WO2017175874A1 - 抗mct5抗体を用いた癌治療用医薬組成物 - Google Patents
抗mct5抗体を用いた癌治療用医薬組成物 Download PDFInfo
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Definitions
- the present invention relates to an antibody conjugate in which a monoclonal antibody recognizing the extracellular domain of MCT5 is conjugated to a compound having activity against hyperproliferative diseases such as cancer against cancers such as breast cancer and colon cancer. It is related with the therapeutic pharmaceutical composition which consists of.
- the present invention also relates to a method of use for treatment by the combined use of an antibody and the compound, or the combined use of an antibody conjugate and the compound.
- Non-patent Document 1 Weigelt B, et al., Nature Reviews, 2005, 5, 591-602). It has also been reported that only 2-3% of patients are judged to have been in remission for over 20 years. That is, for the treatment of breast cancer, development of a new drug that can suppress recurrence or metastasis is desired.
- “Relapse” of cancer refers to the case where a small invisible cancer that could not be removed is left after the primary lesion is removed by surgery, and then re-forms, or it is once treated with drug therapy (anticancer drug treatment) or radiation therapy. This refers to the case where the cancer that has shrunk becomes larger again, and occurs in the vicinity of the first cancer that has developed.
- “metastasis” means that cancer cells reach an organ different from the primary lesion and secondarily produce the same type of cancer. For example, if a breast cancer forms a tumor again in the breast, it is “relapsed” and a secondary cancer that has metastasized to the lung is a breast cancer lung “metastasis” formed by malignant breast cancer cells.
- the mechanism of “recurrence” or “metastasis” is that cancer cells with high invasion ability locally invade another site in the same tissue as the primary lesion or tissue surrounding the cancer, and are left behind even after surgery. Is considered to be a major cause.
- the property of the cancer cell is important.
- the degree of atypicality and the depth of penetration are used.
- the degree of atypia expresses how much cancer cells are morphologically different from normal cells.
- the depth of penetration indicates the depth of cancer.
- cancer cells with high invasive potential have an increased risk of recurrence and metastasis. Therefore, treatments targeting cancer cells with high invasion ability can reduce the number of cases of recurrence and metastasis, and can be expected to reduce the mortality rate.
- Herceptin is known as an antibody drug approved in Japan as a molecular target drug for breast cancer.
- Herceptin is an antibody drug that targets the receptor HER2 (also called ERBB2, Her2 / neu), and has been approved for use as a therapeutic agent for metastatic breast cancer in which HER2 overexpression has been confirmed. It is thought that cells are killed by an action mechanism that kills cancer cells by binding to HER2 and antibody-dependent cytotoxicity (ADCC, Anti-Dependent Cellular Cytotoxicity) by natural killer cells (NK cells) or macrophages. Yes.
- ADCC Anti-Dependent Cellular Cytotoxicity
- Avastin and Erbitux are known as antibody drugs against colorectal cancer. These are antibody drugs targeting growth factors such as VEGF and EGF.
- Avastin is approved for use in advanced and recurrent colorectal cancer that cannot be curatively excised, and binds to VEGF and suppresses angiogenesis by inhibiting binding to the VEGF receptor, which can be applied to tumor tissues. It exerts its anticancer effect with a mechanism of action that eliminates nutrition.
- Erbitux aims to stop the growth of cancer cells by binding to the EGF receptor and preventing cell growth signals from being activated by EGF.
- molecular target drugs represented by antibodies are excellent substances in that they can kill cancer cells if they specifically recognize cancer.
- the antibody also binds to normal cells, it can show serious side effects.
- Herceptin a breast cancer drug, may cause not only headache, asthenia, nausea / vomiting, but also interstitial pneumonia, bone marrow suppression, liver damage, kidney damage, and cerebrovascular disorder.
- tissue staining strongly reacts with normal cardiomyocytes and causes severe heart damage (Non-patent Document 2: British Journal of Cancer, 94, 1016-1020, 2006).
- Herceptin is an antibody drug targeting HER2, there remains a problem that it only shows a response to patients expressing HER2.
- Avastin a colorectal cancer drug, includes bleeding, thrombosis, gastrointestinal perforation, delayed wound treatment, increased blood pressure, etc. Among them, thrombosis and gastrointestinal perforation are life-threatening side effects (non-patented) Document 3: Cancer Research, 57, 4593-4599, 1997). In Arbitux, skin disorders and the like are known as side effects, and although it does not threaten life, itching and white pustules occur, and there is a mental and physical burden on the patient (Non-patent Document 4). : Journal of Clinical Oncology, 22, 1201-1208, 2004). There is also a problem that it does not show an effect on canceration due to a signal change downstream of the EGF receptor (for example, K-ras mutation).
- Cancer cells are characterized by having a high proliferative power and no limit on the number of cell divisions compared to normal cells, and causing invasion and metastasis to surrounding tissues.
- cancer tissues have such properties, but some limited cells have been considered to have such properties.
- some of these cancer cells are common to stem cells such as embryonic stem cells and somatic stem cells, which have self-replicating ability to produce exactly the same cells as themselves and multipotency capable of differentiating into many types of cells. It is believed that these characteristics are responsible for generating the majority of the surrounding cancer cells by differentiation while maintaining the same cells as themselves by self-replication in cancer tissue.
- Some of these cancer cells are called cancer stem cells, and the hypothesis (cancer stem cell hypothesis) that cancer develops and progresses from these stem cell-like cells has been proposed.
- Non-patent Document 5 GASTROENTERROLOGY, 138, 2151-2162, 2010
- antibodies against these are some cancer stem cells. It is said that it is not effective as a therapeutic agent.
- a marker called LGR5 is known. This marker interacts with R-spondin and has a mechanism of activating Wnt / ⁇ -catenin signal, suggesting that it may be used as a cancer stem cell marker (Patent Document 1: Special Table 2010-532169). Publication).
- Cancer stem cells are considered to be a major cause of cancer recurrence and metastasis, and the importance of targeting cancer stem cells in cancer treatment has been pointed out.
- cancer stem cells there is only a small proportion of cancer stem cells in the tumor tissue, and it is considered that a therapeutic drug targeting only cancer stem cells cannot kill the entire cancer cells.
- the development of new therapies that target markers that are highly expressed in cancer stem cells compared to normal tissues and that are also expressed in general cancer cells is an important issue for cancer medicine It is.
- ADC Antibody-Drug Conjugate
- kadcyla (trastuzumab emtansine or T-DM1) obtained by binding herceptin (trastuzumab) and DM1 (faxane-based drug) is approved as a therapeutic agent for HER2-positive advanced breast cancer.
- T-DM1 kadcyla which is an anticancer agent can be effectively killed by incorporation into cells.
- Membrane proteins include not only receptors that bind to ligands, but also transport proteins (hereinafter referred to as transporters) that actively or passively transport low-molecular compounds such as amino acids and sugars. Since these molecules have a role of penetrating molecules inside and outside the cell, it has been considered that no internalization occurs. However, as shown in Non-Patent Document 7 (The Journal of Biological Chemistry, 285, 27289-27301, 2010), SLC6A3 (Sodium dependent dopamine transporter) has been reported to cause constant internalization. There is a possibility that not only the receptor but also the transporter can efficiently transport the drug bound to the antibody into the cell.
- transporters transport proteins
- MCT5 (Monocarbylate transporter 5) is a membrane protein consisting of 487 amino acids, and is registered as NCBI (National Center for Biotechnology Information) Reference Sequences [RefSeq] ID: NM_004696.2, NP_004687.1 (SEQ ID NO: 1: Base) Sequence, SEQ ID NO: 2: amino acid sequence).
- MCT5 is an orphan transporter whose transport substance has not been identified, and is classified into the MCT group based on the homology of amino acid sequences.
- Patent Document 1 Japanese Translation of PCT International Publication No. 2010-532169
- Non-Patent Document 1 Nature Reviews, 5,591-602, 2005
- Non-Patent Document 2 British Journal of Cancer, 94, 1016-1020, 2006
- Non-Patent Document 3 Cancer Research, 57, 4593-4599, 1997
- Non-Patent Document 4 Journal of Clinical Oncology, 22, 1201-1208, 2004
- Non-Patent Document 5 GASTROENTERROLOGY, 138, 2151-2162, 2010
- Non-Patent Document 6 Cancer Research, 68, 9280-90, 2008
- Non-Patent Document 7 The Journal of Biological Chemistry, 285, 27289-27301, 2010
- the present inventors have identified an anti-MCT5 monoclonal antibody that recognizes the extracellular domain of MCT5 highly expressed in a cancer having a higher invasive ability than a normal cancer cell as a cell. When reacted, it was found to internalize into cells through at least the clathrin pathway. That is, the present inventors have found that cancer cells can be effectively killed by conjugating an anti-MCT5 monoclonal antibody and an anticancer agent, thereby completing the present invention.
- the present invention is as follows.
- a pharmaceutical composition for cancer treatment comprising the antibody according to any one of (1) to (5) or the fragment according to (6).
- a pharmaceutical composition for treating cancer comprising a combination of the antibody according to any one of (1) to (5) or the fragment according to (6) and a chemotherapeutic agent, toxin or radioisotope.
- a pharmaceutical composition for treating cancer comprising a complex in which the antibody according to any one of (1) to (5) or the fragment according to (6) is bound to a chemotherapeutic agent, toxin or radioisotope .
- a pharmaceutical composition comprising an MCT5 monoclonal antibody or antigen-binding fragment, and a substance obtained by conjugating the antibody or antigen-binding fragment and an anticancer agent.
- the pharmaceutical composition is particularly useful as a pharmaceutical composition for treating breast cancer and colon cancer.
- MCT5 is highly expressed in cancer cells with high invasion ability compared with normal tissues, cancer cells with high malignancy can be targeted by the present invention, and cancer progression, metastasis, recurrence can be achieved.
- a novel cancer therapeutic agent or therapeutic method that suppresses the disease is provided.
- MCT7 cells that are overexpressed in human breast cancer cells (MCF7-MCT5 cells), MDA-MB231 cells that are highly metastatic human breast cancer cells, HCT116 cells that are human colon cancer cells, anti-MCT5 antibody, It is a figure which shows the result of having analyzed this reaction by the cell immuno-staining.
- MCF7-MCT5 cells analysis was performed using Alexa555-labeled secondary antibody as a secondary antibody, and in MDA-MB231 cells and HCT116 cells, Alexa488-labeled secondary antibody.
- MCF7-MCT5 cells incorporate a gene designed to express EGFP at the same time as MCT5, fluorescence of EGFP was observed at 488 nm excitation. It is the result of having analyzed that the clone number IMI4C4a antibody which is an anti- MCT5 monoclonal antibody bind
- HCT116 cell shows the result of having treated HCT116 cell with Concanavalin A of the density
- HCT116 cells were treated with 0.5 mg / ml Concanavalin A for 1 hour and then reacted with IMI4C4a antibody, 107 antibody, 217 antibody, 221 antibody, 223 antibody, 303 antibody, 306 antibody, and 301 antibody to enter the cell.
- the present invention relates to a monoclonal antibody that binds to MCT5 or an antigen-binding fragment thereof.
- the present invention also relates to the antibody, antigen-binding fragment, and a pharmaceutical composition containing the antibody, a hybridoma cell that produces the antibody, a nucleic acid for producing the antibody and the fragment, a recombinant expression vector, and a cell.
- MCT5 is expressed in breast cancer and colon cancer
- a monoclonal antibody that recognizes MCT5 can be used as a detection agent and diagnostic agent for colon cancer.
- the region of the base sequence 751 to 774 of MCT5 (AATTTAACAGTCTCCAAAAATCAA (SEQ ID NO: 3)) and the region of amino acid residues 251 to 258 (NLTVSQNQ (SEQ ID NO: 4)), which were conventionally considered as intracellular domains, are extracellular. It has been shown to exist.
- the invasion ability of human breast cancer cells overexpressing MCT5 is increased, the anti-MCT5 antibody (clone number IMI4C4a) binds only to a part of the tumor tissue, and the anti-MCT5 antibody positive tissue is a breast cancer tissue
- the rate of expression increases as the progression stage progresses, indicating that MCT5 is specifically expressed in breast cancer with high invasive ability, and that anti-MCT5 antibody can detect breast cancer with high malignancy.
- the IMI4C4a antibody has epitopes at positions 251 to 258 (SEQ ID NO: 4: amino acid sequence) of the MCT5 protein.
- the present invention relates to a pharmaceutical composition containing an antibody-anticancer agent complex in which an anticancer agent is bound to a monoclonal antibody that binds to MCT5 or an antigen-binding fragment thereof.
- MCT5 (Monocarbylate transporter 5) is an orphan transporter that is a multiple-transmembrane membrane protein and whose substance to be transported has not been determined. It is predicted to have a similar function from the homology of the amino acid sequence with MCT1 and MCT4 related to the regulation of lactic acid concentration inside and outside the cell.
- MCT5 which is a membrane protein, is a protein that is overexpressed in cancer tissues, and is highly expressed in highly invasive cancer cells that are a major cause of cancer recurrence and metastasis, rather than normal cancer cells. It is based on the knowledge that it is doing.
- the present invention provides a monoclonal antibody that recognizes the extracellular region of MCT5 or an antigen-binding fragment thereof. Since MCT5 is expressed in cancer cells such as breast cancer and colon cancer, this antibody specifically binds to cancer cells such as breast cancer and colon cancer.
- Anti-MCT5 Antibody of the Present Invention The monoclonal antibody of the present invention (hereinafter also referred to as “anti-MCT5 antibody of the present invention”) can recognize native MCT5. “Native” means that the protein is in an intact three-dimensional structure in the environment of the living body.
- the anti-MCT5 antibody of the present invention can recognize the extracellular region of MCT5.
- the anti-MCT5 antibody of the present invention recognizes at least a part of the three-dimensional structure in the extracellular region of MCT5 as an epitope.
- the extracellular region is encoded by the base sequence (SEQ ID NO: 3) of the base sequence 751-774 of the MCT5 gene and can recognize the region of amino acid residues 251 to 258 (SEQ ID NO: 4).
- the anti-MCT5 antibody of the present invention has a sequence as long as the binding activity to the polypeptide having the amino acid sequence shown in SEQ ID NO: 4 is maintained, that is, as long as the binding target polypeptide has a function as an extracellular region of MCT5.
- the anti-MCT5 antibody of the present invention is a polypeptide having a function as an extracellular region of MCT5, and is encoded by a polynucleotide having the base sequence set forth in SEQ ID NO: 3.
- hybridization can be performed according to a known method (for example, Molecular Cloning 2nd Ed (Cold Spring Harbor Lab. Press, 1989).
- a condition in which a specific hybrid is not formed for example, a condition in which a sodium concentration is 10 mM to 300 mM, preferably 20 mM to 100 mM, and a temperature is 25 ° C.
- the anti-MCT5 antibody of the present invention binds to MCT5, in the above mutant polypeptide, the anti-MCT5 antibody of the present invention can bind to a polypeptide having the amino acid sequence of SEQ ID NO: 4 of the antibody. Binding activity to peptides It is included in the polypeptide which shows that the sex is maintained, that is, has a function as an extracellular region of MCT5.
- Whether the mutant polypeptide has a function as an extracellular domain of MCT5 is determined by forced expression of the mutant polypeptide in animal cells and the like, and whether the invasion ability is increased. This can be confirmed by using an invasion assay.
- the extracellular region of MCT5 is a cell surface site of a marker protein whose expression increases in cancer cells with high invasive ability. Whether the mutant polypeptide has a function as an extracellular domain of MCT5 is determined by immunostaining, ELISA, immunoprecipitation, Western blotting, Flow cytometry, etc. This can be confirmed by comparison.
- the binding between the anti-MCT5 antibody of the present invention and the epitope or mutant polypeptide can be confirmed by ELISA, immunoprecipitation, Western blotting or the like.
- the anti-MCT5 antibody of the present invention also recognizes a protein encoded by an MCT5 mRNA variant. Since it can bind not only to full-length MCT5 but also to mutants partially lacking, it can bind to cancer cells expressing MCT5 extensively.
- an “antibody” is an immunoglobulin molecule composed of two heavy chains and two light chains.
- Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
- This heavy chain constant region consists of three domains CH1, CH2 and CH3.
- Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
- the light chain constant region consists of one domain CL.
- the heavy chain variable region and the light chain variable region further comprise a relatively conserved region called a framework region (FR) and a hypervariable region called a complementarity determining region (CDR).
- FR framework region
- CDR complementarity determining region
- Each VH and VL is composed of three CDRs and four FRs, and are arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxy terminus.
- the anti-MCT5 antibody of the present invention may be full-length or an antigen-binding fragment.
- an “antigen-binding portion” refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (eg, MCT5). It is known that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of “antigen-binding portion” of an antibody include, but are not limited to, Fab, F (ab ′) 2 , Fd fragment, Fv, dAb, CDR, scFv, diabody, and the like.
- Antibody portions such as Fab and F (ab ′) 2 fragments, can be prepared from full length antibodies using conventional techniques, eg, papain or pepsin digestion of the whole antibody, respectively.
- Antibodies and antigen-binding fragments can also be obtained using standard recombinant DNA techniques.
- antibody-like molecules such as an antigen-binding fragment or antibody fragment, a low molecular weight antibody, a recombinant antibody, and an antibody modification product that are part of a monoclonal antibody by various genetic engineering and protein engineering techniques.
- a protein fused with a monoclonal antibody can be prepared.
- multispecific antibodies such as diabodies, chimeric antibodies, humanized antibodies, human antibodies, single chain antibodies, and bispecific antibodies. Any molecule that has the ability to bind to the extracellular region of MCT5 is included in the anti-MCT5 antibody of the present invention.
- MCT5 recognized by the anti-MCT5 antibody of the present invention is a molecular marker that is not expressed in normal cells but highly expressed in cancer cells with high invasive ability in the cell population to be detected. Therefore, the antibody binds to high malignant cancer cells.
- the “cancer cell” in the present invention refers to a cell population having characteristics such as high proliferation ability and the number of cell divisions as compared with normal cells, and infiltration and metastasis to surrounding tissues. .
- “highly invasive cancer cell” refers to a cell having the ability to invade into a site distant from the original location of cancer cells.
- MCF7 cells which are human breast cancer cells
- MCF7-14 cells having enhanced invasive ability from MCF7 cells have high invasive ability and infiltrate from the transplant site to other organs, and thus are cells having high invasive ability (BMC Cancer (2010), 10,414).
- Cells with high invasive ability can be evaluated by an invasive ability test using a Matrigel invasion chamber or the like. Matrigel mimics the basement membrane. By seeding and culturing cells on the upper part of the Matrigel layer, it can be detected as a cell population that easily passes (invades) the Matrigel layer.
- normal cell refers to a cell having a normal function in the activity of a living body or tissue. Normal cells may include somatic stem cells, but are preferably mature cells.
- the anti-MCT5 antibody of the present invention binds strongly to cancer cells with high invasive ability and can bind to cancer cells of one or more cancer types.
- the cancer type and cancer cells are heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, white blood cell, colon, stomach, bone marrow, large intestine and
- One or more cancer types derived from cells or tissues such as peripheral blood mononuclear cells, more preferably cancer cells of breast cancer and colon cancer.
- the anti-MCT5 antibody of the present invention does not bind to normal cells.
- the anti-MCT5 antibody of the present invention does not bind to normal cells.
- at least one or more of, for example, heart, brain, placenta, lung, skeletal muscle, kidney, spleen, thymus, prostate, testis, ovary, small intestine, leukocyte, colon, bone marrow, large intestine and peripheral blood mononuclear cells Does not bind to normal cells.
- the anti-MCT5 antibody of the present invention is preferably an anti-MCT5 monoclonal antibody produced by the following hybridoma.
- Examples of cell lines (hybridomas) that produce the monoclonal antibody of the present invention include “Mouse-Mouse hybridoma IMI4C4a” (hereinafter referred to as “IMI4C4a”) and “Mouse-Mouse hybridoma IMI4C12a” (hereinafter referred to as “IMI4C12a”). .
- the anti-MCT5 antibody of the present invention is a chimeric antibody.
- “Chimeric antibody” means an antibody in which the heavy chain constant region and the light chain constant region are derived from a human, and the heavy chain variable region and the light chain variable region are derived from a non-human (eg, mouse).
- an antibody or antigen-binding fragment in which the constant region is derived from human and the variable region is derived from mouse may be referred to as mouse-human chimeric antibody or antigen-binding fragment.
- the anti-MCT5 antibody of the present invention having these characteristics can be obtained by immunization using a partial protein of the extracellular domain of MCT5.
- MCT5 in the database is a 12-transmembrane protein, and ICD4, which is an antigen of the IMI4C4a antibody, was predicted to be an intracellular domain.
- the number of transmembranes and / or the extracellular domain of MCT5 do not match the database, and at least ICD4 is exposed to the outside of the cell. That is, for a monoclonal antibody that recognizes the extracellular domain of MCT5, it is necessary to identify a region that is the extracellular domain and prepare a monoclonal antibody against that portion.
- the monoclonal antibody which produced the amino acid sequence containing the newly estimated extracellular domain as an antigen can be utilized as an anti- MCT5 antibody.
- the anti-MCT5 antibody of the present invention has the following characteristics. Since the antibody of the present invention is a newly obtained antibody based on the finding that the three-dimensional structure of MCT5 is different from that of the database, native MCT5 on the cell surface can be recognized. In addition, a conventional monoclonal antibody (HPA046986) using an antigen near the epitope of IMI4C4a, which is an anti-MCT5 antibody, cannot recognize native MCT5 and does not bind to MCT5 on the cell surface. In contrast, the recognition site of the antibody of the present invention binds to the extracellular domain of MCT5 and can bind to living cells. Therefore, the anti-MCT5 antibody of the present invention is effective as a therapeutic agent targeting cancer cells that express MCT5.
- HPA046986 monoclonal antibody using an antigen near the epitope of IMI4C4a
- the conventional antibody is a polyclonal antibody, and it has been difficult to continuously produce a homogeneous antibody.
- the antibody of the present invention is a monoclonal antibody, it can be mass-produced with high reproducibility. . From these characteristics, the antibody of the present invention can be used for the treatment of cancer.
- the antibody or antigen-binding fragment against MCT5 is not limited to the mouse monoclonal antibody itself against MCT5 produced by the hybridoma IMI4C4a, but as long as it binds to an epitope recognized by the monoclonal antibody produced by these hybridomas. Included in MCT5 antibody.
- epitope refers to an epitope recognized by the monoclonal antibody produced by the hybridoma (amino acid residues from 196th to 299th in the amino acid sequence of MCT5, as well as a part of these regions). May be).
- the antibody of the present invention may be a recombinant antibody or antigen-binding fragment prepared and expressed by recombinant means.
- the anti-MCT5 antibody of the present invention may be a chimeric antibody, a humanized antibody, or a fully human antibody.
- the recombinant antibodies of the invention can be produced by recombinant expression of heavy and light chains.
- a mouse-human chimeric antibody an antibody gene is isolated from a mouse cell that produces an antibody against the MCT5 protein, and its heavy chain (H chain) constant region is recombined with a human IgG H chain constant region gene. It can be prepared by introduction into tumor cells.
- a humanized antibody can be prepared, for example, by transplanting an antigen-binding site gene of an antibody isolated from a mouse cell producing an antibody against MCT5 protein to a human-derived antibody molecule.
- a human antibody can be prepared by immunizing a mouse in which the immune system is replaced with a human with MCT5 protein.
- a protein in which a monoclonal antibody is fused can be prepared by using an existing gene recombination technique for an antibody variable region that binds to an antigen and other proteins. Alternatively, it can be produced by crosslinking a monoclonal antibody and a protein using a crosslinker.
- a recombinant expression vector having nucleic acids encoding the heavy chain and light chain of the antibody is introduced into a host cell, and the host cell into which the vector has been introduced is cultured.
- the antibody of interest can be recovered from the host cell culture.
- DNA fragments encoding VH and VL can be further manipulated by standard recombinant DNA methods in the art, for example, to produce Fab genes, scFv genes, full-length antibody genes.
- nucleic acid (eg, DNA) encoding VH can be expressed into a full-length heavy chain gene by allowing expression of the DNA encoding VH and DNA encoding the heavy chain constant region (CH1, CH2, and CH3). And can be converted. Nucleic acid sequences of heavy chain constant regions derived from humans, mice, etc. are known in the art.
- Examples of the anti-MCT5 antibody of the present invention include those in which the amino acid sequences of the complementarity determining regions (CDR) 1 to 3 of the heavy chain variable region (VH) include the amino acid sequences shown by SEQ ID NOs: 7, 8, and 9, respectively. Or the amino acid sequence comprising the amino acid sequence and / or the amino acid sequence of the complementarity determining region (CDR) 1 to 3 of the light chain variable region (VL) comprising the amino acid sequences represented by SEQ ID NOs: 12, 13, and 14, respectively What consists of the said amino acid sequence is preferable.
- the anti-MCT5 antibody of the present invention includes, for example, one in which the amino acid sequence of the heavy chain variable region (VH) comprises the amino acid sequence represented by SEQ ID NO: 6 or consists of the amino acid sequence (SEQ ID NO: 5) and / or the light chain variable region (VL) amino acid sequence comprising or consisting of the amino acid sequence shown in SEQ ID NO: 11 (shown in SEQ ID NO: 10) (Encoded by the base sequence) is preferred.
- VH the amino acid sequence of the heavy chain variable region
- VL light chain variable region
- the nucleic acid encoding VH and VL is, for example, a nucleic acid derived from mouse and the nucleic acid encoding the heavy chain and light chain constant regions is derived from human, the antibody or antigen binding of mouse-human chimera Fragments can be obtained.
- the partial or full-length heavy chain gene and light chain gene obtained as described above are inserted into an expression vector.
- the heavy and light chain genes are inserted into separate vectors or both genes are inserted into the same expression vector.
- the antibody gene can be inserted into the expression vector by standard methods.
- the expression vector can also encode a signal peptide that promotes secretion of the antibody chain from the host cell.
- the signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide.
- the expression vector may contain other regulatory sequences, and those skilled in the art can select a regulatory sequence based on a known technique and introduce it into the expression vector.
- the epitope (antigenic determinant) of the anti-MCT5 antibody of the present invention is not limited as long as it is at least a part of MCT5, which is an antigen, but at least a part of the extracellular region of MCT5 is preferable.
- the amino acid sequence of MCT5 represented by SEQ ID NO: 2 is preferably at least part of the region consisting of the 196th to 299th amino acids (SEQ ID NO: 15).
- SEQ ID NO: 16 is more preferred, and particularly preferred is the region consisting of the 251st to 258th amino acids (SEQ ID NO: 4).
- An anti-MCT5 antibody that recognizes the region (binding to the region) has, for example, a high detection sensitivity for MCT5 in a biological sample, and is extremely useful for cancer detection and diagnosis described later.
- isoforms in MCT5 due to differences in mRNA splicing.
- isoform1 SEQ ID NO: 2 having the longest amino acid sequence
- amino acids from position 74 to position 121 of isoform1 lacking the amino acid sequence from the N-terminal to the 62nd amino acid in isoform1
- a peptide having the number 18) isoform 3
- a sequence lacking the amino acid from the N-terminal to the 110th position in isoform 1 and differing from the 111th to 120th amino acid sequences of the isoform 1 SEQ ID NO: 19
- isoform2, isoform3 and isoform4 are common with isoform1 of MCT5 in the region consisting of the 196th to 299th amino acids in the amino acid sequence of isoform1 of MCT5 represented by SEQ ID NO: 2.
- the antibody of the present invention can also detect these three types of isoforms other than isoform1.
- an antibody prepared using an antigen containing SEQ ID NO: 17 can detect isoform2 and isoform5 in addition to isoform1.
- the antibody prepared using SEQ ID NO: 19 can also detect isoform3, isoform4 and isoform6.
- the epitope (antigenic determinant) of the anti-MCT5 antibody of the present invention includes at least a part of the region corresponding to the above region in MCT5 derived from other animals.
- the antibody against MCT5 of the present invention includes an antibody that binds to a site (eg, an epitope) to which the antibody binds, for example, an antibody that binds to a site to which an antibody produced by the hybridoma of the present invention binds.
- One preferred embodiment of the antibody of the present invention is a recombinant antibody.
- the recombinant antibody include, but are not limited to, chimeric antibodies, humanized antibodies, and humanized antibodies.
- a chimeric antibody ie, a human chimeric antibody
- a chimeric antibody is an antibody obtained by linking (joining) a variable region of a mouse-derived antibody to a human-derived constant region (Proc. Natl. Acad. Sci. USA 81.8551). -6855, (1984), etc.), when a chimera is produced, it can be easily constructed by genetic recombination techniques so as to obtain an antibody so linked.
- the heavy chain variable region preferably includes, for example, the amino acid sequence represented by SEQ ID NO: 6 or consists of the amino acid sequence
- the light chain variable region includes, for example, SEQ ID NO: 11
- the thing containing the amino acid sequence shown by or consisting of the said amino acid sequence is preferable.
- the mouse-human chimeric antibody includes an amino acid sequence (SEQ ID NO: 29) in which a heavy chain variable region is bound to a human constant region, and an amino acid sequence (SEQ ID NO: 32) in which a light chain variable region is bound to a human constant region. Or those consisting of the amino acids are preferred.
- CDR grafting refers to transplanting a complementarity determining region (CDR) from a variable region of a mouse antibody into a human variable region, wherein the framework region (FR) is derived from a human and the CDR is derived from a mouse. This is a method for producing a configured variable region. These humanized reshaped human variable regions are then linked to human constant regions. Methods for producing such humanized antibodies are well known in the art (Nature, 321, 522-525 (1986); J. Mol. Biol., 196, 901-917 (1987); Queen C et al. Proc. Natl. Acad. Sci.
- CDRs 1 to 3 of the heavy chain variable region are represented by SEQ ID NOs:
- the amino acid sequences represented by 7, 8 and 9 are preferred, and the amino acid sequences represented by SEQ ID NOs: 12, 13 and 14 are preferred as CDRs 1 to 3 of the light chain variable region.
- a human antibody (fully human antibody) generally has the same structure as a human antibody in the hypervariable region, which is the antigen-binding site of the variable region (V region), the rest of the V region, and the constant region. It is what has. Techniques for producing human antibodies are also known, and methods for producing gene sequences common to humans by genetic engineering techniques have been established.
- the human antibody is, for example, a method using a human antibody-producing mouse having a human chromosome fragment containing the heavy chain (H chain) and light chain (L chain) genes of a human antibody (Tomizuka, K. et al., Nature Genetics). (1977) 16, 133-143; Kuroiwa, Y.
- chimeric antibodies, humanized antibodies, and humanized antibodies can be prepared according to the above-described known methods using a hybridoma or DNA or RNA extracted from the hybridoma as a raw material.
- the protein in which the antibody of the present invention is fused can be prepared by using a known gene recombination method for the variable region of the antibody and other proteins.
- the said fusion protein can be produced by bridge
- the antibody fragment against MCT5 used in the present invention specifically binds to MCT5.
- the antibody fragment means a partial region of the antibody of the present invention, and examples thereof include Fab, Fab ′, F (ab ′) 2 , Fv, diabody (dibodies), dsFv, scFv (single chain Fv), and the like.
- the antibody fragment can be obtained by cleaving the antibody of the present invention with various proteolytic enzymes according to the purpose. For example, Fab can be obtained by treating antibody molecules with papain, and F (ab ′) 2 can be obtained by treating antibody molecules with pepsin. Fab ′ can be obtained by cleaving the disulfide bond in the hinge region of F (ab ′) 2 .
- scFv cDNA encoding the heavy chain variable region (H chain V region) and light chain variable region (L chain V region) of the antibody is obtained, and DNA encoding scFv is constructed. By inserting this DNA into an expression vector and introducing the expression vector into a host organism for expression, scFv can be produced.
- diabody cDNA encoding the H chain V region and L chain V region of the antibody is obtained, and a DNA encoding scFv is constructed so that the length of the amino acid sequence of the peptide linker is 8 residues or less.
- a diabody can be produced by inserting this DNA into an expression vector and introducing the expression vector into a host organism for expression.
- the base sequence of the DNA encoding the heavy chain variable region includes, for example, one comprising the base sequence represented by SEQ ID NO: 5 or consisting of the base sequence, and the DNA sequence encoding the light chain variable region.
- the base sequence include those containing the base sequence represented by SEQ ID NO: 10 and those consisting of the base sequence.
- antibody fragment of the present invention include, but are not limited to, for example, the amino acid sequences of VH CDRs 1 to 3 including the amino acid sequences represented by SEQ ID NOs: 7, 8, and 9, respectively.
- amino acid sequences of VL CDRs 1 to 3 include antibody fragments containing the amino acid sequences represented by SEQ ID NOs: 12, 13, and 14, respectively.
- an antibody fragment containing the amino acid sequence represented by SEQ ID NO: 6 as VH and / or the amino acid sequence represented by SEQ ID NO: 11 as VL can be mentioned.
- An antibody fragment (peptide) containing CDR is constituted by including at least one region of CDR (CDR1 to CDR3) of VH or VL.
- Antibody fragments comprising multiple CDRs can be linked directly or via a suitable peptide linker.
- An antibody fragment containing CDRs constructs DNA encoding the antibody VH and VL CDRs, inserts the DNA into a prokaryotic expression vector or eukaryotic expression vector, and inserts the expression vector into a prokaryotic or eukaryotic expression vector. It can be expressed and produced by introduction into a nuclear organism.
- the peptide containing CDR can also be manufactured by chemical synthesis methods, such as Fmoc method (fluorenylmethyloxycarbonyl method) and tBoc method (t-butyloxycarbonyl method).
- the base sequences encoding VH CDRs 1 to 3 for example, the base sequences represented by SEQ ID NOs: 21, 22, and 23 are preferable, respectively.
- the base sequences encoding VL CDRs 1 to 3 for example, SEQ ID NO: 24, respectively. , 25 and 26 are preferred.
- Binding Affinity Binding affinity can be determined by association constant (KA) and dissociation constant (KD).
- the affinity equilibrium constant (K) is expressed as a ratio of KA / KD. Its binding affinity can be detected as follows.
- the binding affinity has a dissociation constant (KD) of at least 1 ⁇ 10 ⁇ 10 M.
- the dissociation constant is 2 to 5 times, 5 to 10 times, 10 to 100 times, 100 to 1000 times. Alternatively, it has an affinity 1000 to 10,000 times higher.
- the dissociation constant (KD) relating to the binding affinity of MCT5 of the antibody of the present invention is 1 ⁇ 10 ⁇ 10 M, 5 ⁇ 10 ⁇ 11 M, 1 ⁇ 10 ⁇ 11 M, 5 ⁇ 10 ⁇ 12.
- the value may be lower than these KDs and may have high affinity.
- the dissociation constant (KD) of the antibody to be measured for affinity is within about 1 to 100 times the KD of the antibody of the present invention, the antibody is substantially the same as the antibody of the present invention. Is included in the present invention.
- the association constant (KA) and dissociation constant (KD) can be measured using surface plasmon resonance (SPR), and a known apparatus and method for detecting the binding rate in real time and further monitoring can be employed.
- SPR surface plasmon resonance
- Biacore registered trademark
- T200 GE Healthcare
- ProteON XPR36 Bio-Rad
- Reagents for cancer treatment include Chinese hamster ovary cells (CHO cells), COS cells, 293 cells, HeLa cells, 3T3 cells and the like.
- a recombinant expression vector encoding the antibody heavy chain and light chain is introduced into a preferred host cell by a known gene introduction method.
- the obtained transformant is cultured, and the target antibody or antigen-binding fragment is recovered from the culture.
- the antibody or antigen-binding fragment may be purified by a known purification technique.
- the anti-MCT5 antibody of the present invention is a chimeric antibody.
- “Chimeric antibody” means an antibody in which the heavy chain constant region and the light chain constant region are derived from humans, and the heavy chain variable region and the light chain variable region are derived from other than humans (eg, mouse).
- an antibody or antigen-binding fragment in which the constant region is derived from human and the variable region is derived from mouse may be referred to as a human-mouse chimeric antibody or antigen-binding fragment.
- the monoclonal antibodies (including antigen-binding fragments thereof) of the present invention comprise at least one chemotherapeutic agent (including anticancer agent), toxin, or radioisotope.
- chemotherapeutic agent including anticancer agent
- toxin including anticancer agent
- radioisotope can be made into a complex, and such a complex (also referred to as an antibody conjugate of the present invention) is also included in the scope of the present invention.
- an antibody conjugate comprising a monoclonal antibody of the invention and a radioisotope, wherein the antibody conjugate is conjugated to a detectable radioisotope.
- the detectable radioisotopes are, for example, 3 H, 14 C, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho or 153 Sm.
- an antibody conjugate comprising the monoclonal antibody of the present invention and an anticancer agent or toxin, wherein the monoclonal antibody of the present invention forms a complex with the anticancer agent or toxin.
- Antibody conjugates are provided.
- the chemotherapeutic agent is preferably an anticancer agent.
- the toxin is preferably saponin, DM1 (N2′-Deacetyl-N2 ′-(3-mercapto-1-oxopropyl) -maytanine), DM4 (N2′-Deacetyl-N2 ′-(4-mercapto-4-methyl). -1-oxopentyl) -maytansine), MMAE (Monomethyl auristatin E), SN38.
- chemotherapeutic agent is a chemical compound useful in the treatment of cancer.
- Types of chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites, spindle inhibitor plant alkaloids, cytotoxic / antitumor antibiotics, topoisomerase inhibitors, antibodies, light Sensitizers and kinase inhibitors are included.
- Chemotherapeutic agents include compounds used in “targeting therapy” and conventional chemotherapy.
- chemotherapeutic agents include erlotinib (TARCEVA®, Genentech / OSIPharm.), Docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS number 51-21).
- gemcitabine (GEMZAR (registered trademark), Lilly), PD-0325901 (CAS number 391210-10-9, Pfizer), cisplatin (cis-diamine, dichloroplatinum (II), CAS number 15,663-27-) 1), Carboplatin (CAS number 41575-94-4), Paclitaxel (TAXOL (registered trademark), Bristol-Myers Squibb Oncology, Princeton, NJ), Trastuzuma (HERCEPTIN®, Genentech), temozolomide (4-methyl-5-oxo-2,3,4,6,8-pentazabicyclic [4.3.0] nona-2,7,9-triene -9-Carboxamide, CAS No.
- TEMODAR registered trademark
- TEMODAL registered trademark
- Schering Plow Tamoxifen ((Z) -2- [4- (1,2-diphenylbut-1) -Enyl) phenoxy] -N, N-dimethyl-ethanamide, NOLVADEX (R), ISTUBAL (R), VALODEX (R)), and doxorubicin (ADRIAMYCIN (R)), Akti-1 / 2, HPPD And rapamycin.
- chemotherapeutic agents include oxaliplatin (ELOXATIN®, Sanofi), Bortezomib (VELCADE®, Millennium Pharm.), SutentiniB®, SU11248, Pfizer, letrozole (FEMARA®, Novartis), Imatinib mesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO 2007/0444515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPh) , Astra Zeneca), SF-1126 (PI3K inhibitor, Semaphore Pharmaceuticals), BEZ-2 35 (PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787 / ZK 222584 (Novartis), fulvestrant (FASLODEX (R), AstraZeneca), leucovorin (folinic acid), rapamycin (Sirolimus
- dynemicin dynemicin A
- bisphosphonates such as clodronate; esperamicin; as well as neocalcinostatin luminescence Group and related chromoprotein enedyne antibiotic luminophore
- aclacinomycins actinomycin, austramycin, azaserine, bleomycins, cactinomycin, alabicin Carminomycin, cardinophylli (Carzinophilin), including chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and dexoxorubicin ), Epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin
- chemotherapeutic agent includes (i) antihormonal agents that act to regulate or inhibit hormonal effects on tumors, antiestrogens and selective estrogen receptor modulators (SERM), such as tamoxifen (NOLVADEX®) Tamoxifen citrate), raloxifene, droloxifene, 4-hydroxy tamoxifen, trioxyphene, keoxifene, LY11018, onapristone, and FARESTON® Toremifene citrate); (ii) aromatase inhibitors that inhibit aromatase enzymes, which regulate estrogen production in the adrenal glands, eg 4 5) -Imidazoles, aminoglutethimide, MEGASE (R) (megestrol acetate), AROMASIN (R) (exemestane, Pfizer), formestine, fadrozole, RIVISOR (R) (vorozole) )), FEMARA® (Letrozole, Novartis) and
- SERM selective estrogen
- chemotherapeutic agent includes therapeutic antibodies such as alemtuzumab (Campath), bevacizumab (Avastin®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX ( (Registered trademark), Amgen), rituximab (RITUXAN (registered trademark), Genentech / Biogen Idec), pertuzumab (OMNITARG TM, 2C4, Genentech), trastuzumab (HERCEPTIN (registered trademark), Genentech), toxitumab (Bx) An antibody drug conjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth) is included.
- MCT5 monoclonal antibody and humanized monoclonal antibody having therapeutic ability as a chemotherapeutic agent combined therewith include alemtuzumab, apolizumab, acelizumab, atolizumab, bapineuzumab, bevacizumab, bibatuzumab-mertansine, cantuzumab-mertansine, cedelizumab, celtrizumab, Shidofushitsuzumabu, Shidotsuzumabu, daclizumab, eculizumab, efalizumab, epratuzumab, Erurizumabu, Ferubizumabu, fontolizumab, gemtuzumab ozogamicin, Inotsuzumabu ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, Motobizumabu,
- Metal Metabolite is a product produced by metabolism in the body of a specific compound or salt. Metabolites of compounds may be identified using conventional techniques known in the art, and their activity may be determined using tests such as those described herein. Such products may result from, for example, oxidation, reduction, hydrolysis, amidation, deamidation, esterification, esterolysis, enzyme cleavage, etc. of the administered compound. Accordingly, the present invention includes a metabolite of a compound of the present invention, including a compound produced by a method comprising contacting the compound of the present invention with a mammal for a time sufficient to obtain a metabolite.
- the monoclonal antibody of the present invention can be bound (conjugate) directly or indirectly to a chemotherapeutic agent (including an anticancer agent), a toxin or a radioisotope.
- Indirect binding includes binding via a linker and binding via an antibody or antibody fragment that binds to the monoclonal antibody of the present invention.
- an antibody conjugate formed by the binding of an antibody conjugated with a labeling substance such as a fluorescent label and the monoclonal antibody of the present invention is also included in the present invention.
- the monoclonal antibody of the present invention desirably contains an antigen-binding region specific for effector cells having tumoricidal activity or tumor suppressive activity, such as natural killer cells (NK cells) and macrophages.
- tumor killing activity means the activity of destroying or killing tumor cells
- tumor suppression activity means the activity of reducing the number of tumor cells or the activity of suppressing the growth rate of tumor cells.
- ADCC antibody-dependent cellular cytotoxicity
- composition of the present invention in the present invention, a pharmaceutical composition comprising the anti-MCT5 antibody of the present invention (including an antigen-binding fragment) is provided.
- the anti-MCT5 antibody of the present invention recognizes MCT5 expressed in cancer cells.
- the pharmaceutical composition of the present invention containing the anti-MCT5 antibody of the present invention can be used for killing cancer cells expressing MCT5. That is, the pharmaceutical composition of the present invention is useful as a pharmaceutical composition for treating cancer, preferably a pharmaceutical composition for treating colorectal cancer.
- the pharmaceutical composition of the present invention can be used as a cancer therapeutic agent.
- the monoclonal antibody of the present invention may be conjugated with a chemotherapeutic agent, toxin or radioisotope. Accordingly, in another aspect of the invention, a pharmaceutical composition is provided comprising an antibody conjugate of the invention.
- the pharmaceutical composition of the present invention containing the monoclonal antibody of the present invention is preferably a pharmaceutical composition for cancer treatment, preferably It is useful as a pharmaceutical composition for treating colorectal cancer.
- the pharmaceutical composition of the present invention can also be used as a cancer therapeutic agent.
- the cancer therapeutic agent of the present invention includes an anti-MCT5 antibody alone or, as necessary, a pharmaceutically acceptable anticancer agent or carrier appropriately combined with the anti-MCT5 antibody.
- the cancer therapeutic agent of the present invention can be used in combination with other treatment modalities.
- the monoclonal antibody of the present invention recognizes MCT5 expressed in cancer cells, binds to MCT5 on the cell membrane, and then is taken into the cell via an internalization mechanism. Therefore, the pharmaceutical composition of the present invention can be used for killing cancer cells expressing MCT5.
- the chemotherapeutic agent conjugated to the antibody can also be taken up by cancer cells, so that the pharmaceutical composition of the present invention is used in cancer patients. Useful for killing cancer cells.
- cancer treatment includes killing cancer cells, reducing the size of cancer, suppressing or stopping the growth rate of cancer, or suppressing or stopping the progression of cancer. And so on.
- the pharmaceutical composition of the present invention can be a pharmaceutical composition in which the antibody of the present invention is combined with a chemotherapeutic agent, toxin or radioisotope.
- a chemotherapeutic agent to be used in combination, the same chemotherapeutic agent contained in the pharmaceutical composition of the present invention may be used, or a different chemotherapeutic agent may be used.
- a person skilled in the art can appropriately set the dosage and administration method of the anticancer agent.
- the type of cancer to be treated by the pharmaceutical composition of the present invention is not particularly limited as long as it expresses MCT5.
- the pharmaceutical composition of the present invention may be administered orally, parenterally (for example, subcutaneous administration, intradermal administration, mucosal administration, rectal administration, intravaginal administration, topical administration to the affected area, skin administration, etc.), or Examples include direct administration to the affected area.
- parenterally for example, subcutaneous administration, intradermal administration, mucosal administration, rectal administration, intravaginal administration, topical administration to the affected area, skin administration, etc.
- Examples include direct administration to the affected area.
- the dosage of the pharmaceutical composition of the present invention can be appropriately set in consideration of the age, weight, type and progress of disease, administration route, number of administrations, administration period, etc. of the subject of administration (patient). it can.
- the dosage of the pharmaceutical composition of the present invention is generally determined in consideration of the compounding ratio of the active ingredient in the preparation, and the age, body weight, type / progression of the disease, administration route, administration of the administration subject (patient) It can be set as appropriate in consideration of the number of times, the administration period, and the like.
- the active ingredient include a chemotherapeutic agent conjugated with the monoclonal antibody of the present invention, a toxin or a radioisotope.
- parenteral preparation When used as a parenteral preparation, its form is not generally limited. For example, any of intravenous injection (including infusion), intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, etc. There may be.
- the parenteral preparation contains various known excipients and additives according to various forms, and the effect of the active ingredient is impaired. It can be contained in a range that is not.
- water, glycerol, propylene glycol, aliphatic polyalcohols such as polyethylene glycol, and the like can be mentioned.
- the dose (per day) of the parenteral agent is not limited.
- the above-mentioned active ingredient is 1 mg to 15 mg / kg body weight of the subject (patient). It is preferably 2 to 12 mg / day.
- its form is generally not limited, and for example, any of tablets, capsules, granules, powders, pills, troches, liquids for internal use, suspensions, emulsions, syrups, etc. Alternatively, it may be a dry product which is redissolved when used.
- the pharmaceutical composition of the present invention can contain pharmaceutically acceptable additives as necessary.
- pharmaceutically acceptable additives include antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles. , Diluents, carriers, excipients and / or pharmaceutical adjuvants and the like.
- the present invention provides a kit containing the anti-MCT5 antibody of the present invention for treating cancer, for example, colon cancer. If the kit of this invention contains the anti- MCT5 antibody of this invention, the material which comprises it will not be specifically limited.
- the anti-MCT5 antibody of the present invention water, buffer solution, container, syringe, instruction manual and the like may be provided.
- the anti-MCT5 antibody of the present invention is provided in an aqueous solution, a lyophilized state, or the like, and may be prepared in an appropriate state before use.
- the present invention also provides a method for treating cancer, comprising administering the anti-MCT5 antibody of the present invention to a subject (eg, a human).
- a subject eg, a human
- the present invention also provides the anti-MCT5 antibody of the present invention for use in the treatment of cancer.
- the description of the pharmaceutical composition of the present invention can be referred to.
- the present invention also provides a method for treating cancer, comprising administering the monoclonal antibody of the present invention to a subject.
- the monoclonal antibody of the present invention may be conjugated with a chemotherapeutic agent, a toxin or a radioisotope. That is, the therapeutic method of the present invention may include administering the antibody conjugate of the present invention to a subject.
- a further anticancer agent may be used in combination.
- the dosage of the pharmaceutical composition of the present invention can be appropriately set in consideration of the age, weight, type and progress of disease, administration route, number of administrations, administration period, etc. of the subject of administration (patient). it can.
- parenteral preparation When used as a parenteral preparation, its form is not generally limited. For example, any of intravenous injection (including infusion), intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, etc. There may be.
- the parenteral preparation contains various known excipients and additives according to various forms, and the effect of the active ingredient is impaired. It can be contained in a range that is not.
- water, glycerol, propylene glycol, aliphatic polyalcohols such as polyethylene glycol, and the like can be mentioned.
- the dose (per day) of the parenteral agent is not limited.
- the above-mentioned active ingredient is 1 mg to 15 mg / kg body weight of the subject (patient). It is preferably 2 to 12 mg / day.
- its form is generally not limited, and for example, any of tablets, capsules, granules, powders, pills, troches, liquids for internal use, suspensions, emulsions, syrups, etc. Alternatively, it may be a dry product which is redissolved when used.
- Example 1 Cells HCT116 was obtained from DS Pharma and MDA-MB231 was obtained from American Type Culture collection (ATCC accession number HTB-26). The cells described in Japanese Patent Application No. 2014-203162 were used as the cells in which MCT7 was overexpressed in MCF7 cells.
- IMI4C4a Collection of antibody-producing cells Clone No. IMI4C4a, which is a monoclonal antibody that binds to MCT5, is immunized by administering MCT5 or a partial peptide by itself or with a carrier and diluent to a mammal. Can be obtained.
- a region consisting of the 196th to 299th amino acids of MCT5 SEQ ID NO: 32
- This area is hereinafter referred to as “ICD4”.
- the dose of antigen per animal, the type of adjuvant used, the immunization method, and the interval between immunizations are the same as in the production of polyclonal antibodies.
- antibody-producing cells include spleen cells, lymph node cells, peripheral blood cells and the like, with spleen cells or lymph node cells being preferred.
- (Ii) Cell fusion Cell fusion between antibody-producing cells and myeloma cells is performed to obtain hybridomas.
- the fusion operation can be performed according to a known method, for example, the method of Kohler et al.
- myeloma cells to be fused with antibody-producing cells generally available cell lines of animals such as mice can be used.
- the cell line used has drug selectivity and cannot survive in a HAT selection medium (including hypoxanthine, aminopterin, and thymidine) in an unfused state, but can survive only in a state fused with antibody-producing cells. Those having the following are preferred.
- Examples of myeloma cells include P3X63-Ag8, P3X63-Ag8U.
- mouse myeloma cell lines such as SP2 / 0-Ag14, PAI, P3U1, NSI / 1-Ag4-1, NSO / 1, and rat myeloma cell lines such as YB2 / 0.
- the cell fusion between the myeloma cell and the antibody-producing cell is performed by using 1 ⁇ 10 8 to 5 ⁇ 10 8 antibody-producing cells and 2 ⁇ in an animal cell culture medium such as serum-free DMEM or RPMI-1640 medium.
- 10 7 to 10 ⁇ 10 7 myeloma cells are mixed (cell ratio of antibody-producing cells to myeloma cells 10: 1 to 1: 1), and a fusion reaction is performed in the presence of a cell fusion promoter.
- a cell fusion promoter polyethylene glycol having an average molecular weight of 1000 to 6000 dalton, Sendai virus, or the like can be used.
- antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device utilizing electrical stimulation (for example, electroporation).
- the target hybridoma is selected from the cells after cell fusion treatment.
- the cell suspension is appropriately diluted with, for example, 10 to 20% fetal bovine serum-containing RPMI-1640 medium, and then plated on a microtiter plate by a limiting dilution method to calculate approximately 0.3 cells / well.
- a selective medium such as HAT medium is added to each well, and thereafter, the selective medium is appropriately exchanged and cultured.
- Hybridoma screening is not particularly limited, and may be performed according to ordinary methods.
- a part of the culture supernatant contained in the well in which the hybridoma is cultured can be collected and screened by enzyme immunoassay, radioimmunoassay or the like. Specifically, after antigen is adsorbed on a 96-well plate, it is blocked with calf serum, skim milk, or the like.
- the hybridoma cell culture supernatant is reacted with the immobilized antigen at 37 ° C. for 1 hour, peroxidase-labeled anti-mouse IgG is reacted at 37 ° C. for 1 hour, and color is developed using orthophenylenediamine as a substrate.
- screening can be performed by measuring the absorbance at a wavelength of 490 nm.
- the hybridoma producing a monoclonal antibody that is positive by the above measurement method is cloned by a limiting dilution method or the like. Finally, a hybridoma that is a cell producing a monoclonal antibody that specifically binds to MCT5 is established.
- (Iv) Collection of monoclonal antibody As a method of collecting a monoclonal antibody from an established hybridoma, a normal cell culture method or ascites formation method can be employed.
- the hybridoma is cultured in an animal cell culture medium such as RPMI-1640 medium containing 10% fetal bovine serum, MEM medium, or serum-free medium under normal culture conditions (for example, 37 ° C., 5% CO 2 concentration). Culturing for 7 to 14 days, and antibodies are obtained from the culture supernatant.
- an animal cell culture medium such as RPMI-1640 medium containing 10% fetal bovine serum, MEM medium, or serum-free medium under normal culture conditions (for example, 37 ° C., 5% CO 2 concentration).
- ELISA A sample obtained by diluting blood collected from the tail vein of a mouse with PBS ( ⁇ ) to 1 / 160,000 or a culture supernatant of a well in which a hybridoma had grown was collected, and the antibody titer was analyzed. ICD4 was diluted with PBS ( ⁇ ), dispensed to a 96-well ELISA plate (Nunc) at 50 ng per well, and allowed to stand at room temperature for 1 hour to bind to the plate surface.
- TBS-T 25 mM Tris, 150 mM NaCl, 0.05% (v / v) Tween 20, pH 7.4
- PBS-T containing 5% skim milk 300 ⁇ L was added to each well. Dispensing and blocking for 1 hour at room temperature. After washing with TBS-T, the diluted blood or culture supernatant was dispensed onto an ICD4-binding ELISA plate and allowed to react at room temperature for 1 hour.
- FIG. 1 shows the result of analyzing the monoclonal antibody prepared using ICD4 as an antigen by ELISA using the culture supernatant of each hybridoma cell. From these clones, cells were monocloned by the limiting dilution method, and the subsequent experiments were performed.
- FIG. 2 shows the results of staining intact cells with the IMI4C4a antibody.
- MCF7-MCT5 cells when MCT5 is overexpressed, EGFP is also overexpressed at the same time, and therefore, fluorescence by intracellular EGFP is observed with excitation at 488 nm. Therefore, in the case of MCF7-MCT5 cells, anti-mouse IgG polyclonal antibody-Alexa Fluor 555 labeling was used as the secondary antibody. In all cells, the IMI4C4a antibody was shown to be bound to the cell surface.
- HCT116 cells which are human colon cancer cells, were cultured to be 90% confluent. The cells were washed twice with PBS ( ⁇ ), then collected using Accutase (Life Technologies), and suspended in 100 mL of PBS ( ⁇ ) containing 1% FBS so as to be 4 ⁇ 10 5 cells. To this solution, IMI4C4a antibody was added to a final concentration of 10 mg / mL, and reacted on ice for 60 minutes. As a comparison object, cells to which a solution containing no IMI4C4a antibody was added were prepared.
- Example 2 (1) Analysis of internalization Example 1 above. As shown in Fig. 2, it was analyzed whether the IMI4C4a antibody, which is an anti-MCT5 monoclonal antibody, was bound on the cell surface and then taken up into the cell by internalization. Human breast cancer cells SK-BR3, MDA-MB231 and human colorectal cancer cells HCT116 cells were seeded on a glass bottom dish at 1 ⁇ 10 4 cells / well. After culturing for 1 day, the cells were cooled by standing on ice for 15 minutes. After removing the medium, the primary antibody prepared in the medium so that the IMI4C4a antibody was 10 mg / mL was added and reacted on ice for 30 minutes.
- IMI4C4a antibody which is an anti-MCT5 monoclonal antibody
- FIG. 4 shows that the IMI4C4a antibody is taken into cells in any of the cells of SK-BR3, MDA-MB231, and HCT116.
- Trastuzumab shown in the left panel of FIG. 4 is an antibody whose antibody (green fluorescence) has been shown to be internalized.
- the antibody on the cell surface stays on the cell surface for up to 30 minutes, and then is taken into the cell and then quickly taken into Lyssome (red fluorescence), thus showing yellow fluorescence.
- Lyssome red fluorescence
- Endocytosis varies depending on the type and size of the substance to be taken in and the difference in cellular molecules involved, but it is roughly classified into clathrin-dependent endocytosis, caveolae-dependent endocytosis, phagocytosis, and pinocytosis. Has been. Many membrane proteins such as receptors are taken up by clathrin-dependent endocytosis, and an endocytosis inhibitor is used for the analysis.
- ConA concanavalin A
- FIG. 5 shows that pretreatment with ConA inhibits IMI4C4a antibody internalization during the observed period.
- 0 mg / mL in the left panel shows the change over time when ConA is not contained, and the right panel shows the result when ConA is contained.
- pretreatment with ConA it was shown that the antibody remained on the cell surface even during the culture time during which the antibody was originally transported into the cell. From this, it was confirmed that the IMI4C4a antibody bound to MCT5 on the cell membrane was taken up by clathrin-dependent endocytosis.
- trastuzumab which has been analyzed as an antibody to be internalized, undergoes turnover that promptly returns to the outside of the cell, although the antibody bound to the outside of the cell is internalized. Therefore, as shown in the right panel of FIG. 3, it is considered that the internalization could not be clearly detected in the culture for 0 to 30 minutes.
- IMI4C4a since the internalization was clearly detected even after 15 minutes of culture, it is judged that the efficiency of incorporation is extremely good. That is, it is considered that cancer cells can be killed even with a low concentration of antibody-drug conjugate.
- Example 3 Biotinylation of antibody
- the IMI4C4a antibody was biotinylated using EZ-link NHS-LC-LC-Biotin (PIERCE) according to the manual attached to the kit.
- An IMI4C4a antibody prepared to 1 mg / mL in PBS ( ⁇ ) was prepared, and NHS-LC-LC-Biotin powder was dissolved in DMSO and added to the antibody solution. After reacting at room temperature for 30 minutes, 1M amount of 1M Tris-HCl (pH 7.4) was added to inactivate unreacted NHS-LC-LC-Biotin reagent. Unreacted reagents were removed using Amicon Ultra 30 kDa (Millipore), and the solvent was replaced with PBS ( ⁇ ).
- the present invention provides a monoclonal antibody that specifically binds to the extracellular region of MCT5.
- the antibody of the present invention can be used for cancer treatment by specifically binding to a cancer cell expressing MCT5 and transporting the molecule bound to the antibody into the cell.
Abstract
Description
この一方で、乳癌手術においては、術後の生活の質(クオリティ・オブ・ライフ、QOL)を高めることを目的として、温存治療が行われている。2001年の時点でも、全体の40%以上の患者で実施されているが、温存療法を実施した患者の中には再発又は転移のリスクが高い患者も含まれている。
「再発」又は「転移」が起きるメカニズムとしては、浸潤能の高い癌細胞が原発巣と同じ組織内の別の部位又は癌周辺組織に局所的に浸潤し、手術等を行っても取り残されることが大きな原因と考えられる。
乳癌に対する分子標的薬として日本で承認された抗体医薬品としてはハーセプチンが知られている。ハーセプチンは、HER2(ERBB2,Her2/neuとも呼ばれる)という受容体をターゲットとした抗体医薬品であり、HER2過剰発現が確認された転移性乳癌に対する治療薬での使用が承認されており、細胞膜上のHER2に結合し、ナチュラルキラー細胞(NK細胞)やマクロファージなどによる抗体依存性細胞障害(ADCC、Antibody−Dependent Cellular Cytotoxicity)により癌細胞を死滅させる作用機序で細胞を死滅させていると考えられている。
大腸癌に対する抗体医薬品としては、アバスチンやアービタックスが知られている。これらは、VEGFやEGFという増殖因子をターゲットとした抗体医薬品である。アバスチンは治癒切除が不可能な進行・再発の大腸癌での使用が承認されており、VEGFに結合し、VEGF受容体との結合を抑制することで血管新生を抑制して、腫瘍組織への栄養を絶つという作用機序で抗癌作用を発揮する。アービタックスは、EGF受容体に結合し、EGFによる細胞増殖シグナルが働かないようにすることで癌細胞の増殖を停止させるのが狙いである。
特許文献1:特表2010−532169公報
非特許文献1:Nature Reviews,5,591−602,2005
非特許文献2:British Journal of Cancer,94,1016−1020,2006
非特許文献3:Cancer Research,57,4593−4599,1997
非特許文献4:Journal of Clinical Oncology,22,1201−1208,2004
非特許文献5:GASTROENTEROLOGY,138,2151−2162,2010
非特許文献6:Cancer Research,68,9280−90,2008
非特許文献7:The Journal of Biological Chemistry,285,27289−27301,2010
(1) MCT5又はその断片に対するモノクローナル抗体。
(2) MCT5の断片が、MCT5の細胞外領域の断片である(1)に記載の抗体。
(3) MCT5の細胞外領域の断片が、配列番号4で示されるアミノ酸配列からなるものである(1)又は(2)に記載の抗体。
(4) 抗体が、キメラ抗体、ヒト型化抗体又はヒト化抗体である、(1)~(3)のいずれか1項に記載の抗体。
(5) (1)~(4)のいずれか1項に記載の抗体が結合する抗原決定基に結合する抗体。
(6) (1)~(5)のいずれか1項に記載の抗体の断片。
(7) (1)~(4)のいずれか1項に記載の抗体を産生するハイブリドーマ。
(8) (1)~(5)のいずれか1項に記載の抗体又は(6)に記載の断片を含む、癌治療用医薬組成物。
(9) (1)~(5)のいずれか1項に記載の抗体又は(6)に記載の断片と、化学療法剤、トキシン又はラジオアイソトープとの組み合わせを含む、癌治療用医薬組成物。
(10) (1)~(5)のいずれか1項に記載の抗体又は(6)に記載の断片に化学療法剤、トキシン又はラジオアイソトープが結合した複合体を含む、癌治療用医薬組成物。
本発明者は、以前の研究において、MCT5が乳癌及び大腸癌に発現すること、並びに、MCT5を認識するモノクローナル抗体が大腸癌の検出薬及び診断薬として利用できることを見出した。さらに、従来、細胞内ドメインと考えられていたMCT5の塩基配列751~774の領域(AATTTAACAGTCTCACAAAATCAA(配列番号3))、アミノ酸残基251~258の領域(NLTVSQNQ(配列番号4))が、細胞外に存在している事を示した。加えて、MCT5を過剰発現させたヒト乳癌細胞の浸潤能が増加すること、抗MCT5抗体(クローン番号IMI4C4a)は腫瘍組織の一部のみに結合すること、及び抗MCT5抗体陽性組織は、乳癌組織の進行ステージが進むにつれて発現する割合が上昇することから、MCT5は浸潤能の高い乳癌に特異的に発現し、抗MCT5抗体は悪性度の高い乳癌を検出することが出来る事を示した。さらに、IMI4C4a抗体はMCT5タンパク質の251~258番目(配列番号4:アミノ酸配列)をエピトープとしていることを示した。
本発明のモノクローナル抗体(以下「本発明の抗MCT5抗体」ともいう)は、ネイティブなMCT5を認識することができる。「ネイティブな」とは、そのタンパク質が生体内の環境で有するインタクトな立体構造の状態にあることをいう。
本発明の抗体は、MCT5の立体構造がデータベースとは異なる事を見出したことにより、新たに得られた抗体であるために、細胞表面上のネイティブなMCT5を認識することができる。また、抗MCT5抗体であるIMI4C4aのエピトープの近傍を抗原とした従来のモノクローナル抗体(HPA046986)は、ネイティブなMCT5を認識することが出来ず、細胞表面上のMCT5に結合しない。これに対して、本発明の抗体の認識部位はMCT5の細胞外ドメインに結合し、生きた細胞に結合することができる。そのため、本発明の抗MCT5抗体は、MCT5を発現する癌細胞を標的とする治療薬として有効である。
本発明の抗MCT5抗体のエピトープ(抗原決定基)は、抗原であるMCT5の少なくとも一部であればよく限定はされないが、MCT5の細胞外領域の少なくとも一部が好ましく、例えば、配列番号2で示されるMCT5のアミノ酸配列のうち、第196番目~第299番目のアミノ酸からなる領域(配列番号15)の少なくとも一部であることが好ましい。なかでも、第227番目~第258番目のアミノ酸からなる領域(配列番号16)の少なくとも一部がより好ましく、特に好ましくは、第251番目~第258番目のアミノ酸(配列番号4)からなる領域の少なくとも一部である。当該領域を認識する(当該領域と結合する)抗MCT5抗体は、例えば、生体試料中のMCT5の検出感度が高く、後述する癌の検出及び診断の用途に極めて有用なものである。
また、配列番号17を含む抗原を用いて作製した抗体は、isoform1に加え、isoform2及びisoform5も検出することが出来る。さらに、配列番号19を用いて作製した抗体は、isoform3、isoform4及びisoform6も検出することが出来る。
本発明のMCT5に対する抗体には、当該抗体が結合する部位(例えばエピトープ)に結合する抗体、例えば、本発明のハイブリドーマが産生する抗体が結合する部位に結合する抗体が含まれる。
本発明の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、ヒト型化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。
マウス−ヒトキメラ抗体としては、重鎖可変領域をヒトの定常領域と結合させたアミノ酸配列(配列番号29)、軽鎖可変領域をヒトの定常領域と結合させたアミノ酸配列(配列番号32)を含むもの若しくは当該アミノ酸からなるものが好ましい。
さらに、本発明の抗体が融合したタンパク質は、抗体の可変領域とその他のタンパク質を公知の遺伝子組換え方法を用いることにより作製することができる。また、当該融合タンパク質は、モノクローナル抗体と他のタンパク質とをクロスリンカーを用いて架橋することにより作製することができる。
本発明で使用されるMCT5に対する抗体の断片は、MCT5に特異的に結合する。
抗体の断片は、本発明の抗体の一部分の領域を意味し、例えば、Fab、Fab′、F(ab′)2、Fv、diabody(dibodies)、dsFv、scFv(single chain Fv)などが挙げられる。上記抗体断片は、本発明の抗体を目的に応じて各種タンパク質分解酵素で切断することにより得ることができる。
例えば、Fabは、抗体分子をパパインで処理することにより、F(ab′)2は、抗体分子をペプシンで処理することによりそれぞれ得ることができる。また、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することで得ることができる。
diabodyの場合は、抗体のH鎖V領域及びL鎖V領域をコードするcDNAを取得し、ペプチドリンカーのアミノ酸配列の長さが8残基以下となるようにscFvをコードするDNAを構築する。このDNAを発現ベクターに挿入し、当該発現ベクターを宿主生物に導入して発現させることにより、diabodyを製造することができる。
本発明において、重鎖可変領域をコードするDNAの塩基配列としては、例えば配列番号5で示される塩基配列を含むもの又は当該塩基配列からなるものが挙げられ、軽鎖可変領域をコードするDNAの塩基配列としては、例えば配列番号10で示される塩基配列を含むもの又は当該塩基配列からなるものが挙げられる。
結合親和性は、結合定数(KA)及び解離定数(KD)により決定することができる。親和性平衡定数(K)はKA/KDの比で表される。その結合親和性は、以下のようにして検出することができる。
結合親和性は、少なくとも1×10−10Mの解離定数(KD)を有しており、この解離定数に対し、例えば2~5倍、5~10倍、10~100倍、100~1000倍又は1000~10,000倍高い親和性を有する。具体的には、本発明の抗体は、MCT5の結合親和性に関する解離定数(KD)が、1×10−10M、5×10−11M、1×10−11M、5×10−12M、1×10−12M、5×10−13M、1×10−13M、5×10−14M、1×10−14M、5×10−15M、又は1×10−15Mであり、1×10−10M~1×10−13Mであることがより好ましい。あるいはこれらのKDよりも低い値であり高親和性であってもよい。
結合定数(KA)及び解離定数(KD)は、表面プラズモン共鳴(SPR)を用いて測定することができ、結合率をリアルタイムで検出し、さらにモニタリングする公知の機器及び方法を採用することができる(例えばBiacore(登録商標)T200(GEHealthcare社)、ProteON XPR36(Bio−Rad社)など)。
本発明の組換え抗体を発現するのに好ましい哺乳動物細胞としては、チャイニーズハムスター卵巣細胞(CHO細胞)、COS細胞、293細胞、HeLa細胞、3T3細胞などが挙げられる。抗体重鎖及び軽鎖をコードする組換え発現ベクターを公知の遺伝子導入方法によって、好ましい宿主細胞に導入する。得られた形質転換体を培養し、培養物から目的の抗体又は抗原結合断片を回収する。公知の精製技術により、抗体又は抗原結合断片を精製してもよい。
本発明の別の態様において、本発明のモノクローナル抗体(その抗原結合断片を含む)は、少なくとも1種類の化学療法剤(抗がん剤を含む)、トキシン、又はラジオアイソトープをコンジュゲートして複合体とすることができ、そのような複合体(本発明の抗体コンジュゲートともいう)も、本発明の範囲に含まれる。
(triethylenethiophosphaoramide)及びトリメチローロメラミン
(trimethylolomelamine)を含むエチレンイミン類及びメチラメラミン類;アセトゲニン(acetogenins)(特にブラタシン(bullatacin)及びブラタシノン(bullatacinone));カンプトセシン(合成類似体トポテカン(topotecan)を含む);ブリオスタチン;カリスタチン(callystatin);CC−1065(そのアドゼレシン(adozelesin)、カルゼレシン(carzelesin)及びバイゼレシン(bizelesin)合成類似体を含む);クリプトフィシン(cryptophycin)(特にクリプトフィシン1及びクリプトフィシン8);ドラスタチン(dolastatin);デュオカルマイシン(duocarmycin)(合成類似体、KW−2189及びCBI−TM1を含む);エレトロビン(eleutherobin);パンクラチスタチン
(pancratistatin);サルコディクチン(sarcodictyin);スポンジスタチン(spongistatin);クロランブシル、クロルナファジン(chlornaphazine)、チョロホスファミド(cholophosphamide)、エストラムスチン、イホスファミド、メクロレタミン、メクロレタミンオキシドヒドロクロリド、メルファラン、ノベンビチン(novembichin)、フェネステリン(phenesterine)、プレドニムスチン(prednimustine)、トロフォスファミド(trofosfamide)、ウラシルマスタード等のナイトロジェンマスタード;ニトロスレアス(nitrosureas)、例えばカルムスチン(carmustine)、クロロゾトシン(chlorozotocin)、フォテムスチン(fotemustine)、ロムスチン(lomustine)、ニムスチン、ラニムスチン;エネジイン(enediyne)抗生物質等の抗生物質(例えば、カリケアマイシン(calicheamicin)、カリケアマイシンガンマ1I及びカリケアマイシンオメガI1、例えば、Agnew Chem Intl.Ed.Engl.,33:183−186(1994)を参照のこと;ダイネミシン(dynemicin)、ダイネミシンA(dynemicinA);クロドロネート(clodronate)などのビスホスホネート(bisphosphonates);エスペラマイシン(esperamicin);同様にネオカルチノスタチン発光団及び関連色素蛋白エネジイン(enediyne)抗生物質発光団)、アクラシノマイシン(aclacinomysins)、アクチノマイシン、オースラマイシン(authramycin)、アザセリン、ブレオマイシン(bleomycins)、カクチノマイシン(cactinomycin)、カラビシン(carabicin)、カルミノマイシン(carminomycin)、カルジノフィリン(carzinophilin)、クロモマイシン、ダクチノマイシン、ダウノルビシン、デトルビシン(detorubicin)、6−ジアゾ−5−オキソ−L−ノルロイシン、モルフォリノ−ドキソルビシン、シアノモルフォリノ−ドキソルビシン、2−ピロリノ−ドキソルビシン及びデオキシドキソルビシンを含む)、エピルビシン、エソルビシン(esorubicin)、イダルビシン、マセロマイシン(marcellomycin)、マイトマイシンCなどのマイトマイシン(mitomycins)、マイコフェノール酸(mycophenolic acid)、ノガラマイシン(nogalamycin)、オリボマイシン(olivomycins)、ペプロマイシン、ポトフィロマイシン(potfiromycin)、ピューロマイシン、クエラマイシン(quelamycin)、ロドルビシン(rodorubicin)、ストレプトニグリン、ストレプトゾシン、ツベルシジン(tubercidin)、ウベニメクス、ジノスタチン(zinostatin)、ゾルビシン(zorubicin);メトトレキセート及び5−フルオロウラシル(5−FU)のような抗−代謝産物;デノプテリン(denopterin)、メトトレキセート、プテロプテリン(pteropterin)、トリメトレキセート(trimetrexate)のような葉酸類似体;フルダラビン(fludarabine)、6−メルカプトプリン、チアミプリン、チオグアニンのようなプリン類似体;アンシタビン、アザシチジン(azacitidine)、6−アザウリジン(azauridine)、カルモフール、シタラビン、ジデオキシウリジン、ドキシフルリジン、エノシタビン(enocitabine)、フロキシウリジン(floxuridine)のようなピリミジン類似体;カルステロン(calusterone)、プロピオン酸ドロモスタノロン、エピチオスタノール、メピチオスタン、テストラクトン(testolactone)のようなアンドロゲン類;アミノグルテチミド、ミトタン、トリロスタンのような抗副腎剤;フロリン酸(frolinic acid)のような葉酸リプレニッシャー(replenisher);アセグラトン;アルドホスファミドグリコシド;アミノレブリン酸;エニルウラシル(eniluracil);アムサクリン(amsacrine);ベストラブシル(bestrabucil);ビサントレン(bisantrene);エダトラキセート(edatraxate);デフォファミン(defofamine);デメコルシン(demecolcine);ジアジコン(diaziquone);エルフォルニチン
(elfornithine);酢酸エリプチニウム(elliptinium acetate);エポチロン(epothilone);エトグルシド(etoglucid);硝酸ガリウム;ヒドロキシ尿素;レンチナン;ロニダミン(lonidamine);メイタンシン(maytansine)及びアンサマイトシン(ansamitocin)のようなメイタンシノイド
(maytansinoid);ミトグアゾン(mitoguazone);ミトキサントロン;モピダモール(mopidamol);ニトラクリン(nitracrine);ペントスタチン;フェナメット(phenamet);ピラルビシン;ポドフィリン酸(podophyllinic acid);2−エチルヒドラジド;プロカルバジン;PSK(登録商標)多糖類複合体(JHS Natural Products,Eugene,OR);ラゾキサン(razoxane);リゾキシン(rhizoxin);シゾフィラン;スピロゲルマニウム(spirogermanium);テニュアゾン酸(tenuazonic acid);トリアジコン(triaziquone);2,2′,2′′−トリクロロトリエチルアミン;トリコテセン(trichothecenes)(T−2トキシン、ベラキュリンA(verracurin A)、ロリデンA(roridin A)及びアングイデン(anguidine));ウレタン;ビンデシン;ダカルバジン;マンノムスチン(mannomustine);ミトブロニトール;ミトラクトール(mitolactol);ピポブロマン(pipobroman);ガシトシン(gacytosine);アラビノシド(「Ara−C」);シクロホスファミド;チオテパ;6−チオグアニン;メルカプトプリン;メトトレキセート;シスプラチン及びカルボプラチンのようなプラチナ類似体;ビンブラスチン;エトポシド(VP−16);ミトキサントン;ビンクリスチン;ビノレルビン(NAVELBINE(登録商標));ナベルビン(Navelbine);ノバントロン(novantrone);テニポシド;エダトレキセート;ダウノマイシン;アミノプテリン;カペシタビン(XELODA(登録商標),Roche);イバンドロナート(ibandronate);CPT−11;トポイソメラーゼインヒビターRFS2000;ジフルオロメチロールニチン(DMFO);レチノイン酸などのレチノイド類;並びに上述したものの製薬的に許容可能な塩類、酸類及び誘導体が含まれる。
(nilutamide)、ビカルタミド、ロイプロリド、及びゴセレリン;並びにトロキサシタビン(troxacitabine)(1,3−ジオキソランヌクレオシドシトシン類似体);(iv)プロテインキナーゼインヒビター、例えばMEKインヒビター(国際公開2007/044515);(v)脂質キナーゼインヒビター;(vi)アンチセンスオリゴヌクレオチド、特に不粘着性細胞増殖に関係するシグナル伝達経路の遺伝子の発現を抑制するもの、例えばPKC−α、Raf及びH−Ras、オブリメルセン(GENASENSE(登録商標),Genta Inc.);(vii)リボザイム、例えばVEGF発現インヒビター(例えばANGIOZYME(登録商標))及びHER2発現インヒビター;(viii)遺伝子治療ワクチン、例えばALLOVECTIN(登録商標)、LEUVECTIN(登録商標)及びVAXID(登録商標)などのワクチン;PROLEUKIN(登録商標)rIL−2;LURTOTECAN(登録商標)などのトポイソメラーゼ1インヒビター;ABARELIX(登録商標)rmRH;(ix)ベバシズマブ(AVASTIN(登録商標),Genentech)などの抗血管形成剤;並びに上記のものの製薬的に許容可能な塩類、酸類又は誘導体が含まれる。また、「化学療法剤」の定義には、治療的抗体、例として、アレムツズマブ(Campath)、ベバシズマブ(アバスチン(登録商標),Genentech);セツキシマブ(ERBITUX(登録商標),Imclone);パニツムマブ(VECTIBIX(登録商標),Amgen)、リツキシマブ(RITUXAN(登録商標),Genentech/Biogen Idec)、ペルツズマブ(OMNITARG TM,2C4,Genentech)、トラスツズマブ(HERCEPTIN(登録商標),Genentech)、トシツモマブ(Bexxar,Corixia)、及び抗体薬剤コンジュゲート、ゲムツズマブオゾガマイシン(MYLOTARG(登録商標),Wyeth)が含まれる。
ここで、「殺腫瘍活性」とは、腫瘍細胞を破壊又は死滅させる活性を意味し、「腫瘍抑制活性」とは腫瘍細胞の数を減少させる活性又は腫瘍細胞の増殖速度を抑制させる活性を意味する。
エフェクター細胞に対して特異的な抗原結合領域を含む本発明のモノクローナル抗体ががん細胞に結合すると、エフェクター細胞が当該抗体に結合し、抗体依存性細胞傷害(ADCC、Antibody−Dependent Cellular Cytotoxicity)活性によりがん細胞を死滅させることが可能となる。
同様に、このような領域を含む本発明のモノクローナル抗体ががん細胞に結合すると、補体系が活性化し、CDC(Complement−Dependent Cytotoxicity:補体依存性細胞傷害)活性によってがん細胞を死滅させることが可能となる。
本発明において、本発明の抗MCT5抗体(抗原結合断片を含む)を含む医薬組成物が提供される。本発明の抗MCT5抗体は、がん細胞に発現するMCT5を認識する。本発明の抗MCT5抗体を含む本発明の医薬組成物は、MCT5を発現するがん細胞を殺傷するために使用することが可能である。すなわち、本発明の医薬組成物は、がん治療用医薬組成物、好ましくは大腸がん治療用医薬組成物として有用である。また、本発明の医薬組成物は、がん治療剤として使用することができる。
非経口剤として用いる場合、一般にその形態は限定されるものではなく、例えば、静脈内注射剤(点滴を含む)、筋肉内注射剤、腹腔内注射剤、皮下注射剤、坐剤等のいずれであってもよい。
非経口剤として用いる場合、一般にその形態は限定されるものではなく、例えば、静脈内注射剤(点滴を含む)、筋肉内注射剤、腹腔内注射剤、皮下注射剤、坐剤等のいずれであってもよい。
(1)細胞
HCT116はDSファーマより、MDA−MB231はAmerican Type Culture collection(ATCC受託番号HTB−26)より入手した。MCF7細胞にMCT5を過剰発現させた細胞は、特願2014−203162公報に記載された細胞を用いた。
(i)抗体産生細胞の採取
MCT5と結合するモノクローナル抗体であるクローン番号IMI4C4aは、MCT5又は部分ペプチドをそれ自体で、あるいは担体及び希釈剤と共に哺乳動物に投与することにより免疫し得ることが出来る。例えばMCT5の第196番目~第299番目のアミノ酸からなる領域(配列番号32)が利用できる。この領域を以下では「ICD4」と称す。動物1匹当たりの抗原の投与量、用いられるアジュバントの種類、免疫方法、免疫の間隔はポリクローナル抗体の作製と同様である。最終の免疫日から1~30日後、好ましくは2~5日後に、抗体価の認められた個体を選択し抗体産生細胞を採集する。抗体産生細胞としては、脾臓細胞、リンパ節細胞、末梢血細胞等が挙げられるが、脾臓細胞又はリンパ節細胞が好ましい。
ハイブリドーマを得るため、抗体産生細胞とミエローマ細胞との細胞融合を行う。融合操作は既知の方法、例えばKohlerらの方法に従い実施できる。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使用することができる。使用する細胞株としては、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、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P3X63−Ag8、P3X63−Ag8U.1、SP2/0−Ag14、PAI、P3U1、NSI/1−Ag4−1、NSO/1などのマウスミエローマ細胞株、YB2/0などのラットミエローマ細胞株などが挙げられる。
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10~20%のウシ胎児血清含有RPMI−1640培地などで適当に希釈後、マイクロタイタープレート上に限界希釈法で計算上0.3個/well程度まき、各ウェルにHAT培地などの選択培地を加え、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、10日前後から生育してくる細胞をハイブリドーマとして得ることができる。
樹立したハイブリドーマ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。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。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、例えばマウス(BALB/c)の腹腔内にハイブリドーマを約5×106~2×107個投与し、ハイブリドーマを大量に増殖させる。そして、1~2週間後に腹水を採取する。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、アフィニティークロマトグラフィーなどの公知の方法を適宜選択して、又はこれらを組み合わせることにより精製することができる。
マウスの尾静脈より採取した血液をPBS(−)で1/160,000に希釈したサンプル、あるいはハイブリドーマが増殖してきたウェルの培養上清を回収し、抗体の力価を解析した。ICD4をPBS(−)で希釈し、96ウェルのELISAプレート(Nunc社)に1ウェル当たり50ngとなるよう分注して、室温で1時間放置することでプレート表面に結合させた。次に、TBS−T(25mM Tris、150mM NaCl、0.05%(v/v)Tween20、pH7.4)350μLで3回洗浄した後、5%スキムミルクを含むPBS−Tを300μLずつ各ウェルに分注し、1時間、室温でブロッキングした。TBS−Tで洗浄後、希釈した血液あるいは培養上清をICD4結合ELISAプレートに分注し、1時間、室温で反応させた。TBS−Tで洗浄後、ペルオキシダーゼ(以下、PODと記載する)標識抗マウス免疫グロブリン抗体(BETHYL社製)を10,000倍希釈したものを各ウェル100μL分注し、1時間、室温で反応させた。同様の洗浄を行った後、0.5mg/mL PODとなるよう調製したOPD基質を加え、室温、5分間発色させた。1.5Nの硫酸で反応を停止した後、490nmの吸収を、プレートリーダー(モレキュラーデバイス社)を用いて測定した。ICD4を抗原として作製したモノクローナル抗体に関して、それぞれのハイブリドーマ細胞の培養上清を用いてELISAで解析した結果を図1に示した。これらのクローンの中から、限界希釈法により細胞を単クローン化し、以後の実験を行った。
MCF7−MCT5、MDA−MB231及びHCT116細胞を80%コンフルエントとなるよう培養し、Cellmatrix type I−A(新田ゼラチン社)でコートしたGlass Bottom Dish(MATSUNAMI社)上に、1×104cells/wellとなるよう播種した。1日間培養後、上記(2)に記載したIMI4C4a抗体を10μg/mLとなるよう培地に希釈した溶液を細胞へ添加し、氷上で1時間反応させた。10%FBSを含む培地で洗浄した後、二次抗体として抗マウスIgGポリクローナル抗体−Alexa Fluor488標識あるいは抗マウスIgGポリクローナル抗体−Alexa Fluor555標識で氷上、30分間反応させた。培地で3回洗浄した後、PBS(−)で洗浄し、蛍光顕微鏡(BZ−8000、キーエンス社)で解析した。
ヒト大腸がん細胞であるHCT116細胞を90%コンフルエントとなるよう培養した。細胞をPBS(−)で2回洗浄した後、Accutase(Life Technologies社)を用いて回収し、4×105cellsとなるよう1%FBSを含むPBS(−) 100mLに懸濁した。この溶液に、最終濃度10mg/mLとなるようIMI4C4a抗体を添加し、氷上で60分間反応させた。比較対象として、IMI4C4a抗体を含まない溶液を添加した細胞を準備した。細胞をPBS+1%PBSで2回洗浄後、二次抗体として抗マウスIgGポリクローナル抗体−Alexa Fluor488標識を1/1,1000倍に希釈した溶液を添加し、氷上で30分間反応させた1%FBSを含むPBSで2回洗浄後、FACS Calibur(BD社)で解析を行った。結果を図3に示した。
(1)内在化の解析
上記実施例1.で示されるように、抗MCT5モノクローナル抗体であるIMI4C4a抗体が細胞表面上に結合した後、internalizationにより細胞内に取り込まれるかを解析した。
ヒト乳癌細胞であるSK−BR3、MDA−MB231及びヒト大腸癌細胞であるHCT116細胞をGlass Bottom Dish上に、1×104cells/wellとなるよう播種した。1日間培養を行った後、氷上に15分間静置して細胞を冷却した。培地を除去後、IMI4C4a抗体を10mg/mLとなるよう培地で調製した一次抗体を添加し、氷上で30分間反応させた。培地を用いて2回洗浄後、二次抗体として抗マウスIgGポリクローナル抗体−Alexa Fluor488標識で氷上30分反応させた。培地で2回洗浄した後、37℃、5%CO2下で0、15、30、60、120分間培養を行なった後、氷上で冷却した培地で洗浄した。さらに、細胞内のLysosomeを染色するために、PBSに200nMとなるよう調製したLysoTracker DND−99(Life Technologies社)を添加し、室温で1分間反応させた後、PBS(−)で2回洗浄した後に蛍光顕微鏡を用いて解析を行った。結果を図4に示す。
図4左のパネルに示したtrastuzumabは、抗体(緑の蛍光)が内在化されることが明らかにされている抗体である。Trastuzumabの場合は、細胞表面上の抗体は30分までは細胞表面に留まり、その後細胞内に取り込まれた後速やかにLysosome(赤の蛍光)に取り込まれるため、黄色の蛍光を示す。一方、IMI4C4a抗体の場合、培養開始15分後には細胞内への取り込みが観察され、120分の間で継時的に進行する事が示されたが、Lysosomeへの局在化は明確に検出されなかった。
Internalizationは細胞のエンドサイトーシスによって行われる。エンドサイトーシスは、取り込む物質の種類や大きさ、関与する細胞分子の違いによって異なるが大きく分けて、クラスリン依存性エンドサイトーシス、カベオラ依存性エンドサイトーシス、ファゴサイトーシス、ピノサイトーシスに分類されている。
受容体のような膜タンパク質の多くはクラスリン依存性エンドサイトーシスによって取り込まれ、その解析にはエンドサイトーシス阻害剤が利用される。
上記、実施例1(3)に記載されるように、使用した抗体は全てICD4に結合する抗体であることから、301抗体はICD4には結合するが、細胞には結合しない抗体であると考えられた。
(1)抗体のビオチン化
IMI4C4a抗体をEZ−link NHS−LC−LC−Biotin(PIERCE社)を用い、キット付属のマニュアルに従ってビオチン化した。PBS(−)に1mg/mLとなるよう調製したIMI4C4a抗体を準備し、NHS−LC−LC−Biotin粉末をDMSOに溶解させ、抗体溶液に添加した。30分間室温で反応後、1M Tris−HCl(pH7.4)を1/100量添加し、未反応のNHS−LC−LC−Biotin試薬を不活化した。Amicon Ultra 30kDa(Millipore社)を用いて未反応の試薬を除去するとともにPBS(−)に溶媒を置換した。
抗体が細胞内に取り込まれることを利用して、がん細胞の増殖を抑制できるか解析した。培地中に50nMのビオチン化IMI4C4a、303、306及び107抗体を添加し、15,30,50nMのStreptavidin−ZAP(Advanced Targeting Systems社製,Saporin−Streptavidin複合体)を混合し、氷上で1時間反応させた後、前日に2,500cells/wellとなるよう播種しておいたMCF7−MCT5細胞へ添加した。Saporinはリボソーム不活性化活性を有しており、細胞内に取り込まれた場合にのみ細胞増殖を抑制する。溶液を添加後、37℃、5%CO2下で3日間培養を行い、細胞の増殖をWST−8(Roche社)を用いて測定した。図7にその結果を示す。
以上のことから、IMI4C4a抗体は、Antibody−Drug−Conjugateとして細胞を殺傷することに利用できる事が示された。
本発明により、MCT5の細胞外領域に特異的に結合するモノクローナル抗体が提供される。本発明の抗体は、MCT5を発現する癌細胞と特異的に結合し、細胞内へ抗体に結合させた分子を輸送することで癌治療に利用することができる。
配列番号7~9、12~14、21~30:合成配列
Claims (10)
- MCT5又はその断片に対するモノクローナル抗体。
- MCT5の断片が、MCT5の細胞外領域の断片である請求項1に記載の抗体。
- MCT5の細胞外領域の断片が、配列番号4で示されるアミノ酸配列からなるものである請求項1又は2に記載の抗体。
- 抗体が、キメラ抗体、ヒト型化抗体又はヒト化抗体である、請求項1~3のいずれか1項に記載の抗体。
- 請求項1~4のいずれか1項に記載の抗体が結合する抗原決定基に結合する抗体。
- 請求項1~5のいずれか1項に記載の抗体の断片。
- 請求項1~4のいずれか1項に記載の抗体を産生するハイブリドーマ。
- 請求項1~5のいずれか1項に記載の抗体又は請求項6に記載の断片を含む、癌治療用医薬組成物。
- 請求項1~5のいずれか1項に記載の抗体又は請求項6に記載の断片と、化学療法剤、トキシン又はラジオアイソトープとの組み合わせを含む、癌治療用医薬組成物。
- 請求項1~5のいずれか1項に記載の抗体又は請求項6に記載の断片に化学療法剤、トキシン又はラジオアイソトープが結合した複合体を含む、癌治療用医薬組成物。
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US16/091,209 US20190127461A1 (en) | 2016-04-06 | 2017-04-04 | Cancer treatment pharmaceutical composition using anti-mct5 antibody |
EP17779251.2A EP3441465A4 (en) | 2016-04-06 | 2017-04-04 | PHARMACEUTICAL COMPOSITION FOR CANCER TREATMENT OF ANTI-MCT5 ANTIBODIES |
KR1020187031848A KR20180132110A (ko) | 2016-04-06 | 2017-04-04 | 항mct5 항체를 사용한 암 치료용 의약 조성물 |
CN201780028138.4A CN109072229A (zh) | 2016-04-06 | 2017-04-04 | 使用了抗mct5抗体的癌症治疗用药物组合物 |
JP2018510683A JPWO2017175874A1 (ja) | 2016-04-06 | 2017-04-04 | 抗mct5抗体を用いた癌治療用医薬組成物 |
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JP2009213391A (ja) * | 2008-03-10 | 2009-09-24 | Tama Tlo Ltd | ヒト胃がん細胞の分化型と未分化型とを識別するためのdnaプローブ・セット、該dnaプローブ・セットが搭載されたdnaマイクロアレイ及び該dnaマイクロアレイを用いたヒト胃がん用分子診断システム |
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WO2016052756A1 (ja) * | 2014-10-01 | 2016-04-07 | 株式会社オーダーメードメディカルリサーチ | 浸潤能の高い癌細胞の検出用又は診断用試薬 |
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WO2003002137A2 (en) * | 2001-06-27 | 2003-01-09 | DeveloGen Aktiengesellschaft für entwicklungsbiologische Forschung | Trp1, mct, or ftz-f1 homologous proteins involved in the regulation of energy homeostasis |
CN100552027C (zh) * | 2002-10-18 | 2009-10-21 | 株式会社Lg生命科学 | 与癌症相关的基因家族 |
US8771693B2 (en) * | 2009-10-27 | 2014-07-08 | Beth Israel Deaconess Medical Center, Inc. | Methods and compositions for the generation and use of conformation-specific antibodies |
US20110223176A1 (en) * | 2010-03-11 | 2011-09-15 | Abbott Laboratories | Basigin binding proteins |
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JP2009213391A (ja) * | 2008-03-10 | 2009-09-24 | Tama Tlo Ltd | ヒト胃がん細胞の分化型と未分化型とを識別するためのdnaプローブ・セット、該dnaプローブ・セットが搭載されたdnaマイクロアレイ及び該dnaマイクロアレイを用いたヒト胃がん用分子診断システム |
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See also references of EP3441465A4 * |
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US20190127461A1 (en) | 2019-05-02 |
JPWO2017175874A1 (ja) | 2019-02-28 |
EP3441465A4 (en) | 2019-12-04 |
CN109072229A (zh) | 2018-12-21 |
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