed, including hepatocellular carcinoma. It has been desired to develop a therapeutic agent and a therapeutic method for cancer, which can exert a stronger and lasting antitumor effect than an inhibitor.
- the present invention has been made in consideration of the above circumstances, and provides the following combination drugs for cancer treatment and the like.
- a combination drug for treating cancer A substance that inhibits or suppresses the tyrosine kinase activity of fibroblast growth factor receptor 4 (FGFR4), or a prodrug thereof, or a pharmacologically acceptable salt thereof, or a hydrate or solvent thereof.
- the combination drug comprising an antibody against human DLK-1 or an antibody fragment derived from the antibody, which has antitumor activity in vivo.
- a pharmaceutical composition for treating cancer which comprises an antibody against human DLK-1 or an antibody fragment derived from the antibody, which has antitumor activity in vivo.
- FGFR4 fibroblast growth factor receptor 4
- FGFR4 fibroblast growth factor receptor 4
- a pharmaceutical composition for treating cancer including Japanese products. It is used in combination with an antibody against human DLK-1, which has antitumor activity in vivo, or an antibody fragment derived from the antibody. The pharmaceutical composition.
- a pharmaceutical composition for treating cancer which comprises an antibody against human DLK-1 or an antibody fragment derived from the antibody, which has antitumor activity in vivo.
- the treatment is a substance that inhibits or suppresses the tyrosine kinase activity of fibroblast growth factor receptor 4 (FGFR4), or a prodrug thereof, or a pharmacologically acceptable salt thereof, or a hydrate thereof. Including administration of solvates, The pharmaceutical composition.
- FGFR4 fibroblast growth factor receptor 4
- the substance that inhibits or suppresses the tyrosine kinase activity of fibroblast growth factor receptor 4 (FGFR4) is H3B-6527, FGF-401 (lobritinib) or BLU-554 (physogatinib), the above (1).
- the cancer and / or the tumor is hepatocellular carcinoma, lung cancer, uterine body cancer, bile duct cancer, intrahepatic bile duct cancer, esophageal cancer, nasopharyngeal cancer, ovarian cancer, breast cancer,
- the antibody is (A) The amino acid sequences of CDR1 to 3 in the H chain V region are the amino acid sequences represented by SEQ ID NOs: 3 to 5, respectively, and the amino acid sequences of CDR1 to 3 in the L chain V region are, respectively, in order. An antibody, which is the amino acid sequence shown in SEQ ID NOs: 6 to 8. (B) The amino acid sequences of CDR1 to 3 in the H chain V region are the amino acid sequences represented by SEQ ID NOs: 9 to 11 in order, and the amino acid sequences of CDR1 to 3 in the L chain V region are in order, respectively. An antibody, which is the amino acid sequence shown in SEQ ID NOs: 12-14.
- the pharmaceutical composition according to any one of the above. (12) The combination drug according to (1) above, or the combination drug described above, which can suppress the growth of cancer cells or shrink or eliminate a tumor even after the end of administration of the combination drug or the pharmaceutical composition. (2) The pharmaceutical composition according to any one of (5).
- FGFR4 fibroblast growth factor receptor 4
- an antibody against human DLK-1 which has antitumor activity in vivo, or an antibody fragment derived from the antibody.
- a method for treating cancer which comprises administering to a subject an antibody against human DLK-1, which has antitumor activity in vivo, and an antibody fragment derived from the antibody.
- a kit for treating cancer which comprises an antibody against human DLK-1, which has antitumor activity in vivo, or an antibody fragment derived from the antibody.
- a stronger and more sustained antitumor effect than existing tyrosine kinase inhibitors is used in the treatment of cancer, particularly in the treatment of cancer in which FGFR4 is expressed and / or tyrosine kinase activity of FGFR4 is observed. It is possible to provide a therapeutic agent and a therapeutic method that can exert the above-mentioned effects.
- the therapeutic agent and the therapeutic method of the present invention are extremely useful, for example, in that they can exert an effect even on a patient whose therapeutic effect has not been expected so far.
- FIG. 1 In the Hep3B xenograft model, the proliferation group, the HuBA-1-3D (1 mg / kg) administration group, the H3B-6527 (50 mg / kg) administration group, and the HuBA-1-3D (1 mg / kg) + H3B-6527.
- 50 mg / kg is a photograph shown by immunostaining of cell proliferation (Ki-67) and apoptosis (cleave caspase-3) 48 hours after administration in the combined administration group.
- the brown-stained area indicates cells that are proliferating (Ki-67 positive) or apoptotic (cleave caspase-3 positive).
- FIG. 1 In the Hep3B xenograft model, the proliferation group, the HuBA-1-3D (1 mg / kg) administration group, the H3B-6527 (50 mg / kg) administration group, and the HuBA-1-3D (1 mg / kg) + H3B-6527.
- Ki-67 cell proliferation
- FIG. 5A Vehicle, HuBA-1-3D (1 mg / kg), FGFR4 selective inhibitor (H3B-6527 (50 mg / kg), FGF-401 (10 mg / kg), BLU-554 (BLU-554) in the Hep3B Xenograft model. 30 mg / kg), HuBA-1-3D (1 mg / kg) and each FGFR4 inhibitor were administered in combination, and apoptosis (cleaved caspase-3, cleared PARP) and cell proliferation marker (cyclin) in the tumor 48 hours later. It is the figure which Western-blot analysis showed the expression of B1).
- BLU-554.N 3.
- FIG. 5A FIG.
- FIG. 5A shows enhancement of antitumor effect by combined administration of HuBA-1-3D antibody and FGFR4 selective inhibitor (H3B-6527).
- H3B-6527 HuBA-1-3D antibody and FGFR4 selective inhibitor
- FIG. 5C shows the enhancement of antitumor effect by combined administration of HuBA-1-3D antibody and FGFR4 selective inhibitor (H3B-6527).
- FIG. 6 is a diagram showing enhancement of antitumor effect by combined administration of HuBA-1-3D antibody and FGFR4 selective inhibitor (FGF-401).
- FIG. 7 is a diagram showing enhancement of antitumor effect by combined administration of HuBA-1-3D antibody and FGFR4 selective inhibitor (BLU-554).
- (A) Vehicle-administered group ( ⁇ ), HuBA-1-3D alone-administered group ( ⁇ ), BLU-554 alone-administered group ( ⁇ ), HuBA-1-3D and BLU in the Hep3B Xenograft model. It is a figure which showed the average tumor growth in the combination administration group ( ⁇ ) of ⁇ 554. Individuals with tumor volumes reaching 1,500 mm 3 were sacrificed. The graph shows up to the time when at least 7 individuals remained in each administration group (N 8 at the time of grouping). * P ⁇ 0.05 (vs Vehicle group, Dunnett).
- FIG. 8 shows an enhancement of antitumor effect when co-administered with an inhibitor against FGFR1 / FGFR2 / FGFR3 (CH518324 / Debio1347).
- Vehicle administration group ⁇
- HuBA-1-3D single administration ⁇ , ⁇
- CH518324 / Debio1347 single administration
- HuBA-1-3D and CH518324 / Debio1347 It is the figure which showed the tumor growth in the combination administration ( ⁇ , ⁇ ). Individuals with tumor volumes reaching 1,500 mm 3 were sacrificed.
- hdLK-1 is a single-pass transmembrane type I membrane protein consisting of 383 residues of amino acids, and is used for adult cancers such as hepatoblastoma, small cell cancer, pancreatic cancer, and breast cancer, and nerves. It is known to be expressed in childhood cancers such as blastoma, hepatoblastoma, rhabdomyosarcoma, and viral tumors. On the other hand, the expression of DLK-1 is limited to the adrenal gland, pituitary gland, etc. in normal tissues and organs, and is not expressed in most organs, so that it is suitable as a therapeutic target molecule for cancer.
- anti-hDLK-1 monoclonal antibodies have been created that inhibit tumor cell growth including excellent tumor shrinkage effect and induce tumor cell death, and multiple human cancers including human liver cancer cell lines have been created.
- antitumor activity by single administration of the antibody has been shown.
- a sufficient therapeutic effect is expected only by the single administration of the above-mentioned existing tyrosine kinase inhibitor. Sufficient therapeutic effects can be expected even for patients with cancer that cannot (or do not respond).
- the present invention has been completed in this way.
- combination medicine for treating the cancer of the present invention (hereinafter, may be referred to as “combination medicine of the present invention”) is as described above.
- -A substance that inhibits or suppresses the tyrosine kinase activity of fibroblast growth factor receptor 4 (FGFR4) (hereinafter, may be referred to as "FGFR4 inhibitor"), a prodrug thereof, or pharmacologically acceptable thereof.
- FGFR4 inhibitors Obtaining salts, or their hydrates or solvates (hereinafter, may be referred to as "FGFR4 inhibitors, etc.”), and -It is characterized by containing an anti-hDLK-1 antibody having antitumor activity in vivo or an antibody fragment derived from the antibody (hereinafter, may be referred to as "anti-hDLK-1 antibody or the like") as an active ingredient. It is a thing.
- the cancer to be treated is not limited, but a cancer in which FGFR4 is expressed and / or a tyrosine kinase activity of FGFR4 is observed is preferably mentioned.
- hepatocellular carcinoma, lung cancer, uterine body cancer, bile duct cancer, intrahepatic bile duct cancer, esophageal cancer, nasopharyngeal cancer, and ovarian cancer, breast cancer, renal cell cancer, pancreas. Cancer, colon cancer, glioblastoma, and sarcoma including rhabdomyomyoma are preferred.
- the "tumor" in the antitumor activity of the anti-hDLK-1 antibody is not limited, but the same description as that of the cancer to be treated can be applied.
- an FGFR4 inhibitor or the like and an anti-hDLK-1 antibody or the like are used, specifically, for example, an effective amount of the FGFR4 inhibitor or the like and the anti-hDLK-1 antibody or the like is used as a test subject ( Methods for treating cancer, including administration to patients with cancer or patients at risk of developing it (or such non-human mammals), (ii) manufacturing agents for the treatment of cancer.
- Use of FGFR4 inhibitor etc. and anti-hDLK-1 antibody etc. for (iii) use of FGFR4 inhibitor etc. and anti-hDLK-1 antibody etc. for treatment of cancer, and (iv) for treatment of cancer FGFR4 inhibitor and the like and anti-hDLK-1 antibody and the like are also included.
- the treatment of cancer includes, for example, suppression of cancer progression, improvement of prognosis, and / or prevention of recurrence.
- FGFR4 inhibitor and the like which are the active ingredients of the combination drug of the present invention, are not limited as long as they have a function of inhibiting or suppressing the tyrosine kinase activity of FGFR4.
- Preferred FGFR4 inhibitors include FGFR4 selective tyrosine kinase inhibitors such as H3B-6527, FGF-401 (lobritinib), and BLU-554 (physogatinib).
- FGFR4 inhibitor or the like one independently synthesized, extracted, purified or the like may be used, or a commercially available one may be used.
- H3B-6527 N- ⁇ 2-[(6- ⁇ [(2,6-dichloro-3,5-dimethoxyphenyl) carbamoyl] (methyl) amino ⁇ -4. -Pyrimidineyl) amino] -5- (4-ethyl-1-piperazinyl) phenyl ⁇ acrylamide (N- ⁇ 2-[(6- ⁇ [(2,6-Dicholoro-3,5-dimethoxyphenyl) carbamoyl] (methyl)) amino ⁇ -4-pyrimidinyl) amino] -5- (4-ethyl-1-piperazinyl) phenyl ⁇ acrylamide), which is represented by the following structural formula.
- FGF-401 (lobritinib) is N- [5-cyano-4-[(2-methoxyethyl) amino] -2-pyridinyl] -7-formyl-3,4- Dihydro-6-[(4-Methyl-2-oxo-1-piperazinyl) Methyl] -1,8-naphthylidine-1 (2H) -carboxamide (N- [5-cyano-4-[(2-methoxythyl) amino) ] -2-Pyridinyl] -7-formyl-3,4-dihydro-6-[(4-methyl-2-oxo-1-piperazinyl) methyl] -1,8-naphyridine-1 (2H) -carboxamide) Yes, it is represented by the following structural formula.
- the official name (IUPAC name) of BLU-554 (phisogatinib) is N-[(3S, 4S) -3-[[6- (2,6-dichloro-3,5-dimethoxyphenyl) quinazoline-2. -Il] amino] oxane-4-yl] prop-2-enamid (N-[(3S, 4S) -3-[[6- (2,6-dichloro-3,5-dimethoxyphenyl) quinazoline-2-yl) ] Amino] oxan-4-yl] dichloromethane), which is represented by the following structural formula.
- a derivative of the FGFR4 inhibitor can be used together with the FGFR4 inhibitor or in place of the FGFR4 inhibitor.
- the derivative may be any one that is considered to be a derivative of the FGFR4 inhibitor based on the common technical knowledge of those skilled in the art, such as having a chemical structure derived from the FGFR4 inhibitor, and is not limited, but inhibits the tyrosine kinase activity of FGFR4. Alternatively, those having the same inhibitory activity as the FGFR4 inhibitor are preferable.
- the term FGFR4 inhibitor is also meant to include a derivative of the FGFR4 inhibitor.
- the FGFR4 inhibitor used in the present invention includes, for example, those that undergo metabolism such as oxidation, reduction, hydrolysis, or conjugation in vivo, and also undergo metabolism such as redox or hydrolysis in vivo.
- Compounds that produce FGFR4 inhibitors are also included.
- the prodrug refers to a pharmacologically acceptable compound obtained by modifying the parent compound with a group usually used in a prodrug, and is endowed with properties such as improvement in stability and sustainability.
- a prodrug of an FGFR4 inhibitor can be a prodrugizable group (eg, a hydroxyl group, an amino group, or other group) in the compound by a conventional method using a corresponding prodrug-forming reagent such as a halide. ), Then, after introducing a group constituting a prodrug as appropriate according to a conventional method into one or more arbitrary groups, it can be produced by isolation and purification as necessary.
- the group constituting the above prodrug is not limited, but is, for example, lower alkyl-CO-, lower alkyl-O-lower alkylene-CO-, lower alkyl-OCO-lower alkylene-CO-, lower alkyl. -OCO-, lower alkyl-O-lower alkylene-OCO- and the like are preferably mentioned.
- a pharmacologically acceptable salt thereof can be used together with the FGFR4 inhibitor and its prodrug, or in place of the FGFR4 inhibitor and its prodrug.
- the pharmacologically acceptable salt is not limited, and is, for example, an organic sulfonate (for example, methane sulfonate (also referred to as mesylate), trifluoromethane sulfonate, ethane sulfonate, benzene).
- organic carboxylates eg, acetates, trifluoroacetates, maleates, tartrates, fumarates, and citrates
- the FGFR4 inhibitor used in the present invention includes all isomers that may occur due to the structure of the compound (for example, geometric isomers, asymmetric carbon-based optical isomers, rotational isomers, stereoisomers, and tautomers). And a mixture of two or more of these isomers is also included, and the description is not limited to the description of the structural formula for convenience. Further, the FGFR4 inhibitor may be any of S-form, R-form or RS-form, and is not limited.
- the FGFR4 inhibitor may exist in the form of a hydrate or a solvate depending on the type thereof, and in the present invention, the hydrate and the solvate are also included in the FGFR4 inhibitor, and the present invention It can be used as an active ingredient of a combination drug.
- the solvate is not limited, and examples thereof include a solvate with ethanol.
- the content ratio of the FGFR4 inhibitor or the like as an active ingredient is not limited and can be appropriately set, but for example, 0.01 to 99.99 weight with respect to the entire combination drug. It can be in the range of%, preferably 0.01 to 30% by weight, more preferably 0.05 to 20% by weight, and even more preferably 0.1 to 10% by weight. When the content ratio of the active ingredient is within the above range, the combination drug of the present invention can sufficiently exert a therapeutic effect on cancer.
- the combination drug of the present invention may contain any other multikinase inhibitor and the like as long as the effects of the present invention are not significantly impaired.
- the anti-hDLK-1 antibody (antibody against human DLK-1 having antitumor activity in vivo), which is the active ingredient of the combination drug of the present invention, is based on the following description. Can be produced.
- a peptide containing at least a part (all or part) of the amino acid sequence of FA-1) can be used.
- the extracellular region of hDLK-1 refers to a region containing six EGF-like motifs (EGF-1 to EGF-6) as described above, and is the 24th to 244th amino acid sequences shown in SEQ ID NO: 2.
- the region containing the second amino acid preferably the region consisting of the amino acids "24th" to "248th to 285th" (approximately 225 to 262 amino acid residues) in the amino acid sequence shown in SEQ ID NO: 2.
- the above-mentioned "at least a part of the amino acid sequence” is not particularly limited in length, and includes, for example, one or two or more of six EGF-like motifs. Regions are preferred. More preferably, for example, a region containing EGF-1 and EGF-2 (that is, a region consisting of the 24th to 91st amino acids of the amino acid sequence shown in SEQ ID NO: 2), EGF-3 and EGF-4.
- the method for producing the peptide as an antigen may be chemical synthesis or synthesis by a genetic engineering method using Escherichia coli or the like, and a method well known to those skilled in the art can be used.
- chemically synthesizing a peptide it can be synthesized by a well-known method for synthesizing a peptide.
- solid phase synthesis method or the liquid phase synthesis method can be applied to the synthesis.
- a commercially available peptide synthesizer for example, manufactured by Shimadzu Corporation: PSSM-8, etc. may be used.
- a DNA encoding the peptide is designed and synthesized.
- the design and synthesis can be carried out by the PCR method using, for example, a vector containing the full-length hDLK-1 gene as a template and a primer designed so that a desired DNA region can be synthesized.
- a recombinant vector for protein expression is obtained by ligating the above DNA to an appropriate vector, and a transformant is obtained by introducing this recombinant vector into a host so that the target gene can be expressed (Molecular cloning). 4th Ed. Cold Spring Labor Laboratory Press (2012)).
- phages or plasmids that can grow autonomously in the host microorganism are used. Furthermore, animal virus and insect virus vectors can also be used.
- the purified DNA may be cleaved with an appropriate restriction enzyme, inserted into a restriction enzyme site of an appropriate vector DNA, or the like, and ligated to the vector.
- the host used for transformation is not particularly limited as long as it can express the gene of interest. Examples thereof include bacteria (Escherichia coli, Bacillus subtilis, etc.), yeast, animal cells (COS cells, CHO cells, etc.), insect cells or insects. It is also possible to use a mammal such as a goat as a host. Methods for introducing a recombinant vector into a host are known.
- the transformant is cultured, and a peptide used as an antigen is collected from the culture.
- the "culture” means any of a culture supernatant, cultured cells or cultured cells, or a crushed product thereof.
- the peptide is extracted by disrupting the cells or cells.
- the culture medium is used as it is, or the cells or cells are removed by centrifugation or the like. Then, by using a general biochemical method used for isolation and purification of the peptide, for example, ammonium sulfate precipitation, gel filtration, ion exchange chromatography, affinity chromatography, etc.
- a peptide serving as an antigen can also be obtained by in vitro translation using a cell-free synthesis system.
- two methods can be used: a method using RNA as a template and a method using DNA as a template (transcription / translation).
- a cell-free synthesis system commercially available systems such as Expressway TM system (Invitrogen), PURESYSTEM (registered trademark; Post-Genome Research Institute), TNT system (registered trademark; Promega) and the like can be used.
- the peptide obtained as described above can also bind to a suitable carrier protein such as bovine serum albumin (BSA), keyhole limpet hemocianine (KLH), human thyroglobulin, chicken gamma globulin and the like.
- BSA bovine serum albumin
- KLH keyhole limpet hemocianine
- human thyroglobulin chicken gamma globulin and the like.
- the antigen may be a peptide consisting of an amino acid sequence of hdLK-1 (SEQ ID NO: 2) or an amino acid sequence in which one or more amino acids are deleted, substituted or added in the above-mentioned partial sequence thereof.
- one or more amino acids preferably one or several (eg, 1 to 10, more preferably 1 to 5)) of the amino acid sequence of hdLK-1 or a partial sequence thereof are deleted.
- amino acids are substituted with other amino acids, or one or more.
- Peptides consisting of an amino acid sequence to which other amino acids (preferably one or several (eg, 1 to 10, more preferably 1 to 5)) are added can also be used.
- examples of the gene to be introduced into cells and the like include hDLK-1 protein or a partial fragment thereof, or a gene encoding a protein or fragment of a variant thereof.
- a gene having the base sequence shown in SEQ ID NO: 1 or a partial sequence thereof can be used.
- a base sequence encoding a protein that hybridizes with a sequence complementary to the base sequence shown in SEQ ID NO: 1 under stringent conditions and has hDLK-1 activity Alternatively, it is also possible to use a partial array thereof.
- the "stringent condition” is a condition at the time of washing after hybridization, in which the salt (sodium) concentration of the buffer is 10 to 500 mM, the temperature is 42 ° C to 72 ° C, and the above salt is preferable.
- the condition is that the concentration is 50 to 300 mM and the temperature is 55 to 68 ° C.
- a mutation introduction kit using a known method such as the Kunkel method or the Gapped duplex method, for example, a site-directed mutagenesis method, for example, GeneTailorTM Site-Directed Mutagenesis System (manufactured by Invitrogen), It can be performed using TaKaRa Site-Directed Mutagenesis System (Prime STAR (registered trademark) Mutagenesis Basic kit, Mutan (registered trademark) -Super Express Km, etc .: manufactured by Takara Bio Co., Ltd.).
- a site-directed mutagenesis method for example, GeneTailorTM Site-Directed Mutagenesis System (manufactured by Invitrogen), It can be performed using TaKaRa Site-Directed Mutagenesis System (Prime STAR (registered trademark) Mutagenesis Basic kit, Mutan (registered trademark) -Super Express Km, etc .: manufactured by Takara Bio Co., Ltd.).
- the prepared antigen is administered to mammals for immunity.
- Mammals are not particularly limited, and examples thereof include rats, mice, and rabbits, with mice being preferred.
- the dose of the antigen per animal can be appropriately set depending on the presence or absence of an adjuvant.
- the adjuvant include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), aluminum hydroxide adjuvant and the like.
- Immunization can be performed mainly by injecting intravenously, footpads, subcutaneously, intraperitoneally or the like.
- the immunization interval is not particularly limited, and immunization is performed 1 to 10 times, preferably 2 to 3 times, at intervals of several days to several weeks, preferably at intervals of one week.
- the antibody titer was measured by an enzyme-linked immunosorbent assay (ELISA or EIA), radioimmunoassay (RIA), etc., and blood was collected on the day when the desired antibody titer was shown.
- ELISA or EIA enzyme-linked immunosorbent assay
- RIA radioimmunoassay
- Antiserum can be obtained.
- a known method such as sulfur salting out method, ion exchange chromatography, gel filtration chromatography, affinity chromatography, etc. is appropriately selected, or these are used. By combining, it can be purified. After that, the reactivity of the polyclonal antibody in the antiserum is measured by an ELISA method or the like.
- the anti-hDLK-1 antibody of the present invention is not limited, but is preferably a monoclonal antibody.
- the prepared antigen is administered to mammals such as rats, mice and rabbits for immunity.
- the dose of the antigen per animal can be appropriately set depending on the presence or absence of an adjuvant.
- the adjuvant is the same as above.
- the immunization method is the same as described above.
- antibody-producing cells are collected 1 to 60 days after the final immunization day, preferably 1 to 14 days later. Examples of the antibody-producing cells include spleen cells, lymph node cells and peripheral blood cells, and among them, lymph node cells and spleen cells are preferable.
- a hybridoma antibody-producing cell line
- cell fusion between antibody-producing cells and myeloma cells is performed.
- myeloma cells to be fused with the antibody-producing cells generally available cell lines of animals such as mice can be used.
- the cell line to be used it has drug selectivity, cannot survive in HAT selective medium (including hypoxanthine, aminopterin and thymidine) in the unfused state, and can survive only in the state of being fused with antibody-producing cells. It is preferable to have.
- myeloma cells examples include P3-X63-Ag8.653, P3-X63-Ag8 (X63), and P3-X63-Ag8. Examples thereof include mouse myeloma cell lines such as U1 (P3U1), P3 / NS I / 1-Ag4-1 (NS1) and Sp2 / 0-Ag14 (Sp2 / 0).
- the selection of myeloma cells can be made in consideration of compatibility with antibody-producing cells.
- the myeloma cells and the antibody-producing cells are fused.
- Cell fusion was performed in 1 ⁇ 10 6 to 1 ⁇ 10 7 cells / mL antibody-producing cells and 2 ⁇ 10 5 to 2 ⁇ 10 6 cells in serum-free DMEM and animal cell media such as RPMI-1640 medium. Mix with / mL myeloma cells.
- the cell ratio of the antibody-producing cell to the myeloma cell is not limited, but is usually preferably 1: 1 to 10: 1, and more preferably 3: 1.
- a fusion reaction is carried out in the presence of a cell fusion promoter.
- polyethylene glycol having an average molecular weight of 1,000 to 6,000 daltons (D) can be used. It is also possible to fuse antibody-producing cells and myeloma cells using a commercially available cell fusion device that utilizes electrical stimulation (for example, electroporation).
- the target hybridomas are selected from the cells after cell fusion treatment.
- the cell suspension is appropriately diluted with, for example, RPMI-1640 medium containing fetal bovine serum, seeded on a microtiter plate, a selective medium is added to each well, and then the selective medium is appropriately replaced. Perform culture. As a result, cells that grow from about 14 days after the start of culturing in the selective medium can be obtained as a hybrida. Next, it is screened whether or not an antibody that reacts with hdLK-1 is present in the culture supernatant of the grown hybridoma. Screening for hybridomas may follow conventional methods and is not particularly limited.
- a part of the culture supernatant contained in the well grown as a hybridoma can be collected and screened by ELISA, EIA, RIA and the like.
- Cloning of fused cells can be performed by a limiting dilution method or the like.
- An antibody showing strong reactivity with hDLK-1 is determined by flow cytometry or the like, and a hybridoma producing the antibody is selected and established as a clone.
- a normal cell culture method As a method for culturing an established hybridoma and collecting a monoclonal antibody from the obtained culture, a normal cell culture method, an ascites formation method, or the like can be adopted.
- "Culture” means growing a hybridoma in a culture dish or culture bottle, or growing the hybridoma in the abdominal cavity of an animal as described below.
- the hybrid doma is placed in an animal cell culture medium such as RPMI-1640 medium containing 10% bovine fetal serum, MEM medium or serum-free medium under normal culture conditions (for example, 37 ° C., 5% CO 2 concentration). After culturing for 7 to 14 days, the antibody can be obtained from the culture supernatant.
- hybridomas In the case of the ascites formation method, about 1 ⁇ 10 7 hybridomas are administered intraperitoneally to a mammal of the same kind as a mammal derived from myeloma cells, and a large amount of hybridomas are propagated. Then, it is preferable to collect ascites after 2 to 3 weeks.
- known methods such as sulfur salting out method, ion exchange chromatography, gel filtration, affinity chromatography and the like are appropriately selected or combined. Can be purified by.
- the anti-hDLK-1 antibody used in the present invention is an antibody having antitumor activity in vivo.
- the "anti-tumor activity” means an activity that kills a tumor cell (cancer cell) or an activity that inhibits tumor growth.
- tumor angiogenesis inhibitory activity is preferably mentioned.
- Examples of the types of human tumors (tumor cells) in which the anti-hDLK-1 antibody of the present invention can exert antitumor activity include human tumors in which expression of hDLK-1 has been confirmed.
- Confirmation of antitumor activity in vivo can be performed, for example, by using a cancer-bearing mouse in which desired tumor cells are subcutaneously transplanted into a mouse, and administering the antibody obtained as described above to this mouse.
- the antibody may be administered immediately after transplantation of the tumor cells (Prevention model), or may be performed after confirming that the tumor has reached a predetermined volume for transplantation (Treatment model).
- the administration method is not limited in any way, but may be, for example, once every 3 days, 20 mg / kg body weight, intraperitoneal administration.
- the Presence model the presence or absence and level of antitumor activity can be evaluated by the tumor formation frequency and the tumor volume.
- the presence or absence and level of antitumor activity can be evaluated by the tumor volume and the tumor weight.
- the anti-hDLK-1 antibody having antitumor activity in vivo is not particularly limited, but is disclosed in, for example, WO2008 / 056833, WO2009 / 116670, and WO2014 / 054820.
- -1 can be used. More specifically, for example, the following anti-hDLK-1 antibodies (a) to (k) can be mentioned, and at least one of them can be used.
- the amino acid sequences of CDR1 to 3 in the H chain V region are the amino acid sequences represented by SEQ ID NOs: 3 to 5, respectively, and the amino acid sequences of CDR1 to 3 in the L chain V region are, respectively, in order.
- the amino acid sequences of CDR1 to 3 in the H chain V region are the amino acid sequences represented by SEQ ID NOs: 9 to 11 in order, and the amino acid sequences of CDR1 to 3 in the L chain V region are in order, respectively.
- the amino acid sequence of the H chain V region consists of the amino acid sequence shown by SEQ ID NO: 16 (the corresponding base sequence is SEQ ID NO: 15), and the amino acid sequence of the L chain V region is SEQ ID NO: 18 (corresponding base sequence). Is an antibody consisting of the amino acid sequence shown in SEQ ID NO: 17). The CDR sequences in the H chain V region and the L chain V region of the antibody are the same as those of the antibody (a) above.
- the amino acid sequence of the H chain V region consists of the amino acid sequence shown by SEQ ID NO: 20 (the corresponding base sequence is SEQ ID NO: 19), and the amino acid sequence of the L chain V region is SEQ ID NO: 22 (corresponding base sequence). Is an antibody consisting of the amino acid sequence shown in SEQ ID NO: 21). The CDR sequences in the H chain V region and the L chain V region of the antibody are the same as those of the antibody of (b) above.
- the amino acid sequence of the H chain V region consists of the amino acid sequence represented by SEQ ID NO: 24 or 26 (the corresponding base sequence is SEQ ID NO: 23 or 25), and the amino acid sequence of the L chain V region is SEQ ID NO: 28 (the corresponding base sequence is SEQ ID NO: 23 or 25).
- the corresponding base sequence is an antibody (humanized antibody) consisting of the amino acid sequence shown in SEQ ID NO: 27).
- the CDR sequences in the H chain V region and the L chain V region of the antibody are the same as those of the antibody (a) above.
- the amino acid sequence of the H chain V region consists of the amino acid sequence represented by SEQ ID NO: 30, 32, 34 or 36 (the corresponding base sequence is SEQ ID NO: 29, 31, 33 or 35), and the L chain V region.
- the CDR sequences in the H chain V region and the L chain V region of the antibody are the same as those of the antibody of (b) above.
- the amino acid sequence of the H chain V region consists of the amino acid sequence represented by SEQ ID NO: 38, 40, 42 or 44 (the corresponding base sequence is SEQ ID NO: 37, 39, 41 or 43) and the L chain V region.
- the CDR sequences in the H chain V region and the L chain V region of the antibody are the same as those of the antibody of (b) above.
- H An antibody produced by a hybridoma whose accession number is FERM BP-10899.
- I An antibody produced by a hybridoma whose accession number is FERM BP-10707.
- J An antibody produced by a hybridoma whose accession number is FERM BP-10900.
- K An antibody produced by a hybridoma whose accession number is FERM BP-11337.
- the amino acid sequence shown in SEQ ID NO: 32 is obtained by substituting the 24th alanine (A) of the amino acid sequence shown in SEQ ID NO: 30 with glycine (G).
- the amino acid sequence shown in SEQ ID NO: 34 is the one in which the 74th threonine (T) of the amino acid sequence shown in SEQ ID NO: 30 is replaced with lysine (K).
- the 24th alanine (A) of the amino acid sequence shown in SEQ ID NO: 30 is replaced with glycine (G), and the 74th threonine (T) is lysine (T). It was replaced with K).
- the amino acid sequence shown in SEQ ID NO: 40 is the amino acid sequence shown in SEQ ID NO: 38 in which the 24th alanine (A) is replaced with glycine (G).
- the amino acid sequence shown in SEQ ID NO: 42 is obtained by substituting threonine (T) at position 74 of the amino acid sequence shown in SEQ ID NO: 38 with lysine (K).
- the 24th alanine (A) of the amino acid sequence shown in SEQ ID NO: 38 is replaced with glycine (G), and the 74th threonine (T) is lysine ( It was replaced with K).
- the modified antibody with amino acid substitution in the above-mentioned antibodies (f) and (g) is an antibody having a higher avidity (antigen binding activity), and is, for example, a cell. It makes it possible to maintain the binding activity with cancer cells in which the expression level of the antigen on the surface is low. In addition, the modified antibody can maintain long-term stable antigen-binding activity during prescription of liquid preparations and in blood (plasma) of monkeys and humans.
- the hybridoma whose accession number is FERM BP-11337 is referred to as "Mouse-Mouse hybridoma BA-1-3D" and is entrusted on February 1, 2011.
- the hybridoma numbered FERM BP-10707 is referred to as “Mouse-Mouse hybridoma: M3-1" and is dated October 18, 2006, and the hybridoma numbered FERM BP-10899 is "Mouse-Mouse hybridoma DI-”.
- anti-hDLK-1 antibody examples include the above-mentioned various anti-hDLK-1 antibodies that bind to a site (for example, an epitope) to which the above-mentioned various anti-hDLK-1 antibodies bind (recognize).
- Antibodies that can compete with the hdLK-1 antibody are also preferably mentioned.
- the epitope (antigen determinant) of the anti-hDLK-1 antibody is not limited as long as it is at least a part of the antigen hDLK-1, but is not limited, for example, hDLK shown in SEQ ID NO: 2.
- the region consisting of the 24th to 91st amino acids the region containing EGF-1 to EGF-2 of hDLK-1
- the region consisting of the 92nd to 167th amino acids the region consisting of the 92nd to 167th amino acids.
- At least a part of the region containing EGF-3 to EGF-4 of hDLK-1) or the region consisting of the 131st to 244th amino acids (the region containing EGF-4 to EGF-6 of hDLK-1). It is preferable to have. Among them, the region containing EGF-1 to EGF-2 of hDLK-1 is more preferable.
- the anti-hDLK-1 antibody that recognizes the region (binds to the region) has high internalization activity into tumor cells, and is extremely useful for applications such as immunoconjugates described later.
- Recombinant antibody and antibody fragment / recombinant antibody One of the preferred embodiments of the anti-hDLK-1 antibody is a recombinant antibody.
- the recombinant antibody include, but are not limited to, chimeric antibodies and humanized antibodies.
- a chimeric antibody that is, a human chimeric antibody
- a chimeric antibody is an antibody in which a variable region of a mouse-derived antibody is linked (conjugated) to a human-derived constant region (Proc. Natl. Acad. Sci. USA 81,6851). -6855, (1984), etc.), when producing a chimera, it can be easily constructed by gene recombination technology so that an antibody thus linked can be obtained.
- CDRs complementarity determining regions
- FR framework region
- CDR transplantation Reconstructed variable regions
- amino acids preferably 1 to several, more preferably 1 to 2 amino acids
- Amino acid-substituted, modified amino acids are also included.
- the modified humanized anti-hDLK-1 antibody it is possible to make a humanized antibody having even higher avidity (antigen binding activity), for example, binding activity to cancer cells having a low expression level of antigen on the cell surface. It enables the retention of.
- the modified humanized anti-hDLK-1 antibody can also be capable of retaining long-term stable antigen-binding activity during prescription of a liquid preparation and in the blood (plasma) of monkeys and humans.
- the chimeric antibody and the humanized antibody are preferably, for example, those in which the N-glycosidic bond complex type sugar chain in the antibody Fc region is a sugar chain in which fucose is not bound to N-acetylglucosamine at the reducing end of the sugar chain.
- the 1-position of the fucose consists of a recombinant antibody molecule having a sugar chain in the Fc region of the antibody molecule, which is not ⁇ -bonded to the 6-position of N-acetylglucosamine at the reducing end of the N-glycosidic bond complex sugar chain.
- Examples include antibodies. With such an antibody, ADCC activity can be dramatically improved. This point (characteristic of the N-glycosidic bond complex type sugar chain in the antibody Fc region) is also preferable for the above-mentioned polyclonal antibody and monoclonal antibody.
- a fragment of the anti-hDLK-1 antibody can also be used together with the anti-hDLK-1 antibody of the present invention or in place of the anti-hDLK-1 antibody of the present invention.
- the antibody fragment has a binding activity to hdLK-1 and an in vivo antitumor activity, similar to the anti-hDLK-1 antibody of the present invention (including humanized antibodies other than mouse antibodies). It has.
- the antibody fragment means a region of a part of an anti-hDLK-1 polyclonal antibody or an anti-hDLK-1 monoclonal antibody (that is, an antibody fragment derived from the anti-hDLK-1 antibody of the present invention), for example, Fab, Fab'.
- F (ab') 2 Fv (variable fragment of antibody), single chain antibody (H chain, L chain, H chain V region, L chain V region, etc.), scFv, diabody (scFv dimer), Examples thereof include dsFv (disulfide stabilized V region) and peptides containing at least a part of a complementency determination region (CDR).
- Fab has an antigen-binding activity having a molecular weight of about 50,000, in which about half of the N-terminal side of the H chain and the entire L chain are bonded by a disulfide bond among the fragments obtained by treating the antibody molecule with the proteolytic enzyme papain. It is an antibody fragment.
- F (ab') 2 is an antigen bond having a molecular weight of about 100,000, which is slightly larger than the fragment obtained by treating an antibody molecule with the proteolytic enzyme pepsin, in which Fab is bound via a disulfide bond in the hinge region. It is an active antibody fragment. It can also be produced by thioether-bonding or disulfide-bonding Fab, which will be described later.
- Fab' is an antibody fragment having an antigen-binding activity having a molecular weight of about 50,000, which is obtained by cleaving the disulfide bond in the hinge region of F (ab') 2 .
- scFv is a VH-P-VL or VL-P-VH in which one H chain V region (VH) and one L chain V region (VL) are linked using an appropriate peptide linker (P). It is a polypeptide and is an antibody fragment having antigen-binding activity.
- scFv obtains the cDNA encoding VH and VL of the antibody, constructs the DNA encoding scFv, inserts the DNA into a prokaryotic expression vector or a prokaryotic expression vector, and inserts the expression vector into a prokaryote. It can be expressed and produced by introducing it into a living organism or a prokaryote.
- Diabody is an antibody fragment in which scFv is dimerized, and is an antibody fragment having divalent antigen-binding activity.
- the divalent antigen-binding activities can be the same, or one of them can have different antigen-binding activities.
- Diabody obtains the cDNA encoding VH and VL of the antibody, constructs the DNA encoding scFv so that the length of the amino acid sequence of P is 8 residues or less, and expresses the DNA as a prokaryotic expression vector. Alternatively, it can be produced by inserting it into an expression vector for eukaryotes and expressing it by introducing the expression vector into a prokaryote or eukaryote.
- dsFv refers to a polypeptide in which one amino acid residue in each of VH and VL is replaced with a cysteine residue, which is bound via a disulfide bond between the cysteine residues.
- the amino acid residue to be replaced with the cysteine residue can be selected based on the three-dimensional structure prediction of the antibody according to the method shown by Reiter et al. (Protein Engineering, 7, 697-704, 1994).
- dsFv obtain the cDNA encoding VH and VL of the antibody, construct the DNA encoding dsFv, insert the DNA into a prokaryotic expression vector or a prokaryotic expression vector, and insert the expression vector into a prokaryote. It can be expressed and produced by introducing it into a living organism or a prokaryote.
- a peptide containing a CDR comprises at least one region of a CDR (CDR1 to 3) of a VH or VL.
- Peptides containing multiple CDRs can be linked either directly or via a suitable peptide linker.
- the CDR-containing peptide constructs a DNA encoding the VH and VL CDRs of the antibody, inserts the DNA into a prokaryotic expression vector or a prokaryotic expression vector, and inserts the expression vector into a prokaryotic or eukaryotic expression vector. It can be expressed and produced by introducing it into an organism.
- the peptide containing CDR can also be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method).
- the antibody fragment used in the present invention is a part of the antibody Fc region in which the N-glycosidic bond complex sugar chain is a sugar chain in which fucose is not bound to N-acetylglucosamine at the reducing end of the sugar chain in the same shape.
- the antibody fragment may be an antibody fragment containing all of them, or an antibody Fc region in which the above-mentioned antibody fragment and N-glycosidic bond complex sugar chain are sugar chains in which fucose is not bound to N-acetylglucosamine at the reducing end of the sugar chain. It may be a fusion protein with a part or all of. Such an antibody fragment is preferable because it can dramatically improve ADCC activity.
- Specific examples of the antibody fragment used in the present invention are not limited, but include, for example, CDR1 to 3 in the H chain V region and CDR1 to 3 in the L chain V region in the various anti-hDLK-1 antibodies described above. And those including the whole H chain V region and the whole L chain V region.
- the anti-hDLK-1 antibody and antibody fragment used in the present invention may be in the form of a complex with a compound having antitumor activity and / or cell killing activity.
- An antibody molecule or antibody fragment molecule and a compound having antitumor activity and / or cell-killing activity are prepared in advance, and then the compound obtained by combining these is generally referred to as an immunoconjugate. Is called.
- protein toxin as a compound having antitumor activity and / or cell-killing activity is linked with the gene of an antibody or antibody fragment on the gene and expressed as one protein (fusion protein).
- the obtained product is generally referred to as immunotoxin.
- Examples of the compound having antitumor activity include doxorubicin, calikeamycin, mitomycin C, auristatin E and the like.
- Examples of the compound having cell-killing activity include saporin, lysine, Pseudomonas aeruginosa exotoxin, diphtheria toxin and the like, and among them, saporin and Pseudomonas aeruginosa exotoxin are preferably used.
- the method for producing the complex is not limited, and examples thereof include a method of coupling an antibody and a drug by a disulfide bond or a hydrazone bond.
- the anti-hDLK-1 antibody used in the present invention has excellent internalization activity into target tumor cells expressing hDLK-1. Therefore, by compounding compounds having antitumor activity and / or cell-killing activity in advance, these compounds can act directly and highly selectively on tumor cells.
- the complex is extremely excellent in the ability to deliver a drug to a target tumor cell.
- the intracellular internalization activity can be evaluated by fluorescently labeling the antibody with rhodamine or the like and observing the intracellular migration behavior and the localization of the antibody using a fluorescence microscope or the like.
- the content ratio of the anti-hDLK-1 antibody as an active ingredient is not limited and can be appropriately set, but for example, 0.01 to 99.99 for the entire combination drug. It can be in the range of% by weight, preferably 0.01 to 30% by weight, more preferably 0.05 to 20% by weight, and even more preferably 0.1 to 10% by weight. When the content ratio of the active ingredient is within the above range, the combination drug of the present invention can sufficiently exert a therapeutic effect on cancer.
- the combination drug of the present invention contains an FGFR4 inhibitor or the like and an anti-hDLK-1 antibody or the like as active ingredients, respectively, and is another drug used for cancer or tumor (for example, an anticancer agent). May further include.
- the combination drug of the present invention has various routes of administration, specifically, for humans or non-human mammals (eg, mice, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.) as test subjects. Specifically, it can be administered orally or parenterally (for example, intravenous injection (intravenous injection), intramuscular injection, intraperitoneal injection, subcutaneous injection, rectal administration, transdermal administration).
- the combination drug of the present invention can be used alone, but it can be formulated into an appropriate dosage form using a pharmaceutically acceptable carrier by a method commonly used according to the route of administration. can.
- the combination drug of the present invention also includes the form of an antibody-drug complex in which a substance that inhibits or suppresses tyrosine kinase activity is bound to an anti-DLK-1 antibody.
- Examples of the dosage form include tablets, powders, fine granules, granules, coated tablets, capsules, internal liquids, suspensions, emulsions, syrups, troches and the like, and non-oral preparations.
- oral preparations include injections (including infusions), inhalants, ointments, nasal drops, and liposomes.
- the carriers that can be used for the formulation of these formulations include, for example, commonly used excipients, binders, disintegrants, lubricants, colorants, and flavoring agents, as well as stabilizers and emulsifiers, if necessary.
- Absorption promoters Surfactants, pH regulators, preservatives, antioxidants, bulking agents, wetting agents, surface activators, dispersants, buffers, preservatives, solubilizing agents, soothing agents, etc. It is possible to formulate a pharmaceutical product by a conventional method by blending a known ingredient that can be used as a raw material for a pharmaceutical product.
- Non-toxic substances that can be used as the component include, for example, animal and vegetable oils such as soybean oil, beef fat, and synthetic glyceride; hydrocarbons such as liquid paraffin, squalane, and solid paraffin; octyldodecyl myristate, and isopropyl myristate.
- Esther oils such as; higher alcohols such as cetostearyl alcohols and behenyl alcohols; silicon resins; silicon oils; polyoxyethylene fatty acid esters, sorbitan fatty acid esters, glycerin fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene hydrogenated castor oil, and the like.
- Surfactants such as polyoxyethylene-polyoxypropylene block copolymers; water-soluble polymers such as hydroxyethyl cellulose, polyacrylic acid, carboxyvinyl polymers, polyethylene glycol, polyvinylpyrrolidone, and methylcellulose; lower alcohols such as ethanol and isopropanol; Polyhydric alcohols (polyols) such as glycerin, propylene glycol, dipropylene glycol, sorbitol, and polyethylene glycol; sugars such as glucose and sucrose; inorganic powders such as silicic acid anhydride, aluminum magnesium silicate, and aluminum silicate. Inorganic salts such as sodium chloride and sodium phosphate; purified water and the like, all of which may be salts thereof or hydrates thereof.
- water-soluble polymers such as hydroxyethyl cellulose, polyacrylic acid, carboxyvinyl polymers, polyethylene glycol, polyvinylpyrrolidone, and methylcellulose
- excipient examples include lactose, fructose, corn starch, sucrose, glucose, mannitol, sorbit, crystalline cellulose, silicon dioxide and the like
- binder examples include polyvinyl alcohol, polyvinyl ether, methyl cellulose, ethyl cellulose and gum arabic.
- Calcium carbonate, sodium hydrogen carbonate, calcium citrate, dextrin, pectin, carboxymethyl cellulose / calcium and the like are used as lubricants, and examples of the lubricant include magnesium stearate, talc, polyethylene glycol, silica and cured vegetable oil as colorants.
- the flavoring agent for example, cocoa powder, syrup brain, aromatic powder, sardine oil, dragon brain, katsura powder, etc. are preferably mentioned, and all of them are permitted to be added to pharmaceuticals. It may be the salt or a hydrate thereof.
- the dose of the combination drug of the present invention is determined by considering the mixing ratio of the active ingredient in the pharmaceutical product, the age and weight of the subject (patient), the type and progress of the disease, the route of administration, and the number of administrations. (/ 1 day), it can be set in a wide range as appropriate in consideration of the administration period and the like.
- the active ingredient FGFR4 inhibitor or the like and the anti-hDLK-1 antibody or the like may be administered substantially simultaneously, or one of them may be administered first and the other in order. However, it is not limited. Further, these administrations may be administered in a single dose or continuously in an amount contained in the combination drug, and are not limited.
- the combination drug of the present invention is used as a parenteral preparation or an oral preparation
- its form is generally not limited, but in the case of various injectable preparations, for example, the state of a unit dose ampoule or a high dose container, or the state of a lyophilized powder to be redissolved in a solution at the time of use. Can be provided at.
- the parenteral preparation includes various known excipients and additives according to various forms, and the effect of the active ingredient is not impaired. It can be contained in a range.
- water, glycerol, propylene glycol, aliphatic polyalcohols such as polyethylene glycol and the like can be mentioned.
- the dose (per day) of the parenteral preparation is not limited, but for example, in the case of various injections, the active ingredients such as FGFR4 inhibitor and anti-hDLK-1 antibody are generally applied (subjects). , Patients, etc.) Per 1 kg of body weight, 0.01 to 1000 mg, 0.05 to 500 mg, or 0.1 to 50 mg can be taken, or 0.5 to 20 mg can be taken or 1 to 10 mg can be taken. It can be as much as possible.
- its form is generally not limited, and it may be any of the above-mentioned dosage forms, or it may be a dried product to be redissolved when used.
- the oral preparation includes various known excipients and additives according to various forms within a range in which the effect of the active ingredient is not impaired. Can be contained in.
- binders for example, binders (syrup, gum arabic, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone, etc.), fillers (lactose, sugar, cornstarch, potato starch, calcium phosphate, sorbitol, glycine, etc.), lubricants (magnesium stearate, talc, etc.)
- fillers lactose, sugar, cornstarch, potato starch, calcium phosphate, sorbitol, glycine, etc.
- lubricants magnesium stearate, talc, etc.
- examples thereof include polyethylene glycol (polyethylene glycol, silica, etc.), disintegrants (various starches, etc.), and wetting agents (sodium lauryl sulfate, etc.).
- the dose of the oral preparation (per day) is generally 0.05 to 5000 mg per 1 kg of body weight of the target (subject, patient, etc.) to which the active ingredients such as FGFR4 inhibitor and anti-hDLK-1 antibody are applied. , 0.1 to 1000 mg, or 0.1 to 100 mg can be taken, or 0.5 to 50 mg can be taken or 1 to 10 mg can be taken. Further, the mixing ratio of the active ingredient in the oral preparation is not limited, and can be appropriately set in consideration of the number of administrations per day and the like.
- the combination drug of the present invention is an existing FGFR4 selective tyrosine kinase for the treatment of various cancers, particularly hepatocellular carcinoma and other cancers in which FGFR4 is expressed and / or FGFR4 tyrosine kinase activity is observed. It can exert a stronger and more sustained antitumor effect than an inhibitor, and for example, it can suppress the growth of cancer cells or shrink or eliminate a tumor even after the end of administration of the drug. Is. Until now, it is possible to exert a sufficient therapeutic effect even for cancer patients whose therapeutic effect cannot be expected (or unresponsive) by the single administration of the above-mentioned existing tyrosine kinase inhibitor alone. The combination drug of the present invention is extremely useful.
- the combination drug of the present invention may have the effect of delaying, stopping, or reducing the onset of at least one clinical symptom associated with a disease (cancer described above) and is not recognizable by the patient. Including those, it may be one that alleviates, improves or stabilizes at least one physical parameter (eg, weight loss, etc.) associated with a disease (cancer described above).
- compositions (i) to (iv) below.
- a pharmaceutical composition for treating cancer which comprises an antibody against human DLK-1 or an antibody fragment derived from the antibody, which has antitumor activity in vivo. It is used in combination with a substance that inhibits or suppresses the tyrosine kinase activity of FGFR4, or a prodrug thereof, or a pharmacologically acceptable salt thereof, or a hydrate or solvate thereof.
- the pharmaceutical composition is used in combination with a substance that inhibits or suppresses the tyrosine kinase activity of FGFR4, or a prodrug thereof, or a pharmacologically acceptable salt thereof, or a hydrate or solvate thereof.
- Treat cancers comprising substances that inhibit or suppress the tyrosine kinase activity of FGFR4, or prodrugs thereof, or pharmacologically acceptable salts thereof, or hydrates or solvates thereof.
- Is a pharmaceutical composition for It is used in combination with an antibody against human DLK-1, which has antitumor activity in vivo, or an antibody fragment derived from the antibody.
- the pharmaceutical composition is used in combination with an antibody against human DLK-1, which has antitumor activity in vivo, or an antibody fragment derived from the antibody.
- a pharmaceutical composition for treating cancer which comprises an antibody against human DLK-1 or an antibody fragment derived from the antibody, which has antitumor activity in vivo.
- the treatment comprises administration of a substance that inhibits or suppresses the tyrosine kinase activity of FGFR4, or a prodrug thereof, or a pharmacologically acceptable salt thereof, or a hydrate or solvate thereof.
- the pharmaceutical composition comprising substances that inhibit or suppress the tyrosine kinase activity of FGFR4, or prodrugs thereof, or pharmacologically acceptable salts thereof, or hydrates or solvates thereof.
- a pharmaceutical composition for The treatment comprises administration of an antibody against human DLK-1, or an antibody fragment derived from the antibody, which has antitumor activity in vivo.
- the pharmaceutical composition comprises administration of an antibody against human DLK-1, or an antibody fragment derived from the antibody, which has antitumor activity in vivo.
- Kit The present invention can also provide a form of a kit for treating cancer, which comprises an FGFR4 inhibitor or the like and an anti-hDLK-1 antibody or the like as components.
- a kit for treating cancer which comprises an FGFR4 inhibitor or the like and an anti-hDLK-1 antibody or the like as components.
- the FGFR4 inhibitor and the like and the anti-hDLK-1 antibody and the like in the kit may be provided in a dissolved state, for example, in consideration of stability (preservation), ease of use and the like.
- the kit can contain other components as appropriate in addition to the FGFR4 inhibitor and the like and the anti-hDLK-1 antibody and the like.
- a labeling substance for an antibody or a solid phase reagent on which an antibody or a labeling substance thereof is immobilized can be included.
- the antibody-labeled substance means a substance labeled with an enzyme, a radioisotope, a fluorescent compound, a chemiluminescent compound, or the like.
- Various plates, preservatives such as sodium azide, and experimental operation manuals (instructions) may be included.
- the kit may include at least the above-mentioned FGFR4 inhibitor and the like and an anti-hDLK-1 antibody and the like as components. Therefore, all of the essential components for the treatment of cancer may be included in the kit together, or may be provided individually, without limitation.
- Hep3B cells (1x10 6 cells) were transplanted subcutaneously to the right flank of NOD-SCID mice, and when the tumor volume reached 250 to 350 mm 3 , 6 animals in each group were divided into the following 8 groups. The administration was performed on the same day.
- FIG. 1 shows a vehicle administration group, a HuBA-1-3D (1 mg / kg) administration group, an H3B-6527 (50 mg / kg) administration group, and a HuBA-1-3D (1 mg / kg) + H3B-6527 (50 mg / kg).
- the Ki-67 stained image (upper row) and the cleared caspase-3 stained image (lower row) in the combined administration group are shown.
- FIG. 2 shows the results when FGF-401 (10 mg / kg) and HuBA-1-3D (1 mg / kg) were co-administered
- FIG. 3 shows the results with BLU-554 (30 mg / kg). The results when co-administered with HuBA-1-3D (1 mg / kg) are shown.
- the same photographs as those in FIG. 1 were used for the photographs of the Vehicle-administered group and the HuBA-1-3D (1 mg / kg) -administered group in FIGS. 2 and 3.
- Hep3B Xenograft showed a synergistic effect on the inhibition of cancer cell growth in tumors, and further showed that in the combination administration group, cancer cell apoptosis was induced in a wide range of tumors.
- cyclin B1 which is a cell proliferation marker
- HuBA-1-3D (1 mg / kg) administration group a decrease in the expression of cyclin B1 was observed in one individual in which apoptosis was induced. In the remaining two individuals, the expression level of cyclin B1 in the Apoptosis group was equivalent. In the single administration group of the three FGFR4 inhibitors, the expression level of cyclin B1 was not different from that in the Vehicle administration group in any of H3B-6527, FGF-401, and BLU-554.
- Hep3B cells were transplanted subcutaneously to the right flank of NOD-scid mice (Day 0), and when the average tumor volume reached about 100 mm 3 (Day 14), the cells were divided into the following 6 groups on the same day.
- HuBA-1-3D was administered twice a week (Day 14, 18, 22, 25)
- H3B-6527 was administered twice a day (BID) for 5 days a week, with a 2-day washout cycle.
- Medication was given (Day 14, 15, 16, 17, 18, 21, 22, 23, 24, 25).
- Tumor volume was measured twice a week, and individuals who reached 1,500 mm 3 were considered to have completed observation.
- the H3B-6527 + HuBA-1-3D (1 mg / kg) administration group had the H3B-6527 administration group and the HuBA-1.
- the tumor volume was significantly smaller than that of each of the -3D (1 mg / kg) -administered groups ( ** P ⁇ 0.05 by Tukey test).
- tumor regression further progressed from the administration end date (Day 25), and at the time of Day 32, tumor disappearance was observed in 5 out of 8 individuals. ..
- H3B-6527 (50 mg / kg) administration group HuBA-1-3D (1 mg / kg) administration group, H3B-6527 (50 mg / kg) ) + HuBA-1-3D (1 mg / kg) combined administration group of mice was sacrifice and the tumor was excised.
- H3B-6527 (50 mg / kg) administration group and the HuBA-1-3D (1 mg / kg) administration group tumors were confirmed in all 8 individuals, but H3B-6527 (50 mg / kg) + HuBA.
- the tumor weight was significantly smaller than that ( * P ⁇ 0.05 by Takey), and it was confirmed that the combined use of HuBA-1-3D and H3B-6527 enhanced the antitumor effect.
- the above results are shown in FIG. 5C.
- Hep3B cells (1x10 6 cells) were transplanted subcutaneously into the right flank of NOD-SCID mice (Day 0), and were grouped into the following 6 groups at the stage of Day 14 when the average tumor volume exceeded 100 mm 3 and were divided into the following 6 groups on the same day. Administration was started from.
- HuBA-1-3D was administered intraperitoneally once a day for a total of 4 times of Days 14, 17, 21, 24, and FGF-401 was orally administered twice a day, Day 14, 15, ,. 16, 17, 18, 21, 22, 23, 24, 25, a total of 10 times.
- the combined administration of HuBA-1-3D and FGF-401 was performed four times in total on Days 14, 17, 21, and 24. Mice in which the tumor volume of each mouse in each group exceeded 1,500 mm 3 were sacrificed at that time.
- Significant in all administration groups 28 days after cancer cell transplantation (Day 0) (Day 28, 3 days after last administration day (Day 25)) compared to the tumor volume of the Vehickle administration group (N 8). No antitumor effect was observed ( * P ⁇ 0.05 by Dunnett).
- FGF-401 (10 mg / kg) + HuBA-1-3D (0.3 mg / kg) was compared with the FGF-401 (10 mg / kg) administration group.
- Significant antitumor effects were observed in the combination group and the FGF-401 (10 mg / kg) + HuBA-1-3D (1 mg / kg) combination group (P ⁇ 0.05), respectively (P ⁇ 0.05), and HuBA-1-3D (0).
- a significant antitumor effect was observed in the FGF-401 (10 mg / kg) + HuBA-1-3D (0.3 mg / kg) combination group as compared with the .3 mg / kg) administration group (P ⁇ 0. 05).
- the FGF-401 (10 mg / kg) + HuBA-1-3D (0.3 mg / kg) combination group is more resistant.
- the tumor effect was enhanced (P ⁇ 0.05), and the combined administration of HuBA-1-3D and FGF-401 was observed to enhance the antitumor effect.
- FGF-401 (10 mg / kg) + HuBA-1-3D (0.3 mg / kg) was compared with the FGF-401 (10 mg / kg) administration group.
- a significant antitumor effect was observed (P ⁇ 0.05 by Tukey).
- the FGF-401 (10 mg / kg) + HuBA-1-3D (0.3 mg / kg) combination group had a significant antitumor effect ( P ⁇ 0.05 by Tukey), and enhanced antitumor effect was observed by the combined use of FGF-401 and HuBA-1-3D.
- the antitumor effect of the combined administration of FGF-401 (10 mg / kg) + HuBA-1-3D (1 mg / kg) was remarkable, and the tumor disappeared in 7 out of 8 individuals, and 86.2 mm 3 in 1 individual. Only a tumor was confirmed.
- FGFR4 is similar to H3B-6527 and FGF-401 for the purpose of further verifying the effect of enhancing the antitumor activity by the combined administration of the HuBA-1-3D antibody and the FGFR4 inhibitor.
- BLU-554 Cancer Discov. 2019
- HuBA-1-3D was administered intraperitoneally once a day for a total of 4 times of Days 14, 17, 21 and 24, and BLU-554 was orally administered twice a day for Day 14, 15, 16, 17, 18, 21, 22, 23, 24, 25, a total of 10 times.
- the combined administration of HuBA-1-3D and BLU-554 was performed four times in total on Days 14, 17, 21, and 24. Mice in which the tumor volume of each mouse in each group exceeded 1,500 mm 3 were sacrificed at that time.
- the HuBA-1-3D (1 mg / kg) administration group and the HuBA-1-3D (1 mg / kg) + BLU-554 (30 mg / kg) reached 499.4 ⁇ 449.4 mm 3
- CH518324 / Debio1347 is a tyrosine kinase inhibitor that can be orally administered selectively to FGFR, and its tyrosine kinase inhibitory activity (IC 50 ) against four types of FGFR (FGFR1, FGFR2, FGFR3, FGFR4) is 9.3 nM and 7, respectively. It has been reported to be .6 nM, 22 nM, and 290 nM (Mol Cancer Ther (2014); 13 (11); 2547-58).
- HuBA-1-3D was administered intraperitoneally once daily for a total of 4 times of Days 14, 18, 21 and 24, and CH518324 / Debio 1347 was orally administered once daily for Day 14, 15, 16, 17, 18, 21, 22, 23, 24, 25, a total of 10 times.
- the combined administration of HuBA-1-3D and CH518324 / Debio1347 was performed four times in total on Days 14, 18, 21, and 24. Mice in which the tumor volume of each mouse in each group exceeded 1,500 mm 3 were sacrificed at that time.
- the groups were almost completely overlapped, and the HuBA-1-3D (1 mg / kg) administration group ( ⁇ ) and the HuBA-1-3D (1 mg / kg) + CH518324 / Debio1347 (50 mg / kg) combination administration group ( ⁇ ) Tumor formation by day 38 (test end date) almost completely overlapped in both groups, with HuBA-1-3D antibody in combination with CH518324 / Debio1347, a selective tyrosine kinase inhibitor for FGFR1 / FGFR2 / FGFR3. No effect of enhancing the antitumor effect by administration was observed.
- the invention is more potent than existing tyrosine kinase inhibitors in the treatment of cancer, especially in the treatment of cancers in which FGFR4 is expressed and / or FGFR4 has tyrosine kinase activity. Since it is possible to provide a therapeutic agent and a therapeutic method capable of exerting a sustained antitumor effect, the therapeutic agent and the therapeutic method of the present invention can be used, for example, for a patient who has not been expected to have a therapeutic effect so far. Is extremely useful in that it can also be effective.
- SEQ ID NO: 23 Recombinant DNA SEQ ID NO: 24: Synthetic construct (recombinant protein) SEQ ID NO: 25: Recombinant DNA SEQ ID NO: 26: Synthetic construct (recombinant protein) SEQ ID NO: 27: Recombinant DNA SEQ ID NO: 28: Synthetic construct (recombinant protein) SEQ ID NO: 29: Recombinant DNA SEQ ID NO: 30: Synthetic construct (recombinant protein) SEQ ID NO: 31: Recombinant DNA SEQ ID NO: 32: Synthetic construct (recombinant protein) SEQ ID NO: 33: Recombinant DNA SEQ ID NO: 34: Synthetic construct (recombinant protein) SEQ ID NO: 35: Recombinant DNA SEQ ID NO: 36: Synthetic construct (recombinant protein) SEQ ID NO: 37: Recombinant DNA SEQ ID NO: 38: Synthetic construct (recombinant protein) SEQ ID NO: 39: Recombinant DNA SEQ ID NO: 40: Synthetic construct (
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Abstract
Description
切除不能な進行性の肝細胞がんに対する治療薬としては、複数の受容体チロシンキナーゼに対するマルチキナーゼ阻害剤が使用されている。具体的には、第1選択薬としてはソラフェニブやレンバチニブ、第2選択薬としてはレゴラフェニブやカボザンチニブが使用されているが、効果は限定的である。
(1)がんを治療するための組合せ医薬であって、
線維芽細胞増殖因子受容体4(FGFR4)のチロシンキナーゼ活性を阻害又は抑制する物質、若しくはそのプロドラッグ、又はそれらの薬理学的に許容し得る塩、あるいはそれらの水和物若しくは溶媒和物、及び
in vivoで抗腫瘍活性を有する、ヒトDLK−1に対する抗体、若しくは当該抗体に由来する抗体断片
を含む、前記組合せ医薬。
(2)in vivoで抗腫瘍活性を有する、ヒトDLK−1に対する抗体、若しくは当該抗体に由来する抗体断片を含む、がんを治療するための医薬組成物であって、
線維芽細胞増殖因子受容体4(FGFR4)のチロシンキナーゼ活性を阻害又は抑制する物質、若しくはそのプロドラッグ、又はそれらの薬理学的に許容し得る塩、あるいはそれらの水和物若しくは溶媒和物と組み合わせて用いるものである、
前記医薬組成物。
in vivoで抗腫瘍活性を有する、ヒトDLK−1に対する抗体、若しくは当該抗体に由来する抗体断片と組み合わせて用いるものである、
前記医薬組成物。
(4)in vivoで抗腫瘍活性を有する、ヒトDLK−1に対する抗体、若しくは当該抗体に由来する抗体断片を含む、がんを治療するための医薬組成物であって、
前記治療が、線維芽細胞増殖因子受容体4(FGFR4)のチロシンキナーゼ活性を阻害又は抑制する物質、若しくはそのプロドラッグ、又はそれらの薬理学的に許容し得る塩、あるいはそれらの水和物若しくは溶媒和物の投与を含むものである、
前記医薬組成物。
前記治療が、in vivoで抗腫瘍活性を有する、ヒトDLK−1に対する抗体、若しくは当該抗体に由来する抗体断片の投与を含むものである、
前記医薬組成物。
(6)線維芽細胞増殖因子受容体4(FGFR4)のチロシンキナーゼ活性を阻害又は抑制する物質が、H3B−6527、FGF−401(ロブリチニブ)又はBLU−554(フィソガチニブ)である、上記(1)に記載の組合せ医薬、又は上記(2)~(5)のいずれか1項に記載の医薬組成物。
(8)前記がん及び/又は前記腫瘍が、肝細胞がん、肺がん、子宮体がん、胆管がん、肝内胆管がん、食道がん、鼻咽頭がん、卵巣がん、乳がん、腎細胞がん、膵臓がん、結腸がん、及び膠芽腫、並びに横紋筋肉腫を含む肉腫からなる群より選ばれる少なくとも1種である、上記(1)に記載の組合せ医薬、又は上記(2)~(5)のいずれか1項に記載の医薬組成物。
(9)前記抗体は、キメラ抗体又はヒト化抗体である、上記(1)に記載の組合せ医薬、又は上記(2)~(5)のいずれか1項に記載の医薬組成物。
(a)H鎖V領域のCDR1~3のアミノ酸配列が、それぞれ順に、配列番号3~5で示されるアミノ酸配列であり、かつ、L鎖V領域のCDR1~3のアミノ酸配列が、それぞれ順に、配列番号6~8で示されるアミノ酸配列である抗体、
(b)H鎖V領域のCDR1~3のアミノ酸配列が、それぞれ順に、配列番号9~11で示されるアミノ酸配列であり、かつ、L鎖V領域のCDR1~3のアミノ酸配列が、それぞれ順に、配列番号12~14で示されるアミノ酸配列である抗体、
(c)H鎖V領域のアミノ酸配列が配列番号16で示されるアミノ酸配列からなり、かつ、L鎖V領域のアミノ酸配列が配列番号18で示されるアミノ酸配列からなる抗体、
(d)H鎖V領域のアミノ酸配列が配列番号20で示されるアミノ酸配列からなり、かつ、L鎖V領域のアミノ酸配列が配列番号22で示されるアミノ酸配列からなる抗体、
(e)H鎖V領域のアミノ酸配列が配列番号24又は26で示されるアミノ酸配列からなり、かつ、L鎖V領域のアミノ酸配列が配列番号28で示されるアミノ酸配列からなる抗体、
(f)H鎖V領域のアミノ酸配列が配列番号30、32、34又は36で示されるアミノ酸配列からなり、かつ、L鎖V領域のアミノ酸配列が配列番号46で示されるアミノ酸配列からなる抗体、
(g)H鎖V領域のアミノ酸配列が配列番号38、40、42又は44で示されるアミノ酸配列からなり、かつ、L鎖V領域のアミノ酸配列が配列番号46で示されるアミノ酸配列からなる抗体、
(h)受託番号がFERM BP−10899であるハイブリドーマにより産生される抗体、
(i)受託番号がFERM BP−10707であるハイブリドーマにより産生される抗体、
(j)受託番号がFERM BP−10900であるハイブリドーマにより産生される抗体、
及び
(k)受託番号がFERM BP−11337であるハイブリドーマにより産生される抗体からなる群から選択される少なくとも1種である、
上記(1)に記載の組合せ医薬、又は上記(2)~(5)のいずれか1項に記載の医薬組成物。
(12)前記組合せ医薬又は医薬組成物の投薬終了後においても、がん細胞の増殖を抑制し得る又は腫瘍を縮小若しくは消失させ得るものである、上記(1)に記載の組合せ医薬、又は上記(2)~(5)のいずれか1項に記載の医薬組成物。
(13)がんの治療用の薬剤を製造するための、
線維芽細胞増殖因子受容体4(FGFR4)のチロシンキナーゼ活性を阻害又は抑制する物質、若しくはそのプロドラッグ、又はそれらの薬理学的に許容し得る塩、あるいはそれらの水和物若しくは溶媒和物、及び
in vivoで抗腫瘍活性を有する、ヒトDLK−1に対する抗体、若しくは当該抗体に由来する抗体断片
の使用。
in vivoで抗腫瘍活性を有する、ヒトDLK−1に対する抗体、若しくは当該抗体に由来する抗体断片
を被験対象に投与することを特徴とする、がんの治療方法。
(15)線維芽細胞増殖因子受容体4(FGFR4)のチロシンキナーゼ活性を阻害又は抑制する物質、若しくはそのプロドラッグ、又はそれらの薬理学的に許容し得る塩、あるいはそれらの水和物若しくは溶媒和物、及び
in vivoで抗腫瘍活性を有する、ヒトDLK−1に対する抗体、若しくは当該抗体に由来する抗体断片
を含む、がんを治療するためのキット。
(16)前記がん及び/又は前記腫瘍が、FGFR4が発現し及び/又はFGFR4のチロシンキナーゼ活性がみられるがんである、上記(13)に記載の使用、上記(14)に記載の方法、又は上記(15)に記載のキット。
発明の効果
図2:Hep3Bゼノグラフトモデルにおける、Vehicle投与群,HuBA−1−3D(1mg/kg)投与群、FGF−401(10mg/kg)投与群、HuBA−1−3D(1mg/kg)+FGF−401(10mg/kg)併用投与群における、投与48時間後の細胞増殖(Ki−67)およびアポトーシス(cleaved caspase−3)の免疫染色で示した写真である.茶褐色で染色されている部分が、増殖(Ki−67陽性)、またはアポトーシス(cleaved caspase−3陽性)している細胞を示す。
図3:Hep3Bゼノグラフトモデルにおける、Vehicle投与群,HuBA−1−3D(1mg/kg)投与群、BLU−554(30mg/kg)投与群、HuBA−1−3D(1mg/kg)+BLU−554(30mg/kg)併用投与群における、投与48時間後の細胞増殖(Ki−67)およびアポトーシス(cleaved caspase−3)の免疫染色で示した写真である.茶褐色で染色されている部分が増殖(Ki−67陽性)または、アポトーシス(cleaved caspase−3陽性)している細胞を示す。
図4:Hep3Bゼノグラフトモデルにおける、Vehicle、HuBA−1−3D(1mg/kg),FGFR4選択的阻害剤(H3B−6527(50mg/kg),FGF−401(10mg/kg),BLU−554(30mg/kg)、HuBA−1−3D(1mg/kg)と各FGFR4阻害剤とを併用投与し、48時間後における腫瘍における、アポトーシス(cleaved caspase−3,cleaved PARP)、および細胞増殖マーカー(サイクリンB1)の発現をウェスタンブロット解析した図である。(a)H3B−6527,(b)FGF−401,(c)BLU−554.N=3.
図5A:HuBA−1−3D抗体とFGFR4選択的阻害剤(H3B−6527)との併用投与による抗腫瘍効果の増強を示す図である。
具体的には、Hep3Bゼノグラフトモデルにおける,Vehicle投与群(○)、HuBA−1−3D単独投与(△,◇)、H3B−6527単独投与(□)、HuBA−1−3DおよびH3B−6527の併用投与(▲、●)による、移植からDay 32(投与終了から7日後)までの腫瘍成長を示した図である。
図5B:HuBA−1−3D抗体とFGFR4選択的阻害剤(H3B−6527)との併用投与による抗腫瘍効果の増強を示す図である。
具体的には、Day 38(最終投与日(Day 25)から13日後)まで、H3B−6527(50mg/kg)投与群(□)、HuBA−1−3D(1mg/kg)投与群(◇)、H3B−6527(50mg/kg)+HuBA−1−3D(1mg/kg)併用投与群(●)の腫瘍体積の推移を観察した図である。
図5C:HuBA−1−3D抗体とFGFR4選択的阻害剤(H3B−6527)との併用投与による抗腫瘍効果の増強を示す図である。
具体的には、Day 38(最終投与日(Day 25)から13日後)における、H3B−6527(50mg/kg)投与群、HuBA−1−3D(1mg/kg)投与群、H3B−6527(50mg/kg)+HuBA−1−3D(1mg/kg)併用投与群の摘出した腫瘍の、(a)外観の写真と、(b)腫瘍重量のグラフである。
図6:HuBA−1−3D抗体とFGFR4選択的阻害剤(FGF−401)との併用投与による抗腫瘍効果の増強を示す図である。
具体的には、Hep3Bゼノグラフトモデルにおける,Vehicle投与群(○)、HuBA−1−3D単独投与群(△,◇)、FGF−401単独投与群(□)、HuBA−1−3DおよびFGF−401の併用投与群(▲、●)による、腫瘍成長を示した図である。腫瘍体積が1,500mm3に達した個体からサクリファイスした。グラフは、全ての投与群において、全個体(各群N=8)が残っている時点までをグラフで示した。
図7:HuBA−1−3D抗体とFGFR4選択的阻害剤(BLU−554)との併用投与による抗腫瘍効果の増強を示す図である。
具体的には、(A)Hep3Bゼノグラフトモデルにおける,Vehicle投与群(○)、HuBA−1−3D単独投与群(◇)、BLU−554単独投与群(□)、HuBA−1−3DおよびBLU−554の併用投与群(●)における平均の腫瘍成長を示した図である。腫瘍体積が1,500mm3に達した個体からサクリファイスした。グラフは、各投与群(群分け時N=8)において、少なくとも7個体が残っている時点までを示した。*P<0.05(vs Vehicle群、Dunnett).(B)Vehicle投与群、HuBA−1−3D単独投与、BLU−554単独投与、HuBA−1−3DおよびBLU−554の併用投与群の各群の個体毎の腫瘍成長を示した図である。
図8:FGFR1/FGFR2/FGFR3に対する阻害剤(CH518324/Debio1347との併用投与では、抗腫瘍効果の増強を示す図である。
具体的には、Hep3Bゼノグラフトモデルにおける,Vehicle投与群(○)、HuBA−1−3D単独投与(△,◇)、CH518324/Debio1347単独投与(□)、HuBA−1−3DおよびCH518324/Debio1347の併用投与(▲、●)における腫瘍成長を示した図である。腫瘍体積が1,500mm3に達した個体からサクリファイスした。グラフは、全ての投与群において、全個体(各群N=8)が残っている時点までをグラフで示した。
前述のとおり、現在、FGFR4が発現し及び/又はFGFR4のチロシンキナーゼ活性がみられるがんの一例である、肝細胞がんに対しては、FGFR4を標的分子とした5種類の阻害剤が臨床開発段階にあり、そのうちの3種類(FGF−401,H3B−6527,BLU−554)は、低分子のTKI(チロシンキナーゼ阻害剤)で、いずれもFGFR4に特異的なATP結合領域にあるCys552に特異的に結合してキナーゼ活性を阻害する作用機序である。これら低分子チロシンキナーゼ阻害剤については、FGF−19過剰発現または、FGFR4を発現している進行性肝細胞がんの患者を対象とした臨床試験が進行しており、いずれもフェーズIもしくはフェーズI/IIの開発段階であるため薬効についての情報は限られているが、上記の各阻害剤の単独療法での治療効果はいずれも限定的である。
本発明者は、上述した既存のチロシンキナーゼ阻害剤よりも強力かつ持続的な抗腫瘍効果を発揮し得る治療薬を開発するため、ヒト肝細胞がん由来Hep3B細胞株を用いたゼノグラフト治療モデルによる実験及び検討を行った。その結果、上述したようなFGFR4選択的チロシンキナーゼ阻害剤と、ヒトDLK−1(delta−like 1homolog(Drosophila);以下「hDLK−1」ということがある)に対する抗体とを併用投与することにより、それぞれの単独投与による場合と比べて、持続的、かつ著しく強い腫瘍縮小効果を発揮することを明らかにした。なお、線維芽細胞増殖因子受容体1、2、3(FGFR1,2,3)に対する低分子阻害剤と、抗hDLK−1抗体との併用投与では併用による抗腫瘍効果の増強は観察されなかった。
本発明者が見出した、FGFR4選択的チロシンキナーゼ阻害剤と抗hDLK−1抗体との併用医薬・併用療法によれば、前述の既存のチロシンキナーゼ阻害剤の単独投与だけでは十分な治療効果が期待できない(又は不応答な)がんの患者に対しても、十分な治療効果が期待できる。本発明は、このようにして完成された。
本発明のがんを治療するための組合せ医薬(以下、「本発明の組合せ医薬」ということがある。)は、前述の通り、
・線維芽細胞増殖因子受容体4(FGFR4)のチロシンキナーゼ活性を阻害又は抑制する物質(以下、「FGFR4阻害剤」ということがある)、若しくはそのプロドラッグ、又はそれらの薬理学的に許容し得る塩、あるいはそれらの水和物若しくは溶媒和物(以下、「FGFR4阻害剤等」ということがある)と、
・in vivoで抗腫瘍活性を有する抗hDLK−1抗体、若しくは当該抗体に由来する抗体断片(以下、「抗hDLK−1抗体等」ということがある)とを
有効成分として含むことを特徴とするものである。
本発明において、がんの治療としては、例えば、がんの進行抑制、予後改善、及び/又は再発防止等も含まれる。
本発明の組合せ医薬の有効成分である、FGFR4阻害剤等は、FGFR4のチロシンキナーゼ活性を阻害又は抑制し得る機能を有する物質であればよく、限定はされない。FGFR4阻害剤としては、例えば、H3B−6527、FGF−401(ロブリチニブ)、及びBLU−554(フィソガチニブ)等のFGFR4選択的チロシンキナーゼ阻害剤が好ましく挙げられる。このようなFGFR4阻害剤等は、独自に合成、抽出及び精製等したものを使用してもよいし、市販のものを使用してもよい。独自に合成等する場合は、H3B−6527であれば、例えば、WO 2015/057938(compound 108,pages 42−45)の記載等を参照することができ、FGF−401であれば、例えば、WO 2015/059668(Example 83)の記載等を参照することができ、BLU−554であれば、例えば、WO 2015/061572A1の記載等を参照することができる。
当該薬理学的に許容し得る塩としては、限定はされないが、例えば、有機スルホン酸塩(例えば、メタンスルホン酸塩(メシル酸塩ともいう)、トリフルオロメタンスルホン酸塩、エタンスルホン酸塩、ベンゼンスルホン酸塩、トルエンスルホン酸塩、及びカンファースルホン酸塩など)、有機カルボン酸塩(例えば、酢酸塩、トリフルオロ酢酸塩、マレイン酸塩、酒石酸塩、フマル酸塩、及びクエン酸塩など)、アミノ酸塩(例えば、アスパラギン酸塩、及びグルタミン酸塩など)、四級アミン塩、アルカリ金属塩(例えば、ナトリウム塩、及びカリウム塩など)、無機酸塩(例えば、硫酸塩、硝酸塩、過塩素酸塩、リン酸塩、炭酸塩、及び重炭酸塩など)、ハロゲン化水素酸塩(例えば、塩酸塩、臭化水素酸塩、及びヨウ化水素酸塩など)、アルカリ土類金属塩(例えば、マグネシウム塩、及びカルシウム塩など)などが好ましく挙げられる。
本発明の組合せ医薬において、有効成分としてのFGFR4阻害剤等の含有割合は、限定はされず、適宜設定することができるが、例えば、組合せ医薬全体に対して、0.01~99.99重量%の範囲内とすることができ、好ましくは、0.01~30重量%、より好ましくは0.05~20重量%、さらに好ましくは0.1~10重量%の範囲内としてもよい。有効成分の含有割合が上記範囲内であることにより、本発明の組合せ医薬は、がんの治療効果を十分に発揮することができる。
本発明の組合せ医薬の有効成分である、抗hDLK−1抗体(in vivoで抗腫瘍活性を有する、ヒトDLK−1に対する抗体)は、以下の説明記載に基づいて作製することができる。
hDLK−1のアミノ酸配列(配列番号2)の情報は、例えばNCBI(GenBank)のウェブサイト(http://www.ncbi.nlm.nih.gov/)に「Accession number:NP_003827」として公表されている。なお、hDLK−1のアミノ酸配列をコードする塩基配列(配列番号1)の情報は、同ウェブサイトに「Accession number:NM 003836」として公表されている。
抗原としては、hDLK−1のアミノ酸配列の少なくとも一部(全部又は一部)を含むポリペプチド又はペプチド(単にペプチドともいう)を使用することができ、好ましくは、hDLK−1の細胞外領域(FA−1)のアミノ酸配列の少なくとも一部(全部又は一部)を含むペプチドを使用することができる。hDLK−1の細胞外領域は、前述したように6つのEGF様モチーフ(EGF−1~EGF−6)を含む領域を言い、配列番号2に示されるアミノ酸配列のうちの第24番目~第244番目のアミノ酸を含む領域、好ましくは、配列番号2に示されるアミノ酸配列のうちの「第24番目」から「第248~285番目」までのアミノ酸からなる領域(およそ225~262アミノ酸残基)のことを言う。
抗原とするペプチドの作製方法は、化学合成でも、大腸菌などを用いる遺伝子工学的手法による合成でもよく、当業者に周知の方法を用いることができる。
ペプチドの化学合成を行う場合は、ペプチドの合成の周知方法によって合成することができる。また、その合成は、固相合成法及び液相合成法のいずれをも適用することができる。市販のペプチド合成装置(例えば、島津製作所製:PSSM−8など)を使用してもよい。
ベクターには、宿主微生物で自律的に増殖し得るファージ又はプラスミドが使用される。さらに、動物ウイルス、昆虫ウイルスベクターを用いることもできる。組換えベクターの作製は、精製されたDNAを適当な制限酵素で切断し、適当なベクターDNAの制限酵素部位等に挿入してベクターに連結すればよい。形質転換に使用する宿主としては、目的の遺伝子を発現できるものであれば特に限定されるものではない。例えば、細菌(大腸菌、枯草菌等)、酵母、動物細胞(COS細胞、CHO細胞等)、昆虫細胞又は昆虫が挙げられる。ヤギ等の哺乳動物を宿主として使用することも可能である。宿主への組換えベクターの導入方法は公知である。
培養後、目的ペプチドが菌体内又は細胞内に生産される場合には、菌体又は細胞を破砕することによりペプチドを抽出する。また、目的ペプチドが菌体外又は細胞外に生産される場合には、培養液をそのまま使用するか、遠心分離等により菌体又は細胞を除去する。その後、ペプチドの単離精製に用いられる一般的な生化学的方法、例えば硫酸アンモニウム沈殿、ゲル濾過、イオン交換クロマトグラフィー、アフィニティークロマトグラフィー等を単独で又は適宜組み合わせて用いることにより、目的のペプチドを単離精製することができる。
本発明においては、無細胞合成系を用いたin vitro翻訳により抗原となるペプチドを得ることもできる。この場合は、RNAを鋳型にする方法とDNAを鋳型にする方法(転写/翻訳)の2通りの方法を用いることができる。無細胞合成系としては、市販のシステム、例えばExpressway TMシステム(インビトロジェン社)、PURESYSTEM(登録商標;ポストゲノム研究所)、TNTシステム(登録商標;プロメガ社)等を用いることができる。
また抗原は、hDLK−1のアミノ酸配列(配列番号2)又は前述したその部分配列において1又は複数のアミノ酸が欠失、置換又は付加されたアミノ酸配列からなるペプチドであってもよい。例えば、hDLK−1のアミノ酸配列又はその部分配列のうち1又は複数個(好ましくは1個又は数個(例えば1個~10個、さらに好ましくは1個~5個))のアミノ酸が欠失しており、1又は複数個(好ましくは1個又は数個(例えば1個~10個、さらに好ましくは1個~5個))のアミノ酸が他のアミノ酸で置換されており、あるいは、1又は複数個(好ましくは1個又は数個(例えば1個~10個、さらに好ましくは1個~5個))の他のアミノ酸が付加されたアミノ酸配列からなるペプチドを使用することもできる。
本発明において、細胞等に導入するための遺伝子としては、hDLK−1タンパク質若しくはその部分断片又はこれらの変異型のタンパク質又は断片をコードする遺伝子が挙げられる。そのような遺伝子としては、例えば、配列番号1に示される塩基配列又はその部分配列を有するものを使用することができる。
「ストリンジェントな条件」とは、ハイブリダイズさせた後の洗浄時の条件であって、バッファーの塩(ナトリウム)濃度が10~500mMであり、温度が42℃~72℃、好ましくは、上記塩濃度が50~300mMであり、温度が55~68℃での条件をいう。
遺伝子に変異を導入するには、Kunkel法やGapped duplex法等の公知手法により、例えば部位特異的突然変異誘発法を利用した変異導入用キット、例えばGeneTailorTM Site−Directed Mutagenesis System(インビトロジェン社製)、TaKaRa Site−Directed Mutagenesis System(Prime STAR(登録商標)Mutagenesis Basal kit、Mutan(登録商標)−Super Express Km等:タカラバイオ社製)を用いて行うことができる。
調製した抗原を、免疫のため哺乳動物に投与する。哺乳動物は特に限定はされず、例えばラット、マウス及びウサギなどを挙げることができ、なかでもマウスが好ましい。
抗原の動物一匹あたりの投与量は、アジュバントの有無により適宜設定することができる。アジュバントとしては、フロイント完全アジュバント(FCA)、フロイント不完全アジュバント(FIA)、水酸化アルミニウムアジュバント等が挙げられる。免疫は、主として静脈内、足蹠、皮下、腹腔内等に注入することにより行うことができる。また、免疫の間隔については、特に限定されず、数日から数週間間隔、好ましくは1週間間隔で、1~10回、好ましくは2~3回免疫を行う。そして、最終の免疫日から3~7日後に、酵素免疫測定法(ELISA又はEIA)や放射性免疫測定法(RIA)等で抗体価を測定し、所望の抗体価を示した日に採血して、抗血清を得ることができる。上記抗体の採取方法において、抗体の精製が必要とされる場合は、硫安塩析法、イオン交換クロマトグラフィー、ゲル濾過クロマトグラフィー、アフィニティークロマトグラフィー等の公知の方法を適宜選択して、又はこれらを組み合わせることにより、精製することができる。その後は、抗血清中のポリクローナル抗体の反応性をELISA法などで測定する。
・抗体産生細胞の採取
本発明の抗hDLK−1抗体は、限定はされないが、モノクローナル抗体であることが好ましい。
調製した抗原を、免疫のため哺乳動物、例えばラット、マウス及びウサギなどに投与する。抗原の動物一匹あたりの投与量は、アジュバントの有無により適宜設定することができる。アジュバントとしては上記と同様である。免疫手法も前記と同様である。そして、最終の免疫日から1~60日後、好ましくは1~14日後に、抗体産生細胞を採取する。抗体産生細胞としては、脾臓細胞、リンパ節細胞及び末梢血細胞などが挙げられるが、なかでもリンパ節細胞及び脾臓細胞が好ましい。
ハイブリドーマ(抗体産生細胞株)を得るため、抗体産生細胞とミエローマ細胞との細胞融合を行う。抗体産生細胞と融合させるミエローマ細胞として、マウスなどの動物の一般に入手可能な株化細胞を用いることができる。用いる細胞株としては、薬剤選択性を有し、未融合の状態ではHAT選択培地(ヒポキサンチン、アミノプテリン及びチミジンを含む)で生存できず、抗体産生細胞と融合した状態でのみ生存できる性質を有するものが好ましい。
ミエローマ細胞としては、例えば、P3−X63−Ag8.653、P3−X63−Ag8(X63)、P3−X63−Ag8.U1(P3U1)、P3/NS I/1−Ag4−1(NS1)及びSp2/0−Ag14(Sp2/0)等のマウスミエローマ細胞株が挙げられる。ミエローマ細胞の選択は、抗体産生細胞との適合性を適宜考慮して行うことができる。
細胞融合処理後の細胞から目的とするハイブリドーマを選別する。その方法として、細胞懸濁液を、例えばウシ胎児血清含有RPMI−1640培地などに適当に希釈後、マイクロタイタープレート上に播き、各ウェルに選択培地を加え、以後適当に選択培地を交換して培養を行う。その結果、選択培地で培養開始後、14日前後から生育してくる細胞をハイブリーマとして得ることができる。
次に、増殖してきたハイブリドーマの培養上清中に、hDLK−1に反応する抗体が存在するか否かをスクリーニングする。ハイブリドーマのスクリーニングは、通常の方法に従えばよく、特に限定はされない。例えば、ハイブリドーマとして生育したウェルに含まれる培養上清の一部を採取し、ELISA、EIA及びRIAなどによってスクリーニングすることができる。
融合細胞のクローニングは、限界希釈法等により行うことができる。hDLK−1に強い反応性を示す抗体をフローサイトメトリー等により判定し、これを産生するハイブリドーマを選択し、クローンとして樹立する。
樹立したハイブリドーマを培養し、得られる培養物からモノクローナル抗体を採取する方法として、通常の細胞培養法、又は腹水形成法等を採用することができる。「培養」とは、ハイブリドーマを培養皿又は培養ボトル中で生育させること、あるいはハイブリドーマを下記のように動物の腹腔内で増殖させることを意味する。
細胞培養法においては、ハイブリドーマを10%ウシ胎児血清含有RPMI−1640培地、MEM培地又は無血清培地等の動物細胞培養培地中で、通常の培養条件(例えば37℃、5%CO2濃度)で7~14日間培養し、その培養上清から抗体を取得することができる。
腹水形成法の場合は、ミエローマ細胞由来の哺乳動物と同種系動物の腹腔内にハイブリドーマを約1×107個投与し、ハイブリドーマを大量に増殖させる。そして、2~3週間後に腹水を採取することが好ましい。
上記抗体の採取方法において、抗体の精製が必要とされる場合は、硫安塩析法、イオン交換クロマトグラフィー、ゲル濾過、アフィニティークロマトグラフィー等の公知の方法を適宜選択して、又はこれらを組み合わせることにより精製することができる。
本発明に用いる抗hDLK−1抗体は、in vivoで抗腫瘍活性を有する抗体である。
ここで、「抗腫瘍活性」とは、腫瘍細胞(癌細胞)を死滅させる活性又は腫瘍成長を阻害する活性を意味する。本発明において、抗腫瘍活性としては、例えば、腫瘍血管新生阻害活性が好ましく挙げられる。本発明の抗hDLK−1抗体が抗腫瘍活性を発揮しうるヒト腫瘍(腫瘍細胞)の種類としては、hDLK−1の発現が確認されているヒト腫瘍が挙げられる。
in vivoでの抗腫瘍活性の確認は、例えば、所望の腫瘍細胞をマウスの皮下に移植した担癌マウスを用い、このマウスに前記のとおり得られた抗体を投与することにより行うことができる。この場合、抗体の投与は、腫瘍細胞の移植直後から行ってもよいし(Preventionモデル)、移植に腫瘍が所定の体積になったのを確認してから行ってもよい(Treatmentモデル)。投与方法は、何ら限定はされないが、例えば、3日に1回、20mg/kg体重、腹腔内投与としてもよい。Preventionモデルの場合は、腫瘍形成頻度と腫瘍体積により抗腫瘍活性の有無及びレベルを評価することができる。Treatmentモデルの場合は、腫瘍体積及び腫瘍重量により抗腫瘍活性の有無及びレベルを評価することができる。
より具体的には、例えば、以下の(a)~(k)の抗hDLK−1抗体が挙げられ、それらの少なくとも1種を用いることができる。
(a)H鎖V領域のCDR1~3のアミノ酸配列が、それぞれ順に、配列番号3~5で示されるアミノ酸配列であり、かつ、L鎖V領域のCDR1~3のアミノ酸配列が、それぞれ順に、配列番号6~8で示されるアミノ酸配列である抗体。
(b)H鎖V領域のCDR1~3のアミノ酸配列が、それぞれ順に、配列番号9~11で示されるアミノ酸配列であり、かつ、L鎖V領域のCDR1~3のアミノ酸配列が、それぞれ順に、配列番号12~14で示されるアミノ酸配列である抗体。
(d)H鎖V領域のアミノ酸配列が配列番号20で示されるアミノ酸配列(対応する塩基配列は配列番号19)からなり、かつ、L鎖V領域のアミノ酸配列が配列番号22(対応する塩基配列は配列番号21)で示されるアミノ酸配列からなる抗体。なお、当該抗体のH鎖V領域及びL鎖V領域中の各CDR配列は、上記(b)の抗体と同一である。
(f)H鎖V領域のアミノ酸配列が配列番号30、32、34又は36で示されるアミノ酸配列(対応する塩基配列は配列番号29、31、33又は35)からなり、かつ、L鎖V領域のアミノ酸配列が配列番号46(対応する塩基配列は配列番号45)で示されるアミノ酸配列からなる抗体(ヒト化抗体)。なお、当該抗体のH鎖V領域及びL鎖V領域中の各CDR配列は、上記(b)の抗体と同一である。
(h)受託番号がFERM BP−10899であるハイブリドーマにより産生される抗体。
(i)受託番号がFERM BP−10707であるハイブリドーマにより産生される抗体。
(j)受託番号がFERM BP−10900であるハイブリドーマにより産生される抗体。
(k)受託番号がFERM BP−11337であるハイブリドーマにより産生される抗体。
配列番号34に示されるアミノ酸配列は、配列番号30に示されるアミノ酸配列の第74番目のスレオニン(T)がリジン(K)に置換されたものであり、
配列番号36に示されるアミノ酸配列は、配列番号30に示されるアミノ酸配列の、第24番目のアラニン(A)がグリシン(G)に置換され、かつ、第74番目のスレオニン(T)がリジン(K)に置換されたものである。
同様に、上記(g)の抗体に関しては、配列番号40に示されるアミノ酸配列は、配列番号38に示されるアミノ酸配列の第24番目のアラニン(A)がグリシン(G)に置換されたものであり、
配列番号42に示されるアミノ酸配列は、配列番号38に示されるアミノ酸配列の第74番目のスレオニン(T)がリジン(K)に置換されたものであり、
配列番号44に示されるアミノ酸配列は、配列番号38に示されるアミノ酸配列の、第24番目のアラニン(A)がグリシン(G)に置換され、かつ、第74番目のスレオニン(T)がリジン(K)に置換されたものである。
また、上記(h)~(k)の抗体に関しては、受託番号がFERM BP−11337であるハイブリドーマは、「Mouse−Mouse hybridoma BA−1−3D」と称し2011年2月1日付けで、受託番号がFERM BP−10707であるハイブリドーマは、「Mouse−Mouse hybridoma:M3−1」と称し2006年10月18日付で、受託番号がFERM BP−10899であるハイブリドーマは、「Mouse−Mouse hybridoma DI−2−14」と称し2007年8月21日付で、受託番号がFERM BP−10900であるハイブリドーマは、「Mouse−Mouse hybridoma DI−6」と称し2007年8月21日付で、寄託されたものである。
本発明に用い得る抗hDLK−1抗体としては、例えば、上述した種々の抗hDLK−1抗体が結合(認識)する部位(例えばエピトープ)に結合する抗hDLK−1抗体など、上述した種々の抗hDLK−1抗体と競合し得る抗体も好ましく挙げられる。
抗hDLK−1抗体のエピトープ(抗原決定基)は、抗原であるhDLK−1の少なくとも一部であればよく限定はされないが、例えば、配列番号2に示されるhDLK−1のアミノ酸配列中の、第24番目~第91番目のアミノ酸からなる領域(hDLK−1のEGF−1~EGF−2を含む領域)、第92番目~第167番目のアミノ酸からなる領域(hDLK−1のEGF−3~EGF−4を含む領域)、若しくは第131番目~第244番目のアミノ酸からなる領域(hDLK−1のEGF−4~EGF−6を含む領域)の少なくとも一部であることが好ましい。なかでも、hDLK−1のEGF−1~EGF−2を含む領域がより好ましい。当該領域を認識する(当該領域と結合する)抗hDLK−1抗体は、例えば、腫瘍細胞内へのインターナリゼーション活性が高く、後述するイムノコンジュゲート等の用途に極めて有用なものである。
・遺伝子組換え抗体
抗hDLK−1抗体の好ましい態様の一つとして、遺伝子組換え抗体が挙げられる。遺伝子組換え抗体としては、限定はされないが、例えば、キメラ抗体及びヒト化抗体等が挙げられる。
キメラ抗体(すなわちヒト型キメラ抗体)は、マウス由来抗体の可変領域をヒト由来の定常領域に連結(接合)した抗体であり(Proc.Natl.Acad.Sci.U.S.A.81,6851−6855,(1984)等を参照)、キメラを作製する場合は、そのように連結した抗体が得られるよう、遺伝子組換え技術によって容易に構築できる。
上記キメラ抗体及びヒト化抗体は、例えば、抗体Fc領域におけるN−グリコシド結合複合型糖鎖が該糖鎖の還元末端のN−アセチルグルコサミンにフコースが結合していない糖鎖であるものが好ましく、詳しくは、該フコースの1位がN−グリコシド結合複合型糖鎖還元末端のN−アセチルグルコサミンの6位にα結合していない糖鎖を抗体分子のFc領域に有する遺伝子組換え抗体分子からなる抗体が挙げられる。このような抗体であれば、ADCC活性を飛躍的に向上させることができる。なお、この点(抗体Fc領域におけるN−グリコシド結合複合型糖鎖の特徴)は、前述したポリクローナル抗体及びモノクローナル抗体についても同様に好ましい。
本発明においては、抗hDLK−1抗体の断片も、本発明の抗hDLK−1抗体と共に、又は本発明の抗hDLK−1抗体に代えて、用いることができる。ここで、当該抗体断片は、本発明の抗hDLK−1抗体(マウス抗体以外のヒト化抗体等を含む)と同様に、hDLK−1に対する結合活性を有するものであってin vivoで抗腫瘍活性を有するものである。
当該抗体断片としては、抗hDLK−1ポリクローナル抗体又は抗hDLK−1モノクローナル抗体の一部分の領域(すなわち、本発明の抗hDLK−1抗体に由来する抗体断片)を意味し、例えば、Fab、Fab’、F(ab’)2、Fv(variable fragment of antibody)、一本鎖抗体(H鎖、L鎖、H鎖V領域、及びL鎖V領域等)、scFv、diabody(scFv二量体)、dsFv(ジスルフィド安定化V領域)、並びに、相補性決定領域(complementarity determining region:CDR)を少なくとも一部に含むペプチド等が挙げられる。
Fabは、抗体分子をタンパク質分解酵素パパインで処理して得られる断片のうち、H鎖のN末端側約半分とL鎖全体とがジスルフィド結合で結合した、分子量約5万の抗原結合活性を有する抗体断片である。また、抗体のFabをコードするDNAを、原核生物用発現ベクター又は真核生物用発現ベクターに挿入し、該ベクターを原核生物又は真核生物へ導入することにより発現させ、Fabを製造することもできる。
Fab’は、上記F(ab’)2のヒンジ領域のジスルフィド結合を切断した、分子量約5万の抗原結合活性を有する抗体断片である。また、抗体のFab’断片をコードするDNAを、原核生物用発現ベクター又は真核生物用発現ベクターに挿入し、該ベクターを原核生物又は真核生物へ導入することにより発現させ、Fab’を製造することもできる。
scFvは、1本のH鎖V領域(VH)と1本のL鎖V領域(VL)とを適当なペプチドリンカー(P)を用いて連結した、VH−P−VLないしはVL−P−VHポリペプチドで、抗原結合活性を有する抗体断片である。scFvは、抗体のVHおよびVLをコードするcDNAを取得し、scFvをコードするDNAを構築して、該DNAを原核生物用発現ベクター又は真核生物用発現ベクターに挿入し、該発現ベクターを原核生物又は真核生物へ導入することにより発現させて、製造することができる。
dsFvは、VHおよびVL中のそれぞれ1アミノ酸残基をシステイン残基に置換したポリペプチドを、該システイン残基間のジスルフィド結合を介して結合させたものをいう。システイン残基に置換するアミノ酸残基は、Reiterらにより示された方法(Protein Engineering,7,697−704,1994)に従って、抗体の立体構造予測に基づいて選択することができる。dsFvは、抗体のVHおよびVLをコードするcDNAを取得し、dsFvをコードするDNAを構築して、該DNAを原核生物用発現ベクター又は真核生物用発現ベクターに挿入し、該発現ベクターを原核生物又は真核生物へ導入することにより発現させて、製造することができる。
本発明に用いる抗体断片としては、そのままの形状でN−グリコシド結合複合型糖鎖が該糖鎖の還元末端のN−アセチルグルコサミンにフコースが結合していない糖鎖である抗体Fc領域の一部または全部を含む抗体断片でもよく、また、上述した抗体断片とN−グリコシド結合複合型糖鎖が該糖鎖の還元末端のN−アセチルグルコサミンにフコースが結合していない糖鎖である抗体Fc領域の一部または全部との融合タンパク質であってもよい。このような抗体断片であれば、ADCC活性を飛躍的に向上させることができるため、好ましい。
本発明に用いる抗体断片の具体例としては、限定はされないが、例えば、前述した各種抗hDLK−1抗体における、H鎖V領域のCDR1~3とL鎖V領域のCDR1~3とを含むものや、H鎖V領域の全体とL鎖V領域の全体とを含むもの等が挙げられる。
本発明に用いる抗hDLK−1抗体、及び抗体断片は、抗腫瘍活性及び/又は殺細胞活性を有する化合物と複合体を形成した形態であってもよい。なお、予め、抗体分子や抗体断片分子と、抗腫瘍活性及び/又は殺細胞活性を有する化合物とを、それぞれ調製したのち、これらを複合化させて得られたものは、一般に、イムノコンジュゲートと称される。また、遺伝子組換え技術を用い、抗腫瘍活性及び/又は殺細胞活性を有する化合物としてのタンパク質トキシンを、遺伝子上で抗体や抗体断片の遺伝子と連結させて、1つのタンパク質(融合タンパク質)として発現させて得られたものは、一般に、イムノトキシンと称される。
抗腫瘍活性を有する化合物としては、例えば、ドキソルビシン、カリケアマイシン、マイトマイシンC、Auristatin Eなどが挙げられる。殺細胞活性を有する化合物としては、例えば、サポリン、リシン、緑膿菌外毒素、ジフテリアトキシン等が挙げられ、なかでもサポリン及び緑膿菌外毒素が好ましく用いられる。
本発明に用いる抗hDLK−1抗体は、hDLK−1を発現する標的腫瘍細胞内へのインターナリゼーション活性に優れたものである。そのため、予め、抗腫瘍活性及び/又は殺細胞活性を有する化合物を複合化させておくことにより、これら化合物を腫瘍細胞に直接かつ高選択的に作用させることができる。当該複合体は、標的腫瘍細胞への薬剤送達能に極めて優れたものである。
なお、細胞内へのインターナリゼーション活性は、抗体をローダミン等により蛍光標識し、細胞内への移行挙動及び抗体の局在性について蛍光顕微鏡等を用いて観察することにより評価することができる。
本発明の組合せ医薬において、有効成分としての抗hDLK−1抗体の含有割合は、限定はされず、適宜設定することができるが、例えば、組合せ医薬全体に対して、0.01~99.99重量%の範囲内とすることができ、好ましくは、0.01~30重量%、より好ましくは0.05~20重量%、さらに好ましくは0.1~10重量%の範囲内としてもよい。有効成分の含有割合が上記範囲内であることにより、本発明の組合せ医薬は、がんの治療効果を十分に発揮することができる。
本発明の組合せ医薬は、FGFR4阻害剤等と抗hDLK−1抗体等とを有効成分としてそれぞれ含むものであり、癌や腫瘍に対して用いられる他の薬剤(例えば、抗癌剤)をさらに含むものであってよい。
本発明の組合せ医薬は、被験対象としてのヒト、又は非ヒト哺乳動物(例えば、マウス、ラット、ウサギ、ヒツジ、ブタ、ウシ、ネコ、イヌ、サルなど)に対して、種々の投与経路、具体的には、経口、又は非経口(例えば静脈内注射(静注)、筋肉内注射、腹腔内注射、皮下注射、直腸投与、経皮投与)で投与することができる。
従って、本発明の組合せ医薬は、単独で用いることも可能であるが、投与経路に応じて慣用される方法により薬学的に許容し得る担体を用いて適当な剤形に製剤化して用いることができる。また、本発明の組合せ医薬には、チロシンキナーゼ活性を阻害又は抑制する物質を抗DLK−1抗体に結合した抗体−薬剤複合体の形態も含まれる。
これら製剤の製剤化に用い得る担体としては、例えば、通常用いられる賦形剤、結合剤、崩壊剤、滑沢剤、着色剤、及び矯味矯臭剤のほか、必要に応じ、安定化剤、乳化剤、吸収促進剤、界面活性剤、pH調整剤、防腐剤、抗酸化剤、増量剤、湿潤化剤、表面活性化剤、分散剤、緩衝剤、保存剤、溶解補助剤、及び無痛化剤等が挙げられ、医薬品製剤の原料として用いることができる公知の成分を配合して常法により製剤化することが可能である。
本発明の組合せ医薬を、非経口剤又は経口剤として用いる場合について、以下に説明する。
非経口剤として用いる場合、一般にその形態は限定されないが、各種注射剤の場合は、例えば、単位投与量アンプル又は多投与量容器の状態や、使用時に溶解液に再溶解させる凍結乾燥粉末の状態で提供され得る。当該非経口剤には、有効成分となるFGFR4阻害剤等と抗hDLK−1抗体等のほかに、各種形態に応じ、公知の各種賦形材や添加剤を上記有効成分の効果が損なわれない範囲で含有することができる。例えば、各種注射剤の場合は、水、グリセロール、プロピレングリコールや、ポリエチレングリコール等の脂肪族ポリアルコール等が挙げられる。
経口剤として用いる場合、一般にその形態は限定されず、前述した剤形のいずれであってもよいし、使用する際に再溶解させる乾燥生成物にしてもよい。当該経口剤には、有効成分となるFGFR4阻害剤等と抗hDLK−1抗体等のほかに、各種形態に応じ、公知の各種賦形材や添加剤を上記有効成分の効果が損なわれない範囲で含有することができる。例えば、結合剤(シロップ、アラビアゴム、ゼラチン、ソルビトール、トラガカント、ポリビニルピロリドン等)、充填材(乳糖、糖、コーンスターチ、馬鈴薯でんぷん、リン酸カルシウム、ソルビトール、グリシン等)、潤滑剤(ステアリン酸マグネシウム、タルク、ポリエチレングリコール、シリカ等)、崩壊剤(各種でんぷん等)、および湿潤剤(ラウリル硫酸ナトリウム等)等が挙げられる。
経口剤の投与量(1日あたり)は、一般には、有効成分となるFGFR4阻害剤等と抗hDLK−1抗体等を、適用対象(被験者、患者等)の体重1kgあたり、0.05~5000mg、0.1~1000mg、又は0.1~100mg服用できる量とすることができ、あるいは0.5~50mg服用できる量や1~10mg服用できる量とすることもできる。また、経口剤中の有効成分の配合割合は、限定はされず、1日あたりの投与回数等を考慮して、適宜設定することができる。
本発明においては、上記2項で説明したがん治療用の組合せ医薬のほかに、下記(i)~(iv)の医薬組成物に係る発明も包含する。
(i)in vivoで抗腫瘍活性を有する、ヒトDLK−1に対する抗体、若しくは当該抗体に由来する抗体断片を含む、がんを治療するための医薬組成物であって、
FGFR4のチロシンキナーゼ活性を阻害又は抑制する物質、若しくはそのプロドラッグ、又はそれらの薬理学的に許容し得る塩、あるいはそれらの水和物若しくは溶媒和物と組み合わせて用いるものである、
前記医薬組成物。
(ii)FGFR4のチロシンキナーゼ活性を阻害又は抑制する物質、若しくはそのプロドラッグ、又はそれらの薬理学的に許容し得る塩、あるいはそれらの水和物若しくは溶媒和物を含む、がんを治療するための医薬組成物であって、
in vivoで抗腫瘍活性を有する、ヒトDLK−1に対する抗体、若しくは当該抗体に由来する抗体断片と組み合わせて用いるものである、
前記医薬組成物。
前記治療が、FGFR4のチロシンキナーゼ活性を阻害又は抑制する物質、若しくはそのプロドラッグ、又はそれらの薬理学的に許容し得る塩、あるいはそれらの水和物若しくは溶媒和物の投与を含むものである、
前記医薬組成物。
(iv)FGFR4のチロシンキナーゼ活性を阻害又は抑制する物質、若しくはそのプロドラッグ、又はそれらの薬理学的に許容し得る塩、あるいはそれらの水和物若しくは溶媒和物を含む、がんを治療するための医薬組成物であって、
前記治療が、in vivoで抗腫瘍活性を有する、ヒトDLK−1に対する抗体、若しくは当該抗体に由来する抗体断片の投与を含むものである、
前記医薬組成物。
本発明は、FGFR4阻害剤等と抗hDLK−1抗体等とを構成要素として含む、がんの治療用キットの形態を提供することもできる。治療対象となるがんの詳細などについては、上記2項で説明したがん治療用の組合せ医薬に係る記載が適宜採用され得る。
当該キットにおけるFGFR4阻害剤等と抗hDLK−1抗体等は、安定性(保存性)及び使用容易性等を考慮し、例えば溶解した状態で備えられていてもよい。
当該キットは、FGFR4阻害剤等と抗hDLK−1抗体等以外にも、適宜、他の構成要素を含むことができる。例えば、抗体の標識物質、あるいは抗体又はその標識物を固定した固相化試薬などを含めることができる。抗体の標識物質とは、酵素、放射性同位体、蛍光化合物及び化学発光化合物等によって標識されたものを意味する。また、各種バッファー、滅菌水、各種細胞培養容器、各種反応容器(エッペンドルフチューブ等)、ブロッキング剤(Bovine Serum Albumin(BSA),Skim milk,ヤギ血清等の血清成分)、洗浄剤、界面活性剤、各種プレート、アジ化ナトリウム等の防腐剤、及び実験操作マニュアル(説明書)等を含んでいてもよい。
当該キットは、構成要素として少なくとも前述したFGFR4阻害剤等と抗hDLK−1抗体等を備えているものであればよい。従って、がんの治療に必須となる構成要素の全てが共にキットに備えられていてもよいし、個々別々に備えられていてもよく、限定はされない。
・Vehicle群、
・HuBA−1−3D(1mg/kg)投与群
・H3B−6527(50mg/kg)投与群
・FGF−401(10mg/kg)投与群
・BLU−554(30mg/kg)投与群
・HuBA−1−3D(1mg/kg)+H3B−6527(50mg/kg)併用投与群
・HuBA−1−3D(1mg/kg)+FGF−401(10mg/kg)併用投与群
・HuBA−1−3D(1mg/kg)+BLU−554(30mg/kg)併用投与群
HuBA−1−3Dは腹腔内投与を行い、FGFR4阻害剤(H3B−6527,FGF−401,BLU−554)は経口投与を行った。投与から48時間後に、それぞれ各群3匹ずつサクリファイスし、腫瘍を摘出し、免疫染色用に10%ホルマリンで腫瘍を固定したのち、パラフィン包埋切片を作成した。また、ウェスタンブロット用に組織抽出液を調製した。
図1に、Vehicle投与群、HuBA−1−3D(1mg/kg)投与群、H3B−6527(50mg/kg)投与群、HuBA−1−3D(1mg/kg)+H3B−6527(50mg/kg)併用投与群における、Ki−67染色像(上段)とcleaved caspase−3染色像(下段)を示した。Vehicle投与群のHep3Bゼノグラフト腫瘍では、Ki−67で癌細胞の核が染色された増殖している癌細胞が腫瘍全体にわたって観察された。HuBA−1−3D(1mg/kg)投与群、H3B−6527(50mg/kg)投与群において、Ki−67で染色される癌細胞の割合は、Vehicle投与群と比較して変化が見られなかった。それに対して、HuBA−1−3D(1mg/kg)とH3B−6527(50mg/kg)を組み合わせて併用投与した場合、Hep3Bゼノグラフト腫瘍におけるKi−67で染色される癌細胞が、著しく減少していた。癌細胞のアポトーシスについては、Vehicle投与群とH3B−6527(50mg/kg)投与群では、cleaved caspase−3で染色されるアポトーシス細胞は検出されなかったのに対して、HuBA−1−3D(1mg/kg)投与群では、腫瘍の広い範囲で癌細胞のアポトーシスが検出された。HuBA−1−3D(1mg/kg)+H3B−6527(50mg/kg)併用投与群における腫瘍においても、腫瘍の広い範囲で癌細胞のアポトーシスが検出された。
FGF−401(10mg/kg)単独投与群、およびBLU−554(30mg/kg)単独投与群においては、いずれも、Ki−67で染色される癌細胞の割合は、Vehicle投与群と比較して変化が見られなかった。それに対して、HuBA−1−3D(1mg/kg)との併用投与群においては、いずれもKi−67で染色される癌細胞が、著しく減少していた。また、癌細胞のアポトーシスについても、FGF−401(10mg/kg)単独投与群、およびBLU−554(30mg/kg)単独投与群においては,cleaved caspase−3で染色されるアポトーシス細胞は検出されなかったのに対して、HuBA−1−3D(1mg/kg)との併用投与群では、腫瘍の広い範囲で癌細胞のアポトーシスが検出された。
投与48時間後のHep3B腫瘍においては、HuBA−1−3D(1mg/kg)投与群において、3個体中1個体で、アポトーシスマーカー(Cleaved caspase−3とcleaved PARP)の発現が強く誘導されており、HuBA−1−3D投与によってHep3B腫瘍の細胞死が誘導されていることが示された。一方、FGFR4阻害剤(H3B−6527,FGF−401,BLU−554)の投与群では、(a)H3B−6527,(b)FGF−401,(c)BLU−554のいずれの投与群においても、投与したそれぞれ3個体のいずれにおいても、アポトーシスマーカー(Cleaved caspase−3とcleaved PARP)は誘導されなかった。HuBA−1−3DとFGFR4阻害剤との併用投与群においては、それぞれの併用投与群において、3個体いずれにおいてもアポトーシスが誘導された。
以上の結果から、FGFR4選択的阻害剤として使用した3剤(H3B−6527,FGF−401,BLU−554)について、いずれもHuBA−1−3D(1mg/kg)と組み合わせて併用投与した場合、Hep3Bゼノグラフト腫瘍における癌細胞の増殖阻害について相乗的な効果を示し、更に、併用投与群においては癌細胞のアポトーシスが誘導されることが示された。
HuBA−1−3D抗体とFGFR4に対する低分子阻害剤であるH3B−6527(Cancer Res.2017 Dec 15;77(24):6999−7013)の併用投与による抗腫瘍効果を、ヒト肝癌細胞株Hep3Bを用いたゼノグラフト治療モデルで検討した。
・Vehicle投与群(N=8)
・H3B−6527(50mg/kg 体重,1日2回)投与群(N=8)
・HuBA−1−3D(0.3mg/kg 体重)投与群(N=8)
・HuBA−1−3D(1mg/kg 体重)投与群(N=8)
・H3B−6527(50mg/kg 体重)及びHuBA−1−3D(0.3mg/kg 体重)の併用投与群(N=8)
・H3B−6527(50mg/kg 体重)及びHuBA−1−3D(1mg/kg 体重)の併用投与群(N=8)
癌細胞移植から32日後(Day 32、投薬終了後7日)における腫瘍体積は、Vehicle投与群が1377.0±239.1mm3(N=8)であったのに対して、H3B−6527(50mg/kg)投与群では647.8±146.2mm3(N=8、T/C=47%、*P<0.05)、HuBA−1−3D(0.3mg/kg)抗体投与群では1478.4±333.5mm3(N=7)、HuBA−1−3D(1mg/kg)抗体投与群では、204.5±173.0mm3(N=8,T/C=14.9%、*P<0.05)、H3B−6527(50mg/kg)+HuBA−1−3D(0.3mg/kg)併用投与群では473.7±81.6mm3(N=8,T/C=34.4%,*P<0.05)、H3B−6527+HuBA−1−3D(1mg/kg)併用投与群では17.0±24.3mm3(N=8,T/C=1.2%、*P<0.05)となり、Vehicle投与群の腫瘍体積と比較して、H3B−6527(50mg/kg)投与群,HuBA−1−3D(1mg/kg)投与群、HuBA−1−3D(0.3mg/kg)+H3B−6527(50mg/kg)併用投与群,HuBA−1−3D(1mg/kg)+H3B−6527(50mg/kg)併用投与群で、統計的に有意な抗腫瘍効果が観察された。
H3B−6527(50mg/kg)投与群、HuBA−1−3D(1mg/kg)投与群、H3B−6527(50mg/kg)+HuBA−1−3D(1mg/kg)併用投与群の3群間の腫瘍体積を比較したところ、H3B−6527(50mg/kg)+HuBA−1−3D(1mg/kg)併用投与群では、それぞれ単独投与群と比較して、有意な抗腫瘍効果を示した(**P<0.05,Tukey法)。また、併用投与群では、8個体すべての個体で、Day 14の腫瘍体積値と比べて腫瘍の縮小が観察され、そのうち5個体では腫瘍が消失しており、併用投与による強力な抗腫瘍効果が観察された。
以上の結果を図5Aに示す。
以上の結果を図5Bに示す。
以上の結果を図5Cに示す。
次に、HuBA−1−3D抗体とFGFR4阻害剤(FGF−401)との併用投与による抗腫瘍活性の増強効果を、更に検証する目的で、H3B−6527と同様に、FGFR4のアミノ酸番号545から562番目の領域に存在するATP結合領域のアミノ酸番号552番目のシステインに特異的に結合することによって、FGFR4特異的なチロシンキナーゼ活性を阻害する、FGF−401(Mol.Cancer Ther.2019 Dec;18(12):2194−2206)とHuBA−1−3Dの併用投与による抗腫瘍効果を、ヒト肝癌細胞株Hep3Bを用いたゼノグラフト治療モデルで検討した。
・Vehicle投与群(N=8)
・FGF−401(10mg/kg 体重,1日2回)投与群(N=8)
・HuBA−1−3D(0.3mg/kg 体重)投与群(N=8)
・HuBA−1−3D(1mg/kg 体重)投与群(N=8)
・FGF−401(10mg/kg 体重)及びHuBA−1−3D(0.3mg/kg 体重)の併用投与群(N=8)
・FGF−401(10mg/kg 体重)及びHuBA−1−3D(1mg/kg 体重)の併用投与群(N=8)
癌細胞の移植(Day 0)から28日後(Day 28,最終投与日(Day 25)から3日後)において、Vehicle投与群(N=8)の腫瘍体積と比較して、全ての投与群で有意な抗腫瘍効果が観察された(*P<0.05 by Dunnett)。Vehicle群の腫瘍体積は684.7±265.7mm3であったのに対して、FGF−401(10mg/kg)投与群では388.9±94.8mm3(T/C=56.8%,*P<0.05),HuBA−1−3D(0.3mg/kg)投与群では456.3±232.9mm3(T/C=66.6%,*P<0.05),HuBA−1−3D(1mg/kg)投与群では53.5±57.5mm3(T/C=7.8%,8*P<0.05),FGF−401(10mg/kg)+HuBA−1−3D(0.3mg/kg)併用投与群では180.8±109.0mm3(T/C=26.4%,*P<0.05),FGF−401(10mg/kg)+HuBA−1−3D(1mg/kg)併用投与群では14.1±21.4mm3(T/C=2.1%,*P<0.05)であった。
更に、Day 35(最終投与日(Day 25)から10日後)においては、Vehicle投与群(N=8)の腫瘍体積と比較して、FGF−401(10mg/kg)投与群、HuBA−1−3D(1mg/kg)投与群、FGF−401(10mg/kg)+HuBA−1−3D(0.3mg/kg)併用投与群、およびFGF−401(10mg/kg)+HuBA−1−3D(1mg/kg)併用投与群で有意な抗腫瘍効果が観察された(P<0.05 by Dunnett)。
以上の結果を図6に示す。
先の実施例と同様に、HuBA−1−3D抗体とFGFR4阻害剤との併用投与による抗腫瘍活性の増強効果を、更に検証する目的で、H3B−6527、およびFGF−401と同様に、FGFR4のアミノ酸番号545から562番目の領域に存在するATP結合領域のアミノ酸番号552番目のシステインに特異的に結合することによって、FGFR4特異的なチロシンキナーゼ活性を阻害する、BLU−554(Cancer Discov.2019 Dec;9(12):1696−1707)とHuBA−1−3Dの併用投与による抗腫瘍効果を、ヒト肝癌細胞株Hep3Bを用いたゼノグラフト治療モデルで検討した。
・Vehicle投与群(N=8)
・BLU−554(30mg/kg 体重,1日2回)投与群(N=8)
・HuBA−1−3D(1mg/kg 体重)投与群(N:8)
・BLU−554(30mg/kg 体重)及びHuBA−1−3D(1mg/kg 体重)の併用投与群(N=8)
Day 38(最終投与日(Day 25)から13日目)における、Vehicle投与群(N=7)の平均腫瘍体積は1082.1±296.7mm3であったのに対して、BLU−554(30mg/kg)投与群(N=8)では688.8±562.4mm3(T/C=63.7%),HuBA−1−3D(1mg/kg)投与群(N=8)では127.9±95.0mm3(T/C=11.8%,*P<0.05 by Dunnett),HuBA−1−3D(1mg/kg)+BLU−554(30mg/kg)併用投与群(N=7)では56.0±114.5mm3(T/C=5.2%,*P<0.05 by Dunnett)であり、HuBA−1−3D(1mg/kg)+BLU−554(30mg/kg)併用投与群で最も強い抗腫瘍効果が観察された。
以上の結果を図7に示す。
次に、HuBA−1−3D抗体とFGFR4阻害剤との併用投与による抗腫瘍効果の増強作用が、FGFR4阻害剤との併用に特異的であるのかどうかを調べる目的で、FGFR1/FGFR2/FGFR3に対する選択的な低分子チロシンキナーゼ阻害剤であるCH518324/Debio1347(Mol Cancer Ther(2014);13(11);2547−58.)とHuBA−1−3D抗体の併用効果を検討した。CH518324/Debio1347はFGFRに選択的な経口投与可能なチロシンキナーゼ阻害剤で、4種類のFGFR(FGFR1,FGFR2,FGFR3,FGFR4)に対するチロシンキナーゼ阻害活性(IC50)は、それぞれ、9.3nM,7.6nM,22nM,and 290nMであることが報告されている(Mol Cancer Ther(2014);13(11);2547−58)。
・Vehicle投与群(N=8)
・CH518324/Debio1347(50mg/kg 体重,1日1回)投与群(N=8)
・HuBA−1−3D(0.3mg/kg 体重)投与群(N=8)
・HuBA−1−3D(1mg/kg 体重)投与群(N=8)
・CH518324/Debio1347(50mg/kg 体重)及びHuBA−1−3D(0.3mg/kg 体重)の併用投与群(N=8)
・CH518324/Debio1347(50mg/kg 体重)及びHuBA−1−3D(1mg/kg 体重)の併用投与群(N=8)
一方、併用効果については、HuBA−1−3D(0.3mg/kgまたは1mg/kg)とCH518324/Debio1347(50mg/kg)の併用投与群において、いずれの併用の組み合わせにおいても、HuBA−1−3DとCH518324/Debio1347の併用投与群をそれぞれ単独投与群と比較しても抗腫瘍効果の増強は観察されなかった。
以上の結果を図8に示す。
産業上の利用可能性
本発明によれば、がんの治療、特に、FGFR4が発現し及び/又はFGFR4のチロシンキナーゼ活性がみられるがんの治療に関して、既存のチロシンキナーゼ阻害剤よりも強力かつ持続的な抗腫瘍効果を発揮し得る治療薬及び治療方法等を提供することができるため、本発明の治療薬及び治療方法等は、例えば、これまで治療効果が期待できなかった患者に対しても効果を発揮し得るものである点で、極めて有用である。
配列番号24:合成コンストラクト(組換えタンパク質)
配列番号25:組換えDNA
配列番号26:合成コンストラクト(組換えタンパク質)
配列番号27:組換えDNA
配列番号28:合成コンストラクト(組換えタンパク質)
配列番号29:組換えDNA
配列番号30:合成コンストラクト(組換えタンパク質)
配列番号31:組換えDNA
配列番号32:合成コンストラクト(組換えタンパク質)
配列番号33:組換えDNA
配列番号34:合成コンストラクト(組換えタンパク質)
配列番号35:組換えDNA
配列番号36:合成コンストラクト(組換えタンパク質)
配列番号37:組換えDNA
配列番号38:合成コンストラクト(組換えタンパク質)
配列番号39:組換えDNA
配列番号40:合成コンストラクト(組換えタンパク質)
配列番号41:組換えDNA
配列番号42:合成コンストラクト(組換えタンパク質)
配列番号43:組換えDNA
配列番号44:合成コンストラクト(組換えタンパク質)
配列番号45:組換えDNA
配列番号46:合成コンストラクト(組換えタンパク質)
Claims (16)
- がんを治療するための組合せ医薬であって、
線維芽細胞増殖因子受容体4(FGFR4)のチロシンキナーゼ活性を阻害又は抑制する物質、若しくはそのプロドラッグ、又はそれらの薬理学的に許容し得る塩、あるいはそれらの水和物若しくは溶媒和物、及び
in vivoで抗腫瘍活性を有する、ヒトDLK−1に対する抗体、若しくは当該抗体に由来する抗体断片
を含む、前記組合せ医薬。 - in vivoで抗腫瘍活性を有する、ヒトDLK−1に対する抗体、若しくは当該抗体に由来する抗体断片を含む、がんを治療するための医薬組成物であって、
線維芽細胞増殖因子受容体4(FGFR4)のチロシンキナーゼ活性を阻害又は抑制する物質、若しくはそのプロドラッグ、又はそれらの薬理学的に許容し得る塩、あるいはそれらの水和物若しくは溶媒和物と組み合わせて用いるものである、
前記医薬組成物。 - 線維芽細胞増殖因子受容体4(FGFR4)のチロシンキナーゼ活性を阻害又は抑制する物質、若しくはそのプロドラッグ、又はそれらの薬理学的に許容し得る塩、あるいはそれらの水和物若しくは溶媒和物を含む、がんを治療するための医薬組成物であって、
in vivoで抗腫瘍活性を有する、ヒトDLK−1に対する抗体、若しくは当該抗体に由来する抗体断片と組み合わせて用いるものである、
前記医薬組成物。 - in vivoで抗腫瘍活性を有する、ヒトDLK−1に対する抗体、若しくは当該抗体に由来する抗体断片を含む、がんを治療するための医薬組成物であって、
前記治療が、線維芽細胞増殖因子受容体4(FGFR4)のチロシンキナーゼ活性を阻害又は抑制する物質、若しくはそのプロドラッグ、又はそれらの薬理学的に許容し得る塩、あるいはそれらの水和物若しくは溶媒和物の投与を含むものである、
前記医薬組成物。 - 線維芽細胞増殖因子受容体4(FGFR4)のチロシンキナーゼ活性を阻害又は抑制する物質、若しくはそのプロドラッグ、又はそれらの薬理学的に許容し得る塩、あるいはそれらの水和物若しくは溶媒和物を含む、がんを治療するための医薬組成物であって、
前記治療が、in vivoで抗腫瘍活性を有する、ヒトDLK−1に対する抗体、若しくは当該抗体に由来する抗体断片の投与を含むものである、
前記医薬組成物。 - 線維芽細胞増殖因子受容体4(FGFR4)のチロシンキナーゼ活性を阻害又は抑制する物質が、H3B−6527、FGF−401(ロブリチニブ)又はBLU−554(フィソガチニブ)である、請求項1に記載の組合せ医薬、又は請求項2~5のいずれか1項に記載の医薬組成物。
- 前記がん及び/又は前記腫瘍が、FGFR4が発現し及び/又はFGFR4のチロシンキナーゼ活性がみられるがんである、請求項1に記載の組合せ医薬、又は請求項2~5のいずれか1項に記載の医薬組成物。
- 前記がん及び/又は前記腫瘍が、肝細胞がん、肺がん、子宮体がん、胆管がん、肝内胆管がん、食道がん、鼻咽頭がん、卵巣がん、乳がん、腎細胞がん、膵臓がん、結腸がん、及び膠芽腫、並びに横紋筋肉腫を含む肉腫からなる群より選ばれる少なくとも1種である、請求項1に記載の組合せ医薬、又は請求項2~5のいずれか1項に記載の医薬組成物。
- 前記抗体は、キメラ抗体又はヒト化抗体である、請求項1に記載の組合せ医薬、又は請求項2~5のいずれか1項に記載の医薬組成物。
- 前記抗体は、
(a)H鎖V領域のCDR1~3のアミノ酸配列が、それぞれ順に、配列番号3~5で示されるアミノ酸配列であり、かつ、L鎖V領域のCDR1~3のアミノ酸配列が、それぞれ順に、配列番号6~8で示されるアミノ酸配列である抗体、
(b)H鎖V領域のCDR1~3のアミノ酸配列が、それぞれ順に、配列番号9~11で示されるアミノ酸配列であり、かつ、L鎖V領域のCDR1~3のアミノ酸配列が、それぞれ順に、配列番号12~14で示されるアミノ酸配列である抗体、
(c)H鎖V領域のアミノ酸配列が配列番号16で示されるアミノ酸配列からなり、かつ、L鎖V領域のアミノ酸配列が配列番号18で示されるアミノ酸配列からなる抗体、
(d)H鎖V領域のアミノ酸配列が配列番号20で示されるアミノ酸配列からなり、かつ、L鎖V領域のアミノ酸配列が配列番号22で示されるアミノ酸配列からなる抗体、
(e)H鎖V領域のアミノ酸配列が配列番号24又は26で示されるアミノ酸配列からなり、かつ、L鎖V領域のアミノ酸配列が配列番号28で示されるアミノ酸配列からなる抗体、
(f)H鎖V領域のアミノ酸配列が配列番号30、32、34又は36で示されるアミノ酸配列からなり、かつ、L鎖V領域のアミノ酸配列が配列番号46で示されるアミノ酸配列からなる抗体、
(g)H鎖V領域のアミノ酸配列が配列番号38、40、42又は44で示されるアミノ酸配列からなり、かつ、L鎖V領域のアミノ酸配列が配列番号46で示されるアミノ酸配列からなる抗体、
(h)受託番号がFERM BP−10899であるハイブリドーマにより産生される抗体、
(i)受託番号がFERM BP−10707であるハイブリドーマにより産生される抗体、
(j)受託番号がFERM BP−10900であるハイブリドーマにより産生される抗体、
及び
(k)受託番号がFERM BP−11337であるハイブリドーマにより産生される抗体
からなる群から選択される少なくとも1種である、
請求項1に記載の組合せ医薬、又は請求項2~5のいずれか1項に記載の医薬組成物。 - 前記抗体又は抗体断片が、抗腫瘍活性及び/又は殺細胞活性を有する化合物との複合体の形態である、請求項1に記載の組合せ医薬、又は請求項2~5のいずれか1項に記載の医薬組成物。
- 前記組合せ医薬又は医薬組成物の投薬終了後においても、がん細胞の増殖を抑制し得る又は腫瘍を縮小若しくは消失させ得るものである、請求項1に記載の組合せ医薬、又は請求項2~5のいずれか1項に記載の医薬組成物。
- がんの治療用の薬剤を製造するための、
線維芽細胞増殖因子受容体4(FGFR4)のチロシンキナーゼ活性を阻害又は抑制する物質、若しくはそのプロドラッグ、又はそれらの薬理学的に許容し得る塩、あるいはそれらの水和物若しくは溶媒和物、及び
in vivoで抗腫瘍活性を有する、ヒトDLK−1に対する抗体、若しくは当該抗体に由来する抗体断片
の使用。 - 線維芽細胞増殖因子受容体4(FGFR4)のチロシンキナーゼ活性を阻害又は抑制する物質、若しくはそのプロドラッグ、又はそれらの薬理学的に許容し得る塩、あるいはそれらの水和物若しくは溶媒和物、及び
in vivoで抗腫瘍活性を有する、ヒトDLK−1に対する抗体、若しくは当該抗体に由来する抗体断片
を被験対象に投与することを特徴とする、がんの治療方法。 - 線維芽細胞増殖因子受容体4(FGFR4)のチロシンキナーゼ活性を阻害又は抑制する物質、若しくはそのプロドラッグ、又はそれらの薬理学的に許容し得る塩、あるいはそれらの水和物若しくは溶媒和物、及び
in vivoで抗腫瘍活性を有する、ヒトDLK−1に対する抗体、若しくは当該抗体に由来する抗体断片
を含む、がんを治療するためのキット。 - 前記がん及び/又は前記腫瘍が、FGFR4が発現し及び/又はFGFR4のチロシンキナーゼ活性がみられるがんである、請求項13に記載の使用、請求項14に記載の方法、又は請求項15に記載のキット。
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- 2021-10-04 EP EP21877781.1A patent/EP4226945A1/en active Pending
- 2021-10-04 AU AU2021355919A patent/AU2021355919A1/en active Pending
- 2021-10-04 CA CA3198077A patent/CA3198077A1/en active Pending
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CA3198077A1 (en) | 2022-04-14 |
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EP4226945A1 (en) | 2023-08-16 |
KR20230083296A (ko) | 2023-06-09 |
US20230398227A1 (en) | 2023-12-14 |
JPWO2022075482A1 (ja) | 2022-04-14 |
AU2021355919A1 (en) | 2023-05-11 |
TW202228692A (zh) | 2022-08-01 |
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